Microbial-catalysed resolution of sterically demanding cyanohydrins

Article abstract of DOI:10.1016/j.molcatb.2009.12.014

Mycelia containing carboxyl-esterases, a novel source of enzymes, have been investigated in the hydrolytic kinetic resolution of one type of tert-alcohols, α,α-disubstituted cyanohydrins. This class of enzymes was found to be both active and selective towards these tert-alcohols, giving E-values as high as 42.

Full text of DOI:10.1016/j.molcatb.2009.12.014

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microbial-catalysed resolution of sterically demanding cyanohydrins
Monica Paravidinoa,1, Jarle Holta,1,2, Diego Romanoa,b, Neetu Singha, Isabel W.C.E. Arendsa,
Adriaan J. Minnaardc, Romano V.A. Orrud, Francesco Molinarib, Ulf Hanefelda,∗
a Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
b Department of Food and Microbiological Science and Technology, University of Milan, via Celoria 2, 20133 Milan, Italy
c Department of Bio-organic Chemistry, Stratingh Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
d Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycelia containing carboxyl-esterases, a novel source of enzymes, have been investigated in the
hydrolytic kinetic resolution of one type of tert-alcohols, ␣,␣-disubstituted cyanohydrins. This class of
enzymes was found to be both active and selective towards these tert-alcohols, giving E-values as high
as 42.
Received 17 September 2009
Received in revised form 8 December 2009
Accepted 14 December 2009
Available online 29 December 2009
© 2009 Elsevier B.V. All rights reserved.
Keywords:
Enzyme catalysis
Mycelium
Hydrolases
Cyanohydrins
tert-Alcohols
1. Introduction
Enantioselective conversions are even fewer and to date only
three reports on the enantioselective kinetic resolution of steri-
Cyanohydrins are versatile building blocks in organic synthe-
sis and fine chemical industry [1]. Their enantioselective, catalytic
preparation has attracted much attention and both chemical and
enzymatic approaches have been developed. In most cases the
(bio)catalytic methods focus on aldehydes as starting materi-
als since the stereo-differentiation of these prochiral molecules
is relatively easy [2–4]. However, when starting from ketones
the induction of chirality becomes considerably more difficult. In
addition the reaction equilibrium is unfavourable since the prod-
uct, an ␣,␣-disubstituted cyanohydrin, is sterically congested [5].
Indeed, it is a tert-alcohol (Fig. 1). When viewing ␣,␣-disubstituted
cyanohydrins as alcohols, this also opens up a new avenue of
approach towards their enantioselective synthesis, the kinetic res-
olutions of their esters.
cally congested cyanohydrin acetates, derived from ketones, have
been published [7–10]. Bacillus subtilis esterase 2 (BS2), which
displayed excellent enantioselectivity towards other tert-alcohols,
was completely unselective in the hydrolysis of ␣,␣-disubstituted
cyanohydrin acetates [11]. Consequently, a great need for new
enzymes to enantioselectively hydrolyse these ␣,␣-disubstituted
cyanohydrin acetates exists.
Fungal mycelia and cell-bound hydrolases are such a versa-
tile source of new enzymes, that to date has not yet been fully
explored [12–16]. Many microorganisms have cell-bound esterases
and other hydrolases. These enzymes are thus firmly immobilized
[17] to the whole cells and show several technical advantages com-
pared to commercially available enzymes, such as easy scale-up
since only the cells need to be grown and no enzyme purifi-
cation needs to be performed. In addition, the enzymes display
high stability in organic solvents. It has previously been shown
that the catalytic activity for the esterification and ester hydroly-
sis of mycelia of different moulds from various species (Rhizopus
oryzae, Rhizopus javanicus, Rhizopus liquefaciens and Aspergillus
oryzae) is promising [13,16,18]. Similar results were obtained with
cell-bound carboxyl-esterases from Bacillus coagulans [14,15] and
Kluyveromyces marxianus [12]. All these hydrolases hence have
the properties desirable for being catalysts in the enantioselective
hydrolysis of ␣,␣-disubstituted cyanohydrin acetates.
Only few examples of the successful enzymatic hydrolysis of
esters of tert-alcohols have been reported in the literature [6].
∗
Corresponding author. Tel.: +31 15 278 9304; fax: +31 15 278 1415.
