# Probing the Triplet State by Cryogenic Ion Trap IR Spectroscopy Application to Protonated Lumiflavin

Sotaro Ohara, Keisuke Hirata, Gilles Grégoire, Masaaki Fujii, Otto Dopfer, Shun-Ichi Ishiuchi

► To cite this version:

Sotaro Ohara, Keisuke Hirata, Gilles Grégoire, Masaaki Fujii, Otto Dopfer, et al. Probing the Triplet State by Cryogenic Ion Trap IR Spectroscopy Application to Protonated Lumiflavin. Journal of Physical Chemistry Letters, 2025, 16 (49), pp.12514-12519. 10.1021/acs.jpclett.5c02372. hal-05486224

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1 Probing the Triplet State by Cryogenic Ion Trap IR Spectroscopy – Application to
2 Protonated Lumiflavin

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4 Sotaro Ohara,[a] Keisuke Hirata,[a] Gilles Grégoire,* [b, c] Masaaki Fujii,[d, e, f] Otto Dopfer,* [c, g] Shun-ichi

5 Ishiuchi* [a]

6

7 a. Department of Chemistry, School of Science, Institute of Science Tokyo, 2-12-1 4259 Ookayama, Meguro-ku, Tokyo

8 152-8550, Japan

9 b. Université Paris-Saclay, CNRS, Institut des Sciences Moléculaires d'Orsay, 91405 Orsay, France

10 c. Tokyo Tech World Research Hub Initiative (WRHI), Institute of Innovation Research, Institute of Science Tokyo, 4259

11 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan

12 d. Laboratory for Chemistry and Life Science, Institute of Innovative Research, Institute of Science Tokyo, 4259

13 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan

14 e. Research and Development Initiative, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan

15 f. International Research Frontiers Initiative (IRFI), Institute of Innovative Research, Institute of Science Tokyo, 4259,

16 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan

17 g. Institut für Physik und Astronomie, Technische Universität Berlin, Hardenbergstrasse 36, 10623 Berlin, Germany

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19 *Corresponding authors:

20 GG: gilles.gregoire@cnrs.fr, OD: dopfer@physik.tu-berlin.de, SI: ishiuchi.s.aa@m.titech.ac.jp

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24 ABSTRACT

25 Flavins are essential biological cofactors and their protonation isomers (protomers) generated upon
26 photoexcitation play a crucial role in their photochemical reactions. Here, we identify the triplet-state (T<sub>1</sub>)
27 protomer of protonated lumiflavin (LFH<sup>+</sup>) in a cryogenic ion trap by visible-pump infrared-probe
28 photodissociation spectroscopy using H<sub>2</sub>-tagging (H<sub>2</sub>-tagging Vis-IRPD). This approach yields high-
29 resolution IR spectra of cold triplet ions which, along with quantum chemical calculations, enables
30 structural assignments and population analysis of the identified O2-cis@T<sub>1</sub> protomer of LFH<sup>+</sup>. Furthermore,
31 the implementation of a tickle radio frequency technique (Vis-tRF-IRPD) effectively removes non-excited
32 species from the ion trap, allowing vibrational signatures of the T<sub>1</sub> state to be isolated. This work
33 establishes a fundamental platform for monitoring protonation-site switching in photoexcited flavins (S<sub>n</sub> or
34 T<sub>n</sub>) and demonstrates the broader utility of H<sub>2</sub>-tagging Vis-IRPD spectroscopy for electronic-state- and
35 conformation-resolved studies of cold molecular ions.
36
Flavins are a family of tricyclic heterocycles derived from isoalloxazine (Figure 1) and function as essential redox cofactors in diverse biological systems.¹ While previously known for their role in oxidation-reduction reactions, flavins have recently gained attention for their property of absorbing blue light, which provides the basis for their relevance in light-driven processes including photocatalysis and photosensors.²⁻⁴ Upon photoexcitation, flavin molecules may change their protonation site. Calculations have suggested that the proton affinity (PA)⁵ and basicity of the N5 site increases in the S₁ excited state of lumiflavin (from pKₐ = -10.1 in S₀ to pKₐ* = +0.2 in S₁),⁶ potentially enabling proton transfer (PT) to a site that is inaccessible in the electronic ground state. By contrast, the basicity of the N1 site decreases (from pKₐ = -2.8 to pKₐ* = -4.9),⁶ making this site less favorable for protonation upon excitation.

