Enzymatic racemization of amines catalyzed by enantiocomplementary ω-Transaminases

Article abstract of DOI:10.1002/chem.201001602

A strategy for the biocatalytic racemization of primary α-chiral amines was developed by employing a pair of stereocomplementary PLP-dependent ω-transaminases. The interconversion of amine enantiomers proceeded through reversible transamination by a prochiral ketone intermediate, either catalyzed by a pair of stereocomplementary ω-transaminases or by a single enzyme possessing low stereoselectivity. To tune the system, the type and concentration of a nonchiral amino acceptor proved to be crucial. Finally, racemization could be achieved by the cross-transamination of two different amines without a requirement for an external amino acceptor. Several synthetically and industrially important amines could be enzymatically racemized under mild reaction conditions. ω-Transaminases play ping-pong: A biocatalytic protocol for the 'clean' racemization of α-chiral prim-amines was developed by an equilibrium-controlled deamination/amination sequence catalyzed by a pair of (R)- and (S)-ω-transaminases (see scheme).

Full text of DOI:10.1002/chem.201001602

DOI: 10.1002/chem.201001602
Enzymatic Racemization of Amines Catalyzed by Enantiocomplementary
w-Transaminases
[
a]
[a]
[a]
[b]
Dominik Koszelewski, Barbara Grischek, Silvia M. Glueck, Wolfgang Kroutil, and
[
b]
Kurt Faber*
Abstract: A strategy for the biocatalyt-
ic racemization of primary a-chiral
amines was developed by employing a
pair of stereocomplementary PLP-de-
pendent w-transaminases. The inter-
conversion of amine enantiomers pro-
ceeded through reversible transamina-
tion by a prochiral ketone intermedi-
ate, either catalyzed by a pair of ste-
reocomplementary w-transaminases or
by a single enzyme possessing low ste-
reoselectivity. To tune the system, the
type and concentration of a nonchiral
amino acceptor proved to be crucial.
Finally, racemization could be achieved
by the cross-transamination of two dif-
ferent amines without a requirement
for an external amino acceptor. Several
synthetically and industrially important
amines could be enzymatically race-
mized under mild reaction conditions.
Keywords: amines · biocatalysis ·
biotransformations · racemization ·
stereochemistry
[
4]
Introduction
can easily be achieved by mild acid or base catalysis. In
contrast, stereochemically stable compounds, such as secon-
dary alcohols and chiral amines are more difficult to race-
Discovered by Pasteur in 1853, racemization was long re-
garded as an undesired side reaction rather than as an im-
portant tool for the recycling of unwanted stereoisomers de-
rived from kinetic resolution. As a consequence, the con-
trolled racemization of organic compounds has been studied
rather scarcely. It was only recently that the need for
cleanꢁ racemization protocols had been recognized in par-
ticular due to the increasing demand for so-called deracemi-
zation processes, which allow the complete transformation
of a racemate into a single stereoisomeric product in 100%
[5]
mize. In general, the racemization of amines is more diffi-
cult than that of alcohols, because amines are prone to act
[
2j,6]
as strong ligands for catalytically active metal species.
Ef-
ficient catalysts for the racemization of amines are thus
more rare than those for the racemization of alcohols.
Among them, only a few types of catalysts are compatible
with enzymes and thus are applicable to the dynamic kinetic
resolution of amines: 1) palladium metal dispersed on
porous supports, 2) Shvo-type ruthenium complexes, and
Only recently, metal-independent
light-induced radical racemization mediated by trifluoro-
[
1]
ꢀ
[
2,3]
[6e,7]
theoretical yield.
pounds, such as cyanohydrins, hemi
ed carbonyl compounds, or a-aryl-substituted hydantoins,
The racemization of stereolabile com-
3) iridium complexes.
A
H
U
T
N
U
[8]
AHCTUNGTRENNUNGe thanethiol was proposed. The traditional protocol for the
large-scale racemization of amines employs (ex-situ) racemi-
zation by using Ni catalysts at elevated temperatures. Al-
though this method works for sturdy (nonfunctionalized)
substrates, it is inapplicable to amines bearing nitro or halo-
[
a] Dr. D. Koszelewski, B. Grischek, Dr. S. M. Glueck
Austrian Centre of Industrial Biotechnology (ACIB) c/o
Department of Chemistry, Organic and Bioorganic Chemistry
University of Graz, Heinrichstrasse 28
[9]
gen groups, which are prone to reduction.
