Chem. Res. Toxicol. 1995,8, 444-454
444
Direct Synthesis and Characterization of Site-Specific Adenosyl Adducts Derived fiom the Binding of a 3,4-Dihydroxy-1,2=epoxybenzo [c ]phenanthrene Stereoisomer to an 11-mer Oligodeoxyribonucleotide Alfred Laryea,? Monique Cosman,t Jyh-Ming Lin,$ Tongming Liu,? Rajiv Agarwa1,Il Sergey Smirnov,? Shantu Amin,§ Ronald G. Harvey,l Anthony Dipple," and Nicholas E. Geacintov*>t Chemistry Department, 31 Washington Place, New York University, New York, New York 10003, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, American Health Foundation, Valhalla, New York 10595, Ben May Institute, The University of Chicago, Chicago, Illinois 60637, and Chemistry of Carcinogenesis Laboratory, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21 702 Received October 14, 1994@ Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-l,2t-epoxy-1,2,3,4-tetrahydrobenzo[clphenant~ene(B[clPhDE-2, or anti-B[clPhDE) with the single deoxyadenosine (dA)residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[clPhDE-W-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[clPhDE- 3'-dAMP mononucleotide adducts, with those of standards derived fiom the reaction of the enantiomers (+)- and (-)-anti-B[clPhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and 11) were obtained upon reacting racemic antiB[clPhDE with d(CTCTCAC'M'CC). In oligonucleotide adduct I, the lesion is a (+)-trans-antiB[clPhDE-N6-dAresidue, and in oligonucleotide adduct I1 it is a (-)-trans-anti-B[c]PhDE-N6dA residue. These assignments were further confirmed using a standard 32Ppostlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and I1 by spleen phosphodiesterase. The melting points (T,) of duplexes of modified oligonucleotides I and I1 and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[clphenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-antiBIcIPhDE-modified oligonucleotide adducts I and 11; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the %-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct 11, a s observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488-12497, and Cosman et al., Biochemistry, in press].
Introduction
two diastereomeric forms of B[c]PhDE, one in which the 4-OH group and the epoxide oxygen are cis (B[clPhDEBenzo[clphenanthrene (B[cIPh),' like other polycyclic 1, or syn-BkIPhDE), and the other in which these two aromatic hydrocarbons (PAH)(1 -3), can be metabolically groups are trans to one another (B[c]PhDE-2, or anti-Bactivated to highly reactive, mutagenic, and tumorigenic [clPhDE). Each diastereomer can be resolved into a pair diol epoxide derivatives (4-6) that bind to cellular DNA of enantiomers; for example, anti-B[clPhDE can be (7, 8). The most important ultimate mutagenic and resolved into two stereoisomers, the (+)-4S,3R,!2R,1Stumorigenic metabolites of B[clPh are the fjord-region and the (-)-4R,3S,2S,lR-enantiomers (9, 10). The codiol epoxide isomers 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetvalent binding of B[clPhDE to native DNA has been rahydrobenzo[clphenanthrene (BkIPhDE). There are established (6-8),and the biological activities of the various stereoisomers have been investigated by a num* Corresponding author. ber of groups (11-1 7); some of the stereoisomers of B[c]t New York University. i Memorial Sloan-Kettering Cancer Center. PhDE are among the most tumorigenic PAH diol American Health Foundation. epoxides that have been studied (11, 12, 17). 'I NCI-Frederick Cancer Research and Development Center. Ben May Institute, The University of Chicago. Another widely studied PAH diol epoxide is 7,8@Abstractpublished in Advance ACS Abstracts, March 15, 1995. dihydroxy-9,10-epoxy-7,8,9,l0-tetrahydrobenzo[alpyAbbreviations: B[clPh, benzo[clphenanthrene; anti-B[clPhDE (or B[clPhDE-2), 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[cl- rene, or B[a]PDE (18,19); the anti diastereomer has been phenanthrene; SVDP, snake venom phosphodiesterase I; SPD, spleen studied in greater detail than the syn isomer. Unlike phosphodiesterase 11; dG, 2'-deoxyguanosine; dA,2'-deoxyadenosine; anti-B[a]PDE which binds predominantly to deoxygua3'-dAMP, 2'-deoxyadenosine 3'-monophosphate; 3'-dCMP, Y-deoxycytidine 3'-monophosphate; 3'-dTMP, 2'-deoxythymidine 3'-monophosnosine (dG) residues in native DNA (see ref 20, for phate; B[clPhT, 1,2,3,4-tetrahydroxytetrahydrobenzo[c]phenanthrene; example), anti-B[c]PhDE binds more strongly to deoxyPAH, polycyclic aromatic hydrocarbon; PEI-cellulose, poly(ethy1enimine)-cellulose; TEAA, triethylamine acetate. adenosine (dA)residues in native DNA in vitro (7,s) and 0893-228X/95/2708-0444$09.Q0fQ0 1995 American Chemical Society
