Ariel Pérez-Mellor

Ivan Alata

Valéria Lepère

Anne Zehnacker
email: anne.zehnacker-rentien@u-psud.fr

Valeria Lepère

Conformational Study of the Jet-Cooled Diketopiperazine Peptide Cyclo Tyrosyl-Prolyl

  L'archive ouverte pluridisciplinaire

I. Introduction

Diketopiperazine (DKP) dipeptides are cyclic dipeptides, built from the intramolecular peptide bond formation, and are naturally present in natural environments. For example, that built on phenylalanine (Phe), cyclo Phe-Phe, is a decomposition product of the commonly used sweetener Aspartame. Other DKP dipeptides are present in plants or fungi. In particular cyclo tyrosyl-proline (cyclo Tyr-Pro), commonly known as Maculosin, is a natural phytotoxin [START_REF] Stierle | A Host-Specific Phytotoxin for Spotted Knapweed from Alternaria-Alternata[END_REF] .

Several DKP dipeptides, due to their stability and resistance to proteolysis, have been tested for anti-cancerous or antibiotic properties. [START_REF] Milne | The biological activity of selected cyclic dipeptides[END_REF] From a structural point of view, these dipeptides do not possess a terminal charged group, in contrast to linear peptides or -amino acids. Thus, their crystal structure does not involve strong electrostatic interaction between ionic heads, as it is the case for example for solid alanine, [START_REF] Lehmann | Precision Neutron-Diffraction Structure Determination of Protein and Nucleic-Acid Components .1. Crystal and Molecular Structure of Amino-Acid L-Alanine[END_REF] but often rests on hydrogen-bonded networks involving the amide NH and CO, similar to those observed in -sheet structures of proteins. [START_REF] Corey | The Crystal Structure of Diketopiperazine[END_REF] However, protic side chains on the residues can also participate in the hydrogen-bonded network, as found in polymorphs of cyclo Glu-Glu. [START_REF] Mendham | Vibrational spectroscopy and crystal structure analysis of two polymorphs of the diamino acid peptide cyclo(L-Glu-L-Glu)[END_REF] The rigid DKP ring has limited conformational mobility and is usually non-planar because of the steric hindrance brought upon by the substituents. It ranges from pseudo boat or chair to intermediate twisted shapes. [START_REF] Carlson | Theoretical UV circular dichroism of aliphatic cyclic dipeptides[END_REF][START_REF] Milne | Cyclodipeptides -Structure and Conformation of Cyclo(Tyrosyl Prolyl)[END_REF] The residue side chains have more conformational freedom.

Equilibrium between different conformations often happens in solution, which involves different orientations of the side chain. Residues with a bulky aromatic substituent such as Phe, Tyr or Trp, tend to adopt a flagpole position of the substituent 8-9 10 , that is folded over the DKP ring.

We have recently characterized spectroscopically the structure of DKP dipeptides built on two identical aromatic amino acids like Phe or Tyr, both neutral or protonated in the gas phase or in the solid phase. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Vibrational circular dichroism of a 2,5-diketopiperazine (DKP) peptide: Evidence for dimer formation in cyclo LL or LD diphenylalanine in the solid state[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] The idea behind this work is to assess the structural differences between dipeptides having the two residues with the same absolute configuration, i.e. the natural L configuration, or with different, L and D, absolute configurations. Our study concluded that, unless specific interactions are present, the most stable structure is a "folded-extended" geometry, with one of the aromatic ring folded over the DKP ring and the other one extended.

Cyclo LTyr-LTyr stands out by the presence of an additional conformer in which the two aromatic rings are held parallel to each other thanks to an OH…O hydrogen bond formed between the two hydroxyls. This geometry is only possible for cyclo LTyr-LTyr and not for cyclo LTyr-DTyr. Studies under supersonic jet conditions indicate that electronic excitation is localized on one ring only, for both cyclo Phe-Phe and cyclo Tyr-Tyr, as is the charge in the radical cation. Here, we extend our studies to jet-cooled cyclo Tyr-Pro, shown in Figure 1. This system has only one bulky aromatic substituent. Moreover, proline stands out among the amino acids by its N-alkylamide bond when incorporated into a peptide. This results in additional conformational bias induced by the proline cycle fused with the DKP ring. This might modify the competition between optimizing the interaction between the aromatic side chain and the DKP and minimization of steric hindrance, which has been proposed for explaining the geometry of aromatic-containing DKP dipeptides. [START_REF] Deslauriers | NUCLEAR MAGNETIC-RESONANCE STUDIES OF CONFORMATIONS OF CYCLIC DIPEPTIDES[END_REF] Several structural studies have been already devoted to cyclo Tyr-Pro in the condensed phase.

X-ray crystallography concludes that two close conformers of cyclo LTyr-LPro crystallize in the space group P21212, with the tyrosyl side chains folded on the DKP ring in a flattened chair conformation. [START_REF] Milne | Cyclodipeptides -Structure and Conformation of Cyclo(Tyrosyl Prolyl)[END_REF] No data are available for the enantiopure cyclo LTyr-DPro. However, in the racemic mixture, the tyrosine side chain is also folded on the DKP ring, which adopts a twistboat conformation with pseudo C2v symmetry. [START_REF] Hong | Crystal Structure and Spectroscopic Properties of Cyclic Dipeptide: A Racemic Mixture of cyclo(D-Prolyl-L-Tyrosyl) and cyclo(L-Prolyl-D-Tyrosyl)[END_REF] A folded structure similar to that observed in the solid is proposed to explain the electronic circular dichroism (ECD) spectrum of cyclo LTyr-LPro in solution. [START_REF] Li | First-Principles Simulation of Amide and Aromatic Side Chain Ultraviolet Spectroscopy of a Cyclic Dipeptide[END_REF][START_REF] Madison | Cyclic peptides. 14. Conformational energy and circular dichroism of proline-containing cyclic dipeptides[END_REF] This structure is not modified when changing the solvent or the absolute configuration of the residues. The geometry proposed to account for the photo-electron spectrum of cyclo LTyr-LPro in the vapor phase is at odds with the findings in solution and consist of an extended conformer involving a weak NH…π interaction. [START_REF] Arachchilage | Photoelectron spectra and structures of three cyclic dipeptides: PhePhe, TyrPro, and HisGly[END_REF] In this work, we combine conformer-selective vibrational spectroscopy of jet-cooled cyclo LTyr-LPro and cyclo LTyr-DPro, referred to as c-LL and c-LD hereafter, with quantum chemical calculations, to shed light on their gas-phase structure. The size of the system is small enough for full anharmonic calculations, which allows the subtle spectroscopic differences between the two diastereomers to be reproduced theoretically.

II. Theoretical and experimental methods

II-1.

Nomenclature of the Studied Systems

The numbering of the atoms and the angular parameters describing the molecular geometry are

shown in Figure 1. The dipeptides geometry is described by the conformation of the DKP ring itself, that of the proline ring, and the orientation of the aromatic substituent relative to the amide bond. [START_REF] Budesinsky | Structures of cyclic dipeptides: an X-ray and computational study of cis-and trans-cyclo(Pip-Phe), cyclo(Pro-Phe) and their N-methyl derivatives[END_REF] For the aromatic substituent orientation, Young et al, reported three geometries as minima of the potential energy surface. [START_REF] Young | Cyclic peptides. 15. Lanthanide-assisted carbon-13 and proton NMR analysis of preferred side-chain rotamers in proline-containing cyclic dipeptides[END_REF] They are defined by a Newman projection along the C5C1 bond: two gauche geometries, g + and g -, correspond to dihedral angles τ (N C1 C5 C6) of ~ 60° and ~ -60° for the L residue, as shown in Figure 2. The trans t geometry corresponds to τ ~ 180°. The additional parameter I and II describes the orientation of the tyrosine hydroxyl relative to the DKP ring, as indicated in Figure 2. The conformation of proline is determined in a similar manner, by τ' (N C3 C12 C13), defined by a Newman projection along the C14C3 bond.

However, due to the constraints imposed by its cyclic nature, only the g -orientation exists.

The nomenclature used here starts with "c-" denoting the cyclic nature of the dipeptide, followed by the orientation of the tyrosine aromatic side chain g + , g -, or t, followed by I or II for the tyrosyl OH position. The Tyr residue is always of natural absolute configuration L. The second parameter g -represents the orientation of the proline, followed by the subscript D if proline is in the absolute configuration D. When non-specified otherwise, the absolute configuration is the natural L configuration.

II-2. Theoretical Methods

a) Exploration of the conformational landscape and geometry optimisation

The potential energy surface (PES) was first explored using the OPLS-2005 force field combined with the advanced conformational search implemented in the MacroModel program of the Schrödinger package. [START_REF]MacroModel version 9.8[END_REF] Then, the obtained lowest-energy conformers were used as starting points for a relaxed scan along the dihedral angle τ, by steps of ten degrees, which corresponds to an energy variation of less than 2 kcal/mol. The resulting relaxed scan is presented in Figure S1 of the electronic supplementary information (ESI †). All critical points (local minima or transition states) were fully optimized within the frame of the density functional theory (DFT) using the dispersion-corrected functional B3LYP-D3 [START_REF] Grimme | A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu[END_REF][START_REF] Grimme | Effect of the Damping Function in Dispersion Corrected Density Functional Theory[END_REF] associated with the Pople 6-311++g(d,p) split-valence basis set. [START_REF] Frisch | MOLECULAR-ORBITAL METHODS[END_REF] This level of theory satisfactorily reproduces the vibrational spectra of similar systems at an acceptable calculation cost. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF][START_REF] Lepere | Chirality-dependent structuration of protonated or sodiated polyphenylalanines: IRMPD and ion mobility studies[END_REF] The systematic exploration of the PES by means of the relaxed scan does not yield new stable conformations relative to those obtained with the force field exploration.

b) Frequency calculations

The vibrational frequencies were first calculated within the frame of the harmonic approximation at the same level of theory. The absence of imaginary frequency was checked for all local minima found. Full anharmonic calculations were also performed by using the variational perturbation theory. [START_REF] Bloino | A second-order perturbation theory route to vibrational averages and transition properties of molecules: General formulation and application to infrared and vibrational circular dichroism spectroscopies[END_REF][START_REF] Bloino | Aiming at an accurate prediction of vibrational and electronic spectra for medium-to-large molecules: An overview[END_REF][START_REF] Bloino | Anharmonic Effects on Vibrational Spectra Intensities: Infrared, Raman, Vibrational Circular Dichroism, and Raman Optical Activity[END_REF] The harmonic frequencies need to be scaled to account for anharmonicity and basis set incompleteness. [START_REF] Johnson | NIST Computational Chemistry Comparison and Benchmark Database[END_REF] To this end, we used the anharmonic frequencies to define the scaling factor as the range-specific slope of the linear regression between harmonic and anharmonic frequencies, for all the computed structures, as shown in Figure S2 of the ESI †.

