The Total Chemical Synthesis and Biological Evaluation of the Cationic Antimicrobial Peptides, Laterocidine and Brevicidine

Cationic Antimicrobial Peptides, Laterocidine and Brevicidine

Article

The Total Chemical Synthesis and Biological Evaluation of the

Yann Hermant, Dennise Palpal-latoc, Nadiia Kovalenko, Alan J. Cameron, Margaret A. Brimble,*

and Paul W. R. Harris *

Cite This: https://doi.org/10.1021/acs.jnatprod.1c00222

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

ABSTRACT: Antimicrobial resistance is a signiﬁcant threat to

public health systems worldwide, prompting immediate attention

to develop new therapeutic agents with novel mechanisms of

action. Recently, two new cationic non-ribosomal peptides

(

CNRPs), laterocidine and brevicidine, were discovered from

Brevibacillus laterosporus through a global genome-mining

approach. Both laterocidine and brevicidine exhibit potent

antimicrobial activity toward Gram-negative bacteria, including

diﬃcult-to-treat Pseudonomas aeruginosa and colistin-resistant

Escherichia coli, and a low risk of resistance development. Herein,

we report the ﬁrst total syntheses of laterocidine and brevicidine

via an eﬃcient and high-yielding combination of solid-phase synthesis and solution-phase macrolactamization. The crucial

depsipeptide bond of the macrolactone rings of laterocidine and brevicidine was established on-resin between the side-chain hydroxy

group of Thr with Alloc-Gly-OH or Alloc-Ser(tBu)−OH, respectively. A conserved glycine residue within the lactone macrocycle is

exploited for the initial immobilization onto the hyper acid-labile 2-chlorotrityl chloride resin, subsequently enabling an eﬃcient

solution-phase macrocyclization to yield laterocidine and brevicidine in 36% and 10% overall yields, respectively (with respect to

resin loading). A biological evaluation against both Gram-positive and Gram-negative bacteria demonstrated that synthetic

laterocidine and brevicidine possessed a potent and selective antimicrobial activity toward Gram-negative bacteria, in accordance

with the isolated compounds.

he emergence and rapid spread of AntiMicrobial

antimicrobial activities against both Gram-positive and Gram-

negative bacteria (sometimes selectively), including antibiotic-

resistant microbes, such as methicillin-resistant S. aureus

Resistance (AMR) has signiﬁcantly threatened public

health worldwide. In 2018, the World Health Organization

(

WHO) declared AMR to be one of the biggest threats to

(MRSA), vancomycin-resistant Enterococci (VRE), MDR P.

12−14

global health, food security, and development. Globally, at

least 700 000 people die each year from AMR-related

infections and diseases, including infections caused by

multidrug-resistant (MDR) organisms (Staphylococcus aureus,

Escherichia coli, Enterococcus faecium, Streptococcus pneumoniae,

Klebsiella pneumoniae, Pseudomonas aeruginosa, and Mycobacte-

aeruginosa and MDR M. tuberculosis.

A notable example is

teixobactin, a recently discovered macrocyclic Non-Ribosomal

Peptide (NRP) that displays excellent activity against Gram-

positive bacterial pathogens, including MDR strains, such as

MRSA, VRE, and M. tuberculosis. Teixobactin proved

refractory toward bacterial resistance development, thought

to be a result of its ability to simultaneously target a range of

highly conserved critical components of the bacterial cell wall

rium tuberculosis). High rates of morbidity and mortality (in

both humans and animals) due to AMR are a burden to society

and the economy. It is predicted that AMR infections will

result in nearly 10 million deaths per year by 2050 and a total

gross domestic product (GDP) loss of US$100.2 trillion by

via unique modes of action. Numerous research groups,

including our own, have pursued antibiotic development

16−23

programs based upon this promising scaﬀold.

Cationic

050 based on current actions. In particular, there is a

Non-Ribosomal Peptides (CNRPs) are a large and structurally

critical urgency for the development of new antibiotics

targeting Gram-negative bacteria (e.g., E. coli and P.

aeruginosa), given they possess an outer membrane of

lipopolysaccharide (LPS), which forms a permeability barrier

Received: March 7, 2021

to prevent the penetration of antibiotics.

Naturally occurring Anti-Microbial Peptides (AMPs) have

been recognized as potentially useful therapeutic agents against

−11

AMR.

They exhibit rapid, broad-spectrum and potent

XXXX American Chemical Society and

American Society of Pharmacognosy

Journal of Natural Products

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 1. Structure of laterocidine (1) and brevicidine (2). The N-terminal lipid of each compound is shown in a box. The positively charged

ornithine residues are shown in green. The ester bond of the lactone ring is highlighted in blue.

diverse family of AMPs produced by nonribosomal peptide

synthetases in soil-dwelling or plant-associated bacteria, such as

resistant to nearly all other classes of antimicrobial agents,

polymyxins are becoming increasingly relied upon as treat-

24−27

38,39

Pseudomonas, Bacillus, and Streptomyces spp.

Naturally

ments of last resort.

This highlights the urgency for

occurring CNRPs are generally amphipathic, small (12−50

amino acids), carry an overall net positive charge, and possess a

signiﬁcant proportion of cationic and hydrophobic resi-

development of new antibiotics with novel mechanisms of

action and improved safety and eﬃcacy proﬁles.

In 2018, Li et al. developed a global genome-mining

approach based on antibiotics & Secondary Metabolite

Analysis Shell (antiSMASH) for the discovery of

1,12,28

dues.

Possessing these unique chemical and structural

properties enables them to selectively target highly imperme-

able anionic components of the bacterial envelope surface,

such as phosphate groups in LPS of Gram-negative bacteria or

lipoteichoic acid in Gram-positive bacteria, through a

combination of electrostatic and hydrophobic interac-

40,41

CNRPs.

Through the analysis of 7395 bacterial genomes,

the authors identiﬁed two new CNRPs with an antimicrobial

activity against clinically relevant Gram-negative bacterial

strains, coined laterocidine (1) and brevicidine (2), from

Brevibacillus laterosporus strains ATCC 9141 and DSM 25,

6,28−30

tions.

As such, resistance to CNRPs is often minimized,

as it would require multiple mutational events for a bacteria to

signiﬁcantly alter the composition or organization of its cell

membrane or cell wall components to reduce these

respectively (Figure 1). The structure elucidation of

laterocidine (1) and brevicidine (2) by nuclear magnetic

resonance (NMR), Marfey’s, and tandem mass spectrometry

(MS/MS) analyses revealed a new class of CNRPs presenting

N-terminal acylation, an eight-residue exocyclic cationic

segment with three positively charged ornithine residues, and

a C-terminal cyclic depsipeptide. Laterocidine (1) consists of

13 amino acids, a ﬁve-membered cyclic core cyclized via a

8,29,31

interactions.

Additionally, CNRPS have demonstrated

multifaceted mechanisms, and their highly cationic nature can

promote interactions with secondary anionic intracellular

targets, impairing processes such as DNA and protein

synthesis, protein folding, enzymatic activity, and cell wall

synthesis, further mitigating the development of resist-

lactone formation between the hydroxy group of Thr and the

2−36

ance.

