Stereochemical course of baker's yeast mediated reduction of the tri- and tetrasubstituted double bonds of substituted cinnamaldehydes

Article abstract of DOI:10.1002/ejoc.200900827

A comprehensive study of the stereochemical course of baker's yeast mediated reduction of substituted cinnamaldehydes is reported. Hydride addition to the β position of β-methylcinnamaldehydes preferentially afforded isomers of (3S)-3-phenylbutan-1-ol. The reduction of (E)-2,3-dimethyl- cinnamaldehyde (15) produced a mixture of (2S,3S)- and (2R,3S)-2-methyl-3- phenylbutan-1-ol (13 and 14), respectively, with 93 % ee. Conversely (Z)-2,3-dimethylcinnamal-dehyde (16) afforded, a mixture of 13 and 14 with 33 % ee. Accordingly, the reduction of trisubstituted β-methylcinnam-aldehydes 34 and 35 proceeded with the same stereochemical preference and with higher enantioselectivity to give (S) 3-phenylbutan-1-ol (37). In addition, deuterium, incorporation and ²H NMR studies demonstrated that the addition of the second hydrogen atom to the a position proceeded with very low stereochemical control and the overall process is formally a mixture of cis/trans hydrogen addition to the double bond. Alternatively, α-methylcinnamaldehyde is reduced to (S)-2-methyl-3-phenylpropan-1-ol (24) with preferential addition of the hydride to the opposite β face with good stereochemical control of the irons addition of hydrogen to the double bond. Wiley-VCH Verlag GmbH & Co. KGaA.

Full text of DOI:10.1002/ejoc.200900827

FULL PAPER
DOI: 10.1002/ejoc.200900827
Stereochemical Course of Baker’s Yeast Mediated Reduction of the Tri- and
Tetrasubstituted Double Bonds of Substituted Cinnamaldehydes
Giovanni Fronza,[a] Claudio Fuganti,[b] and Stefano Serra*[a]
Keywords: Enzyme catalysis / Hydrogenation / Stereoselectivity / Biotransformations / Aldehydes
A comprehensive study of the stereochemical course of
baker’s yeast mediated reduction of substituted cinnam-
aldehydes is reported. Hydride addition to the β position of
β-methylcinnamaldehydes preferentially afforded isomers of
(3S)-3-phenylbutan-1-ol. The reduction of (E)-2,3-dimethyl-
cinnamaldehyde (15) produced a mixture of (2S,3S)- and
(2R,3S)-2-methyl-3-phenylbutan-1-ol (13 and 14), respec-
tively, with 93% ee. Conversely (Z)-2,3-dimethylcinnamal-
3-phenylbutan-1-ol (37). In addition, deuterium incorpora-
tion and 2H NMR studies demonstrated that the addition of
the second hydrogen atom to the α position proceeded with
very low stereochemical control and the overall process is
formally a mixture of cis/trans hydrogen addition to the
double bond. Alternatively, α-methylcinnamaldehyde is re-
duced to (S)-2-methyl-3-phenylpropan-1-ol (24) with prefer-
ential addition of the hydride to the opposite β face with good
dehyde (16) afforded a mixture of 13 and 14 with 33% ee. stereochemical control of the trans addition of hydrogen to
Accordingly, the reduction of trisubstituted β-methylcinnam-
aldehydes 34 and 35 proceeded with the same stereochemi-
cal preference and with higher enantioselectivity to give (S)-
the double bond.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
tioenriched compounds have been prepared,[2b,2c] some of
which we have used in the stereoselective syntheses of com-
pounds belonging to quite different structural classes.[3]
Introduction
Baker’s yeast mediated reduction of activated olefins is a
process that has been known for a long time. The first stud-
ies in this area go back to the work of Fischer,[1] who de-
scribed in 1940 the reduction of a number of unsaturated
aldehyde and ketone derivatives. This research did not con-
sider the enantioselectivity of this kind of transformation
which was considered a biochemical curiosity until the
1970s when this type of reaction received renewed interest
in the context of the preparation of chiral synthetic interme-
diates to increase the pool of chiral compounds prevalent
in nature.[2a–2c] Indeed, the asymmetric reduction of a
double bond allows the creation of up to two stereogenic
centres and is a pivotal reaction in organic synthesis. In
the field of microbial biocatalysis, baker’s yeast is the most
commonly employed for the reduction of prochiral double
bonds because it is cheap and commercially available and
the transformations usually proceed with high enantio-
selectivity.
Figure 1. Substrates and products of the baker’s yeast mediated
asymmetric reduction of activated alkenes.
To determine the mechanism of the “biohydrogenation”
of carbonyl-activated double bonds using baker’s yeast or
isolated enzymes, a number of isotopic studies[4] have been
performed that pointed to a process involving first the ad-
dition of a hydride [delivered from NAD(P)H] to the car-
bon atom at the β position with respect to the activating
group followed by the addition of a proton from the me-
dium to the α position.
Moreover, for olefins of the type 1–3 the stereochemical
course of the reduction is by now predictable, invariably
affording saturated compounds of the type 4–6, respectively
(Figure 1). Thus, by these means, a large number of enan-
Thus, the overall process is formally an asymmetric trans
hydrogenation. This aspect is very significant because the
asymmetric reduction of olefins using transition-metal cata-
lysts and chiral ligands is a stereospecific cis hydrogena-
tion.[5] Therefore, the two processes could be complemen-
tary and different studies on the use of isolated reductases
[a] C.N.R. Istituto di Chimica del Riconoscimento Molecolare,
Via Mancinelli 7, 20131 Milano, Italy
Fax: +39-02-2399-3180
E-mail: stefano.serra@polimi.it
[b] Dipartimento di Chimica, Materiali ed Ingegneria Chimica del
Politecnico,
Via Mancinelli 7, 20131 Milano, Italy
6160
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biohydrogenation of Double Bonds of Cinnamaldehydes
as catalysts for biohydrogenation have recently been de-
scribed.[6] Moreover, a comprehensive understanding of the
stereochemical course of the biocatalytic reduction might
Results and Discussion
As mentioned above our study of baker’s yeast mediated
afford a number of new synthetic and analytical applica- reduction of methylcinnamaldehydes requires the use of an
tions. The stereoselective bioreduction of tetrasubstituted isomerically pure substrate. Indeed, the stereochemical
olefins is clearly a most challenging synthetic procedure be- course of the reduction could also be affected by the geome-
cause it could afford products not available by chemical try of the double bond. Because isomeric methylcinnamal-
catalytic hydrogenation. In addition, knowledge of the dehydes are not easily separable by the usual laboratory
mechanism of this biochemical transformation, which is methods, we prepared them by starting from the corre-
quite widespread in Nature, would be helpful in studies de- sponding substituted ethyl cinnamates prepared in turn by
signed to determine the origin of substances of practical Horner condensation of acetophenone with the appropriate
interest for the flavour industry.[7]
phosphonate esters and separated by chromatography. The
To the best of our knowledge, the only reported study on desired aldehydes were obtained from the cinnamates by
baker’s yeast reduction of an activated tetrasubstituted ole- LiAlH4 reduction followed by MnO2 oxidation. In this way,
fin relates to a nitroalkene, in which cis hydrogenation pre- methylcinnamaldehydes were obtained in isomerically pure
vailed.[8] This is not the only exception to the generally ac- form and free of any saturated impurity.
cepted anti addition rule mentioned above. Some other ex-
According to the general consideration described above,
amples of cis reduction of carbonyl-activated double bonds Horner condensation of acetophenone (10) with triethyl 2-
mediated by baker’s yeast have been reported.[9] At first phosphonopropionate[11] gave chromatographically separa-
glance, these discordant results have been explained by sup- ble esters 11 and 12 (Scheme 1). These compounds were
posing that a number of different reducing enzymes operate hydrogenated using palladium on charcoal as the catalyst
in a whole-cell-mediated transformation thus affording dif- and the saturated esters obtained were reduced with LiAlH4
ferent results on the basis of their specific enzyme/substrate to give racemic diastereoisomers 13 and 14,[12] respectively,
affinity. However, through deuterium-labelling experiments, which were used as reference materials in the biological re-
we have recently shown[7d] that the chiral α,β-unsaturated duction of 2,3-dimethylcinnamaldehydes. Indeed, the re-
aldehyde perillaldehyde is reduced by baker’s yeast in either duction of esters 11 and 12 with LiAlH4 followed by oxi-
a trans or cis fashion depending upon its absolute configu- dation with MnO2 gave aldehydes 15 and 16, respectively,
ration. More recently,[10] this latter transformation has been which were incubated with fermenting baker’s yeast at a
reinvestigated by using NADPH as the reducing agent and concentration of 2.5 g/L substrate for 4 d.
Ltb4 dehydrogenase as the biocatalyst, which unequivocally
Solvent extraction provided a mixture composed of al-
demonstrated that the same enzyme mediates a net trans lylic alcohols, saturated alcohols and, in the case of 15, the
addition of hydrogen across the double bond of (R)-perillal- C6-C5 methyl-diol 18, formed by reduction of the α-ketol
dehyde, but a cis process for the (S) enantiomer, thus con- of acyloin condensation of C6-C3 starting aldehyde with a
firming the previous findings. Seen together, the results de- C2 unit.[13] The reduction of (E)-aldehyde 15 gave allylic
scribed above would suggest that the “trans addition rule” alcohol 17 (24%), enantio- and diastereoisomerically pure
is not of general validity. Accordingly, we have performed diol (+)-18 (39%) and a mixture of saturated alcohols 13
a detailed (isotopic) study of the stereochemical course of and 14 (18%, ratio 69:31, respectively) both with 93% ee.
baker’s yeast mediated reduction of tetra- and trisubstituted However, the reduction of (Z)-aldehyde 16 did not give a
aldehydes, selecting tetrasubstituted cinnamaldehydes 7c in detectable amount of a diol like 18, the main products be-
addition to trisubstituted 7a and 7b, which have previously ing the allylic alcohol 19 (64%) and a mixture of the satu-
been shown[3c,3e,4a] to provide saturated alcohols of type 8 rated alcohols 13 and 14 (19%, ratio 59:41, respectively),
and 9, respectively (Figure 2). More specifically, we em- both showing 33% ee. The absolute configurations of
ployed methylcinnamaldehydes (Ar = Ph, R1 and/or R2 = alcohols 13 and 14 were determined by chemical corre-
Me), which are easily accessible in good isomeric purity by lation with the known (1,2-dimethylpropyl)benzene
chemical synthesis.
