An exploratory study on the synthesis of heparin-like oligosaccharides by polycondensation

The synthesis of heparin oligosaccharide fragments by autocondensation of suitably protected D - glucosamine α 1 → 4 L -iduronic acid disaccharide constructs containing both glycosyl-donor and glycosyl-acceptor functionalities has been explored. Trichloroacetimidate and n-pentenyl groups have been investigated for anomeric activation. The generation of building blocks equipped with a trichloroacetimidate function is seriously hampered by the presence of the free-OH glycosyl-acceptor group on the same molecule, which favors an intramolecular transesterification reaction. Using an n-pentenyl leaving group, the glycosylation promoter competes as a nucleophile with the OH glycosyl acceptor in the autocondensation process, giving rise to fast termination of the chain reaction, a low yield, and low degree of polymerization. It is concluded that, in this area, polycondensation can hardly compete with elaborate stepwise approaches as a result of the intrinsic low reactivity—both as a glycosyl donor and as glycosyl acceptor—of the D -glucosamine α 1 → 4 L -iduronic acid building blocks.


Introduction
As part of a program on the molecular basis of the activation of fibroblast growth factors (FGFs) by glycosaminoglycans (GAGs) we have developed a modular approach for a completely stereoselective block synthesis of oligosaccharides containing the repeating unit (D-glucosamine α1→4 L-iduronate) of the major sequence of heparin. 1 This approach, which has been extended successfully to solid-phase synthesis using an acceptor-bound strategy, 2 has allowed the

Results and Discussion
2][3] We thought that a polycondensation reaction of a properly selected disaccharide building block as the repeating unit would represent an attractive alternative to the stepwise synthesis for the preparation of regular sequences.The synthesis of polysaccharides using a controlled chemical polymerization has been attempted previously with success in a few cases, 8 and the enzymatic polymerization of a disaccharide building block has been used to synthesize a fragment of the GAG hyaluronan. 9Cationic ringopening-polymerization has been reported from 1,2-ortho-esters 10 and 1,6-anhydrohexoses, 8c and polycondensation reactions have been described from 1,2-cyanoethylene derivatives.8a We envisaged the possibility of generating homogeneously protected heparin oligosaccharide fragments by autocondensation of a suitably protected disaccharide repeating unit as indicated in Chart 1.For their proven efficiency in our stepwise synthesis of heparin oligosaccharides [1][2][3] we first used trichloroacetimidates as leaving groups.Thus, the preparation of a bifunctional trichloroacetimidate carrying both glycosyl-donor and glycosyl-acceptor functionalities was attempted from disaccharide 1 1,3a which is currently used in our stepwise syntheses.Compound 1 was converted into 2 following a well established sequence, 1,3a  Attempted selective installation of the trichloroacetimidate group at the anomeric position of diol 3 by treatment with a slight excess of trichloroacetonitrile in the presence of K 2 CO 3 12 afforded lactone 4 as the main product (Scheme 2).When this reaction was performed with excess of reagent a mixture of 4 and the trichloroacetimidate 5 was formed.The unexpected formation of 4 by intramolecular transesterification of the methyl ester group with the free anomeric hydroxyl group appears to be a consequence of the presence of the free 4'-OH in 3, since this reaction has never before been observed in trichloroacetimidate formation in this series.In this reaction, which involves a conformational inversion of the L-iduronic acid pyranoid ring followed by the base-promoted transesterification of the methyl ester, the 4'-OH may be involved in the activation of the ester function through a hydrogen bridge.In any case, these results demonstrated that trichloroacetimidate anomeric activation is not suitable for straightforward generation of the disaccharide repeating unit and other activation methods had to be investigated.
We then turned our attention to n-pentenyl activation as pentenyl glycosides 13 had been reported to be effective glycosylating agents in the coupling of L-iduronic acid derivatives to the 4-OH group of D-glucosamine units. 14However, the installation of the n-pentenyl group at the anomeric position of a L-iduronic acid derivative has proven to be feasible only after previously activating that position as a bromide or a trichloroacetimidate. 15 Therefore, the known trichloroacetimidate 6 3c was reacted 16 with excess pent-4-en-1-ol to give the n-pentenyl glycoside 7 in 62% yield (Scheme 3).Scheme 3. (i) 4-penten-1-ol, TMSOTf, CH 2 Cl 2 , 47%.
Compound 7 was transformed into the bifunctional building block 9 by trans-acetalation of the benzylidene acetal 17 to give diol 8 followed by selective benzoylation 18 of the primary hydroxyl group in 8 (Scheme 4).Compound 9, endowed with a leaving group at the anomeric position and a free hydroxyl group at the 4'-position, and having a protecting group pattern encoding the negative charge distribution of the regular region of heparin was then submitted to autocondensation conditions.It was expected that the reaction of 9 with a promoter (equimolecular NIS and catalytic Et 3 SiOTf 19 ) would generate a glycosyl carbonium ion 13b that may react with any nucleophile present in the reaction mixture.Under strictly anhydrous conditions, the carbonium ion would react with the 4'-OH group of a second disaccharide building block to give a tetrasaccharide species that itself carries donor-acceptor functionalities.This tetrasaccharide species may give rise to a hexasaccharide or an octasaccharide depending on the concentration of each species, the nucleophilicity of the acceptor OH group, and the concentration of the promoter.
Compound 9 was dissolved in dichloromethane under strictly anhydrous conditions and treated with solid NIS and a catalytic amount of Et 3 SiOTf 19 at 0º C. The reaction was monitored by TLC.When no further evolution was observed the reaction mixture was acetylated by treatment with acetic anhydride and pyridine in the presence of DMAP. 20It was then submitted to extensive TLC fractionation and compounds 10-13 could be isolated and characterized (Scheme 5).The presence of the N-succinyl derivatives 10 and 11 indicated that under the reaction conditions the succinimide nucleophile competes strongly with the 4'-OH group for the NIS-Et 3 SiOTf-generated glycosyl carbonium ion, and this leads to a rapid end of the chain reaction.The formation of N-succinyl glycosides has been reported as a side reaction in the glycosylation of glycosyl acceptors of low reactivity with thioglycosides. 21The fact that Nsuccinyl glycosides are formed at the disaccharide level (compound 11), and the absence of higher oligomers in the reaction mixture, are clear indications of the limited efficiency of this offset for autocondensation reactions.When the reaction was carried out under TMSOTf catalysis with 1.1 eq. of NIS in dichloromethane, a fraction containing tetra-and hexa-saccharide was formed in 11% yield (calculated from (NMR).However, higher oligomers from subsequent glycosylation cycles could never be observed.

