2024 Volume 13 Issue 1 Pages A0160
Intermolecular interactions between aromatic amino acids were investigated by ultraviolet photodissociation spectroscopy of hydrogen-bonded protonated clusters of histidine (His) and tryptophan (Trp) enantiomers in the gas phase. Product ion spectra and photodissociation spectra in the wavelength range of the S1–S0 transition of Trp at several temperatures (8–100 K) were obtained using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. l-Trp detachment forming protonated His was the main pathway. Two bands observed at 288 and 285 nm in the photodissociation spectra of heterochiral H+(d-His)(l-Trp) indicated the coexistence of two types of conformers. The bands at 288 and 285 nm were attributed to the conformers formed from stronger and weaker intermolecular interactions, respectively. In the spectra of homochiral H+(l-His)(l-Trp), only the band due to the stronger interactions was observed at 288 nm. The intermolecular interactions of l-His with l-Trp were stronger than those of d-His with l-Trp.
Histidine (His) is one of the most flexible protein residues and is used to form the active sites of protein enzymes.1,2) This is because the imidazole side chain serves as an acid and base in a living system. His protonation behaviors in protein folding and misfolding have been studied in the pathogenesis of Alzheimer’s disease.3–5) His supplementation suppresses food intake and fat accumulation.6,7) The reactivity and structure of protonated His (H+His) in the gas phase have been investigated using tandem mass spectrometry, photodissociation spectroscopy, and theoretical calculations.1,8–11)
The roles of d-amino acids in biological systems have been revealed using various analytical methods.12–16) d-His was reported to be the substrate of Escherichia coli aminoacyl-tRNA synthetase.17) Enantioselective polymeric transporters were prepared using molecular imprinting techniques with d-His as the template.18) Discrimination of amino acid enantiomers using mass spectrometry without chromatographic separation has been performed by detecting the differences in their chemical and physical properties caused by intermolecular interactions with chiral molecules.19–28) Nuclear magnetic resonance spectroscopy using magnetic resonance acceleration was designed and developed for the structural elucidation of gas-phase ions.29,30)
Chemical properties of inner and surface regions of hydrogen-bonded clusters have been studied by ultraviolet (UV) photodissociation and molecular adsorption analyses in the gas phase.31,32) The NH3+ group, carboxyl group, and indole ring of protonated tryptophan (H+Trp) play an important role in the enantiomer selectivity of chiral crown ether, hexose, and pentose, respectively.33–35) l-Alanine-based tripeptides recognize d-His and d-Trp through protonation, while the differences between aromatic amino acid enantiomers hydrogen-bonded with the tripeptides were not observed in the case of phenylalanine and tyrosine.36)
In this study, temperature-dependent UV photodissociation spectroscopy of hydrogen-bonded protonated clusters of His and Trp enantiomers was performed using a variable-temperature ion trap and a variable-wavelength pulse laser. Based on the results, the intermolecular interactions between aromatic amino acid enantiomers were discussed.
The apparatus consisted of an electrospray ionization source, an octopole ion guide, a quadrupole ion bender, a quadrupole mass filter, a temperature-controlled 22-pole ion trap (8–350 K), and a linear time-of-flight spectrometer. Hydrogen-bonded protonated clusters, generated via electrospray ionization of water solutions containing 0.5 mM amino acids and 0.1% acetic acid, were introduced into the quadrupole mass filter through a metal capillary, a skimmer, an octopole ion guide, and an ion bender. The mass-selected clusters were thermalized in the temperature-controlled ion trap via multiple collisions with helium buffer gas for 50 ms. The temperature of the ion trap was controlled using a cryogenic refrigerator (CH-204SB, Sumitomo, Tokyo, Japan), heater cartridge (HTR-50, Lake Shore, OH, USA), and two silicon diode temperature sensors (DT-670B-CU, Lake Shore). The mass-selected and temperature-controlled clusters were irradiated with a third harmonic of a tunable Ti:sapphire laser (LT-2211T, LOTIS TII, Minsk, Belarus) at 0.4 mJ/pulse, which was not focused to allow a spatial overlap between the ion packets and the laser pulse. Precursor and product ions were orthogonally accelerated to 2.8 keV using two-stage pulsed electric fields and detected using dual microchannel plates (F4655, Hamamatsu Photonics, Shizuoka, Japan) in the linear time-of-flight spectrometer. The delay time between photoexcitation and acceleration was 30 μs, and the repetition rate of the experimental cycle was 10 Hz. UV photodissociation spectra were obtained by monitoring the ion signals as a function of the photoexcitation wavelength.
In the photoexcitation of hydrogen-bonded protonated clusters of His enantiomers and l-Trp (m/z 360), l-Trp detachment from the clusters, which forms protonated His (m/z 156), was the main photodissociation pathway over the measured ranges of wavelength (265–290 nm) and temperature (8–100 K). This indicates that the proton affinity of His is higher than that of Trp.37) Dissociation of the aromatic amino acids and difference of the photoreactivity between enantiomers were not observed in the product ion spectra.