(20).[14] Accordingly, in separate runs, the two mixtures of
diastereoisomeric alcohols were treated with p-toluenesul-
fonyl chloride and the derived tosylates with LiAlH4. In
both cases we obtained samples of (+)-(S)-20, the ees of
which were identical to those measured for the starting
alcohols. These results prove that both aldehydes are re-
duced with preferential hydride delivery to the same face
of the β position of the double bond. However, in the case
of the reduction of (E)-aldehyde 15 the process is highly
enantioselective, whereas the transformation of (Z)-alde-
hyde 16 is less selective. As far as the second step of the
reduction is concerned, that is, the delivery of a proton to
the α position, there is a modest and a low stereocontrol,
respectively.
Figure 2. Substituted cinnamaldehydes and the products of their
baker’s yeast mediated asymmetric reduction.
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Scheme 2. Preparation of 3-chloro-2-methylcinnamaldehyde and its
baker’s yeast mediated asymmetric reduction. Reagents and condi-
tions: i) DMF, POCl3; ii) Baker’s yeast, glucose, water.
were incubated, as above, in tap water containing 1% de-
uteriated water. The chemically identical saturated alcohols
isolated in the two experiments were shown to contain
around 85% of the (2S) enantiomer 24. For spectroscopic
reasons, these materials were converted into the corre-
sponding acetate esters. The 2H NMR spectra of the deriva-
tives of 24 obtained from 22 and 27 in baker’s yeast are
presented in parts A and B of Figure 3, whereas part C
shows the spectrum of the trideuteriated material composed
Scheme 1. Preparation of isomers of α,β-dimethylcinnamaldehydes
and their baker’s yeast mediated asymmetric reduction. Reagents
of
a 1:1 racemic mixture of (1RS,2SR,3RS)- and
and conditions: i) triethyl 2-phosphonopropionate, NaOEt, EtOH; (1SR,2SR,3RS)-[1,2,3-2H3]-2-methyl-3-phenylpropan-1-ol
ii) H2, Pd/C, EtOAc; iii) LiAlH4, Et2O; iv) MnO2, CHCl3, reflux;
(30; Scheme 3), obtained by catalytic reduction of the alde-
v) Baker’s yeast, glucose, water; vi) TsCl, Py, CH2Cl2.
hyde 27 with deuterium gas.
Inspection of spectra A and B indicates that the products
We then examined the transformation of a tetrasubsti- of bioreduction incorporated deuterium atoms at all three
tuted cinnamaldehyde by yeast by using 3-chloro-2-methyl- positions of the side-chain. The deuterium atom present at
cinnamaldehyde (22; Scheme 2), obtained as a 92:8 mixture the 1-position is delivered in the reduction of the aldehydic
of E/Z isomers by the action of phosphoryl chloride and moiety of 2-methyl-3-phenylpropanal, the putative ultimate
DMF on propiophenone (21).[15] The reduction of 22 with intermediate in the process, supposedly mediated by yeast
fermenting baker’s yeast afforded allylic alcohol 23 (53%), alcohol dehydrogenase. If we take into account the result
(S)-2-methyl-3-phenylpropan-1-ol (24; 21%)[16] and the en- obtained in the case of the reduction of cinnamaldehyde,[4a]
antio- and diastereoisomerically pure diol (+)-25 without we can assign the absolute configuration R to the carbon
any trace of the expected saturated chloro alcohol. We in- atom at the 1-position of 24. As far as the deuterium atoms
terpreted the formation of chlorine-free 24 to be the result introduced during the saturation of the double bond are
of a three-step process involving enzyme-mediated satura- concerned, we expect that the deuterium present at the 3-
tion of the double bond of 22 to provide 26, which, by position should have been delivered from the reduced form
spontaneous elimination of hydrochloric acid, yields 27, of the nicotine cofactor, which exchanges hydrogen atom(s)
which is finally reduced to 2-methyl-3-phenylpropan-1-ol, with the deuteriated solvent,[17] whereas the deuterium at
shown to contain around 85% of the (2S) enantiomer 24.
the 2-position derives from the medium.
To verify the above mechanistic proposal for the genera-
Examination of the spectrum of (2S)-24 (0.7 ee; Figure 3,
tion in baker’s yeast of 24 from 22 the following deuterium- B) produced directly from 27 indicates that this material is
labelling experiments were performed. Aldehydes 22 and 27 approximately an 85:15 mixture of (1R,2S,3S)-[1,2,3-2H3]-
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Biohydrogenation of Double Bonds of Cinnamaldehydes
propanal (26; Scheme 2). This deuterium atom is then re-
tained when [2,3-2H2]-26, upon elimination of hydrochloric
acid, provides [3-2H]-27. This product is then reduced and
like the protium counterpart 27 incorporates three deute-
rium atoms. The (3S) deuterium atom present in A (the
second deuterium in that position) is introduced in this way.
Thus, (1R,2S)-[1,2,3,3-2H4]-2-methyl-3-phenylpropan-1-ol
is the prevalent (85%) deuteriated diastereoisomer of 24 de-
rived from 22, whereas the minor one (15%) is the (1R,2R)
isomer. The above-mentioned events are represented in
Scheme 3, including the production of the diol 29 alongside
28 and 24.
To obtain further information on the cryptic stereochem-
istry of the yeast-mediated reduction of substituted cinnam-
aldehydes we investigated in some detail the reduction of
(E)- and (Z)-3-methylcinnamaldehydes (34 and 35), respec-
tively. The condensation of acetophenone (10) with triethyl
phosphonoacetate gave separable esters 31 and 32
(Scheme 4). The double bond of the (E)-ester 31 was satu-
rated with deuterium gas (Pd/C, ethyl acetate) and the re-
sulting product reduced with LiAlH4 to provide racemic
(2RS,3SR)-[2,3-2H2]-33, which was used as a reference com-
pound in subsequent studies. The reduction of esters 31 and
32 with LiAlH4 followed by oxidation with MnO2 gave al-
dehydes 34 and 35, respectively, which were incubated with
fermenting baker’s yeast using the same experimental con-
ditions as described for the reduction of tetrasubstituted al-
dehydes. The reduction of (E)-aldehyde 34 gave allylic
alcohol 36 (65%), enantiopure saturated alcohol (+)-37[18]
(6%) and diastereoisomerically pure diol (–)-38 (19%).
However, the reduction of (Z)-aldehyde 35 did not give a
detectable amount of a diol similar to 38. The main prod-
ucts were allylic alcohol 39 (75%) and the saturated alcohol
(+)-37 showing 90% ee.
Figure 3. 2H NMR spectra of the acetates of 2-methyl-3-phenylpro-
pan-1-ol obtained from labelling experiments: A) bioreduction of
the chloro aldehyde 22 providing the (1R,2S)-[1,2,3,3-2H4] deriva-
tive as the major distereoisomer; B) bioreduction of aldehyde 27
providing the (1R,2S,3S)-[1,2,3-2H3] derivative as the major dia-
stereoisomer; (C) catalytic reduction with deuterium gas of the al-
dehyde 27 affording the racemic mixture (2SR,3RS)-[1,2,3-2H3] of
the acetate of 30.
These results indicate that both aldehydes were reduced
by preferential hydride addition to the same face of the
double bond with an enantioselectivity that ranges from
very high to high in the transformations of the E and Z
regioisomers, respectively. Thus, the mode of reduction of
(E)-34 is identical to that of (E)-15 as far as the direction
of hydride delivery at the β position is concerned and oppo-
site to that of α-methylcinnamaldehyde (27), as revealed by
the deuterium-labelling experiments described above. Con-
sequently, to gain information on the mode of α-proton-
ation in the reduction of 34 we submitted the latter com-
pound to baker’s yeast incubation in the presence of deuter-
iated water and determined the mode of labelling of the
Scheme 3. Baker’s yeast mediated asymmetric reduction of α-meth-
ylcinnamaldehyde and preparation of (2RS,3SR)-2,3-dideuterio-2-
methyl-3-phenylpropan-1-ol. Reagents and conditions: i) baker’s
yeast, glucose, water; ii) D2, Pd/C, EtOAc; iii) H2O.
2
resulting saturated alcohol 37 through H NMR studies.
In the deuterium spectrum of saturated alcohol 37 there
are separate signals for the five hydrogen atoms of the side-
and (1R,2R,3R)-[1,2,3-2H3]-2-methyl-3-phenylpropan-1-ol, chain backbone. The 2H NMR spectra of racemic
as pictorially illustrated in Figure 3. Conversely, (2S)-24 (2RS,3SR)-[2,3-2H2]-33 and (S)-37 obtained in baker’s ye-
formed from the chloro aldehyde 22 bears two deuterium ast/D2O from 34 are presented in Figure 4, C and A.
labels of equal intensity at the benzylic position (Figure 3,
As in the case of 24 from 27 (Figure 3, A) the formation
A). An economic interpretation of this observation is that in baker’s yeast of 37 from 34 led to the incorporation of
of the two diastereotopic deuterium atoms, the one pos- deuterium at all three positions of the side-chain. However,
sessing the (3R) configuration is incorporated into 22 when inspection of C and comparison with A indicates that lab-
the latter is transformed into 3-chloro-2-methyl-3-phenyl- elled (S)-37 isolated from the reduction of 34 is the (1R,3S)-
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Figure 4. 2H NMR spectra of 3-phenylbutan-1-ol obtained from
the labelling experiments: A) bioreduction of aldehyde 34 in H2O/
D2O (1%) to afford the diastereoisomer (1R,3S)-[1,2,2,3-2H4]-37;
B) bioreduction of the deuteriated aldehyde 43 in H2O showing the
lack of sterespecificity at the 2-position of alcohol 44; C) catalytic
reduction (D2–Pd/C then LiAlH4) of the ester 31 providing the ra-
cemic (2RS,3SR)-[2,3-2H2] alcohol 33.
Scheme 4. Preparation of isomers of β-methylcinnamaldehydes and
their baker’s yeast mediated asymmetric reduction as well as the
synthesis of (2RS,3SR)-2,3-dideuterio-3-phenylbutan-1-ol (33).
Reagents and conditions: i) triethyl phosphonoacetate, NaH, THF;
ii) D2, Pd/C, EtOAc; iii) LiAlH4, Et2O; iv) MnO2, CHCl3, reflux;
v) Baker’s yeast, glucose, water.
