Bioreduction of α-Acetoxymethyl Enones: Proposal for an SN2′ Mechanism Catalyzed by Enereductase

Article abstract of DOI:10.1002/adsc.201600601

(Z)-3-Acetoxymethyl-4-R-3-buten-2-ones (R=aryl, alkyl) and (Z)-3-methyl-4-R-3-buten-2-ones (R=aryl) were synthesized and submitted to reduction by the yeast Saccharomyces cerevisiae producing the (R)- and (S)-4-R-3-methybutan-2-ones, respectively. This stereochemistry control strategy was applied in the syntheses of (R)- and (S)-Tropional with moderate to high enantiomeric excesses. Other (Z)-3-acyloxymethyl-4-phenyl-3-buten-2-ones showed similar behavior to the (Z)-3-acetoxymethyl counterpart, and the acylated Morita–Baylis–Hillman adduct 1-acetoxy-2-methylene-1-phenylbutan-3-one produced a mixture of products, with and without the acetoxy group, via three different reaction pathways. In addition to experiments employing whole cells, those in which isolated enereductases were used suggested that the main pathway through which the loss of the acetoxy group occurs during the biocatalytic cascade is an S_N2′-type reaction, rather than formal hydrogen addition followed by acetic acid elimination. Finally, related ethyl enones were reduced enantioselectively by the yeast Candida albicans, producing both (R)- and (S)-reduction products, depending on the presence of the acetoxy group in the starting material. (Figure presented.).

Full text of DOI:10.1002/adsc.201600601

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DOI: 10.1002/adsc.201600601
Bioreduction of a-Acetoxymethyl Enones: Proposal for an SN2’
Mechanism Catalyzed by Enereductase
Bruno R. S. Paula,a Davila Zampieri,a J. Augusto R. Rodrigues,a
and Paulo J. S. Morana,*
a
Institute of Chemistry, University of Campinas, 13084-971 Campinas-SP, Brazil
Fax : (+55)-19-3521-3023; phone: (+55)-19-3521-3048; e-mail: moran@iqm.unicamp.br
Received: June 10, 2016; Revised: August 23, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201600601.
Abstract: (Z)-3-Acetoxymethyl-4-R-3-buten-2-ones ways. In addition to experiments employing whole
(R=aryl, alkyl) and (Z)-3-methyl-4-R-3-buten-2- cells, those in which isolated enereductases were
ones (R=aryl) were synthesized and submitted to used suggested that the main pathway through which
reduction by the yeast Saccharomyces cerevisiae pro- the loss of the acetoxy group occurs during the bio-
ducing the (R)- and (S)-4-R-3-methybutan-2-ones, catalytic cascade is an SN2’-type reaction, rather than
respectively. This stereochemistry control strategy formal hydrogen addition followed by acetic acid
was applied in the syntheses of (R)- and (S)-Tropio- elimination. Finally, related ethyl enones were re-
nalꢀ with moderate to high enantiomeric excesses. duced enantioselectively by the yeast Candida albi-
Other (Z)-3-acyloxymethyl-4-phenyl-3-buten-2-ones cans, producing both (R)- and (S)-reduction prod-
showed similar behavior to the (Z)-3-acetoxymethyl ucts, depending on the presence of the acetoxy group
counterpart, and the acylated Morita–Baylis–Hillman in the starting material.
adduct 1-acetoxy-2-methylene-1-phenylbutan-3-one
produced a mixture of products, with and without Keywords: biotransformation; enantioselectivity;
the acetoxy group, via three different reaction path- enones; enzyme catalysis
Introduction
were shown to occur as follows: after the initial
alkene reduction, dehydrohalogenations produced
The asymmetric biocatalytic reduction of activated al- new enals that underwent further C=C reductions,
kenes is a powerful tool in the production of small forming saturated aldehydes.[7] In another example,
chiral molecules. The bioreduction of several a,b-un- the reduction of polyhalogenated esters by enereduc-
saturated aldehydes,[1] ketones,[2] esters,[3] nitroal- tases resulted in dehalogenated esters, and when sub-
kanes[4] and nitriles[5] using microorganisms or isolated strates bearing halogens in both the a- and b-posi-
Old Yellow Enzymes (OYEs) has been reported as tions were used, a clear distinction could be drawn
the key step in the synthesis of commercially relevant between them: whereas b-halogens were lost in elimi-
compounds. In recent decades, considerable efforts nations, those at the a position in relation to the elec-
have been made to understand the mechanisms by tron-withdrawing group remained in the end prod-
which OYEs catalyze these formal hydrogen addi- uct.[8]
tions.[5b,6]
Recently, our research group showed that some a-
Among activated alkenes, those with a leaving chloromethyl and a-bromomethyl enones exhibit sim-
group at the b-position relative to the electron-with- ilar behavior by participating in reduction–elimina-
drawing group are particularly interesting, because of tion–reduction cascades to produce enantiomerically
their ability to undergo spontaneous elimination reac- enriched saturated ketones as end products.[9] Howev-
tions. In recent years, different research groups er, in these experiments enones with electron donor
have shown that these compounds participate in bio- groups bonded to their aromatic ring underwent rapid
catalytic cascades involving alkene reduction, elimina- solvolysis and could not be reduced by the microor-
tion to produce another activated alkene, and reduc- ganisms. In an attempt to overcome this drawback,
tion of the newly formed C=C bond. For instance, re- we studied the possibility of replacing the halide with
ductions of b-chloroenals by S. cerevisiae or OYEs a less effective leaving group. This article reports the
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results obtained in the bioreduction of a-acetoxy-
methyl enones.
Results and Discussion
The a-acetoxymethyl enones used as substrates in this
study were prepared via three different routes. Com-
pounds 1a–f were prepared using 4-aryl-3-bromo-
methyl-3-buten-2-ones and 3-bromomethy-3-nonanen-
2-one, as previously reported.[9] Enones 1g and 1h
were prepared starting from Knoevenagel condensa-
tion to compounds 2g and 2h, followed by ester re-
Scheme 2. Synthesis of enones 5h–j.
The reduction of enones 1a–g by S. cerevisiae pro-
duction to derivatives 3g and 3h and, finally, carbonyl duced the corresponding (R)-a-methyl ketones in
deprotection. Enones 1i and 1j were prepared by moderate yields and high ees (Table 1). The stereo-
a TiCl4-promoted reaction between methyl vinyl chemical outcome of these reductions is similar to
ketone and aromatic aldehydes,[10] followed by substi- that previously reported for a-halomethyl enones, in
tution of the allylic chloride by a hydroxy group, and which a reduction–elimination–reduction cascade is
its acetylation (Scheme 1). Finally, enones 5h–j were proposed to be taking place.[9] In this case, an OYE-
prepared by via aldol condensation between alde- catalyzed reduction followed by acetic acid elimina-
hydes and butanone (Scheme 2). The synthesis and tion produces an a-methylene ketone, which is then
biotransformation of analogous enones 5a–i was de- reduced by an OYE to the corresponding a-methyl
scribed in a previous study.[9]
ketone. The production of the (R)-isomer is consistent
with the flipped binding mode of a-methylene ketone
to the active site enzymes prior to hydride trans-
fer.[6d,e] These results can be contrasted with previous-
ly reported microbiological reductions of a-methylar-
ylideneacetones, which produce the (S)-isomers of a-
methyl ketones via the same flipped binding mode.[9]
In a previous study, we reported that chloro and
bromo analogues of 1g, which contain an electron-
rich aromatic ring, underwent hydrolysis as the exclu-
sive reaction pathway when subjected to biotransfor-
mation conditions.[9] It is important to note that 1g
did not produce its hydrolysis product during bio-
transformation, indicating that the acetoxy group is
a good balance between being a satisfactory leaving
group for the biocatalytic cascade and stable enough
for solvolysis.
Table 1. Biotransformation of enones 1a–g by S. cerevisiae.[a]
Enone
R
Time [h] Product Yield [%] ee [%]
1a
1b
1c
1d
1e
1f
1g
Ph
16
8
6
(R)-6a 58
(R)-6b 37
(R)-6c 71
(R)-6d 52
(R)-6e 44
96
96
98
89
91
97
94
2-ClC6H4
4-ClC6H4
3-NO2C6H4
3-NO2C6H4 48
n-C5H11
4-MeOC6H4 48
8
6
(R)-6f
61
(R)-6g 57
[a]
Scheme 1. Synthesis of enones 1a–j.
Reactions performed with 0.5 mmol of substrate.
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Table 2. Biotransformation of enone 1a by EREDs.
ERED
Time
[h]
1a
9
10
(R)-6a
ee
[%]
[%][a]
[%][a]
[%][a]
[%][a]
103
6
24
6
24
6
24
6
24
6
24
6
24
6
24
4.4
4.3
0.8
0.6
58.9
57.5
7.0
1.9
1.7
1.1
1.2
0.7
0.8
3.5
3.4
1.2
0.6
48.3
43.5
15.7
12.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.6
93.7
94.0
98.1
98.2
40.4
41.7
89.5
90.4
98.8
99.4
50.1
53.9
37.7
40.6
95
94
110
96
95
91
90
96
94
99
99
98
98
39
36
Scheme 3. Synthesis of ketone 9.
112
Table 3. Biotransformations of enones 1h–j and 5h-j by S.
cerevisiae.[a]
207
6.2
P1-A04
P1-E01
P1-H09
n.d.
n.d.
n.d.
n.d.
45.8
42.3
2.6
0.5
0.9
[a]
Conversion determined by GC.
Given the positive results obtained in the biotrans-
formation of 1a–g by S. cerevisiae, we opted to investi-
gate the biotransformation of 1a by commercially
available enereductases (EREDs). The results are
shown in Table 2. EREDs 103, 110, 207 and P1A04
produced (R)-6a in 6 h of reaction with excellent con-
versions and ees. A low concentration of the proposed
hydrogenation intermediate 9 was observed, whereas
intermediate 10 was not detected. EREDs 112 and
P1-H09 showed poorer conversions of 1a into (R)-6a;
ERED 112 produced (R)-6a at high ee, whereas low
ees of (R)-6a were obtained for ERED P1-H09. Final-
ly, ERED P1-E01 resulted in a mixture of the hydro-
genation product 9 and the biocatalytic cascade prod-
uct (R)-6a, representing the only case, along with P1-
H09, in which a significant amount of 9 was detected.
Enone
Time [h]
Product
Yield [%][b,c]
ee [%][b,c]
5h
1h
5i
1i
5j
1j
72
72
8
48
8
(S)-6h
(R)-6h
(S)-6i
(R)-6i
(S)-6j
(R)-6j
28 [13]
30
46 [59]
42
64
47
93 [>95]
90
97 [>95]
93
75
95
72
[a]
Reactions performed with 0.5 mmol of substrate.
Isolated yields.
[b]
[c]
Yield and ee values in square brackets refer to those in
ref.[11]
In addition, it is noteworthy that only a small amount the ketones (R)-6h–j were obtained in yields of 30–
of 9 underwent elimination, producing 10 over the 47% and 90–95% ee. On the other hand, the reduc-
course of 24 h. Although significantly smaller tion of enones 5h–j, which have no leaving group,
amounts of 9 were produced in the reactions cata- produced the ketones (S)-6h–j with 13–64% yields
lyzed by the other six enzymes, this compound also and 75–97% ee.
remained in the reaction medium in those cases. An
authentic sample of compound 9 was synthesized to transformations, both enantiomers of the chiral fra-
support its identification (Scheme 3). grance Tropionalꢀ were prepared using enones 1j
To illustrate the synthetic potential of these bio-
Since the acetoxy group is an adequate leaving and 5j [Scheme 4, for the (R) enantiomer]. Bioreduc-
group for bioreduction cascades involving enones tions of 1j and 5j were scaled up to 5 mmol of sub-
with electron-rich aromatic rings, enones 1h–j were strate, and (R)- and (S)-6j were obtained with 63%
selected for reduction by S. cerevisiae (Table 3). Thus, and 73% yield, and 96% and 75% ee, respectively.
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spectively, whereas reduced conversion was observed
for substrate 13c and 13d, which contain 4 and 5
carbon alkyl chains, respectively. Poor conversions
were also obtained for substrates 13e and 13f, in
which benzoyl and Boc groups are bonded to the
oxygen atom. However, the ee values were consistent-
ly high and in all cases the (R) isomer was the pre-
dominant product. These results corroborate the pro-
posed biocatalytic pathway, whereby the stereogenic
center of the final product is generated via the enzy-
matic hydrogenation of the intermediate 3-benzyl-3-
buten-2-one.
