Facile formation of an intrastrand cross-link lesion between cytosine and guanine upon pyrex-filtered UV light irradiation of d((Br)CG) and duplex DNA containing 5-bromocytosine

Pyrex-filtered UV light irradiation of d(BrCG) and 5-bromocytosine-containing duplex DNA leads to facile formation of a cross-link lesion between the C5 carbon atom of cytosine and the C8 carbon atom of its adjacent guanine. A similar cross-link lesion has been previously found in the X-ray irradiation mixture of d(CGTA).     

Formation of intrastrand cross-link products between cytosine and adenine from UV irradiation of d((Br)CA) and duplex DNA containing a 5-bromocytosine

Here, we showed that Pyrex-filtered UV light irradiation of d((Br)CA) gave rise to three types of intrastrand cross-link products, that is, d(C[5-N6]A), d(C[5-2]A), and d(C[5-8]A), where the C5 carbon atom of cytosine is covalently bonded to the N6 nitrogen atom, C2, and C8 carbon atoms of adenine, respectively. Furthermore, we demonstrated by LC-MS/MS that the UV irradiation of a 5-bromocytosine-containing duplex oligodeoxynucleotide (ODN) led to the formation of five cross-link products, that is, C[5-N6]A, C[5-2]A, C[5-8]A, A[2-5]C, and A[8-5]C, under both aerobic and anaerobic conditions. LC-MS/MS quantification results showed that the yields for the formation of these cross-link products are different. The presence of molecular oxygen reduces the yields for the formation of all cross-link products except A[2-5]C. To our knowledge, this is the first report about the formation of intrastrand cross-link products between cytosine and adenine in duplex DNA. The chemistry discovered here may facilitate the future preparations of oxidative cross-link lesion-bearing substrates for biochemical and biophysical studies.     

Independent generation of 5-(2'-deoxycytidinyl)methyl radical and the formation of a novel cross-link lesion between 5-methylcytosine and guanine

Reactive oxygen species (ROS) can damage DNA. Although a number of single nucleobase lesions induced by ROS have been structurally characterized, only a few intrastrand cross-link lesions have been identified and characterized, and all of them involve adjacent thymine and guanine or adenine. In mammalian cells, the cytosines at CpG sites are methylated. On the basis of the similar reactivity of 5-methylcytosine and thymine toward hydroxyl radical and the similar orientation of adjacent thymine guanine (TG) and 5-methylcytosine guanine (mCG) in B-DNA, we predict that the cross-link lesion, which was identified in TG and has a covalent bond formed between the 5-methyl carbon atom of T and the C8 carbon atom of G, should also form at mCG site. Here, we report for the first time the independent generation of 5-(2'-deoxycytidinyl)methyl radical, and our results demonstrate that this radical can give rise to the predicted novel intrastrand cross-link lesion in dinucleoside monophosphates d(mCG) and d(GmC). Furthermore, we show that the cross-link lesion can also form in d(mCG) from gamma irradiation under anaerobic conditions.     

Independent generation of the 5-hydroxy-5,6-dihydrothymidin-6-yl radical and its reactivity in dinucleoside monophosphates

Hydroxyl radical is a major reactive oxygen species produced by gamma-radiolysis of water or Fenton reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major product. Here we report the synthesis of all four stereoisomers of 5-hydroxy-6-phenylthio-5,6-dihydrothymidine (T*), which, upon 254 nm UV irradiation, give rise to the 5-hydroxy-5,6-dihydrothymidin-6-yl radical (I). We also incorporated the photolabile radical precursors into dinucleoside monophosphates d(GT*) and d(TT*) and characterized major products resulting from the 254-nm irradiation of these dinucleoside monophosphates. Our results showed that, under anaerobic conditions, the most abundant product emanating from the 254-nm irradiation of d(GT*) and d(TT*) is an abasic site lesion. Products with the thymine portion being modified to thymine glycol and 5-hydroxy-5,6-dihydrothymine were also observed. In addition, we demonstrated that radical I can attack the C8 carbon atom of its 5' neighboring guanine and give rise to a novel cross-link lesion. Moreover, LC-MS/MS results showed that gamma-radiation of d(GT) under anaerobic condition yielded the same type of cross-link lesions.     

