Synthesis of chitobioses with different N-protecting groups

Diploma Thesis, 2007

80 Pages, Grade: 2,1


Table of contents


1. Abstract

2. Introduction

3. Objective

4. Results and discussion

5. Summary / Zusammenfassung

6. Experimental section

7. References


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1. Abstract

The syntheses of different N-protected β-(1→4)-linked disaccharides have been described and discussed in this work. Benzyloxycarbonyl (Cbz), p- nitrobenzyloxycarbonyl (PNZ), phthalimido (Phth), tetrachlorophthalimido (TCP) have been chosen as the amino protecting groups due to their ability of neighbouring group participation during the glycosylation reactions. By using these amino protecting groups the monomeric donors and acceptors were synthesized. For the glycosidic bond formation the trichloroacetimidate strategy has been used. The glycosylation reaction was performed with BF3·OEt2 as a Lewis acid catalyst. Under these reaction conditions three disaccharides have been obtained in good yields.

2. Introduction

This work will focus on carbohydrates, one of the four major classes of natural products. Besides lipids, proteins and nucleic acids the carbohydrates are the largest and most important group of naturally occurring organic compounds.[1] They are the essential constituent of flora and fauna.

The carbohydrates have structural, protective and energy-storing functions; their roles in protein folding, regulation of hormones and enzyme activities, cell signalling, pathogen binding to host tissue and tumor cell metastasis are just a few selected examples.[2],[3],[4]

Glycosides of amino sugars, a special group of glycoconjugates, are the main interest of this work. In nature 2-amino-2-deoxy glycosides are frequently encountered in glycoproteins and glycolipids. However, isolation of these amino glycosides from natural sources is a difficult task that often yields in a heterogeneous mixture of oligosaccharides.[5],[6]

Today the synthesis of oligosaccharides is a major objective because some oligosaccharides of 2-amino-2-deoxy-D-glucose are compounds of biological importance.[7],[8] The β-linked oligosaccharides are compounds which constitute for example building blocks of chitin, chitosan and glycan of glycoproteins such as human milk oligosaccharides or blood group substances contain many derivates of amino glycosides.[9],[10],[11] Also antigen polysaccharides and lipopolysaccharides are often encountered with a β-D-glucosamine residue. [12],[13]

The variety of linkages that a sugar molecule can possess is greater when compared to the other natural biopolymers (peptide, proteins, etc.).[2],[14] The oligosaccharides part of the glycolipids or glycoproteins has more structural information in one building block than the rest of the lipid or protein. This causes severe problems in the synthesis. In general, all sugar function groups that are not involved in the reaction have to be masked before.

Introduction 3

2.1.Structure of a glycoside

Conventional sugars or monosaccharides are the simplest species of carbohydrates. Important polysaccharides are chitin, starch and cellulose. The general empirical formula of saccharides is Cn(H2O)m - but carbohydrates are no hydrates of carbon. They constitute polyalcohol and one primary or secondary hydroxy group is oxidized to an aldehyde group or a keto group.[5],[12]

The carbohydrates are classified in monomers, oligomers and polymers according to the molecular size. Due to the polyolic nature of carbohydrates, a coupling of monosaccharides to oligomers or polymers is characterised by a large number of possibilities. Thus the specific chemical synthesis of oligosaccharides or polysaccharides is much more difficult than that of other biopolymers.

The basic structure (Figure 1) of an O-glycoside is the glycon part of the glycoside and the aglycon that are linked at the anomeric centre.[4]

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The linkage at the anomeric centre can be obtained in two configurations for the D-Glucose; the α-form (the glycosidic O-atom is in an axial position at the anomeric centre) and the β-form (the glycosidic O-atom is in an equatorial position) (Figure 2).

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Introduction 4

A glycoside can possess an acceptor or a donor character. For the formation of glycosidic linkage a nucleophilic acceptor and an electrophilic donor are necessary (Figure 3).

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The glycosidation component is the electrophilic donor with protected hydroxyl groups and a leaving group at the anomeric C atom. A free hydroxyl group is the coupling moiety at the nucleophilic glycosyl acceptor; all other hydroxyl functions are protected.

