Carbohydrate Research 345 (2010) 2048–2059

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Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Conformational preferences of a,a-trehalose in gas phase and aqueous solution

Sandra C. C. Nunes a,*, A. J. Lopes Jesus a,b, M. João Moreno a, M. Ermelinda S. Eusébio a

a Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugalb Faculty of Pharmacy, University of Coimbra, 3004-295 Coimbra, Portugal

a r t i c l e i n f o

Article history:Received 22 March 2010Received in revised form 30 June 2010Accepted 6 July 2010Available online 13 August 2010

Keywords:TrehaloseConformational studyGas phaseAqueous solutionDFTCPCM

0008-6215/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.carres.2010.07.001

* Corresponding author.E-mail address: snunes@qui.uc.pt (S.C.C. Nunes).

a b s t r a c t

This work presents an investigation on the conformational preferences of a,a-trehalose in gas phase andaqueous solution. Eighty-one systematically selected structures were studied at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level, giving rise to 40 unique conformers. The 19 lower energy structuresand some selected other were further re-optimized at the B3LYP/6-311++G(d,p) level. The main factorsaccounting for the conformer’s stability were pointed out and discussed. NBO and QTAIM analyses wereperformed in some selected conformers in order to address the anomeric and exo-anomeric effects aswell as intramolecular hydrogen bonding. The effect of solvent water on the relative stability of the con-formers was accounted for by applying the conductor-like polarizable continuum model, CPCM.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The non-reducing disaccharide a,a-trehalose, formed by two a-D-glucose units held together by a 1,1-glycosidic linkage (Fig. 1), isone of the most chemically stable sugars in nature playing manyimportant biological roles.1,2 For example, it acts as bioprotectorfor living organisms against environmental stresses including ex-treme dehydration, freezing, oxidation, and hyperosmotic pres-sure.2–11 It has also been found to be an effective bioprotectantfor several organisms, allowing them to survive for several yearsin a state of suspended animation under conditions of water defi-ciency.12 Because of these peculiar characteristics, this moleculehas called for great attention and several theories have been devel-oped to explain its anomalous behavior.13–18

As consequence of its high structural flexibility (12 rotatablebonds) and the presence of a great number of OH groups, both in-tra- and intermolecular interactions are highly dependent on theconformation exhibited by this disaccharide. Hence, the explora-tion of the conformational behavior of a,a-trehalose is a crucialstep to better understanding of its biological activity.

Despite some experimental and theoretical studies on the struc-ture of trehalose in vacuum and aqueous solution been pub-lished,19–30 most of them using classical methods (molecularmechanics and molecular dynamics), some doubts and controversystill persist about the most relevant conformers, as well as on theconformational features accounting for their stability. Only in

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one relevant paper ab initio methods were employed24 to obtainthe energy surfaces around the glycosidic linkages for some start-ing structures.

Within this paper we intend to enlighten the structural featuresthat determine the stability order of the conformers of trehalose ingas phase and aqueous solution.

With this purpose, molecular orbital methods have been used toreliably perform a systematic exploration of the conformationalpreferences of this molecule in these two phases, covering a broad

Figure 1. Representation of the molecular structure of trehalose including the atomnumbering scheme for the conformers.

S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059 2049

range of starting structures. The B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) model chemistry was used to obtain the most relevant con-formers that were further re-optimized using the larger basis set.Particular attention was given to identification and characteriza-tion of the conformational features accounting for their stability,namely the dihedral angles around the glycosidic bonds, the rela-tive orientations of the hydroxymethyl groups, and the glycosidicC–O–C angle. Structural features such as the anomeric/exo-ano-meric effects and the intramolecular hydrogen bonding were ratio-nalized in terms of Natural Bond Orbital31–33 (NBO) theory andQuantum Theory of Atoms-In-Molecules34–39 (QTAIM).

The evaluation of the solvent effect on the conformational pref-erences of trehalose was performed in the framework of a contin-uum solvation approach using the conductor-like-polarizablecontinuum model (CPCM).40–43 The solvation Gibbs energy of theconformers was calculated, thus allowing for the estimation oftheir distribution in aqueous solution.

2. Computational methods

2.1. Generation of starting structures and nomenclature

The molecular structure of a,a-trehalose is shown in Figure 1.The existence of 12 independent rotating dihedral angles origi-nates a large number of possible conformations, making unafford-able the exploration of all of them by means of high-levelelectronic structure methods. Therefore, the generation of startingstructures was performed according to the following assumptions:(1) All structures are of a,a-4C1 type. (2) Five orientations aroundeach dihedral of the glycosidic linkage (/ = O5–C1–O1–C01 andw = O05—C01–O1–C1, see Fig. 1 for atom numbering scheme) are as-sumed: gauche+ (G, 60 ± 30�), gauche� (G0, �60 ± 30�), skew+ (S,120 ± 30�), skew� (S0, �120 ± 30�), and trans (T, 180 ± 30�). Thiscorresponds to 25 possible combinations of the central dihedrals(TG, TT, TG0, TS, TS0, etc.). Due to its symmetrical chemical structure(the molecule has a twofold rotational symmetry), the number ofunique conformations is reduced to only 15, since any XY confor-mation around the two central dihedrals (X and Y can be G, G0, T,S or S0) has a symmetry-related YX conformation. This number isfurther reduced to only 10 as some conformations (TG0, G0G0, S0S0,S0G0, S0G) make the two glucopyranose rings to collapse into eachother. (3) Three orientations are assumed for the hydroxymethylgroup, conventionally designated as tg (O6 trans to the ring oxygenO5), gg (C6–O6 gauche to both C5–O5 and C5–C4), and gt (C6–O6

gauche to both C5–O5 and C5–H).44 (4) The present work is con-cerned with input geometries with the exocyclic OH groups ar-ranged in both rings in a retro-clockwise orientation as itcorresponds to the most stable conformation in the isolated a-glucopyranose molecule.44,45 This orientation depends on theglucose ring being in the standard viewing orientation with thering oxygen located at the top right.

Considering all these assumptions, one obtains a total of 81 start-ing conformations to be explored. The structures are labeled by a se-quence of six letters: the two of the middle (capital) refer to theorientation of the glycosidic / and w dihedrals, respectively, whilethe small ones describe the orientation of the hydroxymethyl groupsof the respective rings following the nomenclature given above. Forexample, tgGTgg designates a structure with gauche and trans orien-tations around / and w dihedrals, while the two hydroxymethylgroups assume tg and gg conformations, respectively.

2.2. Gas and aqueous phase calculations

All conformations generated as described above were fully opti-mized at the DFT level using the B3LYP hybrid functional46–48 and

the 6-31G(d) basis set followed by the calculation of the vibra-tional frequencies at the same level. This allowed us to check ifeach optimized structure corresponds to a minimum in the poten-tial energy surface. Since in the previous computational studies ofcarbohydrates it has been demonstrated that the 6-31G(d) basisset gives unreliable conformational energies,49 the energy of eachresulting structure was further evaluated by B3LYP single-pointcalculations employing the 6-311++G(d,p) basis set. Then, a groupof selected conformers, including the 19 lower energy structures(corresponding to an energy range of �10 kJ mol�1 above the low-est energy conformer) and a few selected others, were fully opti-mized at the B3LYP/6-311++G(d,p) level.

