Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins

Abstract

Ultralow molecular weight (ULMW) heparins are sulfated glycans that are clinically used to treat thrombotic disorders. ULMW heparins range from 1500 to 3000 daltons, corresponding from 5 to 10 saccharide units. The commercial drug Arixtra (fondaparinux sodium) is a structurally homogeneous ULMW heparin pentasaccharide that is synthesized through a lengthy chemical process. Here, we report 10- and 12-step chemoenzymatic syntheses of two structurally homogeneous ULMW heparins (MW = 1778.5 and 1816.5) in 45 and 37% overall yield, respectively, starting from a simple disaccharide. These ULMW heparins display excellent in vitro anticoagulant activity and comparable pharmacokinetic properties to Arixtra, as demonstrated in a rabbit model. The chemoenzymatic approach is scalable and shows promise for a more efficient route to synthesize this important class of medicinal agent.

Fig. 1

Chemoenzymatic synthetic schemes of ULMW heparin construct 1 and 2. The synthesis started from disaccharide 3, and it was then elongated to tetrasaccharide 4. Eight additional steps transformed 4 to construct 1 (left column). Steps d through h were combined in sequential one-pot reaction format. Ten additional steps transformed 4 to construct 2 (right column). The recovery yield at each purification step was determined by parallel synthesis of the corresponding radioactively labeled oligosaccharide. KfiA, N-acetyl glucosaminyl transferase of E. coli K5 strain; pmHS2, heparosan synthase-2 of Pasteurella multocida; NST, N-sulfotransferase; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; C₅-epi, C₅-epimerase; 2-OST, 2-O-sulfotransferase; 6-OST, 6-O-sulfotransferase; 3-OST-1, 3-O-sulfotransferase isoform 1. More synthesis details are given in table S3.

Fig. 2

Structural characterization of ULMW heparin construct 1. (A) The DEAE-HPLC profile of a ³⁵S-labeled product. (B) The ESI-MS spectrum of construct 1. Peaks 1 to 3 represent the desulfated signals of quadruply charged ions. Peaks 4 to 7 represent the desulfated signals of triply charged ions. Peaks 8 to 12 represent the desulfated signals of doubly charged ions. (C) The 1D ¹H NMR spectrum of construct 1. Peaks assigned to the anomeric protons of each hexose ring (A to F) are labeled. (D) The 2D correlation spectroscopy spectrum of construct 1 and the corresponding peak assignments of the anomeric protons that resonate as doublets at δ5.48 (d, J = 3.23 Hz, 2H), 5.35 (d, J = 2.94 Hz, 1H), 5.09 (broad doublet, 1H), 4.53 (d, J = 8.11 Hz, 1H), and 4.46 (d, J = 8.07 Hz, 1H) ppm. The small coupling constants (~3 Hz) of the anomeric protons indicate an α linkage, and the large coupling constants (~8 Hz) indicate a β linkage.

Fig. 3

Determination of the anticoagulant activities and pharmacokinetic properties of ULMW heparin construct 1 and 2. (A) shows the anti-Xa activity using a chromogenic substrate. Arixtra and constructs 1 and 2 were incubated with AT (240 nM), factor Xa (5.9 nM), and the peptide substrate (289 μM). The activity of Xa was determined by the rate of increase of the absorbance at 405 nm. The activity without drugs was defined as 100%. Each data point represents the average of four determinations ± SD. (B) The pharmacokinetic profiles in rabbits. Arixtra and constructs 1 and 2 were each independently administered subcutaneously at 120 μg/kg to three rabbits (n = 3) and plasma samples were collected from 0 to 24 hours. The anti-Xa activity of plasma samples was measured against a standard curve (fig. S8). The area under the curve for Arixtra, construct 1, and construct 2 were 457, 473, and 802, respectively. Error bars, mean ± SD.