Synthesis and Investigation of the Abiotic Formation of Pyonitrins A-D

Article abstract of DOI:10.1021/acs.orglett.0c00098

Pyonitrins A-D are recently isolated natural products from the insect-associated Pseudomonas protegens strain, which were isolated from complex fractions that exhibited antifungal activity via an in vivo murine candidiasis assay. Genomic studies of Pseudomonas protegens suggested that pyonitrins A-D are formed via a spontaneous nonenzymatic reaction between biosynthetic intermediates of two well-known natural products pyochelin and pyrrolnitrin. Herein we have accomplished the first biomimetic total synthesis of pyonitrins A-D in three steps and studied the nonenzymatic formation of the pyonitrins using ¹⁵N NMR spectroscopy.

Full text of DOI:10.1021/acs.orglett.0c00098

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Synthesis and Investigation of the Abiotic Formation of Pyonitrins
A−D
Rahul D. Shingare, Victor Aniebok, Hsiau-Wei Lee, and John B. MacMillan*
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ABSTRACT: Pyonitrins A−D are recently isolated natural products from the insect-associated Pseudomonas protegens strain, which
were isolated from complex fractions that exhibited antifungal activity via an in vivo murine candidiasis assay. Genomic studies of
Pseudomonas protegens suggested that pyonitrins A−D are formed via a spontaneous nonenzymatic reaction between biosynthetic
intermediates of two well-known natural products pyochelin and pyrrolnitrin. Herein we have accomplished the first biomimetic
total synthesis of pyonitrins A−D in three steps and studied the nonenzymatic formation of the pyonitrins using 15N NMR
spectroscopy.
Our laboratory has been interested in the study of natural
products that involves nonenzymatic or spontaneous reactions
in their formation, such as the discoipyrrole, bohemamines,
and ammosamides (Figure 1).3 As part of these previous
studies, we have used isotope labeling approaches to validate
the nonenzymatic reactions and further understand the cascade
he pyonitrins are a family of alkaloids recently discovered
Tfrom an insect associated strain of Pseudomonas protegens.
The molecules were identified from a powerful assay platform
to identify microbial-derived natural products with efficacy in
an in vivo model of Candida albicans infection.1 Candida
species commonly cause bloodstream infections that resulted
in 34,800 hospitalizations and 1,700 deaths of patients in the
year 2017 in the United States.2 In the assay, partially purified
fractions were able to inhibit C. albicans growth. Bioassay
guided purification using in vitro antifungal activity led to
isolation of less than 0.5 mg of purified pyonitrin A (1) and B
(2) and only trace quantities of pyonitrin C (3) and D (4).
The in vivo activity could not be fully confirmed due to the
limited quantities.
In addition to the potential in vivo biological activity, our
interest in these molecules was inspired by their proposed
biosynthetic origin. Portions of the pyonitrins are structurally
related to the known microbial metabolites pyrrolnitrin (5)
and pyochelin (6). In the initial publication, Mevers et al.
identified independent and functional biosynthetic gene
clusters for 5 and 6. Based on the organizational structure of
the biosynthetic gene clusters and the qualitative analysis of
intermediates, the proposal was made that aerugaldehyde (7),
a shunt metabolite of the biosynthetic pathway of (6), and an
aminopyrrolnitrin derivative (8) undergo nonenzymatic imine
formation, followed by a spontaneous cascade cyclization/
oxidation (Figure S1).1 Preliminary evidence for the
biosynthetic proposal was provided via a microscale reaction
of 7 and 24 in fermentation media to provide 3.1
Figure 1. Examples of bioactive natural products that utilize one or
more nonenzymatic transformations in their formation.
Received: January 8, 2020
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reactions that take place in the complex chemical environment
of microbial fermentations. Due to the synthetic tractability of
7 and 8, the exciting preliminary biological activity, and the
spontaneous reactivity, we carried out a total synthesis of 1−4
and utilized a 15N-labeled version of 8 to further study the
nonenzymatic imine formation and subsequent spontaneous
cascade.
