Using chemical synthesis to optimise antimicrobial peptides in the fight against antimicrobial resistance

Freda F. Li
  • School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand
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and Margaret A. Brimble
  • Corresponding author
  • School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand
  • Maurice Wilkins Centre for Molecular Biodiscovery, 3 Symonds Street, Auckland 1010, New Zealand
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Abstract

The emergence of multidrug-resistant bacteria has necessitated the urgent need for novel antibacterial agents. Antimicrobial peptides (AMPs), the host-defence molecules of most living organisms, have shown great promise as potential antibiotic candidates due to their multiple mechanisms of action which result in very low or negligible induction of resistance. However, the development of AMPs for clinical use has been limited by their potential toxicity to animal cells, low metabolic stability and high manufacturing cost. Extensive efforts have therefore been directed towards the development of enhanced variants of natural AMPs to overcome these aforementioned limitations. In this review, we present our efforts focused on development of efficient strategies to prepare several recently discovered AMPs including antitubercular peptides. The design and synthesis of more potent and stable AMP analogues with synthetic modifications made to the natural peptides containing glycosylated residues or disulfide bridges are described.

Introduction

Since the discovery of penicillin and the introduction of sulfonamides in 1928, antibiotics have become the most commonly prescribed drugs and have significantly contributed to the reduction in mortality rates due to infections and common bacterial disease [1]. Unfortunately, the utility of these antibiotics has been seriously compromised by the subsequent emergence of resistant bacteria. Such resistance commonly develops as a result of the loss of chemical affinity between the antibiotic and its defined target [2], [3]. Deadly pathogens such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), and Staphylococcus aureus, are now commonly observed to be resistant to front-line antimicrobial treatments and have been labelled as ‘serious threats’ by the Centres for Disease Control and Prevention (CDC) [4], [5]. The problem of increasing bacterial resistance against existing antibiotics is now accepted as a key challenge to global health and is predicted to be responsible for more deaths than cancer by 2050 [2]. Further contributing to this antimicrobial resistance crisis is the limited development of new antibiotics, with only 7 new antibiotics approved from 2003 to 2012 [6], [7]. Indeed, the majority of antibiotics currently in the clinical pipeline are variations on known drug architectures and very few new antimicrobials with novel sites of action have been developed since the 1980s [8], [9].

Natural antimicrobial peptides (AMPs), also known as host-defensive peptides, are produced by multicellular organisms across the phylogenetic spectrum in order to protect the host from invading pathogenic microbes [10]. AMPs exhibit antimicrobial activities against a wide range of pathogenic organisms including Gram-positive and Gram-negative bacteria, fungi and parasites and are considered to be a potential source of novel antimicrobial agents [9], [10], [11]. The majority of AMPs are polypeptides (12–50 amino acids) characterised by an amphipathic secondary structure, composed of a high proportion of hydrophobic and cationic residues [10], [11]. Studies suggest that AMPs appear to act at multiple low affinity targets rather than one defined target to disrupt bacterial membranes and intracellular activities [10], [12]. The cationic properties of AMPs facilitate their initial accumulation onto the negatively charged bacterial cell surface and the hydrophobic portions are responsible for interactions with hydrophobic components of the membrane [10], [12]. Such unique and non-specific mechanisms of action enable AMPs to avoid the common resistance mechanisms observed for conventional antibiotics, making these peptides promising alternatives in the fight against severe infections caused by multi-drug resistant bacteria [11].

To date, more than 2600 AMP sequences from natural sources have been identified and reports of novel AMPs continue to appear in the literature [13]. However, only a few AMPs have been approved for current clinical use and most of these drugs, including polymyxins, gramicidin, tyrothricin and daptomycin, are for topical use [14], [15]. The development of AMPs for therapeutic application has been hampered by several limitations including systemic toxicity of AMPs, their vulnerability to enzymatic degradation and high-cost of production [9], [11]. Thus, much research has focused on modification and development of synthetic analogues of AMPs in order to overcome the aforementioned pharmaceutical limitations.

