Directions of alkylation of deoxyguanosine and deoxyguanylic acid by thio-TEPA

As exemplified with the reaction of well-known anticancer agent thio-TEPA with dGMP, the ability of ethylenimine derivatives to modify this DNA component under the conditions approaching the biological ones was demonstrated for the first time. Modification involves mainly the phosphate group alkylation. In addition, thio-TEPA alkylates the base in dGMP at position 7. The formation of dGMP 7-alkyl derivatives was confirmed by detecting the luminescent products generated in the course of this reaction.     

Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.     

Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase

Vaccinia virus thymidylate kinase, although similar in sequence to human TMP kinase, has broader substrate specificity and phosphorylates (E)-5-(2-bromovinyl)-dUMP and dGMP. Modified guanines such as glyoxal-dG, 8-oxo-dG, O⁶-methyl-dG, N²-ethyl-dG and N⁷-methyl-dG were found present in cancer cell DNA. Alkylated and oxidized dGMP analogs were examined as potential substrates for vaccinia TMP kinase and also for human TMP and GMP kinases. Molecular models obtained from structure-based docking rationalized the enzymatic data. All tested nucleotides are found surprisingly substrates of vaccinia TMP kinase and also of human GMP kinase. Interestingly, O⁶-methyl-dGMP is the only analog specific for the vaccinia enzyme. Thus, O⁶-Me-dGMP could be useful for designing new compounds of medical interest either in antipoxvirus therapy or in experimental combined gene/chemotherapy of cancer. These results also provide new insights regarding dGMP analog reaction with human GMP kinase and their slow recycling by salvage pathway nucleotide kinases.     

Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isozymes

Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.     

Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives

Structural complexes of the eukaryotic translation initiation factor 4E (eIF4E) with a series of N(7)-alkylated guanosine derivative mRNA cap analogue structures have been characterised. Mass spectrometry was used to determine apparent gas-phase equilibrium dissociation constants (K(d)) values of 0.15 microM, 13.6 microM, and 55.7 microM for eIF4E with 7-methyl-GTP (m(7)GTP), GTP, and GMP, respectively. For tight and specific binding to the eIF4E mononucleotide binding site, there seems to be a clear requirement for guanosine derivatives to possess both the delocalised positive charge of the N(7)-methylated guanine system and at least one phosphate group. We show that the N(7)-benzylated monophosphates 7-benzyl-GMP (Bn(7)GMP) and 7-(p-fluorobenzyl)-GMP (FBn(7)GMP) bind eIF4E substantially more tightly than non-N(7)-alkylated guanosine derivatives (K(d) values of 7.0 microM and 2.0 microM, respectively). The eIF4E complex crystal structures with Bn(7)GMP and FBn(7)GMP show that additional favourable contacts of the benzyl groups with eIF4E contribute binding energy that compensates for loss of the beta and gamma-phosphates. The N(7)-benzyl groups pack into a hydrophobic pocket behind the two tryptophan side-chains that are involved in the cation-pi stacking interaction between the cap and the eIF4E mononucleotide binding site. This pocket is formed by an induced fit in which one of the tryptophan residues involved in cap binding flips through 180 degrees relative to structures with N(7)-methylated cap derivatives. This and other observations made here will be useful in the design of new families of eIF4E inhibitors, which may have potential therapeutic applications in cancer.     

The mouse guanylate kinase double mutant E72Q/D103N is a functional adenylate kinase.

Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an important enzyme in nucleotide metabolic pathways. Because of its essential intracellular role, guanylate kinase is a target for a number of cancer chemotherapeutic agents such as 6-thioguanine and 8-azaguanine and is involved in antiviral drug activation. Guanylate kinase shares a similarity in function and structure to other nucleoside monophosphate kinases especially with that of the well-studied adenylate kinase. Amino acid substitutions were made within the GMP binding site of mouse guanylate kinase to alter the polarity of the side chains that interact with GMP as a means of evaluating the role that these residues play on substrate interaction. One of these mutants, E72Q/D103N, was shown by functional complementation and enzyme assays to embody both guanylate kinase activity and a novel adenylate kinase activity.     

