Synthesis and biological activity of position 1 analogs of LH-RH.

(Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.     

The lactam of alpha-guanidinoglutaric acid (1-amidino-2-pyrrolidone-5-carboxylic acid)

alpha-Guanidinoglutaric acid (alpha-GGA) has been reported to occur in the cerebral cortex after epileptic seizures. No physical characteristics of alpha-GGA have been given. A practical procedure for the preparation of alpha-GGA is reported here. alpha-GGA forms a lactam in aqueous solution at 80 degrees C. It is proposed to substitute this lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid (pAGlu), for pyroglutamic acid (pGlu) at the N-terminal position in neuropeptides to modify their biological characteristics. L(+)-Glutamic acid was reacted with S-methylisothiourea (I) at pH 10 in aqueous solution to form L(-)-alpha-guanidinoglutaric acid: mp 165-168 degrees C, [alpha]22D = -22.7 (C = 4, 2 M HCl). alpha-GGA reacted promptly with excess reagent to form a salt, S-methylisothiourea-alpha-guanidinoglutarate: mp 209-210 degrees C, [alpha]22D = -13.0 (C = 4, 2 M HCl). I was removed from the salt with aqueous picric acid, since I readily formed an insoluble picrate, S-methylisothiourea picrate (mp 225-228 degrees C). Alternatively, the salt was added to a cation exchange column, and the alpha-GGA was eluted with molar ammonium acetate buffer, pH 9.5. Its lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid, mp 248-249 degrees C, [alpha]22D = +2.1 (C = 4, 2 M HCl), formed a picrate (mp 196-199 degrees C).     

Saccharopine and 2-aminoadipic acid in Reseda odorata

2(S),2′(S)-N⁶-(2′-Glutaryl)lysine (l-saccharopine) and 2(S)-2-aminoadipic acid have been isolated from Reseda odorata. When traditional isolation procedures are used l-pyrosaccharopine (5(S),5′(S)-N-(5′-amino-5′-carboxy-pentyl)-2-pyrrolidone-5-carboxylic acid) is formed from l-saccharopine by lactamisation. The degree of lactamisation during various isolation steps has been studied, The amino acids were identified by IR and PMR spectroscopy and the configurations established by enzymic and polarimetric analyses. The contents of saccharopine and 2-amino-adipic acid have been determined relative to the total nitrogen content at various stages in the growth cycle of R. odorata.     

Simultaneous determination of 3,4-dihydroxyphenylacetic acid and homovanillic acid in milligram amounts of rat striatal tissue by gas-liquid chromatography

Simultaneous determination of the major metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum has been achieved by gas-liquid chromatography. Striatal tissue from one rat was homogenized in IN HCl and one-tenth of the sample extracted with ethyl ether. After evaporation of the ether, the residue was reacted with a combination of 1-chloro-1,1,3,3,3-pentafluor-2-propanol and pentafluoropropionic anhydride followed by reaction with pentafluoropropionic anhydride. The derivatives were chromatographed on a 3% JXR column and quantitated using electron capture detection. The propionic homologs of DOPAC and HVA served as internal standards. The steady state levels of DOPAC and HVA were found to be 0.90 μg/gm±0.21 S.D. (N=12) and 0.66 μg/gm±0.16 S.D. (N=12) respectively.     

Proton magnetic resonance studies of alpha-keto acids.

Pulsed Fourier transform proton magnetic resonance was used to study alpha-ketoglutaramic, and several other alpha-keto acids in aqueous solutions as a function of pH. Most alpha-keto acids were found to exist in equilibrium with the hydrate (gem-doil). The equilibrium position favors the nonhydrated alphs-keto acid at neutral pH, but at low pH values (below the pKa of the alpha-carboxylic acid group) the hydrate predominates. We found evidence that alpha-ketoglutaric acid exists in a third equilibrium form which is assigned to the lactol. alpha-Ketoglutaramic acid (the alpha-keto acid analog of glutamine) which is known to exist predominantly in a cyclic form at pH 7.0 was shown to exist as a cyclic structure over a wide pH range. However, the cyclic form is an equilibrium mixture of 2-pyrrolidone-5-hydroxy-5-carboxylic and 1-pyrrolin-2-one-5-carboxylic acids.     

Analysis of 5-pyrrolidone-2-carboxylate ester by reverse phase high-performance liquid chromatography

A simple quantitative method for estimating nanomole concentrations of 5-pyrrolidone-2-carboxylic acid (PCA) in tissue homogenates from mouse has been developed using reverse-phase HPLC. PCA was detected as the 4-nitrophenacyl ester which has an absorption maximum at 263 nm, a relatively high stability, and excellent chromatographic separation and detectability. This method offers distinct advantages over other analytical procedures thus far employed for measuring PCA in that the 4-nitrophenacyl derivative of PCA can be readily prepared from deproteinized tissue homogenates and quantitated by HPLC within relatively short time intervals with good precision and specificity.     

