A concise synthesis of carolacton

Article abstract of DOI:10.1021/ol500004k

A synthesis of carolacton, a myxobacterial natural product that has profound effects on Streptococcus mutans biofilms, is reported. The synthesis proceeds via a longest linear sequence of 14 steps from an Evans β-ketoimide and enabled preliminary evaluations of the effects of late-stage intermediates on S. mutans biofilms. These studies suggest that further investigations into carolacton's structure-function relationships are warranted.

Full text of DOI:10.1021/ol500004k

Letter
pubs.acs.org/OrgLett
A Concise Synthesis of Carolacton
Michal S. Hallside,† Richard S. Brzozowski,‡ William M. Wuest,*,‡ and Andrew J. Phillips*,†
†Department of Chemistry, Yale University, New Haven, Connecticut 06511, United States
‡Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, United States
S
* Supporting Information
ABSTRACT: A synthesis of carolacton, a myxobacterial natural product that
has profound effects on Streptococcus mutans biofilms, is reported. The
synthesis proceeds via a longest linear sequence of 14 steps from an Evans β-
ketoimide and enabled preliminary evaluations of the effects of late-stage
intermediates on S. mutans biofilms. These studies suggest that further
investigations into carolacton’s structure−function relationships are
warranted.
ental caries is a chronic health problem that impacts the
1
Dmajority of the global population.
Although culture-
independent sequencing has illuminated the complexity of the
human oral microbiome, Streptococcus mutans remains widely
regarded as a primary etiological agent in caries.2,3 This
facultatively anaerobic, microaereophilic, Gram-positive, bacte-
rium excels in the complex environment of the oral cavity
where stresses including low pH and an oxidative environ-
ment are coupled to variable salivary flow and carbohydrate
supply.4 The physiologic adaptations of S. mutans to these
pressures provide a competitive advantage versus non-
cariogenic commensals that underpins the establishment and
progression of caries.5
Broad-spectrum agents such as fluoride and chlorhexidine
Figure 1. Carolacton structure and an overview of key features in the
synthesis that traces to lactone 4 and β-ketoimide 5.
represent the state-of-the-art in anticariogenic preventative
treatment, although the efficacy of chlorhexidine remains
debated.6 Significant perturbation of the oral microbiome
accompanies their use,7 and there is increasing evidence that
such imbalances can favor pathogens versus commensals.8
Compounds that are species- or phenotype-selective would
allow exploration of the hypothesis that targeting smaller
elements of complex communities may be advantageous.9 In
this light, we were attracted to a report describing the
structure and activity of the myxobacterial metabolite
carolacton (1, Figure 1).10 Initial characterizations showed
that carolacton causes 35−66% cell death within S. mutans
biofilms at concentrations ranging from 5 to 25 ng/mL.
Remarkably, planktonic cultures of S. mutans were not affected
by carolacton, nor were a variety of other bacteria.
The structure and activity of carolacton has attracted much
scientific interest. Kirschning has completed a total synthesis,
and several subunit syntheses have also been reported.11 In
addition, a number of investigations have pursued in more
detail the biology of carolacton’s effects on S. mutans and the
question of its mode-of-action.12 While there is accord on the
differential activity of carolacton for biofilm versus planktonic
cultures of S. mutans, coherency around mode-of-action has
yet to emerge.
In this Letter, we describe a straightforward synthesis of
carolacton that is amenable to an extensive exploration of
structure−function relationships. Key elements of the syn-
thesis are shown in Figure 1, including an assembly strategy
that leverages macrocycle formation by esterification and a
Received: January 1, 2014
© XXXX American Chemical Society
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Letter
ring-closing metathesis, followed by a selective olefin hydro-
genation.
Scheme 2. Synthesis of the C1−C11 Subunit 3
The synthesis of the C12−C19 acid 2 began with known
gulonolactone-derived diol 613 (available in 4 steps, 62%
overall from D-gulonolactone, Scheme 1). Oxidative cleavage
of the diol with sodium periodate on silica14 gave aldehyde 7,
which was subjected to a Z-selective Wittig olefination to yield
