O(6)-allyl protected deoxyguanosine adducts of polycyclic aromatic hydrocarbons as building blocks for the synthesis of oligonucleotides.

We describe a synthetic strategy for the preparation of oligonucleotides using N(2)-alkylated and O(6)-allyl protected deoxyguanosine phosphoramidite building blocks derived from cis- and trans-opened (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and from trans-opened (+/-)-3alpha,4beta-dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene. The appropriately blocked phosphoramidite building blocks were obtained as mixtures of the cis- and trans-opened diol epoxide adducts upon initial reaction of the diol epoxides with O(6)-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine. Key to the present approach is the removal of the O(6)-allyl protecting group utilizing a palladium catalyst prior to release of the constructed oligonucleotide with ammonia from the solid support. The methodology described enables a very convenient access to oligonucleotides containing cis- and trans-N(2)-deoxyguanosine adducts of polycyclic aromatic hydrocarbons in different sequence contexts.     

Synthesis and absolute configuration of cis- and trans-opened cyclopenta[cd]pyrene 3,4-oxide N2-deoxyguanosine adducts: conversion to phosphoramidites for oligonucleotide synthesis

Fluorinated alcohols such as trifluoroethanol (TFE) or hexafluoropropan-2-ol (HFP) catalyze addition of the N2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3) to cyclopenta[cd]pyrene 3,4-oxide (CPPO). The reaction occurs via a carbocation intermediate at C-3 such that cis- and trans-opened dGuo adducts are formed in a combined yield of approximately 37% together with the 4-ketone and a cis-opened solvent adduct. Fluorinated alcohol-mediated regioselective substitution at C-3 of the CPP cis- (11) and trans-3,4-dihydrodiol diacetates (15) with the N2-amino group of 3 proceeded smoothly to give the O6-allyl di-(tert-butyldimethylsilyl) cis- and trans-opened dGuo-adduct acetates (8a,b and 9a,b) in 75-85% yields. The cis-opened adducts predominated (56-70%) from both 11 and 15. Interestingly, trans-3-acetoxy-4-bromo-3,4-dihydro-CPP and 3 in TFE or HFP gave a mixture of 8a,b and 9a,b in 75-85% yield with cis:trans adduct ratios similar to those observed for 11 and 15. This observation is consistent with initial formation of a cyclic acetoxonium intermediate followed by formation of the same carbocation as that derived from 11 or 15. Absolute configurations of 8a,b and 9a,b were assigned by using enantiomerically pure (+)-trans-[3S.4S]-dihydrodiol as the starting material, which afforded a single cis-[3R,4S]-dGuo adduct and a single trans-[3S,4S]-dGuo adduct. The optically active trans-3,4-dihydrodiols were obtained by HPLC separation of their bis-(-)-menthoxyacetates. Their absolute configuration was determined by several empirical methods in addition to application of the exciton chirality CD method to their bis-(p-N,N-dimethylamino)benzoates. Removal of all blocking groups from the protected cis- and trans-opened dGuo adducts (8a,b and 9a,b) in three steps (overall yields of >70%) gave the free dGuo adducts, which are useful markers for DNA-binding studies. Adducts 8a,b and 9a,b were also converted to the appropriately protected phosphoramidites in three steps (overall yields of 72-81%).     

A novel synthesis of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine

Malondialdehyde (MDA) is a mutagenic product of lipid peroxidation and prostaglandin biosynthesis. MDA reacts with DNA bases to produce adducts of deoxyguanosine (M1G), deoxyadenosine (M1A), and deoxycytidine (M1C). A novel synthesis of these MDA nucleoside adducts has been developed, which significantly improves their availability. For the deoxyguanosine adduct, M1G, an amine equivalent to MDA, 4-amino-3-(phenylselenyl)butane-1,2-diol, was reacted with 2-fluoro-O6-(2-(trimethylsilyl)ethyl)-2'-deoxyinosine via a nucleophilic aromatic substitution reaction followed by acid hydrolysis of the O6-protecting group to give an N2-modified deoxyguanosine intermediate. Periodate oxidation of this intermediate under slightly acidic conditions gave M1G in good overall yield via cleavage of the vicinal diol unit and concomitant oxidation of the phenylselenide group to the corresponding selenoxide and syn beta-elimination. M1A and M1C were synthesized by the same strategy starting from 6-chloropurine 2'-deoxyriboside and 1-(2-deoxy-beta-d-erythro-pentofuranosyl)-4-(1H-1,2,4-triazol-1-yl)-2-(1H)pyrimidinone, respectively. An advantage of this approach is that similar chemistry has been shown to be directly applicable to the synthesis of site specifically adducted oligonucleotides containing activated nucleobases such as those used in this study. This strategy may offer an improved synthesis to oligonucleotides containing M1G and a feasible approach to M1A and M1C containing oligonucleotides.     

