Exploiting the enantioselectivity of Baeyer-Villiger monooxygenases via boron oxidation

Article abstract of DOI:10.1016/j.tetasy.2012.05.004

The enantioselective carbon-boron bond oxidation of several chiral boron-containing compounds by Baeyer-Villiger monooxygenases was evaluated. PAMO and M446G PAMO conveniently oxidized 1-phenylethyl boronate into the corresponding 1-(phenyl)ethanol (ee = 82-91%). Cyclopropyl boronic esters were also oxidized but with no enantioselectivity. β-Boryl carboxylic esters were not oxidized by any BVMOs.

Full text of DOI:10.1016/j.tetasy.2012.05.004

Tetrahedron: Asymmetry 23 (2012) 703–708
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Exploiting the enantioselectivity of Baeyer-Villiger monooxygenases via
boron oxidation
Patrícia B. Brondani a, Hanna Dudek b, Joel S. Reis a, Marco W. Fraaije b, Leandro H. Andrade a,
⇑
a Institute of Chemistry, University of São Paulo, Av. Professor Lineu Prestes 748, SP 05508-900, São Paulo, Brazil
b Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The enantioselective carbon–boron bond oxidation of several chiral boron-containing compounds by Bae-
yer-Villiger monooxygenases was evaluated. PAMO and M446G PAMO conveniently oxidized 1-phenyl-
ethyl boronate into the corresponding 1-(phenyl)ethanol (ee = 82–91%). Cyclopropyl boronic esters were
also oxidized but with no enantioselectivity. b-Boryl carboxylic esters were not oxidized by any BVMOs.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 April 2012
Accepted 3 May 2012
Available online 8 June 2012
1. Introduction
they can be converted into other functional groups with retention
of configuration and used as chiral catalysts.9
Baeyer-Villiger monooxygenases (BVMOs) are flavin-containing
and NAD(P)H-dependent enzymes that catalyze the incorporation
of one oxygen atom into organic substrates.1 BVMOs have recently
been shown to be excellent alternatives for performing the oxida-
tion of aldehydes and ketones to their corresponding esters, the
oxygenation of heteroatoms (sulfur, nitrogen, phosphorus, boron,
and selenium) and even epoxidation reactions.2 In many of these
reactions, the transformation of the substrates occurs with high
enantio- and/or regioselectivity, using environmentally friendly
conditions.3 Cyclohexanone monooxygenase (CHMO), which is
one of the most studied BVMOs, has a broad substrate acceptance
and displays moderate thermostability.4 In contrast, phenylace-
tone monooxygenase (PAMO) is remarkably stable but is limited
in substrate range by its preference for aromatic compounds. From
this BVMO, a mutant that has an altered substrate acceptance pro-
file has been generated (M446G PAMO), and accepts a different
range of aromatic ketones and aldehydes while retaining thermo-
stability. 4-Hydroxyacetophenone monooxygenase (HAPMO)3d,5,6
is another BVMO that has been shown to be effective in converting
aromatic ketones.
Based on previous reports of the selectivity of BVMO and the
importance of boron compounds in organic synthesis, herein we
explore the enantioselectivity of BVMOs for oxidative kinetic
resolutions of chiral organoboron compounds (Fig. 1).
2. Results and discussion
2.1. Synthesis of boron substrates for the evaluation of the
enantioselectivity of BVMOs
The set of chiral boron substrates studied is shown in Figure 1.
The organoboron compounds were synthesized according to
Figure 2.
2.2. Evaluation of the enantioselectivity of BVMOs via boron
oxidation
Motivated by our previous result, in which 1-phenylethyl bor-
onate 1a was submitted to oxidative kinetic resolution mediated
by PAMO (Table 1, entry 1),8 we performed the kinetic resolution
of 1a and other boron-containing aromatic compounds 1b and
1c by using PAMO, M446G PAMO, CHMO, and HAPMO (Table 1).
