364
Chem. Res. Toxicol. 1988,1, 364-369
Styrene Oxide as a Stereochemical Probe for the Mechanism of Aralkylation at Different Sites on Guanosine Farida Latif, Robert C. Moschel, Kari Hemminki,+ and Anthony Dipple* Laboratory of Chemical and Physical Carcinogenesis, BRI- Basic Research Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21 701 Received June 27, 1988
The stereochemistry at the a-carbon atom of the styrene moiety in styrene oxide-guanosine products was established, and the stereochemical consequences of reaction of optically active styrene oxide with guanosine were investigated. Inversion of stereochemistry in products a t the a-carbon of styrene oxide decreased in the sequence 7- >> N2- > 06-substituted guanosines. These findings show that an extensively ionized substrate is needed for reaction a t the exocyclic N2 and OBsites on guanosine but that the reactive intermediate is not an ideal planar trigonal carbonium ion. This follows because inversion exceeds retention in both of the exocyclic substituted products.
circular dichroism spectra were recorded on a Jasco Model 5-500 spectropolarimeter. Proton NMR spectra were recorded on a Varian XL 200 instrument interfaced to an Advanced data system. Samples were dissolved in dimethylde sulfoxide or methanold( monomer, have prompted investigations of its reactions with tetramethylsilane as internal standard. Positive ion (+ve) and negative ion (-ve) fast atom bombardment (FAB) mass with nucleic acid components (1, 5, 6). These reports spectra (MS) were obtained with a reversed geometry VG Miindicate that guanine residues react through the N-7cromass ZAB-2F spectrometer interfaced to a VG 2035 data position at the a- and the P-carbons of styrene oxide (1, system. A mixture of glycerol and Nfl-dimethylformamide (1:1 5) and that, in aqueous solution, the exocyclic N2- and v/v) was used as the FAB matrix. HPLC was carried out on an 06-positions of deoxyguanosine are also targets (5, 6). LDC Constametric system with an LKB gradient master. EleReaction at these three sites on guanine residues is a mental analyses were by Galbraith Laboratories, Inc., Knoxville, property previously associated with benzylating agents TN. (7-10). In contrast, simple aliphatic alkylating agents do Preparation of the Diastereomers of 7-(2-Hydroxy-1not usually react with the N2 site (11) and polycyclic phenylethy1)guanosine (aN7I and aN7II). Guanosine (2.8 g) aralkylating agents do not usually react with the Oe site suspended in glacial acetic acid (75 mL) was treated with (*)styrene oxide (12 mL) and stirred for 5 h. The resulting homo(12). Optically active styrene oxide should be a useful geneous solution was diluted with 100 mL of methanol and 900 probe of the mechanism of aralkylation at the N-7, N2,and mL of diethyl ether. The precipitate was collected by filtration, O6 sites on guanosine because, in a single reaction, it should dried, and resuspended in methanol (50 mL). Undissolved be possible to examine the stereochemical consequences guanosine was filtered and the homogeneous solution loaded on of aralkylation at each of these sites. In a previous study a 2.8 X 70 cm Sephadex LH-20 column and eluted at 0.7 mL/min (13),we have established the stereochemistry of the two with methanol. UV absorption was monitored a t 254 nm and pairs of diastereomeric 06-substituted guanosines arising fractions (7 mL) were collected. 