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The Total Synthesis and Structural Reassignment of Chlorofusin

The Total Synthesis and Structural Reassignment of Chlorofusin. Linda Elizabeth Jewell January 22 nd , 2009. Outline. Background Discovery and Structural Assignment Macrocycle Studies Yao’s Synthesis Boger’s Synthesis Conclusions. Discovery and Bioactivity.

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The Total Synthesis and Structural Reassignment of Chlorofusin

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  1. The Total Synthesis and Structural Reassignment of Chlorofusin Linda Elizabeth Jewell January 22nd, 2009

  2. Outline • Background • Discovery and Structural Assignment • Macrocycle Studies • Yao’s Synthesis • Boger’s Synthesis • Conclusions

  3. Discovery and Bioactivity • Cancer is caused by unregulated cell reproduction • p53 is a transcription regulator • It can prevent cancer by “stalling” the cell cycle or inducing apoptosis when DNA damage is detected • Its activity is modulated by a second protein, MDM2 Chéne, P. Nat. Rev. Cancer2003, 3, 102

  4. Background p53 MDM2 Prevents entry into the nucleus Nucleus p53 Prevents p53 from binding to DNA MDM2 p53 Tags p53 to favour degradation DNA MDM2 Adapted from Chéne, P. Nat. Rev. Cancer2003, 3, 102

  5. Discovery and Bioactivity • Disordered regulation of either p53 or MDM2 can lead to the growth of tumours • Disrupting the p53-MDM2 interaction could restore normal function of p53 and halt the growth of tumours • Chlorofusin was discovered in a screening program designed to identify novel antagonists of the p53-MDM2 interaction • KD = 4.7μM Williams et al, JACS, 2001, 123, 554-560

  6. Discovery and Bioactivity • Chlorofusin was isolated by extraction and purified by reverse-phase HPLC • Molecular formula determined by mass spectroscopy as C63H99O19N12Cl Williams et al, JACS, 2001, 123, 554-560

  7. Discovery and Bioactivity • Amino acids were identifying by degradation of the peptide in HCl, purification on an ion exchange resin, and analysis of the resulting fractions by spectrophotometry • Tests identified leucine (x2), asparagine or aspartic acid (x2), alanine (x1) and threonine (x2) • Two nonstandard amino acids were also present Williams et al, JACS, 2001, 123, 554-560

  8. Discovery and Bioactivity • Acetylation of the unknown compound under basic conditions lead to the addition of three acetyl groups • Suggests three acidic hydroxyl groups • Attempted esterification failed • Suggests absence of carboxylic acid functionalities • Compound was stable under acidic and basic conditions Williams et al, JACS, 2001, 123, 554-560

  9. Discovery and Bioactivity • COSY and TOCSY revealed 14 spin systems • A number of CH2 groups, initially obscured by other protons, were also observed • These were assigned to the nonstandard amino acid aminodecanoic acid • Ornithine was identified as the remaining amino acid Williams et al, JACS, 2001, 123, 554-560

  10. Discovery and Bioactivity • The macrocycle was assembled based upon key HMBC and NOESY correlations Williams et al, JACS, 2001, 123, 554-560

  11. Chromophore Assignment – Key NOEs 8 10 4 Williams et al, JACS, 2001, 123, 554-560

  12. Williams’ Structural Assignment • Williams thus proposed that the O-substituents of the chromophore are all syn Williams et al, JACS, 2001, 123, 554-560

  13. Williams’ Structural Assignment Williams et al, JACS, 2001, 123, 554-560

  14. Outline • Background • Discovery and Structural Assignment • Macrocycle Studies • Yao’s Synthesis • Boger’s Synthesis • Conclusions

  15. Determination of Macrocycle Stereochemistry • To unambiguously determine the stereochemistry of the amino acids in the macrocycle, Boger and Searcey simultaneously completed syntheses of the macrocycle • Boger chose a solution-phase synthesis while Searcey pursued a solid-phase approach Boger et al, OrgLett, 2003, 5,5047-5050; Searcey et al, OrgLett, 2003, 5, 50501-5054

  16. Synthesis of D-ADA Boger et al, OrgLett, 2003, 5,5047-5050

  17. Boger’s Peptide Studies D-Leu L-Thr D-Leu D-Ada L-Orn L-Thr L-Ala Boger et al, OrgLett, 2003, 5,5047-5050

  18. Boger’s Peptide Studies D-Ada D-Ada L-Orn D-Leu L-Orn D-Leu L-Thr L-Thr L-Thr L-Thr L-Ala D-Leu L-Ala D-Leu L-Asn D-Asn D-Asn L-Asn Boger et al, OrgLett, 2003, 5,5047-5050

