MARCH/APRIL 1997 Volume 8, Number 2 © Copyright 1997 by the American Chemical Society
COMMUNICATIONS Synthesis of Oligonucleotides Containing 3′-Alkylcarboxylic Acids Using a Palladium Labile Oligonucleotide Solid Phase Synthesis Support Tracy J. Matray, Dong Jin Yoo, Dustin L. McMinn, and Marc M. Greenberg* Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523. Received October 31, 1996X
Oligonucleotides containing a 3′-alkylcarboxylic acid are isolated using a Pd(0)-catalyzed cleavage reaction, in yields that are in most cases within experimental error of those isolated using standard oligonucleotide cleavage conditions (concentrated NH4OH). In contrast to results obtained with photolabile solid phase synthesis supports, no reduction in isolated yields of the oligonucleotides is observed when their length is increased from 20 to 40 nucleotides. The oligonucleotides are characterized by anion exchange HPLC, electrospray mass spectrometry, and enzymatic digestion. When methyl phosphoramidites are employed in the synthesis of the biopolymers, 3 serves as an orthogonal solid phase oligonucleotide synthesis support.
Solid phase oligonucleotide synthesis has advanced to a very efficient state. Scientists working with nucleic acids now have a wide variety of methodologies available for preparing native oligonucleotides, as well as biopolymers containing modified backbones, nucleobases, and sugars (1). These recent advances in solid phase synthesis have led to strategies for the synthesis of oligonucleotide conjugates (2-5). Both naturally occurring and synthetic oligonucleotide conjugates show promise as therapeutic agents and diagnostic probes. For example, it has been shown that oligonucleotide bioconjugates can facilitate the transport of potential antisense agents through cell membranes (6-8). The methodology for synthesizing bioconjugates, particularly those in which the covalent linkage is at the 3′-terminus of the oligonucleotide, has lagged behind the aforementioned aspects of nucleic acid synthesis (9). One approach to bioconjugate formation involves utilizing fully deproX Abstract published in Advance ACS Abstracts, February 15, 1997.
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tected oligonucleotides as substrates. The poor solubility of such substrates in many organic solvents limits the scope of reactions that can be utilized to effect conjugation. Another difficulty associated with this method of oligonucleotide conjugate formation is attributable to side reactions occurring with the exocyclic amines located throughout the biopolymer (10, 11). To ameliorate these limitations, we, and others, have suggested utilizing protected oligonucleotides (retaining their nucleobase and phosphate protecting groups) that contain a single exposed functional group as substrates for nucleic acid conjugation reactions (12-16). Before such a bioconjugation strategy is pursued, it is necessary to develop methodology for cleaving protected oligonucleotides containing a single functional group at their 3′termini from their solid phase supports. We have previously reported on several orthogonal solid phase supports that utilize the o-nitrobenzyl photoredox reaction (12-14). Oligonucleotides containing 3′-hydroxyand 3′-alkylcarboxylic acids and 3′-alkylamines have been © 1997 American Chemical Society
100 Bioconjugate Chem., Vol. 8, No. 2, 1997
Matray et al.
Table 1. Isolated Yields of Fully Deprotected Oligonucleotides Obtained via Pd(0)-Mediated Cleavagea oligonucleotide 8 8 8 9 10 11
phosphate protecting group β-cyanoethyl methyl methyl methyl methyl β-cyanoethyl
reaction time (h)
isolated yieldb (%)
7 7 5 5 5 5
102 ( 7 93 ( 7 93 ( 8 91 ( 13 77 ( 7 105 ( 10
Scheme 1
a Pd(0) cleavage reactions were carried out as described in footnote 2. b Determined as described in footnote 1.
