Conformational Preferences of Modified Nucleic Acid Bases N6-Methyl-N6-(N-Threonylcarbonyl) Adenine and 2-Methylthio-N6-(N-Threonylcarbonyl) Adenine by the Quantum Chemical PCILO Calculations

Abstract

Conformational preferences of the hypermodified nucleic acid bases N⁶-methyl-N⁶-(N- threonylcarbonyl) Adenine, m⁶ tc⁶ Ade, and 2-methylthio-N⁶-(N-threonylcarbonyl) Adenine, mS² tc⁶ Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m⁶ tc⁶ Ade and mS² tc⁶ Ade alike the threonylcar- bonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N⁶-(N-threonylcarbonyl) Adenine, tc⁶ Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc⁶ Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(l) of the adenine base.

The N⁶-methyl protons, in m⁶ tc⁶ Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS² tc⁶ Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N⁶-(Δ²-isopentenyl) Adenine, mS² i⁶ Ade, where the 2-methylthio group orients trans to the C(2)- N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Conformational preferences of anticodon 3'-adjacent hypermodified nucleic acid base cis-or trans-zeatin and its 2-methylthio derivative, cis-or trans- ms(2)zeatin

Conformational preferences of the hypermodified nucleic acid bases N6-(Delta(2)-cis-hydroxyisopentenyl)adenine, cis-io(6)Ade also known as cis-zeatin, and N(6)-(Delta(2)-trans-hydroxyisopentenyl)adenine, trans-io(6)ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms(2)io(6)Ade or cis-ms(2)zeatin, and trans-ms(2)io6Ade or trans-ms(2)zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AM1 and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io(6)Ade, trans-io(6)Ade, cis-ms(2)io(6)Ade and trans-ms(2)io(6)Ade are such that in each of these molecules the isopentenyl substituent spreads away (has "dista" conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io(6)Ade as well as cis-ms(2)io(6)Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io(6)Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms(2)io(6)Ade, possible O-H...S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms(2)io(6)Ade as well as trans-ms(2)io(6)Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this non-obstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i(6)Ade and ms(2)i(6)Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms(2)-zeatin (ms(2)io(6)Ade) modification.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.     

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.     

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).     

UV spectra of adenine methyl and glycosyl derivatives and their transformation induced by amino acid carboxylic groups

UV absorption spectra of adenine, adenosine and their methyl derivatives were studied in dimethylsuloxide (DMSO). Considerable changes in UV spectra of adenine under methylation at the 1 and 3 positions, and adenosine under methylation at the 1 position attested the essential structural reconstruction of adenine purine ring. Ade and m6Ade were shown to form complexes with deprotonated carboxylic group of amino acids (carboxylate-ion) through two H-bonds involving amino group and N7H imino group, tautomeric transition N9H-->N7H being initiated namely by interaction with carboxylate-ion. Considerable changes in UV spectra of m1Ade, m1A, and m3Ade under interaction with neutral carboxylic group of amino acids were interpreted as a result of proton transfer from amino acid to the base.     

Indolylacetic acid and N6-(delta 2-isopentenyl) adenine affect NADH binding to yeast alcohol dehydrogenase and inhibit in vitro the enzymatic oxidation of ethanol

Derivative UV spectroscopic data show that the plant growth substances N6-(delta 2-isopentenyl) adenine (i6Ade) and indolylacetic acid (IAA) can bind to the yeast alcohol dehydrogenase (ADH) and affect coenzyme-enzyme binding. This is confirmed by enzyme kinetics studies. At fixed ethanol concentrations (27.8 and 111.1 mM) and varying NAD+ concentrations (0.033-2 mM), as well as at fixed levels of coenzyme (0.67 and 2 mM), and at varying concentrations of ethanol (1.4-111.1 mM), the rate of ethanol oxidation is significantly inhibited by i6Ade and IAA. The kinetics of the ADH reaction is affected by two inhibition constants (KI and K'I) which correspond to the dissociation constants of complexes EI and ESI, respectively. For i6Ade the KI = 0.52 +/- 0.06 mM and K'I = 0.74 +/- 0.07 mM, and for IAA the KI = 0.88 +/- 0.03 mM and K'I = 0.99 +/- 0.02 mM.     

