Synthesis of 9-Dechlorochrysophaentin A Enables Studies Revealing Bacterial Cell Wall Biosynthesis Inhibition Phenotype in B. subtilis

Article abstract of DOI:10.1021/jacs.0c04917

Chrysophaentin A is an antimicrobial natural product isolated from the marine alga C. taylori in milligram quantity. Structurally, chrysophaentin A features a macrocyclic biaryl ether core incorporating two trisubstituted chloroalkenes at its periphery. A concise synthesis of iso-and 9-dechlorochrysophaentin A enabled by a Z-selective ring-closing metathesis (RCM) cyclization followed by an oxygen to carbon ring contraction is described. Fluorescent microscopy studies revealed 9-dechlorochrysophaentins leads to inhibition of bacterial cell wall biosynthesis by disassembly of key divisome proteins, the cornerstone to bacterial cell wall biosynthesis and division.

Full text of DOI:10.1021/jacs.0c04917

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Communication
Synthesis of 9-Dechlorochrysophaentin A Enables Studies Revealing
Bacterial Cell Wall Biosynthesis Inhibition Phenotype in B. subtilis
Christopher Fullenkamp, Yen-Pang Hsu, Ellen Quardokus, Gengxiang Zhao, Carole A. Bewley, Michael S. VanNieuwenhze, and Gary A. Sulikowski
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c04917 • Publication Date (Web): 31 Aug 2020
Downloaded from pubs.acs.org on August 31, 2020
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9-dechorochyrso No treatment
Total Synthesis
OH
OP
OH
Y
PO
X
HO
iso-dechlorochryso
Cl
C
B
B
O
C
Cl
O
O
9
Z
9
X
9-dechlorochryso
HO
PO
D
PO
A
Cl
D
O
OH
O
A
HO
OP
Cl
16 steps longest linear sequence
29 total steps
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9
Synthesis of 9-Dechlorochrysophaentin A Enables Studies Revealing
Bacterial Cell Wall Biosynthesis Inhibition Phenotype in B. subtilis
Christopher R. Fullenkamp1, Yen-Pang Hsu,4,5 Ellen M. Quardokus,4 Gengxiang Zhao,6 Carole A.
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Bewley6, Michael VanNieuwenhze4,5* & Gary A. Sulikowski1-3*
1Department of Chemistry, Vanderbilt University, Nashville, TN, USA. 2Department of Pharmacology, Vanderbilt
University, Nashville, TN, USA. 3Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN,
USA. 4Department of Chemistry, Indiana University, Bloomington, IN, USA. 5Department of Molecular and Cellular
Biochemistry, IN, USA. 6Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Maryland, USA
ABSTRACT: Chrysophaentin A is an antimicrobial natural product isolated from the marine alga C. taylori in milligram
quantity. Structurally, chrysophaentin A features a macrocyclic biaryl ether core incorporating two trisubstituted
chloroalkenes at its periphery. A concise synthesis of iso- and 9-dechlorochrysophaentin A enabled by a Z-selective RCM
cyclization followed by an oxygen to carbon ring contraction is described. Fluorescent microscopy studies revealed 9-
dechlorochrysophaentins leads to inhibition of bacterial cell wall biosynthesis by disassembly of key divisome proteins,
the cornerstone to bacterial cell wall biosynthesis and division.
Microbial secondary metabolites (natural products) have a chrysophaentins A-H (Figure 1).3 Isolated from the marine
rich history in the discovery and development of antibiotics alga Chrysophaeum taylori, the structures of
for the clinical treatment of infections.1 Not only have these chrysophaentins A-D and F-H were determined to consist
antimicrobial natural products revealed new antibiotic of common halogenated macrocyclic biaryl ethers, while
leads, but they have also identified new antimicrobial chrysophaentin E was assigned an open-chain structure.
