Olivier Martin

Joanne Xie

Agnès Chartier

; Brennan

S ; Tiffany

C ; Moser

H W ; Santos

W ; Shapiro

E G ; Peters

C ; Wagner

J E ; Cornu

G ; Kurtzberg

J ; Wenger

D A ; Kolodny

E H ; Vanier

M T ; Loes

who three years ago took the risk

Thank you Ela! For being my real friend, rescuing me in almost every emergency situation, listening to my problems, sweating together on a gym and the most important: for being the best aunt for my son! I cannot forget Hélène Blin, who changed rainy Orleans for sunny Marseille, but will always be a part of our lab. You are the most warm-hearted person I met in France. You were so kind that you welcomed me for Christmas at your home. For a long time you were the only French I could understand easily! There were many Polish students in my lab, and all of them became my dear friends: crazy Ola who loved climbing and was one of the prettiest brides I've ever seen; Dominika who loved the order and Scandinavia; Roman who enjoyed my cheesecake and is the tallest person I know; Magda who sang and danced with me in the lab and spread her positive energy all day long; Ania and Oksana: two lovely blonds who seduced my son and both Joannas who survived my welcome dinner:)

In our team we were lucky to always have kind and gifted trainees, who made our lab full of laughter. It was nice to meet you: Hélènes, Asma, Lydie and especially Nathanael, Ludovic and Norhane. I will never forget Garry and his true words about the most beautiful NMR spectrum in my career! I should also mention Julien, who does not mind (or at least not a lot) when I am making fun of him and pays me back with interest.

I would like to thank all of you for my good souvenirs from ICOA: Pascal for the rhubarb and NMR advices, Marie, who appreciated my cookies, Aziz for taking care of wearing suitable clothes, Mathieu for his "Salut Anna!", Mikael (the cute guy) for constructive comments on my French and English (hope you are going to finally get your "bronzage mythique"!). Thank you Sandrine, Audréy, Stephanie, Magalie, Romain, Nicolas, Mathieu, Katia, Aleksandra, Aurelien, Oskan, Jeremy, Cyril, Chloé! One of the most trilling experiences during my thesis was the stage in Portland in Doctor Magdalena Petryniak and Greg Potter team in Oregon Health and Science University.

I would like to thank you for your very warm reception and guiding me in my adventure with molecular biology! Hope your nights become calm soon! My deepest thanks go to my family: my Parents Elżbieta and Jerzy, my "little" Sister Agnieszka, my dearest husband Artur and our Son Szymon. They loved me unselfishly and constantly supported me through all my life even if it was not that easy! Thank you for your patience, comprehension and help in every discipline of my life!

~ Table of contents

Glossary Aglycone

The compound remaining after replacement of the glycosyl group from a glycoside by a hydrogen atom. For example, for the galactosylceramide the aglycone is a ceramide.

Allele

One member of a pair (or any of the series) of genes occupying a specific spot on a chromosome (called locus) that controls the same trait.

Apoptosis

Programmed cell death as signalled by the nuclei in normally functioning human and animal cells when age or state of cell health and condition dictates.

Astrocytic gliosis

Also known as astrogliosis or astrocytosis, is an abnormal increase in the number of astrocytes due to the destruction of nearby neurons from CNS trauma, infection, stroke, autoimmune responses, and neurodegenerative disease.

Autophagy

Also known as autophagocytosis is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. Autophagy, if regulated, ensures the synthesis, degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within the autophagosomes, which are then fused with lysosomes and degraded or recycled.

Autosomal

Being encoded by one of the 22 non-sex determining chromosomes.

Cytosol

Also known as an intracellular fluid or cytoplasmic matrix is the liquid found inside cells. It is separated into compartments by membranes.

Cytotoxic

Toxic to cells.

Demyelination

The loss or removal of myelin sheath (e.g. from the nerve fibre).

Dysostosis

A disorder of the development of bone.

Endocytosis

A process in which cell takes in materials from the outside by engulfing and fusing them with its plasma membrane.

Endoplasmic reticulum (ER)

A membrane-bounded organelle that occurs as labyrinthine, interconnected flattened sacs or tubules that is connected to the nuclear membrane, runs through the cytoplasm, and may well extend into the cell membrane.

Endosomes

A membrane-bounded compartment inside eukaryotic cells.

ER quality control system

A quality-control system for 'proof-reading' newly synthesized proteins, so that only native conformers reach their final destinations. Non-native conformers and incompletely assembled oligomers are retained, and, if misfolded persistently, they are degraded.

Eukaryotic cell

A cell that contains a nucleus and other organelles enclosed within membranes.

ex vivo

Outside an organism. It refers to experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions.

Genotype

The genetic make-up of a cell, an organism, or an individual usually with reference to a specific characteristic under consideration

Hematopoietic stem cells

The blood cells that give rise to all the other blood cells.

Hypertrophic cardiomyopathy

A primary disease of the muscle of the heart in which its portion is thickened without any obvious cause.

Hypotonia

A state of low muscle tone (the amount of tension or resistance to stretch in a muscle), often involving reduced muscle strength.

in vitro

Conducted using components of an organism that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms.

in vivo

It is experimentation using a whole, living organism.

Lymphoblasts

A different form of a naive lymphocyte that occurs when the lymphocyte is activated by an antigen (from antigen-presenting cells) and increased in volume by nucleus and cytoplasm growth as well as new mRNA and protein synthesis.

Macrophage

The cells produced by the differentiation of monocytes in tissues. Macrophages function in both non-specific defence (innate immunity) as well as help initiate specific defence mechanisms (adaptive immunity) of vertebrate animals. Their roles are to phagocytise, or engulf and then digest, cellular debris and pathogens, either as stationary or as mobile cells.

Missense mutation

A point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.

Monogenic mutation

A single gene mutation.

Necrotic cell death

Also known as necrosis is a form of cell injury that results in the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma that result in the unregulated digestion of cell components.

Nonsense mutation

A point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. It differs from a missense mutation, which is a point mutation where a single nucleotide is changed to cause substitution of a different amino acid.

Ocular system

An eye and its central visual system.

Oncogenesis

Literally: the creation of cancer. A process by which normal cells are transformed into cancer cells.

Phagocytosis

The process of engulfing a solid particle by a phagocyte or a protist to form an internal phagosome. Phagocytosis is a specific form of endocytosis involving the vesicular internalization of solids such as bacteria, and is, therefore, distinct from other forms of endocytosis such as the vesicular internalization of various liquids. Phagocytosis is involved in the acquisition of nutrients for some cells, and, in the immune system, it is a major mechanism used to remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral particles are all examples of objects that may be phagocytosed.

Phenotype

The composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, behaviour, and products of behaviour (such as a bird's nest). Phenotypes result from the expression of an organism's genes as well as the influence of environmental factors and the interactions between the two.

Phenylketonuria

An autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase (PAH), rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine. When PAH activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate (also known as phenylketone), which can be detected in the urine.

Polysomes

A cluster of ribosomes, bound to a mRNA molecule. Many ribosomes read one mRNA simultaneously, progressing along the mRNA to synthesize the same protein. They may appear as clusters, linear polysomes, or circular rosettes on microscopy, but mainly circular in vivo.

Recessive

A recessive trait only becomes phenotypically apparent when two copies of a gene (two alleles) are present.

Ribosome

A molecule consisting of two subunits that fit together and work as one to build proteins according to the genetic sequence held within the messenger RNA (mRNA). Some ribosomes occur freely in the cytosol whereas others are attached to the nuclear membrane or to the endoplasmic reticulum (ER)

Rough endoplasmic reticulum (RER)

ER bearing many ribosomes on its outer surface giving it a rough appearance; hence, its name. Since RER has ribosomes attached to its surface it is therefore involved in protein synthesis and secretion. It synthesizes and secretes serum proteins (such as albumin) in the liver, and hormones (such as insulin) and other substances (such as milk) in the glands.

Splice-site mutation

A genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing of an intron takes place during the processing of precursor messenger RNA into mature messenger RNA.

Trans Golgi network (TGN)

A major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments.

Transgene expression

Expression of an exogenous gene introduced into the genome of another organism Vacuole A membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain cases they may contain solids which have been engulfed. Vacuoles are formed by the fusion of multiple membrane vesicles and are effectively just larger forms of these. The organelle has no basic shape or size; its structure varies according to the needs of the cell.

Vector

A DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.

NMR

Avant-propos

La maladie de Krabbe est une maladie rare, héréditaire et mortelle, causée par des mutations de la β-galactocérébrosidase (GALC) une glycosidase lysosomale impliquée dans l'hydrolyse de différents galactolipides. Les mutations situées en dehors du domaine catalytique empêchent la GALC d'adopter sa conformation native. L'enzyme mal repliée est ainsi détectée et éliminée de l'organisme. Son substrat non-hydrolysé s'accumule dans le système nerveux central et périphérique entraînant des symptômes mortels avant l'âge de deux ans.

La thérapie chaperon (PCT) est une nouvelle stratégie consistant à administrer, à des concentrations très faibles, une petite molécule ayant des interactions fortes avec l'enzyme.

Cela l'aide à adopter une conformation correcte et à restaurer partiellement l'activité au niveau du lysosome, permettant au substrat en excès d'être hydrolysé. La plupart du temps, les chaperons les plus efficaces sont de puissants inhibiteurs de l'enzyme et les iminosucres sont connus pour inhiber fortement les glycosidases. L'efficacité de cette stratégie a été récemment démontrée pour deux maladies lysosomales: les maladies de Fabry et de Gaucher.

Dans cette optique, l'objectif de mon projet de thèse consistait à synthétiser de nouveaux iminosucres, inhibiteurs potentiels de b-galactocérébrosidase : des imino-Larabinitols, imino-D-galactitols et des composés de type galacto-isofagomine (galacto-IFGs), et à évaluer leur potentiel en tant que chaperons pharmacologiques pour la maladie de Krabbe.

La conception de ces iminosucres a été dictée par la structure de l'état de transition de la réaction catalysée par la GALC, les données de la littérature sur les inhibiteurs de galactosidases et l'expérience de notre groupe dans la synthèse d'iminosucres en tant que chaperons pharmacologiques pour la maladie de Gaucher.

Introduction

I. Lysosomal storage disorders I.1 Lysosome

A human organism is a complicated machine, where the organic and inorganic molecules undergo constantly the catabolic and anabolic processes in order to obtain, accumulate and use energy. To preserve the perfect equilibrium, each cell is equipped with a variety of repair and quality control mechanisms, correcting almost every error. Nevertheless, sometimes the cell balance is disturbed and different pathologies can be observed.

Every cell of our body is composed of diverse organelles, among which the lysosome was originally discovered and described in 1955 by De Duve. 1,2 This small, single membrane, bag-like vesicle, also called the cellular digestive machine, contains at least 7 integral membrane proteins 3 and about 60 soluble hydrolytic enzymes 4 necessary for intracellular digestion. It occurs in almost every eukaryotic cell (common in animals but rare in plants) and its specific functions include digestion of macromolecules (glycosphingolipids, glycogen, oligosaccharides, mucopolysaccharides, glycoproteins and proteins), 5 phagocytosis, endocytosis, or autophagy (Figure 1) 6 and digestion of any other waste materials. It is also responsible for apoptosis 7 and for repair of damage to the plasma membrane by acting as a membrane patch. 8 In white blood cells, lysosome content is carefully released into the vacuole around the bacteria and serves to kill and digest it. Lysosomal functions are dependent on lysosomes fusing with target vacuoles and liberation of digestive enzymes. 9 Uncontrolled release of lysosome contents into the cytoplasm is also a component of necrotic cell death. 10 The pH of lysosome is 4.6-5.0, which is carefully maintained by the proton-pumping ATPases. 11 As all enzymes in the lysosome work best at an acidic pH and as the pH of the cytosol is 7.2, the cell is protected from auto-digestion. Lysosomal proteins are synthesized on membrane-bound polysomes in the rough endoplasmic reticulum (RER). The soluble lysosomal enzymes contain a hydrophobic Nterminal signal peptide, which directs the ribosome towards the endoplasmic reticulum (ER).

After core glycosylation on selected asparagine residues (by transfer of a tetradecasaccharide made of 3 glucose, 9 mannose and 2 N-acetylglucosamine residues, followed by the elaboration of this oligosaccharide by so-called trimming glycosidases), 12 the protein is transported to the trans Golgi network (TGN). The next step is the addition of a phosphomannosyl recognition marker (M6P) that mediates enzyme translocation to the lysosomes, which is divided into two phases (Figure 2): the transfer of a N-acetylglucosamine-1-phosphate from UDP-GlcNAc (catalyzed by N-acetyl-glucosaminyl-1phosphotransferase) to one or more mannose residues on the lysosomal protein 13 and subsequent removal of the N-acetyl-glucosamine residue to generate a phosphoester intermediate. 14,15 Once this active form of an enzyme is created, it forms a complex with one of the two possible receptors: cation dependent or cation independent mannose-6-phosphate receptors (CDM6P-Rc or CIM6P-Rc). 16 Clathrin-coated vesicles that contain the complex 6 Luzio, J. P.; Pryor, P. R.; Bright, N. A. Nat Rev Mol Cell Biol 2007, 8, 622. 12 Kornfeld, R.; Kornfeld, S. Annu Rev Biochem 1985, 54, 631. 13 Little, L.; Alcouloumre, M.; Drotar, A. M.; Herman, S.; Robertson, R.; Yeh, R. Y.; Miller, A. L. Biochem J 1987, 248, 151. 14 Reitman, M. L.; Kornfeld, S. J Biol Chem 1981, 256, 4275. 15 Reitman, M. L.; Kornfeld, S. J Biol Chem 1981, 256, 11977. 16 Ni, X.; Canuel, M.; Morales, C. R. Histol Histopathol 2006, 21, 899. fuse with late endosomes, where due to the acidic pH the enzyme dissociates from the receptor. The released M6P-Rc recycles back to the TGN or move to the plasma membrane to integrate the exogenous ligands. 17 Unlike soluble hydrolases, lysosomal membrane proteins do not require mediation by MP6 receptors. They are transported via the plasma membrane or by a direct intracellular route . The lysosomal storage diseases (LSDs) are a group of genetic, inherited disorders, resulting from impaired activity of one of the lysosomal proteins. There are over 50 [START_REF] Parkinson-Lawrence | [END_REF] such diseases described so far (Table 1). Individually, LSDs have incidence rate of less than 1:100 000 births but overall they occur with incidence rate between 1:5 000-1:8 000 births, 21 which makes them relatively common and represents an important health problem. Moreover, some populations show higher risk for certain LSDs. Among them we can list Ashkenazi Jewish population with greater prevalence of Gaucher, [START_REF] Beutler | The Metabolic and Molecular Bases of Inherited Disease[END_REF] Tay-Sachs and Niemann-Pick diseases, [START_REF] Vallance | [END_REF] or the Finnish population more susceptible to aspartylglucosaminuria 24 and to 17 Lodish, A.; Berk, A.; Zipursky, S. Molecular Cell Biology. 4th edition; W. H. Freeman;: New York, 2000. 18 Linder, M. E.; Deschenes, R. J. Biochemistry 2003, 42, 4311. [START_REF] Alberts | Molecular Biology of the Cell[END_REF] Alberts, B.; Johnson, A.; Lewis, J. In Molecular Biology of the Cell. 4th edition; Garland Science: New York, 2002. [START_REF] Parkinson-Lawrence | [END_REF] Parkinson-Lawrence, E. J.; Shandala, T.; Prodoehl, M.; Plew, R.; Borlace, G. N.; Brooks, D. A. Physiology (Bethesda) 2010, 25, 102. 21 Meikle, P. J.; Hopwood, J. J.; Clague, A. E.; Carey, W. F. JAMA 1999, 281, 249. [START_REF] Beutler | The Metabolic and Molecular Bases of Inherited Disease[END_REF] Beutler, E.; Grabowski, G. In The Metabolic and Molecular Bases of Inherited Disease. 8th ed.; McGraw-Hill: New York, 2001, p 3635. [START_REF] Vallance | [END_REF] Vallance, H.; Ford, J. Crit Rev Clin Lab Sci 2003, 40, 473. 24 Arvio, M.; Autio, S.; Louhiala, P. Acta Paediatr 1993, 82, 587. infantile/juvenile neuronal ceroid lipofuscinosis (NCLs). 25 LSDs are generally monogenic disorders, however, for most of them different mutations have been detected in the same gene in different patients. The mutations (missense, nonsense and splice-site) lead to complete or reduced loss of activity of mutant proteins. Although some enzymes can be synthesized at a normal level, and some of them are also functionally competent, most of them are not correctly folded and as a consequence they are not properly processed and trafficked to the lysosome. Many of them are detected and discarded by the quality control system in the ER.

Most of the dysfunctions observed in LSDs result from a hydrolase deficiency (and subsequent substrate accumulation), but mutations in genes coding for any of the lysosomal proteins or even proteins processing them can lead to those illnesses (Figure 3) 5 . For example, the deficiency of two lysosomal membrane transporter proteins: sialin and cystinosin causes sialic-acid-storage disease and cystinosis respectively. 26,27 It is also known that Danon disease results from mutations in abundant lysosome-associated membrane protein-2 (LAMP2). 28 Moreover, any perturbation of lysosomal enzymes transport through TGN can lead to LSD 5 such as I Cell or pseudo-Hurler disease, where mutations occur in genes coding for N-acetyl-glucosaminyl-1-phosphotransferase (see I.2). In some cases (multiple sulfatase deficiency, 29 galactosialidosis 30 ) the mutation affects only one enzyme, but as it is necessary for the stability or activity of numerous proteins, the deficiency of many enzymes is observed. On the other hand, in illnesses such as NCLs or Batten Disease 31 it was observed that defects in up to 8 distinct genes cause similar pathology by accumulation of similar substances in lysosomes. There are also mutations severe enough to be lethal in early stages of development and because of that they will never be associated with any lysosomal storage diseases.

Being aware of the complexity and the diversity of LSDs it is difficult to find an appropriate classification model. They can be grouped according to the type of material accumulated or on the basis of the deficient enzyme. Table 1 represents all known LSDs classified according to the molecular defect and provides details on both the defective enzyme and the main substrate accumulated. 32 The severity and the type of the disorder depend not only on the enzyme implied but also on the degree to which its activity is compromised. Therefore, a complete or almost complete loss of enzymatic activity leads to the most severe pathology, with an early onset and death. Interestingly, residual enzymatic activity is often sufficient for late onset (juvenile or adult form) and milder symptoms to be observed. 33 The same enzyme defect can lead to different symptoms. In Pompe disease a severe infantile onset of the disorder is expressed by hypotonia, hypertrophic cardiomyopathy and failure to thrive, where the adult onset has no cardiac findings, but proximal muscle weakness and respiratory insufficiency. 34 The nature of the undegraded storage material and the location of its accumulation play also a crucial role by changing the architecture and function of cells, tissues and organs. The cells with a high turnover of enzyme's substrate are usually more concerned than other cells. For example, mucopolysaccharidose type IVA (MPS IVA), called also Morquio A syndrome, results from the impaired activity of N-acetyl-galactosamine-6-sulphatase. This enzyme breakdowns keratan sulfate, mostly found in the skeletal system, which thus implies severe skeletal growth problems, dysostosis and joint disease. 35 Alternatively, the accumulation in the central nervous system (CNS) leads to much more severe disorders than when other tissues are affected. This is the case in Krabbe disease 36 characterized by the deficiency of galactocerebrosidase: the enzyme degrading galactosylceramide. This sphingolipid is highly 31 Cooper, J. D. Curr Opin Neurol 2003, 16, 121. 32 Filocamo, M.; Morrone, A. Hum Genomics 2011, 5, 156. 33 36 Sakai, N. Brain Dev 2009, 31, 485. abundant in neurons and its buildup results in progressive neuroregression. Only in some cases (e.g. Krabbe disease) the accumulated substrate is cytotoxic itself. Usually, its accumulation activates secondary and tertiary biochemical pathways, which have not been well understood until now. Genetic defects that lay at the basis of all LSDs can change cellular processes, alternate signaling pathways and modify gene expression, which leads finally to tissue damage and death. 5 However, even if the pathology development strongly relies on all the factors above and even if the gene mutations are well documented, prediction of illness seriousness and progression is almost never accurate. 5 The clinical course of LSDs is chronic, progressive, and frequently lethal in the late childhood. Although the symptoms associated with those illnesses are often neurological, they can also be multisystemic, with skeletal, CNS, cardiovascular, and ocular system involvement. As mentioned before, there is no clear correlation between the mutation and its phenotypic consequences. This is why next to genetic and molecular analysis, the preliminary (urine and serum) tests and clinical interview are indispensable. Furthermore, it was proved that earlier diagnosis and presymptomatically treatment provide the best outcome. This highlights the need of newborn screening (NBS) for LSDs. The NBS concept was originally developed and used in the early 1960s 37 predominantly for patients suffering from phenylketonuria. Presently, by using tandem mass spectrometry identification of enzyme reaction's products or immunologic protein assays, it is possible to screen several LSDs at once. 38 Although this technique still needs optimization, it gives a hope for early diagnosis and a life-saving therapy. It is already applied in the New York state where the NBS for Krabbe disease has been performed since 2006. 34 The newborn screening has another advantage: it can reveal an illness in parents of sick children, giving them opportunity of early treatment. However, there is also the dark side of the NBS: they are often done without parental consent, which raises psychosocial and ethical concerns.

For most of the LSDs, as soon as they are well diagnosed, immediate therapy is initiated. The vital need of developing existing and finding new therapies for LSDs is due to two facts. Firstly, for many of the lysosomal storage disorders, even if well diagnosed, no specific or definitive treatment is available. Secondly, due to the high regulatory and research costs and lack of competition on LSDs' medicines market, the therapies are very expensive (Table 2). Classically, the treatment consists in symptomatic care of disease manifestations. The therapies applied in LSDs can be roughly divided into those that act on the symptoms and those that act on the cause (Figure 4).

II.2 Hematopoietic stem cells therapy

Although BMT was successful in some cases, it has two major drawbacks: the requirement of matched donors and a high mortality rate. A more recent version of this method uses hematopoietic stem cells from placental cord blood. 43 The objective of hematopoietic stem cells therapy (HSCT) is to engraft healthy cells into a patient without functional enzyme. 44 The donor leukocytes in the host tissue produce and secrete the enzyme, which is then taken up by enzyme-deficient host cells (Figure 5). 45 This principle is called cross-correlation and is a basis of ERT (II.4). Hematopoietic stem cells therapy can be applied in two ways: by transplanting donor healthy cells to a patient or by associating it with ex-vivo gene therapy, where after genetic modifications, the patient own cells are re-grafted to its organism (II.3). 46 The first HSCT for LSDs was performed on Hurler syndrome (MPS I) patient in 1980 47 resulting in biochemical and clinical improvements up to 13 months after. Since then over 900 HSCTs 48 were done (Figure 6). Most of them concern Hurler disease, metachromatic leukodystrophy (MLD) and Krabbe disease. 49 Even though HSCT is a very promising option for treating LSDs at the source of the problem (it is easier to prevent the pathology than to correct it) it has serious limitations. First of all, HSCT is most successful in the neonatal and presymptomatic stage of disease, especially when CNS dysfunction is involved. 51 This requires early diagnosis and transplantation in a young age. Furthermore, hematopoietic stem cells migrate only to several organs (shown in mice) 52 such as bone marrow, liver, spleen, lungs and CNS, and barely reach the skeletal muscles and cartilage (shown in rats 53 ) which results in no therapeutic improvement in skeletal diseases. 54 Finally, like for BMT, the transplant-related mortality rate is about 10-25% 41 and the surgery delays are too long. To limit the risk, careful selection of donor graft source is required. Moreover, the application of cord blood stem cells has reduced the time to transplantation and the danger of the graft-versus-host disease.

II.3 Gene therapy

As the majority of therapies applied to LSDs treat secondary complications of the disease, the somatic gene therapy would be the most attractive and effective approach. 55 It consists in inserting a functional gene to produce a correctly working enzyme and replace the deficient one. The LSDs are monogenic, for many of them the mutation has been identified and a relatively small amount of active lysosomal enzyme can resolve the disease, which make them perfect candidates for such type of therapy. Thanks to a phenomenon known as cross correlation phenomenon genes do not have to be transferred to all cells of the organism, which means that only a small percentage of transducted cells can have a therapeutic effect.

Gene transfer can be applied in vivo and ex vivo. The first method requires a vector (based on disabled adenovirus, adeno-associated virus, lentivirus or herpes virus) 56 carrying the transgene which is injected into circulation or directly into a target tissue. The second one implies in vitro genetic correction of patients cells (often hematopoietic stem cells) followed by re-implantation (Figure 7). 40 Despite a number of animal models 57 and biochemical and clinical progress, 58 very little development has been made in applying this method to humans.

Three human clinical trials of gene therapy for Gaucher disease did not show significant improvements. 59 One of the drawbacks of applying gene therapy to humans is the potential risk of oncogenesis. 60 Additionally, viral vectors can activate inflammatory and immunological responses. 61 The success of gene therapy depends on the level of transgene expression, as well as on the ability of the treatment to cross the blood-brain barrier (BBB) to reach the CNS. Overall, to become a real option to treat LSDs, gene therapy still needs a great deal of optimization on the vectors and procedures safety, specificity and efficiency. 62 which one year later brought in the idea of treatment by replacing defective enzyme by a healthy one. 63 In 1968, Neufeld and coworkers discovered that culturing together cells of patients suffering from Hurler disease (a-iduronidase) and Hunter disease also called MPS II (iduronate sulphatase) leads to correcting of both enzymes' defects. 64 The earliest attempts of ERT were unsuccessful 65 (Pompe disease) owing to the low doses, short treatment duration, the lack of knowledge about receptor-mediated endocytosis and insufficient sources of highly purified enzymes with appropriate markers for targeted uptake. The first victorious ERT was achieved in 1991 66 for type I Gaucher disease, using human placental glucocerebrosidase. The enzyme was 60 Hacein-Bey-Abina, S.; Garrigue, A.; Wang, G. P.; Soulier, J.; Lim, et al. J Clin Invest 2008, 118, 3132. 61 Cheng, S. H.; Smith, A. E. Gene Ther 2003, 10, 1275. 62 Hers, H. G. Biochem J 1963, 86, 11. 63 De Duve, C. Fed Proc 1964, 23, 1045. 64 Fratantoni, J. C.; Hall, C. W.; Neufeld, E. F. Science 1968, 162, 570. 65 Valayannopoulos, V. Handb Clin Neurol 2013, 113, 1851. 66 previously partially deglycosylated, to enhance its uptake by mannose receptor of macrophages (Figure 8). 67 Since then, this therapy was applied to five other 34 Although studies during the last decade have demonstrated the benefits of using ERT, the overall outcome varies from disease to disease. The success of ERT strongly depends on the following factors: accessibility of affected cells, 68 molecular composition and turn-over of the tissue, antibody formation 69 against the therapeutic enzymes and finally, irreversible cell damages impairing intracellular trafficking. 70 As a consequence, ERT has little effect on the brain (due to the BBB), skeletal tissue (slow turn-over of the bones and slow diffusion to cartilage through the matrix), and valvular heart disease in LSD. Animal studies have shown though, that some of the problems associated with cell and tissue accessibility may be overcome by increasing the doses of ERT. 71 However, this approach can increase the immunological response and the therapy cost. Concerning the mild allergic reactions observed during intravenous ERT, they are neutralized with antihistamine and non-steroidal antiinflammatory drugs.

Despite the development mentioned above, low recombinant enzyme uptake by the cells is still the biggest challenge in ERT. It can be resolved by several strategies that will improve enzyme trafficking and recognition by both cells and lysosomes. The first consist in conjugating the enzyme with synthetic oligosaccharides having M6P (see I.2). This idea was tested on a mouse model of Pompe disease (enzyme used: oxime-neo-recombinant human aglucosidase) and has shown decrease of glycogen accumulated during illness in muscles. 72 Another approach is so called glycosylation independent lysosomal targeting, which consist in replacing the enzyme's N-terminal peptide by an N-terminal fragment of insulin-like growth factor 2. Thanks to the high affinity of this signal peptide to M6PR, the modified enzyme is better recognized and more efficiently binds to cell and/or lysosome membrane. 73 Two more solutions for increased enzyme uptake are either to modulate the expression of M6PR on the target cells 74 or to inject enzyme directly to the tissue of interest (e.g. to intracerebrospinal fluid 75 ). However, the latter procedure remains controversial from ethical and practical point of view. Completely different problem lies in passing BBB. No receptor recognition enhancement is successful so far in this case. To overcome this problem, one of the possibilities is to bind the recombinant protein to a monoclonal antibody against an endogenous BBB receptor. 76 This strategy proved to be working in Hunter disease animal models.

Finally, regardless of how optimized and adapted ERT will be, it remains an expensive treatment (see Table 2) and a long-life burden to the patients. 71 Ioannou, Y. A.; Zeidner, K. M.; Gordon, R. E.; Desnick, R. J. Am J Hum Genet 2001, 68, 14. 72 Maga, J. A.; Zhou, J.; Kambampati, R.; Peng, S.; Wang, X.; Bohnsack, R. N.; Thomm, A.; Golata, S.; Tom, P.; Dahms, N. M.; Byrne, B. J.; LeBowitz, J. H. J Biol Chem 2013, 288, 1428. 73 Koeberl, D. D.; Li, S.; Dai, J.; Thurberg, B. L.; Bali, D.; Kishnani, P. S. Mol Genet Metab 2012, 105, 221. 74 Hemsley, K. M.; Hopwood, J. J. Int J Clin Pharmacol Ther 2009, 47 Suppl 1, S118. 75 Pardridge, W. M.; Boado, R. J. Methods Enzymol 2012, 503, 269. 76 Lu, J. Z.; Boado, R. J.; Hui, E. K.; Zhou, Q. H.; Pardridge, W. M. Biotechnol Bioeng 2011, 108, 1954. II.5 Substrate reduction therapy Substrate reduction therapy (SRT) is a drug-based treatment with the objective to diminish lysosomal storage by inhibition of the substrate biosynthesis. 77 As an applied treatment, SRT is used only for glycosphingolipidoses (see Table 1) so far. 78 Those disorders result from accumulation of various glycosphingolipids (GLS), which all have the same first step of biosynthesis (Scheme 1): the addition of a glucose unit to ceramide by ceramidespecific glucosyltransferase (also termed glucosylceramide synthase, GCS). 79 Scheme 1. Biosynthesis of glycosphingolipids in humans. 80 This approach was introduced in the 1970s by Radin 81 and coworkers who identified the first type of glucosyltransferase inhibitors. PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) (Figure 9) and its analogues are powerful inhibitors of GCS, but relatively cytotoxic due to their hydrophobicity. Additionally, they provoke accumulation of free ceramide. New generations of PDMP derivatives proved to be efficient glucosylceramide synthase inhibitors; 82 nevertheless none of them was tested on humans so far. 77

PDMP IC50= 5µM

Figure 9. GCS inhibitors. 83,84,85 NB-DNJ is used in SRT.

Another group of GCS inhibitors includes the N-alkylated iminosugars. The leading compound NB-DNJ (N-butyldeoxynojirimycin, miglustat) was initially designed to be an antiviral agent: 86 by being a good inhibitor of intracellular a-glucosidases I and II, this compound blocks glucosylation of viral proteins and ends virus life cycle. Unfortunately, the 6-month clinical trial 87 has shown no important efficacy of the drug. In 1994, while working on an in vitro model of Gaucher disease, Platt and Butters 84 found that NB-DNJ reduced significantly the endogenous glucosylceramide synthesis. Although this iminosugar is not GCS-specific (it inhibits also a-glucosidase I and II as well as digestive a-glucosidases), the therapy is more efficient than in the HIV case 80 (thanks to cytosolic orientation of ceramidespecific UDP-glucosyltransferase the enzyme is more accessible for the drug). After a series of encouraging assays on animals, 88 the first clinical test was performed on mild-to-moderate type 1 (non-neuronopathic) Gaucher patients. 89 The results of this experiment and follow-up studies lead to registration of miglustat (Zavesca ® , 2003) as a drug for Gaucher type I patients, for whom ERT cannot be applied. In 2009, NB-DNJ was also approved as a drug for Niemann-Pick disease, as it proved to stabilize one of the neurological symptoms of the disorder. 90 On the other hand, even though NB-DNJ was shown to cross BBB in some mouse models, it didn't demonstrate any effect on Gaucher type III patients over a 2-year period of treatment. 91 Despite its benefits to LSDs patients, miglustat has gastrointestinal side-effects 92 and remains an expensive therapy. An alternative to this drug may be eliglustat (Phase III trial in Gaucher type I patients in progress), which due to a higher specificity for ceramide-specific UDP-glucosyltransferase shows minimal side-effects. 93 Other trials of SRT for LSDs are pending: e.g. for MPSs using a soybean isoflavone genistein. 94 To summarize, SRT seems to be a promising approach to treat some LSDs. The advantages include lower cost, oral administration and possibility to treat the disorders with neurological manifestations. However, further investigation is required to find novel compound with higher specificity to be used at lower dosage and to avoid side-effects.

II.6 Pharmacological chaperone therapy

In biology, a chaperone is a protein which helps in the folding of nascent polypeptide chains, refolding of denatured proteins. It also prevents aggregation of surface-exposed hydrophobic parts of the proteins that have problems with folding. Apart from that, chaperones can play an important role in signal transduction, in the maintenance of the organized state of the cytoplasm and other intracellular compartments, in the motions inside the cell, and some other vital functions of the cells. 95 Pharmacological chaperone therapy (PCT) arises from the same principle, with the difference of using low-molecular-weight molecules (PCs) instead of proteins as chaperones. As mentioned previously, LSDs are the genetic disorders resulting from deficiencies of lysosomal proteins. In some cases, the mutant enzymes retain their activity or it is slightly compromised, 96 but as they are often misfolded, they are recognized by the ER quality control system of the cell. 97 In consequence, mutants are polyubiquitinated and translocated to the cytosol, where they are degraded by proteases. 98 However, even small improvement in protein stability or conformation may prevent their 91 Tylki-Szymanska, A.; Groener, J. E.; Kaminski, M. L.; Lugowska, A.; Jurkiewicz, E.; Czartoryska, B. Mol Genet Metab 2011, 104, 627. 92 Reuser, A. J.;Wisselaar, H. A. Eur J Clin Invest 1994, 24 Suppl 3, 19. 93 Lukina, E.; Watman, N.; Arreguin, E. A.; Dragosky, M.; Iastrebner, M.; Rosenbaum, H.; Phillips, M.; Pastores, G. M.; Kamath, R. S.; Rosenthal, D. I.; Kaper, M.; Singh, T.; Puga, A. C.; Peterschmitt, M. J. Blood 2010, 116, 4095. 94 Piotrowska, E.; Jakóbkiewicz-Banecka, J.; Tylki-Szymanska, A.; Liberek, A.; Maryniak, A.; Malinowska, M.; Czartoryska, B.; Puk, E.; Kloska, A.; Liberek, T.; Baranska, S.; Wegrzyn, A.; Wegrzyn, G. Current Therapeutic Research 2008, 69, 166. 95 Ellis, R. J.; Hemmingsen, S. M. Trends Biochem Sci 1989, 14, 339. 96 Fan, J. Q. Biol Chem 2008, 389, 1. 97 Ellgaard, L.;Helenius, A. Curr Opin Cell Biol 2001, 13, 431. 98 Bonifacino, J. S.; Weissman, A. M. Annu Rev Cell Dev Biol 1998, 14, 19. premature degradation and favor further transport to the lysosome. Originally, PCs were believed to enhance the stability of mutant proteins and thanks to this, to avoid their degradation in ER. PCs are designed to selectively bind and stabilize mutant proteins. The concept of this therapy is often considered to be controversial 99 as to restore the enzyme activity, one has to use its own inhibitor. In fact, by binding to the active site of the misfolded protein, the competitive enzyme inhibitor (which has a high affinity for the catalytic domain) acts as a template to re-establish the enzyme's three-dimensional structure, stabilizes it and therefore facilitates its proper trafficking through ER and to the lysosome. 100 As soon as reaching the lysosome, in the presence of undegraded substrate in excess, the inhibitor (in sub-inhibitory concentrations) dissociates from the enzyme and liberated protein can function normally (Figure 10). 101 However, other mechanisms are possible: in 2011, Wei and coworkers 102 showed that even if catalytically defective, the N370S mutant human acid bglucosidase (GCase, most common cause for Gaucher disease) is actually more stable than the wild type enzyme! They have preceded the assays with addition of the GCase competitive inhibitor NN-DNJ, which increased 2-3 folds GCase levels in 24h. They concluded that the major mechanism of this chaperone-mediated enhancement consists in reducing the susceptibility to proteases and decreasing the degradation of the defective enzyme in the lysosome. The studies of Asano 103 on a-galactosidase A showed that, apparently a more potent enzyme inhibitor is a more efficient PC. Ideally, a compound should have weaker inhibitory effects and higher enhancing activity: recent studies have revealed compounds with moderate inhibitory activity having indeed very good activity enhancements. 104 Moreover, it should be noted that PCT can be successful only when mutant enzymes show residual activity. Enzymes which are truncated or carry large deletions cannot be activated by a PC. Thus only disorders resulting from mutations that involve little 3D modifications can be treated by this approach.

The activity above a threshold level (10-15% of native enzyme activity) results in normal substrate turnover, which means that, if delivered to lysosome, even enzymes with small residual activity can reverse the illness.

First chaperone effect was demonstrated by Suzuki et al in 1995 105 on lymphoblasts from patients with Fabry disease using galactose. In their report, they showed a significant increase of a-Gal A levels in cell cultures upon addition of galactose. Moreover, for 7 among 11 tested mutants they observed enzyme activity enhancement. Subsequently, other studies were carried out to provide a proof of principle for other LSDs: Fabry disease, 106 Gaucher disease, 107,108 GM1-109 and GM2-gangliosidoses, 110,111 and Pompe disease. 112,113 Currently 1deoxygalactonojirimycin (DGJ, migalastat, Amigal™) is in Phase III trial for Fabry disease. Isofagomine (IFG) (Figure 11), which was developed by Amicus Therapeutics as a candidate (Plicera ® ) for the treatment of Gaucher disease, was eventually discontinued in phase II trials because it failed to meet efficacy expectations. Another iminosugar 1-deoxynojirimycin (DNJ) is in Phase II trials for Pompe disease (Figure 11, Table 3).114

IFG Isofagomine IC50= 56nM b -glucocerebrosidase DNJ 1-deoxynojirimycin IC50= 1.3µM a 1,4-glucosidase DGJ Migalastat IC50= 4.7nM a -Gal A Figure 11
. Currently tested iminosugars as potential PCs for LSDs. 106,112,115 The pharmacological chaperone therapy arises as a very promising treatment for lysosomal storage disorders mainly because of relatively low cost of drug production, oral administration, small doses used (limited side effects) and finally the possibility of crossing the BBB, thus providing hope for severe LSDs with CNS involvement.

Disease PC Status

Fabry

Galactose Preclinical DGJ (AT1001, Amigal TM ) Phase 3 (psychosine),124 lactosyl ceramide and monogalactosyldiglyceride (Figure 12).

Gaucher

Lactosylceramide Galactosylceramide Galactosylsphingosine

Figure 12. GALC natural substrates.

The gene coding GALC was mapped to chromosome 14q31. 125 Approximately, 80 distinct mutations have been identified in this gene. 126 The most common large deletion ERT was proposed by Lee 141 which resulted in improved GALC activity in CNS and PNS as well as lowered levels of psychosine. This approach, even if efficient (peripheral injections of recombinant GALC every day significantly improved most of the early clinical animal phenotypes 135 ) is not practical due to the necessity of direct brain injections, which are difficult to perform and risky. As shown in Table 3, there is only one PC candidate for Krabbe disease. 33 a-Lobeline (Figure 14) was identified as a weak inhibitor of GALC. In their studies Lee et al 129 demonstrated an important increase of 52% for intracellular and 64%

for extracellular D528N GALC mutant activity when the cells were treated with a-lobeline (240µM). Moreover, a-lobeline passes through the BBB, which is crucial for all drug-based treatment of Krabbe disease. However, despite all the efforts described above, Krabbe disease still remains an untreatable, neurodegenerative illness which is lethal at young age. As mentioned before, even such weak a-Gal A inhibitor as galactose showed a chaperone effect in Fabry disease. As DGJ is about a 120 000-fold more potent competitive inhibitor than galactose, it should be a much better PC as well. Indeed, an increase in enzyme activity was observed in Fabry patient-derived lymphoblasts cultured with 0.2-20µM DGJ.

a-lobeline

Moreover, this state was maintained for five days after drug removal from the medium. 144 Further studies on mouse model TgM/KO (which expresses a human mutant R301Q instead of the endogenous a-Gal A gene) showed a significant increase in enzyme activity in various organs: 4-and 18-fold in heart, 2-and 28-fold in kidney, 1.7-and 1.8-fold in spleen and 3. b-Galactosidase is the enzyme implied in two LSDs. Different mutations in this protein cause either GM1 gangliosidosis or Morquio B syndrome. This is why the molecules that can increase the activity of b-galactosidase were already widely investigated. As for all galactosidases, the first candidate as PC was galactose itself. The test on patients-derived celllines expressing R401H and T393A mutants showed 1.4-and 2.5 fold increase in activity respectively (at 200mM Gal concentration). However, galactose failed to boost S54N mutant activity. 148 Next choices for b-galactosidase inhibitors were DGJ, NB-DGJ and NN-DGJ. The first two compounds were evaluated in mouse cell lines transfected with mutant forms of human b-galactosidase. Both iminosugars promoted increase in enzyme activity: 2-to 6-fold for mutations related to GM1 disease and 1.1-to 1.8-fold for mutations responsible for Morquio B disease. 149 However, those results were obtained at high inhibitor concentration (500µM). Recently, Rigat et al 150 examined inhibitory properties of DGJ, NB-DGJ and NN-DGJ on human lysosomal b-galactosidase and their chaperone effect in cultures of skinfibroblasts derived from patients suffering from either LSDs. They established IC 50 values of 18.4µM, 3.5µM and 0.12µM for the three DGJ derivatives respectively. Moreover, they compared the inhibitory activities of the three compounds on a-galactosidase and found that NB-DGJ and NN-DGJ were weak a-galactosidase inhibitors with <30% and <10% inhibition respectively (at 100µM). These values indicate that the addition of an N-alkyl chain significantly increases the specificity of the DGJ-derivative for b-galactosidase, and that the strength of the inhibition is further increased when the chain is lengthened from 4 to 9 carbon atoms. Further assays were conducted on NN-DGJ and they proved its good chaperone effect on various mutations responsible for GM1 or Morquio syndrome B (up to ~5-fold increase of enzyme activity that would raise the residual activity to about 15% of normal). 148 The most recent group of interesting b-galactosidase inhibitors are so-called sp 2iminosugars developed by Garcia Fernandez and coworkers (Figure 17). 157 These bicyclic derivatives of galactonojirimycin or allonojirimycin were found to be potent and selective inhibitors of various glycosidases. Modifications of the aglycone by introducing heteroatoms (oxygen or sulphur) as well as by changing the length of the imide N-alkyl chain led to selective and potent inhibitors. Two of them (F and G Figure 17) were shown to be good inhibitors for bovine liver b-galactosidase with IC50 of 0.2µM and 0.052µM. The latest experiments on another sp 2 -iminosugar, 6S-NBI-DGJ (Figure 17) confirmed its very good chaperone activity (despite higher IC 50 value compared to the reference compound NOEV). 158 This compound was tested on 88 human b-galactosidase mutants. For 24 of them, the enzyme activity was increased. Moreover, 16 of these mutations were not responding to the NOEV treatment. All of these results indicate that 6S-NBI-DGJ is a promising candidate as PC for a large number of patients suffering from GM1 gangliosidosis.

