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Chem. Res. Toxicol. 1996, 9, 630-637
Improved Strategies for Postoligomerization Synthesis of Oligodeoxynucleotides Bearing Structurally Defined Adducts at the N2 Position of Deoxyguanosine Bart L. DeCorte,† Dimitrios Tsarouhtsis, Satyanarayan Kuchimanchi,‡ Monica D. Cooper, Pamela Horton, Constance M. Harris,* and Thomas M. Harris* Chemistry Department and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received October 18, 1995X
Improved methodology has been developed for preparation of oligodeoxynucleotides bearing adducts on the N2 position of guanine in which the adduction reaction is carried out in homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix. The methodology utilizes a new synthon, 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (3). Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O6 protection is eliminated under very mild conditions following displacement of the 2-fluoro group by amine nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed by removal of the remaining protective groups with NH4OH (60 °C, 8 h) and then 0.1% acetic acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting groups on the exocyclic amino groups.
Introduction Oligodeoxynucleotides containing structurally defined adducts of carcinogens, drugs, and other xenobiotics have recently attracted much interest for structural and biological studies. Major synthetic approaches to them include direct reactions of electrophiles with oligonucleotides (1,2) and assembly of oligonucleotides from adductmodified nucleosides (3-5). Our interest in adducted oligonucleotides involves mutagenic substances. In 1991, we reported a postoligomerization strategy for the synthesis of oligonucleotides containing styrene oxide adducts at the N6 position of adenine. This strategy involves reversal of the customary electrophile-nucleophile relationship of mutagen and DNA, i.e., with an amino alcohol as a replacement for the epoxide in a reaction with a 6-halopurine as a replacement for adenine (6). Similar postoligomerization strategies using other reactive nucleosides have been used by others for different purposes (7,8). This method offers the advantage of being completely regio- and stereospecific and is adaptable to any DNA sequence including those containing multiple deoxyadenosines. The condensation reaction was carried out with the oligonucleotide still attached to the solid support used in its synthesis and with exocyclic amino groups and phosphodiesters fully protected since conditions needed to deprotect the oligonucleotide seemed likely to lead to loss of the halo substituent. The reaction of 6-chloropurine deoxynucleoside, within the immobilized oligonucleotide, worked well for the preparation of * Author to whom correspondence and reprint requests should addressed at the Department of Chemistry, Vanderbilt University, Nashville, TN 37235. † Present address: Janssen Research Foundation, Spring House, PA 19477. ‡ Present address: ChemGenes, Waltham, MA 02154. X Abstract published in Advance ACS Abstracts, March 15, 1996.
0893-228x/96/2709-0630$12.00/0
oligomers containing styrene oxide adducts at the N6 position of deoxyadenosine. Extension of the strategy to weaker nucleophiles required the use of a leaving group better than chloride ion. Fluorine is more reactive than chlorine in aromatic nucleophilic substitution reactions, and 6-fluoropurine was found to have significantly higher reactivity than the 6-chloro species (9). Oligonucleotides bearing adenine N6 adducts of the carcinogenic bayregion diol epoxides of benzo[a]pyrene were prepared by reaction of the 6-fluoropurine oligonucleotides with aminotriols derived from the diol epoxides (10). Our original publication also reported the preparation of oligonucleotides adducted at the N2 position of guanine using 2-fluorodeoxyinosine as the deoxyguanosine synthon (6). However, the procedure proved to be somewhat inferior to that for adenine adducts because the rate of reaction with 2-amino-2-phenylethanol was significantly reduced over the corresponding reaction of 6-halonucleosides, and substantial quantities of byproducts were formed. In spite of these difficulties, pure samples of adducted oligonucleotides could be prepared. However, extensive purification was required due to side reactions, the principal one being nucleophilic attack by the amine at the C4 position of N-acylated cytosine to give N4styrene oxide adducts of deoxycytidine. This observation is in accord with earlier reports on the reactivity of acylated cytosine with amine nucleophiles (7,11). With the more reactive 6-halopurines, the cytosine side reaction was not quantitatively significant. In the present paper we describe synthetic strategies which improve both the yield and purity of oligonucleotides containing guanine adducts.