7-Substituted guanosine products from a- or &substitution of either enantiomer of styrene eluted in fractions 35-50 while unmodified guanosine eluted later oxide. In the present report, we establish the stereoin fractions 60-80. Fractions 35-50 were pooled and evaporated to dryness to afford 0.7 g of 7-substituted guanosines. Diastechemistry of the two diastereomeric N2-substituted guareomers aN7I and aN7II were separated by loading -0.1 g of nosines arising from a-substitution on styrene oxide and the mixture on a 10 X 250 mm Spherisorb ODs-2 column, which of the two pairs of diastereomeric 7-substituted guanosines. was then eluted isocratically with a 0.01 M solution of ammonium We also demonstrate that the predominance of inversion formate, pH 5.3, in methanol/HzO (25:75) at a flow rate of 1.5 over retention of configuration decreases substantially in mL/min. UV absorption was monitored at 254 nm and fractions the sequence 7- >> N2- > 06-substituted guanosines. (3.5 mL) were collected. Diastereomer aN7I eluted in fractions 17-23 and diastereomer “711 in fractions 43-51. Trace amounts Materials and Methods of the isomeric ON71 and ON711 eluted in fractions 15-16 and 34-36, respectively (see below). The appropriatefractions for aN7I Generally labeled [14C]guanosine(specific radioactivity 509 and aN7II were pooled and dried by lyophilization to yield 0.3 mCi/mmol) was obtained from Amersham Searle, Arlington (pH and 0.25 g, respectively, of aN7I and aN7II. aN7I: UV A, Heighta, IL. Guanosine was from P-L Biochemicals Inc., Mil5.3) 258, 282 (sh) nm; NMR (DMSO-d6)6 9.63 ( 8 , 1, H-8), 7.39 waukee, WI, and (*)-styrene oxide, 2,6-dichloropurine,and (R)(s, 5 , C6H5),6.57 (br s, 2, NHz, exchange with DzO),6.42 (dd, 1, and (S)-2-phenylglycinolwere from Aldrich Chemical Co., Mila-CH, Ja,pl= 5.7, Jasz = 3.7 Hz), 5.85 (d, 1,H-1’, J1,,z,= 3.9 Hz), waukee, WI. The diastereomeric (2R)- and (2S)-08-(2-hydroxy4.26 (dd, 1,fi-CH),4.02 (m, l,&CH, Jel& = 9.3 Hz). aN711 UV 2-phenylethyl)guanosines, (1R)- and (1S)-06-(2-hydroxy-l(pH 5.3) 258, 282 (sh) nm; NMR 6 9.63 ( 8 , 1, H-81, 7.40 (s, phenylethyl)guanosines,and (R)- and (S)-l-phenyl-1,2-ethanedioIs , A 5, C&), 6.70 (br s, 2, NHz,exchange with DzO), 6.34 (dd, 1, a-CH, (styrene glycols) were prepared as described earlier (13). (R)- and Ja,@,= 8.6, Jm,s2= 3.9 Hz), 5.84 (d, 1, H-1’, J1,,z, = 3.7 Hz), 4.29 (8)-styrene oxides were prepared from the glycols according to (dd, 1, 0-CH, JB1,8z = 10.3 Hz), 4.01 (m, 1, fi-CH). Eliel and Delmonte (14). UV absorption spectra were recorded Preparation of the Diastereomers of 7-(2-Hydroxy-2on a Hewlett-Packard 8450A diode array spectrophotometer,and phenylethy1)guanosine (BN7I and BN7II). A suspension of guanosine (3 g) in ethanol/H20 (1:l)(400 mL) containing am‘Present address: Institute of Occupational Health, Helsinki 29, monium acetate (0.5 g) was heated until all the guanosine disFinland. solved. (*)-Styrene oxide (4 mL) was then added, and the re-
Introduction The mutagenic (1,2)and carcinogenic (3,4)properties of styrene oxide, a metabolite of the widely used styrene
0893-228~/88/2701-0364$01.50/0 0 1988 American Chemical Society
