Nontargeted Screening of Contaminants of Emerging Concern in the Glen Valley Wastewater Treatment Plant, Botswana.

There is growing concern about the prevalence and impact of contaminants of emerging concern (CECs). The environmental monitoring of CECs has, however, been limited in low- and middle-income countries due to the lack of advanced analytical instrumentation locally. In the present study we employed a nontargeted and suspect screening workflow via liquid chromatography coupled with high-resolution mass spectrometry (HRMS) to identify known and unknown pollutants in the Glen Valley wastewater treatment plant, Botswana, complemented by analysis of groundwater samples. The present study represents the first HRMS analysis of CECs in water samples obtained in Botswana. Suspect screening of 5942 compounds qualitatively identified 28 compounds, including 26 pharmaceuticals and two illicit drugs (2-ethylmethcathinone and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol). Nontargeted analysis tentatively identified the presence of 34 more compounds including (5ξ)-12,13-dihydroxypodocarpa-8,11,13-trien-7-one, 12-aminododecanoic acid, atenolol acid, brilliant blue, cyclo leucylprolyl, decanophenone, DL-carnitine, N,N'-dicyclohexylurea, N4-acetylsulfamethoxazole, NP-003672, and 24 polyethylene glycol polymers. The highest number of detections were in influent wastewater (26 CECs) followed by effluent wastewater (10 CECs) and, lastly, groundwater (4 CECs). Seventeen CECs detected in the influent water were not detected in the effluent waters, suggesting reduced emissions due to wastewater treatment. Two antiretroviral compounds (abacavir and tenofovir) were detected in the influent and effluent sources. This suggests that wastewater treatment plants are a major pathway of chemical pollution to the environment in Botswana and will help inform prioritization efforts for monitoring and remediation that is protective of these key ecosystems. Environ Toxicol Chem 2024;43:52-61. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Photodissociative decay pathways of the flavin mononucleotide anion and its complexes with tryptophan and glutamic acid.

Flavin mononucleotide (FMN) is a highly versatile biological chromophore involved in a range of biochemical pathways including blue-light sensing proteins and the control of circadian rhythms. Questions exist about the effect of local amino acids on the electronic properties and photophysics of the chromophore. Using gas-phase anion laser photodissociation spectroscopy, we have measured the intrinsic electronic spectroscopy (3.1-5.7 eV) and accompanying photodissociative decay pathways of the native deprotonated form of FMN, i.e. [FMN-H]- complexed with the amino acids tryptophan (TRP) and glutamic acid (GLU), i.e. [FMN-H]-·TRP and [FMN-H]-·GLU, to investigate the extent to which these amino acids perturb the electronic properties and photodynamics of the [FMN-H]- chromophore. The overall photodepletion profiles of [FMN-H]-·TRP and [FMN-H]-·GLU are similar to that of the monomer, revealing that amino acid complexation occurs without significant spectral shifting of the [FMN-H]- electronic excitations over this region. Both [FMN-H]-·TRP and [FMN-H]-·GLU are observed to decay by non-statistical photodecay pathways, although the behaviour of [FMN-H]-·TRP is closer to statistical fragmentation. Long-lived FMN excited states (triplet) are therefore relatively quenched when TRP binds to [FMN-H]-. Importantly, we find that [FMN-H]-, [FMN-H]-·TRP and [FMN-H]-·GLU all decay predominantly via electron detachment following photoexcitation of the flavin chromophore, with amino acid complexation appearing not to inhibit this decay channel. The strong propensity for electron detachment is attributed to excited-state proton transfer within FMN, with proton transfer from a ribose alcohol to the phosphate preceding electron detachment.

Photochemical Degradation of the UV Filter Octyl Methoxy Cinnamate Probed via Laser-Interfaced Mass Spectrometry.

