Synthesis and Biochemical Characterization of N1-, N2-,and N7-Guanosine Adducts of Butadiene Monoxide
Rebecca R. Selzer and Adnan A. Elfarra*
Department of Comparative Biosciences and Environmental Toxicology Center, University ofWisconsin, Madison, Wisconsin 53706
Received June 13, 1995X
1,3-Butadiene is a known rodent carcinogen, but the molecular mechanisms of its carcino-genicity are poorly understood. Butadiene monoxide (BM), a known mutagenic metabolite of1,3-butadiene, was previously shown to react with guanosine to yield twoN7-guanine adducts.In the present study, eight guanosine adducts of BM were purified and characterized asdiastereomeric pairs of N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1 and G-3), N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and G-5), N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7),and N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8) on the basis of stability studies andanalyses by UV, 1H NMR, and fast atom bombardment mass spectrometry. While the N7-adducts exhibited half-lives of approximately 50 (G-1 and G-3) and 90 h (G-2 and G-5) uponincubation for 192 h in 100 mM phosphate buffer (pH 7.4) at 37 °C, the N1- and N2-adductsremained stable. When guanosine was reacted with excess BM in phosphate buffer (pH 7.4)at 37 °C, adduct formation exhibited pseudo-first-order kinetics, with the N7-adducts beingformed approximately 10-fold more favorably than the N1- and N2-adducts. When incubationswere carried out at lower BM concentrations, the N7-adducts remained the major detectableadducts, but the N2-adducts were also detectable at equimolar BM and guanosine concentra-tions, and theN1-adducts were detectable at a 5-fold molar excess of BM. These results, whichprovide clear evidence that guanosine can be alkylated at multiple sites following 1,3-butadieneexposure, may aid in the development of useful biomarkers for exposure to 1,3-butadiene. Theresults may also contribute to a better understanding of the molecular mechanisms of 1,3-butadiene-induced carcinogenicity.
Introduction
Exposure of rats and mice to 1,3-butadiene, a chemicalused extensively in the industrial production of syntheticrubber and plastics and detected in automobile exhaustand cigarette smoke, resulted in the formation of tumorsat multiple sites (1, 2). Mice were considerably moresusceptible to 1,3-butadiene-induced carcinogenicity thanrats; however, the biochemical basis for this speciesdifference has not been determined. The InternationalAgency for Research on Cancer has classified 1,3-buta-diene as a “possible” human carcinogen (3), but a recentepidemiological study suggested a definitive increase inthe incidence of various lymphohematopoietic cancers inindustrial workers exposed to 1,3-butadiene (4). Inaddition, an increase in hprt mutant frequency in theperipheral lymphocytes of nonsmoking workers exposedto butadiene (3.5 ( 7.5 ppm) over controls was reported(5). While these results clearly show that current levelsof occupational exposure to 1,3-butadiene may not beadequate to protect workers from the mutagenic/carci-nogenic effects of 1,3-butadiene, the molecular mecha-nisms of 1,3-butadiene-induced carcinogenicity remainunclear.Butadiene monoxide (BM,1 3,4-epoxy-1-butene) is a
major metabolite of 1,3-butadiene both in vivo and invitro by microsomal monooxygenases in both mice and
rats (6-8) and with multiple human cytochrome P450enzymes and human myeloperoxidase (9, 10). Bothenantiomers of BM were formed in rat liver microsomalincubations (11). BM, a mutagen (12) and weak carcino-gen in mouse skin-painting studies (13), was postulatedto be responsible for the mutagenicity of 1,3-butadienein the S9 activated Salmonella typhimurium mutagenic-ity assay (14). Mutants isolated in mice exposed to 1,3-butadiene or BM were found to have both transversionand transition base pair substitutions, as well as both+1 and -1 frame shift mutations (15). In addition,activated K-ras genes were reported in a large numberof tumors induced by 1,3-butadiene exposure (16).Citti et al. studied the reaction of racemic BM with
guanosine in vitro, characterizing two products aftercleavage of the sugar moieties of the adducts (17). Theseadducts were identified as regioisomeric N7-(2-hydroxy-3-buten-1-yl)guanine and N7-(1-hydroxy-3-buten-2-yl)-guanine on the basis of UV and MS data. The NMRspectra of the isolated products were, however, notconclusive with regard to the regiochemistry of the twoproducts (17). Furthermore, because products were onlycharacterized after cleavage of the sugar moiety, thechemical stability of the modified nucleosides was notcharacterized. In addition, because diastereomeric prod-ucts were not resolved, the relative chemical reactivitiesof the two BM enantiomers were not determined.
