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. 2026 Aug 27;17(38):10887–10894. doi: 10.1021/acs.jpclett.6c02005

Solvent-Controlled Product Branching in Riboflavin Uncovered by Time-Resolved Infrared and Transient Absorption Spectroscopy

Meiyue Liu †, Juno Underhill †, Jacob F Jones †, Aditya G Rao †, Caleb J C Jordan †, Somnath Kashid †, James A Smith †, J L Ross Anderson ‡, Adrian J Mulholland †, Basile F E Curchod †, Thomas A A Oliver †,*
PMCID: PMC13618035  PMID: 42917853

Abstract

Riboflavin (vitamin B2) is an important visible chromophore in flavoproteins and a popular photosensitizer in organic photocatalysis. Despite its importance, a complete understanding of riboflavin’s full photocycle remains undetermined, specifically the factors that govern the relative yields of its singlet and triplet excited statesboth playing important roles in electron transfer chemistry. A combination of transient absorption spectroscopy and time-resolved infrared spectroscopy is used to address this problem, tracking the photoexcited states of riboflavin over 9 decades of time under the same experimental conditions. These studies reveal that the triplet quantum yield increases by a factor of 2.7 by merely changing the solvent from DMSO to H2O. Electronic structure calculations show that this enhancement is not likely to originate from a change in the associated singlet and triplet state potential surfaces. Instead, we discern solvent-dependent branching, with more efficient internal conversion in DMSO, likely mediated by its stronger hydrogen bonding acceptor character than water. This strong modulation shows that the triplet branching, and thus the reservoir of long-lived states required for electron transfer reactions, can be carefully tuned by the immediate environment of the flavin chromophore.

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graphic file with name jz6c02005_0005.webp

Riboflavin, more commonly known as vitamin B2, is the archetypal flavin molecule. As the precursor to flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it underpins metabolic electron transfer reactions and contributes to processes that mitigate oxidative stress. − The aromatic isoalloxazine core is the visible chromophore of riboflavin (see chemical structure in Figure (a)) and has a significant absorption cross-section in the visible, peaking at ∼450 nm (Figure (b,c)). The extended π-electron heteroaromatic structure of riboflavin can stabilize large changes in electron density and accommodate shifts in protonation, giving rise to four readily accessible redox states. − Resultantly, flavins are common photoreceptors in LOV (light, oxygen, and voltage)-sensitive domains of proteins, transducing absorbed light into conformational changes that regulate downstream signaling. − Their long-wavelength absorption spans a large portion of the blue part of the visible spectrum, enabling their photoreception function in many natural cryptochromes − and Blue Light Using FAD (BLUF) domain − proteins. Combined, these properties make flavins versatile visible chromophores ,, and redox mediators, ,,, and thus popular photosensitizers or photocatalysts in photoinduced electron transfer reactions. − ,− ,−

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(a) Molecular structures of riboflavin and lumiflavin. The isoalloxazine rings are labeled I, II, and III for ease of reference. (b) Experimental absorption and fluorescence spectra of riboflavin in DMSO. (c) Comparison between the experimental absorption spectrum of riboflavin in water and QM/MM calculations (riboflavin within a shell of 150 QM water molecules) at the LR-TDDFT/TDA/ωB97X-D3/def2-SVP level of theory (broadened with a Gaussian function using a fwhm = 0.165 eV); the latter is normalized to λ = 222 nm. The experimental riboflavin fluorescence spectrum in water is also shown for completeness. The calculated LR-TDDFT/TDA spectrum was shifted by the difference between the LR-TDDFT/TDA and RI-CC2 calculations (0.6 eV). (d) Natural transition orbitals for the two lowest energy π* ← π transitions of riboflavin from a sample snapshot in the QM/MM trajectory (S1 and S3 states in the Franck–Condon region, respectively), where the arrows connect the hole and particle NTOs associated with each electronic excitation.