E-mail addresses: francesco.molinari@unimi.it (D. Romano),
A.J.Minnaard@rug.nl (A.J. Minnaard), rva.orru@few.vu.nl (R.V.A. Orru),
U.Hanefeld@tudelft.nl (U. Hanefeld).
1
Both authors contributed equally to the present work.
Current address: School of Chemistry, University of Leeds, Leeds LS2 9JT, United
2
Kingdom.
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.014

88
M. Paravidino et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
Rhizopus and Aspergillus strains were cultured in 500 mL Erlen-
meyer flasks containing 100 mL of medium and incubated for 48 h
at 28 ◦C on a reciprocal shaker (100 spm). The liquid media con-
tained a basal medium (Difco yeast extract 1 g/L, (NH4)2SO4 5 g/L,
K2HPO4 1 g/L, MgSO4·7H2O 0.2 g/L, pH 5.80) added with Tween 80
(0.5%). Suspensions of spores (1.6 × 104) were used as inoculum.
Mycelium grown for 48 h in submerged cultures was harvested by
filtration at 4 ◦C, washed with phosphate buffer (pH 7.0, 0.1 M) and
lyophilized.
Fig. 1. ␣,␣-Disubstituted cyanohydrins are tert-alcohols.
Kluyveromyces marxianus CBS 1553 was routinely maintained on
malt extract (8 g/L, agar 15 g/L, pH 5.5) and cultured in 2.0 L
Erlenmeyer flasks containing 200 mL of malt broth (pH 6.0) and
incubated for 48 h at 28 ◦C on a reciprocal shaker (100 spm).
Bacillus coagulans NCIMB 9365 was routinely maintained on
Difco nutrient broth (8 g/L, agar 15 g/L, pH 7.0) and cultured
in 2.0 L Erlenmeyer flasks containing 200 mL of CYSP broth
(casitone 15 g/L, yeast extract 5 g/L, soytone 3 g/L, peptone 2 g/L,
MgSO4·7H2O 15 mg/L, FeCl3 115 mg/L, MnCl2 20 mg/L, pH 7.0) and
incubated for 24 h at 45 ◦C on a reciprocal shaker (120 spm).
2. Experimental
CAUTION: All procedures involving hydrogen cyanide were per-
formed in a well-ventilated fume hood equipped with a HCN
detector. HCN-containing wastes were neutralised using commer-
cial bleach and stored independently over a large excess of bleach
for disposal.
2.1. Materials and methods
n-Dodecane (99+%, Sigma–Aldrich), 1,3,5-triisopropylbenzene
(97%, Fluka), phosphate buffer (10 mM, pH 7.0), ethyl acetate
(>99%, Acros Organics), n-heptane (99%, CHROMASOLV® for
HPLC, Sigma–Aldrich), 2-propanol (99.9%, HPLC grade, Fisher Sci-
entific), trifluoroacetic acid (99%, extra pure, Acros Organics),
2,4-di-tert-butylphenol (99%, Sigma–Aldrich), acetic acid (99.5%,
Acros Organics) and (1R, 2R)-(-)-1,2-diaminocyclohexane (98%,
Sigma–Aldrich) were used as received. Racemic ␣,␣-disubstituted
cyanohydrin acetates were prepared according to established pro-
cedures by cyanation, deprotection and acetylation [10]. 1H and
13C NMR spectra were recorded with a Bruker Avance 400 (400 and
100 MHz, respectively) or a Varian Unity Inova 300 instrument (300
and 75 MHz, respectively). Chemical shifts are expressed in parts
per million (ı) relative to tetramethylsilane. Coupling constants J
are expressed in Hertz (Hz). Elemental analyses were performed by
Dr. A. Verwey Analytical Laboratories Rotterdam; HRMS analyses
were performed on a Bruker micrOTOF-Q instrument using Elec-
trospray Ionisation (ESI) in positive ion mode (capillary potential
of 4500 V). Mass spectra were obtained with a Shimadzu GC-
2010 Gas Chromatograph coupled to a Shimadzu GCMS QP-2010S
Gas Chromatographic Mass Spectrometer. Enantiomeric purity was
determined by GC using an enantioselective ␤-cyclodextrin col-
umn (CP-Chirasil-Dex CB 25 m × 0.32 mm) and a Shimadzu Gas
Chromatograph GC-17A equipped with an FID detector and a Shi-
madzu Auto-injector AOC-20i, using He with a linear gas velocity
of 75 cm s−1 as the carrier gas. Alternatively, it was determined
by HPLC using a Waters System (Waters 486 UV detector, Waters
515 pump and Waters 717+ injector) equipped with a Chiralpak
AD-H column from Daicel (4.5 ␮m × 250 mm) with UV-detection
at 215 nm and n-heptane:2-propanol 80:20 + 0.1% trifluoroacetic
acid (TFA) as solvent (flow: 0.5 mL/min). Optical rotations were
measured using a Perkin-Elmer 241 polarimeter.
