Bioconjugate Chem. 2004, 15, 1158−1160
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COMMUNICATIONS Synthesis of Phosphate-Branched Oligonucleotides Petri Heinonen* and Harri Lo¨nnberg Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received July 1, 2004; Revised Manuscript Received September 16, 2004
A solid-phase synthesis for phosphate-branched oligonucleotides is described. The method is based on coupling of a single nucleoside phosphorodiamidite to terminal hydroxyl functions of two solidsupported oligonucleotides. After oxidation of the phosphite triester obtained to a phosphate triester, the third branch is assembled by conventional phosphoramidite chemistry.
Phosphate-branched nucleic acids have recently received interest for the reason that two small nuclear RNAs (U2 and U6 snRNA) participating in splicing of pre-mRNA have been suggested to form an X-shaped phosphotriester structure by an attack of the 2′-hydroxy group of an adenosine residue of U2 snRNA on an ApG phosphodiester bond of U6 snRNA (1). The results obtained by simple ribonucleoside 3′-dialkyl phosphates (2-6) suggest that such a triester, although susceptible to a facile nucleophilic attack by the neighboring 2′hydroxy function of the 3′-linked adenosine moiety, may still exist under physiological conditions. For firm conclusions, studies on reactions of oligomeric substrates that more closely mimic the structure of X-shaped RNA are needed. Unfortunately, no methodology for the solidphase synthesis of phosphate-branched oligonucleotides has been described. The present paper is aimed at partly filling this gap. Appropriately protected nucleoside phosphorodialkylamidites were introduced in late 1980s as alternative building blocks for the solid-phase synthesis of oligodeoxyribonucleotides (7, 8) and their thioate, (7) amidate (8), and triester (7, 9) analogues. In addition, they were shown to form 3′,5′-internucleosidic hydrogen-phosphonate (10) and phosphorodithioate (11) linkages in solution. Since then, phosphorodialkylamidites have rarely found a place in published applications, mainly because of the unfavorable balance between reactivity and hydrolytic stability. These building blocks, however, exhibit one attractive feature. They allow two subsequent couplings and, hence, enable construction of phosphatebranched oligonucleotides. We now report on the use of nucleoside 3′- (1) and 5′-phosphorodiamidites (2, 3) as building blocks in solid-phase syntheses of such oligomers. Phosphorodialkylamidites 1-3 may in principle be used in two different ways to obtain phosphate-branched oligonucleotides. Either two solid-supported oligonucleotides are reacted through their terminal hydroxyl functions with a single phosphorodialkylamidite monomer * To whom correspondence should be addressed. Tel: +358 2 333 7631. e-mail:
[email protected].
(Reaction A in Scheme 1), or an internucleosidic phosphoramidite linkage of a solid-supported oligonucleotide is converted to a phosphite triester by nucleophilic attack of a monomeric nucleoside hydroxyl function (Reaction B in Scheme 1). The rest of the branches are then assembled, after oxidation of the phosphite triester to a phosphate ester, by conventional phosphoramidite chemistry. As mentioned above, five different nucleoside phoshorodialkylamidite building blocks (1-3) were prepared, and the coupling reactions indicated in Scheme 1 were studied. 5′-O-(4,4′-Dimethoxytrityl)thymidine 3′-N,N,N′,N′-tetraethylphosphorodiamidite (1b) and its tetraisopropyl analogue (1c) were prepared by phosphitylating 5′-O(4,4′-dimethoxytrityl)thymidine with an appropriate bis(dialkylamino)chlorophosphine, as described previously (9, 10). The corresponding tetramethylphosphorodiamidite (1a) was obtained by phosphitylating the same nucleoside with tris(dimethylamino)phosphine, using tetrazole as an activator (12, 13). Formation of 1a was accompanied by appearance of a 31P NMR resonance at 138.9 ppm. The compound was not isolated, but the reaction mixture was used immediately for the solidphase synthesis. Phosphitylation of 3′-O-(4,4′-dimethoxytrityl)thymidine and 3′-O-(4,4′-dimethoxytrityl)-2′-Omethyluridine with bis(diethylamino)chlorophosphine gave 2 (31P NMR 135.8 ppm) and 3 (31P NMR 133.7 ppm), respectively. It is essential to note that phosphorodialkylamidites are far more prone to hydrolysis than normal phosphoramidites. The sensitivity to hydrolysis depends on the size of the alkyl group. While chromatographic purification of building block 1a was impossible, compound 1c was
10.1021/bc049845g CCC: $27.50 © 2004 American Chemical Society Published on Web 10/08/2004
Communications
Bioconjugate Chem., Vol. 15, No. 6, 2004 1159
Scheme 1. Key Steps in Branching of Oligonucleotides Using Phosphorodialkylamidites as Building Blocks
Table 1. Structures, Yields, and HPLC/MS Characterization of the Branched Oligonucleotides Synthesized
a According to HPLC. b Hypersil ODS column (4 × 15 mm), 50 mmol L-1 ammonium acetate to 50% MeCN in 50 mmol L-1 ammonium acetate buffer in 30 min. c Applied Biosystems Mariner System 5272, ES ionization mode, negative ion polarity.
