Enduracididine, a rare amino acid component of peptide antibiotics: Natural products and synthesis

Rising resistance to current clinical antibacterial agents is an imminent threat to global public health and highlights the demand for new lead compounds for drug discovery. One such potential lead compound, the peptide antibiotic teixobactin, was recently isolated from an uncultured bacterial source, and demonstrates remarkably high potency against a wide range of resistant pathogens without apparent development of resistance. A rare amino acid residue component of teixobactin, enduracididine, is only known to occur in a small number of natural products that also possess promising antibiotic activity. This review highlights the presence of enduracididine in natural products, its biosynthesis together with a review of analogues of enduracididine. Reported synthetic approaches to the cyclic guanidine structure of enduracididine are discussed, illustrating the challenges encountered to date in the development of efficient synthetic routes to facilitate drug discovery efforts inspired by the discovery of teixobactin.

Free enduracididine (1) was subsequently isolated from the seeds of the legume Lonchocarpus sericeus [3,4]. It was found to inhibit seedling germination of lettuce [5] and did not exhibit any significant effect on the inhibition of protein production in rat hepatoma cells [6]. L- (5) and D-β-hydroxyenduracididine  (6) were first resolved as components of the mannopeptimycin antibiotics, isolated from Streptomyces hygroscopicus LL-AC98 in 2002 [7] and to date, have not been isolated as the free amino acids or observed in any other natural products.
Enduracidin A (7) and B (8) are depsipeptides with the same composition of seventeen amino acids, sixteen of which make up the cyclic core [11,13,19] and are structurally related to the non-enduracididine containing antibiotic, ramoplanin [19]. The enduracidins are active against Gram-positive bacteria, including resistant strains [2,9,20] and Mycobacterium species [21]. No activity was observed against Gram-negative bacteria (except for Neisseria gonorrhoeae), fungi or yeast [9]. The antibacterial activity arises through inhibition of cell wall synthesis [22] by prevention of transglycosylation during peptidoglycan synthesis [23], the same step inhibited by vancomycin [24]. Enduracidin A (7) and B (8) also exhibited inhibition of avian myeloblastosis virus reverse transcriptase but did not suppress replication of HIV cells [25]. Enduracidin A (7) and B (8) have been produced by fermentation industrially and is used as an antibiotic feed additive for pigs [26] and chickens [27] under the trade name enradin ® .

Minosaminomycin
Minosaminomycin (9, Figure 3) was isolated in 1974 from a culture broth of Streptomyces MA514-A1 [28]. It was found to be active against Mycobacteria (M. smegmatis, MIC = 15.6 μg/mL) but only weakly active against all other bacteria tested. The structure was confirmed through degradation and partial synthesis [29,30]. Minosaminomycin (9) inhibits protein synthesis in E. coli more effectively than the related antibiotic kasugamycin (10), however, a different mechanism is operative [31].

Indole metabolite
In 1996, during a screening program for biologically active metabolites from marine ascidians, Riguera et al. identified a small group of amino acid containing compounds in a cytotoxic extract of the ascidian Leptoclinides dubius [32]. Among these compounds was the unique enduracididine-containing bromoindole metabolite 11 ( Figure 4). This was the first time the enduracididine motif had been isolated from a marine source. The exact compound responsible for the observed cytotoxicity was not determined.

Mannopeptimycins
Mannopeptimycins α-ε (12-16, Figure 5) were isolated from Streptomyces hygroscopicus LL-AC98 in 2002 [7] and their structures were elucidated using mass spectrometry and extensive NMR analysis. The absolute stereochemistry was pro-  posed following degradation and nOe studies. The configuration of the β-methylphenylalanine stereocentre was revised from S to R, upon total synthesis [33]. The mannopeptimycins contain a unique sugar substituted hydroxyenduracididine residue (blue, Figure 5).  The mannopeptimycins displayed moderate activity against Gram-positive bacteria, including MRSA, but only exhibited weak activity against Gram-negative bacteria [7] with the primary cellular target deduced to be bacterial cell wall synthesis [34]. Extensive derivatisation of both mannopeptimycin α (12) and β (13) was undertaken to improve the antibacterial activity of the parent natural products (highlighted in blue, Figure 6). An array of ether [35,36], halogenated [36], acetal [37][38][39], benzoxazole [40], thiobenzoxazole [40], ester and carbonate [41] analogues were synthesised and evaluated for antibacterial activity. Only the semisynthetic derivatives possessing hydrophobic groups on the terminal sugar moiety (green) exhibited comparable antibacterial activity to the parent compound and reference antibiotics [42].