* Corresponding author: Dr. Adnan A. Elfarra, Department ofComparative Biosciences, University of Wisconsin School of VeterinaryMedicine, 2015 Linden Dr. W., Madison, WI 53706; telephone, (608)262-6518; fax, (608) 263-3926; internet, [email protected].
X Abstract published in Advance ACS Abstracts, December 1, 1995.
1 Abbreviations: BM, butadiene monoxide; ACN, acetonitrile; FAB/MS, fast atom bombardment mass spectrometry; G-1 and G-3, dia-stereomeric N7-(2-hydroxy-3-buten-1-yl)guanosine; G-2 and G-5, dia-stereomeric N7-(1-hydroxy-3-buten-2-yl)guanosine; G-4 and G-7,diastereomeric N2-(1-hydroxy-3-buten-2-yl)guanosine; G-6 and G-8,diastereomeric N1-(1-hydroxy-3-buten-2-yl)guanosine.
In this study, eight BM-guanosine adducts weresynthesized, purified, and characterized as diastereo-meric pairs ofN7-(2-hydroxy-3-buten-1-yl)guanosine (G-1and G-3),N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 andG-5),N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7),andN1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8).The stability of these adducts in phosphate buffer (pH7.4) at 37 °C was characterized. In addition, the effectof BM concentration on adduct formation and the pseudo-first-order kinetic reaction rates were determined inphosphate buffer (pH 7.4) at 37 °C.
Experimental Procedures
Materials. Racemic BM, trifluoroacetic acid, and deuteriumoxide were obtained from Aldrich Chemical Co. (Milwaukee,WI). Guanosine was obtained from Sigma Chemical Co. (St.Louis, MO). HPLC grade acetonitrile (ACN) was obtained fromEM Science (Gibbstown, NJ). NMR supplies were obtained fromWilmad Glass Co. (Buena, NJ). All other chemicals were of thehighest grade commercially available. Caution: BM is aknown mutagen and carcinogen in laboratory animals and mustbe handled using proper safety measures.Synthesis of BM-Guanosine Adducts. Syntheses of N7-
adducts were carried out as described by Citti et al. (17), withsome modifications. Guanosine (56.6 mg, 0.2 mmol) was dis-solved in 3.5 mL of glacial acetic acid and heated at 60 °C, in a12 mL vial with a screw-top Teflon-coated cap, until theguanosine dissolved. A molar excess of BM (0.161 mL, 2 mmol)was added, and the solution was incubated in a Dubnoff shakingwater bath at 50 °C for 5 h. At 5 h the reaction mixture wasdiluted with an equal volume of acetone and four volumes ofethyl ether. The precipitate, separated by centrifugation anddissolved in 20 mM phosphate buffer (pH 5.5), was analyzedby HPLC. Four peaks, in addition to the starting material, wereresolved.During the time course of BM-guanosine adduct formation
under physiological conditions (see the following), four additionalpeaks were also found to increase with time and became thesole products as pH was increased from 7.4 to 10.0. These N1-and N2-adducts were synthesized by dissolving guanosine (30mg, 0.1 mmol) in 6 mL of 100 mM potassium phosphate buffer,adjusted to pH 10 with KOH and containing 100 mM potassiumchloride with heat (60 °C), and adding an excess of BM (0.3 mL,3.7 mmol). The reaction was incubated for 5 h at 37 °C. At 5h the reaction mixture was extracted twice with four volumesof ethyl ether to remove excess BM. The product was lyophilizedto dryness, dissolved in water, and analyzed by HPLC. Fourpeaks, in addition to guanosine, were resolved.HPLC Purification of BM-Guanosine Adducts. HPLC
