2532 J. Agric. Food Chem., Vol. 44, No. 9, 1996
Lederer
In cu ba tion of P r op yla m in e w ith 3 in Aqu eou s Me-
d iu m a t 37 °C. A solution of propylamine in 0.8 mL of 0.2 N
HCl (14.7 mg/mL) and 1 mL of the respective So¨rensen
phosphate buffer (0.33 M, pH 9.0, 8.0, 7.0, 6.0) were combined,
and 46 mg (0.4 mmol) of 3 was added. The pH was adjusted
to the respective buffer value and the volume filled up to 2
mL. This mixture was incubated for 24 h at 37 °C, lyophilized,
acetylated as described for 12, and analyzed by GLC/MS.
Rea ction of P r op yla m in e w ith 3 u n d er Acid Con d i-
tion s. To 23 mg (0.2 mmol) of 3 in 0.8 mL of ethyl acetate
was added a solution of 47 mg (0.8 mmol) of propylamine in
0.2 mL of water (acidified with hydrochloric acid to pH 1-2).
The resulting emulsion was stirred for 3 h at room tempera-
ture and evaporated to dryness, and the residue was acetylated
as described for 12 and analyzed by GLC/MS. The TIC showed
an additional peak with the same MH+ ion as 12 and the
following data. GLC: tR ) 26.1 min. CI-GLC/MS: m/z 300
(MH+; 2), 240 (100), 199 (24), 169 (7), 149 (17), 112 (7), 97 (34).
Syn th esis of N2-Acetyl-L-lysin e 4-Meth ylcou m a r -7-yl-
a m id e (2). N6-(tert-Butyloxycarbonyl)-L-lysine (13). 13 was
synthesized according to the procedures of Moroder et al.
(1976) and Bayer et al. (1973).
N2-Acetyl-N6-(tert-butyloxycarbonyl)-L-lysine (14). A solution
of 8.4 g (34 mmol) of 13 in 43 mL of 1 N NaOH was cooled to
0 °C in an ice bath. Every 3 min, 0.65 mL of acetic anhydride
and 8.7 mL of 1 N NaOH were added alternately for 0.5 h.
The reaction mixture was stirred for 20 min at room temper-
ature, adjusted to pH 7.0, and extracted three times with 90
mL ether each. The organic layer was discarded, and the pH
of the aqueous phase was adjusted to 3.0 with 2 N HCl. The
solution was extracted seven times with 90 mL of CH2Cl2 each;
the CH2Cl2 phase was washed twice with 50 mL of water each
and dried with anhydrous Na2SO4. The solvent was stripped
off and the crude product dried in vacuo (5 Pa) over KOH. The
1H-NMR analysis of the N2-acetyl-N6-(tert-butyloxycarbonyl)-
L-lysine (14) thus obtained (5.7 g, 19.8 mmol, 58%) showed
the product to still contain 2% acetic acid.
FAB-MS (m-nitrobenzyl alcohol): 599 ([M2Na]+, 11) 577
([M2H]+, 31), 311 ([MNa]+, 51), 289 ([MH]+, 98), 233 (79), 189
(100), 128 (15), 84 (20), 57(9). 1H-NMR (DMSO-d6): δ 12.41
(s, 1H), 8.09 (d, 1H, J ) 7.8 Hz), 6.79 (t, 1H, J ) 5.5 Hz), 4.11
(ddd, 1H, J ) 4.9, 7.8, 8.9 Hz), 2.90 (ddt, 1H, J ) 5.5, 7.5,
(-)14.8 Hz), 2.87 (ddt, 1H, J ) 5.5, 7.5, (-)14.8 Hz), 1.84 (s,
3H), 1.65 (dddd, 1H, J ) 5.0, 6.7, 9.5, (-)13.2 Hz), 1.54 (m,
1H, J ) 5.5, 8.9, (-)13.2 Hz), 1.37 (s, 9H), 1.35 (m, 2H), 1.27
(m, 2H). 13C-NMR (DMSO-d6): δ 173.8, 169.2, 155.5, 77.3,
51.7, 39.5, 30.7, 29.1, 28.2, 22.7, 22.2.
N2-Acetyl-N6-(tert-butyloxycarbonyl)-L-lysine 4-Methylcou-
mar-7-ylamide (16). A mixture of 5.4 g (18.8 mmol) of N2-
acetyl-N6-(tert-butyloxycarbonyl)-L-lysine (14) and 3.25 g (20.6
mmol) of 1,1′-carbonyldiimidazole in 150 mL of anhydrous THF
was kept for 1 h at room temperature (until CO2 formation
had subsided); 2.5 g (14.3 mmol) of 7-amino-4-methylcoumarin
(15) was added and the reaction mixture incubated for 9 days
at 50 °C in a tightly closed tube. Progress of the reaction was
monitored by HPLC equipped with a diode array detector
(DAD). A methanol/0.01 M phosphate buffer (pH 4.0) gradient
was employed (% MeOH(t min)): 5(0)-95(30)-95(40)-5(45)-
5(55); tR(15) ) 18.3 min, tR(16) ) 25.2 min. Transformation
of 15 to 16 had reached 66% after 9 days. The solvent was
stripped off and the residue taken up in 400 mL of ethyl
acetate and extracted twice with 80 mL of 5% aqueous
NaHCO3 each (16 already precipitated during the extraction).
