A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

Abstract

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.

Keywords: carbohydrates; chemical biology; glycosaminoglycans; in silico screening; serpins.

Figure 1

a) The dual‐filter CVLS algorithm used the library of 46 656 HS hexasaccharides. GOLD score was the first filter, while consistency of binding was the second filter. b) Results from the first filter displaying the histogram of number of HS hexasaccharides for every 10 unit change in GOLD score for HCII recognition.

Figure 2

Predicted in silico “specificity” of recognition of HCII and AT by HX1–HX5 (a) and structures of five designed hexasaccharides (b). See the Supporting Information for description of calculation of in silico specificity.

Figure 3

Structures of disaccharide building blocks D1–D11. Of these, D1, D3, D4, D6, D8, D9 and D11 are donor units, while acceptor disaccharides include D2, D5, D7 and D10.

Figure 4

Synthesis of HX2 as a prototypical example of the thioglycoside donor methodology used in the synthesis of designed HS hexasaccharides. See Figures S10–S14 for detailed synthesis of HX1 through HX5 and associated procedures and characterization.

Figure 5

Comparison of the conformational activation of serpins HCII and AT induced by the CVLS‐designed HS hexasaccharides. Conformational activation was studied through increase in rate constant of inhibition of either thrombin or factor Xa, the two primary protease targets of HCII and AT, respectively. Activation of serpin (y‐axis) refers to the ratio of second order rate constant of inhibition in the presence of HX (k _HX) to that in its absence (k _UNCAT).