Specificity of gamma-glutamyl cyclotransferase.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Synthesis and cytotoxicity of salicylate-based poly(anhydride esters)

This paper describes the synthesis and cytotoxicity of poly(anhydride esters) that are composed of several salicylate derivatives, including halogenated salicylates, aminosalicylates, salicylsalicylic acid, and thiolsalicylic acid. The incorporation of these nonsteroidal antiinflammatory drugs (NSAIDs) into a biodegradable polymer backbone yields drug-based polymers that have potential for a variety of applications. The poly(anhydride esters) were synthesized by melt condensation polymerization. The halogenated salicylate derivatives yielded the highest molecular polymers as well as the highest glass transition temperatures. All polymers displayed in vitro degradation lag times from 1 to 3 days, depending on the water solubility of the salicylate derivative. Cell viability and proliferation were determined with L929 fibroblast cells in serum-containing medium to assess the polymer cytotoxicities, which varied as a function of the saliyclate chemistry. Cell morphology was normal for most of the polymers evaluated.     

Directed biosynthesis of novel derivatives of echinomycin by Streptomyces echinatus. I. Effect of exogenous analogues of quinoxaline-2-carboxylic acid on the fermentation

Streptomyces echinatus A8331 cultured on a maltose minimal salts medium normally produces a single antibiotic, echinomycin (quinomycin A), containing two quinoxaline-2-carbonyl chromophores. Echinomycin is powerfully active against experimental tumours and can be assayed by its activity against Gram-positive bacteria. Grown in the presence of aromatic carboxylic acids related to quinoxaline, S. echinatus responds in favourable circumstances by incorporating the added material into analogues of the natural antibiotic having replacement chromophores. Both mono- and bis-substituted derivatives are formed. With quinoline-2-carboxylic acid as precursor, large quantities of analogues are produced, and the time course of synthesis, extraction, purification, assay, and characterization of the derivatives are described. Twenty-two other aromatic acids have been tested as potential substrates for antibiotic analogue biosynthesis. Half of them did not significantly affect growth and echinomycin production. Five appeared to stimulate antibiotic synthesis, while the remainder proved inhibitory. New biologically active antibiotics were detected in cultures supplemented with 7-chloroquinoxaline-2-carboxylic acid; 1,2,4-benzo-as-triazine-3-carboxylic acid; thieno[3,2-b]pyridine-5-carboxylic acid; and 6-methylquinoline-2-carboxylic acid.     

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptaflourobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m×0.32 m I.D. and a 0.52 μm film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.     

Discovery of a highly orally bioavailable c-5-[6-(4-Methanesulfonyloxyphenyl)hexyl]-2-methyl-1,3-dioxane-r-2-carboxylic acid as a potent hypoglycemic and hypolipidemic agent

A series of novel 1,3-dioxane-2-carboxylic acid derivatives containing alkyl chain tether and substituted phenyl group as a lipophilic tail have been prepared as agonists of PPARalpha and gamma. c-5-[6-(4-Methanesulfonyloxyphenyl)hexyl]-2-methyl-1,3-dioxane-r-2-carboxylic acid 13c exhibited potent hypoglycemic and lipid lowering activity with high oral bioavailability in animal models.     

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Studies on dehydro-d-arabino-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

d-erythro-2,3-Hexodiulosono-1,4-lactone 2-arylhydrazones (2) were prepared by condensation of dehydro-d-arabino-ascorbic acid with the desired arylhydrazine. Reaction of 2 with hydroxylamine gave the 2-arylhydrazone 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-d-erythro-glycerol-1-yl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone (5), whereas the unacetylated triazole derivatives were obtained upon reaction of 3 with bromine in water. On treatment of 5 with hydrazine hydrate, 2-aryl-4-(d-erythro-glycerol-1-yl)-1,2,3-triazole-5-carboxylic acid 5-hydrazides (6) were obtained. Acetylation of 6 gave the hexaacetyl derivatives. Similarly, treatment of 5 with liquid ammonia gave the triazolecarboxamides (12). Vigorous acetylation of 12 with boiling acetic anhydride gave tetraacetates, whereas acetylation with acetic anhydride-pyridine gave triacetates. Periodate oxidation of 6 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxylic acid 5-hydrazides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxylic acid 5-hydrazides, characterized as acetates. Similarly, periodate oxidation of 12 gave the triazolealdehyde (15), and reduction of 15 gave the hydroxymethyl derivatives (16). Acetylation of 16 gave the mono- and di-acetates, and, on reaction with o-phenylenediamine, 15 afforded the triazoleimidazole. Controlled reaction of 3 with sodium hydroxide, followed by neutralization, gave 3-(d-erythro-glycerol-1-yl)-4,5-isoxazolinedione 4-arylhydrazones. Reaction of 3 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-d-erythro-2,3-hexodiulosono-1,4-lactone 2-arylhydrazone 3-oximes (21). Compounds 21 were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-d-erythro-glycerol-1-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone on treatment with acetic anhydride.     