The scaling factor for the fingerprint, the ν(CH), and the ν(NH)/ν(OH) region is 0.977, 0.957, 0.952, respectively. The idea behind this approach is to define a set of transferable scaling factors that can be used for similar or larger systems, such as dimers, which we are currently studying. Indeed, mode-dependent scaling factors based on an extensive library of systems improve agreement with the experiment. [START_REF] Gloaguen | Gas-Phase Ir Spectroscopy and Structure of Biological Molecules[END_REF][START_REF] Plowright | Compact Folding of Isolated Four-Residue Neutral Peptide Chains: H-Bonding Patterns and Entropy Effects[END_REF] As mentioned below in the discussion, both anharmonic and scaled harmonic frequencies reproduce the experiment. Thus, we will limit the discussion on the neutral species to the anharmonic frequencies. For the cations, we use the harmonic frequencies scaled as described above. The spectra were simulated by convoluting the calculated frequencies by a Lorentzian shape (FWHM 8 cm -1 ) for better comparison with the experiment. c) Electronic excited state and ionic state

The electronic excited state S1 geometry was optimized at the time-dependent DFT (TD-DFT) level, using the same functional and basis set as S0. The radical cation was optimized at the same level of theory as the neutral by removing an electron from the calculated neutral forms, which reflects the vertical ionization process. The charge distribution was obtained from the Natural Bond Orbital (NBO) analysis. [START_REF] Reed | Intermolecular interactions for a natural bond orbital, donor-acceptor viewpoint[END_REF] The vibrationally-resolved electronic transition was simulated based on the Franck-Condon principle. [START_REF] Santoro | Effective method to compute Franck-Condon integrals for optical spectra of large molecules in solution[END_REF][START_REF] Santoro | Effective method to compute Franck-Condon integrals for optical spectra of large molecules in solution[END_REF] All calculations were performed with the Gaussian 09 package. 36

II-3. Experimental Methods

The experimental set-up combines a supersonic expansion (General Valve) of Argon seeded with the dipeptide put into the gas phase by a homemade laser desorption source, and coupled with a 1m time-of-flight mass spectrometer (Jordan) equipped with MCP detector, as detailed previously. [START_REF] Sen | Conformational analysis of quinine and its pseudo enantiomer quinidine: a combined jet-cooled spectroscopy and vibrational circular dichroism study[END_REF][START_REF] Sen | How do Pseudoenantiomers Structurally Differ in the Gas Phase? An IR/UV Spectroscopy Study of Jet-Cooled Hydroquinine and Hydroquinidine[END_REF] Mass-resolved S0-S1 spectra were obtained by one-color resonance-enhanced two-photon ionization (RE2PI) spectroscopy, using a frequency-doubled dye laser (Sirah) pumped by the second harmonic of a YAG laser (Spectra). Vibrational spectra were obtained using the IR-UV double resonance technique, in the ν(NH) and ν(OH) stretch region. [START_REF] Tanabe | OH Stretching Vibrations of Phenol-(H2O)n(n=1-3) Complexes Observed by Ir-Uv Double-Resonance Spectroscopy[END_REF][START_REF] Pribble | Size-Specific Infrared-Spectra of Benzene-(H2O)(n) Clusters (n=1 through 7) -Evidence for Noncyclic (H2O)(n) Structures[END_REF] Fixing the UV probe on the main vibronic bands of the electronic spectrum and scanning the IR pump in the 3m region allowed for the measurement of mass-resolved conformer-selective vibrational absorption spectra, as depletion of the UV probe-induced ion current.

The IR source was provided by an OPO/OPA system (Laser Vision) and was triggered 80 ns before the UV laser pulse for recording the IR spectrum of the electronic ground state and 50 ns after for recording IR absorption of the ion. A homemade active baseline subtraction scheme was used for monitoring the IR absorption as the difference in ion signal produced by successive UV laser pulses (one without and one with the IR laser). The resolution of the UV laser was ~0.2 cm -1 and that of the IR OPO was 2 cm -1 .

The peptides were purchased from Novopep Limited (Shanghai -China) and used without further purification.

III. Results and Discussion

III-1. Theoretical Results

The calculated minimum-energy conformers only differ by the orientation g + , g -, or t of the aromatic ring and the orientation, I or II, of the hydroxyl. As mentioned earlier, the proline has g -geometry in all the calculated conformers, whatever the absolute configuration. Six stable conformers are therefore expected for each stereoisomer. However, the results for the I and II conformers parallel each other, and the Gibbs energy difference between them is less than 0.2 kcal/mol, which is within the error. Therefore, we will limit the discussion to the II conformers,

shown in Figure 3. Their relative Gibbs energies are listed in Table 1, as well as the angle τ describing the substituent orientation. We must keep in mind however that both I and II forms are expected to be trapped in our experimental conditions, as it is the case for tyrosine itself or for cyclo Tyr-Tyr. [START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Shimozono | Revised conformational assignments and conformational evolution of tyrosine by laser desorption supersonic jet laser spectroscopy[END_REF] The energetic and structural parameters for the conformers with I orientation of the hydroxyl are given in Table S1 of the ESI †. Table S1 also lists the values of all the angles describing the geometry of the two aliphatic rings, the proline and the DKP.

c-LL:

The most stable orientation of the aromatic ring is folded over the DKP ring and corresponds to a c-g 𝐼𝐼 + g -conformer. The extended c-g 𝐼𝐼 -g -conformer is higher in energy by ~ 0.5 kcal/mol and is therefore expected to be populated too. Lastly, the extended c-t 𝐼𝐼 g - conformer is much higher in energy (~ 2 kcal/mol) and is not expected to be populated in our experimental conditions.

As already mentioned for cyclo Phe-Phe and cyclo Tyr-Tyr, the most stable c-g 𝐼𝐼 + g -conformer has a compact geometry, with the phenol folded over the DKP ring. This allows a C14H…π interaction to take place from the CH of proline. The geometry of the DKP ring is quasi-planar and the distance between C14H and the center of the phenol ring is 2.67 Å.

c-t 𝐼𝐼 g -and c-g 𝐼𝐼 -g -conformers both have the DKP ring in pseudo boat conformation and the aromatic ring extended outwards. In c-g 𝐼𝐼 -g -, the tyrosine residue is directed towards the tyrosine NH, resulting to a weak NH…π interaction with a distance from the NH to the center of the phenol ring of 3.30 Å. In c-t 𝐼𝐼 g -, the tyrosine residue is extended towards the proline amide CO.

c-LD:

The results for c-LD parallel those described for c-LL except that all conformers have boat DKP geometry. Again, c-g 𝐼𝐼 + g D -is the most stable geometry and only differs from its c-LL counterpart by the nature of the CH…π interaction, as already observed in cyclo Phe-Phe and cyclo Tyr-Tyr. It consists indeed in a CαH…π interaction in c-g 𝐼𝐼 + g D -. Due to this interaction, the distance between C3H and the center of the phenol ring is short (2.58 Å). The second stable conformer, c-g 𝐼𝐼 -g 𝐷 -, involves an NH…π interaction with a distance between NH and the center of the phenol ring of 3.50 Å, slightly larger than in c-LL. Last, c-t 𝐼𝐼 g 𝐷 -is completely extended and does not involve noticeable non-covalent interactions; the distance between C2O and the center of the ring is 3.88 Å. It should be noted that the energy gap between the most stable cg 𝐼𝐼 + g D -conformer and c-g 𝐼𝐼 -g 𝐷 -or c-t 𝐼𝐼 g 𝐷 -is much larger in c-LD than c-LL and amounts to 1.4 and 3.7 kcal/mol, respectively.

It should be noted that the most stable c-LD conformer is 2.06 kcal/mol lower in energy than the most stable c-LL conformer. This contrasts to the previous findings on neutral or protonated cyclo PhePhe, or neutral cyclo Ty-Tyr, in which homochirality was always favored. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] Among the studied DKP dipeptides, cyclo Tyr-Pro stands out by the cyclic nature of the proline residues, which introduces additional conformational bias. As a result, the NH… interaction,

III-2. Experimental Results

III-2 a. Electronic Spectroscopy

The one-color REMPI electronic spectra of c-LL and c-LD are obtained by monitoring the mass of the parent ion at m/z 260 and are shown in Figure 4. Well-resolved sharp vibronic bands appear in the region of the first ππ* electronic transition of tyrosine, which indicates that the molecules are efficiently cooled down. The lower-energy transition appears at different frequencies for the two diastereomers, 35498 cm -1 for c-LL vs. 35641 cm -1 for c-LD. These values are close to those for tyrosine, which range from 35491 cm -1 to 35650 cm -1 depending on the conformer [START_REF] Shimozono | Revised conformational assignments and conformational evolution of tyrosine by laser desorption supersonic jet laser spectroscopy[END_REF][START_REF] Martinez | The Electronic Spectroscopy of the Amino-Acids Tyrosine and Phenylalanine in a Supersonic Jet[END_REF][START_REF] Inokuchi | Conformation of L-tyrosine studied by fluorescencedetected UV-UV and IR-UV double-resonance spectroscopy[END_REF][START_REF] Grace | The R2PI spectroscopy of tyrosine: A vibronic analysis[END_REF][START_REF] Abo-Riziq | Conformational Structure of Tyrosine, Tyrosyl-glycine, and Tyrosyl-glycyl-glycine by Double Resonance Spectroscopy[END_REF] or to those for cyclo Tyr-Tyr. [START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF] Several vibronic bands appear in these spectra, suggesting strong vibronic activity and/or coexistence of conformers. The spectrum of c-LL shows four origin-like transitions noted A (35498 cm -1 ), B (35532 cm -1 ), C (35592 cm -1 ), D (35673 cm -1 ) in Figure 4. The four of them are followed by a three-membered progression built on a 29 cm -1 mode, as indicated in figure 4. Bands also appear at A+68 cm -1 and B+65 cm -1 , while D is characterized by the presence of a doublet split by 4 cm -1 for each member of the 29 cm -1 progression. The vibronic pattern of c-LD is much simpler than that of c-LL. It mostly comprises two three-membered progressions built on a ~35 cm -1 mode, starting at 35710 cm -1 and 35752 cm -1 (band C and D in Figure 4, respectively), accompanied by two weaker bands, A (35641 cm -1 ), and B (35656 cm -1 ), which also behave like transition origins. In contrast to c-LL, the ν(NH) stretches appear at different frequencies in c-LD, namely, 3429 cm -1 when probing bands C or D and 3418 cm -1 when probing band A and B. Again, this result suggests two families of conformers for c-LD. A shoulder appears at the low-energy side of the ν(NH) stretch of C and D, possibly due to the presence of an overtone or combination band, as already observed for other DKP dipeptides. [START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] The most apparent difference between the two diastereomers is that the ν(NH) stretch is shifted up in energy by ~ 30 cm -1 in c-LD relative to c-LL.

III-2 b. Vibrational Spectroscopy in the electronic ground state and assignment

The harmonic and anharmonic ν(NH) and ν(OH) stretch frequencies are listed in Table 2. The calculated ν(OH) stretch frequency is identical for all the conformers of c-LL (~3650 cm -1 ) like in the experimental spectrum and does not help for the attribution. A tentative assignment can rest only on the ν(NH) stretch region and on thermodynamic considerations. We must also keep in mind that the I and II rotamers, which only differ by the OH orientation, have exactly the same calculated IR spectrum and energy and should appear as pairs in the REMPI spectrum.

The two most stable c-g 𝐼𝐼 + g -/c-g 𝐼 + g -and c-g 𝐼𝐼 -g -/c-g 𝐼 -g -conformers have their ν(NH) at 3400 cm -1 , which compares well with the experimental value of 3398 cm -1 , and are therefore good candidates for the assignment. The calculated ν(NH) frequency is 3426 cm -1 for c-t 𝐼𝐼 g -, which allows discarding this higher-energy structure. The assignment to either c-g 𝐼𝐼 + g -/c-g 𝐼 + g -or c-g 𝐼𝐼 -g -/c-g 𝐼 -g -conformers rests on the relative intensity of the ν(NH) and ν(OH) stretches.

