Femtosecond to Microsecond Observation of Photochemical Pathways in Nitroaromatic Phototriggers Using Transient Absorption Spectroscopy.

The synthetic accessibility and tolerance to structural modification of phototriggered compounds (PTs) based on the ortho- nitrobenzene (ONB) protecting group have encouraged a myriad of applications including optimization of biological activity, and supramolecular polymerization. Here, a combination of ultrafast transient absorption spectroscopy techniques is used to study the multistep photochemistry of two nitroaromatic phototriggers based on the ONB chromophore, O-(4,5-dimethoxy-2-nitrobenzyl)-l-serine (DMNB-Ser) and O-[(2-nitrophenyl)methyl]-l-tyrosine hydrochloride (NB-Tyr), in DMSO solutions on femtosecond to microsecond time scales following the absorption of UV light. From a common nitro-S1 excited state, the PTs can either undergo excited state intramolecular hydrogen transfer (ESIHT) to an aci-S1 isomer within the singlet state manifold, leading to direct S1 → S0 internal conversion through a conical intersection, or competitive intersystem crossing (ISC) to access the triplet state manifold on time scales of (1.93 ± 0.03) ps and (13.9 ± 1.2) ps for DMNB-Ser and NB-Tyr, respectively. Deprotonation of aci-T1 species to yield triplet anions is proposed to occur in both PTs, with an illustrative time constant of (9.4 ± 0.7) ns for DMNB-Ser. More than 75% of the photoexcited molecules return to their electronic ground states within 8 µs, either by direct S1 → S0 relaxation or anion reprotonation. Hence, upper limits to the quantum yields of photoproduct formation are estimated to be in the range of 13-25%. Mixed DMSO/H2O solvents show the influence of the environment on the observed photochemistry, for example, revealing two nitro-S1 lifetimes for DMNB-Ser in a 10:1 DMSO/H2O mixture of 1.95 ps and (10.1 ± 1.2) ps, which are attributed to different microsolvation environments.

Perspective on Theoretical and Experimental Advances in Atmospheric Photochemistry.

Research that explores the chemistry of Earth's atmosphere is central to the current understanding of global challenges such as climate change, stratospheric ozone depletion, and poor air quality in urban areas. This research is a synergistic combination of three established domains: earth observation, for example, using satellites, and in situ field measurements; computer modeling of the atmosphere and its chemistry; and laboratory measurements of the properties and reactivity of gas-phase molecules and aerosol particles. The complexity of the interconnected chemical and photochemical reactions which determine the composition of the atmosphere challenges the capacity of laboratory studies to provide the spectroscopic, photochemical, and kinetic data required for computer models. Here, we consider whether predictions from computational chemistry using modern electronic structure theory and nonadiabatic dynamics simulations are becoming sufficiently accurate to supplement quantitative laboratory data for wavelength-dependent absorption cross-sections, photochemical quantum yields, and reaction rate coefficients. Drawing on presentations and discussions from the CECAM workshop on Theoretical and Experimental Advances in Atmospheric Photochemistry held in March 2024, we describe key concepts in the theory of photochemistry, survey the state-of-the-art in computational photochemistry methods, and compare their capabilities with modern experimental laboratory techniques. From such considerations, we offer a perspective on the scope of computational (photo)chemistry methods based on rigorous electronic structure theory to become a fourth core domain of research in atmospheric chemistry.

Electrochemical Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid-modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p-tau) present two-state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid-tau interaction. The results indicate that p-tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

The Role of Momentum Partitioning in Covariance Ion Imaging Analysis.

We present results from a covariance ion imaging study, which employs extensive filtering, on the relationship between fragment momenta to gain deeper insight into photofragmentation dynamics. A new data analysis approach is introduced that considers the momentum partitioning between the fragments of the breakup of a molecular polycation to disentangle concurrent fragmentation channels, which yield the same ion species. We exploit this approach to examine the momentum exchange relationship between the products, which provides direct insight into the dynamics of molecular fragmentation. We apply these techniques to extensively characterize the dissociation of 1-iodopropane and 2-iodopropane dications prepared by site-selective ionization of the iodine atom using extreme ultraviolet intense femtosecond laser pulses with a photon energy of 95 eV. Our assignments are supported by classical simulations, using parameters largely obtained directly from the experimental data.

Unraveling the Ultrafast Photochemical Dynamics of Nitrobenzene in Aqueous Solution.

