Screening on the use of Kluyveromyces marxianus CBS 6556 growing cells as enantioselective biocatalysts for ketone reductions

Article abstract of DOI:10.1016/j.tetasy.2011.11.014

The versatility of Kluyveromyces marxianus CBS 6556 growing cells in the enantioselective reduction of ketone functionalities to the corresponding alcohols was exploited. In particular, methyl ketones were reduced to (S)-alcohols with ees of up to 96%. Longer chain alkyl ketones afforded, under the same experimental condition, (R)-alcohols with an ee of up to 84%. Interestingly, carbon-carbon double and the triple bonds can also be reduced in the presence of Kluyveromyces marxianus CBS 6556 yeast. A cyclic ketone, such as 2-tetralone, was also quantitatively reduced to its corresponding (S)-alcohol with ee = 76%.

Full text of DOI:10.1016/j.tetasy.2011.11.014

Tetrahedron: Asymmetry 22 (2011) 1985–1993
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Screening on the use of Kluyveromyces marxianus CBS 6556 growing cells
as enantioselective biocatalysts for ketone reductions
⇑
Paola Vitale , Filippo Maria Perna, Maria Grazia Perrone, Antonio Scilimati
Dipartimento Farmaco-Chimico, Università degli Studi di Bari ‘A. Moro’, Via E. Orabona 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The versatility of Kluyveromyces marxianus CBS 6556 growing cells in the enantioselective reduction of
ketone functionalities to the corresponding alcohols was exploited. In particular, methyl ketones were
reduced to (S)-alcohols with ees of up to 96%. Longer chain alkyl ketones afforded, under the same exper-
imental condition, (R)-alcohols with an ee of up to 84%. Interestingly, carbon–carbon double and the tri-
ple bonds can also be reduced in the presence of Kluyveromyces marxianus CBS 6556 yeast. A cyclic
ketone, such as 2-tetralone, was also quantitatively reduced to its corresponding (S)-alcohol with
ee = 76%.
Received 4 October 2011
Accepted 21 November 2011
Available online 4 January 2012
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
have not been extensively investigated as catalysts,8,9 although
they have several interesting features that make this species prom-
Optically active molecules are important building blocks for the
synthesis of many chemicals and bio-active compounds. Biocata-
lyst assisted processes have some advantages over the most con-
ventional asymmetric synthesis, such as chemo-, regio-, and
stereoselectivities, together with very mild reaction conditions.1
Research over recent decades has led to a better understanding
of biological systems and consequently increased their application
into chemical conversion processes, especially in the field of organ-
ic synthesis.2 The use of whole cells for biocatalysed reactions has
been explored since the 1950s but their use can be difficult in large
scale synthesis, due to complicated work-up procedures, high
medium volumes for cells cultures, the low space–time yields
and long reaction times. Despite these disadvantages, whole-cells
are often chosen as biocatalysts because they provide a continuous
source of enzymes and cofactors,3–6 although their depletion can
be a problem. This disadvantage has recently been overcome by ge-
netic engineering, in which new yeast strains have been con-
structed by introducing heterologous genes to simplify the use of
cofactor-requiring enzymes (e.g., enzyme overexpression, and so
on),7 and to improve the overall efficiency (mostly stereoselectivi-
ty), these new techniques were recently implemented for synthetic
applications and already show great promise.
ising for the industrial production of several compounds.
For the past several years, our interests have focused on using
non-conventional yeasts to synthesize novel enantiopure com-
pounds. The thermo-tolerant Kluyveromyces marxianus CBS 6556
was successfully used by us for the stereoselective bioreduction
of prostereogenic keto-esters to prepare clofibric acid analogues
as peroxisome proliferator-activated receptor (PPAR-alpha) li-
gands.10–12 These studies allowed us to also isolate an unknown
NADPH dependent ADH from Kluyveromyces marxianus CBS 6556
yeast strain.13 This novel ADH efficiently mediated the reduction
of 3-oxo esters with a high degree of stereoselectivity, providing
chiral alcohols with an (S) absolute configuration at the newly
formed stereogenic center.
Herein, we report the continuation of such studies, in which
Kluyveromyces marxianus CBS 6556 whole cells were used to ex-
plore the bioreduction of various prochiral ketones, with the aim
to screen the biocatalyst substrate ‘specificity’, as well its regio-
chemo-, and enantioselectivities related to the molecular structure
of the prochiral substrate to be reduced.
2. Results and discussion
The asymmetric reduction of carbonyl compounds with yeast,
in particular Saccharomyces cerevisiae, is probably one of the most
investigated classes of whole-cell biotransformations. Many re-
search groups have focused their attention on looking for ‘non-con-
ventional’ yeasts, compared to the highly investigated S.
cerevisiae.5 Amongst these yeasts, Kluyveromyces marxianus strains
The growing cells of Kluyveromyces marxianus CBS 6556 were
chosen to study the substrate features on which it could be used
as a biocatalyst in a stereoselective reduction. Structurally different
methyl(alkyl)ketones were chosen to check the dependence of ste-
reoselectivity shown by Kluyveromyces marxianus CBS 6556 yeast
on the molecular characteristics of the ketones. Two sets of ketones
were used herein. The first were highly flexible, semi-rigid, and ri-
gid methyl ketones, while the latter were ethyl, n-propyl, or i-butyl
ketones. A cyclic ketone was also used. Fair to good conversions of
⇑
Corresponding author.
E-mail address: paola.vitale@farmchim.uniba.it (P. Vitale).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.11.014

1986
P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
the ketones into the corresponding alcohols were observed,
although the optimization of the reaction time and other experi-
mental conditions is currently in progress.
A summary of results obtained by Kluyveromyces marxianus CBS
6556 growing cells-mediated methyl ketone bioreduction is re-
ported in Scheme 1 and Table 1.
effects also being crucial in the enantioselection of carbonyl
reduction.
The 4-phenyl-3-butyn-2-one 1d reduction to (S)-4-phenyl-3-
butyn-2-ol 2d occurs with lower enantioselectivity (56% ee) and
yield (24%) in 4 days of incubation time. It should be noted that
5% of (E)-4-phenyl-3-buten-2-one was present in the reaction
crude, derived from the triple bond reduction.23
In contrast, the (E)-4-phenyl-3-buten-2-one 1e bioreduction by
Kluyveromyces marxianus CBS 6556 growing cells proceeded with a
complete substrate conversion into the following products
(Scheme 2): (S)-4-phenyl-2-butanol24–27 2b (90% yield and 68%
ee), (S)-4-phenyl-3-buten-2-ol 2e (3% yield and 90% ee) and 4-phe-
nyl-2-butanone 1b (7% yield).