Furthermore, CNRPs often possess macrocyclic

C-terminal Gly , and an isononanoyl (7-methyloctanoyl) lipid

rings and contain nonproteinogenic and D-amino acids,

conferring a large structural diversity and an enhanced

tail (Figure 1). Brevicidine (2) has 12 amino acids, four of

which form a cyclic core cyclized via a lactone bond between

12,29,31

resistance to proteolytic cleavage.

Currently there is a

the α-carboxyl of Ser and the hydroxy group of Thr , and a

conﬁgurationally undeﬁned 4-methylhexanoyl lipid tail (Figure

1).

small number of CNRPs on the market for the treatment of

Gram-negative infections, including polymyxin B (Poly-Rx)

37,38

and polymyxin E/colistin (Xylistin).

Unfortunately, the

To avoid the bottleneck of engineering and expressing large

biosynthetic complexes required to prepare nonribosomally

use of polymyxins as eﬀective antimicrobial agents against

Gram-negative bacteria declined in the 1970s due to their

produced peptides, Kuipers and co-workers recently

designed a strategy to prepare simpliﬁed brevicidine (2)

analogues by a ribosomal synthesis and post-translational

nephrotoxic and neurotoxic side eﬀects. More recently,

however, with the emergence of Gram-negative bacteria

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Scheme 1. Initially Proposed Synthetic Strategy toward Laterocidine (1)

*, 8* = simpliﬁed analogue where all D-AAs are replaced with L-AAs, and the N-terminal fatty acid is n-octanoyl.

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Scheme 2. Succesful Synthetic Route to Prepare Laterocidine (1) and Brevicidine (2)

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Scheme 2. continued

Throughout the scheme the intermediates depicted “a” refer to laterocidine (1), while “b” refers to brevicidine (2). *Macrolactamization reagents

and conditions; (13a) peptide (10 mM in DMF), BOP (5 equiv), HOAt (5 equiv), and DIPEA (10 equiv); (13b) peptide (10 mM in DMF),

PyBOP (5 equiv), and DIPEA (5 equiv).

modiﬁcation. Despite the inability to prepare the key lactone

ring, they were able modify the peptide sequence and prepare

mimics of the lactone ring (thioethers). However, the prepared

analogues were unable to replicate the activity of the native

peptide, and their best candidate was 4- to 16-fold less active

across the test strains. This highlights the importance of

developing eﬃcient chemical syntheses of these new anti-

biotics, allowing atomic precision in the structure modiﬁcation,

which is key to developing clinical drug leads.

Herein, we report an optimized and successful strategy to

prepare both laterocidine (1) and brevicidine (2) in multi-

milligram amounts, exploiting both solid-phase and solution-

phase techniques. The primary structures of both depsipep-

tides, laterocidine (1) and brevicidine (2), were conﬁrmed by

an extensive NMR analysis, and both displayed a potent

activity against E. coli, with laterocidine (1) possessing an

activity equipotent to clinically relevant polymyxin B.

in our previous synthesis of the cyclic lipodepsipeptide

daptomycin.

To establish this synthetic route, we sought to prepare a

simpliﬁed analogue of laterocidine, where all D-amino acid

residues were substituted with their L-isomers and the N-

terminal fatty acid was replaced by the linear n-octanoyl group.

The assembly of the linear sequence 7* (Scheme 1) and the

removal of the allyl ester protection proceeded smoothly as

conﬁrmed by a reversed-phase high-performance liquid

chromatography mass spectrometry (RP-HPLC/MS) analysis

upon resin mini-cleavage. However, a subsequent ring closure

to form the lactone between a Thr side-chain hydroxy group

and Gly was unsuccessful, and only the linear precursor

corresponding to peptidyl-resin 7* was recovered after

cleavage from the resin. A prolonged reaction time and an

increasing excess of the coupling reagents had no eﬀect, and no

macrocyclization was observed.

Second & Third Synthetic Strategies Toward Later-

ocidine. Upon failing to prepare resin 8 * by lactonization, we

redesigned our synthesis (Supporting Information, Scheme

S1), instead harnessing the anticipated ease of lactamization to

aﬀord the macrocycle. The amended strategy also maintained

the late-stage introduction of the ester bond, thus avoiding an

undesired ester aminolysis during repeated piperidine-medi-

ated Fmoc removals.

However, the esteriﬁcation with Alloc-Gly-OH was sluggish,

providing a very poor conversion (ca. 25%) to the desired

product (Figure S22), leading us to abandon the synthetic

route at this stage (as indicated by the red cross in Scheme

S1). We postulated that the presence of the N-terminal fatty

acid might hinder the esteriﬁcation by forming hydrophobic

interactions with the resin or neighboring peptide chains,

reducing the accessibility of the esteriﬁcation site. Therefore,

we modiﬁed our synthetic strategy accordingly (Scheme S2).

In this instance, the esteriﬁcation was undertaken on a

truncated sequence. Unfortunately, little improvement in the

esteriﬁcation was observed under a range of conditions (Table

S1), and only a maximum of 60% conversion to the esteriﬁed

resin (Figure S23) was obtained using long reaction times or

elevated temperatures.

RESULTS AND DISCUSSION

■

Initial Synthetic Strategy Toward Laterocidine.

Several strategies have been employed for the synthesis of

cyclic depsipeptides, which can prove troublesome due to the

3−47

lability of the ester.

An on-resin macrocyclization

approach that involves linking the side chain of reactive

amino acids, such as Ser/Thr/Lys/Asp/Glu, to a solid-phase

resin, and orthogonal protection strategies, has proven to be a

successful approach. For the total syntheses of laterocidine

1) and brevicidine (2), we initially envisioned the preparation

of the linear precursor via a 9-ﬂuorenylmethoxycarbonyl

Fmoc)-based solid-phase peptide synthesis (SPPS) followed

(

by a late-stage on-resin macrocyclization. In contrast to

brevicidine (2), laterocidine (1) comprises an Asn residue in

the C-terminal macrolactone. We anticipated that anchoring

the Asp side chain to a Rink amide linker would provide a

suitable handle for SPPS. Following an acid-mediated detach-

ment of the peptide from the amide-liberating linker, the native

Asn would be generated (Scheme 1).

This synthetic strategy relies on an eﬃcient macrolactoniza-

tion between the hydroxy group of Thr and the α-carboxylic

acid of Gly , and noteworthy, no further postcleavage

modiﬁcations of the synthetic peptide are required. Further-

more, despite the demanding reaction conditions often

required for the macrolactonization, Gly¹³is achiral, thus

avoiding any chance of epimerization upon its activation. The

anchoring of Fmoc-Asp-OAllyl through its side-chain carboxyl

to Rink amide linker 3 would aﬀord resin 4. The subsequent

deprotection of the α-carboxylic acid of resin 4 followed by the

Successful Synthetic Strategy toward Laterocidine

and Brevicidine. The poor conversion to the esteriﬁed Thr

resin in our third strategy suggested that anchoring the peptide

sequence through the side chain of Asn may hinder the

esteriﬁcation. We therefore devised a new site for resin

anchoring and instead performed a solution-phase macro-

lactamization of the linear lipodepsipeptide precursor (Scheme

2).

