Specific Anion Effects on the Optical Rotation of α-Amino Acids - The

Aug 11, 2007 - Changes in optical rotation of some α-amino acids are induced by electrolytes. Such effects on l- and d-enantiomers of a range of amin...
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10510

J. Phys. Chem. B 2007, 111, 10510-10519

Specific Anion Effects on the Optical Rotation of r-Amino Acids Simona Rossi,† Pierandrea Lo Nostro,*,† Marco Lagi,† Barry W. Ninham,†,‡ and Piero Baglioni† Department of Chemistry and CSGI, UniVersity of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy, and Department of Applied Mathematics, Research School of Physical Sciences and Engineering, Institute of AdVanced Studies, Australian National UniVersity, Canberra, ACT 0200, Australia ReceiVed: March 19, 2007; In Final Form: June 29, 2007

Changes in optical rotation of some R-amino acids are induced by electrolytes. Such effects on L- and D-enantiomers of a range of amino acids are explored for sodium salts with varying anion. The amino acids studied were alanine, aspartic acid, glutamic acid, glutamine, proline, threonine, and tryptophan. The anion’s polarizability in solution accounts for the change in [R] only for the halides. Self-association of amino acids in solution and pH changes due to the presence of the electrolytes do not account for the observed variations in optical activity. Specific interactions of anions with the chiral amino acids (Hofmeister effects) and saltinduced perturbations of the amino acid hydration shell appear to be responsible for the effects, and conformational changes in the chiral solutes due to the presence of ionic species are discussed.

Introduction In principle, changes in the properties of amino acids induced by electrolyte solutions can provide information on their interand intramolecular interactions. Through such interactions, electrolytes affect solubility, partitioning, separation, and other phenomena characteristic of amino acids.1,2 Very little work has been done to exploit these possibilities. Only a few, sporadic studies have been carried out to determine the effect of different salts on the solubility and activity coefficients of amino acids.3,4 Optical activity is an extensive property of matter. It is directly related to functionalities like molecular recognition, chirality, and photochirality.5-8 It depends mainly on the anisotropic dielectric properties of the constituent materials involved. Since the original work of Kirkwood, it has been considered to be an internal field effect, due to an asymmetric distribution of molecular dipoles.9,10 In dilute solution the molecular conformation and, related to this, the anisotropic polarizability determine the optical activity of a chiral entity. Moreover, changes in solvent composition have remarkable effects on optical rotation.9,11,12 The conformation, stability, and properties of polymeric macromolecules are determined by both the intra- and intermolecular interactions with other solutes and with the solvent molecules.13 It can be taken that the entire gallimaufry of forcesselectrostatic, van der Waals, hydrogen bonding and hydrophobic interactions, and moresall participate in setting the conformational equilibrium of molecules and macromolecules.14 The biological activity, binding affinity, the folding of proteins, and interactions between them are directly related to the (solvent induced) conformational flexibility of peptides.15 In the gas phase, electrostatic intramolecular interactions between ionized groups determine for the most part the shape and folding of a conformer. That is not so in solution. Thus it has been shown, at least for aspartic acid (Asp) and asparagine (Asn), that hydration forces control the conformational equi* Corresponding author. Fax: +39(055)457-3036. E-mail: pln@ csgi.unifi.it. Internet: http://www.csgi.unifi.it. † University of Florence. ‡ Australian National University.

librium in solution and that these dominate forces due to electrostatic repulsion.13 The effect of low molecular weight cosolutes (electrolytes, sugars, urea, etc.) on the optical rotation of different molecules in aqueous solutions has been described in several reports.16,17 The consensus is that the salt dependence of the optical rotation of organic solutes is due to interactions with specific bonds in the chiral molecule. Such interactions depend on the polarizability of the anion, essentially ion specific dispersion forces. The induced change in local conditions can lead to a change in steric restrictions to rotation, so modifying the molecular conformation. Likewise, it has been observed by Mandelker that optical rotation changes induced by salts in polypeptides are due to structural changes such as a repopulation of rotational states.18 While the optical activity and circular dichroism of peptides and polysaccharides in the presence of electrolytes have been extensively studied in the past, the specific influence of salts on the optical activity of singly dispersed R-amino acids has not been much explored systematically.19 In a previous paper we investigated the effect of some electrolytes on the optical activity and Fourier transform IR spectra of the D- and L-enantiomers of serine and glucose.20 The results were explained with specific interactions/adsorption of the anions at specific bonds in these molecules. In this work we report new measurements of specific optical rotation of D- and L-enantiomers of alanine, aspartic acid, glutamic acid, glutamine, proline, threonine, and tryptophan, in aqueous solutions of a series of some monovalent electrolytes (sodium salts). Qualitatively different changes in specific optical rotation occur with the different salts. We found that the halides induce an anion specific change in [R] that correlates well with the anion’s polarizability, surface tension molar increment, and Gibbs free energy of hydration. These are typical physicochemical parameters that usually reflect the different behavior of ionic species in bulk solutions and at interfaces.21 On the other hand, polyatomic and anisotropic species such as SCN-, NO3-, ClO3-, ClO4-, and H2PO4- produce a more complicated effect, presumably due to their intrinsic anisotropic polarizabilities and

10.1021/jp0721806 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/11/2007

Anion Effects on Amino Acids

J. Phys. Chem. B, Vol. 111, No. 35, 2007 10511

to the presence of acceptor/donor sites available for hydrogen bonding. In all cases the presence of the ions perturbs the dipolar moment of the amino acid molecule, and its interactions (amino acid-amino acid and water-amino acid), producing a significant conformational rearrangement that results in a change of optical activity. Materials and Methods Formulas. When a molecule rotates the plane of polarized light, the rotation of polarization R is given by

R)

πd(nL - nD) λ

(1)

where R is expressed in degrees or radians, d is the distance which the light travels through an optically active sample (liquid or solid), λ is the wavelength of light, and nL and nD are different refractive indices for circularly left- and right-polarized light, respectively.22 At a temperature T and incident wavelength λ, the specific rotation or optical activity is expressed as

[R] λT )

100R cd

(2)

where c is the concentration of the optically active sample (in g/mL), and d is the path length across the cell (in mm).20 If the solvent is an aqueous salt solution, the results need to be corrected for the change in refractive index of the solvent due to the presence of the electrolyte

[R] ) [R]exp

nw2 + 2 ns2 + 2

(3)

Here [R] and [R]exp are the corrected and experimental values and nw and ns are the refractive indices of water and of the aqueous solution of the particular salt at the same T and λ, respectively.17,20 However it is important to recall that such a correction, based on the Lorentz effective field continuum solvent approximation, which attempts to take into account the solvent effect, has been recently questioned. The approximation may lead to significant discrepancies between the calculated and the experimental [R] values, as has been shown by Stephens et al.23,24 The error for [R] was calculated from the experimental errors for R, c, nw, and ns according to the error propagation formulas. Electrolytes and Solutes. Sodium fluoride, chloride, bromide, iodide, nitrate, perchlorate, chlorate, dihydrogen phosphate, thiocyanate, and the D- and L-enantiomers of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), alanine (Ala), proline (Pro), threonine (Thr), and tryptophan (Trp) were purchased from Sigma-Aldrich-Fluka (Milan, Italy) and used without further purification. Buffer solutions at pH 5 and 8 were from Merck (Milan, Italy). Water was purified with a Milli-Q water system (Organex) supplied by Millipore (resistivity >18 MΩ· cm). Sample Preparation, Refractive Index, pH, and Optical Rotation Measurements. The concentrations of the amino acids used in this study are listed in Table 1. These values were chosen to be far from the solubility limits, to avoid changes in the concentration of solute molecules that might contribute to optical activity due to salting out.