Isolation and properties of a glycohydrolase specific for nicotinamide mononucleotide from Azotobacter vinelandii.

A glycohydrolase that catalyzes the irreversible conversion of NMN to nicotinamide and ribose 5-phosphate has been partially purified from a sonic extract of Azotobacter vinelandii. The enzyme is highly specific for NMN. NAD, NADP, nicotinic acid-adenine dinucleotide, nicotinamide riboside and alpha-NMN are not significantly hydrolyzed by this enzyme, nor do they compete with NMN. The enzyme also exhibits an absolute dependence on guanylic acid derivatives with following order of relative effectiveness: GTP, guanosine 5'-tetraphosphate greater than dGTP, GDP, 2'-GMP, 3'-GMP greater than GMP, dGMP. A heat-resistant, nondialyzable factor which could replace the GTP requirement was found in the sonic extract. The Ka for GTP and the Km for NMN in the presence of GTP at 1mm were calculated to be 0.025 mM and 4.5 mM respectively. GMP, dGMP, and dCMP were found to be effective inhibitors of the enzyme when 1 mM GTP was also present. The kinetic data suggest that the binding site for these mononucleotides is distinct from the active site or the GTP binding site. The ability of this enzyme to cleave NMN is suggestive of a metabolic role of the enzyme in selective conversion of NMN to nicotinamide, which, in turn, would be re-utilized by the cell as a precursor of NAD via nicotinic acid.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.     

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins     

Alkylation of nucleic acid components by ethyleneimine derivatives. I. Alkylation of bases

In the course of investigating the reaction conditions of the nucleic acid components alcylation, the interaction of thioTEPA (N,N',N'-triethylenethiophosphoamide) with hydrochloric and perchloric acids was studied, perchloric acid increasing the alkylation products yield. HPLC and UV spectroscopy were used to isolate and identify products of nucleic bases alkylation by ethylenimine and its derivatives (thioTEPA and monoaziridinediethylphosphate). It is shown that under neutral conditions phosphoaminoethylation takes place, whereas under slightly acidic conditions products of aminoethylation are formed.     

Mechanism of the mRNA guanylyltransferase reaction: isolation of N epsilon-phospholysine and GMP (5' leads to N epsilon) lysine from the guanylyl-enzyme intermediate.

The mRNA capping reaction catalyzed by rat liver mRNA guanylyltransferase proceeds through an enzyme-GMP intermediate in which GMP is linked to the enzyme by a phosphoamide linkage. The studies described here show that GMP is bound to the epsilon-amino group of lysine of rat liver guanylyltransferase. The enzyme-[32P]GMP intermediate was digested with pronase to a [32P]GMP-peptide which was then converted to [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. After alkaline hydrolysis of the [32P]phosphoryl-peptide, the major radioactive product co-electrophoresed with the authentic N epsilon-phospholysine on DEAE-cellulose paper. Neither [32P]Nimid-phosphohistidine nor Nguanido-phosphoarginine was detected in the hydrolysates. Furthermore, formation of N epsilon-guanylyl-lysine linkage on the enzyme was more directly shown by isolation of [32P]GMP(5' leads to N epsilon)lysine when the steps of periodate oxidation and beta-elimination were omitted. The results indicate that the nucleophile in the guanylyltransferase to which the guanylyl residue is linked is the epsilon-amino group of a lysine residue. [32P]Phosphoryl-lysine was also isolated from the vaccinia virus capping enzyme-[32P]GMP intermediate. Guanylyltransferase from HeLa cells, wheat germ, Artemia salina and yeast also formed the enzyme-GMP complex and, from the stability of the complex, the linkage between the enzyme and GMP was suggested to be a phosphoamide.     

Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides.

GMP and native DNA were reacted with 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene. It was shown that the 7-halogeno derivatives react on C-8 of guanine. Furthermore the respective amount of arylamidation (covalent linkage on the C-8 of guanine) and arylation (covalent linkage on 2-NH2 groups of C3 of guanine) addition products was determined in both native and denatured DNA-[14C]AAIF. Two G containing deoxytrinucleotides modified by either AAFF or AAIF were studied comparatively by means of circular dichroism, and as a function of several parameters known to affect the conformation of the deoxytrinucleotides. The induced optical activity in fluorofluorene ring seemed to be very sensible to the conformational changes of the deoxytrinucleotides. On the other hand, the AAIF residue exhibit a lower induced optical activity which remained unchanged when the deoxytrinucleotides conformation was affected. The results presented in this paper led us to conclude that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.     

