Blue Fluorophore Research Articles

Citations

Citations

Chemical biology: probes and therapeutics

In the Chemical Biology Minisymposium, cochaired by Alice Ting and Lisa Belmont, the first three talks focused on the development of novel probes for cell biology, while the last three presentations highlighted inhibitors of proteins previously believed to be “undruggable.” Daniel Hochbaum (Cohen lab, Harvard University) described an optical probe for imaging single action potentials using the fluorescence of a rhodopsin protein, Archaerhodopsin 3 (Arch), expressed in cultured rat hippocampal neurons. This voltage indicator exhibited a 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a twofold increase in brightness between −150 mV and +150 mV and a submillisecond response time. Arch detected single electrically triggered action potentials with a signal-to-noise ratio > 10. The mutant ArchD95N lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type. Although it had a slower response (41 ms), ArchD95N resolved individual action potentials. Alice Ting (MIT) presented a novel approach to determining the proteomic composition of subcellular compartments by targeting a promiscuous biotin-conjugating enzyme to subcellular regions. The labeled proteins were enriched and identified by mass spectrometry. This approach was used to determine the proteomes of mitochondria and the endoplasmic reticulum of live mammalian cells without using subcellular fractionation. Katie White, a graduate student in Ting's lab, then described improvements to in vivo fluorophore labeling using mutants of lipoic acid ligase (LplA). She engineered LplA to accept a blue coumarin fluorophore and then used yeast display evolution to evolve LplA into a probe ligase with high activity in the secretory pathway. The LplA variants allowed imaging of intra- or intercellular protein–protein contacts. To expand the scope of bioluminescence imaging, Stephen Miller (University of Massachusetts Medical School) developed new aminoluciferin substrates for firefly luciferase that emit light at longer wavelengths than d-luciferin. Although these substrates were initially limited by product inhibition, this could be ameliorated by mutation of luciferase. Moreover, mutant luciferases were identified that displayed selectivity for these synthetic substrates over d-luciferin. This system has two advantages: it is potentially better suited for in vivo imaging, because tissue is more transparent to light at longer wavelengths, and orthogonal luciferase–luciferin pairs could allow multiplexed bioluminescence imaging. An NMR-based fragment screen for inhibitors of the Ras oncoprotein presented by Guowei Fang (Genentech) identified 25 compounds that inhibit Ras with two distinct mechanisms of action. One class of compounds binds to a small pocket between Switch I and Switch II that expands to accommodate the inhibitor. These compounds competitively inhibit nucleotide exchange by blocking the interaction of RasGDP with its nucleotide exchange factor, SOS. The second class of compounds binds in a pocket created by the interface of the Ras–SOS complex and acts by accelerating nucleotide release. Corey Nislow (University of Toronto) highlighted the power of yeast genetics by screening cell-active, structurally diverse compounds against 1100 heterozygous strains (haploid for essential genes) and 5000 homozygous deletion strains. He identified 55 novel drug targets, including Sec14 and septin. Sec14 coordinates lipid biosynthesis with signaling pathways and was thought to be undruggable. He then showed how chemical genetic profiling revealed that elesclomol, a compound demonstrating efficacy in metastatic melanoma, inhibits the electron transport chain. These approaches will continue to bear fruit as additional targets are confirmed and similar technology is applied to Candida albicans and other organisms. Lisa Belmont described a model for synergy between paclitaxel and navitoclax, a Bcl-2/Bcl-xL inhibitor, in which cells in mitotic arrest slowly degrade Mcl-1, while navitoclax causes acute inhibition of Bcl-xL. Across 50 cancer cell lines, cells with high levels of Bcl-xL relative to Mcl-1 exhibited lowered paclitaxel response and higher paclitaxel/navitoclax synergy. The cell line synergy translated to xenograft studies, and analysis of ovarian cancer tissue from paclitaxel-treated patients demonstrated that high Bcl-xL predicted poor response to paclitaxel. Taken together, the data suggest the paclitaxel–navitoclax combination might be effective in cancers expressing high levels of Bcl-xL.

Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition.

