Bioconjugate Chem. 1996, 7, 564−567
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Direct, Solid Phase Assembly of Dihydropyrroloindole Peptides with Conjugated Oligonucleotides Eugeny A. Lukhtanov, Igor V. Kutyavin,* and Rich B. Meyer Epoch Pharmaceuticals, Inc., 1725 220th Street S.E., Bothell, Washington 98021. Received March 19, 1996X
A new controlled pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotide derivatives carrying a minor groove binding (MGB) agent at the 3′-terminus. The MGB consisted of three repeating 1,2-dihydro-3H-pyrrolo[2,3-e]indole-7-carboxylate (DPI) subunits. The DPI trimer (DPI3) was prepared directly on the CPG support using repeated addition of the DPI subunit. The subunit was protected at the N-3-position with tert-butyloxycarbonyl residue and activated at the 7-carboxy residue by esterification with the 2,3,5,6-tetrafluorophenyl group. A linker, which provided the starting point for oligonucleotide synthesis, was introduced by reaction of the terminal N-3 with p-nitrophenyl 4-[bis(4-methoxyphenyl)phenylmethoxy]butyrate. When used as a support for oligonucleotide synthesis, this modified CPG gave the desired 3′-DPI3-octathymidylate [(dTp)8-DPI3] conjugate in good yield. This conjugate formed hyperstabilized complexes with complementary polyribo- (Tmax ) 35 °C) and polydeoxyriboadenylic (Tmax ) 69 °C) acids. In contrast to the N-carbamoyl derivative reported earlier by us, it demonstrated higher cooperativity of melting transitions.
INTRODUCTION
Oligonucleotides conjugated to specific functional groups are important research tools in the molecular biology and chemistry of nucleic acids. These functional groups can be markers for diagnostic applications or reactive residues for therapeutic purposes. Some of these functional groups are helix-stabilizing agents. For example, covalent attachment of intercalators to the ends of oligonucleotides is a powerful way to enhance oligonucleotide hybridization properties (1-4). Recently, we have developed an alternative hybrid stabilization method based on covalent attachment of a minor groove binding agent (MGB)1 (5, 6). 1-Methylpyrrole-2-carboxamide (MPC) (5) and N-3-carbamoyl 1,2-dihydro-3H-pyrrolo[3,2-e]indole7-carboxylate (CDPI, Figure 1) (6) oligomers were prepared and their hybridization properties examined. Conjugation of the CDPI3 group to either terminus of octathymidylate stabilized its duplex with polydeoxyriboadenylic acid by as much as 44 °C. A similar but lesser effect was observed for d(pT)8 + poly(rA) duplexes. The MPC-oligothymidylate conjugates possessed similar properties in a duplex with poly(dA) but failed to stabilize the (dTp)8 + poly(rA) hybrid, as well as G,C-rich DNADNA duplexes (5). The ODN-CDPI3 conjugates that we reported previously (6) were prepared according to two methods. In the first, the ODN was modified postsynthetically with * Author to whom correspondence should be addressed [telephone (206) 485-8566; fax (206) 486-8336; e-mail kutyavin@ epochpharm.com]. X Abstract published in Advance ACS Abstracts, August 1, 1996. 1 Abbreviations: ODN, oligodeoxyribonucleotide; MPC, 1-methylpyrrole-2-carboxamide; DPI, 1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; CDPI, N-3 carbamoyl 1,2-dihydro-3Hpyrrolo[3,2-e]indole-7-carboxylate; TFP, 2,3,5,6-tetrafluorophenyl; CPG, controlled pore glass; Tmax, melting temperature for an ODN complex, determined at the maximum of derivative of the melting curve; MGB, minor groove binder; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; MMTr and DMTr, 4-monomethoxytrityl and 4,4′-dimethoxytrityl, respectively; tBOC, tert-butyloxycarbonyl; HOBt, 1-hydroxybenzotriazole; placement of the MGB to the right of the ODN denotes 3′-conjugation.
