Synthesis and utility of fluorogenic acetoxymethyl ethers

Lavis, Luke D.; Chao, Tzu‐Yuan; Raines, Ronald T.

doi:10.1039/c0sc00466a

Cited by 87 publications

(126 citation statements)

References 55 publications

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“…We determined the catalytic constants of PLE unmasking of fluorescein-CM 2 (6) and compared them to the unmasking of fluorescein-AM 2 (1) as shown in Fig. S3 B and C. Masked fluorophore 1 was cleaved by PLE with apparent catalytic constants k cat ∕K M ¼ 5.8 AE 0.9 × 10 5 M −1 s −1 (mean AE SE) and K M ¼ 2.7 AE 0.4 μM, in agreement with previously published values (17). The CM derivative 6 was cleaved more slowly, exhibiting apparent catalytic constants of…”

Section: Resultssupporting

confidence: 76%

“…We limited this series to contain small, commercially available, achiral esters with different α-carbon substitution patterns encompassing a range of steric and stereoelectronic properties. Compounds 1-6 show low background fluorescence in the masked form, and hydrolysis of the ester bonds ultimately releases the bright green fluorophore, fluorescein (17). In addition to high contrast, chemical stability is important for accurate determination of cellular esterase activity.…”

Section: Resultsmentioning

confidence: 99%

“…We then tested the generality of this system by synthesizing another masked compound based on the red fluorophore 4-carboxyresorufin (17). We applied the acyloxymethyl masking system to both the phenol and carboxylic acid moieties on this dye (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To find an ester motif resistant to cleavage by endogenous esterases, we synthesized a series of fluorogenic esterase substrates based on the chemically stable fluorescein di(acetoxymethyl ether) (fluorescein-AM 2 , 1, Fig. 1A) (17). Compounds 2-6 were prepared by the efficient reaction of iodomethyl esters with fluorescein in the presence of Ag 2 O (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Selective esterase–ester pair for targeting small molecules with cellular specificity

Tian

Yang

Wysocki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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212

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Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations. cellular imaging | microscopy | enzyme substrates | fluorophores | pharmacological agents C hemical probes are essential tools in biology for measuring and manipulating cellular properties. Optimization of the structural and electronic features of small molecules allows the fine-tuning of molecular recognition specificity for a particular cellular target. Even with high molecular specificity, however, the application of small molecules in complex biological environments is frequently limited by poor cellular specificity. The inability to target small molecules, such as imaging or pharmacological agents, to defined cellular populations can confound the evaluation and control of discrete subsets of cells within a multicellular environment. A general and efficient strategy for cell-specific targeting, combining the molecular specificity of small molecules with the cellular specificity of genetics, would allow intracellular pathways in defined cell types to be selectively probed in complex tissues.An attractive approach for general cell-specific delivery of small molecules employs selective enzyme-substrate pairs. In this strategy, compounds are masked by attachment of a standard, disposable blocking group that is stable to native cellular enzymes, but labile to a specific exogenous enzyme. Expression of such a protein in a genetically defined cell population permits unmasking of the small molecule with cellular specificity. To be useful across experimental paradigms, such a system should utilize an enzyme that unmasks molecules with high efficiency, expresses in different cell types, and exhibits low cellular toxicity. The cognate masking group must be modular, synthetically efficient, and allow molecules to diffuse passively across the cellular membrane, while also exhibiting favorable solubility and stability in aqueous solution.To date only a few enzyme-substrate pairs have been used as targeted delivery systems for small molecules, and none meet all the criteria outlined above. Strategies employing enzymes encoded by common reporter genes (1) have found some success in targeting small molecules (2-4), ...

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Selective esterase–ester pair for targeting small molecules with cellular specificity

Tian

Yang

Wysocki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

163

212

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“…First it was necessary to mask the acidic groups on resorufin as an acetoxymethyl (AM) ester and ether to give resorufin-AM 2 . This probe readily crossed cell membranes; endogenous esterases then cleave the masking groups to regenerate resorufin (27). We observed that resorufin-AM 2 has two regioisomers, cis and trans (SI Appendix, Fig.…”

Section: Structure-based Mutagenesis and Screening For Resorufin Ligasementioning

confidence: 97%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

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Magnetogenese in Wasser, ausgelöst durch einen chemischen Analyten

2013

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International audienceDieser Kurzaufsatz befasst sich mit einem neuen Forschungsfeld: dem Auslösen der Änderung der magnetischen Eigenschaften einer flüssigen Probe bei Zugabe eines chemischen Analyten. Die Kenntnisse über Materialproben, die ihr magnetisches Verhalten bei Modulation physikalischer Umweltparameter ändern, sind umfassend; man kennt sogar einige Beispiele, bei denen dies für flüssige Proben zutrifft. Dagegen sind Fälle von gelösten Molekülen oder Partikeln, die auf diese Weise bei konstanten Bedingungen auf einen chemischen Analyten reagieren, begrenzt. Auch wenn unsere Diskussion Beispiele in organischen Lösungsmitteln einschließt, so liegt doch der Schwerpunkt des Kurzaufsatzes auf Wasser. Dieser soll dem Leser ein Leitfaden zum Entwurf neuer magnetogener Sonden für den Nachweis des chemischen Analyten seiner Wahl sein

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Synthesis and utility of fluorogenic acetoxymethyl ethers

Cited by 87 publications

References 55 publications

Selective esterase–ester pair for targeting small molecules with cellular specificity

Selective esterase–ester pair for targeting small molecules with cellular specificity

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Magnetogenese in Wasser, ausgelöst durch einen chemischen Analyten

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