Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade

Article abstract of DOI:10.1039/d1cy00855b

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases.

Full text of DOI:10.1039/d1cy00855b

Showcasing research from Dr. Caroline E. Paul’s laboratory
in Biocatalysis, Department of Biotechnology, Delft
University of Technology, The Netherlands.
As featured in:
Asymmetric azidohydroxylation of styrene derivatives
mediated by a biomimetic styrene monooxygenase
enzymatic cascade
Artificial nicotinamide coenzyme biomimetics provide an
alternative to the native NAD(P)H to promote biocatalysis,
and can act as an electron donor to reduce flavins in free
solution. In this work, a flavin was reduced to fuel a one-pot
enzymatic cascade for the asymmetric azidohydroxylation
of styrenes. First the asymmetric epoxidation of styrenes
was catalysed by a flavin-dependent monooxygenase with
the coenzyme biomimetic. Then regioselective epoxide ring
opening with azide, tuned with a halohydrin dehalogenase,
allowed access to chiral 1,2-azido alcohols isomers with up to
two stereocenters.
See Caroline E. Paul et al.,
Catal. Sci. Technol., 2021, 11, 5077.
rsc.li/catalysis
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Asymmetric azidohydroxylation of styrene
derivatives mediated by a biomimetic styrene
monooxygenase enzymatic cascade†
Cite this: Catal. Sci. Technol., 2021,
11, 5077
Lía Martínez-Montero,a Dirk Tischler,b Philipp Süss,c Anett Schallmey,d
Maurice C. R. Franssen,
e
a
Frank Hollmann a and Caroline E. Paul
*
Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols,
however their chiral substituted analogues are difficult to access. We established a cascade for the
asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via
enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with
up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene
monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme
biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening,
this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to
>99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded
the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our
biocatalytic cascade to be a starting point for more practical production of these chiral compounds with
two-component flavoprotein monooxygenases.
Received 13th May 2021,
Accepted 13th June 2021
DOI: 10.1039/d1cy00855b
rsc.li/catalysis
Chiral epoxides are highly sought after to access optically
active molecules via ring opening by nucleophiles such as
azide, which affords 1,2-azido alcohols.1 These compounds
are intermediates for chiral aziridines, diamines,2 and
1,2-amino alcohols found in bioactive compounds, chiral
auxiliaries for asymmetric synthesis,3 and give access to click
chemistry.4 Besides azidolysis,5 the synthesis of 1,2-azido
alcohols traditionally involves conversion of β-diols via
sulfites,6 or carbonyl reduction of α-azido ketones.7,8 The
challenge is to allow for a wide array of substituents and
reach enantiopurity. Enzymatic aminohydroxylation of
styrenes was recently described with an engineered
cytochrome c,9 and through enzymatic cascades using
epoxide hydrolases, ADHs and transaminases.10–14 On the
other hand, enzymatic azidohydroxylation of aromatic alkenes
remains to be investigated and applied in vitro.7,15
We envisioned to produce chiral 1,2-azido alcohols from
aromatic alkenes via enzymatic epoxidation with a styrene
monooxygenase (SMO).16 SMO can activate and incorporate
oxygen in alkenes to produce chiral epoxides with high stereo-
and regioselectivity.17–19 SMOs are two-component flavoprotein
monooxygenases: the reductase component StyB (EC 1.5.1.36)
uses nicotinamide adenine dinucleotide (NADH) to reduce
flavin adenine dinucleotide (FAD), which freely diffuses to the
oxygenase component StyA (EC 1.14.14.11) and reduces
molecular oxygen, forming a hydroperoxyflavin that oxidizes
styrene to its chiral epoxide (Fig. 1A).20,21 StyA enzymes accept
a range of aromatic17,19 and aliphatic22,23 alkene substrates,
and can be easily produced recombinantly in E. coli.
