Anomers of Carbohydrates by Diffusion-Ordered NMR Spectroscopy

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 α-PhGlc O β-PhGlcOH α-arbutinO OH 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 13HO * HO 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 α- and β-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 αthe α- andβ 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. of α-Glc and β-Glc. The DOSY spectrum of a 20 mM solution◦ of Glc in D2O at 30 °C revealed that two The DOSYα-Glc and spectrum β-Glc. ofThe a 20 DOSY mM solutionspectrum of of Glc a 20 in mM D2O solution at 30 C of revealed Glc in D that2O at two 30 species°C revealed with that different two species with different diffusion coefficients (D) of 7.6 × 10−10 m2·s−1 and 5.8 × 10−10 m2·s−1 were present, diffusionspecies coefficients with different (D) diffusion of 7.6 × coefficients10−10 m2·s (−D1) andof 7.6 5.8 × 10×−1010 m−2·s10−1m and2·s −5.81 were× 10−10present, m2·s−1 were the present, former the former corresponding to the 1H NMR spectrum of α-Glc, and the latter to the 1H NMR spectrum correspondingthe former corresponding to the 1H NMR to spectrumthe 1H NMR of αspectrum-Glc, and of theα-Glc, latter and to the the latter1H NMR to the spectrum1H NMR spectrum of β-Glc, of β-Glc, as can be seen in Figure 4. We found that the difference between the diffusion coefficients of as canof beβ-Glc, seen as in can Figure be seen4. We in foundFigure that4. We the found difference that the between difference the between diffusion the coefficients diffusion coefficients of α-Glc and of α-Glc and β-Glc was sufficient to separate the two anomeric isomers of Glc by using the DOSY β-Glcα-Glc was sufficientand β-Glc towas separate sufficient the twoto separate anomeric the isomers two anomeric of Glc byisomers using of the Glc DOSY by using technique. the DOSY technique.technique.

OHOH O O HOHO OHOH HOHO OHOH OH OH OHOH β-formβ-form O O OHOH HO HO HO HO H H HO HO HO HO C C OHOH OH OH OH OH O O OHOH O O HOHO HOHO OH OH OH OH α-form α-form

Figure 3. Mutarotation of Glc to interconvert between α-anomer and β-anomer in water. Figure 3. Mutarotation of Glc to interconvert between α-anomer and β-anomer in water.

Figure 4. DOSY spectrum of 20 mM Glc. Figure 4. DOSY spectrum of 20 mM Glc.

Figure 4. DOSY spectrum of 20 mM Glc.

Magnetochemistry 2017, 3, 38 4 of 6

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 13 ( C6)-D-glucopyranose (( C6)Glc), D-galactopyranose (Gal), D-mannopyranose (Man), and cellobiose ◦ (Glcβ(1→4)Glc, Cello) in D2O at 30 C. We had previously confirmed the presence of the α- and 1 1 β-anomers of these glycopyranoses in D2O by H NMR measurements. The individual H NMR spectra of the α- and 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 α- and β-anomers of these glycopyranoses are summarized in Table1, together with the average diffusion coefficients of some of the α- and β-glycopyranose mixtures previously reported. Since the DOSY technique was able to separate the α- and 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.

Reported Diffusion Coefficient Carbohydrate Diffusion Coefficient Entry D (× 10−10) m2·s−1 (Conditions) (Conditions, 30 ◦C, D O) D (× 10−10) m2·s−1 2 [Lit.] α-PhGlc (10 mM) 5.6 - 1 β-PhGlc (10 mM) 5.9 - α-arbutin (10 mM) 5.9 - 2 β-arbutin (10 mM) 5.8 -

Glc (20 mM) - 6.3 (100 mM, H2O) [21] 3 α-anomer 7.6 - β-anomer 5.8 - 13 ( C6)Glc (20 mM) - 4 α-anomer 9 β-anomer 5.4

Gal (20 mM) - 8.7 (100 mM, H2O) [21] 5 α-anomer 10.9 - β-anomer 5.8 - ◦ Man (20 mM) - 7.0 (25 C, H2O) [22] 6 α-anomer 7.4 - β-anomer 6.85 - ◦ Cello (20 mM) - 5.2 (25 C, H2O) [23] 7 α-anomer 5. 5 - β-anomer 5.95 - α-PhGlc (10 mM) 5.77 - 8 α-arbutin (10 mM) 5.49 - β-arbutin (10 mM) 7.2 - 9 β-pNPGlc (10 mM) 5.6 -

2.3. DOSY Separation of a Mixture of Two Kinds of Glycopyranosides Having Similar Aglycon Structures We also investigated the DOSY separation of a mixture of two kinds of glycopyranosides having a similar aglycon structures. The DOSY spectrum of a mixture of 10 mM α-PhGlc and 10 mM α-arbutin ◦ in D2O at 30 C clearly separated the two kinds of glycopyranoside species, which exhibited different diffusion coefficients (D) of 5.77 × 10−10 m2·s−1 (α-PhGlc) and 5.49 × 10−10 m2·s−1 (α-arbutin), as shown in Figure S6. The DOSY spectrum in Figure S7 also evinces the successful separation of a mixture of 10 mM β-arbutin and 10 mM β-pNPGlc, whose diffusion coefficients (D) were 7.2 × 10−10 m2·s−1 and 5.6 × 10−10 m2·s−1, respectively. Magnetochemistry 2017, 3, 38 5 of 6

