Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Article abstract of DOI:10.1002/cbic.201700257

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C₄–C₈) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.

Full text of DOI:10.1002/cbic.201700257

Accepted Article
Title: Photometric characterization of the reductive amination scope
of the imine reductases from Streptomyces tsukubaensis and
Streptomyces ipomoeae
Authors: Philipp Matzel, Lukas Krautschick, and Matthias Höhne
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To be cited as: ChemBioChem 10.1002/cbic.201700257
Link to VoR: http://dx.doi.org/10.1002/cbic.201700257
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10.1002/cbic.201700257
ChemBioChem
COMMUNICATION
Photometric characterization of the reductive amination scope of
the imine reductases from Streptomyces tsukubaensis and
Streptomyces ipomoeae
Philipp Matzel[a], Lukas Krautschick[a] and Matthias Höhne*[a]
Abstract: Imine reductases (IREDs) emerged as promising
enzymes for the asymmetric synthesis of secondary and tertiary
amines starting from carbonyl substrate. Screening the substrate
specificity of the reductive amination reaction is usually performed
by time-consuming GC analytics. We found two highly active
IREDs in our enzyme collection, IR-20 from Streptomyces
tsukubaensis and IR-Sip from Streptomyces ipomoeae that
allowed a comprehensive substrate screening with a photometric
NADPH assay. We screened 39 carbonyl substrates combined
with 17 amines as nucleophiles. Activity data from 663
combinations provide a clear picture about substrate specificity
and capabilities in the reductive amination of these enzymes.
Besides aliphatic aldehydes, the IREDs accepted various cyclic
(C4-C8) and acyclic ketones, preferentially with methylamine. IR-
Sip also accepts a range of primary and secondary amines as
nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-
methylcyclohexanone with dimethylamine or pyrrolidine with high
diastereoselectivity (>94-96%de). The nucleophile acceptor
spectrum depends on the employed carbonyl substrate.
Conversion of well-accepted substrates can also be detected
when crude lysates are employed as enzyme source.
imines formed from the respective ketone and amine in situ
(referred to as reductive amination of ketones).[3]
Especially reductive amination has certain advantages: (i)
it is a one-step reaction, the starting materials are readily
available and the reaction is very modular. The combinations
of structurally different ketone and amine substrates result in
a manifold of possible products, provided enzymes with the
desired substrate specificities are available. Several IREDs
were characterized towards their ability for reductive
amination recently, and a range of model amines were
synthesized with high yields and enantiomeric/diastereomeric
excesses.[7] Also pharmaceutically relevant compounds such
as (R)-rasagiline are accessible, albeit with lower efficiency.[8]
Interestingly, IREDs also accept secondary amines as
nucleophiles, resulting in the formation of tertiary amines.[8]
These new findings highlight the potential of imine reductases
for organic synthesis. However, there are the following
limitations of IREDs: (i) several enzymes show only moderate
enatioselectivities, (ii)
a high amino donor excess is
necessary, and (iii) the reaction in asymmetric reductive
amination relatively slow. This is in contrast to cyclic imine
reduction, which is a comparable fast reaction: Substrate
specificity, catalytic constants and biochemical properties
such as pH- and temperature optimum can be easily
determined using photometric NADPH assays.[7b, 9]
Enantiopure amines are used as important building blocks
and fine chemicals in the pharmaceutical industry.[1] Within the
2]
collection of chemical[1a,
and enzymatic methods, imine
reductase (IRED)-catalyzed reductive amination[1c, 3] emerged as
a promising tool to approach secondary amines (Scheme 1).
Compared to already established enzymatic routes[1b, 4] to primary
amines, e.g. with lipases and transaminases, there are fewer
biocatalytic options that give access to secondary and tertiary
amines: (i) engineered monoamine oxidase variants[1b] allow the
deracemization of amine racemates, and Pictet-Spenglerases
give an entry into the indol and benzylisoquinoline alkaloid
scaffolds by catalyzing a ring closure reaction. Interestingly, also
Scheme 1. IRED-catalyzed reductive amination of the model ketone
R1 R2
R1 R2
O
N
N
R1 R2
N
IRED
H
imine/iminium NADPH NADP+
intermediate
NADP+
NADPH
NADP+
NADPH
NADPH
NADP+
a)
b)
c)
O2
d)
R2
R1
O
H2N R2
NADP+
OH
HN
1,10-disubstituted (spiro) tetrahydroiso-quinolines bearing
a
R1
O
nitrogen substituted quaternary carbon center have been
prepared by this route recently.[5] IREDs offer
a NAD(P)H-
cyclohexanone and possible side reactions that might interfere with a
photometric assay. The consumption of the cofactor NADPH can be
followed photometrically at 340 nm. Whether the imine formation takes
place in solution or in the active site of the enzyme is still not known. The
following site reactions are possible: a) Reduction of cyclohexanone to the
alcohol, b) deamination of substrate amine, c) autooxidation of NADPH, d)
deamination of the product amine. All reactions are equilibrium reactions.