8
010 Graz (Austria)
In contrast to chemical methods, enzymatic racemization
takes place at intrinsic mild reaction conditions—for exam-
ple, room temperature, atmospheric pressure, and physiolog-
ical pH—and is, therefore, more chemoselective and thus
avoids the formation of byproducts. Due to the specificity of
biosynthetic pathways, nature has little need for racemiza-
tion and thus the number of racemases is very limited.
Thus, only a few specialized enzymes are known to catalyze
the racemization of a-hydroxycarboxylic acids, a-amino
[
b] Prof. W. Kroutil, Prof. K. Faber
Department of Chemistry, Organic and Bioorganic Chemistry
University of Graz, Heinrichstrasse 28
8
010 Graz (Austria)
Fax : (+43)316-380-9840
E-mail: kurt.faber@uni-graz.at
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem201001602. It contains detailed
analytical data for the determination of the ketone to amine ratio
and the ee values of amines.
[10,11]
378
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 378 – 383

FULL PAPER
acids, and hydantoins. To the best of our knowledge, no de-
fined enzymes have been described so far for the racemiza-
tion of a-chiral primary amines, which are not a-amino
acids.
Results and Discussion
Inspired by the reversible hydrogen-transfer-like racemiza-
tion mechanism of secondary alcohols catalyzed by transi-
tion-metal catalysis, as well as a consequence of our own re-
search on the enzymatic racemization of a-hydroxycarboxyl-
Scheme 2. Biocatalytic racemization of a-chiral prim-amines 1–6 cata-
lyzed by w-transaminases in presence of an additional amino-acceptor
ketone (7a–h).
[12]
ic acids and sec-alcohols, we envisaged that interconver-
sion of amine enantiomers might be achieved by a reversible
deamination/amination sequence catalyzed by a matching
pair of stereocomplementary w-transaminases (wTA ;
Scheme 1).
tion would be achieved. By following this assumption, we
applied two commercially available wTAs: (S)-selective Vf-
wTA from Vibrio fluvialis and (R)-selective wTA ATA-117
in the presence of the aldehyde form of the cofactor PLP
(
(
1 mm) for the racemization of (S)-1-methoxy-2-propylamine
1) (Scheme 2). The latter is a popular building block for
the synthesis of several widely used herbicides, such as me-
[15]
tolachlor and dimethenamide.
Much to our disappoint-
ment, no racemization could be observed within 24 h and
the expected prochiral ketone methoxyacetone (1a) was
below the detection limit of <0.5% (Table 1, entry 1). Simi-
lar results were obtained in the presence of additional cofac-
tor in its amino form (PMP, 1 and 25 mm).
Table 1. Biocatalytic racemization of (S)-1 in the presence of ketones
[
a]
7
a–h as amino-group transfer mediators.
Scheme 1. Biocatalytic racemization of an a-chiral prim-amine via a non-
chiral ketone intermediate derived from the substrate enantiomers by
employing a pair of stereocomplementary w-transaminases (wTA). The
amino group is reversibly shuttled between the substrate enantiomers via
the cofactor pyridoxal-5’-phosphate (PLP) and pyridoxamin-5’-phosphate
[
a]
Entry
Enzymes
7
(S)-1 ee
Yield ketone
1a [%]
[
b]
[b]
[
%]
1
2
3
4
5
6
7
10
Vf-wTA + ATA-117
Vf-wTA
–
>99.5 (S)
49 (S)
37 (S)
rac (S)
65 (S)
98 (S)
80 (S)
79 (S)
33 (S)
58 (S)
30 (S)
30 (S)
13 (S)
<0.5
50
7a
7a
7a
7b
7c
7d
7e
7 f
7g
7g
7h
7h
(
PMP); L=large, S=small group.
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
Vf-wTA + ATA-117
64
[c]
58
60
9
Recently, wTAs were intensely investigated for their po-
57
21
29
14
40
8
tential to synthesize nonracemic amines by asymmetric ami-
nation of ketones at the expense of an external amino
1
1
[7m,13,14]
12
donor, such as alanine.
