Preparation of enantiomerically enriched aromatic β-hydroxynitriles and halohydrins by ketone reduction with recombinant ketoreductase KRED1-Pglu

Article abstract of DOI:10.1016/j.tet.2016.05.027

A NADPH-dependent benzil reductase (KRED1-Pglu) was used as recombinant enzyme for catalysing the reduction of different functionalised ketones. The reactions were carried out in the presence of a catalytic amount of NADP⁺and an enzyme-coupled transformation (oxidation of glucose catalysed by glucose dehydrogenase), for regenerating the cofactor and thus driving the reaction to completion. KRED1-Pglu showed remarkable versatility, being able to reduce different β-ketonitriles and α-haloketones at different pHs; notably, depending on the nature of the substrate, KRED1-Pglu can be used for efficient and clean enzymatic reduction, avoiding side-reactions due to the pH of the medium. The reduction generally occurred with high enantioselectivity, allowing the preparation of enantiomerically enriched β-hydroxynitriles and halohydrins in high yields; the stereochemical outcome of the reduction followed in all the cases the so-called Prelog's rule.

Full text of DOI:10.1016/j.tet.2016.05.027

Tetrahedron xxx (2016) 1e6
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Tetrahedron
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Preparation of enantiomerically enriched aromatic b-hydroxynitriles
and halohydrins by ketone reduction with recombinant
ketoreductase KRED1-Pglu
Martina L. Contente a, Immacolata Serra a, Francesco Molinari a, Raffaella Gandolfi b,
,
a,
*
Andrea Pinto b , Diego Romano
*
a Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
b Department of Pharmaceutical Sciences (DISFARM), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A NADPH-dependent benzil reductase (KRED1-Pglu) was used as recombinant enzyme for catalysing the
reduction of different functionalised ketones. The reactions were carried out in the presence of a catalytic
amount of NADPþ and an enzyme-coupled transformation (oxidation of glucose catalysed by glucose
dehydrogenase), for regenerating the cofactor and thus driving the reaction to completion. KRED1-Pglu
Received 15 March 2016
Received in revised form 28 April 2016
Accepted 9 May 2016
Available online xxx
showed remarkable versatility, being able to reduce different b-ketonitriles and a-haloketones at dif-
ferent pHs; notably, depending on the nature of the substrate, KRED1-Pglu can be used for efficient and
clean enzymatic reduction, avoiding side-reactions due to the pH of the medium. The reduction generally
Keywords:
Ketoreductase
Stereoselective
Ketone reduction
Pichia glucozyma
Enzymatic reduction
occurred with high enantioselectivity, allowing the preparation of enantiomerically enriched
b-hy-
droxynitriles and halohydrins in high yields; the stereochemical outcome of the reduction followed in all
the cases the so-called Prelog’s rule.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
The most popular example of the utilization of optically pure
b
-hydroxynitriles is the synthesis of serotonin/norepinephrine
Asymmetric hydrogenation of functionalized aromatic ketones
for preparing optically active secondary alcohols is widely used in
the production of drugs.1 Biocatalysis offers an efficient alternative
to conventional chemical methods, ever since enzymes can be
produced in large amounts as recombinant proteins and improved
by enzyme engineering.2 Ketoreductases (KREDs), sometimes also
described as carbonyl reductases (CR) or alcohol dehydrogenases
(ADH), are known to efficiently catalyse the enantiospecific de-
livery of a hydride to prostereogenic carbonyls, thus producing the
desired optically pure alcohols.3e6
reuptake inhibitors for the treatment of depressive or sleep
disorders, anxiety, alcoholism, chronic pain, obesity and anorexia
or bulimia.9,12 Although reduction of aromatic
b-ketonitriles can
be obtained by chemical chiral catalysts,13,14 biocatalysis has
proved to be an efficient and green alternative; biocatalytic re-
duction is mostly performed with isolated enzymes,15e18 since
the use of whole cells is often limited by the occurrence of
adcompetitive ethylation, resulting from non-enzymatic aldol
condensation between aromatic b-ketonitriles and acetaldehyde
(formed by the whole cells in the presence of glucose or ethanol),
followed by reduction of the activated C]C by enoate
reductases.19e23
Optically pure aromatic halohydrins are versatile synthons that
have been used for the synthesis of many pharmaceuticals, in-
cluding drugs for psychiatric disorders, such as (R)-Fluoxetine,24e27
(R)-Duloxetine,27,28 or (R)-Ibutilide, an antiarrhythmic agent.29
Preparation of aromatic halohydrins by enzymatic reduction of
Stereoselective reduction of aromatic ketones functionalized
with
b
-cyano or
b
-halo groups are of particular interest, since the
corresponding chiral
b
-hydroxynitriles7e9 and halohydrins10,11
(b
-
halo alcohols) are key-intermediates in the synthesis of a number of
biologically active compounds.
the corresponding aromatic a-haloketones is also a valid option.
* Corresponding authors. Tel.: þ39 0250319323 (A.P.); tel.: þ39 0250319134
(D.R); e-mail addresses: andrea.pinto@unimi.it (A. Pinto), diego.romano@unimi.it
(D. Romano).
Both microbial cells30e34 and isolated enzymes10,35e37 have proven
effective, although sometimes low yields are observed due to
http://dx.doi.org/10.1016/j.tet.2016.05.027
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
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2
M.L. Contente et al. / Tetrahedron xxx (2016) 1e6
Table 2
possible side-reactions, with formation of 1-phenylethanol (as re-
sult of reductive dehalogenation, followed by reduction) and 1-
phenylethane-1,2-diol (as result of substitution and re-
duction).34,38 Reductive dehalogenation has been detected using
whole microbial cells as an effect of unwanted enzymatic re-
actions,38 whereas substitution can be avoided by tuning the pH of
the biotransformation (i.e., slightly acidic conditions).34
Reduction of 2-alkyl-3-oxo-3-phenylpropanenitriles with KRED1-Pglu
Whole cells of the non-conventional yeast Pichia glucozyma CBS
5766 have been thoroughly exploited for the reduction of aromatic
ketones, often showing good yields and enantioselectivity.39e43
A
versatile benzil ketoreductase (KRED1-Pglu), with high activity and
enantioselectivity towards various functionalized aromatic
ketones,44e46 was identified in the genome of P. glucozyma and
expressed in Escherichia coli.44 In this work we investigated the
reduction of aromatic b-ketonitriles and a-haloketones catalysed
by recombinant KRED1-Pglu for checking its potential as stereo-
selective biocatalyst.
