Diverted total synthesis of the baulamycins and analogues reveals an alternate mechanism of action

Article abstract of DOI:10.1021/acs.orglett.8b00054

The baulamycins were identified as in vitro siderophore biosynthesis inhibitors. Diverted total synthesis was used to construct the natural products and eight strategic analogues, three of which had improved inhibitory activity. Biological testing then revealed that membrane damage is the predominant mode of action in Staphylococcus aureus cells.

Full text of DOI:10.1021/acs.orglett.8b00054

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Diverted Total Synthesis of the Baulamycins and Analogues Reveals
an Alternate Mechanism of Action
Andrew D. Steele,† Guillaume Ernouf,† Young Eun Lee,‡ and William M. Wuest*,†
†Department of Chemistry, Emory University, 1515 Dickey Drive Atlanta, Georgia 30322, United States
‡Department of Chemistry, Temple University, 1901 North 13th Street Philadelphia, Pennsylvania 19122, United States
S
* Supporting Information
ABSTRACT: The baulamycins were identified as in vitro siderophore biosynthesis inhibitors. Diverted total synthesis was used
to construct the natural products and eight strategic analogues, three of which had improved inhibitory activity. Biological testing
then revealed that membrane damage is the predominant mode of action in Staphylococcus aureus cells.
he development of clinical antibiotics has revolutionized
Tmedicine and overall quality of life over the past century;
however, current problems with resistance have made some in
the field question whether we are at the brink of a “post-antibiotic
era”.1 While antibiotic-resistant bacteria are not a newly
discovered phenomenon, current mis- and overuse of antibiotics
has exacerbated the problem.2 With the recent emergence of
multidrug resistant “superbugs”, new antibiotics with novel
mechanisms of action are urgently needed. The strategy of
modifying existing scaffolds such as penicillin, erythromycin, and
tetracycline has only delayed the development of resistance, as
these compounds target essential metabolic pathways, thereby
creating a significant selection pressure. New small molecule
agents that attenuate virulence or target certain pathways that are
upregulated during a pathogen’s host invasion would be a
valuable addition to the current clinical arsenal as they do not
generate such pressures.3
were shown to inhibit bacterial iron acquisition in vitro (Figure
1A).6 The compounds were identified as inhibitors of SbnE and
were able to outcompete citric acid, presumably by mimicking its
structure via the baulamycins’ 1,3,5-triol moiety. The initial
isolation paper showed that the compounds displayed broad-
spectrum whole cell inhibitory activity in both iron-rich media
One such strategy is to interfere with microbes’ uptake of
essential nutrients, for example, iron. Iron acquisition and
utilization is energy-intensive and has therefore evolved to be a
competitive process among microbes in Nature. In an aerobic
human host environment, free iron is in the ferric (3+) state,
which has poor aqueous solubility. Most of this iron is stored in
hemoglobin and ferritin. Iron transport throughout the host is
accomplished by high-affinity transferrin complexes. Bacteria
compete with the host by secreting iron-chelating small
molecules called siderophores that bind iron with even higher
affinity than transferrins. The resultant siderophore−iron
complexes are taken in by the bacterium for its essential
metabolic processes.4 In contrast to “classical” antibiotics that
nonselectively kill an entire population of bacteria, disruption of
iron uptake has been shown to be an effective strategy to reduce a
bacterial population’s growth and virulence.5
Figure 1. (a) Enzymes that the baulamycins were shown to inhibit in
vitro and IC50 values. (b) Originally reported structure of baulamycin A
(with numbering convention) (c) Correct structure as confirmed by
Aggarwal and co-workers, retrosynthetic approach used herein.
It is for these reasons that we were drawn to the baulamycins,
Received: January 5, 2018
compounds isolated in 2014 by Sherman and co-workers, that
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00054
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. (a) Synthetic Approach to the Fragment Corresponding to the Originally Reported Baulamycin Structure (Figure 1B,
a
epi-C14 (−)-1). (b) Revised Route To Access the Left Half Corresponding to the Corrected Baulamycin Structure
a
N.R. = no reaction.
