Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades

Article abstract of DOI:10.1126/science.aac9283

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product.

Full text of DOI:10.1126/science.aac9283

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Science, Trieste, Italy, 2015); http://pos.sissa.it/archive/
conferences/215/037/AASKA14_037.pdf.
7. X. Siemens, J. Ellis, F. Jenet, J. D. Romano, Class. Quantum
Gravity 30, 224015 (2013).
8. S. W. Hawking, Astrophys. J. 145, 544 (1966).
9. P. B. Demorest et al., Astrophys. J. 762, 94
The Parkes radio telescope is part of the Australia Telescope
National Facility, which is funded by the Commonwealth of
Australia for operation as a National Facility managed by CSIRO.
The PPTA project was initiated with support from R.N.M .’ s
Australian Research Council (ARC) Federation Fellowship
(grant FF0348478) and from CSIRO under that fellowship program.
The PPTA project has also received support from ARC through
Discovery Project grants DP0985272 and DP140102578. N.D.R.B.
acknowledges support from a Curtin University research
fellowship. G.H. and Y.L. are recipients of ARC Future Fellowships
(respectively, grants FT120100595 and FT110100384). S.O. is
supported by the Alexander von Humboldt Foundation. R.M.S.
acknowledges travel support from CSIRO through a John Philip
Award for excellence in early-career research. The authors declare
no conflicts of interest. Data used in this analysis can be accessed
via the Australian National Data Service (www.ands.org.au).
2
2
2
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/349/6255/1522/suppl/DC1
Supplementary Text
Figs. S1 to S2
Tables S1 to S8
References (30–54)
(2013).
ACKNOWLEDGMENTS
We thank the observers, engineers, and Parkes Observatory staff
members who have assisted with the observations reported
in this paper. We thank R. van Haasteren for assistance with the
use of the code piccard, E. Thomas for comments on the
manuscript, and I. Mandel for discussions on model selection.
26 March 2015; accepted 12 August 2015
10.1126/science.aab1910
vents for extraction and purification as well as
energy for evaporation and mass transfer (8). As
a consequence, cascade reactions generally pos-
sess elevated atom efficiency and potentially low-
er environmental impact factors (9). The major
challenge is to perform cascade reactions where-
in an oxidative and a reductive step are running
simultaneously. Even more challenging is to car-
ry out a simultaneous interconnected redox-neutral
cascade wherein the electrons liberated in the first
oxidative step are quantitatively consumed in the
subsequent reductive step [(8); for a recent de-
tailed study, see (10)]. This concept is the basis
for the hydrogen-borrowing conversion of al-
cohols (primary or secondary) into amines.
The reducing equivalents (i.e., hydride) liberated in
the first step—the oxidation of the alcohol to the
ketone—are directly consumed in the second
interconnected step—reductive amination of the
ketone.
A number of chemocatalytic hydrogen-borrowing
methods have recently been developed using
ruthenium as well as iridium catalysts (11, 12).
However, the required reaction conditions (e.g.,
high catalyst and cocatalyst loading, low sub-
strate concentration, moderate chemoselectivity,
moderate or total lack of stereoselectivity, the re-
quirement of an excess of substrate, and stringent
temperature and elevated pressure requirements)
complicate the application of these methods on
a large scale (13). Another recently developed
hydrogen-borrowing chemical method involves
the stoichiometric use of Ellman’s enantiopure
sulfinamide auxiliary as the nitrogen donor in
combination with a Ru-Macho catalyst (14). Be-
sides the requirement of the expensive chiral auxi-
liary, the maximum diastereomeric excess was
90%. A reported biocatalytic hydrogen-borrowing
amination of alcohols combining three enzymes—
aw-transaminase (wTA), an alcohol dehydrogenase
(ADH), and the alanine dehydrogenase from
Bacillus subtilis (AlaDH)—also lacks efficiency
because of the requirement for at least 5 equiv-
alents of L- or D-alanine as the sacrificial amine
donor and also as a result of the lower conversion
and chemoselectivity for the amination of secondary
alcohols (15, 16). Another redox-neutral biocatalytic
cascade was applied for the deracemization of man-
delic acid to enantioenriched L-phenylglycine. How-
ever, the method was limited to the conversion of
this specific a-hydroxy acid (17).
