Bypassing Biocatalytic Substrate Limitations in Oxidative Dearomatization Reactions by Transient Substrate Mimicking

Article abstract of DOI:10.1021/acs.orglett.9b01398

Enzymatic oxidative dearomatization is an efficient way to generate chiral molecules from simple arenes. One example is the flavin-dependent monooxygenase SorbC involved in sorbicillinoid biosynthesis. However, SorbC requires a long-chain keto substituent at its phenolic substrate, thus preventing its application beyond the synthesis of natural sorbicillinoids or close structural analogues. This work describes an approach to broaden the accessible product spectrum of SorbC by employing an ester functionality mimicking the natural substrate structure during enzymatic oxidation.

Full text of DOI:10.1021/acs.orglett.9b01398

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Bypassing Biocatalytic Substrate Limitations in Oxidative
Dearomatization Reactions by Transient Substrate Mimicking
‡
Tobias M. Milzarek,‡ Manuel Einsiedler, Hulya Aldemir, Paul M. D’Agostino, Julia K. Evers,
̈
̈
Gesa Hertrampf, Katharina Lamm, Mert Malay, Anke Matura, Jonas I. Muller,
and Tobias A. M. Gulder*
̈
Chair of Technical Biochemistry, Technische Universitat Dresden, Bergstraße 66, 01069 Dresden, Germany
Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich, Technical University of
Munich, Lichtenbergstraße 4, 85748 Garching, Germany
S
* Supporting Information
ABSTRACT: Enzymatic oxidative dearomatization is an efficient way to
generate chiral molecules from simple arenes. One example is the flavin-
dependent monooxygenase SorbC involved in sorbicillinoid biosynthesis.
However, SorbC requires a long-chain keto substituent at its phenolic
substrate, thus preventing its application beyond the synthesis of natural
sorbicillinoids or close structural analogues. This work describes an approach
to broaden the accessible product spectrum of SorbC by employing an ester functionality mimicking the natural substrate
structure during enzymatic oxidation.
The ability to generate molecular complexity from readily
available, simple starting materials is key to the efficient
synthesis of structurally demanding small molecules, such as
natural products. Dearomatization reactions are a synthetic
tool for the rapid functionalization of arenes to give such
valuable synthetic building blocks. It is thus not surprising that
the development of new dearomative transformations has
received significant attention, in particular, over the last two
decades. This has led to a broad repertoire of methods,
including nucleophilic,1 photochemical,2 transition-metal cata-
lyzed,3 and enantioselective dearomatization reactions,4 also
for nonactivated aromatics.5 These techniques have also
significantly contributed to the development of streamlined
routes to complex natural products of diverse biosynthetic
origin.6
monooxygenases. Pioneering work by the Cox and Tang
groups revealed this transformation as the key step in the
biosyntheses of a variety of fungal natural products (Figure 1):
Nature has also developed a broad range of oxidative
dearomatization reactions that can be harnessed as comple-
mentary biocatalytic alternatives to chemical methods.
Impressive examples include aromatic dioxygenases that
catalyze the cis-1,2-addition of both oxygen atoms of molecular
oxygen onto their aromatic substrates, yielding the correspond-
ing 3,5-diene-1,2-diols.7 This enzymatic transformation was
discovered by Gibson et al.8 who also developed a first
recombinant Escherichia coli whole-cell system for its
application in synthetic chemistry.9 The product 3,5-diene-
1,2-diol structural portion can be further modified at the diol
(e.g., by Claisen rearrangements) and the diene (e.g., by
oxidative cleavage and (cyclo-)addition reactions), thus
providing versatile precursors for natural product synthesis.