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

An efficient hole-transporting blue fluorophore 3,6-dipyrenyl-9-ethylcarbazole for undoped organic light-emitting devices

A new blue-emitting carbazole derivative, 3,6-dipyrenyl-9-ethylcarbazole (DPEC), has been designed and synthesized for applications in organic light-emitting devices (OLEDs). Undoped DPEC-based OLEDs give a saturated blue electroluminescence with Commission Internationale de L’Eclairage 1931 coordinates of (0.15, 0.18), and possess high luminous efficiency of 4.75cdA−1. On the other hand, DPEC can also function as a hole-transporting layer (HTL). In particular, by replacing N,N′-diphenyl-1,1′-biphenyl-4,4′-diamine (NPB) as HTL, a simplified bilayer device demonstrated good performance, comparable to that of the trilayer device. This good device performance illustrates that DPEC is a promising candidate as a blue hole-transporting emitter.

Blue Emitting 3 π–2 Spiro Terfluorene–Indenofluorene Isomers: A Structure–Properties Relationship Study

Two novel terfluorenyl derivatives, 2,2'',7,7''-tetrakis(9,9-dioctyl-9H-fluoren-2-yl)dispiro[fluorene-9,11'-indeno-(2,1-a)-fluorene-12',9''-fluorene] ((2,1-a)-DST-IF) and 2,2'',7,7''-tetrakis(9,9-dioctyl-9H-fluoren-2-yl)dispiro- [fluorene-9,6'-indeno-(1,2-b)-fluorene-12',9''-fluorene] ((1,2-b)-DST-IF) have been synthesized by two different synthetic approaches. These terfluorenyl derivatives possess a different central indenofluorene core, namely (2,1-a)-indenofluorene or (1,2-b)-indenofluorene, which imposes two distinct geometry profiles, and different structural environments for the terfluorenyl fluorophores that translates into drastically different optical and electrochemical properties for (2,1-a)-DST-IF and (1,2-b)-DST-IF. These properties have been carefully studied through a combined experimental and theoretical approach. The (2,1-a)-DST-IF isomer has been successfully used as emitting layer in a blue single-layer small-molecule organic light-emitting diode (SMOLED) and appears as the first example of a blue emission arising from intramolecular terfluorenyl excimers. Regarding the importance of terfluorenyl derivatives in organic electronics, the present structure-properties relationship study, may open new avenues in the design of efficient blue fluorophores.

Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells

Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.

Design, Synthesis, and Pharmacological Characterization of Fluorescent Peptides for Imaging Human V1b Vasopressin or Oxytocin Receptors

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys(1),Leu(4),Lys(8)]vasopressin (d[Leu(4),Lys(8)]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu(4),Lys(Alexa 647)(8)]VP (3) and d[Leu(4),Lys(11-aminoundecanoyl-Alexa 647)(8)]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.

Synthesis and photo- and electroluminescence properties of 3,6-disubstituted phenanthrenes: alternative host material for blue fluorophores

Three deep-blue fluorescent 9,10-bis(4-tert-butylphenyl)phenanthrenes with diphenyl, -naphthyl, and -pyrenyl moieties at C3 and C6 positions were synthesized and used as the host for doped blue fluorescent devices; one of these devices reveals excellent external quantum efficiency of 7.7% and current efficiency of 9.8 cd A(-1) with low efficiency roll-off, deep-blue color coordinates (0.14, 0.14) and long operational lifetime.

COLOR TUNABLE ELECTROLUMINESCENCE FROM ORGANIC LIGHT-EMITTING DEVICES BY MANIPULATING EXCITON AND EXCIPLEX EMISSIONS

While exciplex formation is considered to have a negative impact on the performance of organic light-emitting devices (OLEDs), it can be utilized as a simple means for manipulating the emission colors. Here, we explore various important factors influencing the exciplex formation in order to provide a simple way for tailoring the emission color of OLEDs involving exciplex emission. In addition to the well-known effects of the energy difference between electron affinity and ionization potential (IP) of organic materials used, we found that electron mobility and IP of electron-transporting layer (ETL) are also critical factors in controlling the intensity of exciplex emission via the influence on charge accumulation intensity at the organic/organic interface. Interestingly, devices with green, blue, and white electroluminescence colors can be attained by simply replacing different ETL materials in contact with the same blue fluorophore underneath. The underlying mechanism for the color change in OLEDs will be discussed.