43, which, as a consequence of the mode of preparation, is
deuterium-labelled in the β-methyl group. Yeast reduction
of labelled 43 in tap water provided enantiopure (S)-44 and
2
the allylic alcohol 45. The H NMR spectrum of alcohol
[1,2,2,3-2H4] diastereoisomer, which suggests a lack of 44 (Figure 4, B) shows two deuterium signals arising from
stereospecificity in the protonation at the α position in the the methylene group at 1.58 (2-D) and 1.56 ppm (2Ј-D) (ra-
latter conversion.
tio 55:45). This result confirms the above findings, that is,
To clarify this point, specifically labelled [2-2H]-43 was the yeast reduction of 34 to 37 proceeds with stereospecific
synthesized (Scheme 5) and submitted to the yeast re- hydride addition at the β position, but with random proton-
duction.
ation at the α position. As reported above, baker’s yeast
Condensation of acetophenone (10) with ethyl cyanoace- reduction of aldehyde 34 affords in addition to the alcohols
tate gave ethyl (1-phenylethylidene)cyanoacetate[19] as a 36 and (+)-37 the diastereoisomerically pure 5-phenylhex-
mixture of Z/E isomers, which, after hydrolysis of the ester 4-en-2,3-diol [(–)-38].
group and fractional crystallization of the resulting mixture,
The deuterium spectrum of 38 shows incorporation of
afforded pure acid 40.[20] The latter compound was stirred deuterium labels at the 1- and 2-positions (Figure 5). The
with D2O and the solvent was removed under reduced pres- formation of 38 occurs by addition of a C2 unit derived
sure, repeating the operation twice. The acid obtained was from pyruvate onto the carbonyl carbon of 34 to provide
heated at 200 °C until the evolution of carbon dioxide had an α-hydroxy ketone, subsequently reduced to the anti-diol
ceased, thus providing a mixture of nitriles 41 and 42. The actually isolated. The formation of the precursor pyruvate
α,β-unsaturated nitrile 42 was isolated by chromatography and the stereospecific yeast reduction fully accounts for the
and reduced with DIBAH to provide the desired aldehyde incorporation of deuterium in 38.
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Biohydrogenation of Double Bonds of Cinnamaldehydes
The stereochemical assignments of diols 18 and 25 were
made by chemical correlation with the known compound
48 (Scheme 6). Thus, 18 and 25 were converted into the cor-
responding 1,3-dioxolanes 46 and 47, respectively, which,
after ozonolysis and treatment with Ph3P, provided in both
cases the known ketone 48 in enantiopure form. Therefore,
the diols (+)-18 and (+)-25 have the absolute configuration
(2S,3R), the same as the known diols (+)-29[16] and (–)-
38.[21] These experiments have demonstrated that (E)-meth-
ylcinnamaldehydes are good substrates in baker’s yeast me-
diated acyloin condensation and reduction, whereas car-
bon–carbon coupling does not occur with the (Z) iso-
mers.[13]
Scheme 5. Preparation of 2-deuterio-3-methylcinnamaldehyde and
its baker’s yeast mediated asymmetric reduction. Reagents and con-
ditions: i) ethyl cyanoacetate, NH4OAc, AcOH; ii) NaOH, MeOH
then HCl; iii) recrystallization from EtOAc; iv) D2O, stirring then
drying and warming at 200 °C; v) DIBAH, toluene, –60 °C; vi)
Baker’s yeast, glucose, water; vii) MnO2, CHCl3, reflux.
Scheme 6. Chemical correlation of diols (+)-18 and (+)-25 with 1-
[(4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanone. Reagents and
conditions: i) 2,2-dimethoxypropane, PPTS, MeOH; ii) O3,
CH2Cl2/MeOH then Ph3P.
Conclusions
Figure 5. Deuterium spectrum of diol 38 showing incorporation of
deuterium labels at the 1- and 2-positions.
This work further confirms that baker’s yeast mediated
reduction of substituted cinnamaldehydes proceeds with a
stereochemical course strongly dependent on the structure
of the substrate. Aldehydes with a β-methyl substituent are
Finally, note that the modest extent to which α,β-dimeth- invariably reduced with preferential formation of the (3S)-
ylcinnamaldehydes 15 and 16 and β-methylcinnamal- 3-phenylbutan-1-ol isomers. This step is highly enantio-
dehydes 34 and 35 afforded saturated alcohols (19–18 and selective for trisubstituted (E)- and (Z)-β-methylcinnamal-
7–5%, respectively) is in apparent contrast to the results dehydes (34 and 35). In the case of tetrasubstituted alde-
obtained in previous studies[3c,3e] with similar compounds, hydes the reduction of the (E) isomer 15 is again highly
in which the saturated products were obtained in over 50% enantioselective, whereas the same step proceeds with low
yield. This is due to the different experimental conditions enantioselectivity with the (Z) isomer 16.
employed in the latter reductions; we performed the micro-
In the reduction of tetrasubstituted α,β-dimethylcinnam-
bial process on substrates adsorbed on a non-polar resin aldehydes the addition of the second hydrogen atom to the
that slowly released the aldehyde into the aqueous medium α position proceeds with low stereochemical control [7:3
and at the same time adsorbed the transformation prod- and 6:4, respectively, for the (E) and (Z) isomers 15 and
uct(s). The low concentrations of the substrates in the aque- 16], but in a cis fashion. Deuterium-labelling experiments
ous medium favoured the saturation of the double bond revealed that protonation at the α-position in the case of
and reduced the formation of allylic alcohols and methyl- (E)-β-methylcinnamaldehyde (34) proceeds without stereo-
diols. However, in this study we have focused on the stereo- chemical control.
chemical features of the bioreduction. The biotransforma-
Conversely, (E)-α-methylcinnamaldehyde (27) is reduced
tions were performed without resin and thus without opti- with preferential addition of the hydride to the opposite β
mization of the yields, but by using the same conditions for face with fairly good (85:15) anti stereochemical control, as
each experiment.
indicated by deuterium-labelling experiments.
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about 0.008 ppm for nuclei separated by three bonds. For instance,
the partially deuteriated methyl groups of 3-phenylbutan-1-ol show
separate signals for the CH2D, CHD2 and CD3 groups (see parts
B and C in Figure 4).
In future works, the use of enzymes isolated from baker’s
yeast and able to catalyze the reduction of activated double
bonds will clarify in detail the results obtained with the
whole cell system (this work).
Synthesis of Substrates and Reference Materials. Ethyl (2E)- and
(2Z)-2-Methyl-3-phenylbut-2-enoate (11 and 12): Triethyl 2-phos-
phonopropionate (100 g, 420 mmol) was added to a stirred solution
of NaOEt (29 g, 426 mmol) in ethanol (400 mL). After 10 min, ace-
tophenone (42 g, 350 mmol) was added dropwise and the reaction
was heated at reflux for 24 h. After cooling, the mixture was poured
onto ice/water and extracted with diethyl ether (3ϫ200 mL). The
organic phase was washed with brine (2ϫ100 mL), dried (Na2SO4)
and concentrated under reduced pressure. The residue was purified
by chromatography with hexane/diethyl ether (95:5–9:1) as eluent
to afford pure esters 11 (35.1 g, 49%) and 12 (16.7 g, 23%).
11: Colourless oil, 99% purity (by GC). 1H NMR (CDCl3,
400 MHz): δ = 1.34 (t, J = 7.1 Hz, 3 H), 1.75 (q, J = 1.5 Hz, 3 H),
2.25 (q, J = 1.5 Hz, 3 H), 4.26 (q, J = 7.1 Hz, 2 H), 7.11–7.39 (m,
5 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 14.2, 17.3, 23.0, 60.2,
124.9, 126.9, 127.2, 128.3, 143.5, 145.3, 169.9 ppm. GC–MS (EI):
m/z (%) = 204 (100) [M]+, 175 (34), 159 (85), 131 (66), 115 (55),
105 (8), 91 (47), 77 (13).
Experimental Section
General: All moisture-sensitive reactions were carried out under a
static atmosphere of nitrogen. All reagents were of commercial
quality. TLC: Merck silica gel 60 F254 plates. Column chromatog-
raphy (CC): silica gel. GC–MS analyses: HP-6890 gas chromato-
graph equipped with a 5973 mass detector and a HP-5MS column
(30 mϫ0.25 mm, 0.25 µm firm thickness; Hewlett–Packard) with
the following temp. program: 60 °C (1 min)–6 °C/min–150 °C
(1 min)–12 °C/min–280 °C (5 min); carrier gas: He; constant flow
1 mL/min; split ratio: 1:30; tR(11) = 17.45, tR(12) = 16.66, tR(13)
= 14.62, tR(14) = 14.79, tR(15) = 15.19, tR(16) = 15.36, tR(18) =
20.43, tR(20) = 8.51, tR(22, Z isomer) = 15.97, tR(22, E isomer) =
16.06, tR(24) = 13.05, tR(25) = 21.81, tR(31) = 18.16, tR(32) = 15.94,
tR(34) = 15.32, tR(35) = 13.70, tR(37) = 12.92, tR(38) = 17.76,
tR(46) = 20.39, tR(47) = 21.36, tR(48) = 6.35 min. MS (ESI) analy-
ses: Bruker Esquire 3000 PLUS spectrometer (ESI Ion Trap LC/
MSn System). Chiral GC analyses: DANI-HT-86.10 gas chromato-
graph; enantiomer excesses determined on a Chirasil DEX CB col-
umn with the following temp. program: compound 13: 70 °C
12: Colourless oil, 96% purity (by GC). 1H NMR (CDCl3,
400 MHz): δ = 0.82 (t, J = 7.1 Hz, 3 H), 2.02 (q, J = 1.0 Hz, 3 H),
2.08 (q, J = 1.0 Hz, 3 H), 3.83 (q, J = 7.1 Hz, 2 H), 7.10–7.15 (m,
2 H), 7.18–7.30 (m, 3 H) ppm. 13C NMR (CDCl3, 100 MHz): δ
= 13.4, 16.2, 21.5, 59.9, 126.1, 126.7, 126.8, 127.8, 142.7, 144.2,
170.4 ppm. GC–MS (EI): m/z (%) = 204 (100) [M]+, 175 (35), 159
(69), 131 (60), 115 (53), 105 (8), 91 (47), 77 (13).
