Improved synthesis of trinucleotide phosphoramidites and generation of randomized oligonucleotide libraries

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5 - 10 g scale each was developed. The procedure starts with condensation reactions of P- components with N-acyl nucleosides, bearing the 3'-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3'-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.