Stereochemical aspects of the bioreduction of the conjugated double bond of perillaldehyde

Article abstract of DOI:10.1016/j.tetasy.2004.08.025

A study on the regioselective reduction of the conjugate double bond of perillaldehyde is described. The chemical reduction of this substrate was investigated in order to provide a straightforward access to the relevant natural flavour, dihydroperillaldehyde. The biological reduction of both natural (S)-(-)-perillaldehyde and synthetic (R)-(+)-perillaldehyde was accomplished by means of fermenting baker's yeast. The latter microorganism converted, with different diastereoselectivity, the (S)- and (R)-enantiomers into the corresponding trans and cis saturated alcohols, respectively. The origin of the hydrogen atoms added to the double bond was studied by deuterium labelling experiments and ²H NMR measurements that clearly demonstrate a different mechanism of the biohydrogenation of the two enantiomeric forms of perillaldehyde.

Full text of DOI:10.1016/j.tetasy.2004.08.025

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3073–3077
Stereochemical aspects of the bioreduction
of the conjugated double bond of perillaldehyde
Giovanni Fronza, Claudio Fuganti, Matteo Pinciroli and Stefano Serra*
CNR, Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘Adolfo Quilico’ presso Dipartimenti di Chimica,
Materiali ed Ingegneria Chimica ‘Giulio Natta’ del Politecnico, Via Mancinelli 7, I-20133 Milano, Italy
Received 16 June 2004; accepted 23 August 2004
Abstract—A study on the regioselective reduction of the conjugate double bond of perillaldehyde is described. The chemical reduc-
tion of this substrate was investigated in order to provide a straightforward access to the relevant natural flavour, dihydroperill-
aldehyde. The biological reduction of both natural (S)-(ꢀ)-perillaldehyde and synthetic (R)-(+)-perillaldehyde was accomplished
by means of fermenting bakerÕs yeast. The latter microorganism converted, with different diastereoselectivity, the (S)- and (R)-
enantiomers into the corresponding trans and cis saturated alcohols, respectively. The origin of the hydrogen atoms added to the
double bond was studied by deuterium labelling experiments and 2H NMR measurements that clearly demonstrate a different mech-
anism of the biohydrogenation of the two enantiomeric forms of perillaldehyde.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
isms and the stereochemical outcome of the reduction
were investigated extensively.
The sensorial properties of many a,b-unsaturated natu-
ral flavours are frequently very different in comparison
with those showed by their corresponding saturated
analogues. Although the hydrogenation process is often
straightforward, recent legislation1 has meant that fla-
vour substances labelled ÔnaturalÕ may only be prepared
either by physical processes (extraction from natural
sources) or by enzymatic/microbial processes, which in-
volve precursors isolated from nature. Since consumers
prefer natural food components, a number of studies
devoted to their biogeneration from easily available
extractive materials have been developed in the past
years. These latter preparations are especially relevant
when the flavour substances are present in nature in
trace amounts and are not accessible by extraction.
O
O
O
O
O
O
1
2
3
4
HO
HO
O
O
5
6
Scheme 1.
In this context, we have studied different methods of
biohydrogenation of the activated double bonds of some
natural products. Several relevant examples are the cou-
ples (R)-2-decen-5-olide 1 (Massoia lactone)/d-decan-
olide 22 and 4-(4-hydroxyphenyl)-2-but-3-en-2-one
3/4-(4-hydroxyphenyl)-2-butanone (raspberry ketone) 43
(Scheme 1) where both the use of different microorgan-
Recently, 1,2-dihydroperillaldehyde (compounds 5 and
6) was reported for the first time as a natural product.4
The mixture of the trans- and cis-isomers was found in
the essential oil of Enhydra fluctuans. The latter plant
is a species common in south Asia, where it is used in
folk medicine and as a condiment in food.
