Total Synthesis of Ecumicin

Article abstract of DOI:10.1021/acs.orglett.7b03967

The first total synthesis of the potent anti-mycobacterial cyclic depsipeptide natural product ecumicin is described. Synthesis was achieved via a solid-phase strategy, incorporating the synthetic non-proteinogenic amino acids N-methyl-4-methoxy-l-tryptophan and threo-β-hydroxy-l-phenylalanine into the growing linear peptide chain. The synthesis employed key on-resin esterification and dimethylation steps as well as a final macrolactamization between the unusual N-methyl-4-methoxy-l-tryptophan unit and a bulky N-methyl-l-valine residue. The synthetic natural product possessed potent antimycobacterial activity against the virulent H37Rv strain of Mycobacterium tuberculosis (MIC₉₀ = 312 nM).

Full text of DOI:10.1021/acs.orglett.7b03967

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis of Ecumicin
Paige M. E. Hawkins,§ Andrew M. Giltrap,§ Gayathri Nagalingam,‡ Warwick J. Britton,‡
and Richard J. Payne*,§
§School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
‡Tuberculosis Research Program, Centenary Institute, and Sydney Medical School, The University of Sydney, Sydney, NSW 2006,
Australia
S
* Supporting Information
ABSTRACT: The first total synthesis of the potent anti-
mycobacterial cyclic depsipeptide natural product ecumicin is
described. Synthesis was achieved via a solid-phase strategy,
incorporating the synthetic non-proteinogenic amino acids N-
methyl-4-methoxy-L-tryptophan and threo-β-hydroxy-L-phenyl-
alanine into the growing linear peptide chain. The synthesis
employed key on-resin esterification and dimethylation steps
as well as a final macrolactamization between the unusual N-
methyl-4-methoxy-L-tryptophan unit and a bulky N-methyl-L-
valine residue. The synthetic natural product possessed potent
antimycobacterial activity against the virulent H37Rv strain of
Mycobacterium tuberculosis (MIC90 = 312 nM).
uberculosis (TB) is caused by infection with the
bacterium Mycobacterium tuberculosis (Mtb) and was
T
responsible for 1.3 million deaths in 2016, making it the
most deadly infectious disease globally.1 While effective
treatment options are available for TB, this is dependent on
the infection being drug-sensitive.2 Alarmingly, there has been a
steady increase in the number of cases of multiple-drug-
resistant (MDR) and extensively drug-resistant (XDR) TB over
the past decade, where treatment relies on the use of second-
line antibiotics with increased toxicity.1,2 The emerging TB
drug resistance crisis has been fueled by poor compliance to the
long and complex TB treatment regimens (6−24 months of a
cocktail of drugs) as well as the narrow modes of action of the
limited pool of approved antibiotics for TB treatment. As such,
there is an urgent need for new antibiotics which operate via
novel modes of action and that may shorten TB therapy.3,4
Ecumicin (1) is a cyclic depsipeptide natural product from an
actinobacteria Nonomuraea sp. MJM5123 which was discovered
following a high-throughput screening campaign by Gao et al.
(Figure 1).5 The natural product was shown to exhibit potent
antibacterial activity against the virulent H37Rv strain of Mtb
(MIC90 = 160 nM) and crucially retained activity against MDR
and XDR strains of Mtb. Of particular interest is that ecumicin
has been shown to possess in vivo activity with essentially
complete inhibition of Mtb growth in mice after 12 doses at 20
mg kg−1. Structurally, ecumicin is a tridecapeptide consisting of
all L-amino acids, extensive backbone N-methylation, a
dimethylated N-terminus, and two non-proteinogenic amino
acids: N-methyl-4-methoxy-L-tryptophan and threo-β-hydroxy-
L-phenylalanine.
Figure 1. Structure of ecumicin (1) showing synthetic disconnection
strategies; successful strategy (red) and unsuccessful strategy with the
challenging peptide bond formation (blue).
strains followed by gene sequencing.6 Specifically, ecumicin is
thought to stimulate ATPase activity of the chaperone protein
ClpC1 while also invoking a marked decrease in proteolysis by
the ClpC1-mediated ClpP1P2 protease complex. This results in
the uncoupling of ATP hydrolysis from proteolysis. It is
proposed that the accumulation of toxic undegraded proteins is
responsible for the antimycobacterial effects of the natural
product. Importantly, the Clp protease is essential in
mycobacteria but not in other bacteria7,8 which has significant
benefits for ecumicin’s potential use as a TB drug lead.9
Interestingly, a similar mechanism of action has been described
for lassomycin and cyclomarin A, cyclic peptide antibiotics
The molecular target of ecumicin has also been elucidated
Received: December 21, 2017
through the spontaneous generation of ecumicin-resistant Mtb
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03967
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
targeting Mtb; however, the significant structural differences
between the cyclic peptides suggests that the three natural
products target different binding sites on the ClpC1
subunit.10−12
Scheme 1. (A) Synthesis of suitably protected threo-β-
Hydroxy-L-phenylalanine 2 and (B) Synthesis of Suitably
Protected N-Methyl-4-methoxy-L-tryptophan Building Block
4
Given the potent in vitro and in vivo antimycobacterial
activity of ecumicin, together with its novel mechanism of
action, we were interested in the natural product as a target for
total synthesis. Ultimately, we envisaged that a robust synthetic
route to ecumicin would facilitate future analogue generation
which would allow SAR to be generated en route to the
development of potential new leads for the treatment of drug-
resistant TB.
