Oxidation-ActiVe FlaVin Models
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2485
(s, 1H), 8.61 (m, 1H). Anal. Calcd for C30H24N10O4‚H2O: C, 59.4; H,
4.32; N, 23.09. Found: C, 59.82; H, 4.39; N, 22.72.
oxidative decarboxylation was found to take place, giving the
corresponding ketones. It should be noted that it is the first
example of the oxidation of R-hydroxy acids by a flavin model.
The mechanism could be different from that of D-lactate
dehydrogenase, since the present system uses a unique flavin
model. However, similar roles of the metal ion are conceivable
in native D-lactate dehydrogenases, in which Zn2+ is known to
be located in the vicinity of FAD.
3 and 5 were similarly synthesized from 1 and 5-bromomethyl-5′-
methyl-2,2′-bipyridine10 and 6-bromomethyl-6′-methyl-2,2′-bipyridine,12
3. Yield 61%, mp > 300 °C. 1H NMR (200 MHz, CDCl3) δ 1.55 (m,
6H), 2.38 (s, 3H), 3.57 (s, 3H), 4.78 (m, 4H), 5.37 (s, 2H), 7.61 (d, J
) 10.0 Hz, 1H), 8.04 (d, J ) 10.0 Hz, 1H), 8.27 (d, J ) 10.0 Hz, 1H),
8.31 (d, J ) 10.0 Hz, 1H), 8.48 (d, J ) 10.0 Hz, 1H), 8.54 (s, 1H),
8.56 (s, 1H), 8.90 (s, 1H). Anal. Calcd for C31H26N10O4‚H2O: C, 59.99;
H, 4.55; N, 22.65. Found: C, 60.35; H, 4.63; N, 22.65.
1
5: yield 54%, mp > 300 °C. H NMR (200 MHz, CDCl3) δ 1.56
Experimental Section
(m, 6H), 2.68 (s, 3H), 3.57 (s, 3H), 4.80 (m, 4H), 5.53 (s, 2H), 7.17
(d, J ) 8.0 Hz, 1H), 7.42 (d, J ) 8.0 Hz, 1H), 7.68 (m, 1H), 7.81 (t,
J ) 8.0 Hz, 1H), 8.13 (d, J ) 8.0 Hz, 1H), 8.37 (d, J ) 8.0 Hz, 1H),
8.55 (s, 1H), 8.57 (s, 1H). Anal. Calcd for C31H26N10O4‚2H2O: C, 58.30;
H, 4.73; N, 21.93. Found: C, 58.60; H, 4.96; N, 22.05.
Substituted mandelic acids (6) were prepared in fairly good yields
according to the literature procedures.13 Namely, trimethylsilyl ether
of substituted benzcyanohydrins, which were prepared from substituted
benzaldehydes, potassium cyanide, and chlorotrimethylsilane in the
presence of ZnI2 in MeCN, were hydrolyzed in acidic aqueous solution.
6a: mp 108-110 °C (benzene) (lit.13 106.5-108 °C). 6b: mp 145-
147 °C (benzene) (lit.13 145-146 °C). 6d: mp 117-119 °C (benzene)
(lit.13 119-121 °C). 6e: mp 96-97 °C (benzene) (lit.13 113.5-115
°C).
Deuterated mandelic acids were prepared by NaBD4-reduction of
arylglyoxylic acids,25 which were prepared by Pb(CH3CO2)4- oxidation
of the substituted mandelic acids in benzene.26 The D-contents were
determined by comparison with chemical shifts of aryl protons: 86%
(6b) and 83% (6c). The kH/kD values were calculated by correction of
kD values; kD ) (kobs - 0.17kH)/0.83 for 83% D-content.
Determination of Redox Potentials. Cyclic voltammetry was carried
out using a platinum plate (3 mm in diameter, BAS), Ag/Ag+ (0.1 M
tetraethylammonium perchlorate, TEAP, in MeCN), and platinum wire
as working, reference, and counter electrodes in MeCN. A solution of
BDPox (1.0 × 10-3 M in MeCN containing 0.1 M TEAP) and metal
ions (12 µL each, 5.02 × 10-2 M in MeCN) was degassed by bubbling
N2 presaturated with MeCN for 20 min. All potentials were measured
with scan rate 100 mV s-1. MeCN was purified by distillation from
calcium hydride, and TEBP was recrystallized from EtOH-H2O two
times and dried overnight at 100 °C.
