Anomers of Carbohydrates by Diffusion-Ordered NMR Spectroscopy
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magnetochemistry Article Separation of the α- and β-Anomers of Carbohydrates by Diffusion-Ordered NMR Spectroscopy Takashi Yamanoi 1,* ID , Yoshiki Oda 2 and Kaname Katsuraya 3 1 Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan 2 Technology Joint Management Office, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan; [email protected] 3 Department of Human Ecology, Wayo Women’s University, Chiba 272-8533, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-49-271-7958 Received: 31 October 2017; Accepted: 20 November 2017; Published: 22 November 2017 Abstract: This article describes the successful application of the DOSY method for the separation and analysis of the α- and 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. Keywords: DOSY; diffusion coefficient; anomer separation; carbohydrate isomer; glycoside 1. 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 1H 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 [3–8]. 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 [9–14]. 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 [15–20]. 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 α- and β-anomers of carbohydrates. 2. Results and Discussion The DOSY analysis for the isomer separation in a mixture of α- and β-anomers was investigated using several kinds of carbohydrate derivatives of glycopyranosides and glycopyranoses, as shown in Figure1. Magnetochemistry 2017, 3, 38; doi:10.3390/magnetochemistry3040038 www.mdpi.com/journal/magnetochemistry Magnetochemistry 2017, 3, 38 2 of 6 Magnetochemistry 2017, 3, 38 2 of 6 OH OH OH Magnetochemistry 2017, 3, 38 OH 2 of 6 O O HO HO O O OH O O HO HO OH HO HO OH HO OH OH HO OH OH OH O OH OO OH O O O OH HO O HO HO HO HO O HO HO OH HO OH OH O OH O OH α-PhGlc β-PhGlc α-arbutin β-arbutin OH OH HO OH HO α-PhGlc * β-PhGlc α-arbutin β-arbutinOH O * O O O HO HO HO * HO OH HO *OH HO OH HO * * HO HO OH OH OH OH OH* OH OH OH O * O O O HO HO HO * * HO HO 13HO * HO Glc ( C6)Glc* Gal Man OH OH OH OH OH OH OH OH 13 Glc ( C6)GlcOH Gal Man OH O O HO O HO OH O HO OH O NO2 OH HO OH O OH HO OH HO O HO OH O HO O NO 2 OH HO OH OH HO Cello β-pOHNPGlc Cello β-pNPGlc FigureFigure 1.1. Carbohydrate derivatives derivatives used used for for DOSY DOSY analysis analysis on on anomer anomer isomers. isomers. Figure 1. Carbohydrate derivatives used for DOSY analysis on anomer isomers. 2.1.2.1. DOSY DOSY SeparationSeparation ofof thethe aα- and βb-Anomeric-Anomeric Isomers Isomers of of Glycopyranosides Glycopyranosides 2.1. DOSY Separation of the α- and β-Anomeric Isomers of Glycopyranosides TheThe DOSY DOSY separationseparation of the anomeric isomers isomers in in aa mixture mixture of of 10 10 mM mM phenyl phenyl β-glucopyranosideβ-glucopyranoside (β(β-PhGlc)-PhGlc)The andand DOSY 1010 mMmM separation phenylphenyl α of-glucopyranoside the anomeric isomers (α (α-PhGlc)-PhGlc) in a mixture was was firstly of firstly 10 mMinvestigated. investigated. phenyl β-glucopyranoside Figure Figure 2 shows2 shows the DOSY(β-PhGlc) spectrum and 10 ofmM the phenyl mixture α-glucopyranoside of α-PhGlc and (α -PhGlc)β-PhGlc was in Dfirstly2O at investigated. 30 °C,◦ together Figure with 2 shows the the DOSY spectrum of the mixture of α-PhGlc and β-PhGlc in D2O at 30 C, together with the individualthe DOSY 11H NMR spectrum spectra of theof βmixture-PhGlc andof α α-PhGlc-PhGlc and in Dβ-PhGlc2O. In thein DDOSY2O at spectrum,30 °C, together two different with the individual H NMR spectra of β-PhGlc and α-PhGlc in D2O. In the DOSY spectrum, two different speciesindividual with diffusion 1H NMR coefficients spectra of (βD-PhGlc) of 5.9 and × 10α-PhGlc−10 m2·s −in1 andD2O. 5.6 In ×the 10 DOSY−10 m2·s spectrum,−1 could be two identified, different species with diffusion coefficients (D) of 5.9 × 10−10 m2·s−1 and 5.6 × 10−10 m2·s−1 could be identified, whosespecies resonances with diffusion corresponded coefficients to the ( D1H) of NMR 5.9 × spectrum 10−10 m2·s −of1 and β-PhGlc 5.6 × 10and−10 αm-PhGlc,2·s−1 could respectively. be identified, It whose resonances corresponded to the 1H NMR spectrum of β-PhGlc and α-PhGlc, respectively. It was was therebywhose resonances found that corresponded the apparent to differe the 1Hnce NMR between spectrum the ofdiffusion β-PhGlc coefficientsand α-PhGlc, of respectively.