For the most stable folded c-g 𝐼𝐼 + g -conformer, the calculated intensity of ν(OH) is larger than that of ν(NH), as observed for C, D and E. The calculated spectrum of the slightly less stable extended c-g 𝐼𝐼 -g -conformer shows similar intensities for ν(OH) and ν(NH), as observed for A and B. It is therefore tempting to assign bands A and B to c-g 𝐼𝐼 -g -and c-g 𝐼 -g -and bands C and D to c-g 𝐼𝐼 + g -and c-g 𝐼 + g -. Similar reasoning can be followed for the c-LD diastereomer.

Although the intense combination band calculated for c-g 𝐼𝐼 -g 𝐷 -is hardly observed in the experimental spectrum of C or D, we tend to assign the C and D bands to the c-g 𝐼𝐼 -g 𝐷 -and cg 𝐼 -g 𝐷 -conformers family. This assignment rests on the comparison between the calculated and experimental ν(NH) stretch frequencies and thermodynamics considerations, which allow us excluding the c-g 𝐼𝐼 -g 𝐷 -conformer. At this stage, it is not possible to make a definite assignment.

As we shall discuss later on the basis of FFC calvulations, we assign the most intense bands to the c-g 𝐼𝐼 -g 𝐷 -and c-g 𝐼 -g 𝐷 -conformers. The weaker A and B bands may be assigned to the c-g 𝐼𝐼 + g 𝐷 and c-g 𝐼 + g 𝐷 -conformers.

Anharmonic calculations show that the ν(CO) overtone is very close to the ν(NH) stretch and is slightly active. It might gain intensity through Fermi resonance with the ν(NH) stretch and be responsible for the observed shoulder. Lastly, the blue shift of the ν(NH) stretch in c-LD compared to c-LL is well reproduced by the calculations.

III-2 c. Vibrational Spectroscopy of the radical cation

The vibrational spectrum of the radical cation is obtained as a photo-dissociation action spectrum by monitoring the intensity of the fragment ion at m/z 154 (see Figure 7). It shows a single band readily assigned to the ν(OH) stretch at 3563 cm -1 and 3567 cm -1 for c-LL and c-LD, respectively. The ν(OH) stretch frequency is shifted down in energy by about 85 cm -1 compared to the neutral ground state, a value slightly smaller than that observed in phenol (124 cm -1 ), 46- 47 but close to that measured in the radical cation of cyclo Tyr-Tyr (3572 cm -1 ). [START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF] The cation is optimized after removing an electron from the three stable c-g 𝐼𝐼 + g -, c-g 𝐼𝐼 -g -, and c-t 𝐼𝐼 g -conformers. For c-g 𝐼𝐼 + g -and c-g 𝐼𝐼 -g -, the geometry is hardly modified relative to the neutral ground state. The only modification is a small change in the τ angle, from 66 to 69 ° and -60 to -69 ° for c-g 𝐼𝐼 + g -and c-g 𝐼𝐼 -g -, respectively. In contrast, that of c-t 𝐼𝐼 g -changes more, from -153 to -171°. This change in τ corresponds to a rotation of the aromatic ring and in turn optimization of the interaction between CO and the aromatic ring. Indeed, this interaction, which was repulsive in the neutral, is attractive in the ion due to the positive charge on the aromatic ring. This interaction results to additional stabilization of c-t 𝐼𝐼 g -, which is the most stable form in the ion, followed by almost isoenergetic c-g 𝐼𝐼 + g -and c-g 𝐼𝐼 -g -, ~3 kcal/mol higher in energy (see Table S2 of the ESI †). The ν(OH) stretch frequency is 3574 cm -1 for c-g 𝐼𝐼 + g -and 3589 cm -1 for c-g 𝐼𝐼 -g -or c-t 𝐼𝐼 g -. Although c-g 𝐼𝐼 + g -is higher in energy than c-t 𝐼𝐼 g -, its ν(OH) stretch frequency corresponds better to the measured value (3567 cm -1 ).

The same trends are observed for c-LD. c-t 𝐼𝐼 g 𝐷 -is the most stable form, c-g 𝐼𝐼 + g 𝐷 -and c-g 𝐼𝐼 -g 𝐷 being higher in energy by 5.4 and 6.4 kcal/mol. As observed in the neutral, the energy gap between the most stable conformer and the others is larger in c-LD than c-LL. The calculated ν(NH) and ν(OH) stretch frequencies are listed in Table S2 of the ESI †. All the conformers have their ν(OH) stretch frequency at 3585±1 cm -1 , close to the experimental value of 3563 cm -1 . Still, the agreement between calculated and experimental ν(OH) stretch frequencies is not as good as in the electronic ground state, probably because ionization vertical excitation reaches a high-energy region of the PES, with at least 1 eV excess energy after the calculations.

In all the calculated structures, the charge is mostly localized on the phenol ring. For example, the NBO charge on the phenol is 0.74 in c-g 𝐼𝐼 + g -. It is of the same order as observed in cyclo Tyr-Tyr (0.63), except that in cyclo Tyr-Tyr the charge is borne by the extended aromatic ring.

III-2 d. Simulation of The S0-S1 spectrum

The first electronic transition, S0→S1, has ππ * character, as indicated by the electron density difference between S0 and S1 drawn in Figure 8. The transition mostly involves HOMO →LUMO excitation (~70%), accompanied by minor contributions (less than 20%) of HOMO-3→LUMO+1 or HOMO-3→LUMO+2 excitations, as shown in Figure S5 and S6 of the ESI †.

The calculated S0→S1 transition energies and intensities are listed in Table 3. Figure 9 shows the comparison between the experimental electronic spectrum and that simulated from Franck Condon factor calculations. In contrast to the previously studied cyclo Phe-Phe, the inclusion of the Herzberg-Teller term is not necessary, because the electronic transition is dipole allowed.

The calculated origins are overestimated for all conformers; they are therefore shifted to match the experiment.

The lower-energy experimental origin observed for c-LL (A band at 35498 cm -1 ) is assigned to c-g 𝐼𝐼 -g -, which has the lowest calculated origin. All the calculated transitions are therefore shifted by 1204 cm -1 so that the g 𝐼𝐼 -g -origin is superimposed with the A band. The vibronic pattern starting from bands A and B are satisfactorily reproduced by the spectrum calculated for c-g 𝐼𝐼 -g -and c-g 𝐼 -g -, respectively. Moreover, the progressions starting from bands A and B, which correspond to an aromatic ring torsion at 34 cm -1 and the proline torsion at 64 cm -1 , have comparable shapes and intensities, as expected for a pair of conformers that only differ by the orientation of the phenol hydroxyl. They indicate a moderate change in geometry upon electronic excitation. In particular, the puckering modes of DKP and proline are not active. It is interesting to note that the results obtained here (lowest energy transition for the g - conformation of Tyr) parallel those of Shimozono et al. They reported the lower-energy transition for the so-called "B" conformer of Tyr, in which the phenyl is oriented towards the amino group. [START_REF] Shimozono | Revised conformational assignments and conformational evolution of tyrosine by laser desorption supersonic jet laser spectroscopy[END_REF] In contrast, c-g 𝐼 + g -shows extensive geometry change between S1 and S0, mainly due to a rotation of the phenol ring. For this reason, the FC calculations failed and only the stick bar showing the origin transition is indicated in Figure 9. Band E is assigned to a vibronic transition of the same conformer, as it does not appear in any of the other calculated spectra. Band C is tentatively assigned to c-g 𝐼 + g -based on its origin. It should be noted that bands C is followed by several weak bands, which could be due to the activity of low-frequency modes. The S1 geometry of c-g 𝐼𝐼 + g -is slightly different from that in S0. The bands starting from D and above can be tentatively assigned to this conformer based on its calculated origin and vibrational pattern. Lastly, c-t 𝐼𝐼 g -has the highest transition origin with a marked change in geometry between S1 and S0. This observation parallels the results of Shimozono et al., which indicate the highest energy transition for the so-called II conformer of Tyr, in which the phenyl is oriented towards the carboxyl group. [START_REF] Shimozono | Revised conformational assignments and conformational evolution of tyrosine by laser desorption supersonic jet laser spectroscopy[END_REF] The very weak bands at ~350-400 cm -1 above the A origin could be assigned to c-t 𝐼𝐼 g -. However, based on thermodynamics, it is more likely that these bands are due to weak vibronic bands of more stable conformers.

The spectrum of c-LD is simpler. The calculated origin of c-g 𝐼𝐼 -g 𝐷 -is shifted by 1062 cm -1 to reproduce the C band at 35710 cm -1 . The bands C at 35710 cm -1 and D at 35752 cm -1 are then assigned to the origin of c-g 𝐼 -g 𝐷 -and c-g 𝐼𝐼 -g 𝐷 -, respectively. The geometry of c-g 𝐼𝐼 -g 𝐷 -does not change much upon electronic excitation, except the orientation of the phenol ring around C5C1.

This torsion is responsible for the short vibrational progression calculated at 27 cm -1 . The origin transitions of c-g 𝐼 + g 𝐷 -and c-g 𝐼𝐼 + g 𝐷 -are red-shifted compared to c-g 𝐼 -g 𝐷 -and very close to each other. Due to the abrupt change in geometry between S0 and S1, only the origin transition can be computed. These results suggest that bands A at 35641 cm -1 and B at 35656 cm -1 are the origin transitions of c-g 𝐼 + g 𝐷 -and c-g 𝐼𝐼 + g 𝐷 -, respectively. Surprisingly, the bands assigned to c-g 𝐼 + g 𝐷 -and c-g 𝐼𝐼 + g 𝐷 -are very weak in the electronic spectrum, although these structures are calculated to be the most stable. This can be due to fast conformer-specific non-radiative processes. However, due to the closeness of the IR and UV spectra of c-g 𝐼 + g 𝐷 -and c-g 𝐼𝐼 + g 𝐷 -, and c-g 𝐼 -g 𝐷 -and c-g 𝐼𝐼 -g 𝐷 -, the assignment is only tentative.

IV. Conclusion

The two diastereomers of cyclo Tyr-Pro were studied under supersonic expansion conditions.

Despite the presence of two sterically constrained rings, the DKP and the proline, this system shows conformational flexibility. In particular, all the structures appear as pairs related to the phenol hydroxyl isomerism. Two main families are observed experimentally, involving either g + or g -orientation of the aromatic substituent, with proline always in a g + orientation. The cg + g -conformations in which the phenol ring is folded over the DKP ring are the most stable.

The observed structure is the same as detected in the solid. [START_REF] Grant | The structure and conformation of the tryptophanyl diketopiperazines cyclo(Trp-Trp)center dot C2H6SO and cyclo(Trp-Pro)[END_REF] The g + orientation is identical to that observed for the folded aromatic ring in the most stable form of the bichromophoric dipeptides cyclo Ty-Tyr or cyclo Phe-Phe. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] The c-g 𝐼𝐼 + g -/c-g 𝐼 + g -in c-LL are stabilized by a CβH…π interaction involving the CβH of proline. Due to the sterical constraints induced by the proline ring, the interaction is not very strong and the distance between CβH and the center of the ring is large (2.68 Å). These conformers are characterized by extensive change in geometry upon electronic excitation, as expected from the interaction between the aromatic chromophore and the rest of the molecule.

