Total Synthesis and Antimycobacterial Activity of Ohmyungsamycin A, Deoxyecumicin, and Ecumicin

Article abstract of DOI:10.1002/chem.202002408

The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid-phase strategy to assemble the linear peptide precursor, involving a key on-resin esterification and an optional on-resin dimethylation step, before a final solution-phase macrolactamization between the non-proteinogenic N-methyl-4-methoxy-l-tryptophan amino acid and a bulky N-methyl-l-valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC₉₀’s ranging from 110–360 nm and retained activity against Mtb in Mtb-infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.

Full text of DOI:10.1002/chem.202002408

Accepted Article
Accepted Article
Title: Total Synthesis and Antimycobacterial Activity of
Ohmyungsamycin A, Deoxyecumicin, and Ecumicin
Authors: Paige Hawkins, Wendy Tran, Gayathri Nagalingam, Chen-Yi
Cheung, Andrew Giltrap, Gregory Cook, Warwick Britton, and
Richard James Payne
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.202002408
Link to VoR: https://doi.org/10.1002/chem.202002408
01/2020

10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
Total Synthesis and Antimycobacterial Activity of
Ohmyungsamycin A, Deoxyecumicin, and Ecumicin
Paige M. E. Hawkins,[a] Wendy Tran,[a] Gayathri Nagalingam,[b] Chen-Yi Cheung,[c] Andrew M. Giltrap,[a]
Gregory M. Cook,[c] Warwick J. Britton,[b,d] and Richard J. Payne*[a]
[a]
P M. E. Hawkins, W. Tran, Dr. A. M. Giltrap, and Prof. R. J. Payne
School of Chemistry
The University of Sydney
Sydney, NSW 2006 (Australia)
E-mail: richard.payne@sydney.edu.au
Dr. G. Nagalingam and Prof. W. J. Britton
Centenary Institute
The University of Sydney
Sydney, NSW 2006 (Australia)
C.-Y. Cheung and Prof. G. M. Cook
Department of Microbiology and Immunology, School of Biomedical Sciences
University of Otago
PO Box 56, Dunedin, New Zealand
Prof. W. J. Britton
[b]
[c]
[d]
Central Clinical School, Faculty of Medicine and Health
The University of Sydney
Sydney, NSW 2006 (Australia)
Supporting information for this article is given via a link at the end of the document.
Abstract: The ohmyungsamycin and ecumicin natural product
families are structurally related cyclic depsipeptides that display
potent antimycobacterial activity. Herein the total synthesis of
ohmyungsamycin A, deoxyecumicin, and ecumicin are reported,
together with the direct biological comparison of members of these
natural product families against Mycobacterium tuberculosis (Mtb),
the etiological agent of tuberculosis (TB). The synthesis of each of the
natural products employed a solid-phase strategy to assemble the
linear peptide precursor, involving a key on-resin esterification and an
optional on-resin dimethylation step, before a final solution-phase
macrolactamization between the non-proteinogenic N-methyl-4-
use of second-line antibiotics that are more expensive and toxic
than their first-line counterparts.[1, 3a] MDR-TB is considered both
a public health crisis and a threat to health security with only 56%
of global cases treated successfully.[1] Due to this emerging crisis
there has been increased focus on the development of new
antibiotics that target Mtb, i.e. antimycobacterials. Testament to
this, in the last decade the U.S. Food and Drug Administration
fast-tracked the approval of two novel antibiotics: bedaquiline and
pretomanid (the first new TB drugs to be approved in 40 years).[3a,
4]
While this progress is welcoming, owing to poor treatment
compliance during long drug regimens and the ongoing
development of resistance to current and novel drugs, there still
remains an urgent need for new antibiotics which operate via
novel modes of action and that show rapid bactericidal activity
with a view to shortening TB treatment regimens.[3b, 5]
methoxy-L-tryptophan amino acid and a bulky N-methyl-L-valine
residue. The synthetic natural products possessed potent
antimycobacterial activity against Mtb with MIC90’s ranging from 110-
360 nM and retained activity against Mtb in Mtb-infected
macrophages. Deoxyecumicin also exhibited rapid bactericidal killing
against Mtb, sterilizing cultures after 21 days.
Ohmyungsamycin A (1) is a cyclic depsipeptide natural
product that was isolated from
a marine actinobacteria,
Streptomyces sp. SNJ042 from Jeju Island, South Korea, by Um
et al. (Figure 1).[6] Ohmyungsamycin A demonstrated anti-prolific
effects against a range of human cancer cell lines, which were not
replicated against normal cells.[6] More recently, Kim and co-
workers have shown that ohmyungsamycin A (1) is also active
against Mtb (MIC50 = 57 nM).[7] Moreover, the authors also
validated the activity in an in vivo efficacy model of Mtb, using flies
(D. melanogaster) infected with Mycobacterium marinum.[7]
Structurally, ohmyungsamycin A is a dodecapeptide consisting of
Introduction
Tuberculosis (TB) is caused by infection with the bacterium
Mycobacterium tuberculosis (Mtb) and was responsible for 1.5
million deaths in 2018, making it the deadliest infectious disease
globally. It is particularly striking that a quarter of the global
population are latently infected with Mtb, with 10 million new
cases diagnosed in 2018.[1] TB treatment involves intensive daily
administration of various combinations of the four first-line
antibiotics: rifampicin, isoniazid, pyrazinamide, and ethambutol
for a period of six months.[2] While this is effective for drug-
sensitive Mtb, 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.[1, 3] Treatment of drug-
resistant TB is long (up to two years) and complex, relying on the
all
L-amino acids, extensive backbone N-methylation, a
monomethylated N-terminus, and two non-proteinogenic amino
acids: N-methyl-4-methoxy-L-tryptophan and threo-β-hydroxy-L-
phenylalanine.[6]
1
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10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
meanwhile cyclomarin A has been shown to increase proteolysis,
triggering cell death through unregulated protein degradation.[13]
Given the potent in vitro and in vivo antimycobacterial activity
of ohmyungsamycin A (1), this molecule also recently succumbed
to a total synthesis.[14] Suh and co-workers employed a
convergent, solution-phase fragment condensation strategy to
produce ohmyungsamycin A in a 4% overall yield (longest linear
sequence).[14] Herein, we describe the use of a unified solid-phase
synthetic strategy with a late-stage macrolactamization for the
assembly of ohmyungsamycin A (1), ecumicin (3), and the first
total synthesis of deoxyecumin (2). This enabled the direct
comparsion of the structural features that are important for
biological activity, to guide future analogue generation for the
development of potential new TB drug leads that target the ClpC1
chaperone.
O
HN
R2
O
H
N
H
N
Macrolactamization
N
N
H
On-resin esterification
O
O
O
O
O
O
O
N
R1
NH
O
=
N
H
N
N
O
N
H
O
OH
O
O
H
O
N
N
N
R1
=
R1
N
H
N
H
O
O
On-resin
dimethylation
1
2
R2 = H, Deoxyecumicin ( )
R2 = OH, Ohmyungsamycin A ( )
3
R
2 = OH, Ecumicin ( )
Results and Discussion
Figure 1. Structure of the natural products ohmyungsamycin
A (1),
deoxyecumicin (2), and ecumicin (3), showing key synthetic transformations.