For example, for fatty acid photo-decarboxylase (FAP),⁷, ⁸ it has been proposed that a proton is transferred from the fatty acid to the flavin cofactor upon blue-light excitation, specifically targeting the O4/N5 sites. This hypothesis is supported by quantum chemical calculations based on the X-ray structure of FAP,⁷ which suggest that the increase of the PA of the N5 site in the S₁ state facilitates PT that is unfavorable in the S₀ state.⁵ In addition, in electrophotochemistry, flavins have emerged as powerful photoacids capable of donating protons under irradiation with light.⁹ Their involvement in PT can trigger radical formation and C–H bond activation, enabling reaction pathways typically inaccessible by purely thermal or electrochemical methods. Despite these advances, however, no direct experimental evidence has been reported for flavin species protonated at the O4/N5 sites. Thus, the hypothesis of photoinduced switching of the protonation site is theoretically supported but remains experimentally unverified. Motivated by this lack of experimental evidence, our main goal is to identify such O4/N5 protomers using infrared spectroscopy. As a first step, we previously determined the structure of protonated lumiflavin (LFH⁺) it its S₀ state.¹⁰⁻¹³ Under cryogenic ion trap conditions, the O₂-cis protomer (Figure 1) accounts for approximately 90% of the population, while the remaining 10% are N1 protomers.¹⁰

As we move toward identifying the protonation site of the electronically excited state, it is important to note that all previous studies have exclusively focused on the S₁ excited state. However, limiting the analysis to the S₁ state may not provide a comprehensive understanding because flavins are known to undergo highly efficient intersystem crossing (ISC) to the triplet state(s). The phosphorescence quantum yield of flavins ranges from 0.2 to 0.4 in aqueous environments,¹⁴, ¹⁵ indicating that ISC competes strongly with fluorescence or internal conversion (IC). In biological systems, the triplet state of flavins can serve as a relevant intermediate. A prominent example is found in light-oxygen-voltage (LOV) domain photoreceptor proteins, where photoexcitation induces ISC to the triplet state, enabling covalent bond formation with a nearby cysteine residue.¹⁶⁻¹⁸ This light-triggered reaction has been shown to proceed with higher ISC efficiency, supporting the view that triplet-state flavins play an active chemical role in signaling and other biological functions.¹⁹⁻²¹ Therefore, the high ISC efficiency of flavins challenges the current understanding of flavin activity concerning excited-state protonation, namely that excited-state PT (ESPT) occurs in the S₁ state. It remains unclear whether ESPT in S₁ is suppressed by ISC or whether the triplet state itself provides the appropriate conditions for the ESPT to take place. Establishing the accessibility of the triplet
state under cryogenic ion trap conditions provides valuable guidance for any investigation of the excited-state energetics and dynamics of protonation of flavins.

3

In a cryogenic ion trap, UV-pump IR-probe double-resonance (UV-IR) spectroscopy methods employed by the Rizzo²² and Zwier²³ groups have both provided conformer-selective spectroscopic signatures of excited-state peptides, including their triplet states. Despite its utility, this UV-IR approach suffers from several limitations when applied to the investigation of triplet states. First, IR spectra obtained from vibrationally hot species, such as hot triplet-state ions, often exhibit broad and poorly resolved bands that complicate confident spectral and isomer assignments. Second, the technique is inherently restricted to molecules that can undergo efficient IR-induced dissociation, typically via favorable fragmentation channels with low to moderate dissociation thresholds, limiting its general applicability. Third, the observed signal intensities can change depending on the fragmentation pathway being monitored, introducing uncertainty in intensity-based analysis. Moreover, at longer pump-probe delays (micro- to milliseconds), vibrational cooling of hot triplet ions reduces the photodissociation efficiency, making it impossible to accurately quantify excited-state populations in the ion trap.

