Chemistry and Biology of Teixobactin.

A Journal of

Accepted Article Title: Chemistry and Biology of Teixobactin Authors: Qi Zhang This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.201704167 Link to VoR: http://dx.doi.org/10.1002/chem.201704167

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10.1002/chem.201704167

Chemistry - A European Journal

Chemistry and Biology of Teixobactin Chuchu Guo1, Dhanaraju Mandalapu1, Xinjian Ji1, Jiangtao Gao2*, and Qi Zhang1* 1

Department of Chemistry, Fudan University, Shanghai 200433, China

2

College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

*

To

whom

correspondence

should

be

addressed:

E-mail:

[email protected]

and

Abstract Bacterial resistance to existing drugs is becoming a serious public health issue, urging extensive search for new antibiotics. Teixobactin, a cyclic depsipeptide discovered in a screen of uncultured bacteria, shows potent activity against all the tested Gram-positive bacteria. Remarkably, no teixobactin-resistant bacterial strain has been obtained despite extensive efforts, highlighting the great potential of teixobactin as a lead compound in fighting against antimicrobial resistance (AMR). This review summarizes recent progresses in the understanding of many aspects of teixobactin, including chemical structure, biological activity, biosynthetic pathway, and mode of action. We also discuss the different synthetic strategies in producing teixobactin and its analogues, and the structure-activity relationship (SAR) studies.

[Keywords] antimicrobial resistance; lipid II; nonribosomal peptide synthetase; thioesterase; depsipeptide

This article is protected by copyright. All rights reserved.

Accepted Manuscript

[email protected]

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1. Introduction Antibiotic resistance is becoming an increasingly serious threat to human health.[1] Due to the rapid and continuous evolution of antimicrobial resistance (AMR) genes in bacteria, which have been exposed to a much larger number and greater concentration of antimicrobial drugs in the last few decades, it appears that some bacterial pathogens may soon become immune to all

The vast majority of antibiotic scaffolds that are in use today were discovered in the middle of the last century, now referred to as the golden era of antibiotics.[2] However, the rate of antibiotic discovery later drastically declined owing to overmining of soil bacteria, with the repeated identification of the same compounds from time to time.[3] In contrast to the huge number of antibiotic classes found in the golden era, only one new class of antibiotic, daptomycin, was developed into clinical practice in the past five decades.

A possible way out of the current dilemma in antibiotic discovery is to expand the natural product mining scope to encompass the total microbial community. It has been estimated that only 1% or less of the bacteria in the environment are able to grow in laboratory, whereas the majority of microorganisms are only known by molecular fingerprints, leaving a vast number of natural product producers untapped.[4] Of course, the metabolic capability of unculturable strains could be reprogramed by genomics-based technologies, such as pathway heterologous expression and/or enzyme in vitro reconstitution, but a more straightforward strategy is to develop new cultivation approaches to grow microorganisms that resist traditional cultivation methods. Recently, a novel cultivation technique, named iChip, has been developed to assess the antibiotic-producing capacity of unculturable bacteria. Briefly, a soil sample was diluted into chambers that then contact the natural sediment through a semipermeable membrane, and diffusion of nutrients and growth factors through the membrane enables growth of uncultured bacteria in their natural environment.[5] By using this sophisticated technique, Lewis and coworkers isolated teixobactin, a novel peptide natural product from a previously unknown β-proteobacterium Eleftheria terrae.[6] The discovery of teixobactin exceptionally experienced a worldwide echo in the international


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commercially available antibiotics, posing a risk of returning to the pre-antibiotic era of epidemics.

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press, as it raises a great promise for the emergence of future antibiotics without the potential to develop resistance.[7]

Teixobactin is a depsipeptide consisting of eleven amino acid (aa) residues, including seven L-aa

part of the C-terminal tetrapeptide lactone substructure formed by an ester linkage between D-Thr8 and L-Ile11 (Figure 1). Teixobactin shows excellent antimicrobial activity against many notorious Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA, MIC 0.25

μg

mL−1),

vancomycin-intermediate

S.