Because of this synthetic value of dioxygenase products, these
enzymes have since been exploited in many total synthe-
ses.10,11 Another important biosynthetic dearomative reaction
is the oxidative dearomatization of phenols catalyzed by
Figure 1. Oxidative dearomatization of phenolic precursors as a key
step in the biosynthesis of the azaphilone (A), tropolone (B), and
sorbicillinoid (C) natural product families.
for example, aldehyde 1 gets oxidatively dearomatized by AzaH
to give 2, which upon cyclization delivers the azaphilone
derivative azanigerone E (3).12 In tropolone biosynthesis,
TropB transforms aldehyde 4 into 5, which gets further
elaborated to stipidaldehyde (6).13 Sorbicillin (7) is oxidized
by SorbC to the highly reactive sorbicillinol (8), which is
Received: April 22, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b01398
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
further diversified into the large sorbicillinoid natural product
family, for example, by dimerization to yield trichodimerol
(9).14
Scheme 1. Synthesis of the Ester Substrates 12−18 for
SorbC Substrate Screening
We recently showed that SorbC can also be used as a tool
for the in vitro assembly of sorbicillinoids. This led to the first
biocatalytic total syntheses of dimeric15 and further function-
alized sorbicillinoid natural products16 with complex molecular
architectures from synthetically readily available sorbicillin,
thereby significantly improving previous synthetic strategies to
access these compounds and developing first synthetic routes
to some congeners. However, our work and an additional study
evaluating the substrate scope by Narayan et al.17 that also
included AzaH and TropB revealed rather low substrate
promiscuity of these oxidative enzymes. While many
alterations of the substitution pattern at the aromatic portion
of sorbicillin are permitted for SorbC, the sorbyl side-chain (R2
in Figure 1) or a similar long alkyl- or alkenyl ketone
substituent is required to retain high catalytic efficiency and
stereoselectivity.15−17 To enable a more flexible variation of
the sorbicillinoid natural product structures at this position, for
example, for structure-activity relationship studies on this
biomedically rewarding natural product family,18 the avail-
ability of an approach to freely alter this side chain is highly
desirable. In addition, circumventing the need for a long-chain
keto substituent in the substrate would also permit the
application of SorbC in the total synthesis of other compounds
of the many examples of natural products accessible by
oxidative dearomatization.6 While the goal of broadening the
substrate scope might be achieved by protein engineering
approaches, we were interested in developing a straightforward,
simple, alternative approach that exclusively relies on suitable
modifications of the substrate structure.
To facilitate the evaluation of the enantioselectivity of the
SorbC-catalyzed oxidative dearomatization reaction, the
expected oxidation products 26−32 were next chemically
synthesized in racemic, O-acetylated (and thus chemically
stable) form as standards for analysis by high-performance
liquid chromatography (HPLC) on a chiral phase (Scheme 2).
Scheme 2. Chemical Synthesis of Racemic Acetates 26−32
using PIFA as the Oxidant
Given the apparent need of an alk(en)yl ketone substituent
at the substrate of SorbC to permit efficient and stereoselective
conversion to the oxidatively dearomatized product,15−17 we
anticipated that introduction of a moiety mimicking the natural
sorbyl side chain would permit the biocatalytic reaction to
proceed. Potential alternatives resembling the alk(en)yl ketone
could be based on a carboxylic acid, condensed with an
alk(en)ylamine to give the corresponding amide, an alk(en)-
ylthiol leading to the respective thioesters, or an alcohol to give
the corresponding oxoester. The latter was considered the best
option, as oxoesters can be saponified under mild conditions
for further downstream processing when compared to the
amide and in addition should be more easily accommodated
within the active site of the enzyme when compared to the
thioester. To comprehensively assess the substrate promiscuity
of SorbC, the synthesis of substrates with saturated ester
functions ranging from C1 to C6 as well as the ester of crotyl
alcohol bearing a double bond more closely resembling the
sorbyl side chain of the natural substrate was projected. The
precursor to these substrates, 2,4-dimethylresorcinol (10), was
obtained in two steps from methylresorcinol following a
published procedure.15 Carboxylation of 10 was conducted
Hence, esters 12−18 were O-acetylated with acetyl chloride to
give compounds 19−25 in 82−94% yield. Oxidative
dearomatization of 19−25 to the corresponding dienes 26−
32 was achieved with phenyliodine bis(trifluoroacetate)
(PIFA)20 in 24−66% yield.