Phenylacetone 1a was first reduced in the presence of Kluyver-
omyces marxianus CBS 6556 growing cells to the corresponding
optically active 1-phenylpropan-2-ol 2a. After 24 h incubation at
30 °C, a 50% conversion of phenylacetone was obtained; the
product had 92% ee and an (S)-absolute configuration.14,15 1-Phe-
nylpropan-2-ol 2a is an important synthon in the preparation of
L
-deprenyl,16 a monoamine oxidase (MAO)-B selective and irre-
versible inhibitor, used in the treatment of neurodegenerative dis-
orders such as Alzheimer’s and Parkinson’s disease.17,18 However,
the complete phenylacetone conversion required a longer incuba-
tion time (96 h). In this case, the 1-phenylpropan-2-ol isolated had
90% ee. In order to compare homogeneous data, most of the reac-
tions were incubated for 96 h at 30 °C, by using the same growing
cell concentration.19 Under these conditions, 4-phenyl-2-butanone
1b (entry 3) afforded the corresponding 4-phenylbutan-2-ol 2b in
a 74% yield as a racemic mixture (enantiomeric ratio = 51:49). The
bigger distance between the carbonyl moiety and the phenyl
group, comparing the phenylacetone and 4-phenyl-2-butanone, re-
sulted in a less constrained molecule. In fact, it is well known that
most of the ADHs show high stereoselectivity for bulky–bulky sub-
strates and low stereoselectivity for small-bulky substrates.20,21
As a result, no enantioselectivity was observed in the 4-phenyl-
2-butanone reduction mediated by Kluyveromyces marxianus CBS
6556 growing cells. These results can probably be explained by
considering the presence of more than one ADH, operating in the
yeast whole cells, with different stereo-preferences, that are
responsible for the formation of a racemic mixture.
Kluyveromyces marxianus CBS 6556 cells behaved almost in the
same way with 4-phenyl-3-butyn-2-one 1d and (E)-4-phenyl-3-
buten-2-one 1e, because both substrates underwent two subse-
quent reactions: carbonyl and carbon–carbon multiple bond
reduction. A hypothesis for the bioreduction, which takes into ac-
count the observed enantioselectivity, is depicted in Scheme 3.
The 4-phenyl-2-butanol 2b can be formed through two possi-
ble different pathways. Carbon–carbon double bond reduction
followed by reduction of the carbonyl double bond (a + b), or
reduction of the carbonyl followed by the reduction of the car-
bon–carbon double bond (c + d). The reduction of (E)-4-phenyl-
3-buten-2-ol 2e to 4-phenyl-2-butanol under the same experi-
mental conditions, showed that both pathways contributed,
although at a different reaction rates. Hence, the reduction of
(E)-4-phenyl-3-buten-2-one 1e leads to optically active (E)-4-phe-
nyl-3-buten-2-ol 2b (90% ee) (via c) and to 4-phenylbutan-2-one
(via a). The ee (68%) of the final product (S)-4-phenyl-2-buta-
nol24–27 is the result of the sum of the allylic double bond
reductions (via d) and of the carbonyl group reduction by one
non-stereoselective way (via b). These results allowed us to con-
clude that from the two steps b and d, the first (via b) proceeded
with almost no stereoselectivity as happened in the direct reduc-
tion of 4-phenyl-2-butanone (entry 3, Table 1). However, although
the reaction mechanism elucidation requires further studies, these
findings provide evidence for the probable involvement of a yeast
enoate reductase.28,29 This would be, to the best of our knowledge,
the first precedent of the bioreduction of a non-activated double
bond by an enoate reductase.
Next, phenoxyacetone 1c (Table 1, entry 4) was reduced under
the same conditions to afford (S)-1-phenoxy-2-propanol 2c in 24 h,
a 87% yield, with 80% ee.22
By comparing 4-phenyl-2-butanone with phenoxyacetone, the
replacement of a methylene with an oxygen atom allows recovery
of the enantioselectivity. This is probably due to the electronic
These results also highlighted the dependence of the enantiose-
lectivity on the spacer between the aromatic ring and the carbonyl
functionality. In particular, the enantioselectivity in the product
decreased when increasing the distance between the two func-
tional groups. Moreover, with a C2 spacer, the enantioselectivity
observed depended on the C2 hybridization, conjugation possibil-
ity, and conformational degrees of freedom. The enantiomeric
Kluyveromyces marxianus CBS 6556
OH
*
n
O
R2
R2
R1
R1
growing cells
n
2a-g
1a-g
Scheme 1.
Table 1
Bioreduction of prochiral ketones mediated by Kluyveromyces marxianus CBS 6556 whole cells (isolated yields)
Entry
R1
R2
n
Conversiona (%)
Yield (%)
ee (%)
Time (h)
Abs. Config.
1
2
3
4
5
6
7
8
9
CH3
CH3
CH3
CH3
CH3
CH3
CF3
Ph–
Ph–
Ph–
Ph–O–
Ph–C„C–
PhCH@CH–
Ph–
Ph–
Ph–
1
1
2
1
0
0
1
1
0
50
100
80
100
30
100
80
100
70
50 2a
>98b 2a
74 2b
87 2c
24c 2d
3d 2e
75 2f
> 98 2f
67 2g
92
90
—
80
56
24
96
96
24
96
96
24
96
96
(S)
(S)
—
(S)
(S)
(S)
(S)
(S)
(S)
d
68
60
58
48
CF3
CH3
a
b
c
Calculated by 1H NMR diagnostic signals of the unreacted ketone in the reaction crude.
2% of 1-phenylpropan-2-yl acetate was also detected in the reaction crude (See Section 4).
5% of (E)-4-phenyl-3-buten-2-one 1e was also isolated.
d
The main isolated product was the (S)-4-phenyl-2-butanol 2b (90%, ee = 68%), 3% of 4-phenyl-3-buten-2-ol 2e (90% ee) and 7% of 4-phenyl-2-butanone 1b were also
isolated by column chromatography (Scheme 2).

P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
1987
Kluyveromyces marxianus
O
OH
O
OH
CBS 6556
(S)
+
+
(S)
growing cells
1e
2b, 90% yield, ee = 68%
1b, 7% yield
2e, 3% yield, ee = 90%
Scheme 2. Bioreduction of (E)-4-phenyl-3-buten-2-one by Kluyveromyces marxianus CBS 6556.
Table 2
O
O
Bioreduction of phenylacetone analogues by Kluyveromyces marxianus CBS 6556a
a
Entry
Ketone
R2
Yield (%)
ee
Abs. Config.
1
2
3
4
1a
1h
1i
Ph–
>98 2a
>98 2h
93 2ic
90
94
94
92
(S)
(S)b
(S)
(S)
2-FC6H4–
4-FC6H4–
3-CF3C6H4–
good
stereoselectivity
low
c
b
1j
>98 2j
stereoselectivity
a
Incubation conditions: 30 °C, 2000 rpm, 96 h; in all the experiments 100% of
ketone conversion was observed.
OH
OH
b
The absolute configuration was attributed by conversion into the corresponding
d
Mosher’s esters 4h, 5h, Scheme 5; see Sections 2.1 and 4.
c
7% of 1-(4-fluorophenyl)propan-2-yl acetate 3i, was recovered from the reac-
tion mixture (ee = 90%); see Section 4.
Scheme 3. Mechanistic hypothesis of the formation of optically active (S)-4-
phenyl-2-butanol from (E) 4-phenyl-3-buten-2-one by Kluyveromyces marxianus
CBS 6556.
carbonyl compound reductions seems to be predictable on the
basis of the steric hindrance of the substituent of the methyl
ketones. These data confirmed that the Kluyveromyces marxianus
CBS 6556-mediated ketone reduction followed Prelog’s rule, as
for many other ketone bioreductions performed in the presence
of yeasts: the higher steric difference of the substituents linked
to the carbonyl, meant that a higher enantioselectivity was
observed.
E
O
O
O
In order to explore the steric hindrance tolerated for the other
side of the carbonyl functionality, the bioreduction of 1-phenyl-
2-butanone 1k was accomplished. Its complete conversion into
the corresponding alcohol was observed after 72 h of incubation.
