Trehalose in Water Revisited - ACS Publications

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B: Liquids, Chemical and Dynamical Processes in Solution, Spectroscopy in Solution

Trehalose in Water Revisited Alan K. Soper, Maria Antonietta Ricci, Fabio Bruni, Natasha H Rhys, and Sylvia E McLain J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03450 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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The Journal of Physical Chemistry

Trehalose in Water Revisited Alan K. Soper,∗,†,§ Maria Antonietta Ricci,‡ Fabio Bruni,‡ Natasha H. Rhys,¶ and Sylvia E. McLain¶ †ISIS Facility, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot, OX11 0QX, UK ‡Dipartimento di Scienze, Università degli Studi “Roma Tre”, 00146 Roma, Italy ¶Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK §also at: School of Physics and Astronomy, E C Stoner Building, University of Leeds, Leeds, LS2 9JT, UK E-mail: [email protected]

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Abstract Trehalose, commonly found in living organisms, is believed to help them survive severe environmental conditions, such as drought or extreme temperatures. With the aim of trying to understand these properties, two recent neutron scattering studies investigate the structure of trehalose water solutions, but come to seemingly opposite conclusions. In the first study, which looks at two concentrations of trehalose:water mole ratios of 1:100 and 1:25, the conclusion is that trehalose hydrogen-bonds to water rather weakly, and has a relatively minor impact on the structure of water in solution compared to bulk water. On the other hand for the other, using a mole ratio of 1:38, the conclusion is that water structure is rather substantially modified by the presence of trehalose, and that the hydrogen bonding between water and trehalose hydroxyl groups is significant. In an attempt to try to understand the origin of these divergent views, which arise from similar but independent analyses of different neutron diffraction data, we have performed additional X-ray scattering experiments, which are highly sensitive to water structure, at the same trehalose:water concentrations used in the first study, and combined these with empirical potential structure refinement (EPSR) on the previously collected neutron data. The new analysis confirms unequivocally that trehalose does indeed have only a minor impact on the structure of water, at all three concentrations, and forms relatively weak hydrogen bonds with water. Far from being discrepant with the existing literature, our new analysis of the different datasets suggests a natural explanation for the increased glass transition temperature of trehalose compared to other sugars, and hence its enhanced effectiveness as a protectant against drought stress.

Introduction There is a general consensus in the literature on the peculiar role and properties of trehalose, compared to other sugars, as a stabilizer and protective agent against environmental stresses (in particular drought). 1–4 The beneficial role of trehalose should depend on the mechanism 2

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of its interaction with enzymes, proteins, and biomembranes, which are preserved against stressing conditions when embedded in trehalose aqueous matrixes. 5–8 The mechanism by which trehalose achieves this is not precisely known, but is believed to be by one of the following possible mechanisms. Namely either it helps to vitrify the water, preventing the formation of destructive crystalline ice upon cooling, or it prevents protein denaturation upon dehydration through two possible processes. That is, either acting as a water substitute, 9 or segregating water at the surface of a protein. 10 Understanding the peculiarities of the trehalose-water interaction is then considered crucial in order to shed light on this issue. Consequently, several studies dealing with dynamical or thermodynamical properties of trehalose-water solutions, as a function of temperature and concentration, have been published so far. 11–15 In comparison with other carbohydrates solutions, these studies have showed a larger dynamical slowing down of the trehalose-water system and a larger modulus of the heat of solution. Direct measurements of the microscopic structure of trehalose-water solutions have been performed by nuclear magnetic resonance (NMR) studies, looking at the conformation of trehalose in solution, 16 and by neutron diffraction. 17,18 In particular, the latter two papers, starting from neutron total scattering experiments, seemingly claimed different findings for the water structure and degree of hydrogen bonding between water and sugar molecules in the system trehalose in water, the only differences between the studies being the concentration of sugar, and the differing degrees of hydrogen isotope substitution used. Pagnotta et al. measured the solution structure at sugar:water mole ratios of 1:100 and 1:25, with hydrogen isotope substitution on only the hydroxyl and water hydrogen atoms, and found relatively low impact of the trehalose on water structure at either concentration, together with rather weak hydrogen bonding of water molecules to the sugar. On the other hand Olsson et al.,at a mole ratio of 1:38 and with hydrogen isotope substitution on the 14 bound (CH and CH2 ) hydrogen atoms as well as the water and hydroxyl hydrogen atoms claimed to see a marked impact on water structure caused by the sugar, as well as significant hydrogen bonding of water to the sugar, although even in their

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case the hydrogen bonding was notably weaker than that found between water molecules. Olsson et al. claim it is the different deuteriations used between the two experiments that gives rise to the different interpretations. Given that Pagnotta et al. saw relatively marginal impacts on the water structure at both concentrations, which span that used by Olsson et al., it seems unlikely that water structure is strongly perturbed at a mole ratio of 1:38, but not at 1:25 or 1:100. In an attempt to understand these discrepancies, and bearing in mind the importance assigned to the trehalose-water hydrogen bonding in the literature, we have performed additional X-ray total scattering experiments on trehalose-water solutions at the same concentrations as Pagnotta et al., namely trehalose:water mole ratios of 1:25 and 1:100. X-rays are relatively weakly scattered by hydrogen atoms compared to neutrons and so the additional datasets are highly complementary to the existing neutron scattering data. In particular, because of the abundance of water molecules in all of these solutions, the X-ray scattering data are particularly sensitive to the water oxygen radial distribution function, so that substantial changes to the water structure should be readily discernible in the X-ray diffraction data. These new X-ray data were combined with the previous neutron scattering data from Pagnotta et al. in an empirical potential structure refinement (EPSR) 19,20 computer simulation of the structure at both concentrations. The results from these new data and analyses are given below, where it will be seen that the basic conclusions of Pagnotta et al. do not change markedly as a consequence. At the same time, we have reanalyzed the Olsson et al. data, and report here the comparison of this new analysis with the original Olsson et al. text. The new analysis and comparison with the Pagnotta et al. data return a consistent description of the hydrogen bonding interaction between water and trehalose across the entire range of concentrations investigated in previous publications.