Early syntheses and studies of AMPs relied on isolation from natural sources by extraction from a large amount of raw biological sample to obtain small quantities of the pure peptide [16]. Recombinant expression of AMPs requires incorporation of an additional large fusion partner to mask the toxicity of the peptide to the producer cell, which must then be cleaved from the expressed AMP sequence of interest and removed during purification [17]. Chemical synthesis of peptides has quickly become the preferred method for preparing AMPs, of which solid-phase peptide synthesis (SPPS) is most commonly employed to produce small to medium-size peptides (30–50 residues) [18], [19]. The process of SPPS involves anchoring the first amino acid to an insoluble polymer support, then proceeding with peptide chain elongation to ultimately provide the target sequence. The attachment to a solid support via a cleavable chemical linker enables the use of excess reagents and facilitates the effective removal of by-products, eventually affording the desired peptide in both high yield and purity. For the synthesis of larger and more complex AMPs, a combination of SPPS and in-solution fragment condensation or ligation techniques can be implemented [18], [19]. Importantly, chemical synthesis of AMPs provides an additional advantage in that precise modification of the peptide sequence can be achieved by inserting or mutating virtually any natural amino acids, non-natural amino acids or structural fragments in an AMP to modulate its antibiotic potency and investigate structure-activity relationships. Further development of synthetic AMP analogues with improved biological activities and metabolic stability will lead to the successful conversion of natural AMPs into the ‘smart antibiotics’ required for future therapies and industrial applications.

Our group has a strong ongoing interest in the total synthesis of naturally occurring bioactive peptides and the development of methodologies to provide suitable peptide analogues or mimetics with improved pharmacological profiles. This review presents our recent efforts in the synthesis of AMPs, which focus on the development of efficient strategies to access scarce natural AMPs, highlighting synthetic modifications made to peptides containing glycosylated residues or disulfide bridges in order to deliver more potent and stable AMP analogues along the way. Related studies by other research groups towards the synthesis or modification of the target AMP sequence are also described where appropriate.

Synthesis of caenopore-5 and its diselenide bond analogue

The caenopores belong to an ancient family of antimicrobial peptides found in the nematode Caenorhabditis elegans (C. elegans) and share similarities with saposin-like proteins (SAPLIPs) [20]. Currently, 33 caenopores have been identified for the saposin (spp) gene family in C. elegans, among which caenopore-5 is essential for the survival of the nematode in its natural habitat [20], [21]. Caenopore-5 (Cp-5) is a pore-forming AMP exclusively expressed in the intestine of C. elegans and it was found that a spp-5 knockdown led to a significant reduction in offspring production and highly increased numbers of the food bacterium Escherichia coli (E. coli) in the nematode intestine [20], [21]. Recombinant Cp-5 has been shown to be active against both Gram-positive (Bacillus megaterium) and Gram-negative (E. coli) bacteria with minimum inhibitory concentration (MIC) values of 0.05 and 0.1 μM, respectively, and minimum bactericidal concentration (MBC) values of 0.1 and 0.2 μM, respectively [21].

In 2015, we reported the successful synthesis of Cp-5 (1) (Scheme 1) [22]. The 82 residue peptide was constructed by native chemical ligation (NCL) of two smaller polypeptide fragments 2 and 3 that were prepared using Boc SPPS (Boc=tert-butyloxycarbonyl) and folded to give the correct protein structure. The synthesis hinged on a single NCL at the native 36Cys residue to avoid the introduction of a non-native cysteine that would require further chemical manipulation after the ligation step. This approach was shown to be more efficient than using a three-fragment approach with two ligations. The ligated peptide 4 was then subjected to a redox-coupled system to enable a facile thiol/disulfide exchange and promote the formation of the correctly folded protein. The synthetic Cp-5 (1) protein was found to have the same structural features as recombinant Cp-5, as determined by 1H NMR spectroscopy and circular dichroism (CD) experiments. Structural analysis of the antimicrobial function for the peptide fragments 2 and 3, which were previously synthesised en route to Cp-5 (1), revealed that the N-terminal fragment is responsible for the cell permeability activity of the native peptide. The reduced sequence 4 was determined to be inactive compared to the synthetic Cp-5 (1), proving that elements of the secondary structure are crucial for the activity of the protein.

Scheme 1:
Scheme 1:

The chemical synthesis of caenopore-5 (1).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Disulfide bonds play a crucial role in protein folding and impart conformational stability to peptides and proteins, however, they are inherently unstable in reducing environments and are susceptible to disulfide bond exchange reactions with biological thiols such as glutathione [23]. In comparison, diselenide bonds have not been rigorously characterised in proteins and may not exist naturally due to the fact that thioredoxin, the most reducing oxidoreductase in E. coli, would be unable to reduce the diselenide bond [24], [25]. In 2006, Metanis et al. [26] reported the first synthesis and characterisation of selenocysteine (Sec) substitution into an oxidoreductase, glutaredoxin 3, yielding analogues that contain selenenylsulfide and diselenide bonds. The synthetic seleno-glutaredoxin 3 analogues demonstrated physiologically compatible redox potentials and enhanced catalytic efficiency in comparison with their sulfide counterparts. At the time of our reported synthesis of Cp-5 (1), this study was the only example in the literature of using NCL to synthesise an intramolecular diselenide bond analogue of a native protein, which retained its secondary structure with improved activity.

