ABSTRACT
Nuclear magnetic resonance spectroscopy (NMR) is an incredibly powerful technique owing to its quantitative nature, broad dynamic range, and nondestructive nature. Furthermore, the versatility that accompanies NMR’s ability to provide atomic resolution is readily apparent for the study of oligonucleotides. NMR methods can be applied to starting materials, intermediates, drug substances, and drug products. Application of NMR to therapeutic oligonucleotides ranges from methods used
13.1 Introduction .......................................................................................................................... 361 13.2 Nomenclature ........................................................................................................................ 362 13.3 Theory ................................................................................................................................... 362
13.3.1 Chemical Shift .......................................................................................................... 362 13.3.2 NMR Active Nuclei ..................................................................................................364 13.3.3 Scalar and Dipolar Interactions ................................................................................ 365
13.4 Practical Considerations ....................................................................................................... 367 13.4.1 Instrumentation ......................................................................................................... 367 13.4.2 Sample Preparation ................................................................................................... 368 13.4.3 Sensitivity and Resolution ........................................................................................ 369 13.4.4 Water Suppression .................................................................................................... 369 13.4.5 Pulse Sequences ........................................................................................................ 369 13.4.6 Relaxation ................................................................................................................. 371
13.5 NMR Methods Used for the Structural Characterization of Reference Standards and Impurities .............................................................................................................................. 371 13.5.1 Identi‰cation Test...................................................................................................... 372 13.5.2 Structural Characterization ...................................................................................... 373 13.5.3 SiRNA Titration ........................................................................................................ 375 13.5.4 Phosphorothioate Oligonucleotides .......................................................................... 377 13.5.5 Phosphoramidite Starting Materials ......................................................................... 378 13.5.6 Tertiary Structure Determination ............................................................................. 382 13.5.7 Other Applications: LC-NMR, DOSY, and T2-Edited Experiments ....................... 382
13.6 Summary .............................................................................................................................. 383 References ...................................................................................................................................... 383
for the structural characterization of reference standards and impurities to the use of validated methods used for the testing and release of starting materials and products.