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Theory and Methods Theory The basic quantity measured in X-ray or neutron total scattering experiments is, after corrections, the differential scattering cross section (DCS), which depends on a weighted sum of the partial structure factors, Hαβ (Q), between pairs of atom types (in reciprocal or momentum space) and these structure factors are related by Fourier transform to the corresponding site-site radial distribution functions in real space, gαβ (r). For X-rays, which are scattered by electrons, this scattering is, within the independent atom approximation, normalised to the scattering from single atoms, to remove the effect of the finite-sized electron distributions around each atom, and produce the equivalent of the site-site partial structure factors observed in neutron scattering: X 1 cα cβ fα (Q)fβ (Q)(2 − δαβ )Hαβ (Q) 2 α cα fα (Q) αβ

FX (Q) = P

(1)

where cα and fα (Q) are the atomic fraction and X-ray form factor for atoms of type α, and Z Hαβ (Q) = 4πρ



r2 (gαβ (r) − 1)

0

sin Qr dr Qr

(2)

with ρ the total atomic number density for the system. For neutron scattering a similar equation holds, with the Q-dependent X-ray form factors replaced with neutron scattering lengths, bα , which can be isotope dependent, but not Q dependent at the neutron energies used for these experiments. Hence the neutron data are typically not normalised to the single atom scattering:

FN (Q) =

X

cα cβ bα bβ (2 − δαβ )Hαβ (Q)

(3)

αβ

In the neutron scattering case the difference in scattering length between hydrogen (bH = 5

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-3.74 fm) and deuterium (bD = +6.67 fm) is exploited, on the assumption that changing the isotope does not change the structure sufficiently to be noticeable. In the present case the isotope substitution is performed only on the exchangeable hydroxyl hydrogen atoms (H, 8 in total per trehalose molecule) and the water hydrogen atoms (HW), whereas with Olsson et al. they additionally performed isotope substitution on the methyl hydrogen atoms (M, 14 in total per trehalose molecule).

EPSR simulations Molecules of trehalose are first built using the molecular mechanics package within the Jmol program, 21 then structure-refined using the MOPAC-7 semi-empirical molecular orbital package, 22 using the AM1 Hamiltonian method. The structure produced by this method is close to that found in the crystalline dihydrate, 23 in the anhydrous crystal, 24 and also by solution NMR 25 . In the latter case there is little evidence that the molecular conformation changes very much with concentration. An example of the resulting molecule plus atom labels used in the EPSR simulations is shown in Figure 1. Once the trehalose molecule is created, the hydroxyl O*-H bond is allowed to rotate freely about relevant O*-C bond in all subsequent simulations. (Here O* represents one of O, O1, O2 or O3 oxygen atoms on the trehalose molecule.) All other atom positions are constrained by the relevant bond distances, bond angles and dihedral angles. Rotations about the C-O1 bonds at the centre of the trehalose molecule are also constrained by the respective model dihedral angles about these bonds. The Lennard-Jones and Coulomb charges used in the EPSR starting (reference) potential are the same as those used previously, 17,18 and are given in Table 1 for completeness. The EPSR simulation starts with a standard Monte Carlo computer simulation using the assumed reference potential and molecular structures and starting from a completely randomised simulation box (molecules placed at random positions and orientations). Once this has reached equilibrium the empirical potential derived from the difference between the calculated and measured differential cross sections, calculated 6

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Figure 1: Atom labeling for the trehalose molecule used in EPSR analysis. All carbon atoms are labeled ‘C’, the bridging oxygen atom between the two glucose rings is labeled ‘O1’, the bridging oxygen atoms within each glucose ring are labeled ‘O2’, the oxygen atoms attached to CH2 groups are labeled ‘O3’ and the oxygen atoms bonded directly to the ring carbon atoms are labeled ‘O’. Hydrogen atoms are labeled either ‘M’, if they are bonded to a carbon atom, or ‘H’, if they are bonded to an oxygen atom. The latter hydrogen atoms are assumed fully exchangeable with water hydrogen atoms. Water oxygen and hydrogen atoms (not shown here) are labeled ‘OW’ and ‘HW’ respectively.

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according to (1) or (3), is used to perturb the reference potential so that the calculated differential cross sections approach the data as closely as possible 20 . Table 1: Lennard-Jones and Coulomb parameters used in the EPSR reference potential. Atom  [kJ/mol] OW 0.65000 HW 0.00000 O1 0.58576 O2 0.58576 O3 0.71128 O 0.71128 H 0.05000 M 0.12552 C 0.27614

σ [Å] 3.166 0.000 2.900 2.900 3.100 3.100 1.700 1.700 3.500

q [e] -0.8476 0.4238 -0.5000 -0.5000 -0.5000 -0.5000 0.3005 0.0000 0.2580

Table 2: Details of the EPSR simulation boxes used in this work. In all cases the simulation box was cubic. Mole ratio Atomic no. density Box dimension trehalose:water [atoms/Å3 ] [Å] 1:25 0.11538 47.03 1:38 0.10677 42.07 1:100 0.10248 6.57

