Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective, Enzymatic Cycloether Formation on a Gram Scale

Article abstract of DOI:10.1021/acscatal.9b05071

Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles (CSOH), are

Full text of DOI:10.1021/acscatal.9b05071

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Article
Biocatalysts from Biosynthetic Pathways: Enabling
Stereoselective, Enzymatic Cycloether-Formation on A Gram Scale
Tim Hollmann, Gesche Berkhan, Lisa Wagner, Kwang
Hoon Sung, Simon Kolb, Hendrik Geise, and Frank Hahn
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b05071 • Publication Date (Web): 30 Mar 2020
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ACS Catalysis
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Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective,
Enzymatic Cycloether-Formation on A Gram Scale
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0
§
,†
§,‡,†
†
⊥
†
Tim Hollmann , Gesche Berkhan , Lisa Wagner , Kwang Hoon Sung , Simon Kolb , Hendrik
‡
*,‡,†
Geise , Frank Hahn
†Professur für Organische Chemie (Lebensmittelchemie), Fakultät für Biologie, Chemie und Geowissenschaften, Department
of Chemistry, Universität Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
‡Centre for Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 38, 30167 Hannover,
Germany
⊥Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstrasse 7, 38124 Braunschweig, Germany and Institute of
Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstrasse 7, 38106
Braunschweig, Germany and Protein Facility, ILAb Co., Ltd. NP513, The Catholic University of Korea, Bucheon, 420-743,
Republic of Korea
Dedicated to Prof. Peter F. Leadlay on the occasion of his recent retirement.
ABSTRACT: Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular
complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles
(CSOH), are of particular interest for a synthetic application as their use promises to significantly simplify access to these elements.
Here, the biocatalytic characterization of AmbDH3, an enzyme that catalyzes intramolecular oxa-Michael addition (IMOMA) is
reported. This reaction essentially gives access to various types of CSOH with adjacent stereocenters, but is not yet part of the
repertoire of preparative biocatalysis. An in-depth study on the synthetic utility of AmbDH3 was performed, which made extensive
use of complex synthetic precursor surrogates. The enzyme exhibited stability and broad substrate tolerance in in vitro experiments,
which was in agreement with the results of molecular modeling. Its selectivity profile enabled kinetic resolution of chiral
tetrahydropyrans (THPs) under control of up to four stereocenters. After a systematic optimization of the reaction conditions, gram
scale conversions became possible that led to preparative amounts of chiral THP. The synthetic utility of AmbDH3 was finally
demonstrated by its successful application in the key step of a chemoenzymatic total synthesis to the THP-containing phenylheptanoid
(–)-centrolobin. These results highlight the synthetic potential of AmbDH3 and related IMOMA cyclases as a biocatalytic alternative
that further develops the available chemical-synthetic IMOMA methodology.
KEYWORDS: biocatalyst discovery, cyclases, oxa-Michael addition, heterocycles, tetrahydropyrans, natural product
(bio)synthesis, new enzymatic activity
Chiral, saturated oxygen heterocycles (CSOH) are abundant,
often pharmacophoric, structural elements of natural products
INTRODUCTION
9
,10
Natural product biosynthetic pathways are a rich source of
with a strong influence on the molecular shape (Figure 1a).
1
,2
enzymes with unprecedented activities.
Among them,
Their selective synthesis is often hampered by a multitude of
11
enzymes that form complex molecular fragments with multiple
stereocenters, like terpene cyclases, macrocyclizing
densely packed stereocenters. The intramolecular oxa-
Michael addition (IMOMA) basically provides access to CSOH
3
–5
thioesterases or heterocycle-forming cyclases
,
are
with adjacent stereocenters from -branched precursors like 4
1
1–13
particularly attractive for biocatalysis as their selectivity often
significantly exceeds that of analogous chemical-synthetic
methodology. Their biocatalytic application promises to enable
efficient, step-economic access to complex natural products,
(Figure 1b).
Chemical IMOMA, often integrated into efficient tandem
processes, gained growing attention for the stereoselective
synthesis of tetrahydropyrans (THPs) during recent years.
It has been shown that the cis- or trans-THP selectivity of
IMOMA can be influenced by kinetic or thermodynamic
reaction control, by the type of activation (acid, base or metal
12–15
1
,6,7
synthetic drugs and high-value chemicals.