In an attempt to enhance the stability and activity of Cp-5 (1), we developed a seleno-analogue 5 of the native peptide by replacing a disulfide bond in Cp-5 (1) with a more robust diselenide bond (Scheme 2) [27]. Using the two fragment ligation strategy developed earlier for the native Cp-5 (1), 7Cys and 81Cys were selectively replaced with 7Sec and 81Sec, as the disulfide bond formed by these cysteine residues is the most exposed on the peptide surface. The successful conditions identified for the NCL of thioester fragment 6 with cysteinyl fragment 7 required use of 1% v/v PhSH (6 M guanidine–HCl, 0.2 M Na2HPO4) with 5 mM of each fragment at pH 7.5 at 25°C to avoid deselenisation of the sensitive selenocysteine residues. Concurrent formation of the disulfide and diselenide bonds were observed during the ligation reaction although the ligated peptide 8 was unstructured. A separate folding step afforded the structured analogue [7Sec-81Sec]-Cp-5 (5), which exhibited higher permeabilisation activity compared with the native protein. Additionally, CD analysis showed that the temperature at which the folded analogue 5 denatured was almost 25°C higher than the wild type, indicating a dramatic increase in the thermal stability of the modified peptide 5. As a result, substitution of the most exposed disulfide bond with a diselenide bond in [7Sec-81Sec]-Cp-5 (5) significantly aided the folding of the protein and improved both the bioactivity and stability of the seleno-analogue compared with native Cp-5 (1).

Scheme 2:
Scheme 2:

Synthesis of diselenide bond analogue [7Sec-81Sec]-Cp-5 (5).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Synthesis of glycoactive glycocin F and its glyco-mutant analogues

Bacteriocins are ribosomally synthesised AMPs secreted by bacteria to stop or slow the growth of their competing bacterial targets without killing them [28]. One distinctive group of post-translationally modified bacteriocins is the glycocins, which contain one or two monosaccharide moieties linked to the side chains of specific cysteine, serine or threonine residues in the peptide [29]. When the glycosylated residues of bacteriocins are essential for their antibiotic activity, these glycopeptides are also regarded as glycoactive AMPs [28], [29]. Glycocin F (GccF, 9) is a 43 residue glycoactive AMP produced by Lactobacillus plantarum KW30 and contains two interlocked disulfide bonds and two β-linked N-acetyl-glucosamine (GlcNAc) moieties, one attached to the sulfur atom of 43Cys and the other linked to the γ-oxygen of 18Ser [30], [31]. Currently, three bacteriocins containing sulfur-linked glycosylated residues have been reported, namely, sublancin 168 [32], GccF [30] and thurandacins A and B [33], among which only GccF was found to be glycoactive. GccF exhibits bacteriostatic activity against a wide range of Gram-positive bacteria, including Enterococcus, Bacillus, Streptococcus and Lactobacillus species, with L. plantarum strains suspected to be its natural target (IC50<4 pM for L. plantarum ATCC 14917) [30], [31].

The first total synthesis of a biologically active form of GccF (GccF-NH2), identical in structure to the natural peptide GccF except for the C-terminal amidation, was accomplished by our group in 2015 [34]. The peptide GccF-NH2 was synthesised using a three fragment NCL strategy (sequences H2N-1Lys-11Met-COOH, H2N-12Cys-18Ser(β-GlcNAc)-27His-COOH, and H2N-28Cys-43Cys(β-GlcNAc)-COOH) followed by oxidative folding (Method A, Scheme 3). The O-glycosylated amino acid Fmoc-Ser(β-GlcNAc(OAc)3)-OH (Fmoc=9-fluorenylmethoxycarbonyl) and S-glycosylated amino acid Fmoc-Cys(β-GlcNAc(OAc)3)-OH building blocks were prepared and incorporated into the required polypeptide sequences 1012 using Fmoc SPPS with a 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) linker. However, substantial epimerisation at 27His was observed during both loading of the histidine residue onto the HMPB linker and the trans-thioesterification step required to install the thioester-linked fragment 11, resulting in formation of the d-epimer analogue of GccF-NH2. Pleasingly, changing the benzyl alcohol-based HMPB to a 2-chlorotrityl linker reduced the epimerisation significantly and this linker was successfully utilised in the synthesis of native GccF (9), which was found to be more potent than the previously synthesised C-terminal amidated GccF-NH2 [35].