Nitroaromatic compounds are major constituents of the brown carbon aerosol particles in the troposphere that absorb near-ultraviolet (UV) and visible solar radiation and have a profound effect on the Earth's climate. The primary sources of brown carbon include biomass burning, forest fires, and residential burning of biofuels, and an important secondary source is photochemistry in aqueous cloud and fog droplets. Nitrobenzene is the smallest nitroaromatic molecule and a model for the photochemical behavior of larger nitroaromatic compounds. Despite the obvious importance of its droplet photochemistry to the atmospheric environment, there have not been any detailed studies of the ultrafast photochemical dynamics of nitrobenzene in aqueous solution. Here, we combine femtosecond transient absorption spectroscopy, time-resolved infrared spectroscopy, and quantum chemistry calculations to investigate the primary steps following the near-UV (λ ≥ 340 nm) photoexcitation of aqueous nitrobenzene. To understand the role of the surrounding water molecules in the photochemical dynamics of nitrobenzene, we compare the results of these investigations with analogous measurements in solutions of methanol, acetonitrile, and cyclohexane. We find that vibrational energy transfer to the aqueous environment quenches internal excitation, and therefore, unlike the gas phase, we do not observe any evidence for formation of photoproducts on timescales up to 500 ns. We also find that hydrogen bonding between nitrobenzene and surrounding water molecules slows the S1/S0 internal conversion process.

Exploring the ultrafast and isomer-dependent photodissociation of iodothiophenes via site-selective ionization.

C-I bond extension and fission following ultraviolet (UV, 262 nm) photoexcitation of 2- and 3-iodothiophene is studied using ultrafast time-resolved extreme ultraviolet (XUV) ionization in conjunction with velocity map ion imaging. The photoexcited molecules and eventual I atom products are probed by site-selective ionization at the I 4d edge using intense XUV pulses, which induce multiple charges initially localized to the iodine atom. At C-I separations below the critical distance for charge transfer (CT), charge can redistribute around the molecule leading to Coulomb explosion and charged fragments with high kinetic energy. At greater C-I separations, beyond the critical distance, CT is no longer possible and the measured kinetic energies of the charged iodine atoms report on the neutral dissociation process. The time and momentum resolved measurements allow determination of the timescales and the respective product momentum and kinetic energy distributions for both isomers, which are interpreted in terms of rival 'direct' and 'indirect' dissociation pathways. The measurements are compared with a classical over the barrier model, which reveals that the onset of the indirect dissociation process is delayed by ∼1 ps relative to the direct process. The kinetics of the two processes show no discernible difference between the two parent isomers, but the branching between the direct and indirect dissociation channels and the respective product momentum distributions show isomer dependencies. The greater relative yield of indirect dissociation products from 262 nm photolysis of 3-iodothiophene (cf. 2-iodothiophene) is attributed to the different partial cross-sections for (ring-centred) π∗ ← π and (C-I bond localized) σ∗ ← (n/π) excitation in the respective parent isomers.

Omega-3 and -6 Fatty Acids Alter the Membrane Lipid Composition and Vesicle Size to Regulate Exocytosis and Storage of Catecholamines.

The two essential fatty acids, alpha-linolenic acid and linoleic acid, and the higher unsaturated fatty acids synthesized from them are critical for the development and maintenance of normal brain functions. Deficiencies of these fatty acids have been shown to cause damage to the neuronal development, cognition, and locomotor function. We combined electrochemistry and imaging techniques to examine the effects of the two essential fatty acids on catecholamine release dynamics and the vesicle content as well as on the cell membrane phospholipid composition to understand how they impact exocytosis and by extension neurotransmission at the single-cell level. Incubation of either of the two fatty acids reduces the size of secretory vesicles and enables the incorporation of more double bonds into the cell membrane structure, resulting in higher membrane flexibility. This subsequently affects proteins regulating the dynamics of the exocytotic fusion pore and thereby affects exocytosis. Our data suggest a possible pathway whereby the two essential fatty acids affect the membrane structure to impact exocytosis and provide a potential treatment for diseases and impairments related to catecholamine signaling.

Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles.

An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA or protein in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.

Emerging Data Processing Methods for Single-Entity Electrochemistry.

Single-entity electrochemistry is a powerful tool that enables the study of electrochemical processes at interfaces and provides insights into the intrinsic chemical and structural heterogeneities of individual entities. Signal processing is a critical aspect of single-entity electrochemical measurements and can be used for data recognition, classification, and interpretation. In this review, we summarize the recent five-year advances in signal processing techniques for single-entity electrochemistry and highlight their importance in obtaining high-quality data and extracting effective features from electrochemical signals, which are generally applicable in single-entity electrochemistry. Moreover, we shed light on electrochemical noise analysis to obtain single-molecule frequency fingerprint spectra that can provide rich information about the ion networks at the interface. By incorporating advanced data analysis tools and artificial intelligence algorithms, single-entity electrochemical measurements would revolutionize the field of single-entity analysis, leading to new fundamental discoveries.

Characterization of the Reversible Intersystem Crossing Dynamics of Organic Photocatalysts Using Transient Absorption Spectroscopy and Time-Resolved Fluorescence Spectroscopy.