Despite the significance of riboflavin, previous time-resolved studies of flavins have either focused on ‘early-’ (femtoseconds–nanoseconds) − or ‘late-’time (microseconds and longer) − ,− kinetics, but none have reported on the complete photoinduced relaxation cycle of riboflavin in solution. Therefore, key information about the interconversion between singlet and triplet states is missing, e.g., the associated intersystem crossing rate constant or associated quantum yieldvalues that remain disputed in water. As both singlet and triplet states can play significant roles in riboflavin electron-transfer reactions, it is important that these key photophysical parameters are rigorously established. To bridge the many decades of time required to follow the riboflavin photoexcited state evolution, we apply transient absorption (TA) and time-resolved infrared (TRIR) ultrafast spectroscopic experiments capable of seamlessly following photoinduced evolution between 150 fs and 0.1 ms under the same experimental conditions. We chose to contrast the photophysics of riboflavin in water, for biological relevance, and in DMSO because its aprotic nature and high dielectric constant allow the molecule to adopt the same neutral oxidized form as in H2O, and the two solvents have different hydrogen-bonding properties. The experimental photophysical findings are supported by quantum chemical calculations. Combined, our studies demonstrate that small changes in the solvent environment lead to a ∼2.7× variation in the triplet quantum yield, despite only small changes to the intersystem crossing rate. The change in intersystem quantum yield is instead underpinned by the fine balance between multiple competing nonradiative decay pathways, thereby demonstrating that the environment can strongly modulate the photochemical product branching in riboflavin.

The experimental absorption and fluorescence spectra of riboflavin in water and DMSO are similar (Figure (b,c)), exhibiting two main absorption bands centered at ∼450 and ∼350 nm. The calculated absorption spectrum of riboflavin, from quantum mechanical/molecular mechanics (QM/MM) calculations, reproduces the main features in the experimental absorption spectrum (Figure (c)). These are dominated by two long-wavelength maxima at 355 nm (3.49 eV) and 430 nm (2.88 eV), associated with π* ← π transitions, S3 and S1 states, respectively, in the Franck–Condon region. QM/MM calculations also show two 1 nπ* states within this long-wavelength range (the full stick spectra are given in Figure S15). The two lowest energy 1ππ* states are delocalized over the isoalloxazine ring and include only minimal transition density on the ribityl tail (Figure (d)). Conversely, the low-lying 1 nπ* states involve promotions from nonbonding orbitals localized on the nitrogen and oxygen atoms to π* orbitals on the isoalloxazine ring, respectively (Figure S12 in the SI).

The photoexcited state decay of riboflavin is explored with TA spectroscopy probing 9 decades of time in both DMSO and water (Figure (a) and (c), respectively). The full details of our experiment are given in the SI, with the apparatus drawing inspiration from the LIFEtime experiment at the UK’s Central Laser Facility (STFC). , Note that data were acquired with and without dissolved oxygen present, and only the latter is shown in the main text. The negative transients in the TA data at ∼450 nm and ∼555 nm are assigned to ground-state bleach (GSB) and stimulated emission (SE) signals, based on their close correspondence to the respective steady-state spectra. In both solvents, there are also accompanying positive transient features at ∼370, ∼500, and ∼730 nm associated with the riboflavin S1 excited-state absorption (ESA).

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TA spectra at selected time delays for riboflavin in (a) water and (c) DMSO, photoexcited at respective absorption maxima of 445 and 448 nm. Kinetics associated with three representative probe wavelengths for riboflavin are shown in (b) water and (d) DMSO. Overlaid dashed lines are kinetic fits to the data. The data shown are for solutions purged of dissolved oxygen. Data where the dissolved oxygen was not sparged from solutions are shown in Figure S8.

