@article {4185, title = {Synthesis and utility of fluorogenic acetoxymethyl ethers.}, journal = {Chemical Science}, volume = {2}, year = {2011}, month = {2011 Jan 1}, pages = {521-30}, abstract = {

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.

}, issn = {2041-6539}, doi = {10.1039/C0SC00466A}, author = {Lavis, Luke D and Chao, Tzu-Yuan and Raines, Ronald T} } @article {1094, title = {Cellular uptake of ribonuclease A relies on anionic glycans.}, journal = {Biochemistry}, volume = {49}, year = {2010}, month = {2010 Dec 21}, pages = {10666-73}, abstract = {

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.

}, keywords = {Affinity, Animals, Anions, Cattle, Cell Line, Cell Proliferation, CHO Cells, Chondroitin Sulfates, Chromatography, Cricetinae, Cricetulus, Flow Cytometry, Fluorescence, Glycoproteins, Glycosaminoglycans, Heparitin Sulfate, Humans, Mass, Matrix-Assisted Laser Desorption-Ionization, Microscopy, Molecular Weight, N-Acetylneuraminic Acid, Pancreatic, Polysaccharides, Protein Binding, Ribonuclease, Spectrometry}, issn = {1520-4995}, doi = {10.1021/bi1013485}, author = {Chao, Tzu-Yuan and Lavis, Luke D and Raines, Ronald T} } @article {1098, title = {A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells.}, journal = {Bioorganic \& Medicinal Chemistry Letters}, volume = {18}, year = {2008}, month = {2008 Nov 15}, pages = {5864-6}, abstract = {

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 {\textquoteright}trimethyl lock{\textquoteright} 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.

}, keywords = {Cytochrome P-450 CYP1A1, Cytochrome P-450 Enzyme System, Fluorescent Dyes, Humans, Isoenzymes, Molecular Structure, Prodrugs, Rhodamines, Stilbenes, Structure-Activity Relationship, Tetrachlorodibenzodioxin}, issn = {1464-3405}, doi = {10.1016/j.bmcl.2008.06.015}, author = {Yatzeck, Melissa M and Lavis, Luke D and Chao, Tzu-Yuan and Chandran, Sunil S and Raines, Ronald T} } @article {1103, title = {Cytotoxic ribonucleases: the dichotomy of Coulombic forces.}, journal = {Biochemistry}, volume = {46}, year = {2007}, month = {2007 Sep 11}, pages = {10308-16}, abstract = {

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.

}, keywords = {Biomechanics, Cell Death, Endocytosis, Fluorescent Dyes, Humans, K562 Cells, Kinetics, Mutant Proteins, Pancreatic, Protein Structure, Ribonuclease, Secondary}, issn = {0006-2960}, doi = {10.1021/bi700857u}, author = {Johnson, R Jeremy and Chao, Tzu-Yuan and Lavis, Luke D and Raines, Ronald T} } @article {1105, title = {Fluorogenic label for biomolecular imaging.}, journal = {ACS Chemical Biology}, volume = {1}, year = {2006}, month = {2006 May 23}, pages = {252-60}, abstract = {

Traditional small-molecule fluorophores are always fluorescent. This attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single user-designated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

}, keywords = {Fluorescence, Fluorescent Dyes, Hela Cells, Humans, Microscopy, Molecular Probes, Spectrometry}, issn = {1554-8937}, doi = {10.1021/cb600132m}, author = {Lavis, Luke D and Chao, Tzu-Yuan and Raines, Ronald T} } @article {1106, title = {Latent blue and red fluorophores based on the trimethyl lock.}, journal = {Chembiochem: A European Journal of Chemical Biology}, volume = {7}, year = {2006}, month = {2006 Aug}, pages = {1151-4}, keywords = {Animals, Cell Line, Color, Fluorescence, Fluorescent Dyes, Humans, Kinetics, Methylation, Molecular Structure, Tumor, Umbelliferones}, issn = {1439-4227}, doi = {10.1002/cbic.200500559}, author = {Lavis, Luke D and Chao, Tzu-Yuan and Raines, Ronald T} }