In light of the satisfactory results obtained with
enones bearing terminal allylic acetoxy groups, we de-
cided to study the biotransformation of the racemic
acetylated Morita–Baylis–Hillman (MBH) adduct of
benzaldehyde and 3-buten-2-one by S. cerevisiae. Ini-
tially, we expected this enone to follow a similar reac-
tion pathway to that of the a-acetoxymethyl enones
studied up to this point. In other words, a reduction–
elimination sequence would lead to the formation of
Scheme 4. Synthesis of (R)-Tropionalꢀ.
The enantiomerically enriched ketones were convert- a-methylbenzylideneacetone, which would then be re-
ed into the corresponding primary alcohols via a halo- duced to (S)-3-methyl-4-phenyl-2-butanone [(S)-6a].
form reaction, followed by reduction of the mixed an- However, when 14 was reduced by S. cerevisiae, ke-
hydride of the carboxylic acid formed in the reaction. tones (R)-6a and (1R,2R)-15 were isolated from the
The alcohols (R)- and (S)-11 were then transformed reaction medium (Scheme 5). The absolute configura-
into (R)- and (S)-Tropionalꢀ with 94 and 75% ee re- tion of 15 was determined by treating it with an alka-
spectively, via TEMPO-catalyzed oxidation. It is im- line solution of sodium hypobromite, which yielded 3-
portant to underscore that the asymmetric synthesis hydroxy-2-methyl-3-phenylpropanoic acid, and then
of Tropionalꢀ has been reported in the literature: methylating the acid with diazomethane. The relative
both enantiomers of Tropionalꢀ have been synthe- configuration was determined as syn by comparing its
sized by SAMP and RAMP hydrazone alkylations,[12] 1H and 13C NMR spectra with the literature values,[14]
and (S)-Tropionalꢀ via OYE-catalyzed reduction of and the absolute configuration was established by
a,b-unsaturated aldehyde.[13]
Other a-acyloxymethyl enones were also investigat- ture sample.[15]
comparing its optical rotation with that of the litera-
ed as substrates in the bioreduction cascade (Table 4).
It is important to note that although substrate 14
Good conversions were obtained for substrates 13a was racemic, (1R,2R)-15 was produced at a high dia-
and 13b, which have 2 and 3 carbon alkyl chains, re- stereomeric ratio (dr) from the onset of the reaction.
This suggests that only one enantiomer of 14 is re-
duced to the product bearing the b-acetoxy group, as
opposed to reduction of both enantiomers to 15 and
elimination of only one diastereoisomer.
Table 4. Biotransformation of enones 13a–f by S. cerevi-
siae.[a]
Moreover, when the reaction was performed using
a water/hexane biphasic system, three key intermedi-
ates were identified via GC/MS techniques in the re-
Enone R
Time
[h]
Conversion
[%]
(R)-6a ee
[%]
13a
13b
13c
13d
13e
13f
COCH2CH3 24
CO(CH)2CH3 24
CO(CH)3CH3 72
CO(CH)4CH3 72
95
93
77
37
51
78
98
98
97
95
93
97
COPh
Boc
72
72
[a]
Reactions performed with 0.1 mmol of substrate; the
product was not isolated.
Scheme 5. Biotransformation of enone 14 by S. cerevisiae.
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ential conversion of either syn- or anti-15 into 16 did
not occur.
This prompted us to investigate the reactivity of
a potential intermediate b-acetoxy ketone in the cell
medium. Thus, when ketone 9, a possible (though not
detected) intermediate of the reduction of 1a by S.
cerevisiae, was submitted to biotransformation by this
microorganism, a 97:3 mixture of hydrolysis product 8
and AcOH-elimination product 10 was obtained
(Scheme 7).
Scheme 6. Suggested pathways for production of (R/S)-6a
and (1R,2R)-15 in the biotransformation of (R/S)-14 by S.
cerevisiae.
Scheme 7. Biotransformation of ketone 9 by S. cerevisiae.
This result suggests two hypotheses: (i) when the
C=C bond of 1a is hydrogenated by microbial OYEs,
action medium: enone 1a, which is an isomer of 14, 9 undergoes rapid elimination in the intracellular
and enones 10 and 16 (Scheme 6). In this biphasic medium before it can be hydrolyzed or (ii) 9 is not
system, some reaction intermediates are removed formed in the main bioreduction cascade pathway.
from water to the organic solvent. This preliminarily
In order to assess hypothesis (ii), we performed bio-
suggests the three main reaction pathways involved in transformations of 9 using commercially available
the reduction of 14: in addition to the reduction of EREDs; the results are presented in Table 5. All the
(S)-14 to (1R,2R)-15, 14 can undergo isomerization in EREDs studied exhibited significant difficulty in the
the reaction medium to produce 1a, and both 1a and biotransformation of 9. In general, 80–89% and 66–
14 can be reduced via elimination of the acetoxy 77% of 9 still remained after 6 and 24 h of reaction,
groups to produce different enantiomers of 6a. These respectively. The main products detected after 24 h of
diverging pathways also explain the production of reaction were (R)-6a (4.3–12.5%), 10 (4–11%) and 8
(R)-6a with low ee in this biotransformation. In rela- (5–17%). The reduction catalyzed by ERED P1A04
tion to the isomerization of 14 to 1a, it is known that showed an outlying behavior, with a higher quantity
acetates of MBH adducts from aromatic aldehydes of 10 (22.3%) observed after 24 h. Based on these ex-
and methyl propenoate isomerize to the more ther- periments using isolated EREDs, it seems reasonable
modynamically stable acetates, either by intramolecu- to state that compound 9 is not the intermediate of
lar rearrangement or through reagents such as acetate the biotransformation of 1a, since 1a is converted to
ion, which accelerates allylic isomerization.[16]
(R)-6a after 6 h by most EREDs (see Table 2).
In an attempt to study the elimination of the ace-
In summary, the results of the experiment with S.
toxy group of (1R,2R)-15 to produce 16, pure cerevisiae and isolated EREDs indicate that 15 is not
(1R,2R)-15 was placed in an orbital shaker under the an intermediate in the main reaction pathway of the
same biotransformation conditions for up to 96 h. Al- reduction of 14, and the same can be said for 9 in re-
though some elimination occurred, it was sluggish, lation to the reduction of 1a. This means that the sub-
and (1R,2R)-15 remained in the reaction medium. strates 1a and 14 are not biotransformed via C=C
This seems to indicate that (1R,2R)-15 is not an inter- bond hydrogenation followed by acetic acid elimina-
mediate in the production of 16. In another experi- tion. An alternative to the hydrogenation–elimination
ment, a diastereomeric mixture of 15, obtained by re- sequence is an SN2’-type reaction, involving a nucleo-
ducing of 1-phenyl-1-hydroxy-2-methylidene-3-buta- philic attack of the formal hydride from the flavin
none with H2 and Pd/C followed by acetylation with mononucleotide (FMNH2) on the olefinic b-carbon
Ac2O, was submitted to the same biotransformation and concomitant expulsion of the acetoxy group.
conditions in an orbital shaker for up to 96 h. Once (Figure 1).
again, elimination was sluggish, and both diastereoiso-
Thus, it seems that the main bioreduction cascade
mers of 15 persisted in the reaction medium; prefer- pathway for acetoxy enones 1a and 14 is not a hydro-
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Table 5. Biotransformation of ketone 9 by EREDs.
at all. The bakerꢂs yeast-mediated reduction of a-ace-
toxybenzalacetone[17] further substantiates proof to
the proposed SN2’ process, since it does not generate
products lacking the acetoxy group.
It is reasonable to assume that the biotransforma-
tion of (S)-14 mediated by S. cerevisiae produces
(1R,2R)-15 and that of (R)-14 results in (S)-6a
through 16, as shown in Scheme 6. Since these bio-
transformations are catalyzed by an OYE of S. cerevi-
siae, we can assume that (S)-14 shows perfect interac-
tion with the active site of the enzyme, promoting the
formal trans addition of H2 to the C=C bond, produc-
ing (1R,2R)-15. This catalysis may involve proton
donation to the a-carbon of (S)-14 during formal de-
livery of the FMNH2 hydride to the b-carbon. On the
other hand, the a-carbon of (R)-14 should have some
difficulty reaching the proton donor. Thus, the AcO
group is eliminated concomitantly with the hydride
delivery to the a-carbon forming 16 as a product of
the SN2’ reaction.
In this study, the inability of b-acetoxy ketones to
undergo rapid acetic acid elimination allowed us to
detect, isolate and examine these compounds in
detail, and ultimately distinguish between hydrogena-
tion–elimination and allylic substitution pathways.
However, it is unclear whether a-halomethyl enones
and related compounds behave similarly in their bio-
reduction cascades. Given the ease with which b-halo
carbonyl compounds undergo hydrochloric or hydro-
bromic acid elimination in the reaction medium,[7,18]
both hydrogenation–elimination and allylic substitu-
tion pathways should quickly converge to the same in-
termediate, making it more difficult to differentiate
between them. Nevertheless, OYE-catalyzed allylic
nucleophilic substitution may play a role in bioreduc-
tion cascades involving a-halomethyl enones and
other activated alkenes with a halogen in the allylic
position.
ERED
Time
[h]
9
8
10
(R)-6a
ee
[%]
[%][a]
[%][a]
[%][a]
[%][a]
103
6
24
6
24
6
24
6
24
6
24
6
24
6
24
85.4
66.8
86.3
73.2
89.2
76.7
88.0
73.2
80.4
65.9
88.0
66.1
84.5
67.9
5.6
17.3
2.5
10.9
2.2
13.1
1.4
12.3
0.9
5.0
1.0
13.1
2.8
3.9
5.1
6.5
11.0
4.5
5.9
5.3
9.3
12.0
22.3
4.9
8.3
5.6
8.4
5.1
12.5
4.7
4.9
4.1
4.3
5.3
5.2
6.7
6.8
6.1
12.5
7.1
7.7
90
89
96
96
91
89
94
94
97
96
99
98
41
40
110
112
207
P1-A04
P1-E01
P1-H09
16.0
[a]
Conversion determined by GC.
Finally, ethyl enones 17, 19 and 22 were synthesized
(Scheme 8) and submitted to a microorganism screen-
ing, as shown in Table 6. Among the ten yeasts and
fungi screened, the yeast Candida albicans produced
the best results: ketones (S)- and (R)-23 were ob-
tained with 97% ee and 83–93% ee, respectively, de-
pending on the structure of the starting material.
Preparative experiments were therefore performed on
a 1 mmol scale of substrate to produce (S)-23 at
a 79% yield and 95% ee from 17 and (R)-23 at a yield
of 72% and 93% ee from 22. This result is significant
because unlike methyl enones, ethyl enones such as
19 produce 23 at low ee upon reduction by S. cerevi-
Figure 1. SN2’-type reaction mechanism proposed for enones
1a and 14 catalyzed by EREDs.
genation–elimination–hydrogenation
sequence. siae.[19] Also, reductions of ethyl enones such as 17
Rather, it consists of an allylic nucleophilic substitu- catalyzed by isolated OYE1 and OYE2 produce (R)-
tion followed by hydrogenation. The other pathway, 23 at 59 and 76% ee, respectively, with opposite enan-
in which the substrate undergoes formal hydrogen ad- tioselectivity with respect to the reduction of methyl
dition to produce a b-acetoxy ketone, generally plays enones by the same enzymes, since the ethyl enone 17
a minor role in the overall reaction or does not occur cannot fit in the active site with the same orientation
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Table 6. Microorganism screening for the biotransformation
of ethyl enones 17, 19 and 22.[a]
Microorganism
Enone Time Conversion Ketone ee
[h]
[%]
[%]
Saccharomyces
cervisiae
17
19
22
17
19
22
17
19
22
72
24
48
72
24
72
72
72
72
48
24
48
72
48
72
72
72
72
72
48
72
72
8
68
100
100
5
100
7
3
51
5
100
100
100
6
100
9
14
11
5
(S)-23 50
(S)-23 14
(S)-23
n.d.
9
n.d.
Saccharomyces
boulardii
(S)-23 21
n.d.
n.d.
Pichia kluyveri
n.d.
n.d.
(R)-23 89
n.d.
n.d.
Candida albicans 17
(S)-23 97
(R)-23 83
(R)-23 93
19
22
Candida parapsi- 17
losis
n.d.
n.d.
19
22
(S)-23 40
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Trichosporon cu- 17
taneum
n.d.
n.d.
n.d.
n.d.
19
22
17
19
22
Kluyveromyces
marxianus
7
Scheme 8. Synthesis of enones 17, 19 and 22.
