Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
40, Number 1, January 2002, pp. 1-6
Detection of cis–trans Isomers of a Synthetic Peptide Fragment
of Erythropoietin
R.D. Husain[5], J. McCandless[4],
P.J. Stevenson[3], T. Large[4], D.J.S. Guthrie[1], and B. Walker[2]
[1]School of Biology and Biochemistry,
[2]School of Pharmacy, and
[3]School of Chemistry, The Queen’s University of Belfast, 97 Lisburn Road,
Belfast BT9 7BL, N. Ireland, U.K. and
[4]Applied Biosystems Ltd., Kelvin Close, Birchwood Science Park North, Warrington,
Cheshire WA3 7PB, England, U.K.; and
[5]Michigan State University, Department of Biochemistry, East Lansing, MI 48824
The synthesis of the protected fragment t-butoxycarbonyl-alanine-isoleucine-serine(benzyl)-proline
(Pro)-Pro-OH derived from the hormone erythropoietin is described. The analysis
of the peptide by high-pressure liquid chromatography (HPLC) and thin-layer
chromatography (TLC) yields apparently inconsistent results. Although HPLC consistently
indicates the presence of only one component, TLC reveals a number of distinct
species. Because satisfactory amino acid analysis and fast atom bombardment-mass
spectrometry results are obtained, we think it possible that the distinct components
arise from the cis–trans isomerization of the peptide bonds to the prolyl
residues. An analysis using capillary electrophoresis under basic conditions
identifies four components in the final product. Also, under similar conditions
proton nuclear magnetic resonance spectroscopy is able to confirm the presence
of cis and trans isomers. The results from this study demonstrate the usefulness
of each of the four techniques in identifying the isomerism of the standard
amino acid–Pro bond with respect to the peptide’s ionic state.
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