Materials and Methods General Methods. 1H NMR spectra were recorded at 200.13 and 300.13 MHz on a Bruker AC 200 or AC 300 NMR
© 1996 American Chemical Society
Postoligomerization Synthesis of Oligodeoxynucleotides spectrometer with CD3CN, CDCl3, or MeOH-d4 as solvent. 13C NMR spectra were recorded at 75.47 MHz on a Bruker AC 300 NMR spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane or CDCl3 as an internal standard. 31P NMR spectra were recorded at 81.0 MHz on a Bruker AC 200 NMR spectrometer with CD3CN as solvent; 85% H3PO4 was used as the external standard. 19F NMR spectra were recorded at 282.40 MHz on a Bruker AC 300 NMR spectrometer with CH3CN-d3 and MeOH-d4 as solvents; CFCl3 was used as the internal standard. Methylene chloride, pyridine, triethylamine, and diisopropylethylamine were distilled from calcium hydride under a nitrogen atmosphere. 1,4-Dioxane was distilled from sodium under a nitrogen atmosphere. Methanol was distilled from sodium methoxide (5 mol %) under a nitrogen atmosphere. Other chemicals were used as purchased without further purification. Melting points are uncorrected. Thin layer chromatography was performed with silica gel F254 (Merck) as the adsorbent on glass plates. The chromatograms were visualized under UV (254 nm) or by staining with an anisaldehyde/sulfuric acid solution, followed by heating. Column chromatography was performed using silica gel 60, 70230 mesh (Merck). O6-(Trimethylsilylethyl)-2′-deoxyguanosine (2). This compound, first described by Gaffney and Jones (12), was prepared via a Mitsunobu reaction (3,14,15). N2,3′,5′-Triacetyldeoxyguanosine (16) (1, 5.11 g, 13 mmol) was dried in vacuo in a Abderhalden drying apparatus for 24 h at 78 °C. A 1-L, three-necked flask equipped with a reflux condenser was charged with 1, triphenylphosphine (6.82 g, 26.01 mmol), and anhydrous dioxane (430 mL). The suspension was maintained under an argon atmosphere and heated to 100 °C (oil bath temperature). The pale orange solution was stirred for 5 min, and trimethylsilylethanol (3.08 g, 26.0 mmol) and diethyl azodicarboxylate (4.1 mL, 26.0 mmol) were simultaneously added to the solution. The reaction mixture was stirred for 15 min at 100 °C, and allowed to cool to room temperature over a 2 h period. The orange reaction mixture was concentrated in vacuo to obtain a viscous red oil. The oily residue was dissolved in anhydrous methanol (120 mL) and transferred to a solution of sodium methoxide in methanol (0.35 M, 200 mL). The solution was stirred at room temperature under an argon atmosphere for 7 h. Glacial acetic acid (6.23 mL, 109 mmol) dissolved in water (30 mL) was added, and the reaction mixture was concentrated in vacuo to give a red slurry. The residue was suspended in methylene chloride (100 mL) and washed with water (40 mL). The aqueous portion was extracted with CH2Cl2 (5 × 100 mL); the combined organic portions were dried with Na2SO4 and concentrated in vacuo. The red oil was purified by flash chromatography using a gradient of CH2Cl2/MeOH/Et3N (97.8:2.0:0.2 to 94.8:5.0:0.2) to yield 2 as a pale yellow powder (4.23 g, 89%): mp 66-68 °C, TLC Rf 0.41 (CH2Cl2/MeOH, 9:1). 1H NMR (MeOH-d ) δ 8.00 (s, 1H, H8), 6.30 (dd, 1H, J ) 6.1 4 Hz, J ) 2.1 Hz, H1′), 4.61-4.53 (m, 3H, OCH2CH2Si, H3′), 4.03 (q, 1H, J ) 2.7 Hz, H4′), 3.83 (dd, 1H, J ) 3.3, 12.2, H5′), 3.73 (dd, 1H, J ) 12.2, 2.9, H5′′), 2.76 (m, 1H, H2′), 2.34 (m, 1H, H2′′), 1.21 (m, 2H, CH2Si), 0.08 (s, 9H, [(CH3)3Si]. 1H NMR (CDCl3) δ 7.64 (s, 1H, H8), 6.16 (dd, 1H, J ) 8.8 Hz, J ) 5.7 Hz, H1′), 5.04 (s, 2H, NH2), 4.56 (d, 1H, J ) 4.8 Hz, H3′), 4.39 (t, 2H, J ) 8.6 Hz, OCH2CH2Si), 4.07 (br, 1H, H4′), 3.70 (m, 2H, H5′, H5′′), 2.73 (m, 1H, H2′), 2.21 (m, 1H, H2′′), 1.02 (t, 2H, J ) 8.6 Hz, CH2Si), -0.10 (s, 9H, (CH3)3Si). 13C NMR (CDCl3) δ 161.18 (C aromatic), 158.46 (C aromatic), 151.86 (C aromatic), 138.49 (C aromatic), 116.23 (C aromatic), 88.71 (C4′), 86.58 (C1′), 72.15 (C3′), 64.62 (OCH2CH2Si), 62.90 (C5′), 39.85 (C2′), 17.09 (CH2Si), -1.83 [(CH3)3Si]. HRMS-FAB1 (glycerol-TFADMSO matrix) calcd for C15H25N5O4Si 368.1754, found 368.1749 (M + H)+. 1 Abbreviations: DMTr, 4,4′-dimethoxytrityl; FAB, fast atom bombardment mass spectrometry; MALDI-FT, matrix-assisted laser-desorption Fourier transform mass spectroscopy; NPE, p-nitrophenethyl; PAC, 4-tert-butylphenoxyacetyl; SO, styrene oxide; TEAA, triethylammonium acetate; TMSE, trimethylsilylethyl.