For reasons of clarity, arrows indicate only the forward reaction.
dependent[6] reductive approach: reduction of cyclic imine
substrates (referred to as imine reduction) was described first,
[a]
P. Matzel, L. Krautschick, Prof. M. Höhne
Protein Biochemistry
Institut of Biochemistry
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
E-mail: Matthias.Höhne@uni-greifswald.de
On the contrary, characterization in regard to
asymmetric amination is time intensive until now and was
performed by GC/HPLC in previous studies. This hinders to
Supporting information for this article is given via a link at the end of
the document.
followed by the discovery of IRED’s capability to reduce acyclic
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10.1002/cbic.201700257
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get a clear picture about catalytic capabilities of IREDs. Also
the application of protein engineering to improve activity
Using an excess of NADP+, neither the oxidation of
methylamine nor the conversion of N-methylcyclohexane to
cyclohexaneamine could be detected (using GC and the
purpald assay for formaldehyde detection). The reverse
would benefit if
a fast photometric screening assay is
available. However, we were surprised that we could detect a
significant NADPH consumption in a standard photometric
activity assay for the reductive methylamination of
cyclohexanone (Scheme 1) with few enzymes of the IRED
panel. This prompted us to investigate the substrate spectrum
of selected IREDs in a systematic manner (during the revision
of our manuscript, Aleku et al. published the identification of
IRED homologues of fungal origin. These enzymes were also
characterized by a photometric screening and revealed to be
highly efficient enzymes for the reductive amination).[10]
reaction
(deamination
of
N-methylcyclohexane
to
cyclohexanone) was neglectible (< 0.2 % of the reductive
amination rate). From these results and previous works
showing that reaction rates of IRED-catalysed oxidations are
usually much slower compared to reduction, we conclude that
deamination should hardly affect the assay.
Table 1. Activities of IR-Sip in the reductive amination of certain carbonyl
substrates with selected amines. Activities are shown in mU/mg and were
determined by the photometric NADPH assay. Selected combinations were
confirmed by GC-MS analytics (conversions of biocatalysis reactions are
given in brackets.)
Our combinatorial screening includes aldehydes, ketones,
and keto acids in combination with ammonia, primary and
secondary amines as nucleophiles. We choose to investigate
the substrate scope of two promising imine reductases from
Streptomyces tsukubaensis (IR-20)[11] and Streptomyces
ipomoeae (IR-Sip).[12] These enzymes were identified as
efficient IREDs regarding the reductive amination and showed
Specific activity (mU/mg purified IRED)
a
large substrate scope in our previous experiments.
Compared to our earlier unsuccessful attemps to detect
reductive amination photometrically,[12] we observed
Substrate
pair
a
significant decrease of the absorbance at 340 nm when we
incubated purified IR-20 and IR-Sip with NADPH,
cyclohexanone, and methylamine, which are the best
substrates previously published. This indicates NADPH
consumption at a rate of 0.44 µmol min-1mg-1 for IR-Sip and
0.55 µmol min-1mg-1 for IR-20. However, NADPH
consumption does not guarantee that the product is formed in
an equivalent amount. Possible side reactions that would
simulate IRED activity (Scheme 1) are (i) autooxidation of
NADPH by molecular oxygen or oxidation of NADPH
catalyzed by enzyme impurities, (ii) IRED-catalyzed reduction
of the ketone to the alcohol, and (iii) non-enzymatic side
reactions of the substrates (or other trace impurities) leading
to a change of the absorbance. To monitor these unwanted
processes, we performed three negative controls where (a)
enzyme solution, (b) ketone, or (c) amine substrate was
replaced by enzyme storage buffer, DMSO, or CHES-buffer
(pH 9.5), respectively. We observed a signal intensity of
approx. 1-2 % in all controls (see Table S1 and Figure S1 in
the Supporting Information for exemplary graphs of the
NADPH depletion). This means that activities below 15
mU/mg can hardly be detected. The reaction is reversible:
oxidation of the C–N bond leads to deamination via the imine
intermediate, leading to NADPH formation. As secondary or
tertiary amines contain two or three carbon-nitrogen bonds
that might be cleaved by IREDs, deamination could yield
additional amine and ketone compounds that differ from the
original substrates. This would give rise to different side
products (see Scheme S1 in the Supporting Information for
an exemplary collection of 18 possible side products that
might arise in a single reductive amination reaction, if the
enzyme would show a broadly relaxed substrate specificity).
