Synthesis and conformation of a dinucleoside monophosphate

Robert Shapiro, Stephen Ellis, Brian E. Hingerty, and Suse Broyde. Chemical Research in Toxicology 1998 11 (4), 335-341. Abstract | Full Text HTML | P...
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Chem Res T O X L C1988, O ~ . I , 152-159

152

Synthesis and Conformation of a Dinucleoside Monophosphate Modified by Aniline’ Michael D. Jacobson, Robert Shapiro,* Graham R. Underwood, Suse Broyde, Lynne Verna, and Brian E. Hingerty’ Departments of Chemistry and Biology, New York University, New York,New York 10003, and Health and S a f e t y Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received January 19, 1988

T h e modified dinucleoside monophosphate, N- [deoxycytidylyl-(3’-5’)-guanosin-8-yl] aniline (dCprG-An) has been prepared by the phosphotriester synthesis approach, using suitably blocked derivatives of d C p and N-guanosin-8-ylaniline (rG-An). T h e latter compound was synthesized by a route t h a t featured nucleophilic displacement by antiline upon a n 8-bromoguanosine derivative, A number of attempts t o prepare N-(deoxyguanosin-8-yl)aniline(dG-An) by electrophilic substitution, using activated aniline derivatives, failed. Nucleophilic substitution reactions of aniline with 8-bromodeoxyguanosine derivatives afforded only the base, Nguanin-8-ylaniline. T h e conformation of dCprG-An has been studied by CD, proton magnetic resonance, a n d minimized potential energy calculations. A flexible molecule with a mixture of conformers is indicated. Base-base stacked states predominate, in contrast to the case of a dimer containing 4-aminobiphenyl bound t o the 8-position of guanine, where carcinogen-base stacked states are dominant. T h e mutagenic a n d carcinogenic activities of aniline are much less t h a n those of many polycyclic aromatic amines. T h e diminished stacking ability of the aniline ring, as well as the weak electrophilic reactivity of activated aniline derivatives, may be a cause of this weak biological activity.

Introduction We have been investigating the effect of covalently bound aromatic residues on oligonucleotide conformation, with particular emphasis on those derived from carcinogenic and mutagenic aromatic amines. In a recent paper, we compared the properties of a synthetic deoxydinucleoside monophosphate containing the major adduct derived from the carcinogen 4-aminobiphenyl with those of one containing the more mutagenic analogue 2-aminofluorene (1). A combination of spectroscopic studies and minimized potential energy calculations was used to obtain conformational information. We have now extended our comparison to include the parent member of the aromatic amine series, aniline. Human cancer caused by exposure to aromatic amines was first noted in a dye factory that used commercial aniline as a raw material, and the bladder tumors observed were termed “aniline cancer”. Subsequent epidemiological studies cleared aniline of suspicion in this case and implicated various polycyclic amines (2, 3). More recently, aniline has been shown to bind to DNA and induce DNA damage in certain rat strains ( 4 ) . At high concentrations, it induces spleen tumors in these strains, although it is a noncarcinogen in mice ( 5 ) . Mutagenesis tests with aniline in various bacterial species have been negative (4). Aniline has been classified as a weak genomic carcinogen by one group (6) and a noncarcinogen by another (7). Whichever classification is ultimately accepted, aniline is clearly less potent biologically than many of its polycyclic analogues ( 3 ) . Many factors may contribute to this difference. Mutagenesis and especially carcinogenesis are complex multistep processes. When induced by a chemical, the steps include entry of the chemical into a cell, t

Oak Ridge National Laboratory.

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metabolic activation, reaction with the crucial cellular target (presumably DNA), and the interaction of the modified DNA with the repair and replication systems that process it. We have observed that a dinucleoside monophosphate modified by aniline has less tendency to assume novel carcinogen-base stacked conformational states than one containing bound aminobiphenyl. In addition, modified aniline derivatives exhibit less electrophilic reactivity to deoxyguanosine than their polycyclic analogues.

Methods Except where noted, materials and general methodology are as described previously ( 1 ) . HPLC mobile phases used (with C-18 column) are as follows: A, 10-85% methanol (aqueous), linear gradient, 15 min; B, 20-8070 methanol (aqueous), linear gradient, 20 min; C, 1 5 4 5 % methanol (aqueous), linear gradient, 20-min delay, 5-min sweep; D, (with DEAE column) 5-250 mM (NH4)2C03, pH 7.5, linear gradient, 15 min. Preparation of N-Guanin-8-ylaniline(I). Guanine 3-oxide (34) (80 mg, 0.48 mmol) was added to DMSO (30 mL) and DMF (3 mL) at 80 “C. The mixture was cooled to room temperature and stirred. Aniline (0.12 mL, 1.3 mmol) and acetic anhydride (0.08 mL, 0.85 mmol) were added. The solution turned deep rose. The mixture was stirred overnight at room temperature, and the mixture was then evaporated and recrystallized twice from 1 M Abbreviations: dCprG-An,N - [ 2’-deoxycytidylyl-( 3’-5’)-guanosin-8yllaniline; rG-An, N-guanosin-8-ylaniline; dG-An, N-(2’-deoxyguanosin8-y1)aniline; dCprG, 2’-deoxycytidylyl-(3’-5’)-guanosine;dAprA, 2‘deoxyadenylyl-(3’-5’)-adenosine; rApdA, adenylyl-(3’-5’)-2’-deoxyadenosine; dApdA, 2’-deoxyadenylyl-(3’-5’)-2’-deoxyadenosine; dCp, 2’deoxycytidine 3’-phosphate; dCpdG-An, N- [2’-deoxycytidylyl-(3’-5’)-2’deoxyguanosine-8-yl]aniline;rG-An, N-guanoxin-8-ylaniline; dCpdG, 2’-deoxycytidylyl-(3’-5’)-2’-deoxyguanosine;CD, circular dichroism; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; EDTA, ethylenediaminetetraacetic acid; TLC, thin-layer chromatography; rG, guanosine; dC, 2’deoxycytidine; DMT, dimethoxytrityl; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol;iPrG, 2’,3’-O-isopropylideneguanosine.