Octyl methoxycinnamate (OMC) is a common UVA and UVB filter molecule that is widely used in commercial sunscreens. Here, we used gas-phase laser photodissociation spectroscopy to characterise the intrinsic photostability and photodegradation products of OMC by studying the system in its protonated form, i.e., [OMC·H]+. The major photofragments observed were m/z 179, 161, and 133, corresponding to fragmentation on either side of the ether oxygen of the ester group (m/z 179 and 161) or the C-C bond adjacent to the ester carbonyl group. Additional measurements were obtained using higher-energy collisional dissociation mass spectrometry (HCD-MS) to identify fragments that resulted from the breakdown of the vibrationally hot electronic ground state. We found that the m/z 179 and 161 ions were the main fragments produced by this route. Notably, the m/z 133 ion was not observed through HCD-MS, revealing that this product ion is only produced through a photochemical route. Our results demonstrate that UV photoexcitation of OMC is able to access a dissociative excited-state surface that uniquely leads to the rupture of the C-C bond adjacent to the key ester carbonyl group.

Probing the electronic relaxation pathways and photostability of the synthetic nucleobase Z via laser interfaced mass spectrometry.

The photostability of synthetic (unnatural) nucleobases is important in establishing the integrity of new genetic alphabets, and critical for developing healthy semisynthetic organisms. Here, we report the first study to explore the photostability and electronic decay pathways of the synthetic nucleobase, Z (6-amino-5-nitro-2(1H)-pyridone), combining UV laser photodissociation and collisional dissociation measurements to characterise the decay pathways across the region from 3.1-4.9 eV. Photoexcitation across this region produced the m/z 138 ion as the dominant photofragment, mirroring the dominant fragment produced upon higher-energy collisional excitation. Analysis of the ion-yield production curve profile for the m/z 138 ion indicates that it is produced following ultrafast excited state decay with boil off of the OH functional group of Z from the hot electronic ground state. Electronic structure calculations provide physical insight into why this is the dominant fragmentation pathway, since a node in the electron density along the C-OH bond is found for all tautomers of Z. While the dominant decay pathway for Z is consistent with ultrafast excited state decay, we also identify several minor dissociative photochemistry decay pathways, associated with intrinsic photoinstability. The results presented here can be used to guide the development of more photostable synthetic nucleobases.

Photostability of the deprotonated forms of the UV filters homosalate and octyl salicylate: molecular dissociation versus electron detachment following UV excitation.

While common molecular anions show a strong propensity to undergo electron detachment upon UV excitation, this process often occurs in competition with molecular ion dissociation. The factors that affect the balance between these two major possible decay pathways have not been well understood to date. Laser photodissociation spectroscopy of the deprotonated forms of the UV filter molecules, Homosalate (HS) and Octyl Salicylate (OS), i.e. [HS - H]- and [OS - H]-, was used to acquire gas-phase UV absorption spectra for [HS - H]- and [OS - H]-via photodepletion from 3.0-5.8 eV. No photofragmentation (i.e. dissociation of the ionic molecular framework) was observed for either [HS - H]- and [OS - H]- following photoexcitation, revealing that electron loss entirely dominates the electronic decay pathways for these systems. High-level quantum chemical calculations were used to map out the excited states associated with [HS - H]- and [OS - H]-, revealing that the minimum-energy crossing points (MECPs) between the S1 and S0 states are located in elevated regions of the potential energy surface, making internal conversion unlikely. These results are consistent with our experimental observation that electron detachment out-competes hot ground state molecular fragmentation. More generally, our results reveal that the competition between molecular dissociation and electron detachment following anion photoexcitation can be determined by the magnitude of the energy gap between the excitation energy and the MECPs, rather than being a simple function of whether the excitation energy lies above the anion's vertical detachment energy.

Emerging contaminant exposure to aquatic systems in the Southern African Development Community.

