Tailor-made alginate bearing galactose moieties on mannuronic residues: selective modification achieved by a chemoenzymatic strategy.

1-Amino-1-deoxygalactose (12%, mole) has been chemically introduced on a mannuronan sample via an N-glycosidic bond involving the uronic group of the mannuronic acid (M) residues. The unsubstituted M residues in the modified polymer were converted into guluronic moieties (G) by the use of two C-5 epimerases, resulting in an alginate-like molecule selectively modified on M residues. The molecular details of the newly formed polymer, in terms of both composition and molecular dimensions, were disclosed by use of (1)H NMR, intrinsic viscosity, and high-performance size-exclusion chromatography-multiple-angle laser light scattering (HPSEC-MALLS). Circular dichroism has revealed that the modified alginate-like polymer obtained after epimerization was able to bind calcium due to the introduction of alternating and homopolymeric G sequences. The gel-forming ability of this M-selectively modified material was tested and compared with an alginate sample containing 14% galactose introduced on G residues. Mechanical spectroscopy pointed out that the modified epimerized material was able to form stable gels and that the kinetics of the gel formation was similar to that of the unsubstituted sample. In contrast, the G-modified alginate samples showed a slower gel formation, eventually leading to gel characterized by a reduced storage modulus. The advantage of the selective modification on M residues was confirmed by measuring the Young's modulus of gel cylinders of the different samples. Furthermore, due to the high content in alternating sequences, a marked syneresis was disclosed for the modified-epimerized sample. Finally, calcium beads obtained from selectively M-modified alginate showed a higher stability than those from the G-modified alginate, as evaluated upon treatment with nongelling ions.

Chemoenzymatic synthesis of classical and non-classical anticoagulant heparan sulfate polysaccharides.

Heparan sulfate (HS) polysaccharides interact with numerous proteins at the cell surface and orchestrate many different biological functions. Though many functions of HS are well established, only a few specific structures can be attributed to HS functions. The extreme diversity of HS makes chemical synthesis of specific bioactive HS structures a cumbersome and tedious undertaking that requires laborious and careful functional group manipulations. Now that many of the enzymes involved in HS biosynthesis are characterized, we show in this study how one can rapidly and easily assemble bioactive HS structures with a set of cloned enzymes. We have demonstrated the feasibility of this new approach to rapidly assemble antithrombin III-binding classical and non-classical anticoagulant polysaccharide structures for the first time.

Synthesis of the pentadecapeptide sequence of the active site of rabbit muscle triosephosphate isomerase.

The pentadecapeptide fragment, Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Trp-Ala-Ile-Gly-Thr-Gly-Lys, which constitutes a part of the active site of rabbit muscle triosephosphate isomerase has been synthesized. It does not exhibit any catalytic activity typical of triosephosphate isomerase.