Scheme 3:
Scheme 3:

Initially reported (A) and optimised (B) syntheses of glycocin F (9).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

To further prevent the epimerisation of 27His and reduce the total number of steps to prepare GccF, an alternative strategy was developed using a two fragment (sequences H2N-1Lys-11Met-COOH and H2N-12Cys-18Ser(β-GlcNAc)-43Cys(β-GlcNAc)-COOH), single NCL synthesis (Method B, Scheme 3) [35]. This strategy was then employed for the syntheses of two analogues of GccF (14 and 15, Scheme 4), each bearing a single modification at one of the glycoside positions [35]. Three pseudoproline dipeptides, namely 19Gly-20Thr, 25Tyr-26Ser, and 35Ser-36Ser, were incorporated within the peptide chains 16 and 17 to avoid poor coupling yields due to aggregation during long fragment synthesis. The recovered peptides 18 and 19 were then individually ligated with N-terminal fragment 10, followed by oxidative folding to yield two glycomutant analogues 14 and 15 of GccF. Strikingly, the bioactivity increased significantly when the sequence incorporated two S-GlcNAc moieties at position 18 and 43 (in peptide 15), while replacing the S-GlcNAc at position 43 with O-GlcNAc (in peptide 14) resulted in a 10-fold decrease in antibacterial activity compared to native GccF.

Scheme 4:
Scheme 4:

Optimised synthesis of GccF glycomutants 14 and 15.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

To further probe structure-activity relationships of the glycoactive bacteriocin GccF, a variety of its peptide analogues, which contain modifications to the flexible C-terminal tail, interhelical loop and C-terminal sugar of GccF as well as individual disruption of the two nested disulfide bonds, were prepared using our optimised strategy [36]. Detailed structural analysis for the antibacterial function of these GccF analogues revealed that the bacteriostatic activity of GccF is controlled by the interhelical loop, while the glycosylated flexible tail appears to be involved in localising the peptide to its cellular target [36]. A model for GccF-induced bacteriostasis was therefore proposed as that GccF first diffuses through the thick peptidoglycan layer of Gram-positive cells, whereby the C-terminal S-GlcNAc samples its environment to find extracellular targets [36]. Binding to the receptor effectively ‘tethers’ the bacteriocins to the cell and the O-GlcNAc-bearing interhelical loop is then positioned to interact with a nearby target, setting up a rapid signalling response that results in bacteriostasis [36].

Synthesis of natural antitubercular peptides and derivatives

Tuberculosis (TB) is a highly contagious airborne disease caused by the Gram-positive pathogen Mycobacterium tuberculosis (Mtb) which now ranks alongside human immunodeficiency virus (HIV) as a leading cause of death worldwide [37]. Current front-line treatment for TB utilises small organic molecule medicines such as isoniazid, rifampicin, pyrazinamide and ethambutol [38]. However, TB continues to be a significant threat to public health due to the fact that drug resistant Mtb strains have now been detected in most countries and are spreading at an alarming rate [37], [38]. Second-line drugs such as aminoglycosides and capreomycin are commonly employed in drug combination regimens to treat drug resistant TB, and require the patients to endure lengthy treatments to achieve therapeutic effect [38]. The poor compliance due to the long treatment period and harsh side effects can lead to the development of multi-drug resistant (MDR) or extensively drug resistant (XDR) Mtb strains, which are more difficult to treat [38].

Unfortunately, the development of novel anti-TB drugs has been subcritical over the past several decades, with only two new drugs (bedaquiline and demalanid) approved in more than 40 years, implying the obvious and urgent need for the discovery of novel and potent anti-TB compounds [38], [39]. Compared to the small organic molecule counterparts, natural AMPs are attractive starting point for the search of novel anti-TB drug leads as the range of bioactivities exhibited by these peptides often includes anti-TB activity. Several potent anti-TB AMPs that were derived from the natural sources have been reported [40], [41], including callyaerin A [42], trichoderins A and B [43], lassomycin [44], ecumicin [45], wollamide A [46] and teixobactin [47]. Recent work [48], [49], [50], [51] in our research group has also focused on the synthesis of these natural anti-TB AMPs and derivatives, with the objective of providing potential anti-TB drug leads with improved pharmacological properties.

Callyaerin A

Peptides belonging to the callyaerin family are cyclic AMPs derived from the Indonesian marine sponge Callyspongia aerizusa, of which the basic structural unit comprises a cyclic moiety and a linear side chain joined through an unusual (Z)-2,3-diaminoacrylamide (DAA) linkage (e.g. callyaerin A (16), Fig. 1) [42], [52]. Among this family, callyaerin A (16) exhibited the strongest activity against Mtb (MIC90=2 μM) with no observed cytotoxicity in human cells [42], [52]. Together with the DAA unit in the cyclic structure, callyaerin A (16) contains multiple proline residues which provide the additional influence on the topology of the molecule, potentially contributing to the high affinity for protein-protein interactions [52]. The remaining hydrophobic residues in the linear chain of callyaerin A (16) were suggested to be responsible for the bactericidal specificity of the peptide [52].