targets and provided insight into fundamental Chrysophaentins A-D and F-H are presumably derived
microbiology by study of their mechanism of action. For from chrysophaentin E by oxidative cyclization of the C
example, our understanding of bacterial cell wall ring phenol at B ring carbons C16 and C14, respectively
biosynthesis has benefited from the discovery and study of (Figure 1). The most prevalent metabolite, chrysophaentin
beta-lactam and glycopeptide natural products.2 From the A, displayed antimicrobial activity against S. aureus,
perspective of human health, the emergence of antibiotic methicillin-resistant S. aureus (MRSA), E. faecium, and
resistance remains problematic, and further enforces the vancomycin-resistant E. faecium (VREF). Based on its
need for the continued identification and study of structural similarity to zantrin Z1, a reported inhibitor of
antimicrobial natural products.1b
the bacterial cytoskeletal protein FtsZ4, the mode of action
of chrysophaentin A was hypothesized to operate by the
same mechanism.5 Indeed, chrysophaentin A was shown to
inhibit S. aureus and E. coli FtsZ GTPase activity and
polymerization.3,6 Further study of chrysophaentin A was
hindered due to unreliable supply from the producing
marine alga (C. taylori) as well as failed attempts to
produce chrysophaentins by in lab culturing of C. taylori.7
To date, synthetic studies have yielded only chrysophaentin
fragments devoid of the macrocyclic core.8,9 In one
exception, Harrowven and co-workers10 recently described
an approach to chrysophaentin F that produced an
inseparable mixture of isomeric macrocyclic products.
Figure 1. Bio-oxidative cyclization of chrysophaentin E and
structure of chrysophaentin A.
From a retrosynthetic perspective, the macrocyclic biaryl
ether core structure of chrysophaentin A presented an
interesting synthetic challenge as it offers no obvious bond
disconnection (Figure 2). Closure of the macrocyclic biaryl
In 2010 Bewley and co-workers reported on the isolation,
structure elucidation, and antimicrobial activity of
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ether by way of a ring-closing metathesis (RCM) reaction nitro group was reduced to the corresponding aniline and
was anticipated to be difficult as chloro alkenes remain chlorinated under Sandmeyer14 reaction conditions to
problematic substrates in RCM reactions.11 However, a total complete the Northern BC biaryl ether fragment (4).
1
2
3
4
5
6
7
8
synthesis
momentarily avoid the chloro alkene RCM and reduce the
problem to
Z-selective RCM closure (Figure 2).12
of
9-dechlorochrysophaentin
A
would
The synthesis of the Southern AD biaryl ether (10), its
merger with biaryl 4, and the completion of 9-
dechlorochrysophaentins are shown in Scheme 2. First,
phenol 5, available in two steps from 4-bromo-3,5-
a
Following RCM mediated ring closure, a Lewis acid
mediated O to C alkyl migration13 would likely bifurcate
between C5’ and C3’ leading to iso- and 9-
dechlorochrysophaentin A, respectively (Figure 2). Herein,
we describe the total synthesis of iso- and 9-
dechlorochrysophaentin A, their antimicrobial activity, and
preliminary mechanism of action studies revealing a
potentially novel mode of inhibition of bacterial cell wall
biosynthesis.
resorcylic
acid,
was
coupled
with
3-chloro-4-
fluorobenzaldehyde by way of a second SNAr reaction.
Baeyer-Villiger oxidation of the A ring aldehyde of the
coupled product afforded momentarily an aryl formate that
upon treatment with acidic methanol provided phenol 6 in
45-50%. Bromination of phenol 6 (NBS, p-TSA) occurred
selectively at the C5’ carbon and following phenol
protection (i-PrBr, K2CO3), the derived aryl bromide was
coupled with allyl pinacol boronate ester to give 7.15
Attention was then turned to D ring functionalization
starting with reduction of the ester group with DIBAL,
followed by chlorination (TPP•Cl2) to give an intermediate
benzyl chloride that was homologated to alkyne 8 in two-
steps.9 Alkyne 8 was advanced to trisubstituted allyl alcohol
10 starting with homologation to the corresponding
alkynoate. Alkynoates derived by direct acylation of alkyne
8 with chloroformates
9
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Scheme 1. Synthesis of BC biaryl ether 4.
Figure 2. Retrosynthetic analysis of 9-dechlorochrysophaentins.
Our synthesis started with O-alkylation of resorcinol 1
(Scheme 1) followed by a Dakin oxidation of the remaining
aromatic aldehyde to afford the corresponding phenol. The
latter was alkylated with allyl bromide and the resulting
allyl aryl ether was subjected to a Claisen rearrangement to
afford phenol 2.8 A solution of phenol 2 and 5-bromo-2-
fluoronitrobenzene were subjected to nucleophilic-
aromatic substitution reaction conditions to yield biaryl
ether 3 in 93%. The C-ring aryl bromide 3 was fashioned to
corresponding phenol by way of cross-coupling with
(BPin)2 followed by an oxidative workup. Finally, the C-ring
Scheme 2. Synthesis of 9-dechlorochrysophaentins.
