1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

Catalysis by the serine proteinases proceeds via a tetrahedral intermediate whose oxyanion is stabilized by hydrogen-bonding in the oxyanion hole. There have been extensive 13C-NMR studies of oxyanion and tetrahedral intermediate stabilization in trypsin, subtilisin and chymotrypsin using substrate-derived chloromethane inhibitors. One of the limitations of these inhibitors is that they irreversibly alkylate the active-site histidine residue which results in the oxyanion not being in the optimal position in the oxyanion hole. Substrate-derived glyoxal inhibitors are reversible inhibitors which, if they form tetrahedral adducts in the same way as substrates form tetrahedral intermediates, will overcome this limitation. Therefore we have synthesized 13C-enriched substrate-derived glyoxal inhibitors which have allowed us to use 13C-NMR and 1H-NMR to determine how they interact with proteinases. It is hoped that these studies will help in the design of specific and highly potent warheads for serine proteinase inhibitors. Introduction Catalysis by the serine proteases is thought to involve binding of a peptide substrate followed by addition of an active-site serine hydroxy group to a substrate peptide carbonyl carbon to form a tetrahedral intermediate [1–3]. This intermediate can then revert to starting materials or proceed to form an acyl intermediate by expelling the amino acid that provides the nitrogen of the peptide bond being cleaved. The acyl intermediate is then hydrolysed via a second tetrahedral intermediate to yield free enzyme and the second product. With natural substrates, it is the formation and/or breakdown of the first tetrahedral intermediate which is thought to be the ratelimiting step in catalysis. Proteolytic enzymes are thought to achieve their remarkable catalytic efficiency by transitionstate stabilization of tetrahedral intermediate formation and or breakdown. Inhibitors which mimic this tetrahedral intermediate should be tightly bound by the enzyme. Therefore it is not surprising that many proteinase inhibitors have been designed with warheads which either mimic the catalytic tetrahedral intermediate or are able to react with the active-site