Biocatalytic Formal Anti-Markovnikov Hydroamination and Hydration of Aryl Alkenes

Article abstract of DOI:10.1021/acscatal.7b01464

Biocatalytic anti-Markovnikov alkene hydroamination and hydration were achieved based on two concepts involving enzyme cascades: epoxidation-isomerization-amination for hydroamination and epoxidation-isomerization-reduction for hydration. An Escherichia coli strain coexpressing styrene monooxygenase (SMO), styrene oxide isomerase (SOI), ω-transaminase (CvTA), and alanine dehydrogenase (AlaDH) catalyzed the hydroamination of 12 aryl alkenes to give the corresponding valuable terminal amines in high conversion (many ≥86%) and exclusive anti-Markovnikov selectivity (>99:1). Another E. coli strain coexpressing SMO, SOI, and phenylacetaldehyde reductase (PAR) catalyzed the hydration of 12 aryl alkenes to the corresponding useful terminal alcohols in high conversion (many ≥80%) and very high anti-Markovnikov selectivity (>99:1). Importantly, SOI was discovered for stereoselective isomerization of a chiral epoxide to a chiral aldehyde, providing some insights on enzymatic epoxide rearrangement. Harnessing this stereoselective rearrangement, highly enantioselective anti-Markovnikov hydroamination and hydration were demonstrated to convert α-methylstyrene to the corresponding (S)-amine and (S)-alcohol in 84-81% conversion with 97-92% ee, respectively. The biocatalytic anti-Markovnikov hydroamination and hydration of alkenes, utilizing cheap and nontoxic chemicals (O₂, NH₃, and glucose) and cells, provide an environmentally friendly, highly selective, and high-yielding synthesis of terminal amines and alcohols.

Full text of DOI:10.1021/acscatal.7b01464

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Article
Biocatalytic Formal Anti-Markovnikov
Hydroamination and Hydration of Aryl Alkenes
Shuke Wu, Ji LIU, and Zhi Li
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01464 • Publication Date (Web): 29 Jun 2017
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Biocatalytic Formal AntiꢀMarkovnikov
Hydroamination and Hydration of Aryl Alkenes
Shuke Wu, Ji Liu, Zhi Li*
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117585, Singapore
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ABSTRACT: Biocatalytic antiꢀMarkovnikov alkene hydroamination and hydration were
achieved based on two concepts involving enzyme cascades: epoxidationꢀisomerizationꢀ
amination for hydroamination and epoxidationꢀisomerizationꢀreduction for hydration. An
Escherichia coli strain coꢀexpressing styrene monooxygenase (SMO), styrene oxide isomerase
(SOI), ωꢀtransaminase (CvTA), and alanine dehydrogenase (AlaDH) catalyzed the
hydroamination of 12 aryl alkenes to give the corresponding valuable terminal amines in high
conversion (many ≥ 86%) and exclusive antiꢀMarkovnikov selectivity (> 99 : 1). Another E. coli
strain coꢀexpressing SMO, SOI, and phenylacetaldehyde reductase (PAR) catalyzed the
hydration of 12 aryl alkenes to the corresponding useful terminal alcohols in high conversion
(mostly ≥ 80%) and very high antiꢀMarkovnikov selectivity (> 99 : 1). Importantly, SOI was
discovered for stereoselective isomerization of a chiral epoxide to a chiral aldehyde, providing
some insights on enzymatic epoxide rearrangement. Harnessing this stereoselective
rearrangement, highly enantioselective antiꢀMarkovnikov hydroamination and hydration were
demonstrated to convert αꢀmethylstyrene to the corresponding (S)ꢀamine and (S)ꢀalcohol in 84ꢀ
81% conversion with 97ꢀ92% ee, respectively. The biocatalytic antiꢀMarkovnikov
hydroamination and hydration of alkenes, utilizing cheap and nonꢀtoxic chemicals (O2, NH3, and
glucose) and cells, provide an environmentally friendly, highly selective, and highꢀyielding
synthesis of terminal amines and alcohols.
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KEYWORDS: alkenes, antiꢀMarkovnikov, biocatalysis, enzyme cascades, hydration,
hydroamination, Meinwald rearrangement, wholeꢀcell biotransformation
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INTRODUCTION
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AntiꢀMarkovnikov addition to alkenes provides a facile access to various terminal
functionalized chemicals, with huge potential for industrial applications.1,2 The two most
important antiꢀMarkovnikov reactions are hydroamination and hydration to produce terminal
amines and alcohols, respectively, which was listed as one of ten challenges in industrial
catalysis.2 Despite many developments on these reactions over the years, green and efficient
methods are still highly wanted. AntiꢀMarkovnikov hydroamination with CuH or acridinium
photoredox catalyst was recently reported,3–5 but requiring expensive hydride reagent or a long
reaction time. AntiꢀMarkovnikov hydroamination of unactivated olefins with iridium
photocatalyst was reported very recently.6 Current hydroamination processes mainly use primary
or secondary amines to produce secondary or tertiary hydroamination products, while they are
difficult in using NH3 to generate primary amines. On the other hand, antiꢀMarkovnikov
hydration of alkene is very challenging. Formal hydration methods were thus developed, such as
multiꢀstep hydroborationꢀoxidation or oxidationꢀhydrolysisꢀreduction.7,8 However, these methods
either requires borane reagents, or needs high loading of toxic precious metal catalysts. Very
recently, lightꢀmediated antiꢀMarkovnikov hydration was reported with acridinium photoredox
catalyst with a hydrogen atom donor, but it requires a long reaction time with a TON of 230.9
Biocatalysis is a useful tool for chemical synthesis in a greener and more sustainable way.10–15
Nature provides ammoniaꢀlyases and aminomutases for hydroamination of α,βꢀunsaturated acids
to give amino acids.16–18 However, no biocatalytic method has been described for
hydroamination of unfunctionalized alkenes to produce amines. On the other hand, some
hydratases were known for hydration of C=C bonds conjugated to carbonyl groups.19 Terpene
hydratases,20 oleate hydratase21 and decarboxylases22 were reported for hydrating terpenes, 1ꢀ
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decene and hydroxystyrenes, respectively. However, they show Markovnikov selectivity. There
is a lack of a biocatalytic method for antiꢀMarkovnikov hydration of unfunctionalized alkenes to
alcohols.
