title: Use of a temporary "solubilizing" peptide tag for the Fmoc solid-phase synthesis of human insulin glargine via use of regioselective disulfide bond formation.
abstract: Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.
authors: Howard Florey Institute, Department of Biochemistry and Molecular Biology, School of Chemistry, Department of Medicine (AH/NH), The University of Melbourne, Victoria 3010, Australia.
authors: Mohammed Akhter
authors: MA
authors: Hossain
authors: Alessia
authors: A
authors: Belgi
authors: Feng
authors: F
authors: Lin
authors: Suode
authors: S
authors: Zhang
authors: Fazel
authors: F
authors: Shabanpoor
authors: Linda
authors: L
authors: Chan
authors: Chris
authors: C
authors: Belyea
authors: Hue-Trung
authors: HT
authors: Truong
authors: Amy R
authors: AR
authors: Blair
authors: Sof
authors: S
authors: Andrikopoulos
authors: Geoffrey W
authors: GW
authors: Tregear
authors: John D
authors: JD
authors: Wade
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001786:Blood Glucose / Q000032:analysis / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000069036:Insulin Glargine; D049528:Insulin, Long-Acting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D012995:Solubility; D013237:Stereoisomerism
chemical_list: D001786:Blood Glucose; D004220:Disulfides; D007328:Insulin; D049528:Insulin, Long-Acting; D010455:Peptides; D000069036:Insulin Glargine