In water (Figure (a)), the SE decays on a time scale of several nanoseconds, as expected from the known nanosecond fluorescence lifetime. On a commensurate time scale, the S1 ESA at λ > 470 nm makes a marked spectral evolution and by t > 6 ns is replaced by a new triple-peaked positive transient that persists for microseconds. The ESA at λ < 400 nm also evolves on the same time scale, but in a less pronounced way. In DMSO, a similar evolution associated with the S1 ESA and SE are observed and, as expected, decay on a nanosecond time scale. The spectral shifting of the positive transient at λ > 500 nm in H2O observed on the several-nanosecond time scale is not as significant in the DMSO data (Figure (c)). Further, the intensity of the long-lived (microseconds) transient is less pronounced in DMSO than in water, implying a smaller product branching into this species. We assign the microsecond-lived transient to triplet riboflavin, based on the close resemblance to the known triplet absorption spectrum and the strong oxygen sensitivity observed in its kinetics (Figure S8).

To investigate whether the riboflavin photophysics are altered following excitation to the S3 state, instead of the S1 state, the pump wavelength dependence of the TA signal was examined. Within our instrument response (145 fs), no additional transient features were observed compared to direct excitation of the S1 state (see Figure S5), indicating that the S3 → S1 internal conversion is very fast, and the subsequent decay kinetics mimic those following direct S1 state excitation (Figure S7). Our observation of ultrafast internal conversion is consistent with prior experimental studies that examined only the very earliest time kinetics using an instrument response of 50–60 fs.

Given the clear separation of time scales associated with the observed TA spectral evolution, the photoexcited riboflavin kinetics were extracted by examining the temporal profiles at three different probe wavelengths primarily associated with different species (Figure (b) and (d)): (i) 360 nm is dominated by the S1 ESA; (ii) the peak of the GSB at λ ∼ 450 nm monitors the return of molecules back to the ground electronic state and contains some contribution from the S1 ESA; (iii) ∼550 nm is primarily associated with the SE decay, but at later time delays also overlaps with the triplet absorption.

In water, the S1 ESA kinetics at 360 nm are modeled by an exponential rise (50 ± 30 ps) followed by an exponential decay (5.0 ± 0.1 ns). The rise is likely due to vibrational cooling, nuclear reconfiguration in the riboflavin S1 state, or potentially a change in oscillator strength upon excited-state evolution; the latter has been suggested previously. Our theoretical calculations of lumiflavin (structure shown in Figure (a)), which has been shown to be an excellent model system for riboflavin (truncating the ribityl tail at the N10 position), − reveal that the S1 state minimum is stabilized by ∼0.3 eV relative to the Franck–Condon S1 geometry (Figure S17), indicating there is appreciable nuclear reorganization on the S1 state . The nanosecond decay time constant is in excellent agreement with the reported fluorescence lifetime in water (τF = 5.1 ± 0.1 ns). Fits to this probe wavelength exclude the very small amplitude of the overlapping triplet that contributes at longer time delays.

The GSB recovery (kinetics taken at the bleach maximum, λ = 445 nm) is well described by exponential rise (37 ± 7 ps) followed by biexponential decay (5.0 ± 0.1 ns and 23 ± 1 μs). The relative amplitude of the biphasic bleach recovery is directly proportional to the triplet quantum yield (ΦT), as the triplet state decay is the sole contributor to the microsecond phase of the bleach recovery (see SI eqs S2–S4). The associated fits to data return normalized pre-exponential factors of 0.33 and 0.67 for the nanosecond and microsecond decay components. The latter amplitude is thus associated with the triplet quantum yield (using eq S3). Our results align with the upper limit of the wide range of triplet quantum yields previously reported for riboflavin in water (0.38–0.7). ,,,

Similar probe wavelengths were chosen to extract the kinetic behavior of riboflavin in DMSO, with the exact wavelengths adjusted to account for solvent-induced shifts in the transient maxima. The S1 ESA (350 nm) rises with a 7 ± 2 ps time constant and subsequently decays with τ = 3.0 ± 0.1 ns. The GSB (448 nm) recovers biexponentially with time constants of 3.43 ± 0.08 ns and 13 ± 1 μs (Figure (d)). The nanosecond ESA decay and GSB recovery are comparable with the literature fluorescence lifetime (3.2 ± 0.1 ns) in DMSO. A global analysis yielded very similar time constants in both water and DMSO (Figures S9 and S10), demonstrating the validity of the kinetic analysis performed at selected probe wavelengths and confirming that the triplet absorption rises with the decay of the singlet ESA.

