◦
and lyophilized for 36 h. For the free enzyme preparation, crude
hexane 1 : 3) to give 42.5 mg (85%, 99% ee) of (R)-2: mp 135 C
2
+
ꢀR
1
◦
22
extract was applied to a Ni –NTA affinity column (Invitrogen )
and desalting column (Amersham) and the eluent containing pure
BAL was used in the carboligation reactions.
Lit. , 133–134 C for (R) enantiomer; [a] = -112.1 (c 1.5,
D
1
22
CH
3
COCH
3
) [lit. [a]D = -113.8 (c 1.5, CH
3
COCH ). HPLC:
3
1
(Chiralpak AD) R
t
(R) = 27.1 min; R
t
(S) = 34.5 min; H NMR
(
400 MHz, CDCl /CCl
3
4
): d 7.82 (d, J = 7,8 Hz, 2H), 7.44 (t, J =
Activity assays
7.5, 1H), 7.30 (t, J= 7.6), 7.16–7.23 (m, 5H), 5.73 (d, J = 5.9 Hz,
1
3
1
1
7
H), 4.42 (d, J = 5.9 Hz, 1H); C NMR (100 MHz, CDCl
3
/CCl ):
4
One unit of ligase activity of BAL is defined as the amount
98.7, 139.1, 133.8, 133.6, 129.1, 129.0, 128.6, 128.5, 127.7,
6.2.
of enzyme that catalyzes the formation of 1 mmol of benzoin
◦
per minute under the standard conditions (30 C, pH: 7.8).
With free and immobilized system initial rates of BAL, the
catalyzed benzoin formation reactions were determined by HPLC
Synthesis of 2-hydroxy-1,2-diphenylethan-1-one (S)-2 and
(R)-2-hydroxy-1-phenylpropanone [(R)-3] from racemic benzoin
and acetaldehyde
-
1
analysis (Nucleodur C18, 1 mL min , 254 nm, retention time
2 min).
1
1
According to the procedure for the synthesis of (R)-2, rac. benzoin
Immobilization of BAL to superparamagnetic particles
Superparamagnetic g-Fe (maghemite)–silica nanocomposite
particles were synthesized by using the sol–gel method, in which
the surface modification method was also applied for the immo-
(50 mg, 0.23 mmol) and 74 mg (0.7 mmol) acetaldehyde were
dissolved in the 20 mL reaction medium that was prepared for the
2
O
3
9
3
00 mg BAL immobilized resin. The reaction was performed at
0 C and acetaldehyde (0.2 mmol) was added (30 and 120 min).
◦
The reaction was followed by TLC and stopped at 48 h by
adding chloroform (50 mL). The mixture was extracted twice
with chloroform (2 ¥ 50 mL). After drying the collected organic
bilization of 6XHistidine tagged recombinant benzaldehyde lyase
8
(
BAL, EC 4.1.2.38). 185 mg g-Fe
2
3
O nanoparticles were washed
with a lysis buffer (10 mM imidazole, 100 mM NaCl in 50 mM
potassium phosphate buffer) and incubated for a couple of hours
phase over MgSO , removal of the solvent under reduced pressure
4
◦
gave the crude product mixture, which was then purified by flash
column chromatography to give the desired compounds (S)-2 and
at 4 C in order to enable the silica coat of nanoparticles to swell.
Equilibrated resin was settled for 1–2 min by the aid of a magnet,
in which the supernatant was removed by pipetting. 200 mg
lyophilized crude extract was dissolved in a 30 mL lysis buffer.
After sonication, the slurry was centrifugated and the supernatant
was filtered through a 0.45 mm filter. 2 mL of filtered crude was
(
R)-3 (EtOAc–hexane 1 : 3).The determination of the enantiomeric
excess was performed by HPLC analysis (Chiralpak AD, 90 : 10
-
1
hexane–isopropanol, 1 mL min , 254 nm, retention time for (R)-2-
hydroxy-1-phenylpropanone: 12.45, for (S)-benzoin: 33.35). (S)-2:
2
D
2
yield 10.5 mg (43%), 99% ee; [a] = 109.4 (c 1.5, CH
3
22
COCH ).
