Enzymatic halogenation of tryptophan on a gram scale

Article abstract of DOI:10.1002/anie.201408561

Halogenated arenes are important building blocks in medicinal and agrochemistry. Chemical electrophilic aromatic halogenation requires molecular halogen, whereas FADdependent halogenases form halogenated arenes with high regioselectivity while only halide salts and O2 are required. This reaction proceeds at room temperature in aqueous media. However, enzymatic halogenation is considered inefficient, mainly because halogenases are not stable. Thus, the preparative application remained elusive.We were able to show that the long-term stability, hence, the preparative efficiency of the tryptophan-7-halogenase RebH can be significantly improved by immobilization together with the other enzymes required for cofactor regeneration. We established a facile scalable method suitable for the halogenation of tryptophan and its derivatives on a gram scale using a solid, multifunctional, and recyclable biocatalyst; this immobilization strategy might also be applicable for other FAD-dependent halogenases.

Full text of DOI:10.1002/anie.201408561

Angewandte
Chemie
DOI: 10.1002/anie.201408561
Enzymatic Halogenation
Enzymatic Halogenation of Tryptophan on a Gram Scale**
Marcel Frese and Norbert Sewald*
Abstract: Halogenated arenes are important building blocks in
medicinal and agrochemistry. Chemical electrophilic aromatic
halogenation requires molecular halogen, whereas FAD-
dependent halogenases form halogenated arenes with high
regioselectivity while only halide salts and O2 are required.
This reaction proceeds at room temperature in aqueous media.
However, enzymatic halogenation is considered inefficient,
mainly because halogenases are not stable. Thus, the prepara-
tive application remained elusive. We were able to show that the
long-term stability and, hence, the preparative efficiency of the
tryptophan-7-halogenase RebH can be significantly improved
by immobilization together with the other enzymes required
for cofactor regeneration. We established a facile scalable
method suitable for the halogenation of tryptophan and its
derivatives on a gram scale using a solid, multifunctional, and
recyclable biocatalyst; this immobilization strategy might also
be applicable for other FAD-dependent halogenases.
by molecular oxygen. The resulting flavin hydroperoxide
undergoes nucleophilic attack by a halide ion to form
hypohalous acid, which passes through a 10 ꢀ long channel
from the FAD binding site to the tryptophan binding site.[4–7]
Due to the sandwichlike binding of l-tryptophan inside the
active site of the halogenase, only the less activated C7
position becomes available for chlorination or bromina-
tion.[7,8] The conserved lysine residue K79 is oxidized
selectively by hypohalous acid and the halogenation is
brought about by the actual halogenating species, a long-
lived N-haloamine intermediate.[9] Beside the Trp-7 halogen-
ase RebH, C5[10] and C6[11] Trp-halogenases have also been
described in the literature.
Enzyme-catalyzed reactions are well-known for their high
stereo- and regioselectivity.[12,13] Consequently, biocatalysis
has become an emerging field in biotechnology, especially in
terms of green and sustainable chemistry. Moreover, owing to
the high selectivity of enzymatic reactions, the application of
biocatalysts supersedes the necessity of protecting or activat-
ing groups, leading to more efficient synthetic routes. How-
ever, enzymes like halogenases suffer from low stability and
low activity, especially under non-native reaction conditions
in the presence of high substrate concentrations.
Enzyme immobilization has proven to be an amenable
solution to circumvent stability problems,[14] and it also
facilitates recycling and easy removal of the biocatalyst.
Although different immobilization strategies have been
developed in the past, cross-linked enzyme aggregates
(CLEAs) have emerged as carrier-free catalysts with out-
standing benefits owing to their simplicity. Briefly, the enzyme
of choice is precipitated upon addition of ammonium sulfate
or polyethylene glycol and then lysine residues on the enzyme
surface are cross-linked by bifunctional molecules like
glutaraldehyde. The crucial advantage of this methodology
is the possibility of combining purification and immobiliza-
tion in one step, which also makes this approach applicable
for crude protein extracts.
