Studies on taxol biosynthesis. Preparation of 5α-acetoxytaxa-4(20),11-dien-2α,10β-diol derivatives by deoxygenation of a taxadiene tetra-acetate obtained from Japanese yew

The putative metabolite, 5α-acetoxytaxa-4(20),11-dien-2α,10β-diol (7), which is a promising candidate as a biosynthetic pathway triol in taxol biosynthesis, has been prepared by Barton deoxygenation of the C-14-hydroxyl group of a differentially protected derivative of natural 2α,5α,10β-triacetoxy-14β-(2-methyl)-butyryloxytaxa-4(20),11-diene (8), a major taxoid metabolite isolated from Japanese Yew heart wood. The synthetic protocol devised, is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.     

(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers,blocks taxol binding and results in mitotic arrest

Background: The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail.Results: We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolide's cellular target.Condusions: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.     

Studies on taxol biosynthesis. Preparation of taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol by deoxygenation of a taxadiene tetraacetate obtained from Japanese yew

Taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol 6 has been identified as an early stage intermediate involved in the biosynthesis of taxol (Paclitaxel). This compound has been efficiently prepared by Barton deoxygenation of the C-2- and C-14-hydroxyl groups on a derivative semisynthetically prepared from taxa-4(20),11(12)-dien-2 alpha,5 alpha,10 beta-triacetoxy-14 beta-(2-methyl)butyrate (7), a major taxoid metabolite isolated from Japanese yew heart wood. The synthetic methodology is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.     

The aldol–annulation–fragmentation strategy toward the taxoid diterpene framework revisited. Instructive failures in the chemistry of medium ring containing systems

Central intermediate 5 for the taxoid diterpene framework, prepared by the aldol–annulation sequence, permitted the construction of A-secotaxane frameworks incorporating differentiatable olefin and oxygen functionalities suitable for further elaboration. The key BC-subunits 9 and 8 have proven amenable to efficient conversion into both oxa-bridged 7 and its central eight-membered B-ring analogue 6, providing two potential precursors for taxoid construction. Although their further elaboration into 4 was not progressed at this stage, 6 and 7 are potentially useful synthetic intermediates. Extensive structural studies that included 800 MHz ¹H (200 MHz ¹³C) NMR as well as X-ray crystallographic analyses of 7, 17, and 20 have contributed to the unambiguous elucidation of all the complex structures synthesized.     

A convenient preparation of taxoid right-half building blocks

Wieland–Miescher ketone derived unsaturated diols 4 reacted with Pb(OAc)₄ to furnish tricyclic enolether intermediate 6 which upon ozonolysis gave access to useful synthetic intermediates such as bicyclic lactone 7, methyl furanoside 8 and triol 9 (taxoid right-half precursors), depending on the solvent used during the ozonolysis and the nature of the following synthetic operation.     

Design and Synthesis of New Acridone-Based Nitric Oxide Fluorescent Probe

Nitric oxide (NO) is an important signaling molecule involved in a wide range of physiological and pathological processes. Fluorescent imaging is a useful tool for monitoring NO concentration, which could be essential in various biological and biochemical studies. Here, we report the design of a novel small-molecule fluorescent probe based on 9(10H)acridone moiety for nitric oxide sensing. 7,8-Diamino-4-carboxy-10-methyl-9(10H)acridone reacts with NO in aqueous media in the presence of O₂, yielding a corresponding triazole derivative with fivefold increased fluorescence intensity. The probe was shown to be capable of nitric oxide sensing in living Jurkat cells.     

Design and synthesis of an AIE-active fluorogen with red emission and its biological application

A novel triphenylamine-base derivative L containing pyridine and terpyridine was designed and synthesized. Compound L exhibited distinct aggregation-induced emission (AIE) behavior in water–ethanol and also displayed a threefold increase in the intensity of luminescence at 608 nm. Furthermore, confocal microscopy imaging demonstrated that compound L displays low toxicity and brights red fluorescence in mitochondria in living HepG2 cells. Inherent from the mitochondrial-targeting ability of pyridine moiety and the AIE characteristic of triphenylamine group, compound L could be employed as a fluorescent probe in the near-infrared region for living cell imaging.     