Retrosynthetically, we planned to synthesize pyonitrins A−
D via a Pictet−Spengler reaction between 8 and 7, the
biogenesis products of pyrrolnitrin and pyochelin, respectively
(Figure 2).
conditions led to precipitation of 1, we looked to the use of
organic solvent to keep the product in solution. The Pictet−
Spengler reaction of 22 and 7 in DMSO with TFA (1%) at
room temperature for 36 h led to the production of pyonitrin
A in 61% yield. The structure of synthetic pyonitrin A was
confirmed by comparison of our NMR and high resolution
mass spectral data with the literature report (Table S1).8 The
synthesis of the remaining pyonitrins B−D from the
corresponding aminopyrrolnitrins 23, 24, and 25 and 7 was
achieved using the developed Pictet−Spengler reaction
protocol (1% TFA in DMSO solvent for 36 h) (Scheme 2).
Scheme 2. Synthesis of Pyonitrins A−D (1−4)
Figure 2. Retrosynthetic analysis based on biogenesis of pyonitrins
A−D.
Synthesis of 7 was started from condensation of L-cysteine
methyl ester hydrochloride (9) and 2-hydroxy cyanobenzene
(10) to afford thiazoline derivative (11) in 77% yield.4
Dehydrogenation of 11 with bromotrichloromethane and DBU
at −20 °C gave thiazole (12) as a sole product in quantitative
yield.5 The methyl ester was hydrolyzed to the acid using
lithium hydroxide and transformed into a Weinreb amide (13)
using standard conditions. Reduction of 13 with LAH afforded
7 in excellent yield (Scheme 1).
In natural product biosynthesis, the Pictet−Spengler
condensation is typically a highly regulated enzyme catalyzed
process, and 1−4 are the first examples of the nonenzymatic
Pictet−Spengler condensation in natural products.1 Similar to
our previous work on nonenzymatic condensation/cyclization
transformations, such as the discoipyrroles, our goal was to
monitor the formation and disappearance of key intermediates
in the nonenzymatic formation of the pyonitrins in real time.
We wanted to exploit the large chemical shift range of 15N
NMR due to the electronic interactions involving the lone pair
of electrons on the nitrogen atoms.9 To this end, we have
utilized heteronuclear multiple-bond correlation (HMBC)
Scheme 1. Synthesis of Aeruginaldehyde 7
1
NMR experiments to identify H−15N heteronuclear correla-
tion of key intermediates formed in real time. In order to
create a probe for both structural changes and intermolecular
interactions in compounds that contain nitrogen atoms with
changing chemical environments, an 15N-labeled starting
material was required. As a result, we began our NMR study
of the abiotic formation of the pyonitrins with the synthesis
and utilization of 15N-labeled aminopyrrolnitrin (26), whereby
the aniline contained the 15N isotope label.
Synthesis of pyonitrins A−D began with Suzuki−Miyaura
cross-couplings of the pyrrolepinacolboronote esters (16−17)
and the corresponding aniline (14−15) derivatives in the
presence of catalytic palladium diacetate (SPhos, K3PO4, aq.
nBuOH at rt) to obtain compounds 18−21 in 65−90% yield.6
Final preparation of the aminopyrrolnitrin analogs 22−25 with
differing chlorination patterns was achieved by removal of the
silyl protecting group using TBAF. With the coupling partners
in hand we turned our attention to the final Pictet−Spengler
reaction. To mimic the aqueous fermentation conditions, we
attempted the Pictet−Spengler reaction of 22 and 7 in water at
room temperature for 36 h under acidic conditions (10%
TFA).7 Under these conditions, 1 precipitated out of the
aqueous solution with an overall yield of 56%. While
conditions for the Pictet−Spengler reaction could clearly be
optimized, we were looking for conditions that would be
All the NMR experiments were performed using 26 (1
equiv) and 7 (1 equiv) in 1% TFA in 700 μL of DMSO-d6.
