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Asymmetric reductive amination of boron-containing aryl-ketones using ω-transaminases
Tetrahedron: Asymmetry 24 (2013) 1495–1501
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Asymmetric reductive amination of boron-containing aryl-ketones using x-transaminases Joel S. Reis a, Robert C. Simon b,c, Wolfgang Kroutil b,⇑, Leandro H. Andrade a,⇑ a
Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil 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 b
a r t i c l e
i n f o
Article history: Received 19 August 2013 Accepted 4 October 2013
a b s t r a c t The asymmetric reductive amination of aryl-ketones bearing various boron-functionalities (acid, ester or potassium trifluoroborates) was investigated employing enantiocomplementary x-transaminases as catalysts. 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. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Enantiomerically pure amines are valuable chiral building blocks for the synthesis of a broad range of biologically active compounds, such as agrochemicals or pharmaceuticals. Since the activity of these molecules is generally related to one specific absolute configuration, efficient asymmetric methods are required, in particular those that are mild and environmentally benign reaction conditions.1 Numerous synthetic methods have been described to provide chiral amines based on the utilization of metal- and organocatalysts; 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 displayed by enzymes.3 While initially hydrolases were frequently employed in (dynamic)-kinetic resolution processes,4 monoamine oxidases (MAO), 5 amine-dehydrogenases (AmDH) 6 and x-transaminases (x-TA)7 were only applied more recently. The latter ones have successfully been used to prepare a variety of enantiomerically 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⇑ Corresponding authors. Tel.: +55 11 3091 2287 (L.H.A.). E-mail addresses: [email protected] (W. Kroutil), [email protected] (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
ents are also readily incorporated into complex molecules whereas boronic acids and their derivatives can serve as catalysts (e.g., CBSreduction, 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 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 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). 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 often favors the side of the ketone rather than the amine and additional co-product (pyruvate) consumption may hamper the overall efficiency.7 Nevertheless, the shift of the equilibrium is achieved by combining the x-TA catalyzed reductive amination either with an alanine dehydrogenase (AlaDH) or a lactate dehydrogenase (LDH). The AlaDH approach is favored with respect to atom efficiency since the formed co-product (pyruvate) is
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J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501 O O
pinacol, THF B
O
O
2a; para (86%) 2b; meta (76%)
OH B OH 1a; para 1b; meta
O KHF2, MeOH BF3 K
2 h, 0°C
3a; para, 79% 3b; meta, 76%
Scheme 1. Synthesis of aromatic boron-containing ketones 2 and 3.
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 evaluation for the amination of boronic ester 2a as a model substrate was performed in order to identify appropriate x-TAs [substrate concentration: 50 mM (12.5 mg/mL), 1.0 mL total volume]. Depending on the stereoselectivity of the corresponding enzyme, D- or L-alanine was used as the amine source whereas the pyruvate was recy-
cled with an alanine dehydrogenase (AlaDH). Among the different enantiocomplementary x-TAs tested [x-transaminase (origin): Arthrobacter sp., Aspergillus terreus, Hyphomonas neptunium, Chromobacterium violaceum, Bacilus megaterium, Pseudomonas fluorescens, Paracoccus denitrificans, Ralstonia eutropha, Pseudomonas putida, Arthrobacter citreus, and Vibrio fluvialis], some gave reasonable conversions (between 21% and 68%, detailed data not shown) which rendered them suitable for a more detailed investigation: xTAs from Chromobacterium violaceum,19a Pseudomonas fluorescens,19b Arthrobacter citreus,19c (R)-Arthrobacter sp.19d and Aspergillus terreus.8d 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 solubility reasons (Table 1).20 As expected, a significant influence of the co-solvent on the biotransformation 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 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
O
NH2 ω-transaminase, PLP BY2
BY2
O
NH2
OH
OH
1-3 O
recycling via AlaDH (additional NADH recycling)
BY2: para-B(OH)2 = 1a meta-B(OH)2 = 1b para-BPin = 2a meta-BPin = 2b para-BF3K = 3a meta-BF3K = 3b
(R) or (S)-4
O BY2: para-B(OH)2 = 4a meta-B(OH)2 = 4b para-BPin = 4c meta-BPin = 4d para-BF3K = 4e meta-BF 3K = 4f
OH OH 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 B
O
NH2
B
OH
O
2a
O OH
O
O
O
(R) or (S)-4c
O
AlaDH (additional NADH recycling)
Entry
x-Transaminase
Amine 4c DMSO
1 2 3 4 5
(R)-Arthrobacter sp Aspergillus terreus C. violaceum P. fluorescens Arthrobacter citreus
DME
Conv.b [%]
eec [%]
27 89 92 74 33
>99 >99 >99 >99 >99
(S) (R) (R) (S) (S)
Conv.b [%] 2 83 92 59 42
MeOH
DMF
eec [%]
Conv.b [%]
eec [%]
Conv.b [%]
eec [%]
>99 >99 >99 >99 >99
27 74 82 51 51
>99 >99 >99 >99 >99
21 72 79 38 13
>99 >99 >99 >99 >99
(S) (R) (R) (S) (S)
(S) (R) (R) (S) (S)
(S) (R) (R) (S) (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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
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J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501 Table 2 Asymmetric reductive amination of boronic ester 2a using pyruvate removing system (LDH)a O
NH2 ω-transaminase, PLP B
O
B OH
OH
O
2a
O
NH2
(R) or (S)-4c O
O
O
O
LDH OH OH
(additional NADH recycling) O
Entry
x-Transaminase
1 2 3 4 5
(R)-Arthrobacter sp. Aspergillus terreus C. violaceum P. fluorescens Arthrobacter citreus
Conversionb [%]
eec [%]
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 x-TAs (originating from A. terreus and Arthrobacter sp.) gave excellent results affording amine (R)-4c between 89% and 92% (entries 1 and 2) in the presence of 10 vol % DMSO. A further 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 reaction) 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). The reactions were also tested at elevated temperatures (40 °C) (Table 3), leading to lower conversions in comparison to the experiments at 30 °C (Table 1). This could be rationalized by the partial inactivation of the transaminase and/or any other enzyme employed for the cofactor recycling, although the stereochemical outcome remained excellent (ee >99%) in all cases.
