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Boronic acids are widely used for labeling catechols and carbohydrates in analytical (bio)chemistry due to their high binding affinities for diols. Here, we present two asymmetrically substituted Bodipy dyes with a boronic acid at the β-position (BBB). We present a green-emitting BBB, gBBB, and, by expanding the conjugated system of the Bodipy core at α-position, a red-emitting rBBB. Especially, gBBB shows a bathochromic shift of the electronic spectra upon binding to saccharides and polyols, whereas the fluorescence lifetime of rBBB is more sensitive to hydroxy-ligand binding. Moreover, gBBB constantly shows higher binding affinities than rBBB, reaching

The aim of this study was to identify a high‐affinity BODIPY peptidomimetic that targets the prostate‐specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure‐activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu‐CO‐Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA‐positive LNCaP cells. One compound was identified with comparable affinity (IC50=21.5±0.1 nM) to Glu‐CO‐Lys‐Ahx‐HBED‐CC (PSMA‐11) (IC50=18.4±0.2 nM). Radiolabeling was achieved by Lewis‐acid‐mediated 19F/18F exchange in moderate molar activities (∼0.7 MBq nmol−1) and high radiochemical purities (>99 %) with mean radiochemical yields of 20–30 %. Cell internalization of the 18F‐labeled high‐affinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMA‐mediated internalization over time. By fluorescence microscopy, localization of the high‐affinity BODIPY‐PSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a high‐affinity BODIPY‐PSMA conjugate has been identified as a suitable candidate for the development of PSMA‐specific dual‐imaging agents., Have it both ways: By structure–activity relationship studies, a bimodal imaging probe combining fluorescence with nuclear imaging based on BODIPY dyes with high affinity for the prostate‐specific membrane antigen (PSMA) has been identified. The bimodal conjugate demonstrated PSMA‐specific uptake in prostate cancer cells with accumulation in the cell membrane at early time points and additional accumulation in subcellular compartments at later time points.

Despite the outstanding relevance of proton transfer reactions, investigations of the solvent dependence on the elementary step are scarce. We present here a probe system of a pyrene-based photoacid and a phosphine oxide, which forms stable hydrogen-bonded complexes in aprotic solvents of a broad polarity range. By using a photoacid, an excited-state proton transfer (ESPT) along the hydrogen bond can be triggered by a photon and observed via fluorescence spectroscopy. Two emission bands could be identified and assigned to the complexed photoacid (CPX) and the hydrogen-bonded ion pair (HBIP) by a solvatochromism analysis based on the Lippert-Mataga model. The latter indicates that the difference in the change of the permanent dipole moment of the two species upon excitation is ∼3 D. This implies a displacement of the acidic hydrogen by ∼65 pm, which is in quantitative agreement with a change of the hydrogen bond configuration from O-H···O to -O···H-O+.

Silica nanoparticles with embedded fluorescent dyes represent an important class of markers for example in biological imaging. We systematically studied the various incorporation mechanisms of fluorescent xanthene dyes in 30–40 nm silica nanoparticles. An important parameter was the interaction of the dye with the matrix material, either by weak electrostatic or strong covalent interactions, which also has implications on the stability of fluorescence and brightness of the dyes. Factors that can influence leaching of dyes such as the position of the dyes in particles and the intensity of the particle-dye interaction were investigated by using the solvatochromic effect of xanthene dyes and by stationary fluorescence anisotropy measurements. We compared uranine and rhodamine B, which were physically embedded, with modified fluorescein isothiocyanate and rhodamine B isothiocyanate, which were covalently bound to the silica matrix within a usual Stöber synthesis. Systematic leaching studies of time spans up to 4 days revealed that covalent bonding of dyes like fluorescein isothiocyanate or rhodamine B isothiocyanate is necessary for fluorescence stability, since dyes bound by physical interaction tend to leach out of porous silica networks. Coverage of silica particles with hydrophobic protection layers of alkyltrialkoxysilanes or hydrophilic polyethylene glycol (PEG) groups resulted in a better retention of physisorbed dyes and provides the possibility to adapt the particles to the polarity of the medium. Best results were archived with PEG groups, but even small trimethylsilyl (TMS) groups already reduce leaching.