Fig. 1:
Fig. 1:

Structure of callyaerin A (16).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

In 2018, our group reported the first total synthesis of callyaerin A (16) [48]. We envisaged that the DAA moiety could be readily derived from an α-formyl glycine (FGly) residue in precursor 17 (Route A, Scheme 5), which would undergo Schiff base formation by reacting with an N-terminal amino group in the ring, followed by double bond migration. Oxidation of the serine residue in protected peptide 18 to FGly, however, could not be realised possibly due to the poor stability of FGly owing to the acidic α-proton, analogous to what is known for α-amino aldehydes [53]. An alternative strategy [54], [55] by preparing the more stable enol tosylate derivative 19, which could undergo a facile nucleophilic attack by the N-terminus to furnish the required DAA unit, also proved unsuccessful (Route B, Scheme 5).

Scheme 5:
Scheme 5:

Initial approaches to synthesise callyaerin A (16).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

In 2003, William and DeMong reported a 27-step solution-phase synthesis of capreomycin IB, a naturally occurring AMP containing an exocyclic DAA functionality [56]. In their synthesis, an unusual building block α-formylglycine diethyl acetal ethyl ester was utilised as an intermediate which was compatible with various reaction conditions and was later converted to the FGly residue to deliver the required DAA unit. Encouraged by this, we incorporated an Nα-Fmoc-protected variant of this acetal-protected FGly (20) into Fmoc SPPS as a masked equivalent of FGly to afford linear precursor 17 for the synthesis of callyaerin A (16) (Scheme 6). Final in-solution cyclisation of linear peptide 17 was achieved in excellent yield by using dilute acid to minimise the undesirable intermolecular reaction or peptide fragmentation. The Z configuration for the DAA unit of synthetic 16 was confirmed by the full agreement of 1H and 13C NMR data with those reported for the natural product [42].

Scheme 6:
Scheme 6:

Total synthesis of callyaerin A (16).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

For comparison purposes, we further prepared a homodetic analogue 21 of callyaerin A that contains a lactam linkage with the N-terminus instead of a DAA bridge (Fig. 2). Biological evaluation of analogue 21 revealed that it was inactive against Mtb, suggesting the DAA functionality is essential for the anti-TB activity of callyaerin A (16). A variable-temperature NMR study of 16 and 21 indicated that in solution callyaerin A (16) was present as a single conformer stabilised by four hydrogen bonds, while analogue 21 adopted multiple conformations due to the lack of appropriate structural constraints (Fig. 2). The number of amide resonances observed in the 1H NMR spectrum of lactam analogue 21 exceeded the number of amide protons present in the structure, which may be attributed to multiple exchanging rotamers that were in slow transition at 298 K. As the temperature increased, the exchange rate between the different conformers was accelerated and the peaks became broader, thus indicating the transition regime switched from slow to slow-intermediate exchange. However, the proton signals remained broad even when the temperature increased to 80°C. We reasoned that a much higher temperature would be required to force the peptide into fast exchange, which would generate the more resolved proton signals of analogue peptide 21. These results demonstrated the extraordinary structural rigidity imposed by the DAA moiety and highlighted its potential use as a novel cyclic constraint to reduce the conformational flexibility of cyclic AMPs, thereby improving the properties of bioactive peptides.

Fig. 2:
Fig. 2:

Variable-temperature 1H NMR (500 MHz, DMSO-d6) analysis of callyaerin A (16) and its homodetic counterpart 21. (a) The 1H NMR spectra of 16 recorded between 20 and 45°C and the corresponding Tcoeff values calculated for all the amide protons; (b) The 1H NMR spectra of 21 recorded between 25 and 80°C. Only the amide region is shown.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Trichoderin A

Trichoderins are a novel family of peptaibols (peptides containing α-aminoisobutyric acid (Aib) and a C-terminal alcohol) sourced from a marine sponge-derived fungus Trichoderma sp. [43]. This family of AMPs was found to be highly potent against dormant and live Mtb bacilli, with trichoderin A (22, Fig. 3) being most active (MIC=0.12 μg/mL) and exhibiting higher potency against Mtb H37Rv under hypoxic conditions than the first-line anti-TB drug isoniazid (MIC>100 μg/mL) [43]. The antimycobacterial activity of trichoderins has been assigned to the inhibition of the ATP synthesis, although further studies are required to validate this hypothesis [57].

