Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation

Article abstract of DOI:10.1021/acscatal.8b04500

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogena

Full text of DOI:10.1021/acscatal.8b04500

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Article
2
Straightforward Regeneration of FADH Required
for Enzymatic Tryptophan Halogenation
Mohamed Ismail, Lea Schroeder, Marcel Frese, Tilman Kottke,
Frank Hollmann, Caroline Emilie Paul, and Norbert Sewald
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04500 • Publication Date (Web): 04 Jan 2019
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ACS Catalysis
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Straightforward Regeneration of FADH2 Required for
Enzymatic Tryptophan Halogenation
Mohamed Ismail,†,# Lea Schroeder,§ Marcel Frese,† Tilman Kottke,§ Frank Hollmann,‡ Caroline E.
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Paul,¶,‡ and Norbert Sewald*,†
† Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld,
Germany
§ Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld,
Germany
‡ Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands
¶ Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The
Netherlands
# Department of Microbiology, Faculty of Science, Helwan University, Ain Helwan, Helwan, Cairo 11795, Egypt
ABSTRACT: Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties,
e.g. the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical
halogenation. However, the cofactor FADH2 has to be regenerated using a flavin reductase. Consequently, coupled biocatalytic
steps are usually applied for cofactor regeneration. NADH mimics can be employed stoichiometrically to replace enzymatic
cofactor regeneration in biocatalytic halogenation. Chlorination of L-tryptophan is successfully performed using such NADH
mimics. The efficiency of this approach has been compared to the previously established enzymatic regeneration system using two
auxiliary enzymes PrnF and ADH. The reaction rates of some of the tested mimics were found to exceed that of the enzymatic
system. Continuous enzymatic halogenation reaction for reaction scale-up is also possible.
Key words: regioselective chlorination, flavin dependent halogenases, hydride transfer, NADH mimics, enzymatic cofactor
regeneration, FADH2
Halogenated natural products are of high
biological and biochemical relevance. Many halo-
genated metabolites or their derivatives found
their way into pharmaceutical and agricultural
applications.1–4 Although different chemical
halogenation methods are known, most of them
suffer from low regioselectivity and/or
environmental burden.5,6 In view of the
biosynthesis of halogenated metabolites, FAD-
dependent halogenases have become promising
alternatives for the regioselective halogenation of
natural and synthetic molecules using only halide
salts and molecular oxygen under mild reaction
conditions.7–9 However, this enzyme class still
faces several drawbacks that compromise a
wider application in biocatalytic reactions: a low
Figure 1. Enzymatic tryptophan halogenation with cofactor regeneration
comprising a flavin reductase and a dehydrogenase.
activity, combined with a substrate scope that is
mainly limited to certain core structures,10,11 as
well as their dependence on the cofactor
aerocolonigenes,15,16 or SsuE from Thermus thermophilus12 with
FADH2.12 Hence, the enzymatic halogenation reaction requires a
complex cofactor regeneration system that maintains a steady
FADH2 supply. The regeneration systems for this group of
enzymes usually includes two auxiliary enzymes in addition to
the halogenase in an enzyme cascade system (Figure 1).12–14
Enzymatic cofactor regeneration systems for biocatalytic
halogenation rely on the combination of a flavin reductase like
PrnF from Pseudomonas fluorescens,8,12 RebF from Lechevalieria
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either an alcohol dehydrogenase (ADH) or
a
glucose
BNAH reduced the drug to give a product that can access the
malaria parasite.24 Recently the application of NADH mimics
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dehydrogenase (CDX-901). The ADH (e.g. from Rhodococcus
ruber) regenerates NADH from NAD+ by catalyzing the oxidation
was further investigated in biocatalysis.25
of isopropanol to acetone.8
A
glucose dehydrogenase
The use of NADH mimics was first studied with respect to the
functional role of the pyridine ring in NADH,26 followed by
mechanistic investigation with BNAH. The application of NADH
mimics on NADH-dependent enzymes was first reported for the
horse liver ADH,27,28 and further expanded to other enzymes
including HbpA monooxygenase.29 Recently several NADH
mimics were synthesized to replace the natural cofactor
required by ene reductases from the Old Yellow Enzyme family
for biocatalytic reductions.19,25,30,31
regenerates NAD(P)H by catalyzing the oxidation of D-glucose to
D-glucono-1,5-lactone, followed by a non-enzymatic hydrolysis
to gluconic acid.15–17 The complexity of these enzymatic
cascades limits the usability of halogenases in further
applications and combination with other synthetic reactions like
in chemoenzymatic cascades.
