Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-β-carbolines Enabled by “Substrate Walking”

Article abstract of DOI:10.1002/chem.202004449

Stereoselective catalysts for the Pictet–Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-β-carbolines. Although biocatalysts have previously been employed for the Pictet–Spengler react

Full text of DOI:10.1002/chem.202004449

Accepted Article
Accepted Article
Title: Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-β-
carbolines Enabled by ‘Substrate Walking’
Authors: Elisabeth Eger, Jörg Schrittwieser, Dennis Wetzl, Hans Iding,
Bernd Kuhn, and Wolfgang Kroutil
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Chemistry - A European Journal
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COMMUNICATION
Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-β-
carbolines Enabled by ‘Substrate Walking’
[a]
[a]
[b]
[b]
[c]
,[a,d]
Elisabeth Eger, Joerg H. Schrittwieser, Dennis Wetzl, Hans Iding, Bernd Kuhn, Wolfgang Kroutil*
Abstract: Stereoselective catalysts for the Pictet–Spengler reaction
of tryptamines and aldehydes may allow a simple and fast approach
to chiral 1-substituted tetrahydro--carbolines. Although biocatalysts
have previously been employed for the Pictet–Spengler reaction, not
a single one accepts benzaldehyde and its substituted derivatives. To
address this challenge, a combination of substrate walking and
transfer of beneficial mutations between different wild-type backbones
tryptamine and its derivatives, leading to -carbolines as products.
While for norcoclaurine synthases a broad scope of carbonyl
substrates has been reported,[9] it has only recently been shown
that STRs accept small aliphatic aldehydes besides the natural,
highly functionalized aldehyde secologanin (4c, Figure 1) and its
analogues.[
10d]
In contrast to the natural reaction of tryptamine (1)
with secologanin (4c), which gives the (S)-configured product,
(S)-strictosidine, small aldehydes such as isovaleraldehyde led to
the (R)-configured product. Computational methods suggested
the reason for this inverted absolute configuration to be an
inverted binding mode: in the natural reaction the indole ring of
tryptamine is located at the back of the active-site pocket, while in
the reaction with isovaleraldehyde it points to the outside of the
active site.[
was used to develop
a strictosidine synthase from Rauvolfia
serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet–
Spengler condensation of tryptamine and benzaldehyde derivatives.
The double variant RsSTR V176L/V208A accepted various ortho-,
meta- and para-substituted benzaldehydes and produced the
corresponding chiral 1-aryl-tetrahydro--carbolines with up to 99%
enantiomeric excess.
11]
Interestingly, acceptance of benzaldehyde as a substrate has
neither been reported for NCSs nor for STRs. In fact, only very
recently a variant of a norcoclaurine synthase has been shown to
accept aldehydes branched in -position,[9b] but benzaldehyde
was not investigated in this work. An earlier study found no
product formation from benzaldehyde and dopamine by NCS from
Thalictrum flavum.[9d]
1
-Aryltetrahydro--carbolines have been shown to exert various
biological activities: for instance, the phosphodiesterase-5
inhibitor Tadalafil is used to treat erectile dysfunction and
pulmonary
1]
arterial
hypertension;[
1-phenyltetrahydro-

-carboline (3a, Scheme 1) possesses biological activity binding
receptor[ and was proven effective as growth
2]
to the human 5-HT
7
inhibitor of the African cotton leafworm (Spodoptera littoralis) in
[
3]
(A) Wild-type strictosidine synthases (STRs) proved inactive towards benzaldehyde
feeding bioassays. Interestingly, in the latter case and for many
other non-natural tetrahydro--carboline derivatives, mostly
racemic compounds were tested, indicating a lack of suitable
asymmetric methods.
O
NH
2
STR wild type
H
no product
formation
+
N
1
2a
H
An efficient approach for the synthesis of 1-substituted
tetrahydro--carbolines is the Pictet–Spengler reaction, in which
a 2-arylethylamine reacts with a carbonyl compound, usually
forming a six-membered ring.[4-6] Various chemical protocols for
the Pictet–Spengler reaction have been established, including
(B)
Biotransformation with engineered STR variant
STR variant
RsSTR V176L/V208A
NH
NH
2
+
O
N
H
N
buffer, cosolvent
1
many stereoselective approaches.[5,7] In nature this condensation
H
H
R
R
reaction is catalysed by enzymes called Pictet–Spenglerases,[8]
2a–i
(R)-3a–i
which are sub-categorised according to their substrates. For
instance, norcoclaurine synthases (NCSs)[ prefer dopamine as
9]
Substrates: 2a
R =
2b
p-F
2c
2d
2e
2f
2g
2h
2i
o-Br
amine substrate, while strictosidine synthases (STRs)[10] accept
H
m-F
m-Cl m-Br m-OMe m-NO
2
o-F
Non-Substrates:
2j
p-Cl
2k
2l
R =
p-OMe
p-NO
2
[
a]
Dr. Elisabeth Eger, Dr. Joerg H. Schrittwieser, Prof. Wolfgang Kroutil
Institute of Chemistry, Biocatalytic Synthesis
University of Graz, NAWI Graz, BioTechMed Graz
Heinrichstrasse 28/II, 8010 Graz, Austria
Scheme 1. Biocatalytic Pictet–Spengler reaction using a strictosidine synthase
variant to transform benzaldehyde. (A) The wild-type STR does not transform
benzaldehyde. (B) Substrate scope of RsSTR V176L/V208.
