Mechanism of Formation of Ethenoguanine Adducts from 2

Chem. Res. Toxicol. 1994, 7,205-208

205

Mechanism of Formation of Ethenoguanine Adducts from 2-Haloacetaldehydes: 13C-LabelingPatterns with 2-Bromoacetaldehyde F. Peter Guengerich' and Magnus Persmark Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received November 19,1993"

The mechanism of formation of etheno (e) adducts of nucleic acid bases from 2-haloacetaldehydes is generally assumed to occur via initial Schiff base formation resulting from reaction of the aldehyde with an exocyclic amine. We recently revised the IH NMR assignments of the E protons of 1,N2-c-Guo (Guengerich, F. P., Persmark, M. P., and Humphreys, W. G. (1993) Chem. Res. Toxicol. 6,635-648). In that work we also observed a facile and specific exchange of €37 of 1,N 2-c-G~o and H5 of N 2,3-c-G~a with H20. These findings raise questions about the mechanistic conclusions reached on the basis of labeling studies with deuterated ClCHzCHO (Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) J. Org. Chem. 42,3292-3296). BrCH2WHO was prepared from BrCHz13CO~Hand used to prepare 1,N 2-c-G~o(from Guo) and 0 6ethyl-N2,3-c-Gua (from 0 6-ethylGua). The positions of the labels were determined by 1H NMR spectroscopy experiments to be adjacent to the original Gua N2 (exocyclic) atom in both cases, Le., a t C6 in both c products. The labeling patterns are consistent with a mechanism involving initial Schiff base formation from the N2 atom and the aldehyde and subsequent nucleophilic attack of an endocyclic nitrogen on the methylene carbon.

Introduction

cations of the basic strategy involving2-haloacetaldehydes have been published for 1,N %-Guo (31)and N2,3-c-Guo (32).In all of the studies the general mechanism of c adduct formation from aldehydes is considered to involve the initial formation of a Schiff base between the carbonyl and an exocyclic amino group of the nucleic acid base (2, 16,18-27,30).In the case of the formation of l,N6-c-Ade, the mechanism is based upon trapping of N-methyl However, products and deuterium labeling (2,12,33,34). in the case of the e-Gua products, the only basis for the mechanism is labeling studies with ClC2H&H0 (2,30). Sattsangi et al. (30)reported that the upfield (6 7.4) e proton of 1,N 2-e-G~o was reduced in intensity when the synthesis was done using ClC2HCH0 and used this observation as support for the mechanism mentioned above. They assigned this 6 7.4 (upfield) proton as H7 (Chart 1). However, in a recent investigation on the mechanism of formation of c-Gua adducts from 2-halooxiranes, we reassigned the upfield proton (6 7.4) as H6 and the downfield (6 7.6) proton as H7 (Chart 1) (12). The nuclear Overhauser effects with the proton on N5 and the I3Csplitting patterns in [5-l6N1-1,N2-cGuoappear to yield an unambiguous solution (12).The methylene protons of ClCHzCHO are inherently exchangeable with HzO, particularly at slightly basic pH. Moreover, we also showed the facile exchange of the H7 proton in acid and base (12). Our NMR assignments of 0 6-ethyl-N2 , 3 - ~ - Gwere ~ a in agreement with the original report of Sattsangi et al. (301, but in the case of 1,N 2-E-GUO,the previous deuterium work suggested a mechanism of formation of 1,N2-r-Gua not involving the Schiff base between the Gua N2 atom and the carbonyl. Thus the mechanism of 1,N2-e-Gua formation is not clear (Scheme 1). In order to address the question we prepared B ~ C H Z ~ ~ Cand H Oexamined the 1,NZ-e-Guo and (0e-ethyl)-N 2,3-e-Gua products by lH NMR analysis.