Fig. 3:
Fig. 3:

Originally proposed structure of trichoderin A (22) and the unnatural AHMOD residue 23.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

In addition to multiple Aib residues and a C-terminal alcohol, trichoderin A (22) also contains a fatty acyl chain at the N-terminus and the unnatural amino acid, 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD, 23). The main challenge for the synthesis of trichoderin A (22) lies in the presence of sensitive AHMOD residue, the C-terminal alcohol moiety and consecutive sterically hindered Aib residues. Preparations of AHMOD, albeit lengthy and low yielding, have been reported in the literature [58], [59], [60]. In 2012, our group reported the first total synthesis of an AHMOD-containing anticancer peptide culicinin D by incorporating both (6S)- and (6R)-AHMOD into the peptaibol framework and assigned the absolute stereochemistry in the natural product to be (2S,4S,6R)-AHMOD instead of the proposed (2S,4S,6S)-AHMOD [61]. The C-6 position of the AHMOD residue of trichoderin A at the time of its isolation remained unclear and was also assumed to be (S)-configuration by comparison of the NMR data with the structurally similar peptaibol trichopolyn I [43]. As we recently published a simplified chiral pool-based synthesis of AHMOD that can be readily prepared in both (6R)- and (6S)-configurations on large scale [62], incorporation of both isomers into the synthesis of trichoderin A would allow the determination of the absolute stereochemistry of the secondary alcohol in the AHMOD moiety of the natural product.

Our initial strategies for the synthesis of trichoderin A (the proposed structure 22) adopted the method that was previously employed to prepare the anticancer peptaibol culicinin D (Scheme 7) [49]. In order to prevent the undesired β-elimination of the hydroxyl ketone moiety in the AHMOD unit, 2-chlorotrityl-functionalised aminomethyl polystyrene resin was selected for Fmoc SPPS as it is cleavable under mild conditions using hexafluoropropan-2-ol. The difficult couplings of the consecutive Aib residues were achieved with a powerful mixture of the uronium-type coupling reagent 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylmino morpholinomethylene)]methanaminium hexafluorophosphate (COMU) and 2-cyano-2-(hydroxyimino)acetate (Oxyma) to minimise potential Aib deletion. However, only trace amount of the desired product 22 was yielded due to the poor loading of the C-terminal aminoalcohol to the solid support (Route A, Scheme 7). An alternative anchoring strategy was attempted by attaching the amino group of the C-terminal aminoalcohol to the resin rather than the less nucleophilic hydroxyl group (Route B, Scheme 7). Upon completion of the peptide synthesis, a final O,N-intramolecular acyl migration would afford the desired peptaibol 22. The overall yield for Route B was still low, with the desired product 22 formed in a trace amount similar to that observed for Route A.

Scheme 7:
Scheme 7:

Initial synthetic strategies applied to prepare the proposed structure of trichoderin A (22).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Given that the inefficient synthesis was due to the poor attachment of the C-terminal aminoalcohol to the resin, we eventually developed a combined solid-phase and solution-phase strategy to prepare the postulated structure of trichoderin A (22) and its C-6 AHMOD epimer 24 (Scheme 8). A late-stage solution-phase C-terminal coupling was employed to introduce the unprotected aminoalcohol 25 to the peptide chains 26 and 27, prepared using SPPS, respectively, and this proved to be the significant factor to enable the efficient syntheses of the two C-6 AHMOD epimers 22 and 24 of trichoderin A. A detailed comparison of the 1H and 13C NMR data between isolated trichoderin A and the two synthetic peptides 22 and 24 revealed that the stereochemical assignment of the C-6 position of the AHMOD residue of the natural product to be (R) rather than the originally proposed (S)-configuration. Both synthetic trichoderin A epimers 22 and 24 (MIC=9.3 μg/mL) were found to be more potent against Mtb than the first-line anti-TB drug isoniazid (MIC>100 μg/mL). Our improved synthetic strategy therefore provided a valuable platform for further efficient preparations of trichoderin A analogues to enable a comprehensive structure-activity relationship study for the natural peptide.

Scheme 8:
Scheme 8:

Improved synthesis of the proposed structure of trichoderin A (22) and its C-6 AHMOD epimer 24.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Lassomycin

Lasso peptides are natural AMPs found throughout the bacterial domain and belong to the class of ribosomally assembled cyclic peptides that contain unique post-translational modifications [63]. Since the first lasso peptide was discovered in 1991, more than 30 have been reported [63]. These compounds typically consist of a linear C-terminal tail and a macrolactam ring formed between the N-terminal amino group (e.g. Gly, Ser, Cys or Ala) with the side chain of aspartic acid or glutamic acid located 7–9 residues away. Threading of the C-terminal tail through the ring is observed in nearly all discovered lasso peptides, thus yielding a knotted structure (e.g. class II lasso peptide, Fig. 4) which was suggested to render lasso peptides more thermally and proteolytically stable [63].