E-mail: wolfgang.kroutil@uni-graz.at
[
[
[
b]
c]
d]
Dr. D. Wetzl, Dr. H. Iding
Process Chemistry & Catalysis
F. Hoffmann-La Roche Ltd.
When testing heterologously expressed wild-type strictosidine
[
10d,12]
synthases originating from Catharanthus roseus (CrSTR),
Grenzacherstrasse 124, 4070 Basel, Switzerland
[10d,13]
Ophiorrhiza pumila, (OpSTR),
and Rauvolfia serpentina
(RsSTR, PDB: e.g. 2V91)[10d,14] with tryptamine (1) and
benzaldehyde (2a, Scheme 1A), none of them led to any trace of
product formation. Consequently, a new biocatalyst had to be
developed for this type of substrate structure. Since STR has
been shown to enable two binding modes of tryptamine and the
aldehyde substrate (see above),[11] it is not obvious which region
of the active site needs to be modified to improve activity.
Dr. B. Kuhn
Pharma Research & Early Development
F. Hoffmann-La Roche Ltd.
Grenzacherstrasse 124, 4070 Basel, Switzerland
Field of Excellence BioHealth – University of Graz, Graz, Austria
Supporting information for this article is given via a link at the end of
the document.
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Moreover, since benzaldehyde was not converted at all, a
entry 9). In the double variant V176L/V208A, the exchange of
V176 to leucine even increased the conversion of 2a to 24%
(Table 1, entry 10), while other amino acids in this position such
as I, F or M were less beneficial (Table S4).
[
15]
substrate walking approach was followed. The idea is to adapt
the enzyme to a substrate possessing a structure, figuratively
speaking, between the target substrate and a compound known
to be well accepted, e.g. isovaleraldehyde (4b). Adapting the
catalyst for the structural homologue might also induce low activity
for the target aldehyde 2a, and this activity can then be further
improved. As a smaller structural analogue of benzaldehyde, the
To better understand the observed reactivity with respect to
the non-natural aldehydes of the V176L/V208A mutation we
generated a binding mode model (Figures 2–3) based on X-ray
crystal structure overlays and computational refinement (for
computational details, see the Supporting Information). Our most
likely model assumes the inverted binding mode that was found
previously for 4b in OpSTR (PDB: 6s5q).[11] We think that the
exchange V208A creates space in the back of the active-site
pocket, making it better able to accommodate the phenyl moiety
(Figure 2). The substitution of Val176 by the larger leucine residue
leads, in our model, to modified dispersion interactions with the
indole core, which might result in a more favourable positioning of
the substrate for catalysis.

-substituted aldehyde 2-methylbutanal (4a, Figure 1) was
selected, which was accepted by the three STRs investigated,
although the conversions observed after 24 h were low (0.8% for
OpSTR, using 10 mM of 1 and 50 mM of 4a, Table S1). For
comparison, the isomeric aldehyde isovaleraldehyde (4b) is
transformed under the same conditions with 50–63% conversion.
O
H
O
O
O
OGlc
H
H
H
Table 1. Developing a Pictet–Spenglerase for the asymmetric condensation
of benzaldehyde (2a) and tryptamine (1) via identification of hot spots in the
O
2a
4a
4b
MeO
2
C
4c
[
a]
OpSTR backbone and transferring these to RsSTR.
Figure 1. Aldehyde 4a used for substrate walking as structural transformant
between the well accepted substrate 4b and the target substrate benzaldehyde
Entry
Backbone
OpSTR
OpSTR
OpSTR
OpSTR
OpSTR
OpSTR
OpSTR
RsSTR
RsSTR
RsSTR
RsSTR
Mutation
none
Conv.[b] 4a [%]
Conv.[b] 2a [%]
(
2a), which is not accepted by the wild-type enzymes. The structure of the
1
2
3
4
5
6
7
8
9
0.8
0.9
0.9
0.9
0.9
3.5
3.5
1.0
5.5
3.0
10.0
n.c.
n.c.
n.c.
n.c.
n.c.
