Total Synthesis and Biological Investigation of (-)-Promysalin

Article abstract of DOI:10.1021/jacs.5b04767

Compounds that specifically target pathogenic bacteria are greatly needed, and identifying the method by which they act would provide new avenues of treatment. Herein we report the concise, high-yielding total synthesis (eight steps, 35% yield) of promysalin, a natural product that displays antivirulence phenotypes against pathogenic bacteria. Guided by bioinformatics, four diastereomers were synthesized, and the relative and absolute stereochemistries were confirmed by spectral and biological analysis. Finally, we show for the first time that promysalin displays two antivirulence phenotypes: the dispersion of mature biofilms and the inhibition of pyoverdine production, hinting at a unique pathogenic-specific mechanism of action.

Full text of DOI:10.1021/jacs.5b04767

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Communication
Total Synthesis and Biological Investigation of (-)-Promysalin
Andrew D. Steele, Kyle W. Knouse, Colleen E. Keohane, and William M. Wuest
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b04767 • Publication Date (Web): 29 May 2015
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Total Synthesis and Biological Investigation of (-)-Promysalin
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Andrew D. Steele, Kyle W. Knouse, Colleen E. Keohane, and William M. Wuest*
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Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, USA
Supporting Information Placeholder
ABSTRACT: Compounds that specifically target pathogenic bacteria are in great need and identifying the method by which they
act would provide new avenues of treatment. Herein we report the concise, high-yielding total synthesis (8 steps, 35% yield) of
promysalin, a natural product that displays anti-virulence phenotypes against pathogenic bacteria. Guided by bioinformatics,
four diastereomers were synthesized and the relative and absolute stereochemistry was confirmed by spectral and biological
analysis. Finally, we show for the first time, that promysalin displays two anti-virulence phenotypes: the dispersion of mature
biofilms and the inhibition of pyoverdine production hinting at a unique pathogenic-specific mechanism of action.
Advances in culture-independent genome sequencing
coupled with computational analysis methods have revolu-
tionized research into microbiomes – the entirety of the mi-
crobial community in a given system.1 At the same time, the
burgeoning field of microbial ecology has shed light on the
inherent complexity of interrelationships in such ecosys-
tems.2 Studies investigating the coexistence of organisms,
ranging from mutualistic to parasitic, have had profound
impact on our understanding of life, most notably in the hu-
man body. There has been a call for providing small molecule
probes that could be used to deconvolute such systems by
targeting pathogen-specific bacteria. For example, a narrow-
spectrum antibiotic that can specifically target pathovars
without disrupting the remaining population would be of
interest in agriculture. If successful, these compounds would
not only be of value to farming but also to human health (i.e.
oral, gastrointestinal) and other commercial interests.
resides in the rhizosphere of rice plants.8 The natural product
showed unique species-specific bioactivity, most notably
against Pseudomonas aeruginosa (PA), inhibiting growth at
low micromolar concentrations. Promysalin selectively inhib-
its certain PA strains and other Gram-negative bacteria, but
showed no activity against their Gram-positive counterparts.
In contrast, the compound was also shown to promote
swarming of the producing organism, hinting at two discrete
modes of action. The original report characterized the bio-
synthetic gene cluster and proposed a biosynthesis via anno-
tation and the characterization of shunt products. The au-
thors elucidated the structure of promysalin through spec-
troscopic methods; however, no absolute or relative stereo-
chemical assignments were made. Considering the signifi-
cance of PA clinical settings9 (cystic fibrosis, immuno-
compromised patients) and in agriculture, promysalin could
serve as an attractive alternative to current therapies. The
unique bioactivity, unknown mode of action, and structural
ambiguity are what prompted the synthesis reported herein.
A well-studied example of a multi-species community is
the root system of plants, generally termed the rhizosphere
microbiome.3 The predominant players in this arena are the
Pseudomonads, which are comprised of both commensal and
pathogenic species competing for vital resources, either en-
suring or jeopardizing the health of the host. The competi-
tors produce an array of secondary metabolites with unique
bioactivities evolutionarily designed to promote survival.
Functions include siderophores,4 virulence factors,5 biosur-
factants,6 and antibiotics.7 Such species-specific compounds
represent attractive targets for agricultural and medicinal
needs and may shed light on novel virulence targets for fu-
ture drug design.