Number of Number of trehalose molecules water molecules 100 2500 50 1900 30 3000

Three such EPSR simulations are run, namely at trehalose:water mole ratios of 1:25 and 1:100, corresponding to the data of 17 , and at a mole ratio of 1:38, corresponding to the data of 18 . In all cases the same molecular geometries and potential parameters are used. Table 2 gives the details of these simulations. Once a satisfactory fit to the scattering data is achieved, the simulation is run for a further approximately 5000 steps to form average values of the fitted radial distribution function. In this case 1 step consists of 5 attempted translations and rotations of every molecule in the simulation box, with the acceptance ratio set at 0.25. Fairly early in the simulations it became apparent, particularly when fitting the scattering data, that the trehalose molecules had a tendency to cluster, particularly at the lower concentration (mole ratio 1:100). The trehalose clustering produces excess simulated small Q scattering which is not observed in the scattering data, so to prevent this clustering 8

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a minimum allowed separation of 7 Å is imposed on the O1-O1 pairs. Having obtained a box of molecules arranged in a manner compatible with the scattering data, related quantities can be calculated from the simulated radial distribution functions, gαβ (r). In particular the coordination number of atoms of type β around atoms of type α in the distance range R1 to R2 is defined as: Z

R2

Nαβ (R1 , R2 ) = 4πρcβ

r2 gαβ (r)dr

(4)

R1

Furthermore, following, 26 it is possible to define a local density of β atoms around α by dividing this coordination number by the volume which it occupies: Nαβ (0, R) 3 ρ¯αβ (R) = = 3 ρcβ 3 4πR /3 R

Z

R

r2 gαβ (r)dr

(5)

0

At large R this density will go simply to the bulk density of β atoms, ρcβ , but at short distances, comparable to the dimensions of the molecules involved, it will show either excess or depleted density, depending on the extent to which the two species are associated or not at short range. Such a measure will be used below to assess the extent to which water hydrogen-bonds to trehalose, compared to water hydrogen bonding to itself. It has to be said of course that any method used to interpret neutron scattering data of the kind shown here, such as EPSR, may not necessarily produce a unique conclusion. The main problem comes from the fact that weakly weighted site-site partial structure factors, Hαβ (Q), in the total scattering patterns, FN (Q) and FX (Q), will not be well constrained by the data. EPSR partly overcomes this problem by including a reference potential between all atoms which includes the likely atomic overlap forces, dispersion forces, and where relevant Coulomb forces. This may not represent exactly the real forces, but it should give a realistic representation of those forces. In addition the use of hydrogen isotope substitution highlights the hydrogen to other atom interactions (such as the important hydrogen-bonding interactions) while the inclusion of X-ray data in the present case emphasizes the water oxygen 9

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interactions, which dominate in that particular dataset. Compared to many other methods of interpreting experimental data, we believe this is as reasonable approach to the problem of interpreting data as any others that might be used.

Neutron and X-ray scattering data No new neutron scattering data were used in the present work. Instead the previously published neutron data sets were used in a new set of EPSR simulations of trehalose at the three concentrations 1:25, 1:38 and 1:100. To achieve a set of isotopically unique yet chemically similar set of measured data it is important to recognise that trehalose has two types of hydrogen atom, exchangeable and non-exchangeable. The exchangeable hydrogen atoms on the hydroxyl groups are labelled ‘H’ and exchange readily with water hydrogen atoms, HW. On the other hand, there is no exchange for the non-exchangeable ‘M’ hydrogen atoms bound to the carbon atoms. Since this exchange impacts on the scattering equation (3) it is imperative that the trehalose hydrogens which are in exchange with the water solvent are correctly accounted for when computing the neutron weights for each of the partial structure factors, Hαβ (Q), in this equation. Fortunately, this is normally achieved transparently by setting a particular flag, which signals whether an atom is exchangeable or not, when the EPSR simulation is set up. Hence the statement from Olsson et al. 18 to the effect that this exchange is neglected in the original trehalose experiments from Pagnotta et al. 17 is incorrect. For the Pagnotta et al. data,, 17 the original raw neutron counts were processed to correct for attenuation, multiple scattering, container scattering and data normalisation, using the most recent version of the Gudrun data analysis package, 27 , and using the latest iterative method to remove the effects of neutron inelasticity. 28 These data were recorded on the SANDALS diffractometer at ISIS. Table 3 summarises the neutron data sets that are available from that experiment. For the Olsson et al. data, which were processed much more recently, 18 the interference 10

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Table 3: Neutron datasets available from Pagnotta et al. 17 Note that the H and HW atoms are assumed to be fully exchangeable when calculating the weights in equation (3), so the actual deuteriation of H and HW atoms is calculated as the average for these two atom types. For the first three rows where two numbers are shown, the first refers to the 1:25 mole ratio, while the second refers to the 1:100 mole ratios. For the remaining rows, the numbers apply to only the 1:25 mole ratio. Run number

Sample name

35521, 35537 35536, 35539 35518, 35538 35520 35519 35516 35517 35515

H-H-Tre in H2O H-H-Tre in HDO H-H-Tre in D2O H-HD-Tre in HDO H-HD-Tre in D2O H-D-Tre in H2O H-D-Tre in HDO H-D-Tre in D2O

Deuteriation Deuteriation (M atoms) (H and HW atoms) 0.0 0.000, 0.000 0.0 0.431, 0.481 0.0 0.862, 0.962 0.0 0.500 0.0 0.931 0.0 0.138 0.0 0.569 0.0 1.000

differential cross-sections were used as supplied by the authors without further processing. These data were recorded on the NIMROD diffractometer at ISIS. Table 4 summarises the neutron data sets available from Olsson et al. Table 4: Neutron datasets available from Olsson et al. 18 Note that the H and HW atoms are assumed to be fully exchangeable when calculating the weights in equation (3), so the actual deuteriation of H and HW atoms is calculated as the average for these two atom types. These run numbers apply to a mole ratio of 1:38. Run number 38625 38901 38626 38642 38660 38651