(0 min)–1 °C/min–88 °C
tR[(2S,3S)-13] = 27.1, tR[(2R,3R)-13] = 27.7 min; compound 20:
35 °C (0 min)–0.3 °C/min–45 °C (0 min)–30 °C/min–180 °C
(15 min)–30 °C/min–180 °C
(0 min);
(0 min); tR[(+)-(S)-20] = 24.1, tR[(–)-(R)-20] = 25.1 min. Optical ro-
tations: Jasco-DIP-181 digital polarimeter. 1H and 13C NMR spec-
tra: CDCl3 solutions at room temp.; Bruker AC-400 spectrometer
at 400 and 100 MHz, respectively; chemical shifts in ppm relative
to SiMe4 as an internal standard (δ = 0 ppm), J values in Hz. IR
Ethyl (2E)- and (2Z)-3-Phenylbut-2-enoate (31 and 32): Triethyl
phosphonoacetate (90 g, 401 mmol) was added dropwise under ni-
trogen over a period of 2 h to a stirred suspension of NaH (60%
in mineral oil; 16.2 g, 405 mmol) in dry THF (300 mL) at room
temp. A solution of acetophenone (40 g, 333 mmol) in dry THF
(100 mL) was slowly added to the resulting mixture and the reac-
tion mixture was heated at reflux for 3 h. After cooling, the mixture
was poured onto ice/water and extracted with diethyl ether
(3ϫ200 mL). The organic phase was washed with brine, dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by chromatography with hexane/diethyl ether (95:5–
9:1) as eluent to afford pure esters 31 (43 g, 68%) and 32 (9.5 g,
17%).
31: Colourless oil, 99% purity (by GC). 1H NMR (CDCl3,
400 MHz): δ = 1.31 (t, J = 7.1 Hz, 3 H), 2.57 (d, J = 1.3 Hz, 3 H),
4.21 (q, J = 7.1 Hz, 2 H), 6.13 (q, J = 1.3 Hz, 1 H), 7.30–7.40 (m,
3 H), 7.43–7.50 (m, 2 H) ppm. 13C NMR (CDCl3, 100 MHz): δ =
14.3, 17.9, 59.8, 117.2, 126.3, 128.4, 128.9, 142.3, 155.4, 166.8 ppm.
GC–MS (EI): m/z (%) = 190 (80) [M]+, 175 (6), 161 (47), 145 (100),
115 (70), 102 (8), 91 (25), 77 (7).
spectra: Perkin–Elmer 2000 FT-IR spectrometer; ν in cm–1. Melting
points: Reichert apparatus equipped with a Reichert microscope
values are uncorrected.
˜
Acquisition of the 2H NMR Spectra: The 2H NMR spectra were
recorded with a Bruker Avance 500 spectrometer equipped with a
10 mm probe head and a 19F lock channel under CPD (Waltz 16
sequence) proton decoupling conditions at a temperature of 305 K.
The solutions were prepared by dissolving 20–100 mg of material
in CHCl3 or C6H6 (ca. 3.0 mL) adding about C6F6 (40 µL) for the
lock. The spectra were recorded by performing 64–2048 scans de-
pending on the solution concentration and the deuteriation grade
using the following acquisition parameters: 5.4 s acquisition time,
1530 Hz spectral width, 16 K time domain and 1 s delay. The spec-
tra were Fourier transformed with a line-broadening of 0.3 Hz,
manually phased and integrated after accurate correction of the
base line. For partially overlapped signals the peak areas were de-
termined by the deconvolution routine of the Bruker TopSpin
NMR software using a Lorentzian line shape.
32: Colourless oil, 97% purity (by GC). 1H NMR (CDCl3,
400 MHz): δ = 1.06 (t, J = 7.1 Hz, 3 H), 2.16 (d, J = 1.4 Hz, 3 H),
3.98 (q, J = 7.1 Hz, 2 H), 5.90 (q, J = 1.4 Hz, 1 H), 7.16–7.40 (m,
5 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 13.8, 27.0, 59.6,
117.8, 126.8, 127.6, 127.8, 140.8, 155.0, 165.8 ppm. GC–MS (EI):
m/z (%) = 190 (83) [M]+, 175 (6), 161 (49), 145 (100), 115 (74), 102
(9), 91 (28), 77 (9).
The spectra of the products obtained by incubation of the alde-
hydes with baker’s yeast in tap water with 1% D2O show the pres-
ence of molecules with several deuteriated sites. However, due to
the low concentration of D2O they are not polydeuteriated mole-
cules but rather an assembly of monodeuteriated species. For this
reason their 2H NMR spectra do not exhibit mutual isotope effects
on the chemical shifts of the deuterium nuclei. On the contrary,
polydeuteriated molecules were obtained by chemical reduction
(2SR,3SR)- and (2RS,3SR)-2-Methyl-3-phenylbutan-1-ol (13 and
14): A solution of ester 11 (1 g, 4.9 mmol) in EtOAc (50 mL) was
hydrogenated at atmospheric pressure using 10% Pd/C (100 mg) as
the catalyst. After the reaction was complete (1 h), the catalyst was
filtered and the solution was concentrated in vacuo. The residue
2
with their H NMR spectra exhibiting line-broadening and signal
multiplicity due to isotope effects. The isotope shifts are of the
order of 0.02 ppm upfield for nuclei separated by two bonds and
6166
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6160–6171

Biohydrogenation of Double Bonds of Cinnamaldehydes
1
was dissolved in dry ether (80 mL) and then treated with LiAlH4
GC). H NMR (CDCl3, 400 MHz): δ = 1.66 (q, J = 1.4 Hz, 3 H),
(190 mg, 5 mmol). Work-up with 5% aq. HCl (80 mL) afforded an 2.46 (q, J = 1.4 Hz, 3 H), 7.15–7.20 (m, 2 H), 7.29–7.43 (m, 3
oil that was purified by chromatography to give pure alcohol 13
(0.72 g, 90%) as a colourless oil (98% purity by GC). 1H NMR
(CDCl3, 400 MHz): δ = 0.98 (d, J = 6.9 Hz, 3 H), 1.24 (d, J =
H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 12.9, 19.3, 126.7, 127.8,
128.4, 133.2, 142.8, 155.3, 191.7 ppm. GC–MS (EI): m/z (%) = 160
(50) [M]+, 159 (100), 145 (22), 131 (16), 115 (32), 103 (4), 91 (24),
6.9 Hz, 3 H), 1.37 (br. s, 1 H), 1.76–1.88 (m, 1 H), 2.68 (quint., J 77 (10).
= 7.3 Hz, 1 H), 3.27 (dd, J = 6.4, 10.6 Hz, 1 H), 3.44 (dd, J = 5.1,
(2Z)-2-Methyl-3-phenylbut-2-enal (16): The procedure described
10.6 Hz, 1 H), 7.14–7.21 (m, 3 H), 7.23–7.31 (m, 2 H) ppm. 13C
NMR (CDCl3, 100 MHz): δ = 14.1, 18.0, 41.7, 42.2, 66.7, 126.0,
127.4, 128.3, 146.5 ppm. GC–MS (EI): m/z (%) = 164 (11) [M]+,
146 (9), 131 (13), 117 (5), 105 (100), 91 (25), 77 (10).
above was repeated starting from ester 12 (15 g, 73.5 mmol) to af-
ford aldehyde 16 (9.7 g, 82%) as a colourless oil (95% purity by
1
GC). H NMR (CDCl3, 400 MHz): δ = 1.92 (q, J = 1.0 Hz, 3 H),
2.27 (q, J = 1.0 Hz, 3 H), 7.18–7.25 (m, 2 H), 7.31–7.41 (m, 3
H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 11.1, 23.4, 128.2, 128.7,
134.4, 140.2, 157.4, 193.5 ppm. GC–MS (EI): m/z (%) = 160 (57)
[M]+, 159 (100), 145 (22), 131 (17), 115 (32), 103 (4), 91 (25), 78
(10).
The procedure described above was repeated starting from ester 12
(1 g, 4.9 mmol) to afford alcohol 14 (0.73 g, 91%) as a colourless
oil (96% purity by GC). 1H NMR (CDCl3, 400 MHz): δ = 0.79 (d,
J = 6.9 Hz, 3 H), 1.29 (d, J = 7.1 Hz, 3 H), 1.49 (br. s, 1 H), 1.77–
1.89 (m, 1 H), 2.78 (quint., J = 7.1 Hz, 1 H), 3.51 (dd, J = 6.1,
10.7 Hz, 1 H), 3.61 (dd, J = 5.2, 10.7 Hz, 1 H), 7.14–7.21 (m, 3 H),
7.23–7.31 (m, 2 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 14.5,
19.1, 41.5, 41.7, 66.1, 126.0, 127.9, 128.1, 145.4 ppm. GC–MS (EI):
m/z (%) = 164 (20) [M]+, 146 (15), 131 (13), 117 (10), 105 (100), 91
(40), 77 (18).
(2E)-3-Phenylbut-2-enal (34): The procedure described above was
repeated starting from ester 31 (20 g, 105.3 mmol) to afford alde-
1
hyde 34 (12.9 g, 84%) as a colourless oil (97% purity by GC). H
NMR (CDCl3, 400 MHz): δ = 2.56 (d, J = 1.2 Hz, 3 H), 6.38 (dq,
J = 7.6, 1.2 Hz, 1 H), 7.38–7.44 (m, 3 H), 7.50–7.57 (m, 2 H), 10.17
(d, J = 7.6 Hz, 1 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 16.3,
126.2, 127.2, 128.7, 130.0, 140.6, 157.4, 191.0 ppm. GC–MS (EI):
m/z (%) = 146 (44) [M]+, 145 (100), 131 (24), 115 (41), 103 (14), 91
(17), 78 (10).