Enzymatic recycling of the required NADH generally involves
a dehydrogenase and a co-substrate (Fig. 1A), typically added in
large excess. Non-enzymatic approaches have been investigated
by shortcutting NADH/StyB using various photo- or
electrochemical methods to reduce FAD.24–29 In particular,
1-benzyl-1,4-dihydronicotinamide (BNAH) was shown to be
effective and simple to use as a reductant with StyA16,30 and
other two-component flavoprotein monooxygenases such as
halogenases,31 a bacterial luciferase,32 and an FMN-dependent
type II Baeyer–Villiger monooxygenase.33 BNAH thus circumvents
the use of two enzymes (StyB, dehydrogenase) and NADH, for
using oxygenase StyA in biocatalytic reactions (Fig. 1B).
a Department of Biotechnology, Delft University of Technology, Van der Maasweg 9,
2629 HZ Delft, The Netherlands. E-mail: c.e.paul@tudelft.nl
b Microbial Biotechnology, Ruhr-Universität Bochum, Universitätsstr. 150, 44780
Bochum, Germany
c Enzymicals AG, Walther-Rathenau-Straße 49a, 17489 Greifswald, Germany
d Institute for Biochemistry, Biotechnology and Bioinformatics, Technische
Universität Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany
e Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE
Wageningen, The Netherlands
Previous enzymatic cascades involving two-component
SMOs have led to the synthesis of amino acids and other
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cy00855b
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recently isolated SfStyA (see ESI†) to establish its substrate
scope (Table 1). The aromatic alkene substrates explored bore
different substitution patterns: a methyl substituent at the α-
or β-position (1b–d, 1q–r), a ring substituent in para-, meta-
and ortho-positions (halogen atoms and methyl groups 1e–o),
a non-conjugated alkene (allylbenzene 1p) and a heterocyclic
ring (2-vinylpyridine 1s).
Reaction mixtures were extracted after one hour for
conversion comparison. All alkenes except 2-vinylpyridine 1s
were converted to over 3.3 mM epoxide product within one
hour (Table 1, >66% conversion, turnover frequency (TOF)
>1100 h−1). Full conversion was achieved when the reaction
was left to completion (Fig. 3A) and no diol was detected (see
ESI†). The formation of a ketone side product was observed
in two cases, with 1,2-dihydronaphthalene 1q giving 5%
2-tetralone (Table 1 entry 17), and indene 1r leading to 54%
2-indanone (entry 18). The enantiomeric excess (ee) obtained
for oxide products bearing different substituents were mostly
excellent (94 to >99% ee, entry 1, 3–5, 8–9, 11–15, 17–19),
very good (89% ee for α-methylstyrene oxide 2b entry 2) and
moderate for the non-conjugated alkene in allylbenzene 1p,
affording epoxide 2p with 68% ee (entry 16).
This substrate scope shows substituent-position effects on
SfStyA, observed with other characterized StyA enzymes.44
The position of the substituent affected the ee, especially in
the case of bromostyrene: the para-substituted product 2e
has an ee of 96% (S), compared with 81% and 70% ee (S)
obtained for meta- 2f and ortho-substituted 2g, respectively
(Table 1 entry 5–7). This effect was less pronounced with
chlorostyrenes 2h–j (83 to 95% ee, entry 8–10) and even less
with fluorostyrenes 2k–m (94 to >99% ee, entry 11–13).
Methylstyrenes 2n–o (95 to 99% ee, entry 14 and 15) showed
a similar pattern as chlorostyrenes (entry 8 and 9).
Fig. 1 SMO-Catalyzed asymmetric epoxidation of aromatic alkenes:
(A) two-component flavoprotein monooxygenase StyA and reductase
StyB with the required recycled NADH cofactor; (B) oxygenase StyA
with 1-benzyl-1,4-dihydronicotinamide (BNAH) as reductant for direct
FAD reduction.
valuable chiral compounds.10,34–37 Herein we establish the
proof of principle for a one-pot biocatalytic cascade via StyA-
catalyzed epoxidation, mediated by a nicotinamide coenzyme
biomimetic (NCB), followed by either chemical or enzymatic
azidolysis with halohydrin dehalogenases (HHDHs), towards
the desired product isomer (Fig. 2). Our linear artificial
cascade combines two (bio)chemical steps in one pot without
isolation of intermediates,38 and allows for substituents on
the α- or β-carbon to produce tertiary alcohols, an advantage
over other cascades limited from aromatic haloketones.8,39,40
The regioselective ring opening depends on the nucleophile
and epoxide, among other parameters.41
To establish the first step of the cascade, we explored
three available StyA enzymes, recombinantly produced in E.
coli and purified, for the selective asymmetric oxidation of
aromatic alkenes: StyA from Pseudomonas sp. VLB120,42 StyA1
from Rhodococcus opacus 1CP,43 and SfStyA from Sphingopyxis
fribergensis Kp5.2.
