Enzymatic asymmetric synthesis of enantiomerically pure aliphatic, aromatic and arylaliphatic amines with (R)-selective amine transaminases

Article abstract of DOI:10.1002/adsc.201100435

Seven (R)-selective amine transaminases (R-ATAs) recently discovered by an in silico-based approach in sequence databases were produced recombinantly in Escherichia coli and subjected to partial purification by ammonium sulfate precipitation. A range of additives and various buffers were investigated to identify best conditions to ensure good storage stability and stable activity during biocatalysis. All enzymes show pH optima between pH 7.5-9. These R-ATAs were then applied in the asymmetric synthesis of twelve aliphatic, aromatic and arylaliphatic (R)-amines starting from the corresponding prochiral ketones using a lactate dehydrogenase/glucose dehydrogenase system to shift the equilibrium. For all ketones, at least one enzyme was found that allows complete conversion to the corresponding chiral amine having excellent optical purities >99% ee. Variations in substrate profiles are also discussed based on the phylogenetic relationships between the seven R-ATAs. Thus, we have identified a versatile toolbox of (R)-amine transaminases showing remarkable properties for application in biocatalysis. Copyright

Full text of DOI:10.1002/adsc.201100435

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DOI: 10.1002/adsc.201100435
Enzymatic Asymmetric Synthesis of Enantiomerically Pure
Aliphatic, Aromatic and Arylaliphatic Amines with
(R)-Selective Amine Transaminases
Sebastian Schꢀtzle,a Fabian Steffen-Munsberg,a Ahmad Thontowi,a
Matthias Hçhne,a Karen Robins,b and Uwe T. Bornscheuera,*
a
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix
Hausdorff-Str. 4, 17487 Greifswald, Germany
Fax : (+49)-3834-86-794367; e-mail: uwe.bornscheuer@uni-greifswald.de
Lonza AG, Valais Works, 3930 Visp, Switzerland
b
Received: May 29, 2011; Published online: August 25, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100435.
Abstract: Seven (R)-selective amine transaminases cose dehydrogenase system to shift the equilibrium.
(R-ATAs) recently discovered by an in silico-based For all ketones, at least one enzyme was found that
approach in sequence databases were produced re- allows complete conversion to the corresponding
combinantly in Escherichia coli and subjected to par- chiral amine having excellent optical purities >99%
tial purification by ammonium sulfate precipitation. ee. Variations in substrate profiles are also discussed
A range of additives and various buffers were inves- based on the phylogenetic relationships between the
tigated to identify best conditions to ensure good seven R-ATAs. Thus, we have identified a versatile
storage stability and stable activity during biocataly- toolbox of (R)-amine transaminases showing remark-
sis. All enzymes show pH optima between pH 7.5–9. able properties for application in biocatalysis.
These R-ATAs were then applied in the asymmetric
synthesis of twelve aliphatic, aromatic and arylali-
phatic (R)-amines starting from the corresponding Keywords: chiral amines; enantioselectivity; enzyme
prochiral ketones using a lactate dehydrogenase/glu- catalysis; transaminases
Introduction
prostereogenic ketones. Furthermore, these enzymes
usually show excellent enantioselectivity and thus
Optically active amines play an important role in the they are in great demand. In principle, both enantio-
pharmaceutical, agrochemical and chemical industry. mers of a given amine can be produced with a single
They are frequently used as building blocks for the enantioselective amine transaminase having, for in-
synthesis of various biological active compounds used stance, (S)-enantiopreference. A kinetic resolution
as drugs or agrochemicals including, for example, with this enzyme would leave the (R)-enantiomer
drugs for the treatment of Alzheimerꢁs disease[1] and behind, which can be obtained at maximum in 50%
malaria[2] as well as prostate drugs[3] and antitumor yield as the (S)-enantiomer is converted into the
antibiotics.[4] As both enantiomers of a building block ketone. In turn, asymmetric synthesis starting from
are often needed for targets with different absolute the corresponding prochiral ketone would yield the
configuration, an enzyme platform for the production (S)-amine as product in theoretically 100% yield if
of both (R)- and (S)-enantiomers is highly desired.
