Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site

Abstract

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.

Synthesis of Novel Amino Acid–Fipronil Conjugates and Study on Their Phloem Loading Mechanism

“…As shown in Scheme 1 , the cyano group on the pyrazole ring was first hydrolyzed into a reactive carboxyl group [ 23 ], which enables easy chemical modification. Followed by condensation reactions with amino acid esters in the presence of EDC∙HCl and DMAP [ 24 , 25 ], 12 amino acid ester–fipronil conjugates ( 3a – l ) were obtained. Hydrolysis of the methyl ester group in 3a – l with lithium hydroxide [ 10 , 26 ] provided the new amino acid–fipronil conjugates 4a – l in good yields.…”

Bacterial γ-glutamyltranspeptidases, physiological function, structure, catalytic mechanism and application

“…A series of GSH analogous peptidyl phosphorus esters, which are mechanism- based inhibitors of human GGT and EcGGT, were synthesized to evaluate the structural and stereochemical preferences of the Cys-Gly binding site of GGTs. 55) Both enzymes were inhibited strongly and irreversibly by these inhibitors with phenoxide as a good leaving group. It was found that human GGT was highly selective for L-aliphatic amino acid such as L-2-aminobutyrate (L-Cys mimic) at the Cys binding site, whereas EcGGT significantly preferred L-Phe mimic at this site.…”

“…On the other hand, EcGGT was not selective for GSH, but still retained the dipeptide binding site. 55) d) The reaction mechanism of EcGGT deduced from the structure of .-glutamyl-enzyme intermediate.…”

Synthesis and Biological Evaluation of the Antimicrobial Natural Product Lipoxazolidinone A

“…The reaction was stirred for 10 minutes and then cooled to -78 ˚C at which point a solution of 2-butynoic acid (0.50 g, 5.8 mmol) in THF (12 mL) was added and the reaction was stirred at -78 ˚C for 10 minutes and then warmed to -5 ˚C and stirred for 45 minutes. The reaction was poured into cooled (0 ˚C) 1 M HCl (100 mL) and extracted with EtOAc (3 x 30 mL), the combined organic layers were washed with brine (20 mL), dried (MgSO4), filtered, concentrated and purified by chromatography on SiO2 (10% EtOAc:hexanes) to yield 860 mg (81%) of S6 as a colorless oil: 1 N-Methoxy-N,3-dimethyldec-2-enamide (S7). (E)-3-methyldec-2-enoic acid (S6) (120 mg, 0.66 mmol) was dissolved in THF (2.1 mL) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (140 mg, 0.79 mmol) was added followed by the addition of N-methylmorpholine (0.22 mL, 2.0 mmol).…”

“…The reaction was then poured into saturated sodium bicarbonate and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered, concentrated in vacuo, and purified by chromatography on SiO2 (20% EtOAc:hexanes) to yield 61.2 mg (73%) of 16 as a colorless oil: Rf = 0.55 (20% EtOAc:hexanes); 1…”