Oligonucleotide Adducts Research Articles

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine.

Mitomycin C, (MC), an antitumor drug, is a DNA alkylating agent currently used in the clinics. Inert in its native form, MC is reduced to reactive mitosenes, which undergo nucleophilic attack by guanine or adenine bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs). Although ICLs are considered the most cytotoxic lesions, the role of each individual adduct in the drug's cytotoxicity is still not fully understood. Synthetic routes have been developed to access modified oligonucleotides containing dG MC-monoadducts and dG-MC-dG ICL at a single position of their base sequences to investigate the biological effects of these adducts. However, until now, oligonucleotides containing monoadducts formed by MC at the adenine base had not been available, thus preventing the examination of the role played by these lesions in the toxicity of MC. Here, we present a route to access these substrates. Structural proof of the adducted oligonucleotides were provided by enzymatic digestion to nucleosides and high-resolution mass spectral analysis. Additionally, parent oligonucleotides containing a dG monoadduct and a dG-MC-dG ICL were also produced. The stability and physical properties of all substrates were compared via CD spectroscopy and UV melting temperature studies. Finally, virtual models were created to explore the conformational space and structural features of these MC-DNA complexes.

Application of diazonium chemistry in purine modifications: A focused review

Deaminative modifications of purines via diazonium chemistry have long been the cornerstone in modified nucleoside synthesis. The purpose of this review is to bring into focus the impact of dediazoniative methods such as non‐aqueous Sandmeyer halogenations, fluorination, arylation, and carbon‐heteroatom bond formations on natural purines and their nucleosides as versatile tools for purine modifications en route to various nucleoside drugs, drug discovery scaffolds, and adducted oligonucleotides of biomedical interests. Metal free reactions of arenediazonium salts with purine nucleosides for the synthesis of 8‐aryl purines, in particular, 8‐aryl guanosines and photochromic 8‐arylazo purines are also highlighted. The treatise is expected to create broad interest among organic and biological chemists for further explorations in this area.

Synthesis of Oligonucleotides containing the cis-Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA.

Mitomycin C (MC) an antitumor drug and decarbamoylmitomycin C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α, trans) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1"-β-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1'' in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1"-β ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.

Site-Specific Incorporation of N-(2'-Deoxyguanosine-8-yl)-6-aminochrysene Adduct in DNA and Its Replication in Human Cells.

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields two major adducts with 2'-deoxyguanosine (dG), one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding 5'-O-dimethoxytrityl 3'-phosphoramidite, which was used to synthesize a site-specifically adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS) efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%) inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%, respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ, hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8% in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs error-prone bypass.

Synthesis of Oligodeoxynucleotides Containing a C8-2'-Deoxyguanosine Adduct Formed by the Carcinogen 3-Nitrobenzanthrone.

This unit describes the detailed procedure in five parts for the synthesis of the C8-2'-deoxyguanosine-3-aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2'-deoxyguanosine, O6 -benzyl-N2 -DMTr-3'-5'-bisTBDMS-C8-Br-2'-deoxyguanosine, is described in the first part. The synthesis of the reduced carcinogen 3-aminobenzanthrone is detailed in part two. The third part outlines the key step of the adduct formation between the reduced carcinogen and the protected nucleoside by a palladium-catalyzed cross coupling reaction. The final two parts describe phosphoramidite synthesis from the nucleoside-carcinogen adduct followed by its site-specific incorporation into DNA by solid-phase oligonucleotide synthesis. The adducted oligonucleotides are purified by reversed-phase HPLC and characterized by mass spectrometry. © 2017 by John Wiley & Sons, Inc.

Liquid Chromatography – tandem mass spectrometry methods for the analysis of isomeric oligonucleotide adducts

Liquid Chromatography – tandem mass spectrometry methods for the analysis of isomeric oligonucleotide adducts

115 Detection of organic carcinogens in water by nanophotonic method

anti-Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon–DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereosiomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE–deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligoncleotide adducts.

Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column

In this manuscript, an efficient high resolution reversed phase-ion pairing-liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC–MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatographic separation of isomeric oligonucleotides. Ion pairing reagents and mobile phase conditions were evaluated using as model N-acetylaminofluorene [AAF] adducted ss-oligonucleotides (CCC CGA GCA ATC TCA AT). The optimized mobile phase conditions were then applied for the mapping of sites of base modification of AAF adducted 15-base pair oligonucleotide fragments containing codon 135 of the p53 gene and for profiling a complex synthetic oligonucleotide mixture. The optimized method utilizes a monolithic poly(styrene-divinylbenzene) capillary column, triethylammonium bicarbonate as ion pairing reagent and methanol as organic modifier to perform IP-RPLC–ESI-MS/MS separation. The results show that the method is simultaneously applicable not only to oligonucleotide fragments adducted separately by different carcinogens but also to the analysis of multiple adducts in the same oligonucleotide fragment in a single experiment. The method presents itself as a tool for the identification, characterization and mapping of oligonucleotide adducts as biomarkers for DNA damage from carcinogens.

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5'OH-CCT ACC CCT TCC TTG TA-3'OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5'OH-CCT ACC CCT TCC TTG TA-3'OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5'OH-ATGAACCGGAGGCCC-3'OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides.

Single-Stranded Oligonucleotide Adducts Formed by Pt Complexes Favoring Left-Handed Base Canting: Steric Effect of Flanking Residues and Relevance to DNA Adducts Formed by Pt Anticancer Drugs

Platinum anticancer drug binding to DNA creates large distortions in the cross-link (G*G*) and the adjacent XG* base pair (bp) steps (G* = N7-platinated G). These distortions, which are responsible for anticancer activity, depend on features of the duplex (e.g., base pairing) and of the cross-link moiety (e.g., the position and canting of the G* bases). The duplex structure stabilizes the head-to-head (HH) over the head-to-tail (HT) orientation and right-handed (R) over left-handed (L) canting of the G* bases. To provide fundamental chemical information relevant to the assessment of such duplex effects, we examine (S,R,R,S)-BipPt(oligo) adducts (Bip = 2,2'-bipiperidine with S,R,R,S chiral centers at the N, C, C, and N chelate ring atoms, respectively; oligo = d(G*pG*) with 3'- and/or 5'-substituents). The moderately bulky (S,R,R,S)-Bip ligand favors L canting and slows rotation about the Pt-G* bonds, and the (S,R,R,S)-BipPt(oligo) models provide more useful data than do dynamic models derived from active Pt drugs. All 5'-substituents in (S,R,R,S)-BipPt(oligo) adducts favor the normal HH conformer (∼97%) by destabilizing the HT conformer through clashes with the 3'-G* residue rather than through favorable H-bonding interactions with the carrier ligand in the HH conformer. For all (S,R,R,S)-BipPt(oligo) adducts, the S pucker of the 5'-X residue is retained. For these adducts, a 5'-substituent had only modest effects on the degree of L canting for the (S,R,R,S)-BipPt(oligo) HH conformer. This small flanking 5'-substituent effect on an L-canted HH conformer contrasts with the significant decrease in the degree of R canting previously observed for flanking 5'-substituents in the R-canted (R,S,S,R)-BipPt(oligo) analogues. The present data support our earlier hypothesis that the distortion distinctive to the XG* bp step (S to N pucker change and movement of the X residue) is required for normal stacking and X·X' WC H bonding and to prevent XG* residue clashes.