3. Materials and Methods

D-glucose, D-galactose, D-mannose, D-cellobiose, β-arbutin, phenyl α-glucopyranoside, phenyl β-glucopyranoside, and pNP β-glucopyranoside were purchased from Tokyo Chemical 13 Industry Co., Ltd. (Chuo-ku, Tokyo, Japan) and ( C6)-D-glucose was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). α-Arbutin was purchased from Wako Pure Chemical Industries, Ltd. (Chuo-Ku, Osaka, Japan). D2O was purchased from Kanto Chemical Co., INC. (99.8% minimum in D, Chuo-ku, Tokyo, Japan). The NMR spectra were obtained using a JEOL ECA-600 spectrometer (JEOL Ltd., Akishima, Tokyo, Japan), using 20 mM concentrations for the individual samples and 10 mM concentrations for the mixtures. The 1H NMR spectra were recorded at 600 MHz. The chemical shifts were referenced to the solvent values (δ 4.70 ppm for HOD). The spectra were analyzed after 16 scans and 4 dummy scans. The 2D DOSY experiments were performed at 30 ◦C using the bipolar pulse pair and longitudinal eddy current delay sequence. Gradient amplitudes were 20–247 mT/m. The spectral width was 6000 Hz. Bipolar rectangular gradients were used with total durations of 1 to 2 ms. Gradient recovery delays were 0.1 ms. Diffusion times were between 50 and 200 ms. The relaxation delay was 7.0 s. The spectra were analyzed after 256 scans and 16 dummy scans. The spectral analyses were processed by the Delta NMR processing software version 4.3.6. (JEOL USA, Inc., Peabody, MA, USA).

4. Conclusions This article describes the evaluation of the DOSY method for the separation and analysis of the α- and β-anomers of carbohydrates. We found that the α- and β-anomers of carbohydrates having different diffusion coefficients can be separated by using the DOSY technique, and their individual diffusion coefficients can be determined. In addition, the DOSY method was also applicable to the separation of two kinds of glucopyranosides having similar aglycon structures from a mixture.

Supplementary Materials: The following are available online at www.mdpi.com/2312-7481/3/4/38/s1, ◦ Figure S1. DOSY spectrum of a 10 mM α-arbutin and 10 mM β-arbutin mixture in D2O at 30 C, Figure S2. 13 ◦ ◦ DOSY spectrum of 20 mM ( C6)Glc in D2O at 30 C, Figure S3. DOSY spectrum of 20 mM Gal in D2O at 30 C, ◦ Figure S4. DOSY spectrum of 20 mM Man in D2O at 30 C, Figure S5. DOSY spectrum of 20 mM Cello in D2O at ◦ ◦ 30 C, Figure S6. DOSY spectrum of a 10 mM α-PhGlc and 10 mM α-arbutin mixture in D2O at 30 C, Figure S7. ◦ DOSY spectrum of a 10 mM β-arbutin and 10 mM β-pNPGlc mixture in D2O at 30 C. Author Contributions: T.Y. conceived idea of the article and wrote the paper. Y.O. performed the experiments. K.K. conceived and designed the experiments. All authors approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Johnson, C.S. Diffusion ordered nuclear magnetic resonance spectroscopy: Principles and applications. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 203–256. [CrossRef] 2. Antalek, B. Using Pulsed Gradient Spin Echo NMR for Chemical Mixture Analysis: How to Obtain Optimum Results. Concepts Magn. Reson. 2002, 14, 225–258. [CrossRef] 3. Kavakka, J.S.; Kilpeläinen, I.; Heikkinen, S. General Chromatographic NMR Method in Liquid State for Synthetic Chemistry: Polyvinylpyrrolidone Assisted DOSY Experiments. Org. Lett. 2009, 6, 1349–1352. [CrossRef][PubMed] 4. Kavakka, J.S.; Parviainen, V.; Wahala, K.; Kilpeläinen, I.; Heikkinen, S. Enhanced chromatographic NMR with polyethyleneglycol. A novel resolving agent for diffusion ordered spectroscopy. Magn. Reson. Chem. 2010, 48, 777–781. [CrossRef][PubMed] 5. Huang, S.; Wu, R.; Bai, Z.; Yang, Y.; Li, S.; Dou, X. Evaluation of the separation performance of polyvinylpyrrolidone as a virtual stationary phase for chromatographic NMR. Magn. Reson. Chem. 2014, 52, 486–490. [CrossRef][PubMed] 6. Xu, J.; Tan, T.; Kenne, L.; Sandström, C. The use of diffusion-ordered spectroscopy and complexation agents to analyze mixtures of catechins. New J. Chem. 2009, 33, 1057–1063. [CrossRef] Magnetochemistry 2017, 3, 38 6 of 6

