Ultraviolet Photodissociation Research Articles

Formation of N--bearing complex organic molecules in molecular clouds: Ketenimine, acetonitrile, acetaldimine, and vinylamine via the UV photolysis of C_2H_2 ice

Formation of N--bearing complex organic molecules in molecular clouds: Ketenimine, acetonitrile, acetaldimine, and vinylamine via the UV photolysis of C_2H_2 ice

Direct Observation of Gas-Phase Hydroxymethylene: Photoionization and Kinetics Resulting from Methanol Photodissociation.

Carbene species play an integral role in high-energy chemistry, transition-metal-carbene chemistry, catalysis, photolytic formation of carbohydrates, and possibly even the formation of interstellar sugars. In 1921, "reactive formaldehyde"─now known as hydroxymethylene (HCOH)─was first implicated as an intermediate in photocatalytic processes. However, due to its transient nature, direct observation of HCOH has predominantly been attained using cryogenic isolation methods. As a result, HCOH gas-phase reactivity measurements have been limited. We directly observed HCOH using photoionization spectroscopy following UV photodissociation of methanol. Our measurements show it reacts slowly with O2 at room temperature. This work provides evidence for the formation mechanism of HCOH from CH3OH and its subsequent reactivity under gas-phase reaction conditions.

Panda-UV Unlocks Deeper Protein Characterization with Internal Fragments in Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) mass spectrometry unlocks insights into the protein structure and sequence through fragmentation patterns. While N- and C-terminal fragments are traditionally relied upon, this work highlights the critical role of internal fragments in achieving near-complete sequencing of protein. Previous limitations of internal fragment utilization, owing to their abundance and potential for random matching, are addressed here with the development of Panda-UV, a novel software tool combining spectral calibration, and Pearson correlation coefficient scoring for confident fragment assignment. Panda-UV showcases its power through comprehensive benchmarks on three model proteins. The inclusion of internal fragments boosts identified fragment numbers by 26% and enhances average protein sequence coverage to a remarkable 93% for intact proteins, unlocking the hidden region of the largest protein carbonic anhydrase II in model proteins. Notably, an average of 65% of internal fragments can be identified in multiple replicates, demonstrating the high confidence of the fragments Panda-UV provided. Finally, the sequence coverages of mAb subunits can be increased up to 86% and the complementary determining regions (CDRs) are nearly completely sequenced in a single experiment. The source codes of Panda-UV are available at https://github.com/PHOENIXcenter/Panda-UV.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1. Fifty-three payload-containing peptides were identified, ranging in mass from 1.8 to 16.9 kDa, and leading to the unambiguous identification of 46 out of 92 possible conjugation sites. In addition, seven peptides were identified containing multiple payloads. The characterization of these types of heterogeneous peptides represents an important step in unraveling the combinatorial nature of lysine-conjugated ADCs.

Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

Installation protocol for charge transfer dissociation mass spectrometry on ion trapping mass spectrometers.

Charge transfer dissociation (CTD) is a novel fragmentation technique that demonstrates enhanced structural characterization for a wide variety of molecules compared to standard fragmentation techniques like collision-induced dissociation (CID). Alternative fragmentation techniques, such as electron transfer dissociation, electron capture dissociation, and ultraviolet photodissociation, also overcome many of the shortfalls of CID, but none of them are a silver bullet that can adequately characterize a wide variety of structures and charge states of target compounds. Given the diversity of structural classes and their occasional obstinance towards certain activation techniques, alternative fragmentation techniques are required that rely on novel or alternative modes of activation. Herein, we present a step-by-step protocol for the installation of CTD on a quadrupole ion trap mass spectrometer and best practices for optimizing the signal-to-noise ratio and acquisition times for CTD mass spectra. In addition to two CTD installations in the Jackson laboratory, CTD has also been installed, and is currently in operation, on two 3D ion trap mass spectrometers in France: one in the laboratory of Dr. David Ropartz and Dr. Hélène Rogneaux at INRAE in Nantes, and the other in the laboratory of Dr. Jean-Yves Salpin at Université d'Évry Val-d'Essonne, part of the Paris-Saclay University system. Here, we provide a visual protocol to help others accomplish the instrument modification.

Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

N-H and N-C Bond Dissociation Pathways in Ultraviolet Photodissociation of Dimethylamine.

We investigated the interlinked N-H and N-C photochemistry of primary and secondary amines via the state-resolved detection of vibrationally excited CH3 product and H atom product by 200-235 nm dimethylamine photodissociation using resonance-enhanced multiphoton ionization (REMPI) and velocity map imaging (VMI) techniques. The out-of-plane bending (ν2) vibrationally excited CH3 showed a bimodal translational energy distribution that became unimodal with a near-zero product yield at longer photolysis wavelengths (λphotolysis). In contrast, a unimodal distribution was observed for the C-H stretching (νCH) vibrationally excited CH3 products with an almost constant product yield in the examined λphotolysis region. We ascribed the state-specific energy releases of the CH3 products to two reaction pathways based on calculations of the potential energy surface (PES): the direct N-CH3 dissociation pathway and the indirect N-CH3 dissociation pathway via the N-H bond conical intersection. Meanwhile, the H atom product showed a bimodal energy distribution similar to the ammonia photodissociation model, with an excited-state counterproduct channel that became accessible at a shorter λphotolysis. These results suggest that the N-H and N-C bond dissociations are connected, and these dissociations cause different photochemistry between primary/secondary amines and tertiary amines.

Aqueous alkaline phosphate facilitates the non-exchangeable deuteration of peptides and proteins.

The incorporation of deuterium into peptides and proteins holds broad applications across various fields, such as drug development and structural characterization. Nevertheless, current methods for peptide/protein deuteration often target exchangeable labile sites or require harsh conditions for stable modification. In this study, we present a late-stage approach utilizing an alkaline phosphate solution to achieve deuteration of non-exchangeable backbone sites of peptides and proteins. The specific deuteration regions are identified through ultraviolet photodissociation (UVPD) and mass spectrometry analysis. This deuteration strategy demonstrates site and structure selectivity, with a notable affinity for labeling the α-helix regions of myoglobin. The deuterium method is particularly suitable for peptides and proteins that remain stable under high pH conditions.

Nonadiabatic quantum dynamics explores non-monotonic photodissociation branching of N2 into the N(4S) + N(2D) and N(4S) + N(2P) product channels.

Vacuum ultraviolet (VUV) photodissociation of N2 molecules is a source of reactive N atoms in the interstellar medium. In the energy range of VUV optical excitation of N2, the N-N triple bond cleavage leads to three types of atoms: ground-state N(4S) and excited-state N(2P) and N(2D). The latter is the highest reactive and it is believed to be the primary participant in reactions with hydrocarbons in Titan's atmosphere. Experimental studies have observed a non-monotonic energy dependence and non-statistical character of the photodissociation of N2. This implies different dissociation pathways and final atomic products for different wavelength regions in the sunlight spectrum. We here apply ab initio quantum chemical and nonadiabatic quantum dynamical techniques to follow the path of an electronic state from the excitation of a particular singlet 1Σ+u and 1Πu vibronic level of N2 to its dissociation into different atomic products. We simulate dynamics for two isotopomers of the nitrogen molecule, 14N2 and 14N15N for which experimental data on the branching are available. Our computations capture the non-monotonic energy dependence of the photodissociation branching ratios in the energy range 108 000-116 000 cm-1. Tracing the quantum dynamics in a bunch of electronic states enables us to identify the key components that determine the efficacy of singlet to triplet population transfer and therefore predissociation lifetimes and branching ratios for different energy regions.

Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions.

