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3′-H-Phosphonate Synthesis of Chiral Benzo[a]pyrene Diol Epoxide Adducts at N2 of Deoxyguanosine in Oligonucleotides Prema C. Iyer, Haruhiko Yagi, Jane M. Sayer, and Donald M. Jerina* Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, DHHS, Bethesda, Maryland 20892 ReceiVed October 20, 2006
A synthetic route to oligonucleotides containing N2-deoxyguanosine adducts at C-10 of the enantiomeric 7,8-diol 9,10-epoxides of 7,8,9,10-tetrahydrobenzo[a]pyrene in which the epoxide oxygen and the 7-hydroxyl group are trans is described. The present adducts result from the trans addition of N2 of deoxyguanosine to the epoxide at C-10. Our synthesis proceeds via preparation of the 3′-H-phosphonate of a suitably protected deoxyguanosine N2-adduct. The blocking groups consisted of O6-allyl on the deoxyguanosine, acetates on the 7-, 8-, and 9-hydroxyl groups of the hydrocarbon moiety, and dimethoxytrityl on the 5′-hydroxyl group of the sugar. These blocking groups are well suited to oligonucleotide synthesis on solid supports. The free 3′-hydroxyl group of this nucleoside adduct was readily converted to its 3′-H-phosphonate with diphenyl phosphite in pyridine in high yield for both the 10R and 10S isomers. For synthesis of oligonucleotides, the first several nucleotides were incorporated onto the solid support with an automated synthesizer using standard phosphoramidite chemistry. The adducted deoxyguanilic acid residue was introduced as the H-phosphonate in a manual step (80% yield), followed by completion of the sequence on the synthesizer. Although a 10-fold excess of the 3′-Hphosphonate was used in the manual coupling step, as much as 70% of the reactant could be recovered. The 3′-H-phosphonate of the protected 10S nucleoside adduct was converted to the unblocked nucleotide adduct, various salts of which failed to form crystals suitable for X-ray analysis. Although submilligram quantities of this compound have been formed as mixed diastereomers by direct reaction of deoxyguanylic acid with racemic diol epoxide, the present study represents the first actual synthesis of the major DNA adduct formed from benzo[a]pyrene in mammals as its 3′-phosphate. Introduction The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), first isolated from coal tar and characterized by Cook et al. in 1933 (1), is one of the most prevalent environmental carcinogens to which humans are exposed (2). It is metabolized in mammals (3) by oxidation on the angular benzo-ring to give a pair of diastereomeric 7,8-diol 9,10-epoxides (DEs), designated as DE-1 (benzylic 7-hydroxyl group and epoxide oxygen cis) and DE-2 (benzylic 7-hydroxyl group and epoxide oxygen trans). Each of these two DEs exists as a pair of enantiomers. Of these four DE isomers, the (+)-(7R,8S,9S,10R)-DE-2 enantiomer shown predominates on metabolism of the hydrocarbon in mammals (3), is the most extensively bound to DNA, and is also by far the most tumorigenic isomer in animal models (4). Reaction of BaP DE-2 with DNA in cells or with nucleic acids in solution occurs mainly by trans and to a lesser extent cis opening of the epoxide ring at C-10 by the exocyclic 2- and 6-amino groups of the guanine and adenine bases, respectively. The trans-opened deoxyguanosine adduct of (+)-(7R,8S,9S,10R)DE-2 is the major DNA adduct formed from (()-DE-2 in rodent epidermal cells (5) as well as by its reaction with DNA in solution (6). Small oligonucleotides containing DE adducts derived from PAHs are of considerable interest both for their role in mutagenesis via incorrect replication by DNA polymerases and as probes for the function of other critical DNA-processing enzymes. Recent X-ray structures of PAH-adducted oligonucle* To whom correspondence should be addressed. Tel.: (301)-496-4560. E-mail:
[email protected].