Entry
Substrate
R
Yield (%)a
ee (%)
de (%)b
1
2
1i
1j
Me
Et
95
90
>98 (2R,3S)c
>98 (2R,3S)
46 (2S,3S)
66 (2S,3S)d
a
Reaction conditions: 10 mM substrate, 0.1 mM NADPþ, KRED1-Pglu (20 mU/
mL), GDH (1 U/mL), glucose 40 mM in Tris/HCl buffer pH 8.0 (0.05 M, pH 8.0). Re-
action time 24 h. Yield and enantioselectivity determined after purification of the
product.
b
Determined by HPLC analysis.
Absolute configuration was assigned by a comparative study of its MTPA de-
2. Results and discussion
c
rivatives (see Experimental section).
d
2.1. Reduction of aromatic b-ketonitriles
Absolute configuration was assigned by comparison of the specific rotations
with the literature values (see Experimental section).
The recombinant ketoreductase KRED1-Pglu was firstly used to
asymmetrically reduce different aryl -ketonitriles. Bio-
b
transformations were performed at 0.25 mmol-scale, in the pres-
ence of a catalytic amount of NADPþ and an enzyme-coupled system
(glucose/glucose dehydrogenase, BmGDH) for the regeneration of
the cofactor. Yield and stereoselectivity were determined after pu-
rification of products (Table 1). Reduction followed the Prelog rule,47
giving the corresponding (S)-b-hydroxy nitrile; all the substrates
were reduced affording the desired alcohols with high molar con-
versions and excellent enantiomeric excess after 24 h.
Table 1
Scheme 1. Reduction of 1i using NaBH4.
Reduction of aromatic b-ketonitriles with KRED1-Pglu
Firstly, syn racemate (ꢀ)-2i was separated from the anti racemate
(ꢀ)-3i by semi-preparative silica HPLC and their relative configu-
rations were assigned by comparison of the 1H NMR coupling
constants with the ones already reported in the literature.48 The
racemates (ꢀ)-2i and (ꢀ)-3i were, in turn, independently resolved
into their pure enantiomers by semi-preparative chiral HPLC (see
Experimental section). The absolute configuration of each enan-
tiomer was assigned by Mosher’s ester derivatization (see
Experimental section).
Entry
Substrate
Ar
Yield (%)a
ee (%)b,c
1
2
3
4
5
6
7
9
1a
1b
1c
1d
1e
1f
Ph
2-Fur
2-Th
4-CH3Ph
4-OCH3Ph
4-FPh
3-CH3Ph
3-ClPh
95
95
94
93
70
90
90
85
>98
>98
>98
>98
97
95
95
94
1g
1h
a
Reaction conditions: 10 mM substrate, 0.1 mM NADPþ. KRED1-Pglu (20 mU/mL),
GDH (1 U/mL), glucose 40 mM in Tris/HCl buffer pH 8.0 (0.05 M, pH 8.0). Reaction time
24 h. Yield and enantioselectivity determined after purification of the product.
Enzymatic reduction of 1i and 1j took place with high yields, but
with different stereoselectivity (Table 2).
The acidity of the
a-hydrogen causes spontaneous race-
b
Determined by chiral HPLC analysis.
Absolute configuration was assigned by comparison of the specific rotations
misation of the substrate even at pH 8.0, thus favouring a dy-
namic resolution process (Scheme 2).49 Reduction of 1i
proceeded with outstanding stereoselectivity, furnishing syn
(2R,3S)-2i as the only detectable stereoisomer with 95% yield
c
with the literature values.22
after 24 h.
a-Ethyl derivative 1j was reduced with low stereo-
The results observed with a-unsubstituted aromatic b-ketoni-
selectivity and formation of the anti stereoisomer (2S, 3S)-3j as
the main product. The different stereochemical outcome is due to
a different enzymatic recognition of the enantiomers of the
substrates, depending on the nature of the
Namely, reduction of -methyl substituted 1i occurs on the R-
enantiomer furnishing (2R,3S)-2i, whereas (S)-1j is the preferred
substrate, giving (2S,3S)-3j, although with moderate overall
stereoselectivity (Scheme 2).
triles led us to investigate more hindered substrates such as 2-
methyl-3-oxo-3-phenylpropanenitrile 1i and 2-ethyl-3-oxo-3-
phenylpropanenitrile 1j (Table 2). Since no detailed stereochemi-
cal characterization of the reduction products of 1i has been re-
ported in the literature, compound 1i was reduced with NaBH4 and
the four stereoisomers obtained were isolated and fully character-
ized (Scheme 1).
a-substituent.
a
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M.L. Contente et al. / Tetrahedron xxx (2016) 1e6
3
Table 4
Reduction of 2,2,2-trifluoro-1-arylethanones with KRED1-Pglu
Entry
Substrate
R
Yield (%)a
ee (%)b,c
1
2
1o
1p
Ph
2-Th
94
95
>98 (R)
>98 (S)
Reaction conditions: 10 mM substrate, 0.1 mM NADPþ, KRED1-Pglu (20 mU/mL),
GDH (1 U/mL), glucose 40 mM in Tris/HCl buffer pH 8.0 (0.05 M, pH 8.0). Reaction time
24 h. Yield and enantioselectivity determined after purification of the product.
a
Scheme 2. Enzymatic reduction of
caused by racemisation of the substrate.
a-alkyl-b-ketonitriles with dynamic resolution
b
Determined by chiral HPLC analysis.