Tapping this
potential requires a thorough investigation of the synthetically
relevant properties of such enzymes, like substrate specificity,
8
stereoselectivity, stability and reaction scalability.
catalysis), by the configuration of the double bond and by the
Comprehensive studies on these enzymes are often complicated
by high investment into the stereoselective synthesis of
complex precursor surrogates and a significant number of
analogs.
16–21
nature of the acceptor moiety.
Although the reaction
outcome is predictable for some systems (tandem-cross
metathesis-IMOMA with vinylketone intermediates for
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1
5
example reliably leads to cis-THPs with d.e.>5:1) , low to
moderate d.e.s are rather common and have only occasionally
been improved by extensive reaction optimization or product
re-equilibration (see for example22–27). Important studies on the
thioester IMOMA have provided valuable insight into the
reasons for the high substrate-dependence of the cis-trans
selectivity, along with the establishment of guidelines for
its synthetic properties and could for example be employed for
4
1–43
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the synthesis of hard to access medium sized lactones.
Enzymes that catalyze IMOMA have been discovered in the
44,45,46–54
secondary metabolism
and a basic biochemical
characterization has been reported for three enzymes from the
ambruticin, the pederin and the sorangicin biosynthetic
4
4,45,46,55
pathways.
Despite their diverse sequence-based
1
6–19
diastereoselective THP cyclization in specific systems.
annotation, recent studies suggest that all these IMOMA
cyclases share an enzymatic mechanism in which they activate
OH
a
OMe
OMe
OAc
OH
MeOOC
the 7-hydroxyl group of the precursor by acid-base catalysis for
MeO
O
H
N
HO
O
O
45,47,55
attack onto the Michael acceptor.
No in depth-studies on
O
Bryostatin
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
OMe
Pederin
O
3
the biocatalytic potential of IMOMA cyclases are reported to
date.
2
OH
OH
O
O
O
OH
O
O
OH
a: Biosynthesis
O
O
OH
Amb
DH3
Amb
DH3
O
O
H
O
O
OH
OH
O
OH
COOH
OH
6S
COOMe
Salinomycin
ACP
S
ACP
S
7R
dehydration
IMOMA
1
6
7
OH
b
R2
O
OH
OH
6S
7R
3
7
O
*
2
IMOMA
n
O
3
*
R2
*
R3
*
7
X
R3
3R
n
2*
*
O
acid / base catalysis,
metal catalysis,
organocatalysis
X
O
R1
O
ACP
S 2R
O
9
4
R1
Ambruticin S
5
HOOC
ketone, ester or thioester
n=0,1)
8
chiral tetrahydropyran
new stereocenters
(
2
Analytical scale:
b: In vitro studies
R or S
THP synthesis by IMOMA:
(6S,7R)-10: full conv.
(6R,7R)-10: ~50%
Semipreparative scale:
(6S,7R)-10: 87%
Amb
DH3
O
OH
O
-
-
-
substrate-dependent cis-/trans-THP selectivity
little control over product configuration at C-2
mechanistic understanding is to be improved
SNAC
7R
0 R or S
SNAC
O
7R
11
IMOMA
1
(6R,7R)-10: 18%
(
2R,3R)-configured
Figure 1. a) Pharmacologically relevant natural products
containing THPs, which are biosynthesized by IMOMA. b)
IMOMA enables stereoselective synthesis of chiral THPs and other
CSOH. A C-2–C-6-bisbranched precursor is exemplarily shown.
Scheme 1. a) AmbDH3 catalyzes THP formation in ambruticin
biosynthesis. c) Enzymatic in vitro studies on AmbDH3. N-
Acetylcysteamine (SNAC) mimics the natural acyl carrier protein
4
4,45
(
ACP) attachment.
.
Another important issue is the stereocontrol on C-2 of -
branched Michael acceptors. The product configuration at this
position is highly dependent on the molecular environment.
We have previously characterized the unusual bifunctional
polyketide synthase (PKS) domain AmbDH3 by a combination
of enzymatic in vitro experiments, X-ray crystal structure
1
3,28–
3
0
Selectivity has been realized in cases when C-2 was
4
4,45
3
1,32
analysis and site-selective mutagenesis (Scheme 1a).
It
incorporated into rings containing existing stereocenters.
The behavior of non-cyclic systems is scarcely described
nearly all of these systems in the literature are not -branched).