separations of the crude reaction mixtures were performed witha 20 µL injection volume on a Beckman Ultrasphere 5 µm ODSreverse-phase analytical column (250 × 4.6 mm i.d.), using aBeckman gradient-controlled HPLC system (Irvine, CA) equippedwith a Beckman diode array detector (Model 168) and dual-channel UV detection at 260 and 280 nm. Use of a lineargradient program starting at 10 min from 0% to 100% pump Bover 8 min [pump A, 1% ACN (pH 2.5); pump B, 10% ACN (pH2.5)], at a rate of 1 mL/min, resolved the four major peaks fromeach reaction mix, whose retention times on a typical chromato-gram were 22.26, 24.38, 24.77, and 26.53 min for theN7-adductsand 25.53, 27.99, 28.54, and 29.28 min for the N1- and N2-adducts (Figure 1). These peaks were assigned as G-1 throughG-8 in the order of their retention times, respectively. Thepurified alkylated guanosines (see the following) were used toprepare standard curves. The limit of detection attained bystandard curves (0.1-1000 µg/mL) by using this HPLC methodwas 0.25 µg/mL (r values were greater than 0.999) for alladducts.Bulk separation of crude material was accomplished with a
0.5 mL injection volume on a Beckman Ultrasphere 5 µm ODSreverse-phase semipreparative column (250 × 10 mm i.d.) withthe mobile phases described earlier and a linear gradientstarting at 10 min and going from 0% to 75% for pump B over
15 min at a flow rate of 3 mL/min. Adducts were collected fromthe two separate reaction mixes by using a Gilson 203 fractioncollector (Middleton, WI). Peak solutions were fractionatedtwice to obtain greater than 95% purity, as determined byHPLC, and lyophilized to dryness.Identification of BM-Guanosine Adducts. UV spectra
of each peak were obtained with a Beckman diode array detectorat pH 2.5 in the mobile phase described earlier. Positive ionfast atom bombardment mass spectra (FAB/MS) were obtainedby using a Kratos MS-50 ultrahigh-resolution mass spectrom-eter fitted with a Kratos DS-55 data system (Manchester, UnitedKingdom). An Ion Tech FAB gun utilizing xenon as the FABgas was used with a direct insertion FAB probe. Approximately50-100 µg of each adduct was analyzed with a spectrum rangingfrom m/z 50 to 400, using a 3-nitrobenzyl alcohol or glycerolmatrix. All spectra are matrix subtracted. Proton NMR spectraof all eight adducts, dissolved in D2O at concentrations rangingfrom 3 to 15 mg/mL, were obtained on a Bruker spectrometer(Karlsruhe, Germany) at 400 or 500 MHz. N1 and N2-adductspectra were performed at 5 °C in order to shift the water peakdownfield, away from several sugar peaks. Decoupling experi-ments were performed on G-7 and G-8 to confirm protonassignments. All four N1- and N2-adduct NMR spectra werealso obtained in Me2SO-d6 under the same conditions. Chemicalshifts are reported in ppm with the H2O peaks as an internalstandard.BM-Guanosine Adduct Formation at Physiological
Conditions. The reactions of BM and guanosine were carriedout in 4 mL vials capped with Teflon septa. Guanosine (10 mM)and BM (10, 25, 50, and 500 mM, added to the vial viamicrosyringe injection through the septum) were reacted in 3mL of 100 mM phosphate reaction buffer (pH 7.4) containing100 mM potassium chloride in a shaking water bath at 37 °C.Reactions were stopped at 60 min by four consecutive extrac-tions with two volumes of ethyl ether each to remove unreactedBM. Samples were then analyzed for the presence of adductsby HPLC, as described earlier.For the pseudo-first-order kinetic experiment, guanosine (3.1
or 9.6 mM) and BM (750 mM) were reacted as described earlier.Lower concentrations of BM (188 mM) did not produce a linearresponse, possibly due to extensive voltilization and decomposi-tion of BM (8). At the 750 mM BM concentration used in thekinetic study, a large excess of BM should still exist at the endof the reaction, on the basis of the expected loss of BM due tovolatilization and decomposition. Samples (0.25 mL), with-drawn at timed intervals by syringe to minimize the loss of BM,were extracted three times with four volumes of ethyl ether (1mL) to remove unreacted BM before analysis by HPLC wascarried out as described earlier.Controls were performed to determine whether the ether