The phenomenon of photoacidity, i.e., an increase in acidity by several orders of magnitude upon electronic excitation, is frequently encountered in aromatic alcohols capable of transferring a proton to a suitable acceptor. A promising new class of neutral super-photoacids based on pyranine derivatives has been shown to exhibit pronounced solvatochromic effects. To disclose the underlying mechanisms contributing to excited-state proton transfer (ESPT) and the temporal characteristics of solvation and ESPT, we scrutinize the associated ultrafast dynamics of the strongest photoacid of this class, namely tris(1,1,1,3,3,3-hexafluoropropan-2-yl)8-hydroxypyrene-1,3,6-trisulfonate, in acetoneous environment, thereby finding experimental evidence for ESPT even under these adverse conditions for proton transfer. Juxtaposing results from time-correlated single-photon counting and femtosecond transient absorption measurements combined with a complete decomposition of all signal components, i.e., absorption of ground and excited states as well as stimulated emission, we disclose dynamics of solvation, rotational diffusion, and radiative relaxation processes in acetone and identify the relevant steps of ESPT along with the associated time scales. Graphical abstract

Super-photoacids, that is, photoacids with a negative urn:x-wiley:23670932:media:cptc202200041:cptc202200041-math-0001 value in the electronically excited state, can trigger an excited-state proton transfer (ESPT) to the solvent. For the neutral pyranine-derived super-photoacid studied here, even indications for ESPT in acetoneous solution are reported. The characteristics of ESPT in this environment, that is, which intermediates exist and what the impact of cosolvents is, remain unsettled though. In this work, we study ESPT in acetone-water mixtures by steady-state and time-resolved fluorescence spectroscopy. Various effects are observed: First, the addition of water supports the formation of a hydrogen-bonded ground-state complex comprising one water molecule and the photoacid, whose excitation triggers the formation of a hydrogen-bonded ion pair on a sub-ns time scale. Second, water has an overall accelerating effect on the fluorescence dynamics of the involved emitting species, whose contributions are disentangled in a global analysis scheme, enabling the identification of emission from the free photoacid, a photoacid-water complex, a hydrogen-bonded ion pair, and the deprotonated photoacid. At least two water molecules are necessary for ESPT in the environment. Third, additional acidification thwarts an efficient ground-state complex formation of the photoacid and water. However, upon excitation, complexation may occur on a timescale faster than the photoacid's excited-state lifetime, so that emission from a nascent complex emerges.

We present a femtosecond pump-probe UV-Vis broadband transient absorption spectroscopy study of two styryl-substituted BODIPY chromophores with different position of the substituent. The α-substituted isomer shows typical BODIPY-type spectral features, such as sharp absorption and emission bands, a small Stokes shift and an excited-state lifetime in the 4 ns range, which only weakly depends on the solvent. In contrast, β-styryl-BODIPY features much broader steady-state absorption and emission spectra and a larger Stokes shift, particularly in polar solvents. Transient absorption spectroscopy including a complete global kinetic analysis reports a substantial decrease in S1 lifetime to 300 ps for polar solvents upon change from α- to β-substitution. In the case of the α-isomer, TD-DFT calculations identify a typical “cyanine-type” electron rearrangement upon S0 → S1 excitation accompanied by a slight reduction in dipole moment. In contrast, the same transition in the β-isomer shows strong intramolecular charge transfer (ICT) character involving a substantial increase in dipole moment. Assuming a simple energy-gap-law argument, the accelerated nonradiative decay of the β-isomer in polar solvents may be linked to the decrease of the S1(ICT)-S0 energy difference. BODIPY dyes with a conjugated substituent in β-position therefore suffer a substantial loss in fluorescence brightness in polar environments compared with their α-substituted counterparts. This might limit their applicability in fluorescence imaging.

Immobilization procedures, intended to enable prolonged observation of single molecules by fluorescence microscopy, may generate heterogeneous microenvironments, thus inducing heterogeneity in the molecular behavior. On that account, we propose a straightforward surface preparation procedure for studying chemical reactions on the single-molecule level. Sensor fluorophores were developed, which exhibit dual-emissive characteristics in a homogeneously catalyzed showcase reaction. These molecules undergo a shift of fluorescence wavelength of about 100 nm upon Pd(0)-induced deallylation in the Tsuji-Trost reaction, allowing for separate visualization of the starting material and product. Whereas a simultaneous immobilization of dye and inert silane leads to strongly polydisperse reaction kinetics, a consecutive immobilization routine with deposition of dye molecules as the last step provides substrates underlying the kinetics of ensemble experiments. Also, the found kinetics are unaffected by the chemical variation of inert silanes, nearly uniform, and therefore well reproducible. Additional parameters like photostability, signal-to-noise ratio, dye-molecule density, and spatial distribution of dye molecules are, as well, hardly affected by surface modification in the successive immobilization scheme.