easy to handle under normal laboratory conditions, and it withstood silica gel column chromatography. In fact, 1a did not allow reproducible solid-phase synthesis, owing to its extreme hydrolytic instability, although the same compound had successfully been exploited in a related solution-phase synthesis (12, 13). N,N,N′,N′Tetraisopropylphosphorodiamidite (1c), in turn, proved to be too unreactive to be used in a solid-phase synthesis of oligonucleotide triesters. Accordingly, main attention was paid to N,N,N′,N′-tetraethylphosphordiamidite (1b) representing a reasonable compromise between reactivity and hydrolytic stability. The applicability of ethyl-substituted phosphorodiamidites 1b, 2, and 3 was demonstrated by assembling four branched 9-mers (4-7) on a succinyl linker. Accordingly, T3 sequences were first assembled in an inverse (5′f3′) direction, and the support was then treated with 1b or 2 in small portions in excess of tetrazole, which resulted in coupling of a single thymidine phosphorodiamidite to the 3′-hydroxy group of two solid-supported T3chains. After oxidation to a phosphate ester, the third
branched was assembled by the normal or inverted (5′f3′) phosphoramidite chemistry to obtain 4 and 5, respectively (Reaction A in Scheme 1). The HPLC chromatograms of the crude reaction mixtures showed that the desired 9-mers had been formed in a 70% (4) and 50% (5) overall yield (Figure 1, Table 1). A structural analogue of 5 containing dC, dA, and dG in addition to T (6) was obtained similarly in a 35% yield. Expectedly, the reaction of a 5′-phosphorodiamidite (3) with the 3′-hydroxy function of a solid-supported 2′-O-methylribonucleoside was less efficient, but it still allowed preparation of a branched 9-mer (7), having an all-ribonucleoside structure at the branching site, in a pure state in more than 20% yield. Both CPG and Tentagel were used as a support. Somewhat surprisingly the support material and a loading on the support had only a marginal effect on the yield of the phosphorotriester product. The phosphotriesters (4-7) were remarkably stable to the overnight ammonolysis at 55 °C used to cleave the product from the support and to remove the standard benzoyl and isobutyryl protection groups of the base moieties.
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Figure 1. HPLC chromatograms of crude reaction mixtures of phosphate-branched oligonucleotide trimesters obtained by Reaction A in Scheme 1. Hypersil ODS column (4 × 15 mm), 50 mmol L-1 ammonium acetate to 50% MeCN in 50 mmol L-1 ammonium acetate buffer in 30 min.
Recation B in Scheme 1 represents a more flexible approach to the preparation of phosphate-branched oligonucleotides, since each of the three branches may in principle be elongated independently. Oligonucleotides containing isopropyl triester linkages at selected positions have previously been prepared by displacing the diethylamino ligand of an internucleosidic phosphoramidite linkage with 2-propanol (9). Also in our hands the reaction worked; an isopropyl triester was obtained in an 87% yield. Unfortunately, no similar reaction took place on using 3′-O-levulinylthymidine as a nucleophile instead of 2-propanol. Accordingly, this approach was not studied in more detail. ACKNOWLEDGMENT
Financial aid from the Academy of Finland is gratefully acknowledged. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Valadkhan, S., and Manley, J. L. (2003) Characterization of the catalytic activity of U2 and U6 snRNAs. RNA 9, 892904. (2) Kosonen, M., Oivanen, M., and Lo¨nnberg, H. (1994) Hydrolysis and interconversion of the dimethyl esters of 5′-Omethyluridine 2′- and 3′-monophosphates: Kinetics and mechanism. J. Org. Chem. 59, 3704-3708.
