Stereoselective Access to 1-[2-Bromo(het)aryloxy]propan-2-amines Using Transaminases and Lipases; Development of a Chemoenzymatic Strategy Toward a Levofloxacin Precursor

Article abstract of DOI:10.1021/acs.joc.6b01828

Two independent enzymatic strategies have been developed toward the synthesis of enantioenriched 1-[2-bromo(het)aryloxy]propan-2-amines. With that purpose a series of racemic amines and prochiral ketones have been synthesized from commercially available 2-bromophenols or brominated pyridine derivatives bearing different pattern substitutions in the aromatic ring. Biotransamination experiments have been studied using ketones as starting materials, yielding both the (R)- and (S)-amine enantiomers with high selectivity (91-99% ee) depending on the transaminase source. In a complementary approach, the classical kinetic resolutions of the racemic amines have been investigated using Candida antarctica lipase type B as biocatalyst. Ethyl methoxyacetate was found as a suitable acyl donor leading to the corresponding (S)-amines (90-99% ee) and (R)-amides (88-99% ee) with high selectivity in most of the cases. A preparative biotransamination process has been developed for the synthesis of (2S)-1-(6-bromo-2,3-difluorophenoxy)propan-2-amine in 61% isolated yield after 24 h, a valuable precursor of the antimicrobial agent Levofloxacin.

Full text of DOI:10.1021/acs.joc.6b01828

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Article
Stereoselective access to 1-(2-bromo(het)aryloxy)propan-2-
amines using transaminases and lipases. Development of a
chemoenzymatic strategy toward a Levofloxacin precursor.
Ángela Mourelle-Insua, María López-Iglesias, Vicente Gotor, and Vicente Gotor-Fernández
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01828 • Publication Date (Web): 23 Sep 2016
Downloaded from http://pubs.acs.org on September 25, 2016
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Stereoselective access to 1-[2-
bromo(het)aryloxy]propan-2-amines using
transaminases and lipases. Development of a
chemoenzymatic strategy toward a Levofloxacin
precursor
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Ángela Mourelle-Insua,+ María López-Iglesias,+ Vicente Gotor and Vicente Gotor-Fernández*
Organic and Inorganic Chemistry Department, Biotechnology Institute of Asturias (IUBA), University
of Oviedo, Avenida Julián Clavería s/n, 33006 Oviedo, Spain.
+ These authors contributed equally to this work.
Corresponding author: vicgotfer@uniovi.es
Phone: +34 98 5103454. Fax: +34 98 5103446
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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Abstract
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Two independent enzymatic strategies have been developed toward the synthesis of enantioenriched 1ꢀ
[2ꢀbromo(het)aryloxy]propanꢀ2ꢀamines. With that purpose a series of racemic amines and prochiral
ketones have been synthesized from commercially available 2ꢀbromophenols or brominated pyridine
derivatives bearing different pattern substitutions in the aromatic ring. Biotransamination experiments
have been studied using ketones as starting materials, yielding both the (R)ꢀ and (S)ꢀamine enantiomers
with high selectivity (91ꢀ99% ee) depending on the transaminase source. In a complementary approach,
the classical kinetic resolutions of the racemic amines have been investigated using Candida antarctica
lipase type B as biocatalyst. Ethyl methoxyacetate was found as a suitable acyl donor leading to the
corresponding (S)ꢀamines (90ꢀ99% ee) and (R)ꢀamides (88ꢀ99% ee) with high selectivity in most of the
cases. A preparative biotransamination process has been developed for the synthesis of (2S)ꢀ1ꢀ(6ꢀ
bromoꢀ2,3ꢀdifluorophenoxy)propanꢀ2ꢀamine in 61% isolated yield after 24 h, a valuable precursor of the
antimicrobial agent Levofloxacin.
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Keywords: Asymmetric synthesis/ Benzoxazine/ Biocatalysis/ Levofloxacin / Lipases / Transaminases
Introduction
Chiral amines are attractive building blocks for the synthesis of biologically active and high addedꢀ
value products with interest in different chemical industrial sectors.1 Remarkably, the use of
biotransformations provides nowadays a plethora of possibilities for the design of stereoselective routes
toward enantiopure amines and their derivatives.2 Optically active 1ꢀaryloxyꢀpropanꢀ2ꢀamines (Scheme
1, R2= NH2) are particularly attractive nitrogenous compounds, the absolute configuration of their chiral
center having a remarkable importance in their biological profiles.3 From this family, [1ꢀ(2,6ꢀ
dimethylphenoxy)propanꢀ2ꢀamine, also called as mexiletine, has attracted great attention for clinical
purposes due to its properties as antiarrhythmic agent.4 In the last years, the versatility of biocatalytic
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reactions have been demonstrated toward the asymmetric synthesis of the non substituted 1ꢀphenoxyꢀ
propanꢀ2ꢀamine by means of lipaseꢀcatalyzed resolutions,5 and more recently biotransamination
reactions from the corresponding propanones.6 Remarkably, Turner and coꢀworkers reported the
conversion of racemic alcohols into enantiopure amines through a redox selfꢀsufficient enzymatic
cascade using an alcohol dehydrogenase and an amine dehydrogenase, yielding among other chiral
amines, the enantiopure (R)ꢀ1ꢀphenoxyꢀpropanꢀ2ꢀamine in 84% conversion.7
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Scheme 1. Chemoenzymatic routes toward valuable benzoxazine precursors from 1ꢀ(2ꢀnitrophenoxy)ꢀ
propanꢀ2ꢀones (left) and 1ꢀ(2ꢀbromophenoxy)ꢀpropanꢀ2ꢀones (right).
The introduction of additional functionalities provides new opportunities in medicinal and synthetic
chemistry. In this context, we have recently described the versatility of 1ꢀ(2ꢀnitroaryloxy)ꢀpropanꢀ2ꢀ
ones for the synthesis of benzoxazine fragments (Scheme 1), including the preparation of (S)ꢀ(–)ꢀ7,8ꢀ
difluoroꢀ3ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine, a key precursor of the antimicrobial agent
Levofloxacin (Figure 1).8 On the one hand, lipase from Rhizomucor miehei was able to catalyze with
excellent selectivity the resolution of racemic alcohols and acetates through acylation and hydrolysis
reactions, respectively. On the other hand, various alcohol dehydrogenases (ADHs) led to the
production of both (R) and (S)ꢀ1ꢀ(2ꢀnitroaryloxy)ꢀpropanꢀ2ꢀols with different pattern substitution in the
aromatic ring using the evoꢀ1.1.200 ADH and the ADH from Rhodococcus ruber (ADHꢀA),
respectively.
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Figure 1. Structures of (S)ꢀ(–)ꢀ7,8ꢀdifluoroꢀ3ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine (left) and
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the antimicrobial agent Levofloxacin (right).
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Herein, we have carried out a chemoenzymatic route toward benzoxazine precursors, identifying 1ꢀ[2ꢀ
bromo(het)aryloxy]ꢀpropanꢀ2ꢀones as key compounds. Firstly, biotransamination experiments have been
investigated for the production of the corresponding amine enantiomers depending on the transaminase
source. Alternatively, the reductive amination of the ketones can provide access to the corresponding
racemic amines, which will be used as starting materials in lipaseꢀcatalyzed resolutions. The presence of
the amino and bromo substitutions in the aromatic ring opens up the possibility of developing metalꢀ
catalyzed intramolecular cyclization for the production of optically active benzoxazine derivatives such
as (S)ꢀ(–)ꢀ7,8ꢀdifluoroꢀ3ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine, a valuable precursor of
Levofloxacin.
Results and discussion
Two independent strategies were undertaken for the production of enantiopure 1ꢀ[2ꢀ
bromo(het)aryloxy]propanꢀ2ꢀamines: (a) the transaminaseꢀcatalyzed amination of the corresponding 1ꢀ
[2ꢀnitro(het)aryloxy]ꢀpropanꢀ2ꢀones; and (b) the lipaseꢀcatalyzed resolution of the racemic amines.
Bearing this in mind, a general and convergent route to prepare the prochiral ketones 3a-g and racemic
amines 4a-g was performed starting from the corresponding commercially available 2ꢀbromophenols
1aꢀd and pyridinols 1f and 1g (Scheme 2), requiring a previous selective bromination of 3ꢀ
methoxyphenol at the Cꢀ2 position with Nꢀbromosuccinimide in the case of the non commercially
available methoxy derivative 1e.9
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Substrates 1a-g were alkylated using an equimolecular amount of chloroacetone (2) in the presence of
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2
2 equiv of potassium carbonate and catalytic amounts of potassium iodide in refluxing acetone,
obtaining after 2 h the ketones 3a-g in high to quantitative yields. Prochiral ketones 3a-g served as
substrates for biotransamination reactions, but also as starting materials for the synthesis of the racemic
amines through reductive amination using 2 equiv of sodium cyanoborohydride in combination with a
large of excess of ammonium acetate in methanol. Thus, racemic amines 4a-g were obtained after 16 h
at room temperature in low to moderate yields after purification by column chromatography on silica
gel. The use of longer reaction times, or alternatively the palladiumꢀcatalyzed reductive aminations in
the presence of ammonium formate did not provide better results.
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Scheme 2. Chemical synthesis of prochiral ketones 3a-g and racemic amines 4a-g.
