Synthesis of Tailor‐Made Glycoconjugates Showing AT III‐Mediated Inhibition of Blood Coagulation Factors Xa and Thrombin

Abstract

The sulfated glycosaminoglycan heparin binds with high affinity to the plasma protein antithrombin 111 (ATIII), thereby accelerating its inhibitory activity towards factor Xa and thrombin, two serine proteases involved in blood coagulation.".The AT 111-binding region in heparin consists of a unique pentasaccharide (PS) domain,[3. 41 the synthetic counterpart of which was found to accelerate the ATIII-mediated inhibition of factor Xa but not that of t h r~m b i n . [~,~] In the course of studies on the preparation of PS analogues['-' I the molecular requirements for the specific binding of PS to AT 111 became transparent and a three-dimensional model of the interaction of PS and ATIII could be cons t r u~t e d . The next challenge was to extend the concept of ATIII-mediated inhibition of factor Xa by PS towards synthetically feasible derivatives displaying both antifactor Xa and antithrombin activity.It is now well recognized that for ATIII-mediated inhibition of thrombin a heparin fragment comprising at least 18 saccharide units is req~ired,"~] facilitating the binding of AT 111 and thrombin to the same polysaccharide chain (the so-called "bridge" or "template" mechanism) (Fig. 1 ) .[I5] In the formation of the heparin/AT IIT/thrombin ternary complex the unique PS sequence interacts specifically with ATIII, and any sulfated oligosaccharide fragment along the heparin chain interacts in a less specific mode with thrombin." 5 . 16] Our recent model of the heparin/AT III/thrombin ternary complex reveals that heparin analogues may be obtained when a thrombin-binding oligosaccharide is tethered to the nonreducing terminus of the AT IIIbinding pentasaccharide with a spacer about 50 atoms in length (Fig. To this end, glycoconjugate I (see Scheme 3) has been synthesized, which comprises the ATIII-binding PS 1 (AT 111-binding domain, ABD), a linear spacer, and a persulfated maltotrioside moiety (thrombin-binding domain, TBD). For the construction of a pentasaccharide (ABD) containing a functionalized molecular spacer at its nonreducing end, the following key synthetic steps have to be performed (Scheme 1): a) glycosylation of the tetrasaccharide acceptor 3 with 6-0- To synthesize donor 2, methyl 6-0-benzyl-2,3-di-0-methyl-x-D-glucopyranoside[' 91 was treated with tetraethylene glycol ditosylate in D M F in the presence of sodium hydride at 40°C followed by addition of sodium azide to the reaction mixture and elevation of the temperature to 70 "C. Subsequent acetolysis using 1 % sulfuric acid in acetic anhydride at -20 "C and treatment with piperidine and trichloroacetonitrile/cesium carbonate afforded the glycosyl donor 2 in 30 % overall yield.Trimethylsilyl trifluoromethanesulfonate (TMSOTf) mediated coupling of 2 with 3 at -20°C afforded the a-linked pentasaccharide 4 in 65 YO yield after purification (Scheme 1). Concomitant hydrogenolysis of benzyl esters, benzyl ethers, and the azide group of 4 followed by saponification gave pentasaccharide 5 (75 'YO overall yield), which bears an amino function for attaching the ...

Conjugation of ATIII‐Binding Pentasaccharides to Extend the Half‐Life of Proteins: Long‐Acting Insulin

“…The solution was gently stirred and degassed for another 30 min. Then PS 5, 6, or 7 [23] (23 mg, 2.5 equiv) was added as a solid, followed by the addition of NH 2 OH (50 mL, 0.05 m). The reaction mixture was stirred under a nitrogen atmosphere at ambient temperature.…”

“…To assess whether the half-life of the insulin conjugates can be tuned by altering the affinity for ATIII, three different carrier PSs (PS 1 , PS 2 , or PS 3 ) [22,23] were introduced into recombinant human (recH) insulin with a pharmacologically inactive ethylene glycol spacer using thiol-maleimide coupling [24] chemistry (see Scheme 1 and Supporting Information). Thus, the two complementary di-N-Boc-protected [5b, 25] recH insulins 1 or 3 were treated with the succinic ester derivative of g-maleimidobutyric acid (GMB) to give, after acidic removal of the NBoc groups and purification by preparative HPLC, the B1-GMB and B29-GMB insulins 2 and 4, respectively.…”

Heparin mimetics with anticoagulant activity

“…This finding informed the design of tailor‐made glycoconjgugates such as 8 and 9 (Figure ) with the full anticoagulant properties of heparin, consisting of synthetic ABD and TBD domains linked through a molecular spacer. Initially, a nonglycosaminoglycan ABD pentasaccharide (i.e., idraparinux) was connected via a molecular spacer to a persulfated maltotrioside as the TBD . This study revealed that the anticoagulant activity was dependent on all three domains of the conjugate.…”

“…Synthetic tailor‐made glycoconjugates consisting of ABD and TBD through flexible ( 8 ) and rigid ( 9 ) molecular spacers…”

Chemistry and Biology of Heparin Mimetics that Bind to Fibroblast Growth Factors

“…Sulfated pentasaccharide connected to different spacers (charged, neutral, linear, flexible or rigid) were prepared in order to obtain heparin-like oligosaccharides with full anticoagulant properties [94,95]. Hindsgaul and co-workers [98] have shown that evaluation of carbohybrid libraries composed of D-galactopyranose that carry a diverse range of small non-carbohydrate aglycon structures led to the identification of M inhibitors of a galactose binding plant lectin.…”