Modeling linear and cyclic PKS intermediates through atom replacement

Article abstract of DOI:10.1021/ja5064857

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation o

Full text of DOI:10.1021/ja5064857

Article
pubs.acs.org/JACS
Modeling Linear and Cyclic PKS Intermediates through Atom
Replacement
Gaurav Shakya,†,§ Heriberto Rivera, Jr.,‡,§ D. John Lee,‡ Matt J. Jaremko,‡ James J. La Clair,‡
Daniel T. Fox,‡ Robert W. Haushalter,‡ Andrew J. Schaub,† Joel Bruegger,† Jesus F. Barajas,†
Alexander R. White,† Parminder Kaur,† Emily R. Gwozdziowski,‡ Fiona Wong,† Shiou-Chuan Tsai,*,†
and Michael D. Burkart*,‡
†Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine,
California 92697, United States
‡Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California
92093-0358, United States
S
* Supporting Information
ABSTRACT: The mechanistic details of many polyketide
synthases (PKSs) remain elusive due to the instability of
transient intermediates that are not accessible via conventional
methods. Here we report an atom replacement strategy that
enables the rapid preparation of polyketone surrogates by
selective atom replacement, thereby providing key substrate
mimetics for detailed mechanistic evaluations. Polyketone
mimetics are positioned on the actinorhodin acyl carrier
protein (actACP) to probe the underpinnings of substrate
association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the
actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show
strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized
cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time
within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before
and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing
and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable
to a wide variety of PKSs.
structural evidence suggests that the process is dictated by
stabilizing residues within binding pockets of corresponding
synthases,8 but this remains hypothetical. Important questions
remain to be addressed, including: Why must chain elongation
reach full length before reduction and cyclization? How is site-
specific carbonyl reduction achieved? Are linear polyketones
sequestered by the ACP during chain elongation? Access to
elongated, polar mimics of ketide intermediates, such as 4−6
(Figure 1), could enlighten these uncertainties by their study in
complex with PKS proteins for binding, stabilization, and chain
transfer.
Although triketone units can be adapted synthetically, as
demonstrated by Barrett9a−c and Harris,9d,e preparation and
study of these and longer polyketones have been impeded by
inherent reactivity through spontaneous intra- and intermo-
lecular Aldol/Claisen condensations. We identified a practical
solution to this problem by replacing select carbonyls with sulfur
atoms, thereby thwarting spontaneous condensation and at the
Synthetic mimics of complex natural products are important
tools to evaluate the mechanisms of natural product biosynthesis
and to access syntheses that diversify the natural product
templates. For example, the advent of the Stork−Eschenmoser
hypothesis1 was a defining moment in terpene biosynthesis that
enabled synthetic mimickry2,3 to help elucidate the mechanisms
of terpene biosynthesis.4 Comparable intermediates in aromatic
polyketide biosynthesis have not been possible due to the
instability of nascent polyketones, hindering advancement in the
study of PKS mechanisms, timing, and processivity.5 Here we
describe an approach for generating polyketide intermediates via
atom replacement to mimic elongation intermediates in iterative
(type II) polyketide synthases. We demonstrate application of
these probes to evaluate the biosynthesis of actinorhodin,6
showing that these materials can mimic partially elongated and
cyclized substrates attached to the actACP7 through analysis of
intermediate association.
Figure 1 depicts the proposed biosynthesis of actinorhodin
(1). Few intermediates within this pathway have been
identified,5 leading to uncertainties in both the succession and
process of elongation, reduction, and ring closure. Current
Received: July 1, 2014
© XXXX American Chemical Society
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Figure 2. Structures of S atom replaced crypto-C8-actACP (4a). In this
design, select carbonyl groups are replaced by S atoms, therein reducing
number of potential Aldol/Claisen condensations. Access to 4a was
achieved by chemoenzymatic conversion of mimetic 11 to its
corresponding CoA analog by the action of three E. coli enzymes
CoaA, CoaD, CoaE followed by loading on the actACP by Sfp (see
Supporting Information).10−12 Mimetics 12 and 13 and their
corresponding crypto-loaded ACPs were not achieved due to instability.
While the installation of the thioethers in 12 and 13 eliminated select
Aldol/Claisen condensations, it did not ablate it. Ketide units are
denoted by bars and are colored according to replacement of the
carbonyl group with S atom (blue).