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R1
Br-
R1
MeCN,
Na2S2O4
NaHCO3
R1
R2-CH2-Br
N+
N
reflux, 15h
H2O, rt, 3h
N
R2
R2
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1
2
a
b
c
d
BNAH: R = CONH2; R = Ph
1
2
mBU: R = CONH2; R = Bu
1
2
mAC: R = COCH3; R = Ph
1
2
mCN: R = CN; R = Ph
Figure 2. Synthesis of NADH mimics for FADH2
regeneration.17
As cofactor regeneration is a common prerequisite for many
biocatalytic reactions, alternative non-enzymatic regeneration
systems have been proposed and investigated. The direct
regeneration of FADH2 for tryptophan 7-halogenase PrnA was
Figure 3. Enzymatic tryptophan halogenation with cofactor
regeneration by NADH mimics.
As Paul et al. described previously, different synthetic NADH
mimics can be easily obtained from inexpensive starting
materials in two synthetic steps. A pyridine derivative is N-
alkylated under reflux for 15 h to form a pyridinium salt and
subsequently reduced under inert atmosphere with Na2S2O4
forming the NADH mimicking dihydropyridine (Figure 2).19,32
The synthesis is straightforward and does not require any harsh
conditions, toxic reagents, or further purification after the
synthesis.
first
reported
using
the
organometallic
rhodium
complex
bipyridine
pentamethylcyclopentadienyl
([Cp*Rh(bpy)(H2O)]2+) as catalyst and formate as electron donor
without requiring flavin reductase and NADH (additional
regeneration systems are summarized in table S3).18 The use of
a synthetic mimic of the natural cofactor system is a promising
approach. NADH mimics have recently attracted attention for
their implementation as a regeneration system for cofactor-
dependent enzymes.19,20 They are simpler compared to
enzymatic cofactor regeneration system, their application in the
reactions is easy and does not require special apparatus or
preparations making them more robust and easier to control.
Moreover, the reaction is scalable and proceeds in aqueous
medium under mild conditions without affecting the enzymatic
activity in the biocatalysis. For these reasons we became
interested in using NADH mimics as a regeneration system for
flavin-dependent halogenases.
NADH mimics have been applied in organic reductions as
model systems for understanding the mechanism of the natural
biochemical reactions or for their use in organic synthesis, for
instance the stereoselective reduction of pyruvate under mild
conditions using NADH mimics and perchlorate.21 Moreover, the
reduction of thiobenzophenone to benzhydryl mercaptan has
been described using 1-benzyl-1,4-dihydronicotinamide
(BNAH).22 Medicinal applications have also been reported,
where the reduced nicotinamide derivative 1-carbamoylmethyl-
3-carbamoyl-1,4-dihydropyridine was used as a cosubstrate for
activating the NAD(P)H quinone oxidoreductase 2 (NQO2),
which further activates the antitumor prodrug 5-(aziridin-1-yl)-
2,4-dinitrobenzamide with higher efficiency and stability than
the natural reduced nicotinamide riboside.23 Other reports used
the NADH mimics to explain the mechanism of artemisinin’s
antimalaria activity in vitro, where the hydride transfer from
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ACS Catalysis
O
O
and tested with FAD-dependent halogenases (Table 1; RebH:
Figure 5; Thal: Figure 6A; PyrH: Figure S3B).
OH
OH
NH2
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0.6 mol% RebH, 30 mM NaCl,
10 eq NADH mimic, 100 µM FAD
NH2
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N
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H
H
Cl
7-Chlorotryptophan
1 mM Tryptophan
A
1,0x106
7-Chlorotryptophan
8,0x105
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4,0x105
2,0x105
0,0
9
Oxidized
BNAH
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2 h.
0 h.