2.2. N-protecting groups

The general strategy for a regio selective glycosidic bond formation between a donor and an acceptor requires the protection of all other hydroxy functional groups which are not involved in the bond formation. Typical O-protecting groups for this purpose are acetyl, benzyl and tert-butyldimethylsilyl. The protection of the amino group is important for controlling the stereochemistry of the glycosidic bond formation and also increasing the reactivity of the acceptor.[13] Many amino protecting groups have been developed for the glycosylation of 2-amino sugars. The ideal amino protecting group is stable and shows remarkable stereoselectivity for the exclusive formation of β-linkage, sufficient reactivity, and high yields in glycosylation reactions. The most obvious way in which a protecting group can influence the stereoselectivity of a glycosylation is through anchimeric assistance (neighbouring-group participation). Moreover, the protecting group should be easily removable under mild conditions with high yields and at the same time without degradation of the resulting products and other protecting groups.[16] A “classical” protecting group for this purpose is the N-acetamido group, but the oxazolinium intermediate (Figure 4), which is presumably formed during the glycosylation reaction, is rather stable and decreases the reactivity of the glycosyl donor.

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The benzyloxycarbonyl (Cbz), the p-nitrobenzyloxycarbonyl (PNZ), the phthalimido (Phth) and the tetrachlorophthalimido (TCP) groups that have been used in this work (Figure 5) are special bulky groups which cause activation and β-direction through neighbouring group participation during glycosidation.

Introduction 6

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The advantage of these groups is their electron-attracting character, which increases the glycosyl-donor activity.

2.3. N-protected glucosamine as a nucleophilic acceptor

In order to form the β-(1→4)-glycosidic bond the acceptor has to possess the free hydroxyl group in the C4 position. For this purpose the remaining hydroxyl functionalities were protected with suitable protecting groups. According to the literature the acetyl, benzylidene and benzyl groups are widely used for the protection. The tert-butyldimethylsilyl group is used for the protection of the anomeric position due to its ease of formation as well as the selective removal under mild conditions.

With the use of regioselective O-benzylation, the C3 and C6 positions are protected as the corresponding benzyl derivatives from the tertButyldimethylsilyl-2-deoxy-N-protected-D-glucopyranoside.[19] For the 3,6-O-benzyl derivate dibutyltin oxide was used.

First O-stannylene acetal between C6-OH and C4-OH was formed and then a hemiacetal with C3-OH which is stabilized by interaction of the tin atom with carbonyl oxygen of the N-protecting group (Figure 6).[13],[20]

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The benzylation with BnI, which was formed of BnBr in the presence of TBAI, followed. The regioselective benzylation results in 3,6-di-O-benzylether as the glycosyl acceptor.

2.4. N-protected glucosamine as electrophilic donor

An efficient donor sugar requires an active leaving group at the anomeric centre, which can be achieved in two different ways.[3] The first is the activation through anomeric oxygen-exchange reaction; the mode therefore is the classical Koenigs-Knorr method and its modifications and the sulphur based activations for example. The second is the activation through retention of the anomeric oxygen. The phosphate and the phosphite activation are two examples. Another classical method for activation is the trichloroacetimidate method.[3]

Introduction 8

A reaction with electron-deficient nitriles such as trichloroacetonitrile results in O-glycosyl trichloroacetimidates. The trichloroacetimidates are essentially more stable than the chlorides or bromides used for the Koenigs-Knorr reactions for example.

The anomeric hydroxy group has been converted into the trichloroacetimidate using a base catalysed reaction of the protected monomer with trichloroacetonitrile.

The first paper describing this method was published in 1980. Since then more than 1200 different O-glycosyl trichloroacetimidates have been described in more than 1100 papers.[3]

2.5. Disaccharides

The conversion of the very reactive trichloroacetimidate with the nucleophilic acceptor can easily be achieved under mild acid or Lewis acid catalysis glycosidation conditions.[22],[23] The control of the stereochemistry is derived from the anchimeric assistance (neighbouring group participation), the influence of the solvent, thermodynamic or kinetic effects.[3],[24] By using N-protecting groups without neighbouring group participation, the thermodynamically stable α-product is typically obtained.[15] The combination of a good protecting group with neighbouring group participation in C2 and the reverse addition technique of trichloroacetimidiate are utilised to form β-linkage disaccharides.

Objective 9

3. Objective

With this work I will give an insight into the synthesis and the results of protected disaccharides containing different N-protecting groups. D-Glucosamine hydrochloride was chosen as a starting material. Phthaloyl (Phth), tetrachlorophthaloyl (TCP), benzyloxycarbonyl (Cbz) and p- nitrobenzyloxycarbonyl (PNZ) were used for the protection of the amino functionality. The process of creating the dimers and the several synthetic steps towards its formation were compared in this work. The constructed products were characterised with different analytical methods.

