Regioselectivity of the Berberine Bridge Enzyme
FULL PAPER
Unless otherwise noted, reagents and organic solvents were obtained
from commercially available sources and used without further purifica-
tion. Toluene, methanol, and acetonitrile used for anhydrous reactions
were dried over appropriate molecular sieves (4 ꢁ for toluene, 3 ꢁ for
MeOH and MeCN) for at least 48 h. THF used for anhydrous reactions
was distilled from potassium/benzophenone directly before use. For anhy-
drous reactions, flasks were oven dried and flushed with dry argon just
before use. Standard syringe techniques were applied to transfer dry sol-
vents and reagents in an inert atmosphere of dry argon.
ethyl acetate (3ꢃ30 mL). The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure. No further purification
was necessary. Yield: 2.2 g (86%). M.p.: 110–1128C; Rf (hexanes/ethyl
1
acetate (+1 drop of acetic acid) 3:1)=0.13; H NMR (300 MHz, CDCl3):
d=6.70–6.95 (3H, m, Ar), 3.64 (2H, d,
JHF =1.5 Hz, CH2-COOH);
13C NMR (75 MHz, CDCl3): d=173.2, 150.2 (d, JCF =240.3 Hz), 144.8 (d,
J
J
CF =13.2 Hz), 123.6 (d, JCF =4.5 Hz), 122.7 (d, JCF =13.7 Hz), 121.2 (d,
CF =2.7 Hz), 116.3 (d, JCF =2.8 Hz), 33.7 ppm (d, JCF =3.8 Hz).
2-(3-(Benzyloxy)-2-fluorophenyl)acetic acid: The benzylation of 2-(2-
fluoro-3-hydroxyphenyl)acetic acid was performed by using 2.2 g
(12.9 mmol) of substrate. The substrate was dissolved in ethanol (50 mL),
than KOH (1.7 g, 30.8 mmol), benzyl bromide (2.6 g, 16.5 mmol), and
NaI (0.1 g, 0.4 mmol) were added. This reaction mixture was allowed to
react at RT overnight. After control by TLC, the mixture was filtrated
through pad of Celite, and ethanol was removed under reduced pressure.
The residue was redissolved in water (20 mL) and acidified with acetic
acid. The aqueous phase was extracted with ethyl acetate (3ꢃ30 mL),
and the combined organic layers were dried over Na2SO4 followed by the
removal of the solvents under reduced pressure. Purification was per-
formed by using silica-gel column chromatography with hexane/ethyl ace-
tate 3:1 as eluent. Yield after purification: 2.8 g (83%). M.p.: 95–968C;
Rf (hexane/ethyl acetate (+1% acetic acid) 3:1)=0.25; 1H NMR
(300 MHz, CDCl3): d=7.29–7.46 (5H, m, Ar), 6.84–7.08 (3H, m, Ar),
Catalase from bovine liver was purchased from Sigma–Aldrich (Lot.:
81H7146).
Determination of conversion: Conversions were determined by HPLC on
an achiral C18 stationary phase. Eluent: buffer (30 mm HCOONH4,
pH 2.8)/methanol/acetonitrile
67:18:15
(isocratic);
flow
rate:
0.5 mLminÀ1; column temperature: 208C; detection wavelength: 280 nm.
Retention time is given in the Supporting Information.
Representative synthesis of 1n
2,2,2-Trichloro-1-(2-fluoro-3-methoxyphenyl)ethanol:
2-Fluoro-3-meth-
A
ACHTUNGTRENNUNG
fluoro-3-methoxyphenyl)ethanol by using chloroform (6 mL), DMF
(20 mL), and KOH (1.5 g, 26.7 mmol) in methanol (5 mL). 2-Fluoro-3-
methoxybenzaldehyde was dissolved in DMF and chloroform, and the
solution was cooled to about À108C by using a salt–ice bath. KOH in
methanol was added dropwise under an argon atmosphere with cooling.
