Step-Economic Synthesis of Biomimetic β-Ketopolyene Thioesters and Demonstration of Their Usefulness in Enzymatic Biosynthesis Studies

Article abstract of DOI:10.1021/acs.orglett.0c01348

Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic β-ketopolyene thioesters that is based on an Ir-catalyzed reductive Horner-Wadsworth-Emmons olefination. New β-ketotriene and pentaenethioates of pantetheine and N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.

Full text of DOI:10.1021/acs.orglett.0c01348

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Letter
Step-Economic Synthesis of Biomimetic β‑Ketopolyene Thioesters
and Demonstration of Their Usefulness in Enzymatic Biosynthesis
Studies
Johannes Wunderlich, Theresa Roß, Marius Schro
̈
der, and Frank Hahn*
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ABSTRACT: Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to
appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic β-ketopolyene thioesters that is based
on an Ir-catalyzed reductive Horner−Wadsworth−Emmons olefination. New β-ketotriene and pentaenethioates of pantetheine and
N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was
demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.
olyenes are important structural elements of pharmaco-
logically relevant polyketides and related natural products
handling. Finally, there is no efficient, general synthetic access
to this problematic compound class.
In reported syntheses of biomimetic polyene and β-
P
such as the mycolactones, the aurantosides, and the
1
−3
amphotericins.
Assembly-line-linked polyene thioesters are
ketopolyene thioesters, the polyene is introduced either in
4
,10,11
also often involved in intriguing biosynthetic processes, such as
those described for enediyne antibiotics, polycyclic tetramate
the form of a commercially available diene
or by
repetitive three-step elongation sequences of reduction,
4
−7
macrolactams, and the ambruticins.
Studies of such
oxidation, and Horner−Wadsworth−Emmons (HWE) olefi-
12−15
transformations are restricted by the limited access to realistic
substrate surrogates and their analogs.
nation.
The attachment of the β-keto SNAC thioester
fragment was achieved by olefination with activated β-
12−14
Acyl-carrier-protein (ACP)-bound β-ketopolyene thioesters
are the most frequently passed intermediates of polyene-
forming polyketide synthases (PKSs). In nature, they are
stabilized by the environment of the multidomain protein. In
in vitro assays, assembly line attachment is efficiently simulated
using biomimetic pantetheine and N-acetylcysteamine
ketocarbonyl surrogates and subsequent thiolysis
or by
aldol reaction, thiolysis, and Dess−Martin periodinane
4
,11
oxidation, respectively.
To the best of our knowledge,
there are only two literature reports about synthetic
biomimetic polyene thioesters with more than two conjugated
double bonds, a triene and a tetraenethioate of coenzyme A as
9
,15
(
SNAC) thioesters, whereby pantetheines are crucially better
well as a pentaenethioate of SNAC.
In the examples
8
surrogates in systems like fungal PKS. The respective mimics
of ACP-bound β-ketopolyene thioesters tend to undergo
various side reactions in solution, such as cycloadditions,
described, the synthetic aspects represented a rather minor
facet of a biosynthesis study so that the routes were not
optimized. In light of this situation, a strategy that renders
step-economic polyene assembly in conjunction with straight-
forward β-keto thioester introduction is highly desirable. Apart
from basic biosynthetic studies, efficient access to the target
compounds is also an essential component of a chemo-
enzymatic synthesis strategy that makes use of the previously
mentioned polyene-rearranging enzymes.
9
oxidations, or conjugate additions (Figure 1). Furthermore,
the polyene portion lowers the solubility in aqueous buffer, and
the amphipathic overall structure complicates the isolation and
Received: April 18, 2020
Figure 1. Biomimetic polyene thioesters like 1 are prone to several
side reactions.
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.orglett.0c01348
A
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Table 1. Introduction of the β-Ketothioester Fragment
R1
R2
procedure
a
product
scale (mmol)
overall yield (%)
b
all-trans (%)
entry
1
2
3
4
5
SPan-acetonide
SPan-acetonide
SNAC
SPan-acetonide
SNAC
Me
H
H
H
H
i
ii
ii
iii
iii
3a
3b
3c
3b
3c
2.67/0.64
0.50
0.50
0.40
0.50
17
55
58
61
72
67−75
93
86
>95
>95
a
(
i) 1. reduction (DIBAL-H), 2. oxidation (MnO ) (86% over two steps), 3. olefination (4, KHMDS; 51%), 4. thiolysis (HSPan-acetonide, 38%).
2
(
ii) 1. DIBAL-H, 2. MnO (81% over two steps), 3. 5a/5b, LiHMDS; 68% (3b)/71% for 3c. (iii) [IrCl(coe) ] , H SiEt then KOSiMe , 5a/5b.
2
2
2
2
2
3
b
HSPan-acetonide: pantetheine OO′-acetonide; see Table 2 for full structure. After flash chromatography on silica gel.
