doi.org/10.26434/chemrxiv.7751144.v1

NMR Elucidation of “NaCl Effect” in Carbohydrates Conversion Toward aRational PredictionGan Zhu, Hui Li, Yiqun Li, Liuqun Gu

Submitted date: 21/02/2019 • Posted date: 21/02/2019Licence: CC BY-NC-ND 4.0Citation information: Zhu, Gan; Li, Hui; Li, Yiqun; Gu, Liuqun (2019): NMR Elucidation of “NaCl Effect” inCarbohydrates Conversion Toward a Rational Prediction. ChemRxiv. Preprint.

“NaCl Effect” was well known in biomass chemical degradation including carbohydrate transformations, inwhich NaCl had significant positive effect in promoting/catalyzing particular transformation. However, directevidence was very rare in proposed mechanisms to elucidate “NaCl Effect”, here we reported 1H NMRevidences of “NaCl Effect” on different saccharides and at once non-selective bonding of Cl-H was proposedinstead of sequence bonding during deconstruction of hydrogen bonding network based on evidences. Ageneral recommending NaCl usage based on total hydroxyls on saccharides could well explain the best effectof hydrogen bonding destruction in most reported literatures.

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1

NMR Elucidation of “NaCl Effect” in Carbohydrates

Conversion Toward a Rational Prediction

Gan Zhu♀b, Hui Li♀a, Yiqun Li b* and Liuqun Gua*

a Department of Biomedical Engineering, Jinan University; #601, Huangpudadaoxi,

Guangzhou, China

b Department of Chemistry, Jinan University; #601, Huangpudadaoxi, Guangzhou,

China

KEYWORDS. NaCl effect; 1H NMR elucidation; carbohydrate; hydrogen bonding; biomass

depolymerization.

Abstract:

“NaCl Effect” was well known in biomass chemical degradation including carbohydrate

transformations, in which NaCl had significant positive effect in promoting/catalyzing

particular transformation. However, direct evidence was very rare in proposed mechanisms

to elucidate “NaCl Effect”, here we reported 1H NMR evidences of “NaCl Effect” on different

saccharides and at once non-selective bonding of Cl-H was proposed instead of sequence

bonding during deconstruction of hydrogen bonding network based on evidences. A general

recommending NaCl usage based on total hydroxyls on saccharides could well explain the

best effect of hydrogen bonding destruction in most reported literatures.

Alkali chlorides including sodium chloride (NaCl) and potassium chloride (KCl) et. al played

important roles in extracellular fluid of many multicellular organisms; the former is well known

as a condiment and food preservative, and the latter is one of most frequent used fertilizer in

agriculture. In recent decade, research on their use as additives1-6

or promoters7-9

for biomass

chemical degradation including carbohydrate transformations into valuable chemicals were

increasingly popular because they are abundant in nature and are very cheap in cost;

particularly for sodium chloride and “NaCl Effect” (or “salt effect”) was well known in biomass

conversion and carbohydrate chemistry. However, the mechanism of “NaCl Effect” was very

less explored10. No direct evidence mapping interaction between carbohydrates (or cellulose

2

and so on) and NaCl was reported to our best of knowledge probably because of the complex

nature of biomass components and carbohydrates. Here we reported 1H NMR evidences of

“NaCl Effect” on different saccharides and a relatively stable bonding of O(N)-H-Cl was

observed with all hydroxyls of saccharides.

Chloride ion on ionic liquid along with the anion part have been reported being able to break

hydrogen bond in cellulose11,12

by measuring the crystal Root Mean Square Deviation (RMSD)

values in atomic positions of the cellulose bunch and using molecular dynamics simulations.

