Claude Universite

Bernard -Lyon

M François-Noël

Gilly M Le Professeur

Hamda Ben

Hadid M Le Professeur

Philippe Lalle

M Le Professeur

Germain Gillet

M Alain Helleu

Composantes Sante

J Etienne

La Mme

C Professeure

Burillon

D Bourgeois

Vinciguerra

Y Matillon

P Farge

Keywords: Conclusion, General Conclusion, Perspectives, Chapter V : Experimental section, V.1 General methods., 4 Enzymatic assays, 5 Crystallization and structure refinement

V.2 Synthesis and characterization

COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

List of Abbreviations

uridine diphosphate [α] specific rotation λ max maximum absorption ºC degree Celsius

General Introduction

The work presented in this thesis was performed under guidance of Dr. Sébastien VIDAL conducted in Laboratoire de Chimie Organique 2 at Institut de Chimie et Biochimie Moléculaires et Supramoléculaires of Université Claude Bernard Lyon 1 and financially supported by Ministère de l'Enseignement supérieur et de la Recherche France. In 2006, a series of C-glycosyl ethylphosphonophosphate analogues of natural sugar donor were synthesized and evaluated as different glycosyltransferases inhibitors then published by our group. The present work was a continuation of this work. The aim of this project was to explore neutral sugar donor analogues of glycosyltransferase as inhibitors instead of the ionic natural substrates which are not capable of cell membrane penetration.

Compared with glycosidases, the development of inhibitors of glycosyltransferases was relatively slow due to more complex structures and catalytic mechanisms. The importance of glycosyltransferases in biology was presented and the development of glycosyltransferase inhibitors focused on galactosyltransferases (GalT) and O-linked N-acetyl-glucosaminyl transferase (OGT) was reviewed in Chapter I. Two types of nonionic inhibitors of GT with either pyridine moiety or amino-acid derivative as surrogate of the diphosphate unit of the natural UDP-sugar donors of GT were designed and synthesized. The synthetic part of these analogues was presented in Chapters II and III, respectively. The biological evaluation was performed on 6 glycosyltransferases. All the analogues synthesized were evaluated on 5 galactosyltransferases (β-1,4-GalT, α-1,4-GalT, α-1,3-GalT, GTB and a human blood group mutant AAGlyB). The best candidates were further studied with co-crystallization which gave information on the binding mode. Our designed "neutral" inhibitors of the pyridine surrogate type could chelate the manganese cation in the active binding site of the enzyme. The pyridine-containing analogues in the GlcNAc series as natural sugar donor analogues of UDP-GlcNAc were selected for O-linked N-acetyl-glucosaminyl transferase (OGT) assays, enzymatic assays and cell assays. The best inhibitors obtained from GalT and OGT assays were then submitted to an artifical cell membrane penetration test, lipid bilayer penetration test.

The result revealed that the galactosyl pyridine containing derivative possessed a strong interaction with POPC while the glucose derivative did not. All the biological evaluation mentioned above was included in Chapter IV. An overall conclusion was given including possible directions for further optimization to obtain more potent glycosyltransferase inhibitors.

All the work present was finished within three years including two visits (nine weeks totally) to the Carlsberg Laboratory for biological evaluation. All the experiment details were mentioned in Chapter V and the literature references were listed afterwards.

Table of contents

Acknowledgement ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• List of Abbreviations •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• General Introduction •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Table of contents •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Résumé en français ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Chapter I : Introduction -Glycosyltransferases and inhibitors of glycosyltransferases ••••••• I.1 Carbohydrates in biology •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• I.2 Glycosyltransferases ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• I.2.1 Structural features and classification •••••••••••••••••••••••••••••••••••••••••••••••• I.2.2 Catalytic mechanism ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• I.2.3 Glycosyltransferases in biology •••••••••••••••••••••••••••••••••••••••••••••••••••••• I.3 Inhibitors of glycosyltransferases •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• I.3.1 Inhibitors of galactosyltransferases •••••••••••••••••••••••••••••••••••••••••••••••••• I.3.2 Inhibitors of O-linked N-acetylglucosamine transferase (OGT) •••••••••••••••• I.4 Project aim ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• I.4.1 Synthesis of UDP-sugar analogues •••••••••••••••••••••••••••••••••••••••••••••••••• I.4.2 Biological evaluations ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Chapter II : Synthesis of neutral glycosyltransferase inhibitors -Pyridine as a pyrophosphate surrogate ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.1 Introduction••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.1.1 Chemical aspects •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.2 Investigation of glycosylation methods of a "basic" acceptor •••••••••••••••••••••••••• II.2.1 Galactosylation •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.2.2 Modifications on the pyridine-based acceptor •••••••••••••••••••••••••••••••••••• II.2.3 N-Acetylglucosaminyl donors ••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.2.4
Other glycosyl donors (Glc, Man, Fuc, Rha, Lac)

•••••••••••••••••••••••••••••••• II.
3 Synthesis of UDP-sugar analogues based on glycosylation and amide bond chemistries 4 Synthesis of UDP-sugar analogues based on azide-alkyne and amide bond chemistries

••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.
••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• II.5 Conclusion ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••
Chapter III : Design and synthesis of neutral glycosyltransferase inhibitors -Amino-acids as pyrophosphate surrogates

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• III.1 Galactosylation of serine-based acceptors •••••••••••••••••••••••••••••••••••••••••••••••• III.3 Synthesis of serine derivatives containing UDP-Gal mimetics •••••••••••••••••••••••• III.4 Conclusion ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Chapter IV: Inhibition studies towards glycosyltransferases ••••••••••••••••••••••••••••••••••••••• IV.1 Introduction ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• IV.1.1 Radiochemical GalT enzymatic assays ••••••••••••••••••••••••••••••••••••••••••• IV.1.2 Cell assays •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• IV.1.3 Artifical cell membrane penetration test •••••••••••••••••••••••••••••••••••••••••• IV.2 Results and discussion ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• IV.2.1 GalT inhibition studies •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• IV.2.2 OGT inhibition studies •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• IV.2.3 Evaluation of membrane permeation ••••••••••••••••••••••••••••••••••••••••••••

Résumé en français

Les saccharides sont présents à la surface de la cellule et dans le cytosol, libres ou associés à d'autres biomolécules. Ils jouent de nombreux rôles dans les organismes vivants et sont conjugués à d'autres biomolécules pour former des glycolipides ou des glycoprotéines. Ils sont notamment impliqués dans la transduction du signal, la circulation intracellulaire des protéines, l'adhésion ou la reconnaissance ou la différenciation cellulaire, le développement neuronal, l'inflammation, la réponse immunitaire, la métastase, mais aussi l'infection virale ou bactérienne (Figure 1). Au fur et à mesure des élucidations des rôles des saccharides, l'intérêt grandit en chimie, biochimie et biologie pour définir plus exactement le rôle d'un saccharide précis et pour examiner son potentiel thérapeutique. Les inhibiteurs de GTs peuvent être classés en quatre types: les analogues du donneur, analogues de l'accepteur, les analogues bi-substrat et les molécules identifiées par criblage à haut débit. Ce projet de thèse présente la conception, la synthèse, l'évaluation biologique et l'analyse structurale de deux types d'analogues du donneur. L'idée de base est de créer des analogues neutres de NDP-sucres (substrats naturels des GTs) utilisant un motif pyridine ou amino-acide en remplacement du pyrophosphate. La faible biodisponibilité des analogues chargés (anioniques ou cationiques) de NDP-sucres suggère le développement d'analogues neutres pour fournir des inhibiteurs capables de traverser les membranes cellulaires et donc d'envisager des évaluations in cellulo ou in vivo et donc des applications fondamentales en biologie ou appliquées en médecine. Les galactosyltransférases (GalTs) et la O-linked N-acetylglucosamine transferase (OGT) récemment identifiée ont été sélectionnées comme cibles dans ce projet.

La pyridine et la sérine sont les deux motifs sélectionnés pour remplacer le groupement pyrophosphate (Figure 2). L'incorporation d'un motif pyridine devrait permettre de créer des interactions avec le metal (Mn 2+ ) présent dans le site catalytique de l'enzyme et aussi des liaison hydrogène avec l'enzyme (Figure 2A-D). L'autre série d'analogues incorporant un amino-acide (Figure 2E-F) permet d'obtenir une fonction amine ou acide pour une diversification supplémentaire. L'introduction de plusieurs groupes fonctionnels devrait permettre là aussi de créer des interactions favorables avec le métal et l'enzyme. Les saccharides impliqués dans ces synthèses sont donc le galactose, la N-acétyl glucosamine et le glucose. La synthèse des inhibiteurs neutres de GTs s'effectue par une combinaison de liaison O-glycosidique, amide ou triazole. L'une des premières étapes de ces synthèses était la O-glycosylation d'un motif pyridine ou sérine qui a dû être étudiée en détail. Le 6-(hydroxyméthyl)picolinate de méthyle ainsi qu'un dérivé azido-sérine ont été choisis comme substrat de référence pour cette étude. Une série de 20 donneurs de glycosyle en série glucose, galactose, fucose, lactose, mannose, rhamnose and N-acétyl glucosamine et divers groupements activants de la position anomère (acétate, bromure, tricholoracétimidate, thiopyridine) ainsi qu'un nombre de conditions d'activation ont été évaluées (Tableau 1).

Cette étude a permis d'identifier les conditions optimales de glycosylation pour divers monosaccharides et ont pu être appliquées pour la synthèse d'inhibiteurs de GTs. L'évaluation biologique des analogues de l'UDP-Gal et de l'UDP synthétisés a été effectuée sur 5 GalTs incluant la β-1,4-galactosyltransférase (β-1,4-GalT), la α-1,4-galactosyltransférase (α-1,4-GalT), la α-1,3-galactosyltransférase (α-1,3-GalT), la human blood group B galactosyltransférase (GTB), et une galactosyltransférase mutante (AAGlyB) mais aussi la O-linked N-acetylglucosamine transferase (OGT).

La première série d'inhibiteurs basés sur le motif pyridine a montré de meilleures inhibitions dans le cas des O-glycosides que pour les composés incorporant un lien triazole. Le meilleur inhibiteur 7-Gal a une valeur d'IC 50 de 152 μM pour la β-1,4-GalT. Une étude cristallographique de trois inhibiteurs co-cristallisés dans le site catalytique de l'enzyme mutante AAGlyB a démontré l'intérêt du motif pyridine qui oriente le saccharide dans une direction totalement différente du substrat natural (UDP-Gal) de l'enyzme mais qui permet aussi de chélater le cation métallique même s'il est déplacé de quelques angströms de sa position initiale (Figure 5). Des études complémentaires de "docking" moléculaire ont montré que des interactions étaient bien crées entre les motifs pyridine, triazole et le squelette de l'enzyme. Là encore, le monosaccharide pointe dans une direction différente de sa position naturelle et n'interagit presque pas avec l'enzyme (Figure 6). Ces résultats confirment les observations faites sur les études cristallographiques avec AAGlyB. L'étude d'inhibition enzymatique de la seconde série d'inhibiteurs incorporant la sérine a révélé que la nature du lien entre le saccharide et le nucléoside est très important afin d'orienter de façon optimale l'inhibiteur dans le site catalytique. Quasiment tous les composés sont de faibles inhibiteurs (74, 75, 77, 78, 80, 92) et la meilleur inhibition a été mesurée pour le composé 80 avec 38% inhibition pour AAGlyB à 1 mM.

Le concept d'inhibiteur "neutre" au lieu d'inhibiteurs chargés pour une meilleure biodisponibilité et donc de perméation cellulaire a pu être vérifié en utilisant un modèle utilisant les liposomes de POPC pour les deux meilleurs inhibiteurs de GalT (7-Gal) et de l'OGT (35-Glc). Malgré leur similarité structurale, les deux composés ont donné des résultats différents. Le composé 7-Gal a présenté une forte affinité pour la membrane de POPC du liposome sans pour autant pouvoir la traverser alors que le composé 35-Glc ne s'est pas associé au liposome.

Les différents résultats biologiques obtenus pour ces composés ont fourni des informations importantes de relation structure-activité pour la conception rationnelle d'une nouvelle génération d'inhibiteurs de GTs. Des améliorations sont proposées sur la base de la première famille d'analogues utilisant le motif pyridine et en tenant compte des aspects conformationnels des molécules ciblées pour obtenir des interactions optimales avec les enzymes et donc des inhibiteurs potentiellement puissants et sélectifs. It is notable that most of the carbohydrates present in living organisms are conjugated to other important biomolecules to form glycolipids or glycoproteins. These glycoconjugates are involved in numerous biological processes (Figure I-2), such as signal transduction, intracellular trafficking of proteins, cell adhesion, cell recognition, cell differentiation, development of the neuronal network, inflammation, immune response, metastasis and bacterial infection. [1][2][3][4] For example, to solve the worldwide shortage of organs for clinical implantation, xenotransplantation from a pig or baboon is one of the solutions, which is aimed at treating patients who have life-threatening and debilitating illnesses with cells or tissues. Whereas the α-Gal epitope (Figure I-1.c) sets a limit for its real clinical application, the α-gal epitope is abundantly synthesized on glycolipids and glycoproteins in non-primate mammals and New World monkeys. In contrast, in humans, apes and Old World monkeys the gene to generate the related enzyme that could catalyze the biological synthesis was inactivated, which results in the absence of this epitope and, instead, anti-Gal antibody is produced. The subsequent immune response of the antibody, in some transplantation cases, may result in the immediate death of the recipient. A study demonstrated that gene knockout pigs may avoid this immunological problem. 5 As more and more carbohydrates related biological processes are elucidated, there is great interest in biology, biochemistry and chemistry to define the exact biological roles of a given carbohydrate and examine its potential therapeutic applications. After the development of proteomics and genomics, glycomics appear as the next broad scientific challenge to the realm of scientific research. [6][7][8] Figure I-2. Importance of glycoconjugates in biological processes. 1

Chapter I : Introduction -Glycosyltransferases and inhibitors of glycosyltransferases

I.2 Glycosyltransferases

Glycosyltransferases are ubiquitous enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, either with retention or inversion configuration of the anomeric carbon. 9 The donor substrate of glycosyltransferases can be sugar nucleotide derivatives (e.g. UDP-Gal, CMP-NeuAc) and lipid phosphates (e.g. polyprenol phosphates, sugar-1-phosphates). Nucleotide sugar-dependent glycosyltransferases are termed as Leloir enzymes, in honor of 1970' Nobel Prize winner Luis F. Leloir, the scientist who discovered sugar nucleotides. In contrast, the non-Leloir enzymes may utilize lipid phosphates and other glycosides as donors. Leloir enzymes are in charge of most of the glycoconjugates synthesis in cells, especially in mammals. [START_REF] Unligil | Glycosyltransferase structure and mechanism[END_REF] Among an extensive range of nucleotide sugar donors utilized by organisms, only 9 sugar nucleotide donors are used by mammals (Figure I-3). The acceptor substrates of glycosyltransferases have huge varieties, that can be other sugars, lipids, proteins or nucleic acids. [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF] For metal-dependent GTs of the GT-A fold, the pyrophosphate moiety of the donor interacts with cations (e.g., Mn 2+ or Mg 2+ ) with coordination to two aspartate residues within a DXD (aspartate-any residue-aspartate) motif. The metal such as magnesium or manganese assists binding to the (di)phosphate leaving group and is essential for the activity of the enzyme. Also, it is known that some glycosyltransferases (e.g. OGT) exhibit different catalytic modes which do not require a metal.

I.2.1 Structural features and classification

Although glycosyltransferases utilize considerable diversity of donors or acceptors and exhibit product specificity, the 3D structures of GT are highly conserved. [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF] The CAZy database proposes a continuously updated classification of glycosyltransferases based on the sequence of the enzymes. 9,[START_REF] Campbell | A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities[END_REF] A hierarchical system with an increasing functional predictive power from folds, clans, families and subfamilies is presented. 94 Distinct families have been listed in CAZy database up to May 2013. 13 The large size of the GT family also shows the potential infinite diversities of glycosylated products in Nature.

The reported glycosyltransferase structures have been classified into three folds called GT-A, GT-B and GT-C (Figure I-4). The first 3-D structure for GT-A fold was reported for SpsA, an inverting enzyme from Bacillus subtilis. [START_REF] Charnock | Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms[END_REF] The overall architecture of the GT-A fold is composed of two tightly associated β/α/β domains, the sizes of which may vary slightly leading to the formation of a continuous central β-sheet (Figure I-4.a). The first determined 3-D structure for GT-B fold was reported in 1994 for a DNA-modifying β-glucosyltransferase from bacteriophage T4. [START_REF] Vrielink | Crystal-structure of the dna modifying enzyme beta-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose[END_REF] Like the GT-A fold, the architecture of GT-B enzymes consists of two β/α/β Rossmann-like domains. However, the two domains are less tightly associated and face each other with the active-site lying within the resulting cleft ( This structure weakens the value of the GT-C classification because neither the catalytic activity of this truncated protein nor the active site of the soluble C-terminal domain were indicated. GT-A and GT-B folds are the usually adopted folding form according to X-ray structures that have been resolved. It should be noted that not all the enzymes adopting a GT-A or GT-B fold are glycosyltransferases but enzymes with other biological fuctions.

There is no correspondance relation between the overall fold of the enzyme and the stereochemical outcome of the enzymatic reaction. The mechanism of inverting glycosyltransferases is suggested to be a direct S N2 -like displacement. The amino-acid chains in the catalytic site deprotonate the incoming acceptor substrate, assisting the nucleophilic attack from the opposite side of the anomeric center of the sugar donor (Figure I-6a).

No consensus for the mechanism of retaining glycosyltransferases has been reached but rather there are two main mechanisms proposed: double-displacement (2×S N2 ) and internal return (S Ni ). Theoretically, both pathways are possible and the S Ni pathway provides more experimental support. [START_REF] Chan | Nuclear magnetic resonance spectral assignments of alpha-1,4-galactosyltransferase lgtC from Neisseria meningitidis: Substrate binding and multiple conformational states[END_REF][START_REF] Errey | Mechanistic insight into enzymatic glycosyl transfer with retention of configuration through analysis of glycomimetic inhibitors[END_REF] The double-displacement mechanism involves a covalent glycosyl-enzyme intermediate and two subsequent nucleophilic displacements each occurring with inversion of configuration at the anomeric carbon and therefore leading to an overall retention of configuration (Figure I-6b). The other hypothesis is a S Ni mechanism in which the enzyme shielding on the opposite face of the anomeric center prevents an inverting nucleophilic attack while the oxygen atom of phosphate leaving-group acts as a base to deprotonate the acceptor. Via an enzyme-mediated intermediate, the nucleophilic oxygen atom of the acceptor attaches to the anomeric carbon on the same side after phosphate oxygen leaves, resulting in a retention configuration at the anomeric carbon in the glycoside (Figure I-6c).

I.2.3 Glycosyltransferases in biology

Despite the considerable diversity of carbohydrate derivatives in living organisms, the degradation and biosynthesis of these oligosaccharides, polysaccharides and glycoconjugates are catalyzed by glycosidases (also called glycoside hydrolases or glycosyl hydrolases) and glycosyltransferases (GT). It is reported that glycosyltransferases represent approximately 1-2% of an organism's gene products. [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF] For example, in the human genome, more than 250 glycosyltransferases are encoded while in the model plant, Arabidopsis thaliana, approximately 450 genes are related to glycosyltransferases. [START_REF] Roychoudhury | New structures, chemical functions, and inhibitors for glycosyltransferases[END_REF] Although there is a myriad of GT, several of them should be particularly mentioned which are the targets of our synthesized inhibitors and have been tested in our project. I.2.3.1 β-1,4-Galactosyltransferase β-1,4-Galactosyltransferase (β-1,4-GalT) is an inverting enzyme catalyzing the transfer of galactose from UDP-galactose (UDP-Gal) to the 4-position of N-acetylglucosamine residues to form a LacNAc epitope (Figure I-7). [START_REF] Baisch | Enzymatic galactosylation of non-natural glucosamide-acceptors[END_REF] β-1,4-GalT is involved in the biological synthesis of diversified glycoconjugates and it is probably the most well studied enzyme in synthesis and substrate specificity. [START_REF] Koeller | Synthesis of complex carbohydrates and glycoconjugates: Enzyme-based and programmable one-pot strategies[END_REF] Since the recombinant form of β-1,4-GalT from bovine and human origins are commercially available, it is now used as a model for the design of GT inhibitors. I.2.3.2 α-1,4-Galactosyltransferase α-1,4-Galactosyltransferase (α-1,4-GalT) derived from Neisseria meningitidis is responsible for forming a lipooligosaccharide in biosynthesis by transfering an α-galactose from active donor UDP-Gal to a lactose terminal acceptor (Figure I-8). One of the crucial biochemical products is glycosphingolipid globotriaosylceramide (Gb3, CD77 or P k ) which is synthesized by addition of a galactose to lactosylceramide via α-1,4-GalT (Figure I-9). The glycosphingolipid globotriaosylceramide plays an important role in disease spreading process, such as bacterial Haemophilus influenza infection. [START_REF] Erwin | Role of lgtC in resistance of nontypeable Haemophilus influenzae strain R2866 to human serum[END_REF][START_REF] Tzeng | Epidemiology and pathogenesis of Neisseria meningitidis[END_REF][START_REF] Gotschlich | Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide[END_REF] Figure I-8. Enzymatic-catalyzed glycosylation using α-1,4-galactosyltransferase (α-1,4-GalT). . [START_REF] Letts | Differential recognition of the type I and IIH antigen acceptors by the human ABO(H) blood group A and B glycosyltransferases[END_REF] In human ABO(H) blood group system, humans encoded with gene for GTB have blood group B-antigen associated with anti-A antibody, those with GTA generate blood group A-antigen associated with anti-B antibody. The coexistence of antigen and antibody blocks blood transfusion between different blood types. AAGlyB is a mutant of the wild type human blood group glycosyltransferase. The human blood group A and B glycosyltransferases are highly homologous differing in only four critical amino acid residues which are Arg/Gly176, Gly/Ser235, Leu/Met266, and Gly/Ala268, respectively. The name of the mutant AAGlyB gives a clue of the mutant fashion by keeping the first two amino acid residues from GTA and inherit amino acid residue 268 from GTB while mutate amino acid residue 266 to glycine which gives AAGlyB (Arg176, Gly235, Gly266, Ala268). [START_REF] Seto | Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes[END_REF] As for biochemical functions, GTA and GTB catalyze the final addition of a sugar moiety to H-antigen acceptors to produce blood group A and B epitopes. Different from GTB which uses UDP-Gal as a donor substrate transferring α-galactose, GTA utilizes UDP-GalNAc as the donor substrate and transfers GalNAc to the acceptors through an α-linkage. Further observation on OGT targeted proteins indicate that they are both glycosylated and phosphorylated and it exists that direct cross-talk between dynamic O-GlcNAc glycosylation and kinase/protein phosphatase mediated phosphorylation. [START_REF] Hart | Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease[END_REF][START_REF] Wang | Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc[END_REF] The modification site may be O-GlcNAc occurs in the cytosol as other GTs, but also in the nucleus. More than 1,000 substrate proteins have been identified to date and O-GlcNAcylation is involved in almost all aspects of cellular metabolism. [START_REF] Comer | O-glycosylation of nuclear and cytosolic proteins: dynamic interplay between O-GlcNAc and O-phosphate[END_REF][START_REF] Wells | Glycosylation of nucleocytoplasmic proteins: Signal transduction and O-GlcNAc[END_REF] A recent work describing crystal structures of human OGT has given insight into the catalytic domain of this enzyme. [START_REF] Lazarus | Structure of human O-GlcNAc transferase and its complex with a peptide substrate[END_REF] His498 is proposed to act as a catalytic base deprotonating the hydroxyl group of serine or threonine residue of the glycosylated protein while Lys 842 from α-side stabilizes and activates the UDP leaving group resulting the O-GlcNAc glycosylation occurs on the β-side (Figure I-15). [START_REF] Lazarus | Structure of human O-GlcNAc transferase and its complex with a peptide substrate[END_REF] OGT has now been shown to play important roles in cancer, [START_REF] Lynch | Critical role of O-linked beta-N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis and metastasis[END_REF][START_REF] Slawson | O-GlcNAc signalling: implications for cancer cell biology[END_REF] Alzheimer's disease [START_REF] Yuzwa | Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation[END_REF][START_REF] Dias | O-GlcNAc modification in diabetes and Alzheimer's disease[END_REF][START_REF] Lefebvre | Does O-GlcNAc play a role in neurodegenerative diseases?[END_REF][START_REF] Zachara | The emerging significance of O-GlcNAc in cellular regulation[END_REF] and diabetes [START_REF] Zachara | The emerging significance of O-GlcNAc in cellular regulation[END_REF][40] and being a potential therapeutic target. Several reviews have been recently reported for the structure and mechanism elucidation of GT. [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF][START_REF] Breton | Recent structures, evolution and mechanisms of glycosyltransferases[END_REF][START_REF] Wagner | Glycosyltransferases and their assays[END_REF][START_REF] Breton | Structures and mechanisms of glycosyltransferases[END_REF] Design of inhibitors of glycosyltransferases has been accelerated with the developments in glycobiology presenting oligosaccharides as active species (eg. blood groups, antigens) rather than "decorations" of biomolecules. Several reviews on the development of GT inhibitors have been reported recently [START_REF] Roychoudhury | New structures, chemical functions, and inhibitors for glycosyltransferases[END_REF][START_REF] Gloster | Developing inhibitors of glycan processing enzymes as tools for enabling glycobiology[END_REF] according to the biological functions of emerged inhibitors, [START_REF] Gloster | Mechanism, structure, and inhibition of O-GlcNAc processing enzymes[END_REF][START_REF] Jung | Carbohydrate-Baed Drug Discovery[END_REF][START_REF] Qian | Glycosyltransferase inhibitors[END_REF] classification of inhibitors by structural features, [START_REF] Kajimoto | Synthesis of glycosyltransferase inhibitors[END_REF][START_REF] Izumi | Bisubstrate analogues as glycosyltransferase inhibitors[END_REF][START_REF] Zou | C-glycosides and Aza-C-glycosides as potential glycosidase and glycosyltransferase inhibitors[END_REF][START_REF] Compain | Carbohydrate mimetics-based glycosyltransferase inhibitors[END_REF] or focusing on iminosugar-based inhibitors. [START_REF] Whalen | Iminosugar-based glycosyltransferase inhibitors[END_REF][START_REF] Cipolla | Combinatorial approaches to iminosugars as glycosidase and glycosyltransferase inhibitors[END_REF][START_REF] Compain | Design, synthesis and biological evaluation of iminosugar-based glycosyltransferase inhibitors[END_REF] Early research efforts on GT inhibitors could be traced back to 1987 that sugar methylenediphosphonates and nucleoside sugar phosphonic derivatives were synthesized and evaluated as GT inhibitors. [START_REF] Vaghefi | Synthesis of glycopyranosylphosphonate analogs of certain natural nucleoside diphosphate sugars as potential inhibitors of glycosyltransferases[END_REF][START_REF] Vaghefi | Synthesis of certain nucleoside methylenediphosphonate sugars as potential inhibitors of glycosyltransferases[END_REF] It should be specially mentioned that the comparison of compounds' potency from one report to another is risky since it is demonstrated that assays conditions (pH and ionic strength) may influence the results [START_REF] Saotome | Synthesis and enzymatic evaluation of five-membered iminocyclitols and a pseudodisaccharide[END_REF][START_REF] Takayama | Selective inhibition of β-1,4-and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach[END_REF] needless to say the substrates concentrations will affect the experimental IC 50 values measured. With regard to the choice of individual targets, among different types of GT, a representative type of GT named galactosyltransferases (GalT) and a recently identified highly valuable O-linked N-acetylglucosamine transferase (OGT) were selected. According to structure features, inhibitors could be classified into four types, donor substrate analogues, acceptor substrate analogues, bi-substrate analogues and small molecule inhibitors identified from library screening approach or high-throughput screening. The examples presented below are not an exhaustive review of the literature but rather a focused selection of inhibitors based on the enzyme and inhibitor structural features described.

I.3.1 Inhibitors of galactosyltransferases

The UDP-Gal donor substrate of GalT is composed of three parts: a galactose moiety, a diphosphate linker and uridine. Design of donor substrate analogues could be based on modifications of either one or several simultaneous modifications.

I.3.1.1 Modification on the carbohydrate moiety

Using carbocyclic to mimic glycosyl ring is one of the examples of modification on the carbohydrate moiety (A2, Figure I-17). [START_REF] Yuasa | Synthesis of the carbocyclic analog of uridine 5'-(alpha-D-galactopyranosyl diphosphate) (UDP-Gal) as an inhibitor of beta(1->4)-galactosyltransferase[END_REF] The analogue was tested as an effective inhibitor of β-1,4-GalT and verified as a competitive inhibitor of donor substrate with a K i value of 58 μM.

A series of mono-O-methylated UDP-Gal analogues were synthesized by Tsuyoshi Endo et al. [START_REF] Endo | Novel aspects of interaction between UDP-Gal and GlcNAc β-1,4-galactosyltransferase: transferability and remarkable inhibitory activity of UDP-(mono-O-methylated Gal), UDP-Fuc and UDP-Man[END_REF] Among them, the 2-O-methylated UDP-Gal analogue A3 (Figure I-17) showed a good inhibition of β-1,4-GalT with a calculated K i value of 20 μM but was also a good donor substrate of this transferase. This showed a good compatibility of this enzyme towards modification at the 2-position.

Fluorination is another approach to obtain valuable analogues for enzymatic evaluation. [START_REF] Caravano | A new methodology for the synthesis of fluorinated exo-glycals and their time-dependent inhibition of UDP-galactopyranose mutase[END_REF][START_REF] Grizot | Structure of the Escherichia coli heptosyltransferase WaaC: Binary complexes with ADP and ADP-2-deoxy-2-fluoro heptose[END_REF][START_REF] Eppe | Probing UDP-galactopyranose mutase binding pocket: A dramatic effect on substitution of the 6-position of UDP-galactofuranose[END_REF] Fluorinated molecules (e.g. A1, Figure I-17) are attractive for the study of interactions between the fluorine atom and an enzyme in order to better elucidate enzymatic mechanisms. 64 A compound with fluoro-substitution at 2-position (A4, Figure I-17) was prepared by chemoenzymatic method and showed inhibitory effect on β-1,4-GalT and α-1,3-GalT with K i values in the micromolar range. [START_REF] Hayashi | A chemoenzymic synthesis of UDP-(2-deoxy-2-fluoro)galactose and evaluation of its interaction with galactosyltransferase[END_REF][START_REF] Takayama | Chemoenzymic preparation of novel cyclic imine sugars and rapid biological activity evaluation using electrospray mass spectrometry and kinetic Analysis[END_REF] Similar fluoro-substitution at the 6-position, the compound A5 (Figure I-17) showed 75% inhibition using a 10-fold molar excess relative to UDP-Gal for both β-1,4-GalT and α-1,3-GalT 67 which indicated that the presence of fluorine at C-6 does not affect the binding to enzymes.

Based on docking simulation, an analogue with a 6-substitution of a tethered naphthyl moiety (A6, Figure I-17) was designed and evaluated as human recombinant galactosyltransferase (αGalT1) inhibitor. [START_REF] Takaya | Rational design, synthesis and characterization of novel inhibitors for human β1,4-galactosyltransferase[END_REF] It was found that the synthesized analogue A6 is a potent inhibitor (K i 1.86 μM) with respect to UDP-Gal (K m 4.91 μM) and mass spectrometry analysis revealed that the inhibitor could create a covalent bond with the indole ring of Trp310 located near the active site therefore blocking the binding of the respective acceptor substrate. Iminosugar-based mimetics represent a large family in inhibitors design which has been reviewed. [START_REF] Whalen | Iminosugar-based glycosyltransferase inhibitors[END_REF][START_REF] Cipolla | Combinatorial approaches to iminosugars as glycosidase and glycosyltransferase inhibitors[END_REF][START_REF] Compain | Design, synthesis and biological evaluation of iminosugar-based glycosyltransferase inhibitors[END_REF] A simple five-membered galacto-iminocyclitol B1 (Figure I-18) was indicated to have inhibitory activity against β-1,4-GalT. [START_REF] Saotome | Synthesis and enzymatic evaluation of five-membered iminocyclitols and a pseudodisaccharide[END_REF][START_REF] Saotome | Combinatorial library of five-membered iminocyclitol and the inhibitory activities against glyco-enzymes[END_REF] Yong Jip Kim et al. [START_REF] Kim | A rationally designed inhibitor of α-1,3-galactosyltransferase[END_REF] synthesized a series of UDP-galactose mimetics, using galactose-type iminosugar as a replacement of the galactose residue, a vicinal diol linker as a pyrophosphate surrogate and a thioether bond linked to the uridine part (B2, Figure I-18). The mimetics containg three segments showed strong inhibitory activity and moderate selectivity towards α-1,3-GalT among β-1,4-GalT. The inhibition studies indicated that the compound did not show a competitive mode of inhibition against UDP-Gal which means the compound could bind to the free enzyme but also to enzyme-substrate complex. Another type of UDP-Gal mimetics based on D-and L-epimers of iminosugar was synthesized by Guo-Liang Zhang et al. [START_REF] Zhang | Synthesis and evaluation of the analogues for galactosyl donors as inhibitors of β-1,4-galactosyltransferase[END_REF] The uridine nucleoside was attached to an iminosugar moiety via a variable length spacer. The best result was obtained with the L-epimer B3 (Figure I-18) which displayed a moderate inhibition against α-1,3-GalT with an IC 50 value of 320 μM and a weak inhibitory effect towards β-1,4-GalT.

I.3.1.2 Modification on the diphosphate

C-glycoside is another approach for the design of UDP-sugar analogues, which could be taken as a modification on the linker part. The derivative may keep binding affinity to the enzyme while could not be transferred to the respective acceptor, thus, serves as an inhibitor. The synthesis of C-glycosides of UDP-Gal, UDP-GlcNAc and UDP-GalNAc (C1, Figure I- 19) should be mentioned here even biological evaluation data was not provided. [START_REF] Schaefer | Synthesis of novel donor mimetics of UDP-Gal, UDP-GlcNAc, and UDP-GalNAc as potential transferase inhibitors[END_REF] Several years later, our group synthesized a series of C-glycosyl ethylphosphonophosphate analogues of UDP-Glc, UDP-Gal, UDP-GlcNAc and GDP-Fuc (C2, Figure I- 19) and assayed the analogues in vitro. [START_REF] Vidal | Non-isosteric C-glycosyl analogues of natural nucleotide diphosphate sugars as glycosyltransferase inhibitors[END_REF] The IC 50 values of UDP-Gal, GDP-Fuc and UDP-GlcNAc analogues towards different glycosyltransferases, β-1,4-GalT, FUT3 and LgtA, were 40 μM, 2 mM and 3.5 mM respectively. The synthesis of a pair of diastereoisomeric UDP-galactose analogues (C3, Figure I- 19) were reported by V. Kolb et al. [START_REF] Kolb | Specific inhibition of an alpha-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues[END_REF] The target of the study was the glycosylation of Trypanosoma brucei membranes which could cause sleeping sickness and animal African trypanosomiasis. The enzymatic results showed that both diastereoisomers were effective inhibitors towards N-glycan specific α-galactosyltransferase from trypanosomes with K i values 21 μM (R) and 34 μM (S) in respect to the K m value of UDP-Gal 20 μM. Taking into consideration that donor or acceptor substrate analogues containing charged pyrophosphate moiety have limited bioavailability, scientists turned their attention to nonionic linker mimics. Malonic, tartaric, and monosaccharide moieties were employed as mimics of the pyrophosphate linker (D1, Figure I-20). [START_REF] Wang | A search for pyrophosphate mimics for the development of substrates and inhibitors of glycosyltransferases[END_REF] Among the mimics, glucose containing derivative showed inhibitory activity towards the β-1,4-GalT with a K i value of 119.6 μM, whereas the malonic and tartaric linkages appeared to be ineffective as replacements of the pyrophosphate moiety. Copper (I) catalysed modified Huisgen cycloaddition is widely used to rapidly and effectively construct compound libraries. A series of α-glycosides of UDP-sugar mimics were synthesized (D2, Figure I-20) taking triazole as a replacement of pyrophosphate via click chemistry. [START_REF] Yeoh | Probing replacement of pyrophosphate via click chemistry; synthesis of UDP-sugar analogues as potential glycosyl transferase inhibitors[END_REF] These compounds showed no inhibition against β-1,4-GalT up to 4.5 mM. The authors proposed that a bis-triazole moiety may be a more appropriate replacement for pyrophosphate. Another two types of UDP-sugar analogues using amino-acid as pyrophosphate mimics were synthesized and studied as inhibitors against GlfT2, a galactofuranosyltransferase activated in cell wall galactan biosynthesis in Mycobacterium tuberculosis. The first type of mimics is amino acid bridged C-glycoside analogues (D3, Figure I-20). The hypothesis by Vembaiyan et al. [START_REF] Vembaiyan | Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors[END_REF] was that a basic amino-acid may be beneficial for the interaction with the enzyme. Four amino-acids (lysine, glutamine, tryptophan, and histidine) were selected for the study. Among the analogues, histidine containing compound showed the best inhibitory activity with an IC 50 value of 332 μM. The other type of analogue which was initially designed towards MurG, a glycosyltransferase involved in bacterial peptidoglycan biosynthesis, was also evaluated as GlfT2 inhibitors. [START_REF] Trunkfield | Inhibition of Escherichia coli glycosyltransferase MurG and Mycobacterium tuberculosis Gal transferase by uridine-linked transition state mimics[END_REF] In this family of compounds, a functionalized proline was linked via a variable length spacer to the uridine nucleoside. The best activity against GlfT2 among these compounds came from a phenyl modified proline derivative (D4, Figure I-20) with 80% inhibition at 1 mM while at this concentration no inhibition against MurG was observed.

I.3.1.3 Modification on the nucleoside base

Modifications on the nucleoside base are rare since the nucleoside moiety is considered as a necessary part for enzyme binding. Studies on different GalT structures disclosed that these enzymes might have compatibility towards modification at the 5-position of uridine. [START_REF] Gastinel | Crystal structures of the bovine β4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose[END_REF][START_REF] Alfaro | ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes[END_REF] Different types of substituents in this position have been reported. 5-formylthien-2-yl substituted uracil UDP-Gal analogue E1 (Figure I-21) was synthesized and evaluated. [START_REF] Pesnot | Structural and mechanistic basis for a new mode of glycosyltransferase inhibition[END_REF] Enzymatic experiments showed this uracil-substituted analogue had broad inhibitory effect on different GalT including β-1,4-GalT, α-1,3-GalT, α-1,4-GalT, GTB, and human blood group transferase mutant AAGlyB. Crystal structure analysis of the complex with AAGlyB indicated that conformational changes of the enzyme were blocked by the substituent on the uracil ring upon binding, therefore locking the enzyme in an inactive catalytic conformation. Based on pioneering work in this area, Karine Descroix et al. [START_REF] Descroix | The first C-glycosidic analog of a novel galactosyltransferase inhibitor[END_REF] designed and synthesized a C-glycoside derivative bearing a 5-formylthien-2-yl substituent at the 5-position of the uracil base (E2, 21). UDP-Gal 4C-epimerase GalE was selected as the enzymatic target. This analogue gave a K i value at micromolar concentration. Trifluoromethyl is another type of substituent at the 5-position of uracil base (E3, Figure I-21). The synthesis and biological evaluation was reported by Andrew Evitt et al. [START_REF] Evitt | One-step synthesis of novel glycosyltransferase inhibitors[END_REF] The enzymatic evaluation showed that the decoration does not interfere with the recognition and binding with either β-1,4-GalT or α-1,4-GalT. This new type of analogue was determined as inhibitors of the tested enzymes and gave more information on structure-activity relationships for new classes of GT inhibitors design.

I.3.1.4 Other aspects of UDP-sugar mimetics as GalT inhibitors

Enzymatic reactions can be equilibrated processes and the leaving moiety UDP is a natural competitive inhibitor of GT which utilize UDP-sugar as active sugar donor. Design of GT inhibitors based on structure modifications on UDP was also reported. Monosaccharide moieties were employed as mimics of the pyrophosphate linker. [START_REF] Wandzik | Synthesis of 2-deoxy-hexopyranosyl derivatives of uridine as donor substrate analogs for glycosyltransferases[END_REF][START_REF] Ballell | Synthesis and evaluation of mimetics of UDP and UDP-α-D-galactose, dTDP and dTDP-α-D-glucose with monosaccharides replacing the key pyrophosphate unit[END_REF] These mimetics gave weak inhibitors against β-1,4-GalT (F1-F2, Figure I-22). Multivalent effect, which is normally applied in design of high affinity ligands for lectins, 86 was also tested [START_REF] Hatanaka | New polymeric inhibitor of galactosyl transferase[END_REF] on GT by synthesis of poly(uridine 5'-p-styrenesulfonate) (F3, Figure I-22). Enzymatic evaluation showed that this polymeric compound could inhibit the activity of β-1,4-GalT by 75% at 120 μM. The inhibition mechanism of this type of polymeric compound against GT has not been revealed to date and the application potency of synthetic polymeric compound over low-molecular-weight inhibitors still needs to be investigated.

The fact that the UDP moiety provides most of the binding affinity while the carbohydrate group adds the selectivity to the enzyme was verified by Katrin Schaefer et al. [START_REF] Schaefer | A new concept for glycosyltransferase inhibitors: nonionic mimics of the nucleotide donor of the human blood group B galactosyltransferase[END_REF][START_REF] Schaefer | A nonionic inhibitor with high specificity for the UDP-Gal donor binding site of human blood group B galactosyltransferase: Design, synthesis, and characterization[END_REF] Based on docking experiment, two isomers were synthesized and tested by radiolabeled enzymatic assays as human blood group B galactosyltransferase (GTB) inhibitors. In their further work, the addition of an α-Gal moiety to 9-D-arabinityl-1,3,7-trihydropurine-2,6,8-trione F4 did increase the binding affinity to GTB from a K i value of 2.2 mM to 1.5 mM (F5,

Figure I-22)
and more importantly gave a selectivity to GTB over GTA which shares the same acceptor as GTB but uses UDP-GalNAc as an activated donor. A series of 3-GlcNAc or 4-GlcNAc modified peracetylated GlcNAc-1,3-β-Gal-β-O-naphthalenemethanol were synthesized targeting of human β-1,4-GalT 1 which is involved in the glycan biosynthesis in tumor cells. [START_REF] Brown | Deoxygenated disaccharide analogs as specific inhibitors of β1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis[END_REF] The deacetylated compound indicated inhibitory effect in vitro and the acetylated form (H1, Figure I-24) was tested to inhibit sLe X formation in U937 monocytic leukemia cells with an IC 50 value of about 30 μM. This report is one of the rare reports that verified that the designed inhibitor does have activity in vivo. Analysis of acceptor substrate binding site of β-1,4-GalT gave a hint that aromatic groups may be beneficial for substrate binding in Site B. [START_REF] Chung | Acceptor substrate-based selective inhibition of galactosyltransferases[END_REF] A series of acceptor analogues containing either bulky aromatic group or aliphatic chain were then synthesized. Two representative analogues (H2-H3, Figure I- 24) showed strong inhibition and high selectivity between β-1,4-GalT and α-1,3-GalT.

I.3.1.6 Bisubstrate analogues

Bisubstrate analogue means that the derivative contains both features of donor and acceptor substrates. Design and synthesis of this type of inhibitor involve extensive work but a promising means to obain the most active and selective inhibitors for a specific glycosyltransferase. To date, two examples of this type of inhibitor targeting GalT have been reported (I1-I2, Figure I-25). [START_REF] Hashimoto | Synthesis of the first tricomponent bisubstrate analog that exhibits potent inhibition against GlcNAc:β-1,4-galactosyltransferase[END_REF][START_REF] Waldscheck | α(1-3)-Galactosyltransferase inhibition based on a new type of disubstrate analogue[END_REF] Both of them were designed based on a steric rearrangement of donor and acceptor components according to the transition state. As expected, they gave the best inhibition result so far towards their respective enzymatic targets. were found to be effective against β-1,4-GalT. [START_REF] Takayama | Selective inhibition of β-1,4-and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach[END_REF][START_REF] Gao | Specificity of β1,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside[END_REF] Another classic example is a small heterocycle (J3, Figure I-26) obtained from a Molecular Fragment Library which was determined to exhibit weak inhibition against GT with a special mechanism simultaneously interfering with metal ion and substrate binding. 28

I.3.2 Inhibitors of O-linked N-acetylglucosamine transferase (OGT)

O-GlcNAcylation attracts increasing interest from biologists, biochemists and chemists since more and more O-GlcNAcylation relevant physiological and pathological processes are elucidated, including cancer, [START_REF] Lynch | Critical role of O-linked beta-N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis and metastasis[END_REF][START_REF] Slawson | O-GlcNAc signalling: implications for cancer cell biology[END_REF] Alzheimer's disease [START_REF] Yuzwa | Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation[END_REF][START_REF] Dias | O-GlcNAc modification in diabetes and Alzheimer's disease[END_REF][START_REF] Lefebvre | Does O-GlcNAc play a role in neurodegenerative diseases?[END_REF][START_REF] Zachara | The emerging significance of O-GlcNAc in cellular regulation[END_REF] and diabetes. [START_REF] Zachara | The emerging significance of O-GlcNAc in cellular regulation[END_REF][40] The O-GlcNAcylation is a reversible post-translational modification through catalytic formation by O-linked N-acetylglucosamine transferase (OGT) and hydrolysis by N-acetyl β-glucosaminidase (O-GlcNAcase, OGA). [START_REF] Hanover | The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine[END_REF] Unlike other usual GT which are normally located in the cytosol, OGT is also present in the nucleus and mitochondria. More than 1000 substrate proteins have been identified, but no consensus sequence has been identified among them. Therefore, the study of OGT requires the design of potent inhibitors not only for fundamental research but also potential for pharmaceutical applications.

Three different isoforms of OGT have been reported (nuclear, cytoplasmic OGT (ncOGT) and mitochondrial OGT (mOGT) and a short form OGT (sOGT). The difference is the length of their tetratricopeptide repeats (TPRs), 13, 9.5, 3.5 TPRs respectively. 100 These three constructs of human OGT have been successfully expressed in E. coli. with only ΔmOGT which is 50 residues shorter than the known splice variant. 101 There is a few research papers on OGT inhibition and the design strategy is similar to the approaches presented above for GalT. Hajduch et al. and Dorfmueller et al.,separately. 72,[104][105] While the C-glycoside K3 showed weak inhibition on ncOGT, the thioglycoside K4 gave an IC 50 in the micromolar range. Recently, it was shown that a synthetic carbohydrate precursor K5 using a sulfur atom instead of oxygen in the sugar ring could be recruited by the hexosamine biosynthetic pathway (HBP) in cells and consequently inhibited OGT's function with a low micromolar EC 50 . The counterpart of the enzymatic product of HBP (K6, Figure I-27) was synthesized and verified as an inhibitor of OGT in vitro with a K i value of 8 μM. This biosynthetic precursor K5 gives a good example of inhibitor design capitalizing on forming the inhibitor intracellularly therefore acting on the desired enzyme.

I.3.2.1 UDP-sugar donor substrates analogues

Alloxan (K1,

I.3.2.2 Small molecule inhibitors from library or HTS

Recent developments in accurate measurement of O-GlcNAcylation have facilitated the discovery of small-molecule inhibitors of OGT. In late 2005, the Walker's lab used fluorescent UDP-GlcNAc analogues instead of natural sugar donor and the displacement assay was used in a HTS assay of OGT inhibitors. 101 102 Compounds were selected as positives among 64 416 commercial library compounds screened for a hit rate of 0.2% and 19 of these compounds were verified by a radiometric assay. The best two inhibitors selected by this screening displayed with IC 50 values in the micromolar range (L1-L2, Figure I-28). To continue the study, chemical modifications were performed on the best inhibitor selected from the library screening. 106 Mechanistic studies showed that the benzoxazolinone core could functionalize as a diphosphate isostere and a double-displacement mechanism (SN 2 ×SN 2 ) was proposed based on mass spectrometry analysis of inhibitor treated enzyme (L3, Figure I-28). The concentration required for inhibition of O-GlcNAcylation in cells was relatively high, possibly due to decomposition or side reactions. Two years later, instead of donor modification, a peptide acceptor substrate labeled approach was utilized by the Walker's lab and the so-called "protease-protection" assay was used to screen OGT inhibitors. 107 Among 124 226 compounds screened, 84 compounds that inhibited OGT activity by 30% or more were identified for a hit rate of 0.065% and 9 of these compounds were verified by a radiometric assay to have IC 50 values between 0.9 and 20 μM.

I.4 Project aim

Several parameters must be considered for the design of potent and selective GT inhibitors:

(1) the inhibitor must have a sufficient affinity for the enzyme, typically in the micromolar range or at least very similar to the K m value of the natural NDP-sugar substrate;

(2) the inhibitor must be able to penetrate cells and therefore to cross the different cell membranes (e.g. plasmic, nuclear) to reach its target GT in the correct cellular compartment;

(3) the inhibitor must exhibit high specificity for GT among all enzymes (e.g. kinases, phosphatases, peptidase, glycosidases) but also for only one family of GT (e.g. GalT vs FucT).

Despite the tremendous efforts for the design of GT inhibitors, reported analogues which are effective in vivo are still rare and studies in this aim are still challenging. The diphosphate linker is the most problematic residue due to its potential metabolic instability and poor pharmacological properties in particular with respect to cellular permeability. The current project focused on the design and synthesis of neutral inhibitors of GT. The diphosphate unit of NDP-sugars usually interacts with metallic cations. Therefore, surrogate of this moiety should be capable of retaining this property and provide chelating groups for cations. In our study, diphosphate moiety was replaced with neutral functional groups capable of binding to the active site of glycosyltransferases (Figure I-29) and these compounds were evaluated as GT inhibitors.

I.4.1 Synthesis of UDP-sugar analogues

Pyridine containing scaffold and amino acid containing linker were chosen as two types of surrogates for the diphosphate moiety. The carbohydrates used are galactose and N-acetyl glucosamine as the selective natural moieties for the enzymes but also glucose as a negative control. The stereochemistry at the anomeric center of the carbohydrate does not necessarily need to be identical to the natural NDP-sugar since β-anomers could also be identified as GT inhibitors. 108 First, we designed UDP-sugar analogues containing the pyridine moiety (I-IV, Figure I-29). It was reported that heterocyclic moieties containing a pyridine ring conjugated with two symmetric triazoles were capable for chelating metal cation. 109 The incorporation of a pyridine moiety to the diphosphate mimics will enhance the metal-binding and hydrogen bonding capability of this series of analogues. The conjugation of the carbohydrate, pyridine and nucleoside building blocks could be achieved through a combination of glycosylation, Staudinger-Vilarrasa ligation and Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC).

