A Photoactivatable Small-Molecule Probe for the In Vivo Capture of Polyketide Intermediates

Article abstract of DOI:10.1002/chem.201903661

A photolabile carba(dethia) malonyl N-acetylcysteamine derivative was devised and prepared for the trapping of biosynthetic polyketide intermediates following light activation. From the lasalocid A polyketide assembly in a mutant strain of the soil bacterium Streptomyces lasaliensis, a previously undetected cyclised intermediate was identified and characterised, providing a new outlook on the timing of substrate processing.

Full text of DOI:10.1002/chem.201903661

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Title: A photoactivatable small molecule probe for the in vivo capture of
polyketide intermediates
Authors: Manuela Tosin, Samantha Louise Kilgour, and Robert Jenkins
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A photoactivatable small molecule probe for the in vivo capture
of polyketide intermediates
[a]
[a]
[a]
Samantha L. Kilgour, Robert Jenkins and Manuela Tosin*
Abstract: A photolabile carba(dethia) malonyl N-acetylcysteamine
derivative was devised and prepared for the trapping of biosynthetic
polyketide intermediates following light activation. From the lasalocid
A polyketide assembly in a mutant strain of the soil bacterium S.
lasaliensis,
a previously undetected cyclised intermediate was
identified and characterised, providing a new outlook on the timing of
substrate processing.
Polyketide natural products constitute an abundant family of
secondary metabolites comprising prominent pharmaceuticals
and agrochemicals such as fidaxomicin and avermectin.[1] They
are biosynthesized by multifunctional enzymes known as
polyketide synthases (PKSs) through a common pathway: this
involves multiple decarboxylative Claisen condensations of acyl
Figure 1. (A) Premature polyketide chain termination for intermediate capture
via
a nonhydrolysable carba(dethia) malonyl N-acetylcysteamine 2: this
competitively interferes with polyketide chain assembly (dashed pathway) to off-
load enzyme-bound intermediates (3).[ (B) Newly devised photolabile DMNB
probe (4) for polyketide intermediate capture following light activation (h).
Legend: ACP= Acyl Carrier Protein; AT= Acyl Transferase; KS= Ketosynthase;
KR= ketoreductase.
5a]
carrier protein (ACP) or CoA-bound malonyl
units onto
ketosynthase- (KS) bound acyl groups (Fig. 1A). Newly generated
ACP- bound intermediates ‘grow’ in length and chemical
complexity thanks to the action of reductive enzymes (e.g.
ketoreductases, dehydratases, and enoylreductases, ERs) until
the end polyketide product is released from a PKS (typically by
thioesterase-, TE-, mediated hydrolysis/cyclisation); further
enzymatic tailoring ultimately afford the mature bioactive natural
product.
thiotetronates.[5-7] More recently the chemical chain termination
strategy was successfully extended to the study of nonribosomal
peptide synthetases via the use of nonhydrolysable mimics of
PCP-bound amino acids. [8]
In utilising our chemical tools for the investigation of natural
product pathways in Gram-positive bacteria, Gram-negative
bacteria and fungi, [7a] we found that chemical modifications in the
probe structure greatly affect its bioavailability and intermediate
capture ability in vivo. For instance, variation of the N-acyl moiety
of ester-masked probes (1) affects both cell permeation and the
extent of in vivo ester hydrolysis of 1 to generate the active probe
2. [6c] Besides, the use of a labile acetoxymethyl ester as a -
ketoacid protecting group for 2 leads to increased titers of
captured intermediates, likely due to increased amounts of 2
generated in situ. [6c] Nonetheless tool development is still needed
in order to improve our ability to dissect challenging biosynthetic
pathways.
Amongst the most attractive approaches employed to study,
control and modulate biological process is the use of light: indeed
photosensitive groups do not require the use of (bio)chemical
reagents for their cleavage/activation, and this last can be
selectively achieved at biologically non-damaging wavelengths
with high spatio-temporal precision.[9] Several photoactivatable
PKSs are classified as modular or iterative and into different
types on the basis of their structural organization and modus
operandi.[2] A detailed knowledge of PKS biosynthesis pathways
constitutes the basis of rational enzyme engineering for the
production of commodity and high value chemicals. [3] Over time
key insights into PKS workings have been provided by molecular
biology, enzymology, analytical chemistry and structural studies.
[
3b, 4]
Nonetheless challenges in PKS studies remain; these are
mainly associated with limitations in our ability to closely monitor
the biocatalysis in its natural context and thereby determine the
exact mechanism and order of enzymatic transformations.
