Biocatalysis Hot Paper
P ow ering Artificial Enzymatic Cascades with Electrical Energy
Ammar Al-Shameri, Marie-Christine P etric h, Kai junge Puring, Ulf-P eter Apfel,
Bettina M. Nestl, and Lars Lauterbach*
Abstract : W e have developed a scalable platform that employs
electrolysis for an in vitro synthetic enzymatic cascade in
a continuous flow reactor . Both H 2 and O 2 were produced by
electrolysis and transferred through a gas-permeable mem-
brane into the flow system. The membrane enabled the
separation of the electrolyte from the biocatalysts in the flow
system, where H 2 and O 2 served as electron mediators for the
biocatalysts . W e demonstrate the production of methylated
N-heterocycles from diamines with up to 99 % product
formation as well as excellent regioselective labeling with
stable isotopes . Our platform can be applied for a broad panel
of oxidoreductases to exploit electrical energy for the synthesis
of fine chemicals .
T he use of electrical energy to perform biological and
chemical reactions has gained extensive interest in recent
years . Electro-driven reactions offer the advantages of being
clean, easy to tune , and sustainable when coupled with
renewable energy sources . Biological electron transfer reac-
tions are performed predominantly with cofactor -dependent
oxidoreductases . T hese represent a highly interesting and
versatile class of biocatalysts for specific reduction, oxidation,
and oxyfunctionalization reactions in organic synthesis . [1]
Electrical energy has been applied in various biotechnological
approaches to drive whole cells or immobilized enzymes on
electrodes towards cofactor recycling and production of
biofuels and fine chemicals . [2] In this context, electrochemical
water splitting delivers H 2 as an electron mediator required
for redox reactions with H 2 splitting enzymes . However,
performing electro-driven enzymatic cascades with isolated
enzymes (in vitro) is still hampered by the high potentials
required for water splitting, the low pH, and the high
temperature generated in the process . T hese crucial aspects
will eventually lead to denaturation of the metal-dependent
enzymes , unspecific side reactions , and the formation of
undesired reactive oxygen species (ROS). [2c]
W e therefore set out to design a novel platform to perform
an electro-driven in vitro enzymatic cascade . T his one-pot
enzymatic cascade exploits the redox power of H 2 , which is
generated by water splitting, in order to produce N-hetero-
cycles in a flow system. By utilizing a gas-selective permeable
membrane , [3] we were able to decouple the electrochemical
H 2 generation from the enzyme transformations in one setup ,
thereby establishing an unhindered transfer of gases between
the two liquid phases . T his ensured safe H 2 handling by
avoiding the formation of explosive gas mixtures and
rendered the biological system more stable . Furthermore ,
we established an integrated platform to monitor the relevant
parameters within the system on-line .
T he bioelectrochemical system (F igure 1, top) was sub-
sequently validated with a synthetic enzymatic cascade
consisting of an immobilized oxidase , reductase , and hydro-
genase to produce N-heterocycles from diamines in a contin-
uous process . N-heterocycles belong to a highly important
class of compounds , and are found in various natural products ,
biologically active structures , and medicinally relevant com-
pounds . [4] In previous work, we successfully showed that an
N ADPH-dependent imine reductase (IRED) can be com-
bined with an oxygen-dependent diamine oxidase variant
(PuO E203G ) and an N ADP + -reducing hydrogenase in a one-
pot process for the selective synthesis of N-heterocycles . [5]
F or this study , we decided to couple an NADH-dependent
IRED variant [6] from Myxococcus stipitatus with the O 2 -
tolerant, N AD + -reducing hydrogenase (SH) from Ralstonia
eutropha [7] using molecular H 2 as the reductant. W e also
exploited the versatility of this electro-driven approach for
1) the synthesis of methylated N-heterocycles and 2) the
isotopic labeling of N-heteroc ycles , which gave insight into
the reaction mechanism of IREDs . Electrolysis was per -
formed using a pentlandite and Ni catalysts for the hydrogen
evolution reaction (HER) and the oxygen evolution reaction
(OER), respectively . Pentlandite is a Ni- and Fe-rich metal
sulfide . It is a cheap and a sustainable alternative to platinum
for HER in operations performed under conditions poisonous
to other catalysts . [8] T he pentlandite/Ni system was bench-
marked against the more classical Pt system. Notably , both
systems were comparable in terms of efficiency with a variable
dependency of H 2 and O 2 evolution on the pH and voltage
(F igure S1–S3 in the Supporting Information). In preliminary
experiments , we observed that pentlandite could produce
[*] A. Al-Shameri, M.-C. Petrich, Dr . L. Lauterbach
T echnical University of Berlin, Institute of Chemistry
Strasse des 17. Juni 135, 10623 Berlin (Germany)
E-mail : lars.lauterb [email protected]
K. junge Puring, Prof. Dr . U.-P. Apfel
Ruhr-University Bochum, Inorganic Chemistry
Universitaetsstrasse 150, 44780 Bochum (Germany),
and
F raunhofer UMSICHT
Osterfelder Strasse 3, 46047 Oberhausen (Germany)
Dr . B. M. Nestl
Universitaet Stuttgart
Institute of Biochemistry and T echnical Biochemistry
Department of T echnical Biochemistry
Allmandring 31, 70569 Stuttgart (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under :
https ://doi.org/ 10.1002/anie.2020013 02.