The racemization rate is a
[
[
c]
c]
1
1
3
4
function of the stereoselectivity of the chiral catalysts. How-
ever, since most biocatalysts show high stereoselectivity, rac-
emization based on a single enzyme would be exceedingly
15
11
[
a] Reaction conditions: substrate (50 mm), ketone 7a–h (25 mm), 24 h,
[12c]
slow.
This problem can be overcome by employing two
308C, buffer pH 7.0, PLP (1 mm). [b] Determined by GC analysis; the ee
of the starting material (S)-1 was >99.5%. [c] Reaction was performed
for 48 h.
biocatalysts that exhibit opposite stereopreference, which
ensures fast racemization. Due to the intrinsic excellent
regio- and chemoselectivity, enzymatic racemization is usu-
ally ꢀcleanꢁ and avoids the formation of undesired side prod-
ucts.
To prove this general strategy, we tested wTAs for the
racemization of a-chiral primary amines via the prochiral
ketone intermediate (Scheme 2). As wTAs are enantioselec-
tive in general, only one enantiomer of amines 1–6 is deami-
nated to ketones 1a–6a. Consequently, by employing a
second wTA with opposite stereo-preference, ketones 1a–6a
are reaminated yielding the opposite enantiomer of amines
To facilitate the amino-group transfer between substrate
enantiomers, we modified the system by adding an external
ketone as an amino-acceptor co-substrate, which would act
as shuttle thereby enabling the amino-group transfer be-
tween the two stereocomplementary wTAs (Scheme 2). To
avoid problems with the stereorequirements of the wTAs,
we selected symmetric ketones 7a–h. Racemization experi-
ments in the presence of ketones 7a–h were performed by
using (S)-1-methoxy-2-propylamine (1) as the substrate.
1
–6. If both processes are in equilibrium, overall racemiza-
Chem. Eur. J. 2011, 17, 378 – 383
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
379

K. Faber et al.
By taking a 2:1 ratio of amine and amino-acceptor 7a, 7g,
or 7h as a lead, a range of a-chiral prim-amines 2–4 and 6
was racemized by employing a two-enzyme system; optimal
combinations of substrate amine and amino-group acceptor
ketone are depicted in Table 3. In addition to Vf-wTA and
Table 3. Biocatalytic racemization of a-chiral prim-amines 2–4 and 6 by
employing a pair of stereocomplementary wTAs with amino-group ac-
[
a]
ceptor ketones 7a, 7g, and 7h.
The first proof-of-concept was achieved by using a single
moderately stereoselective wTA from V. fluvialis in the pres-
ence of 0.5 equivalents of 7a as an amino acceptor. Within
[a]
Entry Enzymes
Substrate Acceptor
Substrate Ketone
[
b]
[b]
7
ee [%]
(yield [%])
1
2
3
4
5
6
7
8
9
1
1
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
ATA-113 +
ATA-117
CV-wTA +
ATA-117
BM-wTA +
ATA-117
Vf-wTA +
ATA-117
CV-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
(R)-2
(R)-3
(R)-4
(R)-6
(S)-2
(R)-2
(R)-2
(R)-2
(R)-3
(R)-4
(R)-2
(R)-4
(R)-6
7a
7a
7a
7a
7g
7g
7g
7g
7g
7g
7h
7h
7h
92 (R) 2a (22)
81 (R) 3a (15)
23 (R) 4a (78)
26 (R) 6a (98)
22 (S) 2a (2)
24 h, (S)-1 was partially racemized to 49% ee (Table 1,
entry 2). The efficiency of this system was stepwise im-
proved by applying the enantiocomplementary (R)-selective
wTA ATA-117 (ee=37%, entry 3). Complete racemization
of (S)-1 (ee<5%) was achieved when the reaction time was
extended to 48 h (entry 4). Next, further symmetric ketones
were tested for their potential as amino-group acceptors
(
entries 5–13). Among the test candidates, 7a and 7 f–h
proved to be superior with residual ee values for (S)-1 below
5%. By comparing the structures of amino-group accept-
57 (R) 2a (<1)
46 (R) 2a (<1)
31 (R) 2a (14)
rac (R) 3a (91)
16 (R) 4a (<1)
92 (R) 2a (22)
81 (R) 4a (15)
85 (R) 6a (21)
3
ors, ketones bearing free hydroxy groups (7b) or small (7c)
and large (7d, 7e) ketones appear to be suboptimal, where-
as medium-sized compounds (7 f–h) were clearly superior.