Before initiating our synthetic investigation we sought to
reannotate the biosynthetic gene cluster using AntiSMASH
(Scheme 1).10 We postulated that this computational work
would aid in determining the absolute stereochemistry of
dehydroproline thus limiting the synthesis to one enantio-
meric series. This study confirmed that ppgJ encodes for a
truncated Non-Ribosomal Peptide Synthetase (NRPS) mod-
ule containing both an adenylation (A) and thiolation domain
but lacking a condensation domain, reminiscent of sylC
found in P. syringae.11 Upon closer inspection, we were una-
ble to identify any putative epimerase or thioesterase do-
mains contained in either the characterized gene cluster or in
In 2011, De Mot and coworkers isolated a novel metabolite,
promysalin (1), from Pseudomonas putida (PP) RW10S1 which
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the flanking regions. Bioinformatic investigation of the ppgJ
A-domain revealed the
diastereomers in enantiomerically pure form (45-54% over
five steps).
1
2
OH
O
3
4
5
6
7
8
9
Synthesis of the proline-salicylate fragment commenced
OH
PpgG PpgH
CO2H
1) 3, NaHMDS, THF
2) TBSCl, Imidazole
O
HO2C
O
O
C13H27
Xc
Xc
Xc =
PpgM
chorismate
O
myristic acid
OTBS
(R) = (-)-2
(S) = (+)-2
(-)-4 (2R): 73%, 2 steps
(+)-4 (2S): 73%, 2 steps
O
3
N
PKS Module
NRPS Module
Tailoring Enzyme
PpgF
PpgC
*
Bn
(R) = (+)-5
(S) = (-)-5
PpgA
OH
*
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1)
O
O
S
S
PpgJ
N
C711 (10 mol %), CH2Cl2
PhO2S
Ph
iPr
O
2) Pd/C, H2, EtOAc
3) NH4OH, THF
O
C711
iPr
S
5
HO
N
H
N
N
O
2R
8R
C6H13
OH
C6H13
iPr
iPr
Cl
salicylate
proline
H2N
Ru
OTBS
OH
Cl
O
(+)-6a (2R,8R): 74%, 3 steps
(+)-6b (2R,8S): 62%, 3 steps
(-)-6c (2S,8S): 74%, 3 steps
(-)-6d (2S,8R): 62%, 3 steps
PpgL
PpgC
with the ester hydrolysis of the SEM-protected methyl trans-
4-L-hydroxyproline methyl ester and Dess-Martin oxidation
to
O
S
C6H13
16
O
8
N
5
O
PpgN
O
H13C6
O
Scheme 1. Synthesis of Aliphatic Precursors 6a-d
OH
HO
promysalin, 1
H2N
PpgO
O
provide (+)-9 (Scheme 2). At this stage, we sought to develop
a method for the regioselective dehydration of (+)-9 to give
the delicate enamine functionality. To this end, we treated
the ketone with triflic anhydride and 2,6-lutidine to provide
the desired enol-triflate which was cleanly reduced using a
modified Stille reaction furnishing the corresponding
enamine with the desired regiochemistry found in the natural
product.15 Base hydrolysis of the methyl ester ultimately led
to the key coupling fragment (-)-10 in six steps and an overall
yield of 56%.
2
N
O
O
OH
Figure 1. Proposed biosynthesis of promysalin. Polyketide syn-
thase (PKS), NRPS, and Tailoring enzymes (Rieske Iron-Sulfur
cluster, asparagine synthase, chorismate synthase) are depicted.
Stachelhaus code,12 DVQFVAHV, corresponding to the selec-
tive activation of L-proline as previously hypothesized by De
Mot. This exercise led us to the conclusion that the absolute
configuration of the C16-stereocenter should be assigned as
(S). Taken together, we reevaluated the proposed biosynthe-
sis of promysalin, which is depicted in Figure 1. With this in-
formation in hand, we began our campaign to synthesize the
four diastereomers generated from the two unresolved ste-
reocenters (C2 and C8).
EDC-mediated esterification of alcohols 6a-d with (-)-10
proceeded smoothly to give all four diastereomers of fully
protected promysalin. As is the case in many total syntheses,
the final global deprotection proved to be non-trivial. Most
literature methods of SEM-deprotection call for either
BrØnsted or Lewis acidic conditions or fluoride (TBAF or
TASF) at elevated temperatures. Unfortunately, the sub-
strate was unstable to both prolonged heat and/or acid,
providing only trace amounts of the desired product. Unde-
terred, we sought milder deprotection conditions. After
much experimentation we found that 1M TBAF in THF with
DMPU as a co-solvent cleanly removed both silyl protecting
groups in a single operation.16 Our method of SEM-
deprotection provides a straightforward alternative to previ-
ous published precedent as it is performed at ambient tem-
perature, using commercially available TBAF/THF solution,
and with short reaction times (30-60 min).