*
We prepared some samples of synthetic dihydroperill-
aldehydes that were submitted to sensorial evaluation
Corresponding author. Tel.: +39 2 23993076; fax: +39 2 23993080;
e-mail: stefano.serra@polimi.it
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.08.025

3074
G. Fronza et al. / Tetrahedron: Asymmetry 15 (2004) 3073–3077
performed by experienced perfumers. Surprisingly, it
was observed that the latter compound is suitable at a
dilution level of 0.2ppm in order to impart an effective
watery character to fruit formulations. The best applica-
tion seems to be its use in watermelon aroma since the
main part of the flavour sold on the marked is lacking
this feature and shows an undesirable melon side note.
a procedure where perillaldehyde dissolved in a mixture
of ether, anhydrous ammonia and propan-2-ol was
treated with lithium wire. The reaction afforded a 3:1
mixture of cis/trans-isomers of the dihydro derivative even
if on a large scale, complete reduction to p-menthan-
7-ol occurred.
Subsequently, Brestensky and Stryker8 described the
regioselective conjugate reduction of perillaldehyde by
the use of the copper (I) hydride complex [(Ph3P)CuH]6
in the presence of Me3SiCl. This procedure provided the
saturated silyl enol ether, which was then hydrolysed to
give a 2:1 mixture of dihydroperillaldehyde isomers.
Furthermore, the C1 double bond of perillaldehyde9
or perillyl alcohol 910 was reduced by sodium metal
but the products in both cases were the saturated alcoh-
ols 11 and 12.
These observations prompted us to study the bioreduc-
tion of the natural and easy available (S)-(ꢀ) perillalde-
hyde 7 to obtain the corresponding C1 saturated
analogues 5, 6, 11 and 12 (Scheme 2). The key step of
the process is the hydrogenation of the conjugated dou-
ble bond. Otherwise the oxidation/reduction of the alco-
holic functionality could be performed by a number of
biological procedures.5 We selected bakerÕs yeast as the
ÔnaturalÕ reducing agent for this kind of transformation
since it proved to be a versatile tool in the selective
reduction of many a,b-unsaturated aldehydes, ketones
and lactones.6
A further oxidative step to dihydroperillaldehyde was
hence necessary. Therefore, we studied a straightfor-
ward method of reduction that could be applicable on
a large-scale transformation and did not involve the
use of an expensive reducing agent. We found that the
above mentioned selective hydrogenation can be easily
achieved by a one-pot procedure based on the use of a
rhodium (I) complex catalysed hydrosilylation reac-
tion.11 In effect, the treatment of perillaldehyde with tri-
ethylsilane in the presence of a catalytic amount of
(Ph3P)3RhCl (0.3mol%) gave the corresponding satu-
rated silyl enol ethers 13 and 14 in good yields as a 1:1
mixture of isomers (Scheme 3).
O
HO
O
HO
1,2
1,2
reduction
reduction
9
7
8
10
1,4
reduction
1,2 and 1,4
reduction
1,4
reduction
HO
HO
5, 6
5,6
OSiEt3 OSiEt3
b
Et3SiO
a
+
+
9
5
6
7
11
12
5:6:9=52:47:1
Scheme 2. Reduction of the a,b-unsaturated aldehydes 7 and 8.
13
14
15
Herein we report on the study of the bakerÕs yeast medi-
ated reduction of the C1 double bond of natural and
artificial perillaldehyde (S)-(ꢀ)-7 and (R)-(+)-8, respec-
tively. The stereochemistry of the process was investi-
gated by deuterium labelling experiments and by 2H
NMR studies.
Scheme 3. Chemical reduction of (S)-(ꢀ)-perillaldehyde: (a) Et3SiH,
(Ph3P)3RhCl cat, THF; (b) H2O/THF, 5% HCl aq soln; 68% overall.
The only side product of the reaction was a trace
amount of 15 deriving from a 1,2-reduction, whereas
the starting material was consumed completely. The fol-
lowing acidic hydrolysis afforded a 1:1 mixture of trans-
5 and cis-6 dihydroperillaldehyde that were separated
from the silanol by chromatography and/or distillation.
2. Results and discussion
2.1. Chemical reduction of natural perillaldehyde
2.2. BakerÕs yeast mediated reduction of natural
perillaldehyde
First we needed a significant amount of dihydroperill-
aldehyde in order to perform its olfactory evaluation.
Therefore, we explored its preparation by chemical
reduction of the easy available (ꢀ)-perillaldehyde 7.
