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Author
Date
2018Type
- Doctoral Thesis
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yes
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Abstract
Insulin is a protein hormone with a pivotal role in the regulation of numerous
metabolic processes. Malfunction of insulin secretion or reduced sensitivity of the
insulin receptor to its native ligand are the main reasons for diabetes mellitus.
Recombinant technologies allow the large-scale production of some insulin variants,
providing a treatment for millions of patients who rely on the daily administration of
the hormone.
The marketed insulin products are life-saving for the people living with diabetes,
however they are not able to restore completely the healthy glucose metabolism.
Mutations in the protein sequence or chemical modifications could improve the
therapeutical properties of human insulin.
Insulin is a small protein (51 amino acid residues), constructed from two peptidic
chains (A- and B-chain) that are held together by two interchain and one intrachain
disulfide bonds in its folded state. This unique structure makes insulin a challenging
target for classical synthetic approaches.
Inspired by the biosynthesis of insulin, we developed a general method for the
chemical synthesis of insulin variants. We utilized a short, traceless prosthetic
C-peptide for facile folding of linear insulin intermediates; the folding precursors were
assembled by α-ketoacid–hydroxylamine (KAHA) ligation.
The first part of the dissertation focuses on the development of the synthetic
platform for insulin. The KAHA ligation proved to be very efficient in assembling the
highly hydrophobic linear insulin variants. The applied prosthetic C-peptide made it
possible to form of the disulfide bonds in a controlled fashion. Using our synthetic
methodology towards insulin we generated four different variants of the protein
(Glargine – M2, human, mouse, guinea pig) and were pleased to find that the
synthetic proteins possessed comparable biological activities to a recombinant
reference compound.
The KAHA ligation based on the (S)-5-oxaproline monomer proved to be a powerful
tool for protein synthesis. In order to further expand the scope of the reaction, new
hydroxylamine monomers were investigated.
In the following chapter we successfully applied the natural aspartic acid-yielding
(S)-4,4-difluoro-5-oxaproline hydroxylamine monomer on the synthesis of a different
Glargine – M2 insulin variant. We were pleased to see that the new monomer could
operate under standard KAHA ligation conditions (DMSO/H2O with 0.1 M oxalic acid)
and yield the ligated peptide in a comparable yield to the (S)-5-oxaproline monomer.
The last part of the thesis describes the synthesis of an insulin variant containing a
hydroxylamine moiety on the side chain of the incorporated ornithine residue that
readily underwent potassium acyltrifluoroborate (KAT) ligation. By taking advantage
of the incorporated hydroxylamine handle, we could access rhodamine-labeled,
PEGylated and dimerized insulins by KAT ligation.
The synthetic platform developed for insulin proved to be useful for the synthesis of
several biologically active insulin variants – regardless the changes in the amino acid
sequence. Furthermore the method enabled the incorporation of non-canonical
amino acid building blocks for late-stage functionalization of the folded protein. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000301883Publication status
publishedExternal links
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Publisher
ETH ZurichSubject
Chemical protein synthesis; Protein modifications; KAHA ligation; KAT ligation; InsulinOrganisational unit
03861 - Bode, Jeffrey W. / Bode, Jeffrey W.
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ETH Bibliography
yes
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