Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Similarity scores of vibrational spectra reveal the atomistic structure of pentapeptides in multiple basins.

Vibrational spectroscopy combined with theoretical calculations is a powerful tool for analyzing the interaction and conformation of peptides at the atomistic level. Nonetheless, identifying the structure becomes increasingly difficult as the peptide size grows large. One example is acetyl-SIVSF-N-methylamide, a capped pentapeptide, whose atomistic structure has remained unknown since its first observation [T. Sekiguchi, M. Tamura, H. Oba, P. Çarçarbal, R. R. Lozada-Garcia, A. Zehnacker-Rentien, G. Grégoire, S. Ishiuchi and M. Fujii, Angew. Chem., Int. Ed., 2018, 57, 5626-5629]. Here, we propose a novel conformational search method, which exploits the structure-spectrum correlation using a similarity score that measures the agreement of theoretical and experimental spectra. Surprisingly, the two conformers have distinctly different energy and geometry. The second conformer is 25 kJ mol-1 higher in energy than the other, lowest-energy conformer. The result implies that there are multiple pathways in the early stage of the folding process: one to the global minimum and the other to a different basin. Once such a structure is established, the second conformer is unlikely to overcome the barrier to produce the most stable structure due to a vastly different hydrogen bond network of the backbone. Our proposed method can characterize the lowest-energy conformer and kinetically trapped, high-energy conformers of complex biomolecules.

Transition from vehicle to Grotthuss proton transfer in a nanosized flask: cryogenic ion spectroscopy of protonated p-aminobenzoic acid solvated with D2O.

Proton transfer (PT) is one of the most ubiquitous reactions in chemistry and life science. The unique nature of PT has been rationalized not by the transport of a solvated proton (vehicle mechanism) but by the Grotthuss mechanism in which a proton is transported to the nearest proton acceptor along a hydrogen-bonded network. However, clear experimental evidence of the Grotthuss mechanism has not been reported yet. Herein we show by infrared spectroscopy that a vehicle-type PT occurs in the penta- and hexahydrated clusters of protonated p-aminobenzoic acid, while Grotthuss-type PT is observed in heptahydrated clusters, indicating a change in the PT mechanism depending on the degree of hydration. These findings emphasize the importance of the usually ignored vehicle mechanism as well as the degree of hydration. It highlights the possibility of controlling the PT mechanism by the number of water molecules in chemical and biological environments.

Conformational preference of 2-(4-methoxyphenyl)ethanol studied by supersonic jet spectroscopy: Intramolecular OH/π interaction.

A π-type hydrogen bonding between the OH group and the π electron is a crucial factor for the conformational preference of the molecular structure with a flexible group. However, the information on the effect of the substituent on the OH/π interaction is insufficient. The laser-induced fluorescence (LIF) excitation, the dispersed fluorescence (DF), the IR-UV hole-burning, and the IR dip spectra of jet-cooled 2-(4-methoxyphenyl)ethanol were measured for the first time. Almost all bands observed in the spectral region of 35 550-36 500 cm-1 in the LIF excitation spectrum were successfully assigned with the DF and the IR-UV hole-burning spectra coupled with the quantum chemical calculation at M06-2x/6-311G and MP2/6-311G levels. Five conformers were found in the LIF excitation spectrum. The most stable conformer was Ggπ, and the second most stable conformer was Ggπ' (the trans rotamer of the methoxy group for Ggπ). Ggπ and Ggπ' had the OH group directed toward the π electron system of the benzene ring. The OH stretching frequency of Ggπ/Ggπ' of MPE in the IR dip spectra was red-shifted against that of Ggπ of phenylethanol, indicating that the introduction of the methoxy group would enhance the intramolecular OH/π interaction. In addition, the torsional vibration between the benzene ring and the side chain (-CH2CH2OH) (mode 63) was observed in the DF spectra of the Ggπ-00 and Ggπ'-00 band excitation, but their intensities were rather different, resulting from the different orientation of the OH group for each conformer toward the π electron system. The methoxy group would increase the negative charge on the benzene ring and would enhance the intramolecular OH/π interaction through the electrostatic interaction.

Switching of Protonation Sites in Hydrated Nicotine via a Grotthuss Mechanism.

