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2 What is Click Chemistry? Reactions with the following characteristics: Modular, wide in scope Afford high yields w/o purification Stereospecific Generate inoffensive byproducts and operate in a benign solvent
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3 Click Reactions Nucleophilic substitution Cycloadditions “Non-aldol” carbonyl
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4 Click Reactions Nucleophilic substitution Cycloadditions “Non-aldol” carbonyl
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5 [3+2] Dipolar Cycloaddition 1,3-Dipole
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6 Thermal Thermal: 1:1 mixture Kolb. J. Am. Chem. Soc. 2004, 126, 12809
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7 Cu(I) Catalyzed Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596
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8 “this is a very robust catalytic process, which is so insensitive to the usual reaction parameters as to strain credulity” - V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596-2599
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9 Catalytic Cycle
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10 Autocatalytic? Rate acceleration during formation of dendrimers Binding is tetradentate Prevents oxidation and disproportionation Improves catalytic activity Forkin. Org. Lett. 2004, 6, 2853
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11 Optimized Cycloaddition Conditions CuSO 4 – 1mM Ligand ( tris(triazoyl) amine ) – 2mM Reducing agent ( tris(carboethyoxy)phosphine ) – 2mM
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12 Applications Library synthesis in situ inhibitor formation Bioconjugation –Activity based protein profiling (ABPP) –Cell Surface Modification –Non-canonical amino acids
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13 Protein Synthesis DNA RNA Protein Active Modified “Stored ”
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14 Proteomics Genomics –The study of an organism’s genome and use of it’s genes Proteomics –The identification and functional assignment of all proteins in the proteome
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15 Methods for Analyzing the Proteome 2-D Gel Electrophoresis / staining + MS Charge (pI) MW
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16 Methods for Analyzing the Proteome 2-D Gel Electrophoresis / staining + MS LC-MS/MS based –Isotope coded affinity tagging (ICAT) Limitation: primarily measures protein abundance
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17 Activity Based Protein Profiling (ABPP) Proteins analyzed by function
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18 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth
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19 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth
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20 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth
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21 Retrosynthesis of (-)-FR182877 Evans. J. Am Chem. Soc. 2003, 125, 13531 Sorensen J. Am Chem. Soc. 2003, 125, 5393
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22 Synthesis of Fragments: Evans
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23 Macrocycle Formation: Evans
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24 Transannular Diels-Alder: Evans
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25 Activity Based Protein Profiling (ABPP) Cravatt. J. Am Chem. Soc. 2004, 126, 1363
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26 Reporter Tags Biotin Target purification / isolation Avidin chromatography Rhodamine Target detection Fluorescent probe
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27 Synthesis of Rhodamine Tag
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28 Synthesis of Biotin / Rhodamine Tag
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29 Synthesis of tagged FR182877 Prepared: (-)-FR182877 Rhodamine tag (+)-FR182877 Rhodamine tag (-)-FR182877 Rhodamine-Biotin tag Cravatt. Ang. Chem. Int. Ed. 2003, 42, 5480
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30 Mouse Tissue Proteome (-)-FR182877 – Rh tag 0.1 M electrophile 2mg/mL protein
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31 Mouse Tissue Proteome (-)-FR182877 – Rh tag Heat denatured
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32 Mouse Tissue Proteome (-)-FR182877 – Rh tag (+)-FR182877 – Rh tag
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33 Mouse Tissue Proteome (-)-FR182877 – Rh tag (-)-FR182877
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34 Mouse Tissue Proteome (-)-FR182877 – Rh tag (+)-FR182877
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35 Identification of Target Isolated using biotin-rhodamine tagged (-)-FR182877 via avidin chromatography and analyzed by MS Target protein is Carboxyl Esterase-1