The synthetic logic for the assembly of the natural products 1 and
2 was inspired by the solid-phase strategy developed in our total
synthesis of ecumicin (3).[10] The peptide cyclization junction was
Deoxyecumicin (2) was also isolated from a soil actinobacteria
Nonomuraea sp. MJM5123, from Jeju Island, South Korea,
alongside the natural product ecumicin (3) by Gao et al. (Figure
1).[8] Ecumicin has been well studied for its antimycobacterial
activity, displaying promising in vitro activity (MIC90 = 160 nM
against Mtb strain H37Rv) and in vivo activity, inhibiting Mtb
growth in mice after 12 doses at 20 mg kg-1.[9] For this reason,
ecumicin was our first natural product target and in 2018 we
reported the first total synthesis of this molecule and confirmed its
structure (including absolute configuration) and antimycobacterial
activity.[10] Deoxyecumicin has been less well studied although,
like ecumicin, it was shown to exhibit potent activity against Mtb
chosen between the N-Me-L-Val-OH and N-methyl-4-methoxy-L-
tryptophan residues (Figure 2).[10] Although this cyclization
involves an N-methylated N-terminus and possesses significant
steric hindrance at the putative cyclization site, it was envisioned
that the termini could react through pre-organization of the fully
assembled linear peptide chain. Inspection of the intramolecular
hydrogen bonds present in the crystal structure of ecumicin (3)
reported by Gao et al. revealed a β-sheet stabilized by four
intramolecular hydrogen bonds (blue) and two possible n→π*
interactions (green) (Figure 2).[8a] Based on these interactions, it
would appear that the amino sequence between Val-2-Val-3 is
highly turn-inducing and predominantly responsible for the twisted
β-sheet structure of ecumicin. For ecumicin, we had previously
hypothesized that disconnection of the cyclic core in the middle of
strain
H37Rv.[8b]
Deoxyecumicin
and
ecumicin
are
tridecapeptides structurally related to ohmyungsamycin A (1),
with notable distinctions being a dimethylated N-terminus and the
presence of an additional N-methyl-allo-L-isoleucine on its
this turn-inducing sequence (N-Me-L-Val-OH and N-Me-4-OMe-L-
Trp) could be beneficial for macrolactamization and may pre-
organize the peptide for macrolactamization.[10] Given that
ohmyungsamycin A (1) and deoxyecumicin (2) share a near
identical cyclic core, we envisaged that the same strategy would
be effective to generate these natural products.
exocyclic tail component. Additionally, unlike ecumicin and
ohmyungsamycin A, deoxyecumicin lacks the β-hydroxy
functionality on L
-phenylalanine.[8b]
The molecular target of ecumicin (3) is the chaperone protein
ClpC1, which is involved in protein degradation in mycobacteria.
Very recently, Wolf et al. reported a co-crystal structure of
ecumicin bound to the ClpC1 N-terminal domain.[11] Interestingly,
the complex showed a 1:2 target:ligand stoichiometry, with
binding triggering significant conformational changes in the N-
terminus of the protein.[11] Mechanistically, ecumicin is thought to
stimulate ATPase activity of ClpC1, while also invoking a marked
decrease in proteolysis by the ClpC1-mediated ClpP1P2 protease
complex, ultimately leading to 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.[9] Given the structural similarities
between the ecumicin and ohmyungsamycin natural product
families, it is likely that both ohmyungsamycin A (1) and
deoxyecumicin (2) also target ClpC1. Importantly, ClpC1 has
O
HN
Val-2
HO
O
H
H
N
Val-1
O
Macrolactamization
N
N
C
C
O
N
H
O
O
O
O
O
Val-5
O
N
O
N
N
NH
O
N
N
Val-3
H
H
N
N
O
O
N
H
O
OH
Val-4
Figure 2. Structure of ecumicin (3), showing intramolecular hydrogen bonds
(blue), n→π* interactions (green), and macrolactamization site (red).
been recognized as
a promising antimycobacterial target,
because it is only essential in mycobacteria.[12] Three other
natural products have also been shown to target the N-terminal
domain of ClpC1: lassomycin, cyclomarin A, and rufomycin. Like
ecumicin, lassomycin and rufomycin inhibit proteolysis,
2
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10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
We proposed that natural products 1, 2, and 3 could all be
At this point, the solid-phase synthesis diverged and to one pool
obtained and diverged from a single common resin-bound peptide
of resin was coupled Fmoc-L-Val-OH and Boc-N-Me-L-Val-OH
precursor 4. Towards this end, Fmoc-N-Me-L-Val-OH was loaded
sequentially using standard Fmoc-SPPS conditions to provide 6.
onto activated 4-(diphenylhydroxymethyl)benzoic acid (Trityl-
OH)-functionalized polyethylene glycol-based ChemMatrix® resin
to generate resin-bound amino acid 5 (Scheme 1). 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-oxid hexafluorophosphate (HATU) and
1-hydroxy-7-azabenzotriazole (HOAt) as the coupling reagents
and iPr2NEt as the base proceeded readily to afford resin-bound
hexapeptide 4. 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). In particular, the coupling of
A separate pool of resin was elongated by three residues,
specifically, Fmoc-N-Me-allo-
Fmoc- -Val-OH to provide resin-bound 7.
From here, the key on-resin esterification between the
-threonine sidechain and the symmetric anhydride of -Val-OH
L-Ile-OH, Fmoc-L-Val-OH, and
L
L
L
[generated using N,N’-diisopropylcarbodiimide (DIC) with
catalytic 4-dimethylamino)pyridine (DMAP)] was carried out twice,
to provide resin-bound depsipeptide 8 and 9. It should be noted,
that while an Fmoc-protecting group could be used for
depsipeptide 8, an Alloc-protecting group was required for
depsipeptide 9 to provide differentiation between the N-terminal
valine tail and esterified sidechain valine.
With resin-bound depsipeptide 8 in hand, the Fmoc group was
next removed via treatment with piperidine in DMF to afford 10
(Scheme 2). Depsipeptide 9 was also Fmoc-deprotected before
the resulting amine was dimethylated on-resin through a modified
Eschweiler-Clarke reaction using formaldehyde, dilute acetic acid,
and sodium cyanoborohydride. Having successfully generated
the tertiary amine en bloc, the Alloc-protecting group on the
branched valine was removed via treatment with catalytic
palladium(0) tetrakistriphenylphosphine (Pd(PPh3)4), using
phenylsilane as an allyl scavenger to generate resin-bound 11.
Standard Fmoc-SPPS was continued on peptide 10 to afford
12. This included the incorporation of two synthetic amino acid
Fmoc-L-Thr-OH to the hindered N-Me-L
-Thr(OtBu) required
careful monitoring to ensure reaction completion.
O
O
N
building blocks, Fmoc-β-OH-L-Phe-OH and Fmoc-N-Me-4-OMe-
Fmoc
5
L-Trp-OH, the latter prepared via Negishi cross coupling
chemistry (see Supporting Information for details).[10] Two
O
Fmoc-SPPS
OH
separate pools of 11 were also elongated by Fmoc-SPPS to afford
O
the resin-bound linear peptides, 13 (incorporating Fmoc-
L-Phe-
N
O
OH) and 14 (incorporating Fmoc-β-OH- -Phe-OH).
L
Having assembled the target linear peptides 12, 13, and 14,
these were next cleaved from resin using 30%
hexafluoroisopropanol in CH2Cl2, purified using RP-HPLC and
lyophilized to provide the linear peptides as trifluoroacetate salts
in good yield (average yield of 90-93% per step).
O
Fmoc
NH
O
N
H
N
N
O
N
H
O
OtBu
4
With the linear peptides in hand, macrolactamization was next
attempted. A range of conditions were trialed for the cyclization of
HFIP-cleaved linear peptide 12 and 14 in an attempt to minimize
epimerization; however, 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 proved optimal.[10] These conditions were then replicated
for the cyclization of the HFIP-cleaved linear peptide 13.