the equilibrium is efficiently shifted towards product
In principle, various enzymatic routes can be em- formation for which a range of methods are avail-
ployed for the synthesis of optically active primary able.[6] Whereas many (S)-selective amine transami-
amines using hydrolases, oxidoreductases or transfer- nase (S-ATAs) had been discovered and extensively
ases (for further reading see review[5]). All routes investitaged, only a few (R)-amine transaminases (R-
have their advantages and disadvantages, but only ATAs) were known until recently and only one
amine transaminases offer the unique possibility to enzyme (ATA-117, Codexis) was commercially avail-
synthesize enantiomerically pure amines directly from able. During an extensive project aiming for an R-
Adv. Synth. Catal. 2011, 353, 2439 – 2445
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ATA for the industrial synthesis of the drug Sitaglip- MycVan from Mycobacterium vanbaalenii) for the
tin, Codexis and Merck & Co created a variant of asymmetric synthesis of a range of aliphatic, aromatic
ATA-117, which now allows synthesis of the drug on and arylaliphatic amines to elucidate the substrate
industrial scale.[7] Thus, an efficient asymmetric syn- scope of these enzymes originating from various mi-
thesis of (R)-amines was mostly hampered by the lim- croorganisms and for which no natural substrate is
ited access to R-ATAs. Recently, we identified 20 known (Scheme 1). These enzymes were primarily
(R)-amine transaminases by an in silico search strat- chosen from the 17 recently discovered R-ATAs[8] be-
egy in which careful analysis of typical motifs for (S)- cause of their high specific activities towards 2b, 7b
or (R)-selective amino acid transaminases (BCAT, and 9b and sufficient expression levels in E. coli.
branched chain amino acid transaminase; DATA, d-
amino acid transaminase) and certain lyases from the
same fold class led to a complex algorithm.[8] This al- Enzyme Production and Formulation
gorithm was then used in an alignment of 5,000 se-
quences deposited in public databases to filter out all Before the newly discovered R-ATAs could be bio-
known enzymes with BCAT, DATA or lyase activity chemically characterized in more detail and used in
and certain additional criteria to ensure that the re- asymmetric synthesis, methods for their expression in
maining sequences are true amine transaminases and E. coli on sufficient scale and stability studies needed
that they exhibit (R)-enantiopreference. The proteins to be performed, especially as we have noticed before
encoded by the remaining 20 sequences were all that several enzymes were rather unstable after His-
cloned, functionally expressed in E. coli and in pre- tag purification in tricine buffer used for the conduc-
liminary analyses, we could demonstrate that 17 en- tivity assay.[9] For application in biocatalysis purified
zymes were indeed amine transaminases and all had enzymes are not needed as long as it is ensured that
(R)-selectivity (three enzymes could not be expressed the crude cell lysate does not contain any interfering
at sufficiently high levels) as confirmed by determin- background activity, which was never observed for all
ing specific activities towards different amine enantio- R-ATAs expressed in E. coli. Thus, the crude extracts
mers.
containing the seven R-ATAs were only subjected to
In this contribution, we now report further analysis ammonium sulfate precipitation at 60% saturation
of seven of these R-ATAs, which includes expression and could be recovered fully active. Some parts of the
optimization to produce the enzyme in sufficient total protein could be discarded in the cases of
amounts, formulation to achieve stable biocatalysts AspFum and GibZea by fractionated precipitation at
and the determination of pH profiles. Special focus is an ammonium sulfate saturation of 40% beforehand.
given on the application of the enzymes in asymmet- This step was not carried out for the other enzymes as
ric synthesis to identify their potential in industrial it would have caused a reasonable loss of activity.
biocatalysis and hence suitable methods to shift the Next, various additives – commonly used osmolytes
equilibrium needed to be identified too.
