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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Hydrogen Bond Length as a Key to Understanding Sweetness Fabio Bruni, Camilla Di Mino, Silvia Imberti, Sylvia E McLain, Natasha Hazel Rhys, and Maria Antonietta Ricci J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01280 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Hydrogen Bond Length as a Key to Understanding Sweetness F. Bruni,† C. Di Mino,† S. Imberti,‡ S. E. McLain,¶ N. H. Rhys,¶ and M. A. Ricci∗,† †Dipartimento di Scienze, Sezione di Nanoscienze, Universit` a degli Studi “Roma Tre”, Via della Vasca Navale 84, 00146 Roma, Italy. ‡ISIS Neutron and Muon source, STFC, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, United Kingdom. ¶Department of Biochemistry, University of Oxford, South Park Road, Oxford, Oxfordshire OX1 3QU, UK. E-mail: [email protected] Phone: +39 0655737226

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Abstract Neutron diffraction experiments have been performed to investigate and compare the structure of the hydration shell of three monosaccharides, namely fructose, glucose and mannose. It is found that, in spite of their differences with respect to many thermodynamical quantities, bio-protective properties against environmental stresses and taste; the influence of these monosaccharides on the bulk water solvent structure is virtually identical. Conversely, these sugars interact with the neighboring water molecules by forming H-bonds of different length and strength. Interestingly, the sweetness of these monosaccharides, along with that of the disaccharide trehalose, is correlated with the length of these H-bonds. This suggests that the small differences in stereochemistry between the different sugars determine a relevant change in polarity, which has a fundamental impact on the behavior of these molecules in vivo.

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The interaction of sugars with proteins is of great interest for the pharmaceutical and food industries, as these interactions help stabilize biological components in aqueous solutions 1–5 and drive living organisms towards recognition of energy rich nutrients, through the perception of sweet taste. 6 In this regard, it is important to note that the interaction between sugar and proteins occurs in the presence of water, either within a cell or in the saliva. This suggests that water undoubtedly plays an essential role, perhaps through mediation, in the protein-sugar interaction. Relatively recently it has been suggested 7–10 that to elicit sweet taste, a sugar molecule needs to occupy a specific binding pocket within the appropriate receptor, where it associates with a protein via hydrogen bonds (at least two at a donor and an acceptor site, respectively). This bonding is thought to expose a hydrophobic region of the bound sugar to a complementary one within the receptor pocket. What is still unclear is why a particular sugar is perceived sweeter than another, given the almost identical chemical structure of all sugar molecules. The sweetness ranking of a particular sugar neither correlates with the sugar solubility, nor with the number of glucose rings in its structure, and among monosaccharides, sweetness appears to be correlated with apparently minor differences in glucose ring stereochemistry. For instance, in a relative sweetness scale where sucrose has sweetness of 1, single-ringed mannose is considered tasteless, while glucose is 0.47 and fructose is 1.13. 11 Given that all of these interactions necessarily take place in aqueous solution, an understanding at an atomistic level of the sugar-water H-bonding can greatly contribute to a better fundamental understanding of this issue, be designated as a benchmark for new ab-initio simulations 12 and drive the synthesis of new artificial sweeteners. The present study is devoted to revealing the atomistic interactions of the smaller sugars, namely three monosaccharides, with the water molecules surrounding them within an aqueous environment in order to provide insight into sugar-receptor binding in vivo. Neutron diffraction with isotopic substitution, augmented by atomistic computer simulation tools is a powerful method to investigate the hydration structure of small biological molecules, such as peptides, drugs and saccharides, 13–21 as this technique is particularly sen-

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sitive to the O· · · H correlations. The differential selectivity of neutrons for hydrogen and deuterium nuclei 22 allows several isotopically distinct solutions to be measured, where these data are structurally equivalent, yet giving rise to different diffraction patterns. 23,24 Indeed all measured diffraction patterns, Fα (Q), are linear combinations of the same site-site partial structure factors, Sij (Q):

Fα (Q) =

XX i

wij [Sij (Q) − 1]

