Effects of Microsolvating Water on the Stability of Zwitterionic vs. Canonical Diglycine

Abstract

We present calculations for diglycine - $(H_2O)_n$ (n = 0-3) to examine the effects of microsolvating water on the relative stability of the zwitterionic vs. canonical forms of the dipeptide. We calculate the structures, energies and Gibbs free energies of the conformers at wB97XD/6-311++G** and MP2/aug-cc-pvdz levels of theory level of theory. We predict that microsolvation by up to three water molecules does not give thermodynamic stability of the zwitterion relative to the canonical forms. Our calculations also suggest that zwitterionic diglycine - $(H_2O)_3$ is not stable kinetically in low temperature gas phase environment.

Introduction

Dipeptides1 are of fundamental interest as the simplest system containing the peptide bond units. Studying them may help to shed further light to larger polypeptides, allowing us to investigate the geometrical, physiological,2 and spectroscopic3 characteristics of proteins. Dipeptides comprising two amino acids by a peptide bond may be quite different from amino acids in many respects, for example, the effects of microsolvation on the relative stability of canonical vs. zwitterionic conformers. For the case of amino acids, it was proposed that 4-5 water molecules are usually necessary to stabilize the zwitterions of glycine,4 alanine,5 and valine,6 although it has also been proposed that fewer water molecules may be sufficient for amino acids such as arginine7 and lysine8 containing strongly basic side chains.

For dipeptides, on the other hand, the situation may be quite different because of several structural features. First, the presence of a peptide bond may affect the stability of zwitterionic dipeptides vs. canonical conformers. Second, proton transfer between the carboxyl and terminal amino group in dipeptides may be considered to be more difficult because these two functional groups are separated to larger distances in dipeptides than in amino acids. This may affect the kinetic stability of zwitterionic dipeptides, for example, more water molecules needed to relay the proton transfer processes.

Studies of the microsolvation of peptides are quite scarce, probably because of the large nuclear degrees of freedom and the consequent necessity to search over the vast potential energy landscape. As for dipeptides, Lin and co-workers’9 investigation for 13 representative dipeptides seems to be the most comprehensive and systematic work. As far as we know, however, systematic study of the dipeptide – water complexes is still lacking, although some investigations were reported for microsolvation of dialanine.10

Here, we present a quantum chemical study of the structures of diglycine ‒ (H2O)n (n = 0‒3) system to elucidate the relative stability of canonical vs. the zwitterionic forms. The energies and Gibbs free energies (5 K) of zwitterionic diglycine ‒ (H2O)3 are calculated to be higher (> 7.8 kcal/mol) than those of the canonical conformers, indicating that up to three microsolvating water molecules are not sufficient to stabilize the zwitterionic diglycine [GG]. We also examine the canonical ↔ zwitterion pathways to examine how the lowest energy zwitterionic conformer transforms to the canonical form of GG ‒ (H2O)3, focusing on the role of microsolvating water molecules in the process.

 

Computational Methods

We employ the density functional theory (wB97XD11) with the 6‒311++G** basis set and MP2/aug-cc-pvdz12 method, as implemented in the GAUSSIAN 09 set of programs.13 For the zwitterionic GG, we first obtained 7776 conformers using the semi-empirical method PM614 by rotating the five dihedral angles at an interval of 60 degrees. From these structures, we find thirty lowest energy conformers by the wB97XD/ 6‒311++G**method. Most of these conformers converged to canonical forms, but we found a high energy zwitterionic GG of very high energy (~55 kcal/mol above the canonical GG). Structures of GG ‒ (H2O)n (n = 1‒3) clusters were subsequently obtained by allowing an additional water molecule to interact over extensive configuration space of GG ‒ (H2O)n‒1. We find that various initial configurations lead to water molecule bridging the two functional groups in GG, as described below. Stationary structures are confirmed by ascertaining that all the harmonic frequencies are real. Structure of the transition state is obtained by verifying that one and only one of the harmonic frequencies is imaginary, and also by carrying out the intrinsic reaction coordinate analysis along the reaction pathways. Zero point energies (ZPE) are taken into account, and default criteria are used for all optimizations.

Table 1.awB97XD/6‒311++G**. bMP2/aug-cc-pvdz

Figure 1.Structures of the lowest energy conformers of GG (relative Gibbs free energy at 5 K in kcal/mol).

 

Results and Discussion

Diglycine. The lowest energy conformers of GG as presented in Table 1 and Figure 1. The four conformers, (ca-1), (ca-2), (ca-3), (ca-4) agree well with those reported by Lin and co-workers.9 The energies of the three lowest conformers (ca-1), (ca-2) and (ca-3) are quite close (within 0.6 kcal/mol), whereas those of (ca-4) and (ca-5) are higher than that of (ca-1) by ~1.6 kcal/mol. The structures (ca-1), (ca-2), (ca-3), (ca-5) are quite similar, with the terminal ‒NH2, ‒C=O and ‒NH groups in peptide bond positioned in zigzag fashion. They differ mostly in dihedral angles between the peptide bond and the terminal ‒COOH. In (ca-4), the terminal ‒NH2 lies in the vicinity of the ‒C=O group in the peptide bond. We could find only one stationary structure (zw-1) of zwitterionic GG. In this conformer, the proton binds to the terminal amino rather that to the amide bond. Because of the extremely high energy (~55 kcal/mol) relative to canonical structures, however, it will not exist even in low temperature (typically ~5 K) gas phase.

