Characterization of a deoxyguanosine adduct of tetrachlorobenzoquinone: dichlorobenzoquinone-1,N2-etheno-2'-deoxyguanosine

Pentachlorophenol (PCP), a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone. DNA adducts attributable to tetrachloroquinone have been observed previously in vitro and detected in vivo. In addition, an unidentified adduct in these studies coeluted with the product of the reaction of deoxyguanosine (dG) and tetrachlorobenzoquinone (Cl4BQ). We have synthesized, isolated, purified, and characterized the predominant adduct formed from the reaction of dG and Cl4BQ. The preparation of a 13C-labeled version of this adduct facilitated its structural characterization. On the basis of 1H NMR, 13C NMR, MS, IR, UV, and cyclic voltammetry, we propose that the adduct is a dichlorobenzoquinone nucleoside in which two chlorine atoms in Cl4BQ have been displaced by reaction at the 1- and N2-positions of dG. The 1H and 13C NMR chemical shifts are consistent with the dichlorobenzoquinone assignment. In contrast, under standard analytical conditions, LC-MS data are consistent with a reduced hydroquinone structure, similar to what may be expected based on results from other chloroquinones. Data from the present study indicate that this reduction could be occurring in the electrospray ionization source and that the initial product of the reaction of dG and Cl4BQ is a dichlorobenzoquinone. The results of this study contribute to the hypothesis that direct reactions between chlorophenols and DNA may play a role in the toxic effects of chlorophenols and indicate a potential difference in reactivity and biological influence between PCP and other less substituted chlorophenols or phenols.     

1,N2-ethenodeoxyguanosine: properties and formation in chloroacetaldehyde-treated polynucleotides and DNA.

1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.     

Structure of the 1,N2-ethenodeoxyguanosine adduct opposite cytosine in duplex DNA: Hoogsteen base pairing at pH 5.2

The exocyclic 1, N(2)-ethenodeoxyguanosine (1,N(2)-is an element of dG) adduct, arising from the reaction of vinyl halides and other vinyl monomers, including chloroacetaldehyde, and lipid peroxidation products with dG, was examined at pH 5.2 in the oligodeoxynucleotide duplex 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCCATGCG)-3' (X = 1,N(2)-is an element of dG). Previously, X(anti).C(anti) pairing was established in this duplex, containing the 5'-TXG-3' sequence context, at pH 8.6 [Shanmugam, G., Goodenough, A. K., Kozekov, I. D., Harris, T. M., Guengerich, F. P., Rizzo, C. J., and Stone, M. P. (2007) Chem. Res. Toxicol. 20, 1601- 1611]. At pH 5.2, the 1,N(2)-is an element of dG adduct decreased the thermal stability of the duplex by approximately 13 degrees C. The 1,N(2)-is an element of dG adduct rotated about the glycosyl bond from the anti to the syn conformation. This resulted in the observation of a strong nuclear Overhauser effect (NOE) between the imidazole proton of 1,N(2)-is an element of dG and the anomeric proton of the attached deoxyribose, accompanied by an NOE to the minor groove A(20) H2 proton from the complementary strand. The syn conformation of the glycosyl bond at 1,N(2)-is an element of dG placed the exocyclic etheno moiety into the major groove. This resulted in the observation of NOEs between the etheno protons and the major groove protons of the 5'-neighboring thymine. The 1,N(2)-is an element of dG adduct formed a Hoogsteen pair with the complementary cytosine, characterized by downfield shifts of the amino protons of the cytosine complementary to the exocyclic adduct. The pattern of chemical shift perturbations indicated that the lesion introduced a localized structural perturbation involving the modified base pair and its 3'- and 5'-neighbor base pairs. A second conformational equilibrium was observed, in which both the modified base pair and its 3'-neighboring G.C base pair formed tandem Hoogsteen pairs. The results support the conclusion that at neutral pH, in the 5'-TXG-3' sequence, the 1,N(2)-is an element of dG adduct exists as a blend of conformations in duplex DNA. These involve the interconversion of the glycosyl torsion angle between the anti and the syn conformations, occurring at an intermediate rate on the NMR time scale.     

