Diverted Total Synthesis of Carolacton-Inspired Analogs Yields Three Distinct Phenotypes in Streptococcus mutans Biofilms

Article abstract of DOI:10.1021/jacs.7b03879

The oral microbiome is a dynamic environment inhabited by both commensals and pathogens. Among these is Streptococcus mutans, the causative agent of dental caries, the most prevalent childhood disease. Carolacton has remarkably specific activity against S. mutans, causing acid-mediated cell death during biofilm formation; however, its complex structure limits its utility. Herein, we report the diverted total synthesis and biological evaluation of a rationally designed library of simplified analogs that unveiled three unique biofilm phenotypes further validating the role of natural product synthesis in the discovery of new biological phenomena.

Full text of DOI:10.1021/jacs.7b03879

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Communication
The Diverted Total Synthesis of Carolacton-Inspired Analogs Yields
Three Distinct Phenotypes in Streptococcus mutans Biofilms
Amy E. Solinski, Alexander Koval, Richard Brzozowski, Kelly Morrison, Americo J. Fraboni, Carrie Carson,
Anisa Eshraghi, Guangfeng Zhou, Robert Quivey, Vincent A. Voelz, Bettina A. Buttaro, and William M. Wuest
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 May 2017
Downloaded from http://pubs.acs.org on May 13, 2017
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Journal of the American Chemical Society
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The Diverted Total Synthesis of Carolacton-Inspired Analogs Yields
Three Distinct Phenotypes in Streptococcus mutans Biofilms
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Amy E. Solinksi1‡, Alexander B. Koval1‡, Richard S. Brzozowski1‡, Kelly R. Morrison1, Americo J.
Fraboni1, Carrie E. Carson1, Anisa, R. Eshraghi1, Guangfeng Zhou1, Robert G. Quivey, Jr.2, Vincent A.
Voelz1, Bettina A. Buttaro3, William M. Wuest1*
1Department of Chemistry, Temple University, Philadelphia PA 19122; 2Center for Oral Biology, University of Rochester School of Mediꢀ
cine and Dentistry, Rochester NY 14642; 3Department of Microbiology and Immunology, Lewis Katz School of Medicine, Temple Uniꢀ
versity, Philadelphia PA 19140
Supporting Information Placeholder
single most common childhood chronic disease.7 Reꢀ
ABSTRACT: The oral microbiome is a dynamic enviꢀ
cently, WagnerꢀDöbler showed that dental composites
impregnated with carolacton could inhibit S. mutans
biofilm growth8 demonstrating a future role for the natuꢀ
ral product in the development of preventative materials.
ronment inhabited by both commensals and pathogens.
Among these is Streptococcus mutans, the causative
agent of dental caries, the most prevalent childhood disꢀ
ease. Carolacton has remarkably specific activity against
S. mutans, causing acidꢀmediated cell death during bioꢀ
film formation; however, its complex structure limits its
utility. Herein, we report the diverted total synthesis and
biological evaluation of a rationally designed library of
simplified analogs that unveiled three unique biofilm
phenotypes further validating the role of natural product
synthesis in the discovery of new biological phenomena.
This exquisite bioactivity of carolacton has drawn
the interest of the synthetic community as a number of
groups have targeted the natural product.9 Kirschning in
particular has been quite prolific as they have disclosed
both the first total synthesis10 and uncovered useful inꢀ
formation regarding the structureꢀactivity relationship
(SAR) by analog synthesis (Fig. 1).11 These studies eluꢀ
cidated that both drastic (increased ring size) and even
small conformational changes to the twelveꢀmembered
Natural products have served as starting points for both
the discovery of new drugs and the identification of bioꢀ
logical targets for decades.1 An exemplary case of the
former is penicillin, initially discovered in 1928, which
has since been reprogrammed into four new classes of
derivatives all in an effort to overcome resistance.2 Reꢀ
cently, natural products have been postulated to serve as
excellent starting points for narrowꢀspectrum therapeuꢀ
tics and also as chemical probes to help deconvolute
complex microbiomes and aid in their study.3 The oral
microbiome is home to around 1000 species and has
been identified as one of the most diverse biotic habitats
in the human body.4 Therefore, the identification of
small molecules that can specifically target a small subꢀ
set of pathogenic species would further our understandꢀ
ing of their role in the microbiota. Carolacton is a natuꢀ
ral product that displays remarkable bioactivity against
the pathogen Streptococcus mutans whereby it does not
affect the viability of planktonic bacteria but instead
causes acidꢀmediated cell death during biofilm forꢀ
mation at low nanomolar concentration (Fig. 1).5 S. mu-
tans plays a significant role in human health as it is the
causative agent in the formation of dental caries6 – the
carolacton
Planktonic Cells
Adhered Cells
Microcolonies
Biofilm
Kirschning Analog Library
Modifications of Macrocycle
OH
Inactive!