Recent advances in top-down mass spectrometry strategies continue to improve the analysis of intact proteins. 193 nm ultraviolet photodissociation (UVPD) is one method well-suited for top-down analysis. UVPD is often performed using relatively low photon flux in order to limit multiple-generation dissociation of fragment ions and maximize sequence coverage. Consequently, a large portion of the precursor ion survives the UVPD process, dominates the spectrum, and may impede identification of fragment ions. Here, we explore the isolation of subpopulations of fragment ions lower and higher than the precursor ion after UVPD as a means to eliminate the impact of the surviving precursor ion on the detection of low abundance fragment ions. This gas-phase fractionation method improved sequence coverage harvested from fragment ions found in the m/z regions lower and higher than the precursor by an average factor of 1.3 and 2.3, respectively. Combining this gas-phase fractionation method with proton transfer charge reduction (PTCR) further increased the sequence coverage obtained from these m/z regions by another factor of 1.3 and 1.4, respectively. Implementing a post-UVPD fractionation + PTCR strategy with six fractionation events resulted in a sequence coverage of 75% for enolase, the highest reported for 193 nm UVPD.

Mechanism behind polypropylene surface modifications by OH radicals: An experimental study

We quantitatively investigated the roles and mechanisms of OH radicals in polypropylene (PP) surface modification utilizing a previously proposed vacuum ultraviolet photodissociation reactive species supply method. Water contact angle (WCA), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS) were employed to characterize the modifications in surface hydrophilicity and components. The relationship between the WCA decline rate and OH radical density at the PP surface was quantified as (2.1 ± 0.1)[OHs] + (0.5 ± 0.1) degrees/s, where [OHs] is in ppm. The ATR-FTIR and XPS results demonstrated that C–OH, CO, C–O–O, and OCO groups were gradually introduced onto the PP surface at different treatment stages. The formation and behavior of low-molecular-weight oxidized materials (LMWOMs) on the PP surface during OH radical treatment were investigated. A modified layer composed of LMWOMs eventually formed on the PP surface. The physicochemical mechanisms underlying these phenomena at the micro-level were also discussed. A possible surface oxidation model was proposed based on WCA results, surface functional groups, and LMWOMs, indicating that the oxidation of the PP chains by OH radicals was initiated at the topmost surface and advanced in the depth direction.

Role of Triplet States in the Photolysis of Proteogenic Amino Acids.

This investigation delves into the UV photodissociation of pivotal amino acids (Alanine, Glycine, Leucine, Proline, and Serine) at 213 nm, providing insights into triplet-state deactivation pathways. Utilizing a comprehensive approach involving time-dependent density functional calculations (TD-DFT), multi-configurational methods, and ab-initio molecular dynamics (AIMD) simulations, we scrutinize the excited electronic states (T1 , T2 , and S1 ) subsequent to 213 nm excitation. Our findings demonstrate that α-carbonyl C-C bond-breaking in triplet states exhibits markedly lower barriers than in singlet states (below 5.0 kcal mol-1 ). AIMD simulations corroborate the potential involvement of triplet states in amino acid fragmentation, underscoring the significance of accounting for these states in photochemistry. Chemical bonding analyses unveil distinctive patterns for S1 and T1 states, with the asymmetric redistribution of electron density characterizing the C-C breaking in triplet states, in contrast to the symmetric breaking observed in singlet states. This research complements recent experimental discoveries, enhancing our comprehension of amino acid reactions in the interstellar medium.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

Molecular Structure Characterization of Micro/Nanoplastics by 193 nm Ultraviolet Photodissociation Mass Spectrometry.

The degradation of macroplastics results in micro/nanoplastics (MNPs) in the natural environment, inducing high health risks worldwide. It remains challenging to characterize the accurate molecular structures of MNPs. Herein, we integrate 193 nm ultraviolet photodissociation (UVPD) with mass spectrometry to interrogate the molecular structures of poly(ethylene glycol) terephthalate and polyamide (PA) MNPs. The backbones of the MNP polymer can be efficiently dissociated by UVPD, producing rich types of fragment ions. Compared to high-energy collision dissociation (HCD), the structural informative fragment ions and corresponding sequence coverages obtained by UVPD were all improved 2.3 times on average, resulting in almost complete sequence coverage and precise structural interrogation of MNPs. We successfully determine the backbone connectivity differences of MNP analogues PA6, PA66, and PA610 by improving the average sequence coverage from 26.8% by HCD to 89.4% by UVPD. Our results highlight the potential of UVPD in characterizing and discriminating backbone connectivity and chain end structures of different types of MNPs.

