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Accurate Characterization of the Peptide Linkage in the Gas Phase: A Joint Quantum-Chemical and Rotational Spectroscopy Study of the Glycine Dipeptide Analogue Cristina Puzzarini,*,† Malgorzata Biczysko,‡ Vincenzo Barone,*,‡ Laura Largo,§ Isabel Peña,§ Carlos Cabezas,§ and José Luis Alonso*,§ †

Dipartimento di Chimica “Giacomo Ciamician”, Università di Bologna, Via F. Selmi 2, 40126 Bologna, Italy Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa, Italy § Grupo de Espectroscopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Parque Científico UVa, Unidad Asociada CSIC, Universidad de Valladolid, S-47005 Valladolid, Spain ‡

S Supporting Information *

ABSTRACT: Accurate structures of aminoacids in the gas phase have been obtained by joint microwave and quantum-chemical investigations. However, the structure and conformational behavior of α-aminoacids once incorporated into peptide chains are completely different and have not yet been characterized with the same accuracy. To fill this gap, we present here an accurate characterization of the simplest dipeptide analogue (N-acetylglycinamide) involving peptidic bonds. State-of-the-art quantum-chemical computations are complemented by a comprehensive study of the rotational spectrum using a combination of Fourier transform microwave spectroscopy with laser ablation. The coexistence of the C7 and C5 conformers has been proved and energetically as well as spectroscopically characterized. This joint theoretical-experimental investigation demonstrated the feasibility of obtaining accurate structures for flexible small biomolecules, thus paving the route to the elucidation of the inherent behavior of peptides. SECTION: Spectroscopy, Photochemistry, and Excited States

S

replacing a methyl group with hydrogen at either N or the CO moiety has been pointed out in several works (see, e.g., ref 14 for a detailed analysis of 13 model dipeptides); in the former case, only an increased flexibility of the NH2 group with respect to NHCH3 is noted. Therefore, we are left with CH3−CO− NH−CHR−CO−NH2 (hereafter referred to as dipeptide analogue) as the smallest realistic and representative systems. On these grounds, microwave spectra have been recently recorded for the L-alanine (i.e., R = CH3) dipeptide analogue pointing out the contemporary presence of the Ceq 7 (γ-turn) and C5 (β-turn) conformers.15 Both of them are stabilized by a CO···HN intramolecular hydrogen bond, which leads to a seven- or a five-membered ring, respectively. In contrast, in the investigation of the N-acetyl-alanine N′-methylamide (Ac-AlaNHMe), the first rotational study on mimic dipeptide, Lavrich et al. succeeded in identifying only the most stable C7eq conformational form, with the inability to detect the C5 form attributed to its conformational relaxation to Ceq 7 in the supersonic expansion.16 The comparison of the results obtained

tructure−property relationships are at the heart of modern molecular approaches to biology and are the result of a complex and subtle balance of a number of different, both intrinsic and environmental (i.e., relative to the interaction with the solvent, ligands and/or other macromolecular partners), interactions.1,2 Gas-phase studies are therefore mandatory in order to avoid the competition between intra- and intermolecular interactions in tuning the overall conformational behavior.3,4 In the specific case of proteins, the complexity of the polypeptide chain organization and the flexibility of both the backbone and most of lateral chains involve a further balance between local and nonlocal effects. This has stimulated several experimental and computational studies of aminoacids in the gas phase, which have led to a detailed knowledge of their structures and conformational preferences (see, for example, refs 5−12 and references therein). However, aminoacids are not suitable models even for designing local conformational effects in peptides and proteins. The smallest realistic systems are the so-called dipeptide analogues, which contain two peptide linkages (−CO−NH−) because both the amino and carboxyl groups are replaced by amido moieties. Several studies have shown that a methyl linked at N-terminus has negligible effect, whereas this is not the case for the Cterminus (see ref 13 and references therein). The effect of © 2014 American Chemical Society

Received: December 20, 2013 Accepted: January 20, 2014 Published: January 20, 2014 534

