Trimethyl Lock Research Articles

Mass spectrometric analysis of acid-assisted photochemical release of the trimethyl lock system on the monolayers on gold.

We report the acid-assisted photolysis of the trimethyl lock system which has long been harnessed for a variety of applications such as drug delivery, cellular imaging, enzyme activity assays, and surface patterning. By mass spectrometric analysis, we found that photoinduced intramolecular cyclization and the ensuing release of the pendant groups of the trimethyl lock on the self-assembled monolayers proceeded cleanly in the presence of HCl, to give a high yield.

A Quencher‐Fluorophore‐Type Probe for Detection and Imaging of NADPH in Human Breast Cancer Cells

A new fluorogenic trimethyl lock quinone (TLQ) derivative, designated as the quencher‐TLQ‐fluorophore‐type probe (Q‐TLQ‐F), was developed for selective detection of nicotinamide adenine dinucleotide phosphate (NADPH). By taking advantage of the well‐known facile intramolecular ring cyclization reaction (δ‐lactonization) of TLQ that can release a reporter molecule upon reduction, Q‐TLQ‐F was successfully applied for the detection of physiological NADPH generated by glucose‐6‐phosphate dehydrogenase in human breast cancer MDA‐MB‐231 cells.

Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit.

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.

Trimethyl Lock: A Multifunctional Molecular Tool for Drug Delivery, Cellular Imaging, and Stimuli-Responsive Materials.

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.

An instantaneous near-infrared trimethyl lock based fluorescent probe for biothiols with a large Stokes shift

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.

A turn-on fluorescent probe for selective detection of glutathione using trimethyl lock strategy

A novel turn-on fluorescent probe based on trimethyl lock with acrylate recognition moiety and latent fluorophore of 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-NH2) for the detection of glutathione has been developed. The probe can selectively and sensitively detect glutathione in solution, and the limit of detection was calculated to be 26nM. Furthermore, the probe has been successfully used to detect GSH in living cells. The interesting preference of GSH was attributed to the steric repulsion of trimethyl which retarded Cys, Hcy to reach Bürgi-Dunitz angle and allowed more flexible side chain of GSH to attack carbonyl functionality.

Mechanistic Studies of the Photoinduced Quinone Trimethyl Lock Decaging Process.

Mechanistic studies of a general reaction that decages a wide range of substrates on exposure to visible light are described. The reaction involves a photochemically initiated reduction of a quinone mediated by an appended thioether. After reduction, a trimethyl lock system incorporated into the quinone leads to thermal decaging. The reaction could be viewed as an electron-transfer initiated reduction of the quinone or as a hydrogen abstraction-Norrish Type II-reaction. Product analysis, kinetic isotope effects, stereochemical labeling, radical clock, and transient absorption studies support the electron transfer mechanism. The differing reactivities of the singlet and triplet states are determined, and the ways in which this process deviates from typical quinone photochemistry are discussed. The mechanism suggests strategies for extending the reaction to longer wavelengths that would be of interest for applications in chemical biology and in a therapeutic setting.

A General Strategy for Visible-Light Decaging Based on the Quinone Trimethyl Lock

Visible-light triggered quinone trimethyl locks are reported as a general design for long-wavelength photoremovable protecting groups for alcohols and amines. Intramolecular photoreduction unmasks a reactive phenol that undergoes fast lactonization to release alcohols and amines. Model substrates are released in quantitative yield along with well-defined, colorless hydroquinone byproducts. Substituent modifications of the quinone core allow absorption from 400 to 600 nm.

Visible-light-mediated cleavage of polymer chains under physiological conditions via quinone photoreduction and trimethyl lock.

We introduce a click and visible-light triggered unclick approach via thio-bromo reaction and hydroquinone photoreduction/trimethyl lock cleavage for polymer modifications. Both reactions can be carried out in water and at ambient temperature, enabling preparation of bioorthogonal hydrogels for encapsulation and controlled release of various cells.

Inside Cover: Esterase‐Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide (Angew. Chem. Int. Ed. 14/2016)

A series of thioacids is described as hydrogen sulfide prodrugs by B. Wang et al. in their Communication on page 4514 ff. The release takes advantage of a “trimethyl lock”-facilitated lactonization controlled by an esterase trigger that unmasks a hydroxy group as the attacking nucleophile. These hydrogen sulfide prodrugs are stable under physiological conditions and release H2S with tunable release rates by modifying the ester group.

Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 μM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 μM and 0.2 to 1.3 μM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.

Boronic Acid for the Traceless Delivery of Proteins into Cells.

The use of exogenous proteins as intracellular probes and therapeutic agents is in its infancy. A major hurdle has been the delivery of native proteins to an intracellular site of action. Herein, we report on a compact delivery vehicle that employs the intrinsic affinity of boronic acids for the carbohydrates that coat the surface of mammalian cells. In the vehicle, benzoxaborole is linked to protein amino groups via a "trimethyl lock." Immolation of this linker is triggered by cellular esterases, releasing native protein. Efficacy is demonstrated by enhanced delivery of green fluorescent protein and a cytotoxic ribonuclease into mammalian cells. This versatile strategy provides new opportunities in chemical biology and pharmacology.

Syntheses and Antibacterial Activity of N-Acylated Ciprofloxacin Derivatives Based on the Trimethyl Lock.

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.