The high conversions and enantioselectivity obtained with
SfStyA led to its selection for further reactions, as StyA1 is
less stable and gives lower TOFs,16 and StyA affords slightly
lower ee for desired chiral epoxides in vitro (2a 98.0% ee, 2c
97.6% ee,24 compared with >99% ee with SfStyA in both
cases). We were especially interested in substrates leading to
products with two chiral centers: (S)-α-methylstyrene oxide 2b
(Table 1, entry 2), (1S,2S)-1-phenylpropylene oxide 2c and
(1S,2R)-1-phenylpropylene oxide 2d (entry 3 and 4) were
obtained with excellent ee.
Compared with using reductase StyB, NADH and its
recycling system (Fig. 1A), the oxygenase StyA had previously
displayed similar catalytic activity using BNAH as hydride
donor to reduce FAD (Fig. 1B).16 Initially, we screened a panel
of aromatic alkenes 1a–s -mostly styrene derivatives- with the
Next, we determined the time course (Fig. 3A) and various
reaction parameters for the SfStyA-catalyzed epoxidation of
styrene to demonstrate the practical use of the SfStyA/BNAH
system
with
this
oxygenase
component:
enzyme
concentration (Fig. 3B), FAD concentration (Fig. 3C), the
number of BNAH equivalents (Fig. 3D), and hydride donor
(Fig. 3E), which influenced product formation.
Higher enzyme concentration at 50 μM FAD led to higher
product formation (Fig. 3B). FAD concentration was explored
to ensure optimal product formation (Fig. 3C). FAD
concentrations of up to 100 μM led to increased oxide
formation, which translates to FAD reduction as the rate-
limiting step. A plateau was then reached, hence the limiting
Fig. 2 Linear one-pot chemo- or bi-enzymatic cascade from aromatic
alkenes 1 towards chiral 1,2-azido alcohols. First step: StyA, FAD, NCB
to enantiopure epoxides 2; second step: with NaN3 to α-azido alcohol
products 3, or with NaN3 and HHDH to β-azido alcohol products 4.
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Table 1 Biocatalytic asymmetric epoxidation of styrene derivatives with the oxygenase SfStyAa
Entry
1
Product 2
[2] (mM)
TOFb (h−1
1300
)
eec (%)
2a
3.9
>99 (S)
2
3
4
5
2b
2c
2d
2e
4.7
4.7
4.7
4.3
1565
1565
1565
1435
89 (S)
>99 (1S,2S)
>99 (1S,2R)
96 (S)
6
7
2f
4.1
3.3
1365
1100
81 (S)
70 (S)
2g
8
2h
4.7
1565
97 (S)
9
2i
2j
4.7
4.3
1565
1435
>99 (S)
83 (S)
10
11
2k
4.5
1500
>99 (S)
12
13
2l
4.3
4.7
1435
1565
95 (S)
94 (S)
2m
14
15
2n
2o
4.2
4.8
1400
1600
95 (S)
99 (S)
16
17
2p
2q
3.8
1265
1400
68 (S)
4.0 + 0.2
>99 (1S,2R)
18
19
2r
2s
1.9 + 2.2
2.5
1365
835
>99 (1S,2R)
>99 (S)
a
Reaction conditions: [BNAH] = 15 mM, buffer (50 mM tris-SO4 pH 7.0), catalase = 650 U mL−1, [FAD] = 50 μM, [SfStyA] = 3 μM, [alkene 1] = 5
mM, 0.2% v/v dimethyl sulfoxide (DMSO), final volume 1 mL in a 2 mL plastic tube shaken on a thermomixer at 900 rpm and 30 °C, for 1 h.
b
c
Turnover frequency (TOF) = [product]/[enzyme] per hour. Enantiomeric excess of epoxides determined by chiral GC-FID (see ESI†). Average
of duplicates, standard deviation <10%.