Electron density analysis for some selected trehalose conform-ers was performed using the Natural Bond Orbital theory (NBO)and the Quantum Theory Atoms-In-Molecules (QTAIM). NBO anal-ysis was undertaken using the NBO 5.G program50 linked to the GA-MESS software package51 version 22-Feb-2006 (R5). Regarding theQTAIM calculations, the wave function was calculated from theB3LYP/6-311++G(d,p)-optimized geometries using the GAUSSIAN 03program, while the bond critical points (bcp) were located andcharacterized using the EXTREME program52 included in the AIMPACsoftware package.53

The effect of water on the conformational equilibrium of treha-lose has been described using the conductor-like polarizable con-tinuum model (CPCM) as implemented in the GAUSSIAN 03package. The dielectric constant of water at 298.15 K (e = 78.4)was used to simulate the aqueous environment and the cavitywas built according to the United Atom Topological Model(UAHF).54 Details of these calculations can be found in a previousauthors’ work.55 The Gibbs energy of solvation of previously ex-plored structures was obtained through single-point calculationsat the CPCM/B3LYP/6-311++G(d,p) level of theory. The SCF = tightconvergence criterion was used in these calculations.

3. Results and discussion

3.1. Analysis of the gas phase structures

Upon the preliminary calculations using the B3LYP/6-311++G(d,p)//6-31G(d) model chemistry, many of the 81 startingconformations converged to other structures. For example, all SS0

input conformations converged to SG0 and GG0 conformers and allthe S0T conformations did not converge. In addition, most of theSG and TS input conformations converged to GG (or SS) and TGconformers, respectively. A total of 40 unique equilibrium struc-tures were obtained and the results of the calculations are summa-rized in Table 1. Since no imaginary frequencies were found in thevibrational calculation, all the stationary points obtained werecharacterized as minima on the potential energy surface. In Figure 2are displayed the optimized geometries of some representativeconformers within each one of the most relevant families (charac-terized by a common orientation of the glycosidic dihedrals). Asstated in the previous section, dihedrals between 90� and 150�are called S (Skew). However, while in some conformers the valuesof / and w are close to the standard skew orientation, in others, thevalues are too apart, one close to gauche (�90�) and the other closeto trans (�150�). Hence, to avoid ambiguities, the formers arecalled SS and the latter TG (or GT).

A general analysis of the data displayed in Table 1 shows thatthe conformers’ energy is primarily determined by the conforma-tion around the two central dihedrals. All GG, TG, and SS conform-ers are found within a 15 kJ mol�1 energy range above c1 andrepresent the 23 lower energy structures. These three arrange-ments were also found to be the preferred ones through the com-putation of energy scans at different levels of theory.24 The GG

Table 1Glycosidic bond angles (\C1–O1–C01), dihedral angles, and relative electronic energies, DEelec, for the generated conformers of a,a-trehalose optimized at the B3LYP/6-31G(d) leveland their electronic energy evaluated at the 6-311++G(d,p) level

Conf. Labela\ (�) Dihedral anglesb (�) DEelec

c (kJ mol�1)

/ w h1 h01 h2 h02

c1 ggGGgg 114 72.6 72.6 �57.9 �57.9 56.0 56.0 0.0c2 gtGGgg 115 71.1 68.1 58.4 �57.8 �57.2 56.0 1.3c3 tgGGgg 114 74.3 72.3 168.2 �58.0 51.6 56.2 1.5c4 tgGGgt 115 70.9 67.3 168.1 58.2 51.8 �58.1 2.3c5 tgGGtg 114 73.3 73.3 168.3 168.3 51.6 51.6 2.6c6 gtGGgt 115 67.2 67.2 58.2 57.8 �58.2 �58.2 2.6c7 gtTGtg 119 148.9 95.0 59.0 167.4 �57.0 52.3 4.5c8 gtTGgg 119 146.9 95.1 59.2 �58.9 �57.0 57.3 5.0c9 gtSSgt 119 113.9 105.0 59.8 59.9 �61.5 �64.5 5.1c10 tgTGtg 119 149.2 95.5 167.6 167.3 51.2 52.3 5.4c11 ggTGtg 118 149.5 95.7 �58.1 167.6 55.9 52.3 5.7c12 ggTGgg 118 148.2 95.7 �58.2 �59.0 56.0 57.3 6.1c13 tgTGgg 119 147.9 96.2 167.5 �59.0 51.3 57.0 6.3c14 gtGGtg 115 83.7 67.5 59.7 166.3 �54.7 52.4 6.7c15 tgSSgt 119 127.4 98.1 167.6 57.6 51.3 �70.3 6.8c16 gtSSgg 119 98.0 128.1 57.5 �57.8 �70.5 55.5 7.0c17 gtGTtg 119 93.7 154.0 62.4 167.9 �82.6 50.6 7.5c18 gtGTgt 119 91.3 155.5 64.2 59.1 �82.0 �55.1 7.6c19 gtGTgg 119 92.3 155.1 63.6 �58.0 �81.9 55.5 8.3c20 gtTGgt 119 156.6 82.4 59.7 74.9 �55.6 �65.3 12.7c21 ggTGgt 119 155.9 82.3 �58.4 75.2 55.7 �64.7 14.1c22 tgTGgt 119 156.3 82.7 167.7 75.0 50.1 �65.0 14.2c23 (gt)TGgt 119 160.9 89.2 59.5 49.5 �54.6 �45.4 15.8c24 gtGG0gt 122 89.9 �63.5 71.3 61.3 �68.3 �57.2 25.5c25 (gg)TTgt 124 �176.4 178.7 �63.0 59.7 58.8 �50.3 26.3c26 (gg)TTgg 123 �178.4 179.8 �62.8 �56.6 58.2 53.4 26.7c27 (gg)TTtg 123 �176.9 178.5 �62.6 169.3 58.0 49.8 27.3c28 gtGG0gt 123 85.0 �53.6 67.0 61.5 �67.4 �57.1 29.2c29 gtGG0tg 124 82.5 �74.9 72.4 156.3 �81.7 56.1 33.5c30 ggGG0gt 123 87.3 �69.3 �59.7 59.3 61.2 �53.6 34.0c31 tgTT(gt) 124 179.3 �177.1 170.0 60.5 49.3 �48.8 34.1c32 ggSG0gg 122 91.8 �67.4 �57.8 �57.2 56.5 55.7 34.3c33 tgSG0gg 122 91.3 �66.5 168.8 �57.4 51.9 56.7 35.0c34 ggSG0tg 123 91.5 �68.9 �58.0 168.2 59.6 52.7 35.3c35 tgGG0gt 122 86.6 �66.3 169.1 60.5 52.7 �54.3 35.5c36 gtGG0gg 123 90.0 �67.4 58.2 �57.8 �58.1 58.0 36.8c37 tgSG0tg 123 91.6 �67.4 168.9 168.8 52.2 52.1 37.1c38 ggSG0gt 124 105.7 �38.0 �57.2 60.6 55.1 �52.9 40.0c39 tgSG0gt 124 105.8 �37.3 168.5 61.4 50.8 �52.9 40.7c40 tgTT(tg) 124 �178.6 179.7 169.7 168.7 49.5 48.9 42.0

a Parentheses identify rings with the exocyclic OH groups oriented in the clockwise direction.b / = O5C1O1C01, w = O05C01O1C1, h1 = O5C5C6O6, h01 ¼ O05C05C06O06, h2 = C5C6O6H6, h02 ¼ C05C06O06H06.c Values obtained at the B3LYP/6-311++G(d,p)//6-31G(d) level.