(2SR,3RS)-1,2,3-Trideuterio-2-methyl-3-phenylpropan-1-ol (30): A
solution of 2-methylcinnamaldehyde (1 g, 6.8 mmol) in EtOAc
(50 mL) was stirred in the presence of deuterium at atmospheric
pressure using 10% Pd/C (100 mg) as the catalyst. After the reac-
tion was complete (24 h), the catalyst was filtered and the solution
was stirred with water (50 mL) for 1 h. Then the organic phase was
separated, dried (Na2SO4) and concentrated in vacuo. The residue
was purified by chromatography to give alcohol 30 (0.81 g, 78%),
which was quantitatively transformed into the acetyl derivative. 1H
NMR (CDCl3 500 MHz): δ = 0.93 (s, 3 H, CH3), 2.07 (s, 3 H,
COCH3), 2.44 (br. s, 0.9 H, 3-H), 2.73 (d, J = 13.4 Hz, 0.1 H, 3Ј-
H), 3.90 (br. s, 0.4 H, 1Ј-H), 3.94 (br. s, 0.4 H, 1-H), 7.31–7.15 (m,
(2Z)-3-Phenylbut-2-enal (35): The procedure described above was
repeated starting from ester 32 (10 g, 52.6 mmol) to afford aldehyde
1
35 (6.1 g, 79%) as a colourless oil (95% purity by GC). H NMR
(CDCl3, 400 MHz): δ = 2.30 (d, J = 1.3 Hz, 3 H), 6.13 (dq, J =
8.2, 1.3 Hz, 1 H), 7.26–7.33 (m, 2 H), 7.37–7.42 (m, 3 H), 9.47 (d,
J = 8.2 Hz, 1 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 26.3,
128.2, 128.3, 129.0, 129.1, 138.4, 161.8, 193.1 ppm. GC–MS (EI):
m/z (%) = 146 (47) [M]+, 145 (100), 131 (25), 115 (42), 103 (16), 91
(17), 78 (12).
2
5 H, Ph) ppm. H NMR (CHCl3 76.7 MHz): δ = 2.12 (br. s, 1 D,
3-Chloro-2-methylcinnamaldehyde (22): Dry DMF (90 mL) was
added dropwise whilst stirring to phosphorus oxychloride (70 mL,
765 mmol) at 0 °C. The resulting solution was stirred for a further
1 h and then propiophenone (50 g, 373 mmol) was added dropwise.
The mixture was kept at 0 °C for 5 h and then at room temp. for
2 h. The reaction was quenched by the addition of crushed ice and
extracted with diethyl ether (2ϫ200 mL). The organic phase was
washed with saturated aq. Na2CO3 and then with brine and then
dried (Na2SO4). The solvent was removed in vacuo and the residue
distilled (b.p. 78–80 °C/0.1 Torr) to give aldehyde 22 (60.4 g, 90%)
as a pale-yellow oil as a 92:8 mixture (by GC analysis) of E/Z
isomers.
2-D), 2.74 (br. s, 1 D, 3-D), 3.94 (br. s, 0.5 D, 1Ј-D), 3.99 (br. s, 0.5
D, 1-D ppm (see Figure 3, C).
(2RS,3SR)-2,3-Dideuterio-3-phenylbutan-1-ol (33): A solution of es-
ter 31 (1 g, 5.3 mmol) in EtOAc (50 mL) was stirred in the presence
of deuterium at atmospheric pressure using 10% Pd/C (100 mg) as
the catalyst. After the reaction was complete (2 h), the catalyst was
filtered and the solution was concentrated in vacuo. The residue
was dissolved in dry diethyl ether (80 mL) and then treated with
LiAlH4 (200 mg, 5.3 mmol). Work-up with 5% aq. HCl (80 mL)
afforded an oil that was purified by chromatography to give pure
alcohol 33 (0.70 g, 87%). 1H NMR (C6D6 500 MHz): δ = 0.66 (br.
s, 1 H, OH), 1.12 (s, 2.5 H, CH3), 1.66–1.57 (m, 1.32 H, 2-H, 2Ј-
H), 2.73 (quint., J = 7.0 Hz, 0.38 H, 3-H), 3.31–3.23 (m, 2 H, 1-H,
1
(E) Isomer: H NMR (CDCl3, 400 MHz): δ = 2.08 (s, 3 H), 7.39–
7.47 (m, 5 H), 9.48 (s, 1 H) ppm. 13C NMR (CDCl3, 100 MHz): δ
= 13.2, 128.4, 130.0, 130.2, 135.8, 136.3, 154.2, 190.1 ppm. GC–
MS (EI): m/z (%) = 182 (18) [M]+, 181 (42), 180 (52) [M]+, 179
(100), 145 (14), 115 (88), 91 (13), 74 (5), 63 (9).
2
1Ј-H), 7.18–7.05 (m, 5 H, Ph) ppm. H NMR (C6H6 76.7 MHz): δ
= 1.08–1.04 (CH2D + CHD2 + CD3), 1.59 (br. s, 2-D), 2.69 (br. s,
3-D) ppm (see Figure 4, C).
(2E)-2-Methyl-3-phenylbut-2-enal (15): The ester 11 (20 g, 98 mmol)
as a solution in dry diethyl ether (300 mL) was reduced with Li-
AlH4 (3.7 g, 97 mmol) at 0 °C. The reaction was quenched by drop-
wise addition of ethyl acetate (50 mL) followed by the addition of
5% aq. HCl (350 mL). The organic phase was separated and the
aqueous layer extracted with ethyl acetate (2ϫ100 mL). The com-
bined organic phases were washed with brine, dried (Na2SO4) and
concentrated under reduced pressure. The crude allylic alcohol was
dissolved in CHCl3 (250 mL) and treated at reflux with MnO2
(40 g, 460 mmol) for 6 h. The residue obtained upon filtration and
evaporation of the solvent was purified by chromatography to give
pure aldehyde 15 (13.9 g, 89%) as a colourless oil (98% purity by
(1-Phenylethylidene)cyanoacetic Acid (40): Ethyl cyanoacetate (38 g,
336 mmol), acetophenone (40 g, 333 mmol), ammonium acetate
(6 g, 78 mmol) and acetic acid (16 g, 266 mmol) were heated at re-
flux in benzene (100 mL) removing the water formed in a Dean–
Stark apparatus. After cooling, the reaction was quenched by di-
lution with diethyl ether (100 mL) and the addition of water
(300 mL). The organic phase was washed with saturated aq.
Na2CO3 and brine. The solvent was evaporated and the residue was
dissolved in methanol (140 mL) and stirred at room temp. with a
solution of NaOH (13.3 g, 332 mmol) in water (60 mL). After 8 h,
the reaction was diluted with water (300 mL) and extracted with
Eur. J. Org. Chem. 2009, 6160–6171
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6167

G. Fronza, C. Fuganti, S. Serra
FULL PAPER
diethyl ether (100 mL). The organic phase was discarded and the
aqueous phase was acidified by addition of 10% aq. HCl (150 mL)
followed by extraction with ethyl acetate (2 ϫ150 mL). The organic
solvent was dried (Na2SO4) and concentrated under reduced pres-
sure. The residue, consisting of a mixture of isomers, was recrys-
tallized from ethyl acetate to give pure acid 40 (15.5 g, 25%) as
yellow crystals (98% purity by GC analysis of the corresponding
(E)-(2S,3R)-4-Methyl-5-phenylhex-4-ene-2,3-diol [(+)-18]: Colour-
less crystals, 97% purity (by GC); m.p. 78–79 °C. [α]2D0 = +41.5 (c
= 2, CHCl3). 1H NMR (CDCl3, 400 MHz): δ = 1.30 (d, J = 6.3 Hz,
3 H), 1.59 (q, J = 1.4 Hz, 3 H), 2.01 (q, J = 1.4 Hz, 3 H), 2.03 (br.
s, 2 H), 3.94 (quint., J = 6.3 Hz, 1 H), 4.64 (d, J = 6.0 Hz, 1 H),
7.06–7.13 (m, 2 H), 7.19–7.25 (m, 1 H), 7.28–7.36 (m, 2 H) ppm.
13C NMR (CDCl3, 100 MHz): δ = 14.4, 18.5, 20.8, 69.4, 74.7,
126.3, 127.9, 128.2, 130.0, 135.9, 144.7 ppm. MS (ESI): m/z = 229.1
1
methyl ester); m.p. 176–177 °C. H NMR [(CD3)2SO, 400 MHz]: δ
= 2.62 (s, 3 H), 7.45–7.54 (m, 5 H), 13.80 (br. s, 1 H) ppm. 13C [M + Na]+.
NMR [(CD3)2SO 100 MHz]: δ = 22.8, 105.5, 116.5, 127.0, 128.4,
The mixture of alcohol 17 and saturated alcohols 13/14 was dis-
129.9, 140.3, 163.2, 171.0 ppm. MS (ESI): m/z = 210.1 [M + Na]+.
solved in CHCl3 (70 mL) and treated with MnO2 (20 g, 230 mmol),
stirring at reflux for 5 h. The residue obtained upon filtration and
evaporation of the organic phase was purified by column
chromatography using hexane/ethyl acetate (9:1 to 3:1) as eluent to
give recovered aldehyde 15 (2.4 g, 15 mmol) and a mixture of
alcohols 13 and 14 (1.85 g, 18%) as a pale-yellow oil (97% purity
by GC) in a 13/14 ratio of 69:31 (by GC). [α]2D0 = +22.4 (c = 2,
CHCl3); 93% ee (by chiral GC).
2-Deuterio-3-phenylbut-3-enenitrile (41) and 2-Deuterio-3-phen-
ylbut-2-enenitrile (42): A sample of acid 40 (15 g, 80.2 mmol) was
stirred with D2O at 50 °C for 1 h. The mixture was then dried under
reduced pressure and the above procedure was repeated three times.
The deuterium-enriched acid obtained was slowly heated to 200 °C
and then left at this temperature until evolution of carbon dioxide
was complete (1 h). After cooling, the residue was purified by
chromatography using hexane/diethyl ether (95:5–9:1) as eluent to
afford nitriles 41 and 42 (8.7 g, 75%).
Reduction of (2Z)-2-Methyl-3-phenylbut-2-enal (16): The reduction
of aldehyde 16 (5 g, 31.2 mmol) performed according to the general
procedure gave recovered aldehyde 16 (3.6 g, 20 mmol) and a mix-
ture of alcohols 13 and 14 (0.96 g, 19%) as a pale-yellow oil (98%
purity by GC) in a ratio of 13/14 of 59:41 (by GC). [α]2D0 = +8.2 (c
= 2, CHCl3); 33% ee (by chiral GC). No diols were detected in the
reaction mixture.
