Before synthesis of the diastereomer of malacidin A (1a) could commence, appropriately protected building blocks of (2S,3S)-3-MeAsp¹, (2S,3S)-3-MeDap², (2S,3S)-3-HyAsp⁵, (2R,3R)-3-MeAsp⁸ and (2S,4R)-4-MePro¹⁰ and (2E,4Z)-8-methylnona-2,4-dienoic acid were required to facilitate incorporation using solid-phase synthesis.

A robust synthetic strategy toward diastereomer 1a was established by first preparing a simplified analogue (31), wherein all non-canonical amino acids and the unsaturated lipid tail were substituted for the corresponding canonical/commercially available amino acids and decanoic acid, respectively (Figure 2). The synthetic route was inspired by the synthesis of glycinocins A-C reported by the Payne group (Corcilius et al., 2017). It was envisioned that macrocyclization would take place at the same site as the native peptide’s macrolactam bond, i.e. between the β-NH₂ of the Dap² derivative and α-CO₂H of Pro¹⁰. The protected linear precursor was assembled via Fmoc-SPPS on a hyper acid-labile 2-chlorotrityl chloride (2-CTC) polystyrene (PS) resin (Scheme 6). The use of 2-CTC PS resin was also required to prevent diketopiperazine formation upon incorporation of Pro as the C-terminal residue. This synthetic approach required an orthogonally protected Dap² building block, thus, commercially available Fmoc-Dap(Alloc)-OH was used. As the peptide sequence contains an aspartamide-prone Asp⁶Gly⁷ region, dimethoxybenzyl (Dmb)N ^α-protected Gly¹⁰ was used.

The synthesis of 31 commenced with attachment of Fmoc-Pro-OH to 2-CTC resin to give resin-bound 32 (Scheme 6). After capping of unreacted chloride sites with methanol and N,N-diisopropylethylamine (DIPEA), the assembly of orthogonally protected linear precursor 33 was undertaken. Fmoc removal was achieved by treatment with 20% piperidine in N,N-dimethyl formamide (DMF) (v/v). l-Amino acids were coupled using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and DIPEA in DMF for 30 min, and for 2 h when using d-amino acids, Dap², Gly⁷ and decanoic acid. Double coupling was found to be necessary for Fmoc-Dap(Alloc)-OH. Next, Alloc removal from Dap² was carried out using tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) and phenylsilane (PhSiH₃). Next the assembled fully protected linear peptide was cleaved from the resin with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in CH₂Cl₂ (3:7, v/v) for 1 h. Following solvent evaporation the peptidic residue was dissolved in H₂O:CH₃CN (1:4, v/v) and lyophilized. The obtained side chain protected linear precursor 34 was subjected to macrocyclization conditions (peptide concentration 10 mM) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTTM·BF₄) and DIPEA in DMF overnight. No difficulties were observed during the handling and dissolution of the maximally protected peptide. The final side chain deprotection was performed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and H₂O (95:2.5:2.5, v/v/v) for 2 h, followed by purification by reverse-phase high pressure liquid chromatography (RP-HPLC) to give simplified analogue 31 in 9% overall yield.

Late-stage incorporation of the lipid enabled the preparation of five additional lipid tail analogues based on the simplified peptidic core of 31, namely hexanoyl, tetradecanoyl, hexadecanoyl, 4-pentylbenzoyl and 4-phenylbenzoyl analogues 36–40 (Scheme 7). The previously detailed SPPS protocol was used to prepare the common Fmoc protected linear sequence 35, followed by division of the resin for coupling to the various lipid tails. The remaining steps of the synthesis for each analogue were carried out separately using the same conditions as for 31. Analogues 36–40 were thus obtained in 5–17% yield in >96% purity after RP-HPLC purification.

Attention next focused on synthesis of the selected diastereomer of malacidin A 1a. While the same overall strategy could be used, some modification of the synthesis was required to incorporate the (2S,3S)-Fmoc-3-MeDap(Dde)-OH (8), (2S,3S)-Fmoc-3-Hy(TBS)Asp(OtBu)-OH (14) and (2E,4Z)-8-methylnona-2,4-dienoic acid (25) building blocks. The unsaturated nature of the lipid tail precluded use of Alloc as a protecting group on the 3-MeDap² residue sidechain (Keinan and Greenspoon, 1986). (2S,3S)-Fmoc-3-MeDap(Dde)-OH (8) was therefore prepared, as the Dde group could be removed from the fully assembled linear precursor under mild conditions using hydroxylamine hydrochloride/imidazole without affecting the unsaturation of the lipid tail (Díaz-Mochón et al., 2004). Lipid stability investigations (see Supplementary Material Section 5) revealed that partial cis-trans isomerization of the (4Z)-π-bond of the lipid could be minimized by performing the final side chain deprotection in 50% TFA for 30 min. However, removal of the TBS protecting group was incomplete under these conditions and a separate deprotection step was required.