BIOCATALYSIS
Conversion of alcohols to enantiopure
amines through dual-enzyme
hydrogen-borrowing cascades
1,2
2
2
Francesco G. Mutti, *† Tanja Knaus, † Nigel S. Scrutton,
3
1
Michael Breuer, Nicholas J. Turner *
a-Chiral amines are key intermediates for the synthesis of a plethora of chemical
compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of
primary and secondary alcohols that allows for the efficient and environmentally benign
production of enantiopure amines. The method relies on a combination of two enzymes: an
alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating
in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a
structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96%
conversion and 99% enantiomeric excess. Primary alcohols were aminated with high
conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency,
sourcing nitrogen from ammonium and generating water as the sole by-product.
mines are among the most frequently used
chemical intermediates for the production
of active pharmaceutical ingredients, fine
chemicals, agrochemicals, polymers, dyestuffs,
pigments, emulsifiers, and plasticizing agents
ing the past decade. The intrinsic advantage of the
direct amination of an alcohol is that the reagent
and the product are in the same oxidation state;
therefore, theoretically, additional redox equiva-
lents are not required. However, many of these
methods have low efficiency and high environ-
mental impact (e.g., Mitsunobu reaction) (3). The
amination of simple alcohols such as methanol
and ethanol via heterogeneous catalysis requires
harsh conditions (>200°C), and more structurally
diverse alcohols are either converted with extreme-
ly low chemoselectivity or not converted at all (4).
Furthermore, most of the work in this field involves
nonchiral substrates, whereas 40% of the commer-
cial optically active drugs are chiral amines (2).
Increasingly, biocatalytic methods are applied for
the production of optically active amines [e.g., the
lipase-catalyzed resolution of racemic mixtures of
amines or the w-transaminase process; a recent
example uses an engineered enzyme applied to
the industrial manufacture of the diabetes med-
ication Januvia (sitagliptin)] (5–7).
A
(
1). However, the requisite amines are scarce in
nature, and their industrial production mainly
relies on the metal-catalyzed hydrogenation of
enamides (i.e., obtained from related ketone pre-
cursors). This process requires transition metal
complexes, which are expensive and increasingly
unsustainable (2). Moreover, the asymmetric syn-
thesis of amines from ketone precursors requires
protection and deprotection steps that generate
copious amounts of waste. As a consequence, var-
ious chemical processes for the direct conversion
of alcohols into amines have been developed dur-
1
School of Chemistry, University of Manchester, Manchester
Institute of Biotechnology, Manchester M1 7DN, UK.
2
Manchester Institute of Biotechnology, Faculty of Life
Sciences, University of Manchester, Manchester M1 7DN, UK.
3
Multistep chemical reactions in one pot avoid
the need for isolation of intermediates and puri-
fication steps. This approach offers economic as
well as environmental benefits, because it elim-
inates the need for time-consuming intermediate
work-ups and minimizes the use of organic sol-
BASF SE, White Biotechnology Research, GBW/B-A030,
67056 Ludwigshafen, Germany.
*Corresponding author. E-mail: nicholas.turner@manchester.
ac.uk (N.J.T.); f.mutti@uva.nl (F.G.M.) †Present address:
Van ’t Hoff Institute for Molecular Sciences, University of
Amsterdam, Science Park 904, 1098 XH Amsterdam,
Netherlands.
Here, we present a highly enantioselective cat-
alytic hydrogen-borrowing amination of primary
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RESEARCH
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as well as secondary alcohols that requires only
two biocatalysts, namely an ADH and an amine
dehydrogenase (AmDH) (Fig. 1). The redox self-
sufficient cycle uses ammonium ion/ammonia as
the source of the nitrogen and generates only wa-
ter as the by-product. The cascade requires only
catalytic quantities of a nicotinamide coenzyme
that shuttles hydride from the oxidative step to
the reductive step. The method has been success-
fully applied to amination of optically active sec-
ondary alcohols with inversion of configuration,
amination of the corresponding enantiomeric
secondary alcohols with retention of configura-
tion, asymmetric amination of racemic secondary
alcohols, and amination of primary alcohols.
Initially, we examined the catalytic activity of
the amine dehydrogenase variant that was re-
cently obtained by protein engineering of the
wild-type phenylalanine dehydrogenase from
B. badius (Ph-AmDH) (18, 19). The substrate scope
of the Ph-AmDH variant K78S-N277L for the con-
version of a broad range of ketone substrates has
not been reported; only the reductive amination of
para-fluorophenylacetone (2b) was previously de-
scribed, using glucose and glucose dehydrogenase
very similar amino acid arrangement, but in an
inverted conformation. In particular, a tyrosine res-
ence of buffer systems ranging from pH 7 to 8.7.
Although formation of the amine product (R)-3a
was observed, the maximum conversion was 6%.
In particular, accumulation of the ketone interme-
diate 2a was observed (from 61% to 97%; table
S2). However, under these conditions, the concen-
tration of ketone 2a cannot exceed the concentra-
93
189
idue (Tyr for AA-ADH, Tyr for LBv-ADH) is
crucial for the stereoselectivity, as it protrudes into
the active site and forces the substrates to bind
with the larger group in the opposite direction.