The etheno ( e ) l and related extracyclic DNA adducts are the subject of considerable interest (1).2 They are formed from a number of chemical carcinogens, and some have been shown to be mutagenic in studies with defined oligonucleotides [e.g., N 2,3-e-Gua (4, 5 ) and 3,N 4-e-cyt (S)]. Some also appear to be highly persistent in DNA (7) and may pose greater dangers as a result. Our own interest in the 6 adducts has been in relationship to the adducts derived from vinyl halides. In that setting there is considerable evidence to support the view that the e and other DNA adducts are derived directly from the 2-halooxiranes as opposed to the 2-haloacetaldehyde5, which are formed from the oxiranes (8-12). However, DNA can also react with 2-haloacetaldehydes to form c adducts. The in vivo relevance of this reaction has not been demonstrated, but 2-chloroacetaldehyde has been shown to react in vitro with DNA to form N 2,3-e-Gua (13)and 1,N2-cGua (14). The literature contains reports of the formation of e and propeno and related DNA adducts from other compounds, including glyoxal (151,acrolein (161,2-cyanoethylene oxide (17), malonaldehyde and its derivatives (18-21),crotonaldehyde (22,23), and hydroxyalkenals and their epoxides (22,24-27).Although the mechanisms are not well defined, the recent reports of the formation o f t adducts from mucochloric acid (28)and lipid peroxidation products (29)are also of note. The syntheses of the two e-Gua derivatives (1,N 2-c and N2,3-c) were reported by Sattsangi et al. (30). Modifi-

* Author to whom correspondence should be addressed.

Abstract published in Advance ACS Abstracts, March 1, 1994. etheno. For a list of standard abbreviations of nucleosides and their bases now used in this journal, see: Chem. Res. Toxicol. (1994)7 , 7A. * For reviews of the chemistry and biochemistry of the c adducte, see refs 2 and 3. 1 Abbreviations: t,

0893-228x/94/2707-0205$04.60/0

Q

1994 American Chemical Society

206 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

Chart 1. Structures of e-Gua Compounds

B

H

Rib 1,N2

-

~

-

~

~

~

0

N 2,3-~-Gua

Scheme 1. Previous Labeling Patterns for t-Gua Derivatives Formed from ClC2H2CH08 OR

I

9%

H

\

9

0

II

a Path b indicatespotential reactions unresolved by previous NMR assignments.

Experimental Procedures Caution! BrCHzCHO is an alkylating agent and should be used in sealed vessels or with appropriate ventilation. Chemicals. 0 6-EthylGua was prepared by Dr. W. G. Humphreys (formerly of this laboratory) using a described procedure (35).Guo (Sigma Chemical Co., St. Louis, MO) was used without further purification. BrCHzWOzH (99% 13C atomic excess) was obtained from Cambridge Isotopes (Cambridge, MA). The site of labeling was verified by NMR analysis: 'H NMR (C2HC13)b 3.88 (s,2H, -CHz-, JCH = 4.7 Hz). BrCHZW02H (5.0 g, 36 mmol) was stirred in 50 mL of fresh (CzH&O under dry Nz, and a 1.5 molar excess of LiAlH4 (1.54 g, 40 mmol) in (CzH5)ZO was added dropwise over 30 min, at a rate sufficient to maintain gentle reflux. The reaction proceeded with stirring for an additional 1h, after which it was stopped by the addition of a few milliliters of ethyl acetate and then80mLof 10%H a 0 4 (v/v). Theaqueousphasewasextracted with (CzH&O four times, and the combined organic extracts