The switching of the protonation sites in hydrated nicotine, probed by experimental infrared (IR) spectroscopy and theoretical ab initio calculations, is facilitated via a Grotthuss instead of a bimolecular proton transfer (vehicle) mechanism at the experimental temperature (T = 130 K) as unambiguously confirmed by experiments with deuterated water. In contrast, the bimolecular vehicle mechanism is preferred at higher temperatures (T = 300 K) as determined by theory. The Grotthuss mechanism for the concerted proton transfer results in the production of nicotine's bioactive and addictive pyrrolidine-protonated (Pyrro-H+) protomer with just 5 water molecules. Theoretical analysis suggests that the concerted proton transfer occurs via hydrogen-bonded bridges consisting of a 3 water molecule "core" that connects the pyridine protonated (Pyri-H+) with the pyrrolidine-protonated (Pyrro-H+) protomers. Additional water molecules attached as acceptors to the hydrogen-bonded "core" bridge result in lowering the reaction barrier of the concerted proton transfer down to less than 6 kcal/mol, which is consistent with the experimental observations.

Electronic and vibrational spectroscopies of aromatic clusters with He in a supersonic jet: The case of neutral and cationic phenol-Hen (n = 1 and 2).

Van der Waals clusters composed of He and aromatic molecules provide fundamental information about intermolecular interactions in weakly bound systems. In this study, phenol-helium clusters (PhOH-Hen with n ≤ 2) are characterized for the first time by UV and IR spectroscopies. The S1 ← S0 origin and ionization energy both show small but additive shifts, suggesting π-bound structures of these clusters, a conclusion supported by rotational contour analyses of the S1 origin bands. The OH stretching vibrations of the PhOH moiety in the clusters match with those of bare PhOH in both the S0 and D0 states, illustrating the negligible perturbation of the He atoms on the molecular vibration. Matrix shifts induced by He attachment are discussed based on the observed band positions with the help of complementary quantum chemical calculations. For comparison, the UV and ionization spectra of PhOH-Ne are reported as well.

Infrared Spectra of Beauvericin-Alkaline Earth Metal Ion Complexes─Ion Preference to Physiological Ions.

Beauvericin (Bv) is a naturally occurring ionophore that selectively transports ions through cell membranes. However, the intrinsic ion selectivity of Bv for alkaline earth metal ions (M2+) is yet to be established due to inconsistent results from condensed phase experiments. Based on fluorescence quenching rates, Ca2+ appears to be preferred while extraction experiments favor Mg2+. In this study, we apply cold ion trap─infrared spectroscopy to Bv-M2+ coupled with electrospray ionization mass spectrometry. The mass spectrum shows that Bv favors binding to physiologically active ions Mg2+ and Ca2+ although it can form complexes with all four alkaline earth metal ions. Infrared spectroscopy, as measured by the H2 tag technique, reveals that Bv binds Mg2+ and Ca2+ ions by six carbonyl oxygens in the center of its cavity. This observation is supported by theoretical calculations. Other alkaline earth metal ions are bound by three carbonyl groups at the amide face. This difference in configuration is consistent with the binding preferences for the alkaline earth metal ions.

Stepwise hydration of [CH3COOMg]+ studied by cold ion trap infrared spectroscopy: insights into interactions in the magnesium channel selection filters.

The magnesium channel controls Mg2+ concentration in the cell and plays an indispensable role in biological functions. The crystal structure of the Magnesium Transport E channel suggested that Mg2+ hydrated by 6 water molecules is transported through a selection filter consisting of COO- groups on two Asp residues. This Mg2+ motion implies successive pairing with -OOC-R and dissociation mediated by water molecules. For another divalent ion, however, it is known that RCOO-⋯Ca2+ cannot be separated even with 12 water molecules. From this discrepancy, we probe the structure of Mg2+(CH3COO-)(H2O)4-17 clusters by measuring the infrared spectra and monitoring the vibrational frequencies of COO- with the help of quantum chemistry calculations. The hydration by (H2O)6 is not enough to induce ion separation, and partially-separated or separated pairs are formed from 10 water molecules at least. These results suggest that the ion separation between Mg2+ and carboxylate ions in the selection-filter of the MgtE channel not only results from water molecules in their first hydration shell, but also from additional factors including water molecules and protein groups in the second solvation shell of Mg2+.

Does Chiral Sensitivity of a Structure Depend on the Metal Core? Alkali Ion Complexes of Cyclo(Tyr-Tyr).

Alkali metal complexes of cyclic dipeptide cyclo Tyr-Tyr have been studied under cryogenic ion trap conditions. Their structure was obtained by combining Infra-Red Photo-Dissociation (IRPD) and quantum chemical calculations. The structural motif strongly depends on the relative chirality of the tyrosine residues. For residues of identical chirality, the cation interacts with one amide oxygen and one of the aromatic rings only; the distance between the aromatic rings does not change with the nature of the metal. In contrast, for residues of opposite chirality, the metal cation is located in between the two aromatic rings and interacts with both of them. The distance between the two aromatic rings strongly depends on the metal. Electronic spectra obtained by Ultra Violet Photodissociation (UVPD) spectroscopy and analysis of the UV photo-fragments shed light on the excited state deactivation processes, which depend on both the chirality of the residue and that of the metal ion core. Na+ stands out by the presence of low-lying charge transfer states resulting in the broadening of the electronic spectrum.

