Gf b glass fiber strips

Most of the chemicals and solvents were obtained from Sigma Aldrich. Protected amino acids were provided by Trimen Co. (Lodz, Poland) and the MBHA Rink-Amide peptide resin (100–200 mesh, 0.8 mmol/g) by NovaBiochem. Opioid radioligands [³H]DAMGO, [³H]deltorphin-2 and [³H]U-69593 and human recombinant opioid receptors came from PerkinElmer (Krakow, Poland). GF/B glass fiber strips were purchased from Whatman (Brentford, UK). Analytical and semi-preparative RP HPLC was performed using a Waters Breeze instrument (Milford, MA, USA) with a dual absorbance detector (Waters 2487). The HR ESI-MS experiments were performed on a Shimadzu LCMS-IT-TOF (ion trap–time-of-flight) hybrid mass spectrometer (Shimadzu, Japan) equipped with an ESI source connected to a Nexera HPLC system (Shimadzu, Japan) with auto tuning in the positive-ion mode. Peptide solutions (1 μL) were introduced in a 0.2 mL/min flow of mobile phase (water:acetonitrile (1:1) with 0.1% HCOOH).

Most of the chemicals and solvents were obtained from Sigma Aldrich (Poznan, Poland). Protected amino acids were provided by Trimen Co (Lodz, Poland), and MBHA Rink-Amide peptide resin (100–200 mesh, 0.8 mmol/g) was provided by NovaBiochem. Opioid radioligands, [³H]DAMGO, [³H]deltorphin-2, and [³H]U-69593, and human recombinant ORs and NK1 receptor came from PerkinElmer (Krakow, Poland). GF/B glass fiber strips were purchased from Whatman (Brentford, UK). Analytical and semi-preparative RP HPLC was performed using a Waters Breeze instrument (Milford, MA, USA) with a dual absorbance detector (Waters 2487). The ESI-MS experiments were performed on a Bruker FTICR (Fourier transform ion cyclotron resonance) Apex-Qe Ultra 7 T mass spectrometer equipped with a standard ESI source. The instrument was operated in the positive ion mode and calibrated with the Tunemix™ mixture (Agilent Technologies, CA, USA). The structures of peptides were confirmed using a Shimadzu LCMS-IT-TOF (ion trap–time-of-flight) hybrid mass spectrometer with auto-tuning in the positive ion mode.

Standard chemicals were obtained from commercial sources. Purification of the compounds was performed by flash chromatography over silica gel; eluent: cyclohexane/EtOAc 85:15. The purity of the final products was analyzed by reverse-phase (RP) HPLC, performed on Agilent 1100 series apparatus (Agilent, Technologies, Waldbronn, Germany), equipped with a RP column Phenomenex (Torrance, CA, USA) No 00D-4439-Y0 Gemini 3 μm C18 110 Å, LC column 100 mm × 3.0 mm; diode-array detector (DAD) λ = 210 nm and 254 nm; mobile phase: from 9:1 H₂O/acetonitrile (ACN) containing 0.1% formic acid to 2:8 H₂O/ACN containing 0.08% formic acid in 20 min, flow rate 1.0 mL min⁻¹. ESI-MS was done on a MS single quadrupole HP 1100 MSD detector (Agilent). ¹H NMR analysis was performed on a Varian Gemini 400 MHz apparatus (Agilent Technologies); peptide samples were dissolved in CDCl₃ or in DMSO-d6 to the final concentration of 0.01 M and analyzed in 5 mm tubes at rt. Water suppression required the PRESAT presaturation procedure. Chemical shifts (δ) are expressed as p.p.m., using the residual peak of the deuterated solvent as an internal standard (δH DMSO-d6 = 2.50 p.p.m., δH CDCl₃ = 7.27 p.p.m.).
Opioid radioligands, [³H]DAMGO, [³H]deltorphin-2 and [³H]U-69593, and human recombinant opioid receptors came from PerkinElmer (Krakow, Poland). GF/B glass fiber strips were purchased from Whatman (Brentford, UK).