A Green BODIPY‐Based,Super‐Fluorogenic,Protein‐Specific Labelling Agent

We report the development of YC23, a novel green BODIPY‐based dimaleimide derivative that undergoes a fluorogenic addition reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

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.     

Intramolecular Long‐Distance Nucleophilic Reactions as a Rapid Fluorogenic Switch Applicable to the Detection of Enzymatic Activity

Long‐distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence‐quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine‐amino‐acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half‐life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine‐targeting enzymes.     

ROS‐Responsive N‐Alkylaminoferrocenes for Cancer‐Cell‐Specific Targeting of Mitochondria

Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro‐DLCs is proposed based on an N‐alkylaminoferrocene structure. These prodrugs are activated by the reaction with reactive oxygen species (ROS) forming ferrocenium‐based DLCs. Since ROS are overproduced in cancer, the high‐efficiency cancer‐cell‐specific targeting of mitochondria could be achieved as demonstrated by fluorescence microscopy in combination with two fluorogenic pro‐DLCs in vitro and in vivo. We prepared a conjugate of another pro‐DLC with a clinically approved drug carboplatin and confirmed that its accumulation in mitochondria was higher than that of the free drug. This was reflected in the substantially higher anticancer effect of the conjugate.     

A Minimal,Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

The unstrained S‐allyl cysteine amino acid was site‐specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5‐tetrazines by means of an inverse‐electron‐demand Diels–Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre‐targeting approach. The small size, easy chemical installation, and selective reactivity of the S‐allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.     

A Minimal,Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

The unstrained S‐allyl cysteine amino acid was site‐specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5‐tetrazines by means of an inverse‐electron‐demand Diels–Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre‐targeting approach. The small size, easy chemical installation, and selective reactivity of the S‐allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

New 3‐(Heteroaryl)‐2‐iminocoumarin‐based Borate Complexes: Synthesis,Photophysical Properties,and Rational Functionalization for Biosensing/Biolabeling Applications

Members of a series of boron difluoride complexes with 3‐(heteroaryl)‐2‐iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N‐aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two‐step method. These novel fluorogenic dyes belong to the family of “Boricos” (D. Frath et al., Chem. Commun.­ 2013 , 49, 4908–4910) and are the first examples of phenol‐based fluorophores of which the photophysical properties in the green‐yellow spectral range are dramatically improved by N,N‐chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4‐dinitrophenyl (DNP) ethers, which led to a dramatic “OFF‐ON” fluorescence response upon reaction with thiols. Additionally, to expand the scope of these “7‐hydroxy‐Borico” derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and “Borico”‐DOTA‐like scaffolds in an effort to design novel monomolecular multimodal fluorescence‐ radioisotope imaging agents.     

A fluorogenic ‘click’ reaction of azidoanthracene derivatives

Fluorogenic reactions have broad applications in biolabeling, combinatorial synthesis of fluorescent dyes, and materials development. It was recently reported that the highly selective and efficient Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction can be employed in designing new types of fluorogenic reactions. In this study, we report a fluorogenic reaction using anthracene azides as starting materials. The fluorescence of the anthryl core can be greatly inhibited upon introducing electron-donating azido groups in the proximity. Such weakly fluorescent anthracene azides demonstrate high reactivity with a variety of alkynes under the CuAAC conditions producing a strongly fluorescent triazole product with high quantum yields. This reaction can be used in the synthesis and screening of fluorescent dyes combinatorially. Compared with most existing methods, the fluorogenic CuAAC reaction is a much milder and simpler technique to prepare large libraries of fluorescent dyes without further purification. In order to demonstrate the efficiency of using anthracene azides for biolabeling applications, both small molecules and biomolecules including the multialkyne-derivatized cowpea mosaic virus and tobacco mosaic virus had been studied.     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

Development of protein, peptide, and small molecule catalysts using catalysis-based selection strategies

We have developed peptide catalysts and antibody catalysts that catalyze aldol, retro-aldol, and Michael reactions via an enamine mechanism using reaction-based selections with 1,3-diketone derivatives. Nucleophilic amino groups of the catalysts were covalently trapped during the selections. We have also developed fluorogenic substrates that are useful for real-time monitoring of the progress of bond-forming reactions, such as aldol reactions, by an increase in fluorescence. These fluorogenic substrates have been used to monitor peptide-catalyzed, antibody-catalyzed, enzyme-catalyzed, and small molecule-catalyzed reactions. Catalysis-based screening using fluorogenic substrates will accelerate rapid identification of superior catalysts and reaction conditions.     