223
291
308
64
40
91
34
93
114
0
202
412
24
0
66
103
103
0
95
(>40%)
322
(>40%)
12
0
0
0
9
22
0
35
0
0
0
66
0
0
0
0
163
173
43
7
33
0
162
41
0
36
0
0
O
444
(>95%)
16
(34%)
66
(>95%)
8
0
6
O
438
(>95%,
94%de
35
(16%,
96%de
169
(>95%,
96%de
27
0
0
36
0
O
49
0
0
0
15
28
8
0
146
(>80%)
0
0
0
O
F
35
(72%)
0
0
0
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450"
400"
350"
300"
250"
200"
150"
100"
50"
H2
N
H
N
NH3
H2
N
NH2
H2
N
H2
N
450"
375"
300"
225"
150"
75"
heptanal"
3/methyl"cyclohexanone"
cyclohexanone"
H2
N
N
H
4/fluoro"phenylacetone"
1/indanone"
H2
N
N
H
0"
N
H
H2
N
0"
N
H
H2
N
NH2
N
H
Figure 1. Activity plot for the conversion of amines (ammonia, primary and secondary amines) with heptanal (left) and different keto substrates with IR-Sip (right).
Activities are shown in mU/mg and were determined by a photometric screening: 200 µl volume, 200 mM amine, 10 mM heptanal, 0.5 mM NADPH and 0.1-0.15
mg/ml IR-Sip or IR-20. The screeniong was performed for 10 min at 30°C and 340 nm
Figure 2. Activity plot for the conversion of aliphatic aldehydes (left) and aliphatic ketones (right) catalyzed by imine reductase IR-Sip (blue) and IR-20 (red) with
methylamine as co-substrate. Activities are shown in mU/mg and were determined by photometric measurements.
Figure 3. Activity plot for the conversion of cyclic ketones (left) and aromatic ketones (right) catalyzed by imine reductase IR-Sip (blue) and IR-20 (red) with
methylamine as co-substrate. Activities are shown in mU/mg and were determined by photometric measurements.
Based on these initial results we extended the photometric
assay to a sufficient substrate screening. 46 keto substrates
containing aldehydes, aliphatic ketones, cyclic ketones,
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aromatic ketones and keto acids were combined with 18
amines, including ammonia, primary and secondary amines.
Similar to the reductive methylamination of cyclohexanone,
control reactions with most carbonyl substrates were in the
range of 10 mUmg–1. However, control reactions with seven
carbonyl substrates and one amine gave significant
absorbance changes. These substrates were thus withdrawn,
resulting in 663 substrate combinations. Deamination was
only assayed with the substrate amines, as amine standards
of most of the product amines were not available.
longer aldehydes up to octanal, and the methyl ketones
acetone up to 2- nonanone plus the ‘internal‘ ketones 3-
hexanone and 4-heptanone. Both enzymes showed a small
divergent pattern, IR-Sip with an activity maximum for the
conversion of octanal and IR-20 with hexanal as preferred
aldehyde (with methylamine). Similarly, activities were highest
for ketone substrates with chain lengths of 6-8 carbon atoms.
Aldehydes were more reactive than linear ketones, which
might reflect their higher electrophilicity and thus a better
imine formation. Also cyclic ketones appeared to be more
reactive: ≈550 mU/mg was measured with cyclohexanone,
which is the best accepted substrate. Both enzymes showed
a very similar pattern: There is no significant influence if a
methyl group is attached somewhere to the cyclohexanone
ring, but activity drops if a polar substituent (keto, amine or
hydroxyl group) is present in 3-position. Activities also
decrease if the ring size shrinks to cyclobutanone or
increases to cyclooctanone.
Finally, we investigated the conversion of cyclic and
aryl(aliphatic) ketones considering the relevance for these
compounds as building blocks in fine chemistry. The
acceptance of these ketones differ more between the two
tested enzymes (Figure 3). IR-Sip showed reasonable
activities for 4-fluoro phenyl acetone and 4-phenyl-2-
butanone. In contrast to IR-20, just 4-fluoro phenyl acetone
was well converted (97 mU/mg). Except of two more bulky
substrates, acetophenone derivatives were converted, but
with relatively low activity. Based on these findings, we can
say that cyclic substrates, secondary cyclic amines and as
well cyclic ketones, are favored substrates for reductive
amination using IR-20 and IR-Sip. Considering to the
probable natural function in synthetic pathways of antibiotics,
the preference of cyclic substrates is plausible for the
enzymes.
