Does the Residues Chirality Modify the Conformation of a Cyclo

Article pubs.acs.org/JPCA

Does the Residues Chirality Modify the Conformation of a CycloDipeptide? Vibrational Spectroscopy of Protonated Cyclodiphenylalanine in the Gas Phase Published as part of The Journal of Physical Chemistry virtual special issue “W. Lester S. Andrews Festschrift”. Ivan Alata,† Ariel Pérez-Mellor,† Feriel Ben Nasr,†,‡ Debora Scuderi,§ Vincent Steinmetz,§ Fabrice Gobert,§ Nejm-Eddine Jaïdane,‡ and Anne Zehnacker-Rentien*,† †

Institut des Sciences Moléculaires d’Orsay, CNRS, Univ. Paris-Sud, Université Paris-Saclay, F-91405 Orsay, France Laboratoire de Spectroscopie Atomique Moléculaire et Applications, Université de Tunis El Manar, Tunis 1060, Tunisia § Laboratoire de Chimie Physique, CNRS, UMR8000, Univ. Paris-Sud, Orsay F-91405, France ‡

S Supporting Information *

ABSTRACT: The structure of a protonated diketopiperazine dipeptide, cyclodiphenylalanine, is studied by means of infrared multiple photon dissociation spectroscopy combined with quantum chemical calculations. Protonation exclusively occurs on the oxygen site and, in the most stable conformer, results to an intramolecular OH···π interaction, accompanied by a CH···π interaction. Higherenergy conformers with free OH and NH···π interactions are observed as well, due to kinetic trapping. Optimization of the intramolecular interactions involving the aromatic ring dictates the geometry of the benzyl substituents. Changing the chirality of one of the residues has consequences on the CH···π interaction, which is of CαH···π nature for LD, while LL shows a CβH···π interaction. Higher-energy conformers also display some differences in the nature of the intramolecular interactions.

1. INTRODUCTION Chirality is one of the most striking characteristics of life and is ubiquitous in the chemistry of living organisms.1 The homochirality of life has been suggested to happen through initial symmetry breaking, followed by propagation and amplification,2 possibly by autocatalysis.3 It manifests itself by the incorporation of molecules of a specific chirality throughout life-related biological reactions. It is significant however that once the biological processes have stopped, homochirality tends to vanish. Amino acids racemization has indeed been observed in Pleistocene cave bear dentine and is used as a dating method.4 Chirality and stereochemistry play an important role in various biochemical reactions such as protein folding or enzymatic catalysis.1,5,6 Exogenous molecules like drugs also act differently depending on the chirality of their stereogenic centers.7 Numerous artificial drugs must therefore be of a specific chirality to be integrated into the complex biological system of living organisms. The different involvement of two enantiomers or diastereomers in biological reactions rests upon several factors, some of them being related to the environment or the solvent, others being intrinsic to the molecule itself. Gasphase studies enable studying the structural differences between diastereomers without the perturbations caused by the solvent.8−19 Studying each diastereomer in isolated conditions allows understanding the influence of chirality on intra- and © 2017 American Chemical Society

intermolecular interactions, which in turn govern the enantioselectivity of the reactive processes. Many biological molecules are protonated in physiological conditions, which influences their chemical and physical properties as well as the chemical processes they are involved in. Protonation can, for example, serve as a trigger for reversible structural interconversion from extended to helical forms of pyridine-derived oligoamides.20 The structure of protonated polypeptide often differs strongly from that of neutral ones. In particular, protonated polyalanines in the gas phase contrast with neutral ones and fail to adopt a helical conformation, because the location of the charge at the N-terminus destabilizes the helix.21 Chirality-induced structural changes have been studied in small protonated peptides in isolated conditions. Limited differences have been observed between protonated or cationized LL and LD diphenylalanines or tetraphenylalanine.22−24 However, cyclic species seem to show increased sensitivity to stereochemical factors because of the constraints related to the ring. The structure of protonated model systems like 1-amino-2-indanol25 or 4-hydroxyproline diastereomers15 are indeed sensitive to chirality, as also are neutral cyclic Received: June 23, 2017 Revised: August 28, 2017 Published: September 5, 2017 7130

DOI: 10.1021/acs.jpca.7b06159 J. Phys. Chem. A 2017, 121, 7130−7138

Article

The Journal of Physical Chemistry A

Figure 1. Structure of (a) cyclo (LPhe-DPhe) and (b) cyclo (LPhe−LPhe). (c) Protonation on N for cyclo (LPhe-DPhe). (d) Protonation on O, for cyclo (LPhe-DPhe), together with the numeration of the atoms. The chiral centers are indicated by asterisks.