Tedious cascades, like IMOMA–-halogenation–radical
reduction, are thus currently used for the reliable synthesis of
chiral THPs with adjacent stereocenters.
modest stereoselectivity of IMOMA along with the harsh
reaction conditions (e.g. strong base or acid) currently prevent
catalyzes a cascade of dehydration and IMOMA of 6 to the
chiral tetrahydropyran (THP) 9 via 7. In vitro conversion
experiments of the purified domain with synthetic substrate
surrogates showed quantitative, stereospecific transformation
of the natural precursor surrogate (6S,7R)-10 into
(2R,3R,6S,7R)-11 in an analytical scale experiment and 87%
conversion on the semipreparative scale (8 mg / 27 µmol
starting material, Scheme 1b). The C-6 epimer (6R,7R)-10 was
partially converted into (2R,3R,6R,7R)-11, suggesting
substrate tolerance of the enzyme.
(
3
3,34
Taken together, the
1
6
the more frequent use of this reaction in organic synthesis.
Novel variants that merge stereoselectivity and broad
applicability are highly desirable. C-2 stereoselectivity and the
potential to discriminate between C-7 enantiomers/epimers
would for example add valuable new features to the existing
IMOMA methodology. Enzymatic catalysis with its tight
control over extended transition states is particularly well suited
to provide a solution for the problems described above.
RESULTS AND DISCUSSION
Substrate specificity of AmbDH3. A library of precursor
derivatives was stereoselectively synthesized via multistep
sequences (see supporting information). For probing the
substrate selectivity of AmbDH3, the members were
individually incubated with the purified enzyme under the
reported conditions (8-10 mg ≈ 30 µmol starting material, Table
The biosynthesis of CSOH occurs by direct oxidation, by
reduction of hemiacetals or by intramolecular addition of
hydroxyls to electrophiles.
5
,35,36
The synthetic applicability of
44
1
, Figures S1–S26). Enzyme-free reference incubations
such cyclizing enzymes has been successfully studied in some
cases. The bifunctional cytochrome P450 monooxygenase
AurH introduces a tetrahydrofuran (THF) ring in (+)-aureothin
biosynthesis by direct oxidation. Hertweck et al. have
biochemically characterized this enzyme and successfully
applied it in a total synthesis of the natural product as part of an
elegant late-stage kinetic resolution process.
group reported on a PKS domain for vinylogous chain
branching that gives rise to -lactone in rhizoxin
biosynthesis. This enzyme was extensively studied regarding
determined the spontaneous background cyclization. The 7S-
configured compounds (6R,7S)-10 and (6S,7S)-10 were not
accepted, showing the crucial nature of the stereochemistry at
C-7. Various alterations of the polyketide backbone substitution
pattern were tolerated by the enzyme with the conversion
increasing with the steric bulk of the residues on C-6/C-7
results for (6S,7R)-10, 12, 14 and 22) and in the absence of
electron-donating substituents on the acceptor (results for
6S,7R)-10, 18, 20, 22 and 24). The low solubility of 16 and 18
in aqueous buffer prevented their reaction under standard assay
3
7–39
The same
(
a
(
4
0
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conditions. Modest conversion was however observed in small-
scale experiments of these compounds that were carried out at
lower concentration. The enzyme strongly differentiated
between various types of carbonyls in the acceptor (results for
O
O
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SNAC
SNAC
SNAC
O
31
(2R,3R)
>95
n.d.
n.d.
3
0
OH
O
O
OH
(6S,7R)-10, 26 and 28 as well as 34-37). Neither the dethia-oxo
O
<29 (<12)d
SNAC
O
33
2R,3S)
analog 34 nor the other oxo esters 35 and 36 were competent
substrates of AmbDH3. This underlines the aforementioned
dependence of the enzymatic reaction on the electron-density of
the acceptor. Accordingly, the little soluble S-ethyl-enethioate
32
(
Not converted by AmbDH3:
O
OH
O
OH
SNAC
SNAC
2
6 and vinylketone 28 were cyclized by the enzyme by
(
6R,7S)-10
(6S,7S)-10
OH
approximately 40% (see footnotes c, d and f in Table 1).
