تفاعل #2321903

معادلة التفاعل

iodoacetamide

2′-chloro-deoxyguanosine

Iodoacetic anhydride

phosphoramidite

ICN

p-aminobenzoic acid

→

4-iodoacetylaminobenzoic acid

المذيبات

dioxane

ظروف التفاعل

المعالجة

1. 1
   أخرىduring its synthesis

الإجراء التجريبي

An oligonucleotide, 20 bases in length, comprised of 2′-chloro-deoxyguanosine bases, acts as the sequence-specific component of the first reagent. This oligonucleotide is prepared on an automated oligonucleotide synthesizer using phosphoramidite chemistry. A primary amino group is introduced at the 3′-terminus of the oligonucleotide during its synthesis using the reagent 3'Amine O CPG (Clontech Laboratories, Inc., Palo Alto, Calif.). The primary amine at the 3′-terminus of the oligonucleotide is then reacted with SIAB (succinimydl 4-iodoacetyl aminobenzoate, Pierce, Rockford, Ill.) to introduce an iodoacetamide residue, according to Weltman, et al. (Weltman, J. K., et al., “N-Succinimidyl(4-lodoacetyl)Aminobenzoate: A New Heterobifunctional Crosslinker,” Biotechniques 1:148-152 (1983)). Weltman et al. disclose in their “MATERIALS AND METHODS” section the preparation of N-succinimidyl(4-iodoacetyl)amino-benzoate (SIAB). SIAB is prepared as outlined in FIG. 1 of Weltman et al. (figure not shown below). Iodoacetic anhydride, 354 mg (1 mmole) purchased from ICN Pharmaceuticals-K and K Laboratories, is reacted with 68.6 mg (0.5 mmoles) of p-aminobenzoic acid in 7.5 ml of dioxane, for 5 hours at room temperature in the dark, and then for 2 days at 4 degrees. A white flocculent intermediate (4-iodoacetylaminobenzoic acid) is isolated by centrifugation and triturated with 0.5 ml of diethyl ether three times. The resulting white intermediate is dried with hot air with a yield of 160 mg. Dicyclohexycarboiimide (86.2 mg; 0.4 mmoles) is added to a solution of the intermediate (128 mg; 0.4 mmoles) and N-hydroxysuccinimide (48.5 mg; 0.4 mmoles) in 3.35 ml of tetrahydrofuran (THF) and placed at 4 degrees for 20 hours. A precipitate is removed and the supernatant liquid is recovered and evaporated to dryness and triturated with diethyl ether. Pale yellow crystals of SIAB are recovered, yield 135 mg (80%), melting point 172-175 degrees. The yellow crystals of SIAB are recrystallized from methanol and washed twice with diethyl ether, yielding white crystals of SIAB, melting point 194-196 degrees with decomposition. The calculated molecular weight of SIAB is 402 daltons. The iodoacetamide-derivatized oligo(dG) is then reacted with the thiol group on the derivatized Fab′ fragment, thereby forming the first reagent of the invention.

المصدر