L‐Hydroxyprolinol‐Based Cyanine Phosphoramidites and Solid Supports for the Efficient Synthesis of Quantitative Polymerase Chain Reaction Probes

Four cyanine phosphoramidites (Cy3, Cy3.5, Cy5, Cy5.5) and their corresponding solid supports, featuring a versatile L-hydroxyprolinol linker, were prepared. Both sets of reagents demonstrated excellent performance in automated oligonucleotide synthesis: The newly synthesized phosphoramidites, prepared on a multigram scale, enabled efficient incorporation of 5'- and internal labels, while the cyanine-functionalized solid supports facilitated the incorporation of 3'-labels into olisnucleotides. Under mild deblocking conditions, no significant fluorophore degradation was observed. Destabilization effect of the labels was evaluated via fluorescent melting experiments. A model assay demonstrated that Cy5/BHQ2 fluorescent probes synthesized with these reagents performed comparably in quantitative polymerase chain reaction to probes synthesized from commercially available phosphoramidites.