2-benzamidoethyl group - A novel type of phosphate protecting group for oligonucleotide synthesis

A number of 5'-O-(4,4'-dimethoxytrityl)thymidine N,N-diisopropylamino phosp horamidites protected at P(III) with derivatives of 2-benzamidoethanol were synthesized and incorporated into synthetic oligonucleotides. Depending on substitution patterns at the alkyl chain, amido group, and phenyl ring, th e time required for removal of these protecting groups using concentrated a mmonium hydroxide varied from 48 h at 55 degreesC to 25 min at 25 degreesC. Of the 11 groups studied, 2-[N-isopropyl-N- (4-methoxybenzoyl)amino]ethyl- (H) and omega-(thionobenzoylamino)alkyl protections (I and K) were most ea sily removed. Derivatives of the 2-[N-methyl-N-benzoylamino]ethyl group (E- G) demonstrated moderate stability, but those of the 2-(N-benzoylamino)ethy l group (A-C) were the most stable. For the most reactive group, H, a phosp hitylating reagent, bisamidite 60, was synthesized and used in the preparat ion of four deoxynucleoside phosphoramidites 28 and 65-67, plus the 2'-O-(2 -methoxyethyl)-5-methyluridine phosphoramidite 68. All of these novel build ing blocks were successfully tested in the preparation of natural, 20-mer o ligonucleotides and their phosphorothioate analogues. With the model phosph otriester 37, the mechanism of deprotection was studied and revealed, in th e case of group H, a pH-independent formation of the 2-oxazolinium cation 4 7. Under aqueous conditions, 47 gave 54, which in turn was converted in the presence of ammonia to a number of identified products. It is important to note that none of the products formed was reactive toward the oligonucleot ide backbone or nucleic bases. Thus, a general strategy for protection of i nternucleosidic phosphodiester groups is described, which may also find app lication in synthetic organic chemistry of phosphorus(III) and (V).