Fmoc-SPPS
O
O
O
H
N
6: R1
7: R1
=
=
N
O
O
O
Boc
N
O
OH
O
R1
O
NH
O
N
H
N
H
N
N
N
N
Fmoc
O
N
H
H
O
OtBu
Finally, removal of the protecting groups with TFA and
triisopropylsilane in CH2Cl2 and subsequent RP-HPLC purification
Fmoc-L-Val-OH (for 6) or Alloc-L-Val-OH (for 7)
DIC, CH2Cl2, DMAP, DMF, 2 x 16 h
and
lyophilization
afforded
ohmyungsamycin
A
(1),
O
deoxyecumicin (2), and ecumicin (3) in 22-41% yield over the
cyclization and deprotection steps following HPLC purification
(2-5% overall yields over the 24-28 steps).
O
H
N
8
: R1
=
PG
O
O
N
O
N
H
PG = Fmoc
O
O
Boc
N
O
O
R1
O
O
H
N
NH
O
N
H
N
9
: R1
=
N
N
N
Fmoc
O
N
PG = Alloc
H
H
O
OtBu
Scheme 1. Synthesis of resin-bound depsipeptides 8 and 9.
3
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10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
8
9
Importantly, each of the natural products exhibited activity that
was only 1-1.5 orders of magnitude less potent compared with the
frontline TB drug rifampicin which was used as a positive control
in the assay.
1. 10 vol % piperidine, DMF, 2 x 3 min
2. Aq CH2O, AcOH, DMF, 2 h
NaBH3CN, 2 vol % AcOH, DMF, 6 h
3. Pd(PPh3)4, PhSiH3, CH2Cl2, 20 min
10 vol % piperidine, DMF, 2 x 3 min
10
11
O
H
O
Table 1. Activity of synthetic natural products against Mtb strain H37Rv.
N
N
10
: R1
=
=
O
N
O
H2N
O
O
Boc
MIC90 (nM)
N
O
O
O
N
R1
O
O
NH
O
N
H
Ohmyungsamycin A (1)
Deoxyecumicin (2)
Ecumicin (3)
110 ± 5
280 ± 7
360 ± 30
7 ± 0.3
N
11
: R1
N
N
H
O
N
H
O
OtBu
Fmoc-SPPS
Rifampicin
O
H
N
O
R1
=
N
HN
O
R2
O
Boc
O
H
Mtb resides in lung macrophages and as such, the synthetic
natural products were also assessed in an intracellular killing
assay. Pleasingly they all inhibited the growth of Mtb in infected
human THP-1 macrophages (Figure 3). Of these compounds,
deoxyecumicin (2) and ecumicin (3) were most effective, with 1-2
log10 reduction in Mtb growth at 10 µM, providing similar activities
to the frontline TB drug rifampicin. The apparent contrast in the
order of activity compared with those observed against virulent
H37Rv Mtb (Table 1) may reflect differences in the ability of the
natural products to penetrate macrophages.
H
N
N
12
N
R2 = OH,
N
H
H
O
O
O
O
O
O
N
O
O
N
O
N
R1
=
N
H
O
R1
NH
O
N
H
N
N
O
N
H
13
R2 = H,
R2 = OH,
O
OtBu
14
1. 30 vol % hexafluoroisopropanol, CH2Cl2, 3 x 20 min
2. DMTMM.BF4, NMM, DMF, 50 oC, 16 h
3. TFA:CH2Cl2:iPr3SiH (49:49:2 v/v/v), 1 h
O
H
O
N
R1
=
N
HN
H
R2
O
O
6
H
N
H
N
Ohmyungsamycin A (1)
R2 = OH, Ohmyungsamycin A (1)
N
O
N
H
5% (24 steps)
Deoxyecumicin (2)
O
O
O
5
O
O
O
O
O
Ecumicin (3)
O
N
N
N
R1
=
N
Rifampicin
4
3
2
H
R1
O
NH
O
N
H
N
N
N
H
R2 = H, Deoxyecumicin (2)
O
3% (28 steps)
R2 = OH, Ecumicin ( )
OH
3
2% (28 steps)
0
10
0
10
0
10
0 10
Drugs (M)
Scheme 2. Total synthesis of ohmyungsamycin A (1), deoxyecumicin (2), and
ecumicin (3).
Figure 3. Activity of natural products against Mtb in infected human THP-1
macrophages. All experiments were performed in biological triplicate and the
standard error of the mean is shown.
Gratifyingly, spectroscopic data for synthetic natural products 1-3
was consistent with that reported for the isolated natural products
by Um et al. and Gao et al.[6, 8] (see Supporting Information for
comparison data). Having successfully completed the total
synthesis of the three natural products, we next assessed the
antimycobacterial activity by screening against Mtb strain H37Rv
using a resazurin-based assay.[15] The activity of each of the three
synthetic natural products (1-3) against Mtb strain H37Rv was
consistent with those reported for the isolated natural products
(Table 1).[7, 8b, 9] It was interesting to find that ohmyungsamycin A
(1) (MIC90 of 110 nM) displayed slightly better antimycobacterial
activity than the other natural products (MIC90’s ranging from 280-
360 nM). These findings indicate that the shorter Val-Val mono-
methylated N-terminal tail of ohmyungsamycin A provides an
improvement in activity against Mtb, in contrast to the findings
recently reported by Wolf et al.[11] Additionally, our study has
shown that the presence of the non-proteinogenic β-hydroxy
Finally, synthetic deoxyecumicin (2) and ecumicin (3) were
screened in a time-kill kinetic assay to better understand the killing
kinetics of the natural products (Figure 4). For these cell killing
experiments we used Mtb strain mc26230 (ΔRD1 ΔpanCD).[16]
Strain mc26230 (ΔRD1 ΔpanCD) is an avirulent auxotrophic Mtb
mutant that behaves identically to wild-type Mtb strain H37Rv
when grown in pantothenate-supplemented medium, but is
incapable of causing disease even in severe combined
immunodeficiency (SCID) mice.[16] The MIC values for
deoxyecumicin and ecumicin of Mtb strain mc26230 (ΔRD1
ΔpanCD) were comparable to Mtb strain H37Rv. When Mtb strain
mc26230 was challenged with the test compounds at 10 the MIC
(Table 1), deoxyecumicin exhibited rapid bactericidal activity, with
complete sterilization of the Mtb culture after 21 days (Figure 4A).
Isoniazid (INH, 2 µM) was used as a positive control and showed
early bactericidal activity but tolerance to isoniazid emerged after
functionality on
L-Phe is not critical for biological activity.
4
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10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
7 days (Figure 4B), a result that has been observed previously for
INH-treated cultures.[17]
for the development of potential new TB drug leads based on the
sructure of this molecule. Overall, this preliminary work provides
the basis for further structure-activity relationship studies on
synthetic analogues which can be rapidly assembled using the
robust solid-phase route that we have reported here.
Ecumicin demonstrated strong early bactericidal activity with
a three log10 reduction in cells (CFU/mL) at day seven, but this
was followed by the emergence of cells that became resistant to
the compound. INH-treated cells showed a similar pattern of cell
killing followed by the emergence of resistant cells (Figure 4B).
The rapid bactericidal activity exhibited by deoxyecumicin and
the lack of tolerant or resistant mutants arising as observed with
isoniazid, a front line drug for TB treatment, now provides the
impetus to generate analogues of this natural product for the
development of novel TB drug leads.
Experimental Section
In vitro inhibition assay against Mtb
The compounds were originally stored as 10 mM stock solutions in 100%
DMSO. Two-fold serial dilutions of the compounds were made in a 96 well
plate using Middlebrook 7H9 medium supplemented with ADC (Difco
Laboratories, Detroit, MI, USA), 0.5% v/v glycerol and 0.05% v/v Tween-
80. Mtb strain H37Rv was grown to mid-exponential phase to an OD600 of
0.6-0.8 in 7H9 media at 37 °C. On the day of the assay, the culture was
diluted to an OD600 of 0.002 in 7H9 medium and 100 L of bacterial
suspension was added to the 96 well plate containing 100 L of the diluted
compounds. The plate was incubated for 5 days at 37 °C in a humidified
incubator and 30 L of Resazurin (0.02% w/v) and 12.5 L of Tween-80
was added to each well and incubated for a further 24 h. On day 6, the
fluorescence was read using a BMG Labtech Polarstar plate reader
(excitation 530 nm and emission 590 nm). The results are presented as
Mtb survival as a percentage of negative control (no drug controls).
