Regioselective enzymatic halogenation of substituted tryptophan derivatives using the FAD-dependent halogenase RebH

Article abstract of DOI:10.1002/cctc.201301090

Regioselective methods to establish carbon-halide bonds are still rare, although halogenation is considered as a commonly used methodology for the functionalization of organic compounds. The incorporation of halogen substituents by organic synthesis usually requires hazardous conditions, shows poor regioselectivity and results in the formation of unwanted byproducts. In addition, halogenation by electrophilic aromatic substitution (S_EAr) obeys distinct rules depending on electron-withdrawing or -donating groups already present in the aromatic ring. We employed the tryptophan-7-halogenase RebH for regioselective enzymatic halogenation to overcome these limitations. In combination with a tryptophan synthase, an array of C5- and C6-substituted tryptophan derivatives was synthesized and halogenated by RebH. The halogenase is able override these directing effects and halogenates at the electronically unfavored C7-meta-position, even in presence of ortho/para-directing groups. No business as usual: The tryptophan halogenase RebH from Lechevalieria aerocolonigenes is able to halogenate at the electronically unfavored C7-meta-position of C5-substituted tryptophan derivatives, even in presence of deactivating ortho/para-directing groups.

Full text of DOI:10.1002/cctc.201301090

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DOI: 10.1002/cctc.201301090
Regioselective Enzymatic Halogenation of Substituted
Tryptophan Derivatives using the FAD-Dependent
Halogenase RebH
[
a]
Marcel Frese, Paulina H. Guzowska, Hauke Voß, and Norbert Sewald*
Regioselective methods to establish carbon–halide bonds are
still rare, although halogenation is considered as a commonly
used methodology for the functionalization of organic com-
pounds. The incorporation of halogen substituents by organic
synthesis usually requires hazardous conditions, shows poor re-
gioselectivity and results in the formation of unwanted by-
products. In addition, halogenation by electrophilic aromatic
matic ring. We employed the tryptophan-7-halogenase RebH
for regioselective enzymatic halogenation to overcome these
limitations. In combination with a tryptophan synthase, an
array of C5- and C6-substituted tryptophan derivatives was
synthesized and halogenated by RebH. The halogenase is able
override these directing effects and halogenates at the elec-
tronically unfavored C7-meta-position, even in presence of
ortho/para-directing groups.
substitution (S Ar) obeys distinct rules depending on electron-
E
withdrawing or -donating groups already present in the aro-
Introduction
[
10,11]
Halogenated compounds are important bioactive substances
and 258C in aqueous media.
This reaction is catalyzed by
[
1]
[2]
and essential intermediates in the chemical, agrochemical,
FAD-dependent halogenases, one of the major enzyme families
responsible for regioselective halogenation of aromatic or
[
3]
and pharmaceutical industry, owing to their metabolic stabili-
ty and the ease of modification, e.g., by nucleophilic substitu-
tion or metal-catalyzed cross-coupling reactions. However, the
introduction of halogen substituents, especially in mechanisti-
cally less-favored positions, remains a challenge. More than
[12]
hetero-aromatic rings in natural metabolites.
The first
[11]
member of this family, PrnA, was discovered in 2000. Its
close relative, the FAD-dependent tryptophan halogenase
RebH belongs to the biosynthetic pathway of the chlorinated
antitumor agent Rebeccamycin from Lechevalieria aero-
[4]
5000 naturally occurring halogenated compounds are known,
[13]
including molecules with diverse biological activities like hor-
colonigenes. The enzyme is able to chlorinate and brominate
[
5]
mones (e.g. thyroxine ), antibi-
[
6]
otics (e.g. chloramphenicol and
[
7]
chlorotetracycline ), and cyto-
[
8]
statics
(e.g.
cryptophycin ).