An extremely powerful b-galactosidase inhibitor is 4-epi IFG (galacto-IFG), with an IC 50 of 12 nM (enzyme from Aspergillus orizae). Its N-butyl derivative is no longer that potent with IC 50 = 5mM. 159,160 There are no studies of this compound and of its N-butyl derivative as pharmacological chaperones.

GALC is the least explored lysosomal galactosidase. Little is known about inhibitors and the best and only candidate as chaperone so far is a-lobeline (Figure 14), which has been mentioned before.

IV.3 Additional studies

Recently, Kato and coworkers 161 showed that, while DGJ is a competitive nanomolar inhibitor of several a-galactosidases, its enantiomer, L-DGJ is a non-competitive, micromolar one. Moreover, it was found that iminosugars related to fagomine: 2-deoxy-DGJ (4-epifagomine), deoxyadenophorine (1-C-alkyl-2-deoxy DGJ) also showed inhibitory properties towards a-galactosidase. 162 The presence of an ethyl chain at C-1 position of 2-deoxy DGJ slightly enhances the inhibitory properties towards a-galactosidase from coffee bean (IC 50 = 6.4 mM).

Objectives

The aim of my PhD project was to synthesize three families of iminosugar derivatives as potential inhibitors of galactocerebrosidase (GALC): novel imino-L-arabinitols, imino-Dgalactitols and galacto-isofagomines (galacto-IFGs) carrying a C-alkyl chain, and to evaluate them as potential chaperones for Krabbe disease.

The choice of those structures was dictated by the consideration of the transition state

II. Structures of iminosugars of interest

Because of their ability to mimic in their protonated form the transition state of the glycoside hydrolysis reaction, iminosugars are potent glycosidase inhibitors. 166 In general, it has been observed that DNJ-like structures, with nitrogen replacing the ring oxygen atom, are selective and potent inhibitors for a-glycosidase, whereas IFG-like structures, where nitrogen occupies the position of the anomeric carbon, are potent b-glycosidase inhibitors. 167,168 For galactosidases, 4-epi-IFG is indeed a very powerful b-galactosidase inhibitor, but DGJ derivatives are also good inhibitors of both a-and b-galactosidases. Nevertheless, as no inhibitors of GALC are known so far, we decided to investigate both types of structures in our search for potent and selective inhibitors of this enzyme. As shown in Scheme 5, the oxocarbenium ion character of the GALC transition state can be reproduced by creating structures in which the nitrogen atom is at the position of oxygen (DGJ-like) or of C-1 (galacto-IFG-like).

III. Detailed objectives

Our first objective, namely, the synthesis of imino-L-arabinitol derivatives as potential GALC inhibitors, was suggested by the previous work of our group on PC for GCase (glucocerebrosidase). 169,170 Indeed, our group showed that iminosugars having an iminoxylitol core (OH configurations as in glucose, but no CH 2 OH group at C-5, DIX) and carrying a lipophilic C-linked aglycone, were even more powerful inhibitors of GCase than the corresponding imino-D-glucitols (Figure 18). Moreover, Deane and co-workers showed in their X-ray structure of GALC-galactose complex that galactose orientation in the active site is similar to the one of glucose analogues in GCase. 131 This led us to the idea that compounds based on an imino-L-arabinitol structure may be also interesting as inhibitors of GALC. Our synthetic strategy was based on the same principle as for the synthesis of 1-Calkyl DIX derivatives: C-1 chain elongation by the addition of allyltrimethylsilane to an Nprotected arabinopyranosylamine. Further elaboration by cyclization, double bond functionalization and deprotection should afford a small library of 1-C-alkyl imino-Larabinitols (Figure 19). These compounds turned out to be completely devoid of activity as GALC inhibitors.

The second objective was the synthesis of imino-D-galactitols carrying a 1-C-linked lipophilic aglycone. We firstly used a strategy similar to that used for arabinitols, but in spite of intensive optimization, the desired compounds were obtained in very poor yield. However, this synthetic approach was used to obtain a related family of compounds, the C-5 epimer of the desired product namely imino-L-altritols which were functionalized and also submitted to biological tests (Figure 19). To reach the target imino-D-galactitols, we used another strategy, consisting of a C-6 chain elongation by way of Ellman's imine (tert-butanesulfinylimines) formation, followed by Grignard reagents addition. This provided us novel 1-C-alkyl-imino-D-galactitols (Figure 19).

The third type of structure of interest was 1-N-iminosugar derivatives related to Dgalactose (galacto-IFG derivatives). As described by Ichikawa, 160 the parent galacto-IFG is a very potent inhibitor of b-galactosidases. Our goal was to synthesize various derivatives of galacto-IFG carrying substituents at C-1, C-5 and at nitrogen to test them on GALC. The synthetic approach proved very challenging and required several improvements. Our studies led to two main structures: pseudo-galacto-IFG and 5-C-pentyl-pseudo-galacto-IFG.

Although the first one was previously described by Ichikawa, 171 our synthesis was shorter with better overall yield. The final objective of this project was to realize the biological assays of synthesized compounds. Thanks to the collaboration with Dr Asano (Hokuriku University, Japan) our compounds were tested on various galactosidases to determine their inhibitory properties and their selectivity. Experiments have also been performed on human lysosomal GALC in collaboration with Dr Wenger, (Thomas Jefferson University, USA) and further biological investigations on mutant lysosomal GALC will be conducted, in part by myself, in the research group of Dr. M. Petryniak (at the Oregon Health Science University, USA). This known compound175 was prepared in three steps from L-arabinose. L-arabinose was first converted into methyl L-arabinopyranoside 1 in refluxing methanolic HCl. The three hydroxyl groups of 1 were then benzylated under standard conditions to give 2 and then the methyl glycoside was hydrolyzed under strongly acidic conditions to afford 3 in moderate yield.

I.3 Synthesis of protected arabinopyranosylamines 4 and 11

For the preparation of compound 4, 2,3,4-tri-O-benzyl-a,b-L-arabinopyranose 3 was directly submitted to condensation with benzylamine. 176 In the processing, a coevaporation with toluene was used to remove the excess of benzylamine and the crude product 4 was clean enough to be used for further reactions. By contrast, the direct condensation of 3 with benzyl carbamate in the presence of trimethylsilyl trifluoromethanesulfonate resulted in a complex mixture of the desired amine 11 and by-products, with a drastically low yield (22%). To improve the synthesis of 11, hemiacetal 3 was firstly acylated using acetic anhydride in pyridine 177 to give 10 which was then condensed with benzyl carbamate as described previously by our team. 178 This simplified the purification and increased the yield of 11 to 81% over two steps.

I.4 C-1 chain elongation of N-protected glycosylamines

The C-1 chain elongation could be conducted on both glycosylamines, but under different conditions: as described by Nicotra, 174 N-benzyl glycosamines are convenient substrates for chain-extension by the addition of an organometallic reagent (Grignard reagent, R-Li). However, the stereoselectivity of the addition is often modest. Addition of allylmagnesium bromide directly onto 4 gave the open chain product 5 in 81% yield as a 7:3 mixture of diastereoisomers in favour of the S (anti) isomer. Surprisingly, the same reaction with hexylmagnesium bromide failed to give the expected 1-C-hexyl derivative.

The first assay of addition of allyltrimethylsilane to 11 using 0.5 eq of trimethylsilyl trifluoromethanesulfonate as an azaphilic 180 Lewis acid gave the desired amine 12 in 87% yield but difficult to purify. The same reaction with 1 eq of Lewis acid gave much cleaner compound in 71% yield. It is worth mentionning that the reaction did not have to be conducted at -40°C as previously done in our team, 179 but at -20°C. In contrast to the reaction of the N-benzyl glycosamine, the addition of allyltrimethylsilane to the N-Z compound gave selectively the syn isomer (9:1 ratio). Both mixtures of diastereoisomers were not separable at this stage. The difference in the diastereoselectivity of those two reactions comes from different mechanisms. The NHBn group is first deprotonated which favors ring opening to an alkoxide. Then we would expect a mechanism with a chelation-controlled addition onto the imine. This model would however favour the syn isomer (which is generally observed in such additions to glycosylamines). The formation of the anti product in this case is unusual and we do not have a good explanation for its formation. According to the NMR analysis, the major, pseudo a-(S)-isomer 6 is in a well defined 4 C 1 chair conformation, while its minor epimer, the pseudo b-(R)-7 exists in an equilibrium of two chair forms: 4 C 1 and 1 C 4 (Scheme 13). This can be explained by the fact that each of these two conformers of 7 has two axial and two equatorial substituents, the two chair forms being thus of similar stability.

Considering the N-Z protected derivatives, the direct determination of the pseudo anomeric configuration in 14 and 15 was difficult as a result of the low quality of their 1 H NMR spectra due to the presence of rotamers. However, it is known that N-acyl protected piperidines usually favor the conformation in which the substituent a to nitrogen is in axial position. 181,182 We assumed then that the minor, pseudo a (S) isomer 14 would be in respectively. The large coupling constants in 16 (J 1-2 =J 2-3 = 9.3 Hz) suggested that this compound adopted the 4 C 1 conformation, while small constants for 17 We predicted that the first compound (8) would have the same 4 C 1 conformation as his precursor 6. For the other isomer 9, the conformation should be well defined this time as a 1 C 4 chair conformation with the propyl chain in equatorial position. The first hypothesis was confirmed by the analysis of 1 H NMR spectrum, despite its low resolution. For compound 8, both H5 have one large (J 5eq-5ax = 13.75

(J 1-2 = 1.9 Hz, J 2-3 =J 3-4 = 2. 8
Hz) coupling constant, and the other much smaller (J 5eq-4 = 1.5 Hz, J 5ax-4 = 2.75 Hz). This indicates that H4 is equatorial, which confirms the 4 C 1 chair conformation. Compound 9 exhibited broad signals in its 1 H NMR spectrum in MeOH-d 4 and we could not verify its conformation. N-Z Protected imino-L-arabinitols were submitted to further functionalization. We investigated two modes of functionalization designed to add polar substituents to the pseudo aglycone, to mimic more closely the natural substrates of GALC. For this the introduction of one hydroxyl group by hydroboration/oxidation and of two hydroxyl groups by dihydroxylation were envisaged. Another potential modification would be chain elongation by cross metathesis; however, we limited our investigation to the formation of more polar aglycones (Scheme 15). Hydroboration/oxidation reaction of 15 turned out to be a challenging task. Various hydroboration agents were tried, but the reaction was never complete and the yield was moderate (Table 5). The best result was achieved by using catecholborane in the presence of Wilkinson catalyst (Figure 20), which is known to improve hydroboration of alkenes with catecholborane and pinacolborane. 183 According to our experiments, this catalyst is not compatible with 9-BBN, as its addition drastically lowered the yield. The best conditions gave compound 22 in 46% yield and 14% of starting material was recovered. We were aware of the need for additional optimization, but as we obtained enough product to continue, we decided to finish the synthesis and to wait for biological evaluation results before working again on this reaction.

Reagent

Eq Temp Catalyst Yield SM recovered Pinacolborane 6 40°C -- -- 50% Catecholborane 2+2 40°C -- 36% 37% Catecholborane 2 40°C Wilkinson, (2%) 46% 14% 9BBN 2 40°C -- 28% 35% 9BBN 2 40°C
Wilkinson, (2%) 8% 30% Dihydroxylation of 15 was efficiently (73% overall yield) realized using the OsO 4 /NMO system 184 leading to a separable mixture of diastereoisomers 18 and 19. The configurations of the secondary alcohols were determined by circular dichroism analysis 185 with the kind help of Prof. Frelek team (Institute of Organic Chemistry of Polish Academy of Science in Warsaw, Poland). The in situ dimolybdenum methodology of electronic circular dichroism spectroscopy (ECD) has been applied. This straightforward, simple, and reliable approach consists of mixing a nonracemic transparent vic-diol with dimolybdenum tetraacetate acting as an auxiliary chromophore. The transition metal ions, when complexed to an optically active ligand, become involved in the symmetry of the ligand. Thus, the Cotton Effects (CEs) related to electronic transitions of the metal atom are obtained and they are characteristic for the absolute configuration of the compound acting as ligand (in this case 1,2-diol). In general, an application of the helicity rule relating the sign of the O-C-C-O torsional angle with the sign of the CEs arising in the 300-400 nm spectral range allows an unequivocal assignment of the stereostructure of investigated diol units. This is due to the fact that in the chiral Mo-complexes formed in situ the conformational mobility of diols is very much reduced due to the restricted rotations of the remaining acetate ligands still coordinated to the metal atoms. Thus, this reduction or restriction of the conformational freedom makes an absolute configurational assignment possible on the basis of the chiroptical data alone. 184 Schonemann, W.; Gallienne, E.; Compain, P.; Ikeda, K.; Asano, N.; Martin, O. R. Bioorg Med Chem 2010, 18, 2645. 185 Górecki, M.; Jabłońska, E.; Kruszewska, A.; Suszczyńska, A.; Urbańczyk-Lipkowska, Z.; Gerards, M.; Morzycki, J. W.; Szczepek, W. J.; Frelek, J. J Org Chem 2007, 72, 2906.

I.8 Conclusions

In conclusion, we have successfully applied our existing methodology to obtain 1-Csubstituted imino-L-arabinitols. The key step is the diastereoselective addition of allylmagnesium bromide or allyltrimethylsilane which takes place on the open-chain form of a glycosylamine. Moreover, the diastereoselectivity is opposite for N-Bn and N-Z protected amines, which gave thus access to both pseudo anomers of the 1-C-alkyl imino-L-arabinitol derivatives. In addition, this strategy allows the direct functionalization of the allyl substituent using oxidation reactions, leading to a diversity of non-functionalized or functionalized 1-Calkylated iminosugars.

Résumé section I:

Synthèse et évaluation biologique des 1-C-alkyl-imino-L-arabinitols. The synthesis turned out to be very challenging. The first difficulties were faced during the allyltrimethylsilane addition. This reaction gave an inseparable mixture of the desired alcohol and the starting material in low yield (26mix). Despite thorough optimization (time, temperature, number of equivalents of Lewis acid, Lewis acid), we did not find conditions under which the reaction could be completed. Secondly, the last step, intramolecular reductive amination, was inefficient and afforded complex mixtures of two or more deoxy iminosugars or epimers of the expected product even under the mildest conditions. To better understand the reaction mechanism, some of the mixtures were separated by HPLC and characterized by NMR analysis.

Eventually, to overcome the epimerization problems, we decided to abandon the intramolecular reductive amination approach and implement an intramolecular S N 2 type cyclisation as used previously for the arabino series. To achieve this, we had to invert the configuration of the hydroxyl group at C-5 in the addition product (R epimer gives an L-altro final product, while the S epimer gives D-galacto, Scheme 17). Again, this task was a real challenge and only a small amount of the desired alcohol was obtained which could lead eventually to 1-C-propyl-imino-D-galactitol 49. The details of these synthetic studies are provided in the next sections.

II.2 Synthesis of N-Z protected galactopyranosylamine 25 and C-1 chain elongation

According to our experience from the arabino series, we did not try the direct condensation of 2,3,4,6-tetra-O-benzyl-D-galactose with benzyl carbamate but we used the acetylated derivative 24 as the starting material. The reaction with benzyl carbamate worked very well with an overall yield of 80% for two steps (1g scale). The key chain-elongation step, the addition of allyltrimethylsilane to N-Z protected amine surprised us because it did not give the expected product in the first assay. Various conditions were used with different Lewis acids (Table 6) but the reaction was never complete and moreover, the open-chain product was inseparable from starting material 25. As shown in Table 6, one equivalent of TMSOTf as the Lewis acid was not sufficient to promote the reaction (entry 1). An excess of Lewis acid (3 eq) as well as increasing the temperature led to degradation (entry 3 and 5). This indicated clearly that the reaction was extremely sensitive to variations of the conditions and needed to be handled carefully. The reaction time did not seem to play a crucial role. Lewis acids other than trimethylsilyl trifluoromethanesulfonate did not improve the reaction turnover. On the contrary, bismuth (III) trifluoromethanesulfonate did not catalyze the reaction, and zinc trifluoromethanesulfonate caused degradation (entries 6 and7). Triisopropylsilyl trifluoromethanesulfonate seemed to be promising (entry 8), but not as efficient as trimethylsilyl trifluoromethanesulfonate.

Entry Lewis acid Eq Temp Time Conversion a (Yield b ) Comments

1 TMSOTf 1 -20°C 3 days -- SM, degradation 2 TMSOTf 1 -20°C 42h 82% (27%) Low yield 3 TMSOTf 1 0°C 42h 36% important degradation 4 TMSOTf 2 -20°C 29h 85% 50% c of desired alcohol 5 TMSOTf 3 -20°C 42h -- degradation 6 Bi(OTf) 3 2 -20°C 42h -- SM 7 Zn(OTf) 2 2 -20°C 42h -- SM, degradation 8 TIPSOTf 2 -20°C 42h 70% (10%) -- 9 TMSOTf 2 -20°C 2.5 days 85% (30%) -- 10 TMSOTf 2 -20°C 2*2.

5days 90% (59%) Chosen conditions

Table 6. Various conditions used to optimize allyltrimethylsilane addition onto 25. All assays performed in CH 3 CN. a reaction conversion calculated as if only expected alcohol was obtained. b yield calculated for pure alcohol obtained after an acetylation/separation/deacetylation sequence c based on the NMR spectrum of the crude mixture Finally, we decided to carry out the reaction with two equivalents of trimethylsilyl trifluoromethanesulfonate, at -20°C for 2.5 days, then to purify the reaction mixture and repeat the procedure on the mixture, as if it would be pure starting material 25. This approach resulted in a satisfactory yield (~60%) that allowed the continuation of the synthesis.

However, the alcohol thus obtained was inseparable from the residual starting material. To overcome this inconvenience, we acetylated the mixture 26mix, separated the acylated alcohols 37 and 38 from the N-Z protected amine 25 and deacetylated them to obtain pure 39 and 40 in 31% and 3.5% overall yield from 25 respectively (Scheme 18).

The diastereoselectivity of this reaction was fairly good, as the two epimers 37 and 38 were obtained in a ratio of ~8:1. Their configuration cannot be determined precisely at this stage, but later transformation showed that the major epimer 37 is the syn (1R) product.

II.3 Synthesis of ketones 41 and 42

The alcohols 39 and 40 were oxidized with Dess-Martin periodinane 188 in good yields to give ketones 41 and 42. However, as 42 was issued from the minor alcohol 40, it was synthesized for characterization purposes only and was not used further in synthesis. Ketones 41 and 42 were submitted then to cross metathesis 189,190 in order to prepare the final iminosugars with various alkyl chain lengths (Scheme 20). The reaction with 1octene was conducted on a mixture of ketones 41 and 42 to give ketones 43 and 44 and the one with 1-pentene on pure 41 to afford ketone 45. Hoveyda-Grubbs 2 nd generation catalyst 191,192 was used to improve the reaction conversion. While the derivatives with the longer chain were obtained in a good global yield of 72%, compound 45 was obtained in the much lower yield of 43%. This could be explained by the fact that 1-pentene is quite volatile and while the reaction was conducted during the summer, the light alkene could have easily evaporated during manipulation.

X

In both reactions, homodimers were not observed (mass analysis of the crude compound) and only the E isomer was obtained. The ketones were then submitted to the next step, which was intramolecular reductive amination by hydrogenolysis, to obtain cyclic galacto iminosugars.

II.4 Intramolecular reductive amination.

There are several ways to form C-N bonds to afford an iminosugar. As described for the arabino and xylo series, an intramolecular S N 2 reaction is one of the solutions. In the galacto series, due to the presence of the CH 2 OH group at C-5 and the inversion of configuration at this position during cyclisation, this approach cannot be directly applied. The most popular method to achieve cyclisation is reductive amination. 193 In this reaction, the amine reacts with a carbonyl group (aldehyde or ketone) to form a hemiaminal. By losing one molecule of water an imine is created, which is subsequently reduced to an amine (Scheme 21). This reaction generally requires an acidic pH (~5) in order to activate the imine as an iminium salt. A very common reducing agent for reductive amination is sodium cyanoborohydride. This mild hydride donor does not reduce ketones nor aldehydes. 194 Thanks to this method, in a single synthetic operation, a C-N bond can be stereoselectively formed to create a piperidine ring from a ketone and an amine within the same molecule (intramolecular reductive amination). 195 However, the amine function in 41 and related compounds is protected as a benzyl carbamate, which requires its cleavage in a first step. It is well known that hydrogenation is a very efficient and clean way to deprotect N-and O-benzyl groups. 196 Thus, hydrogenation is the ideal way to perform the last steps of imino-galactitols' synthesis.

Reductive amination can also be performed using hydrogen as the reagent. This one-pot reaction can be divided into several stages (Scheme To better understand what did not work in the cyclisation step, we decided to repeat the synthesis of ketone 41 and submit it to reductive amination under various conditions. We have performed hydrogenation in acid, neutral and basic conditions as well as classical reductive amination using sodium cyanoborohydride ( Scheme 24). 198 We first tried the reaction with sodium cyanoborohydride, although this reaction was unlikely to work with the N-Z group (formation of cyclic hemiaminal not favourable, Scheme 19). The reaction failed, we recovered starting material with traces of alcohol resulting from reduction of ketone. These data establish unambiguously that the imino hexitol has a pseudo L-altro configuration (4,5-trans) and that the anomeric configuration is pseudo-b (L-series; 1,2-cis).

Hydrogenation of the iminium salt occurred from the "top face" (trans with respect to the C-1 substituent) and the "anomeric" configuration is R. The second product, pseudo b-L-ido, has undergone epimerisation at C-4, remarkably. A possible mechanism is suggested further below (Figure 24).

Hydrogenation under neutral conditions (Scheme 24) promoted also the intramolecular reductive amination, but the benzyl groups were not cleaved. Remarkably, under these milder conditions, the benzyloxy group at C-3 was also lost, leading again to 3deoxy iminohexitols. In this case however, only the epimers at C-5 were obtained (mixture of pseudo D-galacto and pseudo-L-altro in a 1:1 ratio), without epimerisation at C-4.

Although it is unusual to perform this type of reaction under basic conditions, the treatment of ketone 41 under catalytic hydrogenation conditions in the presence of triethylamine promoted the desired, intramolecular reductive amination (i.e. reduction of allyl group, cleavage of Z-group and reductive amination) to afford the a-1-C-alkyl iminohexitol derivatives with traces only of the product of deoxygenation at C-3. Two major products were obtained, which could not be separated by flash column chromatography. The mixture was debenzylated and samples of both epimers could be isolated by HPLC (column Hypercarb, 250mm x 10mm, H 2 O/formic acid 0.5%, pressure 2.2 bars, debit 4.4mL/min, T=30°C, ELS detection). NMR analysis of the two products revealed that we had finally obtained the desired a-1-C-propyl imino-D-galactitol 49, as well as its L-ido epimer 52 (double epimer at C-4 and C-5) in a 1:1 ratio.

II.5 Synthesis of 1-C-alkyl-imino-D-galactitols by intramolecular S N 2 reaction

In order to try to improve the synthesis of the target compounds, we decided to examine also the approach by intramolecular cyclisation via a S N 2 reaction. Since cyclisation of the original alcohol 39 would give a 1-C-propyl-imino-L-altritol, we attempted to invert its configuration at C-5. However, this turned out to be again a challenging task. The simplest way to achieve this would be to oxidize the alcohol 39 to ketone 41, and to reduce then to the alcohol 46, provided that the stereochemistry of the reduction would be in favor of the L-altro epimer. Another method would be to use the Mitsunobu reaction 199 , wherein the alcohol 39 199 Mitsunobu, O. Synthesis 1981, 1. would be converted into an ester with an inversion of configuration. We investigated a series of hydride reducing agents (entries 1-4, Table 7) and attempted the Mitsunobu reaction (entry 5). The latter was unsuccessful and led to degradation: only half of the starting material was recovered. None of the investigated hydrides gave the desired L-altro product as the major isomer. Reduction with sodium and lithium borohydrides gave an inseparable mixture of both L-altro 39 and D-galacto 46 alcohols in high yield. L-selectride afforded also a mixture of alcohols but with significantly lower yield and poor purity of the crude product. The reaction with sodium borohydride under Luche conditions 200 (entry 3) also gave a mixture of the two epimeric alcohols. Each assay was conducted on a small sample of ketone 41 and none of them gave the desired results, therefore the synthesis was not continued on them. Moreover, 200 Luche, J. L. J Am Chem Soc 1978, 100, 2226. as the epimeric alcohols have very similar NMR spectra, it was difficult to determine their percentage in each mixture. In any event, the highest global yield and the cleanest crude mixture of the two alcohols were obtained by the reduction with sodium borohydride (entry 1). The mixture was then mesylated. S N 2 cyclisation under basic conditions afforded a mixture of the two expected products in a 5:1 ratio which could be separated: compound 48 was isolated in 7% yield and 28 in 32% yield from the mixture of alcohols 39+46.

Hydrogenolysis

II.6 Conclusions

Unanticipated problems and challenges were faced in the synthesis of 1-C-alkyl iminogalactitol derivatives. In the first strategy we observed an unexpected behaviour of the galacto substrate, which led to new 3-deoxy derivatives of the target compound. However, by adjusting the conditions of catalytic hydrogenation from acidic to basic pH, we obrained the desired 1-C-propyl-imino-D-galactitol as a mixture with its L-ido epimer (1:1). On the other hand, the yield and diastereoselectivity of this synthesis towards the desired product is low.

The other approach, by the procedure of double inversion of hydroxyl group Carbosynth turned out to be highly heterogenous and to contain a significant amount of Lsorbose. Therefore we decided to synthesize this compound ourselves. Although 53 has been used as a starting material for various syntheses (sugar derivatives 204 or ligands and catalysts for asymmetric synthesis 205,206,207 ), its synthesis appeared to be a challenging task, due to misleading literature data. Eventually, we obtained 1,2:4,6-di-O-isopropylidene-a-Lsorbofuranose 53, the improved synthesis and correct characterisation of which we described in a recent publication. 208 The newly developed synthetic strategy to access 1-C-alkyl imino- Moreover, better yields were obtained when the reaction was carried out in 2,2-dimethoxypropane as the solvent and when dimethoxyethane was used in small amount to solubilise the tin salt. Initially we have experienced difficulties with this protocol which consisted in refluxing the reaction mixture until complete disappearance of starting material (the solution becomes clear after 3-4 h on a 5 g-scale). In our hands, these conditions led to a mixture of 1,2:4,6-and2,3:4,6-di-O-isopropylidene-a-L-sorbofuranose that could not be separated by crystallization. We used other conditions, but our effort was fruitless (Table 8, entries 1 to 3 and 7). As suggested by Zhao and Shi211 we decreased the reaction temperature to 70°C and stopped the reaction before the solution became clear (Table 8, entries 4 and5).

Although the reaction was not complete (66% of L-sorbose was recovered), we were able to obtain mainly the desired isomer that could be purified by recrystallization from hexane. We have identified a number of errors in literature reports, 212,213 The following step was the inversion of the C-3 hydroxyl group configuration by oxidation/reduction sequence. The lower face of 53 is sterically crowded and, after oxidation, the reducing agent will approach the keto group of 63 from the upper face, which will result in inversion of the configuration at C-3. 201 While having a good experience with Dess-Martin periodinane as an oxidizing agent, we decided to use it. However, it turned out that this reagent required a high purity of 1,2:4,6-di-O-isopropylidene-a-L-sorbose. The oxidation assay on the commercial 53 failed due to its degradation. The quality of crude or even purified by chromatography 53 was not sufficient as no traces of the desired ketone 63 could be obtained. On the other hand, when 53 was obtained by recrystallization, it was clean enough to be oxidized with Dess-Martin periodinane quantitatively. Subsequent reduction with an excess (2.5 eq) of sodium borohydride afforded the crude L-tagato-derivative 64 ready to be used in the next step. As all intermediates, except 53 were used as crude compounds, the yield was calculated after the next step, which was the migration of the isopropylidene to give alcohol 65. The best overall yield for this 4-step sequence from Lsorbose to 65 (including two purifications) was 7%. Confident with those conditions, we performed the first protection step with the difference that we used a longer reaction time to obtain the maximum conversion (reaction stopped when the solution was clear, 3h 45min on 7g scale), and then we repeated the oxidation-reduction sequence on crude 53. As the formed ketone 63 has a different R f from 53 and its isomer, as well as from other by-products, we can oxidize the crude mixture and isolate the desired ketone 63 by flash column chromatography on silica gel. We succeeded to obtain the inverted L-tagato-alcohol 64 which was purified on silica gel and submitted to further reaction to prepare 65. The best overall yield for this 4-step sequence from L-sorbose to alcohol 65 is 12% (purification after reduction). This means that we almost doubled the yield of the previous procedure and avoided difficult purification by recristallization. The disadvantage of this approach is the use of toxic PCC. However with careful handling and proper waste disposal, this method becomes efficient and safe.

III.4 Liberation and oxidation of C-6 hydroxyl group

As the principle of this synthetic strategy is to elongate the chain at C-6 position, we had to liberate it. To achieve this, we capitalized on the differences in stabilities of isopropylidene groups. The acetonide between two vicinal secondary hydroxyl groups (C-3 and C-4) is more stable than the one between primary and secondary hydroxyl groups (C-4 and C-6). In 1,2:4,6-di-O-isopropylidene-a-L-sorbose 53 the C-3 and C-4 hydroxyl groups are in trans orientation thus an isopropylidene acetal between these secondary hydroxyl groups cannot be formed and migration of protecting group is not possible. However, in an L-tagatofuranose, the C-3 and C-4 hydroxyl groups are in cis orientation which favours displacement of acetonide to form a more stable one. This occurs spontaneously in acetone and is accelerated by the addition of a catalytic amount of acid (Scheme 30). The L-tagatofuranose derivative 65 was thus obtained in 73% yield (12% overall yield from L-sorbose). Oxidation with Dess-Martin periodinane was then performed to prepare the key aldehyde intermediate 66. This unstable aldehyde 66 was directly used in the next reaction.

As the reaction is difficult to monitor by TLC, the reaction time was set up experimentally on the basis of the yield of the subsequent reaction. We observed that leaving the reaction overnight leads to degradation of the aldehyde 66, while 4h was not sufficient to oxidize completely the alcohol 65 (recovered after the next step): the reaction time was set up to 6h.

III. Sulfinylimines are known to be excellent "Michael" acceptors due to the strong electron withdrawing N-sulfinyl group. They react with a variety of nucleophiles in a 1,4addition fashion, including oxygen, nitrogen, sulfur, carbon, and phosphorous nucleophiles. 215 Moreover, the sulfinyl group is easily removed under acidic conditions.216 This is why, to introduce the nitrogen into the molecule and then to perform addition of organometallics we decided to use Ellman's imines. 217 The condensation of aldehyde 66 with a racemic mixture of Ellman's amines ((S/R)-tert-butanesulfinamide) in the presence of molecular sieves and dry copper sulfate afforded tert-butanesulfinylimine derivatives 67 and 68. The S and R isomers were separable by silica gel chromatography. We also performed the synthesis of 67 and 68

using each enantiomer of Ellman's amine separately in order to have stereochemically well defined intermediate and to investigate the stereochemistry of the following additions of the Grignard reagents (Scheme 30). Both reactions gave satisfactory yields of 71% and 79%

(from alcohol 65).

III.6 Addition of Grignard reagents

The additions of Grignard reagents were conducted in dry toluene, at 0°C or -78°C

with five equivalents of allyl, hexyl or nonyl Grignard reagent used as solutions in ether or THF. 218 Each reaction was conducted on pure starting imine (67 or 68) and gave from one to three distinct products (Scheme 32). As a result of introducing Ellman's chiral auxiliary, there are two chiral fragments in the molecule that can direct the addition of Grignard reagents. In our previous experience in the xylo series, 202 only the sugar moiety was responsible for the stereochemistry of the organometallic addition, while the configuration of the imine was completely passive.

However, it was reported in the literature that changes in reaction conditions can reverse the diastereoselectivity of organometallics addition to sulfinimines. 219 The results we obtained were sensitive to reagents used as well as the type of organomagnesium reagent involved (Tables 9 and10). As summarized in Table 9 for the addition of allylmagnesium bromide, essentially only one stereoisomer is obtained from both the R-and S-imines, and therefore the reaction is almost exclusively controlled by the sugar moiety. As expected, the stereoselectivity is higher at lower temperature. We are not completely sure about the attribution of the configuration of the allyl chain, as the synthesis was not carried out from these intermediates until the final cyclic compounds and the assignment is based on NMR spectra comparisons.

Entry Imine Reagent

In Table 10 are listed the results for the addition of hexylmagnesium bromide and nonylmagnesium bromide. As regards the addition of a hexyl chain, the experiments were carried out at -78°C and two solutions of hexylmagnesium bromide were used: 0.8N in THF and 2N in Et 2 O. For the assays with the THF solution, only one diastereoisomer was obtained 202 from both imines (entry 1 and 2). In this case it appears that it is also the sugar which controls the stereochemistry of the addition. The same reaction with an Et 2 O solution of hexylmagnesium bromide afforded two different epimeric amines, with the R isomer highly predominant from the R imine and a majority of S isomer from the S imine. This means that changing the cosolvent (Et 2 O instead of THF) decreased the influence of the sugar moiety on the stereochemistry of the addition in favor of that of the chiral sulfinylimine. As regards the addition of a nonyl chain, only one assay for each imine was done at 0°C using the Grignard reagent as a solution in ether. The diastereoselectivity was better for the S imine 67 (1:6 in favor of S isomer) while for the R imine 68, both isomers were obtained in nearly the same yield. As the reactions in the nonyl series were performed at higher temperature, their diastereoselectivity is lower. Interestingly, the addition of nonylmagnesium bromide gave a third product: a compound without any chain 76, which must arise from a Meerwein-Ponndorf-Verley-type reduction. Finally, the isomers were separable by silica gel chromatography, and the low diastereoselectivity of the Grignard reagent addition was not an important problem, as we need to have access to both isomers (a and b) of the final 1-Calkyl-imino-D-galactitols.

Entry

III.7 Deprotection and reductive amination of amine intermediates

As the last step of this synthesis was a ring closure by reductive amination, we needed to cleave first the sulfinyl group to liberate the amine, and then to hydrolyze the isopropylidene acetals to free the anomeric position. Both processes were to be performed under acidic conditions, using HCl, HBr, TFA, AcOH or acid resin. 216,220,221 After successful deprotection, we planned to achieve an intramolecular reductive amination to access the final 1-C-alkyl-imino-D-galactitols. At the beginning we decided to cleave the sulfinyl group and the isopropylidene acetals at the same time by using trifluoroacetic acid in water (Table 11, entry 1). The spectra of the crude product were very difficult to interpret, which could be due to the presence of various possible forms of expected compound A. The mixture was submitted to hydrogenation which resulted in a complicated combination of desired imino-galactitols, their deoxy derivatives and epimers. The NMR was again illegible and the only information we had came from mass spectroscopy analysis.

We decided to repeat the deprotection but stepwise. At first we cleaved the sulfinyl with hydrochloric acid in methanol. It turned out however, that we also removed one of the acetonides which resulted in an unstable intermediate. To remove the second isopropylidene, we used the same conditions as previously. But we realized after hydrogenation that the product must have partially been degraded during the second step and the reaction sequence 216 gave a complex mixture. As MeOH/HCl mixture was efficient enough to cleave isopropylidene also, we decided to use this method to achieve deprotection (Table 11,entry 3). However, this time we formed, as we could have expected, the corresponding methyl glycoside (confirmed by the mass analysis and NMR) and further reductive amination was not possible. Then we tried an acidic ion exchange resin (Dowex50WX8), but again we obtained a complex mixture and we did not achieve the isopropylidene cleavage (Table 11,entry 4).

From those fruitless trials, we realized that the intermediate with a free primary amino group and deprotected anomeric hydroxyl group (an amino hemiketal) must be very unstable and labile. Therefore, in the next assays ( As triflyl azide is a hazardous reagent that can be stored in solution only, we chose to synthesise it according to the safe and convenient protocol reported by Ernst and coworkers. 225 All azido-derivatives 83-86 were synthesised using the same procedure in the presence of catalytic copper (II) sulfate in good yields from 61% to 85% (for two steps).

Unfortunately we did not have time to verify if this methodology could be applied to the 6-Callyl derivatives.

III.9 Deprotection and reductive amination of azido-intermediates

Having azido-intermediates 83-86 in hand, we decided to cleave the isopropylidene groups using Dowex 50WX8 (H + ) resin as described by O'Brien and Murphy. 226 This time we succeeded to obtain the desired free azido hemiketal derivatives 87 and 88, which allowed us to complete the synthesis. Although this reaction is long and sensitive to temperature (at lower temperature the reaction is not complete and very slow, while heating over 65°C leads to elimination reactions), we found optimized conditions (Scheme 35) that led us to very clean samples of azido intermediated 87 and 88 in 61-78% yields. We observed also that for the R isomer this reaction functions better than for the S one. During those assays we completely consumed the nonyl derivatives and further reductive amination reactions were conducted on hexyl-intermediates only (Scheme 35). This time, we did obtain the desired imino-D-galactitols without any deoxy byproducts! The first assay from 87 in methanol under atmospheric hydrogen pressure gave the desired product 89 in a mixture with the corresponding N-methyl-imino-D-galactitol 90 and another epimer in a ratio 5:2.5:2.5 (entry 1, Table 12). When we changed the solvent to isopropanol ,we did not observe any N-methyl by-product, but the desired 89 was in a mixture with its gluco epimer 91 (4:1 ratio approx). The best results were obtained by hydrogenation under a 10 bar pressure in methanol with 10% palladium on carbon as the catalyst (entry 3, Table 12). This time we also observed an important difference in reactivity between the two isomers. Interestingly, less reactive in the previous reaction, the S isomer gave the expected b-

1-C-hexyl-imino-D-

III.11 Conclusions

The alternative synthetic strategy to prepare 1-C-alkyl-imino-D-galactitols from Lsorbose is significantly more efficient than the previous one from D-galactose. We first overcame the difficulties in the synthesis of 1,2:4,6-di-O-isopropylidene-a-L-sorbose 53 and established correct protocols and characterizations for 1,2:4,6-and 2,3:4,6-di-Oisopropylidene-a-L-sorbose isomers. This is important because, for many of sorbfuranose derivatives, correct analytical data were missing in the literature. We then sucessfully applied our previous experience with Ellman's imines to tagato intermediates as precursors of 1-Calkyl iminogalatitol derivatives. The deprotected amine intermediates resulting from this strategy were found to be extremely labile: the conversion of the amino group into an azido group by diazo transfer, an unusual step at the late stage of such synthesis, allowed us to solve this problem and to perform the deprotection step to a clean azido hemiketal in high yield.

The final piperidine ring formation by intramolecular reductive amination upon catalytic hydrogenation led successfully to the desired final products, in appropriate yields, but contaminated by a small amount of an epimer. Some optimization of this last step is still required in order to obtain eventually the desired iminogalactitols free of traces of other epimers.

Résumé section III :

Synthèse alternative des 1-C-alkyl-imino-D-galactitols à partir du Lsorbose: stratégie par élongation de la chaîne en C-6.

Compte tenu des difficultés de la stratégie de synthèse précédente s'appuyant sur une The rest of the synthesis was then conducted on the major R isomer. Using an S N 2 reaction, cyclisation of 27R upon treatment with potassium tert-butoxide afforded protected iminosugar 28 in 47% yield. As shown on Scheme 39, compound 28 was submitted to three further reactions:

i. deprotection by hydrogenation to give 1-C-propyl-imino-L-altritol 30

ii. cross-metathesis with 1-octene and deprotection to give 1-C-nonyl-imino-L-altritol 32

iii. dihydroxylation and deprotection of the resulting epimeric diols 33 and 34 to give 35 and 36

All these compounds were obtained in satisfactory yields. As expected, dihydroxylation of the double bond in 28 was not stereoselective and afforded a mixture of diols 33 and 34, which could be separated. Configurations of the secondary alcohols were determined by circular dichroism analysis in Prof. Frelek's laboratory. The absolute configurations of the newly formed stereogenic centers are (R) in 33 and (S) in 34.

As regards the conformations of the compounds prepared in L-altro series, we observed the same phenomenon as for D-arabino products: the N-acyl protected piperidines favor the conformation in which the substituent a to nitrogen is in axial position. We assumed then, that the major pseudo b (R) isomer of protected imino-L-altritols 28, 31 and 33/34 would be in 4 C 1 chair conformation with three axial substituents: this is confirmed by the large J 2,3

coupling constant in 28 (Figure 27). After selective N-deprotection of 28, we obtained 29 which adopted 1 C 4 conformation, as indicated by the small J 2,3 coupling constants (Figure 27). The deprotected iminosugars 30, 32, 35 and 36 also adopt this 1 C 4 conformation which was confirmed by NMR analysis. All the N-Z protected compounds inverted thus their conformations upon N-protecting group cleavage (from 4 C 1 to 1 C 4 ). This led to a series of new 1-C-alkyl imino-L-altritol derivatives, which can be submitted to biological assays.

