Bioconjugate Chem. 2000, 11, 599−603
599
Preparation of an Imidazole-Conjugated Oligonucleotide Marc Beban and Paul S. Miller* Department of Biochemistry and Molecular Biology, School of Public Health Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received January 22, 2000; Revised Manuscript Received March 31, 2000
Postsynthetic modification of an oligonucleotide with an imidazole functional group was achieved by formation of an amide bond between the functional group and a single 2′-amino-2′-deoxyuridine, d-aU, of the oligonucleotide. The succinimidyl ester of N-glutaryl-histamine was synthesized under anhydrous conditions and added to the oligonucleotide in an acetonitrile-containing buffer at pH 8.0. Formation of the conjugate was assayed by digestion with snake venom phosphodiesterase and bacterial alkaline phosphatase, followed by reversed-phase HPLC to resolve constituent nucleosides. The disappearance of a peak corresponding to d-aU and the appearance of a peak that coelutes with authentic 2′-(Nglutaryl-N′-histaminyl)-2′-deoxyuridine confirmed the formation of the conjugate. Imidazole-conjugated oligonucleotides may have utility as antisense agents capable of hydrolyzing RNA.
INTRODUCTION
Oligonucleotides and their analogues can provide a scaffold of chemically reactive sites to which may be tethered a variety of functional groups. Conjugates between oligonucleotides and functional groups result in hybrid molecules that retain properties of each moiety (Goodchild, 1990). As a result, oligonucleotide-conjugates can be created that have altered biophysical properties (e.g., binding affinity, hydrophobicity, and fluorescence), chemical reactivity (e.g., radical generating or hydrolytic activity), or both (e.g., cross-linking). Some conjugates improve upon the intrinsic qualities of oligonucleotides, such as affinity for their complementary target (Asseline, et al., 1984) whereas others impart new properties such as chemical reactivity (Lee, et al., 1988; Chu and Orgel, 1985; Francois, et al., 1988). Site-specific modifications may be introduced into oligonucleotides either through modified nucleosides during automated synthesis, or through postsynthetic modification of the oligonucleotide (Goodchild, 1990; Agrawal, 1994; Chu and Orgel, 1994). Automated synthesis is advantageous because it allows a controlled, sitespecific introduction of the modification without formation of side products. However, the synthesis of protected nucleoside analogues for automated synthesis may be complicated, and the protection and/or deprotection schemes for the functional group may be incompatible with the chemistry of automated synthesis. Postsynthetic modification is useful in initial attempts at derivatizing oligonucleotides or oligonucleotide analogues. Selective postsynthetic modification of terminal phosphates can be achieved using carbodiimide chemistry to generate activated phosphates such as phosphorimidazolides (Chu, et al., 1983). Subsequent nucleophilic attack by bifunctional primary amines provides a terminal nucleophilic site through which conjugates can be generated by amide or disulfide bond chemistry (Chu and Orgel, 1994). Terminal hydroxyls can be derivatized using N,N′-carbonyldiimidazole. This generates a reactive * To whom correspondence should be addressed. Phone: (410) 955-3489. Fax: (410) 955-2926. E-mail:
[email protected].
imidazole carbamate intermediate of the oligonucleotide (Bonfils, et al., 1992; Komiyama and Inokawa, 1994; Ushuijima, et al., 1998). Subsequent displacement of the imidazole by an amine generates a stable carbamate derivative of the oligonucleotide. It is also possible to derivatize oligonucleotides postsynthetically at internal sites. To do this, it is necessary to have a unique, chemically reactive site in order to avoid formation of undesired adducts. Commercially available modified bases may be incorporated into oligonucleotides at specific sites during automated synthesis. Masked primary amines can be deprotected and derivatized postsynthetically (Haralambidis, et al., 1987). Alternatively, selective derivatization of unmodified bases can be achieved in the presence of base analogues. For instance, the selective, bisulfite-catalyzed transamination of cytosine in an oligonucleotide-containing bisulfite insensitive 5-methylcytosine has been reported (Miller and Cushman, 1992). Additionally, oligonucleotide analogues containing aminoalkyl linkers at an internal site in place of a nucleotide have been derivatized to give conjugates active in cleaving RNA (Reynolds, et al., 1996). In this paper, we describe a simple procedure for preparing an oligodeoxyribonucleotide that is conjugated with an imidazole functional group. The procedure allows placement of the imidazole function at any position within the oligonucleotide. EXPERIMENTAL PROCEDURES
Protected deoxyribonucleoside phosphoramidites and reagents for oligonucleotide synthesis were purchased from Glenn Research. All chemicals and solvents were of reagent grade or better. Reversed-phase high-performance liquid chromatography was carried out on an analytical scale using a Microsorb C18, 5 µm particle size, 100 Å pore size, 4.6 × 150 mm column and on a preparative scale using a Microsorb C18, 5 µm particle size, 100 Å pore size, 10.0 × 250 mm column, both purchased from Rainin Instruments Inc. 1H NMR spectra were acquired on a Bruker AMX 300 spectrometer. Chemical shifts are reported in parts per million (d) and referenced to the proton chemical shifts of deuterated solvent.