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the flavin-binding site, although the affinity for FADH2 is
approximately 8000-fold higher.18,43,45,46 Additionally, FAD
and FADH2 can disproportionate to give FAD radicals,
reacting with molecular oxygen to form hydrogen peroxide,47
leading to a futile loss of electrons.
Concerning the source of electrons, several NADH
regeneration systems have been established with formate
(FDH) or glucose (GDH) dehydrogenases (Fig. 1A). For our
cascade design however, sodium azide can be an inhibitor
for FDH, whereas high excess of glucose is typically used for
GDH (see ESI†), and we aimed to achieve a more efficient
and practical system. As we previously showed that the use of
BNAH allows for cost-effective direct FAD reduction with up
to 85% efficiency,16 we explored other NCBs. Flavin reduction
by NCBs such as BNAH is known to occur through direct
hydride transfer in solution.48,49 Different substituents were
previously shown to affect the overall NCB redox potential
and lead to different FAD reduction rates.31 Therefore, we
screened six NCBs bearing substituents varying from amide,
acetyl, carboxylic acid to nitrile, with either a benzyl, butyl or
alkyl amide chain on the nitrogen, and the classical Hantzsch
ester (HEH, Fig. 3E and 4), as a hydride donor for the
reduction of FAD without interfering with the epoxidation.50
Within one-hour reactions, BNAH displayed the best overall
conversion (3.9 mM), followed by AmNAH (2.6 mM). BCNPH
and HEH proved to be the worst reductants. No product was
observed in the absence of FAD, or enzyme, or BNAH.
Once the biocatalytic asymmetric oxidation process was
established, we proceeded to investigate the use of the chiral
epoxides in a chemo-enzymatic cascade. Azide can cleave
epoxides under mild conditions. With aliphatic epoxides, ring
opening occurs through a bimolecular reaction, an SN2-type
backside attack of the azide on the least substituted carbon of
the protonated epoxide, forming a trans-1,2-azido alcohol as
product. The rate of chemical epoxide ring opening is pH
dependent, the more acidic, the faster the reaction, and also
Fig. 3 Parameters for the SfStyA-catalyzed asymmetric oxidation of
styrene to its oxide 2a: (A) time course; (B) [SfStyA] = 0 to 4 μM; (C)
[FAD] = 0 to 200 μM; (D) BNAH equivalents (1 equiv. = 5 mM); (E) type
of NCBs hydride donor (see Fig. 4 for structures). General reaction
conditions unless otherwise specified: [BNAH] = 15 mM, buffer (50 mM
tris-SO4 pH 7.0), catalase = 650 U mL−1, [FAD] = 50 μM, [SfStyA] = 3
μM, [styrene] = 5 mM, 0.2% v/v DMSO, final volume 1 mL in a 2 mL
plastic tube shaken on a thermomixer at 900 rpm and 30 °C, for 1 h.
Average of duplicates.
Fig. 4 Top: schematic FAD reduction by NCBs. Bottom: chemical
structures of NADH and NCBs (1-benzyl-3-acetyl-1,4-dihydropyridine
BAPH, 1-benzyl-3-cyano-1,4-dihydropyridine BCNPH, 1-butyl-1,4-
dihydronicotinamide BuNAH, 1-carbamoyl-1,4-dihydronicotinamide
AmNAH, Hantzsch ester HEH).
step most likely became the SfStyA-catalyzed epoxidation.
Less product was observed with >100 μM FAD, as previously
reported with other StyA enzymes.16,24,45 The lower product
formation can be ascribed to FAD competing with FADH2 for
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higher temperatures increase the reaction rate (see ESI†).
