Asymmetric reductive amination of boron-containing aryl-ketones using ω-transaminases

Article abstract of DOI:10.1016/j.tetasy.2013.10.004

The asymmetric reductive amination of aryl-ketones bearing various boron-functionalities (acid, ester or potassium trifluoroborates) was investigated employing enantiocomplementary ω-transaminases as catalysts. Under the optimized conditions, high convers

Full text of DOI:10.1016/j.tetasy.2013.10.004

Tetrahedron: Asymmetry 24 (2013) 1495–1501
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Asymmetric reductive amination of boron-containing aryl-ketones
using
x-transaminases
a,
Joel S. Reis a, Robert C. Simon b,c, Wolfgang Kroutil b, , Leandro H. Andrade
⇑
⇑
a Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil
b Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstraße 28, 8010 Graz, Austria
c ACIB GmbH, c/o University of Graz, Heinrichstraße 28, 8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 August 2013
Accepted 4 October 2013
The asymmetric reductive amination of aryl-ketones bearing various boron-functionalities (acid, ester or
potassium trifluoroborates) was investigated employing enantiocomplementary -transaminases as cat-
alysts. Under the optimized conditions, high conversions (up to 94%) and excellent ee’s (up to >99%) were
obtained providing access to both (R)- and (S)-configured amino-aryl boronates under mild reaction
conditions.
x
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
ents are also readily incorporated into complex molecules whereas
boronic acids and their derivatives can serve as catalysts (e.g., CBS-
Enantiomerically pure amines are valuable chiral building
blocks for the synthesis of a broad range of biologically active com-
pounds, such as agrochemicals or pharmaceuticals. Since the activ-
ity of these molecules is generally related to one specific absolute
configuration, efficient asymmetric methods are required, in par-
ticular those that are mild and environmentally benign reaction
conditions.1
reduction, Petasis-reaction).13 Simple aryl- and alkyl boronic acids
have recently gained attention from medicinal chemists since they
were found to be potent and efficient enzyme inhibitors (especially
for proteases).14 Curiously, numerous sophisticated chemical
methods for the synthesis of chiral organoboron compounds have
been described15 in the literature but enzymatic routes still remain
scarce.16
Numerous synthetic methods have been described to provide
chiral amines based on the utilization of metal- and organo-
catalysts; chiral auxiliaries are also routinely employed for this
purpose.2 Biocatalysis has emerged as a further alternative due to
the high regio-, chemo- and stereoselectivities commonly dis-
played by enzymes.3 While initially hydrolases were frequently
employed in (dynamic)-kinetic resolution processes,4 monoamine
oxidases (MAO),5 amine-dehydrogenases (AmDH)6 and
Herein various aromatic ketones bearing a boron-atom in the
form of an acid or pinacol ester were investigated as substrates
for
x-transaminases. Since organotrifluoroborates represent a
more stable and less sensitive alternative to boronic acids,17 the
analogues of potassium salts were also prepared and investigated.
2. Results and discussion
x
-transaminases (x
-TA)7 were only applied more recently. The lat-
All of the boron-containing aryl ketones were synthesized
according to Scheme 1. Boronic esters 2a and 2b were readily
obtained via esterification from the corresponding acids 1a–b16f
whereas the trifluoro salts 3a and 3b were obtained by employing
KHF2 as the fluorine source17d (Scheme 1).
ter ones have successfully been used to prepare a variety of enan-
tiomerically pure amines8 including natural products9 and
pharmaceuticals on an industrial scale.10
In order to extend and demonstrate the feasibility of
x-TAs, we
have focused on the asymmetric reductive amination of aromatic
keto-boronates since they are useful reagents and intermediates
in synthetic organic chemistry: boronates are mainly employed
in transition-metal-catalyzed C–C cross-couplings (e.g.,
Suzuki–Miyaura)11 and can also be converted into other functional
groups such as alcohols, aldehydes, or ketones.12 Boron-substitu-
Prior to us investigating the
x-TA catalyzed reductive
amination of organoboron compounds 1–3, two issues had to be
considered first: the equilibrium in these amination-reactions of-
ten favors the side of the ketone rather than the amine and addi-
tional co-product (pyruvate) consumption may hamper the
overall efficiency.7 Nevertheless, the shift of the equilibrium is
achieved by combining the
either with an alanine dehydrogenase (AlaDH) or
x
-TA catalyzed reductive amination
lactate
a
⇑
Corresponding authors. Tel.: +55 11 3091 2287 (L.H.A.).