Synthesis of Benzo[c]phenanthrene -DNA Adducts in rodent embryo cell cultures (6). The differences in the biological activities of anti-B[alPDE and anti-B[clPhDE may be due to differences in structure and in chemical reactivity, as well as to differences in their modes of binding to native DNA. While the aromatic ring system in B[a]PDE is planar (21,22),the steric crowding in the fiord region of B[clPh (23) causes a severe deviation from planarity in the B[clPhDE molecules. Dipple et al. (7) have suggested that these distortions may be associated with the observed higher chemical reactivities of the B[clPhDE isomers with adenine residues in DNA and that high reactivities with dA in general may be associated with higher tumorigenic activities of PAH diol epoxides. The covalent binding of B[clPhDE to dA residues occurs via its C1-position to the exocyclic amino group (N6)of dA by either cis or trans addition (7, 8). The B[c]PhDE stereoisomers are characterized by differences in their mutagenic activities (13-15). The relationships between the structures of the PAH diol epoxide-DNA adducts that are formed and the biological consequences associated with these lesions are of great interest for understanding the relationships between molecular structure and mutagenic activity and specificity (24, 25). Because of the variety of adducts that are formed when B[c]PhDE reacts with native DNA (7, 8), and the effects of base sequence on reactivity and biological activity (26,271,it is important to generate sitespecific and stereochemically well-defined lesions embedded in DNA sequences of known base compositions for studies of structure-biological activity relationships. Total and direct synthetic strategies can be used for obtaining modified oligonucleotides of known base composition and sequence with site-specifically placed bulky PAH residues that can serve as model DNA sequences (24). Total synthesis approaches have been recently reported for generating covalent B[clPhDE-N6-dA adducts for subsequent site-specific incorporation into oligodeoxynucleotides of defined sequences (28, 29). We have successfully used the direct synthesis method (30, 31) t o synthesize stereochemically defined B[alPDE-N2dG lesions site-specifically placed in oligonucleotides of various sequences (32-36); the conformations of some of these B[ulPDE-N2-dGlesions (G*)in the double-stranded oligodeoxynucleotides d(CCATCG*CTACC).d(GGTAGCGATGG) in aqueous solutions have been established by high resolution NMR techniques (37-39). In this work, we describe a direct synthesis approach that is suitable for generating milligram quantities of (+)and (-)-trans-anti-B[c]PhDE-N6-dA residues (A*) in the oligonucleotide d(CTCTCA*CTTCC) for NMR and other structural studies. The solution NMR characteristics of these lesions in the double-stranded oligonucleotides d(CTCTCA*CTTCC).d(GGAAGTGAGAG)have already been reported (40).2 The stereochemical characteristics of the adducts were established here by a variety of approaches described previously for other PAH diol epoxide-oligonucleotide adducts (30, 31, 36); the HPLC retention times, U V absorbance, and CD characteristics of the covalent B[c]PhDE-N6-3'-dAMP mononucleotide adducts, obtained by enzyme digestion of the oligonucleotide adducts, are compared to those of 3'-dAMP mononucleotide adduct standards (8). The identification of the (+I- and (-)-truns-anti-B[c]PhDE-N6-3'-dAMP adducts were further confirmed by a 32P-postlabelingassay (41) Cosman, M., Laryea, A,, Fiala, R., Hingert, B. E., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J., Biochemistry, in press.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 445 using authentic standards described recently by Canella et al. (42).
Materials and Methods B[clPhDE Synthesis. The synthesis of the racemic antiBLcIPhDE was accomplished by published methods (43, 44). A 7 mM stock solution of anti-B[c]PhDE in tetrahydrofuran (THF) was prepared; the concentrations of B[clPhDE in solution were estimated using the molar extinction coefficient €260 = 4.4 x lo4 M-' cm-l. Caution: This diol epoxide is known to be mutagenic a n d carcinogenic a n d must be handled with utmost care. Oligonucleotide Synthesis. The oligodeoxyribonucleotide d(CTCTCACTTCC) and its complementary strand d(GGAAGTGAGAG) were synthesized by the phosphoramidite method on a Biosearch Cyclone automated DNA synthesizer (MilligenBiosearch Corp., San Rafael, CAI. The crude oligonucleotide products were purified by HPLC methods on a Rainin Dynamax C4 column (Rainin Instrument Co., Woburn, MA) using linear 0.1 M triethylamine acetate (TEAA)/acetonitrile solvent gradient systems. The DNA purity was checked by HPLC on a Dynamax300 A C4 column (Beckman Instruments, Fullerton, CA) using a potassium phosphate/acetonitrile ionic gradient solvent system. The molar extinction coefficients t of the oligonucleotides were based on phosphorus analysis (45);the value for d(CTCTCACTTCC) is €265 = (10.8 i 0.65)x lo4 M-l cm-', and €265 = (10.2 f 0.60)x lo4 M-I cm-l for the complementary strand d(GGAAGTGAGAG1. Reaction of B[c]PhDE-2 with d(CTCTCACT"CC). Typical reaction mixtures were prepared a s follows: 7.5 mL of a 7 mM d(CTCTCACTTCC) solution (concentration expressed in terms of oligonucleotide molecules) in 50 mM Tris-HC1 buffer solution (pH 6.8)was mixed with 2.5 mL of acetone and 28 mL of a 7 mM solution of racemic anti-B[clPhDE i n THF and mixed thoroughly. The initial B[c]PhDE/oligonucleotide molar ratio was 4:l. This solution was agitated and allowed to stand in the dark at room temperature. The contents of the reaction mixture were examined by reverse phase HPLC analysis using a 5 pm Hypersil ODS 250 x 10 mm column with a pore size of 120 A (Keystone Scientific, Bellefonte, PA). A Waters HPLC system with a Waters Model 991 photodiode array detector (Millipore Corp.) was used in all separation and analysis experiments. B[c]PhDE-g'-dAMPMononucleotideAdduct Standards. Mononucleotide