extraction had an effect on adduct recovery. Purified adductwas dissolved 1 mg/mL in 100 mM phosphate reaction bufferand diluted with buffer to concentrations ranging from 5 to 1000µg/mL. Six replicate samples were prepared at each concentra-tion: three were extracted with ether as described earlier andthe other three remained unextracted. Samples were analyzedby HPLC to determine adduct recovery. All samples showedgreater than 90% recovery.Guanosine Adduct Stability Studies. All eight purified
adducts were dissolved individually (100 ppm) in 100 mMphosphate buffer (pH 7.4) containing 100 mM potassiumchloride. These solutions were placed in a shaking water bathat 37 °C; samples were withdrawn at 24 h intervals andanalyzed by HPLC, under the conditions described earlier, todetermine the disappearance of the parent adduct peaks.In other experiments, 3 mL of synthetic N7-adducts (2 mM)
were either boiled for 30 min or incubated at 80 °C with 0.6 mLof 0.1, 1, or 2 N HCl. Aliquots of the acid-incubated solutionswere removed at 5, 90, or 300 min, neutralized with NaOH, andanalyzed by HPLC, using the conditions described earlier, forboth the disappearance of the parent guanosine adduct peaksand the formation of the corresponding guanine adduct peaks(G-1 or G-3 hydrolysis resulted in the detection of a new peakat 18.97 min, and G-2 or G-5 hydrolysis resulted in the detection
of a new peak at 20.76 min). The guanine adduct peaksexhibited UV spectra similar to those reported by Citti et al.(17).Synthetic N1- and N2-adducts were dissolved in 1 M KOH
and incubated at 95 °C for 2 h. Aliquots were withdrawn every30 min, and the pH was adjusted to 2.5 with HCl andimmediately analyzed by HPLC for the disappearance of theparent adduct peaks.
Results
Reaction of racemic BM with guanosine under physi-ological conditions yields eight products, G-1, G-2, G-3,G-4, G-5, G-6, G-7, and G-8 (Figure 1), which wereseparated by HPLC. With absorbance maxima of 260 nmfor G-1 and G-3, 258/260 nm for G-2 and G-5, and 258nm for G-4, G-6, G-7, and G-8, the UV maxima andspectral shapes (data not shown) suggested that all eightproducts were nitrogen-substituted guanosine adducts(18). Identification of the isomers was achieved byexamination of their proton NMR and FAB/MS spectra.Proton NMR spectra of the eight products show the
presence of both the guanosine and BM moieties. Theassignment of protons (Table 1, Figures 2 and 3) wasachieved by comparing the chemical shifts, multiplicities,and integration ratios of the protons in the products withthe published spectra of guanosine (19) and the spectraof 3-butene-1,2-diol, as well as by several decouplingexperiments (data not shown).The differentiating assignments between regioisomers
of the N7-adducts (Figure 3) were based on the expectedgreater downfield chemical shifts of the hydrogens ad-jacent to the positively chargedN7 of guanosine comparedto the hydrogens adjacent to the hydroxyl group. Theproton NMR spectra of G-1 and G-3 appeared nearlyidentical, as did the spectra of G-2 and G-5. Onerepresentative spectrum for each regioisomer is given inFigure 2, while the chemical shifts and J values for allcompounds are given in Table 1. The relative downfieldshifts of the two protons, assigned to H9, in G-1 (4.36,4.50 ppm) and G-3 (4.29, 4.55 ppm) led to the conclusionthat the attached carbon was adjacent to the N7 ofguanosine, identifying these products as the diastereo-mers ofN7-(2-hydroxy-3-buten-1-yl)guanosine (Figure 3).The chemical shifts of the H9 protons of G-2 (3.92, 3.99ppm) and G-5 (3.96, 4.01 ppm) were smaller than thoseof the H9 protons of G-1 and G-3, suggesting that this
