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What is Click Chemistry?

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

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What is Click Chemistry?

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  1. 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

  2. Click Reactions • Nucleophilic substitution • Cycloadditions • “Non-aldol” carbonyl

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

  4. [3+2] Dipolar Cycloaddition 1,3-Dipole

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

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

  7. “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

  8. Catalytic Cycle

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

  10. Optimized Cycloaddition Conditions • CuSO4 – 1mM • Ligand (tris(triazoyl) amine) – 2mM • Reducing agent (tris(carboethyoxy)phosphine) – 2mM

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

  12. Protein Synthesis • DNA • RNA • Protein • Active • Modified • “Stored”

  13. 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

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

  15. 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

  16. Activity Based Protein Profiling (ABPP) • Proteins analyzed by function

  17. (-)-FR182877 • Isolated 1998 from Streptomyces • Found to inhibit tumor cell growth

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

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

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

  21. Synthesis of Fragments: Evans

  22. Macrocycle Formation: Evans

  23. Transannular Diels-Alder: Evans

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

  25. Reporter Tags • Rhodamine • Target detection • Fluorescent probe • Biotin • Target purification / isolation • Avidin chromatography

  26. Synthesis of Rhodamine Tag

  27. Synthesis of Biotin / Rhodamine Tag

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

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

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

  31. Mouse Tissue Proteome (-)-FR182877 – Rh tag (+)-FR182877 – Rh tag

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

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

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

  35. IC50 Determination • Pre-incubate proteome with (+/-)-FR182877 then treat with Rhodamine tagged label • IC50 = 34nM

  36. Activity of Carboxylesterase-1 • Broad spectrum serine hydrolase • Drug and xenobiotic metabolism

  37. Summary • (+)-FR182877 is inactive • (-)-FR182877 target is carboxyl esterase-1 • Potent: IC50 34nM • Selective: 1M gives 20X difference

  38. Limitations • Ideal: measure activity in native environment • Reporter tag limits scope • Bioavailability • Biological activities • Subcellular compartmentalization • Electrostatic interactions

  39. Click Chemistry ABPP in vivo in vitro • General Concept • Dose electrophile tethered azide • Collect sample, homogenize, perform cycloaddition • Isolate and characterize target Cravatt. Chem & Biol.2004, 11, 535

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

  41. “Click Chemistry”-ABPP RG-N3 / Dye-Ξ RG-Ξ/ Dye-N3

  42. “Click Chemistry”-ABPP RG-N3 / Dye-Ξ RG-Ξ / Dye-N3

  43. “Click Chemistry”-ABPP RG-N3 / Dye-Ξ RG-Ξ / Dye-N3

  44. Results • PS-≡ / Rh-N3 reduced background labeling • Improved signal : noise • Allowed detection of low abundant proteins • Successfully measured enzyme activity in vivo

  45. 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

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

  47. Staudinger Reaction

  48. Aza-ylide Traps Biological applications require aqueous solvent

  49. Intramolecular Staudinger

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