coupling of H N-Gly-OAllyl would yield peptidyl-resin 5. A

second round of allyl-deprotection and coupling of H N-Gly-

The revised synthesis of laterocidine (1) began by loading

Fmoc-Gly-OH onto the hyper acid-labile 2-chlorotrityl

chloride resin (2-CTC) using N,N-diisopropylethylamine

OAllyl would subsequently yield peptidyl-resin 6. Following an

iterative Fmoc-SPPS, coupling of the N-terminal isononanoyl

fatty acid, allyl ester deprotection and on-resin macro-

lactonization would aﬀord resin-bound laterocidine (8). A

resin cleavage and global deprotection would then deliver

laterocidine (1) directly. This strategy, employing a late-stage

esteriﬁcation, would circumvent the risk of premature

aminolysis of the ester bond during Fmoc-SPPS, as observed

(DIPEA) in CH

Cl , followed by the capping of any unreacted

sites with a CH Cl solution of MeOH and DIPEA (17:2:1, v/

v/v), for 20 min. Resin 9 was then elongated from the C- to N-

terminus until the inclusion of the Fmoc-Trp (Boc)−OH

residue with repeated cycles of N -Fmoc removal with 20%

piperidine in N,N′-dimethylformamide (DMF) (v/v, 2 × 5

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

min, room temperature (rt)) and coupling of amino acids with

(10%, based on the initial resin loading capacity, ca. greater

than 96% purity, Figure S10).

-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylami-

nium hexaﬂuorophosphate (HCTU) as the coupling reagent,

H and C NMR, MS/MS, and HRMS analyses of synthetic

and DIPEA in DMF for 20 min at rt. During this process, the

Thr residue was introduced without side-chain protection to

facilitate the subsequent esteriﬁcation.

laterocidine (1) and brevicidine (2) were performed.

Pleasingly, the spectroscopic data of the synthetic compounds

were in good agreement with those of the natural compounds

The resin-bound linear precursor 10a was then O-acylated at

the side chain of Thr⁹with Alloc-Gly-OH (Supporting

Information ) using N,N′-diisopropylcarbodiimide (DIC) in

the presence of catalytic 4-dimethylaminopyridine (DMAP) in

DMF to yield depsipeptidyl resin 11a. Gratifyingly, the

esteriﬁcation proceeded to completion after 1 h at rt (ca.

reported by Li et al. (Tables S2 & S3, Figures S11 −S21),

thus conﬁrming the correct structural assignment and total

syntheses of laterocidine (1) and brevicidine (2). As the

conﬁguration of the lipid tail of brevicidine was not resolved by

Li et al., the commercially available racemic (R,S)-4-

methylhexanoyl group was installed in the synthetic material.

The resulting epimer mixture was not separable by achiral RP-

HPLC (Figure S10). However, a subsequent biological

evaluation of synthetic (2) did not reveal any diﬀerence in

potency compared to the putative conﬁgurationally pure

natural compound (vide infra).

6% purity, Figure S24 ); no detectable starting material was

observed, and there was no aminolysis of the ester bond during

subsequent removals of the N -Fmoc protection. The

completion of the amino acid sequence by Fmoc-SPPS to

yield depsipeptidyl resin 12a (ca. 80% purity, Figure S25) was

followed by a selective removal of the Alloc protection via a

treatment with tetrakis(triphenylphosphine)palladium(0) (Pd-

Synthetic laterocidine (1) and brevicidine (2) were tested

for antimicrobial activity against the Gram-positive pathogen S.

aureus and Gram-negative pathogen E. coli by a minimum

inhibitory concentration (MIC) assay (Table 1). Toward E.

(

PPh ) ) and phenylsilane (PhSiH ) in CH Cl for 3 h at rt.

3 4 3 2 2

Upon resin cleavage eﬀected with 20% hexaﬂuoroisopropanol

HFIP) in CH Cl (v/v), the resulting side-chain protected

(

peptide 13a was subjected to macrolactamization in the

presence of benzotriazol-1-yloxytris(dimethylamino)-

phosphonium hexaﬂuorophosphate (BOP), 1-hydroxy-7-aza-

benzotriazole (HOAt), and DIPEA for 5 h at rt in DMF (10

mM). Finally, crude laterocidine (1) was obtained following a

global side-chain deprotection using a cleavage cocktail

solution of triﬂuoroacetic acid (TFA)/triisopropylsilane

Table 1. MIC Assay of Laterocidine (1) and Brevicidine (2)

MIC (μg/mL)

Lit. MIC (μg/mL)

S. aureus

(ATCC

29213)

coli(ATCC

S. aureus

MRSA

compound

25922)

Brevicidine

(synthetic)

>64

TIPS)/H O (95:2.5:2.5, v/v/v, 1 h, rt) (ca. 77% purity,

Brevicidine

>64

(

isolated)

Figure S7 ). Following puriﬁcation by RP-HPLC, laterocidine

1) was aﬀorded in an excellent overall yield (36%, based on

Laterocidine

synthetic)

0.25

>64

(

the initial resin loading capacity, ca. greater than 98% purity,

Figure S8).

Laterocidine

>64

(

isolated)

Amoxicillin

Polymyxin B

Brevicidine (2) was synthesized following the same protocol

0.3

>64

with minor modiﬁcations. In this case, the side chain of Thr

was esteriﬁed with Alloc-Ser(OtBu)−OH (Supporting In-

formation ). To facilitate esteriﬁcation with the more hindered

Alloc-Ser(OtBu)−OH, we made a minor adaption to our SPPS

strategy (with respect to the synthesis of laterocidine). Thus,

coli (ATCC 25922), the synthetic laterocidine (1) and

brevicidine (2) displayed a potent antimicrobial activity

(0.25 μg/mL and 2 μg/mL, respectively), in good agreement

we introduced Trp without side-chain protection to reduce

potential hindrance at the site of esteriﬁcation. The

esteriﬁcation proceeded in 88% conversion following 2 × 1 h

treatments using DIC/catalyst DMAP to yield resin 11b (ca.

with the report by Li et al., who assayed the same strain.

Although a diﬀerent strain of S. aureus was used in the current

study, little or no activity was observed toward this Gram-

positive pathogen (>64 μg/mL), also in agreement with the

0% purity, Figure S26). After the coupling of the N-terminal

Asn residue and acylation with (R,S)-4-methylhexanoic acid,

the Alloc group was removed from depsipeptidyl resin 12b,

and the resin was cleaved with 20% HFIP in CH Cl (v/v) to

original report. As such, the potency of the synthetic

laterocidine (1) was eightfold greater toward E. coli (ATCC

25922) than was reported for the isolated compound, while the

synthetic brevicidine (2) was equally potent to the isolated

compound. The observed increase in potency for synthetic

laterocidine (1) is likely a result of the testing conditions.

yield the side-chain protected peptide 13b. We opted to

substitute the coupling reagent BOP for benzotriazole-1-

yloxytris(pyrrolidino)phosphonium hexaﬂuorophosphate

(

PyBOP) when eﬀecting the solution-phase cyclization for

Whereas Li et al. performed their MIC of the isolated

material in tissue culture-treated plates, which are known to

bind strongly to cationic AMPs (thus reducing their observed

potency), our assays were instead performed in polypropylene

brevicidine (2), due to its improved safety proﬁle.