In c-LD, similar structures c-g 𝐼𝐼 + g 𝐷 -/c-g 𝐼 + g 𝐷 -are observed and are the most stable too. They differ from their c-LL counterpart by the nature and the strength of the stabilizing CH…π interaction, which involves CH…π in c-LD. The CH…π interaction is much stronger in c-LD than in c-LL (CH…π distance ~ 2.57 Å), because it is favored by the boat conformation of the DKP ring.

This may explain that, in contrast with previously studied DKP dipeptide, c-LD is more stable than c-LL. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] In contrast to c-LL, the c-g 𝐼𝐼 + g 𝐷 -/c-g 𝐼 + g 𝐷 -pair appears very weakly in the REMPI spectrum, despite its stability, due to the lower oscillator strength of the transition and possibly non-radiative processes.

The second observed family, c-g -g -, is an extended conformation in which the phenol slightly interacts with the NH group of the tyrosine amino acid. The g -orientation is identical to that observed for the extended aromatic ring in the most stable form of the bichromophoric dipeptides cyclo Ty-Tyr or cyclo Phe-Phe. [START_REF] Alata | Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase[END_REF][START_REF] Bennasr | Stereochemistry-dependent hydrogen bonds stabilise stacked conformations in jet-cooled cyclic dipeptides: (LD) vs. (LL) cyclo tyrosine-tyrosine[END_REF][START_REF] Perez-Mellor | Chirality effects in the structures of jetcooled bichromophoric dipeptides[END_REF] These conformers are observed in both c-LL and c-LD and their electronic spectrum is characterized by vibrational progressions built on the aromatic ring and proline torsions.

Lastly, the c-t g -conformation is too high in energy to be populated in our experimental conditions. In this structure, there is limited interaction between tyrosine and the rest of the molecule. However, it becomes the most stable in the radical cation.

An important finding of this work is that the IR spectroscopy alone would give ambiguous conclusions concerning the assignment. The preliminary assignment resting on IR spectroscopy can be confirmed only by simulating the electronic spectra. Simulation of Franck-Condon progressions has already proven to be very powerful for elucidating structures, for example for isolated protonated species either in jet-cooled conditions or in a cryogenic ion trap. [START_REF] Alata | Electronically excited states of protonated aromatic molecules: benzaldehyde[END_REF][START_REF] Feraud | Photofragmentation Spectroscopy of Cold Protonated Aromatic Amines in the Gas Phase[END_REF] Indeed, the structural differences between the two diastereomers are not very large and mainly consist in the nature of the CH…π interaction in the c-g + g -folded conformers and in the strength of the NH…π interaction in the extended c-g -g -conformers. However, the electronic spectra strongly differ from each other. Studied on protonated cyclo Tyr-Pro and its dimer are currently in progress.

  which is stronger in cyclo LPhe-DPhe or cyclo LTyr-DTyr than in their c-LL counterpart, is weaker in c-LL for cyclo Tyr-Pro. However, c-LL and c-LD show a different geometry of the DKP ring, which counterbalances the effects of NH… interactions. While the DKP ring is almost planar in c-LL, it adopts in c-LD a boat conformation that allows optimizing the CH… interaction. The stronger CH… interaction in c-LD manifests itself by a much shorter CH… distance (2.58 Å in c-LD vs. 2.67 Å in c-LL).

Figures 5 and

 5 Figures 5 and Figure6show conformer-specific vibrational spectra obtained in the region of the ν(NH) and ν(OH) stretches by setting the probe on the electronic transitions labelled A and C for c-LL and A and D for c-LD (see the electronic spectrum in Figure4). The other vibrational spectra are shown in FiguresS3 and S4of the ESI †. All the spectra display identical phenol ν(OH) stretch frequency (3650 cm -1 ), very close to that of tyrosine (3660 cm -1 ).[START_REF] Shimozono | Revised conformational assignments and conformational evolution of tyrosine by laser desorption supersonic jet laser spectroscopy[END_REF] c-LL shows an identical position of the ν(NH) stretching frequency, within the experimental error, for all the bands probed. However, the spectra display different behaviors in terms of relative intensity ν(NH)/ν(OH), which allow classifying them in two categories. Category I (bands A and B) is characterized by similar intensities for the ν(OH) and ν(NH) stretching modes, while ν(OH) is twice as intense as ν(NH) in category II (bands C, D and E). This suggests two families of conformers.

  Identical m/z 154 fragment is observed for both diastereomers. It corresponds to the cleavage of C1C5 (CC of Tyr) bond accompanied by proton transfer from the Tyr part to the peptide ring. It indicates charge migration from the initially ionized phenol to the peptide ring. This transfer must happen after IR absorption because the measured (OH) stretch frequency is characteristic of a phenol radical cation.

Figure 1 : 22 Figure 2 :

 1222 Figure 1: Structure of cyclo Tyr-Pro and atom numbering. The angular parameters defining the geometry are as follows: ϕ1 (C2 N C3 C4) ψ1 (N C3 C4 N) ω1 (C3 C4 N C1) ϕ2 (C4 N C1 C2) ψ2 (N C1 C2 N) ω2 (C1 C2 N C3) B (N C12 C13 C14) C (C12 C13 C14 C3) D (C13 C14 C3 N) E (C14 C3 N C12)

Figure 4 :

 4 Figure 4: one-color REMPI spectra of jet-cooled cyclo Tyr-Pro in the region of the 𝑆 1 (𝜋 * ) ← 𝑆 0 (𝜋 * ) transition of tyrosine for a) c-LL and b) c-LD. The bands labelled with a letter are those probed for recording the ion-dip IR spectrum. The vertical scale is in arbitrary units.

Figure 5 :

 5 Figure 5: Infrared spectra of c-LL in the region of the (NH) and (OH) stretching modes by probing a) band A and b) band C c), d) and e) simulated infrared spectra for the most stable TyrII-containing conformers. The stick bands are as follows: black-fundamentals bands, redovertones and green-combination bands.

Figure 6 :

 6 Figure 6: Infrared spectra of c-LD in the region of the (NH) and (OH) stretching modes by probing a) band A and b) band D c), d) and e) simulated infrared spectra for the most stable TyrII-containing conformers. The stick bands are as follows: black-fundamentals bands, redovertones and green-combination bands.

Figure 7 :

 7 Figure 7: Infrared spectrum of the ion of a) c-LL with the probe set on band B and b) c-LD with the probe set on band D, in the ν(OH) stretch region. The spectra are obtained by photodissociation action spectroscopy by monitoring the m/z 154 photo-fragment.

Figure 8 :

 8 Figure 8: Electron density difference between S1 and S0. The electron density isovalue is set at 0.004 a.u. Positive-blue and negative-red are the signs of the electron density difference.

Table 2

 2 Calculated scaled harmonic (*) and anharmonic () frequencies (cm -1 ) and intensities (km/mol) of the ν(NH) and ν(OH) stretching modes. The scaled factor for the harmonic frequencies is 0.952

	20

Table 3 Details

 3

of the S0→S1 and S0→D0 transitions. VT: Vertical transition. TDMI: Transition Dipole Moment Integral. AT: Adiabatic Transition. OT: Origin of the Transition calculated by correcting the AT by the difference in Zero-Point Energy (ZPE) between S0 and S1. RMSD: Root Mean Square Deviation between the geometry of ground state and the S1 or D0 geometry as appropriate.

Acknowledgments

The research described here has been supported by the ANR contract (ESBODYR ANR-14-CE06-0019-03). This work is supported by a public grant from the "Laboratoire d'Excellence Physics Atom Light Mater" (LabEx PALM) overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX-0039).

We acknowledge the use of the computing center MésoLUM of the LUMAT research federation (FR LUMAT 2764).

TOC Graphic

Supporting Information

Energetic and geometrical parameters of the most stable structures of cyclo Tyr-Pro, as well as the transition states separating them. Scaled harmonic frequencies of the ν(NH) and ν(OH) stretching modes of the radical cation of c-LL and c-LD. Infrared spectra of c-LL and c-LD in the region of the (NH) and (OH) stretching modes. Boat Axial


Ben Feriel

Nasr

Ivan Alata

Debora Scuderi

Valeria Lepère

Valerie Brenner

Nejm-Eddine Jaïdane

Anne Zehnacker
email: anne.zehnacker-rentien@u-psud.fr

Effects of complexation with sulfuric acid on the Photodissociation of Protonated Cinchona Alkaloids in the gas phase

The role of complexation with sulfuric acid on the photodissociation of protonated cinchona alkaloids, namely cinchonidine (Cd), quinine (Qn) and quinidine (Qd), is studied by combining laser spectroscopy with quantum chemical calculations. The protonated complexes are structurally characterized in a room-temperature ion trap by means of Infra-Red Multiple Photon dissociation (IRMPD) spectroscopy in the fingerprint and the (XH) (X=C, N, O) stretch regions.

Comparison with density functional theory calculations including dispersion (DFT-D) unambiguously shows that the complex consists of a doubly protonated cinchona alkaloid strongly bound to a bisulfate HSO4 -anion, which bridges the two protonated sites of the cinchona alkaloid.

UV excitation of the complex does not induce loss of specific photo fragments. Indeed the UVinduced fragmentation pattern is identical to that observed in collision-induced dissociation experiments. This contrasts with the photodissociation observed in the protonated monomer or dimer, for which photo-specific fragments were observed. Analysis of the nature of the first electronic transitions at the second order approximate coupled-cluster level (CC2) explains the difference in behavior of the complex relative to the monomer or dimer towards UV excitation.

Introduction

Exposure of biomolecules like proteins or DNA to UV light leads to structural damages, which in turn alter their biological functions, an exemplary case being mutagenic and carcinogenic effects caused by UV radiations on DNA. [1][2][3] These damages may be increased in the presence of sensitizing drugs. 4 Understanding the protection mechanisms of biomolecules against UV-induced damages has prompted numerous studies, both in the biomolecules themselves and in model systems. 5 For example, the photochemistry and photophysics of individual DNA bases have been intensively studied in solution. [6][7][8][9][10] The solvent, water in a biological context, plays an important role in the deactivation process, by stabilizing polar or charged species, modifying the excited-state energetics, or acting as a thermal bath. Gas-phase experiments are therefore required to get information on the molecule properties without the participation of the solvent. They have been conducted on small biomolecules such as DNA bases [11][12][13] and peptides and allow getting information on the photophysical processes at play in the chromophore. 8,[14][15][16][17][18][19][20][21][START_REF]Gas-Phase IR Spectroscopy and Structure of Biological Molecules[END_REF] Many studies have been devoted to the electronic excited states of protonated biomolecules isolated in the gas phase, either under supersonic expansion conditions, [START_REF] Alata | [END_REF]24 in room-temperature ion traps, 15,[25][26][27][28][29][30][31] and more recently in cryogenic ion traps. [32][33][34][35][36][37][38][39][40] Of special interest are the aza-aromatic molecules due to their complex photophysical behavior related to several close-lying electronic transitions. 29,30,37,41,42 We have recently studied aza-aromatic molecules, namely, cinchona alkaloids. Cinchona alkaloids, among which quinine is the most famous, originate from the bark of Cinchona trees and are widely used in chemistry or medicine. [43][44][START_REF] Song | Cinchona Alkaloids in Synthesis and Catalysis[END_REF][START_REF] Burgi | [END_REF][47][48] However, photosensitizing effects have been observed in the treated patients. 49 This justifies the studies of photophysical and photochemical processes in quinine, in its free base or sulfate forms.

