J L Jimenez Blanco

V M Diaz Perez

C Ortiz Mellet

J Fuentes

J M Garcia

M I Garcia Moreno

J C Diaz Arribas

PhD Thesis, S Silva

Recently, rare sugars have attracted much attention because of their countless applications 11 . D-Psicose might be an excellent sucrose substitute for food products due to its sweet taste, easy processing, and functional properties. 11, 12 Actually, research studies are being conducted in order to known how it can be used in diets to help fighting hyperglycemia, diabetes mellitus and arteriosclerosis diseases. Moreover, it has been shown to have a large number of chemotherapeutical properties 13 .

ACKNOWLEDGEMENTS

First of all, I would like to express my sincerely gratitude to my supervisors, Prof. Amélia Pilar Rauter and Prof. Patrick Rollin, for giving me the opportunity to develop this interesting subject of research. Thank you for your excellent supervision, stimulation, encouragement and support they were essential for the accomplishment of this project. I would also like to thank to Fundação para a Ciência e Tecnologia for the PhD grant SFRH/BD/25891/2005. In a particulary, I want to show my deep gratitude to Prof. Arnaud Tatibouët, for all knowledge transmitted, good advices, availability, patience, sympathy and friendship. It was very important to me and encouraging all along this project.

I want to express my grateful thanks to Prof. Jorge Justino for his great interest, motivation and availability to test my compounds for biological activity. I also want to thank Filipa Vinagre for the antimicrobial activity tests and Margarida Goulart for cytotoxicity tests.

I wish to express my gratitude to Professor Olivier Martin for having accepted me in ICOA, the institute he directs.

I also extend my warmest thanks to Prof. Isabel Ismael from University of Beira Interior and Dr. David Gueyrard from University Claude Bernard -Lyon 1 for having kindly accepted to judge this work. Moreover I would like to thank Professor Sergio Castillón from University of Rovira i Virgili -Tarragona for giving me the honor of participating in this PhD jury.

To all of ICOA, thank you for your kindness and companionship that make the lab a pleasant and enjoyable place to work. Among them I would like to thank in particulary Labo 1: Sandy, Deimas, Sophie, Sandrine, Ana Cristina, Jola, Estelle, Charlotte, Sara, Claudia, Andreia, Filipa, Joana, Séb, Julie, Benita, Vilija, Anthony, Balla and Leo. But also from others labs: Nick-Nick, grand Mathiew, Abdel, Mimi, petit Mathieu, Matteo, Aude, Aurélien, Cleo, Jérôme, Monika, Alexis, Fred, Joana, Stéphane, Catherine, Vincent, Julie Broggi, Mathilde, and the effective personnel Marie Madeleine, Jean-Marie, Nathalie et Yan. From portuguese lab I want to thank Susana, Nuno, Filipa, João, Caio, Ana Catarina, Salta and Rui.

To my friends, Paula, Mi, Pat, Paulo, Tiago, Raposo, Fernando e Carla I want to thank them for their friendship, patience and support during this PhD.

At last but no less important I want to express my biggest gratitude to my family. Muito obrigada aos meus pais e mano, por me terem apoiado nesta decisão tão importante pela sua compreensão, encorajamento nos momentos mais difíceis e por todos os sacrifícios que fizeram para que eu chegasse até aqui. Sem esquecer os meus avós, padrinhos e priminha que também acreditaram em mim e estiveram sempre presentes não só nesta etapa como ao longo de todos os momentos importantes da minha vida. São vocês que me dão força para enfrentar todas as dificuldades e lutar pelos meus objectivos.

En fin un grand merci à Ugo pour son soutien inconditionnel, patience, bon humour… entre autres, mais aussi à ma nouvelle famille Christine, Gérard (et Dudune).

Esta tese é dedicada às pessoas mais importantes da minha vida Mommy, Pappy, Micas, Vó, Vô, Madrinha, Padrinho,

CHAPTER I

Ketoses...ever since until nowadays 1. A KETOSE

A ketose ?

What is it ? Where can we find it ? And what we can do with it?

A ketose is a monosaccharide which contains a ketone function in its structure. The first example, fructose exists in abundance in fruits, honey, vegetables… and has found many applications mainly in fine chemistry and is currently being developed especially in view of its use as raw material. It has applications in several areas, such as food, medicine, cosmetics and pharmaceuticals.

Just as a reminder, monosaccharides are classified according to three different characteristics: the placement of the carbonyl group, the number of carbon atoms in the chain, and its chiral handedness. When the carbonyl group is an aldehyde, the monosaccharide is called aldose. When the carbonyl group is a ketone, the monosaccharide is called ketose, a less common class of sugars. Monosaccharides with three carbon atoms are called trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on… These two systems of classification are usually combined: for example, ribose is an aldopentose (five-carbon aldehyde), and fructose is a ketohexose (six-carbon ketone) (Figure 1). The above four members of this family being the topic of discussion in this PhD thesis, a brief description of each of them seems to be appropriate.

D-Fructose

D-Fructose -D-arabino-hex-2-ulose -is also known as levulose (or "laevulose" -an older common name) thanks to its levorotatory property. It is the most important and the most exploited of all ketoses. This simple sugar can be found in many foods and along with D- glucose and D-galactose is one of the three essential dietary monosaccharides. D-Fructose is accessible in fruits, vegetables and honey, 1 is responsible for the intense sweetness of these natural food and hence the source of its name. In nature it can exist mainly in two forms, either as a free monosaccharide or bound to D-glucose as in sucrose (disaccharide).

It is the sweetest of all naturally occurring carbohydrates and its sweetening power is 173% higher than that of sucrose 2 , the reference sugar. For this reason using D-fructose, to reach the same degree of sweetness a smaller amount is necessary which makes it less expensive, profitable, attractive and explains its commercial uses in foods and beverages. In addition it has little effect on measured blood glucose levels making it recommended in some diets. It can also (a) L. M. Hanover, J. S. White Am. J. Clin. Nutr. 1993, 58, 724;(b) A. E. Benders Dictionary of Nutrition and Food Technology 1990, Butterworths, Boston.

enhance other flavours in the system 2a and can also exhibit a sweetness synergy effect when used in combination with other sweeteners. 3 When in solution, D-fructose forms different cyclic structures in equilibrium. In water at 25ºC, different tautomeric forms can be observed, however, there is a predominant form, the β-D-fructopyranose (73%) followed by β-D-fructofuranose (20%) 4 (Figure 3). Just for curiosity, it is the 5-membered ring form of D-fructose that is the sweetest; the 6membered ring form tastes about the same as typical table sugar. Unfortunately, warming fructose leads to formation of the pyranose form 5 .

Just out of curiosity

It is known that every cell in the body can metabolize D-glucose but on the other hand, D-fructose is almost entirely metabolized in the liver. In United States a team of researchers observed that the liver of rats on a high fructose diet looked like the liver of alcoholics, plugged with fat and cirrhotic. 6 However this cannot be entirely true as a few other tissues (e.g. sperm cells and some intestinal cells) do use D-fructose directly, though in less metabolically significant amounts.

D-Psicose

D-Psicose -D-ribo-hex-2-ulose or D-allulose -is the C-3 epimer of D-fructose. In water at 27ºC, all tautomeric forms are in equilibrium and α-D-psicofuranose is the predominant form (39%) 7 (Figure 4). D-Psicose is an ultralow-energy monosaccharide : with the same amount as sucrose, it liberates only 0.3% of the equivalent metabolic energy. It is known as a ʺrare sugarʺ because it is scarcely found in nature, and even when it occurs, it is in small amounts. The presence of D- psicose can also be detected in some agricultural products or in commercially prepared carbohydrate complexes but always in small quantities. First described by Steiger and Reichstein 8 in 1936, this ketose can also be isolated from psicofuranine, 9 a nucleoside antibiotic which has good antibacterial activity in vivo and antitumor activity, but shows no activity in vitro when assayed by conventional methods 10 (Figure 5). 7 S. C. Köpper, S. Freimund Helv. Chim. Acta 2003, 86, 827. 8 M. Steiger, T. Reichstein Helv. Chim. Acta 1936, 19, 187. 9 V. K. Srivastava, L. M. Lerner J. Org. Chem. 1979, 44, 3368. 10 L. J. Hanka J. Bacteriol. 1960, 80, 30 and references cited therein. Since it is differently metabolized from sucrose, it has a minimal effect on blood glucose and insulin levels. It has shown anti-hyperglycemic properties and is therefore useful in the treatment of diabetes. 16 Its sweetness is about 75-92% higher 14, 17 but with only 38% of the calories comparing to sucrose (in equal weight). It has also been approved as a tooth friendly ingredient. 14

Just out of curiosity

During the past decade, a continuous growing interest on D-tagatose has been observed.

Notably, the fact that D-tagatose was approved by the FDA (U.S. Food and Drug Administration) in 1999 as a low-calorie sugar substitute (1.5 kcal/g as against 4 kcal/g) and also in 2001 "Generally Recognized as Safe" (GRAS) by the FAO/WHO 17 and in 2003 as food additive. 16 Nowadays, it has applications in the food industry as a sweetener, flavour enhancer, humectant, texture agent, thickener, etc... 14, 15 However this rare sugar of easy access (inexpensive production) is not only attractive for food and beverage markets but also for pharmaceutical markets. 14, 18, 19 In 2004, Australian researchers revealed the effects of D-tagatose on blood glucose and insulin responses, animal studies in rats confirmed the hypoglycaemic properties of the product, triggering its development as an anti-diabetic agent in humans. application: another product based on D-tagatose, targeting the treatment of type 2 diabetes is also in phase 3 of testing. 19 The first efficacy studies in humans confirm that orally taken Dtagatose leads to very low insulin and glycemic responses, both in normal and in diabetic patients. Besides, considerable increases on HDL cholesterol and antioxidant properties were also observed. In addition, these studies also highlight the significant benefits of the product on obesity, unlike other anti-diabetic such as sulfonylureas or thiazolidinediones which lead to weight gain. 14, 19 The standard toxicity studies in animals and potential genotoxicity, embryotoxicity and teratogenicity studies demonstrate that the product may be considered riskfree.

L-Sorbose

L-Sorbose -a white crystalline sugar -is the C-5 epimer of D-fructose. It is the most readily large-scale available L-sugar as part of the commercial preparation of vitamin C (Lascorbic acid).

In water at 27ºC, all tautomeric forms are in equilibrium and α-D-sorbopyranose is the largely predominant form (98%) 4 (Figure 7).

SOME KETOHEXOSE CHEMISTRY

On a general point of view, carbohydrates are implicated in a large number of biological pathways and thus have been the subject of numerous researches. Besides those intense studies, working on this topic is not as simple as it could be thought. Of the number of reasons, in water (in solution), one monosaccharide exist as a complex mixture in equilibrium, of different isomers (α-, β-pyrano or furano and acyclic forms) which requires from organic chemists challenging and ingenious approaches to solve or simplify this complexity.

Rare sugars like ketoses have seen light for their uses as healthy alternative foodstuff. 20 The recent discovery of several beneficial healthy properties has potentiated the application of rare sugars in pharmaceutical industry for the treatment of different diseases, such as diabetes and obesity.

Consequently, many attempts to find more effective synthetic accesses to these rare sugars and/or new potential sugar compounds are currently under investigation. However for large scale production it is predicted that biotechnological approaches might be more economically efficient and environmentally friendly. 16 In this part of the chapter, we shall present some recent reports on ketose chemistry: how to synthesis these rare sugars and derivatives, their application as chiral auxiliaries 21 and as synthons in the synthesis of natural compounds 22 , nucleosides 23 and iminosugar analogues 24 .

The kind of molecules synthesized goes from cyclic to acyclic structures, simple to more complex. The purposes are diverse but mainly with a wide range of applications in the biological treatment of various diseases, namely cancer, diabetes, HIV, herpes...

Ketoses as intermediate synthetic tools

Exocyclic vinyl ethers 25

Exocyclic vinyl ether derivatives of sugars are reported in literature as intermediates in various metabolic and biosynthetic pathways and for their potential as synthetic intermediates, for example in the synthesis of enzyme inhibitors, biologically relevant compounds or as monomers for polymerization. Throughout the years, numerous researches have been developed in this area with sugars but most of them with aldoses, the ketose family still remaining scarcely explored.

Latest studies proposed that the biosynthesis of the carbocyclic moiety of natural products such as neplanocin A (a novel cyclopentenyl analogue of adenosine, naturally occurring antibiotic which exhibits significant antitumor activity against leukemia in mice) 26 and aristeromycin (an inhibitor of purine biosynthesis, carboxylic nucleoside antibiotic which exhibits a variety of biological activities 27 ) involved a key intramolecular aldol condensation. As a result Bechor and Albeck proposed that these molecules may engage a common exocyclic vinyl derivative of fructose-6-phosphate (Scheme 1).

Neplanocin A

Aristeromycin

HO

Scheme 1

Consequently, the synthetic strategy relied on two different reactions: a β-elimination or a reductive elimination. However there are some important aspects that should be taken into account like locking the ketohexose sugar in its furano form, the choice of a suitable protection for hydroxyl groups and an adequate leaving group at the C-6 primary position. The 2-C-methyl-branched mannose is the first example of a small molecule that binds to a DC-SIGN receptor. From this viewpoint and due to all the promising advantages and applications of rare sugars, many attempts for finding more effective syntheses and also 'new' monosaccharidic compounds have been made.

Even today, the use of enzymes in organic synthesis transformations is limited because of substrate specificity. However, the evolution and discovery of new enzymes seems to open new horizons for this type of biotechnological process. For instance the procedures developed by Izumori 30 allow the isomerization of all 16 aldohexoses and 8 ketohexoses in water under environmentally friendly conditions.

Herein is presented just an example, the preparation starting from D-fructose of 5-Cmethyl-D-psicose via 5-C-methyl-D-fructose through a combination of chemical and biotechnological steps (Scheme 3). activity. These reactions were also applied with success to other members of the ketose family.

1-Deoxy-D-ketohexoses 31

Rare monosaccharides, like ketoses, seem to be owners of interesting biological activity. D-Tagatose is an anti-hyperglycemic agent and therefore useful in the treatment of diabetes. D-Psicose, readily available from D-fructose by the action of DTE enzyme, has a considerable number of chemotherapeutic properties. And both of them seem to be attractive as healthy food substitutes. 30 (a) K. Izumori J. Biotech. 2006, 124, 717;(b) T. B. Granstrom, G. Takata, M. Tokuda, K. Izumori J. Biosci. Bioeng. 2004, 97, 89;(c) Until now, scarce attention has been given to the synthesis of deoxyketohexoses. The objective of the reported syntheses was to explore the deoxyketohexoses abilities for biological applications. Such new compounds might have potential applications in food chemistry as well.

If they reveal interesting characteristics they might be prepared on a large scale by the biotechnological Izumori's procedures.

The synthetic pathway is quite simple, each diastereomeric D-ketohexose is obtained by methyl lithium addition to a suitably protected lactone of an aldonic acid (Scheme 4).

O O O HOH 2 C O COOH HO HO OH CH 2 OH O O O [Si]OH 2 C OH CH 3 CH 3 HO O HO OH CH 2 OH

1-deoxy-D-tagatose

Scheme 4

For example 1-deoxy-D-tagatose is prepared from D-lyxonic acid. All other D-and Lketohexoses can also be synthesized by the same method starting from the appropriate lactone.

2-C-(Hydroxymethyl)aldoses 32

The Bílik reaction corresponds to the reciprocal interconversion of C-2-epimeric aldoses under molybdic acid catalysis. Taking into account its easiness, this reaction is commonly used to synthesise quite a few rare aldoses on a large scale.

Scheme 5

The reaction mechanism seems to be similar to that described for aldoses, the carbon atoms exchange with their substituents is mediated by a tetradentate dimolybdate complex involving four neighbouring hydroxyl groups. This complex promotes the formation of a new C-1-C-3 bond while simultaneously breaking the C-2-C-3 bond, thereby causing epimerization

with concomitant C-1-C-2 transposition.
Curiously, when boric acid is added in the reaction mixture in L-sorbose and D-fructose series, the equilibrium is considerably changed in 2-C-(hydroxymethyl)-pentoses. Furthermore, these two ketoses also undertake a competitive secondary isomerization process although in small quantity (C).

Di-D-fructose dianhydrides (DFAs) 33

Difructose dianhydrides (DFAs), a single class of spiroketal disaccharides (condensation of two fructose molecules, with formation of two reciprocal glycosidic linkages) comes from thermal and/or acidic activation of sucrose-and/or D-fructose-rich materials. Until their presence was detected in food materials (caramels or chicory) in substantial amount they were just considered as laboratory curiosity. This discovery has completely changed DFA chemistry, by exhaustive and meticulous research which revealed the biological (beneficial prebiotic nutritional functions), nutritional (low-caloric sweeteners) and technological importance of this family of compounds. However, the complexity of the formed DFA mixtures -up to 14 diastereomeric spiroketal cores -makes the evaluation of their individual characteristics a tricky challenge (Figure 8). Improvement of standard methodologies to get access to each individual, isolated and pure compound is fundamental in order to understand their biological role and to develop new applications. With this purpose chemical and enzymatic syntheses have been used as complementary strategies to obtain these compounds and study their properties. For example, they can be chemically synthesised by protic acid activation starting from either unprotected, ring-size blocked (Scheme 6), or conformationally controlled D-fructose precursors and even via the intramolecular aglycon delivery methodology.

Scheme 6

Actually with all the knowledge acquired about DFAs more attractive perspectives can be considered in terms of food processing for enhancement of biological functions. As it was presented 1,5-anhydro-D-fructose is considered a valuable chiral building block to prepared biologically active fine and as well bulk chemicals with potential applications in the food and pharmaceutical industry.

Iminosugars

Azasugars or better "iminosugars" (carbohydrate mimics with a nitrogen replacing the ring oxygen) represent the most important class of glycosyl hydrolase inhibitors 35, 36, 37 . Thanks to their inhibition potencies they are a high promise as probes for structure and useful biological tools for studies on glycoconjugate functions, targeting and turnover 24a . Furthermore these compounds have also applications as chemotherapeutic agents for the treatment of cancer, diabetes and inflammation or viral replication 35, 24a and rare diseases. Actually there are many available iminosugars but the problem is the lack of specificity. 24a Recently however, two iminosugar based drugs have been released on the market. In 1996, Glyset™ for handling with type II diabetes complications and in 2003 Zavesca™ for the treatment of Gaucher's disease, a harsh lysosomal storage disorder. 37 Starting with D-fructose, natural glycosidase inhibitors, DMDP and DGDP, via intramolecular delivery of benzyl protection have been prepared. 36 Further modifications of the inhibitors demonstrated that remarkable specificities in enzyme inhibition can be achieved upon modifications on the nature of the sp 2 -hybridized endocyclic ring nitrogen (Scheme 8). 35 35 M. I. Garcia-Moreno, D. Rodriguez-Lucena, C. Ortiz Mellet, J. M. Garcia Fernandez J. Org. Chem. 2004, 69, 3578; 36

Scheme 8

Starting also from D-fructose a highly stereocontrolled preparation of a DMDP analogue, has been described and used as source of chirality in the preparation of protected derivatives of hyacinthacines, another well-known family of glycosidase inhibitors 38 (Scheme 9). immunodeficiency (HIV). 39 Many of those compounds exhibit antiproliferative, antibiotic and antifungal activities and some have been used as probes for DNA damages 40 as well as in the anti-sense approach and DNA-probe technology with fluorescence properties. 41 It is believed that introducing diversity either onto the carbohydrate or onto the heterocyclic moiety of nucleosides, in methylated bases of the bacteria world, in RNA duplexes 42 or in new antibiotic analogues 43 leads to gifted molecules with therapeutical potential (antiviral and anti-cancer agents). 44 Two general methods for nucleoside preparation have been reported in literature: one is using a glycosylation procedure which may lead to anomeric selectivity problems, 45 the other is based on a multistep process to construct the base from the sugar template. 46 Methods for the synthesis of nucleoside analogues based on saccharidic 1,3-oxazolidine-2-thiones (OZT) have slightly been described. In 1975, Ranganathan developed the synthesis of 9-β-D-arabinofuranosyl-adenine from a D-arabino OZT derivative. 47 A few years later, Imbach obtained nucleoside analogues from D-ribose 48 as well as from αand β-D-xylose 49 and β-Darabinose 50 . Grouiller has also applied this method for the preparation of nucleosides derived from β-D-fructose. 51 Recently, the approach developed in our laboratory involved this thionocarbamate moiety / a sugar derived 1,3-oxazolidine-2-thione (OZT) and anthranilic acid. Application of the process led to new homochiral quinazolinone derivatives and conducts to the synthesis of new base-modified "artificial nucleosides" 46a, 52 (Scheme 10). In summary, starting from natural carbohydrates, a short, simple and original synthetic pathway through OZT derivatives for the preparation of based-modified nucleosides has been disclosed. This pathway as also been extended with success to diverse aldose and ketose series. 46a

Another example of original nucleoside bearing modifications on the sugar ring was reported by Kifli. 44 It refers to a nucleoside with a fused-OZT moiety in the sugar ring. This molecule could be a zidovudine (AZT) or stavudine (d4T) analogue, which are antiviral and anticancer agent analogues 53 (Figure 10). In general OZT moieties onto carbohydrate scaffolds are very useful structures. The synthesis of saccharidic OZTs is an attractive challenge from a chemical point of view but also because of the growing interest aroused by their biological properties. OZTs have shown potential uses as chiral intermediates 21 in various synthetic transformations as synthons in the synthesis of iminosugars, 23 as precursors to nucleosides, 23 as mimics of ketohexose conformations to inhibit the fructose transporter, GLUT5 21, 23d, 23e, 54 or as potential biological agents. 2124c, 55 These two last applications constitute the biological challenge of this PhD.

3.1.9. Glucose and fructose transporters, GLUT5. Potential insecticides.

PhD Objectives.

a) First Part of the Project

The aim of the project is the development a selective inhibitor of a fructose-transporter (protein GLUT 5, a facilitative transporter). Therefore we needed to build up a library of potential GLUT5 inhibitors based on structural modulation of ketohexose structures. We would then expect to extract information of the structure-activity relationship to improve our understanding of the interactions with the protein. For the leading inhibitors further transformation could be made in order to develop novel biochemical tools bearing a photoaffinity label and / or a biochemical tag. This new sensor would be a precious tool in defining the tissue specific patterns of expression of the GLUTs, their response to alterations in diet and hormonal regulation and to disease states.

However, designing an inhibitor of D-fructose transporters is not so simple ! In early studies it was decided to compare the influence of various factors in the recognition process.

These studies required investigation of the positions around D-fructose structure by the synthesis of molecules in which each position was substituted in order to determine the importance of each of the hydroxyl groups in the binding and interaction with GLUT5. Firstly, some studies were investigated using bulky groups. It was concluded that both ring forms (pyrano and furano) were tolerated, 56 thus increasing the difficulty in finding efficient inhibitors (Scheme 11).

Scheme 11

Two With the aim to explore this model of interaction, Girniene-Rousseau et al. 54 have started to develop a method using OZTs moieties to block the D-fructose structure and analogues (Figure 12). The OZT structures can be regarded as analogues of D-fructose in pentose series (derived from D-and L-arabinose, D-ribose and D-xylose), as well as in ketohexose series (derived from D-fructose and L-sorbose). 56 (a) A. Tatibouët, J. Yang, C. Morin, G. D. Holman Bioorg. Med. Chem. 2000, 8, 1825;(b) These first studies have put in evidence that pentose-derived OZTs were not recognized by the protein transporter. Only ketohexose-derived OZTs expressed some inhibition of GLUT5. These inhibition constants showed to be more effective with L-sorbo than for D-fructo derivatives. The oxygenated compounds, 1,3-oxazolidin-2-ones (OZO) were also observed to be better inhibitors than their thio-analogues (OZTs) and even better than D-fructose itself. Above all, these studies only address the furano isomer: nothing has been previously explored on pyrano structures.

Taking into account the results obtained in our laboratory it was decided that in this PhD the approach would follow the same guidelines: designing a molecule that mimics perfectly the ketohexose -either in αand β-pyrano or αand β-furano forms -but avoiding tautomeric problems. 1,3-Oxazolidine-2-thione (OZT) and 1,3-oxazolidin-2-one (OZO) were chosen as cyclic structures able to maintain the geometry and the interaction capability of the original ketohexose structures (Scheme 12). These new molecules (derived from all ketohexoses) would map the recognition pattern for each GLUTs of Class II, in testing only one tautomeric form of a ketohexose at a time.

Scheme 12

According to the chemistry developed in the laboratory, OZT was preferentially chosen.

This structure provides a flexible chemistry and offers a number of advantages compared to their OZO analogues 57, 58 , furthermore, they can easily be converted into their parent 2-oxo 59 .

Previous studies conducted in the laboratory have already shown the chemical potential of this synthon. 57 Anchoring an OZT on a saccharidic backbone can follow two main different pathways. 60 The most important is the condensation of an β-aminoalcohol with a thiocarbonyl source. This method implies control of different parameters from the saccharidic frame, 22, 24a, 24b, 61 for example, requirement of a 1,2-cis relationship between alcohol and amine, and the thermodynamic force imposed by the monosaccharide to created the OZT. When these parameters are satisfied the formation of 1,3-oxazine-2-thiones becomes possible (Scheme 13). In most cases, the reaction of thiocyanic acid with aldoses produces furano bicyclic compounds. This method directly generates OZT moieties under perfect stereochemical control from aldoses without preliminary protections (Scheme 14). Under the same conditions, ketohexoses behave differently and in a much more complicated way. The major problem lies in the capricious ketose chemistry: under acidic conditions, a complex equilibrium settles between tautomeric forms, resulting in a lack of selectivity and therefore frequently leading to the formation of mixtures of both straight-chain and cyclic structures. Indeed, it is possible to predict up to ten different structural types: fused bicyclic structures in furano and pyrano forms, spiro-epimers (α/β mixtures), acyclic chain or even spiro-bis-OZT (Scheme 15).

Scheme 15

The reaction results into

Scheme 16

All of these synthetic pathways will be described in more details along this PhD work.

The choice of each method was carried out according to the type of targeted molecule.

Numerous different structures bearing the OZT or the OZO moieties could be produced from D-fructose itself. When locking is effected at the anomeric carbon, spiro-structures (furanoor pyrano-) as well as fused ones should be attained. On the other hand when locking is effected on the saccharidic backbone, only fused bicyclic structures should be obtained (Figure 13).

Figure 13

Applying to D-fructose and its epimers D-psicose, D-tagatose and L-sorbose, the glycochemical methodologies currently developed in our laboratory, we could give access to a panel of diverse compounds which constitute our library of potential inhibitors.

b) Biological applications of the project

The second part of this project is run in common with our partner, G. Holman (University of Bath). It concerns the fructose transport assays on Chinese hamster ovary (CHO) cells expressing GLUT5, 56, 62 measuring inhibition of D-[ 14 C-U]-fructose uptake by using scintillation counting. 56 This screening technique is to be applied on all our library of compounds in order to define the leading ligands to GLUT5. Among all leading compounds, transport assays against other GLUT should then be applied. The combined results would give a complete map of interaction between synthesized drugs and GLUTs. An in-depth understanding of the recognition site of GLUT5 should be the first result of this approach;

further studies would be enlightened by the finding of inhibitors both selective and specific to GLUT5 (also to Class II family).

In addition, the potential of these molecules as biological agents should be explored. It is reported in the literature that some OZTs exhibit biological properties such as effective antifertility action in rats 63 or herbicidal activity 64 (Figure 14).

Figure 14

It was also observed that other molecules which contained in their skeletons an OZT or similar heterocyclic moieties could be used as insecticide Erreur ! Signet non défini.b , antifungal Erreur ! Signet non défini.c , antimicrobial Erreur ! Signet non défini.c and antibacterial agents Erreur ! Signet non défini.d, Erreur ! Signet non défini.e (Figure 15).

Figure 15

The search for D-fructose mimes aiming at developing new inhibitors selective for a protein carrier and potential bio-active agents has enabled us to improve some of the chemistry of D-fructose and its epimers. We have enlarged the ketose chemistry in order to have access to these new structures. However this is a long, laborious and audacious project that can not be completed in just 4 years of research. Herein we have done the first steps. We have synthesised a panel of molecules, potential inhibitors that are going to be tested. Only later, this kind of biochemical tools and application can be done and used. 64 G. Li, X. Quian, J. Cui, Q. Huang, R. Zhang, H. Guan J. Agric. Food Chem. 2006, 54, 125.

CHAPTER II 3,4-Fused thionocarbamates in pyranose form

1. SYNTHESIS OF 3,4-FUSED THIONOCARBAMATES STARTING FROM

D-FRUCTOSE

In this chapter, the synthesis of 3,4-fused thionocarbamates starting from D-fructose is described and their chemistry explored. The OZT 1 has been prepared as well as the 1,3oxazolidin-2-one (OZO) 2 and the 1,3-oxazoline-2-thione (OXT) 3 (Scheme 17). For both cases, the OZT moiety was the material of choice to access these structures, 65 thanks to Rollin's group experience. 5721, 46a, 52, 54Erreur ! Signet non défini., 62, 66 Scheme 17

Choosing the pathway

As was stated in the introduction (Chapter I -3.1.9.) there are two main routes for the synthesis of thionocarbamates: condensation with a thiocarbonyl source or with thiocyanic acid.

Each approach possesses its own characteristics and is especially suitable depending on the formation of a specific structure. It has to be mentioned that reacting trimethylsilyl 65 (a) M. T. isothiocyanate at the anomeric position still cannot be considered a convincing method because of low yields.

For our purpose, we planned to prepare a fused OZT starting with 1,2:4,5-di-Oisopropylidene-β-D-fructopyranose 4. The direct methods involving condensation of thiocyanic acid with unprotected sugars 23, 54, 67 or with trimethylsilyl reagents 22d can not be used. Both are devoted to the synthesis of OZTs involving the anomeric carbon, i.e. 2,3-fused 5 (first method only) and spiro OZTs 6 (Scheme 18). These methodologies will be discussed later in Chapter IV. Therefore the procedure that seemed to be the most appropriate was the condensation of a β-aminoalcohol with a thiocarbonyl source. 22a, 24a, 24b, 68 However, it was also observed that it might not be a good pathway since the starting material configurations are kept. No matter the route chosen to form the amine substituent, it will be always placed in a 1,2-trans relationship with the alcohol, 66, 69 8 and 10 (Scheme 19). This trans configuration obstructs the OZT formation, due to its intrinsic geometry and to the rigidity of the sugar backbone which are strong enough to hamper the condensation. So, how to achieve the formation of this 3,4-fused OZT ? modifying the configuration ?

A shorter solution was brought by using condensation of thiocyanic acid -not with a free sugar like in Zemplén's method 67a, 67b but with an α-hydroxyketone according to Willems and Vandenberghe. 70 A brief introduction about this methodology seems to be important.

Condensation with HSCN. Zemplén vs Willems-Vandenberghe reaction.

The first method developed for anchoring an OZT onto a saccharidic backbone was originally reported by Zemplén 67a, 67b in 1936 (it will be described in more details in Chapter IV 1.1.; herein we will only illustrate some general conclusions). It was based on the condensation of O-unprotected sugars with thiocyanic acid (generated in situ from potassium thiocyanate and a protic acid) in anomeric position. Since then, several groups of researchers 23, 54, 67c, 67d, 67e, 67f proved to be interested in this issue and therefore different theories were appointed. After a controversy surrounding which thionocarbamate structures were obtained, ideas were finally clear. In both sugars series, aldose and ketose produced bicyclic fused-OZT type structures.

Thus, for aldoses most of the products obtained possess furano backbones, 23, 54, 67d, 67f the thermodynamically more stable entities. For ketoses, things are much more complicated due to the numerous possibilities offered by their chemistry. In fact, as already pointed out previously up to 10 different compounds (fused, spiro OZTs in furano or pyrano forms as well as acyclic structures and OXT) can be expected (Chapter I -3.1.9.).

In 1961, Willems and Vandenberghe 70 described a similar process, involving condensation of HSCN with simple hydroxyacetones, and leading to OXT moieties instead of OZT moieties (Scheme 20). A possible thione-thiol tautomeric equilibrium could exist between the OXT 15 and the oxazole 16. Surprisingly, in literature 70, 71 this kind of reaction has been little investigated.

Scheme 20

Consequently, in our laboratory, Leconte 66a has explored this reaction. His first assays were performed with 1,3-hydroxyacetone and L-erythrulose, for which the formation of chiral

Scheme 22

Thus taking into account the results obtained by Dr. Silva and considering the targeted 3,4-fused OZT, the reaction of Willems and Vandenberghe was chosen as the key-step for this synthesis. This pathway will also allow to check whether the results previously obtained in aldose series could be extended to other carbohydrate series.

Synthesis of a 3,4-fused OZT

For the reasons explained above, the pathway to be followed in order to synthesize the 3,4-fused OZT is : introduction of a keto group in position 3 then condensation with thiocyanic acid (Scheme 23).

Scheme 23

Preparation of the α-hydroxyketone

The first step of this pathway consisted in protecting C-1,2 and C-4,5 hydroxyl groups of D-fructose in order to functionalize position C-3 without any problem. This standard reaction was carried out in acetone, which acted both as reagent and solvent, and using sulfuric acid as

Scheme 25

The next step involved regioselective hydrolysis of the 4,5-O-isopropylidene group. This can be achieved, for example, by using 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in solution, 74 however a more practical alternative method has been reported in literature. 79a, 73 A simple 8:2 acetic acid/water mixture can also be very efficient (Scheme 26). In addition, an easy workupevaporation and co-evaporation of the solvents with toluene -make this procedure very attractive.

The selective 4,5-deprotection is possible because the vicinity of the The stable α-hydroxyketone 24 is thus rather easily obtained in good yields.

Synthesis of the OZT -the Willems-Vanderberghe reaction

The key-step was the OZT formation through a Willems-Vandenberghe reaction, by condensing thiocyanic acid generated in situ with the α-hydroxyketone 24 (Scheme 27).

The solvent was chosen taking into account the results of Silva's investigations 66b (Table 2). From that, the best conditions established involved a In the reaction performed with ethanol, detection of a small amount of 1 was not surprising when considering the mechanism illustrated above. 66b

The yields obtained range from moderate to reasonable, but they never reach the values reported by Silva et al. in aldose series. We can postulate that, even if the cyclization of the isothiocyanate in OZT occurs spontaneously, the fused ring system associates a 5-membered ring to a 6-membered sugar ring which is a disfavored situation (the fusion of two 5-membered rings is usually preferred) (Figure 16). Furthermore the saccharidic backbone being rather rigid, this does not particularly support the OZT formation. This can be considered an original approach for the synthesis from carbohydrate-based α-hydroxyketones of OZTs anchored onto saccharidic backbones.

In terms of manipulation and purification (column chromatography) it can be stated that OZTs are easy to handle as they strongly absorb UV light (λmax in 280-300 nm range), making their detection straightforward. 65a

Deprotection assays

The last step of this pathway was the deprotection of the target molecules (Scheme 30): however these unprotected OZTs are not stable and thus rather difficult to isolate.

O O R 2 O O NH O S OR 1 O OH R 2 O O NH S OR 1 OH R 1 = H, R 2 = H, 1 R 1 = Et, R 2 = H, 26 R 1 = Et, R 2 = Ac, 31 R 1 = H, R 2 = H, 32 R 1 = Et, R 2 = H, 33 R 1 = Et, R 2 = Ac, 34

Scheme 30

In order to remove the isopropylidene group several attempts were made in aqueous acidic conditions (Table 3). The first trials were performed under TLC monitoring with standard 80% aqueous acetic acid: at room temperature no reaction was observed; increasing the temperature to 50°C only resulted in total degradation of the starting material.

Since those first assays did not afford the desired unprotected product 32, the latter molecule was regarded as very sensitive. It was thus decided to work rather with the ethyl protected substrate 26. After disappointing attempts with 80% aqueous AcOH, much harsher conditions were applied, using 90% aqueous trifluoroacetic acid at room temperature. In that case, only traces of the expected compound 33 could be detected.

Finally, 2M hydrochloric acid was applied with a view to obtaining a larger amount of 33, but unfortunately, only degradation was observed. An ultimate assay was performed involving a protected substrate 31 bearing an acetyl group on position C-5 : to our disappointingment however, degradation also occurred in that case.

Meanwhile, a more unusual deprotection with Bi(OTf)3.xH2O was experienced. In this case the expected final molecule 36 would be more stable because of the acetyl protecting groups (Scheme 30). This method has been recently reported by Zhang and coll75 as a facile onepot procedure for the conversion of acetonides into diacetates, and it was applied to our case :

unfortunately OZT 35 could not be transformed into the expected derivative 36.

