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Conjugates composed of C2-18 fatty acid (FA) residues as a molecular carrier and 5-fluorocytosine (5-FC) as an active agent, released upon the action of intracellular esterases on the ester bond between FA and "trimethyl lock" intramolecular linker, demonstrate good in vitro activity against human pathogenic yeasts of Candida spp. The minimal inhibitory concentrations (MIC) values for the most active conjugates containing caprylic (C8), capric (C10), lauric (C12), or myristic (C14) acid residues were in the 2-64 µg mL-1 range, except for these against the least susceptible Candida krusei. The least active conjugates containing C2, C16, or C18 FA were slowly hydrolyzed by esterase and probably poorly taken up by Candida cells, as found for their analogs containing a fluorescent label, Nap-NH2 instead of 5-FC.

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Masked trimethyl lock (TML) systems as molecular moieties enabling the bioresponsive release of compounds or dyes in a controlled temporal and spatial manner have been widely applied for the development of drug conjugates, prodrugs or molecular imaging tools. Herein, we report the development of a novel amino trimethyl lock (H2 N-TML) system as an auto-immolative molecular entity for the release of fluorophores. We designed Cou-TML-N3 and MURh-TML-N3 , two azide-masked turn-on fluorophores. The latter was demonstrated to selectively release fluorescent MURh in the presence of physiological concentrations of the redox-signaling molecule H2 S in vitro and was successfully applied to image H2 S in human cells.

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Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is overexpressed in cancer cells and associated with the drug resistance factor of cancer. The objective of this work is the development of fluorescent probes for the efficient detection of hNQO1 activity in cancer cells, which can be employed for the cancer diagnosis and therapeutic agent development. Herein, we report naphthalimide-based fluorescent probes 1 and 2 that can detect hNQO1. For hNQO1 activity, the probes showed a significant fluorescence increase at 540 nm. In addition, probe 1, the naphthalimide containing a triphenylphosphonium salt, showed an enhanced enzyme efficiency and rapid detection under a physiological condition. The detection ability of probe 1 was superior to that of other previously reported probes. Moreover, probe 1 was less cytotoxic during the cancer cell imaging and readily provided a strong fluorescence in hNQO1-overexpressed cancer cells (A549). We proposed that probe 1 can be used to detect hNQO1 expression in live cells and it will be applied to develop the diagnosis and customized treatment of hNQO1-related disease.

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We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.

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Using trimethyl lock as a useful mean, a selective and sensitive fluorescent probe was developed for the detection of biothiols with an emission in near infrared spectral region (694 nm) and a large Stokes shift (129 nm). The inserted trimethyl lock between the fluorophore and the sensing group, 2, 4-dinitrobenzensulfonate, highly improved the response rate of this probe toward GSH because the unfavorable steric interactions between three methyl groups resulted in an extremely rapid intramolecular lactonization reaction to form a hydrocoumarin. This probe displayed an instantaneous response (within seconds) to GSH at a very low concentration in aqueous medium and the detection limit was as low as 2.5 nM based on S/N = 3. The fluorescence intensity of the solution of probe 1 at 694 nm displayed a good linearity (R = 0.9917) with the concentration of GSH in a range of 0.0-5.0 µM. Importantly, this probe exhibited a good performance in detecting biothiols in serum samples, living cells and organism.

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Trimethyl lock (TML) systems are based on ortho-hydroxydihydrocinnamic acid derivatives displaying increased lactonization reactivity owing to unfavorable steric interactions of three pendant methyl groups, and this leads to the formation of hydrocoumarins. Protection of the phenolic hydroxy function or masking of the reactivity as benzoquinone derivatives prevents lactonization and provides a trigger for controlled release of molecules attached to the carboxylic acid function through amides, esters, or thioesters. Their easy synthesis and possible chemical adaption to several different triggers make TML a highly versatile module for the development of drug-delivery systems, prodrug approaches, cell-imaging tools, molecular tools for supramolecular chemistry, as well as smart stimuliresponsive materials.

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A responsive magnetic resonance (MRI) contrast agent has been developed that can detect the enzyme activity of DT-diaphorase. The agent produced different chemical exchange saturation transfer (CEST) MRI signals before and after incubation with the enzyme, NADH, and GSH at different pH values whereas it showed good stability in a reducing environment without enzyme.

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Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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Several N-acyl ciprofloxacin quinone derivatives based on a trimethyl lock structure were synthesized, and their in vitro antibacterial activity against a panel of clinically relevant bacteria was evaluated. A few new analogues displayed enhanced activity against Gram-positive species compared to the parent drug. Additionally, studies of 8-Cip, which was the most potent compound tested, indicate that it may act through a dual-action mechanism.