This Tyr residue is in a mirror-image position in
the active site of the two ADHs (22, 25).
+
tion of the cofactor NAD (1 mM), and hence the
The amination of alcohol substrate (R)-1a
(20 mM) was carried out initially by combining a
crude cell extract of LBv-ADH with purified His-
accumulation of high levels of 2a was attributed to
the presence of at least one NAD oxidase from the
host organism (Escherichia coli) used for the ex-
pression of the ADH, as previously observed by
other groups (26). The NADH oxidase competed
+
tagged Ph-AmDH in the presence of catalytic NAD
[1 mM; 5 mole percent (mol %)] and in the pres-
(GDH) for cofactor regeneration, and very recently
three other ketones were also tested (20). Hence,
the Ph-AmDH variant K78S-N277L was expressed
and purified as histidine (His)–tagged protein. The
activity of the enzyme was initially studied using
2b as the test substrate in ammonium buffer sys-
tems with a range of different counterions (chloride,
sulfate, acetate, phosphate, borate, citrate, oxalate,
and formate). The pH was also varied from 4 up
to 11.5, depending on the ammonium buffer used
(fig. S5). The highest catalytic activity was observed
at pH 8.2 to 8.8, whereas the optimal buffer was
ammonium chloride. In contrast, previous stud-
ies with this enzyme were carried out at pH 9.6
(
18). Reductive amination of 2b (20 mM) was
carried out at varying concentrations of NH Cl/
NH buffer at pH 8.7 using GDH and glucose for
cofactor regeneration. Quantitative conversion
>99%) was achieved after 12 hours using ~0.7 M
4
3
Fig. 1.Two-enzyme cascade for the hydrogen-borrowing amination of alcohols. In the first oxidative step, the
Prelog AA-ADH and the anti-Prelog LBv-ADH were applied for the oxidation of the (S)- and (R)-configured alcohol
substrates, respectively. The AmDHs used in this study afforded the (R)-configured amines in the second re-
ductive step. Structures of the alcohol substrates explored in this study are shown below the schematic
catalytic cycle. Me, methyl; Et, ethyl. See (27) for detailed structural representations of the alcohol substrates.
(
ammonium buffer (table S1 and fig. S6).
ADHs have been extensively used in biocatal-
ysis for the interconversion of ketones and alco-
hols, and hence a wealth of data is available for
these enzymes (21). Because of the dependence of
Ph-AmDH on nicotinamide adenine dinucleotide
Fig. 2. Kinetics of
asymmetric hydrogen-
borrowing biocatalytic
amination. The
(oxidized form; NAD) as cofactor, we searched for
suitable stereocomplementary NAD-dependent
secondary ADHs that might exhibit high stability
and activity toward a wide range of secondary
alcohols at pH >8.5 as well as tolerance of high
concentrations of ammonium ions. The NAD-
dependent Prelog ADH from Aromatoleum
aromaticum (AA-ADH, previously named as de-
nitrifying bacterium strain EbN1; PDB 2EW8 and
reaction of (S)-1a
(20 mM) gives inverted
(R)-3a using AA-ADH
and Ph-AmDH with
+
catalytic NAD (1 mM;
5 mol %). Concentrations
2EWM) (22) and an engineered anti-Prelog ADH
of the amine product
from Lactobacillus brevis (LBv-ADH; PDB 1ZK4 for
the wild-type enzyme) were selected for this
study (23–25). Analysis of the crystal structures
of the ADHs with bound NAD(P)H (reduced NAD
phosphate) as well as previous docking studies
(solid line, black circles),
ketone intermediate
(dotted line, black
squares), and alcohol
substrate (dashed line,
(
i.e., substrate bound to the enzyme) revealed that
white circles) were monitored over time. As expected, the concentration of the ketone intermediate 2a
remained constant and below the concentration of the nicotinamide coenzyme. See (27) for details.
the active sites of AA-ADH and LBv-ADH possess a
1
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| REPORTS
with the AmDH in the amination step for the
oxidation of the NADH, leading to accumulation
of 2a (27) (fig. S3). Therefore, the LBv-ADH was
purified by ion exchange chromatography and
size exclusion chromatography and was then re-
combined with the purified His-tagged Ph-AmDH
for the alcohol amination reaction (1a concentra-
the ADHs and the His-tagged Ph-AmDH were
separately incubated in the same buffer under
the same conditions. AA-ADH and LBv-ADH
belong to the family of the short-chain dehy-
drogenases/reductases (SDRs). Both ADHs are
homotetramers and possess the characteristic
Ser-Tyr-Lys catalytic triad of the SDRs. Addition-
tag of the Ph-AmDH. As a consequence, enzyme
aggregation and precipitation occurred. We then
used a highly selective recombinant thrombin to
cleave the His tag from the Ph-AmDH (27) (fig. S7).
