A bis-boron boramino acid PET tracer for brain tumor diagnosis.

PURPOSE: Boramino acids are a class of amino acid biomimics that replace the carboxylate group with trifluoroborate and can achieve the 18F-labeled positron emission tomography (PET) and boron neutron capture therapy (BNCT) with identical chemical structure. METHODS: This study reports a trifluoroborate-derived boronophenylalanine (BBPA), a derived boronophenylalanine (BPA) for BNCT, as a promising PET tracer for tumor imaging. RESULTS: Competition inhibition assays in cancer cells suggested the cell accumulation of [18F]BBPA is through large neutral amino acid transporter type-1 (LAT-1). Of note, [18F]BBPA is a pan-cancer probe that shows notable tumor uptake in B16-F10 tumor-bearing mice. In the patients with gliomas and metastatic brain tumors, [18F]BBPA-PET shows good tumor uptake and notable tumor-to-normal brain ratio (T/N ratio, 18.7 ± 5.5, n = 11), higher than common amino acid PET tracers. The [18F]BBPA-PET quantitative parameters exhibited no difference in diverse contrast-enhanced status (P = 0.115-0.687) suggesting the [18F]BBPA uptake was independent from MRI contrast-enhancement. CONCLUSION: This study outlines a clinical trial with [18F]BBPA to achieve higher tumor-specific accumulation for PET, provides a potential technique for brain tumor diagnosis, and might facilitate the BNCT of brain tumors.

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

Highly stable organic photothermal agent based on near-infrared-II fluorophores for tumor treatment.

BACKGROUND: The aim to develop a highly stable near-infrared (NIR) photoinduced tumor therapy agent stems from its considerable potential for biological application. Due to its long wavelength, biological imaging exhibits a high signal-to-background ratio, deep tissue penetration and maximum permissible light power, which can minimize damage to an organism during photoinduced tumor therapy. RESULTS: A class of stable NIR-II fluorophores (NIR998, NIR1028, NIR980, NIR1030, and NIR1028-S) based on aza-boron-dipyrromethene (aza-BODIPY) dyes with donor-acceptor-donor structures have been rationally designed and synthesized by harnessing the steric relaxation effect and intramolecular photoinduced electron transfer (IPET). These fluorophores exhibit an intense range of NIR-II emission, large Stokes shift (≥ 100 nm), excellent photothermal conversion performance, and superior stability against photobleaching. Among the NIR-II fluorophores, NIR998 possesses better NIR-II emission and photothermal conversion performance. NIR998 nanoparticles (NIR998 NPs) can be encapsulated by liposomes. NIR998 NPs show superior stability in the presence of light, heat, and reactive oxygen nitrogen species than that of indocyanine green NPs, as well as a higher photothermal conversion ability (η = 50.5%) compared to other photothermal agents. Finally, under the guidance of photothermal imaging, NIR998 NPs have been proven to effectively eliminate tumors via their excellent photothermal conversion performance while presenting negligible cytotoxicity. CONCLUSIONS: Utilizing IPET and the steric relaxation effect can effectively induce NIR-II emission of aza-BODIPY dyes. Stable NIR998 NPs have excellent photothermal conversion performance and negligible dark cytotoxicity, so they have the potential to act as photothermal agents in biological applications.

Oxidative degradation profile studies of tavaborole by a validated stability indicating RP-UPLC method: Isolation and characterization of novel degradant using 2D-NMR and HRMS.

