Separation of Methylated Peptidic Standards via HPLC

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Separation of Methylated Peptidic Standards via HPLC

  1. 1. SEPARATION OF METHYLATED PEPTIDIC STANDARDS BY HPLC Erin Saucke-Lacelle Supervisors: Dr. Ralf Schirrmacher Dr. Chris Wilds
  2. 2. OBJECTIVES <ul><ul><li>Synthesize a biologically active peptide combining desired features of radio-labelling precursor (rapid synthesis, high selective uptake) </li></ul></ul><ul><ul><li>Synthesize the peptidic standards that represent the most likely mono-methylation products </li></ul></ul><ul><ul><li>Achieve baseline separation of these peptides via HPLC </li></ul></ul>
  3. 3. PREVIOUS WORK <ul><ul><li>In order to investigate radioactive methylation reactions of a model peptide , an HPLC method was developed separating all the methylated peptidic standard compounds.
  4. 4. Identities of radioactive products were determined via HPLC by comparing their retention times to those of mono-methylated standards. (1) </li></ul></ul><ul><ul><li>Octreotide is a biologically active cyclic octapeptide which is used in the assessment of neuroendocrine tumors (2) </li></ul></ul>
  5. 5. PRECURSOR PEPTIDE Peptide Sequence: Trp-Cys-Gly-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr OCTREOTIDE (KIMCHI)
  6. 6. POSSIBLE METHYLATION PRODUCTS
  7. 7. METHODS USED <ul><ul><li>Fmoc solid phase peptide synthesis </li></ul></ul><ul><ul><li>Separation of products by HPLC </li></ul></ul><ul><ul><li>Identification of peptides via MALDI-TOF </li></ul></ul><ul><ul><li>Purification of the desired product by HPLC </li></ul></ul><ul><ul><li>HPLC method optimization to achieve baseline separation of peptides </li></ul></ul>
  8. 8. SOLID PHASE PEPTIDE SYNTHESIS <ul><ul><li>1:1 Piperidine/DMF for removing Fmoc which protects incoming amino acid’s NH 2 terminal
  9. 9. HBTU/DIPEA in DMF for coupling amino acid </li></ul></ul><ul><ul><li>95% TFA for removing side-chain protective groups and cleave peptide from resin once peptide is complete </li></ul></ul>CO 2 _ NHC(R)COOH where R=growing peptide chain FMOC protective group PIP
  10. 10. IDENTIFICATION <ul><ul><li>Separate by HPLC </li></ul></ul><ul><ul><li>Identify by MALDI-TOF (Bruker Instrument, 2,5-dihydroxy benzoic acid matrix, in HPLC solution) </li></ul></ul>PRODUCT PRODUCT product + Na product + Na
  11. 11. PURIFICATION AND SEPARATION <ul><li>H 2 0, ACN. TFA content in both solvents 0.05%
  12. 12. Gradient from 100% H 2 0 to 100% ACN over 40 min
  13. 13. Chromolith Column, 100 x 4.6mm
  14. 14. flow 1.4 mL/min
  15. 15. Agilent 1200 series multi-wavelength UV detector
  16. 16. Raytest gamma detector </li></ul>
  17. 17. SEPARATION <ul><ul><li>Obtain baseline separation of methylated standards </li></ul></ul>33% ACN 39% ACN
  18. 18. CONCLUSIONS <ul><ul><li>Successfully synthesized model octreotide derivative
  19. 19. Synthesized peptides to represent most likely monomethylated standards
  20. 20. This HPLC method sufficiently separates standards in order to clearly identify radiochemistry methylation results </li></ul></ul>
  21. 21. FUTURE WORK <ul><ul><li>Preform methylation reactions of peptides with 11 C-methyl triflate & nonaflate
  22. 22. Isolate and identify products
  23. 23. Hopefully, alkylation will occur at Cys residue to give a single labeled product </li></ul></ul>
  24. 24. REFERENCES <ul><li>E. Saucke-Lacelle, paper presented at the 236 th American Chemical Society National Meeting and Exposition, Washington, DC, 16 August 200 9
  25. 25. R. Maurer, B. H. Gaehwiler, H. H. Buescher, R. C. Hill, D. Roemer, Proc. Natl. Acad. Sci. 79(15) , 4815-7 (1982) </li></ul>
  26. 26. ACKNOWLEDGEMENTS <ul><ul><li>Dr. Ralf Schirrmacher
  27. 27. Dr. Esther Schirrmacher
  28. 28. Dr. Chris Wilds
  29. 29. Ryuhjin Angela Ahn
  30. 30. Grant from Natural Sciences and Engineering Research Council of Canada </li></ul></ul>

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