M. S ; ; Reddy

G D ; Sharma

M U ; Kiran

For this dissertation I would like to thank the committee members: Dr. Christine Gravier-Pelletier, Prof. Arnaud Haudrechy and Prof. Olivier Martin for their time, interest, and comments.

A special acknowledgement goes to Prof. Yunbao Jiang in Xiamen University, who is my science mentor in China. His insightful knowledge, confident prediction and unselfish supporting inspire me move on during these years. Thanks for his support and help in the application of the scholarship, the funding that made my Ph.D. work possible.

I am indebted to all the kind and lovely members in Xie's research group, and all of them make the lab like a family. The group has been a source of friendships as well as good advice and collaborations. Thanks very much for the humor of Dr. Nicolas Bogliotti, the hearty laughter of Claire DEO, the warmth of Dr. Nhi-Ha Nguyen, the kindness of Dr. Olivier Noël, Dr. Laura Nodin, and Chaoqi Lin. Special thanks go to Stéphane Maisonneuve, my best friend in the lab. We shared all the laboratory equipment and supplies and he taught me how to measure the NMR, melting point, optical rotation, etc. He also provided insightful discussions and suggestions about the research. Moreover, he is also a guarantor for me in Crous.

I would like to thank all the faculty members and staff in PPSM: Dr. Rémi Métivier, Prof. Pierre Audebert, Dr. Robert Pansu, Dr. Valérie Alain-Rizzo, Dr. Clémence Allain, Dr. Gilles Clavier, Dr. Cécile Dumas-Verdes, Dr. Valérie Génot, Dr. Carine Julien-Rabant, Prof. Rachel Méallet-Renault, Dr. Laurent Galmiche, Andrée Husson, Christian Jean-Baptiste, Arnaud Brosseau. Thanks to Jacky Formont for handling all the problems of my computer.

I would also like to acknowledge the members of PPSM: Dr. Haitao Zhang, Dr. Yuanyuan Liao, Dr. Eva Julien, Dr. Alex Depauw, Dr. Jia Su, Meriem Stamboul, Dr. Yang Si, Yuan Li, Paul Rouschmeyer, Charlotte Remy, Clarisse Tourbillon, Corentin Pavageau, Marine Louis, Dr. Naresh Kumar, Yayang Tian, Qui Pham and Etienne Barrez. Thanks for your kind-ness to offer a very harmonious study environment.

Words alone cannot express my thanks to Dr. Yanhua Yu, who treats me like a sister. She has been a source of love and energy ever since. Many thanks to Dr. Yibin Ruan, he is my labmate both in Xiamen University and ENS Cachan. He helped me a lot in work and daily life, such as picking me up at the airport, offering me a lot of stuffs. Thanks again to Yuan Li for the help and accompany, which make me feel I am not alone in France. Thanks again to Yayang Tian for her help in daily life. I also would like to thank all my friends in ENS Cachan.

I am forever grateful to the care and support from my close friend, Su Chen. We worked on our manuscript during the same time, so she gave me a lot of understanding, accompanies, and encouragement. It is a precious memory for me forever. Thanks to Yuan Yang for all the help in daily life. I am so lucky to be as your neighbor. Thanks to all my friends in France and in China. A faithful friend is a source of strength, and all of you give me the strength.

Finally, my deepest gratitude goes to my family for their unflagging love and encouragement. Thanks to my parents who raised me with a love of science and supported me in all my pursuits, and thanks to my lovely brother.

Na CHEN ENS Cachan

DMF N,N-Dimethylformamide

DNA

Deoxyribonucleic acid

L Liter

LacCer Lactosylceramide

LG General Introduction

As part of glycoconjugate family, glycolipids and glycopeptides are involved in a variety of important biological, physiological and pathological processes, such as cell-cell interactions, viral and bacterial infections, immune response, cancer progression, etc. Synthesis of glycoconjugate mimics has attracted intensive research interest for biological and pharmaceutical applications, which has heightened the need for developing new types of glycoconjugates.

Aminooxy acids, a class of unnatural amino acids in which the amine group is replaced by an aminooxyl function, have emerged as very attractive scaffolds, since the peptides containing aminooxy acids can easily organize into turns and helices through intramolecular hydrogen bond formation. Furthermore, the N-oxypeptides are easily formed and resistant to chemical and enzymatic hydrolysis. These unique properties make N-oxyamide linkage attractive for the modification of biomolecules.

Recently, our group is interested in synthesizing N-oxyamide-containing compounds. As a continuing research program, we try to introduce the N-oxyamide function into glycolipids and glycopeptides to prepare novel glycoconjugates for studying their potential biological applications. This thesis is structured as follows:

The first chapter introduces the structures and functions of glycoconjugates. After presenting the synthetic strategy and stereochemistry of glycosidic bond, the synthesis and biological applications of glycopeptides/glycoproteins will be described. We will also introduce the classifications, structures, modifications and biological functions of glycolipids.

The second chapter attempts to review the N-O linkage in carbohydrate-based compounds. After introduction of aminooxy acids, the formation and applications of N-glycosyl hydroxylamines, O-glycosyl hydroxylamines and O-amino sugar derivatives will be presented.

The third chapter describes the synthesis of N-oxyamide-linked glycolipids. The glycoaminooxy ester, with both the carboxylic acid and aminoxyl functions attached on the sugar frame, has been prepared as a multifunctional building block. Further derivatization of this glycoaminooxy ester leads to the formation of N-oxyamide-linked glycolipids.

The fourth chapter presents the synthesis of N-oxyamide-linked glycoglycerolipids with one or two lipids chains as novel mimics of glycoglycerolipids and glycosphingolipids.

In the last chapter, we focus on the stereoselective synthesis of O-glycosyl aminooxy esters and their use for the construction of N-oxyamide-linked glycopeptides.

Chapter 1

Structures and Functions of Glycoconjugates I Introduction

Carbohydrates are implicated in a variety of important biological and physiological processes, such as biological transport, cell-cell recognition, activation of growth factors, modulation of the immune system, etc. 1 Carbohydrate-based compounds have been successfully applied in the biopharmaceutical and medical fields, especially in diagnostics, vaccines and therapeutics. 2 There are three major classes of carbohydrates, which can be defined as monosaccharides, oligosaccharides and polysaccharides. Compounds with mono-or oligosaccharides covalently attached to a non-sugar moiety such as lipids, peptides and proteins through glycoside bond, are called glycoconjugates. The major glycoconjugates are glycopeptides, glycoproteins, glycolipids, and proteoglycans.

Many reports supported that the glycans, including oligosaccharides, polysaccharides, and glycoconjugates, can play a very important role in biological recognition processes. 3 Now they could be reasonably added to the 'central dogma molecules' (nucleic acids, proteins), by playing a distinct role in information transfer (Figure 1). 3a,4 H. Chem. Rev. 2000, 100, 4495-4538; (e) Davis, B. G. J. Chem. Soc; Perkin Trans. 1. 1999, 3215-3237. 4 Crick, F. Nature, 1970, 227, 561-563. The branching structures and the stereochemistry of the glycosidic linkage result in the complexity of glycoconjugates. 5 The stereocontrol in the glycoside formation is one of the most important aspects of oligosaccharides and glycoconjugates synthesis. In this chapter, we will focus on the glycosidic bond formation (glycosylation), as well as the structures, functions and synthesis of glycopeptides and glycolipids.

II Formation of glycosidic bonds

The chemistry of glycosylation reactions, which is a common and essential process in all organisms, has been developed most intensively in the past few decades. 6 The glycosylation usually involves the coupling of a glycosyl donor and a suitable protected glycosyl acceptor via initiation using an activator under suitable reaction conditions (Figure 2). Examples of the products of glycosylation reactions are glycopeptides, glycolipids, glycosaminoglycans, oligosaccharides, and polysaccharides.

II.1 Glycosylation strategies

The first glycosylation reaction was reported by Arthur Michael in 1879, which proceeded by the nucleophilic displacement of an anomeric leaving group (Scheme 1a). 7 In 1893, Emil Fischer reported a new approach for the glycosylation reaction, in which an aldose or ketose was coupled with an alcohol or phenol catalyzed by an acid (Scheme 1b). 8 The Koenigs-Knorr glycosylation, a substitution reaction by reacting a glycosyl halide with a conventional alcohol using the halophilic promoter (silver salts, mercury salts, or Lewis acid) to give a glycoside, is one of the oldest and most famous glycosylation reactions (Scheme 1c). 9 Since then, a very large number of glycosylation methods, 6 and the mechanism of gly- 5 Seeberger, P. H. Nat. Chem. Biol. 2009, 5, 368-372. 6 (a) Satoh, H.; Manabe, S. Chem. Soc. Rev. 2013, 42, 4297-4309; (b) Ishiwata, A.; Lee, Y. J.; Ito, Y. Org. Biomol. Chem. 2010, 8, 3596-3608; (c) Zhu, X.; Schmidt, R. R. Angew. Chem. Int. Ed. 2009, 48, 1900-1934; (d) Demchenko, A.V. Handbook of Chemical Glycosylation, Wiley-VCH, Weinheim, 2008; (e) Galonic, D. P.; Gin, D. Y. Nature 2007, 446, 1000-1007; (f) Toshima, K. Carbohydr. Res. 2006, 341, 1282-1297; (g) Demchenko, A. V. Synlett 2003, 1225-1240; (h) Schmidt, R. R.;Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21-123. 7 Michael, A. Am. Chem. J. 1879, 1, 305-312. 8 Fischer, E. Ber. Dtsch. Chem. Ges.1893, 26, 2400-2412. 9 Koenigs, W.; Knorr, E. Ber. Dtsch. Chem. Ges. 1901, 34, 957-981. cosylation 10 have been developed and reviewed. Most of the glycosylation methods involving different glycosyl donors, could be named by the functionalities of the glycosyl donors, such as trichloroacetimidate method, phosphite method, thioglycoside, etc. Galonic et al. have summarized the glycosylation strategies. 6e The general glycosylation approach usually involves the coupling of a selectively protected glycosyl donor with an anomeric leaving group (LG) (1, Figure 3a) and a suitable protected glycosyl acceptor via initiation using a suitable electrophilic activator (EI + ) under suitable reaction conditions to generate the glycoconjugate (3).

The glycosyl donors and the corresponding electrophilic activator for the acetal exchange reactions were listed in Figure 3b. The glycosyl donors mainly contain: anomeric hydroxyl group (4); glycosyl halides including fluorides, chlorides, bromides and iodides (5); trichloroacetimidates (6); phosphates/phosphites (7); esters/carbonates/thiocarbonates (8); aryloxy groups (9); 4-pentenyl glycosides (10); glycosyl sulphides and sulphoxides (11).

The glycal assembly approach has been developed extensively in the synthesis of complex carbohydrates. The 1,2-alkene functionality enables various electrophilic oxidants (El + ) reacting with enol ether nucleophiles (12) to generate a three-membered ring, which is an actived glycosyl donor (13). Opening of the epoxide ring by an appropriate nucleophilic glycosyl acceptor (Nu-H) furnished the corresponding glycoconjugate (14), along with the introduction of functionalities 'Z' at the C2-position (Figure 3c). Various electrophilic oxidants and the correspondingly generated glycosides are lised in Figure 3d. Other methods were also reported for the glycoside bond formation, such as one-pot and automated glycosylation,11 solid-phase oligosaccharide synthesis,12 intramolecular glycoside formation etc. 6b,13

II.2 Stereochemistry

The formation of a glycoside linkage results in the creation of a new stereogenic center, which often leads to a mixture of two anomeric stereoisomers. There are two types of O-glycosides formed through a sp 2 -hybridized oxonium intermediate. The linkages formed are defined as 1,2-cis and 1,2-trans glycosides (Scheme 2). The stereoselective introduction of glycosidic linkages during glycosylation reactions is one of the most challenging aspects that have yet to be completely addressed by the synthetic chemist. 6c,10b,14 Scheme 2: Generic mechanism of the stereoselective formation of O-glycosidic bonds. 10b The nature of the protecting group at 2-position of a glycosyl donor is a major determinant of the anomeric selectivity. 15 A protecting group at C-2, which can perform neighboring group participation during a glycosylation, leads reliably to the formation of 1,2-trans glycosides by a S N 2 mechanism. As shown in Scheme 3, a promoter activates an anomeric leaving group resulting in its departure and the formation of an oxacarbenium ion. Subsequent neighboring group participation by the 2-O-acyl protecting group will give a more stable five-membered cyclic acetoxonium ion intermediate, which can only be formed as a 1,2-cis fused ring system. An alcohol can then attack the anomeric center of the acetoxonium ion intermediate from only one face providing a 1,2-trans-glycoside. Thus, in the case of glucopyranosyl-type donors, -anomers will be obtained whereas mannopyranosides will give the corresponding α-anomers. 16 Scheme 3: Generic mechanism of neighboring group participation in glycosylation reaction.

The presence of a sterically non-assisting, non-participating functionality at the 2-position is often used for the synthesis of 1,2-cis-glycosides. However, the stereocontrolled synthesis of 1,2-cis-glycosidic linkages is considerably more difficult. 6b,17 In this type of glycosylation reaction, the anomeric selectivity will be affected by many factors such as the activation condition, the constitution of the glycosyl donor and acceptor (for example, type of saccharide, leaving group at the anomeric center, protection and substitution pattern), as well as the solvent, temperature, concentration, and even the sequence of addition of the reactants. 18

III Structures and functions of glycopeptides and glycoproteins

III.1 Types of glycosidic linkages

The types of glycosidic bonds are classified according to the identity of the atom of the glycosyl acceptor which binds to the carbohydrate chain, i.e. N-glycoside, O-glycoside, C-glycoside or S-glycoside.

Two main forms of the naturally occurring protein glycosylations are N-and O-glycosides. N-glycoside, the most common type of glycosidic linkage in glycoproteins, is the attachment of a sugar molecule to the nitrogen atom of asparagine (Asn) side chains. In O-glycoside, the saccharide is linked to the hydroxyl group of serine (Ser), threonine (Thr), or tyrosine (Tyr). The majority of motifs are -N-GlcNAc-Asn, α-O-GalNAc-Ser/Thr and -O-GlcNAc-Ser/Thr. Other unusual linkage forms have also been reported, such as α-N-GlcNAc-Asn, Glc-N-Asn, GalNAc-N-Asn, α-O-Fuc-Ser/Thr and O-glycosides of hydroxylysine and hydroxyproline. Structures of some representative N-and O-glycosides are listed in Figure 4. 1a,19 16 Boltje, T. J.; Buskas, T.; Boons, G.-J. Nature Chem. 2009, 1, 611-622. 17 (a) Demchenko, A. V. Curr. Org. Chem. 2003, 7, 35-79; (b) Fairbanks, A. J. Synlett 2003, 1945-1958. 18 Boons, G.-J. Contemp. Org. Synth. 1996, 3, 173-200. 19 Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131-163. Natural C-glycopeptides are also existing. For example, a mannose is attached to a tryptophan (Trp) residue within an extracellular protein. The extremely rare S-glycopeptides, in which saccharides are covalently linked to the sulfur atoms of cysteine (Cys) residue, have been found in human and bacterial peptides. 20 Structures of C-linked and S-linked glycopeptides are presented in Figure 5.

III.2 Biological functions of glycopeptides and glycoproteins

Nowadays glycopeptides and glycoproteins are mainly recognized for their applications in various cellular activities such as cell-surface binding, including growth, differentiation, proliferation, adhesion or fertilization and immune responses. Furthermore, they also play a important role in pathogenic processes such as viral and bacterial infections, tumor growth and metastasis, autoimmune disorders and chronic inflammations. 21 Structurally defined glycopeptides and glycoproteins are useful tools for functional biological studies, because they contain precise informations about the carbohydrate structures and glycosylation sites. Synthetic glycopeptides have also been used as vaccines to induce specific immune responses or for the inhibition of protein-binding events (for example, lectin recognition). In a review paper, Westerlind et al. described the development of glycopeptide anti-tumor vaccines. He also gave a summary on the synthesis of homogenous glycoproteins and glycopeptide dendrimers for the inhibition of microbe binding events. 21 Besides, Ashford et al. have reviewed the recent advance in the synthesis of new glycopeptide antibiotics. 22 Thanks to the important biological functions of glycopeptides and glycoproteins, the development of glycopeptide and glycoprotein mimics attracted a lot of interest. 23 Aussedat et al. designed and chemically synthesized several homogeneous glycoprotein 120 (gp120) V1V2 domain containing two closely spaced high-mannose glycans at Asn 160 and Asn 156 as potential human immunodeficiency virus (HIV)-1-directed vaccines (Figure 6). 2a,24 The synthetic glycopeptides were found to have high affinity to the broadly neutralizing antibodies (bNAbs) PG9 which can effectively neutralize diverse strains of HIV-1, while the oligosaccharides alone were not active. The characterization of the antigenic properties of these glycopeptides and the evaluation of their immunogenicity in animal models are in progress.

Figure 6: Synthetic glycopeptide antigens as PG9 epitope mimics for HIV-1-directed vaccines. 2a,24 21 Westerlind, U. Beilstein J. Org. Chem. 2012, 8, 804-818. 22 Ashford, P. A.; Bew, S. P. Chem. Soc. Rev. 2012, 41, 957-978. 23 Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58-68. 24 Glycopeptoids, in which one or several carbohydrate moieties were attached to N-substituted glycine or -alanine oligomer backbone, have been developed as proteolytically stable glycopeptide mimics (Figure 7). 25 This type of glycoconjugates has greater conformational flexibility relative to glycopeptides and could be used as multivalent glycocluster constructs for targeting carbohydrate-lectin interactions or RNA biomolecules. 26 Different synthetic methods and applications of glycopeptoids have been reviewed by Szekely et al. c) their peptoid analogues. 27 Galan et al have reviewed the recent advances in the preparation of linear and cyclic glycopeptides, and their applications in glycobiology, such as antitumoral vaccines, inhibitors against pathogens and ligands for carbohydrate-binding proteins. 28 Moreover, cyclopeptides could be developed as non-immunogenic carriers with improved resistance against proteolytic degradation. Recently, Pawar et al. reported the chemical synthesis of a series of linear and cyclic glycopeptides (Figure 8) which showed the inhibition activities against wild type HIV-1 C-SA protease.

III.3 Chemical synthesis of glycopeptides and glycoproteins

Structurally defined glycopeptides and glycoproteins have been proved to play an important role in studying their diverse biological phenomena. Various methods, such as chemical or chemoenzymatic synthesis, native chemical ligation (NCL), expressed protein ligation (EPL), as well as several other assembly strategies and enzyme-based routes, have been developed for the preparation of functional glycopeptides and glycoproteins. A number of reviews on the synthesis of glycopeptides and glycoproteins can be found in the literature. 30

III.3.a Retrosynthetic analysis of glycoproteins

Glycoproteins could be considered as polyamino acids-poly/oligosaccharide conjugates. A generalized disconnective analysis of glycoprotein synthesis reported by Gamblin et al. is shown in Scheme 4, in which glycoproteins may be prepared through three strategies. 30c,30e Method I displayed that the glycoproteins could be prepared by linear assembly of preformed glycoamino acids/glycopeptides via native chemical ligation (NCL), enzymatic ligation, solid-phase peptide synthesis (SPPS), etc (disconnection B). The glycoamino acids/glycopeptides could be obtained by amino acids/peptides glycosylation (disconnection A). Convergent protein glycosylation with prepared glycan array represents the disconnection C (II, Scheme 4). Another approach is glycoprotein remodeling, in which the glycoprotein with monosaccharide moiety is synthesized firstly, and then elongation with endoglycosidases or recombinant glycosyltransferases to rebuild the new glycoprotein (III, Scheme 4). 30 (a) Crucho, C. I.; Correia-da-Silva, P.; Petrova, K. T.; Barros, M. T. Carbohydr. Res. 2015, 402, 124-132; (b) Wu, C. Y.; Wong, C. H. Chem. Commun. 2011, 47, 6201-6207; (c) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131-163; (d) Liu, L.; Bennett, C. S.; Wong, C. H. Chem. Commun. 2006, 21-33; (e) Davis, B. G. Chem. Rev. 2002, 102, 579-602; (f)

III.3.b Formation of the glycosidic linkages

The key step of all glycopeptide synthetic strategies is the formation of glycan-amino acid bond (glycosidic linkage). Several reviews have already covered the chemical synthesis of glycosidic linkages. 30c,30e,31 Herein, we list some common methods for the formation of Nand O-glycosidic linkages.

III.3.b (1) Formation of N-glycopeptides

The most common approach for the formation of N-glycosides is via reaction of a protected glycosyl amine (by using a coupling reagent) or glycosyl azide (by Staudinger reaction in the presence of triphenylphosphine (Ph 3 P), by Staudinger ligation with an Asp-derived phosphinothioester, or by phototransamidation strategy 32 ) with a suitably protected Asp derivative (Scheme 5a) to generate an amide bond. Another strategy is the direct glycosylation of the amide side chain of Asn using a glycosyl donor (Scheme 5b). 31

III Structures and functions of glycolipids

III.1 Classification of glycolipids

As an important part of the glycoconjugate family, glycolipids are comprised of lipids covalently attached to one or several monosaccharides. The hydrophilic polar sugar head group and the hydrophobic lipid moiety give their amphiphilic character, which make the glycolipids able to self-insert in cell membranes. 33 Nowadays, glycolipids are well known to play an important role in a variety of important biological phenomena such as cell-cell interactions, viral and bacterial infections, activation and modulation of immune system, signal transduction, cell proliferation, etc. [START_REF] Brandenburg | Glycolipids: Distribution and Biological Function[END_REF] Based on their lipid moieties, glycolipids can be mainly classified as glycoglycerolipids (GGLs), glycosphingolipids (GSLs) and other lipids. [START_REF]IUPAC-IUB, Joint Commission on Biochemical Nomenclature (JCBN)[END_REF] The classification and some representative structures of glycolipids are shown in Figure 9. 33 Hashim, R.; Sugimura, A.; Minamikawa, H.; Heidelberg, T. Liq. Cryst. 2012, 39, 1-17. [START_REF] Brandenburg | Glycolipids: Distribution and Biological Function[END_REF] Brandenburg, K.; Holst, O. 2015, Glycolipids: Distribution and Biological Function. eLS. 1-10. [START_REF]IUPAC-IUB, Joint Commission on Biochemical Nomenclature (JCBN)[END_REF] IUPAC-IUB, Joint Commission on Biochemical Nomenclature (JCBN). Carbohydr. Res. 1998, 312, 167-175. GGLs are glycolipids containing mono-or oligosaccharide moieties linked to the C-3 hydroxyl group of glycerol which may be attached with one or two fatty acid chains.

GSLs are comprised of a ceramide backbone covalently linked to one or several saccharide residues. GSLs can be subdivided as neutral GSLs, and acidic GSLs. In neutral GSLs family, cerebrosides are monoglycosylceramides in which the sugar residue is attached by O-ether linkage to the primary alcohol, while oligoglycosylceramides contain more than one sugar moiety. The acidic GSLs were further divided into sulfoglycosphingolipids and gangliosides containing respectively sulfonic or carboxylic acid functions. 36

III.2 Glycoglycerolipids (GGLs)

III.2.a Structures and functions of natural glycoglycerolipids (GGLs)

GGLs mainly exist in the thylakoid membranes in chloroplasts of higher plants, eukaryotic algae and cyanobacteria. Lesser amounts of GGLs are also found in animals. They demonstrated various biological activities, such as anti-tumor, anti-viral, and anti-inflammatory activities.

The common structures of GGLs extracted from natural products are mainly comprised of three types: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG) (Figure 10). For example, the chemical structures of three major glycolipids extracted from spinach were determined as MGDG, DGDG and SQDG by Mizushina's group. 37 It has been reported that both the MGDG and its monoacyl derivative (MGMG) obtained by hydrolyzing MGDG with a pancreatic lipase, can inhibit the DNA polymerase activities and the growth of NUGC-3 human gastric cancer cells, with a stronger inhibition by MGMG (Figure 10). 38 Further studies showed that the SQDG from spinach can inhibit mammalian DNA polymerase activity, human cultured cell growth and in vivo solid tumor proliferation. Both of the sulfo group and fatty acid moiety potently affected the DNA polymerase inhibition of the SQDG. 39 The natural aminoglycoglycerolipids have also shown good biological activities. From the rhizomes of an herbaceous plant serratula strangulate, Dai et al isolated the aminoglycoglycerolipid a (Figure 11) and other two GGLs. These compounds were reported to exhibit significant antibacterial and antitumor activities. 40 40,41 Another class of 6-O-ether-linked GGLs, nigricanosides A, B and their respective dimethyl ester derivatives A-1, B-1 (Figure 12) were extracted from the green alga Avrain Villea nigricans by Williams et al, which showed potent antimitotic activity and the ability to promote tubulin polymerization. 41 Zhou, B.-N.; Tang, S.; Johnson, R. K.; Mattern, M. P.; Lazo, J. S.; Sharlow, E. R.; Harich, K.; Kingston, D. G. I. Tetrahedron 2005, 61, 883-887. 42 Williams, D. E.; Sturgeon, C. M.; Roberge, M.; Andersen, R. J. J. Am. Chem. Soc. 2007, 129, 5822-5823. Figure 12: Chemical structure of ether-linked GGLs reported by Williams et al. 42 There are a number of naturally existing GGLs which have shown interesting biological activities. In a review paper, Holzl et al. have described the structures and functions of GGLs in plants and bacteria. 43 Very recently, Li and co-workers reviewed the structures and activities of GGLs from marine organisms. 44

III.2.b Modifications of glycoglycerolipids (GGLs)

Various GGLs have been extracted from the natural products and shown potent biological activities. Studies focused on the structure-activity relationships suggested that the biological activities of the glycolipids were influenced by their chemical structures. 44 Consequently, in order to obtain GGLs with the similar or better biological activities, different strategies have been developed for the modification of GGLs and synthesis of their analogues.

Modifications are mainly based on four types: A number of synthetic GGL mimics have been designed and synthesized. For example, with the purpose to obtain better Myt1-kinase inhibitors, Li et al. synthesized series of 6-amino-6-deoxy-glycoglycerolipid analogues with different glycosyl (D-gluco-, 45 D-galacto-, 46 D-manno- 47 ) moieties and lipid chains (Figure 13). Evaluation of the inhibition activity against Myt1-kinase of compounds A1-A8, and B1-B7 showed that the activities of 43 Holzl, G.;Dormann, P. Prog. Lipid. Res. 2007, 46, 225-243. 44 the galactose series were generally better than the corresponding glucose derivatives; and with the same glycosyl moiety, the increasing length of the lipid chains led to the enhancement of the activities. 46 Importantly, among these compounds, the aminogalactoglycerolipids B7 with a branched acyl chain exhibited the best inhibitory activity, and in their following research on the mannose analogues, the C7 with a branched acyl chain showed effective anti-influenza A virus (IAV) activity. 47

Figure 13: Structures of the natural aminoglycolipids A1 and glucose analogues A2-A8, 45 galactose analogues B1-B7, 46 mannose analogues C1-C8. 47 Vetro et al. reported the synthesis of new glucuronosyldiacylglycerol (GlcADG) analogues as 3-phosphorylated phosphatidylinositol (PI3P) analogues (Figure 14) which could target the protein kinase B (also known as Akt). 48 In an in vitro kinase assay, compounds Ia and IIb showed the Akt inhibitory effect, and antiproliferative activity in human ovarian carcinoma cells.

III.3 Glycosphingolipids (GSLs)

III.3.a Structures and functions of natural glycosphingolipids (GSLs)

As a most abundant and diverse class of glycolipids in animals, GSLs are found in the plasma membrane of cells and play key roles in cell-cell communication and the modulation of membrane-protein function. 49 They also could be found in fungi, plants, and invertebrates. The most widely known structures of GSLs are αor -galactosyl ceramide (α-/ -GalCer), in which a galactosyl moiety is linked to an N-acylated sphingosine backbone (ceramide).

A series of glycolipids termed agelasphins (AGLs) was isolated from the marine sponge Agelas mauritianus by Natori et al, which was known as α-GalCer. 50 The diversity of the compositions and lengths on the lipid chains lead to different agelasphins, and some structures are present in Figure 15. The AGLs-9b showed potent in vivo anti-tumor activity against the murine B16 mouse melanoma. In 1991, Harouse et al. have reported that the antibodies against the -GalCer 1 (Figure 16) can inhibit the HIV-1 infection of CD4-negative cell lines in nerve system. 51

III.3.b Modifications of glycosphingolipids (GSLs)

As GGLs, modifications of GSLs have attracted intensive research interest, in particular, since the discovery of KRN 7000 (Figure 17), a synthetic analogue of ASLs-9b. The KRN 7000 was found to possess the similar anti-tumor activity as ASLs-9b. 52 More importantly, it was reported to have potential activities against cancer, malaria, type 1 diabetes, and multiple sclerosis. Its ability to bind to CD1d and to activate invariant natural Killer T (iNKT) cells was also observed. Various efforts were made on the modification of KRN 7000, and have been summarized by Haudrechy and co-workers in 2011. 53 The four types of modifications described in this review paper are listed in Figure 18. 52 Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176-2187. 53 Banchet-Cadeddu, A.; Henon, E.; Dauchez, M.; Renault, J. H.; Monneaux, F.; Haudrechy, A. Org. Biomol. Chem. 2011, 9, 3080-3104. Very recently, by replacing the ring oxygen of the galactopyranose residue with a sulfur atom, a new KRN 7000 structural analogue (a, Figure 19) was designed by Bi and co-workers. 54 Another two 5-thio-analogues b and c, in which the modifications were taken place on both the sugar ring and the lipid chain have also been prepared. In vitro tests of the analogue b showed that it can selectively compress the production of interleukin 4 (IL-4) in the presence of relatively high interferon-(IFN-) production of NKT cells, which may be useful for developing a new potent immunostimulating agent for specific inducing iNKT cells to produce T helper 1 (Th1) cytokines. 54 Guillaume et al. designed and synthesized an aromatic α-GalCer, the 6'-naphthylurea derivative of KRN 7000 (A, Figure 20), which showed a potent Th1 response in both mice and men. 55 Based on this previous research, another two series of α-GalCers analogues B and C with modification on the 6'-position of the galactose were prepared in order to evaluate their biological activities as iNKT cell agonists. 56 Structure of the 6'-naphthylurea derivative A, 6'-carbamate derivative B and 5'-uronamide derivative C is shown in Figure 20. Various analogues of -GalCer were also synthesized and showed inhibition activity to HIV infection. For example, two analogues (C-glycoside 1 and aza-C-glycoside 2, Figure 21) were synthesized by Thota et al. It has been shown that the aza-C-glycoside exhibited a significantly higher affinity to gp120 than GalCer, whereas the C-glycoside was as active as GalCer. 57 58 Thanks to their potent biological activities, such as anti-tumor and anti-HIV, the design and synthesis of glycolipid mimics has become a useful strategy in pharmaceutical and medical research. 59

IV Conclusion

In this chapter, we have given a short literature overview on the structures and functions of glycopeptides/glycoproteins and glycolipids. Many efforts have been made for the synthesis of functional glycoconjugates. Both of the synthetic glycopeptides and glycolipids have shown interesting biological activities.

Our group has been interested in synthesizing N-oxyamide-linked compounds in the past 57 Garg, H.; Francella, N.; Tony, K. A.; Augustine, L. A.; Barchi, J. J.; Fantini, J.; Puri, A.; Mootoo, D. R.; Blumenthal, R. Antivir. Res. 2008, 80, 54-61. 58 Thota, V. N.; Brahmaiah, M.; Kulkarni, S. S. J. Org. Chem. 2013, 78, 12082-12089. 59 (a) Daniotti, J. L.; Vilcaes, A. A.; Demichelis, V. T.; Ruggiero, F. M.; Rodriguez-Walker, M. Front. Oncolo. 2013, 3, 306; (b) Wennekes, T.; van den Berg, R. J.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. Angew. Chem. Int. Ed. 2009, 48, 8848-8869; (c) McReynolds, K. D.; Gervay-Hague, J. Chem. Rev. 2007, 107, 1533-1552. few years. In this thesis, we were interested in preparing novel glycolipids and glycopeptides analogues containing the N-oxyamide linkage. In the next chapter, we will present the properties of the N-O linkage and the functions of the N-O linked compounds.

I Introduction

As described in Chapter 1, glycopeptides and glycolipids play very important biological functions in living organisms. Various methodologies have been developed for the construction of glycoconjugate mimetics which have shown interesting properties. Design and synthesis of new carbohydrate derivatives with various functions are attracting more and more research efforts.

One of the recent research subjects in our group is the synthesis of N-oxyamide-containing biomolecules mimetics. Studies on aminooxy acids by Yang's group showed that aminooxy acid derived peptides can easily organize into turns and helices structures through intramolecular hydrogen bond formation. 60 This unique property makes N-oxyamide linkage attractive for the modification of biomolecules. Furthermore, the N-oxy amide linkage is resistant to chemical and enzymatic hydrolysis, 61 and N-oxy peptides could be readily prepared using classical peptide coupling methods.

There are a large number of compounds containing an oxygen-nitrogen bond, such as hydroxylamine, hydroxamic acid, oxime, nitrone, and N-oxyamide compounds, etc. (Figure 23). In this chapter, we will present the aminooxy acids and N-O linkage in carbohydrate derivatives.

II Aminooxy acids II.1 Structure of aminooxy acids

Aminooxy acids are a class of unnatural amino acids where an aminooxyl function has been introduced in place of the amine group (Figure 24). 60 Li, X.;Wu, Y.-D.;Yang, D. Accounts. Chem. Res. 2008, 41, 1428-1438. 61 L-Canavanine, the first amino acid with a substituted hydroxylamine moiety, was isolated from the non-protein fraction of Jack bean Canvalia ensiformis by Kitagawa and co-workers in 1929 (1, Figure 25). 62 L-Canaline, which contains a free aminooxyl group, was obtained by hydrolysis of the guanidineoxyl group in L-canavanine by a liver ferment (2, Figure 25). 63 The L-canaline was reported to possess the inhibition activities against ornithine-dependent enzyme, and function as a lysine antagonist. It has also shown significant antineoplastic activity in vitro against human pancreatic cancer cells.

62,63

Another natural aminooxy acid derivative isolated by Kuehl Jr et al was D-cycloserine, a cyclic product of 2-amino-3-aminooxypropionic acid, which showed a broad spectrum of antibacterial activity (Figure 26). 62 Kitagawa, M.; Tomiyama, T. J. Biochem. 1929, 11, 265-271. 63 (a) Kitagawa, M.; Yamada, H. J. Biochem. 1932, 16, 339-350; (b) Kitagawa, M.; Monobe, S.-I. J. Biochem. 1933, 18, 333-343. 64 Rosenthal, G. A. Life Sci. 1997, 60, 1635-1641. 65 Kuehl Jr, F. A.; Wolf, F. J.; Trenner, N. R.; Peck, R. L.; Buhs, R. P.; Howe, E.; Putter, I.; Hunnewell, B. D.; Ormond, R.; Downing, G. J. Am. Chem. Soc. 1955, 77, 2344-2345. Two aminooxy analogues of peptides were synthesized by Briggs and Morley (Figure 27). 66 Compared to aspartame, its aminooxy analogue was not sweet. Unlike gastrine, its aminooxy analogue presented no stimulation of gastric acid secretion, however resistant to enzymatic hydrolysis.

II.2 Methods for the introduction of oxyamine function

The reported methods for introducing oxyamine function are mainly divided into two approaches: (i) nucleophilic substitution reaction; (ii) amination of alcohols

II.2.a Nucleophilic substitution reaction

II.2.a (1) Alcohol to oxyamine transformation

This transformation is generally performed using N-hydroxyphthalimide (PhthN-OH) under Mitsunobu-like conditions or using N-protected hydroxylamine derivatives under nucleophilic substitution condition, followed by deprotection to provide the oxyamine product.

The synthesis of free O-substituted hydroxylamine using Mitsunobu reaction was firstly reported by Grochowski and Jurczak. 67 Treatment of a primary or secondary alcohol with PhthN-OH, PPh 3 and diethyl azodicarboxylate (DEAD) furnished the O-alkyl phthalimide. Subsequently, the phthaloyl group was removed by hydrazinolysis to give the free oxyamine (Scheme 7). 68 Scheme 7: Synthesis of oxyamine compounds using Mitsunobu reaction followed by hydrazinolysis.

In the case of second alcohols, the Mitsunobu reaction leads usually to the inversion of 66 Briggs, M. T.; Morley, J. S. J. Chem. Soc., Perkin Trans I. 1979, 2138-2143. 67 Grochowski, E.; Jurczak, J. Synthesis 1976, 682-684. 68 Chemla, P. Tetrahedron Lett. 1993, 34, 7391-7394. configuration (Scheme 8). 69 Scheme 8: Mitsunobu reaction with inversion of configuration.