The current research work reports a study on the degradation profile of tavaborole, which is an oxaborole antifungal drug used to treat infections in the toenails. This work also reports the chemical stability of tavaborole in different stress conditions along with the isolation and characterization of degradation products by high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance techniques. A sensitive and reproducible stability-indicating ultra-performance liquid chromatography method was developed and validated for quantification of tavaborole bulk drug in the presence of degradation products. Significant degradation was observed during oxidative stress conditions using H2 O2 . It was observed that the drug was highly unstable under oxidation stress conditions and thus degradation profiles with various oxidizing reagents were studied. One unknown impurity (DP-1) was formed during peroxide degradation, which was isolated by reverse-phase preparative chromatography. The structure of this degradant was characterized by high-resolution mass spectrometry and multidimensional nuclear magnetic resonance techniques. The structure of this novel impurity DP-1 was identified as [4-fluoro-2-(hydroxymethyl)phenol], which was not reported as a degradant in the literature. An Acquity BEH C18 , 100 × 2.1 mm, 1.7 µm column was used to achieve the desired separation within a shorter runtime of 4.0 min. The method was validated for specificity, precision, linearity and accuracy over the concentration range of 5.0-400 µg ml-1 (r2 -0.9999) and limit of quantitation 5.0 µg ml-1 . This method is compatible with LCMS analysis which enables to identify the unknown impurities formed in the process.

Development and validation of a simple method for the extraction and quantification of crisaborole in skin layers.

Crisaborole is a boron compound recently approved by the US Food and Drug Administration as a 2% ointment for the treatment of mild to moderate atopic dermatitis. This work describes a simple method for the quantification of the drug in the skin layers at the end of in-vitro permeation experiments. Chromatographic separation was carried out on a reverse-phase C18 column using a mixture of trifluoroacetic acid 0.05%-acetonitrile (55:45, v/v) as mobile phase, pumped at 1 ml/min. Column temperature was 35°C and UV detection was performed at 250 nm. The method was linear in the range of concentration from 0.06 to 6 µg/ml (R2 = 1) and was selective, precise and accurate. Depending on the solvent used, the LOQ ranged from 0.014 to 0.030 µg/ml and the LOD from 0.005 to 0.010 µg/ml. The extraction from all the skin layers was quantitative. The developed method was successfully tested in an in-vitro permeation study, proving to be an effective tool in the development of new formulations containing crisaborole.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells.

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.

Faradaic Ion Concentration Polarization on a Paper Fluidic Platform.

We describe the design and characteristics of a paper-based analytical device for analyte concentration enrichment. The device, called a hybrid paper-based analytical device (hyPAD), uses faradaic electrochemistry to create an ion depletion zone (IDZ), and hence a local electric field, within a nitrocellulose flow channel. Charged analytes are concentrated near the IDZ when their electrophoretic and electroosmotic velocities balance. This process is called faradaic ion concentration polarization. The hyPAD is simple to construct and uses only low-cost materials. The hyPAD can be tuned for optimal performance by adjusting the applied voltage or changing the electrode design. Moreover, the throughput of hyPAD is 2 orders of magnitude higher than that of conventional, micron-scale microfluidic devices. The hyPAD is able to concentrate a range of analytes, including small molecules, DNA, proteins, and nanoparticles, in the range of 200-500-fold within 5 min.

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single-Molecule Detection.

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 (<30%), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40-90%). All compounds are characterized by steady-state and time-resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.

A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities.

Fluorophore-tagged pharmacophores for antitumor cytotoxicity: Modified chiral lipidic dialkynylcarbinols for cell imaging.

Chiral lipidic dialkynylcarbinols (DACs), recently highlighted as antitumoral pharmacophores, have been conjugated to difluoroboron-dipyrromethene (bodipy), 7-hydroxy-coumarine, and 7-nitro-benzoxadiazole (NBD) fluorophore motifs through triazole clips. The labeled lipids preserve cytotoxic activity against HCT116 cells, and fluorescence microscopy of the stained cells showed clear signals in the intra-cellular membrane system. While the bodipy conjugate also labels lipid droplets very brightly, as expected, the coumarine and NBD probes appear as promising specific tools for the identification of the intra-cellular targets of DACs' cytotoxicity.

Detection of boronic acid derivatives in cells using a fluorescent sensor.

The detection of boron-containing compounds requires very expensive facilities and/or tedious sample pretreatments. In an effort to develop a convenient detection method for boronic acid derivatives, boron chelating-ligands were synthesized for use as fluorescent sensors. In this paper, the synthesis and properties of fluorescent sensors for boronic acid derivatives are reported.