Thermally activated delayed fluorescence (TADF) emitters are molecules of interest as homogeneous organic photocatalysts (OPCs) for photoredox chemistry. Here, three classes of OPC candidates are studied in dichloromethane (DCM) or N,N-dimethylformamide (DMF) solutions, using transient absorption spectroscopy and time-resolved fluorescence spectroscopy. These OPCs are benzophenones with either carbazole (2Cz-BP and 2tCz-BP) or phenoxazine/phenothiazine (2PXZ-BP and 2PTZ-BP) appended groups and the dicyanobenzene derivative 4DP-IPN. Dual lifetimes of the S1 state populations are observed, consistent with reverse intersystem crossing (RISC) and TADF emission. Example fluorescence lifetimes in DCM are (5.18 ± 0.01) ns and (6.22 ± 1.27) µs for 2Cz-BP, (1.38 ± 0.01) ns and (0.32 ± 0.01) µs for 2PXZ-BP, and (2.97 ± 0.01) ns and (62.0 ± 5.8) µs for 4DP-IPN. From ground state bleach recoveries and time-correlated single photon counting measurements, triplet quantum yields in DCM are estimated to be 0.62 ± 0.16, 0.04 ± 0.01, and 0.83 ± 0.02 for 2Cz-BP, 2PXZ-BP, and 4DP-IPN, respectively. 4DP-IPN displays similar photophysical behavior to the previously studied OPC 4Cz-IPN. Independent of the choice of solvent, 4DP-IPN, 2Cz-BP, and 2tCz-BP are shown to be TADF emitters, whereas emission by 2PXZ-BP and 2PTZ-BP depends on the molecular environment, with TADF emission enhanced in aggregates compared to monomers. Behavior of this type is representative of aggregation-induced emission luminogens (AIEgens).

Photoredox-HAT Catalysis for Primary Amine α-C-H Alkylation: Mechanistic Insight with Transient Absorption Spectroscopy.

The synergistic use of (organo)photoredox catalysts with hydrogen-atom transfer (HAT) cocatalysts has emerged as a powerful strategy for innate C(sp3)-H bond functionalization, particularly for C-H bonds α- to nitrogen. Azide ion (N3-) was recently identified as an effective HAT catalyst for the challenging α-C-H alkylation of unprotected, primary alkylamines, in combination with dicyanoarene photocatalysts such as 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN). Here, time-resolved transient absorption spectroscopy over sub-picosecond to microsecond timescales provides kinetic and mechanistic details of the photoredox catalytic cycle in acetonitrile solution. Direct observation of the electron transfer from N3- to photoexcited 4CzIPN reveals the participation of the S1 excited electronic state of the organic photocatalyst as an electron acceptor, but the N3• radical product of this reaction is not observed. Instead, both time-resolved infrared and UV-visible spectroscopic measurements implicate rapid association of N3• with N3- (a favorable process in acetonitrile) to form the N6•- radical anion. Electronic structure calculations indicate that N3• is the active participant in the HAT reaction, suggesting a role for N6•- as a reservoir that regulates the concentration of N3•.

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size.

We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of 13 C-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an 127 I-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.

Single Exosome Amperometric Measurements Reveal Encapsulation of Chemical Messengers for Intercellular Communication.

In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomes─one of the main extracellular vesicles (EVs)─carrying cell-dependent DNA, mRNA, proteins, etc., play an essential role in cellular communication. Due to experimental limitations, it has been difficult to monitor the real-time release of individual exosomes; this restricts a comprehensive understanding of the basic molecular mechanisms and the functions of exosomes. In this work, we introduce amperometry with microelectrodes to capture the dynamic release of single exosomes from a single living cell, distinguish them from other EVs, and differentiate the molecules inside exosomes and those secreted from LDCVs. We show that, similar to many LDCVs and synaptic vesicles, exosomes released by neuroendocrine cells also contain catecholamine transmitters. This finding reveals a different mode of chemical communication via exosome-encapsulated chemical messengers and a potential interconnection between the two release pathways, changing the canonical view of exocytosis of neuroendocrine cells and possibly neurons. This defines a new mechanism for chemical communication at the fundamental level and opens new avenues in the research of the molecular biology of exosomes in the neuroendocrine and central nervous systems.

Hofmeister Series: From Aqueous Solution of Biomolecules to Single Cells and Nanovesicles.

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems. These important steps have increased our understanding of the Hofmeister effects on cellular exocytosis, vesicular content, and nanovesicle opening. Increasing evidence is firmly establishing that the ions along the Hofmeister series play an important role in living organisms that has often been ignored.

Correlative Cellular Mass Spectrometry Imaging and Amperometry Show Dose Dependent Changes in Lipid Composition and Exocytosis.

Aberrant functioning of the proteasome has been associated with crucial pathologic conditions including neurodegeneration. Yet, the complex underlying causes at the cellular level remain unclear and there are conflicting reports of neuroprotective to neurodegenerative effects of proteasomal inhibitors such as lactacystin that are utilised as models for neurodegenerative diseases. The conflicting results may be associated with different dose regimes of lactacystin and hence we have performed a dose dependent study of the effects of lactacystin to identify concurrent changes in the cell membrane lipid profile and the dynamics of exocytosis using a combination of surface sensitive mass spectrometry and single cell amperometry. Significant changes of negatively charged lipids were associated with different lactacystin doses that showed a weak correlation with exocytosis while changes in PE and PE-O lipids showed dose dependent changes correlated with initial pore formation and total release of vesicle content respectively.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.