(1)

j≥i

where α encompasses all isotopically substituted solutions, i, j all atomic pairs, Q is the exchanged momentum in the neutron-nucleus interaction, and the wij coefficients weight the contribution of the individual i, j pairs, accounting for their concentration and neutron scattering lengths. 22 The structure factors, Sij (Q), being the Fourier transform of the site-site radial distribution functions, gij (r), are the quantities to be extracted from the experiment. Obviously, if the number of atomic components increases the number of partials, Sij (Q)s and gij (r)s, also increases. As a consequence the number of available diffraction patterns may not be enough to extract complete structural information from the experimental data alone. To overcome this problem, here we complement the experiment with a computer simulation, performed by using the Empirical Potential Structure Refinement code (EPSR); 25–27 a Monte Carlo simulation that is constrained by the experimental data. In the following we show and comment on previously published results obtained through the study of aqueous solutions of glucose 19 and mannose 20 at a relative concentration of 1 solute per 50 water molecules, along with new measurements on aqueous solutions of fructose at two concentrations, namely 1 solute molecule per 50 and per 12.5 water molecules, respectively. All experiments have been performed at ambient temperature. Details on sample preparation, experimental method, and EPSR simulations are given in the Supporting Information; only the radial distribution functions relevant to the water-sugar H-bonding are shown and discussed below. These three monosaccharides have been chosen for the following reasons: glucose is one of the most prevalent constituents of many disaccharides and thus 4

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alpha-glucose

beta-glucose

Oring

Oring

Oring

alpha-mannose

beta-mannose

Oring

Oring H11

fructopyranose

fructofuranose

O22 Oring

O22

O11 H22 H22

Figure 1: (Color online) Space filling models of the predominant anomers of the three monosaccharides in aqueous solution and atom labeling. All carbon atoms are labeled C; the OH groups of fructose belonging to the CH2 OH groups are labeled O11 H11 and O22 H22 in the fructo-pyranose and fructo-furanose anomers respectively; all other hydroxyl oxygens and hydrogens are labeled O and H respectively, while the hydrogens bonded to a carbon atom are labeled M.

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can be considered as reference for the hydration of many other compounds; mannose, in spite of minor stereochemical differences compared to glucose, elicits a bitter aftertaste, usually ascribed to its β anomer; 28,29 fructose has been selected because is the sweetest among the group. In the investigated aqueous solutions, fructose has an equilibrium composition comprised of fructo-pyranose and fructo-furanose at relative concentration of about 70% and 22%, respectively (the remaining 8% is a mixture of three other variants, including the acyclic structure, and has not been taken into account in this study), thus adding a degree of complication with respect to the other two sugars. Models of the anomers used to build the EPSR simulation box are shown in Fig. 1, along with the labels used hereafter to identify relevant atomic sites; water atoms are labeled Ow and Hw . In principle, any solute is expected to disturb the structure of water by distorting and breaking the tetrahedral network of H-bonds in the bulk water structure, either because of a competition between water-water H-bonding and water-solute interaction forces (as occurs for electrolyte solutions, 24,31–33 ) or as a result of the volume needed to accommodate the solute 34 within the solution. The comparison, reported in Fig. 2, of the radial distribution functions of the water atomic pairs, namely Ow –Ow , Ow –Hw and Hw –Hw , in the investigated sugar-containing solutions with those of pure water 30 shows that changes of the intensity and position of the first peaks are not large, suggesting minor changes to the bulk water structure. More importantly, these functions are almost identical for all three monosaccharides at the same concentration, suggesting that the differences in thermodynamic, bio-protecting and taste properties of these sugars are not correlated with their influence on the water structure. In particular, looking at the gOw Ow (r) in all of the sugar solutions examined, the intensity of the first peak is higher and the second peak moves to shorter distances, compared to pure water: this is likely due to an excluded volume effect related to the solvation of molecules occupying a larger volume compared to that of water. 34 Thus, it is not surprising that the three monosaccharides have almost the same effect on the water structure, and the slightly larger distortion seen in the case of fructose may be due to the presence of its furanose

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6

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2 0

4 g(r)

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1

2

3

4

5

OwOw

3

fructose solution glucose solution mannose solution pure water

2 HwHw

1 OwHw

0

0

2

4 r(Å)

6

8

Figure 2: (Color online) Site-site radial distribution functions of water in monosaccharide solutions, compared with those of pure water (blue line): 30 data relative to fructose, glucose 19 and mannose 20 at a concentration of 1 sugar molecule per 50 water molecules are reported as red, black and green lines respectively. gHw Hw (r) and gOw Ow (r) functions have been shifted for clarity. The inset shows the comparison of pure water data with those of fructose at two concentrations, namely the already quoted 1:50 and 1:12.5 (magenta line).