otides in complex with DNA polymerases have provided valuable insights into how these adducts may block replication (7, 8) and cause mutations (8, 9). To date, three approaches have been utilized in the synthesis of such oligonucleotides: direct reaction of DEs with a small oligonucleotide, usually containing a single purine base (10-12), reaction of an oligonucleotide containing a halopurine in which the halogen is displaced by the amine group of an amino triol derived from a DE (13, 14), and total synthesis of the desired oligonucleotide utilizing a phosphoramidite of the adducted purine base as a building block (15-17). As discussed elsewhere (18), each of these approaches has its own advantages and disadvantages for preparation of significant (multi-milligram) quantities of pure adducted oligonucleotides. The latter phosphoramidite approach is the most versatile in terms of desired sequence and quantity of product synthesized but suffers occasionally from variable and somewhat unpredictable coupling yields. In our experience, BaP-adducted dG phosphoramidites are particularly prone to suboptimal (generally from 99% pure by HPLC). 1H NMR indicated the presence of 2.67 mol of triethylamine per mol of nucleotide. On the basis of an elemental formula of C30H28N5O10P‚2.67 Et3N, this corresponds to an overall yield for the four steps from 10S-3 to the adducted nucleoside phosphate 10S-4 of 46%. Yields for the individual steps were not evaluated. 1H NMR (shown in Figure 1) (300 MHz, DMSO-d with a trace 6 of D2O) 2.60 (m, 1H, H2′), 2.90 (m, 1H, H2′), 3.55 and 3.67 (each m, each 1H, 2 × H5′), 3.96 (dd, 1H, H8, J ) 8.8 and 1.8), 3.99 (m, 1H, H4′), 4.39 (dd, 1H, H9, J ) 3.4 and 1.8), 5.04 (d, 1H, H7, J ) 8.8), 5.05 (m, 1H, H3′), 6.01 (d, 1H, H10, J ) 3.4), 6.31 (m, 1H, H1′), 7.97-8.50 (m, 9H, H8′′ and eight aromatic protons). Ethyl groups of the triethylammonium salt appeared at δ 1.12 (t, 24H, 8 × CH3, J ) 7.3) and δ 2.97 (q, 16H, 8 × CH2, J ) 7.3) which indicated 2.67 mol of triethylamine per mol of nucleotide. 31P NMR spectra (DMSO-d6 with a trace of D2O) δ 0.079 (d, J P-H-3′ ) 8.4) which collapsed to a single peak on proton decoupling. HRMS (m/ z): calcd. for C30H27N5O10P (anion): 648.1496. Found: 648.1501. Circular dichroism spectrum (in H2O, normalized to 1.0 A279) λ, nm (θobs, mdeg): 241 (-31.6), 250 (68.1), 276 (-10.4), 282 (17.1). Use of a 3′-H-Phosphonate to Incorporate the 10R-B[a]P dG Adduct into an Oligonucleotide. An 11-mer oligonucleotide, 5′-
Phosphonate Synthesis of BaP Adducted Oligonucleotides
Figure 2. HPLC separation of the synthetic 10R BaP-adducted 11-mer oligonucleotide (arrow) from earlier-eluting failure sequences on a Waters XTerra MS C18 column (4.6 × 50 mm) eluted at 1 mL/ min with a gradient that increased the proportion of solvent B in solvent A from 0% to 35% over 20 min, where A is 0.1 M ammonium carbonate buffer (pH 7.2), and B is a 1:1 mixture of A with acetonitrile, adjusted to the same pH. Only the peak at 17.9 min contained the pyrene chromophore at 345 nm and constituted 80% of the integrated trace at 260 nm.
