Phenyl- acetate, binding

Values of /c2 and Kd for the reactions of the cycloamyloses with a variety of phenyl acetates are presented in Table IV. The rate constants are normalized in the fourth column of this table to show the maximum accelerations imposed by the cycloamyloses. These accelerations vary from 10% for p-f-butylphenyl acetate to 260-fold for m-f-butylphenyl acetate, again showing the clear specificity of the cycloamyloses for meta-substituted esters. Moreover, these data reveal that the rate accelerations and consequent specificity are unrelated to the strength of binding. For example, although p-nitrophenyl acetate forms a more stable complex with cyclohexa-amylose than does m-nitrophenyl acetate, the maximal rate acceleration, h/kan, is much greater for the meta isomer. 

Values of /c2, the maximal rate constant for disappearance of penicillin at pH 10.24 and 31.5°, and Ka, the cycloheptaamylose-penicillin dissociation constant are presented in Table VII. Two features of these data are noteworthy. In the first place, there is no correlation between the magnitude of the cycloheptaamylose induced rate accelerations and the strength of binding specificity is again manifested in a rate process rather than in the stability of the inclusion complex. Second, the selectivity of cycloheptaamylose toward the various penicillins is somewhat less than the selectivity of the cycloamyloses toward phenyl esters—rate accelerations differ by no more than fivefold throughout the series. As noted by Tutt and Schwartz (1971), selectivity can be correlated with the distance of the reactive center from the nonpolar side chain. Whereas the carbonyl carbon of phenyl acetates is only two atoms removed from the phenyl ring, the reactive center... 

More recently, Kaiser and coworkers reported enantiomeric specificity in the reaction of cyclohexaamylose with 3-carboxy-2,2,5,5-tetramethyl-pyrrolidin-l-oxy m-nitrophenyl ester (1), a spin label useful for identifying enzyme-substrate interactions (Flohr et al., 1971). In this case, the catalytic mechanism is identical to the scheme derived for the reactions of the cycloamyloses with phenyl acetates. In fact, the covalent intermediate, an acyl-cyclohexaamylose, was isolated. Maximal rate constants for appearance of m-nitrophenol at pH 8.62 (fc2), rate constants for hydrolysis of the covalent intermediate (fc3), and substrate binding constants (Kd) for the two enantiomers are presented in Table VIII. Significantly, specificity appears in the rates of acylation (fc2) rather than in either the strength of binding or the rate of deacylation. 

Fig. 4 Correlation of constants for transition state stabilization (pKxs) and substrate binding (pKs) for the cleavage of meta- and para-substituted phenyl acetates by /3-CD. The substituents are alkyl groups and the four halogens. The two deviant points are for longish p-alkyl groups (n-butyl and n-pentyl). Data from Tables A5.2...

Fig. 5 Correlation of constants for transition state stabilization (p ts) and substrate binding (pKs) for the cleavage of we/o-substituted phenyl acetates by a-cyclodextrin.

At the start of this section the cleavage of meta- and para-substituted phenyl acetates by a- and )S-CD was discussed in detail and a variety of evidence was cited that is consistent with the mechanisms A and B, in Scheme 2. Further support for the view that para-substituents tend to force the phenyl group out of the cavity (Scheme 2B) comes from the different effects that neutral additives (potential inhibitors) have on the cleavage of m- and p-nitrophenyl acetate (mNPA and pNPA). In brief, species which bind to CDs, and inhibit the reaction of mNPA, do not necessarily inhibit that of pNPA (Tee and Hoeven, 1989 Tee et al., 1993b). 