Previous studies have shown that azidolysis of aryl-
substituted epoxides show different regioselectivity, affording
α-azido alcohols as the major products.51,52 The nucleophilic
attack occurs at the α-carbon of the aromatic substituted
epoxide, with complete inversion of configuration (see
ESI†).41,53 The regioselectivity was explained by stabilization
of positive charge formed through delocalization,53 but recent
Table 2 One-pot chemo-enzymatic cascade from styrene derivatives to α-azido alcoholsa
Entry
1
Major product
Conv.b (%)
3 : 4 (%)
3a
>99
98 : 2
2
3
4
5
3b
3c
3d
3e
98c
>99
27
97 : 3d
100 : 0
68 : 32
96 : 4
99
6
7
3f
99
82
96 : 4
98 : 2
3g
8
3h
96
97 : 3
9
3i
3j
92
90
93 : 7
95 : 5
10
11
3k
98
98 : 2
12
13
3l
97
98
91 : 9
94 : 6
3m
14
15
16
3n
3o
3q
98
99
98 : 2
98 : 2
94 : 6
>99
17
18
3r
3s
79
62
96 : 4
53 : 47
a
Reaction conditions (i): [BNAH] = 15 mM, buffer (50 mM tris-SO4 pH 7.0), catalase 650 U mL−1, [FAD] = 50 μM, sodium azide (7 equiv.),
[SfStyA] = 3 μM, [alkene] = 5 mM, 0.2% v/v DMSO, final volume 1 mL in a 2 mL plastic tube shaken on a thermomixer at 900 rpm and 30 °C,
b
d
for 24 h. Determined by chiral GC-FID. Average of duplicates, standard deviation <10%. c 5% diol product observed. Assignment of isomers
ratio not determined.
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studies revealed that electrostatic interaction between the
phenyl ring and the incoming nucleophile play a dominant
role.54
We chose sodium azide salt for its high solubility in water
and determined that the amount needed for fast azidolysis
was seven equivalents with respect to the substrate (see
ESI†). The reactivity and regioselectivity of the azidolysis in
water is controlled by a neutral pH value, 30 °C temperature
and low buffer concentration, thus avoiding diol formation
stemming from the competition of hydroxide or water with
the azide ion (see ESI†).52 Under these conditions, we
achieved full conversion and highly regioselective ring
opening of styrene oxide: the desired α-azido alcohol
(2-azido-2-phenyl-1-ethanol 3a) was formed as the main
Fig. 5 One-pot chemo-enzymatic cascade from styrenes to chiral
α-azido alcohols, 15 mg scale, % isolated yield of product. Reaction
conditions: [BNAH] = 30 mM, buffer (50 mM tris-SO4, pH 7.0), catalase
(650 U mL−1), [FAD] = 50 μM, [SfStyA] = 3 μM, [alkene] = 10 mM, [NaN3]
= 35 mM, final volume 10 mL, shaken in Erlenmeyer flasks on an
incubator shaker at 180 rpm and 30 °C for 22 h.
product with only
a trace amount of regioisomer 4a,
determined by gas chromatography with flame ionization
detection (GC-FID, ratio of 98 : 2 3a : 4a). The linearly
see ESI†)58,59 were screened towards racemic styrene oxide
rac-2a (Table 3), using sodium azide for the selective epoxide
ring opening,58 giving two possible products, 1-azido-2-
phenylethanol 3 or 2-azido-1-phenylethanol 4. To minimize
the uncatalyzed background reaction of sodium azide that
leads to the α-azido alcohols, the reactions with HHDHs were
performed using only one equivalence of sodium azide. In
general, the most active HHDH towards the formation of
β-azido alcohols 4 gives a higher ratio towards the desired
product.
The results of the HHDH screening to obtain the β-azido
alcohol were variable (Table 3): the enzymatic reaction was
clearly outrun by the chemical one with HheD6 (entry 6),
likely due to the low activity of the enzyme, as was the case
with HheA3 (entry 1) and HheD3 (entry 4). The other HHDHs
also showed mixed ratios (entries 2–3 and 6). HheE5 from
Gammaproteobacterium strain IMCC3088 gave the best ratio
of α : β 16 : 84 (entry 7).
increasing
product
formation
with
higher
azide
concentrations can be explained by the SN2 type mechanism.
We then carried out the full azidohydroxylation cascade with
the established SfStyA-catalyzed epoxidation of alkenes 1a–o,
q–s, affording the corresponding (2R)-α-azido alcohols 3 with
good to excellent conversions and regioselectivity (Table 2). The
ee from the SfStyA reaction was retained in all cases.