such as glycerol, ethylene glycol, PEG 1550, trehalose,
sucrose and sarcosine as well as the surfactant Tween
80 – were investigated for their stabilizing effect on
the proteins during freeze-drying and storage as lyo-
philizate and in solution. The best conditions for each
Results and Discussion
In the present study we utilized a toolbox consisting R-ATA along with the activity and yield of lyophili-
of seven (R)-selective amine transaminases (AspTer zate from 400-mL shake flask cultivations are sum-
from Aspergillus terreus, AspFum from Aspergillus fu- marized in Table 1. In most cases sucrose or glycerol
migatus, AspOry from Aspergillus oryzae, PenChry had the best stabilizing effect on the R-ATAs. All en-
from Penicillium chrysogenum, NeoFis from Neosar- zymes were stable for at least three months in lyophi-
torya fischeri, GibZea from Gibberella zeae and
Scheme 1. Chiral (R)-amines obtained from the corresponding prochiral ketones using the amine transaminases.
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Enzymatic Asymmetric Synthesis of Enantiomerically Pure Aliphatic, Aromatic and Arylaliphatic Amines
Table 1. Summary of parameters for the preparation of the
lyophilized biocatalysts. Activities were measured with the ace-
tophenone assay photometrically[10] and protein concentrations
were determined with the BC assay according to the supplier’s
manual.
Enzyme Additive[a] Activity[b] Yield[c] Total ac- Activity
[U/mg]
[mg]
tivity[c]
[U]
[U/mg
protein]
AspTer sucrose
PenChr sucrose
0.74
0.15
1.10
0.62
956
789
757
687
622
574
580
711
122
829
426
759
1415
258
3.94
1.01
3.65
4.83
8.14
18.63
1.95
Scheme 2. Reaction scheme for the asymmetric synthesis of
chiral (R)-amines with the R-ATAs in combination with the
LDH/GDH system to shift the equilibrium towards product
formation.
AspOry
–
AspFum sucrose
NeoFis sarcosine 1.22
GibZea sucrose
MycVan glycerol
2.47
0.44
[a]
2% (w/v) added after ammonium sulfate precipitation.
Activity of lyophilisate.
For lyophilisate obtained from 400 mL cultivation.
of the PLP-cofactor. On the other hand, we showed
in a previous study that TRIS, EPPS and especially
HEPES had an inhibitory effect on the S-ATA from
Rhodobacter sphaeroides compared to phosphate
[b]
[c]
lized form and for at least two weeks in solution at buffer (7b was converted with 71%, 76% and 24%,
48C.
respectively[9]). Therefore, the initial activity towards
7b in four different buffer systems (TRIS, HEPES,
MOPS and BES) was compared to the activity in
50 mM and 100 mM phosphate buffer (Table 2).
Overall, the influence of the different buffer sys-
Finding the Right Reaction System
With stable and active biocatalysts available, the next tems was only moderate (Table 2). Significantly lower
important challenge for a successful asymmetric syn- activity was only observed for PenChr, in 100 mM
thesis of chiral amines was the identification of the phosphate or 50 mM TRIS buffer, whereas higher ac-
best method to shift the equilibrium towards product tivity was found in tricine or HEPES buffer. Alto-
formation. Thus, the use of pyruvate decarboxylase[6b] gether the differences were rather small and hence
and the combination of lactate dehydrogenase (LDH) phosphate buffer was chosen for subsequent experi-
with glucose dehydrogenase (GDH) for cofactor recy- ments as this buffer is suitable for the LDH/GDH re-
cling[6a] were preliminary investigated and the LDH/ cycling system.
GDH system (Scheme 2) gave most satisfying results.
The pH profiles of the R-ATAs were determined
Next, several buffers were investigated to identify using the photometric acetophenone assay.[10] The ma-
the best system for the biocatalysis. Recent studies jority of the enzymes show a pH optimum between 8–
showed that the most often used phosphate buffer is 9, which is also typical for S-ATAs such as the
not always the best choice for transaminases because enzyme from Vibrio fluvialis;[11] AspTer and PenChr
of inhibitory effects of the phosphate ions – assuming- show a sharp optimum at pH 8.5–9 while AspOry,
ly by blocking the binding site of the phosphate group AspFum and NeoFis have a broader optimal range
Table 2. Relative activity of the R-ATAs in different buffer systems. The activity towards 7b was measured spectrophotomet-
rically[10] in the different buffers at pH 8 and was normalized to the activity in 50 mM phosphate buffer (set to 100%). All
values are means from three individual measurements and standard deviations did not exceed 10%.