adduct standards were prepared by reacting (*)-anti-B[clPhDE (and (+)- and (-1-anti-B[c]PhDE) with 3'dAMP in separate reactions employing conditions similar to those described for preparing the B[clPhDE-oligonucleotide adducts. After reacting (k)-anti-B[c]PhDE with 3 ' - U P , reverse phase HPLC analysis yielded four major products (see below). Enzyme Digestion. The nature of the modified base and the stereochemical properties of the adducts were established by enzyme digestion of the B[c]PhDE-modified oligonucleotides. Two exonucleases were employed for this purpose: (1)snake venom phosphodiesterase I (SVPD, from Crotalus adamanteus venom, Pharmacia Molecular Biology Division) and (2)spleen phosphodiesterase I1 (SPD, from bovine spleen, Worthington Biochemicals). In the case of S W D digestion, 3 units of the enzyme were added to about 50 pg of modified or unmodified oligonucleotides i n 1mL of 50 mM Tris-HC1 buffer and 10 mM MgClz (pH 8.5).The reaction mixture was incubated for various time periods at 37 "C as described below. In the case of digestion with SPD, about 50 pg of unmodified oligonucleotide d(CTCTCACTTCC) or oligonucleotide adducts I and I1 was dissolved in 1 mL of 0.15 M sodium acetate buffer solution (pH 6.0) containing 3 units of spleen exonuclease (phosphodiesterase II), and also incubated at 37 "C. In both cases, the digestion products were separated by reverse phase HPLC, using a 0-90% MeOH i n 20 mM Na2HP04 gradient over 60 min. 32P-Postlabelingand TLC Separation. The covalent antiB[clPhDE-3'-nucleotide samples (2 p L ) obtained from SPD digests were concentrated to dryness and then redissolved in 2
Laryea et al.
446 Chem. Res. Toxicol., Vol. 8, No. 3, 1995 ,uL of polynucleotide kinase buffer before the addition of 2 pL of polynucleotide kinase (3 units/pL) and 3 yL of a 10 pCi'pL solution of [y-32PlATP(Amersham Corp., Arlington Heights, IL) as described by Gupta e t al. (41). After 30 min of incubation at 37 "C, 2 ,uL of apyrase (20 milliunitdpl) was added, and the solution was further incubated for a n additional 40 min (42). Each labeled mixture (1p L ) was applied to the PEI-cellulose plates; wicks (46)were attached, and the plates were developed for 18 h in 1.0 M sodium phosphate (pH 6.8) (D1). Separation of the adducts was achieved by developing the PEI-cellulose plates in the D3 and Dq directions. The solvent for development in the D3 direction ( 5 h) consisted of 1.8 M lithium formate and 3.7 M urea (pH 3.4). Development in the Dd direction was with 0.4 M sodium phosphate, 0.25 M Tris, and 3.7 M urea (pH 8.2) for 5 h. Further cleanup of the PEI-cellulose plates was achieved by developing in 0.1 M sodium phosphate (pH 6.8) (Dj) for 15 h. Melting Curve Measurements. The melting profiles were measured as described previously (47, 48) in 20 mM sodium phosphate buffer solution (pH 7.0) containing 100 mM NaCl, with a rate of temperature increase of 0.5 "Umin. NMR Results. All NMR experiments were carried out using either a Varian Unity or Varian Unity Plus 600 MHz instrument using -5-9 mg of t h e duplex d(CTCCCTCA*CTTCC).d(GGAAGTGAGAG) with A* = (+)-trans- o r (-)-trans-anti-B[clPhDE-N6-dA, dissolved in 0.6 mL of 0.1 M NaC1, 10 mM sodium phosphate, and 0.1 M EDTA buffer solution (pH 7.0) a t 25 "C. The temperatures of the samples were calibrated with a n external methanol sample. The phase-sensitive COSY spectra of the adduct duplexes were obtained i n the States-TPPI mode (49) with a 2 s presaturation of the HDO signal between scans and with sweep widths in both D1 and Dz dimensions set to 10 PPm.
Results Large-Scale Synthesis of Adducts Derived from the Binding of (f)-anti-B[c]PhDEto d(CTCTCACTTCC). A series of experiments were conducted in order to identify the optimum conditions with respect to the reaction yields and reaction time; the details are described e1sewhe1-e.~The progress of the reaction was periodically examined by subjecting a 10 pL aliquot to HPLC analysis using an elution gradient of 0-90% MeOW20 mM sodium phosphate buffer (pH 7.0) for 60 min. A typical elution profile of such a sample is shown in Figure 1A. The unmodified 11-mer oligonucleotide elutes first, at about 26 min, followed by the B[clPhDEmodified oligonucleotides which elute between 29 and 32 min. The B[clPhDE hydrolysis products, the trans- and cis-tetraols B[c]PhT (1,2,3,4-tetrahydroxytetrahydroben~~[clphenanthrene), elute at about 46 and 49 min, respectively. The most abundant hydrolysis product is the trans-tetraol(50), which elutes before the cis-tetraol. The diol epoxides B[c]PhDE elute last, at about 54 min (Figure 1A). The complete disappearance of this latter peak marked the end of the reaction. The two poorly resolved peaks eluting between 29 and 32 min (Figure 1A) constitute the two major adducts resulting from the covalent binding of (+)-anti-B[c]PhDE to d(CTCTCACTTCC). The eluates containing these adducts were collected and separated by a second HPLC cycle (Figure 1B). The first major BCcIPhDE- -1igonucleotide product (I) elutes between 56 and 62 min, while the second product (11) elutes between 74 and 80 min. The oligonucleotide adducts eluting earlier (up to 56 min) were not identified in this work. Each of the solutions containing the Laryea, A. (1995) Ph.D. Dissertation, New York University, in preparation.