With respect to halogenases, several studies determined
low kinetic constants with turnover rates kcat of approximately
1.0 minÀ1 and total turnover numbers (TTNs) of less than 200,
which makes the practical synthetic applicability of halogen-
ases elusive.[2,3,6,9,11,15–17] Up to this point, halogenases have
mainly been studied on an analytical scale to determine the
substrate scope,[18] whereas preparative enzymatic halogen-
ation still requires optimization. Recently, Payne et al. used
E. coli lysate containing overexpressed RebH from more than
10 L of cultivation medium to obtain less than 90 mg of
chlorinated tryptophan in moderate yields.[15] In addition,
large amounts of contaminants in E. coli lysate containing
overexpressed halogenases hinder purification of the halo-
genation product. In particular residual chloride ions from the
cultivation media interfere with bromination reactions.
H
alogenation is a common reaction in organic synthesis, yet
the regioselective introduction of halogen substituents at
mechanistically less favored positions remains a challenging
task. Aryl halides are important intermediates in the chem-
ical, agrochemical, and pharmaceutical industries because
they can be modified readily by nucleophilic substitution and
metal-catalyzed cross-coupling reactions.[1] The chemical
incorporation of halogen substituents is an environmentally
hazardous process that requires elemental chlorine or bro-
mine, often in combination with Lewis acids and may also
lead to the formation of byproducts because of ambiguous
regioselectivity. In nature, enzymatic strategies have evolved
for the halogenation of organic metabolites under much
milder conditions, making use of benign halide salts and
oxygen at 258C and pH 7. FAD-dependent halogenases form
the major class of enzymes responsible for regioselective,
carrier-free halogenation in nature. Representatives of this
class are the Trp-7 halogenase PrnA[2] and its close relative
RebH from Lechevalieria aerocolonigenes.[3] l-Tryptophan is
chlorinated regioselectively at the electronically unfavored
C7 position of the indole ring. A flavin reductase supplies
FADH2, which is oxidized in the active site of the halogenase
[*] M. Frese, Prof. Dr. N. Sewald
Fakultꢀt fꢁr Chemie, Organische und Bioorganische Chemie
Universitꢀt Bielefeld
Universitꢀtsstrasse 25, 33615 Bielefeld (Germany)
E-mail: norbert.sewald@uni-bielefeld.de
Homepage: http://www.uni-bielefeld.de/chemie/oc3sewald/
[**] We thank Prof. Dr. Karl-Heinz van Pꢂe for donating the plasmid
encoding for the flavin reductase PrnF, as well as Prof. Dr. Werner
Hummel for donating the plasmid encoding for the alcohol
dehydrogenase.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408561.
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We embarked on a project focusing on the development of
a facile enzymatic halogenation system that can be applied on
a multigram scale without requiring large expression volumes.
Hence, the idea was to concomitantly immobilize l-trypto-
phan-7-halogenase RebH and the necessary auxiliary
enzymes by precipitation and cross-linking in order to
obtain one solid biocatalyst as multifunctional cross-linked
enzyme aggregates, so-called combiCLEAs (Scheme 1). The
Figure 1. A) Influence of different glutaraldehyde concentrations on the
cross-linking of the precipitated enzymes RebH, PrnF, and ADH. The
highest conversion of l-Trp was achieved by using relatively low cross-
linker concentrations of 0.5%. Since glutaraldehyde forms Schiff bases
with primary amines, higher concentrations might lead to the modifi-
cation of the K79 residue that is essential for halogenation activity.[9]
B) Long-term storage experiments show that RebH combiCLEAs are
stable for more than 4 months, whereas free purified RebH showed
a considerable loss of activity after 12 weeks.
200 mg of regioselectively brominated l-tryptophan as the
TFA salt (Figure 2).
Notably, each halogenation reaction stops at a distinct
time, suggesting a limitation, for example, of the cofactor
regeneration by ADH. However, higher 2-propanol concen-
trations or in situ product removal (ISPR) of the produced
acetone[21] did not extend the catalytic activity. Also higher
aeration and additional oxygen supply to the vessel did not
improve the reaction.[22] Decreasing the l-tryptophan con-
centration from 3 to 1 mm and increasing the volume from
30 mL to 750 mL drove the halogenation of 153 mg l-tryp-
tophan to completion within 6 days without the need to
replace the reaction buffer (Figure 3). Since the halogenation
is highly regioselective, a single desalting step using reversed-
phase C18 silica is sufficient for workup, leading to 248 mg of
Scheme 1. Cross-linking of precipitated Trp-7-halogenase RebH, flavin
reductase PrnF, and an alcohol dehydrogenase from Rhodococcus sp.
