Manuel Martin-Lomas

The Activation of Fibroblast Growth Factors by Heparin: Synthesis, Structure, and Biological Activity of Heparin‐Like Oligosaccharides

An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D‐glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right‐hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well‐defined structural models of naturally occurring heparin‐like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half‐maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin‐induced FGF1 dimerization is not an absolute requirement for biological activity.

Inositolphosphoglycan Mediators Structurally Related to Glycosyl Phosphatidylinositol Anchors: Synthesis, Structure and Biological Activity

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.

The Activation of Fibroblast Growth Factors (FGFs) by Glycosaminoglycans: Influence of the Sulfation Pattern on the Biological Activity of FGF-1

Six synthetic heparin‐like oligosaccharides have been used to investigate the effect of the oligosaccharide sulfation pattern on the stimulation of acidic fibroblast growth factor (FGF‐1) induced mitogenesis signaling and the biological significance of FGF‐1 trans dimerization in the FGF‐1 activation process. It has been found that some molecules with a sulfation pattern that does not contain the internal trisaccharide motif, which has been proposed for high affinity for FGF‐1, stimulate FGF‐1 more efficiently than those with the structure of the regular region of heparin. In contrast to regular region oligosaccharides, in which the sulfate groups are distributed on both sides of their helical three‐dimensional structures, the molecules containing this particular sulfation pattern display the sulfate groups only on one side of the helix. These results and the fact that these oligosaccharides do not promote FGF‐1 dimerization according to sedimentation‐equilibrium analysis, confirm the importance of negative‐charge distribution in the activation process and strongly suggest that FGF dimerization is not a general and absolute requirement for biological activity.

The reaction of carbohydrate-derived alkoxyaldehydes with methoxycarbonylmethylenetriphenylphosphorane: stereoselective synthesis of β-unsaturated esters

Abstract The reaction of several carbohydrate-derive alkoxyaldehydes with methoxycarbonylmethylenetriphenylphosphorane afford α, β-unsaturated esters with Z-stereoselectivity. The stereoselectivity depends on the substrate stucture and the nature of the solvent used.

Construction of N‐Glycan Microarrays by Using Modular Synthesis and On‐Chip Nanoscale Enzymatic Glycosylation

An effective chemoenzymatic strategy is reported that has allowed the construction, for the first time, of a focused microarray of synthetic N-glycans. Based on modular approaches, a variety of N-glycan core structures have been chemically synthesized and covalently immobilized on a glass surface. The printed structures were then enzymatically diversified by the action of three different glycosyltransferases in nanodroplets placed on top of individual spots of the microarray by a printing robot. Conversion was followed by lectin binding specific for the terminal sugars. This enzymatic extension of surface-bound ligands in nanodroplets reduces the amount of precious glycosyltransferases needed by seven orders of magnitude relative to reactions carried out in the solution phase. Moreover, only those ligands that have been shown to be substrates to a specific glycosyltransferase can be individually chosen for elongation on the array. The methodology described here, combining focused modular synthesis and nanoscale on-chip enzymatic elongation, could open the way for the much needed rapid construction of large synthetic glycan arrays.

Glycosyl Inositol Derivatives Related to Inositolphosphoglycan Mediators: Synthesis, Structure, and Biological Activity

17 paginas, 12 figuras, 4 esquemas, 7 tablas.-- Supporting information for this article is available on the WWW under http://www.wiley-vch.de/home/chemistry/ or from the author.

Solution NMR structure of a human FGF-1 monomer, activated by a hexasaccharide heparin-analogue

The 3D structure of a complex formed by the acidic fibroblast growth factor (FGF‐1) and a specifically designed synthetic heparin hexasaccharide has been determined by NMR spectroscopy. This hexasaccharide can substitute natural heparins in FGF‐1 mitogenesis assays, in spite of not inducing any apparent dimerization of the growth factor. The use of this well defined synthetic heparin analogue has allowed us to perform a detailed NMR structural analysis of the heparin–FGF interaction, overcoming the limitations of NMR to deal with the high molecular mass and heterogeneity of the FGF‐1 oligomers formed in the presence of natural heparin fragments. Our results confirm that glycosaminoglycans induced FGF‐1 dimerization either in a cis or trans disposition with respect to the heparin chain is not an absolute requirement for biological activity.

