Substrate trapping experiments

Mechanistically there is ample evidence that the Balz-Schiemann reaction is heterolytic. This is shown by arylation trapping experiments. The added arene substrates are found to be arylated in isomer ratios which are typical for an electrophilic aromatic substitution by the aryl cation and not for a homolytic substitution by the aryl radical (Makarova et al., 1958). Swain and Rogers (1975) showed that the reaction takes place in the ion pair with the tetrafluoroborate, and not, as one might imagine, with a fluoride ion originating from the dissociation of the tetrafluoroborate into boron trifluoride and fluoride ions. This is demonstrated by the insensitivity of the ratio of products ArF/ArCl in methylene chloride solution at 25 °C to excess BF3 concentration. 

Menadione (79) has been shown by Krishna et al. to act as a photosensitizer for thymine degradation. Spin-trapping experiments with several nucleosides showed that the photosensitizations occurred by electron transfer from the substrate to the excited triplet state of menadione to form the cation radical of the substrate and the anion radical of menadione, both of which were detected... 

In a still less basic solvent, HFIP, a considerable amount of the rearranged product 23 was obtained even in the presence of base. Interestingly, compound 23 obtained was largely racemized, although 23 was the exclusive product and retained the optical purity of the substrate in a neutral HFIP without added base.12 So, the racemization cannot be ascribed to the intermediate formation of the primary cation 20. Trapping experiments support the formation of cycloheptyne 39 as an intermediate (eq 16).18... 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

An analogous assay using a radiolabeled soft nucleophile would also be required to complement the hard nucleophile radiolabeled cyanide trapping assay. Investigations into radiolabeled glutathione have proved unsuccessful since the material is unstable due to cross-reactions induced by beta radiation. Alternate soft nucleophiles such as cysteine, N-acetyl cysteine and P-mercaptoethanol all have promise as radiolabeled substances for quantitative trapping experiments since they are more stable than GSH and equally nucleophilic, although clearly these would not be substrates for GST. 

In general, mechanistic evidence for a reactive intermediate from trapping experiments needs to be linked to arguments against the introduction of an alternative pathway from the reactant, i.e. to show that an intermediate really has been trapped, not the reactant. A classic case is the hydrolysis of 4-nitrophenyl acetate catalysed by imidazole. The mechanism is nucleophile catalysis and the intermediate (N-acetylimidazolium cation) was trapped by aniline (to give acetanilide) with no kinetic effect, i.e. the aniline does not react directly with the substrate [51]. 

The cysteine residue in the catalytic loop (Cysl2 in LMPTP, Cys215 in PTPIB) is the essential nucleophile for PTPase activity. Experiments show that cysteine to serine mutants are completely inactive but can still bind substrate molecules. The ability to bind substrates without hydrolyzing them is called substrate trapping and has been exploited when searching for native PTPase... 

Conclusively establishing the role of potential intermediates in a biosynthetic pathway is a difficult aspect of biosynthesis. Typically, intermediates accumulate because subsequent enzymatic reactions are slow. Organisms also produce shunt metabolites that are off the main pathway and may not be further metabolized these will also accumulate. Isolation of an intermediate does not, therefore, establish intermediacy. Trapping experiments are sometimes used to overcome these problems. In the pathway A B C, where A is a known precursor of C, labeled A and non-labeled B are fed at the same time. The latter is metabolized to C and labeled B is produced from A Bis then temporarily available for isolation. An alternative approach for microbial metabolites is to mutate the organism or add specific enzyme inhibitors. This may allow intermediates to accumulate. Incorporation of a labeled, potential intermediate into a product does not prove that the intermediate lies on the main biosynthetic pathway. It may simply serve as a substrate for the enzymes involved. Only when each of the enzymes in a pathway has been isolated and characterized, and the substrate specificity determined, can the intermediates in a biosynthetic route be characterized. 

Measure the rates of substrate binding and dissociation by stopped-flow methods or by substrate trapping methods. This is an optional step in that although information on binding rates can be useful, it is not essential for the design of subsequent experiments. 

It is crucial in performing TS analysis to know exactly which step of the reaction the experimental KIEs reflect. Using isotope-trapping experiments, it is possible to demonstrate whether formation of the Michaelis complex, E-S, is kinetically significant, and if necessary, to find conditions where it is not. However, internal steps can also complicate the interpretation of KIEs. These can include, but are not limited to (1) establishment of equilibria between different enzyme-bound intermediates, (2) isotopically insensitive steps, such as conformational changes in the enzyme or substrate, or (3) substrate channeling. 

KIEs were determined for a stem-loop RNA substrate A-IO" and an analogous DNA substrate, dA-10 (Table 8). In both these cases, hydrolysis reaction proceeded through a stepwise Dn An mechanism. Isotope-trapping experiments showed that substrate binding was not kinetically significant however, the experimental KIEs with A-10 as substrate were inconsistent with any mechanism where only chemical steps were kinetically significant, implying equilibrium formation of an RTA ox-ocarbenium ion adenine complex, followed by an isotopically insensitive step. In contrast, the KIEs for dA-10 were consistent with a Dn An mechanism. 

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