A colorimetric and fluorescent turn-on chemosensor for Al and its application in bioimaging

The sensing properties of a boron dipyrromethene derivative 1 containing a N,N-(dimethylamino)styryl group at its α-position and an aniline moiety at meso-position were investigated by steady-state UV-vis absorption and fluorescence spectroscopy, which were found to exhibit wavelength ratiometric and large fluorescence enhancement in the presence of Al³⁺ with specific selectivity over other metal ions in aqueous media. Furthermore, confocal fluorescence microscopy experiments demonstrated that 1 could be used as a fluorescent probe for Al³⁺ in living cells.     

Studies towards the total synthesis of taxoids: a rapid entry into bicyclo[6.4.0]dodecane ring system. Part 2

We report a synthetic route that allows access into A-seco taxoid frameworks embodying the entire carbon skeleton and a large number of oxygen functionalities. The BC-subunit (−)-4 was constructed in five steps from (+)-3, through a step-efficient and stereocontrolled bond construction.     

Single-molecule spectroscopy of fluorescent proteins

The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the single-molecule level, which removes averaging inherent to ensemble studies. In this paper we review how single-molecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.

A naked-eye rhodamine-based fluorescent probe for Fe(III) and its application in living cells

A naked-eye turn-on fluorescent Fe³⁺ probe (RQ6) was developed by linking a new conjugated quinoline fluorescent group to the rhodamine platform. The probe can detect Fe³⁺ with high selectivity over other metal ions. Bioimaging studies indicated that RQ6 was cell permeable and suitable for detecting Fe³⁺ in the living cells by confocal microscopy.     

A highly selective turn-on fluorescent probe for hypochlorous acid based on hypochlorous acid-induced oxidative intramolecular cyclization of boron dipyrromethene-hydrazone

A BODIPY-based fluorescent probe, HBP, was developed for the detection of hypochlorous acid based on the specific hypochlorous acid-promoted oxidative intramolecular cyclization of heterocyclic hydrazone in response to the amount of HOCl. The reaction is accompanied by a 41-fold increase in the fluorescent quantum yield (from 0.004 to 0.164). The fluorescence intensity of the reaction between HOCl and HBP is linear in the HOCl concentration range of 1–8 μM with a detection limit of 2.4 nM (S/N = 3). Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HBP could be used as an effective fluorescent probe for detecting HOCl in living cells.     

Facile preparation of water dispersible red fluorescent organic nanoparticles and their cell imaging applications

Red fluorescent organic nanoparticles (FONs) based on a cyano-substituted diarylethylene and tetraphenylethene derivative conjugated molecule (R-TPE) were facilely prepared via surfactant modification with lecithin for the first time. The obtained R-TPE-LEC FONs were characterized by a series of techniques including fluorescence and UV spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of R-TPE-LEC FONs were further investigated to explore their potential biomedical application. We demonstrated that such red FONs exhibit anti-aggregation-caused quenching property, broad excitation wavelength, high water dispersibility, uniform morphology (40–60 nm), and excellent biocompatibility, making them promising for cell imaging application.     

Studies on Taxol biosynthesis: preparation of taxadiene-diol and triol derivatives by deoxygenation of taxusin

The putative Taxol biosynthesis metabolites, taxa-4(20),11(12)-diene-5α,13α-diol (7), taxa-4(20),11(12)-diene-5α,9α,13α-triol (9), and taxa-4(20),11(12)-diene-5α,10β,13α-triol (10), have been prepared by Barton deoxygenation of the C-9 and C10-hydroxyl groups of protected derivatives of taxusin, a major taxoid metabolite isolated from Yew heart wood. The synthetic protocol devised is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of Taxol.     