aureus

(VISA,

MIC

0.5

μg

mL−1),

vancomycin-resistant Enterococcus (VRE, MIC 0.5 μg mL−1), and penicillin-resistant Streptococcus pneumonia (PRSP, MIC 0.03 μg mL−1), among many others.[6] Remarkably, teixobactin exhibits potent activity against Mycobacterium tuberculosis (Mtb, MIC 0.125 μg mL-1), many of which show resistance to the frontline antibiotic treatments, and is extremely active against Clostridium difficile (MIC 0.05 μg mL-1).[6] Plating on media with a low dose of teixobactin did not produce teixobactin-resistant mutants of S. aureus and M. tuberculosis, and serial passage of S. aureus in the presence of sub-MIC levels over a period of 27 days still failed to yield any resistant strains.[6] Teixobactin has no toxicity against mammalian NIH/3T3 and HepG2 cells at 100 mg mL-1, and shows neither haemolytic activity nor DNA-binding activity.[6] An animal efficacy study using a mice septicemia model showed that teixobactin has a PD50 of 0.2 mg kg-1, which significantly outperforms vancomycin (PD50 2.75 mg kg-1).[6] All these observations demonstrate the phenomenal antibacterial activity of teixobactin and its great potential in drug development.


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and four D-aa residues. Among them is an unusual L-allo-enduracididine (L-allo-End), which is

Figure 1. The chemical structure of teixobactin. D-amino acids are shown in red and L-allo-End is shown in blue.

2. Mode of action The fact that resistance to teixobactin was not detected despite extensive efforts suggests that the target of this compound is likely not an endogenous protein. Because the essential lack of resistance development through mutations has been well known for vancomycin, it appears that teixobactin may act on the same target of vancomycin, lipid II, the precursor of peptidoglycan (Figure 2). This proposal was supported by labeling studies, showing that teixobactin strongly inhibited peptidoglycan biosynthesis but had no effect on label incorporation into DNA, RNA and protein.[6] Addition of purified lipid II prevented teixobactin from inhibiting bacterial growth, suggesting that teixobactin directly interact with lipid II, and stoichiometric analysis showed that it binds to lipid II with a 2:1 ratio.[6] Teixobactin is active against several vancomycin-resistant strains (e.g. VISA and VRE), suggesting that the lipid II binding site of teixobactin is different from that of vancomycin.[6,

8]

Further analysis show that teixobactin likely binds to the

pyrophosphate moiety and the first sugar moiety attached to the lipid carrier (Figure 2).[6] Notably, such a structural motif of lipid II is also present in the wall teichoic acid (WTA) precursor lipid III,[9] suggesting that teixobactin is also an inhibitor of WTA biosynthesis (Figure 2), which also represents an important target for antimicrobial drug development.[10]


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Figure 2. Biosynthesis of peptidoglycan and wall teichoic acid (WTA), showing the chemical structure of lipid II and lipid III and the proposed binding site of teixobactin.

Compared with other lipid II-targeting antibiotics such as vancomycin, teixobactin exhibits impressive bactericidal activity and is good in killing late exponential phase bacterial populations.[6] It is noteworthy that the frequent failure in vancomycin clinic treatment is owing to its poor bactericidal activity,[11] again reflecting the great potential of teixobactin for antibiotic development. The superior bactericidal activity of teixobactin is because of its dual modes of action, which simultaneously inhibit peptidoglycan and WTA biosynthesis. This triggers synergistic effects, resulting in cell wall damage, delocalization of the cell wall degrading proteases antolysins, and subsequent cell lysis.[8] Although teixobactin also efficiently binds lipid I, its contribution to antimicrobial activity is probably less significant, because unlike the surface-exposed lipid II and lipid III, lipid I is intracellular. Teixobactin does not bind mature peptidoglycan, which likely contributes to its activity against dense populations.[8] It is noteworthy that binding to mature peptidoglycan is an important reason of the reduced bactericidal activity of vancomycin at high cell densities, which results in vancomycin-intermediate resistance.[12]

3. Biosynthesis of Teixobactin


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Through a homology search, the biosynthetic gene cluster of teixobactin was identified (GenBank accession number KP006601), which mainly consists of two large non-ribosomal peptide synthetase (NRPS) genes, txo1 and txo2 (Figure 3).[6] The two NRPSs have eleven modules in total, and the in silico predicted substrate specificity of each adenylation domain matches well with the teixobactin structure.[6, 13] Hence teixobactin biosynthesis follows the canonic co-linearity

acid in the nascent peptide chain (Figure 3).[14] The linear peptide chain is then released from the NRPS assembly line by a lactonization reaction between L-Ile11 and D-Thr8. A methyltransferase (MT) domain is present in the first module (module 1), which is responsible for N-methylation of D-Phe1 (Figure 3). Notably, the second NRPS Txo2 contains two tandem thioesterase (TE)

domains at its C-terminus. Such an architecture is rare in NRPS enzymology[14] and only found occasionally.[15] It is noteworthy that module 8, the second module in Txo2, contains an extra condensation (C) domain, and the functions of this extra C domain remain to be investigated. Several genes are adjacent to txo1 and txo2, the functions of which are not clear. These genes are presumably involved in regulation, product exporting, or resistance.