Afterwards, another series of UDP-Gal analogues functionalized with an amino-acid derived linker were synthesized (series V-VI, Figure I-29). The incorporation of an amino-acid residue between the carbohydrate and nucleoside moieties provided a functional amine or acid group which could be substituted with other functional moieties. The introduction of functional groups could favor binding to GT or interact with specific amino-acids in the active sites of GT. Glycosylation reaction conditions with serine derivatives were also investigated in this section. The construction of this library of analogues was conducted through a combination of glycosylation and amide bond chemistries.

I.4.2 Biological evaluations

The neutral inhibitors designed and synthesized were then evaluated for their inhibition activities towards five galactosyltransferases (GalT). The best inhibitors of human blood group transferase mutant AAGlyB were then selected for crystallographic studies in order to obtain the structural features of their interactions with the enzyme at the active site in a co-crystal. Eight UDP-sugar or UDP analogues which were functionalized with a pyridine ring conjugated with two symmetric triazoles were selected for OGT inhibition, enzymatic assays, cell assays and docking studies. The concept of "neutral" inhibitor instead of ionic analogues was finally tested by an artifical cell membrane penetration test on the two best inhibitor candidates against GalT or OGT.

Chapter II : Synthesis of neutral glycosyltransferase inhibitors -Pyridine as a pyrophosphate surrogate

Chapter II : Synthesis of neutral glycosyltransferase inhibitors -Pyridine as a pyrophosphate surrogate

II.1 Introduction

This chapter describes the synthesis of neutral glycosyltransferase inhibitors taking pyridine as a pyrophosphate surrogate. The syntheses of UDP-sugar analogues (7-Gal, 7-Glc, 20, 23) and one UDP analogue (10) (Figure II-1) are based on glycosylation and amide bond chemistries. One key step in this synthetic route is the generation of the glycosidic bond with basic pyridine-containing acceptors which are quenching the Lewis Acid used as a promoter. A careful literature search provided only a handful of reports, hence a systematic study on glycosylation methods involving methyl 6-(hydroxymethyl)picolinate as a model acceptor and 20 glycosyl donors is implemented in this section. (25, 45) using pyridine as a pyrophosphate surrogate. The syntheses are performed in galactose, glucose and N-acetyl glucosamine series. Two synthetic routes, namely pathway A from Sugar-to-Nucleoside and pathway B from Nucleoside-to-Sugar, are applied in the glucose series in order to compare their efficiency. The introduction of a desymmetrized bis-alkyne pyridine intermediate provided access to the "one-pot" synthesis of bis-triazole analogues 43 rapidly through CuAAC conjugations with sequential deprotection of the alkyne groups.

II.1.1 Chemical aspects

Apart from rare synthetic benefits obtained from enzymatic engineering, a large series of compounds were constructed towards glycosyltransferase inhibition based on chemical synthesis. The chemistries involved are mainly phosphonate chemistry, sulphur chemistry, carbohydrate chemistry, "Click" chemistry, amide or ester bond formation chemistry. The main chemical methods, glycosylation, amide bond chemistry and "Click" chemistry, were also used in the present study and briefly reviewed in this section with highlight of mechanism and the influence of key parameters.

II.1.1.1 Glycosylation

Despite the considerable diversity of carbohydrate derivatives in living organisms, the biosynthesis of these oligosaccharides, polysaccharides and glycoconjugates are catalyzed by different GT. Different types of glycosidic bonds, such as O-glycoside, S-glycoside and N-glycoside, are present in Nature. Among them, O-glycosides are the most abundant and important species. The enzymatic syntheses of O-glycosides in living organisms implemented by GT are highly specific and selective. Despite the merit of enzymatic synthesis, the availability of genetically engineered enzyme and complexity of glycoconjugates limit the real application of enzymatic synthesis in most of cases. Besides, UDP-sugars, sugar donors for enzymatic synthesis, are either expensive or difficult to produce in a large scale. [110][111] Thus, chemical synthesis is a major tool to access diversified glycoconjugates to study their biological roles therefore plays a crucial role in carbohydrate chemistry. [112][113][114] The formation of a covalent bond between the anomeric position of a glycosyl donor and a glycosyl acceptor yields a new stereogenic carbon atom (Scheme II-1). The linkage formed may either be axial or equatorial. Efficient, regioselective and stereoselective creation of glycosidic linkages is challenging and sensitive to a large number of factors, such as leaving group at the anomeric center, protecting groups applied for donors and acceptors, promoters or even solvents. The leaving group (LG, Scheme II-1) present at the anomeric position of glycosyl donors requires activation of a promoter (i.e., Lewis or Brønsted acid) in order to generate the reactive oxonium/carbenium ion intermediate which will then undergo a nucleophilic attack by the glycosyl acceptor. In order to control the stereochemical outcome of a glycosylation, the protecting group at the 2-position of the glycosyl donor is a major parameter.

Scheme II-1. Schematic representation of a glycosylation reaction.

Normally, a participating group, typically an acetate or benzoate ester, will form an acetoxonium ion intermediate which would block the attack from the α-face thus forming mainly β-glycoside, also described as 1,2-trans glycoside (a, Scheme II-2). Meanwhile, the formation of the acetoxonium ion intermediate potentially increased the possibility of a byproduct forming, namely orthoester. N-acetyl glucosamine derivates are the optative examples which favor the formation of oxazoline intermediate which are stable enough to be separated (a, Scheme II-2). A non-participating group, such as a benzyl ether, will predominantly gives α-glycoside or 1,2-cis glycoside favoured by the anomeric effect 115 (b, Scheme II-2). Meanwhile, other reaction conditions, promoter, solvent, temperature and even the sequence of addition of reactants, may also influence the outcome of a glycosylation reaction. The solvent has a preference for interaction in axial (MeCN) or equatorial (Et 2 O) position thus favoring the nucleophilic attack of the acceptor on the opposite face (Scheme II-3). 257

Scheme II-2. Schematic representation of a glycosylation reaction with a participating or a non-participating group at the 2-position of glycosyl donor.

Scheme II-3. Schematic representation of the influence of solvent in glycosylations. 257

II.1.1.1.1 Sugar donors

A wide variety of sugar derivatives containing leaving groups at the anomeric carbon are used as sugar donors in glycosylation (Table II-1).

One of the first glycosylation methods published in the 19 th centry was Fischer glycosylation which used directly native sugars as glycosyl donors. This reaction with alcohols in the presence of protic acid is an equlibrated process which results a mixture of kinetic furanosides and thermodynamic pyranosides. Modifications have been made on the original conditions which permit a better control of the ratio. 116 Peracetylated sugars are readily available and have also been used as glycosyl donors. However, the acetyl group is not a good leaving group, so specific conditions are required. Lewis acids, such as SnCl 4 117 and BF Another versatile sugar donor is the 1,2-anhydro glycal derivative. The efficient activation method via oxidation with dimethyldioxirane (DMDO) affords epoxides which are active glycosylating species under regioselective ring opening usually catalyzed by ZnCl 2 . 134 The deactivating-activating methodology could also be used in multisteps programmed synthesis and another potential property of using this method is to provide an unprotected hydroxyl group at the 2-position which could be further converted or functionalized.

The last type of donors which will be discussed in this section is oxazolines. The 2-acetamido-2-deoxy group in N-acetyl-D-glucosamine and N-acetyl-D-galactoamine reacts differently in glycosylation reactions in comparison to their counterparts. The reason related is that the N-acyl group is an excellent participating group. As the leaving group activated, the neighboring group N-acyl participates the formation of an intermediate resulting the stable oxazoline formed (Scheme II-6). The oxazoline can be further activated with TMSOTf 135 to afford the 1,2-trans-glycoside.

Scheme II-6. Formation of oxazoline.

II.1.1.2 Staudinger-Vilarrasa reaction

Amide bond, named also peptide bond, is a chemical covalent bond formed between a carboxylic acid and an amine by condensation reaction. Amide bond is widespread in Nature as found in proteins which are composed by amino-acids oligomerized through amide bonds. The peptide bond is neutral, possessing both hydrogen-bond accepting and donating capabilities and metabolically stable compared to ester bond. An analysis of the Comprehensive Medicinal Chemistry database shows that over 25% of the known drugs bearing the amide bonds. [137][138] Simply mixing an amine and an acid will give a stable organic salt. Direct condensation of the salt could occur under high temperature (160-180°C), 139 but these reaction conditions are not compatible with heat-sensitive functional groups. Thus, activation and appropriate protecting groups are needed. Common activated carboxyl derivative have been used, such as acyl halides, acyl azides, acylimidazoles, anhydrides or esters. 138 Different protecting groups could be applied to the corresponding strategies according to respective synthetic requirement. An amine group could also be obtained from its precursor azido group by combination of the azide with a phosphine (or phosphate) followed by hydrolysis (Scheme II-7) which is named Staudinger reaction or Staudinger reduction. 140

Scheme II-7. Phosphine mediated reaction of converting azide into amine. 140 Later, effort was made to form an amide bond directly between Staudinger iminophosphorane and activated carboxylic acids (Scheme II-8) such as anhydrides, 141-142 acyl halides [143][144] and other activated esters. [145][146][147][148] This general process was named Staudinger-Vilarrasa reaction [149][150][151] in view of the seminal work in this area performed in professor Jaume Vilarrasa's group (University of Barcelona, Spain).

Scheme II-8. Direct formation of amide bond from carboxyl derivatives, organic azides and phosphines.

Based on the previous results obtained in our department, [149][150][151]

II.2 Investigation of glycosylation methods of a "basic" acceptor

An initial step for the synthesis of neutral analogues of UDP-sugar incorporating a pyridine glycoside is the glycosylation of hydroxymethylpyridine derivatives. Surprisingly, a careful survey of the literature for the glycosylation of such glycosyl acceptors did not afford many results and most of these were poor in terms of isolated yields of glycosides (Figure II-3).

Pyridoxine can be glycosylated on the hydroxymethyl groups at positions 4' or 5', or both, and is better known as vitamin B 6 . In these initial reports, the glycosidic bond formation of pyridoxine had been studied by different glycosidases (1-3, Figure II-3). [159][160][161] In another series of experiments, the influence of borate was evaluated in order to obtain a regioselective glucosylation of pyridoxine at the 4'-or 5'-position by microorganisms. [162][163] The glycosyl donors used in these reports were natural (oligo)saccharides, sucrose or dextrins but also chemically modified carbohydrates such as p-nitrophenyl (PNP) or α-bromo activated glycosides. Nevertheless, under the aforementioned conditions using enzymatic or microbial glycosylations, a maximum yield of 40% was achieved. Despite these bio-catalyzed synthetic strategies inspired by natural enzymes or microorganisms did not require extensive protecting group chemistry and could provide the desired glycosides in high anomeric stereoselectivity, the yields obtained remained quite low and did not call for further applications.

In an organic chemistry approach, only a few reports have been identified. The glucuronylation of ciamexon (an immunomodulating agent with application in diabetes) was achieved from the corresponding α-glucuronopyranosyl bromide donor using silver(I) salt as a promoter leading to the desired glucuronide in 45% yield (4, Figure II-3). 164 The reaction conditions were quite sensitive and the use of basic medium (sym-collidine) influenced the reaction affording a large portion of undesired orthoester. This problem could be overcome by using excess of silver triflate (3 equivalents) most probably in order to balance the basicity of the pyridine acceptor. The formation of the orthoester intermediate could not be prevented in the glycosylation between acetobromoglucose and 2-vinyl-5-hydroxymethylpyridine derivative using AgOTf as the promoter 165 in a study for the design of peptide functionalizing agents (5, Figure II-3). Again, an excess of promoter (1.5 equivalents) was used but led to exclusive formation of the orthoester intermediate in 73% yield since the same amount of acceptor was used and its basicity could therefore not be totally balanced. A subsequent rearrangement in the presence of trimethylsilyl triflate (TMSOTf) afforded the desired glycoside in 32% yield corresponding to an overall yield of only 24% over two steps. In another case, isomaltulose was used as a precursor for α-glucosylated hydroxymethylpyridine derivatives (6, Figure II-3). 166 Acid dehydration of isomaltulose afforded a glucosylated furfural which could be transformed into a pyridine moiety through two additional synthetic steps. The overall process did not afford the desired compounds in good yields (< 5%) but represents yet another possibility for the construction of the pyridine scaffold from natural source.

Figure II-3. Enzymatic and chemical approaches to the synthesis of glycosylated hydroxymethylpyridine derivatives. a. Yield reported for galactose derivatives; b. The 4'-regioisomer was also isolated in a 28:72 mixture with the desired 5'-derivative desired; c. Yield calculated over two steps from the data reported; d. Yield calculated from the reported data for the multi-step process.

Based on the literature search, a systematic investigation of the glycosylation was performed taking methyl 6-(hydroxymethyl)picolinate as a model acceptor substrate. 167 A detailed study including 20 glycosyl donors was performed to determine the influence of protecting group and leaving group on the glycosyl donor as well as the nature of the promoter. 41 Different reaction conditions were tested and the optimized glycosylation methods 167 were applied to the synthesis of neutral inhibitors of glycosyltransferases.

II.2.1 Galactosylation

Scheme II-13. Presentation of galactosylation investigated.

The first type of glycosylation investigated employed different galactosyl donors in order to address both the influence of protecting groups on the carbohydrate moiety as well as the different leaving groups at the anomeric position (Scheme II-13). When a Lewis acid was used as promoter, it was added in excess in order to balance the basicity of the pyridine-based acceptor and in almost all cases, an excess of glycosyl donor was used to ensure maximal functionalization of the acceptor.

Scheme II-14. Galactosylation of methyl 6-(hydroxymethyl)picolinate 2a with galactosyl donors 1a-f.

A series of 6 different galactosyl donors 1a-f were synthesized and used in the present study (Scheme II-14). Peracetylated galactose 1a was firstly chosen because of less synthetic steps required for its preparation. An efficient synthetic methodology for glycosylation from peracetylated sugar donors and catalyzed by silver and tin salts was re-investigated by our group. 168 In this case, only a slight spot corresponding to the glycoside could be detected by TLC which was not isolated (Entry 1, Table II-2). Then, we turned to the Koenigs-Knorr glycosylation system using galactopyranosyl bromide 1b as sugar donor (Entries 2-4, Table II-2). In the first two experiments using the common silver triflate as promoter (Entries 2-3, Table II-2), poor yields were obtained regardless of which donor or acceptor was used in excess. Applying the Helferich modification method 169 using mercury(II) cyanide (Entry 4, Table II-2) as the promoter, the expected β-glycoside was formed in 11% yield. In this reaction, a mixture of acetonitrile and dichloromethane was used for optimal solubility of the reactants. These poor results prompted us to turn to more activated donors such as the galactose trichloroacetimidate 1c which was known as a powerful glycosylating agent. [170][171] Boron trifluoride etherate and trimethylsilyl trifluoromethanesulfonate are the two common catalysts used when applying sugar trichloroacetimidate as the active sugar donor. When boron trifluoride etherate was used (Entries 5-10, Table II [172][173] This new methodology was also evaluated (Entry 12-13, Table II-2) but proved rather unsuccessful providing poor yields even at higher temperatures than initially required. We also synthesized N-phenyl-trifluoroacetimidate donor 1d which was recently shown to provide better yields than the standard trichloroacetimidates 174 but the yield of galactoside 3a was not improved (Entry 14, Table II-2).

Benzoate protecting groups were used in some cases because they were more stable than acetates. Therefore, the benzoylated trichloroacetimidate donor 1e [175][176] was used under the best conditions obtained previously (Entry 5, Table II-2). With 1.3 equivalent of donor and prolonged reaction time (Entry 15 and 16, Table II-2), the yield obtained was acceptable (42%), but not improved in comparison to the acetylated donors.

Considering the "arm-disarm" concept in glycosylation, 177-180 the ester protected glycosyl donors (e.g., acetates or benzoates) would be considered as disarmed and lead to poorer reactivity (i.e., longer reaction time and need for stronger promoters) while the ether protected donors (e.g., benzyl) are armed and would provide higher reactivities and better yields for the glycosylation. The 2-thiopyridyl perbenzylated donor 1f 181 was therefore evaluated as a glycosyl donor for this pyridine-based acceptor. Activation by methyl iodide provided a good yield (75%) of the desired galactoside 3c with an α configuration at the anomeric center. The α-anomer was favoured in this case due to the benzyl ether as a non-participating group at the 2-position.

As a conclusion for the galactosylation of 6-(hydroxymethyl)picolinate 2a, the best conditions obtained to afford the desired β-anomer are using the trichloroacetimidate donor 1c catalyzed by boron trifluoride etherate as a promoter in excess (1.5 equivalent) and with only 2 h reaction time at low temperature (-20°C). Better yields, albeit with the opposite configuration at the anomeric center, can be obtained with benzylated armed donors such as 1f.

II.2.2 Modifications on the pyridine-based acceptor

The intrinsic basicity of the pyridine-based acceptor under acid-promoted glycosylation conditions was then investigated (Scheme II-15) through variations on the pyridine moiety with N-oxide derivative 2c and also the corresponding phenyl-based donor 2d.

Scheme II-15. Influence of the pyridine-based acceptor on the galactosylation reaction.

The silylated acceptor 2b was isolated from Entry 11, Table II-2 and reintroduced for galactosylation with the best donor 1c identified in the previous experiments. It is known that the acidity of the promoter can trigger the cleavage of the trimethylsilyl group therefore allowing the glycosylation to proceed. [182][183][184] Only a very small amount of the desired galactoside 3a (< 5%) could be observed by TLC (not isolated) although the donor 1c was totally consumed while the silylated acceptor 2b was still largely unreacted (Entry 18, Table II-3). Afterwards, a common method for masking the basicity of a pyridine ring was applied through oxidation to the N-oxide derivative 2c. 185 In this case, excess of promoter was avoided but only very limited amounts of the desired galactoside 3d could be isolated as an inseparable mixture of α and β anomers along with the hydrolyzed hemiacetal donor and unreacted acceptor (Entry 19, Table II-3). Finally, the influence of basicity of the pyridine ring could be clearly demonstrated by studying the galactosylation of methyl 6-hydroxymethylbenzoate acceptor 2d. 186 When the reaction was performed under the best conditions determined previously, transfer of an acetate from the donor to the acceptor was observed with methyl 6-acetoxymethylbenzoate obtained in 37% isolated yield (Entry 20, Table II-3). Therefore, using a three-fold excess of acceptor provided a very good yield of 79% (Entry 21, Table II-3). While this last result appeared promising, similar conditions, using an excess of donor did not improve the yield when the pyridine-based acceptor 2a was used (Entry 9, Table II-2).

II.2.3 N-Acetylglucosaminyl donors

Design and synthesis of glycosyltransferase inhibitors targeting N-acetylglucosaminyl transferases is part of the present research program. After assessing the parameters influencing the glycosylation reaction in the galactose series, glycosylation methodology towards N-acetylglucosamine (GlcNAc) series was then studied applying sugar donors 1g-m (Scheme II-16).

The isoxazoline donor 1g 187 is commonly employed for glycosylation in the GlcNAc series (also referred as GlcNAcylation). Two different promoters were used, but unsatisfying yields were obtained (Entries 22-23, Table II-4). Then, we turned to 2-deoxy-2-amino-protected GlcNAc donors 1h-m which could be taken as precursors of N-acetyl moieties (Scheme II-16).

Carbamates, such as donors 1h-k, are usually invoked for their good stereocontrol in favour of β-anomers through anchimeric participation while 2-azido-2-deoxy-derivatives 1l-m would lead to α-anomers. THF 191 , the Troc protecting group was replaced by acetate, but the methyl ester was also reduced to alcohol followed by acetylation as evidenced by mass spectrometry (m/z, [M+H] + = 511.1) (a, Scheme II-17). TFA was also tried as a deprotection reagent, but a mixture of anomers was obtained without altering the Troc protecting group (b, Scheme II-17). Then, an allyl-carbamate (Alloc) protecting group was introduced in donors 1j-k [192][193] which provided moderate yields (Entries 27-28, Table II-4). The protecting group transformation from NHAlloc to NHAc was carried out under reported reaction condition Pd(PPh 3 ) 4 /dimethyl malonate in THF 194 followed by acetylation (c, Scheme II-17). Finally, in this case, a quantitative yield was obtained. a. Values are isolated yields. b. Product was isolated as a mixture of β-anomer 3h and 0.3 eq. unreacted acceptor 2a and could not be separated due to similar polarity. Yield was calculated excluding the unreacted acceptor 2a based on 1 H NMR data. c. Product could not be isolated but detected by TLC. A byproduct identified as methyl 6-(acetoxymethyl)picolinate was isolated in 53% yield.

Scheme II-17. Functional group conversion of glucosamine derivatives.

The azido functionality is a general precursor of NHAc groups and its use in GlcNAcylation has two consequences. The azido group is non-participating and therefore the glycoside obtained is usually the α-anomer. The need for the 2-acetamido substituent requires a subsequent reduction/acetylation process which must be included in the synthetic strategy in terms of steps but also stability for the further elaboration of the target molecule. Nevertheless, these types of 2-azido-2-deoxy donors are quite useful and the peracetylated donor 1l [195][196] (prepared from commercially available 2-azido-2-deoxy-D-glucose) was used but provided only moderate yields (Entries 29-30, Table II-4). A transfer of acetyl group was observed from the glycosyl donor 1l to the acceptor 2a affording the methyl 6-(acetoxymethyl)picolinate in 53% isolated yield (Entry 29, Table II-4) when using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the promoter. The 2-azido-2-deoxy trichloroacetimidate donor 1m 197 was then applied. Treated with TMSOTf, only small amounts of the desired product could be separated as the α-glycoside 3i (Entry 31, Table II-4). A very good yield of 82% could then be obtained but as a mixture of both isomers using boron trifluoride etherate as the promoter (Entry 32, Table II-4).

As a short conclusion for the GlcNAcylation section, 7 different donors with various protecting groups at the 2-position under a total of 11 synthetic methodologies were performed. The best conditions found in this series were the use 2-azido-2-deoxy derivatives to obtain the α-anomer while the Alloc carbamate was favored for the preparation of β-glycosides.

II.2.4 Other glycosyl donors (Glc, Man, Fuc, Rha, Lac)

In order to broaden the scope of this glycosylation methodology of pyridine-based acceptors, the reaction was studied with several other glycosyl donors of monosaccharides (glucose, mannose, rhamnose, fucose) and one disaccharide (lactose) (Scheme II-18 II-5). When a 4,6-benzylidene protecting group was introduced to the phenyl thioglycoside 1s, better yields were obtained (Entries 39-40, Table II-5). More interestingly, when this bicyclic system was used, the unusual β-mannoside 3o was favoured. β-Mannoside 3o was isolated as the only glycoside with a yield of 68% using TMSOTf-NIS as promoters. Changing the catalytical system to BSP-Tf 2 O improved slightly the yield although stereoselectivity was poorer. The confirmation of configurations at the anomeric carbon of mannosides 3n and 3o was based on 1 J coupling constants. The 1 J coupling constants measured between the anomeric carbon (C-1) and the anomeric proton (H-1) with values above 170 Hz was assigned for α-mannosides versus values below 160 Hz were measured for β-mannosides. 205 II.2.4.4 Lactose. Another disaccharide, the benzoylated trichloroacetimidate lactosyl donor 1t 206 was reacted with the acceptor 2a using boron trifluoride etherate as a promoter and afforded the desired lactoside 3p in 51% isolated yield (Entry 41, Table II-5). As a conclusion for the investigation of glycosylation methods on pyridine-based acceptors, a series of 20 glycosyl donors derived from glucose, galactose, mannose, fucose, rhamnose and lactose and a large number of activating groups at the anomeric center (e.g., acetate, bromide, tricholoracetimidate, thiopyridine) and a complete set of activating conditions were evaluated.

The pyridine moiety was identified to have a negative influence on the reaction in comparison to the "phenyl-based" donor 2d. Therefore, the basicity of the pyridine ring could be verified as a limitation to the outcome of the glycosylation reaction. The best conditions for the glycosylation of the model donor methyl 6-(hydroxymethyl)picolinate 2a could provide isolated yields of glycosides from 46% to 82% with different type of glycosyl donors. And the optimized glycosylation methods could then be applied to the synthesis of the target neutral inhibitors of glycosyltransferases.

II.3 Synthesis of UDP-sugar analogues based on glycosylation and amide bond chemistries

For synthesis of UDP-sugar analogues containing pyridine as a pyrophosphate surrogate, a combination of glycosylation and Staudinger-Vilarrasa reactions was used and two synthetic routes, namely pathway A from "sugar-to-nucleoside" and pathway B from "nucleoside-to-sugar", were investigated (Schemes II-19 and II-20).

The synthetic route from "sugar-to-nucleoside" (Pathway A, Scheme II- 19), synthesis of Gal derivative and Glc derivative as a negative control, started from dimethyl pyridine-2,6-dicarboxylate, after monoreduction to alcohol 2a, [START_REF] Ballell | Synthesis and evaluation of mimetics of UDP and UDP-α-D-galactose, dTDP and dTDP-α-D-glucose with monosaccharides replacing the key pyrophosphate unit[END_REF] glycosylation of the 6-hydroxymethylpyridine derivative 2a provided the β-glycosides 3j-Glc and 3a-Gal in good yields. 167 The methyl ester attached to the pyridine ring was then selectively unmasked in the presence of acetates to afford the carboxylic acids 4-Glc and 4-Gal. These compounds could then be engaged in a Staudinger-Vilarrasa reaction with 5'-azido-uridine 5 to afford the acetylated amides 6-Glc and 6-Gal. Removal of the acetates gave access to the desired GT inhibitor candidates 7-Glc and 7-Gal.

With similar synthetic methodology, the route from "nucleoside-to-sugar" gives a more flexible approach to introduce the sugar moiety at the end of the synthesis (Pathway B, Scheme II- 19). The mono-ester 2 was converted to the acid 8 which was conjugated with 5'-azido-uridine 5 to afford the amide 9 which underwent glycosylation to provide the glycosides 7-Glc and 7-Gal. The key intermediate compound 9 was also deprotected and evaluated as a uridine-diphosphate (UDP) analogue 10 in the later enzymatic assays since UDP is known as a natural inhibitor of GT and testing UDP analogues will provide information on the influence of the carbohydrate moiety. Similar synthetic routes were then applied to obtain UDP-GlcNAc analogue (Scheme II-20).

Surprisingly, after glycosylation of mono-ester 2 with NH-Alloc trichloroacetimidate 1k, followed by protecting group transformation to glycoside 11 and ester deprotection, the acid derivative 12 did not react with 5'-azido-uridine derivative 5 to afford the desired amide 13.

As a consequence, the unreacted acid 12 and 5'-amino-uridine derivative 5 were recovered.

The other synthetic route was also investigated (Pathway B, Scheme II-20). Although the best glycosylation conditions determined previously were applied to acceptor 9 with NH-Alloc trichloroacetimidate 1k, only traces of the desired glycoside 14 were observed after purification and further confirmed by mass spectrometry (m/z, [M+H] + = 834.2). Thus, the reaction condition to synthesize this type of UDP-GlcNAc analogue must be further optimized. The "sugar-to-nucleoside" approach was then performed to obtain the α-isomer analogue of UDP-Gal (Scheme II-21). For this purpose, the benzylated galactosyl donor 15 was glycosylated with the 6-hydroxymethylpyridine derivative 2a to provide the α-galactoside 3c.

At this point, attempted debenzylation under hydrogenation conditions (Pd/C, H 2 1 to 8 atm.) did not affect the benzyl ethers but rather reduced the pyridine ring to a piperidine as a mixture of diastereoisomers as observed by The influence of the pyridine ring upon binding to the enzyme's catalytic site can also be addressed by the synthesis of a phenyl-based inhibitor candidate 22 in which the pyridine ring was replaced by a benzene moiety (Scheme II-22). Taking hydroxymethylbenzoate 2d as acceptor, the galactoside 3e was obtained with 79% yield. 167 In the case of phenyl derivative 3e, selective deprotection of methyl ester versus acetate appling sodium iodide did not work an left the starting material unchanged. Whereas, after hydrolysis of all ester functions, the acid glycoside 20 reacted with 5'-azido-uridine 5 to afford the partially acetylated amide 21 under Staudinger-Vilarrasa conditions with moderate yield (43%) which was then converted to the hydroxylated GT inhibitor candidate 22.

Scheme II-22. Synthesis of a phenyl-based pyrophosphate analogue through β-galactosylation and Staudinger-Vilarrasa ligation. Reagents and conditions: (a) methyl 5-hydroxymethylbenzoate, BF

II.4 Synthesis of UDP-sugar analogues based on azide-alkyne and amide bond chemistries

In order to use a combination of amide bond and triazole for the design of UDP-sugar analogues, the syntheses were started from commercially available 6-bromopicolinic acid 23 (scheme II-23). Conjugation of acid 23 with 5'-azido-uridine 5 under Staudinger-Vilarrasa reaction conditions afforded the amide intermediate 24 which could also be deprotected to compound 25 and evaluated as an UDP analogue. After functionalization with an alkynyl group through Sonogashira reaction, the alkynylated pyridine derivative 26 was readily converted to the terminal alkyne derivative 27. Conjugation with different glycosyl azides involving glucose 28-Glc, galactose 28-Gal and N-acetyl glucosamine 28-GlcNAc under CuAAC conditions afforded the protected UDP-sugar analogues 29 which were deprotected to the GT inhibitor candidates 30.

In the other synthetic route, with similar strategy but reverse positions of the amide and triazole moieties, another series of GT inhibitor candidates 35 was synthesized. 6-Bromopicolinic acid 23 was reacted with different glycosyl azides 28 to form an amide bond between sugar moiety and the pyridine scaffold. Then, Sonogashira reaction between the bromo-pyridine derivative 31 and trimethylsilylacetylene afforded the alkynylated pyridine derivative 32. Subsequent deprotection of the silyl group, CuAAC conjugation with 5'-azido-uridine 5 and hydrolysis of the acetate protecting groups afforded the desired GT inhibitor candidates 35 (Scheme II-23).

In the syntheses of the last family of GT inhibitors with two triazoles building blocks, two synthetic routes were again investigated in the glucose series in order to compare their efficiency (Scheme II-24). In pathway A, from "Sugar-to-Nucleoside", the commercially available 2,6-dibromopyridine was mono-functionalized with an alkynyl group 37. 216 After deprotection of the silyl group, CuAAC conjugation with glucosyl azide 28-Glc afforded the bromo-intermediate 39. Subsequent Sonogashira reaction to obtain 40 and desilylation provided the desired terminal alkyne 41. Final CuAAC with 5'-azido-uridine 5 followed by removal of the ester protecting groups gave the desired GT inhibitor candidate 43-Glc. With similar chemistry but reverse sequence, first forming a triazole between 5'-azido-nucleoside 5 and mono-terminal alkyne derivative 38, then transforming the functional group from bromo to alkynyl under Sonogashira reaction, then desilylating and CuAAC coupling and final hydrolysis of the protecting groups afforded the GT inhibitor candidate 43-Glc through pathway B. In terms of the total yield, we obtained 43% yield in pathway A over 7 steps while pathway B gave the desired compound 43-Glc in 21% over 5 steps. Even if the yield with the pathway B is slightly lower, the merit of this synthetic route lies in the introduction of different carbohydrate moieties conveniently at the end of the synthetic sequence providing a convenient divergent approach to a family of GT inhibitors. Therefore, pathway B was employed to the synthesis of the N-acetyl glucosamine derivative 43-GlcNAc (Scheme II-24).

Scheme II-23. Synthesis of three different series of neutral GT inhibitors using a combination of Staudinger-Vilarrasa ligation and CuAAC. Reagents and conditions: (a) PMe 3 , DIC, HOBt, THF, r. The idea of "One-pot" synthesis of 2,6-bis-triazolyl-pyridyl-based neutral GT inhibitor by two sequential CuAAC was then tested in the galactose series (Scheme II-25). The introduction of two orthogonally protected alkyne moieties on a multivalent scaffold was reported as a rapid approach to obtain hetero-functionalized compounds through CuAAC conjugations through sequential deprotection of the alkyne groups. 217 In our study, we used the desymmetrized 2,6-bis-acetylene pyridine derivative 48 218 as the key intermediate for the synthesis of our target GalT inhibitor candidate 43-Gal (Scheme II-25).

Scheme II-25. "One-pot" synthesis of a 2,6-bis-triazolyl-pyridyl-based neutral GalT inhibitor using two sequential CuAAC cycloadditions.

Our initial attempt was performed under the conditions described by Aucagne and Leigh. 217 The intermediate 48 was first coupled with 5'-azido-uridine 5 followed by removal of the silyl protecting group by adding silver tetrafluoroborate. Another portion of catalysts and the azido galactose 28-Gal were added to allow the second CuAAC coupling to be proceed (Pathway A, Scheme II-25). We isolated the desired acetylated derivative 42-Gal in 31% yield, although the synthesis reported 217 was almost quantitative (>95%). In this preliminary experiment, we noticed that the silyl orthogonally protecting group was not stable under the reaction condition since a small amount of desilylated intermediate was observed by TLC after the first CuAAC coupling. It is known that the silyl protecting group could be removed by weak base, such as K 2 CO 3 216 and the basic conditions are probably responsible for this partial deprotection. To further improve the reaction condition, we added the azido galactose 28-Gal and more base (DIPEA) after the first CuAAC coupling (Pathway B, Scheme II-25). A slight increase of the temperature from 20°C to 35°C allowed a clean deprotection of the silyl protecting group and the global yield was then increased to 41%. Finally, we obtained an optimal yield of 60% when silver salts were reintroduced for the deprotection concomitantly with the second CuAAC reagents (Pathway C, Scheme II-25). This sequential double-CuAAC synthetic strategy involved three synthetic steps performed with two subsequent additions of reagents in a "one-pot" procedure leading to a global yield of 60% which corresponds to 84% yield for each step. The acetates protecting groups were then removed to afford the target GalT inhibitor candidate 43-Gal.

II.5 Conclusion

Glycosylation is promoted by acid promoters rendering the reactions with basic acceptors challenging. A systematic investigation of glycosylation methods on pyridine-based acceptors was performed, involving (1) a series of 20 glycosyl donors derived from glucose, galactose, mannose, fucose, rhamnose and lactose) (2) a large number of activating groups at the anomeric center (e.g., acetate, bromide, tricholoracetimidate, thiopyridine) and (3) a complete set of activating conditions. The pyridine moiety was identified to have a negative influence on the reaction compared with the glycosylation result with a "phenyl-based" donor 2d. Therefore, the basicity of the pyridine ring could be verified as a limitation to the outcome of the glycosylation reaction. The best conditions for the glycosylation of the model donor methyl 6-(hydroxymethyl)picolinate 2a could provide isolated yields of glycosides from 46% to 82% with different types of glycosyl donors. The optimized glycosylation methods were then applied to the synthesis of neutral inhibitors of glycosyltransferases. Four UDP-sugar analogues (7-Gal, 7-Glc, 20 and 23) and one UDP analogue (10) were synthesized (Figure II-1), including α-and β-galactoside (7-Gal, 7-Glc), one β-glucoside (20) containing a pyridine moiety and one phenyl-based inhibitor candidate 23. These candidates prepared were then evaluated as GalT inhibitors.

The synthesis of another type of GT inhibitors containing eleven candidates was achieved in a minimum number of steps from readily available starting materials using two conjugation techniques: Staudinger-Vilarrasa reaction and azide-alkyne cycloaddition (CuAAC). The syntheses were performed in galactose, glucose and N-acetyl glucosamine series. The galactose-containing derivatives 30-Gal, 35-Gal, 43-Gal were selected as GalT inhibitor candidates while the N-acetyl glucosamine-based compounds 30-GlcNAc, 35-GlcNAc, 43-GlcNAc were evaluated as OGT inhibitors. As negative controls, the glucose derivatives were also synthesized to assess the influence of the carbohydrate moiety on the selectivity and inhibitory properties towards different enzymes. Two synthetic routes, from "Sugar-to-Nucleoside" and from "Nucleoside-to-Sugar", were applied in the synthesis of glucose series in order to compare the efficiency (Table II-6). In both cases, the latter synthetic route afforded a slightly lower yield. However, the advantage of this synthetic route lies in the introduction of different carbohydrate moieties conveniently at the end of the synthetic sequence providing a convenient approach to obtain a family of GT inhibitors. An orthogonally protected bis-alkynyl pyridine was introduced to obtain inhibitor candidates in a minimum number of steps. Two UDP analogues 25 and 45 were also prepared in order to obtain more information about the influence of the carbohydrate on enzymatic binding. Chapter III : Design and synthesis of neutral glycosyltransferase inhibitors -Amino-acids as pyrophosphate surrogates

Chapter III : Design and synthesis of neutral glycosyltransferase inhibitors -Amino-acids as pyrophosphate surrogates

This chapter describes the synthesis of neutral glycosyltransferase inhibitor candidates using amino-acid derivatives as pyrophosphate surrogates. Two types of analogues were designed (Figure III-1), conjugating the uridine moiety through either the amine or carboxylic acid group of the amino-acid linker. The incorporation of an amine, azido or carboxylic acid groups allowed further substitution with other functional moieties favoring binding to GT or interaction with specific amino-acids in the active sites of GT. The key steps in this synthesis were the generation of the glycosidic bond with serine-based derivatives and an amide bond formation afterwards with uridine. Glycosylation of amino-acid residue is a biological phenomenon which has been commonly found in protein post-translational modification 219 and widespread studied in protein chemistry [220][221][222] . Nevertheless, the reaction condition needs to be optimized for each specific case. Therefore, glycosylation conditions with serine derivatives were also investigated in this section.

Figure III-2. Synthesized UDP-Gal and UDP analogues using serine derivatives as pyrophosphate surrogates.

III.1 Galactosylation of serine-based acceptors

In an initial attempt, galactosylation was performed under reported method 223 with fluorenylmethyloxycarbonyl (Fmoc) protected serine. The galactoside 49 was obtained in 45%. Nevertheless, the Fmoc protecting group was not stable under the amide bond coupling reaction with the 5'-amino-uridine 61. Only 13% yield of the desired amide was obtained and 9-methylene-9H-fluorene 52 was isolated as a byproduct of the reaction while 79% percent of the reactant 49 was decomposed (Scheme III-1).

Scheme III-1. Synthesis of amino-acid containing mimetics through glycosylation and amide bond formation.

Reagents and conditions: (a) FmocSerOH, BF The tert-butyloxycarbonyl (Boc) protecting group was then employed in the synthesis. To our disappointment, the galactosylation with free or benzyl protected carboxyl group of N-Boc serine derivatives 53 and 54 and three common galactosyl donors (peracetylated galactose 1a, galactopyranosyl bromide 1b, galactose trichloroacetimidate 1c) (Table III-1), the best yield obtained was 15% by reaction between donor 1c and N-Boc serine benzyl ester 54 under boron trifluoride etherate activation (Entry 6, Table III-1). Then, we turned our attention to an amino-acid precursor such as azido-functionalized serine derivative 57. An efficient synthetic methodology for glycosylation taking peracetylate sugar 1a as donor and silver and tin salts [120][121] was first tried in this case (Entry 7, Table III-2). A moderate yield of 55% for the desired β-glycoside was obtained. The more reactive sugar donor galactose trichloroacetimidate 1c was further examined with no better yield (45%) obtained (Entry 8, Table III-2). Then, the benzoylated sugar donor galactose trichloroacetimidate 1e was evaluated for the glycosylation which could give a better yield of 67% but with difficulty in purification. At this point, the glycosylation method applying the simplest sugar donor 1a affording moderate yield was selected for our synthetic route (Scheme III-2).

III.3 Synthesis of serine derivatives containing UDP-Gal mimetics

Azido-functionalized serine methyl ester 57 was synthesized according to literature method [224][225] starting from methyl serine hydrochloride 55 via a diazo transfer reaction with retention of configuration. 259 Glycosylation under optimized conditions (Table III-2) afforded the glycoside 59 in 55% yield. The amide-bond coupling between acid 60 and silyl ether protected 5'-amino-uridine 61 did not give the desired amide under DCC/HOBt activation. Then, the synthetic strategy was changed to start from 5'-azido-uridine. After protection of the hydroxyl group of 5'-azido-uridine followed by hydrogenation of the azido group, the uridine residue 61 was first condensed with serine derivative 58 before glycosylation. The reaction with amide 63 taking peracetylated sugar 1a as donor and activation by silver and tin salts did not afford the desired glycoside. In contrast, the reaction using the trichloroacetimidate 1c afforded a good yield (61%) of the amide 62 (Scheme III-2).

Then, the two key intermediates azido 62 and amine 65 were prepared for further functionalization. A possible mechanism of this process was proposed as shown in Scheme III-6. After desilylation by 90% TFA, the proton at the stereocenter bearing the triazole was attacked by a base leading to the elimination intermediate Scheme III-6. Proposed mechanism for base-induced elimination followed by Michael-addition. Another interesting phenomenon was observed on NMR spectra of products 81 and 82. Over 10 days of spectrum analysis, the proton attached to the asymmetric carbon linked with the triazole moiety was completely or partially deuterated for products 81 and 82, respectively. The proton and the corresponding tertiary carbon atom did not show up in the respective 1 H NMR and 13 C NMR of products 81 (a, Figure III-3) while the proton and carbon at the same position of product 82 (b, Figure III-3) behaved slightly different since the exchange was slower (>10 days). Deuteration was previously described to cause "losing" signals in the 1 H and 13 C NMR. 226 The integration of the proton resonating at 5.63 ppm was 0.36 which correlated with a weak carbon peak centered at 64.6 ppm of the 13 Another type of serine-based UDP-Gal analogues was synthesized through amide-bond formation between the amino group of the serine backbone and a carboxyl group of a uridine derivative (Scheme III-7). Starting from uridine 87, selective protection of the primary alcohol followed by acetylation afforded the protected uridine 89 in 88% yield for two steps.

After desilylation, the primary alcohol 90 was oxidized to carboxyl derivative 91 under reported method. 227 The sequence of the addition of reactants is crucial for the synthesis of the amide bond (Chapter VI, method I and II of compound

III.4 Conclusion

The syntheses of glycosyltransferase inhibitors using amino-acid derivatives as pyrophosphate surrogates were studied in this chapter. Eight UDP-sugar analogues (74, 75, 77, 78, 80, 81, 82 and 95), three UDP analogues (64, 79, 93) (Figure III-2) have been synthesized. The glycosylation conditions of serine derivatives including Boc protected derivatives and the azido precursors were carefully studied. The optimized conditions were further utilized for analogues syntheses. The incorporation of an amino-acid residue between the carbohydrate and uridine moieties provided a functional amine, azido or acid group to be further substituted with other functional moieties. The introduction of functional groups which could favor binding to GT or interact with specific amino-acids in the active sites of GT was studied including phenyl, hydroxyl or amino group. Unusual byproducts were observed in the final deprotection steps and a possible mechanism of this process was proposed. The inhibitory properties of analogues prepared in this chapter will be further studied in Chapter IV against 5 galactosyltransferases.

Chapter IV: Inhibition studies towards glycosyltransferases

IV.1 Introduction

This chapter describes the evaluation of the synthetic analogues against different glycosyltransferases. A collaboration with Prof. Monica M. Palcic (Carlsberg Laboratory, Copenhagen, Denmark) provided access to five GalTs (β-1,4-galactosyltransferase

(β-1,4-GalT), α-1,4-galactosyltransferase (α-1,4-GalT),
α-1,3-galactosyltransferase (α-1,3-GalT), human blood group B galactosyltransferase (GTB), a mutant of the wild type human blood group glycosyltransferase (AAGlyB)). IC 50 and K i measurements, mechanistic studies and crystallographic data could therefore be obtained. O-linked N-acetylglucosamine transferase (OGT) was the second main enzyme studied in this project through collaboration with Prof. David Vocadlo (Simon Fraser University, Vancouver, Canada). IC 50 and K i measurements were performed as well as docking studies in collaboration with Prof. Mario Pinto (Simon Fraser University, Vancouver, Canada). OGT inhibition studies were also performed in a cell-based assay in collaboration with the group of Prof. Tony Lefebvre (Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille). As outlined in Chapter I, these enzymes are crucial for the biosynthesis in living organisms and the study on these enzymes has potential applications in carbohydrate related disease therapies.

According to structural features and biological targets, the prepared analogues could be divided into three groups, candidates using ( 1) pyridine moiety or ( 2) amino-acid derivatives as pyrophosphate surrogates tested against GalT and candidates using ( 3) pyridine as pyrophosphate surrogate evaluated as OGT inhibitors, listed in The carbohydrates used are galactose and N-acetyl glucosamine as the selective natural substrates for the enzymes. Glucose was also selected as a negative control. The best inhibitors (7-Glc, 7-Gal and 19) of human blood group transferase mutant AAGlyB were then selected for crystallographic studies in order to obtain the structural features of their interactions with the enzyme at the active site. An artifical cell membrane penetration test was also studied in collaboration with Dr. Ofelia Maniti and Prof. Agnes Girard-Egrot in our institute.

IV.1.1 Radiochemical GalT enzymatic assays

Progress in GT enzymatic assays development has been recently reviewed [START_REF] Wagner | Glycosyltransferases and their assays[END_REF] , including radiochemical and chromatographic methods, label-free and fluorescence-based GT assays. The choice of assay depends on individual requirements. The most popular and commonly used radiochemical method was employed in our project for its high sensitivity and well established protocol. The assay is based on modification of the sugar donor by a radioactive element, subsequently quantification of the radioactive-labelled sugar transfer by scintillation counter, as outlined below (Figure IV-4). Tritiated uridine-5'-diphosphogalactose (UDP-[ 3 H]Gal) was used as the radioactive substrate and GalT was used to construct the target oligosaccharide of each GalT. IC 50 and K i could be determined based on the residual radioactivity measurment of the product after workup.

IV.1.2 Cell assays

To evaluate the possible permeation of our designed inhibitor through cell membranes, 3 different cell lines were chosen, MCF7, MCF10A and HeLa cells, with increasing concentrations of inhibitors. The influence on the O-GlcNAc cell cycling was determined after 24h or 48h treatment by Western blot using the anti-O-GlcNAc antibody RL2. In parallel, a known OGT inhibitor Ac-5SGlcNAc (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glycopyranose) was chosen as a positive control of the in cellulo OGT inhibition. 228

IV.1.3 Artifical cell membrane penetration test

Membrane permeation capability of the best inhibitor candidates were evaluated by an artificial cell membrane penetration test. Liposomes, made of phosphatidylcholine (POPC), was taken as a model for the test since POPC represents about 50 % of membrane content. Two methods were used for reciprocal confirmation of the evaluation. In the "out-in" method, prepared liposomes were incubated with inhibitor solutions to evaluate the amount of compound that could penetrate the lipid bilayer to get inside of the liposomes. In the "in-out" approach, liposomes were prepared with the presence of the inhibitor to obtain inhibitor loaded liposomes.

IV.2 Results and discussion

IV.2.1 GalT inhibition studies

IV.2.1.1 Enzymatic evaluations (IC 50 / K i )

The first generation of candidates using pyridine moiety as pyrophosphate surrogate was tested against GalT applying radioactive assays, structures as listed in Figure IV-1. As a preliminary assessment, compounds were evaluated as inhibitors of five galactosyltransferases at a concentration of 1 mM of inhibitor, using K m concentrations of donor and high concentrations of acceptor substrates (Table IV-1). Among the compounds, the derivatives synthesized through glycosylation (7-Glc, 7-Gal, 19, 22) exhibited better inhibition than those obtained through CuAAC (30-Glc, 30-Gal, 30-GlcNAc, 35-Glc, 35-Gal, 35-GlcNAc, 43-Glc, 43-Gal, 43-GlcNAc).

Table IV-1. Enzymatic evaluation of neutral analogues containing pyridine as pyrophosphate surrogate.

a. Analogues which have an inhibition >35% at 1 mM (concentration of donor at K m value and concentration of acceptor at K m value or higher) were selected for IC [START_REF] Zou | C-glycosides and Aza-C-glycosides as potential glycosidase and glycosyltransferase inhibitors[END_REF] measurements. The IC [START_REF] Zou | C-glycosides and Aza-C-glycosides as potential glycosidase and glycosyltransferase inhibitors[END_REF] values were determined by constructing a dose-response curve and examining the effect of 10 different concentrations of each inhibitor ranging from 0 to 3000 μM at concentration of donor of 1/5 th K m value and concentration of acceptor at K m value or higher.

The most potent compounds are glycosides containing a galactose moiety, 7-Gal and 19, the β and α-anomers, respectively. Interestingly, the α-anomer 19 which has the same α-anomeric configuration as the natural UDP-Gal donor was always a weaker inhibitor with 37-97% inhibition in comparison with the β-anomer 7-Gal which inhibited the five enzymes between 80-100%. Compound 7-Glc, the same β-glycoside but bearing a glucose moiety, was a very weak inhibitor of all of the enzymes except for AAGlyB where 47% inhibition was observed.

The bromo-pyridine analogues of UDP, namely compounds 25 and 45, lacking saccharide moieties, were weaker inhibitors, as well as the alcohol derivative 10. This observation illustrates that the introduction of a carbohydrate moiety is important for inhibition of the enzymes.

Compound 22 containing a phenyl moiety rather than pyridine was less effective for the retaining enzymes (GTB, AAGlyB, α-1,4-GalT and α-1,3-GalT) with 19-39% inhibition, while the inverting β-1,4-GalT was inhibited at 78%. Therefore, the influence of the nitrogen atom in the pyridine-containing inhibitors is crucial as rationalized later in the crystallographic studies.

Compound 43-Glc with bis-triazolyl pyridine moieties was a potent inhibitor of β-1,4-GalT (100% inhibition) and a weak inhibitor of the retaining enzymes (30-36%). Surprisingly, the compound 43-Gal bearing a natural galactosyl moiety was a weaker inhibitor than 43-Glc. Compounds 30 and 35 with triazolyl pyridine were also weak inhibitors. The GlcNAc derivatives 30-GlcNAc, 35-GlcNAc and 43-GlcNAc tested generally exhibited weak inhibition against these galactosyltransferases. In these series, the influence of the carbohydrate moiety was less detrimental than for glycosylated compounds, 7, 19 and 22.

The mode of inhibition of the most active compounds 7-Gal was next determined as a competitive inhibitor of UDP-Gal using high concentrations of acceptor and varying the concentration of inhibitor at low and high concentrations of donor. Non-linear plots were observed in Dixon plots for AAGlyB and β-1,4-GalT, suggesting a complex mode of inhibition (Figures V.9.1 and V.6.5A, Chapter V). This complex behaviour was confirmed in Michaelis-Menten plots which showed mixed inhibition with effects on both V max and K m at high concentrations of 7-Gal (Figures V.6 Due to the complex inhibition patterns, applying K i to evaluate the inhibition capability was not feasible. To obtain a relative ranking of the compounds, IC 50 measurements were carried out with selected inhibitors which could exibit at least 35% inhibition at 1 mM concentration. For a better comparison, IC 50 values of uridine, UMP and UDP were also measured. For all enzymes, compound 7-Gal (Table IV-1) was a more potent inhibitor than uridine suggesting there are additional binding interactions of the enzyme with the pyridine scaffold. It was a better inhibitor of β-1,4-GalT and α-1,4-GalT than UMP while somewhat less effective than UMP for α-1,3-GalT and much weaker than UMP for GTB and AAGlyB. The corresponding α-anomer 19 (Table IV-1) was also more effective than uridine, though weaker than UMP. Removal of the nitrogen atom from the pyridine ring or changing the nature of the sugar moiety provided weaker inhibitors 22 and 7-Glc in this glycosylated series (Table IV-1). The introduction of triazole moieties through CuAAC was also detrimental. The neutral UDP analogues 25 and 45 in the control group did not display inhibition towards almost all enzymes tested (Table IV-1).