Other reaction-relevant parameters such as the alanine-concentration 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).
Table 4 Asymmetric reductive amination of 2a by different alanine concentrations using an AlaDH cofactor recycling systema Entry
x-Transaminase
Alanine [mM]
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 C. violaceum C. violaceum
500 250 125 500 250 125 500 250 125
Conversionb [%] 90 86 65 82 77 68 43 60 48
eec [%] >99 >99 >99 >99 >99 >99 >99 >99 >99
(R) (R) (R) (R) (R) (R) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1.0 mM), 2a (25 mM), D- or 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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
Table 3 Asymmetric reductive amination of boronic ester 2a at 40 °C using AlaDH cofactor recycling systema 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 >99 >99 >99 >99
(R) (R) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1 mM), substrate 2a (25 mM), D- or L-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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
Having established a suitable protocol for the enzymatic reductive 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 aminoboronic acids 4a/4b and amino-trifluoroborates 4e/4f into the corresponding pinacol esters 4c/4d seemed to be therefore the method 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
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NH2
NH2 O
(i) BY2
BY2
B BY2 = BF3K: (iii)
(R) or (S)-4
O (R)- or (S)-4 para-BPin = 4c meta-BPin = 4d
BY2: para-B(OH)2 = 4a meta-B(OH)2 = 4b para-BF3K = 4e meta-BF3K = 4f
Scheme 3. Synthesis of amino-boronates 4a,b,e,f and their conversion into the pinacol esters 4c and 4d. (i) x-Transaminase, D- or L-alanine, PLP, NAD+, AlaDH, FDH, 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.
and effective way, but also to convert them into the analogous pinacol esters 4c and 4d in a one pot protocol. The scope of the methodology was then investigated but omitting the x-TAs from A. citreus and P. fluorescens since these enzymes generally gave only moderate conversions. We first examined the (R)-selective x-TA employing the organoboron compounds 1–3 (Table 5). Excellent conversions were observed; in the case of the para-substituted boron species, the pinacol ester 2a was converted by both enzymes with 94% conversion to provide the corresponding amine 4c in perfect enantiomeric 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%).
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 Conv.b [%]
eec [%]
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 >99 >99 >99 >99 n.a.
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.
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 >99 >99 98 98 n.a.
(R) (R) (R) (R) (R)
(S) (S) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1 mM), substrate 1–3 (25 mM), D- or L-alanine (250 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, 30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm) (in the case of boronic salt was not used any organic solvent). b Determined by GC-analysis. c 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.
The meta-substituted boron-derivatives 1b, 2b, and 3b however gave diminished conversions likely due to the enhanced steric hindrance. Nevertheless, the best results regarding the regioisomers were obtained with (R)-Arthrobacter sp., which converted the
meta-derived pinacol-ester 2b in 62% yield (ee >99%), whereas the x-TA from A. terreus was not suitable (entries 10–12). The same trend was also observed for the (S)-selective x-TA from C. violaceum: Even though the para-isomers were converted in up to 68%, the meta-isomers led to lower conversions (e.g., 51%, pinacol ester 2a). Nevertheless, the stereochemical outcome remained excellent throughout with ee’s >98% for all the experiments. 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)-configured amines in up to 94% conversion with perfect stereocontrol (ee’s >99%). The analogous (S)-enantiomers were obtained at diminished conversions when employing the x-TA from C. violaceum, although the enantiomeric purities were outstanding with up to ee >99%. Our results showed that the x-transaminases investigated 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.