Biotransamination experiments were initially explored as they allow the synthesis of (S)ꢀ or (R)ꢀ
amines depending on the enzyme selectivity. In the last decades transaminases (TAs) have emerged as
powerful enzymes for the synthesis of chiral amines starting from racemic amines, but more importantly
from prochiral ketones as they provide access to a desired amine enantiomer in theoretically 100%
yield.10 The most structurally simple ketone was selected as the model compound for an initial enzyme
screening, this is 1ꢀ[2ꢀnitrophenoxy]ꢀpropanꢀ2ꢀone (3a) using a 50 mM substrate concentration.
Different amine donors (L/Dꢀalanine or isopropylamine) and cofactor recycling systems were used
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depending on the transaminase acceptance, which are described in the Experimental Section and
Supporting Information. The temperature was kept at 30 ºC and the reactions were shaken at 250 rpm in
a phosphate buffer 100 mM pH 7.5. Based on our previous experience with the commercially available
transaminases,11 a 2.5% v/v of DMSO was added in the biotransaminations with these TAs to favor the
solubility of the ketone 3a in the reaction medium.
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A set of 37 commercially available transaminases and 4 enzymes overexpressed in Escherichia coli
were tested, including the (S)ꢀselective Arthrobacter citreus12 and Chromobacterium violaceum TAs,13
and the (R)ꢀselective Arthrobacter species14 and Aspergillus terreus TAs.15 The best results are shown
in Table 1, while the results with the total 41 transaminases appear in the Supporting Information (Table
S1). From the entire transaminase set, only 12 displayed complete selectivity toward the formation of
single enantiomers, 10 for the (S)ꢀ4a and 2 for its antipode (Table S1). From all these selective enzymes,
10 of them led to the amine in more than 40% conversion (Table 1). Remarkably, the (S)ꢀselective
enzymes ATAꢀ200 and ATAꢀ256 gave the (S)ꢀ4a with excellent conversions (entries 2 and 5, >96%
conversion), obtaining the maximum value in the (R)ꢀ4a formation with the TA from Aspergillus
terreus (At, 70% conversion, entry 10). For the At TA, an optimization study of the reaction conditions
was performed, which showed that the use of DMSO as cosolvent led to a slightly lower conversion
(65%, entry 11), while a significant improvement was achieved when doubling the amount of enzyme
(96%, entry 12).
The scaleꢀup of the process was performed for 100 mg of ketone 3a employing the enzyme ATAꢀ256
as biocatalyst, obtaining the enantiopure amine (S)ꢀ4a in 60% isolated yield after a purification by
column chromatography (Scheme 3). The absolute configuration of the resulting enantiopure amine (S)ꢀ
4a was assigned by its transformation into the 3ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine using
palladium(II) acetate and xantphos, yielding the benzoxazine (S)ꢀ5 in 66% isolated yield without loss of
the optical purity. Its optical rotation value was compared with the one previously described in the
literature,16 assigning the (S)ꢀconfiguration for the amine 4a obtained through the ATAꢀ256
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biotransformation.
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Table 1. Biotransamination of ketone 3a in phosphate buffer 100 mM pH 7.5 after 24 h at 30 ºC.
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Entry
TA
Amine
donora (%)b
c
ee
(%)b
1
2
TAꢀP1ꢀG06c
ATAꢀ200c
ATAꢀ251c
AT1ꢀ254c
ATAꢀ256c
ATAꢀ260c
ATAꢀP1ꢀB04c
Cv
IPA
IPA
67
97
66
89
99
53
94
77
45
70
65
96
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
3
IPA
4
IPA
5
IPA
6
IPA
7
IPA
8
LꢀAla
DꢀAla
DꢀAla
DꢀAla
DꢀAla
9
ArR sp.
At
10
11
12
Atc
Atd
a
Isopropylamine (IPA) or alanine (Lꢀ or DꢀAla)
was used as amine donors.
Conversion and enantiomeric excess values were
calculated by GC analysis after derivatization of
the amines in the reaction crude with Ac2O.
Absolute configurations appear in parentheses.
c DMSO was added as cosolvent (2.5% v/v).
Double amount of enzyme was used (see
Experimental section).
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d
Scheme 3. Chemoenzymatic synthesis of (S)ꢀ3ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine (5) for the
absolute configuration assignment of the amine 4a.
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With these results in hand, the transaminaseꢀcatalyzed reactions were performed over the
aryloxypropanones 3b-e and hetaryloxypropanones 3f and 3g (Table S2). It must be highlighted that a
low reactivity was observed with all the TAs tested when ketones 3b bearing a cyano functionality in
the Cꢀ4 position and the pyridine derivative 3e were tested as substrates (>45% conversion). In addition,
the formation of sideꢀreaction products was detected in some cases when performing the
biotransamination over 3b. The non heteroaromatic ketones bearing the methyl and the fluoro
substitutions on the Cꢀ4 position (3c and 3d) led to the best selectivities (Table 2, entries 1ꢀ10),
obtaining both amine enantiomers depending on the transaminase source. From the set of commercially
available transaminases the best results were found with the ATAꢀ200, ATAꢀ254, ATAꢀ256 and ATAꢀ
P1ꢀB04, which led to the (S)ꢀamines 4c and 4d, the fluoro substituted one leading in some cases to
almost quantitative conversions (entries 8 and 9). In fact, the 100ꢀmg preparative biotransamination of
ketone 3d with the ATAꢀ256 led to the enantiopure (S)ꢀ4d in 66% isolated yield and very high purity.
For the 5ꢀmethoxy derivative 3e, good to quantitative conversions were achieved although none of the
enzymes provided access to the (S)ꢀ4e in enantiopure form (entries 11ꢀ14). This amine was obtained in
91% ee and 74% isolated yield in the preparative biotransformation when using the TAꢀP1ꢀG06.
Finally, the pyridine ketone 3f led to the (S)ꢀ4f in 95ꢀ97% ee and high to excellent conversion values
(88ꢀ97%, entries 15ꢀ17). Unfortunately, none of the commercially available TAs gave good activities
for the preparation of the (R)ꢀamines, the synthesis of (R)ꢀ4c and (R)ꢀ4d in enantiopure form being
achieved with 91% and 57% conversion, respectively, when using the AtꢀTA (entries 5 and 10).
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Table 2. Biotransamination of ketone 3c-e,g (50 mM) in phosphate buffer 100 mM pH 7.5 after 24 h at
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3
30 ºC.a
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Entry Substrate
R
4ꢀMe
4ꢀMe
4ꢀMe
4ꢀMe
4ꢀMe
4ꢀF
X
TA
c (%)a
77
ee (%)a
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
93 (S)
1
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
ATAꢀ200b
ATAꢀ254b
ATAꢀ256b
ATAꢀP1ꢀB04b
At
ATAꢀ200b
ATAꢀ254b
ATAꢀ256b
ATAꢀP1ꢀB04b
At
ATAꢀ251b
ATAꢀ256b
ATAꢀP1ꢀB04b
TAꢀP1ꢀG06b
ATAꢀ251b
ATAꢀ254b
TAꢀP1ꢀG06b
3c
2
77
3c
3
68
3c
4
64
3c
5
91
3c
3d
3d
3d
3d
3d
3e
3e
3e
3e
3g
3g
3g
6
76
7
4ꢀF
87
8
4ꢀF
98
9
4ꢀF
95
10
11
12
13
14
15
16
17
4ꢀF
57
5ꢀOMe
5ꢀOMe
5ꢀOMe
5ꢀOMe
H
97
87
93 (S)
72
89 (S)
>99
93
91 (S)
95 (S)
H
N
88
97 (S)
H
N
97
97 (S)
a
Conversion and enantiomeric excess values were calculated by GC analysis after derivatization of the
amines in the reaction crude with Ac2O. Absolute configurations appear in parentheses.
b DMSO was added as cosolvent (2.5% v/v).
In the search of an alternatively methodology for the synthesis of enantiopure amines, the hydrolaseꢀ
catalyzed kinetic resolution of the racemic amines 4a-g was attempted. Lipaseꢀcatalyzed acylation is a
common strategy for the resolution of racemic amines and alcohols under mild reaction conditions,17
mainly using esters as acyl donors.18 So at this point, an initial screening of the reaction conditions was
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performed with the less hindered substrate, 1ꢀ(2ꢀbromophenoxy)propanꢀ2ꢀamine (4a) as the model
substrate (Table 3). Candida antarctica lipase type B (CALꢀB)19 was the enzyme of choice based on its
capability to selectively produce nitrogenous compounds, and tetrahydrofuran was used as solvent since
it allows a complete solubility of the amine 4a in 100 mM concentration. Two non activated esters such
as ethyl acetate (6a) and ethyl methoxyacetate (6b) were initially assayed as acyl donors, which resulted
in a similar selectivity but higher reactivity for 6b after 5 h (entries 1 and 2). A notably decrease of the
enantioselectivity was observed over the time when using 6b, so the biotransformations were carried out
with lower amount of the ester (1 or 2 equiv, entries 3 and 4), which led to conversions close to the ideal
50% and very high selectivity in short reaction times. Similarly, the reaction with methyl tertꢀbutyl ether
(MTBE) as solvent occurred very quickly but with less selectivity in comparison with THF. At this
point, the reaction temperature was decreased, leading to both the (R)ꢀamide and the (S)ꢀamine with
excellent selectivity at both 4 and 20 ºC (entries 6ꢀ8), while reducing the loading of the enzyme did not
provide additional benefits (entries 9 and 10). The reaction at 20 ºC and 2 equiv. of 6b (entry 8) was
performed at a 200 mgꢀscale, finding similar results in a highly stereoselective process that allowed the
recovery of the (R)ꢀamide 8a and the remaining amine (S)ꢀ4a in 49% and 47% isolated yield,
respectively (see Experimental section).