• The main focus is the synthesis and analysis of the glycosyl acceptors with different N-protecting groups. A difficulty here is the selective benzylation of C6 and C3 positions in one pot; five steps are necessary to achieve the glycosyl acceptors.
• Another issue is the preparation of the donor. The same protecting groups used for the preparation of acceptors have also been used for the donors. The first three steps were the same shown in the preparation of the acceptor.

Results and discussion 10

4. Results and discussion

4.1. Preparation of 3,4,6-Tri-O-acetyl-N-protected glucosamine derivatives

The four chosen N-protecting groups which behave similar were used because they are stable and thus more or less capable of neighbouring group participation for the formation of β-linkages. But formation of stable oxazolines decreased the reactivity of the glycosyl donors, and according to literature phthaloyl groups are therefore preferred.[3]

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Results and discussion 11

In most literature the Phth and the Cbz group have found widespread use in protecting 2-amino compounds. Recent alternative groups are the TCP group, which behaves similar to the Phth group, and the PNZ group, which behaves similar to the Cbz group during glycosylation reactions, but they can be removed under very mild conditions.

Starting material for the synthesis of the N-protected glucosamine derivatives was the commercially available α-D-Glucosamine hydrochloride, which was neutralised with NaHCO3, sodium acetate trihydrate or NaOMe. The glucosamine was next treated with one equivalent of the protecting group to yield the N-protected compounds GB1, GB8, GB15 and GB22 (Scheme 1). These four products were obtained in good yields between 76-93 %.

After this the N-protected derivatives were treated with an excess of acetic anhydride in pyridine to provide the N-protected per-O-acetyl derivatives in good yields (64-89 %). The dried residues were resuspended in pyridine and treated with acetic anhydride at room temperature and the per-O-acetyl derivates (GB2, GB9, GB16, and GB23) have been obtained. [16],[25],[26],[27]

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Results and discussion 12

The N-Phth and N-TCP protected per-O-acetyl products were obtained as the β-anomer. The tetra-acetate with N-Cbz and N-PNZ groups were isolated as a mixture of anomers. [16] The stereochemistry of all compounds was assigned on the basis of the coupling constant (J) between the anomeric C1 proton and the adjacent C2 proton. For the β-anomer J = 8-10 Hz and for the α-anomer J = 3-5 Hz. The synthesis of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-benzyloxycarbonyl-D-glucopyranoside (GB2) gave an α, β-mixture of 64 % yield. The NMR data showed a larger amount for the β-anomer (3 J1,2 = 9.3 Hz) as for the α-anomer (3 J1,2 = 3.6 Hz). Also a mixture of anomers were obtained in 72 % for the 1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-p-nitrobenzyloxycarbonyl-D- glucopyranoside (GB16) but with a larger percentage of the α-anomer. The regioselective removal of the anomeric O-acetyl group was effected with 1.14 eq. hydrazine acetate in DMF or THF. [28] The parent substance for synthesis of the donors and acceptors gave different results for the anomers in 68-89 % yields (Scheme 2).

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These results demonstrate a major difference of the protecting groups. For the Phth and the TCP group the neighbouring group participation ensured only the β-anomer.

For the benzyloxycarbonyl (Cbz) and the p-nitrobenzyloxycarbonyl (PNZ) N-protecting groups derivate the results show the α-anomers.

4.2. Synthesis of glycosyl acceptor

The synthesis of the acceptor is accomplished by the protection of the anomeric OH group with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole.[17] In the presence of imidazole as a catalyst for silylation, exclusively the β-1-O-TBDMS ether was obtained (Scheme 3). Literature often uses different equivalents of TBDMSCl and imidazole.

Best results were obtained when 1.18 eq. of TBDMSCl and 2 eq. of imidazole were used for the batches. The four β-1-O-TBDMS derivatives (GB4, GB11, GB18 and GB25) were obtained in good yields. The Phth and the TCP products gave the best results with 79 % and 80 % yield. The yields of the Cbz and PNZ protected derivates are lower with 53 % respectively 62 %. The NMR spectra shows explicitly the signals for the tert-butyl group (s, δ = 0.70-0.90) and the two methyl groups which are attached to Si (2s, δ = -0.50- 0.30).

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The silylation of the Cbz protected derivative was examined with different equivalents of TBDMSCl and imidazole. The results showed that 1.18 eq. of TBDMSCl and 2 eq. of imidazole gave the best results.

