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03/15/2024 | Press release | Distributed by Public on 03/14/2024 18:14

Analyze of stereoisomer by NMR

Certain organic compounds possess identical molecular structures but exhibit multiple stereoisomers due to differences in their stereochemistry. These stereoisomers often manifest distinct physical properties, reactivity, and physiological activities from one another. Consequently, it becomes essential to differentiate between them. While enantiomers cannot be discerned directly in solution NMR spectra, derivatization into diastereomers enables their discrimination within the NMR spectrum, facilitating the quantification of their respective ratios. This discussion delves into the analysis of cyclic peptides using 1H and 13C NMR techniques. Specifically, a JNM-ECZL500G spectrometer, configured with a 2H channel extension, and a ROYALPROBETM were employed for these experiments, offering the capability of 13C observation alongside simultaneous 1H and 2H decoupling.

D-substitution and diastereomerization reactions under basic conditions　

Cyclo(L-Pro-L-Ala) is known to isomerize under basic conditions, leading to the formation of DLand LLforms, with deuterium incorporation at the H9 position. [1]

Estimation of DL and LLratio by1H NMR　

Fig. 1 displays the 1H spectra of the cyclic peptide cyclo(L-Pro-L-Ala) dissolved in D2O solution (a) and 0.01M KOD/D2O (b). Fig. 1b illustrates the alteration in the 1H spectral pattern and the absence of the H9 signal. Based on the integral ratio of H3 signals in the LLand DLstereoisomers, the LL:DLratio is estimated to be 40:60.

Fig. 1 : 1H NMR spectra
a) D2O solution, b) 0.01M KOD/D2O solution

Effective observation of 13C signals coupled to 2H

The diastereoisomerization reaction induces significant changes in the 13C spectrum, as depicted in Fig. 2. Each 13C signal shown in Fig. 2a undergoes splitting into two signals in Fig. 2b. Additionally, Fig. 3 presents an expanded region focused on C9. It is difficult to spot any signal in Fig. 3b due to 13C-2H splitting. Through simultaneous 1H and 2H decoupling, Fig. 3c reveals the presence of two signals corresponding to the LLand DLforms. This spectrum provides evidence of proper deuteration of the compound.

Fig. 2 : 13C NMR spectra
a) D2 solution, b) 0.01M KOD/D2 solution

Fig. 3 : Expanded 13C NMR spectra focused on C9
a)D2O solution　13C{1H}
b)0.01M KOD/D2O solution 13C{1H}
c)0.01M KOD/D2O solution 13C{1H}{2H}

Sample courtesy of Prof. Takashi Ishizu
(Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University)

Reference

[1] J. Am. Chem. Soc., 96(12), 3985-3989, 1974.

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