In DMSO, the normalized pre-exponential factors associated with the biexponential GSB recovery are 0.76 and 0.24, for nanosecond and microsecond components, respectively. However, we note that the amplitude of the microsecond bleach recovery can be better estimated when the bleach is fully spectrally separated from any other overlapping feature. Thus, in our TA data, the initial bleach intensity at t = 0 fs will be underestimated due to the overlapping strong oppositely signed S1 ESA, despite being largely unshaped by overlapping ESAs (Figure S4).

A previous TRIR study of riboflavin in DMSO showed that the mid-infrared fingerprint region contains multiple spectrally isolated ground-state bleach vibrations. This prior study only measured to t = 800 ps and was thus unable to capture the intersystem crossing kinetics. Here we use TRIR spectroscopy with the same laser platform as used for TA experiments and can thus examine the multiple phases of bleach recovery and provide a secondary measure of the triplet branching. Our data span 1380–1720 cm–1 composed from two overlapping probe windows (Figure (a,b)) in DMSO-d 6 and DMSO. The vibrational band assignments of riboflavin have been previously determined and are thus not examined here.

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TRIR spectra of riboflavin for the displayed time delays in (a) DMSO-d 6 and (b) DMSO. The respective solvent-subtracted FTIR spectra (inverted) are also overlaid for reference. Kinetic traces at three different probe wavenumbers are displayed for riboflavin dissolved in (c) DMSO-d 6 and (d) DMSO.

The two dominant and isolated bleaches in TRIR data at 1547 and 1711 cm–1 (Figure (b)) in DMSO were simultaneously fitted to biexponential decay functions, yielding lifetimes of 3.04 ± 0.07 ns and 5.0 ± 0.6 μs (fit to data overlaid in Figure (d)). The associated normalized pre-exponential factors are 0.81 and 0.19 for nanosecond and microsecond decay components. The ratio is slightly higher (by 5%) compared to those extracted from the TA data, for the reasons highlighted above.

We note that TRIR data recorded in fully deuterated DMSO yield a shorter riboflavin excited-state lifetime of 2.66 ± 0.03 ns (Figure (c)) compared to regular DMSO; however, the ratio of pre-exponential factors remains similar upon solvent deuteration (0.81 and 0.19 in DMSO and 0.82 and 0.18 in DMSO-d 6). A possible explanation for this difference in riboflavin S1 lifetime is given in the SI and echoes prior work by Maillard et al. and Kubitz et al. TRIR data in H2O/D2O were not acquired due to the order of magnitude lower riboflavin solubility in water and very strong mid-IR solvent absorption. Due to the lack of the comparator TRIR data, our determination of the triplet quantum yield relies .

Comparing the ISC branching in water and DMSO, less than one-quarter of the excited singlet riboflavin molecules intersystem cross to triplet riboflavin (ΦT = 0.24) in the latter solvent, contrasting with our far higher estimated triplet quantum yield of 0.66 for riboflavin in water. This difference in triplet branching is surprisingly not reflected in the respective fluorescence quantum yields (ΦF) of 0.24 ± 0.02 and 0.28 ± 0.04 in DMSO and water, respectively, and in good agreement with prior reports. Using the combination of experimentally directly determined (τF and ΦF) and derived (ΦT) photophysical quantities, k f, k ISC, and k IC can be determined for riboflavin in H2O and DMSO (summarized in Table ) using eqs S1, S4 and S5. The relative ratios of the three derived rate constants in the two solvents (k H2O/k DMSO) are also given in Table , enabling direct comparison of the solvent-dependent rates. The fluorescence rate constant is similar for riboflavin in both solvents and in good agreement with independent estimates from a Strickler–Berg analysis (comparison given in Table S1). The difference in radiative rate constants cannot entirely be rationalized by the change in solvent refractive index and may instead reflect some solvent-dependent mixing between 1 nπ* and the 1ππ* (S1) state as previously suggested by Weigel et al. Changes in hydrogen bonding will modulate the energy of the 1 nπ* state relative to the 1ππ* state, thereby altering the level of mixing. The net result is to change the oscillator strength of the transition, with greater nπ* state (optically dark) character leading to a decrease in the radiative rate constant. Conversely, the nonradiative rate constants, k IC and k ISC, vary, with intersystem crossing dominating nonradiative decay in water, but internal conversion for DMSO.

