Chemiluminescence of a Firefly Luciferin Analogue Reveals that Formation of the Key Intermediate Responsible for Excited State Generation Occurs on a Fully Concerted Step.

The chemiluminescence (CL) reaction of eight different 2-(4-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylate esters with an organic superbase and oxygen was investigated through a kinetic and computational study. These esters are all analogues to the luciferin substrate involved in efficient firefly bioluminescence. The kinetic data obtained from CL emission and light absorption assays were used in the context of linear free energy relationships (LFER); we obtained the Hammett reaction constant ρ = +1.62 ± 0.09 and the Brønsted constant ßlg = -0.39 ± 0.04. These observations from LFER, together with activation parameters obtained from Arrhenius plots, suggest that the formation of the high-energy intermediate (HEI) 1,2-dioxetanone occurs via a concerted mechanism during the rate-determining step of the reaction. Calculations performed using density functional theory support a late transition state for HEI formation within the reaction mechanism pathway, which was described considering geometric parameters, Wiberg bond indices from natural bond order analysis, and the atomic charges derived from the electrostatic potential.

Mechanisms for the Deactivation of the Electronic Excited States of α-(2-Hydroxyphenyl)-N-phenylnitrone: From Intramolecular Proton and Charge Transfer to Structure Twisting and Aggregation.

The search for new prominent chemosensors is significantly related to the rationalization of possible multiple pathways of excited-state deactivation. We have prepared and studied compound α-(2-hydroxyphenyl)-N-phenylnitrone (Nit-OH), observing that Nit-OH is stable in acetonitrile solution under UV-vis light. The experimentally observed 540 nm fluorescence for Nit-OH was shown to be related to excitation at 360 nm from the highest occupied molecular orbital to the lowest unoccupied molecular orbital (HOMO-LUMO transition). Potential energy curves (PECs) for the S1 state of Nit-OH did show that there are structures associated with excited-state intramolecular proton transfer (ESIPT), and the existence of an intramolecular H-bonding was confirmed using X-ray powder diffraction (XRD). Twisted intramolecular charge transfer (TICT) took place following ESIPT, and a nonradiative deactivation at the S1/S0 conical intersection occurred; aggregation-induced emission was observed at 540 nm associated with the formation of a stacked dimer. Anti-Kasha emission from the S2 was proposed based on the dependence of the fluorescence excitation wavelength on Nit-OH concentration. From the calculation of the PEC for the S2 state, we obtained radiative transitions at 379 and 432 nm, similar to the obtained experimental values of 383 and 453 nm. We proposed a Jablonski-like diagram that depicts all experimental and theoretical electronic transitions for Nit-OH, summarizing the unique intricate photophysical behavior of this nitrone derivative.

Evidence for the Formation of 1,2-Dioxetane as a High-Energy Intermediate and Possible Chemiexcitation Pathways in the Chemiluminescence of Lophine Peroxides.

A kinetic study of the chemiluminescent (CL) reaction mechanism of lophine-derived hydroperoxides and silylperoxides induced by a base and fluoride, respectively, provided evidence for the formation of a 1,2-dioxetane as a high-energy intermediate (HEI) of this CL transformation. This was postulated using a linear Hammett relationship, consistent with the formation of negative charge on the transition state of HEI generation (ρ > 1). The decomposition of this HEI leads to chemiexcitation with overall low singlet excited state formation quantum yield (ΦS from 1.1 to 14.5 × 10-5 E mol-1); nonetheless, ΦS = 1.20 × 10-3 E mol-1 was observed with both peroxides substituted with bromine. The use of electron-donating substituents increases chemiexcitation efficiency, while it also reduces the rate for both formation and decomposition of the HEI. Different possible pathways for HEI decomposition and chemiexcitation are discussed in light of literature data from the perspective of the substituent effect. This system could be explored in the future for analytical and labeling purposes or for biological oxidation through chemiexcitation.

Light-Emitting Probes for Labeling Peptides.

Peptides are versatile biopolymers composed of 2-100 amino acid residues that present a wide range of biological functions and constitute potential therapies for numerous diseases, partly due to their ability to penetrate cell membranes. However, their mechanisms of action have not been fully elucidated due to the lack of appropriate tools. Existing light-emitting probes are limited by their cytotoxicity and large size, which can alter peptide structure and function. Here, we describe the available fluorescent, bioluminescent, and chemiluminescent probes for labeling peptides, with a focus on minimalistic options.

The decomposition of triphenylimidazole-para-acetate follows specific base catalysis and can be conveniently followed by fluorescence.

The kinetics of the decomposition reaction of 4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl acetate (1) in basic alcoholic media was investigated, using a simple fluorescence (FL) spectrophotometric procedure. The process was conveniently studied using FL, since the triphenylimidazole-derived ester 1 and its reaction products (the corresponding phenol 2 and phenolate 2- ) are all highly fluorescent (ΦFL  > 37%). By carefully selecting excitation and emission wavelengths, observed rate constants k1 in the order of 10-3 to 10-2  s-1 were obtained from either reactant consumption (λex  = 300 nm, λem  = 400 nm) or product formation (λex  = 350 nm, λem  = 475 nm); these were shown to be kinetically equivalent. Intensity-decay time profiles also gave a residual FL intensity parameter, shown to be associated to the distribution of produced species 2 and 2- , according to the basicity of the medium. Studying the reaction in both methanol (MeOH) and isopropanol (iPrOH), upon addition of HO- , provided evidence that the solvent's conjugate base is the active nucleophilic species. When different bases were used (tBuO- , HO- , DBU and TEA), bimolecular rate constants kbim ranging from 4.5 to 6.5 L mol-1  s-1 were obtained, which proved to be non-dependent on the base pKaH , suggesting specific base catalysis for the decomposition of 1 in alcoholic media.