S1043-1802(96)00041-9 CCC: $12.00
Figure 1. Structure of methyl N-3 carbamoyl 1,2-dihydro3H-pyrrolo[3,2-e]indole-7-carboxylate trimer (7) and its 3′octathymidylate conjugate prepared by approach described in ref 6.
an amine linker at one terminus. Alternatively, a controlled pore glass (CPG) solid support bearing the CDPI3 residue was employed, with the desired conjugate obtained directly after oligonucleotide synthesis. Both approaches require utilization of the 2,3,5,6-tetrafluorophenyl ester of CDPI3 (TFP-CDPI3) (6), an acylating derivative of the MGB. Assembly of this trimeric MGB requires several steps, and preparation of the activated acylating reagent is complicated by its low solubility. Herein, we describe a new, more versatile solid phase approach that avoids the preparation of the complex CDPI3 analog. The approach is based on a combination of solid phase peptide synthesis for assembling the DPI3 segment followed by phosphoramidite chemistry for incorporation of the oligonucleotide sequence. A solid phase method for the preparation of polyamide-oligonucleotide conjugates, previously developed by Haralambidis et al. (8), bears similarities. EXPERIMENTAL PROCEDURES
Reagents. All air- and water-sensitive reactions were carried out under a slight positive pressure of argon. Anhydrous solvents were obtained from Aldrich (Milwaukee, WI). Standard reagents for the β-cyanoethylphosphoramidite coupling chemistry were purchased from Glen Research (Sterling, VA). Enzymes were © 1996 American Chemical Society
Oligonucleotide−Minor Groove Binder Conjugates
obtained from Sigma (St. Louis, MO). Flash chromatography was performed on 230-400 mesh silica gel. UVvisible absorption spectra were recorded in the 200-400 nm range on a Lambda 2 (Perkin-Elmer) spectrophotometer. 1H NMR spectra were run at 20 °C on a Varian Gemini 300 spectrometer; chemical shifts are reported in parts per million downfield from (CH3)4Si. 2,3,5,6-Tetrafluorophenyl 3-[[[(4-Methoxyphenyl)diphenyl]methyl]amino]propyl Butanedioate (1). To a solution of 3-aminopropanol (5.0 g, 67 mmol) in 100 mL of dry CH2Cl2 was added 4-monomethoxytrityl chloride (10.0 g, 33 mmol). After 30 min, the mixture was diluted with 900 mL of CH2Cl2 and washed twice with 1 L of water. The organic phase was dried over Na2SO4 and then concentrated to an oil. The residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to give 8.2 g (70%) of the desired N-(4-monomethoxytrityl)3-aminopropan-1-ol as a pale yellow syrup: 1H NMR (CDCl3, 300 MHz, ppm) 7.43-7.19 (m, 12H, MMTr), 6.82 (d, 2H, J ) 9 Hz, MMTr), 3.88 (t, 2H, J ) 5 Hz, CH2O), 3.78 (s, 3H, OCH3), 2.39 (t, 2H, J ) 6 Hz, CH2N), 1.70 (m, 2H, CH2). To a solution of the above MMTr derivative (4.0 g, 11.8 mmol) in 20 mL of anhydrous CH2Cl2 was added 1.18 g (11.8 mmol) of succinic anhydride, 2 mL (14 mmol) of triethylamine, and 50 µL of 1-methylimidazole. The mixture was stirred for 3 days. 2,3,5,6-Tetrafluorophenyl trifluoroacetate (10) (3.0 g, 11.5 mmol) was added dropwise. After 30 min, the mixture was concentrated and redissolved in 30 mL of hexane/ethyl acetate (3:1). The product was purified by flash chromatography using the same solvent as eluent. The fractions containing pure product were combined and evaporated in vacuo to give 5.7 g (81%) of the title compound as colorless syrup: 1H NMR (CDCl3, 300 MHz, ppm) 7.50-7.15 (m, 12H, MMTr), 6.82 (m, 2H, MMTr), 4.28 (t, 2H, J ) 7 Hz, CH2O), 3.78 (s, 3H, OCH3), 2.96 (t, 2H, J ) 7 Hz, COCH2), 2.72 (t, 2H, J ) 7 Hz, COCH2), 2.21 (t, 2H, J ) 7 Hz, CH2N), 1.82 (m, 2H, CH2). Preparation of DPI3-CPG (6). Derivatization of the CPG. To long-chain aminoalkyl controlled pore glass (Sigma, 500 Å, 4.0 g) were added 1 (1.5 g, 2.5 mmol) and 1.5 mL of triethylamine in 15 mL of anhydrous DMF. After being shaken for 20 h, the CPG was washed with DMF (100 mL), acetone (200 mL), and ether (50 mL) and dried under vacuum. The degree of functionalization was quantitated by spectrophotometric assay of the monomethoxytrityl cation (λ ) 472, ) 60 mM-1cm-1) released upon acid treatment (60% HClO4/methanol, 1:1) of a small amount of CPG. MMTr loading was found to be 41 µmol/g. The residual amino groups were capped with a mixture of pyridine (20 mL) and acetic anhydride (2 mL) for 30 min. The CPG (2) was then washed with acetone (200 mL) and ether (100 mL) and dried under vacuum overnight. DPI3-CPG Synthesis. The CPG (2) (2.0 g) was treated with trifluoroacetic acid (2 × 10 mL), washed with CH2Cl2, neutralized with 20% triethylamine in CH2Cl2, washed with CH2Cl2 again, and dried in vacuo. It was then reacted with a solution of 2,3,5,6-tetrafluorophenyl 3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate (3) (6) (78 mg, 0.33 mmol), triethylamine (0.6 mL), and 1-hydroxybenzotriazole (44 mg, 0.33 mmol) in 6 mL of anhydrous DMSO for 24 h. After washings with 100 mL portions of DMSO, acetone, and ether, the CPG was capped as described above and dried. The deprotection, coupling, and capping steps were repeated two more times, giving rise to the CPG-coupled 3-(tert-butyloxycarbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimer (4).