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tended to undergo isomerization to the corresponding
proved useful in preliminary antimicrobial and
phenotypic studies.
allenoate. The undesired isomerization was avoided by
Fisher esterification of the corresponding alkynoic acid,
the latter derived from alkyne 8 by deprotonation
followed by carbon dioxide quench. Stannyl cupration16
of the derived methyl alkynoate proceeded in 60-70%
yield to afford enoate 9 as a single geometric isomer.
Reduction of enoate 9 to the corresponding allylic
alcohol followed by tin-chloride exchange17 completed
assembly of biaryl ether 10. Coupling of phenol 4 and
allyl alcohol 10 under Mitsunobu conditions, resulted in
smooth merger of the BC and AD fragments. The
coupled product underwent cyclization to macrocycle 11
on treatment with Grubbs Z-selective catalyst (C633)18 in
yields of 65 to 70%. In contrast, ring closure using
second-generation Grubbs catalyst resulted in an equal
mixture of Z- and E-isomeric products. Heating a
dichloroethane solution of aryl ether 11 and BF3•OEt2
afforded a near 1:1 inseparable mixture of isomeric
products from non-selective O to C3’/C5’ alkyl migration.
Removal of the isopropyl protecting groups proceeded
smoothly on treatment with boron trichloride to yield 9-
Table 1. Antimicrobial activities of chrysophaentinsa.
aBacterial strains were obtained from the ATCC and have
the following identifiers: pathogenic strains: S. aureus 25913;
MRSA, methicillin-resistant S. aureus 43300; Enterococcus
faecalis 29212; VRE, vancomycin-resistant-E. faecalis 51299;
non-pathogenic strain: B. subtilis; Each assay was performed
in triplicate.
Aside from a slight loss of potency against VRE, the
bacterial growth inhibition of 9-
dechlorochrysophaentins and congeners compared
favorably to chrysophaentin A when evaluated across a
panel of Gram-positive bacteria (Table 1, Figure S3 and
dechlorochrysophaentin
dechlorochrysophaentin
A
and
iso-9-
A
following HPLC assisted
separation. The isomeric 9-dechlorochrysophaentins
were assigned compound registry numbers VU0849855
and VU0849838, respectively. In the course of our
synthetic studies, we accessed two additional
chrysophaentin congeners, VU0848355 and VU0848354
that incorporate D ring methyl ethers (Scheme 2) and
S4).
dechlorochrysophaentin A and VU0848354), possessing
an unnatural ring substitution pattern, showed
approximately two-fold greater potency relative to their
Unexpectedly,
iso-chrysophaentins
(iso-9-
C
naturally substituted
dechlorochrysophaentin
C
ring counterparts (9-
and VU0848355).
A
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Figure 3. Bacillus subtilis phenotype in response to antimicrobials. A. Structure and activity of the FtsZ inhibitor PC190723. B.
Cell lengthening in response to PC190723 and chrysophaentin congeners VU0848354 and VU0848355. C. Quantification of FDAA
incorporation is inhibited in cells treated withVU0849383 and VU0849855. D. B. subtilis cells treated with VU0849838 or
VU0849855 leads to the dispersion of FtsZ.
As mentioned above, chrysophaentin
A
has been
the rate of cell division. This dynamic process has been
visualized in real-time using high-resolution microscopy
with orthogonally labeled fluorescent protein fusions and
fluorescent D-amino acids (FDAAs).25
proposed to act as an inhibitor of the cytoskeletal protein
FtsZ. As a central player in cell division, inhibition of
FtsZ has been considered an untapped target in the
development of novel antimicrobial agents.5,19 The
synthesis described here allows us to investigate in detail
the mechanism by which the chrysophantins perturb
FtsZ.
Chrysophaentin A was shown to inhibit FtsZ mediated
polymerization in vitro by microscopy and was shown to
bind recombinant FtsZ at the GTP binding site by
Saturation Transfer Difference (STD) NMR.3 We
compared chrysophaentin congeners VU0848354 and
VU0848355 to the well-studied FtsZ inhibitor PC1907723
(Figure 3A).26 Inhibition of FtsZ by PC1907723 is known
to produce an elongated phenotype of rod-like bacteria
such as B. subtilis. We observed cell lengthening when B.
