A Concise Synthetic Strategy Towards the Novel Calcium-dependent Lipopeptide Antibiotic, Malacidin A and Analogues

Malacidin A is a novel calcium-dependent lipopeptide antibiotic with excellent activity against Gram-positive pathogens. Herein, a concise and robust synthetic route toward malacidin A is reported, employing 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis of a linear precursor, including late-stage incorporation of the lipid tail, followed by solution-phase cyclization. The versatility of this synthetic strategy was further demonstrated by synthesis of a diastereomeric variant of malacidin A and a small library of simplified analogues with variation of the lipid moiety.


INTRODUCTION
The present COVID-19 pandemic shows how vulnerable society is to an infectious disease without access to an immediate effective treatment. Somewhat overshadowed by the current situation but equally as urgent, antimicrobial resistance (AMR) represents another ongoing global health crisis. According to World Health Organization (WHO) reports, as many as 2.8 million people contract infections caused by AMR pathogens in the U.S. alone, leading to more than 35,000 deaths annually (Centers for Disease Control and Prevention (U.S.), 2019; World Health Organization, 2019). Similar statistics are observed for Europe (Cassini et al., 2019). Although new stewardship programs and policies to increase AMR awareness and limit the use of existing antimicrobials are being introduced around the world, the existing clinical pipeline does not meet the demand to effectively combat increasing rates of AMR infections (World Health Organization, 2015). Thus, novel antimicrobial agents that can be developed into potential drug candidates are critically needed. Over the last 20 years antimicrobial peptides (AMPs) emerged as a rich yet underexplored source of such compounds. Development of alternative platforms for AMP discovery and methods for synthetic optimizations of natural scaffolds are yielding many promising examples of clinically relevant AMPs (Jin, 2020;Liu et al., 2021).
In 2018, Brady et al. reported the isolation of malacidin A (1) as part of an extensive metagenomic mining study of bacterial DNA obtained from soil samples in search of novel bioactive natural products (Owen et al., 2013;Hover et al., 2018). This novel AMP possesses potent bioactivity against a range of Gram-positive strains including multi-drug resistant pathogens such as methicillinresistant Staphylococcus aureus (MRSA) (minimum inhibitory concentrations (MIC) 0.2-0.8 μg ml −1 ) and vancomycin-resistant Enterococcus faecium (VRE) (MIC 0.8-2.0 μg ml −1 ) (Hover et al., 2018). Malacidin A (1) belongs to a family of calcium-dependent lipopeptide antibiotics (CDLAs) that exhibit their activity upon binding to calcium ions. The CDLA family is represented by several subgroups of potent antibiotics: A21978C complex, which includes the antibiotic of last resort, daptomycin; A54145 complex; calcium dependent antibiotics (CDAs); friulimicins, of which friulimicin B reached Phase I clinical trials; amphomycins, of which, MX-2401 (a semi-synthetic analogue), progressed to latestage preclinical development; glycinocins and, recently, cadasides (Wood and Martin, 2019;Wu et al., 2019). Malacidin A (1) is structurally unique compared to other common CDLA members in that the canonical Asp-AA-Asp-Gly (AA Gly or D-amino acid) calcium-binding motif lacks the spacer residue, AA, and the first Asp residue is replaced by an unusual 3-hydroxy aspartic acid (3-HyAsp) (Hover et al., 2018). Preliminary mechanistic studies of malacidin A revealed binding to lipid II, a different target compared to other CDLAs. Malacidin A (1) was also found to be non-cytotoxic and did not induce resistance after repeated exposure to S. aureus (Hover et al., 2018). These features render 1 an exciting target for development as a novel antibiotic. The key step toward this goal is design of robust synthetic routes that would enable facile access to the lead compound and analogues thereof to establish structure activity relationships (SARs).
Herein, we report a concise synthetic strategy toward malacidin A (1) as demonstrated by the synthesis of a diastereomeric variant and simplified analogues thereof. The key steps involve preparation of the key linear precursor by 9-fluorenylmethoxycarbonyl (Fmoc)-solid-phase peptide synthesis (SPPS), followed by tail-to-side chain solution-phase cyclisation.
Frontiers in Chemistry | www.frontiersin.org August 2021 | Volume 9 | Article 687875 5 (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 Dap 2 building block, thus, commercially available Fmoc-Dap(Alloc)-OH was used. As the peptide sequence contains an aspartamide-prone Asp 6 Gly 7 region, dimethoxybenzyl (Dmb)N αprotected Gly 10 was used.
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. SCHEME 6 | Synthesis of simplified analogue 31.
Frontiers in Chemistry | www.frontiersin.org August 2021 | Volume 9 | Article 687875 8 removal from the side chain hydroxy group of 3-HyAsp 5 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-MeDap 2 residue was carried out under mild conditions using 3.6 M NH 2 OH·HCl and 2.7 M imidazole in NMP/ CH 2 Cl 2 (5:1, v/v) for 4 h. Following cleavage of the resulting peptide sequence from the resin with HFIP:CH 2 Cl 2 (3:7, v/v) and solvent evaporation the crude peptide was dissolved in H 2 O:CH 3 CN (1:4, v/v) and lyophilized. The obtained protected linear peptide 43 was subjected to macrocyclization with DMTTM·BF 4 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:CH 2 Cl 2 :H 2 O: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 H 2 O and CH 3 CN 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 CaCl 2 , 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 CaCl 2 (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 Ca 2+ ions to form the active antibiotic-Ca 2+ 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-HyAsp 5 , that forms part of the calcium binding motif, is highly important to provide efficient coordination to Ca 2+ 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 1 H and 13 C 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) Figure  S12). The 1 H spectrum of daptomycin is known to change upon Ca 2+ binding, demonstrating significant line broadening that is characteristic of aggregation (Ball et al., 2004). To investigate the Ca 2+ binding capability of 1a, the 1 H NMR spectra of 1a was recorded in the presence of CaCl 2 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 Ca 2+ ions, hence the lack of antibacterial activity. Despite these differences, it was observed that the lipid sp 2 proton signals closely matched that of the natural product and no peaks arising from cis-trans isomerization were observed, indicating the tolerance of acidsensitive lipid 25 to the optimized TFA side chain deprotection conditions and HPLC purification protocols.

(Supplementary
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.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.