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The Structure of DNA Dictates Purine Atom Site Selectivity in Alkylation by Primary Diazonium Ions Xuefang Lu, Jacqueline M. Heilman, Patrick Blans, and James C. Fishbein* Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Received May 23, 2005
The 1-propanediazonium ion, generated from N′-nitro-N-nitroso-N-propylguanidine in aqueous solutions, was reacted with the purine nucleosides dGuo and dAdo or single-stranded or double-stranded DNA. After nucleobase liberation by acid hydrolysis, the percent yields of products were determined by LC/MS using either isotopically distinct internal standards in the case of the nucleoside reactions or an internal standard and the ratios of response factors of all other products that were separately determined in the case of the reactions with DNA. In the reactions of nucleosides, products of both n-propylation and iso-propylation at all of the heroatoms were observed. For these reactions, the yields of the three most abundant n-propyl adducts of Gua are in the order O6 > N7 > N2, in the ratio of 9.0/6.4/1, while for Ade, the order of the yields of N-propyl products is N1 > N7 > N3 > N6 in the ratio 2.5/1.8/1.1/1. The ratios of n-propylated to iso-propylated products at each site, Pn/Pi, generally a measure of enhancement of SN2 displacement on the diazonium ion, vary with each heteroatom but by no more than a factor of 6 for Gua and a factor of 3 for Ade. In the reactions with duplex DNA, products of reactions at all sites could not be detected. In addition, much larger selectivities are observed, similar to what has been observed by others in the reactions with ethanediazonium ion. Thus, Pn/Pi ) 30, 21, and 0.9 for N7, O6, and N2 of Gua. Similarly, the values of Pn/Pi are 11 and 8 for N3 and N7 of Ade. Reactions with single-stranded DNA give values of Pn/Pi that are intermediate between the nucleoside reactions and the reactions of duplex DNA in most cases. The factors responsible for the relatively small atom site selectivities intrinsic to the nucleosides are analyzed, and reasons for enhanced SN2 nucleophilicity in duplex DNA are discussed.
Introduction Many nitrosamines are powerful carcinogens (1-3). The activity of simple dialkylnitrosamines is believed to be manifest by the fact that they undergo P450-mediated formation of R-hydroxynitrosamines. These species decompose as indicated in eq 1 to yield diazonium ions that can alkylate DNA directly or through carbocations in certain instances.
It has been known for some time that atom site selectivity in the alkylation of DNA by diazonium ions is different from the alkylation pattern observed with classical alkylating agents such as methane- and ethanesulfonate esters (4, 5). These latter agents react mainly at a few atoms in reactions with DNA, mostly the N7 of Gua. In the case of the corresponding diazonium ions, N7Gua is still preferred, but there is more extensive alkylation of the exocyclic oxygens atoms in DNA (6). The ethyldiazonium ion gives proportionately more of the oxygen atom adducts. The exocyclic amino groups are unreactive with all of the above agents, although it has * To whom correspondence should be addressed. Tel: 410-455-2190. E-mail:
[email protected].
recently been demonstrated that sec-carbocations, derived from sec-diazonium ions, attach to these sites with a propensity comparable with the other heteroatoms (7, 8). The factors that control atom site selectivity in diazonium ion alkylation are of significance because, at least for the methyl- and ethyldiazonium ions, the oxygen atom adducts appear to account for much of the mutation spectra of these compounds (9-12). Because of the biological significance, there has been great interest in understanding the basis for the greater oxygen atom “affinity” of methyldiazonium ion as compared to methanesulfonate esters and of ethyldiazonium ion as compared to methyldiazonium ion. Historically, there was some imprecision in the mechanistic formulations, but it was considered that there was a “component” of SN1 reactivity in the case of diazonium ion substitutions, a greater component in the case of the ethyldiazonium ion (4, 5). It is now firmly established that primary diazonium ions undergo strictly concerted bimolecular SN2 substitution with even weak nucleophiles such as solvent water and acetic acid (13, 14). Thus, a reformulation of the logic would be that the reactivity order combined with a Hammond type effect giving rise to an earlier, less selective, SN2 transition state in going from the sulfonate ester to the methyl- and then ethyldiazonium ion was proposed to account for the observed trend. The earlier transition state is supported by the smaller Swain-Scott s value for the ethyldiazonium ion
10.1021/tx0501334 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005
Purine Atom Site Selectivity in Alkylation