1H NMR spectra were recorded on Varian Gemini-200 (200 MHz)
or a JEOL JAM R-500 (500 MHz) instrument with chemical shifts
from tetramethylsilane. Cyclic voltammograms were recorded on a
Hokuto Denko (HA-301, 1B-104) with an X-Y recorder (Riken
Electronics F-35). Electronic absorption spectra were recorded on a
JASCO Ubset-560 or Shimadzu UV-2200 A spectrophotometer.
Stopped-flow rate measurements were performed with Otuka Electron-
ics RA-401 spectrophotometer. Fluorescence spectra were measured
on a Hitachi 850 fluorescence spectrophotometer. Melting points are
uncorrected. Flash column chromatography was performed by using
Wakogel C-200 (silica gel, 70-150 µm, Wako Pure Chemical Co.).
Elemental analyses were performed at the Center of Instrumental
Analysis of Gunma University. Divalent metal ions used were M(NO3)2‚
6H2O. Acetonitrile and DMF were purified by distillation from calcium
hydride.
Synthesis of BDPox. BDPox (2) was supplied from our previous
study.3d BDPox(1) was prepared by stepwise reactions of N,N′-diethyl-
p-phenylenediamine with 6-chloro-3-methyluracil and 6-chlorouracil.
N,N′-Diethyl-N-(3-methyluracil-6-yl)-N′-(uracil-6-yl)-p-phenylene-
diamine. A mixture of N,N′-diethyl-N-(3-methyluracil-6-yl)-p-phe-
nylenediamine32 (3 g, 10 mmol) and 6-chlorouracil (2.3 g, 15 mmol)
in N,N-diethylaniline (3.6 mL) was heated at 180 °C for 1 h under N2.
After cooling, diethyl ether (20 mL) containing a small amount of EtOH
was added to crush to pieces. The powdery precipitate was collected
by filtration and washed with water (20 mL × 2). Purification was
performed by precipitation from acetic acid and diethyl ether to give
powder. Yield 2.6 g (96%), mp >300 °C. 1H NMR (200 MHz, DMSO-
d6) δ 1.10 (t, 6H), 3.07 (s, 3H), 3.69 (q, 4H), 4.59 (s, 1H), 4.76 (s,
1H), 7.34 (s, 4H), 9.79(s, 1H), 10.11(s, 1H), 10.43 (s, 1H).
Kinetics. (i) Addition of Sulfite Ion. Pseudo-first-order rate
constants were determined by following the absorption increases of
the adduct at 502 nm in MeOH with a stooped-flow aparatus. Namely,
in one reservoir BDPox (1.0 × 10-5 M: 60 µL of 1.0 × 10-3 M in
DMF) and Zn(NO3)2‚6H2O (30-360 µL of 5.0 × 10-3 M in MeOH)
were placed with MeOH (total 3 mL), and NaHSO3 (5.0 × 10-3 M:
30 µL of 1.0 M in H2O) were placed with MeOH (2.96 mL) in another
reservoir. After flowing N2 in the reservoir, the reaction was started.
(ii) BNAH Oxidation. Psusdo-first-order rate constants were
determined by following the absorption increases of BDPox at 640 nm
with a stopped-flow apparatus. In one reservoir, BDPox (1.0 × 10-5
M: 60 µL of 1.0 × 10-3 M in DMF) and Zn(NO3)2‚6H2O (30-180
µL of 5.0 × 10-3 M in MeCN) were placed by a microsyringe with
7,14-Diethyl-3-methylbenzo[1,2-g; 4,5-g′]dipteridine-2,4,9,11-
(3H,7H,10H,14H)-tetraone (1). A mixture of the above diamine (1.8
g, 4.5 mmol), NaNO3 (3.2 g, 38 mmol), and concentrated H2SO4 (0.2
mL) in AcOH (30 mL) was heated at 90 °C for 6 h under N2. After the
mixture cooled, AcOH was evaporated, and the residue was extracted
with hot DMF-CHCl3 solution. Di-N-oxide was obtained by evaporat-
ing the solvent. Without purification, deoxygenation was performed in
DMF (10 mL) at 100 °C for 3 h. After this mixture cooled, DMF was
evaporated, and diethyl ether (100 mL) was added to give the powder
of 1, which was purified by recrystallization from DMF (violet powder).