1012
A
1010
Deoxyecumicin (2)
108
Isoniazid
106
Untreated
104
102
100
0
7
14
21
28
Time (Days)
1012
1010
108
106
104
102
100
Ecumicin (3)
Isoniazid
B
Untreated
Mtb inhibition using infected macrophage
culture
Mtb strain H37Rv (NR-123, BEI Resources) was grown in Middlebrook
7H9 broth medium supplemented with ADC (Difco Laboratories), 0.05%
glycerol and 0.05% Tween-80. Bacterial cultures were grown at 37ꢀ°C, to
mid-exponential phase (OD600 0.4–0.8) and then used to infect a human
macrophage-like cell line (THP-1; ATCC TIB-202). THP-1ꢀcells were
cultured in RPMI-1640 medium (with phenol red, 25ꢀmM HEPES and
0
7
14
21
28
Time (Days)
Figure 4. Effect of test compounds on the cell viability of Mtb strain mc26230.
Test compounds were added to Mtb strain mc26230 cultures grown to an OD600
of 0.5 (time zero, approximately 1  108 CFU/mL). The culture was challenged
with 10 the MIC of the test compound and cell viability monitored for 28 days
by plating onto 7H11 media, and colony forming units determined after four
weeks of incubation at 37°C. All experiments were performed in biological
triplicate and the standard error of the mean is shown.
2ꢀmM L-glutamine) supplemented with 10% FBS (FBS-500) and 0.05ꢀmM
β-mercaptoethanol. 1 × 105 cells/well of THP-1 cells were plated in 96-well
tissue culture plates and differentiated with phorbol myristic acetate (PMA;
100ꢀnM) for 48ꢀh at 37ꢀ°C at 5% CO2. Mtb strain H37Rv was sonicated to
achieve single cell suspension in RPMI-1640 medium and used to infect
differentiated THP-1 cells at a multiplicity of infection of 1 for 4ꢀh at 37ꢀ°C
at 5% CO2. Supernatant was then removed from all wells, THP-1 cells
were washed with 200ꢀμL phosphate buffered saline (PBS) three times and
were subsequently replenished with fresh RPMI-1640 cell culture medium
and incubated for a further 24ꢀh at 37ꢀ°C and 5% CO2. The compounds
were diluted in fresh RPMI-1640 cell culture medium and added to
corresponding wells. Positive controls were dissolved in 100% dimethyl
sulfoxide (DMSO) and after 72ꢀh of incubation at 37ꢀ°C at 5% CO2, tissue
culture medium containing the test compound was removed from the wells
and cells lysed with sterile water containing 0.1% Triton X-100. Cell lysates
were serially diluted, 1:10, and plated on Middlebrook 7H11/OADC
(283010; Difco) agar through to 1:10,000 dilution. Agar plates were then
incubated at 37ꢀ°C for 3–4 weeks, after which the bacteria colonies were
counted and CFU mL−1 of cell lysates were determined.
Conclusion
In summary, we have described the total synthesis of the three
marine actinobacterially-derived cyclic depsipeptide natural
products ohmyungsamycin
A (1), deoxyecumicin (2), and
ecumicin (3) using an efficient solid-phase synthetic strategy with
a late stage macrolactamization step. Access to all three natural
products enabled direct comparison of the antimycobacterial
activities of the family of natural products. Ohmyungsamycin A
was found to be the more active congener against Mtb with an
MIC90 of 110 nM, however, ecumicin and deoxyecumicin
displayed superior activity against intracellular Mtb (within
infected THP-1 cells). Deoxyecumicin also displayed rapid
bactericidal activity against Mtb where it was capable of sterilizing
Kinetics of cell killing by deoxyecumicin (2)
and ecumicin (3)
The avirulent auxotrophic Mtb strain mc26230 (ΔRD1 ΔpanCD) was used
to monitor the kinetics of cell killing by deoxyecumicin (2) and ecumicin (3).
Isoniazid were used as a positive control. Strain mc26230 (ΔRD1 ΔpanCD)
is an avirulent auxotrophic Mtb mutant that behaves identically to wild-type
a bacterial culture after 21 days at a concentration 10 the MIC
This is a particularly encouraging feature that could be harnessed
.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
Mtb when grown in pantothenate-supplemented medium, but is incapable
of causing disease even in severe combined immunodeficiency (SCID)
mice.[16] Growth of M. tuberculosis strain mc26230 was grown in
Middlebrook 7H9 broth supplemented with OADC (0.005% oleic acid,
0.5% bovine serum albumin, 0.2% dextrose, 0.085% catalase), 0.05%
tyloxapol (Sigma) and 25ꢀμg/mL pantothenic acid (PAN). Cultures were
maintained in 10 mL volumes in 30 mL inkwells at 37°C with agitation (140
rpm). When required mc26230 was grown on Middlebrook 7H11 agar
supplemented with OADC and PAN.
confirmed via variable temperature NMR experiments or saturation
transfer NMR experiments. Low resolution mass spectra for novel
compounds were recorded on a Bruker amaZon SL mass spectrometer
(ESI) operating in positive/negative mode or on a Shimadzu 2020 (ESI)
mass spectrometer operating in positive/negative mode. High resolution
mass spectra were recorded on a Bruker-Daltronics Apex Ultra 7.0T
Fourier transform (FTICR) mass spectrometer.
LC-MS was performed on a Shimadzu UPLC-MS instrument with an LC-
M20A pump, SPD-M30A diode array detector and a Shimadzu 2020 (ESI)
mass spectrometer operating in positive mode. Separations on the UPLC
system were performed on a Waters Acquity 1.7 µm, 2.1 x 50 mm (C18)
column. These separations were performed using a mobile phase of 0.1
vol % trifluoroacetic acid in water (Solvent A) and 0.1 vol % trifluoroacetic
acid in MeCN (Solvent B) using linear gradients. Preparative reverse-
phase HPLC was performed using a Waters 500 pump with a 2996
photodiode array detector and a Waters 600 Multisolvent Delivery System.
The MIC values for deoxyecumicin (2), ecumicin (3), and isoniazid were
determined by inoculating Mtb into 96-well plates containing supplemented
Middlebrook 7H9 broth, at a starting OD600 of 0.05, in a total starting
volume of 200 µL. Test compounds were dispensed from a 9-point, 3-fold
dilution gradient to each well, with a maximum of 2% dimethyl sulfoxide
(DMSO). 96-well plates were incubated without shaking at 37°C for 10
days. On day 7, OD600 values were measured in a Varioskan LUX
microplate reader, and MIC values were determined in biological triplicate.
To determine the effects of test compounds on the cell viability of Mtb, cells
were grown in 10 mL volumes in 30 mL inkwells at 37°C with agitation (140
rpm). When cells reached an OD600 of 0.5 (time zero, approximately 1 
108 CFU/mL), the culture was challenged with 10 the MIC of the test
compound and cell viability monitored for 28 days. At various time points
(as indicated), culture was removed from the desired inkwell and diluted
along a four-point ten-fold dilution curve. Five µL was spotted onto 7H11
media, and colony forming units determined after four weeks of incubation
at 37°C. All experiments were performed in biological triplicate and the
standard error of the mean is shown.