Moreover, halogenation is in
many cases essential for biologi-
[
9]
cal activity. Although chemical
halogenations mostly use molec-
ular chlorine or bromine as haz-
ardous reagents, often in a com-
bination with Lewis acids, nota-
bly Nature employs for halogen-
ation benign halide salts togeth-
er with molecular oxygen and
the reaction proceeds at pH 7
Scheme 1. Combination of tryptophan synthase from Salmonella enterica and tryptophan-7-halogenase RebH
from Lechevalieria aerocolonigenes for the regioselective halogenation of substituted tryptophan derivatives.
l-tryptophan regioselectively at the electronically unfavored C7
[
a] M. Frese, P. H. Guzowska, H. Voß, Prof. Dr. N. Sewald
Fakultꢀt fꢁr Chemie
position, while indole or tryptophan usually are halogenated at
[14]
Organische und Bioorganische Chemie, Universitꢀt Bielefeld
Universitꢀtsstr. 25, 33615 Bielefeld (Germany)
Fax: (+49)0521-106-8094
E-mail: norbert.sewald@uni-bielefeld.de
Homepage: http://www.uni-bielefeld.de/chemie/oc3sewald/
the C3 or C2 position respectively (Scheme 1).
Investigations by the groups of van Pꢀe and Walsh, pioneers
in the field of halogenases, have led to the elucidation of the
[15,16]
enzymatic halogenation mechanism.
The proposed mecha-
nism involves an additional flavin reductase, which supplies
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201301090.
FADH . The latter is oxidized by molecular oxygen to form
2
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a flavin peroxide, which is commonly found in Baeyer–Villiger
[
17–19]
monooxygenases.
After nucleophilic attack of the halide
À
À
ion (Cl or Br ) on the flavin peroxide intermediate, hypoha-
lous acid is formed and channeled along a 10 ꢂ long tunnel
from the FADH binding site to the tryptophan binding site.
2
Upon oxidation of the e-amino function of a distinct lysine resi-
due (K79) in the active site, a long-lived N-haloamine inter-
mediate is formed that is responsible for the regioselective
halogenation at the electronically unfavored C7 position of
[
15–17,20]
tryptophan.
Results and Discussion
Establishment of an enzymatic halogenation system
As the regiochemistry of chemical halogenation of substituted
arenes is restricted by electronic substituent effects, we
became interested in the potential of enzymatic halogenation
to overcome these limitations. This study focuses on enzymatic
halogenation by the l-tryptophan-7-halogenase RebH that was
amplified from genomic DNA of L. aerocolonigenes via PCR,
cloned into the expression vector pET28a and overexpressed
[
21]
in E. coli BL21(DE3) pGro7 to establish an in vitro enzymatic
halogenation system. After purification by means of immobi-
lized metal ion affinity chromatography (IMAC), enzyme activi-
ty was determined on an analytical scale in the presence of
[11]
the flavin reductase PrnF from Pseudomonas fluorescens and
[
22]
an alcohol dehydrogenase from Rhodococcus sp. for in situ
cofactor regeneration (Figure 1A). l-Tryptophan was converted
almost quantitatively into 7-chloro-l-tryptophan (1), as identi-
fied both by a HPLC-standard and by mass spectrometry (Fig-
ure 1B). Product formation was analyzed based on the con-
sumption of l-tryptophan in comparison with a calibration
Figure 1. Halogenase activity assay of purified RebH. A) Enzymatic activity
was measured in the presence of the flavin reductase PrnF and an alcohol
dehydrogenase to promote cofactor regeneration. B) The activity of RebH
was monitored by reversed-phase HPLC at l=280 nm. l-Tryptophan
À1
curve (Figure 1C). A catalytic constant of kcat =1.0 min and
(
0.25 mm) is eluted at a retention time of t
=177 s corresponds to 4-nitrophenol as an internal standard. During
30 min, a constant consumption of l-tryptophan was observed. In addition,
a new signal at t =156 s appeared, which was identified as 7-chloro-l-tryp-
r
=134 s, whereas the signal at
a total turnover number (TTN) of 102Æ3 could be extracted
t
r
from these data.
r
tophan (1) based on a standard, as well as by LC-MS. C) Product formation
during the reaction process was calculated based on the decrease in peak
area of the l-tryptophan signal in comparison with a calibration curve.
Halogenation of substituted tryptophan derivatives
As the halogenation of the natural substrate l-tryptophan was
successful with our in vitro system on an analytical scale, the
influence of electron-withdrawing and -donating groups at the
indole ring on the regioselectivity of halogenation by RebH
was further studied. An array of C5- and C6-substituted l-tryp-
tophan derivatives was envisaged to be synthesized enzymati-
Table 1. Total turnover numbers [TTN] for the chlorination of selected
Trp-derivatives.