Scheme 31

Taking into account all the de-acetonation tests conducted, we had to admit that the best result was obtained with 9:1 TFA/H2O at room temperature : no more starting material was detected by TLC after overnight reaction, but only traces of the desired compound could be isolated from degradation products.

Therefore it can be assumed that aqueous TFA solution could be an appropriate hydrolytic system provided that the concentration is carefully established. This assumption is actually supported by literature: Izquierdo 38 reported the use of 60% aqueous TFA for isopropylidene cleavage. The structures reported were similar to ours, bearing isopropylidene groups at the anomeric carbon of a ketose. So we decided to apply these conditions to our

Scheme 32

As can be observed, a more diluted aqueous TFA and a shorter reaction time bring a solution to our deprotection problems, most of the degradation being avoided. However for 26 the ethyl group cleavage was also observed, leading to only one kind of structure.

The hydrolysis product was not isolated but immediately submitted to peracetylation without any preliminary treatment: with this approach we could assure stabilization and better purification and characterization of the targeted product. Not only 5-O-protection was obtained but also N-acetylation, although in a lower proportion. The overall yields were about 64-74%.

Scheme 32).

Final de-O-acetylation was performed in standard Zemplén conditions -sodium methoxide or ammonia in methanol. However this proved to be fruitless because only degradation of the compounds was observed. Therefore in spite of all attempts and due to its instability, the final compound could not be isolated.

Synthesis of a 3,4-fused OZO

A list of methods

Oxidative desulfurization is a type of transformation that can be achieved by direct or indirect procedures using a wide range of reagents and conditions. 78 Herein we are going to do a succinct report on what has been performed in our laboratory.

a) Direct oxidative desulfurization

Pistara 79 reported a variety of methods that allow the transformation of a thiocarbonyl group into a carbonyl group through the use of various organic or inorganic reagents.

These methods were tested in our laboratory first with simple OZTs 80 then with OZTs anchored onto saccharidic backbones 81 but all assays were disappointing (Scheme 33). The attempts to replace the sulfur atom by an oxygen atom on a partly-protected model OZT derived from D-fructose did not lead to the corresponding OZO (Scheme 33). This approach of direct oxidative desulfurization by smooth methods confirms the reluctance of the thionocarbamate function. In our laboratory we thought that the N-H bond needed to be masked in order to perform the oxidative transformation. b) After nitrogen protection Indeed, the transformation was achieved after prior protection of the nitrogen atom. Gueyrard et al. 57b, 80 described the use of meta-chloroperoxybenzoic acid to produce OZOs with excellent yields. Later, Girniene-Rousseau et al. 81 tested this synthetic route and verified that Nprotection of the thionocarbamate was also needed. Consequently applying this approach, she performed S to O transformations (such as 41 to 42, Scheme 34) with good results. This synthetic route gives OZOs in only three steps with good yields. However, it is important to notice that these structures still needed an additional deprotection reaction to generate the OZOs.

c) Via S-alkylation

Another method of oxidative desulfurization has also been performed after preliminary

S-alkylation.

Chemoselective S-alkylations were reported on some simple OZTs, 57b taking advantage of the smooth electrophilic character of various haloalkanes. Preferential alkylation at the sulfur atom was achieved in good yields to form enantiopure S-alkylthiooxazolines.

Coxon 82 and Toth 83 reported that reaction of chloromethane or p-chlorobenzyl chloride with saccharidic OZTs also lead to the corresponding 2-alkylsulfanyloxazolines.

82 R. M.

Scheme 35

We hypothesized that under oxidizing conditions the benzylsulfanyl substituent might be converted into better leaving groups, thus facilitating further hydrolysis to the OZO 44.

Chosen method: via S-alkylation

In our synthesis, we decided to use the S-alkylated intermediate method, in which some changes were introduced comparing to Girniene-Rousseau's procedure 81 . Concerning Sbenzylation, she observed large variations in yields, which means some degradation occurs in the presence of excess NaH. Besides, Fujita 84 reported that in the presence of excess BnBr or under heating, S-benzylated compounds could suffer a rearrangement leading to N-benzylated products. Taking into account these assumptions, the following conditions were fixed: NaHCO3 as much weaker base than NaH and reaction performed at room temperature.

Following those parameters, we obtained the S-benzylated compound 45 in good yield as compared with literature data 46a, 52b 85 (Scheme 36). Moreover, neither N-benzylation nor O-benzylation were observed, even in the presence of BnBr in excess.

Oxidation

The oxidizing agent was chosen based on previous results obtained in our laboratory. 55, 57b, 93 Even if the synthetic pathways were different -N-protection method 55 or S-alkylation 93both used m-CPBA as the oxidant.

Normally, with an excess of m-CPBA, the sulfur function would be oxidized to an unstable sulfoxide or sulfone derivative. This intermediate should be sensitive to a nucleophilic attack of the remaining water in the medium, leading to the formation of an OZO through the expulsion of a sulfur-containing moiety. However in our case, the expected OZO 2 was not detected (Scheme 37). Fearing that the targeted OZO 2 would cause purification problems, due to its strong polarity and lower stability compared to the corresponding OZT, we decided to protect the starting molecule. In standard conditions, 45 was acetylated in 93% overall yield as a mixture of 5-O-acetylated derivative 46 (76%) and 3,5-di-O-acetylated derivative 47 (17%).

Once again oxidation conditions were applied to 46 but even after 24 h at room temperature no transformation was noted. Refluxing the mixture during 24 h did not bring any improvement and the starting material was totally recovered (Scheme38).

Scheme 38

Taking into account the results reported above, we searched for an alternative reagent that could be efficient in this transformation. The oxidizing capacity of hydrogen peroxide is known for being so strong that it is considered as a highly reactive oxygen species. Moreover it is an eco-friendly reagent, its manipulation and storage is quite secure and it can be considered a low cost reagent. Indeed, hydrogen peroxide gave good results, with 72% and 81% yields of

Scheme 39

Tirandaz recently reported a direct oxidative desulfurization of thioamides into amides using H2O2 in the presence of zirconium tetrachloride with good results. 86 However, Tirandaz conditions were not tested, the present part of our project having been completed at the beginning of the PhD.

The structure of the S-benzylated and OZO compounds was ascertained by 13 C NMR experiments. The chemical shifts for quaternary carbons vary from ca 188 ppm (C=S in OZT 1)

to ca 161 ppm (C-SBn in 45 and 46) and ca 155 ppm (C=O in OZOs 2 and 48) (Table 4). On a more pratical aspect, comparing a thiono-derivative with its oxo analogue, one can observe that beyond its higher polarity, an OZO also has a much weaker absorption in UV.

Those aspects importantly interfere with the detection and purification of our compounds.

Synthesis of 3,4-fused OXTs 1.5.1. Previous studies

As was mentioned before in this chapter (1.1. and 1.2.) that reacting an α-hydroxyketone with HSCN would generally produce the hydrated form of an OXT.

While working on acyclic ketols, Silva et al. noticed the formation of OXTs without a trace of hydrated form. 85 So the question was: "is it possible to control this reaction in order to obtain selectively either the hydrated form or the aromatic form ?" and further: "is it possible to eliminate water in order to convert an hydrated form into the related OXT ?"

In the course of their study on dehydration of fused OZT on carbohydrate scaffolds, Silva et al. 85 realized that two major parameters were those she should pay attention to: a)

protection of the thiocarboxamido system to improve stability of the substrate b) protection of the free hydroxyl groups to avoid side-reactions.

After S-benzylation of the hydrated OXT, mesyl chloride 86 was first successfully applied as dehydrating agent on simple structures. But when trying more complex hydrated OXTs anchored to saccharidic backbones, no elimination was observed and the starting material was always recovered (Scheme 40). This lack of reactivity could be attributed to steric hindrance to access the hydroxyl group. With the purpose of forcing the reaction conditions, a more powerful electrophilic reagent like triflic anhydride 87 was used and hydrated OZT forms underwent smooth dehydration.

S-Alkylation. A surprising result.

In this chapter, S-alkylation has already been explored in the course of OZO preparation (chapter II -1.4.2.) (Scheme 33). Although the yield was satisfactory, however we wanted to 86 (a) A. Padwa, H. Zhang J. Org. Chem. 2007, 72, 2570. (b) J. Reeves Org. Lett. 2007, 9, 1879. 87

OXT formation through OZT dehydratation

Having in mind Silva's considerations, we decided that it would be safer to firstly protect the free hydroxyl at C-5. Initially, we tried O-silylation with tert-butyldimethylsilyl chloride but even at reflux for over 24 h no silylation occurred. That could be mostly due to the protective group size and to hindrance at the C-5 position. Next benzoylation (benzoyl chloride in pyridine with a catalytic amount of DMAP) was tested, but the starting material 45 was completely recovered. We then turned to our acetylated derivative 46, which had been previously prepared for the OZO study. 5).

Table 5

Conclusion

In this chapter, we have focused our attention on the synthesis from D-fructose of 3,4fused thionocarbamates on saccharidic backbones (Scheme 44).

O O R 2 O O N O SBn O OH HO OH OH OH D-fructose 24 O O HO OH O O O O HO OR 1 O O NH S R 1 = Et, 26 H, 1 O O R 2 O OH O O NH O R 2 = Ac, 48 H, 2 R 2 = H, 3 Bz, 53 37 O OAc AcO O NH S OAc OAc

Scheme 44

From the results obtained, we can put forward some preliminary assumptions:

Condensation of an α-hydroxyketone anchored onto a sugar scaffold with HSCN favors the formation of fused hydrated OXTs instead of OXTs (as previously observed by Silva in aldose series), while the opposite was observed for acyclic structures.

The 3,4-fused OZT that we have prepared have shown some sensivity and unstability.

Complete deprotection could not be achieved neither under acidic nor basic conditions.

The S-alkylation method allowed us to obtain saccharidic chiral OZOs in good yields in only two steps from OZTs. This is a shorter route compared to Girniene-Rousseau's method.

In partnership with Silva was developed a novel family of molecules, hydrated OXTs and OZOs fused in aldose and ketose series; those have scarcely appeared in literature, at least to our knowledge.

The dehydration method allowed us to obtain saccharidic oxazoles in only two steps from OZTs. However, the thiono group should be masked in order to have good results. OXTs anchored onto carbohydrate backbones constitute an attractive subject that has not yet been much explored in literature.

All of these compounds, hydrated OXT, OZO and OXT analogues, are strictly related to enantiomerically pure OZO, the importance of which has already been repeatedly mentioned.

Some of these molecules have been submitted to biological testing (Chapter VI).

Choice of the pathway

Since the purpose of this chapter was the synthesis of fused OZT moieties, the pathway developed in Chapter II was explored, via the key-step reaction of Willems and Vandenberghe 70 , i.e., condensation of thiocyanic acid with an α-hydroxy keto sugar.

Preparation of α-hydroxyketone

We were keen to develop a short access to an α-hydroxyketone, starting from the fructopyrano derivative 57 (Scheme 2). The di-O-isopropylidene-D-fructopyranose 4 was synthesized following Kim's 79a procedure. Benzylation of the free hydroxyl under standard conditions 88 then selective 4,5-O-isopropylidene hydrolysis using 80% aqueous acetic acid at room temperature 79a, 82 afforded 57 in good overall yield (Scheme 46). The reaction time of this regioselective deprotection could be improved from 15 h to 3 h by carrying out the reaction at

Scheme 47

Herein an example of a successful mono-oxidation of a partially protected D-gluco diol, 60 through treatment with Bu2SnO followed by brominolysis.

Scheme 48

In 1989, Tsuda 93 reported the mono-oxidation of non protected glycosides. He compared two systems -Bu2SnO-Br2 and (Bu3Sn)2O-Br2 -concluding that the second one was better in terms of yield and selectivity. In our case, the optimised conditions for smooth oxidation of glycosides involved an excess of (Bu3Sn)2O in the presence of 3Å molecular sieves in refluxing chloroform, followed by in situ bromolysis of the cooled mixture.

All of these procedures were used for selective oxidation of a secondary OH to the detriment of a primary OH, either on protected or non-protected glycosides. However our objective is the oxidation of a secondary alcohol in the presence of another secondary alcohol.

In 1993, Grindley 94 described that dibutylstannylene ketals could suffer regiospecific oxidations using N-bromosuccinimide (NBS) with good results. This method improved the conversion of carbohydrate diols into α-hydroxyketones. This has been experimented both on terminal (anchored) and internal (secondary) diols in furano and pyrano structures.

Scheme 49

This lack of reactivity might be attributed to non-stannylation of the diol, so that brominolysis could not take place.

As direct formation of an α-hydroxyketone through regioselective oxidation could not be achieved, another way had to be considered.

Since the target was a fused-OZT, we moved to the preparation of a fused bicycle using the condensation of a β-aminoalcohol with a thiocarbonyl source (Scheme 50).

Scheme 50

Thus a brief survey of the methodologies involved seems pertinent.

Condensation with a thiocarbonyl source -CS 2 or CSCl 2

In glycochemistry, the best known method for the synthesis of cyclic thionocarbamates on saccharidic backbones uses amino-sugars as precursors. The first steps are related to the introduction of the amine function on the partial or unprotected saccharidic structure. The reaction generally takes place via an isothiocyanate intermediate which is formed from carbon disulfide or thiophogene to give the desired OZTs or 1,3-oxazine-2-thiones.

The capacity and facility with which this process can occur depends on the size and natural strain of the saccharidic ring.

Herein are described some crucial rules compiled from literature: a) five-member thionocarbamates are preferred against six-membered counterparts b) furanic systems are more pre-arranged to receive bicyclic derivatives than their pyranic tautomers c) the relative arrangement of both reacting groups is extremely important: e.g. trans-diaxial-arranged isothiocyanate and hydroxyl groups in a conformationally rigid pyrano ring do not cyclize to give a saccharidic thionocarbamate. 75a

In literature can be found an exhaustive list with examples of this methodology applied to aldoses and ketoses, distinguishing the location of the amine function, either on the anomeric carbon, or on the carbon skeleton (C-2, C-3, C-5, C-6 and C-1 for ketoses). Here we are going to illustrate some of them.

Glycosylamines

In the course of studies on O-unprotected glycosylthioureas, while introducing an isothiocyanate function at the anomeric position of some aldose series, Fuentes reported important results. 74a, 74b, 74c The traditional method consisted of reacting glycosamines with thiophosgene in a buffered medium to obtain isothiocyanates, which were engaged with assorted amines to form the required thioureas. However, in β-D-gluco and β-D-galacto series, the presence of a mixture of two compounds in equilibrium was detected: the desired isothiocyanate 66 and more surprisingly, the cyclic thionocarbamate 67 (Scheme 51

Scheme 51

The above results seem to be in contradiction with the assumptions outlined for the synthesis of D-fructopyrano-based OZTs in Chapter II 1.1. However, these cases are different:

the saccharidic structures are less rigid, thus OZTs could be formed -remaining however in equilibrium with the isothiocyanate. The existence of similar trans-fused pyrano-carbamates, are reported in the literature. 74d, 95 In β-D-mannose series, only the cis-OZT 69 was isolated (Scheme 52

Scheme 52

This differential behavior is due to aldose initial configuration. In the first two series (D-Glc and D-Gal), hydroxyl groups (at anomeric position and C-2) are in trans-relationship while they are in cis-relationship in β-D-mannose. On the opposite to the cis relation, the formation of the OZT ring with the trans configuration is not so favorable which explains the observed equilibrium. These results agree with the fact that bicyclic compounds can be formed either in a 5 + 5 or 6 + 5 ring fusion, nevertheless in both cases cis isomers are clearly more stable than trans. 74d

Saccharidic templates bearing an amino group

The OZT formation starting from an amine generally arises in a similar way regardless of whether the amino group stands at the anomeric carbon or on the skeleton. The isothiocyanate is preliminary formed from CS2 or thiophosgene, and then by spontaneous cyclization or base-induced reaction (from βand/or γ-hydroxyl groups nearby) different enantiopure OZTs or oxazinethiones can be prepared.

Several studies have resulted in diversified positioning of thionocarbamate structures in a selective manner on saccharide backbones. 22, 24a, 24b, 74a, 74e, 74f, 74g, 74h, 74i Hereafter, some examples from aldose and ketose families are described. Following the general rules, in these cases, the formation of OZTs forced the anomeric position to adopt a cis relationship; no equilibrium involving isothiocyanate was observed.

74a,74b,74c

In the D-manno series, a specific point was observed -the formation of 2,3-cis-fused OZT 75, rather than the 1,2-cis regioisomer 76. This is most likely due to the fact that 74 is expected to be in higher proportion as an α-isomer, which leads to the formation of a trans structure that is less favorable. Simultaneously there is the reduced nucleophilicity of the hydroxyl group in the anomeric position.

C-6

The construction of thionocarbamate on the side chain of the sugar ring has been explored for example with an 7-amino-6,7-dideoxy-α-heptopyranoside, related to the D-gluco series. 74i The amino-alcohol was prepared through cyanide anion displacement from a 6-iodo-Dgluco derivative, followed by nitrile reduction. Further condensation with thiophosgene yielded the isothiocyanate 78. Under acidic conditions the isopropylidene acetal was removed, allowing rearrangement to the pyranose 80. This unprotected isothiocyanate seems to be stable in the absence of base. Final treatment of 80 with triethylamine in DMF at 80ºC forced the sugar ring to return to a furano form and afforded 1,3-oxazine-2 thermodynamic conditions, obtaining (R)-and (S)-cyanohydrin with excellent yields. Aziridines were synthesized in two steps: O-tosylation followed by hydride reduction of the cyano group with spontaneous intramolecular cyclization. As expected, direct acetolysis of aziridines failed:

the ring-opening of a spiro-aziridine often requires strong activation via N-sulfonylation. After obtaining epimeric β-amino alcohols precursors, the last step is the formation of OZT by condensation with thiophosgene under basic conditions.

Exo-OZTs can also be obtained via a cyanohydrin from the 3-keto sugar albeit without stereoselectivity. Reduction of the nitrile by LiAlH4 led to the corresponding amino-alcohol, which was further condensed with thiophosgene to afford the (3R)-spiro-OZT in ca. 30% overall yield. Despite being a shorter pathway, the last two steps gave disappointing yields.

4,5-Fused OZT in β-D-fructopyranose

As mentioned before (Chapter 1.1.) condensation of a β-amino-alcohol with a thiocarbonyl source was the chosen pathway for the preparation of 4,5-fused OZTs.

First Approach a) Structural protection

This approach first involved protection steps in order to functionalize selectively position C-5 and introduce the azide moiety. With respect to D-fructose structure, it was needed to select an efficient selective protection of OH-3 and OH-4. In the extensive list of hydroxyl protective groups, benzyl ethers are very popular. At an early stage, benzylation of OH-3 is straightforward; in contrast regioselective benzylation of equatorial OH-4 over axial OH-5 is much more tricky. Alternatively the fastidious multistep process described by Tatibouët can be envisaged. 62 Recently, Garcia Fernandez 36 reported the use of xylylene bis-ethers, which offer several advantages and can be easily removed by catalytic hydrogenolysis under mild conditions.

However our first approach was based on a synthesis previously developed in our laboratory 62 which involved OH-4 selective benzoylation. It is important to refer that this kind of protection, as 4-O-acylation and silylation, has already been reported in literature. 99

The 3-position of 4 was O-benzoylated before partial de-acetonation 79a, 82 to afford 88. In this reaction the intermediary isothiocyanate is not obtained through the conventional method (condensation of CS2 or thiophosgene onto an amine resulting from azide reduction).

This strategy avoids to handle amines which are known for being frequently difficult to purify 101 (a) M. I. Garcia-Moreno, P. Diaz-Perez, J. M. The yield of 59% is acceptable because (as reported in literature) 101a secondary sugar azides afford lower isothiocyanation yields than primary azides. And even if there is spontaneous cyclization of the isothiocyanate into OZT, the rigidity of the D-fructo backbone hampers its formation. Moreover it was mentioned before (Chapter II 1.3.3.) that formation of a 5 + 6 bicyclic system is not so favored. In order to improve the yield, this reaction was carried out in a sealed tube because of CS2 high volatility.

The mechanism of the reaction is not completely established. Actually, Garcia Fernandez and co-workers 101a proposed a double route: one via an iminophosporane and the other via a phosphazide. Both intermediates suffer a rearrangement to form the isothiocyanate, which cyclizes to give the OZT 93 (Scheme 61). At the end of this chapter a full recount is made on the selective iodination optimizations and studies on some other diol and triol moieties in ketose and aldose series (1.6.).

c) OZT formation

As reactions were optimized, we have prepared an assortment of 4,5-fused OZT with different substituents in C-3 in a view of future biological tests.

The first steps of the sequence were dedicated to preliminary protection. Compounds 95, 88 and 98 were prepared with excellent yields. Selective iodination in C-5 took place with good results for all substrates: 94% for 97 and 70% for 99. It has to be emphasized that compound 98 is a triol which was also successfully submitted to the selective iodination in 65% yield. Further azidation was also achieved in good yields, 62-92%. The last OZT formation step followed the same CS2 protocol which afforded moderate yields (53-62%) albeit completely acceptable for the reasons explained above (Scheme 64).

Scheme 64

Comparing the first and second approach the number of steps (and time consumption)

was reduced, from 7 to 4 in some cases. This novel and more original pathway has allowed the development of an innovative methodology that can be attractive in other fields of carbohydrate chemistry. Scheme 67

An issue at the end of this chapter is dedicated to selective iodination, and this different behavior, between D-fructo and D-psico series will be discussed.

c) Azido and OZT formation

Finally to obtain the OZT there was an intermediary "passage" through the azido derivative followed by isothiocyanate formation and cyclization to give OZT 55. This pathway enabled the synthesis of a 4,5-fused OZT in D-psico series, which will allow a comparison between the two series in biological tests. In addition it was also an opportunity to observe the behavior and to investigate the relatively unexplored chemistry of D-psicose.

Deprotection assays

As mentioned in introduction one of our objectives is the development of a library of

OZTs to be tested as potential GLUT5 inhibitors (Chapter VII). The inhibition capacity is quantified by the interaction of the hydroxyl groups of the molecule and the protein. Therefore, unmasking the hydroxyl groups in our molecules is crucial. Scheme 70

It can be deduced that 2M aqueous HCl might be too strong a medium, which allows deprotection at the anomeric carbon but prevents re-cyclization to the pyrano hemi-ketal. In other respects, it can be noted that these 4,5-OZTs are less sensitive than the 3,4-fused OZTs as less degradation was observed (Chapter II 1.2.4.).

An original reagent, Sc(OTf)3

With the same goal of deprotecting C-1,2 positions, another type of reagent was tested, scandium (III) triflate. 103 From the literature, this kind of reagent seemed to be more adequate

Trifluoroacetic acid conditions

From the literature 38 and the encouraging results obtained for the 3,4-fused OZT in chapter II (1.3.4.), in using 6:4 aqueous TFA for C-1,2 isopropylidene cleavage, we decided to try the same conditions on these substrates.

Scheme 72

In conclusion final compounds 109 and 112 could not be isolated under acidic and basic conditions, due to their instability, but the acyclic derivatives 110 and 111 could be obtained in quite good yields and will be submitted to biological tests (Chapter VI).

Selective Iodination

Iodination is a classical reaction commonly used in organic synthesis. The replacement of a hydroxyl group by iodine has been widely used in carbohydrate chemistry and it has aroused many interest in the last decades. 104 Deoxyhalogeno sugars -especially deoxyiodo -are very useful intermediates in many syntheses, 105,38 of azido-, amino-and thio-deoxy derivatives by substitution, 105 or deoxy sugars by reduction, which allows the preparation of natural compounds of biological importance such as antibiotic substances, cardiac glycosides, and bacterial antigens. 106 Their capacity to create radicals allows its applications in the synthesis of some natural compounds. 105 These versatile precursors can also be used in the conception of carbafuranosides which are analogues of biologically important products. 107 The one-step conversion of diols into olefins, has attracted the attention of a number of chemists and several methodologies were developed. 100,104d,106,108 This kind of modifications unlocks the potential of structural alterations in important biological compounds, like modified aminoglycoside antibiotics, nucleosides or nucleosides analogues. 109 In addition, deoxyiodo sugars also have biological interest as themselves, for example in SPECT medical imaging they are tracers of glucose transport. 110 Or, once iodine is a heavy element (quite radio-opaque) certain organic compounds (iodine-substituted) are thus used in medicine as X-ray radio contrast agents for intravenous injection, and X-ray urology. 111

Scheme 73

The list of methodologies is extended but the most currently used and well known was developed in 1979 by Garegg and Samuelsson. 100, 106 The authors described the synthesis of deoxyiodo sugars from a three-reagent system:

PPh3, imidazole and I2 or tri-iodoimidazole, which gave better yields, in refluxing toluene. The first conversions were done with primary and secondary hydroxyl groups on protected structures (Scheme 74). 100, 106 Scheme 74

Later they improved the reaction conditions by changing the solvent to a mixture of All of these methodologies provide efficient iodination. The substitution proceeds with clean inversion of chiral secondary hydroxyl and there is selective iodination of primary hydroxyl over the secondary. Furthermore, the iodination of the secondary is only possible onto protected structures.

During optimization of our synthetic pathway for the preparation of 4,5-fused OZT we have tried selective iodination with good results (1.3.2. b) and1.4 Scheme 77

At first, we decided to apply the conditions developed by Garegg and Samuelsson 100 and as expected, the major product was an olefin, 114 or 115. But more surprising was the unprecedented formation of a mono-iodinated compound. Taking into account this result we have concentrated our efforts to optimize this reaction in order to improve the synthesis of the iodinated derivatives. An optimization study was carried out involving several parameters: solvent, temperature, reaction time and equivalent number of iodine (Table 6). equivalents was also reduced to one equivalent and this must be precise otherwise olefins will be formed too. At room temperature no conversion was detected, thus the reaction was placed under THF reflux. As a result, the iodinated derivatives 97 and 107 were isolated in good yields, 94% and 56%, respectively. Replacing THF by 1,4-dioxane did not improve the reaction yields.

In both series, the reaction leads to iodination with inversion of the configuration, but not at the same position. It was observed in D-fructo series that iodination occurred at C-5 yielding 97. Quite surprisingly, in D-psico series iodination occurred at C-4 yielding 107 (Scheme 77).

How to explain this behavior ?

A plausible mechanism could be hypothesized (Scheme 78). It is known that during the iodination mechanism an oxyphosphonium intermediate is formed. But in our case, the vicinal alcohol might chelate the phosphorus atom: thus, in the D-fructo case, a tautomeric equilibrium between intermediates 116 and 117 can exist. The nucleophilic attack of iodine then would be on the C-5 position to generate the thermodynamic iodo L-sorbopyranose 97. All substituents are in equatorial position, which is the most stable situation.

In the D-psico case, a similar equilibrium can be postulated together with a conformational equilibrium between 117 and 118. Consequently iodine would be introduced at C-4 with inversion of both configuration and conformation, leading to a D-tagatopyrano unit.

Once again all substituents are in the favored equatorial position. Similarly to the study of diols an optimization was carried out (Table 7). The first tests were applied using the conditions previously developed for diols. Some selectivity was observed but only with low yields. The reaction was then carried out for 72 h but without noticeable improvement. Since the reaction was not complete, it was decided to force the conditions, and the solvent was changed for 1,4-dioxane in order to increase the temperature. The amelioration was significant but starting material still remained in the mixture, even after extending the reaction time. However, if the reaction was performed on larger scale (ca. 1g starting material), the reaction yields for 100 and 120 increased to 63% and 65%, respectively. This might be due to the concentration of the starting material. We have thus optimized the conditions to obtain good yields of a selectively iodinated ketose. It can also be pointed out that no olefin formation is detected.

In comparison with the study on diols, it was interesting to observe that whatever the sugar series -D-fructo or D-psico -only position C-5 is selectively iodinated.

We might propose in these cases that complexation was not the main aspect for the In order to experience the efficacy of this method it was decided to also test it on aldoses and therefore it was applied in D-gluco, D-manno and D-galacto series.

The triol substrates were prepared from methyl β-D-pyranosides, in which the primary hydroxyl was selectively O-silylated under standard conditions to afford 6-O-silylated derivatives 123, 124 and 125 in good yield (Scheme 81).

O R 3 OH OMe HO R 1 O R 3 OTBDMS OMe HO R 1 TBDMSCl, pyridine rt, 15h R 1 =R 3 =OH, R 2 =R 4 =H (D-gluco) R 2 =R 3 =OH, R 1 =R 4 =H (D-manno) R 1 =R 4 =OH, R 2 =R 3 =H (D-galacto) R 1 =R 3 =OH, R 2 =R 4 =H, 123, 86% (D-gluco) R 2 =R 3 =OH, R 1 =R 4 =H, 124, 81% (D-manno) R 1 =R 4 =OH, R 2 =R 3 =H, 125, 80% (D-galacto) R 2 R 4 R 4 R 2

Scheme 81

Then the conditions for selective iodination were applied (Scheme 82 and Table 8). The conditions used were the same as for triol derivatives: I2 (1.0 eq.) in 1,4-dioxane at 100ºC for 4 h. Selective iodination was observed in D-gluco and D-galacto series but high reactivity was detected in D-mannose series. The yields are acceptable about 42 and 45% for 126

and 127, respectively. We thought that these results could be increased if the reaction has been done on a higher scale. The selectivity can be explained taking into account the stability of the final compounds. As previously stated the reaction occurs with inversion of the configuration at the iodinated position.

When it was decided to test selective iodination in D-gluco series it was not expected that it occurs once all substituent are already in equatorial position. And if that happened to it was attended in position C-2 since it known for being the most reactive, for example, in glycosylation reactions or selective O-silylation. However, regarding literature it can be observed a similar behaviour when working with other halogen elements like bromine or chlorine. 116 Under our conditions, the D-mannopyranoside 124 gave an intractable mixture of compounds. The D-galacto derivative 125 behaved as expected, iodination occurring at C-4 and all substituents being equatorial. The iodinated compound 127 belongs to the D-gluco series.

Conclusion

In Taking into account the results obtained we can put forward some preliminary assumptions:

A novel and original synthetic pathway for 4,5-OZTs preparation was developed using selective iodination. In some cases we succeeded in reducing the number of steps from seven to just four.

Functionalization and epimerization of C-5 and C-4 in D-fructo and D-psico series, respectively, was explored via Garegg's methodologies.

In some cases, fused OZTs are very sensitive molecules. Complete deprotection of cyclic structures could not be achieved neither under acidic nor basic conditions. In special acidic conditions the decyclization of the saccharidic backbone was observed, maybe due to the steric effect of the cyclic thionocarbamate.

Some of the chemistry of D-psicose, a "rare sugar", which has not yet been much explored in literature has been further developed.

Some of these molecules have been submitted to biological tests (Chapter VI).

On a more specific point, regarding the selective iodination, it can be assumed that:

Regio and stereoselective iodination on diol and triol carbohydrate templates have been achieved.

The yields of conversion are good to satisfactory depending of the kind of derivative and sugar series that we are working on.

This selectivity can be applied to different sugar families: ketoses and aldoses, but no clear rule could be extracted except the choice of the thermodynamic pathway.

It is a shortened access to OZTs which can be useful for iminosugars preparation once those can be prepared from azido compounds similar to ours. In these approaches we will compare two different methods we have used and improved.

CHAPTER IV

Spiro-thionocarbamates in pyranose form

Scheme 84

Choice of the pathway

The purpose of this chapter is the preparation of spiro-OZTs. Considering the different processes discussed in the previous chapters, the appropriate method seems to be the condensation with thiocyanic acid. Another approach with the use of trimethylsilyl isothiocyanate has more recently been reported in the literature. 22d Between the two approaches, the first one involves several steps to be efficient; the reaction using TMSNCS is more direct but has not shown significant reactivity, low yields (about 10%) being observed. Those two procedures are going to be developed in this chapter and therefore a brief description of both seems to be appropriate.

Condensation with HSCN

Preparation of carbohydrate-derived thionocarbamates by condensation of HSCN on Ounprotected sugars was initially described by Zemplén. The reaction implies the anomeric position and the neighboring hydroxyl group able to cyclize intramolecularly with the transient isothiocyanate. The condensation gives the thermodynamically most stable OZT. Curiously, this pioneering method has been no so well studied. The first assays were performed in two standard sugar families, an aldose 73a (D-glucose) and a ketose 73b (D-fructose), and the products obtained were believed to be OZTs fused on pyrano backbones (Figure 17).

Figure 17

Ten years later, Herbst and Bromund explored other aldose series (D-galactose, D-xylose and L-arabinose) and came to similar structural conclusions (Figure 18). 73c

Figure 18

However, structural determination was not completely accepted and it was an issue of contentious discussions for some time. In 1959, Wickstrom and Wold decided to reconsider the studies previously carried out. 73d Characterization tests such as periodate oxidation, IR and UV spectral measurements were performed to confirm and correct the structures previously postulated. Therefore it was demonstrated that the structures obtained were indeed fused bicyclic OZTs: those arising from aldoses were in furano form while those from ketoses were in pyrano form (Figure 19). This conclusion was additionally confirmed by the first NMR analysis In 1975, Ranganathan reported about nucleosides using D-arabinofurano-derived OZTs as precursors. 23a, 23b In the same lines, Imbach worked with D-xylo-derived bicyclic OZTs. 23c, 23d, 23e Valencia strengthened later this hypothesis: 73f aldo-series originated mostly bicyclic OZTs on furano frames, contrary to the first erroneous interpretations of Zemplén. 73a, 73b But if there is a consensus about the structure obtained with aldoses, the same cannot be said for ketoses. In recent years, the OZT subject aroused the interest of our laboratory and several studies were developed in diverse sugar families. In aldose series, Girniene-Rousseau et al. 46a, 52, 55 agreed with the assumptions previously proposed, essentially concluding that this is the only method which can lead directly to OZTs without preliminary protection of the sugar.

When submitted to HSCN condensation, ketoses behave differently. A major problem lies in the lack of chemical selectivity, probably due to the complexity of the tautomeric equilibria and the predisposition of ketoses to form tertiary oxocarbenium ions under acidic conditions. This often leads to the formation of cyclic and open forms mixtures but also inevitably to degradation. 22c The reaction mechanism is probably similar to that of aldoses, certainly involving initial formation of an intermediate anomeric isothiocyanate, which will subsequently undergo nucleophilic attack of an α-hydroxyl. The first stage is reversible and leads to the formation of the thermodynamically most stable (Scheme 85). From that point, Tatibouët presented a hypothesis that reflects well this complicated ketose chemistry. 66c Indeed, it is possible to predict ten different compounds: fused bicyclic structures in furano and pyrano forms, spiro-epimers (α/β mixtures), acyclic chain or even spirobis-OZT (Scheme 86). Herein, our objective was to improve these selective pre-and post-protections in order to increase chemical yields.

From this standpoint, and as a parallel to the HSCN method, we called for a recently described and not much explored TMSNCS methodology, 22d which might allow a one-step conversion.

Condensation with trimethylsilyl reagents 22d

In 2001, Fuentes reported an unconventional pathway for the synthesis of spiro thionocarbamates. The key-step of the method involves reaction of sugars with trimethylsilyl azide (TMSN3), isothiocyanate (TMSNCS) or cyanide (TMSCN). The mechanism is similar to the one postulated for the condensation with HSCN: after nucleophilic attack at the anomeric position, the isothiocyanate formed undergoes spontaneous cyclization.

a) TMSN3

To synthesize a spiro-D-fructopyrano derivative, TMSN3 was used in the presence of a Lewis acid, TMSOTf. The initially formed azide is hydrogenated into an amine 130, which is subsequently condensed with a thiocarbonyl donor (Im2CS) to furnish the isothiocyanate 131.

Spontaneous cyclization selectively affords in good yield the β-anomer 132 (α:β)=(1:9) (Scheme 89).

Synthesis of spiro-OZTs

As mentioned at the beginning of this chapter, our objective was the synthesis of spiro-OZTs using either the HSCN or the TMSNCS method. In order to avoid the formation of a complex mixtures of different compounds (see § 1.1.1.), we first had to properly protect our ketose moieties. Once we wanted to prepare spiro-OZT OH-3, OH-4 and OH-5 could be protected with a stable protective group and different from the one at the anomeric position.

Ketose protection

Benzyl ethers are among the most popular protecting groups used in glycochemistry.

They can be easily introduced by treatment of an alcohol with benzyl halides in the presence of a base. Such standard conditions (BnBr, NaH) were previously applied, Chapter III 1.1.1., with excellent results.

