A method to accurately determine the extent of solid-phase reactions by monitoring an intermediate in a nondestructive manner.
The application of Fourier transform infrared (FT-IR) spectroscopy to the quantitation of effective nucleoside loading on a solid support, via detection of a reaction intermediate, was developed. 4,5-Dicyanoimidazole (DCI) activator is reacted with a known amount of polystyrene-bound phosphoramidite to form a diisopropylamine-DCI salt complex that is detected by FT-IR spectroscopy at a unique wavenumber of 1104 cm(-1). Based on a linear standard curve, the species' absorbance at 1104 cm(-1) is then directly translated into an appropriate concentration. This concentration characterizes the number of active phosphoramidite sites on the solid support. This method consistently determines the loading extent of nucleosides (deoxy-A/T/G/C) on the solid support to the nearest 0.02 mmol/g. The coupling reaction extent of a deoxy-T derivative with the support-bound phosphoramidite compares well with the nucleoside loading, as determined by the FT-IR-based analytical method. The nucleoside loading was further verified by phosphorus elemental analysis of the 3'-phosphate linker concentration.
Mihaichuk
Jeffery
J
Proligo LLC, Boulder, Colorado 80301, USA.
Tompkins
Christopher
C
Pieken
Wolfgang
W