0 1988 American Chemical Society

Aniline-Modified Dinucleoside Monophosphate

Chem. Res. Toxicol., Vol. 1, No. 3, 1988 153

HCl to yield white crystals (80 mg, 51%). The free base was liberated by addition of NaOH. TLC: R, 0.45 (95% CH3CN). HPLC (program A): tR 15 min. UV (HzO): A, ( e ) [pH 1.81 295 nm (12100), 261 (15200),A, 281 nm; [pH 61 A, 312 nm (15900), 256 (16300), A,, 278 nm; [pH 11.01 A, 300 nm (15400), 251 (10400), A, 263 nm. pK,s: 9.55. Isosbestic point: 323 nm. NMR (DMSO-d,), 6: 10.7, s, 1 H (NH-1); 9.3, s , 1 H (NH aniline); 7.7 d, 2 H (H2, 6); 7.2, t, 2 H (H3, 5); 6.85, t, 1 H (H4). Anal. Calcd for CllHloN60: C, 54.54; H, 4.16; N, 34.69. Found: C, 54.32; H, 4.12; N, 34.44. Alkaline hydrolysis was performed on this material: N guanin-tkylaniline (1mg, 4 pmol) was dissolved in 0.75 mL of DMF with stirring. Aqueous NaOH (0.5 mL, 2 M) was added, and the mixture was heated (16 h, 75 “C). The mixture was neutralized, and HPLC analysis of the resultant mixture (program A) indicated that several products had formed, the major ones being 8hydroxyguanine (35) and aniline.