The proposed mechanism of the reaction between a nucleophile (NuH) and a chiral secondary alcohol in the presence of DIAD and PPh 3 is shown in Scheme 9. Reaction of PPh 3 and DIAD quickly generates a zwitterionic intermediate which is able to deprotonate the nucleophile to form the anionic nucleophile and the phosphonium ion. The alcohol then attacks the phosphonium ion to form an oxyphosphonium salt, which was attacked by the deprotonated nucleophile in an S N 2 manner to yield the final product with inversion of configuration. Different from the S N 2 mechanism of the Mitsunobu reation, the preparation of tertiary alkoxyamine reported by Palandoken et al. involves the displacement of a hydroxyl group via S N 1 mechanism. 70 The tertiary alcohol was treated with stoichiometric amount of BF 3 •Et 2 O and PhthN-OH in CH 2 Cl 2 to give the O-alkyl phthalimide. However, the S N 1 mechanism led to a loss of stereochemistry, and this method is not applicable to secondary alcohol. Hydrazinolysis of the phthalimide group afforded the (tert-alkoxy)amine product in good yield (Scheme 10). Chem. Soc. Jpn. 1967, 40, 935-939; (c) Mitsunobu, O.; Eguchi, M. B. Chem. Soc. Jpn. 1971, 44, 3427-3430. 70

II.2.a (2) Halogen to oxyamine transformation

The S N 2 displacement of halides can also be applied for the introduction of aminooxyl functions, by using N-hydroxyphthalimide, 71

II.2.b Amination of alcohols

The direct electrophilic amination of alcohols, with the advantage of retention of alcohol stereochemistry, provides another approach to prepare the aminooxy product. For example, Choong et al. reported that a substituted oxaziridine can be used as an electrophilic amination reagent for the transformation of an alkoxide nucleophile to the corresponding alkoxylamine (Scheme 13). 74 Scheme 13: Amination of alcohols to alkoxylamines. 74

II.2 α-, -and -aminooxy acids

Compared to amine function (pKa ≈ 10), the oxyamine is a weaker base (pKa ≈ 4.6) due to lone pair repulsion between adjacent nitrogen and oxygen. Likewise, the N-oxy amide bond (pKa 10-15) is more acidic than a regular amide bond (pKa ≈ 20-25) (Table 1). 75 This fact makes NH group in N-oxyamide a better hydrogen bond donor and the carbonyl group good hydrogen bond acceptor. A few family of foldamers has been developed from α-, -and -aminooxy acids. It has been reported that peptides constructed from aminooxy acids can fold into turn (α N-O turns, N-O turns, N-O turns and reverse turns) or helix structure through intramolecular hydrogen bond. These properties are very important for the specific biological functions of biomolecules which are closely related to their secondary structures. The current methodology to analyse the secondary structures mainly includes NMR spectroscopy, circular dichroism (CD) spectroscopy, X-ray crystallography and theoretical calculation, etc.

II.2.a α-Aminooxy acids

Yang and co-workers have reported that α-aminooxy acids induced a strong eight-membered-ring intramolecular hydrogen bond between adjacent residues (called α N-O turn) when it was incorporated into a peptide backbone (Figure 28). The D--aminooxy acids induce a right-handed  N-O turn while the L-enantiomers induce a left-handed N-O turn. 60 They have also shown that the homochiral oligomers of D-α-aminooxy acids adopted a right-handed 1.8 8 helix consisting of consecutive N-O turn. 76 77 The NH stretch region FT-IR spectra of these compounds in CH 2 Cl 2 at room temperature 76 (a) Peter, C.; Daura, X.; van Gunsteren, W. F. J. Am. Chem. Soc. 2000, 122, 7461-7466; (b) Wu, Y.-D.; Wang, D.-P.; Chan, K. W.; Yang, D. J. Am. Chem. Soc. 1999, 121, 11189-11196; (c) Am. Chem. Soc. 1996, 118, 9794-9795. were shown in Figure 30, with the subtraction of the spectrum of pure CH 2 Cl 2 . The spectra of compound A at the concentration of 5 mM showed a single peak at 3400 cm -1 , corresponding to the stretching frequency of the non-hydrogen-bonded NH of an N-oxyamide (A, Figure 30). Two peaks at 3386 and 3198 cm -1 , respectively assigned to the non-hydrogen-bonded amide NH (at the N-terminus) and an intramolecular hydrogen-bonded amide NH (at the C-terminus), were observed from the IR spectra of compound B at 0.5 mM (B, Figure 30). For the IR spectra of compound C, there are two major peaks at 3384 and 3294 cm -1 , and a small shoulder peak at 3427 cm -1 , which were assigned to be the stretching frequency of non-hydrogen-bonded N-oxyamide NH, hydrogen-bonded N-isobutyl amide NH and the non-hydrogen-bonded isobutyl amide NH, respectively (C, Figure 30). From left to right: A (5 mM), maximum at 3400 cm -1 ; B (0.5 mM), maxima at 3386 and 3198 cm -1 ; C (0.5 mM), maxima at 3427 (shoulder), 3384 and 3294 cm -1 . 77 The 1 H NMR chemical shifts of the amide protons for compounds A, B and C tested in CD 2 Cl 2 were listed in Table 2. For compound A, the chemical shift of N-oxy amide NH appeared at 9.74 ppm at 5 mM and without change in diluted concentration. For compound B at or below 1 mM, two singlets at 11.41 and 8.71 ppm were assigned to an intramolecular hydrogen-bonded NH, and a non-hydrogen-bonded NH, respectively. The chemical shifts of the two NH protons in compound C appeared at 8.63 ppm (singlet) and 8.28 ppm (triplet), which corresponded to the N-oxy amide NH and the isobutyl amide NH, respectively. The unusually downfield chemical shift at 8.28 ppm suggested that the C-terminus amide NH was involved in an intramolecular hydrogen bonding. 77 From the 1 H NMR and IR data of the NH in compounds A, B, C, we can suggest the general chemical shifts and FT-IR stretch regions of the hydrogen-bonded and non-hydrogen-bonded NH in amide and N-oxyamide in dichloromethane (Table 3).

Compounds

NMR (ppm)

IR (cm -1 ) Amide Non-hydrogen-bonded NH 6-7 ~3400

Hydrogen-bonded NH >8 ~3300

N-Oxyamide

Non-hydrogen-bonded NH 8-9 ~3400

Hydrogen-bonded NH >10 ~3200 Because of the acidity of their N-oxy amide protons, peptides constructed from α-aminooxy acids can also act as good receptor for anion, like carboxylate and chloride through intermolecular hydrogen bond. 78 For example, Yang and co-workers have developed two cyclic hexapeptides (A and B in Figure 32) as chloride ion receptors through intermolecular hydrogen bond. The cyclic hexapeptide A, which was comprised of alternating D,L-α-aminooxy acids, showed affinities for halides with higher selectivity for Cl -ion than F - and Br -in nonpolar solvent (Figure 32a). 78a Another cyclic hexapeptide B consisting of alternating D-α-amino and D-α-aminooxy acids functioned as an efficient chloride ion receptor (Figure 32b). A cyclic tetrapeptide C was prepared by alternating C-linked carbo--amino acid and (R)-aminooxy acid. 79 NMR and mass spectral studies of this cyclic peptide showed selective and appreciable binding affinity with the chloride ion, meaning that it could be used as a selective chloride ion receptor (Figure 33). 78

II.2.b -Aminooxy acids

-Aminooxy acids are analogues of -amino acids in which the -carbon atom is replaced by an oxygen atom. Thanks to the extra carbon atom in their backbone (compared with α-aminooxy acids), this type of aminooxy acids can provide more substitution patterns, such as 2 -, 3 -, 2,2 -, 2,3 -, and 3,3 -aminooxy acids (Figure 34). Most of them incorporated into peptide backbones induce a rigid nine membered-ring hydrogen bond between adjacent residues, which is called N-O turn (Figure 34).80 For example, the structural properties of the diamide A (Figure 35a) and triamide B (Figure 35d) were studied by X-ray crystallography and 2D NMR spectroscopy. 80b The crystal structures of A and B are shown in Figure 35b and Figure 35e, respectively. In the crystal structure of diamide A, a N-O turn between the C=O i and NH i+2 groups was observed. Another six-membered-ring hydrogen bond between the NH i+2 and NO i+1 groups further stabi-lized this structure. A well-defined helical structure of triamide B was shown in its X-ray structure. Nuclear overhauser effects (NOEs) in 2D NOESY spectra in CDCl 3 at 5 mM were summarized in Figure 35c and Figure 35f, which showed that the N-O turn structures of A and B also existed in CDCl 3 . A cis--2,3 -furanoid sugar aminooxy acid and its short oligomers were designed, synthesized and characterized by Chandrasekhar et al. 81 A series of structure studies by DFT calculation, NMR spectroscopies and MD simulation techniques suggested that the short homo-oligomers building block exhibited rigid ribbon-like secondary structures involving 5/7 bifurcated intramolecular hydrogen-bonded rings (Scheme 14).

II.2.c -Aminooxy acids

Like the construction of α and N-O turns, N-O turns are the 10-membered-ring structures which are formed through intramolecular hydrogen bonds in peptides consisting of -aminooxy acids. It has been reported that -aminooxy peptides can form turn and helix structures incorporating N-O turns. 82 For example, the secondary structures of 4 -Ph aminooxy diamide (Figure 36a) have been investigated by using FT-IR, NMR spectroscopy, and X-ray crystallography. 82a In the FT-IR spectrum, three peaks were observed at 3428 cm -1 , 3388 cm -1 , 3324 cm -1 , which were respectively assigned to the stretching frequencies of the non-hydrogen-bonded amide NH b , non-hydrogen-bonded N-oxyamide NH a and hydrogen-bonded amide NH b (Figure 36b). The stronger NH b peak at 3324 cm -1 suggested NH b was involved in a 10-membered-ring intramolecular hydrogen bond. A strong NOE signal was found between H b and the -proton in 2D-NOESY studies (Figure 36c). X-Ray crystallographic structural analysis showed the presence of N-O turn, involving a 10-membered-ring intramolecular hydrogen bond between the CO i and NH i+2 in this 4 -aminooxy peptides (Figure 36d).

III N-O linkage in sugar chemistry

For the development of glycoconjugates, various strategies have been exploited, by direct conjugation to carbohydrates or by linking the carbohydrate to a bifunctional linker which can be further functionalized. Among different approaches, use of oxyamine allows easy conjugation with carbonyl or carboxylic compounds through oxime, N-glycoside or N-oxy amide bonds. Moreover, the oxyamine function can be easily introduced and oxyamine-derived compounds are quite stable to hydrolytic cleavage. 83 The stability of hydrazine and hydroxylamine-based glycoconjugates in aqueous solution has been studied by Gudmundsdottir et al. 84 It has been proved that the hydrolysis rates accelerated under increasing acidic conditions and all the conjugates became increasingly stable as the pH approaches neutrality, with the half-live varied from 1 to 300 days. Besides, the hydrolysis rates of the glycoconjugates formed from electron-rich monosaccharides were significantly more rapid than those from electron-poor monosaccharides (xylose (fastest) > glucose > N-acetylglucosamine (slowest)) (Figure 37 and Table 4). Twenty years later, Peri et al. developed a general method for the glycosylation of oxyamine with reducing sugars where the oxyamines were used as bifunctional linker for the preparation of neoglycopeptides and neoglycolipids. 86 Thanks to the uniquely high chemical reactivity and specificity of N,O-disubstituted hydroxylamine, the reaction can be performed with unprotected and non-activated reducing sugars like glucose, galactose, mannose, N-acetylglucosamine and lactose in aqueous solution (in buffer at pH 4) or in polar solvent (generally a mixture of acetic acid and DMF). 86 The cyclic nature of the saccharide reducing unit is preserved, and this reaction proceeds chemoselectively and with high -anomeric selectivity without requirement of activation at the anomeric center and protection of functional groups. A proposed mechanism is shown in Scheme 15. The aminooxyl group B selectively reacts with the aldehyde of the reducing sugar A to give an oxy-iminium intermediate C.

Subsequent ring closure results in the formation of the N-(O-methoxy)-glycoside.

Scheme 15: Mechanism of N(OMe)glycoside formation via reaction between reducing sugars and O, N-disubstituted hydroxylamines. 86 Loskot et al. have reported that the aniline derivatives can enhance the rate of acid catalyzed neoglycosylation for a range of sugar substrates up to a factor of 32 relative to the uncatalyzed reaction (Scheme 16). 87 Scheme 16: Nucleophilic catalysis of N(OMe)-neoglycoside formation by aniline derivatives. 87 In the past few years, this chemoselective ligation method has emerged as a unique and simple strategy to prepare various N-O linked glycoconjugates (Scheme 17). The development, the scope, and the utility of the neoglycosylation reaction were reviewed by Goff and Thorson. 88 Scheme 17: Two general strategies for the conjugation of carbohydrates to oxyamine linkers. 88 The relative stability of O-linked and N(OMe)-linked glycosides to glycosidase was compared by Iqbal and co-workers. 89 From their experimental data, they suggested that degradation of aminooxy glycoconjugates in vivo is more likely to arise from acid-catalyzed, rather than glycosidase-catalyzed hydrolysis.

III.1.a (2) Preparation of oxyamine linkers

Much efforts have been devoted for the development of oxyamine linkers. 83,90 For example, Munneke et al. have synthesized a number of N-alkylmethoxyamine bifunctional linkers, which contain a N-methoxyamine functional group for conjugation to carbohydrates and a terminal group (i.e. amine, azide, thiol, or carboxylic acid) for conjugation to the probe of choice. The reported strategies are rapid and efficient for the synthesis of linkers, which can be conjugated to glycans in excellent yield and showed excellent stability towards hydrolytic cleavage (Scheme 18). 83 88 Goff, R. D.; Thorson, J. S. Med. Chem. Comm. 2014, 5, 1036-1047. 89 Iqbal, A.;Chibli, H.;Hamilton, C. J. Carbohydr. Res. 2013, 377, 1-3. 90 Scheme 18: Synthesis of azide-, amine-, thiol-and carboxylic acid-functionalized oxyamine linkers. 83 A multi-functional linker that can readily be attached to the anomeric center of reducing sugars was developed by Prudden et al. (Scheme 19). 90a Due to the lipophilicity of the Fmoc group, the Fmoc-protected linker can be used to purify glycans prepared by chemoenzymatic approaches using reverse phase column chromatography and monitor progress of enzymatic glycosylations by HPLC. Moreover, the free amine, which is helpful for glycan immobilization and array development, can be easily obtained by removing the Fmoc protecting group under mild basic conditions.

III.1.b Applications of neoglycosylation

Thanks to the simplicity and efficiency, the chemoselective neoglycosylation has been demonstrated as an excellent method for the formation of oligosaccharide analogues, glycoconjugates such as glycolipid and glycopeptide, and glycoproteins, as well as glycoarrays. 91 III. 1.b (1) Synthesis of neoglycolipids 91 Peri, F.;Nicotra, F. Chem. Commun. 2004, 623-627. A neoglycosphingolipid (N-LacCer) was prepared as a natural O-lactosylceramide (O-LacCer) mimic, in which the carbohydrate moiety was linked to a N-methoxyaminofunctionalized ceramide (Scheme 20). 92 The synthetic neoglycolipid showed potent inhibition against hydrolysis of natural O-LacCer by a recombinant endoglycoceramidase II from Rhodococcus sp.

Scheme 20: Structure of natural lactosylceramide and synthesis of non-natural N-LacCer by one step glycosylation reaction. 92

III.1.b (2) Synthesis of neoglycopeptides/glycopeptoids

A series of liponeoglycopeptide variants were prepared by neoglycosylation of a N-methoxyamine-appended vancomycin aglycon with 2-,3-,4-or 6-N'-decanoyl or biphenoyl D-glucopyranose (Figure 40). 93 This method led to good yield and excellent stereoselectivity. The activity test against vancomycin-resistant Enterococci (VRE) revealed the 3-or 4-acylated glucose was the optimal one for neoglycopeptide lipidation, while the natural ones favored the attachment of lipid on the 2-position (like teicoplanin). These results demonstrate that the natural O-glycosidic linkage in glycopeptide can be replaced with the neoglycoside N-glycosidic linkage with enhanced bioactivity. 92 Ishida, J.; Hinou, H.; Naruchi, K.; Nishimura, S. Bioorg. Med. Chem. Lett. 2014, 24, 1197-1200. 93 Griffith, B. R.; Krepel, C.; Fu, X.; Blanchard, S.; Ahmed, A.; Edmiston, C. E.; Thorson, J. S. J. Am. Chem. Soc. 2007, 129, 8150-8155. 93 Seo and co-workers reported an efficient synthesis of glycopeptoids via microwave assisted chemoselective glycosylation of N-alkyl oxyamine containing peptoid with unprotected reducing sugars (Scheme 21). 94 The protected N-alkyl aminooxy amine I was synthesized as a bifunctional linker, and then incorporated into oligopeptoids using standard solid phase peptoid synthesis procedures. Oligopeptoids II was then neoglycosylated with unprotected reducing sugars to produce the glycopeptoids III. The increasing conversion and short reaction time of the glycosylation were achieved by employing microwave irradiation. Reducing sugars like D-glucose, D-maltose, D-melibiose, and maltotriose, ligated smoothly to the peptoid with -stereoselectivity in good yield. 94 Seo, J.; Michaelian, N.; Owens, S. C.; Dashner, S. T.; Wong, A. J.; Barron, A. E.; Carrasco, M. R. Org. Lett. 2009, 11, 5210-5213. Scheme 21: Synthesis of glycopeptoids by microwave-assisted chemoselective glycosylation. 94

III.1.b (3) Synthesis of gold glyconanoparticles

The chemoselective oxime coupling was used for the synthesis of gold glyconanoparticles (glyco-AuNPs) by Thygesen et al (Scheme 22). 95 The core-shell AuNPs A carrying reactive aminooxyl groups on the organic shell was prepared firstly. Subsequently, the unprotected reducing glycans and glycopeptide aldehydes were readily anchored through a single chemoselective reaction onto the core-shell AuNPs A to generate the glyco-AuNPs B. This methodology avoids the protection and deprotection of glycans and allows direct evaluation of glycan-binding proteins interactions (colorimetric detection) with high specificity.

Scheme 22: Strategy for nanoparticle functionalization and detection of protein-glycan interactions. 95

III.1.b (4) Study of structure-biological activity relationship

Neoglycorandomization method, based upon the chemoselective formation of glycosidic bonds between reducing sugars and a secondary alkoxylamine-containing aglycon to generate a large combinatorial neoglycoside-based libraries, is a powerful tool in glycobiology and 95 Thygesen, M. B.; Sorensen, K. K.; Clo, E.; Jensen, K. J. Chem. Commun. 2009, 6367-6369. drug discovery to understand the relationship between attached sugars and biological activities in the glycosylated products (Scheme 23). 96 Scheme 23: Principle of neoglycorandomisation. 96 Thorson and co-workers synthesized a number of digitoxin (also known as cardiac glycoside) analogs with different sugar moieties (Figure 41). Some analogs display significantly enhanced Na + /K + -ATPase-inhibitory potency and tumor specific cytotoxicity. 96 By using the same method, Langenhan et al have prepared a new class of digitoxin analogs by varying the nature of N-alkoxy group (Scheme 24). 97 It has been observed that neoglycosides IIa-c and IIIa-c were more potent cytotoxins than the corresponding aglycons (Ia-c), confirming the importance of sugar attachments to the cytotoxicity of cardiac neoglycosides. 96 Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, F. M.; Thorson, J. S. Proc. Natl. Acad. Sci U S A 2005, 102, 12305-12310. 97 Langenhan, J. M.; Engle, J. M.; Slevin, L. K.; Fay, L. R.; Lucker, R. W.; Smith, K. R.; Endo, M. M. Bioorg. Med. Chem. Lett. 2008, 18, 670-673. Scheme 24: Synthesis of digitoxin analogs by oxyamine neoglycosylation. 97 A library of 37 differentially glycosylated triterpenoid betulinic acids (BA) variants were synthesized using neoglycosylation and their biological activities of anticancer and anti-HIV were examined by Goff and Thorson (Scheme 25). 98 The biological test revealed that the desired bioactivities of glycosylated BAs were predominately dictated by the appended sugars, and they were also influenced by the nature of alkoxyamine handle connection to the scaffold (i.e., ester versus amide).

HO

Scheme 25: Synthesis of betulinic acid neoglycosides. 98 Recently, the same group reported the facile synthesis of a library of 34 differentially perillyl alcohol glycosides (PG1-PG34) by neoglycosylation of the perillyl aglycon with dif- 98 Goff, R. D.; Thorson, J. S. Org. Lett. 2009, 11, 461-464. ferent sugars (Scheme 26). 99 In vitro studies show that the antiproliferative activity was dependent on the sugar moiety.

Scheme 26: Synthesis of perillyl alcohol glycosides and their IC 50 value against cell viability of two human cancer cell lines (non-small-cell lung A549 and prostate PC3). 99

III.1.b (5) N-O linkage as an anomeric protecting group

Dasgupta and Nitz reported that the N,O-dimethylhydroxylamine could be used as an anomeric protecting group in carbohydrate synthesis, as the N-methoxy-N-methyl-N--glycosides are tolerant of various protecting group manipulations, such as acylation, alkylation, silylation, acetal formation, and stable under glycosylation conditions. 100 The N-methoxy-N-methyl-N-glycosides were prepared under mildly acidic conditions by reacting with N,O-dimethylhydroxylamine hydrochloride, and then the glycoside was protected with desired protecting groups. Finally, the oxyamino group can be selectively removed with N-chlorosuccinimide (NCS) in more than 88% yield (Scheme 27). Scheme 27: Carbohydrate synthesis using N,O-dimethylhydroxylamine as an anomeric protecting group. 100 99 Nandurkar, N. S.; Zhang, J.; Ye, Q.; Ponomareva, L. V.; She, Q. B.; Thorson, J. S. J. Med. Chem. 2014, 57, 7478-7484. 100 Dasgupta, S.; Nitz, M. J. Org. Chem. 2011, 76, 1918-1921.

III.2 O-Glycosyl hydroxylamine derivatives III.2.a Preparation of O-glycosyl hydroxylamines

The O-glycosyl hydroxylamines, widely used as efficient intermediates for the preparation of glycoconjugates, were firstly reported as a new class of monosaccharides by Nashed et al. 101 The Mitsunobu reaction of PhthN-OH with reducing sugars, followed by hydrazinolysis, has been reported for the synthesis of O-glycosyl hydroxylamines.102 O-Glycopyranosyl-N-hydroxysuccinimides of glucose and lactose have been synthesized, by using peracetylated sugars, peracetylated glycosyl bromides, or benzoylated thioethyl glycosides as glycosyl donors. 103 The phase transfer catalyzed (PTC) condition was also reported and applied for the synthesis of O-glycosyl hydroxylamine with high stereoselectivity (Scheme 28). 104 In this reaction, NHS is glycosylated with protected sugars using tetra-n-butylammonium hydrogen sulfate (TBAHS) as catalyst in a CH 2 Cl 2 /1 M Na 2 CO 3 biphasic system at room temperature. The glycosylation of PhthN-OH using glycosyl fluoride as a donor and BF 3 •Et 2 O as a promoter was described by Dumy and co-workers. 105 Chen et al. reported that both of the αand -galactosyl hydroxylamine can be synthesized from the same precursor using different promoters (Scheme 29). 106 The α-glycoside was prepared by treating the peracetylated galactose with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in acetonitrile at low temperature, while the -glycoside using BF 3 •Et 2 O in CH 2 Cl 2 . The NHS group was cleaved by hydrazinolysis to produce the corresponding O-galactosyl hydroxylamine.

Scheme 29: Synthesis of αand -O-galactosyl hydroxylamine. 106 Reddy et al. described the Ferrier glycosylation of hydroxylamine derivatives promoted by various Lewis acids for the preparation of aminooxy pseudoglycals. 107 For example, the glycosylation of PhthN-OH or NHS with tri-O-protected D-glucal catalyted by zinc(II) chloride gave the corresponding aminooxy pseudoglycal with high -selectivity in good yields (Scheme 30).

Scheme 30: Synthesis of aminooxy pseudoglycal. 107 The first efficient O-glycosylation of hydroxamic acids to prepare glycosyl hydroxamates (i.e. glucosyl, galactosyl, mannosyl, glucuronyl, and ribosyl hydroxamates) in good yield was reported by Thoma et al (Scheme 31). 108 In this reaction, the glycosyl N-phenyl trifluoroacetimidates were used as glycosyl donors and TMSOTf as promoter in CH 2 Cl 2 .

Scheme 31: O-glycosylation of hydroxamic acids. 108 107 Reddy, C.; Rao, Y.; Kumar, T.; Reddy, K.; Chandrasekhar, S. Synthesis 2008, 122-126. 108 Thomas, M.; Gesson, J.-P.; Papot, S. J. Org. Chem. 2007, 72, 4262-4264. Yu's group reported a direct glycosylation of sugar oximes and HONHFmoc using glycosyl ortho-hexynylbenzoates as glycosyl donors catalyzed by PPh 3 AuOTf for the efficient preparation of N-O linked disaccharides and O-glycosyl hydroxylamines (Scheme 32). 109 Furthermore, the N-O linked saccharides could be obtained stereoselectively by reduction of the resulting oxime with NaBH 3 CN/BF 3 .Et 2 O.

III.2.b Oxime-linked glycoconjugates

Thanks to the ready formation 110 and the stability under physiological conditions of the oxime bond, oxime ligation constitutes an attractive route for the conjugation of bioorganic molecules and macromolecules, such as peptide dendrimers, protein-polymer conjugates, bifunctional chelating agents, glycoconjugates, etc.

III.2.b (1) Oxime-linked glycolipids

Two stereospecific oxime-linked galactosylceramide analogues were successfully synthesized by Wang and co-workers (Scheme 33). 106 In this synthesis, both αand -linkages were pre-installed, and then the glycolipids were prepared by oxime ligation of the αand -O-glycosyl hydroxylamines with ceramide aldehyde. Their bioactivity for activating iNKT cells has not been reported. 109 Yu, J.; Sun, J.; Yu, B. Org. Lett. 2012, 14, 4022-4025. 110 Forget, D.;Renaudet, O.;Defrancq, E.;Dumy, P. Tetrahedron Lett. 2001, 42, 7829-7832. Scheme 33: Synthesis of oxime galactosylceramide analogues. 106

III.2.b (2) Oxime-linked glycopeptides/glycoproteins

The oxime-based strategy provides an efficient way for the preparation of complex glycosyl amino acids for peptide synthesis, and the incorporation of glycopeptides into larger, full-length proteins. Marcaurelle et al reported the synthesis of oxime-linked mucin mimics containing the tumor-related Tn and sialyl Tn antigens (Scheme 34). 111 The multiple clustered ketone residues were incorporated into a peptide firstly, and then connected to aminooxy sugars by oxime ligation.

Scheme 34: Synthesis of oxime-linked mucin mimics by Marcaurelle et al. 111 The oxime chemical ligation has also been used for the preparation of homogeneously glycosylated proteins, which are important targets for fundamental research and biopharmaceutical development. Hudak et al described the chemical ligation of aminooxy glycans to recombinantly produced proteins via oxime linkages to construct site-specifically glycosylated variants of the human growth hormone (Scheme 35). 112 The aldehyde tag method was used to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site, and the aminooxy glycans were prepared by glycosylation with N-pentenoyl hydroxamate as key intermediate followed by appropriate chemoenzymatic glycosylations.

Scheme 35: Synthesis of homogeneously glycosylated proteins. 112 A tailored glycosylation method of proteins by a combination of genetic, chemical, and enzymatic methods has been reported by Smith et al (Scheme 36). 113 A genetically encoded aldehyde tag has been installed at the internal glycosylation site of the crystallizable fragment (Fc) of immunoglobulin G1 (IgG1) without perturbing protein structure. The aldehyde groups were then conjugated with aminooxy N-acetylglucosamine by oxime linkages to generate the modified Fc. Subsequent enzymatic transfer of complex N-glycans from the corresponding glycan oxazolines by an EndoS-derived glycosynthase afforded the oxime-linked glycoconjugates.

Scheme 36: Synthesis of oxime-linked glycoconjugates. 113 Dumy, Renaudet and co-workers reported the preparation of a number of oxime-linked glycocyclopeptides. 114 These neoglycocyclopeptides have been developed for various biological applications. For example, disulfide bridged compound P 3 could be used as a good candidate to fight copper overload in the liver (Scheme 37), 114a and neoglycocyclopeptide in Figure 42 for protein-glycan interaction study. 114b,c,g These multivalent neoglycopeptides have also been studied as cancer related T-antigen. 114f Scheme 37: General structure of Cu(I) chelators designed to target hepatocytes. 114a Oxime-ligation has also been employed to conjugate an oxyamine functionalized peptide with glycosyl aldehyde/ketone.

A C-glycoside analogue of the GalNAc (Tn epitope) was synthesized as a non hydrolyzable anticancer vaccine by Peri et al. (Figure 43). 115 Chemoselective convergent synthetic approach was applied for the covalent conjugation of B-epitope with a T cell epitope via an oxime bond. Based on their previous work, Peri's group reported another three neoglycopeptides, in which the full unprotected α-C-glycosyl analogue of N-acetylgalactosamine was conjugated to the segment peptides [328][329][330][331][332][333][334][335][336][337][338][339][340] OVA (Ovalbumin) and [327][328][329][330][331][332][333][334][335][336][337][338][339] OVA by an oxime bond (Figure 44). 116 Compounds A-C could be presented to and recognized by the T cell receptor, with the neoglycopeptide C containing two B-epitopes presenting higher efficiency than B.

III.3 O-Amino sugar derivatives

Carbohydrates and glycoconjugates bearing an aminooxyl function not directly linked to the anomeric carbon have been reported as multifunctional building blocks.

III.3.a Glycosyl aminooxy acids

Synthesis of diversely functionalized carbohydrate building blocks has attracted a great attention for the generation of new carbohydrate mimetics. Glycosyl aminooxy acids, containing an aminooxyl function instead of amino one, are useful building blocks for the synthesis of novel glycopeptides and glycoproteins mimetics.

Two series of O-and N-glycosylated α-aminooxy acids have been prepared by Shin and Lee (Scheme 38). 117 The O-glycosylated α-aminooxy acids were obtained by glycosylation of α-aminooxy acid derivative with glycosyl bromide, while the N-glycosylated one by coupling of glycosyl amine with (S)-malic acid derivative, followed by introduction of phthalimidoxyl function using Mitsunobu reaction.

III.3.b O-Amino sugars and glycoaminooxy acids

The first O-phthalimido sugar derivatives were synthesized by Grochowski et al. in 1976, by using Mitsunobu reaction (Figure 45). 119 Subsequent hydrazinolysis led to the corresponding O-amino sugars which were then used to prepare various oxime derivatives or reduced as O-glycosyl hydroxylamines. 120 Jensen and coll. have developed aminooxy-functionalized D-galactose template for chemoselective ligation with aldehyde-functionalized peptides (Scheme 41). 122 The glycoproteins carrying imidazoyl and pyridyl moieties have been investigated for metal ion-binding studies and for kinetic investigation of esterase activity. 123 Scheme 41: Chemoselective ligation of aminooxy-functionalized D-galactose template with aldehydefunctionalized peptides. 122 Glycoaminooxy acids, with both aminooxyl and carboxyl groups on the sugar frame were first reported by our group. From the readily available α-C-allyl glycoside, Malapelle et al. have prepared the glucopyranoid aminooxy acid as a new type of sugar building block. 124 This glycoaminooxy acid has been successfully converted into an N-oxyamide-linked disaccharide and glycopeptide mimetic (Scheme 42).

Scheme 42: Synthesis of pyranoid glycoaminooxy acid derivatives. 124 As a continuing research program in our group, Gong et al. synthesized D-ribofuranoid glycoaminooxy acid derivatives from 1,2:5,6-di-O-isopropylidene-D-glucose by introducing aminooxyl and carboxylic acid functions onto the 3-and 5-positions of the furanose ring. 125 The obtained glycoaminooxy acid has been successfully used as a building block to synthesize homo-oligomers (dimer to hexamer) as a novel type of oligosaccharide mimetics (Scheme 43a). Furthermore, Song et al also synthesized a pyranoid glycoaminooxy acid with carboxylic and aminooxyl functions onto its 2,6-positions from methyl 3,4-di-O-benzylα-D-glucopyranoside. 126 The obtained glycoaminooxy acid building unit was successfully used to construct the N-oxy amide-linked oligosaccharide mimetics via N-acylation (Scheme 43b). 124 Malapelle, A.; Ramozzi, R.; Xie, J. Synthesis 2009, 888-890. 125 Gong, Y.; Sun, H.; Xie, J. Eur. J. Org. Chem. 2009, 6027-6033. 126 Song, Z.; He, X.-P; Chen, G.-R.; Xie, J. Synthesis 2011, 2761-2766.

Scheme 43: Synthesis of glycoaminooxy acids and their oligomers. 125,126

V Conclusion

Aminooxy acids have emerged as very attractive scaffolds since the discovery of turns and helices structures in peptides containing aminooxy acids through intramolecular hydrogen bonds formation. Improved hydrolytic and metabolic stability have also been demonstrated in N-oxyamide-containing compounds. Furthermore, thanks to its high nucleophilicity, oxyamine can react readily with carbonyl and carboxylic compounds. These interesting properties make oxyamine attractive for the modification of biomolecules.

Carbohydrates bearing oxyamine group can be found in N-or O-glycosyl hydroxylamine and O-amino sugar derivatives. Neoglycosylation and oxime ligation have been proven very efficient for the preparation of various N-O linked glycoconjugates, with interesting biological activities. Glycosyl aminooxy acids and glycoaminooxy acids have been developed as multifunctional building blocks for the construction of novel glycoconjugate mimics. For the PhD work, we were interested in synthesizing orthogonally protected glyco(syl)aminooxy acids for the development of N-oxyamide-linked glycolipids and glycopeptides. Three main parts were included in this thesis. In the Chapter 3, we will focus on the preparation of glycoaminooxy acids and the N-oxyamide-linked glycolipids with lipid chains attached on the sugar frame (Figure 46). In the Chapter 4, a series of the N-oxyamide-linked -O-glycosyl glycerolipids were synthesized as novel mimics of glycoglycerolipids and glycosphingolipids (Figure 47). The preparation of glycosyl aminooxy acid and N-oxyamide-linked glycopeptides was described in Chapter 5 (Figure 47). Chapter 3

Synthesis of Glycoaminooxy Acid and N-Oxyamide-linked

Glycolipids I Introduction

Carbohydrates exist in all the living systems and play key roles in various biological and physiological processes. The rapidly growing importance of carbohydrate-based compounds in biological systems and their potential applications in the biopharmaceutical and medical fields, have attracted intensive research interest in the design and synthesis of new carbohydrate derivatives.

Sugar or glyco-aminooxy acids are sugar amino acid analogues with both a carboxylic acid and aminooxyl function connected to the sugar scaffold (Figure 48). Due to the easily hydrogen bond formation, and the resistance to chemical and enzymatic hydrolysis of the N-oxyamide bond (Chapter 2), this kind of compounds have been developed as multifunctional building blocks for the construction of novel glycoconjugate mimics through N-oxyamide or oxime bond, providing a potent tool for the design of novel functional carbohydrate mimetics. In this work, the benzyl ether was chosen as a protecting group because it is robust and stable to a wide range of acidic and basic conditions. However, the removal of the benzyl ether usually requires radical conditions (catalytic hydrogenolysis or single-electron transfer) or strong acids, which renders it incompatible with O-N and O-glycoside bond. Consequently, it was impossible to remove the benzyl groups of these pyranoid glycoaminooxy acid derivatives.

The substituted benzyl ether protecting group, p-methoxybenzyl ether (PMB), is one of the widely used hydroxyl protecting groups (Figure 50). The electron-rich PMB ether is much less stable to acidic condition than benzyl ether and can be selectively removed by aqueous mineral acid, camphor sulfonic acid (CSA) in methanol, or trifluoroacetic acid (TFA) in CH 2 Cl 2 /water. Nevertheless, their prime virtue is that they can be cleaved oxidatively with 2,4-benzoquinone (DDQ) in CH 2 Cl 2 /water or with ceric ammonium nitrate (CAN) in acetonitrile/water, in which conditions many other protecting groups such as acetals, esters, benzyl ethers, allyl ethers and TBS ethers are stable. Therefore the PMB ether can be used in the synthesis of functionally complex molecules. 127 We decided to synthesize the glycoaminooxy acid 7, in which the 3,4-hydroxyl groups were protected by PMB group, and both aminooxyl and carboxyl group attached on the sugar scaffold. From this multifunctional glycoaminooxy ester 7, we decided to introduce lipid chains to prepare new glycolipid mimics for biological applications. The N-oxyamide linked oligosaccharide mimics could be also constructed for the conformational studies (Scheme 44). In this chapter, we will present the synthesis of glycoaminooxy acid derivatives and N-oxyamide-linked glycolipids.

II Synthesis of glycoaminooxy ester II.1 Retrosynthesis

Our synthetic strategy for the preparation of pyranoid glycoaminooxy ester 7, starting from the commercially availability methyl α-D-glucopyranoside, is shown in Scheme 45. Pyranoid glycoaminooxy ester 7 could be synthesized by introducing aminooxyl and carboxylic functions onto the 2,6-positions of the pyranoid ring. The aminooxyl function could be introduced through Mitsunobu reaction onto the 6-position of 6, which in turn can be prepared through O-alkylation with ethyl bromoacetate and desilylation from the TBS ether 4. The compound 4 could be synthesized by selective protection and deprotection from methyl α-D-glucopyranoside.

II.2 Synthesis of pyranoid glycoaminooxy ester

II.2.a Synthesis of the orthogonally protected glucopyranoside 4

With our synthetic strategy described above, the first goal was the preparation of orthogonally protected glucopyranoside 4. Methyl α-D-glucopyranoside was chosen as the starting material because of its commercially availability and low cost (1kg, 250 euros). The 2-and 6-hydroxy groups were firstly regioselectively protected.

