5092 Biochemistry zyxwvu 1986, 25, 5092-5097 Solid-Phase Syntheses of zyxwv Oligodeoxyribonucleoside Methylphosphonatest Paul S. Miller,* M. Parameswara Reddy,* Akira Murakami, Kathleen R. Blake, Shwu-Bin Lin, and Cheryl H. Agris Division of Biophysics, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21 205 Received December 2, 1985; Revised Manuscript Received April 18, 1986 ABSTRACT: zyxwvutsrqp Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(Np),N, where zyxwvutsr n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5’-(dimeth- oxytrity1)deoxyribonucleoside 3’-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5’-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazo1ide) and can be stored in the reaction solution for up to 2 weeks at 4 OC with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95% complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78%, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5’-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5’-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate. The positions of purine and pyrimidine bases were confirmed by treatment of 5’-end labeled oligomers with acid and hydrazine, respectively. These experiments show that oligodeoxyribonucleoside methylphosphonates can be prepared and characterized by procedures analogous to those used to prepare oligodeoxyribonucleotides. Rant reports from our laboratory have demonstrated that nonionic oligodeoxyribonucleoside methylphosphonates are capable of inhibiting mRNA translation in mammalian cells and certain bacterial cells (Miller et al., 1981, 1983a; Jay- araman et al., 1981; Blake et al., 1985). In addition, oligo- deoxyribonucleoside methylphosphonates complementary to the splice junctions of SV40 and herpes simplex virus precursor mRNA have been shown to selectively inhibit virus protein synthesis in virus-infected cells (Miller et al., 1983a; Smith et al., 1986). These nonionic nucleic acid analogues contain a neutral methylphosphonate linkage that replaces the nega- tively charged phosphodiester internucleotide bond normally found in nucleic acids. The methylphosphonate linkage, which is resistant to hydrolysis by nucleases, enables the oligomer to be taken up intact by mammalian cells in culture and by a cell wall mutant of Escherichia coli. In the course of our biochemical and cell culture experi- ments, we required a procedure that would allow efficient synthesis of oligomers of defined sequence up to 15 nucleoside units in length. In previous papers, we have described the syntheses of oligomers in solution and on a polystyrene support (Miller et al., 1983b,c). In this paper we describe a new solid-phase approach that makes use of protected nucleoside 3’-(methylphosphonic imidazolides) as synthetic intermediates. Using this method, it is possible to prepare 15-mers of defined sequence in sufficient quantities for biochemical experiments. MATERIALS AND METHODS Protected deoxyribonucleosides, protected deoxyribo- nucleoside 3‘-(cyanoethyl diisopropylphosphoramidites), and ‘This research was supported in part by a grant from NIH (GM 31927) to P.S.M. and by a grant to Dr. Paul 0. P. Ts’o from the Albert Szent-Gyorgyi Foundation. ‘Present address: Beckman Instruments, Inc., Palo Alto, CA 94303. derivatized controlled pore glass supports were purchased from American Bionuclep Inc. 5’-Protected deoxyribonucleoside 3’-(o-chlorophenylphosphates) and 1-(2-mesitylenesulfo- nyl)-3-nitro-1,2,4-triazole (MSNT)’ were purchased from Vega Biochemicals Inc. Methylphosphonic dichloride was purchased from Alfa Chemical Co. and distilled under vacuum prior to use. The derivatized polystyrene support was a product of ChemGenes Inc. All solvents were reagent- or HPLC- grade. Tetrahydrofuran and acetonitrile, which were used for washing the support; were dried over a 4-A molecular sieves 24 h prior to use. Anhydrous acetonitrile was prepared by refluxing reagent-grade acetonitrile with anhydrous calcium chloride for 6 h. After distillation, the acetonitrile was further refluxed with calcium hydride and then distilled onto calcium hydride chips in a 10-mL V-vial and sealed with a Teflon-faced silicone septa. Anhydrous tetrahydrofuran was prepared by refluxing dry tetrahydrofuran with calcium hydride for 6 h followed by distillation into sealed 10-mL V-vials containing several chips of calcium hydride. Anhydrous pyridine was prepared as previously described (Miller et al., 1980). High-performance liquid chromatography was carried out on a Whatman ODs-3 column (0.9 X 25 cm) with a gradient of 0.5-3096 or 0.5-35% acetonitrile in 0.1 M ammonium acetate buffer at pH 5.8. The flow rate was 2.5 mL/min, and the total volume of solvent in the gradient was 50 mL. ’ Abbreviations: d-Np(Np),N or d-NpNNNN, an oligodeoxyribo- nucleoside methylphosphonate that terminates with a phosphodiester linkage at its 5’-end; p or italicized bases, 3’-5’-linked methylphosphonate internucleoside bonds; p, an (o-chloropheny1)phosphoryl group; MSNT, 1 -(2-mesitylenesulfonyl)-3-nitro- 1,2,4-triazole;HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; TEAA, tri- ethylammonium acetate; CPG, controlled pore glass; Tris, tris(hydroxy- methy1)aminomethane;EDTA, ethylenediaminetetraacetic acid. 0006-2960/86/0425-5092$01.50/0 0 1986 American Chemical Society