Chem. Res. Toxicol., Vol. 1, No. 6, 1988 365
Aralkylation of Guanosine by Styrene Oxide sulting solution was stirred at 37 OC for 72 h. After evaporation to dryness, the residue was triturated with diethyl ether (150 mL) and the remaining solids were suspended in methanol (50 mL). Unreacted guanosine was fiitered and the solution was loaded on a Sephadex LH-20 column and eluted with methanol as described above. Fractions 35-50 were pooled, dried, and subjected to chromatography on the Spherisorb ODs-2 column, as above. Diastereomer BN7I eluted in fractions 15-16 and diastereomer BN7II in fractions 34-36. Fractions were dried by lyophilization to yield 0.34 and 0.3 g of ON71 and BN711, respectively. BN7I: UV A, (pH 5.3) 258, 282 (sh) nm; NMR (DMSO-d6)6 9.17 (5, 1,H-8), 7.39 (m, 5, c6H5),6.68 (br s, 2, NH2, exchange with DzO), 5.84 (d, 1,H-l’, J1,x = 4.3 Hz), 5.06 (dd, 1, (UGH,Jasl 1.6, J,A = 8.7 Hz), 4.68 (d, 1,@-CH,J,,, = 2, JB A = 13 Hz), 4.24 (dd, 1, B-CH, JB8,a= 9.3, JB@, = 13 Hz). BN7If: UV A,, (pH 5.3) 258, 282 (sh) nm: NMR (bhSO-dd 6 9.20 (s,l, H-8), 7.40 (m, 5, C6Hd, = 6.73 (br s, 2, NH2, exchange with D20), 5.84 (d, 1, H-l’, J1t,2, 4.6 Hz), 5.05 (dd, 1, a-CH, Ja@ 1.8, Ja,p = 9.1 Hz), 4.71 (d, 1, P-CH, J,,+ = 2.4, JBlbz= 13.2kz), 4.20 (d%,l,P-CH, JBz,a= 9.5, Jpl,Bz = 13.1 Hz). Preparation of the Diastereomers of N2-(2-Hydroxy-1phenylethy1)guanosine(aN3 and aN%). To a rapidly stirred solution of guanosine (5 g) in H 2 0 (300 mL) containing Na2C03 (9 g) at 55 OC was added (*)-styrene oxide (10 mL). This suspension was stirred at 55 “C for 8 h and for an additional 72 h at room temperature. The resulting solution was extracted three times with an equal volume of CHCIS. The remaining aqueous phase was adjusted to pH 5.5 with concentrated HC1. This solution was evaporated to dryness and the dry residue treated with 120 mL of methanol/H20 (2:8). Undissolved guanosine was filtered, and 60 mL of the solution was loaded on a 2.8 X 70 cm Sephadex LH-20 column eluted with methanol/H20 (2%). UV absorption was continuously monitored at 254 nm and fractions (10 mL) were collected. Unreacted guanosine eluted in fractions 56-65. The desired N2-substituted products eluted in fractions 70-90. 1-Substituted guanosine products eluted in fractions 95-120. Following identical column chromatography of the second 60 mL of crude product mixtures, the combined fractions 70-90 from both runs were evaporated to dryness and rechromatographed on the LH-20 column equilibrated with methanol/ H20/NH40H(2:8:0.3). The column was eluted with this same solvent. The mixture of diastereomers aN21and aN211eluted in fractions 30-45 (yield 0.6 9). Separation of the individual diastereomers was accomplished by loading -0.15-g portions on a 10 X 250 mm Spherisorb ODs-2 column which was eluted isocratically with 0.01 M ammonium formate in methanol/H20 (3:7), pH 5.3, at a flow rate of 1.5 mL/min. UV absorption was continuously monitored at 254 nm and fractions (3.6 mL) were collected. Diastereomer aN21 eluted in fractions 16-23. Diastereomer aN211eluted in fractions 26-35. Fractions containing the two isomers were separately pooled and dried by lyophilization to yield in total 0.24 g of aN21and 0.26 g of aN211. aN21: UV A- (pH 1)260,282 (sh) nm; A, (pH 6.9) 255,276 (sh) nm; A(PH 13) 260,271 (sh) nm; NMR (DMSO-d6) 6 7.89 ( ~ , lH-8), , 7.31 (m, 5, C6H5),7.10 (d, 1,NH, exchanges with D20,JNHp= 7.2 Hz), = 5.5 Hz), 5.05 (dd, 1, a-CH), 3.72 (m, 1, 5.60 (d, 1, H-l’, J1t,2t B-CH), 3.63 (m, 1, fl-CH);-ve FAB MS, m/z 402 ([M - HI-). Anal. Calcd for ClsH21N506*H20:C, 51.30; H, 5.50; N, 16.62. Found C, 51.22; H, 5.22; N, 16.59. aN211 UV A, (pH 1)260, 282 (sh) nm; A, (pH 6.9) 255, 276 (sh) nm; ,A, (pH 13) 260, 271 (sh) nm; NMR (DMSO-d6)6 7.86 (5, 1, H-8), 7.32 (m, 5, C6H5),7.08 (d, 1,NH, exchanges with D20,J N H =, ~7.4 Hz), 5.61 (d, 1,H-l’, J1t,2t = 5.6 Hz), 4.99 (dd, 1, a-CH), 3.74 (m, 1,8-CH), 3.62 (m, 1, B-CH);-ve FAB MS, m/z 402 ([M - HI-). Anal. Found C, 50.89; H, 5.45; N, 16.43. Preparation of Individual Diastereomers of N2-(2Hydroxy-1-phenylethy1)guanine (aN21’ and aN211’). 2Chloro-6-hydroxypurine(0.1 g), derived from the dichloropurine by hydrolysis (15), and (R)-or (S)-2-phenylglycinol(O.36g) were heated in dimethyl sulfoxide (2 mL) at 80 “C for 4 days. The solution was then poured into ice water (50 mL), and the resultant precipitate was collected, dissolved in 40% methanol, and loaded on a reversephase Altex Ultrasphere column (10 X 250 mm) which was eluted with 40% methanol. The retention time for both a m , derived from (R)-2-phenylglycinol,and aN211’,derived from the S enantiomer, was 33 min. The total yield of aN21’ was 60 mg
-
-
and that of aN211’65 mg. aN21’: UV ,A, (pH 1)253, 279 (sh) (pH 6.9) 250,276 (sh) nm; A,- (pH 13) 275 nm; NMR nm; A, (methanold& 6 7.7 (s, 1,H-8), 7.3 (m, 5, C6H5),5.1 (dd, 1, a-CH, Ja,@, = 6.3,Ja,@= 4.9 Hz), 3.8 (m, 2,j3-CH2);+ve FAB MS, m/z 272 ([M HI+). aN211’: UV A, (pH 1) 253,279 (sh) nm; A, (pH 6.9) 250,276 (sh) nm; A, (pH 13) 275 nm; NMR (methanol-d4) 6 7.7 (s, 1, H-8), 7.3 (m, 5, C6H5),5.1 (dd, 1, a-CH, Jasl = 5.2,Ja,@= 4.8 Hz), 3.8 (m, 2,B-CH2);+ve FAB MS, m / z 272 ([M + HI‘). Aralkylation of [14C]Guanosinein Aqueous Solution by Optically Active Styrene Oxide. (R)-or (S)-styreneoxide (4.2 pg, 0.035 pmol) was added to 0.05 M Tris-HC1 buffer (pH 7, 1 mL) which contained [14C]guanosine(1pCi, -0.55 pg, 0.00196 pmol), and the reaction mixture was kept at 37 “C, usually for 24 h. The reaction mixture was then filtered and mixed with a solution containing guanosine, the four 06-substituted isomers (a061, (YO‘%,B061, and OO6II) the four 7-substituted isomers (aN71, aN711, BN71, and BN7II) and the two N2-substituted isomers (&I and aWII) (0.1 mL). Aliquots of this solution were then separated by using a B e c k ” Ultrasphere ion-pair column (4.6 X 250 mm) eluted at 1 mL/min with a linear gradient changing from 0.012 M sodium phosphate buffer, pH 6, and methanol (85:15) to buffer/methanol(67.532.5) over 21 min. The elution was continued isocratically thereafter for a further 159 min. Fractions (1mL) were collected through moet of the elution, but smaller fractions (0.5 mL) were collected for fractions 26-44. Radioactivity in each fraction was determined by liquid scintillation counting using PCS scintillant (Amersham Searle, Arlington Heights, IL). Retention timw for individual products were as follows: guanosine, 5.5 min, @N71,25min, aN71,28 min, aN21, 31 min; PN711, 33.5 min; aN211,41 min; aN711, 44.5 min; a061, 73.5 min; p061, 125 min; a0611,137.5 min; @II, 144 min.