A new rearrangement process in tert-amyl cation

13C NMR spectroscopy of the 2-methyl-2-butyl-1-13C cation (13C-labeled tert-amyl cation) indicates that interchange of the inside and outside carbons occurs via a barrier of 19.5 +/- 2.0 kcal/mol. A plausible mechanism involves hydride migration in the proposed 2-pentyl cation 4 to form 3-pentyl cation 5. Via the protonated cyclopropane intermediate 6, which undergoes degenerate corner-to-corner hydride shift, the secondary 3-pentyl cation 5' with the label shifted to the central carbon atom is formed. The tert-amyl cation obtained from 5' in the reverse process has the 13C label on an inside carbon atom. All intermediates and transition structures were located on the PES theoretically at the MP2/6-31G(d,p) level of theory. The rearrangement rate of the doubly labeled tert-amyl cation (methyl-13C-butyl-1-13C cation), followed by means of 13C NMR, revealed that the process that interchanges inside and outside carbons has the highest barrier. Comparison of the initial rates revealed that isotopomer 1e arises considerably more slowly than other isotopomers, indicating that in the overall rearrangement process transition structure 5-TS has the highest energy.     

A novel nitroimidazole compound formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-guanosine.

Peroxynitrite reacts with 2',3',5'-tri-O-acetyl-guanosine to yield a novel compound identified as 1-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6). This characterization was achieved using a combination of UV/vis spectroscopy and ESI-MS. Additionally, 1-(beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6a) was synthesized by an independent route, characterized by UV/vis spectroscopy, ESI-MS, and (1)H- and (13)C NMR, and shown to be identical to deacetylated 6. This product is extremely stable in aqueous solution at both pH extremes and is formed in significant yields. These characteristics suggest that this lesion may be useful as a specific biomarker of peroxynitrite-induced DNA damage. We also observed formation of 2',3',5'-tri-O-acetyl-8-nitroguanosine (2',3',5'-tri-O-acetyl-8-NO(2)()Guo), 2-amino-5-[(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (2',3',5'-tri-O-acetyl-Iz), and the peroxynitrite-induced oxidation products of 2',3',5'-tri-O-acetyl-8-oxoGuo. The formation of 6 and 2',3',5'-tri-O-acetyl-8-NO(2)()Guo was rationalized by a mechanism invoking formation of the guanine radical.     

Formation of a N2-dG:N2-dG carbinolamine DNA cross-link by the trans-4-hydroxynonenal-derived (6S,8R,11S) 1,N2-dG adduct

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.     

胞嘧啶与一氧化碳复合物的结构与性质

在B3LYP/6-311+G**水平上对胞嘧啶…CO复合物体系进行了理论计算, 发现了6个能量极小的复合物. 其结合方式是CO的C或O原子与胞嘧啶的N—H键形成氢键, 最稳定的复合物的结合能为-8.72 kJ·mol-1. CO的C原子与胞嘧啶的结合具有更强的优势, C原子结合的复合物中CO的键长缩短, 而O结合的复合物中CO键长伸长. 同时, 对复合物的振动分析发现, 在C原子结合的复合物中CO的伸缩频率蓝移, 而O结合的复合物中CO伸缩频率是红移的.     

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts

The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity.     

2’-脱氧胞苷-5’-磷酸羟基加合物的分子结构与电子结构

使用密度泛函理论(DFT)的B3LYP/DZP++研究了羟基自由基与2’-脱氧胞苷-5’-磷酸(dCMP)的胞嘧啶环加成产物的分子结构与电子结构. 结果表明, dCMP胞嘧啶环中各C原子上的单羟基加合物的相对稳定性顺序为C5>C6>>C4≥C2. 加合物的稳定性、自旋密度、静电势以及dCMP的电子密度、静电势、电荷分布分析表明, dCMP遭遇多个羟基自由基攻击时, 第一个羟基自由基加在dCMP的C5上, 而C6则成为第二个羟基自由基的进攻目标. 反应中一旦形成了C2-位单羟基加合物, 则极有可能在DNA复制过程中引起致命的基因突变, 也可能诱发DNA-DNA以及DNA-蛋白质的链间交联, 引起更复杂的损伤. 相反, C5、C6-位上单羟基加合物的形成对DNA的稳定性不构成直接威胁.     

