Take home message: While GFP enables people to image proteins; with azide, we now can image any other chemicals, such as glycans.
Today, we had the 6th Andrew Braisted Lectureship on campus. I stayed awaken 100% of the time, after a long day at bench. It was awesome! I am totally excited about the work she is doing. All about the imaging.
4:00 pm, MBay, Genentech Hall 106
"Imaging the Glycome"
Carolyn Bertozzi, PhD
Professor of Chemistry and Molecular and Cell Biology, UC-Berkeley; HHMI Investigator
I managed to recall couple of her most important discoveries.
J Am Chem Soc. 2002 Dec 18;124(50):14893-902.
Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation.
Saxon E, Luchansky SJ, Hang HC, Yu C, Lee SC, Bertozzi CR.Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, CA 94720, USA.
The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.
J Am Chem Soc. 2003 Apr 23;125(16):4708-9.
A fluorogenic dye activated by the staudinger ligation.
Lemieux GA, De Graffenried CL, Bertozzi CR.Center for New Directions in Organic Synthesis, Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
Specific labeling of biomolecules with biochemical and biophysical probes is a central element of proteomics research. Here we describe a coumarin-phosphine dye that undergoes activation of coumarin fluorescence upon Staudinger ligation with azides. Since azides can be metabolically incorporated into cellular proteins and oligosaccharides, this dye may be a useful tool for profiling proteins and their posttranslational modifications.
J Am Chem Soc. 2004 Nov 24;126(46):15046-7.
A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems.
Agard NJ, Prescher JA, Bertozzi CR.Department of Chemistry, Howard Hughes Medical Institute, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3 + 2] cycloaddition of azides and alkynes ("click chemistry"). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidation and synthetic challenges. Click chemistry obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3 + 2] cycloaddition between cyclooctynes and azides that proceeds under physiological conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomolecules in vitro and on living cells, with no apparent toxicity.
J Am Chem Soc. 2008 Aug 27;130(34):11486-93.
Second-generation difluorinated cyclooctynes for copper-free click chemistry.
Codelli JA, Baskin JM, Agard NJ, Bertozzi CR.Departments of Chemistry, University of California, Berkeley, California 94720, USA.
The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.
Science. 2008 May 2;320(5876):664-7.
In vivo imaging of membrane-associated glycans in developing zebrafish.
Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR.Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.
She had a very recent review which summarized the things she talked about today.
Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):12-7.
Imaging the glycome.
Laughlin ST, Bertozzi CR.Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Molecular imaging enables visualization of specific molecules in vivo and without substantial perturbation to the target molecule's environment. Glycans are appealing targets for molecular imaging but are inaccessible with conventional approaches. Classic methods for monitoring glycans rely on molecular recognition with probe-bearing lectins or antibodies, but these techniques are not well suited to in vivo imaging. In an emerging strategy, glycans are imaged by metabolic labeling with chemical reporters and subsequent ligation to fluorescent probes. This technique has enabled visualization of glycans in living cells and in live organisms such as zebrafish. Molecular imaging with chemical reporters offers a new avenue for probing changes in the glycome that accompany development and disease.
I had the seminar recorded and presented here. Enjoy the world of azide!
Part I
[video vid_image=http://en.dogeno.us/wp-content/video/VID00073.png filename=/wp-content/video/VID00073.mp4 /]
Part II
[video vid_image=http://en.dogeno.us/wp-content/video/VID00074.png filename=/wp-content/video/VID00074.mp4 longtail=true /]
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