Synthesis and chromatographic properties of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine

Details are reported for the synthesis of S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) from cysteine and 2-bromopropionic acid or 2-bromobutyric acid, respectively. Some analytical data and the behaviour of these two compounds on paper and ion-exchange chromatography are also reported, which allow their identification.     

Oxidative deamination of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine by L-aminoacid oxidase

S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) are oxidatively deaminated by L-aminoacid oxidase with consumption of half a mole of oxygen per mole of substrate in the presence of catalase. This reaction gives rise to the corresponding alpha-ketoacids, identified by some chemical and chromatographic tests and by comparison with synthetic compounds. It has been possible, therefore, to demonstrate that S-(1-carboxyethyl)-thiopvruvic acid (1-CETP) and S-(1-carboxypropyl)-thiopvruvic acid (1-CPTP) are the main products of oxidative deamination of 1-CEC and 1-CPC.     

Oxidative deamination of Se-(1-carboxyethyl)-,Se-(1-carboxypropyl)- and Se-(2-carboxyethyl)-selenocysteine by snake venom L-aminoacid oxidase

Details are reported for the synthesis of Se-(1-carboxyethyl)-selenocysteine (1-CESeC), Se-(1-carboxypropyl)-selenocysteine (1-CPSeC) and Se-(2-carboxyethyl)-selenocysteine (2-CESeC). They can be obtained in pure cristalline form with good yield. Some chromatographic properties, useful for their identification, are described. The three aminoacids are good substrates for snake venom L-aminoacid oxidase, giving the corresponding alpha-ketoacids as reaction products.     

Monitoring exposure to acrylamide by the determination of S-(2-carboxyethyl)cysteine in hydrolyzed hemoglobin by gas chromatography-mass spectrometry

Acrylamide is a potent cumulative neurotoxin in animals and man. In vivo exposure to this electrophile results in the formation of a covalently bound reaction product with cysteine residues in hemoglobin. This adduct yields on acid hydrolysis S-(2-carboxyethyl)cysteine which has been analyzed by capillary gas chromatography with mass spectrometry. Globin isolated from the blood of rats exposed to acrylamide was spiked with an internal standard (globin treated in vitro with d3-acrylamide) and was then hydrolyzed with 6 N HCl. The protein hydrolysate was fractionated on a Dowex 50W H+ ion exchange column and the amino acids in the partially purified extract were determined as N-heptafluorobutyryl methyl esters using an OV-1701 fused silica capillary column. Quantitation was made by chemical ionization (isobutane) selective ion monitoring in which the ions m/z 386 (M-OCH3)+ derived from derivatized S-(2-carboxyethyl)cysteine in the sample and the corresponding ion m/z 389 from the added deuterium-labeled internal standard were monitored. The dose-response relationship between production of hemoglobin adduct and dose of acrylamide (0.1 mg/kg-5 mg/kg) is curved, showing an increasing slope with increasing doses of acrylamide.     

Reversible cyclization ofS-(2-oxo-2-carboxyethyl)-L-homocysteine to cystathionine ketimine

Summary S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N₂ atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer.Abbreviations CK cystathionine ketimine - OCEHC S-(2-oxo-2-carboxyethyl) homocysteine - HPLC high performance liquid chromatography     

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).     

Complexation models of N-(2-carboxyethyl)chitosans with copper(II) ions

The copper(II) complex formation equilibria of N-(2-carboxyethyl)chitosans with three different degrees of substitution (DS = 0.42, 0.92, and 1.61) were studied in aqueous solution by pH-potentiometric and UV-spectrophotometric techniques. It was demonstrated that the complexation model of CE-chitosans depends on DS: the [Cu(Glc-NR(2))(2)] complexes are predominant for two lower substituted samples ("bridge model", log beta(12) = 10.06 and 11.6, respectively), whereas the increase of DS leads to formation mainly of the [Cu(Glc-NR(2))] complexes ("pendant model", log beta(11) = 6.41). As a model for copper complexation with a disubstituted residue of CE-chitosan, the complex of N-methyliminodipropionate [CuMidp(H(2)O)].(H(2)O) was synthesized and structurally characterized by XRD. The unit cell consists of two crystallographically nonequivalent Cu atoms having slightly distorted square pyramidal coordination; Midp constitutes the basal plane of the pyramid and acts as a tetradentate NO(3) chelate-bridging ligand by the formation of two six-membered chelate rings (average Cu-O 1.99 A, Cu-N 2.04 A) and a bridge via carbonyl O atom (average Cu-O 1.99 A), an apical position is occupied by a water molecule (average Cu-Ow 2.30 A).     

Structural and mechanistic studies on N-(2-carboxyethyl)arginine synthase

N²-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the β-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates d-glyceraldehyde-3-phosphate (d-G3P) and l-arginine, and are consistent with the proposed CEAS mechanism in which d-G3P binds at the active site and reacts to form an α,β-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the α-amino group of l-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the l-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative β-amino acid products.     

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

N2-(1-carboxyethyl)methionine. A ''pseudo-opine'' in octopine-type crown-gall tumours.

A novel methionine-containing plasmid-determined compound, N2-(1-carboxyethyl)methionine (NCEM) has been identified in crown-gall tumours induced by octopine-type strains of Agrobacterium tumefaciens. NCEM is probably synthesized by octopine synthase. Cell-free preparations from octopine-type strains of A. tumefaciens can degrade NCEM; however, the bacterium cannot transport the compound into the cell, although these strains can take up and degrade the octopine family of opines.     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.