It is noteworthy that (R)-1-phenyl-2-butanol 2k (ee = 84%) was
isolated.30,31 An opposite absolute configuration was obtained with
respect to the methyl ketone reduction.
increase enantioselectivity
Figure 1. Dependence of the enantioselectivity on the spacer hybridization.
1-Phenyl-2-pentanone 1l was incubated for the reduction, and
we observed both a lower conversion and enantioselectivity. Also
in this case the product had an (R)-absolute configuration,32
revealing a different stereo-preference of Kluyveromyces marxianus
CBS 6556 whole cell catalytic system in the bioreduction of longer
chain alkyl ketones, under the same experimental conditions.
In order to investigate the substrates on which Kluyveromyces
marxianus CBS 6556 yeast cells were capable of inducing ketone
reduction, a cyclic ketone such as 2-tetralone 1m, was reacted. A
high yield of (S)-2-tetralol33–35 2m was obtained with 76% ee
(Table 3).
Finally, the effect of steric hindrance on the stereochemistry of
the bioreduction was explored by incubating 4-methyl-1-phenyl-
2-pentanone 1n and rac-3-phenyl-2-butanone 1o (Table 3, entry
5).
The reduction of racemic 3-phenyl-2-butanone 1o afforded a
diasteromeric mixture of (2S, 3R)- and (2S, 3S)-3-phenyl-2-butanol
2o in 61% yield, with a diastereomeric ratio (2S,3R)/(2S,3S) = 83:17.
(2S,3R)-3-Phenyl-2-butanol was the main stereoisomer (ee =
96%),36,37 whereas the unreacted 3-phenyl-2-butanone was found
to be enriched in the (R)-enantiomer (50% ee), because of a resolu-
tion due to a different reduction reaction rate of the two enantio-
mers of 3-phenyl-2-butanone.38
excess was higher when an
a,b-unsaturated ketone was reduced
(90%), although it decreased to 56% for the alkyne and became
practically null for the saturated ketone reduction (Fig. 1 and
Table 1).
The size of the substituents around the carbonyl is important
for the reduction reaction rate and enantioselectivity shown by
Kluyveromyces marxianus CBS 6556 yeast. In particular, we noted
that the distance between the phenyl and carbonyl groups affected
the product ee: the higher ee was obtained in the phenylacetone
reduction with respect to acetophenone and 4-phenyl-2-butanone
(Table 1).
To broaden the Kluyveromyces marxianus CBS 6556 substrate
applicability study, 1,1,1-trifluoro-3-phenyl-2-propanone 1f was
incubated, and after 24 h it was converted into the corresponding
(S)-alcohol 2f (Table 1, entry 7) with 58% ee and 75% yield.
The enantiomeric excess did not change after 96 h incubation
time (Table 1, entry 8). The fluorinated phenylacetone analogues
1h–j provided, after 96 h incubation with Kluyveromyces marxianus
CBS 6556 yeast, the corresponding alcohols 1-(2-fluorophenyl)-2-
propanol 2h, (S)-1-(4-fluorophenyl)-2-propanol 2i, and (S)-1-(3-
trifluoromethyl)-phenyl)-2-propanol 2j with an ee of up to 94%
(Scheme 4 and Table 2).
Although further studies are required to better define the effect
of the substituent on the aromatic ring and substrates accepted by
Kluyveromyces marxianus CBS 6556, the stereoselectivity of most
The reduction of 4-methyl-1-phenylpentan-2-one gave
4-methyl-1-phenyl-2-pentanol 2n with good enantioselectivity
(84% ee), although its absolute configuration was not assigned.

1988
P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
Table 3
Bioreduction of phenylacetone analogues by Kluyveromyces marxianus CBS 6556 (isolated yields)
a
Entry
1
Substrate
Conversion (%)
Yield 2k–p (%)
87 2k
ee
Abs. Config.
O
90
84
(R)
1k
O
2
3
4
60
100
15
58 2l
>98 2m
10 2n
50
76
84
(R)
(S)
1l
O
1m
O
1n
1o
n.d.
O
96
90
(2S,3R)
(2S,3S)
5
70
61 2o (dr = 83:17)
a
Calculated by 1H NMR diagnostic signals of the unreacted ketone in the reaction crude.
2.1. Mosher’s esters analysis
Both diastereomeric (S)- and (R)-MTPA esters of the optically
active 1-(2-fluorophenyl)propan-2-ol (Scheme 5) 5h and 4h,
respectively, were prepared and studied with regards to their dif-
ferences in the chemical shifts (DSR values) of the diagnostic 1H
NMR data of two esters.
Although we found a stereo-preference of Kluyveromyces marxi-
anus CBS 6556 for the reduction of phenylacetone analogues into
the corresponding (S)-alcohols, Mosher’s ester analysis was used
to deduce the absolute configuration of 1-(2-fluorophenyl)-2-pro-
panol 2h at the new stereogenic center.
D
dSR(CH2) = 2.97ꢀ2.92 = +0.05;
D
dSR(CH3) = 1.35ꢀ1.38 = ꢀ0.03
On the basis of d analysis, performed with reference to CH3
D
Since the (S)-MTPA ester group affects the chemical shifts of the
R1 and R2 groups of the secondary alcohol in a predictable way, the
(R)-MTPA ester group had an opposite effect on d; the absolute
configuration of the stereogenic centre can be reliably deduced
and CH2, the absolute configuration of (S)-1-(2-fluorophenyl)-2-
propanol 2h was deduced.
3. Conclusions
by analyzing the sign of
D
dSR values.39,40
In conclusion, we have found that with Kluyveromyces
marxianus CBS 6556 yeast (Fig. 2), phenylacetone analogues can
be reduced with high enantioselectivity to the corresponding
(S)-alcohols; a reversal of stereoselectivity occurs by increasing
the alkyl chain of the ketone, affording (R)-alcohol enriched
mixtures. Interestingly, whole cells of Kluyveromyces marxianus
proved to be effective for the reduction of multiple bonds, espe-
cially double bonds. Although some aspects need further study, a
new enoate reductase seems to be involved in the bioreduction
of a non-activated CAC double bond.
This study also provides interesting insight on the enzymatic
pool present in Kluyveromyces marxianus CBS 6556 yeast, which
can be further exploited for the stereoselective synthesis of opti-
cally active building-blocks. In particular, this biocatalyst repre-
sents an alternative method to ‘classic’ stereoselective reduction
reaction approaches that often require both high temperatures
and pressures and special equipment, or milder reducing agents
(e.g., silanes)41 with expensive transition metal (Rh, Ru, Ir, Ti, Zn,
Cu, Fe) containing catalysts, complexed with optically active bio-
mimetic ligands such as BINAP, SEGHPHOS, etc.42,43
a) The two MTPA esters have to be described in the conforma-
tional model which successfully correlates the known
results: it shows the carbonyl group syn-coplanar with the
O–CO bond, with the trifluoromethyl (CF3) substituent of
the MTPA moiety and with the methine proton of the sec-
ondary alcohol moiety.
b) After calculation of
nucleus that shows
the OCH3; conversely, the proton with
D
D
d = dSꢀdR for the diagnostic protons, the
d >0, must be shifted downfield from
d <0, must be
D
shifted upfield because it faces the phenyl ring.
1H NMR spectroscopy was used to determine the configuration
on the basis of different chemical shift of
a-methoxy-a-trifluoro-
methylphenylacetic (MTPA) diastereomeric ester protons.