Guengerich and Persmark were dried with anhydrous MgSO4, filtered through paper, and carefully concentrated in vacuo to yield 1.7 g (38%) of BrCHzWH20H: lH NMR (C2HC13)6 1.31 (d oft, 2H, -CHzBr, J = 4.4 Hz), 3.76 and 4.12 (d oft, 2H, -WHzOH, JHH = 5.7 Hz, JCH = 145 Hz). BrCH2l3CH20H(1.65 g, 13 mmol, from above) was dissolved in 5 mL of CHzClz and added to a stirred suspension of 4.23 g (20 mmol) of pyridinium chlorochromate in 50 mL of CHzClZ. The mixture was stirred for 1.5 h at room temperature, during which the color changed from orange to greenish black. The liquid was decanted, and the residue was washed three times with 15 mL of (CzH&O. The combined CHZC12 and (CzH6)zO extracts were mixed and percolated through a 2- X 12-cmFlorisil column [poured from (CzH5)20], which was then washed with (CzH5)zO. The filtrate was carefully reduced to an -10-mL volume in vacuo (30 "C) and extracted with severalsmall portions of 5 mM HzSO4 to give a final volume of 22 mL of the desired BrCHzWHO solution. The concentration of BrCHzCHO in the solution (7.9 mM) was determined by measuring the oxidation of NADH ( € 9 ~=) 6.22 cm-l mM-l) in the presence of excess horse liver alcohol dehydrogenase (9). An aliquot was used to form a 2,4-dinitrophenylhydrazone(36),which did not yield M + H ions (mlz 304,306) in MS (+ fast atom bombardment) but did give the M - Br ion (mlz 225, 48% relative abundance). Formation of e Products. (A) l,N*--t-Guo(30). Guo (47 mg, 0.17 mmol) was mixed with 7 mL of 7.9 mM aqueous BrCHzW H O and 20 mL of 1.0 M sodium acetate buffer (pH 6.4). The mixture was adjusted to pH 6.4, homogenized using a sonicator probe (Branson Sonic Power Co., Danbury, CT, in an amber vial), and sparged with Ar. The vial was sealed with a Teflon liner and shaken in a gyrorotary water bath (100 rpm) at 37 "C ~ min) were for 4 days. Guo (tR 3.9 min) and 1 32-e-Guo( t 6.0 separated on a Beckman Ultrasphere semipreparative octadecylsilane HPLC column (10 pm, 10 X 250 mm, Beckman, San Ramon, CA) using a solvent mixture composed of 22 % CHsOH (v/v) in 50 mM NHIHCOz buffer (pH 5.5) (flow rate 4.0 mL o 515 pg (1.68 fimol), based min-l). The yield of 1 32 - t - G ~was upon spectral analysis (30): UV ,A, 281,226 nm (pH 7.0), 306, 279 nm (pH 11). Salts were removed by repeated lyophilization from HzO. (B) 0 6-Ethyl-N2,3-Gua(30). 0 6-EthylGua (12.8 mg, 0.07 mmol) was dissolved in a mixture of 7 mL of 7.9 mM for BrCH2WHO, 1.0 mL of 1.0 M sodium acetate buffer (pH 4.5), and 5 mL of CH30H. The solution (in amber glass) was sparged with Ar, and the vial was sealed and shaken in a gyrorotary water bath (100 rpm) at 37 "C for 4 days. Separation of 0 6-ethylGua and the product 0 6-ethyl-N2,3-e-Gua was done using the HPLC system described above, except that the CHsOH concentration was reduced to 18% (v/v). Both AB, and Fz25/418 were monitored. 0 6-EthylGua was eluted at a tR of 23 min, and 0 6-ethyl-N2,3t-Gua was eluted at a tR of 25 min (flow rate 4.0 mL min-l). The collected product was analyzed by rechromatography in the same system and by UV spectroscopy (A, 273 at pH 7.0). Two 0 6ethyl-N 2,3-t-Guafractions were pooled. One (1.19 mg, 5.9 pmol) contained -5 % residual 0 6-ethylGua and was used for subsequent analysis. A fraction eluting between the peaks of 0 8ethylGua and 0 6-ethyl-N2,3-t-Gua was also collected; it was estimated to contain 1.15 mg of 0 6-ethyl-N 2,3-t-Gua(5.7 pmol) and 20% residual 0 6-ethylGua. Sal& were removed from these fractions by repeated lyophilization from HzO. Spectroscopy. UV spectra were recorded with a modified Cary 14/0LISsystem (On-LineInstrument Systems,Bogart,GA). Wavelength maxima were determined using the peak finder software. Mass spectra were recorded on a VG 70/250 system using positive ion fast atom bombardment and glycerol as a matrix (VG/Fisons, Manchester, U.K.). NMR spectra were recorded on a Bruker AM-400 instrument (Bruker, Billerica, MA) in the Vanderbilt facility.

-

Results and Discussion Preparation of 13C t-Gua Derivatives. A simple course was used to prepare BrCHPCHO. Commercial

Chem. Res. Toxicol., Vol. 7, No. 2, 1994 201

2- Haloacetaldehydes and Ethenoguanine Formation

A

Table 1. 1H NMR Chemical Shifts and Coupling Constants for Base Protons of ‘42-Labeled l,”’-eGuo and 0 6-ethyl-N?3-eGua*

H2

7

_-

lJ?22-c-Quo H2 7.75 ‘JCH 184 H6 7.01 2JcH 9.2 H7 7.37 0 6-ethyl-N2,3-c-Gua 8.01 H2 7.76 ‘JCH = 187 H5 7.35 2Jch 8.8 H6 a Recorded in (C2Hs)&O. Chemicalshifta are referredto internal (CH&Si at O.OO0 ppm.

I

8.2

~

8.0

I

~

7.8

~

7.6

I

~

7.4

~

7.2

~

7.0

PPm Figure 1. 1H NMR spectra of (A) 1,N2-e-G~o and (B) 0 6-ethyl-

N 2,3-t-Guaformed from BrCH&VHO.