However, performing a triple O-benzylation under these conditions was not so effective.

Actually, a mixture of several compounds was obtained, including mono-, bis-and trisprotected pyrano systems, the column chromatography.of which proved tedious. Terashima has reported improved conditions to realize this reaction by using benzyl chloride (acting both as reagent and solvent), and potassium hydroxide (KOH) at 130ºC for 3 h. 118

Scheme 93

At this stage the ketoses were ready to undergo condensation with HSCN and TMSNCS to give spiro-OZTs without any complex mixture formation.

OZT formation -HSCN

The first method experienced was the condensation with HSCN; once it is a broadly exploited procedure in our laboratory.

Scheme 94

In order to improve the yields, various solvents were tested. This optimization was done taking into account a study developed by Silva et al. 66b about the choice of adequate solvent-acid couple for the synthesis of simple OXTs and OZTs. The best results in D-fructo and D-psico series were obtained with ethanol (Table 1). The anomeric ratio established after deprotection correlated well with the 5:5 α:β-ratio obtained for 142 and 143; for both cases it was not possible to separate the anomers. As can be seen, a low yield of acyclic compounds 146 and 147 could be obtained, albeit in one assay only. The above OZT preparation could be scaled up to 6g starting material.

OZT formation -TMSNCS

TMSNCS is used in a one-pot procedure and stereoselectivity at the anomeric position can be observed. Such selectivity could be explained by the fact that the ketopyrano precursors were already in β form (Scheme 95).

Scheme 95

We first applied the conditions developed by Fuentes, namely TMSNCS, TMSOTf at room temperature. 22d As expected, only traces of the desired compound were detected.

Changing the Lewis acid from TMSOTf to BF3.OEt2 at 0ºC mostly resulted in degradation, whereas working at -78ºC froze the reactivity. Increasing the number of TMSNCS equivalents proved to be more effective. The conditions optimized in D-fructo series -TMSNCS (3.0 eq), BF3.OEt2 (1.0 eq) at room temperature -were subsequently applied to the D-psico series. Major formation of the β anomer was observed for the D-fructo OZT 144 (66% yield, α:β=1:9) and the D-psico OZT 145 (47% yield, α:β=2:8) (Table 10).

Figure 21

These structures were determined by NOESY experiments which showed the constant coupling of long distance between the H-1 and H-6. Furthermore the C-2 chemicals shifts are about 92.6 ppm for both 148 and 149 which is characteristic of α structures.

Synthesis of spiro-OZOs

Notwithstanding our main concern -OZT syntheses -we had to take into consideration the importance of OZOs in the literature 84, 85 and it seemed therefore quite interesting to study the oxidative desulfurization of OZTs 144 and 145, to be able to introduce some biological comparison between both structures.

Oxidative desulfurization can be achieved by three different routes, which have already been introduced in Chapter II 2.1.4.: 1) direct oxidation 2) oxidation after N-protection or 3) oxidation after S-alkylation.

S-alkylation

Since S-alkylation had given good results when applied to 3,4-fused OZT, the same procedure was adopted. Compounds 144 and 145 were S-benzylated using BnBr and NaHCO3

as base in DCM at room temperature (Scheme 96). The results were excellent: indeed the 90% yields obtained were better than reported in literature, 24c, 46a, 52b but more surprisingly this procedure allowed easy column chromatography separation of spiro-epimers. In D-fructo series 150 and 152 were obtained with 48% and 43% yields, and in D-psico series 151 and 153 were obtained with 43% and 47% yields.

Tatibouët initially tried to perform separation of stereoisomers via N-acetylation in D- fructo series but without good results. 119 Only 9% and 16% yields were respectively obtained for α and β isomers.

Oxidation

The oxidizing reagent chosen was 77% m-CPBA, which is currently used in our laboratory to perform this kind of transformation. OZOs 154 155, 156 and 157 were obtained with acceptable yields -65% and 64% in D-fructo series; 69% and 52% in D-psico series (Scheme 97).

Scheme 97

In Chapter II 1.4.3., we have observed that oxidation with m-CPBA was not as efficient as with H2O2. However due to lack of time, no test was effected with this reagent.

Deprotection assays

The complete deprotection of the OZO was effected by catalytic (10% Pd/C) hydrogenolysis (Scheme 98). The unprotected α-isomers 158 and 159 were obtained with low yields -25% and 34% respectively -but the results were better for β isomers 157 and 161, with 68% and 56% yields, respectively. Nevertheless those yields were lower than what can be expected in such de-O-benzylation processes. This could be due to the presence of several benzyl groups: indeed tri-, di-and mono-benzylated compounds were detected on TLC after four days reaction. In order to improve the yields and to accelerate the reaction, a catalytic amount of AcOH can be added, as reported in the literature. 120 Py & coll. described good results with using boron trichloride in DCM for the deprotection of benzyloxy-substituted nitrones. 121 However when applied to our OZO with varying reaction time and temperature, those conditions failed to bring improvement in de-Obenzylation.

Conclusion

In this part of chapter IV we have focused our attention on the synthesis of spiro-pyrano OZTs and OZOs in D-fructo and D-psico series starting from tri-O-benzylated compounds (Scheme 99). From the results obtained, we can report some preliminary statements:

The purpose of pre-selective protection in order to control the products formed was efficient by 3,4,5-O-benzylation it were only obtained spiro-OZTs.

Both methods for spiro-OZT preparation were effective using either HSCN or TMSNCS.

An original and attractive procedure for direct synthesis of spiro-OZTs using TMSNCS has been improved from 10% (literature) to 47-66% of yield.

Stereoselectivity at the anomeric position can be controlled depending on the type of reaction performed. The β-isomer is preferentially formed with TMSNCS .

Spiro-OZTs have been prepared in good yields, but separation of isomers is rather difficult;

nevertheless small amounts of β-isomers in D-fructo and D-psico series were obtained for characterization.

S-alkylation was performed with excellent yields. Furthermore it has allowed perfect separation of the αand β-anomers by simple silica gel column chromatography.

Spiro-OZOs were also prepared by a reaction sequence that allowed the preparation of each isomer separately in acceptable yields.

The de-O-benzylation of the α-isomer was difficult to perform, whereas with the β-isomer acceptable yields were obtained.

Some of these molecules were submitted to biological tests (Chapter VI). Signet non défini. we wanted to develop a specific approach to spiro-OZTs anchored on such frames.

SYNTHESIS OF SPIRO

Synthesis of spiro-OZTs in D-fructo series

Unlike D-psicose, D-fructose is readily available and cheap; among ketohexoses, it is also by far the one whose chemistry is the best documented. Therefore our experiments were first optimized in D-fructo series then applied in the D-psico series.

Choice of the pathway

With the purpose of preparing spiro-OZTs in furano form, we have chosen a preselective protection approach and subsequent OZT formation, which reduced the number of products to four OZTs, instead of a mixture of ten.

Synthesis of spiro-OZTs

The starting 3-O-benzylated D-fructo derivative 57 was prepared following the sequence described in Chapter III 1.1.1. and we have applied a similar approach as that previously used for the tri-O-benzylated derivatives. A two-step protocol involved isopropylidene acid hydrolysis to yield the intermediate 162, which was then condensed with thiocyanic acid. A one-step protocol using TsOH and HSCN also gave the OZTs, but both methods resulted, as expected, in a mixture of four products: both αand β-isomers of spiro-OZTs in furano and pyrano form (Scheme 100). In the present case, it was quite difficult to estimate the isomeric ratio of the compounds; the separation of such a complex mixture was rather tricky. First attempt to separate the OZTs involved acetylation using Ac2O but only a mixture of fully acetylated compounds was obtained. Insertion of an isopropylidene group was then experimented. The purpose was simultaneous protection of OH-4 and OH-5 in the pyrano structures, with a view to inducing a chromatographic difference between the two ring forms.

Two procedures were tested: acetone-dimethoxypropane with CSA and acetone/sulfuric acid.

The best results with the first procedure were 6% and 27% yields of isolated αand β-pyrano forms 163 and 164, while in both attempts unseparated α:β mixtures were respectively obtained in 11% and 24% yields. In other respect, the second procedure allowed isolation in 16% yield of the unprotected furano OZT. Other protection attempts were performed with TBDMSCl: complex mixtures of mono-and di-O-silylated α and β, pyrano and furano forms were obtained, therefore the idea was to selectively protect the primary hydroxyl of furano forms.

Using just one equivalent of TBDMSCl, mono-O-silylated αand β-furano-OZT were obtained in 10% yield (Table 12). The separation of spiro-OZTs in both furano-and pyrano-forms could be achieved albeit with not very encouraging results. Nonetheless the isolated compounds are interesting for characterization and for biological testing (Chapter VI).

b) By S-alkylation

Previously, for the tri-O-benzylated compounds, it was observed that isomers separation was possible using an S-functionalization. After application of nearly quantitative S-benzylation to, effective separation of the epimers 165 and 166 was performed with 52% and 47% yield, respectively (Scheme 102). From that point, we decided to perform S-benzylation on the mixture of four products, in order to check whether a separation of the alkylated products would be facilitated (Scheme 103). Unfortunately, no clean separation of the S-alkylated mixture was achieved.

Synthesis of spiro-OZTs in D-psico series

Although not exceptional, the results obtained in D-fructo series contributed to a better understanding of ketose behavior. Altogether, the synthesis and characterization of interesting new OZTs allowed to enrich our library of thionocarbamates. On another hand, D-psico derivatives have not been much explored yet and that called for further experiments in the D- psico series.

A similar approach was considered and analogous results were achieved (Scheme 105).

Condensation with HSCN led to the formation of a four-compound mixture containing αand β-isomers in furano and pyrano forms. Subsequent isopropylidene protection was performed and both αand β-spiro-OZTs isomers in pyrano form were obtained in 31% yield. The furano unprotected compounds were not isolated.

Scheme 106

From the results obtained, we can put forward some preliminary statements:

Starting with a 3-O-benzylated precursor, both pyrano and furano spiro-OZTs could be prepared.

In both series, the effective isolation of the spiro-OZTs proved to be rather complicated especially with the furano forms.

Once again S-alkylation proved to be an excellent method for the separation of spiro-OZT isomers but was only effective on pyrano entities. Some of these molecules were submitted to biological tests (Chapter VI).

SYNTHESIS OF SPIRO-AND FUSED OZTs BUILT ON D-TAGATO AND D-PSICOFURANO FRAMES

In this chapter we are going to illustrate the thionocarbamate chemistry of another ketose -D-tagatose. This sugar is a C-4 epimer of D-fructose, whose chemistry has not been much explored according to the literature. Furthermore a chemical comparison with D-psicose in furano form will be reported.

Preparation of D-tagatose

D-Tagatose is currently manufactured, but even if its commercial production is already well developed (and on a large scale) it remains extremely expensive: the cost of 1g D-tagatose is ca 200€, while the cost of 100g of D-fructose is around 15€ ! Taking into account this huge difference of price, it looked interesting to prepare D-tagatose from its cheap C-4 epimer.

From D-fructose to D-tagatose series

In Chapter III 1.4.1. we reported the conversion of D-fructose into D-psicose through a simple inversion of the C-3 configuration via an oxidation-reduction sequence.

However, due to the conformational characteristics of D-tagatose, this type of transformation seems to be more tricky in this case, the selective access to C-4 being more complicated. Transposition of the D-psicose synthetic pathway to D-tagatose would require several protection/deprotection steps, which would make the procedure difficult and not very productive (Scheme 107). Furthermore selective oxidation was already tried without success, Chapter III 1.1. The nucleophilic ring cleavage of epoxide 170 was therefore investigated in miscellaneous (basic or acid) conditions, however in most cases no reaction occurred, the starting material 170, always being entirely recovered. Only MeONa in methanol solution at 60ºC effected epoxide opening to give a moderate yield of the L-sorbopyrano compound 174.

The attack and inversion at C-5 might be favored by the stability of the resulting all-equatorial system: indeed a similar result was obtained in previous iodination studies (Chapter III 1.6.1. and 1.6.2.). This regioselective inversion could also be induced by the epoxide geometry, which after minimization (Figure 1) showed that both position C-4 and C-5 are quite similar for the nucleophilic approach. The main difference comes from the benzyl substituent which could hamper the attack in C-4 and thus favor the "anti-Fürst-Plattner" nucleophilic opening.

Conversion of D-tagatose into an OZT

Since it did not seem feasible to synthesize D-tagatose from D-fructose using the epoxide-way, we had to turn to commercially available D-tagatose for OZT synthesis.

Few experiments involving unprotected D-tagatose have been reported. As previously described in Chapter IV 1.

Scheme 111

The same reaction was subsequently performed with a reduced quantity of KSCN in order to avoid the formation of bis-OZTs: as expected, a mixture of several products was obtained (Scheme 112). A post-protection reaction was thus needed to try and separate the compounds. The TBDMS bulky group was chosen to accomplish selective protection, which might facilitate separation. Only 2,3-fused cyclic thionocarbamates in furano and pyrano form were isolated in 53% overall yield for a two step sequence. Three molecules 177-179 isolated in 20%, 22% and 11% yield, respectively were characterized. No spiro or acyclic structure could be detected. Curiously, compound 177 was shown to be a 2,3-fused OZO, indicating that a sulfur/oxygen exchange had occurred, probably resulting from an hydrolysis. The structure of compound 177 was easily assigned by 13

D-Tagatose and D-psicose -OZT Selective Formation on Furanose Form

After some assays with unprotected D-tagatose the selective synthesis of some spiro-OZTs was then considered, implying pre-protection reactions: the reactivity of D-tagatose and D-psicose both in furano form will be compared.

From pyrano to furano form a) D-Tagatose

The protection of D-tagatose with isopropylidene groups leads to a furano form, which is induced by the structural relationship of hydroxyls. Both isomers α and β were obtained even if in different proportions (Scheme 113).

Scheme 113

This protection was performed under acid catalysis using two different reagents.

Standard conditions involved acetone with a catalytic amount of H2SO4, as previously used for D-fructose protection (Chapter II 1.3.1.): a mixture of two separable isomers was obtained: α 180 in 67% and β 181 as traces (2.4 %). The second method used 2,2-dimethoxypropane with PTSA catalysis in a 4:5 toluene-DCM mixture at 60-65ºC for 18h. In this case only the α isomer was produced in 83% yield and both results were in accordance with literature. 124 Although better in yield, the second procedure is more susceptible, because of reaction parameters which have to be scrupulously respected to avoid significant yield reductions. 130b

O-Benzylation

In both ketose series, the primary alcohol was protected as a benzyl ether using standard A second attempt was performed with slightly modified conditions. The two consecutive reactions were carried out in aqueous medium, first effecting deprotection with a 60% TFA solution then condensation with HSCN. For both ketose series, a similar mixture of three compounds was obtained as previously noted. Those mixtures were obtained in good to excellent yields and a major compound was clearly formed, which was supposed to be the 2,3fused OZT (188 or 190) -however this could not be purified (Scheme 118). 15). The conditions using 3 equivalents of TMSNCS were also applied in D-psico series. In this case, the selective formation of the β OZT 192 in 57% of yield was observed, without any side-product resulting from the benzyl migration.

Table 15

PRODUCT CONDITIONS RESULTS

183

Synthesis of OZO

In the D-tagato series, a spiro-OZO was obtained as side-product in the synthesis of OZTs: however the compound only appeared in mixtures. In order to prepare pure OZO, Salkylation of the OZT, followed by oxidative desulfurization had to be performed

S-Alkylation

The reaction was performed as previously described with BnBr in the presence of Diabetes mellitus is a chronic metabolic disorder. Unlike type 1 diabetes, which appears in childhood and results from an inadequate secretion of insulin, type 2 diabetes comes after years while secreted insulin did not sufficiently act on the body tissue to lower blood glucose levels. This pathogenesis is complex, and involves progressive development of insulin resistance and a relative deficiency in insulin secretion, leading to hyperglycemia. The disease is largely associated with obesity, a major cause of insulin resistance. 128 About 75-80% of type 2 diabetics are obese and 20-25% of obeses become diabetics.

To date several oral drugs and insulin are the current available therapies. 128, 129 The two major pharmacological approaches are bisguanides and thiazolidinediones but even though successful, they are often associated with undesirable side effects. A potential new advance is the use of antioxidants however, combined innovative pharmaceutical and non-pharmaceutical strategies are desirable. 127 Diabetes incidence is increasing at an alarming rate and has reached epidemic proportions. According to World Health Organizations (WHO) reports, if it continues to grow as actually in 2030 there will be about 366 millions of patients being more than 90% type 2 diabetes (Figure 23). Furthermore, and always according to WHO, deaths are projected to increase more than 50% over the next decade if appropriate measures are not taken as quick as possible. These diseases underlie many costly health problems in most developed countries.

Managing the long-term complications of the disease, like cardiovascular complications among others, will have a serious impact on public health budgets. 130 Our detailed knowledge of the symptoms of the disease (peripheral insulin resistance, early hyperinsulinemia, late hypoinsulinemia, intolerance to glucose, hyperglycemia, frequent infections, hypertension and cardiac failure) is in sharp contrast with the poor information that is available on its biochemical, cell biological and genetic background. Poor regulation of glucose intake and disposal has a strong polygenic background but the influences of dietary factors and sedentary lifestyle on the aetiology of the disease are gradually being more fully appreciated and understood. An important interdisciplinary study at this stage would be very helpful and would bring an integrated approach to this multi-factorial problem. Only with this kind of inter-cooperation it would be possible to improve better understanding on basis disposal of excess amounts of fat, fructose and glucose, with the aim of taking appropriated measures of prevention and treatment to a long term battle against these diseases.

c) Sugars in human body. GLUTs.

Sugars are the major source of calories at all stages of life and they are essential for supplying our metabolic needs. They are abundant components of diet and can vary from simple to complex carbohydrates. For example, sucrose is broken down in the intestine to produce both glucose and fructose. These sugars are absorbed into the blood stream and act as a supply of nutrient for tissue metabolism. Sugar disposal occurs via uptake into selective tissues that use specialized membrane transporters called GLUTs.

Glucose transporters (GLUT or SLC2A family) are a family of transmembrane proteins found in most mammalian cells which are responsible for glucose homeostasis by facilitating the transmembrane passage of monosaccharides. 131 Since the discovery of the glucose transporters GLUTs 1-5 in the early 80/90s it has generally been assumed that these isoforms alone controlled mammalian glucose homeostasis. However, the project of sequencing of human genome has recently disturbed this notion. It is now believed that there are 8 more un- characterised isoforms, which makes a total of 13 GLUTs. Therefore these protein transporters were divided into three subfamilies namely Class I (GLUT 1-4), Class II ( GLUT 5,7,9 and 11) and Class III (GLUT 6, 8, 10 and 12 and the myo-inositol transporter HMIT 1). 132 We would like to utilize this recent human genome data in post-genomic approaches aimed at understanding the functional roles of the new transporters, there substrates preference tissue distribution, and how they complement and are add to the functionality of GLUTs 1-5. At the moment research must be focused on the molecular function: substrate specificity, affinity, regulation of subcellular localization and mechanism of cellular trafficking. An integrated process starting with pharmacomodulation of inhibitors of transporters to the development of biochemical tool with high selectivity and affinity for a specific GLUT is the first step toward understanding the physiological regulation between GLUTs and glucose homeostasis. In this project, we want to focus our attention on the Class II transporters, in particularly GLUT5, due to its specificity to the transport of D-fructose, which is the second sugar -after D-glucose -most present in human diet. So far it is known that GLUT5 is localized in the mucosal surface in small intestine but also expressed in skeletal muscle 133 , testis, kidney, fat tissue, and brain 134 (Figure 24). Although some digestion occurs in the mouth and stomach, disaccharides and oligosaccharides from any food source remain relatively undigested, for the most part, until entering in the small intestine where the appropriate enzymes break them down into the respective monosaccharides. Absorbed monosaccharides pass through the small intestine into the portal circulation and are transported to the liver, where glucose, galactose, and fructose are phosphorylated. Fructose absorption mechanism in the small intestine is still not completely understood. Some facts propose either active transport, because fructose uptake was observed to occur against a concentration gradient135 or by facilitated transport involving GLUT5 (fructose specific) or GLUT2 (in competition with glucose and galactose) protein transporter136 (Figure 25). Studies have shown that the greatest absorption rate occurs when glucose and fructose are administered in equal quantities 137 . It appears that the GLUT5 transfer rate may be saturated at low levels and absorption is increased through joint absorption with glucose138 (glucosedependent co-transport phenomenon). In addition, fructose transfer activity increases with dietary fructose intake. A GLUT5 insufficiency can generate an excess of fructose carried into the lower intestine 136 which provide nutrients for the existing glut flora, and may cause some illness.

GLUT5 is found in many tissues, its expression levels and activity are clearly regulated under normal conditions and are altered under pathological conditions (diabetes, hypertension, obesity, and inflammation). It also seemed to be induced during carcinogenesis, particularly in the mammary glands. 126 The increasing importance of fructose in human nutrition and disease calls for additional studies that hopefully will increase our understanding of the role of the fructose transporter GLUT5 in health and disease. Despite the importance of dietary fructose in the development of diabetes, and that of GLUT5 in fructose transport, very few studies have investigated the link between diabetes and GLUT5 in these insulin-sensitive organ systems. The abundance and the specificity of GLUT5 suggest that it plays a metabolic important role which is imperfectly understood. The comprehension of the structure and function of this transmembrane protein is indeed crucial. 139 This involves utilization of chemical biology approaches for the design and chemical synthesis of sugar analogues and inhibitors of the GLUT proteins. Thus, specific inhibitors of this protein are reagents of choice to develop chemical probes. 140 We wanted to focus on fructose specific transporters of Class II.

PhD project 1.2.1. Objectives

This part of the project dedicated to biological tests was in charge of our partner Dr. G.

Holman at the University of Bath. The library of compounds synthesized is going to be submitted to a screening to determine the potential GLUT5 inhibitors. The inhibition rate will be defined by measuring inhibition of D-[ 14 C-U]-fructose uptake by using scintillation counting 75a of Chinese hamster ovary (CHO) cells. 62, 75a The results will give a entire knowledge of interaction between synthesized compounds and GLUT5 which will allow a detailed understanding of the recognition site of GLUT5. Furthermore it will determine which inhibitors will be selective and specific to GLUT5 (also to Class II family) and will allow us to prepare photoaffinity labels which will track in vitro and in vivo the localization and physiological behavior of GLUT5 (Class II).141

Figure 26

The bio-chemical device should be composed of three different components: (1) the recognition domain of GLUTs (the structure proposed is based on previous results), ( 2) on a selected position, a photoreactive structure, (3) a long spacer toward the last part of the biochemical tool like biotin or a fluorescent tag (Figure 26). 75b, 142

Previous works

First studies to find potential inhibitors of GLUT5 started with Dr. G. Holman & coll. by blocking all the hydroxyl groups, one at a time, of D-fructose in both ring forms with allylic ethers. 56a At the end, it was concluded that both furano and pyrano forms were tolerated and the two sites O-2 (furanose and pyranose) and O-6 (furanose) could be altered to give informations about the interaction with the protein. Furthermore, postulating that D-fructose is a quasi-symmetrical molecule, they deduced that the binding site could be on both sides of the molecule, the anomeric and the opposite side, which means 3, 4, 5-OH for D-fructopyranose and 1, 2, 3-OH or 3, 4, 6-OH for D-fructofuranose (Figure 27).

Figure 27

In order to investigate this model of interaction Rollin & coll. developed a second approach involving thionocarbamate moieties. OZTs and OZOs were the structures of choice to block the D-fructose skeleton. Erreur ! Signet non défini.

To better understand the recognition process and determine the importance of each of hydroxyl groups in the binding and interaction with GLUT5 a study was made involving diverse parameters like sugar family (aldoses or ketoses) and series (configuration of different hydroxylated sites), nature of the group at C-1 (allyl or benzyl) and type of thionocarbamate (OZT or OZO) (Figure 28).

Figure 28

The results highlighted that aldopentose OZT derivatives were not recognized by the protein transporter and ketohexose OZT and OZO derivatives presented some inhibition.

Moreover, L-sorbo OZO derivatives, with C-1 benzyl group expressed better inhibition constants (Ki) than OZTs, with 1-O-allyl, in D-fructose series than D-fructose itself (Table 16).

Table 16

D-fructose 15.5 ± 2.9 Those studies allowed a better comprehension of the interaction model. It is understood that the C-1 arm in ketoses is important to obtain some inhibition, however this can be substituted and when it is it presents even better results. The fact that C-1 can carry bulky groups means that the binding site could be on the opposite side of the anomeric position, in C-4 and C-6, as previously postulated (Figure 29).

OZT K i [mM] OZO K i [mM]

Figure 29

PhD prospects

In this PhD we have synthesized various molecules that compose a library of potential GLUT5 inhibitors. Among them stands a wide range of thionocarbamates: OZTs, OZOs and OXTs. Those were prepared in different ketose series, as fused or spiro structures, in cyclic or even acyclic frameworks and in furano, but mostly in pyrano form.

In the previous studies the structures of choice which gave the best results were OZTs and OZOs in furano form. This PhD gave the possibility to go more deeply by enlarging it to other ketose family members and specially focus on pyrano forms that have not been tested.

However to date, this biological screening has not yet been realized. Nonetheless, taking into account preceding studies and the interaction models proposed it can be predicted that some of the molecules could be promising inhibitors. Some examples of the molecules synthesized in furano and pyrano forms are presented in Figure 30.

O R 3 O R 2 O O NH OR 1 X 177, R 1 =R 2 =R 3 =TBDMS, X=O (D-tagatose) 178, R 1 =R 3 =TBDMS, R 2 =H, X=S (D-tagatose) 190, R 1 =R 2 =H, R 3 =Bn, X=S (D-psicose)
Figure 30

POTENTIAL ANTIMICROBIAL AGENTS

Biological activity of thionocarbamates

For some decades oxazolidinethione chemistry has received considerable attention once those derivatives exhibit high potentials of biological activities, like antithyroid, 143 antifertility, 63, 144 inhibition of dopamine β-hydroxylase, 145 antibacterial, 146 and insecticidal 147 and herbicidal Erreur ! Signet non défini.a activities (Figure 31).

Antimicrobical activity

The strongly diversified panel of molecules synthesized was tested as potential antimicrobial agents in partnership with Pr. Jorge Justino in Escola Superior Agrária of Instituto Politécnico de Santarém.

Methodology for susceptibility testing

The antimicrobial activity of 19 compounds was screened using the paper disk diffusion method. 149 The microorganisms used in the tests belong to the American Type Culture

Staphylococcus aureus (ATCC 25923).

The culture medium and incubation temperature used for fungal growth was potato dextrose agar and 25ºC, whereas for bacteria, nutrient agar incubated at 37ºC was used. Paper disks of 6.4 mm were placed on the agar and the solution of each substance (300 μg) in DMSO (15 μL) was applied on each disk. Chloramphenicol and actidione were used as antimicrobial controls for bacteria and fungi, respectively. After incubation, the nearest diameter of the inhibition zone was measured. At least two replicates were made.

Antimicrobial activity results

The results for compounds exhibiting antimicrobial activity are presented in mm in diameter and uniform growth in the dish were considered indicative of weak antimicrobial activity, 10-15mm moderate activity, more than 15 mm strong activity.

a) Fused OZTs

Table 1 showed results for OZTs fused to the ketose backbone leading to cyclic and acyclic structures. In general this family of compounds has presented better bactericidal than fungicidal activity ( Table 17). The measurements are expressed in terms of average diameter of inhibition (∅) in mm.

b) Spiro OZTs and OZOs

The second family of molecules tested is composed of the spiro derivatives. In an overall view, spiro structures did not seem to present any significant antimicrobial activity. The measurements are expressed in terms of average diameter of inhibition (∅) in mm.

c) D-Tagato OZTs and OZOs

The last family is dedicated to D-tagato derivatives. The acyclic bis-OZT 175 presented some weak to moderated antimicrobial activity. The 2,3-fused OZTs both in furano (178) and pyrano (179) form did not showed significant results. However 2,3-fused OZO 177 expressed some antibacterial activity for Bacillus cereus and Bacillus subtilis. On the overview this small family of derivitaves did not show effective antimicrobial activities. The measurements are expressed in terms of average diameter of inhibition (∅) in mm.

Cytotoxicity measurements

As some of our molecules have shown interesting antimicrobial activities, it is pertinent to also test their cytotoxicity. This study was also developed in partnership with Pr. Jorge Justino in Escola Superior Agrária of Instituto Politécnico de Santarém.

Methodology for measuring cytotoxicity

Acute cytotoxicity measurements were performed by the MTT method. 150 The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantify metabolically viable cells in all samples. Adherent cells (A549 human lung fibroblasts, 100μL of 5x10 4 cells per mL cell suspension) were seeded onto 96-well plates, allowed to attach for 24 h and exposed to the test compound for the following 24 h. Positive control (hydrogen peroxide)

and negative control (pure solvent) were also included. At 48 h of culture MTT was added to the cells at a final concentration of 0.5 mg/mL, followed by an incubation period of 3 h to allow the formazan crystals to form. After incubation, medium was removed, cells were washed twice to remove traces of medium and un-metabolized MTT, and DMSO (100 μL) was added to each well. Solubilization of formazan crystals was performed by gentle shaking in a 96-well plate shaker for 20 min at room temperature. Absorbance of each well was quantified at 540 nm using 620 nm as reference wavelength on a scanning multiwell spectrophotometer (automated microplate reader).

Cytotoxicity results

The cytotoxicity tests were performed in the family of molecules that showed the best results for antimicrobial activity: fused-OZTs. Five compounds were tested but unfortunately all proved to be relatively toxic to human lung fibroblasts cells (Table 20).

Conclusion

This Chapter was dedicated to the biological screening of our thionocarbamate library.

Taking into account the results obtained we can put forward some preliminary assumptions:

In general protected and fused molecules presented better antimicrobial activities as well as

OZTs compared to OZOs.

The tested molecules exhibited stronger antibacterial activity than antifungal activity.

Compound 54 showed a great activity regarding Pyricularia oryzae (∅ = 25 mm) and a large spectrum of action which will prompt us to continue some pharmacomodulation studies.

Compound 164 presented an impressive selectivity of action against Candida albicans (∅ = 37 mm), much better than the control and also a large spectrum of activity. Several studies of pharmacomodulation will shortly be developed on this compound.

The cytotoxicity tests showed that the compounds were relatively toxic so the next step is to modulate them with a view to lowering their toxicity without a change in activity. A positive aspect is that the cytotoxicity is not directly correlated to the antimicrobial activity.

Future structural modification and biological evaluation will be carried out to explore the full potential of this novel class of antimicrobial compounds.

GENERAL CONCLUSION

This PhD work allowed the exploration of ketoses chemistry, focused on D-psicose and D-tagatose series which are two 'rare sugars' which have not been much explored in literature.

Furthermore some aspects of cyclic thionocarbamate chemistry were investigated and have shown simplified synthesis of various molecules which were submitted to preliminary biological screening.

In Chapter II the preparation of fused bicyclic structures was studied by condensation of an α-hydroxyketone with thiocyanic acid. This procedure favors the formation of hydrated-OZTs over the expected OXTs. Both oxidized and dehydrated analogues were obtained via an S-alkylation. The benzylsulfanyl derivatives were easily oxidized with H2O2 to the corresponding OZOs. This is an improved procedure over the previous N-protection methods.

The dehydration was obtained by reacting the hydroxyl of the S-benzylated derivatives with Tf2O-Et3N in good yields. All of these structures make a new family of molecules littlely described in literature.

Chapter III was dedicated to the synthesis of 4,5-fused OZT via condensation with a thiocarbonyl source. First, for D-fructose series a previous functionalization method was improved by the development of a selective iodination of on polyhydroxylated structures. After optimization of the protocol, the method was also applied with success to D-psicose series. The improvement allowed to reduce the time and number of steps in synthetic pathways. In fact a study in ketoses diols and triols derivatives was performed and it was observed that this methodology can also be extended with good results to some aldohexoses series. Some general rule could be proposed such as the introduction of the iodine was correlated to formation of the thermodynamically most stable compound but in some cases, this simple rule could not be applied.

In Chapter IV we concentrated our efforts toward the synthesis of spiro-OZTs (thus protecting the 3,4&5 hydroxyls of D-psicose and D-fructose series) following two different pathways: a) deprotection of the anomeric position, followed by condensation with thiocyanic acid or b) a one-pot procedure using trimethylsilyl isothiocyanate reagent. This last method was greatly optimized: spiro-OZTs in both D-fructose and D-psicose series were obtained in only one step with controlled stereoselectivity at the anomeric position in favor of the β form with good yields. Separation of stereoisomers α and β of structures in pyranose form was performed via an S-alkylation with impressive and unexpected efficiencies in both series. We were unable to separate the spiro-OZT, but the benzylsulfanyl derivatives allowed us after selective oxidation with m-CPBA to obtain each α and β OZOs.

When the synthesis of spiro-OZT was performed using partially protected sugar frameworks, complex mixtures were obtained. The purification of each molecule by postprotection reaction was re-studied and in some cases these methods have been improved and therefore their yields. However the best method to obtain pure compounds remains the Salkylation of the OZTs.

In Chapter V the chemistry of D-tagatose was explored. Some assays with the unprotected sugar were done and have shown that depending on the equivalent of thiocyanic acid, bis-and fused-OZTs were fairly obtained. D-tagatose is much more sensible than Dfructose or L-sorbose to the number of equivalent of HSCN. Latter two different pathways previously used for the synthesis of spiro-OZT were performed. When condensation with HSCN is applied to partial protected ketose in furanose form 2,3-fused OZTs are majority formed and spiro-OZTs merely in some traces. In the other hand, when TMSNCS method is employed only spiro-OZTs and OZOs are detected. In order to compare the two series of ketoses the same reaction were applied to D-psicose series in furanose form.

Finally, the Chapter VI describes the biological tests that were performed on this panel of compounds. It was planned to test these molecules as potential inhibitors of GLUT5, a protein transporter specific to D-fructose transport. The aim was to find selective inhibitors for the development of the novel biochemical tools. This type of sensor would be very useful to determine the standard tissue-specificity of GLUT5 expression, their response to changes in diet, hormonal regulation and to diseases states, in order to better understand some metabolic disorder likes type 2 diabetes and obesity. Unfortunately these tests have not been made yet.

For antimicrobial screening several compounds presented a certain and interesting activity, in general stronger for bacteria. Among the molecules tested, two of them, 54 and 165, have shown a large spectrum of activities together with excellent results for a specific fungus.

Compound 54 expressed excellent antifungal activity against pyricularia oryzae, considered as an agent of bioterrorism in international listings and 165 for candida albicans known as a causal agent of opportunistic infections in humans that lately has concentrated large attention from health and medicine fields once systemic fungal infections have appeared as main reason of morbidity and mortality in immuno-compromised patients. The next step from these results would be to modulate the leading compounds in order to reduce their quite high toxicities and still maintaining or improving their specific activities.

As could be seen in this PhD we have worked with three of the four ketoses: D-fructose, D-psicose and D-tagatose. But L-sorbose was not forgotten in this project. Some of its chemistry was started however due to lack of time final OZT or OXT compounds were not obtained (Scheme 124). Therefore it was decided to avoid the description in this manuscript of the chemical steps studied toward these objectives. They could be shortly overviewed in the

GENERAL METHODS

In this PhD work, reactions requiring anhydrous conditions were performed in blazed or oven-dried glassware with anhydrous solvents under inert atmosphere (argon). The solvents and reagents were purchased from Acros Organics, Aldrich, Fluka or Merck and distilled following the procedures described by D. D. Perrin, W. L. F. Armarego and D. R. Perrin, Purification of Laboratory Chemicals, Pergamon, Oxford, 1986. The methods used are as follows:

DCM was distilled in the presence of CaH2; THF was distilled in the presence of sodium/benzophenone; Toluene was distilled in the presence of CaH2; DMF (HPLC), DMSO (HPLC) and 1,4-dioxane were dried over molecular sieves 4 Å; MeOH (HPLC) was dried over molecular sieves 3 Å; Chloroform (HPLC) and cyclohexane (HPLC) were used without further purification.

The reactions were monitored by TLC on Silica Gel 60F-254 precoated plates (Merck). Visualization of the compounds was made by UV light (254 nm) and revelation was done either by immersion in a 10% solution of concentrated sulfuric acid in ethanol followed by heating or by contact with dispersed iodine in silica gel.

Flash column chromatography was carried out using Silica gel 60N (spherical, neutral, 40-63μm) (Merck) under nitrogen pressure.