Diglycine ‒ (H2O). Table 1 and Figure 2 present the structures, relative energies, and relative Gibbs free energies of GG ‒(H2O). The energies of the five canonical conformers, (ca-1-1), (ca-1-2), (ca-1-3), (ca-1-4), and (ca-1-5) are also quite close, within 0.5 kcal/mol. Ten canonical conformers presented in Figure 2 exhibit three kinds of interactions between GG and the water molecule. The first is between water and the terminal ‒COOH group ((ca-1-1), (ca-1-2), (ca-1-5), (ca-1-6), (ca-1-7) and (ca-1-8)). In the second type, the water molecule bridges the terminal ‒NH2 and carboxyl ‒C=O group ((ca-1-3), (ca-1-4)). The last type of interactions are due to binding between water molecule and the amide ‒C=O ((ca-1-9), (ca-1-10)). The energy of one of the zwitterionic conformer, (zw-1-1), is closer to that of (ca-1- 1), but still quite higher (~18 kcal/mol). The proton interacts with the terminal amino group. The energies of other zwitterionic conformer are calculated to be higher than the canonical conformers by > 40 kcal/mol, and are not presented here.

Figure 2.Structures of the lowest energy conformers of GG ‒ (H2O) (relative Gibbs free energy at 5 K in kcal/mol).

Figure 3.Structures of the lowest energy conformers of GG ‒ (H2O)2 (relative Gibbs free energy at 5 K in kcal/mol).

Diglycine ‒ (H2O)2. There exist a large number of GG – (H2O)2 conformers with similar energies, some of which are depicted in Figure 3. Ten canonical conformers presented in Figure 3 also show three kinds of interaction between GG and water molecule described above. Compared with the GG ‒ (H2O) complexes, however, the stabilizing effects in GG ‒ (H2O)2 rendered by each type of interactions follow a different pattern. Stabilization by water molecules bridging the peptide ‒C=O group and the carboxyl -OH group ((ca-2- 1), (ca-2-3)) is a bit stronger than those by water molecules binding to the terminal ‒COOH ((ca-2-2), (ca-2-4), (ca-2-6) and (ca-2-10)). The energy of the lowest lying zwitterionic structure (zw-2-1) is now much closer (~8.9 kcal/mol) to that of (ca-2-1), whereas those of other zwitterionic conformers are calculated to be 10-13 kcal/mol higher. The proton does not attach to the amide nitrogen or oxygen atoms in any of these zwitterionic conformers.

Figure 4.Structures of the lowest energy conformers of GG ‒ (H2O)3 (relative Gibbs free energy at 5 K in kcal/mol).

Diglycine ‒ (H2O)3. The structures of GG ‒ (H2O)3 com-plexes are illustrated in Figure 4. The energy of the zwitterionic conformer (zw-3-1) is now a bit closer to that of the lowest energy canonical conformer (ca-3-1), but still quite higher (~7.5 kcal/mol). Figure 5 presents the IR spectra (MP2/aug-cc-pvdz frequencies are scaled by a factor of 0.960415) and the normal modes of the lowest energy conformer of GG ‒ (H2O)3 with zwitterionic and canonical GG core. The IR spectra of (ca-3-1) and (zw-3-1) exhibit widely different features that may be very helpful to discern them experimentally. The conformer (ca-3-1) is featured by intense bands at 3070, 3382, 3492, and 3738 cm‒1, whereas (zw-3-1) is characterized by strong bands at 3145, 2772, 3431, and 3741 cm‒1 with a wide window in the 1500-2750 cm‒1 region. The lower bands at 3070 and 3382 cm‒1 of (ca-5-1) describe the proton transfer modes between ‒COOH, water molecules, and ‒C=O in peptide bond. And also, the bands at 2772 and 3145 cm‒1 of (zw-3-1) exhibit the normal modes resembling the proton transfer between the ‒COOH and –NH3 groups. The bands at 3492 and 3738 cm‒1 of (ca-3-1) and 3431 and 3741 cm‒1 of (zw-3-1) indicate symmetric and anti-symmetric stretching mode of water molecule which stay apart from the other two water molecules.

Figure 5.IR spectra and normal modes of the most intense bands of (a) canonical (ca-3-1) and (b) zwitterionic (zw-3-1) conformers of GG ‒ (H2O)3 (MP2/aug-cc-pvdz frequencies with a scaling factor of 0.9604).

Figure 6.Transformation from the lowest energy zwitterionic (zw-3-1) and canonical (ca-3-1) conformer of GG ‒ (H2O)3 (relative energy and reaction barrier in kcal/mol, ZPE included; wB97XD/ 6-311++g**).

Figure 6 illustrates that the lowest energy zwitterionic conformer (zw-3-1) may transform to the canonical form (ca-3- a) by triple proton transfer mediated by water molecules with the barrier of G‡ = 5.74 kcal/mol. The proton is transferred from the ammonium to the carboxylate, without intervention by the amide group. Since the magnitude of the barrier is low, we suggest that the zwitterionic conformer (zw-3-1) is kinetically not stable, readily isomerizing to the canonical form, once it is formed in low temperature gas phase.

In summary, we predict that microsolvation by up to three water molecules does not yield stable zwitterionic diglycine. The peptide (amide) bond does not seem to help to stabilize the zwitterion as compared with those of amino acids. Further investigation of the solvating effects by more water molecules, and of other types of peptides (especially those containing acidic/basic side chains) will be highly interesting.