Mechanism of Formation of an HNE-Derived Fluorophore
J . Org. Chem., Vol. 64, No. 16, 1999 5743
(-), 168.86 (-), 204.42 (+); HRMS calcd for C12H21NO2
211.1573, found 211.1574. The NMR spectra of the nonfluo-
rescent fraction in CDCl3 indicated it to be the ring-opened
form 1-(p r op yla m in o)-1-n on en e-3,4-d ion e (9b): 1H NMR
(CDCl3) δ 0.87 (t, J ) 6.81 Hz, 3H), 0.95 (t, J ) 7.40 Hz, 3H),
1.23-1.32 (m, 6H), 1.55-1.64 (m, 4H), 3.24 (q, J ) 6.61 Hz,
2H), 5.68 (d, J ) 7.08 Hz, 1H), 7.06 (dd, J ) 13.11 and 7.08
Hz, 1H), 10.42 (br, 1H). Evaporation of the CD3OD solution of
9a and dissolution in CDCl3 revealed its tautomerization to
9b. The combined yield of the two tautomers was 250 mg
(59%).
(E)-4-Oxo-2-p en ten a l (14). Compound 14 was prepared by
a modification of the literature procedure.45 2-Methylfuran (18
mL, 0.2 mol), anhydrous Na2CO3 (42.4 g, 0.4 mol), and
methanol (100 mL) were added into a three-neck round-
bottomed flask. The reaction mixture was cooled to -10 °C,
and a solution of 11.2 mL of bromine (0.22 mol) in 60 mL of
methanol was added dropwise over 1 h. The reaction mixture
was stirred at -10 °C for 3 h, and then the undissolved salts
were removed by filtration. The filtrate was poured into 400
mL of saturated NaCl solution and extracted with CH2Cl2 (4
× 60 mL). The combined CH2Cl2 extracts were dried over
anhydrous Na2SO4. After removing CH2Cl2, the resulting
yellow oil was added into 60 mL of distilled water, stirred for
6 h, and allowed to stand overnight, followed by extraction
with CH2Cl2 (4 × 15 mL). The CH2Cl2 extracts were dried with
Na2SO4, concentrated, and distilled (65-70 °C at 11 Torr) to
afford 8 g (38% based on 2-methylfuran) of 14.
In cu ba tion of (E)-4-oxo-2-p en ten a l (14) w ith P r im a r y
Am in es. Compound 14 (490 mg, 5 mmol) in 10 mL of
acetonitrile was added to 20 mL of an aqueous solution of 50
mmol of either ethylamine (from the 70% aqueous reagent) or
propylamine, which is neutralized to pH 7.4 by adding NaH2-
PO4. The solution was incubated overnight and then extracted
with EtOAc (3 × 20 mL). TLC analysis showed several spots;
the major nonfluorescent product from each incubation reac-
tion was purified by flash chromatography (EtOAc as eluent).
Pyrrole dimer 15a from reaction of 14 with ethylamine: 1H
NMR (CDCl3) δ 1.05 (t, J ) 7.23 Hz, 3H), 1.30 (t, J ) 7.23 Hz,
3H), 1.68 (s, 3H), 1.87 (s, 3H), 3.12 (dq, J ) 14.16 and 7.10
Hz, 1H), 3.33 (dq, J ) 14.10 and 7.13 Hz, 1H), 3.79 (q, J )
7.16 Hz, 2H), 6.08 (d, J ) 5.73 Hz, 1H), 6.11(d, J ) 2.85 Hz,
1H), 6.56 (d, J ) 2.91 Hz, 1H), 6.91 (d, J ) 5.67 Hz, 1H); 13C
NMR (CDCl3) δ 9.72 (-), 13.85 (-), 16.36 (-), 24.27 (-), 34.19
(+), 41.42 (+), 66.69 (+), 106.85 (-), 114.68 (+), 117.78 (-),
123.85 (-), 125.22 (+), 153.81 (-), 170.61 (+). HRMS calcd
for C14H20N2O 232.1577, found 232.1572. Pyrrole dimer 15b
from reaction of 14 with propylamine: 1H NMR (CDCl3) δ 0.81
(t, J ) 7.36 Hz, 3H), 0.87 (t, J ) 7.37 Hz, 3H), 1.38-1.54 (m,
2H), 1.57-1.74 (m, 2H), 1.66 (s, 3H), 1.84 (s, 3H), 2.87-3.04
(m, 1H), 3.12-3.27 (m, 1H), 3.70 (t, J ) 7.28 Hz, 2H), 6.08 (d,
J ) 5.90 Hz, 1H), 6.08 (d, J ) 2.56 Hz, 1H), 6.53 (d, J ) 2.94
Hz, 1H), 6.91 (d, J ) 5.82 Hz, 1H); 13C NMR (CDCl3) δ 9.77,
11.11, 11.64, 21.69, 24.20, 24.37, 41.40, 48.30, 66.63, 106.59,
114.48, 118.71, 123.69, 125.29, 153.74, 170.86; HRMS calcd
for C16H24N2O 260.1890, found 260.1894.