 2020 The Authors. Published by Wiley-VCH V erlag GmbH & Co .
KGaA. This is an open access article under the terms of the C reative
C ommons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
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How to cite: Angew . Chem. Int. Ed. 2020 , 59 , 10929 – 10933
International Edition: doi.org/10.1002/anie.202001302
German Edition: doi.org/10.1002/ange.202001302
10929 Angew . Chem. Int . Ed. 2020 , 59 , 10929 –10933  2020 The Authors. Publish ed by Wiley-VCH V erlag GmbH & Co. KGaA, W einheim

extensive amounts of H 2 at a low voltage of 0.6 V , leading to
displacement of the trace amounts of O 2 generated. T hus , the
required amounts of O 2 could only be generated at high
voltages with Ni as OER. Moreover, the low solubility of the
gases in the electrolyte negatively affected the transfer
efficiency of the gases through the membrane . T hus , increas-
ing the gas concentrations in the electrolyte by applying high
voltages was crucial to enhance the transfer efficiency . [9]
T herefore , we decided to perform the electrolysis at 2 V and
pH 2.0 for Pt/Pt and at 3.5 V and pH 1.3 for pentlandite/Ni.
T he conversion of the model substrate 1,5-diamino-2-
methylpentane ( 1 ) into 3-methylpiperidine ( 2 ) was subse-
quently investigated with in situ produced H 2 and O 2 in the
enzymatic cascade . A mixture of purified enzymes was thus
injected into the circulating system to start the transformation
of the test substrate 1 . F irst results showed the successful
formation of 2 with a production formation of 53 % within
16 hours (F igure S4) independent of the electrodes used. T he
general applicability of the flow reactor was then tested for
the preparation of piperidine ( 6 ) and its derivatives using
immobilized enzymes in the cascade reaction. While SH and
catalase were immobilized on Amberlite FP54 TM , [10] enzyme
carrier EziG TM from EnginZyme [11] was used for immobiliza-
tion of PuO E203G and IRED NADH . T he specific activities per
carrier ranged from 0.64 to 1 U mg  1 (T able S1). T he immo-
bilized enzymes were packed in a column that was integrated
into the flow reactor. T he evolution and consumption of O 2 /
H 2 and N ADH, respectively , were monitored during the
cascade using N -methylcadaverine ( 7 ) as the model com-
pound (F igure 2). T he on-line monitoring enabled the record-
ing and tracking of each step of the catalysis by checking if the
O 2 and H 2 concentrations were sufficient to start/proceed with
the reaction. T he monitoring also gave a comprehensive
picture of whether each enzyme was functioning correctly and
Figure 1. Platform of the electro-driven in vitro enzymatic cascade for the synthesis of N-heteroc ycles from diamines in a flow reactor . Both H 2
and O 2 are delivered by electrolysi s using Pt/pentlandite as HER and Pt/Ni as the OER catalyst in an acidic electrolyte. A gas-permea ble tube
transfers H 2 and O 2 from the electrolysis chamber (250 mL) into the flow system (16 mL). Sensors for H 2 ,O
2 , and temperature and
a spectrophotometer (NADH) were integrated into the flow reactor to monitor on-line the evolution and consumption of H 2 and O 2 and NADH,
respectively . The enzymes of the cascade (left side) were immobilized and packed into a column within the flow system. Electro-driven cascades
were performed in deuterated T ris-HCl buffer (50 m m , pD 8.0) with 5 m m diamine substrate and 2 m m NAD + cofactor in the flow reactor at 22
8

C
for 16 h. O 2 and H 2 were generated by performing electrolysis for 5 hours using Pt/Pt electrodes at 2 V and pH 2.0. Product formation (in %) was
determined by GC-FID. The regioselective labeling was confirmed by 1 H NMR analysis (Figures S10–S18). The labeling yield was determined by
comparing the signals in LC-MS spectra (Figure S6–S9). a) Electrolysis with pentlandite/Ni electrodes at 3.5 V and pH 1.3. b) Substrate
concentration 8 m m .* x D indicates the incorporation of x deuterium atoms into the product. R = methyl.