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 631 2-Fluoro-(O6-trimethylsilylethyl)-2′-deoxyinosine (3). This compound was prepared essentially as described previously for the synthesis of the O6-benzyl (3,6) and O6-(2-nitrophenylethyl) (3) analogues. O6-(Trimethylsilylethyl)-2′-deoxyguanosine (2) (0.53 g, 1.44 mmol), dried in vacuo in an Abderhalden drying apparatus at 64 °C for 20 h, was transferred to a polypropylene conical tube (50 mL) equipped with a stirring bar and a septum under an argon atmosphere. Anhydrous pyridine (5.42 mL) was added to a second polypropylene conical tube (50 mL) equipped with a stirring bar and septum, under an argon atmosphere. Both tubes were placed in a cooling bath (dry ice-acetone), and the temperature was lowered to -35 to -40 °C. Hydrogen fluoride-pyridine (70% solution, 9.65 mL, Aldrich Chemical Co.) was added to the tube containing pyridine over a period of 3 min to generate a 45% hydrogen fluoride-pyridine solution (17). The yield in this reaction is highly dependent upon the quality of the HF/pyridine reagent; deeply colored reagent gave considerable depurination. This solution was stirred under an argon atmosphere for 15 min at -35 to -40 °C. The clear solution was then added slowly to 2 and stirred at -35 to -40 °C for 5 min. tert-Butyl nitrite (0.43 mL, 36 mmol) was added over 5 min to the reaction mixture while maintaining the bath temperature of -35 °C. The red reaction mixture was stirred under argon for 25 min. The solution was cautiously transferred to a K2CO3 solution (23 g in 34 mL H2O) at 0 °C over a 2 min period. The solution was extracted with EtOAc (5 × 40 mL), and the combined organic portions were dried over K2CO3, filtered, and concentrated in vacuo. The red oily residue was placed under high vacuum overnight. Flash chromatography, using a gradient of CH2Cl2/MeOH/Et3N (95:4:1 to 90:9:1) provided 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (3, 0.51 g, 95%): mp 120-122 °C, TLC Rf 0.36 (CH2Cl2/MeOH, 9:1). 1H NMR (MeOH-d4) δ 8.46 (s, 1H, H8), 6.41 (t, 1H, J ) 6.7 Hz, H1′), 4.67-4.72 (m, 2H, OCH2CH2Si), 4.56 (m, 1H, H3′), 4.02 (q, 1H, J ) 3.6 Hz, H4′), 3.81 (dd, 1H, J ) 3.6, H5′′), 3.73 (dd, 1H, J ) 2.9, H5′), 2.77 (m, 1H, H2′′), 2.44 (m, 1H, H2′), 1.26 (m, 2H, CH2Si), 0.1 (s, 9H, [(CH3)3Si]. 1H NMR (CDCl3) δ 8.56 (s, 1H, H8), 6.31 (t, 1H, J ) 6.7 Hz, H1′), 5.35 (d, 1H, J ) 4.2 Hz, CHOH), 4.94 (t, 1H, J ) 5.5 Hz, CH2OH), 4.64 (t, 2H, J ) 8.3 Hz, OCH2CH2Si), 4.40 (m, 1H, H3′), 3.87 (q, 1H, J ) 4.0 Hz, H4′), 3.55 (m, 2H, H5′ and H5′′), 2.68 (m, 1H, H2′), 2.32 (m, 1H, H2′′), 1.20 (t, 2H, J ) 8.3 Hz, CH2Si), 0.08 (s, 9H, (CH3)3Si). 13C NMR (CDCl3) δ 161.8 (d, JC,F ) 18 Hz, C6), 156.0 (d, JC,F ) 209 Hz, C2), 152.7 (d, JC,F ) 18 Hz, C4), 142.4 (d, JC,F ) 2 Hz, C8), 119.5 (d, JC,F ) 5 Hz, C5), 88.0 (C4′), 83.8 (C1′), 70.5 (C3′), 66.1 (OCH2CH2Si), 61.4 (C5′), 39.3 (C2′), 16.9 (CH2Si), -1.4 [(CH3)3Si)] 19F NMR (MeOH-d4) δ -51.55. 19F NMR (CDCl3) δ -50.40. HRMS-FAB (glycerol-TFA-DMSO matrix) calcd for C15H23N4O4SiF 371.1551, found 371.1551 (M + H)+. 5′-O-(4,4′-Dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (4). The dimethoxytritylation of 3 was carried out by standard procedures (18). 2-Fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (3) (200 mg, 0.54 mmol) was dried by treatment with anhydrous pyridine (3 × 10 mL) and placed under vacuum for 90 min. Anhydrous pyridine (20 mL) and diisopropylethylamine (0.56 mL, 3.24 mmol) were added to the flask containing (3), and the mixture was stirred under an argon atmosphere. The solution was cooled to 10 °C, and 4,4′dimethoxytrityl chloride (728 mg, 2.15 mmol) was added. The reaction mixture was allowed to warm slowly to room temperature, and the progress of the reaction was monitored by TLC. After 15 h the reaction mixture was treated with methanol (0.052 mL, 1.3 mmol) and stirred for 10 min to quench the unreacted trityl chloride. The reaction mixture was concentrated in vacuo, and the orange compound was taken up in CH2Cl2 (125 mL) and extracted with 10% aqueous K2CO3 solution (2 × 50 mL). The combined organic layers were dried over K2CO3, filtered, and evaporated. Flash chromatography (CH2Cl2/ MeOH/Et3N, 98:1.5:0.5 to 94.5:5.0:0.5) yielded 5′-O-(dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (4) (305 mg, 84%): TLC Rf 0.29 (CH2Cl2/MeOH, 9.8:0.2). 1H NMR (MeOH-d4) δ 8.29 (s, 1H, H8), 7.34-7.31 (m, 2H, aromatic),