100
10
75
100
(S)-23 32
n.d.
n.d.
as the corresponding methyl enone 16.[6d,e] On the
other hand, the reduction of enone 17 mediated by
isolated OYE3 produces (S)-23 in 89% ee, since
OYE3 better tolerates the increased steric hindrance
of the enone 17 compared to enone 16, via flipped
binding mode of the substrate to the active site of the
enzyme.[6d] Therefore, the reduction of 17 by microor-
ganisms that have both OYE1-3 like S. cerevisiae pro-
duces 23 at low enantioselectivity. The yeast C. albi-
cans has been shown to contain an estrogen binding
protein (EBP) similar to S. cerevisiae OYEs,[20] and
this enzyme, which possibly is similar to OYE3, may
be involved in the reduction of 17 by C. albicans.
Geotrichum can- 17
didum
(S)-23 90
(R)-23 97
19
22
[b]
[b]
[b]
24
72
72
72
72
6
–
–
–
Curvularia lunata 17
7
13
8
13
94
9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
19
22
Aspergillus niger 17
19
22
(S)-23 58
n.d. n.d.
72
[a]
Reactions performed with 0.1 mmol of substrate.
The C=O bond reduction product was the only detected
product.
[b]
Therefore, both ethyl enones 17 and 19 may fit in the Conclusions
active site of this enzyme via the flipped binding
mode producing (S)-23 and (R)-23, respectively, as Several a-acetoxymethyl enones were shown to take
previously proposed for the corresponding methyl part in bioreduction cascades in which the substrate is
enones.[9] The same rationalization may be done for reduced by an OYE with loss of the acetoxy group
G. candidum, since this microorganism produces (S)- producing a new enone that is reduced by an OYE to
23 and (R)-23 at 90 and 97% ee from 17 and 19, re- form an enantiomerically enriched ketone. The ace-
spectively.
toxy group is an adequate leaving group for bioreduc-
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5 min. 1 mL of a compound solution (1 mgmLÀ1) in ethyl
acetate was injected and the retention times (min) are re-
ported for all compounds.
tion cascades involving the enones studied with elec-
tron-rich aromatic rings, avoiding hydrolysis that
occurs with the corresponding a-halomethyl enones.
This stereochemistry control strategy was applied to
the syntheses of (R)- and (S)-Tropionalꢀ with moder-
ate to high enantiomeric excesses. Experiments per-
formed using both S. cerevisiae and isolated EREDs
suggest that the main biocatalytic cascade pathway
for a-acetoxymethyl enones might not be a hydroge-
Chiral GC/FID analyses were obtained on an Agilent
6850 Series GC System, using a Hydrodex chiral capillary
column (30 mꢃ0.25 mm IDꢃ0.25 mm film thickness). Hy-
drogen was used as a carrier gas (1 mLminÀ1), the injector
temperature was 2008C and the detector temperature was
2208C. Three different methods were employed for the anal-
ysis of the bioreduction products. Method A: the column
temperature was held at 808C for 3 min, increased to 1808C
at a rate of 38C minÀ1, and then kept at 1808C for 20 min.
Method B: the column temperature was held at 808C for
3 min, increased to 1808C at a rate of 18C minÀ1, and then
kept at 1808C for 20 min. In all cases, 1 mL of a compound
solution (1 mgmLÀ1) in ethyl acetate was injected and the
retention times (min) are reported for all compounds.
Chiral HPLC analyses were performed on a Shimadzu
LC-20AT, using a Chiralcel OJ-H column from Daicel. A
hexane/3-propanol (99.5%/0.5%) mixture was used as
eluent at a flow of 0.4 mLminÀ1.
nation–elimination–hydrogenation
sequence,
but
rather an allylic nucleophilic substitution followed by
hydrogenation.
Experimental Section
General Methods
Benzaldehyde, 2-chlorobenzaldehyde, 3-nitrobenzaldehyde,
4-nitrobenzaldehyde, hexanal, p-anysaldehyde, o-anysalde-
hyde, piperonal, 3,4-dimethoxybenzaldehyde, 3-buten-2-one,
DABCO, bromine, trifluoroacetic acid, acetic anhydride,
morpholine, diisobutylaluminum hydride, ethyl acetoacetate,
benzyl chloride, lithium aluminum hydride, TEMPO, iodo-
benzene, propionic anhydride, valeric anhydride, hexanoic
anhydride, benzoic anhydride, 3-pentanone, tert-butyl dicar-
bonate, methanesulfonyl chloride, DBU and methyl 3-oxo-
pentanoate were purchased from Sigma–Aldrich. DMAP,
sodium hydride and butyric anhydride were purchased from
Fluka. p-Toluenesulfonic acid was purchased from Alfa–
Aesar. Triethylamine was purchased from Acros. Hydrobro-
mic acid, titanium(IV) chloride and 4-chlorobenzaldehyde
were purchased from Merck. Potassium carbonate, glacial
acetic acid, sodium hydroxide, ethylene glycol, sulfuric acid
and hydrochloric acid were purchased from Synth. Benzal-
dehyde and p-anysaldehyde were distilled before use; all the
other reagents were used as received.
IR spectra were recorded on an FT-IR BOMEM MB-100
from Hartmann & Braun. Melting points were recorded on
a Metler Toledo MP50 Melting Point System. Optical rota-
tion measurements were recorded on a Perkin–Elmer 341
polarimeter with a sodium lamp. ESI-HR-MS analyses were
performed on a Waters Xevo Q-Tof.
General Procedure for the Preparation of a-Acetoxy-
methyl Enones 1a–f
Trifluoroacetic acid (1 mL, 13 mmol) and potassium carbon-
ate (1,52 g, 11 mmol) were added to a round-bottom flask
containing (E)-3-bromomethyl-4-(aryl or alkyl)-3-buten-2-
one (10 mmol) and acetone (10 mL) and the mixture was re-
fluxed for 6 h. After this time, the mixture was cooled to
room temperature and added to a separatory funnel con-
taining water (100 mL). The product was extracted with
ethyl acetate, the organic layer was washed with brine, and
then dried over sodium sulfate. The solvent was removed
under reduced pressure and then dichloromethane (50 mL)
was added to the crude product. The solution was cooled to
08C, and then acetic anhydride (1,135 mL, 12 mmol), trie-
thylamine (1,67 mL, 12 mmol) and DMAP (122 mg,
1 mmol) were added to the mixture. The mixture was stirred
at 08C for 1 hour, and then water (100 mL) was added to
the round-bottom flask. The mixture was further stirred at
room temperature for 15 min, and then the organic layer
was separated. The aqueous layer was extracted with di-
chloromethane, washed with brine, dried over sodium sul-
fate. The solvent was removed under reduced pressure and
the product was purified by column chromatography.
Yeast type II from Saccharomyces cerevisiae was pur-
chased from Sigma (Lot BCBL8059V). All the EREDs used
were from a Kit EREDs purchased from Codexis (Lot
N15006).
Thin-layer chromatography (TLC) analyses were per-
formed with precoated aluminum sheets (silica gel 60
Merck), and column chromatography was carried out on
silica (200–400 mesh, Merck). Detection was performed by
UV inspection (254 nm) for all compounds.
1H NMR spectra were recorded at 250, 400, 500 or
600 MHz, and 13C NMR spectra were recorded at 62.5, 100,
125 or 150 MHz. Chemical shifts are reported in ppm rela-
tive to tetramethylsilane (d=0) in CDCl3, and coupling con-
stants (J) are reported in Hz.
GC/MS analyses were obtained on an Agilent 7890A GC
System with a Hewlett–Packard 5975C mass spectrometer
(70 eV) using an HP-5MS fused silica capillary column
(crosslinked 5% phenyl ethyl siloxane, 30 mꢃ0.25 m IDꢃ
(E)-3-Acetoxymethyl-4-phenyl-3-buten-2-one (1a): Fol-
lowing the general procedure, 1a was obtained as a colorless
oil; yield: 79%. Rt (GC/MS): 5.98 min; MS-EI: m/z (%)=
218 (21) [M+], 176 (28), 175 (55), 159 (14), 158 (88), 157
(18), 143 (15), 133 (34), 130 (11), 129 (23), 117 (19), 116
(26), 115 (100), 103 (12), 77 (14); 1H NMR (CDCl3,
250 MHz): d=2.08 (s, 3H), 2.48 (s, 3H), 4.94 (s, 2H), 7.35–
7.48 (m, 5H), 7.79 (s, 1H); 13C NMR (CDCl3, 62.5 MHz):
d=20.9, 26.1, 58.5, 128.8, 129.5, 129.7, 134.3, 135.4, 145.0,
170.7, 198.3.
0.25 mm film thickness) and helium as
a carrier gas
(1 mLminÀ1). The split ratio was 1:50. The injector tempera-
ture was kept 2708C and detector was kept at 2808C. The
column temperature was held at 808C for 3 min, increased
to 2308C at a rate of 308C minÀ1, increased from 2308C to
2808C at a rate of 258C minÀ1, and then kept at 2808C for
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(E)-3-Acetoxymethyl-4-(2’-chlorophenyl)-3-buten-2-one
(1b): Following the general procedure, 1b was obtained as
a colorless oil; yield: 56%. Rt (GC/MS): 6.40 min; MS-EI:
m/z (%)=218 (14), 217 (100), 175 (27), 158 (11), 157 (76),
149 (12), 115 (24); IR (film): n=1741, 1677, 1435, 1371,
1238, 1029; 1H NMR (CDCl3, 500 MHz): d=2.04 (s, 3H),
2.51 (s, 3H), 4.83 (s, 2H), 7.31–7.47 (m, 4H), 7.88 (s, 1H);
13C NMR (CDCl3, 62.5 MHz): d=20.8, 26.2, 58.5, 127.0,
129.8, 130.2, 130.7, 132.9, 134.1, 137.0, 141.5, 170.5, 198.0;
HR-MS-ESI+: m/z=275.0471 [(M+Na)+], calculated:
275.0451.
Preparation of Ethyl (Z)-2-(2’-Methyl-1’,3’-dioxolan-
2’-yl)-3-(4’-methoxyphenyl)propenoate (2g)
Morpholine (862 mL, 10 mmol) was slowly added to
a
round-bottom flask containing ethyl acetoacetate
(1.276 mL, 10 mmol), p-anisaldehyde (1.36 g, 10 mmol) and
glacial acetic acid (1.144 mL, 20 mmol). The reaction mix-
ture was stirred for 24 h, and then ethyl acetate was added
to the mixture. The solution was washed with water, and the
organic layer was dried over sodium sulfate. The solvent was
removed under reduced pressure, and the residue was used
in the next step without purification.
(E)-3-Acetoxymethyl-4-(4’-chlorophenyl)-3-buten-2-one
(1c): Following the general procedure, 1c was obtained as
a colorless solid; yield: 81%; mp 91.7–93.18C. Rt (GC/MS):
6.67 min; MS-EI: m/z (%)=252 (2) [M+], 211 (26), 210
(44), 209 (68), 194 (26), 192 (81), 151 (24), 149 (55), 116
(20), 115 (100), 114 (20), 75 (20), 70 (20); IR (KBr): n=
The Knoevenagel adduct was dissolved in toluene
(20 mL), and ethylene glycol (1.118 mL, 20 mmol) and p-tol-
uenesulfonic acid (344 mg, 2 mmol) were added to the
round-bottom flask. The mixture was refluxed for 6 h with
water removal by means of a Dean–Stark apparatus. Then,
the mixture was added to a separatory funnel, and washed
with water, 10% sodium bicarbonate and brine. The organic
layer was dried over sodium sulfate, the solvent was re-
moved under reduced pressure and the product was purified
by column chromatography (90:10 hexane/ethyl acetate).
Product 2g was obtained as a colorless oil; yield: 1.975 g
(67%). Rt (GC/MS): 7.18 min; MS-EI: m/z (%)=292 (18)
[M+], 278 (15), 277 (87), 219 (22), 165 (39), 137 (10), 117
(12), 89 (15), 87 (100); IR (film): n=1719, 1607, 1513, 1256,
1
1737, 1656, 1630, 1250, 1222; H NMR (CDCl3, 250 MHz):
d=2.06 (s, 3H), 2.46 (s, 3H), 4.90 (s, 2H), 7.30–7.38 (m,
4H), 7.70 (s, 1H); 13C NMR (CDCl3, 62.5 MHz): d=20.9,
26.1, 58.3, 129.1, 130.8, 132.7, 135.8, 135.9, 143.5, 170.6,
198.0; HR-MS-ESI+: m/z=275.0456 [(M+Na)+], calculated:
275.0451.