+
Results As indicated under Materials a n d Methods, diastereomeric styrene oxide-guanosine products were labeled I and I1 on t h e basis of t h e sequence of their elution from t h e chromatographic systems used for purification. T h e stereochemistries assigned t o each of these products (Figure 1) are based on structure-determining syntheses for t h e 06-,N2-, a n d two 7-substituted products, a n d o n mechanistic considerations for the other two 7-substituted products. T h e stereochemistries of the four O‘hbstituted guanosines were established through their synthesis from the sodium alkoxides of optically active styrene glycol and 2-amino-6-chloropurine riboside, as previously described (13). One a-a n d one /3-substituted diastereomer was obtained from each styrene glycol enantiomer, and since t h e reaction does not break any bonds at the chiral center, the products were assigned the stereochemistry of t h e glycol starting material. T h e N2-substituted products were prepared by reaction of guanosine with styrene oxide in alkaline solution, conditions known t o favor N2-aralkylation (16). Only two N2-substituted guanosines ( a mand aWII) were obtained. T h e similarities in spectral properties for these products suggested a diastereomeric relationship between them. Their NMR spectra indicated they arose from reaction with t h e a-carbon of styrene oxide. For example, t h e doublet at 7.08 or 7.1 p p m for t h e NH proton was consistent with splitting by a single proton on t h e adjacent carbon rather t h a n by two protons, which would be the case for a @substituted product. This was confirmed and t h e stereochemistry established, by preparation of t h e corresponding guanines from optically active 2-phenylglycinol a n d 2-chloro-6-hydroxypurine following the general approach used by Shapiro et al. (15). As illustrated in Figure 2, (S)-2-phenylgycinol can only yield ( l S ) - W (2-hydroxy-l-phenylethyl)guanine, a n d t h e circular dichroism spectrum of this product exhibited a negative long-wavelength band. T h e product from (R)-2-phenylglycinol exhibited a mirror image circular dichroism
366 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
Latif et al.
Table I. Products from Optically Active Styrene Oxides and Guanosine:" Fraction of Total Products ,==CY
CsHs
dCs-
H
+
H*.. /OH
+
C-CYN.7Gw H
qp/*-J
%& N - 7 G w b/ ,cs-cHzoH
%. A
4
HZN
-
k
(R)-styrene oxide (&styrene oxide
+
tgH5 ' R
C6y5 0%0. /
HdCs-CwH
C-H
,/*-
H*..
He.. / N . ~ G w , C r CHzOH
C rC Y O H
5%
C6HS
+
,r /o-
H*..
C
CHIOH
C6Hs
SIR
SIR
SIR
SIR
0.01/0.46 0.01/0.48 0.48/0.01 0.48/0.01
0.31/0.02 0.30/0.02
0.11/0.02 0.11/0.02 0.03/0.10 0.04/0.10
0.04/0.03 0.04/0.03
0.01/0.31 0.01/0.30
0.02/0.04 0.02/0.04
Numbers represent the fraction of total guanosine products formed. Conversion of guanosine to product averaged 5.65% for the two was -12% and -15% of the total (S)-06-guanosineproducts reactions of (R)-styreneoxide and 6.23% for those of @)-styrene oxide. @061 for (S)- and (R)-styrene oxide, respectively, while @0611 was -15% and -21% of the total (R)-06-guanosineproducts for ( S ) -and (R)styrene oxides, respectively.
%* O t/
spectrum (not shown). In order to determine the stereochemistry of the guanosine products, aN21and aN211were each subjected to hydrolysis in 1 N HC1 at 100 "C for 1 h to yield the guanine produds aN21' and aN?II', and their circular dichroism spectra were recorded. These spectra were superimposable on those of the phenylglycinol-derived products, with aN211' giving the same spectrum as the product from (S)-Zphenylglycinol(Figure 2) and aN21' giving the same spectrum as the product from (R)-2phenylglycinol. Thus, the stereochemistry of the N2-substituted guanosines at the a-carbon atom of the styrene moiety is S for aN211 and R for aN21 (Figure 1). The circular dichroism spectra of these nucleosides were found to be very similar to those of the guanines (Figure 3). The 7-substituted guanosines have been described before ( I , 5 , 6 ) ,and the a-and P-substituted products have been discriminated from one another by their distinctive fragmentation patterns in the mass spectrometer ( I , 5 ) . However, the individual stereochemistries have not been elucidated. These stereochemistries were assigned on the basis of the products formed in reactions of optically active styrene oxides with guanosine in aqueous solution (Table I). In these reactions, which were carried out in duplicate, (R)- and (SI-styrene oxides gave a substantial yield of PN7II and PN7I respectively, with only trace amounts of the other P-diastereomer (presumably because of incomplete optical purity in the oxides) being formed. Since formation of these products involved nucleophilic attack on the &carbon atom, no changes in the stereochemistry
h
H'cs-CwH a0 6 1
ycsy H'
C h O h
p 061
%% Nzoup t/ Hc 's-
CYoH
adn
CkHS N-7tuo **. / H'cs-cwH a N-7II
a N-7I
%?/OH c-cl-y+7Guo
H'
p N-71
Figure 1. Stereochemistry of styrene oxideguanosine products. H*. lo\
a N'II
+ 50 I
\H'
L %%
**. /
-
1
N b a
,C-Cl+OH H
S
-05-
/f
200
300 400 Wavelength (nm)
Figure 2. Assignment of stereochemistry of N2-substituted guanosines. The circular dichroism spectra in 1 N HC1 for Rfz-(2hydroxy-1-phenylethy1)guanineare the same when the substituted guanine is derived from acid hydrolysis of aN211or by synthesis from (S)-2-phenylglycinol.