as compared to the methyldiazonium ion measured in reactions with inorganic nucleophiles (15) and a pyridine reporter nucleophile (16, 17). However, it was pointed out that change in selectivity in the change from the methylto ethyldiazonium ion is not uniform at all heteroatoms, a “failure” of the Swain-Scott relationship, and in particular that oxygen atoms have a relatively enhanced reactivity as compared to nitrogen atoms in the latter case (18). This was dubbed “oxyphilicity”. Extensive systematic studies identified a shift from N7Gua to O6Gua as the electrophile becomes more “cationic” (19-23). Although this empirical framework lacks a theoretical underpinning, and thus fails to predict the exocyclic amino group adducts of sec-diazonium ions recently discovered (7, 8), it is part of more global assessment of atom site selectivity by many classes of electrophiles that has proven quite useful. Semiempirical quantum mechanical calculations for reaction of methyl- and ethyldiazonium ions have suggested similar barriers for O6Gua and N7Gua ethylation, a somewhat larger barrier for methylation at the O6 atom as compared to the N7 atom, and in both alkylations a very much larger discrimination against the N2 of dGua (24). This trend appears to be consistent with what is observed in the reactions with DNA. It is not possible to understand what controls atom site selectivity in reactions with DNA without understanding the factors that control selectivity that is intrinsic to the monomers. Even this can be obscured when inherent selectivities are small, because the contribution of different equilibrium constants for association to the various heteroatom sites is overlaid on intrinsic “nucleophilicities” (8). We have undertaken a study of propylation of purine nucleosides and DNA in order to understand what factors dictate atom site selectivity in this reaction. Use of a compound that generates the 1-propane diazonium ion results in both n-propylation and iso-propylation due to the tendency of primary diazonium ions to undergo unimolecular rearrangement (25-28), as in eq 2.
Chem. Res. Toxicol., Vol. 18, No. 9, 2005 1463 (oligo 2) were obtained from Midland Certified Reagent Co. (Midland, TX). 3-Nitro-1-nitroso-1-propylguanidine (PNNG) (98%) and 2-mercaptoethanesulfonic acid sodium salt (MES) (98%) were purchased from Aldrich. The isotopically labeled 15N2-2′-deoxyguanosine and 15N6-2′-deoxyadenosine were prepared as described previously (8). The n-propylated purine adduct standards were synthesized and purified following published procedures and were analyzed for purity (>97%) by 1H NMR and for structure by 1H and 13C NMR (29-32). Standards for the isopropylated purines were available from earlier work (8). Methods. Alkylation Reaction of 15N-Labeled Purine Nucleosides, Single- and Double-Stranded DNA by PNNG. The reaction concentrations of 15N2-2′-deoxyguanosine and 15N62′-deoxyadenosines were 0.40 and 0.38 mM, respectively. The concentration of ss DNA was 0.6 µg/µL. Double-stranded λ-phage DNA/Hind III restriction fragments (0.5 µg/µL) were received as a solution in a storage buffer (pH 7.4, 10 mM tris-HCl, 5 mM NaCl, and 0.1 mM EDTA) and were used directly. Stock solutions of all other substrates were prepared in the same buffer. One milliliter of each substrate solution was mixed with 60 µL of aqueous cacodylate buffer (pH 6.8, 1 M) and 68 µL of MES solution (0.5 M). To the above stirred solution, 120 µL of PNNG (0.25 M) in acetonitrile was added dropwise to a final concentration of 24 mM PNNG, which rapidly underwent MESstimulated decay (33). The reaction mixture was stirred at 22 ( 2 °C for 30 min. Where it was necessary to determine the levels of N1Ade adducts, the reaction was split into equal volumes. One-half was acidified with HCl to a final [HCl] ) 0.1 M and heated to 80 °C for 30 min. The solution was then neutralized with NaOH. For determination of N1Ade adducts, the other half was base hydrolyzed prior to acid hydrolysis and the N1Ade adducts were determined as the difference in N6Ade adducts from the two halves, as described previously (8). Reactions in D2O were carried out by making all stock solutions with 99.6 atom-% D2O. The λ DNA solution, as received, was evaporated and redissolved in D2O through five cycles of evaporation and dissolution. Analysis of Propylpurine and Isopropylpurine Adducts. All reaction mixtures were analyzed on a LC/MS (Waters/Micromass) system using a Phenomenex Luna 5µ C18(2) or a Waters YMC Pro C18 3µ column. The mobile phase was composed of acetonitrile (or methanol)/water (0.01 M-0.02M ammonium acetate buffer, pH 5.3-6.6) gradients. Detection employed single ion monitoring at the relevant masses. Analysis of 15N-Labeled Purine Nucleosides Alkylation Reactions. Synthetic propylpurine and isopropylpurine adducts (14N) were used as internal standards. Aliquots of the reaction mixtures with internal standards were chromatographed and analyzed. The identity and amount of the products of the reaction were determined from the peaks of mass [M(15N)H+], which coeluted with the peaks of the standards [M(14N)H+].