NMR spectra were recorded on 400MHz Bruker Avance2 or 250 MHz Bruker Avance DPX250 spectrometers using TMS as the internal standard. Chemical shifts are reported in parts per million (ppm, δ units). Coupling constants (J) are reported and expressed in hertz (Hz), splitting patterns are designated as: br (broad), s (singlet), d (doublet), dd (double doublet), q (quartet), dt (double triplet), td (triple doublet), ddd (double double doublet), m (multiplet) and t (triplet).

Mass spectra were recorded on Perkin Elmer Sciex API 300 for negative (ISN) and positive (ISP) electrospray ionization.

High resolution mass spectra (HRMS) were recorded with a TOF spectrometer in electrospray ionisation (ESI) mode or in chemical ionisation (CI) mode.

IR spectra were reported on Thermo-Nicolet AVATAR 320 AEK0200713. Melting points were measured using a Buchi 510 capillary apparatus and are uncorrected.

Optical rotations were measured at 20°C with a Perkin Elmer 341 polarimeter (path length 1 dm).

GENERAL PROCEDURES

Protection reactions 2.1.1. Isopropylidene ketal formation a) Method using acetone

A suspension of D-fructose (20 g, 0.11 mol) in dry acetone (0.4 L) was cooled (ice-salt bath) to 0ºC. Then 98% sulfuric acid (1.5 mL) was added dropwise. When addition was completed the reaction mixture was stirred at room temperature until the starting material was entirely dissolved. At this time the reaction mixture was again cooled to 0ºC and triethylamine was added slowly until basic pH. The crystals formed were filtered and the filtrate was concentrated. The solid in the bottom of the flash was dissolved in AcOEt. In a separating funnel, the organic layers were washed with water, brine and finally dried over MgSO4, filtered, and concentrated. The residue was purified by recrystallisation or by silica gel column chromatography.

b) Method using dimethoxypropane

To a 0.2M suspension of the starting material in dry acetone was added 2,2dimethoxypropane in large excess and the chosen acid. The reaction mixture was stirred at room temperature until protection was complete. To eliminate de traces of acid the reaction mixture was made neutral with sodium hydrogen carbonate, filtered and the filtrate concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

c) Method used for tagatose

To a 0.2M suspension of the starting material in a dry mixture of toluene/DCM (4:6) was added p-toluenesulfonic acid (0.03 eq.). The reaction mixture heated until reflux was reached (60-65ºC), then 2,2-dimethoxypropane (4.0 eq.) was added and reflux was maintained until complete protection. After cooling to room temperature the reaction mixture was diluted with AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Acetylation

To a 0.4M solution of the starting material in pyridine (first dried over KOH) was added acetic anhydride (0.5 mL of pyridine). The reaction mixture was stirred at room temperature until acetylation was complete. The solvents were evaporated and co-evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography.

Benzylation a) Method for mono-O-benzylation

To a 0.2M solution of the starting material in dry DCM was added a chosen base. The mixture was cooled to 0ºC and benzyl bromide was added dropwise. The reaction mixture was allowed to reach room temperature and stirred until benzylation was complete. In order to eliminate traces of benzyl bromide the reaction mixture was first diluted in a 1M aqueous solution of sodium hydroxide. Then the resulting solution was extracted with DCM, washed with water, then brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

b) Method for tri-O-benzylation

To the starting "triol", benzyl chloride (16.0 eq.) was added followed by potassium hydroxide (19.5 eq.). The reaction mixture was stirred under reflux until complete protection. After cooling to room temperature the reaction mixture was diluted with chloroform, washed with water, brine, then dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Benzoylation a) Benzoylation at O-3

A 0.8 M solution of the starting material in dry pyridine was cooled to 0ºC. Then benzoyl chloride (1.2 eq.) was added dropwise. When addition was completed the reaction mixture was stirred at room temperature until the starting material was entirely esterified. Pyridine was evaporated and co-evaporated with toluene under reduced pressure. The crude was dissolved in AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

b) Benzoylation at O-4

A 0.3 M solution of the starting material in a dry DCM/pyridine 8:2 mixture was cooled to -12ºC (using crushed ice-salt bath). Then a 3.5 M solution of benzoyl chloride (1.3 eq.) in dry 6:4 DCM/pyridine was slowly added without temperature exceeding 0ºC. When addition was completed DMAP (catalytic amount) was added and the reaction mixture was stirred at room temperature until esterification was complete. The reaction mixture was diluted in DCM, washed with 1M hydrochloric acid, then 1M NaOH and brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Formation of tert-butyldimethylsilyl ethers

To a 0.5M solution of the starting material in dry pyridine or in dry DMF containing imidazole (2.2 eq.) was added tert-butyldimethylsilyl chloride. The reaction mixture was stirred at room temperature until O-silylation was complete. The solvent was evaporated and co-evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography.

Methyl glycosidation

To a 0.3M solution of the starting material in dry MeOH was added sulfuric acid 98% (0.3 eq.). The reaction mixture was stirred at room temperature until glycosidation was complete. Solid K2CO3 in excess was added to neutralize the reaction mixture, which was further filtered and concentrated under reduce pressure. The residue was purified by silica gel column chromatography.

Benzylidene acetal formation

To a 2.5M solution of the starting material in dry DMF was added benzaldehyde (5.0 eq.) and zinc chloride (1.5 eq.). The reaction mixture was stirred at room temperature until protection was complete. It was then diluted with AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Deprotection reactions 2.2.1. 4,5-Isopropylidene cleavage

A 0.4M suspension of the starting material in 80% aqueous acetic acid was stirred at room temperature until deprotection was complete. The solvents were evaporated and coevaporated with toluene under reduced pressure. The desired compound was obtained pure without requiring any further purification.

Cleavage of benzoyl groups

To a 0.1M solution of the starting material in dry MeOH was added a 0.5M methanol solution of MeONa until basic pH. The reaction mixture was stirred at room temperature until deprotection was complete. The solvent was evaporated and co-evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography.

Cleavage of benzyl groups

To a 0.2M solution of the starting material in dry MeOH was added Pd/C (10%) (equal masse of starting material).The reaction mixture was stirred at room temperature under hydrogen atmosphere until deprotection was complete. The catalyst was then removed using Celite ® filtration and the filtrate concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

1,2-Isopropylidene cleavage a) Without ring opening

A 0.3M solution of the starting material in 60% aqueous trifluoroacetic acid was stirred at room temperature until deprotection was complete. The solvents were evaporated and coevaporated with toluene under reduced pressure. The desired compound was obtained without requiring any further purification.

b) With ring opening

To a 0.3M solution of the starting material in THF was added 2M hydrochloric acid (equal volume of the solvent).The reaction mixture was stirred at room temperature until deprotection was complete. The organic layer was then extracted with AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Functionalization reactions 2.3.1. Oxidation a) With PCC

To a 0.3 M solution of the starting material in dry DCM was added PCC (2.0 eq.). The reaction mixture was stirred under reflux until the oxidation was complete. After cooling to room temperature a small amount of silica was added to the mixture, and the solvent removed under reduced pressure. The residue was purified by silica gel column chromatography. In case the final product is not pure enough, an extraction with AcOEt can be effected.

b) With PDC

To 0.3M solution of the starting material in dry DCM was added PDC (1.1 eq.) and acetic anhydride (3.0 eq.). The reaction mixture was stirred under reflux until the oxidation was complete. After cooling to room temperature a small amount of silica was added to the mixture, and the solvent removed under reduce pressure. The residue was purified by silica gel column chromatography. In case the final product it is not pure enough, an extraction with AcOEt can be effected.

Iodination

To a 0.5M solution of the starting material in the chosen dry solvent was added imidazole (3.0 eq.) and triphenylphosphine (1.5 eq.). After cooling to 0ºC iodine (1.0 eq.) was added. The reaction was then stirred at 60-70ºC until the iodination was complete. The solvent was evaporated and co-evaporated under reduced pressure with toluene. The residue was purified by silica gel column chromatography.

Elimination

To a 0.2M solution of the starting material in dry toluene was added imidazole (3.0 eq.) and triphenylphosphine (1.5 eq.). After cooling to 0ºC iodine (1.4 eq.) was added. The reaction was then stirred under reflux until elimination was complete. The solvent was evaporated and co-evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography.

Azide formation

To a 0.3M solution of the starting material in DMSO was added sodium azide (3.0 eq.).

The reaction mixture was stirred at 100°C until azide formation was complete. After cooling to room temperature, water was added and the solution was extracted with AcOEt. The combined organic layers were washed with water, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography.

S-Benzylation

To a 0.3M solution of the starting material in dry DMF was added sodium hydride (60% in mineral oil, 4.0 eq.). The mixture was cooled to 0ºC and benzyl bromide (4.0 eq.) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred until benzylation was complete. In order to eliminate traces of benzyl bromide, the reaction mixture was first washed with 1M aqueous NaOH, then extracted with AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Epoxide formation

A 0.2M solution of the starting material in dry DMF was cooled to 0ºC, then sodium hydride (60% in mineral oil, 3.0 eq.) was added and the mixture was stirred at room temperature until conversion was complete. The reaction mixture was then diluted with AcOEt, washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Epoxide cleavage

To a 0.2M solution of the starting material in dry MeOH was added a 0.5M methanol solution of MeONa until basic pH. The reaction mixture was stirred at 60ºC until epoxide cleavage was complete. The solvent was evaporated and co-evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography.

Synthesis of OZTs

Condensation with thiocyanic Acid

To a 0.3M solution of the starting material in the chosen solvent was added potassium isothiocyanate (4.0 eq.). The reaction was cooled to 0ºC and 37% hydrochloric acid (2.0 eq.) was added dropwise. The reaction mixture was stirred under reflux or at room temperature until complete consumption of the starting material. After cooling to room temperature, if necessary, crushed ice was added and the solution was extracted with AcOEt. The combined organic layers were washed with water, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography.

Condensation with a thiocarbonyl source: CS2

To a 0.3M solution of the starting material in dry 1,4-dioxane were added triphenylphosphine (1.1 eq.) and carbon disulfide (20 eq.). The reaction mixture was stirred at 50°C until complete consumption of the starting material. After cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography.

Using trimethylsilyl isothiocyanate

To a 0.2M solution of the starting material in DCM (with 4Å molecular sieves) was added trimethylsilyl isothiocyanate (3.0 eq.). After cooling to -78°C, boron trifluoride etherate (1.0 eq.) was added. The reaction mixture was stirred at room temperature until complete consumption of the starting material. Saturated aqueous sodium hydrogen carbonate was then added and the organic phase was extracted with DCM, washed with water, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography.

Synthesis of OZOs

Oxydation a) With hydrogen peroxide

To a 0.1M solution of the starting material in DCM was added sodium hydrogenocarbonate (2.0 eq.) and 30% hydrogen peroxide (5.0 eq.). The reaction mixture was stirred at room temperature until oxidation was complete. The residue was concentrated under reduced pressure and purified by silica gel column chromatography.

b) With m-chloroperbenzoic acid

To a 0.2M solution of the starting material in DCM was added sodium hydrogenocarbonate (3.0 eq.) and m-chloroperbenzoic acid 77% (3.0 eq.). The reaction mixture was stirred at room temperature until oxidation was complete. Then it was washed with a saturated solution of sodium metabisulfite and a 0.5M solution of sodium hydrogen carbonate. The organic layer was extracted with DCM washed with water, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography.

Synthesis of OXTs

Dehydration

A 0.03 M solution of the starting material in dry DCM was cooled to -5ºC and DIPEA (4.0 eq.) was added. After stirring for 30 min. at -5ºC triflic anhydride (2.0 eq.) was introduced. The reaction mixture was stirred at room temperature until dehydration was complete. Then crushed ice was added and the solution was extracted with DCM. The combined organic layers were washed with water, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography.

1,2:4,5-Di-O-isopropylidene-β-D-fructopyranose (4)

Starting with D-fructose (15.0 g, 83.8 mmol) and after using G.P. 2.1.1.a) (reaction time 1.5h) the product was crystallized with PE/EA (8:2) to give 4 (12.5 g, 48.0 mmol, 58%) as a colorless solid.

1 H NMR (250MHz, CDCl3) δ: 1.37 (s, 3H, CH3); 1.45 (s, 3H, CH3); 1.52 (s, 3H, CH3); 1.54 (s, 3H, CH3); 2.03 (d,1H,, OH); 3.67 (t, 1H, J3-4=7.1Hz, H-3); 3.98 (d,1H,, H-1a); 4.01 (d,1H,, H-6a); 4.09-4.23 (m, 3H, H-4, H-5, H-6b); 4.19 (d, 1H, J1b-1a=9.0Hz, H-1b). 13 C NMR (62.89MHz, CDCl3) δ: 24. 3,24.6,24.7,26.2 (CH3) Starting with 4 (3.5 g, 13.5 mmol) and using G.P. 2.3.1. b) (reaction time 2h) the residue was chromatographied with PE/EA (8:2) to give 25 (3.4 g, 13.3 mmol, 99%) as a colorless solid.

1 H NMR (250MHz, CDCl3) δ: 1.38 (s, 6H, CH3); 1.44 (s, 3H, CH3); 1.53 (s, 3H, CH3); 3.97 (d, 1H, J1a-1b=9.5Hz, H-1a); 4.10 (d,1H,, H-6a); 4.37 (d,1H,, H-6b); 4.52 (ddd, 1H, J5-6a=1.0Hz, J5-6b=2.2Hz, J5-4=5.5Hz, H-5); 4.59 (d,1H,, H-1b); 4.71 (d, 1H, J4-5=5.5Hz, H-4). 13 C NMR (62.89MHz, CDCl3) δ: 26.2, 26.7, 27. Starting with 25 (2.5 g, 9.6 mmol) and using G.P. 2.2.1. (reaction time 15h) the compound 24 (2.5 g, 11.5 mmol, 99%) was obtained as a colorless solid.

1 H NMR (250MHz, CDCl3) δ: 1.39 (s, 3H, CH3); 1.55 (s, 3H, CH3); 2.65 (brs, 1H, OH-4); 3.62 (brs, 1H, OH-5); 3.99 (dd,1H,, H-6a) 4.00 (d,1H,, H-1a); 4.33 (dd,1H,, H-6b); 4.38-4.41 (m, 1H, H-5); 4.68 (d,1H,, H-1b); 4.73 (d, 1H, J4-5=4.0Hz, H-4). 13 C NMR (62.89MHz, CDCl3) δ: 26.3 (CH3); 26.5 (CH3); 63.4 (C-6); 69.7 (C-1); 73.6 (C-4); 74.3 (C-5); 104.4 (C-2); 113.7 (Cq-isop); 199.1 (C=O).

J. Kang, G. J. Lim, S. K. Yoon, M. Y. Kim J. Org. Chem. 1995, 60, 564; X.-Y. Wu, X. She, Y. Shi J. Am. Chem. Soc. 2002, 124, 8792.

4,5-Dihydro{3-O-ethyl-1,2-O-isopropylidene-β-D-fructopyranoso}[3,4-d]-1,3oxazolidine-2-thione (26)

Starting with 24 (0.40 g, 1.8 mmol) and using G.P. 2.4.1. with ethanol as solvent and at reflux (reaction time 15h) the compound 26 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as an orange oil (0.20 g, 0.65 mmol, 35%).

1 H NMR (400MHz, CDCl3) δ: 1.26 (t, 3H, J=7.2Hz, CH2-CH3); 1.39 (s, 3H, CH3isop); 1.51 (s, 3H, CH3-isop); 2.87 (brs, 1H, OH); 3.56-3.64 (m, 3H, H-6a, CH2-CH3); 3.99 (d,1H,, H-1a); 4.04 (dd,1H,, H-6b); 4.20 (d,1H,

Starting with 24 (0.4 g, 1.8 mmol) and using G.P. 2.4.1. with DMF as solvent and at room temperature (reaction time 15h) the compound 1 was obtained after column chromatography on silica gel with a mixture of PE/EA (5:5) as a cream-colored solid (0.30 g, 1.15 mmol, 59%).

1 H NMR (250MHz, DMSO) δ: 1.30 (s, 3H, CH3); 1.40 (s, 3H, CH3); 3.16 (d,1H,, H-6a); 3.72 (d,, H-1a); 3.73 (dd, J6b-5=5.3Hz, J6b-6a=9.5Hz, H-6b); 4.15 (d,1H,

4,5-Dihydro{5-O-acetyl-3-O-ethyl-1,2-O-isopropylidene-β-D-fructopyranoso}-[3,4-d]-1,3-oxazolidine-2-thione (31)

Starting with 26 (0.2 g, 0.7 mmol) and using G.P. 2.1.2. (reaction time 15h) the compound 31 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as a cream colored oil (0.16 g, 0.5 mmol, 67%).

1 H NMR (400MHz, CDCl3) δ: 1.27 (t, 3H, CH2-CH3); 1.39 (s, 3H, CH3-isop); 1.52 (s, 3H, CH3-isop); 2.14 (s, 3H, O-CO-CH3), 3.53-3.64 (m, 2H, CH2-CH3); 3.75 (dd,1H,, H-6a); 4.01 (d,1H,, H-1a); 4.05 (dd, 1H, J6b-5=5.6Hz, J6b-6a=10.6Hz, H-6b); 4.15 (d,1H,, H-1b); 5.12 (d, 1H, J4-5=4.4Hz, H-4); 5.62 (ddd,1H,, H-5); 8.42 (s, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 15.1 (CH2-CH3); 19.6 (O-CO-CH3); 24.6, 26.0 (CH3-isop); 57.9 (CH2-CH3); 58.9 (C-6); 64. The compound 35 was a side product of the synthesis of compound 31. Compound 35 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a cream oil (60 mg, 0.15 mmol, 23%). 3.65 (dd,1H, J6a-6b=11.0Hz, J6a-5=9.4Hz, H-6a); 3.75 (d,1H,, H-1a); 4.05 (dd, J6b-6a=11.0Hz, J6b-5=5.6Hz, H-6b); 4.44 (d,1H,, H-1b); 5.07 (d,1H,, H-4); 5.69 (ddd, 1H, J5-4= J5-6b=5.6Hz, J5-6a=9.4Hz, H-5). 13 C NMR (100MHz, CDCl3) δ: 14.8 (CH2-CH3,); 19.9 (O-CO-CH3); 24.8, 27.6 (CH3-isop); 36.9 (N-CO-CH3); 59.7 (CH2-CH3); 60. Starting with 26 (0.5 g, 1.64 mmol) and using G.P. 2.2.4. a) (reaction time 2h) the compound 32 was obtained and immediately used for the next reaction without further purification. Starting with 32 and using G.P. 2.1.2. (reaction time 15h) the compound 37 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as a brown oil (0.33 g, 0.91 mmol, 55%).

Starting with 1 (0.5 g, 1.80 mmol) and using G.P. 2.2.4. a) (reaction time 2h) the compound 32 was obtained and immediately used for the next reaction without further purification. Starting with 32 and using G.P. 2.1.2. (reaction time 15h) the compound 37 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as a brown oil (0.32 g, 0.88 mmol, 49%).

1 H NMR (400MHz, CDCl3) δ: 2.09 (s, 6H, O-CO-CH3); 2.15 (s, 3H, O-CO-CH3); 2.17 (s, 3H, O-CO-CH3); 4.28 (dd, 1H, J6a-5=4.4Hz, J6a-6b=12.0Hz, H-6a); 4.39-4.48 (m, 2H, H-5,H-6b); 4.55 (d, 1H, J1a-1b=12.4, H-1a); 4.82 (d,1H,, H-1b); 5.24 (d,1H,, H-4); 8.15 (brs, 1H, NH). 13 C NMR (100MHz,CDCl3) δ: 20.7,20.8,21.5,21.6 (O-CO-CH3); 61.5 (C-1); 61.9 (C-6); 80.2 (C-5); 87.8 (C-4); 101.9 (C-2); 108.6 (C-3); 168. 8, 169.8, 169.9, 170.4 (C=O); 189.2 (C=S). The compound 38 was a side product of the synthesis of compound 37 using both procedures. Compound 38 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a brown oil (0.14 g, 0.31 mmol, 19%) starting from 26 and (0.11 g, 0.25 mmol, 15%) from 1.

HRMS:

1 H NMR (400MHz, CDCl3) δ: 2.08 (s, 3H, O-CO-CH3); 2.10 (s, 6H, O-CO-CH3); 2.16 (s, 3H, O-CO-CH3); 2.78 (s, 3H, N-CO-CH3); 4.30 (dd, 1H, J6a-5=4.0Hz, J6a-6b=12.2. H-6a); 4.42 (dd,1H,. H-6b); 4.58 (dd, 1H, J5-6a=4.6Hz, J5-4=5.6 H-5); 4.74 (d,1H,, H-1a); 5.09 (d, 1H, J1b-1a=12.0Hz, H-1b); 5.15 (d,1H,, H-4).

2-Benzylsulfanyl-4,5-dihydro{1,2-O-isopropylidene-β-D-fructopyranoso}[3,4-d]-1,3oxazoline (45)

Starting with 1 (0.2 g, 0.72 mmol) and using G.P. 2.3.6. with sodium hydrogen carbonate (4.0 eq.) as base and adding BnBr (4.0 eq.) (reaction time 4h) the compound 45 was obtained after column chromatography on silica gel with a mixture of PE/EA (5:5) as a colorless oil (0.2 g, 0.54 mmol, 74%).

Starting with 1 (0.3 g, 1.08 mmol) and using G.P. 1.1.3. a), with NaH (60% in mineral oil) (4.0 eq.) and adding BnBr (4.0 eq.), or triethylamine (1.2 eq.) then addition of BnBr (2.5 eq.), (reaction time 4h), the compound 45 was obtained after column chromatography on silica gel with a mixture of PE/EA (5:5) as a colorless oil (0.28 g, 0.76 mmol, 71%) or (0.27 g, 0.73 mmol, 69%) respectively.

1 H NMR (400MHz, CDCl3) δ: 1.46 (s, 3H, CH3); 1.47 (s, 3H, CH3); 3.63 (d, J1a-1b=9.4Hz, H-1a); 3.91 (d,1H,, H-7a); 3.94 (d,1H,, H-7b); 4.06 (d,1H,, H-6a); 4.18 (d, 1H, J4-5=2.0Hz, H-4); 4.22 (dd, J6b-6a=13.8Hz, H-6b); 4.25 (d,1H,, H-1b); 4.51 (dd, 1H, J5-4=2.0Hz, J5-6=3.6Hz, H-5); 7.24-7.36 (m, 5H, Ph). Starting with 45 (0.13 g, 0.35 mmol) and using G.P. 2.1.2. (reaction time 15h) the compound 47 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as a colorless oil (0.11 g, 0.27 mmol, 76%).

1 H NMR (400MHz, CDCl3) δ: 1.47 (s, 6H, CH3-isop); 2.11 (s, 3H, O-CO-CH3); 2.72 (s, 1H, OH); 3.60 (d,1H,, H-1a); 3.92 (dd,1H,, H-6a); 4.00 (d,1H,, H-6b); 4.06 (d,1H,, H-7a); 4.23 (d,1H,, H-7b); 4.31 (d,1H,, H-1b); 4.55 (t, 1H, J5-4=2.0Hz, H-5); 5.29 (d, 1H, J4-5=2.0Hz, H-4); 7.22-7.37 (m, 5H, Ph). The compound 46 is a side product of the synthesis of compound 47. Compound 46 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as an yellow oil (0.03 g, 0.07 mmol, 17%).

1 H NMR (400MHz, CDCl3) δ: 1.40 (s, 3H, CH3-isop); 1.44 (s, 3H, CH3-isop); 2.07 (s, 3H, O-CO-CH3); 2.11 (s, 3H, O-CO-CH3); 3.61 (d,1H,, H-1a); 3.93 (dd,1H,, H-6a); 4.05-4.11 (m, 2H, H-6b, H-7a); 4.26 (d,1H,, H-7b); 4.35 (d,1H,, H-1b); 4.66 (t, 1H, J5-4=2.3Hz, H-5); 6.23 (d,1H,, H-4); 7.24-7.39 (m, 5H, Ph). Starting with 46 (50 mg, 0.11 mmol) and using G.P. 2.5.1. a) (reaction time 3h) the compound 48 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (31 mg, 0.09 mmol, 81%).

4,5-Dihydro{1,2-O-isopropylidene-β-D-fructopyranoso}[3,4-d]-1,3-oxazoline-2-one (2)

Starting with 45 (16 mg, 0.04 mmol) and using G.P. 2.5.1. a) (reaction time 3h) the compound 2 was obtained after column chromatography on silica gel with a mixture of PE/EA (1:9) as a colorless oil (8 mg, 0.03 mmol, 72%).

1 H NMR (400MHz, CDCl3) δ: 1.46 (s, 6H, CH3-isop); 2.01 (brs, 2H, OH); 3.88-3.94 (m, 3H, H-1a, H-6a, H-6b); 4.23 (brs, 1H, H-4); 4.37 (d,1H, The compound 3 was a side product of the synthesis of compound 45. Compound 3 was obtained (with triethylamine (1.2 eq.) or NaH (60% in mineral oil) (4.0 eq.) as base and adding BnBr (4.0 eq.) or (2.5 eq.) respectively) after column chromatography on silica gel with a mixture of PE/EA (8:2) as a orange oil (20 mg, 0.06 mmol, 8%) or (15 mg, 0.04 mmol, 6%) respectively to the base used.

1 H NMR (400MHz, CDCl3) δ: 1.52 (s, 3H, CH3-isop); 1.56 (s, 3H, CH3-isop); 2.08 (brs, 1H, OH); 4.03 (d,1H,, H-6a); 4.18 (d,1H,, H-1a); 4.30 (dd,1H,, H-6a); 4.40 (s, 2H, H-7); 4.44 (d,1H, Starting with 46 (50 mg, 0.18 mmol) and using G.P. 2.6.1. (reaction time 5 min.) the compound 53 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (33 mg, 0.08 mmol, 70%).

1 H NMR (250MHz, CDCl3) δ: 1.46 (s, 3H, CH3-isop); 1.50 (s, 3H, CH3-isop); 2.04 (s, 3H, O-CO-CH3); 3.96-4.10 (m, 2H, H-5, H-6a); 4.14 (d,1H,, H-1a); 4.27 (dd,1H,, H-6b); 4.33 (s, 2H, H-7); 4.39 (d,1H, Starting with 4 (2.5 g, 9.6 mmol) and using G.P. 2.1.4. a) (reaction time 15h) compound 85 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colourless oil (3.48 g, 9.55 mmol, 100%).

1 H NMR (250MHz, CDCl3) δ: 1.37 (s, 3H, CH3); 1.41 (s, 3H, CH3); 1.51 (s, 3H, CH3); 1.61 (s, 3H, CH3); 3.90 (d,1H,, H-1a); 4.01 (d, 1H, J1b-1a=9.2Hz, H-1b); 4.14 (d,1H,, H-6a); 4.21 (dd,1H,, H-6b); 4.29 (dd,1H,, H-5); 4.46 (dd,1H,, J4-3=7.8Hz H-4); 5.39 (d,1H,, H-3); 7. 2H,; 7. 1H, ;2H,. 13 C NMR (62.89MHz, CDCl3) δ: 26. 3, 26.5, 26.6, 28.

3-O-benzoyl-1,2-O-isopropylidene-β-D-fructopyranose (88)

Starting with 85 (2.8 g, 7.68 mmol) and using G.P. 2.2.1. (reaction time 15h) the compound 88 was obtained as a colorless oil (2.48 g, 7.65 mmol, 100%).

1 H NMR (400MHz, CDCl3) δ: 1.47 (s, 3H, CH3); 1.52 (s, 3H, CH3); 2.74 (brs, 1H, OH); 3.89 (dd,1H,, H-6a); 3.96 (d, 1H, J1a-1b=9.2Hz, H-1a); 4.04-4.09 (m, 3H, H1b, H-5, H-6b); 4.14 (dd, 1H, J4-3=9.6Hz, J4-5=3.6Hz, H-4); 5.40 (d,1H,7.46 (t,2H,, m-Ph); 7.59 (t, 1H, Jp-m=7.6Hz, p-Ph); 8.09 (d,2H,, o-Ph). Starting with 88 (2 g, 6.17 mmol) and using G.P. 2.1.4. b) (reaction time 15h) the compound 89 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as a colorless solid (2.38 g, 5.56 mmol, 90%).

1 H NMR (400MHz, CDCl3) δ: 1.41 (s, 3H, CH3); 1.52 (s, 3H, CH3); 2.24 (brs, 1H, OH); 3.90 (dd,1H,, H-6a); 3.98 (d, 1H, J1a-1b=9.2Hz, H-1a); 4.08 (d,1H,, H-1b); 4.19 (d,1H,, H-6b); 4.33 (m, 1H, H-5); 5.62 (dd,1H,, H-4); 5.87 (d,1H,, H-3); 7.31-7.36 (m, 4H, Hm-Ph); 7.45-7.50 (m, 2H, Hp-Ph); 7.91 (d,2H,, Ho-Ph); 7.97 (d,2H,, Ho-Ph). Starting with 89 (2 g, 4.67 mmol) using G.P. 2.3.3. (reaction time 4h) with dry tetrahydrofuran. The compound 90 was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as a yellow solid (2.26 g, 4.20 mmol, 90%).

1 H NMR (400MHz, CDCl3) δ: 1.41 (s, 3H, CH3); 1.54 (s, 3H, CH3); 1H,J6a,6b=12.8Hz, J6a, 5=2.0Hz, H-6a); 3.99 (d,1H,, H-1a); 2H,J6b,6a=12.8Hz, H-5, H-6b); 5.40 (d, Starting with 90 (2 g, 3.72 mmol) and using G.P. 2.3.6. (reaction time 3h) the compound 91 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless solid (1.43 g, 3.15 mmol, 85%).

1 H NMR (250MHz, CDCl3) δ: 1.44 (s, 3H, CH3); 1.53 (s, 3H, CH3); 3.88 (dd,1H,, H-6a); 4.03 (d,1H,, H-1a); 4.10 (d, 1H, J1b-1a=9.3Hz, H-1b); 4.24 (dd,1H,, H-6b); 4.31-4.32 (m, 1H, H-5); 5.79 (dd,1H,, H-4); 5.88 (d,1H,, H-3); 7.30-7.38 (m, 4H, Hm-Ph); 7.43-7.51 (m, 2H, Hp-Ph); 7.94-8.03 (m, 4H, Ho-Ph). 13 C NMR (62.89MHz, CDCl3) δ: 26.2, 26.5 (CH3); 60.2 (C-5); 62.1 (C-6); 67.2 (C-3); 71.8 (C-4); 71.9 (C-1); 104.8 (C-2); 112.5 (Cq-isop); 128.4 (CH-mPh); 128.6, 128.9 (Cq-Ph); 129.7, 129.9 (CH-oPh); 133. 5,165.7,165.8 (C=O).

A. Tatibouët, M. Lefoix, J. Nadolny, O. R. Martin, P. Rollin, J. Yang, G. D. Holman Carbohydr. Res. 2001, 333, 327.

5-Azido-5-deoxy-1,2-O-isopropylidene-β-D-fructopyranose (92)

Starting with 90 (1.5 g, 3.31 mmol) and using G.P. 2.2.2. (reaction time 15h) the compound 92 was obtained after column chromatography on silica gel with a mixture of PE/EA (3:7) as a colorless solid (0.66 g, 2.69 mmol, 81%).

Starting with 100 (0.5 g, 1.51 mmol) and using G.P. 2.3.6. (reaction time 3 days) the compound 92 was obtained after column chromatography on silica gel with a mixture of PE/EA (3:7) as a colorless solid (0.23 g, 0.94 mmol, 62%).

1 H NMR (400MHz, CDCl3) δ: 1.45 (s, 3H, CH3); 1.49 (s, 3H, CH3); 2.73 (brs, 1H, OH); 3.64 (brs, 1H, OH); 3.73 (brs, 1H, H-3); 3.77 (dd, 1H, J6a-5=1.2Hz, J6a-6b=12.4Hz, H-6a); 3.94-3.96 (m, 2H, H-5, H-6b); 3.97-3.99 (m, 1H, H-4); 4.01 (d,1H,, H-1a); 4.19 (d,1H,, H-1b). Starting with 92 (0.5 g, 2.04 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 93 was obtained after column chromatography on silica gel with a mixture of PE/EA (6:4) as a colorless solid (0.31 g, 1.19 mmol, 59%).

1 H NMR (400MHz, CDCl3) δ: 1.43 (s, 3H, CH3); 1.49 (s, 3H, CH3); 2.73 (d,1H,3.79 (d,1H,, H-6a); 3.79 (t,1H,, H-3); 3.98 (d,1H,, H-1a); 4.11-4.19 (m, 3H, H-1b, H-5, H-6b); 5.00 (dd, 1H, J4-3=5.2Hz, J4-5=8.0Hz, H-4); 7.20 (brs, 1H, NH). 13

3-O-benzyl-5-deoxy-5-iodo-1,2-O-isopropylidene-α-L-sorbopyranose (97)

Starting with 57 (1 g, 3.22 mmol) and using G.P. 2.3.3. (reaction time 4h) with dry tetrahydrofuran as solvent the compound 97 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as yellow solid (1.27 g, 3.02 mmol, 94%).

1 H NMR (250MHz, CDCl3) δ: 1.42 (s, 3H, CH3); 1.49 (s, 3H, CH3); 2.60 (brs, 1H, OH); 3.44 (d,1H,, H-3); 3.84 (dd,1H,, H-6a); 3.85 (d,1H,, H-1a), 3.96 (dd,1H,, H-6b); 3.98 (d,1H,, H-1b); 4.03 (s, 1H, H-4); 4.07 (s, 1H, H-5); 4.72 (d,1H,, H-7a); 4.91 (d,1H,, H-7b); 7.28-7.37 (m, 5H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 26.2, 27.2 (CH3); 30.5(C-5); 65.5 (C-6); 71.9 (C-1); 75.6 (C-7); 76.7 (C-4); 79.6 (C-3); 105.8 (C-2); 112.6 (Cq-isop); 128. 1,128.2,. Starting with 4 (3 g, 11.5 mmol) and using G.P. 2.2.1. (reaction time 15h) the compound 98 was obtained as a colorless solid (2.52 g, 11.5 mmol, 100%).

HRMS:

1 H NMR (400MHz, DMSO-d6) δ: 1.31 (s, 3H, CH3); 1.37 (s, 3H, CH3); 3.46-3.50 (m, 3H, H-3, H-4, H-6b); 3,68 (brs, 1H, H-5); 3.71 (d,1H,, H-6b); 3.73 (d,1H,, H1-a); 4.01 (d,1H,

3-O-benzoyl-5-deoxy-5-iodo-1,2-O-isopropylidene-α-L-sorbopyranose (99)

Starting with 88 (1 g, 3.08 mmol) using G.P. 2.3.3. and tetrahydrofuran as solvent the compound 99 was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as cream solid (0.94 g, 2.16 mmol, 70%).

1 H NMR (400MHz, CDCl3) δ: 1.43 (s, 3H, CH3); 1.51 (s, 3H, CH3); 2.69 (d, JOH-4=4.4Hz, 1H, OH); 3.92 (d,1H,, H-1a); 3.93 (dd, 1H, J6a-5=5.0Hz, J6a-6b=11.2Hz, H-6a); 3.97 (d,1H,, H-1b); 4.04-4.09 (m, 1H, H-5); 4.14 (dd,1H,, H-6b); 4.15-4.22 (m, 1H, H-4); 5.16 (d,1H, Starting with 98 (1 g, 4.54 mmol) using G.P. 2.3.3. (reaction time 4h) and 1,4dioxane as solvent the compound 100 was obtained after column chromatography on silica gel with a mixture of PE/EA (6:4) as cream solid (0.97 g, 2.94 mmol, 65%).

1 H NMR (400MHz, CDCl3) δ: 1.46 (s, 3H, CH3); 1.51 (s, 3H, CH3); 2.10 (d,1H,, OH-3); 2.80 (d, 1H, JOH-4=1.8Hz OH-4); 3.44 (t,1H,, H-3); 3.81 (dt,1H,, H-4); 3.97 (dd,1H,, H-6a); 3.93-3.97 (m, 1H, H-5); 3.98 (d,1H,, H-1a); 4.04 (d,1H,, H-6b); 4.18 (d,1H,, H-1b). 13 C NMR (100MHz, CDCl3) δ: 26.4, 26.7 (CH3); 28.2 (C-5); 66.0 (C-6); 72.1 (C-1); 72.9 (C-3); 77.0 (C-4); 105.7 (C-2); 112.6 (Cq-isop).