The growing production and use of chemicals and the resultant increase in environmental exposure is of particular concern in developing countries where there is rapid industrialization and population growth but limited information on the occurrence of emerging contaminants. Advances in analytical techniques now allow for the monitoring of emerging contaminants at very low concentrations with the potential to cause harmful ecotoxicological effects. Therefore, we provide the first critical assessment of the current state of knowledge about chemical exposure in waters of the Southern African Developmental Community (SADC). We achieved this through a comprehensive literature review and the creation of a database of chemical monitoring data. Of the 59 articles reviewed, most (n = 36; 61.0%) were from South Africa, and the rest were from Botswana (n = 6; 10.2%), Zimbabwe (n = 6; 10.2%), Malawi (n = 3; 5.1%), Mozambique (n = 3; 5.1%), Zambia (n = 2; 3.4%), Angola (n = 1; 1.7%), Madagascar (n = 1; 1.7%), and Tanzania (n = 1; 1.7%). No publications were found from the remaining seven SADC countries. Emerging contaminants have only been studied in South Africa and Botswana. The antiretroviral drug ritonavir (64.52 µg/L) was detected at the highest average concentration, and ibuprofen (17 times) was detected most frequently. Despite being the primary water source in the region, groundwater was understudied (only 13 studies). High emerging contaminant concentrations in surface waters indicate the presence of secondary sources of pollution such as sewage leakage. We identify research gaps and propose actions to assess and reduce chemical pollution to enable the SADC to address the Sustainable Development Goals, particularly Goal 3.9, to reduce the deaths and illnesses from hazardous chemicals and contamination. Environ Toxicol Chem 2022;41:382-395. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Photodegradation of Riboflavin under Alkaline Conditions: What Can Gas-Phase Photolysis Tell Us about What Happens in Solution?

The application of electrospray ionisation mass spectrometry (ESI-MS) as a direct method for detecting reactive intermediates is a technique of developing importance in the routine monitoring of solution-phase reaction pathways. Here, we utilise a novel on-line photolysis ESI-MS approach to detect the photoproducts of riboflavin in aqueous solution under mildly alkaline conditions. Riboflavin is a constituent of many food products, so its breakdown processes are of wide interest. Our on-line photolysis setup allows for solution-phase photolysis to occur within a syringe using UVA LEDs, immediately prior to being introduced into the mass spectrometer via ESI. Gas-phase photofragmentation studies via laser-interfaced mass spectrometry of deprotonated riboflavin, [RF - H]-, the dominant solution-phase species under the conditions of our study, are presented alongside the solution-phase photolysis. The results obtained illustrate the extent to which gas-phase photolysis methods can inform our understanding of the corresponding solution-phase photochemistry. We determine that the solution-phase photofragmentation observed for [RF - H]- closely mirrors the gas-phase photochemistry, with the dominant m/z 241 condensed-phase photoproduct also being observed in gas-phase photodissociation. Further gas-phase photoproducts are observed at m/z 255, 212, and 145. The value of exploring both the gas- and solution-phase photochemistry to characterise photochemical reactions is discussed.

Measurement of the Population of Electrosprayed Deprotomers of Coumaric Acids Using UV-Vis Laser Photodissociation Spectroscopy.

The measurement of deprotonation sites in multifunctional molecules following electrospray ionization is important to better inform a wide range of spectroscopic and photophysical studies that use electrospray to prepare molecular species for study in the gas phase. We demonstrate that low-resolution UV-vis laser photodissociation spectroscopy can be applied in situ to identify the deprotomers of three coumaric acids, trans-para-coumaric acid (CMA), trans-caffeic acid (CA), and trans-ferulic acid (FA), formed via electrospray. Electronic absorption spectra of the deprotonated coumaric acids are recorded via photodepletion and photofragmentation following electrospray from solutions of ethanol and acetonitrile. By comparing the experimental spectra to wave function theory calculations, we are able to confirm the presence of phenoxide and carboxylate deprotomers upon electrospray for all three coumaric acids, when sprayed from both protic and aprotic solvents. Ratios of the phenoxide:carboxylate deprotomers are obtained by generating summed theoretical absorption spectra that reproduce the experimental spectra. We find that choice of electrospray solvent has little effect on the ratio of deprotomers obtained for deprotonated CMA and CA but has a greater impact for FA. Our results are in excellent agreement with previous work conducted on deprotonated CMA using IR spectroscopy and demonstrate that UV photodissociation spectroscopy of electrosprayed ions has potential as a diagnostic tool for identifying deprotomeric species.

