The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N⁴-bis(2-carboxyethyl)deoxycytidine (3,N⁴-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N⁴-BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3,N⁴-bis(2-carboxyethyl)cytosine (3,N⁴-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d₂₇) tri-TMS derivative of 3-CEC. The structure of 3,N⁴-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N⁴-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N⁴-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H₂O at 37°C and pH 7.0–7.5.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.     

The isolation and characterization of 2-carboxyethyl adducts following in vitro reaction of acrylic acid with calf thymus DNA and bioassay of acrylic acid in female Hsd:(ICR)Br mice

Reaction of acrylic acid (AA) at pH 7.0 and 37 degrees C for 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo) and thymidine (dThd) resulted in the formation of 2-carboxyethyl (CE) adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (percent yield after 40 days) were 1-CE-dAdo (5%), N6-CE-dAdo (11%) (via Dimroth rearrangement of 1-CE-dAdo), 3-CE-dCyd (7.5%), 7-CE-Gua (4%), 7,9-bis-CE-Gua (0.9%) (formed by reaction of AA with depurinated 7-CE-Gua during the course of the reaction) and 3-CE-dThd (0.5%). The products isolated following in vitro reaction of AA with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-Ade (9.9), N6-CE-Ade (8.2), 7-CE-Gua (7.2) and 3-CE-Thy (1.9). Compound 3-CE-Cyt was not detected. Thus the adducts formed following in vitro reaction of AA with DNA are identical to those formed by in vitro reaction of the carcinogen beta-propiolactone (BPL) with DNA as reported in an earlier paper. Structures were assigned on the basis of identical UV spectra, Rf values on paper chromatograms and Rt values on HPLC as marker compounds prepared from reactions of BPL with 2'-deoxynucleosides and 2'-deoxynucleotides-5'-monophosphoric acids. AA was assayed for carcinogenic activity by s.c. injection (20 mumol, once a week for 52 weeks) in female Hsd: (ICR)Br mice. Two mice with sarcomas at the site of application were observed out of 30 mice. Malignancies were not observed in solvent and no-treatment controls. The bioassay results reported in this paper and elsewhere in the same strain of mice suggest that AA is a weak carcinogen in female Hsd:(ICR)Br mice.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Biosynthesis and stereochemical configuration of N5-(1-carboxyethyl)ornithine. An unusual amino acid produced by Streptococcus lactis

In a recent communication (Thompson, J., Curtis, M. A., and Miller, S.P.F. (1986) J. Bacteriol. 167, 522-529) we described the purification and characterization of N5-(1-carboxyethyl)ornithine from cells of Streptococcus lactis 133. This unusual amino acid has not previously been found in nature. Radiotracer experiments presented here reveal that exogenous [14C]ornithine serves as the precursor for biosynthesis of [14C]arginine, [14C]N5-(1-carboxyethyl)ornithine, and [14C]N5-acetylornithine by cells of S. lactis K1 during growth in a defined medium lacking arginine. In the absence of both arginine and ornithine, cells of S. lactis K1 can also generate intracellular [14C]N5-(1-carboxyethyl)ornithine from exogenous [14C]glutamic acid. Previously we showed that the properties of N5-(1-carboxyethyl)ornithine prepared from S. lactis were identical to one of the two diastereomers [2S, 7S) or (2S, 7R] present in a synthetic preparation of (2S, 7RS)-N5-(1-carboxyethyl)ornithine. The two diastereomers have now been unambiguously synthesized by an Abderhalden-Haase condensation between (2S)-N2-t-butoxycarbonyl-ornithine and the chiral (2S)-, and (2R)-bromopropionates. By 13C-NMR spectroscopy it has been established that the preparation from S. lactis is exclusively (2S, 7S)-N5-(1-carboxyethyl)ornithine. has been demonstrated in a cell-free extract of S. lactis 133. The requirements for ornithine, pyruvic acid, and NAD(P)H suggest that biosynthesis of N5-(1-carboxyethyl)ornithine occurs via a reductive condensation mechanism. A general survey revealed that N5-(1-carboxyethyl)ornithine was produced only by certain strains of Group N streptococci. These findings may indicate a plasmid locus for the gene(s) encoding the enzyme(s) for N5-(1-carboxyethyl)ornithine biosynthesis.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

On the mechanism of DNA-adenine methylase

Experiments were performed to determine whether EcoRI methylase catalyzes the transfer of the methyl group of S-adenosylmethionine (a) directly to the N6 of adenine in DNA or (b) initially to N1 to give N1-methyladenine followed by isomerization of the N1-methylamino and 6-NH2 to give N6-methyladenine (Dimroth rearrangement). A facile synthesis of highly enriched [6-15N]deoxyadenosine and a dodecamer substrate of EcoRI methylase with [6-15N]adenine in the methylation site are reported. In the product of EcoRI enzymatic methylation, all of the isotope remains at the N6 position of the N6-methyladenine product. It is concluded that, contrary to existing chemical precedent, the methylation occurs by direct transfer from S-adenosylmethionine to the N6 of adenine in DNA.     

