9366 J . Org. Chem., Vol. 61, No. 26, 1996
Fronza et al.
deuterium atmosphere in the presence of 0.9 g of PtO2 at
normal pressure. At the end of the absorption, the filtered
reaction mixture was evaporated and chromatographed on
SiO2 with hexane/AcOEt (7:3) to provide the acetate ester of
4-butylphenol, 4.64 g (60%) [1H NMR (400 MHz, CDCl3) δ 0.92
(3H, s), 1.34 (0.5H, m), 1.58 (1.3H, m), 2.29 (3H, s), 2.60 (2H,
m), 6.99 (2H, m), 7.18 (2H, m); 2H NMR (61.4 MHz, CHCl3) δ
1.33 (1.5 D), 1.56 (0.7 D)] and subsequently, product 6, 4 g
(40%), oil: 1H NMR (400 MHz, C6D6) δ 1.03 (3H, s), 1.65 (1H,
m broad), 1.71 (3H, s), 1.77 (3H, s), 2.34 (1H, dd, J ) 14.0,
9.9), 2.43 (1H, dd, J ) 14.0, 6.6), 6.91 (2H, m), 7.00 (2H, m);
2H NMR (61.4 MHz, C6H6) δ main signals 1.43 (0.9 D), 4.90
(1D); minor signals 1.00 (0.1 d), 1.64 (0.1 D), 2.31 (0.13 D),
of the sequence, at the level of the C-6-C-5 intermediate
26, provides upon hydrolysis and loss of a C-1 unit as
carbon dioxide, the C-6-C-4 unsaturated ketone 3, from
which raspberry ketone (1) is produced. In B. bassiana
Baeyer-Villiger chain shortening of 1 provides C-6-C-2
tyrosol (4), the ubiquitous primary metabolite of shiki-
mate-derived tyrosine, formed from the latter by oxida-
tive decarboxylation to 28 and reduction. This metabolic
operation brings back a product of the shikimate primary
metabolism, i.e., 4, a secondary metabolite of mixed
biosynthetic origin, i.e. 1. Very little is known on the
mechanism that regulates in nature the generation of
flavor materials, apart from the consideration that these
products are formed at the moment of ripening, when
catabolic processes prevail and nothing is known on the
biodegradation of plant-specific odorants. Raspberry
ketone (1), possessing a very low odor threshold and
present in raspberry fruit at ppm level, likely plays some
role in the ecosystem. It is formed through a plant-
specific deviation from the ubiquitous flavonoid-produc-
ing system. However, the present observations indicate
the presence in nature of an enzymic capacity designed
to the biodegradation of 1 to the primary metabolite
tyrosol 4. The enzymic shunt between secondary me-
tabolism (raspberry ketone) and primary metabolism
(tyrosol) relys on a rather sophisticated reaction such as
a Baeyer-Villiger oxidation. This capacity has now been
demonstrated to be present in B. bassiana, but future
experiments might possibly demonstrate that the flavor-
control mechanism in raspberry fruit consists of a
Baeyer-Villiger degradation of highly odorant raspberry
ketone (1) to tyrosol (4), devoid of sensory properties.
Experiments in raspberry plants designed to identify this
enzymic activity are in progress.
2.39 (0.1 D) (see also Figure 1). Anal. Calcd for C14H18O4
C, 67.18; H, 7.25. Found: C, 67.25; H, 7.21.
