Comparison of the isolation of adducts of 2'-deoxycytidine and 2'-deoxyguanosine with phenylglycidyl ether by high-performance liquid chromatography on a reversed-phase column and a polystyrene-divinylbenzene column

2'-Deoxycitidine (dCyd) and 2'-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were developed on analytical and semi-preparative scales using high-performance liquid chromatography with photodiode-array detection on a reversed-phase column and on a polystyrene-divinylbenzene column. The use of the latter column was prompted by decomposition of the preparatively isolated dGuo-PGE adducts on the reversed-phase column. The use of a polystyrene-divinylbenzene column solved this problem and also revealed the presence of one more peak in both the dCyd- and dGuo-PGE reaction mixtures. The adducts of dCyd and dGuo were isolated on preparative reversed-phase and polystyrene-divinylbenzene columns and characterized by UV, fast atom bombardment mass and 360 MHz 1H NMR spectrometry. The adducts of dCyd were the diastereomers of N-3-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine and N4-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine whereas those of dGuo were the two diastereomers of N-7-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine and a third peak which appeared to be mainly N2-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine.     

超高效液相色谱-串联质谱法鉴定与分析乙醛诱导脱氧核糖核苷酸加合物

采用超高效液相色谱-串联质谱法对两种非对映异构体（6S,8S）1,N²-丙基-2'-脱氧鸟苷（ProdG）和（6R,8R）ProdG加合物进行鉴定与分析。通过色谱保留时间及质谱碎裂方式分析,证明乙醛与2'-脱氧鸟苷（dG）反应可形成ProdG加合物。体外实验表明,乙醛能够诱导脱氧核糖核苷酸（DNA）形成ProdG加合物,并且（6R,8R）ProdG的生成量大于（6S,8S）ProdG的生成量。细胞实验结果显示,乙醛暴露能显著提高人肺胚成纤维细胞（MRC5）基因组DNA中ProdG加合物的水平,且ProdG加合物的水平与乙醛的暴露浓度呈正相关。此外,100 μ mol/L的乙醛暴露使（6R,8R）ProdG的含量从（6.4±0.3） 个/10⁸个碱基增加到（127.2±2.7） 个/10⁸个碱基,上调程度大于（6S,8S）ProdG（从（6.5±0.3） 个/10⁸个碱基增加到（115.3±2.5） 个/10⁸个碱基）。该工作为乙醛暴露所引起的DNA加合物水平上升提供了实验依据。     

Characterisation of estrone-nucleic acid adducts formed by reaction of 3,4-estrone-o-quinone with 2'-deoxynucleosides/deoxynucleotides using capillary liquid chromatography/electrospray ionization mass spectrometry

Xenobiotic and endobiotic molecules can react with DNA leading to formation of so-called DNA adducts. This modified DNA can be repaired enzymatically, but, if not, these modifications are believed to be responsible for the initiation of carcinogenic processes. Hence, we studied the interaction of 2'-deoxynucleosides and 2'-deoxynucleotides with 3,4-estronequinone (3,4-E(1)Q), a metabolite of estrone (E(1)) and a supposed carcinogen. These estrone-nucleic acid adducts were analysed by capillary liquid chromatography (CapLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Knowledge of their behaviour from in vitro studies is a prerequisite for detecting adducts in in vivo studies. Our initial attempts to synthesise nucleos(t)ide adducts of 3,4-E(1)Q in an aprotic solvent (dimethylformamide) yielded no adducts. However, under acidic aqueous conditions, adducts were obtained. With dGuo, a dGuo adduct was found in addition to a Gua adduct. Earlier publications on adduct formation in protic solvents failed to report formation of any adduct with dAdo. A N(3)-Ade adduct was reported upon reaction of 3,4-E(1)Q with Ade base and with DNA. With dAdo, we obtained two nucleoside adducts and six Ade adducts due to loss of 2'-deoxyribose. Thus, contrary to general belief that only 2,3-E(1)Q can form stable adducts, we showed formation of substantial amounts of intact DNA adducts with 3,4-E(1)Q in addition to deglycosylated adducts. Adducts were also obtained with dGMP and dAMP, but no phosphate alkylation was found. Adducts of dCyd, dCMP, dThd, and dTMP were not detected. Using chromatographic-MS data a structural relationship between the 2'-deoxynucleoside, 2'-deoxynucleotide and base adducts was found in the various reaction mixtures. The adducts of dGuo and dGMP reaction mixtures were alkylated at the same N(7)-position of the nucleobase, as indicated by the occurrence of a rapid deglycosylation reaction. In dAdo and dAMP reaction mixtures, 14 adducts were detected; their relationships from the LC and MS data reduced the number of structures to six adenine base alkylated adducts with respect to alkylation between N(1), N(3), N(7) and/or N(6) in the adenine and C(1), C(2) and/or C(6) in 3,4-E(1)Q. We could infer, in addition, whether they had an A ring attachment or a C(6) attachment on the estrone moiety.     