Very recently C. Hu and J. H. Clark reported a comprehensive mechanistic study10 on “NaCl

effect” on the thermochemical depolymerization of cellulose and further demonstrated the

strong potential of a cheap and readily available mineral (NaCl) can be used to help improve

the resource efficiencies of lignocellulosic biorefineries. However, mechanism detail on how

NaCl molecular interact with proton of hydroxyl on sugar moiety in cellulose and the

minimum of NaCl required for the best effect of disrupting hydrogen bonding are largely

unknown. Inspired by a sharp difference of 1H NMR shifts of glucosamine in deionized water

and saturated NaCl solution, we envisaged an NMR angle by measuring 1H NMR shifts of

monosaccharides and oligosaccharides might be helpful to understand the mechanism of

“NaCl Effect”.

It was known that solid state NMR and IR spectra both showed no obvious change, especially

in spectral regions characteristic of the ordered and disordered regions of cellulose in the

presence of NaCl10,13

, probably because of cellulose’s poor solubility in water. Hence, water

soluble monosaccharides including D-glucose, D-glucosamine hydrogen chloride, N-acetyl-

D-glucosamine and D-fructose were initially chosen in order to gain some insights on “NaCl

effect” on the intramolecular/intermolecular hydrogen bonding in water via regular 1H NMR

analysis. Variant concentration of 4.8%, 9.1%, 13.0%, 16.7% (all in wt%) and saturated NaCl

solutions were made for comparison study.

Figure 1. 1H NMR Spectra of N-Acetyl-D-glucosamine and Five Peaks Marked for

Tracking.

3

Initially 0.5 mmol of N-acetyl-D-glucosamine was dissolved into 6 mL of NaCl solution with

different concentration and the mixtures continued to stir for 6 hours at room temperature

before one portion was taken out for 1H NMR analysis. Five peaks including hydrogens on 1-

position carbon of both α-anomer and β-anomer14 were marked (Figure 1) in order to track

their changes in different concentration of NaCl solution. After a solution of 0.5 mmol of N-

acetyl-D-glucosamine was stirred in deionized water, protons on 1-position carbon of α-

anomer and β-anomer displayed at δ 5.18 and 4.67 respectively on 1H NMR spectra in D2O,

the former was at left side of D2O peak while the latter was at right side. Significant deshielding

effect was observed for all protons of N-acetyl-D-glucosamine (△δ = 0.26 ppm) in the

presence of 4.8% NaCl solution, which was likely induced by newly formed H-Cl bond (Figures

2 and 3). A remarkable downfield shifting (△δ = 0.18 ppm) on 1H NMR shift between spectra

obtained in 4.8% NaCl solution and that obtained in 9.1% NaCl solution continued. Shift

changes became intangible when further increase of concentration (13.0% from 9.1% or to

16.7% from 13.0% or to saturated NaCl solution from 16.7%) (△δ <= 0.02 ppm). Based on

these data (Figures 2 and 3), it could be concluded that almost completed deconstruction of

intramolecular/intermolecular hydrogen bonding within N-acetyl-D-glucosamine molecular

was achievable in 9.1% NaCl solution and in which relatively stable H-Cl bonding formed.

Figure 2. 1H NMR Spectra of N-Acetyl-D-glucosamine after Mixing with Different

Concentration of NaCl Solutions.

4

Figure 3. 1H NMR Shift Changes of N-Acetyl-D-glucosamine, D-Glucose, D-Fructose and

D-Glucosamine Hydrogen Chloride Correlating with Concentration of NaCl by Tracking

Marked Five/Six Peaks.

In order to gain more clues, similar 1H NMR tracking analysis in different concentration of

NaCl solutions was also performed with other three abundant monosaccharides (D-glucose,

D-fructose and D-glucosamine hydrogen chloride) in nature (Figure 3). Trends of 1H NMR

shift changes of D-glucose in marked six peaks were pretty similar with those observed from

N-acetyl-D-glucosamine; Significant changes were observed upon increase of concentration

of NaCl solution (4.8% from 0% or to 9.1% from 4.8%). No change at all for all marked peaks

occurred in the presence of higher concentration of NaCl solution (13.0%, 16.7% and saturated

solution). For D-fructose, obvious 1H NMR shifting could still be observed for all protons of

six marked peaks when further increase of concentration to 13.0% from 9.1% (△δ = 0.07 ppm).