V. The synthesis of galacto-isofagomine-like iminosugars.

V Although this promising synthetic strategy worked beautifully for IFG itself (glucolike configuration), it failed for galacto-IFG. This was due to the ineffectiveness of all of the assays carried out to substitute the C-4 hydroxyl with a cyano group. Although Dess-Martin periodinane is more expensive then the reagents used for Swern or PCC oxidations, it is less toxic, easier to manipulate and finally gives very clean crude products. The high stereoselectivity of the reduction of the ketone 100 is arising from its bicyclic structure, which ensures a clean addition of the hydride in the exo direction.

V.

V.5 Synthesis of nitro-alcohols 112+113 and dehydration assays

Since the initial synthetic approach to galacto-IFGs by way of cyano-intermediates failed, we considered using nitromethane in order to introduce the C-N fragment 231 by way of its addition to the keto intermediate 100 (Scheme 47). As indicated above, the extension of this strategy to nitroalkanes may provide a means of introducing directly a 5-C-alkyl substituent, and initial assays were performed with nitrohexane. The addition of nitromethane and nitrohexane to the ketone 100 was then investigated. We found that the diastereoselectivity and the yield of the addition of nitromethane depended strongly on the conditions used. When using DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) as a base and acetonitrile as the solvent, the reaction was not diastereoselective and gave an unseparable mixture of 4R and 4S isomers in 1:1 proportion in low yield. This changed drastically when the reaction was conducted in pure nitromethane with triethylamine as a base. One diastereoisomer, 112, was highly predominant, and the yield much improved. Dehydration of the nitroaldols to the corresponding nitroalkenes can be performed under a number of conditions. We decided to investigate first the dehydration under the mild conditions reported by Moffatt et al 232 on the mixture of isomers 112+113 (conditions listed in Table 13). However these conditions did not promote the expected reaction. Among the other conditions investigated, only the last attempt with trifluoroacetic acid in DMSO gave the desired unsaturated product, however, in a mixture with an endocyclic isomer. Hydrogenation of this mixture failed and due to lack of time we did not study further this reaction. As regards the reaction with nitrohexane, we investigated different nitrohexane concentrations, with and without co-solvent, and with triethylamine as a base only. The best results (64% yield) were obtained using a 0.28N solution of SM in neat nitrohexane during 2.5 days. Longer reaction times did not improve the yield. In each case the reaction was not complete and the starting material was recovered. The reaction with nitrohexane is more complex than that with nitromethane, as it can lead up to four diastereoisomers (two chiral carbons created C-4 and C-5). Gratefully, the reaction led to only two major isomers 119+120 in a 65:35 ratio, but their actual configurations cannot be determined at this stage. V.6 Reduction of the nitro-intermediates and cyclisation by reductive amination.

Entry

Entry

The mixtures of epimers 112+113 and 119+120 were submitted to hydrogenation.

During this reaction, the following processes take place: The assignment of configuration at C-4 in 115 and 116 is a difficult problem, as a result of the absence of a proton at C-4, and cannot be determined from NMR data. However, we could consider two arguments to assign a configuration. Addition of nitromethane under CH 3 NO 2 /NEt 3 conditions is likely to give the thermodynamic product (weak base, excess CH 3 NO 2 would give a reversible reaction). The thermodynamic product is probably the one with the CH 2 NO 2 group equatorial (Figure 28). If this is correct, the hydrogenation of 112 should give D-galacto isomer 116. This hypothesis is supported by the biological assays of the deprotected products 117 and 118: the major isomer 118 is a much better inhibitor of GALC than the minor one (see Results and discussion section VI). We deduce that the better inhibitor has a D-galacto-like, and not L-altro-like configuration. This indicates that the compound 118 is most likely the pseudo-Dgalacto epimer.

After these satisfying results with nitromethane derivatives, we applied the same strategy to nitrohexane adducts. This time, the hydrogenation using palladium on carbon or palladium hydroxide did not give the expected product. We realized that, while the benzyl group was cleaved, the reduction of the nitro group is more difficult under these conditions because it is hindered by the presence of the pentyl chain (Table 15). We decided to perform this reaction in two steps: first reduce the nitro group into an amine with Raney nickel, and then deprotect the anomeric position to promote reductive amination (Scheme 49). Under these conditions, we obtained first a clean mixture of two aminoalkyl derivatives (121+122), which was purified and submitted to catalytic hydrogenation using palladium hydroxide as the catalyst in acidic isopropanol. This reaction led to the formation of the desired, 5-C-substitued piperidines (isomers 123+124) which were deprotected using an acidic ion-exchange resin to provide 5-C-pentyl pseudo IFG isomers 125 and 126. While this sequence of reaction led to a clean mixture of epimers, the epimers themselves could not be separated. Pure samples of 125 and 126 were isolated by HPLC (column Hypercarb, 250mm x 10mm, H 2 O/isopropanol 99.5/0.5 formic acid 0.4%, pressure 3.3 bars, debit 4.4mL/min, T=30°C, ELS detection). In the absence of X-ray crystal structure data, the determination of the configuration of 125 and 126 is a difficult problem. However, we can make the following hypothesis: as the reaction of ketone 100 with nitromethane in CH 3 NO 2 /NEt 3 leads to a single diastereoisomer presumed to be the thermodynamic product, the addition of the more hindered nitrohexane is likely to lead to an adduct having the same configuration at C-4 as 112. Thus, the two stereoisomers obtained, 119 and 120, are most probably epimers at C-4', the stereogenic center carrying the nitro group and the alkyl chain (Figure 29). Upon hydrogenation and cleavage of the protecting group, the final products are therefore likely to have a pseudo-D-galacto configuration with a 5-C-pentyl chain in a cis or trans relation with respect to the 4-OH group. Further investigations on the isolated products will be performed in order to ascertain their configuration.

Entry

V.7 Conclusions

Although we were not able to remove the hydroxyl group at the branching position,

Résumé section V:

La synthèse d'iminosucres de type galacto-isofagomine. As shown above, the imino-L-altritols with apolar (30 and 32) or polar (35 and 36) aglycones do not have interesting inhibitory properties on the examined enzymes. However, we observed that longer alkyl chain increases inhibition of GALC (entry 2). Although imino-D-galactitol 49 turned out to be inactive on GALC, it inhibits strongly a-galactosidase A (97%) at 1mM concentration. The most interesting properties were observed for D-galacto IFG analogue 118, with 67% of inhibition of GALC at 1.2 mM.

Initial results are listed in

Entry Inhibitor Concentration

Conclusion

During my PhD, we have effectively realized all presumed goals. We have successfully applied our existing methodology to obtain 1-C-substitued imino-L-arabinitols.

These compounds turned out to be deprived from all inhibitory activity towards GALC and other galactosidases. However, these data confirmed that CH 2 OH group is essential to obtain strong inhibitor-enzyme interactions.

The same synthetic strategy was applied to obtain 1-C-substituted imino-D-galactitols.

Despite numerous unanticipated problems and challenges we faced during this synthesis, we finally obtained a small sample of 1-C-propyl-imino-D-galactitol. This compound did not show inhibitory activity towards GALC. However, it turned out to be a weak inhibitor of agactosidase with an IC 50 of 0.01 mM. This approach was also applied to prepare the 1-Csubstitued imino-L-altritols, but they did not show interesting biological activity.

To overcome the problems faced in the first synthesis of imino-D-galactitols, we used an alternative synthetic strategy by a C-6 chain elongation strategy. This methodology was significantly more successful than the previous one and we obtained samples of both anomers of 1-C-hexyl-imino-D-galactitol.

As the last goal, we have developed an easy and convenient synthesis to prepare pseudo-galacto-IFG derivatives. Pseudo-galacto-IFG is our first compound showing a moderate activity towards GALC with an IC 50 value of 0.45 mM.

As perspectives for this work, we need to optimize the last step of the 1-C-hexylimino-D-galactitol's synthesis to obtain pure samples without epimerisation. It would be also interesting to complete this synthesis with the nonyl and allyl chains in order to prepare 1-Csubstituted-imino-D-galactitols with a longer alkyl chain or more polar aglycone. We realized that the chemistry in the D-galacto series is much more difficult than we expected compared to the D-gluco series. Thus the synthesis of such iminosugars remains a real challenge for organic chemists and will need the development of new and innovative methodologies.

For the newly synthesized C-5-substituted pseudo-galacto-IFGs, we will have to determine the C-5 absolute configuration and also to confirm the suspected configuration at C-4. Our encouraging results on GALC activity with these compounds will lead us to develop other strategies for the synthesis of galacto-IFG derivatives, which do not bear the supplementary hydroxyl group at C-4 and to investigate their biological activities. This family represents the best objective to achieve for the discovery of potent GALC inhibitors and potential pharmacological chaperones for the treatment of Krabbe disease.

Experimental part

I. General

All reactions requiring anhydrous conditions were carried out using oven-dried glassware under an atmosphere of dry Ar. THF was distilled using Glass Technology Dry Solvent Station GTS100. CH 2 Cl 2 was distilled from CaH 2 . CH 3 CN, DMF and pyridine were dried using activated 3Å molecular sieves. Other anhydrous solvents and all reagent-grade chemicals were obtained from commercial suppliers and were used as received. Analytical thin layer chromatography was performed using Silica Gel 60F 254 precoated plates (Merck)

with visualization by ultraviolet light and phosphomolybdic acid or ceric sulfate/ammonium molybdate solutions. Flash chromatography was performed on Silica Gel 60 (230-400 mesh).

Melting points were determined in capillary tubes with a Büchi apparatus and are uncorrected. Optical rotations were measured with a Perkin-Elmer 341 polarimeter in appropriate solvent, at the indicated temperature and at 589 nm sodium line, in a 1 dm cell.

Concentrations are given in g/100 mL. protein of leukocyte or fibroblast sonicate) and distilled water were added up to the total volume of 0.2 mL. The mixture was incubated at 37 o C for 1 or 2 hours. Then, on ice, 0.1 mL of a solution of galactose (0.5 mg/mL in distilled water) and 1.5 mL of C-M (2-1) were added, vortexed and centrifuged at 1200 g for about 3 min. The lower phase was removed with pipette on syringe and saved in waste tube. 1mL of theoretical lower* was added to the assay tube, vortexed and centrifuged as above. Upper phase was carefully removed to scintillation vial, 10 mL of scintillation fluid was added and the 3 H-galactose hydrolyzed from the starting * Theoretical lower is made by taking 100 mL a solution of galactose (0.5 mg/ml) plus 500 mL of C-M (2:1) in a glass stoppered separatory funnel. Shake and let separate. Save lower phase for working up assays. This is a theoretical lower phase. Acetyl chloride (2.4 mL, 33.6 mmol, 0.5 eq) was added to anhydrous methanol (111 mL) at 0°C. The mixture was left stirring for 20 min in an ice-bath. Then solid L-arabinose (10 g, 66.6 mmol) was added. The reaction mixture was heated under reflux overnight. The mixture was allowed to cool down gradually to RT and left in the fridge for 24 h to give crystals of the desired glycoside as a mixture of anomers (4:6 a:b). The crystals were filtered and washed with cold MeOH. After partial concentration of the filtrate the crystallization was repeated. New crystals were collected with the product previously obtained.

TLC:

• DCM/MeOH (8:2);

• R fSM = 0.1; R f1 = 0.43

NMR

Obtained spectrum corresponds to literature data. 233 Yield 59% (6.46 g). 233 Gorin, P. A. J.; Mazurek, M. Can. J. Chem., 1975, 53, 1212-1223. To a solution of crude 2 (39.4 mmol) in 80% aqueous AcOH (32.5 mL), 3N HCl (9.5 mL) was slowly added. The mixture was stirred at 90°C until TLC showed completion of the reaction (20 h). Most of the acetic acid was removed by evaporation under vacuum, and then water (54 mL) and EtOAc (27 mL) were added. The organic phase was separated and the aqueous phase was extracted with EtOAc (3 x 27 mL). The organic phases were combined, washed with saturated NaHCO 3 (60 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a yellowish oil. Flash column chromatography on silica gel (PE/EtOAc 7:3) gave pure product as a white solid.

TLC:

• PE/EtOAc (7:3);

• R f = 0.10

NMR

Obtained spectrum corresponds to literature data. 234 Yield 56% (9.26 g). 234 Violante de Paz Banez, M.; Aznar Moreno, J. A.; Galbis, J. A. J. Carbohydr. Chem., 2008, 27, 120-140. The reaction was conducted under argon at RT. To a solution of compound 3 (1.00 g, 2.38 mmol) in anhydrous CH 2 Cl 2 (2.4 mL) were added p-toluenesulfonic acid monohydrate (0.414 g, 2.4 mmol, 1 eq) and benzylamine (0.78 mL, 7.14 mmol, 3 eq) to give a milky solution. The reaction was left stirring until TLC showed completion (24 h). The reaction mixture was diluted with CH 2 Cl 2 (20 mL) then washed with saturated Na 2 CO 3 (10 mL) and dried over MgSO 4 . After concentration under vacuum and three coevaporations with toluene, the crude product was obtained as a yellow oil which was used without purification in the next reaction.

TLC:

• PE/EtOAc (6:4); The reaction was conducted under argon. To a solution of crude 4 (655 mg, 1.19 mmol) in anhydrous THF (11.9 mL) was slowly added at 0°C a 1M solution of allylmagnesium bromide in Et 2 O (5.95 mL, 5.95 mmol, 5 eq). The reaction was left stirring at RT until TLC showed completion (16 h). The reaction was quenched with saturated NH 4 Cl (10 mL). Then CH 2 Cl 2 (25 mL) was then added. The organic phase was separated, washed successively with a 5% aqueous solution of HCl (15 mL), saturated NaHCO 3 (15 mL), water (15 mL) and dried over MgSO 4 . After concentration under vacuum the crude compound was obtained as a light yellow oil. Flash column chromatography on silica gel (PE/EtOAc 8:2) gave the desired product as a light yellow oil and as a mixture of inseparable diastereoisomers 5S and 5R in a 7:3 ratio.

• R f = 0.83 (1R,1S)-1-C-Allyl-2,

TLC:

• PE/EtOAc (8:2);

• R f = 0.19

HRMS (ESI)

[M+H] + calculated for C 36 H 42 NO 4 m/z = 552.31084; found m/z = 552.31171 Yield 81% (529 mg).

(1S)-1-C-Allyl-2,3,4-tri-O-benzyl-N-benzyl-1,5-dideoxy-1,5-imino-L-arabinitol 6 (1R)-1-C-Allyl-2,3,4-tri-O-benzyl-N-benzyl-1,5-dideoxy-1,5-imino-L-arabinitol 7 The reaction was conducted under argon. Mixture 5 (0.57 g, 1.03 mmol) was dissolved in anhydrous pyridine (10.3 mL) containing 4Å molecular sieves. After 10 min of stirring, methanesulfonyl chloride (0.2 mL, 2.58 mmol, 2.5 eq) was added. The mixture was stirred at 100°C until TLC showed completion of the reaction (1 h), then filtered over celite and the solid was washed with CH 2 Cl 2 . The filtrate was combined and concentrated under vacuum, EtOAc (30 mL) was added and the organic phase was washed with water (3 x 15 mL) then dried over MgSO 4 . After concentration under vacuum and coevaporation with toluene, the crude product was obtained as a dark orange oil. Flash column chromatography on silica gel (PE/EtOAc 95:5) gave the two desired isomers as separated products: 6 as an orange oil (1S, isomer pseudo-a) and 7 as a light yellow oil (1R, isomer pseudo-b).

IR [cm

LRMS (ESI)

[M+H] + = 534.5

pseudo-a isomer : TLC:

• PE/EtOAc (9:1);

• R fa = 0.35 48.36 (C5), 31.65 (C6). (1S)-1-C-Propyl-1,5-dideoxy-1,5-imino-L-arabinitol 8. To a solution of isomer 6 (200 mg, 0.375 mmol) in isopropanol (3.7 mL) were added 1N aqueous HCl (0.74 mL, 0.74 mmol, 2 eq) and 10% Pd/C (56 mg). The reaction mixture was left stirring at RT under a hydrogen atmosphere for 5 days. It was then filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated.

HRMS (ESI) [M+H] + calculated for

After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ) the final product was obtained as brownish crystals. (1R)-1-C-Propyl-1,5-dideoxy-1,5-imino-L-arabinitol 9. To a solution of isomer 7 (66 mg, 0.124 mmol) in isopropanol (1.2 mL) were added 1N aqueous HCl (0.24 mL, 0.24 mmol, 2 eq) and 10% Pd/C (19 mg). The reaction mixture was left stirring at RT under a hydrogen atmosphere for 5 days. Then it was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ) the final product was obtained as a brownish solid.

1 H NMR (400 MHz, CD 3 OD), d 3. 88-3.84 (m, 2H, H3, H4), 3.70-3.68 (m, 1H, H2), 2.89-2.86 (m, 1H, H1), 2.79 (d, 2H, H5, J= 8.0 Hz), 1.53-1.32 (m, 4H, H6, H7), 0.95 (t, 3H, H8, J 8,7 = 7.0 Hz).

13 C NMR(100 MHz, CD 3 OD), d 72.30 (C3), 71.34 (C2), 66.80 (C4), 53.96 (C1), 46.67 (C5), 33.65 (C6), 20.37 (C7), 14.51 (C8).

HRMS (ESI)

[M+H] + calculated for C 8 H 18 NO 3 m/z= 176.128120; found m/z= 176.128340 Yield 68% (14.9 mg).

(1R,1S)-1-C-Allyl-2,3,4-tri-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-L-arabinitol 12. The reaction was conducted under argon at -20°C. To a solution of glycosamine 11 (1.07 g, 1.92 mmol) in anhydrous acetonitrile (19 mL) was added allyltrimethylsilane (2.15 mL, 13.5 mmol, 7 eq). After 10 min of stirring, trimethylsilyl trifluoromethanesulfonate (0.36 mL, 1.98 mmol, 1 eq) was slowly added. The reaction mixture was left stirring overnight. The reaction was quenched with saturated NaHCO 3 (33 mL) at 0°C. Then EtOAc (200 mL) was added. The organic phase separated, then was washed with brine (2 x 70 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a light yellow oil. Flash column chromatography on silica gel (toluene/acetone 95:5, solid loading) gave an unseparable mixture of diastereoisomers 12S (anti)/ 12R (syn) in a ratio of 1:9 approximately, as a colourless oil.

TLC:

• Toluene/Acetone (9:1);

• R f = 0.33 80.81, 80.28, 79.42 (C2, C3, C4), 75.42, 75.27, 71.86 (CH 2 Ph), 66.97 (NHCOOCH 2 Ph), 61.34 (C5), 52.29 (C1), 38.21 (C6). The reaction was conducted under argon at RT. To a solution of isomers 12 (0.82 g, 1.37 mmol) in dry CH 2 Cl 2 (14 mL) were added NEt 3 (0.42 mL, 3.02mmol, 2.2 eq) and 4Å molecular sieves. Then methanesulfonyl chloride was added (0.22 mL, 2.84 mmol, 2.1 eq). The mixture was left stirring until TLC showed completion of the reaction (35 min). The solids were filtered, washed with CH 2 Cl 2 (150 mL) and the filtrate was washed with saturated NH 4 Cl (27 mL). The organic phase was separated, washed with brine (2 x 50 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a yellow oil, which was used without purification in the next reaction.

IR [cm

TLC:

• Toluene/Acetone (9:1); The reaction was conducted under argon at RT. To a solution of crude mesylates 13 (1.37 mmol) in dry THF (14 mL) was added potassium tert-butoxide (0.31 g, 2.76 mmol, 2 eq). The mixture was left stirring until TLC showed completion of the reaction (2.5h). The reaction was quenched with saturated NH 4 Cl (27 mL). EtOAc (150 mL) was added. The organic phase was separated, washed with brine (150 mL), and dried over MgSO 4 . Concentration under vacuum gave the crude product as a yellow oil. Flash column chromatography on silica gel (PE/EtOAc 9:1, solid loading) afforded the two pure desired products as colourless oils. Both isomers were found to exist as mixtures of rotamers (rot a /rot b 1:1).

• R f = 0.53 (1S)-1-C-Allyl-2,3,4-tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-L-arabinitol 14. (1R)-1-C-Allyl-2,3,4-tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-L-arabinitol 15.

IR [cm

LRMS (ESI)

[M+H] + = 578.5, [M+Na] + = 600.5, [M+K] + = 616.0 pseudo-a isomer :

TLC:

• PE/EtOAc (8:2);

• R fa = 0.67 44-127.54 (C aromatic ), 117.67, 117.44 (C8 rot-a+b ), 75.88 (C3 rot a+b ), 75.36, 75.16 (C2 rot-a+b ), 73.45 (C4 rot-a+b ), 73.36-73.11, 71.37, 71.25 (3CH 2 Ph), 67.23 (NCOOCH 2 Ph), 53.65, 52.67 (C1 rot-a+b ), 38.27, 37.77 (C5 rot-a+b ), 34.03, 33.73 (C6 rot-a+b ).

HRMS (ESI)

[M+H] + calculated for C 37 H 40 NO 53.63, 52.98 (C1), 40.03, 39.35 (C5), 29.37 (C6).

HRMS (ESI)

[M+Na] + calculated for C 37 H 39 NNaO 5 m/z= 600.2726; found m/z= 600.2712 Yield 66% (503 mg).

(1S)-2,3,4-Tri-O-benzyl-1-C-propyl-1,5-dideoxy-1,5-imino-L-arabinitol 16. The reaction was conducted at room temperature. To a solution of compound 14 (60 mg, 0.104 mmol) in isopropanol (1.0 mL) were added NEt 3 (3.5 mL, 0.024 mmol, 0.24 eq) and 10% Pd/C (15.6 mg). The mixture was left stirring under a hydrogen atmosphere until TLC showed completion of the reaction (2 h). Then it was filtered through a membrane, the retained catalyst was washed with CH 2 Cl 2 . Concentration under vacuum gave the desired product 16 as a colorless oil. 3.77 (s, 1H, H4), 3.49-3.47 (m, 2H, H2, H3), 3.13 (dd, 1H, H5 ax , J 5ax,4 = 2.9 Hz, J 5ax,5eq = 14.3 Hz), 2.48-2.44 (m, 2H, H1, H5 eq ), 2.00 (br s, 1H, NH), 1.88-1.81 (m, 1H, H6), 1.58-1.47 (m, 1H, H7), 1.36-1.23 (m, 2H, H7,H6), 0.91 (t, 3H, H8, J 8,7 = 7.1 Hz).

13 C NMR (100 MHz, CDCl 3 ), d 138. 98, 138.73, 138.71 3.81-3.80 (m, 1H, H4), 3.43 (dd, 1H, H3, J 3,4 = 2.8 Hz, J 2,3 = 9.3 Hz), 3.29 (t, 1H, H2, J 2,3 = J 2,1 = 9.3 Hz), 3.01 (dd, 1H, H5 ax , J 5ax,4 = 2.9 Hz, J 5ax,5eq = 14.4 Hz), 2.52 (br s, 1H, NH), 2.37 (dd, 1H, H5 eq , J 5eq,4 = 1.1 Hz, J 5eq,5ax = 14.4 Hz), 2.25 (dt, 1H, H1, J 1,6b = J 1,6a = 2.8 Hz, J 1,2 = 9.3 Hz), 1.74-1.66 (m, 1H, H6), 1.48-1.37 (m, 1H, H7), 1.28-1.05 (m, 2H, H7, H6), 0.84 (t, 2H, H8, J 8,7 = 7.3 Hz).

13 C NMR (100 MHz, acetone-d6), d 140. 52, 140.40, 140.20 (1R)-2,3,4-Tri-O-benzyl-1-C-propyl-1,5-dideoxy-1,5-imino-L-arabinitol 17. The reaction was conducted at room temperature. To a solution of 15 (60 mg, 0.104 mmol) in isopropanol (1.0 mL) were added NEt 3 (3.5 mL, 0.024 mmol, 0.24 eq) and 10% Pd/C (15.6 mg). The mixture was left stirring under a hydrogen atmosphere until TLC showed completion of the reaction (2 h). Then it was filtered through a membrane, the retained catalyst was washed with CH 2 Cl 2 . Concentration under vacuum gave product 17 as a light yellow oil. 75.11 (C4), 73.49 (C3), 73.13, 72.66, 71.05 (CH 2 Ph), 53.71 (C1), 44.58 (C5), 33.33 (C6), 19.71 (C7), 14.34 (C8). Compound 15 (465 mg, 0.805 mmol) was dissolved in a mixture of THF, t-BuOH, H 2 O (3 mL: 9 mL: 1.6 mL). Then N-methylmorpholine-N-oxide (113 mg, 0.965 mmol, 1.2 eq) was added. After 5 min of stirring, a 2.5% solution of OsO 4 in t-BuOH (0.82 mL, 0.062 mmol, 0.08 eq) was slowly added. The reaction was left stirring at RT for 16 h. After the addition of a 0.1 N Na 2 S 2 O 5 solution (5 mL) the reaction mixture was stirred for 30 min. CH 2 Cl 2 (20 mL) and H 2 O (10 mL) were added and the organic phase was separated. The aqueous phase was further extracted with CH 2 Cl 2 (20 mL). The combined organic phases were dried over MgSO 4 . Concentration under vacuum gave the crude product as a light yellow oil. Flash column chromatography of the mixture on silica gel (CH 2 Cl 2 /Acetone 9:1) afforded the two pure isomers 18 and 19 as colourless oils.

IR [cm

1 st isomer 18: TLC:

• CH 2 Cl 2 /Acetone (9:1)

• R f1 = 0.63 Note: The configuration of the carbinol chiral center C7 of 18 and 19 was determined in Prf. Frelek laboratory (Institute of Organic chemistry, Polish Academy of Science, Warsaw) from the ECD spectrum of the preformed complexex of these compounds with dimolybdenum tetracetate (see Results and Discussion, section I.6 for details). These results have already been published172 . It is thus established that 18 is the 7R epimer and 19 the 7S epimer.

(1R)-1-C-((2R)-2,3-Dihydroxypropyl)-1,5-dideoxy-1,5-imino-L-arabinitol 20. To a solution of isomer 18 (213 mg, 0.348 mmol) in isopropanol (3.5 mL) were added 1N aqueous HCl (0.7 mL, 0.7 mmol, 2 eq) and 10% Pd/C (52.5 mg). The reaction mixture was left stirring at RT under a hydrogen atmosphere overnight. Then it was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated once to afford a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -), the final product was obtained as a brownish solid.

1 H NMR (400 MHz, CD 3 OD), d 3. 87-3.85 (m, 1H, H3), 3.82 (dd, 1H, H4, J 4,3 = 2.9 Hz, J 4,5 = 7.9 Hz), 3.78-3.72 (m, 1H, H7), 3.66 (dd, 1H, H2, J 2,3 = 1.5 Hz, J 2,1 = 3.9 Hz), 3.50-3.43 (m, 2H, H8), 3.10-3.07 (m, 1H, H1), 2.76 (d, 2H, H5, J 5,4 = 7.9 Hz), 1.64-1.49 (m, 2H, H6). To a solution of isomer 19 (103 mg, 0.168 mmol) in isopropanol (1.7 mL) were added 1 N aqueous HCl (0.34 mL, 0.34 mmol, 2 eq) and 10% Pd/C (25.5 mg). The reaction mixture was left stirring at RT under a hydrogen atmosphere overnight. Then it was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated.

The procedure was repeated twice to afford a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -), the final product was obtained as a light yellow oil.

1 H NMR (400M Hz, CD 3 OD), d 3.89-3.86 (m, 1H, H3), 3.84 (dd, 1H, H4, J 4,3 = 2.9 Hz, J 4,5 = 7.9 Hz), 3.81-3.75 (m, 1H, H7), 3.72-3.71 (m, 1H, H2), 3.51-3.43 (m, 2H, H8), 3.15-3.12 (m, 1H, H1), 2.79 (d, 2H, H5, J 4,5 = 7.9 Hz), 1.66 (ddd, 1H, H6 b , J=3.3 Hz, J 6b-6a =5.9 Hz, J=14.2 Hz), 1.53-1.46 (m, 1H, H6 a ). 53.32 (C1), 40.00 (C5), 29.10, 20.58 (C6, C7). To a solution of compound 22 (135 mg, 0.227 mmol) in isopropanol (0.44 mL) were added 1N aqueous HCl (0.44 mL, 0.44 mmol, 2 eq) and 10% Pd/C (35 mg). The reaction mixture was left stirring at RT under a hydrogen atmosphere overnight. Then it was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated once to give a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -) the final product was obtained as a light yellow oil.

HRMS (ESI)

1 H NMR (400 MHz, CD 3 OD), d 3. 91-3.88 (m, 2H, H3, H4), 3.73-3.72 (m, 1H, H2), 3.58-3.56 (m, 2H, H8), 2.90 (t, 1H, H1, J= 6.3 Hz), 2.81 (d, 2H, H5, J 5,4 = 7.8 Hz), 1.66-1.47 (m, 4H, H7, H6).

13 C NMR (100 MHz, CD 3 OD), d 72.16 (C3/C4), 71.49 (C2), 66.53 (C3/4), 62.91 (C8), 54.43 (C1), 46.39 (C5), 30.44 (67), 28.20 (C7). The reaction was conducted under argon at RT. Crude acetate 24 (1.85 mmol) was dissolved in dry CH 2 Cl 2 (3.75 mL) containing 4Å molecular sieves. Then benzyl carbamate (560 mg, 3.7 mmol, 2 eq) and trimethylsilyl trifluoromethanesulfonate (0.34 mL, 1.85 mmol, 1 eq) were added. The reaction mixture was left stirring for 6 h. Then it was quenched by the addition of NEt 3 (0.26 mL, 1.85 mmol, 1 eq). The reaction mixture was filtered over celite and the solids were washed with CH 2 Cl 2 . The filtrate was concentrated under vacuum and the residue was coevaporated with toluene to afford the crude product as a yellow oil. Flash column chromatography on silica gel (toluene/EtOAc, 95:5) gave the desired product 25 as a mixture of anomers (1:1) and as a dense, slightly yellow oil.

HRMS (ESI)

TLC:

• toluene/Acetone (9:1);

• R f = 0.44 74.99, 74.77, 74.41, 73.47, 72.93 , 72.89, 72.62 (CH 2 Ph), 74.56 (C3/C5), 74.01 (C4a), 70.46 (C4b), 68.01 (C6), 67.06, 66.93 (NHCOOCH 2 Ph). The reaction was conducted under argon. To a solution of 25 (978 mg, 1.45 mmol) in dry acetonitrile (14.5 mL) was added allyltrimethylsilane (1.61 mL, 10.16 mmol, 7 eq). After 10 min of stirring at -20°C, trimethylsilyl trifluoromethanesulfonate (0.53 mL, 2.90 mmol, 2 eq) was slowly added. The mixture was left stirring at -25°C for 48 h. The reaction was then quenched by the addition of saturated NaHCO 3 (60 mL). After extraction with EtOAc (2 x 100 mL), the organic phase was washed with saturated NaCl (2 x 70 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude mixture of products as a light yellow oil. Flash column chromatography on silica gel (toluene/acetone 95:5) afforded a mixture of the addition products 39, 40 and starting material 25 as a colorless oil (ratio1:1). The whole procedure was repeated using the purified mixture as a starting material. Diastereoselectivity of the reaction cannot be determined at this point; however, the excess of one of the diastereoisomers is observed. The sample 26mix thus obtained contains approximately 60% of addition products 40% of residual starting material 25. This mixture was used in the next step.

IR [cm

TLC:

• toluene/Acetone (9:1);

• R f = 0.44

IR, HNRM, CNMR

Due to the fact that the product is in mixture with the starting material, only LRMS was performed to confirm the presence of the desired addition product.

LRMS (ESI)

TLC:

• toluene/acetone (9:1);

• R fR = 0.51; R fS = 0.56

LRMS (ESI)

[M+H] + = 794.5 To a solution of 27R (170 mg, 0.21 mmol) in dry THF (3 mL) was added potassium tertbutoxide (72 mg, 0.64 mmol, 3 eq). The reaction was left stirring for 24h. The reaction was quenched with saturated NH 4 Cl (5 mL). After extraction with EtOAc (25 mL), the organic phase was washed with brine (10 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a yellowish oil. Flash column chromatography on silica gel (toluene/EtOAc, 95:5) gave the pure product as a mixture of rotamers (1:1) and as a yellowish oil.

Yield

TLC:

• toluene/EtOAc (9:1);

• R f = 0.5 54.59, 53.73 (C1, C5), 34.81 (C7). To a solution of compound 28 (69 mg, 0.099 mmol) in isopropanol (1 mL), NEt 3 (3.5 μL, 0.025 mmol, 0.25 eq) and 10% Pd/C (15 mg) were added. The reaction was left stirring under a hydrogen atmosphere overnight. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated to give compound 29 as a light yellow oil.

IR [cm

TLC:

• toluene/acetone (9:1);

• R f = 0.26

HRMS (ESI)

[M+H] + calculated for C 37 H 44 NO 4 m/z= 566.32649; found m/z= 566.32670 Yield Quant. ( 56 To a solution of compound 28 (132 mg, 0.19 mmol) in isopropanol (1.9 mL), 1N HCl (0.38 mL, 0.38 mmol, 2 eq) and 10% Pd/C (38 mg) were added. The mixture was left stirring under a hydrogen atmosphere until TLC showed completion of the reaction (20h). Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated once to afford a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -), the final product was obtained as an orange, amorphous solid. The reaction was conducted under agron at RT. To a solution of compound 28 (100 mg, 0.14 mmol) in dry CH 2 Cl 2 (1 mL) were added Hoveyda-Grubs 2 nd generation catalyst (9 mg, 0.014 mmol, 0.1 eq) and 1-octene (112 mL, 0.72 mmol, 5 eq). The reaction mixture was left stirring overnight. Concentration under vacuum and purification of the crude product by flash column chromatography on silica gel (PE/EtOAc, 9:1) gave compound 31 as a colorless oil.

TLC:

• PE/EtOAc (8:2);

• R f = 0.54 54.26, 53.82 (C1,C5), 33.59 (C7), 32.67, 31.86, 29.59, 29.03, 22.75 (C10-C14), 14.25 (C15). To a solution of compound 31 (80 mg, 0.10 mmol) in isopropanol (1.0 mL), 1N HCl (100 mL, 0.10 mmol, 1 eq) and 10% Pd/C (15 mg) were added. The reaction mixture was left stirring under a hydrogen atmosphere overnight. Then it was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated once to afford a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ), the final product was obtained as a brownish, amorphous solid.

HRMS (ESI)

1 H NMR (400 MHz, CD 3 OD), d 3.89 (t, 1H, H3, J 3,2 = J 3,4 = 3.8 Hz), 3.76-3.69 (m, 3H, 2H6, H4), 3.67 (dd, 1H, H2, J 2,1 = 1.6 Hz, J 2,3 = 3.8 Hz), 2.94-2.91 (m, 1H, H1), 2.80 (dt, 1H, H5, J 5,6a = J 5,6b = 4.0 Hz, J 5,4 = 10.4 Hz), 1.58-1.20 (m, 16H, H7-H14), 0.90 (t, 3H, CH 3 , J= 6.8 Hz).

13 C NMR (100 MHz, CD 3 OD), d 73.18 (C3), 71.73 (C2), 67.61 (C4), 63.07 (C6), 57.67 (C5), 54.02 (C1), 33.06, 31.97, 30.92, 30.73, 30.70, 30.46, 27.28, 23.72 (C7-C14), 14.43 (C15).

HRMS (ESI)

[M+H] + calculated for C 15 Compound 28 (117 mg, 0.17 mmol) was dissolved in a mixture of THF, t-BuOH, H 2 O (0.44 mL: 1.34 mL: 0.24 mL). Then N-methylmorpholine-N-oxide (24 mg, 0.20 mmol, 1.2 eq) was added. After 5 min of stirring, a 2.5% solution of OsO 4 in tBuOH (0.172 mL, 0.013 mmol, 0.08 eq) was slowly added. The reaction was left stirring at RT overnight. After the addition of a 0.1 N solution of Na 2 S 2 O 5 (1 mL), the reaction mixture was stirred for 30 min. CH 2 Cl 2 (5 mL) and H 2 O (2 mL) were added. The aqueous phase was separated and further extracted with CH 2 Cl 2 (5 mL). The combined organic phases were dried over MgSO 4 . Concentration under vacuum gave the crude product mixture as a dark brown oil. Flash column chromatography on silica gel (CH 2 Cl 2 /acetone 98:2, 2% NEt 3 ) afforded the separated products 33 and 34 as colorless oils.

1 st isomer 33:

TLC:

• CH 2 Cl 2 /acetone (9:1);

• R f1 = 0.5 To a solution of 33 (52 mg, 0.07 mmol) in isopropanol (1.0 mL) were added 1N HCl (70 mL, 0.070 mmol, 1 eq) and 10% Pd/C (11 mg). The reaction mixture was left stirring under a hydrogen atmosphere for 48 h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ), the final product 35 was obtained as a brownish, thick oil. To a solution of 34 (25 mg, 0.034 mmol) in isopropanol (0.5 mL) were added 1N HCl (34 mL, 0.034 mmol, 1 eq) and 10% Pd/C (5 mg). The reaction mixture was left stirring under a hydrogen atmosphere 48h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated once to give a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ), the final product 36 was obtained as an orange/brown, thick oil.

1 H NMR (400 MHz, CD 3 OD), d 3.91 (t, 1H, H3, J 3,2 = J 3,4 = 3.7 Hz), 3.83-3.77 (m, 1H, H8), 3.75-3.72 (m, 3H, H4, 2H6), 3.69 (dd, 1H, H2, J 2,1 = 1.2 Hz, J 2,3 = 3.7 Hz), 3.47 (d, 2H, H9, J 9,8 = 5.2 Hz), 3.27-3.24 (m, 1H, H1), 2.85 (dt, 1H, H5, J 5,6a = J 5,6a = 4.0 Hz, J 5,4 = 10.3 Hz), 1.66 (ddd, 1H, H7 b , J= 3.1 Hz, J= 5.5 Hz, J 7b,7a = 14.3 Hz), 1.44 (ddd, 1H, H7 a , J= 8.2 Hz, J= 9.6 Hz, J 7a,7b = 14.3 Hz). To a solution of 26mix (1.45 mmol) in pyridine (2.0 mL) was added Ac 2 O (0.47 mL, 5.03 mmol, 5 eq). The reaction was left stirring at RT until TLC showed completion of the reaction (24 h). After addition of CH 2 Cl 2 (15 mL), the reaction mixture was washed with H 2 O (3 x 5 mL) and the organic phase was dried over MgSO 4 . After concentration under vacuum, the crude product mixture was obtained as a yellowish oil. Flash column chromatography on silica gel (toluene/EtOAc, 95:05) gave the two separated diastereoisomers 37 (R, syn, 358 mg, 33%) and 38 (S, anti, 44 mg, 4%), the mixture of both diastereoisomers (37+38, 80 mg, 7%), all three as yellowish oils, and recovered 25 (236 mg, 24%). All yields are calculated for two steps from 25.

IR [cm

TLC:

• toluene/EtOAc (9:1);

• R f = 0.31 To a solution of 38 (44 mg, 0.058 mmol) in MeOH (5 mL) sodium was added until basic pH.

The mixture was left stirring at RT until TLC showed completion of the reaction (48 h). The mixture was neutralized with Dowex 50WX8 resin (H + ). The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum, to give the crude product 40 as a colorless oil.

TLC:

• toluene/EtOAc (9:1);

• R f = 0.29

Optical rotation

= -10.9 (c= 1.0; CHCl 3 ) Yield 88% (36 mg).

(6R)-6-C-Allyl-1,3,4,5-tetra-O-benzyl-6-(N-benzyloxycarbonyl)amino-keto-l-tagatose 41 The reaction was conducted under argon at RT. To a solution of alcohol 39 (610 mg, 0.85 mmol) in dry CH 2 Cl 2 (8.5 mL) was added Dess-Martin periodinane (434 mg, 1.02 mmol, 1.2 eq). The reaction mixture was left stirring overnight. After concentration under vacuum, the crude product was suspended in a small volume of Et 2 O, the solids were removed by filtration through a membrane and the filtrate was concentrated under vacuum. Simple filtration through a layer of silica gel (toluene/EtOAc, 95:5) gave ketose 41 as a yellowish oil.

IR [cm

LRMS (ESI)

[M+H] + = 714.5, [M+Na] + = 736.5

TLC:

• toluene/EtOAc (9:1);

• R f = 0.31 75.66, 74.77, 73.30, 72.69 The reaction was conducted under argon at RT. To a solution of alcohol 40 (36 mg, 0.05 mmol) in dry CH 2 Cl 2 (0.5 mL) was added Dess-Martin periodinane (26.5 mg, 0.62 mmol, 1.2 eq). The reaction mixture was left stirring overnight. After concentration under vacuum, the crude product was suspended in a small volume of Et 2 O, filtered through a membrane and the filtrate was concentrated under vacuum. Purification by flash column chromatography on silica gel (toluene/EtOAc, 95:5) gave the desired ketose 42 as a yellowish oil.

TLC:

• toluene/EtOAc (9:1);

• R f = 0.29 74.63, 73.54, 73.32, 72.86 (CH 2 Ph, C6), 66.56 (NHCOOCH 2 Ph), 51.18 (C1), 35.54 (C7).

HRMS (ESI)

[M+H] + calculated for C 45 The reaction was conducted under argon at RT. To a solution of a mixture of ketoses 41 and 42 (30 mg, 0.042 mmol) in dry CH 2 Cl 2 (0.21 mL) was added Hoveyda-Grubbs 2 nd generation catalyst (2.6 mg, 0.0041 mmol, 0.1 eq. Then 1-octene (33 µmL, 0.21 mmol, 5 eq) was added.

The reaction was left stirring for 48 h. After concentration under vacuum, the crude product was obtained as a dark brown oil. Purification by flash column chromatography on silica gel (toluene/EtOAc 95:5) gave two separable diastereoisomers 43 and 44 as colorless oils.