RATE CONSTANTS FOR THE DEHYDRATION OF SINGLE AND DOUBLE HYDRATES OF CYTIDYLYL (3''-5'')–CYTIDINE

Abstract— Cytidylyl (3'-5') cytidine (CpC) was irradiated with ultraviolet light (u.v.) to produce the single hydrate (a mixture of C*pC and CpC*) and the double hydrate C*pC* which were separated by electrophoresis. These photoproducts rapidly dehydrate to CpC and deaminate to a mixture of U*pC and CpU*. The rate constants for dehydration and deamination of the hydrates were evaluated for a range of pH values from 3 to 8 at 0°C. It is observed that the rate constants for decay of C*pC* lie between those for C*pC and CpC* for all pH values studied. Both single and double hydrates show minimum stability around pH 4·5 and maximum stability around pH 8. The maximum rate constants for dehydration of C*pC*, C*pC and CpC* are 0·26, 0·145 and 0.35 hr^-1 respectively and the minimum values are 0.024, 0.011 and 0.091 hr^-1 respectively all at 0°C. The rate constants for deamination of C*pC to U*pC for a range of pH values at 0°C were measured. The amount of deamination product varies from about 2 to 10 per cent of the hydrate depending on pH with the maximum amount being produced around pH 8.     

Deamination of the Radical Cation of the Base Moiety of 2′‐Deoxycytidine: A Theoretical Study

Five pathways leading to the deamination of cytosine (to uracil) after formation of its deprotonated radical cation are investigated in the gas phase, at the UB3LYP/6‐311G(d,p) level of theory, and in bulk aqueous solvent. The most favorable pathway involves hydrogen‐atom transfer from a water molecule to the N3 nitrogen of the deprotonated radical cation, followed by addition of the resulting hydroxyl radical to the C4 carbon of the cytosine derivative. Following protonation of the amino group (N4), the C4? N4 bond is broken with elimination of the NH₃^?+ radical and formation of a protonated uracil. The rate‐determining step of this mechanism is hydrogen‐atom transfer from a water molecule to the cytosine derivative. The associated free energy barrier is 70.2 kJ mol^?1.     

Mercury(II) Site-Selective Binding to a DNA Hairpin. Relationship of Sequence-Dependent Intra- and Interstrand Cross-Linking to the Hairpin-Duplex Conformational Transition

