Total synthesis of the aristolochic acids, their major metabolites, and related compounds.

Plants from the Aristolochia genus have been recommended for the treatment of a variety of human ailments since the time of Hippocrates. However, many species produce the highly toxic aristolochic acids (AAs), which are both nephrotoxic and carcinogenic. For the purposes of extensive biological studies, a versatile approach to the synthesis of the AAs and their major metabolites was devised based primarily on a Suzuki-Miyaura coupling reaction. The key to success lies in the preparation of a common ring-A precursor, namely, the tetrahydropyranyl ether of 2-nitromethyl-3-iodo-4,5-methylendioxybenzyl alcohol (27), which was generated in excellent yield by oxidation of the aldoxime precursor 26. Suzuki-Miyaura coupling of 27 with a variety of benzaldehyde 2-boronates was accompanied by an aldol condensation/elimination reaction to give the desired phenanthrene intermediate directly. Deprotection of the benzyl alcohol followed by two sequential oxidation steps gave the desired phenanthrene nitrocarboxylic acids. This approach was used to synthesize AAs I-IV and several other related compounds, including AA I and AA II bearing an aminopropyloxy group at position-6, which were required for further conversion to fluorescent biological probes. Further successful application of the Suzuki-Miyaura coupling reaction to the synthesis of the N-hydroxyaristolactams of AA I and AA II then allowed the synthesis of the putative, but until now elusive, N-acetoxy- and N-sulfonyloxy-aristolactam metabolites.

Lack of recognition by global-genome nucleotide excision repair accounts for the high mutagenicity and persistence of aristolactam-DNA adducts.

Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.

DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells.

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In approximately 50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA-->T and AL-II-dG-->T transversions.

Detoxification of aristolochic acid I by O-demethylation: less nephrotoxicity and genotoxicity of aristolochic acid Ia in rodents.

Ingestion of aristolochic acids (AA) contained in herbal remedies results in aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA I and AA II, primary components in AA, have similar genotoxic potential, whereas only AA I shows severe renal toxicity in rodents. AA I is demethylated to form 8-hydroxy-aristolochic acid I (AA Ia) as a major metabolite. However, the nephrotoxicity and genotoxicity of AA Ia has not yet been determined. AA Ia was isolated from urine collected from rats treated with AA I and characterized by NMR and mass spectrometry. The purified AA Ia was administered intraperitoneally to C3H/He male mice for 9 days and its toxicity was compared with AA I. Using (32)P-postlabeling/polyacrylamide gel electrophoresis, the level of AA Ia-derived DNA adducts in renal cortex was approximately 70-110 times lower than that observed with AA I, indicating that AA Ia has only a limited genotoxicity. Supporting this result, when calf thymus DNA was reacted with AA Ia in a buffer containing zinc dust, the formation of AA Ia-DNA adducts was two-orders of magnitude lower than that of AA I. Histopathologic analysis revealed that unlike AA I, no significant changes were detected in the renal cortex of mice treated with AA Ia. Therefore, the contribution of AA Ia to renal toxicity is minimum. We conclude the metabolic pathway of converting AA I to AA Ia functions as the detoxification of AA I.

Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-Hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers.

N(2)- (4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N(2)-(4-hydroxyphenyl)-2'-dG (N(2)-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N(2)-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N(2)-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

Quantitative determination of aristolochic acid-derived DNA adducts in rats using 32P-postlabeling/polyacrylamide gel electrophoresis analysis.

Aristolochic acids (AA) are nephrotoxic and carcinogenic nitroaromatic compounds produced by the Aristolochiaceae family of plants. Ingestion of these phytotoxins by humans results in a syndrome known as AA nephropathy, characterized by renal tubulointerstitial fibrosis and upper urothelial cancer. After activation by cellular enzymes, AA I and II react with DNA to form covalent adducts and as such represent potential biomarkers for studies of AA toxicity. Using site-specifically modified oligodeoxynucleotides as standards, we have developed a method for quantifying 7-(deoxyadenosin-N(6)-yl) aristolactam-DNA or 7-(deoxyguanosin-N(2)-yl) aristolactam-DNA adducts in tissues of Wistar rats using an assay in which (32)P-postlabeling techniques are coupled with nondenaturing polyacrylamide gel electrophoresis. The limit of detection with this technique is five adducts in 10(9) nucleotides for a 5-microg DNA sample. In contrast to previous reports, we find that the levels of AA adducts in renal tissues of Wistar rats treated p.o. with AA for 1 week with 5 mg/kg/day of AA I or AA II were much higher than that in the forestomach. Highest adduct levels were observed in rats treated with AA II, suggesting that this compound may be more genotoxic than AA I. Treatment of rats with aristolactam I, an end-product of AA I metabolism, resulted in a much lower level of adduction. This study establishes the feasibility of using AA-DNA adducts as intermediate biomarkers of exposure in studies of AA nephropathy and its associated urothelial cancer.

Incorporation of N2-deoxyguanosine metabolic adducts of 2-aminonaphthalene and 2-aminofluorene into oligomeric DNA.

In previous work we described an efficient procedure for the synthesis of the respective N2 and N6 adducts of 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) derived from a series of aminoaryl compounds. We now outline methods for the site-specific introduction into oligomeric DNA of the adducts dG-N2-AN (6), dG-N2-AAN (7), dG-N2-AF (8), and dG-N2-AAF (9) derived from 2-aminonaphthalene (2-AN) or 2-aminofluorene (2-AF). For the 2-AN adduct 7, containing an acetylamino group, the 5'-O-4,4'-dimethoxytrityl- (DMT-) 3'-O-phosphoramidite (14) required for automated DNA synthesis was synthesized in high yield via the sequence 10-->11-->14. On the other hand, introduction of the desacetyl adduct 6 into oligomeric DNA was accomplished via the N-trifluoroacetyl-DMT-phosphoramidite derivative 18. This involved a similar sequence (10-->15-->18) except that the order of the reactions was changed to avoid a decomposition that occurred when the silyl-protected amino derivative 11 was treated with trifluoroacetic anhydride. In the 2-AF series the 5'-O-DMT-3'-O-phosphoramidites 27a and 27b, related to 8 and 9, were prepared by similar methods. Again, however, the order of the reactions was changed to avoid the extreme insolubility associated with the N2-[3-(2-acetylaminofluoren-3-yl)]dG (dG-N2-AAF, 9) adduct that we had noted previously. The incorporation into oligomeric DNA of the acetylamino compounds 7 and 9 proceeded smoothly and in high yield (95-100%). By contrast, the trifluoroacetyl analogues led in both the naphthyl and fluorenyl series to a mixture of oligomers containing the desired free amino adduct (6 or 8) accompanied by the N-acetyl adduct (7 or 9, respectively, after the deprotection step), indicating secondary acetylation by the capping agent acetic anhydride.

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells.

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.