MS: (IS+) m/z: 413 (M+CH3COOH+Na) + .

IR: (neat) ν (cm -1 ): 3556 (OH); 2999 (CH).

5-Azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-β-D-fructopyranose (96)

Starting with 97 (1 g, 2.38 mmol) and using G.P. 2.3.6. (reaction time 3 days) the compound 96 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless solid (0.73 g, 2.17 mmol, 92%).

1 H NMR (400MHz, CDCl3) δ: 1.42 (s, 3H, CH3); 1.48 (s, 3H, CH3); 2.31 (d,1H,, OH); 3.67 (d,1H,, H-3); 3.74 (dd, 1H, J6a-6b=12.4Hz, J6a-5=1.6Hz, H-6a); 3.93-3.95 (m, 1H, H-5); 3.96 (d,1H,, H-1a), 3.99 (dd,1H,, H-6b); 4.03 (d,1H,, H-1b); 4.19 (ddd,1H,, H-4); 4.75 (d,1H,, H-7a); 4.84 (d,1H,, H-7b); 7.32-7.38 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 26.2, 27.0 (CH3); 61.9 (C-6); 62.4 (C-5); 71.7 (C-4); 72.0 (C-1); 73.0 (C-3); 75.8 (C-7); 105.7 (C-2); 112. 128.1,128.3,. Starting with 99 (1 g, 2.30 mmol) and using G.P. 2.3.6. (reaction time 3 days) the compound 101 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless oil (0.60 g, 1.72 mmol, 75%).

1 H NMR (250MHz, CDCl3) δ: 1.42 (s, 3H, CH3); 1.49 (s, 3H, CH3); 3.72-3.80 (m 1H, H-6); 3.93-4.08 (m, 4H, H-1a, H-4, H-5, H6b); 4.20 (d,1H,, H-1b); 5.43 (dd,1H,, H-3); 7. 3H,Ph);2H,Ph). 13 Starting with 96 (0.5 g, 1.49 mmol) using G.P. 2.4.2. (reaction time 15h) the compound 102 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as cream oil (0.28 g, 7.97 mmol, 53%).

1 H NMR (400MHz, CDCl3) δ: 1.42 (s, 3H, CH3); 1.48 (s, 3H, CH3); 3.65 (d,1H,, H-3); 3.87 (d,1H,, H-6a); 3.86 (d,1H,, H-1a); 4.03 (d,1H,, H-1b); 4.09 (dd,1H,.4Hz H-6b); 4.22 (dd,1H,, H-5); 4.70 (d,1H,, H-7a); 4.92 (d,1H,, H-7a); 5.07 (dd, 1H, J4-3=6.8Hz, J4-5=8.0Hz, H-4); 7.27-7.40 (m, 5H, Ph); 8.46 (brs, 1H, NH). Starting with 101 (0.5 g, 1.43 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 103 was obtained after column chromatography on silica gel with a mixture of PE/EA (:) as cream oil (0.32 g, 0.88 mmol, 62%). Starting with 25 (5 g, 19.3 mmol) and using G.P. 2.3.2. (reaction time 3h) the compound 104 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as colorless solid (4.92 g, 18.9 mmol, 98%).

1 H NMR (250MHz, CDCl3) δ: 1.38 (s, 3H, CH3); 1.41 (s, 3H, CH3); 1.50 (s, 3H, CH3); 1.56 (s, 3H, CH3); 2.36 (d,1H,, OH); 3.75 (dd, 1H, J3-OH=6.5Hz, J3-4=4.0Hz, H-3); 4.00 (m, 2H, H-6a, H-6b); 4.04 (d,1H,, H-1a); 4.21-4.25 (m, 1H, H-5); 4.26 (d,1H,, H-1b); 4.45 (dd, 1H, J4-3=4.0Hz, J4-5=6.8Hz, H-4). 13 C NMR (62.89MHz, CDCl3) δ: 25. [START_REF]-N,1-O-carbonyl-β-D-psicopyranosylamine (161) Starting with 157 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 161 was obtained after column chromatography on silica gel with EA as colorless oil[END_REF]26.1,26.3,26.6 (CH3);; 68.9 (C-3); 71.9 (C-5); 72.2 (C-4); 73.1 (C-1); 105.0 (C-2); 109.5 (Cq-isop); 111.2 (Cqisop).

J. Kang, G. J. Lim, S. K. Yoon, M. Y. Kim J. Org. Chem. 1996, 60, 564 Starting with 106 (1 g, 3.22 mmol) using G.P. 2.3.3. (reaction time 4h) and dry tetrahydrofuran as solvent the compound 107 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as cream solid (0.76 g, 1.81 mmol, 56%).

1 H NMR (250MHz, CDCl3) δ: 1.42 (s, 3H, CH3); 1.53 (s, 3H, CH3); 2.86 (d,1H,JOH=6.3Hz, OH); 3.45 (dd,1H,, H-6a); 3.76 (d, 1H, J3-4=7.8Hz, H-3); 3.91-4.00 (m, 1H, H-5); 4.04 (d,1H,, H-1a); 4.09-4.13 (m, 1H, H-4); 4.14 (d,1H,, H-1b); 4.24 (dd, 1H, J6b-5=3.8Hz, J6b-6a=12.3Hz, H-6b); 4.77 (d,1H,, H-7a); 4.82 (d,1H,, H-7b); 7.27-7.38 (m, 5H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 26.2, 27.1 (CH3); 64.6 (C-6); 67.5 (C-4); 69.2 (C-1); 71.2 (C-5); 74.7 (C-7); 79.7 (C-3); 105.6 (C-2); 112. 128.3,128.4,128.5,128.7,; 137.0 (Cq-Ph).

HRMS:

3-O-benzyl-4-deoxy-4-iodo-1,2-O-isopropylidene-β-D-psicopyranose (108)

Starting with 107 (1 g, 2.38 mmol) using G.P. 2.3.5. (reaction time 3 days) the compound 108 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless solid (0.55 g, 1.64 mmol, 69%).

1 H NMR (250MHz, CDCl3) δ: 1.36 (s, 3H, CH3); 1.47 (s, 3H, CH3); 2.52 (d, 1H, JOH=7.0Hz, OH); 3.68-3.73 (m, 1H, H-3); 3.74 (d,1H,, H-6a); 3.84 (d,1H,, H-1a); 1H,3H,, H-1b, H-5, H-6b); 4.71 (d,1H,, H-7a); 4.81 (d, 1H, J7b-7a=11.5Hz, H-7b); 7.25-7.40 (m, 5H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 26. 3,26.8 (CH3); 56.1 (C-6); 62.2 (C-4); 72.1 (C-5); 72.8 (C-1); 75.6 (C-7); 79.4 (C-3); 105.1 (C-2); 112.4 (Cq-isop); 128. 3,128.4,. Starting with 55 (0.5 g, 1.42 mmol) and using G.P. 2.2.4.b) (reaction time 15h) the compound 111 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as brown solid (0.35 g, 1.12 mmol, 79%).

HRMS:

1 H NMR (400MHz, DMSO) δ: 3.28-3.31 (m, 1H, H-6a); 3.37-3.42 (m, 1H, H-6b); 3.82-3.85 (m, 1H, H-5); 4.31 (ddd,2H,, H-1a, H-1b); 4.60 (d,1H,, H-7a); 4.65 (d,1H,, H-3); 4.66 (d,1H,, H-7b); 5.04 (dd,1H, Starting with 112 and using G.P. 2.1.2. (reaction time 15h) the compound 113 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a brown oil (0.43 g, 0.98 mmol, 70%).

1 H NMR (400MHz, CDCl3) δ: 1.30 (s, 3H, O-CO-CH3); 1.31 (s, 3H, O-CO-CH3); 1.40 (s, 3H, N-CO-CH3); 3.60 (dd,1H,.4Hz H-6a); 3.72 (dd,1H,.4Hz H-6b); 3.97 (d,1H,, H-1a); 4.09 (ddd,1H,, H-5); 4.18 (d,1H, Starting with 57 (0.5 g, 1.61 mmol) and using G.P. 2.3.4. (reaction time 1h) the compound 114 was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as orange oil (0.24 g, 0.87 mmol, 54%).

1 H NMR (250MHz, CDCl3) δ: 1.46 (s, 3H, CH3); 1.54 (s, 3H, CH3); 3.90 (d 1H,, H-1a); 4.04 (d,1H,, H-1b); 4.06-4.13 (m, 2H, H-3, H-6a); 4.40 (dd, 1H, J6b-5=3.8z H-6b); 4.57 (d,1H,, H-7a); 4.79 (d,1H,, H-7b); 2H,5H,Ph). 13

3-O-benzyl-4,5-dideoxy-1,2-O-isopropylidene-β-D-glycerohex-4-en-2-ulose (115)

Starting with 106 (0.5 g, 1.61 mmol) and using G.P. 2.3.4. (reaction time 1h) the compound 115 was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as orange oil (0.30 g, 1.09 mmol, 67%).

1 H NMR (250MHz, CDCl3) δ: 1.38 (s, 3H, CH3); 1.53 (s, 3H, CH3); 3.78 (dd,1H,, H-3); 4.13 (s, 2H, H-1); 4.21-4.30 (m, 1H, H-6a); 4.39 (ddd,1H,, H-6b); 4.56 (d,1H,, H-7a); 4.66 (d,1H,, H-7b); 5.98 (ddt,1H,, H-4); 6.09 (ddd, 1H, J5-6a= J5-6b=2.0Hz, J5-3=1.8Hz, J5-4=10.3Hz, H-5); 7.24-7.38 (m, 5H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 26.3, 27.1 (CH3); 61.7 (C-6); 70.3 (C-7); 71.9 (C-3); 72.9 (C-1); 103.9 (C-2); 111.9 (Cq-isop); 122.2 (C-4); 127.8, 127.9, 128.9 (CH-Ph); 131.0 (C-5); 138. 1 H NMR (250MHz, DMSO-d6) δ: 1.29 (s, 3H, CH3); 1.38 (s, 3H, CH3); 3.46 (d,1H,, H-3); 3.60-3.72 (m, 4H, H-4, H-5, H-6a, H6b); 3.88 (d,1H,, H-1a); 3.96 (d,1H,, H-1b); 4.89 (d,1H,, OH-4); 5.01 (d,1H,, OH-3); 5.12 (d,1H,, OH-5). 13 C NMR (62.89MHz, DMSO-d6) δ: 26.3, 26.9 (CH3); 64.8 (C-6); 66.4 (C-4); 68.5 (C-5); 72.0 (C-1); 72.9 (C-3); 105.8 (C-2); 110.6 (Cq-isop).

MS: (IS+) m/z: 243 (M+Na) + IR: (neat) ν (cm -1 ): 3391, 3293 (OH); 2932, 2890 (CH).

3,4-Di-O-acetyl-5-deoxy-5-iodo-1,2-O-isopropylidene-α-L-sorbopyranose (100a)

Starting with 100 (0.5 g, 1.51 mmol) and using G.P. 2.1.2. (reaction time 15h) the compound 100a was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as cream solid (0.60 g, 1.45 mmol, 95%). Starting with 119 (1 g, 4.54 mmol) using G.P. 2.3.3. (reaction time 4h) and 1,4dioxane as solvent the compound 120 was obtained after column chromatography on silica gel with a mixture of PE/EA (6:4) as cream solid (0.94 g, 2.85 mmol, 63%).

1 H NMR (400MHz, CDCl3) δ: 1.40 (s, 3H, CH3); 1.50 (s, 3H, CH3); 2.44 (d,1H,, OH-3); 2.54 (d,1H,, OH-4); 3.83 (t, 1H, J3-4=3.0Hz, H-3); 3.94 (d,1H,, H-6a); 4.01-4.05 (m, 1H, H-4); 4.04 (d,1H,, H-1a); 4.09 (d,1H,, H-6b); 4.12 (d, 1H, J1b-

3,4-Di-O-acetyl-5-deoxy-5-iodo-1,2-O-isopropylidene-α-L-tagatopyranose (120a)

Starting with 120 (0.5 g, 1.51 mmol) and using G.P. 2.1.2. (reaction time 15h) the compound 120a was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as cream solid (0.58 g, 1.40 mmol, 95%).

1 H NMR (400MHz, CDCl3) δ: 1.41 (s, 3H, CH3-isop); 1.49 (s, 3H, CH3-isop); 2.04 (s, 3H, O-CO-CH3); 2.12 (s, 3H, O-CO-CH3); 3.78 (d,1H,, H-1a); 3.93 (d,1H,, H-1b); 4.08 (dd,1H,, H-6a); 4.14-4.23 (m, 2H, H-5, H-6b); 5.17 (d,1H,, H-3); 5.35 (dd, 1H, J4-3=3.2Hz, J4-5=10.8Hz, H-4). 13 C NMR (100MHz, CDCl3) δ: 20.8 (O-CO-CH3); 21.2 (C-5); 26.4, 26.9 (CH3isop); 66.6 (C-6); 72.2 (C-3); 72.7 (C-4); 73.4 (C-1); 103.9 (C-2); 113.6 (Cq-isop); 169.5, 169.9 (C=O). Starting with methyl-β-glucopyranoside (1.0 g, 4.92 mmol) using G.P. 2.1.5. (reaction time 15h), pyridine (in KOH) and tert-butyldimethylsilyl chloride (1.1. eq.) the compound 123 was obtained after column chromatography on silica gel with a mixture of PE/EA (2:8) as colorless solid (1.31 g, 4.25 mmol, 86%). M. Chung, G. Orlova, J. D. Goddard, M. Schlaf, R. Harris, T. J. Beveridge, G. White, F. R. Hallet J. Am. Chem. Soc.2002, 124, 10508.

HRMS:

Methyl 6-O-tert-butyldimethylsilyl-β-D-mannopyranoside (124)

Starting with methyl-β-mannopyranoside (1 g, 4.92 mmol) using G.P. 2.1.5. (reaction time 15h), pyridine (in KOH) and tert-butyldimethylsilyl chloride (1.1. eq.) the compound 124 was obtained after column chromatography on silica gel with a mixture of PE/EA (2:8) as colorless solid (1.23 g, 3.99 mmol, 81%).

1 H NMR (400MHz, CDCl3) δ: 0.07 (s, 6H, Si(CH3)2); 0.88 (s, 9H, C(CH3)3); 3.33 (s, 3H, OMe); 3.49-3.54 (m, 1H, H-5); 3.66 (t, 1H, J4-3=9.3Hz, H-4); 3.75 (dd,1H,, H-3); 3.79 (dd,1H,, H-6a); 3.86-3.87 (m, 1H, H-2); 3.90 (dd,1H,, H-6b); 4.12 (brs, 3H, OH); 4.66 (s, 1H, H-1). 13 C NMR (100MHz, CDCl3) δ: -5.2 (Si(CH3)2); 18.4 (Cq-C(CH3)3); 26.0 (C(CH3)3); 54.8 (OMe); 64.3 (C-6); 69.4 (C-2); 70.6 (C-5); 71.8 (C-3); 71.9 (C-4); 100.9 (C-1).

M. Chung, G. Orlova, J. D. Goddard, M. Schlaf, R. Harris, T. J. Beveridge, G. White, F. R. Hallet J. Am. Chem. Soc.2002, 124, Methyl 6-O-tert-butyldimethylsilyl-β-D-galactopyranoside (125)

Starting with methyl-β-galactopyranoside (1 g, 4.92 mmol) using G.P. 2.1.5. (reaction time 15h), pyridine (in KOH) and tert-butyldimethylsilyl chloride (1.1. eq.) the compound 125 was obtained after column chromatography on silica gel with a mixture of PE/EA (2:8) as colorless solid (1.21 g, 3.92 mmol, 80%). M. Chung, G. Orlova, J. D. Goddard, M. Schlaf, R. Harris, T. J. Beveridge, G. White, F. R. Hallet J. Am. Chem. Soc.2002, 124, 10508.

Methyl 6-O-tert-butyldimethylsilyl-3-deoxy-3-iodo-β-D-allopyranoside (126)

Starting with 123 (0.5 g, 1.62 mmol) using G.P. 2.3.3. (reaction time 4h) and 1,4dioxane as solvent the compound 126 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as cream solid (0.31 g, 0.74 mmol, 45%).

1 H NMR (400MHz, CDCl3) δ: 0.09 (s, 6H, Si(CH3)2); 0.89 (s, 9H, C(CH3)3); 2.85 (d,1H,, OH-2); 3.36 (ddd,1H,, H-5); 3.54 (s, 3H, OMe); 3.59 (ddd,1H,, H-2); 3.65 (d,1H,, OH-4); 3.79-3.85 (m, 1H, H-4); 3.82 (d,1H,, H-6a); 3.90 (dd, 1H, J6b-6a=10.2, H-6b); 3.91-3.95 (m, 1H, H-3); 4.18 (d,1H,, H-1). 13 C NMR (100MHz, CDCl3) δ: -5.3 (Si(CH3)2); 18.3 (Cq-C(CH3)3); 25.9 (C(CH3)3); 38. Starting with 125 (0.5 g, 1.62 mmol) using G.P. 2.3.3. (reaction time 4h) and 1,4dioxane as solvent the compound 127 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as cream solid (0.28 g, 0.67 mmol, 42%).

Starting with 127 (0.2 g, 0.48 mmol) and using G.P. 2.1.2. (reaction time 15h) the compound 127a was obtained after column chromatography on silica gel with a mixture of PE/EA (95:5) as cream solid (0.22 g, 0.44 mmol, 93%).

3,4,5-Tri-O-benzyl-1,2-O-isopropylidene-β-D-fructopyranose (140)

Starting with 98 (5 g, 22.7 mmol) and using G.P. 2.1.3. b) (reaction time 3h) the compound 140 was obtained after column chromatography on silica gel with a mixture of cyclohexane/EA (85:15) as colorless oil (10.2 g, 20.8 mmol, 92%).

1 H NMR (400MHz, CDCl3) δ: 1.43 (s, 3H, CH3); 1.48 (s, 3H, CH3); 3.74-3.83 (m, 3H, H-6a,H-6b, H-5); 3.90-3.96 (m, 2H, H-3, H-4); 3.96 (d,1H,, H-1a); 4.00 (d,1H, 6,127.7,127.8,128.0,128.4,138.6,. Starting with 119 (5 g, 22.7 mmol) and using G.P. 2.1.3. b) (reaction time 3h) the compound 141 was obtained after column chromatography on silica gel with a mixture of cyclohexane/EA (85:15) as colorless oil (10.6 g, 21.6 mmol, 95%).

MS: (IS+)

1 H NMR (400MHz, CDCl3) δ: 1.38 (s, 3H, CH3); 1.52 (s, 3H, CH3); 3.52 (dd, 1H, J5-4=2.8Hz, J5-6=7.2Hz, H-5); 3.55 (d,1H,, H-3); 3.78 (d, 1H, J6a-6b=11.4Hz, H-6a); 3.83 (d,1H,, H-6b); 4.03 (t, 1H, J4-5=2.8Hz, H-4); 4.17 (d,1H,, H-1a); 4.21 (d, 1H, J1b-1a=10. 13 C NMR (100MHz, CDCl3) δ: 25.9, 27.6 (CH3); 62.0 (C-6); 68.6 (C-1); 71.6, 73.6, 73.9 (C-7); 75.6 (C-4); 77.5 (C-3); 106.1 (C-2); 110. 127.6,127.7,127.8,127.9,128.4,128.5 (Ph);138.1,138.5,.

MS: (IS+) m/z: 513 (M+Na) + IR: (neat) ν (cm -1 ): 2986, 2985 (CH); 1496, 1454 (Ph).

3,4,5,Tri-O-benzyl-2-N,1-O-thiocarbonyl-α-D-fructopyranosylamine (148)

Starting with 140 (6 g, 12.2 mmol) and using G.P. 2.2.4. b) (reaction time 24h) the mixture 142 was obtained without further purification. Starting with 142 using G.P. 2.4.1. (reaction time 24h) and ethanol as solvent the mixture 144 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a yellow oil (4.69 g, 9.54 mmol, 78%).

Starting with 140 (1 g, 2.04 mmol) and using G.P. 2.4.3. (reaction time 15h) the compound 144 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a yellow oil (0.66 g, 1.34 mmol, 66%). Some traces of the 148 were isolated: [START_REF]Dihydro-{3-O-benzyl-4-deoxy-1,2-O-isopropylidene-β-D-psicopyranoso}[4,5-d]-1,3oxazoline-2-thione (55) Starting with 108 (1 g, 2.98 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 55 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2)[END_REF]127.7,127.8,127.9,128.1,128.3,137.2,137.7,190.2 (C=S). 6,127.9,128.0,128.1,128.3,128.5,137.1,137.7,190.4 (C=S).

HRMS:

4-((1R,2S,3R)-4-hydroxy-1,2,3-tribenzyloxybutyl)[1,3]oxazole-2-thione (146)

The compound 146 was a side product of the synthesis of compound 144 when using G.P. 2.4.1.. Compound 146 was obtained after column chromatography on silica gel with a mixture of PE/EA (85:15) as a yellow solid (1.20 g, 2.44 mmol, 20%). 6, 127.9, 128.0, 128.2, 128.3 128.5, 128.6, 128.9 (CH-Ph); 136.5 (C-1); 137. 3,137.6, The compound 147 was a side product of the synthesis of compound 145 when using G.P. 2.4.1.. Compound 147 was obtained after column chromatography on silica gel with a mixture of PE/EA (85:15) as a yellow solid (0.90 g, 1.83 mmol, 15%).

1 H NMR (400MHz, CDCl3) δ: 3.23-3.26 (m, 1H, H-5); 3.69 (dd,1H,.0Hz H-6a); 3.80 (dd,1H, Starting with 144 (1 g, 2.03 mmol) using G.P. 2.3.6. (reaction time 3h), sodium hydrogen carbonate (4.0 eq.) as base and benzyl bromide (4.0 eq.) the compound 150 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as cream oil (0.57 g, 0.98 mmol, 48%).

1 H NMR (250MHz, CDCl3) δ: 3.33 (d,1H, Starting with 145 (1 g, 2.03 mmol) using G.P. 2.3.6. (reaction time 3h), sodium hydrogen carbonate (4.0 eq.) as base and benzyl bromide (4.0 eq.) the compound 151 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as cream oil (0.51 g, 0.88 mmol, 43%).

1 H NMR (400MHz, CDCl3) δ: 3.33 (d,1H,, H-6a); 3.42 (d, 1H, J4-5=3.2Hz, J4-3=9.4Hz, H-4); 4.07 (dd,1H,, H-6b); 4.18 (d,1H,, H-3); 4.23 (d,1H, (d, 1H, J7b-7a=12.4, H-7b); 7.17-7.42 (m, 20H, Ph). 13 C NMR (100MHz, CDCl3) δ: 36.5 (SCH2Ph); 62.0 (C-6); 71.2, 72.2 (CH2Ph); 72.3 (C-5); 73.0 (C-1); 75.2 (CH2Ph); 78.4 (C-4); 78.9 (C-3); 90.1 (C-2); 127.6,127.7,128.0,128.2,128.3,128.4,128.5,138.1,138.3,; 170.6 (C-S). Starting with 144 (1 g, 2.03 mmol) using G.P. 2.3.6. (reaction time 3h), sodium hydrogen carbonate (4.0 eq.) as base and benzyl bromide (4.0 eq.) the compound 152 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as cream oil (0.51 g, 0.88 mmol, 43%).

HRMS:

1 H NMR (250MHz, CDCl3) δ: 3.77 (d,1H, 127.9,128.0,128.2,128.4,137.3,137.6, Starting with 145 (1 g, 2.03 mmol) using G.P. 2.3.6. (reaction time 3h), sodium hydrogen carbonate (4.0 eq.) as base and benzyl bromide (4.0 eq.) the compound 153 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as cream oil (0.56 g, 0.96 mmol, 47%).

1 H NMR (400MHz, CDCl3) δ: 3.77 (dd, 1H, J6a-5=2.0Hz, J6a-6b=12.2Hz, H-6a); 3.86 (brs, 1H, H-5); 3.98 (d,1H,, H-6b); 4.00 (d,1H,, H-3); 4.12 (s, 2H, SCH2Ph); 4.15 (dd, 1H, J4-5=3.2Hz, J4-3=9.6Hz, H-4); 4.24 (d,1H,, H-1a); 4.29 (d,1H, 13 C NMR (100MHz, CDCl3) δ: 36.4 (SCH2Ph); 63.0 (C-6); 71.9, 72.3 (CH2Ph); 73.9 (C-5); 75.1 (CH2Ph); 76.6 (C-1); 77.4 (C-3); 80.5 (C-4); 102.7 (C-2); 127. [START_REF]C17H21NO5S [M+H] + 352[END_REF]127.9,128.1,128.5,128.6,; 137.2 (Cq-SBn); 138. 5,138.6,; 169.6 (C-S).

HRMS:

3,4,5,Tri-O-benzyl-2-N,1-O-carbonyl-β-D-fructopyranosylamine (154)

Starting with 150 (1 g, 1.72 mmol) and using G.P. 2.5.1. b) the compound 154 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless oil (0.53 g, 1.11 mmol, 65%).

1 H NMR (250MHz, CDCl3) δ: 3.51 (d,1H,, H-3); 3.71-3.72 (m, 1H, H-4); 3.83-3.88 (m, 1H, H-4); 3.88 (s, 2H, H-1); 4.02 (d, 1H, J6a-6b=10. [START_REF]C17H21NO5S [M+H] + 352[END_REF]128.5,128.8,137.4,137.6,

3,4,5,Tri-O-benzyl-2-N,1-O-carbonyl-β-D-fructopyranosylamine (156)

Starting with 152 (1 g, 1.72 mmol) and using G.P. 2.5.1. b) the compound 156 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as colorless oil (0.52 g, 1.09 mmol, 64%).

1 H NMR (400MHz, CDCl3) δ: 3.62 (dd,1H,, H-6a); 3.80-3.83 (m, 4H, H-3, H-4, H-5; H-6b); 4.38 (d,1H,, H-1a); 4.22 (d,1H,, H-1b); 4.51-4.66 (m, 5H, CH2Ph); 4.83 (d,1H,J=11.8Hz,CH2Ph); 7.26-7.30 (m, 15H, Ph); 8.19 (s, 1H, NH). 13 C NMR (62.89MHz, CDCl3) δ: 61.9 (C-6); 71.4 (C-5); 71.9 (CH2Ph); 72.5 (C-4); 72.8 (C-7); 73.9 (C-1); 74.9 (C-7); 76.1 (C-4); 101.3 (C-2); 127.9, 128.0, 128. [START_REF]Dihydro-{3-O-benzyl-4-deoxy-1,2-O-isopropylidene-β-D-psicopyranoso}[4,5-d]-1,3oxazoline-2-thione (55) Starting with 108 (1 g, 2.98 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 55 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2)[END_REF]128.6,137.5,137.8,138. Starting with 153 (1 g, 1.72 mmol) and using G.P. 2.5.1. b) the compound 157 was obtained after column chromatography on silica gel with a mixture of petroleum ether/ethyl acetate (7:3) as colorless oil (0.43 g, 0.90 mmol, 52%).

1 H NMR (400MHz, CDCl3) δ: 3.50 (d,1H,, H-3); 3.71 (dd, 1H, J6a-6b=10.8Hz, H-6a); 3.83-3.90 (m, 3H, H-4, H-5, H-6b); 4.00 (d,1H,, H-1a); 4.14 (d,1H,, H-1b); 4.26 (d, 1H, J7a-7b=12.0Hz, CH2Ph); 4.43 (d, 1H, J7b-7a=12.0Hz, CH2Ph); 4.47 (d, 1H, J7a-7b=12.0Hz, CH2Ph); 4.56 (d, H, J7b-7a= J7a-7b=12.0Hz, CH2Ph); 4.79 (d, 1H, J7b-7a=11.6Hz, CH2Ph); 6.74 (brs, 1H, NH); 7.06-7.08 (m, 2H, Ph); 7.27-7.37 (m, 13H, Ph);. 13 C NMR (100MHz, CDCl3) δ: 59.1 (C-6); 71.4 (C-5); 71.9 (CH2Ph); 72.4 (C-4); 73.0 (CH2Ph); 73.8 (C-1); 74.0 (CH2Ph); 77.4 (C-3); 102.0 (C-2); 127.7, 128. 1,128.3,128.4,128.6,136.5,137.4,

2-N,1-O-carbonyl-α-D-fructopyranosylamine (158)

Starting with 154 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 158 was obtained after column chromatography on silica gel with EA as colorless oil (53 mg, 0.26 mmol, 25%).

1 H NMR (400MHz, MeOD) δ: 3.66 (dd, 1H, J4-5=3.2Hz, J4-3=9.6Hz, H-4); 3.71-3.74 (m, 3H, H-3, H-6a, H6b); 3.85-3.87 (m, 1H, H-5); 4.21 (d,1H,, H-1a); 4.44 (d,1H,, H-1b). 13 C NMR (100MHz, MeOD) δ: 66.0 (C-6); 69.9 (C-3); 70.3 (C-5); 71.9 (C-4); 73.

2-N,1-O-carbonyl-α-D-psicopyranosylamine (159)

Starting with 155 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 159 was obtained after column chromatography on silica gel with EA as colorless oil (73 mg, 0.36 mmol, 34%).

1 H NMR (250MHz, MeOD) δ: 3.65 (dd, 1H, J4-5=3.0Hz, J4-3=9.8Hz, H-4); 3.71-3.74 (m, 3H, H-3, H-6a, H6b); 3.85-3.88 (m, 1H, H-5); 4.20 (d,1H,, H-1a); 4.44 (d,1H,, H-1b). 13 C NMR (62.89MHz, MeOD) δ: 66.1 (C-6); 69.9 (C-3); 70.4 (C-5); 71.9 (C-4); 74.3 (C-1); 92.8 (C-2); 161.0 (C=O).

IR: (neat) ν (cm -1 ): 3339 (OH); 2962 (CH); 1636 (C=O).

2-N,1-O-carbonyl-β-D-fructopyranosylamine (160)

Starting with 156 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 160 was obtained after column chromatography on silica gel with EA as colorless oil (0.15 g, 0.73 mmol, 68%).

1 H NMR (250MHz, MeOD) δ: 3.74-3.80 (m, 2H, H-3, H-4); 3.81-3.89 (m, 3H, H-5, H-6a, H6b); 4.06 (d,1H,, H-1a); 4.10 (d,1H,, H-1b). (5R,8R,9R,10S) 8,9-(Isopropylidenedioxy)-10-benzyloxy-2-benzylsulfanyl-1,2-dehydro-1-aza-3,6-dioxaspiro [START_REF]Dihydro-{3-O-benzyl-4-deoxy-1,2-O-isopropylidene-β-D-psicopyranoso}[4,5-d]-1,3oxazoline-2-thione (55) Starting with 108 (1 g, 2.98 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 55 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2)[END_REF]5]

decane (166)

Starting with the mixture of the two OZTs 163 and 164 (50mg g, 0.14 mmol) using G.P. 2.3.6. (reaction time 4h), sodium hydrogen carbonate (4.0 eq.) as base and benzyl bromide (4.0 eq.) the compound 166 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as cream solid (29 g, 0.07 mmol, 47%).

1 H NMR (400MHz, CDCl3) δ: 1.36 (s, 6H, CH3); 1.51 (s, 3H, CH3); 3.81 (dd,1H,.6Hz H-6a); 3.94-4.05 (m, 4H, H-1, H-3, H-6b); 4.11 (s, 2H, H-8); 4.20 (dd,1H,, H-5); 4.31 (t, 1H, J4-5=6.2Hz, H-4); 4.64 (d,1H,, H-7a); 4.81 (d,1H,, H-7b); 6.14 (s, 1H, NH); 7.23-7.40 (m, 10H, Ph). 13 C NMR (100MHz, CDCl3) δ: 25.9 (CH3); 27.5 (CH3); 34.4 (C-8); 62.0. (C-6); 72.7 (C-5); 74.0 (C-3); 74.7 (C-4); 75.1 (C-1); 89.3 (C-2); 110.0 (Cq-isop); 127. [START_REF]Dihydro-{3-O-benzyl-4-deoxy-1,2-O-isopropylidene-β-D-psicopyranoso}[4,5-d]-1,3oxazoline-2-thione (55) Starting with 108 (1 g, 2.98 mmol) and using G.P. 2.4.2. (reaction time 15h) the compound 55 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2)[END_REF]128.5,128.8,; 136.9 (Cq-SBn); 138.0 (Cq-isop); 166.4 (C-S). Starting with 56 (0.3 g, 0.86 mmol) and using G.P. 2.4.3. (reaction time 15h) the compound 167 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as a colorless oil (13 mg, 0.03 mmol, 4%). Starting with 97 (1 g, 2.38 mmol) and using G.P. 2.3.7. (reaction time 1h) the compound 170 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless oil (0.65 g, 2.22 mmol, 94%).

HRMS:

1 H NMR (400MHz, CDCl3) δ: 1.44 (s, 3H, CH3); 1.52 (s, 3H, CH3); 3.22 (d,1H,, H-5); 3.38 (d,1H,, H-4); 3.48 (s, 1H, H-3); 3.83 (d, 1H, J1a-1b=8.6Hz, H-1a); 3.86 (d,1H,, H-1b); 4.00 (d,1H,4.16 (d,1H,, H-6b); 4.61 (d,1H,J=11.8Hz,CH2Ph); 4.88 (d,1H,J=11.8Hz,CH2Ph);5H,Ph). Starting with 170 (0.5 g, 1.71 mmol) and using G.P. 2.3.7. (reaction time 24h) the compound 174 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as colorless oil (0.27 g, 0.83 mmol, 49%).

1 H NMR (400MHz, CDCl3) δ: 1.38 (s, 3H, CH3); 1.45 (s, 3H, CH3); 3.24 (ddd,1H,, H-5); 3.29 (d,1H,, H-3); 3.45 (s, 3H, OCH3); 3.56 (t,1H,, H-6a); 3.79 (dd,1H,, H-6b); 3.82 (d,1H,, H-1a); 1H,, H-4); 3.94 (d,1H,4.69 (d,1H,J=11.6Hz,CH2Ph) Starting with 174 (0.2 g, 0.62 mmol) and using G.P. 2.2.1. (reaction time 15h) the compound 174a was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless oil (0.22 g, 0.60 mmol, 95%).

1 H NMR (400MHz, CDCl3) δ: 1.38 (s, 3H, CH3); 1.47 (s, 3H, CH3); 2.03 (s, 3H, O-CO-CH3); 3.33-3.39 (m, 4H, OCH3, H-5); 3.34 (d,1H,, H-3); 3.70 (t,1H,, H-6a); 2H,, H-1a, H-6b); 3.93 (d,1H,, H-1b); 4.58 (d,1H,, H-7a); 4.73 (d, 1H, J7b-7a=11.6Hz, H-7b); 5.41 (t, 1H, J4-3=9.6Hz, H-4) 7.26-7.35 (m, 5H, Ph). Starting with D-tagatose (0.3 g, 1.66 mmol) using G.P. 2.4.1. (reaction time 15h) and potassium thiocyanate (2.0 eq.) the compound 175 was obtained after column chromatography on silica gel with a mixture of PE/EA (4:6) as colorless solid (0.15 g, 0.54 mmol, 33%).

1 H NMR (400MHz, DMSO) δ: 3.35-3.48 (m, 2H, H-6a, H-6b); 3.58 (dd, 1H, J5-4=6.4Hz, J5-6=13.6Hz, H-5); 3.93 (t,1H,, H-4); 4.61 (d, 1H, J1a-1b=10.4Hz, H-1a); 4.66 (d,1H, The compound 176 was obtained together with compound 175 as a colorless solid (0.13 mg, 0.05 mmol, 28%). Starting with D-tagatose (0.3 g, 1.66 mmol) using G.P. 2.4.1. (reaction time 15h) and potassium thiocyanate (1.0 eq.) the compound 177 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless solid (0.18 g, 0.33 mmol, 20%).

1 H NMR (400MHz, CDCl3) δ: 0.05-0.12 (m, 18H, Si(CH3)2); 0.89-0.93 (m, 27H, C(CH3)3); 3.53 (dd,1H,, H-6a); 3.60 (d,1H,, H-1a); 3.63 (d,1H,, H-1b); 3.80-3.84 (m, 1H, H-4, H-5); 3.99 (dd,1H,, H-6b); 4.55 (d,1H,, H-3); 5.24 (brs, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: -5. 5,2); 17. [START_REF]C17H21NO5S [M+H] + 352[END_REF]18.0,25.7,25.8 Starting with D-tagatose (0.3 g, 1.66 mmol) using G.P. 2.4.1. (reaction time 15h) and potassium thiocyanate (1.0 eq.) the compound 178 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless solid (0.16 g, 0.36 mmol, 22%).