Rea ction of 8 w ith n -Bu tyla m in e in Aqu eou s Meth a -
n ol. A solution of n-butylamine (860 mg, 11.8 mmol) in water
(10 mL) was neutralized to pH 4.75 with NaH2PO4. To this
amine solution was added freshly prepared 8 (200 mg, 1.2
mmol) in 10 mL of MeOH, at which point the mixture turned
brown. After 16 h, the mixture was concentrated and extracted
with EtOAc. The water layer was evaporated under reduced
pressure, and the residue was dissolved in a minimal amount
of MeOH. This solution was subjected to semipreparative
HPLC purification. The eluent was monitored for absorbance
at 355-365 nm. Compound 1b was isolated in 17% yield (51
mg) and shown to be identical with that isolated from the
incubation of HNE with butylamine in all respects (NMR,
HRMS). The EtOAc extracts were evaporated, and the crude
residue was purified by flash chromatography (EtOAc as
mobile phase) to afford fluorescent product N-bu tyl-2-p en tyl-
2-m eth oxy-1,2-d ih yd r op yr r ol-3-on e (10) in 45% yield (120
mg). 1H NMR(CD3OD) δ 0.88 (t, J ) 6.89 Hz, 3H), 1.02 (t, J )
7.29 Hz, 3H), 1.04-1.13 (m, 2H), 1.23-1.33 (m, 4H), 1.39-
1.51 (m, 2H), 1.69-1.82 (m, 4H), 3.09 (s, 3H), 3.29 (t, J ) 7.73
Hz, 2H), 5.07 (d, J ) 3.66 Hz, 1H), 8.40 (d, J ) 3.54 Hz, 1H);
13C NMR(CD3OD) δ 14.18(-), 14.36 (-), 21.34 (+), 23.12 (+),
23.56 (+), 32.28 (+), 32.92 (+), 35.59 (+), 45.54 (+), 52.36 (-),
96.77 (+), 97.32 (-), 170.37 (-), 202.52 (+); HRMS calcd for
C
14H25NO2 239.1887, found 239.1885.
(E)-1,1-Dim et h oxy-2-n on en -4-on e (12). A solution of
HNE dimethyl acetal (11) (0.5 g, 2.45 mmol) in 20 mL of dry
CH2Cl2 was added dropwise to a well-stirred solution of
pyridinium chlorochromate (1.06 g, 4.90 mmol) in dry CH2Cl2
containing 1.5 g of NaOAc. After 6 h the mixture was
concentrated, and the dark residue was diluted with 250 mL
of dry ether and filtered through cotton gauze. Upon evapora-
tion of solvent, the residue was purified by flash chromatog-
raphy (EtOAc as eluent) to yield 460 mg (96%) of 12.1H NMR
(CDCl3) δ 0.85 (t, J ) 6.8 Hz, 3H), 1.20-1.32 (m, 4H), 1.53-
1.63 (m, 2H), 2.53 (t, J ) 7.4 Hz, 2H), 3.31 (s, 6H), 4.99 (dd, J
) 3.8 and 1.1 Hz, 1H), 6.33 (dd, J ) 16.6 and 1.1 Hz, 1H),
6.55 (dd, J ) 16.4 and 3.9 Hz, 1H); 13C NMR (CDCl3) δ 13.94
(-), 22.48 (+), 23.72 (+), 31.43 (+), 40.68 (+), 52.98 (-), 101.08
(-), 132.12 (-), 139.91 (-), 200.47 (+); HRMS calcd for
15N-La beled N,N′-P r op yl-2-p en tyl-2-h yd r oxy-1,2-d ih y-
d r op yr r ol-3-on e P r op yl Im in iu m (15N-la beled 1a ). A solu-
tion of 500 mg of 15N-labeled propylamine hydrochloride (5.18
mmol) in 15 mL of water was neutralized to pH 7.30 with Na2-
HPO4. CuSO4‚5H2O (10 mg, 0.04 mmol) and 20 mg of 2,2′-
bipyridine (0.13 mmol) were slowly added, and then 200 mg
(1.30 mmol) of 13 in 10 mL of acetonitrile was added. After 16
h, the reaction solution was extracted with ethyl acetate/
hexane (1:1 v/v), and the water layer was concentrated and
subjected to HPLC isolation. The ex/em 360/430 nm fluorescent
fractions were combined, concentrated, and further purified
by flash chromatography (MeOH as eluent) to afford 98 mg
C
11H20O3 200.1413, found 200.1412.