Since the latter compound shows two double bonds
and one stereogenic centre, this preparation involves
some problems of regio and diastereoselectivity. In fact,
only the conjugated double bond should be reduced and
a mixture of trans/cis-isomers should also be possible.
With an effective synthetic method to dihydroperillalde-
hyde in hand, the next step was looking for an analo-
gous transformation by means of biological reduction.
To the best of our knowledge, only one paper dealing
Compounds 13, 14 and 15 were not isolated. The GC–MS analysis of
the reaction mixture revealed two peaks (13 and 14; tR: 21.10; and
21.38; ratio 51:48% of total) showing M+ 266. A further peak (tR:
21.26; ratio 1% of total) showed M+ 266 and was conceivably due to
compound 15.
Few examples of this regioselective process have been
described in the literature. Murphy and Prager7 reported

G. Fronza et al. / Tetrahedron: Asymmetry 15 (2004) 3073–3077
3075
with microbiological transformation of perillaldehyde is
2.3. Stereochemistry of the bakerÕs yeast mediated
double bond saturation
reported in the literature. This work12 describes a study
on the biotransformation of different monoterpenoids
by Euglena gracilis Z. The latter photosynthetic micro-
organism converts perillaldehyde 7 into a mixture of
perillic alcohol 9, dihydroperillic alcohol 11, 1,2-dihydro-
perillic acid and 8,9-dihydroperillic acid. Furthermore,
the incubation of perillaldehyde 8 gives a similar prod-
uct distribution. These results also show that the
isolated double bond is reduced and an undesirable
over-oxidation of the aldehydic functionality occurs as
a side reaction of the process.
In order to explain the observed behaviour of this dia-
stereoselective reduction, we investigated the origin of
the hydrogen atoms formally added to the conjugate
double bond. To this end, the yeast transformations
were performed in the presence of deuterated water
according to the procedure described above.
Previous works2,3a,14 carried out on the bakerÕs yeast
reduction of a,b-unsaturated carbonyl derivatives
showed that the addition of hydrogen to the double
bond may occur with different stereochemistry. The glo-
bal mechanism of the addition suggests that the hydro-
gen at the b-position originates from the NADPH
enzyme while that at the a-position is picked up from
the medium; the resulting stereochemistry of the reduc-
tion may be anti or syn depending on the substrate.
Accordingly, in our case the labelling experiments
showed that the incorporation occurred at positions 1,
2 and 7 (p-menthane numbering).
As mentioned above, we selected bakerÕs yeast as the
microorganism of choice for the reduction of a,b-
unsaturated aldehydes. It is known that fermenting
bakerÕs yeast stereoselectively reduces the conjugated
double bond of carbonyl compounds with an efficiency
and stereoselectivity dependent on the structural
features of the substrates. We have previously applied
this approach in the enantioselective reduction of
prochiral aldehydes13 and accordingly, extended this
acquired biotechnological methodology to the reduction
of perillaldehyde.
The deuterium at position 1 originates from the solvent
while the deuterium atoms at position 2 and 7 are incor-
porated as hydrides in the reduction step from the
reduced form of the nicotinamide cofactor, which
exchanges the deuterium atoms with the solvent under
the action of a diaphorase.15 The 2H NMR spectra
(Fig. 1) of the mixtures 11/12 obtained from 7 and 8
are reported in Figure 1 and allow some interesting
considerations.
In order to achieve a large-scale preparation of the
reduction products, we performed the microbial reduc-
tion by adsorbing the substrates on a non-polar resin
(XAD 1180). This procedure allowed us to use a high
concentration of substrate (8–15g/L) and the products
were recovered by simple filtration of the resin. Extrac-
tion of the latter with a suitable solvent gave the crude
reaction mixture. We found that incubation of ferment-
ing bakerÕs yeast with (S)-(ꢀ)-perillaldehyde afforded,
almost exclusively, a mixture of perilyl alcohol 9 (51%)
and saturated compounds 11 and 12 (48%) in an 89:11
ratio, respectively (Scheme 4).
