Enantioseparation of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidones on chiral stationary phases based on 3,5-dimethylanilides of N-(4-alkylamino-3,5-dinitro)benzoyl L-alpha-amino acids

Three novel chiral packing materials for high-performance liquid chromatography were prepared by covalently binding of (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]propan-amide (7), (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]-4-methylpentanamide (8), and (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonyl-amino]-2-phenylacetamide (9) to aminopropyl silica. The resulting chiral stationary phases (CSPs 1-3) proved effective for the resolution of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidone derivatives (TR 1-14). The mechanism of their enantioselection, supported by the elution order of (S)-TR 13 and (R)-TR 13 and molecular modeling of the complex of the slower running (S)-TR 13 with CSP 1 is discussed.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

Metabolism of dichloromethylcatechols as central intermediates in the degradation of dichlorotoluenes by Ralstonia sp. strain PS12

Ralstonia sp. strain PS12 is able to use 2,4-, 2,5-, and 3,4-dichlorotoluene as growth substrates. Dichloromethylcatechols are central intermediates that are formed by TecA tetrachlorobenzene dioxygenase-mediated activation at two adjacent unsubstituted carbon atoms followed by TecB chlorobenzene dihydrodiol dehydrogenase-catalyzed rearomatization and then are channeled into a chlorocatechol ortho cleavage pathway involving a chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase. However, completely different metabolic routes were observed for the three dichloromethylcatechols analyzed. Whereas 3,4-dichloro-6-methylcatechol is quantitatively transformed into one dienelactone (5-chloro-2-methyldienelactone) and thus is degraded via a linear pathway, 3,5-dichloro-2-methylmuconate formed from 4,6-dichloro-3-methylcatechol is subject to both 1,4- and 3,6-cycloisomerization and thus is degraded via a branched metabolic route. 3,6-Dichloro-4-methylcatechol, on the first view, is transformed predominantly into one (2-chloro-3-methyl-trans-) dienelactone. In situ (1)H nuclear magnetic resonance analysis revealed the intermediate formation of 2,5-dichloro-4-methylmuconolactone, showing that both 1,4- and 3,6-cycloisomerization occur with this muconate and indicating a degradation of the muconolactone via a reversible cycloisomerization reaction and the dienelactone-forming branch of the pathway. Diastereomeric mixtures of two dichloromethylmuconolactones were prepared chemically to proof such a hypothesis. Chloromuconate cycloisomerase transformed 3,5-dichloro-2-methylmuconolactone into a mixture of 2-chloro-5-methyl-cis- and 3-chloro-2-methyldienelactone, affording evidence for a metabolic route of 3,5-dichloro-2-methylmuconolactone via 3,5-dichloro-2-methylmuconate into 2-chloro-5-methyl-cis-dienelactone. 2,5-Dichloro-3-methylmuconolactone was transformed nearly exclusively into 2-chloro-3-methyl-trans-dienelactone.     

3-Chloro-, 2,3- and 3,5-dichlorobenzoate co-metabolism in a 2-chlorobenzoate-degrading consortium: role of 3,5-dichlorobenzoate as antagonist of 2-chlorobenzoate degradation

A study was made of the metabolic and co-metabolic intermediates of 2- and 3-chlorobenzoate, 2,3- and 3,5-dichlorobenzoate to elucidate the mechanism(s) involved in the negative effects observed on the growth of a chlorobenzoate-degrading microbial consortium in the presence of mixed chlorobenzoates. 2-Chloro-muconate accumulated as the end-product in the cultural broths of the microbial consortium during growth on 2-chlorobenzoate; the same 2-chloromuconate was identified in the reaction mixtures of resting cells pre-grown on 2-chlorobenzoate and exposed to 3-chloro- and 2,3-dichlorobenzoate, while in similar experiments 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate was detected as dead-end product of 3,5-dichlorobenzoate co-metabolism. These results suggest an initial degradative attack by 2-chlorobenzoate induced dioxygenase(s). The role of 3,5-dichlorobenzoate as an antagonist of 2-chlorobenzoate degradation was also studied: in the presence of mixed 2-chloro- and 3,5-dichlorobenzoate, the 3,5-dichlorobenzoate preferential uptake by the resting cells of the chlorobenzoate-degrading consortium was observed. 2-Chlorobenzoate entered the cells only after the complete removal of the co-substrate. In growing cells experiments, the addition of 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate, the 3,5-dichlorobenzoate co-metabolite, to 2-chlorobenzoate exerted the same antagonistic effect of the parent compound, inhibiting both the microbial growth and the degradative process. These data are discussed, allowing us to attribute the inhibitory effects observed to a substrate/co-substrate competition, though other additional causes may not be totally excluded.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