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.     

Chemical modification of N6-(N-threonylcarbonyl) adenosine. Part II. Condensation of the carboxyl group with amines.

Carboxyl group of N6-/N-threonylcarbonyl/adenosine was quantitatively modified with amines/aniline, glycine ethyl ester and ethylenediamine/in the presence of a water-soluble carbodiimide, yielding the respective amides. The reaction was carried out in a water solution of pH about 4 at 20 degrees C and was finished within minutes. The structure of the products was confirmed by UV and PMR spectra, and by chemical reactivity. Under conditions applied for modification of T6A, four common nucleosides and internucleotide linkage of UpA were unreactive, while 5'-AMP was transformed to the respective phosphoramides. At pH 4, the rate of 5'-AMP modification was over 100 times lower than the rate of t6A reaction.     

Stereospecific syntheses of 9-(S)-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA)

9-(S)-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) was prepared from 9-(S)-(2,3-dihydroxypropyl)adenine (DHPA) via its 3-O-chloromethanephosphonate. The latter compound is obtained by treatment of DHPA with chloromethanephosphonyl dichloride and the 3'-isomer separated from its 2'-congener by ion-exchange chromatography. The 3'-isomer is prepared selectively by the same method starting from 2',6-dibenzoyl derivative of DHPA. The 3'-ester is transformed to HPMPA by treatment with aqueous alkali. Alternatively, 9-(S)-(2-hydroxy-3-triphenylmethoxypropyl)-N6-benzoyladenine can be converted to HPMPA by reaction with dialkyl p-tolylsulfonyloxymethane-phosphonates in the presence of NaH followed by successive acid and alkaline treatment.     

N-6-(delta-2-isopentenyl) adenosine: hydrolysis by a mucleosidase isolated from Lactobacillus acidophilus cells.

A nucleosidase activity has been isolated from Lactobacillus acidophilus which rapidly hydrolyses N-6 (delta-2-isopentenyl) adenosine to its corresponding base, N-6(delta-2-isopentenyl) adenine. The activity can be distinguished from the spleen exzyme (EC. 2.4.2.1), a purine nucleoside transferase, on the basis of its substrate specificity, electrophoretic behavior, and nondependence on phosphate. The bacterial enzyme hydrolyzes both inosine and isopentenyl adenosine, giving Km values of 63.3muM and 177 muM respectively. The presence of this enzyme in bacteria counts for the rapid conversion of the parent nucleoside to isopentenyl adenine, which has been observed in these cells. The enzyme thus assumes importance as one of the catabolic activities available to the cell for metabolizing the cytokinin, N-6-(delta-2-isopentenyl) adenosine.     

Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6-15NH2 in adenine nucleotides

A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6-15NH2]adenine nucleotides is described. The method involves formation of the di-t-butyldimethylsilyl (TBDMS) derivative of adenine following isolation of the nucleotide fraction with solid-phase ion-exchange chromatography and subsequent acid hydrolysis of nucleotides to free base. Mass spectra for both adenine-diTBDMS and [6-15NH2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH2 moiety. The base peak (m/z 306) was formed by loss of C4H9 (57) and constitutes approximately one-third of the total ion current. Using selected ion monitoring of the m/z 306/m/z 307 ratio, levels of isotopic abundance of 1.0-50.0 mol% excess could be measured reproducibly with the injection of 10-20 pmol of the adenine-diTBDMS derivative obtained from isolated rat hepatocytes. Confirmation that measured isotopic abundance was referable to labeling of the 6-15NH2 group was obtained by oxidation of adenine to hypoxanthine and determination of enrichment in the hypoxanthine-diTBDMS derivative. The method was used to study the formation of [6-15NH2]adenine nucleotides during the incubation of isolated rat hepatocytes with [15N]alanine. A level of approximately 6.0 mol% excess was observed at 60 min incubation.     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

