Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

Abstract

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by α-methyl-γ,γ-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N- (α-methyl-γ,γ-diphenylallenecarbonyl)-L-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pKa value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism. KeywordsAllene; Cysteine protease; Cysteine protease inhibitor; Oxygen; Radical Cysteine proteases are an important class of peptide-processing enzymes and, as the name implies, are characterized by having a cysteine residue within the active site. 1 These proteases are also ubiquitous enzymes that play important roles in many biochemical processes. 1,2 The presence of cysteine proteases in pathogenic microorganisms and roles in facilitating metastases of tumors are examples of many factors that make selective cysteine protease inhibitors important targets for drug design. 1 As a consequence of the wide therapeutic potential of these compounds, many cysteine protease inhibitors have been developed. 3 In much of the design of these inhibitors, it has been assumed that the catalytic mechanism of *Corresponding author. Tel.: +81 76 434 7555; e-mail: takeuchi@pha.u-toyama.ac.jp. † Deceased, 6 July 2001. ‡ Dedicated to the memory of Professor Toshio Satoh of Tokushima Bunri University who made many lasting contributions to the field of medicinal chemistry.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. cysteine proteases is similar to that of serine proteases. Thus, the cysteinyl and histidinyl residues present in the active site form a thiolate/imidazolium ion pair that promotes the formation of an S-acyl-enzyme intermediate that is necessary for cleavage of the peptide bond. 4 However, despite the similarities of the proposed catalytic mechanism of cysteine and serine proteases, important differences have been revealed. Thus, the replacement of the active site serine with cysteine in two serine proteases (trypsin and subtilisin) abolished activity. 5 This result implies that cysteine proteases have another reaction mechanism, for example, a radical reaction mechanism that has been found in several enzymatic systems. 6 NIH Public AccessAllenyl esters 2 deri...

Horner–Wadsworth–Emmons reaction as an excellent tool in the synthesis of fluoro-containing biologically important compounds

“…93 The preparation of allenyl esters is important in the context of their use for the synthesis of new cysteine protease inhibitors. 94…”