Figure 3. Biosynthetic pathway of teixobactin. Domain analysis were performed by using NRPSpredictor2.[13b] The two TE domain are shown in magenta.


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rule of NRPSs, in which each module is responsible for the incorporation of one specific amino

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Teixobactin contains a non-proteinogenic amino acid L-allo-End with a unique five-membered cyclic guanidine moiety (Figure 1). L-enduracididine (L-End), a diastereoisomer of L-allo-End, has been found in several natural products, including mannopeptimycin and enduracidins (Figure 4), two compounds that also bind lipid II.[16] Early feeding experiments with isotopically labelled

analysis of the mannopeptimycin and enduracidin biosynthetic gene clusters revealed three pairs of enzymes with a high degree of sequence identity: EndP/MppP (80%), EndQ/MppQ (68%), and EndR/MppR (75%).[18] Biochemical and structural studies showed that MppP is a PLP-dependent hydroxylase,

which

catalyzes

the

conversion

of

L-Arg

and

dioxygen

to

produce

2-oxo-4-hydroxy-5-guanidinovaleric acid (1) (Figure 5).[19] MppR shares a high degree of structural

similarity

with

acetoacetate

decarboxylase,

which

converts

1

to

3-[(4R)-2-iminoimidazolidin-4-yl]-2-oxopropanoic acid (2), the ketone form of L-End.[20] The PLP-dependent aminotransferase MppQ then catalyzes the reductive amination of 2 to produce L-End (Figure 5). Despite of the different stereochemistry at C4, biosynthesis of L-allo-End in

teixobactin may follow a similar route to that of L-End. However, no homologous genes of L-End biosynthesis has been found in the teixobactin gene cluster. A possible scenario is that the genes responsible for L-allo-End biosynthesis are located at the other position of the E. terrae chromosome; future studies are awaited to test this hypothesis.


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compounds showed the L-End moiety in enduracidin is derived from L-Arg.[17] Comparative

Figure 4. Natural products containing L-End.[18c] The L-End moieties are highlighted in blue.

Figure 5. Proposed biosynthesis pathway of L-End in mannopeptimycin. The chemical structure of L-allo-End is also shown in blue.

4. Chemical Synthesis of L-allo-End Teixobactin has a molecular scaffold of moderate complexity, which, as detailed in the next section, can be accessed by conventional solid phase peptide synthesis (SPPS). However, the L-allo-End unit is not commercially available, limiting the scalable synthesis of teixobactin for

downstream studies. One of the main challenges in L-allo-End synthesis is to establish the C4 chiral center in a highly stereoselective way, which was not achieved in the previous synthesis


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efforts.[21] To reveal the configuration of the enduracididine moiety of teixobactin, Lewis and coworkers synthesized all the four diastereomers.[22] L-allo-End was obtained by 4 steps from the staring material 3 (Figure 6), which can be easily obtained from the protected L-Asp, as shown by Rudolph and coworkers.[23] However, L-End was also produced as a minor product, with a 1:6

Figure 6. Synthesis of L-allo-End by Lewis and coworkers.[22]

In 2015, Yuan and coworkers reported the first highly stereoselective and scalable synthesis of L-allo-End, which affords L-allo-End by 10 steps from the Boc-protected trans-hydroxyproline in good yield (overall 31%) with more than 50:1 diastereoselectivity.[24] The key step in this synthesis route is the construction of the cyclic guanidine moiety by an intramolecular nucleophilic substitution reaction (Figure 7). The linear intermediate 5 serves as a key intermediate in this synthetic approach, which contains all the requisite chiral centers of the final product L-allo-End. The C2 stereochemistry is derived from the starting material 4, whereas the C4 chiral center is established by inverting the original stereochemistry by an SN2 reaction (Figure 7). The guanine moiety is introduced by using Goodman’s reagent, allowing for the ring closure by an intramolecular nucleophilic substitution of the pendant guanidine after the hydroxyl was converted to a mesylate (Figure 7). A noteworthy feature of this synthetic approach is the using of a bulky tert-butyl to mask the carboxylate, which prevents not only lactone formation from 5 but also the lactamization of 6, securing the efficient synthesis of L-allo-End.


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ratio to L-allo-End (Figure 6).