DNA-protein cross-links between guanine and lysine depend on the mechanism of oxidation for formation of C5 vs C8 guanosine adducts

The reaction between N(alpha)-acetyllysine methyl ester (Lys) and 2'-deoxyguanosine (dGuo) was used to study structural aspects of DNA-protein cross-link (DPC) formation. The precise structure of DPCs depended on the nature of the oxidant and cross-linking reactions in which a series of different oxidation conditions generated a distribution of adducts, principally spirodiiminodihydantoins with lysine appended at the purine position of C5 (5-Lys-Sp), C8 (8-Lys-Sp), or both C5 and C8 (5,8-diLys-Sp). Singlet oxygen oxidation of dGuo produced 5-Lys-Sp exclusively when Rose Bengal or methylene blue was used to photochemically generate (1)O2 in the presence of Lys, whereas riboflavin or benzophenone-mediated photochemistry generated lysine radicals and led to C8 adduct formation, yielding 8-Lys-Sp and 5,8-diLys-Sp. Notably, the yield of dGuo modifications from riboflavin photooxidation increased dramatically in the presence of lysine. Oxidation of deoxyguanosine/lysine mixtures with Na2IrCl6 or sulfate radicals produced both 5-Lys-Sp and 8-Lys-Sp. The same adducts were formed in single and double-stranded oligodeoxynucleotides, and these could be analyzed after nuclease digestion. Adduct formation in duplex DNA was somewhat dependent on the accessibility of lysine to C5 vs C8 of the purine. No adduct formation was detected between lysine and the other nucleobases T, C, or A. Overall, the precise location of adduct formation at C5 vs C8 of guanine appears to be diagnostic of the oxidation pathway.     

Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay

A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.     

Unprecedented monofunctional metalation of adenine nucleobase in guanine- and thymine-containing dinucleotide sequences by a cytotoxic platinum-acridine hybrid agent

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.     

Qualitative study of in vivo melphalan adduct formation in the rat by miniaturized column-switching liquid chromatography coupled with electrospray mass spectrometry

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg(-1)). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua-melphalan adduct was found, although in very low abundance. These first results of the search for in vivo-formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed.     

Spiroiminodihydantoin nucleoside formation from 2'-deoxyguanosine oxidation by [(18)O-labeled] singlet molecular oxygen in aqueous solution

The main singlet molecular oxygen ((1)O(2)) oxidation products of free 2'-deoxyguanosine (dGuo) in aqueous solution were identified as a pair of diastereomeric spiroiminodihydantoin 2'-deoxyribonucleosides (dSp) together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In the present work, evidence is provided from (18)[(1)O(2)] and H(2) (18)O labeling experiments, using HPLC-ESI-MS/MS, that the formation of dSp is explained by the addition of water to a reactive quinonoid intermediate, and a second reaction pathway leading to dSp involves (1)O(2) oxidation of initially generated 8-oxodGuo.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

Synthesis of N2 2'-deoxyguanosine adducts formed by 1-nitropyrene

[reaction: see text] Synthesis of N(2) 2'-deoxyguanosine adducts formed by the ubiquitous carcinogen, 1-nitropyrene, is reported. Various conditions of Buchwald-Hartwig palladium-catalyzed amination are examined. The most convenient synthetic approach involved a straightforward coupling between protected 2'-deoxyguanosine and bromonitropyrenes, which, upon reductive deprotection, provided excellent yield of the two 1-nitropyrene adducts.     

One-electron oxidation of the guanine moiety of 2'-deoxyguanosine: influence of 8-oxo-7,8-dihydro-2'-deoxyguanosine

The influence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) on riboflavin and UVA-mediated one-electron oxidation of an aqueous aerated solution of 2'-deoxyguanosine (dGuo) has been studied. Using labeled experiments, we have demonstrated that, despite not being able to detect significant amounts of 8-oxodGuo upon one-electron oxidation of dGuo, 8-oxodGuo is indeed produced but is further rapidly degraded to oxidized nucleosides. Evidence is provided showing that an efficient electron transfer reaction from 8-oxodGuo to the guanine radical cation or rather its deprotonated form occurs, giving rise to the specific decomposition of 8-oxodGuo together with the restitution of dGuo. It could be concluded that 8-oxodGuo efficiently protects dGuo from decomposition by the one-electron oxidation reaction.     