Our synthetic efforts began with construction of the four
diastereomers of the myristic acid fragment (Scheme 1). We
envisioned utilizing a convergent route wherein cross me-
tathesis followed by hydrogenation would be used to forge
the complete aliphatic chain providing a succinct route to all
four L-proline diastereomers. Beginning with the known
compound (-)-2 available in one step from 5-hexenoic acid
and the phenylalanine-derived Evans’ oxazolidinone,13 dia-
stereoselective oxidation using the Davis oxaziradine fol-
lowed by silyl-protection furnished compound (-)-4 in good
yield. Cross metathesis with known, enantiomerically pure
homoallylic alcohol (+)-5 or (-)-6 in the presence of catalyst
C711,14 subsequent hydrogenation, and ammonolysis pro-
vides diastereomers 6a/b. Analogously, the enantiomeric
series of compounds was synthesized starting with (+)-2
providing 6c/d. This route provides concise access to all four
With the four diastereomers in hand we set out to une-
quivocally define the relative and absolute stereochemistry
through both NMR spectral comparison and biological as-
says. Upon careful examination of the chemical shift differ-
1
ence in the H and 13C NMR spectra of compounds 1a-d, we
identified distinct features that were used to assess the cor-
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rect configurations at C2 and C8. As shown in Figure 2, the
hydrocarbons, was unaffected.17 PP OUS82 has been previ-
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3
4
5
6
7
8
spectral data of compound 1a best correlates to that of the
isolated material when compared to the other diastere-
omers. Key chemical shifts of protons located on C3, C7, C9,
and C19 strongly indicate that (2R,8R,16S) is the proper rela-
tive stereochemical assignment for promysalin. Unfortunate-
ly, without a reported optical rotation nor authentic sample
available we were unable to unequivocally determine the
absolute configuration. We postulated that only the correct
enantiomer would elicit the biological responses reported
previously and, therefore, we sought to recapitulate both the
inhibitory activity with
ously shown to contain enzymes capable of degrading salicy-
late; therefore, it is possible that the bacteria is consuming
promysalin before it is able to elicit a biological response.
Nevertheless, these results indicate that (-)-1a is responsible
for a swarming phenotype in a broad range of closely related
organisms.
The main mode by which bacteria swarm is through bio-
surfactant production.18 We postulated that 1a could be act-
ing either directly as a biosurfactant or as a trigger for biosur-
factant production. In order to differentiate the former from
the latter, we performed a simple surface tension assay. We
found that all four diastereomers displayed similar biosurfac-
tant-properties at equimolar concentrations (Figure S1). It is
well established that amphipathic molecules, like rhamno-
lipids, can
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Scheme 2. Synthesis of Promysalin Diastereomers 1a-d.
O
1) LiOH, THF/H2O
2) 8, HATU, DIEA, DMF
3) DMP, CH2Cl2, H2O (cat.)
OSEM
CO2Me
CO2Me
N
HO
O
CO2Me
7
N
8
OSEM
(+)-9: 85%, 3 steps
•HCl
H
1) Tf2O, 2,6-lutidine
CO2H
2) Pd(OAc)2, PPh3,
Bu3SnH, LiCl, THF
3) LiOH, THF/H2O
N
O
C6H13
OSEM
(-)-10: 66%, 3 steps
O
8
N
O
1) 6, EDC, DMAP, CH2Cl2
2) TBAF, THF/DMPU
O
OH
Promysalin: (-)-1a (2R,8R): 62%, 2 steps
(-)-1b (2R,8S): 66%, 2 steps
(-)-1c (2S,8S): 56%, 2 steps
HO
2
O
(-)-1d (2S,8R): 62%, 2 steps
H2N
compounds 1a-d versus PAO1 and PA14 and the swarming
activity identified in the producing strain.
