Hydrolase-catalysed preparation of chiral a,a-disubstituted cyanohydrin acetates

Article abstract of DOI:10.1002/adsc.200700053

The enzymatic hydrolysis of esters of tertiary alcohols has long been a challenge. In particular their kinetic resolutions have virtually not been addressed. Here we describe the successful kinetic resolution of a,a-disubstituted cyanohydrin acetates, a t

Full text of DOI:10.1002/adsc.200700053

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DOI: 10.1002/adsc.200700053
Hydrolase-Catalysed Preparation of Chiral a,a-Disubstituted
Cyanohydrin Acetates
Jarle Holt,a Isabel W. C. E. Arends,a Adriaan J. Minnaard,b and Ulf Hanefelda,*
a
Biokatalyse en Organische Chemie, Technische Universiteit Delft, Julianalaan 136, 2628 BL Delft, The Netherlands
Fax : (+31)-(0)15-278-1415; e-mail: u.hanefeld@tudelft.nl
Synthetisch Organische Chemie, Stratingh Instituut, Rijksuniversiteit Groningen, Nijenborgh 4, 9747 AG Groningen,
b
The Netherlands
Received: January 24, 2007
Dedicated to Prof. Dr. Liisa T. Kanerva on the occasion of her 60th birthday.
Abstract: The enzymatic hydrolysis of esters of ter-
tiary alcohols has long been a challenge. In particu-
lar their kinetic resolutions have virtually not been
addressed. Here we describe the successful kinetic
resolution of a,a-disubstituted cyanohydrin ace-
tates, a type of protected tertiary alcohols that form
particularly interesting building blocks in organic
synthesis. Utilising Subtilisin A the hydrolysis reac-
tion was (S)-selective, while Candida rugosa lipase
was (R)-selective. With these commercially avail-
able enzymes both enantiomers of the a,a-disubsti-
tuted cyanohydrin acetates are now accessible.
pose.[5] Attempts in this direction have also been per-
formed in the enantioselective hydroxynitrile lyase-
catalysed reactions, but the success is still limited to
few substrates.[6]
A different approach is to view these a,a-disubsti-
tuted cyanohydrins as tertiary alcohols. Their racemic
esters are straightforward to prepare and a kinetic
resolution should allow an uncomplicated access to
the chiral cyanohydrin. However, tertiary alcohols
and their esters are notoriously problematic substrates
for hydrolases, the enzymes of choice for a kinetic
resolution (Figure 1).[7–12] Only few examples of the
Keywords: asymmetric catalysis; cyanohydrins;
enzyme catalysis; hydrolases; kinetic resolution; ter-
tiary alcohols
Cyanohydrins are versatile building blocks in organic
synthesis and in the fine chemical industry. Their
enantioselective, catalytic preparation has attracted
much attention and both chemical and enzymatic ap-
proaches have been developed.[1–4] In most cases the
(bio)catalytic methods focus on aldehydes as starting
materials, since stereodifferentiation of these pro-
chiral molecules is relatively easy. When starting from
ketones the induction of chirality becomes considera-
Figure 1. The enzymatic hydrolysis of tertiary alcohols – i.e.,
of a,a-disubstituted cyanohydrins – is a challenge.
bly more difficult. In addition, the reaction equilibri- successful hydrolysis of these esters are known, enan-
umis less favourable since the product, an a,a-disub- tioselective conversions are even fewer and the appli-
stituted cyanohydrin is sterically congested, indeed it cation for a,a-disubstituted cyanohydrins is very limit-
is a tertiary alcohol. When preparing themenantio-
ed. Only two non-commercial crude enzymes were
pure by HCN addition, a large excess thereof needs described to performthis reaction with limited yields
to be used, independent of whether the reaction was and ees[13–15] and just one successful example (a-tri-
catalysed chemically or with an enzyme.[1–4] Chemical fluoromethyl-a-ethyl cyanohydrin acetate) for Candi-
approaches with activated cyanides such as TMSCN da rugosa lipase (CRL) is known.[16] Taking this as a
have been described in which the product is immedi- starting point we set out for an extensive screening of
ately protected, ensuring a shift of the equilibrium.[3] hydrolases for the kinetic resolution of a,a-disubsti-
Similarly, acid anhydrides can be added for this pur- tuted cyanohydrin acetates.