Preparation of C8-amine and acetylamine adducts of 2'-deoxyguanosine suitably protected for DNA synthesis

[reaction: see text] C8-Amine and acetylamine adducts of 2'-deoxyguanosine were synthesized. Our approach provides solutions for the coupling of aromatic amines to a protected 8-bromo-2'-deoxyguanosine derivative, for the selective acetylation of the coupled adduct at N(8) and for a protecting group scheme preserving the integrity of the base-labile N(8) acetyl group during DNA synthesis.     

Equilenin-2'-deoxynucleoside adducts: analysis with nano-liquid chromatography coupled to nano-electrospray tandem mass spectrometry

The interaction of 4-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupling of miniaturized liquid chromatography (LC) to electrospray tandem mass spectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reaction of 4-hydroxyequilenin with the main 2'-deoxynucleosides were separated on a Hypersyl C(18) BDS nano-HPLC column (15 cm x 75 microm i.d.) at a flow-rate of 300 nl min(-1) using gradient elution with CH(3)OH--0.2% CH(3)COOH in H(2)O. The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution low-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts were found. The nano-HPLC column-switching ES-MS system was tested for its sensitivity on a triple-quadrupole instrument, and detection limits down to 197 fg in the single reaction monitoring mode were obtained for semi-preparatively isolated equilenin--2'-deoxyguanosine adduct.     

Acid-base and metal ion binding properties of guanylyl(3'-->5')guanosine (GpG-) and 2'-deoxyguanylyl(3'-->5')-2'-deoxyguanosine [d(GpG)-] in aqueous solution

The acidity constants of guanylyl(3'-->5')guanosine (GpG(-)) and 2'-deoxyguanylyl(3'-->5')-2'-deoxyguanosine [d(GpG)(-)] for the deprotonation of their (N1)H sites were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO(3)). The same method was used for the determination of the stability constants of the 1:1 complexes formed between Mg(2+), Ni(2+), or Cd(2+) (= M(2+)) and (GG-H)(2-), and in the case of Mg(2+) also of (GG-2H)(3-), where GG(-) = GpG(-) or d(GpG)(-). The stability constants of the M(GG)(+) complexes were estimated. The acidity constants of the H(dGuo)(+) and dGuo species (dGuo = 2'-deoxyguanosine) and the stability constants of the corresponding M(dGuo)(2+) and M(dGuo-H)(+) complexes were also measured. Comparison of these and related data allows the conclusion that N7 of the 5'G unit in GG(-) is somewhat more basic than the one in the 3'G moiety; the same holds for the (N1)(-) sites. On the basis of comparisons with the stability constants measured for the dGuo complexes, it is concluded that M(2+) binding of the GG dinucleoside monophosphates occurs predominantly in a mono-site fashion, meaning that macrochelate formation is not very pronounced. Indeed, it was a surprise to find that the stabilities of the complexes of dGuo or (dGuo-H)(-) and the corresponding ones derived from GG(-) are so similar. Consequently, it is suggested that in the M(GG)(+) and M(GG-H) complexes the metal ion is mainly located at N7 of the 5'G unit since this is the more basic site allowing also an outer-sphere interaction with the C6 carbonyl oxygen and because this coordination mode is also favorable for an electrostatic interaction with the negatively charged phosphodiester bridge. It is further suggested that Mg(2+) binding (which is rather weak compared to that of Ni(2+) and Cd(2+)) occurs mainly in an outer-sphere mode, and on the basis of the so-called Stability Ruler it is concluded that the binding properties of Zn(2+) to the GG species are similar to those of Ni(2+) and Cd(2+).     

Rearrangement of the (6S,8R,11S) and (6R,8S,11R) exocyclic 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal to N2-deoxyguanosine cyclic hemiacetal adducts when placed complementary to cytosine in duplex DNA

trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. The Michael addition of deoxyguanosine to HNE yields four diastereomeric exocyclic 1,N(2)-dG adducts. The corresponding acrolein- and crotonaldehyde-derived exocyclic 1,N(2)-dG adducts undergo ring-opening to N(2)-dG aldehydes, placing the aldehyde functionalities into the minor groove of DNA. The acrolein- and the 6R-crotonaldehyde-derived exocyclic 1,N(2)-dG adducts form interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Only the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Moreover, as compared to the exocyclic 1,N(2)-dG adducts of acrolein and crotonaldehyde, the cross-linking reaction is slow (Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc. 2003, 125, 5687-5700). Accordingly, the chemistry of the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry has been compared with that of the (6R,8S,11R) adduct, when incorporated into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3', containing the 5'-CpG-3' sequence (X = HNE-dG). When placed complementary to dC in this duplex, both adducts open to the corresponding N(2)-dG aldehydic rearrangement products, suggesting that the formation of the interstrand cross-link by the exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry, and the lack of cross-link formation by the exocyclic 1,N(2)-dG adduct of (6R,8S,11R) stereochemistry, is not attributable to inability to undergo ring-opening to the aldehydes in duplex DNA. Instead, these aldehydic rearrangement products exist in equilibrium with stereoisomeric cyclic hemiacetals. The latter are the predominant species present at equilibrium. The trans configuration of the HNE H6 and H8 protons is preferred. The presence of these cyclic hemiacetals in duplex DNA is significant as they mask the aldehyde species necessary for interstrand cross-link formation.     