The cinchona alkaloids, quinine (Qn), quinidine (Qd), cinchonidine (Cd) and cinchonine (Cn) are displayed in Figure 1. They consist of an aliphatic bicyclic tertiary amine, the alkaloid part, which is substituted by a vinyl group at C3 and linked to a planar aromatic quinoline ring via an OH-containing flexible linker C8C9. These natural molecules are classified into pairs, i.e. Qd/Qn and Cd/Cn, which only differ by the chirality of C8 and C9. Qd/Qn differ from Cd/Cn by the presence of a methoxy substitutent on the aromatic ring. Cinchona alkaloids contain two basic nitrogens (Nalk and Narom in Figure 1). The alkaloid nitrogen Nalk is the most basic site and is protonated in our experimental conditions. When not specified otherwise, CdH + denotes cinchonidine protonated on Nalk. Protonation on Narom will be denoted by CdNaromH + . Doubly protonated cinchonidine (noted CdH2 2+ in what follows) is formed in acidic solution (pH below <~4-5). Cinchona alkaloids possess different transitions in the near UV region. The ππ* transition localized on the aromatic ring is optically active while the nπ* transitions involving the quinoline or the alkaloid nitrogen lone pair have much weaker oscillator strength. 50,51 Like for other aza-aromatic molecules, 30 protonation removes the nπ* excited state involving the alkaloid nitrogen lone pair from the transition manifold. This results in high fluorescence quantum yield. 52 We have previously reported that protonated cinchona alkaloids show complex photochemical properties in the gas phase, using MS 3 experiments involving IR characterization of the UV fragments. [53][54][55][56][57] Based on IR spectra of the photofragments, combined with CC2 calculations, we have proposed that UV-induced C8C9 bond cleavage happens through conformation-dependent reactions involving coupled electron and proton transfers. 53 The mechanism is modified in the protonated dimer, for which a photo-induced proton transfer, followed by dissociation of the complex, results to a metastable molecule protonated on the aromatic nitrogen, CdNaromH + . 54,57,58 We extend here these studies to the complexes of cinchona alkaloids with sulfuric acid. Indeed, drugs based on cinchona alkaloids are used under their sulfate form and the abovementioned complexes are spontaneously formed in the electrospray. The complexes are studied by multistage mass spectrometry (MS n ) coupled with laser spectroscopy. We first resort to Infra-Red Multiple Photon Dissociation (IRMPD) spectroscopy, combined with quantum chemical calculations, to determine the structure of the protonated complexes of cinchona alkaloids with sulfuric acid. H2SO4 is a strong di-acid; in water at neutral pH, the most abundant species is SO4 2- and the concentration in H2SO4 is negligible. In contrast, H2SO4 is stable in the gas phase 59,60 while isolated HSO4 -has never been observed. Rare gas matrices are an intermediate case in which H2SO4 is observed when isolated or included in a 1:1 complex with NH3. 61 However, for more basic environments, proton transfer takes place and an ion pair containing HSO4 -is formed.

This study first aims at defining which forms of Cd, CdH + or CdH2 2+ , and sulfuric acid, H2SO4, HSO4 -or SO4 2-, are contained in the protonated complex. We can exclude SO4 2-because there are only two stable protonation sites in the Cd molecule. However, should we describe the complex in terms of CdH + :H2SO4 or CdH2 2+ :HSO4 -? Furthermore, UV irradiation only results to dissociation of the complex into protonated monomers, in contrast to protonated monomers and dimers that yield specific fragments under UV excitation. The second goal of this study is then to explain the absence of specific photofragments of the complex, in terms of structure and nature of the electronic excited states calculated at the CC2 level. The present study focuses first on Cd, and is extended to Qn and Qd, which have a methoxy substitutent on the aromatic ring. Moreover, Qn and Qd differ from each other by the chirality of C8 and C9, which is 8S/9R in Qn, as it is in Cd, and 8R/9S in Qd. We will assess whether the structure of the complexes or their photodissociation mechanism is affected by this change in absolute configuration.

Experimental and theoretical methods: a) Experimental method:

The experimental set-up has been described in details elsewhere. 53 The multistage mass spectrometry (MS n ) experiments were based on a modified Paul ion trap (Bruker, Esquire 3000+). 62 Ions were generated by electrospraying a 10 μM solution of the alkaloids or their sulfate in a 50:50 water/methanol mixture, adding 2 μl of 98% formic acid to assist the protonation. The electrospray ionization (ESI) conditions were those already described for these systems. 57,63 The alkaloids under study as well as their sulfates were obtained from Sigma-Aldrich and were used without further purification.

Collision-induced dissociation (CID) spectra were obtained in a MS 2 experiment by mass selection of the parent in a ±5 Da window. The parent was fragmented in the Paul ion trap by collision with He, applying a radiofrequency (RF) voltage of variable amplitude during 200 ms.

The IRMPD spectra were recorded resorting to a MS 2 scheme involving IR irradiation in the above-mentioned Paul ion trap, equipped with a diamond window. The spectra were recorded in the 800-1800 cm -1 range using the infrared free electron laser (FEL) at the "Centre Laser Infrarouge d'Orsay CLIO". 64 The spectral bandwidth was typically 1% of the central wavelength and the power of the order of 0.8W. The 2800-4000 cm -1 range was covered by a tabletop IR Optical Parametric Oscillator/Amplifier (OPO/OPA) (LaserVision 10 Hz repetition rate, pulses duration 4-6 ns, spectral bandwidth 3 cm -1 , IR power of ~ 200 mW). The IR laser was focused on the center of the trap by a parabolic Oxygen-Free High-Thermal Conductivity (OFHC) copper mirror with focal length of 350 mm. A synchronized CO2 laser (Universal Laser system 10 W at cw operation, centered at λ = 10.6 μm) was co-focused with the FEL or OPO source to enhance the fragmentation yield.

The fourth harmonic (266 nm) of a 10 Hz Nd-YAG laser (Minilite Continuum 500 µJ/pulse, pulse duration 5 ns) was used to electronically excite the systems, hence inducing their photodissociation. The UV laser was slightly focused on the center of the trap by a 1000 mm focal length quartz lens. The protonated complex built from cinchonidine and sulfuric acid (m/z 324) was isolated in a 5 Da window and fragmented by irradiation at 266 nm during 1 s. The photo-fragments were isolated and characterized by IRMPD based on a MS 3 scheme reported previously. 53,54,58 b) Theoretical methods:

The potential energy surface (PES) of the complexes of cinchonidine was first explored with a force field using the Maestro program, a part of the Schrödinger package. [START_REF]MacroModel version 9.8[END_REF] Exploration with two force fields, MMFFs and OPLS2003, generated similar structures. 66 67 These force fields were already used successfully for exploring the potential-energy surface of neutral or protonated quinine as well as its protonated dimer. 57,68 The structures with energy below 21 kJ/mol relative to the most stable one were fully optimized within the frame of the density functional theory (DFT) including empirical dispersion (DFT-D3), 69,70 at the B3LYP-D3/6-31++G(d,p) level [71][72][73] and the harmonic frequencies were calculated at the same level of theory for structures which correspond to true minima. This level is known to yield frequencies in good agreement with experimental values. 70,72,74,75 The "tight" criterion and fine grid were used so that the convergence threshold was set to 6 10 -5 and 1.5 10 -5 au for the maximum displacements and forces, respectively. In order to take into account the anharmonicity, the harmonic frequencies were corrected by mode-dependent scaling factors.

These scaling factors were determined as the average of the ratio of the experimental frequencies to the calculated values of the studied molecules as described in section c).

Basis-set superposition error (BSSE) effects on both the geometry and energetics were taken into account. The geometry optimization was performed considering the BSSE term and the energetic was calculated using the counterpoise method. 76 In what follows, the stability of the complexes is given in terms of Gibbs energy ΔG relative to the most stable structure taken as the zero of the scale. The BSSE corrections does not amount to more of 5% of the relative Gibbs energy and hardly modify the geometry. For example, the NalkH + …O -distance and NalkH + …O -angle are modified by less than 0.1 % in the most stable form of CdH2 2+ :HSO4 -(vide infra). The difference is slightly larger for the CdH + :H2SO4 -complexes, 0.3% and 1% for the most stable forms. Furthermore, BSSE corrections hardly modify the calculated harmonic frequencies of the most stable form of CdH2 2+ :HSO4 -. The BSSE-corrected frequencies differ only by less than 0.1% from the non-BSSE-corrected ones in the ν(XH) range, a discrepancy smaller than the experimental resolution (4 cm -1 ). The discrepancy is slightly larger in the fingerprint region, but remains smaller than 0.3%, except for the β(NalkH + ) mode (0.6%). The intermolecular modes are more affected (1 to 3%). The most modified mode is the hindered rotation of HSO4 -(cogwheel motion) for which the discrepancy between the two calculated frequencies is 5%.

Quinine and quinidine differ from cinchonidine by the presence of a methoxy OCH3 substituent in the aromatic ring. This leads to additional conformational flexibility, with two classes of conformers corresponding to the OCH3 in cis or trans position, as observed in 2methoxynaphthalene. 77 The complexes containing quinine and quinidine were then calculated using those containing cinchonidine as starting points. The chirality of the chiral centers was modified when necessary and the methoxy group was added, in the two possible positions.

The calculations were done using the Gaussian 09 package. 78 Obtained conformers structures were drawn with the Gabedit software. 79 Assignment was based on spectroscopic criteria (good match between calculated and experimental frequencies) and thermodynamics, favoring the most stable structures.

The electronic excited states of the protonated monomers and complexes were calculated at the CC2 level of theory. 80 The calculations were performed using the resolution-of-the-identity (RI) approximation 81 and the correlation-consistent polarized valence double-ζ (cc-pVDZ) basis set. 82 The ground state of the most stable structure assigned to the experiment was calculated at the RI-CC2 level, keeping the DFT-D geometry. Vertical excitation energies were then calculated at the RI-CC2 level considering six excited states. 22 core orbitals were frozen for the protonated cinchonidine monomers and 31 for the complex. The CC2 convergence was set at 10 -7 . This method has been used on numerous protonated organic molecules and satisfactorily reproduces the S10 transition energy. 83 All the calculations were done using the Turbomole V6.6 program package. 84 The difference in density between the ground state and the electronic excited states were calculated using the density analysis of Turbomole and plotted using the VMD software. 85

c) Isolated systems

The vibrational spectroscopy of the molecular sub-units is summarized here. Comparison between the experimental vibrational frequencies determined previously or in this work and those calculated here are used for determining the scaling factors used.

H2SO4 and HSO4 -:

The vibrational spectroscopy of H2SO4 is well known in the gas phase 59,60 and in low-temperature matrices. 86 In the 3 μm region, the infra-red spectrum of H2SO4 vapours shows an intense band at 3609.2 cm -1 assigned to the ν(OH) stretch. 59 The scaling factor in this range is determined as the ratio between experimental and calculated (3755 cm -1 ) frequencies of the H2SO4 ν(OH) stretch, it amounts to 0,961. This scaling factor is of the same order as those defined for the B3LYP functional associated with other Pople basis sets. 87 The experimental spectrum of H2SO4 in the fingerprint region (800-1800 cm -1 ) is dominated by two intense features at 1220.1 and 1464.7 cm -1 assigned to the ν(SO2) symmetric and antisymmetric stretches, respectively. The band located at 1157.1 cm -1 is assigned to the β(SOH) asymmetric bend while that at 834.1 and 891.4 cm -1 are attributed to the S(OH)2 symmetric and asymmetric stretches. As we shall see later, the vibrational modes of the complexes involve stretching and bending motions extensively delocalized on the OH and CO groups. For this reasons, we have treated the stretching and bending modes on the same footing. The scaling factor in the fingerprint region is determined as the average ratio between experimental and calculated frequencies of the β(SO2) and β(OH) bends, and νS(OH)2 and ν(SO2) stretches. The obtained scaling factor is 1,060. This value reproduces well the experimental IR spectra of H2SO4 in the low frequencies region and scaling factors >1 are generally used for SO containing species. [88][89][90] Less is known on the infrared spectroscopy of the bisulfate anion HSO4 -, which has been studied mostly in the condensed phase, for example in sulfuric acid solutions 91,92 or thin films, 93 or in strongly bound complexes under cryogenic matrix 61 or gas-phase conditions. 90 Due to the absence of experimental data in isolated conditions, we will use the same scaling factors as for H2SO4.