Sample name

Deuteriation (M atoms) H-H-Tre in H2O 0.0 H-HD-Tre in HDO 0.0 H-D-Tre in D2O 0.0 D-H-Tre in H2O 1.0 D-HD-Tre in HDO 1.0 D-D-Tre in D2O 1.0

Deuteriation (H and HW atoms) 0.0 0.5 1.0 0.0 0.5 1.0

In addition to the existing neutron data, new X-ray data were measured for the mole ratios of 1:100 and 1:25. In these cases aqueous solutions of trehalose were prepared at the required concentrations and mounted in thin-walled silica glass capillaries of diameter

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2 mm. Data were accumulated on a laboratory X-ray diffractometer using a Ag anode (wavelength 0.5609 Å). They were converted to X-ray interference differential scattering cross section using the methods described in Soper 27 to correct for attenuation, multiple scattering, container scattering and to put on an absolute scale. There are currently no X-ray data at 1:38 mole ratio.

Results Data Fits Figures 2 - 4 show the fits to the respective neutron and X-ray data, where available, for the three concentrations of trehalose in water. Generally, the quality of the fits at all three concentrations are equivalent and similar to what has already been shown in the previous publications. As is usual in these cases, small discrepancies between data and fit, particularly at low Q are apparent: these can arise either from inadequacies in the data fit procedure, or from residual inelasticity effects not fully removed from the neutron data, or from other systematic uncertainties that arise in the conversion of raw neutron and X-ray counts to absolute differential scattering cross section. Generally, however, the simulations reproduce the measurements to a sufficiently accurate level to allow us to draw some general conclusions about the liquid structure in these solutions. It is important to control the amplitude of the empirical potential in an EPSR simulation. There is, unfortunately, no reliable way of determining the best value for this amplitude other than by trial and error. This means the final result will, to an extent, be subjective to the individual performing the simulation. If this is not set large enough then the fit is demonstrably not as good as it could be. If it is set too large, then the fit does not improve, but the simulation starts to reproduce artefacts in the data, such as noise and Fourier truncation effects. For the simulations shown in this paper, the amplitude of the empirical potential was set to 15 kJ/mole for all mole ratios, this value being determined to give the fits shown in these figures, but without obvious artifacts 12

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from the data. It should be noted that the fits presented here in Figure 3 for the Olsson et al. data are equivalent in quality to those shown in Fig. 2 of Olsson et al. 18 Trehalose:water 1:25 9

X−ray

8

35515

7

35519

6

35518

5 F(Q)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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35517

4

35520

3

35536

2

35516

1

35521

0 −1

0

5

10

15

20

Q [1/Å]

Figure 2: EPSR fits to neutron and X-ray interference differential scattering cross sections at trehalose:water mole ratio of 1:25.

Water Structure Figure 5 shows the water-water radial distribution functions at each concentration, plus the corresponding functions for pure water. It can be seen that the underlying pattern of peaks is very similar to that for water for each of the OW-OW, OW-HW and HW-HW functions, at all the measured concentrations. In particular for the 1:38 concentration there is no sign of the splitting of the second peak in the OW-OW function that is reported by Olsson et al. 18 (see Fig. 3 of that paper). It is well known that the behavior of this second peak is a sensitive indicator of changes to the local topology of water structure. 29,30 At all three concentrations, the main impact of the presence of trehalose compared to pure water is the substantial sharpening of the first peak in each of these functions. This does not necessarily imply that the water is more structured at longer ranges in solution compared to the bulk, but it does mean that the main effect of trehalose in solution is to sharpen

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Trehalose:water 1:38 6 38651

5

38626

F(Q)

4

38660

3

38901

2

38642

1

38625

0 −1

0

5

10

15

20

Q [1/Å]

Figure 3: EPSR fits to neutron and X-ray interference differential scattering cross sections at trehalose:water mole ratio of 1:38.

Trehalose:water 1:100 3.5

X−ray

3 2.5

35538

2 F(Q)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.5

35539

1 0.5

35537

0 −0.5

0

5

10

15

20

Q [1/Å]

Figure 4: EPSR fits to neutron interference differential scattering cross sections at trehalose:water mole ratio of 1:100.

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(a) Trehalose:water 1:25 9 8 7

g(r)

6 OW−OW

5 4

OW−HW

3 2

HW−HW

1 0

0

2

4

6 r [Å]

8

10

12

(b) Trehalose:water 1:38 9 8 7

g(r)

6 OW−OW

5 4

OW−HW

3 2

HW−HW

1 0

0

2

4

6 r [Å]

8

10

12

(c) Trehalose:water 1:100 9 8 7 6 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

OW−OW

5 4

OW−HW

3 2

HW−HW

1 0

0

2

4

6

8

10

12

r [Å]

Figure 5: Water-water radial distribution functions (OW-OW, OW-HW and HW-HW) in solutions of trehalose:water at mole ratios of (a) 1:25, (b) 1:38, and (c) 1:100. For each concentration the same functions for pure water are shown as dots. 28 15

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the short-range bonding between water molecules compared to what would happen if this bonding had been diluted by the presence of trehalose in proportion to its concentration. An alternative way of looking at this is to say that the water has to some extent been excluded from the region around the trehalose, causing the water-water interactions to sharpen. 31 A similar sharpening of the first neighbor structure was found in a previous simulation. 32 That paper demonstrates also that this effect is common to several saccharides and cannot explain the peculiarity of trehalose.

