Design and synthesis of glutathione analogues

Date of Completion

January 1997


Chemistry, Biochemistry|Chemistry, Pharmaceutical|Health Sciences, Pharmacy




Glutathione (GSH) conjugation, mediated by the glutathione S-transferases (GSTs), is an important detoxication reaction, although cases are known where glutathione-dependent bioactivation has resulted in increased toxicity. In an effort to understand the mechanism of catalysis a series of GSH analogues with modifications at the $\beta$-position of the L-cysteine moiety were designed and synthesized.^ The initial part of the study was the investigation of the ways and means needed to modify the $\beta$-position of L-serine to produce a variety of substituents without the usual elimination reactions that characterize this system. Our studies led to the synthesis of the $\beta$-azido, $\beta$-amino, $\beta$-ethanolamino, $\beta$-bis-(2-ethanolamino) and the $\beta$-aziridinyl protected L-serine via either the N-trityl or the N,N-dibenzyl protecting groups. Both the N-trityl and the N, N-dibenzyl protecting groups appeared to stabilize the labile $\alpha$-proton of L-serine.^ Several tripeptides were then synthesized by replacing the L-cysteinyl-moiety of GSH with other amino acids, including D-serine, L-alanine, L-threonine, DL-$\alpha$-methyl serine, DL-homoserine, $\beta$-chloro-L-alanine, and $\beta$-amino-propionic acid. These analogs were synthesized to probe the common G-site of the GSTs and to determine the degree of tolerance to cysteinyl modification with respect to catalysis of GSH conjugation. In each case, an ethyl derivative of glycine was used, since such derivatives were more easily synthesized and were known to be tolerated by the GST enzymes.^ Significant progress was made in designing potential affinity labels, including the $\beta$-azido, the $\beta$-(2-chloroethyl)amino, the $\beta$-bis-(2-chloroethyl)amino, and the $\beta$-aziridinyl functionalized GSH at the cysteinyl position. Such compounds were designed to probe the glutathione binding site of the GSTs by covalently binding to (and thus inhibiting) the catalytic "G-site" of the GSTs. Such inhibition may lead to the general inhibition of the whole family of GSTs. Work is ongoing to test these molecules for such inhibitory activity.^ As a separate part of the dissertation, a study was also undertaken to improve the synthesis and isolation of the glutathionyl- and L-cysteinyl-conjugates of acetaminophen. These putative toxic conjugates were synthesized by a phase-transfer catalyzed reaction between the amino acid or peptide and N-acetyl-p-benzoquinone imine (NAPQI). The products were isolated in excellent yields following a single purification step, using reversed-phase flash chromatography (RPFC). Acetaminophen is believed to manifest its toxic effects via NAPQI, involving covalent binding to the cysteinyl residues of essential proteins in the liver and kidney. These conjugates will now be more readily available for further study of these putative protein targets. An improved synthesis of NAPQI is also described. ^