Differences between the mutational consequences of replication of cis- and trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-deoxyguanosine adducts in M13mp7L2 constructs.

The four adducts at N(2) of deoxyguanosine derived from cis-opening at C-10 of four optically active isomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene were incorporated into 5'-TTCGAATCCTTCCCCC [context III(G)] and 5'-GGGGTTCCCGAGCGGC [context IV(G)] at the underlined site. The mutagenic consequences of these lesions in each of the two sequence contexts were examined after ligation of the modified oligonucleotides into single-stranded M13mp7L2 and replication of the vector in SOS-induced Escherichia coli. Total frequencies of base substitution mutations ranged between 14 and 48%. The mutation frequencies were generally higher in context IV(G) than in context III(G), and consisted mainly of G-->T followed by G-->C base substitutions. A substantial number of deletions or insertions of one guanine was also found for all adducts in context IV(G), where the adduct is located at the 3'-end of a run of five guanines. The overall frequencies of base substitution mutations induced by cis-opened adducts were substantially higher than those observed with the trans-opened dGuo adducts in the same sequences [Page et al. (1998) Biochemistry 37, 9127-9137]. Although G-->T base substitutions predominated for both the cis- and trans-opened adducts, the cis-opened dGuo adducts generally resulted in a higher proportion of G-->C [particularly in context III(G)] relative to G-->A, whereas the opposite was true for the trans-opened dGuo adducts. The present results along with previous data indicate that mutagenicity is highly dependent on a combination of sequence context and adduct stereochemistry.     

Synthesis of oligonucleotides containing the N2-deoxyguanosine adduct of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine formed in all cooked meats. IQ has been found to be a potent inducer of frameshift mutations in bacteria and carcinogenic in laboratory animals. Upon metabolic activation, IQ forms covalent adducts at the C8- and N2-positions of deoxyguanosine with a relative ratio of up to approximately 4:1. We have previously incorporated the major dGuo-C8-IQ adduct into oligonucleotides through the corresponding phosphoramidite reagent. We report here the sequence-specific synthesis of oligonucleotides containing the minor dGuo-N2-IQ adduct. Thermal melting analysis revealed that the dGuo-N2-IQ adduct significantly destabilizes duplex DNA.     

Identification of individual benzo[c]phenanthrene dihydrodiol epoxide-DNA adducts by the 32P-postlabeling assay.

Purine deoxyribonucleoside 3'-phosphates were reacted separately with the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. Products resulting from the cis and trans opening of the epoxide ring by the exocyclic amino groups of deoxyadenosine and deoxyguanosine 3'-phosphates were separated by high-pressure liquid chromatography and identified by comparison of the observed circular dichroism spectra with the known spectra for the corresponding nucleoside adducts. The 16 structurally identified benzo[c]phenanthrene-purine deoxyribonucleoside 3'-phosphate adducts were then separately postlabeled according to the Randerath method, and the positions of the individual bisphosphates were mapped by thin-layer chromatography. Chromatographic conditions were developed that allowed separation of the four adducts for 3 of the 4 dihydrodiol epoxide isomers.     

DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide

The characterization of eight benzo[a]pyrene-deoxyribonucleoside adducts derived from reaction of the anti-dihydrodiol epoxide and deoxyguanylic and deoxyadenylic acids is described. It is reported that the epoxide ring is opened by the purine amino groups to yield similar amounts of both cis and trans products. NMR data show that the 7- and 8-hydroxyl groups are pseudodiaxial in the cis products and pseudodiequatorial in the trans products, and we suggest that these products arise from reaction with the diaxial and diequatorial conformers of the dihydrodiol epoxides, respectively. The chiral nature of the interactions of these metabolites with DNA restricts the range of products formed with this macromolecule, and a trans product with deoxyguanosine is the major product formed with either enantiomer of the anti-dihydrodiol epoxide.     

Synthesis and characterization of nucleosides and oligonucleotides with a benzo[a]pyren-6-ylmethyl adduct at adenine N6 or guanine N2

Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best studied. An alternative and interesting pathway to a highly genotoxic electrophile is through alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester 4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct identified from exposure of rats and mice to 4 is the guanine N(2) adduct [2'-deoxy-N(2)-(benzo[a]pyren-6-ylmethyl)guanosine, 5] which is also formed via activation of 2 to a radical cation species by horseradish peroxidase/H(2)O(2) or iodine. To study the biological and structural properties of this adduct and the analogous adenine N(6) adduct (6), a nonbiomimetic synthesis of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation of oligonucleotides containing 5 or 6 at a single site.     

