H. Zheng et al. / Biochemical Pharmacology 70 (2005) 1642–1652
1645
(20 ml) and the mixture was extracted with CHCl3/
2.2.6. L-3-(8-Hydroxyquinolin-5-yl)alanine (7 or M10)
EtOAc (3ꢂ 20 ml, 1:1). The organic layer was washed
and D-3-(8-hydroxyquinolin-5-yl)alanine ethyl ester (8)
Ester 6 (89.2 mg) was dissolved in 5 ml water and the
pH of the solution was adjusted to about 6.4 with 0.2 M Na
OH. a-Chymotrypsin (0.9 mg) was added, and the mixture
was incubated at RT for 6 h with the pH being kept
constant by addition of 0.2 M NaOH. After digestion,
the mixture was lyophilized to dryness and separated by
preparative HPLC [C ; solvent A = water, 0.1%, v/v, TFA;
with brine (2ꢂ 20) and dried over Na SO . Evaporation
2
4
afforded the title compound 4 (170 mg, 59%) as a light
brown solid (crude) mp = 153–154 8C, Rf: 0.25
1
CHCl :MeOH:NH , 9:1:0.25), H NMR (250 MHz,
(
CDCl ): 1.30 (dd, J = 7.1, 7.1 Hz, 6H), 1.86 (s, 3H),
3
3
3
4.03 (s, 2H), 4.27 (m, 4H), 6.46 (s, 1NH), 7.10 (dd,
J = 16.6, 7.9 Hz, 2H), 7.42 (dd, J = 8.7, 4.2 Hz, 1H),
1
8
8
.76 (dd, J = 4.2, 1.1 Hz, 1H).
solvent B = MeCN:water = 3:1, 0.1%, v/v, TFA; t = 18.0
R
min (linear gradient 0–80% B over 55 min)]. L-3-(8-
Hydroxyquinolin-5-yl)alanine 7 (M10) was obtained:
2
.2.4. DL-3-(8-Hydroxyquinolin-5-yl)alanine (5)
Diethyl (8-hydroxyquinolin-5-yl-methyl)-acetamido-
20
27.6 mg, 35%, 100% purity, ½aꢃ = +13.5 (D-ethyl ester
D
was recovered 39.2 mg, 88% recovery). H NMR
1
malonate 4 (8.9 g, 21.9 mmole) was dissolved in 6N
HCl (150 ml) and the resultant mixture was refluxed
for 10 h. The reaction mixture was evaporated to dryness
by removing the solvent in vacuum. The residue was
(250 MHz, D O) 3.49 (m, 1H), 3.65 (m, 1H), 3.95 (m,
2
1H), 7.34 (m, 1H), 7.57 (m, 1H), 8.0 (m, 1H), 8.91 (m, 1H),
+
9.14 (m, 1H); calcd for C H N O m/z [M + H] =
1
2 12 2 3
+
233.24, found [M + H] = 233.26.
redissolved in H O and filtered. The pH of the solution
2
was adjusted to 5–5.5 with 10% NaOH. A yellow pre-
cipitate was collected by filtration, washed thoroughly
with water, re-crystallized from water (pH 5.5) and
washed with acetone to give DL-3-(8-hydroxy quinolin-
2.2.7. D-3-(8-hydroxyquinolin-5-yl)alanine (9 or M9)
D-3-(8-Hydroxyquinolin-5-yl)alanine ethyl ester
8
(33.4 mg) was dissolved in 11 ml of 0.2 M NaOH and
the solution was stirred at RT for 6 h. Water was removed
by lyophilization, and the product was purified by pre-
5
by HPLC [C ; solvent A = water, 0.1%, v/v, TFA; solvent
-yl)alanine 5 (3.8 g, yield 65%) of 100% purity, checked
1
8
B = MeCN:water = 3:1, 0.1%, v/v, TFA; t = 18.2 min
R
parative HPLC [C ; solvent A = water, 0.1%, v/v, TFA;
18
(
linear gradient 0–80% B over 55 min)]. mp = 194 8C
solvent B = MeCN:water = 3:1, 0.1%, v/v, TFA;
tR = 18.4 min (linear gradient 0–80% B over 55 min)] to
give D-amino acid 9 or M9 26.8 mg, 89%, 99.1% purity.