Trehalose-water Structure By comparison the trehalose-water correlations, as signified by the O*-HW and O*-OW radial distribution functions, are relatively weak (see Figures 6 and 7, which also show the the OW-HW and OW-OW functions again for completeness), and indeed much weaker than seen for hydroxyl-water hydrogen bonding in other sugar molecules. 33–35 In all the cases examined here it can be seen that the O*-HW first peak near r = 2 Å is much weaker or non-existent for trehalose-water compared to the corresponding water-water OW-HW peak. A similar comment applies to the O*-OW first peak compared to OW-OW. In addition, in both of the previous papers 17,18 coordination numbers for these peaks are calculate and used to assign numbers of hydrogen bonds between trehalose and water. However such hydrogenbond numbers are subjective in the sense that they depend on which radial cut-off is used and how a hydrogen bond is defined, as demonstrated elsewhere. 18 It should be noted that Olsson et al. in some cases have used a hydrogen bonding definition that extends well beyond the first peak minimum for the radial distribution functions within their work. A more qualitative way of representing the same information is to calculate the local density of water hydrogen atoms around the various oxygen atoms, ρ¯O∗ HW (R) (5), for each of the cases shown in Figure 6. These quantities are shown in Figure 8 for the O*-HW and OW-HW distribution functions. It can be readily seen that at all three concentrations the trehalose-water distributions are significantly depleted out to R ∼ 10 Å compared to the 16

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(a) Trehalose:water 1:25 7 6 OW−HW

g(r)

5

O3−HW

4

O−HW

3

O2−HW

2

O1−HW

1 0

0

2

4

6 r [Å]

8

10

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Figure 6: Oxygen to water hydrogen (HW) radial distribution functions for trehalose:water solutions at mole ratios of (a) 1:25, (b) 1:38, and (c) 1:100. 17

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Figure 7: Oxygen to water oxygen (OW) radial distribution functions for trehalose:water solutions at mole ratios of (a) 1:25, (b) 1:38, and (c) 1:100. 18

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water-water distributions, which approach the bulk limit (dashed lines) for R < 4 Å. Hence it seems the trehalose-water hydrogen bonding is markedly affected by the conformation of the trehalose which inhibits water hydrogen-bonding to itself and so gives rise to a marked excluded volume effect. There can only be one conclusion here, namely that trehalose is significantly under-bonded to water compared to water bonding to itself via hydrogen bonds at all concentrations.

Discussion The present analysis broadly concurs with that of Pagnotta et al. 17 which claimed trehalose was relatively weakly bonded to water, and disagrees with that of Olsson et al. 18 which concluded that trehalose-water hydrogen bonding is abundant, although primarily of the weak kind. It also finds rather weak impacts on water structure (Figure 5); in particular the second peak in the OW-OW function is not greatly perturbed even in the most concentrated solutions. Hence the contention that water structure is substantially modified in the presence of trehalose made by Olsson et al. is not supported here. Nonetheless, both studies agree that there is little if any aggregation of sugar molecules at any of the concentrations studied so far. In attempting to explain the discrepancies with the earlier Pagnotta et al. work, Olsson et al. ascribe the different interpretations to the fact that Pagnotta et al. did not use trehalose with the hydrogen atoms attached to carbon deuteriated, as they had done, so limiting the amount of neutron contrast data available. Whilst it is certainly true that deuteriating these atoms adds valuable extra contrast, the present EPSR simulations, which use exactly the same differential scattering cross sections as used by Olsson et al. can fit those data equally well without a large perturbation to the water structure, and without strong water bonding to the trehalose molecule. The conclusions from the present analysis of the Olsson et al. data at mole ratio 1:38 are in fact in quite close accord with those from Pagnotta et al.

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Figure 8: Local density of water hydrogen atoms around the O, O1, O2, and O3 oxygen atoms on trehalose and OW oxygen atom on the water molecule for trehalose:water solutions at mole ratios of (a) 1:25, (b) 1:38, and (c) 1:100. The horizontal dashed lines show the bulk density of water hydrogen atoms at each concentration.

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at mole ratios of 1:25 and 1:100 to which new X-ray data have now been added. In the present instance the calculated water-trehalose radial distribution functions for the trehalose hydroxyl group, hydrogen bonding to the surrounding water solvent shown in Figs. 6 and 7 of this paper bears close resemblance to the same functions deduced by Olsson et al. in Fig. 6 of 18 , suggesting that the stated differences are more a question of interpretation rather than quantitative. To illustrate this point, Olsson et al. 18 calculate the hydration number of trehalose in solution. They do this using several different criteria for defining a hydrogen bond, which gives rise the values for the hydration number between 4.4 (criterion 2) and 12.8 (criterion 1). To compare with our own simulations we have calculated the hydration number based on the number of water hydrogen atoms (HW) around each of the O1, O2, O and O3 atoms of the trehalose molecule out to a distance of 2.52 Å, and also the H-OW coordination number to the same distance. This distance corresponds approximately to the first minimum of the corresponding radial distribution functions and signifies those water hydrogen and oxygen atoms that are clearly associated with the corresponding oxygen or hydrogen atom on the trehalose molecule, and hence with water-trehalose hydrogen bonds. For comparison the same criterion is used for water hydrogen bonding to itself. These numbers can in turn be used to calculate the occupancy of the water and trehalose hydrogen bond sites. To achieve this we assume a single water molecule has a total of four possible hydrogen bonding sites, namely two H-bond donor sites and 2 H-bond acceptor sites. Assuming O1 and O2 contribute 2 H-bond acceptor sites each, while O and O3 each contribute 2 acceptor and 1 donor H-bond sites each, the maximum number of possible H-bond sites on a trehalose molecule is 3×2+8×3 = 30 sites. Using these numbers we can estimate the probability that a H-bond site on water and trehalose is occupied - the “occupancy” - at each concentration by dividing the actual number of H-bonds found by the maximum number possible. The results are shown in Table 5. The hydration numbers estimated here are in line with those reported by Olsson et al.

