Inhibition of Trypsin with Active-Site-Directed Enzyme-Activated N-Nitrosoamide Substrates

Abstract

A series of active-site-directed enzyme-activated nitrosoamide inhibitors of trypsin has been designed, synthesized, and tested. The inhibitors contain an N-nitrosoamide group that can generate an alkylating agent and a positively charged ammonium ion group at the end of an aliphatic carbon chain that provides specificity. The half-lives of inhibition under normal conditions were 0.6 to 2 min for compounds in series 1. One of the compounds, N-(4-amino-1-butyl)-N-nitrosobenzamide (1b), is a very efficient inhibitor; its partition ratio, k2/kinact, is zero suggesting that it may be a useful titrant for trypsin and related enzymes. The extent of inhibition is substantially decreased by the competitive inhibitor benzamidine, indicating that the inhibitors were operating in the active site. Two modes of inhibition were noted: reversible and irreversible. The N-nitrosoamide inhibitors bind to the trypsin binding pocket guided by the primary specificity. They then acylate the enzyme (at Ser-195), producing a leaving group that generates diazonium ions (or) carbocations in the active site; these react with a proximal carboxylic acid side chain of the enzyme to form a carboxylic acid ester, presumably that of Asp-194. If primary amino groups are present on the alkyl group, the ester (13C NMR delta 67.2 ppm for R1COO13CH2-(CH2)nNH2) rearranges into the amide form (13C NMR delta 62.9 ppm for R1CONH(CH2)n13CH2OH) through an O-->N acyl migration; an irreversibly inhibited enzyme results. A model based on the orientation of the site-specific group of N-(4-amino-1-butyl)-N-nitroso-N'-isobutyryl-D-alaninamide (D2a) and its L antipode in the active site of trypsin is proposed to explain the preferential inhibition of trypsin by the D-isomer. Analysis of the structure--inhibition relationship revealed four factors that determine the inhibition modes of the inhibitors: 1, length of the alkyl group; 2, stability of the acyl-enzyme; 3, the option O--N acyl migration; and 4, chirality.

Application of the O-N Intramolecular Acyl Migration Reaction in Medicinal Chemistry

“…Irreversible trypsin inhibitors were developed by White and Chen [150]. They suggested a formation of 4-aminobutan-1-ol in situ from nitrosoamide inhibitors, and reaction of an acid moiety in the enzyme with the hydroxy group of this aminobutanol, followed by an O-N intramolecular acyl migration to form the corresponding N-(4-hydroxy-1-butyl)carboxyamide, finally resulting in the formation of an irreversibly inhibited trypsin.…”

O−N Intramolecular Alkoxycarbonyl Migration of Typical Protective Groups in Hydroxyamino Acids

“…O−N Intramolecular acyl migration is a well-known rearrangement reaction that has been applied in peptide ligation, the synthesis of “difficult” peptides, − peptidomimetics, − and lactams, , as well as in water-soluble prodrug strategies 7,18-22 and the design of irreversible enzyme inhibitors and S−N intramolecular acyl migration has recently been extensively explored. , This suggests that the concept of the O−N intramolecular acyl migration can be effectively applied to similar rearrangement reactions.…”