D-7
D-2ax+D-2eq
D-1
*
*
HO
HO
*
(A)
(B)
a
a
D-7
10 +
11
+
9
7
8
+
+
12
D-1 D-2ax
b
b
12
11
Scheme 4. BakerÕs yeast reduction of perillaldehyde: (a) bakerÕs yeast,
glucose, water, 4days; (b) MnO2, CHCl3 reflux, 5h.
4.5
4.0
3.5
3.0
2.5
(ppm)
2.0
1.5
1.0
Figure 1. 2H NMR spectra in CHCl3 of (A) mixture of compounds 12
and 11 obtained by bakerÕs yeast reduction of (R)-(+)-perillaldehyde
(asterisks denote signals belonging to the minor trans-isomer 11 and
(B) mixture of compounds 11 and 12 obtained by bakerÕs yeast
reduction of (S)-(ꢀ)-perillaldehyde (only traces of the cis-isomer 12
appear in the spectrum).
Only traces of the starting aldehyde (<1%) were detected
at the end of reaction (4days). The crude mixture was
easily purified by treatment with MnO2 in refluxing
CHCl3 to afford (S)-(ꢀ)-perillaldehyde 7 and alcohols
11 and 12 that were separated by chromatography.
Otherwise, a similar procedure performed on (R)-(+)-
perillaldehyde 8 showed similar results in terms of
effectiveness but very different results with respect to dia-
stereoselectivity. In fact, the ratio of allylic alcohol/satu-
rated alcohols was the same but the trans/cis ratio of the
products deriving from double bond reduction was re-
versed. In this case cis-alcohol 12 was the major diaster-
eoisomer with a cis/trans ratio of 80:20.
Compound 11a (Scheme 5) showed that the deuterium
atoms at positions 1 and 2 at 1.01 and 1.46ppm, respec-
tively, are axially oriented as deduced from the large
coupling constant of 12.5Hz occurring between the cor-
responding hydrogen nuclei in the proton spectrum.
Thus, they conceivably derive from a trans addition of
deuterium to the double bond, where the NADPD
undertook delivery of hydride on the Re face of 7

3076
G. Fronza et al. / Tetrahedron: Asymmetry 15 (2004) 3073–3077
BakerÕs yeast was obtained from Gist-brocades (DSM
HO
D
Bakery Ingredients Italy spa). Natural (S)-(ꢀ)-perillal-
dehyde of undefined origin was commercially available
from Aldrich; (R)-(+)-perillaldehyde was prepared by
MnO2 oxidation of (R)-(+)-perillyl alcohol (Fluka).
OH
D
D
11a
12a
D
Path B
Path A
NADPD
4.2. Chemical reduction of (S)-(ꢀ)-perillaldehyde
O
O
8
A solution of (S)-(ꢀ)-perillaldehyde (25g, 167mmol) in
dry THF (25cm3) was treated under nitrogen with
Et3SiH (28.7cm3, 180mmol) and (Ph3P)3RhCl (0.5g,
0.54mmol). The mixture was stirred and heated at
50ꢁC for 3h. After cooling to rt, the reaction was diluted
with THF (100cm3), water (30cm3) and 5% HCl aq
(10cm3). The stirring was prolonged for a further 2h
and then the mixture diluted with water (500cm3) and
extracted with ether (3 · 150cm3). The combined organ-
ic phases were washed with brine (100cm3), dried over
Na2SO4 and concentrated under reduced pressure. The
residue was purified by chromatography (hexane ! hex-
ane/ethyl acetate 9:1) and bulb to bulb distillation (bp
90–95ꢁC/20Torr) to afford pure dihydroperillaldehyde
(17.2g, 68%) as a 1:1 mixture of trans/cis-isomers 5
and 6, respectively. FT-IR (film) 3085, 2709, 1730,
1645, 1451, 1375, 963, 923, 888, 812; m/z (EI) trans-iso-
mer (tR 10.58): 152 (M+, 55), 137 (10), 134 (16), 121 (86),
119 (63), 109 (76), 105 (28), 95 (56), 93 (45), 91 (50), 81
(100), 79 (79), 77 (30), 67 (93), 55 (49); cis-isomer (tR
10.50): 152 (M+, 23), 137 (5), 134 (12), 121 (29), 119
(21), 109 (42), 105 (12), 95 (28), 93 (25), 91 (22), 81
(100), 79 (48), 77 (15), 67 (68), 55 (36); d 1.12–1.42
(6H, m), 1.52–1.73 (4H, m), 1.66 (3H, s, CH3), 1.72
(3H, s, CH3), 2.00–1.79 (4H, m), 2.18–2.00 (2H, m),
2.32-2.18 (3H, m), 2.45 (1H, m, H-1), 4.62 (1H, br s,
C@CHH), 4.67 (1H, br s, C@CHH), 4.69 (2H, br s,
C@CH2), 9.63 (1H, s, CHO), 9.72 (1H, s, CHO). Anal.