Efficient and colour-stable hybrid white organic light-emitting diodes utilizing electron–hole balanced spacers

High-efficiency two-colour white organic light-emitting diodes (WOLEDs) comprising a newly synthesized iridium complex orange phosphor ((impy)2Ir(acac)) and a blue fluorophor (BD012) have been realized by placing several kinds of thin spacers between two emitters. Hybrid WOLEDs with a spacer composed of a hole-transporting N,N-dicarbazolyl-3,5-benzene (mCP) and an electron-transporting 4,7-diphenyl-1,10-phenanthroline (Bphen) exhibit a high external quantum efficiency (EQE) of up to 8.4% and a negligible colour change (the colour coordinate of (0.39, 0.41) at 1000 cd m−2) with increasing brightness, whereas the device using a hole-transporting mCP spacer shows a relatively low EQE of 6.2% and a large shift of emitting colour with increasing brightness. Device performance is further characterized based on the charge transport behaviour of the spacers inserted between the two emitters.

Triplet Harvesting in Hybrid White Organic Light‐Emitting Diodes

AbstractWhite organic light‐emitting diodes (OLEDs) are highly efficient large‐area light sources that may play an important role in solving the global energy crisis, while also opening novel design possibilities in general lighting applications. Usually, highly efficient white OLEDs are designed by combining three phosphorescent emitters for the colors blue, green, and red. However, this procedure is not ideal as it is difficult to find sufficiently stable blue phosphorescent emitters. Here, a novel approach to meet the demanding power efficiency and device stability requirements is discussed: a triplet harvesting concept for hybrid white OLED, which combines a blue fluorophor with red and green phosphors and is capable of reaching an internal quantum efficiency of 100% if a suitable blue emitter with high‐lying triplet transition is used is introduced. Additionally, this concept paves the way towards an extremely simple white OLED design, using only a single emitter layer.

Blue emitting fluorophores of phenyleneethynylenes substituted by diphenylethenyl terminal groups for organic light-emitting diodes

Phenyleneethynylene motifs substituted by diphenylethenyl groups at both ends were prepared successfully by use of double elimination protocol of β-substituted sulfones for introducing phenyleneethynylene arrays followed by Wittig–Horner reaction for introducing diphenylethenyl moiety. The hybrid blue fluorophores exhibited strong emission even in the solid-state films ( Φ F ≧ 0.60) while, in CHCl 3 solution, incorporation of substituents on the central phenylene unit significantly enhanced emission efficiency up to Φ F = 0.57. The OLED devices with use of these blue fluorophores as an emitting material provided maximum external quantum efficiency of η ext = 2.4%.

Cover Picture: Helquats: A Facile, Modular, Scalable Route to Novel Helical Dications (Chem. Eur. J. 5/2009)

A missing link has been discovered. In their Communication on p. 1072 ff., F. Teplý et al. describe the three-step synthesis of helquats, a novel class of small helical dications representing a missing structural link between helicenes and viologens. Bridging these areas, until now separate, is anticipated to open up unknown scientific territories. The parent helquat is a water-soluble, blue fluorophore and can be prepared by a unique metal-catalyzed [2+2+2] cycloisomerization in water under aerobic conditions.

Helquats: A Facile, Modular, Scalable Route to Novel Helical Dications

As easy as one, two, three: Relying on a facile [2+2+2]-cycloisomerization strategy, we have developed a robust three-step synthetic entry into a novel family of helical extended diquats (helquats). The key metal-catalyzed step proceeds in water under aerobic conditions to produce a water-soluble, blue fluorophore (picture; TfO=triflate). The electrochemical manifold of the parent helquat features two reversible one-electron reduction steps. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Achieving high-efficiency non-doped blue organic light-emitting diodes: charge-balance control of bipolar blue fluorescent materials with reduced hole-mobility

