Aminopropyl-CPG; 500 Å

A general method is described for the solid phase supported synthesis of DNA containing 3'-terminal phosphodiesters modified with linkers bearing either amino, thiol, or hydroxyl groups. These products are all made from a common intermediate, obtained by the reaction of trimellitic anhydride chloride with aminopropyl CPG. The anhydride-derivatized support was then reacted with three appropriate bifunctional spacers, giving DMT-protected hydroxyl solid supports bearing the masked functionality as an ester, amide, or thioester. DNA synthesis was then performed, followed by ammonia cleavage and deprotection, giving the hydroxyl-, amino-, or thiol-functionalized DNA 3'-phosphate diesters, respectively. Test mononucleotides synthesized with each of the new supports were identical with control mononucleotides made with 5'-immobilized nucleosides and alkylhydroxyl, alkylamino, and alkylthio phosphoramidites. The new supports were then used to prepare several 3'-modified oligonucleotides, which were characterized by gel electrophoresis, HPLC, and MALDI mass spectroscopy. The amino- and thiol-functionalized DNAs were conjugated with chromophores, and purification of these products was facilitated by use of reversed phase cartridges.

We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual delta 5-3 beta-hydroxysteroid sulphates. A microcolumn with immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatography (HPLC). The reduced nicotinamide-adenine dinucleotide produced from delta 5-3 beta-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC-immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.