Transesteri®cation of monophenyl phosphonamidatesÐchemical modelling of serine protease inhibition Artur Mucha p and Paweø Kafarski Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocøaw University of Technology, Wybrzez Çe Wyspian Âskiego 27, 50-370 Wroclaw, Poland Received 11 February 2002; revised 10 May 2002; accepted 30 May 2002 Abstract ÐO-Phenyl phosphonamidates have been designed to bind covalently by nucleophilic substitution to the serine residue in the active site of serine proteases, similarly to the diphenyl phosphonates used as standard. The synthesis of these compounds as well as their phosphonylating reactivity towards methanol, which served as mimetic of the serine nucleophile, is described. The stereochemistry of the substitution in basic solutions was studied in some detail. The stability of the phosphonamidates in aqueous solutions and their selectivity in the reaction against alcohols versus thiols proved that they constitute a class of potential inhibitors of serine proteases, as well as valuable tools to investigate the mechanism of inhibition. q 2002 Published by Elsevier Science Ltd. 1. Introduction More than one hundred members form the serine protease familyÐone of the four main superfamilies of enzymes that hydrolyse the amide bond of peptide substrates. Under normal conditions serine proteases are involved in various physiological processes, namely blood coagulation, immune defence, digestion and reproduction. Moreover, their activity is also associated with many pathological disorders and consequently with various human diseases including pulmonary emphysema, rheumatoid arthritis, in¯ammatory diseases and others. This important role makes them potential targets for the development of inhibi- tors in the hope that they might serve as new therapeutic agents against many diseases. The rational design of new inhibitors and drugs demands knowledge of chemical mechanisms of enzymatic catalysis. Generally the proteases belonging to the same family cleave peptide bonds using the same basic mechanism and the same or similar catalytic residues. In the case of the serine family, the catalytic mechanism is based on the presence of three amino acid residues in the catalytic centre, namely Ser195, His57 and Asp102 (chymotrypsin numbering system). The reactive hydroxyl group of Ser195 attacks the carbonyl group of the scissile amide bond of the substrate to form a tetrahedral intermediate stabilised by a speci®c network of hydrogen bonding. In the next step the substrate±enzyme complex collapses to the acyl intermediate, which is subsequently hydrolysed by a water molecule to regenerate the active enzyme and release the product of reaction. The hydrolysis step also proceeds via a tetrahedral transition state. The whole process is facilitated by basic±acidic catalysis coming from the presence of the imidazole ring of His57 and carboxylate residue of Asp102. The synthesis of compounds able to bind covalently to one of the amino acid residues crucial for activity, and thus blocking active-site function, was proven to be one of the most effective strategies in the construction of inhibitors for serine proteases. 1±3 Among phosphorylating agents, the most popular is diisopropyl phosphoro¯uoridate 4 (1), which nonselectively inactivates enzymes of this class, and is considered as a diagnostic tool for them. The structural features of substrate±enzyme selectivity can be assured by the use a-aminoalkylphosphono¯uoridates (2)Ðphosphorus containing analogues of amino acids. 5,6 However, their application is strongly limited by poor stability in water solutions. Although less reactive, diphenyl esters of a-aminoalkylphosphonic acids and their peptide derivatives (3) are resistant to hydrolysis and since the beginning of the last decade they have been intensively exploited as effective and selective inhibitors of various serine proteases. 7±19 They are considered as stable ana- logues of high-energy tetrahedral transition states of peptide hydrolysis able to phosphonylate the serine residue in the active site of enzymes (Fig. 1). The molecular mechanism of the action of diphenyl phos- phonates has not yet been fully elucidated. After noncova- lent binding of the inhibitor, the nucleophilic substitution proceeding on the phosphorus atom leads to the formation of an enzyme-Ser-O-phosphonate mixed ester which can be Tetrahedron 58 (2002) 5855±5863 Pergamon TETRAHEDRON 0040±4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd. PII: S0040-4020(02)00561-6 Keywords: phenyl phosphonates; phosphonamidates; transesteri®cation; serine proteases; inhibitors. p Corresponding author. Tel.: 148-71-3203354; fax: 148-71-3284064; e-mail: amucha@kchf.ch.pwr.wroc.pl