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Here, we present two novel biocatalytic methods for formal antiꢀMarkovnikov hydroamination
and hydration of alkenes, respectively. The methods utilize oneꢀpot cascade biotransformation23–
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and involve two enzyme cascades: epoxidationꢀisomerizationꢀamination for hydroamination
and epoxidationꢀisomerizationꢀreduction for hydration (Scheme 1).
RESULTS AND DISCUSSION
Selection of Enzymes for Target Reactions. To demonstrate the concept, we selected the
transformation of styrenes 1 to phenethylamines 4 and phenethyl alcohols 5 as the model
reactions. While styrene is a simple and easily available alkene, the products are very useful with
many synthetic applications (Table S1 in Supporting Information). Styrene monooxygenase
(SMO)41ꢀ43 is a wellꢀstudied and efficient enzyme, thus it was select selected for the epoxidation
of 1 to (S)ꢀ2. For isomerization of (S)ꢀ2 to 3, a unique styrene oxide isomerase (SOI)39, 44ꢀ46 was
chosen. A ωꢀtransaminase (ωꢀTA)47ꢀ50 and an alanine dehydrogenase (AlaDH)51 were selected
for the amination of 3 to 4. Combining these four enzymes together enables the formal antiꢀ
Markovnikov alkene hydroamination. On the other hand, formal antiꢀMarkovnikov alkene
hydration could be achieved by combining alcohol dehydrogenase (ADH) or aldehyde reductase
(AR)52 (for reducing 3 to 5) with SMO and SOI. The enzymes were coꢀexpressed in whole cells
for practical biotransformation,53 because 1) SMO in whole cells produced much higher
concentration of (S)ꢀ2 (36.3 g L– 1)42 than that produced by SMO in vitro (0.16 g L– 1);43 2) SOI is
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an integral membrane protein;44ꢀ46 3) the cofactor NAD(P)H could be regenerated by cellular
metabolism of glucose.
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Genetic Engineering of Biocatalysts. The SOI gene (styC)44 from Pseudomonas sp. VLB120
was synthesized and cloned together with the SMO genes (styAB)28 to give plasmid pRSFꢀ
StyABC. For hydroamination, CvTA from Chromobacterium violaceum48 is active for amination
of 3a to 4a (Figure S1). The genes of CvTA and AlaDH from Bacillus subtilis51 were
constructed with pRSFꢀStyABC to give pRSFꢀStyABCꢀCvTAꢀAlaDH (Figure S2).
Hydroamination of 1a with E. coli T7 Express host harboring this plasmid gave 20% byproduct
5a (Figure S3), due to the competing reduction by native ADHs and reductases in the host. A
new E. coli RAREꢁMao strain, which was engineered by deletion of maoA-padA genes from E.
coli RARE strain54 (already deleted 6 ADH genes), was used as the host, and the formation of 4a
is much less (3%, Figure S3). To optimize the coꢀexpression of the four enzymes, the StyABC
and CvTAꢀAlaDH was split into two plasmids to generate 12 different strains (Figure S4). The
strain containing pACYCꢀStyABC and pCDFꢀCvTAꢀAlaDH (Figure 1a) gave the best
biotransformation result of 1a to 4a (Figure S5), thus it was named E. coli (StyABCꢀCvTAꢀ
AlaDH) strain and used for hydroamination.
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For hydration, we tested several ADHs and ARs for the reduction of 3a to 5a, and the
phenylacetaldehyde reductase (PAR) from tomato Solanum lycopersicum52 was found to be the
best (Figure S6). Thus, the PAR gene was engineered with StyABC to give plasmid pRSFꢀ
StyABCꢀPAR. The recombinant plasmid was transformed into E. coli T7 Express cells to give E.
coli (StyABCꢀPAR) for hydration of 1 to 5 (Figure 1b).
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Hydroamination of Styrene Derivatives. Hydroamination of 1a at 50–100 mM was
performed with resting cells of E. coli (StyABCꢀCvTAꢀAlaDH), and high conversion to 4a was
achieved for 75 mM 1a (Figure S7). The time course of hydroamination of 80 mM 1a was
investigated (Figure 1c): 1a was quickly converted to 4a in the first 4 h, and a small amount of
2a (up to 8 %) and 3a (up to 2 %) was accumulated initially and then converted. At the end of
reaction (10 h), the amine (4a) was produced in 93 % conversion. Noteworthly, byproduct (5a)
was detected in trace amount (0.6%), indicating the very high chemoseletivity. In addition,
another isomer of 4a, 1ꢀphenyethylamine, was not observed, demonstrating the excellent antiꢀ
Markovnikov regioselectivity.
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To explore the substrate scope of hydroamination, aryl alkenes (1aꢀ1l) were transformed with
the resting cells of E. coli (StyABCꢀCvTAꢀAlaDH) (Table 1). Styrene derivatives with a fluoro
substituent (1b, 1c, 1d), a methyl substituent (1i, 1j) or a methoxy substituent (1k, 1l) were
converted to the corresponding phenethylamines 4 in very high conversion of 93–99 %. Chloro
substituted styrenes (1e, 1f) and bromo substituted styrenes (1g, 1h) were also converted to the
corresponding terminal amines 4 in good to moderate conversion of 86–45 %. The
chemoselectivity of amine over alcohol is also very high (all > 20 : 1, Figure S46–S57), and the
antiꢀMarkovnikov regioselectivity is excellent (all > 99 : 1, Figure S20–S31).
Hydration of Styrene Derivatives. Biocatalytic hydration of 1a at 60–80 mM was carried out
with resting cells of E. coli (StyABCꢀPAR), and high conversion to 5a (>90 %) was obtained
with 60 mM 1a in organic phase (Figure S8). The detail time course of hydration of 60 mM 1a is
shown in Figure 1d: 1a was quickly converted to 5a in the initial 3 h, and only a very small
amount of 2a (0.3 %) and 3a (3 %) were accumulated at initial 1 h. At end of reaction (8 h), the
desired 5a was formed in 93 % conversion with little 1a (2 %) was remained. Very importantly,
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another isomer of 5a, 1ꢀphenyethanol, was not observed, proving the exclusive antiꢀ
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Markovnikov regioselectivity.