Cyclization was therefore eﬀected with PyBOP and DIPEA

in DMF (10 mM) to yield the side-chain protected peptide

4b. Global deprotection and resin cleavage provided crude

plates. Furthermore, our assay conditions also employed

cation-adjusted Mueller-Hinton broth (MHB), which can

brevicidine (2) (ca. 54% purity, Figure S9), and gratifyingly,

complete consumption of the starting material was observed

for the cyclization step. However, byproducts resulting from a

common residual contamination of the PyBOP reagent with

pyrrolidine were frequently observed and may have

consequently reduced the overall yield. A puriﬁcation by RP-

HPLC yielded brevicidine (2) in an acceptable overall yield

antagonize the activity of some cationic AMPs. Pleasingly,

however, both compounds retained potent activity under

physiologically relevant divalent cation concentrations,

although the MIC of brevicidine (2) did not improve despite

the use of polypropylene plates, possibly suggesting later-

ocidine (1) to possess greater salt resistance. Alternatively, the

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

absence of improvement in the activity of brevicidine might be

the consequence of the presence of two epimers in the

synthetic sample, resulting from the use of racemic (R,S)-4-

methylhexanoic acid for the installation of the lipid tail.

Assuming that the antimicrobial activity is entirely attributed to

one isomer, the measured MIC for an equimolar mixture

would be increased twofold. However, the lipid conﬁguration

in brevicidine was not resolved in the original report, and such

a drastic eﬀect on the antimicrobial activity is purely

hypothetical.

software. An Agilent Zorbax 300SB-C3; 3.0 × 150 mm; 3.5 μm (0.3

mL/min) column and a linear gradient was used as speciﬁed. For the

LC-MS analysis, solvent A was H O containing 0.1% formic acid, and

solvent B was MeCN containing 0.1% formic acid.

For puriﬁcations, semipreparative RP-HPLC was performed on a

Dionex UltiMate 3000 with a Phenomenex Gemini C18 110 Å, 10 ×

250 mm, 5 μm column at a ﬂow rate of 4 mL/min. A gradient was

used as speciﬁed, where solvent A was 0.1% TFA in H O, and B was

0.1% TFA in MeCN and using UV−vis detection at 210 nm. Gradient

systems used for semipreparative RP-HPLC were altered according to

the elution time and peak proﬁles obtained from the analytical RP-

HPLC chromatograms. Analytical RP-HPLC was performed on a

Dionex UltiMate 3000 with Phenomenex Gemini C18 110 Å, 4.6 ×

CONCLUSIONS

■

50 mm, 5 μm (1 mL/min) as the column and using a linear gradient

In summary, we have successsfully executed the ﬁrst total

as speciﬁed, where solvent A was 0.1% TFA in H O and B was 0.1%

syntheses of the cationic antimicrobial peptides laterocidine

TFA in MeCN.

(

1) and brevicidine (2), using a solid-phase synthesis of the

All reagents and solvents for peptide synthesis and RP-HPLC were

purchased as synthesis and HPLC grade, respectively, and used

without further puriﬁcation. Ethyl acetate (EtOAc) and petroleum

ether (PET) were purchased from Burdick & Jackson. 2-Chlorotrityl

chloride (2-CTC) polystyrene resin, Fmoc-Ile-OH, Fmoc-Thr-OH,

Fmoc-Trp-OH, Fmoc-Trp(Boc)−OH, Fmoc-Asn(Trt)−OH, Fmoc-

Gly-OH, and Fmoc-D-Tyr(tBu)−OH were purchased from Chempep

Inc. Benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexaﬂuoro-

phosphate (PyBOP), Fmoc-D-Trp(Boc)−OH, Fmoc-D-Orn(Boc)−

OH, and Fmoc-D-Asn(Trt)−OH were purchased from Aapptec.

Fmoc-Orn(Boc)−OH, Fmoc-D-Ser(tBu)−OH, isononanoic acid,

tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ) ), and hexa-

linear lipodepsipeptide and a solution-phase macrolactamiza-

tion. The crucial depsipeptide bond in the macrolactone ring

of laterocidine (1) and brevicidine (2) was established by an

on-resin esteriﬁcation between the side chain of Thr with

Alloc-Gly-OH or Alloc-Ser(tBu)−OH, respectively. Anchoring

the peptides onto the hyper acid-labile 2-chlorotrityl chloride

resin enabled an oﬀ-resin solution-phase macrocyclization to

be performed. A biological evaluation of laterocidine (1) and

brevicidine (2) conﬁrmed their potent activity against a Gram-

negative bacterium, while little to no activity was observed

toward the tested Gram-positive bacterium. Given their potent

and selective activity toward Gram-negative pathogens, these

lipopeptides are promising candidates for further research and

development, and the preparation of analogues of laterocidine

ﬂuoroisopropanol (HFIP) were purchased from AK Scientiﬁc.

CH Cl , sodium hydroxide (NaOH) (AR grade), hydrochloric acid

(HCl), calcium chloride anhydrous (LR), magnesium chloride (AR),

and anhydrous sodium sulfate (Na SO ) were purchased from ECP

Limited. 4-Dimethylaminopyridine (DMAP) was purchased from

(

1) and brevicidine (2) to establish structure−activity-

Novabiochem. H-Ser(tBu)-OMe·HCl, 2-(6-chloro-1-H-benzotriazole-

relationships (SARs) is currently underway and will be

reported in due course.

-yl)-1,1,3,3-tetramethylaminium hexaﬂuorophosphate (HCTU),

N,N-diisopropylethylamine (DIPEA), piperidine, N,N′-diisopropyl-

carbodiimide (DIC), triisopropylsilane (TIPS), phenylsilane, sodium

EXPERIMENTAL SECTION

■

carbonate (Na CO ), (R,S)-4-methylhexanoic acid, allyl chlorofor-

2 3

General Experimental Procedures. Optical rotations were

measured with an Autopol IV automatic polarimeter using the

sodium-D line (589 nm), with the concentration measured in grams

per 100 mL. UV irradiation was performed using a hand-held

Spectroline UV lamp at a peak wavelength of 365 nm. IR spectra were

recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer using a

diamond attenuated total reﬂectance (ATR) sampling accessory.

NMR experiments were recorded at rt in CDCl₃or deuterated

mate, and sodium diethyldithiocarbamate trihydrate were purchased

from Sigma-Aldrich. Triﬂuoroacetic acid was purchased from

Oakwood Chemicals. Octanoic acid was purchased from Honeywell

Fluka. Formic acid, tetrahydrofuran (THF), and diethyl ether (Et O)

were purchased from Macron Fine Chemicals. CDCl and DMSO-d

were purchased from Cambridge Isotopes. Milli-Q high-purity

deionized water (MQ H O) was available from a Sartorius Arium

Pro Ultrapure Water System from Sartorius Stedim Biotech. DMF

(synthesis grade), MeCN (HPLC grade), and Oxoid Mueller Hinton

Broth (MHB) were purchased from Thermo Fisher Scientiﬁc.

Polypropylene 96-well ﬂat bottom plates were purchased from

Greiner Bio-One.