The reaction was was continued for an additional 2 h before quenching
the mixture, still under cooling, to pH 1 with concentrated aqueous HCl
solution. After stirring for additional 30 min at À108C, the reaction was
allowed to warm to RT, and phase separation was performed. The aque-
ous phase was extracted with toluene (30 mL). The combined organic
layers were washed with water (30 mL) and brine (30 mL) and the sol-
vent was removed under reduced pressure. No further purification steps
were necessary. Yield: 8.3 g (93%). Rf (hexanes/ethyl acetate (+1% of
acetic acid) 3:1)=0.23; 1H NMR (300 MHz, CDCl3): d=7.36–7.28 (1H,
m, Ar), 7.06 (1H, t, J=8.1 Hz, Ar), 6.93 (1H, t, J=8.0 Hz, Ar), 5.59
(1H, s, Ar-CH), 3.83 ppm (3H, s, O-CH3); 13C NMR (75 MHz, CDCl3):
5.14 (2H, s, CH2-O), 3.66 ppm (2H, d,
JHF =1.6 Hz, CH2-COOH);
13C NMR (75 MHz, CDCl3): d=173.0, 151.3 (d, JCF =245.2 Hz), 146.7 (d,
J
J
CF =11.0 Hz), 136.9, 128.1, 127.6, 127.2, 123.4 (d, JCF =4.8 Hz), 122.9 (d,
CF =3.1 Hz), 114.3 (d, JCF =1.7 Hz), 70.8, 33.6 ppm (d, JCF =3.9 Hz).
2-(3-(Benzyloxy)-2-fluorophenyl)acetyl chloride: For the synthesis of 2-
(3-(benzyloxy)-2-fluorophenyl)acetyl chloride, 2-(3-(benzyloxy)-2-fluoro-
phenyl)acetic acid (0.91 g, 3.3 mmol) was dissolved in try toluene
(20 mL). To this mixture, oxalyl chloride (0.7 g, 5.4 mmol) and one drop
of DMF were added. The reaction was allowed to proceed for 3 h, than
toluene and remaining oxalyl chloride were removed under reduced pres-
sure. The product, 2-(3-(benzyloxy)-2-fluorophenyl)acetyl chloride, was
directly used for the next step without further purification.
d=152.0, 150.3 (d, JCF =248.1 Hz), 147.1 (d, JCF =11.3 Hz), 125.1 (d, JCF
9.8 Hz), 123.4 (d, JCF =4.6 Hz), 120.8 (d, JCF =1.7 Hz), 113.6 (d, JCF
1.9 Hz), 102.9, 56.2 ppm.
=
2-(3-(Benzyloxy)-2-fluorophenyl)-N-(3,4-dimethoxyphenethyl)-N-methyl-
AHCTUNGTERGaNNUN cetamide: 2-(3-(Benzyloxy)-2-fluorophenyl)-N-(3,4-dimethoxypheneth-
=
yl)-N-methylacetamide was synthesized by the use of 2-(3-(benzyloxy)-2-
fluorophenyl)acetyl chloride (0.95 g, 3.3 mmol) and 2-(3,4-dimethoxy-
phenyl)-N-methylethanamine (0.7 g, 3.3 mmol). 2-(3,4-Dimethoxyphen-
yl)-N-methylethanamine was dissolved in chloroform (10 mL), and
NaOH aqueous solution (3%, 10 mL) was added. This mixture was
cooled in an ice bath, and 2-(3-(benzyloxy)-2-fluorophenyl)acetyl chlor-
ide dissolved in chloroform (10 mL) was added dropwise under cooling.