obtained using these supports. These supports are compatible with commercially available reagents and automated oligonucleotide synthesis protocols. Isolated yields as high as 98% of oligonucleotides containing photodamage below detectable limits have been obtained using commonly available irradiation sources (12, 13).1 A shortcoming of these photolabile synthesis supports was revealed during the synthesis of longer oligonucleotides. A decrease in yield was observed as the length of the biopolymer was increased from 20 to 40 nucleotides (12). This decrease in yield was attributed to greater competition for light by the protected biopolymer with the o-nitrobenzyl chromophore. While yields can be increased by extending the irradiation period, this also increases the amount of photodamage. This observation prompted us to investigate an alternative reaction for the cleavage of protected oligonucleotides from solid phase supports. In designing a new generation of orthogonal oligonucleotide synthesis supports for which the cleavage reaction would not be dependent upon biopolymer length, we chose to utilize the proven Pd(0)-catalyzed allyl transfer reaction (17-21). This reaction has been used successfully for deprotecting the exocyclic amines of nucleobases and the phosphate diesters during oligonucleotide synthesis, as well as an orthogonal linker for peptide synthesis. Prior to commencing the synthesis of an appropriate solid support, we sought to ensure that the conditions employed to cleave the allyloxy linker were orthogonal with respect to the oligonucleotide protecting groups used in commercially available reagents. The stability of amide protecting groups for the exocyclic amines was not a concern. In addition, the 5′-Odimethoxytrityl group of 1 was stable to Pd2(dba)3‚CHCl3
and PPh3 in n-BuNH2/HCO2H (1.2 M) between 25 and 55 °C for up to 7 h. However, contrary to assumptions in a recent paper, the β-cyanoethyl group was cleaved completely within 1 h at 55 °C under these conditions 1 Yields of oligonucleotides obtained from orthogonal solid phase supports are determined via comparison of the isolated yield of oligonucleotide obtained via Pd(0) cleavage and subsequent NH4OH treatment versus that obtained via direct NH4OH treatment of resin-bound oligonucleotide from the same oligonucleotide synthesis.
(16). This problem was alleviated using monomer 2. The O-methyl phosphate triester was shown to undergo only minor (≈10%) decomposition over the course of 7 h at 55 °C in the presence of these reagents. Having established the suitability of the Pd(0)-catalyzed allyl transfer reaction as the basis for an orthogonal solid phase support, 3 was designed to function as a universal solid phase synthesis support for the preparation of oligonucleotides containing 3′-alkylcarboxylic acids (Scheme 1) (14). Support 3 was rapidly assembled in a convergent manner via the coupling of previously reported 4 and 5 (Scheme 2) (14, 22). The long-chain alkylamine controlled pore glass support (LCAA-CPG) was loaded (45 µmol/g) using the trichlorophenyl ester of 7. Oligonucleotides were synthesized on 3 using standard oligonucleotide synthesis cycles, in which (while not necessary) the 5′-O-dimethoxytrityl group was removed prior to cleavage of the oligonucleotide from the support. With the exception of the substitution of tBuOOH in CH2Cl2 for I2 in pyridine/H2O (to guard against iodination of the double bond), all oligonucleotide synthesis reagents used were commercially available (17). Conditions for the cleavage of oligonucleotides from 3 were optimized utilizing the isolation of an eicosameric polythymidylate (8). The yields were determined for fully deprotected, gel-purified oligonucleotides.1 Consequently, cleavage reactions could be carried out on oligonucleotides prepared using β-cyanoethyl or methyl protected phosphoramidites. Initial Pd(0)-catalyzed cleavage reactions were carried out under the conditions prescribed for the removal of phosphate and nucleobase amine protecting groups (17). To obtain tractable material, it was necessary to remove the n-butylamine/formic acid buffer and phosphine reagent prior to treating the cleaved oligonucleotide with NH4OH. However, removal of the large excess of PPh3 via trituration of the residue with hexanes proved difficult. The amorphous organic material obtained was difficult to handle, resulting in erratic yields of isolated 8. Utilization of bis(diphen-
Communications
Bioconjugate Chem., Vol. 8, No. 2, 1997 101
Scheme 2
ylphosphino)ethane (DIPHOS) in place of PPh3 enabled us to employ only 5 equiv of phosphine ligand relative to Pd2(dba)3‚CHCl3.2 Subsequent workup of the protected oligonucleotide yielded a more tractable material. Yields of eicosameric polythymidylate within experimental error of those obtained via direct ammonolysis were achieved from O-β-cyanoethyl phosphate protected oligonucleotides after 7 h at 55 °C (Table 1).1 Eicosameric polythymidylates containing phosphate protecting groups (O-methyl) that are orthogonal with regard to the Pd(0)-mediated cleavage were also isolated in quantitative yields (Table 1). Further optimization of the reaction conditions proved that 5 h was sufficient to effect complete cleavage of the oligonucleotide from the solid support. Electrospray mass spectrometry (ESMS) proved that the alkylcarboxy group remained intact during the various deprotection and purification procedures. Only a small amount of dealkylated material was formed. We believe that the dealkylated material results from formation of the dianion during NH4OH deprotection, followed by intramolecular displacement of the phosphate-terminated oligonucleotide. ESMS could not distinguish between the polythymidylates cleaved directly from 3 with NH4OH and those cleaved with Pd(0), as they differ by only 1 mass unit. However, the two products are separable on anion exchange HPLC, where 8 eluted more than 1 min later than the respective amide obtained from NH4OH treatment of resin-bound oligonucleotide.