The absolute configuration of 1-carboxyethyl substituents on common hexoses by circular dichroism

Determination of the absolute configuration of the 1-carboxyethyl substituent on a monosaccharide by circular dichroism measurements was found to be a sensitive and simple method. It relies on comparison of the spectrum of a 1-carboxyethyl substituted sugar or sugar derivative with the spectra of (R)- and (S)-lactic acid in the region 200-260 nm in which the (R)- and (S)-configuration give negative and positive deltaepsilon, respectively. The oligo- or poly-saccharide containing a 1-carboxyethyl substituted sugar is hydrolyzed to monomers and the 1-carboxyethyl substituted sugar isolated by chromatography. The CD spectrum obtained for the 1-carboxyethyl substituted sugar in water solution at pH 2 is then compared with spectra of (R)- and (S)-lactic acid. The sign for the absorption and a maximum of comparable intensity and appearance around 210 nm, identify the stereochemistry.     

Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway

The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution. The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.     

N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Raman spectroscopy study of acid-base and structural properties of 9-[2-(phosphonomethoxy)ethyl]adenine in aqueous solutions

The acid-base properties of the acyclic antiviral nucleotide analogue 9- [2-(phosphonomethoxy)ethyl] adenine (PMEA) in aqueous solutions are studied by means of Raman spectroscopy in a pH range of 1-11 and compared with the properties of its common adenosine monophosphate counterparts (5'-AMP, 3'-AMP, and 2'-AMP). Factor analysis is used to separate the spectra of pure ionic species (PMEA2-, HPMEA-, H2PMEA, H3PMEA+) in order to determine their abundance, sites of protonation, and corresponding spectroscopic pK(a) values. The characteristic Raman features of the neutral adenine moiety in PMEA2- and HPMEA- species resemble those of neutral adenine in the AMPs, whereas significant differences are observed between the Raman spectra of the N1-protonated adenine of the solute zwitterionic H2PMEA and its N1-protonated AMP counterparts. On the contrary, the spectrum of crystalline H2PMEA, adopting an "anti-like" conformation, is found to be similar to the N1-protonated AMPs in solution. To explain peculiar Raman features a "syn-like" conformation is suggested for N1-protonated PMEA species in aqueous solutions instead of an anti-like one adopted by H2PMEA in crystals or by common AMPs in aqueous solutions. A physical mechanism of the anti-like to syn-like conformational transition of the solute PMEA that is due to adenine protonation and the flexibility of the (phosphonomethoxy)ethyl group is proposed and discussed.     

Isolation using triflic acid solvolysis and identification of N(epsilon)-[(R)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine as a component of the O-specific polysaccharide of Proteus mirabilis O13

An amino acid was released from the O-specific polysaccharide of Proteus mirabilis O13 by acid hydrolysis and identified as N(epsilon)-[(R)-1-carboxyethyl]-L-lysine by comparison with the authentic sample. An amide of this amino acid with D-galacturonic acid was isolated from the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid and characterised by 1H and 13C NMR spectroscopy. These and published data enabled determination of the full structure of the repeating unit of the polysaccharide.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo. S -BMO adducts were the main product and represented 77 % (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6-15NH2 in adenine nucleotides

A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6-15NH2]adenine nucleotides is described. The method involves formation of the di-t-butyldimethylsilyl (TBDMS) derivative of adenine following isolation of the nucleotide fraction with solid-phase ion-exchange chromatography and subsequent acid hydrolysis of nucleotides to free base. Mass spectra for both adenine-diTBDMS and [6-15NH2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH2 moiety. The base peak (m/z 306) was formed by loss of C4H9 (57) and constitutes approximately one-third of the total ion current. Using selected ion monitoring of the m/z 306/m/z 307 ratio, levels of isotopic abundance of 1.0-50.0 mol% excess could be measured reproducibly with the injection of 10-20 pmol of the adenine-diTBDMS derivative obtained from isolated rat hepatocytes. Confirmation that measured isotopic abundance was referable to labeling of the 6-15NH2 group was obtained by oxidation of adenine to hypoxanthine and determination of enrichment in the hypoxanthine-diTBDMS derivative. The method was used to study the formation of [6-15NH2]adenine nucleotides during the incubation of isolated rat hepatocytes with [15N]alanine. A level of approximately 6.0 mol% excess was observed at 60 min incubation.     

Conformational preferences of modified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) adenine and 2-methylthio-N6-(N-threonylcarbonyl) adenine by the quantum chemical PCILO calculations

Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) Adenine, m6tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS 2tc6 Ade alike the threonylcarbonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(1) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS 2tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(delta 2-isopentenyl) Adenine, mS 2i6 Ade, where the 2-methylthio group orients trans to the C(2)-N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP.

Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl)thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine (III) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine (IV) which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-([N-3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (VIII), N6-(N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (IX), N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (XIII), and N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bo-nd derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX, and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.     

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