A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

Abstract A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytic...

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Autores principales:	Kevin Herr, Max Fleckenstein, Martin Brodrecht, Mark V. Höfler, Henrike Heise, Fabien Aussenac, Torsten Gutmann, Michael Reggelin, Gerd Buntkowsky
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3e3cc9a70aab41148a91c6390f29b657
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spelling	oai:doaj.org-article:3e3cc9a70aab41148a91c6390f29b6572021-12-02T16:10:27ZA novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy10.1038/s41598-021-92975-62045-2322https://doaj.org/article/3e3cc9a70aab41148a91c6390f29b6572021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92975-6https://doaj.org/toc/2045-2322Abstract A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.Kevin HerrMax FleckensteinMartin BrodrechtMark V. HöflerHenrike HeiseFabien AussenacTorsten GutmannMichael ReggelinGerd BuntkowskyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kevin Herr Max Fleckenstein Martin Brodrecht Mark V. Höfler Henrike Heise Fabien Aussenac Torsten Gutmann Michael Reggelin Gerd Buntkowsky A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
description	Abstract A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.
format	article
author	Kevin Herr Max Fleckenstein Martin Brodrecht Mark V. Höfler Henrike Heise Fabien Aussenac Torsten Gutmann Michael Reggelin Gerd Buntkowsky
author_facet	Kevin Herr Max Fleckenstein Martin Brodrecht Mark V. Höfler Henrike Heise Fabien Aussenac Torsten Gutmann Michael Reggelin Gerd Buntkowsky
author_sort	Kevin Herr
title	A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
title_short	A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
title_full	A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
title_fullStr	A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
title_full_unstemmed	A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy
title_sort	novel strategy for site selective spin-labeling to investigate bioactive entities by dnp and epr spectroscopy
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/3e3cc9a70aab41148a91c6390f29b657
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A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

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