Phenylthioester 37 was not a competent substrate, showing that
structural aspects play a role, even if the acceptor is sufficiently
activated (Figure S27 and Table S1). While precursor 30 was
fully converted by AmbDH3 into homochiral tetrahydrofuran
O
OH
O
O
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
EtO
EtO
AcHN
34
35
36
O
OH
O
S
SNAC
3
7
38
OH
(
(
THF) 31, oxepane precursor 38 was not a competent substrate
Figure S27 and Table S1). The decarba-oxo analog 32 was
Full spontaneous cyclization observed:
O
NH2
O
OH
partially converted into the 1,4-dioxane 33. Substrates 39 and
0 underwent complete spontaneous cyclization under all tested
SNAC
SNAC
4
39
Br 40
reaction conditions, avoiding their assessment in this study.
a
Carried out under non-optimized conditions (see supporting
b
1
c
Table 1. Substrate surrogates that were reacted with
purified AmbDH3.
information). Determined by H NMR spectroscopy. Conducted
on the 1-4 mg-scale with a substrate concentration of 2-4 mM due
d
to solubility issues. If applicable, the conversion of the
spontaneous IMOMA under standard reaction conditions is shown
in brackets. An e.e. of >95% was determined by H NMR
spectroscopy after reduction and esterification with (S)-Mosher’s
Product
abs. configuration )
conversion
[%]
Substratea
Yield [%]
70h
b
b
e
1
(
O
OH
OH
O
f
acid chloride. Conducted in an Eppendorf tube under shaking to
SNAC
SNAC
SNAC
O
87
18
suppress spontaneous cyclization of 28 that occurred during the
stirred reaction. The d.e. and absolute configuration of the
components in the crude reaction product could not be determined.
purified by semipreparative HPLC. purified by flash
chromatography on silica gel. crude yield.
(
6S,7R)-11
2R,3R)
(6S,7R)-10
(
O
O
g
h
O
SNAC
O
n.d.
i
(6R,7R)-11
(6R,7R)-10
(2R,3R)
Taken together, these results demonstrate a relaxed substrate
specificity of AmbDH3. This might be explained by its origin
as a modular type I PKS domain. The high relevance of protein-
protein interactions in these systems goes along with lower
substrate affinity and lower reaction rates of their individual
O
O
O
OH
SNAC
O
>95
89
n.d.
52g
n.d.
SNAC
13
1
2
(
2R,3R)
O
O
OH
OH
SNAC
SNAC
SNAC
SNAC
O
15
2R,3R)
5
6–59
14
domains compared to free enzymes.
Indeed, the kinetic
(
parameters for the reaction between AmbDH3 and (6S,7R)-10
were determined to be in the range of previously observed
values for reductive loop domains from modular type I PKS
13c
O
17
C5H11
C5H11
1
6
(2R,3R)
-
1
-
(K
m
= 8.2 ± 3.1 mM, kcat= 230 ± 83 s , kcat/K
m
= 28.2 ± 14.8 s
O
O
1
-1
56–59
O
O
OH
OH
OH
*M ; Figure S28).
A semipreparative scale time course
17c
n.d.
SNAC
SNAC
SNAC
SNAC
O
experiment showed a steady and finally quantitative conversion
in an overnight incubation, confirming that the rather slow
reaction is not an issue at all for the use of the enzyme in
biocatalytic reactions (Figures S29–S33). Only 2R,3R- / 3R-
configured products (or equivalent, like (2R,3S)-33) were
observed in the substrate specificity study, showing that the
stereoselectivity of AmbDH3 is conserved among a broad range
of substrates. The enzymatic cyclization gives access to various
types of heterocycles, such as THFs (31) and 1,4-dioxanes (33).
The low solubility of substrates like 16, 18, and 26 in aqueous
buffer was identified as a cause for their low to modest
conversion. This problem might be overcome by adaption of the
reaction conditions (vide infra).
18
19
(
2R,3R)
>90 (32)d
72i
O
21
2R,3R)
20
H
H
(
H
H
O
O
O
24h
58h
SNAC
SNAC
H
SNAC
O
23
2R,3R)
37
22
H
H
(
H
H
O
OH
72 (<6)d,e
H
SNAC
EtS
O
25
(3R)
24
H
H
O
O
OH
43c
25h
O
7
2R,3R)
Molecular modeling. After we had gained a comprehensive
overview of the AmbDH3 substrate specificity, we turned to
link this knowledge with structural data. Some members of the
precursor library were modeled into the active site of the
previously determined X-ray structure of AmbDH3 (PDB ID:
EtS
2
26
(
O
OH
O
>95 (>95)d
39 (0)d,f
n.d.