Continued increase of NaCl concentration led to intangible change on 1H NMR spectra (△δ

<= 0.02 ppm). Interestingly, with D-glucosamine hydrogen chloride stable complex could

only be obtained till NaCl concentration was increased to 16.7% although the shifting of the

marked six peaks was small (△δ = 0.03 - 0.05 ppm) when increase of concentration to 16.7%

from 13.0%. The significant delay to a stable H-Cl bonding was likely due to the increase of

free protons by amine ion moiety of D-glucosamine hydrogen chloride. It worth being noted

that shifting trends of all marked peaks were almost same in all four monosaccharides which

indicated that deconstruction process of hydrogen bonding and generating new hydrogen

chloride bonding occurred at once and were non-selective.

Figure 4. 1H NMR Shift Changes of Sucrose, Trehalose and Stachyose Correlating with

Concentration of NaCl by Tracking Marked Four Peaks.

5

Next two disaccharides (sucrose and trehalose) and one tetrasaccharide (stachyose) were also

selected as targets for 1H NMR comparison study in variant concentration of NaCl solutions

(Figure 4), in order to provide more viable references for NaCl promoted deconstruction of

hydrogen bonding of polysaccharides such as cellulose. The correlation of 1H NMR shifts with

concentration of NaCl solution for sucrose was pretty much similar with that of fructose, in

which an obvious shifting on 1H NMR spectra disappeared after concentration of NaCl was

increased to 13.0%. It was easy to be understand because one molecule sucrose composed of

two monosaccharides (glucose and fructose) and the moiety (fructose) in slow rate

determined the rate of sucrose in deconstruction of hydrogen bonding. An increase of mixing

temperature to 60℃ had positive effect (△δ = 0.11 ppm) on deconstruction of hydrogen

bonding of sucrose in low concentration of NaCl solution (4.8%); however, influence was

intangible in relatively high concentration of NaCl solution (>9.1%) (see supporting

information). As a disaccharide formed by a 1,1-glycosidic bond between two α-glucose units,

trehalose has a stronger hydrogen bonding network than that by D-glucose. Trends of 1H

NMR shift for four marked peaks of trehalose correlating with concentration of NaCl solution

was more like that of sucrose rather than that of D-glucose, and almost a same 1H NMR

spectra was obtained in case that concentration of NaCl solution was over 13.0%. Increase of

disaccharide to tetrasaccharide (stachyose) led to small effect on the correlation between 1H

NMR shift and concentration of NaCl solution. Small shifting for marked four peaks of

stachyose was observed upon increase of concentration of NaCl solution to 16.7% from 13.0%,

meanwhile the change vanished when increase of temperature to 60℃ (see supporting

information).

With all clues in hand, we combed through three main factors including temperature,

6

concentration (NaCl solution) and absolute volume (NaCl solution) which might have effect

on deconstruction of hydrogen bonding network and generation of H-Cl bonds. The

significant shifting difference for all marked peaks of sucrose in the presence of 4.8% NaCl

solution at room temperature and 60℃ indicated the important role of temperature,

particularly an expected more important role for polysaccharides that have poor solubility in

water due to large molecular weight. NaCl concentration was well known to have strong effect

on hydrogen bonding in water due to solvation15-17

; here it was also found to influent

deconstruction of hydrogen bonding network and generation of H-Cl bonds remarkably for

all selected monosaccharides and oligosaccharides. The higher concentration of NaCl solution,

the better effect on deconstruction of hydrogen bonding network based on 1H NMR shifting.

Effect of volume of was also investigated with N-acetyl-D-glucosamine (0.5 mmol) in 9.1 wt%

NaCl solution (Figure 5). Downfield shifting was observed along with increase of volume till

6 mL, and after then bonding network became steady and no change was observed on 1H

NMR spectra. The total usage of NaCl was 10 mmol under the turning point condition (6 mL).

