Synthesis and Study of the Cyclic Lipopeptide Antibiotics Daptomycin and A54145
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Daptomycin (dap) is a clinical antibiotic used to treat infections with multi-drug resistant Gram-positive bacteria. Despite nearly 20 years of clinical use, the action mechanism of this important peptide is not fully understood. However, it is known that dap targets the bacterial membrane to which it binds in a calcium and phosphatidylglycerol (PG, an anionic membrane lipid found in bacteria) dependent manner. More recent works on dap suggested that it may possess a chiral target, but the identity of this chiral target was unknown. A54145D (A5D) is a peptide natural product related to dap in both structure and function. Like dap, A5D requires calcium and PG for membrane insertion. In contrast to dap, A5D is not as greatly inhibited by lung surfactant (LS) and thus A5D was deemed a lead compound for the development of a treatment for community acquired bacterial pneumonia (CAP). To date no clinical drugs have come out of these efforts and no explanation for the difference in the antagonism of dap and A5D in the presence of lung surfactant has been forthcoming. To better elucidate the mechanism of action of these peptides, we set out to determine if they have a chiral target and if this chiral target is a stereoisomer of PG. Preliminary investigations done using synthetically accessible dap analogs and stereochemically impure samples of PG suggested that dap and PG may participate in a chiral interaction, warranting further investigation with dap and stereochemically pure samples of PG. To this end, we aimed to synthesize dap, A5D and their unnatural enantiomers, as well as the 4 stereoisomers of PG. Enroute to a new synthesis of dap and its unnatural enantiomer (ent-dap), we developed a highly efficient, enantiospecific and diastereoselective synthesis of (2S,3R)- and (2R,3S)-methylglutamate (an unusual amino acid found in dap) suitably protected for solid-phase peptide synthesis (SPPS). This route was used to prepare multigram quantities of each aforementioned isomer of methylglutamate. A new synthesis of dap was developed which overcame many of the shortcomings of pervious routes. Key to the development of this synthesis was the elucidation of an unprecedented side reaction involving a central building block. Modification of this building block rendered it stable to Fmoc SPPS and led to the first truly efficient synthesis of daptomycin. This route was used to prepare dap and ent-dap. Bioactivity assays of dap and ent-dap revealed that dap is approximately 85-fold more active than ent-dap strongly supporting the proposal that dap interacts with a chiral molecule as part of its mechanism of action. To determine if this molecule is a stereoisomer of PG, we developed a stereospecific synthesis of PG which gave access to all 4 stereoisomers. Using these lipids, we demonstrated that dap recognizes both stereocenters of PG but is more sensitive to the stereocenter at the headgroup of this lipid. Using circular dichroism and fluorescence, we determined that lipid stereochemistry influences the membrane affinity, backbone conformation and oligomerization of dap. Since these are all predictors of antibacterial activity, we concluded that the chiral target of dap is 2R,2'S-PG – the naturally most abundant stereoisomer of this lipid. The interaction between dap and PG emerged as central to the antibacterial action of dap and thus we set out to establish a structure-activity relationship between these two molecules. To this end, 9 modified PGs were prepared. Modification to the headgroup of PG was found to substantially affect membrane affinity, backbone conformation and oligomerization. The data collected suggests that daptomycin envelops the headgroup of PG and possesses unique binding vi sites for each hydroxyl group at the headgroup of this lipid. Shortening the acyl tails of PG demonstrated that PGs containing two saturated, linear tails of 8 carbons in length are needed for binding at micromolar concentrations. Furthermore, these data demonstrate that incorporation of PGs into a lipid membrane and preassembly of PGs are not perquisites for daptomycin-PG interactions. Dap resistant strains of bacteria often possess mutations to an enzyme involved in the lysinylation of PG converting it to lysyl-PG. Despite considerable efforts by many research groups, it is currently unclear how increased lysyl-PG content can confer dap resistance. Our structure-activity study suggested that lysinylation of PG may confer resistance to dap by effectively masking PG thereby reducing the number of binding sites for dap. To investigate this hypothesis, we developed a stereospecific synthesis of lysyl-PG and explored how the presence of lysyl-PG in PG-containing model membranes affected the affinity and membrane bound state of daptomycin. We found that dap possesses a substantially reduced affinity for lysyl-PG compared to PG confirming the plausibility of the masking hypothesis; however, the collected data does not provide a compelling explanation for observed levels of dap resistance when the quantities of lysyl-PG in resistant strains of bacteria are considered. We aimed to determine whether A5D targets the same stereoisomer of PG as dap by developing a total synthesis of A5D. We achieved the total synthesis of this natural product and I contributed to this effort by developing new routes to the unusual amino acids present in A5D. Key to the synthesis of these building blocks was the development of conditions for the Sharpless asymmetric aminohydroxylation reaction which allowed for the preparation of Fmoc-protected 1,2-aminoalcohols. Through reaction scoping, we were able to demonstrate that these conditions yield products with enantio- and regioselectivities that rival the classical reaction conditions. The scoping study resulted in the discovery of a substrate well-suited for the synthesis of the unusual amino acids present in the unnatural enantiomer of A5D (ent-A5D). Bioactivity studies on A5D and ent-A5D revealed that A5D is 32-fold more active than ent-A5D proving that A5D possesses a chiral target. To determine if this chiral target is a stereoisomer of PG, we prepared a fluorescently labelled analog of A5D using some of the synthetic methodology we previously developed. This analog of A5D was found to have nearly identical antimicrobial activity compared to the natural product. Using this analog, it was determined that PG stereochemistry influences the membrane affinity, backbone conformation and oligomerization of A5D. A5D possessed a strong preference for 2R,2'S-PG and thus it was concluded that 2R,2'S-PG is the chiral target of A5D. LS contains high concentrations of PG; thus, it was hypothesized that PG is responsible for the antagonism of dap and A5D, and the activity difference of these peptides in the presence of LS is due to a difference in how each peptide binds to the different stereoisomers of PG. To investigate this hypothesis, we determined the stereochemical content of PG in mammalian LS using a new approach and found that it consisted solely of 2R,2'S-PG which is preferred by dap and A5D. Binding studies revealed that each peptide binds to this lipid with a μM dissociation constant. Reinvestigation of the claim that dap and A5D possess different activities in the presence of LS were found to contradict previous reports. Indeed, antagonism studies on dap, A5D and analogs thereof demonstrated that each peptide is antagonized equally by LS and the antagonism is fully explained by a sole interaction with PG.
Cite this version of the work
Ryan Moreira (2022). Synthesis and Study of the Cyclic Lipopeptide Antibiotics Daptomycin and A54145. UWSpace. http://hdl.handle.net/10012/18767