Figure 7. Synthesis of L-allo-End by Yuan and coworkers.[24]

Payne and coworkers later reported another way to prepare L-allo-End building block from the protected L-Asp ester as the starting material (Figure 8).[25] In this synthetic effort, the C4 stereochemistry is introduced by a stereoselective ketone reduction using L-selectride, and the minor diastereoisomeric product was removed by flash column chromatography. A variant of Goodman’s reagent was used to install the guanidine moiety, and a following intramolecular cyclization by trifilation of the γ-alcohol in 7 under basic conditions produce the L-allo-End scaffold. The following acidic deprotection and Fmoc protection of α-amine afford the target building block 8, with an overall yield of 21% (Figure 8). By using a similar strategy, Reddy and coworkers achieved gram-scale synthesis of the L-allo-End building block, allowing for the synthesis of the teixobactin macrocyclic core.[26] In this synthetic route, 8 was further derivatized by CbzCl to give fully protected L-allo-End. However, the Cbz protecting group was unable to be completely removed from the final macrocyclized product, and this possibly due to the intramolecular hydrogen bonding between nitrogen atom and the carboxylic acid group on the enduracididine ring.[26]


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Figure

8.

Synthesis

of

L-allo-End

by

Payne

and

coworkers.[25]

DIPEA,

N,N-diisopropylethylamine.

5. Total synthesis of Teixobactin The success in efficient preparation the L-allo-End building block paves the way to access teixobactin by conventional SPPS. Thus far two groups of scientists have reported the total synthesis of teixobactin.[25, 27] Although their synthetic strategies are very different from each other, they both installed the ester linkage between D-Thr8 and L-Ile11 at an early stage, and performed the macrolactamization between L-Thr8 and L-Ala9, a site that is less sterically congested.

Payne and coworkers adopted a linear synthetic strategy involving an early on-resin esterification step, and a key solution-phase macrolactamization step followed by global side-chain deprotection (Figure 9).[25] The initial efforts with 2-chlorotrityl chloride (2-CTC) functionalized polystyrene resin failed in the on-resin esterification step, which is likely owing to the steric bulk of the 2-CTC linker. Hence (4-(hydroxymethyl)-3-methoxyphenoxy)acetic acid (HMPB) functionalized polyethylene glycol-based NovaPEGresin was used, taking advantage of the decreased steric bulk surrounding the loaded amino acids as well as the increased swelling properties provided by the polyethylene glycol-based support.[28] By this way, on-resin esterification step and further


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Fmoc-based amino acid chain extension proceed smoothly to give the intermediate 9. However, conventional Fmoc removal led to the formation of a de-esterified resin-bound peptide, which is likely due to diketopiperazine formation resulted from the nucleophilic attack of the α-amine of the deprotected L-allo-End onto the L-Ile α-carbonyl.[29] This side reaction can be largely avoided by treating the resin with diluted piperidine (10 % in DMF) for a shorter time (30 s) to remove the

add the L-Ala moiety (Figure 9). Teixobactin was finally synthesized with a yield of 3.3% over 24 steps, and its identity has been validated by NMR analysis and antibacterial activity evaluation.[25]

Figure 9. Total synthesis of teixobactin by Payne and coworkers.[25]


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Fmoc group, and the resin was washed rapidly and then treated with the preactivated solution to

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Distinct from the method reported by Payne et al.,[25] Li and coworkers reported a convergent synthesis of teixobactin by utilizing a key Ser-based ligation approach,[27] which was previously developed in their lab (Figure 10).[30] The ligation approach uses a peptide substrate containing a C-terminal salicylaldehyde ester, and a second peptide substrate with an unprotected Ser or Thr

linked intermediate, which can be readily converted to the natural peptidic linkage without isolation (Figure 10).

Figure 10. Salicylaldehyde ester-mediated peptide ligation at serine/threonine sites.[30]

Preparation of the linear hexapeptide salicylaldehyde esters 10 was achieved by conventional Fmoc-SPPS starting from 2-coumarate with a subsequent ozonolysis (Figure 11). Synthesis of the cyclic depsi-pentapeptide 11 started from construction of the ester linkage between Fmoc-Ile-OH and Alloc-D-Thr-OH via solution-phase coupling, and the resulting depsidipeptide was immobilized onto 2-Cl-Trt resin (Figure 11). Using of the two orthogonal amine protecting group (i.e. Fmoc and Alloc) enables extension of the peptide chain both from the N-terminus and C-terminus to produce 11. It is noteworthy that in contrast to the report by Payne et al.,[25] de-esterification of the resin-bound peptide via diketopiperazine formation was not observed in this case, which is possibly because of the steric hindrance of the bulky 2-Cl-Trt resin. 10 and 11


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residue at the N-terminus. The two peptides are then ligated to form an N,O-benzylideneacetal

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was then ligated by the salicylaldehyde ester-mediated coupling reaction to afford teixobactin (Figure 11). To demonstrate the robustness and general applicability of this synthetic strategy, they

Figure 11. Total synthesis of teixobactin by Li and coworkers.[27] PMBCl, para-methoxybenzoyl chloride;

EDCI,

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

(hydroxyimino)cyanoacetate; DIPEA, N,N-diisopropylethylamine.


