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19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR

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Abstract

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

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Acknowledgements

This work was supported by The Danish Council for Independent Research, Technology and Production Sciences (Grant No. 4005-00455), the Danish Council for Independent Research (DFF-1323-00344) and the NovoNordisk Foundation (NNF15OC0016186). We acknowledge laboratory technicians Uraiwan Ngamrabiab Adamsen and Birgitte Simonsen, scholar students Rasmus Svane Pedersen and Zishan Bazai, and postdoc Anna Mette Hansen for assistance with synthesis and purification of peptides. Helene Halkjær Jensen and Malene Brohus are acknowledged for advice on cell culturing. The NMR laboratory at Aalborg University is supported by the Obel Family, Carlsberg and SparNord foundations.

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Authors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark

    Malene V. Christensen, Kenneth T. Kongstad, Dan Staerk & Henrik Franzyk

  2. Section for Biotechnology, Department of Chemistry and Bioscience, Aalborg University, Frederik Bajers Vej 7H, 9220, Aalborg, Denmark

    Teis Esben Sondergaard & Reinhard Wimmer

  3. Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark

    Hanne M. Nielsen

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  1. Malene V. Christensen
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Correspondence to Reinhard Wimmer.

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Christensen, M.V., Kongstad, K.T., Sondergaard, T.E. et al. 19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR. J Biomol NMR 73, 167–182 (2019). https://doi.org/10.1007/s10858-019-00239-3

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