Linking Electronic Relaxation Dynamics and Ionic Photofragmentation Patterns for the Deprotonated UV Filter Benzophenone-4.

Understanding how deprotonation impacts the photophysics of UV filters is critical to better characterize how they behave in key alkaline environments including surface waters and coral reefs. Using anion photodissociation spectroscopy, we have measured the intrinsic absorption electronic spectroscopy (400-214 nm) and numerous accompanying ionic photofragmentation pathways of the benzophenone-4 anion ([BP4-H]-). Relative ion yield plots reveal the locations of the bright S1 and S3 excited states. For the first time for an ionic UV filter, ab initio potential energy surfaces are presented to provide new insight into how the photofragment identity maps the relaxation pathways. These calculations reveal that [BP4-H]- undergoes excited-state decay consistent with a statistical fragmentation process where the anion breaks down on the ground state after nonradiative relaxation. The broader relevance of the results in providing a basis for interpreting the relaxation dynamics of a wide range of gas-phase ionic systems is discussed.

Investigating the mapping of chromophore excitations onto the electron detachment spectrum: photodissociation spectroscopy of iodide ion-thiouracil clusters.

Laser photodissociation spectroscopy (3.1-5.7 eV) has been applied to iodide complexes of the non-native nucleobases, 2-thiouracil (2-TU), 4-thiouracil (4-TU) and 2,4-thiouracil (2,4-TU), to probe the excited states and intracluster electron transfer as a function of sulphur atom substitution. Photodepletion is strong for all clusters (I-·2-TU, I-·4-TU and I-·2,4-TU) and is dominated by electron detachment processes. For I-·4-TU and I-·2,4-TU, photodecay is accompanied by formation of the respective molecular anions, 4-TU- and 2,4-TU-, behaviour that is not found for other nucleobases. Notably, the I-·2TU complex does not fragment with formation of its molecular anion. We attribute the novel formation of 4-TU- and 2,4-TU- to the fact that these valence anions are significantly more stable than 2-TU-. We observe further similar behaviour for I-·4-TU and I-·2,4-TU relating to the general profile of their photodepletion spectra, since both strongly resemble the intrinsic absorption spectra of the respective uncomplexed thiouracil molecule. This indicates that the nucleobase chromophore excitations are determining the clusters' spectral profile. In contrast, the I-·2-TU photodepletion spectrum is dominated by the electron detachment profile, with the near-threshold dipole-bound excited state being the only distinct spectral feature. We discuss these observations in the context of differences in the dipole moments of the thionucleobases, and their impact on the coupling of nucleobase-centred transitions onto the electron detachment spectrum.

Illuminating the Effect of the Local Environment on the Performance of Organic Sunscreens: Insights From Laser Spectroscopy of Isolated Molecules and Complexes.

Sunscreens are essential for protecting the skin from UV radiation, but significant questions remain about the fundamental molecular-level processes by which they operate. In this mini review, we provide an overview of recent advanced laser spectroscopic studies that have probed how the local, chemical environment of an organic sunscreen affects its performance. We highlight experiments where UV laser spectroscopy has been performed on isolated gas-phase sunscreen molecules and complexes. These experiments reveal how pH, alkali metal cation binding, and solvation perturb the geometric and hence electronic structures of sunscreen molecules, and hence their non-radiative decay pathways. A better understanding of how these interactions impact on the performance of individual sunscreens will inform the rational design of future sunscreens and their optimum formulations.

A "one pot" mass spectrometry technique for characterizing solution- and gas-phase photochemical reactions by electrospray mass spectrometry.

