Microotof qiii

Model collagen fragments H-Gly-Hyp-Pro-Ala-Hyp-Pro-OH (1), H-(Pro)₆-OH (2), H-(Pro)₉-OH (3), H-(Hyp)₆-OH (4) and H-(Hyp)₉-OH (5) were synthesized on chlorotrityl resin according the Fmoc/tBu strategy.
Analytical RP-HPLC was performed on a LC Dionex UltiMate 3000 (ThermoFisher Scientific, Waltham, MA, USA), using a Kinetex Reversed Phase C18 column (100 × 4.6 mm). Gradients of 0.1% TFA in H₂O (B) and 0.1% TFA in CH₃CN (A) were used, at a flow rate 0.4 mL/min with UV detection at 220 and 254 nm. Preparative HPLC was done on a CombiFlash, EZPrep, Teledyne ISCO (Lincoln, NE, USA) using a Supelco Discovery BIO Wide Pore C18 column (25 cm × 21.2 mm, 10 mm; Sigma-Aldrich) flow rate 5 mL/min, detection wavelengths 220 and 254 nm, gradient ratio A (0.1% TFA in CH₃CN) and B (0.1% TFA in H₂O) 0:100 to 18:82 in 30 min, followed by an isocratic run for 5 min.
Mass Spectrometry analysis was performed on a Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA, USA).

NMR spectra were measured on a Bruker Avance II Plus (Bruker Corporation, Billerica, MA, USA) spectrometer (700 MHz for ¹H-NMR and 176 MHz for ¹³C-NMR) in CDCl₃ solution. ¹H and ¹³C-NMR spectra were referenced according to the residual peak of the solvent based on literature data. Chemical shifts (d) were reported in ppm and coupling constants (J) were reported in Hz. ¹³C-NMR spectra were proton-decoupled. Flash chromatography was performed using a glass column packed with Baker silica gel (30–60 μm). For TLC, silica gel was used with a 254 nm indicator on Al foils (Sigma-Aldrich, St. Louis, MO, USA). All reagents and solvents were purchased and used as obtained from Sigma-Aldrich (Poznan, Poland). Melting points were obtained using a Büchi SMP-20 apparatus. Mass spectrometry analysis was performed on a Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA, USA) equipped with electrospray ionization mode and a time-of-flight detector (TOF). IR spectra were measured on an FT-IR Alpha Bruker (ATR) instrument in cm⁻¹.