2050 S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059

arrangement was also found to be that of the lowest energy byother authors56 and the one existing in the crystal structure.57

On the other hand, the GG0, SG0, and TT structures, the last one pre-dicted as the global minimum according to Engelsen and Perez20

and Liu et al.,21 have relative energies above 25 kJ mol�1 and there-fore are not expected to contribute significantly to the gas phasestructure. The proximity of the two rings in these structures(Fig. 2), leading to steric repulsions, is certainly a significant contri-bution to their destabilization.

One or two inter-residue intramolecular H-bonds are likely tobe present in all conformers’ families, except the GG one. The val-ues of the O� � �H distances and O–H� � �O angles fall between 1.9 and2.7 Å and between 135� and 160�, respectively.

Interesting differences among families are observed for the gly-cosidic C–O–C angle. For the GG family, this structural parameterassumes values of 114�/115�, increasing to around 119� in TGand SS conformers. Regarding the higher energy conformers, likethose belonging to the GG0, TT, and SG0 families, the values arearound 123�, reflecting the existence of a severe structural stress.

Within each ring, the exocyclic OH groups are always alignedin an acceptor–donor–acceptor sequence, suggesting the forma-tion of a cooperative system of vicinal intramolecular hydrogenbonds. The existence of such weak intramolecular hydrogen

bonds in mono- and disaccharides has been accepted by someauthors24,58–60 but refused by others.61 A more detailed analysisof these interactions based on NBO and QTAIM methods will begiven further ahead on this paper. Another conformational fea-ture of these groups is the maintenance of their input retro-clockwise orientation. The only exception are the conformersbelonging to the TT family where after optimization the exocy-clic OH groups of one of the glucopyranose rings are re-orientedfrom retro-clockwise to clockwise to minimize the repulsionbetween the O2H and O02H groups (see Table 1).

After a general discussion on the results obtained for all theconformers at the B3LYP/6-311++G(d,p)//6-31G(d) level, specialattention will be now dedicated to the three families of conformerswith lower energy, that is, GG, TG, and SS, and particularly to thelowest energy one. In order to obtain more reliable geometriesand energies and also to access the thermochemical quantities at298.15 K, thus allowing for the calculation of the relative popula-tions, additional calculations have been performed. Hence, the 19lower energy structures (c1–c19) and some others, representingthe TT and GG0 families, respectively, were fully re-optimized atthe B3LYP/6-311++G(d,p) level. Some other structures, that willbe used to clarify specific conformational features in the GG familyof conformers, were generated and optimized at the same level.

Figure 2. Optimized geometries (B3LYP/6-31G(d)) of representative trehalose conformers of the GG, TG, SS, GG0 , TT, and SG0 arrangements around the two glycosidicdihedrals.

S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059 2051

The values of most relevant structural parameters optimized atthis level of theory, together with the relative electronic energyvalues, are displayed in Table 2. From the comparison of the dataincluded in Tables 1 and 2, one can see that for most of the con-formers, re-optimization with the larger basis set introduces onlyslight geometry and energy variations. The only exception is c18which converges to c9 after full optimization with the larger basisset.

In Table 3 are shown the values of the enthalpy, entropy, andGibbs energy of conformers c1–c19 at 298.15 K. This table also in-cludes the relative populations of the conformers at the sametemperature.

As can be seen, the enthalpy contributions do not affect signif-icantly the energetic sequence of the conformers given by the elec-tronic energy. When the Gibbs energy is considered the mostpronounced change was the stability increase of c9 as a result ofa high entropy value (26.9 J mol�1 K�1). Oddly, unlike the GG con-formers, no symmetry is found in c9. Given this and the anomalousentropy value, which is suspicious when compared with the valuesfound for the other two conformers of the same family (c15 andc16), we decided to re-optimize c9 by constraining its symmetryto C2. One imaginary frequency was obtained in the vibrational cal-culation which means that it corresponds to a first order saddlepoint rather than a true minimum. Repeating the calculation withthe cc-pVDZ basis set no imaginary frequency has been foundfor the symmetric structure. As these results raise some doubtsabout the nature of this specific conformer in the potential energysurface, it will not be considered either in Table 3 or in the discus-sion hereafter.

As it was found previously, the six lowest energy conformersadopt a GG arrangement around the two glycosidic dihedrals. Atthis level of theory, the values of / and w fall between 66� and73�, which are close to those found in the anhydrous (/ = 60.1�,w = 60.8�) and hydrated crystalline trehalose structures (/

= 61.8�, w = 74.9�).57 In the work of Soares and da Silva56 the GGconformers have values of / and w between 66.7� and 82.1�,respectively. Since in c1, c2, and c5, / = w and both rings havethe same conformation, the conformers have a C2 symmetry. Con-cerning the orientation of the hydroxymethyl groups in this family,one can see that there is no clear preference for a particular orien-tation. This behavior is somehow unexpected since the tg confor-mation is the only one that is likely to be stabilized by anintramolecular hydrogen bond involving non-vicinal OH groups.

Within the TG family, the two central dihedrals adopt values of95� and 145�. This arrangement allows the establishment of an in-ter-residue intramolecular hydrogen bond between the O2H groupand the O05 oxygen atom (see conformers c7 and c8 in Figures 2 and3, respectively). The values of the H� � �O distances and O–H� � �O an-gles are ca. 1.94 Å and 162�, respectively. As it was found in theprevious family of conformers, the conformation of the hydroxy-methyl groups has a small influence on the conformers’ energyas the maximum energy difference among the conformers is2 kJ mol�1.

Regarding the SS family both glycosidic dihedrals assume val-ues close to 110� or 120�, which are close to those reported byCramer et al. (/ = 115�, w = 115�).24 Re-optimization gives rise toslightly more open structures. Despite the relative proximity ofthe two rings, no significant inter-residues hydrogen bonds arefound using the commonly accepted geometric H-bond criteria.62

3.2. Effect of the exocyclic OH groups orientation

In order to have a deeper insight into the influence of the exo-cyclic groups’ orientation on the conformers’ stability, additionalstructures belonging to the GG family have been studied. Two setsof six structures, generated from the six lowest electronic energyones, have been optimized at the B3LYP/6-311++G(d,p) level: cX0

and cX00, where X can take values from 1 to 6. In the first set, the

Table 2Glycosidic bond angle (\C1–O1–C01), dihedral angles, and relative electronic energies, DEelec, for selected conformers of a,a-trehalose in gas phase obtained by full optimization atthe B3LYP/6-311++G(d,p) level

Conf.a Labelb\ (�) Dihedral anglesc (�) DEelec (kJ mol�1)

