Light-Emitting Probes for Labeling Peptides

[Abstract] 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.Estados Unidos. National Institute of General Medical Sciences; R35GM138201Fundação de Amparo à Pesquisa do Estado de São Paulo; 2016/10585-4Fundação de Amparo à Pesquisa do Estado de São Paulo; 2019/15871-3Xunta de Galicia; ED481A-2019/08

Inverse electron transfer in peroxyoxalate chemiexcitation using easily reducible activators

Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron transfer, from the peroxide intermediate to the activator, in its chemiexcitation step. Relative catalytic rate constants and singlet quantum yields were obtained for the peroxyoxalate reaction, using 9-chloro, 9,10-dichloro, 9-cyano and 9,10-dicyanoanthracenes as activators. The linear free-energy correlation of the relative rate constants with the activators' reduction potentials and the dependence of the quantum yields on the released energy confirm, for the first time, the occurrence of this inverse electron transfer.À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Light: a rare reaction product

The production of visible light by chemical reactions constitutes interesting and fascinating phenomena and several reaction mechanisms are discussed to rationalize excited state formation. Most efficient chemiluminescence reactions are thought to involve one or more electron transfer steps and chemiexcitation is believed to occur by radical annihilation. A brief introduction to the general principles of light production and the main known chemiexcitation mechanisms will be given here. Subsequently, recent results on the mechanistic elucidation of efficient chemiluminescence systems, as the peroxyoxalate reaction, the induced decomposition of phenoxy-substituted 1,2-dioxetanes and the catalyzed decomposition of new a-peroxylactones will be discussed.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenadoria para o Aperfeiçoamento de Pessoal do Ensino Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Mechanisms for the Peroxyoxalate System in Aqueous Media and 1,2-Dioxetanone Chemiluminescence