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ensured that no parameter was limiting within the cascade. It
is worth mentioning that such a monitoring system (F igure 1)
had previously only been used in vivo during fermentations .
Notably , we prepared various methylated N-heterocycles
with product formations of up to 99 %. T he results obtained in
the conversion of 1 and 3 were comparable to those from
previous work using native IRED and N ADP + -reducing
SH. [5] Interestingly , in this approach, 1,5-diaminopentane ( 5 )
was poorly converted. T he poor conversion might be
explained by the application of the IRED NADH variant. T he
K M,NADH value of this variant is ten times higher than the K M
value of its wild type for N ADPH. T he k cat value of
IRED NADH is 41 % lower than that of wild-type IRED for
the reduction of the model substrate 2-methylpyrroline . [6] We
have also observed product inhibition on IRED NADH for 2 and
6 (F igure S22). Herein, a scale-up of the reaction was easily
made possible by increasing the reaction volume (up to
300 ml). W e produced 69 mg of 8 and 55 mg of 2 in a 300 mL
and 150 mL setup , respectively , using a threefold and twofold
excess of the immobilized enzymes (T able S2). Furthermore ,
we produced 3 mg of N -methylpyrrolidine from linear
N -methylputrescine (11 % yield) by using the same set of
immobilized enzymes on a 150 mL scale .
T he new setup was then used to test the reusability of the
immobilized enzymes . T he same set of immobilized enzymes
could produce 6 from 5 , with 26 % of the maximum product
formation retained after six cycles (F igure S5). T he total
turnover numbers were very high for all biocatalysts (T ables 1
and S3), demonstrating the operational stability of our
system.
F inally , we studied the labeling of piperidines with stable
isotopes such as deuterium in the electro-driven cascade . T he
preparation of organic compounds labeled with hydrogen
isotopes is of essential importance in the chemical, biological,
and environmental sciences . Hence , deuterated fine chem-
icals are valued molecules for spectroscopic analysis , phar -
maceuticals , and analytical tracing . [12] Herein, we exploited
the unique characteristic of the SH to synthesize deuterated
N AD(D). SH splits H 2 at the hydrogenase module separately
from the N AD + reduction at the diaphorase module. H 2 -
driven labeling can be achieved using D 2 O by providing two
electrons from H 2 splitting and D + from D 2 O-generating
N AD(D). [13] N AD(D) can then be utilized as a cofactor for
various N ADH-dependent reductases such as IRED to
produce deuterated compounds (Scheme 1).