BrCH2l3COzHwas reduced with LiAlH4 to BrCHz13CHzOH. (BrCH213COzH is more readily available than 13Clabeled ClCHZCOzH, and BrCHzCHO is more reactive than ClCHZCHO.) The position of the 13C label was verified by lH NMR spectroscopy. B ~ C H Z ~ ~ C Hwas ~ Ooxidized H to BrCHzWHO with pyridinium chlorochromate and extracted into H20, in which the concentration was analyzed with alcohol dehydrogenase. BrCH213CH0 was reacted with Guo to form 1,N2-eGuo (30)which was isolated by preparative HPLC and identified by its lH NMR and unique UV spectra (30).In order to form N 2,3-e-Guaderivatives in appreciable yield,

I

it is necessary to obstruct the N1 atom by alkylation of the 0 6 atom (30).@-EthylGua was used, and the product was isolated by HPLC. ‘H NMR spectra were recorded directly to avoid acid hydrolysis. W-Labeling Analysis Using 1H NMR Spectroscopy. The 13C NMR signals were too weak to analyze directly, and we utilized the strong l3C-lH splitting in the lH NMR spectra and the known assignments (12). In both cases the upfield e protons show the strong 13C-lH geminal splitting of -180 Hz (Figure 1,Table 1). There are some noticeable impurities in the 1 82-cGuospectrum, but they do not affect the assignments;the other expected lJV2-c-Guo protons were also observed upfield (results not shown). The upfield e protons correspond to H6 in both cases, as previously demonstrated using a variety of approaches (12). This carbon atom is the one adjacent to the nitrogen atom exocyclic to the ring (original N2 atom of Gua) in both cases (Chart 1). Mechanism of Formation of e-Gua Derivatives. The 13C-labeling studies clearly provide evidence that the carbonyl carbon of BrCHzCHO reacts directly with the N2 atom of Gua derivatives to yield both 1,N b G u a and N 2,3-e-Gua. The labeling patterns must be considered unambiguous in light of the use of 13Cand the basis of the spectral assignments. Since both products result from the reaction with BrCHzCHO, it appears reasonable to suggest that initial reaction yields a Schiff base and subsequent halogen displacement by a ring nitrogen yields an imine that can tautomerize to an enamine (Scheme 2). If N1 is blocked by 0 6 alkylation or, in DNA, by hydrogen bonding then formation of N 2,3-e-Guabecomes favored. ~

~

~

I

~

~

Scheme 2. Mechanisms of t-Gua Derivative Formations Inferred from BrCHPCHO Labeling

Y N\,

H

7

H

OR

H

..

~

I

~

~

208 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

Thus the original mechanistic suggestions of Leonard and his associates appear correct (2, 30). The l3C-labelingpatterns seen with 2-haloacetaldehydes may be compared with those reported with 2-halooxiranes (12). 2-Halooxiranes rearrange to form 2-haloacetaldehydes by opening of the oxirane ring and halide migration (37), so that the original halide-substituted carbon of the oxirane becomes the carbonyl carbon (Cl) of the 2-haloaldehyde. The labelingpatterns seen for t-Gua formation from 2-halooxiranes (derived from 2,2-dihaloethanols) cannot be accounted for by such rearrangement to 2-haloacetaldehydes (12). With the 2-halooxiranes, evidence has been presented that the initial attack by Gua is of the endocyclic nitrogen atoms (N1 and N3) on the halide-substituted methylene to form the t-Gua derivatives, which are rather minor products since those resulting from the attack by N7 and N2 atoms on the unsubstituted methylene carbon dominate (12). However, the reaction of Gua and its nucleosides with 2-haloacetaldehydes is slow but eventually yields mainly the c adducts (30).In more physiological settings, these electrophiles are also inactivated by oxidation, reduction, and conjugation (9, 12).

