DECEMBER 2002 VOLUME 15, NUMBER 12 © Copyright 2002 by the American Chemical Society
Communications Efficient Synthesis of the Benzo[a]pyrene Metabolic Adducts of 2′-Deoxyguanosine and 2′-Deoxyadenosine and Their Direct Incorporation into DNA Francis Johnson,* Radha Bonala, Deepak Tawde, M. Cecilia Torres, and Charles R. Iden Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-3400 Received September 14, 2002
A new and efficient method is described for the synthesis in gram quantities of the benzo[a]pyrene (B[a]P) metabolic adducts of 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) substituted, respectively, at the N2- and N6- positions. When the racemic form of the tris(benzoyloxy)amine 5 (related to the notoriously carcinogenic epoxydiol 2) is coupled with the bromoinosine derivative 6 by means of a Buchwald-Hartwig reaction, the expected pair of diastereomers, 7 and 8, is obtained in high (combined) yield. Selective deblocking of this mixture then gave cleanly the pair of diastereomers 9. These were used in the synthesis of a series of DNA oligomers via their 5′-O-DMT-3′-O-phosphoramidites (10) using standard automated methods. Coupling efficiencies were 94-98% at the point of introduction of the xeno-2′deoxynucleoside, and in all cases the mixtures of the two diastereomeric oligomers (DMT-off stage) were easily separated by HPLC. By a similar sequence of reactions beginning with 5 and the protected 6-bromopurine 2′-deoxynucleoside 11, it was possible with equal efficiency to introduce the N6-modified diastereomers (16) of dA into oligomeric DNA. Circular dichroism measurements were used to establish the fundamental configurations at the xeno-2′deoxynucleoside site for each of the oligomers. Mass spectral data in both the dG and the dA series confirmed the presence of the xeno-2′-deoxynucleoside in the oligomers. This was complemented by enzymatic degradation of one of the oligomers from each of the series. In both of these cases, after HPLC separation, circular dichroism measurements on the reisolated xenonucleoside also confirmed its presence in the oligomer.
Introduction Many carcinogenic polycyclic aromatic hydrocarbons are widely distributed in the environment, and their * To whom correspondence should be addressed. Telephone: (631) 632-8862. Fax (631) 632-7394. E-mail:
[email protected].
generation from burning organic matter is well-documented. Mostly these substances require metabolic activation (1-3) before they exhibit genotoxic effects. Extensive investigations have shown that among a variety of oxidative metabolites the epoxydiols 1 and 2 (Figure 1) are perhaps the most potent carcinogens
10.1021/tx0256174 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/21/2002
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Figure 1. Chemical structures of the epoxydiols 1 and 2 and the 2′-deoxynucleosides 3 and 4 with which they were coupled.
Figure 2. Basic structures of the B[a]P-metabolite adducts of dG and dA.
although other isomers also are similarly active. The epoxy group in each of these compounds reacts electrophilically with DNA at a number of electron-rich sites, the most prominent being the N2- and N6-amino groups of 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) residues (4, 5) to give the corresponding adducts (Figure 2). The role that such adducts play in both mutagenesis and carcinogenesis has been, and continues to be, an area of intense scientific investigation. However, these studies have been hampered by two limitations: (a) the difficulty of synthesizing these adducts in large quantity and (b) the problem of incorporating them site-specifically into oligomeric DNA. Two lines of attack have been employed in attempts to solve these problems. In the earlier methods a suitably protected purine 2′-deoxynucleoside having either a fluoro (6-8) or a sulfonyloxy leaving group (9-12) (at locations corresponding to the amino groups in the guanine and adenine series) was allowed to react with a 10-aminotriol related to the benzo[a]pyrene epoxydiols (13-15). These reactions can be used to synthesize the adducts themselves, and in the case of the 2′-deoxyadenosine, DNA oligomers containing the modified adducts could be obtained by a “postsynthetic” method of introduction (10). However, the synthesis of the corresponding adducts in the dG series has proved more difficult (12). In discussing some of the problems associated with previous approaches Ramesha, Kroth, and Jerina (16) have introduced a new method for the preparation of specific adducts in both the dG and dA series. In principle, this consists of reacting an epoxydiol, 1 or 2, with 3 or 4 in trifluoroethanol solution at room temperature (Figure 1). In the dG series, mixtures of C-10 isomers were obtained which could only be separated by HPLC as their triacetates, whereas in the dA series the reactions gave single isomers. Yields varied from 22 to 65%.
