1488 J. Agric. Food Chem., Vol. 49, No. 3, 2001
Torres y Torres and Rosazza
Ta ble 3. Ma ss Sp ectr a l Da ta for 11 Isola ted fr om
Ta ble 2. Yield s (HP LC) of P r od u cts Obta in ed fr om
Rea ction s of p-Cou m a r ic Acid (1) w ith Nitr ite in H2O
over a Ra n ge of p H Va lu es
Rea ction s of p-Cou m a r ic Acid (1) w ith Nitr ite in H2O a n d
H218O
pH
isolated standard 11
11 from 10% H218O reaction
compound
2
3
7a
10a
ion
ion relative
fragment
ion
ion relative
intensity (%)
fragment
ion
(m/z) intensity (%)
3
5
7
11
13
16 ( 5%
-
-
-
-
-
59 ( 7% 25 ( 7%
168
166
165
164
0.03
0.84
9.07
[M + H + 4]+
[M + H + 2]+
[M + H + 1]+
[M + H]+
0.91
20.84
11.01
100
[M + H + 4]+
[M + H + 2]+
[M + H + 1]+
[M + H]+
26 ( 3% 67 ( 5% 23 ( 3%
17 ( 3%
-
1.4 ( 0.4%
6 ( 2%
6 ( 1%
6 ( 1%
-
-
1.4 ( 0.8%
100
a
Results from duplicate analyses.
10.77 (1H, s, OH), 8.30 (1H, d, J ) 2.1 Hz, H-2′), 7.81 (1H, dd,
J ) 2.1, 8.9 Hz, H-6′), 7.67 (1H, d, J ) 16.0 Hz, H-3), 7.25
(1H, d, J ) 8.9 Hz, H-5′), 6.46 (1H, d, J ) 16.0 Hz, H-2), 3.86
HP LC An a lysis of P r od u cts F or m ed a t p H 2. The
reaction was carried out as described above on a 50-mL scale
at pH 2 (8:1 molar ratio of NaNO2/1). The reaction was
monitored by HPLC. After 1, 25, 55, 80, 105, and 130 min,
1-mL volumes of the reaction mixture were sampled and
diluted with 2 mL of MeOH, and 5-µL samples were analyzed.
The peaks were identified by comparison with retention
volumes of the isolated standards. The product yields in
solution were quantitated (from three separate experiments)
from established standard curves, and yields of each compound
in solution after 130 min are given in Table 2.
1
(3H, s, COOCH3); H NMR (CD3OD, 400 MHz) δ 8.18 (1H, d,
J ) 2.4 Hz, H-2′), 7.75 (1H, dd, J ) 2.4, 8.6 Hz, H-6′), 7.63
(1H, d, J ) 15.7 Hz, H-3), 7.02 (1H, d, J ) 8.7 Hz, H-5′), 6.40
(1H, d, J ) 15.8 Hz, H-2), 3.77 (3H, s, COOCH3); 13C NMR
(CDCl3, 150 MHz) δ 166.7 (COOCH3), 156.0 (C-4′), 141.7
(C-3), 136.0 (C-6′), 133.6 (C-3′), 127.0 (C-1′), 124.9 (C-2′), 120.8
(C-5′), 118.8 (C-2), 51.7 (COOCH3); EIMS (70 eV) m/z 223 [M]+
(81), 192 (100), 146 (22), 118 (16), 89 (31); HREIMS m/z
223.0498 (calcd for C10H9NO5, 223.0481).
Rea ction of p-Cou m a r ic Acid (1) w ith Na NO2 a t p H 2
in H218O. A sample of 5 mg (0.02 mmol) of p-coumaric acid
(1) dissolved in 30 µL of MeOH and 22 µL of 6 N HCl were
added to 11 mg (0.16 mmol) of NaNO2 dissolved in 1.0 g of
H218O (10 at. % 18O). The reaction mixture was stirred for 60
min until it became dark yellow. Samples of 5 µL were
withdrawn from the reaction after 0 and 60 min and directly
injected without workup for HPLC analysis. After 60 min, the
pH was adjusted to 8.0 with 300 µL of 1 M NaHCO3 and
extracted with 3 × 0.5 mL EtOAc. Pooled EtOAc extracts were
evaporated to yield 4 mg of a bright orange oil, which was
redissolved in 200 µL of Optima MeOH, and 12 × 15 µL
samples were injected for HPLC purification. Peaks eluting
at Rv 16 mL were collected, pooled, and extracted with three
half-volumes of EtOAc. The EtOAc layers were combined and
evaporated to yield 55 µg of 11 (0.3 µmol, 2% yield).
Rea ction of 4-Hyd r oxyben zen ep r op a n oic Acid (14)
w ith Nitr ite. A sample of 32 mg of 14 dissolved in 300 µL of
MeOH was added to 5 mL of pH 2 distilled H2O containing 57
mg (0.8 mmol) of NaNO2. After 30 and 60 min, 200-µL volumes
of the reaction mixture were sampled and diluted with 400
µL of MeOH, and 5-µL samples were analyzed by HPLC (280
nm), indicating that 15 (Rv 23.0 mL) was present in about 10%
yield. After 60 min, ammonium sulfamate (NH4SO3NH2, 47
mg, 0.4 mmol) was added to the dark orange-red-colored
reaction (20), and it was extracted with 3 × 2.5 mL EtOAc.
