Cy3 phosphoramidite

Two cyanine derivatives, Cy3 and Cy5 phosphoramidites, are the most popular fluorescent labeling reagents for preparing fluorescence-labeled oligonucleotides. Cyanine dyes are normally added once at the 5'-terminus and the MMT group should be removed on the synthesizer. The absorbance of the MMT cation (yellow) is noticeably different from the DMT cation (orange), thus the trityl absorbance can be used to monitor oligo coupling. Cyanine dye phosphoramidites have also been used successfully adjacent to the 3'-terminus. Deprotection of oligos containing cyanine dyes may be carried out with ammonium hydroxide at room temperature, regardless of the base protecting groups on the monomers used. If ammonium hydroxide is used at elevated temperature, Cy3 and Cy3.5 are more stable than Cy 5 and Cy 5.5. However, it is always prudent to use monomers with base labile protecting groups to limit the exposure time to 2 hours or less at 65°C during deprotection.