Unraveling the protonation site of oxazole and solvation with hydrophobic ligands by infrared photodissociation spectroscopy †

Protonation and solvation of heterocyclic aromatic building blocks control the structure and function of many biological macromolecules. Herein the infrared photodissociation (IRPD) spectra of protonated oxazole (HOx) microsolvated by nonpolar and quadrupolar ligands, HOx-Ln with L = Ar (n = 1–2) and L = N2 (n = 1–4), are analyzed by density functional theory calculations at the dispersion-corrected B3LYP-D3/aug-cc-pVTZ level to determine the preferred protonation and ligand binding sites. Cold HOx-Ln clusters are generated in an electron impact cluster ion source. Protonation of Ox occurs exclusively at the N atom of the heterocyclic ring, in agreement with the thermochemical predictions. The analysis of the systematic shifts of the NH stretch frequency in the IRPD spectra of HOx-Ln provides a clear picture of the sequential cluster growth and the type and strength of various competing ligand binding motifs. The most stable structures observed for the HOx-L dimers (n = 1) exhibit a linear NH L hydrogen bond (H-bond), while p-bonded isomers with L attached to the aromatic ring are local minima on the potential and thus occur at a lower abundance. From the spectra of the HOx-L(p) isomers, the free NH frequency of bare HOx is extrapolated as nNH = 3444 3 cm . The observed HOx-L2 clusters with L = N2 feature both bifurcated NH L2 (2H isomer) and linear NH L H-bonding motifs (H/p isomer), while for L = Ar only the linear H-bond is observed. No HOx-L2(2p) isomers are detected, confirming that H-bonding to the NH group is more stable than p-bonding to the ring. The most stable HOx-(N2)n clusters with n = 3–4 have 2H/(n 2)p structures, in which the stable 2H core ion is further solvated by (n 2) p-bonded ligands. Upon N-protonation, the aromatic C–H bonds of the Ox ring get slightly stronger, as revealed by higher CH stretch frequencies and strongly increased IR intensities.


Introduction
2][3][4] In particular, heterocyclic azole compounds have attracted the attention of pharmacologists since their first reported antifungal activity. 5Good solubility in water and high thermal stability with respect to other heteroaromatic systems make these molecules suitable for the synthesis of therapeutic and natural products. 6Among these, the oxazolecontaining amino acids are quite ubiquitous in various naturally occurring peptides, [7][8][9][10][11][12] which possess potential antibiotic and antitumor activity. 13,149][30] For example, protonation of the Ox ring in metamifop, the acetyl-coenzyme A carboxylase (ACCase) inhibitor, governs the binding interactions between metamifop and the carboxyltransferase domain. 30he shape and biochemical function of such biological macromolecules are often regulated by their heterocyclic building blocks, such as oxazole (Ox, C 3 H 3 NO).In the condensed phase, details of their interaction are usually obscured by macroscopic solvent effects, the interaction with other molecules and substrates, and thermal and heterogeneous broadening. 2,3,31On the other hand, interrogation of the relevant small heterocyclic building blocks in the gas phase, i.e., free from interference with the external bulk environment, provides detailed insight into their physical and chemical properties relevant to the function of the heavier biomolecules.To this end, spectroscopy of cold clusters of heterocyclic molecules in supersonic beams gives direct access to the relevant interaction potentials.Herein, we employ infrared photodissociation (IRPD) spectroscopy in a tandem mass spectrometer to determine fundamental properties of protonated oxazole (H + Ox) and its microsolvation interaction with nonpolar (L = Ar) and quadrupolar (L = N 2 ) ligands with the aid of dispersion-corrected density functional theory (DFT) calculations.In a forthcoming paper, we extend these studies to dipolar ligands (L = H 2 O) to characterize the microhydration network.8][39][40][41][42][43][44][45][46] However, no experimental information is available for any neutral Ox-L n clusters, probably because the broad absorption spectrum prevents the application of convenient size-selective resonant ionization techniques. 47,48Photoelectron spectra of Ox reveal that ionization into the planar ground electronic state ( 2 A 00 ) occurs by removal of an electron from a bonding p-orbital localized on the C4-C5 and O1-C2-N3 bonds. 49,50The high-resolution mass-analyzed threshold ionization spectrum of Ox provides an accurate adiabatic ionization energy, and the analysis of the observed vibrational modes confirms the planarity of the Ox + radical cation and illustrates the changes in geometry upon ionization. 50Previous photoelectron imaging of the oxazolide anion indicates selective deprotonation of the Ox ring at the C2 position. 42In contrast to neutral and cationic Ox (+) , only very limited information is available for H + Ox and its clusters.Previous DFT studies indicate that N-protonation of the heterocyclic ring is strongly preferred over O-protonation, 28,29 and the measured proton affinity is tabulated as 876.4 kJ mol À1 . 51,52Thus far, no spectroscopic data are available for H + Ox and its clusters.To this end, our combined IR and DFT studies of H + Ox-L n presented herein provide the first reliable experimental data about the preferred protonation site in H + Ox and a first impression of the intermolecular interaction of this prototypical protonated heterocyclic aromatic molecule with hydrophobic aprotic ligands.