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Addition of Linker. After deprotection of the terminal amino group with trifluoroacetic acid, the CPG was treated with p-nitrophenyl 4-[bis(4-methoxyphenyl)phenylmethoxy]butyrate (5) (8) (0.9 g, 1.7 mmol), triethylamine (0.6 mL), and 1-hydroxybenzotriazole in 6 mL of anhydrous DMSO for 6 h. After washing and drying, the CPG was analyzed for DMTr content and found to have a loading of 29 µmol/g. Residual amino groups were acetylated as above and the CPG (6) was used for DNA synthesis. Oligonucleotide Synthesis and Molar Extinction Coefficients. Trityl-off 3′-DPI3 octathymidylate [(dTp)8CDPI3] was prepared in 1 µmol scale using the modified CPG support (6) (∼50 mg) on an ABI 394 according to the protocol supplied by the manufacturer. For comparison, an octathymidylate bearing a 3′-CDPI3 [(dTp)8CDPI3] was prepared according to our previously described method (6). The concentration of both conjugates was determined in water using the extinction coefficient of 110.1 mM-1cm-1 (6). Thermal Denaturation Studies. Hybrids formed between MGB-tailed (dTp)8 conjugates and complementary polyadenylates were melted in 140 mM KCl, 10 mM MgCl2, and 20 mM HEPES-HCl (pH 7.2) using a Lambda 2 (Perkin-Elmer) spectrophotometer equipped with a PTP-6 automatic multicell temperature programmer. Each conjugate (2 µM) was mixed with sufficient polymer to give a T:A ratio of 1:1. To determine the concentrations of poly(dA) and poly(rA), 260 values of 8.4 and 10.3 mM-1 cm-1 were used (9). Thermal dissociation curves were obtained by heating samples from 0 to 100 °C with a temperature increase of 0.5 °C/min. Before melting, the samples were preheated at 100 °C and cooled to the starting temperature over a 10 min period. The melting temperatures (Tmax values) of complexes were determined from the derivative maxima. Nuclease Digest of (dTp)8-DPI3 Conjugate. To a solution of (dTp)8-DPI3 (∼15 µg) in 50 µL of a buffer containing 50 mM Tris-HCl (pH 8.5) and 10 mM MgCl2 were added RQ1 DNase (5 µL, 1000 U/mL), phosphodiesterase I (5 µL, 100 U/mL), and calf intestine alkaline phosphatase (1 µL, 650 U/mL). The solution was incubated at 37 °C for 30 h. Ten microliter aliquots were taken during this period to monitor the progress of the reaction by HPLC analysis. RESULTS AND DISCUSSION
We have previously prepared a CPG with an attached CDPI3 residue for synthesis of ODNs with this residue on the 3′-terminus and have shown that the residue is stable to the treatments used in automated phosphoramidite chemistry (6). In that approach, the CDPI3 residue was bound to the solid support through a specially designed aminodiol linker which provided attachment points for (a) the CDPI3 residue, (b) the solid support, and (c) the initial 3′-nucleic acid residue. Although this method was efficient for the preparation of the conjugates, drawbacks included the necessity for CDPI3 reagent (TFP-CDPI3) and the complexity of the aminodiol linker. We describe here an alternative approach that combines solid support synthesis of both the trimeric minor groove binding moiety and its attached ODN. The DPI trimer, which is chemically stable to ODN synthesis conditions, is assembled first on the CPG, followed by ODN extension. This approach also provides for conjugation of the ODN through the N-3 amino function of the DPI moiety, as opposed to the C-7 carboxy group in our previously published approach (6). The preparation of the new MGB-bound CPG support is shown on Figure 2. Long-chain alkylamine CPG was
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Figure 2. Synthetic scheme and structure of the (dTp)8-DPI3 conjugate.
