Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity

Histone deacetylases (HDACs) are key enzymatic regulators of many cellular processes such as gene expression, cell cycle, and tumorigenesis. These enzymes are attractive targets for drug development. However, very few simple methods for monitoring HDAC activity have been reported. Here, we have developed a fluorogenic probe, K4(Ac)-CCB, which consists of the histone H3 peptide containing acetyl-Lys and a coumarin fluorophore with a carbonate ester. By the simple addition of the probe to a HDAC solution, enzyme activity was clearly detected through spontaneous intramolecular transesterification, which renders the probe fluorescent. In addition, K4(Ac)-CCB can be applied to the evaluation of HDAC inhibitor activity. This is the first report to demonstrate the monitoring of HDAC activity by using a one-step procedure. Thus, our novel fluorogenic probe will provide a powerful tool for epigenetic research and the discovery of HDAC-targeted drugs.     

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.     

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λ _ex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.     

Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans ‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.     

Lewis acid mediated reactions of 1-cyclopropyl-2-arylethanones with allenic esters: a facile synthetic protocol for the preparation of dihydrofuro[2,3-h]chromen-2-one derivatives

TMSOTf-mediated reactions of 1-cyclopropyl-2-arylethanones with allenic esters afford a novel method for the synthesis of dihydrofuro[2,3-h]chromen-2-one derivatives in moderate to good yields. This process is a sequential reaction involving a nucleophilic ring-opening reaction of the cyclopropane by H(2)O, two intermolecular aldol-type reactions and one intramolecular aldol-type reaction, a cyclic transesterification, dehydration, and aromatization mediated by Lewis acid.     

Near-infrared fluorescence activation probes based on disassembly-induced emission cyanine dye

Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.     

Lewis acid-mediated reactions of 1-cyclopropyl-2-arylethanone derivatives with allenic ester, ethyl acetoacetate, and methyl acrylate

TMSOTf-mediated reactions of 2-aryl-1-(1-phenylcyclopropyl)ethanones 1 with allenic esters afford a novel method for the synthesis of 6-methyl-3a,7-diaryl-3,3a-dihydro-2H-benzofuran-4-one derivatives 2 in moderate yields. In addition, we also found that TMSOTf-mediated reactions of 1-cyclopropyl-2-arylethanones with ethyl acetoacetate can provide the corresponding 2,3-dihydrobenzofuran-4-ol and dihydrofuro[2,3-h]chromen-2-one in moderate yields via a sequential reaction involving a nucleophilic ring-opening reaction of the cyclopropane by H2O, one intermolecular aldol type reaction and two intramolecular aldol type reactions, a cyclic transesterification, dehydration, and aromatization. Moreover, by using methyl acrylate to replace allenic ester, the corresponding 7-aryl-3,5,6,7-tetrahydro-2H-benzofuran-4-one and 5-aryl-3,5,6,7-tetrahydro-2 H-benzofuran-4-one can be formed in moderate to high yields in the presence of Bi(OTf)2Cl. Plausible reaction mechanisms have also been provided on the basis of control experiments.     

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid‐forming proteins partially unfold and assemble into insoluble β‐sheet rich fibrils. Molecular‐level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. In all peptides, three valine residues, known to prefer the β‐sheet conformation, have been incorporated at the solvent‐exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH‐controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α‐helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid‐like fibrils. Peptide B features a glutamate‐based switch functionality and exhibits opposite pH‐dependent folding behavior. In this case, α‐helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.     

Pyrylium‐based dye and charge tagging in proteomics

The pyrylium group is a selective reagent for ε‐amino groups in proteins. In particular, for fluorescence labeling, a number of advantages over traditional N‐hydroxysuccinimidyl ester chemistry were recognized such as the rapid prestaining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py‐1 using liquid chromatography coupled to MS with the aim of determining its specificity and possible side products. Peptides containing no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py‐1 at yields > 30%. Gas phase fragmentation proved both labeling of lysine residues as well as that of the N‐terminus also in peptides that contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label, Py‐1 recommends itself for N‐terminal charge tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a‐ and b‐type ion series were observed for N‐terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red.     

Expanding the catalytic activity of nucleophilic N-heterocyclic carbenes for transesterification reactions

[reaction: see text] Currently, there is a renewed interest in reactions that are catalyzed by organic compounds. Typical organic catalysts for acylation or transesterification reactions are based on either nucleophilic tertiary amines or phosphines. This communication discusses the use of nucleophilic N-heterocyclic carbenes as efficient transesterification catalysts. These relatively unexplored and highly versatile organic catalysts were found to be mild, selective, and more active than traditional organic nucleophiles.     

Guanidine Additive Enabled Intermolecular ortho-Phthalaldehyde-Amine-Thiol Three-Component Reactions for Modular Constructions

Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures. Thus, 12 model peptide-peptide conjugates have been synthesized from unprotected peptides featuring all proteinogenic residues. Besides, 6 peptide-drug conjugates have been prepared in one step, with excellent conversions and isolated yields. In addition, a conjugate product has been further functionalized by utilizing a premodified OPA derivative, demonstrating the versatility and flexibility of this reaction.     

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

We have established a coupled assay system targeting protein l ‐isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1′ position of the caspase‐3 recognition sequence. Following PIMT‐catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase‐3 to give fluorescence activation. High‐throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.     