CdH + , QnH + and QdH + : The conformational landscape of neutral cinchona alkaloids has been extensively studied by Bürgi et al. and Caner et al. [94][95][96] The same nomenclature as for the neutral systems is used for all protonated states, 97 as described previously. 53 The details of the nomenclature are given in the electronic supplementary information (ESI †). Briefly, the structures can be separated in two classes: in the "Closed" structures, the alkaloid is folded over the aromatic ring while in the "Open" structures, it is extended outwards.

The vibrational spectroscopy of gas-phase Cd is not known. However, Qn and Qd have been studied previously under jet-cooled conditions by IR-UV double resonance spectroscopy. 68 The experimental frequency of 3650 cm -1 observed in both molecules, a typical free ν(OH) stretch value, has been assigned to the ν(OH) stretch of the most stable γ-open conformer for which the calculated value is 3821 cm -1 , which leads to a scaling factor of 0.955. The experimental ν(OH) frequency does not change much upon protonation on the alkaloid nitrogen; it is measured at ~3650 cm -1 in CdH + , QnH + , or QdH + by IRMPD spectroscopy. 56 It does not change substantially with the protonation on the aromatic nitrogen and appears at the same value in CdNaromH + . The average scaling factor of the ν(OH) stretch of the protonated species is then taken equal to 0.955, the same value as determined for neutral Qn under supersonic-jet conditions.

The ν(NalkH + ) stretch frequency is sensitive to conformation and hydrogen bond formation. 56 The cinchonidine protonated monomers and dimers (ν(NalkH + ) stretch frequency ranging from 3209 to 3300 cm -1 ) were used to determine the average scaling factor, which amounts to 0.970. 54 The ν(NaromH + ) stretch is experimentally observed at 3470 cm -1 , in the monomer and the dimer. The calculated value does not depend on the conformer and yields a scaling factor of 0.971. We can anticipate from what is known on the isolated systems that free ν(OH) stretches should appear around 3650 cm -1 whatever the protonation state of the molecule. For protonation on Nalk, ν(NalkH + ) is expected at 3280 cm -1 for a free NalkH + and strongly shifted down in energy when it is involved in an intermolecular hydrogen bond. 56 Finally, for protonation on Narom, ν(NaromH + ) is expected around 3470 cm -1 range and is not sensitive to the environment. 58 The standard deviation between calculated and experimental frequencies of the reference structures allow us to assume an error of ~20 cm -1 on the calculated frequencies.

Results: 1. Structural studies

Experimental IRMPD spectra

The IRMPD spectra obtained by electro-spraying a solution of cinchonidine sulfate are shown in Figure 3 and Figure 5. The 3 μm region shows two narrow bands in the region of the free ν(OH).

The presence of two bands in this region allows ruling out the presence of SO4 2-; one of these bands belongs to CdH + and the other one to H2SO4 or HSO4 -. The band at 3652 cm -1 is typical of the free ν(OH) of Cd but is not informative about the protonation site of Cd. The intense band at 3621 cm -1 appears in the range of the free ν(OH) of H2SO4 or HSO4 -, but does not allow deducing whether the system contains H2SO4 or HSO4 -. Finally, a broad band appears at 3396 cm -1 . This range corresponds either to ν(Narom H + ) or to intermolecular hydrogen-bonded ν(OH). We will therefore consider the two species H2SO4 and HSO4 -and calculate both CdH + :H2SO4 and CdH2 2+ :HSO4 - complexes. The fingerprint region is dominated by an intense band centered at 1040 cm -1 followed by congested features spreading up to 1400 cm -1 .

Calculated structure of the CdH + :H2SO4 complex

The most stable structures of the complexes are presented in Figure 2. The most important structural parameters are listed in Table 1 and the relative energies in Table 3.

All the calculated conformations are strongly bound by an intermolecular NH +q … -q' O interaction that links NalkH + and the SO group of sulfuric acid (d=1.84 to 1.88 Å). NBO analysis of the electron density and orbital populations is reported in the ESI † and shows that this interaction is mostly electrostatic in nature. It manifests itself by a substantial red shift of the ν(NalkH + ) stretch frequency at 3291 cm -1 , while the ν(OH) frequencies remain unchanged relative to the bare molecules, reflecting the free nature of the H2SO4 hydroxyls.

In Anti-γ-open', one of the OH groups of H2SO4 interacts with the aromatic ring, more precisely the C6 ring without nitrogen, denoted as aromC in what follows (OH…aromC interaction).

The OH…X distance, where X is the center of the aromC ring, is 3.30 Å. Due to this interaction, the ν(OH) frequency of the interacting OH group of H2SO4 is shifted down in energy to 3429 cm -1 .

The complexes with the Closed conformers are more compact, with shorter distances between H2SO4 and the aromatic ring. Anti-γ-closed and Anti-γ-closed' exhibit an OH…πaromC interaction, with a OH…X distance of 2.58 Å, much shorter than in Anti-γ-open'. In Anti-γ-closed', a strong OH…Narom hydrogen bond bridges H2SO4 and the aromatic nitrogen (d=1.58 Å). This interaction results to a strong down shift of the H2SO4 ν(OH) frequency, down to 2343 cm -1 .

The calculated spectra of the CdH + :H2SO4 structures are shown in Figure 3. The free ν(OH) stretches localized on CdH + and H2SO4 are calculated in the 3550-3650 cm -1 range for all the structures. When one hydroxyl group of H2SO4 interacts with the aromatic π electrons, like in Antiγ-open', its ν(OH) frequency is shifted down to the 3400-3450 cm -1 range. When one hydroxyl group of H2SO4 interacts with Narom, its ν(OH) frequency is shifted further down to ~ 3300 cm -1 in Anti-γ-closed' and 2343 cm -1 in Anti--closed, which is out of our experimental range and is not presented in Figure 3. The ν(NalkH + ) stretch is calculated in the 3170-3270 cm -1 range in all the CdH + :H2SO4 complexes indicating that it is involved in a strong interaction.

In the fingerprint region, the two β(OH) bending modes of H2SO4 are calculated in the 1150-1200 and 1250-1300 cm -1 range. They are the most intense calculated transitions, together with the ν(S-OH) stretch at ~900 cm -1 and the two ν(SO) stretches in the 1300-1450 cm -1 region.

Calculated structure of the CdH2 2+ :HSO4 -complex

The most stable structures of the CdH2 2+ :HSO4 -complex are presented in Figure 4. The structural parameters are listed in Table 2 and the energies in Table 3. Two of these complexes contain the Open form of CdH2 2+ and three the Closed form. They all involve strong electrostatic interactions between the bisulfate anion and the protonated nitrogen atoms. These interactions are mostly electrostatic, like that described above. The electrostatic interactions between two charged species being stronger than that between a charged species and a neutral entity. All the CdH2 2+ :HSO4 -complexes are then much more stable than the corresponding CdH + :H2SO4 structures, the energy difference between them being 7 kcal/mol or more.

The structures containing Open and Closed conformations of CdH2 2+ strongly differ from each other. When CdH2 2+ is Open, stabilization mostly arises from the NalkH +q … -q' O interaction. This ionic interaction is strong, as it can be assessed from the short (1. The complexes with the Closed conformers involve an additional interaction between O -q' of HSO4 -and the NaromH + group of CdH2 2+ , which allows HSO4 -to bridge the two protonated nitrogen atoms. This interaction is not as strong as NalkH +q …O -q' , because of the larger (> 2.3 Å) distance between the two sites. Again, it is purely electrostatic. The geometry of the complex results from a compromise between these two interactions, but that involving the NalkH + group is dominant and dictates the NalkH +q …O -q' distance, quasi identical (1.64 to 1.60 Å) to that calculated for the Open complexes (1.58 to 1.57 Å). The NaromH +q …O -q' interaction stabilizes the complexes containing Closed forms by more than 2 kcal/mol relative to those with Open forms.

The spectra calculated for the Cd2H 2+ : HSO4 -structures are shown in Figure 5. The ν(OH) stretches localized on Cd2H 2+ and HSO4 -are calculated in the 3580-3680 cm -1 range, as expected for free OH, for all the structures, except Anti-β-open in which the Cd2H 2+ ν(OH) is shifted down to 3380 cm -1 because the OH group acts as an hydrogen bond donor to an oxygen of HSO4 -. The spectrum of Syn-γ-open stands out by the presence of an intense transition at 3106 cm -1 due to the aromatic ν(C3'H) stretching mode gaining intensity through the interaction with the HSO4 -ion. The calculated spectrum of all the complexes shows an intense ν(NalkH + ) stretch in the 2570 -2700 cm -1 region, which is out of our experimental range and not shown in Figure 5. 56 The calculated ν(NaromH + ) does not vary much with the structure and is calculated at ~ 3465-3470 cm -1 for the complexes with both Open and Closed structures. It is only shifted down by a few wavenumbers (<5 cm -1 ) in the bridged complexes with the Closed forms, with a slightly lower value in Syn-γ-closed' than Anti-γ-closed, in line with the more compact structure of the former.

Most of the peaks calculated in the fingerprint region correspond to vibrations localized on HSO4 -. Syn-γ-closed', Anti-γ-closed and Anti-β-open have vibrations at 1162, 1246 and 1295 cm -1 that are described in terms of the β(OH) bending motion of HSO4 -coupled with ν(SO) stretches.

Syn-γ-open and Syn-γ-closed have several vibrations between 1170 and 1210 cm -1 that correspond to the β(OH) bending motion of HSO4 -coupled with β(CaliphaticH) and β(CaromaticH) bending motions.

Assignment of the observed complex

Figure 3 compares the experiment to the spectra simulated for CdH + :H2SO4. The calculated spectrum of CdH + :H2SO4 poorly reproduces the experimental data in the high frequencies range as well as in the fingerprint region. In particular, no band is observed in the range of the calculated bonded ν(NalkH + ) (3170-3300 cm -1 range). In the fingerprint region, no strong band is observed above 1350 cm -1 where the two ν(SO) stretches are expected. This result allows ruling out that the observed complex is CdH + :H2SO4.