(E)-3-Acetoxymethyl-4-(3’-nitrophenyl)-3-buten-2-one
(1d): Following the general procedure, 1d was obtained as
a colorless solid; yield: 78%; mp 93.8–95.38C. Rt (GC/MS):
7.43 min; MS-EI: m/z (%)=220 (10), 205 (12), 204 (100),
178 (11), 162 (16), 161 (10), 132 (13), 131 (15), 130 (10), 115
(23); IR (KBr): n=1736, 1661, 1626, 1525, 1355, 1244, 1226;
1H NMR (CDCl3, 400 MHz): d=2.12 (s, 3H), 2.52 (s, 3H),
4.88 (s, 2H), 7.27–8.32 (m, 4H), 8.28 (s, 1H); 13C NMR
(CDCl3, 125 MHz): d=20.8, 26.2, 58.0, 124.1, 124.2, 129.9,
134.9, 136.0, 137.8, 141.8, 148.4, 170.5, 197.6; HR-MS-ESI+:
m/z=286.0699 [(M+Na)+], calculated: 286.0692.
1
1176, 1032; H NMR (CDCl3, 250 MHz): d=1.24 (t, 3H, J=
7.00 Hz), 1.73 (s, 3H), 3.80 (s, 3H), 3.95–4.02 (m, 4H), 4.25
(q, 2H, J=7.00 Hz), 6.77 (s, 1H), 6.85–7.27 (m, 4H);
13C NMR (CDCl3, 125 MHz): d=14.0, 25.7, 55.3, 61.0, 64.6,
108.0, 113.9, 127.2, 129.7, 129.9, 134.5, 159.8, 168.7.
(E)-3-Acetoxymethyl-4-(4’-nitrophenyl)-3-buten-2-one
(1e): Following the general procedure, 1e was obtained as
a colorless solid; yield: 60%; mp 129.3–132.08C; Rt (GC/
MS): 7.54 min; MS-EI: m/z (%)=263 (1) [M+], 222 (11),
221 (100), 220 (28), 205 (13), 204 (95), 203 (26), 188 (13),
178 (15), 174 (19), 162 (21), 161 (28), 132 (27), 131 (19), 116
(14), 115 (31), 103 (10), 89 (10), 77 (11); IR (KBr): n=1737,
1661, 1524, 1349, 1246, 1222, 1024; 1H NMR (CDCl3,
500 MHz): d=2.08 (s, 3H), 2.51 (s, 3H), 4.90 (s, 2H), 7.56
(d, 2H, J=7.00 Hz), 7.76 (s, 1H), 8.29 (d, 2H, J=7.00 Hz);
13C NMR (CDCl3, 150 MHz): d=20.8, 26.3, 58.1, 124.0,
130.1, 138.2, 140.7, 141.4, 148.1, 170.4, 197.6; HR-MS-ESI+:
m/z=286.0702 [(M+Na)+], calculated: 286.0692.
(E)-3-Acetoxymethyl-3-nonen-2-one (1f): Following the
general procedure, 1f was obtained as a colorless oil; yield:
52%. Rt (GC/MS): 5.19 min; MS-EI: m/z (%)=169 (7), 152
(13), 110 (17), 109 (100), 99 (17), 95 (19), 81 (12), 67 (19);
IR (film): n=2958, 2931, 1741, 1674, 1372, 1233, 1027;
1H NMR (CDCl3, 250 MHz): d=0.91 (t, 3H, J=6.50 Hz),
1.29–1.55 (m, 6H), 2.03 (s, 3H), 2.34 (s, 3H), 2.36 (q, 2H,
J=7.50 Hz), 4.84 (s, 2H), 6.89 (t, 1H, J=7.50 Hz);
13C NMR (CDCl3, 100 MHz): d=14.0, 20.9, 22.4, 25.7, 28.4,
29.1, 31.5, 57.0, 136.2, 149.8, 170.9, 198.0; HR-MS-ESI+:
m/z=235.1321 [(M+Na)+], calculated: 235.1310.
Preparation of (E)-2-(2’-Methyl-1’,3’-dioxolan-2’-yl)-3-
(4’-methoxyphenyl)-2-propen-1-ol (3g)
Diisobutylaluminum hydride (2M in toluene, 6 mL,
12 mmol) was slowly added to a round-bottom flask contain-
ing compound 2g (1.46 g, 5 mmol) and CH2Cl2 (10 mL) at
08C under a nitrogen atmosphere. The mixture was stirred
for 6 h at 08C, and then ethyl acetate (10 mL) was added to
the round-bottom flask. The reaction was stirred for 30 min,
and then water (1 mL) was added to the round-bottom
flask. The mixture was further stirred for 10 min, and then
a 10% sodium hydroxide solution (1 mL) was added to the
round-bottom flask. The mixture was further stirred for for
10 min, and then water (2 mL) was added to the round-
bottom flask. After 1 hour, the solid mass was separated by
filtration, the organic layer was washed with water and
brine and then dried over sodium sulfate. The organic sol-
vent was removed under reduced pressure, and the product
was purified by column chromatography (85:15 hexane/ethyl
acetate). Product 3g was obtained as a colorless oil; yield:
668 mg (53%). Rt (GC/MS): 6.56 min; MS-EI: m/z (%)=
206 (53), 163 (40), 146 (34), 145 (100), 121 (37), 115 (21),
108 (72), 103 (25), 91 (27), 77 (26), 55 (40); IR (film): n=
1
3386, 1602, 1512, 1257, 1179; H NMR (CDCl3, 400 MHz):
d=1.60 (s, 3H), 2.46 (brs, 1H), 3.81 (s, 3H), 3.97–4.05 (m,
4H), 4.28 (br, 2H), 6.86–6.90 (m, 3H), 7.35–7.38 (m, 2H);
13C NMR (CDCl3, 125 MHz): d=24.4, 55.3, 58.6, 64.7, 110.6,
113.8, 128.6, 129.0, 130.4, 137.8, 159.1.
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Preparation of (E)-3-Acetoxymethyl-3-(4’-methoxy-
phenyl)-3-buten-2-one (1g)
Preparation of (E)-3-Acetoxymethyl-3-(2’-meth-
oxyphenyl)-3-buten-2-one (1h)
Compound 3g (500 mg, 2 mmol), THF (2 mL) and 10% HCl
(1 mL) were stirred for 10 h in a round-bottom flask. The
product was extracted with diethyl ether, the organic layer
was washed with 10% sodium bicarbonate and dried over
magnesium sulfate. The solvent was removed under reduced
pressure, and then dichloromethane (10 mL) was added to
the crude product. The solution was cooled to 08C, and then
acetic anhydride (227 mL, 2.4 mmol), triethylamine (334 mL,
2.4 mmol) and DMAP (24 mg, 0.2 mmol) were added to the
mixture. The mixture was stirred at 08C for 1 hour, and
then water (10 mL) was added to the round-bottom flask.
The mixture was further stirred at room temperature for
15 min, and then the organic layer was separated. The aque-
ous layer was extracted with dichloromethane, washed with
brine, dried over sodium sulfate. The solvent was removed
under reduced pressure and the product was purified by
column chromatography (95:5 hexane/ethyl acetate). Prod-
uct 1g was obtained as a colorless oil; yield: 411 mg (81%).
Rt (GC/MS): 7.05 min; MS-EI: m/z (%)=248 (15) [M+],
206 (14), 205 (61), 189 (10), 188 (48), 187 (15), 173 (12), 163
(21), 159 (16), 147 (17), 146 (34), 145 (100), 132 (11), 131
(16), 121 (12), 115 (15), 108 (27), 103 (25), 102 (13), 91 (11),
77 (20); IR (film): n=1738, 1666, 1603, 1513, 1372, 1306,
1257, 1179, 1028; 1H NMR (CDCl3, 500 MHz): d=2.10 (s,
3H), 2.47 (s, 3H), 3.85 (s, 3H), 4.97 (s, 2H), 6.94–7.40 (m,
4H), 7.74 (s, 1H); 13C NMR (CDCl3, 125 MHz): d=21.0,
26.0, 55.4, 58.6, 114.4, 126.7, 131.6, 133.2, 145.2, 161.1, 170.9,
198.3.
Following the procedure for preparation of 1g and using 3h
(375 mg, 1.5 mmol), the product 1h was obtained as a color-
less oil; yield: 338 mg (91%). Rt (GC/MS): 6.68 min; MS-
EI: m/z (%)=248 (6) [M+], 218 (14), 217 (100), 205 (32),
188 (21), 173 (20), 163 (22), 159 (11), 157 (41), 146 (14), 145
(44), 131 (31), 121 (10), 115 (24), 108 (10), 91 (17), 77 (16);
IR (film): n=1728, 1669, 1628, 1598, 1488, 1463, 1441, 1377,
1305, 1242, 1115, 1024, 976, 768; 1H NMR (CDCl3,
250 MHz): d=2.06 (s, 3H), 2.48 (s, 3H), 3.87 (s, 3H), 4.89
(s, 2H), 6.92–7.42 (m, 4H), 7.99 (s, 1H); 13C NMR (CDCl3,
62.5 MHz): d=21.0, 26.1, 55.6, 59.0, 110.6, 120.6, 123.3,
130.0, 131.4, 135.2, 141.3, 157.6, 170.7, 198.4.
Preparation of (E)-4-(3’,4’-Dimethoxyphenyl)-3-
hydroxymethyl-3-buten-2-one (4i)
Titanium chloride (660 mL, 6 mmol) was added to a mixture
of 3-buten-2-one (1 mL, 12 mmol) and 3,4-dimethoxybenzal-
dehyde (1.66 g, 10 mmol) and dichloromethane (50 mL).
The mixture was stirred at room temperature under a nitro-
gen atmosphere for 72 h, and then quenched with a 10%
sodium bicarbonate solution at 08C. The organic layer was
separated, washed with brine and then dried over sodium
sulfate. The solvent was removed under reduced pressure,
and then the residue was dissolved in acetone (10 mL). Tri-
fluoroacetic acid (1 mL, 13 mmol) and potassium carbonate
(1.52 g, 11 mmol) were added to the round-bottom flask and
the mixture was refluxed for 3 h. After this time, the mix-
ture was cooled to room temperature and added to a separa-
tory funnel containing water (100 mL). The product was ex-
tracted with ethyl acetate, the organic layer was washed
with brine, and then dried over sodium sulfate. The solvent
was removed under reduced pressure and the product was
purified by column chromatography (80:20 hexane/ethyl
acetate). Product 4i was obtained as a colorless solid; yield:
843 mg (36%); mp 76.5–81.58C. Rt (GC/MS): 7.18 min; MS-
EI: m/z (%)=236 (100) [M+], 221 (11), 128 (10), 205 (11),
193 (20), 187 (16), 176 (21), 175 (64), 162 (15), 161 (35), 160
(11), 151 (20), 147 (14), 139 (17), 138 (48), 133 (11), 131
Preparation of Ethyl (Z)-2-(2’-Methyl-1’,3’-dioxolan-
2’-yl)-3-(2’-methoxyphenyl)propenoate (2h)
Following the procedure for preparation of 2g and using o-
anisaldehyde (1.36 g, 10 mmol), the product 2h was obtained
as a colorless oil; yield: 1.837 g (63%). Rt (GC/MS):
6.81 min; MS-EI: m/z (%)=292 (10) [M+], 278 (10), 277
(61), 219 (21), 165 (20), 89 (11), 87 (100); IR (film): n=
1
1722, 1489, 1465, 1372, 1251, 1200, 1116, 1030, 755; H NMR
(CDCl3, 250 MHz): d=1.13 (t, 3H, J=7.25 Hz), 1.77 (s,
3H), 3.82 (s, 3H), 3.97–4.03 (m, 4H), 4.15 (q, 2H, J=
7.25 Hz), 6.83–7.29 (m, 4H), 7.22 (s, 1H); 13C NMR (CDCl3,
62.5 MHz): d=13.9, 25.6, 55.5, 60.7, 64.5, 108.1, 110.5, 120.3,
124.2, 126.8, 128.8, 129.7, 136.1, 157.2, 168.4.
1
(13), 103 (10), 91 (15), 89 (11), 77 (19), 55 (26); H NMR
(CDCl3, 250 MHz): d=2.47 (s, 3H), 3.92 (s, 3H), 3.93 (s,
3H), 4.50 (s, 2H), 6.90–7.14 (m, 3H), 7.61 (s, 1H); 13C NMR
(CDCl3, 62.5 MHz): d=25.6, 56.0, 57.4, 111.1, 112.8, 123.6,
127.3, 138.3, 143.6, 149.0, 150.5, 201.0.