2-chlorophenyl phosphate triethylammonium salt IX (42 mg, 45 pmol) and 2’,3’-0-isopropylideneguanosine(XIIa) (14 mg,45 pmol) were coevaporated (3 x 1 mL pyridine) and maintained under a vacuum overnight a t room temperature. 1-(Mesitylenesulfonyl)-3-nitro-1,2,4-triazole (X) (75 mg, 253 pmol) was added to the resultant syrup under argon. Pyridine (1mL) was added, upon which the syrup dissolved and the mixture turned yellow. After 5 min, TLC (95% CH3CN aqueous) showed some remaining blocked dCp (Rf0.151, all the iPrG consumed, and a new pair of compounds eluting as a “dumbbell” (R, 0.33) suggestive of a pair of diasteriomers (36). Ice-water was added and, after 20 min, 1 mL of 5% NaHC03 added. The mixture was extracted with CHC1, (4 X 2 mL). The CHC1, layer was dried (MgS04),filtered, and evaporated. HPLC (program A) showed the major product as a mixture of two overlapping peaks ( t R 34 min) which possessed UV spectra identical with a 1:lmixture of blocked dCp/guanosine (A,, 238, 259, 304 nm). These peaks were separated from unPreparation of N-(2’,3’,5’-Tri-O-acetylguanosin-8-y1)- reacted blocked dCp by flash chromatography. The mixture was dissolved in approximately 500 pL of CH&N and applied to a aniline (VII). 8-Bromo-2’,3’,5’-tri-O-acetylguanosine (3.0 g, 6.1 silica gel H column 1.2 X 4.5 cm. Fifteen 5-mL fractions were mol), freshly distilled aniline (6.0 mL, 65 mmol), and aniline collected by adding 5 mL of 95% CH3CN (aqueous) to the column hydrobromide (1.2 g, 6.9 mmol) were suspended in 300 mL of dry and sucking the column dry under vacuum for each. The major isopropyl alcohol in a 500-mL screw-cap bottle. The mixture was product, VIIIa, eluted in fractions 3-7. These fractions were stirred a t 100 “C (60 h), during which time all the solid dissolved, evaporated, and dissolved in 4 mL of dioxane/methanol/0.2 M producing a brown solution. Following the reaction, the isopropyl NaOH (1:1:2). After 1 h no starting material remained (TLC). alcohol was evaporated and the resultant brown mass recrystallized HCl(4 mL, 1M) and methanol (1mL) were added to the solution. several times from ethanol/water to give 1.45 g of crystals, which After 6 h, deblocking was complete (HPLC). The mixture was quickly turned brown on exposure to air (48%). neutralized and extracted (3 X 5 mL) with E t 2 0 . There was a TLC: R, 0.67 (95% CH,CN, aqueous), 0.42 (CHCl,/methanol, single UV-absorbing product in the aqueous extract. All the DMT 9:l). HPLC (program A): t R 22 min. UV, A, (e): [pH 71 280 material was extracted into the ether layer. The aqueous extract nm (22000); [pH 11 270 nm (15300); [pH 121 288 nm (14600). was purified by DEAE HPLC, program D. Approximately 8 mg pK,’s: 3.34, 10.17. Isosbestic points: 266 (pH 2-8), 280 nm (pH of dCprG (VIIIa) was obtained (30%). TLC: R, 0.52 (i8-11). NMR (DMSO-d,) 6: 7.40, d, 2 H (aniline 2, 6); 7.27, t, 2 PrOH/NH,0H/H20, 6:3:1). HPLC (DEAE, program D): tR 8 H (aniline 3, 5); 6.96, t, 1H (aniline 4); 6.21, t, 1H (rG H2’); 6.08, min. d, 1 H (rG Hl’); 5.78, t , 1 H (rG H3’); 4.4, m, 1 H (rG H5’); 4.3, The UV of this purified material was nearly identical with that m, 2 H (rG H4’, H5”); 2.13, s, 3 H (acetyl); 2.06, s, 3 H (acetyl); of rCprG a t pH 1,7, and 12. UV A, (e): [pH 71 254 nm (19 100); 1.98, s, 3 H (acetyl). Mp 155 “C dec. Anal. Calcd for C22H24Ns08: [pH 11 278 nm (22600); [pH 121 269 nm. CD (e): (in MeOH) C, 52.8; H, 4.80; N, 16.80. Found: C, 52.62; H , 4.83; N, 16.53. 275 nm (0.8 X lo4); (in 1mM potassium phosphate p H 7.05) 310 Preparation of N-Guanosin-8-ylaniline. N-(2’,3’,5’-Tri-O275 nm (0.64 X nm (-0.22 X 245 (-0.62 X W4)(see acetylguanosin-8-y1)aniline(300 mg, 0.6 mmol) was dissolved in Figure 5). NMR (D20) 6: 8.06, s, 1 H (G-8); 7.57, d, 1 H (C-6); 30 mL of 0.2 M sodium hydroxide/methanol (1:l)a t room tem6.09, t, 1 H (dC Hl’); 5.93, d, 1 H (C-5); 5.84, d, 1 H (rG Hl’); perature. After 5 min, the mixture was neutralized (HC1) and 4.62, br s , 1 H (dC H3’); 4.53, m, 1 H (rG H3’); 4.27, m, 1 H (rG evaporated. D M F (25 mL) was added, and the mixture was H4’); 4.10, br s, 3 H (rG H5’,5”, dC H4’); 3.66, m, 2 H (dC H5’,5”); filtered. T h e D M F solution was evaporated, and the resultant 2.38, m, 1 H (dC H2’’); 1.77, m, 1 H (dC H2’). rG H2’ was not oil was recrystallized from water/ethanol to produce tan crystals. observed a t 25 “C, presumably because it was buried underneath Homogeneous on TLC: R, 0.15 (95% CH3CN aqueous). Hothe HOD solvent peak. At 45 “C it was observed as a triplet (4.90 mogeneous on HPLC (program A): t R 16 min. UV, A, ( e ) : [pH 71 280 nm (21300); [pH 11 270 nm 15600); [pH 121 288 nm PPd. Enzymatic Digestion. dCprG (0.3 AzW),dissolved in Tris-HC1 (15200). NMR (D20-CD,OD) 6: 7.55, d, 2 H (aniline 2,6); 7.27, (1mL, 5 mM, p H 7.0), was incubated with phosphodiesterase I1 t, 2 H (aniline 3, 5); 6.96, t, 1 H (aniline 4); 6.05, d, 1 H (rG Hl’); (0.25 unit) overnight a t 37 “C. Direct HPLC analysis (program 4.33, m, 1 H (rG H3’); 4.14, m, 1 H (rG H4’); 3.90, m, 2 H (rG A) showed no starting material and three products, identified by H5’,5”). rG-H2’ was not visible a t 25 “C, presumably because UV and coinjection with authentic samples as dCp, dC, and rG it was buried underneath the HOD solvent peak. At 45 “C, it ( t R 2.3, 3.5, and 7.8 min, respectively). The ratio rG:(dC dCp) appeared as a triplet (4.81 ppm). CD (Figure 5) (e, in 1 mM was 1:l. potassium phosphate, pH 7.05): 305 nm (-0.64 X lo4), 280 (0.88 x 10-4). Preparation of dCprG-An (VIIIb). All glassware, pipets, Preparation of N-(2’,3’-0 -1sopropylideneguanosin-8-y1)- and syringes were dried overnight a t 120 “C and cooled to room temperature in a desiccator. N-(2’,3’-O-Isopropylideneaniline (XIIb). p-Toluenesulfonic acid (300 mg, 1.7 mmol) and 2,2-dimethoxypropane (3 mL) were added to a D M F solution guanosin-8-y1)aniline (XIIb) (300 mg, 724 pmol) and blocked dCp containing N-guanosin-8-ylaniline (225 mg, 0.6 mmol). The IX (670 mg, 724 pmol) were mixed together, evaporated (3 x 3 mixture was stirred overnight a t room temperature. The mixture mL pyridine), and maintained under vacuum overnight a t room was evaporated, CHCl, (75 mL) was added, and the solution was temperature. l-(Mesitylenesulfony1)-3-nitro-l,2,4-triazole (X) (1.25 extracted with 3 X 25 mL of 5% sodium bicarbonate. The g, 4.22 mmol) was added to the dry mixture. The reaction was chloroform solution was dried with magnesium sulfate, filtered, carried out under argon as described for dCprG. Pyridine (8 mL) and concentrated to 10 mL. The solution was slowly added to was added by syringe, and the reaction turned reddish brown. 100 mL of hexane with stirring a t 0 “C. The brown precipitate After 20 min, 10 mL of ice-water and 15 mL of 5% NaHCO, were (110 mg) was collected by filtration (45%). TLC: R, 0.45 (95% added. The reaction was extracted (4 X 4 mL CHCI,). The CHC13 (e): was dried (MgSO,), filtered, and evaporated. The CHC13 extract CH3CN aqueous). HPLC (program A): t g 20 min. UV ,A, [pH 71 280 nm (21 100); [pH 11 270 nm (15800); [pH 121 288 nm was concentrated to 1 mL and chromatographed on silica gel H (15400). NMR (DMSO-d,) 6: 7.6, d, 2 H (aniline 2, 6); 7.4, t, 2 (17.5 X 2.5 cm column) (95% CH3CN). Seventy 1-mL fractions H (aniline 3, 5); 6.9, t, 1 H (aniline 4); 6.2, d, 1 H (rG Hl’); 5.2, were collected and analyzed by TLC. Fractions 30-54 contained m, 1 H (rG H2’); 5.1, m, 1 H (rG H3’); 4.3, m, 1 H (rG H4’); 3.9, blocked dCprG-An XIb. They were pooled and evaporated to m, 2 H (rG H5’, H5”); 1.6, s, 3 H (methyl); 1.4, s , 3 H (methyl). a brown oil, which was stored desiccated at -50 “C. This material eluted as a pair of “dumbbells” on TLC (R, 0.61, 95% CH,CN Preparation of dCprG (VIIIa). All glassware was dried overnight a t 120 “C and then cooled to room temperature in a aqueous) and as a pair of overlapping peaks on HPLC (tR 38 min, program A). The UV spectrum of this material was identical with desiccator. Solvents were freshly dried and distilled before each reaction. N-Benzoyl-5’-O-(dimethoxytrityl)deoxycytidin-3’-yl that of a 1:l mixture of blocked dCp and N-guanosin-8-ylaniline

+

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Chem. Res. Toricol., Vol. I , No. 3, 1988

(A,,

208, 238, 264 nm; s, 304 nm).