E-mail addresses: wolfgang.kroutil@uni-graz.at (W. Kroutil), leandroh@iq.usp.br
dehydrogenase (LDH). The AlaDH approach is favored with respect
to atom efficiency since the formed co-product (pyruvate) is
(L.H. Andrade).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.10.004

1496
J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
O
cled with an alanine dehydrogenase (AlaDH). Among the different
enantiocomplementary -TAs tested [ -transaminase (origin):
x
x
O
O
pinacol, THF
Arthrobacter sp., Aspergillus terreus, Hyphomonas neptunium, Chro-
mobacterium violaceum, Bacilus megaterium, Pseudomonas fluores-
cens, Paracoccus denitrificans, Ralstonia eutropha, Pseudomonas
putida, Arthrobacter citreus, and Vibrio fluvialis], some gave reason-
able conversions (between 21% and 68%, detailed data not shown)
B
O
2a; para (86%)
2b; meta (76%)
OH
OH
B
O
which rendered them suitable for a more detailed investigation:
x-
1a; para
1b; meta
TAs from Chromobacterium violaceum,19a Pseudomonas fluores-
cens,19b Arthrobacter citreus,19c (R)-Arthrobacter sp.19d and Aspergil-
lus terreus.8d
KHF2, MeOH
2 h, 0°C
BF3K
3a; para, 79%
3b; meta, 76%
Various water-miscible solvents [1,2-dimethoxyethane (DME),
N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), and
MeOH] were then assayed, since they might be crucial for solubil-
ity reasons (Table 1).20
Scheme 1. Synthesis of aromatic boron-containing ketones 2 and 3.
As expected, a significant influence of the co-solvent on the bio-
transformation was observed (Table 1): in one example, an almost
14 fold increase in conversion could be detected by using 10 vol %
DMSO in comparison to DME using the enzyme from A. citreus
(entry 5). However, the conversions were still poor with 27% at
recycled to an alanine (amine source) whereas the LDH removes it
to form a lactate (Scheme 2).
Both systems have been proven to be efficient.18 A first evalua-
tion for the amination of boronic ester 2a as a model substrate was
performed in order to identify appropriate
centration: 50 mM (12.5 mg/mL), 1.0 mL total volume]. Depending
on the stereoselectivity of the corresponding enzyme, - or -ala-
x-TAs [substrate con-
the maximum. The
x-TA from C. violaceum provided the amine
(S)-4c in moderate to good conversion depending on the solvent
(entry 3): the use of DMF resulted in 38% conversion whereas
D
L
nine was used as the amine source whereas the pyruvate was recy-
O
NH2
ω-transaminase, PLP
BY2
NH2
BY2
O
OH
OH
(R) or (S)-4
1-3
O
O
4a
4b
4c
4d
BY2: para-B(OH)2
meta-B(OH)2
para-BPin
=
=
=
=
recycling via AlaDH
BY2: para-B(OH)2 = 1a
(additional NADH recycling)
1b
2a
2b
meta-B(OH)2
para-BPin
=
=
=
OH
meta-BPin
OH
meta-BPin
para-BF3K = 4e
meta-BF3K = 4f
para BF K = 3a
-
3
meta-BF3K = 3b
O
removing via LDH
(additional NADH recycling)
Scheme 2. Biocatalytical methods to enhance product formation via recycling (AlaDH) or removing (LDH) pyruvate. PLP = pyridoxal-50-phosphate, AlaDH = alanine
dehydrogenase, LDH = lactate dehydrogenase.
Table 1
Asymmetric reductive amination of boronic ester 2a using an AlaDH cofactor recycling system in the presence of 10 vol % organic solventsa
O
NH2
ω-transaminase, PLP
O
O
NH2
O
B
B
OH
OH
O
O
2a
(R) or (S)-4c
O
O
AlaDH
(additional NADH recycling)
Entry
x
-Transaminase
Amine 4c
DMSO
DME
MeOH
Conv.b [%]
DMF
Conv.b [%]
eec [%]
Conv.b [%]
eec [%]
eec [%]
Conv.b [%]
eec [%]
1
2
3
4
5
(R)-Arthrobacter sp
Aspergillus terreus
C. violaceum
P. fluorescens
Arthrobacter citreus
27
89
92
74
33
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
2
83
92
59
42
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
27
74
82
51
51
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
21
72
79
38
13
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
a
Lyophilized E. coli cells containing overexpressed
x-transaminase (20 mg), PLP (1.0 mM), substrate 2a (25 mM), D- or L
-alanine (500 mM), NAD+ (1.0 mM), AlaDH (12 U),
FDH (11 U), ammonium formate (150 mM), and co-solvent (10 vol %), KPi buffer (100 mM, pH 7.0), 30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm).
b
Determined by GC-analysis.
Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
c

J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
1497
Table 2
Asymmetric reductive amination of boronic ester 2a using pyruvate removing system (LDH)a
O
NH2
ω-transaminase, PLP
O
O
O
NH2
B
B
OH
OH
O
O
2a
(R) or (S)-4c
LDH
OH
O
O
OH
(additional NADH recycling)
O
Entry
x
-Transaminase
Conversionb [%]
eec [%]
1
2
3
4
5
(R)-Arthrobacter sp.
