Rowan et al.
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3277
mL). The combined ethereal extracts were dried (MgSO4),
filtered, and concentrated in vacuo to give a crude product
which was subjected to short path distillation (Kugelrohr), and
the fraction distilling at 125-135 °C (760 mmHg) was collected
(140 mg, 76%). Further purification by preparative GC gave
2-methylbutan-1-ol-d3 as a colorless mobile oil: EIMS see
Table 1; 1H NMR δ 0.91 (3H, t, J ) 7.3 Hz, H-4), 1.15 (1H, m,
H-3), 1.43 (1H, m, H-3), 1.51 (1H, m, H-2), 1.71 (1H, bs, OH),
3.51 (2H, m, C-1); 13C NMR δ 11.3 (C-4), 25.7 (C-3), 37.1
(C-2), 68.0 (C-1).
interested in determining in more detail the biosynthetic
interconversions of 2-methylbutanoates. This paper
describes the use of deuterium-labeled substrates to
define the biosynthetic origins and interconversions of
2-methylbutyl and 2-methylbutanoate esters in two
apple cultivars: Red Delicious and Granny Smith.
Differences between the cultivars in the processing of
precursors and a novel biosynthetic product are de-
scribed.
Syn th esis of 2-Meth ylbu tyl-d 3 Aceta te. 2-Methylbutan-
1-ol-d3 (2 µL) was treated with acetic anhydride (5 µL) and
pyridine (5 µL) in a pear-shaped HPLC vial at room temper-
ature overnight. Dilution with ether (0.8 mL) and successive
washing with 10% HCl, saturated NaHCO3, and brine followed
by drying (MgSO4) gave an ethereal solution of the required
compound for GC-MS analysis.
Syn th esis of 2-Meth yl-2-Bu ten yl Aceta te. To a stirred
solution of trans-2-methyl-2-butenal (0.87 g, 10 mmol) in dry
ether (20 mL) was added portionwise lithium aluminum
hydride (0.20 g, 2.0 equiv). After 90 min at room temperature,
saturated NH4Cl (6 drops) was cautiously added, and the
solution was diluted with ether (20 mL) and dried (MgSO4)
and the solvent removed under reduced pressure. Kugelrohr
distillation gave 2-methyl-(2E)-butenol (0.48 g, 54%) as a clear
oil: GC-MS gave EIMS m/z 86 (M+, 41), 71 (100), 68 (12), 67
(14), 57 (11), 55 (13), 53 (26), 43 (33), 41 (44), 39 (27); 1H NMR
(CDCl3) δ 1.52 (3H, dm, J ) ∼1, 5.6 Hz, H-4), 1.59 (3H, s,
2-Me), 2.18 (1H, bs, OH), 3.91 (2H, bs, H-2), 5.47 (1H, m, H-3);
13C NMR (CDCl3) δ 12.9 (C-4), 13.1 (2-Me), 68.7 (C-1), 120.3
(C-3), 135.5 (C-2).
To 2-methyl-(2E)-butenol (0.30 g, 3.5 mmol) in pyridine (0.5
mL) and acetic anhydride (0.5 mL) was added a catalytic
quantity of p-(dimethylamino)pyridine, and the solution was
left at room temperature overnight. The reaction mixture was
diluted with ether (60 mL) and the ethereal solution washed
with 10% HCl (2 × 10 mL), saturated NaHCO3 (2 × 10 mL),
and then brine (1 × 10 mL) before drying (MgSO4) and
evaporation of the solvent under reduced pressure. Kugelrohr
distillation of the residual oil gave 2-methyl-(2E)-butenyl
acetate (0.39 g, 87%) as a clear oil: GC-MS (Carbowax, RI
19.7 min) EIMS see Table 1; 1H NMR (CDCl3) δ 1.56 (3H, dt,
J ) 1.0, ∼5 Hz, H-4), 1.59 (3H, bs, 2-Me), 1.99 (3H, s, OAc),
4.39 (2H, bs, H-1), 5.48 (1H, qm, J ) ∼1.4, 5.5 Hz, H-3). 13C
NMR (CDCl3) δ 13.0 (C-4), 13.4 (2-Me), 20.7 (OAc), 70.1 (C-1),
123.9 (C-3), 130.8 (C-2), 170.7 (CdO). The 2E stereochemistry
was confirmed by NOE.
Syn th esis of 3-Meth yl-2-bu ten yl Aceta te. By acetyla-
tion of 3-methyl-2-buten-1-ol as above: GC-MS (Carbowax,
RI 19.9 min) EIMS m/z 128 (M+, 1.6), 113 (0.4), 86 (16), 71
(22), 69 (29), 68 (81), 67 (48), 53 (25), 43 (100), 41 (54), 39 (20);
1H NMR (CDCl3) δ 1.63 (3H, bs), 1.67 (3H, d, J ) 0.8 Hz),
1.96 (3H, s, OAc), 4.49 (2H, d, J ) 6.2 Hz, H-1), 5.24 (1H, bt,
J ) 6 Hz, H-2); 13C NMR (CDCl3) δ 17.8 (3-Me), 20.7 (OAc),
25.5 (C-4), 61.2 (C-1), 118.6 (C-2), 138.7 (C-3), 170.8 (CdO).
