Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.     

Human Serum Albumin Labelling with a New BODIPY Dye Having a Large Stokes Shift

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.     

α-Acrylaldehyde 3-Pyrrolyl BODIPY as a Specific Optical Sensor for Cysteine and Homocysteine

A new fluorophore, α-acrylaldehyde 3-pyrrolyl BODIPY was synthesized by treating 3-pyrrolyl BODIPY with a mixture of 3-(dimethylamino) acrolein and POCl₃ under Vilsmeier–Haack reaction conditions. The X-ray structure revealed that the fluorophore was almost planar, and the appended pyrrole was in the same plane with a small deviation from the mean plane. We investigated the potential use of α-acrylaldehyde 3-pyrrolyl BODIPY for sensing thiol containing amino acids such as cysteine/homocysteine (Cys/Hcy). Our studies showed that the α-acrylaldehyde- 3-pyrrolyl BODIPY was found to be useful for exclusive sensing of Cys/Hcy and to exhibit different optical signaling responses to Cys and Hcy at physiological pH in aq. CH₃CN (1 : 1 v/v, PBS) medium. The enhancement in optical properties for Cys and quenching in same properties for Hcy was attributed to different binding modes of Cys/Hcy with α-acrylaldehyde 3-pyrrolyl BODIPY.     

新型含双酚衍生物三枝氟硼二吡咯染料的合成及其光物理性能

以三聚氯氰为原料合成含醛基的二酚氧基取代中间体（1）; 1分别与酚衍生物（2a~2e）经取代反应制得三酚氧基中间体（3a~3e）; 3a~3e经缩合、氧化和配位等反应合成了5个新型的含双酚衍生物三枝氟硼二吡咯（BODIPY）荧光染料（4a~4e）,其结构经¹H NMR, ¹³C NMR和HR-MS(ESI)表征。4a~4e的最大吸收波长和发射波长分别位于499 nm和508 nm,荧光量子产率为0.41~0.55,显示出BODIPY荧光核典型的光物理性能。     

Synthesis of fluorescent probes based on stilbenes and diphenylacetylenes targeting β-amyloid plaques

Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in the patients with Alzheimer’s disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a,4a-diaza-s-indacene, BODIPY) with the pharmacophore possessing either a stilbene or a diphenylacetylene moiety. Different polyethylene glycol chain lengths were used as linkers between the fluorophore and the pharmacophore to adjust the lipophilicity of these probes. These compounds exhibit strong fluorescence emission between 665 and 680 nm and have very high extinction coefficients comparable to the parent fluorophore, BODIPY dye.     

An integrated solid-phase extraction system for sub-picomolar detection

A microchip structure etched on a glass substrate for packed column solid-phase extraction (SPE) and capillary electrochromatography (CEC) is described. A 200 microm long, octadecylsilane (ODS) packed column was secured using two different approaches: solvent lock or polymer entrapment. The former method was utilized for SPE while the latter approach was applied for CEC. In SPE, the ODS packed chamber gave a detection limit of 70 fM for a nonpolar BODIPY (493/503) dye when concentrated for 3 min at an electroosmotic flow rate of 4.14 nL/min, compared to 30 pM for this detector without the SPE step. SPE beds showed reproducible, linear calibration curves (R(2) = 0.9989) between 1 and 100 pM BODIPY at fixed preconcentration times. Breakthrough curves for the 330 pL (ODS-packed) bed indicated a capacity for BODIPY dye of 8.1 x 10(-14) mmol, or 0.25 mmol dye per liter of bed. The ODS-chamber could also be used to analyze dilute amino acid and peptide solutions. In the CEC format, two neutral dyes (BODIPY and acridine orange) were baseline-separated in an isocratic run with a theoretical plate count of 84 (420 000 plates/m) and a reduced plate height of about 1. A labeled peptide was also analyzed by CEC, using the acidic eluent (84% acetonitrile, and 26% aqueous trifluoroacetic acid (0.05%)) preferred for peptide separations on ODS-coated silica particles.     

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AA's, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AA's using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AA's yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.     

Efficient Intramolecular Energy Transfer between Two Fluorophores in a Bis‐Branched [3]Rotaxane

A bis‐branched [3]rotaxane, with two [2]rotaxane arms separated by an oligo(para‐phenylenevinylene) (OPV) fluorophore, was designed and investigated. Each [2]rotaxane arm employed a difluoroboradiaza‐s‐indacene (BODIPY) dye‐functionalized dibenzo[24]crown‐8 macrocycle interlocked onto a dibenzylammonium in the rod part. The chemical structure of the [3]rotaxane was confirmed and characterized by ¹H and ¹³C NMR spectroscopy and high‐resolution ESI mass spectrometry. The photophysical properties of [3]rotaxane and its reference systems were investigated through UV/Vis absorption, fluorescence, and time‐resolved fluorescence spectroscopy. An efficient energy‐transfer process in [3]rotaxane occurred from the OPV donor to the BODIPY acceptor because of the large overlap between the absorption spectrum of the BODIPY moiety and the emission spectrum of the OPV fluorophore; this shows the important potential of this system for designing functional molecular systems.     