The tert-butyldimethylsilyl (TBS) group is useful for temporary protection of hydroxyl groups since it can be easily introduced and readily removed under mild conditions (by reaction with fluoride ion or by treatment with acid), [START_REF] Corey | [END_REF] and the TBS ether is stable under a wide range of conditions. The TBS group has been used for the selective protection of the primary hydroxyl groups in diols 129 , nucleosides 130 , and carbohydrate derivatives. Accordingly, TBS was chosen as the protecting group to selectively protect the 2-and 6-positions of the methyl α-D-glucopyranoside. [START_REF] Corey | [END_REF] Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972, 94, 6190-6191. 129 Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 20, 99-102. 130 Ogilvie, K. K.; Iwacha, D. J. Tetrahedron Lett. 1973, 14, 317-319. Halmos et al. have reported the selective protection of methyl α/ -D-gluco/galactopyranoside, and methyl α-D-mannopyranoside, using TBSCl in the presence of imidazole, or Et 3 N and DMAP in DMF. 131 They found that the methyl glycosides examined showed good selectivity in their reactions towards TBSCl depending on the reaction conditions. For example, treatment of methyl α-D-glucopyranoside with 2.2 equivalents of TBSCl in the presence of imidazole (2 eq.) in DMF at room temperature for 24 h, gave 6-O-TBS ether (2%), 2,6-and 3,6-di-O-TBS ether (70% and 11%, respectively), 2,3,6-or 2,4,6-tri-O-TBS ether (9%). When the imidazole (2 eq.) was changed to Et 3 N (2.4 eq.) and DMAP (0.08 eq.), the reaction was much less selective, giving 6-O-TBS ether (2%), 2,6-and 3,6-di-O-TBS ether (38% for each), and tri-O-TBS ether (12%) (Scheme 46). In our case, treatment of methyl α-D-glucopyranoside with 2.8 equivalents of TBSCl in the presence of 0.2 equivalent of DMAP in pyridine at room temperature for 16 h, followed by column chromatography gave the 2,6-O-disilylated compound 1 in 87% yield (Scheme 47). Then, for the protection of 3-and 4-hydroxyl groups of compound 1, PMB was chosen as the protecting group. When the compound 1 was treated with 6 equivalents of sodium hydride (NaH) in anhydrous DMF at 0 °C for 20 min, followed by dropwise addition of 4 equivalents of PMBCl, 132 TLC showed the low conversion rate and the presence of several products which were difficult to separate (Table 5, Entry 1).

As the sequence of addition of reactants may affect the result of the reaction, different methods were attempted to prepare the targeted product. When NaH and PMBCl were added alternately over 40 min (following the method B) to a solution of compound 1 in dry DMF at 131 Halmos, T.; Montserret, R.; Filippi, J.; Antonakis, K. Carbohydr. Res. 1987, 170, 57-69. 132 Morales, J. C.; Zurita, D.; Penadés, S. J. Org. Chem. 1998, 63, 9212-9222. 0 °C, the compound 2 was obtained in 50% yield after stirring at room temperature for 16 h (Entry 2). A better yield of 62% was obtained with the same reactants using method C, in which NaH and PMBCl were added in four portions over 40 min (NaH and PMBCl were added at the same time) (Entry 3).

Hopefully, the best yield of 80% was obtained by using method C, by adding 6 equivalents of NaH and 4 equivalents of PMBCl (1.5 equivalents of NaH and 1 equivalent of PMBCl every 20 min) to a solution of compound 1 in dry DMF under vigorous stirring at 0 °C under argon, followed by stirring at room temperature for 16 Subsequently, desilylation of compound 2 using 3 equivalents of tetra-n-butylammonium fluoride (TBAF) in THF at room temperature, afforded compound 3 with two free hydroxyl groups in 90 % yield (Scheme 48). In order to introduce carboxyl function onto the 2-position, the 6-hydroxyl group was reprotected by TBS. Treatment of 3 with 1.2 equivalents of TBSCl in pyridine at 0 °C in the presence of 0.25 equivalent of DMAP, afforded the TBS ether 4 in excellent yield (99%) after the resulting mixture being stirred at room temperature for 16 h (Scheme 48).

II.2.b Introduction of the carboxylic function through O-alkylation

O-Alkylation reaction has been successfully used for introducing the tert-butoxy carbonylmethyl group onto the 2-position of benzyl analogue of 4. 126 We then tried the similar condition to introduce the ethoxycarbonylmethyl group onto the 2-position of compound 4 (Scheme 49). Compound 4 (50 mg) was treated with NaH in dry DMF (1 mL) at 0 °C, followed by addition of ethyl bromoacetate. However, as shown in Table 6, no O-alkylation of 4 was observed with small amount of NaH (1.1 eq. to 5 eq.) and ethyl bromoacetate (1.1 eq. to 3 eq.) (Entry 1-3). Hopefully, increasing amount of NaH (10 eq.) and ethyl bromoacetate (10 eq.) resulted in 20% conversion (Entry 4). Following the report of Tomaszewski et al., 133 addition of catalytic imidazole (0.2 eq.) and tetrabutylammonium bromide (TBABr) (0.2 eq.) improved the conversion to around 30% (Entry 5). The use of TBAI (3 eq.) instead of imidazole and TBABr, followed by dropwise addition of ethyl bromoacetate, afforded a better conversion of 40% (Entry 6). In order to improving the conversion, the reactions of O-alkylation were performed under various reaction conditions, with a fixed reactants of NaH (10 eq.), BrCH 2 COOEt (10 eq.) and TBAI (3 eq.). As shown in Table 6, the conversion ratio increased to 50% when the BrCH 2 COOEt was added 2 h later than NaH and TBAI (Entry 7), which suggests that the deprotonation of the 2-hydroxyl group of 4 is very slow. The longer deprotonation time (5 h) 133 Tomaszewski, M. J.; Holme, K. R.; Perlin, A. S. Carbohydr. Res. 1991, 217, 237-244. led to the degradation of starting material (Entry 8). Interestingly, when the BrCH 2 COOEt was added dropwise more slowly (during 30 min), the conversion increased to 64% which afforded a satisfactory corrected yield of 93% (Entry 9). These results indicated that controlling the reaction time (both deprotonation and dropping time) is crucial for this reaction.

Entry

Reactants for Entry 9 (*The conversion ratio and corrected yield were calculated after column chromatography).

As described above, the most efficient conversion of 64% was observed when the compound 4 was treated with 10 equivalents of NaH and 3 equivalents of TBAI in dry DMF at 0 °C for 2 h, followed by slow, dropwise addition of 3 equivalents of ethyl bromoacetate. By investigating various amounts of reactants (2 eq., 5 eq., 10 eq.), we found that the effective conversion decreased with decreasing reactants in a fixed reaction time (Entry 9, 10, 11), which suggests that a large excess of NaH and ethyl bromoacetate is necessary for this reaction.

It is worth noting that, when this reaction was performed in small scale (less than 200 mg), TLC showed the presence of starting material and the target product (identified by NMR). However, two new spots along with the target product (44% isolated yield) appeared when this reaction was performed in larger scale (1 g), which means that part of the starting material was converted to side-products (Figure 51). The O-alkylation of 4 was then performed in small scale. Compound 4 was also treated with BrCH 2 COOEt (5 eq.) in THF under microwave heating, in the presence of NaH (5 eq.) and TBAI (3 eq.). After 45 min at 150 °C, TLC showed 20% conversion (Microwave condition: T = 150 º C; P = 2.1-2.3 bars; Stirring: 1000 rpm/min; Vol = 1mL/10 mL; t = 45 min).

We also tried the O-alkylation of 4, following a reported procedure by Feher-Voelger et al., 134 which showed an excellent yield of 89% for the O-alkylation reaction (Scheme 50). Compound A in THF at 0 º C was first treated with NaH (1.2 eq.) for 5 min. Then the mixture was added in portion to a solution of tert-butyl bromoacetate (1.5 eq.) in THF. The compound B was obtained in 89 % yield after 2 h stirring at room temperature. In our case, treatment of 4 with NaH (1.2 eq.), then ethyl bromoacetate (1.5 eq.) in THF led to no conversion.

Scheme 50: O-Alkylation of compound A. 134 Compared to the easy introduction of carboxylic function to the 2-position of compound C in our group, 126 perhaps the incomplete conversion of 4 was due to the increased steric hindrance from the PMB groups (Figure 52). Considering that the difficulty of O-alkylation of 4 may be due to the presence of PMB groups, we designed a novel synthetic route for preparing compound 5 from methyl α-D-glucopyranoside (Scheme 51). The target compound 5 could be prepared by using PMBCl to protect the 3,4-positions of compound E, which could be synthesized by 2-O-alkylation of compound D. The TBS ether D can be obtained through regioselective silylation from methyl α-D-glucopyranoside. The 6-O-silylated compound D was easily prepared by treatment of methyl α-D-glucoside with equimolar amount of TBSCl in pyridine in the presence of 0.2 equivalent of DMAP at room temperature for 24 h (Scheme 52). Unfortunately, 2-O-alkylation of compound D using NaH (1.1 eq.) and BrCH 2 COOEt (3 eq.) in DMF failed to give E, since this reaction gave very low conversion and a number of by-products which were difficult to be separated (Scheme 52).

Scheme 52: Attempted method for synthesizing E.

II.2.c Synthesis of the glycoaminooxy ester 7

With the carboxylic acid derivative 5 in hand, the glycoaminooxy ester was prepared by introducing aminooxyl function to the 6-position of the pyranose ring. Removal of the TBS group was realized by treatment of compound 5 with 3 equivalents of TBAF in THF at room temperature, affording the compound 6 in 73% yield (Scheme 53). The aminooxyl function was introduced to the 6-position of compound 6 via Mitsunobu reaction. Compound 6 was treated with 3 equivalents of PhthN-OH in toluene in the presence of 3 equivalents of PPh 3 at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds). The appearance of a dark red coloring means the success of this Mitsunobu reaction. The reaction finished after 1 h stirring at room temperature to give the target glycoaminooxy ester 7 in its fully protected form in 85% yield (Scheme 54).

III Synthesis of N-oxyamide-linked glycolipid 17 III.1 Retrosynthesis

The target N-oxyamide-linked glycolipid 17 could be synthesized by deprotecting the PMB group of glycolipid 11, which could be obtained by introducing lipid chains to the 2and 6-positions of glycoaminooxy ester 7, or through the intermediate 15 by introducing directly the first lipid chain to the 2-position of compound 4 (Scheme 55). From the obtained glycoaminooxy ester 7, the glycolipid 11 could be obtained by introducing acyl chains onto the 2-and 6-positions. Our synthetic strategy is outlined in Scheme 56. The target product could be synthesized by saponification and esterification from compound 9, which could be obtained by hydrazinolysis of 7 followed by acylation. The phthaloyl group of 7 was first removed by hydrazinolysis. Compound 7 was treated with 2 equivalents of hydrazine in MeOH at room temperature for 2 h to give the oxyamine derivative 8 in 75% yield (Scheme 57). Coupling of the obtained oxyamine 8 with equimolar quantity of palmitic acid in CH 2 Cl 2 in the presence of 2 equivalents each of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl), hydroxybenzotriazole (HOBt) and Et 3 N at room temperature, afforded the glucolipid 9 in 86% yield (Scheme 57). The proton of the N-oxyamide bond appeared at 8.04 ppm in its 1 H NMR spectrum in CDCl 3 solution.

III.2.b Synthesis of glycolipid 11 from 4

Another synthetic strategy for the preparation of glycolipid 11, starting from the compound 4, is shown in Scheme 59. Compound 11 could be synthesized by deprotection and acylation of the O-phthalimido derivative 15, which could be prepared from 13, an esterified product of compound 4. The carboxyl function was first introduced onto the 2-position through O-alkylation with ethyl bromoacetate under phase transfer conditions. 135 Compound 4 was treated with 0.5 equivalent of TBAI in a mixture of 50% aqueous NaOH and toluene (1/1, V/V), followed by dropwise addition of 2 equivalents of ethyl bromoacetate. After the resulting mixture was stirred at room temperature for 48 h, neutralized with acid and purified by column chromatography, a pure product was obtained in 74% corrected yield (conversion: 67%).

Based on the HRMS and NMR spectra ( 1 H NMR, 13 C NMR, Dept135, COSY, HMQC), the obtained molecule was identified to be the compound 12 (Scheme 60). This result suggested that the saponification was performed simultaneously in this basic condition, which removed the ethyl group to afford the carboxylic acid derivative 12. Scheme 60: Synthesis of compound 12.

The glycolipid 13 was then prepared by esterification. Coupling of the obtained carboxylic acid derivative 12 with equimolar quantity of 1-dodecanol in CH 2 Cl 2 in the presence of EDC•HCl/DMAP/Et 3 N, afforded the glycolipid 13 in 76% yield (Scheme 61). In order to introduce the aminooxyl function to the 6-position of the sugar template, the TBS group of compound 13 was removed by 3 equivalents of TBAF in THF at room temperature to give compound 14 in 71% yield (Scheme 62). Subsequently, the aminooxyl function was introduced through Mitsunobu reaction. Compound 14 was reacted with PhthN-OH (3 eq.) in toluene in the presence of PPh 3 (3 eq.) at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds). The appearance of a dark red coloring means the success of this Mitsunobu reaction. The reaction finished after 1 h stirring at room temperature to give the O-phthalimido derivative 15 in excellent yield of 98% (Scheme 62).

III.2.c Deprotection of 11

The deprotection of PMB ethers was carried out using 5% TFA in CH 2 Cl 2 at room temperature for 1 h. The target glycolipid 17 was obtained in 60% yield after purification by column chromatography (Scheme 64). The signal of NH in the N-oxyamide bond of 17 appeared at 8.74 ppm in its 1 H NMR spectrum in CDCl 3 solution. All structures of the synthetic compounds have been fully characterized by 1 H, 13 C NMR and HRMS.

IV Synthesis of glycolipid 23 IV.1 Retrosynthesis

The retrosynthetic analysis for the preparation of glycolipid 23 is depicted in Scheme 65. We chose to prepare 23 from the O-phthalimido derivative 20, on which the acyl chain could be readily ligated at the 6-position through hydrazinolysis and acylation reactions. Compound 20 is easily accessible from 18 which could be prepared by esterification of palmitic acid with 4 bearing a free hydroxyl group at the 2-position on the sugar template.

IV.2 Synthesis of glycolipid 23

The initial effort was to synthesize the compound 18 as shown in Scheme 66. Introduction of the first acyl chain on the 2-position of 4 by condensation with equimolar quantity of palmitic acid in CH 2 Cl 2 in the presence of EDC•HCl/DMAP/Et 3 N at room temperature, produced the glucolipid 18 in excellent yield of 99%. Removal of the TBS group of compound 18 using 3 equivalents of TBAF in THF at room temperature, gave the compound 19 containing a free 6-hydroxyl group in 67% yield (Scheme 67). Then the aminooxyl function was introduced to the 6-position via Mitsunobu reaction. When compound 19, PhthN-OH (3 eq.) and PPh 3 (3 eq.) were dissolved in toluene at 0 o C, followed by dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds) with the appearance of a dark red coloring, the O-phthalimido derivative 20 was produced in 98% yield after 1 h stirring at room temperature (Scheme 67). Finally, removal of the PMB group using 5% TFA in CH 2 Cl 2 at room temperature for 1 h, generated the target compound 23 in 77 % yield after purification by column chromatography (Scheme 69). The signal of NH in the N-oxyamide bond of 23 appeared at 8.55 ppm in its 1 H NMR spectrum in CDCl 3 solution.

Scheme 69: Deprotection of compound 22 to synthesize glycolipid 23.

V Conclusion

From the commercially available methyl α-D-glucopyranoside, we have successfully synthesized glycoaminooxy ester 7, in which the carboxylic acid function was introduced onto the 2-position through O-alkylation and the aminooxyl function was introduced onto the 6-position via Mitsunobu reaction (Scheme 70). It is worth mentioning that the 3-and 4-hydroxyl groups were protected by PMB ether, which can be readily removed without affecting other functional groups.

The orthogonally protected glycoaminooxy ester 7 constitutes a useful multifunctional building block for further derivatization. We have succeeded in the use of glycoaminooxy ester 7 for the efficient synthesis of new glycolipid 17, in which two lipid chains were attached on the 2-and 6-positions of pyranose ring through N-oxyamide and ester bond. This glycolipid 17 can be also prepared by introducing two lipid chains to the intermediate compound 4, from which another type of glycolipid 23 has been easily prepared (Scheme 70). We have also tried to prepare the N-oxyamide-linked oligosaccharide mimics. However, the corresponding carboxylic acid of 7 needed for coupling with the oxyamine derivative 8 was not obtained due to the ring opening side reaction of the phthaloyl group during the saponification. 125 Consequently, a carboxyl protecting group compatible with phthaloyl group should be introduced to accomplish the synthesis of N-oxyamide-linked oligosaccharides.

I Introduction

As described in Chapter 1.III, glycolipids are composed of one or several monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety like an acylglycerol (termed glycoglycerolipids, GGLs) or a ceramide (glycosphingolipids, GSLs). As part of the glycoconjugate family, they play central roles in a variety of important biological processes, such as cell-cell interactions, viral and bacterial infections, immune response, signal transduction, cell proliferation, etc. The design of glycolipid mimics has become a useful strategy in drug discovery.

The synthetic glycolipids have shown interesting biological activities. Modifications have been made on the sugar part, on the configuration and nature of the anomeric bond (C-, S-glycoside), on the polar moiety of the ceramide or glycerol, or on the lipid chains. Reports showed that these minor structural changes affected biological activities of glycolipids. The ability to undergo extensive interlipid hydrogen bonding has been considered to be fundamental for the functions of glycolipids in membranes, through imparting structural integrity to the membranes of the organisms. 36 The N-oxyamide-containing compounds have shown improved metabolic stability, and interesting secondary structures thanks to the easy H-bond formation property of N-oxyamide compounds (Chapter 2). With a continuing interest on N-oxyamide-containing biomolecules, we designed the N-oxyamide-linked glycoglycerolipids, by replacing the ester function in GGLs by a N-oxyamide (Figure 53). This kind of glycolipids, never reported in the literature, could be considered as analogues of both GGL and GSL.

II Synthesis of N-oxyamide-linked glucoglycerolipids II.1 Retrosynthesis

GGLs begin with either glucose or galactose attached in αor -linkage to the 1-hydroxyl of mono-/di-acylglycerol or ceramide. As -linked glucose unit is widely existing in natural glycolipids, we investigated first the synthesis of O-amino -glucoglycerol and N-oxyamide linked glucoglycerolipids. Since the modification of lipid chains have shown very interesting results (Chapter 1), we proposed to introduce different fatty chain in the same position to synthesize a series of compounds to study their biological activities. The N-oxyamide linked glycoglycerolipids with just one lipid chain were also targeted (Figure 54). The synthetic strategy toward N-oxyamide containing glucolipids 41-45 is presented in Scheme 71. It is noteworthy that glucolipids 41-45 have four common structural features, which contain two lipid chains at C1 and C2 positions, N-oxyamide-linkage, -glycosidic bond and the chiral center. The two lipid chains could be ligated at C2 and C3 positions of O-amino -glucoglycerol 33, which can be obtained via desilylation of compound 32. The aminooxyl function of 32 was introduced through Mitsunobu reaction on the glucosylglycerol 30, which was envisaged as a key intermediate. We assumed that the glycosyl donor α-D-glucose pentaacetate with a participating acetyl group at 2'-position could couple with (S)-1,2-di-O-benzyl-glycerol 28 to construct the -glycosidic bond. From D-(+)-mannitol as the chiral source, glycerol 28 could be prepared easily using reported conditions. 136,137 The N-oxyamide-linked glucoglycerolipids with just one lipid chain 50-52 could be synthesized by acylation of the oxyamine derivative 46 with different fatty acids. The oxyamine derivative 46 could be obtained by hydrazinolysis of 32 (Scheme 71). 136 van Boeckel, C. A. A.; Visser, G. M.; van Boom, J. H. Tetrahedron 1985, 41, 4557-4565. 137 Mannock, D. A.; Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids. 1987, 43, 113-127. Scheme 71: Retrosynthesis of N-oxyamide-linked glucoglycerolipids.

II.2 Synthesis of N-oxyamide-linked glucoglycerolipids II.2.a Synthesis of (S)-1,2-di-O-benzyl-glycerol

In order to introduce our required functions, (S)-1,2-di-O-benzyl-glycerol 28 was chosen as the glycosyl acceptor. The benzyl protecting group could be selectively removed in the presence of ester group which was used to protect hydroxyl functions of the glycosyl donor.

The synthesis of (S)-1,2-di-O-benzyl-glycerol 28 was performed following a reported procedure by van Boeckel et al. 136 The commercial D-(+)-mannitol was chosen as the starting material. The conversion of D-(+)-mannitol to 1,2:3,4:5,6-tri-O-isopropylidene-D-mannitol 24 was realized in 90% yield using a procedure developed by Wiggins, 138 by shaking mannitol and equimolar quantity of sulphuric acid in dry acetone for 6 h at room temperature (Scheme 72). 138 Wiggins, L. F. J. Chem. Soc. 1946, 13-14. Scheme 72: Synthesis of 1,2:3,4:5,6-tri-O-isopropylidene-D-mannitol 24.

For the synthesis of 3,4-O-isopropylidene-D-mannitol 25, the triacetonide 24 was firstly treated with 70% AcOH at 40 °C for 1 h 30 min following the reported condition, 137 but this condition only gave 50% yield of 25 together with fully deprotected mannitol (Entry 1, Table 7). No better result was obtained with 50% AcOH (Entry 2). A better yield of 68% was obtained with 30% AcOH at 40 °C during 1 h 30 min (Entry 3). Finally, the best result (80%) was obtained using 30% AcOH at 40 °C during 1 h (Entry 4).

Entry

Conditions Heating time Yield 1 70% acetic acid 1.5 h 50%

2

50% acetic acid 1.5 h 50%

3

30% acetic acid 1.5 h 68% The free hydroxyl functions of 25 were treated with 4.8 equivalents of benzyl bromide in the presence of 6 equivalents of sodium hydride in DMF at room temperature for 15 h, to give 1,2,5,6-tetra-O-benzyl-3,4-O-isopropylidene-D-mannitol 26 in 98% yield. Then, the isopropylidene blocking group of compound 26 was removed by hydrolysis with 1 M aqueous HCl/methanol (1/9, V/V) under reflux for 4 h, affording 1,2,5,6-tetra-O-benzyl-D-mannitol 27 in 93% yield (Scheme 73). Subsequently, oxidative cleavage of the compound 27 with 1.5 equivalents of sodium periodate (NaIO 4 ) in a mixture of H 2 O and MeOH (3/5) at room temperature for 3 h gave 2,3-di-O-benzyl-D-glyceraldehyde, which was sufficiently pure to be used without any further purification. Finally, the desired (S)-1,2-di-O-benzyl-glycerol 28 was obtained by reduction of the obtained aldehyde with 10 equivalents of sodium borohydride (NaBH 4 ) in a mixture of H 2 O and MeOH (1/3) at room temperature for 1 h. After being neutralized with acetic acid and purification by column chromatography, the pure product was produced in excellent yield (98% for two steps) (Scheme 74). 137 Scheme 74: Synthesis of (S)-1,2-di-O-benzyl-glycerol 28.

30% acetic acid 1 h 80%

II.3.2 Synthesis of 2-O-amino--glucoglycerol

In order to have high stereoselectivity and efficiency of glycosylation, we decided to use the Koenigs-Knorr procedure which usually worked well for the synthesis of -glucoside with good yield. With the presence of a participating group at the 2'-hydroxyl function, the per-O-acetyl-α-D-glucopyranosyl bromide was chosen as the glycosyl donor.

Treatment of α-D-Glucose pentaacetate with 8 equivalents of HBr (33% HBr in AcOH) in anhydrous CH 2 Cl 2 at room temperature for 6 h gave the per-O-acetyl-α-D-glucopyranosyl bromide, which was used directly for the glycosylation without further purification (Scheme 75). Condensation of equimolar quantity of (S)-1,2-di-O-benzyl-glycerol 28 with the glucosyl bromide in dry acetonitrile, in the presence of HgBr 2 (0.5 eq.) and Hg(CN) 2 (0.5 eq.), afforded -glucoglycerol derivative 29 after stirring at room temperature overnight. Dropwise addition of glycosyl bromide over a period of 30 min is crucial for the good yield (75% for two steps) (Scheme 75). 136 Scheme 75: Synthesis of -glucoglycerol derivative 29.

The 1 H and 13 C-NMR values for all the protons and carbons were assigned on the basis of Dept-135, COSY, and HMQC spectra. The large coupling constant observed for the anomeric proton of 29 appeared at δ 4.55 (d, J 1,2 = 8.2 Hz), which suggested the stereochemistry of the new glycoside bond being in the -position (the coupling constants for the anomeric proton of α-anomer is about 3 Hz). 139 Compound 29 was then debenzylated by catalytic hydrogenolysis with 10% palladium on carbon (15 wt %) in ethyl acetate containing 5% each of EtOH and AcOH at room temperature, affording 30 in 71% yield (Scheme 76). In order to introduce the oxyamine group on the expected position, TBS was chosen as the protecting group for the regioselective protection of 30. Compound 30 was treated with 1.4 equivalents of TBSCl in pyridine in the presence of catalytic DMAP (0.2 eq.), but the conversion is only about 10% after stirring at room temperature overnight. When additional 1.4 equivalents of TBSCl and 0.2 equivalent of DMAP was added to the reaction mixture for stirring another 24 h, the conversion is still about 10% (Entry 1, Table 8). A better conversion of 50% was obtained with the condition of TBSCl (1.2 eq.) and imidazole (2.5 eq.) in DMF at room temperature overnight (Entry 2). 140 Increasing amount of TBSCl (2.5 eq.) and imidazole (5.0 eq.) led to 100% conversion, but unfortunately, the obtained compound was exclusively disilylated product (Entry 3). 139 Prakash, I.; Chaturvedula, V. S. Molecules 2014, 19, 20280-20294. 140 Wang, X.; Porco, J. A. J. Am. Chem. Soc. 2003, 125, 6040-6041.

Entry

Conditions Results

1

1.4 eq TBSCl, 0.2 eq. DMAP in pyridine, then 1.4 eq TBSCl, 0.2 eq. DMAP ~90% SM 30 + 10% 31 2 1.2 eq TBSCl, 2.5 eq. imidazole in DMF ~50% SM 30 + 50% 31 3 2.5 eq TBSCl, 5 eq. imidazole in DMF 100% disilylated product Bartoszewicz et al. reported an efficient silylating system for primary, secondly and tertiary alcohols, consisting of a silyl chloride/N-methylimidazole/iodine. 141 They speculated a mechanism for this system. Firstly, the reaction of a silyl chloride with a nucleophile catalyst (such as imidazole, N-methylimidazole) forms a reactive intermediate A. Then, the expected silylated product was formed from the reaction of A with an alcohol (R-OH). In the presence of iodine, which was assumed to have a propensity to the formation of polyhalide anions (I 2 Cl -), the concentration of chloride anions could be depleted due to formation of I 2 Cl -. Thus, the equilibrium between a silyl chloride and a nucleophile catalyst could be shifted toward the reactive intermediate A, which is crucial for the formation of silylated product (Scheme 77).

Scheme 77: A putative mechanism of silylation of alcohols in the presence of iodine and a nucleophile catalyst. 141 The silyl chloride/N-methylimidazole/iodine system was attempted for the regioselective silylation of compound 30. Fortunately, the best yield of 96% was obtained by using 1.2 equivalents of TBSCl in THF, promoted by N-methylimidazole (3 eq.) and iodine (3 eq.), at 0 °C for 20 min (Scheme 78). 142 Scheme 78: Regioselective silylation of compound 30.

The oxyamine function was introduced via Mitsunobu reaction. Compound 31 was reacted with PhthN-OH (3 eq.) in the presence of PPh 3 (3 eq.) at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds). The appearance of a dark red coloring means the formation of compound 32 which is accompanied by an inversion of configuration. 143,144 The excellent yield of 90% was obtained after 1 h stirring at room temperature (Scheme 79). To remove the TBS group, compound 32 was firstly treated with 3 equivalents of TBAF in THF at room temperature overnight, but this condition deprotected also acetyl groups leading to a mixture of products. The milder condition (1.5 equivalents of TBAF in THF at room temperature for 2 h) also led to a mixture of products (Scheme 80). We then decided to realize the desilylation under acidic condition (AcCl in MeOH). 145 Catalytic AcCl (0.15 eq.) was added to a stirred solution of 32 in dry MeOH at ice-bath temperature, following by stirring at room temperature for 2 h. 5% Aqueous NaHCO 3 was added to neutralize the reaction mixture after completion of the reaction (monitored by TLC). Hopefully, this condition is efficient for removing only the TBS group, leading to the target compound 33 in 72% yield (Scheme 80). 142 Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721. 143 Schenk, S.; Weston, J.; Anders, E. J. Am. Chem. Soc. 2005, 127, 12566-12576. 144 But, T. Y. S.; Toy, P. H. Chem. Asian J. 2007, 2, 1340-1355 145 Lin, L.; Shen, Q.; Chen, G. R.; Xie, J. Bioorg. Med. Chem. 2008, 16, 9757-9763. Scheme 80: Desilylation of compound 32 using TBAF or AcCl. The oxyamine group was deprotected by hydrazinolysis. The reported methods were used to remove the phthaloyl group (Table 9). 146,147 As shown in Table 9, treatment of 33 with 3 equivalents of methylhydrazine in a mixture of CHCl 3 /MeOH (10/1) after 2 h at room temperature gave two spots on the TLC (Entry 1). Unfortunately, it was very difficult to separate the two products, and NMR of the mixture was complicated. The same two spots were observed on the TLC when we changed the solvent to a mixture of CH 2 Cl 2 /MeOH (1/1, V/V), or CH 2 Cl 2 /EtOH (1/3, V/V) (Entry 2, 3). When the compound 34 was treated with 2 equivalents of hydrazine in MeOH at room temperature for 3 h, a main spot and three other spots were observed on the TLC (Entry 4). After workup, NMR of the mixture showed the missing of acetyl group. As the acetyl group could also be removed by hydrazine 148 , mild conditions (1.1 equivalent of hydrazine, 20 min at 0 °C) were used to deprotect selectively the phthaloyl group to obtain oxyamine 35 in 96% yield (Entry 5) (Scheme 82).

II.2.c Synthesis of glucoglycerolipids

Entry

Conditions Results The most delicate step in this synthesis was the selective removal of the acetate groups of 36-40 in the presence of the fatty ester groups on the glycerol moiety. Hopefully, deacetylation of 36-40 was carried out with 12 equivalents of hydrazine (3 eq. per acetate group) at 50 °C in 85% aqueous ethanol overnight to give the corresponding glucoglycerolipids 41-45 (Scheme 84). Due to their amphiphilic nature, some glycolipids were lost during the workup, leading to low yields [41 (44%), 42 (47%), 43 (43%), 44 (61%), 45 (34%)]. All of the final N-oxyamide-linked glucoglycerolipids 41-45 have been fully characterized by 1 H, 13 C NMR and HRMS. Regretfully, the signal of NH in the N-oxyamide bond could not be observed on the 1 H NMR in CD 3 OD.

1

II.3 Synthesis of N-oxyamide-linked glucoglycerolipids with one lipid chain

From intermediate O-phthaloylamino -glucoglycerol 32, N-oxyamide-linked glucoglycerolipids with just one lipid chain (50-52) could be prepared by deprotection of phthaloyl group and introduction of lipid chains, followed by desilylation and final deacetylation (Scheme 85). Finally, acetyl groups of 47-49 were removed by using catalytic Na (0.3 eq.) in methanol at room temperature for 24 h to give the corresponding (2S)-1-O--D-glucopyranosyl-2--O-acylaminoglycerol 50-52 in excellent yields [50 (89%), 51 (100%), 52 (99%)] (Scheme 88). All of the final N-oxyamide-linked glucoglycerolipids 50-52 have been fully characterized by 1 H, 13 C NMR and HRMS. Once again, the signal of NH in the N-oxyamide bond could not be observed on the 1 H NMR in CD 3 OD.

III Synthesis of N-oxyamide-linked galactoglycerolipids III.1 Retrosynthesis

In the previous section, we have successfully prepared the N-oxy amide-linked glucoglycerolipids. In order to compare the effect of sugar modification, we have decided to synthesize N-oxyamide-linked galactoglycerolipids (Scheme 89). Similar to the synthetic route for N-oxyamide-linked glucoglycerolipids, the N-oxyamide-linked galactoglycerolipids could be prepared easily. The desired galactolipids 65-69 would be obtained by introducing two lipid chains on the 2-O-amino--galactoglycerol 57 which could be prepared through Mitsunobu reaction on galactosylglycerol 54. The galactosylglycerol 54 can be obtained via glycosylation from galactose pentaacetate and dibenzylated glycerol 28 (Scheme 90).

Scheme 90: Retrosynthesis of N-oxyamide-linked galactoglycerolipids.

III.2 Synthesis of N-oxyamide-linked galactoglycerolipids III.2.a Synthesis of 2-O-amino--galactoglycerol

For the synthesis of 2-O-amino--galactoglycerol, the -galactoglycerol derivative was firstly synthesized by glycosylation following the Koenings-Knorr method. -D-Galactose pentaacetate was converted to galactosyl bromide in about 100% yield by means of 8 equivalents of HBr (33% HBr in AcOH) in anhyd CH 2 Cl 2 at room temperature for 6 h (Scheme 91). Glycosylation of the freshly prepared per-O-acetyl-a-D-galactopyranosyl bromide with equimolar quantity of (S)-1,2-di-O-benzyl-glycerol 28 in dry acetonitrile, in the presence of HgBr 2 (0.5 eq.) and Hg(CN) 2 (0.5 eq.), gave -galactoglycerol derivative 53 after stirring at room temperature for 15 h. The galactosyl bromide was added drop by drop under vigorous stirring over a period of 30 min, leading to good yield (75% for two steps) (Scheme 90). The large coupling constant observed for the anomeric proton of 53 appeared at δ 4.54 (d, J 1,2 = 7.9 Hz), which suggested the stereochemistry of the new glycoside bond being in the -orientation. Debenzylation of 53 by catalytic hydrogenolysis with 10% palladium on carbon (0.15 eq.) in ethyl acetate containing 5% each of EtOH and AcOH at room temperature afforded -galactoglycerol 54 in 70% yield (Scheme 92). After debenzylation, regioselective silylation of 54 was realized using the efficient silylating system reported by Bartoszewicz et al, in which the presence of N-methylimidazole and iodine was required as promoter to ensure good reactivity and regioselectivity. 141 The expected compound 55 was obtained rapidly in 90% yield by condensation of -galactoglycerol 54 with 1.2 equivalents of TBSCl in THF at 0 °C for 20 min, promoted by N-methylimidazole (3 eq.) and iodine (3 eq.) (Scheme 93).

Scheme 93: Regioselective silylation of 54 to obtain 55.

The introduction of the aminooxyl function was realized via Mitsunobu reaction.

Treatment of compound 55 with PhthNOH (3 eq.) in the presence of PPh 3 (3 eq.) at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds), led successfully to the compound 56 in 97% yield (Scheme 94). 143,144 Scheme 94: Synthesis of 56 via Mitsunobu reaction.

Removal of TBS group was achieved by adding catalytic AcCl (0.15 eq.) to a stirred solution of 56 in MeOH at 0 °C, followed by stirring at room temperature for 2 h. 5% Aqueous NaHCO 3 was added to neutralize the reaction mixture after completion of the reaction (monitored by TLC). This condition was efficient for preparing the 2-O-phthaloylamino--galactoglycerol 57 in 77% yield, instead of TBAF which deprotected also acetyl groups, leading to a mixture of products (Scheme 95).

III.2.b Synthesis of galactoglycerolipids

The target N-oxyamide-linked galactoglycerolipids could be synthesized by introduction of lipid chains to O-phthaloylamino--galactoglycerol 57 and final deacetylation. Firstly, esterification of the O-phthaloylamino -galactoglycerol 57 with equimolar quantity of palmitic acid in the presence of EDC•HCl/DMAP/Et 3 N, furnished the galactolipid 58 in 93% yield (Scheme 96). Since the acetyl group could also be removed by hydrazine, mild conditions (1.1 eq. hydrazine, 20 min, at 0 o C) was used to deprotect selectively the phthaloyl group to obtain oxyamine compound 59 (93% yield). Then, coupling of the oxyamine 59 with equimolar quantity of different fatty acids in the presence of EDC•HCl (2 eq.), HOBt (2 eq.) and Et 3 N (2 eq.) at room temperature, led successfully to the N-oxyamide-linked galactoglycerolipids 60-64 in good yields [60 (88%), 61 (81%), 62 (78%), 63 (90%), 64 (92%)] (Scheme 97). On the 1 Finally, deacetylation of 60-63 was realized with 12 equivalents of hydrazine (3 eq. per acetate group) at 50 °C in 85% aqueous ethanol to give the corresponding galactoglycerolipids 65-68 [65 (47%), 66 (50%), 67 (52%), 68 (58%)] (Scheme 98). The signal of NH in the N-oxyamide bond could not be observed on the 1 H NMR in CD 3 OD. For the deprotection of glucoglycerolipids with one lipid chain (Chapter 4.II.3), we used 0.3 equivalent of sodium in MeOH at room temperature for 24 h to remove the acetyl group, and the final N-oxyamide-linked glucoglycerolipids 50-52 were obtained in excellent yield. The same condition (0.3 eq. Na in MeOH, at room temperature for 24 h) was used to remove the acetyl group of compound 60. A new deacetylated product was obtained, but unfortunately, it was not the expected product 65 after comparing NMR spetra (Scheme 99 and Figure 55, Figure 56). Based on the HRMS and NMR spectra ( 1 H NMR, 13 C NMR, Dept135, COSY, HMQC), the obtained molecule was identified to be the compound 70 (Scheme 100). The proof of NMR and reaction mechanism will be discussed. In conclusion, both conditions (3 eq. hydrazine per OAc or 0.3 eq. Na/MeOH) could remove acetyl groups, but the transesterification condition with MeONa can lead to the intramolecular transacylation (Scheme 100 and Scheme 102).