Fig. 4:
Fig. 4:

Structure of typical class II lasso peptides and lassomycin (28).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Lassomycin (28, Fig. 4) is a lasso AMP recently isolated from the soil bacterium Lentzea kentuckyensis sp. and was discovered in a screen designed to identify compounds acting specifically against Mtb [64]. Lassomycin exhibited potent activities (MIC=0.8–3 μg/mL) against a variety of Mtb strains, including MDR and XDR isolates, whereby the activities of this peptide arise from targeting ATP-dependent protease ClpC1 in mycobacteria [63], [64]. The structure of lassomycin was reported to lack the characteristic knot structure described for other homologous lasso peptides, as the C-terminal tail does not fold back and pass through the lactam ring but remains essentially unstructured, revealed by solution state NMR analysis [64].

In 2016, Cobb et al. [65] reported the first chemical synthesis of lassomycin (28) and a C-terminal amide congener 29 (Fig. 5), in which the linear peptide sequence was assembled using Fmoc SPPS via side chain anchoring then underwent an in-solution 1Gly-8Asp macrolactamisation to afford the desired product. Attempts to perform the 1Gly-8Asp cyclisation on resin were unproductive due to difficulties in removing an allyl ester protecting group from the side chain of 8Asp [65]. Shortly after the synthesis reported by Cobb et al., our research group established an efficient, primarily solid-phase synthesis of lassomycin (28) using an on-resin lactamisation reaction [50]. A highly acid-labile 2-phenylisopropyl protecting group for the side chain of 8Asp was employed to allow the required selective deprotection for cyclisation. The optimised methodology was then applied to the preparation of a small library of analogues (Fig. 5) including C-terminal modified lassomycin amide 29 and acid 30, as well as cyclic analogues 3133 containing a non-native bond or different macrolactam ring size. However, both the synthetic lassomycin (28) and analogues 2933 prepared by Cobb et al. [65] and our group [50] failed to reveal any significant activity against TB-causative pathogens. Although the reasons for this discrepancy have not yet been fully established, the lack of biological activity for the synthetic lassomycin (28) suggested that the natural product may actually adopt a traditional lasso peptide thread conformation rather than the previously reported unthreaded scaffold [50], [63], [65].

Fig. 5:
Fig. 5:

Chemically synthesised variants 29–33 of lassomycin (28).

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Teixobactin

In 2015, a breakthrough AMP teixobactin (34, Fig. 6), was discovered through screening of uncultured bacteria and was reported to be ineffective against most Gram-negative bacteria but exhibit excellent activities against an array of Gram-positive pathogens, including difficult-to-treat drug-resistant strains methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus and Mtb [66]. Remarkably, in animal studies against MRSA and Mtb, no teixobactin-resistant mutants could be detected, despite rigorous attempts to induce resistance [66]. In vitro studies suggested that teixobactin operates by binding to precursors (lipid II and III) of multiple cell wall biosynthetic pathways whereby the binding motifs are highly conserved in bacteria and cannot easily mutate to impart drug resistance [66]. The unique combination of broad Gram-positive activity with its inability to elicit resistance therefore makes teixobactin an outstanding molecule for antimicrobial therapeutic development.

Fig. 6:
Fig. 6:

Structure of teixobactin (34) with four d-amino acids (red) and l-allo-enduracididine (blue) highlighted.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Structurally, teixobactin (34) is an undecapeptide comprised of a cyclo tetradepsipeptide and a 7-amino acid tail, which contains four d-amino acids and one unusual amino acid l-allo-enduracididine (End). The first total synthesis of teixobactin (34) was achieved by Payne’s group [67] and Li’s group [68] independently in 2016. Since then, more than 200 teixobactin analogues have been synthesised by multiple research groups [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], including our own [51], to allow structure-activity relationship studies and elucidation of the pharmacophore [83], [84], [85]. Although early studies [83] suggested that the presence of the unusual End residue might be important for the potent activity of teixobactin, it represents a key bottleneck in the synthesis and development of teixobactin analogues due to various synthetic challenges [86]. Following the successful synthesis of teixobactin (34), Singh and Taylor et al. [70], [71], [72] and others [68], [73], [74], [75], [76], [77], [83], [84] reported the design and synthesis of several teixobactin analogues by replacing the End residue at position 10 with structurally similar natural amino acids or isosteric building blocks, of which l-Arg10-teixobacin, l-Orn10-teixobacin and l-Lys10-teixobacin (Fig. 7) were active against MRSA but were around 10-fold less potent than teixobactin (34). Recently, our group reported the synthesis of several novel teixobactin analogues containing d-histidine (His), l-citrulline (Cit), l-homoarginine (HoArg) and Nω,Nω-dimethyl-l-arginine (ADMA) as End surrogates (Fig. 7) [51]. Biological evaluation of these teixobactin analogues indicated that the homoarginine containing l-HoArg10-teixobacin also possessed promising antimicrobial activity, comparable to l-Arg10-, l-Orn10- and l-Lys10-teixobacins.