We also employed crude extracts as enzyme source to
measure activity of selected enzymes. As anticipated, this
results in higher levels of background reactions, and thus, the
measurements are not as sensitive. Although the higher
measurement error will increase the possibility to detect false
positive or negative activities, libraries of IRED variants can
be screened if well or moderately accepted substrate pairs
are employed, and the enzymes are sufficiently over-
expressed (see Fig. S3 in the Supporting Information for a
SDS-PAGE gel).
In conclusion, this study gives a comprehensive overview of
the substrate specificity in the reductive amination catalyzed
by the two IREDs IR-20 and IR-Sip, although
enantioselectivity cannot be measured in the reductive
amination mode. Besides selected substrate combinations
that were investigated previously, we detected activity with a
rather broad range of further substrates, ranging from
secondary amines to aldehydes and bulky ketones, which can
be used as precursor for pharmaceutical relevant amines. We
further demonstrated that the photometric assay works when
crude extract is employed as enzyme source and moderately
or well accepted substrate pairs are screened. This opens the
possibility for high-throughput screening in protein
engineering studies.
Table 1 shows activities of exemplary reactions (data of all
combinations are shown in the Supporting Information Table
S2).
Importantly, the screening results are in accordance with the
trends observed in model reactions of previous studies:
substrate combinations known to be active from GC-based
assays of biocatalysis reaction were also active in our study,
and inactive biocatalytic reactions gave no signal in the
photometric assay. Furthermore, we confirmed selected
activities not described previously by GC-MS analytics (see
Table 1 and the Supporting Information Figure S2).
From the screened 17 amines, methylamine and ethylamine
were by far best accepted by both enzymes. IR-Sip shows a
much broader amine nucleophile specificity, also including
secondary amines, which are converted to tertiary amines, as
shown in Figure 1 (conversions with heptanal as carbonyl
substrate). This highlights the more flexible substrate scope
of this enzyme compared to IR-20. Besides pyrrolidine, IR-Sip
converts the seven-membered cyclic azepane with lower
activity, but not 4-methyl piperidine (we could not use
piperidine due chemical regulations). The enantioselectivity
cannot be accessed in the reductive amination direction of the
assay. Therefore we reacted (R)-3-methylcyclohexanone with
methylamine, dimethylamine and pyrrolidine in analytical
scale reactions and obtained the products in 94-96 %de
(Table 1). The asymmetric synthesis of secondary amines
with IREDs was already established.[7c, 8, 10] Our experiments
confirm for the first time that also tertiary amines can be
accessed by IREDs by asymmetric reductive amination with
high stereo selectivity.
Interestingly, the amino donor specificity is affected by the
carbonyl substrate employed (Figure 1), as can be seen by
comparing activities with cyclohexanone and heptanal. In the
reaction with methylamine, heptanal has a lower (65%)
relative activity compared to cyclohexanone (100%). Whereas
some nucleophiles are converted less efficient with heptanal,
such as ammonia and propylamine, several amines including
diethylamine and azepan can only be detected with heptanal
as carbonyl substrate, and also other amines, especially
pyrrolidine, are converted with a 4-6 fold higher relative rate
compared with cyclohexanone. The different activities with
aldehydes and ketones likely reflects different reactivities for
imine formation.[10]
We used five groups of carbonyl substrates to understand the
substrate scope of these enzymes. For the investigated keto
acids pyruvate, γ-aminobutyric acid and phenylpyruvic acid,
we did not detect conversion. Aliphatic aldehydes and
ketones are converted with a similar pattern (Figure 2). We
screened shorter aldehydes down to acetaldehyde as well as
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Experimental Section
thank Prof. Bornscheuer for his continuous support and the
ability to use his lab equipment. PM thanks the Deutsche
Forschungsgemeinschaft for funding (DFG grant number
HO4754/4-1).