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in refs. 15 and 16 furthermore demonstrate the negligible effect on the backbone structure due to the presence of a methyl group bonded to N3, which leaves essentially unchanged the φ and ψ torsional angles. In passing, we note that a subsequent study on the proline dipeptide analogue showed as well only the presence of the C7 conformer.17 However, in all cases, no attempt was made to derive an accurate structural characterization of the peptide linkage. In fact, despite rotational spectroscopy being the method of choice when aiming at highly accurate structural determination, it is a formidable task to extract the desired information from the experimental data, especially when large molecules are considered. On the other hand, by joining the experimental investigation with state-ofthe-art quantum-chemical calculations this aim can be fulfilled. Among the various systems, the glycine dipeptide analogue (R = H) is not only the smallest prototype, but is also essential for protein structure and function.18,19 In light of its importance, we decided to tackle the problem of its structure and conformational behavior. As mentioned above, to elucidate the inherent behavior of dipeptides it is necessary to isolate the system to avoid intermolecular interactions. From a theoretical point of view, we can rely on a computational strategy based on high-level quantum-chemical calculations that we recently developed and successfully applied to the conformational and/or tautomeric investigation of glycine,12,20,21 uracil22 and 2-thiouracil.23 On the other hand, among the possible experimental gas-phase techniques, rotational spectroscopy is the most powerful tool for the identification and characterization of different conformers, as it provides the high resolution necessary to distinguish them without any doubt. In fact, the various conformers show distinctive rotational spectra due to different rotational constants, which in turn depend on the mass distribution in the molecule. In addition, the presence of quadrupolar nuclei (14N) allows one to gain further information as the corresponding nuclear quadrupole-coupling constants are related to the electronic environment and can be decisive in the identification of conformers with similar mass distributions but different intramolecular interactions. In conclusion, the aim of this letter is to provide the first accurate investigation of the molecular structure of N-acetyl-glycinamide (Ac-Gly-NH2; also denoted as glycine dipeptide analogue) and to demonstrate how state-of-the-art quantum-chemical computations can meet microwave spectroscopy toward the characterization of flexible building blocks of biomolecules. To characterize the lowest-energy conformers, a preliminary investigation of the potential energy surface was carried out by means of density functional theory (DFT), using the B3LYP functional24 in conjunction with the SNSD basis set.25 As expected,26−31 the two most stable conformers were found to be the C7 and C5 ones (see Figure 1), which exhibit different folds along the backbone. In the C7 rotamer, one of the terminal amide hydrogens is bonded with the acetyl carbonyl oxygen through an hydrogen bond, thus leading to the formation of a seven-membered ring where the glycine is oriented equatorially. The stabilization of the C5 rotamer is through a weaker hydrogen bond, which engages the carbonyl oxygen and one terminal amide hydrogen of the glycine residue, thus forming a five-membered cycle. The equilibrium structures of the C7 and C5 conformers were accurately evaluated by means of a composite scheme in order to account simultaneously for basis-set and electron-correlation effects. This approach mainly involves second-order Møller−

Figure 1. Molecular structures and atom labeling for the C7 and C5 conformers. Best-estimated bond distances are also reported. For C7, φ = −82.10 deg and ψ = 64.24 deg; for C5, φ = −180.0 deg and ψ = 180.0 deg.

Plesset perturbation theory (MP2)32 calculations, with the coupled-cluster (CC) singles and doubles augmented by a perturbative treatment of triple excitations [CCSD(T)]33 method employed in order to improve the electronic correlation treatment. These computations were carried out in combination with correlation-consistent basis sets, (aug)-ccp(C)VnZ (n = T,Q),34−36 from Dunning’s hierarchies. While a detailed account can be found in refs 12 and 20−23, in the following the composite scheme is briefly summarized. The basis-set truncation effects are taken into account by extrapolating the geometrical parameters r (with r denoting either a distance or a bond angle) to the complete basis set (CBS) limit:37,38 r(CBS) =

n3r(n) − (n − 1)3 r(n − 1) n3 − (n − 1)3

(1)

where n = 4, and thus r(n) and r(n − 1) denote the MP2/ccpVQZ and MP2/cc-pVTZ optimized parameters, respectively. The effects due to core−valence (CV) correlation are included by means of the corresponding correction, Δr(CV), derived as the difference between r(CVTZ,all) and r(CVTZ,fc), the geometries optimized at the MP2/cc-pCVTZ level correlating all and only valence electrons, respectively: Δr(CV) = r(CVTZ, all) − r(CVTZ, fc)