Preparation of (E)-2-(2’-Methyl-1’,3’-dioxolan-2’-yl)-3-
(2’-methoxyphenyl)-2-propen-1-ol (3h)
Preparation of (E)-3-Acetoxymethyl-4-(3’,4’-
dimethoxyphenyl)-3-buten-2-one (1i)
Following the procedure for preparation of 3g and using 2h
(1.46 g, 5 mmol), the product 3h was obtained as a colorless
oil; yield: 468 mg (37%). Rt (GC/MS): 6.76 min; MS-EI; m/
z (%)=250 (2) [M+], 235 (32), 221 (14), 219 (15), 131 (10),
115 (11), 91 (13), 87 (100); IR (film): n=3397, 2941, 1660,
1249, 1025; 1H NMR (CDCl3, 500 MHz): d=3.00 (s, 3H),
2.75 (t, 1H, J=6.00 Hz), 3.81 (s, 3H), 4.00–4.05 (m, 4H),
4.24 (d, 2H, J=6.00 Hz), 6.86–7.45 (m, 4H), 7.02 (s, 1H);
13C NMR (CDCl3, 125 MHz): d=24.5, 55.4, 59.0, 64.7, 110.4,
110.6, 120.4, 125.1, 125.3, 129.0, 130.4, 138.8, 157.3.
Acetic anhydride (343 mL, 3.6 mmol), triethylamine (501 mL,
3.6 mmol) and DMAP (37 mg, 0.3 mmol) were added to
a round-bottom flask containing 4i (3 mmol) and dichloro-
methane (15 mL). The mixture was stirred at 08C for
1 hour, and then water was added to the round-bottom
flask. The mixture was further stirred at room temperature
for 15 min, and then the organic layer was separated. The
aqueous layer was extracted with dichloromethane, washed
with brine, dried over sodium sulfate. The solvent was re-
moved under reduced pressure and the product was purified
by column chromatography (95:5 hexane/ethyl acetate).
Product 1i was obtained as a pale yellow solid; yield:
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628 mg (75%); mp: 84.8–87.38C. Rt (GC/MS): 7.58 min;
MS-EI: m/z (%)=278 (86) [M+], 260 (11), 136 (16), 135
(69), 219 (12), 218 (44), 217 (17), 205 (10), 203 (18), 193
(29), 189 (15), 187 (12), 178 (10), 177 (18), 176 (36), 175
(100), 162 (46), 161 (41), 160 (10), 151 (13), 147 (11), 146
(11), 145 (11), 138 (18), 131 (13), 115 (11), 89 (12), 77 (12);
1H NMR (CDCl3, 600 MHz): d=2.10 (s, 3H), 2.48 (s, 3H),
3.89 (s, 3H), 3.93 (s, 3H), 5.00 (s, 2H), 6.92 (d, 1H, J=
8.40 Hz), 7.00 (d, 1H, J=1.80 Hz), 7.05 (dd, 1H, J=1.80,
8.40 Hz), 7.74 (s, 1H); 13C NMR (CDCl3, 125 MHz): d=
21.0, 26.0, 55.8, 56.0, 58.7, 111.2, 112.3, 123.8, 126.9, 133.3,
145.5, 149.0, 150.7, 170.8, 198.2.
Preparation of (E)-4-(3’,4’-Dimethoxyphenyl)-3-
methyl-3-buten-2-one (5i)
Following the procedure for preparation of 5h and using 3,4-
dimethoxybenzaldehyde (1.66 g, 10 mmol), the product 5i
was obtained as a pale yellow oil; yield: 845 mg (38%). Rt
(GC/MS): 6.55 min; MS-EI: m/z (%)=220 (100) [M+], 219
(34), 205 (65), 190 (11), 189 (64), 177 (27), 162 (11), 147
1
(11), 146 (32), 131 (16), 115 (13), 103 (12), 91 (15); H NMR
(CDCl3, 500 MHz): d=2.09 (d, 3H, J=1.00 Hz), 2.46 (s,
3H), 3.92 (s, 3H), 3.93 (s, 3H), 6.92 (d, 1H, J=7.00 Hz),
6.98 (d, 1H, J=1.50 Hz), 7.07 (dd, 1H, J=1.50, 7.00 Hz),
7.47 (br, 1H); 13C NMR (CDCl3, 100 MHz): d=13.0, 25.8,
55.9, 111.0, 112.9, 123.4, 128.7, 136.1, 139.8, 148.7, 149.6,
200.2.
Preparation of (E)-3-Hydroxymethyl-4-(3’,4’-
methylenedioxyphenyl)-3-buten-2-one (4j)
Preparation of (E)-3-Methyl-4-(3’,4’-methylene-
dioxyphenyl)-3-buten-2-one (5j)
Following the procedure for preparation of 4i and using 3,4-
methylenedioxybenzaldehyde (4.50 g, 30 mmol) the product
4j was obtained as a colorless solid; yield: 3.49 g (53%); mp
75.1–75.98C. Rt (GC/MS): 6.91 min; MS-EI: m/z (%)=220
(100) [M+], 202 (27), 177 (29), 160 (38), 159 (73), 147 (74),
135 (33), 122 (40), 103 (26), 91 (40), 89 (32), 65 (21), 63
(22); 1H NMR (CDCl3, 600 MHz): d=2.46 (s, 3H), 2.67
(brt, 1H, J=6.60 Hz), 4.46 (d, 2H, J=6.60 Hz), 6.02 (s, 2H),
6.86–7.06 (m, 3H), 7.56 (s, 1H); 13C NMR (CDCl3,
125 MHz): d=25.6, 57.3, 101.6, 108.6, 109.7, 125.0, 128.5,
138.5, 143.2, 148.1, 149.0, 201.1.
Following the procedure for preparation of 5h and using pi-
peronal (3.0 g, 10 mmol), the product 5j was obtained as
a pale yellow solid; yield: (2.615 g (64%); mp 91.1–94.08C.
Rt (GC/MS): 6.20 min; MS-EI: m/z (%)=204 (100) [M+],
203 (60), 189 (33), 161 (16), 160 (12), 159 (52), 131 (41), 103
1
(65), 102 (13), 77 (28), 51 (10); H NMR (CDCl3, 600 MHz):
d=2.05 (d, 3H, J=1.80 Hz), 2.43 (s, 3H), 6.01 (s, 2H), 6.85
(d, 1H, J=7.80 Hz), 6.94 (dd, 1H, J=1.20, 7.80 Hz), 6.96 (d,
1H, J=1.20 Hz), 7.42 (br, 1H); 13C NMR (CDCl3,
125 MHz): d=13.0, 25.8, 101.4, 108.4, 109.6, 124.9, 130.0,
136.3, 139.6, 147.8, 148.0, 200.1.
Preparation of (E)-3-Acetoxymethyl-4-(3’,4’-
methylenedioxyphenyl)-3-buten-2-one (1j)
General Procedure for the Biotransformation of 1a–j
and 5h–j by Saccharomyces cerevisiae
Following the procedure for preparation of 1i and using 4j
(15 mmol) the product 1j was obtained as a pale yellow
solid; yield: 3.587 g (91%); mp 105.9–109.28C. Rt (GC/MS):
6.12 min; MS-EI: m/z (%)=236 (23), 176 (21), 161 (12), 135
(100), 131 (13), 77 (11); 1H NMR (CDCl3, 500 MHz): d=
2.10 (s, 3H), 2.46 (s, 3H), 4.95 (s, 2H), 6.03 (s, 2H), 6.85–
6.96 (m, 3H), 7.69 (s, 1H); 13C NMR (CDCl3, 100 MHz):
d=21.0, 26.0, 58.5, 101.6, 108.7, 109.3, 125.1, 128.2, 133.8,
145.1, 148.2, 149.2, 170.8, 198.1.
The yeast Saccharomyces cerevisiae, type II (5.0 g of lyophi-
lized cells), was added to distilled water at 40–428C (50 mL)
in a 125 mL Erlenmeyer flask. The substrate (0.5 mmol) was
adsorbed in filter paper (approximately 2 cm2 per mg of sub-
strate) and added in small pieces to the cell suspension. The
mixture was stirred in an orbital shaker at 308C and
180 rpm and monitored by GC/MS. After the appropriate
amount of time, the reaction mixture was extracted with
ethyl acetate, the organic layer was washed with brine, dried
over sodium sulfate and the solvent was removed under re-
duced pressure. The product was purified by column chro-
matography. The ee values were obtained by chiral GC/FID
analysis and the results are provided in Table 1 and Table 2.
Spectral data for 6a–g matched those already reported else-
where.[9]
(R)- and (S)-3-Methyl-4-(2’-methoxyphenyl)-2-butanone
[(R)- and (S)-6h]: Following the general procedure, the re-
duction of 1h afforded (R)-6h and the reduction of 5h gave
(S)-6h as a colorless oil. Optical rotation of the R isomer:
[a]20: À43 (c 1.95, CHCl3); lit.[21] [a]D23: +57.3 (c 1.1, CHCl3)
forD the S-isomer. Rt (GC/MS): 4.95 min; Rt (GC/FID,
Method B): 57.25 min (R-isomer), 57.88 min (S-isomer);
MS-EI: m/z (%)=192 (36) [M+], 121 (100), 108 (15), 91
(52); 1H NMR (CDCl3, 250 MHz): d=1.05 (d, 3H, J=
6.75 Hz), 2.10 (s, 3H), 2.56 (dd, 1H, J=6.75, 13.00 Hz), 2.89
(sx, 1H, J=6.75 Hz), 3.00 (dd, 1H, J=6.75, 13.00 Hz), 3.82
(s, 3H), 6.82–7.20 (m, 4H); 13C NMR (CDCl3, 150 MHz):
d=16.0, 28.6, 33.9, 46.8, 55.2, 110.2, 120.3, 127.6, 128.0,
130.9, 157.5, 212.6.
Preparation of (E)-3-Methyl-4-(2’-methoxyphenyl)-3-
buten-2-one (5h)
Glacial acetic acid (10 mL) and butanone (1.8 mL, 20 mmol)
were added to a round-bottom flask containing 2-methoxy-
benzaldehyde (1.36 g, 10 mmol). Under stirring, sulfuric acid
(2 mL) was added dropwise and the mixture was stirred at
room temperature for 16 h. Then, the reaction mixture was
neutralized with 10% sodium hydroxide and extracted with
diethyl ether. The organic layer was washed with brine and
dried over magnesium sulfate. The solvent was removed
under reduced pressure, and the product was purified by
column chromatography (95:5 hexane/ethyl acetate). Prod-
uct 5h was obtained as a colorless oil; yield: 1.001 g (55%).
Rt (GC/MS): 5.52 min; MS-EI: m/z (%)=190 (3) [M+], 159
(100), 132 (16), 131 (18), 115 (10), 91 (22); 1H NMR
(CDCl3, 250 MHz): d=1.99 (d, 3H, J=1.25 Hz), 2.47 (s,
3H), 3.87 (s, 3H), 6.91–7.36 (m, 4H), 7.74 (br, 1H);
13C NMR (CDCl3, 100 MHz): d=13.1, 25.9, 55.5, 110.5,
120.2, 124.9, 130.1, 130.3, 135.7, 137.7, 157.4, 200.6.
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(73%). Rt (GC/MS): 6.83 min; MS-EI: m/z (%)=245 (23),
169 (16), 137 (21), 109 (10), 107 (24), 96 (100), 95 (45), 83
(10), 82 (43).
(R)- and (S)-4-(3’,4’-Dimethoxyphenyl)-3-methyl-2-
butanone [(R)- and (S)-6i]
Following the general procedure, the reduction of 1i afford-
ed (R)-6i and the reduction of 5i gave (S)-6i as a colorless
oil. Optical rotation of the R isomer: [a]2D0: À28 (c 1.32,
CHCl3); lit.[22] [a]D20: À35.3 (c 4.3, CHCl3). Rt (GC/MS):
5.92 min; Rt (GC/FID, Method B) 85.59 min (R-isomer),
86.12 min (S-isomer); MS-EI: m/z (%)=220 (26) [M+], 179
Preparation of 4-Phenyl-3-hydroxymethyl-2-butanone
(8)
Lithium aluminum hydride (114 mg, 3 mmol) was added to
a solution of 7 (792 mg, 3 mmol) in THF (3 mL) under a ni-
trogen atmosphere. The mixture was stirred for 3 h, and
then the mixture was cooled to 08C. Water (0.2 mL) was
slowly added to the mixture, and then 10% sodium hydrox-
ide (0.5 mL) was added. After ten minutes, water (0.5 mL)
was added and the mixture was heated to room temperature
and stirred for 30 min. The solid was filtered and the prod-
uct was extracted with diethyl ether. The organic layer was
washed with brine and then the solvent was removed under
reduced pressure. THF (5 mL) and 10% hydrochloric acid
(5 mL) were added to the residue, and the mixture was
stirred for 16 h. Water was added to the round-bottom flask,
and the product was extracted with ethyl acetate. The organ-
ic layer was washed with 10% sodium bicarbonate, brine,
and dried over sodium sulfate. The solvent was removed
under reduced pressure and the product was purified by
column chromatography (90:10 hexane/ethyl acetate). Prod-
uct 8 was obtained as a colorless oil; yield: 279 mg (52%).