OH
OH
O
O
OH
O
OH
O
O
O
O
OMe O
O
O
OMe O
O
OMe O
Ot-Bu
OH
OH
"Pro-Drug" Ester
Inactive!
"Pro-Drug" Ketone Esters
OH
Active!
OH
O
OH
OH
O
O
OMe
OH
O
O
O
O
O
OMe O
O
O
O
O
OMe
OMe
Essential for activity: "CO2H", Ketone, Macrocycle conformation
Wuest Analog Design
DTS Library to
O
OH
OH
Rigidification
Probe Effects of:
O
Macrocycle
Side Chain
Oxidation
State
H
O
O
CO2
n
O
O
O
OMe
CO2
H
Chain
Length
Saturation
OH
O
Carolacton
OH
O
CO2
H
"Carylacton"
Figure 1. S. mutans biofilm lifecycle (top); Kirschning Analog Library
(middle); Overview of the diverted total synthesis (DTS) of arylꢀanalogs;
and modeling of simplified analog and carolacton (bottom).
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Page 2 of 5
macrocycle, for example, introduction of an α,βꢀ
unsaturated ketone or an epimeric allylic alcohol, abolꢀ
ish activity. Further, the side chain ketone was thought
to be essential and “proꢀdrug” ester derivatives affected
biofilm development at concentrations relative to their
rate of hydrolysis indicating that carolacton, and not the
analogs, were responsible for activity. Taken in sum,
earlier SAR work demonstrated that significant changes
to the macrocycle were not tolerated; however, it was
not clear if side chain modifications would be tolerated.
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Our initial synthesis of carolacton was accomplished
in collaboration with the Phillips group in 2014.12 The
strategic design of our synthesis allowed for the lateꢀ
stage derivatization of the natural product scaffold to
elaborate on earlier SAR work – an approach referred to
as Diverted Total Synthesis (DTS) (Fig. 1).13 Both Danꢀ
ishefsky and Myers have beautifully applied DTS to the
development of novel anticancer14 and antibiotic15 comꢀ
pounds, respectively, with improved bioactivity. DTS
endeavors from our laboratory have uncovered new
phenotypes for the antibiotic promysalin16 and we
sought to use our initial total synthesis of carolacton
toward this end. We were inspired by the seminal work
of Chandrasekhar in their disclosure of simplified pladiꢀ
enolide analogs wherein they rigidified the side chain
with an aryl isostere and we determined that a similar
replacement was feasible based on computational modꢀ
eling (Fig. 1).17 We and others18 have been drawn to this
substitution as it would both simplify the synthesis and
effectively evaluate the importance of the elaborate side
chain. Herein we report the DTS of sixteen simplified
carolactonꢀinspired analogs which have resulted in the
discovery of three independent biofilm phenotypes: 1)
inhibition of their formation, 2) inhibition of their matuꢀ
ration, and 3) acidꢀmediated cell death during formation,
akin to carolacton, by an analog coined “carylacton”.
Scheme 1: Diverted Total Synthesis of Carolacton Analogs. (a) K2CO3,
MeOH/ THF, >99% (b) DMSO, (COCl)2, CH2Cl2. 94% (c) CuCN, Allylꢀ
MgBr, THF, 90% (d) 4Å MS, NaOH, PhMe, 83ꢀ85% (e) EDCI, DMAP,
CH2Cl2, 89ꢀ96% (f) Grubbs 2nd Generation Catalyst, CH2Cl2, 78ꢀ99% (g)
Pd/C, H2, 55ꢀ69% (h) TBAF, 64ꢀ94% (i) HCl, THF/MeOH, 50ꢀ82% (j)
DMSO, SO3.pyr, CH2Cl2, 63ꢀ89% (k) t-BuOH, 2ꢀmethylꢀ2ꢀbutene,
NaH2PO4, NaClO2, MeCN, H2O, 65ꢀ96% (l) TFA/ H2O, 59ꢀ79%
thylꢀderivative to interrogate the importance of chain
length. Toward this end, monoprotection followed by
oxidation of the corresponding 1,3ꢀdisubstituted benzene
diols provided the known compounds 4a/b. Roush croꢀ
tylation stereospecifically converts the benzylic aldeꢀ
hydes to the desired alcohols, furnishing structures 5a/b.
