Bioconjugate Chem. 1996, 7, 600−605
600
Interactions of Cytosine Derivatives with T‚A Interruptions in Pyrimidine‚Purine‚Pyrimidine DNA Triplexes Sandeep Verma and Paul S. Miller* Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received May 10, 1996X
The ability of triplex-forming oligopyrimidines to interact with duplex targets which contain a single pyr‚pur interruption in their homopurine triplex binding site was studied. These oligonucleotides contain either N4-(3-carboxypropyl)deoxycytidine (3) or N4-(5-carboxytriazolyl)deoxycytidine (4) to target the pyr‚pur interruption. The 3-carboxypropyl and the 5-carboxytriazolyl groups of these cytosine derivatives are designed to span the major groove of the duplex target. Molecular models suggest that the carboxyl group of 3 or 4 can serve as a hydrogen bond acceptor for the N6-amino hydrogen of A in a T‚A base pair. Additional contacts are possible between the N4-amino hydrogen of 3 or 4 and the O4-carbonyl oxygen of T. UV melting experiments showed that a 15-mer, I(3), containing nucleoside 3 formed a stable triplex with duplex targets containing a single T‚A interruption. The melting temperature of this triplex was 7 °C higher than that of a similar triplex containing a single G‚T‚A triad when a Tris buffer was employed. Oligomer I(3) also formed a triplex of lower stability with a target containing a G‚C base pair but not with targets containing C‚G, U‚A, or A‚T base pairs. A similar 15-mer, II, containing nucleoside 4 was found to be less selective than I(3) in its interaction with duplex targets. Thus, gel mobility shift experiments showed that in addition to interacting with T‚A, oligomer II also formed triplexes with duplex targets containing U‚A and C‚G interruptions. These studies suggest that nucleoside derivatives which can potentially contact both bases of a pyr‚pur interruption might provide a means to extend the range of sequences which can be recognized by triplex-forming oligonucleotides.
INTRODUCTION
Pyrimidine-rich oligodeoxyribonucleotides can interact with a purine-rich sequence of a DNA duplex to form triple helical complexes through the formation of stable C+‚G‚C and T‚A‚T base triads (1-5). However, the ability of natural bases to form stable triads in conventional triplex motifs is greatly reduced by the occurrence of pyrimidine interruptions in an otherwise homopurine strand of a target duplex (4, 5). Several strategies employing natural and non-natural nucleosides have been applied to overcome the recognition barrier posed by these interruptions. In the case of the pyr‚pur‚pyr triplex motif, structural studies show that G can recognize a T‚A base pair (6-9) and T can recognize a C‚G base pair through the formation of a single hydrogen bond with the pyrimidine of the base pair (10). Another approach utilizes synthetic non-natural nucleoside analogs whose structures are designed to position hydrogen bond donor and/or acceptor groups to interact with such inversions. For example, interaction of 1-(2-deoxy-β-Dribofuranosyl)-4-(3-benzamidophenyl)imidazole with duplexes containing pyr‚pur interruption has been described (11). It appears that this analog interacts with T‚A and C‚G base pairs as a result of intercalation (12). We have previously reported triplex formation between oligodeoxyribonucleotides that contain the derivatized nucleosides N4-(3-acetamidopropyl)deoxycytidine (1) and N4-(6-amino-2-pyridinyl)deoxycytidine (2) shown in Figure 1 and target duplexes that contain a single C‚G base pair interruption (13, 14). Studies of molecular models suggested that the acetamidopropyl or aminopyridinyl group could extend across the major groove of the duplex. * Author to whom correspondence should be addressed [telephone (410) 955-3489; fax (410) 955-4392]. X Abstract published in Advance ACS Abstracts, August 15, 1996.