OxymaPure,

ethyl

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also synthesized several teixobactin analogues, which are discussed in below.

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6. Teixobactin analogues and the structure-activity relationship (SAR) analysis Because of the inconvenience to access the L-allo-End building block, many efforts have been made to synthesize teixobactin analogues, in which the L-allo-End10 is replaced by commercially available amino acids. For example, L-Arg contains a similar guanidine moiety and hence has been commonly used as a surrogate of L-allo-End (Table 1), which significantly simplifies the

(which are termed L-Arg10-teixobactins; similar nomenclatures apply to other teixobactin analogues) was reported earlier than teixobactin total synthesis.[31] Several synthesis routes have been developed for producing teixobactin analogues. Thus far more than 110 teixobactin analogues have been made, the structures and antibacterial activities of these analogues are summarized in Table 1 and Table 2, respectively.

Four routes for producing L-Arg10-teixobactin are summarized in Figure 12. Among them route 4 is unique in using aryl hydrazide resin as the solid support, which is stable both in strong acidic and basic conditions, and can be easily removed under mild oxidative conditions. More importantly, the acyl diazene intermediate produced after copper oxidation react in situ with the Arg10 amino group, thereby releasing the cyclized peptide into solution; racemization reaction was also suppressed in this macrocyclization process.[32] In all these four routes, formation of the macrocyclic ring is the last step, and two different cyclization sites were selected. In route 1, 2, and 3, cyclization occurs between Ala9 and Arg10, which minimizes steric hindrance of the side chain groups. In route 3, however, cyclization occurred between Arg10 and Ile11, the latter residue contains a large sec-butyl side chain. Although the yield of this cyclization reaction was not reported, the overall yield of L-Arg10-teixobactin in route 3 is comparable to other routes (Figure 12), suggesting that side chain steric hindrance is not a problem in this case. It is also noteworthy that in route 1, alloc group was used to protect the α-amino group of L-Arg (Figure 12), because using Fmoc group in this case will likely lead to diketopiperazine formation and hence peptide cleavage.[31a]


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synthesis procedure. Indeed, total synthesis of the L-allo-End10 to L-Arg substituent of teixobactin

Figure 12. Four different routines for the synthesis of teixobactin analogues (exemplified by Arg10-teixobactin, TX-1). Route 1, Route 2, Route 3, and Route 4 were reported by Albericio and coworkers,[31a] Singh, Taylor, and coworkers,[31b] Nowick and coworkers,[35] and Su, Fang, and coworkers,[32] respectively. The overall yields are also shown for comparison.


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In contrast to the four routes discussed above, in which formation of the macrocyclic ring was carried out in solution as the last step (Figure 12), Albericio, de la Torre, and coworkers recently reported another route in synthesizing Lys10-teixobactin analogues, in which cyclization was conducted in solid-phase prior to installation of the five N-terminal residues (Figure 13).[33] Briefly, Fmoc-Lys-OAllyl was immobilized onto 2-Cl-Trt resin through the ε-amino of Lys, and the

Alloc-Ile11-OH via esterification with L-Thr8. After removal of the Lys10-Allyl and Ile11-Alloc groups with Pd(0), cyclization were achieved on resin with excellent yields (> 95%). The remaining five N-terminal amino acids were added by using conventional SPPS chemistry (Figure 13).

Figure 13. Synthesis of teixobactin analogues (exemplified by Lys10-teixobactin, TX-1) by Albericio, de la Torre, and coworkers.[33]


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peptide was elongated up to the incorporation of L-Ile6, followed by the introduction of

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Because of the structural similarity between L-Arg and L-allo-End, L-Arg10-teixobactin (TX-1) is the first reported analogue, which shows ～10-fold decreased activity compared to natural teixobactin.[31] However, because of its relatively easy access by conventional SPPS chemistry, it has been commonly used as a model to interrogate the SAR of teixobactin (Table 1 and 2). Initial