The characterization of new photochemical pathways is important to progress the understanding of emerging areas of light-triggered inorganic and organic chemistry. In this context, the development of platforms to perform routine characterization of photochemical reactions remains an important goal for photochemists. Here, we demonstrate a new instrument that can be used to characterise both solution-phase and gas-phase photochemical reactions through electrospray ionisation mass spectrometry (ESI-MS). The gas-phase photochemistry is studied by novel laser-interfaced mass spectrometry (LIMS), where the molecular species of interest is introduced to the gas-phase by ESI, mass-selected and then subjected to laser photodissociation in the ion-trap. On-line solution-phase photochemistry is initiated by LEDs prior to ESI-MS in the same instrument with ESI-MS again being used to monitor photoproducts. Two ruthenium metal carbonyls, [Ru(η5-C5H5)(PPh3)2CO][PF6] and [Ru(η5-C5H5)(dppe)CO][PF6] (dppe = 1,2-bis(diphenylphosphino)ethane) are studied using this methodology. We show that the gas-phase photofragmentation pathways observed for the ruthenium complexes via LIMS (i.e. loss of CO + PPh3 ligands from [Ru(η5-C5H5)(PPh3)2CO]+ and loss of just CO from [Ru(η5-C5H5)(dppe)CO]+) mirror the solution-phase photochemistry at 3.4 eV. The advantages of performing the gas-phase and solution-phase photochemical characterisations in a single instrument are discussed.

Photoproducts of the Photodynamic Therapy Agent Verteporfin Identified via Laser Interfaced Mass Spectrometry.

Verteporfin, a free base benzoporphyrin derivative monoacid ring A, is a photosensitizing drug for photodynamic therapy (PDT) used in the treatment of the wet form of macular degeneration and activated by red light of 689 nm. Here, we present the first direct study of its photofragmentation channels in the gas phase, conducted using a laser interfaced mass spectrometer across a broad photoexcitation range from 250 to 790 nm. The photofragmentation channels are compared with the collision-induced dissociation (CID) products revealing similar dissociation pathways characterized by the loss of the carboxyl and ester groups. Complementary solution-phase photolysis experiments indicate that photobleaching occurs in verteporfin in acetonitrile; a notable conclusion, as photoinduced activity in Verteporfin was not thought to occur in homogenous solvent conditions. These results provide unique new information on the thermal break-down products and photoproducts of this light-triggered drug.

Sodium cationization can disrupt the intramolecular hydrogen bond that mediates the sunscreen activity of oxybenzone.

A key decay pathway by which organic sunscreen molecules dissipate harmful UV energy involves excited-state hydrogen atom transfer between proximal enol and keto functional groups. Structural modifications of this molecular architecture have the potential to block ultrafast decay processes, and hence promote direct excited-state molecular dissociation, profoundly affecting the efficiency of an organic sunscreen. Herein, we investigate the binding of alkali metal cations to a prototype organic sunscreen molecule, oxybenzone, using IR characterization. Mass-selective IR action spectroscopy was conducted at the free electron laser for infrared experiments, FELIX (600-1800 cm-1), on complexes of Na+, K+ and Rb+ bound to oxybenzone. The IR spectra reveal that K+ and Rb+ adopt binding positions away from the key OH intermolecular hydrogen bond, while the smaller Na+ cation binds directly between the keto and enol oxygens, thus breaking the intramolecular hydrogen bond. UV laser photodissociation spectroscopy was also performed on the series of complexes, with the Na+ complex displaying a distinctive electronic spectrum compared to those of K+ and Rb+, in line with the IR spectroscopy results. TD-DFT calculations reveal that the origin of the changes in the electronic spectra can be linked to rupture of the intramolecular bond in the sodium cationized complex. The implications of our results for the performance of sunscreens in mixtures and environments with high concentrations of metal cations are discussed.

Observation of Enhanced Dissociative Photochemistry in the Non-Native Nucleobase 2-Thiouracil.