Insight into the role of the counteranion of an imidazolium salt in organocatalysis: a combined experimental and computational study

N‐Heterocyclic carbenes (NHCs) can serve as very reactive nucleophilic catalysts and exhibit strong basicity. Herein, we initiate a combined experimental and computational investigation of the NHC‐catalyzed ring‐closing reactions of 4‐(2‐formylphenoxy)but‐2‐enoate derivatives 1 to uncover the relationship between the counteranion of an azolium salt, the nucleophilicity and basicity of the carbene species, and the catalytic performance of the carbene species by taking imidazolium salts IPr ? HX (X=counteranion, IPr=1,3‐bis(2,6‐diisopropylphenyl)imidazol‐2‐ylidene) as the representative precatalysts. The plausible mechanisms of IPr‐mediated ring‐closing reactions have been investigated by using DFT calculations. The hydrogen‐accepting ability, assigned as the basicity of the counteranion of IPr ? HX and evaluated by DFT calculations, is correlated with the rate of deprotonation of C2 in IPr ? HX, which could be monitored by the capture of the free carbene formed in situ with elemental sulfur. The deprotonation of C2 in IPr ? HX with a more basic anion gives rise to a higher concentration of the free carbene and vice versa. At a relatively low concentration, IPr prefers to show a nucleophilic character to induce the intramolecular Stetter reaction. At a relatively high concentration, IPr primarily acts as a base to afford benzofuran derivatives. These data comprehensively disclose, for the first time, that the counteranions of azolium salts significantly influence not only the catalytic activity, but also possibly the reaction mechanism.     

Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags

Fluorescent tagging of Cys-containing peptides is presented herein. The procedure follows a two-step sequential reaction scheme using thiol specific bifunctional chemical reporters and fluorogenic labels. Vinyl-sulfone bearing chemical reporters have been synthesized and demonstrated to selectively modify cysteine under physiological conditions in the presence of other nucleophilic amino acids. Bifunctional chemical reporters decorated with a terminal alkyne moiety, suitable for modification with azide containing fluorogenic labels were also synthesized. Such fluorogenic (turn on) labels in combination with these new vinyl-sulfone tags can be used generally in fluorescent modulation schemes of thiol-bearing biomolecules.     

Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity

Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm .     

Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate‐controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition‐state structure and bonding optimally requires the use of theoretical models. In this work, we apply density‐functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′‐O‐transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well‐studied series of phosphate and phosphorothioate mono‐, di‐ and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non‐enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.     

Dramatic increase of quench efficiency in "spacerless" dimaleimide fluorogens

In this post-genomic era, new techniques are needed to cope with the task of assigning functional roles to the huge number of identified putative gene products. We have developed a minimalist labelling strategy based on the use of synthetic fluorogenic probe reagents that fluoresce only after their reaction with a target peptide sequence. The probe reagents have fluorescent cores and bear two maleimide groups, such that their latent fluorescence is quenched by a photoinduced electron transfer (PET) to the pendant maleimide groups, until both of these groups undergo a specific thiol addition reaction. The efficiency of the fluorescence quenching is critical to the practicality of this labelling method, and has been predicted to be related to the intramolecular distance between the fluorophore and the maleimide groups. We have conducted the first direct test of this hypothesis by preparing a series of novel fluorogens that differ only by the spacer moiety separating their coumarin fluorophore and their dimaleimide fragment. A striking correlation was observed between intramolecular distance and the fluorescence enhancement (FE) observed after reaction with two equivalents of thiol. Guided by this observation, we then designed 'spacerless' fluorogens, of which a dansyl derivative shows an FE ratio of >300, the largest recorded for dimaleimide fluorogens. The trends observed herein provide valuable lessons for subsequent fluorogen design, and the novel fluorogens developed in the course of this study are currently being applied to protein labelling applications.     

Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications

The bioorthogonality of tetrazole photoclick chemistry has been reassessed. Upon photolysis of a tetrazole, the highly reactive nitrile imine formed undergoes rapid nucleophilic reaction with a variety of nucleophiles present in a biological system, along with the expected cycloaddition with alkenes. The alternative use of the tetrazole photoclick reaction was thus explored: tetrazoles were incorporated into Bodipy and Acedan dyes, providing novel photo‐crosslinkers with one‐ and two‐photon fluorescence Turn‐ON properties that may be developed into protein‐detecting biosensors. Further introduction of these photo‐activatable, fluorogenic moieties into staurosporine resulted in the corresponding probes capable of photoinduced, no‐wash imaging of endogenous kinase activities in live mammalian cells.     

Design and use of fluorogenic aldehydes for monitoring the progress of aldehyde transformations

We describe the first examples of fluorogenic aldehydes useful for monitoring many types of reactions including aldol reactions, allylations, and reductions. The fluorogenic aldehydes were constructed by covalent combination of a fluorophore and an aldehyde moiety via a linker. In the resulting single molecule, the aldehyde functioned as a quencher of the fluorophore's fluorescence. The reaction product, modified at the aldehyde functionality, no longer served as an effective quencher. The reaction products showed up to approximately 80-fold higher fluorescence than the aldehyde reactants.     

A robust, high-sensitivity stealth probe for peptidases

A robust, modular fluorogenic probe system has been developed which allows the highly sensitive off-ON detection of aminopeptidase activity by releasing an exceptionally photostable, insoluble, phenolic ESIPT fluorophore. The probes generate no false positive signal in over 24 hours, but when activated give a signal within 10 minutes.