Foi sintetizada e caracterizada a spiro-ciclopentil-1,2-dioxetanona, um derivado de 1,2-dioxetanona inédito, de forma a se contribuir com informações acerca da quimiluminescência de peróxidos orgânicos. Estudos mecanísticos da decomposição quimiluminescente de 1,2-dioxetanonas são escassos, principalmente quando comparados à enorme quantidade de resultados existentes sobre a formação de estados excitados na decomposição de 1,2-dioxetanos. A spiro-ciclopentil-1,2-dioxetanona foi preparada pela ciclização do 1-carbóxi-1- hidroperóxiciclopentano com N,N-diciclohexilcarbodiimida, com rendimento de 1,2%, e caracterizada por RMN de 1H e 13C a baixa temperatura e pelo comportamento cromatográfico da solução peroxídica. Evidências para a ocorrência do mecanismo de Luminescência Iniciada Quimicamente por Troca de Elétron (CIEEL) na formação de estados excitados, na sua variante intermolecular, foram obtidas por estudos cinéticos da decomposição quimiluminescente dessa 1,2-dioxetanona por diferentes ativadores (ACTs). Entretanto, a decomposição desse peróxido ocorre com rendimentos de formação de estados excitados singlete baixos (ΦS≈ 10-4 E mol-1), indicando que essa 1,2-dioxetanona também possui baixa eficiência de formação de estados excitados, conforme constatado recentemente para outros derivados. Foram encontradas evidências para a ocorrência de uma transferência de elétron na etapa limitante de velocidade, onde o ACT é formado no estado excitado, com um coeficiente de transferência de elétron de α = 0,19. Ainda, foi observada uma correlação entre a energia livre liberada para a formação de estados excitados e os rendimentos quânticos, nos moldes do mecanismo CIEEL. De acordo com os resultados obtidos nesse trabalho e em trabalhos anteriores, postula-se que o tamanho do substituinte alquílico no anel da 1,2-dioxetanona causa um efeito drástico na eficiência da decomposição catalisada do peróxido na seqüência CIEEL, diminuindo o valor da constante de equilíbrio para a formação inicial do complexo de transferência de carga Em uma segunda parte do trabalho, foi averiguado o mecanismo da reação peróxi-oxalato em meios aquosos, uma vez que a maioria dos trabalhos sobre esta transformação nestes meios possui ênfase na aplicação analítica. Foi observado que as reações dos oxalatos de bis(2,4,6-triclorofenila) (TCPO), moderadamente reativo, e bis(2,4-dinitrofenila) (DNPO), altamente reativo, com H2O2 e catálise por imidazol, ocorrem pelo mesmo mecanismo, em 1,2-dimetoxietano e em misturas aquosas desse solvente. Para TCPO, observou-se que há a formação de um intermediário de catálise nucleofílica, previamente ao ataque nucleofílico do H2O2. Para DNPO, há uma adição nucleofílica direta do H2O2 ao éster, com e sem catálise básica por imidazol. Em meios aquosos, a hidrólise do éster oxálico ocorre paralelamente à peridrólise, que leva à emissão de luz. Entretanto, as constantes de velocidade para a peridrólise do éster são significativamente maiores do que as para a sua hidrólise. Observou-se que a reação de TCPO com H2O2 em meios orgânico e aquoso, catalisada por 2,6-lutidina (2,6-dimetilpiridina), ocorre com essa base agindo como um catalisador não-nucleofílico. Por último, estudou-se a reação de TCPO com H2O2 em tampão carbonato de potássio, um meio essencialmente aquoso, onde a espécie reativa é a base conjugada do H2O2; entretanto, a constante de velocidade da peridrólise se mostrou similar à de hidrólise do éster oxálico. Nessa última parte do trabalho, foi demonstrado que a reação peróxi-oxalato também pode ser conduzida em meios aquosos.Spiro-cyclopentyl-1,2-dioxetanone, a new 1,2-dioxetanone derivate, was synthesized and characterized, in order to contribute to the present mechanistic knowledge on organic peroxide chemiluminescence. Mechanistic studies on the chemiluminescent 1,2-dioxetanone decomposition are highly limited, especially when compared to the huge amount of data available for 1,2-dioxetanes. The spiro-cyclopentyl-1,2-dioxetanone was prepared by 1-carboxy-1-hydroperoxycyclopentane cyclization with N,N-dicyclohexylcarbodiimide, in a 1.2% yield and characterized by low temperature 1H and 13C NMR and the chromatographic behavior of the peroxide solution. Evidence for the occurrence of the Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism for excited states formation, in its intermolecular version, was obtained from kinetic studies of the chemiluminescent decomposition of this 1,2-dioxetanone with different activators (ACTs). However, the decomposition of this peroxide occurs with low yields of singlet excited state formation (ΦS≈ 10-4 E mol-1), showing that also this 1,2-dioxetanone possesses low excitation efficiency, as recently found for other derivatives. Evidence was found for the occurrence of an electron transfer in the rate limiting step, where the ACT is formed in its electronically excited state, with an electron transfer coefficient α= 0.19. Moreover, a correlation between the free energy released upon excited states formation and the quantum yields is observed, in agreement with the CIEEL mechanism. The results obtained in the present work together with previous data, allow to postulate that the size of the alkyl substituent on the 1,2-dioxetanone ring plays a crucial role for the efficiency of the catalyzed peroxide decomposition in the CIEEL sequence, by lowering the equilibrium constant for initial charge transfer complex formation. In a second part of this work, the mechanism of the peroxyoxalate reaction in aqueous media was studied, due to the fact that most of recent work on this system is mainly concerned only to analytical application aspects. The reaction of the moderately reactive bis(2,4,6-trichlorophenyl) (TCPO) and the highly reactive bis(2,4- dinitrophenyl) oxalate (DNPO) with H2O2 and imidazol catalysis, showed to occur by the same mechanism, in 1,2-dimethoxyethane as well as in its aqueous mixtures. For TCPO, the formation of an intermediate due to nucleophilic catalysis by imidazole was observed, preceding the H2O2 nucleophilic attack. For DNPO, direct nucleophilic addition of H2O2 to the ester occurs, with and without imidazole catalysis. In aqueous media, oxalic ester hydrolysis also occurs concomitantly to its perhydrolysis, which leads to light emission. However, the rate constants for perhydrolysis are significantly higher than the hydrolysis rate constants. For the reaction of TCPO with H2O2 in organic and aqueous medium, catalyzed by 2,6-lutidine (2,6-dimethylpyridine), it was shown that this base acts as a nonnucleophilic catalyst. Finally, the reaction of TCPO with H2O2 in potassium carbonate buffer, an essentially aqueous media, was studied, where the reactive species is the H2O2 conjugate base; however, the oxalic ester perhydrolysis rate constant proofed to be similar to its hydrolysis rate constant. In this last part it is also shown that the peroxyoxalate reaction can be carried out in aqueous media

Chemiluminescence-based uphill energy conversion

The conversion of red excitation light into blue emission light (uphill energy conversion) using unstable 1,2-dioxetanes is described. The method is based on 1,2-dioxetane formation by red-light sensitized photooxygenation of adequate alkenes and subsequent blue-light emission due to thermal 1,2-dioxetane cleavage. The energy gain resulting from the chemical energy obtained in the transformation of an alkene into two carbonyl compounds transforms a red-light excitation laser beam into a blue-light chemiluminescence emission, producing thereby a formal anti-Stokes shift of 200-250 nm, opening up a whole spectrum of possible applications

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 (Phi(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 (Phi(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 (Phi(s) approximate to 50%), with the notable exception of the peroxyoxalate reaction (Phi(s) approximate to 60%).Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2006/03420-7, 2005/58320-4, 2012/02428-5, 2007/00684-6, 2011/23036-5]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [304887/2010-2]Coordenadoria de Aperfeicoamento de Pessoal de Ensino Superior (CAPES)Coordenadoria de Aperfeicoamento de Pessoal de Ensino Superior (CAPES

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%)