Fig. 7:
Fig. 7:

Teixobactin (34) and its related key analogues with l-allo-enduracididine (End) surrogates. MIC against different strains of MRSA are included: (a) MRSA ATCC 33591; (b) MRSA-USA300; (c) MRSA SA1124.

Citation: Pure and Applied Chemistry 91, 2; 10.1515/pac-2018-0704

Most recently, both Singh’s group [80] and Li’s group [81] reported that the unnatural End residue can be substituted for non-polar amino acids such as leucine (Leu), isoleucine (Ile) as well as other non-isosteric hydrophobic residues with no loss of activity compared to teixobactin (34) (Fig. 7), indicating that the unusual End residue might be non-essential for the potent activity of the natural product. Inspired by these results, Singh et al. [82] further designed and prepared 10 novel active analogues by finely balancing the increased hydrophobicity in l-Leu10- and l-Ile10-teixobacins with d- or l-arginine mutants in the structure, representing significant progress in the development of in vivo ready simplified teixobactin analogues as promising lead compounds for antibiotic drug discovery.

Conclusions

AMPs are considered as potential therapeutic sources of novel antibiotics which could overcome the critical problem of antimicrobial resistance. However, many potent naturally occurring AMPs contain complex non-canonical amino acids and motifs, which are chemically difficult to synthesise in a robust and efficient manner. Thus, extensive efforts have been carried out by synthetic chemists to overcome these challenges. In this review, we detailed our efforts towards the syntheses of several recently discovered AMPs including highly active anti-TB peptides. Efficient strategies for the syntheses of these AMPs were established by either sequential synthesis or convergent assembly, in both cases taking advantage of the solid-phase strategy. Successful preparations of natural AMPs first served to confirm their structures (e.g. trichoderin A and lassomycin) and then conduct structure-activity relationship studies. Design and syntheses of AMP analogues with improved stability and bioactivity (e.g. caenopore-5 and glycocin F) provided fundamental insights into the antimicrobial mechanisms of action of the native proteins. Studies of AMPs containing unusual structural features (e.g. callyaerin A) revealed potential synthetic modifications that could be applied to improve the properties of other bioactive peptides. Furthermore, significant advances have been made by chemists in the field around the globe, and structurally simplified equipotent analogues of natural AMPs have emerged as promising drug leads (e.g. teixobactin). Highly active AMP derivatives with enhanced pharmacokinetic profiles will likely continue to be synthesised and potentially developed to provide a future generation of ‘super’ antibiotics to fight against the global threat of antimicrobial resistance.

Footnotes

Article note

A special collection of invited papers by recipients of the IUPAC Distinguished Women in Chemistry and Chemical Engineering Awards.

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Pure and Applied Chemistry is the official monthly Journal of the International Union of Pure and Applied Chemistry (IUPAC), with responsibility for publishing works arising from those international scientific events and projects that are sponsored and undertaken by the Union.

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    The chemical synthesis of caenopore-5 (1).

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    Synthesis of diselenide bond analogue [7Sec-81Sec]-Cp-5 (5).

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    Initially reported (A) and optimised (B) syntheses of glycocin F (9).

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    Optimised synthesis of GccF glycomutants 14 and 15.

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    Structure of callyaerin A (16).

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    Initial approaches to synthesise callyaerin A (16).

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    Total synthesis of callyaerin A (16).

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    Variable-temperature 1H NMR (500 MHz, DMSO-d6) analysis of callyaerin A (16) and its homodetic counterpart 21. (a) The 1H NMR spectra of 16 recorded between 20 and 45°C and the corresponding Tcoeff values calculated for all the amide protons; (b) The 1H NMR spectra of 21 recorded between 25 and 80°C. Only the amide region is shown.

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    Originally proposed structure of trichoderin A (22) and the unnatural AHMOD residue 23.

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    Initial synthetic strategies applied to prepare the proposed structure of trichoderin A (22).

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    Improved synthesis of the proposed structure of trichoderin A (22) and its C-6 AHMOD epimer 24.

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    Structure of typical class II lasso peptides and lassomycin (28).

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    Chemically synthesised variants 29–33 of lassomycin (28).

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    Structure of teixobactin (34) with four d-amino acids (red) and l-allo-enduracididine (blue) highlighted.

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    Teixobactin (34) and its related key analogues with l-allo-enduracididine (End) surrogates. MIC against different strains of MRSA are included: (a) MRSA ATCC 33591; (b) MRSA-USA300; (c) MRSA SA1124.