carbon was adjacent to the hydroxyl group. These twoadducts also contain a single proton doublet of triplets,H3, shifted downfield (5.43 and 5.48 ppm, respectively),suggesting that these products have a single proton onthe carbon attached to theN7 of guanosine and thus wereidentified as the diastereomers ofN7-(1-hydroxy-3-buten-2-yl)guanosine (Figure 3). Further evidence for thesestructural assignments was provided by the relativedownfield shifts of the assigned H1 protons of G-2 (6.02ppm) and G-5 (6.05 ppm) compared to those of G-1 (5.82ppm) and G-3 (5.81 ppm) as the H1 protons of G-2 andG-5 are in closer proximity to the deshielding N7 ofguanosine. Assignment of protons from the sugar moi-eties was confirmed by comparison to the decouplingexperiments of G-7 and G-8 to follow. The absence of asignal for the C8 proton in all N7-adduct spectra isconsistent with the previous observation that the C8
protons ofN7-alkylguanosines rapidly exchange with thedeuterium of the solvent (20).G-4 and G-7 were determined to be diastereomeric
pairs of a single regioisomer by nearly identified protonNMR spectra (Figure 2C, Table 1). The adducts weredetermined to be N2-substituted guanosine adducts onthe basis of the presence of only one of the twoN2 protonsat 6.6 ppm when the NMR was performed in Me2SO-d6(data not shown). The alkylation with BM was deter-mined to have taken place at the carbon adjacent to thedouble bond of the BM moiety, as evidenced by thesplitting of the remaining N2 proton into a doublet at 6.6ppm when the spectra of G-4 and G-7 were obtained inMe2SO-d6 (data not shown). This demonstrates couplingto only a single proton on the adjacent carbon, and thusG-4 and G-7 were identified as the diastereomers of N2-(1-hydroxy-3-buten-2-yl)guanosine. Further evidence forthe proton assignments in D2O was obtained by homo-nuclear decoupling of the G-7 sample. Saturation of H1
at 5.87 ppm was used to identify H3 at 4.62 ppm.Saturation of H3, in turn, identified the H9 protons at3.67 and 3.75 ppm. Saturation of H8 (4.13 ppm) of thesugar moiety helped to determine the signals of the H10
protons, while other sugar protons were identified bycomparison with decoupling experiments performed onG-8.Further evidence for the identity of G-4 and G-7 asN2-
adducts was their stability under alkaline conditions. Ifalkylation occurred at the N1, N3, O6, or N7 positions ofguanosine, the adducts would be degradable by incuba-tion in 1 M KOH (100 °C, 2 h); however, adducts attachedat the exocyclic amino groups of purines are stable underthese conditions (21). In this study, the stability ofadducts G-4 and G-7 for 2 h under these conditions (datanot shown) provides further evidence for adducts withsubstitutions at the exocyclic amino group. As expected,adducts G-6 and G-8 were found to be unstable underthese conditions and were fully degraded after 30 min.G-6 and G-8 were identified as N1-substituted gua-
nosine adducts on the basis of NMR and UV data. Again,diastereomeric pairs of a single regioisomer are evidentdue to the near identity of the two spectra (Figure 2D,Table 1). Proton assignments were confirmed by severalhomonuclear decoupling experiments on G-8. Saturationof H3 of G-8 was used to identify H9 and confirm H1.Saturation of various signals originating from the sugarmoiety were also confirmatory. Saturation at 3.85 ppm(H10) affected the signals at 4.24 ppm (H8), saturation at4.76 ppm (H6) affected signals at 4.42 (H7) and 5.91 ppm(H2), and saturation at 4.42 ppm (H7) affected signals at4.24 (H8) and 4.76 ppm (H6).