Rt (GC/MS): 5.02 min; MS-EI: m/z (%)=160 (22), 148
(38), 147 (40), 117 (93), 116 (22), 115 (38), 105 (52), 91
(100), 77 (24), 65 (22).
1
(4), 152 (10), 151 (100), 107 (5), 91 (4); H NMR (CDCl3,
250 MHz): d=1.09 (d, 3H, J=6.75 Hz), 2.08 (s, 3H), 2.52
(dd, 1H, J=6.75, 13.00 Hz), 2.81 (sx, 1H, J=6.75 Hz), 2.94
(dd, 1H, J=6.75, 13.00 Hz), 3.85 (s, 3H), 3.86 (s, 3H), 6.67–
6.80 (m, 3H); 13C NMR (CDCl3, 62.5 MHz): d=16.3, 29.0,
38.7, 49.0, 55.9, 111.3, 112.2, 120.9, 132.3, 148.9, 212.3.
(R)- and (S)-3-Methyl-4-(3’,4’-
methylenedioxyphenyl)-2-butanone [(R)- and (S)-6j]
Following the general procedure, the reduction of 1j afford-
ed (R)-6j and the reduction of 5j gave (S)-6j as a colorless
oil. When the reactions were performed in a 5 mmol scale,
(R)-6j was obtained from 1j in 73% yield, and its enantio-
mer, (S)-6j, was obtained from 5j in 63% yield. The absolute
configuration of the (S)- and (R)-enantiomers of 6j were de-
termined based on the configuration of (S)- and (R)-Tropio-
nal [(R)- and (S)-12j]. Optical rotation of the R isomer:
[a]2D0: À27.1 (c 3.59, CHCl3); optical rotation of the S
isomer: [a]2D0: +22.3 (c 3.08, CHCl3). Rt (GC/MS): 5.65 min;
Rt (GC/FID, Method B): 167.06 min (R isomer), 167.74 min
(S isomer); MS-EI: m/z (%)=206 (29) [M+], 163 (5), 135
Preparation of 3-Acetoxymethyl-4-phenyl-2-butanone
(9)
1
(100), 133 (4), 105 (8), 77 (10); H NMR (CDCl3, 250 MHz):
d=1.08 (d, 3H, J=6.75 Hz), 2.09 (s, 3H), 2.48 (dd, 1H, J=
6.75, 13.25 Hz), 2.77 (sx, 1H, J=6.75 Hz), 2.91 (dd, 1H, J=
6.75, 13.25 Hz), 5.91 (s, 2H), 6.57–6.73 (m, 3H); 13C NMR
(CDCl3, 150 MHz): d=16.2, 28.9, 38.7, 49.0, 100.9, 108.2,
109.3, 121.8, 133.4, 146.0, 147.6, 212.1.
Triethylamine (167 mL, 1.2 mmol), acetic anhydride (115 mL,
1.2 mmol) and DMAP (12 mg, 0.1 mmol) were added to a so-
lution of 8 (178 mg, 1 mmol) in dichloromethane (5 mL) at
08C. The mixture was stirred at 08C for 1 hour, and then
water (10 mL) was added to the round-bottom flask. The
mixture was further stirred at room temperature for 15 min,
and then the organic layer was separated. The aqueous layer
was extracted with dichloromethane, washed with brine,
dried over sodium sulfate. The solvent was removed under
reduced pressure and the product was purified by column
chromatography (95:5 hexane/ethyl acetate). Product 9 was
obtained as a colorless oil; yield: 186 mg (85%). Rt (GC/
MS): 5.51 min; MS-EI: m/z (%)=160 (25), 159 (15), 145
Preparation of Ethyl 2-(2’-Methyl-1’,3’-dioxolan-2’-
yl)-3-phenylpropanoate (7)
Sodium hydride (60% in mineral oil, 440 mg, 5.5 mmol) was
washed with hexane and added to a solution of ethyl acetoa-
cetate (650 mg, 5 mmol) in THF (10 mL) at 08C under a ni-
trogen atmosphere. The mixture was stirred for 1 hour, and
then benzyl chloride (575 mL, 5 mmol) in THF (5 mL) was
slowly added to the round-bottom flask. The mixture was
slowly heated to room temperature, and then stirred for
16 h. Then, methanol (10 mL) and water (10 mL) were suc-
cessively added to the mixture. The product was extracted
with diethyl ether. The organic layer was washed with brine,
dried over magnesium sulfate and then the solvent was re-
moved under reduced pressure. Toluene (10 mL) was added
to the residue, and then (550 mL, 10 mmol) and p-toluene-
sulfonic acid (172 mg, 1 mmol) were added to the round-
bottom flask. The mixture was refluxed for 2 h with water
removal by means of a Dean–Stark apparatus. Then, the
mixture was added to a separatory funnel, and washed with
water, 10% sodium bicarbonate and brine. The organic
layer was dried over sodium sulfate, the solvent was re-
moved under reduced pressure and the product was purified
by column chromatography (90:10 hexane/ethyl acetate).
Product 7 was obtained as a colorless oil; yield: 962 mg
1
(18), 117 (100), 116 (25), 115 (38), 91 (38), 65 (11); H NMR
(CDCl3, 500 MHz): d=2.02 (s, 3H), 2.07 (s, 3H), 2.74 (dd,
1H,J=7.50, 13.50 Hz), 2.93 (dd, 1H, J=7.50, 13.50 Hz), 3.14
(dq, 1H, J=5.50, 7.50 Hz), 4.16–4.24 (m, 2H), 7.14–7.31 (m,
5H); 13C NMR (CDCl3, 125 MHz): d=20.8, 30.6, 34.5, 53.1,
64.1, 126.7, 128.7, 128.8, 138.2, 170.7, 209.1.
Preparation of (R)- and (S)-2-Methyl-3-(3’,4’-
methylenedioxyphenyl)-1-propanol [(R)- and (S)-11]
A solution of sodium hypobromite was prepared by drop-
wise addition of bromine (511 mL, 9.9 mmol) to a solution of
sodium hydroxide (1.56 g, 39 mmol) in water (10 mL) in an
ice/salt bath. After all the bromine was consumed, the
bright yellow solution was diluted with 1,4-dioxane (2 mL),
and added dropwise to a cooled (cold water bath) solution
of (R)-6j (96% ee, 618 mg, 3 mmol) in 1,4-dioxane (20 mL).
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The mixture was stirred for 1 hour. Then, most of the diox-
ane/water mixture was evaporated under reduced pressure,
the residue was diluted with water, and extracted with di-
chloromethane. The aqueous layer was acidified, and then
extracted with dichloromethane. The organic layer from the
second extraction was dried over sodium sulfate and the sol-
vent was removed under reduced pressure to afford the cor-
responding acids. The residue was dissolved in THF (6 mL)
14.00 Hz), 2.61 (sxd, 1H, J=1.50, 7.00 Hz), 3.00 (dd, 1H,
J=7.00, 14.00 Hz), 5.92 (s, 2H), 6.61 (dd, 1H, J=1.50,
7.50 Hz), 6.65 (d, 1H, J=1.50 Hz), 6.73 (d, 1H, J=7.50 Hz),
9.69 (d, 1H, J=1.50 Hz); 13C NMR (CDCl3, 125 MHz): d=
13.2, 36.4, 48.2, 100.9, 108.2, 109.3, 121.9, 132.5, 146.1, 147.7,
204.4.
and kept under a nitrogen atmosphere. The solution was General Procedure for the Acylation of 8 with
cooled to 08C, and then ethyl chloroformate (343 mL,
3.6 mmol) and triethylamine (501 mL, 3.6 mmol) were added
to the mixture, which was stirred for 30 min. Triethylammo-
nium chloride was removed by filtration, and then sodium
borohydride (555 mg, 15 mmol) was added to the mixture.
Methanol was slowly added to the mixture until considera-
ble gas evolution started (adding too much methanol affords
the methyl ester), and then the reaction mixture was stirred
for 1 hour. After that, the reaction was quenched with water
and the product was extracted with dichloromethane. The
organic layer was washed with 10% hydrochloric acid, 10%
sodium bicarbonate, brine and dried over sodium sulfate.
The organic solvent was removed under reduced pressure,
and the product was purified by column chromatography
(80:20 hexane/ethyl acetate). Product (R)-11 was obtained
as a colorless oil; yield: 421.2 mg (72%); 94% ee. Optical
rotation: [a]20: +9.1 (c 3.96, CHCl3); lit.[12] [a]2D2: +11.1 (c
0.98, CHClD3); Rt (GC/MS): 5.66 min; Rt: (HPLC):
45.88 min; MS-EI: m/z (%)=194 (26) [M+], 136 (26), 135
Different Anhydrides: Preparation of 13a–e
Triethylamine (167 mL, 1.2 mmol), the acid anhydride
(1.2 mmol) and DMAP (12 mg, 0.1 mmol) were added to
a solution of (E)-4-phenyl-3-hydroxymethyl-3-buten-2-one
(8,176 mg, 1 mmol) in dichloromethane (5 mL) at 08C. The
mixture was stirred at 08C for 1 hour, and then water
(10 mL) was added to the round-bottom flask. The mixture
was further stirred at room temperature for 15 min, and
then the organic layer was separated. The aqueous layer was
extracted with dichloromethane, washed with brine, dried
over sodium sulfate. The solvent was removed under re-
duced pressure and the product was purified by column
chromatography (95:5 hexane/ethyl acetate).
(E)-4-Phenyl-3-propanoyloxymethyl-3-buten-2-one (13a):
Following the general procedure, 13a was obtained as a col-
orless oil; yield: 145 mg (62%). Rt (GC/MS): 6.32 min; MS-
EI: m/z (%)= 232 (11) [M+], 176 (24), 175 (100), 159 (12),
158 (71), 157 (17), 133 (24), 129 (18), 116 (17), 115 (65), 57
(66); 1H NMR (CDCl3, 500 MHz): d=1.15 (t, 3H, J=
7.50 Hz), 2.35 (q, 2H, J=7.50 Hz), 2.48 (s, 3H), 4.95 (s,
2H), 7.38–7.45 (m, 5H), 7.79 (s, 1H); 13C NMR (CDCl3,
125 MHz): d=9.13, 26.1, 27.5, 58.4, 128.8, 129.4, 129.7,
134.3, 135.5, 144.9, 174.1, 198.3; HR-MS-ESI+: m/z=
233.1194 (M+H)+, calculated: 233.1178.
(E)-3-Butanoyloxymethyl-4-phenyl-3-buten-2-one (13b):
Following the general procedure, 13b was obtained as a col-
orless oil; yield: 213 mg (87%). Rt (GC/MS): 6.64 min; MS-
EI: m/z (%)=246 (6) [M+], 176 (20), 175 (100), 158 (43),
157 (11), 133 (16), 129 (11), 116 (12), 115 (43), 71 (45);
1H NMR (CDCl3, 500 MHz): d=0.96 (t, 3H, J=7.50 Hz),
1.67 (sx, 2H, J=7.50 Hz), 2.32 (t, 2H, J=7.50 Hz), 2.48 (s,
3H), 4.94 (s, 2H), 7.37–7.44 (m, 5H), 7.88 (s, 1H); 13C NMR
(CDCl3, 125 MHz): d=13.7, 18.5, 26.1, 36.1, 58.3, 128.8,
129.5, 129.7, 134.3, 135.5, 144.9, 173.3, 198.3; HR-MS-ESI+:
m/z=247.1329 (M+H)+, calculated: 247.1334.
(E)-4-Phenyl-3-pentanoyloxymethyl-3-buten-2-one (13c):
Following the general procedure, 13c was obtained as a col-
orless oil; yield: 192 mg (74%). Rt (GC/MS): 6.94 min; MS-
EI: m/z(%)=260 (4) [M+], 176 (19), 175 (100), 158 (36),
133 (14), 116 (11), 115 (36), 85 (40), 57 (43); 1H NMR
(CDCl3, 500 MHz): d=0.92 (t, 3H, J=7.50 Hz), 1.36 (sx,
2H, J=7.50 Hz), 1.62 (qn, 2H, J=7.50 Hz), 2.34 (t, 2H, J=
7.50 Hz), 2.48 (s, 3H), 4.94 (s, 2H), 7.38–7.44 (m, 5H), 7.79
(s, 1H); 13C NMR (CDCl3, 125 MHz): d=13.7, 22.2, 26.1,
27.1, 34.0, 58.3, 128.8, 129.5, 129.7, 134.3, 135.5, 144.9, 173.5,
198.3; HR-MS-ESI+: m/z=261.1478 (M+H)+, calculated:
261.1491.