Fmoc-SPPS General Protocols
Fmoc-strategy solid-phase peptide synthesis (Fmoc-SPPS) procedures
were employed on acid-labile 4-(diphenylhydroxymethyl)benzoic acid
(Trityl-OH)-functionalized polyethylene glycol resin (Chem-Matrix) within
fritted syringes. All reagent equivalents are with respect to the amount of
the initial amino acid loaded to resin.
Resin Loading
Trityl-OH Chem-Matrix® resin (0.30 mmol/g) was swelled in CH2Cl2 (0.015
M) and shaken for 20 min. The solvent was discharged and the resin
washed with CH2Cl2 (x 5). Resin was treated with 5% (v/v) thionyl
Materials and Methods for the Synthesis of 1-3
chloride/CH2Cl2 and shaken for
discharged and resin washed with CH2Cl2 (x 5) and 5% (v/v)
iPr2NEt/CH2Cl2 (x 2). The resin was treated with Fmoc-N-Me- -Val-OH (4
eq. in regards to predicted resin loading) and iPr2NEt (8 eq.) in CH2Cl2
(0.05 M) and shaken at room temperature for 16 h. The coupling solution
was discharged and washed with CH2Cl2 (x 5), DMF (x 5) and CH2Cl2 (x
5). The resin was treated with a capping solution of 17:2:1 (v/v/v)
CH2Cl2/iPr2NEt/MeOH and shaken at room temperature for 30 min. The
capping solution was discharged and the resin washed with CH2Cl2 (x 5)
and DMF (x 5).
4 h. The activating solution was
All reactions were carried out under an argon atmosphere and at room
temperature (22 °C) unless the reaction was performed under aqueous
conditions or unless otherwise specified. The reactions carried out at 0 °C
employed a bath of water and ice and reactions carried at -40 °C employed
a bath of dry ice and MeCN. Anhydrous THF, CH2Cl2, MeOH, MeCN, DMF,
and toluene were obtained using a PureSolv® solvent purification system.
The reactions were monitored by thin layer chromatography (TLC) on
aluminium backed silica plates (Merck Silica Gel 60 F254). Visualisation
of TLC plates was undertaken with an ultraviolet (UV) light at  = 254 nm
and staining with solutions of vanillin, ninhydrin, and potassium
permanganate followed by exposure of the stained plates to heat. Silica
flash column chromatography (Merck Silica Gel 60 40 – 63 µm) was
undertaken to purify crude reaction mixtures using solvents as specified.
Fractions were collected manually or with a Biotage Isolera Spektra
automated flash purification system.
L
Resin Loading Determination
The resin-bound peptide was treated with
a solution of 10 vol%
piperidine/DMF (2 x 3 min). The deprotection solution was discharged and
collected and the resin washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x
5). The combined deprotection solutions were made up to 25 mL using 10
vol% piperidine/DMF and diluted 1:100 with 10 vol% piperidine/DMF. The
amount of peptide loaded to resin was determined by measurement of the
UV absorbance at λ = 301 nm of the diluted deprotection solution.
All commercially available reagents were used as obtained from Sigma-
Aldrich, Merck, Acros Organics or AK Scientific. Amino acids, coupling
reagents and Trityl-OH ChemMatrix® resins were obtained from
NovaBiochem, PCAS, GL Biochem, or Mimotopes and peptide synthesis
grade DMF was obtained from Merck or Labscan. All non-commercially
available reagents were synthesized according to literature procedures as
referenced.
Fmoc Deprotection
The resin-bound peptide was treated with
a solution of 10 vol%
piperidine/DMF (2 x 3 min). The deprotection solution was discharged and
the resin washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5).
1H NMR spectra were obtained using a Bruker DRX 300, DRX 400 or DRX
500 at frequencies of 300 MHz, 400 MHz or 500 MHz respectively in CDCl3,
MeOD, or pyridine-d5. Chemical shifts are reported in parts per million
(ppm) and coupling constants in Hertz (Hz). 1H NMR data is reported as
follows: chemical shift values (ppm), multiplicity (s = singlet, d = doublet, t
= triplet, q = quartet, m = multiplet, br = broad), coupling constant(s) and
relative integral. 13C NMR spectra were obtained using a Bruker DRX 400
or DRX 500 at 100 MHz or 125 MHz in CDCl3, MeOD, CD3CN, D2O,
pyridine-d5, DMSO-d6, and acetone-d6 unless otherwise specified. 13C
NMR data is reported as chemical shift values (ppm). Any rotamers were
HATU Coupling Conditions
The resin-bound peptide was treated with a solution of a given amino acid
(1.2-4 eq.), HATU (1.2-4 eq.), HOAt (2.4-8 eq.) and iPr2NEt (2.4-8 eq.) in
DMF (0.05 M in regards to Chem-Matrix or NovaPEG loaded peptide or
0.1 M in regards to polystyrene loaded peptide) and shaken for 8 h at room
6
This article is protected by copyright. All rights reserved.

10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
temperature. The coupling solution was discharged and the resin washed
with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5).
Val-OH and Boc-N-Me-
methylated residues required two treatments with fresh coupling solution.
L
-Val-OH. Amino acid couplings following N-
On-resin Esterification Conditions
The resin-bound peptide (47 μmol) was esterified with Fmoc- -Val-OH
L
using the on-resin esterification conditions. The procedure was repeated
2-3 times until all starting material had converted into product as judged by
UPLC-MS monitoring.
To a solution of Alloc-L-Val-OH or Fmoc-L-Val-OH (10 eq.) in CH2Cl2 (0.06
M) was added DIC (5 eq.). The solution was stirred for 30 min at room
temperature before being concentrated under a stream of N2. The resultant
slurry was dissolved in DMF (0.05 M in regards to Chem-Matrix or
NovaPEG loaded peptide or 0.1 M in regards to polystyrene loaded
peptide) and sucked into the fritted syringe containing the resin-bound
peptide. Subsequently, a solution of DMAP (catalytic, ~6 small crystals) in
DMF (0.1 mL) was sucked up and the resin was shaken at room
temperature for 8 h. The coupling solution was discharged and the resin
washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5). The procedure was
repeated 1-3 times until all starting material had converted into product as
judged by UPLC-MS monitoring.
The resin-bound peptide (47 μmol) was then elongated using HATU
coupling conditions and Fmoc-deprotection conditions incorporating the
following amino acids in turn: Fmoc-threo-β-OH-
amino acid used, 16 h coupling), Fmoc- -Val-OH and Fmoc-N-Me-4-OMe-
-Trp-OH (1.2 eq. of amino acid used, 16 h coupling).
L-Phe-OH (1.2 eq. of
L
L
The resin-bound peptide was cleaved using the resin-cleavage conditions.
The crude protected linear peptide was purified by preparative RP-HPLC
using a Waters X-Bridge 5 μm 19 x 150 mm (C18) column using a gradient
of 60 – 70% MeCN in H2O (0.1% TFA) over 30 min at a flow rate of 7 mL
min-1. The peptide was lyophilized to yield the protected linear peptide as
a trifluoroacetate salt (11.1 mg, 21% over 22 steps from resin loading,
average of 93% per step).
On-resin Dimethylation Conditions
The resin-bound peptide was treated with a solution of 37% aqueous
formaldehyde (10 eq.), acetic acid (10 eq.) in DMF (0.05 M in regards to
loaded peptide). The resin was shaken at room temperature for 2 h at
which point an additional solution of sodium cyanoborohydride (4 eq.) in
2% (v/v) acetic acid/DMF (2.2 mL) was sucked into the fritted syringe. The
resin was shaken for 6 h, the solution was discharged and the resin
washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5). The procedure was
repeated 1-2 times until all starting material had converted into product as
judged by UPLC-MS monitoring.