Ga s Ch r om a togr a p h y (GC). Capillary GC analysis was
carried out on a Hewlett-Packard Model 5890 Series II gas
chromatograph equipped with a flame ionization detector (FID)
and running under the control of Hewlett-Packard ChemSta-
tion software. Separations were achieved using a 30 m × 0.25
mm i.d. Carbowax Econo-Cap capillary column (Alltech), film
thickness 0.25 µm, with a temperature program from 40 (10
min) to 100 °C at 3 °C/min and to 220 °C at 5 °C/min; injector
temperature was 220 °C and detector temperature 250 °C. The
column head pressure was 5 psi of hydrogen. Deuterated
aroma volatiles were partially or fully resolved on GC from
nonlabeled analogues and were characterized as new peaks
not present in controls or in apples treated with nonlabeled
analogues and eluting some 4-30 s before nonlabeled material.
Chemical identity was established by direct comparison with
authentic compounds, from Kovat indices and by GC-MS
identifications.
MATERIALS AND METHODS
Gen er a l. Apples (Malus domesticus Borkh. var. Red Deli-
cious) were obtained from the New Zealand Apple and Pear
Marketing Board from CA storage and maintained at 1 °C
until use. Cultivar Granny Smith apples were obtained from
a local retailer. Red Delicious and Granny Smith apples were
harvested in April and March 1994, respectively, and experi-
ments were performed in the period from November 1994 to
J anuary 1995.
Unlabeled aroma precursors and reagents were obtained
from the Aldrich Chemical Co., Inc. (Milwaukee, WI). Hexyl
2-methylbutanoate was synthesized by acid-catalyzed esteri-
fication of hexanol with 2-methylbutanoic acid. Aroma precur-
sors were of >99% purity by GC. Dry THF and ether were
prepared by distillation from sodium benzophenone blue.
L-Isoleucine-d10, uniformly labeled to 98% isotopic purity, was
obtained from Cambridge Isotope Laboratories, Inc., Woburn,
MA. NMR spectra were recorded on a Bruker AC300 (300
MHz) spectrometer in CDCl3 and referenced to δ 7.24 for 1H
and δ 77.0 for 13C NMR spectra. 13C multiplicities were
determined using the DEPT-135 pulse sequence. Traps of
Tenax TA (Alltech) were conditioned at 250 °C for 4 h under
a stream of oxygen-free nitrogen (30-50 mL/min) before use.
Syn th esis of Eth yl 2-Meth ylbu ta n oa te-d 3 (EMB-d 3).
n-Butyllithium (5.96 mL, 1.93 M in pentane) was added to a
stirred solution of diisopropylamine (1.44 mL, 11.0 mmol) in
dry THF (50 mL) at -78 °C (acetone/dry ice). The solution
was stirred for 5 min, and ethyl butyrate (1.32 mL, 10.0 mmol)
was added dropwise. After stirring for 30 min, iodomethane-
d3 (0.75 mL, 12.0 mmol) was added as a single aliquot, and
the flask was allowed to warm to room temperature over 30
min. The flask contents were poured into a separating funnel
containing 1.0 M HCL (50 mL) and ether (50 mL), and the
aqueous layer was re-extracted with ether (50 mL). The
combined organic phases were washed with water (50 mL) and
then saturated brine (50 mL), dried (MgSO4), filtered, and
concentrated in vacuo on a rotary evaporator with a water bath
of 10 °C, to give a yellow oil (1.50 g). Short path distillation
(Kugelrohr) of the fraction distilling at 130-140 °C (760
mmHg) gave the crude product (0.94 g, 70%). Preparative GC
gave the title compound as a colorless oil: EIMS see Table 1;
1H NMR δ 0.90 (3H, t, J ) 7.5 Hz, H-4), 1.25 (3H, t, J )
7.1 Hz, CH3CH2O), 1.43 (1H, m, H-3), 1.67 (1H, m, H-3), 2.33
(1H, t, J ) 6.8 Hz, H-2), 4.13 (2H, q, J ) 7.1 Hz, CH3CH2O);
13C NMR δ 11.6 (C-4), 14.3 (C-2′), 26.7 (C-3), 40.9 (C-2), 60.1
(C-1′), 176.8 (C-1).
Syn th esis of 2-Meth ylbu ta n oic-d 3 Acid . Ethyl 2-meth-
ylbutanoate-d3 (96 mg, 0.72 mmol) was stirred at room
temperature for 48 h in a 10% aqueous NaOH solution (5.0
mL). The reaction mixture was extracted with ether (2 × 5
mL) and acidified with 1 N HCl (8 mL) and the aqueous phase
re-extracted with dichloromethane (3 × 10 mL). The combined
dichloromethane extracts were dried (MgSO4), filtered, and
concentrated in vacuo to give the title compound as a clear
pungent oil (75.5 mg, quantitative): 1H NMR δ 0.95 (3H, t, J
) 7.4 Hz, H-4), 1.51 (1H, m, H-3), 1.71 (1H, m, H-3), 2.39 (1H,
t, J ) 6.8 Hz, H-2), 9.10 (1H, bs, COOH); 13C NMR δ 11.5
(C-4), 26.5 (C-3), 40.7 (C-2), 183.2 (C-1).
Syn th esis of 2-Meth ylbu ta n ol-d 3. Lithium aluminum
hydride (153 mg, 4.02 mmol) was added in one portion to a
stirred solution of 2-methylbutanoic-d3 acid (0.212 g, 2.01
mmol) in dry ether (15 mL) at room temperature. After 4 h,
the reaction was quenched with saturated aqueous NH4Cl
solution (2 mL) and the mixture extracted with ether (2 × 20
Preparative GC was carried out using a modified HP 5840A
gas chromatograph fitted with a 2 m × 5 mm i.d. stainless
steel column packed with 10% OV-101 on GasChromQ run
isothermally at 150 °C with N2 carrier gas at 100 mL/min.