(IRM) and iron-depleted media (IDM). For Staphylococcus
aureus in particular, the differences between IRM and IDM IC50
values were minimal (86 vs 69 μM for MSSA and 130 vs 130 μM
for MRSA). This discrepancy coupled with ambiguity regarding
the relative and absolute stereochemistry7 served as the ideal
platform to leverage diverted total synthesis (DTS) toward
better understanding the biological mode of action (Figure
1A).8,9
Previous efforts from our laboratory have successfully utilized
DTS to both identify modes of action of natural products and
also uncover unique phenotypes.10−12 With these principles in
mind, we designed a synthesis that would be both flexible,
allowing for the construction of all possible stereoisomers, and
strategic in order to answer specific hypothesis-driven inquiries.
Herein we report the total syntheses and biological investigation
of the baulamycins and eight rationally designed analogues in
whole-cell assays. Our results provide evidence that membrane
damage is the predominant mechanism of action of the natural
products in contrast to previous reports.
Our retrosynthetic analysis led us to propose a late-stage cross-
metathesis and hydrogenation providing fragments (−)-3 and
(−)-4 (Figure 1C). We first began our work toward the originally
proposed structure (Figure 1B) but were able to quickly pivot
upon the disclosure of the correct stereoisomer (Figure 1C)
during the course of this project (vida infra).13 In a forward sense,
the synthesis of the originally reported left fragment began with
known Evans syn aldol product (−)-7. The chiral oxazolidinone
was then removed by transamidation to the corresponding
Weinreb amide, followed by TBS protection yielding (−)-8 in
81% over two steps. Displacement of the amide with
methylmagnesium bromide yielded methyl ketone (−)-9,
which was then converted to the corresponding (−)-diisopino-
campheylborane-derived enolate and treated with acrolein to
yield the β-hydroxyketone (−)-10 as a single diastereomer
resulting from synergistic 1,4-syn-1,5-syn induction. Subsequent
diastereoselective 1,3-syn reduction by NaBH4 in the presence of
Et2BOMe yielded the 1,3-diol, which was then converted to the
corresponding acetonide (−)-11 (Scheme 1A).
At this point, the publication by Goswami and co-workers14
reported that the structure assigned to baulamycin A did not
match the natural compound. A conspicuously aberrant coupling
constant (J1′−14 = 3.7 Hz) in their synthesized molecule did not
match the reported value (J1′−14 = 7.0 Hz). With this discrepancy
in mind, we referred to the literature in regards to Evans syn and
anti-aldol adducts and found that larger coupling constants were
associated with an anti-configuration.15 This hypothesis was
further supported by Aggarwal et al., who published the correct
structure during the course of our synthesis,13 and a more recent
study by Sim and co-workers.16
Our revised route to the corrected structure of (−)-1 began
with an Evans anti aldol reaction between building blocks 5 and 6
to provide the desired aldol adduct (−)-12 as a single
diastereomer (Scheme 1B). Masking the resulting alcohol as
the silyl ether yielded (−)-13 followed by displacement by
lithium ethyl thiolate yielded the corresponding thioester.
Treatment of this intermediate with lithium dimethyl cuprate
afforded methyl ketone (−)-14 in nearly quantitative yield over
two steps. Next, we turned to the key aldol reaction between
methyl ketone (−)-14 and acrolein. The previously selective
conditions ((−)-Ipc2BCl, Et3N) yielded a mixture of diastereo-
meric products (dr = 1:1.3). Undeterred, we separated both
resultant diastereomers by column chromatography and
determined the stereochemical outcome by converting the
secondary alcohol to the corresponding Mosher’s esters 16. This
analysis showed that the previously optimized conditions were
now in favor of the undesired stereoisomer. Surprisingly, when
B
DOI: 10.1021/acs.orglett.8b00054
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
achiral boron reagents were employed, the product ratio was
overturned in favor of the desired secondary alcohol, with a
modest 2:1 selectivity. Gratifyingly, Mukaiyama aldol reaction
between the corresponding silyl enol ether and acrolein
improved the product ratio to 4:1 in favor of the desired (R)-
alcohol (−)-15. The 1,3-syn reduction of the β-hydroxyketone
(−)-15 was then investigated. After much optimization, it was
found that zinc borohydride in toluene provided the desired 1,3-
diol with high diastereoselectivity at C13 (>25:1). The resultant
unstable diol 17 was immediately converted to acetonide (−)-3.
