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.