Aromatic Radical Cations Research Articles

Electrochemical Deconstructive Methoxylation of Arylalcohols-A Synthetic and Mechanistic Investigation.

Herein, we report a mechanistic investigation of a recently developed electrochemical method for the deconstructive methoxylation of arylalcohols. A combination of synthetic, electroanalytical, and computational experiments have been performed to gain a deeper understanding of the reaction mechanism and the structural requirements for fragmentation to occur. It was found that 2-arylalcohols undergo anodic oxidation to form the corresponding aromatic radical cations, which fragment to form oxocarbenium ions and benzylic radical intermediates via mesolytic cleavage, with further anodic oxidation and trapping of the benzylic carbocation with methanol to generate the observed methyl ether products. It was also found that the electrochemical fragmentation of 2-arylalkanols is promoted by structural features that stabilize the oxocarbenium ions and/or benzylic radical intermediates formed upon mesolytic cleavage of the aromatic radical cations. With an enhanced understanding of the reaction mechanism and the structural features that promote fragmentation, it is anticipated that alternative electrosynthetic transformations will be developed that utilize this powerful, yet underdeveloped, mode of substrate activation.

Electrochemical Synthesis of C(sp3)-Rich Heterocycles via Mesolytic Cleavage of Anodically Generated Aromatic Radical Cations.

Herein we report an electrochemical deconstructive functionalization approach for the synthesis of C(sp3)-rich heterocycles. The reaction proceeds via the mesolytic cleavage of anodically generated aromatic radical cations and the trapping of formed carbocation intermediates with internal nucleophiles. The method has been demonstrated across various arylalcohol substrates to access a diverse range of C(sp3)-rich heterocycles including tetrahydrofuran, tetrahydropyran, and pyrrolidine scaffolds (26 examples). The electrochemical method was demonstrated on a 5 mmol scale via single pass continuous flow, which utilized lower supporting electrolyte concentration and exhibited increased productivity in relation to the batch process.

Electrochemical deconstructive functionalization of arylcycloalkanes via fragmentation of anodically generated aromatic radical cations.

This highlight summarises electrochemical approaches for the deconstructive functionalization of arylcycloalkanes via the fragmentation of anodically generated aromatic radical cations. A diverse range of deconstructive functionalization processes is described, including discussion on the electrochemical reaction conditions employed, scope and limitations, and reaction mechanisms, in addition to highlighting future opportunities in this burgeoning area of sustainable synthesis.

A Fenton Approach to Aromatic Radical Cations and Diarylmethane Synthesis.

Manipulating carbon-centered radicals to add to electron-deficient systems is a well-precedented process. By coupling the Fe(II)-mediated Fenton reaction with the Fe(III)-mediated single-electron oxidation of anisolic compounds, we demonstrate how electron-rich carbon-centered radicals can react with electron-rich arenes through a radical-polar cascade pathway. This bioinspired approach produces diarylmethane derivatives from simple unfunctionalized precursors.

Deconstructive Functionalization of Unstrained Cycloalkanols via Electrochemically Generated Aromatic Radical Cations.

Herein we report an electrochemical approach for the deconstructive functionalization of cycloalkanols, where various alcohols, carboxylic acids, and N-heterocycles are employed as nucleophiles. The method has been demonstrated across a broad range of cycloalkanol substrates, including various ring sizes and substituents, to access useful remotely functionalized ketone products (36 examples). The method was demonstrated on a gram scale via single-pass continuous flow, which exhibited increased productivity in relation to the batch process.

EPR Study on the Oxidative Degradation of Phenyl Sulfonates, Constituents of Aromatic Hydrocarbon-Based Proton-Exchange Fuel Cell Membranes.

Sulfonated aromatic hydrocarbon-based ionomers are potential constituents of next-generation polymer electrolyte fuel cells (PEFCs). Widespread application is currently limited due to their susceptibility to radical-initiated oxidative degradation that, among other intermediates, involves the formation of highly reactive aromatic cation radicals. The intermediates undergo chain cleavage (dealkylation/dearylation) and the loss of protogenic sulfonate groups, all leading to performance loss and eventual membrane failure. Laser flash photolysis experiments indicated that cation radicals can also be formed via direct electron ejection. We aim to establish the major degradation pathway of proton-exchange membranes (PEMs). To this end, we irradiated aqueous solutions of phenyl sulfonate-type model compounds with a Xe arc lamp, thus generating radicals. The radicals were trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and the formed adducts were observed by electron paramagnetic resonance (EPR). The formed DMPO spin adducts were assigned and relative adduct concentrations were quantified by simulation of the experimental EPR spectra. Through the formation of the DMPO/•SO3 - adduct, we established that desulfonation dominates for monoaromatic phenyl sulfonates. We observed that diaryl ether sulfonates readily undergo homolytic C-O scission that produces DMPO/•aryl adducts. Our results support the notion that polyphenylene sulfonates are the most stable against oxidative attack and effectively transfer electrons from DMPO, forming DMPO/•OH. Our findings help to identify durable moieties that can be used as building blocks in the development of next-generation PEMs.