Calcd for C10H16O: C, 78.90; H, 10.59. Found: C,
78.80; H, 10.60.
H
H
7
NADPD
NADPD
Path C
HO
D
12b
D
Scheme 5. Proposed mode of labelling of 11 and 12.
(path A). Otherwise, compound 12 was formed as a
mixture of 12a and 12b both deriving from 8 by a trans
addition to the Re face (path B) and syn addition to the
Si face (path C).
3. Conclusion
These results, seen together, can be explained in terms
of the different enantioselectivities of the reducing
enzymes. It is reasonable to assume that (S)-perillalde-
hyde was transformed through a trans addition, in
accordance with the behaviour of the enoyl-CoA medi-
ated reduction. Otherwise, (R)-perillaldehyde was not
a good substrate for the latter enzyme and the biotrans-
formation proceeded with loss of stereocontrol, due
probably to a concomitant operation of different reduc-
ing enzymes.
4.3. BakerÕs yeast mediated reduction of perillaldehyde
4. Experimental
4.3.1. Reduction of natural (S)-(ꢀ)-perillaldehyde.
A
5L open cylindrical glass vessel equipped with a
mechanical stirrer was charged with tap water (2L)
and glucose (150g). Fresh bakerÕs yeast (500g) was
added in small pieces to the stirred mixture and the fer-
mentation allowed to proceed for 2h. Aldehyde 7 (20g,
133mmol), absorbed on the resin XAD 1180 (100g),
was added in one portion. Vigorous stirring was contin-
ued for 4days at room temperature. During this time,
more bakerÕs yeast (100g) and glucose (50g) were added
after 24 and 48h after the fermentation had started. The
resin was then separated by filtration on a sintered glass
funnel (porosity 0, >160lm) and the water phase
extracted again with further resin (50g). The combined
resin crops were extracted with ethyl acetate (4 ·
100cm3) and the acetate solution washed with brine.
The dried organic phase (Na2SO4) was concentrated
under reduced pressure to give an oil (25g). The latter
was dissolved in CHCl3 and treated with MnO2 (40g,
460mmol) with stirring at reflux for 5h. The residue
obtained upon filtration and evaporation of the CHCl3
phase was purified by column chromatography using
4.1. General experimental
TLC: Merck silica gel 60F254 plates. Column chromato-
graphy (CC): silica gel. GC: HP-6890 gas chromato-
graph; diastereoisomer excesses determined on a HP-5
column (30m · 0.32mm; Hewlett Packard) with the
following temp. program 60ꢁ (1min)–6ꢁ/min–150ꢁ
1
(1min)–12ꢁ/min–280ꢁ (5 min); tR given in min. H and
13C spectra: CDCl3 solns at rt; Bruker-ARX 400 spec-
trometer at 400MHz; 2H spectra: CHCl3 solns at rt;
Bruker-ARX 400 spectrometer at 61.4MHz. The 2H
1
spectra were acquired with broad band H decoupling
to suppress the heteronuclear proton/deuterium cou-
pling constants to improve the signal resolution. The
chemical shifts in ppm are relative to internal SiMe4
(= 0ppm), J values inHz. Mass spectra were measured
on a Finnigan-Mat TSQ 70 spectrometer; m/z (rel.%).
IR spectra were recorded on a Perkin–Elmer 2000 FT-
IR spectrometer; films. Microanalyses were determined
on an analyser 1106 from Carlo Erba.