Experimentally we began by carrying out the Pictet−Spengler
condensation with 26 and conducted continuous reaction
monitoring in 30 min intervals for 48 h using 1H−15N HMBC.
26 has a 15N shift of 57.8 ppm and a strong correlation to
aromatic protons of the aniline ring at 6.60 and 7.09 ppm
(Figure 3). Immediately (30 min) after addition of 26 and 7 in
1% trifluoroacetic acid in DMSO-d6, a new, strong signal
appeared at δ 15Ν 70.7 ppm with HMBC correlation to
protons at 6.09, 6.57, 6.95, 6.96, 7.13, and 7.18 ppm suggesting
the formation of a 1,2-dihydroquinoline moiety (28). The
1
1
compatible with NMR studies, such as H−15N HMBC NMR
cross peak with a H−15N shift of 11.00 and 151.73 ppm
spectroscopy, to resolve the reaction pathway. As the aqueous
corresponds to nitrogen of the pyrrole on 28. This was
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Figure 3. (a) Formation of pyonitrin A along with the intermediates. (b) Representative time points from H−15N HMBC experiments.
surprising since we expected to see the formation of the imine
intermediate (27) prior to cyclization. After 90 min, the
1H−15N HMBC correlations showed a new 15N signal at 270
ppm appears with correlations to protons at 8.70, 8.34, 7.78,
7.54 ppm, representing formation of 15N-labeled pyonitrin A
(29). These were the only intermediates observed in the
reaction. We stopped the NMR experiment after 48 h, and the
1
ratio of the product to 28 was determined to be 1:2 via a H
NMR (Figure 3 and video S1). The reaction was allowed to
continue for an additional 48 h to obtain complete conversion
to 29.
When compared to the media fermentation conditions, a 1%
solution of TFA in DMSO-d6 is far too acidic and might
promote the cyclization of 27 to 28. We decided to lower the
percentage of TFA and redo the reaction in order to try and
detect the formation of 27. With samples of 15N-labeled
Figure 4. Real time investigation of imine 27 formation by
1
comparison of H NMR of starting materials 26, 7 and intermediate
28.
aminopyrrolnitrin 26, aeruginaldehyde 7, pyronitrin A, and
1,2-dihydroquinoline 28 we reran the experiment using only
1H NMR. The experimental design was as follows: Using 26 (1
cyclization of the imine drives formation of the pyonitrin A
equiv) and 7 (1 equiv) in 0.05% TFA in DMSO-d6, an array of
from any aminopyrrolnitrin and aeruginaldehyde molecules.
1
1 scan H NMR experiments were run and the spectra were
A number of microbial fermentations lead to the co-
compared against the starting materials and products, including
occurrence of aldehydes and amines. Many of these, however,
the dihydroquinoline intermediate 28 (Figure 4). Interestingly,
as soon as the starting materials were added together, we could
immediately see the formation of 28 and not 27 like we
expected. This might be due to the fact that as soon as the
imine intermediate is formed, the inherent reactivity and
proximity of the imine allow for immediate cyclization to 28.
We believe this is significant in context to the biological
do not lead to the production of a new chemical. A vast
number of aldehyde and amine reactions are in equilibrium
with the imine product. Depending on the conditions, the
equilibrium can lie with the starting materials. In order for
these reactions to proceed efficiently, especially in the case of
starting materials in low concentrations, a strong driving force
is needed to shift the equilibrium toward the imine. In the case
system, since the imine intermediate that is formed from the
reaction of aminopyrrolnitrin and aeruginaldehyde is not
observed in the study. The low probability that two
biosynthetic intermediates come together in the bacterial
milieu to form the imine intermediate makes the inherent
reactivity of the two starting materials imperative for pyonitrin
formation. Interaction between the two starting materials in
the biological system and subsequent imine formation is the
limiting factor in the formation of pyonitrin A. As a result,
potential reversibility of imine formation would hinder
conversion to the final product. The fast and spontaneous
of the pyonitrins, irreversible cyclization causes the equilibrium
to shift toward a novel natural product.