1
2
3
4
5
6
7
8
9
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13
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33
34
35
36
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The assignments of the absolute configurations for the optically active amide 8a and the remaining
amine 4a were performed by measuring the optical rotation of 4a, which was compared with the one
obtained through transaminaseꢀcatalyzed reactions. It was concluded that the lipaseꢀcatalyzed reaction
led to the (R)ꢀ8a and the (S)ꢀ4a, which is also in agreement with the expected configurations
considering the Kazaluskas’ rule.20
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Table 3. Lipaseꢀcatalyzed kinetic resolution of 1ꢀ(2ꢀbromophenoxy)propanꢀ2ꢀamine (4a) using CALꢀ
1
2
3
B.a
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Entry CALꢀBb Solvent
Esterc
T (ºC) t (h) eep (%)d ees (%)d c (%)e
Ef
57
1
2
1:1
1:1
THF
THF
THF
THF
6a (3 equiv)
6b (3 equiv)
6b (1 equiv)
6b (2 equiv)
30
30
30
30
30
4
5
5
95
83
95
93
69
97
96
95
97
96
38
99
28
54
50
52
59
49
51
51
47
50
56
3
1:1
2.5
1.5
1.5
1
95
154
156
72
4
1:1
>99
>99
92
5
1:1
MTBE 6b (2 equiv)
6
1:1
THF
THF
THF
THF
THF
6b (2 equiv)
6b (2 equiv)
6b (2 equiv)
6b (2 equiv)
6b (2 equiv)
>200
>200
>200
195
193
7
1:1
4
2
99
8
1:1
20
20
20
1
99
9
0.5:1
0.5:1
1
86
10
2
96
a
Reaction conditions: 4a (100 mM in THF or MTBE), CALꢀB (ratio w/w), 6aꢀb (1ꢀ3 equiv), 4ꢀ30
ºC, 1ꢀ5 h at 250 rpm.
b Ratio amine 4a: CALꢀB (w/w).
c Equivalents of ester in parentheses.
d Determined by HPLC. Isolated yields in parentheses.
e c = ees/(ees + eep).
f E= ln[(1ꢀc)(1ꢀeep)]/ln[(1ꢀc)(1+eep)].21
Once that adequate reaction conditions were found for the resolution of the racemic amine 4a, these
are 100 mM substrate concentration in THF, 2 equiv of 6b, CALꢀB as enzyme in ratio 1:1 (w/w) with
respect to the substrate and 20 ºC, the kinetic resolution was extended to a significant panel of amines
bearing different pattern substitutions in the phenyl ring (4b-e) but also including pyridine derivatives
such as 4f and 4g. The results are shown in Table 4 and in all cases close to 50% conversion values were
reached (additional information is given in Table S4). The corresponding (R)ꢀamides 8a-g and the
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remaining amines (S)ꢀamines 4a-g were isolated with very high to excellent selectivities in just 45
minutes for the most reactive substrates such as the one with the methoxy substitution in the Cꢀ5
position and the pyridine derivatives (entries 4ꢀ7). Interestingly, the lipaseꢀcatalyzed approach provide
an efficient stereoselective access to all the tested amines, allowing the isolation of enantioenriched 4b
and 4f that were not obtained through transaminaseꢀcatalyzed reactions. The optical rotation values for
the remaining amines were measured and later compared with the ones obtained through
biotransamination experiments with transaminases of known stereospecificity, concluding that the CALꢀ
B catalyzed acylations led to the (R)ꢀmethoxyacetamides 8aꢀg and the (S)ꢀamines 4aꢀg.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Lipaseꢀcatalyzed kinetic resolution of 1ꢀ[2ꢀbromo(het)aryloxy]propanꢀ2ꢀamines (4a-g) using
CALꢀB and ethyl methoxyacetate (6b) in dry THF.a
Entry Amine
R1
H
R2
H
X
Y
t (h)
1
eep (%)b ees (%)b c (%)c
Ed
1
2
3
4
5
6
7
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
95
93
97
95
88
99
98
99
51
50
51
51
53
50
50
>200
91
4a
4b
4c
4d
4e
4f
CN
Me
F
H
1
90
H
1
>99
>99
>99
>99
>99
>200
>200
115
H
1
H
OMe
H
0.75
0.75
0.75
H
>200
>200
H
H
CH
4g
a
Reaction conditions: 4a-g (100 mM in THF), CALꢀB (ratio 1:1 in weight), 6b (2 equiv), 20 ºC,
0.75ꢀ1 h at 250 rpm.
b Determined by HPLC. Isolated yields in parentheses.
c c = ees/(ees + eep).
d E= ln[(1ꢀc)(1ꢀeep)]/ln[(1ꢀc)(1+eep)].21
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The Journal of Organic Chemistry
Once that the versatility of CALꢀB and transaminases was demonstrated in the synthesis of valuable
optically active 1ꢀ[2ꢀbromo(het)aryloxy]propanꢀ2ꢀamines, we expand the possibilities of this
chemoenzymatic strategy toward the synthesis of (S)ꢀ4h. This amine can be effectively cyclized to the
corresponding benzoxazine precursor,22 which is a valuable precursor of the antimicrobial agent
Levofloxacin.23 The synthetic pathway is depicted in Scheme 4 starting from the commercially available
2,3ꢀdifluorophenol (9). First, a selective bromination of the aromatic ring was performed using 2 equiv.
of tertꢀbutylamine and equimolecular amount of bromine in toluene at low temperature, yielding after
column chromatography the 2ꢀbromoꢀ5,6ꢀdifluorophenol (1h) in 91% isolated yield.22a This procedure
improves the results using NBS, which led to a mixture of polybrominated products. Next, the Oꢀ
alkylation proceeded smoothly after 4 h providing the ketone 3h in 90% yield, which was subjected to
the reductive amination reaction with sodium cyanoborohydride and ammonium acetate to yield the
racemic amine 4h. On the one hand, an initial attempt of the stereoselective lipaseꢀcatalyzed acylation
was performed, but this amine resulted to be quite unstable in the reaction medium, both in the absence
or presence of the enzyme. On the other hand, from a set of transaminases (see Table S3) the ATAꢀ256
provided the best results yielding the Levofloxacin precursor (S)ꢀ4h in 99% conversion and 61%
isolated yield after purification by column chromatography on silica gel.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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45
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47
48
49
50
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59
60
Scheme 4. Chemoenzymatic synthetic of the Levofloxacin precursor (S)ꢀ4h.
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1
2
3
Conclusions
4
5
6
7
8
The chemoenzymatic synthesis of optically active 1ꢀ[2ꢀbromo(het)aryloxy]propanꢀ2ꢀamines, which
are valuable precursors of benzoxazine derivatives have been explored by using transaminases or
Candida antarctica lipase type B for the stereoselective reaction step. Starting from commercially
available 2ꢀbromophenols or pyridine derivatives, prochiral ketones have been obtained through a
simple alkylation reaction in good yields. The chemical reductive amination have provided access to the
corresponding racemic amines, using both ketones and amines for extensive biocatalytic reaction
studies. Transaminases have catalyzed the amination of ketones with especially good results in terms of
conversion and selectivity when (S)ꢀselective enzymes were considered. In a complementary approach
starting from the corresponding racemic amines, their lipaseꢀcatalyzed resolution was studied, finding
high to excellent selectivity values for the formation of the remaining (S)ꢀamines and the (R)ꢀ
methoxyacetamides in close to 50% conversion value.
9
10
11
12
13
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17
18
19
20
21
22
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60
The applicability of this synthetic strategy has been demonstrated in the production of a valuable
precursor of the antimicrobial agent Levofloxacin. Therefore, the (S)ꢀ1ꢀ(6ꢀbromoꢀ2,3ꢀ
difluorophenoxy)propanꢀ2ꢀamine has been prepared with complete selectivity and a global 50% yield,
starting from 2,3ꢀdifluorophenol through a threeꢀstep sequence that involves a selective bromination
reaction, alkykation step and final biotransamination process using the commercially available ATAꢀ
256 enzyme.
Experimental section
Synthesis of 2-bromo-5-methoxyphenol (1e).9 Nꢀbromosuccinimide (811 mg, 4.55 mmol) was
added in one portion to a solution of 3ꢀmethoxyphenol (500 ꢁL, 4.55 mmol) in dry CH2Cl2 (90 mL)
under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, and then washed
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The Journal of Organic Chemistry
with water (30 mL). The organic layer was dried over Na2SO4, subjected to filtration, and concentrated
in vacuo. The crude product was purified by column chromatography on silica gel (10% Et2O/hexane),
to afford the bromophenol 1e as a colourless oil (693 mg, 75%). Physical and spectral data were found
to be consistent with those previously reported in the literature.9
1
2
3
4
5
6
7
8
9
General procedure for the synthesis of ketones 3a-g. Chloroacetone (2, 82 ꢁL, 1.03 mmol) was
added to a mixture of potassium carbonate (238 mg, 1.72 mmol), potassium iodide (41 mg, 0.25 mmol)
and the corresponding bromophenol 1a-g (0.86 mmol) in acetone (3 mL) at room temperature, and the
mixture was stirred and heated at 55 ºC for 2 h. After this time, the solution was added to water (5 mL)
and the product was extracted with Et2O (4 × 10 mL). The organic layers were combined, washed with
water (20 mL), dried over Na2SO4 and concentrated in vacuo, isolating the bromacetophenones 3a-g
with high purity without further purification (90ꢀ99%).
