[reaction: see text] CCA-pcb (cytidylyl-(3'5')-cytidylyl-(3'5')-3'(2')-O-(N-(6-D-(+)-biotinoylaminohexanoyl)-L-phenylalanyl)adenosine), a ribosomal P-site substrate, was synthesized by phosphoramidite chemistry in 26 steps with an overall yield of 18%, starting from biotin. The synthesis relies on the judicious selection of orthogonal silyl protecting groups for the 5'-hydroxyls and acid-labile protecting groups (DMTr, AcE, and MeE) at other reactive sites to ensure the intactness of the labile ester. Both 3'-esterification and nucleotide coupling were accomplished by in situ activation with imidazolium ions.