Acylation has previously been shown to increase intestinal permeability of peptide drugs, but detailed investigations of systematic acyl variations are lacking, which would benefit rational new designs of peptide drugs. The in vitro intestinal translocation studies can be further supplemented by measurements of peptide binding to model lipid membranes in order to investigate the influence of membrane binding of acylated peptides on cellular membrane translocation. Glucagon-like peptide-2 is a 33 amino acid peptide, which is secreted from the human intestine following nutrient intake. Therapeutically, GLP-2 stimulates intestinal growth and is employed in the treatment of inflammatory bowel diseases and short bowel syndrome. The plasma half-life of GLP-2 in humans is limited to a few minutes due to extensive renal clearance and rapid enzymatic degradation by dipeptidyl peptidase-4. Furthermore, GLP-2 is presently administered as subcutaneous injections, which compromises patient comfort and compliance, in particular for chronic diseases like Crohn’s. It would be highly beneficial to enable oral administration, and the combined effects of prolonged circulation time, improved enzymatic stability and intestinal permeability may render acylated GLP-2 a suitable candidate for oral drug delivery. Currently, however, there are no reports on the intestinal permeability or oral drug delivery potential of acylated GLP-2. In the present study we synthesized and characterized acylated analogues of GLP-2, with systematically increasing acyl chain length, in order to investigate the effect of the acyl chain on membrane interaction and in vitro intestinal permeability. This was achieved by combining investigations of the interaction with lipid membranes and translocation across an intestinal cell model, as outlined in fig. 1. We hypothesize that the acylation chain length can be optimized for translocation across the intestinal barrier, i.e. a moderate interaction with the lipid cell membrane is beneficial for translocation, whereas a stronger interaction may impair translocation. Acylation is expected to Enzalutamide 915087-33-1 confer membrane affinity to GLP2, as the native peptide is not membrane active. In this regard, GLP-2 was employed as a model peptide, however, the results may be applicable for development of a rational acylation strategy for other peptide drugs. Absorption enhancers are often employed to increase oral peptide absorption, which makes it interesting to investigate how these affect the translocation of acylated peptides. In the present study we included two enhancers with different enhancing mechanism, in order to investigate the effect of the enhancing mechanism. Ethylene glycol-bis-N, N, N, Ntetraacetic acid is a paracellular enhancer which increases transport between the cells by opening of the tight junctions, and sodium dodecyl sulfate is a transcellular enhancer which increases transport through the cells at low concentrations, predominantly by fluidizing the cell membrane. We hypothesize that the effect of paracellular enhancers will not be influenced by acylation, whereas the effect of transcellular enhancers that directly interact with the cell membrane may depend on the peptide-membrane interaction, through altered membrane affinity and/or dynamics of membrane insertion. For EGTA the increase is similar for peptide and analogues, and the dependence on acyl chain length is retained, whereas for SDS the increase is greater for the c16-acylation. These results support the hypothesis that the fluidization of cell membranes caused by SDS are beneficial for the long chain acylation, possibly due to altered membrane insertion.