We present the first study to measure the dissociative photochemistry of 2-thiouracil (2-TU), an important nucleobase analogue with applications in molecular biology and pharmacology. Laser photodissociation spectroscopy is applied to the deprotonated and protonated forms of 2-TU, which are produced in the gas-phase using electrospray ionization mass spectrometry. Our results show that the deprotonated form of 2-thiouracil ([2-TU-H]-) decays predominantly by electron ejection and hence concomitant production of the [2-TU-H]· free-radical species, following photoexcitation across the UVA-UVC region. Thiocyanate (SCN-) and a m/z 93 fragment ion are also observed as photodecay products of [2-TU-H]- but at very low intensities. Photoexcitation of protonated 2-thiouracil ([2-TU·H]+) across the same UVA-UVC spectral region produces the m/z 96 cationic fragment as the major photofragment. This ion corresponds to ejection of an HS· radical from the precursor ion and is determined to be a product of direct excited state decay. Fragment ions associated with decay of the hot ground state (i.e., the ions we would expect to observe if 2-thiouracil was behaving like UV-dissipating uracil) are observed as much more minor products. This behaviour is consistent with enhanced intersystem crossing to triplet excited states compared to internal conversion back to the ground state. These are the first experiments to probe the effect of protonation/deprotonation on thionucleobase photochemistry, and hence explore the effect of pH at a molecular level on their photophysical properties.

Direct Measurement of the Visible to UV Photodissociation Processes for the PhotoCORM TryptoCORM.

PhotoCORMs are light-triggered compounds that release CO for medical applications. Here, we apply laser spectroscopy in the gas phase to TryptoCORM, a known photoCORM that has been shown to destroy Escherichia coli upon visible-light activation. Our experiments allow us to map TryptoCORM's photochemistry across a wide wavelength range by using novel laser-interfaced mass spectrometry (LIMS). LIMS provides the intrinsic absorption spectrum of the photoCORM along with the production spectra of all of its ionic photoproducts for the first time. Importantly, the photoproduct spectra directly reveal the optimum wavelengths for maximizing CO ejection, and the extent to which CO ejection is compromised at redder wavelengths. A series of comparative studies were performed on TryptoCORM-CH3 CN which exists in dynamic equilibrium with TryptoCORM in solution. Our measurements allow us to conclude that the presence of the labile CH3 CN facilitates CO release over a wider wavelength range. This work demonstrates the potential of LIMS as a new methodology for assessing active agent release (e.g. CO, NO, H2 S) from light-activated prodrugs.

Unravelling the Keto-Enol Tautomer Dependent Photochemistry and Degradation Pathways of the Protonated UVA Filter Avobenzone.

Avobenzone (AB) is a widely used UVA filter known to undergo irreversible photodegradation. Here, we investigate the detailed pathways by which AB photodegrades by applying UV laser-interfaced mass spectrometry to protonated AB ions. Gas-phase infrared multiple-photon dissociation (IRMPD) spectra obtained with the free electron laser for infrared experiments, FELIX, (600-1800 cm-1) are also presented to confirm the geometric structures. The UV gas-phase absorption spectrum (2.5-5 eV) of protonated AB contains bands that correspond to selective excitation of either the enol or diketo forms, allowing us to probe the resulting, tautomer-dependent photochemistry. Numerous photofragments (i.e., photodegradants) are directly identified for the first time, with m/z 135 and 161 dominating, and m/z 146 and 177 also appearing prominently. Analysis of the production spectra of these photofragments reveals that that strong enol to keto photoisomerism is occurring, and that protonation significantly disrupts the stability of the enol (UVA active) tautomer. Close comparison of fragment ion yields with the TD-DFT-calculated absorption spectra give detailed information on the location and identity of the dissociative excited state surfaces, and thus provide new insight into the photodegradation pathways of avobenzone, and photoisomerization of the wider class of ß-diketone containing molecules.

Mapping the intrinsic absorption properties and photodegradation pathways of the protonated and deprotonated forms of the sunscreen oxybenzone.