Aspergillus terreus
C. violaceum
P. fluorescens
Arthrobacter citreus
58
81
52
11
4
>99 (R)
>99 (R)
98 (S)
n.d.
n.d.
a
Lyophilized E. coli cells containing overexpressed
x-transaminase (20 mg), PLP (1.0 mM), substrate 2a (25 mM), D- or L
-alanine (500 mM), NAD+ (1.0 mM), LDH (90 U),
GDH (15 U), glucose (150 mM) and DMSO (10 vol %), KPi buffer (100 mM, pH 7.0), 30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm).
b
Determined by GC-analysis.
c
Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide; n.d. = not determined.
74% conversion was achieved in DMSO. On the other hand, both
(R)-selective -TAs (originating from A. terreus and Arthrobacter
Other reaction-relevant parameters such as the alanine-concen-
x
tration were also examined separately and optimized with respect
to the conversion (Table 4). We found that the amount of alanine
could be decreased from 500 to 250 mM and still provide amine
4c in enantiomerically pure form and with good conversions
(60–86%, entries 2, 5 and 8).
sp.) gave excellent results affording amine (R)-4c between 89%
and 92% (entries 1 and 2) in the presence of 10 vol % DMSO. A fur-
ther enhancement of DMSO to 20 or 40 vol % generally gave lower
conversions (between 9% and 66%; data not shown), most likely
due to an inactivation of one of the enzymes by the organic media.
As a result, further studies were performed with only 10 vol %
DMSO since this proved to be the best among the solvents tested.
It should be noted that in all cases, the enantiomeric purities were
excellent with ee’s >99% for the (S)- as well as for the (R)-
enantiomer.
Removing pyruvate (the co-product from the amination reac-
tion) by a lactate-dehydrogenase (LDH) as an alternative to shift
the equilibrium did not enhance the amine formation either, as
demonstrated in a separate study (Table 2, between 4% and 81%
conversion; ee >99% in all cases).
Table 4
Asymmetric reductive amination of 2a by different alanine concentrations using an
AlaDH cofactor recycling systema
Entry
x
-Transaminase
Alanine [mM]
Conversionb [%]
eec [%]
1
2
3
4
5
6
7
8
9
(R)-Arthrobacter sp.
(R)-Arthrobacter sp.
(R)-Arthrobacter sp.
Aspergillus terreus
Aspergillus terreus
Aspergillus terreus
C. violaceum
500
250
125
500
250
125
500
250
125
90
86
65
82
77
68
43
60
48
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
The reactions were also tested at elevated temperatures (40 °C)
(Table 3), leading to lower conversions in comparison to the exper-
iments at 30 °C (Table 1). This could be rationalized by the partial
inactivation of the transaminase and/or any other enzyme em-
ployed for the cofactor recycling, although the stereochemical out-
come remained excellent (ee >99%) in all cases.
C. violaceum
C. violaceum
a
Lyophilized E. coli cells containing overexpressed
x-transaminase (20 mg), PLP
(1.0 mM), 2a (25 mM), - or
D
L
-alanine, NAD+ (1.0 mM), AlaDH (12 U), FDH (11 U),
ammonium formate (150 mM) and DMSO (10 vol %), KPi buffer (100 mM, pH 7.0),
30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm).
b
Determined by GC-analysis.
Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the
c
corresponding acetamide.
Table 3
Asymmetric reductive amination of boronic ester 2a at 40 °C using AlaDH cofactor
recycling systema
Having established a suitable protocol for the enzymatic reduc-
tive amination, we turned our attention to the analytics in order to
determine the enantiomeric purities and conversions in a fast and
reliable fashion for boronic acids 1a–b and trifluoro derivatives 3a–
b. For analytical reasons, the transformation of the formed amino-
boronic acids 4a/4b and amino-trifluoroborates 4e/4f into the cor-
responding pinacol esters 4c/4d seemed to be therefore the meth-
od of choice (Scheme 3): the boronic acids 4a and 4b were readily
esterified with pinacol in THF, while an adapted protocol from
Molander et al. was applied for the potassium trifluoroborates 4e
and 4f.21 This method not only allows us to obtain the boronic
acids 4a/4b from the analogues of trifluoro-salts 4e/4f in a mild
Entry
x-Transaminase
Conv.b [%]
eec [%]
1
2
3
4
5
(R)-Arthrobacter sp.
A. terreus
C. violaceum
P. fluorescens
A. citreus
75
73
50
33
20
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
a
Lyophilized E. coli cells containing overexpressed
(1 mM), substrate 2a (25 mM), - or L
x
-transaminase (20 mg), PLP
D
-alanine (500 mM), NAD+ (1 mM), AlaDH
(12 U), FDH (11 U), ammonium formate (150 mM), KPi buffer (100 mM, pH 7.0) plus
10 vol % DMSO, 40 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm).
b
Determined by GC-analysis.
Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the
corresponding acetamide.
c

1498
J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
BY2 = B(OH)2: (ii)
O
NH2
NH2
O
O
(i)
BY2
BY2
B
BY2 = BF3K: (iii)
(R) or (S)-4
(R)- or (S)-4
para-BPin = 4c
meta-BPin = 4d
para-B(OH)2 = 4a
meta-B(OH)2 = 4b
BY2:
4e
4f
para-BF3K
meta-BF3K
=
=
Scheme 3. Synthesis of amino-boronates 4a,b,e,f and their conversion into the pinacol esters 4c and 4d. (i)
ammonium formate, buffer pH 7.0 plus 10 vol % DMSO, 30 °C, 24 h (as shown in Table 5); (ii) pinacol, THF; (iii) silica, pinacol.
x-Transaminase, D- or L
-alanine, PLP, NAD+, AlaDH, FDH,
and effective way, but also to convert them into the analogous
pinacol esters 4c and 4d in a one pot protocol.
meta-derived pinacol-ester 2b in 62% yield (ee >99%), whereas
the -TA from A. terreus was not suitable (entries 10–12). The
same trend was also observed for the (S)-selective -TA from C.
x
The scope of the methodology was then investigated but omit-
x
ting the
zymes generally gave only moderate conversions.
We first examined the (R)-selective -TA employing the orga-
x
-TAs from A. citreus and P. fluorescens since these en-
violaceum: Even though the para-isomers were converted in up
to 68%, the meta-isomers led to lower conversions (e.g., 51%, pina-
col ester 2a). Nevertheless, the stereochemical outcome remained
excellent throughout with ee’s >98% for all the experiments.
x
noboron compounds 1–3 (Table 5). Excellent conversions were ob-
served; in the case of the para-substituted boron species, the
pinacol ester 2a was converted by both enzymes with 94% conver-
sion to provide the corresponding amine 4c in perfect enantio-
meric purity (ee >99%; entries 1 and 7). Boronic acid 1a and
trifluoro-salt 3a were also readily accepted to furnish products
4a and 4e in good yields (56–94%) and excellent stereoselectivies
(ee >99%).
3. Conclusion
In conclusion, various boron-containing ketones were subjected
to
x-TA catalyzed asymmetric reductive amination for the first
time. We found that not just only boronic esters but also boronic
acids and their trifluoro salts were tolerated, thus opening up a
new access to enantiomerically pure (R)- and (S)-amino-boronates.
Among the various enzymes tested, the ones originating from (R)-
Arthrobacter sp. and A. terreus afforded the corresponding (R)-con-
figured amines in up to 94% conversion with perfect stereocontrol
(ee’s >99%). The analogous (S)-enantiomers were obtained at
Table 5
Scope of the asymmetric reductive amination of aromatic keto-boronates 1–3
employing enantiocomplementary
x
-TAsa
Entry
x
-Transaminase
BY2 (ketone)
Amine 4
diminished conversions when employing the
um, although the enantiomeric purities were outstanding with up
to ee >99%. Our results showed that the -transaminases investi-
x-TA from C. violace-
Conv.b [%]
eec [%]
x
1
2
3
4
5
6
(R)-Arthrobacter sp.
para-B(OH)2 1a
para-Bpin 2a
para-BF3K 3a
meta-B(OH)2 1b
meta-Bpin 2b
meta-BF3K 3b
56
94
70
54
62
n.r.
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
n.a.
gated transformed acetophenone derivatives containing boron
substituents while maintaining perfect stereoselectivity. Further
studies regarding the application of the new amino-boronates are
currently under investigation and will be reported in due course.
7
8
9
10
11
12
A. terreus
para-B(OH)2 1a
para-Bpin 2a
para-BF3K 3a
meta-B(OH)2 1b
meta-Bpin 2b
meta-BF3K 3b
94
94
81
4
5
n.r.
>99 (R)
>99 (R)
>99 (R)
n.d.
n.d.
n.a.
4. Experimental
4.1. General
All starting materials were obtained from commercial suppliers
and used as received unless stated otherwise. GC–MS spectra were
recorded with an Agilent 7890A GC-system, equipped with an Agi-
lent 5975C mass selective detector and a HP-5 MS column
13
14
15
16
17
18
C. violaceum
para-B(OH)2 1a
para-Bpin 2a
para-BF3K 3a
meta-B(OH)2 1b
meta-Bpin 2b
meta-BF3K 3b
68
65
11
30
51
n.r.
>99 (S)
>99 (S)
>99 (S)
98 (S)
98 (S)
n.a.
(30 m ꢀ 0.25 mm ꢀ 0.25
l
m;
helium
as
carrier
gas
[flow = 0.55 mL/min]).
a
Lyophilized E. coli cells containing overexpressed
(1 mM), substrate 1–3 (25 mM), - or
(12 U), FDH (11 U), ammonium formate (150 mM), KPi buffer (100 mM, pH 7.0) plus
10 vol % DMSO, 30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm) (in the case of
boronic salt was not used any organic solvent).
