A Selective Cucurbit[8]uril‐Peptide Beacon Ensemble for the Ratiometric Fluorescence Detection of Peptides

Abstract A convenient supramolecular strategy for constructing a ratiometric fluorescent chemosensing ensemble, consisting of a macrocyclic host (cucurbit[8]uril CB[8]), and a pyrene‐tagged amphiphilic peptide beacon (AP 1), is reported. AP 1 unfolds upon encapsulation of the pyrene termini into the hydrophobic CB[8] cavity. This changes pyrene excimer to monomer emission. Substrates with higher affinity for the CB[8] cavity can displace AP 1 from the ensemble. The released AP 1 folds again to form a pyrene excimer, which allows for the ratiometric fluorescence monitoring of the substrate. In this report, the ensemble capacity for ratiometric fluorescence monitoring of biological substrates, such as amino acid derivatives, specific peptides, and proteins, in aqueous media is demonstrated.


Materials and methods:
Solvents were dried and distilled before use. Millipore water was obtained with a Micropure apparatus from TKA. Lyophilization was carried out with an Alpha 1-4 2D plus freeze drying apparatus from Christ. Analytical TLC was carried out on SiO 2 aluminium foils ALUGRAM SIL G/UV 254 from Macherey-Nagel. Reversed phase column chromatography was done with an Armen Instrument Spot Flash Liquid Chromatography MPLC apparatus with RediSep C-18 Reversed-Phase columns. The purity of the compounds was determined with the help of an HPLC apparatus from Dionex containing the following components: P680 HPLC pump, ASI-100 Automated Sample Injector and UVD 340U detector. A Supelcosil TM LC-18 column (25 cm × 4.6 mm, 5 μm) from Supelco or a YMC ODS-A column (15 cm × 3.0 mm, 5 μm) was utilized. Ultrapure water and HPLC-grade solvents were used as eluents.
Detection was achieved with the help of a UV detector. 1 H-and 13 C-NMR spectra were recorded on DRX 500 MHz spectrometer from Bruker at ambient temperature. The chemical shifts are reported in parts per million (ppm) relative to the deuterated solvent DMSO-d 6 and D 2 O. All mass spectra were received by using a Bruker BioTOF III. Determination of pH values was carried out with a pH-Meter 766 Calimatic from Knick. Fluorescence spectra were obtained with a Varian Cary Eclipse spectrometer. For calculation, software used: MacroModel, V11.4, Schrodinger, LLC and method used: OPLS2005/Water.

General procedure for the synthesis of the peptide:
Fmoc Removal: The Fmoc protecting group was cleavage by treatment with 20% piperidine in DMF (2×6 mL, 5 min each) under microwave radio condition (20 W, 50±5 °C, 5 min).
Then, the resin was washed 3×8 mL with DMF, 3×8 mL with DCM, 3×8 mL with DMF (ca. 5 min each) to remove the last traces of piperidine. A positive Kaiser test confirmed the cleavage of the Fmoc group and the presence of a free amino function.

Standard Fmoc solid phase peptide synthesis techniques (SPPS):
Each amino acid was attached using 0.6 mmol/g loading Fmoc Rink amide resin under microwave radio (20 W, 60±5 °C, 20 min) Then, the resin was washed 3×8 mL with DMF, 3×8 mL with DCM, 3×8 mL with DMF (ca. 5 min each) to remove the last traces of the amino acid. A negative Kaiser test confirmed the attachment of the corresponding amino acid.

S3
Cleavage from the Resin: Cleavage of the product from the resin was achieved by treatment with a mixture of TFA/H 2 O/triisopropylsilane (95:2.5:2.5) for 3 h. The yellow cleavage mixture was collected by filtration and the resin was washed twice with pure TFA (6 mL).
The filtrates were combined and concentrated under vacuum to obtain an oily residue. The peptide was precipitated by adding dry diethyl ether to the oil, following by centrifugation of the mixture. The precipitate was dissolved in water (25 mL), and the mixture was freeze dried in vacuum. The resulting solid was purified by MPLC on C18 reversed phase silica gel (MeOH/water, 0.1 % TFA). Then the product was dissolved three times in HCl aq (0.1 N) plus one time in water and consequently freeze dried to obtain white solid.
Purity of the peptides was checked by HPLC on a RP18-column using water/MeOH (with 0.05% TFA) as solvent.