Download Bioorthoganal Chemistry to Selectively Label Cellular Proteins

Presenters: Dan Wang and Tim Andrews
Angew. Chem. Int. ed. 2009. 48, 6974
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1. Background
2. Unique Amino Acid Sequences
3. Bioorthogonal Reactions
4. Applications of Bioorthogonal Chemical reactions
5. Conclusion
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The ability to observe biomolecules within their cellular environment/processes.
 Use of a markers to visualize processes
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High Selectivity
Fast Reactivity
 10‐4 ‐ 103 M‐1s‐1 Reaction Constants
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Covalent modifications
Restrictive reactions conditions
A
 Structure, environment, pH, temperature, nature of reaction
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Osamu Shimomura first to isolate this protein
 Nobel Prize ‐ 2008 
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Genetically incorporated into protein
http://www.mbl.edu/news/features/shimomura.html
Problems
 can inhibit activity of protein
 Many compounds in the body can’t be modified genetically
▪ nucleic acids, lipids, glycans
Mulvihill. C. Am. Soc. Microbio.
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Most common bioorthogonal reaction
 Specific Amino Acids
 N‐Terminal modifications
 Native Chemical Ligation
 Sequence specific fluorogenic modifications
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Chromophores
Spin Lables
Affinity labels
Catalysts
MRI Contrast Agents
Material Surfaces
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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1CBW
Nanocrystals1
Crosslinkers
Enzymes
Polymers
Radiolabels
1Rockenberger, J.; Scher, E. C.; Alivisatos, A. P. J. Am. Chem. Soc. 1999, 121, 49, 11595
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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Specificity for specific amino acids not achieved for all.
 5 of 20 amino acids can be selectively reacted with
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Predominately targeted nucleophilic amino acid side chains
 Lysine, Cysteine, Aspartic acid, and Glutamic acid
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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One of the most widely used method
Commonly found on surface of proteins
 20 or more Lysines on the surface
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Lack of control for modifications M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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N‐Hydroxysuccinimide‐
activated esters
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Isocyanates and Isothiocyanates
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Aldehydes ‐ reductive alkylation
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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Rarest of the amino acids
Not typically found on the surface of peptides. Can be generated by site‐specific mutations
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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Maleimides
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Iodoacetamide
reagents
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Disulfide Exchange
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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Carboxylate residues react with carbodiimides.
Causes formation of active esters
Can go on to react amines, or undergo O‐
acylisouronium rearrangement
M. B. Francis in Chemical Biology Wiley‐VCH, Weinheim, 2007, p. 593
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Selectivity via primary amine
 Problem: Lysine residues?
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Can be selective for a certain amino acid or any at the N‐terminus.
Dixon, H. B. F. J. Protein Chem. 1984.,3, 1, 99.
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Widely used method of conjugating two peptides (or markers to peptides)
Reaction between a thioester and a cysteine
 Thioester affects the rate of the rearrangement
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Limited to Cysteine N‐
terminal pepetides
Kent, S. B. H. et al. Science. 1994. 266, 776.
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Sletten, E. M. Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48,6974
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Require fluorogenic compounds that don’t interfere with biological function
Bind to sequences on hairpin loops
 CCXXCC – Biarsenic dyes
 SSXXSS – Bisboronic acid Rhodium dyes
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Slight background fluoresnce
Nonspecific binding via hydrophobic interactions
Tsien, R. Y. et al. Science. 1998, 281, 269
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FlAsH‐EDT
ReAsH‐EDT
Tsien, R. Y. et al. Science. 1998, 281, 269
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CCXXCC
+
XX
Tsien, R. Y. et al. Science. 1998, 281, 269
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RhoBo
Found to bind SSXXSS
Originally designed to bind monosaccharides.
Binds proteins more selectively Schepartz, A. et al. J. Am .Chem. Soc. 2009, 131, 438
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• Introducing the chemical reporter to biomolecules such as protein, nucleic acid, lipid or glycan.
• The reaction between the chemical reporter and a probe molecule bearing complementary bioorthogonal functionality.
Bertozzi, C. R. Chem. Soc. Rev. 2010 , 39, 1272
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Selectivity:
Chemical reporter & Probe molecule.
The kinetics of the reaction:
[
Rate = k*
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]*[probe molecule].
labeled biomolecule
Toxicity of probe molecules and other reagents.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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Kinetics: reaction rate enhanced by α–effect.
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Selectivity:  Biological nucleophiles don’t react w/ ketones or aldehydes.