Kluyveromyces marxianus
OH
O
CBS 6556
R2
R2
growing cells
CH3
CH3
1h-j
2h-j
Although a wider range of substrates needs to be investigated to
fully evaluate the steric and electronic effects that affect the yield
Scheme 4.
F
F
F
CH3
OH
CH3
O
CH3
O
(R)-MPTA-Cl
(S)-MPTA-Cl
(S)
CF3
CF3
Ph
(R)
*
O
O
*
?
OCH3
Ph
H3CO
5h
4h
2h
Scheme 5.

P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
1989
R
O
CH3
n
m
EWG on carbonyl
R= EWG
n > 2
m > 2
0 > n > 1
m < 2
-
stereoselectivity
+
size differences between
conformational flexibility
of alkyl chains
the two carbonyl substituents
Figure 2. Structural determinants for the stereoselectivity in the bioreductions mediated by Kluyveromyces marxianus CBS 6556.
and stereoselectivity of this yeast-mediated reduction, this method
could already be used for the preparation of intermediates of more
complex chemical architectures both in organic and pharmaceuti-
cal syntheses.16,44
tration (c) is expressed in g/100 mL. The enantiomeric ratios were
determined by HPLC analysis using a Daicel Chiralcel OD-H column
(250 ꢁ 4.6 mm), or by GC-analyses performed on a Hewlett–Pack-
ard 6890 Series II chromatograph equipped with a Chirasil-DEX CB
Since the reduction of the carbon–carbon triple bond occurs at a
lower rate compared to the reduction of a carbonyl, Kluyveromyces
marxianus CBS 6556 could be a useful catalyst for the synthesis of
optically active propargyl alcohols,45,46 which are valuable for the
preparation of various natural products such as alkaloids, phero-
mones, prostaglandins, steroids, antibiotics, vitamins, and
sesquiterpenes.47
Multiple bond reduction by this yeast could be an interesting
new field of investigation, since its high potential in creating
two stereogenic centers in one reaction, is often achieved in
asymmetric synthesis by homogeneous catalysis and/or by
organocatalysis.48
(250 ꢁ 0.25
lm) capillary column, column head pressure = 18 psi,
He flow 2 mL/min, split ratio 100/1, T (oven) from 90 to 100 °C.
All the chemicals and solvents were commercial grade and puri-
fied further by distillation or crystallization prior to use. The race-
mic alcohols 2a–o were prepared by NaBH4 reduction in EtOH in
91–95% yields.49 All the optically active alcohols 2a–o obtained
from Kluyveromyces marxianus CBS 6556 bioreductions had analyt-
ical and spectroscopic data identical to the racemic compounds
prepared by synthesis, or to those commercially available. The pro-
gress of the reactions was monitored by TLC and/or GC.
4.2. Microorganism and cultures
Further studies are currently in progress by using the resting
cells of the biocatalyst, in order to define the process parameters,
and for a further characterization of the enzymes responsible for
the stereoselective biotransformations, through enzyme isolation
and purification, or immobilization on suitable supports. More-
over, new frontiers in molecular biology could also be helpful in
optimizing the biocatalyst not only for carbonyl bioreduction pro-
cesses, but also for processes involving the multiple bond reduc-
tion and the formation of new carbon–carbon bonds.
Kluyveromyces marxianus CBS 6556 was obtained from public
type culture collections (Centraalbureau voor Schimmelcultures,
Delft, The Netherlands), and was cultivated under aerobic
conditions in a medium containing 0.3% yeast extract, 0.3% malt
extract, 0.5% peptone, 1% glucose. Agar–agar (2%) was added to
the same medium for cell preservation on agar slants.
4.3. Blank experiments
A 1 L flask containing 400 mL of the culture medium was stirred
at 30 °C on an orbital shaker at 200 rpm. Ketones 1a–o (100 mg)
dissolved in 1 mL of ethanol were added. The reaction was moni-
tored by GC analysis and stopped after 4 days. The content of the
flask was extracted with ethyl acetate and analyzed.
4. Experimental
4.1. General methods
Melting points were taken on an electrothermal apparatus. 1H
NMR and 13C NMR spectra were recorded on a Varian Inova
400 MHz spectrometer and chemical shifts are reported in parts
per million (d). 19F NMR spectra were recorded by using CFCl3 as
an internal standard. The absolute values of the coupling constants
are reported. FT-IR spectra were recorded on a Perkin–Elmer 681
spectrometer. GC analyses were performed on a HP 6890 model,
4.4. Bioreduction of 1a–o by using Kluyveromyces marxianus
growing cells: general procedure
Kluyveromyces marxianus CBS 6556 was stored on agar slants at
4 °C. For inoculum preparation, a 250 mL conical Erlen–Meyer flask
containing 100 mL of yeast maintenance medium (YMM) (previ-
ously autoclaved at 121 °C, 1 atm, for 15 min), was inoculated with
a single loopful of the microorganisms from the agar slants. The
flask was then incubated aerobically at 30 °C in a rotary shaker
at 200 rpm for 24 h. Next, the growing cultures were inoculated
(5% v/v) into a 250 mL conical Erlen–Meyer flask containing
100 mL of YMM and incubated for additional 24 h under the same
conditions. These cultures were then used as a final inoculum (1%
v/v) into a 1000 mL flask containing 400 mL of YMM. After 24 h of
incubation at 30 °C in the shaker (200 rpm), a mixture of 100 mg of
the ketone 1a–o dissolved in 1 mL of absolute ethanol was added.
The reactions were monitored by GC, by collecting suspension ali-
quots of 1 mL after 1, 3, 4 and 5 days of reaction from each flask:
after extraction with ethyl acetate (2 mL), the organic phase was
analyzed by GC. After the appropriate conversion, the suspension
Series II by using
a
HP1 column (methyl siloxane; 30 m ꢁ
0.32 mm ꢁ 0.25 m film thickness). Analytical thin-layer chroma-
l
tography (TLC) was carried out on pre-coated 0.25 mm thick plates
of Kieselgel 60 F254; visualization was accomplished by UV light
(254 nm) or by spraying a solution of 5% (w/v) ammonium molyb-
date and 0.2% (w/v) cerium (III) sulfate in 100 mL 17.6% (w/v) aq
sulfuric acid and heating to 473 K until blue spots appeared. Chro-
matography was conducted by using silica gel 60 with a particle
size distribution 40–63 lm and 230–400 ASTM. Petroleum ether
refers to the 40–60 °C boiling fraction. GC–MS analyses were
performed on HP 5995C model and elemental analyses on an
Elemental Analyzer 1106-Carlo Erba-instrument. MS-ESI analyses
were performed on Agilent 1100 LC/MSD trap system VL.
Optical rotation values were measured at 20 °C using a Perkin–
Elmer 341 polarimeter with a cell of 1 dm path length; the concen-

1990
P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
was centrifuged (3000 rpm, 6 min, 4 °C), and the aqueous phase
extracted with ethyl acetate (4 ꢁ 150 ml). The yellow organic
phase was dried over Na2SO4, filtered and evaporated under re-
duced pressure. The residue was purified by silica gel column chro-
matography using petroleum ether and ethyl acetate (90:10 or
80:20) as eluent to yield the desired alcohols.50,51
7.40–7.20 (5H, m, ArH), 6.56 (1H, d, J 15.9, CH), 6.28 (1H, dd, J
15.9, 6.3, CH), 4.52–4.45 (1H, m, CH), 2.40–2.20 (1H, br s, OH, ex-
changes with D2O), 1.38 (1H, d, J 6.4, CH3); dC (100 MHz, CDCl3)
136.7, 133.6, 129.3, 128.6, 127.6, 126.4, 68.8, 23.4; m/z 148 (M+,
61%), 133 (34), 131 (18), 130 (82), 129 (100), 128 (65), 127 (27),
116 (12), 115 (85), 105 (99), 104 (15), 103 (27), 102 (16), 91
(56), 79 (14), 78 (23), 77 (42), 65 (12), 63 (14), 55 (17), 51 (24),
50 (10), 43 (31).