The dry weights were determined after centrifugation of 100 mL
of cultures, cells were washed with distilled water and dried at
110 ◦C for 24 h.
Liquid mycelia preparations (grown on medium and stored in
phosphate buffer at 4 ◦C, used within a short time after prepara-
tion): Bacillus coagulans, Kluyveromyces marxianus.
2.3. General procedure for the hydrolytic kinetic resolution
The reactions were run on a scale of 2 g/L (substrate con-
centration). Cells (40.0 mg) and substrate (4.0 mg) were mixed
together in phosphate buffer (10 mM, pH 7.0, 2.0 mL) and stirred
at 30 ◦C for 6 h. The substrate did not dissolve completely in
the phosphate buffer and gave a turbid mixture. n-Dodecane or
1,3,5-triisopropylbenzene (0.05 or 0.1 mL of a solution with con-
centration 2 g/L in ethyl acetate) was added at the end of the
reaction. n-Dodecane (0.1 mL) was used for substrates 1b, 1c and 1e.
1,3,5-Triisopropylbenzene was used for substrates 1a (0.1 mL), 1d
(0.05 mL), 1f (0.05 mL), 1 h (0.1 mL) and 1 g (0.05 mL). The reaction
mixture was extracted with ethyl acetate (2 × 2.5 mL), dried over
Na2SO4, filtered and concentrated to a volume of 1 mL under N2.
Enantiomeric excess (ee) and conversion (c) was calculated from
chiral GC or HPLC analysis. The experiments where no reaction
occurred were considered as a blank reaction (Table 1). No auto-
hydrolysis of the substrates was observed, even after long periods
of time in phosphate buffer. This is in perfect agreement with a
recent publication on these substrates [10].
2.4. Spectroscopic data for ˛,˛-disubstituted cyanohydrin
acetates
1a: R1 = Ph, R2 = Me: see Ref. [10].
1b: R1 = 4-Cl–C6H4, R2 = Me: 1H NMR (CDCl3, 300 MHz): 1.98 (s,
3H), 2.13 (s, 3H), 7.36–7.49 (m, 4H); 13C NMR (CDCl3, 300 MHz):
20.9, 29.6, 72.7, 117.8 (C≡N), 126.0 (2C), 129.2 (2C), 135.2, 136.8,
168.3 (C O); MS: 225/223 (M+), 208, 183/181, 164, 146, 139, 113,
101; GC: 160 oC: 3.7 min (S) and 4.0 min (R).
2.2. Microorganisms and culture conditions
The following microbial strains have been employed:
1c: R1 = 4-OMe–C6H4, R2 = Me: see Ref. [10].
Rhizopus oryzae CBS 112.07, CBS 391.34 (javanicus), CBS 260.38
(liquefaciens) (Central Bureau voor Schimmelcultures, Utrecht, The
Netherlands).
Aspergillus oryzae MIM (Industrial Microbiology Section, DISTAM,
University of Milan).
Bacillus coagulans NCIMB 9365 (National Collection of Industrial
and Marine Bacteria, Aberdeen, UK).
Kluyveromyces marxianus CBS 1553 (Central Bureau voor Schim-
melcultures, Utrecht, The Netherlands).