Bioreversible Protein Labeling For Cellular Delivery

A growing number of potential chemotherapeutic agents are proteins. A major challenge to the use of proteins as intracellular drugs is their traversal of the negatively charged glycocalyx and cell membrane. Many methods have been developed over the past several decades to try to overcome this obstacle, including liposome carriers, various polymers, cell‐penetrating peptides, and changes to the protein itself. All of these methods have downsides, including cytotoxicity, low delivery yields, and irreversible changes to the proteins. Here, we present two complementary methods of bioreversible protein modification that endow cell permeability, and we demonstrate their efficacy using the green fluorescent protein (GFP). The first method labels lysine residues with a module that combines the trimethyl lock scaffold to enable bioreversibility in the presence of cellular esterases, and a boronic acid moiety to promote cellular entry by increasing contact with the glycocalyx. This modification enables cellular uptake of GFP through endocytosis. The second method uses a fluorenyl diazo compound to label carboxylic acids of the target protein, forming ester bonds that are labile to cellular esterases. The pendant fluorenyl groups endow GFP with high levels of cytosolic delivery. Both strategies enhance delivery of a native protein into a cell, which is critical for clinical utility.

Siderophore-fluoroquinolone conjugates containing potential reduction-triggered linkers for drug release: synthesis and antibacterial activity.

Syntheses of two Siderophore-fluoroquinolone conjugates with a potential reduction triggered linker for drug release are described. The "trimethyl lock" based linker incorporated in the conjugates was designed to be activated by taking advantage of the reductive pathway of bacterial iron metabolism. Electrochemical and LC-MS studies indicated that the linker is thermodynamically reducible by common biological reductants and the expected lactonization proceeds rapidly with concomitant release of the drug. Antibacterial activity assays revealed that conjugates with the reduction-triggered linker were more potent than their counterparts with a stable linker, which suggests that drug release occurs inside bacterial cells.

Nanoporous membranes with electrochemically switchable, chemically stabilized ionic selectivity.

Nanopore size, shape, and surface charge all play important roles in regulating ionic transport through nanoporous membranes. The ability to control these parameters in situ provides a means to create ion transport systems tunable in real time. Here, we present a new strategy to address this challenge, utilizing three unique electrochemically switchable chemistries to manipulate the terminal functional group and control the resulting surface charge throughout ensembles of gold plated nanopores in ion-tracked polycarbonate membranes 3 cm(2) in area. We demonstrate the diazonium mediated surface functionalization with (1) nitrophenyl chemistry, (2) quinone chemistry, and (3) previously unreported trimethyl lock chemistry. Unlike other works, these chemistries are chemically stabilized, eliminating the need for a continuously applied gate voltage to maintain a given state and retain ionic selectivity. The effect of surface functionalization and nanopore geometry on selective ion transport through these functionalized membranes is characterized in aqueous solutions of sodium chloride at pH = 5.7. The nitrophenyl surface allows for ionic selectivity to be irreversibly switched in situ from cation-selective to anion-selective upon reduction to an aminophenyl surface. The quinone-terminated surface enables reversible changes between no ionic selectivity and a slight cationic selectivity. Alternatively, the trimethyl lock allows ionic selectivity to be reversibly switched by up to a factor of 8, approaching ideal selectivity, as a carboxylic acid group is electrochemically revealed or hidden. By varying the pore shape from cylindrical to conical, it is demonstrated that a controllable directionality can be imparted to the ionic selectivity. Combining control of nanopore geometry with stable, switchable chemistries facilitates superior control of molecular transport across the membrane, enabling tunable ion transport systems.

Self‐Immolative Bioluminogenic Quinone Luciferins for NAD(P)H Assays and Reducing Capacity‐Based Cell Viability Assays

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

Fluorogenic Probe for Constitutive Cellular Endocytosis

Endocytosis is a fundamental process of eukaryotic cells that is critical for nutrient uptake, signal transduction, and growth. We have developed a molecular probe to quantify endocytosis. The probe is a lipid conjugated to a fluorophore that is masked with an enzyme-activatable moiety known as the trimethyl lock. The probe is not fluorescent when incorporated into the plasma membrane of human cells but becomes fluorescent upon internalization into endosomes, where cellular esterases activate the trimethyl lock. Using this probe, we found that human breast cancer cells undergo constitutive endocytosis more rapidly than do matched noncancerous cells. These data reveal a possible phenotypic distinction of cancer cells that could be the basis for chemotherapeutic intervention.

ChemInform Abstract: Trimethyl Lock: A Trigger for Molecular Release in Chemistry, Biology, and Pharmacology

AbstractReview: 68 refs.

Trimethyl lock: a trigger for molecular release in chemistry, biology, and pharmacology

The trimethyl lock is an o-hydroxydihydrocinnamic acid derivative in which unfavorable steric interactions between three pendant methyl groups encourage lactonization to form a hydrocoumarin. This reaction is extremely rapid, even when the electrophile is an amide and the leaving group is an amino group of a small-molecule drug, fluorophore, peptide, or nucleic acid. O-Acylation of the phenolic hydroxyl group prevents reaction, providing a trigger for the reaction. Thus, the release of an amino group from an amide can be coupled to the hydrolysis of a designated ester (or to another chemical reaction that regenerates the hydroxyl group). Trimethyl lock conjugates are easy to synthesize, making the trimethyl lock a highly versatile module for chemical biology and related fields.