2''-Deoxy-3,7-dideazaguanosine and related compounds. Synthesis of 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl) and 1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]pyridin-4(5H)-one via direct glycosylation of a pyrrole precursor.

The synthesis of two new analogs of 2'-deoxyguanosine, 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H-pyrrolo[3,2-c] pyridin-4(5H)-one (8) and 6-amino-1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]-pyridin-4(5H)-one (13) has been accomplished by glycosylation of the sodium salt of ethyl 2-cyanomethyl-1H-pyrrole-3-carboxylate (4c) using 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose( 5) and 1-chloro-2,3,5-tri-O-benzyl-alpha-D-arabinofuranose (9), respectively. The resulting blocked nucleosides, ethyl 2-cyanomethyl-1-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro- pentofuranosyl)-1H-pyrrole-3-carboxylate (6) and ethyl 2-cyanomethyl-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)- 1H-pyrrole-3-carboxylate, were ring closed with hydrazine to form 5-amino-6-hydrazino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H- pyrrolo[3,2-c]-pyridin-4(5H)-one (7) and 5,6-diamino-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)-1H- pyrrolo[3,2-c]pyridin-4(5H)-one (11), respectively. Treatment of 7 with Raney nickel provided the 2'-deoxyguanosine analog 8 while reaction of 11 with Raney nickel followed by palladium hydroxide/cyclohexene treatment gave the 2'-deoxyguanosine analog 13. The anomeric configuration of 8 was assigned as beta by proton NMR, while that of 13 was confirmed as beta by single-crystal X-ray analysis of the deblocked precursor ethyl 2-cyanomethyl-1-beta-D-arabinofuranosyl-1H-pyrrole-3-carboxylate (10a).     

Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.     

On the, stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile)

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies.

Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide (S₁₃) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

Endoplasmic Reticulum (ER) Stress-Suppressive Compounds from Scrap Cultivation Beds of the Mushroom Hericium erinaceum

Four compounds were isolated from scrap cultivation beds of the mushroom, Hericium erinaceum. Compounds 1–4 were identified as methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, 2-chloro-1,3-dimethoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxybenzoate, and 4-chloro-3,5-dimethoxybenzaldehyde by an interpretation of the NMR and MS data, respectively. This is the first reported isolation of 1 from a natural source. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.     

A series of heteroleptic complexes of the type fac-[MnL2] [H2L=derivatives of N-(2-hydroxybenzyl)glycine or N-(5-nitro-2-hydroxybenzyl)sarcosine] possessing unusual Mn(III) co-ordination spheres

