Thomas W. Bruice

[40] Novel sulfhydryl reagents

Publisher Summary This chapter focuses on sulfhydryl reagents that have been categorized as blocking and labeling groups, reporter groups, cross-linking groups, and affinity labeling groups. Most of these sulfhydryl reagents deliver groups that fall in the category of “blocking and labeling groups,” which may be either reversible or irreversible. These groups are designed to be structurally and chemically relatively innocuous so that when covalently bound, they act merely to block the activity of or to titrate or label (sometimes isotopically) any number of sulfhydryl groups. A second class of sulfhydryl reagents delivers cross-linking groups. They possess one functionality that reacts initially and selectively with sulfhydryls and a second functionality that reacts (often under altered conditions) with another nearby group, which may or may not be another sulfhydryl. For the other two major categories, reporter groups and affinity labeling groups, there is a scarcity of older reagents that may be similarly classified, which react satisfactorily, and are of any general practical utility.

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO2R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Abstract Derivatives of l -cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l -cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, k cat K m , for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k 2 , arising from an increase in the transition-state free energies of acylation.

[6] Use of secondary α-hydrogen isotope effects in reactions of pyrimidine nucleoside/nucleotide metabolism: Thymidylate synthetase

Publisher Summary This chapter discusses that secondary α-hydrogen isotope effects are useful tools that may aid in ascertaining whether an enzyme-catalyzed reaction or covalent interaction of an inhibitor with an enzyme involves rehybridization of a carbon atom of the ligand at or before the rate-determining step of the reaction. It reviews the catalytic mechanism of thymidylate (dTMP) synthetase, as well as the interaction of 5-fluoro-2-deoxyuridylate (FdUMP) with this enzyme. A salient feature of this mechanism is that a primary event in catalysis involves addition of a nucleophile of the enzyme to the 6-position of the pyrimidine heterocycle to form transient 5, 6-dihydropyrimidine intermediates, in which the 6-carbon is rehybridized, from sp 2 to sp 3 . FdUMP behaves as a quasi-substrate for this reaction, proceeding through the first two steps of the mechanism. The chapter also reviews that kinetic studies indicate that initial attack of thiolate is probably rate determining in this reaction; if so, it may be concluded that the isotope effect observed here is the true kinetic isotope effect of the reaction rather than a pre-equilibrium isotope effect. However, from available evidence, it is not possible to determine whether rehybridization of the 6-carbon of BrdUMP in the enzymic reaction occurs at or before the rate-determining step of the reaction. Results demonstrate that secondary α- hydrogen isotope effects may be of great utility, in demonstrating the existence of transient 5, 6-dihydropyrimidine intermediates, in both chemical and enzymic reactions of pyrimidine nucleosides and nucleotides.