(B)
1
4
I I
0.08 .
0
Unidentified adducts
40
80
1 10
TIME, min Figure 1. (A) HPLC elution profile of a (*)-anti-B[c]PhDEd(CTCTCACTTCC) reaction mixture sampled after a 96 h reaction time. HPLC elution: reverse phase, linear 0-90% (MeOH)/(20 mM sodium phosphate buffer, pH 7.0 buffer solution) in 60 min. (B) Elution profile of the 29-33 min fraction in part A (reverse phase, 20-24% (methanol)/(20 mM sodium phosphate buffer, pH 7.0 buffer solution) in 120 min. The two major products of the covalent binding reaction of (i)-anti-B[clPhDE with d(CTCTCACTTCC) are labeled I and 11. Oligo: unmodified d(CTCTCACTTCC); trans-T and cis-T: trans- and cis-tetraols derived from the hydrolysis of (f)-anti-B[clPhDE, respectively. Each of the two products, I and 11, were collected separately and further purified by reverse HPLC using the following regimen: 0-20% MeOWNaZHP04 buffer (pH 7.0) for 10 min, followed by a gradient of 20-40% MeOWNaZHP04 (pH 7.0) for 60 min, and a final purification step under isocratic conditions using a 22% methanoU20 mM sodium phosphate solution for 120 min.
purified adducts I and I1 were desalted on a G-25 Sephadex column and evaporated to dryness. Small-scale Synthesis of Adducts Derived from the Binding of (+I- and (-)-anti-B[clPhDE Enantiomers to d(CTCTCACTTCC). In order to help characterize the stereochemical properties of adducts I and 11, small quantities of (+)-anti-BLcIPhDE and (-)-antiB[c]PhDE were synthesized. These enantiomers were then used to generate 11-mer oligonucleotide adduct standards derived from the covalent binding of each of these enantiomers with d(CTCTCACTTCC) in separate reaction^.^ These two 11-mer adduct standards coeluted with either adduct I or adduct I1 (concentration ratios 1:1)resulting from the reaction of (f)-anti-B[clPhDEwith d(CTCTCACTTCC) (Figure 2). The product designated as I coelutes with the (+I-anti-B[clPhDE-oligonucleotide adduct, while I1 coelutes with the (-)-anti-B[clPhDEoligonucleotide adducts (the same elution protocols as described in the above paragraph was used). Therefore, adducts I and I1 are products derived from the covalent reaction of (+)- and (-)-anti-B[c]PhDE, respectively, with the oligonucleotide d(CTCTCACTTCC). The purity of the adducts was also checked using denaturing 20% polyacrylamide gels (data not shown);
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 447
Synthesis of Benzo[clphenanthrene-DNA Adducts 2%
(A) From II, 12 h
1.5-
3’-dAMP Adduct
1-
dC 0.5
-
dT \
40 50 60 TIME, min Figure 2. Trace labeled “I+(+)”: HPLC elution profiles of 1:l mixtures of oligonucleotide adduct I and the single major oligonucleotide product obtained from the reaction of (+)-antiB[clPhDE with d(CTCTCACTTCC). Trace labeled “II+(-)”: HPLC elution profiles of 1:l mixtures of oligonucleotide adduct I1 and the single major oligonucleotide product obtained from the reaction of (-)-anti-B[c]PhDE with d(CTCTCACTTCC). Elution programs: 0-20% (methanol)/(2O mM sodium phosphate buffer, pH 7.0) for 10 min, followed by a 20-40% gradient for 60 min. 20
30
both adducts I and I1 yielded single bands that migrated somewhat slower than the unmodified ll-mers. Enzyme Digestion of d(CTCTCAC’ITCC)and Oligonucleotide Adducts I and 11. The stereochemical nature of adducts I and I1 was determined by comparison of the HPLC retention times and the UV and CD characteristics of enzyme digested products with antiB[clPhDE-3’-dAMP mononucleotide adduct standards. A significant difference in the rates of digestion of oligonucleotide adducts I and I1 by each enzyme was noted. Adduct I was more easily digested than adduct I1 by SVPD, while adduct I1 was more easily digested by SPD than adduct I. For example, under the conditions described in Materials and Methods, SVPD easily digested the unmodified oligonucleotideand oligonucleotide adduct I in 12 h; however, adduct I1 was only partially digested under these same conditions as ascertained by HPLC a n a l y ~ i s .Complete ~ digestion of adduct I1 with SVPD could be achieved only upon doubling the enzyme concentration and extending the digestion period to 7 days. These stereoselective enzyme digestion effects are illustrated in Figure 3, which depict the reverse phase HPLC elution profiles of the SPD enzyme digestion products. After a digestion period of 12 h, adduct I1 is completely digested and only the unmodified mononucleotides 3’-dCMP and 3’-dTMP are evident (retention times of about 10 and 16 min, respectively). As expected, the unmodified mononucleotide 3’-dAMP is missing; instead, a new product eluting at 43-44 min is evident (Figure 3A). In contrast to adduct 11, the digestion rate of adduct I by SPD is considerably less efficient, as indicated in Figure 3B,C. In addition to the eluate peaks corresponding to 3’-dCMP, 3’-dTMP, and a B[clPhDE-3’-dAMP mononucleotide adduct with a retention time of -44 min, a single additional peak at 34 min is observed after 12 h of digestion (Figure 3B); this product bears a covalently bound B[c]PhDE chromophore, as ascertained from the absorption spectrum measured with an on-line diode array detector (data not shown). However, it is shorter in length than the full ll-mer adduct I, and we therefore attribute the eluate at 34 min to an SPD enzyme digestion-resistant fragment of oligonucleotide adduct I, similar to those observed in the case of covalent B[a]PDE-oligonucleotide adducts (33). After a digestion period of 7 days, adduct I was eventually completely
(B) From I, 12 h
Partially digested Adduct I
1
U.3
A ‘ I fl4
io
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I
dT 20
30
L 40
’1
I
I
1.51
0.5
50
dC
-
010
20
30
40
50
TIME, min Figure 3. HPLC elution diagrams of enzyme digests of oligonucleotides I or I1 (reverse phase, linear gradient of 0-90% (methanol)/(aO mM sodium phosphate buffer, pH 7.0 buffer solution) in 60 min. (A) Spleen phosphodiesterase digestion of oligonucleotide adduct 11, after a 12 h digestion period. (B) Spleen phosphodiesterase digestion of oligonucleotide adduct I, after a 12 h digestion period. (C) Spleen phosphodiesterase digestion of oligonucleotide adduct I, after a 7 day digestion period.