Yield 0.77 g (41%), mp > 300 °C. UV-vis (CHCl3-DMSO (9:1)).
λ
max (log ꢀ): 370 nm (4.29 M-1 cm-1), 553 (4.2). 1H NMR (200 MHz,
CDCl3-CF3CO2H (5:2 v/v) δ 1.68 (m, 6H), 3.71 (s, 3H), 4.98 (4H,
m), 8.92 (s, 1H), 8.95 (s, 1H).
MeCN (total 3 mL), and BNAH (2.0 × 10-4 M: 24 µL of 5.0 × 10-2
M
in MeCN) was placed with MeCN (2.98 mL) in another reservoir.
7,14-Diethyl-3-(bipyridin-6-ylmethyl)-10-methylbenzo[1,2-g; 4,5-
g′]dipteridine -2,4,9,11-(3H,7H,10H,14H)-tetraone (4). A mixture of
1 (0.20 g, 0.48 mmol), 6-bromomethyl-2,2′-bipyridine11(0.20 g, 0.8
mmol), and K2CO3 (0.20 g, 1.5 mmol) in DMF (150 mL) was stirred
for 4 h at room temperature under N2. After filtration of inorganic salts,
DMF was evaporated under reduced pressure, and CHCl3 (200 mL)
was added. The CHCl3 layer was washed with water and dried over
Na2SO4. After evaporating CHCl3, the residue was recrystallized from
DMF. Yield 0.12 g (43%), mp > 300 °C. UV-vis (CHCl3-DMF 9:1):
(iii) Oxidation of r-Hydroxy Acids. Pseudo-first-order rate con-
stants were determined spectrophotometrically by following the absorp-
tion increases of BDPred at 640 nm in MeCN under anaerobic conditions.
In a typical run, BDPox (1.0 × 10-5 M: 30 µL of 1.0 × 10-3 M in
DMF) and Zn(NO3)2‚6H2O (15-120 µL of 5.0 × 10-3 M in MeCN)
were placed by a microsyringe in a cell part of a Thunberg cuvette
with MeCN, and mandelic acid (2.0 × 10-4 M: 30 µL of 2.0 × 10-2
M in MeCN) was placed in the upper part (the total volume was
adjusted to be 3 mL). The solutions were degassed for 30 min at 25
°C by bubbling N2, prehumidified with MeCN, and just prior to the
end, Et3N (6.0 × 10-4 M: 30 µL of 6.0 × 10-2 M in MeCN) was
added quickly. The reaction was started by a quick mixing by up-and-
down shaking.
1
λmax (log ꢀ) 373 (4.5), 555 (4.5). H NMR (200 MHz, CDCl3) δ 1.55
(m, 6H), 3.57 (s,3H), 4.80 (m, 4H), 5.53 (s, 2H), 7.21 (m, 1H), 7.38
(d, J ) 7.5 Hz, 1H), 7.69 (t, J ) 7.5 Hz, 1H), 7.78 (t, J ) 7.5 Hz, 1H),
8.25 (d, J ) 7.5 Hz, 1H), 8.33 (d, J ) 7.5 Hz, 1H), 8.55 (s, 1H), 8.56
Product Analysis. Formation of benzoyl formate was confirmed
(32) Yoneda, F.; Koga, M.; Tanaka, K.; Yano, Y. J. Heterocycl. Chem.
1989, 26, 1221-1228.
by HPLC and TLC analyses. For HPLC, a solution of 4 (1.0 × 10-5