The synthesis of malacidin analogue 1a began with loading of (2S,4R)-Fmoc-4-MePro-OH (20) onto 2-CTC resin followed by iterative Fmoc-SPPS (Scheme 8). Commercially available amino acids were incorporated using the previously established conditions. Custom building blocks (2, 7, 8, 14, 25) were coupled using (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU)/ethyl cyano(hydroxyimino)acetate (Oxyma Pure)/DIPEA as the coupling reagents to maximize the coupling efficiency and reduce the number of molecular equivalents required for complete coupling (1.2 equiv.). Next, TBS removal from the side chain hydroxy group of 3-HyAsp⁵ residue using tetrabutylammonium fluoride (TBAF) buffered with AcOH (1:1, 15 eq.) was performed on peptidyl-resin 42 followed by Fmoc removal and coupling of the polyunsaturated lipid 25. N ^β -Dde removal from the 3-MeDap² residue was carried out under mild conditions using 3.6 M NH₂OH·HCl and 2.7 M imidazole in NMP/CH₂Cl₂ (5:1, v/v) for 4 h. Following cleavage of the resulting peptide sequence from the resin with HFIP:CH₂Cl₂ (3:7, v/v) and solvent evaporation the crude peptide was dissolved in H₂O:CH₃CN (1:4, v/v) and lyophilized. The obtained protected linear peptide 43 was subjected to macrocyclization with DMTTM·BF₄ and DIPEA at 10 mM dilution. As above, no difficulties were experienced during manipulations of the protected peptide. Gratifyingly, the reaction proceeded smoothly with complete consumption of the starting material in 4.5 h. Finally, side chain removal was carried out using the optimized deprotection cocktail of TFA:CH₂Cl₂:H₂O:TIPS (50:45:2.5:2.5, v/v/v/v) for 30 min with no detectable isomerization of the polyunsaturated lipid. To reduce exposure of the acid-sensitive polyunsaturated lipid to highly acidic TFA during HPLC purification, an eluent system of H₂O and CH₃CN containing 0.1% formic acid was employed, providing 1a in 7% overall yield.

Evaluation of the antibacterial activity of the synthesized analogues 1a, 31 and 36–40 was then undertaken. Unfortunately, no activity toward S. aureus was observed for these analogues using media supplemented with 1.25–1.5 mM CaCl₂, as recommended for biotesting of daptomycin (Wiegand et al., 2008) (Supplementary Tables S2, S3). As native malacidin A (1) showed maximum activity at 15 mM CaCl₂ (Hover et al., 2018), diastereomer 1a was also tested at this concentration but no activity was observed (Supplementary Table S2). This observation indicates that both the presence and absolute configuration of the β-substituents of the non-canonical amino acids are important for the activity of the antibiotic, most likely through their interaction with Ca²⁺ ions to form the active antibiotic-Ca²⁺ complex. This observation is not unusual, as previous reports of SAR studies of A54145 D and daptomycin CDLAs showed that removal or reversal of configuration at even one stereocenter may result in a significant reduction or complete loss of bioactivity (Kralt et al., 2019; Xu et al., 2019). It is likely that orientation of the β-OH of 3-HyAsp⁵, that forms part of the calcium binding motif, is highly important to provide efficient coordination to Ca²⁺ ions. Further SAR studies are required to assess the contribution of the individual non-canonical residues to the antimicrobial activity of malacidin A.

Comparison of the ¹H and ¹³C NMR spectra of diastereomer 1a to the reported spectra of malacidin A (under similar conditions, see SI), showed significant differences in the α-proton region (Hover et al., 2018) (Supplementary Figure S12). The ¹H spectrum of daptomycin is known to change upon Ca²⁺ binding, demonstrating significant line broadening that is characteristic of aggregation (Ball et al., 2004). To investigate the Ca²⁺ binding capability of 1a, the ¹H NMR spectra of 1a was recorded in the presence of CaCl₂ at 1.5 mM and 15 mM (Supplementary Figure S16), concentrations similar to those used in MIC assays for daptomycin (typically 1.25 mM) and malacidin (1) respectively. Disappointingly, no detectable signal shifts or line broadening were observed (Supplementary Figure S14), indicating that 1a fails to interact with Ca²⁺ ions, hence the lack of antibacterial activity. Despite these differences, it was observed that the lipid sp² proton signals closely matched that of the natural product and no peaks arising from cis-trans isomerization were observed, indicating the tolerance of acid-sensitive lipid 25 to the optimized TFA side chain deprotection conditions and HPLC purification protocols.

In summary, seven novel analogues of malacidin A (1) were synthesized using primarily an Fmoc-SPPS-based strategy followed by late stage solution-phase macrolactamization and subsequent side chain deprotection. One diastereomeric analogue of the native sequence, 1a, and six simplified analogues containing all canonical/commercially available amino acids with variations in the lipid tail (31, 36–40) were obtained in good overall yields. Despite the lack of activity observed for these analogues, the concise and versatile synthetic strategy reported herein lays a foundation for further SAR studies of malacidin A. In contrast to the reported synthesis of malacidin A, the synthetic route described herein has improved yields, requires no additional amino acids bearing auxiliary groups to aid cyclization, and involves minimal solution-phase manipulations. The mostly solid-phase strategy also permits a single, final purification step. Additionally, a late stage incorporation of the lipid moiety on resin enables facile preparation of lipid analogues to probe the role of the lipid unsaturation and branching on antibacterial activity.