Chem. Res. Toxicol. 1996, 8, 389-395
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Different Mechanisms of Aralkylation of Adenosine at the 1- and N6-Positions Chengyi Qiant7t and Anthony Dipple*lt Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21 702 Received October 28, 1994@ The eight products resulting from opening of either enantiomer of styrene oxide a t the a- or P-carbon by the 1-or N6-positionsof adenosine were prepared and their configurations assigned. These markers allowed the mechanism of aralkylation of adenosine by styrene oxide to be investigated. It was found that formation of a-substituted products a t the 1-position of adenosine involved total inversion of stereochemistry, whereas a t the N6-position inversion: retention was -6:l. These differences in stereochemistry suggest that a more ionic form of styrene oxide is involved in N6-aralkylation than in 1-aralkylation of adenosine. In the course of these studies, it was found that 1-substituted adenosines a t the a- and P-carbon of styrene oxide undergo Dimroth rearrangement a t neutral pH and 37 "C and that the former compound also deaminates fairly readily under these conditions.
Introduction Styrene oxide, a metabolite of the widely used styrene monomer ( I ) , is both mutagenic (2,3)and carcinogenic (4-61,and these properties have stimulated investigations of its reactions with nucleic acids and their constituents (2,6-10).The epoxide forms adducts at many sites on nucleic acid bases, and reactions with each of the four DNA bases have been described (8). We have previously used optically active styrene oxide as a stereochemical probe for the mechanism of aralkylation of guanosine a t different sites (10). In products a t the a-carbon of styrene oxide, inversion of stereochemistry decreased in the sequence 7- >> N2- > O'bubstituted guanosines, indicating that reaction at the exocyclic N2 and O6 sites occurred through extensively, though not completely, ionized substrate and that reaction a t the ring nitrogen proceeded through displacement on the covalent substrate. Guanine residues in DNA are more extensively modified than other bases by most carcinogens (ll), but carcinogens are known that extensively or exclusively target adenine residues in DNA. The latter include 7,12dimethylbenz[a]anthracene (12),benzo[c]phenanthrene (231,and methylenebis(2-chloroaniline)(14,15). The factors involved in determining sites of reaction on adenine residues have not received much attention but are important for a full understanding of the DNA interactions of chemical carcinogens. In the present study, we have extended some earlier work on the mechanism of alkylation and aralkylation of adenosine (16)by determining the stereochemistry of the 1-and N6-substituted products arising a t the a-carbon of optically active styrene oxide. This study required the preparation, characterization, and configurational assignment of the eight products resulting from opening of the epoxide ring of either styrene oxide enantiomer at the a- or P-carbons by the nitrogens a t the 1- and N6positions of adenosine. Previously, a 1-substituted and 'NCI-Frederick Cancer Research and Development Center. i Present address: Reproductive TechnologyLaboratories, 1245 16th St., Santa Monica, CA 90404. @Abstractpublished in Advance ACS Abstracts, March 1, 1995.
two N6-substituted adducts were described from styrene oxide-deoxyadenosine reactions (81, but specific structural and stereochemical isomers were not identified. Specific N6-substituted stereoisomers have been generated previously (17,18) as components of oligonucleotides, but properties of the individual nucleoside adducts were not available from these studies.
Experimental Section Chemicals were used without further purification. Adenosine, inosine, 6-chloropurine riboside, (R)-and (S)-2-phenylglycinol were purchased from Aldrich Chemical Co. (Milwaukee, WI). Racemic and optically active styrene oxides were from Fluka Chemical Corp. (Ronkonkoma, NY). [14ClAdenosine (specific radioactivity 532 mCL"mo1) was obtained from Amersham Searle (Arlington Heights, IL). HPLC was carried out on a Hewlett-Packard Model 1090 high-pressure liquid chromatograph equipped with a diode array detector. Ultraviolet absorption spectra were recorded either on line or with a Milton Roy Spectronic 3000 diode array spectrophotometer. Circular dichroism spectra were measured on a Jasco Model J500A spectropolarimeter equipped with a data processing system for signal averaging. CD spectra of N6substituted adenosine adducts in methanol were normalized t o 1.0 absorbance unit at am=. Proton NMR spectra and COSY homonuclear 2D spectra were obtained using a Varian VXR5005 instrument. Samples were dissolved in dimethyl-& sulfoxide or methanol-& with tetramethylsilane as internal standard. Positive ion (+vel fast atom bombardment (FAB) mass spectra were obtained with a reverse geometry VG Micromass ZAB-2F spectrometer interfaced to a VG 2035 data system. A mixture of glycerol and N,N-dimethylformamide (1:l viv) was used as the FAB matrix.
Preparation of the Diastereomers of NB-(2-Hydroxy-lphenylethy1)adenosine [N6a(R) and N6a(S)1 and W42Hydroxy-2-phenylethy1)adenosine[N6/3(R)and N6/3(S)I.To a suspension of adenosine (3 g) in 400 mL of ethanoVHz0 (1:l) containing ammonium acetate (0.52 g) was added racemic styrene oxide (11mL). The reaction was stirred a t 37 "C for 72 h. After evaporation to dryness, the residue was triturated with diethyl ether and the remaining solids were suspended in methanol (50 mL). Unreacted adenosine was removed by filtration, and after concentration, the filtrate was loaded on a Sephadex LH-20 column followed by elution with 40% methanol
0893-228x/95/2708-0389$09.00/00 1995 American Chemical Society
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390 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
(50 mL). Unreacted adenosine was filtered off, and the filtrate, in HzO. Absorption of the eluate was monitored continuously after concentration, was separated on a 10 x 250 mm YMC at 254 nm. The sequence of elution was 1-substituted adenosine ODS-AQ column eluted isocratically with 3% tetrahydrofuran adducts followed by unreacted adenosine, followed by N6in 1 0 mM ammonium formate (pH 5.3) using a flow rate of 3.5 substituted adenosine adducts. After concentration of the latter m u m i n . The la(S) adduct eluted at 24-26 min: UV R,, fraction, the four diastereomeric N6-substituted adenosine ad(methanol or Tris-HC1 buffer, pH 7.0) 260 nm; NMR (DMSOducts were separated from each other on a 10 x 250 mm YMC &) 6 8.16 ( s , 1, H-81, 8.09 (s, 1, H-21, 7.39 (dt, 2, Ar-H, ortho), ODS-AQ column (YMC, Wilmington, NC) eluted isocratically 7.36 (dt, 2, Ar-H, metal, 7.29 (tt, 1, Ar-H, para), 6.29 (dd, 1, with 9% tetrahydrofuran in HzO at a flow rate of 2.8 m u m i n . a-CH; Ja,,ja = 7.7 Hz, Ja,pb = 5.6 Hz), 5.76 (d, 1,H-1’; J1,,2,= 6.0 The two a-diastereomers eluted ahead of the two P-diastereoH h 4 . 4 8 (m, 1, H-27, 4.24 (dd, 1,Pa-CH; J,ja,jjb = 11.9 Hz), 4.10 mers. Diastereomer N6a(S)eluted between 61 and 64 min: UV (dd, 1,H-3’; J y , y = 5.0 Hz, Jy,