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Table 5: Estimated hydration number and hydrogen bond site occupancy for trehalose and water in solution Mole ratio 1:100 1:38 1:25

Trehalose water Hydration no. H-bond occupancy Hydration no. 10.0±1 0.33 3.9±0.2 12.8±1 0.43 3.5±0.2 11.9±1 0.40 3.9±0.2

H-bond occupancy 0.97 0.88 0.98

as well as in other experiments 36 and simulations 37 . Hence, given the uncertainties involved in estimating these coordination numbers, there does not appear to be any fundamental discrepancy between our analysis and other experiments and simulations on this point. What is important to note however is that the hydrogen bond site occupancy for water bonding to trehalose is markedly lower than for water bonding to itself. This is the origin of our view that water does not bond as strongly to trehalose as it does to itself. Because the hydrogen bond numbers for trehalose given in Table 5 are similar to those presented by Olsson et al. it would appear that the two sets of simulations concur on this point. It is also to be noted that the simulations of Ekdawi-Sever et al. 37 (figure 14 in that paper), show a very similar trend for the OW-OW radial distribution function between pure water and water in trehalose solution as is seen in our own simulations, Fig. 5. The main peak near r = 2.8 Å grows markedly with increasing sugar concentration in both cases. In addition there is a broad second single peak near r = 4.5 Å in both instances, which is quite unlike the double peak structure seen in Olsson et al., Figure 3. Our results are also consistent with another computer simulation of trehalose in water. 38 The gHW HW (r) found in that work at a concentration of 1.3m (corresponding to a molar ratio of approximately 1:43) is closely similar to the same function in pure water, the principle difference being the main peak heights which increase somewhat in solution compared to pure water, as here in Fig. 5. (See supplementary information of Sapir and Harries 38 ) Indeed there is the statement on page 626 of that work that “...This difference (referring to the slightly shorter H-bond distance between water molecules in solution compared to the pure liquid) indicates that trehalose enhances the H bonding within water molecules ...” However 22

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the subsequent statement in that work, referring to Fig. S4, that trehalose restructures the water tetrahedral network (in spite of the enhanced water-water H-bonding) is not supported by the referenced figure. This figure shows the tetrahedral order parameter q at a molar ratio of 1:43. For the trehalose solutions away from any trehalose oxygen atoms, the difference in value of q between pure water and water in trehalose is only about 2% which is not really symptomatic of a significant “destructuring” of the water. In another simulation paper, Bordat et al. 39 we learn that the phrase “destructuring” actually means something slightly different, namely the increased ability of trehalose to break the water into small clusters as the concentration increases, an effect that has been alluded to more recently in the case of sorbitol in solution. 40 The Sapir et al. simulations 38 also demonstrate marked sugar bonding to itself in solution forming percolating chains above a mole ratio of about 1:36. This might possibly be the case, although in the present EPSR simulations an extra distance constraint was imposed on the trehalose-trehalose interactions, via the O1-O1 minimum distance criterion described above, to restrict such association. This was because if too much trehalose-trehalose association was allowed in the simulations it generated pronounced small Q scattering which is not observed in the data. The present work does not support the claim by Sapir et al. that “trehalose possesses a high propensity to form hydrogen bonds with water”. Comparing it with the polyalcohol, glycerol, for example, (see Fig. 2 of Towey et al. 41 ), or with the hydration of other sugars, such as glucose 33,34 (see Fig.4 of Rhys et al. 33 ), mannose, 33 or cellobiose 35 , the hydrogen bonding of water to trehalose appears relatively mild, whichever version of the EPSR simulations one refers to. We surmise, though we have no proof, that this is a steric effect which arises from the conformation of the trehalose molecule in solution (see below). One possible explanation of this apparent lack of trehalose-water hydrogen bonds is that in the models of trehalose used here there is a significant amount of oxygen-hydrogen intramolecular hydrogen bonding. This can be seen in the O1-H, O2-H and O-H intra-molecular distribution functions, Fig. 9. A distinct peak in the region r = 1.8 Å to r = 3.0 Å can be seen

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Figure 9: Trehalose intra-molecular O*-H radial distribution functions at the three mole ratios, namely (a) 1:25, (b) 1:38, and (c) 1:100. These suggest a significant degree of intramolecular hydrogen bonding can occur in these molecules.