In these reactions we used a cofactor regenerating system by
Recently, we described the first example of the kinetic resolu-
tion of boron-containing chiral compounds mediated by PAMO.7
We have been studying boron-containing compounds because they
are known to be versatile intermediates in organic synthesis. The
C–B bond can be easily oxidized leading to the release of boron
and the formation of a C–O bond, resulting in alcohols, aldehydes,
or ketones, among other reactions.8 Additionally, chiral organobo-
ron compounds are especially important in organic synthesis, since
applying phosphite dehydrogenase (PTDH) and
substrate (phosphite).
a sacrificial
We observed that in all cases CHMO and HAPMO led to the
decomposition of the starting material with no desired product
being observed. PAMO only mediated the kinetic resolution of 1a
(E = 33, Table 1, entry 1). Conversely, M446G PAMO mediated the
oxidation reaction of 1a (1-phenylethyl boronate, E = 9.9) and 1b
(4-methoxy-1-phenylethyl boronate, E = 2.8), but no conversion
of 1c was observed (4-fluoro-1-phenylethyl boronate) (Table 1,
⇑
Corresponding author. Tel.: +55 11 3091 2287; fax: +55 11 3815 5579.
E-mail address: leandroh@iq.usp.br (L.H. Andrade).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.05.004

704
P. B. Brondani et al. / Tetrahedron: Asymmetry 23 (2012) 703–708
BPin
BPin
R1
R2
BPin
R3
CO2R1
R
3a, R = Me, R1= Me
5a, R1 = H, R2 = Ph, R3 = H
R = Ph, R = H, R = H
5c, R1 = H, R2 = H, R3 = Ph
R
1
1
2
3
3b,
R = Me, R = Et
5b,
1a, R = H
3c, R = Me, R1= n-Bu
3d, R = Me, R1= Bn
1b,
1c,
R = OMe
R = F
1
3e,
R = Ph, R = Et
Figure 1. Boron-containing substrates used for the evaluation of the enantioselectivity of BVMOs in an oxidative kinetic resolution process.
O
a
MeO
OP(O)(OEt)2
I
BPin
BPin
b
d
MeO
MeO
c
R
R
I
1a,
1b,
R = H
R = OMe
e
1c, R = F
F
F
a) LDA, THF, (EtO)2P(O)Cl; b) TMSCl, NaI, H2O, CH2Cl2; c) B2Pin2, Pd(PPh3)2Cl2, PhOK, PPh3, toluene;
d) Rh/carbon, H2, toluene; e) TMSCl, NaI, H2O, CH3CN
3a, R = Me, R1= Me
DPEphos
1
3b,
R = Me, R = Et
BPin
CO2R1
f
3c, R = Me, R1= n-Bu
CO2R1
R
3d, R = Me, R1= Bn
R
1
3e,
R = Ph, R = Et
O
PPh2
PPh2
f) CuCl, NaOt-Bu, DPEphos, B2Pin2, MeOH, THF
1
2
3
5a,
R1
R2
BPin
R3
R1
R2
BPin
R3
R = H, R = Ph, R = H
g
5b, R1 = Ph, R2 = H, R3 = H
1
2
3
5c,
R = H, R = H, R = Ph
g
) CH2I2, CH2Cl2, Et2Zn
Figure 2. Synthesis of boron substrates.
entries 2, 4, and 6). It has been reported that the oxidation of the
C–B bond mediated by a monooxygenase occurs with retention
of configuration, similar to non-enzymatic oxidations, resulting
in the formation of the alcohol derivatives.10 PAMO and M446G
PAMO reacted both with the (S)-enantiomer leading to the
(S)-alcohol and the (R)-boronic ester.