10.1021/bc000004t CCC: $19.00 © 2000 American Chemical Society Published on Web 06/13/2000
600 Bioconjugate Chem., Vol. 11, No. 4, 2000
Preparation of Histamine. Histamine was prepared from its dihydrochloride salt (Lancaster Chemical Co.) using strong anion-exchange chromatography. A column containing Amberlite IRA-400 quaternary amine resin (Fisher Chemical Co.) was washed with at least 3 column volumes of 0.1 N sodium hydroxide in 20% aqueous dioxane. The resin was washed with at least 3 column volumes of 0.5 N sodium hydroxide and 0.1 N sodium hydroxide. The resin was then washed with ddH2O until the pH of the eluent was equal to that of the ddH2O. A solution of histamine dihydrochloride in ddH2O was loaded onto the column and eluted with ddH2O. Fractions were pooled and dried under vacuum. Histamine was recovered in 82% yield. The identity of the compound was confirmed by 1H NMR in D2O (H2, 7.35 δ, singlet; H4. 6.70 δ, singlet; H7, 2.77 δ, triplet; H6, 2.52 δ, triplet). A 1 M stock solution in ddH2O was prepared, and the molar extinction coefficient was determined (Σ208nm ) 4540 M-1 cm-1). This stock solution was stored at 4 °C. Preparation of 2′-(N-Glutaryl-N′-histaminyl)-2′deoxyuridine (4). The reaction was carried out in two steps. In the first step, equal volumes of a 100 mM solution of 2′-amino-2′-deoxyuridine and a 500 mM solution of disuccinimidyl glutarate (Pierce Chemical Co.) each in DMSO1 were combined and reacted for 5 min at room temperature. Aliquots were quenched with EDA and saved for RP-HPLC analysis. In the second step, an equal volume of 1 M histamine in DMSO was added to the d-aU/DSG reaction solution and aliquots were quenched with EDA 30, 35, and 60 min later. The bulk of the reaction was quenched with an equal volume of 0.5 N sodium hydroxide. Samples from the EDAquenched reactions were assayed at 260 nm by RP-HPLC using a 10 mL linear gradient of 2 to 6.5% acetonitrile in 50 mM sodium phosphate, pH 5.8, at a flow rate of 1 mL/min. The bulk of the reaction was purified by preparative RP-HPLC using a 40 mL linear gradient of 2 to 6.5% acetonitrile in 50 mM sodium phosphate, pH 5.8, at a flow rate of 2 mL/min. The column was monitored at 280 nm. HPLC purified 4 was desalted on a Microsorb C18 preparative guard column (10 × 65 mm, 5 µm particle size, 100 Å pore size, Rainin Instrument Co.). The conjugate was evaporated to dryness; the residue was dissolved in a solution containing 2% acetonitrile in 50 mM sodium phosphate, pH 5.8; and the solution was loaded onto the column equilibrated with 2% acetonitrile in 50 mM sodium phosphate, pH 5.8. The column was washed with 10 mL of water, and the conjugate was eluted with 50% aqueous acetonitrile. The structure of 4 was confirmed by 1H NMR in D2O: H6 (7.65 δ), H5 (5.81 δ), H1′ (5.69 δ), H3′ (4.15 δ), H4′ (4.00 δ), H5′ (3.65 δ), H2′ (4.40 δ), H2′′ (8.25 δ), H4′′ (7.00 δ), H7′′ (3.25 δ), H6′′ (2.72 δ), H10′′, H12′′ (2.05 δ and 1.95 δ), and H11′′ (1.60 d). The double primes denote the N-glutaryl-histamine portion of the molecule. Preparation of a 2′-Amino-Derivatized Oligonucleotide (10). A 2′-amino-derivatized oligodeoxyribonucleotide, d-AAAGAAAAGAAGAAAU*CG (10), where U* is 2′-amino-2′-deoxyuridine, was synthesized on an ABI 392 automated DNA/RNA synthesizer using stan1Abbreviations: BAP, bacterial alkaline phosphatase; d-aU, 2′-amino-2′-deoxyuridine; ddH2O, distilled, deionized water; DMSO, dimethyl sulfoxide; DSG, disuccinimidyl glutarate; EDA, ethylenediamine; NHS, N-hydroxysuccinimide; RP-HPLC, reversed-phase high-performance liquid chromatography; SVPD, snake venom phosphodiesterase; TFA, trifluoroacetic acid.