systems.13,16 This is why we have undertaken the study of cyclic dipeptides, both in the gas phase and in the condensed phase, with the aim of evidencing chirality-induced structural differences.26 Cyclic dipeptides result from the intramolecular peptide coupling of a linear α-dipeptide.27−29 They all contain a diketopyperazine (DKP) ring and are an important class of dipeptides with applications as active pharmaceutical ingredients.30,31 Cyclic dipeptides are often more stable than their linear counterpart in vivo and therefore promising in terms of therapeutic application.31 The DKP ring conformation depends on the nature of the residues and can be either planar, boat, or chair.32,3334−36 A planar conformation is only observed for dipeptides molecules with small side groups like glycine.37 The conformation remains planar for the slightly larger alanine (Ala) residue when the two residues are of opposite chirality, namely, cyclo LAla-DAla. However, it becomes puckered for cyclo (LAla−LAla).33 Cyclic dipeptides containing a bulky residue like phenylalanine (Phe) and tyrosine have a preferred conformation in which the aromatic ring in the side chain stacks over the DKP ring, which adopts a boat form.38 This work aims at determining the structural and spectroscopic differences due to chirality for the two diastereomers of a protonated DKP dipeptide built on Phe. The studied systems, namely, protonated cyclo (LPhe−LPhe), referred to as c-LLH+, and cyclo (LPhe-DPhe), referred to as cLDH+, are shown in Figure 1. The gas-phase structure of the neutral peptides has been studied already, as well as that of the cyclo (LPhe−LPhe) crystal.26,36 The gas-phase neutral molecules display a boat conformation, with one Phe extended toward the amide nitrogen, while the other one is stacked over the DKP ring. Because of the aromatic nature of phenylalanine, dispersion forces are important in the stabilization of the peptide and may favor OH··· π,39−41 NH···π,42 or CH···π43,44 noncovalent interactions, which have been suggested to play a role in chiral recognition.19,45 The two diastereomer dipeptides are studied by combining Fourier transform ion cyclotron resonance (FTICR) mass spectrometry with laser spectroscopy.46 The vibrational signature of the studied systems is obtained by means of infrared multiple photon dissociation (IRMPD) spectroscopy and compared to quantum chemical calculations. Both fingerprint (1000−1800 cm−1) and OH or NH stretch (3100−3700 cm−1) regions are explored. IRMPD spectroscopy has been shown indeed to be a powerful tool for studying the structure of peptides, for example, protonated peptides,47−50 their complexes with alkali metals47 or divalent and trivalent metals,51,52 their cation or anions,53 as well as their reaction products54 or their derivatives with post-translational modifications.55−57 Studies of DKP peptides are limited to the

protonated model systems, cyclo GlyGly,58,59 cyclo AlaGly,60 and linear PhePhe in interaction with Ca2+.61

2. METHODS 2.1. Experimental Section. The experiment setup has been described in details already and is only briefly summarized here.46 It involved a 7 T FTICR hybrid mass spectrometer (Bruker, Apex Qe) coupled to a Free Electron Laser (FEL) at the Centre Laser Infrarouge d’Orsay (CLIO).62 The protonated species were generated by electrospraying a 100 μM solution obtained by diluting a 1 mM stock solution into a slightly acidified (1% formic acid) mixture of methanol and water (50:50). The cyclic dipeptides were obtained from GeneCust-Luxembourg (98% purity) and used without further purification. Infrared spectra were obtained by monitoring the fragmentation efficiency ϕ= −ln(P/(F + P)) as a function of the IR wavelength, with F being the sum of the abundances of the fragment ions produced by IRMPD and P that of the parent. The 900−2000 cm−1 region was covered resorting to the CLIO FEL.62 The CLIO FEL beam was mildly focused by a 2000 mm Ag-protected spherical concave mirror, with a typical beam waist at the center of the FT-ICR of the order of 1 mm diameter. The CLIO FEL operated at 25 Hz with 8 μs long bunch pulses containing 0.5−3 ps micropulses separated by 16 ns. The spectral bandwidth (full width at half-maximum (fwhm)) was ∼7 cm−1 with pulse energy of 1600 to 900 mW from 900 to 2000 cm−1. The wavelength calibration was ensured by simultaneously recording the spectrum of polystyrene. The irradiation time was 1s. Additional broadband CO2 laser synchronized with CLIO (Universal Laser system 10 W at continuous wave (cw) operation centered at λ = 10.6 μm) was employed to sustain the fragmentation. The CO2 laser pulse length was adjusted to 3.8 ms to avoid photodissociation of the molecule by the CO2 laser alone. The vibrational spectrum in the OH and NH stretch region was recorded resorting to an IR optical parametric oscillator (OPO) laser (LaserVision OPO, 25 Hz, 5−10 mJ/pulse). The irradiation time was 3 s and the CO2 laser pulse length was adjusted to 30 ms. 2.2. Computational Details. Exploration of the potential energy surface (PES) was performed using the OPLS-2005 and MMFFs force fields with the advanced conformational search implemented in the MacroModel suite, a part of the Schrödinger package.63 All the peptide structures found thereby with energy below 20 kJ/mol were optimized within the frame of the density functional theory (DFT) at the b97-d3/TZVPP level of theory, using the resolution of the identity approximation for the evaluation of the electron-repulsion 7131