We also explored the oxidation reaction of b-boronated
carboxylic esters to give b-hydroxy carboxylic esters. b-Hydroxy
carboxylic esters are highly attractive synthetic targets due to
their versatility in organic chemistry. Additionally, optically active
b-hydroxy carboxylic acids are important and interesting building
blocks for the synthesis of biologically active compounds.11
The results obtained with PAMO (Table 1) led us to evaluate the
kinetic resolution of b-boronated carboxylic esters 3a–e mediated
by this monooxygenase (Scheme 1).
Unfortunately, compounds 3a–b were not stable in the Tris-buf-
fer, and even with no enzyme in the reaction medium, we observed
a decomposition of starting material. On the other hand, com-
pounds 3c–e were stable under the reaction conditions but again
no reaction was observed.

P. B. Brondani et al. / Tetrahedron: Asymmetry 23 (2012) 703–708
705
Table 1
Evaluation of the enantioselectivity of the BVMO-catalyzed oxidation of phenylethyl boronates 1a–ca
O2
H2O
OH
BPin
BPin
BVMO
R
R
R
1a-c
(R)-
(R,S)-1a-c
(S)-2a-c
NADP+
NADPH
phosphate
phosphite
PTDH
Entry
1
Substrate
Enzyme (BVMO)
Time (h)
Conv. (%)
ee (S)-2a–c (%)
ee (R)-1a–c (%)
BPin
BPin
PAMO
5
49
91
858
1a
2
3
4
5
6
M446G PAMO
4
24
7
50
82
70
1a
BPin
BPin
PAMO
(À)b
(À)c
(À)c
MeO
1b
1b
M446G PAMO
52
50
47
MeO
BPin
PAMO
24
24
(À)b
(À)c
(À)c
(À)c
(À)c
F
1c
1c
BPin
M446G PAMO
(À)b
F
a
b
c
Tris buffer 50 mM pH 7.5, DMSO (1%), substrate (10 mM), phosphite (20 mM), PTDH (1
No reaction was observed.
No enantiomeric excess was observed.
lM), NADPH (0.2 mM), and BVMO (4 lM).
We also carried out the kinetic resolution of cyclopropyl
boronic esters 5a–c (Table 2). In addition to the importance of
boron-containing substrates, cyclopropyl compounds are versatile
building blocks in the synthesis of natural products due to their
well-defined three-dimensional structures.12
We used three cyclopropyl derivatives with different geome-
tries. CHMO was unable to perform the oxidation of any of these
compounds 5a–c. Conversely, PAMO, M446G PAMO, and HAPMO
catalyzed the reaction, but with no enantioselectivity regardless
of geometry. Even with the phenyl moiety close to the boronic
ester moiety 5c, no enantioselectivity was observed.
3. Conclusion
We have shown that PAMO was able to oxidize aromatic boron
compounds with no substituent in the aromatic ring with great
enantioselectivity. It could also be used to convert cyclopropyl
derivatives but with no enantioselectivity. PAMO was unable to
oxidize 1-phenylethyl boronates with substituents on the aromatic
ring (4-OMe and 4-F) apart from b-borylated carboxylic esters. The
mutant M446G PAMO showed a similar substrate acceptance, but
this BVMO displayed a lower enantioselectivity. We observed that
HAPMO can catalyze cyclopropyl boronic ester oxidations, but with

706
P. B. Brondani et al. / Tetrahedron: Asymmetry 23 (2012) 703–708
O2
H2O
BPin
BPin
OH
1
3a,
R = Me, R = Me
CO2R1
PAMO
CO2R
CO2R1
X
3b, R = Me, R1= Et
R
R
1
R
*
*
1
3c,
R = Me, R = n-Bu
3a-e
(RS)-
4a-e
3a-e
3d, R = Me, R1= Bn
NADP+
NADPH
1
3e,
R = Ph, R = Et
phosphate
phosphite
PTDH
Scheme 1. Attempts to promote the oxidative kinetic resolution of b-boronated carboxylic esters 3a–e mediated by PAMO.
no enantioselectivity. CHMO was unable to oxidize any of the
tested substrates. We can conclude that the substituent on the
aromatic ring of 1-phenylethyl boronates affects the performance
of PAMO and M446G PAMO. Moreover, cyclopropyl boronic esters
were efficiently oxidized to cyclopropyl alcohols by PAMO, M446G
PAMO, and HAPMO, but no enantioselectivity was observed.