41: Obtained as a mixture of partially deuteriated molecules. 1H
NMR (CDCl3, 400 MHz): δ = 3.53 (m, 1 H), 5.54–5.52 (m, 0.6 H),
2
5.64–5.62 (m, 0.6 H), 7.42–7.32 (m, 5 H) ppm. H NMR (CDCl3
76.7 MHz): δ = 3.53 (s), 3.54 (s), 5.60 (s), 5.69 (s) ppm.
42: Obtained as a mixture of partially deuteriated molecules. 1H
NMR (CDCl3, 400 MHz): δ = 2.48–2.44 (m, 1.57 H), 5.65 (br. s,
0.4 H), 7.48–7.37 (m, 5 H) ppm. 2H NMR (CDCl3 76.7 MHz): δ =
2.50–2.46 (CH2D + CHD2 + CD3), 5.68 (br. s, 1 D) ppm.
Reduction of 3-Chloro-2-methylcinnamaldehyde (22): The reduction
of the aldehyde 22 (15 g, 83.3 mmol) performed according to the
general procedure gave recovered aldehyde 22 (8 g, 44.4 mmol), sat-
urated alcohol (–)-24 (2.6 g, 21%) and diol (+)-25 (1.1 g, 6%).
2-Deuterio-3-phenylbut-2-enal (43): DIBAH (21 mL of 1.2  solu-
tion in toluene) was added dropwise under nitrogen to a stirred
solution of nitrile 42 (3 g, 20.8 mmol) in dry toluene (50 mL) at
–60 °C. The reaction was stirred at 0 °C for 3 h, then diluted with
diethyl ether (50 mL) and quenched with a saturated solution of
aq. NH4Cl (50 mL) stirring vigorously for 1 h at room temp. The
reaction was extracted with diethyl ether (2ϫ100 mL) and the
combined organic phases were concentrated under reduced pres-
sure. The residue was purified by chromatography eluting with hex-
ane/diethyl ether (95:5–9:1) to give pure aldehyde 43 (1.81 g, 59%).
1H NMR (CDCl3, 400 MHz): δ = 2.56–2.52 (m, 1.5 H), 6.37 (m,
(S)-2-Methyl-3-phenylpropan-1-ol [(–)-24]: Colourless oil, 96% pu-
rity (by GC). [α]2D0 = –8.5 (c = 1, CHCl3) {ref.[22] [α]2D5 = –10.1 (c =
0.8, CHCl3)}. 1H NMR (CDCl3, 400 MHz): δ = 0.89 (d, J =
6.7 Hz, 3 H), 1.84–1.98 (m, 1 H), 2.11 (br. s, 1 H), 2.38 (dd, J =
8.2, 13.5 Hz, 1 H), 2.74 (dd, J = 6.1, 13.5 Hz, 1 H), 3.42 (dd, J =
6.1, 10.5 Hz, 1 H), 3.48 (dd, J = 5.9, 10.5 Hz, 1 H), 7.11–7.20 (m,
3 H), 7.22–7.29 (m, 2 H) ppm. 13C NMR (CDCl3, 100 MHz): δ =
16.3, 37.6, 39.6, 67.4, 125.7, 128.1, 129.0, 140.6 ppm. GC–MS (EI):
m/z (%) = 150 (22) [M]+, 132 (23), 117 (66), 104 (3), 92 (56), 91
(100), 77 (5), 65 (10), 51 (4), 41 (5).
2
(Z)-(2S,3R)-5-Chloro-4-methyl-5-phenylpent-4-ene-2,3-diol [(+)-25]:
0.4 H), 7.54–7.38 (m, 5 H), 10.15 (s, 1 H) ppm. H NMR (CDCl3
76.7 MHz): δ = 2.54–2.50 (CH2D + CHD2 + CD3), 6.38 (br. s, 1
D) ppm.
Pale-yellow crystals, 98% purity (by GC); m.p. 63–65 °C. [α]2D0
=
1
+8.9 (c = 2, CHCl3). H NMR (CDCl3, 400 MHz): δ = 1.29 (d, J
= 6.4 Hz, 3 H), 1.79 (s, 3 H), 2.70 (br. s, 2 H), 4.08–4.20 (m, 1 H),
4.99 (d, J = 4.9 Hz, 1 H), 7.26–7.43 (m, 5 H) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 15.6, 18.1, 69.3, 74.9, 127.7, 128.2, 128.3,
129.0, 133.5, 138.9 ppm. MS (ESI): m/z = 251.0 [M + Na]+, 249.0
[M + Na]+.
General Procedure for Baker’s Yeast Reduction of Aldehydes. Re-
duction of (2E)-2-Methyl-3-phenylbut-2-enal (15): A 5 L open cylin-
drical glass vessel equipped with a mechanical stirrer was charged
with tap water (3 L) and glucose (100 g). Fresh baker’s yeast
(0.5 kg) was added in small pieces to the stirred mixture and the
fermentation was allowed to proceed for 1 h. A solution of the
aldehyde 15 (10 g, 62.5 mmol) in ethanol (25 mL) was added drop-
wise. The vigorous stirring was continued for 4 d at room tempera-
ture. During this time further baker’s yeast (100 g) and glucose
(20 g) were added after 24 h, and again after 48 h after the fermen-
tation had started. The reaction was then interrupted by the ad-
dition of Celite (100 g) and ethyl acetate (400 mL) followed by fil-
tration through a Buchner funnel through a Celite pad. The aque-
ous phase was extracted with ethyl acetate (3ϫ150 mL) and the
combined organic phases were washed with brine, dried (Na2SO4)
and concentrated under reduced pressure to give an oil (15 g). The
latter was purified by chromatography with hexane/diethyl ether
(95:5–1:1) as eluent to afford a mixture of allylic alcohol 17 and
saturated alcohol 13/14 (4.5 g) followed by diol (+)-18 (5.0 g, 39%).
Reduction of (2E)-2-Methylcinnamaldehyde: The reduction of alde-
hyde 27 (10 g, 68.5 mmol) performed according to the general pro-
cedure gave diol (+)-29 (1.95 g, 15%), recovered aldehyde 27 (5.5 g,
55%) and saturated alcohol (–)-24 (1.2 g, 12%) showing 97% pu-
rity (by GC) and [α]2D0 = –8.9 (c = 1, CHCl3).
(E)-(2S,3R)-4-Methyl-5-phenylpent-4-ene-2,3-diol [(+)-29]: Colour-
less crystals, 98% purity (by GC); m.p. 106–107 °C (ref.[16] m.p.
106–107 °C). [α]2D0 = +31.9 (c = 1, EtOH) {ref.[16] [α]2D0 = +32 (c =
1
1, EtOH}. H NMR (CDCl3, 400 MHz): δ = 1.22 (d, J = 6.4 Hz,
3 H), 1.90 (d, J = 1.2 Hz, 3 H), 2.05 (br. s, 1 H), 2.28 (br. s, 1 H),
3.95–4.05 (m, 1 H), 4.16 (d, J = 4.6 Hz, 1 H), 6.60 (s, 1 H), 7.20–
7.37 (m, 5 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 14.6, 17.3,
68.9, 80.4, 126.6, 127.1, 128.1, 129.0, 136.9, 137.2 ppm. MS (ESI):
m/z = 215.1 [M + Na]+.
6168
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6160–6171

Biohydrogenation of Double Bonds of Cinnamaldehydes
Reduction of (2E)-3-Phenylbut-2-enal (34): The reduction of the al-
dehyde 34 (3 g, 20.5 mmol) performed according to the general pro-
cedure gave recovered aldehyde 34 (1.95 g, 65%), saturated alcohol
(+)-37 (0.185 g, 6%) and diol (–)-38 (0.75 g, 19%).
ganic phase was purified by column chromatography using hexane/
ethyl acetate (9:1 to 3:1) as eluent to give recovered aldehyde 34
(1.3 g, 8.9 mmol) and saturated alcohol (+)-37 (0.13 g, 6%).
1
(1R,3S)-[1,2,2,3-2H4]-3-Phenylbutan-1-ol [(+)-37]: H NMR (C6D6
500 MHz): δ = 0.67 (br. s, 1 H, OH), 1.12 (d, J = 7.0 Hz, 3 H,
CH3), 1.67–1.57 (m, 2 H, 2-H, 2Ј-H), 2.74 (qt, J = 7.0 Hz, 3-H),
3.31–3.23 (m, 2 H, 1-H, 1Ј-H), 7.18–7.05 (m, 5 H, Ph) ppm. 2H
NMR (C6D6, 76.7 MHz): δ = 1.57 (s, 0.43 D, 2Ј-D), 1.59 (s, 0.45
D, 2-D), 2.69 (s, 1 D, 3-D), 3.21 (s, 1 D, 1ЈD), 3.24 ppm (see Fig-
ure 4, A).
(S)-3-Phenylbutan-1-ol [(+)-37]: Colourless oil, 98% purity (by
GC). [α]2D0 = +28.6 (c = 2, CHCl3) {ref.[18] [α]2D5 = +25.5 (c = 1.52,
CHCl3)}. H NMR (CDCl3, 400 MHz): δ = 1.26 (br. s, 1 H), 1.27
1
(d, J = 7.0 Hz, 3 H), 1.85 (q, J = 6.8 Hz, 2 H), 2.82–2.93 (m, 1 H),
3.48–3.61 (m, 2 H), 7.15–7.32 (m, 5 H) ppm. 13C NMR (CDCl3,
100 MHz):
δ = 22.3, 36.5, 41.0, 61.2, 126.1, 126.9, 128.4,
146.9 ppm. GC–MS (EI): m/z (%) = 150 (11) [M]+, 132 (18), 117
1
(E)-(2S,3R)-[1,2-2H2]-5-Phenylhex-4-ene-2,3-diol [(–)-38]: H NMR
(46), 105 (100), 91 (28), 77 (16), 65 (3), 51 (6).
(C6D6 500 MHz): δ = 1.08 (d, J = 6.4 Hz, 3 H, CH3), 1.68 (br. s, 2
H, 2 OH), 1.87 (d, J = 1.4 Hz, 3 H, CH3), 3.78 (qd, J = 6.4, 3.6 Hz,
1 H, 2-H), 4.29 (dd, J = 8.6, 3.9 Hz, 1 H, 3-H), 5.89 (dq, J = 8.6,
(E)-(2S,3R)-5-Phenylhex-4-ene-2,3-diol [(–)-38]: Colourless oil, 96%
purity (by GC). [α]2D0 = –4.8 (c = 2, CHCl3) {ref.[21] [α]2D0 = –6.7 (c
= 1, CHCl3)}. 1H NMR (CDCl3, 400 MHz): δ = 1.19 (d, J =
6.4 Hz, 3 H), 2.12 (d, J = 1.3 Hz, 3 H), 2.15 (br. s, 2 H), 3.94 (dq,
J = 3.7, 6.4 Hz, 1 H), 4.49 (dd, J = 3.7, 8.7 Hz, 1 H), 5.84 (dq, J
= 8.7, 1.3 Hz, 1 H), 7.23–7.36 (m, 3 H), 7.37–7.44 (m, 2 H) ppm.