Figure 5. 1H NMR Spectra of N-Acetyl-D-glucosamine after Mixing with Different

Volume of 9.1 wt% NaCl Solution at Room Temperature.

Concerning frequent optimization on NaCl usage in literatures, we tentatively to propose a

calculation model recommending the best usage of NaCl for deconstruction of hydrogen

bonding network of polysaccharides including starch, chitin and cellulose. Recommended

concentration of NaCl solution ranged from 9% to 15% (wt%) since too high concentration led

to little difference (Figure 4). Recommended absolute amount of NaCl in solution is 3.5 – 4.0

7

equivalent to total hydroxyl of saccharides (including protons on amine moiety if

glucosamine-based saccharides were used) since all the turning points for our selected four

monosaccharides and three oligosaccharides are within this range. For temperature, room

temperature is usually sufficient for monosaccharides and oligosaccharides which are soluble

in water; however, typically increase of temperature was pretty necessary for cellulose

depolymerization according to a recent report10 that “NaCl effect” was only be obvious at

above 210℃.

Stable complexes illustrating interaction of saccharides (D-glucose and N-acetyl-D-

glucosamine) and chloride ions in water were proposed as shown in Figure 6. In which five

chloride ions (here ignoring solvation with water molecular along with sodium anion) bond

with hydrogens on hydroxyls (and acetyl amide) respectively at one time in a non-selective

manner. In another word, the bonding structure of complex in NaCl solution should be same

regardless parameters of concentration, volume and temperature and the only change is

bonding length, according to our observance that all marked peaks changed in a similar trend.

Figure 6. Complexes of D-Glucose and N-Acetyl-D-glucosamine Coordinating with

Chloride Ion in NaCl Solution.

A further exploration on 1H NMR shifting of D-glucose in saline solution showed clear shift

on spectrum (Figure 7) for all six marked peaks (△δ = 0.10 – 0.11 ppm). Such observance

might be of much value to understand carbohydrates in human body via NMR analysis

techniques18.

Figure 7. 1H NMR Spectra of D-glucose after Mixing with Saline Solution (0.9 wt% NaCl)

at Room Temperature.

8

In summary, 1H NMR elucidation of “NaCl effect” of monosaccharides and oligosaccharides

at room temperature was investigated, which provided evidences of prevailing “NaCl effect”

in molecular level. With clues in hand, non-selective hydrogen-chloride bonds formed at

once instead of sequence mechanism was proposed during deconstruction of hydrogen

bonding network of carbohydrates. A general recommending NaCl usage of 3.5 – 4.0

equivalent mole of hydroxyls (including amines or amides) on saccharides in a solution of

concentration ranging from 9.0-15 wt% was proposed based on achieved data; which might

be a reference for deconstruction of hydrogen bonding in polysaccharides (including cellulose

and starch) depolymerization. A clear observance of “NaCl effect” on D-glucose in saline

solution might contribute to understanding of carbohydrates in human body.

ASSOCIATED CONTENT

Supporting Information.

The following files are available free of charge.

AUTHOR INFORMATION

Corresponding Author

*(L. Gu) E-mail: guliuqun@jnu.edu.cn or guliuqun@yahoo.com;

* (Y. Li). E-mail: tlyq@jnu.edu.cn

9

Author Contributions

The manuscript was written through contributions of all authors. All authors have

given approval to the final version of the manuscript.

♀These authors contributed equally.

ACKNOWLEDGMENT

We acknowledge a startup funding from Jinan University to L. Gu (No: 88015155 and

88016607) and a funding of the National Natural Science Foundation of China (No.

21372099) to Y. Li.

References:

[1] Chen, X.; Chew, S. L.; Kerton, F. M.; Yan, N., Direct conversion of chitin into a N-containing furan

derivative. Green Chem. 2014, 16 (4), 2204-2212.

[2] Omari, K. W.; Dodot, L.; Kerton, F. M., A simple one-pot dehydration process to convert N-

acetyl-D-glucosamine into a nitrogen-containing compound, 3-acetamido-5-acetylfuran.