Leveraging our previous endgame strategy, an esterificaꢀ
tion united the side chain alcohols with acid 3. Finally,
cyclization using ringꢀclosing metathesis resulted in the
selective formation of the (E)-olefin, providing the fully
protected macrocyclic analog precursors 6a/b, which
can then be hydrogenated whereby the more sterically
accessible olefin is reduced providing 7a/b. Thus, these
four intermediates bearing the fully protected analog
skeleton represent the main branching points from which
we would synthesize our analog library.
It is well known that slight structural modifications
to macrolactones can result in significant biological outꢀ
comes;19 for example, the simple deletion of a methyl
group is enough to render a molecule inactive, as was
nicely demonstrated by the Andrade lab in their syntheꢀ
ses of desmethyl cethromycin.20 Based on these prior
reports, we sought to fully evaluate all desilyated comꢀ
pounds en route to “carylacton” to better understand the
SAR of such perturbations. We envisioned three logical
branching points that would facilitate the production of
four distinct scaffolds whereby we could evaluate the
role that the acetonide, olefin, and oxidation state play in
bioactivity. Facile deprotection of silylꢀethers 6 and 7
generate Scaffolds A and B, respectively, which differ
by an acetonide group. Scaffold A, bearing the primary
alcohol and acetonide, can then undergo a twoꢀstep oxiꢀ
dation, providing the corresponding acid found in Scaf-
fold C. Finally, the acetonide is treated with trifluoroaꢀ
cetic acid in water to furnish Scaffold D (Scheme 1). In
total, milligram quantities of sixteen analogs were synꢀ
thesized and further evaluated for their biological activiꢀ
ty in S. mutans UA159 assays described below.
Our first generation DTS library focused on conꢀ
serving the connectivity and stereochemistry of the
twelveꢀmembered macrolactone, but significantly simꢀ
plifying the carolacton side chain by introducing an aryl
moiety. We rationally selected 1,3ꢀdisubstituted aryl
motifs to mimic the trisubstituted olefin and chose to
vary both the length and oxidation state of the side
chain. This isosteric substitution would thereby elimiꢀ
nate five synthetic operations while maintaining the
structural integrity of the compound. Toward this end,
we optimized our previously published route to the key
intermediate, acid 3, which now proceeds in 36% overall
yield (a 50% improvement) and provides gramꢀ
quantities in under two weeks from commercial material
(Scheme 1, For more details see SI). With both a reliable
and scalable route to 3 in hand, we turned our attention
to the synthesis of the simplified side chains. We initialꢀ
ly chose two aryl diols to use as side chain precursors: a
pentyl moiety (closely mimicking carolacton) and a meꢀ
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We sought to initially evaluate the biological activiꢀ
through our studies with carolacton. All two analogs
appear to arrest growth at the stage of microcolony forꢀ
mation, which can be seen in the field view by their roꢀ
setteꢀlike pattern in Figure 2B, thereby halting biofilm
maturation. At 4x zoom, an apparent haze is apparent
surrounding the microcolonies (as compared to the
DMSO control) indicating possible increased SYTO9ꢀ
staining extracellular DNA (eDNA) in the extracellular
polymeric substance (EPS). This biofilm phenotype is
distinctly different to that of carolactonꢀtreated cells.
Based on these findings we postulate that this subset of
analogs is affecting S. mutans biofilm growth via sepaꢀ
rate modes of action compared to carolacton and is the
subject of ongoing investigation. Of note, these analogs
contain a carboxylic acid which appears to be important
for bioactivity as the corresponding alcohols are inactive
(Fig. S1) consistent with the findings of Kirschning.11
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ty of the analog library via minimum inhibitory concenꢀ
tration (MIC) and minimum biofilm inhibitory concenꢀ
tration (MBIC) assays. We first wanted to evaluate the
ability of the compounds to affect biofilm formation. As
we expected D4 and carolacton were both inactive,
however, we serendipitously discovered that, comꢀ
pounds C3 and C4 inhibited biofilm adherence (Fig.