Moderation of Oxidative Damage on Aromatic Hydrocarbon-Based Polymers

During the operation of aromatic hydrocarbon-based proton exchange membrane fuel cells, formed radical species attack the membrane. The most deleterious radical formed is HO·, both strongly electrophilic and oxidising. Oligomers of α-methylstyrene sulfonates (PAMSS) were used as model compounds. We report on the complex reaction cascade following the oxidative attack on aromatic cores bearing proton conductive sulfonate groups. UV-absorption bands of initial oxidation products indicate the formation of radical adducts and aromatic cation radicals. Subsequently, a transformation associated with an absorbance build-up at 580 nm is observed, presumably also related to aromatic cation radicals. Build-up and decay are significantly accelerated at high ionic strength levels that are also typical in fuel cells. Increased ionic strength causes phase separation: dynamic light scattering experiments indicate particle formation that is dependent both on chain length and on ionic strength. Aromatic cation radicals are known strong oxidants. With a presumed redox potential of E°((PAMSS-580 nm)·+/PAMSS) ∼ 2 V this oxidizing species should react also with mediocre reductants. Here, Mn(II) was oxidised to Mn(III) with rate constants of (5–10) × 106 M−1s−1. Implications for experimental design of kinetics experiments and understanding chemical mechanisms are discussed.

Impact of substitution on reactions and stability of one-electron oxidised phenyl sulfonates in aqueous solution.

Highly reactive aromatic cation radicals have been invoked lately in synthetic routes and in the degradation pathways of hydrocarbon-based polymers. Changes in the electron density of aromatic compounds are expected to alter the reaction pathway following one electron oxidation through altering the pKa of the formed intermediate cation radical. Electron-donating groups increase its stability, however, little experimental data are known. While, in theory, the cation radical can be repaired by simple electron transfer, electron transfer to or from its deprotonated form, the hydroxycyclohexadienyl radical, will cause permanent modification or degradation. Time-resolved absorption spectroscopy indicates a pKa ≈ 2–3 for the 4-(tert-butyl)-2-methoxyphenylsulfonate (BMPS) radical cation, while its parent compound 4-(tert-butyl) phenylsulfonate (BPS) is much more acidic. The stability of both compounds towards oxidation by HO˙ was evaluated under air at pH 5 and pH 0. At pH 5, both BMPS and BPS are unstable, and superstoichiometric degradation was observed. Degradation was slightly reduced for BPS at pH 0. In contrast, the more electron rich BMPS showed 80% lower degradation. We unambigously showed that in the presence of Ce(iii) and H2O2 at pH 0 both BMPS and BPS could be catalytically repaired via one electron reduction, resulting in further damage moderation.

Reactivity Trends in the Gas-Phase Addition of Acetylene to the N-Protonated Aryl Radical Cations of Pyridine, Aniline, and Benzonitrile.

A key step in gas-phase polycyclic aromatic hydrocarbon (PAH) formation involves the addition of acetylene (or other alkyne) to σ-type aromatic radicals, with successive additions yielding more complex PAHs. A similar process can happen for N-containing aromatics. In cold diffuse environments, such as the interstellar medium, rates of radical addition may be enhanced when the σ-type radical is charged. This paper investigates the gas-phase ion-molecule reactions of acetylene with nine aromatic distonic σ-type radical cations derived from pyridinium (Pyr), anilinium (Anl), and benzonitrilium (Bzn) ions. Three isomers are studied in each case (radical sites at the ortho, meta, and para positions). Using a room temperature ion trap, second-order rate coefficients, product branching ratios, and reaction efficiencies are measured. The rate coefficients increase from para to ortho positions. The second-order rate coefficients can be sorted into three groups: low, between 1 and 3 × 10-12 cm3 molecule-1 s-1 (3Anl and 4Anl); intermediate, between 5 and 15 × 10-12 cm3 molecule-1 s-1 (2Bzn, 3Bzn, and 4Bzn); and high, between 8 and 31 × 10-11 cm3 molecule-1 s-1 (2Anl, 2Pyr, 3Pyr, and 4Pyr); and 2Anl is the only radical cation with a rate coefficient distinctly different from its isomers. Quantum chemical calculations, using M06-2X-D3(0)/6-31++G(2df,p) geometries and DSD-PBEP86-NL/aug-cc-pVQZ energies, are deployed to rationalize reactivity trends based on the stability of prereactive complexes. The G3X-K method guides the assignment of product ions following adduct formation. The rate coefficient trend can be rationalized by a simple model based on the prereactive complex forward barrier height.