In the cases of Ala, Thr, and Ser, the values of the optical rotation R were also recorded as a function of the amino acid concentration for reasons that will be explained. The samples were prepared by weighing the appropriate amount of amino acid, using as solvent the previously prepared salt solutions. All samples were left to stand 1 day before measuring the optical rotation. Each sample was prepared, and measurements were made at least twice. For each sample, R is the average of at least 10 distinct measurements and is shown with its standard deviation in Table 2. A Perkin-Elmer 343 polarimeter was used. All measurements were carried out at 20 ( 0.1 °C, at λ ) 589 nm (sodium D line). The cell path length was 10 cm. All electrolyte concentrations in the cell were 0.5 M. The effect of salt concentration on the specific rotation of L-Ser was also checked for NaCl, NaBr, and NaI at 0.25 and 1 M. The effect of pH on the optical rotation of L-Asp and L-Glu in the presence of NaF and NaH2PO4 (0.05 M) was studied by using a buffer solution at pH 5 and 8. The optical purity of all electrolyte solutions was assessed by measuring their optical rotation. All salts were free from optically active impurities within the limits of accuracy given by the instrument. Refractive index measurements of electrolyte solutions were performed at a constant temperature (20 °C) with a modified Abbe´ refractometer (Atago 3T). pH measurements were carried out with a Crison Basic 20 pHmeter, equipped with a Crison glass electrode and an Ag/AgCl electrode as internal reference. The pHmeter was calibrated with standard buffer solutions at pH 4.0, 7.0, and 9.0. Molecular Dynamics Simulations. The OPLS-AA force field parameters for the amino acids and the TIP4P model for water were used, following the work of Shirts on the hydration of amino acids.25,26 The code used in the simulation is a parallelcompiled version of GROMACS v.3.3.1.27,28 The interatomic potential consists of a Lennard-Jones dispersion (LJ) and an electrostatic (ES) term

U(rij) )

[( ) ( ) ]

qiqj σij + 4ij 4π0rij rij

12

-

σij rij

6

(4)

where i and j are the atomic species located at ri and rj, respectively, rij ) |ri - rj|, qi is the partial charge on atom i, 0 is the permittivity of vacuum, while ij and σij are the standard LJ parameters for energy and size, respectively. The LJ interactions were truncated beyond an atom-atom cutoff distance of 1.4 nm. ES interactions, calculated with the particle mesh Ewald method (PME),29 were truncated at 0.9 nm. Integration of Newton’s equations of motion was performed using the Verlet leapfrog algorithm, and all bonds were constrained at their equilibrium values using the LINear Constraint Solver (LINCS) algorithm,30 while bending and torsion angles were allowed to vary. Simulations were performed using a cubic simulation cell with periodic boundary conditions and in a NPT ensemble (isobaric-isothermal), where temperature and pressure were controlled by coupling the system with an external Berendsen bath (T ) 293 K, P ) 1 bar, isothermal compressibility ) 4.5 × 10-5 bar-1, P coupling constant ) 0.5 ps, T coupling constant ) 0.1 ps).31 The initial configuration of the system was built by placing randomly 65, 78, 91, and 104 amino acid zwitterions in a 222 nm3 cubic box with 7082 water molecules, while velocities were assigned according to the Boltzmann distribution at 293 K. The amino acid concentrations ranged between 0.5 and 0.8 M. All coordinates and velocities were saved every picosecond. An energy minimization of 3000 steps with the Steepest Descent algorithm was performed to stabilize the system and

10512 J. Phys. Chem. B, Vol. 111, No. 35, 2007

Rossi et al.

TABLE 1: Side Chain Residue (-R), Concentration (c, in mol/L and in g/100 mL), Specific Rotation [r]DT for the L-Isomer (from ref 79) at 589 nm at Temperature T and Concentration c′ (in g/100 mL) for the r-Amino Acids Used in This Work (at 25 °C) c R-AA

-R

mol/L

g/100 mL

[R]DT (c′)

Ala Asp Glu Gln

-CH3 -CH2COOH -CH2CH2COOH -CH2CH2CONH2

0.50 0.02 0.025 0.15 (L) 0.137 (D) 0.50 (L)

4.45 0.27 0.37 0.22 0.20 5.76

+2.42 (25 °C; c′ ) 10 in H2O) +25.0 (20 °C; c′ ) 1.97 in 6 M HCl) +31.4 (22.4 °C; c′ ) 10 in 2 M HCl) +6.1 (23 °C; c′ ) 3.6 in H2O)

0.43 (D) 0.20 0.30 0.016

4.95 2.10 3.57 0.33

Pro

-CH2OH -CH(CH3)OH

Ser Thr Trp

-85.2 (20 °C; c′ ) 4 in H2O)

-6.83 (23 °C; c′ ) 10.41 in H2O) -28.3 (26 °C; c′ ) 1.09 in H2O) -31.5 (23 °C; c′ ) 1 in H2O)

TABLE 2: Corrected Optical Rotation Values [r] (in deg‚g-1‚mL‚mm-1), for L- and D-Enantiomers in Pure Water and in the Presence of Different Monovalent Sodium Salts [R] anion

L-Ala

D-Ala

L-Asp

D-Asp

L-Gln

D-Gln

L-Glu

D-Glu

(H2O) FClBrINO3ClO4ClO3H2PO4SCN-

1.80 ( 0.02 1.68 ( 0.02 1.82 ( 0.01 1.86 ( 0.02 1.89 ( 0.02 2.04 ( 0.02 1.78 ( 0.02 1.74 ( 0.02 2.34 ( 0.02 1.67 ( 0.02

-1.84 ( 0.02 -1.70 ( 0.02 -1.84 ( 0.01 -1.86 ( 0.02 -1.89 ( 0.03 -2.04 ( 0.02 -1.79 ( 0.03 -1.76 ( 0.02 -2.35 ( 0.02 -1.67 ( 0.02