C1C2A3T4C5G6*C7T8A9C10C11-3′, in which G6* represents the 10RBaP dG adduct, was prepared by a semiautomated method as follows. The 3′-sequence, 5′-CTACC-3′, was synthesized on a 2-µmol scale on high load (108 µmol/g) controlled-pore glass support, utilizing standard phosphoramidite chemistry on an A ¨ KTAoligopilot 10 automated oligonucleotide synthesizer (GE Healthcare). The terminal dimethoxytrityl (DMT) group was cleaved and collected manually, followed by thorough washing of the beads in the synthesizer cartridge with acetonitrile followed by 1:1 pyridine/ acetonitrile. The 3′-H-phosphonate (10R diastereomer of 3) (21 mg, 18 µmol, ca. 10-fold excess) and 1-adamantanecarbonyl chloride (18 mg, 90 µmol) were treated separately by dissolution in dry pyridine and evaporation in vacuo, and each was dissolved in 125 µL of 1:1 (v/v) pyridine/acetonitrile. The two solutions were mixed for ∼15 s in a syringe and were immediately introduced into the synthesis cartridge. Coupling was allowed to proceed by drawing the solution up and down through the cartridge for 20 min. At the end of this time, the reagent solution was collected, the beads were washed, and the reagent and wash solutions were treated with 1:1 water/triethylamine at 4 °C to regenerate and recover the 3′-Hphosphonate salt. After purification by HPLC, 14 mg of starting material was isolated corresponding to ∼70% recovery. The support-bound 3′-H-phosphonate was oxidized by sequential treatment with 4% I2 in pyridine/water/THF and THF/water/triethylamine solutions (Oxidizers I and II, Glen Research, Sterling, VA) for 2 min each. End capping of failure sequences after oxidation was done by the standard method for phosphoramidite chemistry utilizing acetic anhydride and 1-methylimidazole in pyridine/THF. The DMT group was then removed manually, and the cartridge containing the support-bound oligonucleotide was returned to the synthesizer and the sequence was completed utilizing normal phosphoramidite chemistry. The final DMT group was removed on the synthesizer, the beads were washed with acetonitrile, were dried, and were heated (16 h, 60 °C) in concentrated NH4OH to recover and deprotect the oligonucleotide. The coupling yield of the manual step was approximately 80% on the basis of relative DMT peak areas (recorded by the synthesizer) corresponding to individual A or T residues incorporated prior and subsequent to the manual step. The product as its O6-allyl derivative was subjected to reverse-phase HPLC (Figure 2) for estimation of the yield relative to failure sequences, and the product peak (arrow) was collected for mass determination. ESIMS calcd for C127H153N37O67P10: 3579.6. Found: 3579.3.
Results and Discussion Synthesis of 3′-Phosphates. The present synthesis started with the 10S BaP dG adduct, corresponding to trans opening of the (+)-(7R,8S,9S,10R)-DE-2 isomer, as its O6-allyl 7,8,9-
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triacetate, N2-{10S-(7R,8S,9R-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenyl}-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′deoxyguanosine 1 (18) (Scheme 1), which was converted to 2 by standard procedures (cf. 22). Reaction of 2 with diphenyl phosphite (23) provided the 3′-H-phosphonate, which was isolated as its triethylammonium salt 3 in 97% yield. Structure of the hydrogen phosphonate was established by 31P NMR, which showed the requisite large P-H coupling constant (599.67 Hz). Although it was possible to remove the allyl group by standard methods using tetrakis(triphenylphosphine)palladium(0) catalyst and an excess of morpholine as a scavenger for the allyl group in dichloromethane (18), the reaction was very slow (3-4 days) and required a large excess of catalyst. Furthermore, isolation of the desired product from byproducts of the reaction was difficult because of low solubility of the product. An improved procedure employed a polymer-bound tetrakis(triphenylphosphine)palladium(0) catalyst, which permitted isolation of the product simply by filtration and washing of the resin. Following removal of the dimethoxytrityl group, I2 oxidation (21) of the resulting nucleoside H-phosphonate gave the corresponding 3′-phosphate. Finally, the acetate groups were removed to give the desired nucleoside 3′-monophosphate adduct as its triethylammonium salt 4 in excellent yield (150 mg after purification, overall 57% in four steps). The final product was characterized by 31P and 1H NMR (Figure 1) as well as by HRMS. Because of their low solubility and difficult chromatographic properties, reaction intermediates were not isolated but were tracked by changes in retention time on HPLC and HRMS or ESIMS of the HPLC purified fractions. PAH DE-adducted nucleoside 3′-phosphates have previously been prepared and characterized in submilligram quantities by direct alkylation of the nucleoside 3′-phosphates with DEs in aqueous solution (24, 25). Although amounts adequate for use as chromatographic standards could be obtained by this method, it is not satisfactory for the preparation of significant quantities of the desired 3′-monophosphates because of extensive hydrolysis of the DEs as well as lack of cis/trans stereospecificity. In a different approach but on a similarly small