In order to measure the enantiomeric ratios,52 the correspond-
ing acetates of the alcohols 2h–i, 2o were quantitatively obtained
by acetylation with acetic anhydride/Et3N. The absolute configura-
tions of alcohols 2a–g, 2k–m, 2o obtained from the bioprocess
were determined by the comparison of their specific rotations with
those previously reported in the literature or by comparison of the
retention times with previously published data.
4.4.6. (S)-1,1,1-Trifluoro-3-phenyl-2-propanol 2f49
75–98% Yield; er = 80:20, (GC-Chirasil-DEX CB, T = 100 °C) (R)-
isomer: 3.89 min, (S)-isomer: 4.06 min;49 mmax (neat) 3400, 3067,
3034, 2932, 1606, 1497, 1391, 1274, 1166, 1129, 1099, 1031,
919, 882, 803, 749, 702 cmꢀ1; dH (400 MHz, CDCl3) 7.38–7.26
(5H, m, ArH), 4.19–4.10 (1H, m, CH), 3.07 (1H, dd, J 14.01, 2.9,
CH2), 2.85 (1H, dd, J 14.01, 10.3, CH2), 2.19–2.18 (1H, br s, OH, ex-
changes with D2O); dC (100 MHz, CDCl3) 135.6, 129.4, 128.8, 127.2,
4.4.1. (S)-1-Phenyl-2-propanol 2a14,15,25
50–98% Yield; ½a D20
¼ þ20:1 (c 0.6, CHCl3), ee = 92% (HPLC OD-
ꢂ
H, Hexane/IPA = 90:10, 0.6 mL/min, k = 254 nm) (S)-isomer:
8.4 min, (R)-isomer: 10.8 min; mmax (neat) 3368, 3027, 2968,
1
2
3
124.8 (q, JC-F 281.8); 71.5 (q, JC-F 30.8), 36.0 (q, JC-F 1.7); d19F
(CDCl3, 376 MHz) -83.9 (d, J 6.2); m/z 190 (M+, 33%), 92 (9), 91
(100), 65 (10).
2929, 1604, 1496, 1453, 1373, 1118, 1080, 940, 742, 699 cmꢀ1
;
dH (400 MHz, CDCl3) 7.34–7.21 (5H, m, ArH), 4.06–3.99 (1H, m,
CH), 2.80 (1H, dd, J 13.6, 4.8, CH2), 2.69 (1H, dd, J 13.6, 8.1, CH2),
1.63–1.56 (1H, br s, OH, exchanges with D2O), 1.25 (3H, d, J 6.2,
CH3); dC (100 MHz, CDCl3) 138.1, 129.4, 128.5, 126.5, 68.9, 45.7,
22.8; m/z 136 (M+, 1%), 92 (100), 91 (70), 65 (13), 45 (23).
4.4.7. (S)-1-Phenylethanol 2g14,27,56
67% Yield; ½a 2D0
ꢂ
¼ ꢀ22:2 (c 1, CHCl3),14 er = 74:26 (HPLC OD-H,
Hexane/IPA = 90:10,
0.8 mL/min,
k = 254 nm)
(R)-isomer:
7.53 min, (S)-isomer: 8.36 min;54 mmax (neat) 3346, 3080, 3063,
3030, 2973, 2927, 2875, 1602, 1493, 1451, 1410, 1369, 1304,
1262, 1204, 1077, 1029, 1011, 997, 898, 760, 699 cmꢀ1; dH
(400 MHz, CDCl3) 7.39–7.26 (5H, m, ArH), 4.9 (1H, q, J 6.5, CH),
1.93–1.88 (1H, br s, OH, exchanges with D2O), 1.66 (3H, d, J 6.5,
CH3); dC (100 MHz, CDCl3) 145.8, 128.5, 127.4, 125.4, 70.4, 25.1;
m/z 122 (M+, 4%), 107 (10), 105 (1), 79 (10), 78 (2), 77 (6), 51 (2),
43 (1).
4.4.2. (S)-4-Phenyl-2-butanol 2b24–27
74–90% Yield; ee = 68% (HPLC OD-H, Hexane/IPA = 90:10,
0.6 mL/min, k = 254 nm) (R)-somer: 8.2 min, (S)-isomer:
10.6 min);27 mmax (neat) 3350, 3085, 3062, 3026, 2965, 2926,
2860, 1603, 1495, 1454, 1374, 1178, 1128, 1083, 1055, 1031,
954, 745, 698 cmꢀ1; dH (400 MHz, CDCl3) 7.31–7.17 (5H, m, ArH),
3.83 (1H, sextet, J 6.2, CH), 2.80–2.64 (2H, m, CH2), 1.83–1.73
(2H, m, CH2), 1.40–1.38 (1H, br s, OH, exchanges with D2O), 1.23
(3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 142.0, 128.4 (2Cortho
+
4.4.8. (S)-1-(2-Fluorophenyl)-2-propanol 2h57
2Cmeta), 125.8, 67.5, 40.8, 32.1, 23.6; m/z 150 (M+, 8%), 132 (48),
117 (100), 105 (13), 92 (34), 91 (86), 79 (10), 78 (24), 77 (17), 65
(13), 45 (16).
98% Yield; ½a 2D0
ꢂ
¼ þ25:2 (c 1, CHCl3) er = 97:3;
mmax (neat) 3340,
3066, 3040, 2971, 2926, 1616, 1583, 1490, 1453, 1373, 1230, 1180,
1113, 1080, 1050, 936, 856, 826, 753 cmꢀ1; dH (400 MHz, CDCl3)
7.26–7.02 (4H, m, ArH), 4.12–4.04 (1H, m, CH), 2.85 (1H, dd, J
13.6, 5.2, CH2), 2.75 (1H, dd, J 13.6, 7.6, CH2), 1.6 (1H, br s, OH, ex-
changes with D2O), 1.25 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3)
4.4.3. (S)-1-Phenoxy-2-propanol 2c22
87% Yield; ½a 2D0
ꢂ
¼ ꢀ1:8 (c 1, EtOH), ½a D20
ꢂ
¼ þ24:3 (c 1, CHCl3),
0.8 mL/min,
1
3
3
ee = 80%
(HPLC
OD-H,
Hexane/IPA = 90:10,
161.3 (d, JC-F 244.9), 131.7 (d, JC-F 5.3), 128.2 (d, JC-F 8.4), 125.4
2
4
2
k = 254 nm) (R)-somer: 9.8 min, (S)-isomer: 18.6 min); mmax (neat)
3391, 3060, 3040, 2966, 2921, 2873, 1599, 1586, 1495, 1456,
1383, 1330, 1290, 1245, 1170, 1150, 1076, 1042, 953, 935, 863,
810, 752, 691 cmꢀ1; dH (400 MHz, CDCl3) 7.32–6.91 (5H, m, ArH),
4.25–4.17 (1H, m, CH), 3.95 (1H, dd, J 9.2, 7.7, CH2), 3.81 (1H, dd,
J 9.2, 3.3, CH2), 2.43 (1H, br s, OH, exchanges with D2O), 1.30 (3H,
d, J 6.2, CH3); dC (100 MHz, CDCl3) 158.5, 129.5, 121.1, 114.5,
73.2, 66.3, 18.7; m/z 152 (M+, 0.2%), 108 (1), 94 (10), 77 (1).
