Phosphoramidite compound identification and impurity control by Benchtop NMR

Brief background on phosphoramidite: importance, applications, and composition


The development of nucleoside phosphoramidite in 1981 marked a big milestone for the field of oligonucleotide synthesis research. It opened the doors for the development of a wide range of molecular biology applications including gene sequencing methods that enable the sequencing of the human genome.

1,2 Besides playing a critical role in molecular biology applications, oligonucleotides have also been developed to be Active Pharmaceutical Ingredients (APIs).3

Figure 1. Chemical structure of an example phosphoramidite. (The chemical structure was produced using

Phosphoramidites are synthons, or building blocks used for the synthesis of DNA, or RNA oligonucleotides. The chemical structure of a phosphoramidite is shown in Figure 1. Each phosphoramidite is composed of a 5’-O-DMT sugar moiety (2’-deoxyribose for DNA, or 2’-O-protected ribose for RNA), a protected nitrogenous base (adenine, guanine, cytosine, and thymine for DNA, or adenine, guanine, cytosine, and uracil for RNA), and most importantly, a phosphoramidite moiety on the 3’ position of the sugar moiety. The phosphoramidite moiety consists of a 2-cyanoethyl group that protects the second hydroxyl on the phosphorus (III) center, and a diisopropylamino group serves as a leaving group to be removed during the synthesis and links the phosphoramidite to the growing oligonucleotide.1 This phosphorus (III) center is chiral in nature, so the phosphoramidite normally exists as a mixture of two diastereomers

Characterization of phosphoramidite in oligonucleotide synthesis: 1H and 31P NMR in the analysis of phosphoramidite

The synthesis of oligonucleotides is often carried out on solid-supported flow systems that are computer-controlled and fully automated. The reagents are flown through the system sequentially to perform the reactions on the solid support. The correct sequence of phosphoramidite addition in the synthesis of oligonucleotides is critical for the identity of the oligonucleotides. One missing, duplicated, or swapped phosphoramidite can result in the complete loss of the efficacy of a drug or a failed molecular biology application. Therefore, to ensure the integrity of the oligonucleotide synthesis, it is important to have a way to quickly characterize the phosphoramidites before they are added into the synthesis system.

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique that can provide the structural information needed for compound identification. The most widely used nuclei in NMR is 1H. A set of 18 phosphoramidites was analyzed by NMR on the Spinsolve 80 MHz Phosphorous. Their 1D 1H spectra measured at 80 MHz are shown in Figure 2. As seen in this figure, the 1H NMR spectra of phosphoramidites are quite complex with multiple signals ranging from aliphatic (protons on alkyl groups on protecting groups) to aromatic (protons on nitrogenous bases and protecting groups). Analyzing the spectra revealed a region with diagnostic signals that can be used for compound identification. This region spans from 5.0 ppm to 6.4 ppm, with the signals from the anomeric proton of the sugar moiety. In the library of phosphoramidites that we investigated here, this region shows signals with splitting patterns and chemical shifts that are unique to each individual phosphoramidite.

Figure 2. 1D 1H spectra of 18 phosphoramidites measured on a Spinsolve 80 MHz using 16 scans and 30 seconds repetition time, resulting in 8 minutes total measurement time.

Besides 1H NMR, 31P NMR is also very popular with the high natural abundance of the nuclei (100%) and the much larger chemical shift window. For the phosphoramidite analysis, in contrast with 1H NMR, the 31P NMR spectra of phosphoramidites are a lot simpler to interpret. The stack plot in Figure 3 shows 1D 31P spectra at 80 MHz. The phosphoramidite signals resonate in the region between 140 ppm to 155 ppm.

           Figure 3. 1D 31P spectra of 18 phosphoramidites measured on a Spinsolve 80 MHz system.

Each phosphoramidite contains one chiral phosphorus (III) center, therefore phosphoramidites normally exist as a mixture of two diastereomers. Using the proton-decoupled phosphorus NMR pulse sequence, the 31P signals appear as singlets in the zoomed-in spectra (Figure 4a). In case decoupling is not applied, two singlets can be observed in the 31P NMR spectra except in some cases (LNA-T, LNA-A, and DMT-2’’O-MOE-rMeU) where the two signals overlap. As seen in the stack plot (Figure 4a), each phosphoramidite has unique chemical shifts for its 31P NMR signals. The large range of chemical shift and the simplicity of the NMR signals (singlets) makes 31P NMR ideal for rapid and automated compound identification.

A second important application when investigating phosphoramidites is the identification and quantification of potential impurities in the samples. Moreover the analysis of NMR signals stemming from sample degradation and thus to check on long term stability of the phosphoramidites is equally crucial. Figure 4b shows the zoomed region between 50 and -10 ppm with a vertical zoom factor of 100, which can easily be noted by comparing the noise levels. This region is characteristic for different unwanted hydrolyzation products of phosphoramidites.

Figure 4. Zoomed-in 1D 31P spectra of 18 phosphoramidites showing chemical shift region between a) 142 ppm to 155 ppm and b) 50 ppm to -10 ppm (with vertical zoom factor 100).

A database of 1D 31P NMR spectra of phosphoramidites can easily be assembled to aid in an automated compound identification process. 31P NMR provides a straightforward and quick method for characterizing phosphoramidites in the oligonucleotide synthesis process to ensure the correct sequence and the identity of the oligonucleotides.

The Spinsolve software offers a new database plug-in (qIDsolve) where users can add their own substances and create a database following very easy steps. The database gives the opportunity to set up compound categories and add multiple spectra for each of the individual substances.



We would like to thank BioSpring GmbH for providing the phosphoramidite samples.


  1. L. Beaucage, M.H. Caruthers. Tetrahedron Lett., 1981, 22, 1859-62
  2. A. Hughes, A.D. Ellington. Cold Spring Harb Perspect Biol. 2017 Jan; 9(1): a023812
  3. I. Andrews, F.D. Antia, S.B. Brueggemeier, L.J. Diorazio, S.G. Koenig, M.E. Kopach, H. Lee, M. Olbrich, A.L. Watson. J. Org. Chem. 2021, 86, 49-61