Beban and Miller
dard phosphoramidite chemistry. The d-aU residue was introduced using the commercially available protected 2′trifluoroacetamido-2′-deoxyuridine phosphoramidite. The oligonucleotide was deprotected by treatment with concentrated ammonium hydroxide at 65 °C for 5 h and purified by preparative RP-HPLC. Synthesis of the Succinimidyl Ester of N-Glutarylhistamine. An aliquot of the 1 M histamine stock solution in water was evaporated under vacuum, and the residue was evaporated three times with anhydrous methanol. The residue was dissolved in DMSO to give a 1.5 M solution. This solution was combined with 2 M DSG dissolved in DMSO to give a final solution of 0.5 M DSG and 0.2 M histamine. After incubating for 13 h at room temperature, samples were diluted as necessary using 0.08% aqueous trifluoroacetic acid. The reaction was assayed by RP-HPLC at 215 nm using a gradient that consisted of 2 mL of 0.08% TFA followed by 15 mL of a linear gradient of 0 to 30% acetonitrile in 0.08% TFA at a flow rate of 1 mL/min. Aliquots of the reaction mixture were also treated with 5 N sodium hydroxide for 24 h at 37 °C and assayed by RP-HPLC as described above. Preparation of Imidazole Conjugated Oligonucleotide. Ten A260 units (∼50 nmol) of oligonucleotide 10 was dissolved in 5 µL of 50% acetonitrile in 50 mM sodium phosphate, pH 8.0. The oligonucleotide was heated at 37 °C for 15 min, and then combined with 15 µL the succinimidyl ester of N-glutarylhistamine prepared as described above. The solution was incubated overnight at 37 °C. The reaction mixture was then treated with 50 µL of 0.5 N sodium hydroxide for 30 min at 37 °C and then diluted into 50 mM sodium phosphate, pH 5.8. The solution was analyzed by RP-HPLC at 260 nm using 27 mL of a linear gradient of 5 to 15% acetonitrile in 50 mM sodium phosphate, pH 5.8 at a flow rate of 1 mL/min. The imidazole-conjugated oligonucleotide was purified by preparative scale RP-HPLC using 74 mL of a linear gradient of 5 to 15% acetonitrile in 50 mM sodium phosphate, pH 5.8. The column was monitored at 280 nm. The solution containing the purified oligonucleotide was evaporated to dryness; the residue was dissolved in 50 mM sodium phosphate, pH 5.8; and the solution was loaded onto a Microsorb C18 preparative guard column that had been equilibrated with 50 mM sodium phosphate, pH 5.8. The column was washed with 10 mL of water and the oligonucleotide eluted with 50% aqueous acetonitrile. An aliquot containing 0.1 A260 unit of the purified oligonucleotide was evaporated to dryness and the residue was dissolved in 48 µL of a solution containing 10 mM Tris/2 mM magnesium chloride, pH 8.0. The solution was treated with 0.1 unit of snake venom phosphodiesterase and 15 units of bacterial alkaline phosphatase for 18 h at 37 °C. The reaction mixture was diluted into 50 mM sodium phosphate, pH 5.8, and analyzed by RPHPLC using 3 mL of a linear gradient from 0 to 5% acetonitrile in 50 mM sodium phosphate pH 5.8; followed by 5 mL of 5% acetonitrile in 50 mM sodium phosphate, pH 5.8; and followed by 10 mL of a linear gradient of 5 to 50% acetonitrile in 50 mM sodium phosphate, pH 5.8, at 1 mL/min. RESULTS AND DISCUSSION
An oligodeoxyribonucleotide, 10, was prepared that contains a d-aU residue near the 3′-end of the oligonucle-
Technical Notes
Bioconjugate Chem., Vol. 11, No. 4, 2000 601
Scheme 1a
a
(i) DMSO. (ii) 500 mM histamine in DMSO, 4, or EDA, 5, or 250 mM sodium hydroxide, 6. (iii) RP-HPLC.