(d, JC-F 16.0), 124.0 (d, JC-F 3.1), 115.3 (d, JC-F 22.9), 67.9, 38.8,
22.9; d19F (CDCl3, 376 MHz) ꢀ122.1(m); m/z 154 (M+, 1%), 110
(100), 109 (74), 83 (13), 45 (15). The enantiomeric ratio was mea-
sured after conversion of alcohol 2h into the corresponding (S)-1-
(2-fluorophenyl)propan-2-yl acetate 3h: 90% yield; (GC-Chirasil-
DEX CB, T = 100 °C) (S)-isomer: 26.23 min, (R)-isomer: 28.03 min;
mmax (neat) 3040, 2981, 2934, 2873, 1736, 1617, 1586, 1493,
1456, 1373, 1243, 1181, 1133, 1110, 1089, 1057, 1029, 1013,
956, 840, 814, 756, 720 cmꢀ1; dH (400 MHz, CDCl3) 7.24–7.17
(2H, m, ArH), 7.10–6.98 (2H, m, ArH), 5.14 (1H, sestet, J 6.3, CH),
2.94–2.84 (2H, m, CH2), 1.98 (3H, s, COCH3), 1.23 (3H, d, J 6.3,
4.4.4. (S)-4-Phenyl-3-butyn-2-ol 2d45,53,54
24% Yield; ½a 2D0
¼ ꢀ13:5 (c 1, CHCl3), ee = 56% (HPLC OD-H, Hex-
ꢂ
1
ane/IPA = 90:10, 0.8 mL/min, k = 254 nm) (R)-somer: 8.7 min, (S)-
isomer: 18.6 min;54 mmax (neat) 3350, 3053, 2981, 2930, 2866,
2226, 1598, 1489, 1440, 1370, 1330, 1286, 1105, 1073, 1037,
932, 852, 755, 691 cmꢀ1; dH (400 MHz, CDCl3) 7.45–7.27 (5H, m,
ArH), 4.77 (1H, q, J 6.6, CH), 2.02 (1H, br s, OH, exchanges with
D2O), 1.57 (1H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 131.6, 128.3,
128.2, 122.6, 90.9, 84.0, 58.8, 24.4; m/z 145 (M+, 6%), 146 (3), 131
(10), 103 (6), 77 (3).
CH3); dC (100 MHz, CDCl3) 170.5, 161.3 (d, JC-F 245.7), 131.7 (d,
3
2
4
3JC-F 4.6), 128.3 (d, JC-F 7.6), 124.4 (d, JC-F 16.0), 123.8 (d, JC-F
2
3.8), 115.2 (d, JC-F 22.1), 70.4, 34.9, 21.2, 19.4; d19F (CDCl3,
376 MHz) ꢀ122.1 (m); m/z (ESI+): 219 [M+Na]+.
4.4.9. (S)-1-(4-Fluorophenyl)-2-propanol 2i52
93% Yield; ½a 2D0
¼ þ8:6 (c 0.7; CHCl3); mmax (neat) 3346, 3040,
ꢂ
2966, 2920, 1600, 1509, 1416, 1373, 1263, 1220, 1156, 1113,
1096, 1080, 943, 930, 850, 810, 763 cmꢀ1; dH (400 MHz, CDCl3)
7.19–6.97 (4H, m, ArH), 4.03–3.95 (1H, m, CH), 2.76 (1H, dd, J
13.6, 4.8, CH2), 2.67 (1H, dd, J 13.6, 8.1, CH2), 1.6 (1H, br s, OH, ex-
changes with D2O), 1.23 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3)
4.4.5. (S)-(E)-4-Phenyl-3-buten-2-ol 2e25,55
3% Yield; er = 95:5 (HPLC OD-H, Hexane/IPA = 90:10, 0.8 mL/
min, k = 254 nm) (R)-isomer: 10.0 min, (S)-isomer: 13.8 min;27,54
mmax (neat) 3339, 3059, 3026, 2971, 2925, 2873, 1598, 1493,
1449, 1368, 1140, 1069, 967, 748, 693 cmꢀ1; dH (400 MHz, CDCl3)
1
4
3
161.6 (d, JC-F 244.1), 134.1 (d, JC-F 3.8), 130.7 (d, JC-F 7.6), 115.2
(d, JC-F 20.6), 68.8, 44.8, 22.8; d19F (CDCl3, 376 MHz) -121.1 (m);
2

P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
1991
m/z 154 (M+, 4%), 110 (100), 109 (81), 83 (15), 45 (18). The enan-
tiomeric ratio was measured after conversion of alcohol 2i into
the corresponding (S)-1-(4-fluorophenyl)propan-2-yl acetate 3i:52
(5); the enantiomeric ratio was measured after conversion of alco-
hol 2m into the corresponding (S)-1,2,3,4-tetrahydronaphthalen-2-
yl acetate 3m:59 87% Yield; (GC-Chirasil-DEX CB, T = 120 °C) (S)-iso-
mer: 53.20 min, (R)-isomer: 55.28 min; mmax (neat) 3063, 3021,
2938, 2848, 1732, 1496, 1454, 1436, 1366, 1242, 1111, 1034, 910,
747 cmꢀ1; dH (400 MHz, CDCl3) 7.15–7.06 (4H, m, ArH), 5.24–5.17
(1H, m, CH), 3.12 (1H, dd, J 16.5, 4.9, CH), 2.99–2.80 (3H, m,
1CH + 2CH2), 2.06 (3H, s, CH3), 2.05–1.89 (2H, m, CH2).
er = 97:3
(HPLC
OD-H,
Hexane/IPA = 99:1,
0.8 mL/min,
k = 254 nm) (S)-isomer: 19.93 min, (R)-isomer: 21.34 min; mmax
(neat) 3040, 2980, 2933, 2866, 1737, 1602, 1511, 1449, 1373,
1246, 1159, 1134, 1100, 1090, 1057, 1016, 956, 819, 764,
706 cmꢀ1; dH (400 MHz, CDCl3) 7.18–7.13 (2H, m, ArH), 7.00–
6.95 (2H, m, ArH), 5.07 (1H, sestet, J 6.6, CH), 2.88 (1H, dd, J 13.7,
6.8, CH2), 2.73 (1H, dd, J 13.7, 6.4, CH2), 2.0 (3H, s, COCH3), 1.21
4.4.14. 4-Methyl-1-phenyl-2-pentanol 2n60
1
(3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 170.4, 161.6 (d, JC-F
10% Yield; er = 92:8; (HPLC OD-H, Hexane/IPA = 99:1, 0.8 mL/
min, k = 254 nm) major-isomer: 5.57 min, minor-isomer:
6.54 min; mmax (neat) 3557, 3391, 3086, 3063, 3028, 2955, 2929,
2870, 1601, 1496, 1468, 1454, 1385, 1367, 1136, 1081, 1030,
747, 699 cmꢀ1; dH (400 MHz, CDCl3) 7.35–7.30 (2H, m, ArH),
7.27–7.21 (3H, m, ArH), 3.91–3.88 (1H, m, CH), 2.82 (1H, dd, J
13.6, 4.0, CH), 2.63 (1H, dd, J 8.4, 13.6, CH), 1.89–1.79 (1H, m,
CH), 1.52–1.44 (2H, m, CH + 1H, br s, OH, exchanges with D2O),
1.35–1.27 (1H, m, CH), 0.95 (3H, d, J 6.6, CH3), 0.92 (3H, d, J 6.6,
CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5, 126.4, 70.6, 46.0,
44.5, 24.6, 23.4, 22.0; m/z 178 (M+, 0.3%), 145 (1), 121 (5), 117
(2), 103 (6), 93 (8), 92 (100), 91 (42), 77 (4), 69 (11), 65 (7), 43 (7).