leads to multifunctional, recyclable cross-linked enzyme aggregates
(CLEAs), which can be applied for the regioselective halogenation of
l-tryptophan also on the gram scale.
catalyst should be removable by filtration for the recyclable
usage of RebH. E. coli lysate from 1.5 L of culture containing
the overexpressed halogenase RebH was used without any
further purification together with the required auxiliary
enzymes for continuous cofactor regeneration: a flavin reduc-
tase from Pseudomonas fluorescens (PrnF) and an alcohol
dehydrogenase from Rhodococcus sp. (ADH) were added.
After precipitation of all three enzymes by addition of
ammonium sulfate, glutaraldehyde was added at different
concentrations to determine the ideal reagent ratio for cross-
linking. CombiCLEAs produced at low glutaraldehyde con-
centrations showed the highest residual activity (Figure 1A).
Since the proposed halogenation mechanism of RebH
involves a N-haloamine at the K79 residue that is essential
for halogenation activity,[9] the modification of this distinct
residue by glutaraldehyde might be responsible for the
lowered residual activity. However, it is known that excessive
protein modification by glutaraldehyde in general leads to
lower activities.[19] Different cross-linking times did not lead
to a significant decrease in the remaining activity. Under
optimized conditions, an immobilization yield of all three
simultaneously immobilized enzymes of 99% and an activity
recovery of 30% was achieved. Long-term storage experi-
ments proved RebH combiCLEAs to be stable for more than
4 months at 48C, whereas free, purified RebH showed
a considerable loss of activity after 12 weeks (Figure 1B).[20]
RebH combiCLEAs can be recycled at least 10 times for
the halogenation of 3 mm l-tryptophan in a batchwise manner
with an average conversion of 81%, leading to more than
Figure 2. RebH combiCLEAs from 1.5 L of E. coli culture were used in
batch reactions in a final volume of 30 mL containing 3 mm l-trypto-
phan to test their reusability. After each batch reaction, the solid
biocatalyst was removed by centrifugation, washed once with buffer,
and reused for the next batch in new reaction solution. RebH
combiCLEAs can be recycled at least 10 times with an average
conversion of 81%, leading to 204 mg of l-7-bromotryptophan (TFA
salt, 0.52 mmol, 58%). After quantitative halogenation of l-tryptophan,
RebH starts to form the dibrominated species as well.[15] However,
since the product of the first bromination is deactivated for further
electrophilic aromatic substitution, the dibromination is very slow and
occurs to only a very low extent. This issue can be easily avoided by
reaction monitoring and removal of the immobilized biocatalyst.
2
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RebH,[9] the formation of CLEAs might stabilize its quater-
nary structure, which is also known for several other multi-
meric enzymes.[24] Since numerous other tryptophan halogen-
ases like PyrH (Trp-5-halogenase) and PrnA (Trp-7-halogen-
ase) also form dimers in solution,[7,8] the CLEA methodology
is likely to be applicable also for these enzymes to improve
their biocatalytic application. In addition, the further
improvement of the mechanical properties of the combi-
CLEAs by cross-linking the enzymes in presence of different
additives like siloxanes[25,26] and magnetic functionalized
beads[27] might enhance the catalytic efficiency even more.
Although enzymatic halogenation is still in its infancy
because of its limited substrate scope, the preparative
utilization of immobilized RebH for the regioselective
bromination of l-tryptophan on the gram scale laid the
foundation for efficient biocatalytic halogenation. As a con-
sequence, our work now focusses on the expansion of the
substrate scope from tryptophan to other (hetero-)aromatic
rings by means of directed evolution.
Figure 3. A) RebH was shown to brominate 154 mg of l-tryptophan
within 6 days without the need to regenerate the reaction buffer when
higher volumes and lower concentrations were used. A simple desalt-
ing step using RP-silica led to 248 mg of l-7-bromotryptophan·TFA.