Ion Recognition beyond Size Matching: Cooperative Hydration Effect on the K+ Selectivity of Valinomycin over Na+ Revealed by Cryogenic Double Ion Trap Infrared Spectroscopy.

The naturally occurring ionophore valinomycin (VM) selectively transports K+ across the biological membrane, which makes VM a plausible antivirus and antibacterial candidate. The K+ selectivity of VM was rationalized based on a size-matching model despite structural inconsistency between experiments and computations. In this study, we investigated the conformations of the Na+VM complex with 1-10 water molecules using cryogenic ion trap infrared spectroscopy with computational calculations. It shows that the water molecule penetrates the cavity of VM deeply enough to distort the C3-symmetric structure of gas-phase Na+VM, in stark contrast to hydrated clusters of K+VM with C3-symmetric structure, where H2O is located outside the cavity. The high affinity to K+ would be ascribed to minimal hydration-induced structural deformation of K+VM compared to Na+VM. This study highlights a novel cooperative hydration effect on the K+ selectivity and will provide an updated understanding of its ionophoric properties beyond the traditional size-matching model.

Hydration Rearrangement in the 4-Aminobenzonitrile-(H2 O)2 Cluster Induced by Photoionization: The Effect of Solvent-Solvent Interactions.

The interplay between solute-solvent and solvent-solvent interactions plays an essential role in solvation dynamics that has important effects on the mechanism and dynamics of chemical reactions in solution. In this study, the rearrangement of the hydration shell induced by photoionization of a solute molecule is probed in a state- and isomer-specific manner by resonant multiphoton ionization detected IR spectroscopy of the prototypical 4-aminobenzonitrile-(H2 O)2 cluster produced in a molecular beam. IR spectra reveal that the water molecules form a cyclic solvent network around the CN group in the initial neutral state (S0 ). Different from the singly-hydrated cluster, in which either the CN or the NH2 group is hydrated, hydration of the NH2 group is not observed in the dihydrated cluster. IR spectra obtained after ionizing the solute molecule into the cation ground state (D0 ) exhibit features ascribed to both NH-bound and CN-bound isomers, indicating that water molecules migrate from the CN to the NH site upon ionization with a yield depending on the ionization excess energy. Analysis of the IR spectra as a function of the excess energy shows that migration produces two different NH2 solvated structures, namely (i) the most stable structure in which both N-H bonds are singly hydrated and (ii) the second most stable isomer in which one of the N-H bonds is hydrated by a H-bonded (H2 O)2 dimer. The product branching ratio of the two isomers depends on the excess energy. The role of the water-water interaction in the hydration rearrangement is discussed based on the potential energy landscape. Solvation dynamics plays an important role in reaction mechanisms in the condensed phase, where not only solute-solvent solvation but also solvent-solvent interactions have a significant influence on the dynamics. Thus, the investigation of solvation dynamics at the molecular level substantially contributes to our understanding of the reaction mechanism. In this study, the dihydrated cluster of 4ABN was utilized as a model for the first solvation layer to elucidate solvent motions induced by ionization of the solute and the role of W-W interactions for the solvent relaxation.

Cryogenic Ion Spectroscopy of Protonated and Sodiated Methyladenine Derivatives.

Ultraviolet photodissociation (UVPD) spectra of protonated 9-methyladenine (H+9MA), protonated 7-methyl adenine (H+7MA), protonated 3-methyladenine (H+3MA), and sodiated 7-methyladenine (Na+7MA) near the origin bands of the S0-S1 transition were obtained using cryogenic ion spectroscopy. The UV-UV hole burning, infrared (IR) ion-dip, and IR-UV double resonance spectra showed that all the ions were present as single isomers in a cryogenic ion trap. The UVPD spectrum of H+9MA exhibited only a broad absorption band, whereas the spectra of H+7MA, H+3MA, and Na+7MA displayed moderately or well-resolved vibronic bands. Potential energy profiles were computed to understand the reason for the different bandwidths of the vibronic bands in the spectra. The broadening of the bands was correlated with the slopes between the Franck-Condon point and the conical intersection between the S1 and S0 states in the potential energy profiles, thus reflecting the deactivation rates in the S1 state.

