title: Structure-guided RP-HPLC chromatography of diastereomeric α-helical peptide analogs substituted with single amino acid stereoisomers.
abstract: 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 C8 column and a C18 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.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 QianJin Street, Changchun, 130012, China; National Engineering Laboratory for AIDS Vaccine, Jilin University, 2699 QianJin Street, Changchun, 130012, China; School of Life Sciences, Jilin University, 2699 QianJin Street, Changchun, 130012, China.
authors.forename: Yibing
authors.initials: Y
authors.lastname: Huang
authors.forename: Ling
authors.initials: L
authors.lastname: Pan
authors.forename: Lianjing
authors.initials: L
authors.lastname: Zhao
authors.forename: Colin T
authors.initials: CT
authors.lastname: Mant
authors.forename: Robert S
authors.initials: RS
authors.lastname: Hodges
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004912:Erythrocytes; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D017433:Protein Structure, Secondary; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D010455:Peptides
keywords: chiral stereoisomer; hydrophobicity; secondary structure; α-helical peptide