Acknowledgment. This work was supported in part by United States Public Health Service Grants CA44353 and ES00267. M.P. is a postdoctoral trainee supported by USPHS individual fellowship ES05592. We thank Dr. W. G. Humphreys for preparing 06-ethy1Gua. References Singer, B., and Bartach, H. (1986) The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis, IARC Scientific Publications, Lyon, France. Leonard, N. J. (1984) Etheno-substituted nucleotides and coenzymes: fluorescence and biological activity. Crit. Reu. Biochem. 15, 125-199. Leonard, N. J. (1992) Etheno-bridged nucleotides in structural diagnosis and carcinogenesis. Chemtracts: Biochem. Mol. Biol. 3, 273-297. Singer, B., Spengler, S. J., Chavez, F., and Kusmierek, J. T. (1987) The vinyl chloride-derived nucleoside, N 2,3-ethenoguanosine,is a highly efficient mutagen in transcription. Carcinogenesis 8, 745747. Cheng, K. C., Preston, B. D., Cahill, D. S., Dosanjh, M. K., Singer, B., and Loeb, L. A. (1991)The vinyl chloride DNA derivative, N 2,3ethenoguanine, produces G --c A transition in E. coli. Roc. Natl. Acad. Sci. U.S.A. 88,9974-9978. Simha, D.,Palejwala, V. A.,and Humayun,M. 2.(1991)Mechanisms of mutagenesis by exocyclicDNA adducts. Construction and in uitro template characteristics of an oligonucleotide bearing a single sitespecific ethenocytosine. Biochemistry 30,8727-8735. Swenberg, J. A,, Fedtke, N., Ciroussel, F., Barbin, A., and Bartach, H. (1992)Etheno adducts formed in DNA of vinyl chloride-exposed rats are highly persistent in liver. Carcinogenesis 13, 727-729. Guengerich, F. P., and Watanabe, P. G. (1979) Metabolism of [“CIand [BClI-labeled vinyl chloride in uiuo and in uitro. Biochem. Pharmacol. 28, 589-596. Guengerich, F. P., Mason, P. S., Stott, W. T., Fox, T. R., and Watanabe, P. G. (1981) Roles of 2-haloethylene oxides and 2-haloacetaldehydes derived from vinyl bromide and vinyl chloride in irreversible binding to protein and DNA. Cancer Res. 41, 43914398. Guengerich, F. P., and Raney, V. M. (1992) Formation of etheno adducts of adenosine and cytidine from 1-halooxiranes. Evidence for a mechanism involving initial reaction with the endocyclic 114, 1074-1080. nitrogens. J.Am. Chem. SOC. Guennerich.F. P. (1992)Roles ofthevinvlchlorideoxidation products 2-chlirooxirane A d 2-chloroacetaldeh;de in the in vitro firmation of etheno adducts of DNA bases. Chem. Res. Toxicol. 5, 2-5. (12) Guengerich, F. P., Persmark, M., and Humphreys, W. G. (1993) Mechanism of formation of 1,N 2- and N 2,3-ethenoguaninederivatives from 2-halooxiranes: isotopic labeling studies and formation of a hemiaminal derivative of N 2-(2-oxoethyl)guanine.Chem. Res. Toricol. 6,635-648.