digested to the mononucleotide level, however, as shown in Figure 3C. The complete digestion of the unmodified oligonucleotide d(CTCTCACTTCC) required less than 1 h of digestion time and only 1/10 of the amount of enzyme (0.3 unit/mL); the ratio of C:T:A was the expected 6:4:1 in the unmodified oligonucleotide case. However, upon prolonged digestion of adducts I and I1 at the high enzyme concentrations (3 units/mL) that are necessary for the degradation of the modified oligonucleotides to the B[clPhDE-3’-dAMP mononucleotide adduct level, there were deviations from the expected nucleotide ratios. The cause of these discrepancies was not further investigated, but the nucleotide composition of adducts I and I1 was independently verified by NMR methods (40).2 Coelution of Mononucleotide Adducts Obtained from Enzyme Digestion of I and I1 with anti-B[clPhDE-3’-dAMPAdduct Standards. The reverse phase HPLC elution profile of a (f)-anti-B[clPhDE-3’-dAMP reaction mixture is shown in Figure 4 (traces A, dotted lines). From a comparison of elution profiles obtained from adducts prepared from the reaction of the (+I- and (-)-enantiomers of anti-B[clPhDE with 3’-dAMP, and from the UV absorption and CD spectra published by Dipple et al. (7) and Agarwal et al. (8), the adducts were identified (listed in the order of increasing elution times) as the (+)-cis-, (-)-cis-, (-)-trans-, and (+)-truns-B[c]-
448 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Laryea et al.
i
0.5 -
240
,
:
I: (+)-trans from I
400
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Wavelength, nm
A
01
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2: (-)-trans from II I
I
250
I
I
350 300 Wavelength, nm
I
I
400
Figure 5. CD spectra of 43-44 min eluting fractions from SPD enzyme digests of oligonucleotide adducts I and I1 (Figure 4). The CD signal in units of millidegrees is divided by the 2
0.5 0
absorbance a t the 258 nm maximum. Solvent: 1:1 (methanol)/ (20 mM sodium phosphate buffer solution, pH 7.0). Solid line: (+)-trans-anti-B[c]PhDE-N6-3’-dAMP, and dotted line: (-1trans-anti-B[c]PhDE-N6-3’-dAMP adduct.