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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31
32
33
34
35
36
37
38
39
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42
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60
1-(2-Bromophenoxy)propan-2-one (3a). White solid (195 mg, 99% Yield). Rf (40%
EtOAc/Hexane): 0.61. Mp: 67ꢀ68 ºC. IR (NaCl): ν 3450, 3066, 3007, 2919, 1732, 1586, 1575, 1478,
1444, 1431, 1360, 969, 932, 863, 830 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 2.37 (s, 3H), 4.54 (s, 2H),
6.77 (dd, 3JHH = 8.2 Hz, 4JHH = 1.3 Hz, 1H), 6.89 (td, 3JHH = 7.9 Hz, 4JHH = 1.3 Hz, 1H), 7.25 (ddd, 3JHH
4
3
4
13
= 8.4, 7.5 Hz, JHH = 1.6 Hz, 1H), 7.58 (dd, JHH = 7.9 Hz, JHH = 1.6 Hz, 1H) ppm. C NMR (75.5
MHz, CDCl3): δ 27.0 (CH3), 73.8 (CH2), 112.1 (C), 113.1 (CH), 122.9 (CH), 128.6 (CH), 133.7 (CH),
154.1 (C), 205.5 (C) ppm. HRMS (ESI+, m/z): calculated for (C9H9BrNaO2)+ (M+Na)+ 250.9678; found
250.9691.
3-Bromo-4-(2-oxopropoxy)benzonitrile (3b). Yellowish solid (216 mg, 99% Yield). Rf (20%
Et2O/Hexane): 0.33. Mp: 109ꢀ110 ºC. IR (KBr): ν 3364, 3054, 2987, 2306, 1731, 1601, 1440, 1377,
1
3
1322, 923, 896 cmꢀ1. H NMR (300.13 MHz, CDCl3): δ 2.39 (s, 3H), 4.66 (s, 2H), 6.81 (d, JHH = 8.5
3
4
4
13
Hz, 1H), 7.60 (dd JHH = 8.5 Hz, JHH =2.0 Hz, 1H), 7.88 (d, JHH = 2.0 Hz, 1H) ppm. C NMR (75.5
MHz, CDCl3): δ 26.9 (CH3), 73.4 (CH2), 106.4 (C), 112.7 (CH), 112.8 (C), 117.4 (C), 133.1 (CH),
137.1 (CH), 157.6 (C), 203.4 (C) ppm. HRMS (ESI+, m/z): calcd for (C10H8BrNNaO2)+ (M+Na)+
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275.9631; found 275.9625; calculated for (C10H9BrNO2)+ (M+H)+ 253.9811; found 253.9813.
1
2
3
4
5
1-(2-Bromo-4-methylphenoxy)propan-2-one (3c). Yellowish solid (207 mg, 99% Yield). Rf (20%
Et2O/Hexane): 0.26. Mp: 47ꢀ49 ºC. IR (KBr): ν 3055, 2925, 1724, 1606, 1496, 1359, 1290, 947, 882
6
7
8
3
cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 2.28 (s, 3H), 2.36 (s, 3H), 4.51 (s, 2H), 6.66 (d, JHH = 8.3 Hz,
9
3
4
4
13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1H), 7.03 (dd, JHH = 8.3 Hz, JHH = 2.0 Hz, 1H), 7.39 (d, JHH = 2.0 Hz, 1H) ppm. C NMR (75.5
MHz, CDCl3): δ 20.3 (CH3), 27.1 (CH3), 74.1 (CH2), 112.0 (C), 113.2 (CH), 129.1 (CH), 132.9 (C),
134.2 (CH), 152.2 (C), 206.1 (C) ppm. HRMS (ESI+, m/z): calcd for (C10H11BrNaO2)+ (M+Na)+
264.9835; found 264.9827.
1-(2-Bromo-4-fluorophenoxy)propan-2-one (3d). White solid (195 mg, 92% Yield). Rf (20%
Et2O/Hexane): 0.26. Mp: 67ꢀ68 ºC. IR (KBr): ν 3401, 3055, 2306, 1730, 1722, 1594, 1489, 1361, 947,
1
3
4
866 cmꢀ1. H NMR (300.13 MHz, CDCl3): δ 2.36 (s, 3H), 4.52 (s, 2H), 6.75 (dd, JHH = 9.0 Hz, JFH
=
=
4.6 Hz, 1H), 6.98 (ddd, 3JHH = 9.0 Hz, 3JFH = 7.7 Hz, 4JHH = 3.0 Hz, 1H), 7.34 (dd, 3JFH = 7.7 Hz, 4JHH
3
3.0, 1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 27.0 (CH3), 74.5 (CH2), 112.6 (d, JFC = 9.9 Hz, C),
3
2
2
114.2 (d, JFC = 8.6 Hz, CH), 115.0 (d, JFC = 22.9 Hz, CH), 121.0 (d, JFC = 25.8 Hz, CH), 151.0 (d,
1
4JFC = 2.6 Hz, C), 157.4 (d, JFC = 244.5 Hz, C), 205.3 (C) ppm. HRMS (ESI+, m/z): calculated for
(C9H8BrFNaO2)+ (M+Na)+ 268.9584; found 268.9578.
1-(2-Bromo-5-methoxyphenoxy)propan-2-one (3e). White solid (205 mg, 92% Yield). Rf (50%
EtOAc/Hexane): 0.68. Mp: 62ꢀ64 ºC. IR (KBr): ν 2840, 1734, 1722, 1586, 1488, 1421, 1360, 1307,
1201, 1169, 1067, 1025 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 2.32 (s, 3H), 3.73 (s, 3H), 4.47 (s, 2H),
4
4
3
6.30 (d, JHH = 2.7 Hz, 1H), 6.41 (dd, 3JHH = 8.7 Hz, JHH = 2.7 Hz, 1H), 7.39 (d, JHH = 8.7 Hz, 1H)
ppm. 13C NMR (75.5 MHz, CDCl3): δ 26.9 (CH3), 55.6 (CH3), 73.6 (CH2), 101.1 (CH), 102.7 (C), 107.2
(CH), 133.5 (CH), 154.8 (C), 160.2 (C), 205.4 (C) ppm. HRMS (ESI+, m/z): calcd for (C10H11BrNaO3)+
(M+Na)+ 280.9784, found: 280.9792.
1-[(2-Bromopyridin-3-yl)oxy]propan-2-one (3f). White solid (178 mg, 90% Yield). Rf (20%
Et2O/Hex): 0.38. Mp: 73ꢀ74 ºC. IR (KBr): ν 3054, 2986, 1738, 1565, 1415, 1362, 967, 896, 793, 749,
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3
704 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 2.40 (s, 3H), 4.60 (s, 2H), 7.05 (dd, JHH = 8.1 Hz, 4JHH
=
1
2
3
4
5
6
7
8
9
3
3
3
4
1.6 Hz, 1H), 7.23 (dd, JHH = 8.1 Hz, JHH = 4.7 Hz, 1H), 8.06 (dd, JHH = 4.7 Hz, JHH = 1.6 Hz, 1H)
ppm. 13C NMR (75.5 MHz, CDCl3): δ 26.9 (CH3), 73.5 (CH2), 120.0 (CH), 123.4 (CH), 133.0 (C),
142.4 (CH), 151.3 (C), 204.1 (C) ppm. HRMS (ESI+, m/z): calculated for (C8H9BrNO2)+ (M+H)+
229.9811; found 229.9812; calculated for (C8H8BrNNaO2)+ (M+Na)+ 251.9631; found 251.9624.
1-[(3-Bromopyridin-2-yl)oxy]propan-2-one (3g). Brown solid (192 mg, 97% Yield). Rf (20%
Et2O/Hex): 0.20. Mp: 101ꢀ103 ºC. IR (KBr): ν 3055, 2987, 1738, 1656, 1603, 1527, 1422, 1406, 1371,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
970, 856 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 2.31 (s, 3H), 4.76 (s, 2H), 6.14 (t, 3JHH = 7.0 Hz, 1H),
7.18 (dd, 3JHH = 7.0, 4JHH = 1.9 Hz, 1H), 7.78 (dd, 3JHH = 7.0, 4JHH = 1.9 Hz, 1H) ppm. 13C NMR (75.5
MHz, CDCl3): δ 27.7 (CH3), 58.9 (CH2), 106.1 (CH), 116.4 (C), 137.6 (CH), 142.2 (CH), 158.8 (C),
200.1 (C) ppm. HRMS (ESI+, m/z): calculated for (C8H8BrNNaO2)+ (M+Na)+ 251.9631; found
251.9630.
General procedure for the synthesis of racemic 1-[2-bromo(het)aryloxy]propan-2-amines 4a-g.
