Mass Determination of Phosphoramidites

Abstract

Nucleoside phosphoramidites are the most widely used building blocks in contemporary solid-phase synthesis of oligonucleotides. The accurate molecular weight measurements of such molecules, which are acid-labile compounds, may be easily determined by mass spectrometry using a matrix system, triethanolamine/NaCl, on a liquid secondary ion mass spectrometer (LSIMS) or fast-atom bombardment (FAB) MS equipped with a double-focusing mass spectrometer. The present method rapidly and easily measures the accurate molecular weights of various phosphoramidites as adduct ions [M+Na]+ with an average mass error smaller than 0.4 ppm, allowing determination of the formulas of the phosphoramidites in place of elemental analysis. Further, it was found that intensities of molecular-related ions could be enhanced to the highest degree by adjustment of the molar ratio of phosphoramidite and NaCl, fixing the amount of triethanolamine on LSIMS, making the present method a powerful tool for identification of phosphoramidites by mass spectrometry.

Synthesis of Novel C4-Linked C2-Imidazole Ribonucleoside Phosphoramidite and Its Application to Probing the Catalytic Mechanism of a Ribozyme

“…31 P NMR data of 1 indicated a pair of P diastereoisomers at δ 149.2 and 150.0 ppm (CDCl 3 ). We recently reported a reliable MS measurement of nucleoside or non-nucleoside phosphoramidites using a matrix system [triethanolamine (TEOA)−NaCl] on LSIMS or FABMS equipped with a double-focusing mass spectrometer . The present method successfully revealed the molecular-related ion of 1 at m / z 920.4336, leading to the chemical formula C 49 H 64 N 5 O 9 P + Na.…”

“…The resulting mixture was stirred at 40 °C for 20 min and then evaporated. The residual oil was subjected to NH/silica gel chromatography using a solvent system (30% and then 50% EtOAc in hexane) to give 1 (39.1 mg, 72%) as a white foam: R f = 0.73, 0.63 (EtOAc); 1 H NMR (CDCl 3 ) δ 1.11−1.18 (overlapping peaks, 23H), 1.94 (m, 1H), 2.08 (m, 1H), 2.28 (t, 0.8H, J = 6.2 Hz), 2.50−2.90 (m, 5.2H), 3.07 (dt, 1H, J = 10.3, 3.4 Hz), 3.26 (dd, 0.6H, J = 10.3, 3.4 Hz), 3.34 (dd, 0.4H, J = 10.3, 3.4 Hz), 3.46−3.91 (m, 5H), 3.78 (s, 6H), 3.98 (m, 1H), 4.13 (m, 1H), 4.26 (m, 1H), 5.76 (s, 2H), 6.82 (m, 4H), 7.15−7.49 (m, 10H), 7.59 (s, 1H); 31 P NMR (CDCl 3 ) δ 149.2, 150.0; IR (film, cm −1 ) 1735 (CO−O), 2250 (CN); HRMS (FABMS: TEOA+NaCl) calcd for C 49 H 65 N 5 O 9 P (M + H) + 898.4520, found 898.4515; calcd for C 49 H 64 N 5 O 9 PNa (M + Na) + 920.4339, found 920.4336.…”