Nucleic Acids Research, 2000, Vol. 28, No. 3 818-825
© 2000 Oxford University Press
Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group
Centre de Biophysique Moléculaire, UPR 4301 CNRS and Université d’Orléans, Rue Charles Sadron, 45071 Orleans Cedex 02, France
A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (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 fluoresceinylated oligonucleotide; the 5'-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an N
-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate.
* To whom correspondence should be addressed. Tel: +33 2 38 25 55 97; Fax: +33 2 38 63 15 17; Email: asseline@cnrs-orleans.fr
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. d. Diesbach, F. N'Kuli, C. Berens, E. Sonveaux, M. Monsigny, A.-C. Roche, and P. J. Courtoy Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking Nucleic Acids Res., April 1, 2002; 30(7): 1512 - 1521. [Abstract] [Full Text] [PDF]
|
|