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Kjeldsen T, Hogendorf WFJ, Tornøe CW, et al. Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG. ACS Omega. 2020;5(31):19827-19833doi: 10.1021/acsomega.0c02712.
Kjeldsen, T., Hogendorf, W. F. J., Tornøe, C. W., Anderson, J., Hubalek, F., Stidsen, C. E., Sorensen, J. L., & Hoeg-Jensen, T. (2020). Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG. ACS omega, 5(31), 19827-19833. https://doi.org/10.1021/acsomega.0c02712
Kjeldsen, Thomas, et al. "Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG." ACS omega vol. 5,31 (2020): 19827-19833. doi: https://doi.org/10.1021/acsomega.0c02712
Kjeldsen T, Hogendorf WFJ, Tornøe CW, Anderson J, Hubalek F, Stidsen CE, Sorensen JL, Hoeg-Jensen T. Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG. ACS Omega. 2020 Jul 31;5(31):19827-19833. doi: 10.1021/acsomega.0c02712. eCollection 2020 Aug 11. PMID: 32803078; PMCID: PMC7424725.
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ACS Omega. 2020 Jul 31;5(31):19827-19833. doi: 10.1021/acsomega.0c02712. eCollection 2020 Aug 11.

Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG.

ACS omega

Thomas Kjeldsen, Wouter F J Hogendorf, Christian W Tornøe, Jonathan Anderson, Frantisek Hubalek, Carsten E Stidsen, Jan L Sorensen, Thomas Hoeg-Jensen

Affiliations

  1. Novo Nordisk A/S, Novo Nordisk Park H5.S.51, DK-2760 Måløv, Denmark.

PMID: 32803078 PMCID: PMC7424725 DOI: 10.1021/acsomega.0c02712

Abstract

Covalent cross-linking of biomolecules can be useful in pursuit of tissue targeting or dual targeting of two receptors on cell surfaces for avidity effects. Long linkers (>10 kDa) can be advantageous for such purposes, and poly(ethylene glycol) (PEG) linkers are most commonly used due to the high aqueous solubility of PEG and its relative inertness toward biological targets. However, PEG is non-biodegradable, and available PEG linkers longer than 5 kDa are heterogeneous (polydisperse), which means that conjugates based on such materials will be mixtures. We describe here recombinant linkers of distinct lengths, which can be expressed in yeast, which are polar, and which carry orthogonal reactivity at each end of the linker, thus allowing chemoselective cross-linking of proteins. A conjugate between insulin and either of the two trypsin inhibitor peptides/proteins exemplifies the technology, using a GQAP-based linker of molecular weight of 17 848, having one amine at the N-terminal, and one Cys, at the C-terminal. Notably, yeast-based expression systems typically give products with mixed disulfides when expressing proteins that are equipped with one unpaired Cys, namely, mixed disulfides with glutathione, free Cys amino acid, and/or a protein homodimer. To obtain a homogeneous linker, we worked out conditions for transforming the linker with mixed disulfides into a linker with a homogeneous disulfide, using excess 4-mercaptophenylacetic acid. Subsequently, the N-terminal amine of the linker was transformed into an azide, and the C-terminal Cys disulfide was reduced to a free thiol and reacted with halo-acetyl insulin. The N-terminal azide was finally conjugated to either of the two types of alkyne-containing trypsin inhibitor peptides/proteins. This reaction sequence allowed the cross-linked proteins to carry internal disulfides, as no reduction step was needed after protein conjugations. The insulin-trypsin inhibitor conjugates were shown to be stabilized toward enzymatic digestions and to have partially retained binding to the insulin receptor.

Copyright © 2020 American Chemical Society.

Conflict of interest statement

The authors declare the following competing financial interest(s): All the authors are employees of the pharmaceutical company Novo Nordisk A/S, but Novo Nordisk has no direct economic interest in the

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