7. Oda, Y.; Kobayashi, N.; Yamanoi, T.; Katsuraya, K.; Takahashi, K.; Hattori, K. β-Cyclodextrin Conjugates with Glucose Moieties Designed as Drug Carriers: Their Syntheses, Evaluations Using Concanavalin A and Doxorubicin, and Structural Analyses by NMR Spectroscopy. Med. Chem. 2008, 4, 244–255. [CrossRef] [PubMed] 8. Oda, Y.; Matsuda, S.; Yamanoi, T.; Murota, A.; Katsuraya, K. Identification of the inclusion complexation 13 1 13 between phenyl β-D-( C6)-glucopyranoside and α-cyclodextrin using 2D H or C DOSY spectrum. Supramol. Chem. 2009, 21, 638–642. [CrossRef] 9. Haiber, S.; Nilsson, M.; Morris, G.A. Matrix-assisted diffusion-ordered spectroscopy: Application of surfactant solutions to the resolution of isomer spectra. Magn. Reson. Chem. 2012, 50, 458–465. 10. Reile, I.; Aspers, R.L.E.G.; Feiters, M.C.; Rutjes, F.P.J.T.; Tessari, M. Resolving DOSY spectra of isomers by methanol-d4 solvent effects. Magn. Reson. Chem. 2017, 55, 759–762. [CrossRef][PubMed] 11. Gramosa, N.V.; Ricardo, N.M.S.P.; Adams, R.W.; Morris, G.A.; Nilsson, M. Matrix-assisted diffusion-ordered spectroscopy: Choosing a matrix. Magn. Reson. Chem. 2016, 54, 815–820. [CrossRef][PubMed] 12. Adams, R.W.; Aguilar, J.A.; Cassani, J.; Morris, G.A.; Nilsson, M. Resolving natural product epimer spectra by matrix-assisted DOSY. Org. Biomol. Chem. 2011, 20, 7062–7064. [CrossRef][PubMed] 13. Codling, D.J.; Zheng, G.; Stait-Gardner, T.; Yang, S.; Nilsson, M.; Price, W.S. Diffusion Studies of Dihydroxybenzene Isomers in Water−Alcohol Systems. J. Phys. Chem. B 2013, 117, 2734–2741. [CrossRef] [PubMed] 14. Chaudhari, S.R.; Suryaprakash, N. Diffusion ordered spectroscopy for resolution of double bonded cis, trans-isomers. J. Mol. Struct. 2012, 1017, 106–108. [CrossRef] 15. Viel, S.; Capitani, D.; Mannina, L.; Segre, A. Diffusion-Ordered NMR Spectroscopy: A Versatile Tool for the Molecular Weight Determination of Uncharged Polysaccharides. Biomacromolecules 2003, 4, 1843–1847. [CrossRef][PubMed] 16. Groves, P.; Rasmussen, M.O.; Molero, M.D.; Samain, E.; Cañada, F.J.; Driguez, H.; Jiménez-Barbero, J. Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis. Glycobiology 2004, 14, 451–456. [CrossRef][PubMed] 17. Cao, R.; Komura, F.; Nonaka, A.; Kato, T.; Fukumashi, J.; Matsui, T. Quantitative analysis of D-(+)-glucose in fruit juices using diffusion ordered-1H nuclear magnetic resonance spectroscopy. Anal. Sci. 2014, 30, 383–388. [CrossRef][PubMed] 18. Politi, M.; Groves, P.; Chávez, M.I.; Cañada, F.J.; Jiménez-Barberoa, J. Useful applications of DOSY experiments for the study of mushroom polysaccharides. Carbohydr. Res. 2006, 341, 84–89. [CrossRef] [PubMed] 19. Kählig, H.; Dietrich, K.; Dorner, S. Analysis of Carbohydrate Mixtures by Diffusion Difference NMR Spectroscopy. Monatsh. Chem. 2002, 133, 589–598. [CrossRef] 20. Díaz, M.D.; Berger, S. Studies of the complexation of sugars by diffusion-ordered NMR spectroscopy. Carbohydr. Res. 2000, 329, 1–5. [CrossRef] 21. Nagy, L.; Gyetvai, G.; Nagy, G. Determination of the Diffusion Coefficient of Monosaccharides with Scanning Electrochemical Microscopy (SECM). Electroanalysis 2009, 21, 542–549. [CrossRef] 22. Mori, N.; Sugai, E.; Fuse, Y.; Funazukuri, T. Infinite Dilution Binary Diffusion Coefficients for Six Sugars at 0.1 MPa and Temperatures from (273.2 to 353.2). J. Chem. Eng. Data 2007, 52, 40–43. 23. Ihnat, M.; Goring, D.A.I. Shape of the cellodextrins in aqueous solution at 25 ◦C. Can. J. Chem. 1967, 45, 2353–2361. [CrossRef]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).