Guengerich and Persmark (13) Oesch, F., and Doerjer, G. (1982) Detection of N2,3-ethenoguanine in DNA after treatment with chloroacetaldehyde in vitro. Carcinogenesis 3, 663-665. (14) Kusmierik, J. T., and Singer,B. (1992)1,N 2-Ethenodeoxyguanoeine: properties and formation in chloroacetaldehyde-treatedpolynucleotides and DNA. Chem. Res. Toxicol. 5, 634-638. (15) Shapiro, R., Cohen, B. I., Shiuey, S. J., and Maurer, H. (1969) On the reaction of guanine with glyoxal, pyruvaldehyde, and kethoxal, and the structure of the acylguanines. A new synthesis of N 2alkylguanines. Biochemistry 8, 238-245. (16) Sodum,R. S., and Shapiro, R. (1988) Reaction of acrolein with cytosine and adenine derivatives. Bioorg. Chem. 16, 272-282. (17) Guengerich, F. P., Geiger, L. E., Hogy, L. L., and Wright, P. L. (1981)I n uitro metabolism of acrylonitrile to 2-cyanoethyleneoxide, reaction with glutathione, and irreversible binding to proteins and nucleic acids. Cancer Res. 41, 4925-4933. (18) Seto, H., Okuda, T., Takesue, T., and Ikemura, T. (1983) Reaction of malonaldehyde with nucleic acid. I. Formation of fluorescent pyrimido[l,2-a]purin-l0(3i)-onenucleosides.Bull. Chem.SOC. Jpn. 56, 1799-1802. (19) Marnett, L. J., Basu, A. K., OHara, S. M., Weller, P. E.,Rahman, A. F. M. M., and Oliver, J. P. (1986) Reaction of malondialdehyde with guanine nucleosides: formation of adducts containing oxadiazabicyclononeneresidues in the base-pairing region. J.Am. Chem. SOC.108, 1348-1350. (20) Moschel, R. C., and Leonard, N. J. (1976)Fluorescent modification of guanine. Reaction with substituted malondialdehydes. J. Org. Chem. 41, 2941300. (21) Czarnik, A. W., and Leonard, N. J. (1981) Confirmation of the structure of the guanine-methylmalondialdehydereaction product by unequivocal synthesis. J. Org. Chem. 46,815-819. (22) Nair, V., and Offerman, R. J. (1985) Ring-extended products from the reaction of epoxy carbonyl compounds and nucleic acid bases. J. Org. Chem. 50,5627-5631. (23) Eder, E., and Hoffman, C. (1992)Identification and characterization of deoxyguanosine-crotonaldehydeadducts. Formation of 7,8 cyclic adductsand 1JV2,7,8bis-cyclicadducts. Chem. Res. Toxicol. 5,802808. (24) Sodum, R. S., and Chung, F. L. (1991) Stereoselective formation of in uitro nucleicacid adducts by 2,3-epoxy-4-hyd,roxynonanal. Cancer Res. 51, 137-143. (25) Sodum,R. S., and Chung, F. L. (1989) Structural characterization of adducts formed in the reaction of 2,3-epoxy-4-hydroxynonanal with deoxyguanosine. Chem. Res. Toxicol. 2, 23-28. (26) Sodum, R. S., and Chung, F. L. (1988) 1,N 2-Ethenodeoxyguanosine as a potential marker for DNA adducts formation by trans-4hydroxy-2-nonenal. Cancer Res. 48,32+323. (27) Hecht, S. S., Young-Sciame, R., and Chung, F. L. (1992) Reaction with deoxyguanosine: oxygenof a-acetoxy-N-nitrosopiperidine dependent formation of 4-oxo-2-pentenal and a 1,N 2-ethenodeoxyguanosine adduct. Chem. Res. Toxicol. 5, 706-712. (28) Kronberg, L., Sjhholm, R., and Karlsson, S. (1992) Formation of 3,N 4-ethenocytidine,1,N 6-ethenoadenosine,and lfl2-ethenoguanosine in reactions of mucochloricacid with nucleosides. Chem. Res. Toxicol. 5, 852-855. (29) Barbin. A.. El Ghissassi. F.. Nair. J.. and Bartach. H. (1993) Lipid peroxidation leads to formation of 1,N6-ethenoadenine and 3& 4ethenocytosine in DNA bases. Proc. Am. Assoc. Cancer Res. 34,136. (30) Satteangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) 1,”Ethenoguanine and N 2.3-ethenoguanine.Synthesis and comparison of the electronic spectral properties of these linear and angular triheterocycles related to the Y bases. J.Org. Chem. 42,3292-3296. (31) Boryski, J. (1990) 1,”-Ethenoguanosine: three methods of synthesis. Nucleosides Nucleotides 9, 803-813. (32) Kusmierek, J. T., Jensen, D. E., Spengler, S. J., Stolarski, R., and Singer, B. (1987)Synthesis and properties of N 2,3-ethenoguanoaine and N *,3-ethenoguanosine5’-diphosphate. J.Org. Chem. 52,23742378. (33) Sattsangi, P. D., Barrio, J. R., and Leonard, N. J. (1980) 1JeEtheno-bridged adenines and adenosines. Alkyl substitution, fluorescenceproperties, and synthetic applications. J.Am. Chem. SOC. 102,770-773. (34) Singhal, R. P., Landes, P., Singhal, N. P., Brown, L. W., Anevski, P. J., and Toce, J. A. (1989)High-performanceliquid chromatography for trace analysis of DNA and kinetics of DNA modification. BioChromatography 4, 78-88. (35) Balsiger, R. W., and Montgomery,J. A. (1960)Synthesisof potential anticancer menta. Xxv. Preparation of 6-alkoxv-2-amino~urinea. J. Org. Chem. 25,1573-1575: (36) Shriner,R.L.,Fuson,R. C.,andCurtin,D.Y. (1965)Thesystematic Identification of Organic Compounds, 5th ed., p 253, Wiley, New York. (37) McDonald, R. N. (1969) Rearrangements of a-halo epoxides and related a-substituted epoxides, in Mechanisms of Molecular Migrations (Thyagarajan, B. S., Ed.) pp 67-107, Wiley, New York.