Substrate
M/I effect
TTN
[23,24]
cally using a tryptophan synthase from Salmonella enterica.
Briefly, a lysate from a commercially available E. coli strain
ATCC strain 37845) producing recombinant tryptophan syn-
l-tryptophan
–
102Æ3
80Æ3
55Æ9
7Æ4
5-hydroxy-l-tryptophan (2)
+M, -I
+I
+M, -I
+M, -I
(
5-methyl-l-tryptophan (5)
5-fluoro-l-tryptophan (3)
thase was used to condense C5- or C6-substituted indoles with
l-serine in the presence of catalytic amounts of pyridoxal
phosphate to provide the corresponding array of l-tryptophan
derivatives. These compounds were then used as substrates in
RebH halogenation. All of the synthesized derivatives could be
chlorinated, as well as brominated, although the TTN differ sig-
nificantly among all substrates (Table 1). Electron-rich arenes
like 5-hydroxy-l-tryptophan (2) show a TTN similar to the natu-
ral substrate l-tryptophan (TTN=80Æ3 and 102Æ3 respec-
5-bromo-l-tryptophan (4)
traces
tively), whereas arenes with electron-withdrawing groups are
expectedly characterized by much lower TTN, for example, 5-
fluoro-l-tryptophan (3) was converted with a TTN of 7Æ4. The
tendency of the TTN matches the substituent influence on S Ar
E
caused by the mesomeric and/or inductive properties. Upon
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halogenation of 5-bromo-l-tryptophan (4) only traces could be
observed, probably due to the high van der Waals radius of
the bromo substituent in combination with its highly negative
inductive capacity. 5-Methyl-l-tryptophan (5) is converted at
a moderate TTN of 55Æ9, which is in good agreement with
the observed tendency due to its positive inductive effect.
As an alternative to halogenation with purified enzyme, the
reactions were also conducted with E. coli lysate containing
overexpressed RebH. Surprisingly, in many cases the main
product is still halogenated at the electronically unfavored C7
position, even in presence of deactivating and ortho/para-di-
recting groups like the 5-fluoro-substituent. The position of
halogenation in the case of 5-fluoro-l-tryptophan (3) can easily
mainly halogenated at the C6 position. In both cases, dihaloge-
nation occurs in C6 and C7 position for 5-amino (6) and 5-
methyl-l-tryptophan (5), as shown by ROESY-NMR cross-signals
between Cb and C2 and C4 protons at the indole ring. A possi-
ble explanation for the altered regioselectivity for 5-methyl-l-
tryptophan (5) could be a change in the binding position of
the substrate inside RebH in combination with the electronic
preference of the ortho-position. Docking analysis hints toward
a slight rotation of the 5-methyl-derivative in comparison to
the natural substrate l-tryptophan (Figure 3B), whereas no sig-
1
be identified as C7 by using H NMR-spectroscopy (Figure 2).
1
Figure 2. H NMR spectrum of the regioselective halogenation product of 5-
fluoro-l-tryptophan (3). Chlorination takes place in the meta-position of the
fluoro-substituent, an deactivating ortho/para-directing group. This can be
confirmed by the coupling constants of each proton to the C5-fluoro sub-
4
stituent, as well as by the J coupling between both aromatic indole protons
3
4
( JH,F =9.5 Hz, JH,H =2.2 Hz).