The scalable route afforded the key building block (−)-3 in six
steps and 31% overall yield from known material.
Based on the initial ambiguity surrounding the deoxypropi-
onate triad, we sought to use Myers alkylation chemistry to
iteratively investigate the structure in a flexible manner.
Beginning with known iodide (−)-18, a two-step Myers
alkylation/reduction sequence,17 employing pseudoephedrine
amide (+)-19, led to alcohol (+)-20 in 88% yield over two steps
as a single diastereomer. A second Myers alkylation/reduction
sequence after iodination provided alcohol (+)-21 in 64% overall
yield. Sequential Swern oxidation and Wittig reaction then
furnished olefin (−)-4 with no erosion of the α-methyl
stereochemistry (Scheme 2). Benzyl ether (−)-4 was then
converted to the corresponding ethyl ketone (+)-22 to afford a
more convergent route toward baulamycin A and the DTS of
analogues (see Scheme S1). Deoxypropionate precursors are
now readily available via this route in 11 steps and 23% overall
yield from known material.
Having constructed both halves, the cross-metathesis step was
achieved using Grubbs’ second-generation catalyst. The report
by Goswami et al. suggested this disconnection was not feasible;
however, another report by Chandrasekhar et al. claimed the
opposite.14,18 In our hands, multiple additions of catalyst over the
course of the reaction were required to obtain acceptable yields.
From there, hydrogenation and global deprotection in the
presence of HF−pyridine afforded (−)-baulamycin A in a
longest linear sequence of 14 steps, matching the natural product
in all respects ([α]20D −15.1 (c 0.44, MeOH)). Once the correct
structure was verified spectroscopically, we sought to leverage
DTS to investigate the SAR of the baulamycins and investigate
the mechanism of action. From the outset, we were intrigued by
the amphipathic nature of the natural product with its polar
headgroup (two phenols, three hydroxyl groups) and lipophilic
tail. Thus, we postulated that the baulamycins’ broad-spectrum
inhibitory activity in iron-rich media could be due to nonselective
lysis or damage to cell membranes, akin to a detergent.
To test this hypothesis, we systematically altered several of the
natural products’ key structural features. In an effort to alter the
hydrophilic portion, conversion of the phenols to methyl ethers
((−)-23) provided an aromatic system devoid of hydrogen bond
donors (Figure 2). Ketone analogue (−)-24 allowed us to test
the importance of the hydroxyl located at C13. We also
considered the stereochemistry at C14 ((−)-25) and hypothe-
sized that if this epimer retained activity this would lend credence
to our membrane hypothesis, as this drastic structural alteration
is unlikely to be tolerated by an enzyme active site. In a similar
vein, the hydrophobic fragment was converted from the ethyl to
the propyl ketone ((−)-26). As baulamycin A is more active than
B (ethyl vs methyl), we were curious if other extensions would be
tolerated. We also removed all of the stereogenic methyl groups
while either retaining ((−)-27) or removing ((−)-28) the
ketone in an effort to develop a simplified analogue with
improved potency. Finally, we sought to further probe this
hypothesis by either removing the entire hydrophobic portion
((−)-29) or adding a polar component ((−)-30).
Scheme 2. Route to Deoxypropionate Fragment (−)-4 and
Final Steps Leading to (−)-1
Figure 2. Analogue structures and selected biological results. The two MIC values (in μM) refer to IRM and IDM, respectively (S. aureus SH1000).
HC20 concentrations given in μM. Checkmark and X symbols refer to positive and negative uptake results, respectively.
