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.

Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom.

Antimicrobial peptides (AMPs) derived from natural toxins and venoms offer a promising alternative source of antibiotics. Here, through structure-function-guided design, we convert two natural AMPs derived from the venom of the solitary eumenine wasp Eumenes micado into α-helical AMPs with reduced toxicity that kill Gram-negative bacteria in vitro and in a preclinical mouse model. To identify the sequence determinants conferring antimicrobial activity, an alanine scan screen and strategic single lysine substitutions are made to the amino acid sequence of these natural peptides. These efforts yield a total of 34 synthetic derivatives, including alanine substituted and lysine-substituted sequences with stabilized α-helical structures and increased net positive charge. The resulting lead synthetic peptides kill the Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa (PAO1 and PA14) by rapidly permeabilizing both their outer and cytoplasmic membranes, exhibit anti-infective efficacy in a mouse model by reducing bacterial loads by up to three orders of magnitude, and do not readily select for bacterial resistance.

Autonomous Treatment of Bacterial Infections in Vivo Using Antimicrobial Micro- and Nanomotors.

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

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.