Two symmetric double doublet signals are observed for the
1
two protons adjacent to the fluoro substituent in the H NMR
Figure 3. Docking analysis of l-tryptophan compared to 5-hydroxy-l-trypto-
phan (A) and 5-methyl-l-tryptophan (B). 5-hydroxy-l-tryptophan (yellow) is
bound in a similar orientation compared to the natural substrate l-trypto-
phan (black), resulting in a halogenation at the C7 carbon atom, whereas 5-
methyl-l-tryptophan is slightly shifted inside the binding pocket, leading to
a mixture of both C6 and C7 halogenated product. (PDB ID: 2E4G)
spectrum of 7-chloro-5-fluoro-l-tryptophan (11) with two cou-
3
4
pling constants of J =9.5 Hz and J =2.2 Hz. Starting from
H,F
H,H
5
-hydroxy-l-tryptophan (2), the position of chlorination in 9
4
can also be identified as C7, owing to the J coupling of both
aromatic protons in the indole ring. Interestingly, bromination
takes place in the C6 position to give 10, identified by a cross-
signal of the C7 proton and the indole amine functionality in
the ROESY-NMR spectrum. In addition, the C6 and C7 halogen-
ated derivatives are formed together with dihalogenated prod-
uct, if a low concentration of the reactant 5-methyl-l-trypto-
phan (5) and 5-amino-l-tryptophan (6), respectively, is used. In
the case of 5-methyl-l-tryptophan (5), halogenation occurs at
C6 and C7, respectively, to give 13 and 14, as confirmed by
ROESY-NMR cross-signals between the protons of the C5
methyl group and the adjacent protons. Upon chlorination of
nificant change was observable for 5-hydroxy-l-tryptophan (2,
Figure 3A) or 5-amino-l-tryptophan (6).
Owing to this rotation, the C6 carbon atom of the 5-methyl
derivative is in close proximity to the original halogenation po-
sition of the C7 atom of l-tryptophan, resulting in a mixture of
C6 and C7 halogenated product. Although ortho/para-directing
groups in C5 position usually dominate the regioselectivity of
further S Ar reactions, the halogenase is able to override these
E
directing effects for deactivating and activating substituents
like hydroxyl functionalities, whereas strongly activating
groups like amines force the enzyme to halogenate the sub-
strate at the electronically preferred position. Nevertheless,
5-methyl-l-tryptophan (5), a 1:1 ratio between tryptophan de-
rivatives halogenated at C6 or C7 is observed, whereas bromi-
nation prevails at C6 to give 17. 5-Amino-l-tryptophan (6) is
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a combination of steric and elec-
tronic properties of the sub-
strate, as well as the nature of
electrophile (N-chloroamine or
N-bromoamine at K79) seems to
influence the actual position of
the second S Ar.
E
Conclusions
Tryptophan is a precursor mole-
cule e.g., of the mammalian hor-
mones serotonin and melatonin
that regulate several important
physiological processes in the
human body like blood pressure,
mood, as well as the day and
[
25,26]
night rhythm.
Regioselective
halogenation of tryptophan and
its derivatives might lead to
a direct access to intermediates
for the synthesis of potential se-
rotonin and melatonin agonists
and antagonists. In addition,
halogenated amino acids are of
great interest for the synthesis
of peptides, as they can easily
be modified by well-developed
processes, for example, nucleo-
philic substitution, or Pd-cata-
[
1]
lyzed cross coupling reactions.
In parallel to our studies, an en-
zymatic halogenation system
also based on RebH was estab- Figure 4. Halogenation products of tryptophan derivatives by RebH. Halogenation occurs in many cases preferred
in the electronically unfavored C7 position, even in presence of deactivating ortho/para-directing groups like 5-
fluoro.
lished independently by Payne
et al., showing the potential of
RebH for preparative applica-
[
21]
tions.
In combination with the tryptophan synthase from
using a gradient from 5–95% B over 10 min) coupled with
a Waters micromass ZQ2000 ESI-MS. For preparative HPLC purifica-
tion, a LaChrom System (Merck Hitachi) equipped with a Phenom-
enex Jupiter column (10 mm, C , 300 ꢂ, 250ꢃ21.1 mm, eluent A
[
23,24]
Salmonella enterica,
we established an artificial enzymatic
halogenation system based on the halogenase RebH for its
natural substrate l-tryptophan, as well as for C5- and C6-sub-
stituted tryptophan derivatives as non-natural substrates
18
H O/CH CN/TFA=95:5:0.1, eluent
flowrate 10 mL·min using a gradient from 0–35% B over 65 min).