Heinonen and Lo¨nnberg (3) Kosonen, M., and Lo¨nnberg, H. (1995) General and specific acid/base catalysis of the hydrolysis and interconversion of ribonucleoside 2′- and 3′-phosphotriesters: kinetics and mechanisms of the reactions of 5′-O-pivaloyluridine 2′- and 3′-dimethylphosphates. J. Chem. Soc., Perkin Trans. 2, 12031209. (4) Kosonen, M., Hakala, K., and Lo¨nnberg, H. (1998) Hydrolysis and intramolecular transesterification of ribonucleoside 3′-phosphotriesters: the effect of alkyl groups on the general and specific acid-base-catalyzed reactions of 5′-O-pivaloyluridin-3′-yl dialkyl phosphates. J. Chem. Soc., Perkin Trans. 2, 663-670. (5) Kosonen, M., Yousefi-Salakdeh, E., Stro¨mberg, R., and Lo¨nnberg, H. (1998) pH- and buffer-independent cleavage and mutual isomerization of uridine 2′- and 3′-alkyl phosphodiesters: implications for the buffer catalyzed cleavage of RNA. J. Chem. Soc., Perkin Trans. 2, 1589-1595. (6) Kosonen, M., Seppa¨nen, R., Wickmann, O., and Lo¨nnberg, H. (1999) Hydrolysis and intramolecular transesterification of ribonucleoside 3′-phosphotriesters: comparison of structural effects in the reactions of asymmetric and symmetric dialkyl esters of 5′-O-pivaloyl-3′-uridylic acid. J. Chem. Soc., Perkin Trans. 2, 2433-2439. (7) Uznanski, B., Wilk, A., and Stec, W. J. (1987) Deoxyribonucleoside 3′-phosphordiamidites as substrates for solidsupported synthesis of oligodeoxyribonucleotides and their phosphorothioate and dna-triester analogues. Tetrahedron Lett. 28, 3401-3404. (8) Yamana, K, Nishijima, Y., Oka, A., Nakano, H., Sangen, O., Ozaki, H., and Shimidzu, T. (1989) A simple preparation of 5′-O-dimethoxytrityl deoxyribonucleoside 3′-O-phosphorbisdiethylamidites as useful intermediates in the synthesis of oligodeoxyribonucleotides and their phosphorodiethylamidate analogs on a solid support. Tetrahedron 45, 4135-4140. (9) Yamana, K., Nishijima, Y., Negishi, K., Yashiki, T., Nishio, K., Nakano, H., and Sangen, O. (1991) Deoxyribonucleoside 3′-phosphorobisamidites in the synthesis of isopropyl phosphotriester oligodeoxyribonucleotide analogues. Tetrahedron Lett. 32, 4721-4734. (10) Marugg, J. E., Burik, A., Tromp, M., van der Marel, G. A., and van Bom, J. H. (1986) A new and versatile approach to the preparation of valuable deoxynucleoside 3′-phosphite intermediates. Tetrahedron Lett. 27, 2271-2274. (11) Nielsen, J., Brill, W. K.-D., and Caruthers, M. H. (1988) Synthesis and characterization of dinucleoside phosphorodithioates. Tetrahedron Lett. 29, 2911-2914. (12) Lo¨nnberg, T., and Mikkola, S. (2004) Hydrolysis of 2′,3′O-methyleneadenos-5′-yl bis(2′,5′-di-O-methylurid-3′-yl) phosphate, a sugar O-alkylated trinucleoside 3′,3′,5′-monophosphate: Implications for the mechanism of large ribozymes. J. Org. Chem. 69, 802-810. (13) Poija¨rvi, P., Oivanen, M., and Lo¨nnberg, H. (2004) Towards oligonucleotide pro-drugs: 2,2-bis(ethoxycarbonyl) and 2-(alkylaminocarbonyl)-2-cyano substituted 3-(pivaloyloxy)propyl groups as biodegradable protecting groups for internucleosidic phosphoromonothioate linkages. Lett. Org. Chem. 1, 183-188.
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