In our group we have devised a chemical approach for
polyketide biosynthetic investigations based on the use of ‘chain
termination’ probes capable of intercepting PKS biosynthetic
intermediates throughout product assembly. The probes are
carba(dethia) N-acetylcysteamine (NAc) derivatives which mimic
the ACP-malonyl extender units utilised in polyketide formation (2,
Fig. 1) and have been utilised for in vitro and in vivo studies to
shed light on previously unknown details of complex polyketide
formation, such as that of polyethers, macrolides and
moieties have been devised and employed in chemical biology for
10]
a
wide range of applications,[
such as aryl azides,
benzophenones and diaziridines to generate electrophiles for
photoaffinity receptor tagging;[11] phenacyl, benzoin and
nitrobenzyl protecting groups for the ‘uncaging’ of
effector/signaling
conformational switches of gene expression
molecules;[12]
and
azobenzenes
as
[
a]
Samantha L. Kilgour, Robert Jenkins and Dr M. Tosin
Department of Chemistry
[
13]
amongst many
University of Warwick
Library Road, Coventry ,CV4 7AL, UK
E-mail: m.tosin@warwick.ac.uk
others. In the context of natural products, photolabile ‘unnatural’
amino acids have been incorporated into proteins and peptides
via feeding experiments and genetic encoding for protein/peptide
labelling. [14] Furthermore, several medically relevant secondary
Supporting information for this article is given via a link at the end of
the document
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Chemistry - A European Journal
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COMMUNICATION
(A)
(B) m/z
= 572.3922 ꢀ 0.0050
0
h
1
0
1 h
2 h
[
M+Na]
+
1
0
[
M+Na]
+
1
5
20
25
30
35
40
45
t/ min
ms
2
377.2667
1
0
organic
extraction
417.2983
572.3915
C
32
H
55NNaO
6
Exact mass: 572.3922
24 4
C H42NNaO
Exact mass: 417.2975
C H
21
38NaO
4
Exact mass: 377.2668
m/z
2
00
300
400
500
600
700
newly detected captured intermediates
Figure 2. (A) Overview of advanced putative intermediate capture from the lasalocid A polyketide synthase during the fermentation of S. lasaliensis ACP12 (S970A)
n
via the newly devised photolabile probe 4. The stereochemistry of the putative captured species 7-10 has yet to be established. (B) UPLC-HRMS characterization
of the newly detected putative intermediate 10 following light activation of 4.
metabolites bearing photoactive moieties have been chemically
synthesized in order to identify their natural product target and
mechanism of action. [15]
To the best of our knowledge photoactivatable small
molecule have yet to be utilised in the study of secondary
been found to harbor several enzyme-bound biosynthetic
intermediates by us extensively characterised, and to generate
unnatural polyether species such as 7 in the presence of ‘chain
termination’ probes such as 2.[6]
Both liquid and agar-plate cultures of the S. lasaliensis
ACP12 (S970A) strain were supplemented with 4 (without
photolysis), either in a single amount on day 1 of bacterial growth
or in multiple smaller portions throughout days 2 to 5 of
fermentation to achieve a similar final concentration (2.5 mM). 4
was found to be almost quantitatively hydrolysed over 5 days in
organic extracts belonging to ‘day 1’ supplementation
experiments, whereas it remained almost intact in samples
deriving from ‘2-to-5’ daily supplements (Fig. 3S). This indicated
that 4 is a relatively poor substrate for in vivo esterases, unless
incubated in fermentation conditions for prolonged periods of time.
In all the extracts belonging to unphotolysed samples, traces of
the unnatural polyketide 7 and its linear unoxidised precursor 9
(both deriving from the off-loading of intermediates bound to the
lasalocid A synthase) were detected and characterised as
previously reported (Fig. 4S and 5S),6 indicating that even the
presence of a small amount of 2 leads to intermediate capture.
The same in vivo experiments were then carried out subjecting
the bacterial solid and liquid cultures to light irradiation via the in-
house built UVA light box (as detailed in the SI). Culture irradiation
was carried out for up to 2 hours daily over a period of 5 days in
order to minimise disruption on cell growth and metabolism.