(2)

Analogously, the effect of diffuse functions (aug), Δr(aug), is evaluated by the following difference: Δr(aug) = r(augVTZ, fc) − r(VTZ, fc)

(3)

where r(augVTZ,fc) and r(VTZ,fc) are the optimized structures at the MP2 level employing the aug-cc-pVTZ and 535

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Table 1. Experimental and Calculated Spectroscopic Parameters of the C7 and C5 Conformers C7 parameter A/MHz B/MHz C/MHz Pb/uÅ2 N7d χaa/MHz χbb/MHz χcc/MHz N3d χaa/MHz χbb/MHz χcc/MHz μa/D μb/D μc/D Ng σh/kHz ΔEi/cm−1 ΔEZPVm/cm−1 ΔGn/cm−1

experiment 4469.1107(11) 1214.23622(27) 1081.26289(20) 30.9

C5 calculated a

4480.5 1210.6a 1082.9a 31.8c

experiment 5268.86(20) 1012.00118(25) 857.18696(29) 2.9

calculated 5239.0a 1009.4a 856.6a 3.6c

1.2553(38) 0.717(74) −1.972(74)

1.258e 0.752e −2.008e

2.079(65) 1.787(98) −3.865(98)

2.159e 1.748e −3.907e

0.3887(69) 1.593(10) −1.981(10) 32 2.2 0.0l 0.0l 0.0l

0.432e 1.603e −2.035e −3.25f −1.31f −0.70f -

2.113(66) 2.08(12) −4.19(12) 33 2.5 -

1.997e 1.923e −3.920e −2.46f 0.17f 0.0f 392.8 197.4 159.1

a

Best-estimated equilibrium rotational constants (from best-estimated equilibrium structure) augmented by vibrational corrections at the B3LYP/ SNSD level. See text. bPlanar moment of inertia: P = (1/2)(Ia + Ib − Ic). cEvaluated from best-estimated ground-state rotational constants. From equilibrium rotational constants: 31.9 for C7 and 3.1 for C5. dFor atom labeling, see Figure 1. eBest-estimated equilibrium quadrupole-coupling constants augmented by vibrational corrections at the B3LYP/SNSD level. Out-of-plane vibrations of amino group have been excluded from the VPT2 treatment. See text. fBest-estimated equilibrium dipole-moment components augmented by vibrational corrections at the B3LYP/SNSD level. See text. gNumber of fitted transitions. hStandard deviation of the fit. iBest-estimated equilibrium energy difference. See text. lArbitrary fixed to zero. m Best-estimated equilibrium energy difference augmented by zero-point vibrational (ZPV) correction. See text. nGibbs free energy difference based on best-estimated equilibrium energies at T = 298.15 K. The −RT ln 2 factor issuing from a statistical weight of 2 assigned to the C7 conformer is included. See text.

Figure 2. The experimental and computed rotational spectra of the C7 (red) and C5 (blue) conformers in the 6−18 GHz frequency range. In the insets, the comparison between experiment and theory is better shown.

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Figure 3. Comparison of experiment and theory for the hyperfine structure of the J = 31,3 − 21,2 transition of the C5 (left side) and C7 (right side) conformers. The hyperfine components are labeled with the I,F quantum numbers (see text).