Figure 2), was selected for replacement. This involved the
preparation of tetraketide mimetic 11 (Figure 2), which would
be appended onto the actACP using chemoenzymatic methods
developed in our laboratories.11,12 After exploring multiple
approaches, we identified a route using an isoxazole as a tool to
install a protected diketone unit.13 As shown in Scheme 1,
a
Scheme 1. Synthesis of Tetraketide Mimetic 11
Figure 1. Proposed biosynthesis of 1 from holo-actACP (2). One
depiction of the enol/ketol tautomerization states has been shown, in
reality, multiple states likely exist. Gray bars denote ketide units.
same time providing probes to study the effects of length,
polarity, and hydrophobicity upon the PKS elongation and
cyclization process. We strategically substituted carbonyl groups
nonessential for cyclization and/or reduction with sulfur atoms
or isoxazole rings and validated their design via docking
simulations of the probes upon actACP (Supporting Informa-
tion). As shown in Figure 2, the corresponding thioethers would
serve as a chemical “knockout” of the reactive carbonyl moieties.
These probes differ from previously published analogs with the
inclusion of both polyketide mimics and the full 4′-
phosphopantetheine moiety, thus allowing us to evaluate the
significance of ACP−probe interactions. In addition, this atom
replacement design offers the added benefit of facile trans-
formation to sulfoxides or sulfones, offering the ability to install
greater polarity into the same parent compound.
a
Ar = 4-methoxybenzyl.
mimetic 11 was prepared through an eight-step route that began
with commercially available isoxazole 14. Reduction of 14 and
subsequent mesylation afforded 15, which was converted to
thioacetate 16 over the three steps.
Thioacetate 16 was then converted into acid 17 by a three-
step one-pot reaction sequence that began with deacetylation of
the thioacetal, alkylation of the incipient thiolate, and ester
hydrolysis (Scheme 1). Alternatively, addition of thioglycolic
acid to mesylate 15 also provided acid 17 (not shown, see
Supporting Information). Acid 17 was then coupled to
We began by targeting an atom replacement mimetic of the
ACP species bearing tetraketide unit 4 (Figure 1). Of the four
carbonyl groups, the third ketide, as shown in 4a (blue bar,
B
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protected-pantetheinamine 18 using conventional EDAC
coupling conditions to afford amide 19. The synthesis of
mimetic 11 was completed in three steps by removal of p-
methoxyphenylacetal using aq. AcOH, opening of the isoxazole
in 20 by treatment with Mo(CO)6 in refluxing aq. CH3CN, and
conversion of 21 to 11 by treatment with AcOH:H2O:CH3CN
(2:2:1). Using this route, we prepared 11 from isoxazole 14 in
10% overall yield over eight steps.
We then turned our attention to examination of mimetics
bearing chain-elongated ketides as illustrated by heptaketide 12
and octaketide 13 (Figure 2). Our goal was to compare the short
crypto-C8-actACP 4a to the longer crypto-C14 or C16 actACPs
as a means to validate the chain length specificity during
extension of the ketide chain. We began by evaluating the
synthesis of 12 and 13 (Figure 2). As depicted in Scheme 2, the
agent. Conversion of 27 to 29 was readily achieved using t-butyl
4-bromobutanoate (Scheme 2). Coupling acid 29 to 18,
followed by deprotection, completed access to octaketide
mimetic 23 in nine steps from 16 and 24 in 28% overall yield.
Isoxazole opening in 23 again led to an intractable mixture of
products. This indicated that stable mimetics required additional
atom replacement.
Fortunately, the solution arose from 20 (Scheme 1), 22
(Scheme 2), and 23 (Scheme 2). Here, the isoxazole motif not
only served as a synthetic tool but also provided a second type of
atom replacement (Figure 3). In this dual atom replaced model,
Scheme 2. Synthesis of Chain Elongated Mimetics,
a
Heptaketide 22 and Octaketide 23
Figure 3. Structures of dual atom replaced crypto-C8-actACP 4b,
crypto-C14-actACP 5b, and crypto-C16-actACP 6b. Access to 4b, 5b,
and 6b was accomplished by chemoenzymatic attachment of 20
(Scheme 1), 22 (Scheme 2), and 23 (Scheme 2), respectively, to the
actACP.10−12 Ketide units are denoted by bars and are colored
according to replacement of a carbonyl with a sulfur atom (blue) or
replacement of diketide unit with isoxazole (red).
both carbonyl and dicarbonyl units are replaced with thioether
and isoxazole units, respectively. Unlike mimetics 12 and 13,
long chain analogs such as 22 and 23 are readily accessed and
are stable, due to their inability to undergo intramolecular
Aldol/Claisen condensations.
a
Ar = 4-methoxybenzyl.