Determination of plant growth substances in liquid endosperm of immature walnut (Juglans nigra) nuts by an ELISA technique

Plant growth substances (PGSs) were analysed in liquid endosperm of black walnut using HPLC and an ELISA procedure. Of all the PGSs studied, we show no GA₃, low levels of cytokinins (io⁶A, i⁶Ade, i⁶Ado) and ABA, and very high level of IAAAbbreviations ABA Abscisic acid: - Ade Adenine: - GA₃ Gibberellic acid: - IAA Indole-3-acetic acid: - i⁶Ade N⁶(²-1) adenine: - i⁶Ado N⁶(²-isopentenyl adenosine: - io⁶A Zeatin riboside:     

Design of laser pulses for selective vibrational excitation of the N6-H bond of adenine and adenine-thymine base pair using optimal control theory

Time dependent quantum dynamics and optimal control theory are used for selective vibrational excitation of the N6-H (amino N-H) bond in free adenine and in the adenine-thymine (A-T) base pair. For the N6-H bond in free adenine we have used a one dimensional model while for the hydrogen bond, N6-H(A)...O4(T), present in the A-T base pair, a two mathematical dimensional model is employed. The conjugate gradient method is used for the optimization of the field dependent cost functional. Optimal laser fields are obtained for selective population transfer in both the model systems, which give virtually 100% excitation probability to preselected vibrational levels. The effect of the optimized laser field on the other hydrogen bond, N1(A)...H-N3(T), present in A-T base pair is also investigated.      

The crystal structure of N 6 -( 2 -isopentenyl)-2-methylthioadenine, a modified base of tRNA with cytokinin activity

The crystal structure of the title compound, a modified base of tRNA has been determined from three-dimensional x-ray diffraction data. The plane of the isopentenyl side chain is rotated 91° from the plane through the adenine system and the methyl thio group. The substituents on the adenine ring prevent N(1) from hydrogen bonding; the molecule exhibits instead two types of pairing arrangements, one of which is compatible with the Hoogsteen or “reversed” Hoogsteen pairing scheme.     

The reactions of oxo-osmium ligand complexes with isopentenyl adenine and its nucleoside.

We report syntheses of oxo-osmium(VI)bis(ligand) esters of N6-(delta2-isopentenyl) adenine (6-ipAde) and its nucleoside (IPA) which result from the addition of OsO4 to the double bond of the isopentenyl group. A study of the kinetics of these reactions shows that under typical conditions the rates of reaction relative to thymidine are as follows: for OsO4-pyridine: thymidine = 1; 6-ipAde = 4600: for OsO4-2,2'-bipyridyl: thymidine = 380; 6-ipAde = 8600; IPA = 8600. We also report syntheses of osmate esters of IPA in which the osmium is bonded through the 2'-and 3'-hydroxyl groups of the ribose residue.     

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a k_cat/K_M of 3.2 × 10⁷ and 3.0 × 10⁷ s^{? 1}M^{? 1}, respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site‐directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10‐fold decrease in k_cat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828–840. © 2016 Wiley Periodicals, Inc.     

Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3',4'-quinone. Implications for mutagenic activity

A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor initiation involves metabolic formation of 4-catechol estrogens from estradiol (E2) and/or estrone and further oxidation of the catechol estrogens to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal estrogen hexestrol-3',4'-quinone (HES-3',4'-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report here an additional similarity between the natural estrogen E2 and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the N7deoxyguanosine (N7dG) adducts formed by reaction of E2-3,4-Q or HES-3',4'-Q with dG. The half-life of the loss of deoxyribose from the N7dG adducts to form the corresponding 4-OHE2-1-N7Gua and 3'-OH-HES-6'-N7Gua is 6 or 8 h, respectively. The slow cleavage of this glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.