The aqueous layer was discarded and the organic phase
reduced to 150 mL, and kept at 4°C for 24 h. The precipitate
was filtered off, washed twice with 10 mL of ethyl acetate each,
and dried in vacuo (5 Pa), yielding 3.8 g (8.5 mmol, 60%) of
N2-acetyl-N6-(tert-butyloxycarbonyl)-L-lysine 4-methylcoumar-
7-ylamide (16). FAB-MS (m-nitrobenzyl alcohol): 446 ([MH]+,
33), 445(M•+; 21), 346 (100), 57 (59). 1H-NMR (DMSO-d6): δ
10.55 (s, 1H), 8.28 (d, 1H, J ) 7.5 Hz), 7.80 (d, 1H, J ) 2.1
Hz), 7.71 (d, 1H, J ) 8.7 Hz), 7.50 (dd, 1H, J ) 2.1, 8.7 Hz),
6.80 (t, 1H, J ) 5.8 Hz), 6.27 (q, 1H, J ) 1.3 Hz), 4.38 (ddd,
1H, J ) 5.5, 7.5, 8.9 Hz), 2.40 (d, 3H, J ) 1.3 Hz), 1.90 (s, 3H),
2.91 (m, 2H), 1.69 (m, 1H), 1.62 (m, 1H), 1.38 (m, 2H), 1.36 (s,
F igu r e 1. Chemical structure of propylamine (1), N2-acetyl-
L-lysine 4-methylcoumar-7-ylamide (2), trans-4,5-epoxy-trans-
2-hexen-1-ol (3), 9-hydroxy-trans-12,13-epoxy-trans-10-octa-
decenoic acid (4), 13-hydroxy-trans-9,10-epoxy-trans-11-octa-
decenoic acid (5), and 8-hydroxy-cis-11,12-epoxy-all-cis-5,9,-
14-eicosatrienoic acid (6).
(820499) were from Merck (Darmstadt, Germany); trans-2-
trans-4-hexadien-1-ol (18,305-9) was from Aldrich Chemical
Co. (Milwaukee, WI).
Syn t h esis of tr a n s-4,5-E p oxy-tr a n s-2-h exen -1-ol (3).
Acetylation of 3.0 g (30.5 mmol) of trans-2-trans-4-hexadien-
1-ol (7) in a pyridine/acetic anhydride mixture yielded 4.2 g
(30 mmol) of trans-2-trans-4-hexadien-1-ol acetate. A solution
of this acetate in 50 mL diethyl ether was cooled to 0 °C in an
ice bath, 9.4 g of m-chloroperbenzoic acid (≈55%, 30 mmol)
added, and the reaction mixture stirred for 24 h at 0 °C. The
reaction mixture was extracted three times with 40 mL of 10%
aqueous NaOH each and washed three times with 30 mL of
water each (negative starch iodine reaction in the washing
water indicating complete removal of peroxides). The solvent
was stripped off and the residue purified on a silica gel column
(hexane/ethyl acetate, 9 + 1, v:v). Fractions of 10 mL were
collected and tested for trans-4,5-epoxy-trans-2-hexen-1-ol
acetate (10) by TLC (Rf 0.18; solvent mixture as above;
detection, spray reagent 5% H2SO4 in anhydrous ethanol/10
min at 120 °C). All fractions containing 10 were combined
and evaporated to dryness. The colorless oily residue (1.6 g,
10.3 mmol, 34%) was dissolved in 21 mL of methanol, 1.5 g of
potassium carbonate in 9 mL of water added, and the mixture
stirred for 1 h at room temperature. The solution was diluted
with 50 mL of water and extracted five times with 70 mL of
ether each. The combined ether layers were washed twice with
30 mL of water each (the aqueous phase was tested for neutral
reaction), dried with anhydrous Na2SO4, filtered, and concen-
trated. trans-4,5-Epoxy-trans-2-hexen-1-ol (3) was obtained
as a colorless oil: 0.7 g (6.1 mmol, 20%). GLC: tR ) 7.3 min.
CI-GLC/MS: m/z 115 (MH+, 1), 97 (100), 83 (10), 79 (25), 75
(7). 1H-NMR, 13C-NMR (CDCl3): see Table 1.
Syn th esis of 4-(N-Acetyl-N-pr opylam in o)-1,5-diacetoxy-
tr a n s-2-h exen e (12). A mixture of 55 mg (0.5 mmol) of trans-
4,5-epoxy-trans-2-hexen-1-ol (3) and 59 mg (1 mmol) of pro-
pylamine in 0.5 mL of anhydrous THF was heated for 16 h to
100°C in a tightly sealed tube. The solvent was stripped off,
and unreacted starting material removed in vacuo (5 Pa, 2 h).
The residue was acetylated with pyridine/acetic anhydride in
the presence of catalytic amounts of 4-(dimethylamino)pyri-
dine, yielding 90 mg (0.30 mmol, 62%) of 4-(N-acetyl-N-
propylamino)-1,5-diacetoxy-trans-2-hexene (12). GLC: tR
)
27.6 min. CI-GLC/MS: m/z 300(MH+, 1), 240 (100), 212 (2),
199 (2), 180 (4), 138 (3), 110 (4). 1H-NMR, 13C-NMR (CDCl3):
see Table 1.