Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support

Area of Science:

• Solid-state Nuclear Magnetic Resonance (NMR) Spectroscopy
• Oligonucleotide Chemistry
• Biophysical Chemistry

Background:

• Controlled Pore Glass (CPG) is a common solid support for oligonucleotide synthesis.
• 31P NMR is sensitive to the phosphorus environment, crucial for monitoring synthesis.
• Distinguishing subtle structural variations in oligonucleotides is essential for quality control.

Purpose of the Study:

• To evaluate the utility of 31P cross-polarization magic angle spinning (CPMAS) NMR for analyzing oligonucleotides synthesized on CPG.
• To determine the resolution limits of 31P CPMAS NMR for detecting structural and chemical modifications.
• To monitor the oligonucleotide synthetic cycle using 31P CPMAS NMR.

Main Methods:

• Acquisition of 31P CPMAS NMR spectra for various di- and tri-nucleotides on CPG.
• Analysis of spectral differences related to phosphorus oxidation state, protecting groups, and chemical modifications.
• Examination of samples at different stages of the oligonucleotide synthetic cycle.

Main Results:

• 31P CPMAS NMR clearly distinguished phosphorus oxidation states (phosphate vs. phosphate), presence/absence of cyanoethyl protecting groups, and phosphorothioate modifications.
• The technique could not resolve differences in ribose attachment position (2'5' vs. 3'5') or nucleotide bases (adenine, uridine, thymine).
• The cyanoethyl protecting group was observed to be removed during oligonucleotide chain assembly.

Conclusions:

• 31P CPMAS NMR is a valuable tool for assessing key chemical features of oligonucleotides on solid supports.
• The method's limitations lie in resolving structural details distant from the phosphorus atom.
• 31P CPMAS NMR can effectively monitor the deprotection steps in oligonucleotide synthesis.