Formation of a substituted 1,N(6)-etheno-2'-deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2-nonenal

Analysis of the reaction between 2'-deoxyadenosine and 13-hydroperoxylinoleic acid by liquid chromatography/constant neutral loss mass spectrometry revealed the presence of two major products (adducts A and B). Adduct A was shown to be a mixture of two isomers (A(1) and A(2)) that each decomposed with the loss of water to form adduct B. The mass spectral characteristics of adduct B were consistent with the substituted 1, N(6)-etheno-2'-deoxyadensoine adduct 1' '-[3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptan-2' '-one. Adducts A(1), A(2), and B were formed when 2'-deoxyadenosine was treated with synthetic 4-oxo-2-nonenal, which suggested that it was formed by the breakdown of 13-hydroperoxylinoleic acid. A substantial increase in the rate of formation of adducts A(1), A(2), and B was observed when 13-hydroperoxylinoleic acid and 2'-deoxyadenosine were incubated in the presence of Fe(II). Thus, 4-oxo-2-nonenal was most likely formed by a homolytic process. Although adducts A(1), A(2), and B were formed in the reaction between 4-hydroxy-2-nonenal and 2'-deoxyadenosine, a number of additional products were observed. This suggested that 4-hydroxy-2-nonenal was not a precursor in the formation of 4-oxo-2-nonenal from 13-hydroperoxylinoleic acid. This study has provided additional evidence which shows that 4-oxo-2-nonenal is a major product of lipid peroxidation and that it reacts efficiently with DNA to form substituted etheno adducts.     

Formation of the 1,N2-glyoxal adduct of deoxyguanosine by phosphoglycolaldehyde, a product of 3'-deoxyribose oxidation in DNA

Oxidation of deoxyribose in DNA results in the formation of a variety of electrophilic products that have the potential to react with nucleobases to form adducts. We now report that 2-phosphoglycolaldehyde, a model for the 3'-phosphoglycolaldehyde residue generated by 3'-oxidation of deoxyribose in DNA, reacts with dG and DNA to form the diastereomeric 1,N2-glyoxal adducts of dG, 3-(2-deoxy-beta-D-erythro-pentofuransyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one. The glyoxal adducts were the predominant species formed under biological conditions (pH 7.4 and 37 degrees C), with several minor fluorescent adducts, including 1,N6-ethenoadenine. The adducts were fully characterized by HPLC, mass spectrometry, and UV and NMR spectroscopy. The reaction of 2-phosphoglycolaldehyde with dG occurred with a rate constant of 10(-6) M(-1) s(-1) compared to the rate constants of 0.08 and approximately 10(-9) M(-1) s(-1) for the reactions of glyoxal and glycolaldehyde with dG, respectively. The kinetic results rule out contamination of 2-phosphoglycolaldehyde preparations with glyoxal as the basis for our observations. The rate constant for the formation of glyoxal from 2-phosphoglycolaldehyde (10(-8) s(-1)) is consistent with glyoxal generation being the rate-limiting step in the formation of dG adducts in reactions with 2-phosphoglycolaldehyde. Mechanistic studies were also undertaken to define the basis for the different oxidation states of glyoxal and 2-phosphoglycolaldehyde. Although 2-phosphoglycolaldehyde produced a weak ESR signal consistent with generation of hydroxyl radicals and it caused DNA strand breaks at high concentrations, the formation of the glyoxal adducts of dG was insensitive to radical quenchers (e.g., sorbitol) and independent of molecular oxygen. In contrast, the formation of glyoxal-dG adducts with glycolaldehyde was dependent on molecular oxygen and quenched by sorbitol, and the glycolaldehyde-glyoxal rearrangement produced a strong ESR signal characteristic of alkyl radicals. These observations are consistent with a model in which glyoxal is generated from 2-phosphoglycolaldehyde by a nonradical, oxygen-independent mechanism that is currently under investigation. Our results provide a mechanistic basis for the observation by Murata-Kamiya et al. [(1995) Carcinogenesis 16, 2251-2253] that oxidation of DNA with the Fe(II)-EDTA complex results in the formation of the glyoxal adducts of dG.     

trans,trans-2,4-decadienal-induced 1,N(2)-etheno-2'-deoxyguanosine adduct formation