Synthesis of cholesterol analogues bearing BODIPY fluorophores by Suzuki or Liebeskind-Srogl cross-coupling and evaluation of their potential for visualization of cholesterol pools.

We report a synthetic route to BODIPY-cholesterol conjugates in which the key steps were Suzuki or Liebeskind-Srogl cross-coupling of cholesterol phenyl moieties with structurally diverse BODIPY scaffolds. All conjugates feature single-bonded and hydrophobic linkages between the fluorophore and sterol that are devoid of heteroatoms. Using HeLa cells, we show that these BODIPY-cholesterol analogues can be used simultaneously with the parent BODIPY-cholesterol for cell imaging and flow cytometry. The BODIPY-cholesterol analogues exhibit similar cellular localization in HeLa cells and show similar cholesterol efflux properties from THP-1 cells to HDL acceptors. These results demonstrate that the red-shifted BODIPY-cholesterol analogues behave in a manner similar to unlabeled cholesterol and are useful probes for simultaneous visualization of intracellular cholesterol pools and for monitoring cholesterol efflux from cells to extracellular acceptors.

Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

Flow-injection MS/MS for gas-phase chiral recognition and enantiomeric quantitation of a novel boron-containing antibiotic (GSK2251052A) by the mass spectrometric kinetic method.

The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flow-injection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the Cu(II)-bound trimeric complex [Cu(II)(A)(ref*)2-H](+) (A = GSK2251052A or its R-enantiomer, ref* = L-tryptophan) that gives rise to Cu(II)-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC-MS/MS and LC-UV methods as well as the method using chiral derivatization followed by LC-MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development.

Fluorescent ligand for human progesterone receptor imaging in live cells.

We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

Fast mass spectrometry detection of tryptophan-containing peptides and proteins by reduction with pyridine-borane.

In this note, we describe a method devised to detect, by means of mass spectrometry (MS), tryptophan-containing peptides and proteins using pyridine-borane. This reagent selectively reduces tryptophan residues, converting them to 2,3-dihydro-tryptophan, thereby enabling quantitation of tryptophans.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Life cycle assessment of a novel tannin-boron association for wood protection.

In these studies of fix boron compounds, associations between tannins and boron (TB) in the form of boric acid appear to be of interest. These TB associations allow the use of boron at very low levels (in compliance with EU restrictions, 2008/58/EC) and limit boron leaching which maintains biological resistance and fire retardant properties. As a consequence, TB wooden products present an extended service life compared to boron compounds alone and were designed to be environmentally-friendly wood protection systems. A follow-up of tannin­boron use identified the environmental impacts using a life cycle assessment (LCA). This LCA was performed on tannin­boron preservative products as well as several industrial preservative-treated timbers and concrete used in the landscape. Cr-containing inorganic salt and an alkaline copper quaternary preservative formulation, as well as concrete, have been used as referential materials to compare the environmental footprint with the tannin­boron treated system. A model was created with life cycle stages used to calculate inputs and outputs during raw material extraction, supplier transportation, manufacturing process, distribution, disposal transportation and processing. Tannin production data were based on Vieira et al. in the field of condensed tannin extraction. However, the extracted tannin in the extraction yield, the inorganic salt, and the process applied are not perfectly comparable with the extraction conditions industrially applied for the Mimosa (Acacia mearnsii) extract which is the major constituent of the TB formulations. The latter is counter-current water extracted without any chemicals or with a limited amount of NaHSO3 or Na2SO3 (at 0.5 % to 1 %) - at a temperature of 70-90 °C. Unfortunately these parameters cannot be elaborated by the LCA program because there is no data available for the production of Na bisulphite or Na bicarbonate in the LCI data used. Other input data were sourced from the ecoinvent v3.8 database. The ReCiPe midpoint method was used to assess the environmental footprint and the CED method was chosen to analyze a general view of the energy-related environmental impacts in the life cycle. Overall, the results demonstrated that tannin­boron preservatives can be regarded as a low-environmental impact formulation. Additionally, an economic analysis of the development of a commercially-viable tannin­boron preservative would now be timely.

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.