4.57 ( 0.15 -11.11 ( 0.30 8.64 ( 0.28 8.47 ( 0.16 8.13 ( 0.26 8.13 ( 0.21 7.54 ( 0.31 7.52 ( 0.27 0.51 ( 0.36 8.72 ( 0.23

-4.46 ( 0.18 11.54 ( 0.20 -8.18 ( 0.21 -8.46 ( 0.19 -7.89 ( 0.25 -8.16 ( 0.21 -8.15 ( 0.31 -7.74 ( 0.24 -0.45 ( 0.36 -8.54 ( 0.25

6.37 ( 0.06 6.29 ( 0.06 6.24 ( 0.04 6.23 ( 0.05 6.24 ( 0.06 6.41 ( 0.04 5.95 ( 0.05 6.07 ( 0.05 6.28 ( 0.04 5.91 ( 0.05

-6.33 ( 0.06 -6.24 ( 0.05 -6.18 ( 0.05 -6.17 ( 0.05 -6.13 ( 0.05 -6.27 ( 0.08 -5.84 ( 0.06 -5.99 ( 0.05 -6.13 ( 0.05 -5.86 ( 0.03

11.54 ( 0.14 -3.50 ( 0.16 11.39 ( 0.26 11.48 ( 0.14 11.80 ( 0.18 11.53 ( 0.14 11.39 ( 0.28 11.74 ( 0.17 7.90 ( 0.17 12.11 ( 0.27

-11.63 ( 0.18 3.24 ( 0.23 -11.90 ( 0.26 -11.75 ( 0.14 -12.00 ( 0.23 -11.73 ( 0.20 -11.95 ( 0.28 -12.01 ( 0.18 -8.12 ( 0.17 -11.91 ( 0.16

[R] anion

L-Ser

D-Ser

L-Pro

D-Pro

L-Thr

D-Thr

L-Trp

D-Trp

(H2O) FClBrINO3ClO4ClO3H2PO4SCN-

-7.59 ( 0.06 -6.99 ( 0.06 -6.88 ( 0.06 -6.68 ( 0.06 -6.45 ( 0.07 -6.70 ( 0.06 -6.54 ( 0.06

7.49 ( 0.06 7.00 ( 0.06 6.89 ( 0.06 6.69 ( 0.06 6.55 ( 0.07 6.68 ( 0.06 6.63 ( 0.06

-7.20 ( 0.06 -6.44 ( 0.08

7.20 ( 0.06 6.60 ( 0.08

-84.61 ( 0.37 -84.63 ( 0.44 -85.14 ( 0.25 -84.96 ( 0.38 -84.72 ( 0.38 -85.42 ( 0.25 -86.13 ( 0.47 -85.34 ( 0.38 -85.12 ( 0.38 -85.63 ( 0.38

84.57 ( 0.46 85.02 ( 0.44 85.17 ( 0.25 85.11 ( 0.38 84.79 ( 0.38 85.29 ( 0.26 86.20 ( 0.39 85.40 ( 0.38 85.30 ( 0.38 85.66 ( 0.38

-28.52 ( 0.14 -28.82 ( 0.16 -28.65 ( 0.10 -28.37 ( 0.13 -27.84 ( 0.13 -27.70 ( 0.09 -28.62 ( 0.16 -28.38 ( 0.14 -28.61 ( 0.14 -28.43 ( 0.14

28.50 ( 0.16 28.78 ( 0.15 28.63 ( 0.14 28.44 ( 0.14 27.81 ( 0.14 27.72 ( 0.09 28.62 ( 0.16 28.49 ( 0.14 28.67 ( 0.14 28.46 ( 0.14

-30.15 ( 0.29 -25.50 ( 0.31 -29.05 ( 0.21 -28.73 ( 0.18 -28.64 ( 0.29 -30.33 ( 0.28 -29.75 ( 0.38 -30.31 ( 0.29 -26.15 ( 0.32 -29.55 ( 0.27

30.64 ( 0.23 25.72 ( 0.24 28.64 ( 0.25 28.70 ( 0.23 28.83 ( 0.27 30.22 ( 0.28 28.99 ( 0.38 29.92 ( 0.31 26.00 ( 0.31 29.02 ( 0.25

reduce the thermal noise. Finally, each molecular dynamics simulation was run for 2 ns using a 2 fs integration time step: the first nanosecond was not considered in the data treatment and taken as equilibration. Results In aqueous solution, all amino acids possess a zwitterionic structure: +H3N-CRH(R)-COO-. The residues, R, of the amino acids (AA) investigated here are listed in Table 1. Apart from Gly, all AA are chiral and possess a nonzero optical rotation. They are totally transparent at the yellow D-line of sodium (589 nm), so that their R is traditionally measured at this wavelength. [R] is strongly dependent on the lowest allowed electronic excitation, related to the n f π* transition of the carboxylate functional group.32 In aromatic amino acids, such as Trp, the π f π* transition has an additional effect on the chiroptical properties.32 The values of [R] in pure water and in the presence of electrolytes, corrected via eq 3, are listed in Table 2. The first

row shows the value of [R] in pure water at 20 °C at 589 nm obtained in this study. The optical activities show up specific anion effects. For some amino acids, salts induce remarkable changes. For other amino acids, the value of [R] remains almost unchanged. The table shows also that the changes in [R] induced by salts are the same for both the D- and L-enantiomers. Hence we consider the changes in [R] induced by the electrolytes for the L-enantiomers only. We examine the results in the ligth of three other physicochemical properties that reflect characteristic traces of “Hofmeister fingerprints”.33-37 These are the ionic polarizability in solution F (designated F instead of R, to avoid confusion with the optical activity), the surface tension molar increment σ ) ∂(∆γ)/∂c,38 and the Gibbs free energy of hydration ∆Ghydr. Sorting the anions according to the values of their polarizabilities in solution and surface tension molar increments,34-36 we obtain the following sequence: for F, I- > SCN- > ClO4> Br- > ClO3-, NO3- > Cl- > F- ; for σ, ClO4- < SCN-, ClO3- < I- < NO3- < Br- < Cl- < H2PO4- < F-.

