Automated Chemical Synthesis of Long Oligoribonucleotides Using 2'-0-silylated Ribonucleoside S'-O-phosphoramidites on a Controlled-pore Glass Support: Synthesis of a 43-nucleotide Sequence Similar to the 3'-half Molecule of an Escherichia Coli Formylmethionine tRNA

Abstract

The synthesis of silyl-protected ribonucleoside 3'-0-phosphoramidites and the preparation of the controlled-pore glass supports needed for the solid-phase chemical synthesis of oligoribonucleotides are described. These reagents were evaluated in the synthesis of a series of oligomers consisting of the pentadecameric homopolymers of adenosine, cytidine, guanosine, uridine, and various sequences of mixed-base composition. Different reactivities were observed for the morpholino- and (diisopropylamino)phosphoramidites under actual synthesis conditions, and these were attributed to the relative ease of their activation by tetrazole as determined by31P NMR. A comparison of the methyl- and cyanoethyl-protected phosphoramidites indicated that, although the cyanoethyl group was easier to remove, there was no significant difference between the two in terms of the quality of the final sequence. The purity of the 3'-phosphoramidites is easily determined by NMR. A full protocol for the deprotection, handling, and purification of synthetic oligoribonucleotides is also described. The results of these studies were applied to the successful chemical synthesis of the 43-mer 5'-CAU AAC CCG AAG AUC GUC GGU UCA AAU CCG GCC CCC GCA ACC A-3 corresponding to the 3'-terminus of a formylmethionine (fMet) tRNA of Escherichia coli, in which the modified bases have been substituted by their unmodified parent nucleosides. The 43-mer was fully characterized by polyacrylamide gel electrophoresis, enzymatic RNA sequencing, HPLC analysis of an enzymatic digest, and terminal nucleotide analysis, both 5' and 3', using enzymatic digestion followed by two-dimensional chromatography. The effectiveness of alkylsilyl ethers for the protection of the 2'-hydroxyl of the ribose ring, when used in conjunction with the phosphoramidite method for the formation of the phosphotriester linkages in RNA synthesis, is clearly established. © 1987, American Chemical Society. All rights reserved.