General Methods for Peptide Synthesis. Peptides were

synthesized manually by a standard 9-ﬂuorenylmethyloxycarbonyl

(Fmoc) solid-phase peptide synthesis (SPPS) and solution-phase

peptide synthesis at rt.

dimethyl sulfoxide (DMSO-d ) on either a Bruker AVANCE 400

spectrometer operating at 400 MHz for H nuclei and 100 MHz for

C nuclei or a Bruker AVANCE 500 spectrometer operating at 500

MHz for H nuclei and 125 MHz for C nuclei. Chemical shifts were

reported on the δ scale from tetramethylsilane (TMS) relative to the

solvent in which the sample was analyzed (CDCl : δ 0.00 (TMS), δ

7.16; DMSO-d : δ_H2.50, δ_C39.52). Structural assignments were

achieved with the aid of correlated spectroscopy (COSY) and

heteronuclear single quantum correlation (HSQC).

General Method 1: Loading of the First Amino Acid to the

−

High-resolution mass spectra (HRMS) were obtained using a

Bruker micrOTOF-QII mass spectrometer. Tandem mass spectrom-

etry (MS/MS) was recorded on a Waters QSTAR XL Quadrupole-

Time-of-Flight instrument with capillary scale RP-HPLC columns for

chromatographic separations.

Analytical thin-layer chromatography (TLC) was performed on

Kieselgel F254 200 μm (Merck) silica plates and visualized using UV

or by staining with potassium permanganate or vanillin followed by a

heating of the plate. Column chromatography was performed using

Grace Davison Discovery Sciences, Davisil LC60A 40−63 Micron

Chromatographic Silica Media, with the indicated eluent.

2-CTC Resin. Polystyrene 2-CTC resin (118 mg, 0.85 mmol·g , 0.1

mmol) was swollen in CH

drained, and a solution of Fmoc-Gly-OH (89.2 mg, 0.3 mmol) and

DIPEA (105 μL, 0.6 mmol) in CH Cl /DMF (1:1, v/v) was added.

The resin was agitated for 4 h at rt, after which time unreacted sites

were capped by an addition of a mixture of CH Cl , MeOH, and

DIPEA (17:2:1, v/v/v) and agitation for 20 min at rt. The resin was

ﬁltered and washed with CH Cl (3 × 5 mL) and DMF (3 × 5 mL).

Cl (5 mL) for 10 min. The solvent was

A sample of dried resin (∼2 mg) was treated with a solution of 20%

piperidine in DMF (v/v, 1 mL) and agitated at rt for 10 min. After

centrifugation, 200 μL of the supernatant was diluted with 1.8 mL of

DMF, and the absorbance of dibenzofulvene was measured at 290 nm

to determine the experimental loading.

Analytical liquid chromatography with tandem mass-spectrometry

LC-MS) was performed on an Agilent Technologies 1260 inﬁnity

(

LC connected to an Agilent Technologies 6120 quadrupole MSD

spectrophotometer. Data processing was performed on OpenLAB

General Method 2: Removal of N -Fmoc Protecting Group.

To the swelled N -Fmoc-protected peptidyl resin was added a

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

solution of 20% piperidine in DMF (v/v, 3 mL) followed by agitation

for 2 × 5 min at rt. The resin was ﬁltered, drained, and washed with

DMF (3 × 5 mL). After the incorporation of the ester bond (General

method 4), 5% formic acid was added to 20% piperidine in DMF, (v/

v/v, 3 mL), and the reaction time was modiﬁed to 2 × 1 min at rt.

amino group following General method 5. The removal of the Alloc

protecting group was performed by General method 6. The protected

peptide was cleaved from the resin using General method 7 to aﬀord

the crude product 13a as an amorphous oﬀ-white solid (152 mg, 76%

based on resin loading). The crude side-chain protected peptide 13a

(152 mg, 0.068 mmol) was dissolved in DMF (6.8 mL) together with

BOP (150 mg, 0.34 mmol), HOAt (58 mg, 0.34 mmol), and DIPEA

(118 μL, 0.68 mmol) to reach a ﬁnal peptide concentration of 10

mM. The reaction mixture was shaken at room temperature for 5 h.

General Method 3: Coupling of Amino Acids. The N -Fmoc

amino acid (0.4 mmol) and HCTU (157 mg, 0.38 mmol) were

dissolved in DMF (3 mL), DIPEA (174 μL, 1 mmol) was added, and

the solution was transferred to the swelled peptidyl resin (0.1 mmol).

Coupling was performed for 20 min at rt before the resin was washed

with DMF (3 × 5 mL). The reaction completion was monitored by

The crude cyclized peptide was precipitated by an addition of H O

(40 mL) and recovered by centrifugation. The resulting material

underwent a global deprotection using General method 8. Crude

laterocidine (1) was puriﬁed by semipreparative RP-HPLC using

Dionex UltiMate 3000 on a Phenomenex Gemini C18 110 Å, 10 ×

the Kaiser test.

General Method 4: On-Resin Esteriﬁcation. To the swelled

peptidyl resin (0.1 mmol) was added a solution of N -Alloc-protected

−

amino acid (0.3 mmol), DIC (40 μL, 0.3 mmol), and DMAP (1.2 mg,

250 mm, 5 μm column using a gradient of 15−65% B at 1% B·min

.01 mmol) in DMF (3 mL). The reaction was performed for 1 h at

over 40 min, 4 mL/min at rt. Fractions were collected and analyzed

by electrospray ionization mass spectrometry (ESI-MS) and analytical

RP-HPLC. Fractions identiﬁed with the correct m/z were combined

and lyophilized to aﬀord 1 as a white amorphous solid (59.6 mg, 36%

rt, and the resin was washed with DMF (3 × 5 mL). The reaction

completion was monitored by RP-HPLC/MS analysis on a small

sample of cleaved peptide.

General Method 5: Coupling of The Lipid Tail. To the

peptidyl resin (0.1 mmol) was added a solution of fatty acid (0.15

mmol), HATU (46 mg, 0.12 mmol), and DIPEA (105 μL, 0.6 mmol)

in DMF (3 mL). The resin was agitated for 1 h at rt and drained

before being washed with DMF (3 × 5 mL). The coupling

overall yield based on initial 0.1 mmol scale), >98% purity); t 29.9

min; NMR, Table S2 ; HRMS (ESI+) m/z 535.6249 (calcd for

20.5

[C H N O +3H] , 535.6243).; [α] −14.9 (c 0.067, MeOH)

113 19 18

(a literature value was not reported).

Synthesis of Brevicidine (2). Fmoc-Gly-OH (89 mg, 0.3 mmol)

completion was conﬁrmed by the Kaiser test.

was loaded onto 2-chlorotrityl chloride polystyrene resin (112 mg,

−

General Method 6: Removal of Allyl Ester and Allylox-

ycarbonyl (Alloc) Protecting Groups. The peptidyl resin (0.1

mmol) was swelled in CH Cl before the addition of a solution of

0.89 mmol·g , 0.1 mmol) following General method 1. The loading

eﬃciency was 67% as determined by an Fmoc release. The removal

of the N -Fmoc protecting group was performed using General

Pd(PPh ) (29 mg, 0.025 mmol) and PhSiH (123 μL, 1 mmol) in

method 2. A solid-phase peptide synthesis was performed by coupling

Fmoc-Ile-OH, Fmoc-Thr-OH, and Fmoc-Trp-OH following General

method 3. Alloc-Ser(tBu)−OH (73.5 mg, 0.3 mmol) was coupled to

the Thr side-chain hydroxyl group using General method 4.