A number of ring-extended DNA adducts resulting from the reaction of alpha,beta-unsaturated aldehydes, or their epoxides, with DNA bases have been characterized in recent years. These adducts may lead to miscoding during DNA replication, resulting, if not repaired, in mutations that can contribute to cancer development. trans,trans-2, 4-Decadienal (DDE) is one of the highly cytotoxic aldehydes endogenously formed from lipid peroxidation. To evaluate its DNA damaging potential, we have investigated the reaction of DDE with 2'-deoxyguanosine (dGuo) in the presence of peroxides. Three stable adducts were isolated by reverse-phase HPLC. Adduct A1, 3-(2-deoxy-beta-D-erythro-pentafuranosyl)-5,9-dihydro-9H-imidazo[2 , 1-i]purin-9-hydroxy, is a tautomer of 1, N(2)-etheno-2'-deoxyguanosine, a well-known reaction product of epoxy aldehydes with dGuo. Two new diasteroisomeric products, A2-1 and A2-2, 1-?[3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-5, 9-dihydro-9H-imidazo[2,1-i]purin-9-hydroxy]-7-yl?-2-one-3-octanol, were isolated and characterized on the basis of their spectroscopic features as 1,N(2)-etheno adducts possessing a carbon side chain with a carbonyl and a hydroxyl group. The proposed reaction mechanism for the formation of adducts A2 involves DDE double epoxidation and hydrolysis of the C4 epoxy group prior to nucleophilic addition of the exocyclic amino group of dGuo to C1 of the aldehyde, followed by cyclization via nucleophilic attack on the C2 epoxy group by N-1 and elimination of H(2)O. After treatment of calf thymus DNA with DDE, formation of adducts A1 and A2 was detected by the LC/ESI/MS-MS technique. These results can contribute to a better understanding of the chemical structures of adducts resulting from the reaction of aldehydes with nucleic acid bases, a necessary step in assessing the genotoxic risks associated with this class of compounds.     

Synthesis of N2,3-ethenodeoxyguanosine, N2,3-ethenodeoxyguanosine 5'-phosphate, and N2,3-ethenodeoxyguanosine 5'-triphosphate. Stability of the glycosyl bond in the monomer and in poly(dG,epsilon dG-dC)

The synthesis of a new modified etheno-2'-deoxyguanosine is reported. N2,3-Ethenodeoxy-guanosine (epsilon dGuo) is a product in double-stranded DNA treated with the carcinogen vinyl chloride in vivo or its metabolite chloroacetaldehyde in vitro. The lability of its glycosyl bond has, however, interfered with its isolation from DNA. The synthesis, starting with O6-benzyl-2'-deoxyguanosine 5'-phosphate, reacted with bromoacetaldehyde, could only be accomplished in slightly alkaline media, which prevented significant loss of the sugar. The 5'-phosphate also decreased the lability of the glycosyl bond. The resulting compound, when deprotected, was converted to N2,3-ethenodeoxyguanosine 5'-triphosphate, as well as the corresponding nucleoside. Fluorescence, UV, and 1H NMR data were consistent with the assigned structures and almost identical with those of the previously synthesized much more stable ribo analogues [Ku?mierek et al. (1987) J. Org. Chem. 52, 2374-2378]. A systematic study of the pH-dependent glycosyl bond cleavage gave a t1/2, 37 degrees C, pH 6, of approximately 3.5 h for the nucleoside and 7-10 h for the nucleotides. Comparison, under the same conditions, of stability of the glycosyl bond in poly(dG,epsilon dG-dC) showed an increased stability of 2 orders of magnitude, t1/2 = approximately 600 h. The rate of sugar loss was, in all cases, greatly decreased at higher pH's, over the range of pH 5-9. These stability data indicate that when slightly alkaline conditions can be used, studies on incorporation of epsilon dGuo into polymers for in vitro mutagenesis studies are possible.     

1,N 2-Cyclic deoxyguanosine adducts and guanine adducts of 2-haloacroleins. Isolation,characterization, isomerization and stability