Determined by GC-analysis.
Determined by chiral GC-analysis after derivatization to the corresponding
acetamides (Sections 4.5–4.7). n.r.: no reaction; n.a.: not accessible, n.d.: not
determined.
x-transaminase (20 mg), PLP
L
-Lactate dehydrogenase (LDH) from rabbit muscle [lyophilized
D
L
-alanine (250 mM), NAD+ (1 mM), AlaDH
powder, 136 U mgꢁ1 protein (one unit will reduce 1.0
lmol of
pyruvate to
L
-lactate per min at pH 7 at 25 °C), catalog no.
61309] was purchased from Sigma–Aldrich. Glucose dehydroge-
b
nase [GDH; lyophilized powder, 25 U mgꢁ1 (one unit will oxidize
c
1 lmol b-D-glucose to D-glucono-d-lactone per min at pH 8.0 and
37 °C), catalogue no. B-4] was purchased from X-zyme (Düsseldorf,
Germany). Formate dehydrogenase (FDH) from Candida boidinii
[aqueous buffer solution with glycerol, 215 U mLꢁ1 (one unit will
The meta-substituted boron-derivatives 1b, 2b, and 3b however
gave diminished conversions likely due to the enhanced steric hin-
drance. Nevertheless, the best results regarding the regioisomers
were obtained with (R)-Arthrobacter sp., which converted the
oxidize 1.0 lmole of formate to CO2 per min at pH 7.6 at 37 °C),
catalogue no. FDH 002] and b-NAD free acid were purchased from
Codexis. Lyophilized Escherichia coli cells containing overexpressed
x
-transaminases (x L-alanine
-TA)22 as well as recombinant

J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
1499
dehydrogenase (AlaDH) were prepared and purified as described
recently.22c
4.2.2. Synthesis of potassium trifluoroborates 3a and 3b17d
To a 50 mL round-bottomed flask containing the appropriated
boronic acid (6.1 mmol, 1.0 g) and MeOH (10 mL) was added a
solution of KHF2 (31 mmol, 2.41 g) in distilled water (7 mL) at
0 °C. The mixture was stirred for 2 h, at 0 °C, after which the sol-
vent was completely removed under reduced pressure. The crude
solid was washed with hot acetone (3 ꢀ 20 mL) and filtered off.
In a beaker under heating, the volume of acetone was reduced by
half. Next, Et2O (0.5 mL) was added and the solid was precipitated
on an ice bath. The solid was filtered using Büchner apparatus and
4.2. Synthesis of boron-containing ketones
The boron-containing ketones 1a and 1b are commercially
available from Sigma–Aldrich. Ketones 2a,b, 3a,b and the standard
chiral amines 4c and 4d were synthesized as previously
described.16e,f,17d
4.2.1. Synthesis of pinacol boronic esters 2a and 2b16f
dried under high vacuum. Data for organoboron compound 3a: 1
H
To a 50 mL round-bottomed flask containing anhydrous THF
(30 mL) were added the appropriate acetylphenyl boronic acid
(5 mmol, 820 mg) and the pinacol (5 mmol, 590 mg). This solution
was evaporated under reduced pressure at 40 °C. The addition
(30 mL) and evaporation of THF were repeated (usually twice) until
TLC analysis indicated complete conversion. The crude product
was purified by column chromatography (PE/EtOAc = 8:2) to afford
the pinacol esters as white solids. Data for organoboron compound
2a: white solid (mp = 66.3–67.7 °C); 1H NMR (200 MHz, CDCl3) d
[ppm] = 7.91 (m, 5H); 2.62 (s, 3H); 1.36 (s, 12H). 13C NMR
(50 MHz, CDCl3) d [ppm] = 198.4, 139.0, 134.9, 127.2, 84.2, 26.7,
NMR (200 MHz, DMSO-d6) d [ppm] = 7.71 (d, J = 7.8 Hz, 2H), 7.45
(d, J = 7.8 Hz, 2H), 2.51 (s, 3H). 13C NMR (50 MHz, DMSO-d6) d
[ppm] = 198.0, 134.1, 131.3 (d, J = 1.6 Hz), 126.2, 26.4. FT-IR (KBr)
m
max = 2944, 1655, 1398, 1285, 1206, 974, 824, 758, 650, 602,
484 cmꢁ1 Data for organoboron compound 3b: 1H NMR
.
(200 MHz, DMSO-d6) d [ppm] = 7.93 (s, 1H), 7.66 (m, 1H), 7.58
(m, 1H), 7.24 (m, 1H), 2.52 (s, 3H). 13C NMR (50 MHz, DMSO-d6)
d [ppm] = 198.7, 136.3 (d, J = 1.6 Hz), 135.0, 131.1 (d, J = 1.7 Hz),
126.4, 124.9, 26.5. FT-IR (KBr)
mmax = 2930, 1680, 1578, 1359,
1086, 901, 806, 701, 627, 609, 595 cmꢁ1
.