The second generation candidates incorporating amino-acid derivatives as pyrophosphate surrogates (Figure IV-2) was tested against the same five GalT applying radioactive assays. As a preliminary assessment, compounds were evaluated as inhibitors of galactosyltransferases at a concentration of 1 mM of inhibitor, using K m concentrations of donor and high concentrations of acceptor substrates (Table IV-2 a. Analogues which have an inhibition >35% at 1 mM were selected for K i measurements at concentration of donor at K m value and concentration of acceptor at K m value or higher. b. For compounds with which mixed inhibition modes were observed, IC [START_REF] Zou | C-glycosides and Aza-C-glycosides as potential glycosidase and glycosyltransferase inhibitors[END_REF] values were determined by constructing a dose-response curve and examining the effect of 10 different concentrations of each inhibitor ranging from 0 to 3000 μM at concentration of donor at 1/5 th K m value and concentration of acceptor at K m value or higher.

*.

Mixture of two diastereoisomer.

Most of these compounds were identified as competitive inhibitors. K i measurements were applyed to selected inhibitors which could exhibit at least 35% inhibition at 1 mM concentration (Table IV-2 It should be noted that among the second generation candidates, four inhibitors (77, 80, 93 and 95) are either anionic or cationic compounds at physiological pH. Compound 77 posses an amino group on the glycine residue while compound 80 directly on the serine backbone. Without bearing a galactose residue, compound 93 is an UDP analogue but could also serve as a GT inhibitor. As for enzymatic activity, the ionic groups on these four compounds displayed no beneficial or detrimental effect towards inhibition of GTs.

IV.2.1.2 Crystallographic studies with AAGlyB

To better understand the basis for the complex inhibition patterns of the first generation of candidates using pyridine moiety as pyrophosphate surrogate, the well characterized AAGlyB enzyme which readily crystallizes was used to investigate the mode of binding of 7-Gal, 19 and 7-Glc. Crystals of the apo-protein were soaked with the compounds and their structures were solved and refined on the ESRF synchrotron fqcilities in Grenoble (France).

The protein structures in all three crystals are essentially identical and closely resemble that in PDB_ID:2RJ7 for the AAGlyB mutant, 229 which includes an UDP-Gal molecule in the binding site. All three inhibitors (7-Gal, 7-Glc and 19) are bound to the protein in a nearly identical manner. The uridine groups overlap very closely with those in 2RJ7, with the linker to the pyridine ring also in a conformation resembling that of the first phosphate group in UDP-Gal (Figure IV-5). The conformation of the pyridine ring deviates strongly from that of the phosphates in UDP-Gal, with the ring extending roughly perpendicularly to and away from uridine. In any case, the pyridine ring projects the hexopyranoses to a different position compared to the conformation observed for 2RJ7 (Figure IV-5). The pyridine ring in the inhibitors is roughly occupying the space of side chains of Lys346, Arg352 and, to a lesser extent, His348 in which the basic side chains of Lys346 and Arg352 are originally coordinated the pyrophosphate and stabilize it as a leaving group. The amide and anomeric oxygens, together with the pyridine nitrogen, chelate the Mn 2+ ion which is displaced from its position in GTA/GTB by more than 2Å. As a result, the Mn 2+ ion is not bound to both side chains of Asp211 and Asp213 but rather only Asp213 through the carboxylic oxygen.

Only one conformation of the glucose anomeric oxygen was observed for 7-Glc while two alternative positions were observed for 7-Gal of which only one can chelate the manganese. The sugar moieties are not visible in the electron density map, most likely disordered between several conformations, and hence could not been modeled. In the case of the α-galactoside 19, two alternative conformations of the anomeric oxygen were also observed. In the conformation not chelating the manganese, the galactose group is visible with weak electron density and is modeled with reduced occupancy (Figure IV-6). The identification of the galactose moiety suggests that hexoses are present in the crystals but in disordered conformations preventing their modeling and crystallographic description. The manganese cation is affected by the chelating inhibitors and displaced from its normal position by a 4 Å. In crystal structures of GTA and GTB, the Mn 2+ is bound to the carboxylic groups of Asp211 and Asp213, as well as to the pyrophosphate of UDP or UDP-Gal in a tetra-coordinated structure. In 7-Glc/AAGlyB complex, the original position is occupied by a water molecule which is then involved in the coordination sphere of the displaced manganese and hydrogen bonded to the amide carbonyl of the inhibitors, Asp211 and Asp213 (Figure IV-7). The Mn 2+ cation itself has a longer ligand distances and is heptacoordinated with the amide and anomeric oxygens, the pyridine nitrogen and three water molecules. The coordination sphere can be described as a pentagonal bipyramid with three of the equatorial positions occupied by the pyridine nitrogen and its adjacent carboxylic oxygens at 2.3 Å, 2. Compounds 79, 80 and 81 of the second generation of candidates using amino acid as pyrophosphate surrogate have been soaked with apo form of AAGlyB to gain more Structure Activity Relationship (SAR) information on inhibitors design. The results obtained still need some refinements and discussions with our collaborators and will be reported in due course in our coming publications.

IV.2.2 OGT inhibition studies

IV.2.2.1 Enzymatic evaluation

Eight UDP-GlcNAc or UDP analogues functionalized with a pyridine ring conjugated with one or two triazoles (

IV.2.2.2 Docking studies

To gain insight into the binding mode of these compounds to OGT, 4 compounds 43-Glc, 45, 35-Glc, and 35-GlcNAc were selected for docking experiments using the known structure of OGT in complex with UDP-GlcNAc (PDB_: 4GZ5). The best ranking pose of each OGT inhibitor was selected for docking (Figure IV-9). The uridine moieties of the four inhibitors showed nearly perfect overlap, almost identical to the binding mode of UDP-GlcNAc. Enzymatic studies revealed that this series of compounds could exhibit inhibitory effect on human OGT with the best inhibition of 19% observed for 35-Glc at 50 μM. Docking study confirmed that the uridine moieties of these inhibitors could bind to OGT identical to the natural substrate of UDP-GlcNAc and polar interactions were created between triazole linkers and OGT. But cell assay suggested that these compounds may not inhibit OGT in vivo. So an artifical cell membrane penetration test was performed for the most potent compound 35-Glc to evaluate its permeation capability.

IV.2.3 Evaluation of membrane permeation

Membrane penetration capability of the best inhibitor candidates against GalT or OGT ( 7 In method I, prepared liposomes were incubated with synthesized inhibitor candidate in a shaking incubator. Then, liposome-complex fraction was separated from free inhibitors by G-25 column (size exclusion chromatography, large molecules, liposome fraction in our case, leave the column first). UV absorption at λ max was measured for each fraction to evaluate the amount of inhibitor incorporated into the liposomes. Incubation was performed for liposome-inhibitor complex fractions. Further separation was performed to evaluate the amount of inhibitor released by gradient of concentration. The opposite approach was performed to confirm the previous results. In method "in-out" II, liposomes were prepared with inhibitor solution to incorporate the inhibitor in the liposomes. Liposomes were separated from excess inhibitors by G-25 column. The liposome complex fraction was further incubated. After 24 hours, the mixture was purified by G-25 to evaluate the amount of inhibitor released from the liposomes. For compound 7-Gal, in Method I, after incubation with the inhibitor solution (1×10 -2 M), the liposome fraction was readily separated from the free inhibitors (Figure IV-17a, fraction 4 for liposomes complex and fractions 7-11 for free inhibitors). Analysis of the UV absorption spectra of the complex fraction indicated that about 2.3% of the compound was complexed with liposomes (Figure IV-17b, black line: spectrum of liposome only; red line: spectrum of fraction 4). Further incubation of the fraction 4 at 37°C for 24 hours was performed but no free inhibitor could be separated from this fraction (Figure IV-17c) indicating that inhibitors could interact with liposomes but cannot be released, probably sequestered at the surface of the liposomes.

In comparison, Method II was performed. After the preparation of liposomes with inhibitors solution (1×10 -2 M), free inhibitors were separated from the liposome complex fraction (Figure IV-18a, fraction 4 for liposomes complex and fractions 7-11 for free inhibitor). UV spectra analysis of this fraction indicated that a fraction of the compound was indeed captured inside of the liposomes (Figure IV-18b). Further incubation and separation was carried out but no apparent free inhibitor fraction could be isolated from the complex fraction 4 (Figure IV-18c).

Together with the results obtained from Method I, this phenomenon revealed that the inhibitor 7-Gal could interact and be associated with liposomes. The complex formed is relatively stable therefore the affinity of 7-Gal towards liposomes was demonstrated.

a. b. c. A sulfonylurea anti-hyperglycaemic drug (Glibenclamide ® ) with a molecular wight of 494 g.mol -1 similar to our analogues, was reported to cross phospholipid bilayers. 258 It was taken as a positive control for this study. Nevertheless, separation from the liposome fractions was unsuccessful and this molecule could therefore not be used as a positive control for this study.

IV.3 Conclusion

A total of 26 analogues with either pyridine moiety or amino-acid derivatives as pyrophosphate surrogate were evaluated as GalT inhibitors. In the first series of pyridine containing compounds, the derivatives synthesized through glycosylation exhibited better inhibition than those obtained through CuAAC. The enzymatic assays combined with the crystallographic study of the best three inhibitors revealed that:

(1) the uracil and ribose moieties are crucial for binding;

(2) the introduction of a carbohydrate moiety is important for selectivity and inhibition of the enzymes;

(3) the pyridine based linker between the ribose and hexoses has multiple effects, including orienting the hexoses away from the galactose position in UDP-Gal, displacing the manganese from its original position by chelating and preventing the folding of the C-terminal tail of AAGlyB keeping it in a closed form; (4) the inhibitors displayed mixed binding modes, neither competitive nor noncompetitive inhibitors.

The enzymatic study of the second series of amino-acid derivatives indicated that:

(1) an appropriate design of neutral linker between the ribose and hexoses is important to orient the molecule to the proper binding site;

(2) the introduction of a hydrophobic moiety on the backbone of linker could increase the binding affinity with GalT;

(3) the decoration of a hydrogen donating group could contribute to the formation of hydrogen bond therefore increasing the binding affinity.

Among the derivatives synthesized, 8 UDP-sugar or UDP analogues were selected for OGT inhibition, including enzymatic assays and cell assays. The enzymatic evaluation indicated that this series of compounds has minimal inhibition effect at 50 μM and the best inhibitor has a K i value of 422 μM. Further study showed that these compounds did not inhibit OGT function in cells. Docking studies showed that the polar interactions formed between triazole linkers and amino-acid backbone of OGT could mimic the binding function of β-phosphate of UDP-GlcNAc whereas the sugar heads of this series of compounds were pointing into a direction diffent from the original galactopyranose pocket.

The concept of "neutral" inhibitor instead of ionic analogues was finally verified by an artificial cell membrane penetration test on the two best inhibitor candidates against GalT or OGT (7-Gal and 35-Glc). Despite minor structural modifications, the two compounds behaved differently in this model. Compound 7-Gal showed binding affinity towards liposome while 35-Glc showed no apparent binding or penetrating capability.

General Conclusion

The study in this thesis presented the design, synthesis, biological evaluation and structural analysis of glycosyltransferase inhibitors. The design rationale was to make analogues of NDP-sugar natural substrates incorporating a "neutral" pyridine or amino-acid derivative as the pyrophosphate surrogate. Due to the poor bioavailability of the charged native substrates, neutral inhibitors were investigated in order to provide cell permeable substrates for potential in cellulo or in vivo applications as fundamental tools for biology or even as potential drugs.

The design and synthesis of 26 GT inhibitors were performed using a combination of conjugations through O-glycoside, amide bond or triazole functionalities.

(1) A systematic investigation of glycosylation methods on pyridine-or serine-based acceptors was performed. The optimized conditions were used to synthesize neutral analogues of enzyme natural substrates. ( 2) Two conjugation techniques, Staudinger-Vilarrasa reaction and azide-alkyne cycloaddition (CuAAC), were applied to obtain inhibitors candidates in a minimum number of steps. Two synthetic routes, from "Sugar-to-Nucleoside" and from "Nucleoside-to-Sugar", were applied in the glucose series in order to compare their efficiency. An orthogonally protected bis-alkynyl pyridine was introduced in "one-pot" synthetic strategy to increase the efficiency. ( 3) Unusual byproducts were observed in the final deprotection steps of the serine-based analogues and a possible mechanism for this process was proposed.

The inhibition evaluation towards five galactosyltransferases and one GlcNAc transferase (OGT) revealed moderate inhibition. Nevertheless, structural analysis of the most potent compounds in complex with a glycosyltransferase revealed that the designed "neutral" pyridine linker could indeed chelate with manganese although displacing it just a few Å from its original position. The sugar head-group was oriented away from the position found in the related complex with UDP-Gal indicating a new binding mode. Eight UDP-GlcNAc or UDP analogues functionalized with a pyridine ring conjugated with one or two triazoles were selected for OGT inhibition evaluation. Enzymatic studies revealed that this series of compounds could exhibit inhibitory effect on human OGT. Docking study confirmed that the uridine moieties of these inhibitors could bind to OGT identical to the natural substrate of UDP-GlcNAc and polar interactions were created between triazole linkers and OGT. Nevertheless, cell assay suggested that these compounds may not inhibit OGT in vivo.

The concept of "neutral" inhibitor instead of ionic analogues was finally examined by an artificial cell membrane penetration test on the two best inhibitor candidates against GalT or OGT (7-Gal and 35-Glc). Despite minor structural modifications, the two compounds behaved differently in this model. Compound 7-Gal showed apparent binding affinity towards liposome while 35-Glc showed no apparent binding or penetrating capability.

Perspectives

Crystal data analysis on the binding mode of these inhibitors provided useful structure-activity relationship information for future rational design (Figure P-1).

The 4-position of pyridine is 4 Å away from the side chain of Trp181. This position would be ideally substituted by an aliphatic or heterocyclic hydrophobic group to create Van der Waals contacts or stacking interactions with the nearby amino-acid side chain.

The glucose or galactose moiety of the inhibitor is most likely disordered. Since the function of the exo-anomeric oxygen is involved in chelation with manganese, an ester or amide bond instead of a glycosidic bond may perform better.

In the co-crystal, the glycosidic oxygen is in the proximity of the fucose residue of the acceptor and the carbon atom adjacent to the pyridine is only 3.6 Å away from O2 of the acceptor's fucose residue. This position would be ideally substituted with a linker of about three atoms attached to the acceptor to generate a bisubstrate inhibitor. This should result in an increase in both affinity and specificity of the inhibitors.

Considering the different orientation of the inhibitor synthesized in comparison to the natural substrate, more consideration should be given to the conformational plasticity of the designed compounds but not only the conformation of the targeted protein. Yet, a fine balance should be considered. If the structure of the compound is too rigid (e.g. the pyridine group), the conformational space may not orientate the carbohydrate moiety towards the proper position. Whereas, if there is too much flexibility, the molecule could in principle adopt the desired conformation but then large penalty may be required. Chapter V : Experimental section General protocol A for Staudinger-Vilarrasa conjugation. The acid (0.466 mmol, 1 eq.) and HOBt (0.839 mmol, 1.8 eq.) were co-evaporated with toluene (3×5 mL) and THF (3×5 mL).

The mixture was dried under vacuum for 1 h. The mixture was dissolved in dry THF (4 mL) under argon and cooled to 0°C. DIC (0.839 mmol, 1.8 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed and the reaction stirred at r.t. for 30 min. Meanwhile, the azide (0.699 mmol, 1.5 eq.) was dissolved in dry THF (4 mL) under argon and cooled to 0°C. PMe 3 (0.932 mmol, 2 eq.) was added and the reaction was stirred at 0°C. After 30 min, the solution was transferred into the flask containing the acid/HOBt solution at 0°C. The flask was washed with THF (4 mL) and the solution was transferred. The resulting reaction mixture was stirred at 0°C for 1 h then allowed to reach r.t. and stirred for an additional 16 h. The reaction mixture was diluted with water (60 mL), extracted with EtOAc (4×60 mL). The combined organic layers were washed with satd Na 2 CO 3 (60 mL), H 2 O (60 mL) and brine (60 mL), dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (PE to EtOAc) to afford the desired amide.

General protocol B for Sharpless CuAAC conjugation.

To a solution of the alkyne (0.19 mmol, 1 eq.) and the azide (0.19 mmol, 1 eq.) in tBuOH/H 2 O (1/1, 5.6 mL / 280 μL) were added CuSO 4 (0.11 mmol, 0.6 eq.) and sodium ascorbate (0.228 mmol, 1.2 eq.). The reaction was stirred at 35°C for 24 h then diluted with water (20 mL) and extracted with CH 2 Cl 2 (3×30 mL).

The combined organic layers were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography to afford the desired triazole.

General protocol C for Meldal CuAAC conjugation. iPr 2 NEt (0.036 mmol, 0.25 eq.) was added into a flask containing the azide (0.14 mmol, 1 eq.), the alkyne (0.14 mmol, 1 eq.) and CuI (0.01 mmol, 0.1 eq.) in DMF (2 mL). The reaction was stirred at r.t. overnight. After 24 h, the solution was diluted with EtOAc (50 mL), washed with satd Na 2 CO 3 (2×25 mL) and H 2 O (30 mL). The combined aqueous layers were extracted with EtOAc (3×30 mL). The combined organic layers were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography to afford the desired triazole.

General protocol D for Sonogashira reaction. The bromo-arene (1.0 mmol, 1 eq.), Pd(PPh 3 ) 4 (0.1 mmol, 0.1 eq.) and CuI (0.1 mmol, 0.1 eq.) were dissolved in toluene (25 mL) and the solution was degassed with argon. Then, trimethylsilylacetylene (3.0 mmol, 3 eq.) and diisopropylamine (2.2 mmol, 2.2 eq.) were added. The reaction was stirred for 48 h at r.t. protected from light then poured into satd NH 4 Cl (100 mL). The aqueous layer was extracted with CH 2 Cl 2 (3×100 mL). The combined organic layers were washed with H 2 O (100 mL) and brine (100 mL), dried (Na 2 SO 4 ) and concentrated. The residue was then purified by silica gel column chromatography to afford the desired product.

General protocol E for the Zemplén deacetylation. In a flask containing the acetylated compound (1.0 eq.) in MeOH (3 mL) was slowly added solid NaOMe until pH ≥ 9. The reaction was stirred at r.t. for 3 h. DOWEX 50W×2 Resin (Fluka, 50-100 mesh, H + Form) was added to neutralize the reaction until pH~6. Then, the reaction mixture was filtrated and concentrated in vacuum to afford the corresponding deprotected compound.

General protocol F for desilylation. TBAF (1.4 eq.) was added dropwise into a flask containing the trimethylsilylethynyl derivative (1.0 eq) in MeOH (4 mL). The reaction was stirred at r.t. for 4 h. Then, the reaction was poured into water (50 mL), extracted with CH 2 Cl 2 (4 ×50 mL). The combined organic layers were dried over Na 2 SO 4 . The solvent was evaporated and the residue was purified to afford the corresponding deprotected compound.

General protocol G for the synthesis of compounds 74-75 and 77-81.

The protected compound (1.0 eq.) was solubilized with CH 2 Cl 2 (0.5 mL). Then, 90% TFA (2.2 mL) was added dropwise. After addition, the reaction was stirred at r.t. overnight. MeOH (3 mL) and H 2 O (5 mL) were then added into the reaction. The solvent was removed by evaporation and the residue was used directly for the reaction of next step. Aq. LiOH solution (1.2 eq. for each acetate group, in 1 mL of H 2 O) was added dropwise into a solution of the residue solubilized in THF (2 mL) at r.t. After addition, the reaction mixture was stirred at r.t. for 2 h. Then, the reaction mixture was concentrated and the residue was purified by C18 reverse phase column chromatography (100% H 2 O to 100% MeOH) to afford the corresponding deprotected compound.

V.2 Synthesis and characterization

Methyl 6-(hydroxymethyl)picolinate (2a) 230 Sodium borohydride (390 mg, 10.3 mmol, 1.0 eq.) was slowly added at 0°C to a stirred solution of dimethyl pyridine-2,6-dicarboxylate (2.00 g, 10.3 mmol, 1.0 eq.) in MeOH/CH 2 Cl 2 (100 mL, 7:3). The reaction mixture was stirred at room temperature for 3 h. Then, the reaction was neutralized by adding satd aq. NH 4 Cl solution (40 mL). After extraction with CH 2 Cl 2 (3×50 mL), the combined organic layers were dried (Na 2 SO 4 ) and the solvent was evaporated. The resulting crude residue was purified by silica gel column chromatography (PE:EtOAc, 6:4 to 1:1) to afford compound 2a as white solid (970 mg, 5.8 mmol, 56% In a 3-necked 500 mL round-bottomed flask equipped with a mechanical stirrer was placed dimethyl isophthalate (20.00 g, 0.103 mol, 1.0 eq.) dissolved in acetone (200 mL). To this mixture was added dropwise over 20 min a solution of NaOH (4.33 g, 0.108 mol, 1.05 eq.) in MeOH (40 mL). The resulting milky suspension was stirred at r.t. overnight. Then, another portion of NaOH (0.433 g, 0.011 mol, 0.1 eq.) was added into the reaction and the suspension was stirred for another 5 h. The solvent was removed under vacuum and the white precipitate thus obtained was dissolved in water (400 mL). Concentrated HCl (15 mL) was added dropwise until pH~1. Then, the suspension was filtered; the collected precipitate was washed with water (4×100 mL) and dried under vacuum at 65°C for 24 h to give white solid 18.25 g. The obtained monomethyl isophthalate was used directly without further purification. In a 3-necked 500 mL round-bottomed flask equipped with a magnetic stirrer and a low temperature thermometer under a nitrogen atmosphere was placed monomethyl isophthalate (5.00 g, 0.027 mol, 1.0 eq.) dissolved in dry THF (125 mL). Then, this solution was placed in an ice-water bath and a solution of BH 3 •SMe 2 (2M in THF, 70 mL, 0.14 mol, 5.0 eq.) was added dropwise over 90 min to maintain the temperature in the solution below 7°C. After another 15 min, the cooling bath was removed and the solution was allowed to reach ambient temperature. After 5 h, the reaction was carefully quenched (strong gas evolution) with small pieces of ice while cooling in an ice-water bath. When the gas evolution ceased, brine (50 mL) was added and the resulting mixture was extracted with diethyl ether (3×100 mL 2-2 Pure SnCl 4 (0.15 mL, 1.22 mmol, 5.0 eq.) was added dropwise (within 60 min -syringe pump) at 0 o C (an ice/water bath) to a stirred solution of donor 1a (143 mg, 0.366 mmol, 1.5 eq.), silver trifluoroacetate (135 mg, 0.61 mmol, 2.5 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (4 mL). The reaction mixture was stirred at 0 o C with protection of light for 48 h. Satd NaHCO 3 aq. solution (10 mL) was added into the reaction and the solution was vigorously stirred for 15 min. The suspension was filtered and the filtrate was extracted with CH 2 Cl 2 (3×20 mL). The organic layers were combined, concentrated. No glycosylated product was observed.

Entry 2, Table 2-2

Distilled CH 2 Cl 2 (5 mL) was added to a 10 mL Ar-flushed flask containing methyl 6-(hydroxymethyl)picolinate (2a) (61 mg, 0.366 mmol, 1.5 eq.), donor 1b (100 mg, 0.244 mmol, 1.0 eq.) and AgOTf (94 mg, 0.366 mmol, 1.5 eq.) with protection of light. The reaction was stirred overnight at r.t.. The reaction mixture was diluted with CH 2 Cl 2 (100 mL), filtered through celite. The organic phase was washed with satd aq. NaHCO 3 solution (50 mL) and brine (50 mL). The organic phase was dried (MgSO 4 ), filtered and concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc, 4:1 to 2:3) to afford compound 3a as white foam (10 mg, 0.020 mmol, 8%).

Entry 3, Table 2-2

Distilled CH 2 Cl 2 (5 mL) was added into a 10 mL Ar-flushed flask containing methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.), donor 1b (250 mg, 0.61 mmol, 2.5 eq.) and AgOTf (94 mg, 0.366 mmol, 1.5 eq.). The reaction was stirred overnight at r.t. with protection of light. The reaction mixture was diluted with CH 2 Cl 2 (100 mL), filtered through celite. The organic phase was washed with satd aq. NaHCO 3 solution (50 mL), brine (50 mL) and dried (MgSO 4 ), filtered and evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:2) to afford compound 3a as white foam (22 mg, 0.044 mmol, 18%).

Entry 4, Table 2-2

A solution of donor 1b (150 mg, 0.366 mmol, 1.5 eq.) in anhydrous CH 2 Cl 2 (1 mL) was added dropwise under argon with protection of light to a solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and Hg(CN) 2 (246 mg, 0.978 mmol, 4.0 eq.) in dry CH 3 CN (1 mL). The resulting mixture was vigorously stirred overnight.

The reaction mixture was diluted with CH 2 Cl 2 (5 mL) and filtered through celite. The solid was washed with CH 2 Cl 2 . The organic phase was washed with satd aq. NaHCO 3 solution (50 mL) and brine (50 mL). Then, the organic phase was dried (MgSO 4 ), filtered and concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 3:2) to afford compound 3a as white foam (13.5 mg, 0.027 mmol, 11%).

Entry 5, Table 2-2

Boron trifluoride etherate (62 μL, 0.488 mmol, 2.0 eq.) was added dropwise at -20 o C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and donor 1c (180 mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2 (3 mL). The reaction mixture was stirred at -20 o C for 2 h. Triethylamine (0.2 mL, 1.4 mmol, 5.7 eq.) was injected to quench the reaction. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 3:2) to afford compound 3a as white foam (56 mg, 0.113 mmol, 46%). mL) . The organic layers were washed with H 2 O, dried (MgSO 4 ). The solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3a as white foam (17 mg, 0.034 mmol, 14%).

Entry 6,

Entry 14, Table 2-2

Boron trifluoride etherate (11.4 μL, 0.09 mmol, 1.0 eq.) was added dropwise at -20 o C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and donor 1d (190 mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2 (3 mL). The reaction mixture was stirred at -20 o C for 2 h. Triethylamine (0.2 mL, 1.4 mmol, 5.7 eq.) was added into the reaction mixture at -20 o C to quench the reaction. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 6:4) to afford compound 3a as white foam (39 mg, 0.078 mmol, 32%).

Entry 18, Table 2-3

Trimethylsilyl trifluoromethanesulfonate (9 μL, 0.05 mmol, 0.5 eq.) was added dropwise at -20°C to a stirred solution of donor 1c (74 mg, 0.15 mmol, 1.5 eq.), methyl 6-(trimethylsilyloxymethyl)picolinate (2b) (24 mg, 0.1 mmol, 1.0 eq.) and 4Å MS (200 mg -300 mg) in freshly distilled CH 2 Cl 2 (3 mL). After 80 min., another portion of TMSOTf (9 μL, 0.05 mmol, 0.5 eq.) was added into the reaction. After another 100 min, the starting material 1c was consumed. Triethylamine (0.2 mL, 1.4 mmol, 14.0 eq.) was injected at -20°C to quench the reaction. The reaction mixture was filtered and the solid was washed with CH 2 Cl 2. The solvent was evaporated. The desired glycosylated compound 3a was obtained in ~ 5% yield as estimated by TLC. (5 mL) under argon in the presence of 4Å MS (200 mg -300 mg). Then, the mixture was cooled down to -20°C. Trimethylsilyl trifluoromethanesulfonate (3.3 μL, 0.018 mmol, 0.2 eq.) was added at -20°C, then, methyl 6-(hydroxymethyl)picolinate (2a) (15 mg, 0.090 mmol, 1.0 eq.) dissolved in freshly distilled CH 2 Cl 2 (1.5 mL) was added. The reaction mixture was stirred at -20°C for 30 min. Three news portions of TMSOTf were successively added (3.3 μL, 0.018 mmol, 0.2 eq. after 1.5 h then 6.6 μL, 0.039 mmol, 0.4 eq. after 2.5 h and 16.5 μL, 0.090 mmol, 1.0 eq. after 4.5 h) and stirring was maintained for 16 h at -20°C. Triethylamine (0.2 mL, 1.4 mmol, 15.5 eq.) was injected at -20°C to quench the reaction. After filtration of the reaction mixture, the solid was washed with CH 2 Cl 2 . The solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 7:3) to afford compound 3b as white foam (22 mg, 0.030 mmol, 33%).

Entry 16, Table 2-2

Boron trifluoride etherate (11.4 μL, 0.09 mmol, 1.0 eq.) was added dropwise at -20°C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (15 mg, 0.09 mmol, 1.0 eq.) and donor 1e (87 mg, 0.117 mmol, 1.3 eq.) in freshly distilled CH 2 Cl 2 (3 mL). The reaction mixture was stirred at -20°C for 1 h. Then, BF 3 •Et 2 O (11.4 μL) was added again and after another 2 h, more BF 3 •Et 2 O (11.4 μL) was added. After 17 h, Et 3 N (0.2 mL, 1.4 mmol, 15.5 eq.) was added at -20°C into the reaction mixture to quench the reaction. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 7:3) to afford compound 3b as white foam (28 mg, 0.038 mmol, 42% 2-2 In a dried round bottom flask containing donor 1f (618 mg, 0.896 mmol, 1.0 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (164 mg, 0.896 mmol, 1.0 eq.) in CH 2 Cl 2 (1 mL) was added methyl iodide (0.18 mL, 2.68 mmol, 3.0 eq.). Then, the reaction mixture was stirred at 50°C at presence of 4Å MS (200 mg -300 mg). After 22 h, another portion of methyl iodide (0.09 mL, 1.34 mmol, 1.5 eq.) was added. After another 55 h, the reaction mixture was filtered through celite, washed with CH 2 Cl 2 and evaporated to dryness. The residue was purified by silica gel column chromatography (PE:EtOAc, 4:1 to 3:1) to afford compound 3c as white foam (463 mg, 0.672 mmol, 75% 23Boron trifluoride etherate (24 μL, 0.03 mmol, 0.6 eq.) was added at -20 o C to a stirred solution of methyl 6-(hydroxymethyl)picolinate N-oxide (2c) (56 mg, 0.31 mmol, 1.0 eq.) and donor 1c (344 mg, 0.70 mmol, 2.25 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After 3 h, Et 3 N (0.2 mL, 1.4 mmol, 4.5 eq.) was added at -20°C to quench the reaction. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc, 1:1 to MeOH:EtOAc, 5:95) to afford 19 mg of a mixture containing the deactivated donor:unreacted acceptor 2c:α anomer of 3d:β anomer 3d (1:1:0.46:1) as white foam (7% yield calculated for 3d).

β anomer: R f = 0.23 (PE:EtOAc = 1:3); 23Boron trifluoride etherate (7 μL, 0.056 mmol, 0.2 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1c (209 mg, 0.424 mmol, 1.5 eq.) and methyl 3-hydroxymethylbenzoate (2d) (47 mg, 0.283 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After 2 h, Et 3 N (0.2 mL, 1.4 mmol, 4.9 eq.) was injected into the reaction mixture. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 5:1 to 3:1) to afford compound 3e as a white foam (62 mg, 0.125 mmol, 44%) and acetylated acceptor (22 mg, 0.106 mmol, 37%).

Entry 21, Table 2-3

Boron trifluoride etherate (25 μL, 0.20 mmol, 0.2 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1c (500 mg, 1.01 mmol, 1.0 eq.) and methyl 3-hydroxymethylbenzoate (2d) (503 mg, 3.03 mmol, 3.0 eq.) in freshly distilled CH 2 Cl 2 (8 mL). After 1 h, Et 3 N (0.2 mL, 1.4 mmol, 1.4 eq.) was injected into the reaction mixture. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 5:1 to 3:1) to afford compound 3e as white foam (397 mg, 0.800 mmol, 79% Then, the reaction was stirred at 60°C in a sealed vessel. After 2 h, additional CSA (116 mg, 0.5 mmol, 1.0 eq.) was added into the reaction. After another 3.5 h, Et 3 N (1 mL, 7 mmol, 14.0 eq.) was injected into the reaction mixture to quench the reaction. Then, the solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 1:1 to EtOAc) to afford compound 3f as white foam (41 mg, 0.083 mmol, 17%). II II-4 Trimethylsilyl trifluoromethanesulfonate (54 μL, 0.3 mmol, 1.0 eq.) was added dropwise at -20°C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (50 mg, 0.30 mmol, 1.0 eq.) and donor 1h (172 mg, 0.33 mmol, 1.1 eq.) in freshly distilled CH 2 Cl 2 (5 mL). After 1.5 h, additional TMSOTf (54 μL, 0.3 mmol, 1.0 eq.) was added. After another 1 h, the reaction temperature was increased to 0°C. After another 2.5 h, more TMSOTf (54 μL, 0.3 mmol, 1.0 eq.) was added into the reaction mixture at -20°C and the reaction mixture was stirred at -20°C for another 17 h. Then, Et 3 N (0.2 mL, 1.4 mmol, 4.7 eq.) was injected into the reaction mixture at -20°C. The solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3g as white foam (47 mg, 0.075 mmol, 25%).

Entry 23, Table

Entry 25, Table II-4

Trimethylsilyl trifluoromethanesulfonate (54 μL, 0.3 mmol, 1.0 eq.) was added dropwise at -20°C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (50 mg, 0.30 mmol, 1.0 eq.) and donor 1i (206 mg, 0.33 mmol, 1.1 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After 1 h, another fraction of donor 1i (93 mg, 0.15 mmol, 0.5 eq.) and TMSOTf (54 μL, 0.3 mmol, 1.0 eq.) were added to the reaction. After another 3.5 h, Et 3 N (0.2 mL, 1.4 mmol, 4.7 eq.) was injected into the reaction mixture at -20°C to quench the reaction. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3g as white foam (90 mg, 0.143 mmol, 48%). II (3 mL). After 1 h, additional TMSOTf (46 μL, 0.244 mmol, 1.0 eq.) was added to the reaction. After another 1 h, another portion of TMSOTf (46 μL, 0.244 mmol, 1.0 eq.) was added to the reaction. After 1.5 h, the reaction temperature was increased to r.t. After another 15.5 h, Et 3 N (0.2 mL, 1.4 mmol, 5.7 eq.) was injected into the reaction mixture. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3h as white foam (36 mg, 0.067 mmol, 27%).

Entry 26, Table

Entry28, Table II-4

Boron trifluoride etherate (76 μL, 0.60 mmol, 2.0 eq.) was added dropwise at -20°C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (50 mg, 0.30 mmol, 1.0 eq.) and 3,4,6-tri-O-acetyl-2-allyloxycarbonylamino-2-deoxy-α-D-glucopyranosyl trichloroacetimidate (1k) (176 mg, 0.33 mmol, 1.1 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After 1 h, additional BF 3 •Et 2 O (38 μL, 0.30 mmol, 1.0 eq.) was added to the reaction. Then, the reaction mixture was stirred at -20°C. After 16 h, Et 3 N (0.2 mL, 1.4 mmol, 4.7 eq.) was injected into the reaction mixture. The solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3h as white foam (77 mg, 0.143 mmol, 48%) contaminated with 0.13 eq. of methyl 6-(hydroxymethyl)picolinate (2a 7.8 eq.) was injected into the reaction mixture. The yield of the desired product was about 5% as estimated by TLC and the major product of the reaction was the acetylated acceptor (53%).

Entry 30, Table II-4

Boron trifluoride etherate (61 μL, 0.48 mmol, 2.0 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1l (133 mg, 0.36 mmol, 1.5 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (40 mg, 0.24 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 2.5 h, another portion of BF 3 •Et 2 O (61 μL, 0.48 mmol, 2.0 eq.) was added to the reaction and the reaction temperature was increased to r.t. After 16 h, Et 3 N (0.2 mL, 1.4 mmol, 5.8 eq.) was injected into the reaction mixture. Then, the solvent was evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc, 4:1 to 3:1) to afford compound 3i α as white foam (40 mg, 0.08 mmol, 35%).

Entry 31, Table II-4

Trimethylsilyl trifluoromethanesulfonate (92 μL, 0.48 mmol, 2.0 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1m (171 mg, 0.36 mmol, 1.5 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.24 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 2 h, Et 3 N (0. 2mL, 1.4 mmol, 5.8 eq.) was injected into the reaction mixture. Then, the solvent was evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc, 4:1 to 2:1) to afford compound 3i α as white foam (20 mg, 0.04 mmol, 17%).

Entry 32, Table II-4

Boron trifluoride etherate (62 μL, 0.48 mmol, 2.0 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1m (171 mg, 0.36 mmol, 1.5 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.24 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 2 h, another portion of BF 2, 165.9, 165.6, 165.3, 165.2, 158.3, 147.0, 137.7, 133.6, 133.5, 133.4, 133.3, 129.92, 129.91, 129.86, 129.6, 129.2, 128.82, 128.79, 128.53, 128.51, 128.4, 124.9, 124.1, 101.2 (C 1 ), 72.9, 72.5, 72.2, 72.0, 69.6, 63.1, 53 N (0.2 mL, 1.4 mmol, 5.7 eq.) was injected into the reaction mixture. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3l as white foam (43 mg, 0.098 mmol, 39% (1.5 mL). The mixture was stirred at -20 °C for a total of 4 h and the addition of TMSOTf was according to the following: 3.3 μL (0.2 eq.) after 0.5 h, 3.3 μL (0.2 eq.) after 1.5 h and finally 3.3 μL (0.2 eq.) after 2.5 h. After 1.5 h, the reaction was complete, the reaction mixture was then warmed to room temperature and diluted with 30 mL CH 2 Cl 2 and washed with satd aq. NaHCO 3 solution (2×25 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×25 mL) and the combined organic layers were washed with brine (25 mL), dried (Na 2 SO 4 ) and concentrated. The crude product was purified by silica gel column chromatography (Hexane:EtOAc, 2:1) to afford compound 3m as white foam (37 mg, 0.06 mmol, 66% 7, 133.5, 133.3, 130.1, 129.8, 129.4, 129.3, 129.2, 128.7, 128.6, 128,4 2345To a solution of donor 1r (173 mg, 0.23 mmol, 1.3 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (30 mg, 0.18 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (10 mL) at -20°C was added BF 3 •OEt 2 (6.5 μL, 0.036, 0.2 eq.). The reaction was stirred for a total of 22.5 h, and the addition of BF 3 •OEt 2 was according to the following: 6.5 μL (0.2 eq.) after 1 h, 6.5 μL (0.2 eq.) after 2.5 h, 13 μL (0.4 eq.) after 4 h, and finally another 32.5 μL (1.0 eq.) after 6.5 h, for a total of 2 equivalents. After 16 h, the reaction was complete, the reaction mixture was diluted with CH 2 Cl 2 (25 mL) and washed with satd aq. NaHCO 3 solution (2×25 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×25 mL), after which the combined organic layers were washed with brine (30 mL), dried (Na 2 SO 4 ) and concentrated. The crude product was purified by silica gel column chromatography (PE:EtOAc =2:1) to yield compound 3n as white foam (64 mg, 0.086 mmol, 48%).

Entry 38, Table 2-5

To a solution of donor 1r (173 mg, 0.23 mmol, 1.3 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (30 mg, 0.18 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (10 mL) at -20°C was added TMSOTf (6.5 μL, 0.036 mmol, 0.2 eq.). The reaction was stirred for a total of 22.5 h, and the addition of TMSOTf was according to the following: 6.5 μL (0.2 eq.) after 0.5 h, 19.5 μL (0.6 eq.) after 2 h, and finally another 33 μL (1.0 eq.) after 5 h, for a total of 2 equivalents. After 17.5 h the reaction was complete, the reaction mixture was diluted with CH 2 Cl 2 (25 mL) and washed with satd aq. NaHCO 3 solution (2×25 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×25 mL), after which the combined organic layers were washed with brine (30 mL), dried (Na 2 SO 4 ) and concentrated. The crude product was purified by silica gel column chromatography (PE:EtOAc, 2:1) to yield compound 3n as white foam (30 mg, 0.04 mmol, 22% 7, 122.6, 133.4, .133.2, 130.0, 129.9, 129.3, 128.7, 128.6, 128.5, 124.8 Entry 39, Table 2345To a solution of donor 1s (126 mg, 0.23 mmol, 1.3 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (30 mg, 0.18 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (10 mL) was added at -40°C NIS (63 mg, 0.28 mmol, 1.56 eq.) and TMSOTf (6.5 μL, 0.036 mmol, 0.2 eq.). The reaction was stirred for a total of 5.5 h, and the addition of TMSOTf was according to the following: 6.5 μL (0.2 eq.) after 1 h, 19.5 μL (0.2 eq.) after 2 h, and finally another 33 μL (1.0 eq.) after 3 h, for a total of 2 equivalents. After 2.5 h the reaction was complete, the reaction was quenched by adding satd aq. NaHCO 3 solution (10 mL). The reaction mixture was diluted with CH 2 Cl 2 (50 mL), warmed to room temperature and washed with satd aq. NaHCO 3 solution (2×50 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×50 mL), after which the combined organic layers were washed with brine (50 mL), dried (Na 2 SO 4 ) and concentrated.

The crude product was purified by silica gel column chromatography (PE:EtOAc, 2:1) to yield compound 3oβ as slightly yellow amorphous solid (73 mg, 0.12 mmol, 68%).

Entry 40, Table 2-5

A solution of donor 1s (89 mg, 0.16 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (5 mL) was cooled down to -60°C and BSP (39.2 mg, 0.19 mmol, 1.2 eq.), TTBP (58.1 mg, 0.24 mmol, 1.5 eq.) and Tf 2 O (39 μL, 0.21 mmol, 1.3 eq.) were added. The mixture was stirred at -60°C for 15 min after which 1-octene (56 μL, 0.32 mmol, 2.0 eq.) was added and the reaction mixture was stirred for another 15 min after which the activation was complete (confirmed by TLC). The reaction mixture was cooled down to -78°C after which methyl 6-(hydroxymethyl)picolinate (2a) (30 mg, 0.18 mmol, 1.15 eq.) in freshly distilled CH 2 Cl 2 (2 mL) was added. The reaction mixture was stirred at -78°C for 3 h and then quenched by adding P(OEt) 3 (0.2 mL, 1.2 mmol, 7.5 eq.). The reaction mixture was diluted with CH 2 Cl 2 (25 mL) and washed with satd aq. NaHCO 3 solution (25 mL). The water layer was extracted with CH 2 Cl 2 (2×25 mL) and the combined organic layers were washed with brine (25 mL), dried (Na 2 SO 4 ) and concentrated. The crude product was purified by silica gel column chromatography (hexane:EtOAc, 1:1) which yielded both anomers 3o as slightly yellow amorphous solid in a α:β -1:4.4 ratio (75 mg, 0.13 mmol, 80%). 6, 138.0, 137.6, 134.6, 134.5, 130.7, 129.6, 129.4, 129.0, 128.8, 128.4, 128.3, 128.2, 128.1, 127.8, 127.6, 127.5, 126.5, 126.2, 126.2 (arom. C) 4, 138.3, 137.6, 128.9, 128.7, 127.7, 127.6, 126.1, 124.7 3.74 (m, 2H), 3.70 -3.60 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 165. 9, 165.6, 165.53, 165.45, 165.41, 165.3 (s, 2C), 164.9, 158.2, 146.9, 137.6, 133.6 -133.3 (m, 7C), 130.0 -128.3 (m, 32C), 124.7, 124.0, 101.0 (C 1 or C 1' ), 100.9 (C 1 or C 1' ), 76.0, 73.2, 72.8, 72.0, 71.8, 71.8, Then, following the general protocol A, compound 6-Glc was prepared from compound 4-Glc (60 mg, 0.124 mmol, 1.0 eq.) and compound 5 (66 mg, 0.186 mmol, 1.5 eq.). The crude product was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 6-Glc as white foam (60 mg, 0.076 mmol, 61%

6-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxymethyl)-N-(2',3'-di-O-acetyl-uridin-5' -yl)picolinamide (6-Gal)

A mixture of methyl 6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxymethyl)picolinate (3a-Gal) (305 mg, 0.61 mmol, 1.0 eq.) and NaI (562 mg, 3.75 mmol, 6.1 eq.) in dry pyridine (9.5 mL) was stirred at 120°C under an argon atmosphere. After 5 h, the reaction was finished.

Pyridine was evaporated and the residue was co-evaporated with toluene (3×15 mL). The residue was solubilized with CHCl 3 (10 mL), filtered through celite, washed with CHCl 3 , dried over Na 2 SO 4 . Then, the solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc = 1:1 to 100% EtOH) to afford compound 4-Gal (225 mg, 0.466 mmol, 76%).

Then, following the general protocol A, compound 6-Gal was prepared from compound 4-Gal (225 mg, 0.466 mmol, 1.0 eq.) and compound 5 (247 mg, 0.699 mmol, 1.5 eq.). The crude product was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 6-Gal as white foam (116 mg, 0.147 mmol, 31%

6-(β-D-Glucopyranosyloxymethyl)-N-(uridin-5'-yl)picolinamide (7-Glc)

Prepared according to general protocol E from compound 6-Glc (100 mg, 0.13 mmol, 1.0 eq.).

The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 30% MeOH/H 2 O) to afford compound 7-Glc as white foam (44 mg, 0.081 mmol, 63%

6-(β-D-Galactopyranosyloxymethyl)-N-(uridin-5'-yl)picolinamide (7-Gal)

Prepared according to general protocol E from compound 6-Gal (116 mg, 0.146 mmol, 1.0 eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 30% MeOH/H 2 O) to afford compound 7-Gal as white foam (79 mg, 0.146 mmol, quant.).

[α] D 20 = +24.0 (c = 0.

6-(Hydroxymethyl)-N-(2',3'-di-O-acetyl-uridin-5'-yl)picolinamide (9)

6-(Hydroxymethyl)picolinic acid ( 8) (606 mg, 3.96 mmol, 2.0 eq.) and HOBt (936 mg, 6.93 mmol, 3.5 eq.) were co-evaporated with toluene (3×15 mL) and THF (3×6 mL). The mixture was dried with vacuum for another 1 h. Then, the mixture was dissolved in dry THF (10 mL) under argon and cooled to 0°C. DIC (1.08 mL, 6.93 mmol, 3.5 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed and the reaction was stirred at r.t. for 30 min. At the same time, 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine ( 5) (699 mg, 1.98 mmol, 1.0 eq.) was dissolved in dry THF (10 mL) under argon and cooled to 0°C. PMe 3 (3.96 mL, 1 M in THF, 3.96 mmol, 2.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C. After 30 min, the solution was transferred into the flask containing the acid. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed, the reaction was stirred at r.t. for 16 h. Then, the reaction mixture was diluted with water (100 mL), extracted with EtOAc (9×100 mL). The combined organic layers were washed with satd Na 2 CO 3 (3×100 mL) solution, H 2 O (100 mL) and brine (100 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried with vacuum. The residue was purified by silica gel column chromatography (EtOAc to EtOAc:MeOH = 95:5) to afford compound 9 as white foam (156 mg, 0.338 mmol, 17%

6-Hydroxylmethyl-N-(uridin-5'-yl)picolinamide (10)

Prepared according to general protocol E from compound 9 (33 mg, 0.07 mmol, 1.0 eq.

Pd(PPh 3 ) 4 was added into a solution of (6-methoxycarbonylpyrid-2-yl)methyl 3,4,6-tri-O-acetyl-2-allyloxycarbonylamino-2-deoxy-β-D-glucopyranoside (3h) (23 mg, 0.043 mmol, 1.0 eq.) and dimethyl malonate (0.05 mL, 0.43 mmol, 10.0 eq.) in dried oxygen-free THF (2 mL) under argon. The reaction mixture was stirred under argon for 23 h. Pyridine (2 mL) and acetic anhydrate (1 mL) were added into the flask and the reaction was stirred at r.t. overnight. Then, the reaction mixture was concentrated, co-evaporated with toluene (2×5 mL) and ethanol (2×5 mL

O-acetyl-α-D-galactopyranosyloxymethyl)picolinate ( 16) (Adapted from Ref. [214][215]234 ) Methyl 6-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyloxymethyl)picolinate (3c) (50 mg, 0.072 mmol, 1.0 eq.) was dissolved in EtOAc (2 mL) and then a solution of NaBrO 3 (130 mg, 0.864 mmol, 12.0 eq.) in H 2 O (2 mL) was added to the well stirred two phases system. An aqueous solution of Na 2 S 2 O 4 (85% pure, 150 mg, 0.864 mmol, 12.0 eq.) in H 2 O (3 mL) was added dropwise over 10 min at 25 o C. After addition, the color of the reaction mixture changed from colorless to orange. Then, the reaction was stirred strongly at 25 o C for 3 h. Na 2 S 2 O 3 •5H 2 O (214 mg, 0.864 mmol, 12.0 eq.) was added into the reaction and stirred for 10 min; the color was disappeared. Then, the reaction mixture was concentrated. Pyridine (3 mL) and Ac 2 O (1 mL) were added and the reaction was stirred at r.t. overnight. The reaction mixture was filtrated and the solid was washed with EtOAc. The filtrate was concentrated and co-evaporated with toluene (2×5 mL) and ethanol (2×5 mL). Then, the residue was purified by silica gel column chromatography (PE:EtOAc = 9:1 to PE:EtOAc = 1:1) to afford compound 16 as white foam (15 mg, 0.03 mmol, 42% A mixture of methyl 6-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyloxymethyl)picolinate ( 16) (103 mg, 0.21 mmol, 1.0 eq.) and NaI (191 mg, 1.27 mmol, 6.1 eq.) in dry pyridine (3 mL) was stirred at 120°C under an argon atmosphere. After 5 h, the reaction was finished. Pyridine was evaporated and the residue was co-evaporated with toluene (3×10 mL). The residue was solubilized with CHCl 3 (10 mL), filtered through celite, washed with CHCl 3 , dried over Na 2 SO 4 . Then, the solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc = 1:1 to EtOH) to afford compound 17 as yellow foam (99 mg, 0.21 mmol, quant.).

