BOODMTrONNNNHNNNHNONNNNONPEN44B=Thy45,48B= 46,49B= 47,50B= PORN(iPr)2NPEOCHNPEOCNPEOCNO2NO2OONPE= NPEOC=45-47R=CE44,48-50R=NPE
Figure 12. p-nitrophenylethyl (NPEOC/NPE) protected nucleoside phosphoramidites.
Oligonucleotides [d(GCTPrAGC) and d(GCTBuAGC)] bearing ammonia sensitive O-4-propyl (TPr) and O-4-butylthymidines (TBu) have been synthesized on a solid phase using p- nitrophenylethyl (NPE/NPEOC) nucleobase protections (45-47, Figure 12).54-56 The oligonucleotides are assembled on o-nitrophenylcarbonate derivatized resin (9) and then two-step deprotection procedure is carried out. The cyanoethyl groups of the phosphates are first selectively removed by a mixture of 40% triethylamine in pyridine and then the resins are subjected to a mixture of 0.5 M DBU in pyridine, which cleaves the p-nitrophenylethyl protections and concomitantly releases the target oligonucleotides. As a contrast to acrylonitrile, the released p-nitrostyrene is not so strong alkylation agent, but the clear limitation of this procedure is the linker choice, since the product is released to a strong basic media.
More recently cyanoethyloxycarbonyl protections have been applied for the synthesis of oligonucleotides bearing (5R)-5,6-dihydro-5-(1-oxo-2-phenylethyl)thymidine unit (cf. 55 in Figure 13).57 This dihydropyrimidine is prone to a retrocondensation reaction in alkaline condition (e.g. 50 mM K2CO3 in anhydrous MeOH and 0.5 M DBU in CH3CN used to cleave the Pac and NPEOC protections). The standard phosphoramidite coupling procedure is used for the chain assembly, applying a temporal 5’-silyl protection. Demethylation of the phosphotriesters is accomplished with a treatment of disodium-2-carbamoyl-2-cyanoethylene-1,1,-dithiolate trihydrate (dccdt) (57), the cyanoethyl carbamates are removed by a mixture of triethylamine in DMF (1:1, v/v, 16 h at 55°C) (58) and then the fully deprotected oligonucleotide is released by photolysis. It may be notable that the unsubstituted cyanoethyloxycarbonyl group may be used for the guanine base (54), but the more base labile a,a-dimethylated derivative is required for adenine (53) and cytosine (52).