24.8. FT-IR (KBr)
m
max = 2988, 1680, 1359, 1093, 1016, 857, 832,
4.3. General procedure for the reductive amination using an
AlaDH cofactor recycling system
654, 599 cmꢁ1. Data for organoboron compound 2b: white solid
(mp = 50.7–52.5 °C). 1H NMR (200 MHz, CDCl3) d [ppm] = 8.36 (s,
1H), 8.06 (dt, J = 7.8 and 1.6 Hz, 1H), 7.99 (dt, J = 7.4 and 1.2 Hz,
1H), 7.47 (t, J = 7.8 Hz, 1H), 2.64 (s, 3H), 1.36 (s, 12H). 13C NMR
Lyophilized cells of E. coli containing the corresponding overex-
pressed
x-transaminase (20 mg) were rehydrated in a potassium
(50 MHz, CDCl3)
128.0, 84.1, 26.7, 24.8. FT-IR (KBr)
979, 851, 668 cmꢁ1
d
[ppm] = 198.3, 139.3, 136.5, 134.7, 130.7,
phosphate buffer (pH 7.0, 100 mM) containing PLP (1.0 mM),
mmax = 2978, 1712, 1384, 1144,
NAD+ (1.0 mM), ammonium formate (150 mM), FDH (11 U), AlaDH
.
(12 U), and D- or L-alanine (500 mM) at room temperature for
O
NH
O
B
O
(S)-4a-Ac
O
NH
O
B
O
rac-4a-Ac
O
NH
O
B
O
(R)-4a-Ac
Figure 1. GC analysis of the acetylated form of compound 4a (4a-Ac).

1500
J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
30 min. The substrate (25 mM, dissolved in 100
l
L DMSO) was
vacuum and heating (Centrifuge Genevac EZ2 plus) for 1.5 h. The
L) and the superna-
added afterward and the reductive amination carried out at 30 °C
in an Eppendorf ThermomixerÒ (700 rpm) for 24 h (horizontal posi-
tion). After 24 h, the reactions were stopped by the addition of sat-
resultant solid was washed with THF (4 ꢀ 500
l
tant was filtered on a Celite pad. Pinacol (1.05 equiv) was then
added and the THF was evaporated by centrifugation under vacuum
and heating. The addition (1.5 mL) and evaporation of THF were re-
peated (twice). This final content was submitted to an acetylation
protocol (Section 4.7) in order to determine the ee (Section 4.9).
urated Na2CO3 (200
lL). The residue was extracted with EtOAc
(2 ꢀ 500
l
L). The combined organic layers were dried over MgSO4
and an aliquot withdrawn for further analysis (Sections 4.8 and 4.9).
4.4. General procedure for the reductive amination using an
LDH cofactor recycling system
4.6. General procedure to convert the potassium organotrifluoro-
borates 4e,f into their respective pinacol esters 4c,d after enzy-
matic reaction
Lyophilized cells of E. coli containing the corresponding overex-
pressed
phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM), NAD+
(1 mM), glucose (150 mM), LDH (90 U), GDH (15 U), and - or -ala-
nine (500 mM) at room temperature for 30 min. The substrate 2a
(25 mM, dissolved in 100 L DMSO) was added afterward and
the reductive amination carried out at 30 °C in an Eppendorf Ther-
momixerÒ (600 rpm) for 24 h (horizontal position). After the indi-
cated time, the reaction was stopped by the addition of saturated
x
-transaminase (20 mg) were rehydrated in a potassium
The enzymatic reactions were carried out according to the con-
ditions described in the footnotes of the Tables. After the reaction
time, the crude mixture was centrifuged and the supernatant was
transferred to a flask charged with pinacol (1.05 equiv) and silica
(1.3 equiv) under an argon atmosphere. After 2 h of stirring, the
reaction was extracted with EtOAc (2 ꢀ 2 mL). This final content
was submitted to an acetylation protocol (Section 4.7) in order to
determine the ee (Section 4.9).
D
L
l
Na2CO3 (200
lL). The residue was extracted with EtOAc
(2 ꢀ 500
l
L). The combined organic layers were dried over MgSO4
4.7. General procedure for the derivatization of aminoboronic
pinacol esters 4c,d to the corresponding acetamides
and an aliquot withdrawn for GC analysis (Sections 4.8 and 4.9).
4.5. General procedure to convert the boronic acids 4a,b into
their respective pinacol esters 4c,d after enzymatic reaction
The crude aminoboronic pinacol esters 4c,d were dissolved in
EtOAc (500
lL) and treated with pyridine (50 lL) and acetic anhy-
dride (50 L) after extraction of the biotransformation. The acety-
l
The enzymatic reactions were carried out as described above.