Experimental and computational methods
IRPD spectra of mass-selected H + Ox-L n clusters are recorded in the CH, NH, and OH stretch range (2650-3600 cm À1 ) in a tandem quadrupole mass spectrometer coupled to an electron ionization (EI) source and an octupole ion guide. 53,54Briefly, H + Ox-L n clusters are produced in a pulsed supersonic plasma expansion utilizing electron and chemical ionization close to nozzle orifice.The expanding gas mixture is generated by seeding vapor of Ox (Sigma-Aldrich, 98%) heated to 328 K in Ar (or N 2 ) and 5% H 2 in He in a 2 : 1 ratio at a backing pressure of 10 bar.Adding H 2 to the expansion gas strongly enhances the yield of H + Ox, 55,56  Conceivable isomers of H + Ox and its H + Ox-L n clusters are calculated at the B3LYP-D3/aug-cc-pVTZ level of DFT theory to assign the measured IRPD spectra and characterize the intermolecular interaction potential. 579][60] Neutral Ox is also computed to establish the influence of protonation on the geometric and vibrational properties.Fully relaxed potential energy surface calculations are performed during the search for stationary points, and their nature as minima or transition states are verified by harmonic frequency analysis.Harmonic intramolecular vibrational frequencies are subjected to a linear scaling factor of 0.9636, derived from a comparison of computed CH and OH stretch frequencies of neutral Ox and water, respectively, to the measured values. 38,61We consider here also the water modes for optimizing the scaling factor, because we address in a forthcoming paper the vibrational spectroscopy of microhydrated H + Ox-(H 2 O) n clusters using the same experimental and computational procedure.Harmonic IR stick spectra are convoluted with a Gaussian line shape (FWHM = 10 cm À1 ) for convenient comparison to the experimental spectra.All relative energies (E 0 ) and dissociation energies (D 0 ) are corrected for harmonic zero-point vibrational energy.Gibbs free energies are evaluated at 298 K (G 0 ).Previous experience with the chosen computational level illustrates that basis set superposition errors are smaller than 1% and thus not considered here. 58,60The atomic charge distribution and second-order perturbation energies (E (2) ) of donor-acceptor orbitals involved in the H-bonds are evaluated using the natural bond orbital (NBO) analysis. 62Further characterization of the H-bond is obtained from noncovalent interaction (NCI) calculations by evaluating the reduced gradient of the electron density, s(r) B |grad(r)|/r 4/3 , as a function of the electron density r oriented by the sign of the second eigenvalue l 2 of the Hessian, r* = r sign(l 2 ). 63,64The relative strengths of the H-bonding interactions are estimated by comparing the respective r* values.

Results and discussion
The IRPD spectra of H + Ox-L n recorded between 2950 and 3600 cm À1 are summarized in Fig. 1, and the positions, widths, and vibrational and isomer assignments of the transitions observed (A-D, X) are listed in Table 1, along with the computed frequencies and IR intensities.The considered spectral range covers the OH, NH, and CH stretch fundamentals (n OH/NH/CH ), which are sensitive to the protonation site and the ligand binding site and bond strength.The positions, band shapes, and relative intensities of bands A-C occurring in the 3300-3450 cm À1 strongly vary with cluster size and type of ligand, suggesting their assignments to free and bound n NH modes.In contrast, peaks D1 and D2 observed in the 3150-3220 cm À1 range are relatively insensitive to the ligand type and cluster size and thus can be assigned to aromatic n CH modes not involved in ligand bonding.In the following, we discuss the structural, energetic, and vibrational properties of neutral Ox, H + Ox, and various H + Ox-L n isomers relevant for the detailed analysis of the experimental spectra.Cartesian coordinates of all relevant optimized structures are provided in the ESI.†

