Amidine Protecting Groups for Oligonucleotide Synthesis

Abstract

Amidines have been investigated for protection of deoxyadenosine, deoxycytidine, 5-methyldeoxycytidine, cytidine, and deoxyguanosine. These amidine-protected nucleosides and their 5’-bis(p-anisylphenyl)methyl derivatives were prepared in high yield (79-100%) by simple one-flask procedures, converted to their respective phosphoramidites [3’-0-((diiso-propylamino)methoxyphosphino)] with bis(diisopropylamino)methoxyphosphine, and used in situ for synthesizing d(G-G-G-A-A-T-T-C-C-C). This deoxyoligonucleotide was recognized by EcoRI restriction endonuclease, suggesting that amidines can be used to generate biochemically reactive DNA. Deprotection can be routinely performed by using concentrated aqueous ammonia. However, either ethylenediamine/phenol/water or aqueous ammonia containing ammonium acetate enhances amidine deprotection rates severalfold. In addition to their intrinsic ease of preparation, amidines were shown to have certain advantages when compared to amide-protected nucleosides. (1) Deoxyadenosine when protected with amidines was approximately 20-fold more resistant to depurination than 6-N-benzoyldeoxyadenosine under certain acidic conditions used for synthesizing DNA. (2) Unlike amide-protected cytidine and deoxycytidine, amidine derivatives of this nucleoside are not susceptible to attack on C-4 by primary amines. Thus these amines can be used in conjunction with amidine-protected cytidine and deoxycytidine. (3) The high-yield synthesis of completely protected nucleosides using amidines makes these reagents ideal for syntheses involving modified bases usually available in limited quantities. This concept was demonstrated with 5-methyldeoxycytidine. © 1986, American Chemical Society. All rights reserved.