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corresponding to such intra-molecular hydrogen bonding. Calculation of the coordination number associated with these peaks gives 1.9 ± 0.1 H atoms around O1, 0.5 ± 0.1 H atoms around O2, and 0.8 ± 0.1 H atoms around O at a mole ratio of 1:25. (There is no peak in the O3-H intra-molecular distribution at this distance as the oxygen atom is too far away from other H atoms in the same molecule to form hydrogen bonds.) Similar numbers are found at the other concentrations. The presence of such pronounced intra-molecular hydrogen bonding would certainly act as a deterrent to water molecules approaching the respective oxygen atoms on trehalose too closely. It should be noted that the glycosidic linkage ester oxygen O1 between the two rings on trehalose shows less hydrogen-bonding to the surrounding water solvent for 1:100 and 1:25 solutions than was previously reported by Pagnotta et al.. 17 Conversely, at the 1:38 concentration in the current analysis the same O1 oxygen atom shows slightly more water hydrogen bonding compared with that reported previously. 18 This is likely a consequence of the molecular conformation which results in intramolecular hydrogen bonding as delineated above. It is notable that the inclusion of X-ray data, which was not available in the previous investigations, apparently results in a lower number of O1-HW hydrogen bonds. This may, in part, be due to the much stronger weighting of the OW-OW distribution in the X-ray data compared to the neutron data, which in turn gives rise, in the EPSR simulation, to enhanced water structure at the expense of water accessing the glycosidic oxygen. However, overall, this is a relatively minor effect and will not strongly impact the lack of pronounced hydroxyl-oxygen to water bonding observed in the present analyses. Seemingly contrary to these observations, on the basis of a Raman scattering study of trehalose aqueous solutions, Branca et al. 42 argue there is a marked impact of trehalose on water bonding compared to sucrose and maltose. In particular they split the main OH vibration peak into an “open” and “closed” component, the “open” peak corresponding to the tetrahedral water network, and “closed” component which corresponds “to the O-H vibration of H2 O molecules that have a partially developed hydrogen bond (distorted bond)”. They

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analyse the relative area of each peak. At a mole fraction of 0.09 (corresponding to molar ratio of 1:10) the “closed” component for trehalose has significantly larger relative area than for either sucrose or maltose at the same concentration. It is on this basis apparently they claim that the “greater bioprotective action of trehalose on biological structures is to be connected with its greater destructuring effect on the tetrahedral H-bond network of water”. On the other hand at mole fractions of 0.16 and 0.038, corresponding approximately to molar ratios of 1:5 and 1:25 respectively, the differences between the three sugars are less obvious and could be ascribed to experimental uncertainties (which are not stated). When comparing that work with the present work, one has to be aware that the lowest concentration studied in that work corresponds to the highest mole ratio studied here: at this concentration the effect of trehalose on the Raman scattering compared to the other sugars is not so obvious as at higher concentrations. The principle effect of increased concentration seen in the present work is the enhancement of water-water correlations at low r as the concentration increases, Fig. 5. These could simply be the effect of excluded volume effects 43 resulting from the water becoming increasingly confined in small volumes by the sugar molecules, as surmised by Bordat et al. 39 and Sapir et al.. 38 If so, then it is perhaps unsurprising that the Raman spectra from these solutions are blue shifted as the water pockets become increasingly isolated with increasing sugar concentration: the lack of strong H-bonding to trehalose compared to other sugars in solution might have precisely that effect. To summarise this discussion, based on the cases cited above, it does not appear that the reluctance of water to hydrogen-bond to trehalose in these solutions that is observed in the combined neutron diffraction and EPSR analyses presented in this work is necessarily inconsistent with what has been presented in previous studies of trehalose and other sugars in water. Whereas it has frequently been claimed that trehalose hydrogen bonds strongly to water, we find little evidence for a propensity to form such bonds, at least in the concentration range studied here, which is lower than that used in some other studies. This leads to

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the following proposal which might explain why trehalose is apparently the most effective protectant against drought stress found in living organisms. According to Green et al., 11 trehalose has the highest glass transition temperature of all the common sugars, and it is the glass transition on cooling that prevents ice forming and allows the organism to go into a stationary state from which it can recover on heating. Similarly, anhydrobiotic organisms survive severe dehydration at ambient temperature thanks to high sugar concentrations in the cytoplasm of their cells. 44–46 Several authors have alluded to the possibility that sugars in solution have the ability to segregate water into small “pockets” at high enough concentrations, 38–40 , and so inhibit ice formation and allow the glassy state. Equally it is well known that when you initially pressurize low temperature water, thereby breaking some hydrogen bonds, the diffusivity initially increases. 47 If that were to happen in sugar solutions, the effect would surely be to lower the glass transition temperature rather than raise it. Hence our proposal here is that trehalose bonds less strongly to water than the other sugars, while still remaining in solution and causing it to become segregated, and it is this property that raises the glass transition temperature compared to the other sugars. This proposal is borne out by all the neutron scattering studies that have been performed on these sugars to date.

Conclusion The present study of trehalose in water at three mole ratios, 1:25, 1:38 and 1:100, which is largely a reanalysis of existing neutron scattering data, with added new X-ray scattering data at two of the concentrations, 1:25 and 1:100, concludes that, like Pagnotta et al. 17 , trehalose is relatively weakly hydrogen-bonded to water in solution compared to water hydrogen-bonding to itself and compared to other sugars. At the same time the pronounced perturbation to water structure seen by Olsson et al. 18 is not supported by this new analysis, which maintains the view of Pagnotta et al. that water is relatively unperturbed by the presence of trehalose

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in solution. This is in contrast to glycerol and other sugars such as glucose, which certainly do have a strong interaction with water. 33,34,41 Finally, as far as the question of trehalose-water interaction is concerned, we feel confident in concluding that trehalose forms less and weaker hydrogen bonds than other carbohydrates (and sugars in particular) and relatively weakly perturbs the tetrahedral arrangement of water molecules. This statement is not sufficient to discriminate between the proposed mechanisms of preservation against environmental stress. Yet a weak hydrogen bonding interaction of trehalose with water, thus favouring trehalose-trehalose interactions, is in agreement with the observation that the trehalose system possesses significantly higher glass transition temperatures than any of the other disaccharide sugars at the same water content. 11 At the same time, the origin of the observed dynamical slowing down of the solution can be considered as a consequence of the increased propensity of trehalose-water solutions to form a glass, not necessarily of pronounced hydrogen bonds between solute and solvent.