244.1), 133.2 (d, JC-F 3.1), 130.7 (d, JC-F 7.6), 128.6 (d, JC-F 38.1),
115.1 (d, JC-F 21.4), 71.3, 41.3, 21.2, 19.3; d19F (CDCl3, 376 MHz)
ꢀ121.1 (m); m/z (ESI+): 219 [M+Na]+.
4.4.10. (S)-1-[3-Trifluoromethyl)-phenyl]-2-propanol 2j58
98% Yield; ½a 2D0
¼ þ19:4 (c 1, CHCl3), er = 96:4 (HPLC OD-H,
ꢂ
Hexane/IPA = 99.8:0.2, 0.8 mL/min, k = 254 nm) (S)-isomer:
32.01 min, (R)-isomer: 35.40 min; mmax (neat) 3351, 3073, 3046,
2973, 2926, 1613, 1596, 1490, 1450, 1376, 1333, 1200, 1160,
1123, 1073, 940, 906, 866, 796, 750, 703 cmꢀ1; dH (400 MHz,
CDCl3) 7.51–7.40 (4H, m, ArH), 4.10–4.02 (m, 1H, CH), 2.84 (1H,
dd, J 13.6, 5.1, CH2), 2.77 (1H, dd, J 13.6, 7.9, CH2), 1.6 (1H, br s,
OH, exchanges with D2O), 1.26 (3H, d, J 6.2, CH3); dC (100 MHz,
CDCl3) 139.5, 132.8 (q, JCꢀF 1.3), 130.7 (q, JCꢀF 32.6), 128.8, 126.0
(q, JCꢀF 3.8), 124.1 (q, JCꢀF 272.4), 123.3 (q, J 3.8), 68.6, 45.3, 23.0;
d19F (CDCl3, 376 MHz) ꢀ66.9 (s); m/z 204 (M+, 1%), 185 (12), 160
(100), 159 (27), 140 (45), 109 (15), 91 (28), 45 (25).
4.4.15. (2S,3R)-3-Phenyl-2-butanol 2o36
51% Yield; ½a 2D0
ꢂ
¼ þ19:55 (c 1.4, EtOH),61,62 ee = 96%;
mmax (neat)
3366, 3028, 2966, 1601, 1494, 1452, 1377, 1260, 1101, 913, 799,
700 cmꢀ1; dH (400 MHz, CDCl3) 7.36–7.32 (2H, m, ArH), 7.27–
7.21 (3H, m, ArH), 3.89–3.81 (1H, m, CH), 2.68 (1H, quintet, J 7.1,
CH), 1.48–1.40 (1H, br s, OH, exchanges with D2O), 1.27 (3H, d, J
7.1, CH3), 1.23 (3H, d, J 6.3, CH3); dC (100 MHz, CDCl3) 143.5,
128.6, 128.0, 126.7, 72.3, 47.9, 20.6, 17.8; m/z 150 (M+, 0.1%), 135
(2), 106 (84), 105 (39), 103 (14), 91 (100), 79 (13), 78 (13), 77
(19), 65 (3), 51 (5); (2S,3S)-3-phenyl-2-butanol 2o.36 10% Yield;
4.4.11. (R)-1-Phenyl-2-butanol 2k30,31
87% Yield; ½a 2D0
ꢂ
¼ ꢀ30:0 (c 1, CHCl3), er = 92:8, (HPLC OD-H,
Hexane/IPA = 90:10,
0.5 mL/min, k = 254 nm) (S)-isomer:
10.42 min, (R)-isomer: 12.31 min; mmax (neat) 3390, 3084, 3068,
3028, 2963, 2934, 2876, 1604, 1495, 1454, 1337, 1183, 1122,
1079, 1016, 975, 911, 851, 742, 699 cmꢀ1; dH (400 MHz, CDCl3)
7.35–7.22 (5H, m, ArH), 3.80–3.70 (1H, m, CH), 2.85 (1H, dd, J
13.6, 4.4, CH2), 2.66 (1H, dd, J 13.6, 8.4, CH2), 1.65–1.47 (2H: 1H,
m, CH and 1H: br s, OH, exchanges with D2O), 1.01 (3H, d, J 7.5,
CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5, 126.4, 74.0, 43.6,
29.5, 10.0; m/z 150 (M+, 1%), 121 (7), 103 (8), 93 (8), 92 (100), 91
(58), 77 (6), 65 (9), 59 (11).
½
a 2D0
ꢂ
¼ ꢀ0:1 (c 0.9, EtOH),61,62 ee = 90%; dH (400 MHz, CDCl3)
7.34–7.30 (2H, m, ArH), 7.25–7.19 (3H, m, ArH), 3.93–3.84 (1H,
m, CH), 2.75 (1H, quintet, J 7.1, CH), 1.33 (3H, d, J 7.1, CH3), 1.10
(3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 144.0, 128.4, 127.8, 126.4,
72.3, 47.1, 21.0, 15.9; m/z 150 (M+, 0.1%), 135 (2), 106 (89), 105
(39), 103 (12), 91 (100), 79 (12), 78 (12), 77 (18), 65 (3), 51 (5);
the enantiomeric ratios of both the diasteroisomeric alcohols 2o
were measured after their conversion into the corresponding 3-
phenylbutan-2-yl acetates 3o:32 GC-Chirasil-DEX CB, T = 90 °C,
(2S,3S)-isomer: 63.06 min, (2R,3R)-isomer: 65.33 min), (2S,3R)-iso-
4.4.12. (R)-1-Phenyl-2-pentanol 2l32
58% Yield; ½a 2D0
ꢂ
¼ ꢀ1:85 (c 0.5, EtOH), ½a D20
ꢂ
¼ ꢀ8:1 (c 0.4, CHCl3),
mer: 57.63 min, (2R,3S)-isomer: 60.86 min; mmax (neat) 3060, 3027,
er = 25:75, (HPLC OD-H, Hexane/IPA = 90:10, 0.8 mL/min,
2960, 2926, 2853, 1733, 1603, 1493, 1450, 1371, 1257, 1240, 1105,
1017, 947, 800, 700 cmꢀ1; dH (400 MHz, CDCl3) 7.32–7.19 (5H, m,
ArH), 5.09 (1H, quintet, J 6.4, CH), 2.93 (1H, quintet, J 7.0, CH),
1.92 (3H, s, CH3), 1.28 (3H, d, J 7.0, CH3), 1.16 (3H, d, J 6.3, CH3);
dC (100 MHz, CDCl3) 171.0, 142.9, 128.1, 128.0, 126.4, 74.3, 44.4,
21.2, 17.3, 16.6.
k = 254 nm) (S)-isomer: 5.78 min, (R)-isomer: 6.69 min; mmax (neat)
3369, 3080, 3060, 3028, 2958, 2927, 2872, 1602, 1550, 1495, 1454,
1123, 1080, 1030, 1015, 843, 737, 699 cmꢀ1; dH (400 MHz, CDCl3)
7.38–7.30 (2H, m, ArH), 7.27–7.21 (3H, m, ArH), 3.90–3.80 (1H,
m, CH), 2.83 (1H, dd, J 13.6, 4.0, CH2), 2.65 (1H, dd, J 13.6, 8.4,
CH2), 1.60–1.37 (5H, m, 2CH2 + br s, OH, exchanges with D2O),
0.94 (3H, d, J 6.6, CH3); dC (100 MHz, CDCl3) 138.6, 129.4, 128.5,
126.4, 72.4, 44.0, 38.9, 18.9, 14.1; m/z 164 (M+, 1%), 121 (4), 103
(5), 92 (100), 91 (44), 65 (7), 55 (15), 43 (7).