S1043-1802(96)00049-3 CCC: $12.00
As a result, the amide or amino hydrogen could provide a hydrogen bond contact with the guanine O-6 of the C‚G base pair. Although both analogs support triplex formation, the N4-(3-acetamidopropyl)deoxycytidine substitution displayed better binding specificity for the C‚G base pair than did N4-(6-amino-2-pyridinyl)deoxycytidine. These studies and examination of molecular models suggested that a similar strategy could be employed in which a hydrogen bond acceptor group could be used to contact the N6-amino group of a T‚A interruption. To test this hypothesis, we have synthesized oligopyrimidines containing N4-(3-carboxypropyl)deoxycytidine (3) or N4(5-carboxytriazolyl)deoxycytidine (4) and have studied their ability to interact with duplex targets. Molecular models suggest that the carboxylate group of 2 or 3 could be suitably positioned as an acceptor for the hydrogen of the N6-amino group of adenine in a T‚A base pair. EXPERIMENTAL PROCEDURES
Protected deoxyribonucleoside phosphoramidites and controlled pore glass supports were purchased from Glen Research. All chemicals used were of reagent grade or better. γ-[32P]ATP was purchased from Amersham Inc., and T4 polynucleotide kinase was purchased from United States Biochemical Corp. Polyacrylamide gel electrophoresis was carried out on 20 × 20 × 0.75 cm gels containing 20% acrylamide and 7 M urea (denaturing conditions) or 15% acrylamide (nondenaturing conditions). The running buffers were either TBE, which contained 89 mM Tris, 89 mM boric acid, and 0.2 mM ethylenediaminetetraacetate buffered at pH 8, or MES, which contained 90 mM 4-morpholineethanesulfonic acid monohydrate (MES monohydrate) and 10 mM magnesium chloride buffered at pH 6.2. The gel loading buffer contained 90% formamide, 0.05% xylene cyanol, and 0.05% bromophenol blue. Reversed phase HPLC was carried out on Microsorb C18 columns purchased from © 1996 American Chemical Society
Pyr‚Pur Triplex Recognition by Cytosine Derivatives
Bioconjugate Chem., Vol. 7, No. 5, 1996 601
Figure 1. Structures of derivatized deoxycytidines and oligonucleotides. C is 5-metyldeoxycytidine, X is nucleoside 1, 2, or 3, and Y‚Z is C‚G, G‚C, T‚A, U‚A, or A‚T.
Rainin Instruments using a linear gradient of acetonitrile in 50 mM sodium phosphate buffered at pH 5.8. Synthesis of N4-(5-Carbomethoxytriazolyl)-3′,5′O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)deoxycytidine (7a). 2′-Deoxyuridine (0.98 g, 4.27 mmol) was dried by coevaporating it with anhydrous pyridine (2 × 10 mL). To the dry nucleoside was added anhydrous pyridine (10 mL), and the solution was cooled on ice. A solution of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.4 g, 4.46 mmol) in anhydrous dichloromethane was added (10 mL) over a 15 min period with continuous stirring under a nitrogen atmosphere. The reaction was stirred at room temperature for 3 h, at which time analytical TLC showed complete disappearance of the starting material (chloroform/ethanol 9/1). The reaction was quenched with methanol (1 mL) and was evaporated using an oil pump to remove excess pyridine. The resulting glassy solid was dissolved in dichloromethane (25 mL) and washed with 1 M NaHCO3 solution (2 × 15 mL). The organic layer was separated and dried over anhydrous MgSO4 and filtered. The solvents were evaporated to give 3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)deoxycytidine as a white foam in 95% yield. The TIPS-protected 2′-deoxyuridine (8 g, 17 mmol) was dried by coevaporating twice with anhydrous pyridine and was dissolved in anhydrous dichloromethane (80 mL). Triethylamine (30 mL) was added followed by mesitylenesulfonyl chloride (10 g, 46 mmol) and 4-(dimethylamino)pyridine (0.52 g, 4 mmol). The reaction was stirred for 2 h under nitrogen at room temperature, at which time analytical TLC (ethyl acetate/hexane 8/2) indicated that the reaction was complete. Excess solvents were evaporated, and the resulting solid was coevaporated with anhydrous pyridine (3 × 25 mL) followed by drying under vacuum to give nucleoside 6. Nucleoside 6 (11.0 g, 17 mmol) was dissolved in anhydrous pyridine (80 mL). 