Despite the good racemization rate for (S)-1 achieved
with ketone 7a, the amount of undesired ketone intermedi-
ate 1a remained high (58%, Table 1, entry 4). To shift the
equilibrium back to the amine side, the influence of the con-
centration of amino-acceptor 7a on the equilibrium between
amine 1 and methoxyacetone 1a was studied (Table 2). The
data reveal the following trends: the highest racemization
rate was achieved by using an amine/ketone ratio of 2:1
0
1
12
1
3
Vf-wTA +
ATA-117
[
7
a] Reaction conditions: substrates 2–4 or 6 (50 mm), ketone 7a, 7g, or
h (25 mm), 48 h, 308C, buffer pH 7.0, PLP (1 mm). [b] Determined by
GC analysis; the ee of the starting material was >99.5%.
(
Table 2, entry 4), whereas the lowest concentrations of un-
desired ketone 1a were obtained at 0.1 and 1.0 equivalents
of amino acceptor 7a (entries 1 and 6). Similar results were
obtained when using 3-pentanone 7g (25 mm) and cyclopen-
tanone 7h (25 mm, data not shown), leading to an ee of
wTA ATA-117, racemization was proven for combinations
of other w-transaminases, that is, commercially available
wTA ATA-113 (Table 3, entry 6) and two wTAs from Chro-
mobacterium violaceum (CV-wTA, entries 7 and 10)
and Bacillus megaterium (BM-wTA, entry 8) expressed in
E. coli. It is noteworthy that amines, for example, 1-phenyl-
<
10% for (S)-1 after 48 h.
[
14e,16]
Table 2. Biocatalytic racemization of (S)-1 by employing Vf-wTA and
[17]
[
a]
wTA ATA-117 with increasing amounts of amine-group acceptor 7a.
[
a]
Entry Enzymes
Acceptor 7a
equiv]
(S)-1 ee Yield ketone 1a
[b] [b]
[
[%]
[%]
A
H
U
G
E
N
N
ethylamine (2), which is susceptible to CÀN cleavage going
in hand with the formation of undesired side products by ap-
plying transition-metal-catalyzed racemization, were inter-
1
2
3
4
5
6
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
0.1
0.2
0.3
0.5
0.7
1.0
38 (S)
17 (S)
25 (S)
rac (S)
28 (S)
76 (S)
14
23
36
51
60
12
[6]
converted in a clean fashion without side reactions.
To shift the equilibrium further towards the amine side,
the influence of an external amine donor 8 and 9 was inves-
[18]
tigated.
24 h of shaking at 308C and left for an additional 24 h
Table 4). 2-Propylamine and formed acetone can be easily
In each case, the amino donor was added after
(
[19]
removed from the reaction medium by evaporation.
As shown in Table 4, the addition of an achiral amine
donor 8 or 9 was crucial to shift the equilibrium towards the
amine side and to suppress the formation of undesired
ketone 1a to <10% with the exception of entry 7. Although
[
a] Reaction conditions: substrate (50 mm), 48 h, 308C, buffer pH 7.0,
PLP (1 mm). [b] Determined by GC analysis; the ee of the starting mate-
rial was >99.5%.
380
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Chem. Eur. J. 2011, 17, 378 – 383

Enzymatic Racemization of Amines
FULL PAPER
Table 4. Biocatalytic racemization of amine (S)-1 in presence of amine
[
a]
donors 8 and 9.
[
a]
Entry Enzymes
Ketone Amine 8, 9
(S)-1 ee Yield ketone
[b] [b]
7
([equiv])
[%]
1a [%]
1
2
3
4
5
6
7
8
9
1
1
1
Vf-wTA +
ATA-117
Vf-wTA +
ATA-117
ATA-113 +
ATA-117
ATA-114 +
ATA-117
ATA-113 +
ATA-117
ATA-113 +
ATA-117
ATA-113 +
ATA-117
ATA-113 +
ATA-117
ATA-113 +
ATA-117
Vf-wTA +
ATA-117
ATA-113 +
ATA-117
ATA-114 +
ATA-117
7a
8 (1)
rac
6
7h
7h
7h
7a
7a
7a
7a
7a
7h
7h
7h
8 (1)
8 (1)
8 (1)
8 (1)
8 (5)
8 (20)
9 (1)
9 (5)
9 (1)
9 (1)
9 (1)
10 (S)
7 (S)
50 (S)
rac
5
3
2
2
Scheme 3. Biocatalytic cross-racemization of two-different a-chiral prim-
amines A and B via ketone intermediates C and D by employing two ste-
reocomplementary w-transaminases.