Subsequent Fmoc-removals were performed using 5% formic acid

and 20% piperidine in DMF (v/v/v) as described in General method

2. A peptide elongation was continued using Fmoc-D-Orn(Boc)−OH,

Fmoc-Orn(Boc)−OH, Fmoc-D-Trp(Boc)−OH, Fmoc-D-Tyr(tBu)−

OH, and Fmoc-D-Asn(Trt)−OH. (R,S)-4-Methylhexanoic acid (26

μL, 0.15 mmol) was coupled to the N-terminal amino group following

General method 5. The removal of the Alloc protecting group was

performed by General method 6. The resin was then split into four

portions, and the following steps were performed taking 0.017 mmol

of the peptidyl resin. The protected peptide was cleaved from the

resin using General method 7 to aﬀord the crude product 13b, which

was dissolved in DMF (1.7 mL) to achieve the ﬁnal concentration of

10 mM. PyBOP (8.8 mg, 0.085 mmol) and DIPEA (14.8 μL, 0.085

mmol) were added to eﬀect cyclization, and the reaction was stirred

for 5 h at rt. To the crude reaction mixture, water was added such

CH Cl (3 mL). The resin was shaken at rt for 3 h and ﬁltered before

being washed with CH Cl (3 × 5 mL), a solution of 0.5% sodium

diethyldithiocarbamate in DMF (m/v, 5 mL, 3 × 1 min), and DMF

(

3 × 5 mL).

General Method 7: Cleavage. The resin-bound peptide was

washed with DMF (3 × 5 mL) and CH Cl (3 × 5 mL) before it was

dried in vacuo. A solution of 20% HFIP in CH Cl (v/v, 10 mL) was

added, and cleavage was performed for 1 h at rt. After the ﬁltration,

the resin was washed with CH Cl (3 × 5 mL), and the combined

ﬁltrates were concentrated under a stream of N . The crude protected

peptide was dissolved in tert-butanol (10 mL) and lyophilized.

General Method 8: Global Deprotection. The cyclized peptide

was treated with a solution of TFA/H O/TIPS (95:2.5:2.5, v/v/v, 10

mL) for 1 h at rt and concentrated under a stream of N . After a

precipitation by the addition of cold Et O (45 mL), the crude peptide

was recovered by centrifugation and washed with cold Et O (2 × 45

mL) before lyophilization.

General Method 9: Resin Mini Cleavage. The peptidyl resin

(

1−5 mg) was treated with a solution of TFA/H O/TIPS (95:2.5:2.5,

that, for every 1 mL of the reaction mixture, 39 mL of H O was

v/v/v, 1 mL) for 30 min at rt and concentrated under a stream of N .

added, and the resulting supernatant centrifuged. The water layer was

After a precipitation by the addition of cold Et O (10 mL), the crude

decanted, and the obtained precipitate was redissolved in H O/

peptide was recovered by centrifugation and washed with cold Et O

MeCN (1:1) + 0.1% TFA and lyophilized. The resulting material

underwent a global deprotection using General method 8. Crude

brevicidine (2) was puriﬁed by semipreparative RP-HPLC using

Dionex UltiMate 3000 on a Phenomenex Gemini C18 110 Å, 10 ×

250 mm, 5 μm column (3 mL/min) using a gradient of 5−95% B at

3% B/min over 65 min, 4 mL/min at rt. Fractions were collected and

analyzed by ESI-MS and analytical RP-HPLC. Fractions identiﬁed

with the correct m/z were combined and lyophilized to aﬀord 2 as a

white solid (2.6 mg, 10% overall yield (based on resin split at 0.017

mmol scale), >96% purity); t_R34.7 min; NMR, Table S3 ; HRMS

(2 × 10 mL) before lyophilization. The resulting peptide was

obtained as a white powder and used to analyze the progress of on-

resin reactions.

Synthesis of Laterocidine (1). Fmoc-Gly-OH (89 mg, 0.3

mmol) was loaded onto 2-chlorotrityl chloride polystyrene resin (118

−

mg, 0.85 mmol.g , 0.1 mmol) following General method 1. The

loading eﬃciency was 89% as determined by Fmoc release. The

removal of the N -Fmoc protecting group was performed using

General method 2. A solid-phase peptide synthesis was performed by

coupling Fmoc-Asn(Trt)−OH, Fmoc-Ile-OH, Fmoc-Thr-OH, and

Fmoc-Trp(Boc)−OH following General method 3. Alloc-Gly-OH

(ESI+) m/z 760.4072 (calcd for [C H N O +2H] , 760.4064);

106 18 17

[α]²+ 6.9 (c 0.058, MeOH) (a literature value was not reported).

0.1

(

48 mg, 0.3 mmol) was coupled to the Thr side-chain hydroxyl group

Minimum Inhibitory Concentration (MIC) Assay. Staph-

ylococcus aureus ATCC 29213 and E. coli ATCC 25922 were grown

in a cation-adjusted Mueller Hinton (MH) broth at 37 °C with

shaking (200 rpm). MIC assays were performed in accordance with

using General method 4 and repeated twice. Subsequent Fmoc-

removals were performed using 5% formic acid and 20% piperidine in

DMF (v/v/v) as described in General method 2. A peptide

elongation was continued using Fmoc-D-Orn(Boc)−OH, Fmoc-Gly-

OH, Fmoc-Orn(Boc)−OH, Fmoc-D-Orn(Boc)−OH, Fmoc-D-Trp-

the CLSI recommended protocol. Brieﬂy, a twofold dilution series

of the test compounds (from 64 to 0.25 μM, ﬁnal) was prepared in

triplicate in polypropylene 96-well plates, using cation-adjusted MH

media. Cultures of bacteria grown for 8 h were diluted accordingly in

(

Boc)−OH, Fmoc-D-Tyr(tBu)−OH, and Fmoc-D-Ser(tBu)−OH.

Isononanoic acid (26 μL, 0.15 mmol) was coupled to the N-terminal

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

fresh media before 50 μL of inoculum was added to each well of the

MIC plate, to achieve a ﬁnal volume of 100 μL with a uniform CFU/

ml of ∼5 × 10 in each well. A growth control (untreated) and

sterility control (noninoculated) well was included for each test

Notes

The authors declare no competing ﬁnancial interest.

REFERENCES

■

compound replicate. Plates were incubated at 37 °C with shaking for

serious, worldwide threat to public health https://www.who.int/

1) Prestinaci, F.; Pezzotti, P.; Pantosti, A. Pathog. Global Health

015 , 109 (7), 309 −318.

2) WHO. WHO’s ﬁrst global report on antibiotic resistance reveals

8 h before the MIC was determined. MIC values were determined as

the lowest concentration at which no growth was observed

consistently across all three biological replicates of the assay and

within triplicates of each test compound.

(

news/item/30-04-2014-who-s-ﬁ rst-global-report-on-antibiotic-

resistance-reveals-serious-worldwide-threat-to-public-health (accessed

2020-10-29).

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jnatprod.1c00222.