Ox and H + Ox monomers
The calculated geometric and vibrational parameters of neutral Ox in its planar 1 A 0 ground state (with C s symmetry) agree satisfactorily with the measurement (Fig. 2 and Table S1 in the ESI †). 38,44Protonation of Ox may occur at any of the aromatic ring atoms, and their structures are shown in Fig. 3 and Fig. S1 in the ESI.† Apart from the O-protomer, H + Ox(O), all protonated structures have C s symmetry.A detailed potential energy surface, illustrating the relative energies of the various protomers and barriers at the transition states for their interconversion, is shown in Fig. 3. Clearly, H + Ox(N) is by far the most stable isomer, and all Fig. 1 IRPD spectra of H + Ox-L n with L = Ar (n = 1-2) and N 2 (n = 1-4) recorded in the H + Ox-L m fragment channel (indicated as n-m).The positions, widths, and vibrational and isomer assignments of the transitions observed are listed in Table 1.
Table 1 Positions, widths (FWHM in parentheses), and suggested vibrational and isomer assignments of the transitions observed in the IRPD spectra of H + Ox-L n clusters with L = Ar (n = 1-2) and L = N 2 (n = 1-4) compared to frequencies of the most stable isomers calculated at the B3LYP-D3/aug-cc-pVTZ level.All values are given in cm À1 Exp.
Calc. other protomers are more than DE 0 = 120 kJ mol À1 higher in energy.The proton affinity of PA = 876.7 kJ mol À1 predicted for H + Ox(N) matches the recommended experimental value of 876.4 kJ mol À1 to better than 1 kJ mol À1 , 52 confirming that the chosen computational level accurately describes the protonation process.][67][68][69][70] All H + Ox protomers can readily be distinguished by their predicted IR spectra (Fig. S2 in the ESI †).For example, the C-protomers have characteristic aliphatic CH 2 stretch modes (n CH 2 , calculated below 2950 cm À1 ), whereas H + Ox(O) and H + Ox(N) can readily be identified by their unique OH (n OH = 3489 cm À1 ) and NH stretch (n NH = 3446 cm À1 ) oscillators, respectively.The spectral assignment given below demonstrates the exclusive production of H + Ox(N), hereafter denoted as H + Ox (if not mentioned otherwise), and thus, we mainly focus on the structural details of this protomer.Formation of the N-H s-bond upon protonation at the N atom has a significant influence on the geometry of the aromatic Ox ring skeleton (Fig. 2).For example, the neighboring N-C2 bond elongates by 25.5 mÅ.On the other hand, the effect on the peripheral C-H bonds is comparatively smaller (Dr CH r 1.5 mÅ).Still, the perturbation is strong enough to increase the average n CH frequency with a concomitant  1 and Fig. S2 in the ESI †).The NBO analysis reveals that the additional proton carries almost half of the positive charge (0.465 e), while the rest is delocalized mainly on the peripheral aromatic hydrogens (Fig. S3 in the ESI †).

