Chymotrypsin mimics

Figure 12.5 (a) Active site of chymotrypsin and (b) a chymotrypsin mimic. 

Scheme 12.8 Partial model transacylase activity in a chymotrypsin mimic.

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

Support for this concept is provided by H NMR studies which have identified a downfield resonance of the hydrogen-bonded proton in this pair at 18 ppm in chymotrypsinogen and chymotrypsin at low pH and at 14.9-15.5 ppm at high pH values.246 247 Similar resonances are seen in the a-lytic protease,248 in sub-tilisin,249 in adducts of serine proteases with boronic acids250 251 or peptidyl trifluoromethyl ketones,252 in alkylated derivative of the active site histidine,253 and in molecular complexes that mimic the Asp-His pair in the active sites of serine proteases.254... 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

Peptide-based polymers 62, containing imidazole, carboxyl, and hydroxymethyl functionalities, have been prepared from optically active 50d and tested as mimics of enzymes, such as chymotrypsin, which have the same functionalities (Scheme 41) [70]. These polymers exhibit markedly higher activities than the corresponding low molecular weight compounds in the hydrolysis of nitrophenyl and dinitrophenyl esters. Increased activities were... 

Figure 4. Enzyme mimic for chymotrypsin. (A) Structure and molecular weights. (Continued on next page.)...

This Highlight is part of an extraordinary story (also a cautionary tale) in the area of biocatalysis. The point of particular interest was the incredible catalytic activity claimed for so-called pepzymes - small synthetic peptides modelled to mimic the active site structures of trypsin and chymotrypsin. One was claimed to hydrolyse a simple peptide (a trypsin substrate) with efficiency comparable to that of the native enzyme. This extraordinary result provoked at least as much scepticism as excitement, and in the following months several groups tried to reproduce the results. They failed, comprehensively. [1,2] Some reasons why this failure came as no surprise were subsequently summarised by Matthews, Craik and Neurath, [3] and by Corey and Corey [4]. The background has been discussed in an Angewandte Review on Enzyme Mechanisms, Models and Mimics. [5]... 

As mentioned in Volume 13 of these Reports, 4-oxoazetidin-2-yl phosphonates and phosphinates (19) can be prepared by Arbusov-like reactions between P compounds and 4-acetoxyazetidin-2-one (20). Acid hydrolysis of (19) yields phosphono- and phosphino-aspartic acids (21) which can be converted into peptides with antibacterial activity. Diastereomeric mixtures of phosphono-dipeptides, which can be prepared from racemic dialkyl 1 -aminoalkylphosphonates, can be separated by ion-exchange chromatography. It appears that it is easier to synthesize phosphonodipeptides from these phosphonates as their P-dialkyl esters rather than as the free phosphonic acids. Phosphonic acid analogues of A-Cbz-alanine and -phenylalanine can be converted into ester and amide fluoridates, e.g., (22, R = OMe or NHCHMeg). These fluoridates are the most potent inhibitors of elastase and chymotrypsin yet reported and seem to mimic the natural substrates of these enzymes. ... 

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