In conclusion, we have accomplished the first biomimetic
total synthesis of antifungal chimeric natural product
pyonitrins A−D in short synthetic sequence. We have utilized
the Suzuki−Miyaura cross-coupling and Pictet−Spengler
reactions as the key steps in our synthesis. The developed
route is scalable and highly amenable for the synthesis of
diverse library of compounds around the pyonitrin scaffold to
study the structure−activity relationship (SAR). The biological
evaluation of the pyonitrins A−D against the Candida albicans
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(c) Pan, E.; Oswald, N.; Life, J. M.; Posner, B. A.; MacMillan, J. B.
Precursor-Directed Generation of Amidine Containing Ammosamide
Analogs: Ammosamides E-P. Chem. Sci. 2013, 4, 482−488.
(4) Tan, F.; Shi, B.; Li, J.; Wu, W.; Zhang, J. Design and Synthesis of
New 2-Aryl-4,5-dihydro-thiazole Analogues: In Vitro Antibacterial
Activities and Preliminary Mechanism of Action. Molecules 2015, 20,
20118−20130.
is under progress. The nonenzymatic nature of pyonitrin
formation was explored using NMR as a sensitive tool. Key
intermediates, such as the 1,2-dihydroquinoline, were shown,
and others, such as the proposed imine intermediate, were not
1
seen. By utilizing H−15N NMR, potential mechanisms of
pyonitrin formation were elaborated and studied and provide a
framework with which other interesting nonenzymatic
reactions can be explored.
(5) Williams, D. R.; Lowder, P. D.; Gu, Y.-G.; Brooks, D. A. Studies
of Mild Dehydrogenations in Heterocyclic Systems. Tetrahedron Lett.
1997, 38, 331−334.
ASSOCIATED CONTENT
* Supporting Information
(6) Morrison, M. D.; Hanthorn, J. J.; Pratt, D. A. Synthesis of
Pyrrolnitrin and Related Halogenated Phenylpyrroles. Org. Lett. 2009,
11, 1051−1054.
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The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c00098.
(7) Saha, B.; Sharma, S.; Sawant, D.; Kundu, B. Water as an Efficient
Medium for the Synthesis of Tetrahydro-β-carbolines via Pictet−
Spengler Reactions. Tetrahedron Lett. 2007, 48, 1379−1383.
General procedures, data tables, NMR spectra, and
additional data (PDF)
Time-lapse video showing the development of the
1H−15N HMBC spectrum during the Pictet−Spengler
reaction of 15N-labeled aminopyrrolnitrin 26 and 7
(MP4)
1
(8) As referenced in the original isolation paper, the H and 13C
chemical shifts were variable depending on the pH of the sample. As
such, the NMR values are not identical.
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(9) Marek, R.; Lycka, A.; Kolehmainen, E.; Sievanen, E.; Tousek, J.
15N NMR Spectroscopy in Structural Analysis: An Update (2001 −
2005). Curr. Org. Chem. 2007, 11, 1154−1205.
AUTHOR INFORMATION
Corresponding Author
■
John B. MacMillan − Department of Chemistry and
Biochemistry, University of California, Santa Cruz, Santa Cruz,
California 95064, United States; orcid.org/0000-0003-
1430-1077; Email: jomacmil@ucsc.edu
Authors
Rahul D. Shingare − Department of Chemistry and
Biochemistry, University of California, Santa Cruz, Santa Cruz,
California 95064, United States
Victor Aniebok − Department of Chemistry and Biochemistry,
University of California, Santa Cruz, Santa Cruz, California
95064, United States
Hsiau-Wei Lee − Department of Chemistry and Biochemistry,
University of California, Santa Cruz, Santa Cruz, California
95064, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c00098
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge NIH Grant R01CA225960 and
R01CA1499833for supporting this work and NIH Grant
S10OD018455 for supporting the acquisition of the Bruker
800 MHz NMR.
REFERENCES
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Chimeric Natural Products Produced by Pseudomonas protegens. J.
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(2) https://www.cdc.gov/drugresistance/pdf/threats-report/
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D
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