Incubation of the two purified ADHs with the Ph-
AmDH devoid of a His tag resulted in visually more
stable systems wherein enzyme aggregation and
precipitation were not observed even after 24 hours.
The hydrogen-borrowing cascade was then re-
peated by combining AA-ADH with the Ph-AmDH
(devoid of His tag) in ammonium chloride buffer
at pH 8.7. AA-ADH is selective for (S)-1a, whereas
Ph-AmDH shows (R)-selectivity in the reduction
of the intermediate 2a; hence, the overall cas-
cade was expected to proceed with inversion of
configuration. The reaction was tested at various
concentrations of ammonium ions to ascertain
the impact on the conversion. Under reaction con-
+
2+
tion 20 mM, NAD 5 mol %). Under these condi-
ally, the LBv-ADH has two Mg sites that are
tions, the concentration of the ketone 2a at the end
of the reaction was between 1.6 and 2.9% (less than
the maximum theoretical value of 5%). Unfortunate-
ly, the final concentration of the amine product
placed at the interphase between the monomeric
units and are crucial for its stability (25). Although
the crystal structure of AA-ADH was reported
without an evident metal ion, a high homolog
was crystallized in a stable form with six addi-
tional divalent cations (28). Furthermore, we noticed
that the stability of LBv-ADH was significantly
3a was also very low (<1%) (table S3). Replacing
LBv-ADH with AA-ADH for the amination of (S)-1a
led to the same results.
2+
During the course of these preliminary experi-
ments, we noticed that solutions containing the
ADH (LBv-ADH or AA-ADH) together with the
His-tagged Ph-AmDH tended to become cloudy
after a few minutes of the reaction, generating an
enzyme precipitate. However, no precipitation
occurred even after more than 24 hours when
improved during purification when Mg was
added into the buffer, indicating a reversible dis-
sociation process of the cation from the enzyme
(27). Therefore, we speculated that enzyme pre-
cipitation in the dual-enzyme cascade might have
been caused by coordination of free divalent cat-
ions, coming from the ADHs, to the His-terminal
+
ditions of (S)-1a = 20 mM, NAD = 1 mM, and
+
4
NH
/NH
3
= 2 M, the conversion of alcohol to
amine reached 85% after 24 hours, with an en-
antiomeric excess (ee) of >99% (R) (table S4).
Table 1. Asymmetric hydrogen-borrowing amination of enantiopure aromatic secondary alcohols 1a to 1n. Reactions were carried out at 30°C for
48 hours. See (27) for experimental details.
Amination of aromatic chiral secondary
alcohols 1a to 1n with inversion of
configuration
Amination of aromatic chiral secondary
alcohols 1a to 1n with retention of
configuration
Asymmetric amination of aromatic
racemic secondary alcohols 1a to 1n
Entry
Substrate
Conversion
%)
ee
Entry
Substrate
Conversion
(%)
ee
Entry
Substrate
Conversion
(%)
ee
(
(%)
(%)
(%)
1
(S)-1a
95
>99(R)
15
(R)-1a
93
>99(R)
29
Rac-1a
81
>99(R)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
...........................................................................................................................................................................................................................................................................................................................................
2
(S)-1b
93
>99(R)
16
(R)-1b
36
>99(R)
30
Rac-1b
66
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
3
(S)-1c
55
97(R)
17
(R)-1c
27
97(R)
31
Rac-1c
47
97(R)
...........................................................................................................................................................................................................................................................................................................................................
4
(S)-1d
65
>99(R)
18
(R)-1d
24
>99(R)
32
Rac-1d
78
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
5
(S)-1e
31
82(R)
19
(R)-1e
14
82(R)
33
Rac-1e
30
82(R)
...........................................................................................................................................................................................................................................................................................................................................
6
(S)-1f
80
>99(R)
20
(R)-1f
85
>99(R)
34
Rac-1f
87
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
7
(S)-1g
92
82(R)
21
(R)-1g
92
83(R)
35
Rac-1g
92
83(R)
...........................................................................................................................................................................................................................................................................................................................................