DOI: 10.1021/acs.jpca.7b06159 J. Phys. Chem. A 2017, 121, 7130−7138

Article

The Journal of Physical Chemistry A

Figure 2. CID-MS2 mass spectrum of c-LLH+ (black ●) and c-LDH+(red ▲) for activation times of (a) 0.2 s and (b) 0.25 s. Both spectra are normalized by the parent peak intensity at m/z 295. The c-LDH+ spectrum is shifted to the right by 4 amu for easier comparison of the intensities observed in the two spectra.

integrals to decrease the calculation time.64 The conformers of the most stable family, namely, those protonated on oxygen (vide infra), were reoptimized using the dispersion-corrected functional B3LYP-D3 associated with the 6-311G++(d, p) basis set. The B3LYP-D3 functional was chosen here, as it was in previous studies,65,66 because it combines the good frequency description of the hybrid functional B3LYP67 and inclusion of empirical dispersion corrections, which might be important for peptides containing aromatic residues.68,69 Indeed, calculations of the interaction energy between neutral or protonated alanine and benzene indicate that methods taking dispersion into account are necessary to reproduce the OH···π, NH···π, and CH···π, interactions.70 To make sure that the whole PES was fully explored, structures were also built from the most stable neutral conformers found previously.26 A proton was added to each of the two oxygens, with the two possible orientations of the OH, and the resulting structure was optimized as described above. The vibrational spectra were simulated by convoluting the harmonic frequencies obtained at the same level of calculation by a Lorentzian line shape (fwhm 10 cm−1). A scaling factor of 0.970 in the 900−1700 cm−1 region and 0.955 in the 3000−3800 cm−1 region was applied to correct the frequencies for anharmonicity and basis set incompleteness. All calculations were performed with the Gaussian 09 packages71 or with the Turbomole 6.6 software.72

fragmentation pattern and efficiency. For an activation time of 0.2 s, loss of CO (m/z 267) and formation of the iminium ion of Phe (m/z 120) are observed as the major fragmentation channels. For longer activation time, sequential fragmentation is observed. Subsequent loss of NH3 and CO from m/z 267 results to the m/z 250 and m/z 222 fragments. The fragmentation pattern described above is typical of DKP dipeptides.73−75 A mechanism has been proposed for model protonated DKP based on glycine and alanine, which involves either fragmentation from the most stable O-protonated tautomer, in the case of cyclo AlaAla, or isomerization to the N-protonated tautomer with subsequent fragmentation, in the case of cyclo GlyGly.76 Minor differences in the fragments relative intensity are observed in c-LDH+ relative to c-LLH+. While the primary fragments (m/z 267, m/z 120) are slightly more intense in cLLH+, the secondary fragments are slightly more intense in cLDH+. This observation indicates that c-LDH+ has slightly larger fragmentation efficiency. 3.2. IRMPD Spectra. The IRMPD spectra of LLCH+ and LDCH+ are presented in Figure 3. Their aspect is similar on the overall spectral range; they show however minor differences. The fingerprint region is dominated by an intense and congested feature in the region of the β(OH) and aliphatic β(CH) bending modes, which looks identical for the two diastereomers, with two maxima at 1270−1310 cm−1 in c-LLH+ and 1280−1312 cm−1 in c-LLH+. The β(NH) range displays two bands of weak intensity at 1440 and 1493 cm−1 in c-LDH+, whose counterpart seems to overlap in c-LLH+, at 1452−1492 cm−1. Last, the ν(CO) stretch region is characterized by two intense bands located at 1674−1734 cm−1 in the c-LDH+

3. RESULTS AND DISCUSSION 3.1. CID Fragments. Mass spectrometry (MS2) collisioninduced dissociation (CID) spectra of c-LLH+ and c-LDH+, obtained in the hexapole collision cell with a collision voltage of −6.5 V, are displayed in Figure 2. Both systems show similar 7132

