Heparin mapping using heparin lyases and the generation of a novel low molecular weight heparin - PubMed (original) (raw)

. 2011 Jan 27;54(2):603-10.

doi: 10.1021/jm101381k. Epub 2010 Dec 17.

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Heparin mapping using heparin lyases and the generation of a novel low molecular weight heparin

Zhongping Xiao et al. J Med Chem. 2011.

Abstract

Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2, or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH(2)CONHCH(2)COOH (where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, Gal is β-d-galactpyranose, and Xyl is β-d-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the undersulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) is afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.

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Figures

Figure 1

Figure 1

Schematic representation of heparin and heparin derived oligosaccharides. Heparin is polydisperse with chains of molecular weight 5,000–40,000 Dalton with a degree of polymerization from dp20 to dp160 (n + m = 4–74). Two under sulfated domains (<2 sulfo groups per disaccharide unit), indicated at the reducing end and in the internal portion (when R = H) of the chain, are heparin lyase 3 cleavable. The linkages labeled with A in 4c, C in 6c, D and E in 6d, and F in 6a, can be digested by heparin lyase 3. The linkages labeled B in 6b and G in 8a, each connected at their non-reducing end to a fully sulfated (6 sulfo groups) tetrasaccharide residue, are resistant to heparin lyase 3.

Figure 2

Figure 2

Chromatograms of heparin lyases produced oligosaccharides for heparin mapping. SAX-HPLC chromatograms are: a. heparin lyase 1 treated heparin; b. heparin lyase 2-treated heparin; and c. heparin lyase 3 treated heparin. The structures determined for each peak are: 2a ΔUA-GlcNAc, 2b ΔUA-GlcNS6S, 2c ΔUA2S-GlcNS, 2d ΔUA2S-GlcNS6S, 2e ΔUA-GlcNS, 2f ΔUA-GlcNAc6S, 2g ΔUA2S-GlcNAc, 2h ΔUA2S-GlcNAc6S, 4a ΔUA2S-GlcNS-IdoA2S-GlcNS, 4b ΔUA2S-GlcNS6S-IdoA2S-GlcNS, 4c ΔUA2S-GlcNS6S-GlcA-GlcNS6S, 4d ΔUA2S-GlcNS6S-IdoA2S-GlcNS6S, 4e ΔUA-Gal-Gal-Xyl-_O_-CH2CONHCH2COOH, 4f ΔUA-GlcNAc6S-GlcA-GlcNS3S, 4g ΔUA-GlcNAc6S-GlcA-GlcNS3S6S, 4h ΔUA-GlcNS6S-GlcA-GlcNS3S6S, 4i ΔUA-Gal-Gal-Xyl-_O_-Ser, 6a ΔUA2S-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S.

Figure 3

Figure 3

1D and 2D NMR spectra of the linkage core tetrasaccharides. Spectra for 4i are: a. 1D 1H-NMR; b. 1H, 1H-COSY; and c. 1H, 13C-HMQC (insert shows the anomeric signals). Spectra for 4e are: d. 1D 1H-NMR; e. 1H, 1H-COSY; and f. 1H, 13C-HMQC (insert shows the anomeric signals). The structures and labeled positions can be found in Figure S4.

Figure 4

Figure 4

Heparin oligosaccharide mapping results based on the quantification of SAX-HPLC data. Heparin (_M_W 12 000 Da) at 83 μM was treated with: a. heparin lyase 1; b. heparin lyase 2; and c. heparin lyase 3. The micromolar concentrations of each product are shown with standard deviations. The structures of these oligosaccharides can be found in Figure 2.

Figure 5

Figure 5

Heparin lyase 1 and heparin lyase 2 action pattern studies using decasaccharide 10a as a model substrate. 10a ΔUA2S-[-GlcNS6S-IdoA2S-]4-GlcNS6S. SAX-HPLC chromatograms of a digestion time course are shown for a. heparin lyase 1 and b. for heparin lyase 2. The red arrows indicate increasing digestion time. PAGE (22% total acrylamide) analyses of 10a treated by heparin lyase 1 and heparin lyase 2 are shown in c. where lane 1 is 4d; lane 2 is 8b ΔUA2S-[-GlcNS6S-IdoA2S-]3-GlcNS6S; lane 3 through lane 8 are 10a incubated with heparin lyase 1 from time-point 0 to time-point 5; lane 9 through lane 13 are 10a treated by heparin lyase 2 from time-point 0 to time-point 5; lane 8 and lane 13 are 10a exhaustively digested by heparin lyase 1 and heparin lyase 2, respectively. The mole percent of each size product (indicated with different symbols) is plotted as a function of percent reaction completion in d. for heparin lyase 1 (dotted lines) and heparin lyase 2 (solid lines).

References

    1. Rabenstein DL. Heparin and heparan sulfate: structure and function. Nat Prod Rep. 2002;19:312–331. -PubMed
    1. Linhardt RJ. Perspective: 2003 Claude S. Hudson Award Address in Carbohydrate Chemistry. Heparin: Structure and Activity. J Med Chem. 2003;46:2551–2564. -PubMed
    1. Toida T, Linhardt RJ. Structure and bioactivity of sulfated polysaccharides. Trends Glycosci Glycobiol. 2003;15:29–46.
    1. Petitou M, Casu B, Lindahl U. 1976–1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie. 2003;85:83–89. -PubMed
    1. Capila I, Linhardt RJ. Heparin-protein interactions. Angew Chemie Int Ed. 2002;41:390–412. -PubMed

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