ChemBioChem 22(23), 3283-3291 (2021). doi:10.1002/cbic.202100276, Published by Wiley-VCH, Weinheim

Intermolecular Proton Transfer (PT) dynamics can be best studied by optical spectroscopy, which can cover the vast timescale spanned by the process. PT in a hydrogen bonding complex between a pyranine-based photoacid and a trialkyl-phosphine oxide is addressed. The photoreaction is traced with the help of femtosecond transient absorption and picosecond-resolved fluorescence. Characteristic kinetics and spectra of the intervening species are isolated by global analysis and spectral decomposition of time-resolved fluorescence. It is found that the shared proton shifts towards the phosphine site upon photoexcitation in acetonitrile. The process occurs on the sub-picosecond timescale, essentially, under solvent control. Despite the ultrafast rate, an equilibrium between the complex and the hydrogen-bonded ion pair (HBIP) is established. Further reaction steps are delayed to the nanosecond timescale, where formation of the excited deprotonated form is observed. The far-reaching consistency between the various methods supports an irreversible Eigen-Weller mechanism in the excited state.

Photoacids are the most convenient way to deliver protons on demand. So far, their photoacidity allows for studying excited-state proton transfer (ESPT) only to protic or strongly basic solvent molecules. The strongest superphotoacids known so far exhibit excited-state lifetimes of their conjugate base on the order of 100 ps before recapturing the proton again. Here, we describe how we developed a new aminopyrene-based superphotoacid with an excited-state lifetime of its conjugate base of several nanoseconds. It will be shown by fluorescence titration and via Förster cycle that the excited-state acidity is as high as concentrated sulfuric acid and thus exceeding any previous photoacidity by several orders of magnitude. Its outstanding chemical stability and fluorescent properties make it suitable for time-resolved proton-transfer studies in concentrated mineral acids and organic solvents of low basicity.

Coumaryl triflate can be used for a wide range of substitution reactions, either under Pd-catalyzed conditions or via direct nucleophilic substitutions, and is therefore an ideal substrate for the fluorescence labeling of functionalized compounds, such as amino acids.

A series of aryl disilenes Tip2SiSi(Tip)Ar (2a–c) and para-arylene bridged tetrasiladienes, Tip2SiSi(Tip)–LU–Si(Tip)SiTip2 (3a–d) are synthesized by the transfer of the Tip2SiSiTip unit to aryl halides and dihalides by nucleophilic disilenides Tip2SiSiTipLi (Tip = 2,4,6-iPr3C6H2, Ar = aryl substituent, LU = para-arylene linking unit). The scope of the nucleophilic SiSi transfer reaction is demonstrated to also include substrates of considerable steric bulk such as mesityl or duryl halides Ar–X (Ar = Mes = 2,4,6-Me3C6H2; Ar = Dur = 2,3,5,6-Me4C6H, X = Br or I). Bridged tetrasiladienes Tip2SiSi(Tip)–LU–Si(Tip)SiTip2 with more extended linking units surprisingly exhibit fluorescence at room temperature, albeit weak. DFT calculations suggest that partial charge transfer character of the excited state is a possible explanation.

Intracellular pH sensing with fluorescent nanoparticles is an emerging topic as pH plays several roles in physiology and pathologic processes. Here, nanoparticle-sized pH sensors (diameter far below 50 nm) for fluorescence imaging have been described. Consequently, a fluorescent derivative of pH-sensitive hydroxypyrene with p

Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper's redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = - 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms. Graphical abstract.

Gold nanocluster (AuNC) synthesis using a well‐distinguished polymer for nanoparticle‐mediated drug delivery paves the way for developing efficient theranostics based on pharmaceutically accepted materials. Gelatin‐stabilized AuNCs are synthesized and modified by glutathione for tuning the emission spectra. Addition of silver ions enhances the fluorescence, reaching also high quantum yield (26.7%). A simplified model can be proposed describing the nanoclusters' properties–structure relationship based on X‐ray photoelectron spectroscopy data and synthesis sequence. Furthermore, these modifications improve fluorescence stability toward pH changes and enzymatic degradation, offering different AuNCs for various applications. The impact of nanocluster formation on gelatin structure integrity is investigated by Fourier transform infrared spectrometry and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy, being important to further formulate gelatin nanoparticles (GNPs). The 218 nm‐sized NPs show no cytotoxicity up to 600 µg mL−1 and are imaged in skin, as a challenging autofluorescent tissue, by confocal microscopy, when transcutaneously delivered using dissolving microneedles. Linear unmixing allows simultaneous imaging of AuNCs–GNPs and skin with accurate signal separation. This underlines the great potential for bioimaging of this system to better understand nanomaterials' behavior in tissue. Additionally, it is drug delivery system also potentially serving as a theranostic system.