1d: R1 = 3,4-di-OMe–C6H3, R2 = Me: 1H NMR (400 MHz, CDCl3):
2.00 (s, 3H), 2.13 (s, 3H), 3.89 (s, 3H), 3.92 (s, 3H), 6.89 (d, J = 8.2 Hz,
1H), 7.02 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H); 13C NMR (400 MHz, CDCl3):
21.1, 31.0, 56.0, 56.1, 73.1, 108.1, 111.1, 117.3 (C≡N), 118.3, 130.5,
149.2, 149.7, 168.4 (C O); elemental analysis calcd for C13H15NO4:
C, 62.6; H, 6.1; N, 5.6; found: C, 62.8; H, 6.2; N, 5.5; HRMS: m/z
calcd for C13H15NNaO4 (M+Na)+ 272.0893, found 272.0887; MS:
249 (M+), 232, 210, 207, 190, 165, 138, 119, 104, 89, 77, 63, 43, 40;

M. Paravidino et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
89
Table 1
129.1 (2C), 129.4, 134.1, 134.6, 168.1 (C O); elemental analysis
calcd for C18H15NO2: C, 77.9; H, 5.4; N, 5.0; found: C, 77.5; H, 5.5; N,
4.4; HRMS: m/z calcd for C18H15NNaO2 (M+Na)+ 300.0995, found
300.0985; MS: 277 (M+), 253, 238, 235, 206, 179, 165, 140, 130, 105,
89, 77, 63, 43, 40; HPLC: column: Chiralpak AD-H, flow: 0.5 mL/min,
UV-detection: 215 nm, solvent: n-heptane:2-propanol 80:20 + 0.1%
TFA: 13.9 min (S) and 15.2 min (R).
Conversions, enantiomeric excesses and E-values for hydrolytic kinetic resolutions
of ␣,␣-disubstituted cyanohydrin acetates catalysed by mycelia.
Substrate
Mycelia strain
Conversion
ee
1% (R)
1% (R)
0% (–)
12% (R)
2% (S)
0% (–)
2% (S)
E
1a
1a
1a
1a
1a
1a
1a
Rhizopus oryzae
Rhizopus javanicus
45%
46%
51%
62%
45%
n.r.
1
1
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
n.d.
1.3
1
n.d.
1
1 g: R1, R2 = Isatinyl: 1H NMR (400 MHz, CDCl3): 2.18 (s, 3H),
2.71 (br s, NH, 1H), 7.32 (dd, J = 7.6 and 7.6 Hz, 1H), 7.51 (dd, J = 8.0
and 8.0 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H); 13C
NMR (400 MHz, CDCl3): 20.2, 70.8, 112.7, 117.5 (C≡N), 120.9, 124.5,
126.5, 132.9, 140.7, 167.9 (C O), 169.8 (C O); elemental analysis
calcd for C11H8N2O3: C, 61.1; H, 3.7; N, 12.9; found: C, 61.2; H,
3.9; N, 12.3; HRMS: m/z calcd for C11H8N2NaO3 (M+Na)+ 239.0427,
found 239.0428; MS: 216 (M+), 188, 172, 157, 146, 118, 102, 90,
75, 63, 43, 40; HPLC: column: Chiralpak AD-H, flow: 0.5 mL/min,
UV-detection: 215 nm, solvent: n-heptane:2-propanol 80:20 + 0.1%
TFA: 13.5 min and 16.9 min.
61%
1b
1b
1b
1b
1b
1b
1b
Rhizopus oryzae
Rhizopus javanicus
54%
55%
66%
48%
n.r.
4% (R)
3% (R)
3% (R)
3% (R)
0% (–)
0% (–)
1% (S)
1.1
1.1
1.1
1.1
n.d.
n.d.
1.1
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
n.r.
34%
1c
1c
1c
1c
1c
1c
1c
Rhizopus oryzae
Rhizopus javanicus
17%
25%
30%
34%
n.r.
3% (R)
1% (R)
2% (R)
18% (R)
0% (–)
0% (–)
15% (S)
1.4
1.1
1.1
2.5
n.d.
n.d.
1.2
1 h: R1 = 2-Thiophenyl, R2 = Me: see Ref. [10].
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
1i: R1 = 2-Furanyl, R2 = Me: 1H NMR (CDCl3, 400 MHz): 2.08 (s,
3H), 2.10 (s, 3H), 6.42 (s, 1H), 6.66 (d, J = 3.0 Hz, 1H), 7.44 (s, 1H); 13C
NMR (CDCl3, 400 MHz): 20.9, 24.8, 66.9, 110.4, 110.9, 116.8 (C≡N),
143.8, 148.1, 168.4 (C O); elemental analysis calcd for C9H9NO3:
C, 60.3; H, 5.1; N, 7.8; found: C, 61.6; H, 5.3; N, 7.6; HRMS: m/z calcd
for C9H9NNaO3 (M+Na)+ 202.0475, found 202.0475; MS: 179 (M+),
164, 151, 137, 120, 110, 95, 76, 65, 43; GC: 140 ◦C: 1.62 min (S) and
1.67 min (R).
n.r.