Anion Effects on Amino Acids

J. Phys. Chem. B, Vol. 111, No. 35, 2007 10513

TABLE 3: Measured Refractive Index (nD20, c ) 0.5 mol/L), Ion Polarizability in Solution (G, Å3),21 Surface Tension Molar Increment (σ, mN‚L/m‚mol),45 and Gibbs Free Energy of Hydration (-∆Ghydr, kJ/mol)39 for the Different Anions anion

nD20

(H2O) FClBrINO3ClO4ClO3H2PO4SCN-

1.3326 1.3358 1.3375 1.3397 1.3438 1.3374 1.3370 1.3378 1.3400 1.3416

a

F

σ

-∆Ghydr

1.36 3.73 5.06 7.46 4.48 5.26 4.50

3a 1.6 1.3 1.0 1.1 1.4 0.55 2.3 0.5

465 340 315 275 300 430 280 465 280

6.47

salt

nD20

NaF, 0.05 M, pH ) 5 NaF, 0.05 M, pH ) 8 NaH2PO4, 0.05 M, pH ) 5 NaH2PO4, 0.05 M, pH ) 8 NaBr, 0.25 M NaBr, 1 M NaI, 0.25 M NaI, 1 M

1.3370 1.3344 1.3374 1.3348 1.3366 1.3473 1.3386 1.3548

From ref 80.

The most polarizable anions induce the smallest values for the surface tension molar increment. Chaotropes, such as iodide, thiocyanate, and perchlorate, are located in the left side of the series, and kosmotropes, such as fluoride and dihydrogen phosphate, on the right side. Similar trends occur for cations. Multivalent species like Mg2+, Al3+, SO42-, and PO43- are kosmotropic.39 The entities F, σ, and ∆Ghydr are directly or indirectly related to the specific “dispersion”, nonelectrostatic forces that ions experience in the solution, via ion-solvent, ionion, or ion-surface interactions.20,21,33-37,40-43 Table 3 lists their values for the different anions, and the experimental refractive indices (n) measured on the electrolyte solutions used in this work. We recall that, depending on the phenomenon investigated, there may be interchanges in the ordering of the effects along the series, andsin some casessan inversion in the trend.33 A major reason for this is that the ionic dispersion interactions depend in leading approximation not only on their polarizabilities but also on the frequency-dependent dielectric properties of solute or substrate with which they interact. Not just the magnitude but the sign of the interaction with, e.g., an air/water interface, or an organic molecule can change.21,33 The qualitative classification of electrolytes into “kosmotropes” and “chaotropes” in bulk water relies on a “water structure” model.44 A kosmotrope is assumed to strengthen the hydrogen bonding of bulk water, and a chaotrope is supposed to weaken such a structural network. The “water structure” model has occasioned some recent debate.45 In fact it reflects the same ion-solvent interactions that are built into a proper theory of the dispersion interactions.46 We will continue to use the familiar terms kosmotrope and chaotrope to distinguish the two classes of ions. A plot of [R]salt/[R]water versus polarizability F generally shows a regular variation for fluoride, chloride, bromide, and iodide. By contrast for Pro and Gln, the ratio [R]salt/[R]water is almost constant (see Figure 1). The halide series suggests a role for dispersion forces that depend on polarizabilities in the ionamino acid interactions. With the exception of Ala, [R]salt/[R]water increases with F from F- to I- for the L-enantiomers. In the case of Ala, Trp, Ser, and Thr, iodide has the largest effect. Results for Asp and Glu are shown in the inset in Figure 1. For

Figure 1. [R]salt/[R]water for Ala (b), Gln (2), Pro ((), Ser ()), Thr (3), and Trp (1) in the presence of fluoride, chloride, bromide, and iodide, as a function of anion polarizabilities F: cf. Table 3. Inset: Asp (0); Glu (O).

TABLE 4: Effect of Salt Concentration (c, in mol/L) on the Corrected Optical Rotation Values [r] (in deg‚g-1‚mL‚mm-1) for L-Ser (2.1 g/100 mL) c (mol/L)

NaCl

NaBr

NaI

0 0.25 0.50 1.00

-7.59 ( 0.06 -7.44 ( 0.06 -6.88 ( 0.06 -6.31 ( 0.07

-7.59 ( 0.06 -7.14 ( 0.07 -6.68 ( 0.06 -5.86 ( 0.08

-7.59 ( 0.06 -6.87 ( 0.08 -6.45 ( 0.07 -4.98 ( 0.07

these amino acids that bear another COO- group in the side chain, the effect of fluoride is dramatically large and leads to a sign inversion in [R]. Interestingly, L-Asp shows a larger effect than L-Glu. This result may be related to the fact that in Glu (that bears two CH2 groups) the side chain COO- ending group is more distant than that in Asp from the carboxylate residue bound to CR.32 Similar trends can be derived by comparing [R]salt/[R]water to the corresponding variations with ion type of surface tension molar increment (σ) and Gibbs free energy of hydration (∆Ghydr) for the halides. We now consider these salt-dependent changes in more detail. The effect of salt concentration in the case of NaCl, NaBr, and NaI on the specific rotation of L-Ser (2.1 g/100 mL) is shown in Table 4 and Figure 2. The data were fitted with a quadratic equation

[R]s ) [R]w(1 + Axc + Bc)

(5)

where [R]s is the specific rotation at salt concentration, c, and [R]w is the value in pure water. The plot shows that NaI has the greatest effect, chloride has the smallest, and bromide is intermediate. A similar dependence was already found in a previous study20 and in other specific ion phenomena, for example the critical micelle concentration of a short chain lecithin,37,47,48 the formation of pseudopolyrotaxanes,49 and the viscosity of aqueous solutions.44,50 This behavior confirms that ion-specific dispersion forces are at play. The parameter A is directly related to electrostatic interactions, that dominate at low c, while B depends on the salt and reflects the dispersion forces (that emerge at higher salt concentrations).

10514 J. Phys. Chem. B, Vol. 111, No. 35, 2007

Figure 2. [R] for L-Ser (2.1 g/100 mL) in the presence of NaCl (b), NaBr (0), and NaI (1) at different salt concentrations. The fitting curves were obtained by fitting the data according to eq 5.

A similar simple correlation is not discernible for the other anions. However, it has to be remarked that for nonspherical and polyatomic ions, such as NO3-, ClO3-, ClO4-, H2PO4-, SCN-, the anisotropy of polarizability F indeed must have a large impact on the properties of ions in aqueous solutions.51 This is indeed so for activity coefficients for these salts that reflect ion-ion interactions in solution.21 This anisotropy in molecular polarizability reflects the peculiar and special hydrogenbonding capacity of a given species. The consequent anisotropic hydration may inhibit or enhance hydration shell interpenetration and therefore anion specific interactions. With this established we now explore further changes in [R] induced by the electrolytes by considering the possible effects of amino acid self-association in aqueous solutions and that of pH changes. Self-association might lead to a change in [R] due to the formation of dimers, and pH changes could, in principle, modify the ionization degree of the amino acid charge groups. Self-Association of Amino Acids. In order to assess whether amino acids self-associate in water, we studied the concentration dependence of R for L-Ala, L-Ala + NaBr (1:1), L-Thr, and L-Ser. The focus on these amino acids is because their side chains (-CH3, -CH(OH)CH3, and -CH2OH, respectively) are progressively more hydrophilic. For Ala, we also checked the effect of adding sodium bromide in equimolar conditions. The results are listed in Table 5 and plotted in Figure 3, as R (in deg) versus c (in mol/L). The trends can be fitted to a quadratic equation. Such behavior is typical of self-associating chiral molecules that form dimers through hydrogen bonding.52 A quantitative treatment of the R/c data has been proposed by Morbidelli et al.53 Basically, for the dimerization equilibrium 2AA h (AA)2 the conservation of mass requires that the overall stoichiometric molar concentration of the amino acid is c )

Rossi et al.