,
Table 2. Excerpt of the Screening for O,O′-Acetonide Deprotection Conditions
entry
R
acid
solvent
T (°C)
scale (μmol)
yield (%)
a
1
2
3
4
5
Me
Me
Me
Me
H
AcOH (33%)
Dowex G36 H
Dowex G36 H
Dowex G36 H
H O
rt
rt
50
60
rt
91
79
86
218
121
<5, hydrolysis
2
+
+
+
H O
2
no reaction
a
H O/MeOH (1:9)
2
50
a
b
H O/MeOH (1:9)
2
83, 26
b
TfOH (5 mol %)
CF CH OH
3
92
2
a
1
b
Content of the product after filtration of the crude product over a column of silica gel according to H NMR. Isolated yield.
We compared methods for the attachment of the β-
ketothioate motif by the olefination of dienoic model systems
a/2b (Table 1). A four-step sequence of reduction, oxidation,
benzoate. This method was not yet assessed for the synthesis
of β-keto enoates, enethioates, or polyenes with more than
three conjugated double bonds.
2
olefination with 4, and subsequent thiolysis led to 3a in an
rHWE between 2b and 5a or 5b, respectively, gave
improved yields of 3b or 3c, respectively, compared with the
three-step process (entries 4 and 5 versus entries 2 and 3,
Table 1). No detectable loss of all-trans content was observed,
showing that cis−trans isomerization is effectively suppressed
under the less basic conditions. The rHWE thus proved to be
compatible with the highly functionalized and protic thiol
components of the thioyl phosphonates 5a or 5b.
overall yield of 17% with a moderate all-trans content (entry
1
2,13
1
).
The low yield and the significant cis−trans isomer-
ization in the terminal step suggested avoiding thiolysis. We
therefore developed the β-keto thioyl phosphonates 5a and 5b,
which contain a protected pantetheine or a SNAC moiety,
1
respectively (Schemes S5−S9). Substituting the olefination-
thiolysis sequence by HWE olefination with 5a and LiHMDS
increased the yield of the pantetheine thioate 3b to 68% (55%
overall yield over three steps) and significantly reduced the
cis−trans isomerization (entry 2). A similar result was
observed for the homologous SNAC thioate 3c (entry 3).
A further improvement was achieved by adaption of a one-
pot−two-step Ir-catalyzed reductive HWE olefination (rHWE)
The removal of the O,O′-acetonide from 3b was expected to
represent a particular challenge due to the isomerization or
cyclization of the polyene potentially occurring under acidic
22
conditions. A screening of various deprotection conditions
was undertaken on the trienes 3a and 3b (Table 2, Table S1).
Strong acid was necessary to achieve exhaustive deprotection
at room temperature. The absence of water and rapid
quenching by neutral buffer minimized the formation of side
products. High isolated yields were reproducibly observed
using 5 mol % TfOH in 2,2,2-trifluoroethanol at room
temperature.
16
that was recently established by Jeon et al. It is based on the
reductive silylation of esters to give stable mixed O,O-silyl
acetals, like 6, which can be activated under benign Lewis basic
1
7−21
conditions.
These undergo clean, highly stereoselective
HWE olefination to trans-configured enoates upon treatment
with phosphonates under deprotonative conditions. Jeon et al.
successfully examined various enoates and ynoates and
accessed a methyl trienoate in only three steps from methyl
The established methodology was applied in the synthesis of
pentaene 1, to which all described problems of this compound
class are particularly applicable (Scheme 1). Starting from ethyl
B
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4−31
2
Scheme 1. Synthesis of the β-Ketopentaene
and tetraketidic SNAC thioesters and a few oxo esters.
Pantetheinethioate 1 Using Iterative rHWE
Studies with precursor surrogates of KR domains from late
PKS modules are largely neglected, probably due to a lack of
specific synthetic access to them.
The MycKRB domain from the mycolactone PKSs MLSA1,
MLSA2, and MLSB stands out for its intriguing specificity
32
profile. It is present in 13 of their 16 PKS modules and acts
on diverse di- to decaketidic substrates with 3-D selectivity
(
predicted for the olefin-forming modules). Despite this
seemingly highly conserved stereoselectivity, enzymatic in
vitro experiments of MycKRB with the diketidic SNAC
thioester S29 (Scheme S18b) gave mixtures of stereoisomers
27
with the unexpected (2L,3L)-S30 as the main product. Such a
reversal of stereoselectivity is not unusual and reflects the finely
24,25,29
balanced energetics of ketone reduction by KR domains.
For MycKRB, it was concluded that the ACP attachment
exercises a major influence on the stereoselectivity by
restricting the modes of substrate orientation during
24,25,27,29
catalysis.
An influence of the polyketide structure
was indicated but not further investigated due to the lack of
suitable substrate surrogates.
