10.1002/adsc.201800299
Advanced Synthesis & Catalysis
megaterium were overexpressed in E. coli BL21 (DE3) 3H); 13C NMR (100 MHz, CDCl3): δ 220.7, 135.9, 116.6,
strains harbouring a specific plasmid, according to standard 54.2, 41.3, 38.7, 37.6, 32.1, 28.2, 26.6, 26.6, 22.4, 13.9;
molecular biology techniques as described in ref. [30]. MS: m/z (%) 194 [M]+ (1), 153 (13), 124 (6), 109 (2), 97
Protein concentrations were determined according to (4), 83 (100).
Bradford test, using bovine serum albumine (BSA) as a
3-(2-Oxopropyl)-2-pentylcyclopentanone (Magnolione®,
trans-28). To a stirred solution of DMF/H2O (7:1, 5.6 mL),
PdCl2 (124 mg, 0.7 mmol) and CuCl (693 mg, 7 mmol, 1
eq.) were added under an O2 atmosphere. The reaction
mixture was stirred for 1 hour to allow oxygen uptake.
Then, 27 (1.36 g, 7 mmol) was added dropwise. The color
of the solution turned from green to black within 10
minutes and returned gradually to green coulor.[34] After 6
hours, the reaction mixture was quenched with a solution of
HCl (0.1 M, 20 mL), filtered on a pad of Celite and washed
with Et2O (30 mL). The organic layer was separated,
standard. CPADH from Candida parapsilosis and READH
from Rhodoccocus erythropolis were purchased from
Julich Chiral Solutions GmbH (now CODEXIS). PLADH
from Parvibaculum lavamentivorans, DRADH from
Deinococcus radiodurans, BYADH from Saccharomyces
cerevisiae, HLADH from horse liver, TBADH from
Thermoanaerobium brockii, KRED (ketoreductase) from
an unspecified source and laccase from Trametes
versicolor (activity ≥ 10 U mg-1) were purchased from
Sigma-Aldrich.
General Procedure for the Oxidative Rearrangement in washed with brine (sat., 20 mL), dried over Na2SO4,
Water (Method A)
filtered and concentrated under vacuum. The crude product
was purified by column chromatography (gradient eluent:
n-Hex/EtOAc from 90:10 up 80:20) to afford 29 as a
colorless oil:1.25 g; 85% yield; 99% purity by GC (tR =
To a solution of a tertiary allylic alcohol (1.0 mmol) in
DMSO (200 L) in a Schlenk tube was added NaOAc
1
-
buffer (50 mM, pH 5.2, 19.8 mL), TEMPO+BF4 (0.2 mmol,
20.05 min), H NMR (400 MHz, CDCl3): δ 2.76-2.09 (m,
8H), 1.73 (m, 1H); 1.53 (m, 2H); 1.45-1.24 (m, 7H); 0.88 (t,
J = 6.80, 3H); 13C NMR (100 MHz, CDCl3): δ 219.5, 207.1,
54.2, 48.4, 37.6, 37.1, 32.0, 30.4, 28.0, 27.3, 26.4, 22.4,
13.9;[35] MS: m/z (%) 210 [M]+ (7), 195 (1), 181 (1), 167
(1), 153 (100), 140 (37), 125 (23).
49 mg) and T. versicolor laccase (4 mg). The reaction
mixture was purged with pure O2 and then stirred at 30 °C
for 24 h under an O2 atmosphere (balloon). Then, HCl (0.1
M) was added to the reaction mixture and the aqueous layer
was washed with Pentane/Et2O (7:3, 10 mL x 2). The
combined organic layers was washed with brine (sat., 20
mL x 1), dried over Na2SO4 and concentrated under 3-(2-Oxopropyl)-2-pentylcyclopent-2-enone (dehydro-
vacuum. Where needed, the crude material was submitted Magnolione®, 29). To a stirred, completely dissolved
to column chromatography purification.
solution of Hg(OAc)2 (1.84 g, 5 mmol) in H2O (5 mL) THF
(5 mL) was added. The solution was bright yellow. Then,
29 (960 mg, 5 mmol) was added dropwise and the reaction
mixture was stirred at room temperature till the solution
faded. Thus, NaOH (6 M, 3 mL) and NaBH4 (0.5 M, 5 mL)
in 3 M NaOH were carefully added, keeping the
temperature below 25 °C. The reaction was stirred for 2
hours till Hg precipitated as a shiny liquid. Hence, the
reaction mixture was filtered on a Celite pad, washed with
EtOAc (20 mL x 2). The organic layer was extracted,
washed with brine (sat., 20 mL), dried over Na2SO4,
filtered and the solvent was evaporated under reduced
pressure. The crude intermediate product was of sufficient
purity to be used in the next step.[36] Then, the intermediate
was dissolved in CH2Cl2 (50 mL), trapped with a CaCl2
valve and the reaction mixture was cooled to 0°C.
