Enzymatic synthesis of glycosaminoglycan heparin

Abstract

Heparin and its low molecular weight heparin derivatives, widely used as clinical anticoagulants, are acidic polysaccharide members of a family of biomacromolecules called glycosaminoglycans (GAGs). Heparin and the related heparan sulfate are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that currently is prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, micro- and nano-display of GAGs, rapid on-line structural analysis, and microarray/microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities. The advent of these new technologies also makes it possible to consider the construction of an artificial Golgi to increase our understanding of the cellular control of GAG biosyntheses in this organelle.

Figure 1

Primary structure and structural variability of a heparin chain with high affinity for antithrombin III (AT). GlcN_p, glucosamine; ldoA_p, iduronic acid; GlcA_p, glucuronic acid; S, sulfo; Ac, acetyl.

Figure 2

Natural and unnatural uridine diphosphate donors.

Figure 3

Schematic synthesis of sulfonated polysaccharides and 3′-phosphoadenosine-5′-phosphosultate (PAPS) regeneration system. (A) The stepwise enzymatic synthesis of sulfonated polysaccharides using heparan sulfate (HS) sulfotransferases. The description of intermediate polysaccharides is shown in the text. Compounds 4a and 4b were prepared by inverting the order of sulfonation steps. 4a was prepared by incubating compound 1 with 2-O-sullotransferase (2-OST) followed by 6-OST, whereas 4b was prepared by incubating compound 1 with 6-OST followed by 2-O5T. (B) The reaction catalyzed by arylsulfotransferase IV to generate PAPS.

Figure 4

A schematic of a heparin glycosaminoglycan chain immobilized to a carbon nanotube.

Figure 5

Solid-phase synthesis of heparin from N-sulfoheparosan. Epi, ???; OST, O-sulfotransferase; AT, antithrombin III.

Figure 6

Simplified drawing of the Golgi. Core protein (gray ball) is biosynthesized in the endoplasmic reticulum (ER), glycosylation is initiated and core protein decorated with oligosaccharide acceptor is transferred in budded transport vesicle along microtubule tracks to the Golgi (some retrieval from the Golgi by the ER is also possible). As the glycoprotein moves through the Golgi stacks (cis to medial to trans), the glycan chain (black line) is elongated and elaborated. Mature glycoprotein exits the trans Golgi in vesicles for transport to the cell surface (some removal of excess or damaged glycoprotein takes place through lysosomal degradation).

Figure 7

Schematic representation of an artificial Golgi.