Scheme 2.

Synthesis of the nucleoside 5′-triphosphates of TAMRA- and FAM-hx-Pa.

Reagents and abbreviations: (a) (i) CuI, tetrakis(triphenylphosphine)palladium, DMF, triethylamine; (ii) N-(2-propynyl)-6-trifluoroacetamidohexanamide; (b) 4,4′-dimethoxytrityl chloride, pyridine; (c) (i) acetic anhydride, pyridine, (ii) dichloroacetic acid, dichloromethane; (d) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one/dioxane, pyridine, tri-n-butylamine, bis(tri-n-butylammonium)pyrophosphate, DMF, then I₂/pyridine, water, NaHSO₃, NH₄OH; (e) R-N-hydroxysuccinimidyl ester (R = 5-carboxyfluorescein or 5-carboxytetramethylrhodamine)/DMF, 0.1 M NaHCO₃-Na₂CO₃ buffer (pH 8.5), then NH₄OH. Ac: acetyl.