/ w h1 h01 h2 h02

c1 ggGGgg 115 71.9 71.9 �58.8 �58.8 60.3 60.3 0.0c2 gtGGgg 115 70.2 66.9 60.7 �58.9 �61.1 60.5 1.3c3 tgGGgg 114 73.0 71.7 165.3 �58.6 52.6 60.5 1.6c4 tgGGgt 115 68.8 67.8 165.1 60.6 52.6 �61.7 2.3c5 tgGGtg 114 70.3 70.3 165.5 165.5 52.4 52.4 2.8c6 gtGGgt 115 66.4 66.4 60.4 60.4 �61.7 �61.7 2.5c7 gtTGtg 118 145.6 94.9 61.1 163.7 �61.1 53.6 4.6c8 gtTGgg 118 143.9 94.9 61.1 �59.8 �60.9 62.2 5.1c9 gtSSgt 118 111.4 106.0 61.9 61.9 �65.5 �68.1 3.6c10 tgTGtg 118 146.1 95.3 164.6 163.8 52.3 53.5 5.6c11 ggTGtg 118 146.5 95.3 �58.8 163.9 60.2 53.5 5.8c12 ggTGgg 118 145.2 95.1 �59.0 �60.0 60.7 62.3 6.1c13 tgTGgg 118 143.3 95.5 164.6 �59.9 52.3 62.0 6.4c14 gtGGtg 115 84.1 67.1 61.3 163.4 �57.9 53.2 7.0c15 tgSSgt 118 119.6 100.7 164.3 61.5 52.7 �73.4 6.1c16 gtSSgg 118 100.8 120.7 60.9 �58.8 �73.5 60.4 6.3c17 tgTGgt 119 144.4 98.5 164.6 61.8 52.2 �80.0 6.4c19 ggTGgt 119 145.1 97.5 �59.0 61.8 60.2 �80.2 6.7c25 (gg)TTgt 124 �177.0 177.5 �64.1 61.8 63.4 �54.1 26.4c36 gtGG0gg 123 92.5 �67.5 62.1 �58.9 �59.8 62.3 36.5c10 ggGGgg 115 80.4 80.4 �57.7 �57.7 57.6 57.6 15.0c100 (gg)GG(gg) 115 78.4 78.4 �64.1 �64.1 60.8 60.8 2.9c20 gtGGgg 114 71.0 99.4 59.6 �57.5 �77.3 56.9 10.1c200 (gt)GG(gg) 116 82.9 73.4 60.0 �64.4 �52.6 60.7 11.8c30 tgGGgg 115 78.1 85.5 164.5 �54.5 51.0 57.1 15.1c300 (tg)GG(gg) 115 78.9 83.6 179.0 �63.5 �77.9 60.5 2.8c40 tgGGgt 114 98.2 71.2 165.5 59.9 50.5 �73.1 9.8c400 (tg)GG(gt) 115 76.2 82.9 �177.3 60.1 179.2 �51.3 12.7c50 tgGGtg 115 78.1 78.1 164.4 164.4 51.4 51.4 14.5c500 (tg)GG(tg) 115 81.4 81.4 �176.8 179.9 �176.8 179.9 5.7c60 gtGGgt 115 78.6 78.6 60.0 60.0 �60.7 �60.7 10.1c600 (gt)GG(gt) 116 78.3 78.3 59.9 59.9 �52.8 �52.8 19.9

a Conformers are numbered according to their electronic energy obtained at the B3LYP/6-311++G(d,p)//6-31G(d) level. Conformer c18 is not shown as it converges to c9after optimization at the B3LYP/6-311++G(d,p) level. Conformers c10 , c20 , c30 , c40 , c5,0 and c60 were obtained from c1, c2, c3, c4, c5, and c6, respectively, by moving the O2H andO02H groups away from central O1 atom. Conformers c100 , c200 , c300 , c400 , c5,00 and c600 were obtained from c1, c2, c3, c4, c5, and c6, respectively, by changing all the exocyclic OHgroups from a retro-clockwise to a clockwise orientation.

b Parentheses are used to identify rings with the exocyclic OH groups oriented in the clockwise direction.c / = O5C1O1C01, w = O05C01O1C1, h1 = O5C5C6O6, h01 ¼ O05C05C06O06, h2 = C5C6O6H6, h02 ¼ C05C06O06H06.

Table 3Electronic energy (DEelec), thermochemical quantities (DH, DS, and DG) at 298.15 K obtained at the B3LYP/6-311++G(d,p) level and relativeBoltzmann populations at the same temperature for the selected conformers of a,a-trehalose in gas phase

Conf.a Label DEelec (kJ mol�1) DH (kJ mol�1) DS (J mol�1 K�1) DG (kJ mol�1) Relative pop.

c1 ggGGgg 0.0 0.0 0.0 0.0 0.37c2 gtGGgg 1.4 1.2 12.3 �2.5 1.00c3 tgGGgg 1.6 1.7 12.3 �2.0 0.82c4 tgGGgt 2.3 2.7 6.9 0.6 0.29c5 tgGGtg 2.8 3.0 �3.4 4.0 0.07c6 gtGGgt 2.5 2.4 4.5 1.0 0.24c7 gtTGtg 4.6 5.0 1.2 4.7 0.06c8 gtTGgg 5.1 4.9 5.6 3.3 0.10c10 tgTGtg 5.6 6.3 �3.0 7.2 0.02c11 ggTGtg 5.8 6.2 �0.2 6.3 0.03c12 ggTGgg 6.1 5.8 5.2 4.3 0.07c13 tgTGgg 6.4 6.5 3.9 5.4 0.04c14 gtGGtg 7.0 7.1 11.0 3.8 0.08c15 tgSSgt 6.1 5.8 8.7 3.2 0.10c16 gtSSgg 6.3 5.5 15.1 1.0 0.25c17 tgTGgt 6.4 6.4 5.0 4.9 0.05c19 ggTGgt 6.7 6.2 11.5 2.8 0.12

a Conformers are numbered according to their electronic energy obtained at the B3LYP/6-311++G(d,p)//6-31G(d) level.

2052 S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059

O2H and O02H groups were oriented away from the O1 atom dis-rupting the concerted relative orientation, whereas in the six latterall exocyclic OH groups were positioned clockwise around bothrings, resulting in a structure that keeps the concerted system ofOH contacts but excludes the O1 atom. The results of these calcu-lations are included in Table 2.

The mean energy difference between the cX0 and cX structuresis ca. 11 kJ mol�1. This energy increase can be attributed to the dis-ruption of two putative vicinal intramolecular hydrogen bonds(O2H� � �O1 and O02H � � �O1) existent in cX, and/or to the electronicrepulsion arisen from the proximity of the lone pairs of the oxygenatoms in cX0. When only one OH group is moved away from O1,

Figure 3. Optimized geometries at the B3LYP/6-311++G(d,p) level for selected conformers of trehalose with the same hydroxymethyl groups orientation.

S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059 2053

which is the case of c14 when compared to c4, the energy increaseis only 5 kJ mol�1, corresponding to roughly half of the values pre-viously found.

When the cX00/cX pairs of the conformers are compared, the en-ergy differences are always positive, indicating a preference for theretro-clockwise orientation of the exocyclic OH groups relative tothe clockwise one. These results also show that the energy increasevaries considerably with the orientation of the hydroxymethylgroups. A value lower than 3 kJ mol�1 is found when these groupsare tg or gg, increasing to 10 or 17 kJ mol�1 when one or bothgroups adopt a gt conformation, respectively. This conformationalchange is also accompanied by an increase of / and w dihedrals inorder to minimize the repulsions between O1 and O2 or O02.