Figure 3. Optical rotation R as a function of the molar concentration c of the amino acid for Ala (O), L-Ala + NaBr 1:1 (0), L-Thr ((), and L-Ser (2). The lines represent the fitting curves obtained from eq 6.

M1 + 2M2, where M1 and M2 are the equilibrium molar concentrations of the monomer and of the dimer, respectively. The self-association constant is calculated as Kd ) M2/M12. The experimental optical rotation is then given by contributions from both monomeric and dimeric species in solution and can be written as R ) µM1 + δM2, where µ and δ (in deg‚L/mol) are the response parameters of the monomer and dimer, respectively. Combining the four relationships it is easy to show that

R ) µM1 + δKdM12 ) 1 [4Kdδc + (2µ - δ)(1 + 8Kdc)1/2 + δ - 2µ] (6) 8Kd from which the different parameters can be extracted for each case by fitting to the experimental data. At low concentration of amino acid, R is proportional to M1, the presence of dimers is negligible, and µ can be obtained from a linear fitting regression. Once µ is known, Kd and δ are obtained by fitting eq 6. The fitting parameters for each case are shown in Table 6. The degree of dimerization η is given by η ) M2/M1 ) KdM1. Molecular Dynamics Simulations. Simulations of Ala, Thr, and Ser water solutions were performed at four different concentrations in order to verify the occurrence and extent of self-association through classical molecular dynamics (MD) calculations. To the best of our knowledge the literature reports only one MD study on glycine in water up to high concentration (1.57 M).54 Other simulation studies focus on hydration free energies,55 on side chain interactions,56,57 or on hydration properties of single amino acid molecules.58 The radial distribution function of oxygen-oxygen water atoms, gOwOw(r), obtained in the simulations for Thr 0.8 M is shown in Figure 4. The comparison is the experimental g(r) and that obtained from the pure TIP4P model water. Their strong similarity confirms

TABLE 5: Optical Rotation (r, in deg) of L-Ala, L-Ala + NaBr (1:1), L-Thr, and L-Ser as a Function of the Amino Acid Concentration (c, in mol/L) L-Ala

c (M) 0.0459 0.0477 0.0708 0.1001 0.1004 0.2009 0.2268 0.3002 0.5000 1.0072 1.5354

R (deg) 0.004 ( 0.0003 0.004 ( 0.0003 0.006 ( 0.0005 0.010 ( 0.0003 0.011 ( 0.0005 0.023 ( 0.0005 0.028 ( 0.0006 0.039 ( 0.0005 0.080 ( 0.0005 0.220 ( 0.0005 0.428 ( 0.0006

L-Ala

c (M) 0.1001 0.3004 0.4997 1.0001 1.5001

+ NaBr R (deg) 0.011 ( 0.0005 0.042 ( 0.0006 0.083 ( 0.0005 0.223 ( 0.0005 0.416 ( 0.0005

L-Thr

c (M) 0.0286 0.0432 0.0651 0.0860 0.1006 0.1608 0.3004 0.5001 0.6213

R (deg) -0.100 ( 0.0007 -0.150 ( 0.0006 -0.223 ( 0.0005 -0.295 ( 0.0003 -0.346 ( 0.0004 -0.550 ( 0.0007 -1.021 ( 0.0005 -1.702 ( 0.0006 -2.110 ( 0.0005

L-Ser

c (M) 0.0100 0.0160 0.0203 0.0309 0.0485 0.1002 0.1998 0.3038

R (deg) -0.010 ( 0.0003 -0.013 ( 0.0005 -0.017 ( 0.0005 -0.026 ( 0.0003 -0.041 ( 0.0007 -0.083 ( 0.0004 -0.162 ( 0.0003 -0.241 ( 0.0004

Anion Effects on Amino Acids

J. Phys. Chem. B, Vol. 111, No. 35, 2007 10515 obtained as a fitting parameter from the following equation that provides the dimer concentration as a function of c

M2 )

Figure 4. Radial distribution functions for water oxygen-oxygen atoms, gOwOw(r), for experimental results (O), TIP4P model (b), and TIP4P for the simulation with 104 Thr molecules (().

TABLE 6: Values of the Fitting Parameters (see eq 6)a L-Ala

µ δ Kd χ2 R2 η (0.3 M) Kd (MD)

0.05 ( 0.01 1.51 ( 0.04 0.23 ( 0.01 1.6 × 10-4 0.99914 6% 0.28

L-Ala

+ NaBr

0.10 ( 0.01 4.5 ( 1.5 0.20 ( 0.01 8.8 × 10-5 0.99933 6%

L-Thr

L-Ser

-3.43 ( 0.01 -6.6 ( 0.1 0.30 ( 0.05 2.6 × 10-5 0.99999 8% 0.30

-0.85 ( 0.01 -0.86 ( 0.09 0.32 ( 0.05 2.7 × 10-6 0.99995 8% 0.32

a µ (in deg‚L/mol), δ (in deg‚L/mol), Kd (in L/mol) for the different amino acids solutions, with the corresponding values of χ2 and R2. η is the dimerization degree in percentage calculated at 0.3 M concentration of the amino acid. Kd (MD) is the dimerization constant calculated by fitting M2 vs c (see eq 7) obtained from molecular dynamics simulations.

the validity of this model and suggests that the presence of the amino acids does not modify the bulk water structure. This holds good for all the three amino acids investigated at concentrations ranging between 0.5 and 0.8 M. An analysis of the intra- and intermolecular hydrogen bonds (cutoff radius 0.35 nm and cutoff angle 30°, see Table 7)54,59 shows that amino acids and water produce a complex network of interactions. On the average, the hydration number Nw for the ammonium group is 2, while the COO- group interacts with five water molecules, and both values decrease with increasing the AA concentration. Furthermore, approximately 10-15% of the amino acid charged groups interact through an intramolecular hydrogen bond. Intermolecular interactions between amino acids can involve one or two hydrogen bonds or multipole-multipole interactions, as depicted in Figure 5 (structures d and c, respectively). MD simulations show that the proportion of AA molecules that establish one or two hydrogen bonds/multipolemultipole interactions with another amino acid moiety increases with concentration (see Table 7, third and fourth columns). Figure 6 shows the concentration of Ala, Ser, and Thr dimers (M2) versus the overall concentration c. Two interacting molecules are considered to form a dimer if they are linked by two H-bonds (with the same cutoffs reported before) or by multipole/multipole interactions, as depicted in Figure 7. Kd is

