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Structure‐guided RP‐HPLC chromatography of diastereomeric α ‐helical peptide analogs substituted with single amino acid stereoisomers
Author(s) -
Huang YiBing,
Pan Ling,
Zhao LianJing,
Mant Colin T.,
Hodges Robert S.,
Chen Yuxin
Publication year - 2014
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3061
Subject(s) - chemistry , peptide , high performance liquid chromatography , diastereomer , chromatography , amino acid , residue (chemistry) , selectivity , reversed phase chromatography , amide , stereochemistry , biochemistry , catalysis
An α ‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo ‐Ile, d ‐Ile and d ‐ allo ‐Ile. We compared differences in peptide retention behavior on a C 8 column and a C 18 column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo ‐Ile‐substituted analogs of a 26‐residue α ‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α ‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo ‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.