3.3. NBO and QTAIM analyses

After the identification of the lowest electronic energy struc-tures within the group of the explored conformations, we will at-

tempt to quantify, whenever possible, the features accounting forthe stability order. The energetic sequence of the conformers is ex-pected to depend on the balance of three main factors: exo-ano-meric effect, establishment of intramolecular hydrogen bonds,and steric effects. The two former effects are addressed in the pres-ent work in terms of NBO and QTAIM theories.

3.3.1. Anomeric and exo-anomeric effectsAlthough several authors argue that the anomeric/exo-anomer-

ic effects are responsible for the greater stability of the gauche ori-entation around the R–O–C–O fragments in disaccharidesstructures,30,63,64 they have never been quantified in trehalose.Both may be rationalized using the natural bond orbital (NBO)analysis, although a different interpretation based on the QTAIMtheory has been recently proposed to explain these effects.65

As the application of the QTAIM analysis for this purpose inlarge-sized molecules such as trehalose is computationally highlydemanding, it has not been considered in this paper.

2054 S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059

Based on the NBO theory, the anomeric effect can be quantifiedfrom the hyperconjugative stabilization energies involving the ringoxygens (O5 and O05) lone pairs and the antibonding C1–O1 and C1–O01 orbitals; whereas the exo-anomeric effect can be evaluated bythe stabilizing interactions between the glycosidic oxygen O1 lonepairs and the r*(C1–O5) and r*(C01—O05) orbitals.

The values of the Lp(O)?r*(C–O) hyperconjugative stabilizationenergies are given in Table 4 for the lowest electronic and Gibbs en-ergy conformers, c1 (ggGGgg) and c2 (gtGGgg), respectively. Thisanalysis was extended to other conformers representative of differ-ent families, sharing the same hydroxymethyl groups orientation:c8 (gtTGgg), c16 (gtSSgg), c25 [(gg)TTgt], and c36 (gtGG0gg). This se-lected group of conformers allows comparing these effects in thethree most populated families with two others (TT and GG0) of ratherhigh energy. As expected, no major differences are found betweenthe values obtained for the Lp(O5)?r*(C1–O1) and LpðO05Þ !r�ðC01—O1Þ stabilization energies. However, the energies corre-sponding to the charge transference from Lp(O1) to the r*(C1–O5)and r*(C01—O05) orbitals are larger for / or w between 70� and 100�,becoming smaller as the dihedral angles increase, as can be seenby comparing the values obtained for c16 (/ = 100.8�, w = 120.7�),c8 (/ = 143.9�, w = 94.9�), and c25 (/ = -177.0�, w = 177.5�). Thus,these interactions clearly disfavor the TT arrangement, being maxi-mized in the GG and GG0 families.

Conformer c36, despite having a considerable anomeric/exo-anomeric stabilizing effect, has a rather high energy (DE = 36 kJmol�1 at B3LYP/6-311++G(d,p) level). The crowding of variousgroups in this structure (see Fig. 3), as in all the GG0 conformers

Table 4Second order perturbation stabilization energies, E2�

i;j , for the orbital intrepresentative conformer of each family

Conf.a LabelbE2�

i;j Exo-anomeric effectc

Lp(O1)?r*(C1–O5) Lp(O1)?r

c1 ggGGgg 13.8 13.8c2 gtGGgg 11.9 12.3c8 gtTGgg 6.2 13.8c16 gtSSgg 12.3 9.4c25 (gg)TTgt 5.1 4.7c36 gtGG0gg 13.5 12.4

a Conformers are numbered according to their electronic energy obtainb Parentheses are used to identify rings with the exocyclic OH groupsc Energy values in kcal mol�1.

Table 5Second order perturbation stabilization energies, E2�

i;j , corresponding to the interactions betwsome selected conformers of trehalose molecule in gas phasea,b

Orbital interaction

c1ggGGgg

c100

(gg)GG(gg)c2gtGGgg

c200

(gt)GG(gg)

Lp(O4)?r*(O6–H)Lp(O6)?r*(O4–H)Lp(O05)?r*(O2–H)Lp(O6)?r*(O02–H)Lp(O06)?r*(O2–H)Lp(O2)?r*(O02–H)Lp(O2)?r*(O3–H) 0.4 0.5Lp(O3)?r*(O4–H) 0.2 0.3Lp(O4)?r*(O3–H) 0.4 0.3Lp(O3)?r*(O2–H) 0.4 0.5Lp(O02)?r*(O03–H) 0.4 0.5

Lp(O03)?r*(O04–H) 0.2 0.2

Lp(O04)?r*(O03–H) 0.4 0.4

Lp(O03)?r*(O02–H) 0.4 0.4

a Conformers are numbered according to their electronic energy obtained at the B3LYPc2, and c3, respectively, by changing all the exocyclic OH groups from a retro-clockwise

b Parentheses are used to identify rings with the exocyclic OH groups oriented in the

in general, seems to overcome the contribution of the stabilizationhyperconjugative effects. For this conformer, repulsive contacts oc-cur between C1H and C03H (dH� � �H = 1.97 Å), C1H/C05H (dH� � �H =2.41 Å), and O2H/O02H (dH� � �H = 2.11 Å), among others. This resultshows that the general accepted idea that the exo-anomeric effectis the feature determining the conformational stability order indisaccharides cannot be taken as a rule.

3.3.2. Intramolecular hydrogen bondsThe existence of intramolecular hydrogen bonds in the treha-

lose conformers has been investigated by NBO and QTAIM analysis.In NBO terms a hydrogen bond between a D–H group and an elec-tronegative atom A corresponds to a hyperconjugative interactiondefined by Lp(A)?r*(D–H). Within the QTAIM theory, the topolog-ical features of the electron distribution and the partitioning of amolecular system into its atomic fragments from the gradient vec-tor field of the electron density q(r) allow to infer on the nature ofthe interactions between bonded atoms including those involvedin hydrogen bonding. According to this formalism, a hydrogenbond can be identified by the presence of a bond critical point(bcp) with a (3, �1) topology between the atoms participating inthe interaction (H� � �A); a value of qbcp within the range [0.002,0.04] a.u.; a positive value of the Laplacian of q(r), r2qbcp, withinthe range [0.015, 0.15] a.u., and mutual penetration of hydrogenand acceptor atoms.35,66,67

With these approaches we intend to shed some light on twopoints: evaluate the contribution of the intramolecular hydrogenbonds between non-vicinal groups to the conformers’ stability

eractions involved in the anomeric and exo-anomeric effects, for a

E2�i;j Anomeric effectc

*(C01—O05) Lp(O5)?r*(C1–O1) Lp(O05)?r*(C01–O1)

12.7 12.715.6 15.514.9 13.314.6 14.812.0 13.315.2 17.0

ed at the B3LYP/6-311++G(d,p)//6-31G(d) level.oriented in the clockwise direction.

een the oxygen lone pairs and the non-vicinal and vicinal OH antibonding orbitals, for

E2�i;j (kcal mol�1)

c3tgGGgg

c300

(tg)GG(gg)c8gtTGgg

c16gtSSgt

c25(gg)TTgt

c36gtGG0gg

2.84.6

7.30.10.3

5.3 3.20.4 0.6 0.5 0.30.3 0.2 0.3 0.3

0.2 0.40.4 0.6

0.5 0.4 0.4 0.3 0.50.2 0.3 0.3 0.4 0.2

0.40.5

/6-311++G(d,p)//6-31G(d) level. Conformers c100 , c2,00and c300 were obtained from c1,to a clockwise orientation.

clockwise direction.