A two-step deblocking procedure was used. One-quarter of the

chromatographed material was dissolved in 5 mL of dioxane, 5 mL of methanol, and 10 mL of 0.2 M NaOH. After 45 min, 20 mL of 1 M HCl and 5 mL of methanol were added. The fully deblocked material was neutralized with sodium hydroxide after 6 h and extracted (3 X 30 mL) with EtzO. The aqueous extract was chromatographed several times on RP HPLC ( t R 20 min, program C; t R 8 min, program A) to give 1-2 mg of purified dCprG-An (VIIIb): yield, 1-3%. Sufficient material for quantitative determination of the extinction coefficient of dCprG-An was not available. The A,, 280 (pH 7) t was calculated to be 26500 (37). The estimated error in this method is &lo%. The values for dCprG-An at other pH values were calculated by scaling them proportionately to this value. UV, A, ( 6 ) : [pH 71 278 nm (26500), 304 (s, 17000); [pH 11 276 nm (26500); [pH 121 277 nm (19900), 300 (s,15500). NMR (DZO) 6: 7.52, d, 1 H ((2-6);7.35, br s, 4 H (aniline 2, 3, 5, 6); 7.10, t, 1 H (aniline 4); 6.08, t, 1 H (dC Hl’); 5.90, d, 1 H (C-5);5.85, d, 1 H (rG Hl’); 5.09, t, 1 H (rG H2’); 4.63, d, 1 H (dC H3’); 4.53, m, 1 H (rG H3’); 4.24, m, 1 H (rG H4’); 4.19, m, 2 H (rG H5’,5”); 4.02, m, 1 H (dC H4’); 3.59, m, 2 H (dC H5’,5”); 2.43, m, 1H (dC H2”); 1.86,m, 1 H (dC H2’). Enzymatic Digestion. dCprG-An (0.03 A260),dissolved in Tris-HC1 (1 mL, 5 mM, pH 7.0), was incubated with phosphodiesterase I1 (0.25 units) overnight at 37 “C. Direct HPLC analysis (program A) showed no starting material and three products, identified by UV and coinjection with authentic samples as dCp, dC, and rG-An ( t R 2.3, 2.8, and 17 min, respectively). The ratio rG-An:(dC + dCp) was 1:l based on the extinction coefficients at 254 and 280 nm of dCp, dC, and rG-An. No starting material remained. Theoretical Aspects. The total energy of the molecule is partitioned into contributions from nonbonded, electrostatic, torsional, ribose and deoxyribose strain, and ribose, deoxyribose, and phosphate anomeric terms, as described previously in detail (3438). These potentials follow the treatment of Olson (39,40) as described earlier. Counterion condensation is greated by reduction of the partial charge on the nonlinking phosphate oxygens, and solvent is treated by a distance-dependentdielectric constant. Aniline was incorporated with the same geometry as 4-aminobiphenyl (32)with all C-H bond lengths set to 1.0A. The partial charge on the hydrogen replacing carbon of biphenyl was assigned a value of -0.02. Torsion potentials about a’ and p’ (Figure 3) are the same as in earlier work (30). Partial charge assignments in the ribose ring differing from deoxyribose were 02’ -0.285, H(02’) 0.131, C2’ 0.114. Ribose geometry and strain energies followed our earlier work on ribodinucleoside monophosphates (41) except that the earlier data have been shifted in phase such that Pold+ P,,, = 216”, for better agreement with experimental values (39). All eight DNA backbone torsion angles, plus the two pseudorotation parameters PI and Pz, which define the conformations of the sugar rings (42),plus the torsions at the carcinogen-base linkage a’ and p’, were simultaneously variable parameters, for a total of 12 degrees of conformational freedom. Minimizations, performed by a modified version of the Powell algorithm (43)were carried to an accuracy of 1” in each parameter at the minimum, with no angle permitted to vary by more than 100” at any step. Starting conformations for both dCprG-An and dCpdG-An were all minimum energy conformationsof the dCpdG adduct with 4-aminobiphenyl ( 1 ) below 3 kcal/mol.

Results and Discussion Attempted Synthesis of N-(Deoxyguanosin-8-y1)aniline by Electrophilic Substitution. Most chemical carcinogens in their ultimate form in vivo are believed to be electrophilic reactants (81, and electrophilic reactions have generally been used to prepare adducts of aromatic amines and nucleosides of DNA in vitro as well. We therefore investigated this route t o prepare a n adduct of aniline and deoxyguanosine. As a preliminary step, we prepared the modified base, N-guanin-8-ylaniline (I) by reaction of guanine 3-oxide with aniline. This path had previously been used for the preparation of N-guanin-8-

Jacobson et al.