H + Ox-L dimers
We consider two major binding sites for Ar and N 2 attachment to H + Ox, namely H-bonding to the acidic NH proton with high positive partial charge and p-bonding to the aromatic ring.For both ligands, the nearly linear NHÁ Á ÁL bonded isomers, H + Ox-L(H), are the global minima (D 0 = 891/1597 cm À1 for L = Ar/N 2 ), while the H + Ox-L(p) isomers are substantially less stable local minima (D 0 = 598/899 cm À1 ).The stronger bonds of N 2 arise from its larger parallel polarizability and additional quadrupole moment, leading to stronger electrostatic, inductive and dispersive forces. 71,72Moreover, the negative sign of the quadrupole moment favors a linear over a T-shaped approach of N 2 . 71,72The difference in the D 0 values between the H-bonded and p-bonded isomers of N 2 is almost 2.5 times larger than for the Ar ligand, owing to the stronger H-bonding affinity of N 2 resulting from its higher proton affinity (PA = 494/369 kJ mol À1 for L = N 2 /Ar). 52As a consequence of the stronger and shorter NHÁ Á ÁL H-bond (R = 2.031/2.420Å), the elongation of the N-H donor bond and corresponding red shift in the H-bonded n NH (n b NH ) are larger for N 2 (Dr NH = 8.3/3.4 mÅ, Dn b NH = À157/À70 cm À1 ).The E (2) and r* values, which both correlate with the strength of the H-bond, are also larger for N 2 (E (2) = 42.2/13.7 kJ mol À1 , Àr* = 0.022/0.013,Fig. S4 and S5 in the ESI †).As expected for such weak H-bonds, the charge transfer from H + Ox to the H-bonded ligand is small and also scales with the interaction energy (Dq = 0.028/0.017e).In contrast to H-bonding to the NH group, p-bonding of L to the aromatic ring has a negligible influence on the properties of the N-H bond (Dr NH r 0.5 mÅ), and thus the free n f NH mode remains nearly unshifted from the monomer (Dn NH = 3/5 cm À1 for Ar/N 2 ).For both major binding motifs, the aromatic C-H bonds and n CH modes are also essentially unaffected.For completeness, we also consider H + Ox-N 2 (CH) isomers, in which N 2 forms linear H-bonds to the aromatic CH protons of H + Ox.The binding energies obtained for H + Ox-N 2 (C2H) and H + Ox-N 2 (C5H) are comparable or weaker than the p-bond (D 0 = 932 and 705 cm À1 ), and their free n f NH modes are predicted around 3450 cm À1 (Fig. S6 and S7 in the ESI †).Any attempt to optimize H + Ox-N 2 (C4H) converges to the H + Ox-N 2 (H) global minimum.
In Fig. 4 the measured IRPD spectra of the H + Ox-L dimers are compared to those calculated for the most stable isomers, H + Ox-L(H) and H + Ox-L(p).The weak transitions A observed at 3447 and 3446 cm À1 for L = Ar and N 2 are attributed to n f NH of the H + Ox-L(p) isomers predicted at 3449 and 3451 cm À1 , respectively.The more intense bands B at 3395 and 3320 cm À1 can readily be assigned to the n b NH modes of the H + Ox-L(H) global minima.The observed red shifts of Dn b NH = À52 and À126 cm À1 are somewhat smaller but consistent with the predicted values (À70/À157 cm À1 ).In addition, the band profile of transition B with a sharp rise on the red side and a long tail on the blue side is characteristic for the excitation of proton-donor stretch modes and thus confirms the given assignments.The large width of such bands arises mainly from sequence hot bands of n b NH with intermolecular modes, which typically occur to higher frequency than the fundamental.The transitions D1/D2 at 3205/3174 and 3207/3172 cm À1 observed for L = Ar and N 2 , respectively, are attributed to the three closelying n CH modes of the H + Ox-L(p) and H + Ox-L(H) isomers, which are predicted in this spectral range with a similar energy spread and intensity ratio.Indeed, as predicted by the calculations,  N-protonation increases the n CH frequencies.A possible assignment of the bands D1 and D2 to the NH bend overtone, which may gain intensity by anharmonic interaction with the intense n NH fundamental, 73,74 can safely be excluded because of the low frequency predicted for the NH bend fundamental (1427 cm À1 for fundamental and 2844 cm À1 for first overtone from anharmonic calculations).For completeness, we also consider an assignment of bands A to combination modes n b NH + n s of the H + Ox-L(H) isomers involving the intermolecular stretch vibration (n s ).This scenario would yield n s frequencies of 73 and 126 cm À1 for L = Ar and N 2 , respectively, which are indeed similar to their harmonic computed values of 70 and 117 cm À1 .However, if that assignment were correct, such transitions should also appear in the spectra of the larger H + Ox-L n clusters, 56,75,76 in disagreement with experiment (Fig. 1).Hence, we strongly favor an assignment of bands A to n f NH of the H + Ox-L(p) isomers.A definitive isomer assignment, e.g., from hole-burning experiments, is beyond the scope of the present work.In conclusion, all major features of the IRPD spectra of the H + Ox-L dimers can readily be reproduced by the spectra predicted for H + Ox-L(H) and H + Ox-L(p).The analysis of the integrated band intensities of bands A and B, along with the predicted oscillator strengths, results in a rough estimate of the population ratio of H : p B 1.5 and B10 for L = Ar and N 2 , respectively, consistent with both the absolute and relative binding energies of the two ligand binding motifs.
In the following, we briefly present arguments for excluding the presence of other protomers and alternative ligand binding sites.In order to test the abundance of H + Ox(C) protomers via their characteristic and intense n CH 2 modes predicted in the 2850-3000 cm À1 range, IRPD spectra of H + Ox-L are recorded down to 2650 cm À1 for both Ar and N 2 .However, no such transitions are observed in this frequency range, indicating that the concentration of H + Ox(C) protomers is below the detection limit (see Fig. S8 in the ESI, † for a comparison with spectra computed for H + Ox(C2)-L dimers).We also computed IR spectra of dimers of the H + Ox(O) protomer (Fig. S8 in the ESI †).Interestingly, the n b OH mode (3201 cm À1 ) of H + Ox(O)-Ar(H) is predicted with high intensity in the vicinity of band D1.However, the corresponding band of H + Ox(O)-N 2 (H) predicted at 2887 cm À1 is completely missing in the measured spectrum.As these n b OH bands of H + Ox(O)-L(H) have enormous IR oscillator strengths, their absence in the IRPD spectra implies that the H + Ox(O) population is negligible (the lack of any n f OH band of this protomer near 3490 cm À1 confirms this view).Thus, in agreement with the thermochemical data in Fig. 3, we detect in the expansion only clusters of the by far most stable H + Ox(N) protomer and will not consider other protomers further.Finally, we may also safely exclude CH-bonded isomers of H + Ox-N 2 .The intense n b CH transition of the most stable of these isomers, H + Ox-N 2 (C2H), is predicted at 3109 cm À1 , and the IRPD spectrum lacks signal in this spectral range (Fig. S8 in ESI †).