B H O/CH CN/TFA=5:95:0.1,
2 3
2
3
À1
(
Figure 4). Halogenation by RebH clearly indicates that the ori-
For very polar analysts (e.g. 5-amino-l-tryptophan) an acetonitrile
entation of substrates inside the binding pocket of enzymes
has a high impact on the resulting product.
free eluent A (H O:TFA 100:0.1) was used. NMR spectra were re-
2
1
13
corded by using a Bruker DRX-500 ( H: 500 MHz, C: 125 MHz) or
Avance 600 ( H: 600 MHz, C: 150 MHz) spectrometer at 298 K and
1
13
chemical shifts are reported relative to residual solvent peaks (D O:
2
Experimental Section
1
1
H: 4.79 ppm, [D ]DMSO: H: 2.50 ppm). High resolution mass spec-
6
trometry experiments were performed using a Fourier Transform
Ion Cyclotron Resonance (FT-ICR) mass spectrometer APEX III
(Bruker Daltonik GmbH, Bremen, Germany) equipped with a 7.0 T,
160 mm bore superconducting magnet (Bruker Analytik GmbH -
Magnetics, Karlsruhe, Germany), infinity cell, and interfaced to an
external (nano)ESI or MALDI ion source. Nitrogen served both as
the nebulizer gas and the dry gas for ESI. Nitrogen was generated
by a Bruker nitrogen generator NGM 11. Argon served as cooling
Chemical analysis and compound isolation
Reactions were monitored by using RP-HPLC (Thermo Scientific
Accela 600 equipped with a Thermo Scientific Hypersil GOLD 3 mm
column (C , 150ꢃ2.1 mm, eluent A H O/CH CN/TFA=95:5:0.1,
1
8
2
3
À1
eluent B H O/CH CN/TFA=5:95:0.1, flowrate 0.7 mLmin using
2
3
a gradient from 0–100% B over 5 min)). LC-MS analysis was accom-
plished by using a Waters Alliance HT equipped with a Waters Sym-
metry 3.5 mm column (C , 100ꢃ2.1 mm, eluent A H O/HCOOH=
n
gas in the infinity cell and collision gas for MS experiments. Scan
accumulation and Fourier transformation were performed with
8
2
À1
1
00:0.1, eluent B CH CN/HCOOH=100:0.1, flowrate 0.4 mL·min
3
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XMASS NT (7.08) on a PC Workstation, for further data processing
DataAnalysis 3.4 was used. Docking experiments were conducted
OD600nm =0.6 by the addition of 100 mm isopropyl-b-d-thioga-
lactopyranoside (IPTG) and 2 mgmL l-arabinose. After induc-
tion, temperature was decreased to 258C and the cells were
harvested after 20 h of incubation by centrifugation (4000ꢃg,
À1
[27]
[28]
using AutoDockVina and AutoDockTools with the crystal struc-
[18]
ture of RebH.
30 min, 48C) and stored at À208C.
Organisms and growth conditions
For the overexpression of the flavin reductase PrnF, the expres-
sion vector pCIBhis-prnF was electroporated into P. fluorescens
BL915 DORF1-4. The resulting strain P. fluorescens BL915 pCIB-
his-prnF was cultivated overnight at 308C in LB medium con-
The Escherichia coli strains DH5a and BL21(DE3) (Novagene)
were grown at 378C using Luria-Bertani (LB) agar or broth with
À1
shaking (150 rpm), supplemented with 100 mgmL ampicillin,
À1
taining 30 mgmL tetracycline. 1 L of LB medium, containing
À1
À1
6
0 mgmL kanamycin and 50 mgmL chloramphenicol where
the appropriate antibiotic, was then inoculated with 10 mL of
overnight culture and cultivated for 3 d at 308C. The cells were
spun down and stored at À208C.
appropriate. Pseudomonas fluorescens strain BL915 DORF1-4
was grown at 308C in LB agar or broth with shaking (150 rpm),
À1
supplemented with 30 mgmL tetracyclin where appropriate.
The bacterial strain Lechevalieria aerocolonigenes (strain C-
For the overexpression of the alcohol dehydrogenase from
Rhodococcus sp., chemically competent E. coli BL21(DE3) were
simultaneously cotransformed with the expression vector
pET21-ADH and pGro7 (Takara), resulting in the strain E. coli
BL21(DE3)-ADH. For the expression, 30 mL overnight culture
3
8383-RK2, DSMZ-No. 44217) was grown on GYM Streptomy-
À1
À1
À1
ces agar (4 gL glucose, 4 gL yeast extract, 10 gL malt ex-
tract, 2 gL CaCO , 12 gL agar, pH 7.2) for 4 days at 288C.