The reaction of the mutagenic 2-haloacroleins, 2-fluoroacrolein, 2-chloroacrolein and 2-bromoacrolein, with nucleosides and 5′-mononucleotides was studied. We found two different regioisomers of 1,N ²-cyclic deoxyguanosine adducts of 2-chloroacrolein and 2-bromoacrolein: type A, the 6-hydroxy, 7-haloadduct in which the OH-substituent is vicinal to the N ²-atom of the guanine moiety and type B, the 8-hydroxy, 7-haloadduct in which the OH-group is adjacent to the N¹-atom of the guanine moiety. The major adducts were the trans pairs of diastereomers of type A and type B in which the 6,7-substituents as well as the 7,8-substituents are in the energetically favoured diaxial position of the newly formed tetrahydropyrimidine ring. In the case of the type A regioisomers, the cis pairs of diastereomers (traces with chloroacrolein and about 4% with bromoacrolein) were also found in which the halosubstituent probably takes the equatorial position. Due to the anomeric effect, the OH-group takes the axial position in both regioisomers. No cis isomers of the type B regioisomers could be isolated. Acid hydrolysis of the deoxyguanosine adducts released deoxyribose, and the respective guanine adducts were isolated and characterized. Besides the vicinal halo, hydroxy adducts, trace amounts of the corresponding dihydroxy adducts were formed by hydrolysis of the chlorine or bromine substituents. The dihydroxy compounds possess the same structures and conformations in the newly formed tetrahydropyrimidine ring as do the halo, hydroxy adducts. Under our conditions no adducts other than those with deoxyguanosine and guanine could be identified. We found, however, indication for the formation of deoxyadenosine adducts when using dimethylsulfoxide as a solvent. No adducts in substantical amounts could be isolated with fluoroacrolein due to its rapid polymerization. Received: 24 February 1994 / Accepted: 12 April 1994     

4-Hydroperoxy-2-nonenal-induced formation of 1,N2-etheno-2'-deoxyguanosine adducts

Analysis of the reaction between 4-hydroperoxy-2-nonenal (HPNE) and 2'-deoxyguanosine (dGuo) by liquid chromatography/mass spectrometry (LC/MS) revealed the formation of 1,N2-etheno-dGuo as well as heptanone-etheno-dGuo and trace amounts of dihydroxyheptane-etheno-dGuo. Identities of the dGuo adducts were confirmed by comparison with authentic standards. The minor dihydroxyheptane-etheno-dGuo adducts could be generated from 2,3-epoxy-4-hydroxynonanal (EHN), the epoxidation product of 4-hydroxy-2-nonenal (HNE). An LC/MS method was developed for the analysis of EHN. No EHN was detected by LC/MS during the decomposition of HPNE. Therefore, the dihydroxyheptane-etheno-dGuo adducts are either generated from a direct reaction between HPNE and dGuo or from another intermediate that cannot be detected by LC/MS. In addition, no HNE-derived hydroxypropano-dGuo adducts were observed. On the basis of these findings, we conclude that HPNE, a primary product of lipid peroxidation, is a major precursor to the formation of 1,N2-etheno-dGuo. We propose that it arises from the reaction of dGuo and HPNE through the intermediate formation of a cyclic hydroxy-ethano-epoxide derivative. The minor amounts of heptanone-ethano-dGuo adducts that were formed from HPNE in the absence of vitamin C suggest that heptanone-etheno-dGuo can be generated directly from HPNE without the intermediate formation of ONE. Therefore, HPNE can be considered as another lipid hydroperoxide-derived bifunctional electrophile with the potential for biological activities that are similar to HNE and ONE.     

Horseradish peroxidase mediates DNA and deoxyguanosine 3'-monophosphate adduct formation in the presence of ochratoxin A

Ochratoxin A (OTA) gives rise to DNA and deoxyguanosine-3'-monophosphate (dGMP) adducts in vitro using mice kidney microsomes in the presence of arachidonic acid. This result points to the involvement of prostaglandin H synthases, which are present at high levels in the kidney, urinary bladder and seminal vesicles, and/or of lipoxygenases in the metabolic activation of OTA to genotoxic compounds. These enzymes have peroxidase activities. Incubation of OTA with DNA in the presence of horseradish peroxidase (HRP) and cumene hydroperoxide at pH 7.4 led to the formation of one major and three minor adducts with a total adduct level of 42 per 10(9) nucleotides. Incubation with dGMP gave a total adduct level of 159 per 10(9) nucleotides. In the presence of H2O2 instead of cumene hydroperoxide, a lower level of adducts was obtained, both with DNA and dGMP. The concentrations of HRP and co-substrate used in this paper were higher than those used by other authors who obtained negative results when they sought DNA adducts of OTA in the presence of HRP and H2O2. The main adduct we obtained with DNA incubated with HRP and OTA had the same chromatographic behaviour as that obtained when DNA or dGMP were incubated with OTA, arachidonic acid and mice kidney microsomes. However, the main adduct obtained with dGMP incubated with HRP and OTA behaved differently. These results show that OTA can be metabolized by a peroxidase to metabolically activated species that bind covalently to DNA and dGMP; however, the main adduct obtained in vitro with HRP and dGMP cannot serve as a model for one of the adducts formed by OTA with DNA because it behaves differently in two chromatographic systems.     