(E)-4-Oxo-2-n on en a l (13). Compound 12 (460 mg, 2.3
mmol) was added to 30 mL of 2 N HCl/acetone (1:2 v/v) with
stirring for 4 h. Then, the reaction mixture was concentrated
and extracted with CH2Cl2 (3 × 10 mL). The combined organic
layer was dried (Na2SO4) and concentrated to afford 350 mg
(100%) of 13.1H NMR (CDCl3) δ 0.89 (t, J ) 6.7 Hz, 3H), 1.27-
1.36 (m, 4H), 1.60-1.70 (m, 2H), 2.68 (t, J ) 7.4 Hz, 2H), 6.76
(dd, J ) 16.5 and 6.5 Hz, 1H), 6.87 (d, J ) 16.2 Hz, 1H), 9.77
(t, J ) 6.7 Hz, 1H); 13C NMR (CDCl3) δ 13.89 (-), 22.43 (+),
23.37 (+), 31.26 (+), 41.21 (+), 137.33 (-), 144.96 (-), 193.45
(-), 200.17 (+); HRMS calcd for C9H14O2 154.0994, found
154.0995.
P r ep a r a tion of 1a fr om 13. A solution of 29.5 mg of
propylamine (0.5 mmol) in 10 mL of water was neutralized to
pH 7.5 with NaH2PO4. To this amine solution were added
CuSO4 (1 mg, 0.004 mmol), 2,2′-bipyridine (1.9 mg, 0.012
mmol), and then 15.4 mg (0.1 mmol) of 13. After 16 h, the
reaction mixture was concentrated to give a brown residue,
which was dissolved in 1 mL of MeOH. This crude sample was
then subjected to semipreparative HPLC to afford 10 mg
(39.4%) of 1a . The NMR, mass, and fluorescence spectra of
this fluorophore were identical with those for the compound
isolated from the incubation of HNE with propylamine.
1
(30%) of [15N]1a . H NMR (D2O) δ 0.81 (t, J ) 6.78 Hz, 3H),
0.93 (t, J ) 7.41 Hz, 3H), 0.94 (t, J ) 7.38 Hz, 3H), 1.23-1.29
(m, 4H), 1.61-1.70 (m, 2H), 1.71-1.80 (m, 2H), 1.80-1.92 (m,
1H), 2.06-2.17 (m, 1H), 3.35 (t, J ) 6.86 Hz, 2H), 3.45 (t, J )
7.43 Hz, 2H), 5.45 (d, J ) 3.42 Hz, 1/2 H), 5.47 (d, J ) 3.21
Hz, 1/2 H), 8.15 (d, J ) 3.40 Hz, 1/2H), 8.18 (d, J ) 3.20 Hz,
1/2 H); 13C NMR (D2O) δ 10.44, 10.63, 13.13, 21.00, 21.38,
21.66, 21.88, 30.44, 35.48, 45.98 (d, J 13C-15N ) 8.85 Hz), 47.13
(d, J 13C-15N ) 8.60 Hz), 87.81, 95.32 (d, J 13C-15N ) 8.48 Hz),
166.74 (d, J 13C-15N ) 15.0 Hz), 176.87 (d, J 13C-15N ) 18.6
Hz); HRMS calcd for C15H2915N2O 255.2222, found 255.2242.
F lu or escen ce Mea su r em en ts for HNE- or HP E-De-
r ived F lu or op h or es a n d Th eir An a logu es a t Differ en t
p H Va lu es. To assess the effect of pH on HNE- or HHE-
(45) Clauson-Kaas, N.; Limbrg, F. Acta Chem. Scand. 1947, 1, 619-
623.