T he labeling was performed with D 2 O in the biotransfor -
mation and H 2 O in the electrolysis units . W e produced various
labeled piperidine derivatives from diamines with up to 99 %
of deuterium labeling. Only a small fraction of the products
was partially labeled (Figure 1). T his inhomogeneity can be
explained by the presence of water traces in D 2 O (purity
98 %), protons released from H 2 splitting (ca. 1 %), residual
water in the gases , remaining H 2 O from immobilized
enzymes , and the natural proton abundance of N AD + .W e
expected to observe labeling with a single D atom at the C6
carbon atom of the imine . Interestingly , we observed further
labeling with two D atoms at C5 (for the MS and NMR
spectra, see F igures S6–S18). W e hypothesize that the deut-
eration at C5 might have been caused either by a possible
keto–enol tautomerization, followed by the oxidation of the
substrate by PuOE 203G , or an imine–enamine tautomerization
with subsequent spontaneous cyclization of the aminoalde-
hyde intermediate . T o examine this hypothesis , we conducted
the isotopic labeling using imines (2-methylpyrroline and 3,4-
dihydroisoquinoline) and IRED NADH . Here , the labeling is
facilitated only by the activity of SH. Mass and NMR spectra
revealed a 85 % labeling corresponding to the C6 carbon
atom of the imine (F igures S19–S21) without any labeling on
further atoms . In addition, we used formate dehydrogenase
Figure 2. On-line monitori ng of H 2 ,O
2 , and NADH during the electro-driven biotransform ation. The concentrations of H 2 (A), O 2 (B), and NADH
(C) were followed during the first hour of the transformation of 7 into 8 as an exemplary biotransformation. The electrolysis was performed for
1 hour (yellow box) until both the H 2 and O 2 concentrations reached a plateau and stopped increasing. Then substrate and NAD + were added
(black arrows). Purple arrows indicate the activity of each enzyme : the activity of putrescine oxidase (PuO E203G ) is indicated by the consumption of
O 2 after adding the substrate (B) ; the activity of imine reductase (IRED NADH ) is shown in the consumption of NADH (C) ; and the activity of
soluble hydrogenase (SH) is shown in the consumption of H 2 (A) and the evolution of NADH (C). The formation of H 2 O 2 was monitored over
time ; no H 2 O 2 formation was detected. The experiment was performed as described in Figure 1.
T able 1 : T otal turnover numbers (TTN n produ ct / n enzyme ) of each biocatalyst
based on the sum of products formed after the six cycles using the same
set of immobilized enzymes.
Biocatalyst SH IRED NADH PuO E203G C atalase
TTN 1.1  10 6 1.6  10 4 1.2  10 4 1.6  10 8
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(FDH) for cofactor regeneration in control experiments . No
labeling was observed when FDH was used. T his finding
indicates that the deuteration at C5 was merely caused by the
keto–enol tautomerization (Scheme 1).
W e determined that only about 4 % of the electrical
energy was used for product formation (see Chapter S2.11 in
the Supporting Information). Wh ile this value seems very low ,
this finding can be rationalized by the low solubility of the
gases in water, reduction of O 2 at the HER, heat production
by overpotentials , inefficient gas transfer through the mem-
brane , and product inhibition of IRED NADH . Our system can
be further optimized by using suitable gas exchange mem-
branes , sophisticated electrodes , and further engineered
biocatalysts with improved features and broader substrate
scopes .
In conclusion, we have designed a scalable platform to
power enzymatic cascades with electricity . W e furthermore
demonstrated the potential of the novel design by producing
various piperidine derivates from diamines . W e extended the
applicability of the system towards performing regioselective
isotopic labeling and provided useful insight into imine
reduction by IREDs . T his platform represents an important
advance in the field of biocatalytic synthesis , and it can be
expanded for powering various cofactor -dependent oxidor -
eductases .
Acknowledgements
A.A-S . and L.L. received funding from the German Research
F oundation (Deutsche F orschungsgemeinschaft, DFG , proj-
ect number 284111627) and the Einstein foundation. L.L. was
funded by the Deutsche F orschungsgemeinschaft (DFG ,
German Research F oundation) under Germanys Excellence
Strategy – EXC 2008/1 – 390540038 – UniSysCat and by the
F onds der Chemischen Industrie . U .-P .A. and K.j.P received
funding from the DFG (Emmy Noether Grant to U .-P .A. ,
AP242/2-1). U .-P .A. was funded by the Deutsche F orschungs-
gemeinschaft (DFG , German Research F oundation) under
Germanys Excellence Strategy – EXC 2033 – 390677874 –
RESOL V as well as by the F raunhofer Internal Programs
under Grant No . Attract 097–602175. W e thank Reinhard
Schçmcker, Gabrielle V etter, Sebastian Kemper, and Marc
Griffel for product analysis by GC, MS , and NMR spectros-
copy , respectively , Oliver Lenz for generous support by
providing access to his lab equipment, Changzhu W u for
providing Amberlite FP A 54, and Enginzyme (Stockholm,
Sweden) for EziG TM .
C onflict of interest
T he authors declare no conflict of interest.
Keywords : electrochemical biocatalysis · hydrogenases ·
imine reductases · isotopic labeling · N-heterocycles
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Accepted manuscript online: March 23, 2020
V ersion of record online: April 28, 2020
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