Synthesis of Isotopically Labeled Flavor Compounds
Geneva, Switzerland) equipped with a splitless injector and
a flame ionization detector (FID). J &W fused silica capillary
columns were employed: DB-5, DB-1701, DB-FFAP, and DB-
Wax (30 m × 0.32 mm, film thickness ) 0.25 µm) using
different oven temperature programs (Lin et al., 1999). The
carrier gas was helium (100 kPa). Linear retention indices (RI)
were calculated according to the method of van den Dool and
Kratz (1963).
GC/ MS. This was performed on a Finnigan MAT 8430 mass
spectrometer (Bremen, Germany). Electron impact (EI) mass
spectra were generated at 70 eV and positive chemical ioniza-
tion (PCI) at 150 eV with ammonia as the reagent gas. Volatile
components were sampled via a Hewlett-Packard HP-5890 gas
chromatograph (Geneva, Switzerland) equipped with a cold
on-column injector. DB-1701 or DB-Wax fused silica capillary
columns were used (Lin et al., 1999). The carrier gas was
J. Agric. Food Chem., Vol. 47, No. 7, 1999 2823
(
filtrate was washed with cold HCl (1 N) until pH 2-3 and then
with saturated NaCl until neutral. The solvent was removed
by distillation on a Vigreux column (50 × 1 cm). The residue
was further distilled under vacuum (30-31 °C, 21 mbar),
obtaining 1.84 g (18.4 mmol, 45% yield) of d -V with a purity
of 97% (GC): GC RI(DB-5) ) 798, RI(DB-1701) ) 885, RI-
+
(FFAP) ) 1129, RI(DB-Wax) ) 1138; MS-EI 100 (34, M ), 99
(7), 86 (2), 85 (30), 84 (5), 83 (4), 82 (19), 81 (13), 80 (4), 72
(11), 71 (75), 70 (21), 69 (8), 68 (5), 58 (8), 57 (45), 56 (18), 55
(11), 54 (8), 53 (5), 52 (4), 44 (9), 43 (78), 42 (100), 41 (31), 40
+
(25), 39 (12); MS-CI (NH
3
) 118 (100, [M + NH
4
] ), 100 (19,
+
M ).
2
[5,6- H
2
]-(Z)-1,5-Octadien-3-ol (d -VI). d -V (1.8 g, 18 mmol)
in dry THF (30 mL) was added dropwise to a stirred solution
of BrMgC (1 M, 2 0 mL) cooled at 10 °C in an ice bath. The
mixture was stirred overnight and then hydrolyzed using a
saturated solution of NH Cl. The sample was extracted with
Et
2
H
3
helium (90 kPa). Relative abundances of the ions are given in
4
+
percent. Selected ion monitoring (SIM) of [M + NH
3
] ions was
2
O (2 × 50 mL). The solvent was removed, and the residue
used for determining the distribution of doubly and triply
deuterated 1-octen-3-ol.
was distilled under vacuum (105 °C, 13-14 mbar, B u¨ chi GKR-
50), resulting in 1.2 g (9.4 mmol, 51% yield) of d -VI with a
purity of 93% (GC): GC RI(DB-5) ) 972, RI(DB-1701) ) 1075,
RI(FFAP) ) 1483, RI(DB-Wax) ) 1486; MS-EI 110 (2), 72 (65),
For the determination of isotopic purity, GC/MS was
performed on a Finnigan SSQ 7000 using PCI at 200 eV and
isobutane as reagent gas (Lin et al., 1999). The samples were
introduced by splitless injection (1 µL) using the DB-1701
capillary column described above. Helium was used as carrier
gas (70 kPa).
71 (23), 70 (8), 58 (8), 57 (100), 56 (7), 55 (6), 43 (12), 42 (11),
+
41 (5); MS-CI (NH
3
) 164 (1, [M + NH
3
+ NH
4
] ), 146 (7, [M +
+
+
+
NH
[5,6- H
4
] ), 128 (100, M or [M + NH
4
- H
2
O] ).
