Chemical reactivity, substrates

There is a growing list of oligomeric enzymes which display the phenomenon of half-of-the-sites reactivity (Levitzki et al. 1971), in which only half of the active sites react with chemically reactive substrate analogues. In such cases, it is necessary to discriminate between the case in which approximately half of the molecules fail to react because of prior denaturation, and cases in which bom fide half-of-the-sites reactivity exists. 

An illustration of this approach may be seen in the studies on streptococcal proteinase (Liu 1967). The activity of this enzyme is dependent upon the presence of a free sulfhydryl group. The active form of the enzyme was first converted to the inactive S-sulfenyl-sulfonate derivative. Treatment of this derivative with a chemically-reactive substrate "analogue, a-N-bromoacetylarginine methyl ester, resulted in the alkylation of a single histidine residue. The sulfhydryl group of the modified enzyme was regenerated by reduction, however, this did not restore enzymatic activity, thus providing presumptive evidence for the involvement of both a cysteinyl and a histidyl residue in the active site of this enzyme. 

The most critical test for the functionality of the covalently bound peptidyl-tRNA is based in its ability to serve as donor substrate for peptide bond formation. This test was performed usually by first allowing a covalent reaction to take place with added nonradioactive affinity probe, and subsequently adding a radioactive acceptor substrate, amino-acyl-tRNA or puromycin. Covalent attachment of radioactivity to the ribosome now indicates that the attached nonradioactive probe served as donor substrate in peptide bond formation. For this test to be meaningful it is absolutely necessary that affinity labeling does not continue after addition of the acceptor substrate. This condition is easily fulfilled with photosensitive probes, but only to a lesser extent with chemically reactive substrates. 

Figure C2.13.7. Change between polymerizing and etching conditions in a fluorocarbon plasma as detennined by tire fluorine-to-carbon ratio of chemically reactive species and tire bias voltage applied to tire substrate surface [36].

Entrapment of biochemically reactive molecules into conductive polymer substrates is being used to develop electrochemical biosensors (212). This has proven especially useful for the incorporation of enzymes that retain their specific chemical reactivity. Electropolymerization of pyrrole in an aqueous solution containing glucose oxidase (GO) leads to a polypyrrole in which the GO enzyme is co-deposited with the polymer. These polymer-entrapped GO electrodes have been used as glucose sensors. A direct relationship is seen between the electrode response and the glucose concentration in the solution which was analyzed with a typical measurement taking between 20 to 40 s. 

A special type of substituent effect which has proved veiy valuable in the study of reaction mechanisms is the replacement of an atom by one of its isotopes. Isotopic substitution most often involves replacing protium by deuterium (or tritium) but is applicable to nuclei other than hydrogen. The quantitative differences are largest, however, for hydrogen, because its isotopes have the largest relative mass differences. Isotopic substitution usually has no effect on the qualitative chemical reactivity of the substrate, but often has an easily measured effect on the rate at which reaction occurs. Let us consider how this modification of the rate arises. Initially, the discussion will concern primary kinetic isotope effects, those in which a bond to the isotopically substituted atom is broken in the rate-determining step. We will use C—H bonds as the specific topic of discussion, but the same concepts apply for other elements. 

Lithium and zinc tert-butyl phenylmethyl sulfoxide (1) and A-phenyl imines 2, in which the substituent R is alkenyl or aryl, react at —78 °C over 2 hours with high anti diastereoselection (d.r. >98.5 1.5)6. Shorter reaction times result in poorer yields, due to incomplete reaction. In contrast, the reaction of the sulfoxide anion with benzaldehyde is complete after 5 seconds, but shows poor diastereoselection. When the substituent R1 or R2 of the imine 2 is aliphatic, the substrates exhibit poor chemical reactivity and diastereoselection. The high anti diastereoselection suggests that if a chelated cyclic transition state is involved (E configuration of the imine), then the boat transition state 4 is favored over its chair counterpart 5. 

Interestingly, significant progress has been made for the hydroamination of more reactive substrates such as styrenes, alkynes, dienes, and allenes. Specifically, highly selective catalysts have been discovered for the synthesis of fine chemicals (pharmaceuticals, natural products, chemical intermediates). In this area however, the problem of catalyst stabiUty can also be questioned in several cases. 

Improve adhesion of dissimilar materials such as polymers to inorganic substrates. Also called primers. Primers generally contain a multifunctional chemically reactive species capable of acting as a chemical bridge. In theory, any polar functional group in a compound may contribute to improved bonding to mineral surfaces. However, only a few organofunc-tional silanes have the balance of characteristics required... 

In some situations, the direct attachment of a large group (such as fluorescamine) to a biologically active substrate can reduce activity. This is due to steric hindrance which can cause a change in conformation or physically block an active site. This condition can be obviated in many cases by attaching the bulky moiety to a spacer arm composed of two or more methylene groups. To this end, a variant of F-D was also synthesized in which a spacer arm of beta-alanine was inserted between the fluorescent and chemically reactive moieties of the reagent. 

The first step in the CSD process is solution preparation, which involves reagent selection (chemical precursors) and solvent choice.1,5-12,16 During solution preparation, other chemical modifiers may also be added to the solution to facilitate or limit chemical reactivity. Also during this stage of the process, identification of appropriate reaction conditions to promote other desired changes in precursor nature or solution characteristics is also considered. The goal for solution preparation is to develop a homogeneous solution of the necessary cation species that may later be applied to a substrate. 

FIGURE 4.18 Structures of the CYP2E1 substrates, chloroform, butadiene, and N,N-dimethylnitrosoamine, and their chemically reactive and toxic metabolites. 

There are at least two factors that could influence the turnover rate, the site of metabolism (hot spot) and the affinity of a compound toward these enzymes the protein/ligand (substrate or inhibitor) interaction and the chemical reactivity of the compound towards oxidation. Because of the interaction of the protein with the potential ligand, certain atoms of the compound could be exposed to the heme group, and depending on the chemical nature of these moieties the oxidative reaction will take place at different rates, for example celecoxib is metabolized by CYP2C9 at the... 

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