À1
À1
3
À1
À1
containing 50 mgmL chloramphenicol and 100 mgmL ampi-
cillin were used to inoculate 1.5 L of LB medium containing
the same antibiotics. After an optical density of OD600nm =0.3
had been reached, the temperature was decreased to 258C.
Cloning and expression
Standard methods were used for agarose gel electrophoresis,
DNA restriction, ligation and transformation of E. coli. Restric-
tion enzymes, T4 DNA ligase and Phusion Polymerase were
supplied by New England Biolabs (Ipswich, MA). Agarose was
obtained from Peqlab (Erlangen, Germany). Plasmid DNA was
extracted employing a QIAprep Spin Miniprep kit (Qiagen,
Hilden, Germany). dNTP mixture was supplied by Fermentas
After 30 min, ZnCl
and l-arabinose were added to final con-
2
À1
centrations of 0.5 mm and 2 mgmL respectively. Expression
of the alcohol dehydrogenase was induced by adding 0.1 mm
IPTG at an OD600nm =0.6. After 16 h, the cells were harvested
and stored at À208C.
(Vilnius, Lithuania).
For the expression of the tryptophan synthase, 1.5 L LB
À1
The kanamycin-resistant plasmid vector pET28a+ was used to
clone the rebH gene and overexpress its product prior to its
subsequent purification. To amplify the rebH gene of the bacte-
rial strain Lechevalieria aerocolonigenes, two primers, forward
medium containing 100 mgmL ampicillin was inoculated with
15 mL of an E. coli pSTB7 (ATCC strain 37845) overnight culture
and grown for 12–16 h at 378C. The cells were harvested by
centrifugation, washed once with 100 mmK
stored at À208C.
HPO pH 7.4 and
4
2
(
5’-GTACGTCATATGTCCGGCAAGATTGACAAG-3’) and reverse
5’-GTCAGCAAGCTTTCAGCGGCCGTGCTGTGCC-3’), with NdeI
(
and HindIII restriction sites, respectively included as underlined
were designed based on the sequence of the 5’ and 3’ ends of
the rebH gene (GenBank accession number AAN01216.1). A
bacterial colony of L. aerocolonigenes was picked, resuspended
in 200 mL PBS Puffer, heated for 20 min at 1008C and immedi-
ately used for colony-PCR. A PCR product of approximately
Tryptophan synthase bioconversion
Bioconversion of indoles with l-serine for the production of
the C5- or C6-substituted tryptophan derivatives were per-
[23]
formed according to Goss et al. in a modified protocol. Brief-
ly, 12.1 mm indole, 12.1 mml-serine and 100 mm pyridoxal
phosphate in 15 mm NH HCO (pH 7.8) were converted with
1.6 kb was obtained and directly purified to prevent degrada-
4
3
tion by bacterial endonucleases. The amplificate was digested
with NdeI and HindIII, purified again and ligated into the ex-
pression vector pET28a+ that had been digested with the
same restriction enzymes. The ligation product pMF-rebH was
transformed into E. coli strain BL21(DE3) pGro7 and selected
utilizing the kanamycin resistance of the pET28a+ vector and
the chloramphenicol resistance of the pGro7 vector, resulting
in the strain E. coli BL21(DE3)-rebH. The correct base sequence
of the inserted gene was confirmed by DNA sequencing (GATC
Biotech, Kçln, Germany).
2
2
mL of E. coli lysate (1.5 L pellet lysed twice by FrenchPress in
0 mL 500 mm Tris-HCl, 5 mm EDTA, 10 mm 2-mercaptoetha-
nol, 1 mm PMSF, and 0.1 mm pyridoxal phosphate), trapped in
a sealed dialysis bag, within two days at 378C into the corre-
sponding tryptophan derivative. The progress of the reaction
was monitored by reversed-phase HPLC at l=280 nm. For an-
alytical data of the substituted tryptophan derivatives, refer to
the Supporting Information.