ChemSusChem 2012, 5 (9), 1767-72.

[3] Hansen, T. S.; Mielby, J.; Riisager, A., Synergy of boric acid and added salts in the catalytic

dehydration of hexoses to 5-hydroxymethylfurfural in water. Green Chem. 2011, 13 (1), 109-114.

[4] Wang, C.; Zhang, Q.; Chen, Y.; Zhang, X.; Xu, F., Highly Efficient Conversion of Xylose Residues

to Levulinic Acid over FeCl3 Catalyst in Green Salt Solutions. ACS Sustainable Chemistry &

Engineering 2018, 6 (3), 3154-3161.

[5] Yang, H.; Wang, L.; Jia, L.; Qiu, C.; Pang, Q.; Pan, X., Selective Decomposition of Cellulose into

Glucose and Levulinic Acid over Fe-Resin Catalyst in NaCl Solution under Hydrothermal Conditions.

Industrial & Engineering Chemistry Research 2014, 53 (15), 6562-6568.

[6] Potvin, J.; Sorlien, E.; Hegner, J.; DeBoef, B.; Lucht, B. L., Effect of NaCl on the conversion of

cellulose to glucose and levulinic acid via solid supported acid catalysis. Tetrahedron Letters 2011,

52 (44), 5891-5893.

[7] Li, M.; Li, W.; Lu, Y.; Jameel, H.; Chang, H.-m.; Ma, L., High conversion of glucose to 5-

hydroxymethylfurfural using hydrochloric acid as a catalyst and sodium chloride as a promoter in

a water/γ-valerolactone system. RSC Advances 2017, 7 (24), 14330-14336.

[8] Jiang, Z.; Budarin, V. L.; Fan, J.; Remón, J.; Li, T.; Hu, C.; Clark, J. H., Sodium Chloride-Assisted

Depolymerization of Xylo-oligomers to Xylose. ACS Sustainable Chemistry & Engineering 2018, 6

(3), 4098-4104.

10

[9] Amoah, J.; Hasunuma, T.; Ogino, C.; Kondo, A., 5-Hydroxymethylfurfural production from salt-

induced photoautotrophically cultivated Chlorella sorokiniana. Biochemical Engineering Journal

2019, 142, 117-123.

[10] Jiang, Z.; Fan, J.; Budarin, V. L.; Macquarrie, D. J.; Gao, Y.; Li, T.; Hu, C.; Clark, J. H., Mechanistic

understanding of salt-assisted autocatalytic hydrolysis of cellulose. Sustainable Energy & Fuels

2018, 2 (5), 936-940.

[11] Y. Li, X. Liu, S. Zhang, Y. Yao, X. Yao, J. Xu and X. Lu, Dissolving process of a cellulose bunch

in ionic liquids: a molecular dynamics study. Phys. Chem. Chem. Phys., 2015, 17, 17894-17905.

[12] B. D. Rabideau and A. E. Ismail, Mechanisms of hydrogen bond formation between ionic

liquids and cellulose and the influence of water content. Phys. Chem. Chem. Phys., 2015, 17, 5767-

5775.

[13] Fan, J.; Bruyn, M. D.; Zhu, Z.; Budarin, V.; Gronnow, M.; Gomez, L. D.; Macquarrie, D.; Clark, J.

H. Microwave-enhanced formation of glucose from cellulosic waste. Chem. Eng. Process., 2013,

71, 37-42.

[14] Usually a mixture of α-anomer and β-anomer both exist due to dissociated equivalent.

[15] Dedonder-Lardeux, C.; Gregoire, G.; Jouvet, C.; Martrenchard, S.; and Solgadi, D. Charge

separation in molecular clusters: dissolution of a salt in a salt-(solvent)n cluster. Chem. Rev., 2000,

100, 4023 – 4037.

[16] Beladjine, S.; Amrani, M.; Zanoun, A.; Belaidi, A,; Vergoten, G. Structure and hydrogen bonding

in aqueous sodium chloride solutions using theoretical water model AB4: effects of concentration.

