This communication describes a novel protein labeling method that uses a single amino-acid tag -N-terminal cysteine residue -and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods. Graphical abstract KeywordsProtein Labeling; Condensation; Terminal Cysteine; Chemical ligation; Live-cell Imaging Site-specific labeling of proteins with molecular tags enables direct visualization of protein dynamics, localization and interactions in single living cells and is a powerful tool for studying structure and function of proteins. [1] Proteins of interest can be labeled by genetic fusions to fluorescent proteins, or chemical reactions with fluorescent dyes. Chemical labeling often employs a receptor protein, for example, a mutant of human O 6 -alkylguanine-* Fax: (+1) 650-736-7925, ; Email: jrao@stanford.edu, Homepage: http://raolab.stanford.edu Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript DNA transferase, [2a] and E. coli dihydrofolate reductase, [2b] that binds to or reacts with its fluorescently tagged ligand. [2] Alternatively, smaller tags such as short peptides can be labeled by selective binding to fluorogenic dyes [3] or by enzyme-catalyzed ligation to fluorescent probes. [4] Water-compatible chemical reactions can also be applied to protein labeling, such as the Staudinger reaction between the azides and triphenylphosphines, [5] the Huisgen cycloaddition or "Click chemistry" between the azides and alkynes, [6] the reaction between aldehydes (or ketones) and aminooxy containing reagents (or hydrazides). [7] Herein, we describe a water-compatible condensation reaction for labeling terminal cysteine residues on proteins in vitro and at the cell surface.N-terminal cysteine has been frequently used in protein engineering for site-specific labeling and modification. [8] Thioesters are commonly used in a ligation reaction with terminal cysteines, which proceeds through thioester and S-to N-acyl exchanges. [9] This native chemical ligation reaction has been successfully applied to protein semi-synthesis and labeling. [10] Our method to label the terminal cysteine on a protein is based on the condensation of 2-cyanobenzothiazole (CBT) and D-cysteine, a reaction used at the last step of the synthesis of D-luciferin a common substrate for firefly luciferase (reaction 1 in Scheme 1). [11] This reaction can proceed smoothly in aqueous solutions. We hypothesized that CBT could react with the terminal cysteine on a protein. If a fluorophore is conjugated to the CBT motif, this reaction should ligate a fluorescent label specifically to the terminal cysteine of the protein (reaction...
Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.
Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in superresolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.Recently, sequential imaging of sparse subsets of photoactivatable/photoswitchable singlemolecule fluorophores has enabled optical imaging beyond the diffraction limit (DL), providing insight into the sub-diffraction world (e.g. PALM, FPALM, STORM). 1-3 These single-molecule superresolution (SR) techniques have provided the impetus for development of new controllable fluorophores with large numbers of emitted photons N, because the achievable resolution scales as . 4 Most previous SR experiments in living cells 5 have used photocontrollable fluorescent proteins. 6-9 However, despite having the advantage of being target-specific, fluorescent proteins on average provide 10-fold fewer photons before photobleaching than good organic fluorophores. 10,11 Small organic fluorophores have the additional benefit of synthetic design flexibility for tuning target specificity, spectral wavelength, solubility, and other desired properties. Therefore, targeted bright organic Here we present a target-specific photoactivatable organic fluorophore for use inside living and fixed cells, 3, based on the commercial HaloTag targeting approach. [20][21][22] This method requires a genetic fusion to the HaloEnzyme (HaloEnz), which forms a covalent linkage to the HaloTag substrate, thus labeling the protein of interest (i.e. a protein-HaloEnzHaloTag-fluorophore covalent unit). Specifically, we present: (i) the basic photophysical properties of a new targeted photoactivatable probe; (ii) proof-of-principle labeling of known structures in fixed and living mammalian cells validated by co-staining with antibodies or co-transfection with fluorescent proteins; (iii) specific SR imaging of microtubules in a mammalian cell with quantification of resolution enhancement; (iv) demonstration of targeted labeling in living bacteria with diffraction-limited imaging; and finally, (v) SR imaging of poorly understood structures inside living bacteria.As molecules with bright emission for single-molecule imaging, dicyanomethylenedihydrofuran (DCDHF) push-pull fluorophores emit millions of photons before photobleaching, and can enter living cells. 15,23 Recently, we reported a photoactivatable DCDHF fluorogen based on photocaging the fluorescence by replacing the amine donor with a poorly-donating but photolabile azide, which can then be converted back to an am...
The development of efficient methods for the facile construction of important molecular architectures is a central goal in organic synthesis. An unprecedented organocatalytic asymmetric cascade Michael-alkylation reaction of alpha,beta-unsaturated aldehydes with bromomalonates has been developed. The process, efficiently catalyzed by chiral diphenylprolinol TMS ether in the presence of base 2,6-lutidine, serves as a powerful approach to the preparation of synthetically and biologically important cyclopropanes in high levels of enantio- and diastereoselectivities. Remarkably, the power of the cascade process is fueled by its high efficiency of the production of two new C-C bonds, two new stereogenic centers, and one quaternary carbon center in one single operation, which otherwise is difficult to achieve by traditional strategies. Moreover, the beauty of the cascade process is further underscored by the nature of the product formation depending on the reaction conditions. With the alternation of base from 2,6-lutidine (1.1 equiv), which is effective for the cyclopropanations, to NaOAc (4.0 equiv), the spontaneous ring-opening of cyclopropanes takes place to lead to stereoselective (E) alpha-substituted malonate alpha,beta-unsaturated aldehydes. A possible reaction mechanism, which involves a Michael-alkylation-retro-Michael pathway, is proposed and verified by experimental studies. This investigation represents the first example of an organocatalyst-promoted ring opening of the cyclopropanes, whereas such reactions have been intensively explored by Lewis acid-based catalysis.
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