C
O
29
28
H
NHAc
2
AcNH
4
5
5
O15) and the distances between C-3, 7-OH and the catalytic
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residues were put in relation to the conversion (Figure S27,
Page 4 of 10
Kinetic resolution. The ability to efficiently differentiate
between various presented stereoisomers forms the basis for
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table S1). A good correlation of the latter with the D215–7-OH
distance was observed. Complex model structures of AmbDH3
with the non-competent substrates (6S,7S)-10, 37 and 38
showed distances of >5 Å and a bent backbone conformation
that orients 7-OH away from D215 so that its activation for a
nucleophilic attack is prevented. In the complex models of all
accepted substrates, these values were in the range of 1.9–3 Å.
Among these, a strict proportionality between distance and in
vitro conversion was not observed, because other effects, like
the low solubility of 16, 18 and 26, interfered. These results
suggest that molecular modeling can be used to estimate the
principle acceptance of substrates by AmbDH3. They also set
the stage for a structure-guided directed evolution of AmbDH3
features.
6
0,61
DKR and DR.
AmbDH3 was incubated with various
stereoisomeric mixtures of 41 and 10 (Table 2). Of rac-41,
AmbDH3 cyclized only (7R)-41, while (7S)-41 remained
unreacted (entry 1, Figures S34, S41 and S42). In incubations
of AmbDH3 with various stereoisomer mixtures of 10, only
(6S,7R)-10 and (6R,7R)-10 were converted into the expected
cis-THPs, albeit to a strongly varying degree (entries 2-6,
Figures S35–S40 and S43-S48). (6S,7R)-10 was converted in
all reactions by at least 80%, irrespective of the ratio of
competing stereoisomers. (6S,7R)-11 was the major
stereoisomer formed in all reactions with (6S,7R)-11:(6R,7R)-
11 ratios of up to 90:10.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Reaction of AmbDH3 with stereoisomeric mixtures of 41 and 10.
Amb
DH3
6
R
O
OH
O
OH
O
6
+
SNAC
SNAC
SNAC
O
R
R
41: R=H
0: R=Me
(7R)-42: R=H
(6R,7R)-11,(6S,7R)-11: R=Me
41: R=H
10: R=Me
1
R=H
Inserted stereoisomers of 41 [%]
Conversion into 42[b]
Product ratio[b]
(7R):(7S)
Entry[a]
7R
7S
S[(7R)+(7S)]
1
50
50
47%
>95:<5
R=Me
Inserted stereoisomers of 10 [%]
Conversion into 11[b]
Product ratio[b]
(6S,7R):(6R,7R) pr/sr[c]
Entry[a]
6S,7R
50
6R,7R
0
6S,7S
0
6R,7S
50
[(6S,7R)+(6R,7R)]
2
3
4
5
6
41%
15%
32%
12%
23%
>95:<5
73:27
90:10
74:26
90:10
b
n.d.
8.1
9.1
8.4
8.5
13
37
37
13
30
30
30
10
10
30
30
30
26
24
24
26
a
The summed up concentration of the precursor stereoisomers was 2 mM for 41 and 4 mM for 10. The conversion of the individual
stereoisomers of 10 was qualitatively estimated by analysis of the chiral HPLC chromatograms. The overall conversion and the ratio of the
1
c
cyclic stereoisomers in the conversions of 41 and 10 were quantified by H NMR analysis in a separate experiment. pr: ratio of (6S,7R)-
1:(6R,7R)-11 in the crude product; sr: ratio of (6S,7R)-10:(6R,7R)-10 in the starting material
1
This selectivity opens the possibility to develop novel types established systems for chemoenzymatic DKR using transfer
of DKR or DR processes to chiral CSOH that would provide
control over up to four stereocenters. Dynamic epimerization of
the secondary alcohol could be achieved by adapting
hydrogenation catalysts or alcohol dehydrogenase-catalyzed
62–70
DR.
Under ketone-epimerizing conditions, the stereocenter
at C-6 could additionally be included.
Table 3. Optimization of the reaction between (6S,7R)-10 with AmbDH3.
O
OH
Amb
DH3
O
SNAC
O
SNAC
conditions:
see table
(
6S,7R)-10
(
6S,7R)-11
Conversion optimizationa
Yield optimization
c (substrate).
Scale
Co-
solvent
Conversion
Scale PhMe
[mg] [vol%] [°C]
T
Yield
[%]f
Entry
Enzymeb
Entry
Workupe
c
d
[mg]
[%]
[mM]
1
2
3
4
5
6
10
30
10
30
50
10
purified
purified
lysate
-
87
<50
>95
93
1[g]
2
50
50
50
50
50
50
10%
10%
10%
5%
37
37
37
37
37
37
8
4
2
4
4
4
A
A
A
A
A
A
26
60
54
66
68
76
-
-
3
lysate
-
-
4
lysate
<50
quant.