Comput. Theor. Chem. 2011, 977, 97 – 102.

[17] Shalit, A.; Ahamed, S.; Savolainen, J. and Hamm, P. Terahertz echos reveal the inhomogeneity

of aqueous salt solutions. Nat. Chem., 2017, 9, 273-278.

[18] Duus, J.; Gotfredsen, C. H.; and Bock, K. Carbohydrate Structural Determination by NMR

Spectroscopy: Modern Methods and Limitations. Chem. Rev., 2000, 100, 4589 – 4614.

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S1

Supporting Information For NMR Elucidation of “NaCl Effect” in Carbohydrates

Conversion Toward a Rational Prediction

Gan Zhu♀b, Hui Li♀a, Yiqun Li b* and Liuqun Gua*

a Department of Biomedical Engineering, Jinan University; #601, Huangpudadaoxi, Guangzhou, China

E-mail: guliuqun@jnu.edu.cn or guliuqun@yahoo.com;

b Department of Chemistry, Jinan University; #601, Huangpudadaoxi, Guangzhou, China

E-mail: tlyq@jnu.edu.cn

♀These authors contributed equally.

Table of Content

General information ----------------------------------------------------------------------------------S2

Preparation for different concentration of NaCl solutions ---------------------------------S2

General procedure for investigation of “NaCl effect” on monosaccharides via 1H NMR

analysis --------------------------------------------------------------------------------------------------S2

General procedure for investigation of “NaCl effect” on oligosaccharides via 1H NMR

analysis---------------------------------------------------------------------------------------------------S3

General procedure for investigation volume impact (9.1% NaCl solution) of “NaCl

effect” on N-acetyl-D-glucosamine via 1H NMR analysis -----------------------------------S3

General procedure for investigation of “NaCl effect” on D-glucose in saline solution

(0.9% NaCl) via 1H NMR analysis ------------------------------------------------------------------S4

1H NMR shifts for marked peaks of monosaccharides and oligosaccharides in

different concentration of NaCl solutions ------------------------------------------------------S4

Marked peaks on 1H NMR spectrum -------------------------------------------------------------S8

1H NMR spectra of monosaccharides and oligosaccharides in different concentration

of NaCl solutions -------------------------------------------------------------------------------------S12

S2

General Information:

D(+)-Glucosamine hydrochloride was purchased from Shanghai Macklin

Biochemical Co., Ltd. Ethanol was purchased from Guangdong Test Agent

Technology Co., Ltd. N-Acetyl-D-glucosamine, glucose, D2O and NaCl were

purchased from Aladdin Industrial Corporation. Fructose was purchased

form Shanghai TCI Chemical Industry Development Co., Ltd. Sucrose and

trehalose were both purchased from Guangzhou Asegene Co., Ltd.

Stachyose（80%） was purchased from Macklin Co., Ltd. All reagents were

used without further purification. Saline solution (medical, 0.9%) was

purchased from Hebei Tiancheng Pharmaceutical Co. Ltd. Deionized water

was used in all experiments. All reagents were used without further

purification. 1H NMR spectra was recorded on Bruker AV-300 (300 MHz)

instrument.

Preparation for different concentration of NaCl solutions:

1. 4.8% NaCl solution: 1 g NaCl was dissolved in 20 mL deionized water.

2. 9.1% NaCl solution: 2 g NaCl was dissolved in 20 mL deionized water.

3. 13.0% NaCl solution: 3 g NaCl was dissolved in 20 mL deionized water.

4. 16.7% NaCl solution: 4 g NaCl was dissolved in 20 mL deionized water.

General procedure for investigation of “NaCl effect” on

monosaccharides via 1H NMR analysis

S3

Monosaccharide (0.5 mmol) was added into different concentration of

NaCl solution (6 mL) and the mixture was stirred for 6 h at room

temperature. After then, 1 mL of the reaction mixture was taken out and

was mixed with some ethanol (for fast evaporation); and the solvent

mixture was evaporated under reduced pressure at 37℃. Removal of

residual solvent in vaccum gave a crude product for 1H NMR to determine

chemical shift.