DOI: 10.1021/acs.jpca.7b06159 J. Phys. Chem. A 2017, 121, 7130−7138

Article

The Journal of Physical Chemistry A

bond atoms will be referred to with a subscript “p” when they are on the protonated part. The carbon atoms of the residue will be denoted by their position relative to the peptide bond, that is, Cα or Cβ. Last, the ipso carbon of the benzene rings will be denoted by the subscript “i”. Two protonation sites can be considered, namely, protonation of the amide oxygen and protonation of the amide nitrogen. For both c-LLH+ and cLDH+ the most stable structures correspond to protonation of the oxygen atom. The most stable conformer protonated on the amide nitrogen is higher in energy, by ∼13 kcal/mol, than the most stable conformer protonated on the amide oxygen. It is therefore unlikely that it is populated in our experimental conditions at room temperature. The most stable N-protonated structures are shown in Figure S1 of the Supporting Information. Moreover, the calculated spectra of the Nprotonated species do not show satisfactory agreement with the experimental results, as shown in Figures S2 and S3 of the Supporting Information. These conclusions are in line with proton affinity measurements resting on the Cooks kinetic method77 that indicate a larger stability of the O-protonated form of cyclic peptides due to the formation of a resonancestabilized cation.78 These results also parallel those deduced from the IRMPD spectra of the most simple DKP dipeptide, cyclo GlyGly.58 The most stable conformers of c-LDH+ and c-LLH+ are presented in Figure 4, and the relative Gibbs energies given in Table 1. The energetic characteristics obtained at the B97-D3/ TZVPP level are identical to those at the B3LYP-D3/6-311+ +g(d,p) level and are summarized in Table S1 of the Supporting Information. In what follows, we will describe the B3LYP-D3 only. The most stable conformation of c-LDH+, CLD0, is stabilized by two interactions involving the aromatic ring. An ionic OpH···π interaction is accompanied by a neutral CαpH···π interaction. These interactions result to short distances of 2.51 (2.53) Å between OpH (CpH) and the center of the aromatic ring. The CLD1 conformation (1.6 kcal/mol) also shows an ionic OpH···π interaction, but the CαpH···π interaction is missing and is replaced by a NpH···π interaction. CLD2 and CLD3 both involve intramolecular NpH···π and

Figure 3. Experimental IRMPD spectrum of (a) c-LDH+ and (b) cLLH+ in the CLIO IR FEL and IR OPO regions.

spectrum, and at 1681−1750 cm−1 in that of c-LLH+. These bands are typical of the amide I vibration mode of protonated DKP rings.59 In the fingerprint region, the main differences between the two diastereomers is the blue shift of several bands of c-LLH+ relative to c-LDH+. The high frequencies region is characterized by two features identical for the two diastereomers: First, a broad and intense absorption that peaks at ∼3250 cm−1 and extends from 3050 to 3350 cm−1. Second, an intense and narrow peak appears at 3415 cm−1. The main differences appear on the one hand in the ν(OH) stretch region, where the spectrum of c-LDH+ shows two peaks at 3592 and 3564 cm−1, while that of c-LLH+ only shows a broadened peak at 3567 cm−1. Last, a peak appears at 3380 cm−1 in the spectrum of c-LDH+, while it is absent in that of c-LLH+. 3.3. Theoretical Results and Assignment. The nomenclature used for the molecule is shown in Figure 1. The peptide

Figure 4. Ground-state geometry of the most stable conformers of c-LDH+ (left) and c-LLH+ (right), optimized at the B3LYP-D3/6-311++g(d,p) level of theory. OH··· π, NH··· π, and CH··· π interaction are highlighted by a red, blue, and black dashed lines, respectively. 7133

DOI: 10.1021/acs.jpca.7b06159 J. Phys. Chem. A 2017, 121, 7130−7138

Article

The Journal of Physical Chemistry A Table 1

CLD0 CLD1 CLD2 CLD3 CLL0 CLL1 CLL2 CLL3

ΔEa B3LYP-D3/6311++g(d,p)

ΔGa B3LYP-D3/6311++g(d,p)

Boltzmann factor (%)

CαpCpNpCα dihedral angleb (deg)

CαCNCαp dihedral angleb (deg)

NCαCβCi dihedral angleb (deg)

NCαpCβpCip dihedral angleb (deg)

d(Np···H)b (Å)

d(OpH)b (Å)

0.0 3.8 4.1 4.8 0.0 3.3 3.9 4.7

0.0 1.6 2.8 3.8 0.0 1.8 3.7 4.3

93 6 1