Coumarin-derived allylic carbonates are found to be suitable electrophiles for the synthesis of fluorescent amino acids via palladium-catalyzed allylic alkylation of chelated glycine enolates. The formation of regioisomers can be suppressed by proper choice of the allyl carbonate.

Time-resolved experiments with pulsed-laser excitation are the standard approach to map the dynamic evolution of excited states, but ground-state kinetics remain hidden or require pump-dump-probe schemes. Here, we exploit the so-called photon antibunching, a purely quantum-optical effect related to single molecule detection to assess the rate constants for a chemical reaction in the electronic ground state. The measurement of the second-order correlation function g((2)), i.e. the evaluation of inter-photon arrival times, is applied to the reprotonation in a Förster-cycle. We find that the antibunching of three different photoacids in the aprotic solvent DMSO significantly differs from the behavior in water. The longer decay constant of the biexponential antibunching tl is linked to the bimolecular reprotonation kinetics of the fully separated ion-pair, independent of the acidic additives. The value of the corresponding bimolecular rate constant, kp = 4 × 10(9) M(-1) s(-1), indicates diffusion-controlled reprotonation. The analysis of tl also allows for the extraction of the separation yield of proton and the conjugated base after excitation and amounts to approximately 15%. The shorter time component ts is connected to the decay of the solvent-separated ion pair. The associated time constant for geminate reprotonation is approximately 3 ± 1 ns in agreement with independent tcspc experiments. These experiments verify that the transfer of quantum-optical experiments to problems in chemistry enables mechanistic conclusions which are hardly accessible by other methods.

Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the "vGFP strategy" based on structural analysis of GFP bound to a single-domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50%), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single-molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single-step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.

Rationale 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. Methods The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. Results Unusual radical molecular cations ([M]+•) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M]+• and [M+H]+ decompositions significantly complicated the interpretation of the MS/MS spectra. Conclusions Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS. Copyright © 2015 John Wiley & Sons, Ltd.

The direct observation of chemical reactions on the single-molecule level is an ultimate goal in single-molecule chemistry, which also includes kinetic analyses. To analyze the lifetime of reaction intermediates, very sophisticated excitation schemes are often required. Here we focus on the kinetic analysis of the ground-state proton transfer within the photocycle of a photoacid. In detail, we demonstrate the determination of the bimolecular rate constant of this process with nanosecond resolution. The procedure relies on the exploration of a purely quantum-optical effect, namely, photon antibunching, and thus on evaluating interphoton arrival times to extract the reaction rate constant.

We describe a fluorogenic ruthenium (II) carbene complex in which the chromophore is directly connected to the metal center. The compound introduces a boron dipyrromethene (BODIPY) moiety into target double bonds by metathesis. Tagging of terminal double bonds is demonstrated on immobilized styrene units on a glass surface. We also show that two compounds with distinguishable fluorescence properties are formed in the model reaction with styrene. The outcome of the metathesis reaction is characterized by 19F-NMR, optical spectroscopy, and, finally, single-molecule trajectories. This labeling scheme, in our perception, is of particular interest in the fields of interfacial science and biorthogonal ligation in combination with super-resolution imaging.

The asymmetric substituted BODIPY dye 4,4-difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indacene crystallizes in three different crystal habits, that is, as needles (I), leaves (II), or microcrystalline sublimed crystals (III). All crystals share the same crystal structure but exhibit varying solid-state fluorescence from yellow-orange to deep red. The crystal structure mainly consists of one-dimensional chains of J-aggregates which leads to excitonic luminescence at λ = 600 nm. The point-dipole approximation is used to calculate the excitonic splitting to a value of 3300 cm–1, which is in good agreement with the experimental observations. The influence of the macroscopic appearance on the luminescence properties is discussed in terms of reflectivity of the surface and reabsorption within the material. It turns out that the long-range order modulates the solid-state luminescence due to the small Stokes shift of the dye.