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 Agilent 5975C mass selective detector and a HP-5 MS column (30 m 0.25 mm 0.25 lm; helium as carrier gas [flow = 0.55 mL/min]). L-Lactate dehydrogenase (LDH) from rabbit muscle [lyophilized powder, 136 U mg1 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 dehydrogenase [GDH; lyophilized powder, 25 U mg1 (one unit will oxidize 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 mL1 (one unit will 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-TA)22 as well as recombinant L-alanine
J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
dehydrogenase (AlaDH) were prepared and purified as described recently.22c 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 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, 24.8. FT-IR (KBr) mmax = 2988, 1680, 1359, 1093, 1016, 857, 832, 654, 599 cm1. 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 (50 MHz, CDCl3) d [ppm] = 198.3, 139.3, 136.5, 134.7, 130.7, 128.0, 84.1, 26.7, 24.8. FT-IR (KBr) mmax = 2978, 1712, 1384, 1144, 979, 851, 668 cm1.
O
O
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 solvent 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 dried under high vacuum. Data for organoboron compound 3a: 1H 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) mmax = 2944, 1655, 1398, 1285, 1206, 974, 824, 758, 650, 602, 484 cm1. 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 cm1. 4.3. General procedure for the reductive amination using an AlaDH cofactor recycling system Lyophilized cells of E. coli containing the corresponding overexpressed x-transaminase (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1.0 mM), 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
NH
B O (S)-4a-Ac
O
O
NH
B O r ac-4a-Ac
O
O
1499
NH
B O (R)-4a-Ac
Figure 1. GC analysis of the acetylated form of compound 4a (4a-Ac).
1500
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30 min. The substrate (25 mM, dissolved in 100 lL DMSO) was added afterward and the reductive amination carried out at 30 °C in an Eppendorf ThermomixerÒ (700 rpm) for 24 h (horizontal position). After 24 h, the reactions were stopped by the addition of saturated Na2CO3 (200 lL). The residue was extracted with EtOAc (2 500 lL). The combined organic layers were dried over MgSO4 and an aliquot withdrawn for further analysis (Sections 4.8 and 4.9).
vacuum and heating (Centrifuge Genevac EZ2 plus) for 1.5 h. The resultant solid was washed with THF (4 500 lL) and the supernatant 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 repeated (twice). This final content was submitted to an acetylation protocol (Section 4.7) in order to determine the ee (Section 4.9).
4.4. General procedure for the reductive amination using an LDH cofactor recycling system
4.6. General procedure to convert the potassium organotrifluoroborates 4e,f into their respective pinacol esters 4c,d after enzymatic reaction
Lyophilized cells of E. coli containing the corresponding overexpressed x-transaminase (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM), NAD+ (1 mM), glucose (150 mM), LDH (90 U), GDH (15 U), and D- or L-alanine (500 mM) at room temperature for 30 min. The substrate 2a (25 mM, dissolved in 100 lL DMSO) was added afterward and the reductive amination carried out at 30 °C in an Eppendorf ThermomixerÒ (600 rpm) for 24 h (horizontal position). After the indicated time, the reaction was stopped by the addition of saturated Na2CO3 (200 lL). The residue was extracted with EtOAc (2 500 lL). The combined organic layers were dried over MgSO4 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 enzymatic reactions were carried out as described above. After reaction, the water was evaporated by centrifugation under
O
O B
O
The enzymatic reactions were carried out according to the conditions 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). 4.7. General procedure for the derivatization of aminoboronic pinacol esters 4c,d to the corresponding acetamides The crude aminoboronic pinacol esters 4c,d were dissolved in EtOAc (500 lL) and treated with pyridine (50 lL) and acetic anhydride (50 lL) after extraction of the biotransformation. The acetylation was performed at 30 °C for 12 h in an Eppendorf ThermomixerÒ (700 rpm). After the indicated time, the reactions
NH
(S)-4b-Ac
O
O B
O
NH
rac-4b-Ac
O
O B
O
NH
(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