The difference in rate and degree of regioselectivity
between azidolysis of trans- and cis-β-methylstyrene oxide (2c
versus 2d) could be explained by the orientation of the phenyl
ring with respect to the epoxide (see ESI†). A further
advantage of this chemo-enzymatic cascade, besides the
ability to carry out the reactions in one pot via chiral
epoxides, is the high reactivity of the epoxide intermediates:
without the need to isolate the epoxide, the aldehyde and
ketone previously observed with the simple biocatalytic
epoxidation (Table 1, entries 17–18) do not have time to form.
Therefore,
we
obtained
full
conversion
with
The screened HHDHs lacked high selectivity for either the
S- or R-enantiomer with racemic styrene oxide, the highest ee
being reached with HheD3 (68% ee of (R)-4, entry 5), hence
the importance of using a selective StyA in the asymmetric
epoxidation step. In our case, the use of SfStyA provided very
good to excellent ee (Table 1), but other StyA enzymes could
be used to obtain the best ee depending on the substituted
substrate.
We set out to further screen selected HHDHs (Enzymicals
screening kit, see ESI†) with (S)-styrene oxide as starting
material (Table 4). HheD4 and HheF led to trace amounts of
diol side product 5 in addition to low ratios (entry 1 and 4),
and once again HheE5 stood out as the most active towards
β-ring opening (entry 3, the higher ratio with respect to
Table 3 is ascribed to the use of a different batch of
lyophilized enzyme) and was selected for further reactions.
The substrates that gave highest conversion, ee and
regioselectivity for the synthesis of α-azido alcohols were
chosen for subsequent experiments. The bi-enzymatic
cascade with SfStyA and HheE5 was thus carried out for
styrene 1a, trans-β-methylstyrene 1c and 4-fluorostyrene 1k.
The trans-β-methylstyrene 1c led to a mixture of α- and
1,2-dihydronaphthalene 1q and 79% conversion with indene
1r (Table 2, entry 16 and 17).
Encouraged by the enantioenriched 2-azido-2-phenyl-1-
ethanol derivatives obtained, several reactions were carried
out on a scale of 15 mg of substrate, increasing the substrate
concentration to 10 mM. The corresponding azido alcohols
obtained were simply extracted with ethyl acetate without
further purification and identified and characterized by NMR
spectroscopy (Fig. 5, ESI†). A higher scale reaction was
performed with trans-β-methylstyrene 1c (56 mg, 10 mM) as
substrate. Using SfStyA, BNAH and FAD, a 92% crude yield
was obtained with >99% ee of (1R,2S)-1-azido-1-
1
phenylpropan-2-ol 3c. H NMR showed a high purity product
after extraction with ethyl acetate (see ESI†).
It should be noted that sodium azide may interfere with
the steady-state kinetics of substrate epoxidation catalysed by
the SMO.55–57 Our one-pot cascade may be more efficient as a
two-step approach, adding the sodium azide in the second
step.
To access the β-azido alcohol isomers, several halohydrin
dehalogenases (HHDHs, EC 3.8.1.2, Enzymicals screening kit,
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Table 3 HHDH-Catalyzed racemic styrene oxide ring openinga
Entry
HHDH
Ratio 3 : 4
(R)-3 eeb (%)
(R)-4 eeb (%)
1c
2
HheA3
HheA5
HheB5
HheD3
HheD5
HheD6
HheE5
None
87 : 13
54 : 46
33 : 67
82 : 18
37 : 63
95 : 5
8
43
44
15
60
<5
22
<5
42
67
27
68
49
<5
<5
<5
3c
4
5c
6
7
16 : 84
98 : 2
8
a
Reaction conditions: buffer (50 mM tris-SO4, pH 7.0), [rac-styrene oxide] = 5 mM, [NaN3] = 5 mM, lyophilized cell-free extract HHDH (10 mg
mL−1), final volume 1 mL in a 2 mL plastic tube shaken on a thermomixer at 900 rpm and 30 °C for 15 h 30 min. Full conversion was observed.
b
Determined by chiral GC-FID. c Diol side product observed in trace amounts (<2%).
