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 phosphodiester and phosphor-othioate 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/micromol 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 phosphor-amidite 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 proced-ure 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 oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.
Aubert
Y
Y
Centre de Biophysique Moléculaire, UPR 4301 CNRS and Université d'Orléans, Rue Charles Sadron, 45071 Orleans Cedex 02, France.
Bourgerie
S
S
Meunier
L
L
Mayer
R
R
Roche
A C
AC
Monsigny
M
M
Thuong
N T
NT
Asseline
U
U
D000577:Amides / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D013058:Mass Spectrometry; D000432:Methanol / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D063088:Phosphoramides; D010756:Phosphoric Acids / Q000737:chemistry; D021382:Protein Sorting Signals; D011725:Pyridines / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013777:Tetrazoles / Q000737:chemistry; D013873:Thionucleotides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics
D000577:Amides; D000588:Amines; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010710:Phosphates; D063088:Phosphoramides; D010756:Phosphoric Acids; D021382:Protein Sorting Signals; D011725:Pyridines; D013438:Sulfhydryl Compounds; D013777:Tetrazoles; D013873:Thionucleotides; C073762:lysyl-aspartyl-glutamyl-leucine; C005438:phosphoramide; C045574:1H-tetrazole; D019800:Phenol; D016650:Fluorescein-5-isothiocyanate; D000432:Methanol