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To overcome these limitations of conventional UV-IR spectroscopy, we combine visible-pump infrared-probe photodissociation (Vis-IRPD) spectroscopy with the H₂-tagging approach²⁴ to study electronically excited LFH⁺ ions in a cryogenic ion trap. This technique only probes ions that have fully relaxed to their vibrational ground state, because the H₂-tag can be attached to molecular ions only after sufficient collisional cooling in the trap. As a result, the experimental IR spectra present sharp transitions of vibrationally cold ions, including those in long-lived electronically excited states such as the triplet state (T₁), thereby enabling direct comparison with quantum chemical predictions even for small vibrational frequency shifts. It is noted that the S₁ state itself does not contribute to the spectra because its lifetime (up to nanoseconds in aqueous solution²⁵) is much shorter than the experimental delay of milliseconds. In addition, the H₂-tagging Vis-IRPD approach provides a linear dissociation response to IR intensity and maintains a consistent performance even at long delay times (up to tens of milliseconds). Importantly, the dissociation efficiency of the H₂-tag is independent of the ion conformation, making it feasible to reliably estimate relative populations of different species, such as LFH⁺ ions differing in protonation site or electronic state, based on the analysis of the intensities of their IRPD signatures.¹⁰, ²⁶ A similar-in-spirit approach was recently reported by the Kappes group, who combined He-tagging with two-color laser spectroscopy (visible-pump, near-IR-probe) to observe the vibronic spectra of the T₃ ← T₁ transition in organic dyes under cryogenic conditions.²⁷ Their results further emphasize the utility of messenger-tagging techniques, as they achieved well-resolved optical spectra even for long-lived triplet states. However, calculations of higher-lying triplet states (e.g., T₃) suffer from reduced accuracy and reliability due to spin-mixing effects and the involvement of multiple electronic configurations, making structural assignments challenging. In contrast, we employ herein a high-resolution IR approach capable of experimentally detecting vibrational fingerprints corresponding to specific electronic states (S₀ or T₁) and structural conformers, leading to reliable protomer-specific assignment and subsequent population

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analysis based on more robust vibrational frequency calculations.

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Figure 2b shows the H₂-tagging IRPD spectra of LFH⁺ measured without Vis excitation, representing vibrational transitions of the protomers observed in the S₀ state. Based on comparison with computed IR spectra (Figure 2a), we have previously reported that the dominant protomer is O2-cis (~90%, shown in blue), while the minor protomer is N1 (~10%, red).¹⁰ Based on these results, we employ mode-specific scaling factors for all the computed IR spectra shown in Figure 2.

8

Upon irradiation with Vis light ($\lambda_{exc}$ = 23448 cm⁻¹, Figure S3), corresponding to vibronic S₁ excitation of the O2-cis protomer,¹⁰,¹¹ new vibrational bands (indicated in light blue) appear in the H₂-tagging Vis-IRPD spectrum recorded at 12 ms delay (Figure 2d). Prominent peaks are observed at 3567 (band A), 1762 (band B), 1570 cm⁻¹ (band C), with a weaker feature at 1510 cm⁻¹ (band D). To assign these new transitions, Figure 2c shows the IR spectra computed for plausible candidates that can be generated without PT: the O2-cis protomer in the T₁ state (O2-cis@T₁, light blue) and the O2-trans protomer in the S₀ state (O2-trans@S₀, green). The formation of O2-cis@T₁ is rationalized by ISC from its S₁ state and that of O2-trans@S₀ by cis → trans rotation of the O2-H bond, respectively, following photoexcitation of the O2-cis protomer of LFH⁺ from S₀. Other potential PT-induced protomers are excluded from consideration due to large deviations of their predicted vibrational patterns (Figure S4). Comparison between the experimental Vis-IRPD spectrum and the IR spectrum calculated for O2-cis@T₁ reveals quantitative agreement. Band A is shifted by +15 cm⁻¹ from the O2-H stretching mode of O2-cis@S₀ (measured at 3552 cm⁻¹), consistent with the predicted blue shift of +19 cm⁻¹ for O2-cis@T₁ (Table S1). Similarly, band B shows a -29 cm⁻¹ red shift from the C4=O4 stretching band of O2-cis@S₀ (measured at 1791 cm⁻¹), in close agreement with the theoretical shift of -28 cm⁻¹ for O2-cis@T₁. Bands C and D are both assigned to intense ring C=N stretching modes of O2-cis@T₁. Collectively, these spectral matches provide compelling evidence that the O2-cis@T₁ protomer is formed via ISC following optical S₁ ↔ S₀ excitation of O2-cis (Figure 3a). Finally, from an energetic perspective, the O2-cis → trans isomerization is unfavorable. As shown in Figure S5, the O2-trans geometry is more than 20 kJ mol⁻¹ higher in energy than that of O2-cis in all S₀, S₁ and T₁ states, indicating that there is no thermodynamic driving force for the rotation. Furthermore, the IR spectra calculated for O2-trans@S₀ and O2-trans@T₁ (Figure 2c) show poor agreement with the observed Vis-IRPD spectrum and the calculated O2-cis form, particularly for the predicted O2-H stretching frequency more than +40 cm⁻¹.