Both conformers contain one methyl top which implies low torsional barriers (84.4 cm−1 and 78.3 cm−1 for the C7 and C5 conformers, respectively), thus resulting in a large A-E splitting of the spectra. Nevertheless, no attempt was made to assign E component lines in the rotational spectra of both conformers because their assignment is not straightforward. However, more important is to note that the derived set of rotational constants for the unperturbed A states is the one that can be used for deriving structural considerations. Therefore, all the present assignments correspond to the unperturbed A states of the C7 and C5 conformers. In the experimental investigation, a chirped pulse Fourier-transform microwave (CP-FTMW) spectrometer equipped with laser ablation vaporization system was first employed to sample swiftly the rotational spectra of the different conformers present in the gas-phase mixture (see Figure 2). Subsequently, the rotational spectra of the C7 and C5 conformers were recorded using a molecular-beam FTMW (LA-MB-FTMW) spectrometer that permits a higher resolution, thus allowing to resolve the hyperfine structure due to the 14N nuclei. This is shown for the J = 31,3 − 21,2 transition of both C5 and C7 in Figure 3: the good agreement between experiment and theory furthermore confirms the accuracy and reliability of our computational strategy. Please, note that all experimental signals are split into Doppler doublets because of the collinear disposition between the supersonic jet and the microwave resonator axes. In this figure, for labeling the hyperfine components, we made use of the I,F quantum numbers coming from the F = J + I coupling scheme, where I being the total nuclear spin quantum number (I = I1 + I2, with I1,2 = 1). All measured hyperfine components (see Tables S1− S2 in the Supporting Information) were analyzed using Watson’s A-reduced Hamiltonian in the Ir-representation45 supplemented by the nuclear quadrupole-coupling term.46 For both rotamers, the spectroscopic parameters determined from the experimental investigation are summarized in Table 1. From these results a very good agreement between experiment and theory is observed for both the rotational and quadrupolecoupling constants, with discrepancies of about 0.2% for the former. For the latter, an agreement within the experimental uncertainties is obtained for N7, which is the relevant nitrogen for the protein backbone structure. The larger differences (on average 5%) noted for N3 can be ascribed to the limited improvement provided by the inclusion of vibrational corrections. This is due to the exclusion of the out-of-plane vibration of the terminal amino group from the perturbative treatment since such a large amplitude motion is not well

cc-pVTZ basis sets, respectively, within the frozen-core approximation. The higher-order correlation energy contributions are derived from the comparison of the geometries optimized at the MP2 and CCSD(T) levels, both with the ccpVTZ basis: Δr(T) = r(CCSD(T)) − r(MP2)

(4)

On the whole, our best-estimated equilibrium structure is determined as r(best) = r(CBS) + Δr(CV) + Δr(aug) + Δr(T)

(5)

As demonstrated in refs 12 and 20−23, the strategy above is able to provide very accurate results, which can be used to straightforwardly derive reliable and accurate equilibrium rotational constants. This is the first step toward the prediction of the rotational spectrum.39−41 Then, the vibrational corrections to rotational constants were obtained by means of vibrational second-order perturbation theory (VPT2),42 within its generalized (GVPT2)43,44 implementation, applied to a cubic force field obtained at the B3LYP/SNSD level of theory. A composite scheme analogous to that employed for the structural determination was also considered for accurately predicting equilibrium nitrogen quadrupole-coupling constants.22,23 The latter were then augmented by the corresponding vibrational corrections computed at the B3LYP/SNSD level. The computed spectroscopic properties are collected in Table 1 and were used to guide the assignment of the rotational spectra of the C7 and C5 conformers. The missing piece of information is the dipole moment, whose components allow us to predict what type of transitions can be observed. By taking advantage of the computations carried out, an accurate estimate for this property was obtained for both conformers using the composite scheme mentioned above. For a quantitative prediction of the dipole moment components in the vibrational ground state, the vibrational corrections, evaluated by means of a perturbative approach at the B3LYP/SNSD level of theory, were added to the equilibrium values. According to the results obtained (Table 1), we expected a strong a-type spectrum for both conformers and a rather weak b-type spectrum only for C7. The predictions of the rotational spectra of the C7 and C5 conformers in the 6−18 GHz frequency range, based on the computed spectroscopic parameters, are depicted in Figure 2 together with the corresponding experimental spectra. The comparison detailed in the insets points out the accuracy of our computations. 537