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 ketones into amines Determination of the conversion by means of GC measurements: column DB-1701 (Agilent J&W, 30 m 0.25 mm 0.25 lm); 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. 4.9. GC analysis for the determination of the enantiomeric excesses Determination of the enantiomeric excess (ee) by GC on a chiral phase: column hydrodex-b-TBDAc (50 m 0.25 mm 0.25 lm; Marcherey–Nagel). Acetylated derivative of compound 4a (4a-Ac) (Fig. 1): temperature program = from 200 (keep for 10 min isotherm) to 230 °C with slope 1.0 °C min1; Rt [(R)-4a-Ac] = 35.9 min, Rt [(S)-4aAc] = 36.3 min. Acetylated derivative of compound 4b (4b-Ac) (Fig. 2): temperature program = starting from 190 (keep for 10 min isotherm) to 230 °C with slope 1.0 °C min1; Rt [(R)-4b-Ac] = 39.3 min, Rt [(S)4b-Ac] = 39.7 min. Acknowledgements 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-COMETFunding Program. References 1. (a) Patel, B. K.; Hutt, A. J. In Chirality in Drug Design and Development; Reddy, I. K., Mehvar, R., Eds.; Marcel Dekker: New York, 2004; pp 127–174; (b) Brooks, W. H.; Guida, W.; Daniel, G. K. Curr. Top. Med. Chem. 2011, 11, 760–770. 2. (a) Nugent, T. C.; El-Shazly, M. Adv. Synth. Catal. 2010, 352, 753–819; (b) Bartoszewics, A.; Ahlsten, N.; Martín-Matute, B. Chem. Eur. J. 2013, 19, 7274– 7302. 3. (a) Faber, K. Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin/ Heidelberg, 2011; (b)Modern Biocatalysis; Fessner, W.-D., Anthonsen, T., Eds.; Wiley-VCH: Weinheim, 2009; (c)Asymmetric Organic Synthesis with Enzymes; Gotor, V., Alfonso, I., García-Uridales, E., Eds.; Wiley-VCH: Weinheim, 2008. 4. (a) Busto, E.; Gotor-Fernández, V.; Gotor, V. Chem. Rev. 2011, 111, 3998–4035; (b) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis-Regioand Stereoselective Biotransformations, 2nd ed.; Wiley-VCH: Weinheim, 2006. 5. Selected example: (a) Li, T.; Liang, J.; Ambrogelly, A.; Brennan, T.; Gloor, G.; Huisman, G.; Lalonde, J.; Lekhal, A.; Mijts, B.; Muley, S.; Newman, L.; Tobin, M.; Wong, G.; Zaks, A.; Zhang, X. J. Am. Chem. Soc. 2012, 134, 6467–6472; (b) Köhler, V.; Bailey, K. R.; Znabet, A.; Raftery, J.; Helliwell, M.; Turner, N. J. Angew. Chem., Int. Ed. 2010, 49, 2182–2184; (c) Dunsmore, C. J.; Carr, R.; Fleming, T.; Turner, N. J. J. Am. Chem. Soc. 2006, 128, 2224–2226. 6. (a) Abrahamson, M. J.; Wong, J. W.; Bommarius, A. Adv. Synth. Catal. 2013, 355, 1780–1786; (b) Abrahamson, M. J.; Vázquez-Figueroa, E.; Woodall, N. B.; Moore, J. F.; Bommarius, A. S. Angew. Chem., Int. Ed. 2012, 51, 3969–3972. 7. Selected review articles: (a) Kroutil, W.; Fischereder, E.-M.; Fuchs, C. S.; Lechner, H.; Mutti, F. G.; Pressnitz, D.; Rajagopalan, A.; Sattler, J. H.; Simon, R. C.; Siirola, E. Org. Proc. Res. Dev. 2013, 17, 751–759; (b) Mathew, S.; Yun, H. ACS Catal. 2012, 2, 993–1001; (c) Malik, M. S.; Park, E.-S.; Shin, J.-S. Appl. Microbiol. Biotechnol. 2012, 94, 1163–1171; (d) Tufvesson, P.; Lima-Ramos, J.; Jensen, J. S.; Al-Haque, N.; Neto, W.; Woodley, J. M. Biotechnol. Bioeng. 2011, 108, 1479– 1493; (e) Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol. 2010, 28, 324–332; (f) Hailes, H. C.; Dalby, P. A.; Lye, G. J.; Baganz, F.; Micheletti, M.; Szita, N.; Ward, J. M. Curr. Org. Chem. 2010, 14, 1883–1893; (g) Ward, J.; Wohlgemuth, R. Curr. Org. Chem. 2010, 14, 1914–1927.
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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. 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. 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. 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– 7694; for the x-TA synthesis of mexiletine see: (e) Koszelewski, D.; Pressnitz, D.; Clay, D.; Kroutil, W. Org. Lett. 2009, 11, 4810–4812. 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. 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. 14. Smoum, R.; Rubinstein, A.; Dembitsky, V. M.; Srebnik, M. Chem. Rev. 2012, 112, 4156–4220. and references cited therein.. 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. 19. (a) Kaulmann, U.; Smithies, K.; Smith, M. E. B.; Hailes, H. C.; Ward, H. M. Enzyme Microb. Technol. 2007, 41, 628–637; (b) Ito, N.; Kawano, S.; Hagesawa, S.; Yasohara, Y. Biosci. Biotechnol. Biochem. 2011, 75, 2093–2095; (c) Pannuri, S.; Kamat, S. V.; Garcia, A. R. M. WO 2006/063336 A2, Cambrex North Brunswick, 2006.; (d) Yamada, Y.; Iwasaki, A.; Kizaki, N. EP 0987332 A1, Kaneka Corporation, 2000. 20. For the application of x-TA in organic solvents, see Mutti, F. G.; Kroutil, W. Adv. Synth. Catal. 2012, 354, 3409–3413. 21. Molander, G. A.; Cavalcanti, N. L.; Canturk, B.; Pan, P.-S.; Kennedy, L. E. J. Org. Chem. 2009, 74, 7364–7369. 22. (a) Koszelewski, D.; Göritzer, M.; Clay, D.; Seisser, B.; Kroutil, W. ChemCatChem 2010, 2, 73–77; (b) Mutti, F. G.; Fuchs, C. S.; Pressnitz, D.; Turrini, N. G.; Sattler, J. H.; Lerchner, A.; Skerra, A.; Kroutil, W. Eur. J. Org. Chem. 2012, 5, 1003–1007; (c) Mutti, F. G.; Fuchs, C. S.; Pressnitz, D.; Sattler, J. H.; Kroutil, W. Adv. Synth. Catal. 2011, 353, 3227–3233.