otide. The d-aU provides a primary amine to which may be tethered functional groups using conjugation chemistry. The general strategy was to generate the activated ester of the functional group to be derivatized, and then introduce that to the d-aU under conditions amenable to amide bond formation. The carboxylate of the Nglutrylhistamine provides a site for generating activated electrophilic centers that can subsequently react with the nucleophilic d-aU to give an amide linkage. Test conjugation reactions were first carried out at the nucleoside level on d-aU itself. The nucleoside served as both a model reaction for coupling to the d-aU containing oligonucleotide and as a way to generate standards for a RP-HPLC assay used to follow the oligonucleotide conjugation reaction. Commercially available disuccinimidyl glutarate, DSG, was used to form the succinimidyl ester of 2′-(N-glutaryl)-2′-deoxyuridine as shown in Scheme 1. This was accomplished by reacting d-aU (1) with a 10fold excess of DSG (2) in dry DMSO. Formation of the NHS ester of 2′-(N-glutaryl)-2′-deoxyuridine (3) was monitored by first quenching aliquots of the reaction with ethylenediamine and analyzing the products by C-18 RPHPLC. This procedure converts the NHS-activated ester of 3 to aminoethyl derivative 5. As shown in Figure 1 (panels A and B), d-aU is resolved from 5 whereas 2′-Nglutaryl-2′-deoxyuridine (6), which would result upon hydrolysis of the succinimidyl ester, coelutes with d-aU under these conditions. In the second step of the reaction, histamine in DMSO was added to a final concentration of 500 mM. This yielded 87% of 2′-(N-glutaryl-N′-histaminyl)-2′-deoxyuridine (4) after 30 min of incubation. As shown in Figure 1 (panels C, D, and E), the product was well resolved from
Figure 1. Reversed-phase chromatograms of the glutaryl derivatives of 2′-amino-2′-deoxyuridine. Solutions of DSG and d-aU in DMSO were combined and aliquots were quenched with EDA 0 min (panel A) or 5 min (panel B) after mixing. The remaining reaction was treated with histamine in DMSO and aliquots were quenched with EDA 0 min (panel C), 5 min (panel D), or 30 min (panel E) after addition of histamine. The reactions were analyzed by RP-HPLC as described in the Experimental Procedures. The peaks are 1, 2′-amino-2′-deoxyuridine (1); 3, the aminoethyl derivative of 2′-(N-glutaryl-N′-histaminyl)-2′deoxyuridine (5); and 4, 2′-(N-glutaryl-N′-histaminyl)-2′-deoxyuridine (4).