When the compound 63 was treated with 0.3 equivalent of Na in MeOH at room temperature for 24 h, the transacylated product was not obtained. Comparison of the 1 H NMR spectra of obtained product with compound 68 shows that one lipid chain was missing. Based on the HRMS and NMR spectra, the obtained molecule was identified to be compound 72 which has just one lipid chain attached (Scheme 103). Once again, comparison of the 13 C NMR spectra revealed that the C I moved to higher field from δ 63.4 ppm in 68 to δ 60.9 ppm in 72, while the C II moved to lower field, from δ 83.8 ppm in 68 to δ 87.4 ppm in 72 (Figure 60).

IV Conclusion

In this chapter, we have successfully achieved the first synthesis of N-oxyamide-linked -glycolipids as novel mimics of glycoglycerolipids and glycosphingolipids.

From readily available D-glucose/galactose pentaacetate and D-mannitol, the -glycosylglycerols 30/54 were synthesized by glycosylation. Through Mitsunobu reactions, the aminooxyl functions were successfully introduced to afford the 2-O-amino -glycoglycerols 33/57. Different lipid chains were easily introduced on hydroxyl and aminooxyl functions to access to a variety of glycolipid structures (Scheme 105). Synthesis of (S)-1,2-di-O-benzyl-glycerol 28 has also been optimised (Scheme 104).

The cytotoxicity of the synthesized glycoglycerolipids against the cancer cell lines H929 and Hela have been evaluated in collaboration with Dr Xiaopeng He at East China University of Science and Technology, showing no significant activity. Scheme 104: Synthesis of (S)-1,2-di-O-benzyl-glycerol 28.

Scheme 105: Synthesis of N-oxyamide-linked glycoglycerolipids.

I Introduction

As described in Chapter 1, glycopeptides and glycoproteins play an important role in various cellular activities and pathogenic processes. The development of glycopeptide and glycoprotein mimics is of great interest. Most of the interesting functions performed by proteins in biological systems, such as molecular recognition, electron transfer and catalysis, are closely related to their unique secondary and tertiary structures. Understanding the secondary structures of the designed molecule is also required for the theoretical and pharmaceutical significance.

Studies on aminooxy-containing peptides showed that they can easily form intramolecular hydrogen bonds with turns and helices structures, and the N-oxyamide linkage is stable to chemical and enzymatic hydrolysis (Chapter 2). We are interested in the synthesis of glycosyl aminooxy acids and peptides, where an aminooxyl function has been introduced in place of the amine group in natural ones (Figure 61). In this chapter, we decided to synthesize the -O-glucosyl 2(R)-and 2(S)-aminooxy esters as novel glycopeptide mimics, in which the aminooxy acid was linked to the glucose unit through O-glycosidic bond. From the multifunctional glycosyl aminooxy esters, we proposed to prepare the N-oxyamide-linked linear as well as the cyclic glycopeptides (Scheme 106).

Scheme 106: Structures of the designed -O-glycosyl aminooxy esters and novel glycopeptides.

II Synthesis of the -O-glycosyl aminooxy esters I.1 Retrosynthesis

I.2 Synthesis of (2R)-3-O-glycosyl aminooxy ester 73

With the (2S)-1-O-glycosyl phthalimidooxy alcohol 33 in hand, the (2R)-3-O-glycosyl aminooxy ester 73 was prepared by oxidation. Treatment of the primary alcohol 33 with 2 equivalents of pyridinium chlorochromate (PCC) in the presence of 10 equivalents of acetic anhydride (Ac 2 O) and 20 equivalents of t-BuOH in CH 2 Cl 2 at room temperature for one night, led successfully to the compound 73 in 61% yield (Scheme 108).

Scheme 108: Synthesis of (2R)-3-O-glycosyl aminooxy ester 73.

I.3 Synthesis of (2S)-3-O-glycosyl aminooxy ester 77

In order to prepare the (2S)-3-O-glycosyl aminooxy ester 77 from compound 31, an epimerization on the C-2 position of (2S)-glycoglycerol 31 to generate the corresponding diastereomer (2R)-glycoglycerol 74 was proposed (Scheme 109). There are several different synthetic methods for the epimerization, including protocols based on the Mitsunobu reaction, sequential oxidation/reduction routes, and enzymatic methods. In our case, for the preparation of the (2S)-3-O-glycosyl aminooxy ester 77 from compound 31, two methods were used. One was based on the Lattrell-Dax epimerization, and the other was based on the Mitsunobu reaction.

I.3.a Epimerization based on Lattrell-Dax method

The Lattrell-Dax method, in which the free hydroxyl group of the carbohydrate compound was firstly converted into a triflate, followed by a nitrite-mediated substitution, is a very efficient and convenient method to generate structures with the inversion of configuration (Scheme 110). 149,150 149 Lattrell, R.; Lohaus, G. Justus Liebigs Ann. Chem. 1974, 901-920. 150 In order to confirm the configuration of compound 74, the 1 H and 13 C NMR spectra of 31 and 74 were compared in Figure 62. It was shown that the H II in both of compounds 31 and 74 appeared as a multiple due to the coupling with two vicinal CH 2 , while its chemical shift moved to lower δ value, from δ 3. 83-3.77 From the obtained diastereomer 74, the synthesis of (2S)-3-O-glycosyl aminooxy ester 77 was carried out in three steps (Scheme 112). The aminooxyl function was first introduced into the C-2 position through Mitsunobu reaction. Treatment of compound 74 with PhthN-OH (3 eq.) in toluene in the presence of PPh 3 (3 eq.) at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds) with the appearance of a dark red coloring, led successfully to the 2-O-phthalimidooxy derivative 75 with inversion of configuration in 88% yield (Scheme 112).

Subsequently, to remove efficiently the TBS group without deprotecting acetyl groups, compound 75 was treated with catalytic AcCl (0.15 eq.) in MeOH for 2 h (monitored by TLC) and then neutralized with 5% NaHCO 3 to afford the corresponding alcohol 76 in 87% yield after purification (Scheme 112).

Finally, oxidation of the primary alcohol with PCC (2 eq.) in the presence of Ac 2 O (10 eq.) and t-BuOH (20 eq.) The 1 H and 13 C NMR values for all the protons and carbons of compounds 73 and 77 were assigned on the basis of Dept-135, COSY, and HETCOR spectra. Comparison of the 1 H NMR spectra of compounds 73 and 77 in CDCl 3 revealed the chemical shift of H-2 moved to higher field, from δ 4.93 ppm in 73 to δ 4.77 ppm in 77, while the anomeric proton H-1' moved to lower field, from δ 4.63 ppm in 73 to δ 5.00-4.97 ppm in 77 (Figure 63). Moreover, there were also different degrees of downfield shifts of the signals of H-3', 4', 5' on the sugar frame (H-3': from δ 5.21 ppm in 73 to δ 5.29-5.20 ppm in 77; H-4': from δ 5.07 ppm in 73 to δ 5.10 ppm in 77; H-5': from δ 3.74 ppm in 73 to δ 3.83 ppm in 77). It was noted that the H-3' of 73 appeared as a t (J = 9.6 Hz) at 5.21 ppm, while the H-3' of 77 appeared as a multiple. From the compared 13 C NMR spectra of 73 and 77, we can observe that the chemical shifts of C-1', 2, 5' in 73 and 77 moved very slightly (Figure 64). In a previous work of our group, 118 we have synthesized the (2S)-and (2R)-C-glucosyl aminooxy ester as novel glycosyl amino acid building blocks from C-allyl glycoside. Based on 1 H NMR spectroscopic data of these two compounds, we had observed that the CH-2 of the (2S)-C-glucosyl aminooxy ester appeared as a t (J = 6.0 Hz) at 4.13 ppm, while the CH-2 of (2R)-C-glucosyl aminooxy ester appeared as a dd (J = 2.8 and 11.0 Hz) at 4.16 ppm (Figure 65). In our case, from the 1 H NMR spectra of compounds 73 and 77, we observed the similar coupling multiplets of the CH-2. The CH-2 of the (2S)-3-O-glucosyl aminooxy ester 77 appeared as a t (J = 3.4) at 4.77 ppm, while the CH-2 of the (2R)-3-O-glucosyl aminooxy ester 73 appeared as a dd (J = 3.5 and 7.0 Hz) at 4.93 ppm (Figure 66). These results help us to confirm the configuration of C-2 stereogenic center of the obtained O-glucosyl aminooxy esters, and may be useful for suggesting the configuration of this type of compounds.

I.3.b Epimerization based on Mitsunobu reaction

The Mitsunobu reaction was often used for the inversion of configuration in secondly alcohols using DEAD or DIAD and PPh 3 , which has been described in Chapter 2. 69 The reaction proceeds with clean stereochemical inversion, and the reaction conditions are compatible with many functional groups, which make it a popular method for the inversion of stereogenic center.

In Chapter 4, the (2S)-2-O-phthalimidooxy derivative 32 has been prepared. We tried to cleave the O-N bond to obtain the compound 74 (Scheme 113). Compound 32 was first treated with 0.03 equivalent of CuSO 4 •5H 2 O in MeOH, 151 but no reaction was observed on the TLC after stirring the mixture at room temperature overnight (Entry 1, Table 10). Treatment of 32 with 0.15 equivalent of azobisisobutyronitrile (AIBN) and 1.1 equivalents of tri-n-butyltin hydride (n-Bu 3 SnH) in benzene under reflux for 2 h led to the degradation of 32 (Entry 2). 152 Finally, hydrogenolysis of 32 (35 mg) with 10% palladium on carbon (35 wt %) in MeOH 125,153 at room temperature afforded 74 in 54% yield (Entry 3). Compound 74 obtained from 32 showed exactly the same spectroscopic data as the compound obtained using the Lattrell-Dax method. When we enlarged the scale of this reaction to 1.4 g, the TLC showed the presence of compound 74. However, the TBS group was lost during the work up and column chromatography to give the compound 78 in 76% yield (Scheme 114). Scheme 114: Hydrogenolysis of compound 32 in large scale.

The compound 78 has been fully characterized by 1 H, 13 C NMR and HRMS. The (2S)-configuration of 78 was confirmed by comparing the NMR spectra with compound 30 (Figure 67). It was clearly observed from 1 H NMR spectra that the chemical shift differences between H III a and H III b in compound 78 is much larger than that of 30. No big difference was observed in the chemical shifts of H I and H II between compounds 78 and 30. As for the O-TBS derivatives 31 and 74, there is also a slight upfield shift of the signal of C III , from δ 72.6 ppm in 30 to δ 71.9 ppm in 78. In Chapter 4, the regioselective silylation of compound (2R)-glucoglycerol 30 was realized by using 1.2 equivalents of TBSCl in THF, promoted by N-methylimidazole (3 eq.) and iodine (3 eq.), at 0 º C for 20 min. However, no reaction was observed when the isomer 78 was treated in the same conditions. Addition of more TBSCl (1.5 eq.) was not helpful for this reaction (Entry 1, Table 11). Treatment of 78 with TBSCl (1.2 eq.) and DMAP (0.2 eq.) in pyridine at room temperature for 16 h failed again to give compound 74 (Entry 2).

Entry

Conditions Results

1

TBSCl (1.2 eq.), N-methylimidazole (3 eq.) and iodine (3 eq.) in THF, 0 º C for 20 min, then TBSCl (1.5 eq.) was added, RT, 2 days

No reaction 2 TBSCl (1.2 eq.) and DMAP (0.2 eq.) in pyridine, RT, 16 h No reaction Due to the failed protection of compound 78 using TBS group, we tried to protect the primary alcohol of 78 with triphenylmethyl (Tr) group. Hopefully, treatment of 78 with 1.2 equivalents of TrCl in pyridine under reflux for 24 h led to the compound 79 in a corrected yield of 92% (Conversion: 62%) (Scheme 115).

The regioselectively tritylated compound 79 allowed the introduction of aminooxyl group onto the C-2 position through Mitsunobu reaction. Treatment of compound 79 with PhthNOH (3 eq.) in toluene in the presence of PPh 3 (3 eq.) at 0 °C, followed by slow dropwise addition of 3 equivalents of DIAD (1 drop every 5 seconds) with the appearance of a dark red coloring, led successfully to the 2-O-phthalimidooxy derivative 80 with inversion of configuration in 97% yield (Scheme 115).

Finally, detritylation of compound 80 on treatment with catalytic AcCl (0.15 eq.) in MeOH at 0 º C to room temperature for 5 In conclusion, the (2S)-3-O-glucosyl phthalimidooxy alcohol 76 has been synthesized through two routes from (2R)-glucoglycerol 31. The total yields (from 31 to 76) of the two synthetic routes were 34% (based on Latterell-Dax epimerization) and 43% (based on Mitsunobu reaction). The desired (2S)-3-O-glucosyl aminooxy ester 77 has been easily obtained by oxidation of compound 76.

III Synthesis of N-oxyamide-linked glycopeptides III.1 Retrosynthesis

For the preparation of the target N-oxyamide-linked linear and cyclic glycopeptides, the disaccharide A was proposed to be synthesized firstly. As shown in Scheme 116, the N-oxyamide-linked disaccharide A could be synthesized by coupling the carboxylic acid derivative 81 with the oxyamine derivative 82. Both compounds 81 and 82 could be prepared from the (2R)-O-glucosyl aminooxy ester 80 by acidic hydrolysis and hydrazinolysis, respectively.

III.2 Synthesis of N-oxyamide-linked glycopeptides III.2.a Attempted synthesis of N-oxyamide-linked disaccharide A

In order to be used in the following coupling reactions, the protecting group of compound 73 was selectively removed. Firstly, removal of the t-Bu group of compound 73 with 10% TFA in CH 2 Cl 2 at room temperature gave the carboxylic acid derivative 81 in 84% yield (Scheme 117).

Scheme 117: Hydrolysis of 73 to prepare 81.

The carboxylic acid derivative 81 could also be synthesized by oxidation of the (2S)-1-O-glycosyl phthalimidooxy alcohol 33 with Jones reagent. 154 Compound 33 was treated with Jones reagent (3 eq.) at room temperature for 16 h, affording compound 81 in 62% yield (Scheme 118).

Scheme 118: Oxidation of 33 to prepare 81.

Hydrazinolysis of 80 with hydrazine (1.1 eq.) under mild conditions (0 º C, 20 min) deprotected selectively the phthaloyl group to give the oxyamine derivative 82 in 93% yield (Scheme 119). It is worthy noted that the purification of 82 by chromatography may lead its partial decomposition due to the instability of O-NH 2 function in silica. A fast column chromatography was necessary to obtain the pure product. With the two monomers in hand, we were interested in the preparation of disaccharide A by coupling the carboxylic acid 81 with oxyamine 82 (Scheme 120). The carboxylic acid 81 was reacted with equimolar quantity of the oxyamine 82 in a mixture of DMF/CH 2 Cl 2 (1/2, V/V) at 0 º C in the presence of EDC•HCl (1.8 eq.) and HOBt (1.8 eq.). After stirring the mixture at room temperature for 24 h, no reaction was observed on the TLC (Entry 1, Table 12). Addition of more coupling reagent failed again (Entry 2). Under the coupling conditions of DCC (2 eq.), HOBt (2 eq.) and Et 3 N (2 eq.) in CH 2 Cl 2 at 0 º C to room temperature for 24 h, the reaction led to the degradation of starting materials (Entry 3). Then, the carboxylic acid 81 was coupled with equimolar quantity of glycine methyl ester (GlyOMe) (Scheme 122). As shown in Table 13, reaction of 81 and GlyOMe•HCl in DMF/CH 2 Cl 2 (1/2, V/V) in the presence of EDC•HCl (1.8 eq.), HOBt (1.8 eq.) and Et 3 N (2.8 eq.) in CH 2 Cl 2 at 0 º C to room temperature for 2 days, led to several products which are difficult to be separated (Entry 1). When compound 81 was treated with 1.1 equivalents of isobutylchloroformate (i-BuOCOCl) and 3 equivalents of Et 3 N in anhydrous CH 2 Cl 2 at -10 º C for 10 min, 155 followed by addition of a solution of GlyOMe•HCl in DMF/CH 2 Cl 2 , a new product was obtained after 18 h stirring at room temperature (Entry 2). The NMR spectrum of the new product showed the absence of phthaloyl group, which mean that the coupling reaction failed to give the expected glycopeptide again. From these results, we speculated that the failure of the coupling reaction of 81 with 82 or GlyOMe may be attributed to the presence of the phthaloyl group which was sterically bulky and might hide the carboxylic acid function. We decided to replace the phthaloyl group with other protecting groups to attempt the synthesis of disaccharide.

III.2.b Replacement of the Phth protecting group with Fmoc

The fluorenylmethoxy carbonyl (Fmoc) protecting group is one of the widely used amino-protecting groups in peptide synthetic processes. It can be readily introduced by reacting the amine with an active 9-fluorenylmethyl carbonate, such as Fmoc-Cl, Fmoc-OSu, or Fmoc-OBt. 156 Most importantly, the Fmoc group has been shown to have an excellent acid stability, which allows the selective removal of other protection groups (e.g. Boc, Ot-Bu) in its presence. It can be easily removed under very mild basic conditions, such as 20% piperidine or 50% morpholine in DMF. [START_REF] Greene | Protective Groups in Organic Synthesis[END_REF] Treatment of the oxyamine 82 with 3 equivalents of Fmoc-OSu in CH 2 Cl 2 at room temperature for 4 days gave the Fmoc protected compound 84 in 79% yield (Scheme 123). Subsequent hydrolysis of 84 with 10% TFA at room temperature overnight afforded the carboxylic acid 85 in 100% yield. Finally, reaction of the obtained carboxylic acid 85 with 1.2 equivalents of glycine t-butyl ester (GlyOtBu) in the presence of EDC•HCl (2 eq.) and HOBt (2 eq.) in CH 2 Cl 2 at 0 º C to room temperature overnight, led successfully to the glycopeptide 86 in 79% yield (Scheme 123). In the 1 H NMR spectrum of 86 in CDCl 3 , there were two new 156 Unfortunately, by using the same coupling conditions, reaction of the carboxylic acid 85 and oxyamine 86 failed to give the desired disaccharide B (Scheme 124). In summary, the glycosyl carboxylic acid 85 can be coupled with GlyOtBu. However its reaction with the glycosyl oxyamine 82 to disaccharide B was unsuccessful.

III.2.c Attempted synthesis of the N-oxyamide-linked disaccharide C

We then changed our mind to synthesize the glycosyl aminooxy ester derivative 89, in which both of the carboxylic acid and oxyamine functions were attached with an amino acid. In this structure, both of the functional carboxylic acid and amine were not adjacent to the C-2, which can decrease the steric hindrance of the coupling reaction for the synthesis of disaccharide C (Scheme 125). The compound 89 could be synthesized by attaching the amino acids Fmoc-Gly-OH and GlyOtBu to the oxyamine and carboxylic acid functions of compound 82, respectively.

Scheme 125: Retrosynthesis of the disaccharide C.

For the preparation of glycosyl aminooxy ester derivative 89, the oxyamine 82 was firstly treated with equimolar quantity of Fmoc-Gly-OH in CH 2 Cl 2 in the presence of EDC•HCl (2 eq.) and HOBt (2 eq.) at 0 º C. After reaction at room temperature overnight, the compound 87 was obtained in 64% yield (Scheme 126). Subsequent hydrolysis of 87 with 10% TFA at room temperature overnight afforded the carboxylic acid 88 in 77% yield. It is interesting to notice that the oxyamide NH signal appeared at 10.5 ppm in the 1 H NMR spectrum of compound 88, suggesting that the oxyamide NH might be involved in an intramolecular hydrogen bonding. Further studies by FT-IR and NMR titration are needed to investigate its secondary structure.

Finally, coupling of the carboxylic acid 88 with equimolar quantity of GlyOtBu in the presence of EDC•HCl (2 eq.) and HOBt (2 eq.) in CH 2 Cl 2 at 0 º C to room temperature, led successfully to the glycopeptide 89 in 48% yield (Scheme 126). In the 1 H NMR spectrum of 89 in CDCl 3 , there were three NH signals appearing at 9.99, 8.78 and 5.56 ppm, which demonstrated the success of the coupling reaction. Le Grel et al. have reported that both of the hydrazide (CO-NH-N) and N-oxy amide linkages (CO-NH-O) displayed the Z/E isomerism, and the chemical shifts of the Z-isomer and E-isomer were different. [START_REF] Grel | [END_REF] In the 1 H NMR spectrum of Ac-NH-OMe, two signals of NH were observed, which reflected the presence of Z-and E-forms of the N-oxyamide bond (Figure 68). In our work, the 1 H NMR spectrum of 87 in CDCl 3 showed a similar feature. We observed two resolved O-NH-CO signals at 9.50 and 8.73 ppm, which suggest the presence of Z/E isomerism of compound 87 (Figure 69).

IV Conclusion

In this chapter, we have successfully prepared the (2R)- As a new class of glycosyl amino acid building blocks, these multifunctionalized glycosyl aminooxy esters could be useful for the preparation of novel glycopeptide mimics through oxime, amide, and N-oxy amide linkage. From the glycosyl aminooxy ester 73, we have succeeded in the synthesis of N-oxyamide-linked glycopeptides 86 and 89 (Scheme 129). The acetyl groups can also be removed by catalytic Na/MeOH, but this condition led to a transacylation reaction, producing new glycolipids. About 15 new glycoglycerolipids have been synthesized. Their activity towards iNKT cells will be tested.

As perspectives, the sulfo-glycoglycerolipids could be obtained by sulfation of obtained glycoglycerolipids. The α-glycoglycerolipids could be also prepared by using our developed methodology.

The (2R) and (2S)-3--O-glycosyl aminooxy ester 73/77 have been successfully prepared as a new class of glycosyl amino acid building blocks, as described in Chapter 5. The (2R)-3--O-glycosyl aminooxy ester 73 was easily prepared by oxidation of the (2S)-1-O-glycosyl phthalimidooxy alcohol 33. Synthesis of (2S)-3--O-glycosyl aminooxy ester 77 has been developed in two synthetic routes, which were based on the Lattrell-Dax epimerization and the Mitsunobu reactions from the same glycoglycerol derivative 31. These multifunctionalized glycosyl aminooxy esters could be useful for the preparation of novel glycopeptide mimics through oxime, amide, and N-oxy amide linkage. From the (2R)-3--O-glycosyl aminooxy ester 73, N-oxyamide-linked glycopeptides 86 and 89 have been successfully synthesized. These oxyamine-functionalized glycopeptides offer possibilities for the development of novel glycopeptides mimics through N-oxy amide bond, oxime ligation or neoglycosylation.

I. General

All commercial available reagents purchased from Sigma-Aldrich, TCI Chemical and Carlo-Erba Reagents, etc. were used without further purification. All solvents for column chromatography were used without purification. The reactions carried out under anhydrous conditions are performed under argon in glassware previously dried in an oven. Methanol is dried over molecular sieve 3Å. Tetrahydrofuran (THF), dimethylformamide (DMF), dichloromethane, acetonitrile and toluene previously were dried through alumina cartridge using a solvent purificator MBRAUN SPS-800. Petroleum ether used refers to the fraction boiling in the range 60-90 o C.

All reactions were monitored by TLC on Silica Gel 60F-254 plates with detection by UV (254 nm or 365 nm) or by spraying with 10% H 2 SO 4 in EtOH and heating about 30 s at 400-600 º C.

All Column chromatography was performed on Silica gel 60 (40-60 μm).

Melting points were determined with a Kofler melting point apparatus.

Optical rotations were measured using a Jasco P-2000 polarmeter at room temperature in a 10 cm, 1 mL cell.

NMR spectra were recorded on a JOEL ESC-400 spectrometer in CDCl 3 or CD 3 OD solution. Chemical shift was given in units of parts per millon related to TMS or solvent proton ( 1 H: CHCl 3 in CDCl 3 , 7.27 ppm; CH 3 OH in CD 3 OD, 3.31 ppm; 13 C: CHCl 3 in CDCl 3 , 77.16 ppm; CH 3 OH in CD 3 OD, 49.00 ppm) as internal reference.

High-resolution mass Spectra (HRMS) were recorded on a Q-TOF MaXis using standard conditions or Bruker Microflex TM MALDI-TOF mass spectrometry.

II. General Procedures Desilylation:

Procedure A: To a solution of silylated compound (1 eq.) in anhydrous THF (15 mL/mmol) under argon, was added TBAF (3 eq.). After stirring at room temperature overnight, the solvent was removed under vacuum. The residue was diluted with EtOAc (25 mL/mmol), washed with saturated aq NH 4 Cl (10 mL/mmol), H 2 O (10 mL/mmol) and brine (10 mL/mmol), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel to afford the silylated compound.

Procedure B: To a solution of silylated compound (1 eq.) in anhydrous MeOH (20 mL/mmol), was added AcCl (0.15 eq.) at 0 º C. The resulting mixture was stirred at room temperature for 2 h under argon. After completion of the reaction, 5% NaHCO 3 was added to neutralize the reaction mixture, and then the solvent was removed under vacuum. The residue was taken up in H 2 O-CH 2 Cl 2 (1/1, 30 mL/mmol) and the aqueous layer was extracted with CH 2 Cl 2 (3×20 mL/mmol). The combined organic layers were washed with brine (20 mL/mmol), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel to afford the desilylated compound.

Mitsunobu Reaction:

Procedure C: To a solution of alcohol (1 eq.), Ph 3 P (3 eq.) and PhthNOH (3 eq.) in toluene (20 mL/mmol) at 0 º C under argon, was added DIAD (3 eq.) dropwise. The resulting mixture was stirred at room temperature for 1 h and then extracted with EtOAc (3×30 mL/mmol). The combined organic layers were washed with saturated aq NaHCO 3 (3×30 mL/mmol), H 2 O (30 mL/mmol) and brine (30 mL/mmol), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel to afford the O-phthalimido compound.

Hydrazinolysis:

Procedure D: To a solution of O-phthalimido compound (1 eq.) in MeOH (20 mL/mmol), was added N 2 H 4 •H 2 O (2 eq.). The mixture was stirred at room temperature for 2 h and then extracted with CH 2 Cl 2 (3×50 mL/mmol). The combined organic layers were washed with saturated aq NaHCO 3 (50 mL/mmol), H 2 O (50 mL/mmol) and brine (50 mL/mmol), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel to afford the O-amino compound.

Procedure E: To a solution of O-phthalimido compound (1 eq.) in anhydrous MeOH (20 mL/mmol), was added N 2 H 4 •H 2 O (1.1 eq.). The resulting mixture was stirred at 0 º C for 20 min under argon and then extracted with CH 2 Cl 2 (3×50 mL/mmol). The combined organic layers were washed with saturated aq NaHCO 3 (50 mL/mmol), H 2 O (50 mL/mmol) and brine (50 mL/mmol), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel to afford the oxyamine compound.

Coupling Reaction for N-oxyamide Formation:

Procedure F: To a solution of carboxylic acid (1 eq.) in anhydrous CH 2 Cl 2 (15 mL/mmol), was added HOBt (2 eq.), EDC•HCl (2 eq.) and Et 3 N (2 eq.) under argon at 0 º C. After stirring for 20 min, the oxyamine derivative (1 eq.) was added. The resulting mixture was stirred at room temperature overnight. The solution was diluted with EtOAc (100 mL/mmol), washed with aq HCl (1N, 2×40 mL/mmol), saturated aq NaHCO 3 (2×40 mL/mmol) and brine (40 mL/mmol), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel to give the N-oxyamide.

Esterification:

Procedure G: To a solution of carboxylic acid (1 eq.) in anhydrous CH 2 Cl 2 (15 mL/mmol), were added EDC•HCl (2 eq.), DMAP (2 eq.) and Et 3 N (2 eq.) under argon at 0 º C. After the mixture being stirred for 20 min, the alcohol (1 eq.) was added. The resulting mixture was stirred at room temperature overnight. The solution was diluted with CH 2 Cl 2 (50 mL/mmol), washed with aq HCl (1N, 2×40 mL/mmol), saturated aq NaHCO 3 (2×40 mL/mmol) and brine (40 mL/mmol), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel to give the ester.

Deacetylation:

Procedure H: To a solution of acetylated compound (1 eq.) in 85% EtOH (50 mL/mmol) was added N 2 H 4 •H 2 O (12 eq.). The mixture was stirred at 50 º C overnight. The solution was poured into ice-cold brine and extracted with CHCl 3 (3×100 mL/mmol). The combined CHCl 3 layers were dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel to give the deacetylated compound.

Procedure I: To a solution of acetylated compound (1eq.) in anhydrous MeOH (20 mL/mmol), was added sodium methoxide (0.3 eq.). After completion of the reaction, cation exchange resin was added to neutralize the solution. The resin was filtered and the filtrate was concentrated to afford the deacetylated compound.

Methyl 2,6-di-O-tert-butyldimethylsilyl-α-D-glucopyranoside (1)

Chemical Formula: C 19 H 42 O 6 Si 2 Exact Mass: 422.2520 g/mol To a solution of methyl α-D-glucopyranoside (0.97 g, 5 mmol) in pyridine (10 mL) under argon at 0 º C, were added TBSCl (2. 1 g, 14 mmol) and DMAP (0.12 g, 1 mmol). After stirring at room temperature for 16 h, the solvent was removed under vacuum, and the concentrated mixture was partitioned between CH 2 Cl 2 (50 mL) and H 2 O (50 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×50 mL). The combined organic layers were dried over MgSO 4 , filtered, concentrated, and purified by column chromatography over silica gel (petroleum ether/EtOAc = 10/1) to afford compound 1 (1.82 g, 86.3%) as a colourless paste. R f = 0.66 (petroleum ether/EtOAc = 5/1). 1 H NMR (400 MHz, CDCl 3 ): δ 4.60 (d, J = 3.7 Hz, 1H, H-1), 3.87-3.74 (m, 3H, H-3,6), 3.63-3.57 (m, 1H, H-5), 3.55-3.45 (m, 2H, H-2,4), 3.37 (s, 3H, OCH 3 ), 2.78 (s, 2H, 2×OH), 0.89, 0.89 (2×s, 18H, 2×t-Bu), 0.12-0.04 (m, 12H, 2×Si(CH 3 ) 2 ). 13 C NMR (100 MHz, CDCl 3 ): δ 100.0 (C-1), 74.1 (C-3), 73.6 (C-2), 71.8 (C-4), 70.8 (C-5), 64.1 (CH 2 ), 55.3 (OCH 3 ), 26.0, 25.9 (CH 3 , t-Bu); 18.5, 18.3 (C q , t-Bu); -4.4, -4.5, -5.3, -5.3 (CH 3 , Si(CH 3 ) 2 ). To a solution of compound 1 (4.5 g, 10.6 mmol) in anhydrous DMF (50 ml) under argon at 0 º C, was added NaH (60%, 2. 55 g, 63.8 mmol) and PMBCl (5.8 ml, 42.4 mmol) in portions over 1 h. After addition, the solution was stirred at room temperature for 16 h. Ice was added to destroy the excess NaH, and then the solvent was evaporated. The residue was diluted with EtOAc (200 mL), washed with saturated aq NH 4 Cl (100 mL), H 2 O (100 mL) and brine (100 mL), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel (petroleum ether/EtOAc = 20/1) to afford compound 2 (5.64 g, 80%) as a colourless paste.

(NaH and PMBCl was added at the same time, 1/4 equivalent every 20 min.) R f = 0.48 (petroleum ether/EtOAc = 10/1).

[α] D = +36.3 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7. 33-7.20 (m, 4H, PMB), 6.90-6.82 (m, 4H, PMB), 4.79-4.40 (m, 4H, 2×OCH 2 ), 4.33 (d, J = 3.6 Hz, 1H, H-1), 3.96 (t, J = 8.9 Hz, 1H, H-3), 3.80, 3.78 (2×s, 6H, 2×OCH 3 ), 3.72-3.59 (m, 2H, H-6), 3.53-3.46 (m, 1H, H-5), 3.31-3.19 (m, 5H, H-2,4, OCH 3 ), 0.94, 0.85 (2×s, 18H, 2×t-Bu), 0.13-0.02 (m, 12H, 2×Si(CH 3 ) 2 ). 13 C NMR (100 MHz, CDCl 3 ): δ 159. 4, 159.1, 130.8, 130.6 (C q , PMB); 130.0, 129.3, 113.8 (CH, PMB); 98.0 (C-1), 80.1 (C-2), 79.2 (C-4), 74.6 (CH 2 ), 74.1 (C-3), 73.3 (CH 2 ), 71.4 (C-5), 62.6 (CH 2 ), 55.4, 54.8 (OCH 3 ); 26.0, 26.0 (CH 3 , t-Bu); 18.4, 18. 3.81, 3.80 (2×s, 6H, 2×OCH 3 ), 3.79-3.67 (m, 2H, H-6), 3.62-3.57 (m, 1H, H-5), 3.42 (t, J = 9.4 Hz, 1H, H-4), 3.36-3.27 (m, 4H, H-2, OCH 3 ), 2.43 (s, 1H, OH), 1.67 (s, 1H, OH). 13 C NMR (100 MHz, CDCl 3 ): δ 159. 7, 159.5, 130.5, 130.1 (C q , PMB); 129.9, 129.9, 114.1, 114.0 (CH, PMB); 97.7 (C-1), 79.4 (C-2), 77.0 (C-4), 74.3 (CH 2 ), 73.5 (C-3), 72.9 (CH 2 ), 70.3 (C-5), 62.1 (CH 2 ), 55.4, 55.3 (OCH 3 ). To a solution of compound 3 (2. 53 g, 5.82 mmol) in pyridine (40 mL) under argon at 0 º C, were added TBSCl (1.09 g, 7.27 mmol) and DMAP (0.18 g, 1.46 mmol). After stirring at room temperature for 16 h, the solvent was removed under vacuum, and the concentrated mixture was partitioned between CH 2 Cl 2 (100 mL) and H 2 O (50 mL). The aqueous layer was extracted with CH 2 Cl 2 (2×50 mL). The combined organic layers were dried over MgSO 4 , filtered, concentrated, and purified by column chromatography over silica gel (petroleum ether/EtOAc = 4/1) to afford compound 4 (3.15 g, 98.7%) as a colourless paste. R f = 0.55 (petroleum ether/EtOAc = 2/1).

J = 9.2 Hz, 1H, H-3),
[α] D = +58.7 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7.32-7.24 (m, 4H, PMB), 6.92-6.84 (m, 4H, PMB)

Methyl 2-O-ethoxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-6-O-tert-butyldimethylsilyl-α-D-glucopyranoside (5)

Chemical Formula: C 33 H 50 O 10 Si Exact Mass: 634.3173 g/mol To a solution of compound 4 (200 mg, 0.36 mmol) in DMF (5 mL) under argon at 0 º C, was added NaH (60%, 144 mg, 3.6 mmol) and TBAI (386 mg, 1. 1 mmol). After stirring at 0 º C for 2 h, ethyl bromoacetate (0.4 mL, 3.6 mmol) was added drop by drop during 30 min, and the resulting mixture was stirred at room temperature for 15 h. The reaction was quenched with ice, and the mixture was extracted with EtOAc (3× 20 mL). The combined organic layers were washed with saturated aq NH 4 Cl (20 mL) and brine (20 mL), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel (petroleum ether/EtOAc = 4/1) to afford compound 4 (72 mg, conversion: 64%) and compound 5 (135 mg, corrected yield: 92.5%) as a colourless paste. R f = 0.55 (petroleum/EtOAc = 3/1).

[α] D = +27.3 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7. 35-7.23 (m, 4H, PMB), 6.94-6.83 (m, 4H, PMB), 4.86 (d, J = 10.5 Hz, 1H, OCH), 4.71-4.47 (m, 6H, OCH, 2×OCH 2 , H-1), 4.22 (q, J = 7.0 Hz, 2H, OCH 2 ), 3.88-3.72 (m, 9H, 2×OCH 3 , H-3,6), 3.59-3.47 (m, 3H, H-2,4,5), 3.29 (s, 3H, OCH 3 ), 1.27 (t, J = 7.1 Hz, 3H, CH 3 ), 0.96-0.86 (m, 9H, t-Bu), 0.09-0.02 (m, 6H, Si(CH 3 ) 2 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170.4 (C=O), 159. 5, 159.3, 130.9, 130.4 (C q , PMB); 129.8, 114.0, 113.9 (CH, PMB); 97.8 (C-1), 83.7, 79.8, 77.4 (CH); 74.8, 73.0 (CH 2 ); 71.4 (CH), 71. 1, 62.4, 60.8 (CH 2 ); 55.4, 54.9 (OCH 3 ); 26.0 (CH 3 , t-Bu), 18.4 (C q , t-Bu), 14.4 (CH 3 ), -5.0, -5.2 (CH 3 , Si(CH 3 ) 2 ).