subtilis was treated with PC190723 at a concentration of 5
FtsZ is a GTP-dependent protein shown to play a critical
role in bacterial cell division.19,20 Only recently has the
central role of FtsZ and other proteins in the
orchestration of cell division been fully appreciated.21 It is
known that in preparation for cell division, FtsZ, along
with FtsA, forms filaments at the new division site that
recruit the peptidoglycan-synthesizing enzymes required
for construction of the septal disk.22 High-resolution
microscopy, along with single-molecule tracking and
real-time, temporal visualization of bacterial cell wall
synthesis has revealed FtsZ’s dynamic role in guiding the
multi-protein complex called the divisome.23 Super-
resolution techniques have revealed FtsZ filaments
display a circumferential, inward movement correlates
with the progressive insertion of new cell wall material.24
FtsZ “treadmilling”, powered by its own GTPase activity,
controls both the rate of new cell wall synthesis as well as
g/mL (10
x
MIC) (Figure 3B). In contrast,
concentrations of VU0848354 and VU0848355 up to 20
M (10 x MIC) did not lead to cell lengthening. Next, we
examined
the
effect
of
PC190723
and
9-
dechlorochrysophaentins on cell wall biosynthesis using
FDAAs. Much to our surprise 9-dechloro- and iso-9-
dechlorochrysophaentin
(a.k.a.
VU0849855
and
VU0849838) (Figure 3C and S1C) and congeners
VU0848354 and VU0848355 (Figure S1A, B) inhibited
incorporation of FDAAs at 10 M (5 x MIC), comparable
to the well-known peptidoglycan inhibitor ampicillin (10
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x MIC) (Figure S1A, B). In contrast FtsZ inhibitor
chrysophaentin A.3 This might explain the inability of
1
2
3
4
5
6
7
8
PC190723 showed minimal inhibition of FDAA
incorporation up to 10 times it MIC as reported earlier
(Figure S1A, B).26 We next examined, the effect of 9-
dechlorochrysophaentins on the localization of FtsZ
using fluorescently labeled FtsZ protein fusion
(mNeonGreen). Treatment with 30 M (5 x MIC)
VU0849855 or 10 M (5 x MIC) VU0849838 lead to
complete delocalization of FtsZ as observed by high-
resolution microscopy (Figure 3D; and SI Movies S1
untreated, S2 VU0849855, and S3 VU0849838). In
parallel microscopy experiments, we observed the
delocalization of mNeonGreen fluorescent protein
fusions of FtsZ, FtsA, and PBP2B from the division plane
upon treatment with natural (isolated from C. taylori)
chrysophaentin A (Figure S2). In contrast to the 9-
cells in early growth stages to localize FtsZ to the new
division site. The inability to reacquire GTP after its
hydrolysis may also explain the dispersion of FtsZ from
the division site after chrysophaentin treatment.
Regardless, B. subtilis cells treated with the structurally
interesting chrysophaentins produce novel phenotype
that is distinct from the well-studied FtsZ inhibitor
PC190723. This family of compounds may reveal a novel
target(s) for antibacterial intervention or serve as
valuable tools to further elucidate key elements of
bacterial cell division. Toward this end, additional
experiments designed to further elucidate the unique
mechanism(s) of action of these novel agents are being
designed and results from these studies will be reported
in due course.
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dechlorochrysophaentins
and
chrysophaentin
A,
PC190723 leads not to the dispersion of FtsZ but
dislocation away from the cell midline, as reported
earlier,26 and in agreement with continued cell wall
synthesis as observed using FDAA labeling (Figure S1B).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
In conclusion, we report a concise and cost-effective
chemical synthesis of iso- and 9-dechlorochrysophaentin
A (a.k.a. VU0849855 and VU0849838) and confirmed
their antimicrobial activity as equal or greater to
Experimental details, additional biological data and
spectral data (PDF)
chrysophaentin
A itself. In experiments aimed at
AUTHOR INFORMATION
defining the mechanism of their antimicrobial activity,
we discovered a novel mode of inhibition of bacterial cell
wall biosynthesis. Our data suggest that the treatment of
B. subtilis cells with chrysophaentin A and congeners
results in the disassembly of the Z-ring and dispersion of
FtsZ from the division plane in cells preparing for
Corresponding Authors
Gary A. Sulikowski – Department of Chemistry, Vanderbilt
University, 12415 Medical Research Building IV, 2213
Garland Avenue, Nashville, TN 37232-0412, United States;
orcid.org/0000-0002-1067-0767; Email:
division.
