Peptide chloromethyl ketones

However, a-chloroketones are powerful alkylating agents and the bound inhibitor attacks His 57 of the catalytic triad system. The reaction is probably more complex than is indicated in the foregoing equation and may involve an epoxy ether intermediate/ Many other peptide chloromethyl ketone inhibitors have been devised/ 1 ... 

Powers, J. C., et al. 1977. Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochim Biophys Acta 485 156. 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

Peptide chloromethyl ketone inhibitors have been developed for almost every serine protease that has been characterized adequately (30). For example, human leukocyte elastase, due to its involvement in emphysema, has been studied extensively with this class of inhibitor (32). The rate at which peptide chloromethyl ketones inhibit elastase is influenced by their interaction with the primary substrate binding site (Si) of the enzyme and by interactions at other subsites. The most effective chloromethyl ketone elastase inhibitor found thus far is MeO-Suc-Ala-Ala-Pro-ValCH2Cl (MeO-Suc- = CH3OCOCH2CH2CO-). This will not inhibit the other major leukocyte protease, cathepsin G (see Table VI). In contrast, Z-Gly-Leu-Phe-CH2C1 (Z = C6H5CH2OCO-) inhibits cathepsin G, but not elastase. Both enzymes can be inhibited with Ac-Ala-Ala-Pr o-V alCH2Cl. 

Figure 3. Reaction of a serine protease with a peptide chloromethyl ketone. The side chain of the Pt residue of the inhibitor is shown interacting with the primary substrate binding subsite (SJ of the enzyme.

Table VI. Specificity of Peptide Chloromethyl Ketone Inhibitors at pH 7.5...

Inhibitors of Thiol and Carboxyl Proteases. Thiol proteases are inactivated by peptide chloromethyl ketones (30) and other alkylating agents. Peptide diazomethyl ketones are much more selective reagents since they do not react with serine proteases as do chloromethyl ketones. Diazoketones have been applied to papain and cathepsin B (48) thus far and it appears that they should be applicable to most thiol proteases. Specificity should be obtainable by changing the peptide sequence of the inhibitor to match that of the enzyme being studied. 

Peptide Fluoromethyl Ketones Fluoroalkyl derivatives of the peptide chloromethyl ketones have been prepared in an attempt to improve specificity by reducing nonspecific alkylation at cysteine residues (Rasnick, D., Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B, Anal. Biochem. 149, 461 65, 1985). Nonspecific reaction with sulfydryl groups such as those in glutathione was reduced there was still reaction with active site cysteine although at a slower rate than with the chloroalkyl derivative (16,200 M s vs. 45,300 1 2 21.9 min. vs. 

Figure 2.3. Mechanism of enzyme inactivation by peptide chloromethyl ketones.

K., Neurath, H. and Woodbury, RG. (1985). Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dc skin proteases and human cathepsin G with peptide-4-nitroanilide substrates and with peptide chloromethyl ketone and sulphonyl fluoride inhibitors. Biochemistry 24, 2048-2058. 

C. P. Dom, and K. Hoogsteen. Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84 A resolution. Proc. Natl. Sci. USA 86 7 (1989). 

Synthesis of peptide chloromethyl ketones can be accomplished simply by coupling of an appropriate peptide or amino acid with an unblocked amino acid chloromethyl ketone. A few dipeptides have been converted directly to the chloromethyl ketone using the mixed anhydride and CH2N2 followed by HCl. ... 

Two representative procedures are presented in detail. The first, preparation of Z-Gly-Leu-PheCH-Cl, involves deblocking of Z-PheCH-Cl and subsequent coupling of the deblocked chloromethyl ketone with Z-Gly-Leu-OH. This compound is an excellent inhibitor of chymotryp-sin and cathepsin G. The second procedure, that for Ac-Ala-Ala-Pro- ValCH-Cl, illustrates the synthesis of a peptide chloromethyl ketone from Boc-Val-OH with a minimum of isolation and purification along the way this compound is an excellent inhibitor of porcine pancreatic and human leukocyte elastase. ... 

P.E. Wilcox, An X-ray crystallographic study of the binding of peptide chloromethyl ketone Inhibitors to subtlllsln BPN, Biochemistry, 11 2439 (1972). 

Imaging/Labeling Applications Actin amino acids antibodies bacteria peptide chloromethyl ketones herbicides , histamine H1/H2 receptor antagonists " lymphocytes metal ions (Fe(III) Pt(IV) ) nanoparticles " nucleic acids/ nucleotides pheomelanin polystyrene latex particles proteins/peptides " risedronate ... 

Williams, E. B. Krishnaswamy, S. Maim, K. G. Zymogen/enzyme discrimination using peptide chloromethyl ketones. J. Biol. Chem. 1989, 264, 7536-7545. 

Imaging/Labeling Applications Amantadine aminoglycosides anti-digoxin baclofen bacteria peptide chloromethyl ketones gabapentin herbicides histamine H1/H2 receptor antagonists 4 5 memantine metal ions (Fe(III)) nanoparticles nucleic acids/ nucleotides " phosphatidylethanolamine " proteins/ peptides " risedronate amino functionalized rhodamine spirolactams ... 

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