Adv. Synth. Catal. 2007, 349, 1341 – 1344
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1341

Jarle Holt et al.
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Table 1. Enzymatic hydrolysis of 1a–d, only reactions where
Given the rather limited knowledge about the abili-
ty of hydrolases to catalyse the enantioselective hy-
drolysis of a,a-disubstituted cyanohydrin esters, a
general screening of lipases, esterases and proteases
was performed [Candida rugosa lipase (CRL), Rhizo-
mucor miehei lipase, Burkholderia cepacia lipase that
is also known as Pseudomonas cepacia lipase immobi-
lised on ceramic particles (BCL), Candida antarctica
lipase A (Novozyme 735, liquid preparation), Candi-
da antarctica lipase A (immobilised on Celite R-633),
Candida antarctica lipase B (Novozyme 435), porcine
pancreas lipase, pig liver esterase, a-chymotrypsin,
Subtilisin A (SubA; type VIII, from Bacillus licheni-
formis)]. To ensure comparability, the same amount
of units was used for all enzymes. As substrates the
readily prepared acetates 1a–d were employed in a
pH stat-controlled reaction (Scheme 1). Under these
activity was observed are given.[a]
Enzyme
Time
Conv. 1
ee 1
E
1a
1a
1a
1b
1c
1d
1d
CRL
BCL
96 h
48 h
72 h
72 h
20 h
20 h
20 h
60%
44%
55%
41%
50%
61%
64%
91% (S)
47% (S)
88% (R)
64% (R)
90% (R)
54% (S)
87% (R)
13
6
18
44
58
8
SubA
SubA
SubA
CRL
SubA
7
[a]
Conditions: 200 U enzyme, 1 mmol substrate, 10 mL of
10 mM phosphate buffer at pH 7.0 and 258C, for details
of the reaction conditions and analysis, see Experimental
Section. The cyanohydrins 2 that are released during the
reactions decompose under the reaction conditions to the
corresponding ketones. The cyanohydrin acetate of 2-ace-
tylfuran was not converted by any of the enzymes.
CAL-A is known for its activity towards the esters of
tertiary alcohols, in particular 2-phenylbut-3-yn-2-ol
acetate. This molecule differs from 1a only by a CꢁC
in the position of the nitrile group, indicating the im-
portance of electronic effects for the enzyme activi-
ty.[7,10,11]
The most notable result is the significant activity
and selectivity of the protease Subtilisin A (SubA) for
all substrates 1a–d. With an enantioselectivity as high
as E=58 for 1c, the stereoselectivity of this enzyme is
remarkable. In addition, SubA displays (S)-enantiose-
lectivity, the opposite to that of lipases CRL and
BCL. It thus complements them, allowing for a flexi-
ble approach towards both stereoisomers. This stereo-
complementarity of SubA to lipases is already known
for secondary alcohols.[19]
Scheme 1. General conditions for the screening of hydrolytic
enzymes.
conditions the conversions can be followed by the ad-
In summary, a new hydrolase-based enantioselec-
dition of base. The unreactive enantiomer of 1a–d can tive approach towards esters of a,a-disubstituted cya-
readily be isolated while the released cyanohydrins nohydrins has been developed, that forms the basis
2a–d tend to liberate HCN and the corresponding for a new approach towards their enantioselective
ketone.[17,18]
preparation. SubA stereoselectively hydrolyses the
Three significant activities were found (Table 1). In (S)-enantiomer of their acetates 1a–d, while CRL and
line with earlier results, CRL is capable of hydrolys- BCL display activity and selectivity for the (R)-enan-
ing tertiary alcohols, including those that have a tiomers.
carbon-carbon triple bond instead of
a nitrile
group.[8,10,12] As observed earlier for a-trifluoromethyl-
a-ethyl cyanohydrin acetate[16] CRL selectively hy-
drolyses (R)-1a and (R)-1d with reasonable enantiose-
lectivity (Eꢀ8). Surprisingly, the other compounds
were not substrates for CRL. BCL only displayed ac-
tivity for 1a; similar to CRL it is modestly R-selective.
