France-Isabelle Auzanneau

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues.

We describe here the synthesis of the allyl Le(a) trisaccharide antigen as well as that of an analogue of the Le(x) trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le(a) analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the alpha-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF(3) x OEt(2) than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF(3) x OEt(2), glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.

Convenient Temporary Methyl Imidate Protection of N-Acetylglucosamine and Glycosylation at O-4

This paper expands on the scope and utility of the temporary conversion of N-acetyl groups to alkyl imidates when attempting to glycosylate at O-4 of N-acetylglucosamine acceptors. The optimized synthesis of alkyl imidate protected glucosamine acceptors at position 4 and carrying various protecting groups at O-3 is described. These imidates were prepared immediately prior to glycosylation by treating the 4-OH acceptors with 0.5 M MeOTf to obtain the corresponding methyl imidates still carrying a free 4-OH group. When preparing these imidates in diethyl ether as the reaction solvent, we observed the unexpected formation of ethyl imidates in addition to the desired methyl imidates. While the 3-O-allyl acceptors were too unstable to be useful in glycosylation reactions, the 3-O-acylated methyl and ethyl imidates of glucosamine were shown to behave well during the glycosylation of the 4-OH with a variety of reaction conditions and various glycosyl donors. Glycosylation of these acceptors was successfully carried out with perbenzylated beta-thioethyl rhamnopyranoside under MeOTf promotion, while activation of this donor under NIS/TMSOTf or NIS/TfOH proved less successful. In contrast, activation of the less reactive perbenzylated alpha-thioethyl and peracetylated beta-thioethyl rhamnopyranosides with NIS/TfOH led to successful glycosylations of the 4-OH. Activation of a peracetylated rhamnosyl trichloroacetimidate by TMSOTf at low temperature also gave a high yield of glycosylation. We also report one-pot glycosylation reactions via alkyl imidate protected acceptor intermediates. In all cases the alkyl imidate products were readily converted to their corresponding N-acetyl derivatives under mild conditions.

Challenging Deprotection Steps During the Synthesis of Tetra- and Pentasaccharide Fragments of the LeaLex Tumor-Associated Hexasaccharide Antigen

We report the convergent synthesis of two novel tetrasaccharide and two novel pentasaccharide fragments of the Le(a)Le(x) TACA: the tetrasaccharides contain neither the galactose at the Le(a) nonreducing end nor the fucose at the Le(x) reducing end; the pentasaccharides only lack the galactose residue at the Le(a) nonreducing end. Two of the analogues were prepared as hexyl glycosides to be used in NMR experiments and as soluble inhibitors in binding studies and two as 6-aminohexyl glycosides to be conjugated to carrier proteins. Our strategy relied on stepwise extensions using excess monosaccharide glycosyl donors (trichloroacetimidates and thioglycosides) in sequential glycosylation reactions. The protecting groups were chosen to limit the number of deprotection steps required to obtain the final derivatives. While this strategy ensured that all glycosylation reactions proceeded in very good yields (70-84%), deprotection of the oligosaccharide intermediates was challenging. Global deprotection using Birch metal dissolving conditions did not remove the tert-butyldiphenylsilyl group, which indeed was incompatible with such reaction conditions. Attempts to remove the TBDPS with tetrabutylammonium fluoride was unsuccessful and led to a complex mixture of compounds that could not be separated. The desired hexyl and aminohexyl tetrasaccharides were finally obtained after four- and five-step deprotection sequences, respectively. Deprotection of the pentasaccharide intermediate to give the hexyl and aminohexyl analogues also led to unexpected results. Indeed, during Zemplén deacylation, a chloroacetamide chlorine atom was displaced by methoxide ions leading to the corresponding methoxyacetamide. Once the chloroacetamide was fully reduced to an acetamide the pentasaccharides were obtained in four and five steps, respectively.

Synthesis of a BSA-Lex glycoconjugate and recognition of Lex analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross reactive analogue

A Le(x) trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF(3)·OEt(2). In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate-mediated coupling. This BSA-Le(x) glycoconjugate displayed 35 Le(x) haptens per BSA molecule. The relative affinity of the anti-Le(x) monoclonal antibody SH1 for the Le(x) antigen and analogues of Le(x) in which the D-glucosamine, L-fucose or D-galactose residues were replaced with D-glucose, L-rhamnose and D-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Le(x) antigen, only the analogue of Le(x) in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Le(x) analogues by the anti-Le(x) mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le(x) antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Le(x) antigen. The identification of a trisaccharide analogue that does not cross-react with Le(x) but still retains the same conformation as Le(x) constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le(x) tumor associated carbohydrate antigen.

How the substituent at O-3 of N-acetylglucosamine impacts glycosylation at O-4: a comparative study.