1
A
adduct I is therefore identified as 5’-d(CTCTCh*Cn’CC), where A* represents the (+)-trans-anti-B[clPhDE-N6-dA residue. TIME, min The 43-44 min eluting fraction of the enzyme digest Figure 4. HPLC elution diagrams of covalent (%)-anti-B[clof adduct 11 coelutes with the (-1-trans-anti-B[clPhDE-3’-dAMP reaction products (elution gradient: same as in Figure 2). Trace A (dotted lines, panels A and B): covalent PhDE-3’-dAMP adduct standard (Figure 4B, trace B). reaction products of (%)-anti-B[clPhDEwith S’-dAMP. 1: (+ITherefore, oligonucleotide I1 is identified as 5-d(CTCTcis-,2: (-)-cis-, 3: (-)-trans-, and 4: (+)-trans-anti-B[clPhDECA*CTTCC), where A* represents the (-)-trans-anti-B[clN6-3’-dAMP adducts. (Panel A) Trace B (solid line): 43-44 min PhDE-N6-dA residue. fraction from SPD enzyme digest of oligonucleotide adduct I (Figure 4)mixed with the (+)-trans-anti-B[c]PhDE-N6-3’-dAMP The CD spectra of the 43-44 min eluates of the enzyme adduct standard. (Panel B) Trace B (solid line): 43-44 min digests of adducts I and I1 are shown in Figure 5; these fraction from SPD enzyme digest of.oligonucleotide adduct I1 (Figure 4) mixed with the (-)-trans-anti-B[c]PhDE-N6-3’-dAMP are nearly mirror images of one another and are identical to the CD spectra of the corresponding (+)- and (-)-transadduct standard. Insert (panel A): Absorption spectra of (+Itrans- (dotted line) and (+)-cis-anti-B[c]PhDE-N6-3’-dAMP adanti-B[c]PhDE-N6-dAnucleoside adducts published by duct standards (solid line). Agarwal et al. (8). The eluate maxima of traces B coincide approximately with the appropriate (+)- or (-)-transPhDE-N6-3’-dAMPadducts. The relative proportions of anti-B[c]PhDE-N6-dAmaxima in traces A in Figure 4; a these four stereochemically different products are similar complete coincidence is not expected because of small to the yields reported for the reaction of (f)-anti-B[cldifferences in elution times observed in different experiPhDE with dA (8), and the order of elution is also similar ments. for these 3’-dAMP and dA adducts (42). These reaction Analysis of S2P-PostlabelingResults. The stereoproducts are characterized by absorption spectra similar chemical assignments of oligonucleotide adducts I and to those exhibited by authentic (+I- and (-)-trans-anti11were further confirmed by the 32P-postlabelingmethod. B[clPhD-3’-dAMP adducts (insert, Figure 4). The (-1In previous studies, Canella et al. (42) synthesized four trans-anti-B[c]PhDE-3’-dAMP and (+)-trarp-anti-B[c]stereoisomeric adducts by reacting anti-B[c]PhDE with PhDE-3’-dAMP adducts were separately collected (peaks 2’-deoxyadenosine 3’-phosphate, as well as the corre3 and 4, respectively, in traces A in Figure 4) and were sponding 2’-deoxyadenosine 3’,[32P15’-bisphosphateisofurther purified by additional HPLC separation steps. mers by subsequent labeling with [y-32PlATP;the thin The purified products exhibited the CD and UV absorplayer chromatography (TLC) patterns of these isomeric tion spectra characteristic of trans adducts (7,8) and were bisphosphates were then compared. The covalent antiused as the adduct standards in coelution experiments B[c]PhDE-2’-deoxyadenosine 3’-phosphate adducts obwith the B[c]PhDE-3’-dAMP adducts isolated from the tained from the enzymatic digestion of oligonucleotide enzyme digests of oligonucleotide adducts I and I1 (the adducts I and I1 were labeled, and their TLC migration products eluting at 43-44 min shown in panels C and characteristics were compared with those of the 32Plabeled standard markers (+)-trans-,(+)-cis-,(-)-trans-, A, respectively, Figure 3). The results of these coelution and ( -)-cis-anti-B[c]PhDE-N6-adenosine bisphosphates experiments are shown in Figure 4, panels A and B. In (markers 1, 2, 3, and 4, respectively). Figure 4A, trace B represents the coelution of an approximately 1:l mixture of the -44 min eluate of the SPD The results obtained upon co-spotting the standard enzyme digest of adduct I and the (+)-trans-anti-B[clmarkers with the mononucleotide adducts obtained from PhDE-3‘-dAMP mononucleotide adduct standard. Only the enzyme digestion of oligonucleotides I and I1 on PEI one peak is observed, which shows that these two plates, which were then subjected to two-dimensional chromatography, are shown in Figure 6. Plates A and samples have the same retention times; their CD and UV B show the spots obtained upon plating the mononuclespectra are also identical to one another. Oligonucleotide 10
20
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Chem. Res. Toxicol., Vol. 8, No.3, 1995 449
Synthesis of Benzo[clphenanthrene-DNA Adducts
1: Oligo
2: Adduct I
1 i
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-10
’
280
320
360
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WAVELENGTH, nm
Figure 6. Autoradiographic results of the 32P-postlabelingof mononucleotide 3’-monophosphate adducts derived from the enzymatic hydrolysis of oligonucleotide adducts I (A) and I1 (B). (C) Cochromatography of bisphosphates derived from mononucleotide adduct I and marker 1((+)-trans-anti-B[clPhDE-N6dA bisphosphate standard). (D) Cochromatography of bisphosphates derived from mononucleotide adduct I and marker 2 ((+)cis-B[c]PhDE-N6-dA bisphosphate standard). (E) Cochromatography of bisphosphates derived from mononucleotide adduct I1 and marker 3 ((-)-trans-anti-B[clPhDE-N6-dA bisphosphate standard). (F)Cochromatography of bisphosphates derived from mononucleotide adduct I1 and marker 4 ((-)cis-anti-B[clPhDEN6-dA bisphosphate standard). The origin is situated at the lower right-hand corner.
