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DNA interstrand cross-links (ICLs) are extremely deleterious and structurally diverse, driving the evolution of ICL repair pathways. Discovering ICL-inducing agents is, thus, crucial for the characterization of ICL repair pathways and Fanconi anemia, a genetic disease caused by mutations in ICL repair genes. Although several studies point to oxidative stress as a cause of ICLs, oxidative stress-induced cross-linking events remain poorly characterized. Also, polycyclic aromatic amines, potent environmental carcinogens, have been implicated in producing ICLs, but their identities and sequences are unknown. To close this knowledge gap, we tested whether ICLs arise by the oxidation of 8-arylamino-2'-deoxyadenosine (ArNHdA) lesions, adducts produced by arylamino carcinogens. Herein, we report that ArNHdA acts as a latent cross-linking agent to generate ICLs under oxidative conditions. The formation of an ICL from 8-aminoadenine, but not from 8-aminoguanine, highlights the specificity of 8-aminopurine-mediated ICL production. Under the influence of the reactive oxygen species (ROS) nitrosoperoxycarbonate, ArNHdA (Ar = biphenyl, fluorenyl) lesions were selectively oxidized to generate ICLs. The cross-linking reaction may occur between the C2-ArNHdA and N2-dG, presumably via oxidation of ArNHdA into a reactive diiminoadenine intermediate followed by the nucleophilic attack of the N2-dG on the diiminoadenine. Overall, ArNHdA-mediated ICLs represent rare examples of ROS-induced ICLs and polycyclic aromatic amine-mediated ICLs. These results reveal novel cross-linking chemistry and the genotoxic effects of arylamino carcinogens and support the hypothesis that C8-modified adenines with low redox potential can cause ICLs in oxidative stress.

There is currently a tremendous interest in developing bioorthogonal "click chemistry" methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels-Alder reactions have received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels-Alder reaction. We synthesized four different building blocks for 3'-terminal, 5'-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels-Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.

Enzymatic ligation of discrete nanoparticle-DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than by double-stranded DNA as in previous experiments. Ligation was verified by agarose gel and small-angle X-ray scattering. This capability was utilized in two ways: first, to create a new class of multiparticle building blocks for nanoscale self-assembly and, second, to develop a system that can amplify a population of discrete nanoparticle assemblies.

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.

A diastereoselective synthesis of the nucleoside adducts corresponding to a cis ring-opening of the carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE-2) by 2'-deoxyadenosine and 2'-deoxyguanosine is described. The key intermediate (+/-)-10alpha-amino-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene was synthesized by a highly diastereoselective dihydroxylation wherein phenylboronic acid was a water surrogate. The resulting boronate ester was converted to a tetraol derivative in which two of the four hydroxyl groups (trans 7, 8) were protected as benzoate esters while the remaining two (cis 9, 10) were free. The cis glycol entity was then subjected to a reaction with 1-chlorocarbonyl-1-methylethylacetate to yield an intermediate chloro monoacetoxy dibenzoate. Displacement of the halide with azide, complete cleavage of the esters, and catalytic reduction of the azide yielded the requisite amino triol. Fluoride displacement from appropriately protected nucleoside derivatives, 6-fluoropurine 2'-deoxyribonucleoside and 2-fluoro-2'-deoxyinosine, by the amino triol then yielded diastereomeric pairs of diol epoxide-adducted 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) nucleosides. Small aliquots of these adducts were separated for characterization purposes. The present approach provides the first diastereoselective synthesis of the cis adducts of BaP DE-2 with 2'-deoxyguanosine as well as the first synthesis of both dA and dG adducts from a common intermediate. An informative analysis of the 1H NMR spectra of the cis adducts synthesized and comparisons to the trans adducts are reported. To gain insight into the diastereoselectivity in the key dihydroxylation step, a computational analysis, including molecular mechanics (MMFF94) and semiempirical AM1 geometry optimizations, yielded results that are in fairly good agreement with the experimental observations.

