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et al. [21]). Among these hirudin analogs, the tetraglycine linker appeared optimal (3–8, K i = 2.3 nM,
Table 2).
Page 260
Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin. Incorporation of a
ketomethylene pseudo peptide bond (3–4) resulted in a noncleavable bivalent inhibitor that retained high
thrombin affinity [24]. Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character
continues to be observed.
Chemically simpler linkers were made using multiple methylene-containing glycine variants [25]. The
dependence of affinity on placement of amide linkage within linkers containing the same number of
atoms indicated some specific thrombin-to-linker interactions (3–13,14,15,16). This was confirmed in
the crystal structure of hirutonin-6:thrombin complex (3–26 [26]) where continuous electron density was
observed for the entire bivalent inhibitor including the linker region.
The extended nature of the fibrinogen recognition site complicates attempts to reduce inhibitor
molecular weight while maintaining affinity. Although of similar molecular weight, substitution of the
sequence -Asp-Tyr-Glu-Pro-lle-Pro-Glu-Glu-Ala-cyclohexylalanine-(D-Glu) for -Asp-Phe-Glu-Glu-lle-
Pro-Glu-Glu-Tyr-Leu-Gin increases affinity an order of magnitude (compare 3–17 and 3–18). Within a
series of bivalent inhibitors, inclusion of sulfated tyrosine, the naturally occurring residue of hirudin,
increases affinity 5 to 6 fold (3–8 compared to 3–11, and 3–1 to 3–2). Only seven residues are present in
one of the smallest bivalent inhibitors (3–26).
Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding
portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (D-
Phe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (3–17, [27]) or 4-tert-butylbenzenesulfonyl-Arg-
(D-pipecolic acid) (3–18, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Argcontaining
inhibitors make similar interactions with the aspartic acid within the S1 specificity pocket,
the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests
that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors.
Recently, a pyridinium methyl ketone bivalent inhibitor capable of forming a reversible covalent
complex with thrombin was synthesized (3–26, [28]). Crystallographic analysis of its complex with
thrombin showed the ketone carbonyl becomes tetrahedrally coordinate by bonding to the side chain of
thrombin's active site residue, Ser195. Substitutions of cyclohexylalanine for phenylalanine (3–4
compared to 3–5) and the cyclohexylalanine-containing fibrinogen recognition peptide for the hirudin
sequence (3–17 compared to 3–18) also contribute to the increased affinity of this bivalent inhibitor.
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