B) According to RP-HPLC analysis, l-tryptophan (tr =128 s) is con-
verted with high selectivity to l-7-bromotryptophan (tr =156 s) without
the formation of any side products.
l-7-bromotryptophan (TFA salt, 0.63 mmol, 84%). There-
fore, we were confident that the halogenation reaction could
also be scaled up to the gram scale. CLEAs from 6 L of E. coli
culture overexpressing RebH were used for the halogenation
of 1 g of l-tryptophan in a final volume of 5 L. Within 8 days,
the reaction proceeded to full conversion, leading to 1.813 g
of l-7-Br-Trp·TFA (4.58 mmol, 92%) in high purity after
simple desalting using a plug of C18 silica.
Since we previously demonstrated the potential of RebH
for the regioselective halogenation of other l-tryptophan
derivatives at the C7 position, even in the presence of
inactivating ortho/para-directing groups like 5-fluoro,[23] we
also applied the RebH combiCLEAs for the halogenation of
l-5-hydroxytryptophan, as well as d-tryptophan (Table 1).
Even for such substrates, the halogenation works on a prep-
arative scale, albeit slightly lower yields were obtained.
Received: August 26, 2014
Published online: && &&, &&&&
Keywords: biocatalysis · enzyme immobilization · halogenases ·
.
regioselectivity · sustainable chemistry
[1] J. Fauvarque, Pure Appl. Chem. 1996, 68, 1713 – 1720.
[2] S. Keller, T. Wage, K. Hohaus, M. Hçlzer, E. Eichhorn, K.
van Pꢁe, Angew. Chem. Int. Ed. 2000, 39, 2300 – 2302; Angew.
Chem. 2000, 112, 2380 – 2382.
[3] E. Yeh, S. Garneau, C. T. Walsh, Proc. Natl. Acad. Sci. USA
2005, 102, 3960 – 3965.
[4] E. Yeh, L. J. Cole, E. W. Barr, J. M. Bollinger, Jr., D. P. Ballou,
C. T. Walsh, Biochemistry 2006, 45, 7904 – 7912.
[5] E. Bitto, Y. Huang, C. A. Bingman, S. Singh, J. S. Thorson, G. N.
Phillips, Jr., Proteins 2008, 70, 289 – 293.
Table 1: Halogenation of tryptophan and 5-hydroxytryptophan by
RebH.[a]
Substrate
Concentration
Product
Conv. [%][b]
[6] S. Flecks, E. P. Patallo, X. Zhu, A. J. Ernyei, G. Seifert, A.
Schneider, C. Dong, J. H. Naismith, K. van Pꢁe, Angew. Chem.
Int. Ed. 2008, 47, 9533 – 9536; Angew. Chem. 2008, 120, 9676 –
9679.
[7] C. Dong, S. Flecks, S. Unversucht, C. Haupt, K.-H. van Pꢁe, J. H.
Naismith, Science 2005, 309, 2216 – 2219.
[8] X. Zhu, W. De Laurentis, K. Leang, J. Herrmann, K. Ihlefeld, K.-
H. van Pꢁe, J. H. Naismith, J. Mol. Biol. 2009, 391, 74 – 85.
[9] E. Yeh, L. C. Blasiak, A. Koglin, C. L. Drennan, C. T. Walsh,
Biochemistry 2007, 46, 1284 – 1292.
l-Trp
d-Trp
5-HO-Trp
1 mm
0.25 mm
0.25 mm
l-7-bromo-Trp
100
57
53
d-7-bromo-Trp
7-chloro-5-HO-Trp[c]
[a] Reaction conditions: 100 mm NAD+, 1 mm FAD, 15 mm Na2HPO4,
5 vol% 2-propanol, and 30 mm NaBr or NaCl in a final volume of 750 mL
Millipore Water, pH 7.4 adjusted with H3PO4, 258C, 6 d. [b] Determined
by RP-HPLC. [c] 6-Chloro-5-HO-Trp is also formed (product ratio 1:3,
verified by 1H NMR spectroscopy).
[10] S. Zehner, A. Kotzsch, B. Bister, R. D. Sꢂssmuth, C. Mꢁndez,
J. A. Salas, K.-H. van Pꢁe, Chem. Biol. 2005, 12, 445 – 452.
[11] C. Seibold, H. Schnerr, J. Rumpf, A. Kunzendorf, C. Hatscher, T.