We applied these atom replacement mimetics to study
actACP substrate sequestration as detected by solution-phase
protein NMR.12,14 We appended mimetics 11, 20, 22, and 23 to
apo-actACP forming the corresponding crypto-C8-actACP 4a
(Figure 2), crypto-C8-actACP 4b (Figure 3), crypto-C14-actACP
5b (Figure 3), and crypto-C16-actACP 6b (Figure 3).12 We then
examined samples of proteins 4a, 4b, 5b, and 6b by solution-
synthesis of 12 began by converting isoxazole 24 to its mesylate
25 and coupling it to 16 using our tandem three-step coupling
process, as illustrated for the elaboration of 16 to 17 (Scheme
1). The resulting bis-isoxazole-acid 26 was then converted into
its corresponding thioacetate 27 in four steps. Again using the
tandem three-step coupling process, we were able to access acid
28 in high yield from 27. The resulting acid 28 was coupled to
protected pantetheinamine 18, which after deprotection,
afforded heptaketide 22 in nine steps from 16 and 24 in 19%
overall yield. Unfortunately, while multiple conditions and
methods were screened, the conversion of 22 (Scheme 1) to 12
(Figure 2) via isoxazole ring opening underwent rapid
condensation providing an intractable mixture.
10
phase H,15N-HSQC NMR (Figure 4a,c). Comparison of
crypto-C8-actACP 4a and isoxazole-containing crypto-C8-
actACP 4b to holo-actACP via chemical shift perturbation
(CSP) returned only very slight perturbations, indicative of a
substrate that is mostly solvent exposed and not sequestered on
the NMR time scale (Figure 4b).12 These results compare
favorably to known analog sequestration, where early inter-
mediates (C6 and smaller) have been shown to not sequester.14c
Octaketide 6b shows ∼30% larger CSPs over 5b in these key
residues, suggesting that the longer substrate, which mimics the
fully mature octaketide polyketone in 6, displays longer
1
Comparable complications arose in efforts to prepare mimetic
13 (Figure 2). Using thioacetate 27 as a central intermediate,
preparation of 13 required only modification of the alkylating
C
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Figure 4. Sequestration analysis of atom replaced mimetics. (a) 1H,15N-HSQC overlays depicting the holo-actACP (2, red) and the crypto-C8-actACP
(4a, pink), crypto-C8-actACP (4b, orange) bearing 11 and 20, respectively. (b) CSP plots obtained from the spectra shown in panel (a). (c) 1H,15N-
HSQC traces depicting overlays of the holo-actACP (2, red), crypto-C14-actACP (5b, green) bearing heptaketide mimetic 22 and crypto-C16-actACP
(6b, blue) bearing the octaketide mimetic 23. (d) CSP plots obtained from the spectra shown in panel (c). The docked actACP structure with these
probes can be found in Figure S34.
residency within the hydrophobic cleft. This pocket is delineated
by CSPs found in the same residues of actACP previously
reported to stabilize aromatic product analogs.12 Minimal CSPs
for 4a and 4b indicate that tetraketide intermediates are not
stabilized by interaction with the actACP, likely favoring further
elongation by the actKS/CLF. This supports the hypothesis that
sequestration of the elongating polyketone is driven by the
comparative energetic stability between the ACP sequestered
substrate and the ACP-KS/CLF complex. These studies suggest
that the mature polyketone is stabilized through association by
the hydrophobic cleft formed by movement of helix III of
actACP during chain elongation.
Next we turned to evaluate the latter, cyclized intermediates,
7−10 (Figure 1), proposed in the biosynthesis of actinorhodin
(1). We realized that this atom replacement methodology could
be uniquely extended for the study of the cyclization events that
occur in type II PKS pathways. These transformations have long
been unclear, as the intractable and spontaneous cyclization of
ketide intermediates (Figure 1) have prevented their prepara-
tion for detailed studies.8a,15 In particular, we were interested in
mimetics that would allow us to probe ACP sequestration as a
potential regulating force of substrate transport between
elongation and initial cyclization, as illustrated by the conversion
of 7 to 8 (Figure 1) by the actinorhodin ketoreductase (KR) as
well as the processivity between KR and the aromatase/cyclase
(ARO/CYC).15b
A central mystery in type II PKS ACP arises during the
processing of highly reactive polyketone intermediates to
cyclized products, as exemplified by the conversion of 6 to 10
(Figure 1). This process includes steps of cyclization, reduction,
and aromatization. We have hypothesized that intermediate
stabilization through sequestration of elongated intermediates
within the ACP may play a role during this transformation. To
test this hypothesis, we recently demonstrated that actACP
binds mature tricyclic product analogs between helices II and
IV.12 Crump and co-workers decisively showed that simple di-
and triketide analogs are not sequestered by actACP,7a,16 but
linear hydrocarbons do bind within the protein. Taken together,
these findings indicate that identity of the intermediates likely
plays a major role in substrate translocation between enzymes.