Facile Syntheses of O2-[4-(3-Pyridyl-4-oxobut-1-yl]thymidine, the Major Adduct Formed by Tobacco Specific Nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in vivo, and Its Site-Specifically Adducted Oligodeoxynucleotides

O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) is the most persistent adduct detected in the lung and liver of rats treated with tobacco specific nitrosamines: N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). It is an important biomarker to assess the human exposure to these carcinogens. The only synthetic method reported for O(2)-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). We have developed for the first time a regioselective and efficient method for the total synthesis of O(2)-POB-dThd and its site-specifically adducted oligonucleotides. The main step in the synthesis of O(2)-POB-dThd was achieved by a novel method. The treatment of O(2)-5'-anhydrothymidine with the sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol gave exclusively the O(2)-alkylated adduct, which was deprotected in one step to furnish the desired O(2)-POB-dThd in excellent yield. The product was characterized by NMR ((1)H and (13)C), high-resolution MS, and HPLC analysis. This work provided for the first time a reliable method for large scale total synthesis of O(2)-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. The O(2)-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of oligodeoxynucleotides by standard methods. The oligomers were characterized by MS and HPLC analysis. These oligomers will facilitate the elucidation of the mutagenic potential of the O(2)-POB-dThd adduct, which will provide further insight into the role of tobacco-specific nitrosamines in inducing cancers in smokers.

Abstract 1700: An improved synthesis and mutagenicity studies of O2-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vivo

Abstract Among the four pyridyloxobutyl (POB)-DNA adducts formed in the lung and liver of rats treated with tobacco specific nitrosamines [N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)], the O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd) has been found to be the most persistent. It is an important biomarker to assess the human exposure to these carcinogens and also serves as a useful precursor for the synthesis of site-specifically modified oligomers required for mutagenicity studies. The only synthetic method reported for O2-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). Our continued efforts have now led to the development of an efficient method for the total synthesis of O2-POB-dThd and its site-specifically adducted oligonucleotides. The synthesis of O2-POB-dThd was achieved by optimizing reaction between O2-5′-anhydrothymidine and sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol, followed by deprotection of the dithiane group. The product was characterized by NMR (1H, 13C, and COSY spectra), MS, and HPLC analysis. This synthetic strategy provided for the first time a reliable method for a gram scale total synthesis of O2-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. O2-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of the oligomer by standard methods. The oligomer was characterized by MS and HPLC analysis. We examined the in vitro kinetics of the synthesized oligomer with the Klenow fragment of E. Coli DNA polymerase I with the exonuclease activity inactivated (Kf(exo-)) and human DNA polymerasesl eta, kappa, and iota). O2-POB-dThd was replicated in a mutagenic manner with with Kf(exo-)and human pol eta and was unreactive with pol kappa and iota. In summary, highly facile syntheses of O2-POB-dThd and its site-specifically adducted oligonucleotide are developed. The study will facilitate elucidation of mechanistic aspects and mutagenic potential of O2-POB-dThd adduct, and will provide further insight into the role it plays in inducing cancers in smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1700.

The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide‐oligonucleotide adducts determined using nanoLC/MS/MS

Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (+/-)-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [(+/-)-anti-benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3') derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational 'hot spots' and have also been reported as chemical 'hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5 >> G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5 >> G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G(7)) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run.

Characterization of the DNA adducts induced by aristolochic acids in oligonucleotides by electrospray ionization tandem mass spectrometry

Metabolic activation of carcinogenic aristolochic acids (AA) produces reactive aristolactam-nitrenium ion intermediates. Electrophilic attack of the aristolactam-nitrenium ion via its C7 position to the exocyclic amino group in the purine bases leads to the formation of DNA adducts. DNA-binding assays have demonstrated that carcinogens show site- and sequence-specificity and the biological consequence is defined by the nature of binding as well as their position in the genome. In this study, electrospray ionization tandem mass spectrometry was applied for the identification and position mapping of DNA adducts in oligonucleotides (ODNs). The developed method was successfully applied for the analysis of unmodified and AA-modified ODNs (5'-TTTATT-3', 5'-TTTGTT-3' and 5'-TACATGTGT-3'). The observation of the modified bases (modified adenine and guanine) together with the complementary product ions ([a(n)-B*(n)](-), w(-)) from the cleavage of the 3' C--O bond adjacent to the modified base in MS/MS analyses readily enabled the identification of the AA-binding site in ODNs.