LRMS (ESI)

[M+H] + = 798.5 , [M+Na] + = 820.5

Yield total 72% (24.2 mg).

1 st isomer 43 TLC:

• toluene/EtOAc (95:5);

• R f1 =0.35 75.64, 75.61, 74.80, 73.31, 72.67 (CH 2 Ph, C6), 66.86 (NHCOOCH 2 Ph), 52.31 (C1), 36.94 (C7), 32.75 (C10), 31.90, 29.58, 29.06, 22.79 (C11-C14), 14.26 (C15). The reaction was conducted under argon at RT. To a solution of pure ketose 41 (200 mg, 0.28 mmol) in dry CH 2 Cl 2 (2.8 mL) was added Hoveyda-Grubbs 2 nd generation catalyst (8.8 mg, 0.014 mmol, 0.05 eq). Then 1-pentene (0.16 mL, 1.46 mmol, 5.2 eq) was added. The reaction was left stirring overnight. Then Hoveyda-Grubbs 2 nd generation catalyst (8.8 mg, 0.014 mmol, 0.05 eq) and 1-pentene (0.1 mL, 0.91 mmol, 3.3 eq) were added again. The reaction was left stirring for 2 more days. After concentration under vacuum, the crude product was obtained as a dark brown oil. Purification by flash column chromatography on silica gel (tol/EtOAc 95:5) gave ketose 45 as a colorless oil.

HRMS (ESI)

TLC:

• toluene/EtOAc (9:1);

• R f = 0.31 75.59, 74.74, 73.24, 72.60 (CH 2 Ph, C6), 66.80 (NHCOOCH 2 Ph), 52.25 (C1), 36.88 (C7), 34.73 (C10), 22.61 (C11), 13.78 (C12).

LRMS (ESI)

[M+Na] + = 778.0 To a solution of ketose 41 (422 mg, 0.59 mmol) in EtOH (6 mL) NaBH 4 was slowly added at 0°C. The reaction mixture was left stirring at RT overnight. Then it was quenched with a 50% solution of NH 4 Cl (4 mL) in an ice bath. EtOAc (10 mL) was added, the organic phase was separated and the aqueous phase was extracted with EtOAc (3 x 15 mL). The combined organic phases were washed with H 2 O (15 mL), brine (15 mL) and dried over MgSO 4 . Concentration under vacuum gave a colorless oil which contained the D-galacto and L-altro epimers 39 and 46 in 9:1 ratio approximately. Purification by flash column chromatography on silica gel (toluene/EtOAc 95:5) afforded an analytical sample of L-altro isomer 46 which is described below. However, the separation was very difficult and the rest of the synthesis was performed on the mixture of diastereoisomers 39 and 46.

HRMS (ESI)

TLC:

• toluene/EtOAc (9:1);

• R f46 = 0.37

• R f39 = 0.32

LRMS (ESI)

[M+Na] + = 738.5 Yield 97% (412 mg) mixture of 39 and 46 (9:1 ratio).

(1R)-1-C-Allyl-2,3,4,6-tetra-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-5-Omethanesulfonyl-D-galactitol 27R and L-altritol 47. The reaction was conducted under argon at RT. To a solution of the mixture of 39 and 46 (ratio 9:1, 500 mg, 0.70 mmol) in dry CH 2 Cl 2 (7 mL), were added Et 3 N (0.21 mL, 1.54 mmol, 2.2 eq) and 4Å molecular sieves. Then methanesulfonyl chloride was added (0.11 mL, 1.47 mmol, 2.1 eq). The reaction was left stirring overnight. The solid was filtered, washed with CH 2 Cl 2 (100 mL) and the filtrate was washed with saturated NH 4 Cl (20 mL). The organic phase was separated, washed with brine (2 x 30 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a mixture of 27R and 47 and as an orange oil.

TLC:

• toluene/acetone (9:1); The reaction was conducted under argon at RT. To a solution of the mixture of 27R + 47 (0.70 mmol) in dry THF (7 mL) was added potassium tert-butoxide (160 mg, 1.40 mmol, 2 eq). The mixture was left stirring for 24h. Reaction was quenched with saturated NH 4 Cl (10 mL). EtOAc was added (50 mL). The organic phase was separated, washed with brine (20 mL), and dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a mixture of epimers 28 and 48 (5:1 ratio) as a yellowish oil. Flash column chromatography on silica gel (toluene/EtOAc, 98:2) gave pure 28 (36 mg, 7%), mixture of both isomers (17 mg, 3%) and pure 48 (163 mg, 33%) as yellowish oils. All yields are calculated for two steps from the mixture of alcohols 39 and 46.

• R f = 0.30 (

48: TLC:

• PE/EtOAc (8:2);

• R f28 = 0.58

• R f48 = 0.64 To a solution of 48 (40 mg, 0.057 mmol) in isopropanol (0.6 mL) were added 1N HCl (60 mL, 0.06 mmol, 1 eq) and 10% Pd/C (9 mg). The reaction was left stirring under a hydrogen atmosphere for 60h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ), the final product 49 was obtained as a colorless solid. To a solution of 41 (94 mg, 0.13 mmol) in isopropanol (1.9 mL) were added glacial AcOH (15 mL, 0.26 mmol, 2 eq) and 10% Pd/C (26 mg). The reaction was left stirring under a hydrogen atmosphere for 20h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. The procedure was repeated twice to afford a fully deprotected compound. After treatment with Amberlite IRA400 resin (OH -) the final product was obtained as an inseparable mixture of 50 and 51 in a 1:1 ratio and as an orange, amorphous solid. HPLC purification (column Hypercarb, 250mm (1R)-1-C-Propyl-1,5-dideoxy-1,5-imino-L-iditol 52. To a solution of 41 (185 mg, 0.26 mmol) in isopropanol (2.6 mL) were added NEt 3 (9 mL, 0.065 mmol, 0.25 eq) and 10% Pd/C (39 mg). The reaction was left stirring under a hydrogen atmosphere overnight. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. Flash column chromatography on silica gel (CH 2 Cl 2 /acetone, 95:5) afforded N-H deprotected cyclic intermediate (63 mg, 0.088 mmol) which was then submitted to further deprotection. It was dissolved in isopropanol (0.88 mL) and then were added 1N HCl (88 mL, 0.088 mmol, 1 eq) and 10% Pd/C (13 mg). The reaction was left stirring under a hydrogen atmosphere for 48h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. After treatment with Amberlite IRA400 resin (OH -) and purification on Dowex 50WX8 resin (H + ), an inseparable mixture of 49 and 52 (1:1 ratio) was obtained as a colorless oil. HPLC purification (column Hypercarb, 250mm x 10mm, H 2 O/formic acid 0.5%, pressure 2.2 bars, debit 4.4mL/min, T=30°C, ELS detection) gave an analytical sample of 52. Yield 26% (14 mg) fort wo steps, from 41 52: 1 H NMR (400 MHz, D 2 O), d 4.05 (t, 1H, H3, J 3,2 =J 3,4 = 3.0 Hz), 3.80-3.39 (m, 1H, H4), 3.77-3.64 (m, 3H, H2, 2H6), 3.08 (dt, 1H, H5, J= 2.0 Hz, J= 6.4 Hz, J= 6.4 Hz), 2.99-2.94 (ddd, 1H, H1, J 1,2 = 1.6 Hz, J 1,7 = 6.0 Hz, J 1,7 = 8.0 Hz), 1.61-1.32 (m, 4H, 2H7, 2H8), 0.94 (t, 3H, H9, J= 7.0 Hz).

13 C NMR (100 MHz, D 2 O), d 69.22, 69.16 (C2, C3), 68.34 (C4), 61.88 (C6), 55.20 (C5), 53.22 (C1), 32.64 (C7), 18.39 (C8), 13.45 (C9). The reaction was conducted under argon. To a suspension of L-sorbose (10 g, 0.056 mol) in 2,2-dimethoxypropane (30 mL) was added SnCl 2 (50 mg, 0.264 mmol, 0.005 eq) in DME (1 mL). The reaction was left stirring 1h at 70°C and then quenched with NEt 3 (0.26 mL, 1.87 mmol, 0.03 eq). The remaining sorbose was removed by filtration and the filtrate was concentrated under vacuum. The residue was dissolved in EtOAc (100 mL) and washed with H 2 O (2 x 50 mL). The organic phase was dried over MgSO 4 and concentrated under vacuum. The crude product was obtained as a yellow oil which crystallized overnight. Recrystallization from n-hexane gave pure 53 as a white solid.

HRMS (ESI)

TLC:

• toluene/EtOAc (5:5);

• R f = 0.46 28.49, 26.33, 25.42, 19.23 (CH 3 iPr).

HRMS (ESI)

[M+Na] + calculated for C To a solution of compound 53 (150 mg, 0.58 mmol) in pyridine (3 mL) was added Ac 2 O (0.27 mL, 2.88 mmol, 5 eq). The reaction was left stirring overnight at RT. CH 2 Cl 2 (40 mL) was then added. The mixture was washed with 1N aqueous HCl (2 x 20 mL), then with water (20 mL) and dried over MgSO 4 . After concentration under vacuum and coevaporation with toluene, the crude product 54 was obtained as a colorless oil, which was used in the next step without purification.

TLC:

• toluene/EtOAc (5:5);

• R f = 0.55 H 2 SO 4 on silica was prepared according to the protocol of Mukhopadhyay and co-workers. 212 To a solution of compound 54 (67 mg, 0.22 mmol) in MeOH (1.2 mL), silica-H 2 SO 4 (22 mg) was added. The reaction was left stirring 1 h at RT. The suspension was filtered through a membrane and the filtrate was concentrated under vacuum to give crude compound. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 95:5) gave pure 55 as a white solid.

TLC:

• CH 2 Cl 2 /MeOH (9:1);

• R f = 0.69

LRMS (ESI) (ESI)

[M+Na] + = 285.0 To a solution of compound 53 (100 mg, 0.38 mmol) in dry DMF (4 mL) at 0°C sodium hydride (60% dispersion in oil, 30.4 mg, 0.76 mmol, 2 eq) was added. When no H 2 evolution ceased (5 min), p-methoxybenzyl chloride (77 mL, 0.57 mmol, 1.5 eq) was added dropwise. The mixture was left stirring overnight at RT. The reaction was quenched with ice water (5 mL). EtOAc (15 mL) was added. The organic phase was washed with water (2 x 7 mL), and saturated NaHCO 3 (7 mL) and then dried over MgSO 4 . After concentration under vacuum, the crude product was purified by flash column chromatography on silica gel (PE/EtOAc 8:2) to afforda pure 56 as a yellowish oil.

HRMS

TLC:

• PE/EtOAc (7:3);

• R f = 0.48 28.02, 26.30, 26.12, 20.28 (CH 3 iPr).

LRMS (ESI)

[M+Na] + = 403.0 H 2 SO 4 on silica was prepared according to the protocol of Mukhopadhyay and co-workers. 212 To a solution of compound 56 (136 mg, 0.36 mmol) in MeOH (1.9 mL), silica-H 2 SO 4 (36 mg) was added. The mixture was left stirring 1 h at RT. The suspension was filtered through a membrane and the filtrate was concentrated under vacuum to give crude compound. Flash column chromatography on silica gel (PE/EtOAc 3:7) afforded pure 57 as a white solid.

HRMS (ESI)

TLC:

• CH 2 Cl 2 /MeOH (9:1)

• R f = 0.67 26.74, 26.31 (CH 3 iPr).

LRMS (ESI)

[M+Na] + = 363.5

HRMS

TLC:

• toluene/EtOAc (5:5);

• R f = 0.69

LRMS (ESI)

[M+Na] + = 243.0 To a solution of 2,3,4,6-diisopropylidene L-sorbofuranose (100 mg, 0.38 mmol) in dry DMF (4 mL), sodium hydride (60% dispersion in oil, 30.4 mg, 0.76 mmol, 2 eq) was added. When H 2 evolution ceased (5 min), p-methoxybenzyl chloride (77 mL, 0.57 mmol, 1.5 eq) was added dropwise. The mixture was left stirring overnight at RT. The reaction was quenched with ice water (5 mL). EtOAc (15 mL) was added. The organic phase was washed with water (2 x 7 mL) and saturated NaHCO 3 (7 mL) and then dried over MgSO 4 . After concentration under vacuum, the crude product was purified by flash column chromatography on silica gel (PE/EtOAc 8:2) to afford pure 61 as a colourless oil.

Yield

TLC:

• PE/EtOAc (7:3);

• R f = 0.50 97.30 (C2), 84.37 (C3), 73.32 (C4), 73.30 (CH 2 (OPMB)), 72.16 (C5), 69.63 (C1), 60.40 (C6), 55.31 (CH 3 O), 28.97, 27.66, 26.60, 18.69 (CH 3 iPr).

LRMS (ESI)

[M+Na] + = 403.0 H 2 SO 4 on silica was prepared according to the protocol of Mukhopadhyay and co-workers. 212 To a solution of compound 61 (127 mg, 0.33 mmol) in MeOH (1.7 mL), silica-H 2 SO 4 (33 mg) was added. The mixture was left stirring 1 h at RT.The suspention was filtered through a membrane and the filtrate was concentrated under vacuum to give crude compound. Flash column chromatography on silica gel (PE/EtOAc 3:7) afforded pure 62 as a white solid.

HRMS (ESI) [M+NH

TLC:

• PE/EtOAc (3:7)

• R f = 0.36 27.23, 26.18 (CH 3 iPr).

LRMS (ESI)

[M+Na] + = 363.5 The reaction was conducted under argon. To a suspension of L-sorbose (7 g, 0.039 mol) in 2,2-dimethoxypropane (21 mL) was added SnCl 2 (35 mg, 0.183 mmol, 0.005 eq) in DME (0.7 mL). The reaction was left stirring 3h45 at 70°C and then quenched with NEt 3 (0.21 mL, 1.51 mmol, 0.04 eq). The remaining sorbose was removed by filtration and the filtrate was concentrated under vacuum. The residue was dissolved in EtOAc (70 mL) and washed with H 2 O (2 x 350 mL). The organic phase was dried over MgSO 4 and concentrated under vacuum to afford the crude 53. The reaction of oxidation was conducted under argon. To a solution of crude 53 (0.039 mol) in dry CH 2 Cl 2 (78 mL) 3Å molecular sieves (12.9 g) were added. The suspension was left stirring for 5 min and then PCC (16.8 g, 0.078 mol, 2 eq) was added. The reaction was left stirring overnight, then Et 2 O (390 mL) and celite were added and the suspention was stirred vigorously. The solids were filtered through celite and washed with Et 2 O. The filtrate was concentrated under vacuum to give crude ketone 63 as a dense, dark brown oil, which was used in the next step without further purification. To solution of crude ketone 63 in EtOH (78 mL) and CH 2 Cl 2 (10 mL, to increase the solubility of starting material mixture) NaBH 4 (3.69 g, 0.098 mol, 2.5 eq) was slowly added at 0°C. The mixture was left stirring overnight. Then it was quenched with a 50% solution of NH 4 Cl (150 mL) at an ice bath. EtOAc (250 mL) was added, the organic phase was separated and the aqueous phase was extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with H 2 O (150 mL), brine (150 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude alcohol 64, which was purified by flash column chromatography on silica gel (PE/EtOAc 8:2) affording the alcohol 64 as a colorless oil.

HRMS

TLC:

• PE/EtOAc (6:4);

• R f = 0.29 28.65, 26.31, 26.06, 19.62 (CH 3 iPr).

HRMS (ESI)

[M+Na] + calculated for To a solution of alcohol 64 (1.64 g, 6.30 mmol) in acetone (63 mL) was added camphorsufonic acid (144 mg, 0.63 mmol, 0.1 eq). The reaction was left stirring 72 h at RT. The reaction was stopped with NEt 3 (96 µL, 0.69 mmol, 0.11 eq) and concentrated under vacuum. EtOAc (200 mL) was added. The mixture was washed with water (2 x 100 mL) and dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a colorless oil. Flash column chromatography on silica gel (PE/EtOAc 8:2) gave the desired alcohol 65 as a white solid.

TLC:

• PE/EtOAc (6:4);

• R f = 0. To a solution of alcohol 65 (1.0 g, 3.85 mmol) in dry CH 2 Cl 2 (39 mL) was added Dess-Martin periodinane (1.96 g, 4.61 mmol, 1.2 eq). The reaction was left stirring for 6 h at RT. The mixture was concentrated under vacuum. The residue was suspended in minimum volume of cold Et 2 O and filtered through a membrane. The filtrate was concentrated under vacuum to give crude aldehyde 66 as a colorless oil, which was used in the next step without purification.

1 H NMR (400 MHz, CDCl 3 ), d 9. 61 (d, 1H, H6, J 6,5 = 1.6 Hz), 5.12 (dd, 1H, H4, J 4,5 = 4.0 Hz, J 4,3 = 5.6 Hz), 4.66-4.61 (m, 1H, H3), 4.35 (dd, 1H, H5, J 5,6 = 1.6 Hz, J 5,4 = 4.0 Hz), 4.35 (d, 1H, H1 b , J 1b,1a = 10.0 Hz), 4.22 (d, 1H, H1 a , J 1a,1b = 10.0 Hz), 1.47, 1.40, 1.29 (s, 3H, CH 3

iPr).

13 C NMR (100 MHz, CDCl 3 ), d 198. 33 (C6), 113.80, 112.81, 112.39 (C IV iPr, C2), 84.77 (C3), 83.35 (C5), 81.41 (C4), 69.29 (C1), 26.51, 26.41, 26.01, 24.77 (CH 3 iPr).

6-(S S )-N-tert-butanesulfinylimino-6-deoxy-1,2:3,4-Di-O-isopropylidene-a-L-tagatofuranose 67 To a solution of crude aldehyde 66 (1.96 mmol) in dry CH 2 Cl 2 (20 mL) were added (S)-Ntert-butanesulfinamide (261 mg, 2.16 mmol, 1.1 eq) and dry CuSO 4 (1.56 g, 9.80 mmol, 5 eq). The reaction was left stirring overnight at RT. The mixture was filtered through a membrane and the filtrate was concentrated under vacuum to give crude product as a yellowish oil. Flash column chromatography on silica gel (PE/ EtOAc 8:2) gave pure 67 as a white solid.

TLC:

• PE/EtOAc (8:2);

• R fS = 0.55 (19 mL) were added (R)-Ntert-butanesulfinamide (257 mg, 2.12 mmol, 1.1 eg) and dry CuSO 4 (1.54 g, 9.63 mmol, 5 eq). The reaction was left stirring overnight at RT. The mixture was filtered through a membrane and the filtrate was concentrated under vacuum to give crude product as a yellowish oil. Flash column chromatography on silica gel (PE/ EtOAc 9:1) gave pure 68 as a colorless oil.

TLC:

• PE/EtOAc (8:2);

• R fR = 0.33 To a solution of sulfinylimine 67 (220 mg, 0.61 mmol) in dry toluene (30 mL), at 0°C a 1N solution of allylMgBr in Et 2 O (3.04 mL, 3.04 mmol, 5 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (20 mL). The mixture was extracted with Et 2 O (3 x 20 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a colorless oil with solids. Flash column chromatography on silica gel (PE/EtOAc 7:3) gave pure 69 as a single diastereoisomer and as a white solid.

TLC:

• PE/EtOAc (8:2);

• R f = 0.08 To a solution of sulfinylimine 68 (133 mg, 0.37 mmol) in dry toluene (18.5 mL) at 0°C, a 1N solution of allylMgBr in Et 2 O (1.84mL, 1.84 mmol, 5 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (15 mL). The mixture was extracted with Et 2 O (3 x 20 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a colorless oil with solids. Flash column chromatography on silica gel (PE/EtOAc 7:3) gave two diastereoisomers: the major 70 as a white solid and the minor 71 as a colorless oil.

TLC:

• PE/EtOAc (7:3);

• R f70 = 0.34, R f71 = 0.23 To a solution of sulfinylimine 67 (134 mg, 0.37 mmol) in dry toluene (18.5 mL) at -78°C, a 2N solution of hexylMgBr in Et 2 O (0.93 mL, 1.85 mmol, 5 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (10 mL). The mixture was extracted with Et 2 O (3 x 25 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a yellowish oil. Flash column chromatography on silica gel (PE/EtOAc 8:2/7:3) gave two diastereoisomers: the first 72R as a white amorphous solid and the second 72S as a colorless oil.

Melting point T m =95-98°C 6

TLC:

• PE/EtOAc (8:2); To a solution of sulfinylimine 68 (100 mg, 0.28 mmol) in dry toluene (3 mL) at -78°C, a 2N solution of hexylMgBr in Et 2 O (0.7 mL, 1.4 mmol, 5 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (10 mL). The mixture was extracted with Et 2 O (3 x 20 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a yellowish oil. Flash column chromatography on silica gel (PE/EtOAc 8:2) gave two diastereoisomers: the first one 73S as a white solid and the second one 73R as a colorless oil.

• R f R = 0.

TLC:

• PE/EtOAc (7:3);

• R fR = 0.38, R fS = 0.25, 1 st isomer 73S: To a solution of sulfinylimine 67 (138 mg, 0.38 mmol) in dry toluene (19 mL) at 0°C, a 1N solution of nonylMgBr in Et 2 O (1.91 mL, 1.91 mmol, 5 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (15 mL). The mixture was extracted with Et 2 O (3 x 20 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a colorless oil with solids. Flash column chromatography on silica gel (PE/EtOAc 8:2) gave two diastereoisomers: the first 74R as a white solid and the second 74S as a colorless oil.

TLC:

• PE/EtOAc (8:2);

• R fR = 0.18,

• R fS = 0.06 30.78, 29.77, 29.56, 29.49, 29.46, 25.46 (CH 2 nonyl), 26.71, 26.46, 26.22, 25.12 (CH 3 iPr), 22.84 (CH 3 tBu), 14.26 (CH 3 nonyl).

IR [cm

LRMS (ESI)

[M+H] + = 490. To a solution of sulfinylimine 68 (171 mg, 0.47 mmol) in dry toluene (20 mL) at 0°C, a 1N solution of nonylMgBr in Et 2 O (2.40 mL, 2.40 mmol, 5.1 eq) was added dropwise. The reaction was left stirring overnight at RT. Then it was stopped at 0°C with a saturated solution of NH 4 Cl (15 mL). The mixture was extracted with Et 2 O (3 x 20 mL). The combined organic phases were dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a liquid, colorless oil with solids. Flash column chromatography on silica gel (PE/EtOAc 8:2) gave two diastereoisomers 75S and 75R as colorless oils and a byproduct 76 as a white solid.

TLC:

• PE/EtOAc (8:2);

• R fS = 0.26,

• R fR = 0.13 1 st isomer 75S:

LRMS (ESI)

[M+H] + = 364. A solution of acetyl chloride (100 mL, 1.40 mmol, 5 eq) in dry MeOH (4.3 mL) was left stirring for 30 min. Then it was added to 70 (113 mg, 0.28 mmol) and the suspension was left stirring for 20 min. The reaction was stopped with amberlite IRA400 resin (OH -) until basic pH. The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum to give crude 77 as a yellowish oil, which was used in the next step without purification.

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.59. 26.81, 26.78, 26.27, 24.86 (CH 3 iPr).

LRMS (ESI)

[M+H] + = 300.0 * Note: This configuration was determined on the basis of the NMR spectra comparison with hexyl analogues 79 and 80, and may be inverted.

(6S)-6-Amino-6-deoxy-6-C-Hexyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 79 A solution of acetyl chloride (116 mL, 1.62 mmol, 5 eq) in dry MeOH (4.9 mL) was left stirring for 30 min. Then it was added to 72S (145 mg, 0.32 mmol) and the suspension was left stirring for 20 min. The reaction was stopped with amberlite IRA400 resin (OH -) until basic pH. The resin was filtered, washed with MeOH and the filtrate wasconcentrated under vacuum to give crude 79 as an orange oil, which was used in the next step without purification.

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.46. 32.65, 30.86, 30.21, 26.50, 23.59 (CH 2 -hexyl), 26.88, 26.59, 25.95, 24.20 (CH 3 iPr), 14.37 (CH 3 -hexyl).

HRMS (ESI)

[M+H] + calculated for C 18 H 34 NO 5 m/z= 344.243150; found m/z= 344.243334 (6R)-6-Amino-6-deoxy-6-C-Hexyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 80 A solution of acetyl chloride (114 mL, 1.60 mmol, 5 eq) in dry MeOH (5.3 mL) was left stirring for 30 min. Then it was added to 73R (143 mg, 0.32 mmol) and the suspension was left stirring for 20 min. The reaction was stopped with amberlite IRA400 resin (OH -) until basic pH. The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum to give crude 80 as a brownish oil, which was used in the next step without purification.

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.45. 32.53, 30.80, 30.01, 25.85, 23.57 (CH 2 -hexyl), 26.88, 26.58, 26.14, 24.61 (CH 3 iPr), 14.37 (CH 3 -hexyl).

HRMS (ESI)

[M+H] + calculated for C 18 H 34 NO 5 m/z= 344.243150; found m/z= 344.243475 (6S)-6-Amino-6-deoxy-6-C-Nonyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 81 A solution of acetyl chloride (82 mL, 1.15 mmol, 5 eq) in dry MeOH (3.5 mL) was left stirring for 30 min. Then it was added to 74S (113 mg, 0.23 mmol) and the suspension was left stirring for 20 min. The reaction was stopped with amberlite IRA400 resin (OH -) until basic pH. The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum to give crude 81 as a brown solid, which was used in the next step without purification.

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.54. 34.43, 33.05, 30.92, 30.68, 30.65, 30.45, 26.36, 23.74 (CH 2 -nonyl), 26.85, 26.83, 26.30, 24.87 (CH 3 iPr), 14.49 (CH 3 -nonyl). A solution of acetyl chloride (50mL, 0.69 mmol, 5 eq) in dry MeOH (2.1 mL) was left stirring for 30 min. Then it was added to 75R (68 mg, 0.14 mmol), and the suspension was left stirring for 20 min. The reaction was stopped with amberlite IRA400 resin (OH -) until basic pH. The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum to give crude 82 as a yellow solid, which was used in the next step without purification.

HRMS (ESI)

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.51. 33.04, 31.08, 30.61, 30.42, 30.40, 30.36, 26.17, 23.72 (CH 2 -nonyl), 26.91, 26.60, 25.96, 24.64 (CH 3 iPr), 14.44 (CH 3 -nonyl). A stock solution of TfN 3 in toluene (around 0.5 M) was prepared according to Ernst protocol. 225 To crude 79 (0.32 mmol), NaHCO 3 (107 mg, 1.28 mmol, 4 eq), CuSO 4 ·5H 2 O (3.2 mg, 0.013 mmol, 0.04 eq ) and 0.5 mL of water were added. Then MeOH (2.7 mL) and stock solution of TfN 3 (505mL) were added. The reaction was left stirring overnight. After concentration under vacuum, the crude product was obtained as a brown oil with solids. Flash column chromatography on silica gel (PE/EtOAc 9:1) gave pure 83 as a colorless oil.

HRMS (ESI)

TLC:

• PE/EtOAc (9:1);

• R f = 0.59. A stock solution of TfN 3 in toluene (around 0.5 M) was prepared according to Ernst protocol. 225 To crude 80 (0.32 mmol) NaHCO 3 (108 mg, 1.28 mmol, 4 eq), CuSO 4 ·5H 2 O (3.2 mg, 0.0128 mmol, 0.04 eq) and 0.5 mL of water were added. Then MeOH (2.7 mL) and a stock solution of TfN 3 (770 mL) were added. The reaction was left stirring overnight. After concentration under vacuum, the crude product was obtained as a green oil. Flash column chromatography on silica gel (PE/EtOAc 9:1) gave pure 84 as a colorless oil.

TLC:

• PE/EtOAc (9:1);

• R f = 0.50. A stock solution of TfN 3 in toluene (around 0.5 M) was prepared according to Ernst protocol. 225 To crude 81 (0.23 mmol) NaHCO 3 (77 mg, 0.92 mmol, 4 eq), CuSO 4 ·5H 2 O (2.3 mg, 0.0092 mmol, 0.04 eq ) and 0.5 mL of water were added. Then MeOH (1.9 mL) and A stock solution of TfN 3 (533 mL) were added. The reaction was left stirring overnight. After concentration under vacuum the crude product was obtained as a brownish oil with solids. Flash column chromatography on silica gel (PE/EtOAc 9:1) gave pure 85 as a colorless oil.

TLC:

• PE/EtOAc (9:1);

• R f = 0.68. 32.02, 31.87, 29.70, 29.65, 29.62, 29.43, 25.59, 22.82 (CH 2 -nonyl), 26.64, 26.54, 26.17, 25.04 (CH 3 iPr), 14.25 (CH 3 -nonyl).

HRMS (ESI)

[M+Na] + calculated for A stock solution of TfN 3 in toluene (around 0.5 M) was prepared according to Ernst protocol. 225 To crude 82 (0.14 mmol) NaHCO 3 (47 mg, 0.56 mmol, 4 eq), CuSO 4 ·5H 2 O (1.4 mg, 0.0056 mmol, 0.04 eq ) and 0.5 mL of water were added. Then MeOH (1.1 mL) and a stock solution of TfN 3 (337 mL) were added. The reaction was left stirring overnight. After concentration under vacuum, the crude product was obtained as a brownish oil with solids. Flash column chromatography on silica gel (PE/EtOAc 9:1) gave pure 86 as a white solid.

TLC:

• PE/EtOAc (9:1);

• R f = 0.59. 32.03, 30.42, 29.68, 29.57, 29.46, 29.37, 25.90, 22.82, (CH 2 -nonyl), 26.78, 26.18, 25.09 (CH 3 iPr), 14.25 (CH 3 -nonyl). To a solution of the azide 84 (100 mg, 0.27 mmol) in a mixture of acetonitrile/water (1:1) (11 mL) resin Dowex 50WX8 (H + ) (1.94 mg) was added. The reaction was left stirring at 60°C for 4 days. The resin was filtered, washed with acetonitrile/water (1:1) and the filtrate was concentrated under vacuum to give crude product as a brownish solid. Flash column chromatography on silica gel (MeOH/CH 2 Cl 2 9:1) gave pure 88 as colorless oil and a mixture of anomers (7:3).

HRMS (ESI)

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.22. 32.85, 31.50, 31.26, 30.08, 27.10, 27.08, 23.63, (CH 2hexyl), 14.39 (CH 3 -hexyl).

HRMS (ESI)

[M+Na] + calculated for To a solution of 87 (24 mg, 0.08 mmol) in anhydrous MeOH (1.0 mL) was added 10% Pd/C (12mg). The reaction was left stirring under a hydrogen atmosphere for 3 days. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. Purification on Dowex 50WX8 resin (H + ) afforded the final product as a mixture of the desired compound 89 and the N-methyl by-product 90 in a 8:2 ratio and as a yellowish oil. H2 and H3 are under the CD 3 OD signal, but they can be seen on the correlation spectra. The quality of the 1 H NMR spectrum in DMSO-d6 was not good enough to be described. To a solution of 87 (30 mg, 0.10 mmol) in anhydrous MeOH (7.0 mL) was added 10% Pd/C (16 mg). The reaction was left stirring under a 10 bar hydrogen pressure for 4h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. Purification on Dowex 50WX8 resin (H + ) afforded the final product as a yellowish oil. The desired D-galacto compound 89 was obtained as a mixture with around 6% of D-gluco epimer 91. 32.92, 30.66, 26.78, 23.69 (CH 2 -hexyl gluco ), 32.98, 32.95, 30.75, 26.72, 23.69 (CH 2 -hexyl), 14.42 (CH 3 -hexyl gluco ), 14.44 (CH 3 -hexyl).

HRMS (ESI)

[M+H] + calculated for To a solution of 88 (66 mg, 0.23 mmol) in anhydrous MeOH (7.0 mL) was added 10% Pd/C (34mg). The reaction was left stirring under a 10 bar hydrogen pressure for 4h. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated. Purification on Dowex 50WX8 resin (H + ) afforded the final product as a brownish solid. The desired D-galacto compound was obtained in a mixture with around 20% of another epimer.

1 H NMR (400 MHz, CD 3 OD), d 3.90 (t, 01H, H3, J 3,4 = 3.2 Hz), 3.77-3.69 (m, 3H, 2H6, H4), 3.67 (dd, 1H, H2, J 2,1 = 1.6 Hz, J 2,3 = 4.0 Hz), 2. 96-2.87 (1H, H1), 2.81 (dt, 1H, H5, J= 3.6 Hz, J= 3.6 Hz, J= 10 Hz), 1.65-1.24 (m, 10H, CH 2 -hexyl), 0.907 (t, 3H, CH 3 -hexyl, J= 6.6 Hz).

13 C NMR (100 MHz, CD 3 OD), d 73.12 (C3), 71.66 (C2), 67.56 (C4), 63.00 (C6), 57.65 (C5), 54.03 (C1), 32.97, 31.91, 30.58, 27.21, 23.67 (CH 2 -hexyl), 14.43 (CH 3 -hexyl). To a solution of alcohol 65 (158 mg, 0.607 mmol) in dry CH 2 Cl 2 (6 mL) were added NEt 3 (186 mL, 1.34 mmol, 2.2 eq) and 4Å molecular sieves. Then methanesulfonyl chloride (99 mL, 1.28 mmol, 2.1 eq) was added. The reaction was left stirring 1 h at RT. Then it was stopped with a saturated solution of NH 4 Cl (5 mL). The solid was filtered through a cotton and filtrate was extracted with CH 2 Cl 2 (25 mL). The organic phase was washed with brine (7 mL) and dried over MgSO 4 . After concentration under vacuum the crude 94 was obtained as a yellow solid, which was used in the next step without further purification.

HRMS (ESI)

TLC:

• PE/EtOAc (6:4);

• R f = 0.64 13 C NMR (100 MHz, CDCl3), d 113. 25, 112.11, 112.09 (C IV iPr, C2), 85.07 (C3), 79.72 (C4), 76.81 (C5), 69.28 (C1), 68.09 (C6), 37.65 (CH 3 Ms), 26.48, 26.44, 26.03, 24.79 (CH 3 iPr).

LRMS (ESI)

[M+Na] + = 361.0 1,2:3,4-di-O-Isopropylidene-6-O-trifluoromethanesulfonic-a-L-tagatofuranose 95 To a solution of alcohol 65 (190 mg, 0.73 mmol) in dry CH 2 Cl 2 (2.2 mL) was added pyridine (195 mL, 2.41 mmol, 3.3 eq). Then, at -78°C was added trifluoromethanesulfonic anhydride (164 µL, 0.99 mmol, 1.35 mmol). The reaction was left stirring 2 h at ice-water bath temperature. The mixture was extracted with EtOAc (10 mL). The organic phase was washed with brine (5 mL), ice-water (5 mL) and dried over MgSO 4 . After concentration under vacuum the crude 95 was obtained as a yellow oil, which was used in the next step without further purification.

TLC:

• PE/EtOAc To a stirred solution of 101 (415 mg, 1.48 mmol) in CH 2 Cl 2 (8 mL) was added pyridine (0.4 mL, 4.88 mmol, 3.3 eq). Then, at -78°C was dropwise added trifluoromethanesulfonic anhydride (0.33 mL, 2.0 mmol, 1.35 eq). The mixture was left stirring for 2h at 0°C. EtOAc was added (25 mL) and the mixture was washed with brine (7 mL) and ice-water (7 mL) and then dried over MgSO 4 . After concentration under vacuum, the crude triflate was obtained as a yellow oil. The mixture of crude triflate , KCN (482 mg, 7.4 mmol, 5 eq) and 18-crown-6 ether (195 mg, 0.74 mmol, 0.5 eq) were stirred in DMF (30 mL) in a presence of 4Å molecular sieves for 16h at RT. EtOAc was added (20 mL) and the mixture was washed with brine (15 mL) and water (15 mL) and then dried over MgSO 4 . After concentration under vacuum, the crude mixture was obtained as a brown oil. Flash column chromatography on silica gel (toluene/EtOAc 95:5) afforded elimination by-product 102A (200 mg, 52%).

TLC:

• toluene/EtOAc (9:1);

• R f = 0.9 The reaction was conducted under argon. To a solution of alcohol 99 (200 mg, 0.71 mmol) in dry CH 2 Cl 2 (7.1 mL) were added Et 3 N (0.22 mL, 1.57 mmol, 2.2 eq) and 4Å molecular sieves. Then methanesulfonyl chloride (0.12 mL, 1.50 mmol, 2.1 eq) was added. The reaction was left stirring for 4h. The solid was filtered and washed with CH 2 Cl 2 (10 mL). The filtrate was washed with a saturated aqueous NH 4 Cl solution (10 mL). The organic phase was separated, washed with brine (2 x 5 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product 104, which was used in the next step without further purification.

TLC:

• PE/EtOAc (7:3);

• R f = 0.45 The reaction was conducted under argon. To a solution of compound 99 (5.33 g, 0.019 mol) in dry pyridine (20 mL) was added p-toluenesulfonyl chloride (5g, 0.026 mol, 1.38 eq). The reaction was left stirring for 40h. The mixture was diluted with Et 2 O (25 mL) and was washed with 3N aqueous HCl (2 x 25 mL), water (2 x 25 mL) and brine (25 mL). Then the organic phase was dried over MgSO 4 and the solvents were evaporated under vacuum. Flash column chromatography on silica gel (PE/EtOAc 9:1) afforded the pure 105 as a white solid.

TLC:

• PE/EtOAc (8:2);

• R f = 0.43 The reaction was conducted under argon. To a solution of nitromethane (34 mL, 0.63 mmol, 1 eq) in acetonitrile (2.1 mL) was added dropwise DBU (10 mL, 0.067 mmol, 0.1 eq). After 5 min of stirring, this solution was added to ketone 100 (176 mg, 0.63 mmol). The mixture was left stirring overnight. The reaction was quenched with a saturated solution of NH 4 Cl (1 mL). The aqueous phase was separated and extracted with EtOAc (5 mL). The organic phase was washed with brine (2 mL) and dried over Na 2 SO 3 . Concentration under vacuum gave the crude product as a yellow oil. Flash column chromatography on silica gel (PE/EtOAc, 9:1) afforded an inseparable mixture of the 112 and 113 (1:1 ratio) as a colorless oil.

LRMS (ESI)

TLC:

• PE/EtOAc (8:2); 69.53, 68.47 (C4), 65.75 (C5), 62.50 (C5), 26.95, 26.31, 25.49, 25.36 (CH 3 iPr). To a solution of mixture of compounds 112+113 (200 mg, 0.59 mmol) in isopropanol (6 mL) were added glacial acetic acid (0.59 mL, 10.3 mmol, 17 eq) and 10% Pd/C (88 mg). The mixture was left stirring under a hydrogen atmosphere for 2 days. Then it was filtered through a membrane, retained catalyst was washed with methanol and the solvents were evaporated.

IR [cm

After treatment with Amberlite IRA400 resin (OH -) the crude product was obtained as a yellowish oil. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 7:3) gave the two separated diastereoisomers 115 and 116 as colorless oils.

TLC:

• CH 2 Cl 2 /MeOH (6:4);

• R f altro = 0.31, R f galacto = 0.14 To a solution of 116 (28 mg, 0.14 mmol) in a mixture of dioxane/H 2 O (1:1, 1.4 mL) was added Dowex 50WX8 resin (H + ) (190 mg). The mixture was left stirring gently for 1 h. The resin was filtered and washed with MeOH. Then a 0.5N aqueous solution of NH 3 was added to the resin and the mixture was left gently stirring for 30 min. The resin was filtered and washed with a 0.5N aqueous solution of NH 3 . The filtrate was concentrated to give the crude product as a white solid. Purification on Dowex 50WX8 resin (H + ) gave the pure 118 as a white solid. The reaction was conducted under argon. To a solution of crude ketone 100 (1.57 mmol) in nitrohexane (5.3 mL) was added triethylamine (0.61 mL, 4.38 mmol, 2.8eq). The mixture was left stirring for 24h. The reaction was quenched with a saturated solution of NH 4 Cl (15 mL).

The aqueous phase was separated and extracted with EtOAc (40 mL). The organic phase was washed with brine (15 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a liquid oil. Flash column chromatography on silica gel (PE/EtOAc, 92:8) gave an inseparable mixture of the two diastereoisomers (7:3 ratio) 119 and 120 as a colorless oil.

TLC:

• PE/EtOAc (8:2);

• R f = 0.56 31.12, 28.02, 25.99, 22.41 (CH 2 -hexyl MAJ ), 31.17, 27.23, 25.93, 22.45 (CH 2 -hexyl min ), 26.17, 25.36 (CH 3 iPr MAJ ), 26.13, 25.36 (CH 3 iPr min ), 14.00 (CH 3 hexyl min ), 13.98 (CH 3 hexyl MAJ ). To a solution of the mixture of compounds 119+120 (289 mg, 0.71 mmol) in isopropanol (7.1 mL) were added glacial acetic acid (0.71 mL) and Raney Ni (106 mg). The mixture was left stirring under a hydrogen atmosphere for 5 days. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated to give the crude product as a thick, green oil. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 9:1) gave an inseparable mixture of diastereoisomers 121+122 as a slightly green oil.

HRMS (ESI)

TLC:

• CH 2 Cl 2 /MeOH (9:1);

• R f = 0.44 32.95, 30.66, 27.24, 23.60 (3R, 4R, 5S, 6R or S)- 5-Hydroxymethyl-2,3-O-isopropylidene-6-C-pentyl-piperidine-3,4,5-triol 123 To a solution of the mixture of compounds 121 and 122 (26 mg, 0.07 mmol) in isopropanol (1 mL) were added glacial acetic acid (0.1 mL) and 20% Pd(OH) 2 (11 mg). The mixture was left stirring under a hydrogen atmosphere for 2 days. It was not completed. Then it was filtered through a membrane, retained catalyst was washed with MeOH and the solvents were evaporated to give the crude product as a yellowish oil. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 9:1) gave desired 123 almost exclusively as a single diastereoisomer and as a colorless oil.

TLC:

• CH 2 Cl 2 /MeOH (9:1);

• R f = 0.15 33.18, 29.65, 27.48, 23.61 (CH 2 -pentyl), 28.09, 25.80 (CH 3 iPr), 14.40 (CH 3 -pentyl).