2
2
]-(Z)-1,5-Octadien-3-one (d -I). Octadienol d -VI (0.9
Nuclear Magnetic Resonance (NMR) Spectroscopy. Samples
were analyzed in WILMAD 528-PP 5 mm Pyrex NMR tubes
using deuterated chloroform as solvent. If not otherwise
g, 7.0 mmol) was oxidized with PCC to the corresponding
ketone using the procedure described for the oxidation of [5,6-
2
2
H ]hexan-1-ol (Lin et al., 1999). About 270 mg (2.1 mmol, 30%
specified, the NMR spectra were acquired on a Bruker AM-
yield) of d -I was obtained with a purity of 99% (GC). The
1
2
3
60 spectrometer at 360.13 MHz for H and at 90.56 MHz for
C under standard conditions (Lin et al., 1999). All shifts are
isotopic purity of d -I was 98%, with 0.4% of [ H]-I and 1.6%
1
3
2
of [ H
3
]-I: GC RI(DB-5) ) 977, RI(DB-1701) ) 1080, RI(FFAP)
cited in parts per million from the internal tetramethylsilane
TMS) standard. Proton NOE difference spectra (d -I), selective
) 1364, RI(DB-Wax) ) 1371; MS-CI (NH
3
) 144 (100, [M +
+
+
(
NH ] ), 126 (20, M ).
4
homonuclear decoupled spectra (IV and VI), DEPT spectra (d -I
and d -II), and two-dimensional COSY (VI) and HETCOR (d -I
and VI) were acquired as described earlier (Lin et al., 1999)
to make unequivocal assignments (detailed experimental
conditions can be obtained upon request). The SPECINFO on-
line program SPECAL (Lin et al., 1999) was applied to some
of the C NMR spectra for the assignment of equivocal signals.
Chemical and Isotopic Purity. This was determined on the
basis of GC-FID, GC/MS (PCI), and NMR data (Rohwedder,
Syn t h esis of (Z)-1,5-Oct a d ien -3-on e (I). (Z)-3-Hexenal
(V). In analogy to the synthesis of d -V, 3.9 g (38 mmol, 32%
yield) of V was prepared with a purity of 85% (GC) using
commercially available IV (12.0 g, 120 mmol) as starting
material: GC RI(DB-5) ) 798, RI(DB-1701) ) 891, RI(FFAP)
+
) 1141, RI(DB-Wax) ) 1138; MS-EI 98 (20, M ), 97 (7), 84
1
3
(18), 80 (20), 70 (11), 69 (48), 55 (28), 53 (18), 43 (39), 42 (100),
+
+
40 (40); MS-CI (NH
3
) 116 (100, [M + NH ] ), 98 (18, M ).
4
(Z)-1,5-Octadien-3-ol (VI). In analogy to the synthesis of
d -VI, 2.1 g (16.7 mmol, 60% yield) of VI was prepared with a
purity of 97% (GC) using V as starting material (2.9 g, 28
mmol): GC RI(DB-5) ) 977, RI(DB-1701) ) 1077, RI(FFAP)
) 1486, RI(DB-Wax) ) 1488; MS-EI 108 (2), 70 (70), 69 (27),
1
985; Lin et al., 1999). The nondeuterated substances were
analyzed by GC/MS (PCI) for isotope correction of the labeled
compounds.
2
Syn th esis of [5,6- H
2
]-(Z)-1,5-Octa d ien -3-on e (d -I). [3,4-
2H
]-(Z)-3-Hexen-1-ol (d -IV). A solution of 3-hexyn-1-ol (III, 4.0
2
3
57 (100), 55 (45), 42 (17), 41 (43), 39 (15); MS-CI (NH ) 162 (1,
2
+
+
+
g, 41 mmol) in MeO H (100 mL) was deuterated with Lindlar’s
catalyst (200 mg) under normal pressure at room temperature.
