| Assay Detail |
The assay was conducted using MultiScreen-IP Filter Plate, 0.45 µm (Merck) and MultiScreen 96-well Transport Receiver Plate (Merck). 300 µL of 5% DMSO/PBS was added to each well of the acceptor plate. 5 µL of 1% lecithin/dodecane was carefully added on PVDF membrane of each well of the donor plate for preparing an artificial lipid membrane. (The lecithin from soybean was purchased from Alfa Aesar.) 150 µL of compound solution was added to each well of the donor plate. Compound solution consists of a compound in 5% DMSO/PBS and the concentrations of each compound were set to 50 µM and 10 µM for dipeptides and cyclic peptides, respectively. The donor plate was docked on the acceptor plate and the plates were incubated in a box containing a wet paper towel at 25 °C for 18 h and 17 h for dipeptides and cyclic peptides, respectively. After incubation, the concentrations of compounds in donor wells and acceptor wells were determined by reacting with azidocoumarin under copper catalyzed alkyne-azide cycloaddition reaction for dipeptides as described in the previous report,5 or determined by LC-MS for cyclic peptides. From the determined concentration in donor wells and acceptor wells, the effective permeability coefficient (Pe) of each dipeptide was calculated using the following equations: Pe = (-ln[1-CA(t)/Cequilibrium])/(A*(1/VD+1/VA)*t), Cequilibrium = [CD(t)*VD + CA(t)*VA]/(VD + VA). A = filter area (0.3 cm^2) VD = donor well volume (0.15 mL) VA = acceptor well volume (0.30 mL) t = incubation time (s) CA(t) = compound concentration in acceptor well at time t CD(t) = compound concentration in donor well at time t CA(t) and CD(t) were determined by the standard curves generated using 1.0, 0.33, 0.11, 0.037, 0.012 and 0.0041 µM of peptides.
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