632 Chem. Res. Toxicol., Vol. 9, No. 3, 1996 7.22-7.12 (m, 7H, aromatic), 6.75-6.69 (m, 4H, aromatic), 6.38 (t, 1H, J ) 4.8 Hz, H1′), 4.69-4.61 (m, 3H, OCH2CH2Si and H3′), 4.13 (m, 1H, H4′), 3.73 (s, 3H, CH3O), 3.72 (s, 3H, CH3O), 3.41-3.37 (m, 1 H, H5′′) 3.27-3.24 (m, 1H, H5′), 2.97-2.92 (m, 1H, H2′′), 2.50-2.45 (m, 1H, H2′), 1.28-1.24 (m, 2H, CH2Si), 0.08 (9H, s, (CH3)3Si). 13C NMR (CDCl3) δ 162.52 (d, JC,F ) 18 Hz, C6), 158.45 (C aromatic), 157.87 (d, JC,F ) 214 Hz, C2), 152.4 (d, JC,F ) 19 Hz, C4), 144.60 (C aromatic), 140.83 (d, JC,F ) 3 Hz, C8), 135.61 and 135.55 (C aromatic), 129.92 (C aromatic), 127.99 and 127.74 (C aromatic), 126.79 (C aromatic), 120.11 (d, JC,F ) 5 Hz, C5), 113.06 (C aromatic), 86.46 (C4′), 86.40 (CPh(PhOMe)2), 84.57 (C1′), 72.00 (C3′), 66.68 (OCH2CH2Si), 63.77 (C5′), 55.05 (CH3O)2), 40.15 (C2′), 17.46 (CH2Si), -1.55[(CH3)3Si]. 19F NMR (MeOH-d4) δ -51.13. 19F NMR (CDCl3) δ -50.4. HRMS-FAB (glycerol-TFA-DMSO matrix) calcd for C36H41N4O6SiF 673.2858, found 673.2847 (M + H)+. 3′-O-[(N,N-Diisopropylamino)-(2-cyanoethyl)phosphinyl]5′-O-(dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′deoxyinosine (5). 5′-O-(dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (4, 180 mg, 0.26 mmol) was dried by treatment with anhydrous pyridine (2 × 10 mL) and placed under vacuum for 3 h. Anhydrous 1H-tetrazole (21 mg, 0.31 mmol) was added to a flame-dried 25-mL round-bottomed flask and kept under an argon atmosphere. A solution of the tritylated compound in anhydrous CH2Cl2 (5 mL) was injected into the reaction flask followed by 2-cyanoethyl-N,N,N′,N′tetraisopropyl phosphoramidite (125 mg, 0.39 mmol) (19,20). The reaction mixture was stirred under an argon atmosphere at room temperature for 2 h. The mixture was transferred into a saturated solution of NaHCO3 (30 mL) and extracted with CH2Cl2 (5 × 40 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The yellow residue was purified by flash chromatography (CH2Cl2/EtOAc/pyridine, 69:30:1) to yield 3′-O-[(N,N-diisopropylamino)-(2-cyanoethyl)phosphinyl]-5′-O(dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (5, 200 mg, 88%). TLC Rf 0.88 (CH2Cl2-MeOH, 9.8:0.2). 1H NMR (MeOH-d ) δ 8.09 (s, 1H, H8), 7.36-7.32 (m, 2H, H 4 aromatic), 7.27-7.16 (m, 7H, H aromatic), 6.79-6.73 (m, 4H, H aromatic), 6.32 (m,1H, H1′), 4.82 (m, 1H, H3′), 4.65 (m, 2H, OCH2CH2Si), 4.18 (m, 1H, H4′), 3.72 (m, 8H, (CH3O)2 and H5′, H5′′), 3.63-3.56 (m, 2H, N(CHMe2)2), 3.31-3.27 (m, 2H, POCH2), 2.97-2.92 (m, 1H, H2′), 2.63 (t, 1H, J ) 4.4 Hz, CH2CN), 2.58 (m, 1H, H2′′), 2.53 (t, 1H, J ) 4.4 Hz, CH2CN), 1.23 (m, 2H, CH2Si), 1.16 (m, 12H, N(CH(CH3)2)2), 0.09 (9H, (CH3)3Si). 1H NMR (CDCl3) δ 7.98 and 7.96 (s, 1H, H8), 7.40-6.60 (m, 13H, H aromatic), 6.29 (t, 1H, J ) 6.5 Hz, H1′), 4.67 (m , 1H, H3′), 4.58 (t, 2H, J ) 8.5 Hz, OCH2CH2Si), 4.20 (m, 1H, H4′), 3.75 and 3.60 (m, 2H, H5′ and H5′′), 3.67 and 3.66 (s, 6H, (CH3O)2), 3.55 (m, 2H, N(CHMe2)2), 3.50 and 3.30 (m, 2H, POCH2), 2.76 and 2.53 (m, 2H, H2′ and H2′′), 2.37 (t, 2H, J ) 6.3 Hz, CH2CN), 1.18 (t, 2H, J ) 8.5 Hz, CH2Si), 1.09 (m, 12H, (CH(CH3)2)2), 0.04 (9H, (CH3)3Si). 13C NMR (CDCl3) δ 162.40 (d, JC,F ) 18 Hz, C6), 158.30 (s, C aromatic), 157.71 (d, JC,F ) 215 Hz, C2), 152.34 (d, JC,F ) 19 Hz, C4), 144.31 (s, C aromatic), 140.74 (d, JC,F ) 3 Hz, C8), 135.42 and 135.37 (s, C aromatic), 129.85 and 129.81 (s, C aromatic), 127.92 and 127.87 (s, C aromatic), 127.61 (s, C aromatic), 126.66 and 126.62 (s, C aromatic), 120.10 (d, JC,F ) 5 Hz, C5), 117.39 and 117.25 (s, CN), 112.89 (s, C aromatic), 86.27 (s, PhC(PhOMe)2), 85.80 (d, JC,P ) 6 Hz, C4′) and 85.58 (d, JC,P ) 6 Hz, C4′), 84.47 (s, C1′), 73.79 (d, JC,P ) 17 Hz, C3′) and 73.33 (d, JC,P ) 17 Hz, C3′), 66.50 (s, OCH2CH2Si), 63.28 and 63.13 (s, C5′), 58.18 (d, JC,P ) 20 Hz, POCH2) and 58.09 (d, JC,P ) 19 Hz, POCH2), 54.97 and 54.94 (s, (CH3O)2), 43.05 (d, JC,P ) 12 Hz, N(CHMe2)2) and 42.73 (d, JC,P ) 13 Hz, N(CHMe2)2), 39.17 and 39.15 s, C2′), 24.50-24.25 (m, (CH(CH3)2)2), 20.19 (d, JC,P ) 7 Hz, CH2CN) and 20.01 (d, JC,P ) 7 Hz, CH2CN), 17.32 (s, CH2Si), -1.64 (s, (CH3)3Si). 19F NMR (CH3CN-d3) δ -51.13 and -51.17. 19F NMR (CDCl3) δ -50.0 and -50.1. 31P NMR (CH3CN-d3) δ 149.96 and 149.82. 31P NMR (CDCl3) δ 146.6 and 146.5. Oligonucleotides. Oligodeoxynucleotides were synthesized on a Model 391 PCR-Mate DNA Synthesizer (Applied Biosystems) on a 1- or 10-µmol scale using the manufacturer’s
DeCorte et al. standard protocol. Initially, the TCA concentration was reduced from 3% to 0.3%; subsequently syntheses have been performed with the standard 3% trichloroacetic acid solution without significant decrease in yield. Capillary gel electrophoresis was performed on a Beckman P/ACE 2000 instrument using the manufacturer’s ssDNA 100 gel capillary and Tris-borate-urea buffer. Samples were applied at -5 kV and run at -15 kV at 30 °C. Enzymatic digestions were carried out under conditions described by Borowy-Borowski et al. (21) (0.2-0.4 ODs of oligodeoxynucleotide, 0.1 M Tris-HCl, pH 8.2, 2.0 mM MgCl2, 6 µg of snake venom phosphodiesterase, 6 µg of alkaline phosphatase at 37 °C). The digests were analyzed by HPLC (C-18 column, 4.6 × 250 mm) with the following gradient: (A) 0.1 M triethylammonium acetate (TEAA),1 pH 6.5 and (B) CH3CN, 1-10% B over 15 min; 10-20% B over 5 min; hold for 5 min, and then to 100% B over 10 min at a flow rate of 1.5 mL/min. Kinetic Study of Adduction of (S)-2-Amino-2-phenylethanol to Matrix-Bound Modified 11-Mer 6. Individual samples containing 0.2 µmol of beads, 40 µL of solvent (DMSO or dimethylacetamide), and (S)-2-amino-2-phenylethanol (1.37 mg, 10 µmol) in sealed glass tubes were heated in an oil bath at 65, 75, or 85 °C. Tubes were removed daily for 4 days and cooled, and the contents were transferred to a small vial. The supernatant was removed with a pipette, and the beads were washed with methanol (4 × 1 mL) and diethyl ether (4 × 1 mL). After drying the beads were treated with 1 mL of conc. NH4OH for 12 h at 60 °C. The vial was cooled to room temperature and opened, and the contents were evaporated to dryness with a Speed-Vac. HPLC analysis was carried out on an analytical C-18 column, using a gradient of (A) 0.1 M TEAA, pH 7.0, and (B) CH3CN, from 5% to 20% B over 10 min and 20% to 30% B over 20 min with a flow rate of 1.5 mL/min. Adduction of (R)-2-Amino-2-phenylethanol to Modified 11-Mer 6 (Solution Synthesis). Following synthesis of modified 11-mer 6 by the standard protocol (except for the use of 0.3% trichloroacetic acid for detritylation) the beads from four 1-µmol cassettes were suspended in 0.1 M NaOH (5 mL) and stirred slowly for 8 h at room temperature. The beads were allowed to settle, the supernatant was removed, and the solid residue was washed with H2O (5 × 5 mL). The combined aqueous fractions were neutralized cautiously with dilute acetic acid. The solution was lyophilized, and the residual solid (partially deprotected oligonucleotide 7) was transferred into a conical vial (3 mL). DMSO (2.5 mL) and (R)-2-amino-2phenylethanol (0.58 mmol, 80 mg) were added, and the suspension was heated for 2 days at 75 °C with occasional stirring. The reaction vessel was allowed to cool to room temperature, the contents were transferred to a 20 mL vial, and diethyl ether (10 mL) was added. The supernatant was removed, and the residual solid was washed with diethyl ether (4 × 10 mL). The solid was allowed to dry, concentrated NH4OH (10 mL) was added, and the tightly closed vial was heated for 8 h at 60 °C. After cooling to room temperature, the vial was cautiously opened and the excess ammonia was allowed to evaporate for 4 h. The solution was transferred into a conical plastic test tube (50 mL) and lyophilized. The dry material was dissolved in H2O (8 mL) and filtered, and the components were separated by HPLC to give O6-protected oligomer 8. The HPLC separation was carried out on a PRP-1 column (7 × 305 mm, Hamilton) at 40 °C using a gradient of (A) 0.1 M TEAA, pH 7.0, and (B) CH3CN, from 5% to 40% B over 30 min, with a flow rate of 3.0 mL/ min. The fractions containing oligomer 8 were collected, lyophilized, treated with 0.1% acetic acid (5 mL) for 2 h3 and relyophilized. Loss of the O6-TMSE group from the adducted oligonucleotide occurred slowly in neutral aqueous solution even in the absence of acetic acid. O6-Deprotected oligodeoxynucleotide 9 was separated under the same conditions except a gradient from 5% to 20% acetonitrile over 20 min was used. After collection of the peak of interest, the solvent was removed on a lyophilizer, and the samples were desalted on a Bio-Gel P-2 (Bio-Rad Laboratories) column (1.5 × 40 cm) by elution with H2O to give 80 ODs (18%) of oligomer 9. Final
Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 633
Figure 1. Scheme for synthesis of 2-fluoro-O6-TMSE-deoxyinosine (3) and derivatives. purification was accomplished with denaturing polyacrylamide gel electrophoresis. The purified oligomer was characterized by capillary gel electrophoresis, enzymatic digestion, and mass spectroscopy. Synthesis of Fully Deprotected Oligonucleotide 11. The beads from 1.5 1-µmol cassettes of 6, synthesized with phosphoramidites having 4-tert-butylphenoxyacetyl protecting groups (Perseptives/Biosystems), were stirred with 1.5 mL of 0.1 M NaOH for 20 h at rt. The supernatant was removed from the beads and neutralized with 0.1 M acetic acid to pH 7.0. After desalting on a Sephadex G-25 column (H2O elution), 88 ODs of crude 11 was obtained, which was purified by reverse-phase HPLC on an ODS-AQ column (YMC, Inc.), 10 × 250 mm, using a gradient of (A) 0.1 M ammonium formate, pH 6.39, (B) CH3CN, from 10% to 20% B over 30 min, with a flow rate of 3.0 mL/min, to give 28 ODs of purified 11. Synthesis of Oligonucleotides 8 and 9 from 11. Oligonucleotide 11 (8 ODs) and (R)-2-amino-2-phenylethanol (1.6 mg, 0.012 mmol) were dissolved in dry DMSO (0.05 mL) in a conical vial. The solution was heated at 75 °C. Aliquots were removed periodically and analyzed by HPLC. Analysis showed no 11 remained after 24 h; 8 and 9 were present in a ratio of 86:14. After removal of the DMSO in vacuo, the residue was dissolved in a small volume of H2O and dialyzed against H2O to remove excess amino alcohol. Some conversion of 8 to 9 occurred during dialysis, but complete conversion to 9 was accomplished by treatment with 0.1% acetic acid at rt for 2 h. The identity of the product was confirmed by coinjection with 9, prepared by the first procedure described above.