8
(S)-1h
96
>99(R)
22
(R)-1h
94
>99(R)
36
Rac-1h
84
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
9
(S)-1i
17
>99(R)
23
(R)-1i
30
>99(R)
37
Rac-1i
16
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
10
(S)-1j
14
>99(R)
24
(R)-1j
17
>99(R)
38
Rac-1j
16
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
11
(S)-1k
26
>99(R)
25
(R)-1k
33
>99(R)
39
Rac-1k
20
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
12
(S)-1l
12
>99(R)
26
(R)-1l
18
>99(R)
40
Rac-1l
12
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
13
(S)-1m
14
>99(R)
27
(R)-1m
27
>99(R)
41
Rac-1m
19
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
14
(S)-1n
7
>99(R)
28
(R)-1n
14
>99(R)
42
Rac-1n
9
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
Table 2. Asymmetric hydrogen-borrowing amination of aliphatic secondary alcohols 1o to 1s. Reactions were carried out at 30°C for 48 hours. See
(
27) for experimental details.
Amination of aliphatic chiral secondary
alcohols with inversion of configuration
Amination of aliphatic chiral secondary
alcohols with retention of configuration
Asymmetric amination of aliphatic
racemic secondary alcohols
Conversion
%)
ee
Conversion
(%)
ee
Conversion
(%)
ee
Entry
Substrate
Entry
Substrate
Entry
Substrate
(
(%)
(%)
(%)
1
(S)-1o
94
99(R)
6
(R)-1o
91
>99(R)
11
Rac-1o
93
99(R)
.
.
.
.
.
...........................................................................................................................................................................................................................................................................................................................................
2
(S)-1p
95
99(R)
7
(R)-1p
79
99(R)
12
Rac-1p
96
99(R)
...........................................................................................................................................................................................................................................................................................................................................
3
(S)-1q
95
>99(R)
8
(R)-1q
83
>99(R)
13
Rac-1q
95
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
4
(S)-1r
74
>99(R)
9
(R)-1r
73
>99(R)
14
Rac-1r
66
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
5
(S)-1s
88
99(R)
10
(R)-1s
80
99(R)
15
Rac-1s
80
>99(R)
...........................................................................................................................................................................................................................................................................................................................................
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Monitoring the progress of the reaction revealed
a maximum conversion in excess of 93% after
amino acid sequence of a stable chimeric AmDH
(Ch1-AmDH) was recently published, although
its substrate scope and stereoselectivity have not
been elucidated (30). Thus, the Ch1-AmDH devoid
of His tag was combined with the previously se-
lected ADHs for the amination of a much broader
panel of alcohols 1f to 1s. Aromatic substrates 1f
to 1h bearing the phenyl ring in the a position
(Table 1, entries 6, 20, and 34) and b position
(Table 1, entries 7, 8, 21, 22, 35, and 36), relative to
the secondary alcohol, as well as phenylethanol
derivatives 1i to 1n with substituents in the ortho-,
meta-, and para- positions (Table 1, entries 9 to 14,
23 to 28, and 37 to 42), were aminated with 99%
ee (R) and conversions ranging from moderate to
high. The only exception was alcohol 1g, which
was aminated with lower enantioselectivity (82
or 83% ee; Table 1, entries 7, 21, and 35). For this
particular substrate, the progress of ee was moni-
tored as a function of time (table S14 and fig. S12).
The enantiomeric excess of amine 3g remained
constant, demonstrating that longer incubation
times are not detrimental to the stereoselective
outcome of the process. All the aliphatic secondary
alcohols 1o to 1s examined (medium, long, and
branched chain) were aminated with perfect ee
and high conversions up to 96% (Table 2).
The hydrogen-borrowing amination is an ex-
tremely efficient and valuable method for the gen-
eration of optically active amines from alcohols.
However, achiral terminal primary amines are also
in high demand by the chemical industry, espe-
cially for the production of polymers and plas-
ticizing agents (1). To demonstrate the broad
applicability of the methodology, the amination
of different primary alcohols was accomplished
by combining the primary hT-ADH from Bacillus
stearothermophilus (31) with either the Ch1-AmDH
(Table 3, entries 1 to 6) or the Ph-AmDH (Table 3,
entry 7). Quantitative conversion to the amine pro-
duct was obtained with alcohols 1u to 1x.
system uses ammonia as the simplest amine donor
and generates water as the sole innocuous by-
product. Ongoing studies are currently aimed
at extending the substrate scope of the cascade
through further protein engineering of AmDHs
capable of aminating a wide range of more
complex alcohols with elevated stereoselectivity.
Although only enantiopure (R)-configured amines
have been generated to date, the engineering of
stereocomplementary AmDHs (S-selective) starting
from D–amino acid dehydrogenases as scaffolds
will complement the scope of our hydrogen-
borrowing process. Finally, the use of lower
concentrations of ammonia may be possible by
the addition of further enzymes to derivatize the
amine products and hence provide a thermody-
namic driving force for the amination step.
3
days (Fig. 2 and table S5). Increasing the concen-
tration of ammonia up to 4 M led to a slight in-
crease in conversion (95%; Table 1, entry 1, and
tables S6 to S8). Addition of further aliquots of
+
AA-ADH, Ph-AmDH, and NAD after 2 days gave
no further increase in conversion, indicating that
the thermodynamic equilibrium had been reached.