The protected linear peptide (11.1 mg, 6.37 μmol) was cyclized according
to the general cyclization conditions described above. The crude cyclic
peptide was purified by preparative RP-HPLC using a Waters X-Bridge 5
μm 19 x 150 mm (C18) column using a gradient of 70 – 100% MeOH in
H2O (0.1% TFA) over 40 min at a flow rate of 7 mL min-1. The peptide was
lyophilized to give a white fluffy powder of pure ohmyungsamycin A (1) as
a trifluoroacetate salt (2.2 mg, 5% over 24 steps from resin loading, 22%
for the cyclization and deprotection).
Alloc Deprotection
HR-MS: (+ESI) Calc. for C75H119N13O16: 751.9341 [M+2Na]2+, Found:
751.9342 [M+2Na]2+; IR (ATR): max = 3290, 2964, 2929, 1671, 1634, 1542,
1469, 1204, 1140, 723 cm-1; [α]D20 = -47° (c = 0.1 in MeOH); 1H NMR (500
MHz, pyridine-d5): 0.66 (d, 5.8 Hz, 1.5H), 0.67 (d, 5.6 Hz, 1.5H), 0.95 (d,
6.5 Hz, 1.5H), 0.95 (d, 6.5 Hz, 1.5H), 0.97 (d, 6.5 Hz, 1.5H), 1.03 (d, 6.2
Hz, 1.5H), 1.04 (d, 6.2 Hz, 1.5H), 1.12 (d, 6.9 Hz, 1.5H), 1.14 (d, 6.9 Hz,
1.5H), 1.16 (d, 6.5 Hz, 1.5H), 1.20 (d, 6.5 Hz, 1.5H), 1.21 (m, 1.5H), 1.22
(m, 1H), 1.23 (d, 5.8 Hz, 1.5H), 1.27 (d, 6.5 Hz, 1.5H), 1.33 (d, 6.4 Hz, 3H),
1.35 (d, 6.7 Hz, 1.5H), 1.40 (m, 1H), 1.57 (d, 6.1 Hz, 3H), 1.58 (m, 1H),
1.74 (m, 1H), 2.23 (m, 1H), 2.29 (m, 1H), 2.39 (m, 1H), 2.49 (s, 3H), 2.56
(m, 1H), 2.64 (m, 1H), 2.66 (m, 1H), 2.98 (s, 3H), 3.03 (m, 1H), 3.19 (s,
3H), 3.20 (m, 1H), 3.52 (s, 3H), 3.84 (s, 3H), 3.71 (s, 3H), 4.31 (app. dd,
11.2, -13.2 Hz, 1H), 4.37 (d, 5.7 Hz, 1H), 4.44 (app. dd, 4.6, -13.5 Hz, 1H),
4.58 (dd, 4.6, 10.6 Hz, 1H), 4.67 (app. t, 8.6 Hz, 1H), 4.98 (app. t, 9.2 Hz,
1H), 5.06 (m, 1H), 5.20 (app. t, 8.0 Hz, 1H), 5.31 (app. t, 8.0 Hz, 1H), 5.38
(m, 1H), 5.49 (m, 1H), 5.59 (m, 1H), 5.65 (m, 1H), 5.90 (app. d, 10.8 Hz,
1H), 5.93 (app. s, 1H), 6.08 (m, 1H), 6.67 (d, 7.4 Hz, 1H), 7.19 (s, 1H),
7.27 (m, 2H), 7.32 (d, 8.1 Hz, 1H), 7.41 (app. t, 7.4 Hz, 2H), 7.67 (d, 8.1
Hz, 2H), 8.06 (d, 12.4 Hz, 1H), 8.08 (d, 8.7 Hz, 1H), 9.28 (d, 8.7 Hz, 1H),
9.38 (d, 9.9 Hz, 1H), 9.76 (d, 7.4 Hz, 1H), 9.87 (d, 8.7 Hz, 1H), 10.18 (d,
8.7 Hz, 1H), 11.84 (br s, 1H); 13C NMR (125 MHz, pyridine-d5, shifts were
extracted from HSQC and HMBC data): 17.2, 17.6, 18.6, 18.9, 19.1, 19.1,
19.4, 19.5, 19.5, 20.0, 20.1, 20.1, 20.1, 20.1, 20.8, 22.1, 22.2, 23.5, 25.4,
27.5, 29.5, 31.0, 31.3, 31.5, 31.8, 32.3, 32.9, 32.9, 32.9, 35.0, 38.9, 40.4,
41.1, 53.1, 54.7, 54.9, 55.8, 55.9, 58.6, 58.7, 59.6, 60.4, 62.9, 66.8, 68.3,
69.9, 71.0, 71.2, 73.5, 99.9, 106.3, 112.9, 118.8, 122.9, 124.2, 127.3,
127.8, 128.8, 139.5, 143.0, 155.1, 168.6, 169.9, 171.0, 171.7, 173.2, 173.3,
173.3, 173.9, 174.6, 176.0. 1H NMR and 13C NMR data are compared to
the isolated natural product in Table S1. These data is in agreement with
that previously reported by Um et al.[6]
The resin-bound peptide was washed with CH2Cl2 (x 10) and treated with
a solution of Pd(PPh3)4 (0.2 eq.), phenylsilane (20 eq.) in CH2Cl2 (0.018 M
in regards to loaded peptide) and shaken at room temperature for 20 min.
The deprotection solution was discharged and the resin washed with
CH2Cl2 (x 5) and DMF (x 5).
Resin-cleavage Conditions
The resin-bound peptide was washed with CH2Cl2 (x 10) and then treated
with a solution of 30 vol% HFIP/ CH2Cl2 (3 x 20 min). The cleavage solution
was discharged, collected and concentrated under a stream of N2 and
dried in vacuo.
Cyclization Conditions
The protected linear peptide (1 eq.) was dissolved in DMF (0.005 M) and
to this solution was added DMTMM.BF4 (2 eq.) followed by NMM (2 eq.).
The resulting reaction mixture was heated to 50 °C and stirred for 16 h.
The cyclization solution was concentrated under a stream of N2 and to this
residue was added iPr3SiH:TFA:CH2Cl2 (2:49:49, v/v/v, 5 mL) and stirred
at room temperature for 1 h. The deprotection solution was concentrated
under a stream of N2 and in vacuo to afford the cyclic peptide as a crude
solid which was subsequently purified by reversed-phase HPLC.
Synthesis of Ohmyungsamycin A (1)
Fmoc-N-Me-L-Val-OH (0.60 mmol) was loaded to Trityl-OH as per the
general procedures. Resin loading was determined to be 117 μmol.
Synthesis of Deoxyecumicin (2)
The resin-bound linear peptide (117 μmol) was elongated using HATU
coupling conditions and Fmoc-deprotection conditions, incorporating the
Fmoc-N-Me-
L-Val-OH (0.95 mmol) was loaded to Trityl-OH as per the
following amino acids in turn: Fmoc-
L
-Val-OH, Fmoc-N-Me-
L-Leu-OH,
-Thr-(OtBu)-OH, Fmoc-
L-Thr-OH, Fmoc-L-
general procedures. Resin loading was determined to be 190 μmol.
Fmoc- -Val-OH, Fmoc-N-Me-
L
L
7
This article is protected by copyright. All rights reserved.

10.1002/chem.202002408
Chemistry - A European Journal
FULL PAPER
A portion of the resin-bound linear peptide (109 μmol) was elongated using
170.7, 171.6, 171.6, 172.5, 173.2, 173.2, 173.8, 174.0, 174.0, 174.0, 174.6.
1H NMR and 13C NMR data are compared to the isolated natural product
in Table S2. This data is in agreement with that previously reported by Gao
et al.[8b]
HATU coupling conditions and Fmoc-deprotection conditions,
incorporating the following amino acids in turn: Fmoc-
Me- -Leu-OH, Fmoc- -Val-OH, Fmoc-N-Me-
-Thr-(OtBu)-OH, Fmoc-
Thr-OH, Fmoc-N-Me-allo- -Ile-OH, Fmoc- -Val-OH and Fmoc- -Val-OH.