Using the optimized conditions for cleaving methyl phosphate protected polythymidylates, we examined the suitability of the methodology for obtaining oligonucleotides containing all four native nucleotides. We determined that the yields of oligonucleotides prepared on 3 and cleaved via Pd(0) were not strongly dependent upon sequence or length (Table 1).1 Furthermore, enzymatic digestion of heteropolymers prepared on 3 using β-cyanoethyl phosphoramidites that were cleaved with Pd(0) reveals that no extraneous nucleosides are formed, demonstrating that the Pd(0) cleavage process does not damage the biopolymer. In summary, we have utilized Pd(0)-catalyzed cleavage of allyl groups to cleave oligonucleotides, conjugated to alkylcarboxylic acids at their 3′-termini, from their solid phase synthesis supports in very high yield.1 The cleavage reaction is orthogonal to commercially available O-methyl phosphoramidite protecting groups. This methodology should prove to be highly useful for the preparation of more elaborate oligonucleotide conjugates.
2 Typical procedure for palladium(0)-mediated cleavage of CPG-bound oligonucleotides: In a typical procedure, Pd2(dba)3‚ CHCl3 (0.5 mg, 0.5 µmol) was added to a mixture of the appropriate oligonucleotide that was synthesized on 3 (1 mg, ≈0.05 µmol) and 1,2-bis(diphenylphosphino)ethane (1 mg, 2.5 µmol) in THF (170 µL, sparged with N2 for 30 min). After addition of n-butylamine (18 mg, 240 µmol, sparged with N2 for 30 min) and formic acid (11 mg, 240 µmol, sparged with N2 for 30 min), the mixture was vortexed well and heated at 55 °C. The reaction vessel was vortexed approximately every 15-30 min. After the appropriate reaction time, the solution was transferred to a vial and concentrated in vacuo to give a black residue. The residue was dissolved in a 1:1 mixture (by volume) of THF/H2O (1 mL) and evaporated to dryness. This process was repeated a second time. The residue was then triturated with hexanes (2 × 2 mL), dissolved in CH3CN (1 mL), and filtered through a nylon filter (0.45 µm). The filter was washed well with CH3CN (3 mL) and then H2O (3 mL). All of the washings were combined and evaporated to dryness in vacuo. The residue was suspended in concentrated NH4OH and heated for 10 h at 55 °C. After the NH4OH was removed in vacuo, the residue was suspended in formamide loading buffer (70 µL) containing sodium N,N-diethyldithiocarbamic acid (30 mM) and purified by denaturing polyacrylamide gel electrophoresis (20% polyacrylamide). The oligonucleotide was extracted from the gel slice with NaCl (0.2 M) and EDTA (1 mM) and desalted using a reversed phase purification cartridge.
Supporting Information Available: Enzymatic digest of an eicosameric oligonucleotide and an electrospray mass spectrum of 8 (4 pages). Ordering information is given on any current masthead page.
ACKNOWLEDGMENT
This research was supported by the National Science Foundation (CHE-9424040). M.M.G. is a fellow of the Alfred P. Sloan Foundation. We are grateful to Dr. Laurent Bellon (Ribozyme Pharmaceuticals Inc.) for stimulating discussion.
LITERATURE CITED (1) Beaucage, S. L., and Iyer, R. P. (1993) The Synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and Their Applications. Tetrahedron 49, 6123-6194. (2) Truffert, J-C., Lorthioir, O., Asseline, U., Thuong, N. T., and Brack, A. (1994) On-Line Solid Phase Synthesis of Oligonucleotide-Peptide Hybrids Using Silica Supports. Tetrahedron Lett. 35, 2353-2356. (3) de la Torre, B. G., Avin˜o´, A., Tarrason, G., Piulats, J., Albericio, F., and Eritja, R. (1994) Stepwise Solid-Phase Synthesis of Oligonucleotide-Peptide Hybrids. Tetrahedron Lett. 35, 2733-2736. (4) MacKellar, C., Graham, D., Will, D. W., Burgess, S., and Brown, T. (1992) Synthesis and Physical Properties of AntiHIV Antisense Oligonucleotides Bearing Terminal Lipophilic Groups. Nucleic Acids Res. 20, 3411-3417. (5) Haralambidis, J., Angus, K., Pownall, S., Duncan, L., Chai, M., and Tregear, G. W. (1990) The Preparation of Polyamide Oligonucleotide Probes Containing Multiple Non-Radioactive Labels. Nucleic Acids Res. 18, 501-505.