Stable isomeric structures of the pyridine cation (C5H5N•+) and protonated pyridine (C5H5NH+) elucidated by cold ion infrared spectroscopy

Cold ion action spectroscopy is applied to record the gas phase infrared fingerprint spectra of mass-selected C5H5N•+ and C5H5NH+ cations using an infrared free electron laser. The structures of the cations are deduced from the experimental spectra using anharmonic vibrational frequencies from density functional theory calculations. A very good agreement between experimental and theoretical infrared frequencies is observed. The dominant structure of the C5H5N•+ cation is assigned to the aromatic pyridine radical cation form. Additionally, a minor contribution of the lower energetic α-distonic isomer is observed. The C5H5NH+ cation is ascribed to the aromatic pyridinium cation where protonation has taken place on the nitrogen atom. The rare-gas tag used in the action spectroscopic method has a negligible effect on the vibrational frequencies. The observed species, with now accurately determined vibrational frequencies, are good candidates for future rotational spectroscopic studies and infrared observations in astronomical sources such as interstellar clouds or Titan’s atmosphere.

Large Aromatic Hydrocarbon Radical Cation with Global Aromaticity and State-Associated Magnetic Activity

The stable radical cation of large aromatic hydrocarbons remains a challenge, due to the recessive aromatic character and high reactivity. Here we report the design, synthesis, and characterization...

Reaction of Distonic Aryl and Alkyl Radical Cations with Amines: The Role of Charge and Spin Revealed by Mass Spectrometry, Kinetic Studies, and DFT Calculations

AbstractGas‐phase reaction of the aromatic distonic radical cations 4‐Pyr+. and 3‐Pyr+. with amines led to formation of the corresponding amino pyridinium ions 4‐Pyr+NR2 and 3‐Pyr+NR2 through amine addition at the site of the radical, followed by homolytic β‐fragmentation. The reaction efficiencies range from 66–100 % for 4‐Pyr+. and 57–86 % for 3‐Pyr+., respectively, indicating practically collision‐controlled processes in some cases. Computational studies revealed that the combination of positive charge and spin makes nucleophilic attack by the amine at the site of the radical barrierless and strongly exothermic by about 175±15 kJ mol−1, thereby rendering ‘conventional’ radical pathways through hydrogen abstraction or addition onto π systems less important. Exemplary studies with 4‐Pyr+. showed that this reaction can be reproduced in solution. A similar addition/radical fragmentation sequence occurs also in the gas‐phase reaction of amines with the aliphatic distonic radical cation Oxo+C., showing that the observed charge‐spin synergism is not limited to aromatic systems.

Distinguishing Isomeric Aromatic Radical Cations by Using Energy-Resolved Ion Trap and Medium Energy Collision-Activated Dissociation Mass Spectrometry.

Different collision-activated dissociation (CAD) methods were evaluated for their effectiveness at distinguishing several ionized isomeric aromatic compounds by using a linear quadrupole ion trap/orbitrap mass spectrometer. The compounds were ionized by using atmospheric pressure chemical ionization (APCI) with carbon disulfide solvent in the positive ion mode to generate stable molecular ions with limited fragmentation. They were subjected to CAD in the linear quadrupole ion trap (ITCAD) and in an octupole collision cell (medium-energy collision-activated dissociation, MCAD; also known as HCD). Experiments conducted by attempting tovaryion activation times revealed that MCAD and ITCAD occur in the microsecond and millisecond time regimes, respectively. MCAD was found to impart substantially greater internal energies into the molecular ions compared to ITCAD. Accordingly, molecular ions subjected to MCAD favored dissociation via fast σ-bond cleavages, while molecular ions subjected to ITCAD tended to favor rearrangement reactions. MCAD used in the energy-resolved mode (ER-MCAD) enabled the distinction of six ionized isomeric compounds from each other based on modified crossing-point energies (collision energies where the molecular ions and selected fragment ions have an equal abundance). This was not true for ER-ITCAD. Overall, MCAD was superior over ITCAD at the differentiation of isomeric ions, and it provided more detailed structural information.