L-Val-OH, Fmoc-N-
L
L
L
L-
L
L
L
Amino acid couplings following N-methylated residues required two
treatments with fresh coupling solution.
Synthesis of Ecumicin (3)
The resin-bound peptide (109 μmol) was esterified with Alloc-L-Val-OH
Fmoc-N-Me-L-Val-OH (0.60 mmol) was loaded to Trityl-OH as per the
general procedures. Resin loading was determined to be 117 μmol.
using the on-resin esterification conditions. The procedure was repeated
2-3 times until all starting material had converted into product as judged by
UPLC-MS monitoring.
The resin-bound linear peptide (117 μmol) was elongated using HATU
coupling conditions and Fmoc-deprotection conditions, incorporating the
The resin-bound peptide was Fmoc-deprotected and then dimethylated
using the on-resin dimethylation conditions. The procedure was repeated
1-2 times until all starting material had converted into product as judged by
UPLC-MS monitoring.
following amino acids in turn: Fmoc-
Fmoc- -Val-OH, Fmoc-N-Me-
-Thr-(OtBu)-OH, Fmoc-
Me-allo- -Ile-OH, Fmoc- -Val-OH and Fmoc- -Val-OH. Amino acid
L
-Val-OH, Fmoc-N-Me-
L-Leu-OH,
L
L
L
-Thr-OH, Fmoc-N-
L
L
L
couplings following N-methylated residues required two treatments with
fresh coupling solution.
The resin-bound peptide was Alloc-deprotected before 46 μmol of resin
was elongated using HATU coupling conditions and Fmoc-deprotection
The resin-bound peptide (60 μmol) was esterified with Alloc-L-Val-OH
conditions incorporating the following amino acids in turn: Fmoc-
Fmoc- -Val-OH and Fmoc-N-Me-4-OMe- -Trp-OH (1.2 eq. of amino acid
used, 16 h coupling).
L-Phe-OH,
using the on-resin esterification conditions. The procedure was repeated
2-3 times until all starting material had converted into product as judged by
UPLC-MS monitoring.
L
L
The resin-bound peptide was cleaved using the resin-cleavage conditions.
The crude protected linear peptide was purified by preparative RP-HPLC
using a Sunfire OBD 5 μm 19 x 150 mm (C18) column using a gradient of
50 – 60% MeCN in H2O (0.1% TFA) over 40 min at a flow rate of 7 mL
min-1. The peptide was lyophilized to yield the protected linear peptide as
a trifluoroacetate salt (6.0 mg, 7% over 26 steps from resin loading,
average of 90% per step).
The resin-bound peptide Fmoc-deprotected and then dimethylated using
the on-resin dimethylation conditions. The procedure was repeated 1-2
times until all starting material had converted into product as judged by
UPLC-MS monitoring.
The resin-bound peptide was Alloc-deprotected before 48 μmol of resin
was elongated using HATU coupling conditions and Fmoc-deprotection
conditions incorporating the following amino acids in turn: Fmoc-threo-β-
The protected linear peptide (6.0 mg, 3.20 μmol) was cyclized according
to the general cyclization conditions described above. The crude cyclic
peptide was purified by preparative RP-HPLC using a Waters X-Bridge 5
μm 19 x 150 mm (C18) column using a gradient of 70 – 100% MeOH in
H2O (0.1% TFA) over 40 min at a flow rate of 7 mL min-1. The peptide was
lyophilized to give a white fluffy powder of pure deoxyecumicin (2) as a
trifluoroacetate salt (2.2 mg, 3% over 28 steps from resin loading, 41% for
the cyclization and deprotection).
OH-
L
-Phe-OH (1.2 eq. of amino acid used, 16 h coupling), Fmoc-
L
-Val-OH
and Fmoc-N-Me-4-OMe-
coupling).
L-Trp-OH (1.2 eq. of amino acid used, 16 h
The resin-bound peptide was cleaved using the resin-cleavage conditions.
The crude protected linear peptide was purified by preparative RP-HPLC
using a Sunfire OBD 5 μm 19 x 150 mm (C18) column using a gradient of
50 – 60% MeCN in H2O (0.1% TFA) over 30 min at a flow rate of 7 mL
min-1. The peptide was lyophilized to yield the protected linear peptide as
a trifluoroacetate salt (9.4 mg, 9% over 26 steps from resin loading,
average of 91% per step).
HR-MS: (+ESI) Calc. for C83H134N14O16: 803.5034 [M+2Na]2+, Found:
803.5028 [M+2Na]2+; IR (ATR): max = 3383, 2967, 2929, 1679, 1541, 1510,
1442, 1206, 1139 cm-1; 1H NMR (500 MHz, MeOD): 0.21 (d, 6.6 Hz, 1.5H),
0.32 (d, 6.6 Hz, 1.5H), 0.72 (app. t, 7.4 Hz, 3H), 0.76 (d, 6.9 Hz, 3H), 0.87
(d, 7.2 Hz, 1.5H), 0.88 (dd, 6.7 Hz, 3H), 0.91 (d, 6.8 Hz, 1.5H), 0.92 (d, 6.9
Hz, 1.5H), 0.93 (d, 7.3 Hz, 1.5H), 0.98 (m, 3H), 0.98 (m, 2.5H), 1.00 (m,
2.5H),1.02 (d, 6.6 Hz, 1.5H), 1.06 (d, 6.5 Hz, 1.5H), 1.09 (d, 6.7 Hz, 4.5H),
1.27 (m, 1H), 1.29 (m, 1H), 1.31 (d, 6.6 Hz, 3H), 1.43 (m, 1H), 1.96 (m,
2H), 2.04 (m, 1H), 2.15 (m, 1H), 2.16 (s, 3H), 2.18 (m, 1H), 2.37 (m, 1H),
2.40 (m, 1H), 2.59 (m, 1H), 2.92 (s, 3H), 2.84 (app dd, 10.7, -14.2 Hz, 1H),
3.06 (d, 7.7 Hz, 1H), 3.15 (s, 3H), 3.23 (s, 3H), 3.26 (s, 3H), 3.32 (s, 3H),
3.37 (app. dd, 12.4, -14.4 Hz, 1H), 3.54 (app. dd, 10.9, -13.5 Hz, 1H), 3.68
(app. 11.1, -15.8 Hz, 1H), 3.71 (d, 5.7 Hz, 1H), 3.81 (s, 3H), 4.09 (dd, 4.4,
10.9 Hz, 1H), 4.36 (app. t, 9.4 Hz, 1H), 4.40 (app. t, 9.1 Hz, 1H), 4.44 (dd,
3.8, 6.3 Hz, 1H), 4.59 (app. t., 9.1 Hz, 1H), 4.72 (d, 8.8 Hz, 3H), 4.79 (m,
1H), 4.83 (m, 1H), 4.95 (d, 10.9 Hz, 1H), 5.03 (d, 3.6 Hz, 1H), 5.13 (dd,
2.1, 8.8 Hz, 1H), 5.16 (d, 7.6 Hz, 1H), 5.79 (m, 1H), 6.44 (d, 8.1 Hz, 1H),
6.70 (s, 1H), 6.92 (d, 8.1 Hz, 1H), 6.98 (app. t, 8.1 Hz, 1H), 7.09 (d, 7.2
Hz, 2H), 7.16 (d, 7.2 Hz, 1H), 7.20 (m, 2H), 7.70 (d, 8.7 Hz, 1H), 7.80 (d,
9.9 Hz, 1H), 8.53 (d, 9.1 Hz, 1H), 8.66 (d, 7.4 Hz, 1H), 9.02 (d, 9.6 Hz, 1H),
9.13 (d, 9.6 Hz, 1H); 13C NMR (125MHz, MeOD, shifts were extracted from
HSQC and HMBC data): 11.3, 15.2, 16.4, 16.6, 18.9, 18.9, 19.2, 19.2,
19.2, 19.2, 19.3, 19.5, 19.5, 19.5, 19.5, 19.5, 19.8, 21.6, 21.8, 22.2, 23.2,
25.3, 26.2, 26.7, 28.4, 29.6, 31.3, 31.3, 31.6, 31.6, 32.7, 32.8, 33.4, 34.1,
34.0, 37.3, 39.2, 40.3, 40.7, 42.3, 53.3, 55.2, 55.3, 55.5, 56.2, 56.6, 57.1,
59.0, 59.0, 61.5, 62.7, 66.6, 69.3, 70.8, 71.3, 73.7, 99.6, 105.8, 112.2,
118.1, 123.0, 123.6, 127.2, 129.3, 129.9, 139.8, 139.8, 155.0, 166.5, 170.7,
The protected linear peptide (9.3 mg, 4.88 μmol) was cyclized according
to the general cyclization conditions described above. The crude cyclic
peptide was purified by preparative RP-HPLC using a Sunfire OBD 5 μm
19 x 150 mm (C18) column using a gradient of 50 – 60% MeCN in H2O
(0.1% TFA) over 20 min at a flow rate of 7 mL min-1. The peptide was
lyophilized to give a white fluffy powder of pure ecumicin (3) as a
trifluoroacetate salt (2.3 mg, 2% over 28 steps from resin loading, 27% for
the cyclization and deprotection).
