Separation of the α-and β-Anomers of Carbohydrates by Diffusion-Ordered NMR Spectroscopy

This article describes the successful application of the DOSY method for the separation and analysis of the αand β-anomers of carbohydrates with different diffusion coefficients. In addition, the DOSY method was found to effectively separate two kinds of glucopyranosides with similar aglycon structures from a mixture.


Introduction
High-resolution nuclear magnetic resonance (NMR) spectroscopy has become an excellent established tool for determining the molecular structures and conformations of compounds while preserving the sample integrity.In addition, pulsed gradient spin echo (PGSE) NMR is recognized as a powerful technique for the determination of diffusion coefficients and the separation of different species in a mixture on the basis of their diffusion coefficients [1].Diffusion-ordered NMR spectroscopy (DOSY), which displays the PGSE NMR data in a two-dimensional spectrum, is a practical experiment for separating the 1 H NMR spectra of different species [2].In addition to the DOSY separation of a mixture, the DOSY method has been widely used for the characterization of high-molecular-weight polymeric compounds and the identification of supramolecular structures [3][4][5][6][7][8].However, the DOSY method has generally failed to identify the isomeric species of similar size and structure because of their similar diffusion coefficients.Therefore, recent studies on the DOSY technique have focused on developing strategies for the separation of isomeric species [9][10][11][12][13][14].The DOSY method has been also applied to carbohydrate chemistry as a tool for the separation and analysis of mono-, di-, oligo-, and polysaccharides, as well as for the structural analysis of metal-complexed carbohydrates [15][16][17][18][19][20].However, reports on the application of the DOSY method for the separation of carbohydrate anomeric isomers are still scarce.With an aim of increasing the utility and applicability of the DOSY method in carbohydrate chemistry, we tackled its evaluation in the separation and analysis of the αand β-anomers of carbohydrates.

Results and Discussion
The DOSY analysis for the isomer separation in a mixture of αand β-anomers was investigated using several kinds of carbohydrate derivatives of glycopyranosides and glycopyranoses, as shown in Figure 1.

DOSY Separation of the α-and β-Anomeric Isomers of Glycopyranosides
The DOSY separation of the anomeric isomers in a mixture of 10 mM phenyl β-glucopyranoside (β-PhGlc) and 10 mM phenyl α-glucopyranoside (α-PhGlc) was firstly investigated.Figure 2 shows the DOSY spectrum of the mixture of α-PhGlc and β-PhGlc in D2O at 30 °C, together with the individual 1 H NMR spectra of β-PhGlc and α-PhGlc in D2O.In the DOSY spectrum, two different species with diffusion coefficients (D) of 5.9 × 10 −10 m 2 •s −1 and 5.6 × 10 −10 m 2 •s −1 could be identified, whose resonances corresponded to the 1 H NMR spectrum of β-PhGlc and α-PhGlc, respectively.It was thereby found that the apparent difference between the diffusion coefficients of β-PhGlc and α-PhGlc allow the DOSY separation of these glucopyranoside anomers.

DOSY Separation of the α-and β-Anomeric Isomers of Glycopyranoses
Glycopyranoses are known to undergo mutarotation; they interconvert their αand β-anomers in water and an equilibrium mixture of the two forms is achieved.Figure 3 displays the mutarotation of D-glucopyranose (Glc).The 1 H NMR spectrum of Glc at a concentration of 20 mM in D 2 O indicated that the anomer ratio of Glc was ca.1:1.We investigated whether the DOSY method could separate the individual 1 H NMR spectra of the anomeric isomers in an equilibrium mixture of α-Glc and β-Glc.
The DOSY spectrum of a 20 mM solution of Glc in D 2 O at 30 • C revealed that two species with different diffusion coefficients (D) of 7.6 × 10 −10 m 2 •s −1 and 5.8 × 10 −10 m 2 •s −1 were present, the former corresponding to the 1 H NMR spectrum of α-Glc, and the latter to the 1 H NMR spectrum of β-Glc, as can be seen in Figure 4. We found that the difference between the diffusion coefficients of α-Glc and β-Glc was sufficient to separate the two anomeric isomers of Glc by using the DOSY technique.