Figure 1. HPLC chromatogram of the eight products formedby reaction of racemic BM with guanosine. Peaks G-1 and G-3were identified as diastereomers of N7-(2-hydroxy-3-buten-1-yl)-guanosine, G-2 and G-5 as diastereomers of N7-(1-hydroxy-3-buten-2-yl)guanosine, G-4 and G-7 as diastereomers of N2-(1-hydroxy-3-buten-2-yl)guanosine, and G-6 and G-8 asdiastereomers of N1-(1-hydroxy-3-buten-2-yl)guanosine.
The loss of the N1 proton present at 10.5 ppm in theN2-adduct spectra in Me2SO-d6 suggested alkylation attheN1 position (data not shown). In addition, UV spectramatch well with other alkylated N1-guanosine adducts,but not with O6-guanosine adducts (18). When G-6 andG-8 are incubated with 0.1 N HCl at 80 °C, the resultinghydrolysis products do not have the UV spectral shapeand λmax characteristic of N3-alkylated guanines (λmax )263 nm), but rather match those of other N1-alkylatedguanines (λmax ) 252 nm). While the alkylation site onthe BM moiety cannot be ascertained from the NMRspectra, the structures of G-6 and G-8 are tentativelyassigned as the diastereomers of N1-(1-hydroxy-3-buten-2-yl)guanosine on the basis of the following criteria.First, the N2-regioisomers formed under the same condi-tions reacted at the carbon adjacent to the double bondof the BM moiety as evidenced by the NMR results.Second, styrene oxide, an analogue of BM, was reportedto react at the N1 and N2 positions of guanosine to yieldadducts only at the carbon adjacent to the benzene ring(22-24).Mass spectra of all adducts (G-1 to G-8) provided
further evidence for their identity. Mass spectra for eachof the N7-adducts did not yield the expected molecularion (m/z 354 for M, as the N7-adduct already carries apositive charge); however, a fragment at m/z 222,consistent with the loss of the sugar moiety (Figure 4),was seen with all four N7-adducted compounds. This isa common fragmentation pattern of N7-guanosine ad-ducts due to the quaternary nitrogen atN7, which allowsthe adduct to fragment into an ion at BH+ (where B isthe base) rather than the BH2
+ fragment normallyobserved with DNA adducts. G-2 and G-5, as opposedto G-1 and G-3, exhibited a prominent guanine fragment(m/z 152; Figure 4C,D), possibly because of facile cleav-age of the linkage between the allylic carbon of the BMmoiety of G-2 and G-5 and the N7 of the guanine moiety.The G-2 diastereomer shows an m/z 192 fragmentcorresponding to the loss of a CH2dO moiety from themolecule. The fragments at m/z 110 and 124, whichwere observed with G-1, G-2, and G-3, correspond to thepyrimidine and the pyrimidine plus one nitrogen atom
from the adjacent imidazole ring, respectively. The G-5diastereomer exhibited anm/z 136 fragment correspond-ing to the loss of the exocyclic amino group from theguanine fragment (m/z 152).G-4, G-6, G-7, and G-8 adducts did yield the expected
molecular ion (m/z 354 for M + 1) in the mass spectra(Figure 5). The m/z 222 fragment corresponding to theloss of the sugar plus one proton appeared in all fourspectra. The N2-isomers contained anm/z 185 peak notevident in the N1 spectra, which may correspond to theloss of two molecules of water, in addition to the loss ofthe sugar moiety. All four adducts again showed them/z152 peak representing the guanine moiety. Peak G-8 alsocontained m/z 337, which constitutes the loss of ahydroxyl group from the M + 1 peak, and m/z 376,corresponding to the M + Na peak.When guanosine (10 mM) was reacted with varying
