Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5 '-protected thiol function and a 3 '-amino group

A new deprotection procedure enables a medium scale preparation of phosphod iester and phosphorothioate oligonucleotides substituted with a protected t hiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/mu mol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified suppo rt. A linker containing the thioacetyl moiety was manually coupled in two s teps by first adding its phosphoramidite derivative in the presence of tetr azole followed by either oxidation or sulfurization to afford the bis-deriv atized oligonucleotide bound to the support. Deprotection was achieved by t reating the fully protected oligonucleotide with a mixture of 2,2'-dithiodi pyridine and concentrated aqueous ammonia in the presence of phenol and met hanol, This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-en d, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3 '-amino group was reacted with fluorescein isothiocyanate to yield a fluore sceinylated oligonucleotide; the 5'-dithiopyridyl group was then quantitati vely reduced to give a free thiol group which was then substituted by react ion with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjug ate.