H + Ox-L 2 trimers
Guided by the analysis of the H + Ox-L dimer spectra, addition of the second ligand results in the three different structural isomers of H + Ox-L 2 shown in Fig. 5.The planar H + Ox-L 2 (2H) global minimum features an asymmetric bifurcated NHÁ Á ÁL 2 H-bond, whereas in H + Ox-L 2 (H/p) a p-bound ligand is attached to the H + Ox-L(H) dimer.These two isomers have comparable stability, with total binding energies of D 0 (2H) = 1647/2611 cm À1 and In the 2H isomer with the bifurcated H-bond, the two nonequivalent and strongly nonlinear NHÁ Á ÁL bonds are substantially weaker than the linear NHÁ Á ÁL bonds in the dimers.As a result, the N-H bond contracts upon attachment of the second ligand, leading to a significant incremental blue shift in n NH (Dr NH = À1.6/À2.5 mÅ, Dn NH = 36/51 cm À1 for L = Ar/N 2 ).The effect is stronger for N 2 due to its higher H-bonding affinity.For the same reason, the asymmetry between the first and second bond is larger for N 2 .The E (2) energies confirm this view of asymmetric bonding.For example, E (2) = 29.8 and 2.7 kJ mol À1 for the two H-bonds in H + Ox-(N 2 ) 2 (2H), indicating still a substantial H-bond character to the first ligand, while the strongly nonlinear bond to the second ligand has mostly electrostatic character.In addition, the bent H-bond to the first ligand in the 2H isomer is weaker than in the linearly H-bonded dimer (E (2) = 29.8 and 42.2 kJ mol À1 ).Similar differences between linear and bifurcated H-bonds of acidic proton donors to N 2 ligands have previously been reported for indole + -(N 2 ) 2 , pyrrole + -(N 2 ) 2 , and tryptamine + -(N 2 ) 2 cluster cations. 59,77,78n contrast to the 2H isomers, additional p-complexation of the H + Ox-L(H) dimer in the H/p isomer induces only a small perturbation on the N-H bond and leads to a minor incremental blue shift of n b NH (Dr NH = À0.5/À0.9mÅ, Dn NH = 5/13 cm À1 for L = Ar/N 2 ), in line with the slightly smaller E (2) energy of the H-bond (41.1 vs. 42.2kJ mol À1 for L = N 2 ).Hence, the n b NH mode of the H/p isomer appears red shifted from the 2H isomer (Dn NH = À31/À38 cm À1 ) and thus both H-bonded structures can readily be distinguished by their n b NH modes.Finally, the two ligands in the 2p isomer barely influence the NH oscillator, and the associated parameters remain comparable to those of H + Ox (Dr NH = À0.6/À1.0mÅ, Dn NH = 6/10 cm À1 for Ar/N 2 ).
In Fig. 6 the measured IRPD spectra of the H + Ox-L 2 trimers are compared to those calculated for the most stable isomers (2H, H/p, 2p).The experimental H + Ox-Ar 2 spectrum exhibits three bands at 3401 (B), 3204 (D1) and 3175 (D2) cm À1 .Interestingly, band B lies between the predicted n b NH modes of the H/p (3381 cm À1 ) and 2H (3412 cm À1 ) isomers split by 31 cm À1 , which is somewhat larger than the width of band B (25 cm À1 ).Because (i) the calculations overestimate the Dn NH shifts and (ii) the experimental blue shift (Dn NH = 6 cm À1 ) with respect to H + Ox-Ar(H) agrees well with the one predicted for the statistically favored H/p isomer (Dn NH = 5 cm À1 ), we assign band B to the H/p isomer despite its somewhat lower calculated binding energy.The substantially less stable 2p isomer can be excluded because of the absence of any signal near n f NH B 3450 cm À1 .Its population is below 5% considering the achieved signal-to-noise ratio and computed oscillator strengths.This result confirms that the H-bond in H + Ox-Ar 2 is clearly more stable than the p-bond, as already inferred from the n = 1 spectrum and the calculations.According to this scenario, bands D1 and D2 are assigned to the n CH modes of the H/p isomer.
The measured H + Ox-(N 2 ) 2 spectrum displays a triplet structure at 3381 (X), 3357 (C), and 3334 (B) cm À1 in the n b NH range, along with the two n CH bands at 3208 (D1) and 3176 (D2) cm À1 .Compared to the n b NH band of H + Ox-N 2 (H) at 3320 cm À1 , the relative blue shift for band B is smaller than for band C (Dn NH = 14 vs. 37 cm À1 ), and these agree satisfactorily with the computed values of the H/p and 2H isomers (Dn NH = 13 vs. 51 cm À1 ), respectively.The n b NH mode of the H/p isomer has a larger IR oscillator strength (821 vs. 641 km mol À1 ), and this isomer is statistically favored over the 2H isomer (due to the two available p minima).Taking these aspects into account, the higher intensity of band C compared to B may indicate a larger abundance of the 2H isomer, compatible with its higher D 0 value.There is no obvious explanation for the shoulder X, and our currently favored interpretation is a sequence hot band of n b NH of 2H and/or H/p, a conclusion supported by the analysis of the spectra of the colder n = 3 and 4 clusters.Similar to the Ar case, the absence of any weak transition near n f NH B 3450 cm À1 illustrates the lack of the much less stable 2p isomer.The transitions D1 and D2 are then attributed to the n CH modes of the two assigned 2H and H/p isomers.

