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

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

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

Understanding how deprotonation impacts on 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 non-radiative relaxation. The broader relevance of the results in providing a basis for interpreting the relaxation dynamics of a wide range ionic systems is discussed

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

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

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

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 β-diketone containing molecules

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‐CH3CN which exists in dynamic equilibrium with TryptoCORM in solution. Our measurements allow us to conclude that the presence of the labile CH3CN 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, H2S) from light‐activated prodrugs

A “one pot” tool for characterizing solution-phase 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 characterize both solution-phase and gas-phase photochemical reactions through electrospray ionization 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. The advantages of performing the gas-phase and solution-phase photochemical characterizations in a single instrument are discussed

Locating the Proton in Nicotinamide Protomers via Low-Resolution UV Action Spectroscopy of Electrosprayed Solutions

Even in relatively simple molecules, the sites of protonation or deprotonation formed upon electrospray ionization can be controversial. This situation means that it is important to develop new approaches for identifying "protomers" and "deprotomers". In this study, we demonstrate that routine, low-resolution UV laser photodissociation spectroscopy can be applied to identify the gaseous protomers of nicotinamide formed upon electrospray. Nicotinamide is an important biological molecule that possesses multiple protonation sites associated with its pyridine and amide groups. We obtain a gas-phase absorption spectrum for protonated nicotinamide that closely resembles the solution phase spectrum. However, photoexcitation of protonated nicotinamide produces numerous ionic photofragments, and the spectral profiles for production of these photofragments from protonated nicotinamide reveal the existence of two distinctive chromophores, which can be traced to the existence of pyridine and amide protomers. We observe that these protomers are associated with absorption bands centered at 4.96 and 4.73 eV, respectively, with the protomers appearing in an approximate ratio of The fact that the considerably less favorable amide protomer is observed in substantial quantities in the gas phase is surprising given that the pyridine protomer is the lower-energy species in both solution and gas phase. The high amounts of amide protomers observed here can be explained as arising from asymmetric pyridine protomer amide bound dimers, present in solution or in the electrosprayed droplets, which lead to enhanced formation of the unexpected amide-protonated isomers