On-Resin Strategy to Label α-Conotoxins: Cy5-RgIA, a Potent α9α10 Nicotinic Acetylcholine Receptor Imaging Probe

Markus Muttenthaler; Simon T. Nevin; Marco Inserra; Richard J. Lewis; David J. Adams; Paul F. Alewood

doi:10.1071/CH19456

RESEARCH ARTICLE

On-Resin Strategy to Label α-Conotoxins: Cy5-RgIA, a Potent α9α10 Nicotinic Acetylcholine Receptor Imaging Probe

Markus Muttenthaler ^A ^B ^E , Simon T. Nevin ^C , Marco Inserra ^A , Richard J. Lewis ^A , David J. Adams ^D and Paul F. Alewood ^A ^E

+ Author Affiliations

- Author Affiliations

^A Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.

^B Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Vienna 1090, Austria.

^C Centre for Advanced Imaging, The University of Queensland, Brisbane, Qld 4072, Australia.

^D Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia.

^E Corresponding authors. Email: markus.muttenthaler@univie.ac.at; p.alewood@uq.edu.au

Australian Journal of Chemistry 73(4) 327-333 https://doi.org/10.1071/CH19456
Submitted: 13 September 2019 Accepted: 16 October 2019 Published: 3 December 2019

Abstract

In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with tert-butyloxycarbonyl solid phase peptide synthesis (Boc-SPPS), and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5, resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine (100 µM)-evoked current reversibly with an IC₅₀ of 5.0 nM (Hill coefficient = 1.7) for Cy5-RgIA and an IC₅₀ of 1.6 (Hill coefficient = 1.2) for α-RgIA at the α9α10 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in the RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry, allows site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 fluorescence resonance energy transfer (FRET) pairs.

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On-Resin Strategy to Label α-Conotoxins: Cy5-RgIA, a Potent α9α10 Nicotinic Acetylcholine Receptor Imaging Probe

Abstract

References

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