alkene 8. Removal of the benzoate, followed by Swern
oxidation, provided the key lactone 4. In accord with Trost’s
studies,15 exposing 4 to allylmagnesium bromide in the
presence of CuBr·SMe2 resulted in SN2′ opening of the
lactone to produce acid 2 in 60% yield and as a single
diastereomer.
Scheme 1. Synthesis of the C12−C19 Subunit 2 from
Gulonolactone-Derived Diol 6
The stereochemically complex C1−C11 domain was
assembled as shown in Scheme 2. Enolization of the Evans
β-ketoimide 5 under conditions favoring formation of the (E)-
enol borane, followed by reaction with aldehyde 9, produced
10 in 81% yield and with a dr of 5:1.16 Separation of the
diastereomers was not possible. Conveniently, the subsequent
three-step sequence of methylation, reduction, and oxidation−
Wittig olefination not only proceeded in 48% overall yield but
also allowed for isolation of enoate 12 as a single
diastereomer. Silylation of the alcohol gave TBS ether 13.
Introduction of the final two stereocenters was readily
achieved by conversion of the ester to an aldehyde and
application of Leighton’s (R,R)-trans EZ-CrotylMix to give
alcohol 3 (61%, 5:1 dr).17
With routes to the two key subunits secured, it was possible
to investigate the end-game for the synthesis, which began
with a Steglich esterification to connect acid 2 and alcohol 3,
producing a near-quantitative yield of 14 (Scheme 3).
Reaction of 14 with 5.5 mol % of the second-generation
Grubbs catalyst was unsurprisingly selective for the terminal
olefins and led to lactone 15 in 76% yield. Our plans
demanded a selective hydrogenation of the Δ11,12 olefin at this
juncture, a strategy predicated on conformational analysis of
15 suggesting peripheral addition would favor the desired
reduction.18 Pleasingly, this was borne out under operationally
simple conditions: subjecting 15 to 5% Pd/C in EtOH under
a hydrogen balloon resulted in selective reduction of the
Δ11,12 olefin as well as removal of the PMB group (70%
yield). Removal of the TBS ether with TBAF produced diol
16 in 96% yield and set the stage for a simultaneous oxidation
of the primary and secondary alcohols under Swern
conditions and subsequent Lindgren−Pinnick oxidation to
yield keto-acid 17. Removal of the acetonide yielded synthetic
carolacton (1). Analytical characteristics of this material were
in full accord with data reported for the natural product.
Our initial synthetic venture provided multimilligram
amounts of intermediates in the late stages of the synthesis.
We capitalized on this to perform a preliminary biological
evaluation of compounds 16, 17, and carolacton against S.
mutans UA159 grown planktonically and also within a biofilm.
None of the compounds displayed any inhibitory activity
against planktonic cells at concentrations <250 μM. In the
case of carolacton, this is in accord with previous reports.12b
However, when the compounds were incubated with S.
mutans in the presence of biofilm-inducing media, dramatic
morphological and architectural changes to the biofilm matrix
were observed (Figure 2). Confocal microscopy with LIVE/
DEAD staining demonstrated that carolacton (>500 nM) and
16 (>62.5 μM) significantly affected both the integrity and
morphology of the biofilm matrix when compared to the
DMSO control. In contrast, compound 17 had no observable
effect at concentrations <250 μM. While the reasons for the
significant difference in activity between compound 16 and 17
remain unclear at this time, our preliminary investigations
provide credence to the suggestion that a minimal set of
features needed for activity in the carolacton family might be
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Letter
Scheme 3. Subunit Coupling and Completion of the Synthesis
readily identified by synthesis. Future efforts will focus on
expanding the understanding of structure−function relation-
ships in the carolacton class, as well as obtaining a detailed
understanding of the mechanism of action of carolacton on S.
mutans biofilms.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: wwuest@temple.edu.
*E-mail: andrew.phillips@yale.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Yale University (A.J.P.), in part
by the NIH (CA110246, A.J.P.), and Temple University
(W.M.W.). We thank Professor Bettina A. Buttaro (Temple
University) for advice and assistance with confocal microscopy
of S. mutans biofilms, and Dr. Jennifer A. Poole (Yale
University) for mass spectrometry assistance.
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