Chemical Syntheses of 4-O-α and -β-D-galactopyranosyl-D-galactose and 3-O-α- and -β-D-galactopyranosyl-D-galactose

Abstract Reaction of 2,3-di- O -acetyl-1,6-anhydro-β- D -galactopyranose ( 2 ) with 2,3,4,6-tetra- O -acetyl-α- D -galactopyranosyl bromide in the presence of mercuric cyanide and subsequent acetolysis gave 1,2,3,6-tetra- O -acetyl-4- O -(2,3,4,6-tetra- O -acetyl-α- D -galactopyranosyl)-α- D -galactopyranose ( 4 , 40%) and 1,2,3,6-tetra- O -acetyl-4- O -(2,3,4,6-tetra- O -acetyl-β- D -galactopyranosyl)-α- D -galactopyranose ( 5 , 30%). Similarly, reaction of 2,4-di- O -acetyl-1,6-anhydro-β- D -galactopyranose ( 3 ) gave 1,2,4,6-tetra- O -acetyl-3- O -(2,3,4,6-tetra- O -acetyl-α- D -galactopyranosyl)-α- D -galactopyranose ( 6 , 46%) and 1,2,4,6-tetra- O -acetyl-3- O -(2,3,4,6-tetra- O -acetyl-β- D -galactopyranosyl)-α- D -galactopyranose ( 7 , 14%). The anomeric configurations of 4-7 were assigned by n.m.r. spectroscopy. Deacetylation of 4-7 afforded 4- O -α- D -galactopyranosyl- D -galactose ( 8 ), 4- O -β- D -galactopyranosyl- D -galactose ( 9 ), 3- O -α- D -galactopyranosyl- D -galactose ( 10 ), and 3- O -β- D -galactopyranosyl- D -galactose ( 11 ), respectively.

Fucosyltransferases as synthetic tools: glycan array based substrate selection and core fucosylation of synthetic N-glycans.

Two recombinant fucosyltransferases were employed as synthetic tools in the chemoenzymatic synthesis of core fucosylated N-glycan structures. Enzyme substrates were rapidly identified by incubating a microarray of synthetic N-glycans with the transferases and detecting the presence of core fucose with four lectins and one antibody. Selected substrates were then enzymatically fucosylated in solution on a preparative scale and characterized by NMR and MS. With this approach the chemoenzymatic synthesis of a series of α1,3-, α1,6-, and difucosylated structures was accomplished in very short time and with high yields, which otherwise would have required extensive additional synthetic effort and a complete redesign of existing synthetic routes. In addition, valuable information was gathered regarding the specificities of the lectins employed in this study.

Synthesis of heparin-like oligosaccharides on polymer supports.

The biological functions of a variety of proteins are regulated by heparan sulfate glycosaminoglycans. In order to facilitate the elucidation of the molecular basis of glycosaminoglycan-protein interactions we have developed syntheses of heparin-like oligosaccharides on polymer supports. A completely stereoselective strategy previously developed by us for the synthesis of these oligosaccharides in solution has been extended to the solid phase using an acceptor-bound approach. Both a soluble polymer support and a polyethylene glycol-grafted polystyrene resin have been used and different strategies for the attachment of the acceptor to the support have been explored. The attachment of fully protected disaccharide building blocks to a soluble support through the carboxylic group of the uronic acid unit by a succinic ester linkage, the use of trichloroacetimidates as glycosylating agents and of a functionalized Merryfield type resin for the capping process allowed for the construction of hexasaccharide and octasaccharide fragments containing the structural motif of the regular region of heparin. This strategy may facilitate the synthesis of glycosaminoglycan oligosaccharides by using the required building blocks in the glycosylation sequence. Published in 2003.