Table 7Values of the second order stabilizing energy, E2�

i;j , corresponding to all orbitalinteractions involving the O2H and O02H groups used to evaluate the role of theexocyclic OH group orientationa

Orbital interaction E2�i;j (kcal mol�1)

c1 c10 c2 c20 c3 c30

Lp(O1)?r*(O2–H) 0.7 0.8 0.8Lp(O1)?r*(O02–H) 0.7 0.7 0.7Lp(O2)?r*(C1–C2) 6.9 8.8 6.8 9.3 6.8 9.2Lp(O2)?r*(C2–C3) 7.5 2.5 7.6 1.5 7.6 2.0Lp(O2)?r*(C2–H) 2.6 4.2 2.7 5.8 2.7 5.0Lp(O2)?r*(O3–H) 0.4 0.3 0.5 0.3 0.4 0.3Lp(O2)?r*(C3–C4) 1.0 0.6 1.0 0.5 0.9 0.5Lp(O2)?r*(C1–O5) 1.0 1.2 0.9 1.0 1.0 1.1Lp(O02)?r*(C01—C01) 6.9 8.8 7.5 9.0 6.9 9.0

Lp(O02)?r*(C02—C03) 7.5 2.5 6.8 1.3 7.5 2.1

Lp(O02)?rr*(C02–H) 2.6 4.2 2.7 7.4 2.6 4.7

Lp(O02)?r*(O03–H) 0.4 0.3 0.5 0.3 0.5 0.4

Lp(O02)?r*(C03—C04) 1.0 0.6 1.0 0.4 1.0 0.5

Lp(O02)?r*(C01—O05) 1.0 1.2 0.9 1.0 1.0 1.2

r(O2–H)?r*(C05–H) 0.1 0.1r(O2–H)?r*(C2–O2) 0.1 0.1 0.1r(O2–H)?r*(C3–H) 0.1 0.1r(O2–H)?r*(C1–C2) 0.3 0.1 0.3 0.3r(O2–H)?r*(C2–C3) 1.8 2.1 2.0r(O2–H)?r*(C3–C4) 0.1 0.2 0.1r(O2–H)?r*(C2–H) 2.1 0.8 2.1 0.2 2.1 0.6r(O2–H)?r*(C1–O5) 0.2 0.2 0.1 0.2

r(O02–H)?r*(C5–H) 0.1 0.2 0.1

r(O02–H)?r*(C02—O02) 0.1 0.1

r(O02–H)?r*(C03–H) 0.1 0.1

r(O02–H)?r*(C01—C02) 0.3 0.1 0.3 0.3 0.1

r(O02–H)?r*(C02—C03) 1.8 2.1 1.9

r(O02–H)?r*(C03—C04) 0.1 0.2 0.1

r(O02–H)?r*(C02–H) 2.1 0.8 2.0 0.5 2.1 0.7

r(O02–H)?r*(C01—O05) 0.2 0.2 0.2

r(C05–H)?r*(O2–H) 0.1 0.1

S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059 2055

and clarify on the existence of a cooperative H- bond systeminvolving the vicinal exocyclic OH groups.

The existence of hydrogen bonds was investigated in the GGlower energy conformers (c1, c2, and c3) and in their all clockwisecX00 counterparts, as well as on each conformer chosen above torepresent each family.

The values of the hyperconjugative Lp(A)?r*(D–H) interactionenergies are presented in Table 5, while the results of the QTAIManalysis, namely the values of q andr2q at the bond critical points(bcp) that do not correspond to covalent bonds, are given in Table 6.This table also includes the values of the geometrical parameters(O� � �H distance and O–H� � �O angle) commonly used to character-ize H-bonds.

Starting with the non-vicinal OH interactions, one can see thatin the lowest energy family (GG), only the conformers having atg hydroxymethyl group orientation exhibit a hydrogen bond. Itis formed between the hydroxymethyl OH group and the neighbor-ing exocyclic OH group and has similar geometric parameters in alltg conformers. These are exemplified by the values given in Table 6for c3.

As a representative of the TG family we have analyzed con-former c8. This structure exhibits an inter-residue hydrogen bond,established between one of the exocyclic OH groups and the etheroxygen atom of the other glucopyranose ring. Another type of in-ter-residue hydrogen bond involving one exocyclic OH group andthe oxygen atom of the hydroxymethyl group of the other gluco-pyranose ring is present in the SS conformations, although it is avanishing weak one (see values obtained for c15 and c16). TheTT and GG0 families, represented in Tables 5 and 6 by conformersc25 and c36, respectively, present an inter-residue hydrogen bondbetween the exocyclic OH groups O02H and O2H.

As the inter-residue hydrogen bonds have only been identifiedin high energy conformers such as c8, c25, and c36, one can con-clude that they do not play a significant role in the trehalose con-

Table 6Electron density topological analysis properties for the bond critical points locatedbetween the non-bonded H� � �O atoms participating in the establishment of hydrogenbonds and geometrical parameters (distance and angle) for some selected trehaloseconformers

Conf.a qbcp

(u.a.)r2qbcp

(u.a.)d(D–H� � �A)(Å)

\(D–H� � �A)(�)

c3O6–H� � �O4

0.019 0.018 2.08 134.0

c8O2–H� � �O05

0.027 0.023 1.94 163.2

c15O2–H� � �O05

0.007 0.006 2.63 129.9

O6–H� � �O4

0.019 0.018 2.08 134.2

O2–H� � �O06

0.008 0.007 2.49 135.7

c16O02–H� � �O5

0.007 0.006 2.62 130.0

O02–H� � �O6

0.008 0.007 2.50 137.1

c25O02–H� � �O2

0.024 0.023 1.97 143.3

c36O02–H� � �O2

0.015 0.013 2.19 153.7

a Conformers are numbered according to their electronic energy obtained at theB3LYP/6-311++G(d,p)//6-31G(d) level.

r(C1–C2)?r*(O2–H) 0.1 0.1 0.1r(C3–H)?r*(O2–H) 0.1r(C2–C3)?r*(O2–H) 1.4 2.2 1.7r(C2–H)?r*(O2–H) 2.7 0.2 2.6 2.7 0.2

r(C5–H)?r*(O02–H) 0.1 0.1

r(C01—C02)?r*(O02–H) 0.1 0.1 0.1

r(C02—C03)?r*(O02–H) 1.4 1.9 1.6

r(C02–H)?r*(O02–H) 2.7 0.2 2.6 0.2 2.7 0.2

r(C03–H)?r*(O02–H) 0.1

r(C02—O02)?r*(O02–H) 0.1P

(all interactions) 51.8 44.0 50.9 47.5 52.1 45.7

a Conformers are numbered according to their electronic energy obtained at theB3LYP/6-311++G(d,p)//6-31G(d) level. Conformers c10 , c20 , and c30 were obtainedfrom c1, c2, and c3, respectively, by moving the O2H and O02H groups away fromcentral O1 atom.

formational stability. The fact that conformers c8 (TG) and c25 (TT)have a small exo-anomeric effect, and in c25 besides this, the gly-cosidic angle also has quite an unfavorable value seem to prevailover the inter-residue hydrogen bond. For c36, as it has alreadybeen mentioned, the severe structural crowding largely overcomesthe stabilizing effects given by the favorable exo-anomeric effectand hydrogen bond.

