Fast Deprotection of Synthetic Oligodeoxyribonucleotides Using Standard Reagents under Microwave Irradiation

Abstract

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dABz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

“…Generally separations have been performed using C 18 columns (Gaus et al, ; Kurata et al, ; Rentel et al, ) such as YMC (An et al, ; Capaldi et al, ), Acquity BEH (Chen & Bartlett, ; McGinnis et al, ; Smith & Beck, ), Acquity UPLC OST (Roussis, ), Xbridge OST (Roussis, ), XTerra® MS (Farand & Beverly, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Li et al, ), NovaPak (Metelev & Agrawal, ), Inertsil ODS‐2 (Suzuki et al, ) and Zorbax Extend‐C 18 columns (Culf et al, ). XBridge BEH columns have shown higher‐quality separations while retaining long‐term stability and reproducibility when compared with other columns such as Xterra MS, Xbridge OST and Clarity Oligo‐RP columns (Nikcevic et al, ).…”

“…Moreover using a buffering system containing the ion pairing agents and hexafluoroisopropanol and triethylamine (Farand & Beverly, 2008;Fountain et al, 2003;Gilar, 2001;Gilar et al, 2003Gilar et al, , 2002Li et al, 2010;Liu et al, 2012;Nikcevic et al, 2011) and diisopropylethylamine and hexafluoroisopropanol (Chen & Bartlett, 2012b;McGinnis et al, 2013) has provided significant chromatographic separation and higher signal intensity, as well as higher selectivity and chromatographic resolution (Apffel et al, 1997;Basiri & Bartlett, 2014;McGinnis et al, 2012). Nonion-pairing conditions have also been applied using acetonitrile with formic acid or ammonium acetate (Cohen et al, 1993;Culf et al, 2008) or NH 4 HCO 3 , EDTA (Gilar et al, 2002) or ammonium formate Suzuki et al, 1994).…”

“…Furthermore, a comparison of the Kromasil C 18 , C 8 and C 4 RP-HPLC columns showed that the best results for the determination of the major impurities from lipophilic-conjugated PS oligonucleotides were obtained when using the C 4 column (Liu et al, 2012). Better results have been observed when coupling IP-RP-HPLC with mass spectrometry using ESI-MS in the negative ion mode (Capaldi et al, , 2004Culf et al, 2008;Fearon et al, 1995;Gaus et al, 2005;Gilar, 2001;Kurata et al, 2006;Nikcevic et al, 2011;Rentel et al, 2005;Roussis, 2015) and when using higher-resolution or multistage MS n instruments such as ESI-TOF-MS (Chen & Bartlett, 2012b;Farand & Beverly, 2008;Fountain et al, 2003;Gilar et al, 2003;McGinnis et al, 2013), ESI-IT-MS (Farand & Beverly, 2008;Smith & Beck, 2016) and MALDI-TOF-MS (Ball & Packman, 1997;Cui et al, 2008;Li et al, 2010;Liu et al, 2012;Yang et al, 2002). Coupling IP-RP-HPLC with mass spectrometry maximizes both chromatographic separation and mass spectrometric sensitivity and detectability, which enable in depth characterization of the existing impurities.…”

“…An RP‐HPLC method has been developed for the determination of inosine produced from the enzymatic deamination of adenosine (Damha & Ogilvie, ). Moreover RP‐HPLC, ESI‐MS and capillary electrophoresis have been adopted for the investigation of a microwave irradiation method used for fast deprotection of synthetic oligodeoxyribonucleotides and the detection of the resulting deaminated products (Culf, Cuperlovic‐Culf, Laflamme, Tardiff, & Ouellette, ).…”