After complete addition of 2-(3-(benzyloxy)-2-fluorophenyl)acetyl chlor-
ide, the ice bath was removed, and the reaction was allowed to proceed
at RT for 16 h. For work-up, the phases were separated, and the aqueous
phase was extracted with chloroform (3ꢃ10 mL). The combined organic
layers were washed with aqueous HCl solution (1m, 10 mL) and dried
over Na2SO4. Chloroform was removed under reduced pressure, and the
crude product (1.2 g) was purified by silica-gel chromatography (hexanes/
ethyl acetate 1:1). Yield: 1.0 g (70%). M.p.: 104–1068C; Rf (hexanes/
ethyl acetate=1:1)=0.22; NMR spectroscopy revealed that the product
is a mixture of isomers (trans/cis 1.09:1). Based on the peak intensities as
well as the distortionless enhancement by polarization transfer (DEPT),
COSY, and heteronuclear single-quantum coherence (HSQC) spectra,
the NMR signals were assigned to the isomers as follows: 1H NMR
(300 MHz, CDCl3): trans: d=7.39–7.24 (m, 6H, Ar), 6.91–6.59 (m, 6H,
Ar), 5.05 (s, 2H, Ar-CH2-O), 3.78 (s, 3H, O-CH3), 3.77 (s, 3H, O-CH3),
3.62 (s, 2H, CO-CH2-Ar), 3.52 (t, 2H, J=7.5 Hz, Ar-CH2-CH2-N), 2.86
(s, 3H, N-CH3), 2.72 ppm (t, 2H, J=7.5 Hz, Ar-CH2-CH2-N); cis: d=
7.39–7.24 (m, 5H, Ar), 6.91–6.59 (m, 7H, Ar), 5.02 (s, 2H, Ar-CH2-O),
3.77 (s, 3H, O-CH3), 3.76 (s, 3H, O-CH3), 3.46–3.41 (m, 4H, Ar-CH2-
CH2-N, CO-CH2-Ar), 2.91 (s, 3H, N-CH3), 2.66 ppm (t, 2H, J=7.3 Hz,
Ar-CH2-CH2-N); 13C NMR (75 MHz, CDCl3): trans: d=169.8. 150.8 (d,
2-(2-Fluoro-3-methoxyphenyl)acetic acid: For the synthesis of 2-(2-fluoro-
3-methoxyphenyl)acetic acid, oxygen-free ethanol was needed; therefore,
ethanol (60 mL) was purged with argon for 60 min. After purging, di-
phenyl diselenide (9.9 g, 31.8 mmol) was added and solubilized followed
by the addition of NaBH4 (2.4 g, 63.5 mmol) in portions under constant
argon flow. After completed addition, the previously orange solution
turned colorless and was stirred for 30 min at RT. After 30 min, the sub-
strate 2 (8.3 g, 30.2 mmol) was added and dissolved followed by the addi-
tion of NaOH (7.3 g, 181.4 mmol). The reaction was heated to 408C and
left overnight. After all starting material was consumed, as was shown by
TLC, the solvent was removed under reduced pressure. The remaining
solid was dissolved in water (30 mL) and extracted under basic conditions
with ethyl acetate (30 mL). After extraction, the aqueous phase was ad-
justed to pH 1 under cooling with concentrated aqueous HCl solution.
Then extraction with ethyl acetate (5ꢃ30 mL) was performed, and the
combined organic layers were dried over Na2SO4. Ethyl acetate was re-
moved under reduced pressure, and the product was purified by silica-gel
column chromatography by using a gradient of hexanes (to remove the
nonreacted diphenyl diselenide and apolar by-products) and hexanes/ace-
tone 1:1 to elute the product. Yield: 2.8 g (50%). M.p.: 106–1088C; Rf
(hexanes/ethyl acetate (one drop of acetic acid) 3:1)=0.21; 1H NMR
(300 MHz, CDCl3): d=6.89–7.01 (3H, m, Ar), 3.87 (3H, s, O-CH3),
3.69 ppm (2H, s, CH2-COOH); 13C NMR (75 MHz, CDCl3): d=170.9,
150.8 (d, JCF =244.6 Hz), 147.8 (d, JCF =10.8 Hz), 123.8 (d, JCF =4.8 Hz),
123.0 (d, JCF =13.4 Hz), 122.7 (d, JCF =3.1 Hz), 112.3 (d, JCF =1.8 Hz),
55.5 (d, JCF =2.7 Hz), 33.6 ppm (d, JCF =3.8 Hz).
2-(2-Fluoro-3-hydroxyphenyl)acetic acid: 2-(2-Fluoro-3-methoxyphenyl)-
acetic acid (2.8 g, 15.2 mmol) was dissolved in bromic acid (48% in
water, 30 mL) and heated to 1058C by using an oil bath. After a reaction
time of 5 h, the reaction was allowed to cool to RT and extracted with
J
CF =245.1 Hz), 148.9, 147.5, 146.7, 136.6, 131.6, 128.6, 128.1, 127.4, 123.9
(d, JCF =3.0 Hz), 123.6 (d, JCF =8.0 Hz), 122.6 (d, JCF =3.0 Hz), 120.7,
114.0 (d, JCF =1.7 Hz), 112.0, 111.2, 71.3, 55.9, 50.3, 36.4, 33.7, 33.2 ppm.
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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