HR-MS: (+ESI) Calc. for C83H134N14O17: 1600.0124 [M+H]+, Found:
1600.0124 [M+H]+; IR (ATR): max = 3477, 3311, 2962, 2926, 1675, 1632,
1532, 1516, 1467, 1204, 1136, 822 cm-1; UV: (7.3 μM, MeOH, 1 cm quartz
cuvette, 25 °C, background subtracted): ε = 56420 (219 nm), 7339 (263
nm), 6433 (281 nm), 5540 (291 nm) M-1cm-1; 1H NMR (500 MHz, MeOD):
0.17 (d, 6.5 Hz, 1.5H), 0.33 (d, 6.5 Hz, 1.5H), 0.74 (app. t, 7.3 Hz, 3H),
0.76 (d, 6.7 Hz, 3H), 0.86 (d, 6.7 Hz, 1.5H), 0.91 (d, 6.5 Hz, 1.5H), 0.92 (d,
6.7 Hz, 4.5H), 0.93 (d, 6.9 Hz, 1.5H), 0.95 (m, 2.5H), 0.97 (d, 6.4 Hz, 1.5H),
0.98 (d, 6.9 Hz, 1.5H), 0.99 (m, 2.5H), 1.01 (d, 6.9 Hz, 1.5H), 1.06 (d, 6.7
Hz, 1.5H), 1.09 (d, 6.9 Hz, 1.5H), 1.03 (d, 6.8 Hz, 1.5H), 1.08 (d, 6.9 Hz,
1.5H), 1.09 (d, 6.6 Hz, 1.5H), 1.22 (m, 1H), 1.25 (m, 1H), 1.32 (d, 6.5 Hz,
3H), 1.45 (m, 1H), 1.99 (m, 1H), 1.99 (m, 2H), 2.15 (m, 1H), 2.15 (s, 3H),
2.18 (m, 1H), 2.33 (m, 1H), 2.34 (m, 1H), 2.58 (m, 1H), 2.81 (s, 3H), 3.06
(d, 7.6 Hz, 1H), 3.14 (s, 3H), 3.23 (s, 3H), 3.25 (s, 3H), 3.33 (s, 3H), 3.54
8
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10.1002/chem.202002408
Chemistry - A European Journal
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1H), 3.82 (s, 3H), 4.10 (dd, 4.7, 11.2 Hz, 1H), 4.40 (dd, 2.6, 8.9 Hz, 1H),
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(d, 1.2 Hz, 1H), 5.78 (dd, 1.9, 6.6 Hz, 1H), 6.44 (d, 7.7 Hz, 1H), 6.70 (s,
1H), 6.92 (d, 8.4 Hz, 1H), 6.98 (app. t, 8.0 Hz, 1H), 7.20 (m, 1H), 7.24 (m,
2H), 7.26 (m, 2H), 7.72 (d, 8.6 Hz, 1H), 7.85 (d, 9.7 Hz, 1H), 8.21 (d, 7.5
Hz, 1H), 9.02 (d, 9.5 Hz, 2H); 13C NMR (125 MHz, MeOD, shifts were
extracted from HSQC and HMBC data): 11.4, 15.1, 17.0, 17.0, 18.6, 18.9,
19.2, 19.2, 19.2, 19.2, 19.6, 19.6, 19.6, 19.6, 19.6, 19.6, 19.6, 19.6, 19.6,
21.9, 20.1, 20.1, 20.1, 20.1, 20.8, 21.9, 22.2, 23.5, 25.5, 26.5, 26.5, 28.6,
30.0, 31.4, 31.4, 31.8, 31.8, 33.0, 33.6, 33.6, 34.2, 34.2, 39.3, 40.3, 40.8,
41.1, 42.2, 53.3, 54.7, 54.9, 55.5, 55.5, 55.5, 56.3, 56.8, 59.1, 59.1, 59.5,
61.7, 62.9, 66.8, 68.3, 69.8, 71.2, 71.2, 72.8, 73.9, 99.8, 105.9, 112.4,
118.3, 123.2, 123.8, 124.2, 127.0, 128.3, 129.3, 139.9, 142.5, 155.2, 167.8,
169.9, 170.9, 170.9, 171.9, 171.9, 171.9, 173.4, 173.4, 173.4, 174.3,
174.3, 174.7, 174.7. 1H NMR and 13C NMR data are compared to the
isolated natural product in Table S3. These data are in agreement with that
previously reported by Gao et al.[8a]
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Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
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
Synthesis of Fmoc-threo-β-hydroxy-
L-phenylalanine and
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L
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Acknowledgements
We would like to thank the National Health and Medical Research
Council for funding (Project Grant APP1141142 to R. J. P. and W.
J. B. and Investigator Grant APP1174941 to R. J. P.), Dr. Ian Luck
(The University of Sydney) for technical support with NMR
spectroscopy, and Dr. Nick Proschogo (The University of Sydney)
for technical support with mass spectrometry The authors also
acknowledge the Research Training Program (to P. H., W. T., and
A. M. G.) and the John A. Lamberton Research Scholarship for
PhD funding (to P. H. and A. M. G.).
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Keywords: natural products • peptides • solid-phase synthesis •
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9
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Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
O
O
H
N
R1
R
=
HN
Macrolactamization
N
R2
H
O
O
H
N
H
N
Ohmyungsamycin A
2 = OH,
N
O
N
H
On-resin esterification
O
O
O
O
O
O
O
O
O
O
O
N
N
N
R1
=
N
N
Fmoc
R1
H
NH
O
N
O
H
N
N
On-resin
dimethylation
N
H
Deoxyecumicin
R2 = H,
O
Ecumicin
R2 = OH,
OH
A unified strategy towards antimycobacterial natural products: The total synthesis of the cyclic depsipeptide natural products
ohmyungsamycin A, deoxyecumicin, and ecumicin are described. Synthesis was achieved through a unified solid-phase synthetic
strategy and a late-stage, pre-organized macrolactamization at an unusually hindered junction. Access to these natural products
enabled a detailed comparison on their antimycobacterial activity.
Institute and/or researcher Twitter usernames: @PayneResearch
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