Exposure to light irradiation did not affect cell growth and
deprotection of 4 followed by decarboxylation to 2 was achieved
in vivo; the extent of it varied upon culture conditions and was
much more evident in liquid cultures supplemented with daily
doses of 4 (Fig. 6S). Intriguingly, the most interesting results in
terms of biosynthetic intermediate capture came from the
photolysis of liquid cultures supplemented with 4 in day 1. Besides
the previously detected 7 and 9 generated in substantially higher
amounts (Fig. 7S and 8S), organic extract analyses by UPLC-
HRMS revealed the presence of a putative oxidised and cyclized
nonaketide, possibly dehydrated (10, Fig. 2 and 9S). This species
metabolite assembly. Therefore we decided to utilise
a
photolabile protecting group to mask the active -ketoacid moiety
of our polyketide chain termination probes to establish whether 1)
the group could be photolysed in vivo during live bacterial
fermentation, and 2) it could be advantageous in the capture of
biosynthetic intermediates. The photoactive group chosen for our
studies was the 4,5-dimethoxy-2-nitrobenzyl (DMNB) group,
providing straightforward synthetic incorporation and cleavage
upon irradiation at 365 nm, generating a low toxicity by-product
(
o-nitrosobenzaldehyde).[16] The DMNB moiety has been used to
protect alcohols, thiols, selenols, amines, phosphates and
carboxylic acids for biological applications. [12, 15-17] Various
structural modifications of DMNB have been devised to increase
its photolysis quantum yield; this has been shown to be
dependent on the nature of the leaving group in the DMNB
substrate, with esters displaying the lowest quantum yield in
comparison to ethers and amines. [9a]
The preparation of the DMBN probe 4 (Fig. 1) as the first
photoactivable tool for polyketide biosynthetic investigations was
accomplished from -aminobutyric acid 5 in 3 steps (Scheme 1S
and related Supplementary Information, SI). Photolysis studies of
4
in different organic solvent and buffers were performed
employing different light sources. 4 could be quantitatively
photolysed over a period of 2-4 hours in water by irradiation with
either a KiloArc™ Broadband Arc Lamp (1000 W, 2 hours) or with
an in-house built light box containing a circular 22W UVA lamp in
water (4 hours), or in MYM Streptomyces medium (6 hours) at
similar concentrations (0.5- 2.0 mM, SI). We then trialed its use in
vivo during the growth of S. lasaliensis ACP12 (S970A), a model
bacterial strain extensively utilised for the investigation of the
antibiotic lasalocid A assembly in our labs. This mutant strain, for
which the last ACP of the lasalocid A synthase is inactivated, has
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2
was characterized by HR-MS experiments, which displayed an
m/z fragment of 377 characteristic of the lasalocid A polyether part
of the molecule (Fig. 2B); a possibly related species weighing 2Da
less and displaying an m/z 377 fragment was also observed (Fig.
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assembly with second-generation chain termination probes have
allowed us to off-load and capture several putative biosynthetic
intermediates, including partially and fully processed linear
nonaketides such as 8 (Fig. 2).[6c] To date only putative
undecaketides and dodecaketides featuring polyether moieties
had been identified; these would derive from the processing of
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off-loading of a fully processed PKS-bound octaketide from
module 9; or that the tailoring epoxidation-epoxide hydrolysis
cascade might take place on PKS-bound intermediate(s) earlier
than previously envisaged.[6a] Whereas recombinant LasC and
LasB are capable of processing substrate mimics of different
nature and complexity,[18] the nature of the true substrates for
these enzymes in vivo remains debatable. Nonetheless the
detection of 9 and other putative captured species in specific
microorganism fermentation and photolysis conditions exposes
how the timing and the flux of polyketide/polyether intermediate
formation is much susceptible to local and global perturbations
and still holds several intriguing aspects worthy of further
investigation and exploitation.
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In summary we have here obtain novel insights on lasalocid
A polyketide assembly utilising a novel light-activatable tool in vivo.
Further investigations into the modus operandi of 4 and the
development of other light-controlled tools for biosynthetic studies
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[
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Acknowledgements
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The authors gratefully acknowledge BBSRC (project grant
BB/J007250/1 to M. T. and MIBTP PhD studentship to S.L.K.);
EPSRC (DTA studentship to R. J.); the Sadler and Stavros groups
1929-1935; b) R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z.
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(Warwick Chemistry) for the trialling of various light sources; Rod
Wesson (Warwick Chemistry) for building the UVA light box for in
vivo photolysis; Dr Lijiang Song and Philip Aston (Warwick
Chemistry) for assistance with UPLC-MS analysis.
[
[
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n
Keywords: photoactivatable probes · polyketide biosynthesis ·
intermediate capture
565, 112–117.
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Samantha L. Kilgour, R. Jenkins and
Manuela Tosin* Page No. - Page No.
A photoactivatable small-molecule
probe for the in vivo capture of
polyketide intermediates
A 4,5-dimethoxy-2-nitrobenzyl (DMNB)-protected probe was devised and utilised for
the capture of polyketide-derived species in bacterial fermentative cultures.
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