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The scope of hydration was explored by subjecting 12 aryl alkenes (1aꢀ1l) to the resting cells
of E. coli (StyABCꢀPAR) (Table 2). Many substituted 2ꢀphenylethanols were produced in very
high conversion (90–99 %), including fluoro substituted 5b, 5c, 5d, methyl substituted 5i, 5j, and
methoxy substituted 5k, 5l. 2ꢀPhenylethanols with a chloro (5e, 5f) or bromo (5g, 5h) substituent
were formed in good conversion of 89–60 %. Importantly, the antiꢀMarkovnikov regioselectivity
for all the hydration was excellent (> 99 : 1, Figure S33–S44).
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Stereoselective Isomerization of an Epoxide to an Aldehyde. To establish enantioselective
hydroamination and hydration, we chose 1m as the model substrate (Scheme 1b). As SMO was
known for epoxidation of 1m to (S)ꢀ2m43 [we further validated the production of (S)ꢀ2m in 98%
ee with the SMO in our system, Figure S9], the key for the enantioselective cascades is the
isomerization of (S)ꢀ2m. E. coli (SOI) was then used to isomerize (S)ꢀ2m to give (S)ꢀ3m in 91 %
ee at 15ꢀ30 min (Figure 2a). Slow decrease of the ee was observed due to the racemization via
tautomerization in aqueous buffer.55–57 To confirm that the stereoselective isomerization of (S)ꢀ
2m is catalyzed by SOI, the reaction of (S)ꢀ2m was carried out by using E. coli cells without SOI
(control), which did not produce 3m (Figure S10). Furthermore, isomerization of (S)ꢀ2m was
performed with partially purified SOI, which gave (S)ꢀ3m in 95% ee in 15 min (Figure S11). In
comparison, chemical Meinwald rearrangement of (R)ꢀ2m gave the chiral aldehyde in very low
ee.58 Further investigation of the conversion of racꢀ2m with E. coli (SOI) gave nearly racemic
3m [6 % ee (R), Figure S12], indicating that the isomerization of (R)ꢀ2m produced (R)ꢀ3m.
Kinetic resolution of racꢀ2m (20 mM) with E. coli (SOI) was then performed (Figure S12): the
ee of (S)ꢀ2m was 10% at 77% conversion, and the ee of (S)ꢀ2m reached 18% at 91% conversion,
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which corresponds to an E value of 1.2. This suggests that SOI slightly prefers the isomerization
of (R)ꢀ2m over the isomerization of (S)ꢀ2m; on the other hand, the ee of (R)ꢀ3m reached 6% at
35% conversion at 15 min and decreased gradually to nearly zero. A previously proposed
mechanism of SOI involves following steps: enzymatic protonation induces the opening of the
epoxide ring and the formation of a benzyl cation intermediate; it was subjected to proton
abstraction to give an enol, which undergos tautomerization to an aldehyde (Figure S13a).45 We
found now that SOI is stereoselective, thus, If SOI follows this mechanism, (S)ꢀ2m will be firstly
converted to a planar enol intermediate which requires asymmetric tautomerization to give (S)ꢀ
3m. We think, the following mechanism of SOI is also possible: enzymatic Brønsted acid
(glutamic acid or aspartic acid residue) protonates the epoxide and induces a CꢀO cleavage to
form a similar benzyl cation intermediate; a unique stereoselective 1,2ꢀhydrogen shift (Hꢀshift)
takes place to give the chiral aldehyde (S)ꢀ3m (Figure S13b). Currently, we are investigating the
catalytic mechanism of SOI.
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Enantioselective hydroamination. To achieve enantioselective hydroamination, the last step
in the cascade should be fast and enantioselective towards (S)ꢀ3m. For asymmetric
hydroamination of 1m, transaminases CvTA, HnTA from Hyphomonas neptunium,49 and VfTA
from Vibrio fluvialis50 were examined for the transamination of racꢀ3m (Table S2). While CvTA
and VfTA are highly active for transamination of racꢀ3m with Rꢀselectivity, HnTA was less
active but showing the desired Sꢀselectivity towards racꢀ3m. E. coli (StyABCꢀHnTA) and E. coli
(StyABCꢀVfTAꢀAlaDH) were thus engineered (Figure S14, S15). Biotransformation of 1m with
E. coli coꢀexpressing SMO, SOI, and different ωꢀTA (and AlaDH) gave (S)ꢀ4m in 76%
conversion with 51 % ee, 8% conversion with 95 % ee, and 81% conversion with 70 % ee,
respectively (Table 3, entry 1ꢀ3). Considering both conversion and product ee, VfTA was
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selected for directed evolution59–63 to invert its Rꢀenantioselectivity to Sꢀenantioselective towards
racꢀ3m: 12 residues (Figure S16) near the substrate binding site of VfTA were subjected to
iterative saturation mutagenesis64 to give VfTADM (L56A/I259T) as a highly Sꢀenantioselective
mutant (Table S3). Docking of (R)ꢀ or (S)ꢀ4m in the substrate binding pockets of VfTA and
VfTADM explained the reverse of enantiopreference (Figure S17). E. coli (StyABCꢀVfTADMꢀ
AlaDH) was then engineered (Figure S18) for hydroamination of 1m, producing (S)ꢀ4m in 92 %
ee and 84 % conversion (entry 4). During the reaction, (S)ꢀ3m was accumulated only in a small
amount due to the fast conversion of (S)ꢀ3m to (S)ꢀ4m, thus significantly reducing the
racemization (S)ꢀ3m; and (S)ꢀ4m was produced continuously in high ee (Figure 2b). The
biocatalytic hydroamination is much more enantioselective than the CuHꢀcatalyzed method,
which produced N, Nꢀdibenzyl (S)ꢀ4m in 52% ee from 1m.4
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Enantioselective hydration. For asymmetric hydration of 1m, PAR and horse liver ADH
(HLADH, isoenzyme E)55 were studied for the reduction of racꢀ3m (Table S4). While PAR was
almost nonꢀenantioselective towards racꢀ3m, HLADH was highly Sꢀenantioselective. E. coli
(StyABCꢀHLADH) was engineered to coꢀexpress SMO, SOI and HLADH (Figure S16).