otide adducts I and 11; single spots are obtained which show that these products are pure as judged by this assay. Co-spotting of mononucleotide adduct I with marker 1and with marker 2 does not resolve this mixture under the chromatographic conditions employed here (plates C and D, respectively, Figure 6). Because markers 1and 2 produce a single spot on the TLC plates, the specific assignment of (+)-trans or (+)-cis stereochemistry to mononucleotide adduct I had to be based on the CD spectra and HPLC coelution characteristics with the appropriate mononucleotide 3’-phosphate standards (see above);the mononucleotide adduct I was identified as the (+)-trans-B[c]BhDE-N6-3’-dAMP adduct in this manner. Plates E and F show the results of co-spotting the mononucleotide bisphosphate adduct I1 with marker 3 and marker 4,respectively. Plate E exhibits only one spot, suggesting that this adduct is identical to marker 3, which is a (-)-trans-B[c]PhDE-N6-dA bisphosphate derivative. Plate F shows two separate spots with equal intensities, indicating that mononucleotide adduct I1 is different from the marker 4, the (-)-cis-B[clPhDE-N6dA bisphosphate adduct. Spectroscopic Characteristics of the Oligonucleotide Adducts I and 11. The diol epoxide B[clPhDE exhibits a typical phenanthrene-like absorption spectrum
Figure 7. (A) Typical absorption spectra of (1)unmodified oligonucleotide d(CTCTCACTTCC) and (2) anti-B[clPhDE-modified oligonucleotide adduct I. (B) CD spectra of of (1)unmodified oligonucleotide d(CTCTCACTTCC), (2) B[c]PhDE-modified oligonucleotide adduct I, and (3)adduct 11.Solvent: 20 mM sodium phosphate buffer solution, pH 7.0. The CD signals are normalized with respect to the absorbance a t 252 nm (expressed in absorbance units, AU).
with a maximum ai 260 nm; upon hydrolysis to the tetraols B[c]PhT, the maximum shifts to 256 nm (data not shown). The UV absorption spectrum of the unmodified oligonucleotide d(CTCTCACTTCC) exhibits a maximum at 268 nm (Figure 7A). The absorption spectra of adducts I and I1 are indistinguishable from one another and exhibit a single broad band in the 220-310 nm region with a maximum at 259 nm and a weak shoulder in the -302-305 nm region (Figure 7A). These absorption spectra are approximately superpositions of the absorption spectra of B[c]PhT and the oligonucleotide d(CTCTCAC’M’CC) (data not shown). Analysis of the phosphate content of the oligonucleotideadducts (45), and a comparison of these results with the absorbance of the same adducts in solution, leads to an estimate of the molar extinction coefficients of adducts I and I1 of 6259 = (13.0 f 0.8) x lo5 M-l cm-l. The CD spectra of the unmodified oligonucleotide and oligonucleotide adducts I and I1 are depicted in Figure 7B. The CD signals are normalized by dividing the measured values in units of mdeg by the absorbance of the samples at the maxima (259 nm, 1cm path length), as recommended by Weems and Yang (51). The molar ellipticities at the positive CD maxima are about 100 deg M-l cm-l. The CD spectra of oligonucleotide adducts I and I1 closely resemble the superpositions of the CD spectra of the unmodified oligonucleotide and the (+)trans-anti-B[c]PhDE-N6-3’-dAMP or ( -)-trans-anti-B[c]PhDE-N6-3’-dAMP mononucleotides. The CD signal of the (+)-trans-anti-B[c]PhDE-N6-3’-dAMP mononucleotide adduct is negative below about 260 nm and is positive
450 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
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Laryea et al.
(A) UM Duplex
'"1
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-
(C) II Duplex 1.6
1 A
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10
20
30
40
'
io
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70
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T, ' C Figure 8. Melting profiles measured at 260 nm of the unmodified oligonucleotide duplexes d(CTCTCACTTCC)d(GGAAGTGAGAG) and the corresponding duplexes derived from oligonucleotide adducts I and 11. Solvent: 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaC1.
above this wavelength; accordingly, the CD spectrum of oligonucleotideadduct I is more negative at -250 nm and more positive at -280 nm than that of the unmodified oligonucleotide(Figure 7B). In the case of oligonucleotide adduct 11, the CD signal is less negative near 250 nm, and less positive near 290 nm, which is again consistent with an approximate superposition of the CD spectra of the (-)-trans-anti-B[c]PhDE-N6-3'-dAMP mononucleotide adduct and the unmodified oligonucleotide. These CD spectra are useful for distinguishing the two trans-modified oligonucleotide adducts I and I1 from one another. The weak CD signals in the 295-320 nm region, corresponding to the weak optical transition in this region, is assumed to be of an induced nature. Thermal Stabilities of Duplexes. By using the molar extinction coefficients of the modified and unmodified oligonucleotides, 1:1 duplexes of oligonucleotide adducts I and I1 with the complementary strand d(GGAAGTGAGAG) were prepared. The samples were slowly heated to 75 "C, kept at that temperature for 10 min, and then allowed to cool to 4 "C overnight. The melting curves shown in Figure 8 were then recorded. The melting points T,, determined from the maxima in the derivatives of the melting curve (521,were T, = 43.8 f 0.5 "C for the unmodified control duplex, T , = 43.3 i 0.5 "C for the duplex of adduct I, and T , = 43.3 f 0.5 "C for the duplex of adduct 11. These T, values are similar to one another, within experimental error. The hypochromicities measured at the absorption maxima (259 nm) were 16 f 2%, 23 f 2%, and 25 f 2%, respectively. Some NMR Characteristics of Adduct I and I1 Duplexes. While detailed accounts of the NMR spectra of duplexes of adducts I and I1 with their natural complementary strands d(GGAAGTGAGAG) have been provided (40),2 we discuss here some relevant aspects of their NMR characteristics that were not emphasized in these publications (40).2 The full one-dimensional proton NMR spectrum in DzO in the 0.6-9.0 ppm range is
