Shun-Ichi Ishiuchi

IR spectroscopy under cryogenic ion trap conditions shows that the copper II complexes with l -proline and d -tyrosine contain l -proline in its zwitterion form, while no zwitterion is observed when both amino acids have the same absolute configuration.

Shared protons in dicarboxylate motifs are essential biological components. Infrared (IR) spectroscopy is the premier tool to probe the OHO vibrations defining these structures, as vibrational frequencies directly reflect the underlying potential surface. Previously, infrared multiple photon dissociation (IRMPD) studies of deprotonated glutamic acid showed broad features, but their origin─thermal effects, multiple conformers, or intrinsic anharmonicity─remained unknown. To gain a better understanding, we utilized cryogenic ion spectroscopy at 4 K. We discovered that despite a single shared-proton conformer being dominant, broadband features persist, disappearing only upon deuteration. High-dimensional anharmonic calculations (27 modes) show that the OHO vibration is extensively coupled to combination bands and overtones of numerous low-frequency modes (700–1200 cm–1). This demonstrates that shared proton motion is intrinsically linked to the dynamics of the entire molecule rather than being an isolated vibration.

Flavins frequently operate as visible-light photoreceptor in both biological and artificial systems. Previous studies (mostly computational) suggest photoinduced protonation-site switching as the fundamental origin of the flavin photoreactivity. In particular, photoexcitation is predicted to change the preferred protonation site to the opposite side of the tricyclic aromatic flavin ring. However, direct experimental identification of such protonation-site switching by photoexcitation has remained an outstanding challenge. Herein, we directly prove the photoinduced protonation-site switching by combining cryogenic ion-trap infrared spectroscopy with controlled microsolvation by water molecules. Using protonated lumiflavin as prototypical model flavin, we show that S₁ ← S₀ photoexcitation triggers proton transfer catalyzed by a water bridge across the aromatic ring. These results provide a general design principle of flavin photochemistry, with implications for developing biological/artificial photoactive molecules.

Understanding the molecular mechanisms underlying ion transport selectivity is a central issue in supramolecular chemistry and biochemistry. Beauvericin (Bv), a naturally occurring cyclic hexadepsipeptide, selectively transports physiologically relevant Ca²⁺ across cell membranes. Although the 2:1 (Bv-M-Bv) complex has been proposed as a key driver of ion transport, its detailed conformations and contribution to selectivity remain unclear. Here, we systematically investigated the Bv-M-Bv complexes with alkaline-earth and alkali metal ions using cryogenic ion mobility and cryogenic IR spectroscopy. Comparison of collision cross sections, IR spectra, and quantum chemical calculations enabled strongly supported assignments to the stacked, tilted, and shifted conformers, whose relative abundances depend strongly on the size and charge of the encapsulated metal ions. Notably, the Bv-Ca²⁺-Bv complex preferentially adopts the most compact stacked conformation, which may enhance diffusion across cell membranes. These findings provide molecular-level insights into ion transport selectivity governed by structural flexibility.

Metal-ion-monosaccharide interactions play key roles in C-type lectins (CTLs), Ca²⁺-dependent carbohydrate-binding proteins responsible for selective carbohydrate recognition. Although monosaccharides adopt specific coordination motifs with metal ions in CTL binding sites, the factors governing which hydroxyl groups coordinate to the metal ion remain unclear. Here, we investigate the charge-density dependence of these interactions using cryogenic gas-phase infrared spectroscopy of alkali metal ion and Ca²⁺ complexes with methyl α-d-galactopyranoside and methyl α-d-mannopyranoside as ligands for CTLs. The spectra reveal systematic changes in coordination motifs with metal-ion charge density. Lower charge density favors a preferred motif supported by a strong intramolecular hydrogen-bond network, whereas higher charge density favors more highly coordinated structures that disrupt this network. Combined with quantum mechanical cluster calculations, these findings suggest that peptide coordination attenuates the effective charge density of Ca²⁺ in CTLs, thereby promoting formation of the coordination motif relevant to selective carbohydrate recognition.