H
The complex potential energy surface of H + Ox-(N 2 ) 3 is not characterized in detail, and only two relevant structures are optimized (Fig. S9 in the ESI †).Only one calculation is performed for the n = 4 cluster (Fig. S10 in the ESI †).In the most stable 2H/p isomer of H + Ox-(N 2 ) 3 with D 0 = 3482 cm À1 , a p-bound N 2 ligand slightly perturbs the bifurcated 2H trimer, whereas the slightly less stable NH is blue shifted by 11 cm À1 with respect to the 2H isomer, consistent with its predicted shift of 12 cm À1 .Correspondingly, band B at 3348 cm À1 is attributed to the less stable H/2p isomer, whose n b NH blue shift of 14 cm À1 also agrees with the computed value of 12 cm .
The spectrum in the 3-1 channel, which is by a factor 5 weaker than the 3-0 channel, contains in the n b NH range only band C at 3371 cm À1 assigned to the 2H/p isomer.Moreover, the width of this transition is smaller than in the 3-0 spectrum.
The binding energy of this isomer is calculated as D 0 = 3482 cm À1 , i.e. the absorbed photon energy is close to the dissociation energy.Apparently, cold 2H/p clusters can eliminate only two N 2 ligands leading to a narrow n b NH band in the 3-1 channel, while internally warm clusters can eliminate all three N 2 ligands producing the broader n b NH transition in the 3-0 channel.Significantly, the n b NH transition of the H/2p isomer is only detected in the 3-0 channel, because its smaller binding energy calculated as D 0 = 3322 cm À1 allows to fragment all three ligands even for cold clusters.The added intensity of peak C in both fragment spectra is substantially larger than that of peak B. All these experimental results suggest that the 2H/p isomer is indeed more stable than the H/2p isomer, consistent with the calculations.The fact that the branching ratio into the two fragment channels is predicted correctly implies that also the absolute computed binding energies are reliable.The absence of band X in the colder n = 3 and 4 spectra (Fig. 1) is in line with its tentative interpretation as sequence hot band.
The IRPD spectrum of H + Ox-(N 2 ) 4 shown in Fig. 7 is only observed in the H + Ox-N 2 fragment channel (4-1), in line with the computed binding energies for p-bonded and H-bonded N 2 ligands (e.g., D 0 = 4334 cm À1 for the most stable 2H/2p isomer).The spectrum in the n b NH range is dominated by band C at 3376 cm À1 , which is attributed to the 2H/2p isomer by comparison to the n = 3 spectrum.Similarly, its shoulder B at 3358 cm À1 is the signature of a much less abundant H/3p isomer.Both transitions exhibit small incremental blue shifts of Dn b NH B 10 cm À1 typical for p-complexation of H + Ox with N 2 .The n CH bands of the two n = 4 isomers at 3208 (D1) and 3168 (D2) cm À1 are close to the transitions of the n = 1-3 clusters, indicating that all clusters up to n = 4 do not contain any CH-bonded N 2 ligands.

