EFFECT OF RESTRICTED CONFORMATIONAL FLEXIBILITY ON THE PERMEATION OF MODEL HEXAPEPTIDES ACROSS CACO-2 CELL MONOLAYERS

Purpose, To determine how restricted conformational flexibility of hex apeptides influences their cellular permeation characteristics. Method s, Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X Ala]; X = Asp, Asn, Lys) hexapeptides were s ynthesized, and their transport characteristics were assessed using th e Caco-2 cell culture model. The lipophilicities of the hexapeptides w ere determined using an immobilized artificial membrane. Diffusion coe fficients used to calculate molecular radii were determined by NMR. Tw o-dimensional NMR spectroscopy, circular dichroism, and molecular dyna mic simulations were used to elucidate the most favorable solution str ucture of the cyclic Asp-containing peptide. Results, The cyclic hexap eptides used in this study were 2-3 times more able to permeate (e.g., P-app = 9.3 +/- 0.3 x 10(-8) cm/sec, X = Asp) the Caco-2 cell monolay er than were their linear analogs (e.g., P-app = 3.2 + 0.3 X 10(-s) cm /sec, X = Asp). In contrast to the linear hexapeptides, the flux of th e cyclic hexapeptides was independent of charge. The cyclic hexapeptid es were shown to be more lipophilic than the linear hexapeptides as de termined by their retention times on an immobilized phospholipid colum n. Determination of molecular radii by two different techniques sugges ts little or no difference in size between the linear and cyclic hexap eptides. Spectroscopic data indicate that the Asp-containing linear he xapeptide exists in a dynamic equilibrium between random coil and beta -turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two beta-turn s. Conclusions. Cyclization of the linear hexapeptides increased their lipophiticities. The increased permeation characteristics of the cycl ic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of th ese increased lipophilicities. Structural analyses of the cyclic Asp-c ontaining hexapeptide suggest that its well-defined solution structure and, specifically the existence of two beta-turns, explain its greate r lipophilicity.