For improved catalytic efficiency of the cascade, the
+
concentration of NAD was reduced by a factor of
5, to 0.2 mM (1 mol %), which resulted in a slight
drop in conversion to 76% (table S5 and figs. S9
and S10).
Surprisingly, when the same reaction con-
ditions were applied to the amination of (R)-1a
(
20 mM) using LBv-ADH with the Ph-AmDH (mi-
nus His tag), the conversion to amine was <4%
table S9). We speculated that the instability of
(
REFERENCES AND NOTES
the LBv-ADH in ammonium chloride buffer at
pH 8.7 might be the origin of the low conversion,
so we investigated lower pH values. For ammo-
nium chloride buffers, pH values of <8.5 cannot
be attained; hence, ammonium formate buffer
was investigated at various pH values (29). At
pH 8 to 8.5, the amination of (R)-1a (20 mM)
was achieved in 93% conversion and >99% ee
1
.
H. A. Wittcoff, B. G. Rueben, J. S. Plotkin, Industrial Organic
Chemicals (Wiley- Interscience, New York, ed. 2, 2004).
T. C. Nugent, Ed., Chiral Amine Synthesis (Wiley-VCH,
Weinheim, Germany, 2010).
2
.
.
3
E. Fabiano, B. T. Golding, M. M. Sadeghi, Synthesis 1987,
190–191 (1987).
4.
H.-J. Arpe, Industrial Organic Chemistry (Wiley-VCH, Weinheim,
Germany, ed. 5, 2010).
5. F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, J. Prakt. Chem.
39, 381–384 (1997).
3
(Table 1, entry 15). The cascade was then run by
6.
D. I. Sterling, in Chirality in Industry, A. N. Collins,
G. N. Sheldrake, J. Crosby, Eds. (Wiley, Chichester, UK, 1992),
pp. 209–222.
combining both of the stereocomplementary
ADHs with the Ph-AmDH in one pot for the
asymmetric amination of racemic 1a, affording
7. C. K. Savile et al., Science 329, 305–309 (2010).
8.
J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti, W. Kroutil,
Curr. Opin. Chem. Biol. 15, 249–256 (2011).
(R)-3a in >99% ee and 81% conversion (Table 1,
entry 29).
9.
R. A. Sheldon, I. W. C. E. Arends, U. Hanefeld, Green Chemistry
and Catalysis (Wiley-VCH, Weinheim, Germany, 2007).
The hydrogen-borrowing cascade was initially
tested on a limited number of 1-phenyl-2-propanol
derivatives 1a to 1e (Table 1) for amination with
inversion of configuration (entries 1 to 5), retention
of configuration (entries 15 to 19), and asymmetric
amination of racemic alcohols (entries 29 to 33).
Conversion varied from moderate to excellent,
whereas the ee was excellent in almost all cases.
Whereas ADHs generally possess broad sub-
strate specificity, the Ph-AmDH accepts solely
phenylacetone and phenylacetaldehyde deriva-
tives with elevated turnover numbers. Nonethe-
less, the generation of chimeric enzymes through
domain shuffling from different parents can rap-
idly lead to new enzymes with increased activity
or different and extended substrate specificity. The
10. T. Knaus, F. G. Mutti, L. D. Humphreys, N. J. Turner,
N. S. Scrutton, Org. Biomol. Chem. 13, 223–233
(
2015).
1. S. Imm et al., Angew. Chem. Int. Ed. 50, 7599–7603
2011).
1
(
12. Y. Zhang, C.-S. Lim, D. S. B. Sim, H.-J. Pan, Y. Zhao, Angew.
Chem. Int. Ed. 126, 1423–1427 (2014).
1
3. The amination with the Ru catalyst (7) requires 3 mol % of the
metal catalyst, 150°C, ammonia gas under pressure and inert
atmosphere; racemic amines are obtained. The amination with
Ir catalyst (8) requires 5 mol % of the metal catalyst
coordinated to an expensive chiral ligand, 10 mol % of
binaphthalene phosphoric acid as cocatalyst; para-anisidine is
the nitrogen source; at least 33% of the alcohol starting
material is wasted.
To demonstrate the practical application of
the methodology, we carried out bioaminations
of five representative substrates—one for each
structural category reported in Fig. 1—at a pre-
parative scale. Starting from (S)-1a as the alcohol
substrate, the conversion into the amine product
(R)-3a reached 93% after 48 hours. The work-up
consisted of extraction of the unreacted alcohol
and ketone intermediate under acidic conditions,
followed by the extraction of the amine product
under basic conditions (27). The isolated yield of
pure (R)-3a was 85% (99% ee). Following the
same protocol, substrates (S)-1g, (S)-1i, (S)-1q, and
14. N. J. Oldenhuis, V. M. Dong, Z. Guan, J. Am. Chem. Soc. 136,
2548–12551 (2014).