Chem. Res. Toxicol., Vol. 1, No. 6, 1988 367
Aralkylation of Guanosine by Styrene Oxide
- 05 -
200
400
300 Wavelength (nm)
Figure 3. Circular dichroism spectra in methanol of the diastereomeric N-(Zhydroxy-l-phenylethyl)guanosines,aN21and cuN211.
0.0
0.1
0.2
0.3
0.4
(SI - s t o x 0( R ) - S t Ox 0.5
FRACTION OF TDTRL PRODUCTS
Figure 4. Yields and stereochemistry of products from optically active styrene oxides (StOx).
of the chiral a-carbon should arise. The stereochemistry of the products should be, therefore, the same as that of the starting material. As summarized in Figure 1, PN7I was assigned, therefore, S stereochemistry and PN7II was assigned R stereochemistry. Reaction of the optically active styrene oxides with guanosine also yielded appreciable quantities of 7-substituted guanosines involving the a-carbon atom of styrene oxide (Figure 4, Table I). Again, one diastereomer was formed in large excess over the other diastereomer for each enantiomer of styrene oxide. (S)-and (R)-styrene oxide preferentially gave aN7I and aN711, respectively. Because of the large predominance of one diastereomer, this reaction presumably involved bimolecular nucleophilic attack on the back side of the a-carbon atom, leading to inversion of the stereochemistry of the initial epoxide. In this case, aN7I would have R stereochemistry and aN7II would have S stereochemistry (Figure 1). The reaction of guanosine with racemic styrene oxide in aqueous solution gave rise to four 7-substituted guanosines (aN71, aN711, PN71, and PN7II) and four 06substituted guanosines (a061,a0611,p061, and p0611),but to only two N2-substituted guanosines (aN21and aN211). When the products of this reaction were examined at different times (24,48, and 72 h), the yield of 06-substituted guanosines bound through the @-carbonof the styrene moiety (p061and p0611) increased with time to a far greater extent than did any other reaction product. This is consistent with the rearrangement of a06 products to PO6 products observed earlier (13). Therefore, in the summary of data for the optically active enantiomers of styrene oxide (Table I, Figure 4), the yields of 06-substituted products arising from p- and a-substitution of styrene oxide have been summed together. It can be seen from Figure 4 that product yields were similar with each enantiomer so that the chiral ribose residue in guanosine did not have a major effect on the stereochemical course of the reactions. Further, substitution on the a-carbon of
styrene oxide occurred with preferential inversion of configuration at this chiral carbon atom, irrespective of whether the nucleophile was the 7-, the N2-, or the 06position of guanosine. However, there was a quantitative difference in the extent of inversion that occurred. Inversion was very extensive in the "-substituted products (15-30-fold greater than retention), whereas the inverted product was formed only 3.5 or 5.5 times more readily than the retained product for the W-substituted products. The selectivity was reduced further in the 06-substituted products where the preference for inversion was in the range 2:l to 1.5:l. The studies of optically active styrene oxide-guanosine reactions summarized in Table I and Figure 4 were done at pH 7. Less extensive investigations at other pHs were also undertaken. At pH 5.82, the total yield of guanosine products fell to -4% of the initial guanosine for both epoxide enantiomers. The other notable feature was that the yield of 06-substitutedproducts was enhanced somewhat at this pH such that, as a fraction of the total, these products were almost double the values obtained at neutrality. The ratios of inversion to retention of configuration for all the products were not substantially different from the neutrality values, however. A t pH 8, total yields of product rose to -7% for each epoxide enantiomer, and decreased yields of 06-substituted products were more than compensated for by a large increase in N2-substituted products for both enantiomers. The general trends at different pHs were the same for both enantiomers, but specific numbers for each product are not given since duplicate analyses for each enantiomer were not undertaken.
