Thiiranium ion intermediates in the formation and reactions of S-(2-haloethyl)-l-cysteines

Thiiranium ions formed from the cysteine or glutathione conjugates of 1,2-dihaloethanes are believed to be responsible for the genotoxicity of the parent alkyl halides. The conversions of specifically deuterated β-hydroxyethyl sulfides to the corresponding β-haloethyl sulfides are studied to provide direct evidence for the involvement of thiiranium ions in the reactions of the cysteine conjugates of 1,2-dihaloethanes. S-(2-Hydroxyethyl-1, 1-d₂)-l-cysteine is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of the corresponding S-(2-haloethyl)-l-cysteines in concentrated hydrochloric, hydrobromic, or hydroiodic acids without detectable formation of the 2,2-d₂ isomer of the parent hydroxyethyl derivative. Dissolution of S-(2-hydroxyethyl)-l-cysteine in trifluoromethanesulfonic acid yields a compound with the NMR spectral properties of S-(l-cysteinyl)ethyl thiiranium trifluoromethanesulfonate. The organosoluble S-(2-hydroxyethyl-1,1-d₂) benzyl sulfide is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of S-(2-chloroethyl) benzyl sulfide by thionyl chloride or triphenylphosphine: carbon tetrachloride. These results demonstrate the involvement of thiiranium ion intermediates in the conversion of 2-hydroxyethyl sulfides to 2-haloethyl sulfides in halogen acids and a similar symmetrical intermediate in the chlorination reactions effected by thionyl chloride or triphenylphosphine: carbon tetrachloride.     

Enzyme-catalysed reactions between some 2-substituted 5-nitrofuran derivatives and glutathione

1. A glutathione transferase present in rat and human liver supernatant catalyses the reaction of some 2-substituted 5-nitrofuran derivatives with GSH, with formation of a conjugate and release of the nitro group as inorganic nitrite. Some of the substrates undergo the same reaction at a slower rate in the absence of enzyme. Nitrofuran derivatives commonly used as drugs, and five other drugs containing nitro groups, did not react. 2. Substrate activity in the nitrofuran derivatives showed an approximate correlation with the lability of the nitro group to alkali. 3. Optimum pH values ranging from 6.6 to 9.0 were found for the enzymic reaction with various derivatives, the values being influenced by alkali-lability and pK values of the compounds. 4. Tenfold purification of rat liver glutathione S-aryl-transferase resulted in an equal purification of the activities that catalyse the reaction of two of the nitrofuran derivatives with GSH.     

S-(2-chloroacetyl)glutathione, a reactive glutathione thiol ester and a putative metabolite of 1,1-dichloroethylene

Conversion of the toxic vinyl halide 1,1-dichloroethylene (DCE) to S-(2-S-glutathionyl-acetyl)glutathione (GSCH2COSG) involves sequential acylation and alkylation of two glutathione (GSH) molecules by the microsomal DCE metabolite ClCH2COCl. To examine its possible role in DCE biotransformation, we synthesized the putative intermediate S-(2-chloroacetyl)glutathione (ClCH2COSG). In aqueous buffer, ClCH2COSG did not hydrolyze to release GSH, but instead underwent a two-step rearrangement to yield a cyclic product. Product analyses by liquid secondary ion mass spectrometry and 1H-13C heteronuclear correlation nuclear magnetic resonance spectroscopy indicated that rearrangement involved initial transfer of the chloroacetyl moiety from the cysteinyl thiol to the gamma-glutamyl alpha-amine. The cysteinyl thiol then displaced chloride from the 2-chloroacetyl methylene carbon to yield the cyclic product. Incubation of 2 mM ClCH2COSG with 20 mM GSH yielded approximately 4.5-fold more cyclic product than GSCH2COSG. ClCH2COSG alkylated oxytocindithiol and N-acetyl-L-cysteine to yield S-[2-(alkylthio)acetyl]glutathione adducts analogous to GSCH2COSG. S-2-Chloroacetylation products were absent. In reacting with thiols by alkylation and in decomposing by rearrangement, ClCH2COSG displayed properties strikingly different from those of ClCH2COCl. Although much less reactive than its acyl halide precursor, ClCH2COSG may display greater selectivity in covalent modification of cellular targets in DCE intoxication.     

Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)glutathione. Evidence for a role of S-(2-N7-guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.     

Tissue distribution of S-(2-succino)cysteine (2SC), a biomarker of mitochondrial stress in obesity and diabetes

S-(2-succino)cysteine (2SC) is a chemical modification of proteins produced by reaction of fumarate with thiol groups in protein, a process known as succination. We propose to use the name S-(2-succino)cysteine (instead of S-(2-succinyl)cysteine) from this point on. This is to distinguish protein succination (in which fumarate forms a thioether linkage with cysteine residues) from succinylation (in which an ester, thioester or amide bond would be formed). Succination of proteins is increased in muscle of type 1 diabetic rats and in adipose tissue in type 2 diabetic mice. The increase in 2SC is a direct result of tissue accumulation of fumarate in response to nutrient excess and resultant mitochondrial stress in diabetes. In this study, we examine the breadth of succination of tissue proteins in the db/db type 2 model of diabetes. We also determined the extent of succination in epididymal adipocytes of type 1 (Akita, streptozotocin (STZ)) and type 2 (ob/ob, db/db) diabetic mice, in diet-induced obese (DIO) mice, and in the adipose tissue of ground squirrels in various stages of hibernation. While succination was not increased in most tissues (brain, heart, kidney, liver, skeletal muscle) in the db/db model of diabetes, it was increased in all adipose beds of type 2 diabetic and DIO mice in comparison to their controls. Succination was not increased in adipocytes of type 1 diabetic mice. Adipose tissue from hibernating (HIB) 13-lined ground squirrels was also studied to determine if obesity in the absence of hyperglycemia affected succination of proteins. There were no differences in succination of proteins in brown or white adipose tissue over the torpor-arousal cycle. We conclude that 2SC is a biomarker of nutrient excess and mitochondrial stress in adipose tissue, increasing under the hyperglycemic and insulin resistant conditions associated with type 2 diabetes and obesity.     

The synthesis and properties of N6-substituted 2-amino-purine derivatives.

1. The synthesis, ultraviolet absorption spectra, and behaviour in alkali of N6-methoxy-, N6-methyl, hydroxy-, and N6-hydroxy-2-aminopurines have been described 2. N6-Methoxy-2-aminopurine riboside 5'-pyrophosphate has been prepared and used for polymerization with polynucleotide phosphorylase. 3. The copolymer containing N6-methoxy-2-aminopurine riboside and adenosine residues has been obtained; attempts to synthesize the homopolymer have not been successful. 4. All the purine analogues synthesized have been tested and shown to act mutagenically on Salmonella typhimurium TA1530.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

Cytotoxicity of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine in isolated rat kidney cells

S-(1,2-Dichlorovinyl)glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) produced time- and concentration-dependent cell death in isolated rat kidney proximal tubular cells. AT-125 blocked and glycylglycine potentiated DCVG toxicity, indicating that metabolism by gamma-glutamyltransferase is required. S-(1,2-Dichlorovinyl)-L-cysteinylglycine, a putative metabolite of DCVG, also produced cell death, which was prevented by 1,10-phenanthroline, phenylalanylglycine, and aminooxyacetic acid, inhibitors of aminopeptidase M, cysteinylglycine dipeptidase, and cysteine conjugate beta-lyase, respectively. Aminooxyacetic acid and probenecid protected against DCVC toxicity, indicating a role for metabolism by cysteine conjugate beta-lyase and organic anion transport, respectively. DCVC produced a small decrease in cellular glutathione concentrations and did not change cellular glutathione disulfide concentrations or initiate lipid peroxidation. DCVC caused a large decrease in cellular glutamate and ATP concentrations with a parallel decrease in the total adenine nucleotide pool; these changes were partially prevented by aminooxyacetic acid. Both DCVG and DCVC inhibited succinate-dependent oxygen consumption, but DCVC had no effect when glutamate + malate or ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine were the electron donors. DCVC inhibited mitochondrial, but not microsomal, Ca2+ sequestration. These alterations in mitochondrial function were partially prevented by inhibition of DCVG and DCVC metabolism and were strongly correlated with decreases in cell viability, indicating that mitochondria may be the primary targets of nephrotoxic cysteine S-conjugates.     