Next, the ester substrate mimics 12−18 were dissolved in
dimethylformamide (DMF) and submitted to enzymatic
oxidation with SorbC (2 μmol substrate, 2.5 mol % enzyme;
see Supporting Information for experimental details), with an
additional control experiment with sorbicillin (7), prepared by
acylation of 10 with sorbic acid chloride,15 as benchmark
substrate. Substrate conversion was assessed based on
consumption of 7 or 12−18 and quantified over a reaction
time of 120 min using individual calibration curves of the
respective pure substrates (see Supporting Information,
Figures S1−S7 and S10). The natural substrate 7 was fully
consumed within 60 min (Figure S20). While the methyl (12)
and ethyl (13) ester derivatives did not show any significant
conversion to 33 and 34 (Figures S11 and S12), the propyl
ester (14) was selectively oxidized to the desired product 35
with 39% conversion after 120 min (Figure S13). To our
delight, both the butyl (15) and pentyl (16) esters were
efficiently transformed to products 36 and 37 by SorbC in 79%
and 83% yields, respectively, after 120 min (Figures S14 and
S15). The hexyl ester 17 was a less suitable substrate with
reduced 48% consumption after 120 min (Figure S16).
Surprisingly, the crotyl ester derivative 18 was likewise an
19
with KHCO3 to yield 2,4-dihydroxy-3,5-dimethylbenzoic
acid (11) in 44% yield (quantitative yield based on re-isolated
starting material (brsm), Scheme 1). The desired esters 12−18
were obtained by coupling of 11 to the respective alcohols
using N,N′-diisopropylcarbodiimide (DIC) and 4-dimethyla-
minopyridine (DMAP) in yields of 63% (12, methyl ester),
83% (13, ethyl ester), 93% (14, propyl ester), 61% (15, butyl
ester), 73% (16, pentyl ester), 71% (17, hexyl ester), and 59%
(18, crotyl ester).
B
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inferior substrate when compared to 15 and 16, with 51%
conversion after 120 min (Figure S17).
when compared to the purely chemical, racemic acetylation/
PIFA oxidation sequence (cf. Scheme 3, table columns Y1 and
Y2; Table S1).
The stereochemical outcome of the SorbC-catalyzed
oxidative dearomatization of substrates 14−18, all showing
significant conversion, was further evaluated by HPLC analysis
on a chiral phase (Chiralcel OD-RH). To facilitate these
investigations, the biocatalytically formed sorbicillinol inter-
mediates 35−39 of these compounds were O-acetylated using
acetic acid anhydride to give (S)-28 to (S)-32, purified by
semipreparative HPLC, and analyzed in comparison with the
respective synthetic racemic material. For all compounds, the
biocatalytic oxidative dearomatization proceeded with excellent
stereocontrol (no enantiomeric product detectable), exclu-
sively delivering the desired (S)-configured oxidized products,
as exemplarily shown for (S)-29 and (S)-30 in Figure 2 (see
Scheme 3. Biocatalytic Oxidative Dearomatization of
Substrates 12−18 to 33−39 using SorbC and Subsequent O-
Acetylation to Enantiopure (S)-Configured Products 28−
a
32
a
Conversion is given after 120 min. Y1: isolated yields of enantiopure
(S)-product. Y2: isolated yields of rac-product by chemical
acetylation/PIFA oxidation.
For an initial evaluation of the applicability of the ester
substrate mimicking approach, two substrates with altered
substitution at the aromatic core were synthesized. 2-
Methylresorcinol was therefore acetylated at C-4 using acetic
or propionic acid anhydride to give 33 and 34, respectively,
which were reduced to the ethyl- and propyl-substituted
phenols 35 and 36, carboxylated (37 and 38), and esterified
with n-butanol to deliver sorbicillin analogues 39 and 40
analogously to the procedures described above (see Supporting
Information for experimental details). The corresponding
oxidized products were likewise synthetically prepared by O-
acetylation of 39 and 40 to give 41 and 42, followed by PIFA-
mediated oxidation to 43 and 44. Enzymatic oxidative
dearomatization proceeded with 62% and 37% conversion of
39 and 40, respectively, again exclusively producing (S)-43 and
(S)-44 in 25% and 14% isolated yield (Scheme 4; calibration
curves, Figures S8 and S9; substrate conversion analyses,
Figures S18 and S19; yields, Table S1).
Figure 2. Analysis of the stereochemical outcome of oxidative
dearomatization reactions employing SorbC and ester analogues 15
(A) and 16 (B).