C
DOI: 10.1021/acs.orglett.8b00054
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
After completion of our synthetic analogue campaign, we
tested all compounds in three different strains of S. aureus using
both iron-rich and iron-depleted conditions (Figure 2: S. aureus
SH1000 MIC data, for USA300 and ATCC33591, see Table S2).
We used minimum inhibitory concentration (MIC) measure-
ments in contrast to Sherman and co-workers’ IC50 growth data
for reproducibility reasons.6 All analogues were tested alongside
baulamycin A and B and with vancomycin as a positive control. In
all cases, we saw minimal differences in growth inhibition
between IRM and IDM in accordance with Sherman. This again
hinted at an alternative mechanism that did not involve
siderophore biosynthesis or iron acquisition. Compounds with
similar mechanisms in vitro have been shown to display high
(100-fold) differences between IRM and IDM.19 Therefore,
although the baulamycins inhibit SbnE in vitro we do not think
that it is biologically relevant in vivo and instead sought to test
our membrane-targeting hypothesis.
Toward this end, we employed each of our synthetic
compounds in a SYTOX uptake assay. SYTOX exhibits a large
increase in fluorescence when bacterial membranes become
permeabilized, whereby the dye crosses the cell membrane and
complexes with DNA.20 As a positive control we used one of the
most potent quaternary ammonium compounds (12,3,2,3,12)
from our laboratory as a positive control, as this compound is a
well-known inducer of bacterial cell lysis.21a,b We found that
baulamycin A as well as all active analogues tested positive for
membrane disruption via this assay (see the Supporting
Information for details). Baulamycin B was one exception;
however, this discrepancy may be a result of its modest MIC
against S. aureus (500 μM). These results support our hypothesis
that the compounds are potentially inducing membrane damage
and explains their broad-spectrum activity in IRM. Current
studies are underway to investigate the specific mechanism by
which these compounds are able to disrupt membrane
composition and will be reported in due course. Membrane
lytic antibacterial compounds are also known to lyse mammalian
red blood cells; therefore, we tested each compound for
hemolysis activity. Baulamycins A and B did not induce any
hemolysis up to 500 μM, along with the propyl analogue (−)-26.
However, ketone (−)-24 and our most potent compound,
(−)-28, displayed significant hemolytic activity (Figure 2).
Future studies will focus on the optimization of (−)-26, the most
potent compound that did not induce hemolysis.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wwuest@emory.edu.
ORCID
William M. Wuest: 0000-0002-5198-7744
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of General
Medical Sciences (GM119426) and the National Science
Foundation (CHE1755698). The NMR instruments used in
this work were supported by the National Science Foundation
(CHE1531620).
REFERENCES
(1) Alanis, A. J. Arch. Med. Res. 2005, 36, 697.
(2) Ventola, C. L. Pharm. Ther. 2015, 40, 277.
(3) Rossiter, S. E.; Fletcher, M. H.; Wuest, W. M. Chem. Rev. 2017, 117,
■
12415.
(4) Miethke, M.; Marahiel, M. A. Microbiol. Mol. Biol. Rev. 2007, 71,
413.
(5) Litwin, C. M.; Calderwood, S. B. Clin. Microbiol. Rev. 1993, 6, 137.
(6) Tripathi, A.; Schofield, M. M.; Chlipala, G. E.; Schultz, P. J.; Yim, I.;
Newmister, S. A.; Nusca, T. D.; Scaglione, J. B.; Hanna, P. C.; Tamayo-
Castillo, G.; Sherman, D. H. J. Am. Chem. Soc. 2014, 136, 1579.
(7) Tripathi, A.; Schofield, M. M.; Chlipala, G. E.; Schultz, P. J.; Yim, I.;
Newmister, S. A.; Nusca, T. D.; Scaglione, J. B.; Hanna, P. C.; Tamayo-
Castillo, G.; Sherman, D. H. J. Am. Chem. Soc. 2014, 136, 10541.