LRMS (ESI)

[M+H] + = 274.0; Yield 38% (6.8 mg)

Anna BIELA

Synthèse de nouveaux dérivés d'iminogalactitol et d'épiisofagomine comme chaperons pharmacologiques potentiels pour la maladie de Krabbe

Résumé : L'objectif de mon projet de thèse était de synthétiser différentes familles d'iminosucres comme chaperons pharmacologiques potentiels de la b-galactocérébrosidase (GALC). Cette glycosidase lysosomale est responsable de la maladie de Krabbe, une maladie rare et héréditaire qui ne dispose actuellement pas de traitement. La thérapie chaperon est une nouvelle stratégie consistant à administrer, à des concentrations très faibles, une petite molécule ayant des interactions fortes avec l'enzyme. La plupart du temps, les molécules à effet chaperon les plus efficaces sont de puissants inhibiteurs de l'enzyme et les iminosucres sont connus pour inhiber fortement les glycosidases. La conception de ces iminosucres a été dictée par la structure de l'état de transition de la réaction catalysée par la GALC et l'expérience de notre groupe dans la synthèse d'iminosucres en tant que chaperons pharmacologiques pour la maladie de Gaucher.

Dans un premier temps, la synthèse d'imino-L-arabinitols a été réalisée en utilisant une méthodologie d'élongation de chaîne en C1. Puis la même stratégie a été appliquée à la synthèse d'imino-Dgalactitols C-alkylés. Des difficultés rencontrées avec cette méthodologie nous ont alors conduits à développer une autre voie d'accès à ces composés, via un allongement de chaîne en C6. Enfin une dernière famille de composés de type galacto-isofagomine a été préparée. Ces nouveaux composés ont été testés comme inhibiteurs de deux galactosidases lysosomales, afin d'évaluer leur sélectivité et leur potentiel en tant que chaperons pharmacologiques pour la maladie de Krabbe.

Mots clés : iminosucres, b-galactocérébrosidase, maladie de Krabbe

Synthesis of novel iminogalactitol and epi-isofagomine derivatives as potential pharmacological chaperones for Krabbe disease

Abstract: The objective of my PhD was to synthesize different families of iminosugars as potential pharmacological chaperones of b-galactocerebrosidase (GALC). This lysosomal glycosidase is responsible for Krabbe disease, a rare inherited disease that currently has no treatment. Chaperone therapy is a new strategy that consists in the administration, at very low concentrations, of small molecules having strong interactions with the enzyme. Most of the time, the most effective chaperones are potent inhibitors of the enzyme and iminosugars are known to strongly inhibit glycosidases. The design of these iminosugars was dictated by the transition state structure of the reaction catalyzed by GALC and the experience of our group in the synthesis of iminosugars as pharmacological chaperones for Gaucher disease.

As a first stage, the synthesis of imino-L-arabinitols was conducted using a C1 chain extension methodology. Then the same strategy was applied to the synthesis of 1-C-alkyl-imino-D-galactitols.

The difficulties encountered with this methodology led us then to develop another approach to these compounds, by way of a C6 chain extension. Finally a last family of galacto-isofagomine derivatives was prepared. These new compounds have been evaluated as inhibitors of two lysosomal galactosidases, to assess their selectivity and their potential as pharmacological chaperones for Krabbe disease.

Figure 1 .

 1 Figure 1. Specific functions of lysosome. 6 I.2 Synthesis and transport of lysosomal proteins.

Figure 2 .

 2 Figure 2. Transport from the Trans Golgi Network to lysosomes. 19

Figure 3 .

 3 Figure 3. Different basis of LSDs 5

40

 40

Figure 4 .

 4 Figure 4. LSDs therapy strategies. 1-stem cell therapy, 2-gene therapy, 3-ERT, 4-SRT, 5-PCT. 40General treatment includes bone-marrow transplantation41 (BMT) or splenectomy (partial or complete surgical removal of the spleen) applied in Gaucher disease42 . The latter was recommended earlier only in life-saving cases and is no longer used. More important therapeutic approaches are those which can act on accumulating substrate (substrate reduction therapy SRT), on deficient enzyme (enzyme replacement therapy ERT and the novel pharmacological chaperone therapy PCT) or which use directly gene therapy.

Figure 5 .

 5 Figure 5. The concept of cross-correlation in HSCT.

Figure 6 .

 6 Figure 6. Estimated frequencies of HSCTs for various inborn errors in metabolism since 1980. * Other leukodystrophies/white-matter diseases. 50

Figure 7 .

 7 Figure 7. In vivo (direct) and ex vivo (cell-based) gene therapy. Extracted from http://stemcells.nih.gov/info/2001report/pages/chapter11.aspx II.4 Enzyme replacement therapy Pompe disease was discovered in 1963 by Hers, 62 which one year later brought in the

40 Figure 8 .

 408 Figure 8. Macrophage uptake of intravenously administered recombinant GCase.67

Figure 10 .

 10 Figure 10. Principles of pharmacological chaperone therapy. 101

(

  IVS10del30kb) and three others mutations (1538C>T, 1652A>T, 1424delA) together account for about 60% of alleles in European patients (infantile form) and are absent in their Japanese counterparts.127 By contrast, ~30% of Japanese GALC has either 635-646del12insCTC or 1954A>C mutation.128 By preventing a glycosylation, promoting structure destabilization or interfering with binding to activation factors, a large portion of the missense mutations in GALC is likely to result in protein mistargeting or premature degradation. 129 However, some of those mutations 130 have been shown to retain enzyme residual activity. In 2011, Deane and coworkers 131 have resolved the structure of the mouse GALC (83% homology with human GALC) alone and in complex with D-galactose. The protein is composed of 668 residues forming three domains: a triosephosphate isomerase (TIM) barrel, a b-sandwich and a lectin domain (Figure13).

Figure 13 .Scheme 2 .

 132 Figure 13. X-ray structure of mouse GALC 131According to their studies, the active site of the enzyme accommodates only the galactose moiety without any space for the lipid tails of sphingolipids. Compared to other lysosomal enzymes, GALC is the only one so far that possesses a lectin domain, which contributes to the formation of the enzyme-substrate cleft. Moreover, each of the three domains is involved in the substrate binding site.131 Residual GALC activity in Krabbe patients ranges from 0-22% of normal values, with leukocyte enzyme level significantly lower than in healthy controls.118 Interestingly, impaired activity of GALC does not result in accumulation of the primary substrate of the enzyme: galactosylceramide in the CNS, because it is also degraded by G M1 b-galactosidase (ganglioside b-galactosidase). 132 This is why the excess storage of psychosine is predominantly observed.133

Figure 14 .

 14 Figure 14. Structure of a-lobeline.

Figure 15 .

 15 Figure 15. a-Galactosidase inhibitors and PCs Recently, Kato et al. 147 isolated another series of iminosugars, among which the most interesting was 2,5-dideoxy-2,5-imino-D-altritol that binds to a-Gal A with an IC 50 of ~700nM and increases enzyme activity 2-to 9-fold at 50-500µM. However, this interesting work was not continued in further PC application.

(Scheme 3 .Scheme 4 .

 34 Scheme 3. Reaction of hydrolysis promoted by GALC.

Scheme 5 .

 5 Scheme 5. GALC transition state and possible structures of inhibitors.

Figure 19 .

 19 Figure 19. Target and achieved structures of potential GALC inhibitors.

(Scheme 6 .Scheme 7 .

 67 Scheme 6. Synthetic strategy towards 1-C-alkyl-imino-L-arabinitols by C-1 chain extensionThis approach, originally developed by Nicotra, 173,174 consists of four major steps:

Scheme 8 .

 8 Scheme 8. Synthesis of 2,3,4-tri-O-benzyl-a,b-L-arabinopyranose 3.

Scheme 11 .Scheme 12 .

 1112 Scheme 11. Mechanism of the reaction of nucleophilic addition mediated by Lewis acid proposed by Kobayashi.The stereochemistry of this reaction is influenced by the asymmetric environment in a of the electrophilic carbon. We can assume that the formation of a hydrogen bond between oxygen in position 2 and hydrogen of the iminium (Scheme 12) defines the conformation of iminium intermediate. In this case, the Si face is crowded and the nucleophilic attack comes from the Re face resulting in syn product.

  1 C 4 chair conformation with three axial substituents (Scheme 13) and the major, pseudo b (R) isomer 15 would be in 4 C 1 chair conformation. Thus both 14 and 15 have their C-alkyl substituent axial. To confirm this assigment, epimers 14 and 15 were selectively N-deprotected by hydrogenolysis under basic conditions in order to avoid O-debenzylation, to obtain 16 and 17

  Hz) indicated 1 C 4 conformation. Both compounds are formed in the more stable conformation which has the propyl group equatorial, unlike 14 and 15. This means that piperidines 14 and 15 inverted their conformations upon N-protecting group cleavage (from 1 C 4 of 14 to 4 C 1 of 16 and from 4 C 1 of 15 to 1 C 4 of 17). The difference between the conformations of the corresponding isomers in the N-Bn and N-Z protected series is thus due to the presence of the carbonyl group in Z group, which forces the molecule to adopt specific orientation of the substituents a to nitrogen.

Scheme 13 . 8 , 9 , 19 , 20 and 22 .

 13891922 Scheme 13. Intramolecular S N 2-type cyclisation. I.6 Functionalization of double bond and deprotection: preparation of iminosugars 8, 9, 19, 20 and 22.As mentioned before, the N-Bn protected imino-L-arabinitols are not compatible with oxidation reactions of the double bond: the non-deactivated nitrogen atom of the amine could be itself oxidized. The N-Bn glycosylamines were designed to undergo reactions with various Grignard reagents, which, except with the allyl reagent, failed. Compounds 6 and 7 were fully deprotected to prepare pseudo a-(1S)-and pseudo b-(1R)-1-C-propyl-1,5-dideoxy-1,5-imino-L-arabinitols 8 and 9 respectively (Scheme 14). We predicted that the first compound(8)

Scheme 14 .

 14 Scheme 14. Deprotection of iminosugars 6 and 7.

Figure 20 .

 20 Figure 20. Reagents used for hydroboration/oxidation reaction.

Figure 21 .Figure 22 .

 2122 Figure 21. ECD spectra of in situ formed Mo-complexes of 18 (red line) and 19 (blue line) recorded in DMSO. The preferred conformation of the diols in the chiral Mo-complexes is shown on the right. As can be seen in Figure 21, a positive helicity of the diol 18 correlates with positive CEs at 310 and 380 nm whilst the inverse correlation of helicity of the diol subunit and sign of CEs takes place for diol 19. Thus, provided the relative configuration of vic-diol after ligation to the Mo 2 -core to be gauche with preferred antiperiplanar orientation of the hydroxyl group versus bulky substituent, the absolute configuration of the newly formed stereogenic center in 18 is (R) and in 19 (S). Finally, all the above compounds were deprotected by hydrogenolysis under acidic conditions to afford the L-arabino functionalized iminosugars 20, 21 and 23. The presence of the acid in those reactions was necessary to avoid the poisoning effect of the formed free amine on the catalyst. 186

Scheme 16 .Scheme 17 .

 1617 Scheme 16. Synthetic strategy towards 1-C-alkyl-imino-D-galactitols.The final step of the synthesis could not be achieved by intramolecular S N 2 for the following reason. In the galacto open-chain intermediate 26, position C-5 is a secondary alcohol, which under S N 2 conditions will undergo a Walden inversion.187 As a result, by using the substitution approach, we would obtain the L-altro iminosugars instead of the D-galacto isomers (Scheme 17). This was actually also done and will be discussed later in this report.

Scheme 21 .

 21 Scheme 21. Mechanism of reductive amination.

Scheme 22 .Scheme 23 .

 2223 Scheme 22.Processes taking place during reductive amination.

Scheme 24 . 50 Figure 23 .

 245023 Scheme 24. Reductive amination assays.Then we attempted hydrogenation under acidic conditions (Scheme 24). Surprisingly, these conditions were too vigorous, leading unexpectedly to 3-deoxy products. The reaction gave a mixture of 1-C-propyl iminohexitols (1:1 ratio), deoxy at position 3 and having a pseudo-L-ido or pseudo-L-altro configuration. The sample was purified by HPLC (column Hypercarb, 250mm x 10mm, H 2 O/formic acid 0.1%, pressure 2.2 bars, debit 4.4mL/min, T=30°C, ELS detection) and both isomers were separated and described thanks to NMR analysis. The presence of the 3-deoxy position provides a great deal of information on the structure of these products. Thus, for example, coupling constants for 3-deoxy-L-altro epimer are given in the following Figure23:

52 Figure 24 .

 5224 Figure 24. Coupling constants for compounds 49 and 52.These unanticipated difficulties are undoubtedly due to the particular structure/configuration of the galacto substrate. Possible explanation of this particular behaviour is outlined in Figure25.A likely structure and conformation ( 3 H 2 ) of the intermediate iminium cation A is given as a starting point. MM2 calculations of the steric energy of this conformation and of the alternate 2 H 3 conformation indicate that the former one is significantly more stable.Hydrogenation of this iminium ion away from the 3-OBn group would give the desired a-Dgalacto compound (axial orientation of H 2 addition). This is what is mainly observed under basic conditions. In the intermediate, the pseudo-axial C 4 -H bond is allylic with respect to the iminium salt and therefore highly sensitive towards "enolate-like" chemistry and belimination. In this system enamine formation is probably highly favoured: hydrogenation at the enamine stage could provide the a-D-galacto product and also the observed b-L-ido compound (basic conditions). Under acidic conditions, elimination of the 3-OBn group as BnOH would be facilitated by the formation of an a,b-unsaturated iminium salt B. Hydrogenation of this intermediate could lead to the two 3-deoxy products observed, having pseudo-b-L-ido or pseudob-L-altro configurations. "Acidification" of the C4-H bond in the intermediate iminium salt is clearly the factor that distinguishes the galacto series from the gluco series in which such complications are not observed.

Figure 25 .

 25 Figure 25. Proposed mechanism of epimerisation and debenzylation during catalytic hydrogenation.

Scheme 25 .

 25 Scheme 25. Synthesis of 1-C-alkyl-imino-D-galactitols by intramolecular S N 2 reaction.

  configuration at C-5 was not satisfactory either. None of the reduction methods of ketone 41 afforded the desired L-altro alcohol 46 (precursor of D-galacto final product) but gave back in majority the D-galacto intermediate 39. However, the amount of alcohol 46 obtained allowed continuing the synthesis to achieve the first, pure sample of a-1-C-propyl-imino-D-galactitol! Résumé section II: Synthèse des 1-C-alkyl-imino-D-galactitols. Après avoir réalisé avec succès la synthèse des 1-C-alkyl-imino-L-arabinitols, nous sommes passés à notre deuxième objectif : la synthèse des 1-C-alkyl-imino-D-galactitols. Nous avons voulu comparer ces deux familles de composés pour étudier les relations structure-activité, et en particulier l'influence du groupement CH 2 OH. Pour préparer ces dérivés, nous avons appliqué une stratégie de synthèse similaire à celle utilisée précédemment avec trois différences principales : i. comme produit de départ, nous avons utilisé le 2,3,4,6-tétra-O-benzyl-Dgalactopyranose commercial ii. la synthèse a été réalisée uniquement sur les iminosucres N-Z protégés iii. la cyclisation a été réalisée par amination réductrice intramoléculaire, et non par réaction de S N 2 intramoléculaire En effet, il n'est pas possible dans cette série d'utiliser la S N 2 intramoléculaire. Car en série arabino, l'alcool en position C-5 qui subit la substitution est primaire, il n'y a donc pas de modification stéréochimique. Mais dans la série galacto, l'alcool en position C-5 est secondaire, et ferait donc l'objet dans les conditions d'une S N 2 d'une inversion de Walden, ce qui conduirait à l'obtention d'iminosucres de type L-altro et non D-galacto. Malheureusement, cette voie de synthèse s'est avérée très difficile. Les premières difficultés ont été rencontrées lors de l'addition de l'allyltriméthylsilane. Cette réaction a conduit à un mélange inséparable de l'alcool désiré et du produit de départ avec un rendement faible. Malgré une optimisation complète (temps, température, nombre d'équivalents et acide de Lewis), nous n'avons pas trouvé de conditions idéales. Ensuite, lors de l'amination réductrice intramoléculaire, nous avons observé un comportement inattendu du substrat galacto, qui a conduit à l'obtention d'iminosucres deoxy et/ou d'épimères, même dans des conditions douces. Pour mieux comprendre le mécanisme de cette réaction, certains des mélanges ont été séparés par HPLC et caractérisés par RMN. En utilisant des conditions d'hydrogénation catalytique à pH basique, nous avons pu obtenir le 1-C-propyl-imino-Dgalactitol désiré en mélange avec l'épimère L-ido. Mais le rendement et la diastéréosélectivité de cette synthèse restent faibles. Finalement, pour surmonter ces problèmes de désoxygénation et d'épimérisation, nous avons décidé d'abandonner l'amination réductrice intramoléculaire et de mettre en oeuvre une cyclisation de type S N 2. Pour ce faire, il nous fallait inverser la configuration du groupement hydroxyle en position C-5 (l'épimère (R) donnant un produit final de configuration L-altro, l'épimère (S) un produit final de configuration D-galacto). Malheureusement aucune des méthodes de réduction de cétone testées n'a permis d'obtenir majoritairement l'épimère (S) et seulement une faible quantité de 1-C-propyl-imino-D-galactitol a pu être isolée. III. Alternative synthesis of 1-C-alkyl-imino-D-galactitols from L-sorbose: strategy by way of a C-6 chain elongation. III.1 Synthetic strategy Considering the difficulties of the synthetic strategy relying on the C-1 chain elongation, we decided to find another tactic. Inspired by work of Tyler's group 201 in which they synthesised 1-deoxy-galactonojirimycin from L-sorbose, we decided to adapt their approach to the C-6 chain elongation 202 previously used in our team to obtain the desired compounds. Recently published work of Fleet, Kato et al 203 used a similar tactic but started from expensive L-tagatose. Originally, we wanted to start from commercially available 1,2:4,6-di-O-isopropylidene-a-L-sorbofuranose 53. However, the product obtained from

DScheme 26 .Figure 26 .

 2626 Scheme 26. Synthetic strategy towards 1-C-alkyl-imino-D-galactitols from L-sorbose.

Scheme 28 .

 28 Scheme 28. Synthesis of 1,2-and 2,3:4,6-di-O-isopropylidene-a-L-sorbofuranose derivatives. Thus the O-acetyl and O-p-methoxybenzyl derivatives of 53 and 2,3:4,6-di-Oisopropylidene-a-L-sorbofuranose, compounds 54, 56, 58 and 61, were prepared by standard procedures and then submitted to the selective hydrolysis procedure of Mukhopadhyay et al. 214 Compounds 55, 57, 59 and 62 were isolated generally in good yields (21-80%) Comparison of the NMR spectra of synthesized compounds with the spectra provided in supplementary materials by Mukhopadhyay showed clearly that the compounds they have made are not the 1,2-O-isopropylidene derivatives, but the 2,3-O-isopropylidene derivatives. The fact that compound 57 is the correct isomer is further supported by the large chemical shift of the H-3 proton in the 3-O-acetyl derivative 55 (d= 5.12 ppm for 55 and d= 3.77 ppm for 57).

III. 3 Scheme 29 .

 329 Scheme 29. Synthesis of 1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 65.

Scheme 30 .

 30 Scheme 30. Synthesis of aldehyde 66.

Scheme 37 .

 37 Scheme 37. Synthetic strategy to prepare 1-C-alkyl-imino-L-altritolsThe synthetic strategy to prepare 1-C-alkyl-imino-L-altritols starts with the same reactions as for the galacto series; in the last step, instead of a reductive amination, we applied an intramolecular S N 2 cyclisation. As the starting material for this synthesis, we used the mixture of open-chain alcohols containing N-Z protected galactopyranosylamine (26mix), obtained in the first imino-D-galactitols synthesis. It turned out that after mesylation we can also separate the desired alcohol derivatives from the N-Z protected galactopyranosylamine.The advantage of this approach is the fact that we do not have to obtain pure alcohols: purification of the mesylated derivative provides intermediates ready to be used in the last step: cyclisation. The 1-C-alkyl-iminosugar derivatives obtained in this way can be:

Figure 27 .

 27 Figure 27. Coupling constants for compounds 28 and 29.

. 1 Scheme 40 .

 140 Scheme 40. Synthetic strategy to prepare galacto-IFG and its derivatives.

Scheme 41 .Scheme 42 .Scheme 43 .

 414243 Scheme 41. Improved synthetic strategy to prepare galacto-IFG and its derivatives.Thus we changed our tactic to one developed byVasella's team, 230 in which the key step was the regioselective opening of an oxirane ring with diethylaluminum cyanide to provide the 4-cyano derivative (Scheme 41). Improvement and further investigations on this second approach are currently undertaken by other members of our group. This synthesis however, provided the beginning steps of another approach to pseudogalacto-IFGs 117, 118, 125 and 126 (Scheme 42). Inspired by Bols' IFG synthesis,231 we realized that addition of nitromethane onto the previously obtained ketone 100, might provide an intermediate that could serve as a precursor (112, R 2 =H) of galacto-IFG in few steps.Furthermore, use of a nitroalkane with a longer chain in this process opens a direct access to the highly desired 5-C-alkyl galacto-IFG (R 2 ≠ H), by way of an unsaturated intermediate such as B (R 2 ≠ H). This would constitute an efficient synthesis in which the R 2 -C-N fragment of the target compound is introduced in one step.

Scheme 48 .

 48 Scheme 48. Reductive amination of nitromethane adducts.

Figure 28 .

 28 Figure 28. Chair representation of thermodynamic product of nitromethane addition.

Figure 29 .

 29 Figure 29. Proposed structures of obtained IFG-derivatives 125 and 126.

  we successfully achieved the synthesis of pseudo-IFG derivatives in the L-altro and D-galacto series: 4-hydroxy L-altro-IFG 117 and 4-hydroxy D-galacto-IFG 118, as well as 5-C-alkylated derivatives in the D-galacto series: 4-hydroxy-(5R) and (5S)-5-C-pentyl-D-galacto-IFG 125 and 126.

  Le meilleur inhibiteur de b-galactosidase connu à ce jour étant un épimère en position C-4 de l'isofagomine (IFG) : la galacto-IFG (ou 4-epi-IFG), cette structure a constitué une des trois cibles principales de ce projet. Nous avons décidé de synthétiser la galacto-IFG ellemême (comme référence pour les tests biologiques sur les galactosidases lysosomales), mais également des dérivés portant une chaîne alkyle en position C-5. La stratégie de synthèse envisagée comprend les étapes suivantes : i. protection sélective du D-lyxose ii. inversion de la configuration du groupement hydroxyle en C-4 pour obtenir un dérivé en série L-ribo iii. substitution par S N 2 de l'hydroxyle en C-4 par un groupement cyano iv. amination réductrice par hydrogénolyse pour obtenir la galacto-IFG, suivie d'une alkylation réductrice pour préparer des dérivés N-alkylés v. autres réactions sur l'intermédiaire cyano pour préparer des 5-C-alkyl-galacto-IFGs Bien que cette stratégie de synthèse prometteuse ait bien fonctionné pour l'IFG (en série D-gluco), elle a échoué pour la galacto-IFG, en raison de la difficulté à substituer l'hydroxyle en C-4 par un groupement cyano. Nous avons donc suivi la voie de synthèse élaborée par Vasella, dans laquelle l'étape clé est l'ouverture stéréosélective d'un époxyde avec du cyanure de diéthylaluminium permettant l'obtention du dérivé 4-cyano. L'optimisation et d'autres investigations sur cette approche sont actuellement en cours dans notre groupe. Cette voie de synthèse nous a également permis d'envisager une autre méthodologie pour l'obtention de composés de type galacto-IFGs. Par analogie avec la synthèse de l'IFG effectuée par Bols, nous avons envisagé qu'une addition de nitrométhane sur la cétone précédemment obtenue à partir du D-lyxose pourrait fournir un précurseur de la galacto-IFG en quelques étapes. En outre, l'utilisation dans ce processus d'un nitroalcane avec une chaîne plus longue ouvre un accès direct aux très désirés 5-C-alkyl-galacto-IFGs. Bien que nous ne soyons pas parvenus à éliminer le groupement hydroxyle supplémentaire en C-4, nous avons réussi à synthétiser différents dérivés alkylés ou non de la galacto-IFG. VI. Preliminary biological evaluation of the new D-galacto and L-altroconfigured iminosugar derivatives Compounds 30, 32, 35, 36, 49, 117 and 118 (Figure 30) were submitted to preliminary biological assays in Dr Wenger's laboratory (Jefferson School of Medicine, Philadelphia), to investigate their inhibitory activities and selectivity towards lysosomal agalactosidase A (substrate = 4-MU-a-galactoside) and b-galactocerebrosidase (substrate = radiolabeled 3 H-GalCer).

Figure 30 .

 30 Figure 30. Structures of tested compounds.

  1 H and13 C NMR spectra were recorded on Bruker Avance DPX 250 or Bruker Avance 400 spectrometers. Chemical shifts are given in ppm and are referenced to the residual solvent signal or to TMS as internal standard. Carbon multiplicities were assigned by distortionless enhancement by polarization transfer (DEPT) experiments.1 H and13 C signals were attributed on the basis of H-H and H-C correlations.Low-resolution mass spectra were recorded on a Perkin-Elmer Sciex API 3000. Highresolution mass spectra were recorded on a Bruker Q-TOF MaXis spectrometer in Orléans (precision 5 or 6 digits) or on a Waters Q-TOF micro spectrometer in Clermont-Ferrand (precision 4 digits).II. General treatment with basic ion-exchange resin after hydrogenationThe crude compound was dissolved in MeOH. Amberlite IRA400 resin (OH -, 16-50 mesh) was washed thoroughly with H 2 O and then with MeOH before it was added to the solution. The suspension was left stirring for 30 min, the resin was filtered and washed with MeOH. Evaporation of the solvent under reduced pressure gave the desired compound as a free base. III. General procedure for purification on Dowex 50WX8 resin (H + , 50-100 mesh)Approximately 1 mL of resin per 10 mg of compound was used. The resin was first washed in a column with H 2 O and then with MeOH. The product was dissolved in MeOH, the solution was loaded onto the column and washed with MeOH. Then the compound of interest was eluted with a 0.5M aqueous solution of NH 3 . Evaporation of the solvent under reduced pressure gave the desired compound as a free base.IV. General procedure for biological assaysTests of compounds in Dr. Asano's laboratory (Hokuriku University). a-Galactosidase A: The enzyme was prepared according to the method of Ishii 145 . The reaction mixture consisted of 100 mL of 0.15 M sodium phosphate-citrate buffer (pH 4.5), 50 mL of enzyme solution, 50 mL of inhibitor solution, and 200 mL of H 2 O. The enzyme activity was assayed with 2 mM p-nitrophenyl a-D-galactopyranoside as substrate. The reaction was stopped by adding 1 mL of 400 mM Na 2 CO 3 . The released p-nitrophenol was measured spectrophotometrically at 400 nm. Tests of compounds in Dr. Wenger laboratory (Jefferson School of Medicine, Philadelphia). Dissolve 3 H-gal-cer in 6mL 2:1 chloroform-methanol (C-M). Solutions of sodium taurocholate (10mg/mL in C-M, 2:1) and of oleic acid (1mg/mL in C-M, 2:1) were prepared and then equal volumes of them were mixed together to prepapre a mixture A. All solutions should be stored at 4 o C. Into each 10x13 glass tube about 20-25 nmol 3 H-gal-cer and 0.1 mL of mixture A were added. Solvents were evaporated with N 2 or overnight in air. 0.1 mL of citrate-phosphate buffer (0.2 M in phosphate, pH 4.2), enzyme source (usually about 50 μg

- 1 ]

 1 n 3063, 3029 (C-H aromatic ), 2865, 2794 (C-H), 1638 (C=C), 1495, 1452 (C=C aromatic ), 1094, 1071, 1027 (C-O/C-C), 731, 696 (C-H aromatic ).

  C 36 H 40 NO 3 m/z = 534.3008; found m/z = 534

- 1 ]

 1 n 3031 (C-H aromatic ), 2875 (C-H), 1714.5 (C=O), 1498, 1454 (C=C aromatic ), 1210, 1057, 1026.5 (C-C, C-O), 734, 696 (C-H aromatic ). LRMS (ESI) [M+H] + = 596.5, [M+Na] + = 618.0, [M+K] + = 634.5 HRMS (ESI) [M+Na] + calculated for C 37 H 41 NNaO 6 m/z= 618.2832; found m/z= 618.2838 Yield 71% (0.82 g). (1R,1S)-1-C-Allyl-2,3,4-tri-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-5-Omathanesulfonyl-L-arabinitol 13.

- 1 ]

 1 n 3029 (C-H aromatic ), 2926, 2867 (C-H), 1604 (N-H), 1495, 1454 (C=C aromatic ), 1356, 1206, 1091, 1027 (C-O/C-C), 909, 816, 733, 696 (C-H aromatic ). HRMS (ESI) [M+H] + calculated for C 29 H 36 NO 3 m/z= 446.26897; found m/z= 446.26937 Yield Quant. (1R)-2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1-C-((2R)-2,3-dihydroxypropyl)-1,5-dideoxy-1,5-imino-L-arabinitol 18. (1R)-2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1-C-((2S)-2,3-dihydroxypropyl)-1,5-dideoxy-

13 C

 13 NMR (100 MHz, CD 3 OD), d 73.67 (C2), 72.60 (C3), 71.08 (C7), 67.71 (C8), 67.19 (C4), 51.67 (C1), 46.72 (C5), 36.46 (C6).HRMS (ESI)[M+H] + calculated for C 8 H 18 NO 5 m/z=208.11795; found m/z=208.

(

  1R)-1-C-(3-Hydroxypropyl)-1,5-dideoxy-1,5-imino-L-arabinitol 23.

  [M+H] + calculated for C 8 H 18 NO 4 m/z= 192.12303; found m/z= 192.12315 Yield Quant. (43 mg). N-Benzyloxycarbonyl 2,3,4,6-tetra-O-benzyl-a,b-D-galactopyranosylamine 25.

- 1 ]

 1 n 3322 (N-H), 3030 (C-H aromatic ), 2869 (C-H), 1730 (C=O), 1529, 1496, 1453 (C=C aromatic ), 1363, 1250, 1216, 1095, 1040, 1027 (C-O/C-C), 910, 733, 695 (C-H aromatic ). LRMS (ESI) 673.79, [M+H] + = 674.5, [M+Na] + = 696.0, [M+K] + = 712.5 HRMS (ESI) [M+Na] + calculated for C 42 H 43 NNaO 7 m/z= 696.29317; found m/z= 696.29351 Yield 80% (1.00 g). Mixture 26mix of N-Benzyloxycarbonyl 2,3,4,6-tetra-O-benzyl-a,b-D-galactopyranosylamine 25 and (1R,1S)-1-C-allyl-2,3,4,6-tetra-O-benzyl-1-(N-benzyloxycarbonyl) amino-1

Products 39 , 40 :

 3940 [M+H] + = 716.5, [M+Na] + = 738.5 Starting material 25: [M+H] + = 674.0, [M+Na] + = 696.0 (1R,1S)-1-C-Allyl-2,3,4,6-tetra-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-5-Omethanesulfonyl-D-galactitol 27.

  37% (240 mg) (27% for 27R, 10% for mixture of diastereoisomers) and 41% (221 mg) of 25 Corrected yield: 65% All yields are calculated for two steps, from 25.

- 1 ]

 1 n 3063, 3030 (C-H aromatic ), 2864 (C-H), 1694 (C=O), 1495, 1453, 1453 (C=C aromatic ), 1324, 1205, 1092, 1025 (C-O/C-C), 910 (C-H aromatic ).HRMS (ESI)[M+H] + calculated for C 45 H 48 NO 6 m/z= 698.34761; found m/z= 698

  J 2,3 = 3.5 Hz),3.73-3.68 (m, 3H, 2H6, H4),3.33 (dd, 1H, H2, J 1,2 = 1.5 Hz, J 2,3 = 3.5 Hz), 3.23 (ddd, 1H, H5, J 5,6a = 2.9 Hz, J 5,6b = 4.6 Hz, J 5,4 = 10.2 Hz), 3.0 (td, 1H, H1, J 1,2 = 1.5 Hz, J 1,7a = J 1,7b = 7.0 Hz), 1.45-1.36 (m, 2H, H7), 1.28-1.23 (m, 2H, H8), 0.84 (t, 3H, H9, J= 7.2 Hz). 13 C NMR (62.5 MHz, CDCl 3 ), d 138.88-138.51 (C IV aromatic ), 128.41-127.54 (C aromatic ), 76.24 (C2), 75.30 (C4), 73.33, 72.95 (CH 2 Ph), 72.61 (C3), 72.36, 71.74 (CH 2 Ph ), 70.53 (C6), 54.65 (C5), 53.38 (C1), 33.43 (C7), 19.59 (C8), 14.35 (C9).

1 H

 1 NMR (400 MHz, DMSO-d 6 ), d 4.49-4.35 (m, 3H, 3OH), 3.14 (br s, 1H,OH), 3.67 (br s, 1H, H3), 3.47-3.46 (m, 3H, H4, 2H6), 3.38 (br s, 1H, H2), 2.75-2.73 (m, 1H, H1), 2.54-2.50 (m, 1H, H5), 1.29-1.23 (m, 4H, 2H7, 2H8), 0.85 (t, 3H, H9, J= 6.6 Hz).

13 C

 13 NMR (100 MHz, DMSO-d 6 ), d 71.98 (C3), 71.08 (C2), 66.10 (C4), 61.66 (C6), 56.61 (C5), 52.16 (C1), 33.51 (C7), 19.19 (C8), 14.29 (C9). HRMS (ESI) [M+H] + calculated for C 9 H 20 NO 4 m/z= 206.13868; found m/z= 206.13885 Yield 92% (35 mg). (1R)-2,3,4,6-tetra-O-benzyl-N-benzyloxycarbonyl-1-C-(E-Non-3-enyl)-1,5-dideoxy-1

  [M+H] + calculated for C 51 H 60 NO 6 m/z= 782.441515; found m/z= 782.440307

  H 32 NO 4 m/z= 290.232585; found m/z= 290

(

1 H

 1 NMR (400 MHz, CD 3 OD), d 3.90 (t, 1H, H3, J 3,2 = J 3,4 = 3.6 Hz),3.80-3.74 (m, 1H, H8), 3.74-3.69 (m, 3H, H4, 2H6), 3.65 (dd, 1H, H2, J 2,1 = 1.4 Hz, J 2,3 = 3.6 Hz), 3.52-3.42 (m, 2H, 2H9), 3.19 (ddd, 1H, H1, J 1,2 = 1.4 Hz, J 1,7b = 4.5 Hz, J 1,7a = 9.0 Hz), 2.81 (dt, 1H, H5, J 5,6a = J 5,6b = 4.1 Hz, J 5,4 = 10.4 Hz), 1.71-1.53 (m, 2H, 2H7).

13 C

 13 NMR (100 MHz, CD 3 OD), d 74.07 (C2), 73.23 (C3), 71.29 (C8), 67.36 (C9), 67.36 (C4), 63.04 (C6), 57.48 (C5), 51.58 (C1), 37.11 (C7).HRMS (ESI)[M+H] + calculated for C 9 H 20 NO 6 m/z= 238.128514; found m/z= 238

13 C

 13 NMR (100 MHz, CD 3 OD), d 73.12, 73.04 (C2, C3), 72.22 (C8), 67.58 (C9), 67.21 (C4), 62.87 (C6), 57.68 (C5), 53.08 (C1), 35.40 (C7).HRMS (ESI)[M+H] + calculated for C 9 H 20 NO 6 m/z= 238.128514; found m/z= 238

1 H

 1 NMR (400 MHz, CDCl 3 ), d 7.35-7.18 (m, 25H, H aromatic ), 5.67-5.56 (m, 1H, H8), 5.18 (d, 1H, NHCOOCH 2 Ph, J= 10.0 Hz), 5.12 (d, 1H NHCOOCH 2 Ph, J= 12.4 Hz), 5.08 (d, 1H NHCOOCH 2 Ph, J= 12.4 Hz), 5.05-4.94 (m, 2H, 2H9), 43.89 (d, 1H, CH 2 Ph, J= 11.1 Hz), 4.69 (s, 2H, CH 2 Ph), 4.57-4.39 (m, 5H, CH 2 Ph), 4.07-4.04 (m, 1H, H5), 3.97 (dd, 1H, H3, J 3,4 = 2.75 Hz, J 2,3 = 8.6 Hz), 3.90-3.84 (m, 1H, H1), 3.79-3.78 (m, 1H, H4), 3.65 (d, 1H, H2, J 2,3 = 8.6 Hz), 3.56 (dd, 1H, H6 b , J 6b,5 = 5.5 Hz, J 6b,6a = 9.3 Hz), 3.50 (dd, 1H, H6 a , J 6a,5 = 6.6 Hz, J 6a,6b = 9.3 Hz), 2.28-2.15 (m, 2H, 2H7).13 C NMR (100 MHz, CDCl 3 ), d 156.25 (C=O), 138.30-136.63 (C IV aromatic ), 134.40 (C8), 128.51-127.70 (C aromatic ), 118.12 (C9), 81.99 (C3), 79.50 (C2), 77.79 (C4), 75.69-72.75 (CH 2 Ph), 75.52-66.92 (CH 2 Ph), 70.99 (C6), 70.49 (C5), 66.92 (NHCOOCH 2 Ph), 52.13 (C1), 38.11 (C7). HRMS (ESI) [M+H] + calculated for C 45 H 50 NO 7 m/z= 716.35818; found m/z= 716.35865 [M+Na] + calculated for C 45 H 49 NNaO 7 m/z= 738.34012; found m/z= 738

(

(

  6S) and (6R)-6-C-(Non-2-enyl)-1,3,4,5-tetra-O-benzyl-6-(N-benzyloxycarbonyl)amino-keto-

  [M+H] + calculated for C 51 H 60 NO 7 m/z= 798.43643; found m/z= 798.43712 [M+Na] + calculated for C 51 H 59 NNaO 7 m/z= 820.41837; found m/z= 820.41916 [M+K] + calculated for C 51 H 59 NKO 7 m/z= 836.39231; found m/z= 836.39338 Optical rotation = +19.9 (c= 1.0; CHCl 3 ) Yield 37% (12.4 mg). (6R)-6-C-(Hex-2-enyl)-1,3,4,5-tetra-O-benzyl-6-(N-benzyloxycarbonyl)amino-keto-

1 H

 1 NMR (400 MHz, CD 3 OD), d 3.91 (t, 1H, H4, J 4,5 =J 4,3 = 3.2 Hz), 3.84 (dd, 1H, H2, J 2,1 = 4.8 Hz, J 2,3 = 8.4 Hz), 3.74-3.65 (m, 2H, 2H6), 3.60 (dd, 1H, H3, J 3,4 = 3.2 Hz, J 3,2 = 8.4 Hz), 3.09-3.04 (m, 1H, H1), 2.93-2.90 (m, 1H, H5), 1.64-1.22 (m, 4H, 2H7, 2H8), 0.96 (t, 3H, H9, J= 8.0 Hz).

13 C

 13 NMR (100 MHz, CD 3 OD), d 72.82 (C3), 71.48 (C2), 70.21 (C4), 62.67 (C6), 55.87 (C5), 55.30 (C1), 28.88 (C7), 20.84 (C8), 14.49 (C9).HRMS (ESI)[M+H] + calculated for C 9 H 20 NO 4 m/z= 206.138685; found m/z= 206

  [M+H] + calculated for C 9 H 20 NO 4 m/z= 206.138685; found m/z= 206

  [M+Na] + calculated for C 14 H 22 NaO 7 m/z=285.094474; found m/z= 285.094838

  = 7.0 Hz, J 4,3 = 6.0 Hz, J 4,OH = 7.0 Hz), 4.25 (dt, 1H, H5, J 5,6 = 3.25 Hz, J 5,4 = 7.0 Hz), 4.05 (d, 1H, H1, J= 9.25 Hz), 3.97 (d, 1H, H1, J= 9.25 Hz), 3.87 (dd, 2H, H6, J 6,5 = 3.25 Hz, J 6,OH = 6.25 Hz), 3.81 (s, 3H, CH 3 O), 3.77 (d, 1H, H3, J 3,4 = 6.0 Hz), 3.04 (d, 1H, 4-OH, J= 7.25 Hz), 2.20 (t, 1H, CH 2 OH, J= 6.5 Hz), 1.51, 1.46 (s, 3H, CH 3 iPr).

  C 12 H 20 NaO 6 m/z= 283.115209; found m/z= 283.115194 Optical rotation = -60.8 (c= 1.0; CHCl 3 ) Yield 16% (1.64 g) from L-sorbose.

35 1H

 35 NMR (400 MHz, CDCl 3 ), d 4.84 (dd, 1H, H4, J 4,5 = 4.0 Hz, J 4,3 = 6.0 Hz), 4.63 (d, 1H, H3, J 3,4 = 6.0 Hz), 4.27 (d, 1H, H1 b , J 1b,1a = 9.6 Hz), 4.08 (d, 1H, H1 a , J 1a,1b = 9.6 Hz), 4.07 (td, 1H, H5, J 4,5 = 4.0 Hz, J 5,6b = J 5,6a = 5.5 Hz),3.96-3.85 (m, 2H, 2H6), 2.29 (br s, 1H, OH),1.47, 1.43, 1.40, 1.31 (s, 3H, CH 3 iPr).