This rearrangement reaction turns out to be useful in understanding atom site selectivity because the ratio of n-propylation to iso-propylation at any given site is generally a measure of acceleration by a given atom of the SN2 reaction, as described further within. It is found that there are relatively small differences in atom site selectivity in the reactions of nucleosides but that the differences increase in reactions with single-stranded DNA and increase still further in duplex DNA. This data set permits an examination of what factors likely contribute to the observed selectivities.
Experimental Procedures Materials. Double-stranded λ-phage DNA/Hind III restriction fragments were obtained from Invitrogen (Carlsbad, CA). Single-stranded DNA oligomers with sequences of 5′-TACGTCCATTGCACTG-3′ (oligo 1) and 5′-ATGCAGTCCGATCTAG-3′
Analysis of Single-Stranded and Double-Stranded DNA Alkylation Reactions. Preliminary experiments indicated that i-prN6Ade could not be detected as a product of the reaction with double-stranded DNA. Thus, i-prN6Ade was used as an internal standard. The response factors of all other adducts were determined, and the ratios, based on i-prN6Ade, were used to quantify products of the reaction with DNA into which i-prN6Ade had been spiked. Occasional checks before spiking confirmed the absence of i-prN6Ade as a product, and spiking with other standards confirmed the identity of the other adducts. Controls for Analytical Method. Control experiments involved incubation of DNA or purine nucleosides with the products of decomposition of PNNG and MES in buffer, followed by hydrolysis and neutralization. HPLC/MS analysis of these experiments gave profiles with variable background in some regions where certain low yield adducts eluted. This resulted in some variability in precision for these adducts as discussed in the Results section.
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Figure 1. Typical LC/MS chromatograms with single ion monitoring for reactions of the propanediazonium ion with 15N6-dAdo (A and B) and DNA (C and D). (A) Monitoring the mass ) 179 for n-pr-15N6Ade and i-pr-15N6Ade adducts. (B) Monitoring the mass ) 178 for the n-pr-14N6Ade and i-pr-14N6Ade standards. (C) Monitoring the mass ) 194 for n-pr-Gua and i-pr-Gua adducts. (D) Monitoring the mass ) 178 for n-pr-Ade and i-pr-Ade adducts.
Results Typical LC/MS profiles, monitored by employing single ion monitoring, are presented in Figure 1. Panel A presents the raw data for the reaction of 15N-dAdo while panel B is the chromatogram for 14N-dAdo standards. Figure 1C and D are raw data with monitoring for the mass of dG and dA adducts in the reactions with DNA. A comparison of panel A with panel D shows that the
reactions with nucleosides appear to give a more uniform yield of products. Reactions with DNA gave poorer baseline stability, as indicated by control experiments, and the yields for some of the adducts were sufficiently low that their quantification led to significant imprecision. This was especially true in the case of i-pr-N3Gua and n-pr-N2Gua where the percent standard deviations were less than (20%. The percent yields of n-propylated
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Table 1. Percentage Yields of the Products and Ratios of n-Propylation to iso-Propylation at Each Atom Site (Pn/Pi) of Reactions of Purine Nucleosides and DNA with 1-Propane Diazonium Ion in Aqueous Media, 22 ( 2 °C, pH ) 7.0, 10 Vol % Acetonitrilea purine atom adduct
nucleosideb
oligo 1c
oligo 2c
102*% 102*% 102*% yield Pn/Pi yield Pn/Pi yield Pn/Pi
n-pr-N1 i-pr-N1 n-pr-N2 i-pr-N2 n-pr-N3 i-pr-N3 n-pr-O6 i-pr-O6 n-pr-N7 i-pr-N7 total
0.104 0.0149 0.277 0.120d 0.091 0.089d 2.44 0.552 1.78 0.31d 5.77
n-pr-N1 i-pr-N1 n-pr-N3 i-pr-N3 n-pr-N6 i-pr-N6 n-pr-N7 i-pr-N7 total
0.392 0.136 0.186 0.0627 0.160 0.186 0.291 0.094 1.51
7.0 2.3 1.0 4.4 5.7
102*% yield
Pn/Pi
dGua 0.039 6.5 0.032 4.8
