A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5 '-Tamra DNA

6-Carboxy Tamra 1 was conjugated to:4-hydroxypiperidine with BOP and N-meth ylmorpholine, and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl tetraisopropylphosphorodiamidite to give 5 - [N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite)]-Tamra carboxa mide 3. Solutions of 3 were coupled onto the 5'-hydroxyl of solid-phase-sup ported DNA fragments with standard amidite coupling techniques. Cleavage an d deprotection with aqueous tert-butylamine cocktail gave 5-Tamra-functiona lized DNA as well as an additional compound without the Tamra chromophore. A mass spectrum of this product showed the incorporation of tert-butylamine .: The extra product was completely suppressed by including a 5 min acetyla tion step after coupling. A model study of 3 coupled onto thymidine-functio nalized CPG showed similar results. NMR and mass spectra of cleaved product s confirmed;the addition of tert-butylamine to the minor product. Coupling a Tamra active ester onto T CPG; which was previously coupled with N-(4-met hoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) A produc ed. the same major Tamra-bearing product, which coeluted on reverse phase H PLC with the major product generated with 3.