3-Amino-1,2,4-triazole-5carboxylic acid methyl ester (23.4 g, 165 mmol) was added, and the solution was refluxed for 3 h, followed by overnight stirring at 90 °C. Excess pyridine was removed under vacuum, and the resulting brown gummy mass was extracted with dichloromethane (5 × 200 mL). Any remaining insolubles were discarded at this stage, and the organic phase was washed successively with 1 M NaHCO3 (5 × 200 mL) and water (5 × 100 mL). The combined aqueous washings were extracted with dichloromethane (200 mL). The combined organic extract was
dried over anhydrous Na2SO4. After evaporation of solvents, nucleoside 7a was purified by silica gel chromatography using chloroform/methanol (7:3) as eluting solvent (47% yield): 1H NMR (CDCl3) δ 7.83 (H-6, d, J ) 7.77 Hz), 6.11 (H-5, d, J ) 6.06 Hz), 5.72 (H-1′, d, J ) 7.71 Hz), 4.40 (H-3′, m), 4.06 (H-5′,5′′, m), 3.77 (H-4′, -COCH3, m), 2.54 (H-2′, m), 2.35 (H-2′′, m), 1.35 (TIPS, m). Synthesis of N4-(5-Carbomethoxytriazolyl)-5′-O(dimethoxytrityl)-3′-(2-cyanoethyl-N,N-diisopropylamino)deoxycytidine Phosphoramidite (8b). A solution of nucleoside 7a (2 g, 3.37 mmol) and tetrabutylammonium fluoride (8 mL) in anhydrous tetrahydrofuran (15 mL) was stirred at room temperature for 20 min under a nitrogen atmosphere. A solution of pyridine/water/methanol (3/1/1, 8 mL) was added, and the reaction mixture was poured into a stirred solution of Dowex 50WX4-200 (200 mL, pyridinium form) and gently swirled for 30 min. The resin was filtered and was washed with pyridine/water/methanol (3/1/1, 5 × 150 mL). The combined washings were evaporated under reduced pressure, and the resulting yellow residue was coevaporated with anhydrous methanol. N4-(5-Carbomethoxytriazolyl)deoxycytidine was precipitated from its methanolic solution (20 mL) by ether. The resulting yellow powder was dried under vacuum over P2O5. Nucleoside 7b (0.65 g, 1.85 mmol) was coevaporated with anhydrous pyridine (3 × 5 mL), and the residue was redissolved in anhydrous pyridine (15 mL). 4,4′-Dimethoxytrityl chloride (0.75 g, 2.21 mmol) and DMAP (0.025 g, 0.18 mmol) were added, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2 h. Additional 4,4′-dimethoxytrityl chloride (0.035 g, 0.1 mmol) was added, and stirring was continued overnight at room temperature. Methanol (10 mL) was added, and solvents were evaporated under reduced pressure. The resulting gummy mass was dissolved in dichloromethane (45 mL). This solution was washed with 1 M NaHCO3 (4 × 20 mL) followed by water (2 × 25 mL), and the combined aqueous extracts were extracted with dichloromethane (25 mL). The combined organic extract was dried over anhydrous Na2SO4. The solvents were evaporated, and the tritylated nucleoside 7a was precipitated from anhydrous tetrahydrofuran by addition of hexanes. This procedure was repeated three times and gave pure 8a in 83% yield: 1H NMR (CDCl3) δ 8.48 (H-6, d, J ) 7.38 Hz), 7.27 (DMTr, m), 6.85 (DMTr, d, J ) 8.76 Hz),
602 Bioconjugate Chem., Vol. 7, No. 5, 1996