Peroxyoxalate High-Energy Intermediate is Efficiently Decomposed by the Catalyst Imidazole.

The peroxyoxalate reaction is a highly efficient chemiluminescence system, its chemiexcitation process involving the intermolecular interaction between an activator (ACT) and the high-energy intermediate (HEI) of the reaction. Typically, the HEI is generated through the reaction of an oxalate ester with H2 O2 , in the presence of a basic/nucleophilic catalyst, such as imidazole (IMI-H). IMI-H, besides catalyzing the formation of the HEI, is also known to decompose this peroxidic intermediate. Despite that, up to now, no rate constant value has been determined for such significant interaction. Through kinetic measurements, we have observed that IMI-H is roughly four times more efficient than 9,10-diphenylanthracene (DPA), a classic ACT, in catalyzing the decomposition of the HEI by a bimolecular electron transfer reaction through a Chemically Initiated Electron Exchange Luminescence-like process. For instance, when IMI-H and DPA are at the same concentration, 78% of the generated HEI is actually consumed by the nonemissive bimolecular interaction with IMI-H. We have obtained an average singlet excited state formation quantum yield, at infinite ACT concentration, of (5.5 ± 0.5) × 10(-2)  E mol(-1) , determined at five different IMI-H concentrations. This ultimately suggests that the yield of formation of HEI actually does not depend on the IMI-H concentration.

Spectroscopic Studies on the Interaction of Metallic Ions with an Imidazolyl-Phenolic System.

A fluorescent imidazolyl-phenolic compound was applied on the detection of metallic species (Cu(2+), Al(3+), Cr(3+) and Fe(3+)) in a CH3CN/H2O (95/5, v/v) media. The presence and concentration of these cations altered significantly the emission profile of the probe, mainly lowering the signal intensity at 466 nm, while a new emission band around 395 nm appeared (for the trivalent ions). These results were rationalized as a combination of collisional quenching (KSV in the 10(3)-10(4) L mol(-1) range) and formation of a coordinated compound. The later disrupts the Excited State Intramolecular Proton Transfer that regulates the keto-enol tautomerism originally present on the free probe. Since the quenching efficiency and the obtained emission profiles are drastically different for Cu(2+) and Fe(3+) ions, this allows their differential recognition.

Chemiluminescence efficiency of catalyzed 1,2-dioxetanone decomposition determined by steric effects.

The chemiluminescent decomposition of 1,2-dioxetanones (α-peroxylactones), catalyzed by an appropriate fluorescent activator, is an important simple model for efficient bioluminescent transformations. In this work, we report experimental data on the catalyzed decomposition of two spiro-substituted 1,2-dioxetanone derivatives, which support the occurrence of an intermolecular electron transfer from the activator to the peroxide. The low efficiency of the studied systems is associated with steric hindrance during the chemiexcitation sequence, rationalized using the concept of supermolecule formation between the peroxide and the catalyst. This approach explains the difference in the chemiexcitation efficiencies in the decomposition of four-membered cyclic peroxide derivatives: 1,2-dioxetanes, 1,2-dioxetanones, and 1,2-dioxetanedione (the intermediate in the peroxyoxalate reaction), which are the most important model compounds for excited-state formation in chemiluminescence and bioluminescence processes.

Revision of singlet quantum yields in the catalyzed decomposition of cyclic peroxides.

The chemiluminescence of cyclic peroxides activated by oxidizable fluorescent dyes is an example of chemically initiated electron exchange luminescence (CIEEL), which has been used also to explain the efficient bioluminescence of fireflies. Diphenoyl peroxide and dimethyl-1,2-dioxetanone were used as model compounds for the development of this CIEEL mechanism. However, the chemiexcitation efficiency of diphenoyl peroxide was found to be much lower than originally described. In this work, we redetermine the chemiexcitation quantum efficiency of dimethyl-1,2-dioxetanone, a more adequate model for firefly bioluminescence, and found a singlet quantum yield (Φ(S)) of 0.1%, a value at least 2 orders of magnitude lower than previously reported. Furthermore, we synthesized two other 1,2-dioxetanone derivatives and confirm the low chemiexcitation efficiency (Φ(S) < 0.1%) of the intermolecular CIEEL-activated decomposition of this class of cyclic peroxides. These results are compared with other chemiluminescent reactions, supporting the general trend that intermolecular CIEEL systems are much less efficient in generating singlet excited states than analogous intramolecular processes (Φ(S) ≈ 50%), with the notable exception of the peroxyoxalate reaction (Φ(S) ≈ 60%).

Studies on PVP hydrogel-supported luminol chemiluminescence: 2. Luminometer calibration and potential analytical applications.

The use of poly(N-vinyl-2-pyrrolidone) (PVP) hydrogel-supported luminol chemiluminescence (CL) for the automatic determination of hydrogen peroxide and the quantification of the antiradical capacity of Trolox is described. The hydrogel containing luminol and hemin is prepared directly on a 96-well microplate and can be stored for up to 3 months without significant decrease in CL quantum yields. Furthermore, this system can also be used as a secondary light standard for the calibration of microplate luminometers.

Studies on PVP hydrogel-supported luminol chemiluminescence: 1. Kinetic and mechanistic aspects using multivariate factorial analysis.

The chemiluminescent oxidation of luminol by hydrogen peroxide in the presence of hemin is revisited in an UV-C cross-linked PVP hydrogel. Chemiluminescence properties such as initial light intensity (I(0)), area of emission (S) and observed rate constants (k(obs)) are studied, varying the concentration of all reactants using a multivariate factorial approach.