Enzyme purification
For the overexpression of the halogenase RebH, 1.5 L LB
medium was inoculated with an overnight culture of E. coli
BL21(DE3)-rebH with an OD600nm =0.01 and cultivated at 378C
with shaking at 150 rpm. Protein expression was induced at an
All steps of the enzyme purification process were performed at
48C. To purify the His -tagged halogenase RebH, E. coli cells
from 1.5 L culture were resuspended in 30 mL equilibration
6
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buffer (50 mm NaH PO , 300 mm NaCl and 50 mm PMSF pH 8.0
mined by regression of the linear range (usually 15 s after addi-
tion of the enzyme, less than 10% substrate conversion).
2
4
adjusted with NaOH) and lysed twice by FrenchPress. The
crude lysate was centrifuged (10000ꢃg, 30 min, 48C) and fil-
tered through a 0.2 mm Whatman filter. The supernatant was
À1
Determination of RebH activity
loaded on a HisTALON agarose affinity column (0.5 mLmin )
with a bed volume of 1 mL (Clontech, Mountain View, CA) that
had been equilibrated with equilibration buffer before. The
Reactions were assembled in 96-well plates as triplicates in
a final volume of 75 mL from a 3 mL master mix containing
column was washed with 20 mL equilibration buffer
3
0 mm NaCl, 10 mmK HPO4 pH 7.4, 0.1 mm FAD, 0.1 mm
2
À1
(
1 mLmin ) and subsequently washed with 20 mL washing
À1
NADH, 0.25 mml-tryptophan, 5% (v/v) iso-propanol, 2.5 Uml
PrnF, 1 Uml ADH and 10 mm RebH with 0.5 mm 4-nitrophenol
buffer (50 mm NaH PO , 300 mm NaCl, 10 mm imidazole,
À1
2
4
À1
pH 8.0, 1 mLmin ). The protein was eluted with elution buffer
containing 300 mm imidazole. Protein fractions were analyzed
using 10% sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE). The respective protein band was excised
from the gel, de-stained twice with 200 mL 30% acetonitrile
as an internal standard. The reactions were left in an incubator
at 258C and stopped at distinct times by the addition of 75 mL
methanol to each well. The stopped solution was directly
transferred to Eppendorf tubes for removal of precipitated pro-
teins by centrifugation. The resulting supernatant was analyzed
by reversed-phase HPLC. Product formation was calculated
based on the peak area in comparison to a calibration curve of
l-tryptophan and 4-nitrophenol as an internal standard.
0
.1m NH HCO and dried completely for 30 min in a speedvac
4 3
(SpeedVac Concentrator 5301 Eppendorf, Hamburg, Germany).
The proteins were overnight digested in-gel at 378C with
À1
1
5 mL of 6.7 mgmL trypsin (Promega, Madison, WI) in 10 mm
NH HCO3 and subsequently identified by MALDI-ToF mass
4
spectrometry employing a Bruker Ultraflextreme instrument
Determination of Total Turnover Numbers (TTN) for RebH
(
Bruker Daltonics, Bremen, Germany) with peptide mass finger-
Reactions were assembled as triplicates in a final volume of
print. The flavin-reductase PrnF was purified analogously. For
the purification of the alcohol dehydrogenase, a heat precipita-
tion step was used. E. coli cells from 1.5 L of culture were re-
2
1
00 mL containing 1.25 mm substrate, 30 mm NaCl,
0 mmK HPO4 pH 7.4, 0.1 mm FAD, 0.1 mm NADH, 5% (v/v)
2
À1
À1
iso-propanol, 2.5 Uml PrnF, 1 Uml ADH and 5 mm RebH.
The reactions were left in an incubator shaker at 258C and
suspended in 20 mL 100 mmK HPO4 buffer at pH 7.4, lysed
twice by FrenchPress and centrifuged (10000ꢃg, 30 min, 48C).
The crude lysate was heated to 608C in 1.5 mL aliquots for
2
600 rpm for 16 h. At the end of the reaction, proteins were
precipitated by the addition of 200 mL methanol. After centrifu-
gation, the supernatant was analyzed by reversed-phase HPLC.