6.78 (H-5, d, J ) 7.35 Hz), 6.25 (H-1′, t), 4.54 (H-3′, m), 4.17 (H-4′, m), 3.99 (-COCH3, s), 3.80 (-OCH3, s), 3.51 (H-5′,5′′, m), 2.71 (H-2′, m), 2.33 (H-2′′, m). Nucleoside 8a (1 g, 1.5 mmol) was dried and then dissolved in anhydrous dichloromethane (10 mL). N,NDiisopropylethylamine (0.5 mL, 3 mmol) was added under a constant stream of nitrogen, and the solution was cooled in an ice bath. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.5 mL, 1.8 mmol) was added, and the reaction mixture was stirred for 10 min at 0 °C and then at room temperature for 1 h. Additional N,Ndiisopropylethylamine (150 µL) and 2-cyanoethyl N,Ndiisopropylchlorophosphoramidite (100 µL) were added, and stirring was continued for another 1.5 h. The reaction was quenched by addition of methanol (1 mL), and the solvents were evaporated. A gummy mass was obtained which was extracted by ethyl acetate (20 mL). The organic layer was washed with 1 M NaHCO3 (3 × 25 mL) followed by water (3 × 20 mL), and the combined aqueous extracts were extracted with ethyl acetate (2 × 25 mL). The combined organic extracts were dried over anhydrous Na2SO4. The phosphoramidite 8b was purified by silica gel chromatography using a gradient of 0-3% ethanol in dichloromethane containing 0.1% triethylamine. The phosphoramidite was precipitated twice from its ethyl acetate solution by addition of pentanes at -40 °C (47% yield): 31P NMR (CDCl3, 85% phosphoric acid in D2O as external standard) δ 150.07 and 149.80. Syntheses of Oligodeoxyribonucleotides. Oligodeoxyribonucleotides I(C) and II-VI (see Figure 1) were synthesized on nucleoside-derivatized controlled pore glass supports using an Applied Biosystems Model 392 DNA/RNA synthesizer. The oligonucleotides were prepared on 1 µmol scales using commercially available protected 5′-O-dimethoxytrityldeoxyribonucleoside 3′O,N,N-bis(diisopropylamino)-β-cyanoethyl phosphoramidites following standard procedures. The oligomers were deprotected by overnight treatment of the supports with a solution of 50% ammonium hydroxide in pyridine at 55 °C and were purified by C18 reversed phase HPLC using a 0-20% linear gradient of acetonitrile in 50 mM sodium phosphate, pH 5.8. The extinction coefficients of the oligomers were determined after digestion of a known amount of oligomer by snake venom phosphodiesterase as previously described (9). Synthesis of Oligodeoxyribonucleotide I(3). Oligomer I(C) (10 A260 units) was treated with 96 µL of a solution containing 1 M sodium bisulfite and 3 M 4-aminobutyric acid, pH 7.0, at 50 °C for 48 h as previously described (15). Transamination was complete as indicated by reversed phase HPLC analysis. The oligomer was purified by C18 reversed phase HPLC using a 5-15% linear gradient of acetonitrile in 50 mM sodium phosphate, pH 5.8. The oligomer was desalted on a C18 reversed phase cartridge. It was characterized by hydrolysis with a combination of snake venom phosphodiesterase and alkaline phosphatase followed by analysis of the resulting nucleosides by C18 reversed phase HPLC. The expected ratio of nucleosides was obtained, and no deamination of C was observed as evidenced by the lack of deoxyuridine in the digest. Synthesis of Oligodeoxyribonucleotide II. Oligomer II was synthesized using 5′-O-dimethoxytritylprotected 3′-O-β-cyanoethyl N,N-diisopropylphosphoramidites of thymidine, N4-isobutyryldeoxycytosine, and N4(5-carbomethoxytriazolyl)deoxycytidine. A double coupling cycle (10 min/cycle) was used to introduce the latter nucleoside phosphoramidite. The oligomer was deprotected by first treating the support for 15 min with 1 mL of 2 M aqueous sodium carbonate solution. The support
Verma and Miller