Cluster growth
The n NH frequencies observed for the various H + Ox-L n clusters summarized in Fig. 8 show a clear evolution as a function of the     cluster size and the ligand type and binding site, and thus provide a detailed picture of the cluster growth process of the various isomers.The p-bonds are substantially weaker than the H-bonds, and thus H + Ox-L n clusters with only p-bonded ligands (np) are merely observed for the cluster size n = 1.At this binding site the perturbation of the NH group is very small, so that we can accurately estimate the n NH fundamental of bare N-protonated H + Ox as 3444 AE 3 cm À1 , in excellent agreement with the predicted value of 3446 cm À1 .Clearly, the H-bonded H + Ox-L(H) dimers are the global minima on the n = 1 potential, with large incremental red shifts Dn NH = À53 and À122 cm À1 for L = Ar and N 2 , respectively.Further complexation with p-bonded ligands in the H/(n À 1)p isomers induces small incremental blue shifts of Dn NH = +6 cm À1 for Ar (n = 2) and +14, +14, and +10 cm À1 for N 2 (n = 2-4).For L = N 2 , the most stable binding motif for n Z 2 corresponds to the 2H/(n À 2)p isomers with a bifurcated 2H trimer core further solvated by p-bonded ligands.The incremental blue shifts of p-bonding (Dn NH = +11 and +8 cm À1 for n = 3-4) are slightly smaller than for the H/(n À 1)p series because of the weaker bifurcated H-bonds in the 2H/(n À 2)p isomers.