2B). We quantified their activity by measuring the conꢀ
centration at which 50% inhibition of biofilm formation
(MBIC50) occurs and found it to be 63 and 500 ꢀM, reꢀ
spectively (See Figs. S1 and S2 for details). To confirm
that the compounds were specifically targeting biofilms
and not simply killing planktonic cells we performed
MIC assays against planktonic bacteria wherein the meꢀ
dia lacked sucrose. As expected, all 16 analogs and caroꢀ
lacton were ineffective at inhibiting planktonic growth
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(MIC≥500 ꢀM). The discovery of a simplified carolacꢀ
tonꢀinspired analog (C3) available in ten steps that inꢀ
hibits biofilm formation is notable.
At the onset, we postulated that the aryl isostere of
carolacton could serve as a simplified analog, therefore,
we sought to investigate the range of activity for comꢀ
pound D4 (“carylacton”). It has been shown previously
that at nanomolar concentrations of carolacton S. mutans
cells begin to form long chains and become significantly
more spherical than untreated cells within a biofilm. To
our delight, D4 displayed a phenotype consistent to
those of carolacton at concentrations as low as 500 nM
(highlighted by white dotted circles/ovals in Fig. 2C).
This observation lends credence to our earlier hypothesis
that our rationally designed isosteric structure would
maintain biological activity similar to carolacton. Furꢀ
ther testing is needed to validate that D4 is acting by the
same mechanism of action as the natural product and is
currently ongoing. Nevertheless, this work reveals that
significant side chain modifications are tolerated without
the loss of biological activity. It also serves as another
example that metaꢀsubstituted aryl rings can serve as
isosteres of trisubstituted olefins, which are present in a
number of structurally diverse natural products.
The eradication of biofilms is a longstanding chalꢀ
lenge that has inspired many research programs to better
understand their chemical underpinnings. We sought to
screen our analogs in whole cell phenotypic biofilm asꢀ
says to identify unique biological responses that could
potentially unveil new targets or stall biofilm maturation
processes for future microbiological analysis. We raꢀ
tionalized that the structural diversity of our library may
provide leads for probe development and/or proteomic
analysis and wanted to fully evaluate them via microsꢀ
copy. Figure 2 depicts biofilms treated with 500 ꢀM of
carolacton and stained with propidium iodide/SYTO9 to
demonstrate representative phenotypic results. All comꢀ
pounds except C3 and C4 (which inhibit biofilm forꢀ
mation) were able to be imaged at this concentration.
Analogs C1 and D2 shared a common and unique pheꢀ
notypic response that we had not previously seen
The results disclosed herein highlight our DTS enꢀ
deavor that revealed a multitude of compounds that each
elicited distinct biological responses in S. mutans. Figure
3 summarizes the findings of this current bioorganic
exploration. Our work demonstrates that subtle changes
to the macrocyclic ring of carolacton results in substanꢀ
tial phenotypic outcomes (see Fig. S5 for computational
modeling of analogs). We initially discovered that comꢀ
pound C3 inhibits the formation of biofilms at concenꢀ
trations as low as 63 ꢁM. Several molecules have been
shown to inhibit adherence of S. mutans biofilm such as
betulin, vizantin, and quercitrin.21 Surprisingly, we idenꢀ
tified two truncated carboxylateꢀbearing compounds
(C1, D2) that arrested biofilm growth at the microcolony
stage. It is wellꢀestablished that hydrogen fluoride inhibꢀ
its the growth of streptococci by dissipating the bacterial
transꢀmembrane protonꢀmotive force, thereby disrupting
the membraneꢀbound, protonꢀpumping FꢀATPase, and
Figure 2. Carolacton Analogs Elicit a Range of Phenotypic Responses.
A) C3 inhibits biofilm formation with an MBIC50 of 63 ꢀM, “B” = Blank
wells; “C” = S. mutans control. B) Confocal microscopy highlights the
effect of C1 and D2 on the growth of S. mutans biofilm at 500 ꢀM (top:
field view, bottom: 4x zoom). C) Carolacton and D4 (top: field view, botꢀ
tom: 16x zoom) display similar phenotypes at 500 nM. White circles indiꢀ
cate spherical cell morphology and ovals highlight chains of cells. See
Figures S3 and S4 for additional representative images.
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