Hydrophobic labeling of amino acids: transient trapping-capillary/microchip electrophoresis

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.     

Tunable design strategy for fluorescence probes based on 4-substituted BODIPY chromophore: improvement of highly sensitive fluorescence probe for nitric oxide

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is a well-known fluorophore, with a high molar extinction coefficient and high fluorescence quantum efficiency (Phi(fl)). Furthermore, its structure can be modified to change its excitation and emission wavelengths. However, little work has been done on the structural modification of fluorines at the B-4 position with other functional groups. We synthesized 4-methoxy-substituted BODIPY derivatives in satisfactory yields, and found that they exhibited improved solubility in aqueous solution. Moreover, their oxidation and reduction potentials were greatly decreased without any change in their absorbance and fluorescence properties. These features of 4-substituted BODIPYs may be useful for developing novel fluorescence probes based on the intramolecular photoinduced electron transfer (PeT) mechanism, because it is possible to optimize the PeT process precisely by modulating the electrochemical properties of the fluorophore. The value of this approach is exemplified by its application to the development of a highly sensitive and pH-independent fluorescence probe for nitric oxide.     

A near IR di-styryl BODIPY-based ratiometric fluorescent chemosensor for Hg(II)

A novel BODIPY-based near-IR emitting probe as a selective and sensitive fluorophore for Hg(II) is synthesized. This versatile BODIPY fluorophore is functionalized for long wavelength emission at the 3 and 5 positions via a condensation reaction in which two dithiodioxomonoaza-based crown-containing phenyl units are conjugated to the BODIPY core as a chelating unit. This designed fluorophore, employing an ICT sensor can be used effectively to detect Hg(II) cations by way of a hypsochromic shift (∼90 nm) in both the absorption and emission spectra.     

A Galactosidase-Activatable Fluorescent Probe for Detection of Bacteria Based on BODIPY

Pathogenic E. coli infection is one of the most widespread foodborne diseases, so the development of sensitive, reliable and easy operating detection tests is a key issue for food safety. Identifying bacteria with a fluorescent medium is more sensitive and faster than using chromogenic media. This study designed and synthesized a β-galactosidase-activatable fluorescent probe BOD-Gal for the sensitive detection of E. coli. It employed a biocompatible and photostable 4,4-difluoro-3a,4a-diaza-s-indancene (BODIPY) as the fluorophore to form a β-O-glycosidic bond with galactose, allowing the BOD-Gal to show significant on-off fluorescent signals for in vitro and in vivo bacterial detection. This work shows the potential for the use of a BODIPY based enzyme substrate for pathogen detection.     

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli

A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.     

RNA analysis by MEKC with LIF detection

We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.     

Photoswitchable fluorescent dyads incorporating BODIPY and [1,3]oxazine components

We designed and synthesized three compounds incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton and differing in the nature of the linker bridging the two functional components. The [1,3]oxazine ring of the photochrome opens in less than 6 ns upon laser excitation in two of the three fluorophore-photochrome dyads. This process generates a 3H-indolium cation with a quantum yield of 0.02-0.05. The photogenerated isomer has a lifetime of 1-3 μs and reverts to the original species with first-order kinetics. Both photochromic systems tolerate hundreds of switching cycles with no sign of degradation. The visible excitation of the dyads is accompanied by the characteristic fluorescence of the BODIPY component. However, the cationic fragment of their photogenerated isomers can accept an electron or energy from the excited fluorophore. As a result, the photoinduced transformation of the photochromic component within each dyad results in the effective quenching of the BODIPY emission. Indeed, the fluorescence of these photoswitchable compounds can be modulated on a microsecond time scale with excellent fatigue resistance under optical control. Thus, our operating principles and choice of functional components can ultimately lead to the development of valuable photoswitchable fluorescent probes for the super-resolution imaging of biological samples.     

Synthesis and spectroscopic characterisation of BODIPY based fluorescent off-on indicators with low affinity for calcium

Two fluorescent off-on Ca2+ indicators based on APTRA (o-aminophenol-N,N,O-triacetic acid) as low-affinity ligand for Ca2+ and BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) as a fluorophore were synthesized. The new BODIPY-APTRA compounds absorb in the visible spectrum, with absorption maxima from 505 nm to 570 nm, and have fluorescence spectra that span the visible spectrum, with emission maxima ranging from 525 nm to 625 nm dependent on the substituents at the alpha-positions to the nitrogen atoms. The indicators show a large increase of the fluorescence quantum yield upon increasing Ca2+ concentration. The ground-state dissociation constants Kd estimated at 20 degrees C in 100 mM KCl aqueous buffered solution, pH 7.20, for the two complexes with Ca2+ were found to be around 100 microM.     

