Synthesis and EPR studies of 2'-deoxyuridines with alkynyl, rodlike linkages.

Sonogashira coupling of diacetyl 5-ethynyl-2'-deoxyuridine with diacetyl 5-iodo-2'-deoxyuridine gave the acylated ethynediyl-linked 2'-deoxyuridine dimer (3 b; 63%), which was deprotected with ammonia/methanol to give ethynediyl-linked 2'-deoxyuridines (3 a; 79%). Treatment of 5-ethynyl-2'-deoxyuridine (1 a) with 5-iodo-2'-deoxyuridine gave the furopyrimidine linked to 2'-deoxyuridine (78%). Catalytic oxidative coupling of 1 a (O(2), CuI, Pd/C, N,N-dimethylformamide) gave butadiynediyl-linked 2'-deoxyuridines (4; 84 %). Double Sonogashira coupling of 5-iodo-2'-deoxyuridine with 1,4-diethynylbenzene gave 1,4-phenylenediethynediyl-bridged 2'-deoxyuridines (5; 83%). Cu-catalyzed cycloisomerization of dimers 4 and 5 gave their furopyrimidine derivatives. One-electron addition to 1 a, 3 a, and 4 gave the anion radical, the EPR spectra of which showed that the unpaired electron is largely localized at C6 of one uracil ring (17 G doublet) at 77 K. The EPR spectra of the one-electron-oxidized derivatives of ethynediyl- and butadiynediyl-linked uridines 3 a and 4 at 77 K showed that the unpaired electron is delocalized over both rings. Therefore, structures 3 a and 4 provide an efficient electronic link for hole conduction between the uracil rings. However, for the excess electron, an activation barrier prevents coupling to both rings. These dimeric structures could provide a gate that would separate hole transfer from electron transport between strands in DNA systems. In the crystal structure of acylated dimer 3 b, the bases were found in the anti position relative to each other across the ethynyl link, and similar anti conformation was preserved in the derived furopyrimidine-deoxyuridine dinucleoside.

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes.

When positioned opposite to a dA in a DNA duplex, the prototype arylamine-DNA adduct [N-(2'-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called 'wedge' (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290-360 nm range (ICD(290-360 nm)), which supports the W conformation. The ICD(290-360 nm) intensities were the greatest for duplexes with a 3'-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the (19)F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3'-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD(290-360) on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans.

Metallo-nucleosides: synthesis and biological evaluation of hexacarbonyl dicobalt 5-alkynyl-2'-deoxyuridines.

Reactions of 5-alkynyl-2'-deoxyuridines with dicobalt octacarbonyl Co2(CO)8 in THF at room temperature gave hexacarbonyl dicobalt nucleoside complexes (77-93%). The metallo-nucleosides were characterized, including an X-ray structure of a 1-cyclohexanol derivative. In crystalline form, the Co-Co bond is perpendicular to the plane of the uracil base, which is found in the anti position. The level of growth inhibition of MCF-7 and MDA-MB-231 human breast cancer cell lines was examined and compared to results obtained with the alkynyl nucleoside precursors. The cobalt compounds displayed good antiproliferative activities with IC50 values in the range of 5-50 microM. Interestingly, the coordination of the dicobalt carbonyl moiety to 5-alkynyl-2'-deoxyuridines led to a significant increase in the cytotoxic potency for alkyl/aryl substituents at the non-nucleoside side of the alkyne, but in the case of hydrogen (terminal alkyne) or a silyl group, a decrease of the cytotoxic effect was observed. As demonstrated using examples for an active and a low active target compound, the cytotoxicity was significantly influenced by the uptake into the tumor cells and the biodistribution into the nuclei.

5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells.