1
(100), 105 (6), 77 (12), 51 (4); H NMR (CDCl3, 500 MHz):
d=0.90 (d, 3H, J=7.00 Hz), 1.64 (brs, 1H), 1.88 (oc, 1H,
J=7.00 Hz), 2.34 (dd, 1H, J=7.00, 13.50 Hz), 2.67 (dd, 1H,
J=7.00, 13.50 Hz), 3.45 (dd, 1H, J=7.00, 10.50 Hz), 3.51
(dd, 1H, J=7.00, 10.50 Hz), 5.91 (s, 2H), 6.61 (dd, 1H, J=
1.50, 8.00 Hz), 6.67 (d, 1H, J=1.50 Hz), 6.72 (d, 1H, J=
8.00 Hz); 13C NMR (CDCl3, 125 MHz): d=16.4, 37.9, 39.4,
67.5, 100.8, 108.0, 109.5, 121.9, 134.4, 145.7, 147.5.
Starting from (S)-6j (75% ee, 721 mg, 3.5 mmol), the
product (S)-11 was obtained as a colorless oil; yield:
527.8 mg (78%); 74% ee. Optical rotation: [a]2D0: À7.5 (c
3.37, CHCl3); lit.[12] [a]22: À11.0 (c 1.07, CHCl3); Rt (GC/
MS): 5.66 min; Rt (HPLDC): 46.27 min.
Preparation of (R)- and (S)-2-methyl-3-(3’,4’-
methylenedioxyphenyl)propanal – (R)-Tropionalꢀ
and (S)-Tropionalꢀ [(R)- and (S)-12]
TEMPO (15.6 mg, 0.1 mmol) was added to a solution of
(R)- or (S)-11 (96 mg, 0.5 mmol) and PhI(OAc)2 (161 mg,
0.5 mmol) in dry dichloromethane (5 mL). The mixture was
stirred for 3 h, and then the solvent was removed under re-
duced pressure. The product was purified by column chro-
matography (95:5 hexane/ethyl acetate) giving (R)-12 in
68% and (S)-12 in 65% yield. Enantiomeric excesses of the
aldehydes were measured by reducing a small sample of
(R)- or (S)-12 with sodium brohydride in ethanol, and then
analyzing the produced alcohols by chiral HPLC. Optical ro-
tation of the R isomer: [a]2D0: +2.9 (c 3.81, CHCl3); lit.[12]
[a]2D4: +3.3 (c 0.98, CHCl3); optical rotation of the S isomer:
[a]2D0: À2.5 (c 3.49, CHCl3); lit.[12] [a]D24: À2.8 (c 1.07, CHCl3);
Rt (GC/MS): 5.35 min; MS-EI: m/z (%)=192 (34) [M+],
135 (100), 122 (10), 77 (17); 1H NMR (CDCl3, 500 MHz):
d=1.08 (d, 3H, J=7.00 Hz), 2.53 (dd, 1H, J=7.00,
(E)-4-Phenyl-3-hexanoyloxymethyl-3-buten-2-one (13d):
Following the general procedure, 13d was obtained as a col-
orless oil; yield: 166 mg (60%). Rt (GC/MS): 7.32 min; MS-
EI: m/z (%)=274 (4) [M+], 176 (18), 175 (100), 158 (33),
133 (13), 116 (10), 115 (29), 99 (28), 71 (23); 1H NMR
(CDCl3, 500 MHz): d=0.90 (t, 3H, J=7.00 Hz), 1.30–1.33
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(m, 4H), 1.60–1.67 (m, 2H), 2.33 (t, 2H, J=7.50 Hz), 2.48
(s, 3H), 4.94 (s, 2H), 7.38–7.44 (m, 5H), 7.78 (s, 1H);
13C NMR (CDCl3, 150 MHz): d=13.9, 22.3, 24.7, 26.1, 31.3,
34.2, 58.3, 128.8, 129.5, 129.7, 134.3, 135.5, 144.9, 173.5,
198.3; HR-MS-ESI+: m/z=275.1635 (M+H)+, calculated:
275.1647.
Preparation of (1R.2R)-1-Acetoxy-1-phenyl-2-methyl-
3-butanone [(1R,2R)-15]
Yeast from Saccharomyces cerevisiae, type II (20.0 g of
lyophilized cells), was added to distilled water at 40–428C
(200 mL) in a 125 mL Erlenmeyer flask. The substrate
(2.0 mmol) was adsorbed in filter paper (approximately
2 cm2 per mg of substrate) and added in small pieces to the
cell suspension. The reaction mixture was stirred in an orbi-
tal shaker at 308C and 180 rpm and monitored by GC/MS.
After the appropriate amount of time, the reaction mixture
was extracted with ethyl acetate, the organic layer was
washed with brine, dried over sodium sulfate and the solvent
was removed under reduced pressure. The product was puri-
fied by column chromatography (95:5 hexane/ethyl acetate).
Product (1R.2R)-15 was obtained in 14% yield (62.1 mg),
18:1 dr and >99% ee, and product (R)-6a was obtained in
34% yield (110 mg) and 45% ee. Optical rotation: [a]2D0: +15
(c 1.20, CHCl3); Rt (GC/MS): 5.22 min; Rt (GC/FID,
Method A): 30.89 min [(1S,2S) isomer], 31.11 min [(1R,2R)
isomer], 31.38 min [(1S,2R) and (1R,2S) isomers]; MS-EI:
m/z (%)=220 (1) [M+], 178 (10), 177 (79), 160 (69), 159
(26), 149 (13), 145 (11), 118 (37), 117 (79), 115 (27), 107
(100), 106 (10), 105 (89), 91 (19), 79 (18), 77 (26), 72 (43);
1H NMR (CDCl3, 250 MHz): d=1.16 (d, 3H, J=7.00 Hz),
2.02 (s, 3H), 2.08 (s, 3H), 3.05 (qn, 1H, J=7.00 Hz), 6.04 (d,
1H, J=7.00 Hz).7.26–7.34 (m, 5H); 13C NMR (CDCl3,
62.5 MHz): d=12.3, 21.0, 29.6, 52.7, 75.7, 126.7, 128.1, 128.5,
138.9, 169.9, 208.9.
(E)-3-Benzoyloxymethyl-4-phenyl-3-buten-2-one
(13e):
Following the general procedure, 13e was obtained as a col-
orless oil; yield: 104 mg (37%). Rt (GC/MS): 8.15 min; MS-
EI: m/z (%)=280 (1) [M+], 175 (52), 158 (20), 115 (16), 105
1
(100), 77 (24); H NMR (CDCl3, 500 MHz): d=2.52 (s, 3H),
5.20 (s, 2H), 7.38–8.02 (m, 10H), 7.86 (s, 1H). 13C NMR
(CDCl3, 150 MHz): d=26.2, 59.1, 128.4, 128.9, 129.5, 129.7,
129.8, 130.0, 133.0, 134.3, 135.3, 145.1, 166.3, 198.2; HR-MS-
ESI+: m/z=281.1161 (M+H)+, calculated: 281.1178.
Preparation of (E)-2-Benzylidene-3-oxobutyl tert-
Butyl Carbonate (13f)
Boc2O (262 mg, 1.2 mmol) and DMAP (12 mg, 0.1 mmol)
were added to a solution of (E)-4-phenyl-3-hydroxymethyl-
3-buten-2-one (8, 176 mg, 1 mmol) in dichloromethane
(5 mL) at 08C. The mixture was stirred at 08C for 1 hour,
and then water (10 mL) was added to the round-bottom
flask. The mixture was further stirred at room temperature
for 15 min, and then the organic layer was separated. The
aqueous layer was extracted with dichloromethane, washed
with brine, dried over sodium sulfate. The solvent was re-
moved under reduced pressure and the product was purified
by column chromatography (95:5 hexane/ethyl acetate).
Product 13f was obtained as a colorless solid; yield: 103 mg
(37%); mp 130.5–134.28C. Rt (GC/MS): 6.80 min; MS-EI:
m/z (%)=220 (100), 219 (51), 175 (12), 159 (24), 158 (26),
117 (12), 116 (19), 115 (48), 57 (42); 1H NMR (CDCl3,
500 MHz): d=1.51 (s, 9H), 2.47 (s, 3H), 4.94 (s, 2H), 7.39–
7.45 (m, 5H), 7.79 (s, 1H); 13C NMR (CDCl3, 150 MHz):
d=26.1, 27.8, 60.6, 82.4, 128.8, 129.5, 129.7, 134.2, 135.1,
145.4, 153.2, 198.1; HR-MS-ESI+: m/z=277.1442 (M+H)+,
calculated: 277.1440.
The configuration of (1R,2R)-15 was determined by its
transformation in the corresponding b-hydroxy ester. A so-
lution of sodium hypobromite was prepared by dropwise ad-
dition of bromine (17 mL, 0.33 mmol) to a solution of
sodium hydroxide (52 mg, 1.30 mmol) in water (0.3 mL) in
an ice/salt bath. After all the bromine was consumed, the
bright yellow solution was added dropwise to a cooled (cold
water bath) solution of (1R, 2R)-15 (22 mg, 0.1 mmol) in di-
oxane (0.6 mL). The mixture was stirred for 1 hour. After
that, most of the dioxane/water mixture was evaporated
under reduced pressure; the residue was diluted with water
(5 mL), and extracted with CH2Cl2. The aqueous layer was
acidified, and then extracted with CH2Cl2. The organic layer
was dried over Na2SO4 and the solvent was removed under
reduced pressure. The mixture of 3-hydroxy-2-methyl-3-phe-
nylpropanoic acid and a-methylcinnamic acid was then dis-
solved in diethyl ether (0.5 mL) and cooled to 08C. A solu-
tion of CH2N2 in diethyl ether (0.5 mL) was then added to
the carboxylic acid solution, and the mixture was stirred for
30 min. The solvent was removed under reduced pressure,
and the residue was purified by column chromatography in
a glass Pasteur pipette (hexane/EtOAc, 95:5–85:15. Methyl
(2R,3R)-3-hydroxy-2-methyl-3-phenylpropanoate was ob-
tained; yield: 3.8 mg. Optical rotation: [a]2D0: +19 (c 0,38,
CHCl3); lit.[15] [a]D23: +23.1 (c 1.5, CHCl3); Rt (GC/MS):
4.89 min; MS-EI: m/z (%)=194 (7) [M+], 107 (79), 105
Preparation of 1-Acetoxy-1-phenyl-2-methylidene-3-
butanone (14)
Triethylamine (880 mg, 5 mmol), acetic anhydride (567 mL,
6 mmol) and DMAP (60 mg, 0.5 mmol) were added to a so-
lution of 1-phenyl-1-hydroxy-2-methylidene-3-butanone
(880 mg, 5 mmol) in dichloromethane (25 mL) at 08C. The
mixture was stirred at 08C for 1 hour, and then water
(10 mL) was added to the round-bottom flask. The mixture
was further stirred at room temperature for 15 min, and
then the organic layer was separated. The aqueous layer was
extracted with dichloromethane, washed with brine, dried
over sodium sulfate. The solvent was removed under re-
duced pressure and the product was purified by column
chromatography (95:5 hexane/ethyl acetate). Product 14 was
obtained as a colorless oil; yield: 918 mg (84%). Rt (GC/
MS): 5.40 min; MS-EI: m/z (%)=176 (24), 175 (100), 115
1
(19), 88 (100), 79 (51), 77 (36), 57 (22); H NMR (CDCl3,
1
250 MHz): d=1.13 (d, 3H, J=7.25 Hz), 2.80 (dq, 1H J=
3.25, 7.25 Hz), 2.92 (d, 1H, J=3.25 Hz), 3.69 (s, 3H), 5.12 (t,
1H, J=3.25 Hz), 7.30–7.36 (m, 5H); 13C NMR (CDCl3,
62.5 MHz): d=10.7, 46.3, 51.9, 73.6, 125.9, 127.5, 128.3,
141.3, 176.3.
(24), 105 (20), 97 (23), 77 (10); H NMR (CDCl3, 500 MHz):
d=2.09 (s, 3H), 2.31 (s, 3H), 6.07 (d, 1H, J=1.00 Hz), 6.22
(brs, 1H), 6.74 (brs, 1H), 7.27–7.38 (m, 5H); 13C NMR
(CDCl3, 100 MHz): d=21.1, 26.2, 72.5, 125.2, 127.5, 128.2,
128.4, 138.2, 147.8, 169.4, 197.3.
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General Procedure for the Biotransformation of
Enones by EREDs
Preparation of 1-Phenyl-2-methylideno-3-pentanone
(19)
A 500 mL solution of the substrate (25 mg) in DMSO was
added to a solution of GDH-5 (10 mg), NADP+ (2.5 mg)
and glucose (2 equivalents relative to the substrate) in 1M
pH 7 potassium phosphate buffer (4.5 mL). 500 mL of the
resulting mixture were added to a vial containing 5 mg of
the ERED. The reaction was kept in an orbital shaker at
180 rpm and 308C. A 50 mL sample was withdrawn, extract-
ed with ethyl acetate (2ꢃ400 mL) and analyzed by GC/MS
and chiral GC/FID.