:
(2R,3S)-[2,3-2H2] Dia ceta te 10. The racemic diacetate 6
(4 g, 15.9 mmol) was refluxed with 26 mL of a solution of 10%
aqueous NaOH-EtOH (1:1) for 2 h. Once the hydrolysis was
complete (TLC), the reaction mixture was concentrated to a
small volume, diluted with water, acidified with dilute HCl,
and extracted with Et2O (3 × 70 mL) to provide 7: 1H NMR
(400 MHz, CDCl3) δ 1.21 (3H, s), 1.79 (1H, s broad), 1.74 (1.3H,
m), 2.59 (1H, dd, J ) 9.3, 14.0), 2.66 (1H, dd, J ) 6.3, 14.0),
3.81 (0.3 H, m), 5.28 (1H, s broad), 6.74 (2H, m), 7.05 (2H, m);
2H NMR (61.4 MHz, CHCl3) δ 1.71 (1D), 3.81 (1D), deuteration
extent ca. 70%. The dried organic phase was evaporated, and
the residue, diluted with CH2Cl2 (100 mL), was treated with
Ac2O (1.6 mL, 16.95 mmol) and pyridine (1.36 mL). After 12
h the reaction mixture was washed with dilute HCl, 3%
NaHCO3, and water. The residue obtained upon evaporation
of the organic phase was chromatographed with hexane/AcOEt
(8:2) to provide 8, 2.5 g (75%), oil. The latter product (2.5 g,
11.9 mmol) in (CH3)3COCH3 (300 mL) was mechanically stirred
at rt for 50 h with vinyl acetate (48 mL, 519.64 mmol) and
lipase PS Amano (5 g). The course of the reaction was followed
by HPLC analysis. Merck-Hitachi L-6200 apparatus equipped
with UV detector L-4200 with D-2500 integrator. Chiral
stationary phase: Chiracel OD, Daicel, J apan. The elution
conditions were the following: n-hexane/i-PrOH 9/1; flow 0.6
mL/min, the detector was set at 220 nm. The retention times
were 9.9 and 11.2 min for the (R) and (S) enantiomers,
respectively. The filtered reaction mixture was evaporated and
Exp er im en ta l Section
The proton NMR spectra were recorded at 400 MHz at room
temperature, and the deuterium spectra were run at 61.6 MHz
in the gated proton broadband decoupling mode to eliminate
the proton-deuterium coupling constants that cause broaden-
ing of the signals. J values are given in Hz. TLC analyses
were performed on Merck Kieselgel 60 F254 plates. All
chromatographic separations were carried out on silica gel
columns. Due to the variable content of deuterium in the
labeled compounds the elemental analyses supporting the
purity and authenticity of the products were performed on the
corresponding nondeuterated species obtained in the explor-
atory experiments.
chromatographed to afford 10, 1.4 g (46.7%): [R ]21 ) +11.5°
D
(c 0.5, CHCl3); 0.98 ee upon HPLC analysis; 1H NMR (400
MHz, C6D6) δ 1.03 (3H.s), 1.66 (1H, m broad), 1.71 (3H, s),
1.77 (3H, s), 2.34 (1H, dd, J ) 14.0, 9.9), 2.43 (1H, dd, J )
14.0, 6.6), 6.90 (2H, m), 7.00 (2H, m); 2H NMR (61.4 MHz,
C6H6) δ main signals 1.42 (0.85 D), 4.90 (1D); minor signals
0.99 (0.1 d), 1.65 (0.1 D), 2.31 (0.14 D), 2.39 (0.1 D). Subse-
quently, the (2S) product 9 was eluted, 1.1 g (44%). Anal.
Calcd for C14H18O4: C, 67.18; H, 7.25. Found: C, 67.24; H,
7.23.
(Z)-[1,3-2H4]En ol Aceta te 11. Product 1 (8.2 g, 0.05 mol)
in 1,4-dioxane (20 mL) was treated with D2O (30 mL) contain-
ing KOH (2.8 g) under stirring for 48 h. The reaction mixture
was poured into an excess of HCl in crushed ice and Et2O (200
mL). The residue obtained by evaporation of the solvent was
crystallized from cyclohexane to provide [1,3-2H5]-1, mp 81 °C,
8.45 g (100%): 1H NMR (400 MHz, CDCl3) δ 1.80 (2H, s broad),
(Z)-En ol Aceta te (5). A solution of 4-(4-hydroxyphenyl)-
butan-2-one (1) (25 g, 152 mmol) in CCl4 (475 mL) was treated
with Ac2O (288 mL), containing 0.75 mL of 70% perchloric acid.