This activity was unexpected, since earlier studies
with structurally related compounds (CꢁC in place
of a nitrile group)[8,12] revealed no activity. Against
predictions CAL-A did not show any activity. Neither
when immobilised on Celite, nor when used as a
liquid preparation could any activity be detected for
Experimental Section
Materials and Methods
Trimethylsilyl cyanide (Fluka), N-methylmorpholine N-
oxide (97%, Sigma–Aldrich), dichloromethane (anhydrous,
99.8%, Sigma–Aldrich), hydrofluoric acid (48%, Sigma–Al-
drich), acetic anhydride (99+ %, Acros), pyridine (anhy-
drous, 99.8%, Sigma–Aldrich), Candida rugosa lipase (type
VII, 1170 U/mg, Sigma–Aldrich), Subtilisin A (type VIII,
from Bacillus licheniformis, crystallised and lyophilised, 12
U/mg, Sigma–Aldrich), Burkholderia cepacia lipase formerly
this enzyme. This is all the more surprising since known as Pseudomonas cepacia lipase (immobilised on Ce-
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1341 – 1344

Preparation of Chiral a,a-Disubstituted Cyanohydrin Acetates
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ramic particles, 15156 U/g, Fluka), Candida antarctica lipase
A (Novozyme 735, liquid preparation), Candida antarctica
lipase A (Novozyme 735, immobilised on Celite Bio-Cata-
lyst Carrier R-633 fromWorld Minerals according to the lit-
erature[20]), Candida antarctica lipase B (Novozyme 435), a-
chymotrypsin (Sigma–Aldrich), pig liver esterase (Sigma–
Aldrich), porcine pancreas lipase (type II, Sigma–Aldrich),
Rhizomucor miehei lipase (Novo Industri A/S).
All work was performed in the presence of an HCN de-
tector. 1H and 13C NMR spectra were recorded with a
Bruker Avance 400 (400 MHz and 100 MHz, respectively)
or a Varian Unity Inova 300 (300 MHz and 75 MHz, respec-
tively) instrument. Chemical shifts are expressed in parts
per million (d) relative to tetramethylsilane. Coupling con-
stants J are expressed in Hertz (Hz). Mass spectra were de-
termined with a Shimadzu GC-2010 Gas Chromatograph
coupled to a Shimadzu GCMS-QP2010S Gas Chromato-
graphic Mass Spectrometer. Optical rotations were obtained
using a Perkin–Elmer 343 polarimeter (wavelength 589 nm).
3H, CH3), 1.98 (s, 3H, CH3); 13C NMR (300 MHz, CDCl3):
d=168.5 (C=O), 160.2, 130.1 (CꢁN), 126.2 (”2), 118.4,
114.2 (”2), 72.9, 55.4, 29.4, 21.0; MS: m/z=219 (M+), 204,
188, 177, 160, 145, 135, 117, 103, 89, 77, 63, 43.
1
1d: H NMR (400 MHz, CDCl3): d=7.37 (m, 2H, ArH),
7.00 (t, 1H, ArH, J=4.33 Hz), 2.13 (s, 3H, CH3), 2.10 (s,
3H, CH3); 13C NMR (400 MHz, CDCl3): d=168.4 (C=O),
140.7 (CꢁN), 127.3, 126.9, 126.8, 117.6, 69.5, 29.1, 21.1; MS:
m/z=195 (M+), 180, 153, 136, 125, 109, 97, 84, 65, 43.
General Procedure for the Enzymatic Hydrolysis[29,30]
The cyanohydrin acetate 1a–d (1 mmol) was added to a
10 mM phosphate buffer at pH 7 (10 mL) and 258C. The
enzyme (200 U) was added and the pH kept constant by the
addition of a 0.1M NaOH solution with an automatic bu-
rette. At neutral pH, the acetate was extracted with di-
chloromethane, dried over MgSO4 and the solvent was re-
moved under vacuum. The ee of the remaining acetate was
determined by enantioselective GC analysis (see above).
The cyanohydrin released by the enzyme decomposed to the
corresponding ketone. No other by-products were observed.
An enzymatic hydrolysis reaction on a preparative scale
was performed in the same way, using 1a (2.91 mmol) in a
10 mM phosphate buffer at pH 7 (30 mL) and CRL (600 U).
This yielded (S)-1a as a yellow oil with an ee of 67% as de-
termined by enantioselective GC, [a]2D0: +12.2o (c 8.65,
CHCl3). The already fully characterised (R)-1a has [a]2D2:
À15.2o (c 0.34, CHCl3).[22]
The enzyme activity was determined with tributyrin accord-
[21]
ing to a general procedure fromthe literature.
