Enhancement of fluorescent properties of near-infrared dyes using clickable oligoglycerol dendrons

Near-infrared (NIR) fluorescent dyes are gaining increased attention due to their potential to serve as molecular probes for in vivo imaging. Here, we demonstrate that oligoglycerol dendrons effectively enhance the fluorescence properties of an NIR dye by increasing the solubility in water and the prevention of aggregate formation. First- and second-generation oligoglycerol dendrons were conjugated to an NIR dye via a dipolar-cycloaddition (click) reaction. The two new dye conjugates exhibited enhanced NIR fluorescent emission and considerably higher fluorescent quantum yields than the dye alone. The high photostability measured for one of the oligoglycerol-linked dyes, in comparison to commonly used fluorogenic dyes such as Cy5 and Cy7, was validated using fluorescence microscopy of macrophages

A Tale of Switched Functions: From Cyclooxygenase Inhibition to M-Channel Modulation in New Diphenylamine Derivatives

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers

Self-Immolative Chemiluminescence Polymers: Innate Assimilation of Chemiexcitation in a Domino-like Depolymerization

Self-immolative polymers are distinctive materials able to disassemble in a domino-like mechanism from head-to-tail upon a triggering event induced by an external stimulus. We have developed an effective molecular method to intrinsically assimilate a chemiluminescence turn-ON mechanism with a domino-like fragmentation mechanism. A unique molecular unit was synthesized, which could combine the abilities of executing the duel function of quinone-methide elimination and chemiexcitation. Incorporation of this unit as a monomer, results with the first class of stimuli-responsive self-immolative polymers with amplified chemiluminescence output. Responsive groups for various analytes were introduced as a head-trigger during the polymer synthesis. The polymers were demonstrated as chemiluminescence probes for detection of different chemical analytes. The obtained polymers were able to amplify the intensity and the duration of the light emission signal by factors correlated to their length. We anticipate that the chemiluminescence self-immolative polymers described here will find use for various research topics such as signal amplification, light-emitting new materials, and molecular probes with long-lasting light emission and imaging capabilities

Self‐Propagating Amplification Reactions for Molecular Detection and Signal Amplification: Advantages, Pitfalls, and Challenges

Self‐propagating cascade reactions are a recent development for chemosensing protocols. These cascade reactions, in principle, offer low limits of detection by virtue of exponential signal amplification and are initiated by a specific, preplanned molecular detection event. This combination of selectivity for a detection event followed by in situ signal amplification is achieved by exploitation of mechanistic organic chemistry and thus has resulted in various chemosensing protocols that use one or more reagents to achieve the desired selectivity and sensitivity for an assay. Species such as hydrogen peroxide, thiols, and fluoride have been used as active reagents to initiate the first examples of self‐propagating signal amplification reactions, although many other active reagents should be compatible with the approaches. A common feature of the reagents that support the self‐propagating signal amplification reactions is the involvement of quinone methide intermediates resulting from elimination of optical reporters and/or active reagents, where the latter propagates the signal amplification reaction. The early examples of these amplification sequences, however, are slow to reach full signal, thus leaving time for background reactions to generate nonspecific signals. This issue of background has limited practical applications of these self‐propagating signal amplification reactions, as has challenging synthetic routes to the reagents, as well as the potential for other chemical species to interfere with the detection and signal amplification processes. Thus, the goal of this review is to summarize the progress of self‐propagating signal amplification technology, to identify the pitfalls of current designs, and by doing so, to stimulate future studies in this growing and promising research area

Emissive enhancement of the singlet oxygen chemiluminescence probe after binding to bovine serum albumin

16 páginas, 7 figuras y 1 tabla.A chemiluminescence probe for singlet oxygen 1O2 (SOCL) was investigated in phosphate buffer saline (PBS), either in the absence of proteins or containing bovine serum albumin (BSA). In the protein-free PBS, the reactivity of SOCL for methylene blue (MB)-photosensitized 1O2 was found to be moderate or low. The reaction yield increased with temperature and/or concentration of dissolved molecular oxygen. Unexpectedly, the presence of BSA boosted both the emissive nature and the thermal stability of the phenoxy-dioxetane intermediate formed in the chemiexcitation pathway. Isothermal titration calorimetry showed that SOCL has a moderate binding affinity for BSA and that entropy forces drive the formation of the SOCL-BSA complex. A model with two identical and independent binding sites was used to fit the binding isotherm data. Co-operative binding was observed when MB was present. Local viscosity factors and/or conformational restrictions of the BSA-bound SOCL phenoxy-dioxetane were proposed to contribute to the formation of the highly emissive benzoate ester during the chemically initiated electron exchange luminescence (CIEEL) process. These results led us to conclude that hydrophobic interactions of the SOCL with proteins can modify the emissive nature of its phenoxy-dioxetane, which should be taken into account when using SOCL or its cell-penetrating peptide derivative in living cells.This research was funded by MICINN and, grant numbers BFU2007-68107-C02-02/BMC, AGL2016-79589-R, and RTC-2017-6756-2 and Junta de Castilla y León, grant number CSI002A10-2, ERDFPeer reviewe

Multiple event activation of a generic prodrug trigger by antibody catalysis

Chemotherapeutic regimes are typically limited by nonspecific toxicity. To address this problem we have developed a broadly applicable drug-masking chemistry that operates in conjunction with a unique broad-scope catalytic antibody. This masking chemistry is applicable to a wide range of drugs because it is compatible with virtually any heteroatom. We demonstrate that generic drug-masking groups may be selectively removed by sequential retro-aldol–retro-Michael reactions catalyzed by antibody 38C2. This reaction cascade is not catalyzed by any known natural enzyme. Application of this masking chemistry to the anticancer drugs doxorubicin and camptothecin produced prodrugs with substantially reduced toxicity. These prodrugs are selectively unmasked by the catalytic antibody when it is applied at therapeutically relevant concentrations. We have demonstrated the efficacy of this approach by using human colon and prostate cancer cell lines. The antibody demonstrated a long in vivo half-life after administration to mice. Based on these findings, we believe that the system described here has the potential to become a key tool in selective chemotherapeutic strategies

Singlet oxygen detection by chemiluminescence probes in living cells

21 páginas, 4 figurasSinglet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.The research was funded by MCIN/AEI/10.13039/501100011033 (Project n PID2019-107154RB-100) and the regional government of Castilla y León (Project n CSI260P20). The Project ‘CLU-2019–05—IRNASA/CSIC Unit of Excellence’ funded by the Junta de Castilla y León and co-financed by the European Union (ERDF ‘Europe Drives Our Growth’) and the CSIC Interdisciplinary Thematic Platform (PTI) Optimization of Agricultural and Forestry Systems (PTI-AGROFOR) are also acknowledgedPeer reviewe