SEPARATION OF METHYLATED PEPTIDICSTANDARDS BY HPLC

Erin Saucke-LacelleSupervisors:

Dr. Ralf SchirrmacherDr. Chris Wilds

OBJECTIVESSynthesize 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

PREVIOUS WORKIn order to investigate radioactive methylation reactions of a model peptide, an HPLC method was developed separating all the methylated peptidic standard compounds.Identities of radioactive products were determined via HPLC by comparing their retention times to those of mono-methylated standards. (1)

Octreotide is a biologically active cyclic octapeptide which is used in the assessment of neuroendocrine tumors (2)

PRECURSOR PEPTIDE

Peptide Sequence:

Trp-Cys-Gly-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr

OCTREOTIDE(KIMCHI)

POSSIBLE METHYLATION PRODUCTS

METHODS USEDFmoc 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

SOLID PHASE PEPTIDE SYNTHESIS

1:1 Piperidine/DMF for removing Fmoc which protects incoming amino acid’s NH2 terminal

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

FMOC protective group

PIP

CO2_NHC(R)COOH

where R=growing peptide chain

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

PURIFICATION AND SEPARATION

H20, ACN. TFA content in both solvents 0.05%

Gradient from 100% H20 to 100% ACN over 40 min

Chromolith Column, 100 x 4.6mm

flow 1.4 mL/min

Agilent 1200 series multi-wavelength UV detector

Raytest gamma detector

SEPARATIONObtain baseline separation of methylated standards

33% ACN 39% ACN

CONCLUSIONSSuccessfully synthesized model octreotide derivative

Synthesized peptides to represent most likely monomethylated standards

This HPLC method sufficiently separates standards in order to clearly identify radiochemistry methylation results

FUTURE WORKPreform methylation reactions of peptides with 11C-methyl triflate & nonaflate Isolate and identify products

Hopefully, alkylation will occur at Cys residue to give a single labeled product

REFERENCES E. Saucke-Lacelle, paper presented at the 236th American Chemical Society National Meeting and Exposition, Washington, DC, 16 August 2009

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

ACKNOWLEDGEMENTS

Dr. Ralf Schirrmacher

Dr. Esther Schirrmacher

Dr. Chris Wilds Ryuhjin Angela Ahn

Grant from Natural Sciences and Engineering Research Council of Canada