Hg(II) interacted site selectively with only one of three deoxyribooligonucleotides examined; these "oligos" each had a different number of unmatched T residues. Thus, Hg(II) formed an intrastrand T-Hg-T cross-link between the first and fourth T residues of the hairpin, d(GCGCTTTTGCGC) (T4). The DNA strand formed a loop around the Hg, as if the Hg atom had been lassoed. The interactions of Hg(II) with two other oligos, d(ATGGGTTCCCAT) (T2) and d(GCGCTTTGCGC) (T3), were less specific. Previously, we found that at high DNA and salt concentrations, T2 was a mixture of hairpin and duplex forms while T3 and T4 had the hairpin form; modeling studies showed that in the free T4 hairpin the two T's at the ends of the (T)(4) loop form a T.T wobble base pair. Only in T4 are the T residues positioned to form an intrastrand cross-link readily. The Hg(II)-oligo adducts formed as a function of added Hg(II) were investigated by titrations monitored by UV, CD, and (1)H NMR spectroscopy. The appearance of a new set of (1)H signals with the concomitant decay of the free oligo (1)H signals indicated that 1:1 Hg(II):T2, 1.5:1 Hg(II):T3, and 1:1 Hg(II):T4 adducts were formed with Hg(NO(3))(2). In H(2)O, these adducts all had spectra with very downfield signals for the exchangeable TN(3)H and GN(1)H groups, a characteristic of base-paired regions. All upfield N(3)H signals from the (T)(2) and (T)(3) sequences of the free oligo disappeared in the spectra of the 1:1 Hg(II):T2 and 1.5:1 Hg(II):T3 adducts. The disappearance of the NH signals, the UV spectral changes, and the stoichiometries (1:1 Hg(II):T2 and 1.5:1 Hg(II):T3) indicate that these adducts are duplexes containing two and three T-Hg-T interstrand cross-links for T2 and T3, respectively. The (1)H and (13)C signals of the 1:1 Hg(II):T4 adduct in D(2)O were nearly completely assigned by 2D NMR spectroscopy. The spectrum of the adduct in H(2)O had only two of the four original TN(3)H signals from the (T)(4) sequence present in the spectrum of T4; this result is consistent with the presence of a TN3-Hg-TN3 cross-link. The (13)C chemical shift changes upon Hg(II) binding indicated that the TN3-Hg-TN3 cross-link was between the T's at each end of the (T)(4) loop. The NOESY, CD, and UV spectra were all consistent with a hairpin conformation for the 1:1 Hg(II):T4 adduct. A hairpin conformation also appeared reasonable from molecular modeling calculations. In conclusion, the length of the central (T)(n)() sequence influenced the type of T-Hg-T cross-link formed and, in turn, the conformation of the adducts. For (T)(2) and (T)(3), interstrand T-Hg-T cross-linking favored the duplex form. In contrast, for (T)(4), intrastrand T-Hg-T cross-linking stabilized the hairpin form.     

Ultrafast IR spectroscopy of polymeric cytosine nucleic acids reveal the long-lived species is due to a localised state

The decay pathways of UV-excited cytosine polymers are investigated using picosecond time-resolved infrared spectroscopy. Similar yields of a non-emissive (1)nπ* state are found in the single-stranded dC(30) polymer as in the dCMP monomer, but with a longer lifetime in the polymer (80 ps vs. 39 ps). A longer lifetime is also found in the d(CpC) dinucleotide. No evidence of excimer states is observed, suggesting that localised (1)nπ* excited states are the most significant intermediates present on the picosecond timescale.     

d(C4)相关序列形成四分子i-Motif结构的电喷雾质谱研究

设计了与富含胞嘧啶(C)的DNA序列d(C4)相关的DNA序列d(C4), d(TC4), d(AC4), d(T2C4), d(A2C4), d(C4T), d(C4A)和d(TC4T); 采用电喷雾质谱测定发现这些序列形成四分子非共价复合物离子, 根据离子的相对丰度可确定形成四链i-motif结构的数量和可能性; 同时考察了腺嘌呤(A)和胸腺嘧啶(T)在d(C4)序列的5'和3'端对其形成四分子i-motif结构的影响. 结果表明, 在d(C4)的5'端增加A碱基或T碱基更易形成四分子复合物; 5'端含T碱基比含A碱基更利于形成四分子复合物; 而在d(C4)序列中增加2个A碱基或T碱基比增加相应的单个碱基形成了更高丰度的四分子离子峰.     

Stable glucopyranosylpalladium complexes with cis-beta-hydrogen. A six-membered ring metallocycle with an oxygen donor ligand