7 (S)
rac
2
23
3
10 (S)
15 (R)
rac
2
deamination/amination sequence of amine substrate enan-
tiomers via the corresponding prochiral ketone intermediate
is presented. Equilibrium-controlled amino-group transfer is
catalyzed by a pair of stereocomplementary w-transaminases
in the presence of symmetric ketone/amine co-substrates,
which served as amino-group shuttles. Careful adjustment of
the ratio between substrate/amine and ketone/amine shut-
tles suppressed the formation of undesired ketones from the
amine substrate. Finally, cross-racemization by using two dif-
ferent enantiocomplementary chiral amines was introduced.
Since this biocatalytic approach operates under mild reac-
tion conditions, ꢀcleanꢁ isomerization is ensured by avoiding
the formation of side products. At this stage, the enzymatic
process is not (yet) competitive to existing chemical racemi-
zation protocols regarding turnover and scale, but it certain-
ly offers a valuable alternative for amines bearing nitro or
0
1
2
8
rac
4
42 (S)
5
[
2
a] Reaction conditions: substrate (50 mm), ketone 7a or 7h (25 mm),
4 h, 308C, buffer pH 7.0, PLP (1 mm); after 24 h amine donor 8 or 9 was
added and shaking was resumed for an additional 24 h. [b] Determined
by GC analysis; the ee of the starting material was >99.5%.
there was no significant influence of the amino-donor con-
centration (1 or 5 equiv) by using amine group acceptors 7a
and 7h, an excess of 20 equivalents of 2-propylamine 8
seemed to cause inhibition of the wTAs (entry 7).
[9]
Finally, a cross-racemization of two different a-chiral
prim-amines A and B was studied (Scheme 3). The impor-
tant advantage of this procedure is the complete absence of
additional co-substrates, ketone 7 and amines 8 or 9, acting
as amino-group acceptors or donors, respectively, which sig-
nificantly simplifies the racemization protocol by avoiding
the necessity for the separation
halogen groups, for which chemical methods fail. In addi-
tion, it offers the possibility for the development of an enzy-
matic dynamic resolution process of rac-amines in combina-
tion with a lipase-catalyzed amide formation operating
under microaqueous conditions, such as water-in-oil-micro-
[20a,b]
[20c,d]
Ae mulsions
H
U
G
R
N
N
or in bisphasic solvent systems.
of co-substrates. In this proto-
col, the amine-substrates A and Table 5. Biocatalytic cross-racemization of pairs of a-chiral prim-amines A/B by using wTA ATA-117 and a
[
a]
B are equilibrated by the corre- second w-TA.
[
a]
[b]
[b]
[b]
sponding achiral ketone inter- Entry
mediates C and D through the
Enzyme
Substrates A/B
Amine A
Ketone C
(yield [%])
Amine B
(ee [%])
Ketone D
ACHTUNGTRENNUNG( yield [%])
[
b]
A( ee [%])
H
U
G
R
N
U
G
A
H
U
G
R
N
U
G
A
H
U
G
R
N
U
G
combined action of both wTAs.
Overall, rapid racemization
rates were achieved and the un-
desired formation of ketones C
and D remained low (Table 5).
1
2
3
4
5
6
ATA-117 +
Vf-wTA
ATA-117 +
Vf-wTA
ATA-117 +
BM-wTA
ATA-117 +
BM-wTA
ATA-117 +
CV-wTA
(S)-1/(R)-2
(S)-2/(R)-4
(S)-2/(R)-4
(S)-1/(R)-5
(S)-3/(R)-4
(R)-3/(S)-4
(S)-1 (16)
(S)-2 (12)
2 rac
1a (<1)
2a (6)
(R)-2 (37)
4 rac
2a (12)
4a (2)
2a (12)
1a (<1)
3a (<1)
3a (8)
4 rac
4a (2)
1 rac
5 rac
5a (27)
4a (<1)
4a (<1)
3 rac
(R)-4 (26)
(S)-4 (27)
Conclusion
ATA-117 +
CV-wTA
(R)-3 (13)
An enzymatic racemization
strategy for a-chiral prim-
[a] Reaction conditions: amines A and B (50 mm each), wTAs (16 mg), 24 h, 308C, buffer pH 7.0, PLP (1 mm).
amines based on a reversible [b] Determined by GC analysis; the ee of the starting material was >99.5%.