After reaction, the water was evaporated by centrifugation under
lation was performed at 30 °C for 12 h in an Eppendorf
ThermomixerÒ (700 rpm). After the indicated time, the reactions
O
NH
NH
NH
O
B
O
O
O
(S)-4b-Ac
O
O
B
rac-4b-Ac
O
O
B
(R)-4b-Ac
Figure 2. GC analysis of the acetylated form of compound 4b (4b-Ac).

J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
1501
8. Selected examples: (a) Wang, B.; Land, H.; Berglund, P. Chem. Commun. 2013,
161–163; (b) Midelfort, K. S.; Kumar, R.; Han, S.; Karmilowicz, M. J.; McConnell,
K.; Gehlhaar, D. K.; Mistry, A.; Chang, J. S.; Anderson, M.; Villalobos, A.;
Minshull, J.; Govindarajan, S.; Wong, J. W. Protein Eng., Des. Sel. 2013, 26, 25–33;
(c) Steffen-Munsberg, F.; Vickers, C.; Thontowi, A.; Schätzle, S.; Meinhardt, T.;
Svedendahl Humble, M.; Land, H.; Berglund, P.; Bornscheuer, U. T.; Höhne, M.
ChemCatChem 2013, 5, 154–157; (d) Pressnitz, D.; Fuchs, C. S.; Sattler, J. H.;
Knaus, T.; Macheroux, P.; Mutti, F. G.; Kroutil, W. ACS Catal. 2013, 3, 555–559;
(e) Sattler, J. H.; Fuchs, M.; Tauber, K.; Mutti, F. G.; Faber, K.; Pfeffer, J.; Haas, T.;
Kroutil, W. Angew. Chem., Int. Ed. 2012, 51, 9156–9159.
were quenched with a saturated NH4Cl solution and aliquots were
withdrawn for determination of the enantiomeric excess
(Section 4.9).
4.8. GC analysis for the determination of the conversion of keto-
nes into amines
9. (a) Simon, R. C.; Zepeck, F.; Kroutil, W. Chem. Eur. J. 2013, 19, 2859–2865; (b)
Simon, R. C.; Fuchs, C. S.; Lechner, H.; Zepeck, F.; Kroutil, W. Eur. J. Org. Chem.
2013, 3397–3402; (c) Simon, R. C.; Grischek, B.; Zepeck, F.; Steinreiber, A.;
Belaj, F.; Kroutil, W. Angew. Chem., Int. Ed. 2012, 51, 6713–6716.
Determination of the conversion by means of GC measure-
ments: column DB-1701 (Agilent J&W, 30 m ꢀ 0.25 mm ꢀ 0.25
l
m); temperature program: starting from 100 to 280 °C with a
slope of 10 °C/min; Rt (amine 4a) = 10.83 min; Rt (ketone
2a) = 11.17 min; Rt (amine 4b) = 10.46 min; Rt (ketone
2b) = 10.94 min.
10. For an application of an engineered x-TA in industry see: (a) Savile, C. K.; Janey,
J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Javis, W. R.; Colbeck, J. C.; Krebber,
A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.; Hughes, G. J. Science
2010, 329, 305–309; for the synthesis of the dual orexin inhibitor suvorexant
see: (b) Mangion, I. K.; Sherry, B. D.; Yin, J.; Fleitz, F. J. Org. Lett. 2012, 14, 3458–
3461; for CRTH2 antagonist MK-7246 see: (c) Molinaro, C.; Bulger, P. G.; Lee, E.
E.; Kosjek, B.; Lau, S.; Gauvreau, D.; Howard, M. E.; Wallace, D. J.; O’Shea, P. D. J.
4.9. GC analysis for the determination of the enantiomeric ex-
cesses
Org. Chem. 2012, 77, 2299–2309; for the
x-TA catalyzed synthesis of
rivastigmine see: (d) Fuchs, M.; Koszelewski, D.; Tauber, K.; Sattler, J.; Banko,
W.; Holzer, A. K.; Pickl, M.; Kroutil, W.; Faber, K. Tetrahedron 2012, 68, 7691–
Determination of the enantiomeric excess (ee) by GC on a chiral
7694; for the
x-TA synthesis of mexiletine see: (e) Koszelewski, D.; Pressnitz,
phase: column hydrodex-b-TBDAc (50 m ꢀ 0.25 mm ꢀ 0.25
lm;
D.; Clay, D.; Kroutil, W. Org. Lett. 2009, 11, 4810–4812.
Marcherey–Nagel).
11. (a) Martin, R.; Buchwald, S. L. Acc. Chem. Rev. 2008, 41, 1461–1473; (b)
Molander, G. A.; Ellis, N. Acc. Chem. Rev. 2007, 40, 275–286; (c) Miyaura, N.;
Suzuki, A. Chem. Rev. 1995, 95, 2457–2483.