Absolute configuration was assigned by comparison of the specific rotations
c
with the literature values.53,54
2.2. Reduction of aromatic a-haloketones
reduction of substrates whose reactivity strongly depends on the
acidity of the medium. Previously poorly studied substrates, such as
2-alkyl-3-oxo-3-phenylpropanenitriles 1iej, were reduced at pH
The substrate scope of KRED1-Pglu was also studied using the 2-
halo-1-arylethanones 1kel (Table 3). The biotransformation car-
ried out at pH 8.0 furnished low yields. Previous reports show that
enzymatic reduction of 2-halo-1-phenylethanones 1k and 1l pro-
ceeded with high yields only at lower pH, since side-reactions like
halo substitution may occur at higher pHs.34 Thus, the bio-
transformations were carried out in buffers at lower pH; a Na-
acetate buffer (pH 5.0, 0.1 M) proved suited for achieving (R)-2k
and (R)-2l with high enantioselectivity and excellent yields (Table
3); good results were also observed with 2-thienyl derivatives 1m
and 1n. It should be remarked that formation of aromatic halohy-
drins with R-configuration follows Prelog rule (as observed in all
the reductions carried out in this study), and the R-configuration of
the products is merely due to different CIP priority.
8.0 with concurrent racemization of the
a-stereocenter, thus
allowing for the preferential obtainment of (2R,3S)-2i-j as the
major stereoisomer produced. Reduction of 2-halo-1-
arylethanones 1-kel could be carried out at pH 5.0, avoiding
side-reactions. The use of a cheap and easy-to-use system for co-
factor regeneration (glucose dehydrogenase/glucose) allowed for
high yields in all the biotransformations studied. Enzymatic re-
duction occurred with excellent enantioselectivity with all the
substrates, with the exception of 1j, for which the occurrence of an
a-ethyl group limits the enantioselectivity and stereoselectivity.
4. Experimental part
Table 3
4.1. General procedures
Reduction of 2-halo-1-arylethanones with KRED1-Pglu at pH 5.0
All reagents and solvents were obtained from commercial sup-
pliers and used without further purification. Merck SilicaGel 60
F254plates were used for analytical TLC; 1H and 13C NMR spectra
were recorded on a Varian-Gemini 200 spectrometer. Flash column
chromatography was performed on Merck SilicaGel (200e400
mesh). 1H and 13C chemical shifts are expressed in (ppm) and
coupling constants (J) in Hertz (Hz). Rotary power determinations
were carried out using a Jasco P-1010 spectropolarimeter, coupled
with a Haake N3-B thermostat. Elemental analyses were carried out
on a Carlo Erba Model 1106 (Elemental Analyzer for C, H, and N),
and the obtained results are within 0.4% of theoretical values. HPLC
analyses were performed with a Jasco PU-980 pump equipped with
a UV/Vis detector Jasco UV-975 and a OJ-H column (4.6ꢁ250 mm,
Daicel), OD-H column (4.6ꢁ250 mm, Daicel) or Lux Amylose-2
column (4.6ꢁ150 mm, Phenomenex). Preparative HPLC was per-
Entry
Substrate
Ar
X
Yield (%)a
ee (%)b,c
1
2
3
4
1k
1l
1m
1n
Ph
Ph
2-Th
2-Th
Br
Cl
Br
Cl
95
94
92
87
>98
95
90
92
a
Reaction conditions: 10 mM substrate, 0.1 mM NADPþ, KRED1-Pglu (20 mU/
mL), GDH (1 U/mL), glucose 40 mM in acetate buffer (0.1 M, pH 5.0). Reaction time
24 h. Yield and enantioselectivity determined after purification of the product.
b
Determined by chiral HPLC analysis.
Absolute configuration was assigned by comparison of the specific rotations
c
with the literature values.27,51,52
formed with a 1525 Extended Flow Binary HPLC pump equipped
ꢀ
Finally, 2,2,2-trifluoro-1-phenylethanone (1o) and 2,2,2-
trifluoro-1-(thiophen-2-yl)ethanone (1p) were also tested (Table
4), showing high conversion and enantioselectivity. Again the re-
duction of the compound 1o gave the corresponding (R)-alcohol
because of the different CIP priority of the eCF3 group.
with a Waters 2489 UV/Vis detector and Luna 5
m
m C18 (2) 100 A
column (21.2ꢁ220 mm; Phenomenex), Lux Amylose-2 column
(21.2ꢁ220 mm, 5
m
m, Phenomenex) or Kromasil 5-AmyCoat col-
umn (21.2ꢁ250 mm, AkzoNobel) at a flow rate of 15 mL/min.
4.2. Preparation of pure KRED1-Pglu
3. Conclusion
KRED1-Pglu was prepared as recombinant protein in E. coli.25
KRED1-Pglu gene was amplified from the genomic DNA of P. glu-
cozyma by PCR using the following primers, carrying NdeI and
HindIII restriction sites:
The recombinant ketoreductase KRED1-Pglu isolated from the
genome of the non-conventional yeast P. glucozyma CBS 5766
proved to be a versatile biocatalyst for the asymmetric reduction of
aromatic functionalised aromatic ketones. The versatility and
enantioselectivity of KRED1-Pglu is noteworthy, since different
ketonitriles and -haloketones are reduced to the corresponding
Forward: 50-ATACCATATGACGAAGGTGACTGTTGTGAC-30
Reverse: 50-AGAGAAGCTTGGCGTACTCCTTCAACTCTG-30
The amplified gene was then cloned into a pET26b(þ) vector
using the NdeI and HindIII restriction sites. With this cloning
strategy, the resulting protein is expressed with a terminal His6 tag.
b-
a
secondary alcohols with high enantiomeric excesses. Moreover, the
possibility to use this enzyme at different pHs allowed for efficient
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4
M.L. Contente et al. / Tetrahedron xxx (2016) 1e6
The correct construction of the expression plasmid was confirmed
by direct sequencing.
d
¼2.85 (d, J¼6.2 Hz, 2H), 3.04 (s, br s, OH), 5.26 (t, J¼6.2 Hz, 1H),
6.98e7.00 (m, 2H), 7.06e7.07 (d, J¼3.5 Hz, 1H), 7.29e7.32 (m, 1H)
ppm. 13C NMR (75 MHz, CDCl3):
Cultures of E. coli BL21(DE3)Star transformed with the resulting
d
¼28.2, 66.2, 117.1, 124.7, 125.7,
plasmid were grown overnight at 37 ꢂC in LB medium supple-
127.1, 144.5 ppm. Elemental analysis: calcd (%) for C7H7ONS
(153.02): C 54.88; H 4.61, N 9.14; found: C 54.77; H 4.96, N 8.77.