P(MeNCH2CH2)3N: a highly selective reagent for synthesizing trans-epoxides from aryl aldehydes

In contrast to its acyclic analogue P(NMe2)3 (1), which in benzene at room temperature reacts with two aryl aldehyde molecules bearing electron-withdrawing groups to give the corresponding diaryl epoxide as an isomeric mixture (trans/cis ratios: 72/28-51/49), P(MeNCH2CH2)3N (2a) under the same reaction conditions is found to be a highly selective reagent that provides epoxides with trans/cis ratios as high as 99/1. These reactions are faster with 2a, because its phosphorus atom is apparently more nucleophilic than that in 1. Thus, it is found that 2a more easily forms 1:1 and 1:2 adducts with one or two molecules of aldehyde, respectively. These adducts apparently are intermediates in the formation of the product epoxide and the corresponding phosphine oxides of 1 and 2a.     

Metal-intercalator-mediated self-association and one-dimensional aggregation in the structure of the excised major DNA adduct of a platinum-acridine agent

The crystal structure of the excised major DNA monoadduct, [Pt(en)(ACRAMTU-S)(dGuo-N7)]3+ ("dGuo*"; en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation; dGuo = 2'-deoxyguanosine), of a platinum-acridine cytotoxic agent is reported. The adduct dGuo*, previously identified in enzymatic digests of native DNA treated with this drug, is partially deprotonated and dimerizes through formation of a rare GG- mismatch base pair, which is sandwiched between the planar chromophores of the acridine nonleaving groups linked to platinum. NMR evidence exists that indicates that the dimeric form persists in neutral aqueous solution. The one-dimensional pi-stack produced by the dimers in the solid state is reminiscent of a coordinative-intercalative DNA binding mode.     

Synthesis of pyrene and benzo[a]pyrene adducts at the exocyclic amino groups of 2'-deoxyadenosine and 2'-deoxyguanosine by a palladium-mediated C-N bond-formation strategy

Single-electron oxidation of the carcinogenic hydrocarbon benzo[a]pyrene (BaP) is thought to result in a radical cation intermediate and this species has been proposed to cause alkylation at the nitrogens of the purine nucleobases. Although several different nucleoside adducts have been isolated as arising from this mode of metabolic activation, there are no selective, total syntheses of the stable exocyclic amino group adducts formed by the single-electron oxidation of any hydrocarbon with the purine 2'-deoxynucleosides to date. In this paper we disclose the synthesis of the model adducts N(6)-(1-pyrenyl)-2'-deoxyadenosine and N(2)-(1-pyrenyl)-2'-deoxyguanosine as well as the first synthesis of the carcinogen-linked nucleoside derivatives N(6)-(6-benzo[a]pyrenyl)-2'-deoxyadenosine and N(2)-(6-benzo[a]pyrenyl)-2'-deoxyguanosine via a palladium-mediated C-N bond formation. Two different coupling strategies were attempted: coupling of an aryl bromide with a suitably protected nucleoside and the coupling of an arylamine with a suitable halonucleoside. The former had somewhat limited applicability in that only N(6)-(1-pyrenyl)-2'-deoxyadenosine was prepared by this method; on the other hand, the latter was more general. However, there are noteworthy differences in the amination reactions at the C-6 and C-2 positions. Reactions at the C-6 resulted in the competing formation of a 1:2 amine-nucleoside adduct in addition to the desired monoaryl nucleoside. Such a dimer formation was not observed at the C-2. The C-2 adducts, however, displayed an interesting conformational behavior.     

Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent

Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.     

Quantitative detection of N(7)-(2-hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to develop a sensitive and selective method for the quantitative measurement of N(7)-(2-hydroxyethyl)guanine (N(7)-HEG) adducts in DNA obtained from ethylene oxide-exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N(7)-HEG (m/z 196) was monitored. The detection limits for N(7)-HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R(2) = 0.999) was observed for signal intensities obtained by injecting 16 fmol--33 pmol of N(7)-HEG into the HPLC/ESI-MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose-response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N(7)-HEG formation in other biological systems.