Ultraviolet and infrared photodissociation spectra (UVPD and IRPD) of monohydrated N-methyl-dopamineH⁺ NMeDAH⁺(H₂O)₁ are measured by cryogenic ion spectroscopy and compared with those of monohydrated dopamineH⁺ DAH⁺(H₂O)₁ to reveal the methylation effect on intramolecular excited state proton transfer (ESPT). Whereas the UVPD spectrum of DAH⁺(H₂O)₁ is broad due to the proton transfer from the amine chain to the aromatic ring, that of NMeDAH⁺(H₂O)₁ shows sharp vibronic transitions in addition to a broad background signal. This suggests the coexistence of two or more conformers in which the excited state dynamics are very different. Based on conformer-selected IR spectra and theoretical calculations, sharp bands are assigned to two conformers in which a water molecule is inserted between the amine chain and the aromatic ring and prevents NH → π ESPT. This will lead to slow excited state dynamics. This water-inserted conformer is very minor in DAH⁺(H₂O)_1, and its enhancement by methylation is discussed. The broad UVPD background is attributed to the most stable conformer, in which the N-H bond of the amino group directly interacts with the catechol ring. The reaction path for NH → π ESPT in the most stable conformer shows a low barrier of less than 100 meV, which explains the broad UVPD spectrum by fast ESPT. The results confirm the model based on the relationship between the structure of these hydrated complexes and their photo reactivity associated with the excited state proton transfer.

Flavins are essential biological cofactors and their protonation isomers (protomers) generated upon photoexcitation play a crucial role in their photochemical reactions. Here, we identify the triplet-state (T₁) protomer of protonated lumiflavin (LFH⁺) in a cryogenic ion trap by visible-pump infrared-probe photodissociation spectroscopy using H₂-tagging (H₂-tagging Vis-IRPD). This approach yields high-resolution IR spectra of cold triplet ions which, along with quantum chemical calculations, enables structural assignments and population analysis of the identified O2-cis@T₁ protomer of LFH⁺. Furthermore, the implementation of a tickle radio frequency technique (Vis-tRF-IRPD) effectively removes nonexcited species from the ion trap, allowing vibrational signatures of the T₁ state to be isolated. This work establishes a fundamental platform for monitoring protonation-site switching in photoexcited flavins (S_n or T_n) and demonstrates the broader utility of H₂-tagging Vis-IRPD spectroscopy for electronic-state- and conformation-resolved studies of cold molecular ions.

DIYETDYYR is a tryptic peptide from the regulatory loop of insulin receptor, which has the three tyrosine residues need for activation of the protein. In order to spectroscopically differentiate the tyrosine residues, singly protonated DIYETDYYR and its phenylalanine‐substituted analogs were studied by cryogenic ion spectroscopy. By comparing the UV absorption of the peptide at room temperature with those of phenylalanine‐substituted ones, the second and the third tyrosine residues showed UV absorption between 35 000 and 35 200 cm ⁻¹ , while the first tyrosine residue did not. Comparing the infrared (IR) spectra of the peptides at cryogenic temperature, the second tyrosine residue was found to have a hydrogen‐bonded phenolic OH. The IR spectra were explained by density‐functional‐based tight‐binding calculations. The structure of DIYETDYYR was tentatively assigned to the one with a hydrogen bond between the second tyrosine and the C‐terminus.

The replacement of uridine (U) with pseudouridine (ψ) in mRNA vaccines reduces immune inflammation as a result of the molecular recognition of mRNA by one of the Toll-like Receptors (TLR7). This process, which makes the practical application of mRNA vaccines possible, is modulated at the molecular level by the protons shared between the carboxylate group of amino acid residues in TLR7 and the deprotonation sites on U or ψ that determine the charged state in which these molecules exist under physiological conditions. In the gas phase, U is deprotonated at the N3 site, while ψ is deprotonated at the N1 site. This difference originates from the distinct chemical environments of the respective nucleobase moieties rather than the ribose sugar group and can provide the stepping stone toward a molecular level insight into the recognition process in the complex biological environment.

We investigated the structural evolution of protonated phenylalanine ions (H⁺Phe) upon stepwise hydration using cryogenic ion spectroscopy. H⁺Phe ions were generated by electrospray ionization and introduced into a reaction trap containing water vapor at 130-170 K to form hydrated complexes, H⁺Phe(H₂O)_n (n = 1-5). Subsequently, these complexes were cooled in a cryogenic ion trap at 4 K and analyzed by ultraviolet photodissociation (UVPD) spectroscopy. The UVPD spectra exhibited well-resolved vibronic bands near the origin bands of the S₀-S₁ transitions. To determine the number and structures of distinct conformers, we employed IR photodissociation and IR ion-dip spectroscopy in conjunction with quantum chemical calculations. The first water molecule binds exclusively to an NH bond of the protonated NH₃⁺ group, while the second and third water molecules bind either to the remaining NH bonds or to the OH group of the carboxyl terminus. In particular, H⁺Phe(H₂O)₄ exists as a single conformer in which all available hydrogen-bonding sites are occupied, completing the first hydration shell. Water molecules preferentially bind to a specific conformer of bare H⁺Phe, indicating hydration-induced conformational selection. These findings provide detailed insights into site-specific hydration and reveal the progressive conformational changes in H⁺Phe induced by sequential water attachment.