Mechanism-based probes

Liu S, Zhou B, Yang H et al (2008) Aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases. J Am Chem Soc 130 8251-8260... 

Hang HC, Loureiro J, Spooner E, van der Velden AW, Kim YM, Pollington AM, Maehr R, Stambach MN, Ploegh HL (2006) Mechanism-based probe for the analysis of cathepsin cysteine proteases in living cells. ACS Chem Biol 1 713-723... 

The term activity-based protein profiling implies mechanism-based probe/ target reactivity. Photoaffinity labelling approaches represent a complementary technique to mechanism based APBB probes. The use of these photoreactive affinity-based protein profiling probes in proteomic studies are reviewed by Overkleeft et al. 

Selected Biological Aspects. - Phosphonate and phosphinic acids and their derivatives have been widely used in haptens. Examples additional to those already discussed in other sections include the phosphonic acid anion 363 which was an alternative to the preferred but synthetically inaccessible structure 364. The mechanism-based probe 365 has been synthesised and shown to modify a bacterial phosphotriesterase. This strategy for generating a probe is general and should allow the isolation of a host of unique catalysts. ... 

Figure 11 Mechanism-based probes for cysteine protease active site labeling, (a) E-64-mediated inhibition of cysteine proteases via nucleophilic attack of the catalytic cysteine at the epoxy group, (b) E-64-based probe with biotin reporter tag and tyrosine as 125l iodination site for radioactive labeling, (c) Acyloxymethylketone probe for the complementary labeling of cysteine proteases.

Figure 20 Mechanism-based probe for cytochrome P-450 targeting. Oxidative activation of the probe by the enzyme generates a reactive ketene that covalently binds within the active site. 

Mechanism-Based Probes for Profiling Glycan-Processing Enzymes 288... 

Figure 21 Chemical probes for monitoring glycan-processing enzymes, (a) Mechanism-based probe for labeling active exo-glycosidases in cell lysates/ (b) FRET-based reporter for OGT activity in cells. ...

Figure 4 Strategies for the application of CoA analogues. For in vitro studies appropriate analogues are prepared from a suitable precursor, purified, and characterized if necessary, and then normally used as mechanism-based probes and inhibitors (panel A). When CoA analogues are used for the in vitro site-specific modification of proteins by transfer of a reporter label to a carrier protein module using a PPTase enzyme, the analogue can be prepared and used in situ (panel B). In vivo reporter labeling is made possible when cells are provided with suitably modified pantothenamide precursors, which are subsequently transformed into the respective CoA analogues and transferred a carrier protein by the cell s native CoA biosynthesis (CoaADE) and PPTase enzymes. The labeled protein can be recovered from the cells by cell lysis.

Adlhart, C., Chen, P. (2000) Fishing for Catalysts Mechanism-based Probes for Active Species in Solution. Helv. Chim. Acta 83 2192-2196. 

Tanaka K, Miura T, Umezawa N, Urano Y, Kikuchi K, Higuchi T, Nagano T (2001) Rational design of fluorescein-based fluorescence probes. Mechanism-based design of a maximum fluorescence probe for singlet oxygen. J Am Chem Soc 123 2530-2536... 

Together, all the inferences from both computational modeling and simulation (which can reveal novel aspects of the receptor mechanisms, based on the dynamic properties of the proteins) serve as mechanistic working hypotheses for new and more focused experiments. This mode of closely considered interactions and synergy between computational developments and experimental probing of the receptor systems has become a sustained characteristic of current studies of structure-function... 

Glutamine and ATP analogues were useful to probe the reaction mechanism, but these inhibitors are likely to interfere with many other enzymes acting on the same substrates. More recently, analogues of puromycin (22) (Table 5) were synthesized and evaluated as mechanism-based selective inhibitors of H. pylori... 

Arnett and coworkers later examined the reaction of lithium pinacolone enoiate with substituted benzaldehydes in THE at 25 °C. The determination of the heat of reaction indicated that the Hammett p value for the process is 331. Although the aldol reaction was instantaneous in THF at 25 °C, the reaction with o- or p-methylbenzaldehyde could be followed using a rapid injection NMR method in methylcyclohexane solvent at —80 °C. Application of Eberson s criterion based on the Marcus equation, which relates the free energy of ET determined electrochemically and the free energy of activation determined by kinetics, revealed that the barriers for the ET mechanism should be unacceptably high. They concluded that the reaction proceeds via the polar mechanism . Consistent with the polar mechanism, cyclizable probe experiments were negative . The mechanistic discrepancy between the reactions of benzaldehyde and benzophenone was later solved by carbon kinetic isotope effect study vide infraf. ... 

Kent, U.M., Juschyshyn, M.I. and Hollenberg, P.F. (2001) Mechanism-based inactivators as probes of cytochrome P450 structure and function. Current Drug Metabolism, 2, 215-243. 

R(200 ppm of alcohol vapor in air) are 50 and 140 for tin oxide (Sn0x) and palladium-gold/SnOx (PdAu/SnOx) sensors, respectively while the values of R(50 ppm of N0X in air)/R(air) are 60 and 5 for SnOx and PdAu/SnOx sensors, respectively. Possible sensing mechanisms based on XPS, Kelvin probe, Hall and electrical conductivity measurements are also discussed. 

The metalloproteases (MPs) and matrix metalloproteinases (MMPs) are a class of metallohydrolases of particular interest to the pharmaceutical industry due to their role in a number of pathological processes [81-83], The lack of an enzyme-bound nucleophilic residue in the metallohydrolases complicates the design of ABPP probes for this class of enzymes. Rather than mechanism-based and electrophilic probes for ABPP, photoreactive variants of reversible inhibitors of metallohydrolases have been developed [84-86]. These reversible inhibitors usually contain a hydroxamate moiety that is capable of chelating the catalytic zinc ion in a bidentate manner [79, 80]. The hydroxamate moiety was incorporated into the first generation of metallohydrolase ABPP probes along with a benzophenone group capable of covalent bond formation upon UV irradiation (Scheme 4). 

Although these probes show potent, heat-sensitive labeling profiles with purified tyrosine phosphatases, suggestive of specific active site modification, their use in complex proteomes was not reported [109]. Zhang and coworkers introduced a more specific class of tyrosine phosphatase probe [110]. This probe consists of an a-bromobenzylphosphonate moiety that acts as a tyrosine phosphate mimic and a mechanism-based inhibitor of tyrosine phosphatases. The chemistry of the probe mode of action is shown in Scheme 7. 

Possible mechanisms for Cys645 modification by haloacetamide-based probes... 

Many gene products are uncharacterized enzymes that lack a specific class assignment. As discussed in Sect. 2.3, ABPP probes can be used to identify structurally disparate members of enzyme families based on their reactivity with mechanism-based inhibitors. To date, many uncharacterized enzymes have been classified upon their identification via ABPP. For example, the use of a fluorophosphonate probe by Jessani et al. led to the characterization of sialyl acetylesterase - expressed... 

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