yl-1-naphthylamine (9). Compound I was used as a marker to judge the success of the reactions described below (after each had been subjected to acid depurination conditions). The following reactions were run in an effort to prepare an adduct of deoxyguanosine and aniline (or a substituted aniline): (a) deoxyguanosine and N-acetoxyacetanilide, 37 “C, pH 7; (b) 3’,5’-di-O-acetyldeoxyguanosine and N-(tosy1oxy)acetanilide (chloroform-methanol-triethylamine, -23 “C); (c) deoxyguanosine and phenylhydroxylamine, (pH 5 , 37 “C); (d) deoxyguanosine and N-acetoxy-N-(trifluoroacety1)aniline (ethanol-water, p H 7, 50 “C); (e) deoxyguanosine and N-acetoxy-p-methoxyaniline (dimethylformamide-water, 50 “C); (0calf thymus DNA and phenylhydroxylamine (pH 5, 37 “C). In these reactions, no products derived from the nucleoside were observed by HPLC, either directly or after acidic treatment. The only peaks observed (apart from guanine and deoxyguanosine) were decomposition products of the electrophilic reagent (data not shown). These peaks were also present in control reactions lacking the nucleoside. When the substances listed in a above were combined in dimethylformamide at 100 “C, rather than in aqueous solution, four nucleoside products (11-V) were separated by preparative thin-layer chromatography and characterized by their ultraviolet and proton magnetic resonance spectra (data not shown). They were identified as W,03’,05‘-triacetyldeoxyguanosine( I 0), W-acetyldeoxyguanosine, and the two isomeric N2,03’Or5’-diacetyldeoxyguanosines. The products of this reaction resulted from transacetylation, rather than electrophilic substitution upon the guanine ring. Transacetylation has also been observed in a reaction between N,O-diacetyl-1-naphthylhydroxylamine and cytidine (11 ) . Thus no reaction was observed in a number of attempted electrophilic substitution reactions between activated aniline derivatives and guanine nucleosides or guanine in DNA. In most of these cases, the same reaction had succeeded when the activated derivative of a polycyclic aromatic amine was used. The reactions tested by us are not the ones likely to take place in vivo, but the deficiency in electrophilic reactivity is likely to carry over to that situation as well. The covalent binding index for aniline, for example, is much lower than that for 2-aminofluorene (12). Other workers have commented that “the low levels of binding to DNA that we observe could also explain the low genotoxicity and low carcinogenicity of aniline” ( 5 ) . One suggested reaction pathway for electrophilic substitution of activated aromatic amines upon components of DNA involves a nitrenium ion intermediate (13). The single ring of aniline gives less stabilization to nitrenium ions and radicals than do larger ring systems, and this may account for the reduced reactivity of aniline. At least one other factor must be involved, however, for an activated derivative of p-methoxyaniline (which would give a more stable nitrenium ion) also failed to react with guanine in the above studies. Synthesis of N-(2’,3’,5’-Tri-O-acetylguanosin-8y1)aniline (VII) by a Nucleophilic Substitution Reaction. The failure of the above electrophilic reactions led us to consider an alternative nucleophilic route to our product. Aliphatic amines react with 8-haloguanine nucleosides to afford the corresponding alkylamine products (14, 15). One report exists of the use of this reaction with an aromatic amine (16). 2-Aminofluorene was allowed to react with 8-bromo-2’,3’,5’-tri-O-acetylguanosine (VI, Figure 1)in 2-propanol at 160 “C, and the reaction mixture was subjected to acidic depurination to afford N-guanin8-yl-2-aminofluorene.

Chem. Res. Toxicol., Vol. 1, No. 3, 1988 155

Aniline-Modified Dinucleoside Monophosphate 0

n

VI

,

II

N4

VI1

Figure 1. Synthesis of VI1 by nucleophilic substitution. Figure 3. Structure, numbering scheme, and variable conformational angle designations for dCprG-An (VIIIb). The dihedral angles A-B-C-D are defined as follows: x’,Ol’-Cl’-Nl-CG; x, 01’-Cl’-N9-C8; $1, $, C3’-C4’-C5’-05’; $J’,P-O3’-C3’-C4’; 6, C4’-C5’-05’-P; w’, 05’- P-03/43’; W , C5’-05’-P-03’; CY’, N9C8-N-C4; p’, C8-N-C4-C5. The angle A-B-C-D is measured by a clockwise rotation of D with respect to A, looking down the B-C bond. A eclipsing D is 0”. In addition, both sugar puckers are flexible. These are defined by the pseudorotation parameter P ( 4 2 ) . PI is 5’-linked; P2 is 3’-linked.

07

pp.----

I ’

01 00

0

-

0

.I

IO

so

20

40

hours

Figure 2. The effect of the proportions of aniline and its hydrobromide on the yields of VII. The quantity described along the vertical axis corresponds to the mole fraction of product. A value of 1.0 represents 100% yield. Reactions were carried out on 21 wmol of VI in 1 mL of i-PrOH at 100 “C. The quantities

“J? O X ”

of aniline and anilinium hydrobromide (wmol) employed,and the molar ratios were as follows: (a) 195, 21, 9:l; (b) 107, 107, 1:l; (c) 21, 195, 1:9. The reaction mixture was sampled at the appropriate intervals and analyzed by HPLC.

We substituted aniline for 2-aminofluorene and repeated the procedure, omitting, however, the acidic hydrolysis step. Despite this omission, only the modified base I, N-guanin-8-ylaniline was formed. No modified nucleoside was observed. When the reaction was run a t lower temperature, however, a mixture of I and the desired nucleoside product, VI1 (Figure l),was obtained. Product VI1 was identified on the basis of its elemental analysis and spectroscopic properties. We anticipated that the problem of depurination would be more severe in the deoxyribonucleoside series and ran a series of kinetic studies in the above system to optimize the reaction conditions. Depurination was studied with VI and VI1 and substitution with VI. Compound VI1 was found t o be more labile to depurination than VI, so formation of I presumably took place by depurination of VI1 rather than by depurination of VI followed by substitution. (In addition, 8-bromoguanine was found to be unreactive to aniline under these reaction conditions.) Both substitution and depurination were found to be catalyzed by acid. Thus a lag period of several days occurred until enough HBr had been released by substitution to catalyze both reactions. The lag period was extended indefinitely when a hindered tertiary amine, diisopropylamine, was present. It was eliminated, however, when aniline hydrobromide was added to the reaction as a catalyst. The effect of the catalyst was presumably due to protonation a t N-7 of guanine, activating it toward both substitution and glycosyl cleavage. In Figure 2, the effect of varying amounts of catalyst on the yield of VI1 is displayed. A ratio of about 9:l ani1ine:aniline hydrobromide proved optimal. Product yields declined after a time with other ratios, presumably due to depurination. These optimized conditions were then used in a reaction of aniline with 3’,5’-di-O-acetyl-8-bromodeoxyguanosine.