First synthesis of free cholesterol-BODIPY conjugates

Analogues of cholesterol (compounds 1 and 2) and coprostanol (compound 3) containing the BODIPY fluorophore in the aliphatic tail of the free sterol have been synthesized starting with bisnorcholenic acid, cholenic acid 3beta-acetate, and lithocholic acid, respectively. An ester linkage joining the fluorophore to the sterol nucleus interfered with the ability of the fluorescent sterol to pack with phospholipids in monolayers. However, an analogue in which the linker was devoid of polar atoms exhibited a substantially similar physical behavior to cholesterol in model membranes with respect to localization in raft domains.     

Spectrofluorimetric determination of glutathione in human plasma by solid-phase extraction using graphene as adsorbent

An efficient solid phase extraction-spectrofluorimetric method using graphene as adsorbent was developed to sensitively determine glutathione (GSH) in biological samples. Fluorescent probe N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY Fl-C1-IA) was applied for the derivatization of GSH. The procedure was based on BODIPY Fl-C1-IA selective reaction with GSH to form highly fluorescent product BODIPY Fl-C1-IA-GSH, its extraction to the graphene-packed SPE cartridge and spectrofluorimetric determination. Some factors affecting the extraction efficiency, such as the type of the eluent and its volume, sample pH, extraction time, and sample volume were optimized. Comparative studies were also performed between graphene and other adsorbents including C18 silica, graphitic carbon, and multi-walled carbon nanotubes for the extraction of analyte. The calibration graph using the pretreatment system for GSH was linear over the range of 0.5-200 nM. The limit of detection was 0.01 nM (signal-to-noise ratio=3). Relative standard deviation for six replicate determinations of GSH at 80 nM concentration level was lower than 5.0%. The developed method was applied to the determination of GSH in human plasma with recoveries of 92-108%. This work revealed the great potentials of graphene as an excellent sorbent material in the analysis of biological samples.     

New Ca2+ fluoroionophores based on the BODIPY fluorophore

Calcium (Ca(2+)) fluoroionophores are useful in cell-based functional assays of G-protein coupled receptor (GPCR) activation or ion channel modulation. In this paper we describe new calcium probes that improve or overcome certain deficiencies in existing probes. These new fluoroionophores are based on acylation of amino-BAPTA [BAPTA = glycine, N,N'-(1,2-ethanediyl-bis(oxy-2,1-phenylene)) bis(N-(carboxymethyl))] with fluorescent BODIPY(R) propionates [BODIPY = 4,4-difluoro-5,7-dimethyl- 4-bora-3a,4a-diaza-s-indacene]. The resulting probes show high affinity to aqueous calcium solutions, and respond to calcium binding with significant fluorescence increases. The BODIPY fluorophores are uncharged and their fluorescence is pH-insensitive. The wide range of excitation/emission wavelength choices available within the BODIPY fluorophore series allows several different colors of new calcium indicators to be prepared. Cell permeable versions respond well with increasing fluorescence intensities in live cells after calcium influx.     

Metal Complexes of a Boron‐Dipyrromethene (BODIPY)‐Tagged N‐Heterocyclic Carbene (NHC) as Luminescent Carbon Monoxide Chemodosimeters

Several metal complexes with a boron dipyrromethene (BODIPY)‐functionalized N‐heterocyclic carbene (NHC) ligand 4 were synthesized. The fluorescence in [( 4 )(SIMes)RuCl₂(ind)] complex is quenched (Φ=0.003), it is weak in [( 4 )PdI₂(Clpy)] (Φ=0.033), and strong in [( 4 )AuI] (Φ=0.70). The BODIPY‐tagged complexes can experience pronounced changes in the brightness of the fluorophore upon ligand‐exchange and ligand‐dissociation reactions. Complexes [( 4 )MX(1,5‐cyclooctadiene)] (M=Rh, Ir; X=Cl, I; Φ=0.008–0.016) are converted into strongly fluorescent complexes [( 4 )MX(CO)₂] (Φ=0.53–0.70) upon reaction with carbon monoxide. The unquenching of the Rh and Ir complexes appears to be a consequence of the decreased electron density at Rh or Ir in the carbonyl complexes. In contrast, the substitution of an iodo ligand in [( 4 )AuI] by an electron‐rich thiolate decreases the brightness of the BODIPY fluorophore, rendering the BODIPY as a highly sensitive probe for changes in the coordination sphere of the transition metal.