Teixobactin
In early 2015, a new enduracididine-containing antibiotic named teixobactin (17) was reported (Figure 7) [43]. Teixobactin (17) was isolated using the multichannel device, the isolation chip (iChip). The iChip allows a single cell to be delivered to an individual chamber where it can grow. The chambers are covered with a semi-permeable membrane and placed into the microbe's natural environment where nutrients can diffuse into each chamber. This method gives access to cultures of microbes which were previously unobtainable using traditional techniques. Teixobactin (17) exhibits bactericidal activity through binding of Lipid II, a precursor of peptidoglycan, and therefore shows great potential as the foundation for discovery of a new generation of antibiotics to overcome the development of antimicrobial resistance.
Teixobactin (17) was isolated from the β-protobacterium, Eleftheria terrae that belongs to a new genus. Teixobactin (17) demonstrated potent activity against the resistant Gram-positive bacteria, MRSA and vancomycin-resistant Enterococci (VRE), as well as other bacterial species including, Mycobacterium tuberculosis (Mtb) and Clostridium difficile. Remarkably, no teixobactin-resistant mutants of Staphylococcus aureus or M. tuberculosis could be detected after sub-lethal dosing of the compound over a 27 day period [43]. This lack of resistance development may possibly be attributable to the mechanism of action which involves binding to Lipid II, inhibiting one of the membrane-associated steps of peptidoglycan biosynthesis [43,44]. Analogues of teixobactin (17) have undergone biological testing and results show that the L-allo-enduracididine (3, blue, Figure 7) residue is important for potent antibacterial activity [45]. An approximately 10-fold reduction in activity was observed when the enduracididine residue is substituted for L-arginine [46] and almost complete loss of activity was observed when three of the four D-amino acids of this analogue are substituted for their L-counterparts [47].

Synthesis of enduracididine
Although several synthetic approaches to enduracididine and its derivatives have been published, the discovery of teixobactin (17) has reignited interest in the synthesis of this unnatural amino acid. Bamberger cleavage of L-methylhistidine (22) to afford amide 23. Reduction of the double bond and cleavage of the three n-butyryl groups afforded lactam 24. Lactam 24 was opened with base and directly treated with guanylating agent 25 giving tosylguanidine 26 which was unstable upon standing. Immediate treatment with anhydrous HF gave L-enduracididine (1) as a mixture of diastereomers. The synthesis relied on the stereoselective formation of aziridine 27. This key reaction proceeded from allylglycine 28 in 28% yield to give a 7:3 mixture in favour of the S,S diastereomer. Attempted optimisation of the yield and diastereoselectivity afforded no improvement. The synthesis continued with aziridine opening using sodium azide and BF 3 ·OEt 2 in DMF at 65 °C, conditions which were key to prevent undesired intramolecular ring opening. The two diastereomers 29 and 30 could then be separated and elaborated to afford enantiopure pro- route to azide derivatives of β-hydroxyenduracididine [56]. The synthesis hinged on the use of azide 34 as a common intermediate to access both diastereomers. Diacetone D-glucose 35 was converted to azide 34 in 46% yield over twelve steps (Scheme 4). Azide 34 was easily converted to amino azide 36 via a two-step sequence, but conversion of azide 34 to amino azide 37 was more complex and required additional transformations [56].  [58]. Installation of the C-2 stereocentre again began with Garner's aldehyde 48 and Wittig olefination, followed by Sharpless dihydroxylation to stereoselectively afford diol 49 [59,60]. The C-2 epimer was accessed via Still-Gennari olefination of aldehyde 48 to afford the Z-olefin, which underwent dihydroxylation using potassium osmate to afford diol 50 [61,62]. With both diastereomers in hand, conversion to protected amino acids 46 and 47 was effected in four steps. 21% yield in seven steps from diol 49. The C-2 epimer 47 was converted to β-hydroxyenduracididine 52 using the same procedure. The new route proved more efficient than the previous report and provided access to both diastereomers suitably armed with orthogonal protecting groups.