Interaction of purine nucleotides with cobalt-hexammine, cobalt-pentammine and cobalt-tetrammine cations. Evidence for the rigidity of adenosine and flexibility of guanosine and deoxyguanosine sugar conformations.

The interaction of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP) and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) with the [Co(NH3)6]3+, [Co(NH3)5Cl]2+ and [Co(NH3)4Cl2]+ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and 1H-NMR spectroscopy. The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH3) through the N-7 and the PO3(2-) groups of the AMP and via O-6, N-7 and the PO3(2-) of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2'-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine-AMP complexes, whereas a mixture of teh C2'-endo/anti and C3'-endo/anti sugar puckers were observed for the Co(NH3)6-GMP, Co(NH3)5-GMP and a C3'-endo/anti conformer for the Co(NH3)4-GMP complexes. The deoxyribose showed an O4'-endo/anti conformation for the free dGMP anion and a C3'-endo/anti for the Co(NH3)6-dGMP, Co(NH3)5-dGMP and Co(NH3)4-dGMP complexes.     

Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7-9 and is characterized by marked stability. Compared with the human enzyme, the recombinant P. falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the Km values for TMP and dGMP are 22 and 30 microM, respectively. The turnover number kcat(TMP) was found to be 3.4 s(-1), a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study, we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.     

Mapping the functional topography of a receptor.

Photoactivatable derivatives of the alpha-neurotoxin II from Naja naja oxiana are useful tools for investigating the three dimensional architecture of the extra-membrane part of the nicotinic acetylcholine receptor from the electric tissue of Torpedo californica. Three derivatives, carrying an azidobenzoyl group in position Lys-15, Lys-26, and Lys-46, respectively, are shown to react differently within the receptor's quaternary structure. Especially the Lys-26 and Lys-46 derivatives can be used for differentiating between the two nonequivalent alpha-subunits. The Lys-26 derivative is applied for probing the receptor subunits next to the alpha-subunit: the gamma-subunit is shown to be located next to the alpha-subunit binding d-tubocurarine with high affinity. The delta-subunit is the neighbor of the low affinity alpha-subunit. We radioiodinated the toxin derivatives and localized the 125I at the His-31 residue of the toxin. Very little label was found in position Tyr-24, the only tyrosine residue of the toxin, or in position His-4, the only other histidine residue. This result is important for the cleavage experiments necessary in attempts to identify the receptor sequence which reacted with the photolabel.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards.

Nitrogen mustards alkylate DNA primarily at the N7 position of guanine. Using an approach analogous to that of the Maxam-Gilbert procedure for DNA sequence analysis, we have examined the relative frequencies of alkylation for a number of nitrogen mustards at different guanine-N7 sites on a DNA fragment of known sequence. Most nitrogen mustards were found to have similar patterns of alkylation, with the sites of greatest alkylation being runs of contiguous guanines, and relatively weak alkylation at isolated guanines. Uracil mustard and quinacrine mustard, however, were found to have uniquely enhanced reaction with at least some 5'-PyGCC-3' and 5'-GT-3' sequences, respectively. In addition, quinacrine mustard showed a greater reaction at runs of contiguous guanines than did other nitrogen mustards, whereas uracil mustard showed little preference for these sequences. A comparison of the sequence-dependent variations of molecular electrostatic potential at the N7-position of guanine with the sequence dependent variations of alkylation intensity for mechlorethamine and L-phenylalanine mustard showed a good correlation in some regions of the DNA, but not others. It is concluded that electrostatic interactions may contribute strongly to the reaction rates of cationic compounds such as the reactive aziridinium species of nitrogen mustards, but that other sequence selectivities can be introduced in different nitrogen mustard derivatives.     

The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

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.