Successively, DMP (2.33 g, 5.5 mmol) was slowly added
and the mixture was stirred for 24 hours. Hence, the
solution was extracted with CH2Cl2 (30 mL), washed with
brine (50 mL), dried over Na2SO4, filtered and
concentrated under vacuum. The titled product was purified
by column chromatography (gradient eluent: n-Hex/EtOAc
from 90:10 up 70:30) to afford 29 as a colorless oil: 770
General Procedure for the Oxidative Rearrangement in
MeCN (Method B)
To a solution of a tertiary allylic alcohol (1.0 mmol) in
-
MeCN (20 mL) was added TEMPO+BF4 (0.20 mmol, 49
mg) and laccase adsorbed on glass beads (2.5 g,
beads/laccase 50:1). The solution was stirred at 30 °C for
24 h under an O2 atmosphere (balloon). Then, the reaction
mixture was filtered concentrated under reduced pressure
and diluted with Et2O. The organic layer was washed with
brine (sat., 20 mL x 1), dried over Na2SO4 and concentrated
under vacuum. The crude material was submitted to
column chromatography purification.
Synthesis of Magnolione® and dehydro-Magnolione®
3-Allyl-2-pentylcyclopent-2-enone (16b). Method A:
163.2 mg; 85% yield; 99% purity by GC (tR = 18.48 min),
1H NMR (400 MHz, CDCl3): δ 5.86-5.76 (m, 1H), 5.16-
5.12 (m, 2H), 3.17 (d, J = 6.64, 2H), 2.49 (t, J = 3.92, 2H),
2.36 (t, J = 4.72, 2H), 2.18 (t, J = 7.36, 2H), 1.41-1.26 (m,
6H), 0.87 (t, J = 6.56, 3H); 13C NMR (100 MHz, CDCl3): δ
209.7, 170.4, 141.0, 133.2, 117.5, 35.7, 34.2, 31.8, 29.0,
28.2, 23.0, 22.4, 13.9; MS: m/z (%) 192 [M]+ (38), 177
(58), 163 (12), 151 (100), 135 (46); HRMS exact mass
calculated for [M+H]+ (C13H21O) requires m/z 193.1592,
found m/z 193.1587.
1
mg; 74% yield; 99% purity by GC (tR = 20.89 min), H
NMR (400 MHz, CDCl3): δ 3.56 (s, 2H), 2.54 (t, J = 4.76,
2H), 2.39 (qt, J = 2.72, 2H), 2.24 (s, 3H), 2.16 (t, J = 7.52,
2H), 1.39-1.24 (m, 6H), 0.87 (t, J = 6.84, 3H); 13C NMR
(100 MHz, CDCl3): δ 208.9, 203.0, 164.0, 143.1, 45.8, 34.3,
31.7, 30.2, 29.9, 27.9, 23.3, 22.3, 13.8; MS: m/z (%) 208
[M]+ (5), 193 (16), 179 (2), 165 (9), 151 (100), 137 (15);
HRMS exact mass calculated for [M+H]+ (C13H21O2)
requires m/z 209.1542, found m/z 209.1548.
(trans)-3-Allyl-2-pentylcyclopentanone (27). Lithium
(140 mg, 20 mmol) was added to liquid ammonia (60 mL)
at -78 °C. After stirring for 15 minutes, a solution of 16b
(384 mg, 2 mmol) and t-BuOH (0.92 μL, 10 mmol) in THF
Chemo-mutienzymatic Synthesis of 1e
(4 mL) were added at -78 °C and the reaction mixture was To a solution of 1a (1.0 mmol) in DMSO (800 L) in a
stirred for 45 minutes. Then, H2O (40 mL) was carefully Schlenk tube was added NaOAc buffer (50 mM, pH 5.2,
added at the same temperature and the reaction was 19.2 mL), TEMPO+BF4 (0.2 mmol, 47 mg) and T.
-
warmed up to allow ammonia to evaporate. Hence, Et2O versicolor laccase (4 mg). The reaction mixture was purged
(2x40 mL) was added and the combined organic layer was with pure O2 and then stirred at 30 °C under an O2
washed with brine (sat., 50 mL), dried over Na2SO4, atmosphere (balloon). After 24 h the reaction mixture was
filtered and concentrated under reduced pressure.[33] The purged with N2, diluted with a phosphate buffer solution
crude product was purified by column chromatography (50 mM, pH 7, 380 mL), and then glucose (720 mg, 4
(gradient eluent: n-Hex/EtOAc, 95:5) to afford 27 as a mmol), NAD(P)+ (14 mg+14 mg), GDH (4 U mL-1), OYE1
colorless oil: 260 mg; 67% yield; 99% purity by GC (tR (80 μg mL-1), and PLADH (250 μg mL-1) or and READH
1
(trans) = 16.77 min, tR (cis) = 17.40 min ), H NMR (400 (250 μg mL-1), were added. After 48 h, the reaction mixture
MHz, CDCl3): δ 5.82 (m, 1H), 5.11-5.03 (m, 2H), 2.43- was extracted with a pentane/Et2O solvent mixture (7:3, 40
1.72 (m, 8H), 1.51 (m, 4H), 1.27 (m, 4H), 0.88 (t, J = 6.84, mL x 3). The combined organic layers was dried over
8
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