(8) Njardarson, J. n. T.; Gaul, C.; Shan, D.; Huang, X.-Y.; Danishefsky,
S. J. J. Am. Chem. Soc. 2004, 126, 1038.
(9) Wilson, R. M.; Danishefsky, S. J. J. Org. Chem. 2006, 71, 8329.
(10) Solinski, A. E.; Koval, A. B.; Brzozowski, R. S.; Morrison, K. R.;
Fraboni, A. J.; Carson, C. E.; Eshraghi, A. R.; Zhou, G.; Quivey, R. G., Jr.;
Voelz, V. A.; Buttaro, B. A.; Wuest, W. M. J. Am. Chem. Soc. 2017, 139,
7188.
(11) Steele, A. D.; Keohane, C. E.; Knouse, K. W.; Rossiter, S. E.;
Williams, S. J.; Wuest, W. M. J. Am. Chem. Soc. 2016, 138, 5833.
(12) Steele, A. D.; Knouse, K. W.; Keohane, C. E.; Wuest, W. M. J. Am.
Chem. Soc. 2015, 137, 7314.
(13) (a) Wu, J.; Lorenzo, P.; Zhong, S.; Ali, M.; Butts, C. P.; Myers, E.
L.; Aggarwal, V. K. Nature 2017, 547, 436. (b) Lorenzo, P.; Butts, C. P.;
Myers, E. L.; Aggarwal, V. K. Biochemistry 2017, 56, 6177.
(14) Guchhait, S.; Chatterjee, S.; Ampapathi, R. S.; Goswami, R. K. J.
Org. Chem. 2017, 82, 2414.
(15) Harada, K.; Kubo, M.; Horiuchi, H.; Ishii, A.; Esumi, T.; Hioki, H.;
Fukuyama, Y. J. Org. Chem. 2015, 80, 7076.
(16) Sengupta, S.; Bae, M.; Oh, D. C.; Dash, U.; Kim, H. J.; Song, W. Y.;
Shin, I.; Sim, T. J. Org. Chem. 2017, 82, 12947.
(17) Myers, A. G.; Yang, B. H.; Chen, H.; McKinstry, L.; Kopecky, D.
J.; Gleason, J. L. J. Am. Chem. Soc. 1997, 119, 6496.
(18) Paladugu, S.; Mainkar, P. S.; Chandrasekhar, S. Tetrahedron Lett.
2017, 58, 2784.
(19) Neres, J.; Labello, N. P.; Somu, R. V.; Boshoff, H. I.; Wilson, D. J.;
Vannada, J.; Chen, L.; Barry, C. E., 3rd; Bennett, E. M.; Aldrich, C. C. J.
Med. Chem. 2008, 51, 5349.
(20) Roth, B. L.; Poot, M.; Yue, S. T.; Millard, P. J. Appl. Environ.
Microbiol. 1997, 63, 2421.
(21) (a) Jennings, M. C.; Ator, L. E.; Paniak, T. J.; Minbiole, K. P.;
Wuest, W. M. ChemBioChem 2014, 15, 2211. (b) Wuest, W. M.;
Minbiole, K. P. Biscationic and Triscationic Amphiphiles as
Antimicrobial Agents, US20160278375 A1, Sep 29, 2016.
Herein, we report the diverted total synthesis of the
baulamycins in a longest linear sequence of 14 steps and 11%
yield. The high overall efficiency allowed for the expedited
synthesis of eight rationally designed analogues. Whole cell
biological assays revealed a common chemotype that hinted at
membrane lysis as the likely mode of action, which was further
supported by uptake experiments. Future work will require
optimization of the baulamycin scaffold to mitigate nonselective
membrane lysis. The duality of mechanisms that the baulamycins
display demonstrates the importance of supplementing in vitro
data with in vivo assays whenever possible.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.8b00054.
Detailed synthetic procedures and compound character-
ization (PDF)
D
DOI: 10.1021/acs.orglett.8b00054
Org. Lett. XXXX, XXX, XXX−XXX