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry journal homepage: www.elsevier.com/locate/tetasy
Asymmetric reductive amination of boron-containing aryl-ketones using x-transaminases Joel S. Reis a, Robert C. Simon b,c, Wolfgang Kroutil b,⇑, Leandro H. Andrade a,⇑ a
Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil 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 b
a r t i c l e
i n f o
Article history: Received 19 August 2013 Accepted 4 October 2013
a b s t r a c t The asymmetric reductive amination of aryl-ketones bearing various boron-functionalities (acid, ester or potassium trifluoroborates) was investigated employing enantiocomplementary x-transaminases as catalysts. 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. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Enantiomerically pure amines are valuable chiral building blocks for the synthesis of a broad range of biologically active compounds, such as agrochemicals or pharmaceuticals. Since the activity of these molecules is generally related to one specific absolute configuration, efficient asymmetric methods are required, in particular those that are mild and environmentally benign reaction conditions.1 Numerous synthetic methods have been described to provide chiral amines based on the utilization of metal- and organocatalysts; 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 displayed by enzymes.3 While initially hydrolases were frequently employed in (dynamic)-kinetic resolution processes,4 monoamine oxidases (MAO), 5 amine-dehydrogenases (AmDH) 6 and x-transaminases (x-TA)7 were only applied more recently. The latter ones have successfully been used to prepare a variety of enantiomerically 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⇑ Corresponding authors. Tel.: +55 11 3091 2287 (L.H.A.). E-mail addresses: [email protected] (W. Kroutil), [email protected] (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
ents are also readily incorporated into complex molecules whereas boronic acids and their derivatives can serve as catalysts (e.g., CBSreduction, 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 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 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). 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 often favors the side of the ketone rather than the amine and additional co-product (pyruvate) consumption may hamper the overall efficiency.7 Nevertheless, the shift of the equilibrium is achieved by combining the x-TA catalyzed reductive amination either with an alanine dehydrogenase (AlaDH) or a lactate dehydrogenase (LDH). The AlaDH approach is favored with respect to atom efficiency since the formed co-product (pyruvate) is
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J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501 O O
pinacol, THF B
O
O
2a; para (86%) 2b; meta (76%)
OH B OH 1a; para 1b; meta
O KHF2, MeOH BF3 K
2 h, 0°C
3a; para, 79% 3b; meta, 76%
Scheme 1. Synthesis of aromatic boron-containing ketones 2 and 3.
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 evaluation for the amination of boronic ester 2a as a model substrate was performed in order to identify appropriate x-TAs [substrate concentration: 50 mM (12.5 mg/mL), 1.0 mL total volume]. Depending on the stereoselectivity of the corresponding enzyme, D- or L-alanine was used as the amine source whereas the pyruvate was recy-
cled with an alanine dehydrogenase (AlaDH). Among the different enantiocomplementary x-TAs tested [x-transaminase (origin): Arthrobacter sp., Aspergillus terreus, Hyphomonas neptunium, Chromobacterium violaceum, Bacilus megaterium, Pseudomonas fluorescens, Paracoccus denitrificans, Ralstonia eutropha, Pseudomonas putida, Arthrobacter citreus, and Vibrio fluvialis], some gave reasonable conversions (between 21% and 68%, detailed data not shown) which rendered them suitable for a more detailed investigation: xTAs from Chromobacterium violaceum,19a Pseudomonas fluorescens,19b Arthrobacter citreus,19c (R)-Arthrobacter sp.19d and Aspergillus terreus.8d 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 solubility reasons (Table 1).20 As expected, a significant influence of the co-solvent on the biotransformation 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 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
O
NH2 ω-transaminase, PLP BY2
BY2
O
NH2
OH
OH
1-3 O
recycling via AlaDH (additional NADH recycling)
BY2: para-B(OH)2 = 1a meta-B(OH)2 = 1b para-BPin = 2a meta-BPin = 2b para-BF3K = 3a meta-BF3K = 3b
(R) or (S)-4
O BY2: para-B(OH)2 = 4a meta-B(OH)2 = 4b para-BPin = 4c meta-BPin = 4d para-BF3K = 4e meta-BF 3K = 4f
OH OH 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 B
O
NH2
B
OH
O
2a
O OH
O
O
O
(R) or (S)-4c
O
AlaDH (additional NADH recycling)
Entry
x-Transaminase
Amine 4c DMSO
1 2 3 4 5
(R)-Arthrobacter sp Aspergillus terreus C. violaceum P. fluorescens Arthrobacter citreus