Sunscreens provide vital protection against the photodamaging effects of UV radiation, however, many fundamental questions remain about the detailed mechanisms by which they dissipate UV energy. One such issue is the extent to which the pH environment of an organic sunscreen molecule alters its effectiveness, both in terms of ability to absorb UV radiation, and also its potential to photodegrade. Here, we use gas-phase laser photodissociation spectroscopy for the first time to measure the intrinsic UVA-UVC absorption spectra and associated photodegradation products of protonated and deprotonated oxybenzone, away from the complications of bulk mixtures. Our results reveal that protonation state has a dramatic effect on the absorption and photodissociation properties of this sunscreen. While the UV absorption profile of oxybenzone is only modestly affected by protonation across the range from 400-216 nm, deprotonated oxybenzone displays a significantly modified absorption spectrum, with very low photoabsorption between 370-330 nm. Protonated oxybenzone primarily photofragments by rupture of the bonds on either side of the central carbonyl group, producing cationic fragments with m/z 151 and 105. Additional lower mass photofragments (e.g. m/z 95 and 77) are also observed. The production spectra for the photofragments from protonated oxybenzone fall into two distinct categories, which we discuss in the context of different excited state decay pathways. For deprotonated oxybenzone, the major photofragments observed are m/z 211 and 212, which are associated with the ejection of methane and the methyl free radical from the parent ion, respectively. Implications for the suitability of oxybenzone in its protonated and deprotonated forms as an optimum sunscreen molecule are discussed.

Observation of Near-Threshold Resonances in the Flavin Chromophore Anions Alloxazine and Lumichrome.

Lumichrome (LC) is the chromophore of the flavin family of photoactive biomolecules, where key biochemical activity involves interplay between redox and photophysical events. Questions remain about the relationship between the redox status of the ground and excited states and demand an improved understanding of the intrinsic photochemistry. Using anion photodissociation spectroscopy, we have measured the intrinsic electronic spectroscopy (564-220 nm) and accompanying photodegradation pathways of the deprotonated anionic form of LC. Experiments were also performed on alloxazine (AL), which is equivalent to LC minus two methyl groups. We observe a resonance state close to 3.8 eV for both anions for the first time, which we tentatively assign to dipole-bound excited states. For AL this state is sufficiently long-lived to facilitate dissociative electron attachment. Our results suggest that the presence of methyl group rotors at key positions along the molecular dipole may reduce the lifetime of the resonance state and hence provide a structural barrier to valence electron capture, and ensuing molecular dissociation.

Protomer-Dependent Electronic Spectroscopy and Photochemistry of the Model Flavin Chromophore Alloxazine.

Flavin chromophores play key roles in a wide range of photoactive proteins, but key questions exist in relation to their fundamental spectroscopic and photochemical properties. In this work, we report the first gas-phase spectroscopy study of protonated alloxazine (AL∙H⁺), a model flavin chromophore. Laser photodissociation is employed across a wide range (2.34⁻5.64 eV) to obtain the electronic spectrum and characterize the photofragmentation pathways. By comparison to TDDFT quantum chemical calculations, the spectrum is assigned to two AL∙H⁺ protomers; an N5 (dominant) and O4 (minor) form. The protomers have distinctly different spectral profiles in the region above 4.8 eV due to the presence of a strong electronic transition for the O4 protomer corresponding to an electron-density shift from the benzene to uracil moiety. AL∙H⁺ photoexcitation leads to fragmentation via loss of HCN and HNCO (along with small molecules such as CO2 and H2O), but the photofragmentation patterns differ dramatically from those observed upon collision excitation of the ground electronic state. This reveals that fragmentation is occurring during the excited state lifetime. Finally, our results show that the N5 protomer is associated primarily with HNCO loss while the O4 protomer is associated with HCN loss, indicating that the ring-opening dynamics are dependent on the location of protonation in the ground-state molecule.