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described by a forth-order force field. The overall conclusion is that our computation scheme was able to correctly describe the molecular structure of the two conformers, thus confirming the reliability of the geometrical parameters reported in Figure 1 (the complete list is provided in the Supporting Information: Tables S3 and S4 report the geometrical parameters and the Cartesian coordinates of both conformers, respectively). According to our previous studies,12,20−23 conservative uncertainties for the resulting best estimates are 0.001−0.002 Å for bond lengths and 0.05−0.1 degrees for angles. The hyperfine structure originated by the two 14N nuclei provides us with additional information on the geometry. Of particular interest are the values of backbone torsion angles (φ = C8−N7−C1−C2 and ψ = N7−C1−C2−N3; see Figure 1), which describe the conformation of the C7 and C5 rotamers, i. e., reflect the arrangement of the planes defined by the two peptide bonds with reference to the pivotal carbon C1. Since both the amine and amide functional groups are involved in the φ and ψ angles, the corresponding nitrogen quadrupolecoupling constants change by varying the dihedral angle values and can provide additional structural information.15 While interested readers can find in the Supporting Information the discussion concerning the dependence of the nuclear quadrupole coupling constants on the torsional angles and the corresponding pictures showing this dependence in detail, we here only note that the good agreement between experiment and theory mentioned above is a demonstration that our bestestimated geometries are able to well describe the conformational behavior. For C5, the limited dependence of χ’s on the φ and ψ values prevents us from giving the conclusive demonstration of the planarity of its backbone. This can be actually provided by the inspection of the planar moment of inertia, P = (1/2)(Ia + Ib − Ic), where Ia, Ib and Ic denote the moments of inertia (corresponding to the A, B and C rotational constants, respectively). From Table 1, a good agreement of experiment with the corresponding theoretical values is evident. For C5, we note a small value, 2.9 uÅ2 from experiment and 3.6 uÅ2 from theory (which reduces to 3.1 uÅ2 when equilibrium rotational constants are considered), which is due to the contribution of two hydrogens of the methyl group and two hydrogens linked to the carbon of the glycine residue that are out of the plane where the dipeptide chain lies. A nonplanarity of the backbone would lead to a larger P value. The last issue to be addressed is the relative population of C5 and C7. An approach that was found to be reliable in predicting the relative abundance when one species exhibits folded and extended forms coexisting in the gas mixture is based on measuring the relative intensities of several rotational transitions obtained in the CP-FTMW spectra (see, for example, refs 47 and 48). By assuming that the expansion cools down all molecules to the lowest vibrational state, the intensities should be proportional to the concentration of each species in the beam scaled by the square of the dipole moment, μ2·Ni, as determined from ab initio calculations (Table 1).49 On this basis, the population C5/C7 ratio of the Ac-Gly-NH2 conformers was estimated to be 0.32(10). Theoretically, this ratio was evaluated from the computed free energy difference, ΔG, at 298.15 K (see Table 1), and a value of 0.43 obtained. This is in good agreement with the experimental estimate, thus confirming that in our laser ablation experiment the collisional rate in the seeding region is high enough to bring the molecular system to a population distribution close to the equilibrium one

