3552 Nucleic Acids Research, 2005, Vol. 33, No. 11 N, N, N0, N0-Tetraisopropyl-O-2-[(N-formyl-N- methyl)aminoethyl]phosphorodiamidite (8) This phosphinylating reagent (Scheme 2) was prepared following a procedure that had been modiﬁed from its earlier version (2,12). To a stirred solution of 2-(N-formyl- N-methyl)aminoethan-1-ol (3.50 g, 34.0 mmol) and N, N- diisopropylethylamine (35.0 ml, 201 mmol) in anhydrous dichloromethane (20 ml) was added, at 25 C, a solution of bis(N, N-diisopropylamino)chlorophosphine (Aldrich) (9.98 g, 37.4 mmol) in dry dichloromethane (10 ml). Formation of the phosphorodiamidite was monitored by 31P NMR spectroscopy, which revealed over a period of 2 h, complete conversion of bis(N, N-diisopropylamino)chlorophosphine (dP 135.5 p.p.m.) to the desired product as a mixture of rot- amers (dP 118.0 and 118.7 p.p.m.). The suspension was ﬁltered and the ﬁltrate was evaporated to an oil under reduced pres- sure. The material was transferred to a 50 ml round bottom ﬂask, which was then connected to a vacuum-jacketed short path distilling head and a distributing adapter. Vacuum distillation was performed using a heat gun to enable rapid heating without substantial decomposition. A colorless distil- late (bp 145 C at 1 mmHg) was obtained in 67% yield (7.58 g, 22.8 mmol). 1H NMR (300 MHz, C6D6): d 3.56 (m, 2H), 3.53 (sept, J = 6.9 Hz, 2H), 3.49 (sept, J = 6.9 Hz, 2H), 2.30 (m, 2H), 1.80 (s, 3H), 1.63 (m, 4H), 1.23 (d, J = 6.9 Hz, 12H), 1.19 (d, J = 6.9 Hz, 12H). 13C NMR (75 MHz, C6D6): d 15.2, 24.0, 24.1, 24.7, 24.8, 26.2, 31.1 (d, JPC = 9.6 Hz), 34.2, 44.6, 44.7, 64.1 (d, 2JPC = 21.5 Hz). 31P NMR (121 MHz, C6D6): d 118.0, 118.7. EI-HRMS: calcd for C16H36N3O2P (M )+ 333.2545, found 333.2528. General procedure for the preparation of deoxyribonucleoside phosphoramidites 1a–d A properly protected deoxyribonucleoside (9a–d, 2 mmol) was dried under high vacuum for 4 h in a 50 ml round- bottom ﬂask and, then, dissolved in anhydrous MeCN (10 ml). To this solution was added N, N, N0, N0-tetraisopropyl- O-2-[(N-formyl-N-methyl)aminoethyl]phosphorodiamidite (8, 730 mg, 2.2 mmol) followed by 0.45 M 1H-tetrazole in MeCN (4.4 ml, 2 mmol), dropwise, over a period of 0.5 h (Scheme 2). Phosphinylation of suitably protected 20-deoxyribonucleosides was usually complete within 2 h at 25 C with the exception of protected 20-deoxyguanosine, which was allowed to proceed overnight. The reaction mixture was then concentrated under reduced pressure, dissolved in benzene:triethylamine (9:1 v/v), and chromatographed using a column (4 · 10 cm) containing silica gel 60 (230–400 mesh, 20 g) equilibrated in benzene:triethylamine (9:1 v/v). The phosphoramidites were eluted from the column using the equilibration solvent as the eluant. Appropriate fractions were pooled, concentrated, and each of the deoxyribonucleoside phosphoramidites 1a–d was obtained as a white foamy mater- ial. The puriﬁed product was dissolved in 3 ml of benzene and the solution was added to 100 ml of cold ( 20 C) vigorously stirred hexane. The suspension was allowed to settle and most of the supernatant was carefully decanted. The wet material was pulverized under reduced pressure, and then dissolved in 10 ml of benzene. The solution was frozen in a dry-ice/acetone bath, and lyophilized under high vacuum affording triethylamine-free phosphoramidites as white amorphous solids in yields ranging from 70 to 85%. 