Biotransformation of 1m with E. coli (StyABCꢀPAR) gave (S)ꢀ5m in 87 % ee and 85 %
conversion (Table 3, entry 5), whereas the same hydration of 1m with E. coli (StyABCꢀHLADH)
afforded (S)ꢀ5m in 97 % ee and 81 % conversion (entry 6). In comparison with the asymmetric
hydroborationꢀoxidation method [(S)ꢀ5m in 66 % ee from 1m],7 the biocatalytic asymmetric
hydration is much more stereoselective.
Preparative Biotransformation. Hydroamination of 1a, 1d, 1j, and 1k (2.5 mmol each) was
performed with E. coli (StyABCꢀCvTAꢀAlaDH) in a 60ꢀmL system to give the corresponding
phenethylamines in 83ꢀ95 % conversion. Extraction and flash chromatography purification
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afforded 4a, 4d, 4j, and 4k in 78, 68, 71, 82 % isolated yield, respectively. This is corresponding
to 1.7–2.1 mmol product per gram of dry cells. 1a, 1d, 1j, and 1k (2.5 mmol each) were
transformed with E. coli (StyABCꢀPAR) in a 100ꢀmL system to produce the corresponding 2ꢀ
phenylethanols in 87ꢀ99 % conversion. Extraction and flash chromatography purification offered
5a, 5d, 5j, and 5k in 83, 74, 66, 78 % isolated yield, respectively. This is corresponding to 1.6–
2.1 mmol product per gram of dry cells. Preparative asymmetric hydroamination of 1m (0.5
mmol) was demonstrated with E. coli (StyABCꢀVfTATMꢀAlaDH) to give (S)ꢀ4m in 65 %
isolated yield and 90 % ee. Preparative asymmetric hydration of 1m (0.5 mmol) was performed
with E. coli (StyABCꢀHLADH) to give (S)ꢀ5m in 59 % isolated yield and 96 % ee.
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CONCLUSION
Biocatalytic formal antiꢀMarkovnikov hydroamination and hydration of aryl alkenes were
achieved via two enzyme cascades: epoxidationꢀisomerizationꢀamination for hydroamination and
epoxidationꢀisomerizationꢀreduction for hydration. E. coli cells coꢀexpressing 3ꢀ4 enzymes of the
cascades catalyzed the hydroamination and hydration of 12 aryl alkenes, respectively, to give the
corresponding terminal amines and terminal alcohols in high conversion with excellent
chemoselectivity and antiꢀMarkovnikov regioselectivity. Furthermore, highly enantioselective
hydroamination and hydration were also successfully demonstrated, offering a new biocatalytic
access to chiral amines and chiral alcohols. In addition, SOI was proven to be stereoselective,
shedding some light on enzymatic epoxide rearrangement and opening the possibility of using
this enzyme for enantioselective synthesis. To the best of our knowledge, this is the first
biocatalytic antiꢀMarkovnikov hydroamination and hydration of unfunctionalized alkenes. The
biocatalytic antiꢀMarkovnikov hydroamination and hydration with E. coli cells utilize cheap and
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nonꢀtoxic reagents (O2, NH3, and glucose) and cells (biodegradable) to provide a green, highly
selective, and highꢀyielding synthesis of terminal amines and alcohols. Currently, we are
working on exploring and engineering SOI for converting other epoxides, to apply the
biocatalytic cascades for antiꢀMarkovnikov hydroamination and hydration of other alkenes.
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EXPERIMENTAL SECTION
General Procedure for Culturing E. coli Cells for Biotransformation. E. coli strain was
initially inoculated in LB medium (1 mL) with kanamycin (50 mg L–1) [for E. coil (StyABCꢀ
PAR), E. coil (StyABCꢀHLADH)], a mixture of chloramphenicol (50 mg L–1) and streptomycin
(50 mg L–1) [for E. coil (StyABCꢀCvTAꢀAlaDH)] or a mixture of chloramphenicol (50 mg L–1)
and kanamycin (50 mg L–1) [for E. coil (StyABCꢀHnTA), E. coil (StyABCꢀVfTAꢀAlaDH), E.
coil (StyABCꢀVfTADMꢀAlaDH)]. The cells grew for 8–10 h at 37 °C, and then transferred to a
250ꢀmL triꢀbaffled flask with 50 mL M9 medium supplemented with glucose (20 g L–1), yeast
extract (6 g L–1) and appropriate antibiotics. The cells continued to grow at 37 °C and 250 rpm
for about 2 h to reach an OD600 of 0.6, and then IPTG (0.5 mM final concentration) was added to
induce the enzyme expression. The cells further grew at 22 °C overnight (12–13 h) and they
were harvested by centrifugation (4000 g, 10 min).
Hydroamination of 1 to 4 with E. coli (StyABCꢀCvTAꢀAlaDH). The cell pellets of E. coli
(StyABCꢀCvTAꢀAlaDH) were resuspended in NaP buffer (sodium phosphate, 200 mM, pH 8,
2% glucose, 200 mM NH3/NH4Cl) to a cell density of 10 g cdw L– 1. To a shaking flask (100
mL) containing 2 mL of cell suspension, 2 mL nꢀC16H34 containing substrate 1aꢀ1l (20ꢀ80 mM)
was added. The reaction was incubated in a shaking incubator at 250 rpm and 30 °C for 10ꢀ24 h.