In De Mot’s initial report, they surveyed the biological ac-
tivity of >100 bacterial strains through halo diffusion assays
with co-treatment of the producing organism noting qualita-
tive inhibition.8 More specifically, they quantified the IC50
value for PA14 providing a strain by which we could directly
compare. In accordance with their findings, compound (-)-1a
possessed the most potent biological activity of the four
compounds with an IC50 of 125 nM against PA14 (1.8 µM re-
ported) and 1 µM for PAO1 (not reported). Compounds 1b-d
were each ~10-60 times less effective against both strains
(Figure 3A, left). Taken in sum, we propose that the absolute
stereochemistry of promysalin be assigned as (2R,8R,16S)
and depicted by structure (-)-1a (Scheme 2).
1
Figure 2. Comparative H NMR (left) and 13C NMR (right) spec-
tra depicting the absolute Δδppm of compounds 1a-d when com-
pared to the natural product. Red lines indicate Δδppm values of
>0.06 (1H NMR) and >0.4 (13C NMR). For numerical comparison
see Table S1.
disperse and/or eradicate mature biofilms.19 We hypothe-
sized that if compounds 1a-d acted solely as biosurfactants
then all would possess equipotent dispersant activity. To test
this proposal, we grew mature biofilms of both PA14 and
PAO1 for 24 hours and then dosed each trial with varying
concentrations of compounds 1a-d. All diastereomers dis-
persed PA biofilms at 100 µM; however, compound (-)-1a
again showed the most potent biological activity, dispersing
both biofilms at 6.25 and 12.5 µM, respectively (Figure 3A,
In contrast to the activity observed in PA, promysalin has
been shown to increase both swarming and biofilm for-
mation in PP RW10S1. We were curious as to whether the
compound would elicit a swarming response in multiple
strains of PP or solely in the producing organism. As can be
seen in Figure 3B, compound 1a clearly promotes swarming
in the native producing organism and three other species
(two strains of PP and one of P. fluorescens (PF)). Curiously,
the strain PP OUS82, which was originally isolated from oil-
contaminated soil and is known for its ability to cannibalize
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right and Figure S4). These results suggest that promysalin is tural and human health. Current work in our laboratory is
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3
4
5
6
7
8
acting on a specific target.
focused on deciphering the target triggering PA biofilm dis-
persion and whether the molecule is acting directly on pyo-
verdine production or through a pyoverdine-signaling path-
way. The route presented herein allows for the preparation of
gram-quantities of the natural product and analogs to better
understand the specific target of promysalin, all of which will
be reported in due course.
Finally, during the course of these studies we serendipi-
tously observed that 1a inhibited fluorescence in PP KT2440
when compared to either the control or compounds 1b-d
(Figure 3C). Pyoverdine is a siderophore produced by a wide-
range of Pseudomonads and is responsible for their fluores-
cent properties.20 Furthermore, it has been shown that pyo-
verdine-deficient mutants of P. syringae pv. tabaci 6605 ex-
hibited reduced virulence in host tobacco infection.21 Recent
reports have shown that strains deficient in pyoverdine have
increased swarming and biosurfactant phenotypes,22 in ac-
cordance with observations reported herein. Taken together,
this suggests that promysalin is either directly or indirectly
affecting pyoverdine biosynthesis and/or transport in this
strain.
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Supporting Information
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Experimental procedures, characterization data, NMR spectra,
and supporting figures. This material is available free of charge
on the ACS Publications website at http://pubs.acs.org..
AUTHOR INFORMATION
Corresponding Author
In conclusion, we report a concise, stereocontrolled syn-
thesis of the four diastereomers of the L-proline series of the
natural product promysalin guided by bioinformatics. The
compounds were synthesized in a longest linear sequence of
eight
* wwuest@temple.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation
under CHE-1454116 and Temple University through the Un-
dergraduate Research Program (K.W.K.) and a Summer Re-
search Award (W.M.W.). We thank B. A. Buttaro, M. C. Jen-
nings, C. DeBrosse, and C. Ross (Temple University) for in-
valuable assistance, Materia, Inc. for olefin metathesis cata-
lysts, and G. A. O’Toole (Dartmouth Medical School) and R.
De Mot (KU Leuven) for the generous donation of strains.
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1
culminated in compound (-)-1a, identical by H NMR, 13C
NMR, and HRMS to that of the isolated material, which we
propose to be the structure of promysalin. Furthermore, bio-
logical investigations support that the synthesized enantio-
mer is that of the natural product. Finally, we demonstrate,
for the first time, that promysalin disperses established bio-
films and inhibits pyoverdine production, two pathogenic
phenotypes, which may hint at the role the compound plays
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