5. J. H. Sattler et al., Angew. Chem. Int. Ed. 51, 9156–9159
2012).
1
1
1
(
6. When using (R)-selective w-TAs, D-alanine must be used.
D-alanine has a high cost and, moreover, a D-alanine
dehydrogenase is not known. Only L-alanine would be produced
during the reaction. Hence, in the case of (R)-selective w-TAs, the
system works via removal rather than recycling of the pyruvate.
7. V. Resch, W. M. F. Fabian, W. Kroutil, Adv. Synth. Catal. 352,
993–997 (2010).
Table 3. Hydrogen-borrowing amination of
primary alcohols 1t to 1z. Reactions were
carried out at 30°C for 48 hours. See (27) for
experimental details.
1
1
1u were converted to the corresponding amines
8. M. J. Abrahamson, J. W. Wong, A. S. Bommarius, Adv. Synth.
Catal. 355, 1780–1786 (2013).
with 89%, 31%, 95%, and >99% conversion, re-
spectively. The isolated yields of pure (R)-3g, (R)-3i,
(R)-3q, and 3u were 78%, 30%, 91%, and 91%, re-
spectively. The enantiomeric excesses remained the
same as for the experiments on an analytical scale.
Our dual-enzyme hydrogen-borrowing process
enables the asymmetric amination of a broad
range of secondary alcohols to afford the cor-
responding (R)-configured amines in high en-
antiomeric excess. Furthermore, in the majority
of the cases, amination of primary alcohols pro-
ceeded in quantitative conversion. The biocatalytic
Conversion
19. M. J. Abrahamson, E. Vázquez-Figueroa, N. B. Woodall,
J. C. Moore, A. S. Bommarius, Angew. Chem. Int. Ed. 51,
3969–3972 (2012).
Entry
Substrate
1t
(%)
1
8
20. S. K. Au, B. R. Bommarius, A. S. Bommarius, ACS Catal. 4,
.
.
.
.
.
.
.
............................................................................................
4021–4026 (2014).
2
1u
99
............................................................................................
21. M. M. Musa, R. S. Phillips, Catal. Sci. Technol. 1, 1311–1323
(2011).
22. H. W. Höffken et al., Biochemistry 45, 82–93 (2006).
3
1v
99
............................................................................................
4
1w
99
............................................................................................
2
3. The wild-type Lactobacillus brevis ADH is a NADP-dependent
ADH. This enzyme was previously engineered to accept NAD as
cofactor, and this variant was applied in this study.
5
1x
99
............................................................................................
6
1y
61
............................................................................................
7
1z
10
24. W. Hummel, B. Riebel, PCT Int. Appl. WO 9947684
(1999).
............................................................................................
1
528 25 SEPTEMBER 2015 • VOL 349 ISSUE 6255
sciencemag.org SCIENCE

RESEARCH
| REPORTS
2
5. N. H. Schlieben et al., J. Mol. Biol. 349, 801–813
32. K. Niefind, J. Müller, B. Riebel, W. Hummel, D. Schomburg,
J. Mol. Biol. 327, 317–328 (2003).
wrote the manuscript; F.G.M. planned the experiments, expressed
and purified the AmDHs, performed the biocatalytic reactions, and
analyzed the data; T.K. performed the gene cloning of all AmDHs
and purified the ADHs; N.S.S. and M.B. provided intellectual and
technical support; and M.B. and BASF provided the ADHs. We
thank R. Heath for a preliminary kinetic assay of the Ph-AmDH.
(2005).
2
2
2
6. K. Tauber et al., Chemistry 19, 4030–4035 (2013).
7. See supplementary materials on Science Online.
8. A. C. Price, Y.-M. Zhang, C. O. Rock, S. W. White, Structure 12,
ACKNOWLEDGMENTS
The research leading to these results received funding from the
European Union’s Seventh Framework Programme FP7/2007-2013
under grant agreement 266025 (BIONEXGEN). The project leading
to this application has received funding from the European
Research Council under the European Union’s Horizon 2020
research and innovation program (grant agreement no. 638271,
BioSusAmin). T.K. and N.S.S. received funding from UK
Biotechnology and Biological Sciences Research Council (BBSRC;
BB/K0017802/1). N.J.T. is grateful to the Royal Society for a
Wolfson Research Merit Award. Some aspects of the results
reported here are the subject of a provisional patent application.
Author contributions: F.G.M. and N.J.T. conceived the project and
417–428 (2004).