Single‐Molecule Nanocatalysis Shows In Situ Deactivation of Pt/C Electrocatalysts during the Hydrogen‐Oxidation Reaction

By coupling a Pt‐catalyzed fluorogenic reaction with the Pt‐electrocatalyzed hydrogen‐oxidation reaction (HOR), we combine single‐molecule fluorescence microscopy with traditional electrochemical methods to study the real‐time deactivation kinetics of a Pt/C electrocatalyst at single‐particle level during electrocatalytic hydrogen‐oxidation reaction. The decay of the catalytic performance of Pt/C could be mainly attributed to the electrocatalysis‐induced etching or dissolution of Pt nanoparticles. Spontaneous regeneration of activity and incubation period of the Pt electrocatalyst were also observed at single‐particle level. All these new insights are practically useful for the understanding and rational design of highly efficient electrocatalysts for application in fuel cells.     

Fluorescence‐Labeled Bis‐benzamidines as Fluorogenic DNA Minor‐Groove Binders: Photophysics and Binding Dynamics

In recent decades there has been great interest in the design of highly sensitive sequence‐specific DNA binders. The eligibility of the binder depends on the magnitude of the fluorescence increase upon binding, related to its photophysics, and on its affinity and specificity, which is, in turn, determined by the dynamics of the binding process. Therefore, progress in the design of DNA binders requires both thorough photophysical studies and precise determination of the association and dissociation rate constants involved. We have studied two bis‐benzamidine (BBA) derivatives labeled by linkers of various lengths with the dye Oregon Green (OG). These fluorogenic binders show a dramatic fluorescence enhancement upon binding to the minor groove of double‐stranded (ds) DNA, as well as significant improvement in their sequence specificity versus the parent BBA, although with decreased affinity constants. Detailed photophysical analysis shows that static and dynamic quenching of the OG fluorescence by BBA through photoinduced electron transfer is suppressed upon insertion of BBA into the minor groove of DNA. Fluorescence correlation spectroscopy yields precise dynamic rate constants that prove that the association process of these fluorogenic binders to dsDNA is very similar to that of BBA alone and that their lower affinity is mainly a consequence of their weaker attachment to the minor groove and the resultant faster dissociation process. The conclusions of this study will allow us to go one step further in the design of new DNA binders with tunable fluorescence and binding properties.     

Synthesis of 7‐Azido‐3‐Formylcoumarin – A Key Precursor in Bioorthogonally Applicable Fluorogenic Dye Synthesis

Coumarins represent an important group of natural products and a common part of various drugs and fluorescent dyestuffs. Herein, we present the synthesis of a coumarin that can serve as a key starting material in the design and synthesis of bioorthogonally applicable fluorogenic dyes. The synthesis of 7‐azido‐3‐formylcoumarin started from 7‐diallylaminocoumarin. This allyl protected aminocoumarin is otherwise hard to obtain by conventional methods but was conveniently accessed in good yields by a sequential, Wittig‐reaction–UV isomerization process. This sequential approach was studied in more details and applied for the synthesis of a series of substituted coumarins even in one‐pot.     

New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes

The synthesis of a set of tetrazine‐bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through‐bond energy‐transfer‐based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse‐electron‐demand Diels–Alder reaction with proteins modified genetically with strained trans‐cyclooctenes.     

聚酰亚胺对高性能树脂共混体系界面的反应增强

研究了以5-降冰片烯-2,3-二羧酸单甲酯为端基的PMR型聚酰亚胺(POI)作为界面介质对部分相容的聚醚砜/聚苯硫醚(PES/PPS)、聚醚醚酮/聚醚砜(PEEK/PES)共混体系的界面性质、形态结构及结晶行为的影响.结果表明,POI可以有效地增强两相间的界面粘结,显著降低PPS/PES共混物中PPS分散区的尺寸,改善两组分间的相容性.在熔融共混过程中,POI从本体向界面扩散并同PPS,PES产生交联和/或接枝,POI同PPS的反应活性远高于PES,但POI与PES发生反应.POI是PPS结晶的有效成核剂.     

Fluorogenic Behaviour of the Hetero‐Diels–Alder Ligation of 5‐Alkoxyoxazoles with Maleimides and their Applications

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero‐Diels–Alder reaction of 5‐alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions.     

Riboflavin‐Based Fluorogenic Sensor for Chemo‐ and Enantioselective Detection of Amine Vapors

A novel turn‐on fluorogenic chiral sensory system has been developed using a protonated riboflavin and riboflavin‐derived cationic polymer as a fluorophore precursor and a specific amine receptor, respectively, which enables the solid‐state chemo‐ and enantioselective fluorogenic visual detection of primary and secondary amine vapors.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.