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1. 2 What is Click Chemistry? Reactions with the following characteristics: Modular, wide in scope Afford high yields w/o purification Stereospecific.

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Presentation on theme: "1. 2 What is Click Chemistry? Reactions with the following characteristics: Modular, wide in scope Afford high yields w/o purification Stereospecific."— Presentation transcript:

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2 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

3 3 Click Reactions Nucleophilic substitution Cycloadditions “Non-aldol” carbonyl

4 4 Click Reactions Nucleophilic substitution Cycloadditions “Non-aldol” carbonyl

5 5 [3+2] Dipolar Cycloaddition 1,3-Dipole

6 6 Thermal Thermal: 1:1 mixture Kolb. J. Am. Chem. Soc. 2004, 126, 12809

7 7 Cu(I) Catalyzed Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596

8 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

9 9 Catalytic Cycle

10 10 Autocatalytic? Rate acceleration during formation of dendrimers Binding is tetradentate Prevents oxidation and disproportionation Improves catalytic activity Forkin. Org. Lett. 2004, 6, 2853

11 11 Optimized Cycloaddition Conditions CuSO 4 – 1mM Ligand ( tris(triazoyl) amine ) – 2mM Reducing agent ( tris(carboethyoxy)phosphine ) – 2mM

12 12 Applications Library synthesis in situ inhibitor formation Bioconjugation –Activity based protein profiling (ABPP) –Cell Surface Modification –Non-canonical amino acids

13 13 Protein Synthesis DNA RNA Protein Active Modified “Stored ”

14 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

15 15 Methods for Analyzing the Proteome 2-D Gel Electrophoresis / staining + MS Charge (pI) MW

16 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

17 17 Activity Based Protein Profiling (ABPP) Proteins analyzed by function

18 18 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth

19 19 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth

20 20 (-)-FR182877 Isolated 1998 from Streptomyces Found to inhibit tumor cell growth

21 21 Retrosynthesis of (-)-FR182877  Evans. J. Am Chem. Soc. 2003, 125, 13531 Sorensen J. Am Chem. Soc. 2003, 125, 5393

22 22 Synthesis of Fragments: Evans

23 23 Macrocycle Formation: Evans

24 24 Transannular Diels-Alder: Evans

25 25 Activity Based Protein Profiling (ABPP) Cravatt. J. Am Chem. Soc. 2004, 126, 1363

26 26 Reporter Tags Biotin Target purification / isolation Avidin chromatography Rhodamine Target detection Fluorescent probe

27 27 Synthesis of Rhodamine Tag

28 28 Synthesis of Biotin / Rhodamine Tag

29 29 Synthesis of tagged FR182877 Prepared: (-)-FR182877 Rhodamine tag (+)-FR182877 Rhodamine tag (-)-FR182877 Rhodamine-Biotin tag Cravatt. Ang. Chem. Int. Ed. 2003, 42, 5480

30 30 Mouse Tissue Proteome (-)-FR182877 – Rh tag 0.1  M electrophile 2mg/mL protein

31 31 Mouse Tissue Proteome (-)-FR182877 – Rh tag Heat denatured

32 32 Mouse Tissue Proteome (-)-FR182877 – Rh tag (+)-FR182877 – Rh tag

33 33 Mouse Tissue Proteome (-)-FR182877 – Rh tag (-)-FR182877

34 34 Mouse Tissue Proteome (-)-FR182877 – Rh tag (+)-FR182877

35 35 Identification of Target Isolated using biotin-rhodamine tagged (-)-FR182877 via avidin chromatography and analyzed by MS Target protein is Carboxyl Esterase-1

36 36 IC 50 Determination Pre-incubate proteome with (+/-)-FR182877 then treat with Rhodamine tagged label IC 50 = 34nM

37 37 Activity of Carboxylesterase-1 Broad spectrum serine hydrolase Drug and xenobiotic metabolism

38 38 Summary (+)-FR182877 is inactive (-)-FR182877 target is carboxyl esterase-1 Potent: IC 50 34nM Selective: 1  M gives 20X difference

39 39 Limitations Ideal: measure activity in native environment Reporter tag limits scope –Bioavailability –Biological activities –Subcellular compartmentalization –Electrostatic interactions

40 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

41 41 “Click Chemistry”-ABPP Rh-tagged phenylsulfonate labels Phenylsulfonate is a general label for cysteine proteases

42 42 “Click Chemistry”-ABPP RG-N 3 / Dye-Ξ RG-Ξ / Dye-N 3

43 43 “Click Chemistry”-ABPP RG-N 3 / Dye-Ξ RG-Ξ / Dye-N 3

44 44 “Click Chemistry”-ABPP RG-N3 / Dye-Ξ RG-Ξ / Dye-N 3

45 45 Results PS-≡ / Rh-N 3 reduced background labeling –Improved signal : noise –Allowed detection of low abundant proteins Successfully measured enzyme activity in vivo

46 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

47 47 Cell Surface Glycoconjugation Unnatural sugar tolerated by sialic acid biosynthetic pathway Conjugate to azide via Staudinger reaction Bertozzi. Science. 2000, 287, 2007

48 48 Staudinger Reaction

49 49 Aza-ylide Traps Biological applications require aqueous solvent

50 50 Intramolecular Staudinger

51 51 “Traceless” Staudinger

52 52 Phosphine Synthesis Common intermediates allow for diversity

53 53 Synthesis of Non-Natural Sugar Peracylation aids bioavailability

54 54 Modulation of Cell Surfaces in Living Animals 1)Inject Ac 4 ManNaz for 7 Days 1eq Bertozzi. Nature. 2004, 430, 873

55 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

56 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!!

57 57 Acknowledgements Dan Wayner Greg Lopinski John Pezacki Bojana Rakic Jenny Clarke JL Brochu Corinne Stocco

58 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

59 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

60 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

61 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

62 62 Cell Surface Glycoconjugation No toxic effects from dosing unnatural sugar Specific labeling of azido-sugar Dose dependant fluorescence


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