Reactions of Dehydroascorbic Acid
J. Agric. Food Chem., Vol. 44, No. 7, 1996 1631
tegrator were used. Preparative HPLC was performed on a
HiBar column packed with LiChroSorb (RP 18, 250 × 20 mm
i.d., 7 µm particle size).
Rea ction s of DHA w ith P r op yla m in e a n d NR-Acetyl-
lysin e in P h osp h a te Bu ffer in th e P r esen ce a n d Absen ce
of Oxygen . To a solution of DHA (17.4 mg/mL, 0.1 mmol/
mL) in phosphate buffer (pH 7) was added propylamine or NR-
acetyllysine in different ratios. The mixture was adjusted to
pH 7 and heated for different times at 40, 70, or 100 °C.
Diluted samples were injected into the analytical HPLC
system (Figures 1 and 2). For experiments in the absence of
oxygen, diethylentriaminepentaacetic acid (DTPA) was added
(39.3 mg/mL, 0.1 mmol/mL) and nitrogen was bubbled through
the solution during heating.
F igu r e 1. HPL chromatogram of a reaction mixture of DHA
(50 mg, 0.29 mmol) and propylamine (33.9 mg, 0.57 mmol) in
THF (2.0 mL) heated for 1.5 h at 40 °C: UV detection: 0 min,
246 nm; 11.4 min, 278 nm; 15.0 min, 219 nm. Numbers on
top of peaks refer to structures in Figure 6.
2-Deoxy-2-(p r op yla m in o)a scor bic Acid (1). L-Dehy-
droascorbic acid (400 mg, 2.3 mmol) was suspended in 8 mL
of THF and propylamine (300 mg, 5.1 mmol) was added. The
mixture was kept at 40 °C for 1 h in a closed vessel. After the
solvent was removed under reduced pressure, the residue was
dissolved in a mixture of water and methanol (8:2, 4 mL) and
filtered through a Durapore poly(vinylidene fluoride) mem-
brane (0.45 µm). Isolation of 1 was achieved by preparative
HPLC [eluent, water-methanol (95:5); flow rate, 12 mL/min;
UV detection at 246 nm; Injection volume, 0.8 mL]. The
fraction between 13 and 15 min was collected and the aqueous
solution was lyophilized. 1 was obtained as a colorless solid
which rapidly turned red under atmospheric conditions (yield
after purification, 4%): 1H NMR (CD3OD, COSY) δ 0.89 (t,
3H, CH2CH3), 1.57 (m, 2H, CH2CH3), 2.95 (m, 2H, NCH2), 3.58
(m, 2H, CH2O), 3.85 (dt, 1H, CH2OCHO), 4.40 (d, 1H, CHOCd);
13C NMR (CD3OD, COSY, DEPT) δ 11.2 (CH2CH3), 20.5 (CH2-
CH3), 51.8 (NCH2), 63.9 (CH2O), 71.5 (CH2OCHO), 81.3
(CHOCd), 94.3 (+NH2CH2), 175.9 (-OCd), 184.2 (CdO); MS
(m/z, CI) 218 (M + 1); (m/z, EI) 217 (M), 170, 156, 100; UV
λmax (H2O) ) 245.5 (pH 3), 247 (pH 7), 271 nm (pH 11).
Isola tion of Oxa lic Acid Mon op r op yla m id e (2) fr om a
Rea ction Mixtu r e of Deh yd r oa scor bic Acid a n d P r op yl-
a m in e. L-Dehydroascorbic acid (160 mg, 0.92 mmol) was
dissolved in phosphate buffer (pH 7.0, 8 mL) and propylamine
(271 mg, 2.6 mmol) was added. The mixture was adjusted to
pH 7.0 and heated at 100 °C in a closed vessel. After 2 h the
reaction was stopped by freezing the solution. The frozen
reaction mixture was lyophilized to remove the solvent and 1
g of the residue was dissolved in 4 mL of the HPLC eluent.
Each time, 1.0 mL was injected into the preparative HPLC
system [eluent, phosphate buffer (10 mmol KH2PO4, pH 3.0,
adjusted with H3PO4); flow rate, 12 mL/min; UV detection at
220 nm]. The fraction between 23 and 26 min was collected
and lyophilized. To remove the phosphate buffer, the residue
was dissolved in methanol and filtered. The methanol was
evaporated. This procedure was repeated several times. 2 was
obtained as a colorless solid (yield, 25%): mp 113 °C (dec); 1H
NMR (CDCl3) δ 0.90 (t, J ) 7.4 Hz, 3H, CH2CH3), 1.56 (sex, J
) 7.4 Hz, 2H, CH2CH3), 3.28 (m, J ) 7.4 Hz, 2H, NCH2), 7.24
(NH); 13C NMR (CDCl3) δ 11.3 (CH2CH3), 22.4 (CH2CH3), 42.3
(NCH2), 157.6 (OCdO), 160.0 (HNCdO); MS (m/z, CI) 132 (M
+ 1).