Relative activity [%]
Phosphate
(50 mM)
Phosphate
(100 mM)
TRIS
(100 mM)
HEPES
(100 mM)
MOPS
(100 mM)
BES
(100 mM)
Tricine
(100 mM)
AspTer 100
99
76
93
52
100
123
103
104
102
122
104
98
80
99
104
136
105
102
101
119
108
PenChr 100
AspOry 100
AspFum 100
NeoFis 100
GibZea 100
MycVan 100
114
106
103
101
123
104
107
103
101
112
95
106
98
103
107
103
104
101
97
122
109
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between 8–9. GibZea has a rather untypical pH opti-
mum of 7.5 and loses already 30 to 70% activity at
pH 8.5 and 9, respectively, whereas MycVan has a
very broad optimum between pH 7.5–8.5 and still
shows ~80% activity at pH 9 (Figure 1). Nevertheless,
in combination with the LDH/GDH system, prelimi-
nary experiments gave the highest yields at pH 7.5.
Asymmetric Synthesis
Having found suitable ways for stabilizing the en-
zymes and having elucidated the optimal reaction
conditions, the R-ATAs were applied to asymmetric
synthesis to obtain the aliphatic, aromatic and arylali-
phatic amines (Scheme 1) and the results are summar-
ized in Table 3.
These data clearly show that all seven enzymes ex-
hibited excellent selectivity (>99% ee) and in all
cases the (R)-enantiopreference could be confirmed.
Most importantly, for all substrates at least one R-
ATA – and for various compounds several enzymes –
could be identified, which allowed complete conver-
sion of the ketone into the chiral amine. This exceed-
ed our expectations as the ketones represent a rather
diverse set of compounds. In only a few cases, lower
conversions were observed (i.e., 2b, 7b and 9b with
PenChr or GibZea) but further optimization to ach-
ieve higher conversion was considered unnecessary in
light of the other very useful R-ATAs.
Although PenChr and AspTer show a rather high
similarity score in a multiple sequence alignment
(Figure 2), the conversions achieved in biocatalysis
differed considerably between these two R-ATA.
AspTer enabled 100% conversion for 2-aminohexane
2b, -heptane 3b and -nonane 4b, but not for the short-
er 2-aminopentane 1b (~40%) and 2-amino-4-methyl-
pentane 5b (~60%). Substrates with sterically more
demanding groups than alkyl (cyclohexane or phenyl)
next to the ketone function were converted even
worse. These differences will be analyzed in more
detail once a 3-D structure or homology model of
these R-ATAs is available.
Another interesting result is the fact that AspTer
did not give high yields for methylphenylpropiona-
mine 9b and the 4-methoxy derivative 10b (32% and
20%), but good yields of around 80% could be ach-
ieved with the hydroxy derivatives 11b and 12b. This
phenomenon was only observed with AspTer. The
two other enzymes from Aspergillus species (AspOry
and AspFum) turned out to be even better catalysts,
as well as NeoFis. According to their sequence identi-
ty these enzymes are much alike, especially AspFum
and NeoFis with a sequence identity of 96%. AspOry
shares a similarity of 72% and 73%, respectively, and
gave very good to excellent conversions for the ali-
phatic substrates, considerably less conversion for 6a,
Figure 1. pH profiles of the seven R-ATAs. Activities were
measured with the acetophenone assay spectrophotometri-
cally[10] in Davies buffer[12], which is commonly used for pH
profile determinations.
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Enzymatic Asymmetric Synthesis of Enantiomerically Pure Aliphatic, Aromatic and Arylaliphatic Amines
Table 3. Conversion and enantiomeric excess of the amines obtained by asymmetric synthesis from the corresponding ke-
tones. In general, reactions were repeated in triplicates and standard deviation did not exceed 10%.