3
natural substrate, secologanin (4c, Glc = β-D-glucosyl), is shown for comparison.
V147L
Since OpSTR proved to be a synthetically useful catalyst in a
previous study,[10b] the initial mutations were performed using this
scaffold. In a focused library 13 residues inside and around the
active-site pocket were addressed as well as additional residues
identified by MD simulations (Table S7; see also Supporting
Information “Selection of Sites for Mutagenesis”).[16,17] The 83
variants were successfully expressed; however, only 19 variants
were active with aldehyde 4a. Out of these active variants four
displayed minimally higher conversion than the wild type (Table 1,
entries 2–5; Table S2). Next, double variants were prepared by
pairwise combination of the beneficial substitutions in positions
V147, I179 and L290. Additionally, the V147I and I179V
substitutions were combined with amino acid exchanges that had
resulted in similar activity as the wild type (Y76W, F197Y). This
led to the identification of two OpSTR double variants
V147I
I179V
L290I
V147I/I179V
V147I/L290I
none
n.c.
n.c.
3
V176L
1
0
1
V208A
13
1
V176L/V208A
24
[
a]
Reaction conditions: 1 (10 mM), aldehyde (4a or 2a; 50 mM; 2a added as
stock solution in DMSO; final DMSO conc.: 10% v/v), STR preparation
50 mg/mL lyophilised cells), PIPES buffer (0.5 mL; 50 mM, pH 6.1), 35 °C,
(
V147I/I179V, V147I/L290I) displaying a four-fold increase of
conversion for 4a compared to the wild type (Table 1, entries 6,
; data for all double variants: Table S3). When the small library
(
7
650 rpm, 24 h. Conversion determined by GC analysis (4a) or HPLC
analysis (2a). n.c., no conversion. Blank reactions (no enzyme added)
resulted in conversions <0.1%.
of double variants was tested with benzaldehyde (2a), formation
of the desired product 3a was detected for the first time. Thereby
OpSTR V147I/I179V proved to be best (3% conv.; Table 1, entry
6).
Since the residues V147 and I179 seemed to represent hot
spots in OpSTR, we hypothesized that exchanges at the
corresponding positions might also be beneficial for the
strictosidine synthase of Rauvolfia serpentina (RsSTR). Such an
[
18]
approach to identify hot spots in one enzyme and then transfer
them to related enzymes may enable screening a broader overall
sequence space of possibly suitable candidates. The positions
V147 and I179 of OpSTR correspond to residues V176 and V208
[
19]
in RsSTR.
When first investigating single variants for these
positions, V208A allowed already a reasonable conversion of
benzaldehyde under standard assay conditions (13%, Table 1,
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COMMUNICATION
Table 2. Pictet–Spengler reaction between substituted benzaldehydes 2a–i
[
a]
and tryptamine (1) employing RsSTR V176L/V208A.
Entry
Aldehyde 2
Substituent R
H
Conv.[b] [%]
38 ± 1
8 ± 1
e.e.[c] [%]
99 (R)
97 (R)
96
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
p-F
m-F
16 ± 2
29 ± 3
33 ± 3
24 ± 1
21 ± 1
68 ± 2
59 ± 12
m-Cl
98
m-Br
98
m-MeO
98
2g[d]
2h
2i
m-NO
o-F
90
2
Figure 2. Model of (R)-1-phenyl--carboline (3a) in the active site of wild-type
RsSTR (white) and RsSTR V176L/V208A (black). Short non-bonded contacts
between substrate and enzyme are shown as dashed lines.
99 (R)
99 (R)
o-Br
[
a]
Reaction conditions: 1 (10 mM), aldehyde (50 mM), STR preparation
It is also worth to note that RsSTR V176L/V208A is about
twice as active towards the previously reported substrate isovaler-
aldehyde (4b) as the wild-type enzyme, while its specific activity
with the natural substrate secologanin (4c) is reduced to approx.
(
5
50 mg/mL lyophilised cells), MOPS buffer (50 mM, pH 6.1), total volume
00 µL, 35 °C, 650 rpm, 24 h. Conversion determined by HPLC analysis
[b]
on an achiral stationary phase. Reactions were run in triplicates and
conversion is reported as mean ± standard deviation.
excess determined by HPLC analysis on a chiral stationary phase. The
absolute configuration is given in parentheses. Substrate added as stock
[c]
Enantiomeric
58% relative to the wild type (Table S5).