TX-15, produced by replacing the N-Me-D-Phe1 with N-Me-L-Phe, exhibits 32-fold decreased activity compared to TX-1.[31b, 34] Other modifications such as changing D-Thr8 to L-Thr (TX-7), changing D-allo-Ile5 to L-Ile (TX-13), and changing D-Gln4 to L-Gln (TX-14) all result in completely abolished or significantly decreased activities against Staphylococcus aureus (Table 1 and 2).[34-35] Moreover, replacing D-allo-Ile by D-Ile in TX-1 and teixobactin (corresponding to TX-5 and TX-37) also results in 10-fold and 16-fold decreased activity.[27, 36] Intriguingly, TX-34 exhibits comparable activity to its enantiomer TX-1, supporting that only the relative, not the absolute configurations, are important for the antimicrobial activity.[35]

Singh, Taylor and coworkers reported the NMR structures of several teixobactin analogues, in which one or more D-aa resdiues were replaced by their L-isomers.[34] This study indicated that the D-aa residues (D-Gln4 in particular) play important roles in maintaining a highly dynamic and relatively unstructured scaffold, which is essential for activity.[34] Interestingly, Nowick and coworkers showed that TX-33, in which D-Thr8 is replaced by D-diaminopropionic acid (D-Dap), exhibits similar activity with TX-1, suggesting that replacing the lactone oxygen atom with an amide NH does not change the antibacterial activity, nor does removing the methyl group (and hence the C3 stereochemistry) of D-Thr8.[37]

Nowick and coworker also reported a truncated teixobactin analogue (TX-10), which lacks the five N-terminal residues of teixobactin and is not active against all the tested strains.[35] Intriguingly, the antibacterial activity is restored by introducing an N-terminal dodecanoyl group in TX-10 to give TX-11 (also named lipobactin), suggesting that the N-terminal tail of teixobactin, which is highly hydrophobic, is probably responsible for membrane anchoring.[35] Recently, they


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structural modification of TX-1 revealed the vital importance of the stereochemistry. For example,

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also reported the crystal structure of the N-acetylated TX-10, showing that a chloride anion coordinates to three amide NH groups of the cyclic depsipeptide ring.[37] This observation suggesting that the teixobactin macrolactone may bind the anionic pyrophosphate moiety of lipid II, in a way similar to the binding pattern of the lantibiotic nisin.[38] The fact that the enantiomeric isomer TX-34 exhibit a similar activity with teixobactin is consistent with the hypothesis,[37]

the achiral pyrophosphate group of lipid II. The crystal structure of the N-acetylated TX-10 also reveals a hydrogen bond between the amide NH group of Ala9 and the side chain of Ser7, which is likely important for activity, because TX-32 (resulting from changing the Ser7 of L-Arg10-teixobactin to L-Ala) shows significantly diminished activity.[37] This proposal was

further supported by a very recent study, showing that, although TX-73 (resulting from replacing the Ser7 and Ala9 of L-Lys10-teixobactin to L-Lys) exhibits dramatically decreased activity, the activity is partially retained in TX-77 (an L-Lys10-teixobactin analogue in which Ser7 and Ala9 are replaced by a diaminopropionic acid), which likely possess of similar hydrogen bond as observed in the N-acetylated TX-10.[33]

Because the first N-Me-D-Phe1 appears not contribute to the unstructured scaffold of teixobactin, it was proposed the decreased activity of TX-15 (in which the N-Me-D-Phe1 is replaced by N-Me-L-Phe) is possibly owing to its susceptibility to proteolysis.[34] Removal of the methyl group on N-Me-D-Phe1 (TX-27) did not change the activity,[32] whereas addition of a second methyl group onto N-Me-D-Phe1 (TX-105) resulted in slightly decreased activity.[39] Intriguingly, changing the methyl to an acetyl group (TX-4) resulting in more than 600-fold decrease of activity,[40] and substitution of N-Me-D-Phe1 by N-Me-D-Lys also led to a total loss of activity.[33] These observations reflect the intriguing role of the first N-Me-D-Phe as well as the N-terminal tail of teixobactin in interacting with the lipid target.

A lysine scan based on L-Arg10-teixobactin (TX-1) by Albericio, de la Torre, and coworkers showed that replacing any of the four isoleucine residues (i.e. L-Ile2, L-allo-Ile5, L-Ile6, and L-Ile11) by lysine resulted in complete loss of antibacterial activity, suggesting the hydrophobicity of the


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because pyrophosphate group is achiral; amide NH groups on the macrolactone ring may bind to

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isoleucine side chain is important for teixobactin activity. Replacing some other residues such L-Ser3 and L-Gln4 with lysine, however, does not significantly change the activity.[41] Very recently, they reported the synthesis of several additional teixobactin analogues with an increasing number of positive charges.[33] They showed that Lys substitution of Ser3 and D-Gln4, Ser3 and Ala9, and D-Gln4 and Ala9, of L-Lys10-teixobactin (which produced TX-70, TX-71, and TX-72,

TX-73) led to significantly decreased activity.