Figures 5 compares the experiment to the spectra simulated for CdH2 2+ :HSO4 -. In the high frequency region, the band around 3650 cm -1 is assigned to a free ν(OH) stretch of CdH2 2+ . This allows ruling Anti-β-open for the attribution. The strong feature at 3621 cm -1 is attributed to ν(OH) of HSO4 -and is only slightly lower in frequency than the free v(OH), estimated at 3650 cm -1 in HSO4 -. The broader feature around 3400 cm -1 is attributed to the ν(NaromH + ) stretch. Finally, no intense band is experimentally observed in the 3000-3200 cm -1 region corresponding to the ν(CH) stretching modes. Thus, the observed complex cannot be Syn-γ-open. Based on the 3μm region, we can safely assign the observed complex to one of the calculated structures containing a Closed conformer, which all reproduce reasonably well the experimental spectrum. The experimental spectrum obtained in the fingerprint region is also satisfactorily reproduced by those calculated for the Closed structures and most of the observed peaks are assigned to transitions localized on HSO4 -. Moreover, the energetic values listed in Table 3 indicate that the Closed structures are by far more stable than the complexes involving neutral H2SO4, and more stable by several kcal/mol than the Open conformers. Anti-γ-closed, Syn-γ-closed' and Syn-γ-closed are almost iso-energetic within the error. Among them, Syn-γ-closed' displays the best match with the experimental spectrum both in the 3 m and in the fingerprint region. We therefore assume that the observed spectrum is mainly due to Syn-γ-closed', without excluding a contribution of the two other forms.

Extension to quinine and quinindine

The geometries obtained for QnH2 2+ :HSO4 -are shown in Figure 6 and Figure 7. The dihedral angles of the calculated complexes are listed in Table 4 and Table 5 and their energetics in Table 6. The structures obtained for QdH2 2+ :HSO4 -are very similar and are shown in Figure S1.

The energetics and the structural parameters are listed in Tables S1 and Table S2 of the ESI †.

The calculated complexes are similar for the cis and trans structures and resemble those obtained for CdH2 2+ :HSO4 -. The presence of the OCH3 substituent slightly modifies the energetic order between the different geometries and complexes with Open forms become energetically competitive with the complexes with Closed forms. The complexes built from the Closed cis conformers resemble those obtained for CdH2 2+ :HSO4 -, with HSO4 -bridging the two protonated sites of the molecule and showing no interaction with the OH or the OCH3 groups of Qn. Cis and Trans syn-γ-Open show no difference relative to the CdH2 2+ :HSO4 -system. In contrast, the Cis-antiβ-Open structure slightly differs from its counterpart in CdH2 2+ :HSO4 -because HSO4 -interacts not only with the NalkH + group but also with the OCH3 group (distance of 2 Å) and the OH group of QnH2 2+ . These additional hydrogen bonds contribute to the stability to the complex. In Trans-synβ-open, only the additional interaction between HSO4 -and OH is observed.

The IR spectra of all these complexes, the 3μ region and the fingerprint region, are presented in Figures 8 and9. In the 3 μm region, the spectra of the cis/trans QnH2 2+ :HSO4 -complexes parallel those of the CdH2 2+ :HSO4 -complex. In the fingerprint region, the calculated spectra of cis/trans QnH2 2+ :HSO4 -have some transitions in common with CdH2 2+ :HSO4 -. Most of the differences between QnH2 2+ :HSO4 -and CdH2 2+ :HSO4 -are due to modes localized on the methoxy substituent.

The ν(CO) stretch of the OCH3 is calculated between ~ 1110 cm -1 and ~ 1220 cm -1 , with a weak intensity. It is higher in energy and has larger intensity in the trans forms. No particularly intense band stands out in the cis conformers. A very intense β(CaromH) bend is calculated at ~ 1300 cm -1 -1330 cm -1 for all the trans conformers of QnH2 2+ , becoming the most intense in the fingerprint region. The absence of intense transition in the ~ 1400 cm -1 region of the experimental spectrum and the presence of bands above 1650 cm -1 allows discarding the trans conformers.

The results obtained in the high-frequency region parallel those obtained for cinchonidine.

The spectrum of the Closed cis geometries shows better correspondence with the experimental data than those of the Open cis geometries. Moreover, the cis conformers are more stable than the trans.

Cis-Syn-γ-closed, Cis-Syn-γ-closed' and Cis-Anti-γ-closed show good correspondence with the experiment. It is therefore likely that the observed spectrum contains contributions of these three cis Closed forms, whose relative Gibbs energy (1.0-1.5 kcal/mol) is of the order of the expected calculation error. The preference for cis forms results parallel those observed for similar systems like neutral 2-methoxynaphthalene 77 , Naproxene, 98,99 or neutral quinine and hydroquinine. 68,97 No difference is experimentally observed between the complexes Qn and Qd, as it was already the case for the protonated monomers. 56 Qn and Qd therefore behave like true enantiomers in this context.

Photodissociation

Experimental results

The mass spectrum resulting from the photodissociation of CdH2 + HSO4 -at 266 nm is shown in Figure 10, as well as its CID spectrum. The conditions (collision voltage or UV power) have been adjusted so that the total fragmentation yield is the same for the two spectra, namely, 0.33, which allows better comparison between them. The UVPD spectrum is noisier because light scattered from the laser may create stray ions, but the two spectra show identical fragmentation pattern, with the major fragment being the protonated monomer. In contrast with CdH + itself, the CdH2 2+ :HSO4 -complex (m/z 393) does not show any specific UVPD photo-fragment, and the same behavior is observed for Qn or Qd. Figure 10 also shows the CID spectrum of CdH + (m/z 295) produced by UV irradiation of the complex. The fragmentation pattern is the same as observed for CdH + formed in the ESI, with the main channels being water loss (m/z 277) or loss of water and methylamine (m/z 246). The fragmentation efficiency for a given radiofrequency amplitude is of the same order as that of CdH + formed in the ESI. 54

Excited states calculations

Vertical excitation energies and nature of the first three excited states: The energies of the first three vertical electronic transitions of Syn-γ-closed' are listed in Table 7. We have limited the table to the transitions below the energy of the UV laser (4.66 eV). Table 7 also includes the doubly protonated monomer CdH2 2+ for the sake of comparison. However, this doubly charged species is not stable in the gas phase and was not observed in our experimental conditions. Therefore, its PES was not fully explored. The structure of isolated CdH2 2+ presented in the table is a local minimum obtained by optimizing the geometry of CdH2 2+ , starting from the geometry contained in the CdH2 2+ :HSO4 -Syn-γ-closed' complex. Finally, the table includes the results for the corresponding monomers protonated on Nalk or CdNaromH + , the corresponding monomer protonated on Narom, in the same geometry as contained in the complex. In short, the systems will be denoted by CdH2 2+ :HSO4 -, CdH2 2+ , CdH + , and CdNaromH + in what follows. Figure 11 shows the difference between the electronic density of the ground state and that of the first three excited states of CdH + , CdNaromH + , CdH2 2+ and CdH2 2+ :HSO4 -. In the discussion that follows, the canonical occupied HF orbitals delocalized all over the two aromatic rings will be denoted by arom and the unoccupied one by *arom, while the occupied orbitals localized on the aza C6 ring or carbonaceous C6 ring will be denoted by aromN or aromC, respectively. Similarly, the unoccupied ones will be denoted by *aromN or *aromC respectively. The Nakl and Narom lone pairs will be denoted by nNalk and nNarom, respectively. Finally, the canonical HF orbitals localized on the alkaloid C=C group will be denoted by CC or *CC, respectively.

The three lower-energy transitions of CdH + involve aromatic π electrons as well as the lone pair located on Narom, as suggested for quinoline (Figure 11a). 100 The S10 transition mainly involves HOMO-4 (mostly Narom lone pair) → LUMO (π*arom) (68%) and HOMO-3 (mixed Narom lone pair and πarom) → LUMO (24 %) excitations. It is therefore an intramolecular charge transfer (CT) within the aromatic ring, i.e. a narom*arom CT transition. The S20 and S30 electronic transitions are ππ* transitions localized on the aromatic ring. They involve the very same orbitals HOMO-1(πarom)→ LUMO (64%) and HOMO (πarom) → LUMO+1 (π*arom) (30%) for S20 and HOMO-1 →LUMO (85%) for S30 as the so-called Lb and La electronic transitions of naphthalene derivatives. 101 102 68 In CdNaromH + , (Figure 11b), the S10 transition mainly involves HOMO (mostly nNalk) → LUMO (mostly π*arom) (40%) and HOMO-1 (nNalk and πaromC) → LUMO (37 %) excitations. The S10 transition is therefore mainly an intramolecular charge transfer from nNalk to π*arom, i.e. a nalk*arom CT transition. The S20 transition is mainly a naphthalene-like πaromπ*arom transition that involves HOMO-2 (πarom) → LUMO (54%) and HOMO-1 → LUMO (31%) excitations. Finally, the S30 transition involves HOMO-1 → LUMO+1 (π*arom) (44%) and HOMO → LUMO+1 (35%) excitations, it is mainly a naphthalene-like πaromπ*arom transition. For CdH2 2+ , (Figure 11c), both nitrogen atoms are protonated and there is no possibility for CT nπ* transitions involving a nitrogen lone pair. The S10 and S20 transitions are of ππ* character. S10 corresponds to the HOMO-1 (πaromC) → LUMO (π*arom) (94%) excitation, while S20 corresponds to HOMO-2 (πarom) → LUMO (72%) and HOMO-1→LUMO+1 (πarom) (26%), respectively. Interestingly, the S30 transition corresponds to an intramolecular charge-transfer from the alkaloid C=C to the aromatic ring. Indeed, it is a HOMO → LUMO transition (95%) with the HOMO orbital being the π C=C orbital of the C=C double bond.

In the CdH2 2+ :HSO4 -complex also, both nitrogen atoms of CdH2 2+ are protonated and there is no possibility for intramolecular CT involving a nitrogen lone pair (Figure 11d). The presence of HSO4 -completely modifies the nature of the S10 transition compared to CdH2 2+ . At equilibrium distance, the charge transfer transition involving the C=C orbital is out of the manifold of the transitions reached by the UV laser. The C=C orbital corresponds to the HOMO and is involved in none of the first five electronic transitions, and only marginally in S60, which is higher in energy (5.23 eV) than the laser energy. S10 now corresponds to HOMO-3 → LUMO excitation (93%), with the HOMO-3 being mainly described in terms of combination of the lone pairs nO of the oxygen atoms of the three S=O groups of HSO4 -, and the LUMO being a *aromN orbital. The S10 transition can therefore be described in terms of a nO*aromN intermolecular CT from the bisulfate to CdH2 2+ . The S20 and S30 transitions correspond to HOMO-1(aromC) → LUMO (81%) and to HOMO-2 (arom) → LUMO, (61%), respectively. They are of ππ* nature.