Tryptophan
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Time [s]
300
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500
Time [min]
B
Figure 5. Screening of different NADH mimics to regenerate
FADH2 for the halogenase RebH in comparison to the
conventional enzymatic regeneration system ( 3a, 3b,
ESI-Positive mode
7-Chlorotryptophan
3c,
3d,
ERS). The conversion of tryptophan was
monitored with LC-MS. Reactions were done in triplicates
using 1 mM tryptophan, 100 µM FAD, 1.2 mol% RebH, 650 U
-1
mL catalase and 10 mM NADH mimic or PrnF, ADH at pH
7.4 and 25 °C. In case of the ERS, 2.5 U mL-1 flavin reductase
Figure 4. FADH2 regeneration with BNAH (3a) in the
enzymatic halogenation with RebH. (A) RP-HPLC
chromatogram. (B) LC-MS confirms the correct mass of the
product. Reduced 3a was extracted with organic solvent
before the analysis. Reaction conditions: 10 mM Na2HPO4
buffer, pH 7.4, 30 mM NaCl, 650 U mL catalase, 10 mM
NADH mimic, 1 mM tryptophan, 100 µM FAD, 0.6 mol% (6
PrnF,
1
U
mL-1 alcohol dehydrogenase, 5% (v/v)
isopropanol and 100 μ M NAD+ were used instead of the
NADH mimic.
Interestingly, RebH (Figure 5) and Thal (Figure 6) showed the
highest initial reaction rate and fastest conversion with 3a and
3c, even outperforming the enzymatic cofactor regeneration
system (ERS). The reaction proceeded 3.5-4.7 times faster than
with the enzymatic cofactor regeneration system (Table 1,
Figure S5). The highest FADH2 regeneration efficiency was
achieved with 3a, of which the structure most closely resembles
NADH. With RebH, 100% conversion of 1 mM tryptophan was
achieved after only 2 h using 3a, whereas 5 h were needed to
achieve full conversion under optimal reaction conditions using
ERS.8 This is in agreement with previous reports on the NADH
mimic 3a, where it was found to effect 40 times faster hydride
transfer rate than NADH.29 With 3c the reaction rate is
comparable to 3a and 3b with a butyl group at the pyridine
nitrogen initially shows high efficiency, exceeding that of 3c.
However, the reaction with 3b seems to stop at approx. 47%
conversion, most likely due to the instability and rapid oxidation
of this cofactor. Mimic 3d with a nitrile group at the pyridine
ring displays the lowest reaction rate and activity, a common
trend for this mimic, which has a higher redox potential (ca. -
220 mV) compared to the others.35
-1
µM) RebH, final volume 400 µL, 600 rpm at 25 ᵒC.
The application of NADH mimics for the regeneration of
FADH2 required by FAD-dependent tryptophan halogenases
leads to a much simpler enzymatic halogenation system as both
auxiliary enzymes would become redundant (Figure 3). In order
to explore the applicability of NADH mimics as an alternative
cofactor regeneration system for tryptophan halogenases, three
flavin-dependent halogenases with different activities and
regioselectivities were investigated in detail.
The tryptophan 5-halogenase PyrH from Streptomyces
rugosporus,33 the tryptophan 6-halogenase Thal from
Streptomyces albogriseolus,34 and the tryptophan 7-halogenase
RebH from Lechevalieria aerocolonigenes16 were used as model
systems. The purified enzymes RebH and PyrH were first tested
using the standard protocol for the flavin-dependent
halogenation (see SI p. S8), while flavin reductase and alcohol
dehydrogenase usually required for enzymatic cofactor
regeneration were omitted. The halogenation reactions were
performed employing 10 equivalents of the NADH mimic BNAH
(3a, 10 mM). Intriguingly, nearly 50% of tryptophan were
halogenated within 2 h and the reaction proceeded to full
conversion after 24 h (Figure 4, Figure S2).
Hence, the FADH2 regeneration efficiency of the NADH mimics
is strongly affected by the R1 substituent in the first place with
preference for the carboxamide moiety (mimicking the natural
NADH) followed by the acetyl group. On the other hand, the
substituent at the pyridine nitrogen also affects the activity of
the mimic as observed for 3b (Table 1).
In order to identify the NADH mimic with the highest FADH2
regeneration efficiency, several literature-known NADH mimics
3 (BNAH 3a, mBU 3b, mAC 3c, and mCN 3d) were synthesized
The stability of the mimic would also play an important role in
the regeneration of FADH2, in particular in the case of 3b and
3d. This might be attributed to a more rapid consumption of
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these NADH mimics in the reduction of FAD to FADH2. Besides
Thal), final volume 200 µL, shaking at 600 rpm at 25 ᵒC. In case
of the ERS, 2.5 U mL-1 flavin reductase PrnF, 1 U mL-1 alcohol
dehydrogenase, 5% (v/v) isopropanol and 100 μ M NAD+ were
used instead of the NADH mimic.