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For determining the reaction time curve (Figure 1c), 80 µL samples of the two phases were taken
out at 1, 2, 4, 6, and 10 h. 50 µL of nꢀC16H34 was isolated by centrifugation (12000 g, 3 min),
mixed with 950 µL EtOAc [with internal standard: 2 mM benzyl alcohol (BA)], and analyzed by
GCꢀFID for measuring the concentration of 1a, 2a, 3a, and 5a. 20 µL aqueous supernatant was
isolated by centrifugation (12000 g, 3 min), mixed with acid solution (0.5% TFA) and 500 µL
ACN (with 2 mM BA), and then analyzed by HPLC for measuring the concentration of 4a and
5a. For determining the substrate scope (Table 1), 200 µL samples of the two phase were taken
out at the end of reaction. 100 µL nꢀC16H34 was isolated by centrifugation (12000 g, 3 min),
mixed with 900 µL EtOAc (with 2 mM BA), and analyzed by GCꢀFID for measuring the
concentration of 1aꢀ1l and 5aꢀ5l. 100 µL of aqueous supernatant was isolated by centrifugation
(12000 g, 3 min), mixed with 400 µL acid solution (0.5% TFA) and 500 µL ACN (with 2 mM
BA), and then analyzed by HPLC for measuring the concentration of 4aꢀ4l and 5aꢀ5l. At the end
of the reaction, the aqueous phase was subjected to centrifugation (12000 g, 3 min). 500 µL
supernatant was isolated and adjusted to pH > 13 with NaOH solution (10 M). Then 500 µL
EtOAc was used to extract 4aꢀ4l and other amines and analyzed by GCꢀFID for determining the
regioselectivity.
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Hydration of 1 to 5 with E. coli (StyABCꢀPAR). The cell pellets of E. coli (StyABCꢀPAR)
were resuspended in KP buffer (potassium phosphate, 200 mM, pH 8, 2% glucose) to a cell
density of 10 g cdw L– 1. To a shaking flask (100 mL) containing 2 mL cell suspension, 2 mL nꢀ
C16H34 containing substrate 1aꢀ1l (20ꢀ60 mM) was added. The reaction was incubated in a
shaking incubator at 250 rpm and 30 °C for 8 h. For determining the reaction time curve (Figure
1d), 80 µL samples of the two phases were taken out at 0.5, 1, 1.5, 2, 3, 5, and 8 h. 50 µL nꢀ
C16H34 was isolated by centrifugation (12000 g, 3 min), mixed with 950 µL EtOAc (with 2 mM
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BA), and analyzed by GCꢀFID for measuring the concentration of 1a, 2a, 3a, and 5a. 20 µL
aqueous supernatant was isolated by centrifugation (12000 g, 3 min), mixed with 480 µL acid
solution (0.5% TFA) and 500 µL ACN (with 2 mM BA), and then analyzed by HPLC for
measuring the concentration of 5a. For determining the substrate scope (Table 2), 200 µL
samples of the two phases were taken out at the end of reaction. 100 µL nꢀC16H34 was isolated by
centrifugation (12000 g, 3 min), mixed with 900 µL EtOAc (with 2 mM BA), and analyzed by
GCꢀFID for measuring the concentration of 1aꢀ1l and 5aꢀ5l and determining the regioselectivity.
100 µL aqueous supernatant was isolated by centrifugation (12000 g, 3 min), mixed with 400 µL
acid solution (0.5% TFA) and 500 µL ACN (with 2 mM BA), and then analyzed by HPLC for
measuring the concentration of 5aꢀ5l.
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Asymmetric Hydroamination of 1m. The cell pellets of E. coli (StyABCꢀCvTAꢀAlaDH), E.
coli (StyABCꢀHnTA), E. coli (StyABCꢀVfTAꢀAlaDH), or E. coli (StyABCꢀVfTADMꢀAlaDH)
were resuspended in KP buffer (200 mM, pH 8, 2% glucose, 200 mM NH3/NH4Cl) (200 mM ᴅꢀ
alanine instead of NH3/NH4Cl for reaction with HnTA) to a cell density of 20 g cdw L– 1. To a
shaking flask (100 mL) containing 2 mL of cell suspension, 2 mL nꢀC16H34 containing 10 mM
substrate 1m (for reactions with CvTA and VfTA) or 1 mL nꢀC16H34 containing 10 mM substrate
1m (for reactions with HnTA and VfTADM) was added. The reaction was incubated in a shaking
incubator at 250 rpm and 30 °C for 24 h. For determining the end point data (Table 3), 200 µL
samples of the two phases were taken out at 24 h. 100 µL nꢀC16H34 was isolated by
centrifugation (12000 g, 3 min), mixed with 900 µL EtOAc (with 2 mM BA), and analyzed by
GCꢀFID for measuring the concentration of 1m, 2m, 3m and 5m. 100 µL aqueous supernatant
was isolated by centrifugation (12000 g, 3 min), mixed with 900 µL acid solution (0.5% TFA),
and then analyzed by chiral HPLC for measuring the concentration and ee of 4m. The aqueous
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phase was subjected to centrifugation (12000 g, 3 min). 500 µL supernatant was isolated and
adjusted to pH ≥ 13 with NaOH solution (10 M). Then 500 µL EtOAc was used to extract
amines and analyzed by GCꢀFID for determining the regioselectivity. For determining the
reaction time curve (Figure 2b), 40 µL samples of organic phase and 80 µL samples of aqueous
phase were taken out at 1, 2, 5, 10, and 24 h. 20 µL nꢀC16H34 was isolated by centrifugation
(12000 g, 3 min), mixed with 200 µL EtOAc (with 2 mM BA), and analyzed by GCꢀFID for
measuring the concentration of 1mꢀ3m. 50 µL aqueous supernatant was isolated by
centrifugation (12000 g, 3 min), mixed with 450 µL acid solution (0.5% TFA), and then
analyzed by chiral HPLC for measuring the concentration and ee of 4m.