Modification of the electronic transitions when elongating the NH bonds:

Based on calculations of the excited states at the same level as in this work, it was proposed previously that the photoreactivity of the closed forms of CdH + arises from proton transfer from Nalk to Narom. The CdNaromH + formed thereby undergoes electron transfer from the C=C group of the alkaloid to Narom, resulting to the formation of specific photofragments. 53 This was confirmed by the experimental finding that in a similar system, hydroquinine, in which the CC bond is saturated, there is no formation of photofragments. 53 Elongating the NaromH + bond in CdNaromH + lowers the chargetransfer transition that plays a major role in the reactivity of the cinchona alkaloids, like the charge transfer involving CO in tyrosine containing peptides. 38 53 We have therefore calculated the change in electron density for the first three electronic transitions of CdH2 2+ and CdH2 2+ :HSO4 -, when elongating either the NalkH or the NaromH distances. The density difference between the ground state and the first three excited states of the complex is compared to that in CdH2 2+ in Figure 12. In CdH2 2+ , the S10 transition for the NalkH + bond modified from its equilibrium distance (1.023 Å) to 2.000 Å is an intramolecular charge transfer within the alkaloid part of the molecule. It is mostly a HOMO (C=C) → LUMO (*NalkH + ) (96%) transition. It is therefore a CT transition from the C=C orbital to the *NalkH + orbital. 53 For the NaromH + bond elongated from its equilibrium distance (1.018 Å) to 2.000 Å the S10 and S20 are HOMO-1 (arom) → LUMO (*NaromH + ) (96%) and HOMO-2 (arom) → LUMO (*NaromH + ) (98%) transitions. However, the S30 transition is a HOMO (C=C) → LUMO (97%) transition, i.e. a charge transfer from the C=C group localized on the alcaloid. In both cases, one of the first three electronic transitions, namely, S10 when elongating the NalkH + bond or S30, when elongating the NaromH + bond involves charge migration from the C=C orbital to the *NH + . The CdH2 2+ :HSO4 -complex behaves differently from CdH2 2+ . Elongating the NalkH + bond from its equilibrium distance is prevented by the cage effect due to the presence of HSO4 -. Elongating the NaromH bond from its equilibrium value (1.017Å) to 2.000 Å has two consequences. Compared to CdH2 2+ , it modifies the energy order of the transition. For an identical NaromH distance, of 2.000 Å, S20 and S30 in the complex are similar to S20 and S10 in CdH2 2+ , respectively in terms of excitations type and then electron density changes. S10 in the complex is the HOMO-3 → LUMO excitation (90%), with the HOMO-3 being mainly described in terms of combination of the nO of the three S=O groups of HSO4 -, and the LUMO being the NaromH + orbital. It corresponds therefore to an intermolecular CT transition.

We can conclude from these results that the bisulfate anion has a protective effect and prevents photodissociation of the CdH2 2+ :HSO4 -complex in two manners. Firstly, the fact that the two nitrogen atoms, Nalk and Narom, are protonated prevents proton transfer from Nalk to Narom. Moreover, the short hydrogen bond between the bisulfate and Nalk induces a cage effect that prevents motions of the NalkH + . The second effect of the complex formation is that the CT transitions from the C=C orbital of the alkaloid part of the molecule to the *NalkH + or NaromH + orbital are removed from the manifold accessible by the laser. Instead, the first three Sn0 transition involve ππ* excitations and then electron density changes within the quinoline ring, or charge migration from the bisulfate anion to the aza C6 ring, or to the *NaromH + .

Conclusion

This study unambiguously shows that the complexes formed between protonated cinchonidine and sulfuric acid actually contains doubly protonated cinchonidine CdH2 2+ and the bisulfate anion, HSO4 -. The cinchona alkaloid is therefore protonated on both nitrogens, the alkaloid nitrogen Nalk and the aromatic nitrogen Narom. HSO4 -bridges the two protonation sites so that electrostatic interactions between CdH2 2+ and HSO4 -stabilize the doubly protonated species in the gas phase. The same structure as for cinchonidine is observed for quinine and quinidine. No chirality effect is observed in the structure of the complex.

The presence of bisulfate has several consequences on the photophysics of protonated cinchonidine. First, the cinchonidine contained in the complex is doubly protonated, so that the transitions involving the Nalk or Narom lone pair are removed from the manifold. The presence of HSO4 -also has a protective role towards photo-dissociation. It first prevents the mobility of the proton localized on Nalk thanks to the strong electrostatic interaction. Such a cage effect has already been observed in tryptamine, tryptophan, or cinchonidine itself, embedded in cyclodextrine. 53, 103,

Figures and Tables

Table1: Significant calculated dihedral angles (°) and distances (Å) of the most stable conformers of the CdH + H2SO4 complex optimized at the B3LYP-D3/6-31++G(d,p) level. (see text), and of the CdH2 + HSO4 -complex to which the experimental spectra has been assigned, all in the Syn-γ-closed conformation. The transition energy is given relative to the ground state of the corresponding system. Only the transitions below the laser energy (4.66 eV) are considered.

  58 Å) distance between NalkH +q and O -q' , and purely electrostatic. The quinoline ring and HSO4 -are distant by more than 6.0 Å. The Anti-β-open structure involves an additional OH…O -q' hydrogen bond relative to Synγ-open, from the OH of CdH2 2+ to one oxygen atom of HSO4 -. The corresponding OH…O -q' distances are 1.87 and 4.11 Å in Anti-β-open and Syn-γ-open, respectively. This hydrogen bond contributes to the stabilization of Anti-β-open, which is more stable than the Syn-γ-open by ~ 2 kcal/mol.

  -β-open Trans-Syn-γ-open Trans-Syn-γ-closed Trans-Syn-γ-closed' Trans-Anti-γ-closed τ1 (C3' C4' C9 C8)

Figure 1 : 9 9 9 Figure 2 :Figure 3 :

 19923 Figure 1: Structure of the molecules under study

Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 4567 Figure 4: Structure of the five most stable conformers of the CdH2 2+ HSO4 -system optimized at the B3LYP-D3/6-31++G(d,p) level of theory. The Gibbs free energy relative to the most stable calculated complex is given in parentheses in kcal/mol

Figure 8 :

 8 Figure 8: Comparison between the IR experimental spectrum of QnH + H2SO4 (a) and the IR simulated spectra of the most stable Cis conformers: b) Cis-Anti-β-open (Figure 6b), c) Cis-Syn-γopen (Figure 6c), d) Cis-Syn-γ-closed (Figure 6d), e) Cis-Syn-γ-closed' (Figure 6e), f) Cis-Anti-γclosed (Figure 6f). The spectra of the open conformers are in red and those of the closed conformers in blue. Fingerprint region (Top) and high frequencies region (Bottom)

Figure 9 :Figure 10 :Figure 11 :

 91011 Figure 9: Comparison between the IR experimental spectrum of QnH + H2SO4 (a) and the IR simulated spectra of the most stable Trans conformers: b) Trans-Anti-β-open (Figure 7b), c) Trans-Syn-γ-open (Figure 7c), d) Trans-Anti-γ-closed (Figure 7d), e) Trans-Syn-γ-closed (Figure 7e), f) Trans-Syn-γ-closed' (Figure 7f) The spectra of the open conformers are in red and those of the closed conformers in blue. Fingerprint region (Top) and high frequencies region (Bottom)

Figure 12 :

 12 Figure 12: Difference between the electronic density of the ground state and that the first three excited states. The isolvalue is set to 0.005. The red (blue) color codes a decrease (increase) in electron density, respectively: a) CdH2 2+ , with NalkH distance of 2.0 Å b) CdH2 2+ , with Narom H distance of 2.0 Å c) CdH2 2+ HSO4 -with NaromH distance of 2.0 Å

Table 2 :

 2 Significant calculated dihedral angles (°) and distances (Å) of the most stable conformers of the CdH2 + HSO4 -complex optimized at the B3LYP-D3/6-31++G(d,p) level.

	Dihedral angles (°)	Anti-γ-open	Anti-γ-open'	Anti-γ-closed	Anti-γ-closed'
	and distances (Å)
	τ1 (C3'C4' C9 C8)	100		109		68	70
	τ2 (C4' C9 C8 Nalk)	168		164		44	54
	τ3 (N C8 C9 O)	-69		-71		165	175
	T3 (C8 C9 O H)	-179		172		172	179
	dNalkH δ+ … δ-OS		1.88		1.88		1.84	1.87
	dNarom…HOS		7.14		5.47		1.58	3.38
	Dihedral
	angles (°) and	Syn-γ-closed	Syn-γ-closed'	Anti-γ-closed	Anti-β-open	Syn-γ-open
	distances (Å)
	τ1 (C3'C4'C9 C8)	-108		-107	68	100	-83
	τ2(C4'C9C8 Nalk)		72		47	58	165	161
	τ3 (N C8 C9 O)	-165		171	177	-71	-73
	T3 (C8 C9 O H)	172		165	169	108	149
	dNalkH +δ … -δ OS	1.64		1.62	1.6	1.57	1.58
	dNaromH +δ … -δ OS	2.76		2.35	2.66	7.03	6.17

Table 3 :

 3 Calculated relative electronic energy ΔE and Gibbs free energy ΔG including counterpoise correction, for CdH + H2SO4 and CdH2 2+ HSO4 -at the B3LYP-D3/ 6-31++G (d,p) level of theory. Counterpoise correction to the energy BSSE. Natural charges on significant atoms q. The notation used for for CdH + and CdH 2+ is taken from reference55 .

Table 4 :

 4 Significant calculated dihedral angles (°) and distances (Å) of the most stable conformers of the Cis QnH2 2+ HSO4 -complex optimized at the B3LYP-D3/6-31++G(d,p) level of theory.

	Dihedral
	angles (°) and	Cis-Anti-β-open	Cis-Syn-γ-open Cis-Syn-γ-closed Cis-Syn-γ-closed' Cis-Anti-γ-closed
	distances (Å)
	τ1 (C3' C4' C9 C8)	98	-85	-110	-107	74
	τ2 (C4' C9C8Nalk)	162	160	59	47	60
	τ3 (N C8 C9 O)	-74	-73	-178	171	-180
	T3 (C8 C9 O H)	111	151	171	165	170
	dNalkH +δ … -δ OS	1.6	1.58	1.62	1.62	1.64
	dNaromH +δ … -δ OS	7.17	6.19	2.51	2.34	2.51

Table 5 :

 5 Significant calculated dihedral angles (°) and distances (Å) of the most stable conformers of the Trans QnH2 2+ HSO4 -complex optimized at the B3LYP-D3/6-31++G(d,p) level of theory.

Table 6 :

 6 Calculated relative electronic energy ΔE and Gibbs free energy ΔG including counterpoise correction, for the most stable conformers of QnH2 2+ HSO4 -at the B3LYP-D3/ 6-31++G (d,p) level of theory. Counterpoise correction to the energy BSSE.

		103	-87	-109	-108	76
	τ2 (C4' C9 C8Nalk)	166	160	57	46	60
	τ3 (N C8 C9 O)	-71	-74	-180	170	180
	T3 (C8 C9 O H)	106	153	171	166	169
	dNalkH +δ … -δ OS	1.57	1.58	1.62	1.63	1.67
	dNaromH +δ … -δ OS	7.08	6.16	2.49	2.34	2.5

Table 7 :

 7 Energy E0-1,2,3 (eV) of the first three vertical transitions S1,2,30 of the protonated monomer CdH + , of the di-protonated monomer CdH2 +

This explains why the reactivity related to proton mobility, previously proposed for isolated CdH + , is not observed here.53 The second role of the bisulfate is to modify the nature of the electronic transitions. In particular, the CT transitions from C=C orbital to *NalkH + or *NaromH + , which has been supposed to play a role in the photodissociation of CdH + , is higher than the laser energy in the complex.53 This study emphasizes the role of counterions in protecting biomolecules from photodissociation. The influence of complexation by sulfuric acid on the protonated cinchona alkaloid dimers is currently in progress and will be reported in a near future.

Acknowledgment

We thank Dr J. M. Ortega and the CLIO team for technical assistance. We acknowledge the use of the computing center MésoLUM of the LUMAT research federation (FR LUMAT 2764).

The research described here has been supported by the Investissements d'Avenir LabEx PALM contract (ANR-10-LABX-0039-PALM), by the ANR contract (ESBODYR ANR-14-CE06-0019-03), and by the RTRA "Triangle de la Physique" contract (Project N°2013-0562-T, Chiraux IRUV).

We thank Fabrice Gobert for experimental help. FB gratefully acknowledges the support of COST Action CM1405 (MOLIM).

Molecules