Results and Discussion In the search to improve the postoligomerization strategy for guanine adducts, we initially sought ways to increase the reactivity of the guanine synthon. Others also have attempted to find better leaving groups in order to prepare N2-adducted deoxyguanosine. Steinbrecher et al. have examined the use of triflate as the leaving group for synthesis of deoxyguanosine nucleosides; it appears to be more reactive with unhindered amines than the fluoro compound and has been used successfully for the
Figure 2. Kinetics of adduction of (S)-2-amino-2-phenylethanol to matrix-bound 5′-d(GGCAG-X-TGGTG)-3′, where X ) 2-fluoro-O6-TMSE-deoxyinosine (75 °C). Samples were removed from the beads and deprotected with conc. NH4OH (8 h, 60 °C) before HPLC analysis (see Materials and Methods for details).
preparation of N2 adducts of aryl amines (22,23); however, a low yield was obtained in the reaction with the hindered benzo[a]pyrene triol amine (22). We decided to investigate the use of O6-protected 2-fluoro-2′-deoxyinosine in our postoligomerization strategy. Protection at O6 enhances the susceptibility of the 2 position to nucleophilic attack and improves the solubility characteristics of the synthetic intermediates. A number of O6protecting groups have been examined previously (1214); of these, the p-nitrophenethyl has been most widely used in oligonucleotide synthesis (3,14,24,25). Schmid and Behr (26) have recently carried out a postoligomerization synthesis of a spermine-modified oligonucleotide by treatment of matrix-bound oligonucleotide containing 2-fluoro-O6-(p-nitrophenethyl)-2′-deoxyinosine with spermine in methanol at 50 °C; this treatment simultaneously cleaved the oligonucleotide from the beads, removed the protecting groups, and introduced the spermine residues. However, we found the p-nitrophenethyl group to be
634 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Figure 3. HPLC trace of products from reaction of matrixbound oligonucleotide 6 with (S)-2-amino-2-phenylethanol (DMSO, 3 days, 75 °C) after deprotection with conc. NH4OH (8 h, 60 °C). A′ and A contain cytosine side product with and without O6-TMSE on dG at the primary adduction site.
somewhat sensitive to base-catalyzed elimination during the extended treatment required for displacement of fluorine with hindered amines. Ultimately, we chose the trimethylsilylethyl group; this protective group is stable to base but readily cleaved by fluoride ion. Gaffney and Jones had previously explored TMSE for protection of O6 in deoxyguanosine during solid-phase synthesis of DNA but found the group to have poor stability, undergoing facile acid-catalyzed cleavage (13). Cleavage occurs by protonation of the ring which facilitates fragmentation of the TMSE group. We anticipated the group would be more satisfactory as a protective group for 2-fluoro-2′-deoxyinosine because the fluoro-substituted purine would be more stable to acid due to its decreased basicity.
DeCorte et al.
O6-TMSE-2′-deoxyguanosine (2) was prepared by Mitsunobu condensation of N2,3′,5′-triacetyldeoxyguanosine (1) with trimethylsilylethanol (Figure 1) (14,15). After removal of the acetyl groups by alkaline hydrolysis, the 2-amino group was replaced with fluoro using tert-butyl nitrite in HF/pyridine (17). Cleavage of the TMSE group was avoided by the use of low temperature; the reaction gave fluoro nucleoside 3 in 95% yield. When the stability of nucleoside 3 in the standard DNA synthesizer detritylation solution (3% trichloroacetic acid in methylene chloride) was examined, slow depurination, without loss of the TMSE group, was observed over a period of hours. Inasmuch as the deblocking cycle on the synthesizer is of the order of minutes (2.5-3.0 min for a 10 or 15 µmol synthesis), we decided to continue toward oligonucleotide synthesis with the expectation that depurination would not be a serious problem, at least with reasonably short sequences. Cyanoethyl phosphoramidite 5 was prepared by alkylation of the 5′ position of 3 with 4,4′-dimethoxytrityl chloride (18) followed by phosphitylation with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (19,20). The phosphoramidite was then used to prepare oligonucleotides by solid-phase synthesis. An 11-mer, d(GGCAGXTGGTG) where X ) 3, was prepared and, while still fully protected and immobilized on the controlpore glass beads, was treated with 50 equiv of (S)-2amino-2-phenylethanol in DMSO at 65 °C. Aliquots were removed daily, treated with conc. NH4OH to remove the oligonucleotide from the beads and all protecting groups except the TMSE group, and assayed by HPLC. The results are shown in Figure 2, and a representative HPLC trace is shown in Figure 3. The starting material was two-thirds gone after day 1 and had fully disappeared by day 3. The desired guanine adduct (8) represented ∼50% of the mixture after day 1 and peaked at
Figure 4. Scheme for solution synthesis of 5′-d(GGCAG-GN2-SO-GGGTG)-3′ (9). SO ) (R) adduct of styrene oxide.
Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 635
Figure 6. Capillary gel electrophoresis of 9.