 Endogenous aldehydes and ketones compete with the desired reaction.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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Azide:
 Totally absent from biological system.
 Small.
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Potential cross‐reaction:
 Azide & Thiol: slow
 Phospine and disulfide bond: not favored under physiological condition, especially when triaryl‐
phospine is used.
Bertozzi, C. R. Science 2000, 287, 2007
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Kinetics:
 Major limitation. Slow.  High [phosphine] is needed.
 Can be improved by increasing the nucleophilicity
of phosphine reagents, which unfortunately, also result in the susceptibility of phosphine oxidation.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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1,3‐dipole [3+2] cycloaddition reaction.
Require high temperature or pressure, not compatible with living systems.
Sharpless improved the reaction by using copper(I) catalyst.
CuAAC is often referred to as “click chemistry”.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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The 1st report of CuAAC as a bioconjugation
strategy in vivo.
J. Am. Chem. Soc. 2003, 125, 3192.
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Methods to activate alkynes:
 Metal catalysis – toxicity of copper.
 Ring strain.
Bertozzi, C. R Angew. Chem. Int. Ed. 2009, 48, 6074.
Krebs, A. Chem. Ber. 1961, 94, 3260. •Metabolic stable.
•Biocompatible.
•Similar sensitivity to copper activated click reaction.
•Driving force:
Releasing of the ring strain.
Electron‐withdrawing effect of F substitution.
Bertozzi C. R. PNAS, 2007, 104, 16793.
Bertozzi C. R . J. Am. Chem. Soc. 2010, 132, 9516.
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Proteins:
 Metabolic labeling.
 Genetic encoding.
 Activity Based Protein Profiling (ABPP).
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Glycans, Lipids, Nucleic Acid:
 Metabolic labeling.
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Metabolic labeling:
 Residue specific global modification.
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Genetic encoding:
 Site specific modification.
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Activity Based Protein Profiling (ABPP):
 Mechanism based modification.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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Reports can be found for replacement of almost any amino acid with an unnatural derivative.
Methionine surrogates: Tirrell, D. A. J. Am. Chem. Soc. 2005, 127, 14150.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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Site specific introduction of unnatural amino acids.
Low yield.
Labor intensity.
Schultz, P. G. Angew. Chem. Int. Ed. 2005, 44, 34.
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ABPP allows for the study of specific classes of enzymes based on their catalytic mechanism.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074.
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Warhead: electrophilic group (covalently bind with enzyme).
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Affinity probes:
 Used to be large molecules such as biotin or fluorophore.
 In 2003, Cravatt group reported the application of bioorthogonal reaction to ABPP.
Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.
Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.
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Glycans, lipids and nucleic acids can be modified by metabolic labeling with biosynthetic precursors.
Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6074..
Mitchison, T. J. PNAS 2008, 105, 2415.
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Bioorthogonal chemical reactions are very powerful tools to monitor biomolecules
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Bountiful methods to perform these reactions, ranging the scope of biomolecules
out there
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Questions ?
Tsien laboratory ‐ http://www.tsienlab.ucsd.edu/
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Sletten, E. M. Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48,6974
M. B. Francis in Chemical Biology (Eds: S. L. Schreiber, T. Kapoor, G. Wess), Wiley‐VCH, Weinheim, 2007, p. 593
Mulvihill. C. Am. Soc. Microbio. http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=707
Dawson. P. E. Muir. T. W. Clark‐Lewis, I. Kent, S. B. H. Science. 1994. 266, 776.
Dixon, H. B. F. J. Protein Chem. 1984.,3, 1, 99.
Halo. T. L. Appelbaum, J. Hobert, E. M. Balkin, D. M. Schepartz, A. J. Am .Chem. Soc. 2009, 131, 438
Griffin, B. A. Adams, S. R. Tsien, R. Y. Science. 1998, 281, 269
Nobelprize.org
http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE%20‐%20PLATE%20‐%20Beach.jpg
Science 2000, 287, 2007.
Angew. Chem. Int. Ed. 2001, 40, 2004.
J. Am. Chem. Soc. 2003, 125, 3192.
PNAS 2007, 104 , 16793.
J. Am. Chem. Soc. 2010, 132, 9516.
J. Am. Chem. Soc. 2005, 127, 14150.
Angew. Chem. Int. Ed. 2005, 44, 34.
J. Am. Chem. Soc. 2003, 125, 4686.
PNAS 2008, 105, 2415.
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