5
2%
lysate
PhMe
6
1%
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7
8
30
50
lysate
lysate
PhMe
PhMe
>95
94
7
8
9
100
100
100
50
1%
1%
1%
1%
1%
1%
37
30
25
30
30
30
4
4
4
4
4
4
A
A
A
B
C
D
62
66
64
80
80
76
1
2
3
4
5
6
7
8
1
0
11
50
1
2
50
a
b
Reaction conditions: “enzyme”, 8 mM of (6S,7R)-10, HEPES buffer (pH 6.8), 37 °C, 16 h. “purified”: 5 mg/mL of affinity-purified
enzyme. “lysate”: The equivalent amount of cell pellet as for “purified” (2 g pellet / 50 mg substrate) was sonicated and the supernatant used
directly after centrifugation. The substrate was dissolved in the organic co-solvent and added to the cell lysate (10vol% co-solvent). The
conversion was determined by H NMR spectroscopy. Workup comprised triple extraction of the crude reaction mixture with EtOAc (A),
accompanied by previous treatment with CaCl and proteinase K (B), CaCl and papain (C) or CaCl , proteinase K and papain (D),
respectively. The yield was determined after column chromatography on silica gel. Identical conditions as in entry 8 of “Conversion
optimization”.
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
e
2
2
2
f
g
Figure 2. a) Upscaling of the reaction to the preparative scale (conditions as in Table 3, right, entry 10). b) Conversion of (6S,7R)-10 by
AmbDH3 after various substrate-free preincubations of the enzyme.
Upscaling of the enzymatic reaction. Lack of scalability is
a common problem of enzymatic transformations. In order to
enable the preparative-scale synthesis of chiral THP building
blocks we performed an optimization of the cyclase reaction
conditions (Table 3). Scaling up the reaction between AmbDH3
and (6S,7R)-10 under the previously described conditions led
to a significant decline in conversion for reaction batches above
10 mg (33.3 µmol) starting material (entries 1 and 2). An
upscaling to 50 mg batches with a conversion of 94% became
possible by using the cell-free extract instead of the isolated
enzyme and by adding 10% of toluene to the reaction (entries
absence of substrate for 16 h at 30 °C, interrupted by resting
periods of 8 h at 4 °C. In the respective ultimate cycle, (6S,7R)-
10 was added and the conversion determined after 16 h reaction.
The conversion remained on a high level independent of the
number of substrate-free preincubations. Lyophilization or
freeze-thawing of AmbDH3 also had no detrimental effect on
the conversion (Figures S59–S68 and Tables S2–S3).
4
4
Chemoenzymatic total synthesis. Pyran-containing
diarylheptanoids are plant natural products with antibiotic,
antiprofilerative and antioxidative activity.
Centrolobine (47) is a member of this class that shows no
further structural similarities to the ambruticins and thus
represents an optimal model system to demonstrate the
71,72
()-
3
–8). Reducing the substrate concentration from 8 to 4 mM and
fine tuning of the solvent amount gave 76% yield on the 50 mg
scale (Table 3, right, entries 1–6). At higher quantities,
increased precipitation of the enzyme led to lower yield (entry
suitability of AmbDH3 for general chemoenzymatic synthesis
73–76
(Scheme 2, for reported total syntheses see for example
).
7
). This problem was addressed by reducing the reaction
Racemic 44 was synthesized in two steps from bromoacetal 42.
Enzymatic cyclization of 44 on a reaction scale of 20 mg (57
µmol) starting material resolved the enantioenriched syn-THP
with a conversion of 40% and an isolate yield of 33%. The
SNAC moiety was exchanged with a 4-hydroxyphenyl group
by Liebeskind-Srogl coupling. Ketone reduction using LiBH
followed by EtSiH and TFA treatment led to a mixture of (–)-
centrolobine (46) and (+)-centrolobine (ent-46). The
enantiomeric ratio of 89:11 in favor of 46 suggests that
AmbDH3 exhibits comparable selectivity for the precursor
temperature and by treating with a proteinase before extractive
workup (entries 7–12). An isolated yield of 80% was
reproducibly obtained on the 50 mg scale along with nearly
complete recovery of the inserted organic material (entries 10
and 11). The established conditions allowed a further upscaling
to batches of 1 g (3.32 mmol) starting material (Figure 2a,
Figure S48). 83% Conversion and 60% isolated yield (15%
recovery of (6S,7R)-10) after one round of workup were
observed. Further recovery of organic material was possible
after repetitive proteinase treatment of the precipitate. The
scalability and clean course of the reaction shows that this
enzymatic transformation is suitable for producing preparative
batches of chiral heterocyclic building blocks under
operationally simple conditions.
