Abstract
A specific labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the disintegrin protein barbourin in the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for the nuclear spin hyperpolarization method of parahydrogen induced polarization (PHIP). The PHIP-label was synthesized and inserted into the disulfide bridge of eptifibatide via reduction of the peptide and insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide-containing biomolecules and preserves their tertiary structure with a minimum of change. HPLC and MS spectra prove the successful insertion of the label. 1H-PHIP-NMR experiments yield a factor of over 1000 as lower limit for the enhancement factor. These results demonstrate the high potential of the labeling strategy for the introduction of site selective PHIP-labels into biomolecules’ disulfide bonds.
Highlights
A specific labeling strategy for bioactive molecules is presented for eptifibatide an antiplatelet aggregation inhibitor, which derives from the disintegrin protein barbourin in the venom of certain rattlesnakes
We demonstrate by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI–MS) that the intercalator 2 can be incorporated into the disulfide bridge of the peptide
The crude reaction product was directly used in the following substitution reaction with p-thiocresol as a nucleophile yielding the bisulfide 6 which, after recrystallization was isolated in 63% yield
Summary
A specific labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the disintegrin protein barbourin in the venom of certain rattlesnakes. Illustration of the Intercalation-hydrogenation strategy to boost signal intensity in NMR experiments using the ethynyl-containing label 2. We point out the high value of the label for PHIP-NMR spectroscopy by hydrogenation of the ethynyl fragment (Scheme 1).
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