R-ATA
AspTer
PenChr
AspOry
AspFum
NeoFis
GibZea
MycVan
[b]
[b]
[b]
[b]
[b]
[b]
[b]
Substrate c[a]
eep
c[a]
eep
c[a]
eep
c[a]
eep
c[a]
eep
c[a]
eep
c[a]
eep
[%]
[%ee] [%]
E
E
[%ee] [%]
G
G
ACHTUNGTRENNUNG[%ee]
1b
2b
3b
4b
5b
6b
7b
8b
47
>99
>99
>99 15
>99 12
>99
>99
>99
>99
>99
>99
>99
>99
1
1
–
–
>99
>99 77
>99 79
–
–
–
–
–
–
–
92
>99
>99
>99
>99
>99
>99
>99
>99 93
>99 >99
99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99 59
>99 87
>99 74
>99
>99 92
>99
>99
>99
>99
>99
>99
>99
>99 30
>99 14
>99 43
>99 23
>99 32
>99 16
>99 66
>99 88
>99 69
>99 65
>99 68
>99 56
>99 42
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
64
30
3
17
32
20
83
9
1
0
2
2
1
4
2
53
39
8
92
>99
>99
98
>99
>99
>99
>99
>99
>99
4
3
9
7
5
6
68
99
6
99
9b
>99
>99
>99
>99
>99
>99
>99
>99 89
>99 72
>99 71
>99 85
>99
>99
>99
>99
10b
11b
12b
>99
>99
77
>99 94
[a]
Conversion as determined from the product analysis by HPLC.
% enantiomeric excess of product.
[b]
compounds, moderate yields for 6a and 7a and did
not like 8a at all.
Bringing together the observed performances
during biocatalysis and the degree of relationship as
shown in the phylogenetic tree (Figure 2), several cor-
relations attract attention. MycVan, for example, is a
good biocatalysis not far from the performance of
AspOry, AspFum and NeoFis, which show a high
degree of relationship. Although GibZea shows a
higher similarity towards these good biocatalysts such
as MycVan, it showed only a rather poor perfor-
mance. Interestingly, AspTer and PenChr are also
strongly related, but showed very different perform-
ances regarding the conversions achieved. However,
they both prefer aliphatic substrates with a chain
length of at least six carbon atoms over shorter sub-
strates, yielded only very small amounts of 7b, and
both achieved higher conversions with at least one hy-
droxy derivative of 10b as with 10b itself
Figure 2. Phylogenetic tree of the investigated R-ATAs. The
figure shows all 21 hits from the database search and the
biocatalysts used in this study are named. DATA, ADCL (4-
amino-4-deoxychorismate lyase) and BCAT are the other
three members of the PLP-fold type IV.
Conclusions
We have successfully applied the recently discovered
R-ATAs to asymmetric synthesis of various amines
with >99% conversion and excellent enantiomeric
purities of >99% ee. Looking back to our first pre-
7a and 8a and again very good conversions for the ar- liminary experiments with conversions of <40%,[8]
ylaliphatic substrates 9–12a. MycVan is the outsider one may conclude that producing biocatalysts in the
of the investigated proteins, with similarity scores not right formulation, optimization of the reaction system
exceeding 40%. The phylogenetic tree (Figure 2) as well as using a broad set of substrates is as impor-
shows that MycVan has the highest degree of relation- tant as finding new enzymes.
ship to the assumed ancestor BCAT. MycVan ach-
ieved good yields for the aliphatic and arylaliphatic
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FULL PAPERS
ganic phase was dried over Na2SO4 and the extracted
amines were derivatized to the trifluoroacetamides by
adding a 20-fold excess of trifluoroacetic anhydride. After
5 min at room temperature and purging with nitrogen to
remove excess anhydride, residual trifluoracetic acid and
solvent, the derivatized compounds were dissolved in 50 mL
ethyl acetate and 0.6 mL of this solution were injected into a
Hewlett Packard 5890 Series II gas chromatograph equipped
with an octakis-(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-
g-cyclodextrin column (50 mꢄ0.25 mm) for analyzing 8b or
Experimental Section
All chemicals were of analytical grade purity and obtained
from Sigma Aldrich (Munich, Germany), ABCR (Karlsruhe,
Germany) or Acros (Geel, Belgium).
