Residual benzamide contamination in synthetic oligonucleotides observed using UV resonance Raman spectroscopy

Spectroscopic methods based on Raman scattering have for many years employed synthetic oligonucleotides in a broad range of applications, either as probes or as model analytes for biophysical investigations. Benzamide is commonly used as a protecting group in the phosphoramidite synthesis of oligonucleotides and, while standard desalting used after synthesis yields sufficiently pure reagents for most assay reactions or other routine uses of the oligomer, it does not completely remove benzamide. We show that the 1609 cm−1 band of residual benzamide contamination can interfere with certain nucleic acid bands, particularly when using excitation wavelengths near 244 nm where the benzamide band is strongly enhanced. For example, the 1609 cm−1 band of benzamide could obscure (or be mistaken as) a weak vibration attributed to an NH2 scissoring. The extent of benzamide contamination in desalted preparations varies considerably among different commercial sources, and hence caution is advised when making direct comparisons of ultraviolet Raman data of oligonucleotides from different sources. Copyright © 2010 John Wiley & Sons, Ltd.