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To further validate the formation of the T₁ species of the O2-cis protomer, we apply herein a new and enhanced spectroscopic approach by incorporating a tickle radio frequency (tickle RF)²⁸⁻³² pulse into the H₂-tagging Vis-IRPD spectroscopy, referred to as Vis-tRF-IRPD. In this approach, LFH⁺ ions which retain their H₂-tag after Vis excitation are actively ejected from the ion trap (see Supporting Information for experimental details). As a result, only photoexcited LFH⁺ ions that have lost their H₂-tag remain trapped. These bare ions are subsequently re-cooled through collisions with the H₂/He buffer gas and re-tagged with H₂ before the IR laser pulse is introduced at a delay of 12 ms. This procedure allows us to selectively
probe the photoexcited population, effectively eliminating contributions from non-excited species, which are present in the Vis-IRPD spectrum in Figure 2d. The resulting Vis-tRF-IRPD spectrum (Figure 2e) is dominated by vibrational transitions attributed to the O2-cis@T₁ protomer, including bands at 3567 (band A, O2-H stretch), 3390 (band E, N3-H stretch), 1759 (band B', C4=O4 stretch), 1567 (band C', ring C=N stretch), and 1510 cm⁻¹ (band D, ring C=N stretch). In addition, weak residual signals from the O2-cis@S₀ protomer remain, suggesting minor contributions from species that after photoexcitation undergo (i) IC from the S₁ state or (ii) reverse ISC from T₁ into S₀, respectively. The effective removal of non-excited species by the tickle RF pulse confirms that the IR spectrum exclusively originates from photoexcited LFH⁺ ions. These findings provide direct spectroscopic evidence for the generation of the vibrationally relaxed triplet state protomer under cryogenic ion trap conditions.

To assess the stability of this triplet state, we perform time-resolved Vis-IRPD measurements in which the delay between Vis excitation and IR probing is varied (Figure S6). The characteristic O2-H bands of the O2-cis@T₁ protomer are retained with nearly constant intensity even at the maximum trapping time of 48 ms. This result indicates that the T₁ state persists for hundreds of milliseconds or longer, similar to theoretical calculations for neutral isoalloxazine, which predict a radiative lifetime of approximately 116 ms.³³ Such an extended lifetime is likely attributed to the quenching-free nature of the cryogenic ion trap environment, where collisional deactivation by molecular oxygen or triplet-triplet annihilation are not possible. In addition, to estimate the population of the T₁ state, we compare the IRPD intensities of the O2-H stretching bands assigned to O2-cis@T₁ and O2-cis@S₀ (Figure S7). From this analysis, approximately 30% of the LFH⁺ ions are found to undergo photoexcitation (S₁ ← S₀) followed by ISC (Tₙ ← S₁), ultimately reaching the T₁ state.

