Mass Determination of Phosphoramidites

Shinya Harusawa1, Mihoyo Fujitake1, Takushi Kurihara1, Zheng‐yun Zhao2, David M.J. Lilley2

1 Osaka University of Pharmaceutical Sciences, Osaka, Japan, 2 University of Dundee, Dundee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.11
DOI:  10.1002/0471142700.nc1011s26
Online Posting Date:  October, 2006
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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.

Keywords: nucleoside; phosphoramidite; molecular weight; mass spectrometry; matrix

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Table of Contents

  • Basic Protocol 1: Accurate Mass Measurement of Phosphoramidites Using Triethanolamine/NaCl
  • Alternate Protocol 1: Optimized Mass Measurement of Phosphoramidites Using Triethanolamine/NaCl
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Accurate Mass Measurement of Phosphoramidites Using Triethanolamine/NaCl

  • Phosphoramidite to be analyzed (see )
  • Chloroform (>99% [GC]; Aldrich or TCI)
  • Triethanolamine (TEAO; FABMS and LSIMS grade; TCI)
  • Saline (0.9% NaCl in ultrapure water)
  • 2‐µL disposable capillary pipets (e.g., Minicaps; Hirshmann Laborgeräte)
  • Small glass tube
NOTE: Phosphoramidites S.1a‐d were synthesized in the laboratory (Araki et al., , ). Phosphoramidites in Figures 10.11.4 10.11.6 are available from Cruachem ( S.2a, S.2b, S.2d, S.2f, and S.3c), Proligo ( S.2c, S.2e, S.2g, S.3d, S.3f, and S.3h), Glen Research ( S.3a, S.3b, S.3e, S.3g, S.5a 5d, S.6a d, S.7‐16, and S.18), Dharmacon ( S.4a‐d), and Amersham Biosciences ( S.17). Synthesis of S.1e and S.1f is unpublished. Matrices composed of glycerol (G), m‐nitrobenzyl alcohol (NBA), diethanolamine (DEOA), 3:1 dithiothreitol/dithioerythritol (magic bullet, MB), triethanolamine (TEOA), or 1:1 dithiothreitol/α‐thioglycerol (DTT/TG) can be purchased from TCI (Tokyo Kasei Kogyo). Chloroform and TEOA may be purchased from various suppliers.

Alternate Protocol 1: Optimized Mass Measurement of Phosphoramidites Using Triethanolamine/NaCl

  • Microbalance (0.001 mg accuracy; e.g., Mettler ME30)
  • 1‐mL measuring safety pipet (e.g., Pyrex, Iwaki glass)
  • 0.2‐ to 2‐µL, 0.5‐ to 10‐µL, and 5‐ to 50‐µL micropipets (e.g., Finnpipette; Thermo Labsystems)
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Literature Cited

Literature Cited
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