O-acetyl-α-D-galactopyranosyloxymethyl)picolinic acid ( 17) (123 mg, 0.25 mmol, 1.0 eq.) and HOBt (101 mg, 0.75 mmol, 3.0 eq.) were co-evaporated with toluene (3×10 mL) and THF (3×5 mL). The mixture was dried with vacuum for another 1 h. Then, the mixture was dissolved in dry THF (4 mL) under argon and cooled to 0°C. DIC (0.12 mL, 0.75 mmol, 3.0 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed and the reaction was stirred at r.t. for 30 min. At the same time, 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine 5 (176 mg, 0.50 mmol, 2.0 eq.) was dissolved in dry THF (4 mL) under argon and cooled to 0°C. PMe 3 (0.75 mL, 1 M in THF, 0.75 mmol, 3.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C. After 30 min, the solution was transferred into the flask containing the acid. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed. The reaction was stirred at r.t. for 16 h. Then, the reaction mixture was diluted with water (60 mL), extracted with EtOAc (4×60 mL). The combined organic layers were washed with satd Na 2 CO 3 (60 mL), H 2 O (60 mL) and brine (60 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried with vacuum. The residue was purified by silica gel column chromatography (100% PE to PE:EtOAc = 20:80) to afford compound 18 as white foam (54 mg, 0.068 mmol, 27%

6-(α-D-Galactopyranosyloxymethyl)-N-(uridin-5'-yl)picolinamide (19)

Prepared according to general protocol E from compound 18 (30 mg, 0.038 mmol, 1.0 eq.

3-β-D-Galactosyloxymethylbenzoic acid (20)

LiOH (134.4 mg, 3.2 mmol, 4.5 eq.) was solubilized in H 2 O (2 mL), then, added dropwise into a solution of methyl 3-(2,3,4,6-tetra-O-acetyl-β-D-galactosyloxymethyl)benzoate (3e) (349 mg, 0.70 mmol, 1.0 eq.) in THF (4 mL). Then, the reaction mixture was stirred at r.t. for 2 days. DOWEX 50W×2 Resin (Fluka, 50-100 mesh, H + Form) was added to neutralize the reaction until pH~3. The reaction mixture was filtrated and concentrated to afford compound 20 as white foam (205 mg, 0.653 mmol, 93% 3-β-D-Galactosyloxymethylbenzoic acid ( 20) (33 mg, 0.105 mmol, 1.0 eq.) and HOBt (43 mg, 0.315 mmol, 3.0 eq.) were co-evaporated with toluene (3×5 mL) and THF (3×5 mL). The mixture was dried with vacuum for another 1 h. Then, the mixture was dissolved in dry THF (2 mL) under argon and cooled to 0°C. DIC (0.05 mL, 0.320 mmol, 3.0 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed; the reaction was stirred at r.t. for 30 min. At the same time, 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine ( 5) (74 mg, 0.21 mmol, 2.0 eq.) was dissolved in dry THF (2 mL) under argon and cooled to 0°C. PMe 3 (0.3 mL, 1 M in THF, 0.3 mmol, 3.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C. After 30 min, the solution was transferred into the flask containing the acid. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed and the reaction was stirred at r.

3-(β-D-Galactopyranosyloxymethyl)-N-(uridin-5'-yl)benzamide (22)

Prepared according to general protocol E from compound 21 (27 mg, 0.043

6-Bromo-N-(2',3'-di-O-acetyl-uridin-5'-yl)picolinamide (24)

6-Bromopicolinic acid ( 23) (49 mg, 0.24 mmol, 1.2 eq.) and HOBt (48 mg, 0.36 mmol, 1.8 eq.) were co-evaporated with toluene (3×5 mL) and THF (3×8 mL). The mixture was dried with vacuum for another 20 min. Then, the mixture was dissolved in dry THF (2 mL) under argon and cooled to 0°C. DIC (56 μL, 0.36 mmol, 1.8 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed; the reaction was stirred at r.t. for 30 min. At the same time, 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine (5) (70 mg, 0.20 mmol, 1.0 eq.) was dissolved in dry THF (2 mL) under argon and cooled to 0°C. PMe 3 (0.36 mL, 1 M in THF, 0.36 mmol, 1.8 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C for 30 min. Then, the solution was transferred into the flask containing the acid. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed and the reaction was stirred at r.t. for 21 h. The reaction mixture was diluted with water (30 mL), extracted with EtOAc (3×40 mL), the combined organic layers were washed with satd Na 2 CO 3 solution (3×30 mL), H 2 O (30 mL) and brine (30 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried with vacuum.

The residue was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 24 as yellow foam (37 mg, 0.073 mmol, 37%

6-Bromo-N-(uridin-5'-yl)picolinamide (25)

In a flask containing 6-bromo-N-(2,3-di-O-acetyl-5-deoxy-uridin-5-yl)picolinamide ( 24) (64 mg, 0.125 mmol, 1.0 eq.) were added MeOH (2.5 mL), H 2 O (1. 25 mL) and Et 3 N (1.25 mL) and the reaction mixture was stirred at r.t. After 5 days, the reaction was finished. Then, the reaction mixture was concentrated and co-evaporated with toluene (3×5 mL). The residue was purified by silica gel column chromatography (100% EtOAc to 30% MeOH/EtOAc) to afford compound 25 as white foam (15 mg, 0.035 mmol, 28%

6-Ethynyl-N-(2',3'-di-O-acetyl-uridin-5'-yl)picolinamide (27)

Compound 26 was prepared according to general protocol D from compound 24 (436 mg, 0.85 mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (PE to PE:EtOAc = 1:1) to afford a mixture (496 mg) containing compound 26 and the mixture was used directly for the reaction of next step.

Then, following the general protocol F, compound 27 was prepared from compound 26 (a mixture, 496 mg). The crude product was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 27 as yellow foam (193 mg, 0.42 mmol, two steps 50%). R f = 0.17 (PE: EtOAc, 1:2); [α] D = +34.9 (c = 1. To a solution of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (4.59 g, 11.2 mmol, 1.0 eq.), TBAHS (3.80 g, 11.2 mmol, 1.0 eq.) and NaN 3 (2.91 g, 51.4 mmol, 4.6 eq.) in CH 2 Cl 2 (46 mL) was added satd aq. NaHCO 3 (46 mL). The two phase mixture was vigorously stirred at r.t. overnight. Then, EtOAc (300 mL) was added into the reaction. The organic phase was separated and then successively washed with satd aq. NaHCO 3 (100 mL), H 2 O (2×100 mL) and brine (100 mL). The combined organic phase was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure to afford compound 28-Gal as white foam (4.166 g, 11.2 mmol, quant.). R f = 0.4 (PE:EtOAc = 2:1);

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl chloride 236

2-Acetamido-2-deoxy-D-glucopyranose (5.00 g, 22.6 mmol, 1.0 eq.) was added to stirred acetyl chloride (10 mL) and the resulting suspension was stirred magnetically at r.t. overnight. Dichloromethane (40 mL) was added into the reaction and the resulting solution was poured into a mixture of ice (40 g) and water (10 mL) with stirring. The organic layer was immediately separated and run into a mixture of saturated aq. NaHCO 3 (40 mL) and ice with stirring, the neutralization being completed in a separating funnel. The organic layer was then separated and dried over MgSO 4 for 10 min. The organic layer was then filtered with suction and the residue was washed with CH 2 Cl 2 (15 mL). The resulting deep yellow solution was concentrated to about 10 mL under vacuum. Anhydrous Et 2 O (50 mL) was added and the product crystallized 5 min later. The pale yellow solid was then filtered, washed with anhydrous ether (2×15 mL) and allowed to dry with vacuum for 5 min. A mixture of α and β anomers (α:β = 2:1) was afforded (4.627 g, 12.67 mmol, 56%). R f = 0.71, 0.54 (EtOAc, two spots for α and β anomers, respectively); 1 H NMR (300 MHz, CDCl 3 ) δ = 6.23 -6.13 (m, 1H), 5.79 (d, J = 8.6 Hz, 0.65H, NH α anomer ), 5.55 (d, J = 9.1 Hz, 0.35H, NH β anomer ), 5. 37 -5.16 (m, 2H), 4.52 (m, 1H), 4.33 -4.20 (m, 2H), 4.10 -3.93 (m, 1H), 2.21 -1.91 (m, 12H). 2 mmol, 1.0 eq.) and NaN 3 (1.304 g, 20 mmol, 1.8 eq) in DMF (20 mL) was heated at 80°C for 16 h. Then, the heating was stopped; ice was added into the reaction and the reaction mixture was extracted with EtOAc (3×90 mL). The organic layer was washed with satd NaHCO 3 solution (80 mL), H 2 O (2×80 mL) and brine (80 mL). The organic phase was dried over NaSO 4 , filtered and evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc 7, 170.2, 170.1, 169.8, 169.7, 169.4, 164.7, 162.7, 150.2, 149.3, 148.5, 148.3, 140.8 (C 6Uri ), (28-GlcNAc) (53 mg, 0.14 mmol, 1.4 eq.) 1, 163.4, 150.4, 149.4, 148.6, 147.7, 141.5 (C 6Uri Prepared according to general protocol E from compound 29-Glc (30 mg, 0.036 mmol, 1.0 eq.).

2-Acetamido

The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 50% MeOH/H 2 O) to afford compound 30-Glc as white foam (16 mg, 0.028 mmol, 78% (C 1Glc ), 82.1, 79.4, 76.3, 73.6, 72.8, 70.9, 69.4, 60.9 (C 6Glc ), 40.3 (C 5Rib 5, 168.4, 166.6, 153.3, 148.5, 147.7, 146.9, 141.9 (C 6Uri 82.2, 79.4, 73.8, 73.5 (C 2Rib ), 71.0, 69.7, 61. 6-Bromopicolinic acid ( 23) (812 mg, 4.02 mmol, 3.0 eq.) and HOBt (633 mg, 4.69 mmol, 3.5 eq.) were co-evaporated with toluene (3×15 mL) and THF (3×6 mL). The mixture was dried with vacuum for another 1 h. Then, the mixture was dissolved in dry THF (8 mL) under argon and cooled to 0°C. DIC (720 μL, 4.69 mmol, 3.5 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed; the reaction was stirred at r.t. for 30 min. At the same time, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoseyl azide (28-Glc) (500 mg, 1.34 mmol, 1.0 eq.) was dissolved in dry THF (8 mL) under argon and cooled to 0°C. PMe 3 (2.7 mL, 1 M in THF, 2.68 mmol, 2.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C for 30 min.

When there was no bubbling (~30 min), the solution was transferred into the flask containing the acid. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed and the reaction was stirred at r. 8, 170.3, 170.2, 169.6, 163.6, 149.6, 141.0, 139.7 6-Bromopicolinic acid ( 23) (648 mg, 3.21 mmol, 3.0 eq.) and HOBt (506 mg, 3.70 mmol, 3.5 eq.) were co-evaporated with toluene (3×15 mL) and THF (3×8 mL). The mixture was dried with vacuum for another 1 h. Then, the mixture was dissolved in dry THF (8 mL) under argon and cooled to 0°C. DIC (576 μL, 3.70 mmol, 3.5 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed; the reaction was stirred at r.t. for 30 min. At the same time, 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (28-Gal) (400 mg, 1.07 mmol, 1.0 eq.) was dissolved in dry THF (8 mL) under argon and cooled to 0°C. PMe 3 (2.1 mL, 1 M in THF, 2.14 mmol, 2.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C for 30 min.

When there was no bubbling (~30 min), the solution was transferred into the flask containing the acid. The reaction was stirred with an ice-bath for 1 h, then, the ice-bath was removed and the reaction was stirred at r.t. for 16 h. The reaction mixture was diluted with water (100 mL), extracted with EtOAc (3×150 mL), the combined organic layers were washed with satd Na 2 CO 3 solution (3×100 mL), H 2 O (100 mL) and brine (100 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried with vacuum. The residue was purified by silica gel column chromatography (PE to PE:EtOAc = 1:1) to afford compound 31-Gal as yellow foam (579 mg, contaminated with 1,3-Diisopropylurea, according to Synlett. 2010, 8, 1276-1280): 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) = 1.14 (d, J = 6.4 Hz, 12H), 3.87 -3.80 (m, 2H); 13 C NMR (75 MHz, CDCl 3 ) δ (ppm) =23. 5, 42.2, 157.0). R f = 0.45 (PE:EtOAc = 1:1); 6-Bromopicolinic acid ( 23) (812 mg, 4.02 mmol, 3.0 eq.) and HOBt (633 mg, 4.69 mmol, 3.5 eq.) were co-evaporated with toluene (3×15 mL) and THF (3×6 mL). The mixture was dried under vacuum for another 1 h. Then, the mixture was dissolved in dry THF (8 mL) under argon and cooled to 0°C. DIC (720 μL, 4.69 mmol, 3.5 eq.) was added dropwise at 0°C. After addition, the ice-bath was removed and the reaction was stirred at r.t. for 30 min. At the same time, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside azide (28-GlcNAc) (500 mg, 1.34 mmol, 1.0 eq.) was dissolved in dry THF (8 mL) under argon and cooled to 0°C. PMe 3 (2.7 mL, 1 M in THF, 2.14 mmol, 2.0 eq.) was added into the flask at 0°C and the reaction was stirred at 0°C for 30 min. When there was no bubbling (~30 min), the solution was transferred into the flask containing the acid. Then, the color of the reaction mixture changed from transparent to orange. The reaction mixture was stirred with an ice-bath for 1 h, then, the ice-bath was removed and the reaction was stirred at r.t. for 16 h. The reaction mixture was diluted with water (150 mL), extracted with EtOAc (3×200 mL), the combined organic layers were washed with satd Na 2 CO 3 solution (3×100 mL), H 2 O (100 mL) and brine (100 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried under vacuum. The residue was purified by silica gel column chromatography (PE to PE:EtOAc = 3:7) to afford compound 31-GlcNAc as yellow foam [678 mg, 95%, with 0.17 eq. contaminant 1,3-diisopropylurea, according to Synlett. 2010, 8, 1276-1280, data of 1,3-diisopropylurea: 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) = 1.14 (d, J = 6.4 Hz, 12H), 3.87 -3.80 (m, 2H); 13 C NMR (75 MHz, CDCl 3 ) δ

(ppm) = 23.5, 42.2, 157.0 ). R f = 0.19 (PE:EtOAc = 1:2)]; 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.71 (d, J = 9.3 Hz, 1H, NHCO), 8.11 (dd, J = 7.5, 0.7 Hz, 1H, H Pyr ), 7.74 -7.58 (m, 2H, 2 H Pyr ), 6.16 (d, J = 9.1 Hz, 1H, NHAc), 5.36 (dd, J = 9.7, 9.3 Hz, 1H, H 1 ), 5.22 -5.13 (m, 2H, H ),78.0 (C 1 ),74.0 (C 5 ),73.4 (C 3 ),68.0 (C 4 ),61.9 (C 6 ),53.3 (C 2 ),23.1 (COCH 3 ),20 5.20 -5.11 (m, 2H, H 2Glc , H 4Glc ), 4.30 (dd, J = 12.5, 4.3 Hz, 1H, H 6aGlc ), 4.08 (dd, J = 12.5, 1.9 Hz, 1H, H 6bGlc ), 3.87 (ddd, J = 10.1, 4.3, 1.9 Hz, 1H, H 5Glc ), 2.07 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.01 (s, 3H, COCH 3 ), 1.96 (s, 3H, COCH 3 ), 0.30 (s, 9H, SiMe 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170. 8, 170.2, 170.1, 169.6, 164.4, 148.8, 142.1, 137.6 Prepared according to general protocol D from compound 31-Gal (543 mg, 1.0 mmol, 1.0 eq.).

The crude product was purified by silica gel column chromatography (100% PE to 30% EtOAc/PE) to afford compound 32-Gal as yellow foam (512 mg, 0.93 mmol, 93%). R f = 0.43 (PE:EtOAc = 1:1); 1 H NMR (400 MHz, CDCl 3 ) δ = 8.59 (d, J = 9.8 Hz, 1H, NHCO), 8.10 (dd, J = 7.8, 0.9 Hz, 1H, H pyr ), 7.79 (t, J = 7.8 Hz, 1H, H pyr ), 7.59 (dd, J = 7.8, 0.9 Hz, 1H, H pyr ), 5.49 -5.42 (m, 2H, H 1 , H 4 ), 5.35 (t, J = 9.7 Hz, 1H, H 2 ), 5.17 (dd, J = 9.7, 3.4 Hz, 1H, H 3 ) Prepared according to general protocol F from compound 32-Gal (496 mg, 0.55 mmol, 1.0 eq.).

The crude product was purified by silica gel column chromatography (100% PE to 35% EtOAc/PE) to afford compound 33-Gal as yellow foam (207 mg, 0.435 mmol, 79% Prepared according to general protocol B from compound 33-Glc (62 mg, 0.13 mmol, 1.0 eq.) and compound 5 (46 mg, 0.13 Prepared according to general protocol B from compound 33-Gal (88 mg, 0.185 mmol, 1.0 eq.) and compound 5 (65 mg, 0.185 mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (100% PE to 50% EtOAc/PE) to afford compound 34-Gal as white foam (100 mg, 0.121 mmol, 64%). R f = 0.17 Prepared according to general protocol E from compound 34-Glc (96 mg, 0.12 mmol, 1.0 eq.).

The 1, 168.5, 166.9, 153.4, 147.9, 147.6, 146.8, 141.8 (C 6Uri In a 50 mL flask containing 2,6-dibromopyridine (36) (1 g, 4.2 mmol, 3.0 eq.), CuI (14 mg, 0.07 mmol, 0.05 eq.) and Pd(PPh 3 ) 4 (81 mg, 0.07 mmol, 0.05 eq.) in toluene (5.6 mL) was bubbled with argon. Then, diisopropylamine (0.56 mL, 4.0 mmol, 2.9 eq.) and trimethylsilyacetylene (0.2 mL, 1.4 mmol, 1.0 eq.) were injected into the reaction. In a 10 mL round-bottom flask containing 2-bromo-6-(trimethylsilyl)ethynylpyridine ( 37) (750 mg, a mixture as mentioned above) was added K 2 CO 3 (1.426 mg, 10.6 mmol), THF (5 mL) and MeOH (5 mL). The resulting suspension was stirred at r.t. for 2 h. Then, the reaction mixture was poured into H 2 O (50 mL), extracted with EtOAc (4×50 mL). The combined organic layers were washed with brine (50 mL). The organic phase was dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (PE:CH 2 Cl 2 = 9:1 to PE:CH 2 Cl 2 = combined organic phases were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 42-Gal as yellow foam (37 mg, 0.0434 mmol, 31%). (Pathway B) DIPEA (6 μL, 0.036 mmol, 0.25 eq.) was added into a flask containing 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine (5) (50 mg, 0.14 mmol, 1.0 eq.), 2-ethynyl-6-(2-trimethylsilylethynyl)pyridine ( 48) (28 mg, 0.14 mmol, 1.0 eq.) and CuI (2.7 mg, 0.01 mmol, 0.1 eq.) in DMF (2 mL). The reaction was stirred at r.t. with protection of light for 24 h. CuI (5.4 mg, 0.02 mmol, 0.2 eq.), 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside azido (28-Gal) (64 mg, 0.17 mmol, 1.2 eq.) and DIPEA (18 μL, 0.108 mmol, 0.75 eq.) were added into the reaction and the reaction mixture was stirred at 35°C under argon with protection of light for 23 h. The reaction mixture was then diluted with EtOAc (30 mL), washed with satd aq. Na 2 CO 3 solution (2×25 mL) and H 2 O (30 mL). The combined aqueous phase was extracted with EtOAc (3×30 mL). The combined organic phases were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 42-Gal as yellow foam (49 mg, 0.0574 mmol, 41%). (Pathway C) DIPEA (6 μL, 0.036 mmol, 0.25 eq.) was added into a flask containing 2',3'-di-O-acetyl-5'-azido-5'-deoxy-uridine (5) (50 mg, 0.14 mmol, 1.0 eq.), 2-ethynyl-6-(2-trimethylsilylethynyl)pyridine ( 48) (28 mg, 0.14 mmol, 1.0 eq.) and CuI (2.7 mg, 0.01 mmol, 0.1 eq.) in DMF (2 mL). The reaction was stirred at r.t. with protection of light for 17 h. AgBF 4 (55 mg, 0.28 mmol, 2.0 eq.) and 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside azido (28-Gal) (64 mg, 0.17 mmol, 1.2 eq.) were added into the reaction and the reaction mixture was stirred at 35°C under argon with protection of light for 16 h. Then, the reaction mixture was diluted with EtOAc (30 mL), washed with satd aq. Na 2 CO 3 solution (2×25 mL) and H 2 O (30 mL). The combined aqueous phase was extracted with EtOAc (3×30 mL). The combined organic phases were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 42-Gal as yellow foam (72 mg, 0.0844 mmol, 60%

(50 mg, 0.142 mmol, 1.0 eq.), 2-bromo-6-ethynylpyridine (38) (31 mg, 0.17 mmol, 1.2 eq.) and CuI (14 mg, 0.017 mmol, 0.5 eq.) in DMF (2 mL). The reaction was heated for 15 min by microwave irradiation at 100 o C. Then, the reaction suspension was diluted with ethyl acetate (25 mL), washed with satd aq. Na 2 CO 3 solution (2×25 mL) and H 2 O (30 mL). The organic phase was dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc = 2:3 to PE:EtOAc = 1:2) to afford compound 44 as white foam (72 mg, 0.135 mmol, 95%). R f = 0.16 (PE:EtOAc = 2:3); [α] , 5.44 (dd appears as t, J = 6.5 Hz, 1H, H 3Rib ), 5.34 (dd, J = 6.5, 4.0 Hz, 1H, H 2Rib ), 4.85 (dd, J = 14.5, 3.4 Hz, H 5aRib ), 4.78 (dd, J = 14.5, 6.5 Hz, 1H, H 5bRib ), 4.50 (ddd appears as td, J = 6.5, 3.4 Hz, 1H, H 4Rib ), 2.12 (s, 3H, COCH 3 ), 2.10 (s, 3H, COCH 3 ); 13C NMR (100 MHz, CDCl 3 ) δ = 169.9 (COCH 3 ), 169.8 (COCH 3 ), 162.7, 151.1, 150.1, 147.4, 141.9, 141.6

2-Bromo-6-[1-(uridin-5'-yl)-1H-1,2,3-triazol-4-yl]pyridine (45)

Prepared according to general protocol E from compound 44 (10 mg, 0.019 mmol, 1.0 eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 30% MeOH/H 2 O) to afford compound 45 as white foam (7 mg, 0.0156 mmol, 82%). R f = 0.18 (EtOAc); [α] Prepared according to general protocol D from compound 44 (30 mg, 0.056 mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (60% EtOAc/PE to 70% EtOAc/PE) to afford compound 46 as yellow foam (22 mg, 0.04 mmol, 71%

2-Ethynyl-6-[1-(2',3'-di-O-acetyl-uridin-5'-yl)-1H-1,2,3-triazol-4-yl]pyridine (47)

Prepared according to general protocol F from compound 46 (20 mg, 0.036 mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (100% PE to 100% EtOAc) to afford compound 47 as yellow foam (9 mg, 0.0188 mmol, 52%). R f = 0.25 (PE:EtOAc, 1:3); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 169. 83, 169.76, 150.6, 150.0, 148.1, 142.1, 141.5 (C 6Uri

2-Ethynyl-6-(2-(trimethylsilyl)ethynyl)pyridine (48) 218

In a 100 mL round-bottom flask containing 2,6-dibromopyridine (36) (5 g, 21.1 mmol, 1.0 eq.), Pd(PPh 3 ) 4 (1.22 g, 1 mmol, 0.05 eq.) and CuI (190 mg, 1 mmol, 0.05 eq.) in toluene (100 mL) was degassed with argon. Then, trimethylsilylacetylene (9 mL, 63.3 mmol, 3.0 eq.) and diisopropylamine (8.4 mL, 59.9 mmol, 2.8 eq.) were injected. The reaction was stirred at r.t. with protection of light for 42 h. Then, another portion of Pd(PPh 3 ) 4 (294 mg, 0.24 mmol, 0.01 eq.), CuI (50 mg, 0.26 mmol, 0.01 eq.), trimethylsilylacetylene (0.36 mL, 2.53 mmol, 0.12 eq.) and diisopropylamine (0.4 mL, 2.85 mmol, 0.14 eq.) were added into the reaction. After 48 h, the reaction mixture was filtered and the filtrate was poured into satd aq. NH 4 Cl solution (250 mL), extracted with CH 2 Cl 2 (3×300 mL). The combined organic layers were washed with H 2 O (200 mL) and brine (200 mL). The organic phase was dried (MgSO 4 ) and the solvent was evaporated. The residue was then purified by silica gel column chromatography (5% CH 2 Cl 2 /PE to 30% CH 2 Cl 2 /PE) to afford 2,6-bis(2-trimethylsilylethynyl)pyridine (5.6 g, 20.68 mmol, 98%). R f = 0.73 (PE:EtOAc, 2:1); 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) = 7.62 -7.55 (m, 1H, H Pyr ), 7.40 -7.35 (m, 2H, 2 H Pyr ), 0.25 (s, 18H, SiMe 3 ). A solution of 2,6-bis(2-trimethylsilylethynyl)pyridine (2.189 g, 8.1 mmol, 1.0 eq.) in MeOH (90 mL) was degassed with argon then 1 M aq. KOH (20 μL) was added. The reaction mixture was stired at r.t. under argon. After 100 min, the reaction mixture was poured into pentane (300 mL), washed successively with H 2 O (3×100 mL) and brine (100 mL). The aqueous layer was Imidazole-1-sulfonyl azide hydrochloride ( 56) 224 Sulfuryl chloride (16.1 mL, 200 mmol, 1.0 eq.) was added dropwise into an ice-cooled suspension of NaN 3 (13.0 g, 200 mmol, 1.0 eq.) in MeCN (200 mL). After addition, the reaction was stirred at r.t. overnight. Imidazole (25.9 g, 380 mmol, 1.8 eq.) was added portion-wise into the ice-cooled mixture and the resulting slurry was stirred at r.t. for 3 h. Then, the reaction mixture was diluted with EtOAc (400 mL), washed with H 2 O (3×200 mL), satd aq. NaHCO 3 (3×200 mL), dried (MgSO 4 ) and filtered. A solution of HCl in EtOH [obtained by dropwise addition of AcCl (21.3 mL, 300 mmol) to ice-cooled dry ethanol (75 mL)] was added dropwise to the filtrate with stirring. The mixture was cooled to 0°C, filtered and the solid was washed with EtOAc (3×100 mL) to give the imidazole-1-sulfonyl azide hydrochloride salt ( 56 57) [224][225] Imidazole-1-sulfonyl azide hydrochloride ( 56) (250 mg, 1.2 mmol, 1.2 eq.) was added to a solution of L-serine methyl ester hydrochloride salt (156 mg, 1.0 mmol, 1eq.), K 2 CO 3 (346 mg, 2.5 mmol, 2.5 eq.) and CuSO 4 •5H 2 O (2.5 mg, 10 μmol, 0.01 eq.) in MeOH (5 mL). After addition, the reaction was stirred at r.t. for 5 h. Then, the reaction mixture was concentrated, diluted with H 2 O (10 mL), extracted with EtOAc (4×10 mL), dried over Na 2 SO 4 . The organic phase was concentrated and the residue was purified by silica gel column chromatography (10% EtOAc/PE to 20% EtOAc/PE) to afford compound 57 (89 mg, 0.61 In a flask containing compound 50 (224 mg, 0.45 mmol, 1.0 eq.) and 10% Pd/C (49 mg) was added solvent MeOH (5 mL). The atmosphere was changed first to argon then hydrogen with vacuum. The reaction was stirred at r.t. for 4 h. Then, the reaction mixture was filtered through celite, washed with methanol. The combined methanol solution was dried (Na 2 SO 4 ),

Methyl (2S)-2-azido-3-hydroxypropanoate (

N-[2',3'-Di-O-(tert-butyldimethylsilyl)-uridin-5'-yl]-(2S)-2-azido-hydroxypropanamide

In a flask containing compound 50 (150 mg, 0.30 mmol, 1.0 eq.) and 10% Pd/C (34 mg) was added the solvent MeOH (5 mL). The air inside of the flask was changed with argon, then, filled with H 2 . The reaction was stirred at r.t. for 4h. Then, the reaction mixture was filtrated and washed with MeOH. The organic solution was concentrated and the residue was used directly without further purification. compound 58 (27 mg, 0.21 mmol, 1.5 eq.) and HOBt (40 mg, 0.25 mmol, 1.2 eq.) were co-evaporated with toluene (3×5 mL), then, freshly distilled THF (4 mL) was added. The solution was then transferred into a flask containing the amine 61 (150 mg, 0.30 mmol, 1.0 eq.), DCC (52 mg, 0.25 mmol, 1.2 eq.) and DMAP (5 mg, 0.06 mmol, 0.2 eq.). The resulting reaction mixture was stirred at r.

N-[2',3'-Di-O-(tert-butyldimethylsilyl)-uridin-5'-yl]-O-(2,3,4,6-tetra-O-acetyl-β-D-galacto pyranosyl)serinamide (65)

In a flask containing compound 62 (240 mg, 0.026 mmol, 1.0 eq.) and 10% Pd/C (29 mg) was added solvent MeOH (10 mL). The atmosphere in the flask was changed to argon, then, exchanged with hydrogen. The reaction was stirred at r.t. After 5 h, the reaction mixture was filtered through celite, washed with methanol. The combined methanol solution was concentrated to afford a crude product 65 as white foam. The crude product was used directly for the reaction of next step without further purification.

Methyl (2S

N-[2',3'-Di-O-(tert-butyldimethylsilyl)-uridin-5'-yl]-N'-[N''-(tert-butoxycarbonyl)glyciny l]-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)serinamide (69)

Freshly distilled CH 2 Cl 2 (4 mL) was added into a flask containing the amine 65 (57 mg, 0.064 mmol, 1.0 eq.), N-Boc-Gly-OSu (21 mg, 0.077 mmol, 1.2 eq.) and DCC (16 mg, 0.077 mmol, 1.2 eq.). The resulting reaction mixture was stirred at r.t. for 16 h. Then, the reaction mixture was concentrated and the residue was purified by silica gel column chromatography (66% EtOAc/PE) to afford the desired amide 69 as white foam (39 mg, 0.037 mmol, two steps 58%). R f = 0.22 (PE:EtOAc, 1:2); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.84 (s, 1H, NH Uri ), 7.61 (s, 1H, NHCO), 7.24 (d, J = 7.9 Hz, 1H, H 6Uri ), 6.64 (d, J = 7.8 Hz, 1H, NHCO CH2NHBoc ), 5.73 (dd, J = 7.9, 1.5 Hz, 1H, H 5Uri ), 5.50 (bs, 1H, NH Boc ), 5.37 (bd, J = 2.7 Hz, 1H, H 4Gal ), 5.20 (d, J = 6.5 Hz, 1H, H 1Rib ), 5.12 (dd, J = 10.5, 7.8 Hz, 1H, H 2Gal ), 5.00 (dd, J = 10.5, 2.7 Hz, 1H, H 3Gal ), 4.76 -4.68 (m, 1H, H αSer ), 4.56 -4.46 (m, 2H, H 1Gal , 0.89 (s, 9H, SiCMe 3 ), 0.84 (s, 9H, SiCMe 3 ), 0.08 (s, 3H, SiCH 3 ), 0.07 (s, 3H, SiCH 3 ), 0.05 (s, 3H, SiCH 3 ), -0.05 (s, 3H, SiCH 3 ); 13 C NMR (100 MHz, -5'-yl)-2-(4-phenyl-1,2,3-triazol-1-yl)-3-hydroxy-propanamide (81) Prepared according to general protocol G from compound 71 (17 mg, 0.017 mmol, 1.0 eq.). The crude product was purified by C18 reverse phase column chromatography (100% H 2 O to 100% MeOH) to afford compound 81 (8 mg, 0.017 mmol, quant.) as mixture of two diastereomers. 1

N-(Uridin

N-(Uridin-5'-yl)-(2S)-2-(4-phenyl-1,2,3-triazol-1-yl)-3-methoxy-propanamide (82)

Compound 71 (20 mg, 0.020 mmol, 1.0 eq.) was solubilized with CH 2 Cl 2 (0.5 mL). Then, 90% TFA (2.2 mL) was added dropwise. After addition, the reaction mixture was stirred at r.t. overnight. MeOH (3 mL) and H 2 O (5 mL) were then added into the reaction mixture. The solvent was removed by evaporation and the residue was used directly for the reaction of next step. Aq. Et 3 N (1 mL) was added dropwise into a solution of the residue solubilized in MeOH/H 2 O (2 mL/1 mL) at r.t. After addition, the reaction mixture was stirred at r.t. for 24 h. Then, the reaction mixture was concentrated and the residue was purified by C18 reverse phase column chromatography (100% H 2 O to 100% MeOH) to afford compound 82 (7 mg, 0.015 mmol, 75%) as mixture of two diastereomers. 1 H NMR (500 MHz, CD 3 OD) δ (ppm) = 8.46 (d, 1H, H triazole ), 7.87 -7.80 (m, 2H, Ph), 7.64 -7.59 (m, 1H, H 6Uri ), 7.47 -7.41 (m, 2H, Ph), 7.37 -7.32 (m, 1H, Ph), 5.76 -5.69 (m, 2H, H 5Uri , 3.42 -3.37 (s, 3H, OCH 3 ); 13 C NMR (125 MHz, CD 3 OD) δ (ppm) = 168. 67, 168.63 (2s, 2C), 166.12, 166.10 (2s, 2C,), 152.4, 148.8, 143.48, 143.43 BAIB (709 mg, 2.2 mmol, 2.2 eq.), TEMPO (32 mg, 0.2 mmol, 0.2 eq.) and compound 90 (328 mg, 1.0 mmol, 1.0 eq.) were added into a flask and a mixture of solvent CH 3 CN:H 2 O (1:1) was added at -5°C. After addition, the reaction was stirred at -5°C with protection of light. After 6 h, another fraction of BAIB (355 mg, 1.1 mmol, 1.1 eq.) and TEMPO (64 mg, 0.6 mmol, 0.6 eq.) were added into the reaction and the reaction was stirred at -5°C with protection of light for another 16 h. Then, the reaction mixture was concentrated and the resulting yellow gum was triturated with diethyl ether, filtrated and washed with Et 2 O (5×5 mL). The highly hygroscopic yellow solid was then purified by reverse phase colomn (10% MeOH/H 2 O to 100% MeOH) to afford the product 91 as white foam (277 mg, 0.81 mmol, 81%

N-[(2S)-1-Hydroxy-4-oxa-3-oxo-pent-2-yl]-1-uracyl-2,3-di-O-acetyl-β-D-ribofuranurona mide (92)

(Method I) Compound 91 (122 mg, 0.35 mmol, 1.0 eq.) and HOBt (67 mg, 0.43 mmol, 1.2 eq.) were co-evaporated with toluene (3×8 mL). Then, to a stirred solution of compound 91 (122 mg, 0.35 mmol, 1.0 eq.), HOBt (67 mg, 0.43 mmol, 1.2 eq.), L-serine methyl ester hydrogen chloride (83 mg, 0.54 mmol, 1.5 eq.) and DCC (88 mg, 0.43 mmol, 1.2 eq.) in freshly distilled MeCN (5 mL) was added triethylamine (44 mg, 0.073 mL, 0.54 mmol, 1.5 eq.) dropwise at -5°C. After addition, the reaction temperature was slowly increased to r.t. and the reaction was stirred at r.t. for 24 h. Then, the reaction mixture was filtrated, washed with CH 2 Cl 2 and concentrated. The resulting mixture was purified by silica gel column chromatography (80% EtOAc/PE to 100% EtOAc) to afford the desired amide 92 (57 mg, 0.129 mmol, 36%) and a methyl ester of 91 as a byproduct (30 mg, 0.084 mmol, 24%). (Method II) Compound 91 (85 mg, 0.25 mmol, 1.0 eq.), DCC (78 mg, 0.375 mmol, 1.5 eq.) and HOBt (59 mg, 0.375 mmol, 1.5 eq.) were co-evaporated with toluene (3×8 mL). Then, the mixture was solubilized with MeCN (3 mL). The solution was transformed to a stirred solution of L-serine methyl ester hydrogen chloride (98 mg, 0.625 mmol, 2.5 eq.) and triethylamine

V.3 Cloning, expression and purification of GTs

The mutant enzyme was cloned and expressed in E. coli using standard mutagenesis techniques [START_REF] Seto | Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes[END_REF][242][243] . All other GalT were expressed and purified as previously described [243][244][245] apart from β-1,4-GalT, which was obtained commercially (Sigma-Aldrich). Both GTB and AAGlyB were purified by ionexchange and affinity chromatography 246 and yielded ~10-15 mg pure protein per liter cell culture. At the end of purification, excess UDP was removed from the eluted protein solution by dialysis in buffer (50 mM MOPS pH 7, 0.1 M NaCl, 1 mM DTT, 5 mM MnCl 2 ) before concentrating the protein to 15-17 mg/mL. Protein stock containing 5 mM MnCl 2 ,50 mM MOPS pH 7, 100 mM NaCl and 1 mM DTT were stripped of metal using buffer with 50 mM EDTA and solvent exchanged into 50 mM MOPS pH7, 100 mM NaCl and 1 mM DTT by serie dilutions and concentrations using an Amicon Ultra centrifugal concentrator (Millipore, 10 kDa cutoff). Final concentration of the protein stock was approximately 12 mg/mL. The recombinant protein OGT was purified by HisTrap (Amersham) column with gradient imidazole elution. Pure protein was pooled, concentrated and subsequently digested with recombinant enterokinase to cleave the GSTs (glutathione S-transferase) tag which was subsequently removed using column chromatography on glutathione-agarose resin.

V.4 Enzymatic assays

GalT: Radiochemical assay: Radiochemical enzyme assays were performed in a final volume of 15 μL, containing the corresponding enzyme, radioactive labeled UDP- [START_REF] Jorgensen | A glycosyltransferase inhibitor from a molecular fragment library simultaneously interferes with metal ion and substrate binding[END_REF][START_REF] Pesnot | Structural and mechanistic basis for a new mode of glycosyltransferase inhibition[END_REF]247 The inhibition assays at 1 mM and the IC 50 values with β-1,4-GalT, GTB, α-1,4-GalT, α-1,3-GalT and AAGlyB were determined in analogous fashion using the requisite acceptor. The inhibition assays at 1 mM inhibitor and various GT were carried out at concentration of donor at K m value and concentration of acceptor at K m value or higher. The IC 50 values were determined by constructing a dose-response curve and examining the effect of 10 different concentrations of each inhibitor ranging from 0 μM to 3000 μM at concentration of donor at near 1/5 th K m value and concentration of acceptor at K m value or higher. The lowest calculated R 2 value from the fitted lines of each experiment was 0.95. 500 μM acceptor for α-1,3-GalT, 2 mM acceptor for α-1,4-GalT, 500 μM acceptor for β-1,4-GalT, 500 μM acceptor for GTB and 20 μM acceptor for AAGlyB were used. For all the assays, inhibitors were dissolved in MilliQ H 2 O, except compounds 24, 34-Gal, 44 and 49, for which a maximum of 3% final concentration of DMSO/H 2 O were use due to poor solubility with a corresponding 3% F.C. of DMSO/H 2 O no inhibitor assays as control.

OGT:

The ability of small molecule to inhibit OGT activity was assessed using radiolabelled [ 3 H]-UDP-GlcNAc (American Radiolabel) as the donor and recombinant nup62 as the acceptor. For IC 50 assays, 8 μM nup62, 1 μM UDP-GlcNAc (about 1/5th of K m value of donor, constant specific activity of 0.14 Ci/mmol of [ 3 H]-UDP-GlcNAc), 200 nM hOGT and various concentrations of small molecule inhibitors were used. For K i evaluation, 10 μM nup62, 1 μM UDP-GlcNAc (constant specific activity of 0.8 Ci/mmol of [ 3 H]-UDP-GlcNAc) or 50 μM UDP-GlcNAc (constant specific activity of 0.35 Ci/mmol of [ 3 H]-UDP-GlcNAc), 100 nM hOGT, and various concentrations of small molecule, 1000, 500, 250, 125, 62.5 and 31.25 μM, were used for the assays. Reactions were initiated by the addition of enzyme by pipette and incubated at 37 o C for 1 h (a time for which linear rates are obtained using these assay conditions). Reactions were placed on ice after 1 hr of incubation, then immediately applied to a 1.5 by 3 cm piece of nitrocellulose membrane (BioRad) and allowed to air dry. The quantity of protein loaded onto each piece of nitrocellulose was at least ten times less than the binding capacity of the membrane (as detailed in the manufacturer's protocol). The membranes were washed with four consecutive large volumes (100 mL) of PBS and air dried. The pieces of membrane were loaded into scintillation vials, 4 mL of scintillation fluid (Amersham) was added, and the levels of tritium were quantified using a liquid scintillation counter (Beckman LS6000). All assays were done in triplicate (2 minute for sample counting). The K i value is calculated from an equation generated based on the establishment of Dixon plot of each inhibitors and where it is intersect with the 1/Vmax.

V.5 Crystallization and structure refinement

The metal free protein was crystallized at 20 °C by the sitting drop method with drops containing 1.6 μL of protein stock and 1.6 μL of reservoir solution containing 13% or 15% PEG 3350, 50 mM or 150 mM ammonium sulfate and 50 mM MOPS pH7. Seeding was performed with a horse hair from previous similar drops, and the drops were allowed to equilibrate over 500 μL of reservoir solution. Crystals of the apo form of the protein grew to a final size of approx 200 μm in one week. Drop wells containing the best crystals were opened, 2.4 μL of 50 mM MOPS pH 7, 50 mM MnCl 2 and 100 mM inhibitor was added followed by 0.8 μL of 200 mM α-L-Fucp-(1→2)-β-D-Galp-O-(CH 2 ) 7 -CH 3 solution. The drops were mixed, resealed, allowed to equilibrate for 45 minutes, then, opened again and 3 μL of reservoir solution mixed 1:1 with glycerol added as cryoprotectant. After one minute the crystals were mounted in Mitegen loops and flash frozen in liquid nitrogen. Diffraction data for crystals with each of inhibitors 7-Gal, 7-Glc and 19 were collected in Beamline ID23-2 at ESRF at a wavelength of 0.873 Å with a crystal to detector of 167.5 mm with 1° oscillations, 180 images per crystals and exposure times of 0.1 to 0.5 seconds per image. Diffraction data was integrated and scaled with XDS 248 . The structures were refined using PDB_ID:3IOI 81 as the starting model followed by rigid body refinement in lieu of molecular replacement. Geometry descriptions for the inhibitors were made with Sketcher from the CCP4i suite 249 . Refinement was carried on with Refmac5 250 , model building with Coot 251 and figures were rendered with Pymol. A full atom anisotropic model was used for 19 and 7-Glc, while TLS + isotropic atoms were used for 7-Gal. Data collection and refinement statistics are listed in Table V-1. The atomic coordinates and structure factors have been deposited at the Protein Data Bank with the following accession codes: 4KC2, 4KC4 and 4KC1. Human mammary epithelial MCF10A cells were grown in the Mammary Epithelial cell Growth Medium (MEGM) composed of Mammary Epithelial Basal Medium (MEBM) supplemented with 13 mg/mL bovine pituitary extract (BPE), 10 μg/mL human epidermal growth factor (hEGF), 0.5 mg/mL hydrocortisone, and 5 mg/mL insulin (Lonza). Cells were grown at 37 °C in a humidified atmosphere enriched with 5% CO 2 .

To test the various compounds, cells were grown in a 24-wells plate in complete medium. When cells reached 70% confluence, medium was changed and the compound was added at the indicated concentrations (from 50 μM to 500 μM) for 24 h or 48 h. The OGT potent inhibitor Ac-5SGlcNAc (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glycopyranose) was used as a positive control (50 μM, 100 μM) (gift of Pr. David Vocadlo).

V.7.2 Cell lysis and Western blotting

After treatment, cells were lysed on ice for 10 min in 100 μL lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton-X100) containing the complete protease inhibitors cocktail (Roche Diagnostics, Meylan, France), 50 mM sodium fluoride (Sigma-Aldrich) and 100 μM orthovanadate (Sigma-Aldrich). Cell lysate was centrifuged at 20,000 ×g for 10 min and supernatant was collected. Protein concentration was measured using the microBCA protein assay kit (Pierce, Fisher Scientific, Illkirch, France). Proteins (20 μg) were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Hybond ™-C EXTRA, GE Healthcare, Orsay, France). Membranes were blocked in 5% (w/v) nonfat dry milk in Tris-Buffered Saline with 0.05% (v/v) Tween 20 (TBS-T) and probed with primary antibodies directed against O-GlcNAc (RL2, 1:4000) (Ozyme, Montigny le Bretonneux, France), and GAPDH (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. After 3 washes in TBS-T, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG-HRP linked, 1:30,000, GE Healthcare) for 1 h at room temperature. After several washes, blots were developed using enhanced chemiluminescence (ECL Prime Reagent, Hyperfilm TM MP; GE Healthcare, Velizy-Villacoublay, France).

V.8 Permeation assays

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) solution (stock solution, 25 mg/mL in chloroform, Avanti Polar Lipids) was dried in a round bottom flask, and dispersed with buffer (PBS buffer containing inhibitors (0.01 M) or PBS buffer). The suspension was vortexed for 5-10 min until an emulsion was formed. Then, the emulsion was submitted to 5 freeze/thaw cycles (5 min in liquid N 2 / 10 min in water bath at 37°C / 1 min vortex) to yield unilamellar structures, followed by extrusion to the desired size of ca. 200 nm. To check inhibitor penetration across lipid membrane, liposomes were incubated overnight at 37°C for 24 h in a shaking incubator. Separation of liposomes from free inhibitors was performed using gel filtration chromatography (G-25 column -GE Healthcare). Liposomes (200 nm in diameter) are eluted in the 4 mL void volume of the column. Small size molecules penetrated inside column gel, and were eluted later with more than 6 mL. After 30 mL of elution, UV absorption at 260 nm and UV spectra of 230 -400 nm regions were measured for each fraction using Tecan Infinite M200 reader.

Test on 7-Gal: Method I. Prepared liposomes (size 200 nm, final concentration of 5 mg/mL in PBS) were incubated with 7-Gal (final concentration of 1×10 -2 M in PBS) in a final volume of 1 mL at 37°C for 24 h in a shaking incubator. Then, liposome complex were separated from free 7-Gal by G-25 column. After 30 mL elution, UV absorption at 260 nm and UV spectra of the ultraviolet region (230 -400 nm) were measured for each fraction by Tecan Infinite M200 reader. Further incubation was performed on liposome-7-Gal complex fraction (tube 4). After separation, UV absorption at 260 nm was measured for each fraction. Method II. Liposomes were prepared (size 200 nm, final concentration of 5 mg/mL in PBS) with 7-Gal/PBS solution (final concentration of 1×10 -2 M in PBS) to a final volume of 1 mL. Then, liposome complex were separated from free 7-Gal by G-25 column immediately. UV absorption at 260 nm and UV spectra of the ultraviolet region (230 -400 nm) was measured for each fraction by Tecan Infinite M200 reader. The liposome complex fraction (tube 4) was incubated at 37°C in a shaking incubator. After 24 h, the mixture was encounter to another separation. UV absorption at 260 nm was measured for each fraction.

Test on 35-Glc: Method I. Prepared POPC liposomes (size 200 nm, final concentration of 5 mg/mL in PBS) were incubated with 35-Glc (final concentration of 1×10 -2 M in PBS) in a final volume of 500 μL at 37°C for 24 h in a shaking incubator. Then, liposome complex were separated from free 35-Glc by G-25 column. After 30 mL elution, UV absorption at 253 nm, 300 nm and UV spectra of 230 -450 nm regions were measured for each fraction by Tecan Infinite M200 reader. Further incubation was performed on complex fraction (tube 3). After separation, UV absorption at 253 nm, 300 nm and UV spectra of 230 -450 nm regions were measured for each fraction. Method II. Liposomes were prepared (size 200 nm, final concentration of 5 mg/mL in PBS) with 35-Glc/PBS solution (final concentration of 1×10 -2 M in PBS) to a final volume of 1 mL. Then, liposome complex were separated from free 35-Glc by G-25 column immediately. UV absorption at 253 nm, 300nm and UV spectra of the ultraviolet region (230 -400 nm) was measured for each fraction by Tecan Infinite M200 reader. The liposome complex fraction (tube 3) was incubated at 37°C in a shaking incubator. After 24 h, the mixture was encounter to another separation. UV absorption at 253, 300 nm was measured for each fraction.

V.9 Docking studies

The O-GlcNAc transferase (OGT) crystal structure (PDB: 4GZ5) was protonated by Molprobity 252 , and chain B was extracted after the removal of water, ions, and substrate to serve as the model macromolecule in the docking study. All four OGT inhibitors were built with the ribose and GlcNAc (Glc) ring conformations identical to those of the natural substrate in the crystal structure. 253 The macromolecule and four inhibitors were then processed by AutoDockTools to remove non-polar hydrogens. 254 A grid box centered on the position of the natural substrate was constructed using 24×24×24 points and a spacing of 1 Å. Subsequently, each inhibitor was docked into OGT by AutoDock Vina, 255 which automatically calculates the grid maps and clusters the results, with the value of the exhaustiveness parameter being 32. Glycosyltransferase is an important class of enzyme in living organisms responsible for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. As more and more carbohydrate related biological processes are elucidated, there is great interest to define the biological roles of a given carbohydrate and examine its potential therapeutic applications. The present study reports the design, synthesis, biological evaluation and structural analysis of two novel types of glycosyltransferase donor substrate analogues. The design rationale is to make analogues of sugar nucleotide diphosphate substrates, but incorporating a 'neutral' pyridine or amino-acid moiety as the pyrophosphate surrogate in order to provide cell permeable substrates for potential in cellulo or in vivo applications. The syntheses of "neutral" GTs inhibitors were performed using a combination of conjugations through O-glycoside bond, amide bond or triazole functionalities. A total number of 26 "neutral" GT inhibitors were prepared. The evaluation of inhibition towards five galactosyltransferases and one GlcNAc-transferase (OGT) revealed moderate inhibitions in the micromolar range. More interestingly, co-crystallyzation could be achieved for the most potent compounds in complex with a glycosyltransferase. The designed 'neutral' pyridine linker could chelate the manganese cation involved in the enzyme catalytic site. Whereas the sugar head-group was oriented away from the position found in the related complex with natural substrate (UDP-Gal)

Faculté

  Techniques des Activités Physiques et Sportives Observatoire des Sciences de l'Univers de Lyon Polytech Lyon Ecole Supérieure de Chimie Physique Electronique Institut Universitaire de Technologie de Lyon 1 Institut Universitaire de Formation des Maîtres Institut de Science Financière et d'Assurances Directeur : M. le Professeur F. DE MARCHI Directeur : M. le Professeur F. FLEURY Directeur : Mme le Professeur H. PARROT Directeur : M. N. SIAUVE Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur A. GOLDMAN Directeur : M. le Professeur H. BEN HADID Directeur Administrateur provisoire : M. N. LEBOISNE 7

Figure 1 .