Functional Expression and Preparation of
Lyophilized Transaminases
Expression of the transaminases was carried out as de-
scribed elsewhere.[8] The crude extract was stirred on ice and
supplemented with 60% saturation of (NH4)2SO4 for precip-
itation of R-ATAs and stirred for 30 min. In the cases of
AspFum and GibZea a fraction of the proteins from the
crude extract was precipitated by adding 40% saturation of
(NH4)2SO4 from the supernatant, stirred and centrifuged be-
forehand. The transaminase containing pellet was dissolved
in 10 mL sodium phosphate buffer (50 mM, pH 7.5) contain-
ing 0.1 mM PLP and 2% additive (w/v) (Table 1) prior to
lyophilization. Protein concentrations were determined
using the BC assay kit (Uptima, Montlucon, France).
with
a
heptakis-(2,3-di-O-acetyl-6-O-tert-butyldimethyl-
silyl)-b-cyclodextrin column (25 mꢄ0.25 mm) for 1–7b and
9–10b.
Chiral analysis of 11–12b (Table 4) was performed by ca-
pillary electrophoresis on a PACE-MDQ system equipped
with a fused silica capillary (50 mm inner diameter) as re-
ported previously.[6b]
Table 4. Details of chiral analysis.
Amine[a] Temperature Retention time Retention time
Transaminase Activity Assay
[8C]
(S) [min]
(R) [min]
Activity measurements were performed in triplicate as de-
scribed.[10] The pH profiles were determined using 100 mM
Davies buffer and 10 mL (from a 10 mgmLÀ1 solution of lyo-
philisate in deionized water) protein solution were added.
The acetophenone assay was also used to evaluate the
buffer acceptance. For this, seven different buffers (sodium
phosphate 50 mM pH 8, phosphate 100 mM pH 8, Tris
100 mM pH 8, BES 100 mM pH 8, HEPES 100 mM pH 8,
MOPS 100 mM pH 8 and tricine 100 mM pH 8) were chosen
and enzyme solution was added before measuring activity.
1b
2b
3b
4b
5b
6b
7b
8b
125
130
140
130
130
150
170
220
185
185
CE
CE
3.2
3.3
2.7
6.3
2.4
2.3
7.6
7.0
2.3
5.6
3.7
3.5
3.5
3.8
3.0
8.5
2.9
2.4
8.3
6.3
2.5
6.1
3.9
3.7
9b
10b
11b
12b
Asymmetric Synthesis
All reactions were carried out in 0.5 mL scale in 1.5 mL Ep-
pendorf tubes containing 30 mgmLÀ1 transaminase lyophili-
sate, 90 U lactate dehydrogenase, 15 U glucose dehydrogen-
ase, 50 mM 1a–13a, 250 mM d-alanine, 150 mM d-glucose,
1 mM NADH and 0.1 mM PLP in sodium phosphate buffer
(100 mM, pH 7.5). While incubating at 308C and 1000 rpm
in an Eppendorf shaker, samples (150 mL) were taken after
distinct time periods and analyzed by HPLC for conversion
and gas chromatography or capillary electrophoresis for de-
termination of optical purity (% eeP). Promising reactions
were repeated in triplicate with 30 mgmLÀ1 TA lyophilisate
and 150 mL were analyzed by HPLC whereas another
150 mL were used for chiral analysis.
[a]
For 1b–10b the retention times refer to the trifluoracetic
acid derivative.
Acknowledgements
The authors thank the Enzymaticals AG (Greifswald, Ger-
many) for the larger scale production of the amine transami-
nases.
References
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phase C18 column (LiChrospherꢃ). Detection took place
with a UV detector set at 245 nm and an evaporating light
scattering detector (see Supporting Information).
The chiral analysis of 1–10b was performed by gas chro-
matography. A 150 mL sample was supplemented with 15 mL
10M NaOH and extracted with 400 mL ethyl acetate. The or-
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