An assessment of the relative reactivities of the 4-OH of N-acetylglucosamine acceptors bearing simple protecting groups, beta-linked or alpha-linked D or L sugars at O-3 is presented, using a per-O-acetylated alpha-D-glucosyl trichloroacetimidate donor under activation by BF(3) x OEt(2). The presence of either an acyl or carbonate protecting group at O-3 did not impact the reactivity at O-4 with all glycosylations proceeding successfully. On the other hand, the presence of peracetylated sugars at O-3 of N-acetylglucosamine acceptors did impact the reactivity of the 4-OH. The acceptors with an alpha-D-Man, beta-D-Gal, or beta-D-Glc at O-3 reacted promptly. In comparison, the acceptors bearing a beta-L-Fuc, alpha-L-Fuc, or alpha-L-Rha underwent glucosylation slowly, and unreacted acceptor was recovered from the reaction mixtures. Systematic searches carried out on the disaccharide acceptors and trisaccharide products carrying either a peracetylated beta-D-Gal or beta-L-Fuc at O-3 of the glucosamine residue suggest that, for these two acceptors, a conformational reorientation necessary around the fucosidic linkage contributes to the lower reactivity of the beta-fucosylated acceptor. The acceptors bearing a beta-linked D-Gal, D-Glc, or L-Fuc residue at O-3 each gave trisaccharide products that were mostly stable in the reaction conditions. In contrast, the alpha-linked residues at O-3 were rather unstable in these reaction conditions and the degradation of either the acceptors or trisaccharide products led to low glycosylation yields. In these later reactions, it was impossible to clearly assess which of the acceptor or product underwent degradation as comigration and detection issues prevented us from following these glycosylations by TLC or RP-HPLC. In contrast, the glycosylation of an acceptor carrying an alpha-linked perbenzylated L-Fuc residue at O-3 could be easily monitored by RP-HPLC. The data obtained when monitoring this glycosylation showed that the acceptor underwent prompt glycosylation but a decrease in the absorbance peak corresponding to the trisaccharide along with the appearance of a peak corresponding to a perbenzylated fucose hemiacetal indicated that the trisaccharide product was unstable in the reaction conditions.

An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products

Finger millet is an ancient African cereal crop, domesticated 7000 years ago in Ethiopia, reaching India at 3000 BC. Finger millet is reported to be resistant to various fungal pathogens including Fusarium sp. We hypothesized that finger millet may host beneficial endophytes (plant-colonizing microbes) that contribute to the antifungal activity. Here we report the first isolation of endophyte(s) from finger millet. Five distinct fungal species were isolated from roots and predicted taxonomically based on 18S rDNA sequencing. Extracts from three putative endophytes inhibited growth of F. graminearum and three other pathogenic Fusarium species. The most potent anti-Fusarium strain (WF4, predicted to be a Phoma sp.) was confirmed to behave as an endophyte using pathogenicity and confocal microscopy experiments. Bioassay-guided fractionation of the WF4 extract identified four anti-fungal compounds, viridicatol, tenuazonic acid, alternariol, and alternariol monomethyl ether. All the purified compounds caused dramatic breakage of F. graminearum hyphae in vitro. These compounds have not previously been reported to have anti-Fusarium activity. None of the compounds, except for tenuazonic acid, have previously been reported to be produced by Phoma. We conclude that the ancient, disease-tolerant crop, finger millet, is a novel source of endophytic anti-fungal natural products. This paper suggests the value of the crops grown by subsistence farmers as sources of endophytes and their natural products. Application of these natural chemicals to solve real world problems will require further validation.

The flexibility of the LeaLex Tumor Associated Antigen central fragment studied by systematic and stochastic searches as well as dynamic simulations

The solution conformational behavior of the Tumor-Associated Carbohydrate Antigen Le(a)Le(x) central fragment: methyl alpha-L-fucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->3)-beta-D-galactopyranoside was studied using three computational methods: a rigid systematic search as implemented in Sybyl, a stochastic search as implemented in MOE2004, and dynamics simulations using the SANDER module of AMBER9. Our results illustrate the complementarity of these methods to identify energetically relevant conformations and flexible linkages. In particular, the beta-GlcNAc-(1-->3)-Gal linkage was shown to be extremely flexible adopting a wide range of orientations around two energy minima. The modeling results were validated by comparison of theoretical distances, derived from the simulations, with experimental measurements obtained from 1D selective ROESY buildup curves on the synthetic fragment.

Synthesis of LeaLex oligosaccharide fragments and efficient one-step deprotection

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le(a)Le(x). While the linear lacto-N-triose I: beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-[alpha-l-Fucp-(1-->3)]-beta-D-GlcNAcp-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 degrees C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF(3).OEt(2) to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na-NH(3)) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.

Synthesis of Lewis X and three Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose, respectively

Three analogues of the Le(x) trisaccharide: alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-GlcNAcp as well as the Le(x) trisaccharide itself were synthesized as methyl glycosides. In the analogues, either only the fucose residue is replaced by rhamnose or both the N-acetylglucosamine and the fucosyl residues are replaced by glucose and rhamnose, respectively. Our synthetic strategy relied on the use of lactoside and 2-azido lactoside derivatives as disaccharide acceptors, which were submitted to either fucosylation or rhamnosylation. Our results confirm that the reactivity of lactose in protection and glycosylation reactions is greatly affected by (1) the structure of the aglycone and (2) the presence of an azido substituent at C-2 of the glucose moiety. Thus, a methyl lactoside acceptor was easily glycosylated at O-3 with perbenzylated beta-thiophenyl fucoside and rhamnoside to give anomerically pure alpha-fucosylated and alpha-rhamnosylated trisaccharides, respectively. In contrast, the same reactions on a 2-azido methyl lactoside acceptor led to the formation of anomeric mixtures. While the alpha- and beta-fucosylated 2-azido trisaccharides could be separated by RP-HPLC, such separation was not possible for the rhamnosylated anomers. The desired rhamnosylated trisaccharide was finally obtained anomerically pure using an isopropylidene-protected rhamnosyl donor. The deprotection sequences also showed that the presence of a 2-azido substituent at C-2 of the glucose residue conferred stability to the vicinal fucosidic linkage at C-3. To test their relative affinity for anti-Le(x) Abs the Le(x) analogues will be used as competitive inhibitors against methyl Le(x). In addition, their conformational behavior will be studied by NMR spectroscopy and molecular modeling experiments.

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose, respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.