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Treatment of the β-1-O-TBDMS derivative with sodium methoxide in dry methanol resulted in a 3,4,6-O-unprotected derivative (Scheme 3). The reaction obtained in good yields for the Cbz group (71 %, GB5), Phth group (81%, GB12) and the TCP group (78 %, GB26). The N-PNZ-protected 3,4,6-O-unprotected (GB19) derivative gave only 47 % yield and 40 % starting material were recovered. The reaction conditions have not been optimised.

The regioselective benzylation of C3 and C6 position of trihydroxy glucosamine derivatives was developed by R. R. Schmidt.[2] Using Bu2SnO and benzyl bromide in the presence of tetrabutylammoniumiodide the reaction gave a mixture of 3,4,6-tri-O-benzylglucoderivative (a), 6-O-benzylglucopyranoside

(b) and the desired 3,6-di-O-benzyl product (c) (Figure 7). [13],[19]

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The literature developed from Robina[13] describes a direct benzylation with 6 eq. benzyl bromide in a basic medium for 6 days that resulted in a mixture as well. The order of the reactivity for these hydroxyl groups were C6-OH > C4-OH > C3-OH, because the C3-OH group is relatively less reactive.[13] The tert-butyldimethylsilyl 3,4,6-O-unprotected derivate was refluxed in toluene with dibutyltin oxide.[20] The reaction mixture was heated until the colour changed to red or black. Then the solution was treated with benzyl bromide and TBAI. The regioselective benzylation resulted in different outcomes for all four N-protecting derivates.

The results of the benzylation reaction for the Cbz group (GB6):

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The benzylation with 2 eq. and 1.1 eq. dibutyltin oxide gave a mixture of the N-Cbz-protected-6-O-benzylglucosamine and the 3,6-di-O-derivate (GB6). These two derivates were isolated and GB6 was obtained in 45 % and the 6-O-derivative in 30 % yield.

The reaction with tert-butyldimethylsilyl 2-deoxy-2-N-phthalimido-β-D-glucopyranoside, 2 eq. of dibutyltin oxide and 3eq. of BnBr resulted in a mixture of N-Phth-3,4,6-tri-O-benzyl compound (20 %), 3,6-di-O- benzylglucosamine (54 %, GB13) and 6-O-benzylglucosamine (10 %).

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The desired product 3,6-di-O-benzyl-2-N-phthalimido-β-D-glucopyranoside GB13 was isolated and developed into yellow-orange crystals with a melting point of 110°C. The reaction with only 1.1 eq. dibutyltin oxide, 2 eq. TBAI and 3 eq. BnBr resulted in 3,6-di-O-benzyl-2-N-phthalimido-β-D-glucopyranoside (GB13) in 45 % yield and the 6-O-benzylglucosamine derivate in 40 % yield. For the PNZ and the TCP protected sugars only the 6-O-benzyl derivates were isolated, that means the tert-butyldimethylsilyl 6-O-benzyl-2-deoxy-2-N-nitrobenzyloxycarbonyl-β-D-glucopyranoside (GB20) with 42 % and the tert-butyldimethylsilyl 6-O-benzyl-2-deoxy-2-N-tetrachlorophthalimido-β-D- glucopyranoside (GB27) with 41 %. The remaining residue was isolated and later used as the raw material. The NMR spectra of GB20 and GB27 show that only the position C6 has a benzyl group. For GB20 the NMR signals δ = 4.84 for the two protons and δ = 71.71 for the carbon of the CH2-group of the benzyl group. The NMR signals for GB27 are δ = 4.61 for the two protons and δ = 74.21 for the carbon. The mass spectrum supports the NMR data.

4.3. Synthesis of glycosyl trichloroacetimidates

The “classical” trichloroacetimidate strategy was used for the synthesis of the four different donors. Their ease of formation and their high glycosyl donor properties released under mild acid catalysis has led to extraordinarily wide application.

The anomeric hydroxy group has been converted into the trichloroacetimidate using a base catalysed reaction of the N-3,4,6-O-protected sugar with trichloroacetonitrile in dry DCM (Figure 8).[3],[27]

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Base catalysis is important because it allows the formation of pure α- or β-anomers when the appropriate base has been used (Scheme 4).[21]

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Synthesis of chitobioses with different N-protecting groups
University of Potsdam
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Synthesis, N-protecting
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Gerit Brüning (Author), 2007, Synthesis of chitobioses with different N-protecting groups, Munich, GRIN Verlag,


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