DOSY Separation of the α-and β-Anomeric Isomers of Glycopyranoses
Glycopyranoses are known to undergo mutarotation; they interconvert their α-and β-anomers in water and an equilibrium mixture of the two forms is achieved.Figure 3 displays the mutarotation of D-glucopyranose (Glc).The 1 H NMR spectrum of Glc at a concentration of 20 mM in D2O indicated that the anomer ratio of Glc was ca.1:1.We investigated whether the DOSY method could separate the individual 1 H NMR spectra of the anomeric isomers in an equilibrium mixture of α-Glc and β-Glc.The DOSY spectrum of a 20 mM solution of Glc in D2O at 30 °C revealed that two species with different diffusion coefficients (D) of 7.6 × 10 −10 m •s −1 and 5.8 × 10 −10 m 2 •s −1 present, the former corresponding to the 1 H NMR spectrum of α-Glc, and the latter to the 1 H NMR spectrum of β-Glc, as can be seen in Figure 4. We found that the difference between the diffusion coefficients of α-Glc and β-Glc was sufficient to separate the two anomeric isomers of Glc by using the DOSY technique.Next, the DOSY separation of the anomeric isomers in a mixture of 10 mM α-arbutin (p-hydroxyphenyl α-glucopyranoside) and 10 mM β-arbutin in D2O at 30 °C was similarly investigated.The two glucopyranoside anomers-which exhibited diffusion coefficients (D) of 5.9 × 10 −10 m 2 •s −1 (α-arbutin) and 5.8 × 10 −10 m 2 •s −1 (β-arbutin), respectively-were also successfully separated by the DOSY technique, as shown in Figure S1.

DOSY Separation of the α-and β-Anomeric Isomers of Glycopyranoses
Glycopyranoses are known to undergo mutarotation; they interconvert their α-and β-anomers in water and an equilibrium mixture of the two forms is achieved.Figure 3 displays the mutarotation of D-glucopyranose (Glc).The 1 H NMR spectrum of Glc at a concentration of 20 mM in D2O indicated that the anomer ratio of Glc was ca.1:1.We investigated whether the DOSY method could separate the individual 1 H NMR spectra of the anomeric isomers in an equilibrium mixture of α-Glc and β-Glc.The DOSY spectrum of a 20 mM solution of Glc in D2O at 30 °C revealed that two species with different diffusion coefficients (D) of 7.6 × 10 −10 m 2 •s −1 and 5.8 × 10 −10 m 2 •s −1 were present, the former corresponding to the 1 H NMR spectrum of α-Glc, and the latter to the 1 H NMR spectrum of β-Glc, as can be seen in Figure 4. We found that the difference between the diffusion coefficients of α-Glc and β-Glc was sufficient to separate the two anomeric isomers of Glc by using the DOSY technique.In order to confirm the applicability of the DOSY method for the determination of the diffusion coefficients of both anomers of glycopyranose showing mutarotation, the DOSY spectra of several kinds of glycopyranoses were measured.The DOSY measurements were performed using 20 mM solutions of ( 13 C 6 )-D-glucopyranose (( 13 C 6 )Glc), D-galactopyranose (Gal), D-mannopyranose (Man), and cellobiose (Glcβ(1→4)Glc, Cello) in D 2 O at 30 • C. We had previously confirmed the presence of the αand β-anomers of these glycopyranoses in D 2 O by 1 H NMR measurements.The individual 1 H NMR spectra of the αand β-anomers were successfully separated in all the DOSY spectra, and their corresponding diffusion coefficients (D) were thereby obtained.The DOSY spectra are shown in Figures S2-S5, and the αand β-anomers of these glycopyranoses are summarized in Table 1, together with the average diffusion coefficients of some of the αand β-glycopyranose mixtures previously reported.Since the DOSY technique was able to separate the αand β-anomers of glycopyranoses, it seems to be a reliable method for the estimation of their individual diffusion coefficients.

Table 1 .
Diffusion coefficients of carbohydrate anomers measured in this study and their reported values.