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Asymmetric Hydration of 1m. The cell pellets of E. coli (StyABCꢀPAR) or E. coli (StyABCꢀ
HLADH) were resuspended in KP buffer (200 mM, pH 8, 2% glucose) to a cell density of 15 g
cdw L– 1. To a shaking flask (100 mL) containing 2 mL cell suspension of E. coli (StyABCꢀ
PAR), 2 mL nꢀC16H34 containing substrate 1m (10 mM) was added. To a shaking flask (100 mL)
containing 2 mL cell suspension of E. coli (StyABCꢀHLADH), 40 µL substrate 1m (250 mM in
EtOH) was added to reach a final concentration of 5 mM. The reaction was incubated in a
shaking incubator at 250 rpm and 30 °C for 24 h. At the end of reaction, 300 µL samples of the
two phases were taken out. 100 µL nꢀC16H34 was isolated by centrifugation (12000 g, 3 min),
mixed with 900 µL EtOAc (with 2 mM BA), and analyzed by GCꢀFID for measuring the
concentration of 1m, 2m, 3m and 5m. Another 100 µL nꢀC16H34 was isolated by centrifugation
(12000 g, 3 min), mixed with 900 µL nꢀhexane, and analyzed by chiral HPLC for determining
the ee of 5m. 100 µL aqueous supernatant was isolated by centrifugation (12000 g, 3 min),
mixed with 400 µL acid solution (0.5% TFA) and 500 µL ACN (with 2 mM BA), and then
analyzed by HPLC for measuring the concentration of 5m.
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Preparative Hydroamination. The cell pellets of E. coli (StyABCꢀCvTAꢀAlaDH) were
resuspended in NaP buffer (200 mM, pH 8, 2% glucose, 200 mM NH3/NH4Cl) to form a 50ꢀmL
suspension (20 g cdw L–1) in a triꢀbaffled flask (250 mL). 10 mL of nꢀC16H34 containing
substrate 1a, 1d, 1j, or 1k (2.5 mmol each) was added into the flask to start the reaction at 250
rpm and 30 °C. To monitor the reaction, samples were taken out at different time. Extra glucose
(0.5%) and NH3/NH4Cl (50 mM) were added into the system at 8 h. At the end of reaction (24
h), the mixture was adjusted to pH ≤ 2 with concentrated HCl, and the nꢀC16H34 and the cells
were removed by centrifugation (4000 g, 15 min). The aqueous solution was collected and
washed with 50 mL of nꢀhexane to remove organic impurities and residues of nꢀC16H34. The
aqueous solution was adjusted to pH ≥ 12 with NaOH solution (10 M), then saturated with NaCl,
and extracted with EtOAc (50 mL) for 3 times. The EtOAc was combined and dried by adding
anhydrous Na2SO4. The EtOAc was evaporated by using a rotary evaporator. The crude product
was purified by flash chromatography on a silica gel column with CH2Cl2: MeOH: triethylamine
of 100: 5: 1 as the eluent (Rf ≈ 0.3). The collected fractions were subjected to GCꢀFID analysis to
confirm the purity. The organic solvent in the desired fractions was removed by evaporation and
vacuum pumping.
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Phenethylamine 4a was obtained as light yellow oil: 236 mg, 78% yield from 1a. H NMR
(400 MHz, CDCl3): δ = 7.24–7.11 (m, 5H), 2.90 (t, J = 6.8 Hz, 2H), 2.68 (t, J = 6.8 Hz, 2H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 138.6, 127.8, 127.4, 125.2, 42.4, 38.8 ppm.
4ꢀFluorophenethylamine 4d was obtained as light yellow oil: 237 mg, 68% yield from 1d. 1H
NMR (400 MHz, CDCl3): δ = 7.20–6.89 (m, 4H), 2.87 (t, J = 6.8 Hz, 2H), 2.65 (t, J = 6.8 Hz,
2H) ppm; 13C NMR (100 MHz, CDCl3): δ = 160.5 (d, J = 242 Hz), 134.3 (d, J = 3 Hz), 129.1 (d,
J = 8 Hz), 114.2 (d, J = 21 Hz), 42.5, 38.0 ppm.
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4ꢀMethylphenethylamine 4j was obtained as light yellow oil: 240 mg, 71% yield from 1j. 1H
NMR (400 MHz, CDCl3): δ = 7.18–7.00 (m, 4H), 2.87 (t, J = 6.8 Hz, 2H), 2.64 (t, J = 6.8 Hz,
2H), 2.25 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ = 135.6, 134.6, 128.1, 127.7, 42.6, 38.6,
20.0 ppm.
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3ꢀMethoxyphenethylamine 4k was obtained as light yellow oil: 309 mg, 82% yield from 1k.
1H NMR (400 MHz, CDCl3): δ = 7.19–7.12 (m, 1H), 6.73–6.67 (m, 3H), 3.72 (s, 3H), 2.90 (t, J
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= 6.8 Hz, 2H), 2.66 (t, J = 6.8 Hz, 2H) ppm; C NMR (100 MHz, CDCl3): δ = 158.7, 140.3,
128.4, 120.2, 113.6, 110.4, 54.1, 42.3, 38.8 ppm.
The cell pellets of E. coli (StyABCꢀVfTADMꢀAlaDH) were resuspended in KP buffer (200
mM, pH 8, 2% glucose, 200 mM NH3/NH4Cl) to form a 10ꢀmL suspension (20 g cdw L–1) in a
flask (250 mL). 5 mL nꢀC16H34 containing substrate 1m (0.05 mmol) was added into the flask to
start the reaction at 250 rpm and 30 °C. 10 parallel experiments were performed together. To
monitor the reaction, samples were taken out at different time. Extra glucose (0.5%) and
NH3/NH4Cl (50 mM) were added into the system at 5 h. At the end of reaction (24 h), the
reaction mixture was combined, adjusted to pH ≤ 2 with concentrated HCl, and the nꢀC16H34 and
the cells were removed by centrifugation (4000 g, 15 min). The aqueous solution was collected
and washed with 50 mL of nꢀhexane to remove organic impurities and residues of nꢀC16H34. The
aqueous solution was adjusted to pH ≥ 12 with NaOH solution (10 M), then saturated with NaCl,
and extracted with EtOAc (100 mL) for 3 times. The EtOAc was combined and dried by adding
anhydrous Na2SO4. The EtOAc was evaporated by using a rotary evaporator. The crude product
was purified by flash chromatography on a silica gel column with CH2Cl2: MeOH: triethylamine
of 100: 5: 1 as the eluent (Rf ≈ 0.3). The collected fractions were subjected to GCꢀFID analysis to
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confirm the purity. The organic solvent of the desired fractions was removed by evaporation and
vacuum pumping.