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.     

Methyl 2-(2-(4-formylphenoxy)acetamido)-2-substituted acetate derivatives: a new class of acetylcholinesterase inhibitors

A new class of inhibitors of acetylcholinesterase (methyl 2-(2-(4-formylphenoxy)acetamido)-2-substituted acetate derivatives) is described. Compounds 4b and 4i were found to be more potent than galanthamine in inhibiting acetylcholinesterase.     

Facile synthesis of (R)N-2-hydroxyacyl-L-cysteine derivatives: (R)N-2-hydroxyacyl transfer from enzymatically-synthesized (R)S-2-hydroxyacylglutathione derivatives to L-cysteine

Summary N-(R)-2-Hydroxyacyl-L-cysteine derivatives were conveniently synthesized by the reaction of the corresponding S-(R)-2-hydroxyacyl-glutathione with cysteine. The (R)2-hydroxyacyl group was transferred from the S-glutathionyl moiety to S-cysteinyl, forming the corresponding (R)S-2-hydroxyacylcysteine; this rearranged to the (R)N-hydroxyacylcysteine. These compounds have anti-proliferative activity associated with the inhibition ofde novo pyrimidine synthesis.Abbreviations TRIS tris(hydroxymethyl) aminomethane - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Inhibition of octopus glutathione transferase by Meisenheimer complex analog, S-(2,4,6-trinitrophenyl) glutathione

The tight binding of Meisenheimer intermediate with octopus digestive gland glutathione transferase was analyzed with 1,3,5-trinitrobenzene, which forms a trapped Meisenheimer complex with glutathione because there is no leaving group at the ipso carbon. By steady-state enzyme kinetic analysis, an inhibition constant of 1.89 ± 0.17 M was found for the transient formed, S-(2,4,6-trinitrophenyl) glutathione. The above inhibition constant is 407-fold smaller than the K _m value for the substrate (2,4-dinitrochlorobenzene). Thus, S-(2,4,6-trinitrophenyl) glutathione is considered to be a transition-state analog. The tight binding of this inhibitor to the enzyme provides an explanation for the involvement of the biological binding effect on the rate enhancement in the glutathione transferase-catalyzed S_NAr mechanism.     

Comparison of solution and crystal properties of Co(II)-substituted human carbonic anhydrase II

The visible absorption of crystals of Co(II)-substituted human carbonic anhydrase II (Co(II)-HCA II) were measured over a pH range of 6.0-11.0 giving an estimate of pK_a 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pK_a units than the pK_a near 7.2 for Co(II)-CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4 M citrate in the precipitant solution used in the crystal growth. A pK_a of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)-HCA II in solution containing 0.8 M citrate. Citrate is not an inhibitor of the catalytic activity of Co(II)-HCA II and was not observed in crystal structures. The X-ray structures at 1.5-1.6 Å resolution of Co(II)-HCA II were determined for crystals prepared at pH 6.0, 8.5 and 11.0 and revealed no conformational changes of amino-acid side chains as a result of the use of citrate. However, the studies of Co(II)-HCA II did reveal a change in metal coordination from tetrahedral at pH 11 to a coordination consistent with a mixed population of both tetrahedral and penta-coordinate at pH 8.5 to an octahedral geometry characteristic of the oxidized enzyme Co(III)-HCA II at pH 6.0.