Chem. Eur. J. 2011, 17, 378 – 383
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
381

K. Faber et al.
Experimental Section
Acknowledgements
This study was performed in cooperation with BASF AG (Ludwigshafen,
Germany) and K. Ditrich, B. Hauer, R. Stꢃrmer, K. Baldenius, and M.
Bonnekessel are cordially thanked for helpful hints and discussions. Fi-
nancial support by the FFG, SFG, and the Province of Styria is gratefully
acknowledged.
Substrates and reference compounds: Enantiopure amines 1–6 were ob-
tained from BASF (Ludwigshafen, Germany), ketones 1a–6a and 7b–h,
amines 8 and 9, PLP, and PMP were purchased from Sigma–Aldrich.
Ketone 7a was purchased from ABCR (Karlsruhe, Germany).
Enzymes: (R)-Selective wTA ATA-117 (102907WW), (S)-selective wTA
ATA-103 (=Vf-wTA from Vibrio fluvialis, 2100208MW), and ATA-114
(
1100708MW, CV-wTA) were obtained from Codexis. Lyophilized wTAs
from Chromobacterium violaceum and Bacillus megaterium (B-wTA)
were prepared by over-expression in E. coli BL21(DE3)/pET21a as pre-
[
1] a) E. L. Eliel, S. H. Wilen, M. P. Doyle, Basic Organic Stereochemis-
try, Wiley, New York, 2001, p. 277; b) E. J. Ebbers, G. J. A. Ariaans,
J. P. M. Houbiers, A. Bruggink, B. Zwanenburg, Tetrahedron 1997,
AHCTUNGTRENNUNG
[
14a]
viously reported.
5
3, 9417–9476.
General procedure for racemization without amine acceptor 7 (Table 1,
entry 1): Vf-wTA (8 mg), ATA-117 (16 mg), and PLP (final concentration
1
1
(
workup was performed as follows: the pH was adjusted to 12 by using
aq. NaOH (10m, 100 mL) and the resulting mixture was extracted with
EtOAc (500 mL) and centrifuged to achieve phase separation. The organ-
[
2] For reviews, see: a) K. Faber, Chem. Eur. J. 2001, 7, 5004–5010;
b) O. Pꢄmies, J.-E. Bꢅckvall, Chem. Rev. 2003, 103, 3247–3261;
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General procedure for racemization with amine acceptors 7a–h (Tables 1
entries 2–15), 2, and 3)
2 4
SO and filtered.
(
(
2
Vf-wTA and ATA-117: Vf-wTA (8 mg), ATA-117 (16 mg), and PLP (final
concentration 1 mm) were dissolved in phosphate buffer (100 mm, pH 7.0,
total volume 1 mL). The reaction was started by the addition of enantio-
pure (S)-1 (50 mm, 5 mL, ee>99.5%) and ketones 7a–h (25 mm). After
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4 h of shaking (130 rpm) at 308C, workup was performed as described
above.
CV-wTA and ATA-117: Lyophilized cells E. coli BL21 AHCUTNGRNEUNG( DE3)/pET21a-CV-
wATA-CV (100 mg) were rehydrated for 30 min in phosphate buffer
pH 7.0, total volume 1 mL). ATA-117 (16 mg) and PLP (final concentra-
(
tion 1 mm) were added. The reaction was started by the addition of enan-
tiopure (S)-1 (50 mm, 5 mL, ee>99.5%) and ketones 7a–h (25 mm). After
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4 h of shaking (130 rpm) at 308C, workup was performed as described
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8 mg), ATA-117 (16 mg), and PLP (final concentration 1 mm) were dis-
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Received: June 7, 2010
Published online: November 30, 2010
Chem. Eur. J. 2011, 17, 378 – 383
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
383