5-Cyanoamino-4-imidazolecarboxamide 4a (R = CH2-O-CH2-CH2-OH) has been synthesized, purified, and fully characterized by MS, MS/MS, HRMS, IR spectroscopy, and by 1H and 13C NMR spectroscopy. It is shown that cyclization of 4a yields the guanine 6a and the isoguanine 12a. Our findings provide experimental evidence in support of our hypothesis that the formation of oxanine and xanthine in nitrosative guanine deamination may proceed via pyrimidine ring-opened intermediates. The observed formation of 6a from the amide 4a (XH2 = NH2) shows that, in analogy, oxanine can be formed from 3 (XH = OH). The formation of 12a from 4a reveals for the first time the possibility that oxanine might be formed by a second pathway that involves electrocyclic reaction of 3. Finally, the new chemistry suggests the possibility for a new dG-to-dG cross-link.
The results are reported of mass-spectrometric studies of the nucleobases adenine 1h (1, R ϭ H), guanine 2h, and cytosine 3h. The protonated nucleobases are generated by electrospray ionization of adenosine 1r (1, R ϭ ribose), guanosine 2r, and deoxycytidine 3d (3, R ϭ deoxyribose) and their fragmentations were studied with tandem mass spectrometry. In contrast to previous EI-MS studies of the nucleobases, NH 3 elimination does present a major path for the fragmentations of the ions [1h ϩ H] ϩ , [2h ϩ H] ϩ , and [3h ϩ H] ϩ . The ion [2h ϩ H Ϫ NH 3 ] ϩ also was generated from the acyclic precursor 5-cyanoamino-4-oxomethylenedihydroimidazole 13h and from the thioether derivative 14h of 2h (NH 2 replaced by MeS). The analyses of the modes of initial fragmentation is supported by density functional theoretical studies. Conjugate acids 15-55 were studied to determine site preferences for the protonations of 1h, 2h, 3h, 13h, and 14h. The proton affinity of the amino group hardly ever is the substrate's best protonation site, and possible mechanisms for NH 3 elimination are discussed in which the amino group serves as the dissociative protonation site. The results provide semi-direct experimental evidence for the existence of the pyrimidine ring-opened cations that we had proposed on the basis of theoretical studies as intermediates in nitrosative nucleobase deamination. [1,2]. This chemistry has been studied extensively because of the dietary and environmental exposure of humans to these substances [3][4][5]. Toxicological studies of deamination became more significant when it was recognized that endogenous nitric oxide [6,7] causes nitrosation [8,9], and that this process is accelerated by chronic inflammatory diseases [10,11]. It has been known for a long time that deamination of adenine 1, guanine 2, and cytosine 3 (Scheme 1) results in the formation of hypoxanthine, xanthine, and uracil, respectively, and these products are thought to result from DNA base diazonium ions 4-6, respectively, by direct nucleophilic dediazoniation. The discovery of oxanine formation [12][13][14] in the nitrosative deamination of guanine challenged the generality and completeness of this mechanism. Theoretical studies revealed that unimolecular dediazoniation of guaninediazonium ion 5 is accompanied or immediately followed by pyrimidine ringopening [15,16] and that cytosine-catalysis promotes the process [17,18]. The resulting 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazole is a highly reactive intermediate and undergoes acid-catalyzed 1,4-addition via cyano-N or imino-N protonated 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazoles, 9 and 10, respectively [19].Labeling studies support this reaction mechanism for oxanine formation [20]. Moreover, we synthesized 5-cyanoamino-4-imidazolecarboxamide and studied its cyclization chemistry [21] and its proficiency for cross-link formation [22]. The unimolecular dediazoniation of the diazonium ions of adenine and cytosine can proceed without ring-opening but the cations 7 and 11 formed in this...
The cross-link dG-to-dG is an important product of DNA nitrosation. Its formation has commonly been attributed to nucleophilic substitution of N2 in a guaninediazonium ion by guanine, while recent studies suggest guanine addition to a cyanoamine derivative formed after dediazoniation, deprotonation, and pyrimidine ring-opening. The chemical viability of the latter mechanism is supported here by the experimental demonstration of rG-to-aG formation via rG addition to a synthetic cyanoamine derivative. Thus, all known products of nitrosative guanine deamination are consistent with the postulate of pyrimidine ring-opening. This postulated mechanism not only explains what is already known but also suggests that other products and other cross-links also might be formed in DNA deamination. The study suggests one possible new product: the structure isomer aG(N1)-to-rG(C2) of the classical G(N2)-to-G(C2) cross-link. While the formation of aG(N2)-to-rG(C2) has been established by chemical synthesis, the structure isomer aG(N1)-to-rG(C2) has been assigned tentatively based on its MS/MS spectrum and because this assignment is reasonable from a mechanistic perspective. Density functional calculations show preferences for the amide-iminol tautomer of the classical cross-link G(N2)-to-G(C2) and the amide-amide tautomer of G(N1)-to-G(C2). Moreover, the results suggest that both cross-links are of comparable thermodynamic stability, and that there are no a priori energetic or structural reasons that would prevent the formation of the structure isomer in the model reaction or in DNA.
TMC-incorporated carbon nitride (CN) with hexagonal and quadrangle honeycomb-like structure and having periodic lattice defects linked by –CONH– bond was synthesized through combining the high calcination with the chemical condensation of melamine and 1,3,5-benzenetricarbonyl trichloride. The obtained CN has a tri-s-triazine ring and benzene ring skeleton, which makes it have excellent mechanical and thermal stability. The BET specific surface area was enhanced to about 125.6 m2/g, and the mean pore size is about 3.43 nm. This CN exhibited an excellent adsorption-enhanced photocatalytic performance.Graphical abstract Electronic supplementary materialThe online version of this article (10.1186/s11671-018-2654-7) contains supplementary material, which is available to authorized users.
CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.
A discussion of nitrosative deamination of cytosine 1 is presented that argues for the formation of 6 by diazotization of 1 to cytosinediazonium ion 2 and its electrostatic complex 3, dediazoniation to 4 <--> 5, and amide-bond cleavage to 6. The reaction channels available to 6 include hydrolytic deglycation to 3-isocyanatoacrylonitrile 7, water addition to carbamic acid 9 with the possibility for re-closure to uracil 13, water addition to carbamic acid 9, and decarboxylation to 3-aminoacrylonitrile 10. With a view to the instability of the carbamic acid 9, the carbamate models ethyl (Z)-2-cyanovinylcarbamate 14 and (Z)-2-cyano-1-tert-butylvinylcarbamate 20 were studied. Acid-catalyzed hydrolysis of 14 leads to 2-amino-carbonylphenylcarbamate 15, and its cyclization yields the benzo-fused uracil quinazoline-2,4-dione 16. In contrast to the aromatic system 14, acid-catalyzed cyclization cannot compete with oligomerization in the case of 20, and 5-tert-butyluracil 22 is accessible only with base-catalysis. It is shown that 23, the parent of 10, also easily polymerizes. The experimental results provide a rationale as to why 9, 10, and 12 would have escaped detection in in vitro studies: they would have oligomerized. In contrast to the in vitro experiments, the oligomerizations of 9, 10, or 12 clearly are not relevant in vivo because of low monomer concentrations. With the exclusion of recyclization and of oligomerization in vivo, attention thus needs to focus on (Z)-3-aminoacrylonitrile 10 as the most likely deamination product of cytosine aside from uracil.
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