The relative intensity between the product ion (m/z 156) and the precursor ion (m/z 360) in the product ion spectra of the clusters was measured in the wavelength range of 265–290 nm. It was reported that the broad feature in the electronic spectrum of protonated Trp was ascribed to the excited states with short lifetime and the first peak to the S1–S0 origin transition was 284.5 nm.38,39) Figure 1A shows the UV photodissociation spectrum of heterochiral H+(d-His)(l-Trp) at 8 K. The broad feature and two bands at 288 and 285 nm were observed in the wavelength range of the S1–S0 transition in the spectrum. The protonated His in the product ion spectrum and the broad feature of the photodissociation spectrum suggest that the proton interacts with both His and Trp in the cluster.
The band at 288 nm was red-shifted compared to the S1–S0 transition of protonated Trp. The redshifts of the S1–S0 origin transitions induced by intra- and inter-molecular interactions were observed in the electronic spectra of protonated dipeptides containing aromatic amino acids and protonated l-Trp dimer.40–42) In the report for H+(l-Trp)2, two distinguishable bands at 289 and 286 nm were assigned to the S1–S0 transitions of the compact conformer formed from strong cation–π interactions and the flexible extended conformer, respectively, using temperature-dependent photodissociation spectroscopy and time-dependent density functional theory calculations.42) Based on the results, the two bands shown in Fig. 1A indicate that two types of conformers of H+(d-His)(l-Trp) have coexisted at 8 K. The bands at 288 and 285 nm were attributed to the conformers formed from stronger and weaker intermolecular interactions, respectively.
In the UV photodissociation spectrum of homochiral H+(l-His)(l-Trp) at 8 K shown in Fig. 1B, the band at 288 nm was observed, which corresponds to the S1–S0 transition of the conformer formed from stronger intermolecular interactions. The band at 285 nm, which was observed as the S1–S0 origin transitions of H+(l-Trp), H+(l-Trp)2, H+(l-Ala)(d-Trp), and H+(l-Ala)(l-Trp), was not observed in the spectrum of H+(l-His)(l-Trp).32,38,42) This indicates that the intermolecular interactions between l-His and l-Trp in the cluster were strong, leading to the formation of one type of conformer in the gas phase.
Temperature-dependent UV photodissociation spectroscopy in the wavelength range of the S1–S0 transitions was performed to investigate the temperature dependence of the conformers of heterochiral H+(d-His)(l-Trp) and homochiral H+(l-His)(l-Trp). Figure 2 shows the UV photodissociation spectra of H+(d-His)(l-Trp) at 8 K, 30 K, and 100 K. The spectrum at 8 K was also shown for comparison. The two bands were still observed at 100 K. The relative intensity of the band at 288 nm increased as the temperature increased, compared to that of the band at 285 nm, indicating the conformational changes to the conformers with stronger intermolecular interactions. In the case of H+(d-His)(l-Trp), the conformer formed from stronger intermolecular interactions was stable at higher temperatures.
Furthermore, only the broad band at 288 nm was observed in the spectra of homochiral H+(l-His)(l-Trp) at 30 and 100 K, as shown in Fig. 3. The width of the band increased as the temperature increased. The full widths at half maximum at 8 and 100 K were approximately 250 and 500 cm−1, respectively. Several conformers formed from stronger intermolecular interactions could contribute to the broad band at 288 nm. In the case of H+(l-Trp)2, the relative intensity of the red-shifted band decreased exponentially with increasing temperature and the band at 285 nm became dominant above 70 K, indicating that the entropic effect caused the most stable compact conformer, which was formed from strong cation–π interactions, to undergo conformational changes to flexible extended conformers at higher temperatures.42)
The temperature-dependent UV photodissociation spectroscopy of the hydrogen-bonded protonated clusters shows two results: The intermolecular interactions between His and Trp are stronger than those in the Trp dimer. The intermolecular interactions of l-His with l-Trp are stronger than those of d-His with l-Trp. For alanine, leucine, and isoleucine, the stronger intermolecular interactions in homochiral amino acid clusters containing Trp compared to heterochiral clusters have been reported as in the case of His using the enantiomer-selective reactions, UV photodissociation spectroscopy of higher excited states, and gas-phase water adsorption on the surface of hydrogen-bonded protonated clusters.32,43) To reveal the relationships between the intermolecular interactions and enantiomer selectivity of amino acids, it is necessary to determine the hydrogen-bonded structures using ion mobility spectrometry, conformer-selective laser spectroscopy, and theoretical calculations.
UV photoexcitation experiments of hydrogen-bonded heterochiral H+(d-His)(l-Trp) and homochiral H+(l-His)(l-Trp) clusters were performed at several temperatures. l-Trp detachment from the clusters was the main pathway and the difference in the photoreactivity such as enantiomer-selective reactions was not observed in the product ion spectra. The UV photodissociation spectra of H+(d-His)(l-Trp) showed the coexistence of two types of conformers formed from stronger and weaker intermolecular interactions. In the photodissociation spectra of H+(l-His)(l-Trp), only the band due to the stronger interactions was observed. The intermolecular interactions in homochiral clusters were stronger than those in heterochiral clusters.
This work was supported by JSPS KAKENHI Grant Number 23K04668.
Mass Spectrom (Tokyo) 2024; 13(1): A0160