The ee was measured by chiral HPLC on an OJ-H column (n-hex-
ane/2-propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼27.0, t(R)¼
mented with 25 mg/mL kanamycin. The seed culture was then di-
luted into a stirred fermenter containing 4.0 L of cultivation
medium (Terrific Broth, 12 g/L bacto-tryptone, 24 g/L yeast extract,
4 g/L glycerol, 2.3 g/L KH2PO4, 9.4 g/L K2HPO4, pH 7.2) to an initial
OD600nm of 0.05. Cultivation was carried out in batch-mode at 37 ꢂC,
250 rpm stirring and 250 L/h aeration rate. Cells were grown until
OD600nm reached the value of 0.8. The cultures were induced for
28
28
32.3 min; [
CHCl3)].50
a
]
¼ꢃ19.4 (c¼1.0, CHCl3) [lit. [
a
]
¼ꢃ16.71 (c¼1.01,
D
D
4.3.4. (S)-3-Hydroxy-3-(p-tolyl)propanenitrile (2d). Oil. Rf¼0.58
(CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
20 h with IPTG (isopropyl-b-D-thiogalactopyranoside) to a final
concentration of 0.5 mM. Cells were then harvested by centrifu-
gation at 4500 rpm for 30 min, washed once with 20 mM phos-
phate buffer at pH 7.0 and stored at ꢃ20 ꢂC. Protein purification was
carried out with cells suspended in 50 mM TriseHCl, 100 mM NaCl,
6 mM imidazole, pH 8.0 buffer. Proteins were extracted by soni-
cation (5 cycles of 30 s each, in ice, with 1 min interval) and cell
debris were harvested by centrifugation at 15,000 rpm for 30 min at
d
¼2.32 (s, 3H), 2.74 (d, J¼6.2 Hz, 2H), 2.12 (s, br s, OH), 4.94 (t,
J¼6.2 Hz, 1H), 7.19 (d, J¼7.9 Hz, 2H), 7.27 (d, J¼7.9 Hz, 2H) ppm.
13C NMR (75 MHz, CDCl3):
d
¼21.1, 27.9, 69.4, 117.5, 125.5, 129.4,
138.3, 138.4 ppm. Elemental analysis: calcd (%) for C10H11ON
(161.08): C 74.51; H 6.88, N 8.69; found: C 74.24; H 7.21, N 8.66.
The ee was measured by chiral HPLC on an OJ-H column (n-
hexane/2-propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼19.1, t(R)¼
29
28
4
ꢂC. The enzyme was purified by affinity chromatography with
22.3 min; [
CHCl3)].50
a
]
¼ꢃ58.3 (c¼1.0, CHCl3) [lit. [
a
]
¼ꢃ55.68 (c¼0.85,
D
D
HIS-SelectÒ Nickel Affinity Gel. Briefly, the column was equilibrated
with 50 mM TriseHCl, 100 mM NaCl, 6 mM imidazole, pH 8.0 and
the crude extract loaded; column was then washed with 50 mM
TriseHCl, 100 mM NaCl, 6 mM imidazole; finally, the adsorbed
enzyme was eluted with 50 mM TriseHCl, 100 mM NaCl, 250 mM
imidazole, pH 8.0.
4.3.5. (S)-3-Hydroxy-3-(4-methoxyphenyl)propanenitrile (2e). Oil.
Rf¼0.50 (CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz,
CDCl3):
d
¼2.56 (s, br s, OH), 2.71e2.74 (m, 2H), 3.80 (s, 3H),
4.96 (t, J¼6.2, 1H), 6.90 (m, 2H), 7.30 (m, 2H) ppm. 13C NMR
(75 MHz, CDCl3)
d
¼27.9, 55.3, 69.7, 114.3, 117.5, 126.9, 133.2,
4.3. General method for the enzymatic reduction
159.9 ppm. Elemental analysis: calcd (%) for C10H11O2N
(177.08): C 67.68; H 6.26, N 7.90; found: C 66.89; H 6.44, N
7.67. The ee was measured by chiral HPLC on an OJ-H column
(n-hexane/2-propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼43.7,
The enzymatic reaction was performed biotransformations at
25 mL-scale, using an enzyme-coupled system (glucose and glu-
cose dehydrogenase from Bacillus megaterium) for cofactor recy-
cling. Biotransformations were carried out by addition of 10 mM
28
28
t(R)¼45.2 min; [
a
]
¼ꢃ50.5 (c¼1.0, EtOH) [lit. [
a
]
¼ꢃ41.34
D
D
(c¼0.55, EtOH)].50
substrate dissolved in 250 m
L of DMSO, 0.1 mM NADPþ, KRED1-Pglu
(20 mU/mL), GDH (1 U/mL), glucose 40 mM in Tris/HCl buffer pH
8.0 (0.05 M, 25 mL) at 30 ꢂC. Biotransformations of 1ken were also
performed also in acetate buffer (NaAB 0.1 M, pH 5.0). The reaction
mixture was kept under stirring at 30 ꢂC until completion and then
extracted with 20 mL of EtOAc; the aqueous phase was extracted
twice more with 15 mL of EtOAc. The organic phases were collected
and dried over Na2SO4 and the solvent was evaporated. The crude
residue was purified by flash chromatography.