1 H NMR (400MHz, CDCl3) δ: -0.01-0.02 (m, 12H, Si(CH3)2); 0.80-0.82 (m, 18H, C(CH3)3); 2.16 (d,1H,, OH-4); 3.59 (dd, 1H, J6a-5=2.0Hz, J6a-6b=12.8Hz, H-6a); 3.65 (d,1H,, H-1a); 3.68 (d, 1H, J1b-1a=11. Starting with D-tagatose (0.3 g, 1.66 mmol) using G.P. 2.4.1. (reaction time 15h) and potassium thiocyanate (1.0 eq.) the compound 179 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless solid (40 mg, 0.18 mmol, 11%).

1 H NMR (400MHz, CDCl3) δ: 0.07-0.15 (m, 12H, Si(CH3)2); 0.90 (m, 9H, C(CH3)3); 0.94 (m, 9H, C(CH3)3); 2.13 (d,1H,, OH-5); 3.71 (d,1H,, H-1a); 3.75 (d,1H,, H-1b); 3.78 (dd,1H,, H-6a); 3.89 (dd, 1H, J4-3=2.8Hz, J4-5=7.2Hz H-4); 3.95-3.98 (m, 1H, H-5); 4.06 (dd,1H,, H-6b); 4.85 (d,1H,, H-3); 7.01 (brs, 1H, NH). Starting with D-tagatose (1 g, 5.55 mmol) using G.P. 2.1.1. a) (reaction time 15h) and camphorsulfonic acid (1.0 eq.) the compound 180 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless solid (0.97 g, 3.72 mmol, 67%).

Starting with D-tagatose (1 g, 5.55 mmol) using G.P. 2.1.1. b) (reaction time 18h) the compound 193 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless solid (1.20 mg, 4.61 mmol, 83%). (d,1H,, H-1a); 4.26 (d,1H,, H-1b); 4.62 (d,1H,, H-3); 4.83 (dd, 1H, J4-5=4.0Hz, J4-3=5.8Hz, H-4). Starting with D-tagatose (1 g, 5.55 mmol) using G.P. 2.1.1. a) (reaction time 15h) the compound 181 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless solid (34 mg, 0.13 mmol, 2.4%). Starting with 104 (2 g, 7.68 mmol) using G.P. 2.1.1. b) (reaction time 3h) and add perchloric acid (0.17 eq.) at 5ºC the compound 182 was obtained after column chromatography on silica gel with a mixture of PE/EA (7:3) as colorless oil (1.48 g, 5.69 mmol, 95%).

1 H NMR (250MHz, CDCl3) δ: 1.33 (s, 6H, CH3); 1.40 (s, 3H, CH3); 1.44 (s, 3H, CH3); 1.50 (s, 3H, CH3); 3.33 (dd,1H,.5Hz H-6a); 3.59-3.74 (m, 2H, H-6b, OH); 4.05 (d,1H,4.26 (t,1H,, H-5); 4.32 (d,1H,4.64 (d,1H,; 4.88 (dd, 1H, J4-5=1.0Hz, J4-3=6.0Hz, H-4). 13 C NMR (62.89MHz, CDCl3) δ: 24. 5, 25.8, 25.9, 26.

6-O-benzyl-1,2:3,4-di-O-isopropylidene-α-D-tagatofuranose (183)

Starting with 180 (1 g, 3.84 mmol) using G.P. 2.1.3. a) (reaction time 15h), (reaction time 15h), dichloromethane as solvent, sodium hydride (60% in mineral oil) (1.5 eq.) as base and benzyl bromide (1.5 eq.) the compound 183 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless oil (1.04 g, 2.97 mmol, 77%).

1 H NMR (400MHz, CDCl3) δ: 1.21 (s, 6H, CH3); 1.30 (s, 3H, CH3); 1.31 (s, 3H, CH3); 1.40 (s, 3H, CH3); 3.60 (dd,1H,, H-6a); 3.73 (dd,1H,, H-6b); 3.97 (d,1H,, H-1a); 4.07-4.11 (m, 1H, H-5); 4.18 (d,1H,, H-1b); 4.46 (d, 1H, J=12.0Hz, CH2Ph); 4.50 (d,1H,, H-3); 4.55 (d, 1H, J=12.0Hz, CH2Ph); 4.70 (dd, 1H, J4-5=3.8Hz, J4-3=5.8Hz, H-4); 7.16-7.28 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 26.0, 26. 1,26.5,26.6 (CH3); 68.0 (C-6); 69.3 (C-1); 73.3 (CH2Ph); 78.4 (C-5); 80.2 (C-4); 85.2 (C-3); 111.6, 111.9 (Cq-isop); 112.7 (C-2); 127.6,127.7,. Starting with 182 (2 g, 7.68 mmol) using G.P. 2.3.9. (reaction time 15h), dichloromethane as solvent, sodium hydride (60% in mineral oil) (1.5 eq.) as base and benzyl bromide (1.5 eq.) the compound 184 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as colorless oil (2.23 g, 6.36 mmol, 83%).

1 H NMR (400MHz, CDCl3) δ: 1.31 (s, 6H, CH3); 1.36 (s, 3H, CH3); 1.42 (s, 3H, CH3); 1.44 (s, 3H, CH3); 3.52 (dd,1H,, H-6a); 3.57 (dd,1H,, H-6b); 4.04 (d,1H,, H-1a); 4.28 (d,1H, 111.6,127.8,128.5,128.9,.

S. Mio, Y.Kumagawa, S. Sugai Tetrahedron 1991, 47, 2133.

6-O-benzyl-2,N-3,O-thiocarbonyl-β-D-tagatofuranosylamine (288)

Starting with 183 (0.5 g, 1.42 mmol) and using G.P. 2.2.4. a) (reaction time 15h) the mixture 186 was obtained without further purification. Starting with 186 using G.P. 2.4.1. (reaction time 15h) and water as solvent the compound 188 was obtained after column chromatography on silica gel with a mixture of PE/EA (4:6) as a cream oil (0. 13 g, 0.42 mmol, 30%). This fused OZT was obtained as the major compound in a mixture with two spiro-OZTs. Starting with 184 (0.5 g, 1.42 mmol) and using G.P. 2.2.4. a) (reaction time 15h) the mixture 185 was obtained without further purification. Starting with 185 using G.P. 2.4.1. (reaction time 15h) and water as solvent the compound 190 was obtained after column chromatography on silica gel with a mixture of PE/EA (4:6) as a cream oil (0.25 g, 0.80 mmol, 57%).

1 H NMR (250MHz, CDCl3) δ: 3.61-3.64 (m, 1H, H-6a); 3.73-3.80 (m, 3H, H-6b, H-1; 3.92-3.95 (m, 1H, H-3); 4.11-4.15 (m, 1H, H-4); 4.53 (s, 2H, CH2Ph); 5.06-5.08 (m, 1H, H-5); 7.52-7.34 (m, 5H, Ph); 8.68 (brs, 1H, NH). 13 C NMR (62.89MHz, CDCl3) δ: 61.4 (C-1); 67.5 (C-6); 70.8 (C-4); 72.9 (CH2Ph); 78.8 (C-3); 84.9 (C-5); 98.3 (C-2); 127. [START_REF]-N,1-O-carbonyl-β-D-psicopyranosylamine (161) Starting with 157 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 161 was obtained after column chromatography on silica gel with EA as colorless oil[END_REF]127.3,127.9,128.1,; 137.9 (Cq-Ph); 188.7 (C=S).

6-O-benzyl-2,N-3,O-thiocarbonyl-α-D-tagatofuranosylamine (191)

Starting with 183 (0.3 g, 0.86 mmol) using G.P. 2.4.3. (reaction time 15h) and trimethylsilyl thiocyanate (3.0 eq.) the compound 191 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (0.12 g, 0.34 mmol, 39%).

1 H NMR (250MHz, CDCl3) δ: 1.32 (s, 3H, CH3); 1.35 (s, 3H, CH3); 3.62 (dd,1H,, H-6a); 3.76 (dd,1H,, H-6b); 3.84 (d,1H,, H-1a); 3.96 (d,1H, Starting with 183 (0.3 g, 0.86 mmol) using G.P. 2.4.3. (reaction time 15h) and trimethyl silyl thiocyanate (3.0 eq.) the compound 193 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (49 mg, 0.15 mmol, 17%).

1 H NMR (400MHz, CDCl3) δ: 1.28 (s, 3H, CH3); 1.38 (s, 3H, CH3); 3.65-3.70 (m, 1H, H-6a); 3.76 (dd,1H,, H-6b); 4.05 (m, 1H, H-5); 4.36 (d,1H,, H-1a); 4. 2H,CH2Ph);4H, H-1b, H-3, CH2-Ph); 4.77-4.80 (m, 1H, H-4); 7.26-7.36 (m, 5H, Ph); 7.97 (s, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 24.9, 26.0 (CH3); 67.9 (C-6); 70.8 (C-1); 73.7 (CH2Ph); 79.2 (C-5); 80.3 (C-4); 84.9 (C-3); 98.9 (C-2); 113.6 (Cq-isop); 127.9, 128.1, 128.6 (CH-Ph); 137.9(Cq-Ph); 159.5 (C=O).

6-O-benzyl-2,N-3,O-thiocarbonyl-β-D-psicofuranosylamine (192)

Starting with 184 (0.3 g, 0.86 mmol) using G.P. 2.4.3. (reaction time 15h) and trimethyl silyl thiocyanate (5.0 eq.) the compound 192 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (0.13 g, 0.37 mmol, 42%). The compound 194 was a side product of the synthesis of compounds 191 and 193. Compound 194 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (62 mg, 0.15 mmol, 21%).

1 H NMR (400MHz, CDCl3) δ: 1.31 (s, 3H, CH3); 1.41 (s, 3H, CH3); 3.65 (dd,1H,, H-6a); 3.79 (dd,1H,, H-6b); 3.87 (d,1H,, H-1a); 3.98 (d,1H,, H-1b); 4.22-4.26 (m, 1H, H-5); 4.53 (d,1H,J=12.2Hz,CH2Ph);4.59 (d,1H,, H-3); 4.63 (d,1H,J=12.2Hz,CH2Ph); 4.79 (dd, 1H, J4-5=4.0Hz, J4-3=6.0Hz, H-4); 7.26-7.36 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 24.9, 26.

1,2-Di-O-benzyl-3,4-O-isopropylidene-α-D-tagatofuranose (195)

The compound 195 was a side product of the synthesis of compound 191. Compound 195 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (45 mg, 0.11 mmol, 13%).

1 H NMR (250MHz, CDCl3) δ: 1.31 (s, 3H, CH3); 1.40 (s, 3H, CH3); 3.64-3.71 (m, 1H, H-6a); 3.80 (dd,1H,, H-6b); 4.22 (s, 2H, CH2Ph); 4.32 (d,1H,, H-1a); 4.33-4.37 (m, 1H, H-5) ; 4.52-4.54 (m, 3H, H-1b, H-3, CH2Ph); 4.67 (d,1H,J=12.5Hz,CH2Ph);4.88 (dd,1H,, J4-5=6.0Hz, H-4); 7.25-7.36 (m, 10H, Ph). Starting with 195 (0.1 g, 0.28 mmol) using G.P. 2.4.3. (reaction time 15h) and trimethyl silyl thiocyanate (5.0 eq.) the compound 198 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a colorless oil (0.10 g, 0.23 mmol, 83%).

1 H NMR (250MHz, CDCl3) δ: 1.31 (s, 3H, CH3); 1.40 (s, 3H, CH3); 3.68 (dd,1H,, H-6a); 3.79 (dd,1H,, H-6b); 4.22 (s, 2H, CH2Ph); 4.33 (d,1H,, H-1a); 4.36-4.38

LIGAÇÃO SELECTIVA DE TIONOCARBAMATOS CÍCLICOS EM CETOHEXOSES

Uma simples reacção como a condensação de ácido tiociânico com uma cetohexose pode levar à formação de 10 oxazolidinetionas (OZTs) ou oxazolinetionas (OXTs) diferentes. A questão é "como lidar com esta química tão complexa?" O controlo cuidadoso das condições de reacção associado a sequências de purificação incluindo protecções selectivas e diversas funcionalizações podem certamente ser úteis. Um jogo químico subtil e estimulante foi desenvolvido entre pré-e pós-protecções selectivas e reacções originais de funcionalização. Neste trabalho de doutoramento, dedicámo-nos a estudar novas tácticas de síntese e de purificação para aceder a OZTs e oxazolidinonas (OZOs) ligadas a cetohexose. Estes trabalhos foram aplicados à série D-fructo e seus epímeros, D-psico e D-tagato, cuja química tem sido pouco explorada em comparação com outras séries de monossacarídeos. Deste modo foi possível um melhor controlo das abordagens químicas para aceder a estes tionocarbamatos sacarídicos e assim valorizar estas moléculas para as quais o interesse tanto do ponto de vista biológico como químico é importante. Nestes estudos, foi desenvolvida uma biblioteca de formas tautoméricas fixas de compostos híbridos que foram submetidos a testes biológicos. Assim, este grupo de compostos foi objecto de testes antimicrobianos cujos resultados foram muito interessantes. Além disso, os melhores inibidores de GLUT5 serão utilizados no desenvolvimento de novas ferramentas bioquímicas para uma melhor compreensão dos papéis desempenhados por este transportador de D-frutose em relação à diabetes tipo 2 e à obesidade. Oxazolidinetionas; oxazolidinonas; cetohexoses; D-fructose; D-psicose; D-tagatose; tionocarbamatos Ana Catarina SIMÃO

ANCRAGES SELECTIFS SUR CETOHEXOSES DE THIONOCARBAMATES CYCLIQUES

Une réaction simple de condensation de l'acide thiocyanique avec un cétohexose peut conduire à la formation d'une dizaine de 10 oxazolidinethiones (OZTs) ou oxazolinethiones (OXTs) distinctes. La question que nous nous sommes posée a été "comment gérer une chimie aussi complexe?" Un contrôle attentif des conditions de réaction, associé à des séquences de purification comportant des protections sélectives et diverses fonctionnalisations, peuvent certainement être utiles. Un jeu chimique subtil et stimulant a été développé entre des pré-et post-protections sélectives et des réactions originales de fonctionnalisation. Dans ce travail de thèse, nous avons mis à l'étude de nouvelles tactiques de synthèse et de purification pour accéder à des OZTs et des oxazolidinones (OZOs) ancrées sur des charpentes des cétohexoses. Ces travaux ont été mis en application notamment en série D-fructo et dans les séries épimères D-psico et D-tagato dont la chimie ont été peu explorée en comparaison d'autres séries monosaccharidiques. Nous avons ainsi mieux maîtrisé les approches chimiques permettant d'accéder à ces thionocarbamates saccharidiques et ainsi de valoriser ces molécules dont l'intérêt d'un point de vue biologique et chimique est important. Dans ces études, nous avons élaboré une bibliothèque de formes tautomères fixes de composés hybrides qui ont été soumis à des essais biologiques. Ainsi, ce groupe de composés a fait l'objet de tests antimicrobiens, livrant des résultats très intéressants. Par ailleurs, les meilleurs inhibiteurs de GLUT5 seront utilisés dans le développement de nouveaux outils biochimiques pour une meilleure compréhension des rôles joués par ce transporteur du D-fructose en relation au le diabète de type 2 et de l'obésité. Oxazolidinethiones; oxazolidinones; cétohexoses; D-fructose; D-psicose; D-tagatose; thionocarbamates

SELECTIVE ANCHORING OF CYCLIC THIONOCARBAMATES ON KETOHEXOSES

A simple condensation reaction of thiocyanic acid with a ketohexose can provide a library of up to 10 different molecules composed of oxazolidinethiones (OZTs) or oxazolinethiones (OXTs). The question arising is "how to manage such a complex chemistry?" Careful control of the reaction conditions associated with purification sequences (involving selective protection or functionalization) can certainly help. A subtle and challenging chemical game could be played between selective pre-and post-protections and original functionalizing reactions. In this PhD work, we disclose the development of new chemical and purification tactics to access OZTs and oxazolidinones (OZOs) anchored onto ketohexose scaffolds, namely D-fructo and its epimers D-psico, D-tagato, whose chemistry has been poorly investigated in comparison with other monosaccharidic systems. Furthermore we have improved our understanding about saccharidic thionocarbamates chemistry for a better valorization from both chemical and biological point of view. We have generated a library of fixed tautomeric forms of hybrid compounds to be submitted to biological tests. This panel of compounds was subjected to antimicrobial screening and some molecules have shown very efficient effects. Moreover, the leading inhibitors of GLUT5 will be used in the development of biochemical tools for a better comprehension of the role of this D-fructose transporter in relation to type 2 diabetes and obesity. oxazolidinethiones; oxazolidinones; ketohexoses; D-fructose; D-psicose; D-tagatose; thionocarbamates

1 .

 1 Conclusion ..................................................................................................................................... Choice of the pathway ................................................................................................................. 1.1.1. Preparation of α-hydroxyketone ........................................................................................ a) Selective oxidation .................................................................................................................1.2. Condensation with a thiocarbonyl source -CS2 or CSCl2 ....................................................... 1.2.1. Glycosylamines ..................................................................................................................... 1.2.2. Saccharidic templates bearing an amino group................................................................ a) Aldoses .................................................................................................................................... b) Ketoses ..................................................................................................................................... 1.2.3. Saccharidic thionocarbamates via aziridine or epoxide ................................................... 1.3. 4,5-Fused OZT in β-D-fructopyranose....................................................................................... 1.3.1. First Approach ....................................................................................................................... a) Structural protection .............................................................................................................. b) Iodination and azide Formation .......................................................................................... c) OZT Formation ....................................................................................................................... 1.3.2. Second approach ................................................................................................................... a) Selective tosylation ................................................................................................................. b) Selective iodination ................................................................................................................ c) OZT formation ........................................................................................................................ 1.4. Fused OZT in β-D-psicopyranose ........................................................................................... 1.4.1. D-Fructose similar approach ............................................................................................... a) From D-fructo to D-psico series ............................................................................................ b) Iodination ................................................................................................................................ c) Azido and OZT formation .................................................................................................... 1.5. Deprotection assays ..................................................................................................................... 1.5.1. Hydrochloric acid conditions .............................................................................................. a) D-Fructose ............................................................................................................................... b) D-Psicose ................................................................................................................................. 1.5.2. An original reagent, Sc(OTf)3 .............................................................................................. 1.2.3. Trifluoroacetic acid conditions ............................................................................................ 1.6. Selective Iodination ...................................................................................................................... 1.6.1. Diol derivatives ..................................................................................................................... 1.6.2. Triol derivatives .................................................................................................................. a) Ketoses ................................................................................................................................... b) Aldohexoses .......................................................................................................................... 1.7. Conclusion ................................................................................................................................... CHAPTER IV -Spiro-thionocarbamates in pyranose form 1. SYNTHESIS OF SPIRO OZTs and OZOs IN 3,4,5-tri-O-BENZYLATED β-D-FRUCTO-AND β-D-PSICOPYRANO-DERIVATIVES ................................................................................111 1.1. Choice of the pathway ................................................................................................................111 1.1.1. Condensation with HSCN ..................................................................................................111 1.1.2. Condensation with trimethylsilyl reagents ......................................................................116 a) TMSN3 .....................................................................................................................................116 b) TMSNCS .................................................................................................................................117 c) TMSCN ...................................................................................................................................117 1.2. Synthesis of spiro-OZTs .............................................................................................................

CHAPTER 1 . 1 .

 11 4. Conclusion ...................................................................................................................................125 2. SYNTHESIS OF SPIRO OZTs IN 3-O-BENZYLATED β-D-FRUCTO-AND β-D-PSICOPYRANO AND -FURANO DERIVATIVES .....................................................................127 2.1. Synthesis of spiro-OZTs in D-fructo series ...............................................................................127 2.1.1. Choice of the pathway ........................................................................................................127 2.1.2. Synthesis of spiro-OZTs .......................................................................................................127 2.1.2. Isolation of the products .....................................................................................................128 a) By protection ..........................................................................................................................128 b) By S-alkylation ......................................................................................................................129 2.1.3. An unexpected OZT ............................................................................................................130 2.2. Synthesis of spiro-OZTs in D-psico series ................................................................................131 2.3. Conclusion ...................................................................................................................................132 Preparation of D-tagatose ..........................................................................................................135 1.1.1. From D-fructose to D-tagatose series ................................................................................135 a) Epoxide formation ................................................................................................................136 b) Epoxide opening ...................................................................................................................137 1.2. Conversion of D-tagatose into an OZT ....................................................................................139 1.3. D-Tagatose and D-psicose -OZT Selective formation on furanose form ...........................140 1.3.1. From pyrano to furano form ..............................................................................................140 a) D-Tagatose ..............................................................................................................................140 b) D-Psicose ................................................................................................................................141 1.3.2. O-Benzylation .......................................................................................................................142 1.3.3. Isopropylidene selective cleavage .................................................................................... 1.3.4. OZT formation ..................................................................................................................... a) Two-step reaction -HSCN .................................................................................................. b) One-step reaction -TMSNCS ............................................................................................ 1.4. Synthesis of OZO ........................................................................................................................ 1.4.1. S-Alkylation ......................................................................................................................... 1.4.2. Oxidation .............................................................................................................................. 1.5. Conclusion ................................................................................................................................... CHAPTER VI -Biological Screening 1. GLUT5 POTENTIAL INHIBITORS ............................................................................................. 1.1. Role of D-fructose in human life .............................................................................................. a) Sugars in Life ............................................................................................................................. b) Diabetes ..................................................................................................................................... c) Sugars in human body. GLUTs. ............................................................................................. d) GLUT5........................................................................................................................................ 1.2. PhD project .................................................................................................................................. 1.2.1. Objectives ............................................................................................................................. 1.2.2. Previous works .................................................................................................................... 1.2.3. PhD prospects ...................................................................................................................... Biological activity of thionocarbamates .................................................................................. 2.2. Antimicrobical activity .............................................................................................................. 2.2.1. Methodology for susceptibility testing ............................................................................ 2.2.2. Antimicrobial activity results ............................................................................................ a) Fused OZTs ........................................................................................................................... b) Spiro OZTs and OZOs ......................................................................................................... c) D-Tagato OZTs and OZOs .................................................................................................. 2.3. Cytotoxicity measurements ...................................................................................................... 2.3.1. Methodology for measuring cytotoxicity ........................................................................ 2.3.2. Cytotoxicity results ............................................................................................................. 2.4. Conclusion ................................................................................................................................... GENERAL CONCLUSION ................................................................................................................. EXPERIMENTAL PART ...................................................................................................................... General procedures ........................................................................................................................... Compounds description ...................................................................................................................

Figure 1 2The 2 -

 12 Figure 1

  Figure 3

  Figure 4

  Figure 5

  Figure 6

  Figure 7

  Scheme 2

  Scheme 3

  It was reported in the middle of the 90s that submitting 2-C-(hydroxymethyl)-D-fructose to those conditions led to remarkable structural modifications. The treatment with this catalyst induces highly stereospecific carbon-skeleton rearrangements to give equilibrium mixtures containing 2-C-(hydroxymethyl)-D-ribose or D-hamamelose. Later, new improvements were brought and the same kind of reaction was applied in other ketose series giving analogous results (A) (B), with the sole exception of D-psicose (Scheme 5).32 Z. Hricovíniová-Bíliková, M. Hricovíni, M. Petrušová, A. S. Serianni, L. Petruš Carbohydr.Res. 1999, 319, 38.

  Figure 8

Figure 9

 9 Figure 9

  Scheme 9

  Scheme 10

Figure 10 52

 10 Figure 10

  sites, O-2 (pyrano and furano) and O-6 (furano) could be modified and addressed a visualization of important interactions with the protein. These interactions may occur because D-fructofuranose is a relatively symmetrical molecule and for that reason, the binding site can either arise in anomeric position or on the other side of the molecule. Hence D-fructopyranose appears to present to GLUT5 transporter by hydroxyl 3, 4, 5 recognition (Figure 11).

  Figure 11

  Figure 12

  Scheme 13

  Scheme 14

  62 A.

  Scheme 18

  Scheme 19

  Scheme 21

  Scheme 29

  Figure 16

  Scheme 33

  78 K. Bahrami, M. M. Khodaei, Y. Tirandaz Synthesis 2009, 3, 369 and references cited therein. 79 A. Corsaro, V. Pistara Tetrahedron 1998, 54, 15027. 80 D. Gueyrard PhD Thesis, Université d'Orléans 2000. 81 J. Girniene PhD Thesis, Université d'Orléans 2003.

  Scheme 34

  Scheme 37

OZOs 2

 2 and 48, respectively (Scheme 39). Despite of its high polarity OZO 2 could also be isolated in good yield.

  Scheme 40

Silva

  Scheme 41

Submitting

  Scheme 43

1. SYNTHESIS OF 4 , 5 -

 45 Scheme 45

  Scheme 46

  92 D.David, A. Thieffry J. Chem. Soc. Perkin Trans. I 1979, 1568. 93 Y. Tsuda, M. Hanajima, M. Naohisa, Y. Okund, K. Kanemitsu Chem. Pharm. Bull. 1989, 37, 2344. 94 X. Kong, T. B. Grindley J. Carbohydr. Chem. 1993, 12, 557.However in our case, when submitting diol 57 to the above conditions, no oxidation was been observed, the starting material being recovered unchanged (Scheme 49).

  Scheme 53

3 .

 3 Scheme 54

Finally, OH- 4

 4 Scheme 58

  Scheme 59

  by silica gel chromatography: a) amino group introduced via an azide precursor, b) isothiocyanate formation and c) OZT cyclisation -three reactions in a one-pot procedure.

  Scheme 61

  Scheme 63

1. 4 .

 4 Scheme 65

1. 5 . 1 .

 51 Scheme 69

  Scheme 71

  It was really difficult to purify the mixture obtained, so the deprotected mixture was directly peracetylated. The di-O-acetylated compound 113 was obtained with good yield after the two-step reaction. No trace of the acyclic form was observed. Zemplén de-O-acetylation was finally applied but only degradation was observed (Scheme 72).

  For a long time iodination has been being exhaustively studied and many conditions and reagents have been tested.There are mainly two different pathways to perform iodination: a) by a two-step reaction, where in general sulfonate esters e.g., mesylate, tosylate or triflate, are used, 112 as good leaving groups or b) by a one step reaction which means directly from the hydroxyl group (Scheme 73). In these case a phosphorus-based reagent 113 such as commonly PPh3. The iodine sources are various, like N-iodosuccinimide, 114 tetraiodomethane, 112a imidazole and I2 or tri-

  Scheme 75

  .1. b)). As far as we know, selective iodination of secondary polyhydroxylated unprotected structures is unprecedented. This is an important improvement over the original conditions developed by Garegg and Samuelsson.1.6.1. Diol derivativesInitially we focused our attention on two vicinal diol structures 55 and 106 in D-fructo and D-psico series (Scheme 77).

  Scheme 78

  Scheme 80

  Scheme 82

  Scheme 83

1 .

 1 SYNTHESIS OF SPIRO OZTs and OZOs IN 3,4,5-tri-O-BENZYLATED β-D-FRUCTO-AND β-D-PSICOPYRANO-DERIVATIVES In the last two chapters, the synthesis of fused thionocarbamates was described. Herein we report the preparation of spiro-OZTs and OZOs in D-fructo and D-psico series (Scheme 84).

  Figure19

  As it was argued by Zemplén, Wickstrom and Wold, the condensation of HSCN with ketoses leads to the formation of pyrano structures. In 1988, Grouiller et al. raised a controversy by defending the formation of a mixture of fused pyrano and furano OZTs (Figure 20).23e

Figure 20

 20 Figure 20

  61a

  Scheme 85 : The green arrows represent the formation of spiro-or fused-OZTs and the red illustrate the attack of OH-5 or OH-6 that could lead to the formation of furano-or pyrano-rings -acyclic structures might also be formed.

  Scheme 86

(

  Scheme 89

  Scheme 92

  Full protection of triols provided the tri-O-benzylated compounds 140 and 141 in excellent yields (Scheme 93). 118 N. Nakajima, M. Matsumoto, M. Kirihara, M. Hashimoto, T. Katoh, S. Terashima Tetrahedron 1996, 52, 1177.Previously mentioned triols 98 and 119 were prepared following the steps described in Chapter III 1

  The reaction took place in two stages: a) cleavage of isopropylidene group under acidic conditions b) condensation with HSCN followed by spontaneous cyclization to give spiro-OZTs 144 and 145. This two-step reaction is a simple process since intermediates 142 and 143 were not isolated and immediately engaged in the following transformation. The result was a non selective conversion leading to α:β mixtures of spiro-OZTs 144 and 145 and open-chain OXTs 146 and 147 (Scheme 94).

4 .

 4 Comparison of methodsIt can be noted that both methods are efficient for the synthesis of spiro-OZTs. Despite of slightly superior yields obtained with the HSCN condensation, the use of TMSNCS brings the advantages of a one-step procedure and better stereoselectivity in favor of the β-anomers.In either D-fructo or D-psico series, separation of the anomeric forms was challenging: only traces of both α isomers 148 and 149 could be isolated for characterization (Figure21). This separation was only possible through a very slow and meticulous silica gel column chromatography.

  Scheme 96

  Scheme 98

  Scheme 99

  OZTs IN 3-O-BENZYLATED β-D-FRUCTO-AND β-D-PSICOPYRANO AND -FURANO DERIVATIVES Using a 3,4,5-O-protection of the starting material allowed selective preparation of spiro-OZTs in pyrano form. Once that early studies developed in our laboratory have revealed that spiro-OZTs in furanose form bearing a benzyl group show improved biological properties, Erreur !

2 .

 2 Scheme 100

  Scheme 102

3 .

 3 Scheme 103

  Scheme 105

  Scheme 107

  Scheme 112

(

  Scheme 114

  Scheme 115

  Scheme 118

  Scheme 120

  Scheme 121

Figure 23 128

 23 Figure 23

Figure 24 132

 24 Figure 24

  Figure 25

Figure 31 143Figure 32 1, 3 -

 31323 Figure 31

  148

Figure 33 148

 33 Figure 33

4 , 5 -

 45 Fused OZTs (96, 106, 107 and 55) have shown more antimicrobial activity than 3,4-fused OZTs[START_REF]-N,1-O-carbonyl-β-D-psicopyranosylamine (161) Starting with 157 (0.5 g, 1.05 mmol) and using G.P. 2.2.3. the compound 161 was obtained after column chromatography on silica gel with EA as colorless oil[END_REF]. A further analysis show that cyclic 4,5-fused OZT with a protective group in position C-3 (106, 107 and 55) expressed an overall better activity results than the unprotected D-fructose 96. The presence of a benzyl group in place of a benzoyl group did not significantly affect the activity. These compounds (106 and 107) expressed interesting antifungal activity for Candida albicans and a slight bactericidal activity against Bacillus cereus and Bacillus subtilis. Comparing D-fructose (96, 106 and 107) and the only member of the cyclic D-psicose series (55), the latter showed a better antimicrobial activity. The fact that this compound exhibits a greater activity could be due either to a different configuration at the C-3 or to the localization of nitrogen atom. This compound 55 showed a large spectrum of activity with a stronger bacterial activity. Furthermore it expressed excellent antifungal activity against Pyricularia oryzae, which is considered in international listings as a bioterrorism agent.From comparing cyclic (2, 106, 107, 96 and 55) and acyclic (114 and 115) structures, it can be said that cyclic structures gave better results. Once again D-psicose series (115) showed superior activity as compared to D-fructose (114).

  ; 59.1 (C-6); 68.7 (C-3); 70.6 (C-1); 71.6 (C-5); 75.6 (C-4); 102.8 (C-2); 107.7, 110.2 (Cq-isop). J. Kang, G. J. Lim, S. K. Yoon, M. Y. Kim J. Org. Chem. 1995, 60, 564; G. D. K. Kumar, S. Baskaran J. Org. Chem. 2005, 70, 4520.Starting with 4 (1.5 g, 5.8 mmol) and using G.P. 2.3.1. a) (reaction time 3 days) the residue was chromatographied with PE/EA (8:2) to give 25(1.2 g, 4.6 mmol, 79%) as a colorless solid.

  3 (CH3); 60.2 (C-6); 70.1 (C-1); 76.0 (C-4); 78.1 (C-5); 104.3 (C-2); 110.8, 114.0 (Cq-isop); 197.1 (C-3). J. Kang, G. J. Lim, S. K. Yoon, M. Y. Kim J. Org. Chem. 1995, 60, 564; J.-C. Lee, S. W. Chang, C.-C. Liao, F.-C. Chi, C.-S. Chen, Y.-S. Wen, C.-C. Wang, S. S. Kulkareni, R. Puranik, Y.-H. Liu, S.-C. Hung Chem. Eur. J. 2004, 10, 399.

  , H-1b); 4.58-4.63 (m, 1H, H-5); 5.01 (d, 1H, J4-5=3.6Hz, H-4); 8.80 (s, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 15.1 (CH2-CH3); 25.6, 26.9 (CH3-isop); 58.7 (CH2-CH3); 62.5 (C-6); 64.1 (C-5); 71.2 (C-1); 83.0 (C-4); 91.5 (C-3); 103.2 (C-2); 113.7 (Cq-isop); 189.6 (C=S). HRMS: Calculated for C12H19NO6S [M+H] + 306.1019, found: 306.1011. IR: (neat) ν (cm -1 ): 3177 (OH); 2984 (CH); 1484, 1256, 1015 (N-CS-O).

  , H-1b); 4.33-4.44 (m, 1H, H-5); 4.62 (d, 1H, J4-5=3.3Hz, H-4); 5.47 (d, 1H, JOH-5=6.0Hz, OH-5); 7.08 (s, 1H, OH-3); 10.65 (s, 1H, NH). 13 C NMR (62.89MHz, DMSO) δ: 25.7, 26.7 (CH3); 62.0 (C-6); 62.1 (C-4); 70.5 (C-1); 87.3 (C-5); 88.0 (C-3); 103.2 (C-2); 112.1 (Cq-isop); 188.2 (C=S). HRMS: Calculated for C10H15NO6S [M+H] + 278.0707, found: 278.0698. IR: (neat) ν (cm -1 ): 3477 (OH); 3209 (NH); 2994 (CH), 1489, 1372, 1012 (N-CS-O).

  2 (C-5); 70.2 (C-1); 79.3 (C-4); 90.6 (C-3); 102.3 (C-2); 113.2 (Cq-isop); 169.4 (C=O); 188.7 (C=S). MS: (IS+) m/z: 348 (M+H) + ; 370 (M+Na) + . IR: (neat) ν (cm -1 ): 2987, 2942 (CH); 1752 (C=O); 1483, 1255, 1015 (N-CS-O).

1 H

 1 NMR (400MHz, CDCl3) δ: 1.22-1.26 (m, 6H, CH2-CH3, O-CO-CH3); 1.35 (s, 3H, CH3-isop); 1.51 (s, 3H, CH3-isop); 2.78 (s, 3H, N-CO-CH3); 3.42-3.50 (m, 1H, CH2-CH3a); 3.56-3.63 (m, 1H, CH2-CH3b);

  5 (C-6); 65.3 (C-5); 71.1 (C-1); 79.5 (C-4); 91.6 (C-3); 100.2 (C-2); 111.8 (Cq-isop); 156.0, 157.4 (C=O); 188.7 (C=S). MS: (IS+) m/z: 412 (M+Na) + . IR: (neat) ν (cm -1 ): 2985 (CH); 1754 (C=O); 1473, 1245, 1023 (N-CS-O).

  Calculated for C15H19NO10S [M+Na] + 428.0627, found: 428.0643. IR: (neat) ν (cm -1 ): 2928 (CH); 1741 (C=O); 1775, 1369, 1042 (N-CS-O).

13 C

 13 NMR (100MHz, CDCl3) δ: 20.7, 20.8, 21.1, 21.7 (O-CO-CH3); 27.0 (N-CO-CH3); 60.4 (C-1); 62.1 (C-6); 80.5 (C-5); 84.0 (C-4); 102.8 (C-2); 108.6 (C-3); 167.0, 168.6, 169.9, 170.2, 170.3 (C=O); 185.3 (C=S). HRMS: Calculated for C17H21NO11S [M+Na] + 470.0733, found: 470.0751. IR: (neat) ν (cm -1 ): 2885 (CH); 1722 (C=O); 1453, 1370, 1057 (N-CS-O).