Enzymatic
hydrolysis reactions were performed on an automated Dosi-
mat pH-stat from Metrohm. The amount of 0.1M NaOH is
equivalent to the conversion. Enantiomeric purity was deter-
mined by GC using an enantioselective b-cyclodextrin
column (CP-Chirasil-Dex CB 25 m”0.32 mm) using a Shi-
madzu Gas Chromatograph GC-17A equipped with an FID
detector and a Shimadzu Auto-injector AOC-20i, using He
with a linear gas velocity of 75 cmsÀ1 as the carrier gas. All
analyses were performed isothermally. The temperature pro-
grams and retention times are: 1a: 1108C (20 min):
12.03 min (S) and 14.03 min (R); 1b: 1108C (20 min):
16.18 min (S) and 17.30 min (R); 1c: 1508C (10 min):
7.66 min (S) and 8.04 min (R); 1d: 1308C (10 min): 4.93 min
(S) and 5.07 min (R).
The absolute configuration of 1a was determined by com-
parison with optical rotation[22] and elution order of the
enantiomers (as their TMS ethers) by enantioselective
GC.[23,24] Conversion of the enantiopure acetate to its TMS
ether was performed according to a procedure from the lit-
erature.[25]
(S)-1a (ee 67%) was converted into the corresponding
TMS ether of (S)-2a;[25] [a]D20: À1.89o (c 8.75, CHCl3), litera-
ture for TMS ether of (R)-2a[24] [a]D20: +18.5o (c 1.25,
CHCl3). The sequence of enantiomers of the TMS ethers of
2a in the enantioselective GC was identical to those de-
scribed in the literature.[23] The enantiopurity as determined
by enantioselective GC of the TMS ether of (S)-2a was ee
66%.
For 1b–d the enzymatic hydrolysis was performed with
Subtilisin A on a larger scale (2.8 mmol substrate) and the
resulting enantioenriched (R)-1b–d had the following optical
rotations and ee values (determined by enantioselective
GC): (R)-1b [a]2D0: À15.36o (c 11.4, CHCl3), ee 70%; (R)-1c
[a]D20: +0.28o (c 12.9, CHCl3), ee 62%; and (R)-1d [a]2D0:
+3.71o (c 14.3, CHCl3), ee 8%.
Racemic cyanohydrin acetates were prepared by silylcya-
nation, deprotection and acylation according to the litera-
ture,[26–28] the spectroscopic data are in accordance with the
literature:
1
1a[22]: H NMR (300 MHz, CDCl3): d=7.53–7.51 (d, 2H,
Acknowledgements
ArH, J=7.5 Hz), 7.38–7.41 (m, 3H, ArH), 2.14, (s, 3H,
CH3), 1.98 (s, 3H, CH3); 13C NMR (300 MHz, CDCl3): d=
168.3 (C=O), 138.2 (CꢁN), 129.2, 128.9 (”2), 124.2 (”2),
118.2, 73.2, 29.7, 20.9; MS: m/z=189 (M+), 175, 166, 147,
130, 120, 103, 91, 77, 63, 43.
J. H. gratefully acknowledges the National Research School
Combination Catalysis (NRSC-Catalysis) for financial sup-
port. The authors thank Dr. Lars Veum for suggestions and
fruitful discussions.
1b: 1H NMR (400 MHz, CDCl3): d=7.42–7.46 (d, 2H,
ArH, J=7.54 Hz), 7.26–7.40 (m, 3H, ArH), 2.29 (dq, 1H,
CH2, J=7.25 Hz), 2.13 (s, 3H, CH3), 2.09 (dq, 1H, CH2, J=
7.25 Hz), 1.03 (t, 3H, CH3, J=7.35 Hz); 13C NMR
(400 MHz, CDCl3): d=168.3 (C=O), 137.0 (CꢁN), 129.0,
128.8 (”2), 124.8 (”2), 117.2, 78.0, 35.8, 20.9, 8.4; MS: m/z=
204 (M+ +1), 191, 175, 161, 144, 135, 117, 105, 84, 75, 61, 40;
anal. calcd. for C12H13NO2 (203.25): C 70.92, H 6.45, N 6.89;
found: C 70.5, H 6.1, N 6.7.
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Adv. Synth. Catal. 2007, 349, 1341 – 1344
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Adv. Synth. Catal. 2007, 349, 1341 – 1344