modified with a specially designed reagent 1 to provide an ammonia-cleavable linker bearing an MMTr-protected amino group. The amino group was deprotected with trifluoroacetic acid and reacted with the activated DPI block (3), giving the solid support-bound DPI fragment with N-3-tBOC group as a new terminus. Capping with a mixture of pyridine and acetic anhydride, to eliminate possible DPIn-1 contamination, concluded each coupling cycle. The deprotection, coupling, and capping were repeated two more times to give a CPG (4) with the bound tBOC-DPI3 residue. A DMTr-protected primary hydroxy group, the starting point for oligonucleotide synthesis, was introduced by removal of the last tBOC group, treatment with 5, and capping with acetic anhydride in pyridine. The DMTr loading for this CPG (6) was found to be 29 µmol/g, reflecting an average 92% coupling yield during DPI3 assembly. The modified CPG (6) was then used for automated octathymidylate synthesis, using the standard cyanoethyl N,N′-diisopropylphosphoramidite chemistry. The trityl group was removed after the last coupling to simplify HPLC purification. The 3′-DPI3-modified octathymidylate [(dTp)8-DPI3] was deblocked and cleaved from the CPG by aqueous ammonia treatment and isolated by reversed-phase HPLC. A chromatographic profile of the reaction mixture is shown in Figure 3A. The reaction mixture contained only one major peak, the product. As had been observed earlier (6) for CDPI3 conjugates, the addition of the hydrophobic MGB residue to the octathymidylate significantly increased the retention time
Figure 3. HPLC profiles of crude synthetic (A) and HPLC purified (B) (dTp)8-DPI3 conjugate. (C) HPLC analysis of nuclease digest of the conjugate with a mixture of RQ1 DNase, phosphodiesterase I, and alkaline phosphatase at 37 °C for 30 h in a buffer containing 50 mM Tris-HCl (pH 8.5) and 10 mM MgCl2. Chromatography was performed on a Rainin C18 column (4.5 × 150 mm) in a gradient of acetonitrile (0-60%) in 0.1 M triethylammonium acetate buffer (pH 7.5). Flow rate was 1 mL/ min. Dual wavelength detection was used: fine line, 260 nm; bold line, 340 nm.
relative to that of the unmodified oligonucleotide. To further confirm the conjugation site, the purified conjugate (Figure 3B) was subjected to nuclease digestion using a mixture of RQ1 DNase, phosphodiesterase I and alkaline phosphatase. This gave free thymidine and dTp-DPI3 as the final products. The hydrolysis of the last few phosphodiester bonds progressed slowly during this digestion (Figure 3C), explaining the presence of the faster eluting products. These were identified as products of incomplete digestion on the basis of their characteristic UV spectra; the latest eluting peak had absorbance at 350 and 260 nm corresponding to (DPI)3 and one dT residue, respectively, while the next three small, faster eluting peaks (Figure 3C) had progressively more absorbance at 260 nm (data not shown). A second addition of digestion enzymes did not reduce these peaks. The physical properties of the isolated (dTp)8-DPI3 conjugate were similar to those reported for the CDPI3 analog (6). Besides the almost identical chromatographic mobilities, both types of conjugates had similar UVvisible spectra (Figure 4). A slight long wavelength shift
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Oligonucleotide−Minor Groove Binder Conjugates
°C, see Figure 5), which gave an effect of MGB-assisted stabilization of 22 °C. These data show that the polarity of the conjugation (N-3 vs C-7) of the MGB in the minor groove does not dramatically affect the stability of the hybrid. The N-3 carbamoyl residue seems to be nonessential for the stabilization, as we found in the previous investigation (6). This new solid phase approach for the preparation of oligonucleotides with conjugated DPI oligomers is equally applicable for the preparation of longer oligonucleotides and derivatives with MGBs of various length and structure. A major advantage over the solution phase methodology is elimination of the synthesis and purification of DPI oligomers (3-mer, 4-mer), which are complicated by the low solubility of the oligomers. Following the MGB assembly, an oligonucleotide sequence can be efficiently incorporated, allowing the direct preparation of the desired conjugate. Use of conventional 3′-phosphoramidite chemistry, which is described in this study, leads to 3′-type of conjugation. However, commercial availability of 5′-phosphoramidites (Glen Research) enables preparation of 5′-conjugates with the same solid support, making the method more versatile. Figure 4. UV-visible spectra of 10 µM (dTp)8-CDPI3 (1) and (dTp)8-DPI3 (2) in 10 mM Tris-HCl (pH 7.5).