Structure of adduct X, the last unknown of the six major DNA adducts of mitomycin C formed in EMT6 mouse mammary tumor cells

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC-DNA adducts. We identified the last unknown of these ("adduct X") as a guanine N(2) adduct of 2, 7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N(2). The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-(3)H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 microg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM-guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC-DNA adducts, the 2,7-DAM-DNA adducts are not cytotoxic.     

Coupling products of nucleosides with the glyoxal adduct of deoxyguanosine

Glyoxal is a widely dispersed environmental mutagen that reacts with DNA and deoxyguanosine to give primarily the 1,N(2)-guanine adduct, i.e., 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purin-9-one. Kasai et al. have reported [Kasai, et al. (1998) Carcinogenesis 19, 1459-1465] additional minor reactions of glyoxal to give bis-nucleosides of unknown structure involving glyoxal conjugation of dG with dA, dC, and dG itself. Reaction conditions have been modified to give large increases in the yields of the adducts, which has permitted structural characterization utilizing chemical and spectroscopic techniques. The glyoxal conjugates of dG with dA and dC are imidazo[1,2-a]purines involving displacement of the 6-hydroxyl group of the dG conjugate by the exocyclic amino groups of dA and dC. The dG conjugate is a symmetrical fusion of two imidazo[1,2-a]purines in which both the 6- and the 7-hydroxyl groups of the dG conjugate have been replaced. The glyoxal conjugates are formed as pairs of diastereomers. The dC and dA have a trans orientation of substituents at C6 and C7; the adduct of dG has a cis orientation. The absolute configurations of the individual diastereomers have been tentatively assigned based on comparison of their CD spectra with configurationally assigned diastereomers of the crotonaldehyde adduct of deoxyguanosine.     

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydro-11-methylbenzo[a]pyrene and its reaction with DNA

Substitution of a methyl group in the bay region can enhance the tumorigenicity of polycyclic aromatic hydrocarbons such as chrysene, benz[a]anthracene, and others. This phenomenon has been related to facile DNA adduct formation of bay region diol epoxides with a methyl group and epoxide ring in the same bay. While anti-7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and its DNA adduct formation have been studied extensively, it is not known whether a methyl substituent in the bay region alters the reactivity of DNA in this system. This is of interest because 11-methylbenzo[a]pyrene, which has a bay region methyl group, is more tumorigenic than benzo[a]pyrene. To examine the question, we have devised and employed an efficient synthesis based on photochemical cyclization, and prepared anti-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene, the likely ultimate carcinogen of 11-methylbenzo[a]pyrene. We have then reacted anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydro-11-methylbenzo[a]pyrene with calf thymus DNA and found that it gives three major adducts. These were identified as having resulted from cis- and trans-ring opening of the (S,R,R, S)-enantiomer and from trans-ring opening of the (R,S,S, R)-enantiomer. The standard deoxyguanosine adduct markers were prepared, and their structures were tentatively assigned on the basis of their CD and 1H NMR spectra. The adduct distribution of anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydro-11-methylbenzo[a]pyrene is quite different from that observed in the reaction of DNA with the corresponding diol epoxides of benzo[a]pyrene or with 5-methylchrysene. The heterogeneity of adducts obtained with anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydro-11-methylbenzo[a]pyrene thus may be related to the enhanced tumorigenicity of 11-methylbenzo[a]pyrene.     

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic characterization of syn-5-methylchrysene 1,2-dihydrodiol 3,4-epoxide-deoxyribonucleoside adducts

Eight deoxyribonucleoside adducts formed from reactions of syn-5-methylchrysene 1,2-dihydrodiol 3,4-epoxide with DNA or with nucleotides were characterized spectroscopically. The adducts arose from both cis and trans opening of the epoxide ring at C4 by the amino group of either deoxyadenosine or deoxyguanosine residues. NMR data indicated that the partially saturated 1,2,3,4-ring of the hydrocarbon residue adopted a boatlike conformation, with the purine residue being disposed pseudoaxially in all adducts. The cis and trans assignments for epoxide ring opening were readily made from coupling constants. Quantitatively, cis adducts predominated over trans adducts in both DNA and nucleotide reactions. Whereas deoxyadenylic acid appeared to trap this dihydrodiol epoxide more efficiently than deoxyguanylic acid, reaction with DNA led to more extensive reaction with deoxyguanosine than with deoxyadenosine residues.     