DME
Conv.b [%]
eec [%]
27 89 92 74 33
>99 >99 >99 >99 >99
(S) (R) (R) (S) (S)
Conv.b [%] 2 83 92 59 42
MeOH
DMF
eec [%]
Conv.b [%]
eec [%]
Conv.b [%]
eec [%]
>99 >99 >99 >99 >99
27 74 82 51 51
>99 >99 >99 >99 >99
21 72 79 38 13
>99 >99 >99 >99 >99
(S) (R) (R) (S) (S)
(S) (R) (R) (S) (S)
(S) (R) (R) (S) (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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
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J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501 Table 2 Asymmetric reductive amination of boronic ester 2a using pyruvate removing system (LDH)a O
NH2 ω-transaminase, PLP B
O
B OH
OH
O
2a
O
NH2
(R) or (S)-4c O
O
O
O
LDH OH OH
(additional NADH recycling) O
Entry
x-Transaminase
1 2 3 4 5
(R)-Arthrobacter sp. Aspergillus terreus C. violaceum P. fluorescens Arthrobacter citreus
Conversionb [%]
eec [%]
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 x-TAs (originating from A. terreus and Arthrobacter sp.) gave excellent results affording amine (R)-4c between 89% and 92% (entries 1 and 2) in the presence of 10 vol % DMSO. A further 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 reaction) 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). The reactions were also tested at elevated temperatures (40 °C) (Table 3), leading to lower conversions in comparison to the experiments at 30 °C (Table 1). This could be rationalized by the partial inactivation of the transaminase and/or any other enzyme employed for the cofactor recycling, although the stereochemical outcome remained excellent (ee >99%) in all cases.
Other reaction-relevant parameters such as the alanine-concentration 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).
Table 4 Asymmetric reductive amination of 2a by different alanine concentrations using an AlaDH cofactor recycling systema Entry
x-Transaminase
Alanine [mM]
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 C. violaceum C. violaceum
500 250 125 500 250 125 500 250 125
Conversionb [%] 90 86 65 82 77 68 43 60 48
eec [%] >99 >99 >99 >99 >99 >99 >99 >99 >99
(R) (R) (R) (R) (R) (R) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1.0 mM), 2a (25 mM), D- or 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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
Table 3 Asymmetric reductive amination of boronic ester 2a at 40 °C using AlaDH cofactor recycling systema 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 >99 >99 >99 >99
(R) (R) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1 mM), substrate 2a (25 mM), D- or L-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. c Determined by chiral GC-analysis after derivatization with Ac2O/pyridine to the corresponding acetamide.
Having established a suitable protocol for the enzymatic reductive 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 aminoboronic acids 4a/4b and amino-trifluoroborates 4e/4f into the corresponding pinacol esters 4c/4d seemed to be therefore the method 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
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J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501 BY2 = B(OH)2: (ii) O
NH2
NH2 O
(i) BY2
BY2
B BY2 = BF3K: (iii)
(R) or (S)-4
O (R)- or (S)-4 para-BPin = 4c meta-BPin = 4d
BY2: para-B(OH)2 = 4a meta-B(OH)2 = 4b para-BF3K = 4e meta-BF3K = 4f
Scheme 3. Synthesis of amino-boronates 4a,b,e,f and their conversion into the pinacol esters 4c and 4d. (i) x-Transaminase, D- or L-alanine, PLP, NAD+, AlaDH, FDH, 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.
and effective way, but also to convert them into the analogous pinacol esters 4c and 4d in a one pot protocol. The scope of the methodology was then investigated but omitting the x-TAs from A. citreus and P. fluorescens since these enzymes generally gave only moderate conversions. We first examined the (R)-selective x-TA employing the organoboron compounds 1–3 (Table 5). Excellent conversions were observed; in the case of the para-substituted boron species, the pinacol ester 2a was converted by both enzymes with 94% conversion to provide the corresponding amine 4c in perfect enantiomeric 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%).
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 Conv.b [%]
eec [%]
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 >99 >99 >99 >99 n.a.
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.
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 >99 >99 98 98 n.a.
(R) (R) (R) (R) (R)
(S) (S) (S) (S) (S)
a Lyophilized E. coli cells containing overexpressed x-transaminase (20 mg), PLP (1 mM), substrate 1–3 (25 mM), D- or L-alanine (250 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, 30 °C, 24 h in an Eppendorf ThermomixerÒ (700 rpm) (in the case of boronic salt was not used any organic solvent). b Determined by GC-analysis. c 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.