HRMS (ESI) m/z:

[M+Na] + Calcd for C 33 H 50 NaO 10 Si 657.3071; Found 657.3065.

Methyl 2-O-ethoxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-α-D-glucopyranoside (6)

Chemical Formula: C 27 H 36 O 10 Exact Mass: 520.2308 g/mol Desilylation of compound 5 (0.50 g, 0.79 mmol) according to the general procedure A and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) afforded compound 6 (0.3 g, 73%) as a colourless paste. R f = 0.48 (petroleum/EtOAc = 1/2).

[α] D = +34.7 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7.39-7.25 (m, 4H, PMB), 6.95-6.84 (m, 4H, PMB), 4.88 (d, J = 11.0 Hz, 1H, OCH), 4.74-4.36 (m, 6H, OCH, 2×OCH 2 , H-1), 4.21 (q, J = 7.0 Hz, 2H, OCH 2 ), 3.92-3.77 (m, 7H, 2×OCH 3 , H-3), 3.76-3.62 (m, 2H, H-6), 3.60-3.46 (m, 3H, H-2,4,5), 3.29 (s, 3H, OCH 3 ), 1.27 (t, J = 7.1 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170.3 (C=O), 159. 6, 159.5, 130.5, 130. The phthalimido group was introduced at the 6-postion of compound 6 (0.3 g, 0.58 mmol) using Mitsunobu reaction according to the general procedure C. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 3/1) afforded compound 7 (0.33 g, 85%) as a colourless paste. R f = 0.47 (petroleum ether/EtOAc: 1/1).

[α] D = +47.3 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7. 86-7.72 (m, 4H, Phth), 7.35-7.26 (m, 4H, PMB), 6.90-6.82 (m, 4H, PMB)

Methyl 6-O-amino-2-O-ethoxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-α-D-glucopyranoside (8)

Chemical Formula: C 27 H 37 NO 10 Exact Mass: 535.2417 g/mol Hydrazinolysis of compound 7 (67 mg, 0.1 mmol) according to the general procedure D and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) afforded compound 8 (40 mg, 75%) as a colourless paste. R f = 0.25 (petroleum ether/EtOAc: 1/1).

[α] D = +30.3 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7. 38-7.22 (m, 4H, PMB), 6.97-6.82 (m, 4H, PMB), 4.91-4.35 (m, 7H, 3×OCH 2 , H-1), 4.20 (q, J = 7.0 Hz, 2H, OCH 2 ), 3. 93-3.72 (m, 9H, 2×OCH 3 , H-3,6), 3.72-3.65 (m, 1H, H-5), 3.57-3.45 (m, 2H, H-2,4), 3.31 (s, 3H, OCH 3 ), 1.26 (t, J = 7.1 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170.3 (C=O), 159. 5, 159.4, 130.6, 130.2 (C q , PMB); 130.0, 129.9, 114.0, 113.9 (CH, PMB); 98.0 (C-1), 83.5, 79.5, 76.8 (CH); 74.8, 74.4, 73.0, 70.

Methyl 2-O-ethoxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-6-O-palmitoylamino-α--D-glucopyranoside (9)

Chemical Formula: C 43 H 67 NO 11 Exact Mass: 773.4714 g/mol Compound 9 was prepared from compound 8 (70 mg, 0.13 mmol) and palmitic acid according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 3/2) afforded compound 9 (86 mg, 86%) as a white solid. R f = 0.48 (petroleum ether/EtOAc: 1/1).

[α] D = +39.7 (c 0.1, CHCl 3 ). M.p. 75 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 8.04 (s, 1H, NH), 7.39-7.21 (m, 4H, PMB), 6.97-6.81 (m, 4H, PMB), 4.95-4.35 (m, 7H, 3×OCH 2 , H-1), 4.20 (q, J = 7.0 Hz, 2H, OCH 2 ), 4.11-3.99 (m, 2H, H-6), 3.88-3.68 (m, 8H, 2×OCH 3 , H-3,5), 3.61-3.46 (m, 2H, H-2,4), 3.31 (s, 3H, OCH 3 ), 2.43-1.92 (m, 2H, CH 2 ), 1.66-1.54 (m, 2H, CH 2 ), 1.45-1.17 (m, 27H, 12×CH 2 , CH 3 ,) 0.88 (t, J = 6.4 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 7, 170.2 (C=O); 159.6, 159.4, 130.3, 130.2 (C q , PMB); 129.8, 114.0, 113.9 (CH, PMB); 98.1 (C-1), 83.4, 79.5, 76.3 (CH); 74.7, 73.0, 70.9 (CH 2 ); 69.5 (CH), 60.9 (CH 2 ), 55.5, 55.4 (OCH 3 ); 33.4, 32.0, 29.8, 29.6, 29.5, 25.5, 22.8

Methyl 2-O-carboxylmethyl-3,4-di-O-(4-methoxybenzyl)-6-O-palmitoylamino-α-D-glucopyranoside (10)

Chemical Formula: C 41 H 63 NO 11 Exact Mass: 745.440 g/mol To a solution of compound 9 (74 mg, 0.096 mmol) in MeOH (2 mL), was added LiOH (23 mg, 0.96 mmol). The mixture was stirred at room temperature for 48 h and then extracted with CH 2 Cl 2 (3×10 mL). The combined organic layers were washed with aq HCl (1N, 10 mL) and brine (10 mL), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel (CH 2 Cl 2 / MeOH: 25/1) to afford compound 10 (59 mg, 83%) as a white solid. R f = 0.33 (CH 2 Cl 2 / MeOH: 25/1).

[α] D = +62.0 (c 0.1, CHCl 3 ). M.p. 115 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 8.14 (s, 1H, NH), 7. 35-7.15 (m, 4H, PMB), 6.95-6.77 (m, 4H, PMB), 4.82-4.52 (m, 5H, 2×OCH 2 , H-1), 4.44 (d, J = 17.4, OCH), 4.36 (d, J = 17.4, OCH), 4.20-3.93 (m, 2H, H-6), 3.81, 3.79 (2×s, 6H, 2×OCH 3 ), 3.76-3.57 (m, 3H, H-3,4,5), 3.49 (dd, J = 9.5, 3.1 Hz, 1H, H-2), 3.30 (s, 3H, OCH 3 ), 2.43-1.95 (m, 2H, CH 2 ), 1.68-1.52 (m, 2H, CH 2 ), 1.36-1.06 (m, 24H, 12×CH 2 ), 0.88 (t, J = 6.4 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 172.1, 171.0 (C=O); 160.0, 159.7 (C q , PMB); 130.4, 130.2 (CH, PMB); 129.5, 128.5 (C q , PMB); 114.2, 114.1 (CH, PMB); 97.2 (C-1), 82.2, 78.0, 77.4 (CH); 74.9, 74.3, 72.6, 70.5 (CH 2 ); 69.5 (CH), 55.6, 55.4 (OCH 3 ); 33.4, 32.0, 29.8, 29.6, 29.5, 25.4, 22.8 4, 32.0, 29.8, 29.6, 29.5, 29.4, 28.7, 26.0, 25.5, 22.8 [α] D = +23.0 (c 0.1, CHCl 3 ).

Methyl 2-O-dodecyloxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-α-D-glucopyranoside (14)

Chemical Formula: C 37 H 56 O 10

Exact Mass: 660.3873 g/mol Desilylation of compound 13 (74 mg, 0.096 mmol) using TBAF according to the general procedure A and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) afforded compound 14 (45 mg, 71%) as a colourless paste.

R f = 0.10 (petroleum ether/EtOAc = 3/1).

[α] D = +56.7 (c 0.1, CHCl 3 ).

Methyl 2-O-dodecyloxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-6-O-phthalimido-α-D-glucopyranoside (15)

Chemical Formula: C 45 H 59 NO 12

Exact Mass: 805.4037 g/mol

The phthalimido group was introduced at the 6-postion of compound 14 (55 mg, 0.083 mmol) using Mitsunobu reaction according to the general procedure C. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 3/1) afforded compound 15 (66 mg, 98%) as a colourless paste.

R f = 0.77 (petroleum ether/EtOAc: 1/1).

[α] D = +65.7 (c 0.1, CHCl 3 ). 55.6, 55.3 (OCH 3 ); 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 28.7, 26.0, 22.8 33.3, 32.1, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 28.6, 25.9, 25.4, 22.8 (CH 2 ); 14.3 (CH 3 ).

HRMS (ESI) m/z:

[M+Na] + Calcd for C 37 H 71 NaNO 9 696.5027; Found 696.5025.

Methyl 3,4-di-O-(4-methoxybenzyl)-2-O-palmitoyl-6-O-tert-butyldimethylsilyl-α-D-glucopyranoside (18)

Chemical Formula: C 45 H 74 O 9 Si

Exact Mass: 786.5102 g/mol Esterfication of compound 4 (0.62 g, 1. 13 mmol) with palmitic acid according to the general procedure G and purification by column chromatography over silica gel (petroleum ether/EtOAc = 8/1) afforded compound 18 (0.88 g, 99%) as a colourless paste.

R f = 0.78 (petroleum ether/EtOAc: 3/1).

[α] D = +26.0 (c 0.1, CHCl 3 ).

Methyl 3,4-di-O-(4-methoxybenzyl)-2-O-palmitoyl-α-D-glucopyranoside (19)

Chemical Formula: C 39 H 60 O 9

Exact Mass: 672.4237 g/mol Desilylation of compound 18 (0.82 g, 1.04 mmol) according to the general procedure A and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) afforded compound 19 (0.48 g, 67%) as a white paste.

R f = 0.13 (petroleum ether/EtOAc = 3/1).

[α] D = +23.7 (c 0.1, CHCl 3 ). 34.7, 32.1, 29.8, 29.7, 29.5, 29.4, 25.1, 22.8 55.7, 55.4 (OCH 3 ); 34.7, 32.1, 29.8, 29.7, 29.5, 29.4, 25.1, 22.8 34.7, 32.0, 29.8, 29.6, 29.5, 29.4, 25.1, 22.8 34.7, 33.5, 32.1, 29.8, 29.7, 29.5, 29.4, 25.1, 22.8

. 13 C NMR (100 MHz, CDCl 3 ): δ 175.0, 171.9 (C=O); 100.0 (C-1), 75.5 (CH 2 ), 75. 0, 71.4, 70.7, 68.1 (CH); 55.7 (OCH 3 ), 34.6, 33.2, 32.1, 29.8, 29.6, 29.5, 29.4, 29.2, 25.3, 25.1, 22 (m, 2H, H-6'), 3.99-3.69 (m, 3H, H-2,3), 3.65 (ddd, J = 10.0, 4.5, 2.3 Hz, 1H, H-5'), 3.59-3.57 (m, 2H, H-1), 2.07, 2.04, 2.03, 2.01 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 9, 170.5, 169.6, 169.5 (C=O); 138.5, 138.3 To a solution of compound 30 (2 g, 4.74 mmol), N-methylimidazole (1.14 mL, 14.22 mmol) and iodine (3.61 g, 14.22 mmol) in anhydrous THF (20 mL) M.p. 72 ºC.

1 H NMR (400 MHz, CDCl 3 ): δ 7. 87-7.74 (m, 4H, Phth), 5.22-5.15 (m, 1H, H-3'), 5.03 (t, J = 10. 1 Hz, 1H, H-4'), 4.97 (m, 1H, H-2'), 4.64 (d, J = 7.8 Hz, 1H, H-1'), 4.32-4.26 (m, 1H, H-2), 4.22 (dd, J = 12.3, 4.9 Hz, 1H, H-6'a), 4.14-4.06 (m, 2H, H-3a,6'b), 4.01 (dd, J = 11.6, 6.3 Hz, 1H, H-3b), 3.83-3.66 (m, 3H, H-1,5'), 2.07, 2.04, 1.99, 1.97 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 8, 170.3, 169.6, 169.5 (C=O), 164.6 (C=O, Phth); 135.0, 128.8 (C q ); 124.0 (Phth), 100.9 (C-1'), 88.1 (CH), 72.8 (C-3'), 71.9 (C-5'), 71.1 (C-2'), 68.5 (C-4'), 67.8, 61.9, 60.4 (CH 2 ); 20.8, 20.7, 20.7 (OAc). ), 0.88 (t, J = 6.9 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 173. 5, 170.8, 170.4, 169.6, 169.5, 163.6 (C=O); 134.8, 128.9 (C q ); 123.8 (CH), 100.9 (C-1'), 84.5 (CH), 72.9 (C-3'), 72.0 (C-5'), 71.1 (C-2'), 68.5 (C-4'), 67.8, 62.4, 62.0, 34.1, 32.1, 29.8, 29.8, 29.6, 29.5, 29.4, 29.3, 24.9, 22.8 (CH 2 ); 20.9, 20.8, 20.7, 14.3 (CH 3 68.5 (C-4'), 68.0, 62.0, 61.9, 34.3, 32.1, 29.8, 29.6, 29.5, 29.4, 29.3, 25.1, 22.8 (CH 2 ); 20.9, 20.8, 20.7, 14.2 (CH 3 (m, 6H, 2×CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 174. 4, 170.8, 170.3, 169.6 (C=O); 100.7 (C-1'), 82.2 (CH), 72.5 (C-3'), 72.1 (C-5'), 71.5 (C-2'), 68.4 (C-4'), 67.9, 61.8, 61.4, 34.2, 33.2, 32.1, 31.5, 29.8, 29.8, 29.6, 29.5, 29.4, 29.2, 25.2, 25.0, 22.8, 22.5 (CH 2 ); 20.9, 20.7, 14.3, 14.1 (CH 3 34.3, 34.2, 32.2, 32.0, 29.8, 29.6, 29.5, 29.4, 29.2, 25.9, 24.9, 22.8 (CH 2 ); 20.9, 20.8, 20.7, 14.2, 14.1, 12.1 (CH 3 68.4 (C-4'), 67.8, 61.8, 61.3, 34.2, 33.3, 32.0, 29.8, 29.6, 29.5, 29.4, 29.2, 25.5, 24.9, 22.8 (CH 2 ); 20.8, 20.7, 14.2 (CH 3 61.8, 61.3, 34.2, 33.3, 32.0, 29.9, 29.8, 29.6, 29.5, 29.4, 29.2, 27.3, 25.5, 25.0, 22.8 0, 63.3, 62.7, 34.9, 33.7, 33.1, 32.4, 30.8, 30.6, 30.5, 30.2, 26.3, 25.9, 23.7, 23.4 0, 63.3, 62.7, 34.9, 33.8, 33.1, 32.9, 30.8, 30.6, 30.5, 30.2, 30.2, 26.6, 25.9, 23.8, 23.7 34.8, 33.3, 33.1, 30.8, 30.6, 30.5, 30.2, 26.9, 25.9, 23.7, 23.7 9, 63.0, 62.5, 34.7, 33.6, 32.8, 30.5, 30.3, 30.2, 30.0 26.3, 25.6, 23.4 83.7, 78.0, 77.9, 75.0, 71.6 (CH); 69.0, 63.4, 62.7, 34.9, 33.8, 33.1, 30.8, 30.7, 30.5, 30.4, 30.2, 28.2, 26.6, 26.0, 23.8 61.8, 60.4, 33.0, 31.7, 29.1, 29.0, 25.3, 22.7 (CH 2 ); 21.0, 20.8, 20.7, 14.1 (CH 3 61.8, 60.4, 33.0, 32.0, 29.7, 29.6, 29.4, 29.3, 25.4, 22.8 (CH 2 ); 21.1, 20.9, 20.7, 14.2 (CH 3 61.8, 60.4, 33.0, 32.0, 29.8, 29.6, 29.4, 29.2, 29.0, 27.3, 25.3, 22.7 (CH 2 ); 21.0, 20.8, 20.7, 14.2 (CH 3 ), 0.90 (t, J = 6.9 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CD 3 OD): δ 173.9 (C=O), 104.7 (C-1'), 87.4, 78.0, 75.0, 71.6 (CH); 69.0, 62.7, 60.9, 33.7, 33.1, 30.8, 30.6, 30.5, 30.4, 30.2, 26.6, 23.7 0, 62.7, 60.9, 33.7, 33.1, 30.8, 30.6, 30.5, 30.3, 30.2, 28.1, 26.6, 23.7 4.44-4.30 (m, 1H, H-2), 4.28-4.11 (m, 3H, H-3a,6'), 4.05 (dd, J = 11.6, 6.3 Hz, 1H, H-3b), 4.01-3.92 (m, 1H, H-5'), 3.84 (dd, J = 13.8, 2.8 Hz, 1H, H-1a), 3.73 (dd, J = 13.8, 4.1 Hz, 1H, H-1b), 2.16, 2.10, 2.07, 1.99 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 6, 170.4, 170.3, 169.8 (C=O) ), 0.88 (t, J = 6.9 Hz, 3H, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 173. 7, 170.6, 170.4, 170.4, 169.6 (C=O); 101.8 (C-1'), 79.1 (CH), 71.0 (C-3'), 70.7 (C-5'), 68.9 (C-2'), 68.4 (CH 2 ), 67.1 (C-4'), 62.8, 61.3, 34.3, 32.1, 29.8, 29.6, 29.5, 29.4, 29.3, 25.1, 22.8 (CH 2 ); 20.8, 20.8, 14.3 (CH 3 -4'), 61.4, 61.2, 34.1, 33.1, 31.9, 31.4, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 25.1, 24.9, 23.9, 22.7, 22.4 (CH 2 ); 21.0, 20.9, 20.7, 20.6, 14.2, 14.0 (CH 3 ). 4.42 (dd, J = 12.4, 3.2 Hz, 1H, H-1a), 4.26-4.09 (m, 4H, H-2,1b,6'), 4.06-3.98 (m, 1H, H-3a), 3.97-3.90 (m, 1H, H-5'), 3.71 (dd, J = 9.8, 7.4 Hz, 1H, H-3b) (m, 6H, 2×CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 174. 3, 170.8, 170.5, 170.3, 170.1 (C=O); 101.2 (C-1'), 82.4

HRMS (ESI)

(CH), 70.9 (C-5'), 70.6 (C-3'), 69.1 (C-2'), 68.2 (CH 2 ), 67.0 (C-4'), 61.4, 61.3, 34.2, 33.2, 32.0, 31.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.0, 25.5, 24.9, 22.8, 22.7 (CH 2 ); 21.0, 20.8, 20.7, 14.2 (CH 3 ). 4.42 (dd, J = 12.4, 3.2 Hz, 1H, H-1a), 4.26-4.08 (m, 4H, H-2,1b,6'), 4.05-3.98 (m, 1H, H-3a), 3.96-3.89 (m, 1H, H-5'), 3.71 (dd, J = 10.1, 7.3 Hz, 1H, H-3b) (m, 6H, 2×CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 174. 3, 170.8, 170.5, 170.3, 170.2 (C=O); 101.3 (C-1'), 82.4

(CH), 70.9 (C-5'), 70.6 (C-3'), 69.1 (C-2'), 68.2 (CH 2 ), 67.0 (C-4'), 61.4, 61.3, 34.2, 33.3, 32.0, 29.8, 29.6, 29.5, 29.4, 29.2, 25.5, 25.0, 22.8 (CH 2 ); 21.1, 20.8, 20.7, 14.3 (CH 3 (m, 6H, 2×CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 174. 3, 170.8, 170.5, 170.3, 170.1 (C=O); 130.1, 129.8 (CH=CH); 101.2 (C-1'), 82.4 (CH), 70.9 (C-5'), 70.6 (C-3'), 69.1 (C-2'), 68.2 (CH 2 ), 67.0 (C-4'), 61.4, 61.2, 34.2, 33.2, 32.0, 29.8, 29.6, 29.4, 29.2, 27.3, 25.5, 24.9, 22.8 (CH 2 ); 21.0, 20.8, 20.6, 14.2 (CH 3 ). [α] D = -23.3 (c 0.1, MeOH).

1 H NMR (400 MHz, CD 3 OD): δ 4.29 (d, 1H, d, J = 7.8 Hz, H-1'), 4.03 (dd, J = 11.0, 4.0 Hz, 1H, H-1a), 4.01-3.95 (m, 1H, H-2), 3.85-3.81 (m, 1H, H-4'), 3.80-3.71 (m, 3H, H-1b,6'), 3.71-3.61 (m, 2H, H-3), 3.58-3.46 (m, 3H, H-2',3',5') ). 13 C NMR (100 MHz, CD 3 OD): δ 175.9, 173.8 (C=O); 105.4 (C-1'), 87.5, 76.8, 74.9, 72.5, 70.3 (CH); 69.0, 62.5, 61.0 34.8, 33.6, 33.1, 32.4, 30.8, 30.7, 30.6, 30.5, 30.4, 30.2, 26.3, 26.0, 23.7, 23.4 (CH 2 ); 14. 5, 14.3 (CH 3 ). 1H, H-1a), 4.01-3.96 (m, 1H, H-2), 3.83 (d, J = 2.8 Hz, 1H, H-4'), 3.81-3.71 (m, 3H, H-1b,6'), 3.71-3.62 (m, 2H, H-3), 3.59-3.46 (m, 3H, H-2',3',5') 13 C NMR (100 MHz, CD 3 OD): δ 173.9 (C=O), 105.4 (C-1'), 87.4, 76.8, 74.9, 72.5, 70.3 (CH); 69.0, 62.5, 60.9, 33.7, 33.1, 30.8, 30.6, 30.5, 30.4, 30.2, 26.6, 23.7 13 C NMR (100 MHz, CDCl 3 ): δ 170. 7, 170.2, 169.6, 169.5, 165.8 (C=O), 162.9 (C=O, Phth); 134.7 (CH, Phth), 128.8 (C q ), 123.7 (CH, Phth), 100.7 (C-1'), 85.1 (CH), 83.5 (C q , t-Bu), 72.8 (C-3'), 71.8 (C-5'), 71.0 (C-2'), 68.3 (C-4'), 67.7, 61.9 (CH 2 ); 27.8, 20.8, 20.7, 20.6 (CH 3 [α] D = +7.7 (c 0.1, CHCl 3 ). 3.79-3.69 (m, 2H, H-2,5'), 3.66 (dd, J = 10.1, 5.5 Hz, 1H, H-3b,) 3.63-3.55 (m, 2H, H-1), 2.08, 2.04, 2.02, 2.00 (4×s, 12H, 4×OAc), 0.88 (s, 9H, t-Bu), 0.05 (s, 6H, Si(CH 3 ) 2 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 8, 170.4, 169.6, 169.5 (C=O) M.p. 115 º C.

1 H NMR (400 MHz, CDCl 3 ): δ 7. 84-7.72 (m, 4H, Phth), 7.42-7.13 (m, 15H, Tr), 5.17 4.38-4.30 (m, 1H, H-2), 4.25 (dd, J = 12.4, 4.6 Hz, 1H, H-6'a), 4.11 (dd, J = 12.4, 2.3 Hz, 1H, H-6'b), 4.06 (dd, J = 12.4, 3.6 Hz, 1H, H-3a), 3.96 (dd, J = 12.4, 4.1 Hz, 1H, H-3b), 3.76-3.66 (m, 1H, H-5'), 3.58 (dd, J = 11.4, 4.1 Hz, 1H, H-1a), 3.48 (dd, J = 11.4, 6.0 Hz, 1H, H-1b), 2.05, 2.01, 1.98, 1.85 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 9, 170.4, 169.6, 169.5 (C=O), 163.6 (C=O, Phth); 143.7 (C q ), 134.6 (CH, Phth), 129.1 (C q ), 128.7, 127.9, 127.2 (CH, Tr); 123.6 (CH, Phth), 101.0 (C-1'), 87.3 (C q , Tr), 86.2 (CH), 72.9 (C-3'), 71.8 (C-5'), 71.1 (C-2'), 68.5 (C-4'), 67.8, 62.5, 62.0 (CH 2 ); 20.9, 20.7, 20.6 (OAc). M.p. 218 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 7. 96-7.81 (m, 4H, Phth), 5.17 (t, J = 9.4 Hz, 1H, H-3'), 5.01-4.88 (m, 3H, H-2,2',4'), 4.58 (d, J = 7.8 Hz, 1H, H-1'), 4.52 (d, J = 12.4 Hz, 1H, H-3a), 4.23-4.06 (m, 3H, H-3b,6'), 3.70 (ddd, J = 10.0, 4.7, 2.6 Hz, 1H, H-5'), 2.10, 2.09, 2.01, 1.99 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 9, 170.3, 169.8, 169.6, 168.4 (C=O) Hz, 1H, H-3'), 5.08 (t, J = 9.9 Hz, 1H, H-4'), 5.02 (m, 1H, H-2'), 4.66-4.50 (m, 4H, H-1',2, CH 2 -Fmoc), 4.39-4.14 (m, 4H, H-3a,6', CH-Fmoc), 3.89-3.81 (m, 1H, H-3b), 3.78-3.70 (m, 1H, H-5'), 2.08, 2.06, 2.04, 2.03 (4×s, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 9, 170.8, 170.3, 169.6, 169.1, 159.8 (C=O); 143.0, 141.5 (C q ); 128.2, 127.4, 125.1, 125.0, 120.3 (CH, Fmoc) (5 mL), were added EDC•HCl (0.28 g, 1. 88 mmol) and HOBt (0.25 g, 1.88 mmol) under argon at 0 º C. After being stirred for 20 min, Fmoc-Gly-OH (0.28 g, 0.94 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the completion of the reaction, the solution was concentrated to a syrup which was purified by column chromatography over silica gel (petroleum ether/EtOAc: 1/2) to afford compound 87 (0.47 g, 64%) as a white powder. R f = 0.50 (petroleum ether/EtOAc: 1/3).

[α] D = +7.7 (c 0.1, CHCl 3 ). M.p. 77 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 9. 50 and 8.73 (2×s, 1H, NH), 7.84-7.28 (m, 8H, Fmoc), 5.63 (s, 1H, NH-Fmoc) 5.22 (t, J = 9.6 Hz, 1H, H-3'), 5.17-4.98 (m, 2H, H-2',4'), 4.65 (d, J = 6.0 Hz, 1H, H-1' ) 4.55-4.48 (m, 1H, H-2), 4.40 (d, J = 6.8 Hz, CH 2 ), 4.34-4.11 (m, 5H, H-6', CH 2Fmoc ,CH Fmoc ), 3.96-3.87 (m, 2H, CH 2 ), 3.77-3.69 (m, 1H, H-5'), 2.08, 2.05, 2.02, 1.99 (4×s, 12H, 4×OAc), 1.48 (s, 9H, t-Bu). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 8, 170.3, 169.9, 169.8, 169.5, 167.2, 156.6 (C=O); 143.9, 141.4 (C q ); 127.8, 127.2, 125.2, 120.1 (CH, Fmoc); 100.6 (C-1'), 83.6 (CH), 77.4 (C q ), 72.7 (C-3'), 72.0 (C-5'), 71.2 (C-2'), 68.4 (C-4'), 67.8, 67.4, 61.8 (CH 2 ), 47.2 (CH), 42.6 (CH 2 ), 28.1, 20.8, 20.7 (CH 3 ). M.p. 166 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 10.5 (s, 1H, NH), 8.80 (broad s, 1H, COOH), 7.81-7.20 (m, 8H, Fmoc), 5.63 (s, 1H, NH-Fmoc), 5.23 (t, J = 9.6 Hz, 1H, H-3'), 5.12-4.90 (m, 2H, H-2',4'), 4.63-4.53 (m, 1H, H-2), 4.49 (d, J = 6.4 Hz, 1H, H-1'), 4.46-4.30 (m, 3H, H-3a, CH 2 ), 4.28-4.08 (m, 3H, H-6', CH Fmoc ), 3.99-3.89 (m, 2H, CH 2Fmoc ), 3.89-3.78 (m, 1H, H-3b) 3.73-3.65 (m, 1H, H-5'), 2.12-1.91 (m, 12H, 4×OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 171. 5, 171.0, 170.2, 169.8, 169.6, 157.0 (C=O); 143.7, 141.3, (C q ); 127.9, 127.2, 125.2, 120.1 (CH, Fmoc); 101.2 (C-1'), 85.5 (CH), 72.0 (C-3',5'), 71.6 (C-2'), 68.9 (CH 2 ), 68.3 (C-4'), 67.6, 61.8 (CH 2 ); 47.0 (CH), 42.0 (CH 2 ), 20.9, 20.8, 20.7, 20.6 (CH 3

N-{(2R)-2-N-(9-Fluorenylmethoxycarbonylaminomethylcarbonyl)aminooxy-3-[(2′,3′,4′,6 ′-tetra-O-acetyl--D-glucopyranosyl)oxy]propanoyl}-glycine tert-butyl ester (89)

Chemical Formula: C 40 H 49 N 3 O 17 Exact Mass: 843.3062 g/mol To a solution of compound 88 (183 mg, 0.25 mmol) in anhydrous CH 2 Cl 2 (5 mL), were added EDC•HCl (96 mg, 0.5 mmol) and HOBt (68 mg, 0.5 mmol) under argon at 0 º C. After being stirred for 20 min, GlyOtBu (42 mg, 0.25 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the completion of the reaction, the solution was concentrated to a syrup which was purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 100/3) to afford compound 89 (100 g, 48%) as a white powder. R f = 0.25 (petroleum ether/EtOAc: 1/3).

[α] D = -9.0 (c 0.1, CHCl 3 ). M.p. 90 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 9.99 (s, 1H, NH), 8.78 (s, 1H, NH), 7.84-7.29 (m, 8H, Fmoc), 5.57 (t, J = 5.1 Hz,1H, NH-Fmoc), 5.27 (t, J = 9.6 Hz, 1H, H-3'), 5.13-4.97 (m, 2H, H-2',4'), 4.60-4.38 (m, 5H, H-1', 2×CH, CH 2Fmoc ), 4.30-4.15 (m, 3H, H-6', CH Fmoc ), 4.02-3.90 (m, 4H, 2×CH 2 ), 3.84-3.76 (m, 1H, CH), 3.75-3.69 (m, 1H, H-5'), 2.10, 2.07, 2.04, 1.97 (4×s, 12H, 4×OAc), 1.47 (s, 9H, t-Bu). 13 C NMR (100 MHz, CDCl 3 ): δ 171. 7, 171.0, 170.2, 169.6, 168.6, 168.0, 167.6, 156.5 (C=O); 143.8, 141.4 (C q ); 127.9, 127.2, 125.2, 120.1 (CH, Fmoc); 101.1 (C-1'), 86.2 (CH), 82.3 (C q ), 72.1, 72.0 (CH); 69.8 (CH 2 ), 68.3 (CH), 67.4, 61.7 (CH 2 ), 47.1 (CH), 42.1, 41.8 (CH 2 ), 28.1, 21.1, 20.9, 20.7 (CH 3

Figure 1 :

 1 Figure 1: A modified central dogma of biological information flow. 3a

  Figure 1: A modified central dogma of biological information flow. 3a

  1 (a) Wolfert, M. A.; Boons, G.-J. Nat. Chem. Boil. 2013, 9, 776-784; (b) Kolarich, D.; Lepenies, B.; Seeberger, P. H. Curr. Opin. Chem. Biol. 2012, 16, 214-220; (c) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376; (d) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. 2 (a) Fernandez-Tejada, A.; Canada, F. J.; Jimenez-Barbero. J. Chem. Eur. J. 2015, 21, 10616-10628; (b) Fernandez-Tejada, A.; Canada, F. J.; Jimenez-Barbero, J. ChemMedChem 2015, 10, 1291-1295; (c) Hudak, J. E.; Bertozzi, C. R. Chem. Biol. 2014, 21, 16-37; (d) Galan, M. C.; Benito-Alifonso, D.; Watt, G. M. Org. Biomol. Chem. 2011, 9, 3598-3610; (e) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046-1051; (f) Freeze, H. H.; Westphal, V. Biochimie 2001, 83, 791-799; (g) Butters, T. D.; Dwek, R. A.; Platt, F. M. Chem. Rev. 2000, 100, 4683-4696. 3 (a) Wang, L. X.; Davis, B. G. Chem. Sci. 2013, 4, 3381-3394; (b) Cecioni, S.; Imberty, A.; Vidal, S. Chem. Rev. 2014, 115, 525-561; (c) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-2364; (d) Herzner, H.; Reipen, T.; Schultz, M.; Kunz,

Figure 2 :

 2 Figure 2: General chemical glycosylation. PG = protecting group, LG = leaving group.

Scheme 1 :

 1 Scheme 1: (a) Michael, (b) Fisher and (c) Koenigs-Knorr glycosylation reactions. 10b

Figure 3 :

 3 Figure 3: Glycosylation methods reviewed by Galonic et al. (a), (b): Glycosylation of acetal-derived glycosyl donors. (c), (d): Glycosylation with glycal donors. Lg = leaving group; El + = electrophilic promoter (blue); Nu-H = the acceptor (green); M = metal; R = various substituents; X = various leaving groups; Z = various functionalities. 6e

Figure 4 :

 4 Figure 4: Structure of O-linked glycosyl serine and threonine, and N-linked glycosyl asparagine in glycopeptides. 1a

Figure 5 :

 5 Figure 5: Structure of C-and S-linked glycopeptides.

  (a) Aussedat, B.; Vohra, Y.; Park, P. K.; Fernández-Tejada, A.; Alam, S. M.; Dennison, S. M.; Jaeger, F. H.; Anasti, K.; Stewart, S.; Blinn, J. H.; Liao, H.-X.; Sodroski, J. G.; Haynes, B. F.; Danishefsky, S. J. J. Am. Chem. Soc. 2013, 135, 13113-13120; (b) Alam, S. M.; Dennison, S. M.; Aussedat, B.; Vohra, Y.; Park, P. K.; Fernández-Tejada, A.; Stewart, S.; Jaeger, F. H.; Anasti, K.; Blinn, J. H. Proc. Natl. Acad. Sci. USA 2013, 110, 18214-18219.

27

 27

Figure 7 :

 7 Figure 7: (a) Similarities between N-to-C glycopeptides and C-to-N glycopeptoids; (b) typical naturally occurring N-and O-linked glycosylated amino acids in peptides and (c) their peptoid analogues.27

29

 29

Figure 8 :

 8 Figure 8: Synthetic linear and cyclic glycopeptides reported by Pawar et al.29

Scheme 4 :

 4 Scheme 4: Glycoprotein retrosynthetic analysis reported byGamblin et al. 30c

Scheme 5 :Scheme 6 :

 56 Scheme 5: Methods for preparation of N-linked glycopeptides. (a) Amide bond formation; (b) Direct glycosylation of an Asn residue. 31

Figure 9 :

 9 Figure 9: Classification and representative structures of glycolipids.

Figure 10 :

 10 Figure 10: Chemical structure of MGDG, DGDG, SQDG and MGMG. R 1 to R 7 = acyl chains.37

  Another two aminoglycoglycerolipids (b and c, Figure11) were isolated from marine alga by Zhou et al. and exhibited good inhibitory activity against myelin transcription factor 1 (Myt1)-kinase with an IC 50 value of 0.12 μg/mL.41

Figure 11 :

 11 Figure11: Chemical structure of the natural 6-amino-6-deoxy-α-glycoglycerolipids.40,41

( a )

 a Substitution and variation of the sugar moiety; (b) Nature of the glycoside bond and anomeric configuration; (c) Position of the glycerol linkage to the sugar; (d) Variation on the length and location of the lipid chains.

Figure 14 :

 14 Figure14: Structure of natural GlaADG, PI3P and their analogues.48

Figure 15 :

 15 Figure 15: Chemical structure of the natural AGLs from marine sponge. 50

Figure 16 :

 16 Figure 16: Chemical structure of the natural -GalCer in nerve system. 51

Figure 17 :

 17 Figure 17: Chemical structure of the synthetic α-GalCer analogue KRN 7000. 52

Figure 18 :

 18 Figure 18: Four types of modifications to access KRN 7000 analogues reviewed by Haudrechy and co-workers. 53 (a) R 1 , R 2 , R 3 = OH or substituent group, R 4 = CH 2 OH or substituent group; (b) X = CH 2 , O, NH, S, etc; W = CH 2 , =CH-, etc; (c) Y, Z = CH 2 , CH-OH, CH-NH 2 , etc; (d) R 5 , R 6 = lipid chains.