Interestingly, L-Lys10-teixobactin (TX-35), in which L-allo-End is replaced by L-Lys, exhibits ～ 4 fold higher activity than L-Arg10-teixobactin (TX-1).[35] L-Orn10-teixobactin (TX-38), L-DAB10-teixobactin (TX-67), and L-DAP10-teixobactin (TX-68), in which L-allo-End is replaced

by L-ornithine, L-2,4-diaminobutyric acid (DAB), and L-1,3-diaminopropionic acid (DAP), respectively, show similar activities to TX-1,[42] whereas L-His10-teixobactin (TX-36) shows ～5 fold decreased activity compared to TX-1.[32] Several analogues including L-NorArg10-teixobactin (TX-65), L-HoArg10-teixobactin (TX-66), and L-GAPA10-teixobactin (TX-69) were produced by direct

guanidinylation

of

the

corresponding

amine

precursors

by

using

pyrazole-1-carboximidamide and Et3N (Figure 14).[42] No apparent difference in antibacterial activity was observed between the amine derivatives (i.e. TX-35, TX-38, TX-67, and TX-68) and their corresponding guanidinylated analogues (i.e. TX-66, TX-1, TX-65, and TX-69),[42] demonstrating the considerable tolerance for the substitution of the L-allo-End10 residue. Notably, replacing the L-allo-End10 residue with 4-amino-L-Pro (Amp), which incorporates conformational restriction to the new analogue TX-91, led to a complete loss of activity. Similar observations were also made for TX-92 and TX-93, in which steric hindrance was introduced into the guanidine moiety of TX-1.


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respectively) retained the activity, whereas Lys substitution of L-Ser7 and L-Ala9 (which produced

Figure 14. Direct guanidinylation in the synthesis of teixobactin analogues.[42]

Very recently, Nowick and coworkers reported an alanine scan of L-Lys10-teixobactin (TX-35), showing that position 3 (L-Ser) of teixobactin tolerates modification without loss of antibiotic activity.[43] Except for position 1 (N-Me-D-Phe) and position 6 (L-Ile), both of which are highly sensitive to substitution, all other positions tolerate modification to some extends.[43] Remarkably, TX-101 produced by replacing L-allo-End with L-Ala retains most of the activity, opening the door to the development of more teixobactin analogues. The authors also noted a positive correlation between the poor aqueous solubility of teixobactin analogues and the antibiotic activity,[43] which is consistent with the fact that teixobactin binds lipid targets.

7. Outlook Since the ground-breaking discovery of teixobactin in 2015, a large body of researches, including total synthesis, SAR, and mode of action, have been conducted, and apparently, these efforts are just a start. The remarkable antibacterial activity of teixobactin and its low toxicity in mammalian cells offers an appealing working platform for synthetic chemists. Although thus far all the synthesized analogues are less active compared to teixobactin, several analogues (e.g. TX-35 and TX-66) possess desirable activities, which may serve as leads for more extensive modifications. In the meantime, the detailed interaction between teixobactin and its lipid target is currently unclear, and future structural analysis will be of great significance in guiding rational design for better


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antibiotics. Another important aspect in future studies is biosynthetic investigation of teixobactin, which largely lags behind chemical synthesis studies. Knowledge from these studies may help to produce teixobactin and its analogues in large scale at much less expense by biotechnology efforts, such as pathway engineering and chemoenzymatic transformations. It should be pointed out that several barriers need to be overcome before the clinical use of teixobactin, such as the

extensively investigated.

Natural products continue to serve as valuable starting points in the identification of lead compounds for chemistry programs involved in drug discovery. The great promise of these studies is further highlighted by the fact that the known natural products likely represent only a tip of iceberg of the total natural product biosynthetic capacity, as the majority of microorganisms is only characterized by genomic analysis and thus far resist cultivation. With the ever-increasing development of microbial and biochemical technologies, mining of these untapped pools of microorganisms for novel antibiotics, as exemplified by the iChip technology in teixobactin discovery, will certainly be a rewarding effort for future antibiotic drug therapy.