Isola tion of Oxa lic Acid Dip r op yla m id e (3) fr om a
Rea ction Mixtu r e of Deh yd r oa scor bic Acid a n d P r op yl-
a m in e. L-Dehydroascorbic acid (600 mg, 3.4 mmol) was
suspended in 6 mL of THF and propylamine (1116 mg, 18.9
mmol) was added. The mixture was allowed to stand for 1 h
at 40 °C in a closed vessel. After removal of the solvent under
reduced pressure, the residue was dissolved in 100 mL of
water. The aqueous solution was extracted with ethyl acetate
(3 × 40 mL). The collected organic layers were dried over
anhydrous sodium sulfate. After evaporation of ethyl acetate,
the residue was dissolved in a mixture of water and methanol
(8:2, 3 mL). For purification, this solution was injected into
the preparative HPLC system [eluent, water-methanol (45:
55); flow rate, 9 mL/min; UV detection at 220 nm; injection
volume, 1.0 mL]. The fraction between 20 and 23 min was
collected. Methanol was removed under reduced pressure and
the aqueous solution was lyophilized. 3 was obtained as a
colorless solid (yield after purification, 15%): mp 162 °C (dec);
1H NMR (CDCl3) δ 0.95 (t, J ) 7.4 Hz, 3H, CH2CH3), 1.61 (m,
2H, J ) 7.4 Hz, CH2CH3), 3.27 (m, J ) 7.4 Hz, 2H, NCH2),
7.51 (NH); 13C NMR (CDCl3) δ 11.7 (CH2CH3), 22.9 (CH2CH3),
41.8 (NCH2), 160.4 (CdO); MS (m/z, CI) 173 (M + 1); (m/z,
EI) 172, 144, 143, 131, 115; UV λmax ) 217 nm (UV spectrum
was directly taken from the diode array detector).
Syn th eses of Refer en ce Oxa lic Acid Am id es. Oxalic
Acid Monopropylamide (2). Oxalyl chloride (4.3 mL, 50 mmol)
was stirred at -17 °C while ice-cold propylamine (4.1 mL, 50
mmol) was added (very carefully) during 2 h. After an
additional 1 h, 50 mL of ice water was added slowly and the
reaction mixture was adjusted to pH 13 with NaOH (1 N). The
aqueous solution was extracted with ethyl acetate to remove
the oxalic acid diamide and adjusted to pH 1.5 with HCl (2
N). After extraction of the solution with ethyl acetate, the
combined organic layers were dried over anhydrous sodium
sulfate. The solvent was removed under reduced pressure and
2 was obtained as a slightly yellow solid which recrystallizes
from CHCl3 in needles. The spectral data were identical with
those of 2 described above (yield, 10%): Anal. Calcd for
C5H9O3N: C, 45.78; H, 6.92; N, 10.68. Found: C, 45.82; H,
6.81; N, 10.55.
Oxalic Acid Dipropylamide (3). Propylamine (1 mL, 12.2
mmol) was stirred at -17 °C. Oxalyl chloride (0.5 mL, 5.8
mmol) was added in small portions during 1 h. The mixture
was stirred for an additional 1 h, and 20 mL of ice water was
added. The resulting suspension was extracted three times
with ethyl acetate (20 mL). The combined organic layers were
reextracted with HCl (0.1 N, 20 mL) and NaOH (0.1 N, 20
mL) and dried over anhydrous sodium sulfate. After removal
of ethyl acetate under reduced pressure, a colorless solid was
obtained (yield, 90%). The spectral data were identical with
those of 3 described above. Anal. Calcd for C8H16O2N2: C,
55.77; H, 9.37; N, 16.27. Found: C, 55.58, H, 9.67; N, 16.19.
RESULTS AND DISCUSSION
When DHA is reacted with primary aliphatic amines
or NR-acetyllysine, several reaction products can be
separated by HPLC and observed using UV detection
(Figures 1 and 2). Major products are 3-deoxy-3-
(alkylamino)ascorbic acids (4) with a characteristic
absorption maximum at 278 nm. The propylamine and
NR-acetyllysine derivatives were identified by comparing
retention times and UV spectra with those of authentic
samples. 4 was previously isolated from reaction mix-
tures of AA and aliphatic amines (Pischetsrieder et al.,
1995). It was proposed that the 3-hydroxyl group of AA
is substituted by the amine leading to 4. If DHA is the
educt, however, another reaction mechanism can be
envisaged. A Schiff base of the 3-keto group of DHA
can be formed, which is then reduced to give the product
4 (Figure 3). It can be assumed that reductones or
aminoreductones, which are formed during the degra-
dation of DHA [e.g., pentose reductone (Wisser et al.,
1968)], are the compounds that reduce the intermediate
Schiff base. Another possible pathway is the reaction