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reaction of enzyme-bound FADH2 with O2 eventually leading to
halogenation, FADH2 also reacts with molecular oxygen freely in
solution. This uncoupling reaction forms hydrogen peroxide and
consumes NADH mimic in a nonproductive manner. In addition,
the accumulation of H2O2 may compromise the stability of the
mimic and halogenase activity.20 Catalase was added to the
reactions in order to prevent H2O2 accumulation.36
Notably, PyrH displays a lower reaction rate and activity
compared to RebH and Thal with the NADH mimics but a higher
reaction rate with the ERS. However, both regeneration
reactions are assumed to proceed via reduction of FAD in
solution independent of the halogenase. Subsequently, FADH2 is
rapidly bound to the enzyme.37 Recently, the direct regeneration
of enzyme-bound FAD in PyrH by photochemical regeneration
using a reducing agent (EDTA) and light has been reported.38 In
order to test whether 3a might also directly reduce FAD inside
the halogenase, PyrH was reconstituted with FAD and washed
thoroughly to remove free FAD from the sample.
Table 1: Initial reaction rates (v0) for different NADH
mimics and ERS with different flavin-dependent
halogenases RebH, Thal and PyrH.
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NADH mimic 3a
3b
3c
ERS
Initial reaction rate (v0) [× 10-3 µmol min-1]*
Enzyme
RebH
Thal
The spectrum of the sample shows a distinct fine structure in
the band at 450 nm (Figure 7) and a shift of the band at 373 nm
to 350 nm (Figure S6), which is characteristic for protein-bound
FAD. Bleaching of the band at 450 nm is observed upon addition
of 3a to the oxygen-free solution, giving proof of FADH2
formation.39
6.01 ±0.003 4.95 ±0.012 4.83 ±0.016
7.62 ±0.001 6.88 ±0.005 6.81 ±0.008
1.26 ±0.002
1.87 ±0.001
4.51 ±0.002
PyrH
4.07 ±0.001 4.29 ±0.003
2.23 ±0.01
* Reaction conditions: 10 mM Na2HPO4 buffer, pH 7.4, 30 mM
NaCl, 650 U mL-1 catalase, 10 mM NADH mimic, 1 mM tryptophan,
100 µM FAD, 1.2 mol% tryptophan halogenase (RebH, PyrH or
A
B
100
100
80
60
40
20
0
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60
40
20
0
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Time [min]
Time [min]
Figure 6. Application of different Thal protein concentrations (A: 12 µM (1.2 mol%), B: 2.4 µM (0.24 mol%) using NADH
mimics for flavin cofactor regeneration ( 3a, 3b, 3c, 3d, 3b activity was not detected in (B)). Error bars represent
the mean values ± standard deviation from three independent reactions. Reaction conditions: 10 mM Na2HPO4 buffer, pH
-1
7.4, 30 mM NaCl, 650 U mL catalase, 10 mM NADH mimic, 1 mM tryptophan, 100 µM FAD, final volume 200 µM, shaking at
600 rpm at 25 ᵒC.
Within 23 min, the conversion is complete. After admission of
oxygen to the cuvette, FADH2 is consumed forming FAD within
in the protein as evidenced by the same fine structure as prior to
addition of mimic 3a (Figure 7).
These results indicate that FAD might be slowly reduced by
the mimic directly inside the enzyme PyrH, which would explain
the observed differences between the two regeneration
approaches of the halogenases (Table 1) by their different
affinities to FAD. It should be noted that we cannot exclude from
these experiments a reduction via catalytic amounts of free FAD
in solution which is involved in a rapid binding equilibrium with
the enzyme.
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ACS Catalysis
As expected, the reaction rate was almost proportional to the
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7
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FAD concentration (Figure 8A). Continuous increase in FAD
concentration showed an increased rate of 3a oxidation.