Can Ag+ Permeate through a Potassium Ion Channel? A Bottom-Up Approach by Infrared Spectroscopy of the Ag+ Complex with the Partial Peptide of a Selectivity Filter.

Silver and silver ions have a long history of antimicrobial activity and medical applications. Nevertheless, the activity of Ag+ against bacteria, how it enters a cell, has not yet been established. The K+ channel, a membrane protein, is a possible route. The addition of a channel inhibitor (4-aminopyridine) to modulate the Ag+ uptake could support this view. However, the inhibitor enhances the uptake of Ag+, the opposite result. We have applied cold ion trap infrared laser spectroscopy to complexes of Ag+ and Ac-Tyr-NHMe (a model for GYG) which is a portion of the selectivity filter in the K+ channel to consider the question of permeation. With support from quantum chemical calculations, we have determined the stable conformations of the complex. The conformations strongly suggest that Ag+ would not readily permeate the K+ channel. The mechanism of the unexpected enhancement by the inhibitor is discussed.

Cation-responsive cavity expansion of valinomycin revealed by cryogenic ion trap infrared spectroscopy.

Valinomycin (VM) is a natural K+-selective ionophore that transports K+ through the cell membrane. VM captures K+ in its central cavity with a C3-symmetric ß-turn-like backbone. Although the binding affinity is drastically decreased for the VM-sodium (Na+VM) complex with respect to K+VM, VM holds relatively high affinity to Rb+ and Cs+. The high affinity for larger ions irrespective of ionic size seems to conflict with the expected optimal size matching model and raises questions on what factors determine ion selectivity. A combination of infrared spectroscopy with supporting computational calculations reveals that VM can accommodate larger Rb+ and Cs+ by flexibly changing its cavity size with the elongation of its folded ß-turn-like backbone. The high affinity to Rb+ and Cs+ can be ascribed to a size-dependent cavity expansion. These findings provide a new perspective on molecular recognition and selectivity beyond the conventional size matching model.

Hydration-induced protomer switching in p-aminobenzoic acid studied by cold double ion trap infrared spectroscopy.

Para-Aminobenzoic acid (PABA) is a benchmark molecule to study solvent-induced proton site switching. Protonation of the carboxy and amino groups of PABA generates O- and N-protomers of PABAH+, respectively. Ion mobility mass spectrometry (IMS) and infrared photodissociation (IRPD) studies have claimed that the O-protomer most stable in the gas phase is converted to the N-protomer most stable in solution upon hydration with six water molecules in the gas-phase cluster. However, the threshold size has remained ambiguous because the arrival time distributions in the IMS experiments exhibit multiple peaks. On the other hand, IRPD spectroscopy could not detect the N-protomer for smaller hydrated clusters because of broad background due to annealing required to reduce kinetic trapping. Herein, we report the threshold size for O → N protomer switching without ambiguity using IR spectroscopy in a double ion trap spectrometer from 1300 to 1800 cm-1. The pure O-protomer is prepared by electrospray, and size-specific hydrated clusters are formed in a reaction ion trap. The resulting clusters are transferred into a second cryogenic ion trap and the distribution of O- and N-protomers is determined by mid-IR spectroscopy without broadening. The threshold to promote O → N protomer switching is indeed five water molecules. It is smaller than the value reported previously, and as a result, its pentahydrated structure does not support the Grotthuss mechanism proposed previously. The extent of O → N proton transfer is evaluated by collision-assisted stripping IR spectroscopy, and the N-protomer population increases with the number of water molecules. This result is consistent with the dominant population of the N-protomer in aqueous solution.

Conformer-selective Photodynamics of TrpH+ -H2 O.

The photodynamics of protonated tryptophan and its mono hydrated complex TrpH+ -H2 O has been revisited. A combination of steady-state IR and UV cryogenic ion spectroscopies with picosecond pump-probe photodissociation experiments sheds new lights on the deactivation processes of TrpH+ and conformer-selected TrpH+ -H2 O complex, supported by quantum chemistry calculations at the DFT and coupled-cluster levels for the ground and excited states, respectively. TrpH+ excited at the band origin exhibits a transient of less than 100 ps, assigned to the lifetime of the excited state proton transfer (ESPT) structure. The two experimentally observed conformers of TrpH+ -H2 O have been assigned. A striking result arises from the conformer-selective photodynamics of TrpH+ -H2 O, in which a single water molecule inserted in between the ammonium and the indole ring hinders the barrierless ESPT reaction responsible for the ultra-fast deactivation process observed in the other conformer and in bare TrpH+ .