Enzyme preparation was done as described previously.[7c, 12] Activities
were determined by a continuous photometric NADPH assay: in
contrast to NADP+, NADPH absorbs light at a wavelength of 340 nm
(in the microplate with a path length of 5 mm, a value of ε340 = 3.2
mM-1 was determined in a calibration experiment. For calculation of
enzyme activity, the slope of a control reaction without enzyme was
substracted. One U corresponds to a NADPH consumption of 1
µmol/min. The screening was performed under the following reaction
conditions: 200 mM amine buffer (the pH of the aqueous amine
solution was adjusted with HCl to pH 9.5), 10 mM ketone, 0.5 mM
NADH and 0.1-0.15 mg/ml enzyme concentration. For an efficient
screening, a master deep-well plate contained every keto substrate as
duplicate in DMSO at a concentration of 200 mM. The reaction
mixture was prepared on a microtiter plate, 10 µl of each substrate
was added and the reaction was followed for 10 min at 30°C after
addition of the enzyme. For establishing the assay using purified
enzymes and crude extract, 7 replicates were measured and a
detection limit of 15 mU/mg was found. For the subsequent in-depth
characterization, measurements were performed in duplicates. The
Deamination of the high-concentrated substrate amines were assayed
separately by adding enzyme solution and NADP+ to the amine
buffers and obtained the absorbance at 340 nm. An increase of
absorbance, which would indicate an undesirable deamination, was
not observed.
Keywords: enzyme catalysis, amines, imine reductase
reductive amination, photometric assay
[1]
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For confirming selected activities with GC,
biotransformations were performed with purified enzymes for
1
ml scale
a
[5]
[6]
[7]
B. R. Lichman, J. X. Zhao, H. C. Hailes, J. M. Ward, Nat Commun
2017, 8.
reaction time of 20 h under the following conditions: 200 mM amine
buffer, pH 9.5, 10 mM carbonyl substrate, 0.5 mg/mL purified IRED,
and for recycling of NADPH, the reaction contained 0.1 mg/mL of
glucose dehydrogenase (Codexis GDH-105), 60 mM D-glucose, and
0.5 mM NADPH. The reaction was stopped with concentrated sodium
hydroxide (end concentration 1.7 M). 240 µl of the solution was
extracted twice with 120 µL ethyl acetate before analysis in GC-MS.
The GC analytics was performed on a BPX-5 column with following
program: 60°C hold for 10 min, 220°C (10°C/min) hold for 5.5 min,
260°C (5°C/min). The inlet pressure and temperature was set to 78.9
kPa and 300°C. Chromatograms are shown in the Supporting
Information. For analysing stereoselectivity, we synthesized the
racemic product standards according to an established protocol.[13]
The 1H and 13C-NMR spectra and GC-MS analysis confirmed the
product identity. Separation of the diastereomers was achieved by GC
(please see Fig. S4 in the Supporting Information).
M. Gand, C. Thöle, H. Müller, H. Brundiek, G. Bashiri, M. Höhne, J.
Biotechnol. 2016, 230, 11-18.
a) P. N. Scheller, M. Lenz, S. C. Hammer, B. Hauer, B. M. Nestl,
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Applied Microbiology and Biotechnology 2016, 100, 10509-10520;
c) D. Wetzl, M. Gand, A. Ross, H. Müller, P. Matzel, S. P. Hanlon, M.
Müller, B. Wirz, M. Höhne, H. Iding, ChemCatChem 2016, 8, 2023–
2026.
[8]
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Acknowledgements
[11] D. Wetzl, M. Berrera, N. Sandon, D. Fishlock, M. Ebeling, M. Müller,
S. Hanlon, B. Wirz, H. Iding, ChemBioChem 2015, 16, 1749-1756.
[12] M. Gand, H. Müller, R. Wardenga, M. Höhne, J. Mol. Catal. B. 2014,
110, 126-132.
We thank our partners of the IRED research consortium Dr.
Dennis Wetzl and Dr. Hans Iding (Hoffman-La Roche Ltd.),
Prof. Dr. Michael Müller, and Prof. Dr. Dörte Rother for
inspiring and fruitful discussions. We also thank Ina Menyes
and Gabriele Thede for analytical support, and Dr. Bettina
Appel for assistance with the product purification. We like to
[13] K. A. Neidigh, M. A. Acery, J. S. Williamson, S. Bhattacharyya, J.
Chem. Soc., Perkin Trans. 1, 1998, 2527-2531
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P. Matzel, L. Krautschick, M. Höhne
Page No. – Page No.
Photometric characterization of the
reductive amination scope of the
imine reductases from Streptomyces
tsukubaensis and Streptomyces
ipomoeae
Two enzymes, 663 combinations: The substrate scope in the reductive amination catalyzed
by two promising IREDs was established in a rapid photometric NADPH assay. Substrates
ranging from aldehydes to ketones, and from primary to secondary amines are accepted
giving access to various secondary and tertiary amine products.
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