Synthesis and Analysis of Daptomycin Analogues

Abstract

Daptomycin (Dap) is a naturally occurring, membrane-active, calcium-dependent cyclic lipodepsipeptide antibiotic (cLPA) which is used as a last-resort antibiotic to treat serious infections caused by Gram-positive (G+) bacteria including Staphylococcus aureus (S. aureus) and vancomycin-resistant enterococci (VRE). The appearance of Dap-resistant (Dap-R) bacteria with increasing frequency has motivated the search for Dap analogues that are active against Dap-R bacteria. Recently, it has been shown that appending hydrophobic groups to tryptophan (Trp) or kynurenine (Kyn) yielded some Dap analogues with improved activity and were active against Dap-R bacteria. Chapter 2 of this thesis describes the synthesis and evaluation of Dap analogues with hydrophobic modifications to the side chain of the D-asparagine (Asn) residue at position 2 to determine if appending hydrophobic groups to D-Asn2 will also result in Dap analogs with improved activity. Eight Asn derivatives were synthesized containing alkyl or hydroxyl groups appended to the primary amide nitrogen of the D-Asn side chain. Dap analogues containing these D-Asn derivatives at position 2 were synthesized using Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis (SPPS). Dap analogs containing methyl (Me), ethyl (Et), n-propyl (Pr), n-butyl (Bu) and n-hexyl (Hex) on the D-Asn exhibited minimum inhibitory concentration (MIC) values that were 2–4-fold higher than Dap while the n-octyl (Oct) and piperidinyl (Pip) analogs had MIC values that were 8- and 32-fold greater than Dap, respectively. These results demonstrate that the activity of Dap cannot be improved by appending hydrophobic groups to D-Asn2 and suggest that D-Asn2 may not be closely associated with the cell membrane. These results also show that the primary amide of D-Asn2 is not essential for activity while the presence of at least one hydrogen on the nitrogen of the D-Asn2 side chain is very important to activity. Membrane insertion studies using model membranes and fluorescence spectroscopy revealed that the hexyl and octyl analogues were able to insert into membranes even in the absence of Ca²⁺ consistent with their much-increased hydrophobicity compared to Dap. In chapter 3, we wished to determine if it is possible to convert Dap into a Zn+2-dependent antibiotic by substituting the two aspartate (Asp) residues in Dap’s calcium-binding motif, Asp7 and Asp9, with Nγ-hydroxyasparagine (Asn(OH)), an amino acid that has a hydroxamic acid side chain. Hydroxamic acids, known for strong Zn²⁺ chelation, have been used in medicinal chemistry to improve metal-dependent interactions. The synthesis of an Asn(OH) building block with the hydroxamic acid side chain protected with a trityl (Trt) group (Fmoc-Asn(OTrt)-OH) was achieved following a multi-step route starting from Fmoc-Asp(tBu)-OH. Attempts to synthesize Dap analogues containing Asn(OH) at positions 7 or 9 using this building block via Fmoc SPPS failed. However, the synthesis of a Dap analogue containing Asn(OH) at position 2 was successful indicating that incorporation of this residue using this building block is sequence dependent. A new Asn(OH) building block containing a protecting group that is smaller than the Trt group, the dimethoxybenzyl (DMB) group (Fmoc-Asn(ODMB)-OH), was prepared. Attempts to prepare the target peptides using this new building block also failed as were attempts using an Asn(OH) building block with the hydroxamic acid side chain unprotected.