1. Summary of Experimentally Determined and Derived Photophysical Quantities for Riboflavin in H2O and DMSO .

Experimentally Derived Parameter H 2 O DMSO
Fluorescence lifetime, τF/ns 5.1 ± 0.1 3.2 ± 0.1
S1 lifetime/ns 5.0 ± 0.1 3.0 ± 0.1
Fluorescence quantum yield, ΦF 0.28 ± 0.04 0.24 ± 0.02
Triplet quantum yield, ΦT 0.66 ± 0.03 0.24 ± 0.04
Fluorescence rate constant, k F/s–1 (5.6 ± 0.8) × 107 (8.0 ± 0.7) × 107
ISC rate constant, k ISC/s–1 (1.3 ± 0.1) × 108 (8.3 ± 1.4) × 107
IC rate constant, k IC/s–1 (1.0 ± 1.1) × 107 (1.7 ± 0.2) × 108
k F(H2O/DMSO) 0.7 ± 0.1
k ISC(H2O/DMSO) 1.6 ± 0.2
k IC(H2O/DMSO) 0.06 ± 0.07
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a

Unless otherwise stated, the values given were determined in this study.

b

From ref .

c

Determined from TA measurements, and these time constants were used to calculate the associated rate constants.

Quantum chemical calculations on lumiflavin were used to investigate the molecular origin of the experimentally derived intersystem crossing rate constants in riboflavin. The S0 and S1 minimum energy geometries of lumiflavin were obtained using DFT/ωB97X-D3/def2-SVP and LR-TDDFT/TDA/ωB97X-D3/def2-SVP, respectively, including an implicit conductor-like polarizable continuum solvent model (CPCM) for both H2O and DMSO. A geodesic interpolation pathway was then constructed to connect the S0 and S1 state minimum energy structures, and electronic energies were obtained with RI-CC2/def2-TZVP for S0 and S1, as well as T1, T2, and T3 states in H2O and DMSO (see the SI for full details).

The interpolation pathways shown in Figure indicate that the electronic energies of riboflavin are similar in H2O and DMSO in the vicinity of the S1 (ππ*) minimum. The triplet state with 3 nπ* character, T3 in the present case, which would allow a nonzero spin–orbit coupling with the S1 state (ππ*), is substantially higher in energy for both solvents. In the gas phase, the 3 nπ* state is lower in energy and nearly degenerate with the S1 (ππ*) state in the Franck–Condon region (see Figure S18). These observations are reminiscent of the earlier work by Salzmann et al. on three flavin-related compounds, highlighting that the 3 nπ* state is destabilized in an aqueous environment. Our calculations confirm that a similar behavior can be expected for riboflavin in DMSO, rationalizing the similar ISC rate constant determined from experiments (see Table ). Salzmann et al. explained the nonzero intersystem crossing for flavin in aqueous solution by coupling between S1 (ππ*) and a low-lying 3ππ* state (in the present case, the T2 state), mediated by spin–orbit vibronic coupling. Their intersystem crossing rates for flavin in aqueous solution obtained with DFT/MRCI were on the order of 107–108 s–1, in close agreement with the experimentally determined rate constants in the present study for H2O and DMSO.