Simultaneous quantification of homovanillic acid and 5-hydroxyindoleacetic acid in cerebrospinal fluid by mass fragmentography

A mass fragmentographic technique for the simultaneous quantification of HVA and 5-HIAA in CSF was described. Deuterium labelled analogues of the acids were used as internal standards. Derivatives of the compounds were prepared by reacting CSF extracts with a mixture of pentafluoropropanol and pentafluoropropionic anhydride. Fragments set into focus were the molecular ion of the HVA derivatives and M⁺ ?177 of the 5-HIAA derivatives. The experimental error was below 7% for both acids and the recovery of authentic compounds added to CSF was 93–96%. With an AVA allowing simultaneous recording of four mass numbers, both acids could be determined concomitantly. The technique would be appropriate for analysis of large numbers of samples especially when it is of interest to determine the relation between levels of HVA and 5-HIAA in CSF.     

Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation and chiro-optical characterization of polyaniline doped with (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA)

Optically active polyaniline (PANI) salts were readily generated in solution via the enantioselective acid doping of neutral emeraldine base (EB) form of PANI with either (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA) in dimethylsulfoxide (DMSO) and dimethylformamide (DMF) solvents. Strong mirror imaged circular dichroism (CD) spectra were obtained for the deep green polymer solutions obtained with (+) or (-) PCA, suggesting that the acid doping is enantioselective, with one helical screw of the polymer chain being preferentially produced depending on the nature of enantiomer. It was observed that molar concentration of PCA as well as nature of solvent plays a very important role in the generation of optically active PANI. The generated optically active PANI did not show any loss of optical activity up to 200 h.     

Novel regioselective hydroxylations of pyridine carboxylic acids at position C2 and pyrazine carboxylic acids at position C3

We have previously described the isolation of the new bacterial species, Ralstonia/Burkholderia sp. strain DSM 6920, which grows with 6-methylnicotinate and regioselectively hydroxylates this substrate in the C2 position by the action of 6-methylnicotinate-2-oxidoreductase to yield 2-hydroxy-6-methylnicotinate (Tinschert et al. 1997). In the present study we show that this enzymatic activity can be used for the preparation of a series of hydroxylated heterocyclic carboxylic acid derivatives. The following products were obtained from the unhydroxylated educts by biotransformation using resting cells: 2-hydroxynicotinic acid, 2-hydroxy-6-methylnicotinic acid, 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid, 3-hydroxypyrazine-2-carboxylic acid, 3-hydroxy-5-methylpyrazine-2-carboxylic acid and 3-hydroxy-5-chloropyrazine-2-carboxylic acid. Thus the respective educts were all regioselectively mono-hydroxylated at the carbon atom between the ring-nitrogen and the ring-carbon atom carrying the carboxyl group. In contrast to its relatively broad biotransformation abilities, the strain shows a limited heterocyclic nutritional spectrum. It could grow only with three of the seven transformed educts: 6-methylnicotinate, 2-hydroxy-6-methylnicotinate and 5-methylpyrazine-2-carboxylate. 2-Hydroxynicotinate, 2-hydroxy-6-chloronicotinate, 2-hydroxy-5,6-dichloronicotinate, 3-hydroxypyrazine-2-carboxylate and 3-hydroxy-5-chloropyrazine-2-carboxylate were not degraded by the strain. Therefore, unlike 6-methylnicotinate-2-oxidoreductase, which has a broad substrate spectrum, the second enzyme of the 6-methylnicotinate pathway seems to have a much more limited substrate range. Among 28 aromatic heterocyclic compounds tested as the sole source of carbon and energy, only pyridine-2,5-dicarboxylate was found as a further growth substrate, and this was degraded by a pathway which did not involve 6-methylnicotinate-2-oxidoreductase. To the best of our knowledge the microbial production of 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid and 3-hydroxy-5-methylpyrazine-2-carboxylic acid have not been reported before. Strain DSM 6920 is so far the only known strain which allows the microbial production of both these compounds and 3-hydroxypyrazine-2-carboxylic acid and 3-hydroxy-5-chloroypyrazine-2-carboxylic acid. Received: 18 June 1999 / Received revision: 30 August 1999 / Accepted: 3 September 1999