Combinatorial one-pot chemoenzymatic synthesis of heparin

Abstract

Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal-derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin's characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

Keywords: Bioengineered heparin; Liquid chromatography–mass spectrometry; Nuclear magnetic resonance spectroscopy; One-pot synthesis; United States Pharmacopeia.

Figure 1

The chemical structure of typical heparin repeating disaccharides.

Figure 2

Extracted ion chromatogram (EIC) of terasaccharide analysis of BRP samples a. Porcine intestinal heparin T1 = 0.8 %; T2 = 0.4 %; T3 = 0.3 %; T4 = 3.0 %; T5 = 0.5 %; Total = 5.0 % (Fu et al., 2013); b. Bovine lung heparin T1 = 0.2 %; T2 = 1.7 %; T3 = 1.2 %; T4 = 0.9 %; T5 = 0.8 %; Total = 4.8 %; c. Bioengineered heparin 16 (T1 = 0.4 %; T1’ = 0.2 %; T2 = 0.0 %; T3 = 0.4 %; T4 = 0.4 %; T5 = 0.4 %; T5’ = 0.4 %; Total = 2.2 %) d. Bioengineered heparin 17 (T1 = 1.0 %; T1’ = 0.2 %; T2 = 0.0 %; T3 = 0.2 %; T4 = 1.0 %; T5 = 0.4 %; T5’ = 0.6 %; Total = 3.4 %). The fractions identified are T1 (m/z = [477.4]²⁻, Calculated molecular mass = 956.8, Theoretical molecular mass = 956.1, Sequence = ΔUA-GlcNAc6S-GlcA-GlcNS3S), T1’ having the same mass as T1, but of undetermined structure, T2 (m/z = [496.6]²⁻, Calculated molecular mass = 994.4, Theoretical molecular mass = 994.0, Sequence = ΔUA-GlcNS-GlcA-GlcNS3S6S), T3 [m/z = [496.6]²⁻, Calculated molecular mass = 994.4, Theoretical molecular mass = 994.0, Sequence = ΔUA-GlcNS6S-GlcA-GlcNS3S], T4 (m/z = [517.4]²⁻, Calculated molecular mass= 1036.8, Theoretical molecular mass = 1036.0, Sequence = ΔUA- GlcNAc6S-GlcA-GlcNS3S6S), T5 (m/z = [536.3]²⁻, Calculated molecular mass = 1074.6, Theoretical molecular mass = 1074.0, Sequence = ΔUA-GlcNS6S-GlcA-GlcNS3S6S), T5’ having the same mass as T5, but of undetermined structure.

Figure 3

Two-dimensional HSQC spectra of porcine heparin (blue), 17 (red) and one- dimensional of ¹H spectra of 17. (A, glucosamine; I, iduronic acid; G, glucuronic acid)

Figure 4

A. Molecular weight properties of 16 and 17 as determined by size exclusion chromatography. New USP requirements for M_w (15000 < M_w < 19000) are depicted by solid lines. B. In vitro anti-IIa and anti-Xa activity of 16 and 17. Solid line marks the minimum anti-IIa activity of 180 IU/mg required by USP. Molecular weight and anticoagulant activity values for USP heparins were reported previously (Zhang et al., 2011).