Figure 5. (A) HPLC trace of mixture of partially deprotected oligonucleotide (7) after removal from the solid support with 0.1 M NaOH (room temperature, 8 h). (B) HPLC trace of 7 after treatment with (R)-2-amino-2-phenylethanol (DMSO, 2 days, 75 °C) and conc. NH4OH (8 h, 60 °C).
57% after day 2 dropping to 15% by day 4. The drop was due in part to loss of the TMSE group from the adducted oligonucleotide to give fully deprotected 9, a process which is in no way deleterious. It is noteworthy that loss of TMSE from unadducted oligonucleotide was not detected. The sum of the protected and deprotected species was ∼70% of the mixture at day 3. The major side product contained both cytosine and guanine adducts and also underwent deprotection with time.2 The experiment was repeated at 75 and 85 °C where both processes occurred faster and in dimethylacetamide where the reactions were slightly slower. In no case were conditions found which provided any better differentiation of the rates of the desired and the undesired adduction reactions. The sequence under investigation contained only one cytosine; cytosine adduction would be an even more serious problem in oligonucleotides containing several residues. The conclusion to be drawn from the above experiments is that O6 protection does not enhance the rate of 2-fluoro-2′-deoxyinosine adduction sufficiently to avoid undesirably large amounts of cytosine adduction. As an alternative approach for circumventing the cytosine problem, the possibility was examined that the protective groups could be removed from cytosine without concurrent destruction of 3. Nucleoside 3 was found to be quite stable in the presence of aqueous 0.1 M NaOH at 25 °C; even after 12 h, decomposition was minimal. However, these conditions were sufficient to deprotect N4-benzoylcytosine. Significantly, hydroxide attack occurred exclusively on the benzoyl group; no evidence could be found for formation of deoxyuridine. The 11mer oligonucleotide containing 3 was freshly synthesized, and the beads were treated with 0.1 M NaOH for 8 h at 25 °C, followed by neutralization to pH 7 (Figure 4).
HPLC analysis of the supernatant showed the product to be a complex mixture of oligodeoxynucleotides (7) due to partial removal of protective groups from the exocyclic amino groups of the bases. The mixture (panel A in Figure 5) was too complex to warrant separation of the individual components. Without any attempts at purification, the oligonucleotide mixture was treated with (R)2-amino-2-phenylethanol in dry DMSO (2 days, 75 °C), followed by concentrated NH4OH to complete removal of protective groups to give 8; panel B of Figure 5 shows the HPLC trace of unpurified 8 in which there is no evidence of contamination by the side product oligomers seen in the matrix-bound synthesis (Figure 3). After HPLC purification, 8 was treated with 0.1% acetic acid (2 h, rt) to remove residual TMSE groups3 to give adducted 11-mer 9 in good yield and high purity. Adducted oligonucleotide 9 was further purified by PAGE before evaluation by capillary gel electrophoresis (Figure 6) which showed it to be homogeneous and by enzyme digestion with snake venom phosphodiesterase and alkaline phosphatase (Figure 7) to give the appropriate nucleosides: dC (1.0); dG (5.65); T (1.93), dA (0.81, sum of dA + dI); dGN2-SO (1.08); theory: dC (1.0); dG (6), T(2), dA (1.0), dGN2-SO (1.0). Adducted cytidine was not detected. Mass spectroscopic analysis (MALDIFT, 3-hydroxypicolinic acid matrix, Mr ) 3574.4, measured mass ) 3573.8) confirmed the constitution of the oligonucleotide.
2 Enzymatic digestion of side products showed loss of cytosine and appearance of a new peak coincident with authentic dCN4-SO.
3 The trimethylsilylethyl protecting group can also be removed with 1.0 M tetrabutylammonium fluoride in THF.
Figure 7. Enzymatic digest of 9. SO ) (R) adduct of styrene oxide. Deoxyinosine arises from contamination of the snake venom phosphodiesterase with adenosine deaminase. See Material and Methods for HPLC conditions.
636 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
DeCorte et al.
Figure 8. Scheme for solution synthesis of 5′-d(GGCAG-GN2-SO-GGGTG)-3′ (9) via fully deprotected oligonucleotide 11. SO ) (R) adduct of styrene oxide.
The solution-phase reaction procedure provided an unexpected benefit over the previously described solidphase adduction reaction; the solution reaction of 2-amino2-phenylethanol was faster than that with immobilized oligonucleotides, a consideration which becomes particularly important with more hindered amines. NMR quantities (150-200 ODs from 10-µmol reactions) of several related styrene oxide-adducted oligonucleotides have been prepared by this route. These yields can be compared to the 250-300 ODs of nonadducted 11-mer that have been obtained from 10 µmol syntheses performed by commercial suppliers; the yields are at least 10-fold better than those obtained from our initial synthetic strategy. In a third strategy (Figure 8), we have found that the fully deprotected 11-mer containing 3 can be prepared if the oligonucleotide is synthesized using phosphoramidites having the exocyclic amino groups protected with the more labile PAC-type acyl groups (27-31). As others have found (30,31), it is important that the capping step on the synthesizer be carried out with a PAC-type anhydride rather than the normal acetic anhydride; otherwise, acetyl groups can be introduced on guanine N2; the N-acetyl groups are not as easily removed as the PAC and can remain after removal of PAC protection. Treatment of the matrix-bound PAC-protected oligonucleotide (10) with 0.1 M NaOH at rt for 8-24 h released the oligonucleotide from the matrix and deprotected both the phosphates and the exocyclic amino groups. The 2-fluoro substituent remained intact as well as the O6-TMSE group. Fully deprotected oligonucleotide 11 was isolated and purified before further reaction; during the process slight loss of the O6-TMSE group was observed. Mass spectroscopic analysis of 11 confirmed its constitution (MS (electrospray) calculated Mr ) 3556.5; observed ions 1777.3 (M - 2H)/2z, 1184.4 (M - 3H)/3z,
and 888.2 (M - 4H)/4z representing a measured mass ) 3556.5). When purified 11 was reacted with (R)-2-amino2-phenylethanol (DMSO, 75 °C) for 24 h, only O6-TMSE adducted oligonucleotide 8 and fully deprotected product 9 were observed; no 11 was detected. This is in contrast to the matrix-bound reaction where 30% starting material remained after 24 h at 75 °C. In the case of the N2-styrene oxide adducts, this procedure has no significant advantage since the adducts are stable to the hot NH4OH treatment needed to remove the traditional acyl protecting groups. However, the procedure could be very useful for preparing labile guanine N2 adducts and also for preparing oligonucleotides containing 2-fluorodeoxyinosine residues (for 19F NMR use, for example). This methodology could also be used for preparation of [2-N15] dG-containing oligonucleotides; the expensive isotopic label could be introduced at the final step of the synthesis by treatment of the fully deprotected oligonucleotide with 15NH4OH. Furthermore, reactions carried out in solution using fully deprotected, purified oligonucleotides are far easier to monitor than those utilizing matrix-bound or the soluble partiallyprotected oligonucleotides. In work reported elsewhere (32), we have used this methodology for preparation of oligonucleotides containing N2-dG-N2-dG cross-links. In summary, we feel that these new strategies of carrying out oligonucleotide adduction reactions in solution, on either partially or fully deprotected oligonucleotides, should find many useful applications.