■

(3) New report calls for urgent action to avert antimicrobial

resistance crisis https://www.who.int/news/item/29-04-2019-new-

report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis

(

accessed 2020-10-29).

4) Toner, E.; Adalja, A.; Gronvall, G. K.; Cicero, A.; Inglesby, T. V.

Health Secur. 2015 , 13 (3), 153 −155.

5) Chokshi, A.; Sifri, Z.; Cennimo, D.; Horng, H. J. Glob. Infect. Dis.

019 , 11 (1), 36.

(6) Dadgostar, P. Infect. Drug Resist. 2019 , 12 , 3903 −3910.

7) Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K. M.;

Unsuccessful synthetic strategies toward laterocidine.

Supplementary ﬁgures and schemes. Synthetic proce-

dures and characterization of synthetic compounds. RP-

HPLC proﬁles, spectroscopic data, and NMR assign-

ments of synthetic laterocidine and brevicidine (PDF)

(8) Pushpanathan, M.; Gunasekaran, P.; Rajendhran, J. Antimicro-

Wertheim, H. F. L.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I.

M.; Goossens, H.; Greko, C.; So, A. D.; Bigdeli, M.; Tomson, G.;

Woodhouse, W.; Ombaka, E.; Peralta, A. Q.; Qamar, F. N.; Mir, F.;

Kariuki, S.; Bhutta, Z. A.; Coates, A.; Bergstrom, R.; Wright, G. D.;

Brown, E. D.; Cars, O. Lancet Infect. Dis. 2013 , 13 (12), 1057 −1098.

bial Peptides: Versatile Biological Properties https://www.hindawi.

com/journals/ijpep/2013/675391/ DOI: 10.1155/2013/675391

AUTHOR INFORMATION

Corresponding Authors

Margaret A. Brimble − School of Chemical Sciences, The

University of Auckland, Auckland 1142, New Zealand;

School of Biological Sciences and Maurice Wilkins Centre for

Molecular Biodiscovery, The University of Auckland,

Auckland 1142, New Zealand; orcid.org/0000-0002-

■

(

accessed Apr 27, 2020).

9) Kruse, T.; Kristensen, H.-H. Expert Rev. Anti-Infect. Ther. 2008 , 6

6), 887 −895.

10) Magana, M.; Pushpanathan, M.; Santos, A. L.; Leanse, L.;

086-4096 ; Email: m.brimble@auckland.ac.nz

Auckland 1142, New Zealand; orcid.org/0000-0002-

Paul W. R. Harris − School of Chemical Sciences, The

University of Auckland, Auckland 1142, New Zealand;

School of Biological Sciences and Maurice Wilkins Centre for

Molecular Biodiscovery, The University of Auckland,

Fernandez, M.; Ioannidis, A.; Giulianotti, M. A.; Apidianakis, Y.;

Bradfute, S.; Ferguson, A. L.; Cherkasov, A.; Seleem, M. N.; Pinilla,

C.; de la Fuente-Nunez, C.; Lazaridis, T.; Dai, T.; Houghten, R. A.;

Hancock, R. E. W.; Tegos, G. P. Lancet Infect. Dis. 2020 , 20 (9),

e216 −e230.

(11) Lei, J.; Sun, L.; Huang, S.; Zhu, C.; Li, P.; He, J.; Mackey, V.;

Coy, D. H.; He, Q. Am. J. Transl. Res. 2019 , 11 (7), 3919 −3931.

(12) Hancock, R. E. Expert Opin. Invest. Drugs 2000 , 9 (8), 1723 −

579-4543 ; Email: paul.harris@auckland.ac.nz

Authors

Yann Hermant − School of Chemical Sciences, The University

of Auckland, Auckland 1142, New Zealand; School of

Biological Sciences and Maurice Wilkins Centre for Molecular

Biodiscovery, The University of Auckland, Auckland 1142,

New Zealand

729.

13) Nguyen, L. T.; Haney, E. F.; Vogel, H. J. Trends Biotechnol.

011 , 29 (9), 464 −472.

14) Yeaman, M. R.; Yount, N. Y. Pharmacol. Rev. 2003 , 55 (1), 27 −

15) Ling, L. L.; Schneider, T.; Peoples, A. J.; Spoering, A. L.;

Brimble, M. A. Thiol-Ene Enabled Chemical Synthesis of Truncated

Dennise Palpal-latoc − School of Chemical Sciences, The

University of Auckland, Auckland 1142, New Zealand;

Maurice Wilkins Centre for Molecular Biodiscovery, The

University of Auckland, Auckland 1142, New Zealand

Nadiia Kovalenko − School of Chemical Sciences, The

University of Auckland, Auckland 1142, New Zealand;

School of Biological Sciences, The University of Auckland,

Auckland 1142, New Zealand

Alan J. Cameron − School of Chemical Sciences, The

University of Auckland, Auckland 1142, New Zealand;

School of Biological Sciences and Maurice Wilkins Centre for

Molecular Biodiscovery, The University of Auckland,

Auckland 1142, New Zealand

Engels, I.; Conlon, B. P.; Mueller, A.; Schaberle, T. F.; Hughes, D. E.;

Epstein, S.; Jones, M.; Lazarides, L.; Steadman, V. A.; Cohen, D. R.;

Felix, C. R.; Fetterman, K. A.; Millett, W. P.; Nitti, A. G.; Zullo, A. M.;

Chen, C.; Lewis, K. Nature 2015 , 517 (7535), 455 −459.

(16) Yim, V. V.; Cameron, A. J.; Kavianinia, I.; Harris, P. W. R.;

S-Lipidated Teixobactin Analogs. Front. Chem. 2020, 8.

DOI: 10.3389/fchem.2020.00568

(17) Schumacher, C. E.; Harris, P. W. R.; Ding, X.-B.; Krause, B.;

Wright, T. H.; Cook, G. M.; Furkert, D. P.; Brimble, M. A. Org.

Biomol. Chem. 2017 , 15 (41), 8755 −8760.

(18) Jin, K.; Sam, I. H.; Po, K. H. L.; Lin, D.; Ghazvini Zadeh, E. H.;

Chen, S.; Yuan, Y.; Li, X. Nat. Commun. 2016 , 7 (1), 12394.

19) Gunjal, V. B.; Reddy, D. S. Tetrahedron Lett. 2019 , 60 (29),

909 −1912.

20) Giltrap, A. M.; Dowman, L. J.; Nagalingam, G.; Ochoa, J. L.;

Linington, R. G.; Britton, W. J.; Payne, R. J. Org. Lett. 2016 , 18 (11),

788 −2791.

21) Yang, H.; Chen, K. H.; Nowick, J. S. ACS Chem. Biol. 2016 , 11

7), 1823 −1826.

(22) Yang, S.-H.; Hermant, Y. O. J.; Harris, P. W. R.; Brimble, M. A.

Eur. J. Org. Chem. 2020 , 2020 (8), 944 −947.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jnatprod.1c00222

Funding

The authors acknowledge the Ministry of Business, Innovation,

and Employment (MBIE Endeavor Grant No. UOAX2010)

for generous ﬁnancial support and the Maurice Wilkins Centre

for Molecular Biodiscovery. A.J.C. thanks the Lottery Health

Research for a Postdoctoral Fellowship.