[
d]
Analysis of the optical purity of the obtained product 3a
solution (500 mM) in DMSO. Final DMSO conc. 10% v/v.
revealed an e.e. of 99% with (R)-configuration. This absolute
configuration is in line with the previously observed
stereochemical outcome of RsSTR-catalysed Pictet–Spengler
reactions of small aldehydes.[10b] Furthermore, the observed
absolute configuration and the site of mutation that created space
at the back of the active-site pocket (V208A) to accommodate 2a
also support a substrate binding mode in which the indole ring of
tryptamine points out of the active site, as previously suggested
by computational studies.[
11]
To elucidate the substrate scope of RsSTR V176L/V208A,
substituted benzaldehyde derivatives were tested and revealed a
tolerance of the variant for various meta-substituents, including
halogens (F, Cl, Br), methoxy and nitro groups (substrates 2c–
2
g). The best conversion was achieved with the m-bromo
derivative (2e, Table 2, entry 5). Presence of a fluorine atom was
also accepted in para-position (substrate 2b, Table 2, entry 2),
while larger substituents like chloro, methoxy, or nitro (2j–2l) were
not tolerated there.
The reason that meta-substituents are well accepted while
there is a clear limitation in para-position is most likely steric
hindrance, as a model of (R)-1-phenyl--carboline (3a) in the
active site of RsSTR V176L/V208A revealed free space in meta-
position and restriction in para-position (Figure 3).
Since the ortho-position is closest to the carbaldehyde moiety,
which is the site of reaction, it was expected that ortho-substitution
would not be tolerated. However, it turned out that o-fluoro- (2h)
and even o-bromobenzaldehyde (2i) are well-accepted
substrates. Unexpectedly, the conversion even improved
significantly for the o-Br derivative (2i) compared to the
unsubstituted 2a, reaching up to 68% (Table 2, entry 9).
Figure 3. Model of (R)-1-phenyl--carboline (3a) in the active site of RsSTR
V176L/V208A with surface of the enzyme showing the space available in meta-
position in contrast to the limited space in para-position. The catalytically
essential active-site residue Glu309 is shown in black.
The products 3b–i were obtained in optical purities of 96–99%
e.e., the only exception being 3g, which was formed in 90% e.e.
This reduced optical purity can be ascribed to the substantial non-
enzymatic background reactivity of 1 with 2g (2.1% conversion in
24 h). The background reactivity of the other aldehydes is
significantly lower (≤0.6%).
Optimisation of the reaction conditions (see Supporting
Information) allowed to improve the ratio of tryptamine (1) to
aldehyde 2 to 1:1.25 at a tryptamine concentration of 40 mM. Four
selected biotransformations (2a,b,h,i) were carried out on
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Chemistry - A European Journal
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COMMUNICATION
preparative scale (5 mmol 1) to confirm/establish the absolute
configuration (Table S6). The products were isolated in 4–31%
yield and the optical purities obtained were between 96 and 98%
e.e. Analysis of the optically enriched tetrahydro-β-carbolines by
optical rotation, circular dichroism (CD) spectroscopy and HPLC
showed that all of them possess (R)-configuration (Table S15).
Summarising, a Pictet–Spenglerase was developed for the
stereoselective reaction of tryptamine with benzaldehyde
derivatives. Since benzaldehyde was not accepted at all by the
investigated wild-type enzymes, a substrate walking strategy was
applied, whereby suitable hot spots identified in one STR
backbone (OpSTR) were transferred to another (RsSTR). The
RsSTR variant V176L/V208A turned out to accept a broad scope
of benzaldehyde derivatives, particularly those substituted in
meta- and ortho-position, allowing to obtain (R)-configured
products with up to 99% e.e. The suitable catalyst was created by
testing a rather small library of variants (~100) by combining
rational design, single-site saturation and hot-spot transfer to
other backbones. The concept and the catalyst developed open
new approaches for the synthesis of important bioactive 1-
aryltetrahydro--carbolines in optically enriched form.
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Keywords: Biocatalysis • Asymmetric Catalysis • Pictet–
Spengler Reaction • Enzyme Engineering • -Carbolines
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Entry for the Table of Contents
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E. Eger, J. H. Schrittwieser, D. Wetzl,
H. Iding, B. Kuhn, W. Kroutil*
Page No. – Page No.
Asymmetric Biocatalytic Synthesis of
1-Aryltetrahydro-β-carbolines
Enabled by ‘Substrate Walking’
Walking to new frontiers: Taking the challenge that not a single biocatalyst was
available for the Pictet-Spengler reaction with benzaldehyde derivatives, modern
tools of enzyme engineering were applied in a joined forces approach, to create a
catalyst displaying the desired activity yielding optically enriched products with up to
99% e.e.
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