Anthracene-BODIPY cassettes: syntheses and energy transfer

Compounds based on the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) framework are excellent fluorescent markers. When BODIPY dyes of this type are conjugated to functionalities that absorb at relatively short wavelengths, those functionalities can, in some molecules, transmit the absorbed energy to the BODIPY which then fluoresces. In such cases the BODIPY fragment acts as an acceptor while the other group serves as a donor. Energy transfer efficiencies in such donor-acceptor cassette systems must vary with the relative orientation of the two components, and with the structure of the linkers that attach them. This study was designed to probe these issues for the special case in which the linkers between the donor and acceptor fragments are conjugated. To do this, the cassettes 3-10 were prepared. Electrochemical studies were performed to provide insight into the degree of donor-acceptor conjugation in these systems. X-ray Crystallographic studies on single crystals of compounds 7 and 9 revealed the favored conformations of the donor and acceptor fragments in the solid state. Absorption, fluorescence, and time-resolved fluorescence spectra of the compounds were recorded, and quantum yields for the cassettes excited at the donor lambda(max) were measured. Fluorescence steady-state anisotropy data were determined for cassettes 3 and 9 to provide information about the mutual direction of the transition dipole moments.     

Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time τ(1)=4.3 ns for the cationic and τ(2)=3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01)=2.4×10(8) s(-1)) and neutral (k(02)=3.0×10(8) s(-1)) forms of II.     

Optical,Redox, and DNA‐Binding Properties of Phenanthridinium Chromophores: Elucidating the Role of the Phenyl Substituent for Fluorescence Enhancement of Ethidium in the Presence of DNA

The phenanthridinium chromophores 5‐ethyl‐6‐phenylphenanthridinium ( 1 ), 5‐ethyl‐6‐methylphenanthridinium ( 2 ), 3,8‐diamino‐5‐ethyl‐6‐methylphenanthridinium ( 3 ), and 3,8‐diamino‐5‐ethyl‐6‐(4‐N,N‐diethylaminophenyl)phenanthridinium ( 4 ) were characterized by their optical and redox properties. All dyes were applied in titration experiments with a random‐sequence 17mer DNA duplex and their binding affinities were determined. The results were compared to well‐known ethidium bromide ( E ). In general, this set of data allows the influence of substituents in positions 3, 6, and 8 on the optical properties of E to be elucidated. Especially, compound 4 was used to compare the weak electron‐donating character of the phenyl substituent at position 6 of E with the more electron‐donating 4‐N,N‐diethylaminophenyl group. Analysis of all of the measurements revealed two pairs of chromophores. The first pair, consisting of 1 and 2 , lacks the amino groups in positions 3 and 8, and, as a result, these dyes exhibit clearly altered optical and electrochemical properties compared with E . In the presence of DNA, a significant fluorescence quenching was observed. Their binding affinity to DNA is reduced by nearly one order of magnitude. The electronic effect of the phenyl group in position 6 on this type of dye is rather small. The properties of the second set, 3 and 4 , are similar to E due to the presence of the two strongly electron‐donating amino groups in positions 3 and 8. However, in contrast to 1 and 2 , the electron‐donating character of the substituent in position 6 of 3 and 4 is critical. The binding, as well as the fluorescence enhancement, is clearly related to the electron‐donating effect of this substituent. Accordingly, compound 4 shows the strongest binding affinity and the strongest fluorescence enhancement. Quantum chemical calculations reveal a general mechanism related to the twisted intramolecular charge transfer (TICT) model. Accordingly, an increase of the twist angle between the phenyl ring in position 6 and the phenanthridinium core opens a nonradiative channel in the excited state that depends on the electron‐donating character of the phenyl group. Access to this channel is hindered upon binding to DNA.     

Reaction‐Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples

Herein, two compounds ( 1 a and 1 b ) were rationally constructed as novel reaction‐based fluorescent probes for CN^? by making use of the electron‐withdrawing ability of the cyano group that was formed from the sensing reaction. Notably, this design strategy was first employed for the development of fluorescent CN^? probes. The experimental details showed that probe 1 a exhibited a fluorescence turn‐on response to CN^?, whereas other anions, biological thiols, and hydrogen sulfide gave almost no interference. The detection limit of probe 1 a for CN^? was found to be 0.12 μM . The sensing reaction product of 1 a with CN^? was characterized by NMR spectroscopy and mass spectrometry. TD‐DFT calculations demonstrated that the formed cyano group drives the intramolecular charge transfer (ICT) process from coumarin dye to the cyano group and thus the original strong ICT from the coumarin dye to the 3‐position pyridyl vinyl ketone substituent is weakened, which results in recovery of coumarin fluorescence. The practical utility of 1 a was also examined. By fabricating paper strips, probe 1 a can be used as a simple tool to detect CN^? in field measurements. Moreover, probe 1 a has been successfully applied for quantitative detection of endogenous CN^? from cassava root.     