4
3
(7R)-44 as for the natural substrate surrogate (see section
“
Kinetic resolution”). The substrate tolerance and
stereoselectivity as well as the scalability of the AmbDH3
reactions, positively impact the outcome of this enantioselective
synthetic sequence so that the need for stereoselective chemical
transformations is avoided.
Stability of AmbDH3. In order to investigate the stability of
the enzyme, we reacted AmbDH3 with (6S,7R)-10 after
repetitive, substrate-free exhaustion cycles (Figure 2b, Figures
S49–S58). The enzyme was incubated up to nine times in the
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OH
O
centrifugation (5,000 g, 4 °C, 15 min). For purification, 1 g of
PPh3
anisaldehyde
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
SNAC
cells was suspended in 10 mL HEPES buffer (pH 6.8, 25 mM
HEPES, 100 mM NaCl) and cell disruption was performed on
ice by sonication (45% amplitude, 10 cycles, 30 s sonication,
MgBr
THF, 2 h, rt
acidic workup
toluene, 100 °C, 24 h
9% (non-optimized)
O
4
2
43
82%
OMe
Amb
DH3
0 mg scale
3
0 s pause). After centrifugation (10,000 g, 4 °C, 30 min) the
O
OH
O
2
obtained crude lysate was filtered (0.45 μm, cellulose acetate)
and the target protein purified by FPLC equipped with a
HisTrap™ FF Ni-NTA column. The column was washed with
washing buffer (30 mM Tris, 500 mM NaCl, 10 mM imidazole,
10% glycerol, pH 7.5). Elution of the target protein could be
observed during a linear gradient elution with elution buffer (30
mM Tris, 500 mM NaCl, 500 mM imidazole, 10% glycerol, pH
7.5) from 0 to 100% in 25 mL. The protein-containing fractions
were united and the elution buffer replaced by HEPES buffer
(25 mM HEPES, 100 mM NaCl, pH 6.8) using PD-10 desalting
column. If necessary, the purified enzyme was concentrated and
immediately used for activity assays.
SNAC
SNAC
O
4
4
45
OMe
33%
OMe
B(OH)2
OH
OH
THPO
, CuTC,
Pd2dba3, P(o-furyl)3
4 2
LiBH , Et O, rt, 1.5 h,
then Et3SiH, TFA, rt, 1 h
THF, 60 °C, 24 h
70%
7
1%
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
46
(-)-centrolobine (47)
e.r. (47:ent-47): 89:11
OMe
OMe
Scheme 2. Chemoenzymatic total synthesis of (–)-centrolobine
using AmbDH3 for cis-THP-formation in the key step.
CONCLUSION
Semipreparative scale conversions. Assays for
determination of substrate tolerance were carried out in a total
volume of 4 mL containing 8-10 mM of substrate and 5.0-5.4
mg/mL of AmbDH3 in HEPES buffer (pH 6.8, 25 mM HEPES,
We report our investigations on AmbDH3, a member of the
recently discovered, but biocatalytically not exploited group of
IMOMA cyclases. AmbDH3 exhibits relaxed substrate
specificity and conserved stereoselectivity along a broad range
of substrates, like enethioates and vinylketones, providing
access to various types of CSOH like THPs, THFs and 1,4-
dioxanes. The high stereoselectivity of AmbDH3 on C-2 and
the efficient discrimination between individual C-6 and C-7
stereoisomers go beyond the scope of the current synthetic
methodology for IMOMA. These features were exploited in the
resolution of chiral THPs with up to four asymmetric centers
from racemic precursor mixtures and might form the basis for
the development of multienzyme or hybrid catalytic systems for
DKR or DR to CSOH. Upscaling of the enzymatic cyclization
succeeded, providing access to preparative amounts of chiral
THP. The enzyme was successfully applied in the
enantioselective total synthesis of the THP natural product (–)-
centrolobine.