However, FAD concentrations exceeding that of 3a resulted in
no further improvement. The same was observed with
increasing concentration of 3a at a fixed FAD concentration
(data not shown). A steady FADH2 supply is crucial for the
biohalogenation reaction; however, its accumulation results in
H2O2 formation. As the coupling efficiency of the halogenation
reaction is rather low, a certain ratio of FAD:3a is required in
order to assure a continuous biohalogenation without shifting
the equilibrium towards H2O2 formation. Therefore, different
FAD concentrations were tested in the biohalogenation reaction
with 3a. As expected lower FAD concentrations (20, 50 µM)
showed higher conversion of tryptophan, while using 1:1 molar
ratio of FAD:3a did not result in any product formation (Figure
8B). This phenomenon was also observed previously with
styrene monooxygenase, where the higher FAD concentration
results in aerobic reoxidation of FADH2 and consequently H2O2
formation instead of the product chlorotryptophan. 31
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Figure 7. UV/vis spectra monitoring the reduction of FAD
bound to PyrH after addition of 3a. Binding of FAD to PyrH
is characterized by a fine structure between 450 and 470
nm (highlighted in grey). Reaction conditions: 40 µM PyrH,
10 mM BNAH (3a), FAD:protein ratio 1:2, 50 mM phosphate
buffer at pH 8.3 and 20 °C, anaerobic.
A
As the activity of PyrH is much lower than RebH and Thal
when combined with the NADH mimics, halogenation in the
presence of NADH mimics as regeneration system might not
only depend on the affinity of FAD to the protein and the
concentration of the flavin cofactor, but also on the enzymatic
activity. Different concentrations of Thal were used for testing
the different NADH mimics (Figure 6). Halogenation of L-
tryptophan was faster with 12 µM Thal (1.2 mol%) compared to
halogenation at a 5 times lower halogenase concentration
(2.4 µM Thal; 0.24 mol%).
0,012
0,010
0,008
0,006
0,004
0,002
0,000
Full conversion could not be obtained in the latter case with
the tested NADH mimics. This indicates that the halogenation
reaction using NADH mimics as a FAD cofactor regeneration
system is halogenase-dependent, where the halogenase is rate-
limiting. The FADH2 formed reacts with oxygen to the flavin
hydroperoxide, which either generates hypohalous acid for
halogenation or H2O2. Based on the substrate conversion and the
used amounts of mimic with RebH and PyrH, the coupling
efficiency was calculated to be less than 1 %. Therefore,
increasing the halogenase load or engineering the existing
halogenase for higher efficiency would increase the
halogenation rate by directing the excess FADH2 towards
halogenation instead of H2O2 formation.31 The same reactions
were performed with higher PyrH concentrations (12 µM, 1.2
mol% and 24 µM, 2.4 mol%, Figure S3C). The reaction rate and
conversion using the NADH mimics increased upon doubling
enzyme concentration, but still remained lower than in the
reactions catalyzed by 1.2 mol% of RebH and Thal.
0
50
100
150
200
250
300
FAD [µM]
B
100
80
60
40
20
0
1
10
20
50
100
500 1000 5000
FAD [µM]
Figure 8. A) Hydride transfer reaction between 3a and FAD.
The reaction was monitored by measuring the oxidation of
3a at 360 nm. Reaction conditions: 50 mM Tris-HCl buffer,
pH 7.4, 30 mM NaCl, 150 µM 3a, 5-300 µM FAD in 1 mL
volume. B) Biohalogenation reaction using 3a at variable
FAD concentrations. Reaction conditions: 50 mM Tris-HCl
buffer, pH 7.4, 30 mM NaCl, 10 mM 3a, 1-5000 µM FAD, 1.2
Since 3a had been identified as the best NADH mimic for
enzymatic halogenation with RebH and Thal, reaction conditions
and NADH mimic concentration were further optimized for
RebH and PyrH, because RebH and Thal have nearly similar
activities and reaction rates. 50 mM Tris-HCl buffer, pH 7.4 at
25 °C and 20 mM 3a were found as optimal conditions for both
RebH and PyrH. Using tryptophan 7-halogenase RebH after
prolonged reaction period, a dichlorinated tryptophan was
formed as side product. This side reaction can be avoided by
monitoring the progress of the reaction and though the enzyme
load applied to the reaction (Figure S7, Table S1).
-1
mol% RebH, 650 U mL catalase, 25 °C for 1.5 h in 200 µL
volume.
In order to prove that the NADH mimic regeneration system is
also suitable for enzymatic halogenation using immobilized
halogenases, we employed this system for the chlorination of L-
tryptophan. RebH and PyrH were immobilized as cross-linked
enzyme aggregates (CLEAs) as described previously, but
without the addition of the auxiliary enzymes.40,41 At a 20 mM
In order to investigate FAD reduction by 3a independently
from the halogenation, the reaction was performed at fixed
concentration of either FAD or 3a, while altering the other one
and the oxidation of 3a was measured via absorption at 360 nm.