Rigid, conjugated, fluoresceinated thymidine triphosphates: syntheses and polymerase mediated incorporation into DNA analogues

Syntheses of a unique set of energy transfer dye labeled nucleoside triphosphates, compounds 1-3, are described. Attempts to prepare these compounds were only successful if the triphosphorylation reaction was performed before coupling the dye to the nucleobase, and not the other way around. Compounds were prepared as both the 2'-deoxy (a) and 2',3'-dideoxy- (b) forms. They feature progressively longer rigid conjugated linkers connecting the nucleobase and the hydroxyxanthone moiety. UV spectra of the parent nucleosides 12-14 show that as the length of the linker increases so does the absorption of the donor in the 320-330 nm region, but with relatively little red-shift of the maxima. Fluorescence spectra of the same compounds show that radiation in the 320-330 nm region results in predominant emission from the fluorescein. When the linker is irradiated at 320 nm, the only significant emission observed corresponds to the hydroxyxanthone part of the molecules at 520 nm; this corresponds to an effective Stokes' shift of 200 nm. As the absorption at 320-330 nm by the linker increases with length, so does the intensity of the fluorescein emission. A gel assay was used to gauge relative incorporation efficiencies of compounds 1-3, dTTP, ddTTP, and 6-TAMRA-ddTTP. Throughout, the thermostable polymerase TaqFS was used, as it is the one most widely applied in high throughput DNA sequencing. This assay showed that only compounds 3 were incorporated efficiently; these have the longest linkers. Of these, the 2'-deoxy nucleoside 3 a was incorporated and did not prevent the polymerase from extending the chain further. The 2',3'-dideoxy nucleoside 3 b was incorporated only about 430 times less efficiently than ddTTP under the same conditions, and caused chain termination. The implications of these studies on modified sequencing protocols are discussed.     

The chemistry of fluorescent bodipy dyes: versatility unsurpassed

The world of organic luminophores has been confined for a long time to fairly standard biological labeling applications and to certain analytical tests. Recently, however, the field has undergone a major change of direction, driven by the dual needs to develop novel organic electronic materials and to fuel the rapidly emerging nanotechnologies. Among the many diverse fluorescent molecules, the Bodipy family, first developed as luminescent tags and laser dyes, has become a cornerstone for these new applications. The near future looks extremely bright for "porphyrin's little sister".     

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.     

New cyanine-oligonucleotide conjugates: relationships between chemical structures and properties

Because the influence of the chemical structure of monomethine cyanine-oligo-2'-deoxyribonucleotide (ODN) conjugates on their binding and fluorescence properties has remained largely undetermined, we synthesized and studied a wide range of conjugates with various structural patterns. Different cyanine dyes such as thiocyanine, quinocyanine, and thiazole orange isomers were obtained. In the case of unsymmetrical cyanines, the linker was attached to either the quinoline or the benzothiazole nucleus. The influence of the ODN counterpart was evaluated by linking the cyanines to the 5'-end or to an internucleotidic phosphate. In the first case, the influence of neighboring nucleic bases was studied, whereas in the second, the stereochemical configuration at the phosphorus atom bearing the cyanine was investigated. We report here on relationships between the structures of the dyes and conjugates and some of their properties, such as the stability and fluorescence changes observed on their hybridization with the target sequence. This study provides useful information towards the design of ODN-cyanine conjugates.     

Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy**

Formylation of 2,6-dichloro-5-R-nicotinic acids at C-4 followed by condensation with 3-hydroxy-N,N-dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with “small molecules” provided specific labeling (covalent and non-covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two-color STED microscopy with a 775 nm STED laser.     