XI I

I

I

X

n

CH,O

FI

XI

1) O H

-

2) H’

HO

iH Vlll

a) R Hb) R = PhNH-

Figure 4. Synthesis of dCprG (VIIIa) and dCprG-An (VIIIb).

A mixture of I (60%) and 8-bromodeoxyguanosine was produced. No nucleoside product was observed in this reaction or in others in which the conditions or acid catalyst were varied. T h e desired aniline-substituted nucleoside product presumably depurinated as rapidly as it was formed. Synthesis of dCprG (VIIIa) and N-[Deoxycytidylyl-(3’-5’)-guanosin-8-yl]aniline (VIIIb, dCprGAn). Our difficulties in preparing an aniline-modified deoxyguanosine derivative caused us to alter our synthetic target. Earlier studies of the mixed dinucleoside mono-

Jacobson e t al.

156 Chem. Res. Toxicol., Vol. I , No. 3, 1988

phosphates dAprA and rApdA had indicated that the conformation of each resembled that of the homodimer which contained the 5'-sugar of the mixed dimer. Thus, dAprA resembled dApdA (17). We therefore undertook the synthesis of VIIIb (Figures 3 and 4) with the expectation that its conformation would be a good model for that of the all-deoxy dimer. One possibility for the synthesis of VIIIb involved the introduction of aniline by replacement of bromine as the last step. T o test this possibility, we exposed dCp and 8-bromoguanosine to the conditions we had devised for the substitution reaction. Significant amounts of glycosyl cleavage took place with both compounds under those conditions. We opted then for an alternative, in which VIIIb was prepared by combination of suitably substituted monomers. The route selected is given in Figure 4. It involved solution phosphotriester synthesis (18)with conventional blocking groups and condensing agent but with the amino group of guanine unprotected. It has been suggested that protection of cytosine is essential with the phosphotriester method but that guanine protection is not necessary (18). We were concerned with the synthetic complications involved in protecting a guanine ring that had an additional amino group a t the 8-position and also by the alkaline lability of the imidazole ring displayed by guanine nucleosides modified by aminofluorene (19). Cytosine deacylation has been observed to proceed 100 times more rapidly than that of guanine, however, when methanolic sodium hydroxide was the reagent employed (20). With this reagent, we found the half-life for debenzoylation of N-benzoyldeoxycytidine to be 8 min, while that for decomposition of N-guanosin-8-ylaniline was about 9 h. The decomposition, presumably due to opening of the imidazole ring, was accompanied by a change in the ultraviolet spectrum (19) and the formation of a more polar product upon analysis by HPLC. Thus cytosine protection was feasible. This procedure was incorporated into our route. To test the route, and to obtain an unmodified mixed dimer for comparison purposes, we first attempted the synthesis of dCprG, as illustrated in Figure 4. Analysis of the reaction mixture by HPLC indicated the presence of a major product whose properties were consistent with those expected for the blocked dimer XIa. After alkaline and acidic deblocking steps, a new substance was produced, to which structure VIIIa was assigned on the basis of ultraviolet, nuclear magnetic resonance, and enzymatic hydrolysis studies (see the Methods section). This sequence was then applied to the synthesis of the desired dimer VIIIb (dCprG-An). The modified 0acylated nucleoside VI1 was first deacylated to afford N-guanosin-8-ylaniline (rG-An), and this substance was converted to its isopropylidine derivative, XIIb. The blocked dCp component IX and XIIb were combined with a condensing agent, and the reaction mixture was subjected to alkaline and acidic deblocking steps (Figure 4). A complex mixture was produced, from which VIIIb could be isolated in a yield that varied from 1-3% on separate runs, considerably less than the 30% yield obtained with dCprG. A combination of nuclear magnetic resonance and ultraviolet spectroscopy and enzymatic hydrolysis was used to confirm the structure of the product. To explore the reasons for the low yield, the various steps of the procedure were followed chromatographically. We established that the alkaline deblocking step was accompanied by cleavage of the phosphotriester link, giving IX and XIIb, and the acidic treatment afforded some phosphodiester cleavage to dCp and N-guanosin-8-ylaniline

-ob

i

1

1 ; #

'

;

\ -'

-I 0 nm

Figure 5. (a) Circular dichroism spectra of rG and rG-An in 1 mM phosphate buffer, pH 7.05, 25 O C . (b) Circular dichroism spectra of dCprG, l/z(dC + rG-An), and dCprG-An under the above conditions.

