Raspberry Ketone Extractive from Taxus baccata
J. Agric. Food Chem., Vol. 47, No. 3, 1999 1151
65% excess of the S enantiomer. Analysis conditions: Merck-
Hitachi L-6000 pump with L-4000 UV detector (254 nm), 9:1
hexane--propanol at 0.6 mL/min; RT, R enantiomer ) 29 min
and S enantiomer ) 31 min.
(B) With CrO3 in Sulfuric Acid/ Acetone. Carbinol 2 (1.66 g,
10 mmol) in acetone (30 mL) was treated portionwise, at 5-10
°C and under stirring, with an aqueous solution, containing,
in 100 mL, CrO3 (26.72 g) and concentrated sulfuric acid (23
mL), until the color no longer turned to green. After the
addition of a few drops of methanol the reaction mixture was
partially evaporated under vacuum at low temperature and
diluted with ice water. The mixture was extracted with ethyl
acetate (2 × 100 mL), and the organic phase was washed with
3% NaHCO3. Evaporation of the dried solution and column
chromatography on SiO2 of the residue provided raspberry
ketone 1 (1.1 g, 67%), mp 80 °C (from cyclohexane).
F igu r e 1. Numbering of the positions of the raspberry ketone
1 and of the carbinol 2 used in the spectra and in the tables.
Con ver sion of Ra sp ber r y Keton e 1 in to Ben zoic Acid
(6). Raspberry ketone 1 (1.64 g, 10 mmol) in acetone (60 mL)
was treated at reflux under stirring with 1-chloro-5-phenyltet-
razole (1.85 g, 11 mmol) in the presence of 4 g of finely
powdered potassium carbonate for 8 h (Musliner and Gates,
1971). The reaction mixture was filtered and evaporated to
dryness. The crude residue was partitioned between water (100
mL) and ethyl acetate (2 × 200 mL). The residue obtained upon
evaporation of the dried organic phase was purified by SiO2
column chromatography with increasing amounts of ethyl
acetate in hexane. The 4′-(5-phenyltetrazolyl) derivative 4 was
isolated as amorphous solid (2.6 g, 80%): 1H NMR δ 2.15 (3H,
s), 2.78 (2H, m), 2.93 (2H, m), 7.29 (5H, m), 7.57 (2H, m), 7.80
(2H, m); EI-MS m/z 309 (M+ + 1, 25), 265 (M+ - MeCO, 2),
223 (11), 180 (10), 117 (64), 91 (82), 77 (100), 65 (60); FT-IR
(Nujol) ν (cm-1) 1500, 1543, 1595, 1709, 3448. Anal. Calcd for
F igu r e 2. Natural abundance 2H spectrum in methanol of
extractive and synthetic (window) carbinol 2: (a) signal of
water and methanol hydroxyl groups; (b) signal of methanol
methyl group; (c) signal of tert-butyl sulfide used as internal
(D/H) standard. The peaks are numbered according to Table
1 and Figure 1.
C
17H16N4O2: C, 66.22; H, 5.19; N, 18.16. Found: C, 66.34; H,
5.17; N, 18.21.
present results with those previously acquired (Fronza
et al., 1998) allowed a precise distinction of the origin,
i.e., natural extractive, natural biogenerated from 3, and
synthetic, of different raspberry ketone samples, as
indicated in Figure 3.
The latter product 4 (2.5 g, 8.4 mmol) in ethyl acetate (50
mL) was hydrogenated at normal pressure and room temper-
ature in the presence of 10% Pd/C (0.5 g). At the end of the
adsorption, the filtered reaction mixture was evaporated and
4-phenylbutan-2-one 5 (1 g, 80%) was obtained after separation
from 5-phenyltetrazolone by SiO2 chromatography: 1H NMR
δ 2.13 (3H, s), 2.76 (2H, m), 2.90 (2H, m), 7.25 (5H, m); EI-MS
m/z 149 (M+ + 1, 28), 148 (M+, 100), 133 (M+ - Me, 18), 105
(M+ - MeCO, 58), 91 (42); FT-IR (neat) ν (cm-1) 1718. Anal.
Calcd for C10H12O: C, 81.04; H, 8.10. Found: C, 81.11; H, 8.08.
Ketone 5 (1 g, 6.7 mmol) in 50 mL of tap water, containing
5 g of K2CO3, was treated at 60-70 °C with a 5% KMnO4
solution portionwise, until the reaction mixture was no longer
discolored. After cooling, the reaction mixture was filtered and
acidified with 5% hydrochloric acid and extracted twice with
ethyl acetate. The dried organic phase was concentrated under
vacuum, to give the crude benzoic acid 6. The latter was
crystallized twice from water, mp 122-123 °C, undepressed
with an authetic sample (0.3 g, 36%).