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concentration of 3a, full conversion of 0.5 mM tryptophan was
tryptophan 7-halogenase RebH to produce 126.7 mg (74.9 %
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6
7
8
achieved within 5 h, a relatively shorter time compared to the
enzymatic cofactor regeneration system (Figure S8, Table S2).
Notably, the initial reaction rates of each of RebH and PyrH
under the same reaction conditions and enzyme concentrations
are 1.3-1.9 times faster using 3a compared to the ERS, which has
also been found when using the purified enzymes (Figure S9).
This proves the utility of the NADH mimics as a valuable
yield) over the course of 8 days. Due to the simplicity of the
NADH mimic regeneration system, it could be employed in the
course of halogenases engineering via directed evolution. In
particular, the mimics might show potential when improving
halogenase thermostability, because the auxiliary enzymes lack
the required stability at elevated temperature. Halogenase
engineering might improve the coupling efficiency that
eventually would improve the performance of NADH mimics
applied together with FAD dependent halogenases. This will
facilitate and improve the applicability of FAD dependent
halogenases for chemical biotransformation.
alternative
regeneration
system
for
FAD-dependent
halogenases.19,29
9
With most of the synthetic mimics, the overall reaction
proceeds faster than the enzymatic cofactor regeneration
system (Figure 5, Figure 6A, Figure S9). However, after an initial
phase rapid consumption of the NADH mimics and a significant
decline in the halogenation reaction rates was detected using
immobilized enzymes.
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ASSOCIATED CONTENT
Supporting Information
Continuous addition of the mimics is one approach to
overcome this problem. Incremental addition of the substrate
and the NADH mimic 3a was selected after several attempts for
scaling up the reaction using the immobilized enzymes. It was
possible to run the reactions for up to 24 days using RebH
CLEAs and 15 days using PyrH CLEAs without the necessity of
exchanging the reaction buffer or adding more halogenase or
cofactor.
Details for the synthesis of the NADH mimics, enzymatic
reactions using mimics and ERS and semi-preparative scale
reaction are available in the Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website.
A semi-preparative scale reaction using the cross-linked
enzyme aggregates of RebH with continuous addition of 3a as
the sole regeneration factor was performed. 5 equivalents of 3a
were initially applied for biocatalytic chlorination of 10 mg L-
tryptophan (0.05 mmol) by RebH CLEAs in 500 ml reaction
buffer. Based on the consumption of the reactants, L-tryptophan
and 3a were added incrementally in the course of the reaction
(Figure S10; Scheme S3). After 8 days, 102 mg L-tryptophan (0.5
mmol) were chlorinated to 7-chlorotryptophan by using 17.8
molar equivalents of 3a and RebH CLEAs obtained from 6 L of E.
coli culture. For the product purification, Boc-protection was
conducted, followed by extraction with dichloromethane.
Finally, a yield of 74.9% of Boc-7-chlorotryptophan was
obtained (Figure S11). Currently, the low coupling efficiency of
tryptophan halogenases represents a major drawback for the
applicability of the preparative scale reaction. Because of the
competing H2O2 formation, a large percentage of NADH mimic is
being consumed.
AUTHOR INFORMATION
Corresponding Author
* Prof. Dr. Norbert Sewald (norbert.sewald@uni-bielefeld.de)
Organic and Bioorganic Chemistry, Department of Chemistry,
Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
C.E.P. acknowledges a NWO Veni grant number 722.015.011.
M.I. acknowledges funding from the German Academic exchange
service (DAAD) and Bielefelder Nachwuchsfonds. T.K.
acknowledges a Heisenberg fellowship by the DFG (KO3580/4-
2).
This result, in comparison to the previous report on the
upscaling of the enzymatic halogenation reaction,41 has revealed
the usability of the NADH mimic regeneration system for the
scale up reaction comparable to the reported results using ERS.
FAD-dependent halogenases such as RebH can further be
function for prolonged periods (>20 days) using the NADH
mimics regeneration system; most likely the instability of the
auxiliary enzymes impedes longer reaction times using ERS.
Halogenated products or by-products of the auxiliary enzyme
(ADH) may have a detrimental effect on the catalytic activity of
the enzymes (product/by-product inhibition). This provides
another attractive advantage of the NADH mimic over the ERS.
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