Figure 19 :

 19 Figure 19: Structure of the 5-S-substituted KRN 7000 analogues.54

Figure 20 :

 20 Figure 20: Structure of the 6'-naphthylurea (A), 6'-carbamate (B) and 5'-uronamide derivatives (C) of α-GalCer.56

Figure 21 :

 21 Figure 21: Structure of the C-glycosidic analogues of -GalCer. 57

Figure 22 :

 22 Figure 22: Structure of -C-GalCer analogue synthesized by Thota et al.58

Figure 23 :

 23 Figure 23: Examples of compounds containing N-O linkage.

Figure 24 :

 24 Figure 24: Chemical structure of amino acids and aminooxy acids.

64

 64

Figure 25 :

 25 Figure 25: Chemical structure of L-canavanine and L-canaline.

65

 65

Figure 26 :

 26 Figure 26: Chemical structure of D-cycloserine. 65

Figure 27 :

 27 Figure 27: Aminooxy analogues of aspartame and gastrine.

Scheme 9 :

 9 Scheme 9: Proposed mechanism of Mitsunobu reaction with the inversion of configuration. 69a

Scheme 10 :

 10 Scheme 10: Preparation of tertiary alkoxyamine reported by Palandoken et al.70

Figure 28 :

 28 Figure 28: Structure of α-aminooxy acid, α N-O turn and 1.8 8 helix. 76

Figure 29 :

 29 Figure 29: Structures of three diamides compounds for NMR and IR study.77

Figure 31 :

 31 Figure31: Structure of the tripeptide D.77

  78b

Figure 32 :

 32 Figure 32: (a) Structure of cyclic hexapeptide A and the amide NH region of overlaid 1 H NMR spectra of free A and the 1:1 mixture of A with (n-Bu) 4 NF, Ph 4 PCl, and Ph 4 PBr, respectively (3 mM in CDCl 3 at -30 °C); 78 a (b) Structure of cyclic hexapeptide B and the amide NH region of overlaid 1 H NMR spectra of free B and 1:1 mixtures of B with Ph 4 PCl, Ph 4 PBr, or Bu 4 NNO 3 (4 mM in CD 2 Cl 2 at 298 K). 78b

Figure 33 :

 33 Figure 33: Structures of cyclic tetrapeptide C and the chemical shift of amide protons of free C and a mixtures of C with anions (Cl -, Br -, I -).79

Figure 34 :

 34 Figure 34: Structures of various substitution patterns of -aminooxy acids and N-O turn. 80

Figure 35 :

 35 Figure 35: (a) and (d): Structures of diamide A and triamide B; (b) and (e): X-Ray structures of A and B; (c) and (f): NOEs of A and B observed in CDCl 3 at 5 mM (s, strong NOE; M, medium NOE) .80b

Figure 36 :

 36 Figure 36: (a) Chemical structure of 4 -Ph aminooxy peptide; (b) N-H stretch region FT-IR data of 4 -Ph aminooxy peptide in 2 mM CH 2 Cl 2 ; (c) NOE signal observed in CDCl 3 at 5 mM; (d) X-Ray structure. 82a

Figure 37 :

 37 Figure 37: Nine glycoconjugates synthesized and subjected to hydrolysis experiments.84

  0 and immediately analyzed by HPLC.84 Based on the position of the N-O linkage, the N-O linked carbohydrates are mainly divided into three types, such as N-glycosyl hydroxylamine, O-glycosyl hydroxylamine and O-amino sugar derivatives (Figure38).

Figure 38 :

 38 Figure 38: Chemical structure of N-glycosyl hydroxylamine, O-glycosyl hydroxylamine and O-amino sugar derivatives.

Figure 39 :

 39 Figure 39: Chemical structure of N-(4-biphenyl)-N-hydroxyl-D-glucuronosylamine. 85a

  (a) Prudden, A. R.; Chinoy, Z. S.; Wolfert, M. A.; Boons, G. J. Chem. Commun. 2014, 50, 7132-7135; (b) McReynolds, K. D.; Dimas, D.; Le, H. Tetrahedron Lett. 2014, 55, 2270-2273.

Scheme 19 :

 19 Scheme 19: Synthesis of bifunctional linker and lactose conjugation reported byPrudden et al. 90a

Figure 40 :

 40 Figure 40: Chemical structure of natural glycopeptide antibiotics vancomycin and teicoplanin, and synthetically modified liponeoglycopeptides.93

Figure 41 :

 41 Figure 41: Structure of digitoxin (a) and its neoglycoside analogs (b).

Scheme 28 :

 28 Scheme 28: Phase transfer catalyzed (PTC) synthesis of O-glycosyl hydroxylamine.104

Scheme 32 :

 32 Scheme 32: Preparation of N-O linked saccharides(a) and O-glycosyl hydroxylamines(b).109

Figure 42 :

 42 Figure 42: Oxime-linked neoglycocyclopeptides reported by Dumy,

Figure 43 :

 43 Figure 43: Chemical structure of the synthetic vaccine. 115

Figure 44 :

 44 Figure 44:Chemical structure of neoglycopeptides as potential anticancer vaccines.116

Scheme 38 :

 38 Scheme 38: Synthesis of N-and O-glycosylated α-aminooxy acids.117

Figure 45 :Scheme 40 :

 4540 Figure 45: Structure of the first O-phthalimido sugar derivatives.119

Figure 46 :

 46 Figure 46: General structure of glycoaminooxy acids and N-oxyamide-linked glycolipids.

Figure 47 :

 47 Figure 47: General structure of N-oxyamide-linked glycoglycerolipids, glycosyl aminooxy acids and N-oxyamide-linked glycopeptides.

Figure 48 :

 48 Figure 48: Structure of sugar amino acids and glycoaminooxy acids.

Figure 49 :

 49 Figure 49: Chemical structure of the developed glycoaminooxy acid. 126

Figure 50 :

 50 Figure 50: Structure of PMB.

Scheme 44 :

 44 Scheme 44: Structures of the designed glycoaminooxy acid derivatives and novel glycoconjugates.

Scheme 45 :

 45 Scheme 45: Retrosynthesis of pyranoid glycoaminooxy ester 7.

Scheme 46 :

 46 Scheme 46: Various products of the silylation of methyl α-D-glucopyranoside.131

Scheme 47 :

 47 Scheme 47: Synthesis of the 2,6-O-disilylated compound 1.

Scheme 48 :

 48 Scheme 48: Synthesis of compound 4.

Scheme 49 :

 49 Scheme 49: Synthesis of 5 through O-alkylation.

Figure 51 :

 51 Figure 51: The TLC plates of the O-alkylation of 4.

Figure 52 :

 52 Figure 52: Structure of compounds A and 4.

Scheme 51 :

 51 Scheme 51: Retrosynthesis of compound 5.

Scheme 53 :

 53 Scheme 53: Desilylation of 5 to synthesize 6.

Scheme 54 :

 54 Scheme 54: Synthesis of glycoaminooxy ester 7 via Mitsunobu reaction.

Scheme 55 :

 55 Scheme 55: Retrosynthesis of N-oxyamide-linked glycolipid 17.

Scheme 56 :

 56 Scheme 56: Retrosynthesis of glycolipid 11 from 7.

Scheme 57 :

 57 Scheme 57: Synthesis of compound 9.

Scheme 58 :

 58 Scheme 58: Synthesis of compound 14.

Scheme 59 :

 59 Scheme 59: Retrosynthesis of glycolipid 11 from 4.

Scheme 61 :

 61 Scheme 61: Esterification of 12 to synthesize compound 13.

Scheme 62 :

 62 Scheme 62: Synthesis of O-phthalimido derivative 15.

Scheme 64 :

 64 Scheme 64: Deprotection of compound 11 to synthesize glycolipid 17.

Scheme 65 :

 65 Scheme 65: Retrosynthesis of glycolipid 23.

Scheme 66 :

 66 Scheme 66: Esterification of 4 to obtain the glucolipid 18.

Scheme 67 :

 67 Scheme 67: Synthesis of O-phthalimido derivative 20.

Figure 53 :

 53 Figure 53: Structure of GGLs, GSLs and N-oxyamide-linked glycolipids.

Figure 54 :

 54 Figure 54: Structure of N-oxyamide-linked glucoglycerolipids.

Scheme 73 :

 73 Scheme 73: Synthesis of 1,2,5,6-tetra-O-benzyl-D-mannitol 27.

Scheme 76 :

 76 Scheme 76: Debenzylation of 29 affording -glucoglycerol 30.

Scheme 79 :

 79 Scheme 79: Synthesis of 32 via Mitsunobu reaction.

From 2 -

 2 O-phthaloylamino--glucoglycerol 33, N-oxyamide-linked glucoglycerolipids could be prepared by introduction of lipid chains and final deprotection. Firstly, the chosen palmitoyl chain was introduced by esterification. Coupling equimolar quantity of palmitic acid with O-phthaloylamino--glucoglycerol 33 in the presence of EDC•HCl/DMAP/Et 3 N, afforded the glucolipid 34 in 98% yield (Scheme 81).

Scheme 81 :

 81 Scheme 81: Synthesis of 34 by esterification.

Scheme 84 :

 84 Scheme 84: Deacetylation of 36-40 to obtain final N-oxyamide-linked glucoglycerolipids 41-45.

Scheme 85 :

 85 Scheme 85: Retrosynthesis of N-oxyamide-linked glucoglycerolipids with one lipid chain.

Scheme 86 :Scheme 87 :

 8687 Scheme 86: Deprotection of the oxyamine of 32 to obtain 46.

Scheme 88 :

 88 Scheme 88: Deacetylation of 47-49 to obtain 50-52.

Scheme 89 :

 89 Scheme 89: Structures of N-oxyamide-linked galactoglycerolipids.

Scheme 91 :

 91 Scheme 91: Synthesis of -galactoglycerol derivative 53.

Scheme 92 :

 92 Scheme 92: Debenzylation of 53 affording -galactoglycerol 54.

Scheme 95 :

 95 Scheme 95: Desilylation of 56 affording 2-O-phthaloylamino--galactoglycerol 57.

Scheme 96 :

 96 Scheme 96: Synthesis of 58 by esterification.

Scheme 97 :

 97 Scheme 97: Synthesis of N-oxyamide-linked galactoglycerolipids 60-64.

Scheme 98 :

 98 Scheme 98: Deacetylation of 60-63 to obtain final N-oxyamide-linked galactoglycerolipids 65-68.

Scheme 99 :

 99 Scheme 99: Deacetylation of 60 using Na/MeOH.

Scheme 100 :

 100 Scheme 100: Formation of compound 70.

Figure 55 :

 55 Figure 55: 1 H NMR spectra of compounds 65 and 70 in CD 3 OD.

Figure 56 :

 56 Figure 56: 13 C NMR spectra of compounds 65 and 70 in CD 3 OD.

Scheme 101 :Scheme 102 :

 101102 Scheme 101: A proposed mechanism for the formation of compound 70.

Figure 57 : 1 H

 571 Figure 57: 1 H NMR spectra of compounds 67 and 71 in CD 3 OD.

Figure 58 :

 58 Figure 58: 13 C NMR spectra of compounds 67 and 71 in CD 3 OD.

Scheme 103 :

 103 Scheme 103: Deprotection of compound 63 with Na/MeOH.

Figure 59 : 1 H

 591 Figure 59: 1 H NMR spectra of compounds 68 and 72 in CD 3 OD.

Figure 60 :

 60 Figure 60: 13 C NMR spectra of compounds 68 and 72 in CD 3 OD.

Figure 61 :

 61 Figure 61: Structure of O-glycopeptides and its aminooxy analogues.

Scheme 107 :

 107 Scheme 107: Retrosynthesis of the O-glycosyl aminooxy esters.

Scheme 109 :

 109 Scheme 109: Epimerization of 31 to synthesize 74.

Scheme 110 :

 110 Scheme 110: Lattrell-Dax epimerization.

Scheme 111 :

 111 Scheme 111: Synthesis of 74 through Latterell-Dax epimerization.

  ppm in 31 to δ 3.79-3.73 ppm in 74. Moreover, the chemical shift difference between the diastereotopic protons (H III a: δ 3.84 ppm, J = 10.1, 4.6 Hz; H III b: δ 3.66 ppm, J = 10.1, 5.5 Hz) in compound 74 was consistently larger compared to 31 (H III : δ 3.77-3.72 ppm). A very small variation was observed in the chemical shift of C III on 13 C NMR spectra of 31 and 74. The C III moved slightly to upfield from δ 71.7 ppm in 31 to δ 71.4 ppm in 74.

Figure 62 :

 62 Figure 62: 1 H and 13 C NMR spectra of compounds 31 and 74 in CDCl 3 .

Figure 63 : 1 H

 631 Figure 63: 1 H NMR spectra of compounds 73 and 77 in CDCl 3 .

Figure 64 :

 64 Figure 64: 13 C NMR spectra of compounds 73 and 77 in CDCl 3 .

Figure 65 :

 65 Figure 65: Structures of C-glycosyl aminooxy esters. 118

Figure 66 :

 66 Figure 66: Structures of (2S)-and (2R)-3-O-glucosyl aminooxy esters.

Figure 67 :

 67 Figure 67: 1 H and 13 C NMR spectra of compounds 30 and 78 in CDCl 3 .

Scheme 116 :

 116 Scheme 116: Retrosynthesis of the N-oxyamide-linked linear and cyclic glycopeptides.

Scheme 119 :

 119 Scheme 119: Deprotection of the oxyamine of 73.

Scheme 124 :

 124 Scheme 124: Attempted synthesis of the disaccharide B.

Scheme 126 :

 126 Scheme 126: Synthesis of N-oxyamide-linked glycopeptide 89.

Figure 68 :

 68 Figure 68: 1 H NMR spectrum of Ac-NH-OMe in CDCl 3 .

Figure 69 : 1 HScheme 127 :

 691127 Figure 69: 1 H NMR spectrum of compound 87 in CDCl 3 .

  3--O-glycosyl aminooxy ester 73 by oxidation of the (2S)-1-O-glycosyl phthalimidooxy alcohol 33. Synthesis of (2S)-3--O-glycosyl aminooxy ester 77 has been developed in two synthetic routes, which were based on the Lattrell-Dax epimerization and Mitsunobu reactions from the same glycoglycerol derivative 31 (Scheme 128).

Scheme 128 :

 128 Scheme 128: Synthesis of (2R)-and (2S)--O-glycosyl aminooxy esters.

  Ref.: Halmos, T.; Montserret, R.; Filippi, J.; Antonakis, K. Carbohydr.Res. 1987, 170, 57-69.

  3 (C q , t-Bu);-3.7, -4.1, -5.0, -5.3 (CH 3 , Si(CH 3 ) 2 ).HRMS (ESI)m/z: [M+Na] + Calcd for C 35 H 58 NaO 8 Si 2 685.3568; Found 685.3562. 2(5.64 g, 8.52 mmol) using TBAF according to the general procedure A and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) afforded compound 3 (2.53 g, 68.4%) as a white solid. R f = 0.23 (petroleum ether/EtOAc = 1/1).1 H NMR (400 MHz, CDCl 3 ): δ 7.33-7.25 (m, 4H, PMB), 6.92-6.85 (m, 4H, PMB), 4.82 (d, J = 11.0 Hz, 1H, OCH), 4.67-4.57 (m, 3H, OCH, OCH 2 ), 4.54 (d, J = 3.7 Hz, 1H, H-1), 4.05 (t,

  Ref.: Gathirwa, J. W.; Maki, T. Tetrahedron 2012,68, 370-375. (Known structure)

  , 4.80(d, J = 11.0 Hz, 1H, OCH), 4.66-4.56 (m, 4H, OCH, OCH 2 , H-1), 4.04 (t, J = 9.2 Hz, 1H, H-3),3.84-3.73 (m, 8H, 2×OCH 3 , H-6), 3.55 (ddd, J = 9.9, 4.3, 2.1 Hz, 1H, H-5), 3.41 (t, J = 9.4 Hz, 1H, H-4), 3.36-3.27 (m, 4H, H-2, OCH 3 ), 2.40 (broad s, 1H, OH), 0.90 (s, 9H, t-Bu), 0.65-0.45 (m, 6H, Si(CH 3 ) 2 ).13 C NMR (100 MHz, CDCl 3 ): δ 159.6, 159.4, 130.9, 130.3 (C q , PMB); 129.8, 129.7, 114.1, 114.0 (CH, PMB); 97.4 (C-1), 79.6 (C-2), 77.4 (C-4), 74.3 (CH 2 ), 73.7 (C-3), 72.8 (CH 2 ), 71.2 (C-5), 62.5 (CH 2 ), 55.4, 55.0 (OCH 3 ); 26.1 (CH 3 , t-Bu), 18.5 (C q , t-Bu), -5.0, -5.2 (CH 3 , Si(CH 3 ) 2 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 29 H 44 NaO 8 Si 571.2703; Found 571.2698.

  2 (C q , PMB); 130.0, 129.8, 114.0 (CH, PMB); 98.1 (C-1), 83.4, 79.7, 76.5 (CH); 74.7, 73.0, 70.9 (CH 2 ); 70.5 (CH), 62.0, 60.9 (CH 2 ); 55.4, 55.2 (OCH 3 ); 14.3 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 27 H 36 NaO 10 543.2206; Found 543.2201.

  , 4.98(d, J = 10.1 Hz, 1H, OCH), 4.81(d, J = 10.5 Hz, 1H, OCH), 4.70 (d, J = 11.9 Hz, 1H, OCH), 4.56 (d, J = 9.6 Hz, 1H, OCH), 4.55-4.35 (m, 5H, OCH 2 , H-1,6), 4.21 (q, J = 7.0 Hz, 2H, OCH 2 ),3.87-3.72 (m, 9H, 2×OCH 3 , H-3,4,5), 3.57 (dd, J = 9.3, 3.5 Hz, 1H, H-2), 3.35 (s, 3H, OCH 3 ), 1.27 (t, J = 7.1 Hz, 3H, CH 3 ).13 C NMR (100 MHz, CDCl 3 ): δ 170.2 (C=O), 163.3 (C=O, Phth); 159.5, 159.4 (C q ); 134.5 (CH, Phth), 130.6, 130.2 (C q ); 130.1, 129.9 (CH, PMB); 129.0 (C q ), 123.6 (CH, Phth), 114.0, 113.9 (CH, PMB); 98.2 (C-1),83.4, 79.2 (CH); 76.5 (CH 2 ), 76.1 (CH),74.9, 73.1, 71.0 (CH 2 ); 69.2 (CH), 60.8 (CH 2 ), 55.6, 55.3 (OCH 3 ); 14.3 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 35 H 39 NaO 12 688.2370; Found 688.2374.

9 (

 9 CH 2 ); 69.5 (CH), 60.8 (CH 2 ), 55.4, 55.3 (OCH 3 ); 14.3 (CH 3 ). HRMS (ESI) m/z: [M+K] + Calcd for C 27 H 37 KNO 10 574.2055; Found 574.2057.

  (CH 2 ); 14.3 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 41 H 63 NaNO 11 768.4299; Found 768.4297. of compound 10 (52 mg, 0.07 mmol) with 1-dodecanol according to the general procedure G and purification by column chromatography over silica gel (petroleum ether/EtOAc = 2/1) afforded compound 11 (40 mg, 63%) as a white solid. (b) Compound 11 could also be prepared from compound 16 (20 mg, 0.03 mmol) and palmitic acid according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 11 (24 mg, 88%) as a paste. R f = 0.56 (petroleum ether/EtOAc: 1

  12(148 mg, 0.24 mmol) with 1-dodecanol according to the general procedure G and purification by column chromatography over silica gel (petroleum ether/EtOAc = 5/1) afforded compound 13 (144 mg, 76%) as a white paste. R f = 0.63 (petroleum ether/EtOAc: 4/1).

  Ref.: van Boeckel, C.; Visser, G.; van Boom, J. Tetrahedron 1985, 41, 4557-4565. (b) Mannock, D. A.; Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113-127. (c) Janwitayanuchit, W.; Suwanborirux, K.; Patarapanich, C.; Pummangura, S.; Lipipun, V.; Vilaivan, T. Phytochemistry 2003, 64,

  mmol) using AcCl according to the general procedure B and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1) gave compound 33 (0.59 g, 72%) as a white powder. R f = 0.23 (petroleum ether/EtOAc: 1/1).[α] D = -17.3 (c 0.1, CHCl 3 ).

  ) m/z: [M+Na] + Calcd for C 31 H 43 NNaO 14Si 704.2351; Found 704.2346. 56 (1.45 g, 2.13 mmol) using AcCl according to the general procedure B and purification by column chromatography over silica gel (petroleum ether/EtOAc = 2/3) gave compound 57 (0.93 g, 77%) as a white solid. R f = 0.39 (petroleum ether/EtOAc: 1/2).[α] D = -17.7 (c 0.1, CHCl 3 ). M.p. 66 º C.1 H NMR (400 MHz, CDCl 3 ): δ 7.97-7.78 (m, 4H, Phth), 5.40 (d, J = 3.2 Hz, 1H, H-4'), 5.23 (dd, J = 10.6, 8.2 Hz, 1H, H-2'), 5.03 (dd, J = 10.6, 3.2 Hz, 1H, H-3'), 4.65 (d, J = 7.8 Hz, 1H, H-1'),

  ) m/z: [M+Na] + Calcd for C 25 H 29 NNaO 14 590.1486; Found 590.1483. 58(50 mg, 0.062 mmol) by hydrazine under mild conditions according to the general procedure E and purification by column chromatography over silica gel (petroleum ether/EtOAc = 2/1) afforded compound 59 (39 mg, 93%) as a white paste. R f = 0.44 (petroleum ether/EtOAc: 1/1).[α] D = -53.0 (c 0.1, CHCl 3 ).

  HRMS (ESI) m/z:[M+Na] + Calcd for C 39 H 67 NNaO 14796.4459; Found 796.4456. prepared from compound 59 (135 mg, 0.20 mmol) and octanoic acid(32 μL, 0.20 mmol) according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 61 (127 mg, 81%) as a yellowish paste. R f = 0.56 (petroleum ether/EtOAc: 1/1).[α] D = -52.0 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 8.89 (s, 1H, NH), 5.41 (t, J = 3.2 Hz, 1H, H-4'), 5.21 (dd, J = 10.1, 8.3 Hz, 1H, H-2'), 5.05 (dd, J = 10.5, 3.2 Hz, 1H, H-3'), 4.54 (d, J = 7.8 Hz, 1H, H-1'),

  HRMS (ESI) m/z:[M+Na] + Calcd for C 41 H 71 NNaO 14824.4772; Found 824.4769. prepared from compound 59 (170 mg, 0.25 mmol) and palmitic acid(64 mg, 0.25 mmol) according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 63 (204 mg, 90%) as a white solid. R f = 0.29 (petroleum ether/EtOAc: 2/1). [α] D = -38.0 (c 0.1, CHCl 3 ). M.p. 45 º C. 1 H NMR (400 MHz, CDCl 3 ): δ 8.86 (s, 1H, NH), 5.41 (t, J = 2.8 Hz, 1H, H-4'), 5.21 (dd, J = 10.4, 8.3 Hz, 1H, H-2'), 5.05 (dd, J = 10.5, 3.2 Hz, 1H, H-3'), 4.53 (d, J = 7.8 Hz, 1H, H-1'),

  HRMS (ESI) m/z: [M+Na] + Calcd for C 51 H 89 NNaO 14 962.6181; Found 962.6176. 60 (94 mg, 0.12 mmol) with Na/MeOH according to the general procedure I led to compound 70 (70 mg, 96%) as a yellowish paste. R f = 0.27 (CH 2 Cl 2 /MeOH: 5/1).

  HRMS (ESI) m/z: [M+Na] + Calcd for C 31 H 59 NaNO 10 628.4037; Found 628.4034. 63 (50 mg, 0.055 mmol) with Na/MeOH according to the general procedure I led to compound 72 (25 mg, 68%) as a yellowish solid. R f = 0.44 (CH 2 Cl 2 /MeOH: 5/1). [α] D = -25.7 (c 0.1, MeOH). M.p. 91 º C. 1 H NMR (400 MHz, CD 3 OD): δ 4.29 (d, 1H, d, J = 7.4 Hz, H-1'), 4.04 (dd, J = 11.0, 4.0 Hz,

  , 2.13 (t, J = 7.4 Hz, 2H, CH 2 ), 1.68-1.56 (m, 2H, CH 2 ), 1.42-1.22 (m, 24H, 12×CH 2 ), 0.95-0.85 (m, 3H, CH 3 ).

1 H

 1 NMR (400 MHz, CDCl 3 ): δ 5.20 (t, J = 9.6 Hz, 1H, H-3'), 5.06 (t, J = 9.7 Hz, 1H, H-4'), 4.99 (dd, J = 9.6, 8.1 Hz, 1H, H-2'), 4.55 (d, J = 8.2 Hz, 1H, H-1'), 4.23 (dd, J = 12.4, 5.0 Hz, 1H, H-6'a),4.13 (dd, J = 12.4, 2.3 Hz, 1H, H-6'b), 3.84 (dd, J = 10.1, 4.5 Hz, 1H, H-3a),

  ; 101.3 (C-1'), 72.8 (C-3'), 72.0 (C-5'), 71.4 (CH 2 ), 71.4 (C-2'),70.6 (CH), 68.5 (C-4'), 63.6, 62.1 (CH 2 ); 26.0, 20.8, 20.7 (CH 3 ); 18.4 (C q , t-Bu), -5.3 (CH 3 , Si(CH 3 ) 2 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 23 H 40 NaO 12 Si 559.2187; Found 559.2182. was introduced to compound 79 (0.37 g, 0.56 mmol) via Mitsunobu reaction according to the general procedure C. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 3/1) afforded compound 80 (0.44 g, 97%) as a white solid. R f = 0.83 (petroleum ether/EtOAc: 1/1). [α] D = -3.3 (c 0.1, CHCl 3 ).

  HRMS (ESI) m/z:[M+K] + Calcd for C 44 H 43 KNO 14848.2321; Found 848.2323. a solution of compound 80 (190 mg, 0.3 mmol) in CH 2 Cl 2 (6 mL), was added TFA(0.6 mL). The resulting mixture was stirred at room temperature overnight. After the reaction finished, most of the solvent was evaporated, and the residue was dried under high vacuum (oil pump) until no acidic smell could be detected. The residue was purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 50/1) to give compound 81 (146 mg, 84%) as a white solid. (b) The carboxylic acid derivative 81 also could be synthesized by oxidation of the (2S)-1-O-glycosyl phthalimidooxy alcohol 33 with Jones reagent. The Jones reagent was prepared by dissolving 2.67 g of chromium trioxide (CrO 3 ) in 2.3 mL of concentrated sulfuric acid, and then the mixture was diluted to 10 mL with water. Compound 33 was treated with Jones reagent (3 eq.) at room temperature for 16 h, affording compound 81 in

  ) m/z:[M+H] + Calcd for C 25 H 28 NO 15582.1459; Found 582.1453. of compound 84 (124 mg, 0.17 mmol) in CH 2 Cl 2 (3 mL), was added TFA (0.3 mL). The resulting mixture was stirred at room temperature overnight. After the reaction finished, most of the solvent was evaporated, and the residue was dried under high vacuum (oil pump) until no acidic smell could be detected. The residue was purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 20/1) to give compound 85 (114 mg, 100%) as a white powder. R f = 0.39 (CH 2 Cl 2 /MeOH: 20/1).[α] D = -12.0 (c 0.1, CHCl 3 ). M.p. 90 º C.1 H NMR (400 MHz, CDCl 3 ): δ 8.06 (s, 1H, NH),7.87-7.30 (m, 8H, Fmoc), 5.25 (t, J = 9.4

  HRMS (ESI) m/z:[M+K] + Calcd for C 38 H 46KN 2 O 16 825.2484; Found 825.2486. of compound 87 (246 mg, 0.3 mmol) in CH 2 Cl 2 (4 mL), was added TFA (0.4 mL). The resulting mixture was stirred at room temperature overnight. After the reaction finished, most of the solvent was evaporated, and the residue was dried under high vacuum (oil pump) until no acidic smell could be detected. The residue was purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 25/1) to give compound 88 (175 mg, 77%) as a white powder. R f = 0.35 (CH 2 Cl 2 /MeOH: 20/1).[α] D = +4.0 (c 0.1, CHCl 3 ).

  42

halides was reported by Cao et al. Treatment of the obtained glycosyloxy succinimides with an excess of hydrazine hydrate gave the corresponding O-glycosyl hydrox- ylamine (Scheme 12). 72a Scheme 12: Synthesis

  N-hydroxysuccinimide (NHS),72 or other N-protected hydroxylamine derivatives 73 as nucleophile reagents (Scheme 11). For example, Jones et al. reported that the alkyl iodide and bromides can be easily converted to N-protected of O-glycosyl hydroxylamine by glycosylation and hydrazinolysis.

	aminooxy	compounds	by	treatment	with	commercially	available
	N-(tert-butyloxycarbonyl)hydroxylamine (BocNHOH) and 1,8-diazabicycloundec-7-ene
	(DBU). 73c Removal of the Boc group gave the free oxyamine (Scheme 11c).
		Scheme 11: Displacement of halogens to prepare the aminooxy derivatives. 71,72,73
	Successful stereoselective synthesis of glycosyloxysuccinimides via glycosylation of
	NHS with glycosyl

Table 1 :

 1 The pKa value of amine, oxyamine, amide, N-oxyamide reported in literatures.75

	74 Choong, I. C.; Ellman, J. A. J. Org. Chem. 1999, 64, 6528-6529.
	75 (a) Bissot, T. C.; Parry, R. W.; Campbell, D. H. J. Am. Chem. Soc. 1957, 79, 796-800; (b) Bordwell, F. G. Acc. Chem. Res.
	1988, 21, 456-463; (c) Bordwell, F. G.; Fried, H. E.; Hughes, D. L.; Lynch, T. Y.; Satish, A. V.; Whang, Y. E. J. Org. Chem.
	1990, 55, 3330-3336.

H-Bond Figure 30: (a) N-H stretch region FT-IR data of diamide in CH 2 Cl 2 at room temperature after subtraction of the spectrum of pure CH 2 Cl 2 .

	O		O		O		O			O		O
	t-Bu	N H	O	NEt 2	t-Bu	N H	O	N H	OMe	t-Bu	N H	O	H N	Bu-i
														3427 cm -1
	3400 cm -1		3386 cm -1 3198 cm -1	3384 cm -1 3294 cm -1
			AB					C

Table 2 :

 2 Chemical shifts of amide protons for compounds A, B and C in CD 2 Cl 2 at 25 °C. A at 5 mM, B and

	C at 0.25 mM. 77

Table 3 :

 3 The general chemical shifts and FT-IR stretch regions of the hydrogen-bonded and nonhydrogen-bonded NH in amide and N-oxyamide.

	Three amide NH signals at 9.25, 8.28, and 5.23 ppm were observed in the 1 H NMR
	spectra of the tripeptide D at or below 1 mM, which were assigned to N-oxy amide NH, ala-
	nine NH, and valine carbamate NH, respectively. The α N-O turn (the eight-membered-ring
	intramolecular hydrogen bond Val-C=O…HN-Ala) in tripeptide D was evidenced from its
	unusually downfield δ (Ala-NH) of 8.28 ppm (Figure 31). 77

Table 4 :

 4 Half-lives in aqueous solution of glycoconjugates 9-17. 2 mM sample in 20 mM NaOAc (pH 4.0, or pH 5.0), or in 20 mM Na 2 HPO 4 (pH 6.0) was incubated at 37 °C. 200 μL samples were taken out at different time intervals and quenched at 4 °C with the addition of 400 μL of 200 mM Na 2 HPO 4 buffer at pH 7.

  h (Entry 4).

	C)
		1.5 eq. NaH	1.5 eq. NaH
		1 eq. PMBCl	1eq.PMBCl
		OTBS			OTBS
	HO	O			PMBO	O
	HO	TBSO OMe			PMBO	TBSO OMe
		1	1.5 eq. NaH	1.5 eq. NaH	2
			1 eq. PMBCl	1 eq. PMBCl
		Entry	Reactants Addition Sequence	Results
		1	Method A	Several products
		2	Method B (during 40 min)	50% yield
		3	Method C (during 40 min)	62% yield
		4	Method C (during 80 min)	80% yield

Table 5 :

 5 Essays

for the synthesis of compound 2. General condition: NaH (6 eq.) and PMBCl (4 eq.) in DMF, 0 °C to RT,

16 h.

Table 6 :

 6 Attempted

conditions for O-alkylation of 4. All the reactions were performed with 50 mg of 4 in 1 mL DMF, from 0 °C (30 min) to room temperature (15 h). The conversion was determined by TLC except

Table 7 :

 7 Preparation of 3,4-O-isopropylidene-D-mannitol 25.

Table 8 :

 8 Different conditions for the synthesis of 31.

Table 9 :

 9 Attempted conditions for the phthaloyl deprotection of 34. Subsequently, acylation of the obtained oxyamine 35 with equimolar quantity of different fatty acids under usual conditions led successfully to the N-oxyamide-linked glucoglycerolipids 36-40 in good yields [36 (82%), 37 (82%), 38 (64%), 39 (76%), 40 (87%)] (Scheme 83). The 1 H NMR spectra of 36-40 in CDCl 3 showed the NH signals appearing in the range of 8.81-8.87 ppm, indicating the formation of N-oxyamide bond.

Scheme 82: Deprotection of 34. Scheme 83: Synthesis of N-oxyamide-linked glucoglycerolipids 36-40.

  Comparison of the 1 H NMR spectra of compounds 68 and 72 in CD 3 OD (Scheme 70) reveals that the chemical shift of H I and H II moved to higher field (H I : from δ 4.37-4.23 ppm in 68 to δ 3.71-3.62 ppm in 72; H II : from δ 4.22-4.15 ppm in 68 to δ 4.01-3.96 ppm in 72).

  in CH 2 Cl 2 at room temperature for one night, led successfully to the (2S)-3-O-glycosyl aminooxy ester 77 in 55% yield (Scheme 112).

	AcO AcO	O OAc AcO	O	H	OTBS OH	PhthN-OH 3 eq. PPh 3 3e q. DIAD 3 eq. toluene 0 o CtoRT,1h	AcO AcO	OAc O AcO	O	H	NPhth O OTBS
						88%
		74					75
							AcC l 0.15 e q.	MeOH 0 o CtoRT,2h
										87 %
						PCC 2 eq.
	AcO AcO	OAc O AcO	O	H	NPhth O OtBu O	Ac 2 O1 0e q . t-BuOH 20 eq. CH 2 Cl 2 RT, overnight	AcO AcO	O OAc AcO	O	H	OH NPhth O
						55%
		77						76
		Scheme 112: Synthesis of (2S)-3-O-glycosyl aminooxy ester 77.

(2R)-C-glucosyl aminooxy ester (2S)-C-glucosyl aminooxy ester

		OAc			OAc
	BnO	O		BnO	O
	BnO	BnO	ONH 2	Bn O	Bn O	ONH 2
			CO 2 t-Bu			CO 2 t -Bu
	4.13 (t, J =6. 0Hz)	4.16 (dd, J = 11.0, 2.8 Hz)

Scheme 113 :

 113 Conversion of compound 32 to 74.

	Entry	Conditions	Results
	1	CuSO 4 •5H 2 O (0.03 eq.) in MeOH, RT, overnight	No reaction
	2	AIBN (0.15 eq.) and n-Bu 3 SnH (1.1 eq.) in benzene, Reflux, 2 h	Degradation
	3	10% Pd/C (35 wt %) and H 2 in MeOH, RT, overnight	54% of 74

Table 10 :

 10 Attempted conditions for the synthesis of compound 74.

151 Momiyama, N.; Yamamoto, H. J. Am. Chem. Soc. 2003, 125, 6038-6039

.

152

Kim, S.; Lee, T. A.; Song, Y. Synlett 1998, 471-472.

153

Zhang, P.; Li, C.; Yang, H.; Wei, H.; Xia, Z.; Ma, D.; Chen, H.;

Wang, K.; Li, X. Carbohydr. Res. 2014, 398, 36-39.

Table 11 :

 11 Attempted regioselective silylation of compound 78.

  h furnished the corresponding alcohol 76 in 71% yield (Scheme 115).

		OAc						OAc
	AcO AcO	O AcO	O	H	OH OH	TrCl 1.2 eq. Pyridine reflux, 24 h	AcO AcO	O AcO	O	H	OH OTr
		78			92%		79
							PhthN-OH 3 eq.			toluene
							PPh 3 3 eq.	0 o CtoRT,1h
							DIAD 3 eq.
	AcO AcO	AcO O OAc	O	H	OH NPhth O	MeOH 0 o CtoRT,5h AcCl 0.15 eq.	AcO AcO	O OAc AcO	O	H	OTr NPhth O
		76				71 %		80

97%

Scheme 115: Synthesis of compound 76.

Scheme 120 :

 120 Attempted synthesis of the disaccharide A.

	Entry	Coupling conditions	Results
	1	EDC•HCl (1.8 eq.), HOBt (1.8 eq.) in DMF/CH 2 Cl 2 (1/2, V/V), 0 º C to RT, 24 h	No reaction
		EDC•HCl (2 eq.), HOBt (2 eq.) and Et 3 N ( 2 eq.) in CH 2 Cl 2 , 0 º C to
	2	RT, 24 h, then EDC•HCl (2 eq.), HOBt (2 eq.), Et 3 N ( 2 eq.), reflux,	No reaction
		24 h
	3	DCC (1.2 eq.), HOBt (1.2 eq.) and Et 3 N ( 2 eq.) in CH 2 Cl 2 , 0 º C to RT, 24 h	Degradation

Table 12 :

 12 Coupling conditions for preparing the disaccharide A.In order to understand the reason of the failure of coupling reaction, we firstly tried to couple the oxyamine 82 with a simple carboxylic acid. Coupling of the equimolar quantity of octanoic acid with the oxyamine 82 in the presence of EDC•HCl/HOBt/Et 3 N in CH 2 Cl 2 at 0

º C to room temperature overnight, led successfully to the glycolipid 83 in 84% yield (Scheme 121). The 1 H NMR spectrum of 83 in CDCl 3 showed the NH signal appearing at 8.88 ppm, indicating the formation of N-oxyamine bond. Scheme 121: Coupling reaction of 82 with octanoic acid.