As demonstrated above, the presence of polar residues such as
D62 and D63 on helix III may stabilize the polar nature of
polyketone intermediates and may be better suited to the
environment of the actACP interior cavity. But what about the
monocyclic product of the KR? Is this species directly routed to
the ARO/CYC domain or does it return to the actACP pocket
between enzymatic transformations? To address these ques-
tions, the preparation of elongated monocyclic probes is critical
for further insight into the role of sequestration in type II PKS
biosynthesis.
To this end, analogs 8a−8c (Figure 5) were proposed as
probes that mimic the structural identity of putative
intermediates as a means of evaluating actACP sequestration.
D
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a
Analog 8a offered the most simplified mimetic by providing a
single aromatic ring with six ketide units. Analog 8b and 8c more
closely resembled the putative intermediate 8 (Figure 1) with
the addition of an upstream diketide, where the ketide units
extended from six in 8a and 8b to eight in 8c, respectively. Our
first goal in this effort was the preparation of 8a−8c for
phosphopantetheine attachment to 15N-enriched actACP for
solution-phase protein NMR studies of substrate sequestration.
Scheme 3. Synthesis of Mimetic 30
a
Ar = 4-methoxyphenyl.
mimetics 31 and 32 that provide approximate isosteric
placement of this additional diketide moiety. As depicted in
Figure 5, these materials expanded on our use of an isoxazole as
a diketide mimetic.
We then turned to the preparation of 8b and 8c from the
respective mimetics 31 and 32 (Figure 5). The synthesis of
mimetics 31 (Scheme 4) and 32 (Scheme 5) arose through
preparation of acid 41 as a central intermediate. Application of a
Vilsmeier−Haak formylation to 3717 produced crystalline 38 in
excellent yield. Aldehyde 38 was converted to its corresponding
oxime 39, which was then subjected to a 1,3-dipolar cyclo-
addition with isopropenyl acetate to afford isoxazole 40.
Preparation of acid 41 was then completed by hydrolysis
under basic conditions.
Two additional steps were required to complete the synthesis
of mimetic 31 (Scheme 4). Acid 41 was coupled to protected-
pantetheinamine 18 to deliver amide 42. Global deprotection
under conventional hydrogenation conditions provided 31 in
15% overall yield from 37. While mimetic 31 contained the
missing diketide unit from 30 (Figure 5), its chain length did not
a
Scheme 4. Synthesis of Mimetic 31
Figure 5. Structures of atom replaced crypto-actACP 8a, 8b, and 8c.
Access to 8a, 8b, and 8c was achieved by chemoenzymatic conversion
of mimetics 30, 31, and 32, respectively, to their corresponding CoA
analogs by the action three E. coli enzymes CoaA, CoaD, CoaE,10
followed by loading on the actACP by Sfp (see Supporting
Information).11 Mimetic 30 contains N and S atom replacement
(blue bars) and bears structural truncation (black-outlined bars). In
mimetics 31 and 32, 1,3-dicarbonyl units are replaced by isoxazole (red
bars). Two different length substrates were examined. Shaded gray bars
denote ketide units.
We began with the preparation of 8a by application of
hexaketide mimic 30. A general strategy for the synthesis of 30
involves the treatment of m-aminophenol (33) with thiodigly-
colic anhydride (34) to provide aromatic acid 35 (Scheme 3).
Acid 35 was then coupled to protected pantetheinamine 18 to
afford 36. Amide 36 was then deprotected by using aqueous
acetic acid to deliver mimetic 30 in 19% overall yield from 33.
While 30 was readily accessed, it lacks two of the terminal
ketides (black-outlined boxes, Figure 5). We next explored
a
Ar = 4-methoxyphenyl.
E
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match that of the putative intermediate 8 (Figure 5). We
therefore prepared a third mimetic 32 (Scheme 5) through the
addition of a six-carbon spacer.