Acknowledgement The authors thank Christoffer Olsson and Jan Swenson for the numerical values of their neutron differential scattering cross sections at a trehalose:water mole ratio of 1:38, and for their helpful comments on the present manuscript. FB and MAR gratefully acknowledge the Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314 - 337 LEGGE 232/2016).

References (1) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose. Science 1984, 223, 701–703.

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(2) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Aurell Wistrom, C. Stabilization of Dry Phospholipid Bilayers and Proteins by Sugars. Biochem. J. 1987, 242, 1–10. (3) Crowe, L. M. Lessons from Nature: The Role of Sugars in Anhydrobiosis. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2002, 131, 505 – 513. (4) Leopold, A. C. Membranes, Metabolism, and Dry Organisms; Cornell University Press, Ithaca, NY, 1986. (5) Carpenter, J. F.; Crowe, J. H. An Infrared Spectroscopic Study of the Interactions of Carbohydrates with Dried Proteins. Biochemistry 1989, 28, 3916–3922. (6) Zentella, R.; Mascorro-Gallardo, J. O.; Van Dijck, P.; Folch-Mallol, J.; Bonini, B.; Van Vaeck, C.; Gaxiola, R.; Covarrubias, A. A.; Nieto-Sotelo, J.; Thevelein, J. M. et al. A Selaginella lepidophylla Trehalose-6-Phosphate Synthase Complements Growth and Stress-Tolerance Defects in a Yeast tps1 Mutant. Plant Physiol. 1999, 119, 1473–1482. (7) Sampedro, J. D.; Uribe, S. Trehalose-Enzyme Interactions Result in Structure Stabilization and Activity Inhibition. The Role of Viscosity. Mol. Cell. Biochem. 2004, 256, 319 – 327. (8) Chiantia, S.; Giannola, L. I.; Cordone, L. Lipid Phase Transition in Saccharide-coated Cholate-Containing Liposomes: Coupling to the Surrounding Matrix. Langmuir 2005, 21, 4108–4116. (9) 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. (10) Corradini, D.; Strekalova, E. G.; Stanley, H. E.; Gallo, P. Microscopic Mechanism of Protein Cryopreservation in an Aqueous Solution with Trehalose. Sci. Rep. 2013, 3, 1218. 29

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(11) Green, J. L.; Angell, C. A. Phase Relations and Vitrification in Saccharide-Water Solutions and the Trehalose Anomaly. J. Phys. Chem. 1989, 93, 2880–2882. (12) Belton, P. S.; Gil, A. M. IR and Raman Spectroscopic Studies of the Interaction of Trehalose with Hen Egg White Lysozyme. Biopolymers 1994, 34, 957–961. (13) Allison, S. D.; Chang, B.; Randolph, T. W.; Carpenter, J. F. Hydrogen Bonding between Sugar and Protein is Responsible for Inhibition of Dehydration-Induced Protein Unfolding. Arch. Biochem. Biophys. 1999, 365, 289–298. (14) Miller, D. P.; de Pablo, J. J. Calorimetric Solution Properties of Simple Saccharides and Their Significance for the Stabilization of Biological Structure and Function. J. Chem. Phys. B 2000, 104, 8876–8883. (15) Cordone, L.; Cottone, G.; Giuffrida, S. Role of Residual Water Hydrogen Bonding in Sugar/Water/Biomolecule Systems: a Possible Explanation for Trehalose Peculiarity. J. Phys. Condens. Matter 2007, 19, 205110. (16) Batta, G.; Kover, K. E.; Gervay, J.; Hornyak, M.; Roberts, G. M. Temperature Dependence of Molecular Conformation, Dynamics, and Chemical Shift Anisotropy of α − αTrehalose in D2 O by NMR Relaxation. J. Am. Chem. Soc. 1997, 119, 1336–1345. (17) 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? J. Phys. Chem. B 2010, 114, 4904–4908, PMID: 20297794. (18) Olsson, C.; Jansson, H.; Youngs, T.; Swenson, J. Structure of Aqueous Trehalose Solution by Neutron Diffraction and Structural Modeling. J. Phys. Chem. B 2016, 120, 12669–12678, PMID: 27973816. (19) Soper, A. Empirical Potential Monte Carlo Simulation of Fluid Structure. Chem. Phys. 1996, 202, 295–306. 30

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(20) 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. (21) Jmol: an Opena-Source Browser-Based HTML5 Viewer and Stand-alone Java Viewer for Chemical Structures in 3D. http://www.jmol.org/. (22) Stewart, J. P. Stewart Computational Chemistry - MOPAC Home Page. http:// openmopac.net/. (23) Brown, G. M.; Rohrer, D. C.; Berking, B.; Beevers, C. A.; Gould, R. O.; Simpson, R.; IUCr, The Crystal Structure of α,α-trehalose Dihydrate from Three Independent X-ray Determinations. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1972, 28, 3145–3158. (24) Jeffrey, G. A.; Nanni, R. The Crystal Structure of Anhydrous α,α-Trehalose at -150o . Carbohydr. Res. 1985, 137, 21–30. (25) Duda, C. A.; Stevens, E. S. Trehalose Conformation in Aqueous Solution from Optical Rotation. J, Am. Chem. Soc. 1990, 112, 7406–7407. (26) Soper, A. K. Radical Re-appraisal of Water Structure in Hydrophilic Confinement. Chem. Phys. Lett. 2013, 590, 1–15. (27) Soper, A. GudrunN and GudrunX: Programs for Correcting Raw Neutron and X-ray Diffraction Data to Differential Scattering Cross Section; RAL Rep. RAL-TR-2011-013, STFC Rutherford Appleton Laboratory: Didcot, UK, 2011. (28) 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.

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