ACKNOWLEDGMENT
This work was funded by Grant GM52774 from the National Institutes of Health, USPHS. We thank Dr. Vladimir Gorn for oligonucleotide synthesis. LITERATURE CITED
Figure 5. Differential melting curves for the complexes (dTp)8-DPI3 + poly(rA) (1), (dTp)8-DPI3 + poly(dA) (2), (dTp)8CDPI3 + poly(rA) (3), and (dTp)8-CDPI3 + poly(dA) (4) in 140 mM KCl, 10 mM MgCl2, and 20 mM HEPES-HCl (pH 7.2). The concentration of each ODN was 2 µM, and the A:T ratio was 1:1.
for the DPI3 residue was most likely due to the difference in structure of the linker and the end capping groups of the core MGB part (compare structures in Figures 1 and 2). As had been already observed in case of the (dTp)8CDPI3 conjugate, (dTp)8-DPI3 also formed hyperstabilized complexes with complementary polyribo- and polydeoxyriboadenylic acids (Figure 5). Both of these conjugates exhibited the same hybrid stability when bound to poly(dA) (Tmax ) 68-69 °C), although the (dTp)8-DPI3 derivative demonstrated a much higher cooperativity of melting transition than the CDPI3 analog. The melting temperature of (dTp)8-DPI3 bound to poly(rA) (Tmax ) 35 °C) was 4 °C higher than the corresponding value of the CDPI3 conjugate (Tmax ) 31
(1) Letsinger, R., and Schott, M. E. (1981) Selectivity in binding a phenantridinium-dinucleotide derivative to homopolynucleotides. J. Am. Chem. Soc. 103, 7394. (2) Asseline, V., Delarue, M., Laucelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T., and Helene, C. (1984) Nucleic acid binding with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides. Proc. Natl. Acad. Sci. U.S.A. 81, 3297. (3) Benimetskaya, L. Z., Bulychev, N. V., Kozionov, A. L., Koshkin, A. A., Lebedev, A. V., Novozhilov, S. Yu., and Stockman, M. I. (1989) Site-specific laser modification (cleavage) of oligonucleotides. Biopolymers 28, 1129. (4) Lokhov, S. G., Podyminogin, M. A., Sergeev, D. S., Silnikov, V. N., Kutyavin, I. V., Shishkin, G. V., and Zarytova, V. P. (1992) Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides. Bioconjugate Chem. 3, 414. (5) Sinyakov, A. N., Lokhov, S. G., Kutyavin, I. V., Gamper, H. B., and Meyer, R. B., Jr. (1995) Exceptional and selective stabilization of A-T rich DNA-DNA duplexes by N-methylpyrrole carboxamide peptides conjugated to oligonucleotides. J. Am. Chem. Soc. 117, 4995. (6) Lukhtanov, E. A., Kutyavin, I. V., Gamper, H. B., and Meyer, R. B., Jr. (1995) Oligodeoxyribonucleotides with conjugated dihydropyrroloindole oligopeptides: preparation and hybridization properties. Bioconjugate Chem. 6, 418. (7) Boger, D. L., Coleman, R. S., and Invergo, B. J. (1987) Studies on the total synthesis of CC-1065: preparation of a synthetic, simplified 3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2e]indole dimer/trimer/tetramer (CDPI dimer/trimer/tetramer) and development of methodology for PDE-I dimer methyl ester formation. J. Org. Chem. 52, 1521. (8) Haralambidis, J., Dunkan, L., Angus, K., and Tregear, G. W. (1990) The synthesis of polyamide-oligonucleotide conjugate molecules. Nucleic Acids Res. 18, 493. (9) Riley, M., Maling, B., and Chamberlin, M. J. (1966) Physical and chemical characterization of two- and three-stranded adenine-thymidine and adenine-uracil homopolymer complexes. J. Mol. Biol. 20, 359. (10) Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A. A., Scholler, J. K., and Meyer, R. B., Jr. (1993) Facile preparation of nuclease resistant 3′ modified oligodeoxynucleotides. Nucleic Acids Res. 21, 145.
BC960041D