Table 1. A summary of teixobactin analogues. NorArg, norarginine; HoArg, homoarginine; DAB, L-2,4-diaminobutyric acid; DAP, L-1,3-diaminopropionic acid; GAPA, L-2-amino-3-guanidinoaminopropionic acid; Chg: cyclohexylglycine; Amp: 4-aminoproline; Tmg: 2-amino-4-(tetra-methylguanidino)butanoic acid; Mmg: 2-amino-4-(dimethylmorphilinoguanidino)butanoic acid. De: decanyl; Dec: decanoyl; Tm: tetramethylguanidino; TX-11 was also named lipobactin.[35]


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bioavailability issue. The mode of action and the possible resistant mechanism also await to be

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Table 1 (continued).


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Table 2. Teixobactin SAR studies. Minimal inhibitory concentration (MIC) values are shown in

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μg/ml. NI, no inhibition; MRSA, methicillin-resistant S. aureus.


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References

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Acknowledgements This work is supported by grants from the National Key R&D Program of China (2016 Y F A0501302), from National Natural Science Foundation of China (1500028 and 31670060), and from the Open Fund of Key Laboratory of Glycoconjugate Research, Fudan University, Ministry of Public Health, and from the Chemical Structure Association Trust.


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Biographical Information

the Department of Chemistry at Fudan University. She entered Fudan University in 2014, and joined Prof. Qi Zhang’s group in 2016 to investigate novel chemistry in natural product biosynthesis.

Dhanaraju Mandalapu was born and raised in Tummagudem, India. He received his master’s degree from Andhra University in 2010. He joined Dr. V L Sharma’s group at CSIR-Central Drug Research Institute in 2012 and obtained his PhD in 2016. His doctoral research focused on medicinal chemistry of biologically active compounds. He joined Prof. Qi Zhang’s group at Fudan University in 2017 as postdoctoral fellow to study chemoenzymatic synthesis of pharmaceutically interesting natural products.

Xinjian Ji was born and raised in Nantong, China. He received his BSc in pharmacology from Nanjing Tech University in 2013. In 2014, He joined Prof. Qi Zhang’s group at Fudan University to pursue his PhD degree in chemical biology. His doctoral work focuses on natural product biosynthesis and the catalytic mechanism of radical SAM enzymes.


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Chuchu Guo was born and raised in Lanzhou, China. She is currently an undergraduate student in

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Jiangtao Gao received his BSc from Ocean University of China and MSc in Marine Biology from

program in the School of Pharmacy at the University of Mississippi and received PhD in pharmaceutical sciences in 2010. Following this he was a postdoctoral fellow at University of Illinois at Urbana and Champaign, working with Prof. Wilfred A. van der Donk on phosphonate and phosphinate antibiotics. In 2015 he joined Fujian Agriculture and Forestry University as a full professor and started his independent career. His research interests include discovery of novel antibiotics from nature by genome mining.

Qi Zhang was born and raised in Mianyang, China. He obtained his BSc in chemistry from Fudan University in 2003 and his PhD in 2010 from Shanghai Institute of Organic Chemistry (SIOC). After three and a half years of postdoctoral work with Prof. Wilfred A. van der Donk at University of Illinois at Urbana-Champaign, he took up his current position in the Department of Chemistry at Fudan University in 2014. His research interests include natural product biosynthesis, mechanistic enzymology, enzyme engineering, and chemical biology.


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the Institute of Oceanology, Chinese Academy of Sciences. In 2005 he joined the graduate

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Table of Contents (TOC)


Digital biology and chemistry.

Chemistry and biology of prions.

Total synthesis of teixobactin.

Chemistry and Biology of Self-Cleaving Ribozymes.

The chemistry and biology of mycolactones.

The chemistry and biology of theta defensins.

Aptamers and SELEX in Chemistry & Biology.

Fungal phenalenones: chemistry, biology, biosynthesis and phylogeny.

Neurotrophic natural products: chemistry and biology.

Elucidation of the Teixobactin Pharmacophore.

Computational systems biology methods in molecular biology, chemistry biology, molecular biomedicine, and biopharmacy.

The chemistry and biology of febrifugine and halofuginone.

Chemistry and biology of protein and inositol phosphorylation.

Chemistry and biology of the immunophilins and their immunosuppressive ligands.

Retracted: Chemistry and biology of essential oils of genus boswellia.

Physics, chemistry and biology of functional nanostructures III.

Recent advances in the chemistry and biology of pyridopyrimidines.

Recent advances in the chemistry and biology of benzothiazoles.

ROSics: chemistry and proteomics of cysteine modifications in redox biology.

Physics, chemistry and biology of functional nanostructures II.

Supramolecular chemistry at the interface of biology, materials and medicine.

Natural product synthesis at the interface of chemistry and biology.

Coalition of Biology and Chemistry for Ameliorating Antimicrobial Drug Discovery.

Chemistry and biology of the enterobacterial common antigen (ECA).