Discussion It is now well established that the common factor among most chemicals that initiate the carcinogenic process is the fact that they are, or they give rise through metabolism to, reactive entities capable of modifying the cellular genome (17-19). If this reactivity is responsible for tumorinitiating activity, it is obviously important to understand how the reactivity of these entities controls or directs their reaction with the genome. We have begun such investigations by examining the basis for the reaction of alkylating agents with the 7- and 06-positionsof guanine residues (11,20) and the reaction of polycyclic aralkylating agents with the N2-position of guanine residues (12). We have established that benzylating agents are useful models because they react with all three of these sites on guanine residues in aqueous solution (7). By monitoring changes in extents of reaction at these three sites in guanosine with changes in reactivity of the benzylating agent [brought about by changes in solvent (7), leaving group (7,8), or para substituent (IO)],we have developed and supported a rationalization for the site selectivities exhibited by the alkylating and arakylating agents (7).Thus, we have argued that reaction with the exocyclic O6 and N2 sites of guanosine (as opposed to the ring nitrogen site) is favored by changes in substrate structure or reaction medium that tend to advance carbon-leaving group bond breakage, i.e., to increase the SN1 character of the reaction. Second, if the substrate structure is such that developing charge is localized on the reaction center [;.e., when the center is hard (21)],reaction with exocyclic oxygen is favored, whereas when charge can be delocalized [i.e., when the center is soft (21)],reaction is directed to the exocyclic N2 site (7). In the present investigation, we have sought to test this rationalization further by using an optically active sub-
368 Chem. Res. Toxicol., Vol. 1, No. 6, 1988
strate, styrene oxide, so that, in addition to product yields, product stereochemistrycan also be evaluated. Simple s N 2 displacements on epoxides usually occur at the less branched carbon atom, i.e., the 0-carbon atom in styrene oxide (22). Here, and in previous reports (I, 5), it is clear that 7-substituted guanosines arise principally by attack at the 0-carbon of styrene oxide but that similar attack at the a-carbon is also substantial. This finding suggests that some positive charge resides on the a-carbon because it is known that increasing charge at the a-carbon reduces the steric preference for attack at the P-carbon atom (22). Optically active styrene oxide was converted predominantly to a single diastereomer by attack at the a-carbon by the 7-position of guanosine (Figure 4). This indicated that almost complete inversion had occurred and that ionization of the carbon-oxygen bond in the epoxide was far from complete when bond formation occurred. For the N2 group of guanosine, product was formed from reaction at the a-carbon, but no reaction on the P-carbon of the epoxide was detected. This observation suggested that reaction with this nucleophile required substrate. with some considerable ionic character and, in concert with this, the N2 product was more racemized than the 7-substituted product. Although both a- and @-substituted products resulting from reaction at the 06-positionof guanosine were detected, the @-substitutedproducts arose from the asubstituted products by rearrangement without change in stereochemistry at the a-carbon (13). The 06-substituted products were somewhat more racemized than the N2substituted products (Figure 4),suggesting that carbonoxygen bond breakage was further advanced when the 06-substituted,rather than the N2-substituted, products were formed. In these reactions with styrene oxide, the degree of retention or inversion of configuration varied with the particular product being considered. Thus, in the same reaction vessel some products (e.g., the 7-substituted guanosines attached to the 0-carbon of the styrene moiety) arise from an essentially SN2reaction mechanism, while others, derived by attack at the a-carbon, arise from a reaction with considerable s N 1 character, judged by the extensive racemization that has occurred (e.g., the 06substituted guanosines). Moreover, racemization, and, therefore, SN1character in the reactions leading to them, increases along the a-substituted product series 7-