4.3.6. (S)-3-Hydroxy-3-(4-fluorophenyl)propanenitrile
(2f). Oil.
Rf¼0.50 (CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz,
CDCl3):
d
¼2.70 (d, J¼6.2 Hz, 2H), 2.97 (s, br s, 1H, OH), 4.99 (t,
J¼6.2 Hz, 1H), 7.02e7.08 (m, 2H), 7.32e7.37 (m, 2H) ppm. 13C NMR
(75 MHz, CDCl3):
d
¼28.0, 69.2, 115.8 (d, J¼21.9 Hz), 117.3, 127.4 (d,
J¼8.1 Hz), 136.8 (d, J¼2.3 Hz), 162.7 (d, J¼246.4 Hz) ppm. Elemental
analysis: calcd (%) for C9H8ONF (165.06): C 65.45; H 4.88, N 8.48;
found: C 65.26; H 5.19, N 8.01. The ee was measured by chiral HPLC
on an OJ-H column (n-hexane/2-propanol¼90:10, 1.0 mL/min,
24
4.3.1. (S)-3-Hydroxy-3-phenylpropanenitrile
(2a). Oil.
Rf¼0.62
220 nm), t(S)¼20.4, t(R)¼24.8 min; [
a]
¼ꢃ35.5 (c¼1.0, CHCl3) [lit.
D
24
(CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
[a
]
¼ꢃ34.83 (c¼0.9, CHCl3)].50
D
d
¼2.74 (d, J¼6.1 Hz, 2H), 5.01 (t, J¼6.1 Hz, 1H), 7.36e7.39 (m, 5H)
ppm. 13C NMR (75 MHz, CDCl3):
d
¼27.9, 70.0, 117.4, 125.5, 128.8,
4.3.7. (S)-3-Hydroxy-3-(m-tolyl)propanenitrile (2g). Oil. Rf¼0.58
(CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
128.9, 141.1 ppm. Elemental analysis: calcd (%) for C9H9ON (147.07):
C 73.45, H 6.16 N 9.52; found: C 73.41; H 6.16, N 9.45. The ee was
measured by chiral HPLC on an OJ-H column (n-hexane/2-
propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼24.5, t(R)¼31.1 min;
d
¼2.36 (s, 3H), 2.71 (d, J¼6.2 Hz, 2H), 2.85 (s, br s, OH), 4.95 (t,
J¼6.2, 1H), 7.13e7.19 (m, 3H), 7.24e7.29 (m, 1H) ppm. 13C NMR
(75 MHz, CDCl3)
d
¼21.4, 27.9, 70.0, 117.5, 122.6, 126.2, 128.8,
20
20
[
a]
¼ꢃ58.5 (c¼1.0, EtOH) [lit. [
a
]
¼ꢃ57.7 (c¼2.6, EtOH)].22
129.5, 138.6, 141.1 ppm. Elemental analysis: calcd (%) for
10H11ON (161.08): C 74.51; H 6.88, N 8.69; found: C 74.24; H 7.21,
D
D
C
4.3.2. (S)-3-(2-Furyl)-3-hydroxypropanenitrile (2b). Oil. Rf¼0.49
(CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
N 8.66. The ee was measured by chiral HPLC on an OJ-H column
(n-hexane/2-propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼18.9,
20
20
d
¼2.57 (s, br s, OH), 2.88 (d, J¼6.2 Hz, 2H), 5.05 (t, J¼6.2 Hz, 1H),
t(R)¼23.1 min;
[
a
]
¼ꢃ62.5 (c¼1.0, CHCl3) [lit.
[
a
]
¼ꢃ59.3
D
D
6.37e6.41 (m, 2H), 7.41e7.42 (m, 1H) ppm. 13C NMR (75 MHz,
CDCl3):
(c¼0.8, CHCl3)].22
d
¼24.9, 63.8, 107.5, 110.6, 116.9, 142.9, 152.8 ppm. Elemental
analysis: calcd (%) for C7H7ON2 (137.05): C 61.31; H 5.14, N 10.21;
found: C 61.24; H 5.52, N 10.58. The ee was measured by chiral HPLC
4.3.8. (S)-3-Hydroxy-3-(3-chlorophenyl)propanenitrile
(2h). Oil.
Rf¼0.53 (CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz,
on an OJ-H column (n-hexane/2-propanol¼90:10, 1.0 mL/min,
CDCl3):
d
¼2.72 (d, J¼6.2 Hz, 2H), 3.09 (s, br s, OH), 4.98 (t, J¼6.2,
20
220 nm), t(S)¼21.9, t(R)¼25.6 min; [
a
]
¼ꢃ40.2 (c¼1.0, EtOH) [lit.
1H), 7.25e7.28 (m, 1H), 7.29e7.30 (m, 2H), 7.31e7.37 (m, 1H) ppm.