was then treated with 1 mL of 5 M aqueous ammonium hydroxide solution at room temperature for 15 min. The solvents were evaporated, and the crude oligomer was partially purified by C18 reversed phase HPLC using a 2-15% linear gradient of acetonitrile in 50 mM sodium phosphate, pH 5.8. The oligomer was then desalted using a C18 Sep-Pak cartridge and phosphorylated using γ-[32P]ATP and T4-polynucleotide kinase. The phosphorylated oligomer migrated as a single band on a 20% polyacrylamide gel. The portion of the gel containing the oligomer was excised, crushed, and extracted by incubation overnight with 0.1 M aqueous sodium chloride solution at 37 °C. The phosphorylated oligomer was desalted on a preequilibrated C18 Sep-Pak cartridge. Melting Experiments. Melting experiments were carried out in one of the following buffers: 0.1 M sodium chloride, 20 mM magnesium chloride, and 50 mM 4-morpholinepropanesulfonic acid (MOPS), pH 6.2 or 7.0; or 0.1 M sodium chloride, 20 mM magnesium chloride, and 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris), pH 7.0. A solution containing 2.0 µM preformed duplex was mixed with an equal volume of 2.0 µM triplexforming oligomer at room temperature. The solutions were then incubated overnight at 4 °C. These solutions were loaded into cuvettes at 0 °C, and the absorbance versus temperature profile was recorded using a Cary 3E UV-vis spectrophotometer fitted with a thermostated cell block and temperature controller. The cell block was continuously purged with dry nitrogen to prevent moisture condensation at low temperatures. The solutions were heated at a rate of 0.5 °C/min, and the absorbance at 260 nm was measured. Gel Mobility Shift Analysis. Gel mobility shift experiments were performed on 15% polyacrylamide gels under nondenaturing conditions using 90 mM 4-morpholineethanesulfonic acid (MES), pH 6.2, and 10 mM magnesium chloride as the running buffer. The concentrations of labeled oligomer and the target duplexes were fixed at 1 and 10 µM, respectively, in a total reaction volume of 5 µL. A 1 µL aliquot of 50% aqueous glycerol solution was added just before the samples were loaded onto the pre-equilibrated gels. The gels were run at a constant voltage of 150 V and at constant temperature. The gels were dried, autoradiographed, and scanned on an Epson scanner using the Scantastic program. RESULTS
Triplex Formation by an Oligonucleotide Containing N4-(3-Carboxypropyl)-2′-deoxycytidine. The interaction of N4-(3-carboxypropyl)deoxycytidine (3) with pyr‚pur interruptions was studied using oligonucleotide I(3), the sequence of which is shown in Figure 1. This oligomer was prepared by bisulfite-catalyzed transamination of oligomer I(C) in the presence of 4-aminobutyric acid. As previously reported (15), this reaction selectively modifies cytosine but not 5-methylcytosine residues in oligonucleotides. The reaction was carried out at pH 7.2 at 50 °C. Under these conditions essentially no deamination takes place and the conversion of I(C) to I(3) is almost quantitative. The interaction of I(3) with duplex target III‚IV (Y‚Z) was studied by UV melting experiments. In this target system, the base pairs at position Y‚Z were varied to assess the sequence specificity of the triplex-forming oligomer. As shown in Figure 2 oligomer I(3) forms a stable triplex with III‚IV (T‚A) in Tris buffer at pH 7.0 as indicated by the presence of two transitions. The first transition corresponds to dissociation of I(3) from the triplex I‚III‚IV (3‚T‚A) and the second to dissociation of
Bioconjugate Chem., Vol. 7, No. 5, 1996 603
Pyr‚Pur Triplex Recognition by Cytosine Derivatives
Scheme 1. Synthesis of Protected Phosphoramidite of N4-(5-Carbomethoxytriazolyl)deoxycytidine
Figure 2. Melting curve of triplex I‚III‚IV (3‚T‚A). The melt was carried out in a buffer containing 0.1 M sodium chloride, 20 mM magnesium chloride, and 50 mM Tris at pH 7.0. The concentration of each oligomer was 1 µM. Table 1. Triplex Melting Temperatures Tm, °Ca X
Y
3 3 3 3 3
T U C A G
3
T
Z
Tris
Triplex I‚III‚IV (X‚Y‚Z) A 22 A