 1 Figure 1. Implications des oligosaccharides dans divers processus biologiques. 1 La dégradation et la biosynthèse des oligosaccharides, polysaccharides et glycoconjugués est catalysée par les glycosidases et glycosyltransférases (GTs) respectivement. De nombreux travaux de recherche ont été consacrés au développement d'inhibiteurs de GTs. Ces inhibiteurs peuvent servir de sondes moléculaires pour étudier les fonctions biologiques des glycoconjugués et des GTs et envisager des applications biomédicales.

Figure 2 .

 2 Figure 2. Structures et approches synthétique des analogues d'UDP-sucre.

β

  La conjugaison du monosaccharide, du bras espaceur (pyridine ou serine) et du nucléoside (uridine) utilisant des liaisons O-glycosidique, amide ou triazole a fourni une large famille inhibiteurs « neutres » de GTs (Figure3-4).

Figure 3 .

 3 Figure 3. Structures des inhibiteurs « neutres » utilisant la pyridine pour remplacer le motif pyrophosphate et testés pour l'inhibition de GTs.

Figure 4 .

 4 Figure 4. Structures des inhibiteurs « neutres » utilisant la sérine pour remplacer le motif pyrophosphate et testés pour l'inhibition de GTs.

Figure 5 .

 5 Figure 5. Coordination du manganèse (sphère rose) dans le complexe 7-Glc/AAGlyB (PDB_ID:4KC1). 7-Glc est représenté en vert. Une autre conformation pour le composé 19 est représentée en cyan.

Figure 6 .

 6 Figure 6. Superposition des composés et comparaison avec le substrat naturel UDP-GlcNAc complexés avec l'OGT humaine (gris: UDP-GlcNAc; rouge: 43-Glc; bleu: 45; jaune: 35-Glc; cyan: 35-GlcNAc).

Figure I- 1 .

 1 Figure I-1. Structure of glucose, ribose, deoxyribose and α-Gal epitope

Figure I- 3 . 9

 39 Figure I-3. 9 Sugar nucleotide donors used by mammals.

Figure I- 4 .

 4 b). The third glycosyltransferase fold termed as GT-C was identified in 2008 as a soluble C-terminal domain of the Pyrococcus furiosius oligosaccharyltransferase STT3. 16 It is a novel architecture with α-helical domain surrounded by three β-sheet rich domains (Figure I-4.c).

Figure I- 4 .

 4 Figure I-4. General types of folds for classification of glycosyltransferases: (a) GT-A, (b) GT-B, and (c) GT-C. 17

I. 2 . 2 18 Figure I- 5 .

 22185 Figure I-5. Enzymatic reaction with either inverting or retaining glycosyltransferases.

Figure I- 6 .

 6 Figure I-6. Proposed mechanism for (a) inverting glycosyltransferases (b) retaining glycosyltransferases -double-displacement mechanism and (c) retaining glycosyltransferases --internal return mechanism.[START_REF] Breton | Recent structures, evolution and mechanisms of glycosyltransferases[END_REF]

Figure I- 7 .

 7 Figure I-7. Enzymatic-catalyzed glycosylation using β-1,4-galactosyltransferase (β-1,4-GalT).

Figure I- 9 . 5 Figure I- 10 .

 9510 Figure I-9. Structure of glycosphingolipid globotriaosylceramide. I.2.3.3 α-1,3-Galactosyltransferase α-1,3-Galactosyltransferase (α-1,3-GalT) is a retaining transferase catalyzing the α-galactosylation at the 3-OH position of a terminal β-linked galactose using UDP-Gal as an activated donor (Figure I-10). The resulting α-Gal epitope bearing a Galα-(1,3)-LacNAc-OR terminal (Figure I-1. c) is believed to be the main barrier in xenotransplantation which may cause hyperacute rejection. 5

Figure I- 11 .

 11 Figure I-11. Biofuction of human blood group galactosyltransferase and structure of human blood type. I.2.3.5 Human blood group glycosyltransferase mutant (AAGlyB)

Figure I- 12 .

 12 Figure I-12. Biofuction of human blood group galactosyltransferase mutant (AAGlyB). I.2.3.6 O-linked N-acetylglucosamine transferase A reversible O-GlcNAcylation of various nuclear and intracellular proteins is mediated by two enzymes, O-linked-N-acetyl-glucosaminase (OGA) and O-linked N-acetyl-glucosaminyl transferase (OGT) (Figure I-13).The O-linked N-acetylglucosamine transferase (OGT) is an inverting enzyme that uses UDP-GlcNAc as an active donor to transfer a GlcNAc residue to the side chain amino-acids serine or threonine of proteins.

Figure I- 13 .

 13 Figure I-13. Reversible O-GlcNAcylation mediated by OGA and OGT.

31 Figure I- 14 .

 3114 FigureI-14. Complex interaction between O-GlcNAc glycosylation and phosphorylation.[START_REF] Comer | O-glycosylation of nuclear and cytosolic proteins: dynamic interplay between O-GlcNAc and O-phosphate[END_REF]

Figure I- 15 .

 15 Figure I-15.Proposed catalytic mechanism for the human OGT.[START_REF] Breton | Recent structures, evolution and mechanisms of glycosyltransferases[END_REF][START_REF] Lazarus | Structure of human O-GlcNAc transferase and its complex with a peptide substrate[END_REF]

Figure I- 16 .

 16 Figure I-16. Structure of Ethambutol and Miglustat.

Figure I- 17 .

 17 Figure I-17. Representative GalT inhibitors with modification on sugar moiety.

Figure I- 18 .

 18 Figure I-18. Representative iminosugar based mimetics of GalT inhibitors.

Figure I- 19 .

 19 Figure I-19. Representative C-glycoside mimetics of GalT inhibitors.

Figure I- 20 .

 20 Figure I-20. Representative neutral GalT inhibitors.

Figure I- 21 .

 21 Figure I-21. Representative mimetics modification on nucleoside base.

Figure I- 22 .I. 3 . 1 . 5 93 Figure I- 23 .

 223159323 Figure I-22. Representative UDP-sugar mimetics as GalT inhibitors.I.3.1.5 Acceptor substrate analogues as GalT inhibitorsA common strategy to design inhibitors based on acceptor substrate analogues is to synthesize the sugar deoxygenated or modified compounds at the accepting position of the relative natural acceptor (G1-G4 and H1-H3, Figure I-23, I-24). Then, the acceptor substrate analogue may bind in the active site of the enzyme competing with the natural acceptor therefore abolishing the sugar transfer. Systematic research was performed and published by Ole Hindsgaul et al.[START_REF] Hindsgaul | Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases[END_REF] in 1991. Eight acceptor analogues on which the hydroxyl group undergoing glycosylation was replaced by hydrogen were synthesized and evaluated against eight different GT including FucT, GalT, GlcNAcT, GlcNAcT-V and SialylT. Half of the acceptor analogues showed no inhibitory effect towards the respective enzyme including the acceptor analogue (G1, Figure I-23) for β-1,4-GalT. This indicated that such hydroxyl groups are critical for enzymatic activity. All six possible deoxy and deoxyfluoro analogues of human boold group transferases acceptor (α-L-Fucp-(1,2)-β-D-Galp-O-(CH 2 ) 7 CH 3 ) were synthesized by Todd L. Lowary et al. 91 to study the compatibility and inhibitory effect on GTA and GTB. The study indicated that both the enzymes have no tolerance on modification at the 4-position of Gal moiety. The 6-deoxy and 6-deoxy-6-fluoro compounds are substrates for both enzymes while the 3-deoxy and 3-deoxy-3-fluoro compounds serve as competitive inhibitors, e.g. the 3-deoxy derivative (G2, Figure I-23) possess a K i value of 14 μM. A 4-deoxygenated acceptor analogue of α-1,4-GalT (G3, Figure I-23) was determined as an inhibitor of the enzyme with a K i value of 16 mM similar to the K m value (20 mM) of the natural acceptor and the analogue had been successfully co-crystallized with α-1,4-GalT. 92 Modification of the hydroxyl group normally undergoing galactosylation could also give an inhibitor of GT. A.-C. Helland et al. reported that an acceptor analogue, methyl 3'-amino-3'-deoxy-N-acetyllactosaminide (G4, Figure I-23)in which the sugar accepting position was replaced by an amino group, is a selective inhibitor against α-1,3-GalT with a mode of inhibition which is not competitive.[START_REF] Helland | Methyl 3-amino-3-deoxy-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside: an inhibitor of UDP-D-galactose: β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-D-glucose (1→3)-α-D-galactopyranosyltransferase[END_REF]

Figure I- 24 .

 24 Figure I-24. Representative sugar-aromatic mimetics of the acceptor substrates.

Figure I- 25 .

 25 Figure I-25. Bisubstrate analogues as GalT inhibitors. I.3.1.7 Small molecule inhibitors from library or HTS Progress in rapid and efficient detection systems facilitates identification of small molecule inhibitors from large libraries or high-throughput screening. Simple small molecules such as organic bisphosphonates (J1, Figure I-26) and bivalent imidazolium salts (J2, Figure I-26) were found to be effective against β-1,4-GalT. 58, 98 Another classic example is a small heterocycle (J3, Figure I-26) obtained from a Molecular Fragment Library which was

Figure I- 26 .

 26 Figure I-26. Representative small molecule inhibitors from library or high-throughput screening.

Figure I- 27 )

 27 and benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) (K2, Figure I-27) were early determined compounds which could interfere with O-GlcNAcylation. 102-103 Alloxan could selectively induce cell-death of insulin-producing cells in the pancreas, but the high toxicity of alloxan to different cell lines limits its further application. BADGP has been shown to be able to block the O-GlcNAcylation, but no evidence on its direct activity on OGT has been reported. Modification on the anomeric oxgen of UDP-GlcNAc, C-glycoside (K3, Figure I-27) and thioglycoside (K4, Figure I-27) were reported by

Figure I- 27 .

 27 Figure I-27. Representative UDP-GlcNAc analogues as inhibitors of OGT.

  The structures of 3 hits were given by the author as examples without mentioning individual activities (L4-L6, Figure I-28).

Figure I- 28 .

 28 Figure I-28. Representative OGT inhibitors identified from HTS.

Figure I- 29 .

 29 Figure I-29. Structures of the neutral UDP-sugar analogues designed and synthesized.

Figure II- 1 .

 1 Figure II-1. Target UDP-sugar analogues using pyridine as a pyrophosphate surrogate.

Figure II- 2 .

 2 Figure II-2. Target UDP-sugar analogues using pyridine as a pyrophosphate surrogate.

3 •

 3 Et 2 O (2 eq.) 51% 3p, β only a. Values are isolated yields. b. Product was isolated as a mixture of β-anomer 3k and 0.2 eq. unreacted acceptor 2a and could not be separated due to similar polarity. Yield was calculated excluding the unreacted acceptor 2a based on 1 H NMR data. c. Configuration at the α-anomeric center was confirmed by the 1 J C-1,H-1 = 172.4 Hz value. d. Configuration at the β-anomeric center was confirmed by the 1 J C-1,H-1 = 155.5 Hz value. e. Configuration at the α-anomeric center was confirmed by the 1 J C-1,H-1 = 171.5 Hz value.

Figure III- 1 .

 1 Figure III-1. Designed glycosyltransferase inhibitors using amino-acid derivatives as pyrophosphate surrogates.Eight UDP-sugar analogues(74, 75, 77, 78, 80, 81, 82 and 95) and three UDP analogues(64, 79 and 93) (Figure III-2)were synthesized based on our design. The key steps in this synthesis were the generation of the glycosidic bond with serine-based derivatives and an amide bond formation afterwards with uridine. Glycosylation of amino-acid residue is a biological phenomenon which has been commonly found in protein post-translational modification 219 and widespread studied in protein chemistry[220][221][222] . Nevertheless, the reaction condition needs to be optimized for each specific case. Therefore, glycosylation conditions with serine derivatives were also investigated in this section.

  C NMR (Figure III-4). The integration inferior to 1 and clear correlation with its carbon atom by HSQC indicated that the protons on the serine backbone of products 81 and 82 are acidic enough to be exchangeable. a. b.

Figure III- 3 .

 3 Figure III-3. Comparision of 1 H NMR spectra (500 MHz, CD 3 OD) of 81 and 82 measured immediately (~ 60 min) and several days later.

Figure III- 4 .

 4 Figure III-4. NMR data (500 MHz, CD 3 OD) of the deprotected product 82.

92 ).

 92 Adding a base to the mixed pre-dried reaction mixture (method I) gave 36% yield of the desired amide 92 and 24% of methyl ester resulting from the transesterification from 55 to 91. Adding a mixture of 91/HOBt in acetonitrile into the pre-base treated methyl serine ester 55/DCC in acetonitrile avoided the formation of transesterified methyl ester thus afforded amide the 93 in 90% yield. Galactosylation of the amide 93 using the galactose trichloroacetimidate 1c catalyzed by boron trifluoride etherate afforded the protected analogue 94 in 40% yield. Hydrolysis of the analogue 94 gave a UDP-Gal analogue 95.

  Figure IV-1, Figure IV-2 and Figure IV-3, respectively.

Figure IV- 1 .

 1 Figure IV-1. Structures of neutral UDP-Gal and UDP analogues as GalT inhibitor candidates using pyridine as a pyrophosphate surrogate.

Figure IV- 2 .

 2 Figure IV-2.Structures of neutral UDP-Gal and UDP analogues as GalT inhibitor candidates using amino-acid derivatives as a pyrophosphate.

Figure IV- 3 .

 3 Figure IV-3. Structures of neutral UDP-GlcNAc and UDP analogues as OGT inhibitor candidates using pyridine as a pyrophosphate surrogate.

Figure IV- 4 .

 4 Figure IV-4. Radiochemical GT assay.[START_REF] Wagner | Glycosyltransferases and their assays[END_REF]

  .2 and V.6.6, Chapter V). Compound 7-Gal was also a mixed inhibitor of α-1,4-GalT (Figure V.6.8, Chapter V), α-1,3-GalT (Figure V.6.10, Chapter V) and GTB (Figure V.6.12, mixed at high inhibitor concentrations, competitive at low inhibitor concentrations, Chapter V). Non-linear Dixon plots were also obtained from evaluation of compound 19 with β-1,4-GalT (Figure V.6.3, Chapter V) and AAGlyB (Figure V.6.5B, Chapter V). Michaelis-Menten plots confirmed compound 19 was a mixed inhibitor of β-1,4-GalT at high concentrations (Figure V.6.4, Chapter V), competitive with UDP-Gal for AAGlyB (Figure V.6.7, Chapter V), α-1,4-GalT (Figure V.6.9, Chapter V), mixed inhibitor of α-1,3-GalT and GTB at high concentrations (Figures V.6.11 and V.6.13, Chapter V). The determination of the inhibition mechanism for the two best inhibitors towards five GalTs was summarised in Table IV-3.

  ). For compounds with which mixed inhibition modes were observed by Michaelis-Menten plots (Figure V.6.14 -Figure V.6.16, Chapter V), IC 50 values were determined by constructing a dose-response curve.

Figure IV- 5 .

 5 Figure IV-5. Superposition of all three inhibitors and UDP-Gal from PDB_ID:2RJ7. 2RJ7 is shown with gray carbons, 7-Gal with yellow carbons, 19 with cyan carbons, 7-Glc with green carbons. 7-Gal and 19 adopted a second conformation for the anomeric oxygen colored transparent white and shown inside the pink circle; the sugars, likely disordered, are not visible in the electron density for those conformations. For UDP-Gal, the phosphates and the galactose can be seen in the back in orange and gray. Also sticks of Asp211 and Asp213 are included with the same color code. The large spheres are the manganese cations, colored as the corresponding carbons. Also the waters in the coordination spheres are shown as smaller spheres for all three inhibitors.

Figure IV- 6 .

 6 Figure IV-6. Electron density for compound 19, manganese in the AAGlyB structure (PDB_ID:4KC4). The anomeric oxygen is observed in two alternative conformations, but the galactose could only be modeled for one of them. The second conformation, with only the oxygen visible as a transparent rod, is marked with the pink circle. The orange mesh is electron density from the 2F o -F c map contoured at 1.0 σ, while the gray mesh is the same map contoured at 0.4 σ. The side chain in the lower part is from Asp213 chelating the Mn 2+ cation.

  5 Å and 2.3 Å respectively (Figure IV-7). These three atoms, the pyridine ring and the manganese are coplanar. The last two equatorial positions in the manganese's coordination sphere are occupied by water molecules at 2.3 Å and 2.4 Å, respectively. The two axial positions are filled by a water molecule at 2.3 Å and a carboxylic oxygen from Asp213 at 2.2 Å. The pyridine ring is in Van der Waals contact with the hydroxyl oxygen of Tyr126 which has a stacking interaction with uracil. The hydroxyl group is roughly in the center of the π electronic cloud of the pyridine.

Figure IV- 7 .

 7 Figure IV-7. Coordination sphere of Mn 2+ in the 7-Glc complex (PDB_ID:4KC1). 7-Glc is shown in thick sticks with green carbons. The alternative conformation of α-galactoside 19 is also presented in thin sticks with blue carbons, showing the galactose. The manganese is shown in pink. AAGlyB is shown as a semitransparent surface representation colored according to the closest atom and containing a stick model of the protein with yellow carbons.

Figure IV- 8 )

 8 were selected for OGT inhibition evaluation. A preliminary radioactive OGT assay was performed at 50 μM concentration, typical concentration used for the best OGT inhibitors. The compounds exhibit only a limited inhibitory effect on human OGT at 50 μM with the best inhibition of 19% observed for 35-Glc (Figure IV-8). Analogues displaying more than 10% inhibition at 50 μM were then subjected to IC 50 measurements (Table IV-3) which gave IC 50 values of 336 μM, 708 μM and 1608 μM for 35-Glc, 45 and 43-Glc, respectively. The best compound among this series is 35-Glc, a glucose derivative linked to the pyridine moiety by an amide bond and connected to the nucleoside part through a triazole moiety. Further kinetic study showed that this analogue was a competitive inhibitor with a K i value of 422 μM (Figure VI.9.17, chapter VI).

Figure IV- 8 .

 8 Figure IV-8. Inhibition activity of the UDP-Sugar and UDP analogues against OGT at 50 μM.

Figure IV- 9 .Figure IV- 10 .

 910 Figure IV-9. Overview of the best docking poses in comparison with the natural substrate UDP-GlcNAc (grey: OGT, grey sticks: UDP-GlcNAc; red sticks: 43-Glc; blue sticks: 45; yellow sticks: 35-Glc; cyan sticks: 35-GlcNAc). Different regions of UDP-GlcNAc are labeled in black text. The triazole linkers of 43-Glc, 45, and 35-Glc shared the same orientation and location, with N2 forming a hydrogen bond with the hydroxyl group of Thr922 and N3 interacting with the nitrogen on the backbone of Thr921 (Figure IV-10). These polar interactions between triazoles and amino-acid residues of OGT resembled the polar interactions between β-phosphate of UDP-GlcNAc and OGT (shown with grey dashed line in Figure IV-10). This interaction was absent in the model of 35-GlcNAc which may explain why this derivative showed weaker inhibitory activity.

Figure IV- 11 .IV. 2 . 2 . 2

 11222 Figure IV-11. Inhibitor binding within the Glc/GlcNAc portion (Grey sticks: UDP-GlcNAc; red sticks: 43-Glc; blue sticks: 45; yellow sticks: 35-Glc; cyan sticks: 35-GlcNAc.). Dashed lines indicate hydrogen bonds from different inhibitors. The acetamido group of UDP-GlcNAc and 35-GlcNAc are labeled in cyan text. The bromopyridine moiety of compound 45 partially overlapped with the GlcNAc ring of UDP-GlcNAc. In comparison, the three derivatives 43-Glc, 35-Glc and 35-GlcNAc adopted an orientation perpendicular to that of the natural substrate UDP-GlcNAc. In the Glc series, both 43-Glc and 35-Glc could form hydrogen bonds with His498, His499 and Lys634, whereas the GlcNAc derivative 35-GlcNAc was involved in a single hydrogen bond interaction with His499 (Figure IV-11). The acetamido group of 35-GlcNAc showed no direct interaction with the enzyme backbone.As a short conclusion, the uridine moieties of these four inhibitors showed identical binding mode to the natural substrate of UDP-GlcNAc. The polar interactions formed between triazole linkers and OGT could mimic the binding function of β-phosphate of UDP-GlcNAc whereas the carbohydrate moieties of 43-Glc, 35-Glc and 35-GlcNAc were oriented to a diffent direction far from the original pyranose pocket. The bromo-pyridine UDP analogue 45, among the four compounds applied for docking experiment, exhibited the best mimicry of the natural substrate GlcNAc in terms of binding geometry. These modeling studies therefore reveal why the glucose derivatives (35-Glc, 43-Glc) exhibited relatively better inhibitory effect compared to the glucosamine counterpart 35-GlcNAc.

Figure IV- 12 .

 12 Figure IV-12. MCF7 cells were treated for 24 hours (A) or 48 hours (B) with DMSO (vehicle), inhibitors 43-GlcNAc, 35-GlcNAc, 30-GlcNAc, 43-Glc, 35-Glc, 30-Glc, 25 or Ac-5SGlcNAc at indicated concentrations. O-GlcNAc level was examined by immunoblotting using RL2 antibody. GAPDH was used as loading control. Representative results of 3 independent experiments.

Figure IV- 13 .

 13 Figure IV-13. MCF7, MCF10A and HeLa cells were treated for 24 hours (A) or 48 hours (B) with DMSO (vehicle), 35-Glc or Ac-5SGlcNAc at indicated concentrations. O-GlcNAc level was examined by immunoblotting using RL2 antibody. GAPDH was used as loading control. Representative results of 2 independent experiments.

-Gal and 35 -

 35 Glc, respectively) were evaluated by an artifical cell membrane penetration test. Liposomes made of phosphatidylcholine (POPC, Figure IV-14) were used as a model and UV-vis absorption was used to quantify the amount of inhibitors present in solution. Two methods were used in the evaluation, named method I and II as shown in Scheme IV-1.

Figure IV- 14 .Figure IV- 15 .

 1415 Figure IV-14. Structure of POPC and analogues evaluated in membrane permeation experiments.

Figure IV- 16 .

 16 Figure IV-16. UV-titration of 35-Glc. For UV-titration, UV absorption of 35-Glc at concentrations of 1×10 -6 M, 1×10 -5 M, 1×10 -4 M, 1×10 -3 M in 1×PBS buffer were measured in duplicats. Linear regression fit of 35-Glc was taken at 253 nm (λ max = 253 nm and a shoulder peak at 300 nm).

  Figure IV-17. a. UV absorption at 260 nm; b. UV spectra of liposome complex fraction (fraction 4, in red), compared with liposome only (in black). c. UV absorption at 260 nm (second separation).

Figure P- 1 .

 1 Figure P-1. Binding mode of 7-Glc. The protein is represented as a semitransparent surface with a stick model iand yellow carbons, 7-Glc is represented as a thick stick model with green carbons. Acceptor α-L-Fucp-(1→2)-β-D-Galp-O-(CH 2 ) 7 -CH 3 is represented with thin sticks and cyan carbons. The manganese is the purple sphere and the white dashed lines indicate its coordination ligands, which include three water molecules (small red spheres). Black arrows indicate the distances mentioned in the text.

  0.26 (PE:EtOAc = 1:2); [α] D 20 = -14.0 (c = 0.5, CH 2 Cl 2

( 1 -

 1 Methoxycarbonylbenz-3-yl)methyl 2,3,4,6-tetra-O-acetyl-β-D-galactoside (3e) Entry 20, Table

  2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl chloride (4.093 g, 11.

  = 1:2) to afford compound 28-GlcNAc as white foam(1.908 g, 5.1 mmol, 46%). R f = 0.38 (PE:EtOAc = 1:2); 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) = 5.51(d, J = 8.7 Hz, 1H), 5.31 -5.06 (m, 2H), 4.75 (d, J = 9.3 Hz, 1H), 4.28 (dd, J = 12.5, 4.8 Hz, 1H), 4.20 -4.13 (m, 1H), 3.78 (ddd, J = 10.0, 4.

  Prepared according to general protocol C from compound 28-Glc (27 mg, 0.072 mmol, 1.0 eq.) and compound 27(33 mg, 0.072 mmol, 1.0 eq.

  Prepared according to general protocol E from compound 29-GlcNAc (41 mg, 0.05 mmol, 1.0 eq.

  -ESI-MS (positive mode) m/z: calcd. for C 25 H 30 N 8 N a O 11 [M+Na] + 641

  .88 (COCH 3 ), 20.86 (COCH 3 ), 20.7 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 20 H 25 BrN 3 O 9 , [M+H] + 530.0769, found 530.0776. 6-(2-(Trimethylsilyl)ethynyl)-N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)picolinamide (32-Glc) Prepared according to general protocol D from compound 31-Glc (310 mg, 0.58 mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (100% PE to 30% EtOAc/PE) to afford compound 32-Glc as yellow foam (288 mg, 0.526 mmol, 90%). R f = 0.43 (PE:EtOAc, 1:1); [α] D = -35.4 (c = 2.4, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.57 (d, J = 9.9 Hz, 1H, NHCO), 8.09 (dd, J = 7.8, 0.8 Hz, 1H, H Pyr ), 7.79 (t, J = 7.8 Hz, 1H, H Pyr ), 7.58 (dd, J = 7.8, 0.8 Hz, 1H, H Pyr ), 5.49 (d, J = 9.6 Hz, 1H, H 1Glc ), 5.34 (t, J = 9.5 Hz, 1H, H 3Glc ),

  (C PyrH ), 130.7 (C PyrH ), 122.1 (C PyrH ), 102.9 (C alkyne ), 96.6 (C alkyne ), 78.3 (C 1Glc ), 73.9 (C 5Glc ), 73.4 (C 3Glc ), 70.7 (C 2Glc or C 4Glc ), 68.2 (C 2Glc or C 4Glc ), 61.8 (C 6Glc ), 20.9 (COCH 3 ), 20.73 (COCH 3 ), 20.72 (COCH 3 ), 20.69 (COCH 3 ), -0.2 (s, 3C, SiMe 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 25 H 33 N 2 O 10 Si [M+H] + 549.1899, found 549.1898.

  (PE:EtOAc = 1:4); [α] D = +62.4 (c = 0.4, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ = 9.57 (s, 1H, NH Uri

  triazol-4-yl]pyridine(43-GlcNAc)

  mL, 1.065 mmol, 7.5 eq.) was added into a microwave tube containing 2',3'-di-O-acetyl-5'-azido-uridine

  (C 6Uri ), 139.4 (C PyrH ), 127.4 (C PyrH ), 124.6 (C triazoleH ), 119.0 (C PyrH ), 103.6 (C 5Uri ), 91.9 (C 1Rib ), 80.4 (C 4Rib ), 73.0 (C 2Rib ), 70.8 (C 3Rib ), 51.6 (C 5Rib ), 20.6 (COCH 3 ), 20.5 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 20 H 20 BrN 6 O 7 , [M+H] + 535.0571, found 535.0568.

13 C

 13 NMR (100 MHz, CDCl 3 ) δ (ppm) = 169.83 (COCH 3 ), 169.78 (COCH 3 ), 162.7, 150.4, 150.0, 148.2, 142.9, 141.5 (C 6Uri ), 137.2 (C PyrH ), 127.1 (C PyrH ), 124.5 (C triazoleH ), 119.9 (C PyrH ), 103.7 (C≡C), 103.6 (C≡C), 95.4 (C 5Uri ), 91.6 (C 1Rib ), 80.4 (C 4Rib ), 73.0 (C 2Rib ), 70.8 (C 3Rib ), 51.6 (C 5Rib ), 20.56 (COCH 3 ), 20.55 (COCH 3 ), -0.1 (s, 3C, SiMe 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 25 H 29 N 6 O 7 Si, [M+H] + 553.1862, found 535.1850.

  )(26.3 g, 125.4 mmol, 63%). 1 H NMR (300 MHz, D 2 O) δ (ppm) = 9.37 (t, J = 1.4 Hz, 1H), 8.06 -7.98 (m, 1H), 7.61 (dd, J = 2.1, 1.2 Hz, 1H).

  ), 103.0 (C Uri ), 89.5 (C 1Rib ), 80.4 (C 4Rib ), 73.4 (C 2Rib ), 73.0 (C 3Rib ), 20.3 (COCH 3 ), 20.1 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd for C 13 H 14 N 2 NaO 9 , [M+Na] + 365.0592, found 365.0589.

  [ 3 H]Gal, the requisite acceptor α-L-Fucp-(1→2)-β-D-Galp-O-C 8 H 17 , β-D-GlcNAc-O-(CH 2 ) 8 -CO 2 Me or β-D-Lac-O-(CH 2 ) 8 -CO 2 Me as acceptor substrates and inhibitor (0 to 3 mM). The reaction mixture was incubated in MOPS (50 mM), MnCl 2 (20 mM), pH 7.0, bovine serum albumin (1 mg.mL -1 ) for a certain time at 37°C (a time for which linear rates are obtained using these assay conditions) and the reaction was quenched with cold water (400 μL). The enzymatic product was isolated by purification with Sep-Pak RC C 18 cartridges (Waters). Radioactivity was measured by using a Beckman Coulter TM LS 6500 multi-purpose scintillation counter. The K m values for UDP-Gal and the requisite acceptor α-L-Fucp-(1→2)-β-D-Galp-O-C 8 H 17 , β-D-GlcNAc-O-(CH 2 ) 8 -CO 2 Me or β-D-Lac-O-(CH 2 ) 8 -CO 2 Me with different GT were determined previously by a standard radiochemical Sep-Pak assay.

1 .

 1 Figure V.6.1. Non-Linear Dixon Plots of 7-Gal Inhibition of β-1,4-GalT

Figure V. 6 . 2 .Figure V. 6 . 2 .

 6262 Figure V.6.2. Michaelis-Menten Plots of 7-Gal Inhibition of β-1,4-GalT

FigureFigure V. 6 . 3 .

 63 Figure V.6.3. Non-Linear Dixon Plots of 19 Inhibition of β-1,4-GalT

FigureFigure V. 6 . 4 .

 64 Figure V.6.4. Michaelis-Menten Plots of 19 Inhibition of β-1,4-GalT

Figure 5 .

 5 Figure V.6.5. Non-Linear Dixon Plots of 7-Gal and 19 Inhibition of AAGlyB

Figure 6 .

 6 Figure V.6.6. Michaelis-Menten Plots of 7-Gal Inhibition of AAGlyB

Figure V. 6 . 7 .Figure V. 6 . 7 .

 6767 Figure V.6.7. Michaelis-Menten Plots of 19 Inhibition of AAGlyB

Figure V. 6 . 8 .Figure V. 6 . 8 .

 6868 Figure V.6.8. Michaelis-Menten Plots of 7-Gal Inhibition of α-1,4-GalT

FigureFigure V. 6 . 9 .

 69 Figure V.6.9. Michaelis-Menten Plots of 19 Inhibition of α-1,4-GalT

Figure V. 6 .Figure V. 6 . 10 .

 6610 Figure V.6.10. Michaelis-Menten Plots of 7-Gal Inhibition of α-1,3-GalT

Figure V. 6 . 11 .

 611 Figure V.6.11. Michaelis-Menten Plots of 19 Inhibition of α-1,3-GalT

Figure 12 .

 12 Figure V.6.12. Michaelis-Menten Plots of 7-Gal Inhibition of GTB

Figure V. 6 .Figure V. 6 . 13 .

 6613 Figure V.6.13. Michaelis-Menten Plots of 19 Inhibition of GTB

Figure 14 .

 14 Figure V.6.14. Michaelis-Menten Plots of 81 Inhibition of AAGlyB

Figure 15 .

 15 Figure V.6.15. Michaelis-Menten Plots of 81 Inhibition of GTB

Figure V. 6 .Figure V. 6 . 16 .

 6616 Figure V.6.16. Michaelis-Menten Plots of 90 Inhibition of α-1,3-GalT

Figure V. 6 . 17 .Figure V. 6 . 17 .

 617617 Figure V.6.17. Radioactive OGT assay for K i determination of 35-Glc

Tableau 1 .

 1 Conditions optimales de glycosylation pour les motifs pyridine et sérine.

	Donneur	Accepteur	Rapport Donneur: Accepteur	Temp. Temps	Promoteur	Rendement Rapport α : β
						1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	46% β
						1:1	50 o C, 78 h	MeI (4.5 eq.), 4Å MS	75% α
						1.1:1	-20°C, 3 h	BF 3 •Et 2 O (2 eq.)	66% β
						1.5:1	-20°C, 2 h; rt, 16 h	BF 3 •Et 2 O (4 eq.)	82% 72:28
		HO	N 2a	O	OMe	1.3:1	-20°C, 22.5 h	TMSOTf (2 eq.), 4Å MS	61% β
						1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	39% β
						1.3:1	-20 o C, 4 h	TMSOTf (0.8 eq.)	66% α
						1:1.15	-60°C, 0.5 h; -78°C, 3 h	BSP (1.2 eq.), TTBP (1.5 eq.), Tf 2 O (1.3 eq.), 1-octene (2 eq.)	80% 19:81
						1:1	-20 o C, 4 h	BF 3 •Et 2 O (2 eq.)	51% β
		HO	2d	O	OMe	1:3	-20°C, 1 h	BF 3 •Et 2 O (0.2 eq.)	79% β
		HO	OBn NHBoc O	2.5:1	-20 o C, 9 h	BF 3 •Et 2 O (0.5 eq.)	15% β
		HO		OMe

-16). 41

  [START_REF] Breton | Recent structures, evolution and mechanisms of glycosyltransferases[END_REF][START_REF] Lazarus | Structure of human O-GlcNAc transferase and its complex with a peptide substrate[END_REF] Among the 317 marketed drugs, 71 enzymes are targeted and two GT inhibitors are found which are Ethambutol, an inhibitor of mycobacterial arabinotransferases used as an antibacterial drug, and Miglustat, a ceramide glucosyltransferase inhibitor used for treatment of Gaucher's disease (FigureI

I.3 Inhibitors of glycosyltransferases

Based on the roles of carbohydrates in living organisms in numerous biological processes, there is great interest in determining the exact implications of a given glycoconjugate in these processes. Glycosyltransferase inhibitors may serve as molecular probes for investigations of biological functions of glycoconjugate and selective glycosyltransferase inhibitors may have biomedical applications. An analysis of the FDA Orange Book in 2005 indicates that 317 marketed drugs are enzyme inhibitors.

  3

  •Et 2O, 118 were well applied to activate the glycosyl esters. While two or three component catalytic systems, eg. SnCl 4 -AgClO 4 or SnCl 4 -CF 3 CO 2 Ag,[119][120][121] were also applicable in this case which could reduce the amount of Lewis acids required.

	methods uses sulfenyl derivatives under Lewis acid catalysis, such as 1-benzenesulfinyl
	piperidine (BSP)/triflic anhydride (Tf 2 O). 130 The alkylating agent MeI 131 is also applicable for
	activating thioglycosides which are normally 2-pyridyl thioglycosides and afford
	1,2-cis-glycosides with good stereoselectivities.
	Glycosylations with glycosyl bromides and chlorides catalyzed by heavy metal salts were
	known as the Koenigs-Knorr reaction. 122 Ag 2 O and Ag 2 CO 3 were the frequently used
	promoters 123 and Hg(CN) 2 /HgBr 2 were the Helferich variant. 124 Later, AgOTf was employed
	for a better solubility and was examined as one of the most efficient heavy metal
	promoters. 125 A different type of promoter for this kind of donor was in situ anomerization.
	Et 4 NBr or Bu 4 NBr was used in combination with base to convert the less active α-glycosyl
	bromides into their highly reactive β-anomers which underwent rapid S N2 substitution
	resulting in α-glycosides. 126 The drawback of this method is its limitation to only glycosyl
	halides whereas the advantage is the high α-stereoselectivity.
	Glycosyl trichloroacetimidates were developed in Richard R. Schmidt's group and known as
	Schmidt donors. They are relatively stable under basic or neutral conditions but readily
	reactive under acidic activation. BF 3 •Et 2 O and TMSOTf were frequently used
	promoters. 127-128 Glycosyl trifluoroacetimidates were also effective sugar donors and had been
	introduced into glycosylation. 129
	The merit of thioglycosides is their stability. They are relatively shelf stable, tolerant to
	diverse reaction conditions, readily converted into other types of donors and more
	interestingly relative activity between different substituents can be used to program multistep
	glycosylations. The activation process (Scheme II-4) is initiated by an electrophile which
	forms a sulfonium ion with the thioglycoside making a better leaving group. Loss of the
	sulfonium ion part leads to the common oxonium/carbenium ion intermediate which will then
	reacts with a glycosyl acceptor resulting in the desired glycoside. One of the activating

Table II -1. Frequently

 II used sugar donors and activating systerms.

	thus providing the active glycosylating intermediate. This method is highly recommended for
	the synthesis of β-mannopyranosides.
	Entry	Donor	Promoter	Ref.
	Hemiacetal		BF 3 •Et 2 O, FeCl 3	116
			SnCl 4	117
	Glycosyl ester		BF 3 •Et 2 O SnCl 4 -AgClO 4	118 119
			SnCl 4 -CF 3 CO 2 Ag	120-121
			Ag 2 O, Ag 2 CO 3	123
	Glycosyl bromides		Hg(CN) 2 , HgBr 2 AgOTf	124 125
			Et 4 NBr, Bu 4 NBr	126
			BF 3 •Et 2 O	127
	Imidates		TMSOTf	128
			TMSOTf	129
			BSP, Tf 2 O	130
	Thioglycosides
			MeI	131
			NIS, TfOH	132
	O-Glycosides
			DTBMP, Tf 2 O	133
	Glycals		DMDO, ZnCl 2	134
	Oxazolines		TMSOTf	135
		Scheme II-4. Activation of thioglycosides by electrophic reagents.
	O-Glycosides containing specific leaving group precursors could also serve as sugar donors.
	n-Pentenyl glycosides introduced by Fraser-Reid 136 are good examples of this type of donors.
	n-Pentenyl glycosides could be remotely activated by halogen derivatives, especially
	iodinated species, eg. NIS-TfOH 132 , by forming an iodonium intermediate followed by
	intramolecular cyclization (a, Scheme II-5). Another example of this kind of O-glycosides
	activated by remoted activation is 2'-carboxybenzyl glycoside (b, Scheme II-5). 133 The
	2'-carboxybenzyl glycoside reacts with Tf 2 O in the presence of DTBMP forming a lactone,

Scheme II-5. Remote activation of n-pentenyl glycosides a and 2'-carboxybenzyl glycosides b.

the activation method of carboxylic acids used in the present work is a combination of diisopropyl carbodiimide (DIC) and hydroxybenzotriazole HOBt (Scheme II-9). DIC reacts with the carboxylic acid to form

	Scheme II-11. Proposed mechanism for CuAAC by K. B. Sharpless et al. 156 (Pathway A) and Valery V. Fokin et
	al. 157 (Pathway B).
	Later that year, a dinuclear copper intermediate involved in the mechanism was proposed by
	Valery V. Fokin (Pathway B, Scheme II-11). 157 Followed by further research, this group
	provided more experimental evidence supported their mechanistic interpretation in 2013
	(Scheme II-11). 158 Real-time monitoring of the cycloaddition process indicated that two atoms
	of copper are required to promote the process while without adding exogenous copper catalyst
	the Cu-acetylide is not reactive. Further crossover experiments employing isotopically enriched
	copper revealed that the two coppers involved contribute equally to the process indicating a
	rapid exchange during the cycloaddition step (Scheme II-12). Therefore, a dinulear copper
	intermediate mechanism is now clearly demonstrated for the CuAAC.
	an
	O-acylisourea. N-acylurea could be formed by acetyl transfer, but this side reaction could be
	avoided by adding HOBt. The intermediate ester generated is relatively stable but still reactive.
	Then, nucleophilic attack by iminophosphorane or amine affords the amide derivative. DIC was
	chosen rather than DCC for mainly one reason that DIC is a liquid having a better dispension in Scheme II-12. Dinuclear copper intermediated identified in the mechanism for CuAAC proposed by Valery V.
	the reaction than the crystalline DCC. HOBt was added in order to avoid side reactions and also Fokin et al. 158
	suppress the possible racemization. Commercially available HOBt is stabilized by water as
	monohydrate crystal due to the explosive property of anhydrous HOBt. This hydrated HOBt
	requires careful treatment (coevaporation with toluene) before use for reactions to obtain
	anhydrous conditions.

-2), the

  best result in this respect was obtained under standard conditions with 46% yield (Entry 5

, Table II-2).

  Modifications on the reaction conditions were made, but addition of molecular sieves (Entry 6

, Table II-2), prolongation of reaction time (Entry 7, Table II-2), changing the solvent to acetonitrile (Entry 8, Table II-2), excess of acceptor (Entry 9, Table II-2) or larger proportions of donor (Entry 10, Table II-2) did

  not improve the yields. Trimethylsilyl trifluoromethanesulfonate was also investigated as a promoter for this glycosylation, but the main product isolated was the silylated acceptor

2b (Entry 11, Table II-2). A

	relatively neutral glycosylation condition compared to
	boron trifluoride etherate, was reported by the Schmidt group recently using
	difluoro(phenyl)borane (PhBF 2 ) as the glycosylation promoter.

Table II -2. Galactosylation of methyl 6-(hydroxymethyl)picolinate 2a with galactosyl donors 1a-f.

 II

	Entry	Donor	Ratio Donor:Acceptor	Temp. Time	Promoter	Yield a	Product Ratio α : β
	1
			1.5:1	0 o C to r.t., 16 h SnCl 4 (5 eq.) / CF 3 CO 2 Ag (2.5 eq.) <5% b	-/-
	2		1:1.5	r.t., 18 h	AgOTf (1.5 eq.)	8%	3a, β only
	3		2.5:1	r.t., 26 h	AgOTf (1.5 eq.)	18%	3a, β only
	4		1.5:1	r.t., 16 h	Hg(CN) 2 (4 eq.)	11% c	3a, β only
	5		1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	46%	3a, β only
	6		1.25:1	-20 o C, 4 h	BF 3 •Et 2 O (2 eq.), 4Å MS	34%	3a, β only
	7		1.5:1	-20 o C, 16 h	BF 3 •Et 2 O (2 eq.), 4Å MS	32%	3a, β only
	8		1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	36% d	3a, β only
	9		1:3	-20 o C, 2.5 h	BF 3 •Et 2 O (2 eq.)	<5% b	-/-
	10		3:1	-20 o C, 16 h	BF 3 •Et 2 O (3 eq.)	42%	3a, β only
	11		1.5:1	-20 o C, 1 h	TMSOTf (0.5 eq.), 4Å MS	5% e	3a, β only
	12		2:1	-78 o C, 3 h	PhBF 2 (2 eq.)	16%	3a, β only
	13		1.5:1	-20 o C, 2 h	PhBF 2 (2 eq.)	14%	3a, β only
	14
			1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	32%	3a, β only
	15		1.3:1	-20°C, 22 h	TMSOTf (2 eq.), 4Å MS	33%	3b, β only
	16		1.3:1	-20°C, 20 h	BF 3 •Et 2 O (3 eq.)	42%	3b, β only
	17
			1:1	50 o C, 78 h	MeI (4.5 eq.), 4Å MS	75%	3c, α only
	a. Values are isolated yields. b. Product could not be isolated but was detected by TLC. c. The glycosylation was
	performed in CH 3 CN/CH 2 Cl 2 (1/1, v/v). d. The glycosylation was performed in CH 3 CN. e. The main product
	isolated was methyl 6-(trimethylsilyloxymethyl)picolinate 2b.

Table II -3. Influence of the pyridine-based acceptors 2b-d with the galactosyl donor 1c.

 II

	Entry	Donor	Ratio Donor:Acceptor	Temp. Time	Promoter	Yield a	Product Ratio α : β
	18		1.5:1	-20 o C, 3 h	TMSOTf (1 eq.), 4Å MS	<5% b	3a
	19		2.25:1	-20 o C, 3 h	BF 3 •Et 2 O (0.6 eq.)	7%	3d c , 32:68
	20		1.5:1	-20°C, 2 h	BF 3 •Et 2 O (0.2 eq.)	37% d	3e, β only
	21		1:3	-20°C, 1 h	BF 3 •Et 2 O (0.2 eq.)	79%	3e, β only
	a. Values are isolated yields. b. Product was not isolated but identified by TLC in a very small proportion. c.
	Product was isolated as an inseparable mixture of hydroxylated donor, unreacted acceptor, -and -anomers. d.
	Acetylated acceptor (methyl 6-acetoxymethylbenzoate) was also isolated in 37% yield.

Table II - 4 .

 II4 Glycosylation in the GlcNAc series with methyl 6-(hydroxymethyl)picolinate 2a.

	Entry	Donor	Ratio Donor:Acceptor	Temp. Time	Promoter	Yield a	Product Ratio α : β
	22		1.1:1	60°C, 5.5 h	CSA (1.2 eq.), 4Å MS	17%	3f, β only
	23		1.5:1	20°C, 22 h	BF 3 •Et 2 O (3 eq.), 4Å MS 20%	3f, β only
	24		1.1:1	-20°C, 22 h	TMSOTf (3 eq.)	25%	3g, β only
	25		1.6:1	-20°C, 4.5 h	TMSOTf (2 eq.)	48%	3g, β only
	26		1.1:1	-20°C, 3 h	BF 3 •Et 2 O (2 eq.)	66%	3g, β only
	27		1.5:1	-20°C, 3.5 h; r.t., 15.5 h	TMSOTf (3 eq.)	27%	3h, β only
	28		1.1:1	-20°C, 17 h	BF 3 •Et 2 O (3 eq.)	48%	3h b , β only
	29		1.5:1	-20°C, 2.5 h; r.t., 16 h	TMSOTf (4 eq.)	<5%	3i c
	30		1.5:1	-20°C, 2.5 h; r.t., 16 h	BF 3 •Et 2 O (4 eq.)	35%	3i, α only
	31		1.5:1	-20 o C, 2 h	TMSOTf (2 eq.)	17%	3i, α only
	32		1.5:1	-20°C, 2 h; r.t., 16 h	BF 3 •Et 2 O (4 eq.)	82%	3i, 72:28

36, Table II-5). II.2.4.3 Mannose. Two

). Scheme II-18. Glycosylation of methyl 6-(hydroxymethyl)picolinate 2a with mono-and disaccharidic glycosyl donors 1n-t. II.2.4.1 Glucose. Two glucosyl donors, acetylated trichloroacetimidate 1n 198 and benzoylated trichloroacetimidates 1o, 199 were evaluated. The yields obtained were better (Entry 33-34, Table II-5) than the results previously observed in the galactose series (Entries 2-13 and 21-22, Table

II-2)

. II.2.4.2 Fucose and Rhamnose. Glycosylation using 6-deoxyhexoses such as L-fucose is also important due to the relevant biological implications of such carbohydrates in cancer and bacterial infections.

200

The acetylated trichloroacetimidate fucosyl donor 1p 201 was therefore reacted under the best conditions elaborated in the galactose series leading to a moderate isolated yield of 39% of fucoside 3l (Entry 35, Table

II-5

). The rhamnosyl donor 1q 202 was studied in a collaboration with Reko Leino's research group in Åbo University (Finland). Catalyzing by TMSOTf, the benzoylated rhamnosyl trichloroacetimidate 1q afforded a better yield of 66% in comparison with the acetylated fucosyl trichloroacetimidate donor 1p (Entry mannosyl donors, benzoylated trichloroacetimidate mannosyl donor 1r 203 and bicyclic containing armed donor 1s,

204

were evaluated with Prof. Leino's team. Under activation of BF 3 •Et 2 O and TMSOTf, the trichloroacetimidate donor 1r afforded the desired α-mannoside 3n in moderate yields (Entries 37-38, Table

Table II -

 II

	Entry	Donor	Ratio Donor:Acceptor	Temp. Time	Promoter	Yield a	Product Ratio α : β
	33		1.5:1	-20°C, 22 h	BF 3 •Et 2 O (3 eq.)	55% 3j, β only
	34		1.3:1	-20°C, 22.5 h	TMSOTf (2 eq.), 4Å MS	61% 3k b , β only
	35		1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (2 eq.)	39% 3l, β only
	36		1.3:1	-20 o C, 4 h	TMSOTf (0.8 eq.)	66% 3m, α only
	37		1.3:1	-20°C, 22.5 h	BF

5. Glycosylation of methyl 6-(hydroxymethyl)picolinate 2a with mono-and disaccharidic glycosyl donors 1n-t. 3 •Et 2 O (2 eq.) 48% 3n c , α only 38 1.3:1 -20°C, 22.5 h TMSOTf, (2 eq.) 22% 3n c , α only 39 1.3:1 -40°C, 5.5 h TMSOTf, (2 eq.), NIS (1.56 eq) 68% 3o d , β only

  DDQ, 209-210 Ph 3 C + BF 4 -211 , NBS 212 and (n-Bu 4 N) 2 S 2O 8 213 were also tested but with either no reaction or decomposition of the starting material. Finally, the benzyl ethers were successfully removed by bromination/oxidative cleavage conditions[214][215] and the crude mixture was then reacetylated to provide the acetyl-protected compound 16 in 42% yield. After hydrolysis to the acid derivative 17 and formation of an amide bond to compound 18, another pyridine containing UDP-Gal analogue 19 was obtained after deacetylation. This analogue could provide information about the importance of the configuration at the anomeric center towards GalT inhibition.

1

H NMR and mass spectrometry (m/z, [M+H] + = 696.

2)

. Similar phenomenon was reported by Hironao Sajiki et al.

[207][208]

that hydrogenolysis of benzyl ether with Pd/C could be selectively inhibited at the presence of pyridine. Other reaction conditions such as Scheme II-21. Synthesis of the α-galactoside inhibitor candidate through glycosylation and Staudinger-Vilarrasa ligation. Reagents and conditions: (a) MeI, CH 2 Cl 2 , 50°C, 78h, 75%; (b) i) NaBrO 3 , Na 2 SO 4 , EtOAc/H 2 O, r.t., 2h then ii) Ac 2 O, C 5 H 5 N, r.t., 3h, 42% for two steps; (c) NaI, C 5 H 5 N, 120°C, 5h, quant.; (d) PMe 3 , DIC, HOBt, THF, r.t., 16h, 27%; (e) NaOMe, MeOH, 63%.

  3

Table II - 6 .

 II6 Comparison of two different synthetic strategies.