158.80. The reduction of the vinyl boronic ester was carried out
in a similar way to procedures described in section 4.2 to give
2-[1-(4-methoxyphenyl)ethyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane 1b. Yellow oil,90%. 1H NMR (200 MHz, CDCl3): d 1.12 (s, 12H),
1.24 (s, 3H), 2.28 (q, J = 7.8 Hz, 1H), 3.70 (s, 3 H), 6.70–7.14 (m, 4H).
13C NMR (50 MHz, CDCl3): d 17.33, 24.57, 55.16, 83.18, 113.74,
128.58, 136.99, 157.22. LRMS (EI) m/z (%) = 286 (M+, 4), 206 (5),
178 (4), 151(3), 136 (18), 135 (100), 134 (20), 121 (12), 119 (12),
4. Experimental
4.1. General
105 (9), 103 (8), 91 (20), 77 (16), 65 (8). FT-IV (film, cmÀ1
3004, 2919, 1661, 1437, 1407, 1316, 1021.
) mmax:
4.4. Synthesis of 2-[1-(4-fluorophenyl)ethyl]-4,4,5,5-tetrame-
thyl-1,3,2-dioxaborolane 1c8,17
Recombinant phenylacetone monooxygenase (PAMO) from
Thermobifida fusca, M446G PAMO mutant, 4-hydroxyacetophenone
monooxygenase (HAPMO) from Pseudomonas fluorescens and
cyclohexanone monooxygenase (CHMO) from Acinetobacter sp.
were overexpressed and purified as previously described.13
Phosphite dehydrogenase was obtained as described before.14
Phosphite and NADPH were purchased from Sigma–Aldrich.
High performance liquid chromatography (HPLC) analyses for
measurement of enantiomeric excesses were performed on a Shi-
madzu (diode array detector SPD-M1OA, auto injector SIL-10AD e
Column Oven CTO-10A).
The intermediate iodide was synthesized according to the liter-
ature16a and used without purification in the coupling reaction,
which was carried out according to the literature16e,f to give
2-[1-(4-fluorophenyl)vinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane. Yellow solid, 60%. 1H NMR (200 MHz, CDCl3): d 1.25 (s, 12H),
6.88–6.98 (m, 2H), 7.36 (d, J = 5.4 Hz, 2H), 7.40 (d, J = 5.6 Hz, 2H).
13C NMR (50 MHz, CDCl3): d 24.86, 83.95, 114.79, 115.22, 129.35,
129.53, 129.63, 159.05 (d, J = 240.5 Hz). The reduction of the vinyl
boronic ester was carried out in a similar way to procedures de-
scribed in section 4.2 to give 2-[1-(4-fluorophenyl)ethyl]-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane 1c. Yellow oil, 87%. 1H NMR
(200 MHz, CDCl3): d 1.24 (s, 12H), 1.30 (s, 3 H), 1.61–1.81 (m,
1H), 6.94–7.47 (m, 4H). 13C NMR (50 MHz, CDCl3): d 14.13, 24.79,
83.88, 112.11, 114.72, 128.62, 130.51, 156.51 (d, J = 233.5 Hz).