amounts of BM (10, 25, 50, and 500 mM) for 1 h in 100mM phosphate buffer (pH 7.4) at 37 °C, the N7- and N2-adducts were detected at all BM concentrations (Figure6). However, the levels of the N7-adducts detected weresignificantly higher than those of the N2-adducts. TheN1-adducts were detected only at 50 (5-fold molar excess)and 500 (50-fold molar excess) mM BM. Concentrationsof BM lower than 10 mM did not lead to the detection ofany of the N1- and N2-guanosine adducts by the HPLCmethod, as the amounts of adducts formed were belowthe limits of detection. At an unlimiting BM concentra-tion (750 mM), product formation exhibited linear timedependency for all adducts, from 0 to 90 min, whetherreactions were carried out at 3.1 or 9.6 mM guanosine(Figure 7). Indeed, the observed linear increase in ratewith increasing guanosine concentration provided evi-dence for the reaction being carried out under pseudo-first-order kinetics. The calculated rate constants for theformation of G-1, G-2, G-3, G-4, G-5, G-6, G-7, and G-8were 2.63 × 10-2, 2.61 × 10-2, 2.48 × 10-2, 3.26 × 10-3,2.54 × 10-2, 3.23 × 10-3, 4.73 × 10-3, and 3.53 × 10-3
h-1, respectively.The stability of all of the guanosine adducts was
investigated by determining the percent loss of the parentadduct after incubation in phosphate buffer adjusted to
Table 1. 500 MHz 1H NMR Data for Guanosine Adducts of BM in D2O (J Values Reported in Hertz)
a Proton numbering refers to assignments given in Figure 3. Multiplicities can be found on the spectra in Figure 2. b NA: J values notavailable from spectra. c Two chemical shifts are given corresponding to the two protons of the methylene group. d Two J values aregiven corresponding to the two protons of the methylene group.
pH 7.4 at 37 °C. The N1- and N2-adducts appeared to becompletely stable for the 192 h of incubation, while N7-adducts had half-lives of approximately 50 h for G-1 andG-3 and 90 h for G-2 and G-5 (Figure 8). Hydrolysis ofN7-adducts to the guanine adduct can be enhanced byhigh temperatures and acidic conditions. For example,boiling of the N7-guanosine adducts for 30 min at pH 7.4resulted in partial loss (65%) of the sugar moiety. Whenthe guanosine adducts were dissolved with 0.1 N HCl andsamples were heated for 5 min at 80 °C, virtually noguanine adduct was produced. However, the guanosineadducts were fully converted to the guanine adducts byincubation with 1 N HCl at 80 °C for 90 min.
Discussion
The results presented in this article demonstrate thatracemic BM reacted with guanosine to yield diastereo-meric pairs ofN7-(2-hydroxy-3-buten-1-yl)guanosine (G-1and G-3),N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and
G-5),N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7),andN1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8).The N7-adducts exhibited half-lives ranging from 48 to96 h at 37 °C in phosphate buffer (pH 7.4), which is
Figure 2. 500 MHz proton NMR spectra for (A) N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1), (B) N7-(1-hydroxy-3-buten-2-yl)-guanosine (G-5), (C) N2-(1-hydroxy-3-buten-2-yl)guanosine (G-7), and (D) N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6). Peaknumbers correspond to assignments in Figure 3.
Figure 3. Chemical structures of deuterated (A)N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and G-5), (B) N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1 and G-3), (C)N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8), and (D) N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7). Proton numbering is based onchemical shifts of the first spectrum solved (G-5).