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form. Although fructo-furanose has a smaller ring compared to that of fructo-pyranose, it may require a larger volume in the solution due to the presence of two CH2 OH groups. The inset of Fig. 2 reports the comparison with pure water of the two fructose concentrations investigated, namely 1 sugar molecule per 50 and per 12.5 molecules of water, respectively. Here, a more severe distortion of the g(r)s at the highest fructose concentration is visible, in particular for the position of the second peak of the gOw Ow (r), that is the signature of the tetrahedral order in pure water. 35 This is expected as the number of water molecules needed to completely hydrate a monosaccharide is of the order of 10, 20 thus at the highest fructose concentration two solute molecules are likely separated by at most two layers of water molecules, resulting in a larger perturbation of the solvent structure compared to the less concentrated solutions. In addition, a more severe excluded volume effect is expected. These data compare well with those resulting from Molecular Dynamics simulations of Roberts and Debenedetti 36 , provided that similar concentrations are compared. Fig. 3A confirms that all sugars exhibit a potential hydrophobic interaction site for eventual binding to the taste receptor, as evidenced by a clear lack of hydrogen bonding with the ether oxygen on the saccharide ring (Oring ); the gOring Hw (r) increases smoothly from r ∼ 1.5 ˚ A towards a broad maximum at ∼ 5.7 ˚ A. In the case of fructose, conversely to what was done in previous works on the other two monosaccharides, 19,20 the hydroxymethyl groups (-CH2 OH) have been distinguished from all other OH groups in the EPSR simulation and are labeled O11 H11 and O22 H22 for the pyranose and furanose anomers, respectively. The correlations of these fructose-specific sites with water sites (Hw and Ow ) are shown in Fig.3A and 4A, respectively. Inspection of these figures shows that the O11 H11 and O22 H22 groups make somewhat shorter H-bonds with water, compared to the other OH sites on fructose. Indeed, the gH11 Ow (r), gH22 Ow (r), gO11 Hw (r) and gO22 Hw (r) functions have a first peak (the H-bond peak) centered at ∼ 1.77 ˚ A, while the peaks of the gOHw (r) and gHOw (r) functions are found at ∼ 1.8 ˚ A, for both fructopyranose and fructo-furanose solutes. Figs. 3B and 4B show a magnified view of the gOHw (r)

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2.5

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2.0

g(r)

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0.6

OHw

1.5

0.4

1.0 OringHw

0.5 0.0

0.2

A 1

2

3

4 5 r(Å)

6

7

0.0 1.0 1.5 2.0 2.5 3.0 3.5 r(Å)

8

Figure 3: (Color online) Site-site radial distribution functions of water hydrogens and monosaccharide oxygens at concentration of 1 sugar per 50 water molecules. Data relative to fructose solutions are reported in red and blue for the two anomeric forms respectively; data for glucose solution are reported in black and those for mannose solution in green. A) The radial distribution function of water hydrogens and oxygens on the monosaccharide rings, labeled Oring are shown at the bottom; those of water hydrogens and oxygens of the hydroxyl groups, labeled O (see Fig.1 and its caption), are vertically shifted by 1, and those of water hydrogens and oxygens of the CH2 OH groups, labeled O11 and O22 , are vertically shifted by 2. Note that the differences between the last two curves are within the experimental uncertainty. The vertical dotted line marks the position of the H-bond peak between water hydrogens and the O11 and O22 sites. B) A close-up of the radial distribution function of water hydrogens and oxygens of the hydroxyl groups (OHw ).

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and gHOw (r) functions, respectively. This shows that the H-bond peak in the case of both fructose anomers appears at the same position and has almost the same intensity as the peak found in the case of glucose solutions. Instead mannose forms longer (∼ 1.95 ˚ A) and thus weaker bonds with water, particularly at the O sites: we note also that the intensity of the corresponding peak is strongly depressed in this case. The structure of the hydration shell of fructose does not sensibly depend on the solute concentration (see Supporting Information). At the bottom of Fig. 4A, we show the hydration of the M sites, which are not involved in H-bonds, as expected, being bonded to a carbon atom.