Using Density Functional Theory Based Methods to Investigate the Photophysics of Polycyclic Aromatic Hydrocarbon Radical Cations: A Benchmark Study on Naphthalene, Pyrene and Perylene Cations

AbstractUnrestricted DFT (UDFT), time‐dependent DFT (TDDFT) and spin‐flip TDDFT (SF‐TDDFT) were used to investigate the potential energy surfaces of the ground and first two electronic excited states of the naphthalene, pyrene and perylene radical cations. In particular, conical intersections (which play a central role in the photophysics of these cations) were located with these DFT‐based methods. The results are consistent with accurate multiconfigurational wavefunction‐based ab initio methods. These show that naphthalene and pyrene cations can quickly relax nonradiatively from their excited states back down to the original ground state species through easily accessible conical intersections, but the perylene cation cannot do so, due to the absence of any accessible funnels between the lowest excited state and the ground state, leaving radiative decay as the most probable photophysical pathway. This study paves the way for using computationally efficient density functional theory (DFT)‐based methods in future investigations of the photophysics of much larger polycyclic aromatic hydrocarbons, for which multiconfigurational wavefunction‐based methods become prohibitively expensive.

Structure and Reactivity of Aromatic Radical Cations Generated by FeCl3.

This paper describes the isolation and characterization of an aromatic radical cation generated by FeCl3. X-ray crystallographic analysis and kinetic studies reveal the mechanism of the generation of aromatic radical cation. In the solid state, a tight ion-pair of a radical cation with FeCl4- is observed. Leveraging the efficient generation of the radical cation-FeCl4- ion pair, we explore a radical cation-induced cycloaddition of trans-anethole initiated by catalytic amount of FeCl3. Both [4+2] cycloaddition and [2+2] cycloaddition with a broad substrate scope are also described. Moreover, a 100 g-scale reaction is demonstrated with the use of 1 mol % of FeCl3 as a simple and a highly active initiator.

Spectroscopic study of radical cation species formed on sulfated TiO2 upon benzene adsorption

Sulfated TiO2 is well known for its high acidity, which is due to the presence of sulfate ions that create strong Brönsted and Lewis acid sites. The Lewis acid sites can also exhibit oxidizing properties. As previously observed for sulfated ZrO2, the greater the strength of the acid site, the greater the oxidative potential.The oxidative ability can be evaluated by the interaction with a substance with high ionization potential such as benzene. Benzene was adsorbed on sulfated TiO2 at room temperature, and the species formed from the interaction was investigated by Raman and electron spin resonance (ESR) spectroscopy. Resonance Raman spectra of adsorbed benzene showed bands that are distinct from bands observed in the spectra of neutral molecules. Furthermore, from the absorption spectra in the visible and near-infrared it was possible to identify these species as phenylene oligomer radical cations. Also, aromatic radical cations were detected by ESR spectra. Although they were present in small amounts, these cations may be responsible for the bands observed in the Raman spectra because of the resonance Raman effect signal enhancement.