Our synthesis commenced with the preparation of two
suitably protected non-proteinogenic amino acid building
blocks: Fmoc-threo-β-hydroxy-L-phenylalanine 2 (obtained in
nine steps from L-phenylalanine methyl ester 3 using a synthetic
sequence similar to that previously reported by Crich and
Banerjee,13 see Scheme 1A) and Fmoc-N-methyl-4-methoxy-L-
tryptophan 4 (Scheme 1B). Synthesis of 4 took inspiration
from the work of Tanaka et al.14 and employed a key Negishi
cross-coupling reaction between β-iodo-L-alanine 7 and
substituted 3-bromoindole 8 (Scheme 1B). β-Iodo-L-alanine 7
was synthesized from Cbz-L-Ser-OH (9) by tert-butyl
protection of the α-carboxylate followed by iodination. The
critical Negishi cross coupling was achieved by reaction of
bromoindole 8 (synthesized in two steps from 4-methox-
yindole; see the Supporting Information (SI) for details) with
the in situ generated organozinc iodide of 7, catalytic
Pd2(dba)3, and the Q-Phos ligand. This provided the protected
4-methoxy-L-tryptophan 10 in 75% yield following flash column
chromatography. Next, N-methylation of 10 was achieved by
treatment with methyl iodide in the presence of silver oxide to
provide 11 in 69% yield. From here, only protecting group
manipulations remained to generate target building block 4:
reduction of the tosylated indole with magnesium, hydro-
genation of the Cbz protecting group, acidic tert-butyl ester
deprotection,15 and finally Fmoc protection of the resulting
amine provided Fmoc-N-methyl-4-methoxy-L-tryptophan 4 in
75% yield over the four steps. It should be noted that the
protecting groups were carefully chosen to preserve the chiral
integrity of the α-center of the amino acid, as it was found that
even mildly basic reagents used to remove base-labile
protecting groups resulted in racemization (details not shown).
With the suitably protected amino acids 2 and 4 in hand, we
focused our efforts on the synthesis of the linear peptide. It was
envisaged that the linear peptide could be synthesized on-resin
using Fmoc-SPPS including key on-resin esterification and N-
terminal dimethylation steps. We proposed that the polypep-
tide could be grown on a hyper-acid-labile resin such that the
side-chain-protected polypeptide could be released from the
resin, enabling a solution-phase macrolactamization and final
acidic deprotection to furnish ecumicin. Due to the hindered
nature of the peptide bonds within the natural product, there
were no obvious cyclization junctions as all sites possess bulky
β-branched amino acids.16 However, disconnection between
threo-β-OH-L-Phe-OH and L-Val (Figure 1, blue) was initially
chosen as the site for macrolactamization as it used an N-
terminal amino acid without N-methylation, and the C-terminal
threo-β-OH-L-Phe-OH could be masked as an oxazolidine
(through synthetic manipulation of building block 2 to prevent
epimerization; see the SI for synthetic details).17 While
oxazolidine-protected Fmoc-threo-β-OH-L-Phe-OH was suc-
cessfully loaded onto the resin and elongated to a resin-
bound tetrapeptide, which included the precious N-methyl-4-
methoxy-L-tryptophan amino acid, the peptide chain could not
be further extended due to steric hindrance in the coupling
between resin-immobilized N-Me-L-Val and Fmoc-L-Val-OH
(Figure 1, blue, see the SI for the synthetic scheme).
Unfortunately, we were unable to forge this amide bond
despite trialing an exhaustive set of conditions including
microwave irradiation, N-silylation,18 acid fluorides,19 and
even solution-phase condensation.
Because of the difficulty in achieving the above coupling, a
new retrosynthetic strategy was developed. We theorized that
the bulky amino acids were sterically occluding the reaction
site, and so preorganizing this peptide may allow the reactive
centers to react by proximity. As such, a new cyclization
junction was chosen between the N-Me-L-Val-OH and N-
methyl-4-methoxy-L-tryptophan residues. While this cyclization
used an N-methylated N-terminus and possessed significant
B
DOI: 10.1021/acs.orglett.7b03967
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
steric hindrance at the putative cyclization site, it was
envisioned that the termini could react through preorganization
of the fully assembled linear peptide chain. Toward this end,
Fmoc-N-Me-L-Val-OH was loaded onto activated 4-
(diphenylhydroxymethyl)benzoic acid (trityl-OH) function-
alized polyethylene glycol-based ChemMatrix resin to provide
resin-bound amino acid 12 (Scheme 2). A PEG-based resin was
chosen for the improved swelling properties that were deemed
important given the highly lipophilic nature of the peptide.