86%
1d
1d
1d
1d
1d
1d
1d
Rhizopus oryzae
Rhizopus javanicus
28%
38%
23%
56%
36%
89%
97%
16% (S)
36% (S)
18% (S)
59% (S)
49% (S)
91% (S)
94% (S)
2.8
5.5
5
5
23.5
3
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
2.2
2.5. Synthesis of catalyst [(R, R)-salen)Ti(ꢀ-O)]2 (4)
1e
1e
1e
1e
1e
1e
1e
Rhizopus oryzae
Rhizopus javanicus
47%
48%
53%
63%
n.r.
2% (R)
1% (R)
1% (R)
23% (R)
0% (–)
0% (–)
0% (–)
1.1
1
1
1.6
n.d.
n.d.
n.d.
The bimetallic Ti-salen complex 4 was synthesized according
to literature procedures. Spectroscopic data are in agreement with
those reported in literature [19,20]. [␣]23D = −209 (c 1.06, CHCl3;
lit [19] = −267 (c 0.0125, CHCl3)).
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
n.r.
n.r.
2.6. General procedure for the synthesis of enantioenriched
O-TMS-cyanohydrins (S)-3
1f
1f
1f
1f
1f
1f
1f
Rhizopus oryzae
Rhizopus javanicus
n.r.
0% (–)
11% (R)
1% (R)
75% (S)
1% (S)
n.d.
1.2
1
2.4
1
72%
76%
86%
59%
82%
70%
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
The Ti(salen) catalyst 4 (0.5 mol%) was dissolved in dry DCM
(1.5 mL) under N2, then ketone (1 equiv.) and TMSCN (1.5 equiv.)
were added. The mixture was stirred at RT for 24 h. The crude mix-
ture was then filtered through a silica pad eluting with the solvents
indicated for every product [19,21].
31% (R)
12% (R)
1.4
1.2
1g
1g
1g
1g
1g
1g
1g
Rhizopus oryzae
Rhizopus javanicus
46%
47%
89%
68%
51%
n.r.
57%
9
37
2
78%
70%
90%
90%
0%
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
Synthesis of (S)-3b: According to the General Procedure,
182 mg (1.18 mmol) of 4-chloroacetophenone afforded 297 mg
of 3b (quantitative yield). Purification by flash chromatography
was performed using heptane/EtOAc 5:1 as eluent. GC: 40 ◦C
(20 min)–10 ◦C/min–250 ◦C (20 min): 33.42 min (major, S) and
33.49 min (minor, R); ee = 59%; [␣]23D = −17.3 (c 1.93, CHCl3); (R)-
3b: [␣]25D = +29.5 (ee 92%) [22]. 1H NMR (400 MHz, CDCl3): ı 0.21
(s, 9H), 1.85 (s, 3H), 7.39 (ABAꢀBꢀ system, J = 8.4 Hz, 2H), 7.49 (ABAꢀBꢀ
system, J = 8.4 Hz, 2H) ppm. 13C NMR (100 MHz, CDCl3): ı 1.06 (3C),
33.52, 71.07, 121.25, 126.09 (2C), 128.84 (2C), 134.62, 140.73 ppm.
Synthesis of (S)-3c: According to the General Procedure, 240 mg
(1.61 mmol) of 4-methoxyacetophenone afforded 281 mg of 3c
(70%). Purification by flash chromatography was performed using
PE/EtOAc 8:2 as eluent. GC: 105 ◦C: 75.6 min (major, S) and
77.3 min (minor, R); ee = 46%; [␣]23D = −9.21 (c 1.61, CHCl3); (R)-
3c: [␣]25D = +21.8 (ee 89%) [23]. 1H NMR (400 MHz, CDCl3): ı 0.17
(s, 9H), 1.86 (s, 3H), 3.84 (s, 3H), 6.92 (ABAꢀBꢀ system, J = 8.8 Hz,
2H), 7.47 (ABAꢀBꢀ system, J = 8.8 Hz, 2H) ppm. 13C NMR (100 MHz,
CDCl3): ı 1.09 (3C), 31.45, 55.35, 71.28, 113.87 (2C), 121.82, 126.06
(2C), 133.04, 159.77 ppm.
7
42
n.d.
1.6
24%
6%
1h
1h
1h
1h
1h
1h
1h
Rhizopus oryzae
Rhizopus javanicus
76%
80%
79%
90%
92%
n.r.
