Job/Unit: O31352 /KAP1 Date: 05-11-13 18:27:13 Pages: 6 SHORT COMMUNICATION DOI: 10.1002/ejoc.201301352 Synthesis of Short Oligodeoxyribonucleotides by Phosphotriester Chemistry on a Precipitative Tetrapodal Support Vyacheslav Kungurtsev, [a] Pasi Virta, [a] and Harri Lönnberg* [a] Keywords: Oligonucleotides / Synthetic methods / Protecting groups / Soluble supports Short oligodeoxyribonucleotides have been assembled from appropriately protected nucleoside 3'-(benzotriazol-1-yl 2- chlorophenyl phosphate) derivatives on hundred-milligram scales on a soluble tetrakis-O-(4-azidomethylphenyl)pentae- rythritol support bearing four 3'-O-(4-pentynoyl)thymidines. Small-molecule reagents and waste were removed by two precipitations of the support-bound growing oligonucleo- tides with methanol, the first after removal of the 5'-protect- Introduction Oligonucleotides are usually prepared on a solid support by the so-called phosphoramidite strategy, which is based on formation of internucleosidic P–O bonds at the P III level, followed by oxidation of the resulting phosphite triester to the phosphate triester immediately after each coupling step. [1–3] Virtually the same chemistry is used in lab-scale synthesis [4] and in large-scale production. [5] For in-house preparation of short oligonucleotides on hundred-milligram scales, which on a lab-scale synthesizer requires dozens of repetitions, the application of a similar strategy in solution on a soluble support has been attempted. [6–15] The need for oxidation after each coupling step unfortunately increases the number of steps in the coupling cycle. Whereas this is not a problem with solid-supported synthesis, for which waste and excess monomeric blocks and activators can be removed by simple washing, it considerably complicates the synthesis in solution. In fact, the “outdated” phosphotri- ester strategy, [16,17] based on coupling of P V blocks and hence avoiding the oxidation step, might offer a more straightforward procedure for synthesis in solution. This chemistry has previously been applied to the synthesis of oligonucleotides on a soluble poly(ethylene glycol) sup- port [18,19] by using either 1-hydroxybenzotriazole [20–22] or 1- (mesitylsulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) [18] acti- vation. Among these methods, the approach based on cou- pling of prefabricated 3'-(benzotriazol-1-yl 2-chlorophenyl [a] Department of Chemistry, University of Turku, 20014 Turku, Finland E-mail: harlon@utu.fi http://www.utu.fi/en/units/sci/units/chemistry/research/ orgchem/bioorganic/Pages/home.aspx Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201301352. Eur. J. Org. Chem. 0000, 0–0 © 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 ing group and the second after coupling. The building blocks were obtained by one-step phosphorylation of commercially available protected nucleosides with bis(benzotriazol-1-yl) 2- chlorophenyl phosphate. Removal of the phosphate protect- ing groups from the completed chain with (E)-2-nitrobenzal- doxime followed by conventional ammonolysis allowed pre- cipitation of the oligonucleotide as the sodium salt with eth- anol. phosphate) building blocks appears particularly interesting. We have now applied this method to the preparation of short oligodeoxyribonucleotides on our recently reported soluble support, [12] tetrakis-O-(4-{4-[3-(thymidin-3'-yl)-3- oxopropyl]-1,2,3-triazol-1-ylmethyl}phenyl)pentaerythritol (1; Figure 1). The usefulness of this support stems from its quantitative precipitation with MeOH. Accordingly, each coupling cycle contains only two precipitations, one after removal of the 5'-protecting group and the second after the coupling. All the small-molecule compounds remain in solution, while the support precipitates quantitatively. By ammonolytic release, nearly homogeneous heterotrimers were obtained in 70% isolated yield and a pentamer in 55 % yield. In spite of the simplicity of the synthesis, the yields favorably compete with the previously described ap- proaches. Interestingly, a related branched core structure has previously been used for immobilization of prefabri- cated dimers and trimers as building blocks of nanoscale objects. [23,24] Figure 1. Structure of the soluble support and the building-blocks employed. Results and Discussion The tetravalent nucleoside cluster 1 has been shown to be a promising soluble support for the synthesis of oligonu-