D
20
[
a]
¼ꢃ38.6 (c¼1.3, CHCl3)].22
13C NMR (75 MHz, CDCl3)
d¼27.9, 69.2, 117.2, 123.8, 125.8, 128.8,
D
130.2, 134.8, 143.2 ppm. Elemental analysis: calcd (%) for C9H8ONCl
(181.03): C 59.52; H 4.44, N 7.71; found: C 59.65, H 4.74, N 7.32. The
ee was measured by chiral HPLC on an OJ-H column (n-hexane/2-
4.3.3. (S)-3-(2-Thieyl)-3-hydroxypropanenitrile (2c). Oil. Rf¼0.52
(CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
Please cite this article in press as: Contente, M. L.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.027

M.L. Contente et al. / Tetrahedron xxx (2016) 1e6
5
propanol¼90:10, 1.0 mL/min, 220 nm), t(S)¼20.0, t(R)¼23.1 min;
isopropanol¼98:2, 1.0 mL/min, 220 nm), t(S)¼30.1, t(R)¼33.5 min;
20 20
20
20
[
a]
¼ꢃ55.5 (c¼1.0, CHCl3) [lit. [
a
]
¼ꢃ56.8 (c¼1.3, CHCl3)].22
[a
]
¼þ32.1 (c¼1.0, CHCl3) [lit. [
a]
D
¼þ28.5 (c¼0.53, CHCl3)].53
D
D
D
4.3.9. (2R,3S)-3-Hydroxy-2-methyl-3-phenylpropanenitrile [(2R,3S)-
4.3.15. (R)-2,2,2-Trifluoro-1-phenylethanol (2o). Oil. Rf¼0.48 (n-
2i]. Oil. Rf¼0.58 (CH2Cl2/n-hexane/EtOAc¼4:1:1). 1H NMR
hexane/EtOAc¼7:3). 1H NMR (300 MHz, CDCl3):
¼2.59 (s, br s, OH),
d
(300 MHz, CDCl3):
d
¼1.25 (d, J¼6.6 Hz, 3H), 2.93 (dq, J¼6.6, 6.6 Hz,
5.0 (q, J¼6.6 Hz, 1H), 7.42e7.47 (m, 5H) ppm. 13C NMR (75 MHz,
1H), 4.73 (d, J¼6.6 Hz, 1H), 7.26e7.44 (m, 5H) ppm. 13C NMR
CDCl3)
d
72.6 (q, J¼32.3 Hz), 123.9 (q, J¼282.2 Hz), 127.4, 128.6,
(75 MHz, CDCl3)
d
¼14.7, 34.6, 75.1, 121.1, 126.3, 128.8, 140.1 ppm.
129.6,133.9 ppm. Elemental analysis: calcd (%) for C8H7OF3 (176.04)
C 54.55, H 4.01; found: C 54.17, H 4.20. The ee was measured by
Elemental analysis: calcd (%) for C10H11ON (161.08) C 74.51, H 6.88,
N 8.69; found: C 74.65, H 7.22, N 8.46. The ee and de were measured
by chiral HPLC on Lux Amylose-2 column (n-hexane/iso-
chiral HPLC on an OD-H column (n-hexane/isopropanol¼98:2,
21
0.8 mL/min, 220 nm), t(S)¼35.4, t(R)¼45.2 min; [
a
]
¼ꢃ26.5 (c¼1.0,
D
21
propanol¼95:5, 1.0 mL/min, 216 nm), t(2R,3S)¼16.8 min.
CHCl3) [lit. [
a]
¼ꢃ20.41 (c¼0.48, CHCl3)].54
D
20
[
a]
¼ꢃ12.0 (c¼0.2 in CHCl3).
D
4.3.16. (S)-2,2,2-Trifluoro-1-thienylethanol (2p). Oil. Rf¼0.45 (n-
4.3.10. (2S,3S)-2-(1-Hydroxy-1-phenylmethyl)butanenitrile [(2S,3S)-
hexane/EtOAc¼7:3). 1H NMR (300 MHz, CDCl3):
¼2.87 (s, br s, OH),
d
3j]. Solid;
mp
68e70
ꢂC.
Rf¼0.59
(CH2Cl2/n-hexane/
¼1.05 (t, J¼7.7 Hz,
EtOAc¼4:1:1). 1H NMR (300 MHz, CDCl3):
d
5.26 (q, J¼6.15 Hz, 1H), 7.03e7.09 (m, 1H), 7.20 (d, J¼3.50),
7.34e7.42 (m, 1H) ppm. 1H NMR (300 MHz, CDCl3):
J¼34.6 Hz) 123.7 (q, J¼282.2 Hz), 127.0, 127.1, 127.5, 136.1 ppm. El-
emental analysis: calcd (%) for C6H5OF3S (182.00) C 39.56, H 2.77;
found: C 39.49, H 3.10. The ee was measured by chiral HPLC on an
d¼69.5 (q
3H), 1.51e1.70 (m, 2H), 2.76e2.83 (m, 1H), 4.79 (d, J¼6.2 Hz, 1H)
7.33e7.56 (m, 5H) ppm. 13C NMR (75 MHz, CDCl3)
d
¼10.4, 24.9,
76.6, 127.0, 128.1, 128.7, 140.7 ppm. Elemental analysis: calcd (%)
for C11H13ON (175.10) C 75.40, H 7.48, N 7.99; found: C 75.23, H
7.32, N 7.89. The ee and de were measured by chiral HPLC on OD-
H column (n-hexane/isopropanol¼95:5, 0.8 mL/min, 216 nm),
t(2R,3S)¼26.9 min, t(2S,3S)¼28.6 min, t(2S,3R)¼34.2 min,
OD-H column (n-hexane/isopropanol¼98:2, 0.8 mL/min, 220 nm),
25
t(S)¼36.4, t(R)¼47.2 min;
[
a]
¼þ24.2 (c¼1.0, CHCl3) [lit.