13 C

 13 NMR (100MHz, CDCl3) δ: 25.3, 25.4 (CH3); 34.5 (C-7); 62.4 (C-6); 64.1 (C-4); 69.5 (C-1); 76.4 (C-5); 81.6 (C-3); 106.6 (C-2); 111.5 (Cq-isop); 126.4, 127.6, 128.0 (CH-Ph); 136.1 (Cq-Ph); 160.9 (C-SBn). HRMS: Calculated: 368.1168; Found: 368.1155. IR: (neat) ν (cm -1 ): 3394 (OH); 2988, 2928 (CH), 1618, 1064, 699 (N-C=S-O); 1495, 1454 (Ph).

13 C

 13 NMR (100MHz, CDCl3) δ: 21.1 (O-CO-CH3); 26.3, 26.4 (CH3-isop); 35.4 (C-7); 65.0 (C-6); 65.5 (C-4); 70.5 (C-1); 76.0 (C-5); 81.3 (C-3); 108.0 (C-2); 112.6 (Cq-isop); 127.4, 128.5, 129.0 (CH-Ph); 137.2 (Cq-Ph); 160.8 (C-SBn); 171.0 (C=O). HRMS: Calculated for C19H23NO7S [M+H] + 410.1273, found: 410.1268. IR: (neat) ν (cm -1 ): 3453 (OH); 2990, 2928 (CH); 1745 (C=O); 1621, 1063, 987 (N=CS-O); 1496

13 C

 13 NMR (100MHz, CDCl3) δ: 21.1, 22.8 (O-CO-CH3); 25.7, 29.8 (CH3-isop); 35.6 (C-7); 64.4 (C-6); 65.1 (C-4); 71.2 (C-1); 75.6 (C-5); 88.3 (C-3); 108.0 (C-2); 112.7 (Cq-isop); 127.5, 128.6, 129.0 (CH-Ph); 137.2 (Cq-Ph); 167.9 (C-S); 170.2 (C=O). HRMS: Calculated for C21H25NO8S [M+H] + 452.1379, found: 452.1394. IR: (neat) ν (cm -1 ): 2991, 2927 (CH); 1749 (C=O); 1627, 1080, 698 (N=C-S-O); 1495, 1454 (Ph); 876, 845 (CH3).

1 H

 1 NMR (400MHz, CDCl3) δ: 1.40 (s, 3H, CH3-isop); 1.42 (s, 3H, CH3-isop); 2.07 (s, 3H, O-CO-CH3); 2.11 (s, 3H, O-CO-CH3);3.83-3.88 (m, 1H, H-6a); 3.94-4.02 (m, 2H, H-1a, H-4); 4.27 (d, 1H, J1a-1b=9.2Hz,H-1b); 4.42 (d, 1H, J6b-6a=10.0Hz, H-6b); 4.55-4.60 (m, 1H, H-5), 7.41 (brs, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 21.0, 21.3 (O-CO-CH3); 26.0, 26.3 (isop-CH3); 65.1 (C-6); 70.2 (C-4); 74.6 (C-1); 80.4 (C-5); 86.4 (C-3); 107.6 (C-2); 113.5 (Cqisop); 170.2, 171.1 (O-C=O-CH3); 178.0 (C=O). IR: (neat) ν (cm -1 ): 3255 (OH); 2933, 2842 (CH); 1685 (C=O).

  , H-1b); 4.59 (m, 1H, H-5); 7.25 (brs, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 25.1, 25.2 (CH3); 62.0 (C-6); 63.4 (C-4); 69.1 (C-1); 76.5 (C-5); 99.1 (C-3); 106.3 (C-2); 112.2 (Cq-isop); 168.4 (C=O). MS: (IS+) m/z: 279 (M+NH4) + ; 284 (M+Na) + . IR: (neat) ν (cm -1 ): 3268 (OH); 2923, 2853 (CH); 1686 (C=O).

  , H-1b); 4.56-4.58 (m, 1H, H-5); 7.26-7.41 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 26.4, 27.1 (isop-CH3); 36.9 (C-7); 61.4 (C-5); 67.6 (C-6); 72.7 (C-1); 101.4 (C-2); 112.4 (Cq-isop); 128.0, 128.8, 129.3 (CH-Ph); 135.5 (C-3); 136.2 (Cq-Ph); 148.8 (C-4); 161.9 (C-SBn). MS: (IS+) m/z: 372 (M+Na) + . IR: (neat) ν (cm -1 ): 3329 (OH); 2988, 2932 (CH); 1062, 698 (N=CS-O); 1479, 1454 (Ph).

  , H-1b); 7.23-7.40 (m, 5H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 21.6 (O-CO-CH3); 25.3, 26.0 (CH3-isop); 30.1 (C-7); 62.2 (C-5); 64.6 (C-6); 72.9 (C-1); 105.2 (C-2); 112.6 (Cq-isop); 128.7, 129.2, 129.3 (CH-Ph); 133.7 (C-3); 135.4 (Cq-Ph); 143.6 (C-4); 162.7 (C-S); 177.0 (C=O). IR: (neat) ν (cm -1 ): 3330 (OH); 2987, 2933 (CH); 1062, 699 (N=CS-O); 1489, 1454 (Ph).

  0 (CH3); 60.6 (C-6); 70.7 (C-1); 71.8 (C-5); 73.9 (C-3); 75.2 (C-4); 104.0 (C-2); 109.8 (Cq-isop); 112.3 (Cqisop); 128.6, 129.6, 130.0 (CH-Ph); 133.5 (Cq-Ph); 166.1 (C=O).

F

  . W.Lichtenthaler, W. Doleschal, S. Hahn Liebigs Ann. Chem. 1985, 2454; H. Tian, S. She, Y. Shi Org. Lett. 2001, 35, 715.

13 C

 13 NMR (100MHz, CDCl3) δ: 25.5, 25.6 (CH3); 63.1 (C-6); 68.8 (C-5); 69.4 (C-4); 70.3 (C-3); 71.1 (C-1); 103.6 (C-2); 111.3 (Cq-isop); 127.7 (m-Ph); 128.4 (Cq-Ph); 129.1 (o-Ph); 132.8 (p-Ph); 166.7 (C=O).

F

  . W.Lichtenthaler, W. Doleschal, S. Hahn Liebigs Ann. Chem. 1985, 2454; H. Tian, S. She, Y. Shi Org. Lett. 2001, 35, 715.

13 C

 13 NMR (100MHz, CDCl3) δ: 26.4, 26.7 (CH3); 64.2 (C-6); 67.3 (C-3); 68.4 (C-5); 72.1 (C-1); 72.9 (C-4); 105.0 (C-2); 112.5 (Cq-isop); 128.6 (CH-mPh); 129.2 (Cq-Ph); 129.9 (CH-oPh); 133.5 (CH-pPh); 165.8, 166.2 (C=O).A. Tatibouët, M. Lefoix, J. Nadolny, O. R. Martin, P. Rollin, J.Yang, G. D. Holman Carbohydr. Res. 2001, 333, 327.

  , H-3); 5.96 (dd, 1H, J4-5=10.1Hz, H-4); 7.31-7.36 (m, 4H, m-Ph); 7.45-7.50 (m, 2H, p-Ph); 7.91 (d, 2H, o-Ph); 7.97 (d, 2H, o-Ph). 13 C NMR (100MHz, CDCl3) δ: 26.1, 26.7 (CH3); 22.0 (C-5); 65.9 (C-6); 71.0 (C-3); 71.8 (C-1); 74.3 (C-4); 104.6 (C-2); 112.8 (Cq-isop); 128.6 (m-Ph); 129.2 (Cq-Ph); 129.9 (o-Ph); 133.5 (p-Ph); 165.8, 166.2 (C=O).A. Tatibouët, M. Lefoix, J. Nadolny, O. R. Martin, P. Rollin, J.Yang, G. D. Holman Carbohydr. Res. 2001, 333, 327.

13 C

 13 NMR (100MHz, CDCl3) δ: 26.3, 26.5 (CH3); 61.9 (C-5); 62.1 (C-6); 69.4 (C-3); 72.0 (C-1); 72.1 (C-4); 105.7 (C-2); 112.3 (Cq-isop).A. Tatibouët, M. Lefoix, J. Nadolny, O. R. Martin, P. Rollin, J.Yang, G. D. Holman Carbohydr. Res. 2001, 333, 327.

  C NMR (100MHz, CDCl3) δ: 26.1, 26.5 (CH3); 55.2 (C-5); 60.3 (C-6); 68.3 (C-3); 73.1 (C-1); 82.0 (C-4); 103.3 (C-2); 112.3 (Cq-isop); 175.1 (C=S). HRMS: Calculated for C10H15NO5S [M+H] + 262.0749, found: 262.0743. IR: (neat) ν (cm -1 ): 3438 (OH); 3265 (NH); 2987 (CH); 1492, 1226, 1060 (N-CS-O).

  , H1-b); 4.56 (d, 1H, OH-5); 4.64 (d, 1H, OH-3); 4.76 (d, 1H, OH-4). 13 C NMR (100MHz, DMSO-d6) δ: 26.2, 27.0 (CH3); 64.5 (C-6); 67.4 (C-4); 68.9 (C-5); 70.3 (C-1); 71.1 (C-3); 106.4 (C-2); 110.6 (Cq-isop). MS: (IS+) m/z: 243 (M+Na) + IR: (neat) ν (cm -1 ): 3344 (OH); 2988, 2938, 2897 (CH). E. Kaji, E. Kurimoto, R. Saiga, A. Matsuura, K. Harada, T. Nishino Heterocycles 2005, 66, 453.

13 C

 13 NMR (100MHz, CDCl3) δ: 26.3, 26.8 (CH3); 29.3 (C-5); 65.8 (C-6); 72.0 (C-1); 72.3 (C-3); 74.5 (C-4); 104.8 (C-2); 112.7 (Cq-isop); 128.7 (CH-mPh); 129.2 (CH-oPh); 130.1 (CH-pPh); 133.8 (Cq-Ph); 166.4 (C=O). HRMS: Calculated for C16H19IO6 [M+Na] + 457.0124, found: 457.0133. IR: (neat) ν (cm -1 ): 3537 (OH); 2993, 2944 (CH); 1708 (C=O); 1600, 1454 (Ph).

HRMS:

  Calculated for C16H21N3O5 [M+Na] + 358.1379, found: 358.1377. IR: (neat) ν (cm -1 ): 3541 (OH); 2993, 2909 (CH); 2119 (N3); 1457, 1371 (Ph).

13 C

 13 NMR (100MHz, CDCl3) δ: 26.0, 26.7 (CH3); 56.3 (C-5); 58.9 (C-6); 72.1 (C-1); 73.8 (C-7); 74.1 (C-3); 83.8 (C-4); 103.5 (C-2); 112.7 (Cq-isop); 128.1, 128.3, 128.5 (CH-Ph); 137.1 (Cq-Ph); 189.6 (C=S). MS: (IS+) m/z: 374 (M+Na) + IR: (neat) ν (cm -1 ): 2989, 2934 (CH); 1497, 1312, 1072 (N-CS-O); 1454 (Ph).

1 H

 1 NMR (250MHz, CDCl3) δ: 1.38 (s, 3H, CH3);1.49 (s, 3H, CH3); 4H, H-1, H-6);5.15 (t, 1H,, H-4);5.26-5.35 (m, 1H, H-5); 5.52(d, 1H,, H-3); 7.34-7.66 (m, 3H, Ph); 7.97-8.09 (m, 2H, Ph); 8.23 (brs, 1H, NH). 13 C NMR (62.89MHz, CDCl3) δ: 26.1, 26.4 (CH3); 55.8 (C-5); 59.2 (C-6); 68.8 (C-3); 72.3 (C-1); 80.6 (C-4); 105.3 (C-2); 112.6 (Cq-isop); 128.7, 130.0, 130.1, 130.6 (CH-Ph); 133.8 (Cq-Ph); 168.3 (C=O); 189.9(C=S). MS: (IS+) m/z: 388 (M+Na) + IR: (neat) ν (cm -1 ): 2989 (CH); 1722 (C=O); 1506, 1316, 1067 (N-CS-O); 1453 (Ph).

  Calculated for C16H21IO5 [M+Na] + 443.0331, found: 443.0334. IR: (neat) ν (cm -1 ): 3543, 3449 (OH); 2987, 2921 (CH); 1497, 1453, 1407 (Ph).

  , H-4); 5.15 (t, 1H, JOH-6=5.6Hz, OH-6); 5.21 (t, 1H, JOH-1=5.6Hz, OH-1); 7.29-7.40 (m, 5H, Ph); 10.07 (s, 1H, NH). 13 C NMR (100MHz, DMSO) δ: 58.4 (C-5); 61.2 (C-6); 66.8 (C-1); 73.1 (C-7); 81.7 (C-3); 82.4 (C-4); 127.8, 127.9, 128.3 (CH-Ph); 137.4 (Cq-Ph); 187.4 (C=O); 208.0 (C=S). MS: (IS+) m/z: 334 (M+Na) + IR: (neat) ν (cm -1 ): 3307 (OH); 2918, 2850 (CH); 1716 (C=O); 1461, 1376, 1048 (N-CS-O).

  Starting with 55 (0.5 g, 1.42 mmol) and using G.P. 2.2.4. a) (reaction time 15h) the compound 112 was obtained and immediately used for the next reaction without further purification.

  , H-1b); 4.41-4.57 (m, 3H, H-3, H-7); 4.70 (1H, dd, J4-5=4.0Hz, J4-3=5.5Hz, H-4); 7.16-7.28 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 25.0 (N-CO-CH3), 26.1, 26.5 (O-CO-CH3); 68.0 (C-6); 69.3 (C-1); 73.3 (C-7); 78.4 (C-5); 80.2 (C-4); 85.2 (C-3); 100.0 (C-2); 112.6 (Cq-isop); 127.6, 127.7, 128.3 (CH-Ph); 138.3 (Cq-Ph); 189.2 (C=S). MS: (IS+) m/z: 460 (M+Na) + IR: (neat) ν (cm -1 ): 2927 (CH); 1740 (C=O); 1776, 1370, 1041 (N-CS-O).

  C NMR (62.89MHz, CDCl3) δ: 26.1, 27.4 (CH3); 61.7 (C-6); 70.4 (C-3); 71.2 (C-7); 72.1 (C-1); 102.5 (C-2); 112.1 (Cq-isop); 123.8, 127.7 (C-4), (C-5); 127.9, 128.0, 128.5 (CH-Ph); 138.1 (Cq-Ph). I. Izquierdo, M. T. P. López-Espinosa, Anales de Química 1990, 86 (5), 554.

1 H

 1 NMR (250MHz, DMSO-d6) δ: 1.43 (s, 3H, CH3-isop); 1.49 (s, 3H, CH3isop); 2.06 (s, 3H, O-CO-CH3); 2.08 (s, 3H, O-CO-CH3);3.84 (d, 1H,, H-1a); 3.89-4.04 (m, 3H, H1-b, H5, H-6a); 4.12(d, 1H,, H6-b); 4.97 (d, 1H, J3-4=9.7Hz H-3); 5.48 (d, 1H, J4-3=9.7Hz H-4). 13 C NMR (62.89MHz, DMSO-d6) δ: 20.8, 21.0 (O-CO-CH3); 22.3 (C-5); 26.1, 26.2 (CH3-isop); 65.8 (C-6); 70.7 (C-3); 71.9 (C-1); 74.0 (C-4); 104.2 (C-2); 112.9 (Cq-isop); 169.5, 130.3 (C=O). HRMS: Calculated for C13H19IO7 [M+Na] + 437.0073, found: 437.0063. IR: (neat) ν (cm -1 ): 2985 (CH); 1741 (C=O); 906 (CH3).

  (100MHz, CDCl3) δ: 26.5, 26.8 (CH3); 29.7 (C-5); 65.9 (C-6); 68.3 (C-1); 73.4 (C-3); 73.6 (C-4); 96.0 (C-2); 112.6 (Cq-isop). MS: (IS+) m/z: 413 (M+CH3COOH+Na) + IR: (neat) ν (cm -1 ): 3556 (OH); 3377, 3243, 3129 (CH).

1 H

 1 NMR (400MHz, CDCl3) δ: 0.08 (s, 6H, Si(CH3)2); 0.89 (s, 9H, C(CH3)3);3.29- 3.35 (m, 2H, H-2, H-5);3.44 (t, 1H,, H-4); 3.51-3.54 (m, 4H, OMe, H-3); 3.82 (dd, 1H, J6a-5=5.4Hz, J6a-6b=10.8Hz, H-6a); 3.89 (dd, 1H, J6b-5=4.0Hz, J6b-6a=10.8Hz, H-6b); 4.18 (d, 1H, J1-2=7.6Hz, H-1); 4.22 (brs, 1H, OH-3, OH-4); 4.78 (brs, 1H, OH-2). 13 C NMR (100MHz, CDCl3) δ: -5.3 (Si(CH3)2); 18.4 (Cq-C(CH3)3); 26.0 (C(CH3)3); 57.0 (OMe); 64.1 (C-6); 71.8 (C-2); 73.5 (C-4); 75.4 (C-3); 76.6 (C-5); 103.5 (C-1).

1 H

 1 NMR (400MHz, MeOD) δ: 0.10 (s, 6H, Si(CH3)2); 0.92 (s, 9H, C(CH3)3); 3.39 (s, 3H, OMe);3.69 (dd, 1H,7Hz H-3); 3.74-3.83 (m, 4H, H-2, H-5, H-6a, H-6b); 3.88 (d, 1H, J4-3=3.3Hz, H-4); 4.68 (d, 1H, J1-2=3.9Hz, H-1). 13 C NMR (100MHz, MeOD) δ: -5.2 (Si(CH3)2); 19.2 (Cq-C(CH3)3); 26.4 (C(CH3)3); 55.6 (OMe); 63.9 (C-6); 70.3 (C-2); 70.8 (C-4); 71.7 (C-3); 72.4 (C-5); 101.5 (C-1).

1 H

 1 NMR (400MHz, CDCl3) δ: 0.09 (s, 3H, Si(CH3)2); 0.10 (s, 3H, Si(CH3)2); 0.92 (s, 9H, C(CH3)3); 2.06 (s, 3H, O-CO-CH3); 2.10 (s, 3H, O-CO-CH3); 3.39 (s, 3H, OMe); 3.95-4.04 (m, 4H, H-4, H-5, H-6a, H-6b); 4.78 (dd, 1H, J2-1=3.6Hz, J2-3=10.0Hz, H-2); 4.95 (d, 1H, J1-2=3.6Hz, H-1); 5.61 (t, 1H, J3-2=J3-4=10.0Hz, H-3). 13 C NMR (100MHz, CDCl3) δ: -5.2, -5.0 (Si(CH3)2); 18.5 (Cq-C(CH3)3); 20.9, 21.1 (O-CO-CH3); 25.6 (C-4); 26.1 (C(CH3)3); 55.5 (OMe); 64.3 (C-6); 71.9 (C-2); 72.6 (C-3); 72.9 (C-5); 97.3 (C-1); 169.6, 170.3 (C=O). MS: (IS+) m/z: 469 (M+K) + . IR: (neat) ν (cm -1 ): 2932 (CH); 1755 (C=O); 1218 [Si(CH3)2].

  , H-1b); 4.57-4.78 (m, 5H, CH2Ph); 5.04 (d, 1H, J=11.6Hz, CH2Ph); 7.27-7.41 (m, 15H, Ph). 13 C NMR (100MHz, CDCl3) δ: 26.4, 27.3 (CH3); 61.5 (C-6); 71.5, 71.9 (CH2Ph); 72.2 (C-1); 73.5 (C-5); 75.1 (C-4); 75.5 (CH2Ph); 80.2 (C-3); 106.0 (C-2); 112.0 (Cq-isop); 127.

1 H

 1 NMR (400MHz, CDCl3) δ: 3.47-3.52 (m, 2H, H-3, H-5); 3.73-3.78 (m, 2H, H-6); 4.05 (s, 1H, CH-4); 4.42-4.68 (m, 6H, H-1 , CH2Ph); 4.77 (d, 1H, J=11.4Hz, CH2Ph); 4.85 (d, 1H, J=11.4Hz, CH2Ph); 7.26-7.35 (m, 15H, Ph); 8.80 (brs, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 60.5 (C-6); 71.5 (C-1); 72.6 (CH2Ph); 74.2 (C-4); 74.5 (C-5); 74.6 (CH2Ph), (CH2Ph); 75.7 (C-3); 92.6 (C-2); 127.

  MS: (IS+) m/z: 514 (M+Na) + IR: (neat) ν (cm -1 ): 2886 (CH); 1494, 1321, 1046 (N-CS-O); 1454 (Ph).

1 H

 1 NMR (400MHz, CDCl3) δ: 3.52-3.53 (m, 1H, H-5); 3.70-3.75 (m, 1H, H-6a); 3.77-3.81 (m, 1H, H-4); 3.86-3.88 (m, 1H, H-6b); 4.27 (d, 1H, J=11.6Hz, CH2Ph); 4.41-4.62 (m, 5H, H-3, CH2Ph); 4.79 (d, 1H, J=12.0Hz, CH2Ph); 6.40 (s, 1H, H-1); 7.22-7.36 (m, 15H, Ph); 9.89 (brs, 1H, NH). 13 C NMR (100MHz, CDCl3) δ: 60.2 (C-6); 71.1 (C-3); 71.7, 72.7, 72.9 (CH2Ph); 77.1 (C-5); 79.1 (C-4); 110.6 (C-2); 127.

  Calculated for C35H35NO5S [M+H] + 582.2314, found: 582.2308. IR: (neat) ν (cm -1 ): 3030, 2884 (CH); 1589, 1090, 696 (N-CS-O); 1495, 1453 (Ph).

  Calculated for C35H35NO5S [M+H] + 582.2314, found: 582.2308. IR: (neat) ν (cm -1 ): 3030, 2921 (CH); 1600, 1091, 697 (N=CS-O); 1495, 1453 (Ph).

13 C

 13 NMR (62.89MHz, CDCl3) δ: 60.5 (C-6); 71.6 (C-5); 72.0 (CH2Ph); 72.4 (C-4); 73.8 (C-1); 74.0, 74.2 (CH2Ph); 77.8 (C-3); 92.1 (C-2); 127.

13 C

 13 NMR (62.89MHz, MeOD) δ: 63.0 (C-6); 72.8 (C-1); 74.1 (C-5); 77.0, 80.2 (C-3), (C-4); 101.3 (C-2); 163.0 (C=O).

  Calculated for C24H27NO5S [M+H] + 442.1688, found: 442.1678. IR: (neat) ν (cm -1 ): 3030, 2886 (CH); 1694, 1093, 697 (N-CS-O); 1495, 1454 (Ph).

1 H

 1 NMR (400MHz, CDCl3) δ: 1.35 (s, 6H, CH3); 1.49 (s, 3H, CH3); 1.68 (brs, 1H, OH); 1.94 (brs, 1H, OH);3.28 (dd, 1H,, H-6a); 3.70-3.81 (m, 3H, H-1, H-6b); 4.15 (dd, 1H, J5,6a=1.6Hz, J5-4=7.6Hz, H-5); 4.26 (s, 2H, CH2Ph); 4.61 (d, 1H, J3-4=2.0Hz, H-3); 4.66 (dd, 1H, J4-3=2.0Hz, J4-5=7.6Hz, H-4); 7-26-7.40 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 24.3, 26.1 (CH3); 36.5 (CH2Ph); 61.6 (C-6); 66.6 (C-1); 69.7 (C-4); 70.3 (C-5); 75.5 (C-3); 99.3 (C-2); 109.6 (Cq-isop); 128.0, 128.8, 129.2 (CH-Ph); 136.3 (Cq-Ph); 170.1 (C=S). MS: (IS+) m/z: 392 (M+Na) + IR: (neat) ν (cm -1 ): 3342 (OH); 3029, 2943 (CH); 1495, 1314, 1065, (N-CS-O); 1453 (Ph).

13 C

 13 NMR (100MHz, CDCl3) δ: 25.9, 27.2 (CH3); 49.9 (C-5); 52.2 (C-4); 59.1 (C-6); 70.4 (C-3); 72.0 (C-1), (CH2Ph); 101.0 (C-2); 112.6 (Cq-isop); 128.2, 128.3, 128.7 (CH-Ph); 137.1 (C-8). MS: (IS+) m/z: 315 (M+Na) +

  ; 4.92 (d, 1H, J=11.6Hz, CH2Ph); 7.24-7.35 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 26.2, 27.2 (CH3); 58.6 (OCH3); 60.5 (C-6); 71.8 (C-1); 75.0 (C-4); 75.1 (CH2Ph); 78.0 (C-3); 79.8 (C-5); 105.0 (C-2); 112.3 (Cqisop); 127.9, 128.0, 128.6 (CH-Ph); 138.2 (Cq-Ph).

13 C

 13 NMR (100MHz, CDCl3) δ: 21.3 (O-CO-CH3); 26.1. 27.1 (CH3); 58.5 (OCH3); 60.8 (C-6); 71.7 (C-1); 74.7 (C-7); 75.0 (C-4); 76.9 (C-3); 78.2 (C-5); 105.1 (C-2); 112.6 (Cq-isop); 127.7, 127.9, 128.6 (CH-Ph); 137.9 (Cq-Ph); 170.0 (C=O). MS: (IS+) m/z: 389 (M+Na) + . IR: (neat) ν (cm -1 ): 2987, 2934, 2890 (CH); 1742 (C=O).

  , H-1b); 4.67-4,70 (m, 2H, H-3, OH-6); 4.75 (d, 1 H, JOH-4=8.8Hz, OH-4); 4.90 (d, 1 H, JOH-5=5.6Hz, OH-5); 10.94 (s, 1H, NH); 11.00 (s, 1H, NH). 13 C NMR (100MHz, DMSO) δ: 61.3 (C-6); 66.5 (C-4); 69.8 (C-5); 75.9 (C-1); 84.4 (C-2); 82.5 (C-3); 186.7, 187.8 (C=S). HRMS: Calculated for C8H12N2O5S2 [M+Na] + 303.0085, found: 303.0092. IR: (neat) ν (cm -1 ): 3214 (OH); 2923 (CH); 1471, 1372, 1081 (N-CS-O).

1 H

 1 NMR (400MHz, DMSO) δ: 3.72-3.85 (m, 3H, H-5, H-6); 3.98-4.04 (m, 3H, H-1, H-4); 4.64 (s, 1H, H-3); 4.72 (s, 1H, OH); 5.18 (d, 1H, OH); 5.36 (d, 1H, OH); 10.96 (s, 1H, NH); 11.00 (s, 1H, NH). MS: (IS+) m/z: 303 (M+Na) + . IR: (neat) ν (cm -1 ): 3225 (OH); 2943 (CH); 1475, 1354, 1071 (N-CS-O).

  Calculated for C25H53NO6Si3 [M+H] + 548.3259, found: 548.3261. IR: (neat) ν (cm -1 ): 2929, 2886 (CH); 1773, 1739 (C=O); 1388 (C(CH3)3); 125 (Si(CH3)2); 678 (Si-C).

  0Hz, H-1b);3.73-3.77 (m, 1H, H-4);3.79-3.83 (m, 1H, H-5);3.89 (dd, 1H, J6b-5=5.2Hz, J6b- 6a=12.8Hz, H-6b); 4.89(d, 1H,, H-3); 7.23 (brs, 1H, NH).13 C NMR (100MHz, CDCl3) δ: -5.3, -5.2, -4.5, -4.4 (Si(CH3)2); 18.1, 18.3 (t-CH3); 25.8, 25.9 (C(CH3)3); 64.3 (C-1); 67.9 (C-6); 70.4 (C-5); 71.8 (C-4); 82.3 (C-3); 93.0 (C-2); 189.6 (C=S). HRMS: Calculated for C19H39NO5Si2 [M+H] + 450.2166, found: 450.2170. IR: (neat) ν (cm -1 ): 2930, 2857 (CH); 1463, 1390 (N-C=S-O); 1360 (C(CH3)3

  Calculated for C19H39NO5SSi2 [M+H] + 450.2166, found: 450.2179. IR: (neat) ν (cm -1 ): 3357 (OH); 2929, 2856 (CH); 1462, 1223, 1051 (N-C=S-O); 1361 (C(CH3)3); 1256 (Si(CH3)2); 669 (Si-C).

1 H

 1 NMR (400MHz, CDCl3) δ: 1.30 (s, 3H, CH3); 1.39 (s, 3H, CH3); 1.42 (s, 3H, CH3); 1.46 (s, 3H, CH3); 2.38 (brs, 1H, OH-6); 3.85-3.94 (m, 2H, H-6); 4.04-4.08 (m, 1H, H-5); 4.06

  13 C NMR (100MHz, CDCl3) δ: 24.8, 26.0, 26.5 (CH3); 61.1 (C-6); 69.3 (C-1); 79.0 (C-5); 80.5 (C-4); 85.5 (C-3); 111.8, 111.9 (Cq-isop); 113.0 (C-2). Y. Bechor, A. Albeck Tetrahedron 2008, 64, 2080.

1 H

 1 NMR (400MHz, CDCl3) δ: 1.36 (s, 3H, CH3); 1.40 (s, 3H, CH3); 1.46 (s, 3H, CH3); 1.53 (s, 3H, CH3); 3.28 (d, 1H, JOH-6=9.0Hz, OH-6); 3.68-3.83 (m, 2H, H-6); 3.95-4.03 (m, 1H, H-3); 4.01 (d, 1H, J1a-1b=9.5Hz, H-1a); 4.12-4.16 (m, 1H, H-1b, H-5); 4.27-4.31 (m, 1H, H-4). 13 C NMR (100MHz, CDCl3) δ: 25.6, 26.8, 26.9, 27.5 (CH3); 64.5 (C-6); 67.8 (C-1); 73.4 (C-5); 76.5 (C-4); 76.6 (C-3); 102.8 (C-2), 110.2, 112.4 (Cq-isop). HRMS: Calculated for C12H20O6 [M+Na] + 283.1158, found: 283.1164. IR: (neat) ν (cm -1 ): 3511 (OH); 2988, 2951 (CH).

  0 (CH3); 63.3 (C-6); 69.5 (C-1); 86.2 (C-5); 85.3 (C-3); 81.4 (C-4); 111.2, 111.9 (Cq-isop); 113.1 (C-2).S.Mio, Y.Kumagawa, S. Sugai Tetrahedron 1991, 47, 2133.

  MS:(IS+) m/z: 373 (M+Na) + IR: (neat) ν (cm -1 ): 2985, 2937 (CH); 1455 (Ph).

  , H-1b); 4.27-4.31 (m, 1H, H-5); 4.53 (d, 1H, J=12.0Hz, CH2Ph); 4.58 (d, 1H, J=12.0Hz, CH2Ph); 4.59 (d, 1H, J3-4=5.8Hz, H-3); 4.75 (dd, 1H, J4-3=5.8Hz, H-4); 7.25-7.40 (m, 5H, Ph). 13 C NMR (100MHz, CDCl3) δ: 25.3, 26.5, 26.6, (CH3); 70.0 (C-1); 71.0 (C-6); 73.5 (CH2Ph); 82.7 (C-4); 84.0 (C-5); 85.3 (C

  , H-1b); 4.18-4.25 (m, 1H, H-5); 4.48-4.63 (m, 3H, H-3, CH2Ph); 4.74-4.78 (dd, 1H, J4-3=4.0Hz, J4-5=6.0Hz, H-4); 7.24-7.33 (m, 5H, Ph); 8.85 (brs, 1H, NH). IR: (neat) ν (cm -1 ): 2925 (CH); 1453 (Ph); 1088, 698 (N-CS-O).

1 H

 1 NMR (400MHz, CDCl3) δ: 1.33 (s, 3H, CH3);1.46 (s, 3H, CH3); 2H, 3H, H-1, H-5); 4. 1H, CH2Ph); 2H, H-3, CH2Ph); 4.81 (dd, 1H, J4-3=6.0Hz, J4-5=10.4Hz, H-4); 6.56 (brs, 1H, NH), 7.26-7.36 (m, 5H, Ph).13 C NMR (100MHz, CDCl3) δ: 25.3, 26.6 (CH3); 70.9 (C-6); 73.5 (CH2Ph); 74.0 (C-1); 83.6 (C-4); 84.1 (C-5); 85.7 (C-3); 109.7 (C-2); 112.8 (Cq-isop); 127.9, 128.5, 128.7, 191.4 (C=S). IR: (neat) ν (cm -1 ): 2926, 2855 (CH); 1592, 1045, 698 (N-CS-O); 1495, 1454 (Ph).

  1 (CH3); 60.0 (C-1); 68.0 (C-6); 73.5 (C-7); 78.3 (C-5); 80.5 (C-4); 83.4 (C-3); 104.3 (C-2); 113.1 (Cq-isop); 127.7, 127.9, 128.5 (CH-Ph); 138.3(Cq-Ph). MS: (IS+) m/z: 310 (M+NH4) + ; 315 (M+Na) + IR: (neat) ν (cm -1 ): 2936 (CH); 1454 (Ph).

13 C

 13 NMR (62.89MHz, CDCl3) δ: 26.2, 25.1 (CH3); 36.4 (CH2Ph); 68.2 (C-6); 73.2 (C-1); 73.6 (CH2Ph); 72.7 (C-5); 81.2 (C-4); 85.3 (C-3); 108.3 (C-2); 112.7 (Cqisop); 127.8, 128.0, 128.5, 128.7, 129.2 (CH-Ph); 136.9, 138.2 (Cq-Ph). MS: (IS+) m/z: 423 (M+Na) + . IR: (neat) ν (cm -1 ): 2932 (CH); 1495, 1454 (Ph).

  (m, 1H, H-5);4.53-4.59 (m, 3H, H-1b, H-3, CH2Ph); 4.67(d, 1H, J=7.8Hz, CH2Ph); 4.89-4.91 (m, 1H, H-4); 7.26-7.37 (m, 10H, Ph). 13 C NMR (62.89MHz, CDCl3) δ: 25.1, 26.2 (CH3); 36.4 (SCH2Ph); 68.2 (C-6); 73.2 (C-1); 73.6 (C-7); 78.7 (C-5); 81.2 (C-4); 85.5 (C-3); 108.4 (C-2); 112.7 (Cqisop); 127.8, 128.0, 128.5, 128.7, 129.2 (CH-Ph); 138.2 (Cq-Ph); 163.5 (Cq-SBn). MS: (IS+) m/z: 442 (M+H) + ; 464 (M+Na) + IR: (neat) ν (cm -1 ): 2921, 2851 (CH); 1592, 1049, 698 (N-CS-O); 1496, 1454 (Ph).

  Patty e Ugo. Pela sua amizade, amor, paciência, atenção, carinho e dedicação… "The productive scientist must be a traditionalist who enjoys playing intricate games by preestablished rules in order to be a successful innovator who discovers new rules and new pieces with which to play them." Oxidation ................................................................................................................................ 1.5. Synthesis of 3,4-fused OXTs ....................................................................................................... 1.5.1. Previous studies .................................................................................................................... 1.5.2. S-Alkylation. A surprising result. ....................................................................................... 1.5.3. OXT formation through OZT dehydratation .................................................................... 1.6.