at the temperature of the carrier gas.50 In view of accurately establishing the energy difference between the two conformers, both the electronic energies and thermodynamic contributions were computed at high accuracy.12,21 Best-estimated equilibrium energies were obtained by means of the composite scheme employed for the molecular structure determination. On top of the best energy difference, the evaluation of thermodynamic properties was carried out beyond the doubleharmonic approximation. The proper treatment of lowfrequency torsional motions and anharmonic effects was assured by the computation of the vibrational corrections with a Hindered-Rotor Anharmonic Oscillator (HRAO) model,51−53 within the vibrational second-order perturbation theory (VPT2) coupled with the Simple Perturbation Theory (SPT).53,54 Within the HRAO model, the methyl group vibrations were discarded from the VPT2 treatment and considered as one-dimensional hindered rotations. The results, given in Table 1, show that at the equilibrium the electronic energy difference is about 400 cm−1, which approximately halves once moving to the vibrational ground state and is further reduced upon consideration of the Gibbs free energy. This leads to the C5/C7 ratio in good agreement with experiment mentioned above, while resorting to harmonic approximation would lead to 1:1 ratio. It should be noted that Gibbs free energy is computed also taking into account the presence of two equivalent C7 conformers. [The presence of two equivalent substituents (H) at C1 has as a consequence that the C7 conformer has an equivalent (isoenergetic) counterpart obtained by reversing the signs of the φ and ψ angles. This is not the case for C5 because of the planarity of its backbone.] In conclusion, a state-of-the-art quantum-chemical approach allowed us to guide the first experimental study of the simplest dipeptide model and to complement the experimental data, thus leading to a complete and accurate structural, thermodynamic, and spectroscopic characterization. In our opinion, in addition to the intrinsic interest of the results obtained, the most interesting outcome of this work is the promise that integrated experimental and computational studies enable the characterization of flexible medium-sized molecular systems of biological and/or technological interest with an accuracy reached so far only for small rigid molecules.

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EXPERIMENTAL AND COMPUTATIONAL SECTION A sample of N-acetylglycinamide (m.p. 136−140 °C) was used without further purification, and prepared by mixing the compound powder with a commercial binder. The mixture was pressed to form cylindrical rods, which were placed in a laser ablation nozzle55 to be vaporized using a 20 ps Nd:YAG laser (12 mJ/pulse). The vaporized sample was then seeded in the Ne carrier gas at backing pressure of 18 bar, to expand adiabatically into the vacuum chamber, and probed by either a broadband CP-FTMW56−58 or LA-MB-FTMW55 spectrometer. MP2 and CCSD(T) calculations were carried out with the quantum-chemical CFOUR program package,59 while DFT and VPT2 computations were performed employing a locally modified version of the Gaussian suite of programs for quantum chemistry.60

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1−S2: List of measured hyperfine components for the C5 and C7 conformers, respectively. Tables S3−S4: Complete 538