102 Bioconjugate Chem., Vol. 8, No. 2, 1997 (6) Jones, D. S., Hachmann, J. P., Osgood, S. A., Hayag, M. S., Barstad, P. A., Iverson, G. M., and Coutts, S. M. (1994) Conjugates of Double-Stranded Oligonucleotides With Poly(ethylene glycol) and Keyhole Limpet Hemocyanin: A Model for Treating Systemic Lupus Erythematosus. Bioconjugate Chem. 5, 390-399. (7) Leonetti, J-P., Degols, G., and Lebleu, B. (1990) Biological Activity of Oligonucleotide-Poly(L-lysine) Conjugates: Mechanism of Cell Uptake. Bioconjugate Chem. 1, 149-153. (8) Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-Conjugated Oligonucleotides: Synthesis, Properties, and Activity as Inhibitors of Replication of Human Immunodeficiency Virus in Cell Culture. Proc. Natl. Acad. Sci. U.S.A. 86, 6553-6556. (9) Goodchild, J. (1990) Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of Their Synthesis and Properties. Bioconjugate Chem. 1, 165-187. (10) Ghosh, S. S., and Musso, G. F. (1987) Covalent Attachment of Oligonucleotides to Solid Supports. Nucleic Acids Res. 15, 5353-5372. (11) Bischoff, R., Coull, J. M., and Regnier, F. E. (1987) Introduction of 5′-Terminal Functional Groups Into Synthetic Oligonucleotides For Selective Immobilization. Anal. Biochem. 164, 336-344. (12) McMinn, D. L., and Greenberg, M. M. (1996) Novel Solid Phase Synthesis Supports For The Preparation of Oligonucleotides Containing 3′-Alkyl Amines. Tetrahedron 52, 38273840. (13) Venkatesan, H., and Greenberg, M. M. (1996) Improved Utility of Photolabile Solid Phase Synthesis Supports For The Synthesis of Oligonucleotides Containing 3′-Hydroxyl Termini. J. Org. Chem. 61, 525-529. (14) Yoo, D. J., and Greenberg, M. M. (1995) Synthesis of Oligonucleotides Containing 3′-Alkyl Carboxylic Acids Using Universal Photolabile Solid Phase Synthesis Supports. J. Org. Chem. 60, 3358-3364.
Matray et al. (15) Matray, T. J., Yoo, D. J., and Greenberg, M. M. (1996) Palladium Labile Oligonucleotide Synthesis Supports. Abstracts of Papers, 211th National Meeting of the American Chemical Society, New Orleans, LA, Division of Organic Chemistry Abstract 119, American Chemical Society, Washington, DC. (16) Zhang, X., and Jones, R. A. (1996) Solid-Phase Synthesis of Peptide Aminoalkylamides Using An Allyl Linker. Tetrahedron Lett. 37, 3789-3790. (17) Hayakawa, Y., Wakabayashi, S., Kato, H., and Noyori, R. (1990) The Allylic Protection Method in Solid-Phase Oligonucleotide Synthesis. An Efficient Preparation of SolidAnchored DNA Oligomers. J. Am. Chem. Soc. 112, 16911696. (18) Seitz, O., and Kunz, H. (1995) A Novel Allylic Anchor for Solid-Phase Synthesis-Synthesis of Protected and Unprotected O-Glycosylated Mucin-Type Glycopeptides. Angew. Chem., Int. Ed. Engl. 34, 803-805. (19) Kunz, H., and Dombo, B. (1988) Solid Phase Synthesis of Peptides and Glycopeptides on Polymeric Supports With Allylic Anchor Groups. Angew. Chem., Int. Ed. Engl. 27, 711713. (20) L-Williams, P., Merzouk, A., Guibe´, F., Albericio, F., and Giralt, E. (1994) Solid-Phase Synthesis of Peptides Using Allylic Anchoring Groups 2. Palladium-Catalysed Cleavage of Fmoc-Protected Peptides. Tetrahedron Lett. 35, 4437-4440. (21) Kaljuste, K., and Unde´n, A. (1996) Solid-Phase Synthesis of Peptide Aminoalkylamides Using an Allyl Linker. Tetrahedron Lett. 37, 3031-3034. (22) Nicolaou, K. C., Prasad, C. V. C., Somers, P. K., and Hwang, C-K. (1989) Activation of 6-Endo Over 5-Exo Hydroxyl Epoxide Opening. Stereoselective and Ring Selective Synthesis of Tetrahydrofuran and Tetrahydropyran Systems. J. Am. Chem. Soc. 111, 5330-5334.
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