Acknowledgment. We thank Dr. Robert Hettich (Oak Ridge National Laboratory) for obtaining MALDIFT mass spectra on oligonucleotide 9 and Dr. Ajai Chaudhary and Brian Nobes (Center for Mass Spectrometry, Department of Pharmacology, Vanderbilt University, USPHS RR-05805) for the electrospray mass spec-
Postoligomerization Synthesis of Oligodeoxynucleotides
trum of oligonucleotide 11 and FAB mass spectra. Generous support of this project by USPHS Grants ES00267, ES05355, and ES05509 is gratefully acknowledged.
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Chem. Res. Toxicol., Vol. 9, No. 3, 1996 637 (16) Gaffney, B. L., Marky, L. A., and Jones, R. A. (1984) Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine. Biochemistry 23, 56865691. (17) Robins, M. J., and Uznanski, B. (1981) Nucleic acid related compounds. 34. Non-aqueous diazotization with tert-butyl nitrite. Introduction of fluorine, chlorine and bromine at C-2 of purine nucleosides. Can. J. Chem. 59, 2608-2611. (18) Jones, R. (1984) In Oligonucleotide Synthesis, A Practical Approach; Gait, M. J., Ed.; IRL Press, Washington, DC, pp 22-34. (19) Barone, A. D., Tang, J.-Y., and Caruthers, M. H. (1984) In situ activation of bis-dialkylaminophosphinessA new method for synthesizing deoxyoligonucleotides on polymer supports. Nucleic Acids Res. 12, 4051-4061. (20) Bannwarth, W., and Trzeciak, A. (1987) A simple and effective chemical phosphorylation procedure for biomolecules. Helv. Chim. Acta 70, 175-186. (21) Borowy-Borowski, H., Lipman, R., Chowdary, D., and Tomasz, M. (1990) Duplex oligodeoxyribonucleotides crosslinked by mitomycin C at a single site: synthesis, properties, and crosslink reversibility. Biochemistry 29, 2992-2999. (22) Steinbrecher, T., Wameling, C., Oesch, F., and Seidel, A. (1993) Activation of the C-2 position of purine by the trifluoromethanesulfonate group: Synthesis of N2-alkylated deoxyguanosines. Angew. Chem., Int. Ed. Engl. 32, 404-406. (23) Scheer, S., Steinbrecher, T., and Boche, G. (1994) A selective synthesis of 4-aminobiphenyl-N2-deoxyguanosine adducts. Environ. Health Perspect. (Suppl. 6) 102, 151-152. (24) Eritja, R., Acedo, M., Avino, A., and Fabrega, C. (1995) Preparation of oligonucleotides containing non-natural base analogues. Nucleosides Nucleotides 14, 821-824. (25) Avino, A., Garcia, R. G., Marquez, V. E., and Eritja, R. (1995) Preparation and properties of oligodeoxynucleotides containing 4-O-butylthymine, 2-fluorohypoxanthine and 5-azacytosine. Bioorg. Med. Chem. Lett. 5, 2331-2336. (26) Schmid, N., and Behr, J.-P. (1995) Recognition of DNA sequences by strand replacement with poly-amino-oligonucleotides. Tetrahedron Lett. 36, 1447-1450. (27) Ko¨ster, H., Kulikowski, K., Liese, T., Heikens, W., and Kohli, V. (1981) N-Acyl protecting groups for deoxynucleosides. Tetrahedron 37, 363-369. (28) Schulhof, J. C., Molko, D., and Teoule, R. (1987) The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups. Nucleic Acids Res. 15, 397-416. (29) Wu, T., and Ogilvie, K. K. (1988) N-Phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase synthesis of oligoribonucleotides: Synthesis of a ribozyme sequence. Tetrahedron Lett. 29, 4249-4252. (30) Chaix, C., Molko, D., and Teoule, R. (1989) The use of labile base protecting groups in oligoribonucleotide synthesis. Tetrahedron Lett. 30, 71-74. (31) Sinha, N. D., Davis, P., Usman, N., Perez, J., Hodge, R., Kremsky, J., and Casale, R. (1993) Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facilitating N-deacylation, minimizing depurination and chain degradation. Biochimie 75, 13-23. (32) Tsarouhtsis, D., Kuchimanchi, S., DeCorte, B. L., Harris, C. M., and Harris, T. M. (1995) Synthesis of oligonucleotides containing interchain cross-links of bifunctional pyrroles. J. Am. Chem. Soc. 117, 11013-11014.
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