1.0

A

3.0

B H11Ow H22Ow

2.5

0.8

2.0

0.6 HOw

1.5

g(r)

g(r)

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0.4

1.0 MOw

0.5 0.0

0.2

0

2

4 r (Å)

6

8

fructopyranose fructofuranose glucose mannose

0.0 1.0 1.5 2.0 2.5 3.0 3.5 r(Å)

Figure 4: (Color online) Site-site radial distribution functions of water oxygens and monosaccharide hydrogens at concentration of 1 sugar per 50 water molecules. Data relative to fructose solutions are reported in red and blue for the two anomeric forms respectively; data for glucose solution are reported in black and those for mannose solution in green. A) The radial distribution function of water oxygens and methyl hydrogens, labeled M, are shown at the bottom; those of water oxygens and hydrogens of the hydroxyl groups, labeled H, are vertically shifted by 1, and those of water oxygens and hydrogens of the CH2 OH groups, labeled H11 and H22 , are vertically shifted by 2. The vertical dotted line marks the position of the H-bond peak between water oxygens and the H11 and H22 sites. B) A close-up of the radial distribution function of water hydrogens and oxygens of the hydroxyl groups (HOw ).

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While all monosaccharides expose hydrophilic sites to the surrounding water solvent via the OH groups as well as one hydrophobic region corresponding to the -C-O-C oxygen site on the pyranose or furanose ring, as required by the atomic model for the sweet taste (“sweetness triangle”), 7–10,20 we find that the affinity of these sugar molecules for water, as measured by the H-bond length, is different. To put this result on a more quantitative basis, we show in Fig. 5 the correlation between the sweetness intensity (data taken from Portmann and Birch 11 ) and the average distance, rHB , between Ow · · · H and O· · · Hw H-bonded sites (The average has been calculated taking into account all oxygen and hydrogen sites on the sugars and weighting by the relative concentrations of anomers). The sweetness and rHB of the disaccharide trehalose 13 have also been included in the plot as a comparison, with trehalose as the least sweet of these sugars. Since it is a disaccharide, we report here the rHB value obtained for the solution of 1 trehalose molecule per in 100 water molecules, to adjust for the number of molecules per sugar ring as in the monosaccharide solutions considered in the present study. This figure suggests a clear correlation between sweetness and length of the sugar-water hydrogen bond. This may in fact explain why the investigated monosaccharides have different taste in spite of their similarity and why trehalose, that is made by two glucose rings is not as sweet as two sugar molecules, but almost tasteless. In conclusion, following our analysis of neutron diffraction with isotopic substitution experiments on three monosaccharide solutions within the EPSR Monte Carlo code, 25–27 we suggest that the influence of these sugars on the structure of water as a solvent cannot explain the differences in their taste. We propose instead that the differences in taste can be understood by measurement of the H-bonding interactions between water and sugars. Indeed, while all the investigated monosaccharides satisfy the “triangle of sweetness” model 7,8,10 for compounds eliciting a sweet taste, their sweetness intensity is markedly different and decreases as the length of the H-bonds with water (and likely with the protein at the receptor surface) increases. This implies that not only the spatial arrangement of H-bond donors and acceptors within the molecule and their number is important in determining the taste of a

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sweetness (arb. units)

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F 50 40 30 20

G

10

M T

0 1.7

1.8

1.9

2.0

2.1

rHB (Å)

Figure 5: Sweetness intensity as function of the average length of the hydrogen bonds between the sugars and water. Points are labeled by the initials of the sugars: F, G, M, and T for the disaccharide trehalose. The red curve is a guide for the eyes.

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sugar, but that the strength of the H-bonding interaction is the most important determinant of taste. In this framework, the case of trehalose is intriguing. This solute consists of two glucose rings, although linked differently compared to all other disaccharides, yet its sweetness is considerably lower compared to the investigated monosaccharides. This observation indicates not only that a single glucose ring is enough to activate the sweet taste signal transduction to the brain, but also that a disaccharide is required to fold in order to fit into the receptor pocket. Interestingly, a recent Raman investigation of aqueous solutions of the three monosaccharides studied here 37 confirms that glucose and fructose interact strongly with water, while the two anomers of mannose have a different behavior, with β-mannose interacting very weakly with water. The observed differences in the H-bonding with water suggest that the small differences in the stereochemistry of these sugars likely determines relevant changes of the electronic clouds of these molecules, which would be interesting to investigate by quantum simulations. Finally, we notice that the almost negligible, yet similar, perturbation brought by the investigated carbohydrates to the tetrahedral structure of water cannot be taken as a key to explain the differences in terms of bio-protection (both against dehydration and temperature stress) and glass transition temperatures observed for the solutes. In particular, as far as the bio-protection mechanism is concerned, the hypotheses that carbohydrates act as water substitutes 38 or by segregating water at the surface of a protein 39 are better supported by our data.