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA gives four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine; background levels of these adducts have been detected in animal tissue. Stereospecific syntheses of these four adducts at the nucleoside level have been accomplished. In addition, a versatile strategy for their site-specific incorporation into oligonucleotides has been developed. These adducts are destabilizing as measured by melting temperature when compared to an unadducted strand. The thermal destablization of the adducted 12-mers ranged from 5 to 16 degrees C and is dependent on the absolute stereochemistry of the adduct. The HNE adducts were also examined for their ability to form interstrand DNA-DNA cross-links when incorporated into a CpG sequence. We find that only one of the HNE stereoisomers formed interstrand DNA-DNA cross-links.

The ability of the carcinogenic fungal toxin Ochratoxin A (OTA, 1) to react with deoxyguanosine (dG) has been assessed using electrospray mass spectrometry and NMR. Photoexcitation of OTA (100 muM) in the presence of 50 mol equiv of dG led to the isolation and identification of the C8-deoxyguanosine nucleoside adduct 4. Importantly, the same adduct was formed upon oxidative activation of OTA using horseradish peroxidase (HRP)/H2O2 or the transition metals Fe(II) and Cu(II), as evidenced by mass spectrometry. Because the mutagenicity and subsequent carcinogenicity of OTA are believed to stem from oxidative DNA damage (strand scission and oxidative base products) and formation of guanine-specific DNA adducts, the adduct 4 confirms the ability of OTA to react covalently with dG and has important implications for the mechanism of action of OTA and other chlorophenolic toxins that undergo oxidation to yield phenoxyl radicals. The C8 position of dG is susceptible to radical attack, as was amply proven through formation of the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine. The adduct 4 is the first structurally characterized nucleoside adduct of a chlorophenolic toxin, and its formation has important implications for the mutagenicity of phenolic xenobiotics.

Methyl Red H aggregate of predetermined size is successfully synthesized from the DNA conjugate involving multiple Methyl Red moieties in sequence. In the single stranded state, hypsochromicity monotonically increases with the number of incorporated dyes: the peak maximum of the conjugate involving six Methyl Reds appears at 415 nm, and the shift is as great as 69 nm (3435 cm(-)(1)) with respect to the monomeric transition. This large hypsochromicity accompanied by the narrowing of the band clearly demonstrates that H aggregate is formed in the single strand. H aggregation is further promoted at higher ionic strength. Upon addition of complementary DNA below the T(m), however, this H band disappears and a new peak appears at 448 nm, indicating that aggregated structure is changed by the duplex formation. This spectral change is completely reversible so that the H band at 415 nm appears again above T(m). Thus, aggregated structure can be reversibly controlled by the formation and dissociation of the DNA duplex.

We here report our studies on the conjugation of photoreactive Ru(2+) complex to oligonucleotides (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru(2+) complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H(2)O)](2+)-ODN (tpy = 2,2':6',2' '-terpyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH(3)CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl](+) complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl(-) ligand by acetonitrile in the coordination sphere of the Ru(2+) label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru(2+)-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODNs form highly stabilized ODN.DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degrees C) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of the CH(3)CN ligand to give the corresponding aqua complex, [Ru(tpy)(dppz)(H(2)O)](2+)-ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru(2+)-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Thus, when [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand, giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru(2+) centers at both 3'- and 5'-ends. For ODNs carrying only one Ru(2+) complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru(2+)-ODN (22%) > 5'-Ru(2+)-ODN (9%) > middle-Ru(2+)-ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru(2+) complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru(2+)-ODN (VI.T) (DeltaT(m)(b) = 7 degrees C) < 5'-Ru(2+)-ODN (V.T) (DeltaT(m)(b) = 16 degrees C) < middle-Ru(2+)-ODN (VII.T) (DeltaT(m)(b) = 24.3 degrees C, Table 2). This shows that, with the rigidly packed structure, as in the duplex with middle-Ru(2+)-ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthunium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.