As stressed before, several theoretical studies on carbohydratesrefer to the existence of a concerted structure of intramolecularhydrogen bonds between the exocyclic OH groups that tend to in-crease the stability of some orientations over others by means of acooperative effect.45,58–60 However, serious objections have beenraised by different authors on the existence of OH bonds betweenthe vicinal groups.61,68–70 Taking into account the geometricparameters obtained for the optimized conformers of trehalose(distance and angle values around 2.45 Å and 105�, respectively)

Table 8Relative Gibbs energy in solution, Gsol,rel, solvation Gibbs energy, DGsolv, relative population of each conformer in both phases, component terms of the solvation energyconcerning electrostatic, DGelec, repulsion, DGrep, dispersion, DGdisp and cavity formation, DGcav for trehalose conformers obtained at the CPCM/B3LYP/6-311++G(d,p)a,b

Gas phase Aqueous solution

Rel. pop. Gsol,relc DGsolv DGelec DGrep DGdisp DGcav Rel. pop.

c1 0.37 0.0 �153.0 �188.3 58.6 �187.2 163.9 0.57c2 1.00 �1.4 �151.9 �187.8 58.1 �186.5 164.2 1.00c3 0.82 2.5 �148.5 �184.3 59.3 �188.0 164.4 0.21c4 0.29 8.0 �145.6 �180.1 58.5 �187.1 163.0 0.02c5 0.07 14.3 �142.7 �176.1 59.4 �188.3 162.2 0.00c6 0.24 11.1 �143.0 �178.3 57.0 �185.4 163.7 0.01c7 0.06 10.9 �146.8 �184.3 63.1 �187.8 162.2 0.01c8 0.10 2.75 �153.5 �191.7 62.9 �187.7 163.0 0.19c10 0.02 13.8 �146.4 �181.9 62.9 �188.7 161.3 0.00c11 0.03 8.4 �150.9 �187.5 62.5 �188.1 162.2 0.02c12 0.07 0.1 �157.2 �194.7 62.4 �188.0 163.1 0.55c13 0.04 6.3 �152.0 �188.5 63.0 �188.8 162.2 0.04c14 0.08 4.9 �152.0 �187.6 61.3 �188.5 162.7 0.08c15 0.10 9.0 �147.2 �185.0 64.5 �190.0 163.3 0.01c16 0.25 1.4 �152.6 �191.5 64.1 �189.2 164.0 0.33c17 0.05 10.2 �147.7 �185.0 63.8 �188.8 162.3 0.01c19 0.12 2.5 �153.3 �191.6 63.4 �188.2 163.2 0.21

a Gibbs energy values are expressed in kJ mol�1.b Conformers are numbered according to their electronic energy obtained at the B3LYP/6-311++G(d,p)//6-31G(d) level.c Gsol = Ggas + DGsolv.

Figure 4. Representation of the solvation Gibbs energy of trehalose conformers together with its electrostatic and non-electrostatic components.

2056 S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059

these could only be considered as very weak hydrogen bonds. Sinceno bcps have been identified between the vicinal OH groups andthe values shown for the hyperconjugative Lp(O)?r*(O–H) areconsistently low, Table 5, no evidence of hydrogen bonds betweenthe vicinal groups are found to exist.

Although no hydrogen bond could be identified between theexocyclic OH groups, the energy of the cX0 structures (obtainedby disruption of the OH concerted orientation) is considerablyhigher than that of cX. In order to explain the preference for theconcerted orientation of the exocyclic OH groups, an NBO analysiswas performed on c1, c2, and c3 as well as on their cX0 counter-parts. Having in mind that the main difference between cX andcX0 conformers relies on the O2H and O02H orientation, all hyper-conjugative interactions involving these groups with energyP0.1 kcal were computed and are shown in Table 7. As expected,the Lp(O1)?r*(O2–H) and Lp(O1)?r*(O02–H) energies in cX0 arenegligible. Also, all the stabilizing interactions involving the O2

and O02 lone pairs are lower in cX0 than in cX conformers. Thecharge transference from r(O2–H) or r(O02–H) has identical valuesin all the conformers, despite the acceptor orbitals being different.The charge transference for the corresponding antibonding orbitalsalways favors the cX conformers.

As conclusion, the sum of all hyperconjugative interactions dis-played in Table 7 stabilizes the cX structures over the cX0 ones. Thismeans that the concerted exocyclic OH groups’ orientation maxi-mizes the orbitals overlap that is involved in hyperconjugativeinteractions, thus stabilizing the overall molecule.

3.4. Influence of the solvent in the stability order of theconformers

Most of the papers dealing with the investigation of trehaloseaqueous solution structure concern molecular dynamics studiesusing different molecular mechanics, carbohydrate force fields,

c1 c2 c3 c4 c5 c6 c7 c8 c10 c11 c12 c13 c14 c15 c16 c17 c19

-158

-156

-154

-152

-150

-148

-146

-144

-142

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e Po

pula

tion

Pgas Psol

ΔGsolv (kJ/m

ol)

Figure 5. Relative populations of trehalose conformers, c1–c19, in gas phase, Pgas, and in aqueous solution, Psol, and their respective values of the Gibbs energy of solvation,DGsolv (-j-).

Table 9Heteronuclear coupling constant, 3JH,C, values, in Hz, for the conformers of trehaloseand their respective dihedral angles, in degrees

Conf.a /H1 /H013JH,C

b

(/H1)

3JH,Cb

(/H01)Rel. pop.sol.

c1 �44.8 �44.8 2.94 2.94 0.57c2 �49.8 �46.5 2.49 2.79 1.00c3 �43.6 �44.9 3.05 2.93 0.21c4 �47.8 �49 2.67 2.56 0.02c5 �46.2 �46.2 2.82 2.82 0.00c6 �52.1 �52.1 2.28 2.28 0.01c7 30.2 �21.4 4.24 4.88 0.01c8 28.5 �21.3 4.37 4.89 0.19c10 31.1 �21 4.17 4.91 0.00c11 31.4 �21 4.14 4.91 0.02c12 30 �21.2 4.26 4.90 0.55c13 28.1 �20.7 4.41 4.93 0.04c14 �32.6 �49.9 4.04 2.48 0.08c15 3.67 �16.2 5.58 5.18 0.01c16 �16.1 4.85 5.19 5.56 0.33c17 �18.1 29.2 5.08 4.32 0.01c19 �19.1 30 5.02 4.26 0.213JH,C weighted

mean3.6 3.7

a Conformers are numbered according to their electronic energy obtained at theB3LYP/6-311++G(d,p)//6-31G(d) level. /H1 = H1C2O1C01; /H01 ¼ C1O1C01H01.

b 3JH,C determined by Karplus equation.