LRMS (EI) m/z (%) = 248 (M+, 54), 247 (27), 232 (26), 205 (30),
204 (31), 191 (36), 174 (25), 162 (45), 149 (44), 148 (100), 147
4.2. Synthesis of 4,4,5,5-tetramethyl-2-(1-phenylethyl)-1,3,2-
dioxaborolane compound 1a15
This compound was synthesized according to the literature.15
Clear oil, 80%. 1H NMR (200 MHz, CDCl3):d 1.19–1.35 (m, 15H),
2.36–2.46 (m, 1H), 7.12–7.26 (m, 5H). 13C NMR (50 MHz, CDCl3):
d 16.99, 24.54, 24.58, 83.26, 125.02, 127.74, 128.24, 144.92. LRMS
(EI) m/z (%) = 232 (M+, 60), 217(28), 174(14), 146(22), 132(74),
117(30), 105(100), 91(14), 83(64), 77(15), 59(10). FT-IV (film,
(90), 123 (35), 77 (34), 58 (31), 50 (18). FT-IV (film, cmÀ1
2957, 2918, 2850, 1736, 1635, 1380, 1233, 1181, 835.
) mmax:
cmÀ1
) mmax: 3065, 3029, 2981, 1459, 1351, 1146.
4.5. Synthesis of b-borylated carboxylic esters 3a–e
4.3. Synthesis of 2-[1-(4-methoxyphenyl)ethyl]-4,4,5,5-tetrame-
thyl-1,3,2-dioxaborolane 1b16
These compounds were synthesized according to the litera-
ture11 and the spectroscopic data are in the literature as well.18
The intermediate diethyl 1-(4-methoxyphenyl)vinyl phosphate
was synthesized according to the literature.16a Orange oil, 75%. 1H
NMR (200 MHz, CDCl3): d 1.34 (t, J = 7.2 Hz, 6H), 3.82 (s, 3H), 4.07–
4.20 (m, 4H), 5.10–5.17 (m, 2 H), 6.88 (d, J = 9 Hz, 2 H), 7.52
(d, J = 9 Hz, 2 H). 13C NMR (50 MHz, CDCl3): d 16.04, 25.47, 55.18,
64.28, 64.40, 95.36, 113.56, 122.27, 126.50, 152.05, 160.11. The
intermediate iodide was synthesized according to the literature16d
and used without purification in the coupling reaction, which was
carried out according to the literature16e,f to give 2-[1-(4-methoxy-
phenyl)vinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. Clear oil,
83%. 1H NMR (200 MHz, CDCl3): d 1.29 (s, 12 H), 3.77 (s, 3H),
5.94 (d, J = 2.8 Hz, 1H), 5.99 (d, J = 2.6 Hz, 1H), 6.8 (d, J = 8.6 Hz,
2H), 7.17 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H). 13C NMR
(50 MHz, CDCl3): d 24.77, 55.20, 83.69, 112.08, 113.56, 128.21,
4.6. Synthesis of cyclopropyl boronic esters 5a–c19
These compounds were synthesized according to the
literature.19
4.6.1. 4,4,5,5-Tetramethyl-2-(2-phenylcyclopropyl)-1,3,2-dioxa-
borolane 5a
Clear oil, 82%. 1H NMR (200 MHz, CDCl3): d 0.25–0.33 (m, 1H),
0.86–1.03 (m, 2H), 1.29 (s, 12H), 2.04–2.10 (m, 1H), 7.03–7.22
(m, 5H). 13C NMR (50 MHz, CDCl3): d 15.05, 21.86, 24.78, 83.31,
125.57, 127.01, 128.19, 128.52, 143.31. LRMS (EI) m/z (%): 245
(13), 244 (M+, 80), 243 (17), 186 (16), 145 (100), 144 (49), 143
(45), 126 (50), 117 (90), 116 (55), 115 (52), 101 (30), 91 (29), 85

P. B. Brondani et al. / Tetrahedron: Asymmetry 23 (2012) 703–708
707
Table 2
Evaluation of the enantioselectivity of the BVMO-catalyzed oxidation of cyclopropyl boronic esters 5a–c oxidationa
O2
H2O
R1
R2
OH
R3
R1
R2
BPin
R3
R1
R2
BPin
R3
BVMO
*
*
5a-c
5a-c
6a-c
NADP+
NADPH
phosphate
phosphite
PTDH
Entry
1
Substrate
Enzyme
Time (h)
Conv. (%)c
45
H
BPin
H
PAMO
24
16
24
24
24
16
24
24
24
16
24
24
Ph
5a
5a
5a
5a
5b
5b
5b
5b
H
BPin
H
2
M446G
HAPMO
CHMO
PAMO
49
Ph
H
BPin
H
3
50
Ph
H
BPin
H
4
(À)b
51
Ph
Ph
H
BPin
H
5
Ph
H
BPin
H
6
M446G
HAPMO
CHMO
PAMO
46
Ph
H
BPin
H
7
55
Ph
H
BPin
H
8
(À)b
50
H
H
BPin
9
Ph
5c
H
H
BPin
Ph
10
11
12
M446G
HAPMO
CHMO
51
5c
5c
5c
H
H
BPin
Ph
56
H
H
BPin
Ph
(À)b
a
b
c
Tris buffer 50 mM pH 7.5, DMSO (1%), substrate (10 mM), phosphite (20 mM), PTDH (1
No reaction was observed.