(

  6R * )-6-C-Allyl-6-(S S )-N-tert-butanesulfinylamino-6-deoxy-1,2:3,4-di-O-isopropylidene-a-

  -(S S )-N-tert-butanesulfinylamino-6-deoxy-(6R)-6-C-Hexyl-1,2:3,4-di-O-isopropylidene-a-Ltagatofuranose 72R and 6-(S S )-N-tert-butanesulfinylamino-6-deoxy-(6S)-6-C-Hexyl-1

6 -

 6 (S R )-N-tert-butanesulfinylamino-6-deoxy-(6S)-6-C-Hexyl-1,2:3,4-di-O-isopropylidene-a-Ltagatofuranose 73R and 6-(S R )-N-tert-butanesulfinylamino-6-deoxy-(6R)-6-C-Hexyl-1

  S S )-N-tert-butanesulfinylamino-6-deoxy-(6R)-1,2:3,4-di-O-isopropylidene-6-C-nonyl-a-Ltagatofuranose 74R and 6-(S S )-N-tert-butanesulfinylamino-6-deoxy-(6S)-6

  Opticalrotation = -5.4 (c= 1.0; CHCl 3 ) 6-(S R )-N-tert-butanesulfinylamino-6-deoxy-(6S)-1,2:3,4-di-O-isopropylidene-6-C-nonyl-a-Ltagatofuranose 75S and 6-(S R )-N-tert-butanesulfinylamino-6-deoxy-(6R)-6

  [M+H] + calculated for C 21 H 40 NO 5 m/z= 386.290100; found m/z= 386.290223 (6R)-6-Amino-6-deoxy-6-C-Nonyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 82

  [M+H] + calculated for C 21 H 40 NO 5 m/z= 386.290100; found m/z= 386.290210 (6S)-6-Azido-6-deoxy-6-C-hexyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 83

IR

  C 21 H 37 N 3 NaO 5 m/z= 434.262542; found m/z= 434.262558

1 H

 1 NMR (400 MHz, CD 3 OD), d 4.17 (d, 0.3H, H3 min , J 3,4 = 3.2 Hz), 4.16 (d, 0.7H, H3 MAJ , J 3,4 = 3.2 Hz), 4.10 (t, 0.7H, H4 MAJ , J 4,5 = 3.6 Hz, J 4,3 = 3.2 Hz), 4.07 (dd, 0.3H, H4 min , J 4,5 = 3.6 Hz, J 4,3 = 3.2 Hz), 3.99 (dd, 0.3H, H5 min , J 5,4 = 3.6 Hz, J 5,6 = 8.6 Hz), 3.80 (dd, 0.7H, H5 MAJ , J 5,4 = 3.6 Hz, J 5,6 = 9.4 Hz), 3.71-3.66 (m, 1H, H6 MAJ , H1 b min ), 3.61-3.56 (m, 0.3H, H6 min ), 3.54 (br s, 0.3H, H1 a min ), 3.51 (br s, 1.4H, 2H1 MAJ ), 1.71-1.59 (m, 1H, CH 2 -hexyl), 1.59-1.22 (m, 9H, CH 2 -hexyl), 0.96-0.86 (m, 3H, CH 3 -hexyl).

  C 12 H 23 N 3 NaO 5 m/z= 312.152992; found m/z= 312.153197 IR [cm -1 ] n 3362 (O-H), 2927, 2858 (C-H), 2100 (N 3 ), 1457, 1262, 1137, 1044 (C-O/C-C).

(

  C 12 H 26 NO 4 m/z= 248.185635; found m/z= 248.

  [M+H] + calculated for C 12 H 26 NO 4 m/z= 248.185635; found m/z= 248

1 H

 1 NMR (400 MHz, CDCl 3 ), d 4.82 (dd, 1H, H4, J 4,5 =4.0 Hz, J 4,3 =5.6 Hz), 4.62 (d, 1H, H3, J 3,4 = 5.6 Hz), 4.48 (dd, 1H, H6 b , J 6b,5 = 4.4 Hz, J 6b,6a = 11.2 Hz), 4.38 (dd, 1H, H6 a , J 6a,5 = 7.2 Hz, J 6b,6a = 11.2 Hz), 4.27 (d, 1H, H1 b , J 1b,1a = 10.0 Hz), 4.25-4.22 (m, 1H, H5), 4.05 (d, 1H, H1 a , J 1a,1b = 10.0 Hz), 3.07 (s, 3H, CH 3 Ms), 1.46 (s, 3H, CH 3 iPr), 1.42 (s, 3H, CH 3 iPr), 1.40 (s, 3H, CH 3 iPr) , 1.30 (s, 3H, CH 3 iPr).

102A 1 H

 1 NMR (400 MHz, CDCl 3 ), d 7.38-7.23 (m, 5H, H aromatic ), 4.92 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.72-4.70 (m, 1H, H4), 4.66 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.53 (d, 1H, H1, J= 6.4 Hz), 4.41- 4.35 (m, 2H, H5 b , H2), 4.26-4.20 (m, 1H, H5 a ), 1.48, 1.45 (s, 3H, CH 3 iPr). 13 C NMR (100 MHz, CDCl 3 ), d 147.19 (C3), 137.22 (C IV aromatic ), 128.38-127.77 (C aromatic ), 112.07 (C IV iPr), 100.29 (C1), 90.14 (C4), 73.76 (C2), 70.09 (CH 2 Ph), 63.73 (C5), 26.

1 H

 1 NMR (250 MHz, CDCl 3 ), d 7.43-7.26 (m, 5H, H aromatic ), 5.03 (s, 1H, H1), 4.72 (d, 1H, CH 2 Ph, J= 11.8 Hz), 4.74-4.65 (m, 1H, H4), 4.52 (d, 1H, CH 2 Ph, J= 11.8 Hz), 4.31-4.26 (m, 1H, H3), 4.21 (dd, 1H, H2, J 2,1 = 1.25 Hz, J 2,3 = 5.5 Hz), 3.85-3.70 (m, 2H, 2H5), 3.14 (s, 3H, CH 3 (Ms)), 1.55, 1.36 (s, 3H, CH 3 iPr).

(

  3R, 4R, 5R)-5-Hydroxymethyl-2,3-O-isopropylidene-piperidine-3,4,5-triol 115 (3R, 4R, 5S)-5-Hydroxymethyl-2,3-O-isopropylidene-piperidine-3

1

  st isomer 115 (L-altro-like) 1 H NMR (250 MHz, CD 3 OD), d 4.41-4.35 (m, 1H, H2), 4.21 (d, 1H, H3, J 2,3 = 6.0 Hz), 3.52 (d, 1H, H6 b , J= 11.0 Hz), 3.42 (d, 1H, H6 a , J= 11.0 Hz), 3.35-3.28 (m, 1H, H1 eq ), 3.16 (dd, 1H, H1 ax , J 1ax,2 = 4.75 Hz, J 1ax,1eq = 14.25 Hz), 3.02 (d, 1H, H5 b , J 5b,5a = 13.25 Hz), 2.85 (d, 1H, H5 a , J 5a,5b = 13.25 Hz), 1.55, 1.38 (s, 3H, CH 3 iPr).

1 H

 1 NMR (400 MHz, D 2 O), d 4.00-3.96 (m, 1H, H2), 3.81 (br s, 1H, H3), 3.63 (d, 1H, H6, J= 12.0 Hz), 3.52 (d, 1H, H6, J =12.0 Hz), 2.84 (dd, 1H, H1 b , J 1b,2 = 3.6 Hz, J 1b,1a = 12.8 Hz), 2.66 (d, 1H, H5 b , J= 15.6 Hz), 2.61-2.56 (m, 2H, H1 a , H5 a ).

13 C

 13 NMR (100 MHz, D 2 O), d 73.75 (C4), 69.94 (C3), 66.00 (C2), 64.32 (C6), 45.44 (C5), 44.25 (C1). HRMS (ESI) [M+H] + calculated for C 6 H 14 NO 4 m/z= 164.091734; found m/z= 164.091799 [M+Na] + calculated for C 6 H 13 NNaO 4 m/z= 186.073679; found m/z= 186

  [M+NH 4 ] + calculated for C 21 H 35 NO 7 m/z= 427.243878; found m/z= 427.243845 [M+Na] + calculated for C 21 H 31 NNaO 7 m/z= 432.199273; found m/z= 432.199176 Yield 60% (384 mg) Benzyl 2,3-O-isopropylidene-4-C-(1-aminohexyl)-b-L-ribopyranoside 121+122

  Keywords: iminosugars, b-galactocerebrosidase, Krabbe disease ICOA Université d'Orléans Pôle de chimie, BP 6759 rue de Chartres 45067 Orléans cedex 2

Table 1 .

 1 List of lysosomal storage disorders with the deficient enzyme and main storage material.

32

II. Therapies for LSDs II.1 General

Table 2 .

 2 Currently licensed treatments for LSDs.39

Table 3 .

 3 Examples of pharmacological chaperones for LSDs 114 sphingolipids containing terminal b-galactose such as D-galactosylsphingosine

and other galactosidases by Fan and coworkers. 103 As shown in Table 4, the most potent inhibitors of this enzyme are DGJ and a-galacto-

	IV. Galactosidases inhibitors microorganism) for the biological assays, as the inhibition properties vary very significantly
	between different a-galactosidases (DGJ: 270-fold higher IC 50 value for a-Gal A then for a-
	IV.1 General galactosidase from E. coli).
	Enzyme inhibitors represent almost 47% of the drugs in clinical use today. 142 The two most famous: aspirin and amoxicillin act on cyclooxygenase (to fight down the pain, fever IC 50 a for a-galactosidases Inhibitor Human Coffee bean A. niger b E. Coli c
	and inflammation) and penicillin-binding proteins (to stop bacterial infection) respectively. DNJ 830 320 NI NI
	As described previously (chapter II.5 and II.6), enzyme inhibitors are crucial to implement manno-DNJ NI d 420 NI NI
	LSDs therapies such as SRT and PCT. Several LSDs are caused by galactosidase deficiencies allo-DNJ NI NI NI NI
	(Fabry, GM1, Morquio B and Krabbe diseases) and thus potent and selective inhibitors of the galacto-DNJ (DGJ) 0.04 0.003 1.0 11
	corresponding enzymes are needed. A number of galactosidase inhibitors have already been gulo-DNJ NI 400 350 NI
	reported, which can be divided into three groups: structures of fagomine/isofagomine type, 2-deoxy-DGJ 250 8.0 NI NI
	DNJ type or novel structures of NOEV type which are carbocyclic. a-homonojirimycin (a-HNJ) NI NI NI	NI
	a-manno-HNJ	464	NI	NI	NI
	IV.2 Use of galactosidase inhibitors as PCs for galactosidase-linked LSDs a-allo-HNJ 4.3 50 NI NI
	a-Galactosidase A (a-Gal A), the enzyme implied in Fabry disease is the most a-galacto-HNJ (a-HGJ) 0.21 0.06 5.8 32
	thoroughly studied target for PCT. The first assays were conducted simply with D-galactose N-methyl-DGJ 96 1.8 55 950
	itself by Suzuki and coworkers, and led to the first evidence that the concept of N-ethyl-DGJ 306 13 NI NI
	pharmacological chaperone could work, albeit with high concentrations of galactose. 105 N-propyl-DGJ 301 20 NI NI
	N-butyl-DGJ Further experiments 143 consisting in three months of intravenous galactose infusions to 300 21 370 NI N-hydroxyethyl-DGJ 520 32 NI NI cardiac Fabry patients resulted in remarkable improvement in patients' health. After two years b-1-C-butyl-DGJ 24 0.5 365 NI
	of treatment the patient did not require cardiac transplantation. Then, as DNJ proved to be a
	good inhibitor of acid a-glucosidase, a number of galacto-analogues such as DGJ and other
	epimers were tested on human a-Gal A homonojirimycin (a-HGJ, Figure 15) with IC 50 values equal to 0.04mM and 0.21mM
	respectively. DGJ was the first example of a PC for an LSD and is now in phase III clinical
	trials. Generally, N-alkylation (e.g. N-butyl-DGJ), deoxygenation at C-2 and epimerization at
	C-3 of DGJ lowered or abolished the inhibition of a-Gal A. Fan's group investigations
	highlight also the importance of the choice of the enzyme source (human, plant or
	142 Copeland, R. A. Methods Biochem Anal 2005, 46, 1.
	105 Okumiya, T.; Ishii, S.; Takenaka, T.; Kase, R.; Kamei, S.; Sakuraba, H.; Suzuki, Y. Biochem Biophys Res
	141 Lee, W. C.; Courtenay, A.; Troendle, F. J.; Stallings-Mann, M. L.; Dickey, C. A.; DeLucia, M. W.; Dickson, Commun 1995, 214, 1219.
	D. W.; Eckman, C. B. FASEB J 2005, 19, 1549. 143 Frustaci, A.; Chimenti, C.; Ricci, R.; Natale, L.; Russo, M. A.; Pieroni, M.; Eng, C. M.; Desnick, R. J. N Engl
	33 Boyd, R. E.; Lee, G.; Rybczynski, P.; Benjamin, E. R.; Khanna, R.; Wustman, B. A.; Valenzano, K. J. J Med J Med 2001, 345, 25.
	Chem 2013, 56, 2705. 103 Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O. R.; Fan, J. Q. Eur J Biochem 2000,
	129 Lee, W. C.; Kang, D.; Causevic, E.; Herdt, A. R.; Eckman, E. A.; Eckman, C. B. J Neurosci 2010, 30, 5489. 267, 4179.

Table 4 .

 4 Inhibition

	of a-galactosidases by DGJ derivatives. IC 50 values (i.e. inhibitor concentration giving 50% inhibition) were determined by variation of inhibitor concentrations. All constants are expressed in mM. 103 a K m of a-Gal A was
	determined as 0.17 mM with p-nitrophenyl-a-D-galactopyranoside. b Aspergillus niger. c Escherichia coli. d NI: Inhibition
	<50% at 1000µM

R= Figure 16. b-Galactosidase inhibitors and PCs A wide series of other DGJ derivatives (Figure 16) was synthesized and tested by

	is ~12-fold). Further analogs of DGJ built on an azepane scaffold have been reported by
	Sinaÿ, Blériot and coworkers: for example, the DGJ homolog and b-galactose mimick E
	(Figure 16) exhibits a good activity as an inhibitor of bovine liver b-galactosidase (Ki = 5.7
	mM). 154
	The most examined inhibitor of lysosomal b-galactosidase is carbagalactosylamine
	R = H C 4 H 9 NB-DGJ DGJ NOEV (Figure 16). It was developed by Ogawa and Suzuki 155 as a chemical chaperone for DGJ derivative A
	C 9 H 19 NN-DGJ Morquio B disease and GM1 gangliosidosis. Assays on 50 different cell lines expressing DGJ derivative B
	various mutant b-galactosidases responsible for these diseases showed >3-fold increase of
	DGJ derivative activity for 17 of them, among which 10 reached >10% of normal activity (at 0.2-2µM). 156
	C These encouraging results were confirmed on a mouse model carrying human R201C
	DGJ derivative mutation responsible for GM1 gangliosidosis. Immunostaining showed reduction in
	accumulated storage material in various regions of the brain. 109	D
	While NOEV itself shows good inhibitory activity towards lysosomal b-galactosidase
	DLHex-DGJ from bovine liver with IC 50 = 0.87 mM, its derivative carrying a dodecyl instead of an octyl
	group demonstrates even higher potency: IC 50 = 0.01 mM.
	DGJ homolog	NOEV
	E
	DGJ derivative	6S-NBI-DGJ
	G
	Wrodnigg and co-workers. The nonafluoro-t-Bu ether A increases b-galactosidase activity
	1.6-fold at 30µM (patient-derived fibroblasts). 151 Compounds B and C enhance 5.5-fold the
	mutant enzyme activity at 100µM and D gives a 4.8-fold improvement in activity at 2µM. 152
	The same group synthesized and tested DL-Hex DGJ on various mutant b-galactosidases
	derived from GM1 and Morquio B patients. 153 They established the IC 50 value to be 6µM and
	showed significant enzyme activity enhancements for 7 among 13 mutants tested (best result
	Caciotti, A.; Donati, M. A.; d'Azzo, A.; Salvioli, R.; Guerrini, R.; Zammarchi, E.; Morrone, A. Eur J Paediatr
	Neurol 2009, 13, 160.
	149 Tominaga, L.; Ogawa, Y.; Taniguchi, M.; Ohno, K.; Matsuda, J.; Oshima, A.; Suzuki, Y.; Nanba, E. Brain
	Dev 2001, 23, 284.
	150 Rigat, B. A.; Tropak, M. B.; Buttner, J.; Crushell, E.; Benedict, D.; Callahan, J. W.; Martin, D. R.; Mahuran,
	D. J. Mol Genet Metab 2012, 107, 203.

DGJ derivative F Figure 17. Examples of sp 2 iminosugars

activity Figure 18. Potent

  GCase inhibitors synthesised previously by our group.

	D-glucose	N-nonyl-DNJ IC50= 0.66 mM	a-1-C-nonyl-DNJ IC50= 0.27 mM	a-1-C-nonyl-DIX IC50= 7 nM
		2 to 2.5-fold increase of	1.7-fold increase of	1.8-fold increase of
		N370S mutant GCase activity	N370S mutant GCase	N370S mutant GCase activity

10 NH2CO2Bn, TMSOTf dry CH2Cl2 4A mol. sieves 2h 81% for 2 steps NH2Bn, PTSA dry CH2Cl2 24h Quant. NH2CO2Bn, TMSOTf dry CH2Cl2 4A mol. sieves overnight 22% Scheme 9. Synthesis of protected arabinopyranosylamines 4 and 11.

	Ac2O
	pyridine
	3h
	Quant.
	3	4 R1= Bn 11 R1= Z

4 and 11 (Scheme 10).

	excellent diastereoselectivity. 179 Both procedures were investigated from the glycosylamines
	5
	7:3 (S:R)
	1:9 (S:R)
	In our group, we have
	shown that N-COOBn glycosylamines can be submitted to chain-elongation by the reaction
	with a silylated nucleophile (e.g. allyltrimethylsilane) in the presence of a Lewis acid, with
	176 Lay, L.; Nicotra, F.; Paganini, A.; Pangrazio, C.; Panza, L. Tetrahedron Lett 1993, 34, 4555.
	177 Lucero, C. G.; Woerpel, K. A. J Org Chem 2006, 71, 2641.
	178 Liautard, V.; Pillard, C.; Desvergnes, V.; Martin, O. R. Carbohydr Res 2008, 343, 2111.
	174 Cipolla, L.; Lay, L.; Nicotra, F.; Pangrazio, C.; Panza, L. Tetrahedron 1995, 51, 4679.

11 12 X AllylMgBr, dry THF 0°C up to RT overnight 81% HexylMgBr, dry THF 0°C up to RT overnight AllylTMS, dry CH3CN -20°C overnight 71% 4 Scheme 10

. C-1 chain elongation of N-protected glycosylamines.

  Kobayashi proposed the mechanism of the reaction of nucleophilic addition mediated by Lewis acid (Scheme 11). The first step is glycosylamine opening, where the open-chain form is stabilised by coordination of trimethylsilyl group of Lewis acid to oxygen and nitrogen charge compensation by triflate anion. Then nucleophile attacks iminium carbon, the Lewis acid catalyst is recovered and during aqueous treatment the O-TMS group is hydrolyzed to free alcohol.

15 51% 22% 22 19 18 23 21 20

Table 5

 5

	H2, Pd/C,
	1N HCl
	iPrOH
	overnight
	quant.
	OsO4, NMO,
	THF, tBuOH, H2O,
	overnight
	H2, Pd/C, 1N HCl	H2, Pd/C, 1N HCl
	iPrOH	iPrOH
	overnight	overnight
	96%	57%

Scheme 15. Functionalization of double bond.

Table 5 .

 5 Different conditions for the hydroboration of 15. All reactions were conducted in THF.

	Pinacolborane	Catecholborane	9-Borabicyclo[3.3.1]nonane 9-BBN	Wilkinson catalyst

183 Evans, D. A.;Fu, G. C.; Hoveyda, A. H. J Am Chem Soc 1988, 110, 6917.

Table 6

 6

		NH2CO2Bn,
	Ac2O	TMSOTf
	pyridine	dry CH2Cl2
	overnight	4A mol. sieves
		6h
		80% for 2
	2,3,4,6-tetra-O-benzyl-D-galactose	24	25
			25
			26MIX
			1) Ac2O, pyridine
			overnight
			2) separation

steps 33% from 25 40 4% from 25 MeOH, Na(cat) 88% 37 38 39 MeOH, Na(cat) 94% Scheme

18. Synthesis of N-Z protected galactopyranosylamine 25 and C-1 chain elongation.

DMP, dry CH2Cl2 61% 41c 42 41 40 39 DMP, dry CH2Cl2 94% Scheme

19. Synthesis of ketones 41 and 42.

It should be noted that these ketones exist exclusively in the open-chain form: the nucleophilic character of the NHZ group appears to be too weak to give any amount of the corresponding cyclic hemiaminal (41c on Scheme

19) 41 44 Hoveyda-Grubbs 2 nd generation catalyst

	1-octene,
	H. Grubbs II
	CH2Cl2
	48h
	72%
	1

-pentene, H. Grubbs II CH2Cl2 3 days 43% 45 43 41+42 Scheme 20

. Chain extension via cross metathesis.

188

Dess, D. B.;

Martin, J. C. J Org Chem 1983, 48, 4155.

Table 7 .

 7 of 48 afforded the desired 1-C-propyl-imino-D-galactitol 49 in 5% overall yield from ketone 41. Conditions and yields of various attempts to inverse C-5 hydroxyl group configuration.

	Entry	Reagent	Eq	Time	Yield	Comments
	1	NaBH 4	2 overnight 97% (86%) a mixture of two alcohols, attempt of separation
	2	L-selectride	6	24h	--(47%) a	complex mixture, purification needed
	3	NaBH 4 , CeCl 3	1/1	30min	71% (39%) a	mixture of two alcohols, attempt of separation
	4	LiBH 4	2	2h	90% --	mixture of two alcohols, no purification
	5	PH 3 P, DEAD p-nitro-benzoic acid	2/2 2	5 days	54% of SM	degradation

a Yield after purification

Table 8 Dess-Martin dry CH2Cl2 L-sorbose Scheme

 8

27. Synthesis of alcohol intermediate 65 from L-sorbose. 209 Reichstein, T.; Grüssner, A. Helv Chim Acta 1934, 17, 311. 210 Chen, C.-C.; Whistler, R. L. Carbohydr Res 1988, 175, 265.

Table 8 .

 8 Various conditions to prepare 1,2:4,6-di-O-isopropylidene-a-L-sorbose 53. a The solvent is underlined, while the reactive or co-solvent is in italics. b Yield of first step, after purification, does not mean pure 53. c Yield after 4 steps (pure 65 from L-sorbose).

d Not determined.

  5 Condensation of aldehyde 66 with sulfinylamines

		CuSO4 anh	OR
		dry CH2Cl2
		36h
	66	71%	67 S-imine	R-imine 68
		Scheme 31. Condensation of aldehyde 66 with sulfinylamines.

  Addition of Grignard reagents to Ellman's imines.

		RMgBr 5eq
		toluene
		0°C/-78°C
	S/R-imine	59%-93% R= allyl hexyl nonyl	S/R-amine	69, 70, 71: R= allyl 72, 73: R= hexyl 74, 75: R= nonyl 76: R= H
	67/68		69-76
	Scheme 32.

Table 9 .

 9 Yields and diastereoselectivity of allylmagnesium bromide addition to sulfinimines 67 and 68.

Table 10 .

 10 Yields and diastereoselectivity of hexyl-and nonylmagnesium bromide additions to sulfinimines 67 and 68.

70: R= allyl 72, 73: R= hexyl 74, 75: R= nonyl 77: R= allyl 79, 80: R= hexyl 81, 82: R= nonyl A 77 79-82 89, 92

Table 11 ATable 11 BTable 11 C

 111111 Scheme 33. Deprotection and reductive amination of amine intermediates.

Table 11 ,

 11 entries 5 and 6) we shortened the time for sulfinyl cleavage and we tried to deprotect the isopropylidene groups during hydrogenation under acidic conditions in order to promote reductive amination as soon as the labile intermediate A is formed. This time we reached completely deprotected compounds, but the reductive amination still did not work.

Table 11 .

 11 Summary of conditions used for the sulfinyl group cleavage and further reductive amination.All of the assays above were conducted on different amines with allyl, hexyl or nonyl chain at the C-6 position. None of them was satisfactory and we had to revise the synthetic scheme. As noted by Tyler: 201 this type of aminohemiketal is particularly unstable and degrades easily upon acidic acetonide cleavage conditions. Additionally, after the isopropylidene deprotection step, we could not determine the composition of the reaction mixture and could not give a reason for the inefficacy of the chosen synthetic approach.Inspired by work of Vasella222 and Wong 223 we realized that the synthesis could be successful, if we temporarily "protected" the amine as an azido group and we decided to transform the amine into an azide.

	Entry	A	B	C	Comments
	1	TFA:H 2 O (3:1), 4.5h	H 2 , Pd/C, HCl 1N, iPrOH	Low yield, desired compound in an unseparable mixture with deoxy products.
	2	MeOH:HCl, 2h	TFA:H 2 O (3:1), 5 h	H 2 , Pd/C, AcOH gl 10%, iPrOH	Cleavage of one of the isopropylidenes during first step, unstable product. Degradation after second step.
	3	MeOH:HCl, 2h	MeOH:HCl 1 h	H 2 , Pd/C, AcOH gl 10%, iPrOH	During second step, formation of methyl glycoside. Traces of desired compound.
	4	Resin H +	--	Degradation, not efficient for isopropylidene cleavage.
	5	MeOH:HCl, 30 min	TFA:H 2 O (3:1 or 1:1), 6 h then H 2 , Pd/C	Fully deprotected compound, reductive amination does not work. Degradation
	6	MeOH:HCl, 15 min	H 2 , Pd(OH) 2 , HCl 1N, 3 eq, iPrOH	Fully deprotected compound, traces of desired compound.
	7	MeOH:HCl, 20 min, then resin OH -	1) HCl 1N, 3 eq iPrOH, 2) Resin H +	NaBH 3 CN, AcOH gl , MeOH	Clean crude compound after first step. Cleavage with HCl does not work. Reaction repeated with resin H + works! Reductive amination gives mixture of desired compound and deoxy derivatives.

During all these attempts, the desulfinylation with MeOH/HCl gave a compound without 1,2-O-isopropylidene as a by-product. To avoid further degradation during concentration, we neutralized the reaction mixture with a basic resin. As a result, we obtained a clean crude amine-intermediate quantitatively. Further successful cleavage of III.8 Synthesis of azido-derivatives In 1972 Cavender and Shiner 224 described the first example of diazo transfer from triflyl azide to primary amines. This method was successfully applied in carbohydrate chemistry by Vasella in 1991 222 and optimised by use of copper (II) by Wong 223 . We decided to employ this method to convert the hexyl and nonyl-amino-derivatives 79-82 into the corresponding azido compounds 83-86 (Scheme 34

). 83, 84: R= hexyl 85, 86: R= nonyl 79, 80: R= hexyl 81, 82: R= nonyl 72, 73: R= hexyl 74, 75: R= nonyl TfN3, NaHCO3 CuSO4*5H2O RT overnight MeOH:HCl 20 min resin OH - Scheme 34. Synthesis

of azido-derivatives.

Table 12 Table 12 Scheme 35 .

 121235 Deprotection and reductive amination of azido-intermediates 83 and 84.

	Entry Isomer	Solvent	Catalyst	Time	H 2 pressure	Comments
	1		MeOH anh Pd/C 10% overnight	atm	68% yield, desired compound with N-methylated derivative
	2	S iPrOH Pd/C 10% overnight atm	59% yield, 20% of gluco epimer
	3		MeOH	Pd/C 10%	4h	10 bars	64% with 6% of gluco epimer only
	4		iPrOH	Pd/C 5%	6h	atm	50%, illegible mixture,
	5		Dioxane/ H 2 O	Pd/C 10%	5.5h	atm	45%, mixture of epimers
	5	R MeOH anh Pd/C 10% overnight atm	63%, illegible mixture
	6		iPrOH	Pd/C 10%	14h	atm	low yield, illegible mixture
	7		MeOH	Pd/C 10%	4h	10 bars	71%, 20% of another epimer

The last, final step of this synthesis was the intramolecular reductive amination by hydrogenation of azido intermediates 87 and 88. During this reaction, firstly the azide was reduced to an amine that reacted with the open chain keto form, according to the previously described mechanism (Scheme 21).

Table 12 .

 12 Various conditions of reductive amination via catalytic hydrogenation.

Hydrogenation NaN 3 , DMF RT 23h 76% Tf 2 O, piridine CH 2 Cl 2 -78°C-->0°C 2h NaN 3 , DMF 80°C+90°C 18h+24h 69% MsCl, NEt 3 CH 2 Cl 2 4Amol sieves 1h Scheme 36.

  galactitol 89 in 64% yield contaminated with a very small amount of gluco epimer 91 (6% only). The R isomer gave a-1-C-hexyl-imino-D-galactitol 92 in 71% yield, but the sample contains a small amount of a second product that is probably also the gluco epimer 93. Nevertheless, the quality of the NMR spectrum did not allow identifying precisely this other iminosugar. Separation of epimers by HPLC to obtain very pure samples for biological assays are in progress.While working on the synthesis of 1-C-substitued imino-D-galactitols, we decided to verify if this strategy could provide an improved synthesis of DGJ. Starting from L-sorbose we could reach DGJ in 8 steps (Scheme 36). As we already had the L-tagatofuranose 65 we envisaged to substitute its C-6 hydroxyl group with an azide, to cleave the isopropylidene groups and to perform the reductive amination by hydrogenation. To transform the hydroxyl group into an azide, we used the triflate intermediate 95 which was very reactive and gave the desired azide 96 in 76% yield (from alcohol 65). Cleavage of the acetonides using Dowex 50WX8 (H + ) worked in 61% yield. However, for the last step, we faced the same problems as during the synthesis of substituted DGJs. We tried hydrogenation under atmospheric pressure in isopropanol with palladium hydroxide, but recovered starting material. On the same sample we performed hydrogenation in dry methanol with 10% palladium on carbon, which gave an illegible mixture of epimers. In the last assay we tried to silylate the primary hydroxyl group as suggested by Tyler, 201 but the reaction did not work. During those optimizations, we ran out of the precursors 65 and 96 and we could not further investigate this synthesis. Synthesis of DGJ from alcohol 65.

	III.10	Synthesis of DGJ
		94	96
	65
		95
		Dowex50W X8
		CH 3 CN/H 2 O 65°C 71h	X
		61%
	96	97	DGJ

  Synthesis of protected 1-C-alkyl-imino-L-altritols via S N 2 cyclisation.

			27
			10%
		25	1) MsCl, Et3N 2) separation
			37%
		26mix		t-BuOK
				dry THF
				overnight
				47%
			27R		28
			27%
	28	31 Scheme 38. ( )5 30 H2, Pd/C, 1N HCl 92% 1-Octene, H. Grubbs II dry THF overnight 74%	H2, Pd/C, 1N HCl iPrOH overnight quant.	32
			OsO4, NMO,
		THF, tBuOH, H2O,
			overnight
				33/34
				49% /29%
			H2, Pd/C, 1N HCl		H2, Pd/C, 1N HCl
			iPrOH		iPrOH
			overnight		overnight
			57%		89%
				35/36
		Scheme 39. Deprotection and functionalization of the double bond.

IV.3 Functionalization of the double bond.

Tf 2 O, pyridine, dry CH 2 Cl 2 , -78°C--->0°C 2h 2) KCN, Bu 4 NCl*H 2 O, Et 2 O, H 2 O, RT overnight X SM KCN, 18-crown-6 DMF 4A mol. sieves 3days 1) Tf 2 O, pyridine, dry CH 2 Cl 2 , -78°C--->0°C 2h 2) KCN, 18-crown-6, DMF, RT overnight MsCl, NEt 3 , CH 2 Cl 2 , 4A mol. sieves 4h Scheme 44.

  3 Attempted substitution reactions to introduce a CN group As summarized in Scheme 44, several methods were tried to introduce the cyano group into the molecule. The first one consisted in performing an S N 2 reaction of activated alcohol 101 with potassium cyanide. Neither the triflate of 101, nor its mesylate 103 reacted with potassium cyanide to give the desired compound. The triflate favored elimination reactions and the mesylate was unreactive. Different phase transfer agents or use of molecular sieves did not help either. Different approaches to introduce cyano group into intermediate 101.Starting from protected D-lyxopyranoside 99, we tosylated the free 4-hydroxyl group and subsequently cleaved the isopropylidene acetal under acidic conditions. The resulting diol was treated with potassium tert-butoxide to give the corresponding L-ribo epoxide. The hydroxyl group at C-2 was then protected with a bulky triisopropylsilyl group. The key step of this strategy is the regioselective oxirane ring opening with diethylaluminum cyanide to give the desired C-4 cyano intermediate, which is favoured by the presence of a large group at O-2, as shown byVasella. 230 This step was optimised by a trainee Lydie Mbele, and the cyano intermediate 109 was obtained in good yield. The synthesis was completed by Sophie Front in our group, which afforded the free galacto-IFG as a reference compound.

	X
	101 99 107 1) 99 TsCl, MsCl, NEt3, CH2Cl2, 4A mol. sieves 4h 1) Tf2O, pyridine, dry CH2Cl2, -78°C--->0°C 2h 2) KI, DMF, X X 108 105 pyridine 40h 2,6-lutidine, TIPSOTf DMF 82% 91%	C C 103 104 45°C 3h30 73% Et 2 AlCN Et 2 O aq. AcOH 110°C 1h20	102 102 KI, DMF 4A mol. sieves X 50°C + 100°C 18h + 20h Pd(OH) 2 /C H 2 HCl MeOH 106 109 dry THF C tBuOK 45 min 69% for 2 steps	SM 111
	RT + 110°C
	overnight + 2 days
	Scheme 45. Alternative approaches to introduce cyano group into intermediate 101.
	V.4 Modified synthesis of cyano-derivative
	After these numerous fruitless assays, we decided to use Vasella's approach 230 to

Another approach was to perform a double S N 2 from 99 by way of an iodointermediate (Scheme 45) which might make the elimination reaction less favorable. Since during two S N 2 reactions, the configuration of C-4 would be changed twice, we had to start from D-lyxo alcohol 99. However, none of the attempted reactions to replace the 4-hydroxyl group by iodine was successful. The assay by way of a triflate gave degradation only, while the mesylate intermediate was very stable and gave only traces of the desired compound (identified by mass spectroscopy).

prepare the cyano derivative, by way of the opening of an oxirane intermediate (Scheme 46). Scheme 46. Synthesis of cyano-derivative 109 using Vasella's approach.

Table 13 Table 14 CH 3

 13143

	CH 3 NO 2 1.1eq
	DBU 0.1 eq
	CH 3 CN
	overnight
	27%
	NO 2
	NEt 3 3eq
	24h
	91%
	mixture of two dias

Scheme 47. Synthesis of nitro-alcohols 112, 113, 119 and 120 and dehydration assays.

Table 13 .

 13 Various

dehydration conditions used on nitro-alcohols 112 and 113.

232

Albrecht, H. P.; Moffatt, J. G. Tetrahedron Lett 1970, 1063.

Table 14 .

 14 Different conditions used for nitrohexane addition to ketone 100.

Table 15 .

 15 Various conditions used to reduce the nitro group and perform reductive amination. All reactions were done in 0.1N solution of isopropanol, at atmospheric pressure of hydrogen and room temperature.

		Conditions	Time	Comments
	1	H 2 , Pd/C, AcOH 10%	2 days	--
	2	H 2 , Pd/C, HCl 1N, 1eq	5 days	isopropylidene partially hydrolyzed
	3	H 2 , Pd(OH) 2 , AcOH 10%	3 days	--
	4	H 2 , Pd(OH) 2 , HCl 1N, 1eq	6 days	isopropylidene partially cut off

Table 10 123+124 119+120

 10119+120

	mixture of two dias
	Raney Ni
	AcOH 10%
	iPrOH (0.1N)
	4days
	85%
	Pd(OH)2 AcOH 10% iPrOH (0.1N) 64%	Dowex 50W X8 dioxane/H2O (1:1), 1h 60%
	Scheme 49. Reductive amination of nitrohexane adducts.

Table 16 : Entry Inhibitor α-gal GALC

 16

	1	30	8%	0%
	2	32	59%	24%
	3	35	56%	0%
	4	36	69%	0%
	5	49	97%	0%
	6	117	13%	0%
	7	118	0%	67%

Table 16 .

 16 Percent inhibition at 1 mM final inhibitor concentration (or 1.2mM for 117 and 118).

[mM] Enzyme % inhibition IC 50 [mM]

	1		1.2		71%
	2		0.6		58%
	3	118	0.24	GALC	31%	0.45
	4		0.12		22%
	5		0.024		13%
	6		1		99%
	7 8	49	0.1 0.01	α-gal	91% 51%	0.01
	9		0.001		10%

Table 17 .

 17 Percent of inhibition at different concentrations for 49 and 118.

	For 49 and 118 further investigations were carried out by Dr. Wenger to estimate the
	IC 50 values (Table 17).

Even though these results show that none of the tested compounds are potent inhibitor of GALC, they gave us very important information about structure-activity relationships. It is clear that the imino D-galactitols are good a-galactosidase inhibitors, and apparently alkyl chain elongation enhances inhibitory properties. IFG-related compounds are much better bgalactosidase inhibitors: pseudo galacto IFG 118 is a promising candidate as an inhibitor of lysosomal GALC, but further structural modifications have to be done to increase both the selectivity and inhibitory properties.

Evaluation of the latest compounds as well as further investigations on 118 will be performed in the Laboratory of Dr. Petryniak at Oregon Health and Science University in October 2013.

  calculated for C 36 H 40 NO 3 m/z = 534.3008; found m/z = 534.2988

	1 H NMR (400MHz, CDCl 3 ), d 7.37-7.19 (m, 20H, H aromatic ), 6.09-5.99 (m, 1H, H7), 5.14-5.09 (m, 2H, H8), 4.93 (d, 1H, CH 2 Ph, J= 11 Hz), 4.63 (d, 1H, CH 2 Ph, J= 11 Hz), 4.63 (d, 1H, CH 2 Ph, J= 12 Hz), 4.55 (d, 1H, CH 2 Ph, J= 12 Hz), 4.53 (d, 1H, CH 2 Ph, J= 12.5 Hz), 4.29 (d,1H, CH 2 Ph, J= 12.5 Hz), 4.15 (d, 1H, NCH 2 Ph, J= 13.2 Hz), 3.90 (t, 1H, H2, J 2,1/3 = 8 Hz), 3.70 (br s, 1H, H4), 3.53 (dd, 1H, H3, J 3,4 = 2.8 Hz, J 3,2 = 8 Hz), 3.16 (d, 1H, NCH 2 Ph, J= 13.2 Hz), 3.01 (dd, 1H, H5 b , J 5b,4 = 4.9 Hz, J 5b,5a = 12.9 Hz), 2.69-2.68 (m, 2H, H6), 2.49-2.47 (m, 1H, H1), 1.93 (d, 1H, H5 a , J 5a,5b = 12.9 Hz). 13 C NMR (100 MHz, CDCl 3 ), d 139.50-138.66 (C IV aromatic ), 135.36 (C7), 128.25-127.01 (C aromatic ), 116.94 (C8), 82.26 (C3), 77.14 (C2), 74.54, 71.60, 69.92 (CH 2 Ph), 71.13 (C4), 63.69 (C1), 57.03 (NCH 2 Ph), 50.13 (C5), 31.62 (C6). HRMS (ESI) = +60.5 (c= 1.3; CHCl 3 ) Yield 53% (0.29 g). pseudo-b isomer : TLC: • PE/EtOAc (9:1); • R fb = 0.27; 1 H NMR (400MHz, CDCl 3 ), d 7.34-7.21 (m, 20H, H aromatic ), 5.82-5.71 (m, 1H, H7), 5.04-4.98 (m, 2H, H8), 4.70 (d, 1H, CH 2 Ph, J= 12.3 Hz), 4.56 (d, 1H, CH 2 Ph, J= 12.3 Hz), 4.56-4.47 (m, 3H, CH 2 Ph), 4.43 (d, 1H, CH 2 Ph, J= 12.3 Hz), 3.88-3.72 (m, 5H, H4, H3, H2, NCH 2 Ph), 3.03 (br s, 1H, H1), 2.78-2.68 (m, 2H, H5), 2.51-2.37 (m, 2H, H6). 13 C NMR (100 MHz, CDCl 3 ), d 139.48-138.84 (C IV aromatic ), 137.19 (C7), 128.39-126.92 (C aromatic ), 116.28 (C8), 77.59 (C2 or C3), 75.00 (C3 or C2), 73.50 (C4), 72.90-71.13 (CH 2 Ph), [M+H] Optical rotation 59.29 (C1), 57.21 (NCH 2 Ph),

+

  5 m/z= 578.29010; found m/z= 578.29032 CDCl 3 ), d 156.23 (C=O), 138.77-136.77 (C IV aromatic ), 135.09 (C7), 128.41-127.57 (C aromatic ), 117.12 (C8), 78.26 (C3), 76.46 (C2), 73.27 (CH 2 Ph), 72.57 (C4), 72.31, 70.82 (CH 2 Ph), 67.45 (NCOOCH 2 Ph),

	Yield
	8% (61 mg).
	pseudo-b isomer :
	TLC:
	• PE/EtOAc (8:2);
	• R fb = 0.51;
	1 H NMR (400MHz, CDCl 3 ), d 7.38-7.00 (m, 20H, H aromatic ), 5.74 (br s, 0.5H, H7 rot-b ), 5.60 (br
	s, 0.5H, H7 rot-a ), 5.14-5.07 (m, 2H, NCOOCH 2 Ph), 5.03-4.91 (m, 2H, H8), 4.85-4.46 (m,
	7.5H, CH 2 Ph, H1, H5 b-rot-a or rot-b ), 4.31-4.20 (m, 0.5H, H5 a-rot-a or rot-b ), 4.12-4.07 (m, 1H, H2),
	3.74-3.69 (m, 1H, H4), 3.55 (dd, 1H, H3, J 3-2 = 10.1 Hz, J 3-4 = 3.3 Hz), 2.71 (br s, 1H, H5 a ),
	2.58 (br s, 1H, H6 b ), 2.22-2.13 (m, 1H, H6 a ).

13 C NMR (100 MHz,

  NMR (400 MHz, CDCl 3 ), d 7.40-7.26 (m, 15H, H aromatic ), 4.97 (d, 1H, CH 2 Ph, J= 10.8 Hz), 4.70 (s, 2H, CH 2 Ph), 4.64-4.57 (m, 3H, CH 2 Ph),

1 H

  (C IV aromatic ), 128.49-127.63 (C aromatic ), 84.36, 81.14 (C2, C3), 75.60 (CH 2 Ph), 73.83 (C4), 71.81, 71.51 (CH 2 Ph), 60.23 (C1), 47.06 (C5), 34.62 (C6), 19.22 (C7), 14.44 (C8).