G. Fronza et al. / Tetrahedron: Asymmetry 15 (2004) 3073–3077
3077
hexane–ethyl acetate (9:1–3:1) as the eluent to give a
mixture of 11 and 12 (9.9g, 48%) as a pale yellow oil.
The diastereomeric ratio was 91:9 (determined by
NMR analysis). FT-IR (film) 3338, 1644, 1449, 1375,
1093, 1035, 970, 886; m/z (EI) trans-isomer (tR 12.10):
was dissolved in CHCl3 and treated with MnO2 (40g)
stirring at reflux for 5h. The residue obtained upon filtra-
tion and evaporation of the CHCl3 phase was purified by
column chromatography using hexane–ethyl acetate
(9:1–3:1) as the eluent to give a mixture of 11 and 12
(5.7g, 46%) as a pale yellow oil. The diastereomeric ratio
154 (M+, 26), 136 (28), 121 (91), 108 (48), 107 (74), 93
1
2
(100), 81 (50), 79 (85), 67 (61), 55 (29), 41 (30);
H
was 89:11 (determined by NMR analysis). H NMR of
NMR of 11 d 1.01 (2H, qd, J ca. 12.5 and 3.6, H-2ax,
6ax), 1.46 (1H, m, H-1), 1.71 (3H, t, J 1.2, CH3), 1.86
(1H, m, H-2eq, 6eq), 1.22 (2H, qd, J ca. 13 and 3.6,
H-3ax, 5ax), 1.82 (1H, m, H-3eq, 5eq), 1.47 (1H, m,
H-4), 3.47 (2H, d, J 6.2, CH2OH), 4.67 (2H, m,
@CH2). The spectral assignment is based on the values
of vicinal coupling constants and the chemical shift
correlation experiments (COSY). Anal. Calcd for
C10H18O: C, 77.87; H, 11.76. Found: C, 77.90; H, 11.70.
11 d 1.01 (D-2ax), 1.46 (D-1), 3.47 (D-7 = CD2OH).
Low signals due to the occurrence of the cis-isomer 12
are present in the spectrum (Fig. 1).
4.4.2. Reduction of synthetic (R)-(+)-perillaldehyde.
The same procedure described above was performed
on (R)-(+)-perillaldehyde 8 (10g, 67mmol) to give a
79:21 mixture of cis/trans-isomers 12 and 11 (4.4g,
2
43%). H NMR of 12 d 1.53 and 1.62 (D-2ax + D-2eq),
1.79 (D-1), 3.60 (D-7 = CD2OH). Strong signals due to
the occurrence of the trans-isomer 11 are clearly visible
in the spectrum (Fig. 1).
4.3.2. Reduction of synthetic (R)-(+)-perillaldehyde.
The same procedure described above was per-
formed on (R)-(+)-perillaldehyde 8 (10g, 67mmol) to
give an 80:20 mixture of cis/trans-isomers 12 and 11
(4.2g, 40%). FT-IR (film) 3335, 1645, 1450, 1373,
1093, 1053, 1032, 951, 886; m/z (EI) cis-isomer 12 (tR
12.40): 154 (M+, 13), 136 (32), 121 (65), 108 (41), 107
(84), 93 (100), 81 (61), 79 (96), 67 (70), 55 (34), 41
Acknowledgements
The authors would like to thank COFIN-MURST for
partial financial support.
1
(34); H NMR of 12 d 1.53 and 1.62 (4H, m, H-2ax,
6ax and H-2eq, 6eq, not assigned), 1.47 and 1.54 (4H,
m, H-3ax, 5ax and H-5eq, 5eq, not assigned), 1.74 (3H,
t, J 1.2Hz, CH3), 1.99 (1H, m, H-4), 3.60 (2H, d,
J 7.3Hz, CH2OH), 4.72 (2H, m, @CH2). The spectrum
was very crowded and could not be completely analysed.
The assignment above is based on the chemical shift
correlation experiments (COSY) and selective total
correlation spectroscopy (TOCSY-1D). Anal. Calcd
for C10H18O: C, 77.87; H, 11.76. Found: C, 78.00; H,
11.65.
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