8% (R)
0% (–)
7% (R)
12% (R)
28% (R)
0% (–)
1.1
n.d.
1.1
1.1
1.3
n.d.
1.1
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianusa
Kluyveromyces marxianusb
Bacillus coagulans
88%
10% (S)
All enzymes tested showed no activity towards substrate 1i.
a
Lyophilised.
Freshly prepared.
b
HPLC: column: Chiralpak AD-H, flow: 0.5 mL/min, UV-detection:
215 nm, solvent: n-heptane:2-propanol 80:20 + 0.1% TFA: 13.4 min
(R) and 14.6 min (S).
1e: R1 = Ph, R2 = Et: see Ref. [10].
1f: R1 = Ph, R2 = CH CH–Ph: 1H NMR (CDCl3, 300 MHz): 2.17
(s, 3H, CH3), 6.33 (d, J = 15.9 Hz, 1H), 6.94 (d, J = 15.9 Hz, 1H),
7.29–7.61 (m, 10H, ArH); 13C NMR (CDCl3, 300 MHz): 21.1, 76.4,
116.4 (C≡N), 125.3, 125.6 (2C), 127.2 (2C), 128.5, 128.6, 128.8 (2C),
Synthesis of (S)-3d: According to the General Procedure, 365 mg
(2.03 mmol) of 3,4-dimethoxyacetophenone afforded 396 mg of
3d (70%). Purification by flash chromatography was performed

90
M. Paravidino et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
using PE/EtOAc 1:1 as eluent. GC: 40 ◦C (20 min)–10 ◦C/min–250 ◦C
(20 min): 33.1 min (major, S) and 33.2 min (minor, R); ee = 19%;
[␣]23D = −6.13 (c 1.26, CHCl3). 1H NMR (400 MHz, CDCl3): ı 0.18 (s,
9H), 1.87 (s, 3H), 3.91 (s, 3H), 3.92 (s, 3H), 6.87 (d, J = 8.4 Hz, 1H), 7.04
(s, 1H), 7.25 (d, J = 8.4 Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3): ı
1.07 (3C), 33.51, 55.93, 55.96, 71.41, 108.04, 110.81, 117.11, 121.72,
134.50, 149.02, 149.28 ppm.
Synthesis of (S)-3f: According to the General Procedure, 422 mg
(2.03 mmol) of trans-chalcone afforded 584 mg of 3f (94%). Purifi-
cation by flash chromatography was performed using PE/EtOAc
1:1 as eluent. [␣]23D = +5.93 (c 1.30, CHCl3). 1H NMR (400 MHz,
CDCl3): ı 0.27 (s, 9H), 6.22 (d, J = 15.8 Hz, 1H), 7.03 (d, J = 15.8 Hz,
1H), 7.62–7.23 (m, 10H) ppm. 13C NMR (100 MHz): ı 1.31, 75.09,
119.70, 125.50 (2C), 127.09 (2C), 128.70 (2C), 128.75 (2C), 128.79,
128.89, 129.73, 130.93, 135.14, 140.39 ppm.
Scheme 1. Hydrolytic kinetic resolution of ketone-derived cyanohydrin acetates
catalysed by mycelia.
(S)-1d: Following the General Procedure, 1d (125 mg, 83% from
3d) was obtained as a yellow oil. GC: 160 ◦C: 19.9 min (major, S)
and 20.2 min (minor, R); ee = 14%; [␣]23D = −1.34 (c 1.57, CHCl3).
Analytical data as given for rac-1d.
(S)-1f: Following the General Procedure, 1f (151 mg, 75% from
3f) was obtained as a yellow oil. HPLC: 13.8 min (major, S) and
15.2 min (minor, R); ee = 19%; [␣]23D = +0.59 (c 1.25, CHCl3). Ana-
lytical data as given for rac-1f.
2.7. Synthesis of (S)-1b
202 mg (0.8 mmol) of 3b were dissolved in acetonitrile
(2 mL) under N2. Acetic anhydride (150 ␮L, 2 equiv.) and scan-
dium(III)triflate (4 mg, 0.011 equiv.) were added and the mixture
was stirred at RT for 26 h. After removal of the solvent in vacuo
the crude product was purified twice through flash chromato-
graphy using PE/EtOAc 8:2 as eluent to afford 109 mg of 1b (61%).