Wage, A. J. Ernyei, C. Dong, J. H. Naismith, K.-H. van Pꢁe,
Biocatal. Biotransform. 2006, 24, 401 – 408.
[12] C. M. Clouthier, J. N. Pelletier, Chem. Soc. Rev. 2012, 41, 1585 –
1605.
[13] H. Grçger, Y. Asano, U. T. Bornscheuer, J. Ogawa, Chem. Asian
J. 2012, 7, 1138 – 1153.
[14] R. A. Sheldon, S. van Pelt, Chem. Soc. Rev. 2013, 42, 6223 – 6235.
[15] J. T. Payne, M. C. Andorfer, J. C. Lewis, Angew. Chem. Int. Ed.
2013, 52, 5271 – 5274; Angew. Chem. 2013, 125, 5379 – 5382.
[16] K. Muffler, A. R. Kuetchou Ngnigha, R. Ulber, Chem. Ing. Tech.
2010, 82, 121 – 127.
The practical applicability of FAD-dependent halogen-
ases for preparative regioselective halogenation has been
questioned because of low kinetic constants in combination
with poor total turnover numbers, which limited RebH to
analytical application or to low yields. Prohibitively extensive
efforts were previously necessary to halogenate 100 mg of
l-tryptophan, for example, by using 12 L of E. coli culture
containing overexpressed RebH. By using RebH-PrnF-ADH
combiCLEAs, we have established a facile enzymatic regio-
selective halogenation process that is easily scalable up to
gram scale. For the first time, an active FAD-dependent
halogenase was co-immobilized with all auxiliary enzymes
necessary for halogenation. Owing to the dimeric structure of
[17] C. B. Poor, M. C. Andorfer, J. C. Lewis, ChemBioChem 2014, 15,
1286 – 1289.
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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.
Angewandte
Communications
[18] M. Hçlzer, W. Burd, H.-U. Reißig, K.-H. van Pꢁe, Adv. Synth.
Catal. 2001, 343, 591 – 595.
[19] D. I. Perez, F. van Rantwijk, R. A. Sheldon, Adv. Synth. Catal.
2009, 351, 2133 – 2139.
[20] In addition, it is worth mentioning that extensive washing in
combination with a buffer free of potassium ions is essential for
proper CLEA halogenation using E. coli BL21(DE3) lysate
owing to a high tryptophanase activity (EC 4.1.99.1); this K+-
dependent enzyme would degrade the substrate l-tryptophan.
See also: T. Honda, M. Tokushige, J. Biochem. 1986, 100, 679 –
685.
[22] Since FADH2 reacts immediately with oxygen in the solvent, the
p(O2) of the reaction solution decreases rapidly within 30 min to
nearly 0%. However, the reaction proceeds several days at this
low p(O2). Notably, low p(O2) conditions seem to increase the
catalytic activity of RebH.[3]
[23] M. Frese, P. H. Guzowska, H. Voß, N. Sewald, ChemCatChem
2014, 6, 1270 – 1276.
[24] L. Wilson, L. Betancor, G. Fernꢃndez-Lorente, M. Fuentes, A.
Hidalgo, J. M. Guisꢃn, B. C. C. Pessela, R. Fernꢃndez-Lafuente,
Biomacromolecules 2004, 5, 814 – 817.
[25] R. A. Sheldon, Appl. Microbiol. Biotechnol. 2011, 92, 467 – 477.
[26] R. A. Sheldon, Org. Process Res. Dev. 2011, 15, 213 – 223.
[27] R. A. Sheldon, M. J. Sorgedrager, Non-Leachable Magnetic
Cross-Linked Enzyme Aggregate, WO2012/023847A2, 2012.
[21] K. Schroer, E. Tacha, S. Lꢂtz, Org. Process Res. Dev. 2007, 11,
836 – 841.
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Communications
Enzymatic Halogenation
M. Frese, N. Sewald*
One for all, all for one: The combined
immobilization of the tryptophan-7-halo-
genase RebH, a flavin reductase, and an
alcohol dehydrogenase as a cross-linked
enzyme aggregate leads to a solid multi-
functional biocatalyst that can be used for
an easily scalable regioselective synthesis
of C7-halogenated tryptophan on a gram
scale.
&&&& — &&&&
Enzymatic Halogenation of Tryptophan
on a Gram Scale
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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