1 / 62

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.

elvin
Télécharger la présentation

What is Click Chemistry?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related