Fmoc-Dab(Dde)-OH

The antifungal antibiotic aureobasidin A (AbA) is a cyclic depsipeptide composed of eight amino acids and a hydroxy acid. New Ab analogs were produced by feeding various amino acids to Aureobasidium pullulans R 106 c-712 in a chemically-defined medium containing glucose and ammonium sulfate. The constituent amino acids of AbA at positions 3 (L-phenylalanine), 4 (N-methyl-L-phenylalanine), 5 (L-proline), 6 (L-allo-isoleucine) and 8 (L-leucine) were replaced by respective analogous amino acids such as o-fluoro-L-phenylalanine, 4-hydroxy-L-proline, L-norleucine and L-norvaline, resulting in the production of eight new Ab analogs. This is the first paper to describe amino acid replacements at positions 3, 5 and 8. L-[1-13C]-Valine exogenously added was incorporated into the three valine-related moieties of AbA at positions 2, 7 (both N-methyl-L-valine) and 9 (beta-hydroxy-N-methyl-L-valine), but these moieties were never replaced by exogenous amino acid analogs. The comparative antifungal activities of AbA and the eight new Ab analogs were determined.

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

The 1H and 13C NMR spectra of aureobasidins A and E were analyzed by a variety of 2D NMR techniques. Two isomers of aureobasidin A existed as an equilibrium mixture in deuteriochloroform. The isomerism was associated with cis-trans rotation of the amide bond between N-methylphenylalanine and proline. Almost all of the aureobasidin E was found in deuteriochloroform as one conformer; the amide bond between beta-hydroxy-N-methylphenylalanine and proline was in the cis-conformation. Experiments with the NOE made identification of the conformation of the amide bonds of aureobasidins A and E possible.