The reaction mixture was kept overnight at rt, concentrated
to remove the majority of Ac2O, diluted with CH2Cl2 , and
washed several times with 3% NaHCO3. The dried organic
phase was chromatographed on SiO2 with hexane/AcOEt (8:
2) to provide the (Z)-enol acetate 5, 10 g (40%), oil: 1H NMR
(400 MHz, CDCl3) δ 1.93 (3H, q, J ) 1.1), 2.17 (3H, s), 2.25
(3H, s), 3.26 (2H, d, J ) 7.5), 5.17 (1H, tq, J ) 7.5, 1.1), 6.97
2
6.40 (1H, s broad), 5.75 (2H, m), 7.02 (2H, m); H NMR (61.4
MHz, CHCl3) δ 2.11 (3D), 2.70 (2D), deuteration extent ca.
90%. The latter material was converted into the (Z)-enolace-
tate (11) as reported for 5 in 30% yield. (Z)-isomer: 1H NMR
(400 MHz, CDCl3) δ 2.17 (3H, s), 2.28 (3H, s), 3.26 (2H, s
13
(2H, m), 7.18 (2H, m); C NMR (100.6 MHz, CDCl3) δ 19.7
2
broad), 5.16 (0.3H, t, J ) 7.4), 6.98 (2H, m), 7.18 (2H, m); H
(CH3), 20.8 (CH3), 21.2 (CH3), 31.3 (CH2), 115.5 (CH), 121.6
(2CH), 129.4 (2CH), 137.8 (C), 146.0 (C), 149.2 (C), 169.1 (C),
169.6 (C). Sample 5 contains about 10% of the (E)-isomer: 1H
NMR (400 MHz, CDCl3) δ 1.94 (3H, q, J ) 1.0), 2.10 (3H, s),
2.26 (3H, s), 3.37 (2H, d, J ) 7.7), 5.31 (1H, tq, J ) 7.7, 1.0),
NMR (61.4 MHz, CHCl3) δ 1.91 (3D), 5.19 (1D), deuteration
extent ca. 70%. (E)-Isomer (ca. 10% in mixture with the (Z)-
isomer): 1H NMR (400 MHz, CDCl3) δ 2.12 (3H, s), 2.28 (3H,
sr, 3.37 (2H, s broad), 5.30 (ca. 0.3H, t, J ) 7.8), 7.0 (2H, m),
7.22 (2H, m). 2H NMR (61.4 MHz, CHCl3) δ 1.91 (3D), 5.33
(2D), deuteration extent ca. 70%.
13
7.00 (2H, m), 7.21 (2H, m); C NMR (100.6 MHz, CDCl3) δ
15.4 (CH3), 21.1 (CH3), 21.2 (CH3), 32.2 (CH2), 116.0 (CH),
121.7 (2CH), 129.3 (2CH), 137.6 (C), 146.8 (C), 149.3 (C), 169.1
(C), 169.6 (C). Anal. Calcd for C14H16O4 : C, 67.73; H, 6.50.
Found: C, 67.64; H, 6.48.
(2SR,3SR)-[1,3-2H4]-Dia ceta te 12. The conversion of 11
into 12 was performed exactly as reported above for 5, but
using hydrogen gas instead of deuterium. Product 12: 1H
NMR (400 MHz, C6D6) δ 1.45 (1H, m broad), 1.71 (3H, s), 1.77
(3H, s), 2.34 (1H, dd, J ) 14.0, 6.6), 2.43 (1H, dd, J ) 14.0,
(2SR,3RS)-[2,3-2H2]Dia ceta te (6). The (Z)-enol acetate 5
(10 g, 40.3 mmol) in AcOEt (100 mL) was stirred at rt in