50-O-(4,40-dimethoxytrityl)-30-O-(N, N-diisopropylamino)[2- (N-formyl-N-methyl)aminoethoxy]phosphinyl-20-deoxy- thymidine (1a). 31P NMR (121 MHz, C6D6): d 148.4, 148.3, 148.2. FAB-HRMS: calcd for C41H53N4O9P (M + Cs)+ 909.2604, found 909.2544. N4-benzoyl-50-O-(4,40-dimethoxytrityl)-30-O-(N, N-diisopro- pylamino)[2-(N-formyl-N-methyl)aminoethoxy]phosphinyl- 20-deoxycytidine (1b). 31P NMR (121 MHz, C6D6): d 149.0, 148.9, 148.5, 148.4. FAB-HRMS: calcd for C47H56N5O9P (M + Na)+ 888.3714, found 888.3745. N6-benzoyl-50-O-(4,40-dimethoxytrityl)-30-O-(N, N-diisopro- pylamino)[2-(N-formyl-N-methyl)aminoethoxy]phosphinyl- 20-deoxyadenosine (1c). 31P NMR (121 MHz, C6D6): d 148.9, 148.8, 148.1. FAB-HRMS: calcd for C48H56N7O8P (M + Na)+ 912.3827, found 912.3843. N2-isobutyryl-50-O-(4,40-dimethoxytrityl)-30-O-(N, N-diiso- propylamino)[2-(N-formyl-N-methyl)aminoethoxy] phosphi- nyl-20-deoxyguanosine (1d). 31P NMR (121 MHz, C6D6): d 149.0, 143.9, 143.7. FAB-HRMS: calcd for C45H58N7O9P (M + Na)+ is 894.3933, found 894.3978. Proton-decoupled 31P NMR spectra were recorded at 7.05 T (300 MHz for 1H) using deuterated solvents and 85% phos- phoric acid in deuterium oxide as an external reference. The NMR spectrometer was run at 25 C and chemical shifts d are reported in parts per million. Low- and high-resolution FAB mass spectra were acquired from samples dissolved in either 4-nitrobenzyl alcohol or a mixture of dithiothreitol and dithioerythritol (3:1, v/v), and bombarded with 8 keV fast cesium ions. A mass calibration standard of cesium iodide, or a mixture of cesium iodide and sodium iodide, was used. Accurate mass measurements were performed on [M + H]+ or on [M + Na]+ ions, which were obtained by addition of aqueous sodium iodide to the sample matrix. Solid-phase oligonucleotide synthesis Solid phase synthesis of d(GPS(FMA)CPS(FMA)TPS(FMA)- APS(FMA)GPS(FMA)APS(FMA)CPS(FMA)GPS(FMA)TPS(FMA)TPS(FMA)- APS(FMA)GPS(FMA)CPS(FMA)GPS(FMA)T) [CpG ODN fma1555], d(GPS(FMA)CPS(FMA)TPS(FMA)APS(FMA)GPS(FMA)APS(FMA)- GPS(FMA)GPS(FMA)TPS(FMA)TPS(FMA)APS(FMA)GPS(FMA)GPS(FMA)- GPS(FMA)T) [ODN fma1556], where PS(FMA) stands for a thermolytic 2-(N-formyl-N-methyl)aminoethyl phosphoro- thioate triester function, and d(GPSCPSTPSAPSGPSAPSCPS GPSTPSTPSAPSGPSCPSGPST) [CpG ODN 1555], where PS stands for a phosphorothioate diester function, was performed on a scale of 1 mmol using a succinyl long chain alkylamine controlled-pore glass (Succ-LCAA-CPG) support func- tionalized with 50-O-DMTr-dT as the leader nucleoside. The syntheses were carried out using an ABI 392 DNA/ RNA synthesizer and phosphoramidites 1a–d or commercial 2-cyanoethyl deoxyribonucleoside phosphoramidites as 0.1 M solutions in dry MeCN. The reaction time for each phosphor- amidite coupling step was 180 s. With the exception of the deblocking solution, all ancillary reagents necessary for the