No enantiomeric excess was observed.
lM), NADPH (0.2 mM), and BVMO (4 lM).
(25), 83 (27). FT-IV (film, cmÀ1
1624, 1449, 1419, 1353, 1323, 1212, 1144, 969, 852, 750, 697.
)
m
max: 3081, 3061, 3026, 2978, 2930,
7.00–7.20 (m, 5H). 13C NMR (50 MHz, CDCl3): d 1.02, 8.86, 21.72,
24.35, 24.74, 82.88, 125.67, 127.62, 128.76, 140.70. LRMS (EI) m/z
(%): 245 (15), 244 (M+, 95), 243 (20), 229 (13), 186 (20), 185
(18), 145 (100), 144 (45), 143 (40), 128 (30), 126 (45), 117 (87),
4.6.2. 4,4,5,5-Tetramethyl-2-(2-phenylcyclopropyl)-1,3,2-
dioxaborolane 5b
116 (70), 115 (65), 101 (30), 91 (28), 83(25). FT-IV (film, cmÀ1
)
Clear oil, 84%. 1H NMR (200 MHz, CDCl3): d 0.28–0.41 (m, 1H),
0.78 (s, 6 H), 0.91 (s, 6H), 0.95–1.06 (m 1H), 1.14–1.22 (m, 2H),
mmax: 3083, 3060, 3026, 2978, 2927,1603, 1409, 1322, 1223,
1144, 1028, 859, 699.

708
P. B. Brondani et al. / Tetrahedron: Asymmetry 23 (2012) 703–708
4.6.3. 4,4,5,5-Tetramethyl-2-(1-phenylcyclopropyl)-1,3,2-
dioxaborolane 5c
The analysis of 5a–c was carried out on Column ChiralpakÒ
AD-H, n-heptane, flow rate: 0.25 mL/min, UV detection: 220 nm.
Retention time of 5a: tR = 20.67 and 22.09; retention time of 5b:
tR = 19.78 and 20.81; retention time of 5c: tR = 17.55 and 19.77.
Clear oil, 75%. 1H NMR (200 MHz, CDCl3): d 0.87–0.92 (m, 2H),
1.07–1.12 (m, 2H), 1.20 (s, 12H), 7.2–7.51 (m, 5H). 13C NMR
(50 MHz, CDCl3): d À4.02, 13.31, 24.57, 83.29, 125.19, 127.14,
127.93, 128.89, 144.79. LRMS (EI) m/z (%): 245 (15), 244 (M+, 90),
243 (17), 229 (16), 187 (55), 171(18), 145 (97), 144 (77), 143
(75), 129 (29), 117 (100), 116 (65), 115 (59), 105 (50), 101 (60),
Acknowledgments
We thank CNPq, CAPES and FAPESP for their financial support.