D
25
[a
]
¼þ27.6 (c¼0.88, CH3OH)].55
D
20
28
t(2R,3R)¼36.4 min. [
a
]
¼ꢃ32.5 (c¼1.0, CHCl3) [lit. [
a
]
¼ꢃ46.4
D
D
(c¼0.5, CHCl3)].51
4.4. Chemical preparation of (2R*,3S*)-3-hydroxy-2-methyl-3-
phenylpropanenitrile [( )-2i] and (2S*,3S*)-3-hydroxy-2-
methyl-3-phenylpropanenitrile [( )-3i]
4.3.11. (R)-2-Bromo-1-phenylethanol (2k). Oil. Rf¼0.57 (n-hexane/
EtOAc¼7:3). 1H NMR (300 MHz, CDCl3):
d
¼2.70 (s, br s, OH),
3.52e3.67 (m, 2H), 4.92 (d, J¼8.5 Hz, 1H), 7.38e7.32 (m, 5H) ppm;
To a solution of 2-methyl-3-oxo-3-phenylpropanenitrile 1i
(100 mg, 0.63 mmol) in EtOH (4 mL) was added NaBH4 (6.0 mg,
0.16 mmol) and the reaction mixture was kept under agitation at
room temperature. After 1 h, the reaction was evaporated under
vacuum and the crude residue was purified by flash chromatogra-
phy (n-hexane/EtOAc 9/1) to give a mixture of (ꢀ)-2i and (ꢀ)-3i
(95 mg, 0.59 mmol). (ꢀ)-2i and (ꢀ)-3i were separated by pre-
13C NMR (75 MHz, CDCl3):
d¼40.2, 73.8, 125.9, 128.4, 128.7,
140.3 ppm. Elemental analysis: calcd (%) for C8H9OBr (197.97) C
47.79, H 4.51; found: C 47.47, H 4.76. The ee was measured by chiral
HPLC on an OD-H column (n-hexane/isopropanol¼97:3, 1.0 mL/
25
min, 220 nm), t(S)¼16.9, t(R)¼19.3 min; [
a
]
¼ꢃ45.5 (c¼1.0, CHCl3)
D
20
[lit.29
[
a]
¼ꢃ39.5 (c¼0.592, CHCl3)].52
D
parative HPLC under the following conditions: Luna 5
mm C18
4.3.12. (R)-2-Chloro-1-phenylethanol (2l). Oil. Rf¼0.56 (n-hexane/
(21.2ꢁ250 mm, Phenomenex); n-hexane/isopropanol¼95:5;
15 mL/min; 220 nm. Retention times of (ꢀ)-2i and (ꢀ)-3i were 12.5
and 13.5 min, respectively.
EtOAc¼7:3).1H NMR (300 MHz, CDCl3):
¼2.65 (d, J¼3.0 Hz,1H), 3.65
d
(dd, J¼11.0, 8.5 Hz, 1H), 3.76 (dd, J¼11.0, 3.0 Hz, 1H), 4.90 (dt, J¼8.5,
3.0 Hz, 1H), 7.30e7.40 (m, 5H) ppm. 13C NMR (75 MHz, CDCl3)
d 50.9,
(ꢀ)-2i: 1H NMR (300 MHz, CDCl3):
¼1.25 (d, J¼6.6 Hz, 3H), 2.93
d
74.1, 126.1, 128.5,140.0 ppm. Elemental analysis: calcd (%) for C8H9OCl
(156.61) C 61.35, H 5.79;found:C 61.40, H 6.11. The eewas measured by
(dq, J¼6.6, 6.6 Hz, 1H), 4.73 (d, J¼6.6 Hz, 1H), 7.26e7.44 (m, 5H)
ppm. 13C NMR (75 MHz, CDCl3)
140.1 ppm.
d
¼14.7, 34.6, 75.1, 121.1, 126.3, 128.8,
chiral HPLC on an OD-H column (n-hexane/isopropanol¼97:3,1.0 mL/
20
(ꢀ)-3i: 1H NMR (300 MHz, CDCl3):
d¼1.28 (d, J¼6.6 Hz, 3H), 3.02
min, 220 nm), t(S)¼18.9, t(R)¼20.3 min; [
a
]
¼ꢃ48.5 (c¼1.0, cyclo-
D
20
hexane) [lit. [
a
]
¼ꢃ50.7 (c¼0.225, cyclohexane)].52
(dq, J¼5.7, 6.6 Hz, 1H), 4.80 (d, J¼5.7 Hz, 1H), 7.34e7.43 (m, 5H)
D
ppm. 13C NMR (75 MHz, CDCl3)
128.7, 139.6 ppm.
d¼13.5, 34.0, 74.5, 120.8, 126.3,
4.3.13. (R)-2-Bromo-1-thienylethanol (2m). Oil. Rf¼0.49 (n-hexane/
EtOAc¼7:3). 1H NMR (300 MHz, CDCl3):
d
¼2.64 (s, br s, OH),
3.66e3.80 (m, 2H), 4.65e4.69 (m, 1H), 6.94e7.01 (m, 3H) ppm. 13
C
4.4.1. Preparative chiral HPLC resolution of (ꢀ)-2i. Preparative chiral
HPLC conditions: Lux Amylose-2 column (21.2ꢁ250 mm, Phe-
nomenex); n-hexane/isopropanol 9:1; 15 mL/min 220 nm; Re-
tention times of (ꢃ)-2i and (D)-2i were 19.9 and 21.2 min,
NMR (75 MHz, CDCl3)
d 39.5, 67.2, 124.7, 125.4, 126.7, 142.5 ppm.
Elemental analysis: calcd (%) for C6H7OBrS (205.94) C 34.80, H 3.41;
found: C 34.90, H 3.75. The ee was measured by chiral HPLC on an
OD-H column (n-hexane/isopropanol¼98:2, 1.0 mL/min, 220 nm),
respectively.
20
20
t(S)¼29.8, t(R)¼31.7 min;
[
a]
¼þ30.1 (c¼1.0, CHCl3) [lit.
(ꢃ)-2i: [
a
a
]
]
¼ꢃ12.0 (c¼0.2 in CHCl3);
¼þ12.0 (c¼0.2 in CHCl3).
D
D
[a]
D
¼ꢃ32.3 (c¼1.0, CHCl3; S-enantiomer)].27
20
20
D
(þ)-2i: [
4.3.14. (R)-2-Chloro-1-thienylethanol (2n). Oil. Rf¼0.46 (n-hexane/
4.4.2. Preparative chiral HPLC resolution of (ꢀ)-3i. Preparative chi-
ral HPLC conditions: Kromasil 5-AmyCoat column (21.2ꢁ250 mm,
AkzoNobel); n-hexane/isopropanol 95:5; 15 mL/min, 220 nm; Re-
tention times of (þ)-3i and (ꢃ)-3i were 18.2 and 22.0 min,
EtOAc¼7:3). 1H NMR (300 MHz, CDCl3):
d
¼2.75 (s, br s, OH),
3.70e3.83 (m, 2H), 5.12e5.20 (m, 1H), 6.96e7.06 (m, 1H), 7.28e7.33
(m, 2H) ppm. 13C NMR (75 MHz, CDCl3):
127.3, 143.6 ppm. Elemental analysis: calcd (%) for C6H7OClS
(161.99) C 44.31, H 4.34; found: C 43.99, H 4.73. The ee was mea-
sured by chiral HPLC on an OD-H column (n-hexane/
d
¼50.9, 70.7, 125.2, 125.8,
respectively.
