Dichloro-diphenyl-fluorescein, SIMA (HEX) exhibits virtually identical absorbance and emission spectra to HEX. SIMA (HEX) is much more stable to basic deprotection conditions than HEX and oligonucleotides can be deprotected using ammonium hydroxide at elevated temperatures and even ammonium hydroxide/methylamine (AMA) at room temperature or 65°C for 10 minutes. SIMA absorption maximum was 3 nm blue-shifted compared to HEX at pH 7. The absorbance is broader, so the extinction coefficient is smaller than that of HEX, but when exciting at 500 nm where the absorbance was normalized, the emission was still 90% of HEX and the emission was red-shifted by 5 nm. A second SIMA (HEX) product, SIMA (HEX)-dT, can be used to introduce SIMA (HEX) in the synthetic oligonucleotide sequence, usually as a replacement for the native dT linkage. Again, this product is fully compatible with deprotection schemes using ammonium hydroxide at elevated temperatures or AMA at room temperature and 65°C.
Coupling: 10 minute coupling time recommended
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.