To a solution of the corresponding ketone 3a-g (0.43 mmol) in dry MeOH (1.4 mL), ammonium acetate
(335 mg, 4.34 mmol) and sodium cyanoborohydride (55 mg, 0.87 mmol) were successively added under
inert atmosphere. The mixture was stirred at room temperature during 16 h y, and after this time H2O
(15 mL) was stopped to quench the reaction. The solution was acidified with a few drops of
concentrated aqueous HCl and extracted with Et2O (3 × 15 mL). The organic layers were discarded and
the aqueous phase basified with 2ꢀ3 pellets of NaOH, and extracted with Et2O (3 × 20 mL). The organic
layers were combined, dried over Na2SO4 and concentrated in vacuo. The reaction crude was purified
by column chromatography on silica gel (10% MeOH/CH2Cl2), to afford the racemic amines (31ꢀ68%).
1-(2-Bromophenoxy)propan-2-amine (4a). Yellowish oil (36 mg, 36% Yield). Rf (10%
MeOH/CH2Cl2): 0.28. IR (NaCl): ν 3356, 2965, 2929, 2227, 1658, 1614, 1597, 1487, 1369, 1295, 975,
855, 730 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.22 (d, 3JHH = 6.6 Hz, 3H), 2.25 (brs, 2H), 3.22ꢀ3.57
(m, 1H), 3.74 (dd, 2JHH = 8.9 Hz, 3JHH = 7.4 Hz, 1H), 3.96 (dd, 2JHH = 8.9 Hz, 3JHH = 4.0 Hz, 1H), 6.75ꢀ
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3
4
3
4
6.92 (m, 2H), 7.24 (td, JHH = 8.3 Hz, JHH = 1.6 Hz, 1H), 7.53 (dd, JHH = 7.9 Hz, JHH = 1.6 Hz, 1H)
ppm. 13C NMR (75.5 MHz, CDCl3): δ 19.4 (CH3), 46.5 (CH), 75.2 (CH2), 112.3 (C), 113.4 (CH), 122.1
(CH), 128.5 (CH), 133.3 (CH), 155.1 (C) ppm. HRMS (ESI+, m/z): calculated for (C9H13BrNO)+
(M+H)+ 230.0175; found 230.0178.
1
2
3
4
5
6
7
8
9
4-(2-Aminopropoxy)-3-bromobenzonitrile (4b). Yellowish oil (44 mg, 40% Yield). Rf (10%
MeOH/CH2Cl2): 0.33. IR (NaCl): ν 3364, 3358, 3104, 2227, 1750, 1596, 1494, 1461, 1398, 1397, 886,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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53
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55
56
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58
59
60
816, 751, 715, 672 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.24 (d, 2JHH = 6.6 Hz, 3H), 2.12 (brs, 2H),
2
3
2
3
3.42ꢀ3.53 (m, 1H), 3.82 (dd, JHH = 8.8, JHH = 7.3 Hz, 1H), 4.01 (dd, JHH = 8.8, JHH = 4.1 Hz, 1H),
3
4
6.93 (d, 3JHH = 8.6 Hz, 1H), 7.58 (dd, JHH = 8.5, JHH = 2.1 Hz, 1H), 7.83 (d, 4JHH = 2.1 Hz, 1H) ppm.
13C NMR (75.5 MHz, CDCl3): δ 19.5 (CH3), 46.1 (CH), 75.5 (CH2), 105.3 (C), 112.7 (C), 112.8 (CH),
117.7 (C), 133.1 (CH), 136.6 (CH), 158.6 (C) ppm. HRMS (ESI+, m/z): calculated for (C10H12BrN2O)+
(M+H)+ 255.0128; found 255.0139.
1-(2-Bromo-4-methylphenoxy)propan-2-amine (4c). Yellowish oil (33 mg, 31% Yield). Rf (10%
MeOH/CH2Cl2): 0.35. IR (NaCl): ν 3355, 2925, 2932, 1663, 1486, 1373, 1287, 933, 796, 782, 685 cmꢀ1.
3
1H NMR (300.13 MHz, CDCl3): δ 1.22 (d, JHH = 6.6 Hz, 3H), 2.27 (s, 3H), 2.60 (brs, 2H), 3.37ꢀ3.48
(m, 1H), 3.72 (dd, 2JHH = 8.9, 3JHH = 7.4 Hz, 1H), 3.94 (dd, 2JHH = 8.9, 3JHH = 4.0 Hz, 1H), 6.78 (d, 3JHH
3
4
4
13
= 8.4 Hz, 1H), 7.03 (dd, JHH = 8.4, JHH = 2.1 Hz, 1H), 7.35 (d, JHH = 2.1 Hz, 1H) ppm. C NMR
(75.5 MHz, CDCl3): δ 19.3 (CH3), 20.2 (CH3), 46.4 (CH), 75.3 (CH2), 112.0 (C), 113.5 (CH), 128.9
(CH), 131.8 (C), 133.6 (CH), 152.9 (C) ppm. HRMS (ESI+, m/z): calculated for (C10H15BrNO)+ (M+H)+
244.0332; found 244.0322.
1-(2-Bromo-4-fluorophenoxy)propan-2-amine (4d). Yellowish oil (42 mg, 39% Yield). Rf (10%
MeOH/CH2Cl2): 0.23. IR (NaCl): ν 3410, 3012, 2979, 1687, 1433, 1326, 1290, 947, 867, 753, 701 cmꢀ1.
3
1H NMR (300.13 MHz, CDCl3): δ 1.22 (d, JHH = 6.6 Hz, 3H), 2.43 (brs, 2H), 3.33ꢀ3.53 (m, 1H), 3.71
(dd, 2JHH = 8.8, 3JHH = 7.4 Hz, 1H), 3.93 (dd, 2JHH = 8.8, 3JHH = 4.0 Hz, 1H), 6.83 (dd, 3JHH = 9.1, 4JHF
=
3
4
13
4.8 Hz, 1H), 6.93ꢀ7.00 (m, 1H), 7.28 (dd, JHF = 7.8, JHH = 3.0 Hz, 1H) ppm. C NMR (75.5 MHz,
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CDCl3): δ 19.2 (CH3), 46.4 (CH), 75.8 (CH2), 112.4 (d, 3JFC = 9.9 Hz, C), 114.1 (d, 3JFC = 8.5 Hz, CH),
114.8 (d, 2JFC = 22.6 Hz, CH), 120.3 (d, 2JFC = 25.8 Hz, CH), 151.7 (d, 4JFC = 2.4 Hz, C), 156.7 (d, 1JFC
= 243.3 Hz, C) ppm. HRMS (ESI+, m/z): calculated for (C9H12BrFNO)+ (M+H)+ 248.0081; found
248.0085.
1
2
3
4
5
6
7
8
9
1-(2-Bromo-5-methoxyphenoxy)propan-2-amine (4e). Colourless oil (74 mg, 66% Yield). Rf (50%
EtOAc/Hexane): 0.38. IR (NaCl): ν 3368, 2330, 2174, 1591, 1489, 1467, 1306, 1283, 1203, 1170, 1061,
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11
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14
15
16
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53
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59
60
1023, 828 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.24 (d, 3JHH = 6.6 Hz, 3H), 2.68 (brs, 2H), 3.39ꢀ3.52
(m, 1H), 3.70ꢀ3.79 (m, 1H) overlapped signal with 3.77 (s, 3H), 3.95 (dd, 2JHH = 8.9 Hz, 3JHH = 4.0 Hz,
3
4
4
3
1H), 6.39 (dd, JHH = 8.7 Hz, JHH = 2.7 Hz, 1H), 6.49 (d, JHH = 2.7 Hz, 1H), 7.38 (d, JHH = 8.7 Hz,
1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 15.3 (CH3), 48.4 (CH), 55.9 (CH3), 69.9 (CH2), 102.1 (CH),
103.1 (C), 108.8 (CH), 133.3 (CH), 154.5 (C), 160.3 (C) ppm. HRMS (ESI+, m/z): calculated for
(C10H15BrNO2)+ (M+H)+ 260.0281, found: 260.0275.
1-[(2-Bromopyridin-3-yl)oxy]propan-2-amine (4f). Yellowish oil (32 mg, 32% Yield). Rf (10%
MeOH/CH2Cl2): 0.28. IR (NaCl): ν 3330, 3321, 2960, 2929, 2227, 1645, 1596, 1563, 1538, 1494, 1447,
1416, 849, 795, 726, 680 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.19 (d, 3JHH = 6.5 Hz, 3H), 1.78 (brs,
2
3
2
3
2H), 3.35ꢀ3.48 (m, 1H), 3.72 (dd, JHH = 8.6, JHH = 7.5 Hz, 1H), 3.93 (dd, JHH = 8.6, JHH = 4.1 Hz,
1H), 7.11 (dd, 3JHH = 8.1, 4JHH = 1.6 Hz, 1H), 7.18 (dd, 3JHH = 8.1, 3JHH = 4.6 Hz, 1H), 7.96 (dd, 3JHH
=
4.6, 4JHH = 1.6 Hz, 1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 19.8 (CH3), 46.2 (CH), 75.7 (CH2), 119.9
(CH), 123.5 (CH), 133.2 (C), 141.5 (CH), 152.3 (C) ppm. HRMS (ESI+, m/z): calculated for
(C8H12BrN2O)+ (M+H)+ 231.0128; found 231.0123.