13C NMR (CDCl3, 100 MHz): δ = 16.5, 17.5, 70.4, 72.5, 125.6,
125.8, 127.5, 128.3, 139.7, 142.8 ppm. GC–MS (EI): m/z (%) = 174
(17) [M – H2O]+, 159 (1), 131 (100), 115 (16), 103 (4), 91 (35), 77
(6).
2
1.3 Hz, 1 H, 4-H), 7.30–7.07 (m, 5 H, Ph) ppm. H NMR (C6D6,
76.7 MHz): δ = 1.06 (s, CH3), 3.75 (s, 2-D) ppm (see Figure 5).
Reduction of (2Z)-3-Phenylbut-2-enal (35): The reduction of the al-
dehyde 35 (2 g, 13.7 mmol) performed according to the general pro-
cedure gave recovered aldehyde 35 (1.2 g, 8.2 mmol) and saturated
alcohol (+)-37 (0.11 g, 5%). No diol was detected in the reaction
mixture.
(1R,2S)-[1,2,3,3-2H4]-2-Methyl-3-phenylpropyl Acetate: 1H NMR
(CDCl3 500 MHz): δ = 0.94 (d, J = 6.7 Hz, 3 H, CH3), 2.07 (s, 3
H, COCH3), 2.13 (m, 1 H, 2-H), 2.47 (dd, J = 7.8, 13.4 Hz, 1 H,
3Ј-H), 2.75 (dd, J = 6.4, 13.4 Hz, 3-H), 3.93 (dd, J = 6.4, 10.8 Hz,
1 H, 1Ј-H), 3.98 (dd, J = 6.2, 10.8 Hz, 1 H, 1-H), 7.15–7.31 (m, 5
Reduction of (2Z)-3-Phenylbut-2-enal (35): The reduction of alde-
hyde 35 (3.2 g, 21.9 mmol) performed according to the general pro-
cedure gave recovered aldehyde 35 (2.4 g, 16.4 mmol) and saturated
alcohol (+)-37 (0.16 g, 5%) showing 97% purity (by GC) and
[α]2D0 = +25.0 (c = 2, CHCl3). No diol was detected in the reaction
mixture.
2
H, Ph) ppm. H NMR (CHCl3 76.7 MHz): δ = 2.13 (br. s, 1 D, 2-
D), 2.47 (br. s, 0.52 D, 3Ј-D), 2.75 (br. s, 0.48 D, 3-D), 3.94 (br. s,
0.75 D, 1Ј-D), 3.98 (br. s, 0.25 D, 1-D) ppm (see Figure 3, A). The
minor signal (1-D, ca. 25%) belongs to the isotopomer (1R,2R)-
[1,2,3-2H3]-2-methyl-3-phenylpropan-1-ol acetate.
Reduction of 2-Deuterio-3-phenylbut-2-enal (43): The reduction of
aldehyde 43 (1 g, 6.8 mmol) performed according to the general
procedure gave saturated alcohol (+)-44 (75 mg, 7%) and recovered
aldehyde 43 (0.59 g, 4 mmol).
(1R,2S,3S)-[1,2,3-2H3]-2-Methyl-3-phenylpropyl Acetate: 1H NMR
(CDCl3 500 MHz): δ = 0.94 (d, J = 6.7 Hz, 3 H, CH3), 2.07 (s, 3
H, COCH3), 2.13 (m, 1 H, 2-H), 2.47 (dd, J = 7.8, 13.4 Hz, 1 H,
3Ј-H), 2.75 (dd, J = 6.4, 13.4 Hz, 3-H), 3.93 (dd, J = 6.4, 10.8 Hz,
1 H, 1Ј-H), 3.98 (dd, J = 6.2, 10.8 Hz, 1 H, 1-H), 7.15–7.31 (m, 5
(3S)-[2,2-2H2]-3-Phenylbutan-1-ol [(+)-44]: 1H NMR (C6D6
500 MHz): δ = 0.57 (br. s, 1 H, OH), 1.13–1.07 (m, 2.3 H, CH3 +
CH2D + CHD2), 1.67–1.57 (m, 1.8 H, 2-H, 2Ј-H), 2.76–2.70 (m, 1
H, 3-H), 3.31–3.23 (m, 2 H, 1-H + 1Ј-H), 7.18–7.05 (m, 5 H,
Ph) ppm. 2H NMR (C6D6, 76.7 MHz): δ = 1.08–1.04 (CH2D +
CHD2 + CD3), 1.56 (s, 2Ј-D), 1.59 (s, 2-D) ppm (ratio 2-D/2Ј-D =
55:45; see Figure 4, B).
2
H, Ph) ppm. H NMR (CHCl3 76.7 MHz): δ = 2.13 (br. s, 1 D, 2-
D), 2.47 (br. s, 0.85 D, 3Ј-D), 2.75 (br. s, 0.15 D, 3-D), 3.94 (br. s,
0.85 D, 1Ј-D), 3.98 (br. s, 0.15 D, 1-D) ppm (see Figure 3, B). The
minor signals (ca. 15%) belong to the isotopomer (1R,2R)-[1,2,3-
2H3]-2-methyl-3-phenylpropan-1-ol acetate.
General Procedure for Baker’s Yeast Reduction of Aldehydes in the
Presence of D2O. Reduction of (2E)-3-Phenylbut-2-enal (34): A 3 L
open cylindrical glass vessel equipped with a mechanical stirrer was
charged with tap water (1 L), D2O (13 mL) and glucose (50 g).
Fresh baker’s yeast (0.2 kg) was added in small pieces to the stirred
mixture and the fermentation was allowed to proceed for 1 h. A
solution of aldehyde 34 (2 g, 13.7 mmol) in ethanol (10 mL) was
added dropwise. The vigorous stirring was continued for 3 d at
room temperature. During this time further baker’s yeast (50 g) and
glucose (10 g) were added after 24 h, and again after 48 h after the
fermentation had started. The reaction was then interrupted by the
addition of Celite (50 g) and ethyl acetate (200 mL) followed by
filtration through a Buchner funnel through a Celite pad. The
aqueous phase was extracted with ethyl acetate (2ϫ100 mL) and
the combined organic phases were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure to give an oil
(3.5 g). The latter was purified by chromatography with hexane/
diethyl ether (95:5–1:1) as eluent to afford a mixture of allylic
alcohol 36 and saturated alcohol (+)-37 (1.4 g) followed by diol
(–)-38 (0.48 g, 18%). This mixture was dissolved in CHCl3 (50 mL)
and treated with MnO2 (5 g, 57.5 mmol), stirring at reflux for 5 h.
The residue obtained upon filtration and evaporation of the or-
General Procedure for the Determination of the Absolute Configura-
tions of Alcohols 13 and 14: A solution of p-toluenesulfonyl chloride
(0.63 g, 3.3 mmol) in CH2Cl2 (5 mL) was added dropwise to a
stirred solution of the mixture of alcohols 13 and 14 {0.5 g,
3 mmol; [α]2D0 = +22.4 (c = 2, CHCl3)} in pyridine (5 mL). After
4 h, the mixture was diluted with diethyl ether (90 mL) and washed
in turn with 1  aq. HCl solution (100 mL), saturated NaHCO3
solution (50 mL) and brine. The organic phase was dried (Na2SO4)
and concentrated in vacuo. The residue was dissolved in dry diethyl
ether (20 mL) and added dropwise to a solution of LiAlH4 (0.15 g,
3.9 mmol) at reflux in diethyl ether (50 mL). After 1 h, the reaction
was cooled (0 °C) and quenched by dropwise addition of water
(1 mL) and a 20% aqueous solution of NaOH (5 mL). The diethyl
ether layer was separated, washed with brine, dried (Na2SO4) and
the solvent evaporated in vacuo. The residue was purified by
chromatography with hexane/diethyl ether (95:5) as eluent and the
product was further purified by bulb-to-bulb distillation to afford
pure (+)-20 (0.39 g, 86%).
(S)-(1,2-Dimethylpropyl)benzene [(+)-20]: Colourless oil, 98% pu-
rity (by GC). [α]2D0 = +28.4 (c = 2.5, CCl4) {ref.[14a] [α]2D3.5 = –27.2
Eur. J. Org. Chem. 2009, 6160–6171
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6169

G. Fronza, C. Fuganti, S. Serra
FULL PAPER
(c = 2.39, CCl4)} for the (R) isomer. 1H NMR (CDCl3, 400 MHz):
The above described compound was ozonized to give (–)-48 (0.31 g,
δ = 0.75 (d, J = 6.8 Hz, 3 H), 0.93 (d, J = 6.8 Hz, 3 H), 1.23 (d, J 84%; 98% purity by GC). [α]2D0 = –81.9 (c = 1.5, CHCl3).
= 7.0 Hz, 3 H), 1.70–1.83 (m, 1 H), 2.41 (quint., J = 7.2 Hz, 1 H),
7.11–7.18 (m, 3 H), 7.22–7.30 (m, 2 H) ppm. 13C NMR (CDCl3,
[1] F. G. Fischer, Angew. Chem. 1940, 53, 461–471.
100 MHz): δ = 18.7, 20.1, 21.1, 34.4, 46.9, 125.7, 127.6, 128.0,
[2] a) D. Seebach, H.-O. Kalinowski, Nachr. Chem. Tech. Lab.
147.1 ppm. GC–MS (EI): m/z (%) = 148 (55) [M]+, 115 (9), 105
1976, 24, 415; b) R. Csuk, B. I. Glänzer, Chem. Rev. 1991, 91,
(100), 91 (31), 77 (25), 65 (4), 51 (7).
49–97; c) S. M. A. De Wildeman, T. Sonke, H. E. Schoemaker,
O. May, Acc. Chem. Res. 2007, 40, 1260–1266.
The procedure described above was repeated starting from the mix-
ture of alcohols 13 and 14 (0.45 g, 2.8 mmol) showing [α]2D0 = +8.2
(c = 2, CHCl3) to give (+)-20 (0.33 g, 80%; 96% purity by GC).