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Lett. 2015 , 17 (24), 6182 −6185.

Article

(

23) Jad, Y. E.; Acosta, G. A.; Naicker, T.; Ramtahal, M.; El-Faham,

(51) Alsina, J.; Barany, G.; Albericio, F.; Kates, S. A. Lett. Pept. Sci.

1999 , 6 (4), 243 −245.

A.; Govender, T.; Kruger, H. G.; de la Torre, B. G.; Albericio, F. Org.

(

M.; Pianet, I.; Josten, M.; Sahl, H.-G.; Silby, M. W.; Loper, J. E.;

Gross, H. J. Nat. Prod. 2019 , 82 (2), 301 −308.

(

C. K.; Shaffer, B. T.; Elbourne, L. D. H.; Stockwell, V. O.; Hartney, S.

L.; Breakwell, K.; Henkels, M. D.; Tetu, S. G.; Rangel, L. I.; Kidarsa,

T. A.; Wilson, N. L.; van de Mortel, J. E.; Song, C.; Blumhagen, R.;

Radune, D.; Hostetler, J. B.; Brinkac, L. M.; Durkin, A. S.; Kluepfel, D.

A.; Wechter, W. P.; Anderson, A. J.; Kim, Y. C.; Pierson, L. S.;

Pierson, E. A.; Lindow, S. E.; Kobayashi, D. Y.; Raaijmakers, J. M.;

Weller, D. M.; Thomashow, L. S.; Allen, A. E.; Paulsen, I. T. PLoS

Genet. 2012 , 8 (7), e1002784.

(52) Wiegand, I.; Hilpert, K.; Hancock, R. E. W. Nat. Protoc. 2008 , 3

(2), 163 −175.

24) Jahanshah, G.; Yan, Q.; Gerhardt, H.; Pataj, Z.; Lämmerhofer,

(53) Bowdish, D. M. E.; Hancock, D. J. D. and R. E. W. A Re-

evaluation of the Role of Host Defence Peptides in Mammalian

Immunity https://www.eurekaselect.com/79060/article

DOI: 10.2174/1389203053027494 (accessed 2021-03-01).

(54) Al-Ayed, K.; Ballantine, R. D.; Zhong, Z.; Li, Y.; Cochrane, S.;

Martin, N. Total Synthesis of the Brevicidine and Laterocidine Family

of Lipopeptide Antibiotics. ChemRxiv . Cambridge, Cambridge Open

Engage; 2021; DOI: 10.26434/chemrxiv.13660949.v1.

(55) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.

Biochem. 1970 , 34 (2), 595 −598.

25) Loper, J. E.; Hassan, K. A.; Mavrodi, D. V.; Davis, E. W.; Lim,

56) Eissler, S.; Kley, M.; Bächle, D.; Loidl, G.; Meier, T.; Samson,

D. J. Pept. Sci. 2017 , 23 (10), 757 −762.

57) M100Ed30 | Performance Standards for Antimicrobial

Biol. 2017 , 63 (10), 20 −32.

26) Tajbakhsh, M.; Karimi, A.; Fallah, F.; Akhavan, M. M. Cell. Mol.

27) Payne, J. A. E.; Schoppet, M.; Hansen, M. H.; Cryle, M. J. Mol.

BioSyst. 2017 , 13 (1), 9 −22.

28) Hancock, R. E. W.; Lehrer, R. Trends Biotechnol. 1998 , 16 (2),

(

Susceptibility Testing, 30th Edition https://clsi.org/standards/

products/microbiology/documents/m100/ (accessed 2020-12-20).

2 −88.

29) Hancock, R.; Rozek, A. FEMS Microbiol. Lett. 2002 , 206 , 143.

30) Bahar, A. A.; Ren, D. Pharmaceuticals 2013 , 6 (12), 1543 −

1575.

31) Straus, S. K.; Hancock, R. E. W. Biochim. Biophys. Acta,

Biomembr. 2006 , 1758 (9), 1215 −1223.

32) Le, C.-F.; Fang, C.-M.; Sekaran, S. D. Intracellular Targeting

Mechanisms by Antimicrobial Peptides. Antimicrob. Agents Chemother.

017, 61 (4). DOI: 10.1128/AAC.02340-16

33) Powers, J. H. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin.

Microbiol. Infect. Dis. 2004 , 4 , 23 −31.

34) Kragol, G.; Lovas, S.; Varadi, G.; Condie, B. A.; Hoffmann, R.;

Otvos, L. Biochemistry 2001 , 40 (10), 3016 −3026.

35) Patrzykat, A.; Friedrich, C. L.; Zhang, L.; Mendoza, V.;

Hancock, R. E. W. Antimicrob. Agents Chemother. 2002 , 46 (3), 605 −

14.

36) Wenzel, M.; Rautenbach, M.; Vosloo, J. A.; Siersma, T.;

W.; Behrends, J. C.; Bechinger, B.; Hamoen, L. W. The Multifaceted

Aisenbrey, C. H. M.; Zaitseva, E.; Laubscher, W. E.; van Rensburg,

Antibacterial Mechanisms of the Pioneering Peptide Antibiotics

Tyrocidine and Gramicidin S. mBio 2018, 9 (5). DOI: 10.1128/

mBio.00802-18

37) Tally, F. P.; DeBruin, M. F. J. Antimicrob. Chemother. 2000 , 46

4), 523 −526.

38) Shatri, G.; Tadi, P. Polymyxin. In StatPearls; StatPearls

Publishing: Treasure Island, FL, 2020.

Nucleic Acids Res. 2011 , 39 , W339 −346.

39) Mogi, T.; Kita, K. Cell. Mol. Life Sci. 2009 , 66 (23), 3821.

(40) Li, Y.-X.; Zhong, Z.; Zhang, W.-P.; Qian, P.-Y. Nat. Commun.

(

018 , 9 (1), 1 −9.

41) Medema, M. H.; Blin, K.; Cimermancic, P.; de Jager, V.;

Zakrzewski, P.; Fischbach, M. A.; Weber, T.; Takano, E.; Breitling, R.

(42) Zhao, X.; Li, Z.; Kuipers, O. P. Cell Chem. Biol. 2020 , 27 (10),

016 , 11 (10), 1924 −1947.

262 −1271e4.

43) Pelay-Gimeno, M.; Albericio, F.; Tulla-Puche, J. Nat. Protoc.

44) Kjeldsen, F.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2011 , 22

8), 1441 −1452.

45) Choudhary, A.; Raines, R. T. ChemBioChem 2011 , 12 (12),

801 −1807.

46) Alcaro, M. C.; Sabatino, G.; Uziel, J.; Chelli, M.; Ginanneschi,

M.; Rovero, P.; Papini, A. M. J. Pept. Sci. 2004 , 10 (4), 218 −228.

47) Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2002 , 4 (19), 3219 −

221.

A. Chem. - Eur. J. 2019 , 25 (62), 14101 −14107.

48) Davies, J. S. J. Pept. Sci. 2003 , 9 (8), 471 −501.

49) Xu, B.; Hermant, Y.; Yang, S.-H.; Harris, P. W. R.; Brimble, M.

50) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990 , 31

2), 205 −208.