Chemical synthesis, heparin

Petitou M and van Boeckel CAA (1992) Chemical synthesis of heparin fragments and analogues. Prog Chem Org Nat Prod 60, 143-210. 

Compounds known to behave in this way in vivo are listed in recent reviews in this Series.1 2 The structures of some of the /3-D-glucopyranosiduronic acids isolated from urine have been proved by chemical synthesis.3 A few similar derivatives of flavones and triterpenes have been isolated from plants. D-Glucuronic acid also occurs in mammalian tissues as a constituent of acid mucopolysaccharides (aminodeoxypolysaccharides, containing uronic acid), such as hyaluronic acid, chondroitinsulfate, and heparin,4 and it is a direct precursor of L-ascorbic acid in plants and mammals.6 It is present in many of the plant polysaccharides classified as hemicelluloses6 and gums,7 and it has also been found in certain bacterial polysaccharides.4... 

Heparin/heparan, hyaluronan, and chondroitin are three prevalent glycosaminoglycans. Vertebrates use glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Chemical synthesis of naturally occurring polysaccharides is considered to be impractical. Most polysaccharides, especially those from bacteria origins, are obtained by purification from natural sources or from cell culture, enzymatic approaches have been increasingly applied to obtain some structures. 

Chemical synthesis of heparin fragments and analogues has been used for biological studies and to define structure-activity relationships [46]. For example, the heparin-antithrombin m interaction [47,48,49,50,51,52,53], which is responsible for the anticoagulant activity of heparin, has been studied in detail by using synthetic fragments. The interaction of heparin with FGF-1, FGF-2, platelets [54,55,56,57,58,59,60,61,62,63,64], and the Herpes Simplex virus, has also been studied by using synthetic specimens. 

A customary approach to research concerning the biological activity of a macromolecule focuses on its very detailed chemical structure. If smaller portions of such a molecule exhibit the desired activity, one can more easily duplicate its detailed structure or vary and examine the significance of one or the other structural detail. Heparin fragments of 5 to 7 sugar units have indeed been shown to be active (jj.). However, the structural/chemical complexity is still so large that the creation of structural variants or duplicates by chemical synthesis requires an extraordinarily large number of synthetic steps (12.13). ... 

The total synthesis of a series single glyco-mimetics of heparin was recently announced (169). One structurally optimized oligosaccharide heparin mimetic is 10 times more potent in vivo than both standard heparin and low molecular weight heparins and is also devoid of the undesired side effect, thrombocytopenia, that is associated with heparin treatment. Thus, chemical synthesis was employed to optimize the length and the charge of the synthetic oligosaccharides. 

G. Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide 0-(2-deoxy-2-sulfamido-6-0-sulfo-alpha-D-glucopyranosyl)-... 

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

This process starts with the synthesis of novel chemical compounds. Substances with complex structures may be obtained from various sources, e.g., plants (cardiac glycosides), animal tissues (heparin), microbial cultures (penicillin G), or human cells (urokinase), or by means of gene technology (human insu-Un). As more insight is gained into structure-activity relationships, the search for new agents becomes more clearly focused. 

The most representative example utilizing this approach is the synthesis of the ultralow molecular weight (ULMW) heparin construct 27 (Scheme 9.2), a heptasac-charide that has a very similar structure to the FDA-approved anticoagulant drug fondaparinux. Fondaparinux is currently synthesized chemically through 50 steps... 

While this approach afforded gram quantities of a heparin-like polysaccharide with anticoagulant activity, unnatural saccharide units, such as 3-0-sulfo-D-glucuronic acid, were present in their product, suggesting a limitation in the selectivity of chemical sulfonation/desulfonation in HS synthesis. 

PPy is a conductive polymer with several biomedical applications owing to its good biocompatibility and the ability of cells to attach, differentiate and proliferate on its surface [148,149]. In one study, the attachment, proliferation and differentiation of rat MSCs on PPy surfaces was shown to be comparable to those of regular tissue culture plastic surfaces [150]. The synthesis of PPy involves either electrochemical or chemical polymerization, with the admicellar polymerization technique enabling the uniform deposition of thin PPy films from a few to 100 nm thick [151]. Moreover, the attachment of cells to the PPy surface can be improved, for example, via the adsorption of fibronectin or the incorporation of Arg-Gly-Asp (RGD)-containing peptides [152]. Immobilization of the glycosaminoglycans heparin and hyaluronic acid on PPy surfaces was also shown to maintain bone marrow-derived MSC cultures and to induce differentiation successfully into mature osteoblasts [153]. 

---