Synthesis and characterization of oligodeoxynucleotides containing an N1 beta-hydroxyalkyl adduct of 2'-deoxyinosine.

Hydroxyethyl adducts arising by the reactions of simple epoxides at the N1 position of adenine nucleosides can deaminate to give the inosine analogues which, if formed in DNA, are suspected of being highly mutagenic. A method has been developed for synthesis of oligonucleotides containing N1-adducted 2'-deoxyinosines. The 2'-deoxyinosine adduct of 3,4-epoxy-1-butene was prepared from (+/-)-4-acetoxy-3-bromo-1-butene and tetraisopropyldisiloxanediyl-protected 2'-deoxyinosine with base. The 2'-deoxyinosine derivative was then incorporated into the oligodeoxynucleotide sequence 5'-d(CGGACXAGAAG)-3' (X = N1-(1-hydroxy-3-buten-2-yl)-2'-deoxyinosine).     

Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA: formation of cyclic deoxyguanosine adducts and formaldehyde cross-links

We investigated the reactions of formaldehyde plus acetaldehyde with dGuo and DNA in order to determine whether certain 1,N(2)-propano-dGuo adducts could be formed. These adducts-3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-(3H)-one (1) and 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (3a,b)-have been previously characterized as products of the reaction of acrolein with dGuo and DNA. Adduct 1 predominates in certain model lipid peroxidation systems [Pan, J., and Chung, F. L. (2002) Chem. Res. Toxicol. 15, 367-372]. We hypothesized that this could be due to stepwise reactions of formaldehyde and acetaldehyde with dGuo, rather than by reaction of acrolein with dGuo. The results demonstrated that adducts 1 and 3a,b were relatively minor products of the reaction of formaldehyde and acetaldehyde with dGuo and that there was no selectivity in their formation. These findings did not support our hypothesis. However, substantial amounts of previously unknown cyclic dGuo adducts were identified in this reaction. The new adducts were characterized by their MS, UV, and NMR spectra as diastereomers of 3-(2'-deoxyribosyl)-6-methyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (10a,b). Adducts 10a,b were apparently formed by addition of formaldehyde to N1 of N(2)-ethylidene-dGuo, followed by cyclization. An analogous set of four diastereomers of 3-(2'-deoxyribosyl)-6,8-dimethyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (12a-d) were formed in the reactions of acetaldehyde with dGuo. These products are the first examples of exocyclic dGuo adducts of the pyrimido[1,2-a]purine type in which an oxygen atom is incorporated into the exocyclic ring. Formaldehyde-derived adducts were the other major products of the reactions of formaldehyde plus acetaldehyde with dGuo. Prominent among these were N(2)-hydroxymethyl-dGuo (9) and the cross-link di-(N(2)-deoxyguaonosyl)methane (13). We did not detect adducts 1, 3a,b, or 10a,b in enzymatic hydrolysates of DNA that had been allowed to react with formaldehyde plus acetaldehyde. However, we did detect substantial amounts of the formaldehyde cross-links di-(N(6)-deoxyadenosyl)methane (17), with lesser quantities of (N(6)-deoxyadenosyl-N(2)-deoxyguanosyl)methane (18), di-(N(2)-deoxyguanosyl)methane (13), and N(6)-hydroxymethyl-dAdo (19). Schiff base adducts of formaldehyde and acetaldehyde were also detected in these reactions. These results demonstrate that the reactions of formaldehyde plus acetaldehyde with dGuo are dominated by newly identified cyclic adducts and formaldehyde-derived products whereas the reactions with DNA result in the formation of formaldehyde cross-link adducts. The carcinogens formaldehdye and acetaldehyde occur in considerable quantities in the human body and in the environment. Therefore, further research is required to determine whether the adducts described here are formed in animals or humans exposed to these agents.     

Formation of 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine in reactions of mucochloric acid with nucleosides.

Mucochloric acid, a genotoxic hydroxyfuranone found in chlorinated drinking water, was reacted with cytidine, adenosine, guanosine, and uridine. HPLC analyses with UV detection at 290 nm showed that one major product peak was formed in the reactions of cytidine, adenosine, and guanosine. The products formed in reactions carried out at 90 degrees C for 24 or 45 h were isolated by HPLC and characterized by 1H and 13C NMR spectroscopy, direct inlet chemical ionization mass spectrometry, and UV absorbance spectroscopy. The structures of the products were assigned to 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine. In reactions performed at 37 degrees C and pH 7.0 the same compounds were detected after a reaction time of 7 days. No observable reaction took place between mucochloric acid and uridine.     