A New Highly Selective and Sensitive Assay for Fluorescence Imaging of .OH in Living Cells: Effectively Avoiding the Interference of Peroxynitrite

A new nonredox fluorescent probe to realize the imaging of hydroxyl radicals (^.OH) in living cells was designed and synthesized. The structure comprised the fluorescent dye boron dipyrromethene (BDP) and a 2,2,6,6‐tetramethyl‐1‐piperidinoxyl (TEMPO) unit. This probe could rapidly respond to ^.OH with a detection limit of 18 pM , and it possessed superior photostability and pH insensitivity. Other reactive oxygen species (ROS) and relevant intracellular components did not interfere. In particular, the important problem of ONOO^? interference was efficiently avoided. An MTT assay proved that the probe was not very cytotoxic. The probe could penetrate into intact cell membranes to selectively detect intracellular ^.OH without causing cellular damage in living mice macrophages, normal human liver cells. and human hepatoma cells. These advantageous characteristics make the fluorescent probe potentially useful as a new candidate to detect ^.OH in broad biosystems.     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem‐crossing rates. They perform very well both in conventional and in subdiffraction‐resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground‐state depletion with individual molecular return), as well as in single‐molecule‐based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore‐containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC‐MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High‐resolution GSDIM images and live‐cell STED‐FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.     

Modified Isoindolediones as Bright Fluorescent Probes for Cell and Tissue Imaging

New L -shaped fluorophores possessing five conjugated rings have been synthesized through a four-step procedure involving diketopyrrolopyrrole synthesis and its double N-alkylation, followed by trimethylsilyl bromide-mediated rearrangement to thieno[2,3-f]isoindole-5,8-dione and an intramolecular Friedel–Crafts reaction. In comparison with the parent isoindolediones and π-expanded diketopyrrolopyrroles, these new dyes show red-shifted absorption and emission (up to ≈630 nm). Their structural rigidity is responsible for both the observed small Stokes shifts and large fluorescence quantum yields. Tissue imaging studies revealed that these new dyes show advantageous features including minimal autofluorescence interference and pronounced solvent-sensitive emission. Interestingly, there is a fundamental difference between a dye possessing an amino group and its analog bearing an N-alkyl substituent. The former dye under two-photon excitation at 900 nm gives bright images whereas its N-alkylated counterpart does not. A new type of membrane localization has been discovered by an N-alkylated isoindoledione possessing a benzofuryl substituent. In spite of the fact that the fluorescence quantum yield of this dye in a range of solvents is rather low, it does stain cell membranes exclusively. This new mode of cellular staining opens the door towards further development of membrane staining dyes.     

Iron(III)‐Selective Chelation‐Enhanced Fluorescence Sensing for In Vivo Imaging Applications

A “turn‐on” pattern Fe³⁺‐selective fluorescent sensor was synthesized and characterized that showed high fluorescence discrimination of Fe³⁺ over Fe²⁺ and other tested ions. With a 62‐fold fluorescence enhancement towards Fe³⁺, the probe was employed to detect Fe³⁺ in vivo in HeLa cells and Caenorhabditis elegans, and it was also successfully used to elucidate Fe³⁺ enrichment and exchange infected by innexin3 (Inx3) in hemichannel‐closed Sf9 cells.     

Photophysics of New Water‐Soluble Terrylenediimide Derivatives and Applications in Biology

The photophysical properties of three new water‐soluble terrylenediimide (WS‐TDI) derivatives are investigated and their utilization in biological experiments is demonstrated. Each of these dyes can be excited in the far red region of the visible spectrum, making them good candidates for in‐vivo studies. Single‐molecule techniques characterize their photophysics that is, the number of emitted photons, blinking characteristics and survival times until photobleaching takes place. All three dyes exhibit bright fluorescence, as well as an extremely high resistance against photodegradation compared to other well‐known fluorophores. Due to their different characteristics the three new WS‐TDI derivatives are suitable for specialized biological applications. WS‐TDI dodecyl forms non‐fluorescent aggregates in water which can be disrupted in a hydrophobic environment leading to a monomeric fluorescent form. Due to its high lipophilicity WS‐TDI dodecyl anchors efficiently in lipid bilayers with its alkyl chain and hence can be ideally used to image membranes and membrane‐containing compartments in living cells. In contrast, the positively charged WS‐TDI pyridoxy is a new type of chromophore in the WS‐TDI family. It is fully solubilized in water forming fluorescent monomers and is successfully used to label the envelope of herpes simplex viruses. Finally, it is shown that a WS‐TDI derivative functionalized with N‐hydroxysuccinimide ester moiety (WS‐TDI/NHS ester) provides a versatile reactive dye molecule for the specific labelling of amino groups in biomolecules such as DNA.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Development of Photoactivated Fluorescent N‐Hydroxyoxindoles and Their Application for Cell‐Selective Imaging

Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N‐hydroxyoxindoles formed through the intramolecular photocyclization of substituted o‐nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra‐high fluorescence enhancement (up to 800‐fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure–activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE‐based fluorescent probes with desired photophysical and biological properties. As a proof‐of‐concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365–405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs).