With all peptides in hand we first set out to estimate their passive membrane permeability using the immobilized artificial membrane (IAM) chromatography technique. An IAM column is packed with a reverse phase like material modified with phospholipids to mimic a cellular membrane. After analysis of a compound its retention times is compared to a set of calibration compounds to yield a CHI-IAM value. A higher value indicates a stronger membrane interaction which can be correlated to membrane permeability. Previously, the technique has been applied to both peptidic and non-peptidic macrocycles. The CHI-IAM values indicated that SA-B1 maintained the same capacity to diffuse into a phospholipid membrane as Sanguinamide A (SA-A1) itself. This is interesting especially since the design introduced an extra HBD as well as increased the macrocycle size, both of which are often detrimental for membrane permeability. Surprisingly, the N-oxide variant (SA-B2) had a similar CHI-IAM value as the benzene analogue (SA-B3) in this experiment. Although the values were not as high as SA-B1, the compounds still performed better than the negative control SA-A3. For all extended analogues (SA-C and SA-D series) the membrane affinity was strongly decreased indicating that macrocycle flexibility cannot be too high. To further evaluate the passive membrane permeability of our compounds we used the Caco-2 assay in the presence of a cocktail of transporter inhibitors. The permeability was measured Scheme 5: Synthesis of cyclic peptides in A to B direction and the values correlated well with the CHI-IAM values (Fig. ). One exception was compound SA-B2 which shows significantly lower membrane permeability in Caco-2 cells in comparison to compound SA-B3 even though the CHI-IAM values are similar. It has previously been described that the membrane interaction of charged molecules could be overestimated by IAM chromatography as they do not only interact with the lipid part of the stationary phase but also the charged phosphatidylcholine group which carries both a positive and a negative charge. Besides this difference, it is also evident that Sanguinamide A (SA-A1) has a higher permeability in the Caco-2 assay in comparison to the IAM chromatography.