LiAlH4 (380 mg, 10 mmol) was added to a solution of 18
(2.64 g, 10 mmol) in dry THF (10 mL) under an N2 atmos-
phere. The mixture was stirred for 3 h at room temperature
and after that the mixture was cooled to 08C. Water
(0.5 mL) was slowly added and 10% NaOH solution (1 mL)
was also slowly added. After 10 mmin, water (2 mL) was
added and the mixture was heated to room temperature.
After the mixture had been stirred for 30 min, MgSO4 was
added and the resulting mixture was filtered. 10% HCl
(10 mL) was added to the filtrate and the resulting mixture
was stirred for 16 h. After that, the reaction product was ex-
tracted with EtOAc, the organic phase was washed with
10% NaHCO3 solution, brine and dried with MgSO4. The
solvent was removed under reduced pressure giving an oil
that was treated with benzene (10 mL), MsCl (851 mL,
11 mmol) and DBU (3.29 mL, 22 mmol). The resulting mix-
ture was refluxed for 2 h and then water (10 mL) was added
and the mixture stirred for 10 min. The organic phase was
washed three times with water, brine and dried with
Na2SO4. After the removing the solvent under reduced pres-
sure, the product was purified by column chromatography
(hexane/EtOAc, 95:5) giving 19 as a colorless oil; yield:
1.05 g (60%). Rt (GC/MS): 4,58 min; MS-EI: m/z (%)=174
(50) [M+], 173 (26), 146 (10), 145 (84), 118 (10), 117 (100),
116 (28), 115 (82), 91 (36), 65 (11), 57 (15); 1H NMR
(CDCl3, 250 MHz): d=1.08 (t, 3H, J=7.25 Hz), 2.71 (q, 2H,
J=7.25 Hz), 3.60 (s, 2H), 5.60 (s, 1H), 6.07 (s, 1H), 7.15–
7.29 (m, 5H); 13C NMR (CDCl3, 62.5 MHz): d=8.3, 31.0,
37.1, 125.0, 126.2, 128.4, 129.1, 139.2, 148.2, 201.9.
Preparation of (E)-1-Phenyl-2-methyl-1-penten-3-one
(17)
Gaseous HCl, generated by addition of 35% HCl to CaCl2,
was bubbled into a solution of benzaldehyde (1.02 mL,
10 mmol) and 3-pentanone (1.22 mL, 20 mmol). The mixture
was stirred for 72 h, and then added to a separatory funnel
containing diethyl ether and water. The organic layer was
separated, washed with 10% sodium bicarbonate, brine and
dried over magnesium sulfate. The solvent was removed
under reduced pressure and the product was purified by
column chromatography (95:5 hexane/ethyl acetate). Prod-
uct 17 was obtained as a colorless oil; yield: 1.27 g (73%).
Rt (GC/MS): 5.00 min; MS-EI: m/z (%)=174 (49) [M+],
173 (17), 145 (84), 117 (100), 116 (16), 115 (82), 91 (29);
1H NMR (CDCl3, 250 MHz): d=1.18 (t, 3H, J=7.25 Hz),
2.07 (d, 3H, J=1.25 Hz), 2.85 (q, 2H, J=7.25 Hz), 7.26–7.52
(m, 5H), 7.53 (q, 1H, J=1.25 Hz); 13C NMR (CDCl3,
62.5 MHz): d=8.9, 13.2, 30.8, 128.4, 128.8, 129.7.136.0, 137.2,
138.2, 203.0.
Preparation of Methyl (Z)-2-(2’-Ethyl-1’,3’-dioxolan-
2’-yl)-3-phenyl-2-propenoate (20)
Preparation of Methyl 2-(2’-Ethyl-1’,3’-dioxolan-2’-
yl)-3-phenylpropanoate (18)
Morpholine (2.585 mL, 30 mmol) was slowly added to
a round-bottom flask containing methyl 3-oxopentanoate
(3.790 mL, 30 mmol), benzaldehyde (3.060 mL, 30 mmol)
and glacial acetic acid (3.430 mL, 60 mmol). The reaction
mixture was stirred for 24 h, and then ethyl acetate was
added to the mixture. The solution was washed with water,
and the organic layer was dried over sodium sulfate. The
solvent was removed under reduced pressure, and the resi-
due was dissolved in toluene (60 mL). Ethylene glycol
(3.355 mL, 60 mmol) and p-toluenesulfonic acid (517 mg,
3 mmol) were added to the round-bottom flask. The mixture
was refluxed for 6 h with water removal by means of
a Dean–Stark apparatus. Then, the mixture was added to
a separatory funnel, and washed with water, 10% sodium bi-
carbonate and brine. The organic layer was dried over
sodium sulfate, the solvent was removed under reduced
pressure and the product was purified by column chroma-
tography (95:5 hexane/ethyl acetate). Product 20 was ob-
tained as a colorless oil; yield: 6.084 g (77%). Rt (GC/MS):
6.26 min; MS-EI: m/z (%)=234 (15), 233 (100), 121 (37),
103 (38), 102 (10), 101 (39), 57 (12).
NaH (60% in mineral oil, 880 mg, 22 mmol) was added to
a solution of methyl 3-oxopentanoate (2.6 g, 20 mmol) in
dry THF (40 mL) at 08C under an N2 atmosphere in a two-
neck round-bottom flask (250 mL). The resulting mixture
was stirred for 1 h at 08C, and then BnCl (2.3 mL, 20 mmol)
in THF (20 mL) was slowly added. After that, the mixture
was heated to room temperature under stirring for 16 h.
Then, MeOH (10 mL) and H2O (10 mL) were added and
the product was extracted with ether. The organic phase was
washed with brine and dried with MgSO4 and the solvent
was removed under reduced pressure giving an oil that was
dissolved in PhMe (40 mL) in a two-neck round-bottom
flask (250 mL). Ethylene glycol (2.2 mL, 40 mmol) and p-
TsOH (344 mg, 2 mmol) were added and the resulting mix-
ture was refluxed for 4 h in a Dean–Stark apparatus to
remove water. After that, the resulting mixture was washed
with a 10% aqueous solution of NaHCO3, water and brine.
The organic phase was dried with Na2SO4, the solvent re-
moved under reduced pressure and the product purified by
column chromatography (hexane/EtOAc, 95:5) giving 18 as
a colorless oil; yield: 3.210 g (61%). Rt (GC/MS): 6,24 min;
MS-EI: m/z (%)= 235 (4), 159 (5), 131 (3), 113 (4), 101
(100), 91 (17), 57 (15).
Preparation of (E)-2-(2’-Ethyl-1’,3’-dioxolan-2’-yl)-3-
phenyl-2-propen-1-ol (21)
Diisobutylaluminum hydride (2M in toluene, 24 mL,
48 mmol) was slowly added to a solution of compound 20
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(5.24 g, 20 mmol) in dichloromethane (20 mL) at 08C under
a nitrogen atmosphere. The mixture was stirred for 6 h at
08C, and then ethyl acetate (50 mL) was added to the
round-bottom flask. The reaction was stirred for 30 min, and
then water (5 mL) was added to the round-bottom flask.
The mixture was further stirred for 10 min, and then 10%
sodium hydroxide solution (5 mL) was added to the round-
bottom flask. The mixture was further stirred for 10 min,
and then water (15 mL) was added to the round-bottom
flask. After 1 hour, the solid mass was separated by filtra-
tion, the organic layer was washed with water and brine and
then dried over sodium sulfate. The organic solvent was re-
moved under reduced pressure, and the product was puri-
fied by column chromatography (85:15 hexane/ethyl ace-
tate). Product 21 was obtained as a colorless oil; yield:
2.704 g (58%). Rt (GC/MS): 6.34 min; MS-EI m/z (%)=205
(100), 115 (17), 101 (26), 57 (11); 1H NMR (CDCl3,
600 MHz): d=0.94 (t, 3H, J=7.80 Hz), 1.90 (q, 2H, J=
7.80 Hz), 2.71 (brs, 1H), 3.97–4.03 (m, 4H), 4.25 (s, 2H),
6.89 (s, 1H), 7.25–7.41 (m, 5H); 13C NMR (CDCl3,
62.5 MHz): d=7.8, 29.9, 58.7, 64.7, 112.5, 127.4, 128.3, 129.0,
129.9, 136.1, 138.2.
The reaction mixture was stirred in an orbital shaker at
308C and 180 rpm and monitored by GC/MS. After 48 h,
the reaction mixture was extracted with ethyl acetate, the
organic layer was washed with brine, dried over sodium sul-
fate and the solvent was removed under reduced pressure.
The product was purified by column chromatography (95:5
hexane/ethyl acetate). Product (S)-23 was obtained as a col-
orless oil; yield: 139.3 mg (79%); 95% ee. Product (R)-23
was obtained as a colorless oil; yield: 126.5 mg (72%); 93%
ee. Optical rotation of the R isomer: [a]2D0: À65.1 (c 3.86,
CHCl3); Optical rotation of the S isomer: [a]2D0: +63.0 (c
3.20, CHCl3); lit.[22] [a]D23: +70.9 (c 1.10, CHCl3); Rt (GC/
MS): 4.40 min; Rt (GC/FID, Method A): 22.43 min (S
isomer), 23.66 min (R isomer); MS-EI: m/z (%)=176 (20)
1
[M+], 147 (39), 119 (28), 91 (100), 57 (36); H NMR (CDCl3,
600 MHz): d=0.97 (t, 3H, J=7.20 Hz), 1.08 (d, 3H, J=
7.20 Hz), 2.25 (dq, 1H, J=7.20, 17.40 Hz), 2.43 (dq, 1H, J=
7.20, 17.40 Hz), 2.57 (dd, 1H, J=7.20, 13.80 Hz), 2.84 (sx,
1H, J=7.20 Hz), 2.97 (dd, 1H, J=7.20, 13.80 Hz), 7.12–7.28
(m, 5H). 13C NMR (CDCl3, 62.5 MHz): d=7.6, 16.6, 35.2,
39.3, 47.9, 126.2, 128.4, 128.9, 139.9, 214.8.
Preparation of (E)-2-Acetoxymethyl-1-phenyl-1-
penten-3-one (22)
Acknowledgements
10% Hydrochloric acid (10 mL) was added to a solution of
21 (2.34 g, 10 mmol) in THF (10 mL). The reaction mixture
was refluxed for 2 h, and then the water was added to the
round-bottom flask and the product was extracted with
ethyl acetate. The organic layer was washed with 10%
sodium bicarbonate, brine, and dried over sodium sulfate.
The solvent was removed under reduced pressure, the prod-
uct was dissolved in dichloromethane (50 mL) and the solu-
tion was cooled to 08C. Triethylamine (1.67 mL, 12 mmol),
acetic anhydride (1.15 mL, 12 mmol) and DMAP (122 mg,
0.1 mmol) were added to the mixture. The mixture was
stirred at 08C for 1 hour, and then water (100 mL) was
added to the round-bottom flask. The mixture was further
stirred at room temperature for 15 min, and then the organic
layer was separated. The aqueous layer was extracted with
dichloromethane, the extract was washed with brine and
dried over sodium sulfate. The solvent was removed under
reduced pressure and the product was purified by column
chromatography (95:5 hexane/ethyl acetate).Product 22 was
obtained as a colorless oil; yield: 1.656 g (71%). Rt (GC/
MS): 6.27 min; MS-EI: m/z (%)=232 (15) [M+], 190 (24),
189 (40), 173 (12), 172 (79), 161 (53), 143 (100), 133 (51),
128 (10), 117 (51), 116 (21), 115 (85), 103 (11), 77 (12), 57
(46); 1H NMR (CDCl3, 600 MHz): d=1.19 (t, 3H, J=
7.20 Hz), 2.08 (s, 3H), 2.84 (q, 2H, J=7.20 Hz), 4.95 (s,
2H), 7.37–7.43 (m, 5H), 7.79 (s, 1H); 13C NMR (CDCl3,
62.5 MHz): d=0.4, 21.0, 31.1, 58.8, 128.8, 129.4, 129.6, 134.3,
134.9, 143.7.170.8, 201.0.
Support from CNPq: proc. 304730/2013-0, 301811/2014-8,
143099/2006-0 and FAPESP: proc. 2014/00108-9, 2012/
08385-6 is gratefully acknowledged.
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18 Bioreduction of a-Acetoxymethyl Enones: Proposal for an
SN2’ Mechanism Catalyzed by Enereductase
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Bruno R. S. Paula, Davila Zampieri,
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