To investigate the origin of such a high ISC efficiency, coupled-cluster calculations are performed in the singlet and triplet states of O2-cis, including geometry optimization and frequency calculations of the ground and excited states to estimate their excitation energies. Single point excitation energies of the low-lying excited states (S₁, S₂, S₃, T₁, T₂) at the optimized geometry of S₁ state are reported in Table 1, Figure 3b and details of the calculations are available in the Supporting Information. The zero-energy reference is consistently taken as the energy of the S₀ state at its optimized geometry. At the ground state minimum of the O2-cis protomer, the ¹ππ* state is the lowest singlet excited state (S₁) with an adiabatic excitation energy of 2.87 eV (23158 cm⁻¹, corrected for harmonic zero-point vibrational energy), only 30 cm⁻¹ above the experimental band origin, as already reported in our previous publications.¹⁰, ¹¹ At the optimized geometry of S₁, the second excited singlet state (S₂) with ππ* character (HOMO-1 to LUMO) is predicted +0.49 eV above S₁, while the third excited singlet state (S₃) has n₀π* electronic character lying more than +1.2 eV above S₁. Geometry optimization of the ¹n₀π* state leads to a decrease of its adiabatic excitation energy along with a mixing between the lone pair orbitals of O₂, N₁, and N₅, which nevertheless remains +0.54 eV above S₁. Because of this large energy gap, the ¹n₀π* state is not expected to be involved in the deactivation of the ¹ππ* state in the vicinity of its band origin. However, at the optimized geometry of the S₁ state, the first two triplet states have the same electronic configurations (³ππ*) as S₁ (HOMO to LUMO).
transition) and S₂ (HOMO-1 to LUMO). T₁ lies -0.48 eV below S₁ whereas the energy difference between S₁ and T₂ is very small, within 0.005 eV. Although spin-orbit coupling between states of the same symmetry is formally zero for this planar molecule according to the El-Sayed rules,³⁴ the T₂/S₁ degeneracy and spin-vibronic coupling involving out-of-plane deformations of the aromatic ring may enhance the ISC rate by orders of magnitude.³⁵ For instance, in psoralen molecule, a coumarin derivative obtained by addition of a fused furan ring, ISC outcompetes the fluorescence and triplet formation becomes the dominant deactivation process.³⁶

In summary, we demonstrate herein that H₂-tagging visible-pump infrared-probe photodissociation (Vis-IRPD) spectroscopy enables the direct observation of the triplet state of protonated lumiflavin (LFH⁺) under cryogenic ion trap conditions. Upon visible excitation, new IR transitions appear that closely match those calculated for the O2-cis@T₁ protomer, supporting the scenario that upon S₁ excitation the electronically ground-state species (O2-cis@S₀) undergoes ISC and relaxes subsequently into the vibrational ground state of the T₁ electronic state. Moreover, we implement a tickle RF approach into the spectroscopy method (Vis-tRF-IRPD), which removes non-excited ions from the trap, allowing us to isolate the IR signature of the photoexcited population and more clearly confirm the formation of the O2-cis@T₁ protomer. More generally, this H₂-tagging Vis-IRPD approach can overcome the limitations of conventional UV-IR methods, offering a broadly applicable strategy for detecting triplet state species of various molecular ions. For flavins, this work provides a fundamental platform for future investigations of ESPT pathways (in Sₙ or Tₙ). Our findings show that photoexcitation alone does not induce PT from the O₂ site, indicating that additional factors such as microsolvation³⁷ may be necessary to lower the reaction barrier and promote a protonation-site switch driven by changes in proton affinity. A deeper investigation of such proton-transfer dynamics will contribute to mechanistic insight into the photochemical reactivity of biological flavoproteins such as FAP, which may inspire the development of solar-to-fuel conversion strategies based on photoactivated enzymes.³⁸, ³⁹

## Associated contents

### Supporting information

Experimental (S-1) and computational (S-2) methodology, vibronic spectra of LFH⁺ with/without H₂-tagging (S-3), calculated vibrational frequencies of all LFH⁺ protomers (S-4), energy values of O₂ protomers for cis → trans isomerization (S-5), time-resolved Vis-IRPD spectra (S-6), and population analysis (S-7).

## Acknowledgements

This work is supported in part by KAKENHI (JP21H04674, JP21K14585) and the Core-to-core program of JSPS (JPJSCCA20210004, JPJSCCA2024002), World Research Hub Initiative in Institute of Science Tokyo, and Deutsche Forschungsgemeinschaft (DFG, DO 729/10). The computations are performed at the Research Center for Computational Science, Okazaki, Japan, and the HPC resources from the "Mésocentre" computing center of CentraleSupélec and École Normale Supérieure Paris-Saclay, supported by CNRS and Région Île-de-France (http://mesocentre.centralesupelec.fr/) and the HPC
1 resources MAGI from Université Sorbonne Paris Nord, France.