Starting with 5-iodo-2'-deoxyuridine, a series of 5-alkynyl-2'-deoxyuridines (with n-propyl, cyclopropyl, 1-hydroxycyclohexyl, p-tolyl, p-tert-butylphenyl, p-pentylphenyl, and trimethylsilyl alkyne substituents) have been synthesized via the palladium-catalyzed (Sonogashira) coupling reaction followed by a simplified isolation protocol (76-94% yield). The cytotoxic activity of modified nucleosides against MCF-7 and MDA-MB-231 human breast cancer cells has been determined in vitro. 5-Ethynyl-2'-deoxyuridine, the only nucleoside in the series containing a terminal acetylene, is the most potent inhibitor with IC(50) (microM) 0.4+/-0.3 for MCF-7 and 4.4+/-0.4 for MDA-MB-231.

Examination of the long-range effects of aminofluorene-induced conformational heterogeneity and its relevance to the mechanism of translesional DNA synthesis.

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the "B-DNA" major groove, S is "stacked" into the helix with base-displacement, and W is "wedged" into the minor groove. Here, we conducted (19)F NMR, CD, T(m), and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T(m) results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.

Conformation-specific recognition of carcinogen-DNA adduct in escherichia coli nucleotide excision repair.

We report a systematic and quantitative structure-function relationship study of the major N-[deoxyguanosin-8-yl]-2-aminofluorene adduct (AF) derived from the prototype carcinogen 2-aminofluorene and its derivatives. The AF adduct is known to exist in two distinct conformational motifs, depending upon the location of the hydrophobic fluorine moiety: major groove binding "B type" (B) conformation (AF-dGanti) and base-displaced "stacked" (S) conformation (AF-dGsyn). The AF-induced S/B conformational heterogeneity is sequence-dependent and follows a typical two-site dynamic chemical exchange. The population of S conformation decreases in the order of 3'-G > A > C > T, indicating the importance of the purine flanking bases in promoting the stacking structure. Line-shape analysis showed that the S/B interconversion energy barriers (DeltaG) are in the narrow 14-16 kcal/mol range. The energy differences of the two conformers are relatively small (<0.5 kcal/mol), suggesting a possibility for a facile adduct conformation switch in the active site of a polymerase. The S/B equilibrium modulates the efficiency of Escherichia coli UvrABC-based nucleotide excision repair (NER) in a conformation-specific manner. The 19F NMR/NER results indicate greater repair susceptibility for the base-displaced S conformer, which lacks a Watson-Crick base pair at the lesion site. These findings represent the first of its kind quantitative structure-function work relating NER activity to a specific adduct conformer and will lead to a better understanding of how bulky DNA adducts are accommodated by the repair protein.

Induced circular dichroism characteristics as conformational probes for carcinogenic aminofluorene-DNA adducts.

We report novel induced circular dichroism (ICD) characteristics for probing the conformational heterogeneity induced by the arylamine carcinogen 2-aminofluorene, namely, B type (B), stacked (S), and wedged (W) conformers. CD experiments were conducted with five different aminofluorene-modified DNA duplexes (I-V). An intense positive ICD was observed for the W conformeric I in the 290-360 nm range (ICD(290)(-)(360nm)). This was in contrast to the negative ICD(290)(-)(360nm) exhibited by the mostly B conformeric V (17% S/83% B). Duplex IV, which adopts an approximately equal mixture of S (53%) and B (47%), exhibited low ellipticities along the baseline. The magnitude of the positive ICD for I was significantly greater than that observed for II (70% S/30% B). While the ICD(290)(-)(360nm) of the W conformeric III showed no changes in intensity with increasing temperature from 10 to 35 degrees C, dramatic changes were observed for I across the same temperature range. Dynamic (19)F NMR results revealed that I exists in an 85:15 mixture of W and S/B conformers. The dramatic intensity changes observed for I are consistent with the presence of a W/B heterogeneity because of its susceptibility to result in a large difference on the magnitude of the ICD(290)(-)(360nm). In conclusion, the sign and magnitude of the ICD(290)(-)(360)(nm) are sensitive conformational markers for studying arylamine-induced conformational heterogeneity. The temperature-dependent ICD(290)(-)(360nm) data, coupled with (19)F NMR spectroscopy, provide valuable information about conformational distribution and dynamics, which are important factors that affect mutational outcomes.