side products and from the incompletely reacted succinimidyl ester 3. The product was readily purified by preparative-scale RP-HPLC, and was characterized by 1H NMR in D O. 2 Although the reaction shown in Scheme 1 worked well when applied to the nucleoside, d-aU, it could not be applied successfully to the derivatization of d-aU contained oligonucleotide 10. To generate the oligonucleotide conjugate, it was necessary to first generate the succinimidyl ester of N-glutarylhistamine (8) as shown in Scheme 2. Histamine (7) was combined with DSG (2) to give the activated ester of 8, and bis-histaminylglutarate (9). The progress of the reaction was assayed by RPHPLC using an acetonitrile gradient in 0.08% trifluoroacetic acid. As shown by the chromatograms in Figure 2, this gradient is capable of separating the activated ester of 8 from bis-histaminylglutarate (9), and from histamine (7) and DSG (2) as well. Treatment of the crude reaction with sodium hydroxide confirmed the identity of the peaks in the chromatogram. Thus as expected, 9 is stable to sodium hydroxide whereas 8 disappears and an earlier eluting peak is evident. Oligonucleotide 10 was coupled with 8, generated in situ as described above, in a 50% acetonitrile/sodium phosphate buffer at pH 8.0 as shown in Scheme 3. The crude reaction mixture was then analyzed by RP-HPLC
602 Bioconjugate Chem., Vol. 11, No. 4, 2000 Scheme 2
after quenching with sodium hydroxide. The chromatogram of the crude reaction mixture (Figure 3, panel A) showed the formation of a new peak eluting with a retention time of 15 min. This peak was purified by preparative RP-HPLC; rechromatographed to confirm its purity (Figure 3, panel B), and coinjected with underivatized oligonucleotide to confirm that it was a distinct species (Figure 3, panel C). The purified conjugated oligonucleotide was hydrolyzed to its component nucleosides by digestion with a combination of snake venom phosphodiesterase and bacterial alkaline phosphatase. The digest was analyzed by RPHPLC using conditions that are capable of resolving the
Figure 2. Formation of the succinimidyl ester of N-glutarylhistamine. Solutions of DSG and histamine in DMSO were mixed and aliquots were assayed directly 1 h (panel A) or 13 h (panel B) after mixing. Alternatively the reactions were quenched with sodium hydroxide 1 h (panel C) or 13 h (panel D) after mixing. The reactions were analyzed by RP-HPLC as described in the Experimental Procedures. The peak labeled 8 is the succinimidyl ester of N-glutarylhistamine and the peak labeled 9 is bis-histaminylglutarate.
Beban and Miller Scheme 3a
a (i) 10 in 50% acetonitrile/sodium phosphate buffer, pH 8.0 combined with 8 in DMSO. (ii) SVPD/BAP. (ii) RP-HPLC.
expected nucleosides as shown in Figure 4, panel A. As expected, digestion of underivatized oligonucleotide 10 produced d-C, d-G, d-A, and d-aU (see panel B) whereas the digest of the derivatized oligonucleotide lacked the peak corresponding to d-aU and showed the appearance of a new peak that eluted between d-G and d-A (see panel C). Coinjection of the oligonucleotide conjugate digest with authentic d-aU and 2′-(N-glutaryl-N′-histaminyl)2′-deoxyuridine (4) standards confirmed that this new peak is 2′-(N-glutaryl-N′-histaminyl)-2′-deoxyuridine. Site specific hydrolysis of RNA has been mediated by oligonucleotides covalently modified with functional groups designed to mimic the active site of RNAse A. Chemical
Figure 3. Reaction of oligonucleotide 10 with the succinimidyl ester of N-glutarylhistamine. Panel A shows the crude reaction mixture; panel B shows the purified conjugated oligonucleotide and panel C a coinjection of 10 and the conjugated oligonucleotide. The reactions were analyzed by RP-HPLC as described in the Experimental Procedures.
Technical Notes
Bioconjugate Chem., Vol. 11, No. 4, 2000 603 ACKNOWLEDGMENT
The research described in this report was supported by a grant from the National Institutes of Health, GM57140. LITERATURE CITED
Figure 4. Enzymatic digestion of the imidazole-conjugated oligonucleotide. Panel A shows a chromatogram of a mixture of d-aU, d-C, d-G, 2′-(N-glutaryl-N′-histaminyl)-2′-deoxyuridine, 4, (indicated by the arrow), and dA standards; panel B shows a chromatogram of the digest of oligonucleotide 10; panel C shows a chromatogram of the digest of the imidazole-conjugated oligonucleotide and panel D shows a coinjection of conjugated oligonucleotide digest and d-aU and (4). The reactions were analyzed by RP-HPLC as described in the Experimental Procedures.