Antioxidant Strategies for Hydrocarbon-Based Membranes

Perfluoroalkylsulfonic acid (PFSA) ionomers are commonly used as electrolyte membrane in low temperature fuel cells and membrane water electrolyzers. The quest for alternative membranes based on hydrocarbon chemistry has, however, not subsided and these materials continue to be of interest. Their versatile chemistry provides design flexibility and offers the prospect of tailoring membrane chemistry and architecture to specific requirements. Typical hydrocarbon ionomers are thermally stable polymers that contain aromatic units in the main chain, which may be linked by a variety of functional groups (e.g., polysulfones, polyphenylenes). They are of high interest because of their expected lower cost, intrinsically lower gas permeability, and higher glass transition temperatures compared to PFSA membranes [1]. The lower reactant permeability in particular is extremely attractive: it enables the use of thinner membranes, which leads to cost reduction and increased power density. Furthermore, the lower gas permeability leads to a lower radical formation rate through reaction of H2 and O2 on the catalyst surface. However, polyaromatic membranes are generally and intrinsically more susceptible to attack and degradation induced by oxidizing radicals [2]. In particular, aromatic units present in most of these polymers react readily and rapidly with radical intermediates, e.g. HO·, which are formed in the fuel cell in the presence of H2, O2 and the noble metal catalyst. Radical attack triggers chain scission and polymer decomposition. The objective of this study is to investigate, implement and assess strategies to mitigate radical induced degradation of aromatic hydrocarbon based proton exchange membranes for fuel cells. Other than in PFSA membranes, where HO· radicals have a lifetime on the order of microseconds, their lifetime is expected to be significantly shorter, i.e. ~1 ns, in polyaromatic membranes due to the fast reaction of HO· with the aromatic units. Therefore, scavenging of HO· using cerium-ions or ceria nanoparticles is not likely to be an effective approach. Alternative radical scavengers, such as hindered phenols, which are also used as antioxidants in plastics, may achieve a higher scavenging yield, yet in order to sustain stabilization over thousands of hours, the radical quenching needs to be a catalytic process, as it is the case with the redox cycling ability of Ce and Mn ions used as antioxidants in PFSA ionomers [3]. An alternative approach is to target the intermediates formed upon attack of the ionomer by HO·. Some intermediates, such as aromatic radical cations, may be sufficiently long lived for suitable repair chemistry to take effect [4]. In this study, we present a kinetic study of possible pathways for damage transfer and ionomer repair reactions and highlight requirements for polymer intermediate lifetimes and kinetics of repair and regeneration reactions of additives. One challenge is to stabilize the ionomer intermediates sufficiently long (milliseconds range) to allow repair reactions to take place. Another challenge is to ensure the kinetics of repair reactions are sufficiently fast and the additive can be repeatedly regenerated. Evidently, the chemistry of the polymer and the additive needs to be adequately tuned and side-reactions minimized. Furthermore, additives incorporated into the membrane merely by doping may be leached out over time, calling for strategies to tether the functional motifs to the polymer chain or an inorganic filler for immobilization. (Figure: Schematic representation of an antioxidant strategy for hydrocarbon based proton exchange membranes based on the ‘damage transfer’ and ‘repair’ concept. the ionomer P is attacked by HO·, the damage inflicted to the polymer is then transferred to an additive (M, mediator), which undergoes a subsequent repair reaction with H2O2.)

Radiation-thermal decomposition of lignin: Products and the mechanism of their formation (Review)

The mechanism of the radiation-thermal conversion of lignin including the formation of aromatic radical cations and their fragmentation resulting in the appearance of phenoxyl radicals is considered. The multipath formation of phenoxyl radicals occurs with the participation of the reactions of molecules with electrons and small radicals (•Н and •СН3) and electronic excitation relaxation processes. Phenoxyl radicals are characterized by smaller thermal stability in comparison with that of parent macromolecules. The further thermally stimulated decomposition of these radicals results in the release of monohydric and dihydric phenols from a polymeric chain. The most effective liberation of phenols takes place on the surface of lignin particles, whereas the formation of wood charcoal with the participation of unsaturated products dominates in the bulk. The formation of dihydric phenols is intensified in the presence of alkanes in the irradiated sample; this fact is indicative of an important role of •Н and •СН3 radicals in the formation of monomeric phenol products.