Na+ Selective Binding by Beauvericin and Its Mechanism Studied by Mass-Coupled Cold Ion Trap Infrared Spectroscopy.

Beauvericin (Bv) is a cyclic hexadepsipeptide mycotoxin that selectively transports ions across cell membranes. Characterization of its intrinsic ion affinity has been complicated by different previous results in condensed phases and biological membranes. We report the marked specificity between alkali metal ions by Bv using experimental and computational methods. Mass spectrometry shows Bv readily binds all five alkali ions; however, the complex with Na+ is the most abundant species, indicating a strong binding preference. Gas phase infrared spectroscopy and theoretical calculations show that Li+, K+, Rb+, and Cs+ are coordinated by three amide carbonyl oxygens on the N-methylamino-l-phenylalanyl face. Selectivity for Na+ is achieved as Bv sequesters Na+ in the center of its cavity formed by three amide carbonyl and three ester carbonyl groups, a configuration unique among alkali metal ions. This finding provides insight into the correlation between selectivity and conformation of Bv, essential for development of this mycotoxin.

Do Stereochemical Effects Overcome a Charge-Induced Perturbation in Isolated Protonated Cyclo(Tyr-Tyr)?

Two diastereomers of the protonated diketopiperazine (DKP) dipeptide cyclo(Tyr-Tyr), namely, cyclo(LTyr-LTyr)H+ and cyclo(LTyr-DTyr)H+, are studied in a cryogenic ion trap by means of IR photodissociation spectroscopy combined with quantum chemical calculations. The two diastereomers have similar structures in which one of the rings is folded over the DKP ring and the other one is extended in a trans geometry, allowing a strong OH+···π interaction to take place. This contrasts to the observation of a stacked geometry for neutral cyclo(LTyr-LTyr) only under supersonic expansion conditions that do not exist for cyclo(LTyr-DTyr). In the protonated form, the strength of the OH+···π interaction is different for the two diastereomers, resulting in a ∼110 cm-1 difference in the ν(OH+) frequency and a smaller but clearly identifiable difference in the protonated amide ν(NH) frequency. Stereochemical effects are therefore still evidenced despite the strong perturbation due to the excess charge.

A bottom-up approach to the ion recognition mechanism of K+ channels from laser spectroscopy of hydrated partial peptide-alkali metal ion complexes.

K+ channels allow selective permeation of K+, but not physiologically abundant Na+, at almost diffusion limit rates. The conduction mechanism of K+ channels is still controversial, with experimental and computation studies supporting two distinct conduction mechanisms: either with or without water inside the channel. Here, we employ a bottom-up approach on hydrated alkali metal complexes of a model peptide of K+ channels, Ac-Tyr-NHMe, to characterize metal-peptide, metal-water, and water-peptide interactions that govern the selectivity of K+ channels at a molecular level. Both the extension to the series of alkali metal ions and to temperature-dependent studies (approaching physiological values) have revealed the clear difference between permeable and non-permeable ions in the spectral features of the ion complexes. Furthermore, the impact of hydration is discussed in relation to the K+ channels by comparisons of the non-hydrated and hydrated complexes.

Structure of Gas Phase Monohydrated Nicotine: Implications for Nicotine's Native Structure in the Acetylcholine Binding Protein.

We report a joint experimental-theoretical study of the never reported before structure and infrared spectra of gas phase monohydrated nicotine (NIC) and nornicotine (NOR) and use them to assign their protonation sites. NIC's biological activity is strongly affected by its protonation site, namely, the pyrrolidine (Pyrro-NICH+, anticipated active form) and pyridine (Pyri-NICH+) forms; however, these have yet to be directly experimentally determined in either the nicotinic acetylcholine receptor (nAChR, no water present) or the acetylcholine-binding protein (AChBP, a single water molecule is present) but can only be inferred to be Pyrro-NICH+ from the intermolecular distance to the neighboring residues (i.e., tryptophan). Our temperature-controlled double ion trap infrared spectroscopic experiments assisted by the collisional stripping method and high-level theoretical calculations yield the protonation ratio of Pyri:Pyrro = 8:2 at 240 K for the gas phase NICH+···(H2O) complex, which resembles the molecular cluster present in the AChBP. Therefore, a single water molecule in the gas phase enhances this ratio in NICH+ relative to the 3:2 for the nonhydrated gas phase NICH+ in a trend that contrasts with the almost exclusive presence of Pyrro-NICH+ in aqueous solution. In contrast, the Pyri-NORH+ protomer is exclusively observed, a fact that may correlate with its weaker biological activity.