1
decompose). H NMR (250 MHz, D O) 3.42 (m, 1H),
(
2
3
.58 (m, 1H), 3.93 (dd, J = 7.2, 7.2 Hz, 1H), 7.22 (d,
20
1
J = 8.0 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.94 (dd,
J = 8.7, 5.4 Hz, 1H), 8.84 (d, J = 5.1 Hz, 1H)., 9.06 (d,
1
J = 8.7 Hz, 1H); C NMR (100 MHz, DMSO) 32.14,
½aꢃ = ꢄ10.3. H NMR (250 MHz, D O) 3.40 (s, 1H), 3.55
D
(s, 1H), 3.91 (s, 1H), 7.15 (s, 1H), 7.45 (s, 1H), 7.91 (s, 1H),
2
3
8.80 (s, 1H), 9.02 (s, 1H), calcd for C H N O m/z
2 12 2 3
1
+
[M + H] = 233.24, found [M + H] = 233.26.
+
5
1
3.11, 111.03, 121.27, 127.46., 129.76, 133.35, 138.13,
47.46, 152.55, 170.30. Mass spectrometry: calculated
+
+
for C H N O m/z [M + H] = 233.24, found [M + H]
2 12 2 3
2.3. Detection of free radical intermediates
1
=
233.25.
ꢁ
Hydroxyl radical ( OH) was produced by the photolysis
of H O as previously described [35]. Formation of the
2.2.5. DL-3-(8-Hydroxyquinolin-5-yl)alanine ethyl
ester (6)
To a stirred slurry of DL-3-(8-hydroxyquinolin-5-yl)ala-
nine 5 (2.19 g, 7.04 mmole) in absolute ethanol (26 ml) at
0
in N2, was added dropwise thionyl chloride (1.1 ml,
2
2
ꢁ
hydroxyl radical ( OH) was followed by electron spin
ꢁ
resonance (ESR) of the spin adduct (DMPO– OH). A
ꢁ
typical 60 ml incubation mixture for trapping OH radicals
contained 50 mM DMPO. For H O photolysis, the irra-
8C, protected from atmospheric moisture by CaCl tube
2
2 2
diation source was a KL1500 electronic projector lamp
(Schott, Germany). Samples were irradiated directly inside
the ESR cavity by white light with an optical fiber. The
4
.1 mmole). The reaction mixture was stirred at 0 8C for
3
0 min, and at RT for 30 min, and then refluxed overnight.
ꢁ
The solution was evaporated to dryness in vacuum. The
residue was dissolved in absolute ethanol and evaporated to
dryness. To ensure completeness of the esterification, the
whole procedure was repeated. The ester 6 (yellow solid,
1.67 g, 92% yield), shown by HPLC to contain about 1%
free amino acids, was further purified by Flash column
ESR spectra of DMPO– OH were recorded in a Bruker
electron spin resonance ER200D-SRC spectrometer (Bru-
ker, Karlsruhe, Germany) in a quartz flat cell of 60 ml. The
standard instrumental parameters were as follows: micro-
wave frequency, 9.7 GHz; incident microwave power,
10 MW; center of the field, 3480; scan range, 100 G; field
1
ꢄ5
chromatography (CH Cl :MeOH:AcOH, 9:1.5:1.5). H
NMR (250 MHz, D O) 0.94 (dd, J = 7.2, 7.2 Hz, 3H),
modulation, 1 G; receiver gain, 6.3 ꢂ 10 ; and time
2
2
ꢁ
constant, 1.3 s. The ESR spectrum of DMPO– OH con-
sisted of a quartet (1:2:2:1) with hyperfine splitting con-
2
3
.63 (m, 2H), 4.01 (m, 2H), 4.34 (dd, J = 7.7, 7.5 Hz,
H), 7.31 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.2 Hz 1H), 8.01
dd, J = 8.7, 5.4 Hz, 1H), 8.93 (d, J = 5.4 Hz, 1H), 9.08 (d,
1
stants of a = a = 14.9 G. The amplitude of the third peak
N
H
ꢁ
(
in the quartet of the ESR spectrum of DMPO– OH was
used for further calculations.
J = 8.7 Hz, 1H).