Two stable glucopyranosylpalladium complexes, chloro[1,3-dimethyl-5-(3,4,6-tri-O-acetyl-2-deoxy-alpha-D -arabinohexopyranosyl)-2,4(1H,3H)-pyrimidinedionnato] (triphenylphosphine)-palladium and the corresponding triphenylarsine analog, were studied using fast atom bombardment mass spectrometry, 1H, 13C and 31P nuclear magnetic resonance, UV and IR spectroscopy to establish structures for these complexes. The data obtained indicate that the pyranosyl ring is in a chair conformation in which palladium (C2'), acetoxy (C3' C4') and acetoxymethyl (C5') are equatorial and 1,3-dimethyl-2,4(1H,3H) pyrimidinedion-5-yl (C1') is axial. The palladium(II) ion is encompassed in a six-membered ring metallocycle in which C2' of the glucopyranosyl ring and the oxygen of the C4 carbonyl of the pyrimidinedionyl group occupy adjacent ligand sites. The other two ligand sites on square planar palladium are occupied by triphenylphosphine (or triphenylarsine) cis to C2' and trans to carbonyl oxygen, and chloride trans to C2' and cis to oxygen. This stable metallocycle has three unusual features, a cis-beta-hydrogen, a six-membered Pd-containing ring and an oxygen donor ligand. Its surprising stability is due to conformational barriers to the proper alignment of Pd with pyranosyl ring substituents required for elimination reactions.     

Identification of the alpha and beta anomers of 1-(2-deoxy-D-erythro-pentofuranosyl)-oxaluric acid at the site of riboflavin-mediated photooxidation of guanine in 2'-deoxyguanosine and thymidylyl-(3'-5')-2'-deoxyguanosine

Products of riboflavin-mediated photosensitization of 2'-deoxyguanosine (dG) and thymidylyl-(3'-5')-2'-deoxyguanosine (TpdG) by 350-nm light in oxygen-saturated aqueous solution have been isolated and identified as 1-(2-deoxy-beta-D-erythro-pentofuranosyl) oxaluric acid (beta-dOx) and thymidylyl-(3'-5')-1-(2-deoxy-beta-D-erythro-pentofuranosyl) oxaluric acid (Tpbeta-dOx), respectively. In aqueous solution the modified beta-deoxyribonucleoside is slowly converted to the alpha-anomer, generating alpha-dOx and Tpalpha-dOx. These modified nucleosides and dinucleoside monophosphates have been isolated by HPLC and characterized by proton and carbon NMR spectroscopy, fast atom bombardment mass spectrometry, and enzymatic analyses. Both alpha-dOx and Tpalpha-dOx slowly convert back into the modified beta-deoxyribonucleoside, indicating that the furanosidic anomers are in dynamic equilibrium. Relative to TpdG, the rate of hydrolysis of Tpbeta-dOx and Tpalpha-dOx by spleen phosphodiesterase is greatly reduced. Hot piperidine (1.0 M, 90 degrees C, 30 min) destroys Tpbeta-dOx and Tpalpha-dOx. Riboflavin-mediated photosensitization of TpdG in D2O instead of H2O has no detectable effect on the yield of Tpbeta-dOx, suggesting that oxaluric acid is generated through a Type-I reaction mechanism, likely through the intermediary on initially generated 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Synthesis of C3' modified nucleosides for selective generation of the C3'-deoxy-3'-thymidinyl radical: a proposed intermediate in LEE induced DNA damage

DNA damage pathways induced by low-energy electrons (LEEs) are believed to involve the formation of 2-deoxyribose radicals. These radicals, formed at the C3' and C5' positions of nucleotides, are the result of cleavage of the C-O phosphodiester bond through transfer of LEEs to the phosphate group of DNA oligomers from the nucleobases. A considerable amount of information has been obtained to illuminate the identity of the unmodified oligonucleotide products formed through this process. There exists, however, a paucity of information as to the nature of the modified lesions formed from degradation of these sugar radicals. To determine the identity of the damage products formed via the 2',3'-dideoxy-C3'-thymidinyl radical (C3'(dephos) sugar radical), phenyl selenide and acyl modified sugar and nucleoside derivatives have been synthesized, and their suitability as photochemical precursors of the radical of interest has been evaluated. Upon photochemical activation of C3'-derivatized nucleosides in the presence of the hydrogen atom donor tributyltin hydride, 2',3'-dideoxythymidine is formed indicating the selective generation of the C3'(dephos) sugar radical. These precursors will make the identification and quantification of products of DNA damage derived from radicals generated by LEEs possible.