4.5. Mosher’s esters synthesis39
Mosher’s ester analysis was used to deduce the absolute config-
uration of 1-(2-fluorophenyl)propan-2-ol (2h) at the new stereo-
genic center. Both diastereomeric (S)- and (R)-MTPA esters of the
optically active 1-(2-fluorophenyl)propan-2-ol (Scheme 5) 5h and
4h, respectively, were prepared and analyzed (Scheme 5).
To a stirred solution of optically active 1-(2-fluorophenyl)-2-
propanol 2h (10.0 mg, 65 mmol), DMAP (8.0 mg, 65 mmol), and
dry Et3N (0.108 mL, 0.780 mmol) in dry dichloromethane (3 mL)
at room temperature, was added (S)-MTPA-Cl (0.024 mL,
0.130 mmol, 2.0 equiv). The reaction progress was monitored by
TLC on silica gel (petroleum ether/EtOAc = 9:1). After complete
conversion of the alcohol (15 h), the reaction mixture was
quenched by the addition of water (5 mL). The aqueous layer was
4.4.13. (S)-2-Tetralol 2m33–35
98% Yield; ½a 2D0
¼ ꢀ36:5 (c 0.5, CHCl3), er = 88:12; mmax (neat)
ꢂ
3339, 3060, 3018, 2926, 2840, 1688, 1603, 1580, 1495, 1453,
1437, 1361, 1340, 1291, 1232, 1112, 1049, 963, 842, 813,
745 cmꢀ1; dH (400 MHz, CDCl3) 7.15–7.08 (4H, m, ArH), 4.21–4.12
(1H, m, CH), 3.13–3.07 (1H, m, CH), 3.00–2.93 (1H, m, CH), 2.90–
2.75 (2H, m, CH2), 2.11–2.04 (1H, m, CH), 1.89–1.80 (1H, m, CH),
1.79–1.72 (1H, br s, OH, exchanges with D2O); dC (100 MHz, CDCl3)
135.6, 134.2, 129.5, 128.6, 125.9, 125.8, 67.2, 38.4, 31.5, 27.0; m/z
148 (M+, 12%), 131 (12), 130 (100), 129 (42), 128 (12), 115 (27),
105 (14), 104 (59), 103 (18), 91 (16), 78 (14), 77 (10), 65 (5), 51

1992
P. Vitale et al. / Tetrahedron: Asymmetry 22 (2011) 1985–1993
18. Vaglini, F.; Pardini, C.; Cavalletti, M.; Maggio, R.; Corsini, G. U. Brain Res. 1996,
741, 68–74.
extracted with two additional portions of CH2Cl2 (5 mL), and the
combined organic layers were dried (Na2SO4), filtered, and concen-
trated in vacuo. The crude product mixture was purified by silica-
gel chromatography (petroleum ether/ethyl acetate = 9:1) to give
(2R)-1-(2-fluorophenyl)propan-2-yl 3,3,3-trifluoro-2-methoxy-2-
phenylpropanoate 4h (19.5 mg, 80% Yield) as a semi-solid; (R)-
MTPA ester dH (400 MHz, CDCl3) 7.43–7.30 (5H, m, ArH), 7.21–
7.15 (1H, m, ArH), 7.08–6.92 (3H, m, ArH), 5.44 (1H, m, CH), 3.47
(3H, s, OCH3), 2.96 (1H, dd, J 13.9, 7.7, CH), 2.89 (1H, dd, J 13.9,
5.6, CH), 1.39 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 165.9, 161.2
19. Absolute ethanol was used as the solvent for the substrate solubilization, due
to the yeast tolerance to ethanol, and also because of the noteworthy yeast
regeneration of the cofactor NAD(P)H through the ethyl alcohol oxidation into
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assigned by comparison with the specific rotation value in two reference
solvents (chloroform and ethanol): Hoff, B. H.; Waagen, V.; Anthonsen, T.
Tetrahedron: Asymmetry 1996, 7, 3181–3186.
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24. Nogawa, M.; Shimojo, M.; Matsumoto, K.; Okudomi, M.; Nemoto, Y.; Ohta, H.
Tetrahedron 2006, 62, 7300–7306.
1
3
3
(d, JC-F 245.7), 131.6 (d, JC-F 4.6), 129.4, 128.5 (d, JC-F 7.6),
4
2
1
128.3, 127.1, 123.9 (d, JC-F 3.8), 123.6 (d, JC-F 16.0), 123.2 (q, JC-
2
288.4), 115.2 (d, JC-F 22.1), 73.3, 55.3, 34.8, 29.7, 19.6; d19F
F3
(CDCl3, 376 MHz) -75.9 (s), -122.0 (m); m/z 189 (C9H8F3O+, 91%),
137 (27), 136 (31), 109 (100), 105 (11), 91 (6).
25. Musa, M. M.; Ziegelmann-Field, K. I.; Vieille, C.; Zeikus, J. G.; Phillips, R. S. J. Org.
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4.5.1. (2S)-1-(2-Fluorophenyl)propan-2-yl 3,3,3-trifluoro-2-
methoxy-2-phenylpropanoate 5h
(19.0 mg, 79% Yield) as a colorless oil; (S)-MTPA ester mmax
(neat) 3060, 2961, 2927, 2877, 1747, 1620, 1587, 1495, 1455,
1383, 1261, 1169, 1120, 1107, 1018, 801, 758, 713 cmꢀ1; dH
(400 MHz, CDCl3) 7.42–7.15 (7H, m, ArH), 7.08–7.00 (2H, m,
ArH), 5.42 (1H, m, CH), 3.39 (3H, s, OCH3), 3.03–2.90 (2H, m,
CH2), 1.35 (3H, d, J 6.2, CH3); dC (100 MHz, CDCl3) 166.1, 161.3 (d,
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1JC-F 245.7), 132.5, 132.0 (d, JC-F 4.6), 129.6, 128.8 (d, JC-F 8.4),
4
1
128.5, 127.5, 124.3 (d, JC-F 3.8), 124.1, 123.2 (q, JC-F3 288.4),
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Acknowledgements
37. Cram, D. J. J. Am. Chem. Soc. 1949, 71, 3863–3870.
This work was financially supported by National Project PRIN
‘Stereoselezione in Sintesi Organica, Metodologie ed Applicazioni’,
MiUR (Rome), and by the University of Bari. Thanks are due also to
Dr. Fabio Plasmati and Mrs. Luciana Volpe for their contribution to
Section 4.
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