Bioactivation mechanisms of haloalkene cysteine S-conjugates modeled by gas-phase, ion-molecule reactions.

Glutathione conjugate formation plays important roles in the detoxification and bioactivation of xenobiotics. A range of nephrotoxic haloalkenes undergo bioactivation that involves glutathione and cysteine S-conjugate formation. The cysteine S-conjugates thus formed may undergo cysteine conjugate beta-lyase-catalyzed biotransformation to form cytotoxic thiolates or thiiranes. In the studies presented here, cysteine conjugate beta-lyase-catalyzed biotransformations were modeled by anion-induced elimination reactions of S-(2-bromo-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, S-(2-chloro-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, and S-(2-fluoro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester in the gas phase. Examination of these processes in the gas phase allowed direct observation of the formation of cysteine S-conjugate-derived thiolates and thiiranes, whose formation is inferred from condensed-phase results. The cysteine S-conjugates of these haloethenes exhibit distinctive patterns of mutagenicity that are thought to be correlated with the nature of the products formed by their cysteine conjugate beta-lyase-catalyzed biotransformation. In particular, S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine is mutagenic, whereas the chloro and fluoro analogues are not. It has been proposed that the mutagenicity of S-(2-bromo-1,1, 2-trifluoroethyl)-L-cysteine is correlated with the greater propensity of the bromine-containing cysteine S-conjugate to form a thiirane compared with those of the chlorine- or fluorine-containing conjugates. The ease of thiirane formation is consistent with the gas-phase results presented here, which show that the bromine-containing conjugate has a greater propensity to form a thiirane on anionic base-induced elimination than the chloro- or fluoro-substituted analogues. The blocked cysteine S-conjugates were deprotonated by gas-phase ion-molecule reactions with hydroxide, methoxide, and ethoxide ions and then allowed to decompose. The mechanisms for these decompositions are discussed as well as the insights into the bioactivation of these cysteine S-conjugates provided by the further decompositions of thiolate intermediates.

Apparent proton affinities of highly charged peptide ions.

The apparent proton affinities (PA) of various charge states of three highly basic peptides [(KAP)10, (KAP)8, (KAA)8] were measured by the "bracketing" method. For all three peptides the apparent PA decreases as the charge state increases and the magnitude of the decrease is consistent with an increase in coulombic repulsion in the highly protonated species. Based on a simple electrostatic model, theoretical PAs were predicted for each charge state and the values for (KAP)10 and (KAP)8 were within 10 kcal/mol of the experimental values. The maximum charge state of these peptides was observed in all cases even when the most volatile solvent was sufficiently basic to deprotonate that charge state in the gas phase. In solution (KAP)8 exhibits a random coil secondary structure while (KAA)8 exhibits an α-helix structure. Comparison of measured and calculated apparent PAs suggests that (KAP)8 retains its solution random coil structure in the gas phase and (KAA)8 retains the solution compact α-helix structure in the lower charge states but opens up to a ß structure in the gas phase to minimize electrostatic repulsions in higher charge states.

Fourier-transform ion cyclotron resonance mass spectrometric evidence for the formation of alpha-chloroenethiolates and thioketenes from chloroalkene-derived, cytotoxic 4-thiaalkanoates.

The cytotoxicity of chloroalkene-derived cysteine S-conjugates is thought to be associated with the formation of alpha-chloroenethiolates and thioketenes as reactive intermediates. Recent studies indicate that the formation of 1,2-dichloroethenethiolate, which may give rise to chlorothioketene, is a key step in the bioactivation of 5,6-dichloro-4-thia-5-hexenoic acid (Fitzsimmons et al. (1995) Biochemistry 34, 4276-4286). We report here the use of Fourier-transform ion cyclotron resonance mass spectrometry to provide the first direct evidence for the formation of alpha-chloroenethiolate and thioketene species from a cytotoxic 4-thiaalkanoate. The bioactivation of 5,6-dichloro-4-thia-5-hexenoic acid involves conversion to the corresponding CoA thioester 5,6-dichloro-4-thia-5-hexenoyl-CoA and subsequent processing by the fatty acid beta-oxidation pathway. It has been proposed that the bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA involves loss of 1,2-dichloroethenethiolate, followed by loss of chloride to form chlorothioketene. 1,2-Dichloroethenethiolate and related alpha-chloroalkenethiolates have not been observed directly in aqueous solution. Fourier-transform ion cyclotron resonance mass spectrometric experiments show that S-propyl 5,6-dichloro-4-thia-5-hexenethioate reacts in the gas phase with base (hydroxide ion) to release 1,2-dichloroethenethiolate, which is observed directly in the mass spectrum of the products of the gas-phase reaction. Furthermore, the elimination of chloride from 1,2-dichloroethenethiolate on collision-induced decomposition is facile and provides evidence for chlorothioketene formation. Preliminary evidence for the formation of 1,2-dichloroethenethiolate and chlorothioketene from S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine methyl ester was also obtained. These observations support the intermediacy of alpha-chloroenethiolates and chlorothioketenes in the bioactivation of cytotoxic, chloroalkene-derived 4-thiaalkanoates and cysteine S-conjugates and demonstrate the utility of Fourier-transform ion cyclotron mass spectrometry in studying the formation of reactive intermediates.

Radiative stabilization of trimethylsilyl adduct ions.

Ketones and phenol react with trimethylsilyl ions to form adduct ions by radiatively or collisionally stabilized addition reactions, in contrast to aliphatic alcohols and ethers, which react with trimethylsilyl ions to form adduct ions by a rapid two-step process. Secondorder rate constants for the addition of trimethylsilyl ions to acetone were independent of pressure from 3×10(-7) to 50×10(-7) tort at room temperature; consequently, the adduct ions, [M+73](+), are formed primarily by radiatively stabilized addition in these ion cyclotron resonance experiments.

Superelastic collisions of electrons with excited Mn(+).

Excited Mn(+) ions formed by electron ionization of Mn2(CO)10 are deexcited in superelastic electron-ion collisions. The ions are held in the trap of a Fourier transform ion cyclotron resonance spectrometer and subjected to bombardment by an electron beam of varying energy. The population of excited Mn(+) ions after exposure to the beam is monitored by examining reaction of the trapped Mn(+) ions with Cr(CO)6. Charge transfer to form Cr(CO) 6 (+) is exothermic and efficient only for excited Mn(+). It is found that deexcitation is read if y observable for electrons with energies less than 2 eV.