	TFA ABBREVIATIONS LIST trifluoroacetic acid TBDMSCl tert-butyldimethylsilyl chloride 1.4.3.		U
	Tf2O Å TLC Ac THF Ac2O AcOH TsCl TMS	trifluoromethanesulfonic anhydride A thin layer chromatography Angstrom tetrahydrofuran acetyl acetic acid 4-toluenesulfonyl chloride acetic anhydride trimethylsilyl	UV Im2CS IR J υ	I ultraviolet diimidazolyl thione Infra red V coupling constant J wave number
	Bn BnBr BzCl Boc BPSE	B benzyl benzyl bromide benzoyl chloride tert-butoxycarbonyle 1,2-bis-(phenylsulfonyl)ethylene	m M m-CPBA Me MHz	M multiplet molar (g.dm -3 ) meta-chloroperbenzoic acid methyl Megahertz
	C Celsius degree(s) NMR carbon spectrum wave number CORREL NMR correlation 1 H-13 C ºC 13 C cm -1 COSY NMR correlation 1 H-1 H CSA camphosulfonic acid	min mL mmol MS MsCl MW m/z	minute(s) millilitre(s) milimol mass spectroscopy mesyl chloride molecular weight mass under charge
	δ	chemical shift in ppm		N
	d dd ddd dt	D doublet doublet doublet doublet doublet doublet doublet triplet	NBS NMR NOESY Nu	N-bromosuccinimide Nuclear Magnetic Resonance Nucleor Overhauser Effect Spectroscopy nucleophile
	DCC DCM DDQ DIEA	N-dicyclohexylcarbodiimide dichloromethane 2,3-dichloro-5,6-dicyanobenzoquinone diisopropylamine	OXT OZO OZT	O 1,3-oxazoline-2-thione 1,3-oxazolidin-2-one 1,3-oxazolidine-2-thione
	DMAP DMF DMP DMSO	4-(N,N-dimethylamino)pyridine dimethylformamide 2,2-dimethoxypropane dimethylsulfoxide	PCC PDC Pd/C	P pyridinium chlorochromate pyridinium dichromate palladium under carbon
	EA eq. Et	E ethyl acetate equivalent(s) ethyl	PE Ph ppm PTSA	petroleum ether phenyl parts per million para-toluene sulfonic acid
	g GLUT5	Thomas S. Kuhn in The Essential Tension (University of Chicago Press, 1977) R Rf retention factor protein transporter specific for G gram(s) rt room temperature
		fructose transport		S
		H	s	singulet
	h 1 H Hz	hour(s) NMR proton spectrum Hertz	t-Bu	T tert-butyl

  .118 1.2.1. Ketose protection .................................................................................................................118 1.2.2. OZT formation -HSCN.......................................................................................................119 1.2.3. OZT formation -TMSNCS ..................................................................................................120 1.2.4. Comparison of methods......................................................................................................122 1.3. Synthesis of spiro-OZOs .............................................................................................................123 1.3.1.

S-alkylation ...........................................................................................................................123 1.3.2. Oxidation ..............................................................................................................................124 1.3.3. Deprotection assays .............................................................................................................124 1.

  19 In United States a pharmaceutical company has already developed a new product with a view to including it in the treatment of intestine cancer taking into account its restorative properties on the intestinal flora. The non absorbed D- tagatose (nearly 75% of the total amount) acts as a prebiotic.19 But this is not the only

16 N. A. Jones, S. F. Jenkinson, R. Soengas, M. Fanefjord, M. R. Wormald, R. A. Dwek, G. P. Kiran, R. Devendar, G. Takata, K. Morimoto, K. Izumoric, G. W. J. Fleet Tetrahedron: Asymmetry 2007, 18, 774. 17 Agency Response Letter GRAS Notice No. GRN 000078. 18 T. W. Donner, J. F. Wilber, D. Ostrowski Diabetes, Obesity and Metabolism 1999, 1 (5), 285. 19 Y. Lu, G. V. Levin, T. W. Donner Diabetes, Obesity and Metabolism 2008, 10, 109.

1982, 38

	, 1163; (b) R. Masuda, M. Hojo, T. Ichi, S.
	Sasano, T. Kobayashi, C. Kuroda Tetrahedron Lett. 1991, 32, 1195; (c) M. Moreno-Mañas, I. Padros J. Heterocycl Chem.
	1993, 30, 1235; (d) A. Corsaro, V. Pistarà Tetrahedron 1998, 54, 15027.
	60 For a general survey, cf A. C. Simão, J. Rousseau, S. Silva, A. P. Rauter, A. Tatibouët , P. Rollin Thionocarbamates on
	carbohydrate scaffolds -from synthesis to bioactivity in Specialist Periodical Reports -Carbohydr. Chem. 2009, 35, 127.
	61 (a) J. M. Garcia Fernandez, C. Ortiz Mellet Sulfur Reports 1996, 19, 61; (b) J. M. Garcia Fernandez, C. Ortiz Mellet,
	Adv. Carbohydr. Chem. Biochem. 2000, 55, 35.

Brown, P. Kocienski Synlett 1994, 6, 415; (d)

M. T.

Crimmins, B. W. King, E. A. Tabet J. Am. Chem. Soc. 1997, 119, 7883;

(e) M. T. Crimmins, B.W. King, J. Am. Chem. Soc. 1998, 120, 9084; (f) M. T. Crimmins, E. A. Tabet J. Am. Chem. Soc. 2000, 122, 5473; (g) Y. C. Wang, D. W. Su, C. M. Lin, H. L. Tseng, C. L. Li, T. H. Yan Tetrahedron Lett. 1999, 40, 3577; (h) Y. Nagao, M. Yagi, T. Ikedo, E. Fujita Tetrahedron Lett. 1982, 23, 201; (i) Y. Nagao, T. Kumagai, S. Yamada, E. Fujita, Y. Inoue, Y. Nagase, T. Abe J. Chem. Soc. Perkin Trans 1 1985, 2361. 59 (a) K. A. Jorgesen, A. B. Ghattas, S. O. Lawesson Tetrahedron

  Tatibouët, M. Lefoix, J. Nadolny, O. R. Martin, P. Rollin, J.Yang, G. D. Holman Carbohydr. Res. 2001, 333, 327.

			S	O
	S		O	N	H N
	O	NH
	antifertility agent O F 3 C	N	herbicidal activity Cl

63 G. A. Youngdale, G. W. Duncan, D. E. Emmert, D. Lednicer J. Med. Chem. 1966, 9, 155.

spiro-bis-OZTs 19 was observed. Hence he decided to use substrates with only one α-hydroxyl and thereby only OXT structures 20 resulted (Scheme 21).

71 (a) G. Lacasse, J. M. Mucowki Can. J. Chem. 1972, 50, 3082; (b) R. Gompper, H. Herlinger Chem. Ber. 1956, 89, 1762.

Table 1

 1

	catalyst. Compound 4 was easily isolated after recrystallization with an acceptable yield
	according to literature 72 (Scheme 24).
			OH				O	O
	HO	O OH	OH OH	acetone, H 2 SO 4 rt, 1.5h	O O	O	OH 4
		D-fructose				58%
				Scheme 24
	Oxidation of secondary alcohols into ketones represents an important transformation in
	organic chemistry. The list of reagents able to carry out this conversion is considerable. Among
	72 (a) J. Kang, G. J. Lim, S. K. Yoon, M. Y. Kim J. Org. Chem. 1995, 60, 564; (b) N. Nieto, P. Molas, J. Benet-Buchholz, A.
	Vida-Ferran J. Org. Chem. 2005, 70, 10143.

  ).

					Table 1
		O	O		O	O
	O	OH	oxidizing agent	O
	O O			O O	O
		4			25

Table 2

 2

						SOLVENT	R	YIELD
	O OH O O HO	O 24	KSCN, HCl, solvent rt, 15h	O O NH O HO S	O OR R = Et, 26 H, 1	EtOH THF/DMF DMF	Et, 26 H, 1 H, 1	35% 31% 59%
			Scheme 27
	As expected from Dr. Silva's previous observations in aldose series, 78 we have obtained a
	hydrated-OXT. In position C-3 stands a hydroxyl or an ethoxy group, depending on the solvent
	used.
	How does it happen ?
	When performing the reaction in an aprotic solvent like DMF, the medium does not
	interferes in OZT formation. The reaction occurs under acidic conditions : the carbonyl group of
	24 can first be protonated to the oxonium ion 27 which further reacts with thiocyanic acid to
	form an isothiocyanate 28. This intermediate undergoes spontaneous attack of the OH-4 to
	produce OZT 1 (Scheme 28).

Table 3

 3

	R1	R2	R3	CONDITIONS	RESULTS
	H	H	H	AcOH/H 2 O (8:2), rt, 15 h	starting material
	H	H	H	AcOH/H 2 O (8:2), 50ºC, 15 h	degradation
	Et	H	H	TFA/H 2 O (9:1), rt, 15 h	traces
	Et	H	H	2M HCl, THF, rt, 15 h	degradation
	Et	Ac	H	2M HCl, THF, rt, 15 h	degradation

  89, 92 Oxidation takes place on the secondary hydroxyl group in good yield (Scheme 48).

	HO BnO	O OH OBn BnO 60	O BnO	OH O BnO OBn 61

  ). No signal in the NMR spectrum accounts for the presence of isothiocyanate.

	HO HO HO	O OH	NH 2	CSCl 2 buffered medium	HO HO HO	O O	NH S
		68				69

Table 6

 6

	D-FRUCTOSE	D-PSICOSE

When Garegg's conditions were applied, i.e., excess I2 in refluxing toluene, the olefin was obtained in reasonable yields. To avoid this product, our first modification was to decrease the temperature but no reaction (and no degradation) was detected. Consequently, it was decided to replace toluene by THF for solubility problems. But most importantly, the number of I2

Table 7

 7

	D-FRUCTOSE	D-PSICOSE

Table 8 STARTING MATERIAL PRODUCT

 8

D-glucose, 123 D-allose, 126 45% D-mannose, 124 D-mannose, no selectivity D-galactose, 125 D-glucose, 127 42%

Table 10

 10

	D-FRUCTOSE	D-PSICOSE

Table 12

 12

	R	CONDITIONS	RESULTS
	Ac	Ac 2 O, pyridine, rt, 15h	mixture of the acetylated products
			pyranose α=5% pyranose β=9%
		dimethoxypropane, CSA, acetone, rt, 15h	mixture pyranose α and β 24% furanose α and β not protected 18%
		TBDMSCl (3.0eq), imidazole,	mixture of products mono and di-silylated
		THF, rt, 15h	α and β in pyranose and furanose forms
	TBDMS
		TBDMSCl (1.0eq), imidazole, THF, rt, 15h	furanose α and β mono-silylated 10%

α and β in pyranose and furanose forms isopropylidene acetone, H 2 SO 4 , rt, 15h α-pyranose 163=6% β-pyranose 164=27% mixture pyranose α and β 11% furanose α and β not isolated

Table 13 REACTIFS

 13

	RESULTS

  1.1., reacting HSCN with an unprotected ketose can result in the formation of ten different OZTs.123 Surprisingly however, applying the standard conditions previously used in other ketohexose series, only acyclic bis-OZTs 175 and 176 were isolated in 61% overall yield and no other thionocarbamate was detected (Scheme 111). Fortunately, the two spiro-epimers could be isolated separately, but the configuration of the newly formed stereogenic carbon could not be established for lack of crystallinity of both epimers.

							S
	OH	OH		OH	OH	HN	O
	O D-tagatose OH OH HO	KSCN (2.0 eq.), HCl, H 2 O 55ºC, 15h	OH	O	176, 28% S 175, 33% NH

Table 14 PRODUCT CONDITIONS RESULTS D-tagatose

 14

Table 17 ,

 17 3 and Table19: they are expressed in terms of average diameter of inhibition (∅) in mm for the active compounds (300 μg). For comparison purposes, the control substances (chloramphenicol or actidione) are also shown for doses of 30 μg and 300 μg. Zones less than 10

149 (a) A. W. Bauer, W. M. Kirby, J. C. Sherris, M. Turck The American Journal of Clinical Pathology 1966, 45, 493; (b) CLSI, Performance standards for antimicrobial disk susceptibility tests 2006, 9 th edition, M2-A9, Clinical and Laboratory Standards Institute, USA; (c) F. V. M. Silva, M. Goulart, J. Justino, A. Neves, F. Santos, J. Caio, S. Lucas, A. Newton, D. Sacoto, E. Barbosa, M. S. Santos, A. P. Rauter Bioorg. Med. Chem. 2008, 16, 4083.

Table 17

 17

					FUNGI				BACTERIA
				Botrytis	Candida	Pyricularia	Rhizopus	Bacillus	Bacillus	Enterococcus	Staphylococcus
				spp.	albicans	oryzae	spp.	cereus	subtilis	faecalis	aureus
		O	O
	O	OH	-	-	-	-	8-10	8-10	8-10	8-10
	O NH HO	2
	S

  Spiro-OZT 148 showed no antimicrobial activitiy against any of the microbes tested.Herein, D-psicose series (155, 157 and 159) do not present better results than D-fructose (154, 156 and 158) like previously observed for fused-OZTs. Comparing α (154 and 153) and β (156 and 157) spiro-OZOs, α isomer showed to have better activity. Unexpectedly, compound 164, a β spiro-OZT protected with an isopropylidene group instead of benzyl groups, presented a large spectrum of activity with extraordinary activity against Candida albicans, twice higher than the control (Table3). This fungus is considered as a causal agent of opportunistic oral and genital infections in humans. Moreover systemic fungal infections have emerged as important causes of morbidity and mortality in immuno-compromised patients -AIDS, cancer chemotherapy, organ or bone marrow transplantation -a reason why Candida albicans has aroused great interest of research in health and medicine fields.

The results were similar for both protected (148, 154, 156, 155 and 157) and unprotected structures (158 and 159).

Table 18

 18

									FUNGI			BACTERIA
								Botrytis	Candida	Pyricularia	Rhizopus	Bacillus	Bacillus	Enterococcus	Staphylococcus
								spp.	albicans	oryzae	spp.	cereus	subtilis	faecalis	Aureus
							S
			O OBn HN O	-	-	-	-	-	-	-	-
		BnO OBn	148
					O
							O
		O		NH OBn	8-10	8-10	8-10	8-10	-	-	-	-
	C	BnO OBn			154
	O						O
	M P		O OBn HN O	-	-	-	-	-	-	-	-
	O	BnO OBn	156
	U				O
	N D	HO OH O	158 OH NH	O	< 6.4	< 6.4	< 6.4	< 6.4	< 6.4	8	15	< 6.4
	S
					O
							O
		O		NH	-	-	-	-	-	-	-	-
		BnO OBn	OBn	155
							O
			O HN		O	-	-	-	-	-	-	-	-
		BnO OBn	OBn	157

Table 19

 19

								FUNGI				BACTERIA
							Botrytis	Candida	Pyricularia	Rhizopus	Bacillus	Bacillus	Enterococcus	Staphylococcus
							spp.	albicans	oryzae	spp.	cereus	subtilis	faecalis	Aureus
						S
	C	OH	OH	HN
	O M	OH	O	S NH 175 O	< 6.4	9	< 6.4	< 6.4	< 6.4	9	12	< 6.4
	P
	O	RO		O	OR
	U		RO		O	NH	-	-	-	-	-	-	-	-
	N	O R=TBDMS 177

Table 20 COMPOUND

 20

															OH
		O O NH O HO S OH O 2	HN O	O S	O OH 96	O	HN O	O S	O OBz O 107	O NH OBn O S O	O 55	HO	O S HN	O	OBn 114
	IC50 (mg/mL)	0,027	0,029		0,071	0,007			0,003
	DMSO							0,449
	CONTROL
	H 2 O 2							0,002

  Calculated for C21H25NO8S [M+Na] + 443.0331, found: 443.0334.

	1,2-O-isopropylidene-β-D-fructopyranose (98)
	O	O
		O
	HN	OH
	O
	S
	MW=261.29g.mol -1
	C 10 H 15 NO 5 S
	Yield=59%
	Rf=0.2 PE/EA(6:4)
	mp=170-173ºC
	[α] 20 D =-186 (c=0.27
		MeOH)
	O	O
		O
	I	OBn
	HO
	MW=420.24g.mol -1
	C 16 H 21 IO 5
	Yield=94%
	Rf=0.26 PE/EA(9:1)
	mp=123-125ºC
	[α] 20 D =+51 (c=0.14
	CHCl3)
	IR: (neat) ν (cm -1 ): 3429 (OH); 2921, 2851 (CH); 1455, 1373 (Ph).

o-Ph).

	, H-
	3); 7.46 (t, 2H, m-Ph); 7.59 (t, 1H, p-Ph); 8.09 (d, 2H,

872345-40-9]

  . . Izquierdo, M. T. Plaza, M. Rodriguez, F. Franco, A. Martos Tetrahedron 2005, 61, 11697.

	O	O	O O	O
	O		O OBn
	HN O			OBz
		O
	S MW=350.41g.mol -1
	C 19 H 26 O 6 MW=365.40g.mol -1 C 17 H 19 NO 6 S [139764-73-1]
	Yield=100% Yield=62% Rf=0.59 PE/EA(7:3) Rf=0.57 PE/EA(7:3)
	[α] 20 D =-137 (c=0.39
			CHCl3)
	O		O
		O	O	O
	HO		O OBn
	OH MW=310.34g.mol -1 HO O O
	C 16 H 22 O 6
	MW=260.28g.mol -1 [
	C 12 H 20 O 6 [18422-54-3] Yield=100%
	Rf=0.42 PE/EA(5:5)
	Yield=98%
	Rf=0.24 PE/EA(7:3)

I

  Calculated for C16H21N3O5 [M+Na] + 358.1379, found: 358.1384.

	(4R,5R) 4-[(1S)-1-Benzyloxy-2-oxo-3-hydroxypropyl]-4,5-dihydro-5-hydroxymethyl-1,3-
	oxazoline-2-thione (111)
		O		O
	O	O O
	I		O	OBn
	O	HO	OBn
	MW=420.24g.mol -1 NH
	S	C 16 H 21 IO 5
	MW=351.42g.mol -1 Yield=56% C 17 H 21 NO 5 S
	Rf=0.22 PE/EA(8:2)
	mp=98-101ºC Yield=54%
	[α] 20 Rf=0.28 PE/EA(7:3) D =+21 (c=0.54 mp=147-158ºC CHCl3) [α] 20 D =-110 (c=0.14
			CHCl3)
		O		O
	HO HO	N 3 O		O OH OBn OBn
	MW=335.36g.mol -1 C 16 H 21 N 3 O 5 HN O
		S
	Yield=69% MW=311.35g.mol -1
	Rf=0.20 PE/EA(9:1) C 14 H 17 NO 5 S
	mp=118-121ºC [α] 20 Yield=70% D =-13 (c=0.52 Rf=0.12 PE/EA(5:5) CHCl3) mp=58-61ºC
	MS: (IS+) m/z: 334 (M+Na) + .
	IR: (neat) ν (cm -1 ): 3514 (OH); 2980 (CH); 2086 (N3); 1454, 1377 (Ph). IR: (neat) ν (cm -1 ): 3306 (OH); 1727 (C=O); 1520, 1074 (N-CS-O); 1455 (Ph).

  3 (Cq-Ph). Starting with 104 (5 g, 19.2 mmol) and using G.P. 2.2.1. (reaction time 15h) the compound 119 was obtained as colorless solid (4.21 g, 19.1 mmol, 100%).

	1,2-Di-O-isopropylidene-β-D-psicopyranose (119)
	O	O
	O
	OBn
	MW=276.33g.mol -1
	C 16 H 20 O 4
	[132369-48-3]
	Yield=54%
	Rf=0.68 PE/EA(8:2)
	O	O
	O
	OBn
	MW=276.33g.mol -1
	C 16 H 20 O 4
	Yield=67%
	Rf=0.67 PE/EA(8:2)
	[α] 20 D =-240 (c=5.53
		CHCl3)
	MS: (IS+) m/z: 299 (M+Na) +
	IR: (neat) ν (cm -1 ): 2934, 2884 (CH); 1496, 1454 (Ph).

  10508.

	TBDMSO	O	OMe
	HO	OH
		OH
	MW=308.44g.mol -1
	C 13 H 28 O 6 Si
	[74264-88-3]
	Yield=86%
	Rf=0.69 PE/EA(2:8)
	TBDMSO	O	OMe
	HO	OH
		OH
	MW=308.44g.mol -1
	C 13 H 28 O 6 Si
	[74264-88-3]
	Yield=81%
	Rf=0.68 PE/EA(2:8)

  m/z: 513 (M+Na) +

	TBDMSO	O	OMe
	I	OAc
		OAc
	MW=502.41g.mol -1
	C 17 H 31 IO 7 Si
	Yield=93%
	Rf=0.62 PE/EA(8:2)
	mp=93-96ºC
	[α] 20 D =+58 (c=0.23
	MeOH)
	O	O
		O
	BnO	OBn
	BnO
	MW=490.59g.mol -1
	C 30 H 34 O 6
	[138528-01-5]
	Yield=92%
	Rf=0.61 PE/EA(9:1)
	[α] 20 D =-69 (c=0.34
	CHCl3)

IR: (neat) ν (cm -1 ): 2987, 2932 (CH); 1496, 1454 (Ph).

  Calculated for C28H29O5S [M+H] + 492.1845, found: 492.1863. Starting with 141 (6 g, 12.2 mmol) and using G.P. 2.2.4. b) (reaction time 24h) the mixture 143 was obtained without further purification. Starting with 143 using G.P. 2.4.1. (reaction time 24h) and ethanol as solvent the compound 145 was obtained after column chromatography on silica gel with a mixture of PE/EA (9:1) as a yellow oil (4.21 g, 8.56 mmol, 70%).

	3,4,5,Tri-O-benzyl-2-N,1-O-thiocarbonyl-α-D-psicopyranosylamine (149)
	O	O
		O
	BnO	OBn
	BnO
	MW=490.59g.mol -1
	C 30 H 34 O 6	Starting with 141 (1 g, 2.04 mmol) and using G.P. 2.4.3. (reaction time 15h) the
	[153289-45-3]	compound 145 was obtained after column chromatography on silica gel with a
			mixture of PE/EA (9:1) as a yellow oil (0.47 g, 0.96 mmol, 47%).
	Yield=95%
	Rf=0.60 PE/EA(9:1)	Some traces of the 149 were isolated:
	[α] 20 D =-21 (c=0.57	1 H NMR (400MHz, CDCl3) δ: 3.43 (d, 1H, J3-4=2.4Hz, H-3); 3.47 (ddd, 1H, J5-
	CHCl3)	6a=2.0Hz, J5-4=5.2Hz, J5-6b=10.0Hz, H-5); 3.71-3.82 (m, 2H, H-6); 4.06 (brs, 1H, H-
			4); 4.49 (d, 2H, J1a-1b=3.6Hz, H-1a, H-1b); 4.54-4.57 (m, 3H, CH2Ph); 4.69 (d, 1H,
			J=11.8Hz, CH2Ph); 4.79 (d, 1H, J=11.8Hz, CH2Ph); 4.87 (d, 1H, J=11.2Hz,
			CH2Ph); 7.25-7.35 (m, 15H, Ph); 8.22 (brs, 1H, NH).
			13 C NMR (100MHz, CDCl3) δ: 60.7 (C-6); 71.7 (C-1); 72.6 (CH2Ph); 74.2 (C-4);
			74.3 (C-5); 74.7 (CH2Ph), (CH2Ph); 75.7 (C-3); 92.6 (C-2); 127.
	O	O
		NH	S
	BnO	OBn
	BnO
	MW=491.60g.mol -1
	C 28 H 29 O 5 S
	[785803-51-2]
	Yield=78%
	Rf=0.50 PE/EA(7:3)
	O	O
		NH	S
	BnO	OBn
	BnO
	MW=491.60g.mol -1
	C 28 H 29 O 5 S
	[α] 20 D =-7 (c=2.64
	CHCl3)

IR: (neat) ν (cm -1 ): 2885 (CH); 1494, 1322, 1047 (N-CS-O); 1454 (Ph).

-hydroxy-1,2,3-tribenzyloxybutyl)[1,3]oxazole-2-thione (147)

  ; 171.1 (C=S).

	4-((1S,2S,3R)-4
		O	O
			NH	S
	BnO		OBn
		BnO
	MW=491.60g.mol -1
	C 28 H 29 NO 5 S
	Yield=70%
	Rf=0.49 PE/EA(7:3)
		O	O
			NH	S
	BnO		OBn
		BnO
	MW=491.60g.mol -1
		C 28 H 29 NO 5 S
	[α] 20 D =-11 (c=0.54
		CHCl3)
	OH	OBn	O	S
	OBn OBn	N H
	MW=491.60g.mol -1
		C 28 H 29 NO 5 S
	Yield=20%
	Rf=0.31 PE/EA(8:2)
	mp=103-105ºC
	[α] 20 D =+25 (c=0.56
		CHCl3)
	MS: (IS+) m/z: 492(M+H) + ; 514 (M+Na) +

IR: (neat) ν (cm -1 ): 3271 (OH); 3029, 2923 (CH); 1627 (C=C); 1514, 1306, 1080 (N-CS-O); 1453 (Ph).

  Calculated for C28H29NO5 [M+H] + 492.1845, found: 492.1833.

	0Hz, H-6b); 4.08 (dd, 1H,
	J4-3=3.2Hz, J4-5=8.0Hz, H-4); 4.31 (d, 2H, J=12.0Hz, CH2Ph); 4.47 (d, 1H, J3-
	4=3.2Hz, H-3); 4.54 (d, 1H, J=11.8Hz, CH2Ph); 4.58 (d, 1H, J=11.8Hz, CH2Ph);
	4.75 (d, 1H, J=11.2Hz, CH2Ph); 4.81 (d, 1H, J=11.2Hz, CH2Ph); 6.50 (s, 1H, H-
	1); 7.25-7.38 (m, 15H, Ph); 10.0 (brs, 1H, NH).
	13 C NMR (100MHz, CDCl3) δ: 59.4 (C-6); 70.9 (C-3); 71.1, 71.4, 75.5 (CH2Ph);
	77.6 (C-5); 79.0 (C-4); 127.1 (C-2); 128.0, 128.2, 128.3, 128.4, 128.7, 128.8 (CH-
	Ph); 135.4 (C-1); 136.7, 137.3, 137.4 (Cq-Ph); 179.3 (C=S).
	IR: (neat) ν (cm -1 ): 3030, 2874 (CH); 1501, 1453 (Ph); 1471, 1028, 1027 (N-CS-O).

HRMS:

  ; 177.2 (C-S).

	(5R,8R,9R,10R) 8,9,10-Tribenzyloxy-2-benzylsulfanyl-1,2-dehydro-1-aza-3,6-
	dioxaspiro[4,5]decane (153)
	O	O
		N	SBn
	BnO	OBn
	BnO
	MW=581.72g.mol -1
	C 35 H 35 NO 5 S
	Yield=43%
	Rf=0.21 PE/EA(8:2)
	[α] 20 D =-16 (c=1.02
		CHCl3)
	O	O
		N	SBn
	BnO	OBn
	BnO
	MW=581.72g.mol -1
	C 35 H 35 NO 5 S
	Yield=43%
	Rf=0.21 PE/EA(8:2)
	[α] 20 D =-16 (c=1.02
	CHCl3)
	MS: (IS+) m/z: 604 (M+Na) + .

IR: (neat) ν (cm -1 ): 3030, 2921 (CH); 1600, 1091, 697 (N=CS-O); 1495, 1453 (Ph).

  ; 161.5 (C=O). Calculated for C28H26NO6 [M+H] + 476.2073, found: 476.2083. Starting with 151 (1 g, 1.72 mmol) and using G.P. 2.5.1. b) the compound 155 was obtained after column chromatography on silica gel with a mixture of PE/EA (8:2) as colorless oil (0.53 g, 1.11 mmol, 65%). Calculated for C28H26NO6 [M+H] + 476.2073, found: 476.2073.

	3,4,5,Tri-O-benzyl-2-N,1-O-carbonyl-α-D-psicopyranosylamine (155)
	O	O
		N	SBn
	BnO	OBn		1 H NMR (400MHz, CDCl3) δ: 3.64 (d, 1H, J6a-6b=10.8Hz, H-6a); 3.78-3.85 (m,
	BnO			4H, H-3, H-4, H-5, H-6b); 4.09 (d, 1H, J1a-1b=9.6Hz, H-1a); 4.20 (d, 1H, J1b-
	MW=581.72g.mol -1 C 35 H 35 NO 5 S	1a=9.6Hz, H-1b); 4.55-4.67 (m, 5H, CH2Ph); 4.85 (d, 1H, J=11.6Hz, CH2Ph); 7.24-7.34 (m, 15H, Ph); 8.40 (brs, 1H, NH).
	Yield=43% Rf=0.21 PE/EA(8:2) [α] 20 D =-16 (c=1.02 CHCl3)	13 C NMR (62.89MHz, CDCl3) δ: 62.2 (C-6); 71.9 (C-5); 72.6 (C-4); 72.7 CH2Ph; 72.8 (C-1); 73.9 CH2Ph; 75.7 CH2Ph; 78.5 (C-3); 90.3 (C-2); 127.8, 127.9, 128.0, 128.2, 128.5 (CH-Ph); 137.6, 137.9, 138.0 (Cq-Ph); 159.9 (C=O).
	IR: (neat) ν (cm -1 ): 3031, 2874 (CH); 1756 (C=O); 1496, 1454 (Ph).
	O	O
		NH	O
	BnO	OBn
	BnO
	MW=475.53g.mol -1
	C 28 H 26 NO 6
	Yield=65%
	Rf=0.36 PE/EA(6:4)
	[α] 20 D =+52 (c=0.19
	CHCl3)

HRMS:

IR: (neat) ν (cm -1 ):3031, 2874 (CH); 1760 (C=O); 1496, 1454 (Ph).

HRMS:

  0 (Cq-Ph); 159.6 (C=O).

	3,4,5,Tri-O-benzyl-2-N,1-O-carbonyl-β-D-psicopyranosylamine (157)
	O	O
		NH	O
	BnO	OBn
	BnO
	MW=475.53g.mol -1
	C 28 H 26 NO 6
	Yield=69%
	Rf=0.35 PE/EA(6:4)
	[α] 20 D =+25 (c=0.59
	CHCl3)
	O	O NH	O
	BnO	OBn
	OBn
	MW=475.53g.mol -1
	C 28 H 29 NO 6
	Yield=43%
	Rf=0.47 PE/EA(6:4)
	[α] 20 D =-39 (c=1.13
	CHCl3)
	HRMS: Calculated for C28H26NO6 [M+H] + 476.2073, found: 476.2092.
	IR: (neat) ν (cm -1 ): 2872 (CH); 1752 (C=O); 1496, 1454 (Ph).

  ; 157.5 (C=O). Calculated for C28H26NO6 [M+H] + 476.2073, found: 476.2083.

	IR: (neat) ν (cm -1 ): 3031, 2874 (CH); 1753 (C=O); 1496, 1454 (Ph).

HRMS:

173934-39-9]

  . W.Lichtenthaler, J. Klotz, F. Flath Liebigs Ann. 1995, 2069.

	O	O
		NH	O
	BnO	OBn
	BnO
	MW=475.53g.mol -1
	C 28 H 26 NO 6
	Yield=52%
	Rf=0.48 PE/EA(6:4)
	[α] 20 D =-16 (c=2.73
	CHCl3)
	O	O
		NH	O
	HO	OH
	HO
	MW=205.17g.mol -1
	C 7 H 11 NO 7
	[
	Yield=25% Rf=0.3 EA	(C-1); 92.4 (C-2); 162.1 (C=O).	4

F

  . W.Lichtenthaler, J. Klotz, F. Flath Liebigs Ann. 1995, 2069.

	O	O O	NH O NH O O O	O
	HO HO		OH OH NH	S
	HO HO O	OBn
	MW=205.17g.mol -1 MW=205.17g.mol -1 O
	C 7 H 11 NO 7 C 7 H 11 NO 7 MW=351.42g.mol -1
	Yield=25% C 17 H 21 NO 5 S
	Rf=0.3 EA Yield=56% Yield=6%
	Rf=0.3 EA Rf=0.29 PE/EA(6:4)
	mp=65-68ºC
	[α] 20 D =-116 (c=0.30
			CHCl3)
	O		O
			NH	S
	O		OBn
	O	O	O
	NH MW=351.42g.mol -1	O
	HO C 17 H 21 NO 5 S HO OH O O
	MW=205.17g.mol -1 Yield=27% N SBn
	C 7 H 11 NO 7 Rf=0.33 PE/EA(8:2) OBn O [17372968-5] mp=60-63ºC O
	Yield=25% Rf=0.3 EA D =-130 (c=0.22 [α] 20 MW=441.54g.mol -1 CHCl3) C 24 H 27 NO 5 S
	Yield=52%
	Rf=0.29 PE/EA(8:2)
	mp=73-76ºC
	MS: (IS+) m/z: 374 (M+Na) + [α] 20 D =0.6 (c=0.24
	CHCl3)
	IR: (neat) ν (cm -1 ): 2983, 2935 (CH); 1495, 1454 (Ph); 1066, 698 (N-CS-O).

FMS: (IS+) m/z: 442(M+H) + ; 464 (M+Na) + IR: (neat) ν (cm -1 ): 2923, 2880 (CH); 1607, 1081, 697 (N-CS-O); 1494, 1454 (Ph).

E. J. Prisbe, J. Smejkal, J. P. H. Verheyden, J. G. Moffatt J.Org. Chem. 1976, 41 , 1836.

N. A. Jones, D. Rao, A. Yoshihara, P. Gullapalli, K. Morimoto, G. Takata, S. J. Hunter, M. R. Wormald, R. Dwek, K. Izumori, G. W. J. Fleet Tetrahedron:Asymmetry 2008, 19, 1904 and references cited therein.

M. I. García-Moreno, J. M. Benito, C. Ortiz Mellet, J. M. García Fernández Molecules 2008, 13, 1640 and references cited therein.

The second method is the oldest one but curiously the least used. It results from the condensation of thiocyanic acid (generated in situ from potassium thiocyanate and hydrochloric acid) on an anomeric-free sugar to give essentially the thermodynamically more stable OZT.

Q.-P. Wu, M.-X. Zhou, X.-D. Xi, D. Song, Y. Wang, H.-X. Liu, Y.-Z. Li, Q.-S. Zhang Tetrahedron Lett. 2008, 49, 2714-2718.

M.-K. Chung, G. Orlova, J. D. Goddard, M. Schlaf, R. Harris, T. J. Beveridge, G. White, F. R. Hallett J. Am.Chem. Soc. 2002, 124, 10508.

F. W. Lichtenthaler, J. Klotz, F.-J. Flath Liebigs Ann.Chem. 1995Chem. , 2069. .

(a) K. Krohn, D. Gehle, U. Flörke Eur. J. Org. Chem. 2005, 4557; (b) M. Samadi, C. Munoz-Letelier, S. Poigny, M. Guyot Tetrahedron Lett. 2000, 41, 3349.

(a) I. Izquierdo Cubero, M. T. P. López-Espinosa Carbohydr. Chem. 1986, 5, 299; (b) Y. Bechor, A. Albeck Tetrahedron 2008, 64, 2080.

S. Mio, Y. Kumagawa, S. Sugai Tetrahedron 1991,47, 2133.

V.Douard, R. P. Ferraris Am. J. Physiol. Endocrinol. Metab. 2008, 295, 227 and references cited therein.

J. L. Evans, J. F. Youngren, I. D. Goldfine Trends in Endocrinology and Metabolism 2004, 15.

M. H. Stipanuk Biochemical, Physiological, and Molecular Aspects of Human Nutrition 2006, W. B. Saunders, Philadelphia, PA. 2 nd Edition.

A. E.Buchs, S. Sasson, H. G. Joost, E. Cerasi Endocrinology 1998, 139 (3), 827.

T. Fujisawa, J. Riby, N. Kretchmer Gastroenterology 1991, 101, 360.

K. Ushijima, T. Fujisawa, J. Riby, N. Kretchmer Journal of Nutrition 1991, 125, 2156.

(a) M. I.Borges-Walmsley, A. R. Walmsley Trends Microbiol. 2001, 2, 71; (b) V. H. L. Lee Eur. J.Pharm. Sci. 2000, 11, S41.

M. Hashimoto, J.Yang, G. D. Holman ChemBioChem. 2001, 2, 52.

G. Dorman, G. D. Prestwich Trends Biotechnol. 2000, 18, 64.

(a) R. L. Freshney Culture of Animal Cells: A Manual of Basic Techniques 2000 Wiley-Liss: New York; (b) J. A. Plumb, R. Milroy, S. B. Kaye Cancer Res. 1989, 49, 4435.

C NMR (100MHz, CDCl3) δ: 59.0 (C-5); 63.5 (C-6); 65.5 (C-1); 74.2 (C-7); 82.0 (C-3); 95.7 (C-4); 128.8, 128.9 (CH-Ph); 135.3 (Cq-Ph); 190.0 (C=S); 203.0 (C=O).

CHAPTER V 2,3-Fused and spiro thionocarbamates in furanose form

Considering the results obtained we can put forward some preliminary assumptions:

The synthesis of D-tagatose via C-5 epimerization of D-fructose was not efficient.The best approach seems to be from the literature but the overall yield is still quite low.

The epoxide opening under basic or acidic conditions was not an efficient transformation.

Nonetheless, the reaction was regioselective with the formation of the L-sorbopyrano derivative.

In comparison with other unprotected ketoses, D-tagatose appears more susceptible to the number of equivalents of thiocyanic acid.

When the same reaction was realized on protected derivatives with KSCN, the 2,3-fused OZT was formed preferentially while with TMSNCS, spiro structures are mainly formed.

The separation of this mixture of three compounds, specially fused and spiro-OZTs, was more difficult than in other ketose series.

CHAPTER VI