Acknowledgement This work has been performed within the Agreement No.0018318 (02/06/2014) between STFC and CNR, concerning collaboration in scientific research at the spallation neutron source ISIS and with partial financial support of CNR. Beamtime awarded by ISIS under

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RB numbers 810202, 1520061 and 1520065 is gratefully acknowledged. SEM and NHR thank The Leverhulme Trust (UK) for the provision of research funding (RPG-2015-135). CDM, FB and MAR gratefully acknowledge the Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314 337 LEGGE 232/2016).

Supporting Information Available Sample preparation. Details about the neutron diffractometer and data collection. EPSR code and simulation box. Comparison between fructose solution data at two concentrations. This material is available free of charge via the Internet at http://pubs.acs.org/.

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(6) Chaudhari, N.; Roper, S. D. The Cell Biology of Taste. The Journal of Cell Biology 2010, 190, 285–296. (7) Shallenberger, R. S.; Acree, T. E. Molecular Theory of Sweet Taste. Nature 1967, 216, 480 – 482. (8) Kier, L. B. A Molecular Theory of Sweet Taste. J. Pharm. Sci. 1972, 61, 13941397. (9) Nofre, C.; Tinti, J.-M. Sweetness Reception in Man: the Multipoint Attachment Theory. Food Chem. 1996, 56, 263–274. (10) Eggers, S. C.; Acree, T. E.; Shallenberger, R. S. Sweetness Chemoreception Theory and Sweetness Transduction. Food Chem. 2000, 68, 4549. (11) Portmann, M.-O.; Birch, G. Sweet Taste and Solution Properties of α,α−trehalose. Journal of the Science of Food and Agriculture 1995, 69, 275–281. (12) Stubbs, J. M.; Marx, D. Glycosidic Bond Formation in Aqueous Solution: On the Oxocarbenium Intermediate. Journal of the American Chemical Society 2003, 125, 10960–10962, PMID: 12952477. (13) Pagnotta, S. E.; McLain, S. E.; Soper, A. K.; Bruni, F.; Ricci, M. A. Water and Trehalose: How Much Do They Interact with Each Other? The Journal of Physical Chemistry B 2010, 114, 4904–4908, PMID: 20297794. (14) O’Dell, W. B.; Baker, D. C.; McLain, S. E. Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution. PloS ONE 2012, 7, e45311. (15) Silva-Santisteban, A.; Steinke, N.; Johnston, A. J.; Ruiz, G. N.; Carlos Pardo, L.; McLain, S. E. On the Structure of Prilocaine in Aqueous and Amphiphilic Solutions. Phys. Chem. Chem. Phys. 2017, 19, 12665–12673.

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(16) Tavagnacco, L.; Brady, J. W.; Bruni, F.; Callear, S.; Ricci, M. A.; Saboungi, M. L.; Cesro, A. Hydration of Caffeine at High Temperature by Neutron Scattering and Simulation Studies. The Journal of Physical Chemistry B 2015, 119, 13294–13301, PMID: 26421842. (17) Johnston, A. J.; Busch, S.; Pardo, L. C.; Callear, S. K.; Biggin, P. C.; McLain, S. E. On the Atomic Structure of Cocaine in Solution. Phys. Chem. Chem. Phys. 2016, 18, 991–999. (18) Steinke, N.; Genina, A.; Lorenz, C. D.; McLain, S. E. Salt Interactions in Solution Prevent Direct Association of Urea with a Peptide Backbone. The Journal of Physical Chemistry B 2017, 121, 1866–1876, PMID: 28134523. (19) Maugeri, L.; Busch, S.; McLain, S. E.; Pardo, L. C.; Bruni, F.; Ricci, M. A. StructureActivity Relationships in Carbohydrates Revealed by their Hydration. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861, 1486–1493. (20) Rhys, N. H.; Bruni, F.; Imberti, S.; McLain, S. E.; Ricci, M. A. Glucose and Mannose: A Link between Hydration and Sweetness. The Journal of Physical Chemistry B 2017, acs.jpcb.7b03919. (21) Steinke, N.; Genina, A.; Gillams, R. J.; Lorenz, C. D.; McLain, S. E. Proline and Water Stabilization of a Universal Two-Step Folding Mechanism for -Turn Formation in Solution. Journal of the American Chemical Society 2018, 140, 7301–7312. (22) Sears, V. F. Neutron Scattering Lengths and Cross Sections. Neutron News 1992, 3, 26–37. (23) McLain, S. E.; Benmore, C. J.; Siewenie, J. E.; Urquidi, J.; Turner, J. F. C. On the Structure of Liquid Hydrogen Fluoride. Angew. Chem.-Int. Ed. 43, 1952–1955.