1-[(3-Bromopyridin-2-yl)oxy]propan-2-amine (4g). Yellowish oil (67 mg, 68% Yield). Rf (10%
MeOH/CH2Cl2): 0.21. IR (NaCl): ν 3419, 3093, 2345, 2177, 1706, 1648, 1583, 1530, 1427, 1396, 1327,
976, 866, 855 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.16 (d, 3JHH = 6.5 Hz, 3H), 2.15 (brs, 2H), 3.39ꢀ
3
3
3.52 (m, 1H), 3.70 (dd, 2JHH = 12.9 Hz, JHH = 8.1 Hz, 1H), 4.08 (dd, 2JHH = 12.9 Hz, JHH = 4.7 Hz,
3
3
4
3
1H), 6.08 (t, JHH = 7.0 Hz, 1H), 7.37 (dd, JHH = 7.0 Hz, JHH = 1.9 Hz 1H), 7.74 (dd, JHH = 7.0 Hz,
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4JHH = 1.9 Hz, 1H) ppm. C NMR (75.5 MHz, CDCl3): δ 20.9 (CH3), 45.8 (CH), 58.4 (CH2), 105.8
(CH), 116.7 (C), 138.1 (CH), 141.7 (CH), 159.3 (C) ppm. HRMS (ESI+, m/z): calculated for
(C8H11BrN2NaO)+ (M+Na)+ 252.9947; found 252.9945.
13
1
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8
Synthesis
of
racemic
N-[1-(2-bromophenoxy)propan-2-yl]acetamide
(7a).
4ꢀ
9
Dimethylaminopyridine (8.0 mg, 0.066 mmol), triethylamine (72.2 ꢁL, 0.983 mmol) and acetic
anhydride (72.2 ꢁL, 0.655 mmol) were successively added to a solution of the racemic amine 4a (75
mg, 0.328 mmol) in dry CH2Cl2 (2 mL). The reaction was stirred at room temperature for 1 hour and
after this time the solvent removed by distillation under reduced pressure. The crude was purified by
column chromatography on silica gel (5% MeOH/CH2Cl2), to afford the racemic acetamide 7a as a
white solid (86 mg, 96%). Rf (10% MeOH/CH2Cl2): 0.65. Mp: 67ꢀ68 ºC. IR (KBr): ν 3053, 2987, 1768,
10
11
12
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1662, 1608, 1498, 1267, 914, 896, 761, 721 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.37 (d, 3JHH = 6.9
Hz, 3H), 2.01 (s, 3H), 3.97ꢀ4.07 (m, 2H), 4.35ꢀ4.47 (m, 1H), 6.13 (d, 1H, 3JHH = 7.7 Hz), 6.82ꢀ6.93 (m,
2H), 7.26 (ddd, 3JHH = 8.2, 3JHH = 7.4, 4JHH = 1.6 Hz, 1H), 7.53 (dd, 3JHH = 7.8, 4JHH = 1.6 Hz, 1H) ppm.
13C NMR (75.5 MHz, CDCl3): δ 18.0 (CH3), 23.8 (CH3), 45.0 (CH), 72.1 (CH2), 112.7 (C), 114.0 (CH),
127.8 (CH), 129.1 (CH), 133.7 (CH), 155.3 (C), 170.4 (C) ppm. HRMS (ESI+, m/z): calcd for
(C11H15BrNO2)+ (M+H)+: 272.0281 found: 272.0287; calcd for (C11H14BrNNaO2)+ (M+Na)+: 294.0100
found: 294.0103.
General procedure for the synthesis of racemic methoxyacetamides 8a-g. 4ꢀ
Dimethylaminopyridine (1.5 mg, 0.012 mmol), triethylamine (25 ꢁL, 0.18 mmol) and methoxyacetyl
chloride (11 ꢁL, 0.12 mmol) were successively added to a solution of the corresponding racemic amine
4a-g (0.06 mmoles) in dry CH2Cl2 (2.1 mL). The reaction was stirred at room temperature for 1 hour
and after this time the solvent removed by distillation under reduced pressure. The crude was purified
by column chromatography on silica gel (5% MeOH/CH2Cl2), to afford the corresponding
methoxyacetamide 8a-g (85ꢀ99%).
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N-[1-(2-bromophenoxy)propan-2-yl]-2-methoxyacetamide (8a). White solid (18 mg, 98% Yield).
Rf (10% MeOH/CH2Cl2): 0.79. Mp: 52ꢀ53 ºC. IR (KBr): ν 3409, 3054, 2983, 2937, 2829, 1678, 1586,
1
2
3
4
5
6
1
1574, 1483, 1266, 986, 932, 896, 738, 704, 555 cmꢀ1. H NMR (300.13 MHz, CDCl3): δ 1.32ꢀ1.60 (m,
7
8
9
3H), 3.42 (s, 3H), 3.89ꢀ3.92 (m, 2H), 4.03ꢀ4.05 (m, 2H), 4.43ꢀ4.51 (m, 1H), 6.77ꢀ6.93 (m, 3H), 7.18ꢀ
7.33 (m, 1H), 7.45ꢀ7.59 (m, 1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 18.1 (CH3), 44.4 (CH), 59.6
(CH3), 72.0 (CH2), 72.3 (CH2), 112.8 (C), 113.8 (CH), 122.7 (CH), 129.0 (CH), 133.7 (CH), 155.2 (C),
169.5 (C) ppm. HRMS (ESI+, m/z): calculated for (C12H17BrNO3)+ (M+H)+ 302.0386; found 302.0381;
calculated for (C12H16BrNNaO3)+ (M+Na)+ 324.0206; found 324.0204.
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N-[1-(2-bromo-4-cyanophenoxy)propan-2-yl]-2-methoxyacetamide (8b). White solid (17 mg, 87%
Yield). Rf (10% MeOH/CH2Cl2): 0.63. Mp: 102ꢀ103 ºC. IR (KBr): ν 3417, 3012, 2182, 1696, 1669,
1
3
1605, 1510, 1456, 1298, 1267, 965, 868, 852 cmꢀ1. H NMR (300.13 MHz, CDCl3): δ 1.42 (d, JHH
=
=
3
6.9 Hz, 3H), 3.43 (s, 3H), 3.85ꢀ3.97 (m, 2H), 4.07ꢀ4.16 (m, 2H), 4.45ꢀ4.53 (m, 1H), 6.75 (d, 1H, JHH
3
3
4
4
6.9 Hz), 6.97 (d, JHH = 8.5 Hz, 1H), 7.60 (dd, JHH = 8.5, JHH = 2.1 Hz, 1H), 7.85 (d, JHH = 2.1 Hz,
1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 17.8 (CH3), 43.7 (CH), 59.2 (CH3), 71.7 (CH2), 71.8 (CH2),
105.6 (C), 112.9 (C), 113.0 (CH), 117.7 (C), 133.2 (CH), 136.7 (CH), 158.4 (C), 169.3 (C) ppm. HRMS
(ESI+, m/z): calculated for (C13H16BrN2O3)+ (M+H)+ 327.0339; found 327.0335; calculated for
(C13H15BrN2NaO3)+ (M+Na)+ 349.0158; found 349.0164.
N-[1-(2-bromo-4-methylphenoxy)propan-2-yl]-2-methoxyacetamide (8c). White solid (16 mg,
85% Yield). Rf (10% MeOH/CH2Cl2): 0.67. Mp: 64ꢀ65 ºC. IR (KBr): ν 3419, 3093, 2345, 2177, 1698,
3
1623, 1597, 1419, 1265, 896, 751, 706 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.41 (d, JHH = 6.8 Hz,
3H), 2.29 (s, 3H), 3.43 (s, 3H), 3.85ꢀ3.97 (m, 2H), 4.00ꢀ4.04 (m, 2H), 4.58ꢀ4.35 (m, 1H), 6.80 (d, 3JHH
=
8.3 Hz, 1H), 6.95 (d, 1H, 3JHH = 7.9 Hz), 7.00ꢀ7.13 (m, 1H), 7.37 (d, 4JHH = 2.0 Hz, 1H) ppm. 13C NMR
(75.5 MHz, CDCl3): δ 17.7 (CH3), 20.2 (CH3), 44.0 (CH), 59.3 (CH3), 71.8 (CH2), 71.9 (CH2), 112.1
(C), 113.5 (CH), 128.9 (CH), 132.1 (C), 133.7 (CH), 152.8 (C), 169.1 (C) ppm. HRMS (ESI+, m/z):
calculated for (C13H19BrNO3)+ (M+H)+ 316.0543, found: 316.0532; calculated for (C13H18BrNNaO3)+
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(M+Na)+ 338.0362, found 338.0360.