Pleasingly, extension of the peptide chain using standard Fmoc-
SPPS conditions with 1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5b]pyridinium 3-oxide hexafluorophosphate
(HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) as the
coupling reagents and iPr2NEt as the base proceeded readily to
afford resin-bound nonapeptide 13. It should be noted that
couplings to N-methylated residues required two treatments
with the coupling solution to ensure complete conversion (as
judged by UPLC−MS analysis of a small amount of cleaved
peptide).
Scheme 2. Total Synthesis of Ecumicin (1)
At this point, the key on-resin esterification between the L-
Thr side chain and the symmetric anhydride of Alloc-L-Val-OH
[generated using N,N′-diisopropylcarbodiimide (DIC) with
catalytic 4-(dimethylamino)pyridine (DMAP)] was carried out,
affording resin-bound depsipeptide 14 following two treat-
ments. Next, the Fmoc group was removed via treatment with
piperidine in DMF, followed by an on-resin dimethylation of
the uncovered amine through a modified Eschweiler−Clarke
reaction. Having successfully dimethylated the N-terminus of
the resin-bound peptide, the Alloc protecting group on the
branched L-Val was removed via treatment with Pd(PPh3)4 and
phenylsilane in CH2Cl2 to provide 15. From here, standard
Fmoc-SPPS was continued to afford the resin-bound linear
peptide 16. Notably, β-OH-L-Phe-OH building block 2 was
coupled without the oxazolidine protecting group used in the
initial synthetic strategy as at this late stage in the synthesis the
secondary alcohol was no longer exposed to reaction conditions
that could lead to the formation of side products. Subsequently,
the linear peptide 16 was cleaved from resin using 30%
hexafluoroisopropanol in CH2Cl2, purified using RP-HPLC,
and lyophilized to provide the linear peptide as a trifluoroacetic
acid (TFA) salt in 9% yield over 28 steps from resin-bound 12
(average of 92% per step).
With the linear peptide in hand, macrolactamization was next
carried out. Pleasingly, the use of 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium tetrafluoroborate
(DMTMM·BF4) and N-methylmorpholine (NMM) in a dilute
solution of DMF at 50 °C for 16 h effected the cyclization with
neglible epimerization (as measured by HPLC). Removal of the
protecting groups with TFA and triisopropylsilane in CH2Cl2
and subsequent RP-HPLC purification and lyophilization
afforded ecumicin 1 as the TFA salt in 27% yield over the
cyclization and deprotection steps following HPLC purification
(2.4% overall yield over the 30 steps). It is important to note
that monitoring of the macrolactamization via UPLC−MS
showed complete conversion of the linear peptide to the cyclic
peptide (with very few byproducts), and the lower than
expected isolated yield was likely owing to the amphoteric
nature of the natural product which had a broad elution profile
during HPLC purification (see the SI). Gratifyingly, spectro-
scopic data for synthetic ecumicin 1 were consistent with those
reported for the isolated natural product by Gao et al.5 Having
completed the total synthesis of ecumicin, we next assessed the
antimycobacterial activity of the natural product by screening
against the virulent H37Rv strain of Mtb using a resazurin-
based assay.20 The activity of the synthetic natural product 1
C
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Organic Letters
Letter
(8) Raju, R. M.; Goldberg, A. L.; Rubin, E. J. Nat. Rev. Drug Discov.
2012, 11, 777.
against virulent H37Rv Mtb was consistent with the isolated
natural product with 1 possessing a MIC90 against Mtb H37Rv
of 312 31 nM (control: rifampicin MIC90 = 7 0.3 nM; see
the SI for raw data).
In summary, we report here the first total synthesis of the
potent antimycobacterial cyclic depsipeptide natural product
ecumicin (1). This work now lays the foundation for the
generation of synthetic analogues of ecumicin in order to
generate SARs against the chaperone protein ClpC1, the
putative target of the natural product that is essential for
mycobacterial viability. This may ultimately stimulate the
discovery of ecumicin analogues as new TB drug leads that
operate through a novel mechanism of action to currently
employed therapies.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b03967.
(18) Huang, Y.; Feng, W.-H. Chin. Chem. Lett. 2016, 27, 357.
(19) Wenschuh, H.; Beyermann, M.; Winter, R.; Bienert, M.;
Ionescu, D.; Carpino, L. A. Tetrahedron Lett. 1996, 37, 5483.
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288.
Experimental procedures for synthesis, antibacterial
screening, and characterization data and NMR spectra
of ecumicin (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: richard.payne@sydney.edu.au.
ORCID
Richard J. Payne: 0000-0002-3618-9226
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Dr. Ian Luck (The University of Sydney) for
technical support for NMR spectroscopy and Dr. Nick
Proschogo (The University of Sydney) for technical support
for mass spectrometry. We also acknowledge the Australian
Postgraduate Award and the John A. Lamberton Research
Scholarship for PhD funding (P.M.E.H. and A.M.G.).
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D
DOI: 10.1021/acs.orglett.7b03967
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