General procedure for investigation of “NaCl effect” on oligosaccharides

via 1H NMR analysis

Sucrose (107.0 mg, 0.31 mmol, Mw: 342.3 g/mol, 8 OHs/molecular) or

trehalose (107.0 mg, 0.31 mmol, Mw: 342.3 g/mol, 8 OHs/molecular) or

stachyose (148.8 mg, 0.18 mmol, 80%, Mw: 666.6 g/mol, 14

OHs/molecular) was added into different concentration of NaCl solution

(6 mL) and the mixture was stirred for 6 h at room temperature or 60℃;

After then, 1 mL of the reaction mixture was taken out and was mixed with

some ethanol (for fast evaporation); and the solvent mixture was

evaporated under reduced pressure below 50℃ . Removal of residual

solvent in vaccum gave a crude product for 1H NMR to determine chemical

shift.

General procedure for investigation volume impact (9.1% NaCl solution)

S4

of “NaCl effect” on N-acetyl-D-glucosamine via 1H NMR analysis

N-Acetyl-D-glucosamine (0.5 mmol) was added into 9.1 wt% NaCl solution

(1.5 mL, or 3 mL or 6 mL or 12 mL) and the mixture was stirred for 6 h at

room temperature. After then, 1 mL of the reaction mixture was taken out

and was mixed with some ethanol (for fast evaporation); and the solvent

mixture was evaporated under reduced pressure at 37℃. Removal of

residual solvent in vaccum gave a crude product for 1H NMR to determine

chemical shift.

General procedure for investigation of “NaCl effect” on D-glucose in

saline solution (0.9% NaCl) via 1H NMR analysis

D-glucose (0.5 mmol) was added into 9.1 wt% NaCl solution (1.5 mL, or 3

mL or 6 mL or 12 mL) and the mixture was stirred for 6 h at room

temperature. After then, 1 mL of the reaction mixture was taken out and

was mixed with some ethanol (for fast evaporation); and the solvent

mixture was evaporated under reduced pressure at 37℃. Removal of

residual solvent in vaccum gave a crude product for 1H NMR to determine

chemical shift.