Advantageously, acid 41 could be used to prepare a chain-
elongated mimetic 32 (Scheme 5). This began by preparing
amide 43 by coupling N-Boc-1,6-hexanediamine to 41. Boc-
deprotection followed by coupling of amine 44 to protected
pantothenic acid 45 afforded amide 46. A two-step sequence
involving deprotection of the benzyl ethers in 46, followed by
removal of the PMP acetal in 47 under acidic conditions,
provided mimetic 32 in 13% overall yield from 41.
a
Scheme 5. Synthesis of Mimetic 32
We then loaded mimetics 30-32 to 15N-enriched actACP
chemoenzymatically as previously reported10−12 to generate
crypto-actACP 8a, 8b, and 8c, respectively. Next, we subjected
crypto-actACP 8a−8c to solution-phase protein NMR studies
(Figure 6a). CSPs were observed (Figure 6b), which compare
the crypto-actACP 8a−8c to holo-actACP. We found that the
crypto-actACP loaded with cyclized hexaketide mimetic 30
showed slight CSPs; the crypto-actACP loaded with cyclized
mimetic 31 showed moderate CSPs; and the crypto-actACP
loaded with cyclized mimetic 32 showed large CSPs in residues
important to the interior sequestration cavity of actACP (Figure
6).12
On the NMR time scale, the fully elongated, cyclized
octaketide 32 had much higher residence time in the actACP
interior cavity than 30 or 31. The strongest perturbations were
observed in residues located at the end of helix III and the
following loop, which corroborate with the sequestration
residues that we previously reported in emodin-crypto-ACP.12
Interestingly, only species 8c showed moderate periodic
perturbations throughout residues in helix II and those
a
Ar = 4-methoxyphenyl.
Figure 6. Sequestration analysis of atom replaced cyclic intermediates. (a) 1H,15N-HSQC traces depicting overlays of the holo-actACP (2, red) and the
crypto-actACP (8a, orange) bearing hexaketide mimetic 30 (Figure 5). (b) 1H,15N-HSQC traces depicting overlays of the holo-actACP (2, red), crypto-
actACP (8b, green) bearing hexaketide mimetic 31 and crypto-actACP (8c, brown) bearing octaketide mimetic 32. (c) CSP plots obtained from the
spectra shown in panels (a and b).
F
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Notes
immediately preceding it. This information mirrored our study
on linear polyketide mimetics (Figure 4), which showed the
strongest sequestration of the fully elongated mimetic 32.
In this study, we have shown that selective replacement of
carbonyl groups with heteroatoms facilitates access to a diverse
class of polyketide mimetics that can be used to interrogate
polyketide biosynthetic enzymes. Inherent to this methodology,
these atom replacement mimetics can be chemoenzymatically
conjugated to ACP through the corresponding CoA analogs,
which provide the native scaffold to study these processes. While
no probe could ever exactly mimic the properties of natural
polyketone intermediates, this work demonstrates that mole-
cules that are much more polar than fatty acids14c or tricyclic
aromatics12 do associate with actACP, that longer chains
experience more residence time, and that cyclic intermediates
demonstrate the longest residence times. Our data, combined
with docking simulation of these probes (Figure S34), strongly
support that binding of both linear and cyclized polyketide
mimetics is not observed unless the mimetics are of sufficient
length. Taken together, these observations suggest that until the
polyketide has reached its terminal length, no preferential
sequestration by the actACP stabilizes the intermediate.
However, at full elongation, polyketide binding with the actACP
could facilitate release from the ketosynthase and assist transfer
to the KR for the first cyclization and reduction steps.15b
Binding of the first cyclized, reduced intermediate by the
actACP would then occur to facilitate release from the KR,
followed by delivery to the ARO/CYC.
In this study, the goal of these atom replacement mimetics
was to help understand the comparative binding and
stabilization of polyketone intermediates by ACPs. These
mimetics were not designed to serve as functional substrates
of PKS enzymes but instead to mimic the length, polarity, and
hydrophobicity found in natural intermediates. As with all
mimetics, the match was not perfect. In these examples, the C−
S bonds are considerably longer than those of their anticipated
ketides. The actual enol-keto states of natural polyketones
remain unknown, and the isoxazole moiety in part restricts
conformational freedom. These caveats aside, the establishment
of polyketide mimietics provides an excellent tool for inter-
rogating the iterative processes polyketide biosynthesis.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. X. Huang (UCSD), Dr. A. Mrse (UCSD), Dr. Y.
Su (UCSD), and Dr. P. Dennison (ICI) for assistance with
collection of NMR and MS data. We also thank Prof. C. A.
Townsend (Johns Hopkins), Prof. F. Ishikawa (UCSD), Prof. C.
D. Vanderwal (UCI), and Prof. A. R. Chamberlin (UCI) for
helpful advice. We also thank Prof. M. P. Crump for your advices
from full structural determinations as well as helpful discussions.
Funding was provided by National Institutes of Health
GM100305 and GM095970.
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ASSOCIATED CONTENT
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S
* Supporting Information
Copies of spectral data, synthetic methods, and experimental
procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
sctsai@uci.edu
mburkart@ucsd.edu
Author Contributions
§G.S. and H.R. contributed equally.
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