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(S)ꢀβꢀmethylphenethylamine 4m was obtained as light yellow oil: 45 mg, 67% yield from
1m, 91% ee, [α]D20: –31o (c 1.0, CHCl3) {literature65 [α]D20: –35.3o (c 0.5, CHCl3), 95% ee}. 1H
NMR (400 MHz, CDCl3): δ = 7.33–7.19 (m, 5H), 2.87–2.60 (m, 3H), 1.25 (d, J = 6.8 Hz, 3H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 144.9, 128.6, 127.4, 126.4, 49.4, 43.4, 19.3 ppm.
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Preparative Hydration. The cell pellets of E. coli (StyABCꢀPAR) were suspended in KP
buffer (200 mM, pH 8, 2% glucose) to form a 50ꢀmL suspension (20 g cdw L–1) in a triꢀbaffled
flask (500 mL). 50 mL nꢀC16H34 containing substrate 1a, 1d, 1j, or 1k (2.5 mmol each) was
added into the flask to start the reaction at 250 rpm and 30 °C. To monitor the reaction, samples
were taken out at different time. Extra glucose (0.5%) was added into the system at 8 h. At the
end of reaction (24 h), the cells were removed by centrifugation (4000 g, 15 min). The nꢀC16H34
was extracted with water (50 mL) for 5 times. The water and aqueous solution were combined,
saturated with NaCl, and extracted with EtOAc (100 mL) for 3 times. The EtOAc was combined
and dried by adding anhydrous Na2SO4. The EtOAc was evaporated by using a rotary
evaporator. The crude product was purified by flash chromatography on a silica gel column with
nꢀhexane: EtOAc of 5: 1 as the eluent (Rf ≈ 0.3). The collected fractions were subjected to GCꢀ
FID analysis to confirm the purity. The organic solvent of the desired fractions was removed by
evaporation and vacuum pumping.
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Phenethyl alcohol 5a was obtained as colorless oil: 253 mg, 83% yield from 1a. H NMR
(400 MHz, CDCl3): δ = 7.33–7.21 (m, 5H), 3.84 (t, J = 6.6 Hz, 2H), 2.86 (t, J = 6.6 Hz, 2H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 138.7, 129.2, 128.8, 126.7, 63.9, 39.4 ppm.
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4ꢀFluorophenethyl alcohol 5d was obtained as colorless oil: 259 mg, 74% yield from 1d. H
NMR (400 MHz, CDCl3): δ = 7.26–6.97 (m, 4H), 3.83 (t, J = 6.6 Hz, 2H), 2.83 (t, J = 6.6 Hz,
2H) ppm; 13C NMR (100 MHz, CDCl3): δ = 161.9 (d, J = 243 Hz), 134.4 (d, J = 3 Hz), 130.6 (d,
J = 8 Hz), 115.5 (d, J = 21 Hz), 63.8, 38.5 ppm.
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4ꢀMethylphenethyl alcohol 5j was obtained as colorless oil: 225 mg, 66% yield from 1j. H
NMR (400 MHz, CDCl3): δ = 7.25–7.11 (m, 4H), 3.83 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.6 Hz,
2H), 2.32 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ = 136.2, 135.5, 129.5, 129.1, 64.0, 38.9,
21.2 ppm.
3ꢀMethoxyphenethyl alcohol 5k was obtained as colorless oil: 297 mg, 78% yield from 1k.
1H NMR (400 MHz, CDCl3): δ = 7.26–7.21 (m, 1H), 6.83–6.77 (m, 3H), 3.85 (t, J = 6.4 Hz, 2H),
3.80 (s, 3H), 2.84 (t, J = 6.6 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3): δ = 160.0, 140.3, 129.8,
121.5, 115.0, 112.0, 63.7, 55.4, 39.4 ppm.
The cell pellets of E. coli (StyABCꢀHLADH) were suspended in KP buffer (200 mM, pH 8,
2% glucose) to form a 10ꢀmL suspension (20 g cdw L–1) in a flask (250 mL). 200 µL substrate
1m in EtOH (250 mM, 1m amount 0.05 mmol) was added into the flask to start the reaction at
250 rpm and 30 °C. 10 parallel experiments were performed together. To monitor the reaction,
samples were taken out at different time. Extra glucose (0.5%) was added into the system at 5h.
At the end of reaction (24 h), the reaction mixture was combined and subjected to centrifugation
(4000 g, 15 min) to remove the cells. The supernatant was saturated with NaCl, and extracted
with EtOAc (50 mL) for 3 time. The EtOAc was combined and dried by adding anhydrous
Na2SO4. The EtOAc was evaporated by using a rotary evaporator. The crude product was
purified by flash chromatography on a silica gel column with nꢀhexane: EtOAc of 5: 1 as the
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eluent (Rf ≈ 0.3). The collected fractions were subjected to GCꢀFID analysis to confirm the
purity. The organic solvent of the desired fractions was removed by evaporation and vacuum
pumping.
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(S)ꢀ2ꢀphenylꢀ1ꢀpropanol 5m was obtained as colorless oil: 40 mg, 59% yield from 1m, 96%
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ee, [α]D20: –12o (c 1.0, CHCl3) {literature66 [α]D22: –11.6o (c 0.21, CHCl3), 95% ee}. H NMR
(400 MHz, CDCl3): δ = 7.36–7.23 (m, 5H), 3.70 (d, J = 6.8 Hz, 2H), 2.95 (sex, J = 6.8 Hz, 1H),
1.28 (d, J = 6.8 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ = 143.7, 128.6, 127.5, 126.7, 68.7,
42.5, 17.6 ppm.
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Scheme 1. a) Formal antiꢀMarkovnikov hydroamination and hydration of aryl alkenes using O2
and NH3 via epoxidationꢀisomerizationꢀamination cascade, and using O2 via epoxidationꢀ
isomerizationꢀreduction cascade, respectively. b) Enantioselective hydroamination and hydration
of αꢀmethylstyrene via the enzyme cascades.
