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116 Publications

Showing 101-110 of 116 results
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    12/01/11 | Advances in the chemistry of small molecule fluorescent probes.
    Wysocki LM, Lavis LD
    Current Opinion in Chemical Biology. 2011 Dec;15(6):752-9. doi: 10.1016/j.cbpa.2011.10.013

    Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.

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    11/18/11 | Facile and general synthesis of photoactivatable xanthene dyes.
    Wysocki LM, Grimm JB, Tkachuk AN, Brown TA, Betzig E, Lavis LD
    Angewandte Chemie. 2011 Nov 18;50:11206-9. doi: 10.1002/anie.201104571

    Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, “leuco” intermediates.

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    02/01/11 | Histochemistry: live and in color.
    Lavis LD
    The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 2011 Feb;59:139-45. doi: 10.1369/0022155410395760

    Histochemistry (chemistry in the context of biological tissue) is an invaluable set of techniques used to visualize biological structures. This field lies at the interface of organic chemistry, biochemistry, and biology. Integration of these disciplines over the past century has permitted the imaging of cells and tissues using microscopy. Today, by exploiting the unique chemical environments within cells, heterologous expression techniques, and enzymatic activity, histochemical methods can be used to visualize structures in living matter. This review focuses on the labeling techniques and organic fluorophores used in live cells.

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    01/01/11 | Synthesis and utility of fluorogenic acetoxymethyl ethers.
    Lavis LD, Chao T, Raines RT
    Chemical Science. 2011 Jan 1;2(3):521-30. doi: 10.1039/C0SC00466A

    Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

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    12/21/10 | Cellular uptake of ribonuclease A relies on anionic glycans.
    Chao T, Lavis LD, Raines RT
    Biochemistry. 2010 Dec 21;49(50):10666-73. doi: 10.1021/bi1013485

    Bovine pancreatic ribonuclease (RNase A) can enter human cells, even though it lacks a cognate cell-surface receptor protein. Here, we report on the biochemical basis for its cellular uptake. Analyses in vitro and in cellulo revealed that RNase A interacts tightly with abundant cell-surface proteoglycans containing glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, as well as with sialic acid-containing glycoproteins. The uptake of RNase A correlates with cell anionicity, as quantified by measuring electrophoretic mobility. The cellular binding and uptake of RNase A contrast with those of Onconase, an amphibian homologue that does not interact tightly with anionic cell-surface glycans. As anionic glycans are especially abundant on human tumor cells, our data predicate utility for mammalian ribonucleases as cancer chemotherapeutic agents.

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    10/07/09 | Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells.
    Watkins RW, Lavis LD, Kung VM, Los GV, Raines RT
    Organic & Biomolecular Chemistry. 2009 Oct 7;7(19):3969-75. doi: 10.1039/b907664f

    Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme-substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse-chase experiments, high-content screening, and numerous other protocols.

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    07/01/09 | Onconase cytotoxicity relies on the distribution of its positive charge.
    Turcotte RF, Lavis LD, Raines RT
    The FEBS Journal. 2009 Jul;276(14):3846-57. doi: 10.1111/j.1742-4658.2009.07098.x

    Onconase (ONC) is a member of the ribonuclease A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is now in Phase IIIb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity, despite the apparent absence of a cell-surface receptor protein. Endocytosis and cytotoxicity do, however, appear to correlate with the net positive charge of ribonucleases. To dissect the contribution made by the endogenous arginine and lysine residues of ONC to its cytotoxicity, 22 variants were created in which cationic residues were replaced with alanine. Variants with the same net charge (+2 to +5) as well as equivalent catalytic activity and conformational stability were found to exhibit large (> 10-fold) differences in toxicity for the cells of a human leukemia line. In addition, a more cationic ONC variant could be either much more or much less cytotoxic than a less cationic variant, again depending on the distribution of its cationic residues. The endocytosis of variants with widely divergent cytotoxic activity was quantified by flow cytometry using a small-molecule fluorogenic label, and was found to vary by twofold or less. This small difference in endocytosis did not account for the large difference in cytotoxicity, implicating the distribution of cationic residues as being critical for lipid-bilayer translocation subsequent to endocytosis. This finding has fundamental implications for understanding the interaction of ribonucleases and other proteins with mammalian cells.

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    11/15/08 | A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells.
    Yatzeck MM, Lavis LD, Chao T, Chandran SS, Raines RT
    Bioorganic & Medicinal Chemistry Letters. 2008 Nov 15;18(22):5864-6. doi: 10.1016/j.bmcl.2008.06.015

    A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a ’trimethyl lock’ that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k(cat)/K(M)=8.8x10(3)M(-1)s(-1) and K(M)=0.09microM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.

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    04/18/08 | Ester bonds in prodrugs.
    Lavis LD
    ACS Chemical Biology. 2008 Apr 18;3(4):203-6. doi: 10.1021/cb800065s

    A recent study challenges the oft-held notion that ester bonds in prodrug molecules are cleaved rapidly and completely inside cells by endogenous, nonspecific esterases. Structure-activity relationship studies on acylated sugars reveal that regioisomeric compounds display disparate biological activity, suggesting that ester bonds can persist in a cellular context.

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    03/20/08 | Bright ideas for chemical biology.
    Lavis LD, Raines RT
    ACS Chemical Biology. 2008 Mar 20;3:142-55. doi: 10.1021/cb700248m

    Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular "core" dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

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