(additional side reactions were also noted, but the products were not characterized). No phosphodiester or triester cleavage had been observed in the preparation of dCprG. A number of variations on the synthesis of VIIIb were explored, including different ratios of the reactant, varying concentrations of the deblocking reagents, different times of exposure to them, and alternative deblocking procedures. The overall yield of VIIIb was not improved by any of these steps, however. Circular Dichroism Spectra. In certain instances, the spectra of carcinogen-modified dinucleoside monophosphates have differed dramatically from those of the sum of the constituent monomers, or the unmodified dimer. For example, an increase of an order of magnitude in the amplitude of the principal Cotton effect was observed in some cases with dimers modified by binding of N-acetyl-2-aminofluorene at the 8-position of guanine (21, 22). In certain dimers containing a 2-naphthyl group bound to the amino group of cytosine, a virtual inversion of the CD spectrum was observed after modification (23). These effects were largely abolished when the spectra were run in methanol or a t higher temperature. The results were interpreted in terms of a novel conformation for the modified dimers, with significant carcinogen-base stacking. The amplitude of the effect observed with N-acetyl-2aminofluorene modification was diminished by two-thirds when the corresponding dimers containing 2-aminofluorene were examined (24). Dimers containing bound 4-aminobiphenyl exhibited an increase in CD amplitude only 50% greater than the constituent monomers (1). It was suggested that carcinogen-base stacking played a decreasing role as one progressed along this series. In the current study, the spectrum of the modified monomer, N-guanosin-8-ylaniline was obtained first (Figure 5a). It was similar to those of 8-(alkylamino)-

Aniline-Modified Dinucleoside Monophosphate

5.902 (-.030)

7.518 (-.OS01 3.592 (-.072)

Chem. Res. Toxicol., Vol. 1, No. 3, 1988 157

fi0 I

HOYC

H

(-.003) 6.083

4.023 (-.080)" 4.629 (+.003)

I

(+.049) 2.428

H'

(+.089) 1.857

H

O=P-O-

I

4.192 (+.oa9)

0 4 2 3 5 (-.035)

4 5 2 9 (0)

H

(-.037) 7.351

$ +

H (+.004) 5.846

H

H (+.I551 5.088

HO

OH

Figure 6. NMR chemical shifts of dCprG-An in D20 (pH 7.05) relative to CH,OD (3.340 ppm) are listed adjacent to their respective protons. In parentheses are the chemical shift values minus the chemical shift values of the respective protons for unmodified dCprG. The values for the aniline protons represent the modified dimer minus those for the modified monomer in D20. The experimental error of these values was f0.005 ppm. The value marked by * was obtained at 45 O C and the remainder at 25 "C. guanine nucleosides, whose spectra have been interpreted in terms of a possible high anti conformation (25) or a flexible syn-anti mixture (26,27). It also resembles that of N-(deoxyguanosin-8-yl)-4-aminobiphenyl ( I ) , suggesting a similar conformation. The spectra of the unmodified mixed dimer dCprG very closely resembled that of the homodimer dCpdG (28), which was consistent with a similarity in the conformations of the two compounds. This result was in accord with earlier observations made in the adenine series of dinucleoside monophosphates (16) and lent support to our assumption that VIIIb would be an appropriate model for the all-deoxy dimer. In Figure 5b, the CD spectra of dCprG-An (VIIIb), its constituent monomers, and dCprG are compared. They differ in some details, but no enhancement of the positive Cotton effect in VIIIb, of the type displayed with other aromatic amine substituents and discussed above, is observed. On the other hand, an enhanced negative Cotton effect relative to the unmodified dimer (or the constituent monomers) is present. The differences between the spectra of VIIIb and dCprG almost disappeared when both were taken in methanol (data not shown). This suggested that stacking interactions contributed to the modest differences observed between the modified and unmodified spectra in neutral aqueous solution. Proton Nuclear Magnetic Resonance (NMR) Spectra. T h e chemical shifts of the nonexchangeable protons of VIIIb are displayed in Figure 6. The assignments were made with the help of decoupling experiments as described earlier (1) and two-dimensional J-correlated NMR spectroscopy (COSY). T h e aromatic region of the spectrum displayed a one-proton triplet assigned to the aniline 4-proton, while the remainder of the aniline protons appeared as a broad multiplet. T h e changes in chemical shift produced by aniline substitution of dCprG are also presented (in parentheses) in Figure 6. Positive numbers represent downfield shifts (deshielding effects) produced by aniline substitution; negative numbers signify upfield shifts (shielding effects).

T h e numbers given for the aniline protons represent modified dimer minus modified monomer. The largest chemical shift difference observed was for H2 of the guanosine unit, +0.16. This is comparable to the value of +0.19 obtained for the corresponding proton in an aminobiphenyl-modified dimer (1). In the case of 8-substituted guanosines, the magnitude of this change has been related to the proportion of syn conformation (27). Thus, the sh ft difference of +0.58 for 8-bromoguanosine, relative to guanosine, was taken to represent an almost entirely syn conformation for the former compound. Our own result for aniline modification, and the earlier one for aminobiphenyl modification, may be interpreted in terms of a modest increase in the syn conformation for guanine in dCprG-An, relative to dCprG. The cytosine ring 5- and 6-protons of VIIIb displayed shift differences of -0.03 and -0.05, respectively. The corresponding values for aminobiphenyl-modified dCpdG were -0.26 and -0.20 ( I ) . In dApdG modified by (acety1amino)fluoreneand by aminofluorene, the shift differences of the adenine ring protons were -0.60, -0.60, and -0.30, -0.44, respectively (24,29). These values were considered to be consistent with an increased population of conformers which place these protons within a shielding region of an aromatic ring. The magnitude of the change was taken as a measure of the stacking effect of the aromatic amine and the neighboring base ( I ) . The values observed in the present case are smaller than those cited above and suggest only a slight stacking interaction of cytosine with aniline. This conclusion is reinforced by a consideration of the shift differences for the aniline ring, in which the modified dimer is compared with the modified monomer. The values observed, -0.02 to -0.04, suggested less amine-cytosine stacking than was noted for the aminobiphenyl analogue, where values of -0.11 to -0.16 were determined for the ring proximal to guanine (1). Theoretical Results: Comparison with Experiment. The conformation of VIIIb was investigated by using minimized semiempirical potential energy calculations. Calculated minimum energy conformations within 3 kcal/mol of the global minimum were grouped according to stacking type and guanine glycosidic torsion (anti or syn), and approximate statistical weights were computed as described previously (30). The results for the four conformational classes were the following: (a) guaninecytosine stack with anti guanine, 38% (the global minimum, 2870,belonged to this class); (b) guanine-cytosine stack with guanine syn, 18%;(c) aromatic amine-cytosine stack with guanine high anti, 4%; (d) aromatic aminecytosine stack with guanine syn, 39%. The lowest energy conformation for each category is shown in Figure 7 . In addition, unstacked conformers accounted for 170of the population. Tables I-M to IV-M (supplementary material) present conformational details of these forms. Calculations of the same type were also made for the aniline-modified homodimer dCpdG. The conformer populations corresponding to the categories described above were the following: (a) 4370, including the global minimum; (b) 10%; (c) 4%; (d) 43%. These calculations further support our assumption that dCprG-An and dCpdG-An have similar conformations. It is interesting to compare these results with the conformational mixture previously computed for 4-aminobiphenyl-modified dCpdG (1): (a) 1 7 % ; (b) 2 % ; (c) 23%; (d) 58%, including the global minimum. Some striking differences exist between this compound and the anilinemodified dimers. In the aminobiphenyl case, 81% of the low energy conformers exhibit aromatic amine-cytosine