2
9. The Ph-AmDH tolerates different buffers well (i.e., ammonium
chloride, formate, borate, citrate, acetate, oxalate). However, the
2+
SUPPLEMENTARY MATERIALS
homotetrameric LBv-ADHs contains two Mg centers; therefore,
strong chelators such as oxalate, citrate, and acetate must be
www.sciencemag.org/content/349/6255/1525/suppl/DC1
Materials and Methods
Figs. S1 to S12
Tables S1 to S20
References (33–36)
2+
avoided as buffer salts. Removal of these Mg ions inactivates the
enzyme (32).
3
0. B. R. Bommarius, M. Schürmann, A. S. Bommarius, Chem.
Commun. 50, 14953–14955 (2014).
1. R. Cannio, M. Rossi, S. Bartolucci, Eur. J. Biochem. 222,
3
30 June 2015; accepted 14 August 2015
10.1126/science.aac9283
345–352 (1994).
energy components of the system by storing the
electro-active species outside the battery container
itself (3–5). In a flow battery, the power is gen-
erated in a device resembling a fuel cell, which
contains electrodes separated by an ion-permeable
membrane. Liquid solutions of redox-active spe-
cies are pumped into the cell, where they can be
charged and discharged, before being returned to
storage in an external storage tank. Scaling the
amount of energy to be stored thus involves sim-
ply making larger tanks (Fig. 1A). Existing flow
batteries are based on metal ions in acidic solu-
tion, but challenges with corrosivity, hydrogen evo-
lution, kinetics, materials cost and abundance, and
efficiency thus far have prevented large-scale com-
mercialization. The use of anthraquinones in an
acidic aqueous flow battery can dramatically
BATTERIES
Alkaline quinone flow battery
1
2
2
1
1
Kaixiang Lin, Qing Chen, Michael R. Gerhardt, Liuchuan Tong, Sang Bok Kim,
3
3
1
1,2
Louise Eisenach, Alvaro W. Valle, David Hardee, Roy G. Gordon,
Michael J. Aziz, * Michael P. Marshak
*
2
1,2
*
Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem
between the intermittent supply of these renewable resources and variable demand. Flow
batteries permit more economical long-duration discharge than solid-electrode batteries by
using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based
on redox-active organic molecules that are composed entirely of Earth-abundant elements
and are nontoxic, nonflammable, and safe for use in residential and commercial environments.
The battery operates efficiently with high power density near room temperature. These results
demonstrate the stability and performance of redox-active organic molecules in alkaline
flow batteries, potentially enabling cost-effective stationary storage of renewable energy.
1
Department of Chemistry and Chemical Biology, Harvard
University, 12 Oxford Street, Cambridge, MA 02138, USA.
2
he cost of photovoltaic (PV) and wind elec-
tricity has dropped so much that one of the
largest barriers to getting most of our elec-
tricity from these renewable sources is their
intermittency (1–3). Batteries provide a means
to store electrical energy; however, traditional, en-
closed batteries maintain discharge at peak power
for far too short a duration to adequately regulate
wind or solar power output (1, 2). In contrast, flow
batteries can independently scale the power and
Harvard John A. Paulson School of Engineering and Applied
Sciences, 29 Oxford Street, Cambridge, MA 02138, USA.
3
Harvard College, Cambridge, MA 02138, USA.
Corresponding author. E-mail: aziz@seas.harvard.edu (M.J.A.);
*
gordon@chemistry.harvard.edu (R.G.G.); michael.marshak@colorado.
edu (M.P.M.)
T
Fig. 1. Cyclic voltammetry of electrolyte and cell schematic. (A) Schematic
of cell in charge mode. Cartoon on top of the cell represents sources of
electrical energy from wind and solar. Curved arrows indicate direction of
electron flow and white arrows indicate electrolyte solution flow. Blue ar-
row indicates migration of cations across the membrane. Essential com-
ponents of electrochemical cells are labeled with color-coded lines and
text. The molecular structures of oxidized and reduced species are shown
on corresponding reservoirs. (B) Cyclic voltammogram of 2 mM 2,6-DHAQ
(dark cyan curve) and ferrocyanide (gold curve) scanned at 100 mV/s on
glassy carbon electrode; arrows indicate scan direction. Dotted line rep-
resents CV of 1 M KOH background scanned at 100 mV/s on graphite foil
electrode.
SCIENCE sciencemag.org
25 SEPTEMBER 2015 • VOL 349 ISSUE 6255 1529

Conversion of alcohols to enantiopure amines through dual-enzyme
hydrogen-borrowing cascades
Francesco G. Mutti et al.
Science 349, 1525 (2015);
DOI: 10.1126/science.aac9283
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