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Although this method is a significant improvement over previous procedures, the lack in the dG series of specificity in the opening of the epoxide ring and the problems of separation make it difficult to prepare large quantities of the pure isomers. For this reason in our own approach to the synthesis of both the dG and dA adducts, we elected to start with an aminotriol derivative in the benzo[a]pyrene series (as had been done in earlier work) because of the better control of the stereochemistry at the 9 and 10 positions. The coupling of the nucleosides to the hydrocarbon moiety is the most vexing step in this area of purine chemistry, but we would now like to report a complete solution to the problem through the use of the Buchwald-Hartwig amination reaction (17-20). In previous work, we had shown (21-23) that this reaction could be employed to synthesize, in excellent yield, a variety of N2-substituted dG derivatives, starting with a series of amino compounds and 2-bromo-O6-benzyl-3′,5′bis-O-tert-butyldimethylsilyl-2′-deoxyinosine (6). The latter compound was prepared in 75% yield by adapting the methods used by Robins and Uznanski (24) for the synthesis of a series of 2-halopurine nucleosides of the ribo-class. Lakshman and his associates (25), using a series of simple arylamines, also have reported the synthesis of a number of N6-arylated derivatives of dA by means of the Buchwald-Hartwig procedure. However, none of these derivatives were subsequently incorporated into oligomeric DNA.
Results and Discussion We have now found that under the conditions of the Buchwald-Hartwig reaction, compound 6 reacts with the racemic form of the 10-aminotribenzoate 5 (26) to give essentially an equimolar mixture (27a) of the expected diastereomers 7 and 8 in a combined yield of ∼87%. (Scheme 1). This coupling yield represents a significant advance in the synthesis of such adducts because it allows the preparation of gram quantities of these substances directly and in a short period of time. The mixture of diastereomers was easily purified from undesired materials by chromatography on silica gel. All physical data including mass spectral information confirmed that only 7 and 8 were present after purification. Although it was possible to separate these diastereomers by chromatography we elected to simplify the overall process by using the mixture of isomers in the preparation of oligomeric DNA. Removal of the 6-O-benzyl group from the purine moiety was then accomplished by hydrogenolysis over a palladium catalyst. This did not affect either the 10amino or the 7-benzoyloxy groups on the B[a]P residue, despite the fact that both occupy benzylic locations. Deblocking of the silyl protecting groups was effected cleanly by anhydrous HF in an excess of pyridine, there being no evidence of any cleavage at the glycosidic linkage. Conversion of the resulting mixture of diastereomers 9 to the desired 5′-O-DMT-3′-O-phosphoramidites 10 by standard methods was accomplished easily, the overall yield being ∼60%. When this mixture of four diastereomers was employed in a standard automated protocol for DNA synthesis, the coupling efficiencies at the point of introduction of the modified nucleoside varied from 94 to 98%. In all cases the coupling time allowed was 30 min. The oligomers, containing the B[a]P-N2-dG adduct, that were synthesized are denoted in Table 1 by
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Chem. Res. Toxicol., Vol. 15, No. 12, 2002 1491 Scheme 1
Table 1. Sequence and Mass Spectral Data of the Synthesized DNA Oligomers mass m/z calculated
entry
oligomer sequence G* ) B[a]P-N2-dG A* ) B[a]P-N6-dA
1 2 3 4 5 6 7 8 9 10 11 12 13
5′-GAG GTG CG*T GTT TGT-3′ 5′-GTG AGG CG*C TGC CCC-3′ 5′-ATG AAC CG*G AGG CCC-3′ 5′-GAG GTG AG*T GTT TGT-3′ 5′-GAG GTG GG*T GTT TGT-3′ 5′-GAG GTG TG*T GTT TGT-3′ 5′-GTG AGG AG*C TGC CCC-3′ 5′-GTG AGG GG*C TGC CCC-3′ 5′-GTG AGG TG*C TGC CCC-3′ 5′-GAG GTG [5Me]CG*T GTT TGT-3′ 5′-CCT TCG* CTA CTT TCC TCT CCC TTT-3′ 5′-TCC TCC TCA* CCT CTC-3′ 5′-CCG GAC A*AG AAG C-3′
4972.1 4872.0 4889.0 4996.1 5012.0 4987.1 4896.0 4912.0 4887.0 4986.1 7409.7 4676.9 4279.7
entries 1-11. All of these oligomers were synthesized at the 1.0 µmol scale on a Perkin-Elmer/Applied Biosystems (Foster City, CA) 394 DNA Synthesizer.
observed (-)-trans
(+)-trans
4971.4 4871.6 4888.7 4996.1 5012.3 4987.5 4896.6 4912.7 4887.7 4986.8 7408.4 4677.3 4280.4
4971.6 4871.8 4888.8 4996.3 5012.4 4987.6 4896.7 4912.8 4887.7 4986.2 7408.1 4677.3 4280.3
The purification of the DNA was accomplished in two stages. In the first the deprotected oligomer having a terminal DMT group was separated from failure se-
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purified by HPLC. This double purification technique was found to be superior to the single-stage method in which the DMT is removed while the DNA is still on the column. In effect the purification is simpler and less work intensive. It would also be noted that little depurination at the xenonucleotide site was observed during DNA synthesis and purification, provided that the final deprotection with 80% acetic acid (removal of the terminal DMT group) was limited to a brief period (