	1 H NMR (400 MHz, acetone-d6), d 7.31-7.09 (m, 15H, H aromatic ), 4.82 (d, 1H, CH 2 Ph, J= 11.2
	Hz), 4.60-4.53 (m, 3H, CH 2 Ph) 4.47 (d, 1H,CH 2 Ph, J= 11.2 Hz), 4.46 (d, 1H, CH 2 Ph, J= 11.8
	Hz),

  NMR (400 MHz, CDCl 3 ), d 7.36-7.17 (m, 15H, H aromatic ), 4.78 (d, 1H, CH 2 Ph, J= 12.3 Hz), 4.56 (d, 1H, CH 2 Ph, J= 12.3 Hz), 4.55 (d, 1H, CH 2 Ph, J= 12.2 Hz), 4.49 (d, 1H, CH 2 Ph, J= 12.2 Hz), 4.43 (d, 1H, CH 2 Ph, J= 11.8 Hz), 4.36 (d, 1H, CH 2 Ph, J= 11.8 Hz), 3.86 (t, 1H,

	13 C NMR (100 MHz, CDCl 3 ), d 139.05, 138.84, 138.36 (C IV aromatic ), 128.46-127.65 (C aromatic ),
	77.18 (C2),

1 H H3, J 3,2 = J 3,4 = 2.8 Hz), 3.71 (ddd, 1H, H4, J 4,3 = 2.8 Hz, J 4,5eq = 6.3 Hz, J 4,5ax = 9.3 Hz), 3.37 (br d, 1H, H2, J 2,3 = 2.8 Hz), 3.04-2.97 (m, 2H, H5), 2.93 (dt, 1H, H1, J 1,2 = 1.9 Hz, J 1,6b = 6.9 Hz, J 1,6a = 6.9 Hz), 1.98 (br s, 1H, NH),

1.43-1.26 (m, 3H, H6, H7), 1.20-1.10 (m, 1H, H7)

, 0.86 (t, 3H, H8, J 8,7 = 7.3 Hz).

  [M+H] + calculated for C 37 H 42 NO 7 m/z= 612.29558; found m/z= 612.29569 [M+Na] + calculated for C 37 H 41 NNaO 7 m/z= 634.27750; found m/z= 634.27766

	2 nd isomer 19:
	TLC:
	• CH 2 Cl 2 /Acetone (9:1)
	• R f2 = 0.53
	1 H NMR (400 MHz, CDCl 3 ), d 7.34-7.24 (m, 20H, H aromatic ), 5.16-5.06 (m, 2H,
	NCOOCH 2 Ph), 4.80 (d, 1H, CH 2 Ph, J= 11.4 Hz), 4.73-4.49 (m, 6H, H1, CH 2 Ph), 4.28-4.25
	(m, 1H, H5 b ), 4.12-4.09 (m, 1H, H2), 3.76-3.64 (m, 3H, H4, H7, H8 b ), 3.51-3.44 (m, 2H, H3,
	H8 a ), 2.86 (d, 1H, H5 a , J 5a,5b = 15.0 Hz), 2.04-2.00 (m, 1H, H6), 1.60-1.52 (m, 1H, H6).
	13 C NMR (100 MHz, CDCl 3 ), d 156.76 (C=O), 138.63-136.28 (C IV aromatic ), 128.70-127.68
	(C aromatic ), 78.42 (C3), 76.32 (C2), 73.61 (CH 2 Ph), 72.64, 72.61 (C4, C7), 71.44, 71.12
	(CH 2 Ph), 68.02 (NCOOCH 2 Ph), 66.29 (C8), 52.29 (C1), 40.79 (C5), 28.51 (C6).
	HRMS (ESI)
	Yield
	22% (106 mg).
	1 H NMR (400 MHz, CDCl 3 ), d 7.40-7.24 (m, 20H, H aromatic ), 5.19 (d, 1H, NCOOCH 2 Ph, J=
	12.2 Hz), 5.09 (d, 1H, NCOOCH 2 Ph, J= 12.2 Hz), 4.92-4.90 (m, 1H, H1), 4.73-4.55 (m, 6H,
	CH 2 Ph), 4.27 (d, 1H, H5 b , J 5b,5a = 14.6 Hz), 4.19 (dd, 1H, H2, J 2,1 = 6.5 Hz, J 2,3 = 10.0 Hz),
	3.97 (s, 1H, OH), 3.69 (s, 1H, H4), 3.53-3.49 (m, 4H, H3, H7, H8), 2.68 (d, 1H, H5 a , J 5a,5b =
	14.6 Hz), 2.34-2.33 (m, 1H, OH), 1.89-1.82 (m, 1H, H6), 1.48 (t, 1H, H6, J= 13.1 Hz).
	13 C NMR (100 MHz, CDCl 3 ), d 157.32 (C=O), 138.65-136.11 (C IV aromatic ), 128.73-127.64
	(C aromatic ), 78.40 (C3), 75.80 (C2), 73.31, 72.76 (CH 2 Ph), 72.48 (C4), 71.25 (CH 2 Ph), 68.28
	(NCOOCH 2 Ph), 68.25 (C7), 66.73 (C8), 50.41 (C1), 40.71 (C5), 27.85 (C6).
	HRMS (ESI) [M+H] + calculated for C 37 H 42 NO 7 m/z= 612.29558; found m/z= 612.29541 [M+Na] + calculated for C 37 H 41 NNaO 7 m/z= 634.27750; found m/z= 634.27752
	Yield
	51% (252 mg).

  The reaction was conducted under argon. To a solution of compound 15 (208 mg, 0.360 mmol) in dry THF (0.8 mL) were added catecholborane (1 N solution in THF, 0.72 mL, 2 eq) and Wilkinson's catalyst (6.5 mg, 0.007 mmol, 0.02 eq). The reaction mixture was heated at 40°C for 4 days, then cooled down to RT and diluted with EtOH (0.72 mL). 2N Aqueous NaOH (0.72 mL) and 30% H 2 O 2 (0.72 mL) were added. The reaction mixture was left stirring at RT for 2.5 days. The aqueous phase was then extracted with Et 2 O (2 x 10 mL). The combined organic phases were washed successively with 0.1 N Na 2 S 2 O 3 (2 x 10 mL), 2 N NaOH (2 x10 mL), H 2 O (10 mL), saturated NH 4 Cl (10 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a brown oil. Flash column chromatography on silica gel (PE/AcOEt 8:2/5:5) gave pure product 22 as a colorless oil.

	(1R)-2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1-C-(3-hydroxypropyl)-1,5-dideoxy-1,5-
		imino-L-arabinitol 22.
		6	8
	5	1	7	C37H41NO6
	4	2
	3			MW=595.74 g/mol
	TLC:
	• PE/EtOAc (8:2);
	• R f = 0.095
	1 H NMR (400 MHz, CDCl 3 ), d 7.34-7.24 (m, 20H, H aromatic ), 5.10 (br s, 2H, NCOOCH 2 Ph), HRMS (ESI) 4.79-4.24 (m, 8H, CH 2 Ph, H1, H5 b ), 4.09 (br s, 1H, H2), 3.73-3.53 (m, 3H, H4, H8), 3.55 (dd, [M+H] + calculated for C 8 H 18 NO 5 m/z= 208.11795; found m/z= 208.11787 1H, H3, J 3,4 = 2.8 Hz, J 3,2 = 10.1 Hz), 2.76-2.73 (m, 1H, H5 a ), 1.86-1.15 (m, 4H, H6, H7).
	Optical rotation = -11.3 (c= 1.00; MeOH) 13 C NMR (100 MHz, CDCl 3 ), d 156.46 (C=O), 138.81-136.57 (C IV aromatic ), 128.61-127.55
	(C aromatic ), 78.30 (C3), 76.65 (C2), 73.29 (CH 2 Ph), 72.62 (C4), 72.26, 70.91 (CH 2 Ph), 67.70
	Yield (NCOOCH 2 Ph), 62.62 (C8),
	57% (20 mg).

13 C NMR (100 MHz, CD 3 OD), d 72.53 (C3), 72.39 (C2), 71.91 (C7), 67.54 (C8), 67.16 (C4), 52.88 (C1), 46.79 (C5), 34.85 (C6).

  [M+H] + calculated for C 37 H 42 NO 6 m/z= 596.30066; found m/z= 596.30055 [M+Na] + calculated for C 37 H 41 NNaO 6 m/z= 618.28261; found m/z= 618.28206 [M+K] + calculated for C37H 41 KNO 6 m/z= 634.25655; found m/z= 634.25590

	Yield
	46% (98 mg).

  The reaction was conducted under argon at RT on the sample of 26mix previously obtained. To a solution of 26mix (0.8 mmol) in dry CH 2 Cl 2 (7 mL) were added Et 3 N (0.21 mL, 1.54 mmol) and 4Å molecular sieves. Then methanesulfonyl chloride was added (0.11 mL, 1.47 mmol). The reaction was left stirring overnight. The solids were filtered, washed with CH 2 Cl 2 (100 mL) and the filtrate was washed with saturated aqueous NH 4 Cl (20 mL). The organic phase was separated, washed with brine (2 x 30 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a mixture of 27R/S and starting material 25 in a ratio 6:4 as a yellowish oil. Flash column chromatography on silica gel (toluene/EtOAc, 95:5) afforded the first, major addition product 27R (175 mg, 27%), the mixture of diastereoisomers 27R and 27S (65 mg, 10%), both as yellowish oils, and recovered 25 (221 mg, 41%). All yields are calculated for two steps from 25.

									C46H51NO9S
	6	5	4	3	2	1	7	8	9
									MW= 793.96 g/mol

  NMR (250 MHz, CDCl 3 ), d 7.31-7.16 (m, 20H, H aromatic 4.68 (d, 1H, CH 2 Ph, J= 12.3 Hz), 4.56-4.34 (m, 7H, CH 2 Ph), 3.77 (t, 1H, H3,

1 H

  NMR (100 MHz, CDCl 3 ), d 157.19 (C=O), 138.52-136.30 (C IV aromatic ), 128.69-127.62 (C aromatic ), 75.58 (C3), 75.36 (C2), 73.96 (CH 2 Ph), 73.76 (C4), 73.12-72.29 (CH 2 Ph), 71.41 (C8), 69.85 (C9), 68.09 (NCOOCH 2 Ph), 66.36 (C6), 55.22 (C5), 52.23 (C1), 35.55 (C7). [M+H] + calculated for C 45 H 50 NO 8 m/z= 732.353094; found m/z= 732.352445 [M+Na] + calculated for C 45 H 49 NNaO 8 m/z= 754.335038; found m/z= 754.333740 The configuration of the carbinol chiral center C7 of 33 and 34 was determined in Prf.Frelek laboratory (Institute of Organic chemistry, Polish Academy of Science, Warsaw) from the ECD spectrum of the preformed complexex of these compounds with dimolybdenum tetracetate (see Results and Discussion, section I.6 for details). These results have already been published 172 . It is thus established that 33 is the 7S epimer and 34 the 7R epimer.

	2 nd isomer 34:
	TLC:
	• CH 2 Cl 2 /acetone (9:1);
	• R f2 = 0.33
	1 H NMR (400 MHz, CDCl 3 ), d 7.43-7.08 (m, 25H, H aromatic ), 5.15 (d, 1H, NCOOCH 2 Ph, J=
	12.4 Hz), 5.09 (d, 1H, NCOOCH 2 Ph, J= 12.4 Hz), 4.79 (d, 1H, CH 2 Ph, J= 11.2 Hz), 4.68-
	4.58 (m, 6H, H1, H5, CH 2 Ph), 4.53 (d, 1H, CH 2 Ph, J= 12 Hz), 4.36 (d, 1H, CH 2 Ph, J= 12
	Hz), 4.29 (d, 1H, CH 2 Ph, J= 12 Hz), 4.24-4.20 (m, 1H, H2), 3.90-3.87 (m, 2H, H4, OH 8 ),
	3.78-3.68 (m, 2H, H8, H3), 3.59-3.56 (m, 1H, H6 b ), 3.39-3.34 (m, 3H, H6 a , 2H9), 2.25 (br s,
	1H, OH 9 ), 2.17-2.13 (m, 1H, H7 b ), 1.35-1.22 (m, 1H, H7 a ).
	HRMS (ESI)
	Optical rotation
	= +11.2 (c= 1.0; CHCl 3 )
	Yield
	29% (36 mg)
	Note:
	1 H NMR (400 MHz, CDCl 3 ), d 7.42-7.19 (m, 25H, H aromatic ), 5.17 (d, 1H, NCOOCH 2 Ph, J=
	12.4 Hz), 5.12 (d, 1H, NCOOCH 2 Ph, J= 12.4 Hz), 4.92-4.83 (m, 1H, H1), 4.72-4.51 (m, 7H,
	H5, CH 2 Ph), 4.39-4.24 (m, 4H, CH 2 Ph, H2, OH 8 ), 3.87 (s, 1H, H4), 3.70 (dd, 1H, H3, J 3,4 =
	2.8 Hz, J 3,2 = 10.4 Hz), 3.59-3.41 (m, 3H, H8, 2H9), 3.33-3.31 (m, 2H, 2H6), 2.31-2.28 (m,
	1H, OH 9 ), 1.91 (m, 1H, H7 b ), 1.35-1.31 (m, 1H, H7 a ).
	13 C NMR (100 MHz, CDCl 3 ), d 158.00 (C=O), 138.67-136.16 (C IV aromatic ), 128.72-127.65
	(C aromatic ), 75.52 (C3), 75.22 (C2), 74.04 (C4), 73.24-72.36 (CH 2 Ph), 69.80 (C6), 68.36
	(NCOOCH 2 Ph), 68.25 (C8), 66.63 (C9), 55.16 (C5), 50.97 (C1), 32.95 (C7).
	HRMS (ESI) [M+H] + calculated for C 45 H 50 NO 8 m/z= 732.353094; found m/z= 732.352425
	Optical rotation
	= +53.8 (c= 1.0; CHCl 3 )
	Yield
	49% (60 mg)

13 C

  To a solution of 37 (690 mg, 0.91 mmol) in MeOH (25 mL) sodium was added until basic pH. The mixture was left stirring at RT until TLC showed completion of the reaction (22 h). The mixture was neutralized with Dowex 50WX8 resin (H + ). The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum, to give the crude product 39 as a colorless oil.

	2 nd isomer 38: (1R)-1-C-Allyl-2,3,4,6-tetra-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-D-galactitol
								39.
	TLC:
	• toluene/EtOAc (9:1);
	• R f2 = 0.35	6	5	4	3	2	1	7	8	9	C45H49NO7
	1 H NMR (250 MHz, CDCl 3 ), d 7.34-7.21 (25H, H aromatic ), 5.76-5.55 (m, 1H, H8), 5.55-5.44 MW= 715.87 g/mol
	(m, 1H, H5), 5.32 (d, 1H, NHCOOCH 2 Ph, J= 8.8 Hz), 5.04-4.95 (m, 4H, NHCOOCH 2 Ph,
	2H9), 4.66-4.46 (m, 8H, CH 2 Ph), 4.12-3.99 (m, 1H, H4), 3.99-3.94 (m, 1H, H1), 3.83 (t, 1H,
	H3, J 3-2 = J 3-4 = 5.2 Hz), 3.71-3.59 (m, 3H, 2H6, H2), 2.35-2.11 (m, 2H, H7), 1.97 (s, 3H, CH 3 )
	Yield
	4% (from 25, 44 mg)
	Corrected yield: 5.5 %
	IR [cm -1 ] n 3434 (N-H), 3063, 3030 (C-H aromatic ), 2864 (C-H), 1716 (C=O), 1497, 1453
	(C=C aromatic ), 1209, 1061, 1026 (C-C /C-O), 912 (C-H), 734 (C-H aromatic ).
	HRMS (ESI) [M+H] + calculated for C 47 H 52 NO 8 m/z= 758.36874; found m/z= 758.36901 [M+NH 4 ] + calculated for C 47 H 55 N 2 O 8 m/z= 775.39529; found m/z= 775.39510 [M+Na] + calculated for C 47 H 51 NNaO 8 m/z= 780.35069; found m/z= 780.35070
	1 st isomer 37:
	TLC:
	• toluene/EtOAc (9:1);
	• R f1 = 0.41
	1 H NMR (400 MHz, CDCl 3 ), d 7.30-7.22 (m, 25H, H aromatic ), 5.65-5.55 (m, 1H, H8), 5.41 (q,
	1H, H5, J= 4.9 Hz), 5.15 (d, 1H, NHCOOCH 2 Ph, J= 10.1 Hz), 5.10-5.04 (m, 2H,
	NHCOOCH 2 Ph), 5.00-4.92 (m, 2H, 2H9), 4.87(d, 1H, CH 2 Ph, J=11.0 Hz), 4.68-4.41 (m, 7H,
	CH 2 Ph), 3.91-3.89 (m, 1H, H4), 3.86-3.83 (m, 2H, H1, H3), 3.69-3.59 (m, 3H, 2H6, H2),
	2.30-2.12 (m, 2H, 2H7), 1.92 (s, 3H, CH 3 ).
	13 C NMR (100 MHz, CDCl 3 ), d 170.66 (C=O OAc), 156.27 (C=O Z), 138.76-130.71 (C IV aromatic ), 134.56 (C8), 128.50-127.57 (C aromatic ), 117.88 (C9), 81.00 (C3), 79.54 (C2), 78.57
	(C4), 75.43-73.24 (CH 2 Ph), 71.74 (C5), 68.90 (C6), 66.84 (NHCOOCH 2 Ph), 52.43 (C1),
	38.35 (C7), 21.26 (CH 3 ).
	Yield
	33% (from 25, 358 mg). Corrected yield: 43%.

-1 ] n 3422 (N-H), 3063, 3031 (C-H aromatic ), 2868 (C-H), 1718 (C=O), 1498, 1454, (C=C aromatic ), 1235, 1055, 1026 (C-O/C-C), 913, 848, 733 (C-H aromatic ).

  calculated for C 45 H 50 NO 7 m/z= 716.35818; found m/z= 716.35843 [M+NH 4 ] + calculated for C 45 H 53 N 2 O 7 m/z= 733.38473; found m/z= 733.38499 [M+Na] + calculated for C 45 H 49 NNaO 7 m/z= 738.34012; found m/z= 738.34048

	92 (m, 1H,
	H3), 3.84 (d, 1H, H4, J= 5.9 Hz), 3.72 (t, 1H, H2, J= 4.4 Hz), 3.60-3.50 (m, 2H , 2H6), 2.90
	(d, 1H, OH, J= 6.4 Hz), 2.38-2.08 (m, 2H, 2H7).
	13 C NMR (100 MHz, CDCl 3 ) δ 156.39 (C=O), 138.17-136.88 (C IV aromatic ), 134.41 (C8),
	129.03-126.95 (C aromatic ), 117.84 (C9), 79.53 (C3), 78.68 (C2), 77.49 (C4), 74.47, 73.91,
	73.37, 72.62 (CH 2 Ph), 71.21 (C6), 69.45 (C5), 66.51 (NHCOOCH 2 Ph), 51.13 (C1), 35.67
	(C7).
	HRMS (ESI)
	[M+H]

1 H NMR (400 MHz, CDCl 3 ) δ 7.

36-7.18 (m, 25H, H aromatic ), 5.72-5.64 (m, 1H, H8)

, 5.48 (d, 1H, NHCOOCH 2 Ph, J= 8.4 Hz), 5.12-4.92 (m, 4H, NHCOOCH 2 Ph, 2H9), 4.84 (d, 1H, CH 2 Ph, J= 10.8 Hz,), 4.69 (d, 1H, CH 2 Ph, J= 10.9 Hz), 4.61 (d, 1H, CH 2 Ph, J= 11.3 Hz),

 5H, CH 2 Ph), 1H, H5), 1H, H1),

  (C6, CH 2 Ph), 66.91 (NHCOOCH 2 Ph), 51.94 (C1), 38.11 (C7). [M+H] + calculated for C 45 H 48 NO 7 m/z= 714.34253; found m/z= 714.34268 [M+NH 4 ] + calculated for C 45 H 51 N 2 O 7 m/z= 731.36908; found m/z= 731.36904 [M+Na] + calculated for C 45 H 47 NNaO 7 m/z= 736.32447; found m/z= 736.32467

	HRMS (ESI)
	Optical rotation
	= +32.2 (c= 1.0; CHCl 3 )
	Yield
	94% (570 mg).

  H 48 NO 7 m/z= 714.34253; found m/z= 714.34203 [M+NH 4 ] + calculated for C 45 H 51 N 2 O 7 m/z= 731.36908; found m/z= 731.36922 [M+Na] + calculated for C 45 H 47 NNaO 7 m/z= 736.32447; found m/z= 736.32408 [M+K] + calculated for C 45 H 47 NKO 7 m/z= 752.29841; found m/z= 752.29778

  [M+H] + calculated for C 48 H 54 NO 7 m/z= 756.39005; found m/z= 756.38948 [M+Na] + calculated for C 48 H 53 NNaO 7 m/z= 778.37215; found m/z= 778.37142 [M+K] + calculated for C 48 H 53 NKO 7 m/z= 794.34601; found m/z= 794.34536

	(1R)-1-C-Allyl-2,3,4,6-tetra-O-benzyl-1-(N-benzyloxycarbonyl)amino-1-deoxy-L-altritol 46.
																		C45H49NO7
	6	5	4	3	2	1	7	8	9	6	5	4	3	2	1	7	8	9
																		MW= 715.87 g/mol
				39									46
	Optical rotation
	= +24.3 (c= 1.3; CHCl 3 )
	Yield
	43% (91 mg).

  calculated for C 45 H 48 NO 6 m/z= 698.347132; found m/z= 698.347615 [M+Na] + calculated for C 45 H 47 NNaO 6 m/z= 720.328986; found m/z= 720.329559

	(1R)-1-C-Propyl-1,5-dideoxy-1,5-imino-D-galactitol 49.
	6		7	9
	5		1	8	C9H19NO4
	4	3	2		MW=205.26 g/mol
	1 H NMR (250 MHz, CDCl 3 ), d 7.32-7.25 (m, 25H, H aromatic ), 5.72-5.61 (m, 1H, H8), 5.09-
	4.97 (m, 4H, 2H9, NCOOCH 2 Ph), 4.81 (d, 1H, CH 2 Ph, J= 11.75 Hz), 4.75-4.15 (m, 9H, H1,
	H5, CH 2 Ph), 4.15-3.44 (m, 4H, H2, H4, 2H6), 3.60 (dd, 1H, H3, J 3,2 = 9.0 Hz, J 3,4 = 2.9 Hz),
	2.69-2.22 (m, 2H, H7).
	13 C NMR (62.5 MHz, CDCl 3 ), d 155.35 (C=O), 139.05-138.24 (C IV aromatic ), 136.62 (C IV aromatic ), 135.27 (C8) 128.50-127.42 (C aromatic ), 116.92 (C9), 80.20 (C3), 77.04, 74.73 (C4,
	C2), 74.09, 73.12, 73.06 (CH 2 Ph), 68.18, 67.21 (NHCOOCH 2 Ph, C6), 55.29, 54.35 (C5, C1),
	29.82 (C7).
	HRMS (ESI)
	[M+H] Optical rotation
	= -1.3 (c= 1.0; CHCl 3 )
	Yield
	48 7% (36 mg) from 39 and 46
	28 32% (163) from 39 and 46

+

  x 10mm, H 2 O/formic acid 0.1%, pressure 2.2 bars, debit 4.4mL/min, T=30°C, ELS detection) gave analytical samples of both 3-deoxy compounds 50 and 51.

	Yield
	68% (17 mg)
	HRMS (ESI) [M+H] + calculated for C 9 H 20 NO 3 m/z= 190.143770; found m/z= 190.144172
	50:
	1 H NMR (400 MHz, CD 3 OD), d 3.84-3.77 (m, 2H, H2, H6 b ), 3.71 (ddd, 1H, H4, J 4,3eq = 4.8
	Hz, J 4,3ax = 9.6 Hz, J 4,5 = 9.2 Hz), 3.67 (dd, 1H, H6 a , J 6a,5 = 6.0 Hz, J 6a,6b = 11.2 Hz), 3.66 (br s,
	1H, NH), 2.59-2.53 (m, 1H, H1), 2.42 (ddd, 1H, H5, J 5,6b = 3.6 Hz, J 5,6a = 6.0 Hz, J 5,4 = 9.2 Hz),
	2.19 (ddd, 1H, H3 eq , J 3eq , 2 = 3.6 Hz, J 3eq , 4 = 4.8 Hz, J 3eq , 3ax = 13.2 Hz), 1.50 (ddd, 1H, H3 ax ,
	J 3ax , 2 = 2.6 Hz, J 3ax , 4 = 9.6 Hz, J 3ax , 3eq = 13.2 Hz), 1.56-1.32 (m, 4H, 2H7, 2H8), 0.95 (t, 3H,
	3H9, J= 7.0 Hz).
	13 C NMR (100 MHz, CD 3 OD), d 68.27 (C2), 65.30 (C4), 64.85 (C5), 63.19 (C6), 59.94 (C1),
	42.17 (C3), 34.96 (C7), 20.36 (C8), 14.58 (C9).
	51:
	1 H NMR (400 MHz, CD 3 OD), d 3.89-3.86 (m, 1H, H4), 3.77-3.74 (m, 1H, H2), 3.66 (dd, 1H,
	H6 b , J 6b,5 = 6.4 Hz, J 6b,6a = 10.8 Hz), 3.63 (dd, 1H, H6 a , J 6a,5 = 6.4 Hz, J 6a,6b = 10.8 Hz), 2.71 (dt,
	1H, H5, J 5,4 = 1.6 Hz, J 5,6 = J 5,6 = 6.4 Hz), 2.42 (ddd, 1H, H1, J 1,2 = 1.6 Hz, J 1,7 = 5.6 Hz, J 1,7 =
	8.0 Hz), 2.16 (dt, 1H, H3 eq , J 3eq,4 = J 3eq,2 = 3.2 Hz, J 3eq,3ax = 14.4 Hz), 1.71 (dt, 1H, H3 ax , J 3ax,4 =
	J 3ax,2 = 3.0 Hz, J 3eq,3ax = 14.4 Hz), 1.63-1.54 (m, 1H, H7), 1.49-1.35 (m, 3H, H7, 2H8), 0.96 (t,
	3H, 3H9, J= 7.2 Hz).

  12 H 20 NaO 6 m/z= 283.115209; found m/z= 283.115171

	3-O-Acetyl-1,2:4,6-di-O-isopropylidene-a-L-sorbofuranose 54
	5		2
	6	4	3	1	C 14 H 22 O	7
					MW = 302.33 g/mol
	Optical rotation
	= -30.9 (c= 1.0; acetone)
	Yield
	16% (2.275 g)
	Corrected yield: 46%

  NMR (400 MHz, CD 3 OD), d5.12 (d, 1H, H3, J 3,4 = 5.2 Hz), 4.40 (t, 1H, H4, J~ 5 Hz, J 4,5 = 5.6 Hz), 4.20 (d, 1H, H1 b , J= 9.2 Hz), 4.16 (q, 1H, H5, J~ 5 Hz), 4.05 (d, 1H, H1 a , J=9.2 Hz), 3.75 (dd, 1H, H6 b , J 6b-5 = 4.4 Hz, J 6a,6b = 12.0 Hz), 3.71 (dd, 1H, H6 a , J 6a-5 = 5.2 Hz, J 6a,6b = 12.0 Hz), 2.10 (s, 3H, OAc), 1.44 (s, 3H, CH 3 iPr), 1.30 (s, 3H, CH 3 iPr).

	13 C NMR (100 MHz, CD 3 OD), d 171.90 (C=O from OAc), 112.11, 109.58 (C IV iPr, C2),
	80.48 (C5), 80.44 (C3), 74.77 (C4), 73.13 (C1), 61.79 (C6), 26.49, 26.31 (CH 3 iPr), 20.67
	(CH 3 O).

1 H

  NMR (250 MHz, CDCl 3 ), d 7.29 (d, 2H, H aromatic , J= 8.5 Hz), 6.87 (d, 2H, H aromatic , J= 8.75 Hz), 4.71 (d, 1H, CH 2 (OPMB), J= 11.8 Hz), 4.61 (d, 1H, CH 2 (OPMB), J= 11.8 Hz),

	4.26 (dd, 1H, H4, J 4,3 = 1.8 Hz, Hz, J 4,5 = 3.5 Hz), 4.19 (br q, 1H, H5, J= 3.5 Hz), 4.14 (d, 1H,
	H1 b , J= 9.5 Hz), 4.02 (d, 1H, H1 a , J= 9.5 Hz), 3.96 (dd, 1H, H6 b , J 6b,5 = 3.5 Hz, J 6b,6a = 12.8
	Hz), 3.84 (dd, 1H, H6 a ,J 6a,5 = 3.5 Hz, J 6a,6b = 12.8 Hz), 3.80 (s, 4H, CH 3 O, H3), 1.54, 1.48,
	1.38, 1.34 (s, 3H, CH 3 iPr).
	13 C NMR (100 MHz, CDCl 3 ), d 159.42, 129.84 (C IV aromatic ), 129.61, 113.85 (C aromatic ), 110.94, 110.80 (C IV iPr), 97.96 (C2), 84.55 (C3), 74.01 (C4), 73.27 (CH 2 (OPMB)), 72.67 (C1), 72.14
	(C5), 60.59 (C6), 55.31 (CH 3 O),

1 H

  [M+H] + calculated for C 20 H 29 O 7 m/z= 381.190780; found m/z= 381.190771 [M+Na] + calculated for C 20 H 28 NaO 7 m/z= 403.172724; found m/z= 403.172306

	3-O-p-Methoxybenzyl-1,2-O-isopropylidene-a-L-sorbofuranose 57
	5		2		C	17 H 24	O	7
	6	4	3	1	MW = 340.15 g/mol
	Optical rotation
	= -18.7 (c= 1.9; CHCl 3 )
	Melting point
	T m = 98-99°C
	Yield
	93% (136 mg)

  NMR (250 MHz, CDCl 3 ), d 7.30 (d, 2H,H aromatic , J= 8.5 Hz), 6.89 (d, 2H,H aromatic , J= 8.7 Hz), 4.74 (d, 1H, CH 2 (OPMB), J= 11.8 Hz), 4.68 (d, 1H, CH 2 (OPMB), J= 11.8 Hz), 4.54 (dt, 1H, H4, J 4,5

1 H

  [M+NH 4 ] + calculated for C 17 H 28 NO 7 m/z=358.186029; found m/z= 358.185917 [M+Na] + calculated for C 17 H 24 NaO 7 m/z=363.141424; found m/z= 363.141514 Cl 2(50 mL) was added. The mixture was washed with 1N agueous HCl (2 x 20 mL), then with water (20 mL) and dried over MgSO 4 . After concentration under vacuum and coevaporation with toluene, the crude product was obtained as a colorless oil, which was used in the next step without purification.

	1-O-Acetyl 2,3:4,6-di-O-isopropylidene-a-L-sorbofuranose 58
	5		2
	6	4	3	1	C	14 H 22	O7
					MW = 302.33 g/mol
	To a solution of 2,3:4,6-di-O-isopropylidene-a-L-sorbofuranose (200 mg, 0.77 mmol) in
	pyridine (4 mL) was added Ac 2 O (0.36 mL, 3.84 mmol, 5 eq). The reaction was left stirring
	overnight at RT. CH 2
	Optical rotation
	= -57.6 (c= 0.9; MeOH)
	Melting point
	T m = 119-120°C
	Yield
	21 % (25 mg)

  H 2 SO 4 on silica was prepared according to the protocol of Mukhopadhyay and co-workers.212 To a solution of acetate 58 (100 mg, 0.33 mmol) in MeOH (1.7 mL), silica-H 2 SO 4 (33 mg) was added. The reaction was left stirring 4 h at RT. The suspension was filtered through a membrane and the filtrate was concentrated under vacuum to give crude mixture of compound 59 and 60. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 95:5) gave pure products 59 and 60 as yellow oils. H NMR (250 MHz, CD 3 OD), d 4.39 (s, 1H, H3), 4.29 (ddd 1H, H5, J 5,4 = 2.8 Hz, J 5,6b = 5.3 Hz, J 5,6a = 6.3 Hz),4.13 (d, 1H, H4, J 4,5 = 2.8 Hz), 3.81 (dd, 1H, H6 b , J 6b,5 = 5.3 Hz, J 6a,6b = 11.5 Hz), 3.73 (dd, 1H, H6 a , J 6a,5 = 6.3 Hz, J 6a,6b = 11.5 Hz),3.68 (s, 2H, 2H1), 1.48, 1.36 (s, 3H, CH 3 iPr).

	1-O-Acetyl-2,3-O-isopropylidene-a-L-sorbofuranose 59 and 2,3-O-isopropylidene-a-L-
					sorbofuranose 60
	C MW = 262.27 g/mol O 7 11 H 18 13 C NMR (62.5 MHz, CD 3 OD), d 115.54, 113.12 (C IV iPr, C2), 86.79 (C3), 83.33 (C5), 75.65 O MW = 220.22 g/mol 6 C 2 2 9 5 H 16 5 (C4), 63.78 (C1), 61.13 (C6), 27.77, 26.86 (CH 3 iPr).
	6	4	3	1	6	4	3	1
		59					60
	Compound 59:
	TLC:
	• CH 2 Cl 2 /MeOH (9:1);
	• R f = 0.50
	1 H NMR (400 MHz, CDCl 3 ), d 4.56 (d, 1H, H1 b , J 1b,1a = 12.0 Hz), 4.43 (s, 1H, H3), 4.33 (d, 1 H NMR (250 MHz, CD 3 OD), d 4.46 (d, 1H, H1 b , J 1b,1a = 11.8 Hz), 4.40 (s, 1H, H3), 4.28 1H, H4, J 4,5 = 2.0 Hz), 4.17 (d, 1H, H1a, J 1a,1b = 12.0 Hz), 4.12 (q, 1H, H5, J~2 Hz), 4.10-4.00 (ddd 1H, H5, J 5,4 = 2.8 Hz, J 5,6b = 5.0 Hz, J 5,6a = 6.25 Hz), 4.17 (d, 1H, H4, J 4,5 = 2.8 Hz), 4.09 (m, 2H, 2H6), 2.11 (s, 3H, OAc), 1.51, 1.44, 1.39, 1.37 (s, 3H, CH 3 iPr). (d, 1H, H1 a , J 1a,1b =11.8 Hz), 3.80 (dd, 1H, H6 b , J 6b,5 = 5.0 Hz, J 6a,6b = 11.8 Hz), 3.74 (dd, 1H,
	13 C NMR (100 MHz, CDCl 3 ), d 169.97 (C=O from OAc), 112.56, 112.16 (C IV iPr), 97.28 H6 a , J 6a,5 = 6.25 Hz, J 6a,6b = 11.8 Hz), 2.07 (s, 3H, OAc), 1.47, 1.36 (s, 3H, CH 3 iPr).
	(C2), 84.55 (C3), 73.11 (C4), 72.34 (C5), 63.42 (C1), 60.04 (C6), 28.82, 27.34, 26.26, 18.57 13 C NMR (100 MHz, CD 3 OD), d 171.98 (C=O from OAc), 113.67, 113.23 (C IV iPr, C2), (CH 3 iPr), 20.76 (CH 3 CO). 87.00 (C3), 83.41 (C5), 75.76 (C4), 64.49 (C1), 61.13 (C6), 27.72, 26.77 (CH 3 iPr), 20.75
	(CH 3 CO). HRMS (ESI) [M+H] + calculated for C 14 H 23 O 7 m/z= 303.143829; found m/z= 303.140610 [M+NH 4 ] + calculated for C 14 H 26 NO 7 m/z= 320.170379; found m/z= 320.170543 [M+Na] + calculated for C 14 H 22 NaO 7 m/z= 325.125774; found m/z= 325.125708 LRMS (ESI) [M+Na] + = 285.0
	Yield
	Optical rotation 34% (30 mg)
	= -13.9 (c= 1.0; CHCl 3 )
	Yield
	Quant. (233 mg) Compound 59:
	TLC:
	• CH 2 Cl 2 /MeOH (9:1);
	• R f = 0.31

1

  4 ] + calculated for C 20 H 32 NO 7 m/z= 398.217329; found m/z= 398.217460 [M+Na] + calculated for C 20 H 28 NaO 7 m/z= 403.172724; found m/z= 403.172497

	1-O-p-Methoxybenzyl-2,3-O-isopropylidene-a-L-sorbofuranose 62
	5		2
	6	4	3	1	C	17 H 24	O	7
					MW = 340.15 g/mol
	Optical rotation
	= -4.8 (c= 1.0; CHCl 3 )
	Yield
	87% (127 mg)

  NMR (400 MHz, CDCl 3 ), d 7.24 (d, 2H, H aromatic , J= 8.5 Hz), 6.86 (d, 2H, H aromatic , J= 8.6 Hz), 4.60 (d, 1H, CH 2 (OPMB), J= 11.3 Hz), 4.51 (d, 1H, CH 2 (OPMB), J= 11.3 Hz), 4.40 (br s, 1H, H3), 4.33 (td, 1H, H5, J 4,5 = 2.8 Hz, J 5,6 = 5.2 Hz), 4.16 (dd, 1H, H4, J 4,5 = 2.7 Hz, J 4,OH = 10.6 Hz), 3.89 (br t, 2H, H6, J 6,5 = J 6,OH = 5.6 Hz), 3.84 (d, 1H, 4-OH, J= 10.

	6 Hz), 3.80 (d,
	1H, H1 b , J 1b,1a = 10.0 Hz), 3.79 (s, 3H, CH 3 O), 3.61 (d, 1H, H1 a , J 1a,1b = 10.0 Hz), 2.63 (t, 1H,
	CH 2 OH, J= 6.1 Hz), 1.50, 1.30 (s, 3H, CH 3 iPr).
	13 C NMR (100 MHz, CDCl 3 ), d 159.63 (C IV aromatic ), 129.66 (C aromatic ), 128.82 (C IV aromatic ), 114.05 (C aromatic ), 112.72, 112.41 (C IV iPr, C2), 86.82 (C3), 81.87 (C5), 75.31 (C4), 73.67
	(CH 2 (OPMB)), 71.13 (C1), 61.09 (C6), 55.29 (CH 3 O),

1 H

  [M+H] + calculated for C 17 H 25 NO 7 m/z= 341.159480; found m/z= 341.159301 [M+Na] + calculated for C 17 H 24 NaO 7 m/z= 363.141424; found m/z= 363.141371

	1,2:4,6-Di-O-isopropylidene-a-L-tagatofuranose 64
	5		2		C 12 H 20 O 6
	6	4	3	1	MW = 260.28 g/mol
	Optical rotation
	= +4.2 (c= 1.4; MeOH)
	Melting point
	T m = 60-61°C
	Yield
	80% (90 mg)

  To a solution of crude aldehyde 66(1.93 mmol) in dry CH 2 Cl 2

	6-(S R )-N-tert-butanesulfinylimino-6-deoxy-1,2:3,4-Di-O-isopropylidene-a-L-tagatofuranose
				68
	5		2		C16H27NO6S
	6	4	3	1	MW = 361.16 g/mol
	1 H NMR (400 MHz, CDCl 3 ), d 8.02 (d, 1H, H6, J 6,5 = 4.2 Hz), 4.99 (dd, 1H, H4, J 4,5 = 4.2 Hz,
	J 4,3 = 6.0 Hz), 4.73 (t, 1H, H5, J 5,6 = J 5,4 = 4.2 Hz), 4.65 (d, 1H, H3, J 3,4 = 6.0 Hz), 4.33 (d, 1H,
	H1 b , J 1b,1a = 10.0 Hz), 4.16 (d, 1H, H1 a , J 1a,1b = 10.0 Hz), 1.47, 1.41, 1.39, 1.27 (s, 3H,
	CH 3 iPr), 1.23 (s, 9H, CH 3 tBu).
	13 C NMR (100 MHz, CDCl 3 ), d 166.07 (C6), 113.48, 112.52, 112.18 (C IV iPr, C2), 85.04 (C3), 82.04 (C4), 80.88 (C5), 69.40 (C1), 57.48 (C IV tBu), 26.57, 26.38, 26.11, 25.08
	(CH 3 iPr), 22.58 (CH 3 tBu).
	HRMS (ESI) [M+H] + calculated for C 16 H 28 NO 6 S m/z= 362.163185; found m/z= 362.163434 [M+Na] + calculated for C 16 H 27 NNaO 6 Sm/z= 384.145129; found m/z= 384.145380
	Optical rotation
	= +227.2 (c=1.2; CHCl 3 )
	Melting point
	T m = 98-100°C
	IR [cm -1 ] n 2986 (C-H), 1631 (N=C), 1457, 1373, 1209, (C-O/C-C), 1071, 1033 (S=O), 854.
	Yield
	71 % (510 mg) for two steps from 65.