Interestingly,
cells
treated
with
9-
gary.a.sulikowski@vanderbilt.edu
dechlorochyrsophaentins when in their early growth
stages do not appear to be able to localize FtsZ at the
new division site. Furthermore, diminished FDAA
incorporation in treated cells suggests that new cell wall
synthesis and/or cell wall modification are being
suppressed.
Michael S. VanNieuwenhze—Department of Chemistry,
Indiana University, Simon Hall MSB1, 212 S. Hawthorne Dr.,
Bloomington, IN 47405-7003; orcidid.org/0000-0001-6093-
5949; Email: mvannieu@indiana.edu
Ellen M. Quardokus – orcidid.org/0000-0001-7655-4833
When compared to data obtained with PC190723, our
data are suggestive of a unique mechanism of action. For
example, B. subtilis cells treated with PC190723 show
some dislocation of FtsZ from the division plane whereas
those treated with chrysophaentin A and congeners show
complete dispersion of FtsZ form the division site.
Moreover, PC190723-treated cells are still able to
incorporate new cell wall material whereas those treated
with the chrysophaentins show no evidence for
incorporation of new cell wall material. Interestingly, as
noted above, cells treated with the chrysophaentins in
early growth stages appear unable to localize FtsZ at the
new division site. It is tempting to speculate that the
chrysophaentins may inhibit the ability of FtsZ to bind
GTP, which is required for proper FtsZ polymerization
for treadmilling and consistent with the GTP-
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Funding
C. R. F. acknowledges the support of the Vanderbilt
Chemistry Biology Interface (V-CBI) training program (T32
GM065086). M. S. V. gratefully acknowledges support from
the National Institutes of Health (GM113172 and GM136365),
and C. A. B. is supported by the Intramural Research
Program of the National Institutes of Health (NIDDK).
Notes
The authors declare no competing financial interests.
competitive
binding
originally
reported
for
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Activity of the Chrysophaentins and their Synthetic Fragments.
Mar. Drugs 2012, 10, 1103-1125.
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
We gratefully acknowledge Professor Robert Grubbs
(Caltech) for supplying catalyst C633 and Professor Jared
Shaw (UC-Davis) for helpful discussion.
10. Vendeville, J. B.; Matters, R. F.; Chen, A. Q.; Light, M. E.;
Tizzard, G. J.; Chai, C. L. L.; Harrowven, D. C., A Synthetic
Approach to Chrysophaentin F. Chem. Commun. 2019, 55,
4837-4840.
11. (a) Sashuk, V.; Samojlowicz, C.; Szadkowska, A.; Grela, K.,
Olefin Cross-metathesis with Vinyl Halides. Chem. Commun.
2008, 2468-2470. (b) Chao, W. C.; Weinreb, S. M., The First
Examples of Ring-closing Olefin Metathesis of Vinyl Chlorides.
Org. Lett. 2003, 5, 2505-2507.
12. Herbert, M. B.; Grubbs, R. H., Z-Selective Cross Metathesis
with Ruthenium Catalysts: Synthetic Applications and
Mechanistic Implications. Angew. Chemie., Int. Ed. 2015, 54,
5018-5024.
13. (a) Lawson, K. V.; Rose, T. E.; Harran, P. G., Template-
constrained Macrocyclic Peptides Prepared from Native,
Unprotected Precursors. Proc. Natl. Acad. Sci. U.S.A. 2013, 110,
E3753-E3760. (b) Lawson, K. V.; Rose, T. E.; Harran, P. G.,
Template-induced Macrocycle Diversity through Large Ring-
forming Alkylations of Tryptophan. Tetrahedron 2013, 69,
7683-7691. (c) Rose, T. E.; Lawson, K. V.; Harran, P. G., Large
Ring-forming Alkylations provide Facile access to Composite
Macrocycles. Chem. Sci. 2015, 6, 2219-2223.
ABBREVIATIONS
RCM, ring-closing metathesis;
temperature-sensitive mutant
Bis(pinacolato)diboron; NBS, N-bromosuccinimide; p-TSA,
p-Toluenesulfonic acid; iPr-Br, isopropyl bromide; DIABL,
diisobutylaluminium hydride; TPP·Cl2, triphenylphosphine
dichloride; STD NMR, saturation transfer difference
nuclear magnetic resonance; MRSA, methicillin-resistant
FtsZ, Filamenting
Z; (BPin)2,
9
10
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12
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Staphylococcus
aureus;
VRE,
vancomycin-resistant
Enterococcus faecalis; MIC, minimum inhibitory
concentration; FDAAs, fluorescently labeled D-amino
acids.
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