shown in Figure 9 (elsewhere (40): only the region above 5.1 ppm is shown). Narrow and well-resolved resonances are evident throughout the entire spectrum, indicating that there is no significant broadening due to multiple conformations or excessive flexibility at or near the binding site due to loss of hydrogen bonding. The coupling between protons in the benzo[clphenanthrenyl residues in oligonucleotide I and I1 duplexes provides information about the benzylic ring conformations. Expanded contour plots of the phase-sensitive COSY spectra correlating the three-bond vicinal protonproton coupling of the benzo[clphenanthrenyl protons of the (-1- and (+)-trans-anti-B[clPhDE-dA.dT 11-mer duplexes in DzO buffer, pH 7.0 a t 25 "C, are plotted in Figure 10. The COSY crosspeak and chemical shift assignments of the benzo[clphenanthrenyl protons of both adduct duplexes are provided in the caption to Figure 10. The numbering schemes of the protons on the B[c]PhDE residue are indicated in Figure 9, while the numbering scheme of the different nucleotide residues in the duplex is indicated below: 5'-d(Cl--n---C3---T4---C5--A*6-C7--T8---T9-~ClO--Cll)
3'-d(G22-A21-G20-A19-Cil8-T17-G16-A15-A14-G13-G12)
The COSY crosspeaks between the B[clPh(H2) and B[clPh(H3) protons of both adduct duplexes and between B[clPh(H5) and B[c]Ph(H6) protons of the (+)-trans-antiB[clPhDE-N6-dA.dT 11-mer duplex I are located close to the diagonal and thus are not shown. As expected, strong COSY crosspeaks are observed for the three-bond vicinal proton-proton coupled phenanthrenyl protons located on the same aromatic ring, i.e., the crosspeaks between B[c]Ph(H5) and B[clPh(H6) protons of the (-)-trans-antiadduct I duplex (peak E, Figure 10A, and peak D, Figure 10B, respectively). The solid lines which connect the crosspeaks labeled A, B, and C in Figure 10, panels A and B, show the through-bond connectivities among the four B[clPh(H9),B[cIPh(HlO), B[cIPh(Hll), and B[clPh(H12) protons located on the same phenanthrenyl aromatic ring. The coupling pattern and intensities of these crosspeaks are more complex than that observed between the B[clPh(H5)-B[c]Ph(HG) and B[cIPh(H7)-B[clPh(HS) protons because the BlcIPh(H10) proton is not only coupled to the B[clPh(H9) proton (peak C, Figure 10A,B), but also to the B[c]Ph(11)proton (peak B, Figure 10A,B), which in turn is coupled to the B[c]Ph(H12) proton (peak C, Figure 10A,B). Similar complex COSY patterns are observed for the benzylic ring B[c]Ph(Hl), B[c]Ph(H2), B[clPh(H3), and B[clPh(H4) protons of both adduct duplexes (peak G, Figure 10A, and peak F, Figure 10B, corresponding to the adducted (-1- and (+)-stereoisomers, respectively) and B[clPh(H3)-B[clPh(H4) protons (peak F, Figure 10A, and peak E, Figure 10B, corresponding to the adducted (-1- and (+)-stereoisomers, respectively). Some of the features of the full 1-D NMR spectrum (Figure 9) are noteworthy and provide some important information about adduct conformations. The nucleic acid purine H8, pyrimidine H6, and adenosine H2 base protons of the adduct duplexes resonate between 6.8 and 9.0 ppm. The most downfield resonance (at 8.52 ppm, peak 1 in Figure 9A, and at 8.62 ppm, peak 1 in Figure 9B), observed in both of the 1-D NMR spectra, corresponds to the H8 proton of the modified adenosine (dA*6) residue; this moderate downfield chemical shift reflects the loss of stacking interactions between this B[c]PhDEmodified adenosine residue and the neighboring dC7
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 451
Synthesis of Benzo[clphenanthrene-DNA Adducts
B
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residue (40A2By contrast, the most upfield nucleic acid base resonances observed in the proton spectra of both duplex adducts (at 6.98 ppm, peak 2, Figure 9A, and at 6.97 ppm, peak 2, Figure 9B) are assigned to the H6 proton of the partner thymidine residue (dT17) located on the complementary strand opposite B[clPh-dA*6, which is due to ring current effects associated with the benzo[clphenanthrenyl residues which are stacked over the dT17 residues (40);2 consistent with such stacking interactions, the dT17 methyl protons of both adduct duplexes also resonate upfield (at 1.30 ppm, peak 3, Figure 9A, and at 0.94 ppm, peak 3, Figure 9B) relative to the dT17-CH3 resonances of the four thymidine methyl protons (1.5-1.8 ppm) which are located 2 or more base pairs away from the binding site.
Discussion Reaction Yields. Using the reaction conditions described, typical initial yields of anti-B[clPhDE-modified oligonucleotides by trans addition to dA were 10 f 2% based on the amount of oligonucleotide in the solution (the yield based on the amount of B[clPhDE initially added is 4 times lower, or %2.5%). The yield of oligo-
through-bond connectivities of the benzo[clphenanthrenyl protons (three-bond coupling). (A) (-)-trans-Oligonucleotide adduct I1 duplex (3.8-4.5 ppm, and 6.0-7.6 ppm to 5.0-7.3 ppm regions). (B) (+)-trans-Oligonucleotideadduct I duplex (4.04.5 uum and 5.7-6.9 uum to 4.8-6.5 Dum repions). The crosspeaks A-G in (A) ariassigned as follo6i: A, BklPh(Hl2)B[clPh(Hll): B. BTclPh(HlO)-BTclPh(Hll): C. BTclPh(Hl0iB[clPh(H9); D, B[~lPh(H5)-B[clPh