20
(þ)-3i: [
a
]
]
¼þ18.5 (c¼0.2 in CHCl3);
¼ꢃ18.5 (c¼0.2 in CHCl3).
D
20
D
(ꢃ)-3i: [
a
Please cite this article in press as: Contente, M. L.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.027

6
M.L. Contente et al. / Tetrahedron xxx (2016) 1e6
16. Ankati, H.; Zhu, D.; Yang, Y.; Biehl, E. R.; Hua, L. J. Org. Chem. 2009, 74,
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4.4.3. General procedure for the synthesis of Mosher’s esters. (S)-
MTPA (31.5 mg, 0.13 mmol) was added to a solution of the alcohol
(20 mg, 0.12 mmol) in anhydrous CH2Cl2 at 4 ꢂC. The reaction
mixture was kept at 4 ꢂC under stirring and DCC (29.2 mg,
0.14 mmol) and DMAP (4.7 mg, 0.038 mmol) were added; the re-
action was left at room temperature for 6 h, then filtered and the
filtrate was concentrated under vacuum. The crude residue was
purified by flash chromatography (n-hexane/EtOAc 9:1).
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(S)-MTPA ester of (L)-2i: 1H NMR (300 MHz, CDCl3):
d
¼1.20 (d,
J¼6.6, 3H), 3.09 (dq, J¼6.6, 6.6 1H), 3.47 (s, 3H), 6.00 (d, J¼6.6, 1H),
7.30e7.50 (m, 10H) ppm.
(S)-MTPA ester of (D)-2i: 1H NMR (300 MHz, CDCl3):
d
¼1.20 (d,
J¼6.6, 3H), 3.09 (dq, J¼6.6, 6.6, 1H), 3.64 (s, 3H), 5.85 (d, J¼6.6, 1H),
7.16e7.20 (m, 2H), 7.27e7.41 (m, 8H) ppm.
In (S)-MTPA ester of (ꢃ)-2i the eOCH3 signal (3.48 ppm) is
further upfield respect to the eOCH3 signal (3.64 ppm) of (S)-MTPA
ester of (D)-2i. Based upon the already assigned relative configu-
rations of 2i, these data are in agreement with an absolute con-
figuration of (2R,3S) for enantiomer (ꢃ)-2i and (2S,3R) for
enantiomer (þ)-2i.
33. Janeczko, T.; Dymarska, M.; Kostrzewa-Sus1ow, E. Int. J. Mol. Sci. 2014, 15,
22392e22404.
(S)-MTPA ester of (D)-3i: 1H NMR (300 MHz, CDCl3):
d
¼1.29 (d,
34. Aguirre-Pranzoni, C.; Bisogno, F. R.; Orden, A. A.; Kurina-Sanz, M. J. Mol. Catal. B:
Enzym. 2015, 114, 19e24.
35. Zhu, D.; Hyatt, B. A.; Hua, L. J. Mol. Catal. B: Enzym. 2009, 56, 272e276.
36. Rodríguez, C.; Borze˛cka, W.; Sattler, J. H.; Kroutil, W.; Lavandera, I.; Gotor, V.
Org. Biomol. Chem. 2014, 12, 673e681.
37. Xu, G. C.; Yu, H. L.; Shang, Y. P.; Xu, J. H. RSC Adv. 2015, 5, 22703e22711.
38. Utsukihara, T.; Okada, S.; Kato, N.; Horiuchi, C. A. J. Mol. Catal. B: Enzym. 2007,
45, 68e72.
J¼6.6, 3H), 3.19 (dq, J¼5.7, 6.6, 1H), 3.58 (s, 3H), 5.99 (d, J¼5.7, 1H),
7.22e7.26 (m, 2H), 7.31e7.45 (m, 8H) ppm.
(S)-MTPA ester of (L)-3i: 1H NMR (300 MHz, CDCl3):
d
¼1.26 (d,
J¼6.6, 3H), 3.17 (dq, J¼5.7, 6.6, 1H), 3.46 (s, 3H), 5.96 (d, J¼5.7, 1H),
7.32e7.44 (m, 10H) ppm.
ꢁ
ꢁ
39. Hoyos, P.; Sansottera, G.; Fernandez, M.; Molinari, F.; Sinisterra, J. V.; Alcantara,
A. R. Tetrahedron 2008, 64, 7929e7936.
In (S)-MTPA ester of (ꢃ)-3i the eOCH3 signal (3.46 ppm) is
further upfield respect to the eOCH3 signal (3.58 ppm) of (S)-MTPA
ester of (D)-3i. Based upon the already assigned relative configu-
rations of 3i, these data are in agreement with an absolute con-
figuration of (2S,3S) for enantiomer (ꢃ)-3i and (2R,3R) for
enantiomer (þ)-3i.
40. Gandolfi, R.; Cesarotti, E.; Molinari, F.; Romano, D. Tetrahedron 2009, 20,
411e414.
€
41. Husain, S. M.; Stillger, T.; Dunkelmann, P.; Lodige, M.; Walter, L.; Breitling, E.;
€
€
Pohl, M.; Burchner, M.; Krossing, I.; Muller, M.; Romano, D.; Molinari, F. Adv.
Synth. Catal. 2011, 353, 2359e2362.
42. Fragnelli, M. C.; Hoyos, P.; Romano, D.; Gandolfi, R.; Alcantara, A. R.; Molinari, F.
ꢁ
Tetrahedron 2012, 68, 523e528.
43. Contente, M. L.; Molinari, F.; Zambelli, P.; De Vitis, V.; Gandolfi, R.; Pinto, A.;
Romano, D. Tetrahedron Lett. 2014, 55, 7051e7053.
References and notes
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Please cite this article in press as: Contente, M. L.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.027