4Kdc + 1 - x8Kdc + 1 8Kd

(7)

The values of Kd obtained from MD simulations are listed in Table 6 (last row). pH. The fact that apart from the sodium halides there is no clear correlation of optical activity with the usual Hofmeister series might suggest that salt-dependent pH effects might be a factor.33 In order to check the effect of salts on the pH of aqueous solutions of amino acids, the pH of Asp, Ser, and Pro solutions was measured in pure water and in the presence of NaBr, NaF, and NaH2PO4 all at 0.5 M. The three amino acids were chosen because of their nature: Asp has an anionic side chain, Ser has a polar but neutral side chain, and Pro has a quite rigid and less polar residue. The results, listed in Table 8, indicate that NaBr leads to a small reduction of pH with respect to pure water, in agreement to our previous findings.33 Sodium fluoride and sodium dihydrogen phosphate modify the pH in a more significant way. The former increases the pH of the Asp solution by 2.2 units, that of Ser of 0.9 units, and that of Pro of 0.7 units. H2PO4- instead decreases the pH more consistently for Pro (1.9 units) and Ser (1.4 units) than for Asp (0.8). When the pH of each solution is compared to the [R]salt/[R]water ratio, it is clear that only in the case of Asp can the optical rotation variation be directly related to the pH change. This effect is probably due to the presence of a carboxylate group in the side chain of aspartic acid, whose pKa can be modified by the presence of electrolytes. Instead, in the case of Ser and Pro, the [R]salt/[R]water ratio and the pH are not correlated. Interestingly, F- and H2PO4- have opposite effects on [R] of L-Asp and L-Glu. The specific rotation for these two AA was then measured in buffer solutions at pH 5 and 8 (at the same ionic strength), in the presence of small amounts of sodium fluoride and sodium dihydrogen phosphate, i.e., 0.05 M. The results are reported in Table 9. The data indicate that in the presence of F-, [R] decreases in going from pH 5 up to 8, both for L-Asp and for L-Glu. Instead, the presence of H2PO4- produces a significant increment of the specific rotation. This result shows that the observed variation is not simply due to a pH effect but also depends on the nature of the added electrolyte. In fact, starting from an initial salt concentration of 0.05 M, the final concentration of fluoride ions at pH 5 and 8 is practically the same (the pKa for HF is about 3.15). Instead for NaH2PO4, since the pKa for the H2PO4-/HPO42- equilibrium is about 7.21, at pH 5 the dominant species is dihydrogen phosphate, but at pH 8 the divalent anion prevails with a concentration of [HPO42-] ) 0.043 M. The formation of HPO42- will change the intermolecular interactions between ions and AA zwitterions as depicted in Figure 5e. A competition effect between buffer anions and added salt anions cannot be excluded at this time.33,60 This issue will be addressed in a future study by comparing the specific rotation of a given AA at different concentrations of buffer and electrolyte. These observations taken together seem to indicate that the pH change induced by the dissolved electrolytes cannot be linked straightforwardly to the measured variation in [R]. Discussion In summary, monovalent electrolytes, with fixed cationshere sodiumsmodify the optical activity of amino acids differently.

10516 J. Phys. Chem. B, Vol. 111, No. 35, 2007

Rossi et al.

Figure 6. Dimers concentration M2 (mol/L) as a function of the amino acid total concentration (c in mol/L) for Ala (O), Thr ((), and Ser (4). The lines represent the fitting curves obtained from eq 7.

Figure 5. Intramolecular (a and b) and intermolecular (c and d) interactions between amino acids in solution. (e) Interactions between a H2PO4- anion with two amino acid molecules.

The effects must be related to intrinsic properties of each specific anion, amino acid, and their interactions. Self-Association of Amino Acids. Intermolecular hydrogen bonding and electrostatic interactions lead to self-association in amino acids,61 and largely reduce water accessibility to the side chains.62 Moreover, the formation of self-associated pairs in solution can produce a nonlinear concentration dependence of [R], known as the “Horeau effect”.63 The self-association process between amino acids requires a partial dehydration of the groups involved,64 as depicted in Figure 8. The direct interactions between the charged or polar groups of an amino acid molecule provide an exothermic contribution, while the partial dehydration is accompanied by an endothermic effect.65 MD simulations confirm that increasing the concentration results in partial dimerization of the amino acid and partial dehydration of the charged groups (see Tables 6 and 7), through the

Figure 7. Snapshot of a simulation showing the formation of a dimer from two threonines. Hydrogens, carbons, oxygens, and nitrogens are white, light blue, red, and deep blue spheres, respectively. H-bonds are depicted as white dotted lines; water molecules are hidden for clarity.

formation of hydrogen bonds and/or multipole-multipole interactions. And indeed the concentration dependence of R shown in Figure 2 indicates that amino acid molecules in aqueous solutions self-associate partially. The monomeric AA and dimeric (AA)2 species in equilibrium contribute to the overall optical rotation with a response factor (µ and δ, respectively) as shown in eq 6. The dimerization parameter is small for the

TABLE 7: H Bond Analysis from MD Simulations, Averaged over 1 ns of Run (cutoffs: rmax ) 0.35 nm, θmax ) 30°)a

c

intramolecular H bonds (%)

single AA-AA intermolecular interactions

0.497 0.594 0.690 0.785

12.5 8.4 11.3 12.3

0.103 0.123 0.177 0.256

double AA-AA intermolecular interactions, M2

Nw (NH3+)

Nw (COO-)

1.0 1.7 3.4 5.1

2.2

5.3

2.1

5.0

1.2 3.0 3.7 6.4

2.2

5.2

2.0

5.1

2.0 4.3 5.5 7.7

2.2

5.3

2.0

5.1

Ala

Thr 0.492 0.586 0.680 0.771

11.7 15.8 13.6 14.7

0.097 0.143 0.178 0.267

0.495 0.591 0.687 0.781

10.3 10.0 15 13

0.134 0.187 0.246 0.313

Ser

a

Total concentration of amino acid (c, mol/L), percentage of intramolecular hydrogen bonding in amino acid molecules, concentration (mol/L) of amino acid molecules that interact with another AA, concentration (M2, in mmol/M) of amino acid molecules that form “dimers” with another AA, number of water molecules (Nw) that solvate the charged residues.

Anion Effects on Amino Acids

J. Phys. Chem. B, Vol. 111, No. 35, 2007 10517

Figure 8. Interactions between two amino acid molecules involving the ammonium (+) and the carboxylate (-) charged groups. Partial dehydration occurs during the association process and solvating molecules re-enter in the bulk phase.

