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

Autor:	Richard J. Lewis, Marco Inserra, Simon T. Nevin, Paul F. Alewood, David J. Adams, Markus Muttenthaler
Rok vydání:	2020
Předmět:	chemistry.chemical_classification 0303 health sciences technology industry and agriculture Peptide General Chemistry 7. Clean energy Article 03 medical and health sciences Nicotinic acetylcholine receptor 0302 clinical medicine Nicotinic agonist Förster resonance energy transfer chemistry Biophysics medicine Peptide sequence 030217 neurology & neurosurgery Acetylcholine 030304 developmental biology Macromolecule Acetylcholine receptor medicine.drug
Zdroj:	Aust J Chem Australian Journal of Chemistry
ISSN:	0004-9425
DOI:	10.1071/ch19456
Popis:	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 (100µM)-evoked current reversibly with an IC50 of 5.0nM (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 invivo 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.
Databáze:	OpenAIRE
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