Synthesis of L-allo-enduracididine by Ling et al.:
In 2014, Ling et al. filed a patent for their discovery of teixobactin (17) which included details of the structural elucidation. To confirm the configuration of the amino acids that are found in teixobactin, advanced Marfey's analysis was performed, requiring samples of known absolute stereochemistry for comparison (Scheme 11) [64]. The synthesis of L-allo-enduracididine (3) was reported to begin with nitro alcohol 61 and afforded the free amino acid in four steps via key intermediate 4-hydroxyarginine 62. The synthesis of nitro alcohol 61 was not described but its preparation has been reported [65]. All four diastereomers were synthesised for comparison with the isolated enduracididine sample. [66]. The C-4 stereocentre was installed through inversion of the hydroxy group of proline derivative 64 via mesylation and azide displacement to afford 65. Oxidation installed the required carbonyl group which allowed reductive ring opening to afford alcohol 66. Azide reduction, installation of the guanidine motif using Goodman's reagent (67) [67] and cyclisation afforded 68. Protecting group manipulation then afforded protected L-allo-enduracididine 63 with a free acid moiety available for peptide coupling over nine steps in 32% overall yield.  [68]. Their synthetic strategy started from silylated serinol 70 to which the mannosyl unit was attached to afford glycosylamine 71, prior to construction of the cyclic guanidine motif and amino acid functionality. With glycosylamine 71 in hand, attention turned to installation of the guanidine moiety. Treatment of 71 with isothiourea 33 followed by mesyl chloride afforded cyclic guanidine 72 in 70% yield. Silyl deprotection, Swern oxidation and Still-Gennari olefination afforded Z-alkene 73. Diastereoselective dihydroxylation of 73 followed by treatment with 1,1'-thiocarbonyldiimidazole (TCDI) and sodium azide afforded azide 69 over eight steps in 5.5% from silylated serinol 70. The reported route was the most efficient of the many investigated however, the exact sequence of functional group installation was important in order to obtain high yields.

Synthesis of Minosaminomycin by Kondo et al.:
The only total synthesis of minosaminomycin (9) to date was reported in 1977 by Kondo et al. (Scheme 14) [69]. Enduracididine (1) was prepared using the method reported by Shiba et al. [54] and was coupled with the isocyanate formed in situ from protected leucine 74 affording urea 75. Coupling of 75 with amino sugar 76 and global deprotection afforded minosaminomycin (9) in three steps from enduracididine (1). It should be noted that the diastereomer (2R-isomer) of 9 was also synthesised starting from D-enduracididine. Biological testing of both compounds revealed that the 2R-isomer exhibited 80% lower bacteriostatic activity against Mycobacterium smegmatis ATCC 607 compared to the parent natural product.
The synthesis of key amino acids 81 and 82 was based on an aldol reaction between protected aldehyde 83 and glycine 84 (Scheme 16). The reaction yielded 85 and 86 which proved to be easily separated by chromatography. The C-3 stereochemistry of the addition products 85 and 86 was rationalised by the Felkin-Ahn model, and the inability of diastereomer 85 to cyclise due to unfavourable steric interactions. Conversion of aldol products 85 and 86 to acetals 87 and 88, respectively, was achieved using standard transformations and Goodman's reagent (67) [67] was used to install the guanidine moiety. bacterial activity [35,38,39,41]. The most difficult challenge involved the preparation of the N-α-mannosyl-D-β-hydroxyenduracididine unit. N-Mannosylation was complicated by steric hindrance around the reaction site and poor compatibility of the cyclic guanidine motif with Lewis acids.
Initial attempts to glycosylate cyclic guanidine 91 using an array of donors under Lewis acidic or basic conditions failed to provide access to N-mannosylguanidine 92 (Scheme 18). However, gold(I) mediated [71] N-mannosylation using orthoalkynyl benzoate 93 finally afforded N-mannosylated 92 in 87% yield [70]. Application of these conditions to afford the fully functionalised amino acid 94 was unsuccessful. However, Attempts to utilise the same procedure reported by Doi et al. [33] to provide the amino acid L-β-hydroxyenduracididine 98 were unsuccessful. An alternative route to L-β-hydroxyenduracididine based on the synthesis reported by Oberthür et al. [58] afforded L-β-hydroxyenduracididine 98 in 7% yield over twelve steps from 99 (Scheme 20). In the final stages of the synthesis (Scheme 21), benzyl protected mannosyl D-β-hydroxyenduracididine 97 was coupled with H-Ser(Bn)-OAllyl to afford dipeptide 100. Unmasking of the amino and alcohol functionalities and peptide coupling with L-β-hydroxyenduracididine 98 afforded tripeptide 101 with no loss of the sugar group. This then completed the synthesis of the key fragment 101 and both mannopeptimycin α (12) and β (13) could be accessed in a further six steps.