	Entry	Sugar-to-Nucleoside Number of steps yield (%)	Nucleoside-to-Sugar Number of steps yield (%)
	7-Glc (Scheme II-19)	5	6	5	2
	43-Glc (Scheme II-24)	7	43	5	21
		70 71

  3

Table III -

 III

	Entry	Donor	Acceptor	Ratio Donor: Acceptor	Temp. Time	Promoter
	1		R = H	1:1.2	0°C to r.t., 24 h	BF 3 •Et 2 O (3 eq.), CH 3 CN
	2		R = Bn	2.5:1	0°C to r.t., 48 h	SnCl 4 (5 eq.)/CF 3 CO 2 Ag (2.5 eq.) CH 2 Cl 2
	3		R = H	1:2.8	0°C to r.t., 22 h	I 2 (1.5 eq.), CH 3 CN
	4		R = Bn	1:1.7	0°C to r.t., 24 h	I 2 (1.5 eq.), CH 3 CN
	5		R = Bn	2:1	0°C to r.t., 53 h	AgOTf (1 eq.), CH 2 Cl 2
	6		R = Bn	2.5:1	-20 o C, 9 h	BF 3 •Et 2 O (0.5 eq.), CH 2 Cl 2

1. Glycosylation of N-Boc serine derivatives with different galactosyl donors 1a-c.

Table III - 2 .

 III2 Glycosylation study of serine precursor derivative 57 with different galactosyl donors 1a, 1c and 1e.

			Ratio	Temp.		Yield a
	Entry	Donor	Donor:		Promoter		Product
				Time		(%)
			Acceptor
					SnCl 4 (5 eq.)/AgCO 2 CF 3 (2.5 eq.)
	7		1.5:1	0°C, 17 h		55	59, β only
					CH 2 Cl 2
	8		1.5:1	-20 o C, 2 h	BF 3 •Et 2 O (0.2 eq.), CH 2 Cl 2	45	59, β only
	9		1.5:1	-20 o C, 2.5 h	BF 3 •Et 2 O (0.2 eq.), CH 2 Cl 2	66	66, β only

a. Isolated yield. b. Product was obtained as a mixture of the glycoside and the decomposed sugar donor. Yield was calculated based on the glycoside in the mixture.

  Functionalization of amino-acid containing mimetics through amide bond chemistry and CuAAC. For comparison with the triazole linkage, amide-bond chemistry was applied to the intermediate amine 65 for the coupling to afford terminal methyl, phenyl, acetoxymethyl or Boc protected amine, compounds 67-70 (Scheme III-3). of the derivatives sequentially by 90% TFA in CH 2 Cl 2 /H 2 O for silyl ethers and Boc followed by hydrolysis with base were performed. The derivatives with free amino group (compound 65) or amide derivatives 67-70 provided the desired products with yields ranging from 52% to quantitative with exception of derivative 68 which gave 61% yield of the desired compound with 35% of a conjugated alkene byproduct 76 (Scheme III-4). nucleophilic Michael-addition byproducts on alkene intermediates were found as only products for these cases. Two different deprotection systems were applied for derivative 71.In the parallel reaction in which smoother conditions for the deacetylation were used (Et 3 N/H 2 O/MeOH), a pair of diastereoisomers 82 was obtained resulting from the addition of methanol to the alkene byproduct. In cases of derivatives 72-73 containing nucleophilic amino group of 72 and hydroxyl group of 73, the products were more complex. Possible intermolecular additions might occur resulting in oligomeric species or even intramolecular additions. Due to the small quantity of 72 and 73 and the uncertainty of the composition, these two products 83 and 84 were not tested in enzymatic assays.

	Deprotection After deprotection of silyl ether by 90% TFA, the expected desilylated intermediate was
	Reagents and conditions: (a) equals to (g) of Scheme III-2; (b) Ac 2 O, Pyridine, r.t., 16 h, 21% for two steps; (c) isolated which was confirmed by 1 H proton spectra and HR-MS (m/z, [M+H] + = 789.2523,
	[M+Na]

Scheme III-2. Synthesis of amino acid containing mimetics through glycosylation and Staudinger-Villarasa ligation. Reagents and conditions: (a) K 2 CO 3 , CuSO 4 •5H 2 O, MeOH, r.t., 5 h, 61%; (b) LiOH, THF/H 2 O, r.t., 2h, quant.; (c) SnCl 4 (5 eq.)/ CF 3 CO 2 Ag (2.5eq.), CH 2 Cl 2 , 0 o C, 17h, 55% for 59; BF 3 •Et 2 O, CH 2 Cl 2 , -20 o C, 2h, 61% for 62; (d) NaI, Pyridine, 120 o C, 5h; (e) DCC, HOBt, DMAP, THF, r.t., 16h, 30% for 63; (f) TBDMSCl, imidazole, pyridine, r.t., 40 h, 93%; (g) H 2 , Pd/C, MeOH, r.t., 4h, 85% for 61 or PPh 3 , THF/H 2 O, r.t., 16h for access of 67 in Scheme III-3; (h) 90% TFA, CH 2 Cl 2 /H 2 O, quant. Scheme III-3. benzoyl chloride, DIPEA, CH 2 Cl 2 , r.t., 16 h, 82% for two steps; (d) Boc-Gly-OSu, DCC, CH 2 Cl 2 , r.t., 16 h, 58% for two steps; (e) acetoxyacetyl chloride, DIPEA, CH 2 Cl 2 , r.t., 16 h, 43% for two steps; (f) phenylacetylene, DIPEA, DMF, microwave, 100 o C, 15 min, quant.; (g) N-Boc-propargylamine, DIPEA, DMF, microwave, 100 o C, 15 min, 58%; (h) propargyl acetate, CuI, DIPEA, DMF, microwave, 100 o C, 15 min, 56%. The azido 62 was functionalized by CuAAC with terminal phenyl, acetoxymethyl or Boc protected amine, compounds 71-73 (Scheme III-3). Scheme III-4. Deprotection of amino-acid containing UDP-Gal mimetics. Reagents and conditions: a. 1. 90% TFA, CH 2 Cl 2 /H 2 O, r.t., 16 h; 2. LiOH, THF/H 2 O, r.t., 2 h, 34% -quantative for two steps.

In comparison (Scheme III-5), the alkene byproduct 79 was obtained quantitatively from azido derivative 62. Furthermore, for derivatives containing a triazole moiety (compounds 71-73), + = 811.2355). Then, the product was divided into two fractions. In the first one, LiOH/H 2 O/THF was employed for deacetylation. The amount of LiOH was fixed at 1.2 eq. for each acetate group and the pH of the reaction medium was adjusted to 9~10. A pair of diastereoisomers 81 was obtained resulting from an addition of H 2 O at the alkene byproduct. Scheme III-5. Deprotection of amino-acid containing mimetics. Reagents and conditions: (a) 1. 90% TFA, CH 2 Cl 2 /H 2 O, r.t., 16 h; 2. LiOH, THF/H 2 O, r.t., 2 h or Et 3 N, MeOH/H 2 O, r.t., 24 h for 82, 75%-quant. for two or three steps.

Table III - 3 .

 III3 Deprotection of serine-based derivatives.

	Entry	Reactant	Product b	Yield a (%)
	10			quant.
	11			quant.
	12			56%
				61% for 75,
	13			35% for 76
	14			34%
	15			56%
				quant. for 81,
	16			75% for 82 c
	17			-/-
	18			-/-
	a.Isolated yields. b. Without further indication, the reaction conditions for deprotection are as follows: 1. 90%
	TFA, CH 2 Cl 2 /H 2 O, r.t., 16 h; 2. LiOH, THF/H 2 O, r.t., 2 h; c. The reaction conditions for deprotection are as
	follows: 1. 90% TFA, CH 2 Cl 2 /H 2 O, r.t., 16 h; 2. LiOH, THF/H 2 O, r.t., 2 h for 81 or Et 3 N, MeOH/H 2 O, r.t., 24 h
	for 82.
	Table III-4. Summary of deprotection of serine-based derivatives.
	Compound
	Elimination	√	√	< 36%
	Michael addition	×	√	×

Table IV - 3 .

 IV3 Determination of the inhibition mechanism for the two best inhibitors towards five GalTs.

	Entry	Inhibitor	7-Gal		19
		Concentration	Low conc.	High conc.	Low conc.	High conc.
	1	β-1,4-GalT	noncompetitive dominated a	mixed	noncompetitive dominated b	mixed
	2	GTB	competitive dominated e	mixed	competitive dominated c	mixed
	3	α-1,4-GalT	mixed	mixed	mixed	mixed
	4	α-1,3-GalT	mixed	mixed	competitive dominated d	mixed
	5	AAGlyB	competitive dominated a	mixed	competitive dominated c	mixed
	a. conc. threshold = 300 μM; b. conc. threshold = 400 μM; c. conc. threshold = 600 μM;
	d. conc. threshold = 500 μM; e. conc. threshold = 200 μM.

75, 77, 78, 80 and 95) were

  very weak inhibitors of all of the enzymes except for AAGlyB where 38% inhibition was observed for compound 80. The vinyl azide UDP analogue 79 gave inhibitions from 15% to 66% against the five GalT tested while the corresponding diastereomers 64 with addition of water displayed less inhibition. Substitution of the azido group of compound 64 by a carboxylic acid group in the UDP analogue 93 exhibited better inhibition. Heterocyclic modification on the linker of derivatives 81 and 82 provided more potent inhibitors than the parent compound 64. The methoxy compound 82 was relatively weaker with 0-24% inhibition in comparison with the hydroxyl compound 81 which inhibited the five enzymes between 21-47%.

).

Surprisingly, almost all of the glycosides (compounds 74,

Table IV - 2 .

 IV2 Enzymatic evaluation of neutral analogues containing amino-acid derivatives as pyrophosphate surrogate.

Table IV - 3 .

 IV3 IC 50 measurements of neutral analogues containing pyridine derivatives as pyrophosphate surrogates.

	Inhibitor	IC 50 (μM)
	35-Glc	336
	45	708
	43-Glc	1608

  All chemicals were obtained from commercial sources and used without further purification. Dichloromethane, acetonitrile and methanol were distilled over CaH 2 , CaH 2 and Mg/I 2 , respectively. All reactions were performed under an argon atmosphere. Thin-layer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F 254 (Merck). TLC plates were inspected by UV light (λ = 254 nm) and developed by treatment with a mixture of 10% H 2 SO 4 in EtOH/H 2 O (1:1 v/v) followed by heating. Silica gel column chromatography was performed with silica gel Si 60(40-63 μm). NMR spectra were recorded at 293 K, unless otherwise stated, using a Bruker 400 or 500 MHz spectrometer. Chemical shifts are referenced relative to deuterated solvent residual peaks. The following abbreviations are used to explain the observed multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet and bd, broad doublet. Complete signal assignments were based on 1D and 2D NMR (COSY, HSQC and HMBC correlations). High resolution (HR-ESI-QToF) mass spectra were recorded using a Bruker MicroToF-Q II XL spectrometer. Optical rotations were measured using a Perkin Elmer polarimeter and values are given in 10 -1 deg.cm 2 .g -1 .

V.1 General methods.

  Pyr ), 7.53 (d, J = 7.8 Hz, 1H, H Pyr ), 4.86 (d, J = 5.3 Hz, 2H, OCH 2a ), 4.00 (s, 3H, OMe), 3.41 (t, J = 5.3 Hz, 1H, OH).

	Methyl 6-(trimethylsilyloxymethyl)picolinate (2b)
	Product isolated from glycosylation (Entry 11, see page 108). 1 H NMR (400 MHz, CDCl 3 ), δ
	(ppm): 7.99 (d, J = 7.7 Hz, 1H, H Pyr ), 7.84 (t, J = 7.7 Hz, 1H, H Pyr ), 7.72 (d, J = 7.7 Hz, 1H,
	H Pyr ), 4.89 (s, 2H, OCH 2 ), 3.98 (s, 3H, OMe), 0.17 (s, 9H, SiMe 3 ); 13 C NMR (100 MHz,
	CDCl 3 ), δ (ppm): 166.0 (C=O), 162.0 (C Pyr ), 147.1 (C Pyr ), 137.7 (C PyrH ), 123.61 (C PyrH ), 123.55
	(C PyrH ), 65.5 (OCH 2 ), 53.1 (OMe), -0.5 (s, 3C, SiMe 3 ); HRMS m/z: calcd. for C 11 H 18 NO 3 Si,
	[M+H] + 240.1050, found 240.1048; for C 11 H 17 NNaO 3 Si, [M+Na] + 262.0870, found 262.0864.
	Methyl 3-hydroxymethylbenzoate (2d) 231

). R f = 0.16 (EtOAc:PE = 1:1); 1 H NMR (300 MHz, CDCl 3 ), δ (ppm): 8.04

(d, J = 7.8 Hz, 1H

, H Pyr ), 7.85

(t, J = 7.8 Hz,

1H, H Methyl 6-(hydroxymethyl)picolinate N-oxide (2c)

Methyl 6-(hydroxymethyl)

picolinate (2a) (200 mg, 1.2 mmol, 1.0 eq.) was dissolved in chloroform (4 mL) at 0°C. Then, meta-chloroperoxybenzoic acid (340 mg, 1.4 mmol, 1.2 eq.) was added. The reaction mixture was allowed to warm to r.t. and stirred overnight. The reaction was quenched with satd aq. Na 2 S 2 O 3 solution (10 mL) and the mixture was stirred for 2 h. Then, the mixture was extracted with CHCl 3 (11×20 mL). The organic phases were combined, dried (MgSO 4 ), filtered and concentrated to dryness. The resulting crude residue was purified by silica gel column chromatography (MeOH:CH 2 Cl 2 :CH 3 COOH, 4:95.5:0.5) to afford compound 2c as white needles (196 mg, 1.07 mmol, 89%). R f = 0.29 (MeOH:CH 2 Cl 2 :CH 3 COOH = 4:95.5:0.5); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 7.58 (dd, J = 7.8, 2.0 Hz, 1H, H Py ), 7.48 (dd, J = 7.8, 2.0 Hz, 1H, H Py ), 7.35 (t, J = 7.8 Hz, 1H, H Py ), 4.82 (s, 2H, OCH 2 ), 4.01 (s, 3H, OMe); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 162.1 (C=O), 151.3 (C Pyr ), 141.8 (C Pyr ), 126.3 (C PyrH ), 126.0 (C PyrH ), 125.6 (C PyrH ), 61.4 (CH 2 OH), 53.6 (OMe); HRMS m/z: calcd. for C 8 H 9 NNaO 4 , [M+Na] + 206.0424, found 206.0424.

  ). The combined organic extracts were washed with diluted bleach (50 mL, original solution 9.6% bleach diluted 10 times), brine(50 mL) and dried (MgSO 4 ). The solvent was removed under vacuum to give an oil which contained a small amount of white precipitate. Diethyl ether(20 mL) was added and the solid was removed by filtration and washed with Et 2 O (2×10 mL). The filtrate was concentrated to yield pale yellow oil (4.48 g). The crude oil was then purified by silica gel column chromatography (PE:EA, 4:1 to 7:3) to afford compound 2d as colorless oil(3.77 g, 0.023 mmol, 85%). R

f = 0.17 (EtOAc:PE = 1:4); 1

H NMR (300 MHz, CDCl 3 ), δ (ppm): 8.00 (s, 1H, H Ar ), 7.93 (dd, J = 7.7, 1.4 Hz, 1H, H Ar ), 7.61 -7.51 (m, 1H, H Ar ), 7.40 (t, J = 7.7 Hz, 1H, H Ar ), 4.71 (s, 2H, OCH 2 ), 3.89 (s, 3H, OMe).

Table 2 -2

 2 picolinate (2a)(41 mg, 0.244 mmol, 1.0 eq.) and BF 3 •Et 2 O (62 μL, 0.488 mmol, 2.0 eq.) in freshly distilled CH 2 Cl 2(2 mL) at the presence of 4Å MS (200 mg -300 mg). The reaction mixture was stirred overnight at -20 o C. Triethylamine (0.2 mL, 1.4 mmol, 5.7 eq.) was injected to quench the reaction. The reaction mixture was filtered, the solid was washed with CH 2 Cl 2 and the organic phase was concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 3:2) to afford compound 3a as white foam (40 mg, 0.080 mmol, 32%).Boron trifluoride etherate (31 μL, 0.244 mmol, 2.0 eq.) was added dropwise at -20

	Distilled CH 2 Cl 2 (3 mL) was added into a Ar-flushed flask containing methyl
	6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.), donor 1c (148 mg, 0.30 mmol,
	1.25 eq.) and 4Å MS (200 mg -300 mg). The reaction mixture was cooled down to -20 o C. After
	30 min, BF 3 •Et 2 O (62 μL, 0.488 mmol, 2.0 eq.) was introduced via a syringe. After 4 h, Et 3 N
	(0.2 mL, 1.4 mmol, 5.7 eq.) was injected to quench the reaction. The reaction mixture was
	filtrated, the solid was washed with CH 2 Cl 2, and the combined organic phases were
	concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to

3:2)

to afford compound 3a as white foam

(42 mg, 0.085 mmol, 34%)

.

Entry 7, Table 2-2

A solution of donor 1c (180 mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2

(1 mL)

was added dropwise (within 60 min-syringe pump) at -20 o C to a stirred solution of methyl 6-(hydroxymethyl)o C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (61 mg, 0.366 mmol, 3.0 eq.) and donor 1c (60 mg, 0.122 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2

(3 mL)

. The reaction mixture was stirred at -20 o C for 1.5 h. Another portion of BF 3 •Et 2 O (31 μL, 0.244 mmol, 2.0 eq.) was added to the reaction at -20 o C. After 1 h, Et 3 N (0.2 mL, 1.4 mmol, 5.7 eq.) was injected to quench the reaction. The reaction mixture was filtered and the solid was washed with CH 2 Cl 2 . The organic phase was concentrated. The desired glycosylated product was about 5%, estimated by TLC.

Entry 10, Table 2-2

Boron trifluoride etherate (62 μL, 0.488 mmol, 2.0 eq.) was added dropwise at -20 o C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and donor 1c (362 mg, 0.732 mmol, 3.0 eq.) in freshly distilled CH 2 Cl 2

(3 mL)

. The reaction mixture was stirred at -20 o C for 2 h. Another portion of BF 3 •Et 2 O (31 μL, 0.244 mmol, 1.0 eq.) was added to the reaction mixture, and stirring was maintained at -20 o C for another 14 h. Triethylamine (0.2 mL, 1.4 mmol, 5.7 eq.) was injected to quench the reaction. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3a as white foam (51 mg, 0.103 mmol, 42%). (0.2 mL, 1.4 mmol, 5.7 eq.) was injected at -20°C to quench the reaction. The reaction mixture was filtered and the solid was washed with CH 2 Cl 2. The solvent was evaporated, then, the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 6:4) to afford compound 3a (~6 mg, 0.012 mmol, 5%). The main product was the silylated acceptor methyl 6-(trimethylsilyloxymethyl)picolinate (2b) (30 mg, 0.126 mmol, 51%).

Table 2 - 2

 22 To a solution of methyl 6-(hydroxymethyl)picolinate (2a)(41 mg, 0.244 mmol, 1.0 eq.) in CH 2 Cl 2 (2 mL) was added PhBF 2 (60 μL, 0.48 mmol, 2.0 eq.) at room temperature. The mixture was cooled to -78°C. A cooled solution (-78°C) of donor 1c (240 mg, 0.488 mmol, 2.0 eq.) in CH 2 Cl 2 (2 mL) was added into the reaction. The reaction was stirred at -78°C for 3 h then quenched with satd aq. NaHCO 3 solution (25 mL) and extracted with CH 2 Cl 2. The organic layers were washed with H 2 O and dried (MgSO 4 ). Cl 2(1.5 mL) was added into the reaction mixture. Then, the reaction was stirred at -20°C for 2 h, quenched with satd aq. NaHCO 3 solution (30 mL) and extracted with CH 2 Cl 2 (3×30

	The solvent was evaporated, the residue was

Table 2 - 2

 22 Hz, 1H, H 1 ), 4.16 -4.12 (m, 2H, H 6 ), 4.00 (s, 3H, OMe), 3.95 (dd, J = 6.4, 6.4 Hz, 1H, H 5 ), 2.16 (s, 3H, COCH 3 ), 2.05 (s, 6H, 2×COCH 3 ), 1.99 (s, 3H, COCH 3 );13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 170.6 (C=O), 170.4 (C=O), 170.2 (C=O), 169.7 (C=O), 165.7 (CO 2 Me), 158.3 (C Pyr ), 147.2 (C Pyr ), 138.0 (C PyrH ), 124.6 (C PyrH ), 124.2 (C PyrH ), 101.2 (C 1 ), 71.8 (OCH 2 ), 71.1 (C 5 ), 70.9 (C 3 ), 69.0 (C 2 ), 67.2 (C 4 ), 61.6 (C 6 ), 53.2 (OMe), 20.9 (COCH 3 ), 20.8 (COCH 3 ), 20.8 (COCH 3 ), 20.7 (COCH 3 ); HRMS m/z: calcd. for C 22 H 28 NO 12 , [M+H] + 498.1606, found 498.1604. Donor 1e (87 mg, 0.117 mmol, 1.3 eq.) was dissolved in freshly distilled CH 2 Cl 2

	(6-Methoxycarbonylpyrid-2-yl)methyl	2,3,4,6-tetra-O-benzoyl-β-D-galactopyranoside
	(3b)
	Entry 15,

); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 8.04 (d, J = 7.7 Hz, 1H, H Pyr ), 7.86 (t, J = 7.7 Hz, 1H, H Pyr ), 7.65 (d, J = 7.7 Hz, 1H, H Pyr ), 5.41 (bd, J = 3.3 Hz, 1H, H 4 ), 5.34 (dd, J = 10.5, 7.9 Hz, 1H, H 2 ), 5.15

(d, J = 13.8 Hz, 1H, OCH 2a ), 5.04 (dd, J = 10.5, 3.3 Hz, 1H, H 3 ), 4.86 (d, J = 13.8 Hz, 1H, OCH 2b ), 4.65 (d, J = 7.9

  ). .2, 101.6 (C 1 ), 72.0 (OCH 2 ), 71.7 (C 5 ), 71.6 (C 3 ), 69.9 (C 2 ), 68.2 (C 4 ), 62.2 (C 6 ), 53.2 (OMe); HRMS m/z: calcd. for C 42 H 36 NO 12 , [M+H] + 746.2232, found 746.2221.

	R f = 0.21 (PE:EtOAc = 2:1); [α] D 20 = +10.0 (c = 0.8, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ
	(ppm) = 8.17 -7.19 (m, 23H, H Ar ), 6.05 -5.98 (m, 1H, H 4 ), 5.92 (dd, J = 8.4 Hz, 1H, H 2 ), 5.68
	-5.59 (m, 1H, H 3 ), 5.24 (d, J = 14.2 Hz, 1H, OCH 2a ), 5.07 -4.96 (m, 2H, H 1 , OCH 2b ), 4.64 (dd,

J = 10.4, 5.9 Hz, 1H, H 6a ),

4.44 (dd, J = 10.4, 4.8 Hz, 1H, H 6b ), 4.38

(bd, J = 5.9 Hz, 1H, H 5 ), 3.99 (d, J = 1.5 Hz, 3H, OMe);

13

C NMR (100 MHz, CDCl 3 ), δ (ppm) = 166.2 (C=O), 165.7 (C=O), 165.4 (C=O), 158.3 (C=O), 146.9 (C=O), 138.

1, 133.8, 133.6, 133.5 (s, 2C), 130.2 (s, 2C), 129.92 (s, 4C), 129.89 (s, 2C), 128.8 (s, 2C), 128.63 (s, 2C), 128.58 (s, 2C), 128.4 (s, 2C), 125.1, 124

  ).

	R f = 0.24 (PE:EtOAc = 3:1); [α] D 20 = +46.0 (c = 0.2, CH 7 Hz, 2H, 2× CH 2Ph ),
	4.13 (dd, J = 10.3, 3.1 Hz, 1H, H 2 ), 4.08 -4.00 (m, 3H, H 3 , H 4 , H 5 ), 3.99 (s, 3H, OMe), 3.62 -
	3.55 (m, 1H, H 6a ), 3.48 (dd, J = 9.1, 5.5 Hz, 1H, H 6b ); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm):
	165.9 (CO 2 Me), 159.2, 147.3, 138.84, 138.79, 138.7, 138.0 (C PyrH ), 137.8, 128.6 (s, 2C), 128.51
	(s, 2C), 128.49 (s, 2C), 128.36 (s, 2C), 128.27 (s, 2C), 128.1 (s, 2C), 128.0 (s, 2C), 127.9, 127.8,
	127.69, 127.65, 127.6 (s, 2C), 124.9 (C PyrH ), 124.0 (C PyrH ), 98.0 (C 1 ), 79.2 (C 3 or C 4 or C 5 ), 76.7
	(C 2 ), 75.0 (s, 2C, C 3 or C 4 or C 5 , OCH 2 ), 73.9 (OCH 2 ), 73.7 (OCH 2 ), 73.1 (OCH 2 ), 70.0
	(OCH 2 ), 69.8 (C 3 or C 4 or C 5 ), 68.7 (C 6 ), 53.1 (OMe); HRMS m/z: calcd. for C 42 H 44 NO 8 ,
	[M+H] + 690.3061, found 690.3052.

2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 8.01 (d, J = 7.4 Hz, 1H, H Pyr ), 7.79 (d, J = 7.7 Hz, 1H, H Pyr ), 7.71 (t, J = 7.7 Hz, 1H, H Pyr ), 7.44 -7.27 (m, 20H, H Ar ), 4.99 -4.73 (m, 7H, H 1 , OCH 2Pyr , 4×CH 2Ph ), 4.67

(d, J = 11.9 Hz, 1H, CH 2Ph ), 4.59 (d, J = 11.4 Hz, 1H, CH 2Ph ), 4.43 (dd, J = 14.0, 11.

  1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 8.36 (dd, J = 7.7, 1.1 Hz, 1H, H Pyr ), 8.15 (dd, J = 7.7, 1.1 Hz, 1H, H Pyr ), 8.06 (t, J = 7.7 Hz, 1H, H Pyr ), 5.44 -

	5.38 (m, 1H, H 4 ), 5.34 (dd, J = 10.5, 7.9 Hz, 1H, H 2 ), 5.19 -5.13 (m, 1H, OCH 2aPyr ), 5.10 -5.04
	(m, 1H, H 3 ), 4.87 (d, J = 16.4 Hz, 1H, OCH 2bPyr ), 4.69 (d, J = 7.9 Hz, 1H, H 1 ), 4.17 -4.12 (m,
	2H, H 6 ), 4.07 (s, 3H, OMe), 4.02 -3.93 (m, 1H, H 5 ), 2.19 -1.96 (m, 12H, COCH 3 ); HRMS m/z:
	calcd. for C 22 H 27 NNaO 13 , [M+H] + 536.1375, found 536.1350; MS m/z: [M+H] + 514.2,
	[M+Na] + 536.1.

  ).

	R f = 0.27 (PE:EtOAc = 2:1); [α] D 20 = -31.8 (c = 0.4, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 7.98 -7.90 (m, 2H, 2 H Ar ), 7.46 (d, J = 7.6 Hz, 1H, H Ar ), 7.40 (t, J = 7.6 Hz, 1H, H Ar ), 5.36 (bd, J = 3.4 Hz, 1H, H 4 ), 5.26 (dd, J = 10.4, 8.0 Hz, 1H, H 2 ), 4.97 (dd, J = 10.4, 3.4 Hz, 1H, H 3 ), 4.91 (d, J = 12.6 Hz, 1H, OCH 2aPh ), 4.65 (d, J = 12.6 Hz, 1H, OCH 2bPh ), 4.51 (d, J = 8.0 Hz, 1H, H 1 ), 4.21 -4.06 (m, 2H, H 6 ), 3.91 -3.85 (m, 4H, OMe, H 5 ), 2.13 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 1.95 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 170.5 (C=O), 170.3 (C=O), 170.2 (C=O), 169.6 (C=O), 166.8 (COOMe), 137.3 (C Ar ), glucopyranoside (3f) Entry 22, Table II-4 Methyl 6-(hydroxymethyl)picolinate (2a) (84 mg, 0.5 mmol, 1.0 eq.) and (1R)-(-)-camphorsulfonic acid (23.2 mg, 0.1 mmol, 0.2 eq.) were dissolved in freshly distilled CH 2 Cl 2 (3 mL) containing activated 4Å molecular sieves. The mixture was stirred at r.t. for 30 132.2 (C (6-Methoxycarbonylpyrid-2-yl)methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-min, after which donor 1g (186 mg, 0.56 mmol, 1.1 eq.) in CH 2 Cl 2 (1 mL) was added dropwise.

Ar-H ), 130.4 (C Ar ), 129.2 (C Ar-H ), 128.70 (C Ar-H ), 128.65 (C Ar-H ), 100.0 (C 1 ), 70.9 (C 3 ), 70.8 (C 5 ), 70.1 (OCH 2 ), 68.8 (C 2 ), 67.1 (C 4 ), 61.3 (C 6 ), 52.2 (OMe), 20.7 (s, 3C, 3×COCH 3 ), 20.6 (COCH 3 ); HRMS m/z: calcd. for C 23 H 28 NaO 12 , [M+Na] + 519.1473, found 519.1466.

-4

  C=O), 170.8 (C=O), 169.5 (C=O), 165.5 (C pyr ), 156.0 (C=O), 154.9 (C=O), 147.2 (C pyr ), 137.9 (C PyrH ), 124.6 (C PyrH ), 124.2 (C PyrH ), 101.5 (C 1 ), 95.6 (CCl 3 ), 74.5 (CH 2 CCl 3 ), 72.9 (C 3 ), 72.3 (C 5 ), 70.3 (OCH 2 ), 68.5 (C 4 ), 62.2 (C 6 ), 56.3 (C 2 ), 53.2 (OMe), 20.9 (COCH 3 ), 20.8 (COCH 3 ), 20.7 (COCH 3 ); HRMS m/z: calcd. for C 23 H 28 Cl 3 N 2 O 12 , [M+H] + 629.0702, found 629.0697.

	Boron trifluoride etherate (38 μL, 0.3 mmol, 1.0 eq.) was added dropwise at -20°C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (50 mg, 0.30 mmol, 1.0 eq.) and donor 1i (206 mg, 0.33 mmol, 1.1 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After 2 h, additional BF 3 •Et 2 O (38 μL, 0.3 mmol, 1.0 eq.) was added to the reaction. After another 1 h, Et 3 N (0.2 mL, 1.4 mmol, 4.7 eq.) was injected into the reaction mixture at -20°C to quench the reaction. Then, the solvent was evaporated and the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 1:1) to afford compound 3g as white foam (124 mg, 0.197 mmol, 66%). R f = 0.32 (PE:EtOAc = 1:1); [α] D 20 = -10.2 (c = 1.2, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 8.03 (d, J = 7.8 Hz, 1H, H pyr ), 7.82 (t, J = 7.8 Hz, 1H, H pyr ), 7.47 (d, J = 7.8 Hz, 1H, H pyr ), 6.34 (d, J = 9.1 Hz, 1H, NH), 5.25 -5.19 (m, 1H, H 3 ), 5.12 (t, J = 9.6, 9.6 Hz, 1H, H 4 ), deoxy-β-D-glucopyranoside (3h) Entry 27, Table II-4 Trimethylsilyl trifluoromethanesulfonate (46 μL, 0.244 mmol, 1.0 eq.) was added dropwise at -20°C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and 1,3,4,6-tetra-O-acetyl-2-allyloxycarbonylamino-2-deoxy-α-D-glucopyranose (1j) (158 5.06 -5.03 (m, 2H, H 1 , OCH (6-Methoxycarbonylpyrid-2-yl)methyl 3,4,6-tri-O-acetyl-2-allyloxycarbonylamino-2-mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2

2a ), 4.92

(d, J = 15.0 Hz, 1H, OCH 2b ), 4.70 -4.57 (m, 2H, CH 2

CCl 3 ), 4.26 (dd, J = 12.3, 4.9 Hz, 1H, H 6a ), 4.15 -4.12 (m, 1H, H 6b ), 4.02 (s, 3H, OMe), 3.94 (dd, J = 10.1, 9.1 Hz, 1H, H 2 ), 3.72 (ddd, J = 9.6, 4.9, 2.2 Hz, 1H, H 5 ), 2.03 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 );

13

C NMR (100 MHz, CDCl 3 ), δ (ppm): 123 170.9 (

  ).

	R f = 0.14 (PE:EtOAc = 1:1), 1 H NMR (300 MHz, CDCl 3 ), δ (ppm): 7.97 (d, J = 7.8 Hz, 1H,
	H Pyr ), 7.78 (t, J = 7.8 Hz, 1H, H Pyr ), 7.54 (d, J = 7.8 Hz, 1H, H Pyr ), 5.87 -5.71 (m, 1H,
	CH=CH 2 ), 5.60 (s, 1H, NH), 5.18 -4.79 (m, 7H, H 1 , H 3 , H 4 , OCH 2Pyr , C=CH 2 ), 4.46 (s, 2H,
	COOCH 2 ), 4.21 (dd, J = 12.3, 5.0 Hz, 1H, H 6a ), 4.06 (dd, J = 12.3, 2.0 Hz, 1H, H 6b ), 3.94 (s, 3H,
	OMe), 3.88 -3.75 (m, 1H, H 2 ), 3.68 (ddd, J = 9.7, 5.0, 2.0 Hz, 1H, H 5 ); HRMS m/z: calcd. for
	C 24 H 31 N 2 O 12 , [M+H] + 539.1872, found 539.1862.

Table II - 4

 II4 Trimethylsilyl trifluoromethanesulfonate(65 μL, 0.36 mmol, 2.0 eq.) was added dropwise at -20°C under argon to a stirred solution of donor 1l (103 mg, 0.28 mmol, 1.5 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (30 mg, 0.18 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 2.5 h, another portion of TMSOTf (65 μL, 0.36 mmol, 2.0 eq.) was added to the reaction and the reaction temperature was increased to r.t. After 16 h, Et 3N (0.2 mL, 1.4 mmol,

  3

(6-Methoxycarbonylpyrid-2-yl)methyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (3j) Entry 33, Table 2-5

  silica gel column chromatography (PE:EtOAc, 4:1 to 2:1) to afford compound 3i as a α:β 72:28 mixture of anomers(95 mg, 0.20 mmol, 82%). R f = 0.38 (PE:EtOAc = 1:1); α anomer[α] D[START_REF] Errey | Mechanistic insight into enzymatic glycosyl transfer with retention of configuration through analysis of glycomimetic inhibitors[END_REF] = +126.9 (c = 1.0, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 8.06 (d, J = 7.7 Hz, 1H, H pyr ), 7.91 (t, J = 7.7 Hz, 1H, H pyr ), 7.76 (d, J = 7.7 Hz, 1H, H pyr ), 5.53 (dd, J = 10.6, 9.3 Hz, 1H, H 3 ), 5.13 (d, J = 3.5 Hz, 1H, H 1 ), 5.10 -5.03 (m, 1H, H 4 ), 4.99 (d, J = 13.9 Hz, 1H, OCH 2aPyr ), 4.82 (d, J = 13.9 Hz, 1H, OCH 2bPyr ), 4.31 -4.24 CDCl 3 ), δ (ppm): 8.06 (d, J = 7.8 Hz, 1H, H pyr ), 7.89 (t, J = 7.8 Hz, 1H, H pyr ), 7.75 (d, J = 7.8 Hz, 1H, H pyr ), 5.17 (d, J = 13.9 Hz, 1H, OCH 2aPyr ), 5.03 -4.88 (m, 3H, H 3 , H 4 , OCH 2bPyr ), 4.55 (d, J = 8.1 Hz, 1H, H 1 ), 4.25 (dd, J = 12.3, 5.0 Hz, 1H, H 6a ), 4.08 (dd, J = 12.3, 1.9 Hz, 1H, H 6b ), 4.00 (s, 3H, OMe), 3.71 -3.65 (m, 1H, H 5 ), 3.65 -3.59 (m, 1H, H 2 ), 2.08 (s, 3H, COCH 3 ), 2.06 (s, 3H, COCH 3 ), 2.01 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 170.7 (C=O), 170.1 (C=O), 169.8 (C=O), 165.6 (C Pyr ), 157.8 (C=O), 147.3 (C Pyr ), 138.1 (C PyrH ), 124.9 (C PyrH ), 124.4 (C PyrH ), 101.5 (C 1 ), 72.6 (C 3 or C 4 ), 72.2 (s, 2C, OCH 2 , C 5 ), 68.4 (C 3 or C 4 ), 64.0 (C 2 ), 62.0 (C 6 ), 53.2 (OMe), 20.9 (COCH 3 ), 20.8 (COCH 3 ), 20.7 (COCH 3 ); HRMS m/z: calcd. for C 20 H 24 N 4 NaO 10 , [M+Na] + 503.1385, found 503.1362. Boron trifluoride etherate (31 μL, 0.244 mmol, 1.0 eq.) was added dropwise at -20°C under argon to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (41 mg, 0.244 mmol, 1.0 eq.) and donor 1n (180 mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2 (3 mL). After addition, the reaction mixture was stirred at -20°C. After 1 h, another 1eq. of BF 3 •Et 2 O (31 μL) was added. After another 2 h, another 1.0 eq. of BF 3 •Et 2 O (31 μL) was added. After 17 h, Et 3 N (0.2 mL, 1.4 mmol, 5.7 eq.) was added at -20°C into the reaction mixture to quench the reaction. 3H, COCH 3 ), 1.99 (s, 3H, COCH 3 ), 1.98 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 170.7 (C=O), 170.3 (C=O), 169.5 (C=O), 169.4 (C=O), 165.6 (C=O), 158.2 (C Pyr ), 147.2 (C Pyr ), 137.8 (C PyrH ), 124.6 (C PyrH ), 124.1 (C PyrH ), 100.6 (C 1 ), 72.7 (C 3 ), 72.0 (C 5 ), 71.8 (OCH 2Pyr ), 71.4 (C 2 or C 4 ), 68.4 (C 2 or C 4 ), 62.0 (C 6 ), 53.1 (OMe), 20.79 (COCH 3 ), 20.75 (COCH 3 ), 20.67 (COCH 3 ), 20.65 (COCH 3 ); HRMS m/z: calcd. for C 22 H 28 NO 12 , [M+H] + 498.1606, found 498.1604. Donor 1o (87 mg, 0.117 mmol, 1.3 eq.) was dissolved in freshly distilled CH 2 Cl 2 (5 mL) under argon at presence of 4Å MS (200 mg -300 mg). Then, the mixture was cooled down to -20°C. TMSOTf (3.3 μL, 0.018 mmol, 0.2 eq.) was added at -20°C, then, methyl 6-(hydroxymethyl)picolinate (2a) (15 mg, 0.09 mmol, 1.0 eq.) dissolved in freshly distilled CH 2 Cl 2 (1.5 mL) was added. The reaction mixture was stirred at -20°C for 30 min. Another 3.3 μL of TMSOTf was added. After 1 h, another portion of TMSOTf (3.3 μL, 0.018 mmol, 0.2 eq.

	(m, 1H, H 6a ), 4.08 (ddd, J = 8.3, 4.4, 2.1 Hz, 1H, H 5 ), 4.02 (dd, J = 12.4, 2.1 Hz, 1H, H 6b ), 3.47
	(dd, J = 10.6, 3.5 Hz, 1H, H 2 ), 2.08 (s, 3H, COCH 3 ), 2.07 (s, 3H, COCH 3 ), 2.01 (s, 3H,
	COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 170.7 (C=O), 170.2 (C=O), 169.7 (C=O),
	165.5 (C Pyr ), 157.4 (C=O), 147.4 (C Pyr ), 138.3 (C PyrH ), 124.8 (C PyrH ), 124.4 (C PyrH ), 97.7 (C 1 ),
	70.6 (s, 2C, C 3 , OCH 2 ), 68.5 (C 4 ), 68.1 (C 5 ), 61.8 (C 6 ), 61.1 (C 2 ), 53.1 (OMe), 20.8 (COCH 3 ), (6-Methoxycarbonylpyrid-2-yl)methyl 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranoside (3k)
	20.8 (COCH 3 ), 20.7 (COCH 3 ); β anomer [α] D 20 = -29.0 (c = 1.0, CH 2 Cl 2 ); 1 H NMR (400 MHz, Entry 34, Table 2-5
	The reaction mixture was concentrated and the residue was purified by silica gel column
	chromatography (PE:EtOAc, 9:1 to 5:5) to afford compound 3j as white foam (64 mg, 0.129
	mmol, 53%).
	R f = 0.26 (PE:EtOAc = 1:1); [α] D 20 (c = 0.9, CH 2 Cl 2 ) = -18.9; 1 H NMR (400 MHz, CDCl 3 ), δ

•Et 2 O (62 μL, 0.48 mmol, 2.0 eq.) was added to the reaction and the reaction temperature was increased to r.t. After 16 h, Et 3

N (0.2 mL, 1.4 mmol, 5.8 eq.)

was injected into the reaction mixture. Then, the solvent was evaporated. The residue 125 was purified by (ppm): 8.01 (d, J = 7.7 Hz, 1H, H Pyr ), 7.83 (t, J = 7.7 Hz, 1H, H Pyr ), 7.59 (d, J = 7.7 Hz, 1H, H Pyr ), 5.19 (dd, J = 9.4, 9.4 Hz, 1H, H 3 ), 5.14 -5.03 (m, 3H, H 2 , H 4, OCH 2aPyr ), 4.83 (d, J = 14.0 Hz, 1H, OCH 2bPyr ), 4.65 (d, J = 7.8 Hz, 1H, H 1 ), 4.21 (dd, J = 12.3, 4.9 Hz, 1H, H 6a ), 4.07 (dd, J = 12.3, 1.6 Hz, 1H, H 6b ), 3.96 (s, 3H, OMe), 3.74 -3.67 (m, 1H, H 5 ), 2.03 (s, 3H, COCH 3 ), 2.00 (s, )

was added. After 2.5 h, TMSOTf (6.6 μL, 0.039 mmol, 0.4 eq.) was added. Then, after 2 h, another 1.0 eq. TMSOTf (16.5 μL) was added and the reaction mixture was stirred at -20°C for another 16 h. Et 3 N (0.2 mL, 1.4 mmol, 15.5) was injected at -20°C to quench the reaction. The reaction mixture was filtered and the solid was washed with CH 2 Cl 2 . The solvent was evaporated, then, the residue was purified by silica gel column chromatography (PE:EtOAc, 9:1 to 7:3) to afford compound 3k as white foam (41 mg, 0.055 mmol, 61%) contaminated with 0.15 eq. of acceptor 2a. R f = 0.29 (PE:EtOAc = 2:1); 1 H NMR (400 MHz, CDCl 3 ), δ (ppm): 7.98 -7.15 (m, 23H, H Ar ), 5.85

(t, J = 9.7 Hz, 1H, H 3 ), 5.69 -5.55 (m, 2H, H 2

, H 4 ), 5.07

(d, J = 14.3 Hz, 1H, OCH 2aPyr ), 4.94 (d, J = 7.8 Hz, 1H, H 1 ), 4.87 (d, J = 14.3 Hz, 1H, OCH 2bPyr ), 4.55 (dd, J = 12.2, 2.9 Hz, 1H, H 6a ), 4.41 (dd, J = 12.2, 5.2 Hz, 1H, H 6b ), 4.16 -4.08 (m, 1H, H 5 ), 3.90 (s, 3H, OMe)

;

13

C NMR (100 MHz, CDCl 3 ), δ (ppm): 166.

  .1; HRMS m/z: calcd. for C 42 H 36 NO 12 , [M+H] + 746.2232, found 746.2221.

	(6-Methoxycarbonylpyrid-2-yl)methyl 2,3,4-tri-O-acetyl-β-L-fucopyranoside (3l)

Entry 35, Table 2-5

Boron trifluoride etherate (62 μL, 0.488 mmol, 2.0 eq.) was added dropwise at -20°C under argon into a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a)

(41 mg, 0.

244 mmol, 1.0 eq.) and donor 1p (159 mg, 0.366 mmol, 1.5 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 2 h, Et 3

  ). R f = 0.34 (PE:EtOAc = 1:1); [α] D 20 = +26.0 (c = 0.1, CH 2 Cl 2 ); 1 H NMR (500 MHz, CDCl 3 ), δ (ppm): 8.03 (d, J = 7.8 Hz, 1H, H Pyr ), 7.85 (t, J = 7.8 Hz, 1H, H Pyr ), 7.64 (d, J = 7.8 Hz, 1H, H Pyr ), 5.31 (dd, J = 10.4, 7.9 Hz, 1H, H 2 ), 5.24 (bd, J = 3.5 Hz, 1H, H 4 ), 5.14 (d, J = 14.1 Hz, 1H,

	OCH 2aPyr ), 5.03 (dd, J = 10.4, 3.5 Hz, 1H, H 3 ), 4.85 (d, J = 14.1 Hz, 1H, OCH 2bPyr ), 4.60 (d, J =
	7.9 Hz, 1H, H 1 ), 3.99 (s, 3H, OMe), 3.83 (bd, J = 6.3 Hz, 1H, H 5 ), 2.18 (s, 3H, COCH 3 ), 2.03 (s,
	3H, COCH 3 ), 1.98 (s, 3H, COCH 3 ), 1.21 (d, J = 6.3 Hz, 3H, H 6 ); 13 C NMR (125 MHz, CDCl 3 ),
	δ (ppm): 170.9 (C=O), 170.4 (C=O), 169.9 (C=O), 165.8 (C pyr ), 158.6 (COOMe), 147.3 (C Pyr ),
	138.0 (C PyrH ), 124.7 (C PyrH ), 124.2 (C PyrH ), 100.9 (C 1 ), 71.6 (OCH 2 ), 71.4 (C 3 ), 70.4 (C 4 ), 69.6
	(C 5 ), 69.2 (C 2 ), 53.2 (OMe), 21.1 (COCH 3 ), 20.93 (COCH 3 ), 20.86 (COCH 3 ), 16.2 (C 6 ); HRMS
	m/z: calcd. for C 20 H 25 NNaO 10 , [M+Na] + 462.1371, found 462.1352.

(6-Methoxycarbonylpyrid-2-yl)methyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranoside (3m) Entry 36, Table

2-5

To a solution of donor 1q (72 mg, 0.12 mmol, 1.3 eq.) in freshly distilled CH 2 Cl 2 (5 mL) under argon at -20 °C was added 4 Å MS (200 mg -300 mg) and TMSOTf (3.3 μL, 0.02 mmol, 0.2 eq.) and methyl 6-(hydroxymethyl)picolinate (2a) (15 mg, 0.09 mmol, 1.0 eq.) dissolved in CH 2 Cl 2

  ).

	R f = 0.27 (PE:EtOAc = 2:1); [α] D 20 = -44.5 (c = 1.2, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ
	(ppm): 8.25 -7.00 (m, 23H, H Ar ) 6.21 (dd, J = 10.2, 10.1 Hz, 1H, H 4 ), 6.01 (dd, J = 10.2, 3.3 Hz,
	1H, H 3 ), 5.86 (dd, J = 3.3, 1.8 Hz, 1H, H 2 ), 5.32 (d, J = 1.8 Hz, 1H, H 1 ), 5.15 (d, J = 13.7 Hz,
	1H, OCH 2aPyr ) and 4.95 (d, J = 13.7 Hz, 1H, OCH 2bPyr ), 4.70 (dd, J = 12.2, 2.5 Hz, 1H, H 6a ),
	4.51 (dd, J = 12.2, 3.8 Hz, 1H, H 6b ), 4.55 (ddd, J = 10.1, 3.8, 2.5 Hz, 1H, H 5 ), 4.02 (s, 3H,
	OMe); 13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 166.3 (C=O Bz ), 165.7 (C=O Bz ), 165.6 (CO 2 Me),
	165.5 (s, 2C, 2×C=O Bz ), 157.6 (C Pyr ), 147.6 (C Pyr ), 138.3 (C PyrH ), 133.

  Hz, 1H, OCH 2bPyr ), 4.69 (d, J = 12.1 Hz, 1H, OCH 2Ph ), 4.30 (dd, J = 10.0, 9.4 Hz, 1H, H 4 ), 4.22 (dd, J = 10.3, 4.9 Hz, 1H, H 6a ), 4.04 (dd, J = 10.0, 3.2 Hz, 1H, H 3 ), 4.00 (s, 3H, OMe), 3.93 (dd, J = 3.2, 1.6 Hz, 1H, H 2 ),3.89 (dd, J = 10.4, 10.3 Hz, 1H, H 6b ), 3.84 (ddd, J = 10.4, 9.4, 4.9 Hz, 1H, H 5 );13 C NMR: (150 MHz, CDCl 3 ), δ (ppm): 165.6 (CO 2 Me), 158.0 (C Pyr ), 147.5 (C Pyr ), 137.8 (C PyrH ), 138.

), δ (ppm): 8.05 (dd, J = 7.8, 1.0 Hz, 1H, H Pyr ), 7.82

(t, J = 7.8 Hz, 1H, H Pyr ), 7.44 (dd, J = 7.8, 1.0 Hz, 1H, H Pyr ), 7.55 -7.47 and 7.40 -7.25 (m, 15

H, H Ar ), 5.66 (s, 4,6-O-CHPh), 4.95 (d, J = 1.6 Hz, 1H, H 1 ), 4.90

(d, J = 13.7 Hz, 1H, OCH 2aPyr ), 4.87 (d, J = 12.1 Hz, 1H, OCH 2Ph ), 4.84 (d, J = 12.3 Hz, 1H, OCH 2Ph ), 4.75 (d, J = 12.3 Hz, 1H, OCH 2Ph ), 4.69 (d, J = 13.7

  J = 12.2 Hz, 1H, OCH 2Ph ), 4.90 (d, J = 12.2 Hz, 1H, OCH 2Ph ), 4.82 (d, J = 13.9 Hz, 1H, OCH 2bPyr ), 4.70 (d, J = 12.4 Hz, 1H, OCH 2Ph ), 4.59 (d, J = 12.4 Hz, 1H, OCH 2Ph ), 4.58 (bs, 1H, H 1 ), 4.24 (dd, J = 10.5, 4.8 Hz, 1H, H 6a ), 4.21 (dd, J = 9.9, 9.3 Hz, 1H, H 4 ), 4.00 (bd, J = 3.0 Hz, 1H, H 2 ), 3.95 (s, 3H, OMe), 3.88 (dd, J = 10.5, 10.1 Hz, 1H, H 6b ), 3.60 (dd, J = 9.9, 3.0 Hz, 1H, H 3 ),3.32 (ddd, J = 10.1, 9.3, 4.8 Hz, 1H, H 5 );13 C NMR (100 MHz, CDCl 3 ), δ (ppm): 165.6 (CO 2 Me), 158.6 (C Pyr ), 147.2 (C Pyr ), 137.8 (C PyrH ), 138.