The MycKRB-containing PKS modules MLSB4−7 consec-
sorbate (2b), the rHWE with phosphonate 10 was applied
twice, yielding tetraene 9 (43 and 47% yields). It is noteworthy
that an inseparable side product was initially copurified along
with 8 and 9, which was annotated as an alkoxysilane
compound based on H NMR analysis (Figure S1). This
side product was detrimental for the HWE olefination and
utively build up the tetraene of the mycolactone side chain
(
Scheme S18b). Synthetic compound 7b represents a closer
mimic of the advanced intermediates S25−S28 than S29 and
was therefore subjected to an in vitro reaction with purified
MycKRB. The analysis of the crude assay product by UPLC-
1
1
MS, UV−vis, and H NMR spectroscopy showed that the
coincided with residual CH Cl that went into the reaction.
2
2
ketone in 7b was reduced by 66% into a secondary alcohol
Attempts to suppress its formation by varying the stoichiom-
etry of the reactants did not lead to a significant improvement.
Ultimately, the volatiles were fully removed by repetitive
(
Scheme 2). The crude assay product was derivatized by basic
Scheme 2. In Vitro Reaction of 7b with MycKRB and
Configurational Analysis
azeotropic evaporation with Et O after the reduction step, with
2
a substantial loss of the silyl acetal being accepted.
The established one-pot elongation with 5a gave the
protected 11 with a yield of 36% and no detectable cis−
trans isomerization. Acetonide deprotection under the
established conditions proceeded with an excellent yield of
9
2% after flash chromatography on silica gel. Overall, 1 was
accessed in only four steps from commercially available ethyl
sorbate (2b) in an overall yield of 7%. In comparison with this,
traditional routes would require at least 12 linear steps and
would suffer from significant cis−trans isomerization (Scheme
4
,11−14
S17).
To the best of our knowledge, this is the first
described example of a synthetic polyunsaturated β-keto
pantetheinethioester.
Finally, we aimed to demonstrate the utility of these
compounds for studies on biosynthetic enzymes. Ketoreduc-
tase (KR) domains are the first processing domain in the
reductive loop of canonical PKS modules. They catalyze the
NADPH-dependent reduction of ACP-bound β-ketothioester
intermediates (S21 in (Scheme S18) that result from
polyketide chain elongation. KR domains are categorized
according to the absolute configuration of their products
thioester cleavage, esterification, and hydrogenation on Pd/C
to transform 12 into a stable methyl β-hydroxydecanoate (13)
for determination of the enantiomeric excess. Chiral GC
analysis of the crude hydrogenation product showed an ee of
99.3% in favor of the D- (or (R)) enantiomer, which was
33
(S22). Downstream domains are often incompatible with
annotated on the basis of the specific rotation of the sample.
falsely configured KR products, resulting in assembly line
blockage and premature hydrolysis of PKS intermediates.
Analysis using the chiral derivatization agent 2-(anthracen-9-
yl)-2-methoxyacetic acid (9-AMA) confirmed the D-config-
uration and an ee of >95% (Figures S8−S11, Tables S2 and
23
Structure−function relationships of KR domains are therefore
of eminent importance for rational PKS engineering. Previous
in vitro studies of isolated KR domains uncovered relaxed
substrate specificity in conjunction with strongly substrate-
34
S3). This high D-selectivity of MycKRB is in stark contrast
with the moderate L-selectivity observed in the experiments
27
with SNAC-diketides. This result complements the previous
study from Weissman et al. and strengthens the idea that
besides the effects arising from the ACP attachment and
protein−protein interactions, the interaction of the KR domain
2
4−27
dependent stereoselectivity.
Whereas a substantial
number of KR domains have already been tested, the range
of polyketidic substrate surrogates is still restricted to di-, tri-,
C
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and the polyketide portion can play a crucial role in the KR
stereoselectivity. It suggests that studies of KR domains from
late PKS modules should, in general, be carried out with
substrate surrogates that mimic at least the most characteristic
structural features of the polyketide portion to ensure the
significance of the results for subsequent experiments on the
level of whole PKS modules or multimodular assembly lines.
In summary, we present an iterative, highly step-economic
strategy for the synthesis of biomimetic β-keto polyenethioest-
ers. The key to this strategy was the application of the rHWE
for both the polyene assembly and the introduction of polar
SNAC and pantetheine β-keto thioate fragments, respectively.
The utility of such compounds in enzymatic in vitro studies
was demonstrated by an assay of the KR domain MycKRB
with 7b, which reproduced the natural stereoselectivity in
contrast with a previous experiment with SNAC-diketides.
This underpins the general importance of substrate mimics
that resemble the specific structural features of a biosynthetic
precursor to obtain significant results for PKS chemical biology
from KR in vitro studies. Our work is the first reported study of
an isolated KR domain using a complex pentaketidic substrate
surrogate.
Marie Curie program of the European Union (project number
293430) is gratefully acknowledged. We thank the MS facility
of the Faculty BCG, University of Bayreuth and the
“Nordbayerische NMR-Zentrum”.
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