PAPER
Chemoenzymatic Synthesis of (S)-8-O-Methylmellein by Candida parapsilosis Carbonyl Reductase
2047
Scheme 2
ted with a HP 5973 mass selective detector (EI, 70 eV) and a HP-
5MS column. HRMS and microanalyses were performed at the an-
alytical department, “ Kekulé-Institut für Organische Chemie und
Biochemie” (University of Bonn, Germany).
(S)-2-(2-Hydroxypropyl)-6-methoxybenzonitrile (6)
Nitrile 5i (200 mg, 1.1 mmol), NAD+ (35 mg, 50 µmol) and sodium
formate (17 g, 250 mmol) were dissolved in 100 mM triethanol-
amine buffer (250 mL, pH 7). The solution was adjusted to pH 7 and
FDH- (200 µL, 57 U/ml), and CPCR-soln (200 µL, US = 14.2 U/
mL) were added. The progress of the reaction was controlled by
GCMS while additional CPCR (200 µL) was added every 12 h (be-
cause of denaturing and inhibiting effects to the enzyme) up to a to-
tal of 1 mL (14.2 U). After a total reaction time of 96 h, the solution
was acidified with 2M HCl (50 mL) and extracted with CH2Cl2 (3 x
50 mL). The organic layer was dried (Na2SO4) and evaporated to
give 6 (180 mg, 90%), 92% purity (GCMS). This crude product was
used without further purification in the hydrolysis step.
2-(2-Oxopropyl)benzonitrile (5h);21 Typical Procedure
A solution of (i-Pr)2NH (0.7 mL, 5.0 mmol) and BuLi (3.1 mL,
5.0 mmol; 1.6 M in hexane) in THF (15 mL) was stirred under N2
at r.t. for 5 min and then cooled to –78°C. 2-Methylbenzonitrile
(586 mg, 5.0 mmol) dissolved in THF (15 mL) was added and the
red solution was stirred for 10 min. Amide 4 (0.55 mL, 5.5 mmol)
was then added dropwise, stirring was continued for 1 h at –78°C
before adding 2 M HCl (15 mL). The solution was warmed up to r.t.,
additional 2 M HCl (50 mL) was added and the mixture extracted
with CH2Cl2 (3 x 50 mL). The organic layer was dried (Na2SO4) and
evaporated. Flash column chromatography (EtOAc) yielded the
nitrile 5h (580 mg, 73%) as a yellow oil, Rf = 0.61.
1H NMR: δ = 2.23 (s, 3H, CH3), 3.95 (s, 2H, CH2), 7.4-7.6 (m, 4H,
ArH).
13C NMR: δ = 30.2 (CH3), 48.7 (CH2), 113.3 (ArCq), 117.9 (CN),
127.8, 131.0, 132.8, 133.0 (ArCH), 138.2 (ArCq), 203.7 (CO).
IR : ν = 3370, 2985, 2220, 1725, 1600, 1585, 1480, 1440, 1285,
1215, 1080, 775 cm-1.
1H NMR : δ = 1.21 (d, 3H, JH,H = 6.2Hz, CH3), 2.80-2.95 (m, 2H,
CH2), 3.85 (s, 3H, OCH3), 4.05 (m, 1H, CH), 6.75-6.95 (m, 2H,
ArH), 7.45 (m, 1H, ArH).
13C NMR : δ = 23.3 (CH3), 44.1 (CH2), 56.2 (OCH3), 68.3 (CHOH),
102.6 (ArCq), 109.1 (CN), 116.0 (ArCq), 122.7, 133.8, 144.7
(ArCH), 162.0 (ArCq).
GCMS: tR = 8.59 min, m/z (%) = 159 (17) [M+], 117 (100), 89 (50),
63 (34), 51 (12).
GCMS: tR = 10.56 min, m/z (%) = 191 (M+, 1.6%), 147 (100), 118
(43), 104 (15), 89 (12), 77 (14), 63 (4.6), 51 (5.5).
(S)-8-O-Methylmellein (1)6
2-Methoxy-6-(2-oxopropyl)benzonitrile (5i)
Compound 5i was prepared using 3 (740 mg, 5.0 mmol). Purifica-
tion by flash column chromatography (PE:EtOAc, 1:1) afforded 5i
(684 mg, 72%) as a yellow oil, Rf = 0.36.
Compound 6 (166 mg, 0.86 mmol) was dissolved in 32% HCl/
MeOH (1:1, v/v; 40 mL). The solution was heated for 12 h under re-
flux, while the progression was controlled by GCMS. After com-
plete conversion the product was extracted with CH2Cl2 (3 x
50 mL). The organic layer was dried (Na2SO4) and evaporated. The
residue was purified by flash column chromatography (PE:EtOAc,
1:1) to afford 1 (156 mg, 94%) as a colorless solid (ee > 99.5%); mp
IR : ν = 2220, 1725, 1588, 1485, 1445, 1330, 1290, 1165, 1085, 770,
738 cm-1.
1H NMR: δ = 2.20 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 3.88 (s, 2H,
CH2), 6.80 (m, 2H, ArH), 7.40 (m, 1H, ArH).
13C NMR: δ = 30.1 (CH3), 48.7 (CH2), 56.2 (OCH3), 102.8 (ArCq),
109.9 (ArCH), 115.5 (CN), 122.6, 134.0 (ArCH), 140.0, 161.8
(ArCq), 203.7 (CO).
= 87°C (Lit.6 mp = 86.5-87.5°C); [α]26 = +257 (c =0.5, CHCl3)
D
[Lit.6 [α]26D = +261 (c = 0.52, CHCl3)].
1H NMR : δ = 1.39 (d, 3H JH,H = 6.4 Hz, CH3), 3.80 (d, 2H JH,H
4.7 Hz, CH2), 3.86 (s, 3H, OCH3), 4.47 (m, 1H, CH), 6.75 (m, 2H,
ArH), 7.39 (m, 1H, ArH).
=
GCMS: tR = 10.41 min, m/z (%) = 189 (16) [M+], 147 (100), 118
(31), 104 (11), 89 (14), 76 (15), 63 (8.6), 51 (7.5).
13C NMR : δ = 21.0 (CH3), 36.4 (CH2), 56.6 (OCH3), 74.5 (CH),
111.2 (ArCH), 114.0 (ArCq), 119.6, 134.9 (ArCH), 142.3, 161.5
(ArCq), 163.1 (CO).
HRMS : m/z Calcd. for C11H11NO2 :189.0784. Found:189.0787.
Anal.Calcd for C11H11NO2: C, 69.8; H, 5.9; N, 7.4. Found: C, 69.4;
H, 5.9; N, 7.9.
GC (180°C): tR = 83.68 min.
Synthesis 1999, No. 12, 2045–2048 ISSN 0039-7881 © Thieme Stuttgart · New York