Differentiation of isobaric cross-linked peptides prepared via maleimide chemistry using MALDI-MS and MS/MS.

The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS) are useful for differentiating these isobaric species. Four cross-linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross-linked peptides were purified using RP-HPLC (reversed-phase high-performance liquid chromatography) and differentiated using MALDI-MS, MALDI-MS/MS, and ultraviolet photodissociation. A cysteinyl peptide-containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI-MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI-MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. The thiosuccinimide (but not thiazine) form of the cross-linked peptide yielded individual constituent peptides using MALDI-MS and MALDI-MS/MS, showing specific 1,4-elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form.

Primary and Secondary Processes in the Ultraviolet Photodissociation of CpCo(CO)2 (Cyclopentadienylcobalt Dicarbonyl).

We investigated the photodissociation dynamics of CpCo(CO)2 (cyclopentadienylcobalt dicarbonyl) in metal-to-ligand charge transfer (MLCT) bands. By employing DFT calculations, the absorption band (210-240 nm) was characterized as a charge transfer from the Co center to the Cp (cyclopentadienyl, C5H5) ligand. Ion imaging was utilized to analyze the CO fragments and coordinatively unsaturated complexes (CpCoCO, CpCo, and CoC3H3) across the entire MLCT band. Measuring the production yields of individual unsaturated complexes as a function of photolysis wavelength by considering wavelength dependence indicated the involvement of several photochemical pathways: the first photodissociation and sequential dissociation of CpCo(CO)2, and the second photodissociation of unsaturated intermediates within the pulse duration of the photolysis laser. The recoil velocity shifts of CpCo and CoC3H3 were attributed to the onset of the sequential dissociation of CpCoCO. Evidence for the second photodissociation of CpCoCO was obtained through the matching of linear momenta between the CO(v = 0, 1) and CpCo fragments. The DFT calculations performed to determine the electronic structures and potential energy curves for photoinduced CO loss in CpCo(CO)2 and CpCoCO supported our interpretation of the experimental results. This study presents a practical approach to selectively detecting specific processes among the mixture of products and intermediates when photolyzing transition-metal carbonyls, as their concurrent generation is unavoidable in laser-based experiments.

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC–MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic’s (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

Structural Characterization of the Metalized Radical Cations of Adenosine ([Ade+Li-H]•+ and [Ade+Na-H]•+) by Infrared Multiphoton Dissociation Spectroscopy and Theoretical Studies.

Nucleoside radicals are key intermediates in the process of DNA damage, and alkali metal ions are a common group of ions in living organisms. However, so far, there has been a significant lack of research on the structural effects of alkali metal ions on nucleoside free radicals. In this study, we report a new method for generating metalized nucleoside radical cations in the gas phase. The radical cations [Ade+M-H]•+ (M = Li, Na) are generated by the 280 nm ultraviolet photodissociation (UVPD) of the precursor ions of lithiated and sodiated ions of 2-iodoadenine in a Fourier transform ion cyclotron resonance (FT ICR) cell. Further infrared multiphoton dissociation (IRMPD) spectra of both radical cations were recorded in the region of 2750-3750 cm-1. By combining these results with theoretical calculations, the most stable isomers of both radicals can be identified, which share the common characteristics of triple coordination patterns of the metal ions. For both radical species, the lowest-energy isomers undergo hydrogen transfer. Although the sugar ring in the most stable isomer of [Ade+Li-H]•+ is in a (South, syn) conformation similar to that of [Ado+Na]+, [Ade+Na-H]•+ is distinguished by the unexpected opening of the sugar ring. Their theoretical spectra are in good agreement with experimental spectra. However, due to the flexibility of the structures and the complexity of their potential energy surfaces, the hydrogen transfer pathways still need to be further studied. Considering that the free radicals formed directly after C-I cleavage have some similar spectral characteristics, the existence of these corresponding isomers cannot be ruled out. The findings imply that the structures of nucleoside radicals may be significantly influenced by the attached alkali metal ions. More detailed experiments and theoretical calculations are still crucial.