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(12) Barone, V.; Biczysko, M.; Bloino, J.; Puzzarini, C. The Performance of Composite Schemes and Hybrid CC/DFT Model in Predicting Structure, Thermodynamic and Spectroscopic Parameters: The Challenge of the Conformational Equilibrium in Glycine. Phys. Chem. Chem. Phys. 2013, 15, 10094−10111. (13) Bhattacharya, A.; Bernstein, E. R. Influence of Turn (or Fold) and Local Charge in Fragmentation of the Peptide Analogue Molecule CH3CO-Gly-NH2 Following Single-Photon VUV (118.22 nm) Ionization. J. Phys. Chem. A 2011, 115, 10679−10688. (14) Yu, W.; Xu, X.; Li, H.; Pang, R.; Fang, K.; Lin, Z. Extensive Conformational Searches of 13 Representative Dipeptides and an Efficient Method for Dipeptide Structure Determinations Based on Amino Acid Conformers. J. Comput. Chem. 2009, 30, 2105−2121. (15) Cabezas, C.; Varela, M.; Cortijo, V.; Jiménez, A. I.; Peña, I.; Daly, A. M.; López, J. C.; Cativiela, C.; L., A. J. The Alanine Model Dipeptide Ac-Ala-NH2 Exists As a Mixture of Ceq 7 and C5 Conformers. Phys. Chem. Chem. Phys. 2013, 15, 2580−2585. (16) Lavrich, R. J.; Plusquellic, D. F.; Suenram, R. D.; Fraser, G. T.; Walker, A. R. H.; Tubergen, M. J. Experimental Studies of Peptide Bonds: Identification of the C7eq Conformation of the Alanine Dipeptide Analog N-Acetyl-alanine N′-Methylamide from Torsion− Rotation Interactions. J. Chem. Phys. 2003, 118, 1253−1265. (17) Cabezas, C.; Varela, M.; Alonso, J. L. Probing the γ-Turn in a Short Proline Dipeptide Chain. Chem. Phys. Chem. 2013, 14, 2539− 2543. (18) Yan, B.; Sun, Y. Glycine Residues Provide Flexibility for Enzyme Active Sites. J. Biol. Chem. 1997, 272, 3190−3194. (19) Mousavi, A.; Hotta, Y. Glycine-Rich Proteins - A Class of Novel Proteins. Appl. Biochem. Biotechnol. 2005, 120, 169−174. (20) Barone, V.; Biczysko, M.; Bloino, J.; Puzzarini, C. Glycine Conformers: A Never-Ending Story? Phys. Chem. Chem. Phys. 2013, 15, 1358−1363. (21) Barone, V.; Biczysko, M.; Bloino, J.; Puzzarini, C. Characterization of the Elusive Conformers of Glycine from State-of-the-Art Structural, Thermodynamic and Spectroscopic Computations: Theory Complements Experiment. J. Chem. Theory Comput. 2013, 9, 1533− 1547. (22) Puzzarini, C.; Barone, V. Extending the Molecular Size in Accurate Quantum-Chemical Calculations: The Equilibrium Structure and Spectroscopic Properties of Uracil. Phys. Chem. Chem. Phys. 2011, 13, 7189−7197. (23) Puzzarini, C.; Biczysko, M.; Barone, V.; Peña, I.; Cabezas, C.; Alonso, J. L. Accurate Molecular Structure and Spectroscopic Properties of Nucleobases: A Combined Computational-Microwave Investigation of 2-thiouracil as a Case Study. Phys. Chem. Chem. Phys. 2013, 15, 16965−16975. (24) Becke, D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (25) Double and triple-ζ basis sets of SNS and N07 families, are available for download (2012); visit http://dreamslab.sns.it (accessed February 1, 2013). (26) Grenie, Y.; Avigon, M.; Garrigou-Lagrange, C. Molecular Structure Study of Dipeptides Isolated in an Argon Matrix by Infrared Spectroscopy. J. Mol. Struct. 1975, 24, 293−307. (27) Cheam, T. C.; Krimm, S. Ab Initio Force Fields of Glycine Dipeptide in C5 and C7 Conformations. J. Mol. Struct. 1989, 193, 1− 34. (28) Gould, I. R.; Cornell, W. D.; Hillier, H. A Quantum Mechanical Investigation of the Conformational Energetics of the Alanine and Glycine Dipeptides in the Gas Phase and in Aqueous Solution. J. Am. Chem. Soc. 1994, 116, 9250−9256. (29) Perczel, A.; Császár, A. G. Toward Direct Determination of Conformations of Protein Building Units from Multidimensional NMR Experiments I. A Theoretical Case Study of For-Gly-NH2 and For-L-Ala-NH2. J. Comput. Chem. 2000, 21, 882−900. (30) Lee, H.; Song, J.-W.; Choi, Y.-S.; Ro, S.; Yoon, C.-J. The Energetically Favorable Cis Peptide Bond for the AzaglycineContaining Peptide: For-AzGly-NH2 Model. Phys. Chem. Chem. Phys. 2001, 3, 1693−1698.

list of the best-estimated geometrical parameters and Cartesian coordinates for both conformers, respectively. Figures S1−S8: Pictures showing the dependence of nuclear quadrupolecoupling constants of C7 and C5 (for N3 and N7) on the torsional φ and ψ angles. These figures are preceded by the corresponding discussion. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by Italian MIUR (PRIN 2009, FIRB) and by the University of Bologna (RFO funds). In Spain, this research was supported by the Ministerio de Ciencia e Innovación (Grant CTQ 2010-19008), Consolider Ingenio 2010 (CSD 2009-00038), Junta de Castilla y Leó n (VA070A08). The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. ERC-2012-AdG-320951-DREAMS. The authors gratefully thank the high performance computer facilities of the DREAMS center (http://dreamshpc.sns.it) for providing computer resources. The support of the COST CMTS-Action CM1002 “COnvergent Distributed Environment for Computational Spectroscopy (CODECS)” is also acknowledged.

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