Structural characterization of adducts formed in the reaction of 2,3-epoxy-4-hydroxynonanal with deoxyguanosine

Six adducts were isolated by reverse-phase high-performance liquid chromatography from the reaction of deoxyguanosine at 50 degrees C in pH 7.0 buffer with the epoxide of trans-4-hydroxy-2-nonenal, a major alpha,beta-unsaturated aldehyde from lipid peroxidation. These adducts were designated as adducts 1-6. Structures of these adducts were fully characterized by spectroscopic methods such as UV, proton NMR, MS, and CD and by chemical reactions. Adduct 1 was previously identified as 1,N2-ethenodeoxyguanosine. Adducts 2, 3, 5, and 6 are four diastereomeric 1,N2-ethanodeoxyguanosine derivatives possessing two five-membered fused rings at the 1- and N2-positions of guanine. The systematic name of these adducts is 3-(2-deoxy-beta-D-erythropentofuranosyl)-3,5,5a,7,8,8a-hexahydro-8 -hydroxy-7- pentyl-10H-furo[2',3':4,5]imidazo[1,2-a]-purin-10-one. Acid hydrolysis of adducts 5 and 6 yielded the corresponding guanine products which were identical in all respects except having opposite CD. Similar results were obtained with adducts 2 and 3, suggesting they are two pairs of enantiomers. The stereochemical characteristics of these adducts were elucidated. Adduct 4 was characterized by spectroscopic methods and chemical reactions as 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,9-dihydro-7-(1,2- dihydroxyheptyl)-9H-imidazo[1,2-a]purin-9-one, a 1,N2-ethenodeoxyguanosine derivative. Upon mild base treatment, adducts 2, 3, 5, and 6 were readily converted to adduct 1. The mechanisms for the formation of these adducts and the conversion of adducts 2, 3, 5, and 6 to adduct 1 are discussed.     

Synthesis, characterization, and comparative 32P-postlabeling efficiencies of 2,6-dimethylaniline-DNA adducts

2,6-Dimethylaniline (2,6-diMeA) is a ubiquitous environmental pollutant that is used in industry as a synthetic intermediate. It is also found in tobacco smoke and as a major metabolite of lidocaine. Although the potential carcinogenicity of 2,6-diMeA in humans is presently uncertain, this aromatic amine has been classified as a rodent carcinogen. In addition, it is known to form hemoglobin adducts in humans, which indicates a profile of metabolic activation similar to that of typical arylamine carcinogens. Like other aromatic amines, 2,6-diMeA has been shown to yield N-(deoxyguanosin-8-yl)-2,6-dimethylaniline (dG-C8-2,6-diMeA) as a major DNA adduct in vitro. In this study, we show that 2,6-diMeA yields an unusual pattern of DNA adducts. In addition to dG-C8-2,6-diMeA, we have isolated two new adducts, 4-(deoxyguanosin-N(2)-yl)-2,6-dimethylaniline (dG-N(2)-2,6-diMeA) and 4-(deoxyguanosin-O(6)-yl)-2,6-dimethylaniline (dG-O(6)-2,6-diMeA), from the reaction of N-acetoxy-2,6-dimethylaniline with deoxyguanosine. A similar reaction conducted with deoxyadenosine yielded 4-(deoxyadenosin-N(6)-yl)-2,6-dimethylaniline (dA-N(6)-2,6-diMeA). All four adducts were detected in DNA reacted with N-acetoxy-2,6-dimethylaniline, with the relative yields being 46% for dA-N(6)-2,6-diMeA, 22% for dG-N(2)-2,6-diMeA, 20% for dG-O(6)-2,6-diMeA, and 12% for dG-C8-2,6-diMeA. This product profile contrasts markedly with the usual pattern of adducts obtained with aromatic amines, where C8-substituted deoxyguanosine products typically predominate. We further analyzed the kinetics of the T(4) polynucleotide kinase (PNK)-catalyzed phosphorylation of the C8 and N(2) deoxyguanosine 3'-phosphate adducts from 2,6-diMeA. The kinetic parameters obtained with these two structurally different adducts are compared to those determined with the parent nucleotide (dG3'p), and with (+/-)-anti-10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene 3'-phosphate, the major adduct derived from the environmental pollutant benzo[a]pyrene. The results indicate that all the adducts were labeled with lower efficiencies than dG3'p, stressing the likely underestimation of adduct levels in typical 32P-postlabeling protocols. Nonetheless, the N(2) adducts derived from 2,6-diMeA and benzo[a]pyrene were both labeled with higher efficiencies than the C8 adduct derived from 2,6-diMeA, with the benzo[a]pyrene adduct being the best substrate for PNK. Thus, the data suggest that N(2) adducts from dG3'p are intrinsically better substrates than their C8 analogues for PNK, and that bulkier aromatic fragments may favor the enzyme-substrate interaction during the labeling step.     

Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N(6) of deoxyadenosine

We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N(6). Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts. To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the adducted M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control. In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations induced. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported diol epoxide adducts in the same sequence. Remarkably, this (9S,10R)-adduct with cis orientation of the dAdo residue and the 9-OH group gave the highest mutational frequency of all the B[a]P adducts studied in this sequence, including the diol epoxide adducts. With the present adducts, A --> T transversions predominated, with smaller numbers of A --> G transitions and even fewer A --> C transversions.     

Benzo[a]pyrene diol epoxide forms covalent adducts with deoxycytidylic acid by alkylation at both exocyclic amino N(4) and ring imino N-3 positions

The carcinogen 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) alkylates DNA at dGuo, dAdo, and dCyd. dCyd adducts, formed in small amounts, elute near the more abundant dGuo adducts. We isolated the dCyd adducts formed with dCMP. Each BPDE enantiomer forms three major adducts with dCMP, two cis and one trans. The trans adduct and one of the cis adducts form by alkylation at exocyclic N(4), while the second cis adduct is a dUrd adduct formed by alkylation at ring N-3 followed by deamination. Epoxide ring-opening geometries were assigned on the basis of halide and temperature effects on adduct yield, the sign of the major CD band, and benzo ring proton NMR coupling constants. One of each set of cis adducts is fluorescent (FL), and the other is nonfluorescent (NF). The trans and FL cis adducts have fluorescence quantum yields 40-50% of that of the BPDE hydrolysis product. The long wavelength UV maxima of the FL and NF cis adducts are red-shifted 1 and 3 nm relative to the trans adduct. (1)H NMR deuterium exchange experiments indicate that in the trans and FL cis adducts N(4)-H is coupled to C10-H. Adduct formation experiments with methyl-protected Cyd derivatives show that NF cis adducts result from alkylation at N-3. MS results, pK(a) measurements, and dUrd alkylation experiments indicate that the N-3 dCyd adducts spontaneously deaminate to dUrd adducts. NMR coupling constants show that in the NF cis adduct the C7 and C8 substituents are quasi equatorial and the C9 substituent is quasi axial, unlike in other cis BPDE adducts. (1)H NOESY spectra of the (-)-BPDE NF cis adduct reveal that it exists in two conformers. Molecular modeling shows that the conformers result from two low-energy conformations of very similar energies with the pyrimidine in opposite orientations, separated by significant barriers to rotation of the uracil moiety.     

Fluorescence characteristics of site-specific and stereochemically distinct benzo[a]pyrene diol epoxide-DNA adducts as probes of adduct conformation

Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N(2)-dG) and adenine ([BP]-N(6)-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N(2)-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]-N(6)-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849].     

Direct synthesis and identification of benzo[a]pyrene diol epoxide-deoxyguanosine binding sites in modified oligodeoxynucleotides.

Adducts derived from the reaction of the benzo[a]pyrene metabolite model compound (+)-anti-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-BPDE] with the single-stranded oligodeoxynucleotide 5'-d(TATGCGTAT) were obtained according to direct synthesis techniques described earlier [Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990) Carcinogenesis 11, 1667-1672]. Four major adducts, involving trans and cis addition (trans/cis adduct ratio approximately 4.5) of (+)-BPDE to the exocyclic amino groups of guanines G4 and G6 (the numbers denote the positions of the guanines counted from the 5'-side) were obtained. These adducts can be separated from one another by reverse-phase high-performance liquid chromatography methods. The site of BPDE binding on either G4 or G6 can be determined from the electrophoresis band patterns on 20% polyacrylamide gels of the BPDE-modified oligonucleotides subjected to the G+A and G Maxam-Gilbert strand cleavage reactions [Maxam, A. M., and Gilbert, W. (1980) Methods. Enzymol. 65, 499-560]. The electrophoresis gel band patterns are different for unmodified DNA and the two different BPDE-modified oligonucleotides because (1) the strand cleavage fragments bearing BPDE residues migrate slower than the corresponding fragments derived from the unmodified oligonucleotide and (2) strand cleavage tends to be inhibited on the 5'-sides of BPDE-modified guanines in the G+A, but not the G reaction.