1H NMR shifts for marked peaks of monosaccharides and

oligosaccharides in different concentration of NaCl solutions

S5

D-Glucose

Concentration 1 2 3 4 5 6

0.0% 5.143 4.551 3.841 3.665 3.408 3.161

4.8% 5.341 4.757 4.017 3.845 3.616 3.355

9.1% 5.633 5.061 4.276 4.129 3.926 3.664

13.0% 5.647 5.075 4.287 4.14 3.939 3.677

16.7% 5.644 5.071 4.283 4.136 3.936 3.674

saturated 5.647 5.075 4.287 4.14 3.939 3.676

Glucosamine hydrochloride

Concentration 1 2 3 4 5 6

0.50% 5.431 4.924 3.913 3.474 3.312 3.997

1.90% 5.519 5.022 3.992 3.555 3.395 3.093

N-acetyl-glucosamine

Concentration 1 2 3 4 5

0 5.18 4.67 3.84 3.65 3.46

4.8% 5.42 4.96 4.08 3.87 3.71

9.1% 5.6 5.14 4.26 4.05 3.89

13% 5.62 5.17 4.29 4.06 3.92

16.7% 5.62 5.17 4.28 4.06 3.92

saturated 5.64 5.18 4.30 4.07 3.93

Concentration 1 2 3 4 5 6

0.50% 5.17 4.579 3.865 3.69 3.437 3.187

2.40% 5.311 4.726 3.99 3.816 3.584 3.325

Concentration 1 2 3 4 5 6

0 5.45 4.93 3.89 3.52 3.31 3.01

4.8% 5.64 5.15 4.05 3.71 3.53 3.23

9.1% 5.8 5.31 4.23 3.86 3.70 3.41

13% 5.89 5.41 4.32 3.95 3.82 3.52

16.7% 5.94 5.44 4.35 3.98 3.86 3.56

saturated 5.93 5.45 4.35 3.99 3.85 3.57

Concentration 1 2 3 4 5

0.50% 5.162 4.672 3.831 3.649 3.439

S6

N-acetyl-glucosamine in 9.1 wt%

volume 1 2 3 4 5

1.5ml 5.314 4.857 4.006 3.775 3.601

3ml 5.364 4.911 4.054 3.823 3.659

6ml 5.6 5.14 4.26 4.05 3.89

12ml 5.583 5.137 4.257 4.05 3.886

Fructose

Concentration 1 2 3 4 5 6

0 4.091 4.025 3.895 3.784 3.706 3.555

4.8% 4.204 4.133 4.011 3.892 3.818 3.666

9.1% 4.467 4.382 4.278 4.143 4.089 3.928

13% 4.543 4.453 4.355 4.216 4.16 4.002

16.7% 4.543 4.456 4.354 4.216 4.159 4.002

saturated 4.543 4.461 4.359 4.22 4.167 4.006

Sucrose (room temperature)

Concentration 1 2 3 4

0 5.362 4.176 3.994 3.412

4.8% 5.515 4.325 4.145 3.569

9.1% 5.756 4.562 4.377 3.813

13% 5.823 4.63 4.441 3.88

16.7% 5.826 4.633 4.445 3.884

saturated 5.827 4.634 4.446 3.885

60℃

1 2 3 4

0 5.351 4.166 3.984 3.401

4.8% 5.624 4.432 4.251 3.68

9.1% 5.725 4.532 4.348 3.781

13% 5.823 4.63 4.442 3.881

16.7% 5.82 4.627 4.439 3.878

1.90% 5.227 —— 3.889 3.684 3.498

Concentration 1 2 3 4 5 6

0.50% 4.081 4.017 3.883 3.77 3.693 3.538

2.40% 4.2 4.127 4.007 3.886 3.813 3.66

S7

saturated 5.822 4.629 4.44 3.88

Trehalose (room temperature)

Concentration 1 2 3 4

0 5.145 3.828 3.613 3.39

4.8% 5.416 4.119 3.909 3.684

9.1% 5.538 4.253 4.043 3.817

13% 5.601 4.323 4.112 3.886

16.7% 5.597 4.319 4.108 3.882

saturated 5.6 4.322 4.111 3.885

60℃

Concentration 1 2 3 4

0 5.139 3.822 3.607 3.384

4.8% 5.287 3.98 3.769 3.545

9.1% 5.543 4.259 4.049 3.823

13% 5.592 4.314 4.103 3.877

16.7% 5.595 4.317 4.106 3.88

saturated 5.598 4.32 4.109 3.883

Stachyose (room temperature)

Concentration 1 2 3 4

0 5.371 4.931 4.175 3.607

4.8% 5.568 5.144 4.369 3.815

9.1% 5.721 5.314 4.52 3.982

13% 5.822 5.425 4.621 4.094

16.7% 5.842 5.437 4.642 4.115

saturated 5.848 5.451 4.648 4.122

60℃

Concentration 1 2 3 4

0 5.393 4.951 4.197 3.627

4.8% 5.586 5.163 4.387 3.834

9.1% 5.721 5.313 4.52 3.98

13% 5.843 5.446 4.642 4.114

16.7% 5.843 5.446 4.641 4.114

saturated 5.848 5.452 4.648 4.121

S8

Marked peaks on 1H NMR spectrum

S9

S10

S11

S12

1H NMR spectra of D-(+)-Glucose

S13

1H NMR spectra of D-(+)-Glucosamine hydrochloride

S14

1H NMR spectra of Frucrose

S15

1H NMR spectra of sucrose

S16

S17

1H NMR spectra of trehalose

S18

S19

1H NMR spectra of stachyose

S20

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