TABLE 8: pH of L-Asp (0.02 M), L-Ser (0.20 M), and L-Pro (0.50 M) in Pure Water, NaBr, NaF, and NaH2PO4 (0.5 M) (H2O) NaBr NaF NaH2PO4

H2O

L-Asp

L-Ser

L-Pro

5.6 5.4 8.6 4.2

2.9 2.7 6.7 3.7

5.8 5.6 7.1 4.4

6.4 6.1 7.5 4.5

TABLE 9: Effect of pH on the Corrected Optical Rotation Values [r] (in deg‚g-1‚mL‚mm-1) for L-Asp (0.28 g/100 mL) and L-Glu (0.38 g/100 mL) in the Presence of NaF and NaH2PO4 (0.05 M) in Buffer Solutions NaF pH 5

NaH2PO4 pH 8

pH 5

pH 8

-12.55 ( 0.28 -16.63 ( 0.25 -11.99 ( 0.33 -5.93 ( 0.26 L-Glu -0.64 ( 0.13 -3.67 ( 0.15 -3.71 ( 0.18 5.09 ( 0.20

L-Asp

three AA and nearly constant. The results parallel those of Vliegen and Abel for solutions of amino acids in acidic water solutions66,67 and are confirmed by the molecular dynamics simulations. Since the dimerization constant Kd is relatively small (at least in the case of Ala, Thr, and Ser) and is not greatly influenced by the addition of NaBr, a partial self-association process cannot be considered a major determinant of the specific salt effect on [R]. The results indicate that the effect of anions cannot be simply due to a polarizability effect or to a partial association of amino acids in solution or to pH changes. It must involve other mechanisms. In an attempt to obtain a better insight into mechanism, we have in what follows taken into consideration the different intraand intermolecular interactions that involve solutes and solvent molecules and the conformational changes in the chiral solutes that can be promoted by the dissolved electrolytes. We will examine these issues successively. Interactions. Although in all amino acids the charged groups interact in the same manner with water and ions, the hydration of the specific side chains affects the solvation shells of the NH3+ and COO- groups. Therefore, each AA interacts with the solvent and other solutes in its own specific way.68 Figure 1 shows that the effect of salts depends also on the nature of the AA. In fact the presence of a more or less hydrophobic/ hydrophilic side chain determines the interactions that an amino acid molecule establishes with water and, through this, with other cosolutes in solution. Our data indicate that the maximum effect on the optical activity is produced by the most hydrophilic amino acids. There are several kinds of interactions known that are presumed to affect the properties of amino acids in an aqueous environment (see Figure 5). They involve particular features of

the amino acids, water molecules, and electrolytes: (i) intramolecular hydrogen bonding (parts a and b); (ii) intermolecular hydrogen bonding (parts c and d); (iii) hydration of the charged and neutral lateral groups; (iv) ion-dipole interactions. We will briefly describe the roles of these. In water solution amino acids are stable in the zwitterionic form, +H3N-CH(R)-COO-, with an intramolecular hydrogen bond linking the ammonium group hydrogen and the carboxylate residue (Figure 5a). The strength of such an interaction depends on the ionization constants of these two groups.8 When the side chain R carries a suitable polar moiety, as in Ser, Asp, or Glu, another intramolecular hydrogen bond can be established with the charged groups as depicted in Figure 5b.12,69,70 Intermolecular hydrogen bonding between two individual amino acid molecules is formed through the interaction of the charged (Figure 5c,d) or neutral polar residues that belong to each amino acid. In particular, these include the interactions between a COO- and a NH3+, between a COO- and a polar R group, or between a NH3+ moiety with R. In the case of Gln, the interaction involves also the side chain amide residues. On the other hand, the presence of hydrophobic moieties in the side chain (such as CH3 in Ala) perturbs the behavior of amino acids in solution.1,68 Finally, amino acids and water interact through hydrogen bonding and dipolar moments that induce strong anisotropic hydration forces.71 Thus, for example, McLain et al. have found that in the Glu molecule each carboxylate oxygen atom forms an average of three hydrogen bonds with the surrounding water, with one of these hydrogens being shared between the two oxygen atoms on each COO-. Each NH3+ hydrogen forms a single hydrogen bond with the water molecules.2 Our simulation for Ala, Thr, and Ser indicates that two and five water molecules surround the ammonium and carboxylate groups, respectively (see Table 7, fifth and sixth columns), for AA concentrations ranging between 0.5 and 0.8 M. The existence of ion-pair complexes between amino acids and salts has been invoked in the interpretation of the activity coefficients of some amino acids in the presence of electrolytes at different concentrations.72,73 The formation of ion pairs between the amino acid and the electrolyte (M+X-) can be illustrated as -

OOC-CHR-NH3+ + M+X- h [M+(-OOC-CHR-NH3+)X-]

The formation of the complex produces a reduction in the dipole moment of the amino acid and then a partial weakening of dipole-dipole (AA-AA) and ion-dipole (AA-ion) forces. The nature of the ions, i.e., the specific salt effect related to their intrinsic properties (such as polarizability), emerges then more clearly. Other more complex interactions, such as those between the ions and the charge resonance of the carboxylate that leads to a variation of its ionization constant pKa, may also be at play. And this probably occurs in the cases of Asp and Glu, which bear a COO- residue in the side chain, as the pH change of an Asp solution in the presence of F- and H2PO4- shows. Furthermore, as depicted in Figure 5e, H2PO4- can act as a polydentate center and establish up to three hydrogen bonds with amino acid molecules. Also fluoride-based species can behave in similar ways as suggested by previous reports.74,75 The other polyatomic ions, all chaotropic, interact with each AA in a specific way, depending on their activity coefficient and on the more or less hydrophobic nature of the side chain