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36 IC 50 Determination Pre-incubate proteome with (+/-)-FR182877 then treat with Rhodamine tagged label IC 50 = 34nM
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37 Activity of Carboxylesterase-1 Broad spectrum serine hydrolase Drug and xenobiotic metabolism
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38 Summary (+)-FR182877 is inactive (-)-FR182877 target is carboxyl esterase-1 Potent: IC 50 34nM Selective: 1 M gives 20X difference
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39 Limitations Ideal: measure activity in native environment Reporter tag limits scope –Bioavailability –Biological activities –Subcellular compartmentalization –Electrostatic interactions
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40 Click Chemistry ABPP General Concept –Dose electrophile tethered azide –Collect sample, homogenize, perform cycloaddition –Isolate and characterize target in vivo in vitro Cravatt. Chem & Biol. 2004, 11, 535
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41 “Click Chemistry”-ABPP Rh-tagged phenylsulfonate labels Phenylsulfonate is a general label for cysteine proteases
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42 “Click Chemistry”-ABPP RG-N 3 / Dye-Ξ RG-Ξ / Dye-N 3
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43 “Click Chemistry”-ABPP RG-N 3 / Dye-Ξ RG-Ξ / Dye-N 3
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44 “Click Chemistry”-ABPP RG-N3 / Dye-Ξ RG-Ξ / Dye-N 3
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45 Results PS-≡ / Rh-N 3 reduced background labeling –Improved signal : noise –Allowed detection of low abundant proteins Successfully measured enzyme activity in vivo
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46 in vivo Bioconjugation Cu(I) catalyzed [3+2] dipolar cycloaddition valuable tool for addition of tag in vitro Cu(I) is toxic in vivo ligation requires biocompatible reagents
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47 Cell Surface Glycoconjugation Unnatural sugar tolerated by sialic acid biosynthetic pathway Conjugate to azide via Staudinger reaction Bertozzi. Science. 2000, 287, 2007
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48 Staudinger Reaction
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49 Aza-ylide Traps Biological applications require aqueous solvent
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50 Intramolecular Staudinger
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51 “Traceless” Staudinger
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52 Phosphine Synthesis Common intermediates allow for diversity
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53 Synthesis of Non-Natural Sugar Peracylation aids bioavailability
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54 Modulation of Cell Surfaces in Living Animals 1)Inject Ac 4 ManNaz for 7 Days 1eq Bertozzi. Nature. 2004, 430, 873
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55 Results No adverse affects associated with Ac 4 ManNAz or Phos-Flag Ac 4 ManNAz stable to serum esterases Labeled glycoproteins only observed in groups dosed with Ac 4 ManNAz Majority of azides ligated in vivo
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56 Conclusions Identified carboxylesterase-1 as target of (-)-FR182877 Cu(I) catalyzed [2+3] dipolar cycloaddition allows for in vivo labeling of proteins Staudinger ligation in a living animal!!
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57 Acknowledgements Dan Wayner Greg Lopinski John Pezacki Bojana Rakic Jenny Clarke JL Brochu Corinne Stocco
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58 [3+2] Cycloaddition of Biomolecule CuSO 4 (mM) Ligand (mM) TCEP (mM) Loading %/60 ---3 -2.0 <2 1.02.0-<2 1.0-2.017 1.02.0 100 2.0 5.080 Sharpless. J. Am. Chem. Soc. 2003, 125, 3192
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59 [3+2] Cycloaddition of Biomolecule CuSO 4 (mM) Ligand (mM) TCEP (mM) Loading %/60 ---3 -2.0 <2 1.02.0-<2 1.0-2.017 1.02.0 100 2.0 5.080 Sharpless. J. Am. Chem. Soc. 2003, 125, 3192
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60 (-)-FR182877 Tethered to reporter tag via cycloaddition Specifically labels one protein ~70kDa Only natural enantiomer is active Target identified as Carboxylesterase-1 –Potent: 34nM –Selective: >20X over other enzymes
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61 “Click” – Activity Based Protein Profiling Orientation: Electrophile–≡ / Tag–N 3 –Copper alkylidyne causes non-specific labelling Low abundance enzymes observed Proteins labeled in vivo
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62 Cell Surface Glycoconjugation No toxic effects from dosing unnatural sugar Specific labeling of azido-sugar Dose dependant fluorescence
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