scale, racemic 10amino 7,8,9-triols corresponding to cis and trans opening of (()-BaP DE-2 were allowed to react with a phosphate-protected 6-fluoropurine analogue of deoxyadenosine 3′-phosphate to give the desired N6-dA adducts for use as analytical standards (26). To our knowledge, the analogous reaction utilizing a halogenated analogue of deoxyguanosine 3′-phosphate has not been reported. The present use of 3′-H-phosphonates as key intermediates provides a much improved route whereby the 3′-phosphate of a PAH-adducted deoxyguanosine could be prepared on a scale suitable for crystallographic or other physical studies. In addition to improved ease of handling of the H-phosphonate, this synthetic route offers the advantage of eliminating a separate step for removal of the phosphate protecting group. Adducted 3′-H-Phosphonates as Intermediates for Oligonucleotide Synthesis. Although the use of nucleoside 3′-Hphosphonates for incorporation into oligonucleotides is a wellestablished procedure (19, 20), it has largely been supplanted by the phosphoramidite approach (27) for automated oligonucleotide synthesis on solid supports. The improved stability of the H-phosphonate building blocks relative to phosphoramidites made this approach particularly attractive in light of the synthetic challenges presented by our adducted nucleoside derivatives. In the present study, we synthesized an 11-mer oligonucleotide containing a 10R BaP dG adduct using standard phosphoramidite chemistry to introduce the unmodified nucleotide residues and a 3′-H-phosphonate to introduce the 10R BaP
314 Chem. Res. Toxicol., Vol. 20, No. 2, 2007
Iyer et al. Scheme 1a
a Reagents and conditions: (i) 7% HF in pyridine, (ii) DMT+BF -, lutidine, Li CO , (iii) diphenyl phosphite, (iv) polymer-bound tetrakis(triphenylphos4 2 3 phine)palladium, morpholine, (v) 3% dichloroacetic acid in CH2Cl2, (vi) I2 in pyridine, (vii) 1:1 NH4OH:MeOH followed by triethylamine.
DE-dG adduct. The same approach was also successfully utilized with the 10S diastereomer. As had been noted previously for unadducted nucleosides, we have found that the present hydrocarbon adducted 3′-H-phosphonates are more stable, more soluble, and more easily purified than their corresponding phosphoramidite intermediates. An additional advantage is the ease of recovery of the excess H-phosphonate reagent utilized in oligonucleotide synthesis, which can be recovered intact either manually or by diversion of the reagent waste stream to a collection port on the synthesizer. In contrast, recycling of phosphoramidites, although possible (28), is less satisfactory because the material is recovered as the free 3′-hydroxy compound which must be purified and reconverted to the desired phosphoramidite. Thus, the present H-phosphonate approach may also prove useful for the introduction of other modified nucleotide residues where efficient recovery of excess monomer reagent is desirable because of the expense or labor required for its synthesis. Several experimental details of the present oligonucleotide synthesis deserve special mention. We have demonstrated that it is possible to use a “mixed” approach in which the unadducted nucleotides are incorporated as their phosphoramidites by automated synthesis, and only the modified residue is introduced as the H-phosphonate. This obviates the need to reprogram and optimize the synthesizer for H-phosphonate chemistry, since the critical H-phosphonate coupling step can be done manually. However, the H-phosphonate diesters are not stable to the standard conditions used for end capping of failure sequences following subsequent phosphoramidite couplings. Thus, oxidation to the phosphodiester must take place immediately following the H-phosphonate coupling step and not in a single step after completion of the entire oligonucleotide sequence, although this is a common practice (20) when all residues are introduced as H-phosphonates. Although the nucleoside H-phosphonates are stable and easy to handle in the absence of an activator, the coupling reaction in the presence of adamantanecarbonyl chloride is highly sensitive to water and other solvent impurities. For use in our experiments, pyridine and acetonitrile used as solvents for the manual coupling step were freshly distilled each day (from KOH and CaH2, respectively) and were stored briefly over molecular sieves. Although efficient coupling utilized a significant molar excess of the H-phosphonate monomer over the support-bound oligonucleotide, the ease with which the
unused reagent can be recovered largely eliminates this potential drawback when using valuable, specialized H-phosphonate monomers. Acknowledgment. The authors are indebted to Dr. Isabella L. Karle, Naval Research Laboratories, Washington, DC for microscopic examination of crystals of the sodium salt of the nucleotide adduct 10S-4 from ethanol:water. Crystals grew as prisims but were too small for X-ray analysis. This research was supported by the Intramural Research Program of the NIH (National Institute of Diabetes and Digestive and Kidney Diseases).
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Phosphonate Synthesis of BaP Adducted Oligonucleotides
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