To continue with the Kolbe reaction, it has been shown that carbon anodes strongly favour the carbonium ion pathway (Koehl, 1964) at least for simple alkanecarboxylic acids. Also, for phenyl-acetic acid and 1-methylcyclohexylacetic acid the same tendency towards carbonium ion formation on carbon anodes was observed, the phenomenon being explained as due to the presence of paramagnetic centres in carbon. These would bind the initially formed radicals, impede their desorption and hence promote the formation of carbonium ions via a second electron transfer (Ross and Finkelstein, 1969). However, cases of Kolbe oxidations in which no dependence on anode material was noticeable have been found more recently (Brennan and Brettle, 1973 Eberson and Nilsson, 1968a Sato et al., 1968). Actually, the nature of the carbon material determines the yield of products formed via the radical versus carbonium ion pathway (Brennan and Brettle, 1973). Yields of the... 

Another important research direction is the mimieking of enzymes and the construction of selective catalysts. For these purposes, the polymer is imprinted with the desired reaetion-product or better, a molecule which resembles the transition state of the reaction adducts. If the educts bind specifically to the recognition site, they become confined into these micro-reactors and are supposed to react faster and more defined than outside the cavities [445]. Examples for reactions in the presence of such synthetic enzymes can be found in [452,453,454,455,456,457] (cf Figure 40c). First positive results have been reported, e.g. an synthetic esterase , increasing the rate of alkaline hydrolysis of substituted phenyl-(2-(4-carboxy-phenyl)-acetic esters for 80 times [488] and Diels-Alder catalysis fiuic-tional holes containing titanium lewis-acids [489]... 

Antibodies raised to the transition state analogue (Fig. 7.3) will bind to the transition state of the hydrolysis reaction, lowering the activation energy and therefore catalyzing the reaction. These antibodies were trapped at the surface of a pH electrode using a dialysis membrane [Fig. 7.4(a)]. The reaction (Eq. 7.17) produces a change in local pH at the surface of the electrode, since acetic acid is one of the products. The measured pH therefore decreases as the phenyl acetate concentration increases in the external solution, since the steady-state concentration of acetic acid in the reaction layer increases [Fig. 7.4(b)]. 

Figure 7. Binding of p- and m-substituted phenyl acetates by a cyclodextrin...

This turns out to be the case, but what we are really looking for in a good enzyme is a cooperative eflFect. We already know that the nucleophilic functional group is a good catalyst for the deacylation of p-nitro-phenyl acetate, but what we want to see is how much help we get from the hydrophobic binding. 

In the studies of the hydrolyses of substituted phenyl acetates by a- or jS-cyclodextrins, it was observed that meta-substituted phenyl esters were more rapidly hydrolyzed than the corresponding para-isomers, a phenomenon termed m ta-selectivity. This observation indicates that the binding mode is probably asymmetric. This effect is apparently dependent on the depth of the cavity, and Fujita et al. (177) showed that appropriate simple modifications of j8-cyclodextrin such as capping the host, for instance, can alter this selectivity and leads to conversion of the well-established metaselectivity to para-selectivity. 

Cyclohepta-amylose is able to bind non-polar substrates (e.g. ferrocene, fluorobenzene, anisole, pyridine, toluene, and 4-t-butylcyclohexanol) in polar, non-aqueous media e.g. DMSO and DMF). Significant rate increases were found for cycloamylose-promoted processes e.g. the deacylation of 3-t-butyl-phenyl acetate) in non-aqueous and in mixed e.g. 60% DMSO) solvents. 

Johnson (1969b, 1970) assumed that phosphorylable sites would be serine-containing proteins. Several car-boxylesters were tested to find a selective substrate able to interact in the same site (able to reduce the speed of [ P]DFP labeling on the mipafox binding fraction). Phenyl-phenyl acetate was selected as that substrate. In other studies, PV was observed to be more selective and has been used for decades for testing the target site, the neurotoxic esterase, and was later called the NTE. NTE has been monitored as the PVase activity resistant to 40 pM paraoxon (20 min) ("B" activity) and sensitive to 40 pM paraoxon plus 60-150 pM mipafox ("C" activity), with NTE as the difference between the activity in condition B and condition C. 

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