The authors also would like to thank Dayvson J. Palmeira for the
artwork of Graphical Abstract.
85 (42), 83 (44). FT-IV (film, cmÀ1
) mmax: 3081, 3059, 2978, 2932,
2867, 1685, 1391, 1318, 1145, 849, 699.
4.7. General procedure for enzymatic oxidation reactions
To a flask (2 mL, EppendorfÒ containing a solution of the start-
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ing material (1 M in DMSO, 5
7.5 (50 mM, 440 L), phosphite solution (500 mM, 20
(100 mM, 10 L), PTDH (100 M, 5 L), and enzyme (BVMO)
(100 M, 20 L). Reactions were shaken at 200 rpm and 30 °C
lL) were added Tris/HCl buffer at pH
l
lL), NADPH
l
l
l
l
l
(PAMO and M446G PAMO mutant) or 150 rpm and 25 °C (HAPMO
and CHMO) for the time established. The reactions were stopped,
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lyzed by chiral HPLC. Control experiments in the absence of an en-
zyme were performed for all substrates tested, and no reaction was
observed.
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4.8. General procedure for chemical oxidation of boronic esters
1a–c
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To a flask (2 mL, EppendorfÒ) containing enantiopure boronic
ester 1a–c (0.1 mmol) were added a 2 M NaOH solution (1 mmol)
and H2O2 30% (3 mmol). The reaction was shaken for 3 h at room
temperature. After this period, the reaction was extracted with
EtOAc (3 Â 0.5 mL), dried over Na2SO4, and analyzed by chiral
HPLC.
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4.9. Absolute configuration
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Rychlewski, L.; Fraaije, M. W. Appl. Environ. Microbiol. 2011, 77, 5730–5738.
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The absolute configuration of chiral alcohols 2a–c was estab-
lished by comparing HPLC chromatograms with the patterns de-
scribed in the literature.20 The chiral boron compounds 1a–c
were transformed into the corresponding alcohols 2a–c (see Sec-
tion 4.8) to determine their absolute configurations. The absolute
configuration of compounds 5a–c could not be determined because
the bio-oxidations were not enantioselective.
4.10. Determination of the enantiomeric excess (ee)
The enantiomeric excesses of compounds 2a–c and 5a–c were
measured by chiral HPLC analysis. Compounds 1a–c were trans-
formed into the compounds 2a–c, and then the ee was collected
(see Section 4.8).
17. Kamiya, N.; Chikami, Y.; Ishii, Y. Synlett 1990, 675–676.
18. J. S. Reis, L. H. Andrade, Unpublished results.
The analysis of 2a was carried out on Column ChiracelÒ OD,
n-heptane/iso-propanol 95:5, flow rate: 1 mL/min, UV detection:
254 nm, retention times: tR = 5.00 min (R) and tR = 5.92 min (S).
The analysis of 2b was carried out on Column ChiracelÒ OD-H,
n-heptane/iso-propanol 97:3, flow rate: 0.8 mL/min, UV detection:
227 nm, retention times: tR = 40,54 (S) and 41,93 (R).
19. Pietruszka, J.; Witt, A. J. Chem. Soc., Perkin Trans. 1 2000, 4293–4300.
20. (a) Patti, A.; Pedotti, S.; Sanfilippo, C. Chirality 2007, 19, 344–351; (b)
Belmessieri, D.; Joannesse, C.; Woods, P. A.; MacGregor, C.; Jones, C.;
Campbell, C. D.; Johnston, C. P.; Duguet, N.; Concellón, C.; Bragg, R. A.; Smith,
A. D. Org. Biomol. Chem. 2011, 9, 559–570.
The analysis of 2c was carried out on Column ChiralpakÒ AS-H,
n-heptane/iso-propanol 98.5:1.5, flow rate: 0.5 mL/min, UV detec-
tion: 254 nm, retention time: tR = 20,11 (S) and 21,51 (R).