Acetylated derivative of compound 4a (4a-Ac) (Fig. 1): temper-
ature program = from 200 (keep for 10 min isotherm) to 230 °C
12. (a) Morrill, C.; Funk, T. W.; Grubbs, R. H. Tetrahedron Lett. 2004, 45, 7733–7736;
(b) Hall, D. G. Boronic Acids: Preparation, Aplications Aplications in Organic
Synthesis and Medicine; Wiley-VCH: Weinheim, Germany, 2005.
13. For organoboron compounds as catalysts see: (a) Dimitrijevic´, E.; Taylor, M. S.
ACS Catal. 2013, 3, 945–962; (b) Georgiou, I.; Ilyashenko, G.; Whiting, A. Acc.
Chem. Res. 2009, 42, 756–768. and references cited therein; for organoboron
compounds in the Petasis–Borono–Mannich reaction see: (c) Candeias, N. R.;
Montalbano, F.; Cal, P. M. S. D.; Gois, P. M. P. Chem. Rev. 2010, 110, 6169–6193.
and references cited therein.
with slope 1.0 °C minꢁ1
; Rt [(R)-4a-Ac] = 35.9 min, Rt [(S)-4a-
Ac] = 36.3 min.
Acetylated derivative of compound 4b (4b-Ac) (Fig. 2): temper-
ature program = starting from 190 (keep for 10 min isotherm) to
230 °C with slope 1.0 °C minꢁ1; Rt [(R)-4b-Ac] = 39.3 min, Rt [(S)-
4b-Ac] = 39.7 min.
14. Smoum, R.; Rubinstein, A.; Dembitsky, V. M.; Srebnik, M. Chem. Rev. 2012, 112,
4156–4220. and references cited therein..
Acknowledgements
15. For some examples see: (a) Zhao, L.; Ma, Y.; He, F.; Duan, W.; Chen, J.; Song, C. J.
Org. Chem. 2013, 78, 1677–1681; (b) Ibrahem, I.; Breistein, P.; Córdova, A.
Angew. Chem., Int. Ed. 2011, 50, 12036–12041; (c) Park, J. K.; Lackey, H. H.;
Ondrusek, B. A.; McQuade, D. T. J. Am. Chem. Soc. 2011, 133, 2410–2413; (d)
Chen, I.-H.; Kanai, M.; Shibasaki, M. Org. Lett. 2010, 12, 4098–4101.
16. (a) Reis, J. S.; Andrade, L. H. Tetrahedron: Asymmetry 2012, 23, 1294–1300; (b)
Brondani, P. B.; Dudek, H.; Reis, J. S.; Fraaije, M. W.; Andrade, L. H. Tetrahedron:
Asymmetry 2012, 23, 703–708; (c) Brondani, P. B.; de Gonzalo, G.; Fraaije, M.
W.; Andrade, L. H. Adv. Synth. Catal. 2011, 353, 2169–2173; (d) Barcellos, T.;
Tauber, K.; Kroutil, W.; Andrade, L. H. Tetrahedron: Asymmetry 2011, 22, 1772–
1777; (e) Andrade, L. H.; Barcellos, T.; Santiago, C. G. Tetrahedron: Asymmetry
2010, 21, 2419–2424; (f) Andrade, L. H.; Barcellos, T. Org. Lett. 2009, 11, 3052–
3055.
17. (a) Darses, S.; Genet, J.-P. Chem. Rev. 2008, 108, 288–325. and references cited
therein; (b) Molander, G. A.; Ellis, N. Acc. Chem. Res. 2007, 40, 275–286. and
references cited therein; (c) Stefani, H. A.; Cella, R.; Vieira, A. S. Tetrahedron
2007, 63, 3623–3658; (d) Molander, G. A.; Biolatto, B. J. Org. Chem. 2003, 68,
4302–4314.
18. (a) Wang, B.; Land, H.; Berlund, P. Chem. Commun. 2013, 161–163; (b)
Cassimjee, K. E.; Branneby, C.; Abedi, V.; Wells, A.; Berglund, P. Chem. Commun.
2010, 5569–5571; (c) Truppo, M. D.; Turner, N. J.; Rozzell, J. D. Chem. Commun.
2009, 2127–2129; (d) Koszelewski, D.; Lavandera, I.; Clay, D.; Guebitz, G. M.;
Rozzell, D.; Kroutil, W. Angew. Chem., Int. Ed. 2008, 47, 9337–9340; (e) Höhne,
M.; Kühl, S.; Robins, K.; Bornscheuer, U. T. ChemBioChem 2008, 9, 363–365; (f)
Yun, H.; Yang, Y.-H.; Cho, B.-K.; Hwang, B.-Y.; Kim, B.-G. Biotechnol. Lett. 2003,
25, 809–814.
We are grateful for the financial support by Fapesp, CNPq and
Capes. R.C.S. was supported by the Austrian BMWFJ, BMVIT, SFG,
Standortagentur Tirol and ZIT through the Austria FFG-COMET-
Funding Program.
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