1. SEPARATION OF METHYLATED PEPTIDIC STANDARDS BY HPLC Erin Saucke-Lacelle Supervisors:Dr. Ralf Schirrmacher Dr. Chris Wilds

2. OBJECTIVES

• Synthesize a biologically active peptide combining desired features of radio-labelling precursor (rapid synthesis, high selective uptake)

• Synthesize the peptidic standards that represent the most likely mono-methylation products

• Achieve baseline separation of these peptides via HPLC

3. PREVIOUS WORK

• In order to investigate radioactive methylation reactions of a model peptide , an HPLC method was developed separating all the methylated peptidic standard compounds.

4. Identities of radioactive products were determined via HPLC bycomparing their retention timesto those of mono-methylated standards. (1)

• Octreotideis a biologically active cyclic octapeptide which is used in theassessment of neuroendocrine tumors(2)

5. PRECURSOR PEPTIDE Peptide Sequence:Trp-Cys-Gly-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr OCTREOTIDE (KIMCHI) 6. POSSIBLE METHYLATION PRODUCTS 7. METHODS USED

• Fmoc solid phase peptide synthesis

• Separation of products by HPLC

• Identification of peptides via MALDI-TOF

• Purification of the desired product by HPLC

• HPLC method optimization to achieve baseline separation of peptides

8. SOLID PHASE PEPTIDE SYNTHESIS

• 1:1 Piperidine/DMF for removing Fmoc which protects incoming amino acids NH 2terminal

9. HBTU/DIPEA in DMF for coupling amino acid

• 95% TFA for removing side-chain protective groups and cleave peptide from resin once peptide is complete

CO 2 _ NHC(R)COOH where R=growing peptide chain FMOC protective group PIP 10. IDENTIFICATION

• Separate by HPLC

• Identify by MALDI-TOF (Bruker Instrument, 2,5-dihydroxy benzoic acid matrix, in HPLC solution)

PRODUCT PRODUCT product + Na product + Na 11. PURIFICATION AND SEPARATION

H 2 0, ACN. TFA content in both solvents 0.05%

12. Gradient from 100% H 2 0 to 100% ACN over 40 min 13. Chromolith Column, 100 x 4.6mm 14. flow 1.4 mL/min 15. Agilent 1200 series multi-wavelength UV detector 16. Raytest gamma detector 17. SEPARATION

• Obtain baseline separation of methylated standards

33% ACN 39% ACN 18. CONCLUSIONS

• Successfully synthesized model octreotide derivative

19. Synthesized peptides to represent most likely monomethylated standards

20. This HPLC method sufficiently separates standards in order to clearly identify radiochemistry methylation results 21. FUTURE WORK

• Preform methylation reactions of peptides with11 C-methyl triflate & nonaflate

22. Isolate and identify products

23. Hopefully, alkylation will occur at Cys residue to give a single labeled product 24. REFERENCES

E. Saucke-Lacelle, paper presented atthe 236 thAmerican Chemical Society National Meeting and Exposition, Washington, DC, 16 August 200 9

25. R. Maurer, B. H. Gaehwiler, H. H. Buescher, R. C. Hill, D. Roemer,Proc. Natl. Acad. Sci. 79(15) , 4815-7 (1982) 26. ACKNOWLEDGEMENTS

• Dr. Ralf Schirrmacher

27. Dr. Esther Schirrmacher

28. Dr. Chris Wilds 29. Ryuhjin Angela Ahn 30. Grant from Natural Sciences and Engineering Research Council of Canada