Inorganic Reactions

Abstract

Antisense oligonucleotides (AOs) have shown great promise as agents for inhibiting gene expression. 1 In principle, AOs interfere in a sequence-specific manner with processes such as translation of mRNA into protein. In recent years, significant advances have been made in chemical modifications of AOs that can enhance both their stability and their potency. 2 One of the main focal points of the research has been the complete replacement of natural phosphodiester (PdO) backbone with synthetic linkages. 3 Among the various surrogates of the PdO backbone studied in our group, we have selected methylene (methylimino) (MMI) as a linkage of choice for advanced studies and for incorporation into AOs. 4 The MMI linkage is achiral and neutral, readily incorporated into AOs, and stable under physiological conditions (Figure 1). AOs containing MMI linkages hybridize to the complementary RNA with high affinity and base-pair specificity. NMR and modeling studies have indicated that the 3′-CH 2 group of the MMI linkage shifted the sugar conformation to a desired 3′-endo pucker, thus helping the AOs to preorganize into a preferred A-geometry for duplex formation. 5 Biological studies showed that incorporation of MMI linkages into a phospho-rothioate (PS) AOs substantially improved the pharmacological properties of the parent oligomer. 6 Our prior incorporation of the MMI linkage into AOs has been achieved by a nucleosidic phophoramidite dimer, creating alternate PdO or PdS/MMI linkages. This procedure, therefore, does not enable the synthesis of oligonucleosides 7a that are uniformly modified with MMI linkages. This communication reveals a flexible synthetic strategy for constructing AOs containing the MMI backbone in any desirable configuration with the PdO and/or PdS backbone. We have accomplished the synthesis of essential nucleosidic building blocks (1-8), thus enabling us to construct chimeric 7b AOs as potential drugs. The solution phase (SP) methodology described herein is simple to manipulate. The couplings are efficient, and the process is transferable to solid-support (SS) synthesis, which can be further automated. As a demonstration, chimeric oligomers have been assembled on SS utilizing a standard DNA synthesizer. To prepare an oligonucleoside connected via MMI linkages only, four essential nucleosidic units (1, 2, 5, 6) were synthesized. 2′-Deoxy-5′-O-phthalimidonucleosides 8 1a-d served as a precursor for the 3′-terminal unit. The nucleosides 1a-d were successfully anchored onto the SS (CPG) via a succinyl linker 9 in good yield (∼35-40 µmol/g). To avoid the side reaction of incoming 3′-CHO nucleosides (5 or 6) with unprotected NH 2 groups left on the CPG, methylation (HCHO/NaBH 3 CN/AcOH) of the SS provided fully protected CPG units 2a-d. Alternatively , CPG units 2a-d can be prepared from the commercial CPG loaded with 5′-O-DMT deoxynucleosides in three steps. For example, CPG anchored with 5′-O-DMT thymidine was treated with acid to remove the DMT group, followed by a Mitsunobu reaction, 10 and capping off the CPG NH 2 groups via methylation provided 2a (30 µmol/g). The bifunctional units 4a-d were prepared from 3′-C-styrene nucleosides 11 3a-d. Mitsunobu reaction 12 of 3a-d provided the 5′-O-phthalimido-3′-C-styrene nucleosides 4a-d in excellent yields. One-pot oxidative cleavage (OsO 4 /NaIO 4) of 4a-d gave 5a-d, generating the 3′-CHO functionality. Syntheses of the 5′-terminal units 6a,c,d have been published. 11 Preparation of 2′-deoxycytosine derivative 6b was accomplished via triazolation and amination procedures. 13 Nucleoside 7 was prepared from 3a via 5′-O-FMOC protection 14 and oxidative cleavage of the 3′-C-styrene group. Phosphitylation 14 of 1a furnished 8 in 70% yield. SP synthesis of T 4 was accomplished in the following manner. Coupling 4 of 9 with 5a gave an oxime dimer (12, R) Phth, R′) TPS, n) 1, B) T), which on hydrazinolysis (H 3 CNHNH 2) furnished 5′-O-NH 2-oxime dimer 14 (R) NH 2 , R′) TPS, n) 1, B) T). Another round of coupling of 5a with 14, followed by hydrazinolysis, provided an oxime trimer (14, R) NH 2 , R′) TPS, n) 2, B) T), which coupled with 6a to give an oxime tetramer 15 (n) 3, B) T). Reduction of 15 gave 16 (R) R′) TPS, R′′) H, n) 3), which on methylation followed by TBAF treatment gave MMI tetramer 17 (n) 3, B) T) in 79% overall yield. Coupling of 3′-CHO nucleosides with the 5′-O-NH 2 nucleosides was quick and almost quantitative , thus allowing the manual synthesis of 17 in <8 h. Tetramers T 3 C and T 2 CT were assembled in a similar manner in high yields utilizing appropriate building blocks. The general (1) Selected books and reviews published in 1995: (a) Therapeutic Applications of Oligonucleotides; Crooke, S. T., Ed.; R. G. (5) (a) Mohan, V.; Griffey, R. H.; Davis, D. R. Tetrahedron 1995, 51, 8655. (b) In addition, substitution of the O3′ by a CH 2 group reduces the ring gauche effects and may enhance conformational stability. See: Roughton, A. L.; Portmann, S.; Benner, S. A.; Egli, M. J. Am. Chem. Soc. 1995, 117, 7249. (6) Sanghvi, Y. S.; Bellon, L.; Morvan, F.; Hoshiko, T.; Swayze, E.; Cummins, L.; Freier, S.; Dean, N.; Monia, B.; Griffey, R.; Cook, P. D. Nucleosides Nucleotides 1995, 14, 1087. (7) (a) We refer to modified oligonucleotides that lack the phosphorus atom in the backbone linkage as oligonucleosides. (b) Chimeric AOs are oligomers that contain more than one type of modifications to create a gap for RNase H activity. (8) Perbost, M.; Hoshiko, T.; Morvan, F.; Swayze, E.; Griffey, R. H.; Sanghvi, Y.S.