Synthesis of teixobactin by Payne et al.:
The first syntheses of the teixobactin framework were completed by Albericio et al. [46] and Singh et al. [47]. These syntheses substituted the enduracididine residue for the more readily available L-arginine. The total synthesis of the full teixobactin structure was completed in 2016 by Payne et al. [72] using Fmoc solid-phase peptide synthesis (SPPS). The key to the synthesis was access to the protected L-allo-enduracididine residue 102 (Scheme 22). The synthesis of this building block was achieved using a com-bination of reported procedures beginning with protected aspartic acid 103. Using a protocol reported by Rudolph et al. [65] nitro alcohol 61 was accessed in two steps. Following procedures described in the patent filed by Ling et al. [64], alcohol 61 was converted to Boc-protected L-allo-enduracididine 68.
Protecting group exchange afforded the Fmoc protected L-alloenduracididine 102 in seven steps and 17% yield from acid 103.
The synthesis of the natural teixobactin (17)  peptide 106 to piperidine led to de-esterification. Final Fmoc removal of 107 and cleavage from the resin afforded linear peptide 108 which underwent macrolactamisation using 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMM·BF 4 ) and acid-mediated global deprotection to afford teixobactin (17) in 3.3% yield over twenty four steps.  [64,65]. The remaining NH of 68 was protected using Cbz-Cl before acid 111 was afforded after a two-step, deprotectionreprotection sequence. Fully protected enduracididine 111 was then coupled with L-isoleucine methyl ester 112 to give dipeptide 113. Cleavage of the N-Boc group and coupling with dipeptide 114 afforded protected linear precursor 115. Cleavage of the TBS and methyl ester protecting groups afforded seco-acid 116. However, during the hydrolysis step, two of the three Cbz groups were cleaved from the enduracididine residue, and the position of the remaining Cbz and CO 2 H could not be determined. It was decided that final deprotection of the remaining enduracididine protecting groups would take place after formation of the macrocycle.

Conclusion
The recent interest in teixobactin has resulted from its clinically unexploited mode of action, potent activity against resistant strains of bacteria and favourable pharmacokinetics. Structure-activity relationship studies of teixobactin suggest that the rare non-proteinogenic amino acid enduracididine, is a key residue for potent antibacterial activity. This observation has driven the need for new synthetic routes to enduracididine. However, current syntheses are cumbersome and inefficient. A robust and scalable synthetic route to an orthogonally protected enduracididine derivative suitable for solid phase peptide synthesis would greatly facilitate antibiotic drug development focused on a teixobactin inspired lead structure. Efficient access to enduracididine will enable ongoing structure-activity relationship studies of teixobactin and other lead compounds, for the development of much needed antibiotic drug candidates.