S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059 2057

and also different potential energy functions to describe the waterensemble.19–21,23 Within the scope of the used methods and of theconsidered starting structures, the authors tried to conclude on thepreferred trehalose glycosidic dihedrals in solution (for instance, /= 93�, w = 93�,19 / = 60�, w = 70� and / = 56�, w = 65�20 and / = 66�,w = 66�21) and on the dynamics of water in the vicinity of treha-lose. Two other works report quantum mechanics studies on theaqueous solution of trehalose using a continuum model of solva-tion.24,56 However, in one of the works24 only two conformersare considered, and in the other, a low theory level is used to studya restricted group of conformers being / = 72.7�, w = 77.8� foundfor the lowest energy structure.56

In the present work, to access the solvent effect on the relativestability of the trehalose conformers, the solvation Gibbs energiesof the conformers c1–c19 were computed at the CPCM/B3LYP/6-311++G(d,p) level. The Gibbs energy of solvation (DGsolv) corre-sponds to the variation of this function in the transfer of one moleof solute from 1 M ideal gas phase to 1 M ideal dilute solution. Itcan be equated as DGsolv = Gsol � Ggas, where Ggas and Gsol are theGibbs energies in the gas phase and aqueous solution, respectively.

The dielectric continuum methodology allows the decomposi-tion of DGsolv into the following terms: DGsolv = DGcav + DGrep +DGdisp + DGelec, where DGcav stands for the cavity formation, DGrep

and DGdisp for the repulsion and dispersion interactions, respec-tively, and DGelec for the electrostatic contribution.

The results of the CPCM calculations are presented in Table 8.For a better visualization of the results, the most relevant dataare also graphically represented in Figures 4 and 5. The solvationGibbs energy values and the electrostatic and non-electrostaticterms are represented in Figure 4, whereas the relative populationsof the conformers in gas and aqueous solution phases, togetherwith the solvation Gibbs energy values, are displayed in Figure 5.

In Figure 4 it is clearly evidenced that the most significant dif-ferences among the conformers occur in the electrostatic termand that the trend observed for the solvation energy is mainly dic-tated by the variations in the electrostatic component. The solva-tion energy values range from �157 to �142 kJ mol�1 and themean values for each solvation term are: DGcav = (163.0 ± 0.8)kJ mol�1, DGdisp = (�188.0 ± 1.1) kJ mol�1, DGrep = (61.5 ± 2.4)kJ mol�1, DGnon-elec = (36.4 ± 1.5) kJ mol�1, and DGelec = (�186.1 ±5.0) kJ mol�1, reflecting the influence of the electrostatic term inthe conformers discrimination.

The data presented in Table 8 and represented in Figure 5 showthat, in general terms, the interaction with the solvent increases theweight of the TG family, while the GG and SS families remain withalmost the same relative population. Another point to be stressed isthe fact that although the most stable conformer in gas phase (c2) isalso the most stable one in aqueous solution, the weight of manyconformers varies considerably upon solvation. In fact, within thestructures obtained in gas phase only eight are meaningful in aque-ous solution: c1, c2, c3, c8, c12, c14, c16, and c19. With the excep-tion of c3 and c14, their population increases on going from gas tosolution and they are among those presenting the most negativevalues of solvation Gibbs energy (�157 to �152 kJ mol�1).

For the conformers investigated, about 60%, including the high-est abundant ones, have glycosidic dihedrals within 67� and 74�values, which were also found as minima in aqueous solution byother authors as referred above20,21,56 and close to what was foundin crystalline trehalose.57 The calculation of the heteronuclear

2058 S. C. C. Nunes et al. / Carbohydrate Research 345 (2010) 2048–2059

spin-coupling constant across the glycosidic angle 3JH,C has beenused by various authors to validate the conformational mapsobtained for carbohydrates in aqueous solution.20,56,71,72 This con-stant may be estimated by the Karplus equation73 3JH,C =5.7 cos2/H � 0.6 cos /H + 0.5, where /H = H1C1O1C01 and the valuesobtained for the conformers investigated are presented in Table 9.The comparison of these values with those obtained experimen-tally (3JH,C = (3.3 ± 0.5) Hz,27 3JH,C = 3.19 Hz72) rules out a high pre-dominance in the solution of conformers with dihedrals otherthan GG. The weighted mean values of the 3JH,C obtained are 3JH,C

(/H1) = 3.6 Hz and 3JH,C (/H01) = 3.7 Hz which compares favorablywith the experimental values, supporting the glycosidic dihedralsdistribution sampled in Table 8.

The hydroxymethyl groups’ orientation seems to play a key rolein the interaction with the solvent. Despite the very different glyco-sidic dihedrals of these conformers, all structures whose populationincreases upon solvation have the hydroxymethyl groups in the ggor gt orientation (c1, c8, c12, c16, and c19). In all conformers havinga tg-oriented hydroxymethyl group the population in solution de-creases relatively to the gas phase (c3, c4, c5, c7, and c15) or is keptin a low value (c10, c11, c13, c14, and c17). This result is in goodagreement with what was found in research works concerning glu-cose in aqueous solution.74–76 It is worth to remind that in crystal-line trehalose also 57 the hydroxymethyl groups are gt, gg oriented.

The existence of the O6–H� � �O4 and/or O06–H� � �O04 hydrogen bondin conformers with the hydroxymethyl group in the tg arrange-ment, as identified by QTAIM and NBO analysis, seems to disfavorsolvation. On the contrary, the presence of stronger inter-residuehydrogen bonds involving the exocyclic OH groups in other con-formers (e.g., c8, c12, and c19) does not affect the solvation process.

4. Conclusion

In this work the conformational preferences of a,a-trehalosewere investigated for a,a-4C1 structures with the retro-clockwiseexocyclic OH groups arrangement, considering five orientationsaround each dihedral of the glycosidic linkage combined with allthe three commonly explored orientations of the hydroxymethylgroups. For a selected group of conformers exhibiting differentconformational features, the exo-anomeric effect was determinedfrom an NBO analysis and the intramolecular hydrogen bondscharacterized by geometric parameters, NBO, and QTAIM analysis.

The conformers’ energy was found to be mainly determined by theconformation around the two central dihedrals, being the GG, TG, andSS conformers preferred over the GG0, TT, and SG0 ones. Steric effects,resulting from the different central dihedral orientation, seem toovercome the stabilizing contributions resulting either from theexo-anomeric effect or from the inter-residue intramolecular bonds.

Within each ring, the exocyclic OH groups must be arranged in aconcerted orientation in order to maximize the orbitals overlapthat are involved in hyperconjugative interactions, thus stabilizingthe overall molecule, and minimizing the repulsive interactions be-tween oxygen lone pairs and hydroxyl hydrogen atoms. From theNBO and QTAIM analyses no evidence was found for the existenceof intramolecular hydrogen bonds between these vicinal groups.

The stability of the conformers in aqueous solution was foundto be dependent on the hydroxymethyl groups’ orientation, clearlydisfavoring the tg conformers, bearing an intramolecular hydrogenbond, relatively to the gg and gt ones.

Acknowledgment

The authors acknowledge Fundação para a Ciência e a Tecnolo-gia (FCT), Lisbon, for the financial support: Project PTDC/QUI/64565/2006.

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