The meta-substituted boron-derivatives 1b, 2b, and 3b however gave diminished conversions likely due to the enhanced steric hindrance. Nevertheless, the best results regarding the regioisomers were obtained with (R)-Arthrobacter sp., which converted the
meta-derived pinacol-ester 2b in 62% yield (ee >99%), whereas the x-TA from A. terreus was not suitable (entries 10–12). The same trend was also observed for the (S)-selective x-TA from C. violaceum: Even though the para-isomers were converted in up to 68%, the meta-isomers led to lower conversions (e.g., 51%, pinacol ester 2a). Nevertheless, the stereochemical outcome remained excellent throughout with ee’s >98% for all the experiments. 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)-configured amines in up to 94% conversion with perfect stereocontrol (ee’s >99%). The analogous (S)-enantiomers were obtained at diminished conversions when employing the x-TA from C. violaceum, although the enantiomeric purities were outstanding with up to ee >99%. Our results showed that the x-transaminases investigated 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.
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 Agilent 5975C mass selective detector and a HP-5 MS column (30 m 0.25 mm 0.25 lm; helium as carrier gas [flow = 0.55 mL/min]). L-Lactate dehydrogenase (LDH) from rabbit muscle [lyophilized powder, 136 U mg1 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 dehydrogenase [GDH; lyophilized powder, 25 U mg1 (one unit will oxidize 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 mL1 (one unit will 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-TA)22 as well as recombinant L-alanine
J. S. Reis et al. / Tetrahedron: Asymmetry 24 (2013) 1495–1501
dehydrogenase (AlaDH) were prepared and purified as described recently.22c 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 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, 24.8. FT-IR (KBr) mmax = 2988, 1680, 1359, 1093, 1016, 857, 832, 654, 599 cm1. 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 (50 MHz, CDCl3) d [ppm] = 198.3, 139.3, 136.5, 134.7, 130.7, 128.0, 84.1, 26.7, 24.8. FT-IR (KBr) mmax = 2978, 1712, 1384, 1144, 979, 851, 668 cm1.
O
O
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 solvent 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 dried under high vacuum. Data for organoboron compound 3a: 1H 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) mmax = 2944, 1655, 1398, 1285, 1206, 974, 824, 758, 650, 602, 484 cm1. 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 cm1. 4.3. General procedure for the reductive amination using an AlaDH cofactor recycling system Lyophilized cells of E. coli containing the corresponding overexpressed x-transaminase (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1.0 mM), 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
NH
B O (S)-4a-Ac
O
O
NH
B O r ac-4a-Ac
O
O
1499
NH
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 lL DMSO) was added afterward and the reductive amination carried out at 30 °C in an Eppendorf ThermomixerÒ (700 rpm) for 24 h (horizontal position). After 24 h, the reactions were stopped by the addition of saturated Na2CO3 (200 lL). The residue was extracted with EtOAc (2 500 lL). The combined organic layers were dried over MgSO4 and an aliquot withdrawn for further analysis (Sections 4.8 and 4.9).
vacuum and heating (Centrifuge Genevac EZ2 plus) for 1.5 h. The resultant solid was washed with THF (4 500 lL) and the supernatant 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 repeated (twice). This final content was submitted to an acetylation protocol (Section 4.7) in order to determine the ee (Section 4.9).
4.4. General procedure for the reductive amination using an LDH cofactor recycling system
4.6. General procedure to convert the potassium organotrifluoroborates 4e,f into their respective pinacol esters 4c,d after enzymatic reaction
Lyophilized cells of E. coli containing the corresponding overexpressed x-transaminase (20 mg) were rehydrated in a potassium phosphate buffer (pH 7.0, 100 mM) containing PLP (1 mM), NAD+ (1 mM), glucose (150 mM), LDH (90 U), GDH (15 U), and D- or L-alanine (500 mM) at room temperature for 30 min. The substrate 2a (25 mM, dissolved in 100 lL DMSO) was added afterward and the reductive amination carried out at 30 °C in an Eppendorf ThermomixerÒ (600 rpm) for 24 h (horizontal position). After the indicated time, the reaction was stopped by the addition of saturated Na2CO3 (200 lL). The residue was extracted with EtOAc (2 500 lL). The combined organic layers were dried over MgSO4 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 enzymatic reactions were carried out as described above. After reaction, the water was evaporated by centrifugation under