Samples were taken and analyzed by GC periodically to
monitor the process of deuteration that was complete after 3.5
h. The reaction was stopped, and the catalyst was removed
by filtration. After removal of MeOH, the residue was distilled
under vacuum (51-52 °C, 12 mbar), giving rise to 3.7 g (36
mmol, 88% yield) of a colorless oil of d -IV with a purity of 98%
[M + NH
3
+ NH
2
4
] ), 144 (7, [M + NH ] ), 126 (100, M or [M
4
+
+ NH - H O] ).
4
(Z)-1,5-Octadien-3-one (I). The octadienol VI (2.0 g, 16 mmol)
was oxidized to I in the same way as described for the oxidation
2
of [5,6- H
2
]hexan-1-ol (Lin et al., 1999). About 370 mg (3.0
mmol, 19% yield) I was obtained with a purity of 95% (GC):
GC RI(DB-5) ) 979, RI(DB-1701) ) 1085, RI(FFAP) ) 1365,
+
RI(DB-Wax) ) 1371; MS-CI (NH
3
) 142 (100, [M + NH
4
] ), 124
1
2
+
(
1
2
GC): H NMR (400 MHz, C HCl
3
) δ 3.62 (t, J ) 6.8 Hz, 2H,
), 2.30 (t, J ) 6.8 Hz, 2H, 2-CH ), 2.07 (q, J ) 7.7 Hz,
H, 5-CH ), 0.98 (t, J ) 7.7 Hz, 3H, 6-CH
(28, M ).
2
2
-CH
2
2
Syn th esis of [1- H1;2,2- H1;1]-1-Octen -3-on e (d -II). [1-
1
3
2H1;2,2- H1;1]-1-Octen-3-ol (d -VIII). In a 200 mL three-neck
2
2
3
); C NMR (100
2
2
2
MHz, C HCl
3
) δ 134.8 (s 1:1:1, J C H ) 23 Hz, 3-C H), 124.4 (s
reactor fitted with a reflux condenser and a thermometer was
:1:1, J C2H ) 24 Hz, 4-C H), 62.0 (t, 1-CH
0.4 (t, 5-CH ), 14.0 (q, 6-CH ). These spectra, compared to
2
), 30.4 (t, 2-CH
),
2
1
2
2
2
suspended 2.0 g (48 mmol) of LiAl H
4
in anhydrous THF.
2
3
Compound VII (5.0 g, 40 mmol) dissolved in anhydrous THF
(10 mL) was slowly added to the magnetically stirred solution.
The mixture was refluxed under nitrogen. GC analysis indi-
cated complete reduction of VII to d -VIII after 18 h. On cooling
in an ice bath, 20 mL of heavy water was added drop by drop,
followed by 80 mL of aqueous H SO (4 N) to dissolve any
2 4
insoluble residues. The organic phase was separated from the
water phase, and the aqueous solution was then extracted with
those acquired with a 360 MHz instrument of commercial
nondeuterated IV (data not shown), unambiguously proved the
above molecular structure and deuteration pattern.
2
[
3,4- H
2
]-(Z)-3-Hexenal (d -V). Collin’s reagent was prepared
(25.2 g, 0.25 mol) to a stirred solution composed
of anhydrous pyridine (40 g, 0.5 mol) and CH Cl (600 mL).
by adding CrO
3
2
2
The mixture was stirred for 15 min at a temperature not
exceeding 25 °C. A solution of d -IV (4.2 g, 41 mmol) in
Et
washed successively with saturated solutions of NaHCO
× 10 mL) and NaCl (2 × 10 mL) and then dried over
anhydrous Na SO . After removal of the solvent by evaporation
2
O (3 × 50 mL). The combined organic solutions were
anhydrous CH
2
Cl
2
(10 mL) was added in one portion. After
3
(2
an additional 15 min of stirring at room temperature, the
solution was decanted from the residue and the latter was
2
4
washed with dry Et
layers were filtered through a short pad of Florisil, and the
2
O (2 × 100 mL). The combined organic
and distillation under vacuum (59-61 °C, 10 mbar), 4.8 g (37
mmol, 92% yield) of a colorless oil of d -VIII with a purity of