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Geodesic interpolation pathways between the S0 and S1 (ππ*) lumiflavin minima in (a) H2O and in (b) DMSO. Electronic energies for the singlet and triplet states presented were obtained with RI-CC2/def2-TZVP using COSMO for implicit solvation, based on optimized geometries obtained with the (LR-TD)­DFT/TDA/ωB97X-D3/def2-SVP/CPCM level of theory (see computational details in SI).

As the experimental analysis in Table indicates, a nonradiative S1 → S0 pathway must be highly competitive with intersystem crossing and thus further modulates the triplet quantum yield. Electronic structure calculations indicate that it is not likely to originate from changes in the underlying potential energy surfaces (see Figures S16 and S17). Considering the hydrogen-bonding properties of the two solvents, water can act as both a hydrogen-bond donor and acceptor (as illustrated by analyzing our QM/MM output for riboflavin in water, see Figure S13 and Table S2), whereas DMSO acts only as a hydrogen-bond acceptor. Notably, DMSO has a hydrogen-bond acceptor strength more than twice that of water, and water is an equally strong hydrogen-bond donor and acceptor, placing the two solvents at contrasting extremes of hydrogen-bonding character. Thus, we speculate that H-bonding between the flavin N3–H group (on isoalloxazine ring III) and DMSO solvent molecules somehow mediates internal conversion to S0. As water is a weaker H-bond acceptor, this pathway is not as favored and is almost completely turned off (as evident by the close-to-unity sum of fluorescence and triplet yields), giving rise to the significant contrast in internal conversion rates, k IC(H2O/DMSO) = 0.06 ± 0.07, and thus altered triplet yield.

The excited-state photochemistry of riboflavin (vitamin B2) in DMSO and water was studied with a combination of TA and TRIR spectroscopy, tracking kinetics over nine decades of time, and QM/MM calculations. This approach enabled us to determine the entire photocycle for riboflavin in solution and demonstrate that the triplet quantum yield is strongly dependent on the solvent environment and ∼2.7× larger in water than DMSO. We conclude that the main difference in triplet branching, instead, originates due to a competitive S1 → S0 internal conversion pathway that is likely enhanced by the hydrogen-bonding acceptor nature of DMSO with the riboflavin N3–H imide bond. H2O is a weaker hydrogen-bond acceptor, and thus this pathway is less favored in water, which in turn significantly impacts the triplet quantum yield. This remarkable sensitivity to solvent in the important riboflavin molecule likely underpins why it and related flavin derivatives are common cofactors in multiple different proteins but can be tuned by the surrounding environment to serve different photophysical functions.

Supplementary Material

jz6c02005_si_001.pdf (9MB, pdf)

Acknowledgments

TAAO thanks Greg Greetham for discussions about the LIFEtime instrument at the Central Laser Facility, STFC. ML, JU, AGR, SK, JAS, JLRA, AJM, and TAAO thank BBSRC for funding (BB/W003449/1). JFJ, CJCJ, JAS, BFEC, and TAAO acknowledge funding from EPSRC for program grant EP/V026690/1. TAAO is grateful to the Royal Society for support via a University Research Fellowship (UF1402310, URF\R\201007) and Research Fellows Enhancement Award (RF\ERE\210045). BFEC acknowledges funding from EPSRC for program grant EP/X026973/1. ML, JFJ, and JU acknowledge PhD funding from the Colin Western Bequeath, University of Bristol Scholarship, and EPSRC doctoral training award, respectively.

The data underlying this study are openly available at Zenedo at doi: 10.5281/zenodo.22001301.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.6c02005.

  • Experimental and theoretical methods; Additional TA and TRIR data and analysis; Further computational data and analysis (PDF)

#.

M.L. and J.U. contributed equally to this work.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jz6c02005_si_001.pdf (9MB, pdf)

Data Availability Statement

The data underlying this study are openly available at Zenedo at doi: 10.5281/zenodo.22001301.

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