Con ver sion of 2 in to 4-P h en ylbu ta n -2-ol (8). The se-
quence leading from 1 to 5 reported above was repeated on 2
to provide, in an analogous manner, carbinol 8, oil, purified
by bulb-to-bulb vacuum distillation: 1H NMR (250 MHz,
CDCl3) δ 1.21 (3H, d, J ) 6.5 Hz), 1.70-1.83 (2H, m), 2.57-
2.88 (2H, m), 3.82 (1H, m), 4.83 (1H, s, OH), 7.15-7.33
(5H, m).
Con ver sion of Ra sp ber r y Keton e 1 in to 4-Bu tylp h en ol
(10). Raspberry ketone 1 (1.64 g, 10 mmol) in methanol (50
mL) was treated at reflux for 4 h with toluene-4-sulfonic acid
hydrazide (2 g, 11 mmol). NaBH4 (0.76 g, 20 mmol) was added
under stirring to the cooled reaction mixture. The reaction
mixture was boiled for 4 h and then concentrated to a small
volume under vacuum. The residue was partitioned between
ice water and ethyl acetate (300 mL). The residue obtained
upon evaporation of the organic phase was chromatographed
on SiO2 to provide, from increasing amounts of ethyl acetate
in hexane, 4-butylphenol (10), purified by bulb-to-bulb vacuum
distillation, 0.75 g (50%): 1H NMR (250 MHz, CDCl3), δ 0.91
(3H, t, J ) 7.4 Hz), 1.33 (2H, m), 1.55 (2H, m), 2.53 (2H, t, J
) 7.7 Hz), 4.75 (1H, s br), 6.74(2H, d, J ) 8.5 Hz), 7.03 (2H, d,
J ) 8.5 Hz).
EXPERIMENTAL PROCEDURES
Isola tion of 1 a n d 2 fr om Com m er cia l T. ba cca ta Ex-
tr a ct fr om th e Hym a la ya n Region of In d ia . SiO2 column
chromatography of the extract with increasing amounts of
ethyl acetate in hexane provided different fractions enriched
in 1 and 2, respectively. The operation was repeated several
times to eventually provide raspberry ketone 1, mp 80 °C (from
cyclohexane), undepressed with an authetic sample from
Aldrich, and carbinol 2, thick oil which solidified on standing:
[R]D20 -13.6° (c 1, EtOH); 1H NMR (CH3OH, the same solution
used for the deuterium measurements) δ 1.10 (3H, d, J ) 6.5
Hz), 1.61 (2H, m), 2.49 (2H, m), 3.66 (1H, qt, J ) 6.5 Hz), 6.65
(2H, d, J ) 8.6 Hz), 6.93 (2H,d, J ) 8.6 Hz), 8.91 (1H, s br).
Oxid a tion of 2 to 1 by Biologica l a n d Ch em ica l Mea n s.
(A) With Candida boidinii Cultures. Candida boidinii CBS
2428, grown routinely on MA slants (Malt Extract Broth Oxoid
2%, agar 1.5%), was seeded in a 300 mL Erlenmeyer flask
containg 50 mL of MPGB medium (malt 2%, peptone 0.5%,
glucose 1%, pH ) 6.5) and grown in a rotatory incubator (140
rpm) at 28 °C for 24 h. The 24 h old culture was inoculated
(10%) into a 7.5 L jar fermentor (Chemap), containing 5 L of
MPGB medium at pH ) 6.5. The temperature was maintained
at 28 °C, and the stirring, at 500 rpm. At 12 and 22 h,
respectively, was added 0.1% methanol, and at 24 h, after
substitution of the air flow for oxygen, the carbinol 2, 5 g. The
incubation was continued for 24 h. The transformation mixture
was treated with ethyl acetate, 1 L, and filtered under vacuum
on a Celite pad. The organic phase was separated, and the
aqueous phase was extracted with ethyl acetate (2 × 0.7L).
The residue obtained upon evaporation of the dried combined
organic extract was submitted to SiO2 (200 g) chromatography
to provide raspberry ketone 1, mp 80 °C (from cyclohexane),
2.8 g (55%), and unreacted carbinol 2, thick oil, 1.5 g (30%),
shown to contain, at the HPLC analysis on Chiracel OD, ca.