1
2
3
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N-[1-(2-bromo-4-fluorophenoxy)propan-2-yl]-2-methoxyacetamide (8d). White solid (19 mg,
99% Yield). Rf (10% MeOH/CH2Cl2): 0.64. Mp: 80ꢀ81 ºC. IR (KBr): ν 3409, 3054, 2986, 1733, 1678,
6
7
8
9
1527, 1492, 1265, 985, 909, 896, 865, 748, 705 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.41 (d, 3JHH
=
=
3
4
10
11
12
13
14
15
16
17
18
19
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6.8 Hz, 3H), 3.43 (s, 3H), 3.91 (m, 2H), 4.01 (m, 2H), 4.38ꢀ4.54 (m, 1H), 6.86 (dd, JHH = 9.1, JHF
4.7 Hz, 1H), 6.99 (m, 3JHH = 9.1, 3JHF = 7.8, 4JHH = 3.0 Hz, 1H), 7.26ꢀ7.34 (m, 1H) ppm. 13C NMR (75.5
MHz, CDCl3): δ 17.6 (CH3), 44.0 (CH), 59.2 (CH3), 71.9 (CH2), 72.4 (CH2), 112.5 (d, 3JFC = 9.5 Hz, C),
2
2
114.0 (d, 3JFC = 8.5 Hz, CH), 114.8 (d, JFC = 22.6 Hz, CH), 120.4 (d, JFC = 26.3 Hz, CH), 151.5 (C),
1
156.8 (d, JFC = 243.8 Hz, C), 169.2 (C) ppm. HRMS (ESI+, m/z): calculated for (C12H16BrFNO3)+
(M+H)+ 320.0292, found 320.0287; calculated for (C12H15BrFNNaO3)+ (M+Na)+ 342.0112, found
342.0117.
N-[1-(2-bromo-5-methoxyphenoxy)propan-2-yl]-2-methoxyacetamide (8e). White solid (17 mg,
87% Yield). Rf (10% MeOH/CH2Cl2): 0.71. Mp: 67ꢀ68 ºC. IR (KBr): ν 3420, 2934, 2401, 2111, 1670,
1639, 1589, 1423, 1257, 1023, 934, 829 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.41 (d, 3JHH= 6.8 Hz,
3H), 3.43 (s, 3H), 3.79 (s, 3H), 3.91 (d, 3JHH = 5.6 Hz, 2H), 4.02ꢀ4.12 (m, 2H), 4.36ꢀ4.61 (m, 1H), 6.43
3
4
4
3
(dd, JHH = 8.6 Hz, JHH = 2.7, 1H), 6.50 (d, JHH = 2.7 Hz, 1H), 6.91 (d, 1H, JHH = 7.3 Hz), 7.41 (d,
4JHH = 8.6 Hz, 1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 17.7 (CH3), 43.9 (CH), 55.6 (CH3), 59.3
(CH3), 71.8 (CH2), 71.9 (CH2), 100.9 (CH), 103.0 (C), 106.9 (CH), 133.1 (CH), 155.5 (C), 160.2 (C),
169.2 (C) ppm. HRMS (ESI+, m/z): calculated for (C13H18BrNNaO4)+ (M+Na)+ 354.0311, found
354.0316.
N-{1-[(2-bromopyridin-3-yl)oxy]propan-2-yl}-2-methoxyacetamide (8f). White solid (18 mg, 99%
Yield). Rf (10% MeOH/CH2Cl2): 0.57. Mp: 70ꢀ80 ºC. IR (KBr): ν 3400, 2933, 1667, 1651, 1563, 1530,
1447, 1418, 1294, 796, 727 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.43 (d, 3JHH = 6.8 Hz, 3H), 3.44 (s,
3
3H), 3.85ꢀ3.97 (m, 2H), 4.03ꢀ4.12 (m, 2H), 4.44ꢀ4.54 (m, 1H), 6.85 (d, 1H, JHH = 7.9 Hz), 7.17ꢀ7.25
(m, 2H, H4), 8.02 (dd, 3JHH = 4.4, 4JHH = 1.8 Hz, 1H) ppm. 13C NMR (75.5 MHz, CDCl3): δ 17.6 (CH3),
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43.7 (CH), 59.3 (CH3), 71.6 (CH2), 71.9 (CH2), 119.9 (CH), 123.5 (CH), 133.2 (C), 141.7 (CH), 152.0
(C), 169.3 (C) ppm. HRMS (ESI+, m/z): calculated for (C11H15BrN2NaO3)+ (M+Na)+ 325.0158, found:
325.0145.
1
2
3
4
5
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7
8
9
N-{1-[(2-bromopyridin-3-yl)oxy]propan-2-yl}-2-methoxyacetamide (8g). White solid (16 mg, 89%
Yield). Rf (10% MeOH/CH2Cl2): 0.65. Mp: 138ꢀ139 ºC. IR (KBr): ν 3478, 3319, 2922, 1649, 1596,
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11
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1537, 1494, 1378, 1297, 975, 848, 760, 657 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.30 (d, 3JHH = 6.2
3
Hz, 3H), 3.39 (s, 3H), 3.86ꢀ3.96 (m, 2H), 3.97ꢀ4.08 (m, 2H), 4.21ꢀ4.39 (m, 1H), 6.10 (t, JHH = 7.2 Hz,
3
4
13
1H), 6.95 (d, 1H, 3JHH = 6.9 Hz), 7.25ꢀ7.37 (m, 1H), 7.73 (dd, JHH = 7.2, JHH = 1.9 Hz, 1H) ppm. C
NMR (75.5 MHz, CDCl3): δ 18.1 (CH3), 45.9 (CH), 53.9 (CH2), 59.4 (CH3), 71.8 (CH2), 106.2 (CH),
116.6 (C), 137.3 (CH), 141.8 (CH), 159.6 (C), 169.9 (C) ppm. HRMS (ESI+, m/z): calculated for
(C11H16BrN2O3)+ (M+H)+ 303.0339, found 303.0342; calculated for (C11H15BrN2NaO3)+ (M+Na)+
325.0158, found: 325.0165.
General procedure for the enzymatic kinetic resolution by acylation of racemic amines 4a-g. Ethyl
methoxyacetate (6b, 23.5 ꢀL, 0.20 mmol) and CALꢀB (ratio 1:1 in weight amine/enzyme) were added
to a suspension containing the corresponding racemic amine 4a-g (0.10 mmol) in dry THF (0.1 M, 1
mL) under inert atmosphere. The reaction was shaken at 20 ºC and 250 rpm for the necessary time
(0.75ꢀ1 h) to achieve a good kinetic resolution (see Tables 3 and 4). The reaction was followed by
HPLC analysis until around 50% conversion was reached. The enzyme was filtered off, washed with
CH2Cl2 (3 x 5 mL) and the solvent evaporated under reduced pressure. The crude reaction was purified
by column chromatography on silica gel (eluent gradient 5ꢀ10% MeOH/CH2Cl2), to afford the
corresponding optically active methoxyacetamides (R)ꢀ8a-g (88ꢀ99% ee) and amines (S)ꢀ4a-g (90ꢀ>99%
ee).
20
Optical rotation values for the (R)ꢀmethoxyacetamides 8a-g: [α]D +35.4 (c 1, EtOH) for (R)ꢀ8a in
93% ee; [α]D20 +36.4 (c 1, EtOH) for (R)ꢀ8b in 93% ee; [α]D20 +31.8 (c 1, EtOH) for (R)ꢀ8c in 96% ee;
20
[α]D20 +25.3 (c 0.5, EtOH) for (R)ꢀ8d in 93% ee; [α]D20 +12.6 (c 1, EtOH) for (R)ꢀ8e in 88% ee; [α]D
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+19.8 (c 1, EtOH) for (R)ꢀ8f in 99% ee; [α]D20 –50.6 (c 1, EtOH) for (R)ꢀ8g in 83% ee.
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Optical rotation values for the (S)ꢀamines 4a-g: [α]D +6.4 (c 0.5, EtOH) for (S)ꢀ4a in >99% ee;
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[α]D –30.5 (c 0.5, EtOH) for (S)ꢀ8b in 90% ee obtained by chemical derivatization of (S)ꢀ4b; [α]D
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+6.4 (c 1, EtOH) for (S)ꢀ4c in >99% ee; [α]D20 + 2.5 (c 1, EtOH) for (S)ꢀ4d in >99% ee; [α]D20 +13.6 (c
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1, EtOH) for (S)ꢀ4e in 91% ee; [α]D +4.2 (c 1, EtOH) for (S)ꢀ4f in >99% ee; [α]D +31.3 (c 0.5,
EtOH) for (S)ꢀ4g in >99% ee.
Scale-up of the enzymatic kinetic resolution by acylation of racemic amine 4a. Ethyl
methoxyacetate (6b, 204.0 ꢀL, 1.74 mmol) and CALꢀB (200 mg, ratio 1:1 in weight amine/enzyme)
were added to a suspension containing the corresponding racemic amine 4a (200 mg, 0.87 mmol) in dry
THF (0.1 M, 8.7 mL) under inert atmosphere. The reaction was shaken at 20 ºC and 250 rpm for 45
minutes to achieve a 51% conversion with excellent selectivity. The enzyme was filtered off, washed
with CH2Cl2 (3 x 15 mL) and the solvent evaporated under reduced pressure. The crude reaction was
purified by column chromatography on silica gel (eluent gradient 5ꢀ10% MeOH/CH2Cl2), to afford the
corresponding optically active methoxyacetamide (R)ꢀ8a (93% ee, 49% isolated yield) and amine (S)ꢀ4a
(>99% ee, 47% isolated yield).
General procedure for the biotransamination of ketones 3a-h employing isopropylamine as
amino donor. A solution of ketone 3a-h (0.025 mmol, 50 mM) in DMSO (12.5 µL) was added to a
suspension of a commercial TA (2 mg) in phosphate buffer 100 mM pH 7.5 (500 µL) containing PLP (1
mM) and isopropylamine (1 M). The mixture was shaken at 30 °C and 250 rpm for 24 h. Then, the
reaction was quenched by adding an aqueous NaOH 4 M solution (400 µL) and extracted with EtOAc (3
x 500 µL). The organic phases were combined and dried over Na2SO4. Reaction crude was analyzed
through GC to determine conversion values, and later the enantiomeric excess after an in situ
derivatization. For the methoxy derivative 4e, HPLC was employed to determine de ee value of the
enantioenriched amine.