β-PhGlc and It thereby found that the apparent difference between the diffusion coefficients of β-PhGlc and α-PhGlc α-PhGlcwas allowthereby the found DOSY that separation the apparent of these differe glucopyranosidence between the anomers. diffusion coefficients of β-PhGlc and allowα the-PhGlc DOSY allow separation the DOSY of separation these glucopyranoside of these glucopyranoside anomers. anomers. Figure 2. DOSY spectrum of a 10 mM β-PhGlc and 10 mM α-PhGlc mixture. FigureFigure 2. DOSY 2. DOSY spectrum spectrum of a of 10 a mM 10 mM β-PhGlc β-PhGlc and and 10 10mM mM α-PhGlc α-PhGlc mixture. mixture. Magnetochemistry 2017, 3, 38 3 of 6 Magnetochemistry 2017, 3, 38 3 of 6 Next, the DOSY separation of the anomeric isomers in a mixture of 10 mM α-arbutin Next, the DOSY separation of the anomeric isomers in a mixture of 10 mM α-arbutin (p-hydroxyphenylNext, the αDOSY-glucopyranoside) separation of andthe anomeric10 mM βisomers-arbutin in in a Dmixture2◦O at 30of °C10 mMwas αsimilarly-arbutin (p-hydroxyphenyl α-glucopyranoside) and 10 mM β-arbutin in D2O at 30 C was similarly investigated. investigated.(p-hydroxyphenyl The two glucopyrα-glucopyranoside)anoside anomers—which and 10 mM βexhibite-arbutind indiffusion D2O at coefficients 30 °C was (D similarly) of 5.9 × The two glucopyranoside anomers—which exhibited diffusion coefficients (D) of 5.9 × 10−10 m2·s−1 10−10 investigated.m2·s−1 (α-arbutin) The two and glucopyr 5.8 ×anoside 10−10 manomers—which2·s−1 (β-arbutin), exhibite respectively—wered diffusion coefficients also successfully (D) of 5.9 × (α-arbutin) and 5.8 × 10−10 m2·s−1 (β-arbutin), respectively—were also successfully separated by the separated10−10 mby2·s the−1 ( DOSYα-arbutin) techniqu and e,5.8 as ×shown 10−10 inm2 Figure·s−1 (β-arbutin), S1. respectively—were also successfully DOSYseparated technique, by the as shownDOSY techniqu in Figuree, S1.as shown in Figure S1. 2.2. DOSY Separation of the α- and β-Anomeric Isomers of Glycopyranoses 2.2. DOSY Separation of athe α- andb β-Anomeric Isomers of Glycopyranoses Glycopyranoses areare knownknown toto undergo undergo mutarotation; mutarotation; they they interconvert interconvert their theirα- α and- andβ-anomers β-anomers in in water Glycopyranosesand an equilibrium are known mixture to undergo of the mutarotation; two forms they is interconvertachieved. Figure their α -3 and displays β-anomers the waterin and water an equilibriumand an equilibrium mixture of mixture the two of forms the istw achieved.o forms Figureis achieved.3 displays Figure the mutarotation3 displays the of mutarotation of D-glucopyranose1 (Glc). The 1H NMR spectrum of Glc at a concentration of 20 mM in D-glucopyranosemutarotation of (Glc). D-glucopyranose The H NMR (Glc). spectrum The 1H of NMR Glc atspectrum a concentration of Glc at ofa concentration 20 mM in D2 ofO 20 indicated mM in D2O indicated that the anomer ratio of Glc was ca. 1:1. We investigated whether the DOSY method that theD2O anomer indicated ratio that of the Glc anomer was ca. ratio 1:1. of We Glc investigated was ca. 1:1. whetherWe investigated the DOSY whether method the couldDOSY separatemethod could separate the individual 1H NMR spectra of the anomeric isomers in an equilibrium mixture of the individualcould separate1H NMR the individual spectra of 1H the NMR anomeric spectra isomers of the anomeric in an equilibrium isomers in mixture an equilibrium of α-Glc mixture and β-Glc.