The mononuclear manganese(III) complexes [C₅H₁₀NH₂][MnL₂] [L²⁻=a substituted N-(2-hydroxybenzyl)glycinate (hbg²⁻) viz. 3,5-dibromo- (3,5-Br-hbg²⁻), 3,5-dichloro- (3,5-Cl-hbg²⁻), 3-methyl-5-chloro- (3,5-Me,Cl-hbg²⁻), 5-bromo- (5-Br-hbg²⁻), 5-chloro- (5-Cl-hbg²⁻), 5-nitro- (5-NO₂-hbg²⁻) or N-(5-nitro-2-hydroxybenzyl)sarcosine (5-NO₂-hbs²⁻)] have been synthesised by reaction of the appropriate ligand with manganese(II) perchlorate under ambient conditions in a 2:1 molar ratio using piperidine as base. The structures of three of these complexes, [C₅H₁₀NH₂][Mn(3,5-Cl-hbg)₂] (2), [C₅H₁₀NH₂][Mn(5-NO₂-hbg)₂] (6) and [C₅H₁₀NH₂][Mn(5-NO₂-hbs)₂] (7) have been elucidated by single-crystal X-ray crystallography and each displays two similar, independent [MnL₂]⁻ ions in the asymmetric unit linked via piperidinium cations through hydrogen bonding. The ligands co-ordinate in a facial tridentate fashion with the three donor atoms being the phenolate and carboxylate oxygens and the amine nitrogen. The geometry at the Mn centres is compressed rhombic octahedral consistent with a pseudo-Jahn–Teller compression along the Mn–O(phenolate) axis. Mean bond lengths are in the ranges 1.886–1.889 Å for the Mn–O(phenolate), 2.062–2.125 Å for the Mn–O(carboxylate) and 2.091–2.184 Å for the Mn–N(amine) distances. The magnetic susceptibility and electronic and IR spectroscopic data are discussed with reference to the crystal structures.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

On the stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile).

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies. Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S13) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

Synthesis and HPLC chiral recognition of regioselectively carbamoylated cellulose derivatives

Four regioselective-carbamoylated cellulose derivatives having two different substituents at 2-, 3-, and 6-position were prepared and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. Investigations showed that the nature and arrangement of the substituents significantly influenced the chiral recognition abilities of the heterosubstituted cellulose derivatives and each derivative exhibited characteristic enantioseparation. Some racemates were better resolved on these derivatives than the corresponding homogeneously substituted cellulose derivatives including a commercial CSP, Chiralcel OD. Racemic compounds shown in this study were most effectively discriminated on cellulose 2,3-(3-chloro-4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) and 2,3-(3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate).     

Studies on plant growth-regulating substances. XL. 2-Chloro-3-phenylpropionitriles

The complete series of chloro-, dichloro-, methyl-, dimethyl- and chloro-methyl-ring substituted 2-chloro-3-phenylpropionitriles and several related compounds have been prepared. Their auxin activities have been assessed in the wheat coleoptile, pea segment and pea curvature tests, and the results, which are discussed in relation to molecular structure, support the suggestion by other workers that the observed activity is due to rapid uptake of the nitriles by the plant tissues, followed by metabolism to the corresponding phenylacetic acid. The high growth promoting activity of 2-chloro-3-(3-chloro-2-methyl-phenyl)propionitrile (orthonil) is confirmed but 2-chloro-3-(2,3-dichloro-phenyl)propionitrile and 2-chloro-3-(2,3-dichlorophenyl)butyronitrile are shown to be the most active members of the series.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Novel 2′-Deoxy Pyrazine C-Nucleosides Synthesized VIA Palladium-Catalyzed Cross-Couplings

Abstract

The palladium-catalyzed cross-couplings of 2-chloro-3,5-diamino-6-iodopyrazine (1a) and methyl 3-amino-6-iodopyrazine-2-carboxylate (1b) with 1,4-anhydro-3,5-O-bis[(tert-butyl)dimethylsilyl]-2-deoxy-D-erythro-pent-1-enitol (2) followed by desilylation and stereospecific reduction of the 2′-deoxy-3′-keto adduct leads to the formation of 2-chloro-6-(2-deoxy-ß-D-ribofuranosyl)-3,5-diaminopyrazine (4a) and methyl 3-amino-6-(2-deoxy-ß-D-ribofuranosyl)pyrazine-2-carboxylate (4b) in 58% yield and 21% yield, respectively. These are the first syntheses of the heretofore unknown 2′-deoxy pyrazine C-nucleosides and demonstrate the utility of a convergent approach for the synthesis of pyrazine C-nucleosides.     

Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid.

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.