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General procedure for the biotransamination of ketones 3a,c-d employing alanine as amino
donor and alanine dehydrogenase as regeneration system. To a suspension of ketones 3a-d (0.05
mmol, 50 mM) in a 100 mM phosphate buffer pH 7 (440 µL) were successively added ammonium
formate (100 µL of 1.5 M solution in a 100 mM phosphate buffer pH 7; final concentration 150 mM),
alanine (250 µL of 1 M solution in phosphate buffer 100 mM pH 7; final concentration 250 mM), NAD+
(100 µL of 10 mM solution in a 100 mM phosphate buffer pH 7; final concentration 1 mM), PLP (100
µL of 10 mM solution in a 100 mM phosphate buffer pH 7; final concentration 1 mM), lyophilized cells
of E. coli containing overexpressed transaminases (20 mg), formate dehydrogenase (FDH, 2.6 mg, 11
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U) and alanine dehydrogenase (AlaDH, 10 µL, 11 U).
Dꢀ or Lꢀ alanine were used as amine donor
depending on the (R) or (S)ꢀtransaminase selectivity, respectively. The resulting mixture was shaken at
30 °C and 250 rpm for 24 h. After this time the reaction was quenched by adding aqueous NaOH 4 M
(400 µL), extracted with ethyl acetate (3 x 500 µL), and organic phases were combined and dried with
Na2SO4. Reaction crude was analyzed through GC to determine conversion values and, after an in situ
derivatization the enantiomeric excess was calculated.
Synthesis of 6-bromo-2,3-difluorophenol (1h).22a Bromine (119 ꢁL, 2.31 mmol) was added
t
dropwise over 3 minutes to a cooled (ꢀ30 ºC) solution of BuNH2 (485 ꢁL, 4.61 mmol) in dry toluene
(5.8 mL) until formation of a yellow solution. The mixture was cooled to ꢀ78 ºC and after 10 minutes, a
solution of 2,3ꢀdifluorophenol (9, 300 mg, 2.31 mmol) in dry CH2Cl2 (0.6 mL) was added dropwise
over 5 minutes. The mixture was allowed to warm slowly to room temperature over 4 h, stirring the
resulting mixture for additional 1.5 h at this temperature. The mixture was diluted with EtOAc (10 mL)
and washed with an aqueous HCl 1 M solution (2 × 10 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo. The reaction crude was purified by column chromatography on silica gel
(15% Et2O/hexane), to afford the bromophenol 1h as a colourless oil (439 mg, 91%). Physical and
spectral data were found to be consistent with those previously reported in the literature.22a
Synthesis of 1-(6-bromo-2,3-difluorophenoxy)propan-2-one (3h). Chloroacetone (82 ꢁL, 1.03
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Page 26 of 30
mmol) was added to a mixture of potassium carbonate (238 mg, 1.72 mmol), potassium iodide (41 mg,
0.25 mmol) and the bromophenol 1h (111.9 mg, 0.86 mmol) in acetone (3 mL) at room temperature,
and the mixture was stirred and heated at 55 ºC for 2 h. After this time the solution was added to water
(5 mL) and the product was extracted with Et2O (4 × 10 mL). The organic layers were combined,
washed with water (20 mL), dried over Na2SO4 and concentrated in vacuo, isolating the
bromacetophenone 3h as an orangish oil (205 mg, 90%). Rf (20% EtOAc/Hexane): 0.44. IR (NaCl): ν
3461, 3094, 2921, 1741, 1724, 1612, 1588, 1485, 1462, 1429, 1360, 1297, 1208, 1179, 1058, 1019, 991,
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1
5
975, 881, 801 cmꢀ1. H NMR (300.13 MHz, CDCl3): δ 2.35 (s, 3H), 4.64 (d, JFH = 0.9 Hz, 2H), 6.74ꢀ
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6.92 (m, 1H), 7.26 (ddd, JHH = 9.2 Hz, JFH = 5.4, JFH = 2.4 Hz, 1H) ppm. 13C NMR (75.5 MHz,
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CDCl3): δ 26.7 (CH3), 77.5 (d, JFC = 4.8 Hz, CH2), 111.1 (d, JFC = 3.6 Hz, C), 112.9 (d, JFC = 18.4
1
Hz, CH), 127.2 (dd, 3JFC = 7.4 Hz, 4JFC = 4.2 Hz, CH), 144.5 (dd, JFC = 251.8 Hz, 2JFC = 14.8 Hz, C),
2
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144.8 (d, JFC = 7.3 Hz, C), 150.8 (dd, JFC = 250.3 Hz, 2JFC = 11.5 Hz, C), 204.1 (C) ppm. HRMS
(ESI+, m/z): calcd for (C9H7BrF2NaO2)+ (M+Na)+ 286.9490, found: 286.9480.
Synthesis of 1-(6-bromo-2,3-difluorophenoxy)propan-2-amine (4h). To a solution of the ketone 3h
(80.0 mg, 0.43 mmol) in dry MeOH (1.4 mL), ammonium acetate (335 mg, 4.34 mmol) and sodium
cyanoborohydride (55 mg, 0.87 mmol) were successively added under inert atmosphere. The mixture
was stirred at room temperature during 14 h. The reaction crude was purified by column
chromatography on silica gel (10% MeOH/CH2Cl2), to afford the racemic 4h as a colourless oil (63 mg,
55%). Rf (10% MeOH/CH2Cl2): 0.41. IR (NaCl): ν 3243, 3091, 2335, 1613, 1489, 1457, 1294, 1209,
1055 cmꢀ1. 1H NMR (300.13 MHz, CDCl3): δ 1.26 (d, 3JHH = 6.6 Hz, 3H), 3.28 (brs, 2H), 3.42ꢀ3.57 (m,
1H), 3.90ꢀ4.02 (m, 1H), 4.16 (ddd, 2JHH = 9.3 Hz, 3JHH = 3.9 Hz, 5JFH = 1.3 Hz, 1H), 6.82 (dt, 3JFH = 9.2
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Hz, JHH = 7.6, JFH = 7.6 Hz, 1H), 7.25 (ddd, JHH = 7.6 Hz, JFH = 4.9, JHF = 2.5 Hz, 1H) ppm. C
4
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NMR (75.5 MHz, CDCl3): δ 15.1 (CH3), 48.7 (CH), 74.3 (d, JFC = 4.6 Hz, CH2), 111.7 (d, JFC = 3.8
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Hz, C), 113.8 (d, JFC = 18.4 Hz, CH), 127.2 (dd, 3JFC = 7.4 Hz, 4JFC = 4.2 Hz, CH), 144.3 (dd, JFC
=
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10.1 Hz, 3JFC = 1.8 Hz, C), 145.0 (dd, 1JFC = 252.5 Hz, JFC = 14.5 Hz, C), 150.7 (dd, JFC = 250.8 Hz,
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2JFC = 11.3 Hz, C) ppm. HRMS (ESI+, m/z): calcd for (C9H11BrF2NO)+ (M+H)+ 265.9987, found:
265.9989.
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Preparative biotransamination of ketones 3a,d,e,h. In a Falcon tube, the TA (35 mg, ATAꢀ256 for
ketones 3a,d,h and TAꢀP1ꢀG06 for 3e) was suspended in a phosphate buffer 100 mM pH 7.5 (8.8 mL)
containing PLP (1 mM) and isopropylamine (1 M). Then, a solution of ketones 3a, 3d, 3e or 3h (0.44
mmol, 50 mM) in EtOH (220 µL) was added. The mixture was shaken at 30 °C and 250 rpm for 24 h.
Then, the reaction was quenched by adding an aqueous NaOH 4 M solution until pH ≈ 10 (~2 mL) and
extracted with EtOAc (3 x 15 mL). The organic phases were combined, dried over Na2SO4, filtered and
the solvent was removed under reduced pressure. The reaction crude was purified by column
chromatography (MeOH/CH2Cl2 mixtures), yielding the amines (S)ꢀ4a,d,e,h in moderate to good yields
(60ꢀ74%) and good to excellent enantiomeric excess (91ꢀ>99% ee). [α]D20= +4.8 (c 0.4, EtOH) [for (S)ꢀ
4h in >99% ee].
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Acknowledgments. Financial support of this work by the Spanish Ministerio de Ciencia e Innovación
(MICINN, Project CTQꢀ2013ꢀ44153ꢀP) and the Gobierno del Principado de Asturias (Project FCꢀ15ꢀ
GRUPIN14ꢀ002) are gratefully acknowledged. M. L.ꢀI. and A. M.ꢀI. thanks Fundación para el Fomento
en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT) and MICINN for
predoctoral fellowships, respectively.
Supporting Information Available. Enzyme activity screenings, biotransformation reaction course,
1
analytical separations, and H, 13C and DEPT NMR spectra for described organic compounds are
available free of charge via the Internet at http://pubs.acs.org.
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16. Experimental value for (S)ꢀ5: [α]D +11 (c = 0.1, CHCl3); Literature data [α]D ꢀ17.6 (c 0.5,
CHCl3) in reference 7.
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