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N(2)-alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH(2)(6-benzo[a]pyrenyl) (BP) and O(6)-alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O(6)-PobG, which caused almost complete blockage. Steady-state kinetic parameters (k(cat)/K(m)) were similar for insertion of dCTP opposite G and N(2)-G adducts but were severely reduced opposite the O(6)-G adducts. REV1 showed apparent pre-steady-state burst kinetics for dCTP incorporation only opposite N(2)-BPG and little, if any, opposite G, N(2)-benzyl (Bz)G, or O(6)-BzG. The maximal polymerization rate (k(pol) 0.9 s(-1)) opposite N(2)-BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N(2)-BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((S(p))-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N(2)-BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N(2)-adduct at G but not to O(6)-adducts.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.

Fluorinated Alcohol Mediated Displacement of the C10 Acetoxy Group of Benzo[a]pyrene-7,8,9,10-tetrahydrotetraol Tetraacetates: A New Route to Diol Epoxide−Deoxyguanosine Adducts

We describe a novel trifluoroethanol (TFE) or hexafluoropropan-2-ol (HFP) mediated substitution reaction of the bay-region C10 acetoxy group in four stereoisomeric 7,8,9,10-tetraacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (tetraol tetraacetates, two pairs of cis and trans isomers at the 9,10 positions) by the exocyclic N2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3). The tetraacetates are derived from cis and trans hydrolysis of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P DE-1) and of (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P DE-2) at C-10 followed by acetylation. Excellent yields and high regioselectivity were observed. Similar cis/trans product ratios were observed for each set of cis and trans tetraol tetraacetates derived from DE-1 ( approximately 75/25) and from DE-2 (approximately 67/33) in HFP. This strongly suggests that the substitution proceeds via an SN1 mechanism involving a carbocation intermediate that is common to the cis and trans tetraacetates. Since it is likely that the cis and trans products from 3 arise from different conformations of the carbocation, its lifetime must be sufficiently long to permit conformational equilibration before its capture by the purine nucleophile. The corresponding reaction of (+/-)-9alpha-bromo-7beta,8alpha,10beta-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with 3 in HFP was highly regio- and stereoselective and gave exclusively trans 10beta-adducts. This newly developed substitution reaction provides an attractive alternative synthetic strategy for the preparation of polycyclic hydrocarbon adducted oligonucleotide building blocks.

Separation and Sequencing of Isomeric Oligonucleotide Adducts Using Monolithic Columns by Ion-Pair Reversed-Phase Nano-HPLC Coupled to Ion Trap Mass Spectrometry

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (+/-)-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards.

Simultaneous analysis of four stereoisomers of anti-benzo[ a]pyrene diol epoxide–deoxyguanosine adducts in short oligodeoxynucleotides using reversed-phase high-performance liquid chromatography

anti-Benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide ( anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[ a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon–DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereosiomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE–deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligoncleotide adducts.

Application of pressure assisted electrokinetic injection technique in the measurements of DNA oligonucleotides and their adducts using capillary electrophoresis–mass spectrometry

The identification and measurement of negatively charged DNA oligonucleotides and their benzo[a]pyrene-7,8,9,10-tetrahydro-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts by capillary zone electrophoresis (CZE) hyphenated mass spectrometry (MS) system using an on-line enrichment technique, the constant pressure assisted electrokinetic injection (PAEKI), is described here. With optimized PAEKI conditions, an on-line sample concentration power of 300–800 times could be reached for both single-stranded (ss) and double-stranded (ds) oligonucleotides during a 90-s PAEKI injection. The detection limits using single quadrupole MS in the scan mode were 0.01–0.03 μM for ss and 0.04–0.08 μM for ds oligonucleotides, respectively. The relative standard deviations at 1 μM of oligonucleotides were from 7.6 to 15.8%. A dynamic linear calibration range of about two orders of magnitude were observed. Good mass spectra of oligonucleotides and BPDE–oligonucleotide adducts at low micromolar levels could be obtained using single quadrupole MS which could be a helpful tool in DNA adducts studies.