2

3 **Notes**

4 The authors declare no competing financial interest.

5

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## Tables

<table><caption>**Table 1.** Energies [eV] of the low-lying singlet and triplet states at the S<sub>1</sub> optimized geometry of the O2-cis protomer of LFH<sup>+</sup>. (zero energy taken at S<sub>0</sub> optimized geometry)</caption><thead><tr><th></th><th>S<sub>0</sub></th><th>T<sub>1</sub> (³π<sub>h</sub>π<sup>*</sup>)</th><th>S<sub>1</sub> (¹π<sub>h</sub>π<sup>*</sup>)</th><th>T<sub>2</sub> (³π<sub>h-1</sub>π<sup>*</sup>)</th><th>S<sub>2</sub> (¹π<sub>h-1</sub>π<sup>*</sup>)</th><th>S<sub>3</sub> (¹nπ<sup>*</sup>)</th></tr></thead><tbody><tr><td>S<sub>1</sub> geometry</td><td>0.28</td><td>2.50</td><td>2.98</td><td>2.98</td><td>3.47</td><td>4.18</td></tr></tbody></table>

## Figures

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Structure of lumiflavin (LF). The clockwise atom numbering is according to IUPAC. The substituent of N10 site defines the specific flavin molecule: methyl = lumiflavin (LF). The six considered protomers of protonated lumiflavin (LFH<sup>+</sup>) are distinguished by the protonation site and orientation: N1; O2-cis; O2-trans; O4-cis; O4-trans; N5.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>b) IRPD (VisOFF)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>c) Computed IR spectra</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d) Vis-IRPD</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e) Vis-tRF-IRPD</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** (a, c) IR spectra computed for LFH⁺ protomers considering H₂-tagging at the respective O–H and N–H groups. The two H₂-tags are specifically attached to the O–H or N–H sites. The IR spectra are computed using density functional theory (DFT) at the ωB97X-D/cc-pVTZ level. Mode-specific scaling factors are employed; 0.950 for O–H and N–H stretching, 0.952 for C=O stretching and 0.960 for aromatic C=N stretching-dominated (coupling with N–H in-plane-bending) modes. (b, d, e) IRPD spectra of H₂-tagged LFH⁺ ions measured by IRPD, Vis-IRPD, Vis-tRF-IRPD spectroscopy, respectively. Color coding</figcaption></figure>
corresponds to the various protomers, and overlapping bands are partitioned according to approximate

ratios.

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<figure><img src="image_8.png" /><figcaption>b) Calculated energy levels</figcaption></figure>

<figure><img src="image_9.png" /></figure>

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**Figure 3.** (a) Schematic energy level diagram illustrating Vis-IRPD spectroscopy. Initially, the ion trap contains predominantly the O2-cis protomer of LFH⁺ along with a minor population of the N1 protomer. The wavelength of the VIS laser is fixed to the vibronic transition of the O2-cis protomer (blue arrow, λ<sub>exc</sub> = 23448 cm⁻¹). Among the optically excited species, those undergoing intersystem crossing (ISC) from the S₁ state to higher triplet states (T<sub>n</sub>) subsequently relax via internal conversion and vibrational cooling to the T<sub>1</sub> (ν = 0) state. During the IR probing stage, the ion trap contains three species: (i) O2-cis@T<sub>1</sub> protomers, (ii) O2-cis@S<sub>0</sub> protomers (either not absorbing or having relaxed back to the S<sub>0</sub> state), (iii) N1@S<sub>0</sub> protomers (which do not absorb in the considered visible range<sup>10</sup>). The IR laser (red arrows) is scanned across the fundamental vibrational transitions of all species (ν = 0 → 1). (b) Difference energies [eV] between S<sub>1</sub> and S<sub>0</sub>, and T<sub>1</sub>, and T<sub>2</sub> at the optimized geometry of O2-cis@S<sub>1</sub>. Computations at the SCS-CC2/aug-cc-pVDZ level (Table 1 and Figure. S8).
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