  19. Determination of Macrocycle Stereochemistry Boger et al, OrgLett, 2003, 5,5047-5050

  20. Bioactivity of Macrocycle • Interestingly, the macrocycle alone displayed no inhibition of the p53-MDM2 complex in either in vivo and in vitro tests • Boger also tested a number of structures including chromophore mimics unsuccessfully Boger et al, OrgLett, 2003, 5,5047-5050; Searcey et al, OrgLett, 2003, 5, 50501-5054

  21. Outline • Background • Discovery and Structural Assignment • Macrocycle Studies • Yao’s Synthesis • Boger’s Synthesis • Conclusions

  22. Yao’s Retrosynthesis Yao et al, JACS, 2007, 129, 6400-6401

  23. Yao’s Retrosynthesis Yao et al, JACS, 2007, 129, 6400-6401

  24. Yao’s Retrosynthesis Yao et al, JACS, 2007, 129, 6400-6401

  25. Yao’s Synthesis Yao et al., Tet, 2005, 61,11882-11886

  26. Yao’s Synthesis Yao et al., Tet, 2005, 61,11882-11886

  27. Yao’s Synthesis Yao et al, JACS, 2007, 129, 6400-6401

  28. Asymmetric Oxidation / Cyclization Porco et al, JACS, 2005, 127, 9342-9343

  29. Asymmetric Oxidation / Cyclization Porco et al, JACS, 2005, 127, 9342-9343

  30. Yao’s Synthesis Yao et al, JACS, 2007, 129, 6400-6401

  31. Regioselective Chlorination SO2

  32. Yao’s Synthesis Yao et al, JACS, 2007, 129, 6400-6401

  33. Yao’s Model Studies D-Ada D-Leu S L-Thr D-Leu D-Asn L-Asn D-Ada D-Leu R L-Thr D-Leu D-Asn L-Asn Yao et al, JACS, 2007, 129, 6400-6401

  34. Yao’s Model Studies • Using their assignment of the 1st stereocentre, Yao synthesized the remaining diastereomers Yao et al, JACS, 2007, 129, 6400-6401

  35. Addition-Elimination

  36. Addition-Elimination 2

  37. Yao’s Model Studies Yao et al, JACS, 2007, 129, 6400-6401

  38. Yao’s Stereochemical Assignment A B S R S C D Yao et al, JACS, 2007, 129, 6400-6401

  39. Yao’s Synthesis D-Ada D-Leu L-Thr Chlorofusin D-Leu D-Asn L-Asn Yao et al, JACS, 2007, 129, 6400-6401

  40. Yao’s Synthesis • Yao also synthesized the Williams-proposed (S,S,S) isomer, which clearly did not fit the NMR data as well as his (S,R,S) isomer 12 14 11 8 17 10 18 16 1 13 12 14 11 8 17 10 18 16 1 13 Yao et al, JACS, 2007, 129, 6400-6401

  41. Summary of Yao’s Synthesis • 14 steps, 2.6% yield (not accounting for peptide synthesis) • NMR data suggested that a revision of chromophore stereochemistry was required S S S R S S Williams’ structure Yao’s structure

  42. Outline • Background • Discovery and Structural Assignment • Macrocycle Studies • Yao’s Synthesis • Boger’s Synthesis • Conclusions

  43. Boger’s Retrosynthesis Boger et al, JACS, 2008, 130,12355-12369

  44. Boger’s Retrosynthesis Standard peptide synthesis Boger et al, JACS, 2008, 130,12355-12369

  45. Boger’s Retrosynthesis Boger et al, JACS, 2008, 130,12355-12369

  46. Boger’s Total Synthesis 90% yield TMSI 120oC, μW 95% yield Boger et al, JACS, 2008, 130,12355-12369

  47. Boger’s Total Synthesis Boger et al, JACS, 2008, 130,12355-12369

  48. Boger’s Total Synthesis 90% yield TMSI MeCN 120oC, μW 95% yield 3 Boger et al, JACS, 2008, 130,12355-12369

  49. Boger’s Total Synthesis (racemic mixture) Boger et al, JACS, 2008, 130,12355-12369

  50. Boger’s Chromophore Studies Boger et al, JACS, 2008, 130,12355-12369

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