10518 J. Phys. Chem. B, Vol. 111, No. 35, 2007 of the amino acid. For example, in the case of Ala [R] increases in the presence of NO3- and decreases with SCN-. This effect parallels the lowering of Ala solubility in the presence of guanidinium thiocyanate 1 M,76 and the increment of its solubility with KCl and NaNO3.73 Conformations and Optical Activity. More or less polarizable ions can modify internal molecular vibrational states. For conformationally flexible species, such as most AA, the value of [R] is a weighted average over all the thermally populated conformations.23,32 Pecul et al. have observed that conformational states can modify the optical rotation of Ala and Pro.11 The orientation of the NH3+ and particularly of the COO- group with respect to the side chain can change the optical rotation in those amino acids.11 The importance of conformational structure on the optical rotation has been confirmed more recently for Ser and Cys, on the basis of theoretical simulations and experiment.77 Zwitterionstructuressstabilizedbywatermoleculess involve the formation of intramolecular hydrogen bonds, and the sign of optical rotation can vary for different structures.11,12,32,77 Salt-induced conformational changes in aspartic acid solutions were also detected by NMR experiments in different pH conditions, and the results indicate that a significant ion specific effect takes place.78 These observations support the hypothesis that the effect of salts on the optical activity is the result of a set of specific interactions between the ionic species and the chiral molecule, which results in a perturbation of the intra- and intermolecular hydrogen bonding between the constitutive moieties. The saltinduced perturbation of all the interactions that involve amino acids in solution may produce conformational changes in the chiral solutes that modify their optical activity. However, further work is necessary in order to confirm this hypothesis. Conclusions The study of interactions between amino acids, water, and electrolytes is relevant to the understanding of protein properties such as folding, binding, structural stability, and physiological functions in aqueous environments. In this paper we have reported the effect of different sodium salts on the optical activity [R] of aqueous solutions of a set of amino acids (AA). In summary our results show the following: (a) Salts affect optical activity of R-amino acids in solution. The effect depends strongly on the nature of the electrolyte and on the specific AA. (b) For the halides, polarizability, surface tension molar increment, and Gibbs free energy of hydrationsall related to dispersion (nonelectrostatic) NES forcesscharacterize the specific influence of salts on [R]. (c) For the other anisotropic and polyatomic anions (SCN-, ClO3-, ClO4-, NO3-, and H2PO4-) the salt effect is more complicated, because these species can act as acceptors or donors for hydrogen bonding and coordination. (d) Electrolytes perturb the hydration of amino acid molecules and therefore AA-water interaction. The partial self-association process that occurs in water solutions of amino acids does not seem to be responsible for the observed changes in [R]. (e) The salt-induced pH change in AA solutions correlates with the variation of [R] in the case of aspartic acid, but no direct correlation can be established for amino acids bearing neutral side chains, such as Ser and Pro. (f) Fluoride and dihydrogen phosphate modify the value of [R] for L-Asp and L-Glu in opposite ways, also in the presence of a buffer at pH 5 and 8. This evidence indicates that the

Rossi et al. observed phenomenon is not simply a pH effect but is related to the specific kind of electrolyte. In conclusion, this work suggests that electrolytes perturb the extensive set of different interactions (dipole-dipole, iondipole, hydrogen bonding, solvation, hydrophobic interactions) that involve amino acids molecules, water, and electrolytes and, therefore, modify their optical activity. Acknowledgment. We wish to thank the reviewers for their criticism and suggestions. The authors are grateful to Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI, Italy) and Ministero dell’Universita` e Ricerca (PRIN2003) for partial financial support. This paper is dedicated to the memory of Prof. Enzo Ferroni (1921-2007), our mentor and founder of CSGI. References and Notes (1) Banerjee, T.; Kishore, N. J. Solution Chem. 2005, 34, 137-153. (2) McLain, S. E.; Soper, A. K.; Watts, A. J. Phys. Chem. B 2006, 110, 21251-21258. (3) Xu, X.; Pinho, S. P.; Macedo, E. A. Ind. Eng. Chem. Res. 2004, 43, 3200-3204. (4) Ferreira, L. A.; Macedo, E. A.; Pinho, S. P. Ind. Eng. Chem. Res. 2005, 44, 8892-8898. (5) Yamamoto, C.; Okamoto, Y. Bull. Chem. Soc. Jpn. 2004, 77, 227257. (6) Norde´n, B. J. Mol. EVol. 1978, 11, 313-332. (7) Avalos, M.; Babiano, R.; Cintas, P.; Jime´nez, L.; Palacios, J. C. Chem. Commun. 2000, 887-892. (8) Nishino, H.; Kosaka, A.; Hembury, G. A.; Matsushima, K.; Inoue, Y. J. Chem. Soc., Perkin Trans. 2 2002, 582-590. (9) Kirkwood, J. G. J. Chem. Phys. 1937, 5, 479-491. (10) Fitts, D. D.; Kirkwood, J. G. Proc. Natl. Acad. Sci. U.S.A. 1957, 43, 1046-1052. (11) Pecul, M.; Ruud, K.; Rizzo, A.; Helgaker, T. J. Phys. Chem. A 2004, 108, 4269-4276. (12) Kundrat, M. D.; Autschbach, J. J. Phys. Chem. A 2006, 110, 41154123. (13) Cacace, M. G.; Santin, M.; Sada, A. J. Chromatogr. 1990, 510, 41-46. (14) Kimura, T.; Matubayasi, N.; Sato, H.; Hirata, F.; Nakahara, M. J. Phys. Chem. B 2002, 106, 12336-12343. (15) Huang, F.; Nau, W. M. Angew. Chem., Int. Ed. 2003, 42, 22692272. (16) Lo Nostro, P.; Ninham, B. W.; Milani, S.; Lo Nostro, A.; Pesavento, G.; Baglioni, P. Biophys. Chem. 2006, 124, 208-213. (17) Schleich, T.; von Hippel, P. H. Biopolymers 1969, 7, 861-877. (18) Mandelkern, L.; Clark, D. S.; Dechter, J. J. Macromolecules 1980, 13, 533-541. (19) Katzin, L. I.; Kresheck, G. C. Arch. Biochem. Biophys. 1968, 126, 418-425. (20) Lo Nostro, P.; Ninham, B. W.; Milani, S.; Baglioni, P. Biopolymers 2006, 81, 136-148. (21) Kunz, W.; Lo Nostro, P.; Ninham, B. W. Curr. Opin. Colloid Interface Sci. 2004, 9, 1-18. (22) Rabek, J. F. Experimental Methods in Polymer Chemistry; Wiley: Chichester, 1980. (23) Stephens, P. J.; Devlin, F. J.; Cheeseman, J. R.; Frisch, M. J. J. Phys. Chem. A 2001, 105, 5356-5371. (24) Stephens, P. J.; Devlin, F. J.; Cheeseman, J. R.; Frisch, M. J.; Mennucci, B.; Tomasi, J. Tetrahedron: Asymmetry 2000, 11, 2443-2448. (25) Shirts, M. R.; Pitera, J. W.; Swope, W. C.; Pande, V. S. J. Chem. Phys. 2003, 119, 5740-5761. (26) Shirts, M. R.; Pande, V. S. J. Chem. Phys. 2005, 122, 134508:113. (27) Lindhal, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7, 306-317. (28) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. Comput. Phys. Comm. 1995, 91, 43-56. (29) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577-8593. (30) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 18, 1463-1472. (31) Berendsen, H. J. C.; Postma, J. P. M.; Di Nola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684-3690. (32) Kundrat, M. D.; Autschbach, J. J. Phys. Chem. A 2006, 110, 12908-12917.

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