	, 124.4 (C PyrH ) 124.1
	(C PyrH ), 101.4 (4,6-OCH Ph ), 99.1 (C 1 , 1 J C-1,H-1 = 171.5 Hz), 79.1 (C 4 ), 76.2 (s, 2C, C 2 , C 3 ), 73.7
	(OCH 2Ph ), 73.3 (OCH 2Ph ), 69.6 (OCH 2Pyr ), 68.8 (C 6 ), 64.6 (C 5 ), 53.0 (OMe). β-anomer: R f =
	0.38 (Hexane:EtOAc = 1:1); [α] D 20 = -111.1 (c = 1.8, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ
	(ppm): 8.00 (dd, J = 7.7, 1.0 Hz, 1H, H Pyr ), 7.79 (dd, J = 7.8, 7.7 Hz, 1H, H Pyr ), 7.56 (dd, J = 7.8,
	1.0 Hz, 1H, H Pyr ), 7.50 -7.20 (m, 15 H, H Ar ), 5.58 (s, 1H, 4,6-OCHPh), 5.10 (d, J = 13.9 Hz, 1H,
	OCH 2aPyr ), 4.97 (d,

Methoxycarbonylpyrid-2-yl)methyl 2,3,4,6,2',3',6'-hepta-O-benzoyl-β-D- lactopyranoside (3p) Entry 41, Table 2-5

  (C PyrH ), 124.0 (C PyrH ), 101.8 (C 1 , 1 J C-1,H-1 = 155.5Hz), 101.5(4,6-OCH Ph ), 78.7 (C 4 ), 78.0 (C 3 ), 76.1 (C 2 ), 75.0 (OCH 2Ph ), 72.6 (OCH 2Ph ), 72.0 (OCH 2Pyr ), 68.2 (C 6 ), 67.7 (C 5 ), 53.0 (OMe); HRMS m/z: calcd. for C 35 H 36 NO 8 , [M+H] + 598.2441, found 598.2432; C 35 H 35 NO 8 Na, [M+Na] + 620.2260, found 620.2264; C 35 H 35 NO 8 K, [M+K] + 636.2000, found 636.2042.Boron trifluoride etherate (38 μL, 0.30 mmol, 2.0 eq.) was added dropwise at -20°C to a stirred solution of methyl 6-(hydroxymethyl)picolinate (2a) (25 mg, 0.15 mmol, 1.0 eq.) and donor 1t (175 mg, 0.15 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (2 mL). After 4 h, Et 3 N (0.2 mL, 1.4 mmol, 9.3 eq.) was injected into the reaction mixture at -20°C. Then, the solvent was evaporated, the residue was purified by silica gel column chromatography (PE:EtOAc, 7:3 to EtOAc) to afford compound 3p as white foam (89 mg, 0.073 mmol, 51%).

	(6-R f = 0.19 (PE:EtOAc = 2:1); [α] D 20 = +28.1 (c = 4.3, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ), δ
	(ppm): 8.15 -6.89 (m, 38H), 5.74 (t, J = 9.5 Hz, 1H), 5.78 -5.60 (m, 2H), 5.51 (t, J = 8.9 Hz,
	1H), 5.31 (dd, J = 10.3, 2.9 Hz, 1H), 4.97 (d, J = 14.3 Hz, 1H, OCH 2aPyr ), 4.84 -4.71 (m, 3H,
	OCH 2bPyr , H

1 , H 1' ), 4.

52 -4.36 (m, 2H)

, 4.21

(t, J = 9.5, 9.5 Hz, 1H), 3.85 (s, 3H, OMe), 3.83 -

  ). (Pathway B) BF 3 •Et 2 O(20 μL, 0.164 mmol, 1.0 eq.) was added dropwise to a stirred solution of 6-(hydroxymethyl)-N-(2',3'-di-O-acetyl-uridin-5'-yl)picolinamide (10)(76 mg, 0.164 mmol, 1.0 eq.) and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate (1n-Glc) (121 mg, 0.246 mmol, 1.5 eq.) in freshly distilledCH 2 Cl 2 (3 mL) at -20 o C. After 1 h, the reaction temperature was increased to 0 o C. After another 1 h, another portion of BF 3 •Et 2 O (20 μL, 0.164 mmol, 1.0 eq.) was added into the reaction and 1 mL freshly distilled MeCN and 1 mL freshly distilled THF were added into the reaction for solubility reason. Then, the reaction mixture was stirred at r.t. for 16 h. Et 3N (0.2 mL, 1.44 mmol, 8.8 eq.) was injected to quench the reaction. Then, the reaction mixture was concentrated and the residue was purified by silica gel NH Uri ), 8.35 (t, J = 6.0 Hz, 1H, NHCO), 8.08(d, J = 7.7 Hz, 1H, H Pyr ), 7.86 (t, J = 7.7 Hz, 1H, H Pyr ), 7.53 (d, J = 7.7 Hz, 1H, H Pyr ), 7.33 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.79 (d, J = 4.4 Hz, 1H, H 1Rib ), 5.72 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.42 (dd, J = 6.2, 4.4 Hz, 1H, H 2Rib ), 5.33 (bd, J = 6.2 Hz, 1H, H

	column chromatography (PE:EtOAc = 9:1 to PE:EtOAc = 1:9) to afford compound 6-Glc as
	white foam (43 mg, 0.054 mmol, 33%).
	R f = 0.45(EtOAc); [α] D 20 = +2.7 (c = 1.1, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.17
	(s, 1H, 5Glc , H 6aGlc ,
	H 6bGlc ), 2.08 (s, 3H, COCH 3 ), 2.08 (s, 3H, COCH 3 ), 2.05 (s, 3H, COCH 3 ), 2.01 (s, 3H, COCH 3 ),
	2.01 (s, 3H, COCH 3 ), 2.00 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.8
	(COCH 3 ), 170.4 (COCH 3 ), 169.8 (COCH 3 ), 169.8 (COCH 3 ), 169.6 (COCH 3 ), 169.5 (COCH 3 ),
	164.8 (NHCO), 162.9 (C=O Uri ), 156.3 (C Pyr ), 150.1 (C=O Uri ), 148.6 (C Pyr ), 141.0 (C 6Uri ), 138.4
	(C PyrH ), 124.4 (C PyrH ), 121.5 (C PyrH ), 103.3 (C 5Uri ), 100.3 (C 1Glc ), 90.0 (C 1Rib ), 80.2 (C 4Rib ), 73.1
	(C 2Rib ), 72.7 (C 3Glc ), 72.0 (C 5Glc ), 71.4 (C 2Glc or C 4Glc ), 71.3 (OCH 2 ), 70.6 (C 3Rib ), 68.4 (C 2Glc or
	C 4Glc ), 61.8 (C 6Glc ), 40.3 (C 5Rib ), 20.85 (COCH 3 ), 20.79 (COCH 3 ), 20.73 (COCH 3 ), 20.70
	(COCH 3 ), 20.58 (COCH 3 ), 20.56 (COCH 3 ); HRMS m/z: calcd. for C 34 H 41 N 4 O 18 , [M+H] +
	793.2410, found 793.2390.

3Rib ), 5.25

(t, J = 9.5 Hz, 1H, H 3Glc ), 5.16 -5.08 (m, 2H, H 2Glc

, H 4Glc ), 4.97 (d, J = 13.6 Hz, 1H, OCH 2a ), 4.80 -4.70 (m, 2H, OCH 2b , H 1Glc ), 4.28 -4.34

(m, H 4Rib ), 4.25 (dd, J = 12.4, 4.3 Hz, 1H, H 5aRib ), 4.17 (dd, J = 12.4, 2.2 Hz, 1H, H 5bRib ), 3.91 -3.75 (m, 3H, H

  Pyr ), 150.1 (C=O Uri ), 148.6 (C Pyr ), 140.9 (C 6Uri ), 138.4 (C PyrH ), 124.4 (C PyrH ), 121.4 (C PyrH ), 103.3 (C 5Uri ), 100.9 (C 1Gal ), 89.8 (C 1Rib ), 80.2, 73.0, 71.2 (OCH 2 ), 70.9, 70.8, 70.6, 69.0, 67.1, 61.3 (C 6Gal ), 40.4 (C 5Rib ), 20.9 (COCH 3 ), 20.82 (COCH 3 ), 20.80 (COCH 3 ), 20.7 (COCH 3 ), 20.60 (COCH 3 ), 20.58 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 34 H 41 N 4 O 18 [M+H] + 793.2410, found 793.2405.

	). R f = 0.14 (PE:EtOAc, 1:4); [α] D 20
	= +3.7 (c = 0.9, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.03 (s, 1H, NH Uri ), 8.38 (t, J
	= 6.2 Hz, 1H, NHCO), 8.09 (d, J = 7.5 Hz, 1H, H Pyr ), 7.86 (t, J = 7.5 Hz, 1H, H Pyr ), 7.55 (d, J =
	7.5 Hz, 1H, H Pyr ), 7.34 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.81 (d, J = 4.4 Hz, 1H, H 1Rib ), 5.73 (dd, J =
	8.1, 2.0 Hz, 1H, H 5Uri ), 5.41 (dd, J = 6.3, 4.3 Hz, 2H), 5.36 -5.30 (m, 2H), 5.09 (dd, J = 10.5,
	3.4 Hz, 1H), 4.99 (d, J = 13.6 Hz, 1H, OCH 2a ), 4.77 (d, J = 13.6 Hz, 1H, OCH 2b ), 4.73 (d, J =
	8.0 Hz, 1H, H 1Gal ), 4.32 (dd, J = 10.2, 5.8 Hz, 1H, H 4Rib ), 4.22 -4.10 (m, 2H, H 6aGal , H 6bGal ),
	4.98 -4.02 (m, 1H, H 5Gal ), 3.92 -3.77 (m, 2H, H 5aRib , H 5bRib ), 2.16 (s, 3H, COCH 3 ), 2.09 (s, 3H,
	COCH 3 ), 2.08 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 ), 1.99 (s, 3H,
	COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.6 (COCH 3 ), 170.4 (COCH 3 ), 170.3
	(COCH 3 ), 169.84 (COCH 3 ), 169.83 (COCH 3 ), 169.76 (COCH 3 ), 164.9 (NHCO), 162.8
	(C=O Uri ), 156.4 (C

  J = 7.7 Hz, 1H, H Pyr ), 7.74 (d, J = 7.7 Hz, 1H, H Pyr ), 7.64 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.82 (d, J = 3.7 Hz, 1H, H 1Rib ), 5.70 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.04 (d, J = 13.1 Hz, 1H, OCH 2a ), 4.91 (d, J = 13.1 Hz, 1H, OCH 2b ), 4.63 (d, J = 7.9 Hz, 1H, H 1Glc ), 4.40 (bd, J = 4.2 Hz, H, H 2Rib ), 4.29 -4.21 (m, 2H, H 3Rib , H 4Rib ), 3.89 (dd, J = 12.3, 1.8 Hz, 1H, H 6aGlc ), 3.87 -3.77 (m, 2H, H 5aRib ,

	). [α] D 20 =
	+12.0 (c = 0.1, H 2 O); 1 H NMR (500 MHz, D 2 O) δ (ppm) = 8.03 (t, J = 7.7 Hz, 1H, H Pyr ), 7.97
	(d, H 5bRib ), 3.72 (dd, J = 12.3, 5.5 Hz, 1H, H 6bGlc ), 3.53 -3.47 (m, 1H, H 3Glc ), 3.47 -3.36 (m, 3H,
	H 2Glc , H 4Glc , H 5Glc ); 13 C NMR (125 MHz, D 2 O) δ (ppm) = 167.2 (CONH), 166.0 (C=O Uri ), 156.2
	(C Pyr ), 151.4 (C=O Uri ), 148.3 (C Pyr ), 142.0 (C 6Uri ), 139.1 (C PyrH ), 125.9 (C PyrH ), 121.6 (C PyrH ),
	102.1 (C 5Uri ), 102.0 (C 1Glc ), 90.5 (C 1Rib ), 82.0 (C 4Rib ), 76.0 (C 4Glc or C 5Glc ), 75.8 (C 3Glc ), 73.13
	(C 2Glc or C

2Rib ),

73.10 (C 2Glc

or C 2Rib ), 71.4 (OCH 2 ), 70.5 (C 3Rib ), 69.6 (C 4Glc or C 5Glc ), 60.7 (C 6Glc ), 40.3 (C 5Rib ); HRMS m/z: calcd. for C 22 H 28 N 4 NaO 12 , [M+Na] + 563.1596, found 563.1591.

  1, H 2 O); 1 H NMR (400 MHz, D 2 O) δ (ppm) = 7.96 (t, J = 7.7 Hz, 1H, H Pyr ), 7.88 (d, J = 7.7 Hz, 1H, H Pyr ), 7.69 (d, J = 7.7 Hz, 1H, H Pyr ), 7.58 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.73 (d, J = 3.6 Hz, 1H, H 1Rib ), 5.63 (d, J = 8.1 Hz, 1H, H 5Uri ), 4.99 (d, J = 13.3 Hz, 1H, OCH 2a ), 4.84 (d, J = 13.3 Hz, 1H, OCH 2b ), 4.50 (d, J = 6.7 Hz, 1H, H 1Gal ), 4.33 (dd, J = 3.9, 3.6 Hz, 1H, H 2Rib ), 4.18 (t, J = 4.3 Hz, 2H), 3.89 (s, 1H), 3.80 -3.57 (m, 7H); 13 C NMR (100 MHz, D 2 O) δ (ppm) = 166.9 (NHCO), 166.2 (C=O Uri ), 156.6 (C Pyr ), 151.6 (C=O Uri ), 148.1 (C Pyr ), 142.4 (C 6Uri ), 139.9 (C PyrH ), 126.2 (C PyrH ), 121.9 (C PyrH ), 103.0 (C 5Uri), 102.9 (C 1Gal ), 90.9(C 1Rib ), 82.3, 75.6, 73.5 (C 2Rib ), 73.1, 71.3 (OCH 2 ), 71.1, 70.9, 69.0, 61.3 (C 6Gal ), 40.8 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 22 H 29 N 4 O 12 [M+H] + 541.1776, found 541.1752.

	6-(Hydroxymethyl)picolinic acid (8) (Adapted from Ref. 186 )
	A solution of methyl 6-(hydroxymethyl)picolinate (2a) (220 mg, 1.3 mmol, 1.0 eq.) and satd
	aq. NaOH (0.2 mL) in MeOH (4 mL) was heated for 10 min by microwave irradiation at 100°C.
	Then, the reaction mixture was neutralized by DOWEX 50W×2 Resin (Fluka, 50-100 mesh,
	H + form), filtrated and concentrated to afford compound 8 as white solid (205 mg, 1.3 mmol,
	quant.). 1 H NMR (300 MHz, (CD 3 ) 2 SO) δ (ppm) = 7.85 (s, 2H, 2H Pyr ), 7.54 (s, 1H, H Pyr ), 4.60
	(s, 2H, OCH

2 ); ESI-MS (positive mode) m/z: [M+H] + 154.0.

  J = 7.7 Hz, 1H, H Pyr ), 7.78(t, J = 7.7 Hz, 1H, H Pyr ), 7.32 (d, J = 7.7 Hz, 1H, H Pyr ), 7.22 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.69 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.52 -5.45 (m, 2H, H 2Rib , H 3Rib ), 5.43 -5.35 (m, 1H, H 1Rib ), 4.78 (s, 2H, OCH 2a , OCH 2b ), 4.35 (dd, J = 6.9, 3.5 Hz, 1H, H 4Rib ), 3.96 (dd, J = 14.8, 6.9 Hz, 1H, H 5aRib ), 3.70 (dd, J = 14.8, 3.5 Hz, 1H, H 5bRib ), 2.06 (s, 3H, COCH 3 ), 2.04 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.2 (COCH 3 ), 169.8 (COCH 3 ), 165.2 (NHCO), 162.8 (C=O Uri ), 158.3 (C Pyr ), 151.0 (C=O Uri ), 148.6 (C Pyr ), 142.7 (C 6Uri ), 137.9 (C PyrH ), 123.2 (C PyrH ), 121.2 (C PyrH ), 103.1 (C 5Uri ), 94.2 (C 1Rib ), 79.8 (C 4Rib ), 74.0 (C 2Rib or C 3Rib ), 69.7 (C 2Rib or C 3Rib ), 64.1 (OCH 2 ), 39.9 (C 5Rib ), 20.6 (COCH 3 ), 20.4 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 20 H 23 N 4 O 9 [M+H] + 463.1460, found 463.1462.

	). R f = 0.51 (CH 2 Cl 2 :MeOH, 9:1); [α] D 20 = -41.3 (c = 0.7, CH 2 Cl 2 ); 1 H
	NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.68 (s, 1H, NH Uri ), 8.73 (dd, J = 6.9, 3.4 Hz, 1H, NHCO),
	8.04 (d,

  ). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 50% MeOH/H 2 O) to afford compound 10 as white foam (27 mg, 0.07 mmol, quant.). 1 H NMR (400 MHz, D 2 O) δ (ppm) = 7.98 (t, J = 7.6 Hz, 1H, H Pyr ), 7.91 (d, J = 7.6 Hz, 1H, H Pyr ), 7.62 (d, J = 7.6 Hz, 1H, H Pyr ), 7.53 (d, J = 7.8 Hz, 1H, H 6Uri ), 5.82 (d, J = 2.7 Hz, 1H, H 1 ), 5.59 (d, J = 7.8 Hz, 1H, H 5Uri ), 4.75 (s, 2H, CH 2 OH), 4.34 (dd, J = 4.0, 2.7 Hz, 1H, H 2 ), 4.26 -4.17 (m, 2H, H 3 , H 4 ), 3.85 -3.73 (m, 2H, H 5a , H 5b ); 13 C NMR (100 MHz, D 2 O) δ (ppm) = 181.9, 167.7, 159.4, 148.4, 141.6 (C Uri ), 139.4 (C Pyr ), 124.8 (C Pyr ), 121.5 (C Pyr ), 102.7 (C Uri ), 90.8 (C 1 ), 82.2 (C 3 or C 4 ), 73.6 (C 2 ), 70.9 (C 3 or C 4 ), 64.4 (CH 2 OH), 40.6 (C 5 ); HR-ESI-MS (positive mode) m/z: calcd. for C 16 H 18 N 4 O 7 [M+Na] + 401.1068, found 401.1057.

  ). The residue was purified by silica gel chromatography (60% EtOAc/PE to 100% EtOAc) to afford compound 11 as white foam (21 mg, quant.). R f = 0.18 (60% EtOAc/PE); [α] D 20 = +10.0 (c = 0.3, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.02 (d, J NHCOCH 3 ); 13 C NMR (101 MHz, CDCl 3 ) δ (ppm) = 171.1, 170.9, 170.6, 169.5, 165.6, 158.3, 147.0, 138.0 (C PyrH ), 124.8 (C PyrH ), 124.1 (C PyrH ), 101.3 (C 1Gal ), 73.2 (C 3Gal or C 4Gal ), 72.2 (C 5Gal ), 70.3 (OCH 2 ), 68.5 (C 4Gal or C 3Gal ), 62.2 (C 6Gal ), 54.2 (C 2Gal ), 53.1 (CO 2 CH 3 ), 23.2 (NHCOCH 3 ), 20.87 (COCH 3 ), 20.84 (COCH 3 ), 20.73 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd for C 22 H 29 N 2 O 11 , [M+H] + 497.1766, found 497.1756.

	= 7.8 Hz, 1H, H Pyr ), 7.83 (t, J = 7.8 Hz, 1H, H Pyr ), 7.51 (d, J = 7.8 Hz, 1H, H Pyr ), 6.60 -6.53 (m,
	1H, NHAc), 5.20 -5.00 (m, 3H, H 3Gal , H 4Gal , OCH 2a ), 4.96 -4.85 (m, 2H, H 1Gal , OCH 2b ), 4.26
	-4.16 (m, 2H, H 2Gal , H 6aGal ), 4.12 -4.06 (m, 1H, H 6bGal ), 4.00 (s, 3H, CO 2 Me), 3.69 (ddd, J =
	9.3, 4.6, 2.1 Hz, 1H, H 5Gal ), 2.06 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 ), 2.00 (s, 3H, COCH 3 ),
	1.87 (s, 3H, Methyl 6-(

  (OCH 2 ), 68.2 (C 2 ), 68.1 (C 4 ), 67.7 (C 3 ), 66.9 (C 5 ), 61.7 (C 6 ), 53.2 (OMe), 20.93 (COCH 3 ), 20.86 (COCH 3 ), 20.83 (COCH 3 ), 20.80 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 22 H 28 NO 12 [M+H] + 498.1606, found 498.1601.

	). R f = 0.43 (PE:EtOAc = 1:2); [α] D 20 = +54.5 (c = 0.4, CH 2 Cl 2 ); 1 H
	NMR (500 MHz, CDCl 3 ) δ (ppm) = 8.07 (d, J = 7.8 Hz, 1H, H pyr ), 7.89 (t, J = 7.8 Hz, 1H, H pyr ),
	7.67 (d, J = 7.8 Hz, 1H, H pyr ), 5.48 (dd, J = 3.4, 1.0 Hz, 1H, H 4 ), 5.45 (dd, J = 10.7, 3.4 Hz, 1H,
	H 3 ), 5.26 (d, J = 3.6 Hz, 1H, H 1 ), 5.22 (dd, J = 10.7, 3.6 Hz, 1H, H 2 ), 4.97 (d, J = 13.9 Hz, 1H,
	OCH 2a ), 4.77 (d, J = 13.9 Hz, 1H, OCH 2b ), 4.30 (dd, J = 6.8, 1.0 Hz, 1H, H 5 ), 4.15 -4.10 (m,
	1H, H

6a ),

4.06 (dd, J = 11.3, 6.8 Hz, 1H, H 6b

), 4.01 (s, 3H, OMe), 2.15 (s, 3H, COCH 3 ), 2.08 (s, 3H, COCH 3 ), 2.04 (s, 3H, COCH 3 ), 2.00 (s, 3H, COCH 3 ); 13 C NMR (125 MHz, CDCl 3 ) δ (ppm) = 170.6 (COCH 3 ), 170.38 (COCH 3 ), 170.37 (COCH 3 ), 170.26 (COCH 3 ), 165.7 (CO 2 Me), 158.1 (C pyr ), 147.6 (C pyr ), 138.0 (C pyrH ), 124.5 (C pyrH ), 124.4 (C pyrH ), 96.6 (C 1 ), 70.7

  Pyr ), 149.9 (C=OUri ), 149.3 (C Pyr ), 141.3 (C 6Uri ), 138.4 (C PyrH ), 125.2 (C PyrH ), 121.9 (C PyrH ), 103.2 (C 5Uri ), 96.6 (C 1Gal ), 90.7(C 1Rib ), 80.4, 73.2, 70.8 (OCH 2 ), 70.4, 68.3, 68.1, 67.5, 66.9, 61.7 (C 6Gal ), 40.3 (C 5Rib ), 20.84 (COCH 3 ), 20.82 (COCH 3 ), 20.80 (COCH 3 ), 20.79 (COCH 3 ), 20.62 (s, 2C, COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 34 H 41 N 4 O 18 [M+H] + 793.2410, found 793.2404; calcd. for C 34 H 40 N 4 NaO 18 [M+Na] + 815.2230, found 815.2223.

	).
	R f = 0.56 (EtOAc); [α] D 20 = +18.1 (c = 0.9, CH 2 Cl 2 ); 1 H NMR (500 MHz, CDCl 3 ) δ (ppm) =
	8.45 (s, 1H, NH Uri ), 8.40 (t, J = 6.1 Hz, 1H, NHCO), 8.16 -8.13 (m, 1H, H Pyr ), 7.89 (t, J = 7.8
	Hz, 1H, H Pyr ), 7.56 (dd, J = 7.8, 0.7 Hz, 1H, H Pyr ), 7.34 (d, J = 8.2 Hz, 1H, H 6Uri ), 5.75 -5.70
	(m, 2H, H 5Uri , H 1Rib ), 5.48 -5.38 (m, 5H), 5.16 (dd, J = 10.8, 3.7 Hz, 1H), 4.79 (d, J = 13.0 Hz,
	1H, OCH 2a ), 4.72 (d, J = 13.0 Hz, 1H, OCH 2b ), 4.36 -4.29 (m, 2H, H 4Rib , H 5Gal ), 4.10 -4.06 (m,
	1H, H

6aGal ), 4.00

(dd, J = 11.3, 6.6 Hz, 1H, H 6bGal ), 3.90 -3.85 (m, 2H, H 5aRib

, H 5bRib ), 2.15 (s, 3H, COCH 3 ), 2.11 (s, 3H, COCH 3 ), 2.09 (s, 3H, COCH 3 ), 2.01 (s, 3H, COCH 3 ), 2.00 (s, 3H, COCH 3 ), 1.98 (s, 3H, COCH 3 ); 13 C NMR (125 MHz, CDCl 3 ) δ (ppm) = 170.7 (C=O), 170.6 (C=O), 170.4 (C=O), 170.2 (C=O), 169.9 (C=O), 169.7 (C=O), 164.9 (NHCO), 162.5 (C=O Uri ), 156.1 (C

  ). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 50% MeOH/H 2 O) to afford compound 19 as white foam (13 mg, 0.024 mmol, 63%). [α] D 20 = +181 (c = 0.1, H 2 O); 1 H NMR (500 MHz, D 2 O) δ (ppm) = 8.05 (t, J = 7.7 Hz, 1H, H Pyr ), 7.99 (d, J = 7.7 Hz, 1H, H Pyr ), 7.77 (d, J = 7.7 Hz, 1H, H Pyr ), 7.65 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.83 (d, J = 3.7 Hz, 1H, H

	1
	(C 6Uri ), 139.1 (C PyrH ), 125.9 (C PyrH ), 121.5 (C PyrH ), 102.0 (C 5Uri ), 98.5 (C 1Gal ), 90.5 (C 1Rib ), 82.1,
	73.1, 71.2, 70.5, 69.8 (OCH 2 ), 69.4, 69.2, 68.3, 61.0 (C 6Gal ), 40.2 (C 5Rib ); HR-ESI-MS (positive
	mode) m/z: calcd. for C 22 H 28 N 4 NaO 12 [M+Na] + 563.1596, found 563.1581.

1Rib ), 5.69 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.13 (d, J = 3.7 Hz, 1H, H 1Gal ), 4.88 (d, J = 13.1 Hz, 1H, OCH 2a ), 4.82 (d, J = 13.1 Hz, 1H, OCH 2b ), 4.42 (dd, J = 4.1, 3.7 Hz, 1H, H 2Rib ), 4.29 -4.23 (m, 2H), 4.01 -3.77 (m, 6H), 3.71 -3.62 (m, 2H, H 6aGal , H 6bGal ); 13 C NMR (125 MHz, D 2 O) δ (ppm) = 167.3 (NHCO), 166.0 (C=O Uri ), 156.6 (C Pyr ), 151.4 (C=O Uri ), 148.3 (C Pyr ), 142.

  ArH ), 102.3 (C 1 ), 75.5 (C 2 or C 4 or C 5 ), 73.1 (C 2 or C 4 or C 5 ), 71.1 (C 2 or C 4 or C 5 ), 71.0 (OCH 2 ), 68.9 (C 3 ), 61.2 (C 6 ); HR-ESI-MS (positive mode) m/z: calcd. for C 14 H 18 NaO 8 [M+Na] + 337.0894, found 337.0894.

	). [α] D = -20.0 (c = 0.1, H 2 O); 1 H NMR (400 MHz, D 2 O)
	δ (ppm) = 7.89 (s, 1H, H Ar ), 7.81 (d, J = 7.7 Hz, 1H, H Ar ), 7.58 (d, J = 7.7 Hz, 1H, H Ar ), 7.40 (t,
	J = 7.7 Hz, 1H, H Ar ), 4.87 (d, J = 12.1 Hz, 1H, OCH 2a ), 4.68 (d, J = 12.1 Hz, 1H, OCH 2b ), 4.37

(d, J = 7.1 Hz, 1H, H 1 ), 3.86 (bd, J = 2.5 Hz, 1H, H 3 ), 3.78 -3.66 (m, 2H, H 6a

, H 6b ), 3.62 -3.48 (m, 3H, H 2 , H 4 , H 5 ); 13 C NMR (100 MHz, D 2 O) δ (ppm) = 170.6 (CO 2 H), 137.6 (C Ar ), 133.8 (C ArH ), 130.2 (C Ar ), 129.7 (s, 2C, 2 C ArH ), 129.2 (C

  t. for 16 h. The reaction mixture was diluted with water(30 mL); the aqueous phase was washed with CH 2 Cl 2 (2×30 mL). Then, the combined organic layer weas washed with H 2 O (30 mL). The combined aqueous phase was concentrated and dried with vacuum.

	The residue was purified by
	C18 reverse phase column chromatography (H 2 O to H 2 O:MeOH = 7:3) to afford compound 21
	as white foam (28 mg, 0.045 mmol, 43%). R f = 0.55 (MeOH:EtOAc, 4:6); 1 H NMR (500 MHz,
	D 2 O) δ (ppm) = 7.82 (s, 1H, H Ar ), 7.74 (d, J = 7.7 Hz, 1H, H Ar ), 7.66 (d, J = 7.7 Hz, 1H, H Ar ),
	7.63 (d, J = 8.1 Hz, 1H, H 6Uri ), 7.54 (t, J = 7.7 Hz, 1H, H Ar ), 5.85 (d, J = 3.6 Hz, 1H, H 1Rib ), 5.80
	(d, J = 8.1 Hz, 1H, H 5Uri ), 5.59 (dd, J = 6.6, 3.6 Hz, 1H, H 2Rib ), 5.43 (bd, J = 6.6 Hz, 1H, H 3Rib ),
	4.99 (d, J = 12.1 Hz, 1H, OCH 2a ), 4.81 (d, J = 12.1 Hz, 1H, OCH 2b ), 4.47 (d, J = 7.5 Hz, 1H,
	H 1Gal ), 4.46 -4.41 (m, 1H, H 4Rib ), 3.93 (bd, J = 3.3 Hz, 1H, H 4Gal ), 3.84 -3.74 (m, 4H, H 6aGal ,
	H 6bGal , H 5aRib , H 5bRib ), 3.68 (dd, J = 7.8, 4.5 Hz, 1H, H 5Gal ), 3.63 (dd, J = 9.8, 3.3 Hz, 1H, H 3Gal ),
	3.58 (dd, J = 9.8, 7.5 Hz, 1H, H 2Gal ), 2.16 (s, 3H, COCH 3 ), 2.13 (s, 3H, COCH 3 ); 13 C NMR (125
	MHz, D 2 O) δ (ppm) = 172.61 (COCH 3 ), 172.59 (COCH 3 ), 170.7 (CO 2 H), 166.0 (C=O Uri ),
	151.0 (C=O Uri ), 143.4 (C 6Uri ), 137.5 (C Ar ), 133.5 (C Ar ), 132.2 (C ArH ), 129.1 (C ArH ), 127.0
	(C ArH ), 126.9 (C ArH ), 102.3 (C 5Uri ), 102.0 (C 1Gal ), 90.7 (C 1Rib ), 79.5 (C 4Rib ), 75.2 (C 5Gal ), 73.1
	(C 2Rib ), 72.8 (C 3Gal ), 70.8 (s, 2C, C 2Gal , OCH 2 ), 70.6 (C 3Rib ), 68.6 (C 4Gal ), 61.0 (C 6Gal ), 39.9
	(C 5Rib ), 19.9 (COCH 3 ), 19.8 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for
	C 27 H 34 N 3 O 14 [M+H] + 624.2035, found 624.2037; calcd. for C 27 H 33 N 3 NaO 14 [M+Na] +
	646.1855, found 646.1848.

  mmol, 1.0 eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 30% MeOH/H 2 O) to afford compound 22 as white foam (17 mg, 0.032 mmol, 73%). [α] D = +8.0 (c = 0.2, H 2 O); 1 H NMR (500 MHz, D 2 O) δ (ppm) = 7.84 (s, 1H, H Ar ), 7.78 -7.74 (m, 1H, H Ar ), 7.69 -7.65 (m, 2H, H Ar , H 6Uri ), 7.56 (t, J = 7.7 Hz, 1H, H Ar ), 5.82 (d, J = 3.7 Hz, 1H, H 1Rib ), 5.77 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.01 (d, J = 12.0 Hz, 1H, OCH 2a ), 4.83 (d, J = 12.1 Hz, 1H, OCH 2b ), 4.48 (d, J = 7.6 Hz, 1H, H 1Gal ), 4.43 (dd, J = 4.7, 3.7 Hz, 1H, H 2Rib ), 4.27 -4.22 (m, 2H, H 3Rib , H 4Rib ), 3.95 -3.92 (m, 1H, H 4Gal ), 3.87 -3.75 (m, 4H, H 5aRib , H 5bRib , H 6aGal, H 6bGal ), 3.71 -3.67 (m, 1H, H 5Gal ), 3.63 (dd, J = 9.9, 3.3 Hz, 1H, H 3Gal ), 3.58 (dd, J = 9.9, 7.6 Hz, 1H, H 2Gal ); 13 C NMR (125 MHz, D 2 O) δ (ppm) = 170.9 (CONH), 166.1 (C=O Uri ), 151.4 (C=O Uri ), 142.2 (C 6Uri ), 137.5 (C Ar ), 133.6 (C Ar ), 132.2 (C ArH ), 129.1 (C ArH ), 127.0 (C ArH ), 126.9 (C ArH ), 102.1 (C 5Uri ), 101.9 (C 1Gal ), 90.9 (C 1Rib ), 81.9 (C 4Rib ), 75.2 (C 5Gal ), 73.1 (C 2Rib ), 72.8 (C 3Gal ), 70.8 (OCH 2 or C 2Gal ), 70.8 (OCH 2 or C 2Gal ), 70.5 (C 3Rib ), 68.6 (C 4Gal ), 61.0 (C 6Gal ), 40.6 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 23 H 29 N 3 NaO 12 [M+Na] + 562.1643, found 562.1648.

  = +27.6 (c = 1.6, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.25 (s, 1H, NHUri ), 8.20 -8.10 (m, 2H, CONH, H Pyr ), 7.72 (t, J = 7.7 Hz, 1H, H Pyr ), 7.62 (d, J = 7.7 Hz, 1H, H Pyr ), 7.35 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.84 (d, J = 4.5 Hz, 1H, H 1Rib ), 5.82 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.42 - 5.34 (m, 1H, H 2Rib ), 5.29 (dd, J = 6.1, 4.7 Hz, 1H, H 3Rib ), 4.32 (dd, J = 10.0, 4.7 Hz, 1H, H 4Rib ), 4.01 -3.87 (m, 1H, H 5aRib ), 3.77 (ddd, J = 14.4, 4.7 Hz, 1H, H 5bRib ), 2.10 (s, 3H, COCH 3 ), 2.07 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.1 (s, 2C, 2×COCH 3 ), 163.8 (NHCO) , 163.3 (C=O Uri ), 150.8 (C Pyr ), 150.5 (C=O Uri ), 141.1 (C Pyr ), 140.9 (C 6Uri ), 140.3 (C PyrH ), 131.5 (C PyrH ), 122.0 (C PyrH ), 104.0 (C 5Uri ), 89.8 (C 1Rib ), 80.6 (C 4Rib ), 73.3 (C 2Rib ), 71.0 (C 3Rib ), 40.7 (C 5Rib ), 20.9 (COCH 3 ), 20.8 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 19 H 20 BrN 4 O 8 [M+H] + 511.0459, found 511.0462.

). R f = 0.24 (PE:EtOAc, 1:3);

[α] D

  calcd. for C 15 H 15 BrN 4 NaO 6 [M+Na] + 449.0067, found 449.0046.

	). R f = 0.59 (MeOH:EtOAc = 1:4); [α] D
	= +10.2 (c = 0.4, DMSO); 1 H NMR (500 MHz, CD 3 OD) δ (ppm) = 8.10 (dd, J = 7.8, 0.9 Hz,
	1H, H Pyr ), 7.88 (t, J = 7.8 Hz, 1H, H Pyr ), 7.78 (dd, J = 7.8, 0.9 Hz, 1H, H Pyr ), 7.74 (d, J = 8.1 Hz,
	1H, H 6Uri ), 5.84 (d, J = 4.7 Hz, 1H, H 1Rib ), 5.73 (d, J = 8.1 Hz, 1H, H 5Uri ), 4.19 (dd, J = 5.4, 4.7
	Hz, 1H, H 2Rib ), 4.16 -4.11 (m, 1H, H 4Rib ), 4.08 (t, J = 5.4 Hz, 1H, H 3Rib ), 3.80 (dd, J = 14.1, 5.9
	Hz, 1H, H 5aRib ), 3.71 (dd, J = 14.1, 4.7 Hz, 1H, H 5bRib ); 13 C NMR (125 MHz, CD 3 OD) δ (ppm)
	= 166.1 (C=O Uri ), 165.6 (NHCO), 152.3 (C Pyr ), 152.1 (C=O Uri ), 142.8 (C Pyr ), 142.0 (C 6Uri ),
	141.6 (C PyrH ), 132.5 (C PyrH ), 122.6 (C PyrH ), 103.1 (C 5Uri ), 91.6 (C 1 ), 83.9 (C 4 ), 74.9 (C 2 ), 72.5
	(C 3 ), 42.1 (C

5 ); HR-ESI-MS (positive mode) m/z:

  7, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.41 (s, 1H, H NHUri ), 8.37 -8.29 (m, 1H, NHCO), 8.16 (d, J = 7.8 Hz, 1H, H Pyr ), 7.84 (t, J = 7.8 Hz, 1H, H Pyr ), 7.61 (d, J = 7.8 Hz, 1H, H Pyr ), 7.38 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.86 (d, J PyrH ), 130.4 (C PyrH ), 122.4 (C PyrH ), 103.7 (C 5Uri ), 89.2 (C 1Rib ), 82.3 (C≡CH), 80.3 (C 4Rib ), 78.5 (C≡C), 73.0 (C 2Rib ), 70.6 (C 3Rib ), 40.2 (C 5Rib ), 20.6 (COCH 3 ), 20.5 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 21 H 21 N 4 O 8 [M+H] + 457.1354, found 457.1363.

	= 4.5 Hz, 1H, H 1Rib ), 5.81 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.39 -5.33 (m, 1H, H 2Rib ), 5.31 -5.26 (m,
	1H, H 3Rib ), 4.31 (dd, J = 10.0, 5.1 Hz, 1H, H 4Rib ), 4.01 -3.90 (m , 1H, H 5aRib ), 3.74 (dd, J = 14.4,
	4.6 Hz, 1H, H 5bRib ), 3.28 (s, 1H, C≡CH), 2.09 (s, 3H, COCH 3 ), 2.06 (s, 3H, COCH 3 ); 13 C NMR
	(100 MHz, CDCl 3 ) δ (ppm) = 169.8, 164.2, 163.1, 150.3, 149.7, 141.0, 140.6 (C 6Uri ), 138.0
	(C

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl azide (28-Gal)

235

  in tBuOH/H 2 O (4 mL/200 μL) were added CuSO 4 (100 μL, 0.1 M in H 2 O, 0. 01 mmol, 0.1 eq.), sodium ascorbate (200 μL, 0.1 M in H 2 O, 0.02 mmol, 0.2 eq.) and DIPEA (0.1 mL, to adjust pH value around 8). Then, the reaction mixture was stirred at 38°C. After 3 h, the reaction mixture was concentrated and the residue was purified by silica gel column chromatography (60% EtOAc/PE to 10% MeOH/EtOAc) to afford compound 29-GlcNAc as white foam (41 mg, 0.05 mmol, 50%). R f = 0.10 (EtOAc); [α] D

	= -19.3 (c = 0.7, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.90 (s, 1H, H 3Uri ), 8.63 (t, J
	= 6.0 Hz, 1H, CONH), 8.59 (s, 1H, H triazole ), 8.18 (d, J = 7.8 Hz, 1H, H Pyr ), 8.12 (d, J = 7.8 Hz,
	1H, H Pyr ), 7.91 (t, J = 7.8 Hz, 1H, H Pyr ), 7.36 (d, J = 8.1 Hz, 1H, H 6Uri ), 7.08 (d, J = 8.9 Hz, 1H,
	NHAc), 6.31 (d, J = 9.8 Hz, 1H, H 1GlcNAc ), 5.74 (d, J = 4.7 Hz, 1H, H 1Rib ), 5.65 -5.55 (m, 2H,
	H 5Uri , H 3GlcNAc ), 5.51 (dd, J = 5.7, 4.7 Hz, 1H, H 2Rib ), 5.43 (t, J = 5.7 Hz, 1H, H 3Rib ), 5.27 (t, J =
	9.6 Hz, 1H, H 4GlcNAc ), 4.60 (dd, J = 10.6, 9.8 Hz, 1H, H 2GlcNAc ), 4.41 -4.27 (m, 2H, H 4Rib,
	H 6aGlcNAc ), 4.19 -4.11 (m, 2H, H 5GlcNAc, H 6bGlcNAc ), 4.02 (dd, J = 14.3, 5.6 Hz, 1H, H 5aRib ), 3.79
	(dd, J = 14.3, 4.4 Hz, 1H, H 5bRib ), 2.13 -1.99 (m, 15H, 5×COCH 3 ), 1.76 (s, 3H, NHAc); 13 C
	NMR (100 MHz, CDCl 3 ) δ (ppm) = 171.6 (COCH 3 ), 170.9 (COCH 3 ), 170.8 (COCH 3 ), 170.0
	(COCH 3 ), 169.8 (COCH 3 ), 169.7 (COCH 3 ), 165.

  ), 138.6 (C PyrH ), 123.3 (C PyrH ), 122.0 (s, 2C, C PyrH , C triazoleH ), 103.2 (C 5Uri ), 90.5 (C 1Rib ), 86.0 (C 1GlcNAc ), 80.4 (C 4Rib ), 75.0 (C 5GlcNAc ), 72.7 (C 2Rib ), 72.3 (C 3GlcNAc ), 70.6 (C 3Rib ), 68.3 (C 4GlcNAc ), 62.1 (C 6GlcNAc ), 54.1 (C 2GlcNAc ), 40.2 (C 5Rib ), 22.85 (COCH 3 ), 20.82 (COCH 3 ), 20.75 (COCH 3 ), 20.74 (COCH 3 ), 20.60 (COCH 3 ), 20.55 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 35 H 41 N 8 O 16 [M+H] + 829.2635, found 829.2610.

galactopyranosyl-1H-1,2,3-triazol-4-yl)-N-(uridin-5'-yl)picolinamide (30-Gal)

  C=O Uri ), 148.1 (C Pyr ), 147.5 (C Pyr ), 146.9 (C triazole ), 141.2 (C 6Uri ), 139.4 (C PyrH ), 123.7 (C triazoleH ), 123.5 (C PyrH ), 121.9 (C PyrH ), 102.7 (C 5Uri ), 90.9 (C 1Rib ), 88.7 (C 1Gal ),82.0, 78.8, 73.6, 73.4, 71.0, 70.1, 69.0, 61.4 (C 6Gal ), 40.3 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 23 H 27 N 7 NaO 11 [M+Na] + 600.1661, found 600.1658.

	6-(1-β-D-Prepared according to general protocol E from compound 29-Gal (42 mg, 0.05 mmol, 1.0 eq.).
	The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 50%
	MeOH/H 2 O) to afford compound 30-Gal as white foam (23 mg, 0.04 mmol, 80%). [α] D = +98.5
	(c = 0.2, H 2 O); 1 H NMR (400 MHz, D 2 O) δ = 8.59 (s, 1H, H triazole ), 7.81 -7.65 (m, 3H, H Pyr ),
	7.34 (d, J = 7.7 Hz, 1H, H 6Uri ), 5.77 (d, J = 2.5 Hz, 1H, H 1Rib ), 5.70 (d, J = 9.1 Hz, 1H, H 1Gal ),
	5.31 (d, J = 7.7 Hz, 1H, H 5Uri ), 4.30 -4.21 (m, 2H), 4.19 -4.01 (m, 4H), 3.93 -3.71 (m, 4H),
	3.65 -3.56 (m, 1H, H 5bUri ); 13 C NMR (100 MHz, D 2 O) δ = 172.12 (C=O Uri ), 166.3 (NHCO),
	156.1 (
	); HR-ESI-MS
	(positive mode) m/z: calcd. for C 23 H 27 N 7 NaO 11 [M+Na] + 600.1661, found 600.1640.

  t. for 16 h. The reaction mixture was diluted with water (150mL), extracted with EtOAc (3×200 mL), the combined organic layers were washed with satd Na 2 CO 3 solution (3×100 mL), H 2 O (100 mL) and brine (100 mL). The organic phase was dried (Na 2 SO 4 ), concentrated and dried with vacuum. The residue was purified by silica gel column chromatography (PE to PE:EtOAc = 1:1) to afford compound 31-Glc as yellow foam (552 mg, 1.04 mmol, 78%). R f = 0.45 (PE:EtOAc, 1:1); [α] D = -31.1 (c = 2.0, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ = 8.46 (d, J = 9.7 Hz, 1H, NHCO), 8.12 (dd, J = 7.7, 0.8 Hz, 1H, H Pyr ), 7.71 (t, J = 7.7 Hz, 1H, H Pyr ), 7.64 (dd, J = 7.9, 0.8 Hz, 1H, H Pyr ), 5.44 (dd, J = 9.7, 9.6 Hz, 1H, H 1Glc ), 5.35 (t, J = 9.5 Hz, 1H, H 3Glc ), 5.19 -5.09 (m, 2H, H 2Glc , H 4Glc ), 4.30 (dd, J = 12.5, 4.3

	Hz, 1H, H 6aGlc ), 4.14 -4.04 (m, 1H, H 6bGlc ), 3.88 (ddd, J = 10.1, 4.3, 2.1 Hz, 1H, H 5Glc ), 2.07 (s,
	3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 ), 1.97 (s, 3H, COCH 3 ); 13 C NMR (100
	MHz, CDCl 3 ) δ (ppm) = 170.

  PyrH ), 78.8 (C 1 ), 72.6 (C 5 ), 71.2 (C 3 ), 68.3 (C 2 ), 67.3 (C 4 ), 61.4 (C 6 ), 20.80 (s, 2C, COCH 3 ), 20.74 (COCH 3 ), 20.69 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 20 H 24 BrN 2 O 10 [M+H] + 531.0609, found 531.0610.

	1 H NMR (400 MHz,
	CDCl 3 ) δ (ppm) = 8.48 (d, J = 9.5 Hz, 1H, NH), 8.13 (d, J = 7.7 Hz, 1H, H Pyr ), 7.71 (t, J = 7.7
	Hz, 1H, H Pyr ), 7.64 (d, J = 8.7 Hz, 1H, H Pyr ), 5.46 (bd, J = 3.4 Hz, 1H, H 4 ), 5.41 (d, J = 9.4 Hz,
	1H, H 1 ), 5.33 (dd, J =10.1, 9.4 Hz, 1H, H 2 ), 5.17 (dd, J = 10.1, 3.4 Hz, 1H, H 3 ), 4.16 -4.05 (m,
	3H, H 6a , H 6b , H 5 ), 2.18 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 ), 2.00 (s, 3H, COCH 3 ), 1.97 (s,
	3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.52 (C=O), 170.47 (C=O), 170.3
	(C=O), 170.1 (C=O), 163.5 (NHCO), 149.7 (C Pyr ), 141.0 (C Pyr ), 139.7 (C PyrH ), 131.7 (C PyrH ),
	121.8 (C

  3 , H 4 ),4.39 (dd, J = 9.7, 9.1 Hz, 1H, H 2 ), 4.28 (dd, J = 12.5, 4.3 Hz, 1H, H 6a ), 4.10 (dd, J = 12.5, 2.0 Hz,1H, H 6b ), 3.91 -3.59 (m, 1H, H 5 ), 2.10 -2.01 (m, 9H, 3×COCH 3 ), 1.85 (s, 3H, COCH 3 );

	13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 171.7 (COCH 3 ), 171.0 (COCH 3 ), 170.9 (COCH 3 ),
	169.4 (COCH 3 ), 164.0 (CONH), 149.7 (C Pyr ), 141.1 (C Pyr ), 139.6 (C PyrH ), 131.6 (C PyrH ), 121.7
	(C PyrH

  Pyr ), 141.2 (C Pyr ), 137.8 (C PyrH ), 130.6 (C PyrH ), 122.5 (C PyrH ), 82.0 (C≡CH), 78.6 (C≡CH), 78.4 (C 1 ), 72.5 (C 5 ), 71.3 (C 3 ), 68.3 (C 2 ), 67.3 (C 4 ), 61.4 (C 6 ), 20.71 (s, 2C, COCH 3 ), 20.66 (COCH 3 ), 20.62 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 22 H 24 N 2 NaO 10 [M+Na] + 499.1323, found 499.1307.

