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bioorthogonal click

Kalle Kipper, Ebba Gregorsson Lundius, Vladimir Curic, Ivana Nikic, Edward A Lemke, Manfred Wiessler, Johan Elf
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells...
October 24, 2016: ACS Synthetic Biology
Man Kyu Shim, Hong Yeol Yoon, Ju Hee Ryu, Heebeom Koo, Sangmin Lee, Jae Hyung Park, Jong-Ho Kim, Seulki Lee, Martin G Pomper, Ick Chan Kwon, Kwangmeyung Kim
Recently, metabolic glycoengineering with bioorthogonal click reactions has focused on improving the tumor targeting efficiency of nanoparticles as delivery vehicles for anticancer drugs or imaging agents. It is the key technique for developing tumor-specific metabolic precursors that can generate unnatural glycans on the tumor-cell surface. A cathepsin B-specific cleavable substrate (KGRR) conjugated with triacetylated N-azidoacetyl-d-mannosamine (RR-S-Ac3 ManNAz) was developed to enable tumor cells to generate unnatural glycans that contain azide groups...
October 20, 2016: Angewandte Chemie
D Madea, T Slanina, P Klán
A bioorthogonal 'catch and photorelease' strategy, which combines alkyne-azide cycloaddition between p-hydroxyphenacyl azide and alkyne derivatives to form a 1,2,3-triazole adduct and subsequent photochemical release of the triazole moiety via a photo-Favorskii rearrangement, is introduced. The first step can also involve photorelease of a strained alkyne and its Cu-free click reaction with azide.
October 14, 2016: Chemical Communications: Chem Comm
Jorin Hoogenboom, Nathalja Berghuis, Dario Cramer, Rene Geurts, Han Zuilhof, Tom Wennekes
BACKGROUND: Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling...
October 10, 2016: BMC Plant Biology
Catherine Su Hui Teo, Remigiusz A Serwa, Peter O'Hare
We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection...
October 2016: PLoS Pathogens
Kathrin Elisabeth Witzke, Kristin Rosowski, Christian Müller, Maike Ahrens, Martin Eisenacher, Dominik A Megger, Jürgen Knobloch, Andrea R Koch, Thilo Bracht, Barbara Sitek
Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins...
October 3, 2016: Journal of Proteome Research
Pablo Mateos-Gil, Sebastian Letschert, Sören Doose, Markus Sauer
Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions...
2016: Frontiers in Cell and Developmental Biology
Arne H Smits, Annika Borrmann, Mark Roosjen, Jan C M van Hest, Michiel Vermeulen
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts...
October 17, 2016: ACS Chemical Biology
Jing Li, Jiajia Wang, Liuqing Wen, He Zhu, Shanshan Li, Kenneth Huang, Kuan Jiang, Xu Li, Cheng Ma, Jingyao Qu, Aishwarya Parameswaran, Jing Song, Wei Zhao, Peng George Wang
O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification...
October 14, 2016: ACS Chemical Biology
Borja Belda-Palazón, Alejandro Ferrando, Rosa Farràs
The development of novel bioorthogonal reactives that can be used to tag biomolecules in vivo has revolutionized the studies of cellular and molecular biology. Among those novel reactive substances, amino acid analogs can be used to label nascent proteins, thus opening new avenues for measuring protein translation rates in vivo with a limited manipulation of the sample. Here, we describe the use of Click-chemistry to tag and separate newly synthesized proteins in mammalian cells that can be used, coupled with western analysis, to estimate the translation rate of any protein of interest...
2016: Methods in Molecular Biology
Aleksandra Pałasz
This review is an endeavor to highlight the progress in the inverse-electron-demand hetero-Diels-Alder reactions of 1-oxa-1,3-butadienes in recent years. The huge number of examples of 1-oxadienes cycloadditions found in the literature clearly demonstrates the incessant importance of this transformation in pyran ring synthesis. This type of reaction is today one of the most important methods for the synthesis of dihydropyrans which are the key building blocks in structuring of carbohydrate and other natural products...
June 2016: Top Curr Chem (J)
Heebeom Koo, Jeong Heon Lee, Kai Bao, Yunshan Wu, Georges El Fakhri, Maged Henary, Seok Hyun Yun, Hak Soo Choi
A critical limitation of bioorthogonal click chemistry for in vivo applications has been its low reaction efficiency due to the pharmacokinetic barriers, such as blood distribution, circulation, and elimination in living organisms. To identify key factors that dominate the efficiency of click chemistry, here a rational design of near-infrared fluorophores containing tetrazine as a click moiety is proposed. Using trans-cyclooctene-modified cells in live mice, it is found that the in vivo click chemistry can be improved by subtle changes in lipophilicity and surface charges of intravenously administered moieties...
October 2016: Advanced Healthcare Materials
Alexander Mertsch, Sebastian Letschert, Elisabeth Memmel, Markus Sauer, Jürgen Seibel
The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.
September 1, 2016: Zeitschrift Für Naturforschung. C, A Journal of Biosciences
Josephin M Holstein, Lea Anhäuser, Andrea Rentmeister
The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells...
August 26, 2016: Angewandte Chemie
András Herner, Qing Lin
In the last decade and a half, numerous bioorthogonal reactions have been developed with a goal to study biological processes in their native environment, i.e., in living cells and animals. Among them, the photo-triggered reactions offer several unique advantages including operational simplicity with the use of light rather than toxic metal catalysts and ligands, and exceptional spatiotemporal control through the application of an appropriate light source with pre-selected wavelength, light intensity and exposure time...
February 2016: Top Curr Chem (J)
Sukwon Jung, Chang-Hyung Choi, Chang-Soo Lee, Hyunmin Yi
Functionalized polymeric microparticles possess significant potential for controlled drug delivery and biosensing applications, yet current fabrication techniques face challenges in simple and scalable fabrication and biofunctionalization. For programmable manufacture of biofunctional microparticles in a simple manner, we have developed robust micromolding methods combined with biopolymeric conjugation handles and bioorthogonal click reactions. In this focused minireview, we present detailed methods for our integrated approaches for fabrication of microparticles with controlled 2D and 3D shapes and dimensions toward controlled release, and for biomacromolecular conjugation via strain promoted alkyne-azide cycloaddition (SPAAC) and tetrazine-trans-cyclooctene (Tz-TCO) ligation reactions utilizing a potent aminopolysaccharide chitosan as an efficient conjugation handle...
July 1, 2016: Biotechnology Journal
Brendon E Cook, Pierre Adumeau, Rosemery Membreno, Kathryn E Carnazza, Christian Brand, Thomas Reiner, Brian J Agnew, Jason S Lewis, Brian M Zeglis
In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO)...
August 17, 2016: Bioconjugate Chemistry
Christian Nienberg, Anika Retterath, Kira-Sophie Becher, Thorsten Saenger, Henning D Mootz, Joachim Jose
Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α...
2016: Pharmaceuticals
Christoph Denk, Dennis Svatunek, Severin Mairinger, Johann Stanek, Thomas Filip, Dominik Matscheko, Claudia Kuntner, Thomas Wanek, Hannes Mikula
A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles...
July 20, 2016: Bioconjugate Chemistry
Raffaella Rossin, Sander M J van Duijnhoven, Wolter Ten Hoeve, Henk M Janssen, Laurens H J Kleijn, Freek J M Hoeben, Ron M Versteegen, Marc S Robillard
The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice...
July 20, 2016: Bioconjugate Chemistry
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