Scheme 122 :

 122 Coupling reaction of 81 with GlyOMe.

	Entry	Conditions	Results
	1	EDC•HCl (1.8 eq.), HOBt (1.8 eq.) and Et 3 N (2.8 eq.) in DMF/CH 2 Cl 2 (1/2, V/V), 0 º C to RT, 2 days	Several products
	2	i-BuOCOCl (1.1 eq.), Et 3 N (3 eq.) in CH 2 Cl 2 , -10 º C to RT, 18 h 155	Undesired product

Table 13 :

 13 Attempted conditions for coupling 81 with GlyOMe.

  Chinchilla, R.; Dodsworth, D. J.; Nájera, C.; Soriano, J. M. Tetrahedron Lett. 2001, 42, 7579-7581. 157 Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 4th edition. 2006, Chapter 7. NH signals appearing at 8.75 and 8.32 ppm, which demonstrated the formation of the amide bond.

Scheme 123: Synthesis of the glycopeptide 86.

  (CH 2 ); 14.3, 14.2 (CH 3 ). HRMS (ESI) m/z: [M+K] + Calcd for C 43 H 67 KNO 11 812.4351; Found 812.4353.

  .28 mmol) in toluene (10 mL), was added ethyl bromoacetate(0.13 mL, 1.14 mmol) dropwise. After stirring at room temperature for 3 days, the solution was concentrated under vacuum. The residue was diluted with EtOAc (50 ml), washed with saturated aq HCl (1N, 3×20 mL), H 2 O (20 m) and brine(20 mL), dried over MgSO 4 , filtered, evaporated, and purified by column chromatography over silica gel (CH 2 Cl 2 / MeOH: 100/3) to afford compound 4 (103 mg, conversion: 67%) and compound 12 (170 mg, corrected yield: 74%) as a white powder. R f = 0.25 (CH 2 Cl 2 / MeOH: 100/3). [M+Na] + Calcd for C 31 H 46 NaO 10 Si 629.2758; Found 629.2759.

	Methyl 2-O-carboxylmethyl-3,4-di-O-(4-methoxybenzyl)-6-O-tert-butyldimethylsilyl-α-
	-D-glucopyranoside (12)
	Chemical Formula: C 31 H 46 O 10 Si
	Exact Mass: 606.2860 g/mol
	To a mixture of compound 4 (0.31g, 0.57 mmol), aqueous NaOH (5 mL, 50%) and TBAI (104
	mg, 0[α] D = +64.0 (c 0.1, CHCl 3 ).
	M.p. 138 º C.
	1 H NMR (400 MHz, CDCl 3 ): δ 7.38-7.20 (m, 4H, PMB), 6.99-6.82 (m, 4H, PMB), 4.79-4.46
	(m, 5H, 2×OCH 2 , H-1), 4.40 (d, J = 17.4 Hz, OCH), 4.32 (d, J = 18.4 Hz, OCH), 4.02-3.75
	(m, 8H, 2×OCH 3 , H-6), 3.71 (t, J = 8.9 Hz, H-3), 3.61-3.43 (m, 3H, H-2,4,5), 3.30 (s, 3H,
	OCH 3 ), 0.91 (s, 9H, t-Bu), 0.16-0.02 (m, 6H, Si(CH 3 ) 2 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 172.1 (C=O), 159.9, 159.7 (C q , PMB); 130.2, 130.0 (CH,
	PMB); 129.5, 128.6 (C q , PMB); 114.2, 114.1 (CH, PMB); 96.8 (C-1), 82.0, 78.5, 77.6 (CH);
	74.8, 72.4 (CH 2 ); 71.5 (CH), 70.5, 61.9 (CH 2 ); 55.4, 55.1 (OCH 3 ); 26.0 (CH 3 , t-Bu), 18.4 (C q ,
	t-Bu), -5.0, -5.2 (CH 3 , Si(CH 3 ) 2 ).
	HRMS (ESI) m/z:
	(CH 2 ); 14.3 (CH 3 ).
	HRMS (ESI) m/z: [M+Na] + Calcd for C 53 H 87 NaNO 11 936.6177; Found 936.6179.

  [M+Na] + Calcd for C 43 H 70 NaO 10 Si 797.4636; Found 797.4639.

	13 C NMR (100 MHz, CDCl 3 ): δ 170.4 (C=O), 159.4, 159.3, 130.7, 130.3 (C q , PMB); 129.8,
	113.8 (CH, PMB); 97.7 (C-1), 83.6, 79.7, 76.8 (CH); 74.7, 73.0 (CH 2 ); 71.3 (CH), 70.9, 65.0,
	62.3 (CH 2 ); 55.3, 54.9 (OCH 3 ); 32.0, 29.7, 29.6, 29.4, 29.3, 28.7 (CH 2 ); 26.0 (CH 3 , t-Bu),
	25.9, 22.8 (CH 2 ); 18.4 (C q , t-Bu), 14.2 (CH 3 ), -5.1, -5.3 (CH 3 , Si(CH 3 ) 2 ).

1

H NMR (400 MHz, CDCl 3 ): δ 7.

50-7.19 (m, 4H, PMB), 7.05-6.80 (m, 4H, PMB), 5.00-4.32 (m, 7H, 3×OCH 2

, H-1), 4.10 (t, J = 6.7 Hz, 2H, OCH 2 ), 4.04-3.68

(m, 9H, 2×OCH 3 , H-3,6)

,

3.64-3.44 (m, 3H, H-2,4,5)

,

3.26 (s, 3H, OCH 3

), 1.70-1.54 (m, 2H, CH 2 ), 1.52-1.17 (m, 18H, 9×CH 2 ), 1.02-0.82 (m, 12H, t-Bu, CH 3 ), 0.16-0.02 (m, 6H, Si(CH 3 ) 2 ).

HRMS (ESI) m/z:

  [M+Na] + Calcd for C 37 H 56 NaO 10 683.3771; Found 683.3775.

	13 C NMR (100 MHz, CDCl 3 ): δ 170.4 (C=O), 159.5, 159.4, 130.4, 130.1 (C q , PMB); 130.0,
	129.8, 113.9 (CH, PMB); 98.0 (C-1), 83.3, 79.6, 76.4 (CH); 74.7, 73.0, 70.8 (CH 2 ); 70.5
	(CH), 65.0, 61.9 (CH 2 ); 55.3, 55.2 (OCH 3 ); 32.0, 29.7, 29.6, 29.4, 29.3, 28.7, 25.9, 22.8
	(CH 2 ); 14.2 (CH 3 ).

1

H NMR (400 MHz, CDCl 3 ): δ 7.

40-7.22 (m, 4H, PMB), 7.00-6.81 (m, 4H, PMB), 4.91-4.35 (m, 7H, 3×OCH 2

, H-1), 4.10 (t, J = 6.8 Hz, 2H, OCH 2 ), 3.91-3.73 (m, 7H, 2×OCH 3 , H-3'), 3.73 (dd, J = 11.9, 2.8 Hz, 1H, H-6a), 3.65 (dd, J = 11.9, 3.2 Hz, 1H, H-6b),

3.57-3.43 (m, 3H, H-2,4,5)

, 3.26 (s, 3H, OCH 3 ), 1.79 (s, 1H, OH),

1.65-1.55 (m, 2H, CH 2

), 1.40-1.18 (m, 18H, 9×CH 2 ), 0.85 (t, J = 6.8 Hz, 3H, CH 3 ).

HRMS (ESI) m/z:

  (CH 2 ); 14.3 (CH 3 ). HRMS (ESI) m/z: [M+K] + Calcd for C 45 H 59 KNO 12 844.3674; Found 844.3678. [M+Na] + Calcd for C 37 H 57 NaNO 10 698.3880; Found 698.3884. To a solution of compound 11 (29 mg, 0.03 mmol) in CH 2 Cl 2(1 mL), was added TFA (0.05 mL, 5% in CH 2 Cl 2 ). The mixture was stirred at room temperature for 1 h under argon and then extracted with CH 2 Cl 2 (3×10 mL). The combined organic layers were washed with brine, dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (petroleum ether/EtOAc: 1/1) to afford compound 17 (12 mg, 60%) as a white solid.

	Methyl 6-O-amino-2-O-dodecyloxycarbonylmethyl-3,4-di-O-(4-methoxybenzyl)-α-D-glu-Methyl 2-O-dodecyloxycarbonylmethyl-6-O-palmitoylamino-α-D-glucopyranoside (17)
	copyranoside (16)
	Chemical Formula: C 37 H 71 NO 9
	Chemical Formula: C 37 H 57 NO 10
	Exact Mass: 673.5129 g/mol
	Exact Mass: 675.3982 g/mol
	Hydrazinolysis of compound 15 (58 mg, 0.071 mmol) according to the general procedure D
	and purification by column chromatography over silica gel (petroleum ether/EtOAc = 1/1)
	afforded compound 16 (35 mg, 73%) as a yellowish paste.
	R f = 0.39 (petroleum ether/EtOAc: 1/1).
	[α] D = +45.3 (c 0.1, CHCl 3 ).
	[α] D = +80.0 (c 0.1, CHCl 3 ).
	M.p. 70 º C.
	1 H NMR (400 MHz, CDCl 3 ): δ 8.74 (s, 1H, NH), 4.80 (d, J = 3.2 Hz, 1H, H-1), 4.48 (s, 2H, 13 C NMR (100 MHz, CDCl 3 ): δ 170.4 (C=O), 159,5, 159.4, 130.5, 130.2 (C q ); 130.1, 129.9, OCH 2 ), 4.21-4.08 (m, 4H, OCH 2 , H-6), 3.88 (t, J = 9.3 Hz, 1H, H-4), 3.76-3.63 (m, 2H, 114.0 113.9 (CH, PMB); 98.1 (C-1), 83.5, 79.4, 76.7 (CH); 74.8, 74.3, 73.1, 70.9 (CH 2 ); 69.4 H-2,5), 3.51 (t, J = 9.0 Hz, 1H, H-3), 3.42 (s, 3H, OCH 3 ), 2.09 (t, J = 7.4 Hz, 2H, CH 2 ), (CH), 65.0 (CH 2 ), 55.4, 55.3 (OCH 3 ); 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 28.7, 26.0, 22.8 (CH 2 ); 14.3 (CH 3 ). 1.70-1.56 (m, 4H, 2×CH 2 ), 1.38-1.19 (m, 42H, 21×CH 2 ), 0.92-0.82 (m, 6H, 2×CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 173.8, 171.8 (C=O); 99.7 (C-1), 83.8 (CH), 75.9 (CH 2 ), 71.8,
	70.3 (CH); 69.3, 65.9 (CH 2 ); 55.6 (OCH 3 ),

1

H NMR (400 MHz, CDCl 3 ): δ 7.

34-7.24 (m, 4H, PMB), 6.93-6.84 (m, 4H, PMB), 4.93-4.34 (m, 7H, 3×OCH 2

, H-1), 4.13 (t, J = 6.8 Hz, 2H, OCH 2 ),

3.91-3.74 (m, 9H, 2×OCH 3 , H-3,6)

,

3.72-3.65 (m, 1H, H-5), 3.56-3.45 (m, 2H, H-2,4)

, 3.30 (s, 3H, OCH 3 ), 1.69-1.57 (m, 2H, CH 2 ), 1.39-1.17 (m, 18H, 9×CH 2 ), 0.88

(t, J = 6.8 Hz, 3H, CH 3

).

HRMS (ESI) m/z:

R f = 0.44 (petroleum ether/EtOAc: 1/1).

  [M+Na] + Calcd for C 45 H 74 NaO 9 Si 809.5000; Found 809.5005.

	13 C NMR (100 MHz, CDCl 3 ): δ 172.7 (C=O), 159.4, 159.3, 130.4, 130.3 (C q , PMB); 129.5,
	129.4, 113.9 (CH, PMB); 97.8 (C-1), 77.7, 76.0 (CH); 73.8 (CH 2 ), 73.5 (CH), 72.6 (CH 2 ),
	71.2 (CH), 62.2 (CH 2 ), 55.4, 55.0 (OCH 3 ); 34.7, 32.1, 29.8, 29.6, 29.5, 29.4 (CH 2 ); 26.1
	(CH 3 , t-Bu), 25.1, 22.8 (CH 2 ); 18.5 (C q , t-Bu), 14.3 (CH 3 ), -5.0, -5.2 (CH 3 , Si(CH 3 ) 2 ).

1

H NMR (400 MHz, CDCl 3 ): δ 7.

25-7.14 (m, 4H, PMB), 6.94-6.81 (m, 4H, PMB), 5.53 (t, J = 9.6 Hz, 1H, H-3), 4.61-4.47 (m, 5H, 2×OCH

2 , H-1), 3.90-3.74 (m, 8H, 2×OCH 3 , H-6), 3.68-3.62 (m, 1H, H-5), 3.52 (t, J = 9.6 Hz, 1H, H-4), 3.43-3.36 (m, 1H, H-2), 3.33 (s, 3H, OCH 3 ), 2.26-2.18 (m, 2H, CH 2 ), 1.68-1.55 (m, 2H, CH 2 ), 1.50-1.16 (m, 24H, 12×CH 2 ), 0.98-0.82 (m, 12H, t-Bu, CH 3 ), 0.11-0.05 (m, 6H, Si(CH 3 ) 2 ). HRMS (ESI) m/z:

  (CH 2 ); 14.3 (CH 3 ).HRMS (ESI) m/z:[M+Na] + Calcd for C 55 H 91 NaNO 10 948.6541; Found 948.6542. Cl 2 ). The mixture was stirred at room temperature for 1 h under argon and then extracted with CH 2 Cl 2 (3×20 mL). The combined organic layers were washed with brine, dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 100/3) to afford compound 23 (85 mg, 77%) as a white powder. R f = 0.28 (CH 2 Cl 2 /MeOH: 100/3).

	Methyl 2-O-palmitoyl-6-O-palmitoylamino-α-D-glucopyranoside (23)
	Chemical Formula: C 39 H 75 NO 8
	Exact Mass: 685.5493
	To a solution of compound 22 (150 mg, 0.16 mmol) in CH 2 Cl 2 (4 mL), was added TFA (2 mL,
	5% in CH 2 [α] D = +84.7 (c 0.1, CHCl 3 ).
	M.p. 96 º C.
	1 H NMR (400 MHz, CDCl 3 ): δ 8.55 (s, 1H, NH), 5.14 (t, J = 9.7 Hz, 1H, H-3'), 4.77 (d, 1H, J
	= 3.6 Hz, 1H, H-1'), 4.25-4.13 (m, 2H, H-6), 3.96 (t, J = 9.6 Hz, 1H, H-4), 3.83-3.73 (m, 1H,
	H-5), 3.67-3.56 (m, 1H, H-2), 3.43 (s, 3H, OCH 3 ), 2.41 (t, J = 7.6 Hz, 2H, CH 2 ), 2.09 (t, J =
	7.3 Hz, 2H, CH 2 ), 1.73-1.57 (m, 4H, 2×CH 2 ), 1.45-1.19 (m, 48H, 24×CH 2 ), 0.94-0.84 (m, 6H,

  The mixture was stirred at 40 º C. After 1 h, the solution was evaporated and residual AcOH removed by repeated co-evaporation with toluene. Dry acetone was added and the mixture was thoroughly stirred with excess anhydrous K 2 CO 3 and then filtered. The insoluble material (remaining mannitol) was washed with acetone and the combined filtrates evaporated. The residue was then dissolved in a small volume of EtOAc and allowed to crystallize at room temperature to give pure product 25(5.89 g, 80.1%) as a crystal. R f = 0.43 (CH 2 Cl 2 /MeOH: 10/1). C NMR (100 MHz, acetone-d 6 ): δ 109.8 (C q ), 80.8, 74.2 (CH); 64.6 (CH 2 ),27.3 (CH 3 ). The suspension was stirred for 0.5 h at 0 º C in an ice bath. BnBr (15.11 mL, 127 mmol) was then added and the stirring continued at room temperaturefor 15 h. Ice was added to destroy the excess NaH, the solution was then concentrated, diluted with saturated aq NH 4 Cl and the product extracted with EtOAc (200 mL). The extract was washed with water, brine, dried over MgSO 4 and evaporated. Purification by column NaIO 4(8.56 g, 40 mmol in 150 mL water). The reaction mixture was stirred for 3 h at room temperature, after which time TLC analysis revealed the oxidation to be complete (R f = 0.52, petroleum ether/EtOAc: 3/1). The reaction mixture was diluted with MeOH (200 mL) and cooled. The precipitate was filtered off and to the filtrate was added NaBH 4(9.84 g, 260 mmol). After 1 h, the reaction mixture was treated with AcOH and the solution evaporated to a small volume which was diluted in CH 2 Cl 2 (300 mL), dried over MgSO 4 and concentrated. CN) 2(0.66 g, 2.6 mmol) in dry acetonitrile(20 mL) was added dropwise over a period of 1 h, a solution of glycosyl bromide in acetonitrile (10 mL). The reaction solution was stirred at room temperature overnight, and then concentrated to an oil which was dissolved in CH 2 Cl 2 (100 mL) and washed with saturated aq NaHCO 3 (2×30 mL) and water (30 mL). The dried (MgSO 4 ) organic layer was concentrated. Purification by column chromatography (pe-

	3,4-O-Isopropylidene-D-mannitol (25) 1,2,5,6-Tetra-O-benzyl-3,4-O-isopropylidene-D-mannitol (26) 1,2,5,6-Tetra-O-benzyl-D-mannitol (27) (2S)-1,2-Di-O-benzyl-glycerol (28) (2R)-1,2-Di-O-benzyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)glycerol (29)
	Chemical Formula: C 9 H 18 O 6 Chemical Formula: C 37 H 42 O 6 Chemical Formula: C 34 H 38 O 6 Chemical Formula: C 17 H 20 O 3 Chemical Formula: C 31 H 38 O 12
	Exact Mass: 272.1412 g/mol
	Exact Mass: 222.1103 g/mol Exact Mass: 582.2981 g/mol Exact Mass: 542.2668 g/mol Exact Mass: 602.2363 g/mol
	To a solution of compound 27 (14.10 g, 26 mmol) in MeOH (250 mL), was added an aq solu-
	To a stirred solution of 30% aq AcOH (200 mL), was added D-mannitol triacetonide 24 (10 g, 33.1 mmol). 1 H NMR (400 MHz, acetone-d 6 ): δ 4.73 (s, 2H, 2×OH), 3.96-3.90 (m, 2H, 2×CH), 3.77-3.54 (m, 8H, 2×CH, 2×CH 2 , 2×OH), 1.32 (s, 6H, 2×CH 3 ). NaH (60%, 6.36 g, 159 mmol) was added to a stirred solution of compound 25 (5.88 g, 26.49 mmol) in dry DMF (200 mL). chromatography (petroleum ether/EtOAc: 15/1) afforded compound 26 (15.10 g, 98%) as a colorless syrup. R f = 0.31 (petroleum ether/EtOAc: 10/1). 1 H NMR (400 MHz, CDCl 3 ): δ 7.41-7.17 (m, 20H, H-Ph), 4.73 (d, J = 11.8 Hz, 2H, 2×CH), Compound 26 (15.10 g, 25.96 mmol) was treated with 1M aq HCl/MeOH (1:9, v/v, 250 mL) under reflux for 4 h, when TLC showed a new single spot. Excess solid NaHCO 3 was added and the solution evaporated to give an oil which was dissolved in EtOAc (300 mL), dried over MgSO 4 and evaporated. Purification by column chromatography (petroleum ether/EtOAc: 5/1) afforded compound 27 (13.08 g, 93%) as a colorless syrup. R f = 0.12 (petroleum ether/EtOAc: 5/1). 1 H NMR (400 MHz, CDCl 3 ): δ 7.34-7.26 (m, 20H, H-Ph), 4.72 (d, J = 11.5 Hz, 2H, 2×CH), 4.59 (d, J = 11.5 Hz, 2H, 2×CH), 4.54 (s, 4H, 2×CH 2 ), 3.96 (m, 2H, 2×CH), 3.80-3.65 (m, 6H, 2×CH 2 , 2×CH), 3.04 (s, 1H, OH), 3.02 (s, 1H, OH). 13 C NMR (100 MHz, CDCl 3 ): δ 138.3, 138.1 (C q ); 128.5, 128.1, 127.9, 127.8 (CH-Ph); 79.3 To a solution of α-D-glucose pentaacetate (2 g, 5.12 mmol) in anhydrous CH 2 Cl 2 (15 mL) was added HBr (33% in AcOH, 7 mL, 41 mmol) at 0 º C. The mixture was stirred at room temper-ature under argon for 6 h, and then the solution neutralized with aq NaHCO 3 . The resulting 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide was extracted with CH 2 Cl 2 (100 mL), dried over MgSO 4 and evaporated to give a syrup. To a solution of (2S)-1,2-di-O-benzyl-glycerol (28) (1.41 g, 5.2 mmol), HgBr 2 (0.94 g, 2.6 mmol) and tion of Purification by column chromatography (petroleum ether/EtOAc: 3/1) afforded compound 28 (13.87 g, 98%) as a colorless syrup. R f = 0.24 (petroleum ether/EtOAc: 3/1). 1 H NMR (400 MHz, CDCl 3 ): δ 7.38-7.26 (m, 10H, H-Ph), 4.74-4.53 (m, 4H, 2×CH 2 ), Hg(troleum ether/EtOAc: 3/1) afforded compound 29 (2.30 g, 75%) as a colorless paste. 3.79-3.57 (m, 5H, 2×CH 2 , CH), 2.05 (s, 1H, OH). (CH); 73.6, 73.2, 70.3 (CH 2 ); 70.1 (CH). 4.57 (d, J = 11.7 Hz, 2H, 2×CH), 4.48-4.46 (m, 4H, 2×CH 2 ), 4.24-4.15 (m, 2H, 2×CH), 3.81-3.55 (m, 6H, 2×CH 2 , 2×CH), 1.35 (s, 6H, 2×CH 3 ). Ref.: van Boeckel, C.; Visser, G.; van Boom, J. Tetrahedron 1985, 41, 4557-4565. (b) Mannock, D. A.; 13 C NMR (100 MHz, CDCl 3 ): δ 138.4, 138.1 (C q ); 128.6, 128.6, 128.0, 127.9, 127.8 (CH-Ph); R f = 0.39 (petroleum ether/EtOAc: 2/1).
	Ref.: van Boeckel, C.; Visser, G.; van Boom, J. Tetrahedron 1985, 41, 4557-4565. (b) Mannock, D. A.; Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113-127. (Known structure) 78.1 (CH); 73.7, 72.3, 70.3, 63.0 (CH 2 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7.37-7.28 (m, 10H, H-Ph), 5.18 (t, J = 9.4 Hz, 1H, H-3'), 5.09 13 C NMR (100 MHz, CDCl 3 ): δ 138.6, 138.5 (C q ); 128.5, 128.4, 128.0, 127.7, 127.6 (CH-Ph); Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113-127. (Known structure) 109.9 (C q ); 79.4, 78.6 (CH); 73.4, 72.9, 70.7 (CH 2 ); 27.3 (CH 3 ). Ref.: van Boeckel, C.; Visser, G.; van Boom, J. Tetrahedron 1985, 41, 4557-4565. (b) Mannock, D. A.; (t, J = 9.7 Hz, 1H, H-4'), 5.00 (dd, J = 9.5, 8.2 Hz, 1H, H-2'), 4.65 (s, 2H, CH 2 ), 4.57-4.50
	Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113-127. (Known structure) (m, 3H, H-1', CH 2 ), 4.28-4.09
	Ref.: (a) van Boeckel, C.; Visser, G.; van Boom, J. Tetrahedron 1985, 41, 4557-4565. (b) Mannock, D. A.;
	Lewis, R. N.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113-127. (Known structure)

13

  under argon at 0 º C, was added TBSCl(0.86 g, 5.69 mmol). The reaction mixture was stirred for 20 min at 0 º C, then quenched with saturated aq Na 2 S 2 O 3 and diluted with EtOAc (50 mL). The aqueous layers were extracted with EtOAc (2×50 mL). The combined organic layers were dried over MgSO 4 , filtered, concentrated, and purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 100/1) to afford compound 31(2.43 g, 95.7%) as a yellowish paste.[M+H] + Calcd for C 31 H 44 NO 14Si 682.2531; Found 682.2526.

	(2R)-2-O-Phthalimido-1-O-tert-butyldimethylsilyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glu-
	copyranosyl)glycerol (32)
	Chemical Formula: C 31 H 43 NO 14 Si
	Exact Mass: 681.2453 g/mol
	The phthalimido group was introduced to compound 31 (2.10 g, 3.91 mmol) via Mitsunobu
	reaction according to the general procedure C. Purification by column chromatography over
	silica gel (petroleum ether/EtOAc: 3/1) afforded compound 32 (2.39 g, 90%) as a colourless
	paste.
	R f = 0.53 (petroleum ether/EtOAc: 1/1).
	[α] D = -13.3 (c 0.1, CHCl 3 ).
	R f = 0.74 (CH 2 Cl 2 /MeOH: 100/3). 1 H NMR (400 MHz, CDCl 3 ): δ 7.83-7.70 (m, 4H, Phth), 5.16 (t, J = 9.6 Hz, 1H, H-3'), 5.02
	(t, J = 9.8 Hz, 1H, H-4'), 4.92 (dd, J = 9.4, 8.1 Hz, 1H, H-2'), 4.66 (d, J = 7.8 Hz, 1H, H-1'), [α] D = -9.7 (c = 0.1, CHCl 3 ). 4.39-4.41 (m, 1H, H-2), 4.21 (dd, J = 12.3, 4.8 Hz, 1H, H-6'a), 4.15-4.06 (m, 2H, H-3a,6'b),
	1 H NMR (400 MHz, CDCl 3 ): δ 5.20 (dd, J = 12.0, 6.9 Hz, 1H, H-3'), 5.05 (t, J = 9.7 Hz, 1H, 3.93 (dd, J = 11.7, 6.2 Hz, 1H, H-3b), 3.89 (d, J = 4.7 Hz, 2H, H-1), 3.69 (ddd, J = 10.0, 4.7,
	H-4'), 4.98 (dd, J = 9.6, 8.1 Hz, 1H, H-2'), 4.54 (d, J = 7.9 Hz, 1H, H-1'), 4.25-4.11 (m, 2H, 2.2 Hz, 1H, H-5'), 2.06, 2.01, 1.99, 1.96 (4×s, 12H, 4×OAc), 0.77 (s, 9H, t-Bu), -0.02, -0.03
	H-6'), 3.82-3.68 (m, 4H, H-2,3,5'), 3.62-3.58 (m, 2H, H-1), 2.08, 2.04, 2.01, 1.99 (4×s, 12H, (2×s, 6H, Si(CH 3 ) 2 ).
	4×OAc), 0.88 (s, 9H, t-Bu), 0.05 (s, 6H, Si(CH 3 ) 2 ). 13 C NMR (100 MHz, CDCl 3 ): δ 170.8, 170.4, 169.6, 169.6 (C=O), 163.7 (C=O, Phth); 134.5,
	13 C NMR (100 MHz, CDCl 3 ): δ 170.7, 170.3, 169.5, 169.4 (C=O); 101.4 (C-1'), 72.8 (C-3'), 129.0 (C q ), 123.6 (Phth), 100.8 (C-1'), 87.0 (C-2), 73.0 (C-3'), 71.8 (C-5'), 71.2 (C-2'), 68.4
	71.9 (C-5'), 71.7 (CH 2 ), 71.4 (C-2'), 70.5 (CH), 68.5 (C-4'), 63.7, 62.0 (CH 2 ); 25.9, 20.8, (C-4'), 68.0, 62.0, 61.9 (CH 2 ); 25.8, 20.8, 20.8, 20.7 (CH 3 ); 18.2 (C q , t-Bu), -5.6 (CH 3 ,
	20.7 (CH 3 ); 18.3 (C q , t-Bu), -5.4 (CH 3 , Si(CH 3 ) 2 ). Si(CH 3 ) 2 ).
	HRMS (ESI) m/z: [M+H] + Calcd for C 23 H 41 O 12 Si 537.2367; Found 537.2362 Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721. HRMS (ESI) m/z: Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

  m/z:[M+H] + Calcd for C 25 H 30 NO 14 568.1666; Found 568.1661.

	(2R)-1-O-Palmitoyl-2-O-phthalimido-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)g
	lycerol (34)
	Chemical Formula: C 41 H 59 NO 15
	Exact Mass: 805.3885 g/mol
	Esterficationof compound 33 (0.58 g, 1.02 mmol) and palmitic acid according to the general
	procedure G and purification by column chromatography over silica gel (petroleum
	ether/EtOAc = 2/1) afforded compound 34 (1.05 g, 98%) as a white solid.
	R f = 0.54 (petroleum ether/EtOAc: 1/1).
	[α] D = -11.0 (c 0.1, CHCl 3 ).
	M.p. 65 ºC.
	1 H NMR (400 MHz, CDCl 3 ): δ 7.87-7.74 (m, 4H, Phth), 5.20 (t, J = 9.5 Hz, 1H, H-3'), 5.06
	(t, J = 9.7 Hz, 1H, H-4'), 4.96 (dd, J = 9.2, 7.8 Hz, 1H, H-2'), 4.68 (d, J = 8.2 Hz, 1H, H-1'),
	4.62-4.53 (m, 1H, H-2), 4.44-4.31 (m, 2H, H-1), 4.25 (dd, J = 12.3, 4.8 Hz, 1H, H-6'a),
	4.18-4.07 (m, 2H, H-3a,6'b), 4.01 (dd, J = 11.8, 5.8 Hz, 1H, H-3b), 3.73 (ddd, J = 9.9, 4.7, 2.4
	Hz, 1H, H-5'), 2.38-2.24 (m, 2H, CH 2 ), 2.10, 2.05, 2.02, 1.99 (4×s, 12H, 4×OAc), 1.65-1.52
	(m, 2H, CH 2 ), 1.35-1.20 (m, 24H, 12×CH
	Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

  2

  ). HRMS (ESI) m/z: [M+H] + Calcd for C 41 H 60 NO 15 806.3963; Found 806.3957.

	(2R)-2-O-Amino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)glycer
	ol (35)
	Chemical Formula: C 33 H 57 NO 13
	Exact Mass: 675.3830 g/mol
	Hydrazinolysis of compound 34 (0.38 g, 0.47 mmol) by hydrazine under mild conditions ac-
	cording to the general procedure E and purification by column chromatography over silica
	gel (petroleum ether/EtOAc = 3/2) afforded compound 35 (0.31 g, 96%) as a paste.
	R f = 0.40 (petroleum ether/EtOAc: 1/1).
	[α] D = -46.7 (c 0.1, CHCl 3 ).
	1 H NMR (400 MHz, CDCl 3 ): δ 5.22 (t, J = 8.7 Hz, 1H, H-3'), 5.06 (t, J = 9.6 Hz, 1H, H-4'),
	4.96 (dd, J = 12.7, 4.9 Hz, 1H, H-2'), 4.57 (d, J = 7.8 Hz, 1H, H-1'), 4.39-4.06 (m, 4H,
	H-1,6'), 4.04-3.90 (m, 2H, H-3a,2), 3.79-3.65 (m, 2H, H-3b,5'), 2.32 (t, J = 7.5 Hz, 2H, CH 2 ),
	2.09, 2.07, 2.03, 2.01 (4×s, 12H, 4×OAc), 1.68-1.57 (m, 2H, CH 2 ), 1.39-1.20 (m, 24H,
	12×CH 2 ), 0.88 (t, J = 6.7 Hz, 3H, CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 173.9, 170.8, 170.4, 169.5 (C=O); 101.2 (C-1'), 80.1 (CH),
	72.8 (C-3'), 72.0 (C-5'), 71.3 (C-2'),
	Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

  ). HRMS (ESI) m/z:[M+H] + Calcd for C 33 H 58NO 13 676.3908; Found 676.3903.

	(2R)-2-O-Hexanoylamino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranos
	yl)glycerol (36)
	Chemical Formula: C 39 H 67 NO 14
	Exact Mass: 773.4562 g/mol
	Compound 36 was prepared from compound 35 (153 mg, 0.23 mmol) and hexanoic acid (29
	μL, 0.23 mmol) according to the general procedure F for N-oxyamide formation. Purification
	by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound
	36 (145 mg, 82%) as a paste.
	R f = 0.60 (petroleum ether/EtOAc: 1/1).
	[α] D = -35.7 (c 0.1, CHCl 3 ).
	1 H NMR (400 MHz, CDCl 3 ): δ 8.83 (s, 1H, NH), 5.24 (t, J = 9.5 Hz, 1H, H-3'), 5.09 (t, J =
	9.7 Hz, 1H, H-4'), 5.02 (dd, J = 9.6, 7.8 Hz, 1H, H-2'), 4.57 (d, J = 8.0 Hz, 1H, H-1'), 4.42
	(dd, J = 12.2, 3.2 Hz, 1H, H-1a), 4.32-4.09 (m, 4H, H-2,1b,6'), 4.03-3.96 (m, 1H, H-3a),
	3.81-3.68 (m, 2H, H-3b,5'), 2.39-2.33 (m, 2H, CH 2 ), 2.17-2.01 (m, 14H, 4×OAc, CH 2 ),
	1.73-1.57 (m, 4H, 2×CH 2 ), 1.39-1.21 (m, 28H, 14×CH 2 ), 0.96-0.86
	Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

  ). HRMS (ESI) m/z: [M+H] + Calcd for C 39 H 68 NO 14 774.4640; Found 774.4634.

	(2R)-2-O-(2-Ethylhexanoyl)amino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-gluco
	pyranosyl)glycerol (38)
	Chemical Formula: C 41 H 71 NO 14
	Exact Mass: 801.4875 g/mol
	Compound 38 was prepared from compound 35 (151 mg, 0.22 mmol) and 2-ethylhexanoic
	acid (35 μL, 0.22 mmol) according to the general procedure F for N-oxyamide formation.
	Purification by column chromatography over silica gel (petroleum ether/EtOAc: 3/1) afforded
	compound 38 (114 mg, 64%) as a paste.
	R f = 0.47 (petroleum ether/EtOAc: 1/1).
	[α] D = -38.0 (c 0.1, CHCl 3 ).
	47-4.39 (m,
	1H, H-1a), 4.31-4.10 (m, 4H, H-2,1b,6'), 4.03-3.96 (m, 1H, H-3a), 3.78-3.69 (m, 2H,
	H-3b,5'), 2.36 (t, J = 7.2 Hz, 2H, CH 2 ), 2.10, 2.09, 2.04, 2.02 (4×s, 12H, 4×OAc), 1.95-1.85
	(m, 1H, CH), 1.69-1.57 (m, 4H, 2×CH 2 ), 1.52-1.39 (m, 2H, CH 2 ), 1.37-1.20 (m, 28H,
	14×CH 2 ), 0.95-0.84 (m, 9H, 3×CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 174.4, 173.7, 170.8, 170.4, 170.2, 169.6 (C=O); 100.7 (C-1'),
	82.2 (CH), 72.5 (C-3'), 72.0 (C-5'), 71.5 (C-2'), 68.3 (C-4'), 67.9, 61.8, 61.3 (CH 2 ); 45.7
	(CH),
	Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

1

H NMR (400 MHz, CDCl 3 ): δ 8.87 (s, 1H, NH), 5.25 (t, J = 9.4 Hz, 1H, H-3'), 5.09 (t, J = 9.7 Hz, 1H, H-4'), 5.01 (t, J = 8.7 Hz, 1H, H-2'), 4.57

(d, J = 7.7 Hz, 1H, H-1'), 4.

  ). HRMS (ESI) m/z: [M+H] + Calcd for C 41 H 72 NO 14 802.4953; Found: 802.4947.

	(2R)-1-O-Palmitoyl-2-O-palmitoylamino-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranos
	yl)glycerol (39)
	Chemical Formula: C 49 H 87 NO 14
	Exact Mass: 913.6127 g/mol
	Compound 39 was prepared from compound 35 (189 mg, 0.28 mmol) and palmitic acid (72
	mg, 0.28 mmol) according to the general procedure F for N-oxyamide formation. Purifica-
	tion by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded com-
	pound 39 (192 mg, 76%) as a white solid.
	R f = 0.57 (petroleum ether/EtOAc: 1/1).
	[α] D = -42.3 (c 0.1, CHCl 3 ).
	M.p. 74 o C.
	J = 12.2, 3.3 Hz, 1H, H-1a), 4.32-4.08 (m, 4H, H-2,1b,6'), 4.03-3.96 (m, 1H, H-3a), 3.78-3.69
	(m, 2H, H-3b,5'), 2.36 (t, J = 7.5 Hz, 2H, CH 2 ), 2.18-2.01 (m, 14H, 4×OAc, CH 2 ), 1.68-1.58
	(m, 4H, 2×CH 2 ), 1.40-1.22 (m, 48H, 24×CH 2 ), 0.93-0.81 (m, 6H, 2×CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 174.4, 171.2, 170.8, 170.2, 169.5 (C=O); 100.7 (C-1'), 82.1
	(CH), 72.5 (C-3'), 72.1 (C-5'), 71.4 (C-2'),

Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721.