We found an unusual way in improving electroluminescence efficiency of blue organic light-emitting diodes (OLEDs). Two electron deficient 4,5-diazafluorene- or di(2,2′-pyridyl)-containing blue fluorophores, PhSPN2N2DPV (4,5-diaza-2′-diphenylamino-7′-(2,2″-diphenylvinyl)-9,9′-spirobifluorene) and PhFpy2py2DPV (N-[7-(2,2-diphenylvinyl)-9,9′-di(2,2″-pyridyl)-2-fluorenyl]-N,N-diphenylamine), were synthesized and characterized for non-doped blue OLEDs. Whereas PhFpy2py2DPVOLED performs ordinarily, PhSPN2N2DPVOLED outperforms previously known PhSPDPV (2-diphenylamino-7-diphenylvinyl-9,9′-spirobifluorene) OLED significantly: maximum external quantum efficiency of ∼5% (4.6% at 20 mA cm−2) and the peak electroluminance of 60510 cd m−2 (1810 cd m−2 at 20 mA cm−2) versus 3.4% (2.9% at 20 mA cm−2) and 33020 cd m−2 (910 cd m−2 at 20 mA cm−2) ofPhSPDPVOLED. We attribute the superior performance of PhSPN2N2DPVOLED to the good charge balancing, which is in turn due to the very low hole mobility of PhSPN2N2DPV. The experimental results reveal that the electron-deficient moiety, 4,5-diazafluorene or di(2,2′-dipyridyl), increases electron affinity but reduces the hole mobility. Electron mobility, determined by time-of-flight (TOF) method, is 5 × 10−5 and 5 × 10−4 cm2V−1 s−1 (at an electric field of 4.9 × 105 V cm−1) for PhSPN2N2DPV and PhFpy2py2DPV, respectively. Surprisingly, they are not higher than 8 × 10−4 cm2V−1 s−1 of nonpolar PhSPDPV. On the other hand, hole mobility is 2 × 10−6 and 2 × 10−4 cm2V−1 s−1 for PhSPN2N2DPV and PhFpy2py2DPV, respectively, and they are both significantly lower than 6 × 10−3 cm2V−1 s−1 of PhSPDPV. For PhSPN2N2DPV and PhFpy2py2DPV bipolar blue fluorophores, we have demonstrated that electron-transporting and light-emitting functions involve different molecular halves. The design of such molecular halves greatly facilitates the optical and electronic optimizations of fluorophores for high-performance OLEDs.

A Multifunctional Iridium‐Carbazolyl Orange Phosphor for High‐Performance Two‐Element WOLED Exploiting Exciton‐Managed Fluorescence/Phosphorescence

AbstractBy attaching a bulky, inductively electron‐withdrawing trifluoromethyl (CF3) group on the pyridyl ring of the rigid 2‐[3‐ (N‐phenylcarbazolyl)]pyridine cyclometalated ligand, we successfully synthesized a new heteroleptic orange‐emitting phosphorescent iridium(III) complex [Ir(L1)2(acac)] 1 (HL1 = 5‐trifluoromethyl‐2‐[3‐(N‐phenylcarbazolyl)]pyridine, Hacac = acetylacetone) in good yield. The structural and electronic properties of 1 were examined by X‐ray crystallography and time‐dependent DFT calculations. The influence of CF3 substituents on the optical, electrochemical and electroluminescence (EL) properties of 1 were studied. We note that incorporation of the carbazolyl unit facilitates the hole‐transporting ability of the complex, and more importantly, attachment of CF3 group provides an access to a highly efficient electrophosphor for the fabrication of orange phosphorescent organic light‐emitting diodes (OLEDs) with outstanding device performance. These orange OLEDs can produce a maximum current efficiency of ∼40 cd A−1, corresponding to an external quantum efficiency of ∼12% ph/el (photons per electron) and a power efficiency of ∼24 lm W−1. Remarkably, high‐performance simple two‐element white OLEDs (WOLEDs) with excellent color stability can be fabricated using an orange triplet‐harvesting emitter 1 in conjunction with a blue singlet‐harvesting emitter. By using such a new system where the host singlet is resonant with the blue fluorophore singlet state and the host triplet is resonant with the orange phosphor triplet level, this white light‐emitting structure can achieve peak EL efficiencies of 26.6 cd A−1 and 13.5 lm W−1 that are generally superior to other two‐element all‐fluorophore or all‐phosphor OLED counterparts in terms of both color stability and emission efficiency.