Radical Cation Diels-Alder By Electrocatalyst

In recent years, redox reactions with single electron transfer have received significant attention from many chemists. Organo-catalysts and photo-catalysts have been growing as a promising approach to electron path-ways. The electrochemical method is also playing roles as one of the most straightforward processes. With this method, we designed intermolecular carbon-carbon bond forming reactions, in which electron is the key reagent and the catalytic amount of electricity is enough for the full conversion. For example, radical cation Diels-Alder reaction with aromatic "redox tag" is in accordance with it. In this reaction, the chain cycle is the key factor to realize the significant decrease of consumed electricity. Our success leads to the development of the environmentally friendly and extreme high productivity synthetic method to produce the compound which has useful chemical structure for natural compounds and pharmaceuticals. Our investigation commenced with the reaction between trans-anethole (1) and isoprene (2), using electrocatalytic approach. In the reaction, it is understood well that the radical cation (3 ・ +) is generated, accompanying by chain process (Fig. 1). From cyclic voltammetry investigation, it is indicated that 1 (Ep ox = 1.07 V vs. Ag/AgCl) is anodically oxidized selectively in the presence of 2 (Ep ox = 1.83 V vs Ag/AgCl). Based on this, we demonstrate that the aromatic radical cation, which is given by the intermolecular [4 + 2] cycloaddition between radical cation 1 ・ + and 2, gives rise to a radical cation chain cycles and completion of the reaction with the catalytic amount of electricity. Here, we envisioned that the aromatic ring of 1might work as a redox tag to accomplish an electrocatalytic Diels-Alder reaction. Hence, we carried out anodic oxidation of 1in the presence of 2 equiv. of 2 at 1.00 V vs. Ag/AgCl. Beyond our expectation, only 0.1 F/mol of electricity was sufficient to make the reaction completed in excellent yield. This result suggests that the aromatic radical cation 3 ・ + was reduced predominantly by 1, comparing the reduction at the electrodes. Furthermore, the investigation of Cyclic Voltammetry shows that the oxidation peak of Diels-Alder adduct (3) is 1.40 V vs Ag/AgCl, which is sufficiently higher than that of 1. It can explain that radical cation 3 ・ + would oxidize 1 preferably, leading to the chain propagation. Based upon the mechanistic comprehension, we confirmed the applicable range of substrates, which are dienophile and diene, in the electrocatalytic Diels-Alder reaction. It showed that the moderate range of dienes was available, but the scope of styrene structures was limited. To obtain deeper insight into the mechanism, we conducted density functional theory (DFT) calculations. The calculation results demonstrate that the Dies-Alder reaction is proceed in stepwise mechanism. Besides, we speculated that the combination of electrolyte and solvent had an important role in this reaction. Although we have tried to use a variety of electrolytes and solvents, we have not found better media than LiClO4 / Nitromethane which leads this reaction to the excellent yield and faraday efficiency. Considering that using tetrabutylammonium perchlorate did not work in this reaction, we assume that Li+ has the important role. With the investigation of 7Li NMR and the heat of solution, it is implied that the solvation of ions is the key factor to achieve the electrocatalytic C-C bond forming reaction. Then, we turned out interest to how much we could improve the electro-energy efficiency. As we demonstrated above, only 0.1 F/mol, was enough to complete the reaction. However, we postulated that it still has room to be encouraged. We screened several factors; type of cell, type of electrodes, electrode’s surface area, reaction temperature, concentration of starting materials, concentration of electrolyte, etc… Surprisingly, it turned out that the concentration of substrates had the significant impact on the efficient production. Based upon the optimized condition, we have succeeded in that only 0.015 F/mol realized the full conversion. It is demonstrated by GC-MS monitoring that from 0.005 F/mol to 0.01 F/mol, the average number of chain cycle is 106. This optimized condition has the potential of having a big impact on chemical and pharmaceutical production. Our electrochemical approach realizes the environmentally friendly and extreme high productive synthetic method; requiring only a small amount of solvent and electrolyte, high electro-energy efficiency and recyclable. Taking advantage of these points, we demonstrate that we can scale up the production of the compounds that have useful chemical structure for natural compounds and pharmaceuticals. It is noted that electro chemical approach is promising for not only green but also economical chemistry. Figure 1

Chiral photochemical scissors: Toward site specific cleavage of proteins with light

Abstract A powerful strategy to photocleave the protein backbone at a single site, developed over nearly two decades, is described here. The remarkable ability to target one or even a couple of sites on a large protein with a small molecule, under photochemical control, is a considerable challenge. Systematic modification of the structure of the photoreagent provides valuable information on the binding site recognition as well as the mechanism of the photocleavage reaction. Some factors that impact the photocleavage reaction include the exact location of the probe binding site on the protein, conformations of the bound probe, protein size, functional groups present on the probe that interact with the protein surroundings either in a favorable or unfavorable manner, overall charge on the photoreagent, and photophysical/photochemical properties of the probe. The protein photocleavage studies were preceded by detailed binding studies by a variety of spectroscopic methods. Methods as simple as absorption and fluorescence spectroscopies or more sophisticated circular dichroism spectroscopy were used. Conclusions that are most consistent with the binding data indicated a single binding site on most proteins, irrespective of the probe or the protein, with only one exception noted so far. Such high selectivity for targeting a binding site on a biomolecule by a small molecule is remarkable. Photocleavage studies and laser flash photolysis studies were used to probe the photochemical peptide bond cleavage reactions, which indicated the important role of aromatic cation radicals in the mechanistic path. Our work focused mainly on few generic proteins serum albumin, lysozyme, and avidin; but may be extended to other proteins with appropriate modifications. This article strives to provide some guidance on such design strategies for other proteins or other photoreagents. Molecular modeling studies clearly supported our conclusions and the methods described here could be useful in a rational design of novel photoreagents to target desired sites on proteins in future studies.