Conclusions
From this exploratory study it can be concluded that the glucosamine α1→4 L-iduronate building block is not a suitable candidate for self-condensation, because it has low reactivity both as a glycosyl donor and as a glycosyl acceptor.In fact, uronic acids had a poor performance under the classical tritylcyanoethylidene polycondensation conditions. 22The use of a trichloroacetimidate function for anomeric activation seems to be precluded since the presence of a free 4'-OH in the disaccharide building block leads to intramolecular transesterification of the methyl ester with the free anomeric hydroxyl group during the attempted selective installation of the trichloroacetimidate group in the anomeric position.The use of more stable leaving groups, such as the n-pentenyl group, for anomeric activation allows protecting-group manipulation, but suffers from the fact that equimolecular amounts of promoter are required.Being a nucleophile itself, the promoter competes with the acceptor alcohol in the polycondensation process, which results in fast chain-reaction termination and a low yield and degree of polymerization.Since the preparation of the starting disaccharide building block involves a laborious process, the synthesis of homogeneous heparin oligosaccharide fragments by self-condensation does not seem to compete with the sequential assembly of disaccharide building blocks using classical deprotection-glycosylation strategies.

Experimental Section
General Procedures.Tetrahydrofuran, toluene, 1,4-dioxane and diethyl ether were distilled from sodium under nitrogen prior to use.Dichloromethane, and acetonitrile were distilled from calcium hydride and used immediately after distillation.DMF, pyridine and methanol were commercial high-grade solvents (SDS, Prolabo, France) and stored under Argon over molecular sieves (4Å).All reagents were purchased from Aldrich, Fluka, Lancaster, Panreac, Acros, or SDS.All reactions were carried out under argon in pre-dried glassware, unless otherwise stated.Analytical thin layer chromatography (TLC) was performed on silica gel 60 F 254 pre-coated on aluminum plates (Merck, Darmstadt) and the compounds detected by staining with phosphomolybdic acid/EtOH or with anisaldehyde solution [anisaldehyde (25 ml) with sulfuric acid (25 ml), ethanol (450 ml) and acetic acid (1ml)] with detection by heating above 200ºC.Flash-column chromatography was carried out on silica gel 60 (0.2-0.5 mm, 0.2-0.063mm or 0.040-0.015mm; Merck, Darmstadt) under a pressure of 0.3-0.8bar.Preparative TLC purifications were carried out on Merck 20x20 cm silica-gel 60 F 254 -plates.Gel-permeation chromatography used Sephadex LH-20 and G-25 gels (medium and fine) from Pharmacia, on Pharmacia columns equipped with a fraction collector.Optical rotations were determined with a Perkin-Elmer 341 polarimeter at 589 nm and r.t. 1 H-and 13 C-NMR, as all two-dimensional spectra were acquired on Bruker DPX-300, DRX-400 and DRX-500 spectrometers, and chemical shifts are given in ppm (δ) relative to tetramethylsilane as internal reference or relative to D 2 O. FAB MS were measured by the Mass Spectrometry Service, Facultad de Química, Seville, with a Kratos MS-80 RFA spectrometer, MALDI-TOF-MS were recorded on a Hewlett Packard 2030A system, usually with 2,5-dihydroxybenzoic acid (DHB-matrix).Elemental analysis was performed on a Leco CHNS-932 apparatus, after drying the sample over phosphorus pentoxide.