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SMO = styrene monooxygenase, SOI = styrene oxide isomerase, ωꢀTA = ωꢀtransaminase,
AlaDH = ʟꢀalanine dehydrogenase, ADH = alcohol dehydrogenase, AR = aldehyde reductase.
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Figure 1. a) E. coli (StyABCꢀCvTAꢀAlaDH) coꢀexpressing SMO, SOI, CvTA and AlaDH for
hydroamination. b) E. coli (StyABCꢀPAR) coꢀexpressing SMO, SOI and PAR for hydration. c)
Hydroamination of 80 mM styrene 1a to 2ꢀphenylethylamine 4a with E. coli (StyABCꢀCvTAꢀ
AlaDH) cells (10 g cdw L–1) in NaP buffer (200 mM, pH 8, 2 % glucose, 200 mM NH3/NH4Cl)
and nꢀC16H34 (1:1) at 30 °C. 1% Glucose and 100 mM NH3/NH4Cl were added at 4 h. d)
Hydration of 60 mM styrene 1a to 2ꢀphenylethanol 5a with E. coli (StyABCꢀPAR) cells (10 g
cdw L–1) in KP buffer (200 mM, pH 8, 2 % glucose) and nꢀC16H34 (1:1) at 30 °C.
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Figure 2. a) Enantioselective conversion of 20 mM (S)ꢀ2m to (S)ꢀ3m with E. coli (SOI) cells (1
g cdw L−1) in KP buffer (200 mM, pH 8) and nꢀC16H34 (1:1) at 30 °C. b) Asymmetric formal
hydroamination of 5 mM 1m to (S)ꢀ4m with E. coli (StyABCꢀVfTADMꢀAlaDH) cells (20 g cdw
L−1) in KP buffer (200 mM, pH 8, 2 % glucose, 200 mM NH3/NH4Cl) and nꢀC16H34 (2:1) at 30
°C.
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TABLES.
Table 1. AntiꢀMarkovnikov hydroamination of styrenes with E. coli (StyABCꢀCvTAꢀAlaDH).a
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Sub.
X
Conv. to 4 (%) b Chemoselectivity Regioselectivity
b
4 : 5 b
96 : 4
99 : 1
99 : 1
98 : 2
98 : 2
98 : 2
97 : 3
98 : 2
97 : 3
98 : 2
99 : 1
96 : 4
2ꢀNH2 : 1ꢀNH2
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
H
98
1a
1b
1c
1d
1e
1f
oꢀF
94
mꢀF
> 99
96
pꢀF
mꢀCl
pꢀCl
mꢀBr
pꢀBr
mꢀMe
pꢀMe
mꢀOMe
pꢀOMe
86
76
45
1g
1h
1i
60
93
99
1j
> 99
94
1k
1l
a
20 mM 1 was used under the same reaction condition of Figure 1c for 10 h, except 24 h for
1eꢀ1h. b Determined by HPLC and GC analysis.
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Table 2. AntiꢀMarkovnikov hydration of styrenes with E. coli (StyABCꢀPAR).a
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Sub.
X
Conv. to 5 (%) b
Regioselectivity
2ꢀOH : 1ꢀOH b
> 99 : 1
H
> 99
90
1a
1b
1c
1d
1e
1f
oꢀF
> 99 : 1
mꢀF
94
> 99 : 1
pꢀF
98
> 99 : 1
mꢀCl
pꢀCl
mꢀBr
pꢀBr
mꢀMe
pꢀMe
mꢀOMe
pꢀOMe
89
> 99 : 1
78
> 99 : 1
83
> 99 : 1
1g
1h
1i
60
> 99 : 1
99
> 99 : 1
99
> 99 : 1
1j
> 99
94
> 99 : 1
1k
1l
> 99 : 1
a
b
20 mM 1 was used under the same reaction condition of Figure 1d for 8 h. Determined by
HPLC and GC analysis.
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Table 3. Asymmetric antiꢀMarkovnikov hydroamination and hydration of 1m.a
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Entry Catalyst
Product
Conv. to prod. Regioselecꢀ ee (%)
(%) b
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8
tivity b
> 99 : 1
> 99 : 1
> 99 : 1
> 99 : 1
99 : 1
c
1
2
3
4
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6
E. coli (StyABCꢀCvTAꢀAlaDH)
(S)ꢀ4m
(S)ꢀ4m
(S)ꢀ4m
51
95
70
92
87
97
E. coli (StyABCꢀHnTA)
E. coli (StyABCꢀVfTAꢀAlaDH)
81
84
85
81
E. coli (StyABCꢀVfTADMꢀAlaDH) (S)ꢀ4m
E. coli (StyABCꢀPAR)
(S)ꢀ5m
(S)ꢀ5m
E. coli (StyABCꢀHLADH)
> 99 : 1
a
5ꢀ10 mM 1m was used for biotransformation with E. coli cells for 24 h (see experimental
b
c
section for details). Determined by HPLC and GC analysis. Determined by chiral HPLC
analysis.
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ASSOCIATED CONTENT
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Supporting Information.
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The following files are available free of charge.
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Supporting tables, supporting figures, additional experimental section, GC chromatograms,
HPLC chromatograms, and NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: chelz@nus.edu.sg. Phone: +65ꢀ65168416.
ORCID
Shuke Wu: 0000ꢀ0003ꢀ0914ꢀ9277
Zhi Li: 0000ꢀ0001ꢀ7370ꢀ2562
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was financially supported by GlaxoSmithKline (GSK) and Singapore Economic
Development Board (EDB) through Green and Sustainable Manufacturing grants (Project No.
279ꢀ000ꢀ331ꢀ592 and 279ꢀ000ꢀ348ꢀ592) and Ministry of Education of Singapore through an
AcRF Tier 1 Grant (Project No. Rꢀ279ꢀ000ꢀ477ꢀ112). We thank Prof. Kristala L. J. Prather
(MIT) for providing the E. coli RARE strain. We also thank Prof. Sven Panke (ETH Zurich), Dr.
Joseph P. Adams (GSK), and Dr. Radka Snajdrova (GSK) for helpful discussion.
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