Photoacids on the basis of pyrenol have been extensively studied in the past 60 years. As their photophysical properties strongly depend on the substituents at the aromatic scaffold, we introduced two reactive moieties with different electronic coefficients thus creating multi-wavelength fluorescent probes. One probe is capable of monitoring two orthogonal transformations by four fluorescence colors, distinguishable even by the naked human eye. Another derivative can act as a three-color sensor for a wide range of different pH values. Both the presented compounds allow for mimicking of fundamental and advanced two-input logic operations due to the multi-wavelength emission. Furthermore, these compounds can process information in a logically reversible way (Feynman gate).

The combination of the two complementary imaging modalities (19) F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual (19) F MRI-fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T1 and the transverse relaxation times T2 , both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual (19) F MRI-fluorescence agent was also successfully imaged in a mouse post-mortem within a 9.4 T small-animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing.

Pechmann condensation is a straightforward protocol for the synthesis of alkynyl- and azido-substituted 7-(dialkylamino)coumarins, which can be coupled to amino acid and peptide derivatives by copper-catalyzed [3+2]-cycloaddition (click reaction) in high yield. This allows the introduction of these efficient fluorescence labels into biologically relevant molecules.

International audience; New types of fluorescent platinum nanoclusters (λmax.= 570 nm) have been synthesized using glutathione for ligand etching. These nanoclusters are mainly in the oxidation state PtI (90%) exhibiting an intense fluorescence signal (QY∼17%) in the yellow region and lead to the formation of blue-emitting species for a long etching time process.

A principal objective in life sciences is the visualization of biochemical processes. Fluorescence-based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real-time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu(2+) ) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu(2+) binding. We demonstrate that this technique is selective for Cu(2+) . We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu(2+) . Cu(2+) uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu(2+) distribution in vivo is demonstrated in prokaryote and eukaryote cells.

The understanding of cellular processes and functions and the elucidation of their physiological mechanisms is an important aim in the life sciences. One important aspect is the uptake and the release of essential substances as well as their interactions with the cellular environment. As green fluorescent protein (GFP) can be genetically encoded in cells it can be used as an internal sensor giving a deeper insight into biochemical pathways. Here we report that the presence of copper(II) ions leads to a decrease of the fluorescence lifetime (τ fl) of GFP and provide evidence for Forster resonance energy transfer (FRET) as the responsible quenching mechanism. We identify the His6-tag as the responsible binding site for Cu2 + with a dissociation constant K d = 9 ±2 μM and a Forster radius R 0 = 2.1 ±0.1 nm. The extent of the lifetime quenching depends on [Cu2 + ] which is comprehended by a mathematical titration model. We envision that Cu2 + can be quantified noninvasively and in real-time by measuring τ fl of GFP.

Starch was oxidized with TEMPO for the synthesis of water-soluble copolymers of glucuronic acid and glucose. The carboxylate groups of these copolymers were conjugated with pteroic acid as cell-specific ligand for targeting to cancer cells. Stable spherical nanoparticles (NPs) were formulated mixing aqueous solutions of the anionic copolymers and of a cationic thioether of β-cyclodextrin (β-CD). Particle size distributions of NPs were investigated with DLS as the function of the charge ratio of the constituents. The smallest and most uniform particles with a diameter of about 130 nm were generated at a charge ratio of anion/cation close to 1, preferably 1.2. Stabilities and particle size distributions of these starch NPs were very satisfactory. The starch/β-CD NPs could be loaded with hydrophobic guest molecules like 1,4-dihydroxyanthraquinone (DHA), which served as a model for the important class of anthracycline antibiotics used in cancer therapy.

The stabilities of the inclusion compounds of three chemotherapeutic agents, camptothecin (CPT), docetaxel (DOC) and idarubicin (IDA), plus a model compound 1,4-dihydroxyanthraquinone (DHA) with several β-cyclodextrin (β-CD) derivatives were investigated by solubility measurements, isothermal titration microcalorimetry and fluorescence anisotropy measurements. Ionic heptakis-(6-deoxy-6-thioethers) of β-CD were found to exhibit very high binding potentials for these drugs making them to good candidates for advanced drug delivery.

The synthesis and properties of a ratiometric fluorescent dye system as a probe for the detection of oxidizing species are described. The dye consists of a strongly fluorescent Bodipy label attached to a styryl moiety. The oxidation of the exocyclic double bond by agents for syn- and anti- dihydroxylation leads to a visible change in fluorescence colour from orange to green. IR spectroscopy provides evidence for the generation of the epoxide in the reaction with peroxides, whereas a ketone and a product of unknown structure are produced in the reaction with osmiumtetroxide, each in contrast to the expected hydroxylations. Based on these first attempts, we discuss the significance of fluorescence spectroscopy to track chemical reactions at low concentrations and give a perspective of how present shortcomings can be overcome. Copyright © 2009 John Wiley & Sons, Ltd.