3
diluted to 5 mL and incubated with magnetic resin by gentle mixing
2
D
2
1
◦
(R)-3: Viscous oil: [a] = 83.5 (c 2.0, CHCl
3
), Lit. [a] : 85.1 (c
(
90 rpm) at 4 C for 20 min to immobilize the histidine tagged
D
1
2
1
3
.0, CHCl
3
). H NMR (400 MHz, CDCl
3
/CCl
4
): d 7.90 (dd, J =
BAL. The amount of crude loaded was estimated according to
the saturation concentration for the resin. After the protein was
immobilized, the resin was settled as described before and washed
with a lysis buffer twice.
The amount of immobilized BAL was determined by measuring
the protein content in the eluted enzyme solution (200 mM
imidazole, 100 mM NaCl, eluted protein in 50 mM potassium
phosphate buffer (pH: 7.8) and by the colorimetric method at
.4, 8.2 Hz, 2H), 7.40–7.60 (m, 3H), 5.13 (q, J = 6.0 Hz, 1H),
.80 (br.s, 1H), 1.41 (d, J = 6.0 Hz, 3H); C NMR (100 MHz,
/CCl
): d = 202.7, 134.4, 134.0, 128.9, 128.7, 69.2,
1
3
CDCl
2.0.
3
4
2
Synthesis of (R)-2-hydroxy-3,3-dimethoxy-1-
9
(4-methoxyphenyl)propan-1-one (R)-5
5
95 nm by using the SIGMA Bradford reagent with bovine serum
11
According to the procedure for the synthesis of (R)-2, p-
anisaldehyde (50 mg, 0.36 mmol) and dimethoxy acetaldehyde
albumin as a standard. SDS page analysis was performed by the
method of Leammly (1970).
14
(112 mg, 1 mmol) were dissolved in the 30 mL reaction medium
that was prepared for the 900 mg BAL immobilized resin added
and incubated at 30 C via gentle shaking (90 rpm). After 2 h,
Representative reaction with superparamagnetic particles:
synthesis of 2-hydroxy-1,2-diphenylethan-1-one (R)-2
◦
1
8.7 mg (0.18 mmol) dimethoxy acetaldehyde was added to the
BAL immobilized resin was equilibrated with a reaction buffer
containing 0.25 mM TPP, 2.5 mM MgSO4, 25% DMSO in
medium. The reaction was followed by TLC and was stopped at
24 h. The reaction mixture was extracted with chloroform (3 ¥
5
0 mM potassium phosphate buffer at pH: 7.8. The mixture was
50 mL). After drying the collected organic phase over MgSO ,
removal of the solvent under reduced pressure gave the crude
product, which was then purified by flash column chromatography
4
◦
incubated via gentle shaking (90 rpm), at 25 C for 1 min, and
then the reaction was started by adding benzaldehyde (50 mg,
0
.5 mmol). The reaction was monitored by HPLC (Nucleodur
(EtOAc–hexane 1 : 3) to give 72 mg (83%, 99% ee) (R)-5 as a
-
1
22
C18, 1 mL min , 254 nm, retention time 12 min., 45% acetonitrile,
.5% acetic acid, and 54.5% water). After 40 min. (checked by
TLC), the reaction mixture was extracted with chloroform (3 ¥
viscous oil. [a]D = -13.7 (c 0.5, CHCl
3
); HPLC (Chiralcel OD,
0
UV detection at 254 nm, 97 : 3 hexane–2-propanol, flow rate
-
1
1
0.8 mL min ): R
t
(R)= 40.7 min; R
t
(S)= 45.3 min; HNMR: 3.30
5
0 mL).
(s, 3H), 3.39 (s, 3H), 3.62 (d, J = 7.2, 1H), 3.82 (s, 3H), 4.33 (d,
After drying the collected organic phase over MgSO , removal
4
J = 3.5, 1H), 5.0 (dd, J = 3.5, 7.2, 1H), 6.92 (d, J = 8.7, 2H), 7.97
1
3
of the solvent under reduced pressure gave the crude product,
which was then purified by flash column chromatography (EtOAc–
(d, J = 8.7, 2H); C NMR: 55.4, 55.7, 56.7, 73.9, 106.4, 113.9,
128.3, 132.1, 164.4, 197.0.
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1658–1664 | 1663