	). R f = 0.38
	(PE:EtOAc = 1:1); [α] D 20 = -26.5 (c = 2.2, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.60
	(d, J = 9.8 Hz, 1H, NHCO), 8.12 (d, J = 7.8 Hz, 1H, H Pyr ), 7.81 (t, J = 7.8 Hz, 1H, H Pyr ), 7.63 -
	7.56 (m, 1H, H Pyr ), 5.49 -5.38 (m, 2H, H 4 , H 1 ), 5.31 (dd, J = 10.2, 9.7 Hz, 1H, H 2 ), 5.15 (dd, J
	= 10.2, 3.4 Hz, 1H, H 3 ), 4.16 -4.00 (m, 3H, H 5 , H 6 ), 3.23 (s, 1H, C≡CH), 2.15 (s, 3H, COCH 3 ),
	1.99 (s, 3H, COCH 3 ), 1.97 (s, 3H, COCH 3 ), 1.93 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 )
	δ (ppm) = 170.5 (C=O), 170.30 (C=O), 170.25 (C=O), 167.0 (C=O), 164.0 (CONH), 149.0
	(C 6-Ethynyl-N-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)picolinamide
	(33-GlcNAc)
	Prepared according to general protocol F from compound 32-GlcNAc (250 mg, 0.46 mmol, 1.0
	eq.). The crude product was purified by silica gel column chromatography (100% PE to 70%
	EtOAc/PE) to afford compound 33-GlcNAc as yellow foam (144 mg, 0.30 mmol, 66%). R f =
	0.21 (PE:EtOAc = 1:3); [α] D = -56.4 (c = 2.2, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) =
	8.80 (d, J = 9.6 Hz, 1H, NHCO), 8.13 (d, J = 7.7 Hz, 1H, H Pyr ), 7.80 (t, J = 7.7 Hz, 1H, H Pyr ),
	7.57 (d, J = 7.7 Hz, 1H, H Pyr ), 6.45 (d, J = 9.3 Hz, 1H, NHAc), 5.45 (dd, J = 9.7, 9.6 Hz, 1H,
	H 1 ), 5.28 -5.19 (m, 1H, H 3 ), 5.26 -5.09 (m, 1H, H 4 ), 4.42 (dd, J = 9.7, 9.3 Hz, 1H, H 2 ), 4.25 (dd,
	J = 12.5, 4.3 Hz, 1H, H 6a ), 4.09 (dd, J = 12.5, 2.0 Hz, 1H, H 6b ), 3.85 (ddd, J = 9.8, 4.3, 2.0 Hz,
	1H, H 5 ), 3.20 (s, 1H, C≡CH), 2.05 (d, 6H, 2 ×COCH 3 ), 2.02 (s, 3H, COCH 3 ), 1.81 (s, 3H,
	COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 171.5 (COCH 3 ), 170.9 (s, 2C, 2×COCH 3 ),
	169.4 (COCH 3 ), 164.6 (NHCO), 149.0 (C Pyr ), 141.3 (C Pyr ), 137.7 (C PyrH ), 130.5 (C PyrH ), 122.4
	(C PyrH ), 81.9 (C≡CH), 79.6 (C 1 ), 78.5 (C≡CH), 73.9 (C 5 ), 73.5 (C 3 ), 68.2 (C 4 ), 62.0 (C 6 ), 53.2
	(C 2 ), 23.1 (COCH 3 ), 20.8 (s, 2C, 2×COCH 3 ), 23.7 (COCH 3 ); HR-ESI-MS (positive mode) m/z:
	calcd. for C 22 H 25 N 3 NaO 9 [M+Na] + 498.1483, found 498.1487.

  mmol, 1.0 eq.). The crude product was purified by silica gel column chromatography (100% PE to 80% EtOAc/PE) to afford compound 34-Glc as white foam(90 mg, 0.109 mmol, 83%). R f = 0.17 (PE:EtOAc = 1:4);[α] D = +14.1 (c = 0.7, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.55 (s, 1H, NH Uri ), 8.90 (d, J = 9.1 Hz, 1H, NHCO), 8.53 (s, 1H, H triazole ), 8.29 (d, J = 7.7 Hz, 1H, H Pyr ), 8.06 (d, J = 7.7 Hz, 1H, H Pyr ), 7.94 -7.87 (m, 1H, H Pyr ), 7.13 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.74 -5.63 (m, 2H, H 5Uri , H 1Rib ), 5.57 -5.29 (m, 4H, H 1Glc , H 3Glc , H 2Rib , H 3Rib ), 5.19 -5.09 (m, 2H, H 2Glc , H 4Glc ), 4.92 (dd, J = 14.3, 2.8 Hz, 1H, H 5aRib ), 4.85 -4.75 (m, 1H, H 5bRib ), 4.57 -4.49 (m, 1H, H 4Rib ), 4.35 (dd, J = 12.6, 3.1 Hz, 1H, H 6aGlc ), 4.12 -4.01 (m, 1H, H 6bGlc ), 3.96 -3.89 (m, 1H, H 5Glc ), 2.13 -1.93 (6s, 18H, 6×COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 171.4 (COCH 3 ), 170.7 (COCH 3 ), 170.2 (COCH 3 ), 169.82 (COCH 3 ), 169.80 (COCH 3 ), 169.7 (COCH 3 ), 164.8 (NHCO), 163.0 (C=O Uri ), 150.2 (C=O Uri ), 149.0 (C Pyr ), 148.1 (C Pyr ), 147.8 (C triazole ), 141.9 (C 6Uri ), 138.6 (C PyrH ), 124.7 (C triazoleH ), 123.2 (C PyrH ), 121.7 (C PyrH ), 103.2 (C 5Uri ), 92.2 (C 1Rib ), 80.6 (C 4Rib ), 78.9 (C 1Glc ), 73.8 (C 5Glc ), 73.1 (C 3Glc or C 2Rib or C 3Rib ), 72.6 (C 3Glc or C 2Rib or C 3Rib ), 71.

	1 (C 3Glc
	or C 2Rib or C 3Rib ), 70.4 (C 2Glc or C 4Glc ), 68.5 (C 2Glc or C 4Glc ), 61.8 (C 6Glc ), 51.7 (C 5Rib ), 20.80
	(COCH 3 ), 20.74 (s, 2C, COCH 3 ), 20.69 (COCH 3 ), 20.52 (COCH 3 ), 20.47 (COCH 3 );
	HR-ESI-MS (positive mode) m/z: calcd. for C 35 H 40 N 7 O 17 [M+H] + 830.2475, found 830.2458.

  ), 8.99 -8.89 (m, 1H, NHCO), 8.58 (s, 1H, H triazole ), 8.29 (d, J = 7.8 Hz, 1H, H Pyr ), 8.06 (d, J = 7.8 Hz, 1H, H Pyr ), 7.92 (t, J = 7.8 Hz, 1H, H Pyr ), 7.14 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.72 (d, J = 3.7 Hz, 1H, H 1Rib ), 5.65 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.51 -5.42(m, 3H, H 2Rib , H 3Rib , H 4Gal ), 5.38 -5.30 (m, 2H, H 1Gal , H 2Gal ), 5.23 (dd, J = 9.4, 4.2 Hz, 1H, H 3Gal ), 4.92 (dd, J = 14.4, 3.6 Hz, 1H, H 5aRib ), 4.77 (dd, J = 14.5, 7.2 Hz, 1H,

6-[1-(Uridin-5'-yl)-1H-1,2,3-triazol-4-yl]-N-(β-D-galactopyranosyl)picolinamide (35-Gal)

  Hz, 1H, H 4Glc );13 C NMR (125 MHz, (CD 3 ) 2 SO) δ (ppm) = 164.0, 149.4, 149.0, 146.6, 140.9 (C 6Uri ), 139.1 (C PyH ), 125.6 (C triazoleH ), 122.2 (C PyH ), 121.3 (C PyH ), 102.3 (C 5Uri ), 90.0 (C 1Rib ), 81.3 (C 4Rib ), 79.8 (C 1Glc ), 78.9 (C 5Glc ), 78.0 (C 3Glc ), 71.9 (C 2Glc ), 71.4 (C 2Rib ), 70.3 (C 3Rib ), 70.1 (C 4Glc ), 61.1 (C 6Glc ), 51.6 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 23 H 27 N 7 NaO 11 [M+Na] + 600.1661, found 600.1684.

	6-[(1-Uridin-5'-yl)-1H-1,2,3-triazol-4-yl]-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)pi
	colinamide (35-GlcNAc)
	residue was purified by C18 reverse phase column chromatography (100% H 2 O to 50% Prepared according to general protocol E from compound 34-GlcNAc (104.7 mg, 0.126 mmol,
	MeOH/H 2 O) to afford compound 35-Glc as white foam (59 mg, 0.102 mmol, 85%). [α] D = 1.0 eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to
	+27.5 (c = 0.08, DMSO); 1 H NMR (500 MHz, (CD 3 ) 2 SO) δ (ppm) = 8.97 (s, 1H, H triazole ), 8.15 30% MeOH/H 2 O) to afford compound 35-GlcNAc as white foam (66 mg, 0.11 mmol, 85%). R f
	(d, J = 7.8 Hz, 1H, H Py ), 8.01 (t, J = 7.8 Hz, 1H, H Py ), 7.92 (d, J = 7.8 Hz, 1H, H Py ), 7.12 (br, 1H, = 0.50 (EtOAc:MeOH = 2:3); [α] D = +19.5 (c = 0.8, MeOH); 1 H NMR (400 MHz, D 2 O) δ
	H 6Uri ), 5.57 (d, J = 5.4 Hz, 1H, H 1Rib ), 5.33 (d, J = 7.7 Hz, 1H, H 5Uri ), 4.91 (d, J = 9.0 Hz, 1H, (ppm) = 8.44 (s, 1H, H triazole ), 7.83 -7.69 (m, 3H, H Pyr ), 7.00 (d, J = 7.9 Hz, 1H, H 6Uri ), 5.69 (d,
	H 1Glc ), 4.71 (dd, J = 14.4, 6.1 Hz, 1H, H 5aRib ), 4.68 -4.60 (m, 1H, H 5bRib ), 4.11 (dd, J = 9.2, 6.1, J = 2.2 Hz, 1H, H 1Rib ), 5.38 (d, J = 7.9 Hz, 1H, H 5Uri ), 5.11 (d, J = 9.6 Hz, 1H, H 1GlcNAc ), 4.87 -
	4.3 Hz, 1H, H 4Rib ), 3.86 (dd, J = 9.2, 4.8 Hz, 1H, H 3Rib ), 3.78 (s, 1H, H 2Rib ), 3.59 (bd, J = 10.8 4.68 (m, 2H, H 5Rib ), 4.34 -4.28 (m, 1H, H 4Rib ), 4.19 -4.09 (m, 2H, H 2Rib , H 3Rib ), 4.04 -3.89 (m,
	Hz, 1H, H 6aGlc ), 3.31 -3.23 (m, 2H, H 2Glc , H 6bGlc ), 3.22 -3.11 (m, 2H, H 3Glc , H 5Glc ), 3.07 (t, J = 2H, H 2GlcNAc , H 6aGlcNAc ), 3.79 (dd, J = 12.4, 4.8 Hz, 1H , H 6bGlcNAc ), 3.75 -3.65 (m, 1H,
	H 3GlcNAc ), 3.65 -3.50 (m, 2H, H 4GlcNAc , H 5GlcNAc ), 1.85 (s, 3H, NHCOCH 3 ); 13 C NMR (100 9.1 Prepared according to general protocol E from compound 34-Gal (92.6 mg, 0.11 mmol, 1.0 MHz, D 2 O) δ (ppm) = 175.
	eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to
	50% MeOH/H 2 O) to afford compound 35-Gal as white foam (50 mg, 0.087 mmol, 78%). [α] D 20
	= +39.8 (c = 1.2, H 2 O); 1 H NMR (400 MHz, D 2 O) δ (ppm) = 8.29 (s, 1H, H triazole ), 7.83 (m, 3H,
	H Pyr ), 6.78 (d, J = 7.8 Hz, 1H, H 6Uri ), 5.73 (s, 1H, H 1Rib ), 5.18 (d, J = 7.8 Hz, 1H, H 5Uri ), 5.13 (d,
	J = 9.0 Hz, 1H, H 1Gal ), 4.85 -4.70 (m, 2H, H 5aRib , H 5bRib ), 4.37 -4.29 (m, 1H), 4.23 -4.17 (m,
	1H), 4.04 -3.77 (m, 7H); 13 C NMR (100 MHz, D 2 O) δ (ppm) = 172.0 (C=O Uri ), 167.2 (NHCO),
	156.3 (C=O Uri ), 148.0 (C Pyr ), 147.7 (C Pyr ), 146.8 (C triazole ), 140.6 (C 6Uri ), 139.6 (C PyrH ), 126.2
	(C triazoleH ), 123.7 (C PyrH ), 122.2 (C PyrH ), 102.8 (C 5Uri ), 91.3 (C 1Rib ), 80.7 (C 1Gal ), 80.2, 77.2, 73.7,
	72.7, 70.2, 69.4, 69.2, 61.5 (C 6Gal ), 50.6 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for
	C 23 H 27 N 7 NaO 11 [M+Na] + 600.1661, found 600.1640.

  ), 139.4 (C PyrH ), 126.1 (C triazoleH ), 123.3 (C PyrH ), 122.0 (C PyrH ), 102.5 (C 5Uri ), 91.4 (C 1Rib ), 80.7 (C 4Rib ), 79.8 (C 1GlcNAc ), 78.0 (C 5GlcNAc ), 74.2 (C 3GlcNAc ), 72.9 (C 2Rib or C 3Rib ), 70.4 (C 2Rib or C 3Rib ), 70.2 (C 4GlcNAc ), 61.1 (C 6GlcNAc ), 54.6 (C 2GlcNAc ), 51.2 (C 5Rib ), 22.4 (NHCOCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 25 H 30 N 8 NaO 11 [M+Na] + 641.1926, found 641.1911.

	2-Bromo-6-ethynylpyridine (38) 216

  The reaction mixture was stirred overnight. Then, the reaction mixture was filtered, washed with CH 2 Cl 2 . The filtrate was poured into satd aq. NH 4 Cl (50 mL) solution, extracted with CH 2 Cl 2 (4×50 mL). The extract was washed with H 2 O (40 mL) and brine(40 mL), dried over MgSO 4 , concentrated, the residue was purified by silica gel column chromatography (PE:CH 2 Cl 2 = 9:1 to PE:CH 2 Cl 2 = 9:2) to

	afford	a	mixture	(851	mg)	containing	2,6-dibromopyridine	36	and
	2-bromo-6-(trimethylsilyl)ethynylpyridine 37. The mixture was used directly for the reaction
	of next step without further purification.

-galactopyranosyl-1H-1,2,3-triazol-4-yl)-6-[1-(uridin-5'-yl)-1H-1,2,3-triazol-4-yl] pyridine (43-Gal) In

  calcd. for C 36 H 40 N 9 O 16 [M+H] + 854.2588, found 854.2569. 1H, H Pyr ), 7.12 (d, J = 8.0 Hz, 1H, H 6Uri ), 5.71 (d, J = 9.1 Hz, 1H, H 1Glc ), 5.55 (d, J = 2.2 Hz, 1H, H 1Rib ), 5.44 (d, J = 8.0 Hz, 1H, H 5Uri ), 4.73 (bs, 1H, H 5aRib ), 4.70 (bd, J = 5.3 Hz, 1H, H 5bRib ), 4.28 (bs, 1H, H 4Rib ), 4.22 (dd, J = 5.2, 2.2 Hz, 1H, H 2Rib ), 4.08 -4.03 (m, 1H, H 3Rib ), 3.95 (bd, J = 10.8 Hz, 1H, H 6aGlc ),3.88 (dd, J = 9.2, 9.1 Hz, 1H, H 2Glc ), 3.80 -3.70 (m, 3H, H 3Glc , H 4Glc , H 6bGlc ), 3.59 (bd, J = 9.4 Hz, 1H H 5Glc );13 C NMR (125 MHz, D 2 O) δ (ppm) = 166.1, 151.5, 149.0, 148.8, 147.8, 147.6, 142.6 (C 6Uri ), 139.4 (C PyrH ), 125.7 (C triazoleH ), 123.7 (C triazoleH ), 120.5

	). 20 = -13.4 (c = 1.1, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ R f = 0.34 (PE:EtOAc = 1:3); [α] D (ppm) = 9.07 (s, 1H, NH Uri ), 8.48 (s, 1H, H triazole ), 8.36 (s, 1H, H triazole ), 8.10 (d, J = 7.8 Hz, 2H, 2 H Pyr ), 7.88 (t, J = 7.8 Hz, 1H, H Pyr ), 7.04 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.93 (d, J = 9.3 Hz, 1H, H 1Gal ), 5.74 -5.61 (m, 3H, H 5Uri , H 1Rib , H 2Gal ), 5.58 (bd, J = 3.3 Hz, 1H, H 4Gal ), 5.48 -5.39 (m, 2H, H 2Rib , H 3Rib ), 5.30 (dd, J = 10.3, 3.3 Hz, 1H, H 3Gal ), 4.90 -4.75 (m, 2H, H 5aRib , H 5bRib ), 4.55 (bd, J = 4.2 Hz, 1H, H 4Rib ), 4.32 -4.26 (m, 1H, H 5Gal ), 4.23 -4.16 (m, 2H, H 6aGal , H 6bGal ), 2.25 (s, 3H, COCH 3 ), 2.11 (s, 3H, COCH 3 ), 2.10 (s, 3H, COCH 3 ), 2.04 (s, 3H, COCH 3 ), 2.02 (s, 3H, COCH 3 ), 1.91 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.6 (C=O), 170.3 (C=O), 170.0 (C=O), 169.8 (C=O), 169.8 (C=O), 169.5 (C=O), 162.6 (C=O Uri ), 150.1 (C=O Uri ), 149.8(C Pyr ), 149.6 (C Pyr ), 148.6 (C triazole ), 148.3 (C triazole ), 141.4 (C 6Uri ), 138.1 (C PyrH ), 124.3 (C triazoleH ), 121.0(C triazoleH ), 120.0 (C PyrH ), 119.9 (C PyrH ), 103.6 (C 5Uri ), 91.6 (C 1Rib ), 86.5 (C 1Gal ), 80.2 (C 4Rib ), 74.3 (C 5Gal ), 72.9 (C 3Gal or C 2Rib or C 3Rib ), 71.0 (C 3Gal or C 2Rib or C 3Rib ), 70.9 (C 3Gal or C 2Rib or C 3Rib ), 68.1 (C 2Gal ), 67.0 (C 4Gal ), 61.4 (C 6Gal ), 51.4 (C 5Rib ), 20.9 (COCH 3 ), 20.8 76.5 (C 3Glc or C 4Glc ), 73.2 (C 2Glc ), 73.1 (C 2Rib ), 70.4 (C 3Rib ), 69.7 (C 5Glc ), 61.2 (C 6Glc ), 51.3 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 24 H 28 N 9 O 10 , [M+H] + 602.1954, found 602.1956. 2-(1-β-D1 H NMR (500 MHz, (CD 3 ) 2 SO) δ (ppm) = 11.37 (s, 1H, NH Uri ), 8.77 (d, 2H, H triazole ), 8.00 (s, 3H, H Pyr ), 7.61 (d, J = 6.3 Hz, 1H, H 6Uri ), 5.80 -5.73 (m, 1H, H 1Gal ), 5.67 -5.51 (m, 2H, H 1Rib , H 5Uri ), 5.54 (bs, 1H, OH), 5.43 (bs, 1H, OH), 5.34 (bs, 1H, OH), 5.08 (bs, 1H, OH), 4.87 -4.67 (m, 4H, H 5aRib , H 5bRib , OH, OH), 4.28 (bs, 1H, H 4Rib ), 4.17 -4.00 (m, 3H, H 2Rib , H 3Rib , H 2Gal ), 3.85 -3.74 (m, 2H, H 4Gal , H 5Gal ), 3.65 -3.48 (m, 3H, H 3Gal , H 6aGal , H 6bGal ); 13 C NMR (125 MHz, (CD 3 ) 2 SO) δ (ppm) = 162.8, 150.5, 149.8, 149.7, 147.1, 141.0 (C 6Uri ), 138.2 (C Pyr ), 124.1 (C triazoleH ), 121.9 (C triazoleH ), 118.4 (C Pyr ), 101.9 (C 5Uri ), 89.1 (C 1Rib ), 88.4 (C 1Gal ), 81.5 (C 4Rib ), 78.4 (C 4Gal or C 5Gal ), 73.5 (C 3Gal ), 72.0 (C 2Gal ), 70.5 (C 2Rib or C 3Rib ), 69.5 (C 2Rib or C 3Rib ), 68.4 (C 4Gal or C 5Gal ), 60.4 (C 6Gal ), 51.4 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for (COCH 3 ), 20.6 (COCH (C PyrH ), 120.3 (C PyrH ), 102.6 (C 5Uri ), 92.3 (C 1Rib ), 88.3 (C 1Glc ), 81.1 (C 4Rib ), 79.6 (C 3Glc or C 4Glc ), C 24 H 28 N 9 O 10 , [M+H] + 602.1954, found 602.1956.

3 ), 20.54 (COCH 3 ), 20.51 (COCH 3 ), 20.47 (COCH 3 )

; HR-ESI-MS (positive mode) m/z: a flask containing compound 42-Gal (61 mg, 0.071 mmol, 1.0 eq.) were added MeOH (4 mL), H 2 O (2 mL) and Et 3 N (10 mL) and the reaction mixture was stirred at r.t. for 11 days. Then, the reaction mixture was concentrated and co-evaporated with toluene (3×5mL) to afford compound 43-Gal as white foam (42 mg, 0.070 mmol, quant.)

[α]

D = +28.5 (c = 0.2, DMSO);

  α] D = -28.8 (c = 0.08, DMSO); 1 H NMR (400 MHz, D 2 O:CD 3 OD = 2:1) δ (ppm) = 8.72 (s, 1H, H triazole ), 8.55 (s, 1H, H triazole ), 8.06 -7.98 (m, 1H, H Pyr ), 7.91 -7.82 (m, 2H, H Pyr ), 7.21 (d, J = 8.0 Hz, 1H, H 6Uri ), 5.94 (d, J = 9.7 Hz, 1H, H 1GlcNAc ), 5.78 (d, J = 3.3 Hz, 1H, H 1Rib ), 5.60 (d, J = 8.0 Hz, 1H, H 5Uri ), 4.94 (dd, J = 14.8, 3.5 Hz, 1H, H 5aRib ), 4.86 (dd, J = 14.8, 5.7 Hz, 1H, H 5bRib ), 4.40 (ddd, J = 9.9, 5.7, 3.5 Hz, 1H, H 4Rib ), 4.34 (t, J = 9.7 Hz, 1H, H 2GlcNAc ), 4.28 (dd, J = 5.6, 3.3 Hz, 1H, H 2Rib ), 4.22 -4.15 (m, 1H, H 3Rib ), 3.96 (dd, J = 12.4, 1.8 Hz, 1H, H 6aGlcNAc ),

	3.89 -3.68 (m, 4H, H 3GlcNAc , H 4GlcNAc , H 5GlcNAc , H 6bGlcNAc ), 1.82 (s, 3H, NHCOCH 3 ); 13 C NMR
	(100 MHz, D 2 O: CD 3 OD = 2:1) δ (ppm) = 174.7, 150.0, 149.8, 148.3, 141.8 (C 6Uri ), 140.1
	(C PyrH ), 126.4 (C triazoleH ), 123.9 (C triazoleH ), 121.2 (s, 2C, 2×C PyrH ), 103.6 (C 5Uri ), 92.5 (C 1Rib ),
	87.6 (C 1GlcNAc ), 81.6 (C 4Rib ), 80.2 (C 3GlcNAc or C 4GlcNAc or C 5GlcNAc ), 74.7 (C 3GlcNAc or C 4GlcNAc or
	C 5GlcNAc ), 73.9 (C 3GlcNAc or C 4GlcNAc or C 5GlcNAc ), 71.4 (C 2Rib ), 70.5 (C 3Rib ), 61.6 (C 6GlcNAc ), 56.6
	(C 2GlcNAc ), 52.2 (C 5Rib ), 22.6 (NHCOCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for
	C 26 H 30 N 10 NaO 10 [M+Na] + 665.2039, found 665.2023.

Prepared according to general protocol E from compound 42-GlcNAc

(35 mg, 0.

04 mmol, 1.0 eq.). The residue was purified by C18 reverse phase column chromatography (100% H 2 O to 80% MeOH/H 2 O) to afford compound 43-GlcNAc as white foam (24 mg, 0.036 mmol, 91%).

[

  D = +54.6 (c = 0.7, CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCl 3 ) δ = 9.02 (s, 1H, NH Uri ), 8.28 (s, 1H, H triazole ), 8.12 (d, J = 7.8 Hz, 1H, H Pyr ), 7.63 (t, J = 7.8 Hz, 1H, H Pyr ), 7.41 (d, J = 7.8 Hz, 1H, H Pyr ), 7.06 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.75 (dd, J = 8.1, 2.0 Hz, 1H, H 5Uri ), 5.64 (d, J = 4.0 Hz, 1H, H 1Rib

  D = +51.0 (c = 0.3, MeOH); 1 H NMR (500 MHz, CD 3OD) δ (ppm) = 8.50 (s, 1H, H triazole ), 8.03 (d, J = 7.8 Hz, 1H, H Pyr ), 7.77 (t, J = 7.8 Hz, 1H, H Pyr ), 7.53 (d, J = 7.8 Hz, 1H, H Pyr ), 7.40 (d, J = 8.0 Hz, 1H, H 6Uri ), 5.76 (d, J = 3.6 Hz, 1H, H 1Rib ), 5.63 (d, J = 8.0 Hz, 1H, H 5Uri ), 4.89 (dd, J = 14.5, 3.7 Hz, 1H, H 5aRib ), 4.81 (dd, J = 14.5, 6.6 Hz, 1H, H 5bRib ), 4.31 (ddd, J = 6.6, 6.6, 3.7 Hz, 1H, H 4Rib ), 4.18 -4.13 (m, 2H, H 2Rib , H 3Rib ); 13 C NMR (125 MHz, CD 3 OD) δ (ppm) = 165.96 (C=O Uri ), 152.24 (C Pyr ), 152.07 (C=O Uri ), 147.96 (C triazole ), 143.20 (C 6Uri ), 142.94 (C Pyr ), 141.06 (C PyrH ), 128.52 (C PyrH ), 126.16 (C triazoleH ), 119.92 (C PyrH ), 103.05 (C 6Uri ), 93.23 (C 1Rib ), 82.91 (C 4Rib ), 74.22 (C 2Rib or C 3Rib ), 71.98 (C 2Rib or C 3Rib ), 52.63 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd. for C 16 H 16 BrN 6 O 5 , [M+H] + 451.0360, found 451.0359.

  ). R f = 0.42 (PE:EtOAc = 1:3); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.92 (s, 1H, H 3Uri ), 8.33 (s, 1H, H triazole ), 8.12 (d, J = 7.7 Hz, 1H, H Pyr ), 7.73 (t, J = 7.7 Hz, 1H, H Pyr ), 7.41 (d, J = 7.7 Hz, 1H, H Pyr ), 7.06 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.75 (d, J = 8.1 Hz, 1H, H 5Uri ), 5.65 (d, J = 4.1 Hz, 1H, H 1Rib), 5.45 (dd appears as t, J = 6.5 Hz, 1H, H 3Rib ), 5.30 (dd, J = 6.5, 4.1 Hz, 1H, H 2Rib ), 4.84 (dd, J = 14.5, 3.2 Hz, 1H, H 5aRib ), 4.76 (dd, J = 14.5, 6.8 Hz, 1H, H 5bRib ), 4.48 (ddd appears as td, J = 6.8, 6.5, 3.2 Hz, 1H, H 4Rib ), 2.12 (s, 3H, COCH 3 ), 2.09 (s, 3H, COCH 3 ), 0.27 (s, 9H, SiMe 3 );

  1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.70 (s, 1H, NH Uri ), 8.34 (s, 1H, H triazole ), 8.17 (d, J = 7.8 Hz, 1H, H Pyr ), 7.77 (t, J = 7.8 Hz, 1H, H Pyr ), 7.43 (d, J = 7.8 Hz, 1H, H Pyr ), 7.04 (d, J = 8.1 Hz, 1H, H 6Uri ), 5.75 (dd, J = 8.1, 2.1 Hz, 1H, H 5Uri ), 5.65 (d, J = 4.1 Hz, 1H, H 1Rib ), 5.42 (t, J = 6.5 Hz, 1H, H 3Rib ), 5.30 (dd, J = 6.5, 4.1 Hz, 1H, H 2Rib ), 4.85 (dd, J = 14.5, 3.3 Hz, 1H, H 5aRib ), 4.78 (dd, J = 14.5, 6.5 Hz, 1H, H 5bRib ), 4.49 (ddd, J = 6.5, 6.5, 3.3 Hz, 1H, H 4Rib ), 3.20 (s, 1H, C≡CH), 2.13 (s, 3H, COCH 3 ), 2.09 (s, 3H, COCH 3 );

  ), 137.4 (C Pyr ), 127.0 (C Pyr ), 124.5 (C triazoleH ), 120.3 (C Pyr ), 103.6 (C 5Uri ), 91.6 (C 1Rib ), 82.8 (C≡CH), 80.4 (C 4Rib ), 77.4 (C≡CH), 72.9 (C 2Rib ), 70.7 (C 3Rib ), 51.5 (C 5Rib ), 20.57 (COCH 3 ), 20.55 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd. for C 22 H 21 N 6 O 7 [M+H] + 481.1466, found 481.1464.

  ). Cl 2(3 mL). The reaction mixture was stirred at 0 o C for 17 h with protection of light. Then, satd aq. NaHCO 3 solution (10 mL) was added into the reaction. The resulting mixture was vigorously stirred for 15 min. The suspension was filtered. The solution was extracted with CH 2 Cl 2 (3×20 mL). The organic layers were combined, dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel column chromatography (PE:EtOAc = 9:1 to PE:EtOAc = 3:1) to afford compound 59 as white foam (90 mg, 0.189 mmol, 55%). BF 3 •Et 2 O (6 μL, 0.042 mmol, 0.2 eq.) was added dropwise into a stirred solution of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl trichloroacetimidate (152 mg, 0.31 mmol, 1.5 eq.) and methyl (2S)-2-azido-3-hydroxypropanoate (57) (30 mg, 0.207 mmol, 1.0 eq.) in freshly distilled CH 2 Cl 2 (3 mL) at -20 o C. The reaction mixture was stirred at -20 o C for 2 h. The reaction mixture was poured into satd aq. Na 2 CO 3 (10 mL) solution and extracted with CH 2 Cl 2 (4×10 mL). The combined organic layers were dried (Na 2 SO 4 ) and evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc = 9:1 to PE:EtOAc = 3:1) to afford compound 59 as white foam (44 mg, 0.093 mmol, 45%). Me), 20.8 (COCH 3 ), 20.71 (COCH 3 ), 20.69 (COCH 3 ), 20.6 (COCH 3 ); HR-ESI-MS (positive mode) m/z: calcd for C 18 H 25 N 3 NaO 11 , [M+Na] + 498.1330, found 498.1311.

	Methyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy)-(2S)-2-azido-3-propanoate (59)
	(Entry 7, Table III-2) SnCl 4 (1.725 mL, 1 M in CH 2 Cl 2 , 1.725 mmol, 5.0 eq.) was added
	dropwise (within 120 min via syringe pump) at 0 o C to a stirred solution of
	β-D-galactopyranosyl pentaacetate 1a (202 mg, 0.517 mmol, 1.5 eq.), silver trifluoroacetate
	(190 mg, 0.863 mmol, 2.5 eq.) and methyl (2S)-2-azido-3-hydroxypropanoate (57) (50 mg,
	0.345 mmol, 1.0 eq.) in freshly distilled CH 2 R f = 0.19 (PE:EtOAc = 3:1); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 5.35 (bd, J = 3.4 Hz, 1H,
	H 4 ), 5.14 (dd, J = 10.4, 7.9 Hz, 1H, H 2 ), 4.98 (dd, J = 10.4, 3.4 Hz, 1H, H 3 ), 4.55 (d, J = 7.9 Hz,
	1H, H 1 ), 4.21 -4.04 (m, 3H, H 6 , H βa ), 4.00 (t, J = 4.4 Hz, 1H, H α ), 3.92 -3.82 (m, 2H, H βb , H 5 ),
	3.78 (s, 3H, CO 2 Me), 2.11 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 2.01 (s, 3H, COCH 3 ), 1.94 (s,
	3H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ = (ppm) 170.5 (COCH 3 ), 170.3 (COCH 3 ), 170.2
	(COCH 3 ), 169.5 (COCH 3 ), 168.4 (CO 2 Me), 101.2 (C 1 ), 70.9 (C 5 ), 70.8 (C 3 ), 68.8 (C β ), 68.4
	(C 2 ), 67.0 (C 4 ), 61.3 (C α ), 61.3 (C 6 ), 53.0 (CO 2

(Entry 8, Table

III-2)

  t. for 16 h. Then, the reaction mixture was concentrated and the residue was purified by silica gel column chromatography (60% EtOAc/PE) to afford the desired amide 63 as white foam (53 mg, 0.09 mmol, 30%). R 6Uri ), 5.77(d, J = 4.6 Hz, 1H, H 1Rib ), 5.73 (d, J = 8.1 Hz, 1H, H 5Uri ), 4.23 (dd, J = 5.0, 4.6 Hz, 1H, H 2Rib ), 4.06 -3.99 (m, 3H, H 3Rib , H 4Rib , H α ),3.94 (dd, J = 11.4, 4.3 Hz, 1H, H βa ), 3.81 (dd, J = 11.4, 6.6 Hz, 1H, H βb ), 3.64 -3.50 (m, 2H, H 5Rib );13 C NMR (100 MHz, CD 3 OD) δ = 170.9, 166.1, 152.3, 143.3 (C 6Uri ), 102.9 (C 5Uri ), 92.2 (C 1Rib ), 83.7 (C α ), 74.7 (C 2Rib ), 72.2 (C 3Rib or C 4Rib ), 66.2 (C 3Rib or C 4Rib ), 63.6 (C β ), 41.9 (C 5Rib ); HR-ESI-MS (positive mode) m/z: calcd for C 12 H 16 N 6 NaO 7 , [M+Na] + 379.0973, found 379.0962.

	0.08
	(s, 3H, SiCH 3 ), 0.08 (s, 3H, SiCH 3 ), 0.05 (s, 3H, SiCH 3 ), 0.00 (s, 3H, SiCH 3 ); 13 C NMR (100
	MHz, CDCl 3 ) δ (ppm) = 169.0 (C=O), 163.7 (C=O), 150.6 (C=O), 143.4 (C Uri ), 102.6 (C Uri ),
	94.9 (C 1Rib ), 83.8 (C 4Rib ), 73.1 (C 2Rib ), 72.9 (C 3Rib ), 64.6 (CHN 3 ), 63.1 (CH 2 OH), 41.1 (C 5Rib ),
	25.9 (CMe 3 ), 25.8 (CMe 3 ), 18.1 (CMe 3 ), 18.0 (CMe 3 ), -4.3 (SiCH 3 ), -4.5 (SiCH 3 ), -4.66
	(SiCH 3 ), -4.73 (SiCH 3 ); HR-ESI-MS (positive mode) m/z: calcd for C 24 H 44 N 6 NaO 7 Si 2 ,
	[M+Na] + 607.2702, found 607.2674.
	N-(Uridin-5'-yl)-2-azido-3-hydroxypropanamide (64) (Adapted from Ref 240-241 )
	A solution of compound 63 (48 mg, 0.082 mmol, 1.0 eq.) in CH 2 Cl 2 (1 mL) was stirred at r.t.
	Then, 90% TFA (4.4 mL) was added dropwise. After addition, the reaction mixture was stirred
	at r.

f = 0.1 (PE:EtOAc, 2:3); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 9.70 (s, 1H, NH Uri ), 7.36 -7.29 (m, 2H, NHCO, H Uri ), 5.77 (dd, J = 8.0, 2.0 Hz, 1H, H Uri ), 5.36 (dd, J = 5.5, 2.5 Hz, 1H, H 1Rib ), 4.54 (bd, J = 5.5 Hz, 1H, H 2Rib ), 4.13 -4.06 (m, 2H, H 4Rib , H α ), 4.04 -3.91 (m, 3H, H 3Rib , H β ), 3.72 -3.61 (m, 1H, H 5aRib ), 3.51 -3.39 (m, 1H, H 5bRib ), 0.90 (s, 9H, CMe 3 ), 0.86 (s, 9H, CMe 3 ), t. overnight. MeOH (3 mL) and H 2 O (5 mL) were then added into the reaction mixture. The solvent was removed by evaporation and the residue was purified by silica gel column chromatography (EtOAc to 10% MeOH/EtOAc) to afford compound 64 as white foam (L:D = 5:1, 31 mg, 0.087 mmol, quant.). 1 H NMR (400 MHz, CD 3 OD) δ = 7.68 (d, J = 8.1 Hz, 1H, H

)-2-azido-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyloxy)propanoate (66)

  Cl 2(3 mL) at -20 o C. After addition the reaction mixture was stirred at -20 o C for 2.5 h. Then, the reaction mixture was poured into satd aq. Na 2 CO 3 (10 mL) solution and extracted with CH 2 Cl 2 (4×10 mL). The combined organic layers were dried (Na 2 SO 4 ) and evaporated. The residue was purified by silica gel column chromatography (PE:EtOAc, 85:15) to afford product 66(95 mg, 0.131 mmol, 66%) as a mixture of the glycoside and the decomposed sugar donor. Yield was calculated based on the identified glycoside in the mixture from 1 H NMR data.reaction mixture was concentrated and the residue was purified by silica column chromatography (66% EtOAc/PE) to afford the desired amide 68 as white foam (54 mg, 0.054 mmol, two steps 81%). R f = 0.19 (PE:EtOAc, 2:3); 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) = 8.60 (s, 1H, NHUri ), 7.87 -7.83 (m, 2H, Ph), 7.57 -7.50 (m, 1H, Ph), 7.49 -7.42 (m, 2H, 2×Ph), 7.30 (d, J = 8.1 Hz, 1H, H 6Uri ), 7.22 (d, J = 7.0 Hz, 1H, NH Bz ), 5.65(d, J = 8.1 Hz, 1H, H 5Uri ), 5.39 -5.35 (m, 2H, H 1Rib , H 4Gal ), 5.16 (dd, J = 10.4, 7.9 Hz, 1H, H 2Gal ), 5.01 (dd, J = 10.4, 3.4 Ph ), 128.8 (s, 2C, 2×C Ph ), 127.4 (s, 2C, 2×C Ph ), 102.5 (C 5Uri ), 101.6 (C 1Gal ), 94.4 (C 1Rib ), 84.4 (C 4Rib ), 73.0 (C 2Rib ), 72.9 (C 5Gal or C 3Rib ), 71.1 (C 5Gal or C 3Rib ), 70.8 (C 3Gal ), 69.1 (C β ), 69.0 (C 2Gal ), 67.0 (C 4Gal ), 61.3 (C 6Gal ), 53.3 (C α ), 41.5 (C 5Rib ), 25.94 (CMe 3 ), 25.85 (CMe 3 ), 20.9 (COCH 3 ), 20.8 (COCH 3 ), 20.71 (COCH 3 ), 20.69 (COCH 3 ), 18.1 (CMe 3 ), 18.0 (CMe 3 ), -4.3 (SiCH 3 ), -4.5 (SiCH 3 ), -4.6 (SiCH 3 ), -4.9 (SiCH 3 ); HR-ESI-MS (positive mode) m/z: calcd for C 45 H 68 N 4 NaO 17 Si 2 , [M+Na] + 1015.4010, found 1015.3982.

	BF 3 •Et 2 O (6 μL, 0.042 mmol, 0.2 eq.) was added dropwise into a stirred solution of 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl trichloroacetimidate (225 mg, 0.30 mmol, 1.5 eq.) and methyl (2S)-2-azido-3-hydroxypropanoate (57) (28 mg, 0.20 mmol, 1.0 eq.) in freshly H 2Rib ), 4.28 (dd, J = 10.5, 4.7 Hz, 1H, H βa ), 4.15 -4.10 (m, 3H, H 4Rib , H 6Gal ), 3.99 -3.87 (m, 3H, H βb , H 5Gal , H 3Rib ), 3.69 -3.58 (m, 1H, H 5aRib ), 3.56 -3.47 (m, 1H, H 5bRib ), 2.11 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 1.98 (s, 3H, COCH 3 ), 1.97 (s, 3H, COCH 3 ), 0.90 (s, 9H, CMe 3 ), 0.84 (s, 9H, CMe 3 ), 0.10 (s, 3H, SiCH 3 ), 0.08 (s, 3H, SiCH 3 ), 0.06 (s, 3H, SiCH 3 ), 0.03 (s, 3H, SiCH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) = 170.6, 170.3, 170.2, 170.1, 167.3, 162.8, 150.2, 143.0 distilled CH 2 Hz, 1H, H 3Gal ), 4.90 -4.84 (m, 1H, H α ), 4.58 (d, J = 7.9 Hz, 1H, H 1Gal ), 4.47 -4.42 (m, 1H, (C 6Uri ), 133.5, 132.3 (C

  , H 2Rib ),4.23 (dd, J = 10.6, 5.6 Hz, 1H, H βaSer ), 4.16 -4.06 (m, 3H, H 6Gal , H 4Rib ), 3.98 -3.82 (m, 6H, H 5Gal , H 5aRib , H 3Rib , H αGly , H βbSer ), 3.29 -3.19 (m, 1H, H 5bRib ), 2.13 (s, 3H, COCH 3 ), 2.06 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 1.97 (s, 3H, COCH 3 ), 1.46 (s, 9H, 3×CH 3Boc

  H NMR (500 MHz, CD 3 OD) δ (ppm) = 8.49 (d, 1H, H triazole ), 7.86 -7.82 (m, 2H, Ph), 7.60 (t, J = 8.0 Hz, 1H, H 6Uri ), 7.46 -7.41 (m, 2H, Ph), 7.37 -7.32 (m, 1H, Ph), 5.74 -5.68 (m, 2H, H 5Uri , H 1Rib ), 4.29 -4.17 (m, 3H, H 2Rib , H β ), 4.09 -3.98 (m, 2H, H 3Rib , H 4Rib ), 3.68 -3.61 (m, 1H, H 5aRib ), 3.59 -3.53(m, 1H, H 5bRib

  ). (H α does NOT show up);13 C NMR (125 MHz, CD 3 OD) δ (ppm) = 169.05, 169.00 (2s, 2C), 148.7, 143.43, 143.39 (2s, 2C, C 6Uri ), 131.72, 131.71 (2s, 2C, C PhH ), 130.0 (s, 2×C PhH ), 129.4 (s, C PhH ), 126.7 (s, 2×C PhH ), 122.34, 122.29 (2s, 2C, C triazole ), 103.15, 103.09 (2s, 2C, C 5Uri ),93.11, 93.02 (2s, 2C, C 1Rib ), 83.65, 83.58 (2s, 2C, C 4Rib ), 74.66, 74.55 (2s, 2C, C 2Rib ), 72.13, 72.10 (2s, 2C, C 3Rib ), 62.99, 62.88 (2s, 2C, CH 2 OH), 42.13, 42.10 (2s, 2C, C 5Rib

  ). (C α does NOT show up); HR-ESI-MS (positive mode) m/z: calcd for C 20 H 22 N 6 NaO 7 , [M+Na] + 481.1442, found 481.1427.

  , H 1Rib ), 5.65 -5.59 (m, 0.36H, H α ), 4.27 -4.18 (m, 1H, H 2Rib ), 4.15 -4.09 (m, 1H, H βa ), 4.07 -3.97 (m, 3H, CH βb , H 3Rib , H 4Rib ), 3.67 -3.59 (m, 1H, H 5aRib ), 3.60 -3.52(m, 1H, H 5bRib

  (2s, 2C, C 6Uri ), 131.63,131.61, 130.0 (s, 2×C Ph ), 129.4 (C Ph ), 126.7 (s, 2×C Ph ), 122.28, 122.21 (2s,

  ). R f = 0.5 (EtOAc); 1 H NMR (400 MHz, D 2 O) δ (ppm) = 7.99 (d, J = 8.0 Hz, 1H, H 6Uri ), 6.11 (d, J = 5.2 Hz, 1H, H 1Rib ), 5.88 (d, J = 8.0 Hz, 1H, H 5Uri ), 5.73 -5.65 (m, 1H, H 3Rib ), 5.51 (bd, J = 5.2 Hz, 1H, H 2Rib ), 4.79 (bs, 1H, H 4Rib ), 2.17 (s, 3H, COCH 3 ), 2.12 (s, 3H, COCH 3 ); 13 C NMR (100 MHz, D 2 O) δ (ppm) = 172.7 (s, 2C, COCH 3 ), 172.4 (CO 2 H), 166.3 (C=O), 151.8 (C=O), 143.1 (C Uri

Table V - 1 .

 V1 Data collection for crystallography and refinement statistics. As assessed by Molprobity 252 . Numbers in brackets are for the high resolution shell. #Rmerge = Σ hkl Σ i |I i (hkl) -<I(hkl)>| / Σ hkl Σ i I i (hkl). +Rwork = Σ hkl ||F obs | -|F calc || / Σ hkl |F obs |. §Rfree = Σ hkl ||F obs | -|F calc || / Σ hkl |F obs | calculated using a random set containing 3% of the reflections that were not included throughout structure refinement.

	Inhibitor	7-Gal	19	7-Glc
	Space group	C222 1	C222 1	C222 1
	Unit cell parameters a=55.88 Å,	a=55.93 Å,	a=55.85 Å,
		b=149.90 Å,	b=149.87 Å,	b=149.87 Å,
		c=80.03 Å,	c=80.10 Å,	c=80.00 Å,
		α=β=γ=90°	α=β=γ=90°	α=β=γ=90°
	Resolution	1.7 Å (1.9 Å)	1.6 Å (1.8 Å)	1.5 Å (1.7 Å)
	Unique reflections	35437	42400	51220
	Completeness	100.0% (100.0%)	99.9% (100.0%)	99.9% (100.0%)
	Redundancy	7.3	5.7	5.7
	Rmerge	14.3% (66.4%)	9.3% (64.3%)	8.5% (58.7%)
	I/σI	11.44 (3.02)	11.92 (2.87)	14.47 (2.99)
	R work	17.6%	14.6%	14.1%
	R free	20.5%	19.2%	18.1%
	Av. B factor	13.9 Å 2	20.0 Å 2	16.2 Å 2
	R.M.S.D. bonds	0.006 Å	0.007 Å	0.006 Å
	R.M.S.D. angles	1.06°	1.16°	1.12°
	Ramachandran plot	97.2% / 99.6%	97.9% / 100.0%	97.6% / 100%
	(% in favored / allowed
	regions)
	PDB_ID	4KC2	4KC4	4KC1

*

  Synthèse et évaluation biologique d'inhibiteurs neutres de glycosyltransférases __________________________________________________________________________________ RESUMELes glycosyltransférases sont responsables de la biosynthèse d'oligosaccharides, de polysaccharides et de glycoconjugués. Etant donné le nombre croissant de processus biologiques reliés aux saccharides, il existe un grand intérêt pour la compréhension des rôles biologiques des ces saccharides et étudier leurs applications thérapeutiques potentielles. Ce projet concerne la conception, la synthèse, l'évaluation biologique et l'analyse structurale de deux nouveaux types d'inhibiteurs de glycosyltransférases analogues du substrat donneur naturel. L'analogie repose sur l'incorporation d'une unité pyridine ou amino-acide neutre comme mime du motif pyrophosphate afin de fournir des substrats capables de pénétrer les cellules pour des applications potentielles in cellulo ou in vivo. Les synthèses d'inhibiteurs neutres de GTs ont été réalisées en utilisant une combinaison de réactions de conjugaison créant une liaison O-glycosidique, amide ou triazole. Au total, 26 inhibiteurs neutres de GTs ont ainsi été synthétisés. L'évaluation de l'inhibition pour cinq galactosyltransférases et une GlcNAc-transférase (OGT) a révélé des inhibitions modestes de l'ordre du micromolaire. La co-cristallisation des meilleurs inhibiteurs avec l'une de ces galactosyltransférases a démontré que le motif pyridine neutre chélate le cation manganèse impliqué dans le site catalytique de l'enzyme. Cependant, le motif galactose est orienté vers l'extérieur du site catalytique et loin de la position initiale du substart naturel (UDP-Gal) et indique donc un nouveau mode de liaison. Le concept d'inhibiteur neutre a aussi été examiné sur un système modèle de membrane pour leur perméation membranaire. __________________________________________________________________________________ MOTS-CLES : glycosylation, carbohydrate, inhibiteur, glycosyltransférase, chimie médicinale

	__________________________________________________________________________________
	TITLE :
	Synthesis and biological evaluation of neutral glycosyltransferase inhibitors
	__________________________________________________________________________________
	ABSTRACT

TITRE :

  indicating a new binding mode. The concept of 'neutral' inhibitor was examined by an artificial cell membrane penetration test. __________________________________________________________________________________ KEYWORDS : glycosylation, carbohydrate, inhibitor, glycosyltransferase, medicinal chemistry __________________________________________________________________________________ DISCIPLINE : Chimie Organique __________________________________________________________________________________ UMR-CNRS 5246, ICBMS, Laboratoire de Chimie Organique 2, Batiment Curien -CPE, 43 boulevard du 11 novembre 1918, F-69622 Villeurbanne CEDEX.

Acknowledgement

First of all I would like to thank my supervisor, Dr. Sébastien Vidal, for giving me an opportunity to be a member of Laboratoire de Chimie Organique 2 at Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Université Claude Bernard Lyon 1, and for providing guidance and direction over the years. He always encouraged me and gave me

N-[2',3'-Di-O-(tert-butyldimethylsilyl)-uridin-5'-yl]-N'-acetyl-O-(2,3,4,6-tetra-O-acetyl-β -D-galactopyranosyl)-serinamide (67)

Compound 62 (47 mg, 0.051 mmol, 1.0 eq.) and PPh 3 (41 mg, 0.153 mmol, 3.0 eq.) were solubilized in THF (2 mL), then, H 2 O (0.5 mL) was added and the reaction mixture was stirred at r.t. for 17 h. Then, the reaction was concentrated, pyridine (2 mL) and acedic anhydride (1 mL) were added into the flask. The resulting solution was stirred at r.t. for 16 h. MeOH (10 mL) was added and stirring continued for another 15 min. The resulting reaction mixture was co-evaporated with toluene (3×10 mL) and the residue was purified by silica gel column chromatography (50% EtOAc/PE to 100% EtOAc) to afford compound 67 as white foam (10 mg, 0.011 mmol, 21%). R f = 0.1 (PE:EtOAc, 2:3); 1 H NMR (500 MHz, CDCl 3 ) δ (ppm) = 8.68 (s, 1H, NH Uri ), 7.39 (d, J = 8.1 Hz, 1H, H 6Uri ), 7.09 (t, J = 5.6 Hz, 1H, NHCO), 6.50 (d, J = 7.1 Hz, 1H, NHAc), 5.77 (dd, J = 8.1, 1.9 Hz, 1H, H 5Uri ), 5.42 (d, J = 5.6 Hz, 1H, H 1Rib ), 5.40 -5.37 (m, 1H, H 4Gal ), 5.14 (dd, J = 10.5, 7.9 Hz, 1H, H 2Gal ), 5.02 (dd, J = 10.5, 3.4 Hz, 1H, H 3Gal , 2.13 (s, 3H, COCH 3 ), 2.08 (s, 3H, COCH 3 ), 2.05 (s, 3H, COCH 3 ), 2.03 (s, 3H, COCH 3 ), 1.99 (s, 3H, COCH 3 ), 0.91 (s, 9H, CMe 3 ), 0.87 (s, 9H, CMe 3 ), 0.10 (s, 3H, SiCH 3 ), 0.09 (s, 3H, SiCH 3 ), 0.08 (s, 3H, SiCH 3 ), 0.02 (s, 3H, SiCH 3 ); 13 C NMR (125 MHz, CDCl 3 ) δ (ppm) = 170. 7, 170.4, 170.3, 170.2, 170.09, 170.06, 167.9, 162.8, 150.3, 142.9 (C 6Uri ), 132.6, 131.0, 128.9, 102.6

N-[2',3'-Di-O-(tert-butyldimethylsilyl)-uridin-5'-yl]-N'-benzoyl-O-(2,3,4,6-tetra-O-acetyl -β-D-galactopyranosyl)serinamide (68)

Benzoyl chloride (9 μL, 0.08 mmol, 1.2 eq.) was added dropwise into a flask containing a solution of the amine 65 (59 mg, 0.067 mmol, 1.0 eq.) and DIPEA (15 μL, 0.086 mmol, 1.3 eq.) in freshly distilled CH 2 Cl 2 (4 mL). The reaction mixture was stirred at r.t. for 16h. Then, the

N-(Uridin-5'-yl)-N'-glycinyl-O-β-D-galactopyranosylserinamide (77)

Prepared according to general protocol G from compound 69 (39 mg, 0.037 mmol, 1.0 eq.). The crude product was purified by C18 reverse phase column chromatography (100% H 2 O to 100% MeOH) to afford compound 77 as white foam (7 mg, 0.013 mmol, 34% over 2 steps). [α]

N-(Uridin-5'-yl)-N'-hydroxyacetyl-O-β-D-galactopyranosylserinamide (78)

Prepared according to general protocol G from compound 70 (26 mg, 0.026 mmol, 1.0 eq.). The crude product was purified by C18 reverse phase column chromatography (100% H 2 O to 100% MeOH) to afford compound 78 as white foam (8 mg, 0.015 mmol, 56% over 2 steps).