Scheme 4. Synthesis of Ethyl- and Propyl-Substituted
Substrate Analogues 41 and 42 and Their Chemical and
Biocatalytic Oxidation to Sorbicillinol Analogues 43 and 44
also Figures S21 and S22, Table S1). The activity of SorbC
toward the respective precursors 15 and 16 (of 29 and 30,
respectively) was assayed in comparison with the native
substrate sorbicillin (7) by monitoring nicotinamide adenine
dinucleotide (NADH) consumption. The preferred substrate
was 7 (107 U/mg enzyme), with an approximately fourfold
reduced activity for both 15 (27.5 U/mg) and 16 (23.3 U/
mg).
The acetylated sorbicillinol analogues (S)-28 to (S)-32 were
primarily produced to facilitate their stereochemical analysis, as
typical downstream-processing for the total synthesis of
sorbicillinoid natural products directly utilizes unprotected
sorbicillinol.15,16 Although acetylation yields were thus not
optimized, the overall yields of the biocatalytic procedure to
enantiopure products delivered similar to increased yields
C
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Irrespective of the substrate employed, HPLC analyses of all
biocatalytic oxidation reactions revealed the presence of an
additional product peak with a retention time between
oxidized product and substrate, exemplarily depicted for the
synthesis of (S)-36 in Figure 3. The relative amount of this
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tobias.gulder@tu-dresden.de
ORCID
Tobias M. Milzarek: 0000-0001-7512-9093
Paul M. D’Agostino: 0000-0002-8323-5416
Tobias A. M. Gulder: 0000-0001-6013-3161
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Figure 3. Characterization of the additional product peak observed in
biocatalytic oxidation reactions, exemplarily for the conversion of 15
to (S)-36: identification of a new bisorbicillinol analogue 45.
We thank Prof. Dr. T. Gulder (TU Munich, Biomimetic
Catalysis) for providing the Chiralcel HPLC column, the
group of Prof. Dr. E. Brunner (TU Dresden, Bioanalytical
Chemistry) for HRMS measurements, and Dr. T. Lubken and
̈
his team (TU Dresden, Organic Chemistry I) for measuring
NMR spectra. T.M.M. thanks the Stiftung der Deutschen
Wirtschaft for funding. K.L. thanks the Studienstiftung des
Deutschen Volkes for her scholarship. We thank the DFG for
generous financial support of the work in our laboratory
(Emmy Noether Program (GU 1233/1-1)).
product increased with prolonged reaction times. On the basis
of our experience with the synthesis of dimeric sorbicillinoid
natural products15,16 we thus suspected these compounds to
correspond to the respective bisorbicillinol analogues.
Optimization of the turnover to this compound by switching
the employed cosolvent from DMF to acetone and extended
stirring for 6 h facilitated the isolation and in-depth structural
characterization of this product, indeed validating it to be
bisorbicillinol analogue 45, derived of Diels-Alder cyclo-
addition reaction of 2 equiv of (S)-36. The enzymatic
oxidative dearomatization reaction with ester substrate mimics
is thus not only a clean transformation but additionally permits
typical downstream dimerization processing toward the
sorbicillin natural product family.
In conclusion, we herein developed a methodology to
circumvent the inherent structural requirements of SorbC
toward its substrate to broaden the substrate scope of
biocatalytic oxidative dearomatization. Our work revealed
that the enzyme accepts ester analogues of its natural substrate
sorbicillin (7), while retaining catalytic efficiency and stereo-
control. This work constitutes an important breakthrough in
overcoming substrate specificity and limitations in such
biocatalytic transformations and will thus facilitate its broader
application in synthetic organic chemistry. In particular, it sets
the stage for the preparation of unnaturally functionalized
sorbicillinoid natural products with flexible alterations at the
sorbyl side chain. This will permit future biomedical evaluation
of sorbicillinoid analogues with diverse substituents at this
position that can readily be introduced after biocatalytic
synthesis of the respective reactive sorbicillinol derivatives. In
addition, this work paves the way for the utilization of SorbC
as a catalyst for the synthesis of natural products beyond the
sorbicillinoid family.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b01398.
Experimental procedures, supplementary figures, and
NMR spectra (PDF)
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