  IV tBu),32.03, 31.77, 29.70, 29.63, 29.44, 25.78, 22.82 (CH 2 nonyl), 26.68, 26.57, 26.22, 24.89 (CH 3 iPr), 22.80 (CH 3 tBu),14.25 (CH 3 nonyl). NMR (400 MHz, CDCl 3 ), d 4.77 (dd, 1H, H4, J 4,5 = 3.2 Hz, J 4,3 = 5.6 Hz), 4.61 (d, 1H, H3, J 3,4 = 5.6 Hz), 4.25 (d, 1H, H1 b , J 1b,1a = 9.6 Hz), 4.05 (d, 1H, H1 a , J 1a,1b = 9.6 Hz),3.92 (dd, 1H, H5, J 5,4 = 3.2 Hz, J 5,6 = 8.4 Hz), 3.78 (d, 1H, NH, J= 3.2 Hz), 3.72-3.67 (m, 1H, H6), 1.87-1.76 calculated for C 25 H 48 NO 6 S m/z= 490.319686; found m/z= 490.319794 [M+Na] + calculated for C 25 H 47 NNaO 6 S m/z= 512.301630; found m/z= 512.301529

	2 nd isomer 75R:
	(m, 1H, CH 2 nonyl), 1.74-1.63 (m, 1H, CH 2 nonyl), 1.45, 1.41, 1.40 (s, 3H, CH 3 iPr), 1.29-
	1.20 (m, 26H, CH 3 iPr, CH 2 nonyl, CH 3 tBu), 0.94-0.84 (m, 3H, CH 3 nonyl).
	13 C NMR (100 MHz, CDCl 3 ), d 113.05, 111.71, 111.55 (C IV iPr, C2), 85.66 (C3), 80.38 (C5), 79.96 (C4), 69.42 (C1), 55.67 (C IV tBu), 53.30 (C6), 32.01, 29.97, 29.89, 29.65, 29.61, 29.42,
	24.46, 22.79 (CH 2 nonyl), 26.64, 26.44, 26.18, 24.94 (CH 3 iPr), 22.77 (CH 3 tBu), 14.22 (CH 3
	nonyl).
	HRMS (ESI)
	[M+H] Optical rotation
	= -64.4 (c= 1.0; CHCl 3 )
	Yield
	29 % (68 mg)
	3 rd product: 76
	5		2		C16H29NO6S
	6	4	3	1	MW = 363.47 g/mol
	1 H NMR (400 MHz, CDCl 3 ), d 4.94 (dd, 1H, H4, J 4,5 = 3.6 Hz, J 4,3 = 5.6 Hz), 4.56 (d, 1H, H3,
	J 3,4 = 5.6 Hz), 4.21 (d, 1H, H1 b , J 1b,1a = 9.6 Hz), 3.99 (d, 1H, H1 a , J 1a,1b = 9.6 Hz), 3.96 (dd, 1H,
	H5, J 5,4 = 3.6 Hz, J 5,6 = 9.2 Hz), 3.68 (d, 1H, NH, J= 5.6 Hz), 3.46-3.39 (m, 1H, H6), 1.84-1.69
	(m, 3H, CH 2 nonyl), 1.61-1.48 (m, 1H, CH 2 nonyl), 1.44, 1.41, 1.38 (s, 3H, CH 3 iPr), 1.34-
	1.22 (m, 24H, CH 2 nonyl, CH 3 iPr, CH 3 tBu), 0.90-0.86 (m, 3H, CH 3 -nonyl). TLC:
	• PE/EtOAc (8:2); 13 C NMR (100 MHz, CDCl 3 ), d 112.48, 111.91, 111.67 (C IV iPr, C2), 85.11 (C3), 81.85 (C5), • R f = 0.05 1 H NMR (400 MHz, CDCl 3 ), d 4.80 (dd, 1H, H4, J 4,5 = 4.0 Hz, J 4,3 = 6.0 Hz), 4.60 (d, 1H, H3, J 3,4 = 6.0 Hz), 4.24 (d, 1H, H1 b , J 1b,1a = 9.9 Hz), 4.17-4.12 (m, 1H, H5), 4.02 (d, 1H, H1 a , 80.41 (C4), 69.50 (C1), 56.07 (C6), 55.99 (C HRMS (ESI) J 1a,1b = 9.9 Hz), 3.55 (t, 1H, NH, J=6.4 Hz), 3.53-3.46 (m, 1H, H6 b ), 3.39-3.23 (m, 1H, H6 a ), [M+H] + calculated for C 25 H 48 NO 6 S m/z= 490.319686; found m/z= 490.319736 [M+Na] + calculated for C 25 H 47 NNaO 6 S m/z= 512.301630; found m/z= 512.301462 1.45, 1.41, 1.39, 1.30 (s, 3H, CH 3 iPr), 1.22 (s, 9H, CH 3 tBu).
	Optical rotation 13 C NMR (100 MHz, CDCl 3 ), d 112.98, 111.96, 111.79 (C IV iPr, C2), 85.40 (C3), 80.19 (C4), 78.72 (C5), 69.41 (C1), 55.96 (C IV tBu), 44.96 (C6), 26.78, 26.56, 26.16, 24.96 (CH 3 iPr), = -53.0 (c= 1.0; CHCl 3 ) 22.67 (CH 3 tBu).
	Yield
	32 % (74 mg)

1 H +

  calculated for C 18 H 32 N 3 O 5 m/z= 370.233648; found m/z= 370.233650 [M+NH 4 ] + calculated for C 18 H 35 N 4 O 5 m/z= 387.260197; found m/z= 387.260265 [M+Na] + calculated for C 18 H 31 N 3 NaO 5 m/z= 392.215592; found m/z= 392.215360

	(6S)-6-Azido-6-deoxy-6-C-nonyl-1,2:3,4-di-O-isopropylidene-a-L-tagatofuranose 85
	5		2		C21H37N3O5
	6	4	3	1	MW = 411.54 g/mol
					55-
	1.21 (m, 9H, CH 2 -hexyl), 1.50, 1.42, 1.41, 1.30 (s, 3H, CH 3 iPr), 0.89 (t, 3H, CH 3 -hexyl, J=
	6.4 Hz).
	13 C NMR (100 MHz, CDCl 3 ), d 112.97, 111.95, 111.72 (C IV iPr, C2), 85.47 (C3), 82.14 (C5),
	79.93 (C4), 69.46 (C1), 61.80 (C6), 31.75, 30.40, 29.00, 25.85, 22.71 (CH 2 -hexyl), 26.76,
	26.16, 25.06 (CH 3 iPr), 14.19 (CH 3 -hexyl).
	HRMS (ESI)
	[M+H] Optical rotation
	= -21.8 (c= 1.5; CHCl 3 )
	IR [cm -1 ] n 2989, 2931, 2859 (C-H), 2098 (N 3 ), 1381, 1372, 1263, 1209, 1075, 1029 (C-O/C-
	C).
	Yield
	85% (100 mg) for two steps, from 73R

1 H NMR (400 MHz, CDCl 3 ), d 4.70 (dd, 1H, H4, J 4,5 = 3.6 Hz, J 4,3 = 6.0 Hz), 4.60 (d, 1H, H3, J 3,4 = 6.0 Hz), 4.28 (d, 1H, H1 b , J 1b,1a =9.6 Hz), 4.07 (d, 1H, H1 a , J 1a,1b = 9.6 Hz), 3.90 (dd, 1H, H5, J 5,4 =3.6 Hz, J 5,6 = 9.2 Hz), 3.64-3.56 (m, 1H, H6),1.69-1.55 (m, 1H, CH 2 -hexyl), 1.+

  [M+NH 4 ] + calculated for C 21 H 41 N 4 O 5 m/z= 429.307147; found m/z= 429.307176 [M+Na] + calculated for C 21 H 37 N 3 NaO 5 m/z= 434.262542; found m/z= 434.262603

	Yield
	61% (35 mg) for two steps, from 75R

  NMR (100 MHz, CD 3 OD),d 106.56 (C2 min ), 104.22 (C2 MAJ ), 84.20 (C5 MAJ ), 83.19 (C5 min ), 80.17 (C3 min ), 73.30 (C4 min ), 73.01 (C4 MAJ ), 72.03 (C3 MAJ ), 65.76 (C6 MAJ ), 64.08 (C6 min ), 63.94 (C1 min ), 63.74 (C1 MAJ ),

13 C

  NMR (400 MHz, CD 3 OD), d 3.98-3.97 (m, 0.2H, H4 N,methyl ), 3.85 (br s, 0.8H, H4), 3.83-3.81 (m, 0.4H, 2H6 N,methyl ),3.63 (d, 1.6H, 2H6, J 6,5 = 6.5 Hz), 3.56 (t, 0.2H, H2 N,methyl , J 2,3 = 9.2 Hz),3.32-3.28 (m, 1.6H, H2, H3), 3.24 (dd, 0.2H, H3 N,methyl , J= 3.2 Hz, J 3,2 = 9.2 Hz), 2.69 (t, 0.8H, H5, J 5,6 = 6.5 Hz), 2.40-2.32 (m, 0.8H, H1), 2.25 (s, 0.6H, N-CH 3 ), 2.23-2.19 (m, 0.2H, H5 N,methyl ), 1.95-1.77 (m, 1H, H1 N,methyl , CH 2 -hexyl), 1.77-1.68 (m, 0.4H, CH 2hexyl N,methyl ), 1.57-1.45 (m, 08H, CH 2 -hexyl), 1.43-1.26 (m, 8H, CH 2 -hexyl, CH 2hexyl N,methyl ), 0.96-0.84 (m, 3H, CH 3 -hexyl, CH 3 -hexyl N,methyl ). MHz, CD 3 OD), d 77.45 (C3), 77.15 (C3 N,methyl ), 74.06 (C2), 71.92 (C4 N,methyl ), 70.99 (C2 N,methyl ), 70.79 (C4), 69.08 (C1 N,methyl ), 67.97 (C5 N,methyl ), 63.51 (C6), 63.05 (C6 N,methyl ), 61.38 (C1), 60.87 (C5), 37.43 (N-CH 3 ), 33.05 (CH 2 -hexyl N,methyl ), 32.99 (CH 2hexyl), 32.95 (CH 2 -hexyl), 30.99 (CH 2 -hexyl N,methyl ), 30.76 (CH 2 -hexyl), 29.78 (CH 2hexyl N,methyl ), 26.73 (CH 2 -hexyl), 25.92 (CH 2 -hexyl N,methyl ), 23.75 (CH 2 -hexyl N,methyl ), 23.69 (CH 2 -hexyl), 14.43 (CH 3 -hexyl N,methyl ), 14.42 (CH 3 -hexyl). [M+H] + calculated for C 12 H 26 NO 4 m/z= 248.185635; found m/z= 248.185964 [M+H] + calculated for C 13 H 28 NO 4 m/z= 262.201285; found m/z= 262.201324

	HRMS (ESI)
	Yield
	14.5 mg
	Note:

1 H

13 C NMR (100

  NMR (400 MHz, CD 3 OD), d 3.93 (dd, 0.06H, H6 b,gluco , J 6b,5 = 2.8 Hz, J 6b,6a = 10.8 Hz),3.86 (d, 0.94H, H4, J 4,5 = 1.2 Hz), 3.65 (d, 1.88H, 2H6, J 6,5 = 6.4 Hz), 3.50 (dd, 0.06H, H6 a,gluco , J 6a,5 = 8.0 Hz, J 6a,6b = 10.8 Hz),3.35-3.29 (m, 1.88H, H2, H3), 3.20 (t, 0.06H, H3 gluco , J 3,4 =J 3,2 = 9.2 Hz), 3.11 (t, 0.06H, H4 gluco , J 4,3 =J 4,5 = 9.2 Hz), 2.99 (t, 0.06H, H2 gluco , J 2,3 =J 2,1 = 9.2 Hz), 2.69 (dt, 0.94H, H5, J 5,4 = 1.2 Hz, J 5,6a = J 5,6b = 6.4 Hz), 2.57-2.52 (m, 0.06H, H5 gluco ), 2.41-2.31 (m, 1H, H1, H1 gluco ), 1.92-1.80 (m, 1H, CH 2 -hexyl), 1.58-1.44 (m, 1H, CH 2 -hexyl), 1.44-1.24 (m, 8H, CH 2 -hexyl), 0.912 (t, 1H, CH 3 -hexyl, J= 6.6 Hz). MHz, CD 3 OD), d 80.61 (C3 gluco ), 77.45, 74.07 (C3, C2), 76.65 (C2 gluco ), 73.67 (C4 gluco ), 70.80 (C4), 63.52 (C6), 63.67 (C6 gluco ), 62.48 (C5 gluco ), 61.37 (C1), 60.86 (C5), 60.75 (C1 gluco ),

1 H

13 C NMR (100

  NMR (400 MHz, CDCl 3 ), δ 7.85 (d, 2H, H aromatic , J= 8.3 Hz), 7.39-7.27 (m, 7H, H aromatic ), 4.92 (d, 1H, H1, J 1,2 = 1.2 Hz) ,4.72 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.49 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.41 (ddd, 1H, H4, J 4,3 = 6.8 Hz, J 4,5b = 5.2 Hz, J 4,5a = 9.6 Hz), 4.18 (dd, 1H, H3, J 3,4 = 6.8 Hz, J 3,2 = 5.2 Hz), 4.11 (dd, 1H, H2, J 2,1 = 1.2 Hz, J 2,3 = 5.2 Hz), 3.85 (dd, 1H, H5 b , J 5b,4 =5.2 Hz, J 5b-5a = 11.6 Hz), 3.71 (dd, 1H, H5 a , J 4,5a = 9.6 Hz, J 5a-5b = 11.6 Hz), 2.44 (s, 3H, CH 3 Ts), 1.24, 1.14 (s, 3H, CH 3 iPr). CDCl 3 ), d 144.98-133.08 (C IV aromatic ) 129.76-128.07 (C aromatic ), 109.65 (C IV iPr), 96.34 (C1), 76.98 (C4), 75.62 (C2), 74.52 (C3), 69.36 (CH 2 Ph), 58.58 (C5), 27.33-26.140 (CH 3 iPr), 21.61 (CH 3 Ts).

1 H 13 C NMR (100 MHz,

  CDCl 3 ), d 7.40-7.25 (m, 5H, H aromatic ), 5.01 (d, 0.5H, H1, J 1,2 = 1.75 Hz), 4.85 (d, 0.5H, CH 2 Ph, J= 12.0 Hz), 4.78 (d, 0.5H, CH 2 Ph, J= 11.75 Hz), 4.68 (d, 0.5H, H1, J 1,2 = 3.75 Hz), 4.61 (d, 1H, CH 2 NO 2 , CH 2 Ph, J= 12.0 Hz), 4.59 (d, 0.5H, CH 2 NO 2 , J= 11.25 Hz), 4.56 (d, 0.5H, CH 2 Ph, J= 11.75 Hz), 4.49 (d, 0.5H, CH 2 NO 2 , J=12.0 Hz) 4.40 (d, 0.5H, CH 2 NO 2 , J= 11.25 Hz), 4.29 (dd, 0.5H, H3, J= 1.25 Hz, J 2,3 = 6.2 Hz), 4.24 (dd, 1H, H2, J 2,1 = 3.75 Hz, J 2,3 = 6.2 Hz), 4.17-4.11 (m, 1.5H, H2, H3, OH), 3.92 (dd, 0.5H, H5 b , J= 1.1 Hz,J 5b,5a = 12.1 Hz), 3.84-3.79 (m, 1.5H, H5 a , H5 b , H5 a ), 3.07 (br s, 0.5H, OH),1.53, 1.39, 1.36, 1.33 (s, 6H, CH 3 iPr). MHz, CDCl 3 ), d 136.57, 136.30 (C IV aromatic ), 128.64-128.24 (C aromatic ), 110.52, 110.47 (C IV iPr), 98.68 (C1), 97.11 (C1), 79.71 (CH 2 NO 2 ), 78.86 (CH 2 NO 2 ), 76.24 (C3), 74.48 (C2), 74.38 (C3), 72.88 (C2), 70.46 (CH 2 Ph), 69.88 (CH 2 Ph),

• R f = 0.21 Note: Signals for 112 are underlined. Signals for 113 are in italics. 1 H NMR (250 MHz, 13 C NMR

(62.5

  -1 ] n 3427 (O-H), 2988, 2935 (C-H), 1550 (NO 2 ), 1497, 1455, (C=C aromatic ), 1379 (NO 2 ), 1246, 1217, 1161, 1136, 1065, 1019 (C-O/C-C), 739, 699, (C-H aromatic ). [M+H] + calculated for C 16 H 22 NO 7 m/z= 340.139078; found m/z= 340.1399429 [M+NH 4 ] + calculated for C 16 H 25 NO 7 m/z= 357.165628; found m/z= 357.165800 [M+Na] + calculated for C 16 H 21 NNaO 7 m/z= 362.121023; found m/z= 362.121138 [M+K] + calculated for C 16 H 21 KNO 7 m/z= 378.094960; found m/z= 378.094935

	HRMS (ESI)
	Yield
	27% (56 mg)

13 C NMR

  (62.5 MHz, CD 3 OD), d 110.51 (C IV iPr), 74.17 (C3),71.72 (C2),69.87 (C4), 66.81 (C6), 49.49 (C5), 45.77 (C1), 26.72, 25.76 (CH 3 iPr). [M+H] + calculated for C 9 H 18 NO 4 m/z= 204.123034; found m/z= 204.123362 [M+Na] + calculated for C 9 H 17 NNaO 4 m/z= 226.104979; found m/z= 226.105209 H NMR (400 MHz, CD 3 OD), d 4.34 (dt, 1H, H2, J 2,3 = J 2,1b = 5.6 Hz, J 2,1a = 7.0 Hz), 4.11 (dd, 1H, H3, J 3,5 = 1.2 Hz, J 3,2 = 5.6 Hz), 3.59 (d, 1H, H6 b , J= 11.6 Hz), 3.48 (d, 1H, H6 a , J= 11.6 Hz), 3.21 (dd, 1H, H1 b, J 1b,2 = 5.6 Hz, J 1b,1a = 13.2 Hz), 2.85 (d, 1H, H5 b , J= 13.4 Hz), 2.78 (d, 1H, H5 a , J= 13.4 Hz), 2.74 (dd, 1H, H1 a , J 1a,2 = 7.0 Hz, J 1a,1b = 13.2 Hz), 1.48, 1.35 (s, 3H, CH 3 iPr). 13 C NMR (100 MHz, CD 3 OD), d 110.66 (C IV iPr), 75.64 (C3), 71.91 (C2), 71.63 (C4), 66.33 (C6), 46.76 (C5), 45.84 (C1), 27.93, 25.67 (CH 3 iPr). [M+H] + calculated for C 9 H 18 NO 4 m/z= 204.123034; found m/z= 204.123371

	(3R, 4R, 5S)-5-Hydroxymethyl-piperidine-3,4,5-triol 118
	5		1	C6H13NO4
	4	3	2
	6			MW=163.17 g/mol
	HRMS (ESI)
	Yield
	23% (28 mg)
	HRMS (ESI)
	Yield
	31% (37 mg)

1

  NMR (400 MHz, CDCl 3 ), 7.47-4.28 (m, 5H, H aromatic ), 5.05 (br s, 1H, H1),4.75 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.60-4.51 (m, 1H, H6), 4.54 (d, 1H, CH 2 Ph, J= 11.6 Hz), 4.26 (d, 0.7H, H3 MAJ , J= 6.4 Hz), 4.17-4.09 (m, 1.35H, H2 MAJ , H3 min , H2 min ), 3.88 (d, 0.3H, H5 b min , J= 12.4 Hz), 3.81 (d, 0.7H, H5 b MAJ , J= 12.0 Hz), 3.73 (d, 0.7H, H5 a MAJ , J= 12.0 Hz), 3.58 (d, 0.3H, H5 a min , J= 12.4 Hz), 3.04 (s, 0.7H, OH MAJ ), 2.96 (s, 0.3H, OH min ), 2.16-1.95 (m, 1.3H, H7 b MAJ , 2H7 min ), 1.72-1.65 (m, 0.7H, H7 a MAJ ), 1.55 (s, 0.9H, CH 3 iPr min ), 1.54 (s, 2.1H, CH 3 iPr MAJ ), 1.37 (s, 0.9H, CH 3 iPr min ), 1.36 (s,2.1H, CH 3 iPr MAJ ), 1.30-1.29 (m, 6H, CH 2hexyl), 0.95-0.82 (m, 3H, CH 3 -hexyl). NMR (100 MHz, CDCl 3 ), d 136.65 (C IV aromatic ), 128.69-128.28 (C aromatic ), 110.24 (C IV iPr MAJ ), 110.15 (C IV iPr min ), 96.60 (C1 MAJ ), 96.40 (C1 min ), 92.71 (C6 min ), 92.68 (C6 MAJ ), 74.35 (C2 MAJ ), 74.32 (C2 min ), 72.52 (C3 min ), 72.03 (C3 MAJ ), 69.74 (CH 2 Ph MAJ ), 69.66 (CH 2 Ph min ), 69.49 (C4 MAJ ), 69.05 (C4 min ), 61.26 (C5 min ), 60.79 (C5 MAJ ),

1 H

13 C

  NMR (400 MHz, CD 3 OD), d 7.43-7.28 (m, 5H, H aromatic ), 4.90 (d, 1H, H1, J 1,2 = 3.3 Hz); 4.79 (d, 1H, CH 2 Ph, J= 11.90 Hz), 4.61 (d, 1H, CH 2 Ph, J= 11.90 Hz), 4.36 (d, 1H, H3, J 3,2 = 5.8 Hz), 4.13 (dd, 1H, H2, J 2,1 = 3.3 Hz, J 2,3 = 5.8 Hz), 3.78 (d, 1H, H5 b , J 5b,5a = 12.0 Hz), 3.57 (d, 1H, H5 a , J 5a,5b = 12.0 Hz), 2.96 (dd, 0.7H, H6 MAJ , J= 2.4 Hz, J= 9.6 Hz), 2.81-2.79 (m, 0.3H, H6 min ), 1.69-1.26 (m, 14H, CH 2 -hexyl, CH 3 iPr), 0.95 (t, 3H, CH 3 -hexyl, J= 6.6 Hz). MHz, CD 3 OD), d 138.63 (C IV aromatic ), 129.41, 129.19, 128.90 (C aromatic ), 110.96 (C IV iPr MAJ ), 110.63 (C IV iPr min ), 99.73 (C1 min ), 99.56 (C1 MAJ ), 76.33 (C2), 76.09 (C3), 71.20 (C4), 70.76 (CH 2 Ph), 64.84 (C5 min ), 63.52 (C5 MAJ ), 58.39 (C6 min ), 57.59 (C6 MAJ ),

1 H

13 C NMR (100

  (CH 2 -hexyl MAJ ),31.66, 27.48 (CH 2 -hexyl min ), 26.92 (CH 3 iPr min ), 26.84, 25.96 (CH 3 iPr MAJ ), 14.39 (CH 3 -hexyl) HRMS (ESI) [M+H] + calculated for C 21 H 34 NO 5 m/z= 380.243150; found m/z= 380.243272

	Yield
	85% (228 mg)

  NMR (400 MHz, CD 3 OD), d 4.31-4.27 (m, 1H, H2), 4.23 (d, 1H, H3, J= 6.0 Hz), 3.61 (s, 2H, 2H6), 3.16 (dd, 1H, H1 b , J 1b,2 = 5.4 Hz, J 1b,1a = 13.2 Hz), 2.77-2.74 (m, 1H, H5), 2.71 (dd, 1H, H1 a , J 1a,2 = 7.6 Hz, J 1a,1b = 13.2 Hz), 1.61-1.20 (m, 14H, CH 2 -pentyl, CH 3 iPr), 0.92 (t, 3H, CH 3 -pentyl, J= 7.0 Hz). MHz, CD 3 OD), d 110.36 (C IV iPr), 76.95 (C3), 73.32 (C4), 72.29 (C2), 65.58 (C6), 56.60 (C5), 46.73 (C1),

1 H

13 C NMR (100

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H-gal-cer was counted. The specific activity of the enzyme was calculated as nmol/h/mg protein.145 Ishii, S.; Yoshioka, H.; Mannen, K.; Kulkarni, A. B.;Fan, J. Q. Biochim Biophys Acta 2004, 1690, 250.

Zhao, G-L. ; Yu, Z-Y.; Li, Y.; Pang, L-N.; Wang, J-W. Chin. J.Chem.,2008, 26, 158-164.

Biela, A.; Oulaïdi, F.; Gallienne, E.; Górecki, M.; Frelek, J.; Martin, O. R. Tetrahedron 2013,69, 3348.

C NMR (100 MHz, CD 3 OD), d 67.95 (C2), 67.25 (C4), 64.14 (C6), 62.37 (C5), 60.65 (C1), 38.10 (C3), 35.38 (C7), 20.04 (C8),

14.57 (C9).

Rajput, V. K.;Roy, B.; Mukhopadhyay, B. Tetrahedron Lett 2006, 47, 6987.

Melting point

Titz, A.;Radic, Z.;Schwardt, O.; Ernst, B. Tetrahedron Lett 2006, 47, 2383.

Anastasi, C.; Buchet, F. F.; Crowe, M. A.; Helliwell, M.; Raftery, J.; Sutherland, J. D. Chem.Eur. J. 2008Eur. J. , 14, 2375. .

Ichikawa, Y.; Igarashi, Y. Tetrahedron Lett 1995, 36, 4585.

nd isomer 116 (D-galacto-like):

Acknowledgements Acknowledgements ..................................................................................................................... Table of contents ........................................................................................................................

Abbreviations

The reaction was conducted under argon. Crystalline 1 (6.46 g, 39.4 mmol) was dissolved in dry DMF (120 mL) at RT. After 15 min of stirring in an ice bath, sodium hydride (60% dispersion in oil, 9.49 g, 237 mmol, 4.7 eq) was added portionwise. When H 2 evolution ceased (30 min), benzyl bromide (22 mL, 184 mmol, 6 eq) was slowly added. The reaction mixture was left stirring overnight at RT. The reaction was then quenched with ice-water (170 mL). EtOAc (300 mL) was added. The organic phase was washed with water (3 x 150 mL), with saturated NaHCO 3 (150 mL) and then dried over MgSO 4 . After concentration under vacuum the crude product was obtained as a yellow oil, which was used without purification in the next reaction.

TLC:

• PE/EtOAc (8:2);

• R f = 0.25

NMR

Obtained spectrum corresponds to literature data. 175 1-O-Acetyl 2,3,4-tri-O-benzyl-a,b-L-arabinopyranose 10. The reaction was conducted at RT. To a solution of compound 3 (1.0 g, 2.38 mmol) in pyridine (4.8 mL), Ac 2 O was added (1.2 mL, 12.8 mmol, 5.4 eq). The mixture was left stirring until TLC showed completion of the reaction (3 h). CH 2 Cl 2 (48 mL) was added. The mixture was washed with water (3 x 18 mL) and dried over MgSO 4 . After concentration under vacuum and coevaporation with toluene, the crude product was obtained as a colorless oil, which was used in the next step without purification.

TLC:

• PE/EtOAc (8:2);

• R f = 0.28 N-Benzyloxycarbonyl 2,3,4-tri-O-benzyl-a,b-L-arabinopyranosylamine 11. The reaction was conducted under argon at RT. Crude 10 (2.38 mmol) was dissolved in dry CH 2 Cl 2 (2.4 mL) containing 4Å molecular sieves. After 5 min of stirring, benzyl carbamate (0.72 g, 4.76 mmol, 2 eq), then trimethylsilyl trifluoromethanesulfonate (0.44 mL, 2.43 mmol, 1.0 eq) were added. The mixture was left stirring until TLC showed completion of the reaction (2 h). The reaction was quenched by the addition of NEt 3 (0.33 mL, 2.37 mmol, 1 eq). The solids were filtered over celite, washed with CH 2 Cl 2 and the filtrate was concentrated to give the crude product as a dark orange oil. Flash column chromatography on silica gel (toluene/acetone 95:5) gave the mixture of anomers (11a:11b 4:6) as a colourless oil.

TLC:

• PE/EtOAc (9:1);

• R f1 = 0.27; R f2 = 0.

IR [cm

TLC:

NMR

Obtained spectrum corresponds to literature data. 75.65, 74.62, 73.32, 72.80 (CH 2 Ph, C6), 66.52 (NHCOOCH 2 Ph), 34.33 (C7), 32.73 (C10), 31.88, 29.51, 29.03, 22.79 (C11-C14), 14.28 (C15). 55.70 (C6), 32.61, 31.89, 29.35, 25.56, 22.86 (CH 2hexyl), 26.68, 26.51, 26.13, 24.84 32.60, 32.02, 29.69, 29.67, 29.42, 25.58, 22.80 (CH 2 nonyl), 26.67, 26.51, 26.12, 24.83 (CH 3 iPr), 22.85 (CH 3 tBu), 14.24 (CH 3 nonyl).

HRMS (ESI)

HRMS (ESI)

[M+H] + calculated for To a solution of the azide 83 (50 mg, 0.14 mmol) in a mixture of acetonitrile/water (1:1) (5.6 mL) resin Dowex 50WX8 (H + ) (750 mg) was added. The reaction was left stirring at 85°C for 48h. The resin was filtered, washed with acetonitrile/water (1:1) and the filtrate was concentrated under vacuum to give crude product as a brownish solid. Flash column chromatography on silica gel (MeOH/CH 2 Cl 2 9:1) gave pure 87 as a colorless oil and as a mixture of anomers (7:3).

TLC:

• MeOH/CH 2 Cl 2 (9:1);

• R f = 0.28. 32.93, 32.63, 30.30, 30.27, 26.97, 26.82, 23.65 (CH 2hexyl), 14.40 (CH 3 -hexyl).

HRMS (ESI)

[M+Na] + calculated for To a solution of crude 95 (0.73 mmol) in DMF (7.3 mL) was added NaN 3 (95 mg, 1.46 mmol, 2 eq). The reaction was left stirring overnight at RT. After extraction with CH 2 Cl 2 (15 mL), the organic phase was washed with saturated NaHCO 3 (7 mL), water (2 x 7 mL) and dried over MgSO 4 . Concentration under vacuum afforded the crude product as a yellow solid. Flash column chromatography on silica gel (PE/EtOAc 95:5) gave pure 96 as a colorless oil.

TLC:

• PE/EtOAc (9:1);

• R f = 0.48 To a solution of 96 (159 mg, 0.56 mmol) in a mixture of acetonitrile/water (1:1) (22 mL), resin Dowex 50WX8 (H + ) (4.03 g) was added. The reaction was left stirring at 65°C for 70h. The resin was filtered, washed with MeOH and the filtrate was concentrated under vacuum to give crude product as a brownish solid. Flash column chromatography on silica gel (PE/EtOAc 9:1) gave pure 97 as a colorless oil and a mixture of anomers (7:3).

TLC:

• CH 2 Cl 2 /MeOH (9:1);

• R f = 0.097 To a solution of D-lyxose (20.0 g, 0.13 mol) in benzyl alcohol (37 mL) was added ptoluenesulfonic acid (201 mg, 1.10 mmol, 0.008 eq). The reaction was left stirring for 78h at 60°C. Then the mixture was cooled down to RT, concentrated under vacuum and coevaporated with toluene (3 x 30 mL) to give a soft white solid. This residue was slurred in a mixture of hexane/CH 2 Cl 2 (2:1) (180 mL). The solid was filtered, then stirred with cold Et 2 O (25 mL), filtered again and dried under vacuum to give the desired compound 98 as a white solid. The filtrate was concentrated and the procedure was repeated three times.

TLC:

• CH 2 Cl 2 /MeOH (9:1)

• R f = 0.58

NMR

The obtained spectrum corresponds to literature data 227 . The reaction was conducted under argon. To a solution of compound 98 (9.62 g, 0.04 mol) in acetone (128 mL) were added dimethoxypropane (16.7 mL, 0.14 mol, 3.5 eq) and ptoluenosulfonic acid (150 mg, 0.8 mmol, 0.02 eq). The reaction was left stirring overnight at RT. The mixture was concentrated under vacuum and the residue was diluted with EtOAc (120 mL). The solution was washed with a saturated solution of NaHCO 3 (40 mL), water (2 x 40 mL) and dried over MgSO 4 . Concentration under vacuum gave crude product as a colorless oil. Flash column chromatography on silica gel (PE/EtOAc 85:15/5:5) afforded compound 99 as a white solid.

Yield

TLC:

• toluene/EtOAc (8:2);

• R f = 0.22

NMR

The obtained spectrum corresponds to literature data 227 Yield 57% (6.46 g). The reaction was conducted under argon. To a solution of alcohol 99 (1.0 g, 3.57 mmol) in anhydrous CH 2 Cl 2 (36 mL) was added Dess-Martin periodinane (1.82 g, 4.28 mmol, 1.2 eq). The reaction was left stirring overnight. The mixture was concentrated under vacuum. The residue was suspended in the minimal volume of cold Et 2 O and filtered through a membrane. The filtrate was concentrated under vacuum to give the crude product as a colorless oil with white solids. Rapid filtration on silica gel (PE/EtOAc 8:2) gave the desired ketone 100 as a colorless oil.

TLC:

• PE/EtOAc (8:2);

• R f = 0.61

NMR

The obtained spectrum corresponds to literature data 228 .

LRMS (ESI)

[M+H] + = 279.0 To a solution of ketone 100 (2.2 g, 7.91 mmol) in ethanol (40 mL) at 0°C was added sodium borohydride (598 mg, 15.8 mmol, 2 eq). The reaction was left stirring at 0 °C for 1h and then at RT for 5h. Then it was stopped with 50% aqueous solution of NH 4 Cl (40 mL). EtOAc was added (30 mL). The aqueous phase was separated and washed with EtOAc (3 x 50 mL). The combined organic phases were washed with water (30 mL), brine (30 mL) and dried over MgSO 4 . After concentration under vacuum, the crude product was obtained as an amorphous, white solid. Crude compound was dissolved in hot Et 2 O (6 mL), then petroleum ether was slowly added (3 mL). The solution was left at 0 °C for about 1 h. White crystals of 101 were filtered and washed with hexane. The filtrate was concentrated and the procedure was repeated once.

Yield

TLC:

• PE/EtOAc (6:4);

• R f = 0.44

NMR

The obtained spectrum corresponds to literature data 228 .

LRMS (ESI)

[M+H] + = 281.0

[M+Na] + = 303.0 Yield 91% (2.0 g). The reaction was conducted under argon. To a solution of alcohol 101 (200 mg, 0.71 mmol) in dry CH 2 Cl 2 (7.1 mL) were added NEt 3 (0.22 mL, 1.57 mmol, 2.2 eq) and 4Å molecular sieves. Then methanesulfonyl chloride (0.12 mL, 1.50 mmol, 2.1 eq) was added. The reaction was left stirring for 4h. The solid was filtered and washed with CH 2 Cl 2 (10 mL). The filtrate was washed with a saturated aqueous NH 4 Cl solution (10 mL). The organic phase was separated, washed with brine (2 x 5 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product 103, which was used in the next step without further purification.

TLC:

• PE/EtOAc (7:3);

• R f = 0.28 Compound 105 (7.45 g, 0.017 mol) was dissolved in glacial acetic acid (28 mL) and the solution was heated at 110°C for 5 min. Then water (7 mL) was added and the mixture was heated for an additional 80 min. The solvents were evaporated under vacuum and the residue was dissolved in diethyl ether (80 mL). The organic phase was washed with H 2 O (10 mL), saturated NaHCO 3 (2 x 10 mL) and dried over MgSO 4 . The solvents were evaporated under vacuum to give compound 106, which was used in the next step without further purification.

LRMS (ESI)

TLC:

• PE/EtOAc (5:5);

• R f = 0.27 A cold solution of compound 106 (6.38 g, 16.19 mmol) in dry tetrahydrofuran (40 mL) was added at 0°C under argon to a suspension of potassium tert-butoxide (2.18 g, 19.40 mmol, 1.2 eq) in tetrahydrofuran (160 mL). The reaction was stirred for 45 min at RT and a saturated NH 4 Cl solution (60 mL) was then added. After 30 min of stirring, the mixture was extracted with EtOAc (2 x 160 mL). The organic phase was dried over MgSO 4 and the solvents were evaporated under vacuum. Flash column chromatography on silica gel (PE:EtOAc 5:5) afforded the pure compound 107 as a white crystalline solid.

TLC:

• PE/EtOAc (5:5);

• R f = 0.41 Compound 107 (1.84 g, 8.28 mmol) was dissolved under argon in dry dimethylformamide (18 mL). 2,6-Lutidine (2.2 mL, 18.88 mmol, 2.3 eq) was added, the mixture was cooled down to 0°C and triisopropylsilyltriflate (2.23 mL, 8.28 mmol, 1 eq) was added. The reaction was stirred at RT overnight and then diluted with EtOAc (60 mL). The organic phase was washed with 2N aqueous HCl (2 x 10 mL), water (10 mL), brine (10 mL) and dried over MgSO 4. Concentration under vacuum and flash column chromatography on silica gel (PE/EtOAc 95:5) afforded the pure compound 108 as a colorless oil.

TLC:

• PE/EtOAc (95:5);

• R f = 0.14 The reaction was conducted under argon. To a cold solution of compound 108 (2.54 g, 6.71 mmol) in dry diethyl ether (40 mL) was added dropwise a 1M solution of diethylaluminum cyanide in toluene (7.26 mL, 7.26 mmol, 1.1 eq). The mixture was stirred for 3h30 at 45°C then 1h at RT and then cooled to -40°C. A saturated NH 4 Cl solution (14 mL) was added dropwise and the mixture was stirred for 2h at RT. The solid was filtered and washed with EtOAc (2 x 10 mL). The filtrate was evaporated under vacuum and the residue was purified by flash column chromatography on silica gel (toluene/acetone 99.5:0.5) to give the pure compound 109 as a colorless oil.

TLC:

• PE/EtOAc (95:5);

• R f = 0.65 To a solution of 109 (1.585 g, 3.91 mmol) in tetrahydrofuran (50 mL) was added at 0°C a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran (7.5 mL, 7.5 mmol, 1.9 eq). The mixture was stirred for 3h30 at RT and the solvent was removed under vacuum. The residue was purified by flash column chromatography on silica gel (CH 2 Cl 2 /acetone 9:1) to give the pure compound 110 as a white solid.

TLC:

• CH 2 Cl 2 /acetone (9:1); To a solution of compound 110 (134 mg, 0.54 mmol) in MeOH (9 mL) were added a 12N solution of HCl (45 µL, 0.54 mmol, 1 eq) and 20% palladium hydroxide on carbon (117 mg). The reaction was stirred for 48h under a hydrogen atmosphere. Then the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated to give the expected galacto-isofagomine 111 (62 mg) containing 10% of Nmethyl-galacto-isofagomine. A sample of pure galacto-isofagomine 111 could be obtained by recrystallization from EtOH.

NMR

The obtained spectrum corresponds to literature data. 159

HRMS (ESI)

[M+H] + calcumated for C 6 H 14 NO 3 m/z= 148.096820; found m/z= 148.096935. The reaction was conducted under argon. To a solution of ketone 100 (567 mg, 2.04 mmol) in nitromethane (4.1 mL) was added triethylamine (0.85 mL, 6.10 mmol, 3 eq). The reaction was left stirring for 24 h and then was quenched with a saturated solution of NH 4 Cl (10 mL). The aqueous phase was separated and extracted with EtOAc (50 mL). The organic phase was washed with brine (15 mL) and dried over MgSO 4 . Concentration under vacuum gave the crude product as a beige solid. Flash column chromatography on silica gel (PE/EtOAc, 9:1) gave 112 as a white solid, containing about 8% of the other diastereoisomer.

TLC:

• PE/EtOAc (8:2);

• R f = 0.21 To a solution of 115 (36.8 mg, 0.18 mmol) in a mixture of dioxane/H 2 O (1:1, 1.8 mL) was added Dowex 50WX8 resin (H + ) (250 mg). The mixture was left stirring gently for 1 h. The resin was filtered and washed with MeOH. Then 0.5N aqueous solution of NH 3 was added to the filtrate and the mixture was left gently stirring for 30 min. The resin was filtered and washed with a 0.5N aqueous solution of NH 3 . The filtrate was concentrated to give the crude product as a white solid. Purification on Dowex 50WX8 resin (H + ) gave the pure 117 as a white solid. To a solution of the mixture of compounds 121 and 122 (228 mg, 0.6 mmol) in isopropanol (6 mL) were added glacial acetic acid (0.6 mL) and 20% Pd(OH) 2 (91 mg). The mixture was left stirring under a hydrogen atmosphere for 2 days. Then it was filtered through a membrane, retained catalyst was washed with MeOH and the solvents were evaporated to give the crude product as a thick, yellow oil. Flash column chromatography on silica gel (CH 2 Cl 2 /MeOH 9:1) gave a mixture of diastereoisomers 123+124 (7:3) as a colorless oil.

TLC:

• CH 2 Cl 2 /MeOH (9:1);

• R f = 0.15 To a solution of iminosugar 123 (18.4 mg, 0.067 mmol) in a mixture of dioxane/H 2 O (1:1, 1 mL) was added Dowex 50WX8 resin (H + ) (99 mg). The mixture was left stirring gently for 1h. The resin was filtered and washed with MeOH. Then a 0.5N aqueous solution of NH 3 was added to the resin and mixture was left stirring gently for 30 min. The resin was filtered and washed with a 0.5N aqueous solution of NH 3 . The filtrate was concentrated under vacuum to give 125 as a colorless oil.

1 H NMR (400 MHz, CD 3 OD), d 3. 95-3.82 (m, 2H, H2, H3), 3.67 (d, 1H, H6 b , J 6b,6a = 11.2 Hz), 3.53 (d, 1H, H6 a , J 6a,6b = 11.2 Hz), 2.85-2.69 (m, 3H, H5, 2H1), 1.61-1.43 (m, 2H, CH 2pentyl), 1.43-1.16 (m, 6H, CH 2 -pentyl), 0.97-0.85 (m, 3H, CH 3 -pentyl).

13 C NMR (100 MHz, CD 3 OD), d 75.54 (C4), 73.11 (C3), 67.47 (C2), 66.49 (C6), 55.84 (C5), 46.61 (C1), 33.18, 29.45, 27.70, 23.62 (CH 2 -pentyl), 14.42 (CH 3 -pentyl). Yield 56% (8.8 mg) (3R, 4R, 5S, 6RS)- 5-Hydroxymethyl-6-C-pentyl-piperidine-3,4,5- To a solution of a mixture of compounds 123+124 (106 mg, 0.39 mmol) in a mixture of dioxane/water (1:1, 4 mL) was added Dowex 50WX8 resin (H + ) (550 mg). The mixture was left stirring gently for 1h. The resin was filtered and washed with MeOH. Then a 0.5N aqueous solution of NH 3 was added and the mixture was left stirring gently for 30 min. The resin was filtered and washed with a 0.5N aqueous solution of NH 3 . The filtrate was concentrated under vacuum to give the crude product as a mixture of two diastereoisomers 125 and 126 (7:3) and as a colorless oil. Pure samples of 125 and 126 were isolated by HPLC (column Hypercarb, 250mm x 10mm, H 2 O/isopropanol/formic acid 99.5/0.5/0.4, pressure 3.3 bars, debit 4.4mL/min, T=30°C, ELS detection). 13 C NMR Only the major diastereoisomer 125 is fully visible on this spectrum and is very similar to the 13 C spectrum of compound 125.

HRMS (ESI)

[M+H] + calculated for C 11 H 24 NO 4 m/z= 234.169985; found m/z= 234.170167 Yield 60% (54 mg)