The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to ΔOD = 10−7 absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.

In single-molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S(1), in contrast to previous findings.

We highlight our recent achievements in the design of fluorescent dyes for the investigation of chemical reactions on the single-molecule level. These fluorophores are tailored to undergo changes in their photophysical properties upon chemical transformations. Three examples are presented: electrophilic aromatic substitution, phosphoester cleavage, and oxidation of double-bonds. Thin-layer chromatography and fluorescence correlation spectroscopy are used to separate and characterize the different reaction products, respectively. We are planning to develop more fluorescent synthons which enable us to perform single-molecule chemistry of various reactions.

Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert–Prakesh reagent of dimethyl-, tetramethyl- and pentamethyl-substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (

We study the photophysical behavior of 8 mutants of Green Fluorescent Protein (GFP) using fluorescence correlation spectroscopy (FCS) on the single molecule level and double resonance excitation of bulk samples. Experimental data reported here and the previously pub- lished data on the RH/Requilibrium and fluorescence quantum yields FFl (Jung et al., 2005; Bio- phys J 88:1932-1947) are analyzed with respect to single molecule as well as conventional fluores- cence microscopy. The fraction of GFP molecules in a dark state, (D), reduces the effective absorp- tion cross section under photostationary conditions. The determination of the excitable fraction (B) and its fluorescence quantum yield FFl gives the effective brightness Feff. Our results show that in its wavelength range, eGFP is, among the GFPs, the best fluorophore for most microscopic applica- tions. However, in the red shifted YFP-proteins, there is still potential for improvement, since a pronounced dark state population is detectable in all mutants investigated so far. We propose to use the mutant T203Y/E222Q in imaging studies, whenever the expression yield is not a limiting factor. In FCS experiments, where the useful concentration range of the expressed molecules is re- stricted to concentrations below micromolarity, our data suggest the use of wt-GFP or mutant T203Y, as these represent photochemical buffers. Both mutants might surpass the limitations given by out-of-focus bleaching in live cell microscopy. Microsc. Res. Tech. 69:175-185, 2006. V C 2006 Wiley-Liss, Inc.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis.The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments.Unusual radical molecular cations ([M](+•)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+•) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra.Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.

Joint involvement occurs in most patients with systemic lupus erythematosus (SLE), and severe lupus arthritis is often refractory to conventional treatments. Anakinra is used in the treatment of rheumatoid arthritis, but its therapeutic potential has not been proved in patients with SLE.To determine the safety/tolerability and efficacy of anakinra in patients with SLE with leading joint involvement.In patients with SLE with active polyarthritis and no other uncontrolled systemic/organ manifestations, 100 mg/day anakinra was self administered subcutaneously for 3 months. Disease activity was assessed by VAS, number of swollen/tender joints, ECLAM score, and serological and immunological measures.Four patients with SLE were studied; anakinra was safe in all four patients and no drug related serious adverse events occurred. A subjective benefit was seen in all patients and a trend towards better activity measures after 4 weeks. After an initial response, one patient left the study because of an arthritic flare after 6 weeks.In this study anakinra was apparently safe and well tolerated and led to clinical and serological improvement. Anakinra might be an interesting alternative in individual patients with lupus arthritis not responding to conventional treatments.

In a 65-year-old female patient, B-mode sonography detected a single focal lesion in the right liver lobe with a diameter < 3 cm. Histopathologic examination revealed a low differentiated hepatocellular carcinoma (HCC; G3). Tumor staging was performed by CT (computed tomography) scan and Resovist MRI (magnetic resonance imaging). Both examinations found a single liver lesion without signs of additional focal hepatic lesions. In addition, phase-inversion sonography in the late phase was performed using the ultrasound contrast agent Levovist. This examination of late-phase Levovist uptake detected more than five additional focal hepatic lesions in the right liver lobe, which were invisible by CT scan and Resovist MRI. This finding of multiloculated HCC was very important to decide on the patient's correct therapy. While liver transplantation is the treatment of choice in single HCC < 3 cm, it is contraindicated in multicentric HCC. In the patient described here, hemihepatectomy of the right liver lobe was performed. The histopathologic examination of the resected liver confirmed the diagnosis of multicentric HCC, which was noninvasively diagnosed only by contrast-enhanced sonography, but not by CT scan or MRI.

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.