moieties containing two imidazoles, or two amines, or an imidazole and an amine, tethered to an oligonucleotide, have been used to specifically target RNA, and result in site-specific hydrolysis (Reynolds, et al., 1996; Ushijima, et al., 1998). Modeling studies indicate that an imidazole, tethered to the d-aU residue of oligonucleotide 10 by the eight atom glutaryl linker can be positioned in the minor groove of a duplex formed by 10 and a complementary RNA oligonucleotide. In this conformation, the imidazole is in close enough proximity to a 2′-hydroxyl of the RNA backbone to abstract a proton of the hydroxyl. Such proton abstraction in conjunction with other functional groups could promote site-specific cleavage of the RNA target. Experiments to test this possibility are currently in progress.
Agrawal, S. (1994) Functionalization Of Oligonucleotides With Amino Groups And Attachment OF Amino Specific Reporter Groups. In Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates (S. Agrawal, Ed.) Humana Press, Inc., Totowa. Asseline, U., Delarue, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T., and Helene, C. (1984) Nucleic Acid-Binding Molecules With High Affinity And Base Sequence Specificity: Intercalating Agents Covalently Linked To Oligodeoxynucleotides. Proc. Natl. Acad. Sci. U.S.A. 81, 3297-3301. Bonfils, E., Depierreux, C., Midoux, P., Thuong, N. T., Monsigny, M., and Roche, A. C. (1992) Drug Targeting: Synthesis And Endocytosis Of Oligonucleotide-Neoglycoprotein Conjugates. Nucleic Acids Res. 20, 4621-4629. Chu, B. C. F., and Orgel, L. E. (1985) Nonenzymatic SequenceSpecific Cleavage Of Single-Stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 82, 963-967. Chu, B. C. F., and Orgel, L. E. (1994) Postsynthesis Functionalization Of Oligonucleotides. in Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates (S. Agrawal, Ed.) Humana Press, Inc., Totowa. Chu, B. C. F., Wahl, G. M., and Orgel, L. E. (1983) Derivatization Of Unprotected Polynucleotides. Nucleic Acids Res. 11, 6513-6522. Francois, J.-C, Saison-Behmoaras, T., Chassignol, M., Thuong, N. T., Sun, J., and Helene, C. (1988) Periodic Cleavage Of Poly(dA) By Oligothymidylates Covalently Linked To The 1,10-Phenanthroline-Copper Complex. Biochemistry 27, 22722276. Goodchild, J. (1990) Conjugates Of Oligonucleotides And Modified Oligonucleotides: A Review Of Their Synthesis And Properties. Bioconjugate Chem. 1 (3), 165-191. Haralambidis, J., Chai, M., and Tregear, G. W. (1987) Preparation Of Base-Modified Nucleosides Suitable For Non-Radioactive Label Attachment And Their Incorporation Into Synthetic Oligodeoxyribonucleotides. Nucleic Acids Res. 15, 4857-4876. Komiyama, M., and Inokawa, T. (1994) Selective Hydrolysis Of tRNA By Ethylenediamine Bound To A DNA Oligomer. J. Biochem. 116, 719-720. Miller, P. S., and Cushman, C. D. (1992) Selective Modification Of Cytosines In Oligodeoxyribonucleotides. Bioconjugate Chem. 3, 74-79. Reynolds, M. A., Beck, T. A., Say, P. B., Schwartz, D. A., Dwyer, B. P., Daily, W. J., Vaghefi, M. M., Metzler, M. D., Klem, R. E., and Arnold, L. J. (1996) Antisense Oligonucleotides Containing An Internal Non-Nucleotide-Based Linker Promote Site-Specific Cleavage of RNA. Nucleic Acids Res. 24, 760-765. Ushijima, K., Hidetaka, G., Kazumi, H., Shirakawa, M., Kagosima, K., Takai, K., and Takaku, H. (1998) Site-Specific Cleavage Of tRNA By Imidazole And/Or Primary Amine Groups Bound At The 5′-End Of Oligodeoxyribonucleotides. Biochim. Biophys. Acta 1379, 217-233.
BC000004T