GC: 160 ◦C: 8.71 min (major, S) and 9.17 min (minor, R): ee = 58%:
[␣]23D = +6.7 (c 1.49, CHCl3). Analytical data as given for rac-1b.
3. Results and discussion
The mycelia and other whole cells known for cell-bound
carboxyl-esterase activity (the yeast Kluyveromyces marxianus and
the bacterium Bacillus coagulans) were investigated as potential
catalysts for the enantioselective hydrolysis of a novel class of tert-
alcohol esters, namely ␣,␣-disubstituted cyanohydrin acetates 1.
All of the different strains were tested either as lyophilized mycelia
or freshly prepared mycelia grown on medium [12–16,18].
The hydrolytic kinetic resolutions were performed in a phos-
phate buffer (10 mM, pH 7.0) with a substrate concentration of 2 g/L
at 30 ◦C (Scheme 1) analogous to our recently described screen-
ing of commercially available hydrolases [10]. Given the fact that
the whole cells buffer the pH of the reaction mixture, the conver-
sion could not be monitored by the addition of base. In addition,
the products formed in the reaction, the free cyanohydrins 2a–i,
proved to be unstable and decomposed under the reaction condi-
tions to their corresponding ketones, accompanied by the release
of HCN (Scheme 1). Consequently, (S)-acetates are isolated for (R)-
selective enzymes and vice versa. In order to measure the degree
of conversion, n-dodecane and 1,3,5-triisopropylbenzene, respec-
tively, were added as internal standards at the end of the reaction,
but before the mixtures were subjected to work-up. As substrates
a wide range of structurally diverse ␣,␣-disubstituted cyanohydrin
acetates 1a–i were employed (Fig. 2). This allowed a full evaluation
not only of the activity of the mycelia, but also of their substrate
specificity.
2.8. Cleavage of the TMS-group
Synthesis of (S)-2c: 69 mg of (S)-3c were dissolved in 1 M HCl
(3 mL) and the mixture was allowed to stir at RT for 16 h. The crude
product was extracted with diethyl ether (3 × 10 mL), dried over
sodium sulphate and evaporated under reduced pressure (63 mg,
quantitative yield). (S)-2c was immediately acylated as described
in Section 2.9.
Synthesis of (S)-2d: 162 mg of (S)-3d were dissolved in 1 M HCl
(3 mL) and the mixture allowed to stir at RT for 16 h. The crude
product was extracted with diethyl ether (3 × 10 mL), dried over
sodium sulphate and evaporated under reduced pressure (133 mg,
quantitative yield). (S)-2d was immediately acylated as described
in Section 2.9.
Synthesis of (S)-2f: 246 mg of (S)-3f were dissolved in MeOH
(5 mL). 48% aqueous HF (67 ␮L, 2 equiv.) were added and the mix-
ture was allowed to stir at RT for 16 h. A 3:1 DCM/water mixture
(20 mL) was added. After stirring vigorously for 30 min, the phases
were separated and the aqueous layer was extracted with DCM
(3 × 15 mL). The organic layer was dried over sodium sulphate and
evaporated under reduced pressure to afford (S)-2f (186 mg, 98%)
as a yellow oil. The crude cyanohydrin was immediately acylated
as described in Section 2.9.
2.9. General procedure for the acetylation of cyanohydrins
All strains of the mycelium-bound carboxyl-esterases were
found to be active towards most of the substrates, except the 2-
furanyl derivative 1i. In addition to activity, moderate to good
selectivity was alsoobserved. The E-values forthehydrolytic kinetic
resolution were in the range 1–42 (Table 2), which is in the
same range as reported earlier in the literature for tert-alcohols
[10,11]. The highest selectivity was shown by almost all mycelia for
the dimethoxy-substituted compound 1d and for indole-2,3-dione
derived cyanohydrin 1 g. For these two substrates, all mycelia were
found to be (R)-selective, leaving thus the (S)-acetate unchanged.
For other substrates, a mild enantiopreference (E < 4) for the (S)-
acetate was observed. The enantiopure cyanohydrin acetates were
isolated by solvent extraction with low to moderate enantiomeric
excesses (full details in Table 1). The differences between freshly
The crude cyanohydrin 2c, 2d, or 2f (1 equiv.) was dissolved
under nitrogen in dry DCM (5 mL). Pyridine (7 equiv.) and acetic
anhydride (5 equiv.) were added and the mixture was stirred at
RT until completion. The reaction mixture was then washed with
1 M HCl (3 × 10 mL), brine (1 × 10 mL) and then dried over sodium
sulphate. Evaporation of the solvents under reduced pressure
afforded the crude cyanohydrin acetate. No further purification was
required.