Concluding remarks
In summary, IRPD spectra of H + Ox-L n with L = Ar (n r 2) and L = N 2 (n r 4) are analyzed in the informative CH, NH, and OH stretch range with dispersion-corrected DFT calculations.Significantly, the IRPD spectra correspond to the first spectroscopic detection of H + Ox and its clusters in the gas phase.They provide a reliable determination of the preferred protonation site and a first impression of the interaction of this fundamental protonated heterocyclic molecule with hydrophobic ligands.H + Ox ions produced by chemical ionization in a plasma containing H 2 are exclusively protonated at the most basic N position, and protonation at the much less favorable O and C atoms (E 0 4 120 kJ mol À1 ) is not observed.Size-dependent shifts in the NH stretch frequency of H + Ox-L n provide a clear picture of the ligand binding sites and corresponding bond strengths and the sequential microsolvation process including the formation of solvation subshells.The nonpolar Ar and quadrupolar N 2 ligands prefer H-bonding to the acidic NH proton of H + Ox to p-bonding at the aromatic ring.From the spectra of the H + Ox-L(p) dimers, the NH stretch frequency of bare H + Ox is accurately extracted as 3444 AE 3 cm À1 .Similarly, the CH stretching frequencies are extracted as 3205 AE 5, 3180 AE 10, and 3170 AE 10 cm À1 , which indicate a strengthening of the C-H bonds upon N-protonation of Ox.The most stable H + Ox-L n clusters with n Z 2 have a H + Ox-L 2 (2H) trimer core with an asymmetric bifurcated NHÁ Á ÁL 2 H-bond of two nonequivalent ligands to the NH proton.Further solvation in these 2H/(n À 2)p clusters occurs at the p binding sites.A less stable H/(n À 1)p isomer series is also observed for L = Ar and N 2 , in which p-bonded ligands are attached to a H + Ox-L(H) dimer core with a linear NHÁ Á ÁL H-bond.The microsolvation of H + Ox with hydrophobic ligands reported herein differs substantially from the microsolvation with polar hydrophilic ligands, as inferred from the analysis of IRPD spectra microhydrated H + Ox-(H 2 O) n clusters reported in a forthcoming publication.

Fig. 3
Fig. 3 Potential energy surface for proton migration between various protomers of H + Ox calculated at the B3LYP-D3/aug-cc-pVTZ level.All energies (E e in kJ mol À1 ) are without zero-point energy correction.

Fig. 4
Fig. 4 Comparison of IRPD spectra of H + Ox-L (L = Ar and N 2 ) to the linear IR absorption spectra of N-protonated H + Ox and various H + Ox-L isomers obtained at the B3LYP-D3/aug-cc-pVTZ level.The stick spectra are convoluted with Gaussian line profiles with FWHM = 10 cm À1 .

Fig. 5
Fig.5Optimized geometries of various H + Ox-L 2 isomers with L = Ar and N 2 calculated at the B3LYP-D3/aug-cc-pVTZ level.Binding energies (D 0 ) and bond lengths are given in cm À1 and Å, respectively.Values in parentheses correspond to relative energies and free energies in cm À1 (E 0 , G 0 ).

Fig. 6
Fig. 6 Comparison of IRPD spectra of H + Ox-L 2 (L = Ar and N 2 ) to the linear IR absorption spectra of various H + Ox-L 2 isomers calculated at the B3LYP-D3/aug-cc-pVTZ level.The stick spectra are convoluted with Gaussian line profiles with FWHM = 10 cm À1 .

Fig. 7
Fig. 7 Experimental IRPD spectra of H + Ox-(N 2 ) n with n = 3-4 compared to the linear IR absorption spectra of two isomers of H + Ox-(N 2 ) 3 calculated at the B3LYP-D3/aug-cc-pVTZ level.The stick spectra are convoluted with Gaussian line profiles with FWHM = 10 cm À1 .

Fig. 8
Fig.8Plot of experimental n NH frequencies obtained from the IRPD spectra of H + Ox-L n with L = Ar (n = 1-2) and L = N 2 (n = 1-4) as a function of cluster size (Table1).The p and H (and H/(n À 1)p) isomers are indicated by open and filled circles, respectively, while the 2H/(n À 2)p isomers are indicated by crosses.The value for bare H + Ox is extrapolated from the H + Ox-Ar(p) data point.
IR , 10 Hz, 2-5 mJ per pulse, bandwidth B1 cm À1 ) emitted from an optical parametric oscillator laser pumped by a Nd:YAG laser.Calibration of n IR to better than 1 cm À1 is achieved by a wavemeter.Resonant vibrational excitation leads to the loss of one or more weakly bound ligands.The resulting H + Ox-L m fragment ions (m o n) are mass-selected by the second quadrupole and monitored with a Daly detector as a function of n IR to derive the IRPD spectrum of H + Ox-L n .
suggesting that H 3 + serves as major protonating agent for Ox (although we cannot exclude other ions such as H + He, H + L, or H 2 + ).The desired H + Ox-L n parent clusters are mass-selected in the first quadrupole and irradiated in the adjacent octupole ion guide with a tunable IR laser pulse (n