Synthesis of environmentally sensitive 2′-deoxyguanosine containing solvatochromic pyrene fluorophore

The synthesis and photophysical property of novel solvatochromic pyrene derivative, Apa5 and fluorescent guanine ^ApaG8 were described. These newly synthesized fluorescent pyrene derivatives exhibited solvent polarity dependent fluorescence at longer wavelengths. Such environmentally sensitive pyrene derivatives can be used as a reporter probe that is sensitive to the changes in the microenvironment around DNA either in vitro or in vivo.     

Matched and mismatched pairings in B-secotaxane construction: a structure elucidation study

The synthesis of the basic B-seco taxoid skeleton was achieved through a C10–C11 coupling of the A-ring segment (14S)-1 with the C-ring segments (10S) and (10R)-α-alkoxyorganolithium reagents, prepared in situ from 2 and 6 through nBuLi mediated transmetallation. The matched reactions of (14S)-1 with (10S)-2 and (10S)-6 displays an outstanding diastereoselectivity providing 12 and 34, respectively, as single isomers and hence allowing a convenient entry to highly functionalized taxoid diterpene frameworks. Significant mismatching was observed with the (10R)-epimer of 2 and 6 yielding little, if any, diastereoselectivity. The structures of B-secotaxanes were assigned on the basis of spatial proximity effects in the proton NMR spectrum. Assignment of the C10/C11 stereochemistry was made possible through conversion of the B-secotaxoid frameworks, derived from the matched and mismatched adducts, to the corresponding cyclic acetals. Configurational stability of α-alkoxyorganolithium derivatives was verified in all the cases investigated. Structure elucidation of these adducts was essential for the successful C1–C2 bonding via an intramolecular aldol reaction, given the fact that adducts containing a β-MOM substituent at C10 would be disfavored for such an endeavor.     

Facile fabrication of aggregation-induced emission based red fluorescent organic nanoparticles for cell imaging

A cyano-substituted diarylethlene derivative aggregation-induced emission （ALE） dye with two amino end-groups and 4,4＇-（hexafluoroisopropylidene）diphthalic anhydride were facilely incorporated into red fluorescent organic nanoparticles （FONs） via room temperature anhydride ring-opening polymerization under an air atmosphere. These obtained RO-HFDA FONs were characterized by a series of techniques including gel permeation chromatography, Fourier transform infrared spectroscopy, size distribution and zeta potential measurements, UV-Vis absorption spectrum, fluorescent spectroscopy and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of RO-HFDA FONs were further investigated to explore their potential biomedical application. We demonstrated that such FONs showed high water dispersibility, stable uniform spherical morphology （150-200 nm）, broad excitation band （350-605 nm）, intense red fluorescence （627 nm） and excellent biocompatibility, making them promising for cell imaging applications.     

Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (～1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.     

Phenoxy-benzothiadiazole dyes: Synthesis,photophysical properties and preliminary application in OLEDs

The synthesis of a phenoxy-benzothiadiazole (BTD) derivative is reported for the first time. This derivative was employed as an intermediate in the obtention of two novel highly fluorescent aryl-phenoxy-BTD dyes. Their photophysical properties were evaluated in solution and the results indicate a strong intramolecular charge transfer (ICT) character in the excited state. The luminophore 3a, possessing a donor-BTD-phenoxide architecture, exhibits superior fluorescence quantum yield (0.67) than the designed acceptor-BTD-phenoxide dye (0.06). After electrochemical and photoluminescence characterization of thin films of 3a, an OLED with the configuration ITO/PEDOT:PSS/3a/TPBi/LiF/Al was constructed. This device displayed a green emission centered at 547?nm, with CIE coordinates of (0.40, 0.55). For comparison, an OLED using a previously reported luminescent BTD-pyridyl derivative was also constructed. The OLED made with the phenoxy-BTD derivative operated using less current density and led to higher irradiance and electrical stability, indicating the high potential of ArO-BTD dyes for future application in electroluminescent devices.     

血清白蛋白对紫杉醇的化学修饰

采用两种方法由牛血清白蛋白和人血清白蛋白修饰紫杉醇,提高了水溶性.红外和紫外数据证实了修饰结果.