O
O B
O
The enzymatic reactions were carried out according to the conditions 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). 4.7. General procedure for the derivatization of aminoboronic pinacol esters 4c,d to the corresponding acetamides The crude aminoboronic pinacol esters 4c,d were dissolved in EtOAc (500 lL) and treated with pyridine (50 lL) and acetic anhydride (50 lL) after extraction of the biotransformation. The acetylation was performed at 30 °C for 12 h in an Eppendorf ThermomixerÒ (700 rpm). After the indicated time, the reactions
NH
(S)-4b-Ac
O
O B
O
NH
rac-4b-Ac
O
O B
O
NH
(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
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 ketones into amines Determination of the conversion by means of GC measurements: column DB-1701 (Agilent J&W, 30 m 0.25 mm 0.25 lm); 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. 4.9. GC analysis for the determination of the enantiomeric excesses Determination of the enantiomeric excess (ee) by GC on a chiral phase: column hydrodex-b-TBDAc (50 m 0.25 mm 0.25 lm; Marcherey–Nagel). Acetylated derivative of compound 4a (4a-Ac) (Fig. 1): temperature program = from 200 (keep for 10 min isotherm) to 230 °C with slope 1.0 °C min1; Rt [(R)-4a-Ac] = 35.9 min, Rt [(S)-4aAc] = 36.3 min. Acetylated derivative of compound 4b (4b-Ac) (Fig. 2): temperature program = starting from 190 (keep for 10 min isotherm) to 230 °C with slope 1.0 °C min1; Rt [(R)-4b-Ac] = 39.3 min, Rt [(S)4b-Ac] = 39.7 min. Acknowledgements 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-COMETFunding Program. References 1. (a) Patel, B. K.; Hutt, A. J. In Chirality in Drug Design and Development; Reddy, I. K., Mehvar, R., Eds.; Marcel Dekker: New York, 2004; pp 127–174; (b) Brooks, W. H.; Guida, W.; Daniel, G. K. Curr. Top. Med. Chem. 2011, 11, 760–770. 2. (a) Nugent, T. C.; El-Shazly, M. Adv. Synth. Catal. 2010, 352, 753–819; (b) Bartoszewics, A.; Ahlsten, N.; Martín-Matute, B. Chem. Eur. J. 2013, 19, 7274– 7302. 3. (a) Faber, K. Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin/ Heidelberg, 2011; (b)Modern Biocatalysis; Fessner, W.-D., Anthonsen, T., Eds.; Wiley-VCH: Weinheim, 2009; (c)Asymmetric Organic Synthesis with Enzymes; Gotor, V., Alfonso, I., García-Uridales, E., Eds.; Wiley-VCH: Weinheim, 2008. 4. (a) Busto, E.; Gotor-Fernández, V.; Gotor, V. Chem. Rev. 2011, 111, 3998–4035; (b) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis-Regioand Stereoselective Biotransformations, 2nd ed.; Wiley-VCH: Weinheim, 2006. 5. Selected example: (a) Li, T.; Liang, J.; Ambrogelly, A.; Brennan, T.; Gloor, G.; Huisman, G.; Lalonde, J.; Lekhal, A.; Mijts, B.; Muley, S.; Newman, L.; Tobin, M.; Wong, G.; Zaks, A.; Zhang, X. J. Am. Chem. Soc. 2012, 134, 6467–6472; (b) Köhler, V.; Bailey, K. R.; Znabet, A.; Raftery, J.; Helliwell, M.; Turner, N. J. Angew. Chem., Int. Ed. 2010, 49, 2182–2184; (c) Dunsmore, C. J.; Carr, R.; Fleming, T.; Turner, N. J. J. Am. Chem. Soc. 2006, 128, 2224–2226. 6. (a) Abrahamson, M. J.; Wong, J. W.; Bommarius, A. Adv. Synth. Catal. 2013, 355, 1780–1786; (b) Abrahamson, M. J.; Vázquez-Figueroa, E.; Woodall, N. B.; Moore, J. F.; Bommarius, A. S. Angew. Chem., Int. Ed. 2012, 51, 3969–3972. 7. Selected review articles: (a) Kroutil, W.; Fischereder, E.-M.; Fuchs, C. S.; Lechner, H.; Mutti, F. G.; Pressnitz, D.; Rajagopalan, A.; Sattler, J. H.; Simon, R. C.; Siirola, E. Org. Proc. Res. Dev. 2013, 17, 751–759; (b) Mathew, S.; Yun, H. ACS Catal. 2012, 2, 993–1001; (c) Malik, M. S.; Park, E.-S.; Shin, J.-S. Appl. Microbiol. Biotechnol. 2012, 94, 1163–1171; (d) Tufvesson, P.; Lima-Ramos, J.; Jensen, J. S.; Al-Haque, N.; Neto, W.; Woodley, J. M. Biotechnol. Bioeng. 2011, 108, 1479– 1493; (e) Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol. 2010, 28, 324–332; (f) Hailes, H. C.; Dalby, P. A.; Lye, G. J.; Baganz, F.; Micheletti, M.; Szita, N.; Ward, J. M. Curr. Org. Chem. 2010, 14, 1883–1893; (g) Ward, J.; Wohlgemuth, R. Curr. Org. Chem. 2010, 14, 1914–1927.
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