Structure of the 1,N2-etheno-2'-deoxyguanosine adduct in duplex DNA at pH 8.6

The structure of the 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondG) adduct, arising from the reaction of vinyl chloride with dG, was determined in the oligonucleotide duplex 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCCATGCG)-3' (X=1,N(2)-epsilondG) at pH 8.6 using high resolution NMR spectroscopy. The exocyclic lesion prevented Watson-Crick base-pairing capability at the adduct site and resulted in an approximately 17 degrees C decrease in Tm of the oligodeoxynucleotide duplex. At neutral pH, conformational exchange resulted in spectral line broadening near the adducted site, and it was not possible to determine the structure. However, at pH 8.6, it was possible to obtain well-resolved (1)H NMR spectra. This enabled a total of 385 NOE-based distance restraints to be obtained, consisting of 245 intra- and 140 inter-nucleotide distances. The (31)P NMR spectra exhibited two downfield-shifted resonances, suggesting a localized perturbation of the DNA backbone. The two downfield (31)P resonances were assigned to G(7) and C(19). The solution structure was refined by molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints, using a simulated annealing protocol. The generalized Born approximation was used to simulate solvent. The emergent structures indicated that the 1,N(2)-epsilondG-induced structural perturbation was localized at the X(6).C(19) base pair, and its 5'-neighbor T(5).A(20). Both 1,N(2)-epsilondG and the complementary dC adopted the anti conformation about the glycosyl bonds. The 1,N (2)-epsilondG adduct was inserted into the duplex but was shifted towards the minor groove as compared to dG in a normal Watson-Crick C.G base pair. The complementary cytosine was displaced toward the major groove. The 5'-neighbor T(5).A(20) base pair was destabilized with respect to Watson-Crick base pairing. The refined structure predicted a bend in the helical axis associated with the adduct site.     

Untersuchungen an 1,3-Thiazinen, 11. Mitt. 3-Acyl-4-oxo-2-thioxo-tetrahydro-1,3-thiazine,neuartige cyclische N,N-Bis-acyl-dithiourethane

1,3-Thiazines, XI: 3-Acyl-4-oxo-2-thioxotetrahydro-1,3-thiazines, a new Type of Cyclic N,N-Bisacyldithiourethanes The 4-oxo-2-thioxotetrahydro-1,3-thiazines 1 and 2 were reacted with acid chlorides in the presence of triethylamine yielding the 3-acyl derivatives 3 and 4 . These compounds were shown by hydrolysis, alcoholysis and aminolysis to be acylating agents.     

In vivo formation of N7-guanine DNA adduct by safrole 2',3'-oxide in mice

Safrole, a naturally occurring product derived from spices and herbs, has been shown to be associated with the development of hepatocellular carcinoma in rodents. Safrole 2',3'-oxide (SFO), an electrophilic metabolite of safrole, was shown to react with DNA bases to form detectable DNA adducts in vitro, but not detected in vivo. Therefore, the objective of this study was to investigate the formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SFO-Gua) resulting from the reaction of SFO with the most nucleophilic site of guanine in vitro and in vivo with a newly developed isotope-dilution high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method. N7γ-SFO-Gua and [(15)N(5)]-N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine ([(15)N(5)]-N7γ-SFO-Gua) were first synthesized, purified, and characterized. The HPLC-ESI-MS/MS method was developed to measure N7γ-SFO-Gua in calf thymus DNA treated with 60μmol of SFO for 72h and in urine samples of mice treated with a single dose of SFO (30mg/kg body weight, intraperitoneally). In calf thymus DNA, the level of N7γ-SFO-Gua was 2670 adducts per 10(6)nucleotides. In urine of SFO-treated mice, the levels of N7γ-SFO-Gua were 1.02±0.14ng/mg creatinine (n=4) on day 1, 0.73±0.68ng/mg creatinine (n=4) on day 2, and below the limit of quantitation on day 3. These results suggest that SFO can cause in vivo formation of N7γ-SFO-Gua, which may then be rapidly depurinated from the DNA backbone and excreted through urine.