1
00 mM NaCl). After incubation at 37 °C for 16 h, the sample
was extracted with 3 * 5 mL EtOAc and the solvent was
removed under reduced pressure. The product was dissolved in
CDCl
conversion.
1
3
and analyzed by H NMR spectroscopy to determine the
Preparative scale enzymatic conversions. For preparative
scale assays, 2.0 g cell pellet per 50 mg substrate were resolved
in HEPES buffer (1 mL per 0.1 g pellet, 25 mM HEPES,
100 mM NaCl, pH 6.8) and cells were disrupted by sonication
(10 cycles, amplitude 45%, 0.02 kJ/s, 30 s output, 30 s break).
After centrifugation (10,000 g, 30 min, 4 °C) the crude lysate
was directly used for the enzyme assays without further
purification. The substrate was dissolved in toluene (1vol%),
the volume filled up to give a final substrate concentration of
4
mM and the crude lysate was added. After stirring at 30 °C
and 175 rpm for 16 h proteinase K (4 mg per 50 mg substrate)
and CaCl (finale concentration 1 mM) were added and the
This work is one of the rare examples in which isolated PKS
domains were investigated regarding their performance in
semi)preparative enzymatic synthesis. Future work will aim to
2
(
reaction was stirred at 37 °C for 2 h. The phases were separated
and the aqueous phase was extracted with EtOAc. The
combined organic phases were washed with saturated NaCl
solution, dried over MgSO and concentrated under reduced
4
pressure. The conversion was determined by integration of the
appropriate signals in the H NMR spectrum. Purification by
2 2
flash chromatography on silica gel (CH Cl :EtOAc / 2:1  0:1)
gave the product (6S,7R)-11 as colorless solid. Variations of
this procedure were implemented as described in Table 3.
improve the reaction conditions to expand the scope to
hydrophobic substrates and more common Michael acceptors
like S-alkylthioesters and vinylketones. The reported AmbDH3
structure will form a solid base for improving relevant
properties by enzyme engineering.77 Our work paves the way
for the characterization of IMOMA cyclases from other
biosynthetic pathways to expand the scope of biocatalysts and
accessible CSOH types. Incorporation of IMOMA cyclases into
(chemo)enzymatic cascades might expand the product spectrum
to other types of chiral heterocycles like piperidines. Finally,
the combined results on the AmbDH3 structure-function
relationship and its substrate specificity profile are an important
milestone on the way to a rational engineering of cyclase
activities into existing multimodular assembly lines in the frame
1
AUTHOR INFORMATION
Corresponding Author
*E-mail: frank.hahn@uni-bayreuth.de
4
5
Author Contributions
‡T.H. and G.B.: These authors contributed equally to this work.
of directed biosynthesis approaches.
EXPERIMENTAL SECTION
Notes
Enzyme expression and purification. The plasmid
ambDH3_pET28a(+) was transformed into chemically
competent E. coli BL21(DE3) cells. A single colony was used
for inoculation in dYT medium with kanamycin (final
concentration 0.1 mM) for plasmid selection. Cultures were
grown to an OD600 of 0.5 at 37 °C, induced with 0.1 mM IPTG
and shaken at 15 °C for 22 h. Cells were harvested by
The authors declare no competing financial interest
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information material is available free of charge at
the ACS website.
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1
. Methods and materials, Synthetic procedures, Studies on the
Docking calculations,
Tetrahydropyrans: Experimental and Computational Investigation of
the Mechanism of a Thioester Oxy-Michael Cyclization. Chem. Sci.
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1
1
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substrate tolerance of AmbDH3,
Kinetic analysis, Time course experiment, Kinetic resolution,
Upscaling of the enzymatic reaction, Stability studies.
2
(
2
. NMR spectra for all synthetic compounds and assay products.
ACKNOWLEDGMENT
Funding from the Emmy Noether program of the DFG (HA 5841/2-
1
program of the European Union (project number 293430), the
Graduate School MINAS (doctoral stipend to G.B.) and the
Deutsche Bundesstiftung Umwelt (doctoral stipend to L.W.) are
gratefully acknowledged. We thank the MS facilities and NMR
facilities of the OCI Hannover and the Faculty BCG, University of
Bayreuth as well as the Nordbayerisches NMR-Zentrum.
This paper is dedicated to Prof. Peter F. Leadlay on the occasion of
his recent retirement.
), a Sachbeihilfe of the DFG (HA 5841/4-1), the Marie Curie
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