Table 4 HHDHs-Catalyzed (S)-styrene oxide ring openinga
Entry
HHDH
Ratio 3 : 4
(R)-3 eeb (%)
(S)-4 eeb (%)
(R)-5 (%)
1
2
3
4
HheD4
HheE4
HheE5
HheF
63 : 37
26 : 74
6 : 94
>99
>99
>99
>99
>99
>99
>99
>99
8
<1
<1
5
60 : 40
a
Reaction conditions: buffer (50 mM tris-SO4 pH 7.0), HHDH (10 mg), [NaN3] = 5 mM, [(S)-styrene oxide] = 5 mM, final volume 1 mL in a 10
mL glass vial shaken on an incubator shaker at 180 rpm and 30 °C for 15 h. Full conversion was observed. b Determined by chiral GC-FID.
β-azido alcohol 4c, which can be ascribed to the poor activity
of HheE5 towards the epoxide 3c.60 Further screening efforts
with HHDHs did not display a more active enzyme, therefore
there is room for enhancing HHDH enzymes with this type of
substrate.
enantio- and regioselectivity (Fig. 6). With the discovery and
engineering of HHDHs, we can expect variants with higher
selectivity in the near future.
Conclusions
Pleasingly, substrates 1a and 1k afforded the corresponding
β-azido alcohols 4a and 4k with good to excellent conversion,
In conclusion, we explored the substrate scope of a new two-
component flavoprotein styrene monooxygenase SfStyA for
the asymmetric oxidation of styrene derivatives, leading
mostly to high conversions and ee. This study further
demonstrates the use of the oxygenase component of two-
component flavoprotein monooxygenases with a practical
flavin reduction system. Through this biocatalytic
asymmetric epoxidation with SfStyA, we established a linear
artificial one-pot chemo- and bi-enzymatic cascade to achieve
the asymmetric azidohydroxylation of styrenes. The epoxide
ring opening was achieved with sodium azide either
chemically, or catalyzed by
a halohydrin dehalogenase,
leading to high purity of either azidoalcohol isomers with
retention of the StyA selectivity. The latest discovery of (R)-
selective StyA oxygenases allows access to the other
isomers.23,61,62
We anticipate our biocatalytic cascade to expand the use
of oxygenase StyA for the production of amino alcohols63 and
Fig.
6 One-pot bi-enzymatic cascade to β-azido alcohols. %
conversions. Reaction conditions: [BNAH] = 15 mM, buffer (50 mM
tris-SO4 pH 7.0), catalase = 650 U mL−1, [FAD] = 50 μM, [alkene] = 5
mM, [SfStyA] = 3 μM, [NaN3] = 5 mM, HheE5 (20 mg), final volume 1
mL in a 10 mL glass vial shaken on an incubator shaker at 180 rpm and
30 °C for 22 h.
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Catalysis Science & Technology
styrenyl aziridines. Furthermore, the use of alternative Conflicts of interest
nucleophiles, such as halides, cyanate, etc.,64 can expand the
There are no conflicts to declare.
portfolio of chiral enantiopure products obtained. In the
future we expect the use of peroxygenases will be attractive
once evolved to achieve high enantioselectivity.65
Acknowledgements
L. M. M. was funded through the COST Action CM13003
Systems Biocatalysis. D. T. acknowledges the Federal Ministry
for Innovation, Science and Research of North Rhine-
Westphalia (grant number PtJ-TRI/1141ng006). C. E. P.
acknowledges a NWO VENI grant (No 722.015.011). The
halohydrin dehalogenases (HHDHs) were kindly provided by
Enzymicals AG. We thank Prof. A. Schmid for providing the
StyA plasmid, and A. H. Westphal, R. van Oosten and E. van
der Klift for technical assistance.
Experimental details
All biocatalytic reactions were performed on an Eppendorf
ThermoMixer
C
(Eppendorf, Hamburg, Germany) and
followed by gas chromatography with flame ionization
detection (GC-FID). Analyses were carried out on a Shimadzu
GC-2010 GC-FID. Products were confirmed by reference
standards and GC-MS. Product concentrations were obtained
with a calibration curve equation using 5 mM dodecane as
an internal standard.
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