separation of methylated peptidic standards via hplc
TRANSCRIPT
- 1. SEPARATION OF METHYLATED PEPTIDIC STANDARDS BY HPLC Erin Saucke-Lacelle Supervisors:Dr. Ralf Schirrmacher Dr. Chris Wilds
2. OBJECTIVES
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- Synthesize a biologically active peptide combining desired features of radio-labelling precursor (rapid synthesis, high selective uptake)
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- Synthesize the peptidic standards that represent the most likely mono-methylation products
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- Achieve baseline separation of these peptides via HPLC
3. PREVIOUS WORK
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- 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)
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- 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
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- Fmoc solid phase peptide synthesis
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- Separation of products by HPLC
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- Identification of peptides via MALDI-TOF
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- Purification of the desired product by HPLC
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- HPLC method optimization to achieve baseline separation of peptides
8. SOLID PHASE PEPTIDE SYNTHESIS
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- 1:1 Piperidine/DMF for removing Fmoc which protects incoming amino acids NH 2terminal
- 9. HBTU/DIPEA in DMF for coupling amino acid
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- 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
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- Separate by HPLC
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- 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
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- Obtain baseline separation of methylated standards
33% ACN 39% ACN 18. CONCLUSIONS
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- 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
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- 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
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- 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