[α]2D0 = +10.5 (c = 2.5, CCl4).
[3] a) C. Fuganti, S. Serra, A. Dulio, J. Chem. Soc. Perkin Trans.
1 1999, 279–282; b) C. Fuganti, S. Serra, J. Chem. Soc. Perkin
Trans. 1 2000, 97–101; c) C. Fuganti, S. Serra, J. Chem. Soc.
Perkin Trans. 1 2000, 3758–3764; d) S. Serra, C. Fuganti, Tetra-
hedron: Asymmetry 2001, 12, 2191–2196; e) A. Abate, E.
Brenna, C. Dei Negri, C. Fuganti, S. Serra, Tetrahedron: Asym-
metry 2002, 13, 899–904; f) E. Brenna, C. Fuganti, S. Ronzani,
S. Serra, Can. J. Chem. 2002, 80, 714–723; g) S. Serra, C. Fu-
ganti, F. G. Gatti, Eur. J. Org. Chem. 2008, 1031–1037.
[4] a) C. Fuganti, D. Ghiringhelli, P. Grasselli, J. Chem. Soc.,
Chem. Commun. 1975, 846–847; b) B. Sedgwick, C. Morris, J.
Chem. Soc., Chem. Commun. 1980, 96–97; c) G. Fronza, C.
Fuganti, P. Grasselli, M. Barbeni, Tetrahedron Lett. 1992, 33,
6375–6378; d) G. Fronza, C. Fuganti, P. Grasselli, A. Mele, A.
Sarra, G. Allegrone, M. Barbeni, Tetrahedron Lett. 1993, 34,
6467–6470; e) G. Fronza, C. Fuganti, P. Grasselli, S. Lanati,
R. Rallo, S. Tchilibon, J. Chem. Soc. Perkin Trans. 1 1994,
2927–2930; f) Y. Kawai, M. Hayashi, Y. Inaba, K. Saitou, A.
Ohno, Tetrahedron Lett. 1998, 39, 5225–5228.
General Procedure for the Determination of the Absolute Configura-
tion of Diols (+)-18 and (+)-25: A solution of diol (+)-18 (2 g,
9.7 mmol) in methanol (20 mL) and 2,2-dimethoxypropane
(30 mL) was treated with pyridinium p-toluenesulfonate (0.1 g,
0.4 mmol) stirring at room temp. for 12 h. The reaction was then
diluted with CH2Cl2 (100 mL), washed with saturated aq. Na2CO3
and brine, dried (Na2SO4) and concentrated under reduced pres-
sure. The residue was purified by chromatography with hexane/di-
ethyl ether (95:5–9:1) as eluent to afford pure (–)-46 (2.2 g, 92%).
(4S,5R)-2,2,4-Trimethyl-5-[(E)-1-methyl-2-phenylpropenyl]-1,3-dioxo-
lane [(–)-46]: Colourless oil, 98% purity (by GC). [α]2D0 = –34.6 (c
= 2, CHCl3). 1H NMR (CDCl3, 400 MHz): δ = 1.19 (d, J = 6.4 Hz,
3 H), 1.41 (s, 3 H), 1.53 (s, 3 H), 1.58 (q, J = 1.3 Hz, 3 H), 1.93 (q,
J = 1.3 Hz, 3 H), 4.54 (quint., J = 6.6 Hz, 1 H), 5.17 (d, J = 7.2 Hz,
1 H), 7.06–7.12 (m, 2 H), 7.17–7.25 (m, 1 H), 7.26–7.35 (m, 2
H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 16.1, 16.8, 20.6, 24.8,
27.0, 74.1, 77.7, 107.6, 126.2, 128.0, 128.2, 129.6, 133.5, 144.9 ppm.
GC–MS (EI): m/z (%) = 246 (1) [M]+, 202 (55), 187 (63), 171 (26),
156 (18), 144 (24), 129 (100), 115 (15), 103 (5), 86 (15), 77 (7).
[5] W. S. Knowles, Angew. Chem. 2002, 114, 2096–2107; Angew.
Chem. Int. Ed. 2002, 41, 1998–2007.
[6] a) R. Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin. Chem.
Biol. 2007, 11, 203–213; b) C. Stueckler, M. Hall, H. Ehammer,
E. Pointner, W. Kroutil, P. Macheroux, K. Faber, Org. Lett.
2007, 9, 5409–5411; c) A. Müller, R. Stürmer, B. Hauer, B.
Rosche, Angew. Chem. 2007, 119, 3380–3382; Angew. Chem.
Int. Ed. 2007, 46, 3316–3318; d) M. Hall, C. Stueckler, H.
Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B. Hauer, R.
Stuermer, W. Kroutil, P. Macheroux, K. Faber, Adv. Synth. Ca-
tal. 2008, 350, 411–418; e) H. S. Toogood, A. Fryszkowska, V.
Hare, K. Fisher, A. Roujeinikova, D. Leys, J. M. Gardiner,
G. M. Stephens, N. S. Scrutton, Adv. Synth. Catal. 2008, 350,
2789–2803.
A solution of 46 (2.1 g, 8.5 mmol) in CH2Cl2 (100 mL) and MeOH
(20 mL) was treated with a stream of ozone at –78° until the ap-
pearance of a persistent blue colour. Nitrogen was then bubbled
through the solution until it turned colorless and the reaction was
treated with a solution of Ph3P (3 g, 11.4 mmol) in CH2Cl2
(30 mL). The solution obtained was gradually warmed to room
temp. and then concentrated at reduced pressure. The residue was
purified by chromatography with hexane/diethyl ether (95:5 to 8:2)
as eluent and the product was further purified by bulb-to-bulb dis-
tillation to afford pure (–)-48 (0.95 g, 71%).
[7] a) G. Fronza, C. Fuganti, M. Mendozza, R. Rigoni, S. Servi,
G. Zucchi, Pure Appl. Chem. 1996, 68, 2065–2071; b) G.
Fronza, C. Fuganti, G. Pedrocchi-Fantoni, S. Serra, G. Zucchi,
C. Fauhl, C. Guillou, F. Reniero, J. Agric. Food Chem. 1999,
47, 1150–1155; c) H.-L. Schmidt, R. A. Werner, W. Eisenreich,
Phytochem. Rev. 2003, 2, 61–85; d) G. Fronza, C. Fuganti, M.
Pinciroli, S. Serra, Tetrahedron: Asymmetry 2004, 15, 3073–
3077; e) E. Brenna, G. Fronza, C. Fuganti, F. G. Gatti, V.
Grande, S. Serra, C. Guillou, F. Reniero, F. Serra, J. Agric.
Food Chem. 2005, 53, 9383–9388.
1-[(4S,5S)-2,2,5-Trimethyl-1,3-dioxolan-4-yl]ethanone [(–)-48]: Colour-
less oil, 98% purity (by GC). [α]2D0 = –83.7 (c = 1.5, CHCl3) {ref.[16]
[α]2D0 = –80 (c = 1, CHCl3)}. 1H NMR (CDCl3, 400 MHz): δ = 1.16
(d, J = 6.4 Hz, 3 H), 1.38 (s, 3 H), 1.60 (s, 3 H), 2.21 (s, 3 H), 4.36
(d, J = 7.7 Hz, 1 H), 4.52 (dq, J = 7.7, 6.4 Hz, 1 H) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 15.8, 24.8, 27.0, 28.1, 73.7, 83.1, 109.7,
209.5 ppm. GC–MS (EI): m/z (%) = 143 (32) [M – Me]+, 115 (100),
99 (51), 74 (9), 59 (96), 43 (85).
[8] Y. Kawai, Y. Inaba, N. Tokitoh, Tetrahedron: Asymmetry 2001,
12, 309–318.
[9] a) G. Fogliato, G. Fronza, C. Fuganti, S. Lanati, R. Rallo, R.
Rigoni, S. Servi, Tetrahedron 1995, 51, 10231–10240; b) G.
Fronza, C. Fuganti, M. Mendozza, R. S. Rallo, G. Ottolina,
D. Joulain, Tetrahedron 1996, 52, 4041–4052.
[10] D. J. Bougioukou, J. D. Stewart, J. Am. Chem. Soc. 2008, 130,
7655–7658.
[11] G. Gallagher, R. L. Webb, Synthesis 1974, 122–124.
[12] a) K. Ishihara, N. Hanaki, H. Yamamoto, J. Am. Chem. Soc.
1993, 115, 10695–10704; b) Q. Zhu, Y. Lu, Org. Lett. 2008, 10,
4803–4806; c) E. Reyes, J. L. Vicario, L. Carrillo, D. Badìa, U.
Uria, A. Iza, J. Org. Chem. 2006, 71, 7763–7772.
[13] a) C. Fuganti, P. Grasselli, Chem. Ind. (London) 1977, 983; b)
M. Breuer, B. Hauer, Curr. Opin. Biotechnol. 2003, 14, 570–
576.
The transformation of diol (+)-25 (0.6 g, 2.6 mmol) performed ac-
cording to the general procedure gave ketal (–)-47 (0.62 g,
2.3 mmol, 88%).
(4R,5S)-4-[(Z)-2-Chloro-1-methyl-2-phenylvinyl]-2,2,5-trimethyl-
1,3-dioxolane [(–)-47]: Colourless oil, 97% purity (by GC). [α]2D0
=
1
–66.4 (c = 1.5, CHCl3). H NMR (CDCl3, 400 MHz): δ = 1.23 (d,
J = 6.3 Hz, 3 H), 1.42 (s, 3 H), 1.53 (s, 3 H), 1.75 (s, 3 H), 4.67
(quint., J = 6.5 Hz, 1 H), 5.36 (d, J = 7.2 Hz, 1 H), 7.27–7.40 (m,
5 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 15.7, 17.5, 24.7, 26.9,
74.0, 78.4, 108.0, 126.8, 128.3, 128.3, 129.1, 133.3, 138.8 ppm. GC–
MS (EI): m/z (%) = 266 (Ͻ1) [M]+, 251 (3), 222 (20), 187 (100),
173 (16), 159 (14), 145 (5), 129 (57), 115 (19), 86 (4), 77 (4).
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Eur. J. Org. Chem. 2009, 6160–6171

Biohydrogenation of Double Bonds of Cinnamaldehydes
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Published Online: October 23, 2009
Eur. J. Org. Chem. 2009, 6160–6171
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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