EtOAc extracts were dried over Na2SO4 and evaporated to
yield 28 mg of a red mixture. The mixture was separated over
40-µm C18 reversed-phase silica gel (12.3 × 1.2 cm, H2O/
MeOH 9:1-6:4). One fraction contained 23 mg of unreacted
14 (TLC, system A, Rf 0.35, yellow), whereas a second gave 2
mg of a yellow solid that was characterized as 15 (Rf 0.63,
yellow, 9 µmol, 5%).
4-Hyd r oxy-3-n itr oben zen ep r op a n oic Acid (15). Yellow
solid; UV (MeOH) λmax (log ꢀ) 215 (4.14), 275 (3.69), 358 (3.37)
nm; 1H and 13C NMR data were nearly identical to those
reported (21, 22); EIMS (70 eV) m/z 211 [M]+ (13), 193 (21),
175 (17), 152 (100), 151 (36), 147 (36), 106 (15), 72 (60), 55
(17); HREIMS m/z 211.0482 (calcd for C9H9NO5, 211.0481).
P r ep a r a tion of 3-(4-Hyd r oxyp h en yl)-2-p r op en oic Acid
Meth yl Ester (16) (23). H2SO4 (1 mL) was added to anhydrous
1 (1.0 g, 6 mmol) dissolved in 23 mL of anhydrous MeOH and
refluxed under N2 for 2 h at 45 °C. The reaction was monitored
by TLC (system E, Rf 0.57, dark orange), which indicated that
3-(4-hydroxyphenyl)-2-propenoic acid methyl ester (16) was
produced in about 80% yield after 3.5 h. The reaction mixture
(30 mL) was poured over 24 g of crushed ice and stirred until
the ice dissolved and the solution became milky white. The
pH was adjusted to 8.0 with 78 mL of 1 M NaHCO3, and the
solution was extracted with 3 × 130 mL CH2Cl2. Pooled organic
extracts were dried over Na2SO4 and concentrated to afford
920 mg (5.2 mmol) 16 in 86% yield. Recrystallization from
MeOH/H2O provided 838 mg (4.7 mmol) white needles (78%
overall yield) of 16 that gave a melting point of 134.5-136 °C
[lit. 137 °C (24)], NMR spectral properties corresponding to
previous reports (24, 25), and HREIMS m/z 178.0630 (calcd
for C10H10O3, 178.0630).
An a lysis of 18O-La beled 11 by HP LC ESIMS. The
unlabeled standard 11 and the reaction product were each
dissolved to concentrations of 1 mg/mL in Optima MeOH.
Samples of 5 µL were resolved over a Versapack C18, 10µ
column connected to a Hewlett-Packard Series 1100 LC/MSD
instrument and ionized under API-ES conditions. The per-
centage incorporation of 18O was determined by comparing
peak areas for ions of the unlabeled standard and the com-
pound isolated from the reaction in H218O, 10 atom % 18O
(Table 3).
Rea ction of 1 w ith Nitr ite a t p H 7 a n d 10. The same
method was used for reaction of p-coumaric acid (1) with nitrite
at both pH 7 and pH 10. NaNO2 (552 mg, 8.0 mmol) was
dissolved in 50 mL of distilled H2O, pH 7.0. p-Coumaric acid
(1, 165 mg, 1.0 mmol) dissolved in 2 mL of MeOH was added
to the NaNO2 solution. The color became bright yellow within
seconds of the addition of 1. A 1-mL sample of the reaction
mixture was diluted with 2 mL of MeOH, and injection of 5
µL of the MeOH solution for HPLC analysis indicated that 13
(Rv 21.3 mL) was gradually produced over 150 min to a
maximum of 1% yield. TLC analysis (system D) showed five
spots after 30 min and through 150 min (Rf 0.50-0.10), with
about 70% of 1 remaining unreacted. After 150 min, the
reaction mixture was extracted with 3 × 25 mL EtOAc. Pooled
EtOAc extracts were dried over Na2SO4 and concentrated to
yield 38 mg of a yellow-orange oil. The oil was separated over
7 g of 40-µm silica gel (19.0 × 1.1 cm, CH2Cl2/MeOH 98:2-
96:4), giving three fractions (TLC system D). Evaporation of
the first fraction afforded a bright yellow powder (1.5 mg, 8
µmol), which was characterized as 13 (1%, Rf 0.49, yellow),
the second gave 3 mg (16 µmol) of 7 (2%, Rf 0.24, yellow), and
the third fraction contained 13 mg of unreacted 1 (Rf 0.40,
orange). The yields (HPLC) of the products in reactions at pH
7 and pH 10 were very similar: 13 (1%), 7 (23% at pH 7, 17%
at pH 10) (Table 2).
Rea ction of 3-(4-Hyd r oxyp h en yl)-2-p r op en oic Acid
Meth yl Ester (16) w ith Nitr ite. Reaction of 354 mg (2.0
mmol) of 16 with 553 mg (8.0 mmol) of NaNO2 at pH 2 as
before, but in a 2:1 (v/v) mixture of distilled water/acetone,
afforded after extraction and chromatography 104 mg of 3-(4-
hydroxy-3-nitrophenyl)-2-propenoic acid methyl ester (17, Rf
0.86, TLC system E, yellow, 0.47 mmol, 23%). The bright
yellow powder gave a melting point of 146.5-148.0 °C [cor-
rected, lit. 142-144 °C, (26)]; 1H NMR (CDCl3, 360 MHz) δ