158 Chem. Res. Toxicol., Vol. I , No. 3, 1988

Jacobson et al.

w a

b

C

d

Figure 7. Stereo views: (a) Global minimum energy conformation of dCprG-An with anti guanine and guanine-cytosine stacking. Torsion angles in degrees are x’ = 57, +1 = 5 5 , $ ’ = 191, w’ = 273, = 289, = 163, = 63, x = 54, PI = 76, P2 = 159, 01’ = 198, and p’ = 199. (b) Lowest energy conformation of dCprG-An with syn guanine and guanine-cytosine stacking. AE = 0.6 kcal/mol. Torsion angles in degrees are x’ = 31, #1= 300, 4’ = 182, w’ = 60, w = 259, 4 = 167, = 299, x = 226, Pi = 171, Pz = 19, 01’ = 270, and p’ = -5. (c) Lowest energy conformation of dCprG-An with (high) anti guanine and aniline-cytosine stacking. AE = 1.2 kcal/mol. Torsion angles in degrees are x’ = 55, +1 = 60, 4’ = 188, W‘ = 48, w 64, 4 = 188, = 67, x = 168, PI = 207, P2 = 29, a’ = 232, and p’ = 2. (d) Lowest energy conformation of dCprG-An with syn guanine and aniline-cytosine stacking. AE = 0.4 kcal/mol. Torsion angles in degrees are x’ = 50, IC1 = 59, $ ‘ = 211, w’ = 318, w = 259, $ = 170, # = 57, x = 237, PI = 41, Pz = 102, a’ = 59, and p’ = 1.

+

+

stacking; only 43% of the low-energy conformers did so with VIIIb. The global energy minimum in the biphenyl case had syn guanine with aminobiphenyl-cytosine stacking; the global minjmum in the anline case had guanine anti with guanine-cytosine stacking. These results are in accord with the CD and NMR studies which indicate considerably less aniline-cytosine stacking than aminobiphenyl-cytosine stacking. The calculations further indicate that aniline-modified dCpdG and aminobiphenyl-modified dCpdG are similar in one respect: they both have about the same amount of syn guanine conformers. This conclusion again agrees with the inference drawn from the NMR spectra. The aniline and aminobiphenyl series differ most markedly in the stability of guanine anti (or high anti), aromatic amine-base stacked conformers. In the aminobiphenyl case, two structures of this type below 3 kcal/mol were calculated, each 0.15 kcal/mol above the global minimum and comprisine 11% of the total conformer population. The corresponding conformers in the aniline case were examined and found to contribute 4% and 0%

to the population. The aminobiphenyl structures exhibited a stacking interaction between the phenyl ring more distant from guanine and the cytosine ring, which presumably helped stabilize them. The corresponding aniline conformers lacked the second aromatic ring and could not incorporate this feature.

Conclusions Two significant differences between aniline and the more biologically active polycyclic aromatic amines have been noted in this work. Derivatives of aniline have been found to exhibit weaker electrophilic reactivity to DNA and nucleosides than the analogous derivatives of the polycyclic amines. This difference in reactivity may well carry over to the electrophilic intermediates involved in binding to nucleic acids in vivo and account in part for the limited biological activity of aniline. Further, conformational differences have been noted between dinucleoside monophosphates containing bound aniline and those with polycyclic amine residues. The aniline-containing dimer had a higher proportion of base-base stacked states and less of the novel carcinogen-base stacked states than did the dimers modified by polycyclic amines. Although specific conformations observed a t the dimer level may not necessarily be those observed in DNA, the differences in stacking abilities displayed may well persist a t the polymer level and play some role in defining the mutagenic and carcinogenic effectiveness of a particular alteration. It has been suggested that a carcinogen-base stack a t the replication fork plays a role in the mutagenesis by aromatic amines (31-33). If this should be the case, the lesser tendency of aniline to assume such states may also contribute to its lack of biological potency. Acknowledgment. This work was supported by DOE Contract DE-AC02-91ER60015, USPHS Grant 1 R / 1 CA 28038-07, awarded by the National Cancer Institute, DHHS, BRSG Grant RR07062, awarded by the Biomedical Research Support Grant Program, Division of Research Resources, NIH, National Science Foundation Grant DMB8416009, and by the Office of Health and Environmental Research, U S . Department of Energy under Contract DEAC05-840R21400 with Martin-Marietta Energy Systems, Inc. (B.E.H.). Supplementary Material Available: Tables of minimum energy conformations of dCprG-An with anti and syn guanine and G-C stacking and with high anti and syn guanine and An-C stacking (4 pages). Ordering information is given on any current masthead page.

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