(S)-1c: Following the General Procedure, 1c (63 mg, 45% from
3c) was obtained as a yellow oil. GC: 160 ◦C: 11.5 min (major, S)
and 11.8 min (minor, R); ee = 27%; [␣]23D = −1.37 (c 1.14, CHCl3).
Analytical data as given for rac-1c.

M. Paravidino et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
91
Fig. 2. Ketone-derived cyanohydrin acetates tested in the hydrolysis.
prepared and lyophilised Kluyveromyces marxianus as for activity
and selectivity towards some substrates highlight the importance
of the formulation for complex mixtures of enzymes such as whole
cells. For example, the lyophilisation process may be responsible
for the deactivation of some hydrolases.
The enantioselectivity of mycelia towards compounds 1b, 1c,
1d, and 1f was determined by the chemical preparation of enan-
tioenriched samples of these cyanohydrin acetates. In this way,
the absolute configuration could be assigned to the enantiomer
obtained via the kinetic resolution. The corresponding ketones
were converted into their enantioenriched O-TMS-cyanohydrins
(3b, 3c, 3d, and 3f) by a Ti(salen)-complex mediated cyanosily-
lation [19]. Utilising the (R, R)-salen-bimetallic catalyst 4, (S)-3b
was prepared with an ee of 59% and an optical rotation [␣]23 of
D
−17.3. (R)-3b is known to have [␣]25D = +29.5 with an ee of 92%
[22]. Similarly, (S)-3c was obtained with an ee of 46% and an opti-
cal rotation [␣]23 of −9.21, while (R)-3c is known to have [␣]20
D
D
of +21.8 when ee = 89% [23]. Sc(III)triflate then catalysed the direct
conversion of (S)-3b into the corresponding acetate, with retention
of configuration [24] (Scheme 2).
The unknown TMS-cyanohydrins 3d and 3f were prepared in the
same manner, assuming that the enantiopreference by the catalyst
4 towards the corresponding ketones was the same. This was fur-
thermore confirmed upon conversion of the enantioenriched silyl
derivatives 3c and 3d into their acetates. When analyzed by chi-
ral GC with the same method, 1b, 1c, and 1d all showed the same
order of elution, the major (S)-enantiomer eluting first. This was
also the case for 1a, the absolute stereochemistry of which was
determined prior to this study [10]. It is noteworthy that direct
acetylation must be replaced by a two step procedure (cleavage
of TMS, acetylation) for derivatives 3c, 3d, and 3f because they all
underwent Lewis acid catalysed rearrangements or deprotection
Scheme 2. Preparation of (S)-1b.
Table 2
Activity towards the different substrates, selectivity and E-values for hydrolytic
kinetic resolutions of ␣,␣-disubstituted cyanohydrin acetates catalysed by mycelia.
Mycelium
Activity
Selectivity, Ea,b
Rhizopus oryzae
1a–e, 1g–h
1a–h
1a–h
1a–h
1a, 1d, 1f–h
1g, 9
1d, 5.5 (S), 1g, 37
1d, 5 (S)
1d, 5 (S), 1g, 7
1d, 23.5 (S), 1g, 42
Rhizopus javanicus
Rhizopus liquefaciens
Aspergillus oryzae
Kluyveromyces marxianus
(lyophilized)
Kluyveromyces marxianus
(freshly prepared)
Bacillus coagulans
1d, 1f
–
–
1a–d, 1f–h
a
Only E-values above 4 are reported. All details are given in Table 1.
In brackets the absolute configuration of the isolated cyanohydrin acetate.
b
Scheme 3. Synthesis of (S)-1c,d,f.

92
M. Paravidino et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 87–92
in the presence of Sc(III)triflate (Scheme 3). No enantioselective
synthesis was available for compound 1 g.
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cyanohydrin acetates. All seven strains tested proved to be both
active and selective towards these tert-alcohols, with the exception
of 1i, giving E-values up to 42 as the best result. Furthermore, the
most enantioselective mycelia proved to be (R)-selective towards
the tested substrates. The enzymatic toolbox to access sterically
congested enantiopure cyanohydrins has thus been expanded.
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