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(24) McLain, S. E.; Imberti, S.; Soper, A. K.; Botti, A.; Bruni, F.; Ricci, M. A. Structure of 2 molar NaOH in Aqueous Solution from Neutron Diffraction and Empirical Potential Structure Refinement. Phys. Rev. B 2006, 74, 094201. (25) Soper, A. Empirical Potential Monte Carlo Simulation of Fluid Structure. Chem. Phys. 1996, 202, 295–306. (26) Soper, A. K. Partial Structure Factors from Disordered Materials Diffraction Data: An Approach Using Empirical Potential Structure Refinement. Phys. Rev. B 2005, 72, 104–204. (27) Soper, A. K. Radical Re-appraisal of Water Structure in Hydrophilic Confinement. Chem. Phys. Lett. 2013, 590, 1–15. (28) Steinhardt, R. G.; Calvin, A. D.; Dodd, E. A. Taste-Structure Correlation with α-DMannose and β-D-Mannose. Science 1962, 135, 367–368. (29) Stewart, R. A.; Carrico, C. K.; Webster, R. L.; Steinhardt, R. G. Physicochemical Stereospecificity in Taste Perception of -D-Mannose and -D-Mannose. Nature 1971, 234, 220. (30) Soper, A. K. The Radial Distribution Functions of Water as Derived from Radiation Total Scattering Experiments: Is There Anything We Can Say for Sure? ISRN Phys. Chem. 2013, 2013, 1–67. (31) Imberti, S.; Botti, A.; Bruni, F.; Cappa, G.; Ricci, M. A.; Soper, A. K. Ions in Water: The Microscopic Structure of Concentrated Hydroxide Solutions. The Journal of Chemical Physics 2005, 122, 194509. (32) Mancinelli, R.; Botti, A.; Bruni, F.; Ricci, M. A.; Soper, A. K. Perturbation of Water Structure Due to Monovalent Ions in Solution. Phys. Chem. Chem. Phys. 2007, 9, 2959–2967. 17

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(33) Mancinelli, R.; Botti, A.; Bruni, F.; Ricci, M. A.; Soper, A. K. Hydration of Sodium, Potassium, and Chloride Ions in Solution and the Concept of Structure Maker/Breaker. The Journal of Physical Chemistry B 2007, 111, 13570–13577. (34) Soper, A. K. The Excluded Volume Effect in Confined Fluids and Liquid Mixtures. Journal of Physics: Condensed Matter 1999, 9, 2399–2410. (35) Soper, A.; Ricci, M.-A. Structures of High-Density and Low-Density Water. Physical Review Letters 2000, 84, 2881–2884. (36) Roberts, C. J.; Debenedetti, P. G. Structure and Dynamics in Concentrated, Amorphous CarbohydrateWater Systems by Molecular Dynamics Simulation. The Journal of Physical Chemistry B 1999, 103, 7308–7318. (37) Ruggiero, L.; Sodo, A.; Bruni, F.; Ricci, M. A. Hydration of Monosaccharides Studied by Raman Scattering. Journal of Raman Spectroscopy 2018, in press. (38) Sola-Penna, M.; Meyer-Fernandes, J. R. Stabilization against Thermal Inactivation Promoted by Sugars on Enzyme Structure and Function : Why Is Trehalose More Effective Than Other Sugars? Arch. Biochem. Biophys. 1998, 360, 10–14. (39) Corradini, D.; Strekalova, E. G.; Stanley, H. E.; Gallo, P. Microscopic Mechanism of Protein Cryopreservation in an Aqueous Solution with Trehalose. Scientific Reports 2013, 3, 1218.

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