Product formation was calculated based on the decrease in
peak area of the reactant in comparison to a calibration curve
of each substrate.
2
0 min in a thermomixer (Eppendorf, Hamburg, Germany). De-
naturated protein was removed by centrifugation and the
crude extract was stored at À208C. Protein concentrations
were determined in Bradford assays (Applichem, Darmstadt,
Germany) with bovine serum albumin (BSA) as standard.
General procedure for enzymatic halogenation reactions
using E. coli lysate
Determination of PrnF activity
The volumetric activity of the elution fractions of the Flavin re-
ductase PrnF was determined as triplicates by monitoring the
decrease of absorption at l=340 nm due to the oxidation of
For the halogenation of l-tryptophan and its derivatives, E. coli
lysate from 1.5 L of culture, containing the overexpressed halo-
genase RebH, was used. The cells were lysed by FrenchPress in
+
+
À1
À1
NADH+H to NAD (e=6.3 mLmmol cm ) in a final volume
of 1 mL containing 20 mL of the diluted elution fractions (dilu-
a final volume of 30 mL lysis buffer containing 30 mm NaCl or
À1
NaBr, 10 mmK HPO pH 7.4, 50 mm PMSF, 100 mgmL ampicil-
À1
2
4
tion 1:100, usually ~30 mgmL ), 10 mmK HPO pH 7.4, 20 mm
2
4
lin, 10 mm FAD, 20 mm DTT, 1 mm NADH, 5% (v/v) iso-propa-
FAD and 240 mm NADH. The stock solutions were preequilibrat-
ed to 258C and pH 7.4 and temperature was held constant at
À1
À1
nol, 2.5 Uml PrnF, 1 Uml ADH and an excess of substrate (2
to 6 mm), whereas DTT, NADH, iso-propanol, PrnF, ADH and
the substrate were added after lysis and centrifugation
258C in a tempered Jasco V-630 photometer. The conversion
rate of the substrate was determined by regression of the
linear range (usually 15 s after addition of the enzyme, less
than 10% substrate conversion).
(
30 min, 10000 x g, 48C) of the crude lysate. The supernatant
was transferred into an Erlenmeyer flask (50 mL) and incubated
at 258C in an orbital shaker at 100 rpm. The reaction was
monitored by reversed-phase HPLC and stopped at 16–24 h,
depending on the reaction process. The reaction solution was
transferred into a sealed dialysis bag and the product was re-
moved by step-wise dialysis against Millipore water containing
0.1% of TFA (2x 500 mL for 4 h, 1x 500 mL overnight). The
combined dialysis fractions were pooled, the solvent was re-
moved at reduced pressure and the remaining liquid contain-
ing the halogenated tryptophan derivative was purified by
Determination of ADH activity
The volumetric activity of the diluted crude extract of the alco-
hol dehydrogenase after heat precipitation (20 mL, 1:100 dilu-
tion) was determined as triplicates in a final volume of 1 mL
+
containing 250 mm NAD , 10 mmK HPO pH 7.4 and 20% (v/v)
2
4
iso-propanol. The conversion rate of the substrate was deter-
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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preparative HPLC. For the analytical data of the halogenated
tryptophan derivatives, refer to the Supporting Information.
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We thank Prof. Dr. Karl-Heinz van Pꢂe for donating the plasmid
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for donating the plasmid encoding for the alcohol dehydrogen-
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[
[
[
[
2
Keywords: biocatalysis
·
enzymes
·
halogenase
·
regioselectivity · tryptophan
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Published online on && &&, 0000
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 0000, 00, 1 – 8
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7
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These are not the final page numbers!

FULL PAPERS
M. Frese, P. H. Guzowska, H. Voß,
N. Sewald*
&& – &&
Regioselective Enzymatic
Halogenation of Substituted
Tryptophan Derivatives using the FAD-
Dependent Halogenase RebH
No business as usual: The tryptophan
halogenase RebH from Lechevalieria
aerocolonigenes is able to halogenate at
the electronically unfavored C7-meta-
position of C5-substituted tryptophan
derivatives, even in presence of deacti-
vating ortho/para-directing groups.
ꢁ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 0000, 00, 1 – 8
&
8
&
ÞÞ
These are not the final page numbers!