Figure 4. FAB/MS of the diastereomers of N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1 and G-3) and N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and G-5). The peak atm/z 222 correspondsto the peak expected from the sugar-cleaved guanine adduct:(A) G-1; (B) G-3; (C) G-2; (D) G-5.
similar to the 50 h half-life reported by Citti et al. forthe spontaneous depurination of these guanine adductsin DNA (17). The N1- and N2-adducts remained stableunder these conditions, as is typical for alkylationproducts formed at these positions.When adduct formation was monitored over a range
of BM concentrations, N7-adducts were detected atequimolar BM and guanosine concentrations. The N2-adducts were also detected under these conditions, butthe N1-guanosine adducts were not detected until BM
concentrations were 5-fold greater than that of gua-nosine. The roughly linear formation of adducts over theBM concentration range tested and the consistent ratiosof formation of the individual adducts suggest that thereis no threshold BM concentration for N1-, N2-, and N7-adduct formation near the range tested. The inabilityto detect theN1-adducts at BM concentrations lower than50 mM is a consequence of the detection limit of theassay.Adduct formation in the presence of excess BM exhib-
ited pseudo-first-order kinetics, but different rates wereobserved for the formation of N7- vs N1- and N2-adducts.Similar to the results obtained at lower BM concentra-tions (Figure 6), the N7-adduct rates of formation wereapproximately 10-fold higher than those of theN1- orN2-adducts (Figure 7). These results provide evidence formultiple reaction sites for BM with guanosine and
Figure 5. FAB/MS of the diastereomers of N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7) and N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8). The peak atm/z 354 correspondsto the expected M + 1 peak: (A) G-4; (B) G-7; (C) G-6; (D) G-8.
Figure 6. Effect of BM concentration on the formation of eightBM-guanosine adducts. BM was reacted with 10 mM gua-nosine for 60 min at 37 °C in phosphate buffer (pH 7.4). Valuesrepresent the means ( SD of the results obtained from threeexperiments.
Figure 7. Rates of BM-guanosine adduct formation at pH 7.4at 37 °C. To guarantee pseudo-first-order kinetic conditions,the BM concentration (750 mM) was much higher than theguanosine concentrations (A, 3.1 mM; B, 9.6 mM). Valuesrepresent the results obtained from several experiments.
Figure 8. Stability of BM-guanosine adducts in 100 mMphosphate buffer containing 100 mM KCl (pH 7.4) at 37 °C.
indicate similar chemical reactivity of the two BMenantiomers.While the N7-adducts were the major adducts formed
when guanosine was reacted with BM, the formation ofN1- and N2-adducts may also be of toxicological signifi-cance. The N1 and N2 positions of guanosine/2′-deoxy-guanosine are involved in the hydrogen bonding of nucleicacids. In addition, our results showed that these adductsare far more stable than the N7-adducts, in the absenceof enzymatic repair. For many chemicals, studies havedemonstrated the rapid repair of N7- and N2-adducts inhuman cells (25, 26). However, much less has beenreported about the repair ofN1-guanosine adducts. Thus,it is possible that the formation of N2- and N1- BM-guanosine adducts could be at least partially responsiblefor the mutagenicity/carcinogenicity of 1,3-butadiene.Detection of N7-guanosine-BM adducts by 32P-post-labeling methods has been unsuccessful (27). Thus, theidentification of N1- and N2-guanosine adducts may alsobe important for the use of these sensitive biomonitoringtechniques.In conclusion, this article presents conclusive evidence
for guanosine alkylation at multiple sites by BM. WhiletheN7-adducts were formed preferably to the N1- andN2-adducts, the N1- and N2-adducts were much more stablethan theN7-adducts. Identification of these adducts maycontribute to a better understanding of the molecularmechanisms of 1,3-butadiene-induced carcinogenicity.
Acknowledgment. The authors thank Dr. LauraLerner for helpful comments. This research was sup-ported by NIH Grant ES06841 from the National Insti-tute of Environmental Health Sciences. R.R.S. wassupported by a National Science Foundation GraduateResearch Fellowship. NMR spectra were determined atthe National Magnetic Resonance Facility at Madison,WI, which is supported in part by NIH Grant RR02301.
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