The synthesis of is shown in The Boc diaminopropanoic

The synthesis of is shown in . The ---Boc--diaminopropanoic acid--butyl ester () fragment was synthesized in four steps from Boc--asparagine (). The key step in this synthesis was a Hofmann rearrangement of the terminal carboxamide, which was performed according to the elegant protocol of Zhang that employs iodosobenzene diacetate (PIDA) as the oxidant. The primary amine thus formed was subjected to Cbz protection and the free carboxylic acid was esterified with -BuOH and DCC to give . Hydrogenolysis of the Cbz group afforded amine . Formation of the urea linkage between and -cysteine(S–S--Bu) allyl ester hydrochloride ( was accomplished through the use of carbonyl diimidazole (CDI). The S–S--butyl-protecting group for cysteine was chosen from the viewpoint of orthogonal deprotection. Pd catalyzed deprotection of the allyl ester in urea dipeptide gave the free acid which was coupled with glycine -butyl ester hydrochloride in the presence of EDC, HOBt, and -methylmorpholine (NMM), to give the tripeptide in 78% yield. Reductive cleavage of the disulfide protection in by tributylphosphine provided the thiol . -Bromophenylhydroxylamine (, an unstable solid) was synthesized by a Rh-catalyzed transfer hydrogenation (’diimide reduction’) of 4-bromo nitrobenzene. Insertion of a carbonyl between thiol and -bromophenylhydroxy amine () was accomplished by sequentially treating with phosgene (to form the thiochloroformate in situ) and then with in dichloromethane. Under these conditions a 51% yield of the thiocarbamate was obtained. Global deprotection of with trifluoroacetic acid afforded the target molecule . Compound was examined for its ability to inhibit yeast glyoxalase-I. Initial rates were determined by following the increase in UV Camptothecin synthesis at 240nm (0.05M phosphate buffer, pH 6.6) which corresponds to the isomerization process and formation of --lactoyl glutathione (). Methylglyoxal, GSH, , and buffer were added to the cell and allowed to equilibrate at 30°C for 6min (to allow formation of hemimercaptal) before addition of the enzyme. The concentrations of hemimercaptal were calculated using the dissociation constant 3.1×10M as previously determined for this equilibrium reaction. It has been shown previously that is a tight-binding competitive inhibitor of glyoxalase-I (=1.2±0.2μM). Similarly the urea analog was found to be a tight-binding competitive inhibitor with =2.19±0.57μM. The for the hemimercaptal of glutathione and methylglyoxal as determined by this assay was 0.35mM, in agreement with the literature value. Results of these experiments confirm that the urea isostere is well tolerated by glyoxalase-I. We then tested the stability of the urea linkage in toward γ-glutamyltranspeptidase. Compound was incubated at 37°C with equine kidney γ-glutamyltranspeptidase (γ-GT, Sigma) in 200mM AMPD buffer at pH 8.4 in the presence of 40mM of gly–gly as an acceptor peptide for the released glutamic acid. The course of this incubation was monitored by thin-layer chromatography. Excellent separation of the metabolic products was obtained on silica gel TLC plates that were developed with -butanol/acetic acid/water (12:5:3) and sprayed with a solution of fluorescamine in acetone. Glx-I inhibitors PBBG () and were used as references in this assay. The results of this experiment indicate that PBBG () and were significantly degraded after 15min of incubation with γ-GT. TLC of incubation aliquots containing PBBG () and showed a new higher spot corresponding to complete degradation to the respective dipeptides (cysteinylglycine) and a new lower spot corresponding to γ-glutamylglycylglycine, the other expected product from the transpeptidase reaction. The identities of these spots were confirmed by comparison with authentic samples of these dipeptides. Compound after 1h showed a further degradation of cysteinylglycine dipeptide by release of the hydroxylamine portion (a higher spot). In contrast to the above, compound did not show any degradation even after incubation for 15h, thus confirming its stability against γ-GT mediated cleavage.