Influence of Molecular Dipoles on the Photoluminescence and Electroluminescence of Dipolar Spirobifluorenes

AbstractThis Full Paper investigates a series of strongly fluorescent donor–acceptor‐substituted spirobifluorene compounds, red 2‐diphenylamino‐7‐(2,2‐dicyanovinyl)‐9,9′‐spirobifluorene (DCV), green 2‐diphenylamino‐9,9′‐spirobifluorene7‐carxoxaldehyde (CHO), and blue 2‐diphenylamino‐7‐(2,2‐diphenylvinyl)‐9,9′‐spirobifluorene (DPV), together with their spiro‐linked “dimeric” analogs, 2DCV, 2,2′‐bis(diphenylamino)‐9,9′‐spirobifluorene‐7,7′‐dicarboxaldehyde (2CHO), and 2,2′‐bis(diphenylamino)‐7,7′‐bis(2,2‐diphenylvinyl)‐9,9′‐spirobifluorene (2DPV), respectively. The emission optical density and, hence, the intensity of photoluminescence (PL) or electroluminescence (EL) of the “dimeric” analogs is presumed to increase, which is beneficial for organic light‐emitting diode (OLED) applications. The physical properties, including the dipole moments obtained from quantum chemistry calculations, emission solvatochromism, fluorescence quantum yield (Φf) as well as the EL of these six spirobifluorene compounds have been examined in detail. We found that Φf as well as OLED performance (EL efficiency and intensity) of the strongly dipolar DCV decrease significantly in the “dimeric” analog 2DCV, but less so in the moderately dipolar CHO and 2CHO, and only slightly in the weakly dipolar DPV and 2DPV. This is parallel to the intramolecular dipole moment, which is large for 2DCV, medium for 2CHO, and very small for 2DPV. Here, we show for the first time systematically that the luminescence intensity is closely correlated with the local electric field induced by the molecular dipole. A strong electric field may facilitate radiationless decay channels with a charge‐transfer nature, leading to a high quenching rate. Consistent with this conclusion, which is derived from the red DCV/2DCV and green CHO/2CHO, our new blue fluorophore DPV with an essentially zero dipole moment has successfully achieved one of the best electrofluorescent blue OLEDs. At the same time, by doping the highly dipolar DCV into an isolated environment with the low‐polarity Alq3 as the host matrix, we obtained a very high performance of saturated yellow OLEDs as well, This is possibly due to the reduction of emission‐quenching dipoles from the neighboring molecules. Our results have provided an important insight in designing luminescent materials, as follows: molecular dipole moments should be kept at a low magnitude to avoid quenching induced by a strong local electric field in the chromophore.

Cell‐permeant cytoplasmic blue fluorophores optimized for in vivo two‐photon microscopy with low‐power excitation

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.

Role of Xanthurenic Acid 8-O-β-d-Glucoside, a Novel Fluorophore that Accumulates in the Brunescent Human Eye Lens¶

We have been able to identify a blue fluorophore from the low-molecular weight soluble fraction of human adult nondiabetic brunescent cataract lenses as xanthurenic acid 8-O-β-d-glucoside (XA8OG) (excitation = 338 nm and emission = 440 nm). To determine the role of this fluorophore in the lens, we have examined its photophysical and photodynamic properties. We found XA8OG to have a fluorescence quantum yield (ϕ) of 0.22 and a major emission lifetime of 12 ns. We found it to be a UVA-region sensitizer, capable of efficiently generating singlet oxygen species but little of superoxide. We also demonstrated that XA8OG oxidizes proteins when irradiated with UVA light, causing photodynamic covalent chemical damage to proteins. Its accumulation in the aging human lens (and the attendant decrease of its precursor O-β-d-glucoside of 3-hydroxykynurenine) can, thus, add to the oxidative burden on the system. XA8OG, thus, appears to be an endogenous chromophore in the lens, which can act as a cataractogenic agent.

Latent Blue and Red Fluorophores Based on the Trimethyl Lock

Latent Blue and Red Fluorophores Based on the Trimethyl Lock