A Modular Approach to a Library of Semi‐Synthetic Fucosylated Chondroitin Sulfate Polysaccharides with Different Sulfation and Fucosylation Patterns

Abstract

Fucosylated chondroitin sulfate (fCS)-a glycosaminoglycan (GAG) found in sea cucumbers-has recently attracted much attention owing to its biological properties. In particular, a low molecular mass fCS polysaccharide has very recently been suggested as a strong candidate for the development of an antithrombotic drug that would be safer and more effective than heparin. To avoid the use of animal sourced drugs, here we present the chemical transformation of a microbial sourced unsulfated chondroitin polysaccharide into a small library of fucosylated (and sulfated) derivatives thereof. To this aim, a modular approach based on the different combination of only five reactions was employed, with an almost unprecedented polysaccharide branching by O-glycosylation as the key step. The library was differentiated for sulfation patterns and/or positions of the fucose branches, as confirmed by detailed 2D NMR spectroscopic analysis. These semi-synthetic polysaccharides will allow a wider and more accurate structure-activity relationship study with respect to those reported in literature to date.

A Study for the Access to a Semi-synthetic Regioisomer of Natural Fucosylated Chondroitin Sulfate with Fucosyl Branches on N-acetyl-Galactosamine Units

“…Fuc branching regioselectivity between GalNAc O-4 and O-6 positions was measured by integration of DEPT-HSQC spectra of fCS-i-iii, assuming that the signals to be compared displayed similar 1 J CH coupling constants and that a difference of around 5-8 Hz from the experimental set value did not cause a substantial variation in the integrated peak volumes [52,53]. In particular, it was calculated the ratio of the signals at δ H/C 5.31/98.5 and 5.77/97.7 ppm, corresponding to 6-O-and 4-O-α-linked CH-1 Fuc atoms respectively (Figures S12-S14), as previously assigned by 2D-NMR spectroscopy [29]. The obtained data reveal a decrease in 6-O vs. 4-O-regioselectivity with the increase of the glycosylation temperature from −30 • C to 25 • C, even if in all cases the relative amount of 6-O-linked α-Fuc branches was much higher with respect to the 4-O-linked ones (see Table 1).…”

“…A differentiation between GalNAc and GlcA hydroxyls in Escherichia coli O5:K4:H4 sourced chondroitin could be easily done by protection of the diol on GalNAc units with a benzylidene ring [30], followed by acetylation of GlcA diol. This two-step sequence has been demonstrated to work very well on both chondroitin itself [31] and its derivative with GlcA carboxyl unit protected as methyl ester [29] to give polysaccharide derivatives 1 and 2, respectively (Scheme 1). It is very well known in synthetic carbohydrate chemistry that benzylidene rings placed on 1,3-diols, such as on O-4,6 positions of galacto-configured sugars, can be removed employing different kinds of reactions.…”

“…The last reaction has been already applied on chondroitin derivatives 1 and 2. Together with the additional chemical steps, it has allowed for the first semi-synthetic method for accessing non-animal sourced CS-A,C [31] and non-regioselectively fucosylated fCS [29,33], respectively. In order to gain a higher Fuc branching regioselectivity in the semi-synthesis of fCS polysaccharides, alternative methods for benzylidene cleavage based on hydrolytic or reductive reactions were here investigated (Scheme 1).…”

“…The reductive opening methods already developed for the cleavage of benzylidene rings on monosaccharides or short oligosaccharides are based on borane-or silane-type reagents. Among the several variants that have been reported [32], we focused our attention on those showing the following features, necessary for an application on chondroitin polysaccharide derivative 2: [29] (i) a high regioselectivity on galacto-configured sugars, and (ii) the possibility to avoid very low temperatures (e.g., −78 • C) for gaining a satisfying regioselectivity in benzylidene opening. The latter feature is desirable because it is highly unlikely that polysaccharide 2 is soluble in any solvent at very low temperatures.…”

“…This last polysaccharide shares the same linear backbone with CS but without any sulfate [28]. Such approach allowed the obtainment of a collection of semi-synthetic fCS polysaccharides differing for the sulfation pattern and the position of Fuc branches [29]. Some of these products exhibited a promising activity in some preliminary anticoagulant assays, such as AT-dependent activity against factor Xa and HC-II-mediated anti-factor IIa activity.…”