The use of phosphorothioates as DNA analogues useful in antisense-based therapy is well established [1] and led to the development of Vitravene as the first antisense drug. [2] Several other phosphorothioate oligonucleotides (PS-Oligos) are in clinical trials. Although Stec et al. described an elegant oxathiaphospholane-based approach [3] for preparing diastereomerically pure phosphorothioates, it has not been used for large-scale production of PS-Oligos, which are still used as a mixture of about 10 6 diastereomers. The use of cyclic Nacylphosphoramidites as promising monomers for the stereocontrolled synthesis of phosphorothioates was recently reported by Beaucage et al. [4] Previously, we developed cyclic phosphoramidites [5] such as sugar-derived oxazaphosphorinanes, [5b,c] indolooxazaphosphorines (a), [5e, f] and indoleimidazoles [5i] for the stereoselective synthesis of PS-Oligos. Here we report on the use of promising indolooxazaphosphorine precursors derived from tryptophan which do not require a difficult chromatographic separation and may form the basis of a practical process.
As demonstrated in the indolooxazaphosphorine approach, [5f] chiral auxiliary a led to the stereoselective synthesis of phosphorothioate in solution. When it was applied to solidphase synthesis, a b-elimination caused rearrangement to give imine 3 d (5.00 g, 12.9 mmol) in CH 2 Cl 2 (10 mL) was added by syringe to the catalyst solution at 0 8C. Stirring the mixture for 10 min gave an orange solution to which was rapidly added ethyl diazoacetate (1.484 mL, 14.2 mmol) by syringe. Some bubbling was noted after the addition. The reaction was allowed to proceed for 6 h at 0 8C and then for 14 h at room temperature (22 8C). The reaction mixture was transferred to a 500-mL flask, diluted with hexanes (250 mL), and then the volatiles were removed under vacuum to give the crude aziridine 3 d as an off-white solid. The 1 H NMR spectrum of this material revealed 3 d with cis:trans ! 50:1 and indicated that < 1 % of 4 d and 5 d were formed. Purification of 3 d by column chromatography on silica gel with a mixture of ethyl acetate:hexanes (3:7) gave aziridine 3 d (5.20 g, 11 mmol) as a white solid in 85 % yield. The optical purity of this material was determined to be 96 % ee by HPLC analysis (OD-H column). Crystallization from hexanes:CH 2 Cl 2 (10:1, 300 mL) gave 3 d (4.43 g) with 99 % ee. A second crop was taken but found to be only 90 % ee.