1

H NMR (400 MHz, CDCl 3 ): δ 8.86 (s, 1H, NH), 5.24 (t, J = 9.5 Hz, 1H, H-3'), 5.09 (t, J = 9.7 Hz, 1H, H-4'), 5.02

(t, J = 9.6, 7.8 Hz, 1H, H-2'), 4.57 (d, J = 7.9 Hz, 1H, H-1'), 4.42 (dd,

  Calcd for C 49 H 88 NO 14 914.6205; Found 914.6199.

	(2R)-2-O-Oleoylamino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)
	glycerol (40)
	Chemical Formula: C 51 H 89 NO 14
	Exact Mass: 939.6283 g/mol
	Compound 40 was prepared from compound 35 (151 mg, 0.22 mmol) and oleic acid (62 mg,
	0.22 mmol) according to the general procedure F for N-oxyamide formation. Purification by
	column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 40
	(180 mg, 87%) as a white solid.
	R f = 0.52 (petroleum ether/EtOAc: 1/1).
	[α] D = -35.3 (c 0.1, CHCl 3 ).
	M.p. 57 ºC.
	=
	7.9 Hz, 1H, H-1'), 4.42 (dd, J = 12.2, 3.3 Hz, 1H, H-1a), 4.32-4.08 (m, 4H, H-2,1b,6'),
	4.03-3.96 (m, 1H, H-3a), 3.78-3.69 (m, 2H, H-3b,5'), 2.36 (t, J = 7.5 Hz, 2H, CH 2 ), 2.20-1.98
	(m, 16H, 4×OAc, 2×CH 2 ), 1.71-1.57 (m, 4H, 2×CH 2 ), 1.39-1.21 (m, 46H, 23×CH 2 ),
	0.95-0.82 (m, 6H, 2×CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 174.5, 170.8, 170.4, 170.3, 169.6 (C=O); 130.1, 129.8
	(CH=CH); 100.7 (C-1'), 82.2 (CH), 72.5 (C-3'), 72.1 (C-5'), 71.4 (C-2'), 68.4 (C-4'), 67.8,

). HRMS (ESI) m/z: [M+H] + Ref.: Chen, N.; Xie, J. J. Org. Chem. 2014, 79, 10716-10721. 1 H NMR (400 MHz, CDCl 3 ): δ 8.83 (s, 1H, NH), 5.43-5.32 (m, 2H, CH=CH), 5.24 (t, J = 9.5

Hz, 1H, H-3'), 5.09 (t, J = 9.7 Hz, 1H, H-4'), 5.02 (dd, J = 9.6, 8.0 Hz, 1H, H-2'), 4.57

(d, J

  Calcd for C 33 H 64 NO 10 634.4530; Found 634.4525.

	(2R)-2-O-(2-Ethylhexanoyl)amino-3-O--D-glucopyranosyl-1-O-palmitoylglycerol (43)
	Chemical Formula: C 33 H 63 NO 10
	Exact Mass: 633.4452 g/mol
	Deacetylation of compound 38 (85 mg, 0.11 mmol) with hydrazine according to the general
	procedure H and purification by column chromatography over silica gel (CH 2 Cl 2 /MeOH:
	20/1) led to compound 43 (29 mg, 43%) as a paste.
	R f = 0.39 (CH 2 Cl 2 /MeOH: 10/1).
	[α] D = -29.7 (c 0.1, MeOH).
	1 H NMR (400 MHz, CD 3 OD): δ 4.37-4.21 (m, 3H, H-1',1), 4.20-4.12 (m, 1H, H-2), 4.05 (dd,
	13 C NMR (100 MHz, CD 3 OD): δ 175.7, 175.2 (C=O); 104.9 (C-1'), 83.9, 78.0, 77.9, 75.0,
	71.5 (CH); 69.0, 63.2, 62.7 (CH 2 ); 46.6 (CH),

(CH 2 ); 14.5 (CH 3 ).

HRMS (ESI) m/z: [M+H] +

Ref.: Chen, N.; Xie, J. J.

Org. Chem. 2014, 79, 10716-10721

. J = 11.1, 3.9 Hz, 1H, H-3a), 3.85

(d, J = 11.7 Hz, 1H, H-6'a)

,

3.75 (dd, J = 11.3, 6.2 Hz, 1H, H-3b), 3.71-3.60 (m, 1H, H-6'b), 3.44-3.24 (m, 3H, H-3',4',5'), 3.22-3.15 (m, 1H, H-2')

, 2.34 (t, J = 7.3 Hz, 2H, CH 2 ), 2.03-1.90

(m, 1H, CH), 1.70-1.50 (m, 4H, 2×CH 2

), 1.49-1.17 (m, 30H, 15×CH 2 ), 1.06-0.81

(m, 9H, 3×CH 3

).

  [M+Na] + Calcd for C 25 H 41NNaO 13 586.2476; Found 586.2474.

	(2S)-2-O-Palmitoylamino-1-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)glycerol(48)
	Chemical Formula: C 33 H 57 NO 13
	Exact Mass: 675.3830 g/mol
	Coupling of oxyamine compound 46 (135 mg, 0.245 mmol) with palmitic acid according to
	the general procedure F led to the N-oxyamide compound which was used directly for the
	next step without purification. Desilylation of this crude compound using TBAF according to
	the general procedure A and purification by column chromatography over silica gel (petro-
	leum ether/EtOAc = 1/1) afforded compound 48 (78 mg, 47%, two steps) as a white solid.
	R f = 0.14 (petroleum ether/EtOAc = 1/1).
	[α] D = -66.7 (c 0.1, CHCl 3 ).
	M.p. 117 º C.
	4.25
	(dd, J = 12.8, 4.6 Hz, 1H, H-6'a), 4.14 (dd, J = 12.4, 1.8 Hz, 1H, H-6'b), 4.01-3.91 (m, 2H,
	H-1a, 2), 3.74-3.67 (m, 1H, H-5'), 3.61-3.48 (m, 3H, H-1b,3), 2.16-1.96 (m, 14H, 4×OAc,
	CH 2 ), 1.68-1.56 (m, 2H, CH 2 ), 1.34-1.17 (m, 24H, 12×CH 2 ), 0.85 (t, J = 6.9 Hz, 3H, CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 172.6, 171.1, 170.8, 170.2, 169.6 (C=O); 101.2 (C-1'), 89.0
	(CH), 72.2 (C-3'), 72.0 (C-5'), 71.8 (C-2'), 69.4 (CH 2 ), 68.3 (C-4'),

).

HRMS (ESI) m/z:

1 H NMR (400 MHz, CDCl 3 ): δ 9.19 (s, 1H, NH), 5.24 (t, J = 9.6 Hz, 1H, H-3'), 5.06 (t, J = 9.8 Hz, 1H, H-4'), 5.00 (dd, J = 9.6, 7.8 Hz, 1H, H-2'), 4.48

(d, J = 7.8 Hz, 1H, H-1'),

  [M+Na] + Calcd for C 33 H 57NNaO 13 698.3728; Found 698.3734.

	(2S)-2-O-Oleoylamino-1-O-(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)glycerol (49)
	Chemical Formula: C 35 H 59 NO 13
	Exact Mass: 701.3986 g/mol
	Coupling of oxyamine compound 46 (110 mg, 0.20 mmol) with oleic acid according to the
	general procedure F led to the N-oxyamide compound which was used directly for the next
	step without purification. Desilylation of this crude compound using TBAF according to the
	general procedure A and purification by column chromatography over silica gel (petroleum
	ether/EtOAc = 1/1) afforded compound 49 (69 mg, 49%, two steps) as a white solid.
	R f = 0.16 (petroleum ether/EtOAc = 1/1).
	[α] D = -72.7 (c 0.1, CHCl 3 ).
	M.p. 85 º C.
	30-4.11 (m, 2H,
	H-6'), 4.03-3.91 (m, 2H, H-1a,2,), 3.76-3.67 (m, 1H, H-5'), 3.63-3.48 (m, 3H, H-1b,3),
	2.24-1.93 (m, 16H, 4×OAc, 2×CH 2 ), 1.69-1.57 (m, 2H, CH 2 ), 1.46-1.12 (m, 22H, 11×CH 2 ),
	0.85 (t, J = 6.6 Hz, 3H, CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ): δ 172.6, 171.1, 170.8, 170.1, 169.5 (C=O); 130.1, 129.8
	(CH=CH); 101.1 (C-1'), 88.9 (CH), 72.1 (C-3'), 72.0 (C-5'), 71.8 (C-2'), 69.4 (CH 2 ), 68.3
	(C-4'),

).

HRMS (ESI) m/z:

1 H NMR (400 MHz, CDCl 3 ): δ 9.21 (s, 1H, NH), 5.42-5.28 (m, 2H, CH=CH), 5.24 (t, J = 9.6 Hz, 1H, H-3'),

5.10-4.96 (m, 2H, H-2',4'), 4.48 (d, J = 7.8 Hz, 1H, H-1'), 4.

  [M+Na] + Calcd for C 35 H 59NNaO 13 724.3884; Found 724.3878.

	(2S)-3-O--D-Glucopyranosyl-2-O-palmitoylaminoglycerol (51)
	Chemical Formula: C 25 H 49 NO 9
	Exact Mass: 507.3407 g/mol
	Deacetylation of compound 48 (48 mg, 0.071 mmol) with Na/MeOH according to the general
	procedure I led to compound 51 (36 mg, 100%) as a yellowish paste.
	R f = 0.62 (CH 2 Cl 2 /MeOH: 5/1).
	[α] D = +45.0 (c 0.1, CHCl 3 ).
	1 H NMR (400 MHz, CD 3 OD): δ 4.33 (d, J = 7.8 Hz, 1H, H-1'), 4.04 (dd, J = 11.0, 4.1 Hz,
	1H, H-1a), 4.01-3.94 (m, 1H, H-2), 3.91-3.84 (m, 1H, H-6'a), 3.78-3.60 (m, 4H, H-1b,3,6'b),
	3.40-3.24 (m, 3H, H-3',4',5'), 3.21 (dd, J = 9.1, 7.9 Hz, 1H, H-2'), 2.12 (t, J = 7.3 Hz, 2H,
	CH 2 ), 1.66-1.55 (m, 2H, CH 2 ), 1.38-1.21 (m, 24H, 12×CH

).

HRMS (ESI) m/z:

  2

  (CH 2 ); 14.5 (CH 3 ).

	(2S)-3-O--D-Glucopyranosyl-2-O-oleoylaminoglycerol (52)
	Chemical Formula: C 27 H 51 NO 9
	Exact Mass: 533.3564 g/mol
	Deacetylation of compound 49 (49 mg, 0.07 mmol) with Na/MeOH according to the general
	procedure I led to compound 52 (37 mg, 99%) as a yellowish paste.
	R f = 0.51 (CH 2 Cl 2 /MeOH: 5/1).
	[α] D = -22.3 (c 0.1, CHCl 3 ).
	1 H NMR (400 MHz, CD 3 OD): δ 5.40-5.29 (m, 2H, CH=CH), 4.33 (d, J = 7.8 Hz, 1H, H-1'),
	4.04 (dd, J = 11.1, 4.1 Hz, 1H, H-1a), 4.01-3.94 (m, 1H, H-2), 3.91-3.83 (m, 1H, H-6'a),
	3.78-3.60 (m, 4H, H-1b,3,6'b), 3.39-3.24 (m, 3H, H-3',4',5'), 3.21 (dd, J = 9.1, 7.8 Hz, 1H,
	H-2'), 2.12 (t, J = 7.4 Hz, 2H, CH 2 ), 2.07-1.97 (m, 4H, 2×CH 2 ), 1.66-1.55 (m, 2H, CH 2 ),
	1.41-1.19 (m, 20H, 10×CH 2 ), 0.90 (t, J = 6.6 Hz, 3H, CH 3 ).
	HRMS (ESI) m/z: [M+Na] + Calcd for C 25 H 49 NNaO 9 530.3305; Found 530.3306.

13

C NMR (100 MHz, CD 3 OD): δ 173.9 (C=O),

130.9, 130.8 (CH=CH)

; 104.7 (C-1'),

87.3, 78.0, 75.0, 71.6 (CH)

; 69.

  AcOH, 3.52 mL, 20.48 mmol) at 0 º C. The mixture was stirred at room temperature under argon for 6 h, and then the solution neutralized with aq NaHCO 3 in an ice bath. The resulting 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was extracted with CH 2 Cl 2 (100 mL), dried over MgSO 4 , filtered and evaporated to give a syrup. To a solution of (2S)-1,2-di-O-benzyl-glycerol 28(1.39 g, 5.12 mmol), HgBr 2(0.92 g, 2.56 mmol) and Hg(CN) 2(0.65 g, 2.56 mmol) in dry acetonitrile (10 mL) was added dropwise over a period of 1 h, a solution of glycosyl bromide in acetonitrile (5 mL). The reaction solution was stirred at room temperature for 15 h, and then concentrated to an oil which was dissolved in CH 2 Cl 2 (100 mL) and washed with saturated aq NaHCO 3 (2×30 mL) and water (30 mL). The dried (MgSO The reaction mixture was stirred for 20 min at 0 ºC, then quenched with saturated aq Na 2 S 2 O 3 and the solution was concentrated. The residue was diluted with EtOAc (200 mL), washed with saturated aq Na 2 S 2 O 3 (2×50 mL), H 2 O (50 mL) and brine(50 mL), dried over MgSO 4 , filtered, concentrated, and purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 100/1) to afford compound 55(3.20 g, 90%) as a colourless paste. R f = 0.73 (CH 2 Cl 2 /MeOH: 100/3). ,3b,5'),2.12, 2.05, 2.04, 1.96 (4×s, 12H, 4×OAc), 0.79 (s, 9H, t-Bu), 0.00, -0.01 (2×s, 6H, Si(CH 3 ) 2 ).

	(2R)-1,2-Di-O-benzyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-galactopyranosyl)glycerol (53) (2R)-1-O-(2′,3′,4′,6′-Tetra-O-acetyl--D-galactopyranosyl)glycerol (54) (2S)-1-O-tert-Butyldimethylsilyl-3-O-(2',3',4',6'-tetra-O-acetyl--D-galactopyranosyl)gly (2R)-2-O-Phthalimido-1-O-tert-butyldimethylsilyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-gala
	cerol (55) ctopyranosyl)glycerol (56)
	Chemical Formula: C 31 H 38 O 12 Chemical Formula: C 17 H 26 O 12
	Chemical Formula: C 23 H 40 O 12 Si Chemical Formula: C 31 H 43 NO 14 Si
	Exact Mass: 602.2363 g/mol Exact Mass: 422.1424 g/mol
	Exact Mass: 536.2289 g/mol Exact Mass: 681.2453 g/mol
	To a solution of -D-galactose pentaacetate (2 g, 5.12 mmol) in CH 2 Cl 2 (15 mL) was added A mixture of compound 53 (7 g, 11.63 mmol) and 10% Pd/C (1 g) in EtOAc (100 mL) con-
	(CH 2 ); 14.5 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 27 H 51 NNaO 9 556.3462; Found 556.3463. 4.56-4.46 (m, 3H, H-1', OCH 2 ), 4.19-4.08 (m, 2H, H-6'), 3.98 (dd, J = 9.8, 4.2 Hz, 1H, H-3a), 3.86 (td, J = 6.5, 0.8 Hz, 1H, H-5'), 3.78-3.68 (m, 2H, H-2,3b), 3.63-3.55 (m, 2H, H-1), 2.15, t-Bu), -5.4 (CH 3 , Si(CH 3 ) 2 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 17 H 26 NaO 12 445.1322; Found 445.1321. 70.9, 70.8, 70.5, 68.9, 67.1 (CH); 63.7, 61.4 (CH 2 ); 25.9, 20.8, 20.7, 20.6 (CH 3 ); 18.3(C q , 10.5, 7.9 Hz, 1H, H-2'), 4.99 (t, J = 10.5, 3.4 Hz, 1H, H-3'), 4.67-4.62 (m, 2H, OCH 2 ), 20.7 (OAc). 13 C NMR (100 MHz, CDCl 3 ): δ 170.4, 170.2, 170.1, 169.5 (C=O); 101.8 (C-1'), 71.6 (CH 2 ), 1 H NMR (400 MHz, CDCl 3 ): δ 7.38-7.27 (m, 10H, H-Ph), 5.40-5.34 (m, H-4'), 5.20 (dd, J = taining 10 mL each of EtOH and AcOH was vigorously shaken under H 2 at room temperature overnight. The catalyst was filtered off, the filtrate evaporated. Purification by column chro-13 C NMR (100 MHz, CDCl 3 ): δ 170.6, 170.4, 170.3, 170.0 (C=O); 102.0 (C-1'), 72.8 (CH 2 ), 71.0 (C-5'), 70.7 (C-3'), 70.5 (CH), 68.9 (C-2'), 67.1 (C-4'), 63.5, 61.5 (CH 2 ), 20.9, 20.8, To a solution of compound 54 (2.8 g, 6.64 mmol), N-methylimidazole (1.59 mL, 19.92 mmol) and iodine (5.05 g, 19.92 mmol) in anhydrous THF (30 mL) under argon at 0 ºC, was added H-6'), 3.93-3.85 (m, 1H, H-5'), 3.80-3.65 (m, 3H, H-2,3), 3.61-3.53 (m, 2H, H-1), 2.54 (s, 1H, OH), 2.11, 2.01, 2.01, 1.93 (4×s, 12H, 4×OAc), 0.85 (s, 9H, t-Bu), 0.02 (s, 6H, Si(CH 3 ) 2 ). The phthalimido group was introduced to compound 55 (1.68 g, 3.13 mmol) via Mitsunobu reaction according to the general procedure C. Purification by column chromatography over HBr (33% in [α] D = -15.0 (c 0.1, CHCl 3 ). matography (CH 2 Cl 2 /MeOH: 100/3) afforded compound 54 (3.45 g, 70%) as a colourless paste. R f = 0.22 (CH 2 Cl 2 /MeOH: 100/3). [α] D = +3.0 (c 0.1, CHCl 3 ). 99 (s, 2H, 2×OH), 2.17, 2.08, 2.07, 2.00 (4×s, 12H, 4×OAc). silica gel (CH 2 Cl 2 /MeOH: 100/0.5) afforded compound 56 (2.06 g, 97%) as a yellowish paste. R f = 0.60 (CH 2 Cl 2 /MeOH: 100/1). [α] D = +4.0 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7.88-7.71 (m, 4H, Phth), 5.36 (dd, J = 3.4, 1.0 Hz, 1H, H-4'), 5.16 (dd, J = 10.6, 7.8 Hz, 1H, H-2'), 5.00 (dd, J = 10.6, 3.6 Hz, 1H, H-3'), 4.65 (d, J = 8.2 Hz, 1H, H-1'), 4.46-4.39 (m, 1H, H-2), 4.18-4.08 (m, 3H, H-3a,6'), 3.98-3.88 (m, 4H, TBSCl (1.1 g, 7.30 mmol). 05 (m, 2H, H-1
	2.04, 1.98, 1.95 (4×s, 12H, 4×OAc). HRMS (ESI) m/z: [M+Na] + Calcd for C 23 H 40 NaO 12 Si 559.2187; Found 559.2188.
	13 C NMR (100 MHz, CDCl 3 ): δ 170.5, 170.4, 170.3, 169.5 (C=O); 138.6, 138.3 (C q ); 128.5,
	127.8 (CH-Ph); 101.2 (C-1'), 77.0 (CH), 73.6, 72.3 (CH 2 ); 71.0 (C-3'), 70.8 (C-5'), 69.9, 69.3
	(CH 2 ); 69.0 (C-2'), 67.2 (C-4'), 61.4 (C-6'), 20.8, 20.7 (OAc).
	HRMS (ESI) m/z: [M+Na] + Calcd for C 31 H 38 NaO 12 625.2261; Found 625.2261.

4 ) organic layer was concentrated. Purification by column chromatography (petroleum ether/EtOAc: 3/1) afforded compound 53 (2.30 g, 75%) as a colorless paste. R f = 0.20 (petroleum ether/EtOAc: 3/1).

1

H NMR (400 MHz, CDCl 3 ): δ 5.40 (dd, J = 3.7, 0.9 Hz, 1H, H-4'), 5.20 (dd, J = 10.5, 7.

8

Hz, 1H, H-2'), 5.03 (dd, J = 10.5, 3.2 Hz, 1H, H-3'), 4.51 (d, J = 8.2 Hz, 1H, H-1'), 4.21-4.10 (m, 2H, H-6'), 3.99-3.92 (m, 1H, H-5'),

3.90-3.75 (m, 3H, H-2,3), 3.70 (dd, J = 11.4, 3.7 Hz, 1H, H-1a), 3.61 (dd, J = 11.4, 4.6 Hz, 1H, H-1b)

, 2.

[α] D = -7.3 (c 0.1, CHCl 3 ).

1

H NMR (400 MHz, CDCl 3 ): δ 5.34

(d, J = 3.5 Hz, 1H, H-4'), 5.16 (dd, J = 10.5, 8.0 Hz, 1H, H-2'), 4.97 (dd, J = 10.5, 3.3 Hz, 1H, H-3'), 4.47 (d, J = 7.8 Hz, 1H, H-1'), 4.13-4.

  H NMR (400 MHz, CDCl 3 ): 5.39(d, J = 3.2 Hz, 1H, H-4'), 5.21 (dd, J = 10.5, 7.8 Hz, 1H, H-2'), 5.00 (dd, J = 10.5, 3.2 Hz, 1H, H-3'), 4.53 (d, J = 8.2 Hz, 1H, H-1'), 4.41-4.33 (m, 1H, H-2), 4.28-4.09 (m, 4H, H-1,6'), 3.97 (dd, J = 11.4, 5.0 Hz, 1H, H-3a),3.93-3.87 (m, 1H, H-5'), 3.77 (dd, J = 11.2, 6.2 Hz, 1H, H-3b), 2.31 (t, J = 7.6 Hz, 1H, CH 2 ), 2.16, 2.06, 2.04, 1.99 (4×s, 12H, 4×OAc), 1.71-1.57 (m, 2H, CH 2 ),1.35-1.22 (m, 24H, 12×CH

1

  2

  ). HRMS (ESI) m/z: [M+Na] + Calcd for C 33 H 57 NNaO 13 698.3782; Found 698.3715. Chemical Formula: C 39 H 67 NO 14 Exact Mass: 773.4562 g/mol Compound 60 was prepared from compound 59(115 mg, 0.17 mmol) and hexanoic acid (21 μL, 0.17 mmol) according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 60 (115 mg, 88%) as a colourless paste. R f = 0.55 (petroleum ether/EtOAc: 1/1).[α] D = -62.0 (c 0.1, CHCl 3 ). H NMR (400 MHz, CDCl 3 ): δ 8.86 (s, 1H, NH), 5.37 (t, J = 3.2 Hz, 1H, H-4'),5.16 (dd, J = 10.3, 8.6 Hz, 1H, H-2'), 5.01 (dd, J = 10.5, 3.2 Hz, 1H, H-3'), 4.50 (d, J = 7.8 Hz, 1H, H-1'), 4.36 (dd, J = 12.2, 3.5 Hz, 1H, H-1a), 4.24-4.04 (m, 4H, H-2,1b,6'), 4.02-3.95 (m, 1H, H-3a), 3.93-3.87 (m, 1H, H-5'), 3.67 (dd, J = 10.1, 7.2 Hz, 1H, H-3b), 2.31 (t, J = 7.6 Hz, 2H, CH 2 ), 2.15-1.93 (m, 14H, 4×OAc, CH 2 ), 1.65-1.53 (m, 4H, 2×CH 2 ), 1.35-1.11 (m, 28H, 14×CH 2 ), 0.91-0.79 (m, 6H, 2×CH 3 ).

	(2R)-2-O-Hexanoylamino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-galactopyran
	osyl)glycerol (60)

1 13 C NMR (100 MHz, CDCl 3 ): δ 174.2, 170.8, 170.4, 170.2, 170.1 (C=O); 101.2 (C-1'), 82.3 (CH), 70.8 (C-5'), 70.6 (C-3'), 69.1 (C-2'), 68.2 (CH 2 ), 66.9 (C

  , 2.36 (t, J = 7.3 Hz, 2H, CH 2 ), 2.19-1.97 (m, 14H, 4×OAc, CH 2 ), 1.69-1.57 (m, 4H, 2×CH 2 ), 1.38-1.20 (m, 32H, 16×CH 2 ), 0.91-0.84

  , 2.36 (t, J = 7.6 Hz, 2H, CH 2 ), 2.18-1.98 (m, 14H, 4×OAc, CH 2 ), 1.68-1.57 (m, 4H, 2×CH 2 ), 1.36-1.17 (m, 48H, 24×CH 2 ), 0.91-0.83

  ). HRMS (ESI) m/z: [M+Na] + Calcd for C 49 H 87 NNaO 14 936.6024; Found 936.6024. Chemical Formula: C 51 H 89 NO 14 Exact Mass: 939.6283 g/mol Compound 64 was prepared from compound 59(115 mg, 0.17 mmol) and oleic acid(48 mg, 0.217 mmol) according to the general procedure F for N-oxyamide formation. Purification by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) afforded compound 64 (147 mg, 92%) as colourless paste. R f = 0.56 (petroleum ether/EtOAc: 1/1).[α] D = -42.0 (c 0.1, CHCl 3 ). H NMR (400 MHz, CDCl 3 ): δ 8.84 (s, 1H, NH), 5.41 (t, J = 3.2 Hz, 1H, H-4'), 5.39-5.29(m, 2H, CH=CH), 5.21 (dd, J = 10.6, 7.8 Hz, 1H, H-2'), 5.05 (dd, J = 10.6, 3.6 Hz, 1H, H-3'), 4.53 (d, J = 7.8 Hz, 1H, H-1'), 4.42 (dd, J = 12.4, 3.2 Hz, 1H, H-1a), 4.26-4.09 (m, 4H, H-2,1b,6'), 4.05-3.98 (m, 1H, H-3a), 3.96-3.89 (m, 1H, H-5'), 3.71 (dd, J = 10.5, 7.3 Hz, 1H, H-3b), 2.36 (t, J = 7.6 Hz, 2H, CH 2 ), 2.18-1.96 (m, 16H, 4×OAc, 2×CH 2 ), 1.68-1.57 (m, 4H, 2×CH 2 ), 1.38-1.17 (m, 46H, 23×CH 2 ), 0.91-0.84

	(2R)-2-O-Oleoylamino-1-O-palmitoyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl--D-galactopyranosy
	l)glycerol (64)

1

  , 2.31 (t, J = 7.5 Hz, 2H, CH 2 ), 2.12 (t, J = 7.4 Hz, 2H, CH 2 ), 1.68-1.55 (m, 4H, 2×CH 2 ),1.43-1.23 (m, 28H, 14×CH 2 ), 0.97-0.86(m, 6H, 2×CH 3

  (CH 2 ); 14.5 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 25 H 49 NNaO 9 530.3305; Found 530.3311. Chemical Formula: C 29 H 35 NO 15 Exact Mass: 637.2007 g/mol To a solution of compound 33 (0.83 g, 1.46 mmol) in CH 2 Cl 2 (30 mL), were added PCC (98%, 0.63 mg, 2.92 mmol),Ac 2 O (1.38 ml, 14.63 mmol) and t-BuOH (2.80 mL, 29.27 mmol). The resulting mixture was stirred at room temperature and then extracted with CH 2 Cl 2 (3×30 mL). The combined organic layers were washed with brine (30 mL), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) to give compound 73 (0.57 g, 61%) as a white solid. R f = 0.44 (petroleum ether/EtOAc: 1/1).[α] D = +2.0 (c 0.1, CHCl 3 ). H NMR (400 MHz, CDCl 3 ): δ 7.91-7.76 (m, 4H, Phth), 5.21 (t, J = 9.6 Hz, 1H, H-3'), 5.07(t, J = 9.6 Hz, 1H, H-4'), 4.99 (dd, J = 9.6, 7.8 Hz, 1H, H-2'), 4.93 (dd, J = 7.3, 3.6 Hz, 1H, H-2), 4.63 (d, J = 7.8 Hz, 1H, H-1'), 4.30-4.09 (m, 4H, H-3,6'), 3.74 (ddd, J = 10.0, 4.6, 2.2 Hz, 1H, H-5'), 2.11, 2.08, 2.03, 2.00 (4×s, 12H, 4×OAc), 1.47 (s, 9H, t-Bu).

	tert-Butyl (2R)-2-phthalimidooxy-3-[(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)oxy]-
	propanoate (73)

M.p. 64 º C.

1

  ). HRMS (ESI) m/z: [M+Na] + Calcd for C 29 H 35 NNaO 15 660.1904; Found 660.1899. Chemical Formula: C 23 H 40 O 12 Si Exact Mass: 536.2289 g/mol To a solution of compound 31 (54 mg, 0.1 mmol) in CH 2 Cl 2(2 mL) and pyridine (0.05 mL) at -40 º C under argon, was added dropwise trifluoromethanesulfonic anhydrous(34 μL, 0.2 mmol). After 1 h 30 min, the mixture was diluted with CH 2 Cl 2 (20 mL), washed with aq HCl (1N, 10 mL) and brine (20 mL), dried over MgSO 4 , filtered, evaporated. The residue containing triflate derivative was treated with NaNO 2 (69 mg, 1 mmol) in DMF/H 2 O (2 mL, 1/1). After stirring at room temperature overnight, the mixture was diluted with H 2 O (10 mL), and the product was extracted with CH 2 Cl 2 (3×15 mL). The combined organic layers were washed with brine (15 mL), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH: 100/0.5) to give compound 74 (24 mg, 44%) as a yellowish paste. R f = 0.35 (CH 2 Cl 2 /MeOH: 100/1).

	(2R)-1-O-tert-Butyldimethylsilyl-3-O-(2',3',4',6'-tetra-O-acetyl--D-glucopyranosyl)-glyc
	erol (74)

2′,3′,4′,6′tetra-O-acetyl--D-glucopyranosyl)oxy]propanoate (87)

  ; 101.4 (C-1'),86.4 (CH), 72.3 (C-3'), 72.1 (C-5'), 71.7 (C-2'), 68.9 (CH 2 ),68.3 (C-4'), 61.8 (CH 2 ), 46.9 (CH), 21.0, 20.9, 20.7 (CH 3 ). HRMS (ESI) m/z: [M+Na] + Calcd for C 32 H 35 NNaO 15 696.1904; Found 696.1906. Chemical Formula: C 38 H 46 N 2 O 16 Exact Mass: 786.2847 g/mol To a solution of compound 82 (0.48 g, 0.94 mmol) in anhydrous CH 2 Cl 2

	tert-Butyl	(2R)-2-N-(9-fluorenylmethoxycarbonylaminomethylcarbonyl)aminooxy-
	3[(

  ). HRMS (ESI) m/z: [M+K] + Calcd for C 34 H 38 N 2 NaO 16 753.2119; Found 753.2115.

  ). HRMS (ESI) m/z: [M+K] + Calcd for C 40 H 49 N 3 NaO 17 866.2960; Found 866.2962.

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Acknowledgement

This dissertation is accomplished in the "Laboratoire de Photophysique et Photochimie Supramoléculaires et Macromoléculaires (PPSM)'' in École Normale Supérieure de Cachan (ENS Cachan). Here I must express my sincere gratitude to all the people for giving me the help to make this dissertation possible and particularly the directors of PPSM Prof. Keitaro Nakatani, Prof. Fabien Miomandre and Dr. Isabelle Leray.

First and foremost I wish to thank my advisor, Prof. Joanne Xie, for her help both in work and in life. She offered me the opportunity to work in ENS Cachan and gave me very professional guidance in this dissertation. I am deeply grateful of her help in the completion of this manuscript. Her gamut of knowledge, infectious enthusiasm and high efficient in the work will always influence me. In daily life, she is so kind to me and invited me to her house for lunch or dinner several times.

Abbreviations

General Conclusion and Perspectives

Experimental Section tert-Butyl (2S)-2-phthalimodooxy-3-[(2′,3′,4′,6′-tetra-O-acetyl--D-glucopyranosyl)oxy] propanoate (77)

Chemical Formula: C 29 H 35 NO 15 Exact Mass: 637.2007 g/mol To a solution of compound 76 (193 mg, 0.34 mmol) in CH 2 Cl 2 (10 mL), were added PCC (98%, 147 mg, 0.68 mmol), Ac 2 O (0.32 ml, 3.4 mmol) and t-BuOH (0.64 mL, 6.8 mmol). The resulting mixture was stirred at room temperature and then extracted with CH 2 Cl 2 (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (petroleum ether/EtOAc: 2/1) to give compound 77 (120 mg, 55%) as a colourless paste. R f = 0.53 (petroleum ether/EtOAc: 1/1).

[α] D = +16.0 (c 0.1, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 7. 88-7.75 (m, 4H, Phth), 5.29-5.20 (m, 1H, H-3'), 5.10 (t, J = 9.9 Hz, 1H, H-4'), 5.02-4.95 (m, 2H, H-1',2'), 4.77 (t, J = 3.4 Hz, 1H, H-2), 4.33-4.09 (m, 4H, H-3,6'), 3.83 (ddd, J = 10.0, 4.1, 2.2 Hz, 1H, H-5'), 2.12, 2.09, 2.03, 2.00 (4×s, 12H, 4×OAc), 1.48 (s, 9H, t-Bu). 13 C NMR (100 MHz, CDCl 3 ): δ 170. 9, 170.3, 169.8, 169.6, 165.2 (C=O), 163.3 (C=O, Phth); 134.9 (CH, Phth), 128.9 (C q ), 123.8 (CH, Phth), 100.8 (C-1'), 84.6 (CH), 83.4 (C q , t-Bu), 73.1 (C-3'), 71.7 (C-5'), 71.1 (C-2'), 68.5 (C-4'), 67.5, 62.0 (CH 2 ); 28.0, 20.8, 20.7 (CH 3

(2R)-3-O-(2',3',4',6'-tetra-O-acetyl--D-glucopyranosyl)-1-O-triphenylmethylglycerol (79)

Chemical Formula: C 36 H 40 O 12 Exact Mass: 664.2520 g/mol To a solution of compound 78 (0.45 g, 1.07 mmol) in pyridine (10 mL), was added TrCl (0.36 g, 1. 28 mmol). The mixture was refluxed for 24 h and then cooled to room temperature. The solution was concentrated under vacuum to a syrup. The residue was taken up in H 2 O-CH 2 Cl 2 (1/1, 100 mL) and the aqueous layer was extracted with CH 2 Cl 2 (3×30 mL). The combined organic layers were washed with brine (50 mL), dried over MgSO 4 , filtered, evaporated and purified by column chromatography over silica gel (CH 2 Cl 2 / MeOH: 100/1) to afford compound 78 (0.17 g, conversion: 62%) and compound 79 (0.4 g, corrected yield: 92%) as a white powder.

SUMMARY

As part of glycoconjugate family, glycolipids and glycopeptides are involved in a variety of important biological, physiological and pathological processes, such as cell-cell interactions, viral and bacterial infections, immune response, cancer progression, etc. Synthesis of glycoconjugate mimics has attracted intensive research interest for biological and pharmaceutical applications. This thesis was devoted to the synthesis of N-oxyamide-linked glycolipids and glycopeptides, since N-oxyamide-containing compounds have shown improved metabolic stability, and interesting secondary structures due to the easy H-bond formation property of N-oxyamide compounds.

From methyl α-D-glucopyranoside, the 2,6-functionalized pyranoid glycoaminooxy acid derivatives have been successfully prepared as a multifunctional building block for further derivatization to new N-oxyamide glycolipids. From glucose or galactose pentaacetate and (S)-1,2-di-O-benzyl-glycerol, we have successfully achieved the first synthesis of N-oxyamide-linked -glycolipids with one or two lipids chains, as novel mimics of glycoglycerolipids and glycosphingolipids.

In addition, the (2R) and (2S)-3--O-glycosyl aminooxy esters have been stereoselectively synthesized from (2R)--glycoglycerol, with Mitsunobu reaction and Lattrell-Dax epimerization as key steps.

N-Oxyamide-linked glycopeptides have been prepared from the orthogonally protected glycosyl aminooxy esters.

RESUME

Les glycoconjugués comme les glycolipides et les glycopeptides sont impliqués dans de nombreux processus biologique, physiologique et pathologique, tels que les interactions cellule-cellule, les infections virales et bactériales, les réponses immunitaires, le cancer, etc. Ces propriétés ont suscité de recherche intensive pour la synthèse de mimes de glycoconjugés pour des applications en biologie et en pharmacie. Cette thèse est consacrée à la synthèse de glycolipides et de glycopeptides liés par la liaison N-oxy amide qui possède une meilleure stabilité métabolique et une facilité de formation de liaison H conduisant à des structures secondaires très intéressantes.

Les dérivés de pyranoid glycoaminooxy acides fonctionnés en positions 2 et 6 du sucre sont synthétisés à partir du methyl α-D-glucopyranoside, puis transformés en N-oxyamide glycolipides. En tant que nouveaux mimes de glycoglycérolipides et glycosphingolipides, les N-oxyamide -glycolipides possédant une ou deux chaines lipidiques sont préparés à partir du glucose ou du galactose penta-acétate et de (S)-1,2-di-O-benzyl-glycérol.

De plus, une synthèse stéréosélective de (2R) et de (2S)-3--O-glycosyl aminooxy esters a été réalisée à partir du (2R)--glycoglycérol, avec la réaction de Mitsunobu et l'épimérisation de Lattrell-Dax comme étapes clés. Les N-oxyamide glycopeptides ont pu être préparés à partir de glycosyl aminooxy esters orthogonalement protégés.