An orthogonal biocatalytic approach for the safe generation and use of HCN in a multistep continuous preparation of chiral O-acetylcyanohydrins

Article abstract of DOI:10.1055/s-0035-1560644

An enantioselective preparation of O-acetylcyanohydrins has been accomplished by a three-step telescoped continuous process. The modular components enabled an accurate control of two sequential biotransformations, safe handling of an in situ generated hazardous gas, and in-line stabilization of products. This method proved to be advantageous over the batch protocols in terms of reaction time (40 min vs 345 min) and ease of operation, opening up access to reactions which have often been neglected due to safety concerns.

Full text of DOI:10.1055/s-0035-1560644

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An Orthogonal Biocatalytic Approach for the Safe Generation and
Use of HCN in a Multistep Continuous Preparation of Chiral O-Ace-
tylcyanohydrins
Aischarya Brahma1,a,b
Biagia Musio1,b
st biotransformation
2nd biotransformation
chemical protection
1
Uliviya Ismayilovac
Nikzad Nikbinb
Sonja B. Kamptmann2,b
Petra Siegertc
Günter E. Jerominc
Steven V. Ley*b
Martina Pohla
CalB
HCN
5 bar
AtHNL
OAc
*
A
Ar
CN
NCCOOEt
B
C
ArCHO
Py/Ac2O
a IBG-1: Biotechnology, Forschungszentrum Jülich, 52425 Jülich, Germany
b Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK
svl1000@cam.ac.uk
c Institute of Nano- and Biotechnologies (INB), Aachen University of Applied Sciences, 52428 Jülich, Germany
Received: 03.08.2015
Accepted after revision: 03.09.2015
Published online: 29.09.2015
ic strategies in flow9 have been reported so far in contrast
to discrete single-step biotransformations.10 The applica-
tion of hydroxynitrile lyases (HNLs) to access chiral cyano-
hydrins from aldehydes or ketones and HCN has a long tra-
dition in bioorganic chemistry as well as for the production
of respective intermediates for synthesis.11 Over the last
few years, the (R)-selective hydroxynitrile lyase from Arabi-
dopsis thaliana AtHNL has been characterized thoroughly
with respect to its application for the preparation of chiral
cyanohydrins.12 The crucial reaction parameters affecting
its activity and enantioselectivity have been identified,
with a special regard to the pH, the temperature and the
amount of water in the reaction medium.13 Despite the im-
portance of the biocatalyzed hydrocyanation for the prepa-
ration of optically pure amino alcohols as chiral building
blocks,14 this reaction still suffers from safety limitations
related to the handling of the toxic and volatile hydrogen
cyanide (HCN).15 Some HCN surrogates are under investiga-
tion, among which are trialkylsilyl cyanide,16 acetone cy-
anohydrin,17 diethyl cyanophosphonate,18 benzoyl cya-
nide,19 and acyl cyanide.20 A relatively cheap and less toxic
cyanide source, ethyl cyanoformate (ECF), has enjoyed
some successes in asymmetric cyanation of aldehydes, ke-
tones, imines and olefins.21 To the best of our knowledge no
biocatalyzed hydrolysis of ECF has been reported in litera-
ture so far. Thus, we decided to test the efficiency of differ-
ent commercially available lipases for this transformation,
and found that CalB immobilized on acrylic resin (Novo-
zyme 435) in methyl tert-butyl ether (MTBE) showed the
best efficiency for this transformation.22 A packed-bed reac-
tor, PBR (microbore column 3 mm/100 mm), equipped with
a back pressure regulator (5 bar), containing different
amounts of CalB, was fed with a 1 M solution of ECF in
MTBE (Scheme 1).
DOI: 10.1055/s-0035-1560644; Art ID: st-2015-d0610-l
Abstract An enantioselective preparation of O-acetylcyanohydrins has
been accomplished by a three-step telescoped continuous process. The
modular components enabled an accurate control of two sequential
biotransformations, safe handling of an in situ generated hazardous gas,
and in-line stabilization of products. This method proved to be advanta-
geous over the batch protocols in terms of reaction time (40 min vs 345
min) and ease of operation, opening up access to reactions which have
often been neglected due to safety concerns.
Key words Arabidopsis thaliana, novozyme 435, biocatalysis, flow,
telescoped, HCN, cyanohydrins
Biocatalysis has been gaining in popularity over the past
few years as a practical and more sustainable alternative to
conventional synthetic methods,3 especially where chemo-,
regio- and stereoselectivity are key issues.4 However, bio-
catalytic multistep approaches to chiral fine chemicals are
still rare.5 This is partially due to the high demands of ap-
propriate reaction parameters for different enzymes, which
can be difficult to implement on scale. Recently, continuous
processing technologies have been shown to be a powerful
concept in order to tune reaction conditions in a very pre-
cise manner,6 enhance the sustainability and facilitate the
scale-up of the chemical processes involving hazardous re-
agents.7 This becomes even more important during those
multistep approaches, where the use of modular compo-
nents for the downstream processing together with auto-
mated in-line analytics allow the control and coordination
of all the stages of the processes.7b,8 In spite of these advan-
tages, surprisingly few applications to multistep biocatalyt-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 262–266

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A. Brahma et al.
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Novozyme
435 CalB
O
HCN
Et
O
CN
1 M
10 cm
5 bar
Scheme 1 Flow set-up of ECF hydrolysis
By adding a very small amount of ethanol or a potassi-
um phosphate buffer solution (Kpi, pH 6.5) to the reaction
medium, almost complete ECF hydrolysis (95–97%) could
be achieved with a residence time of 17.5 minutes (Table 1,
entries 4 and 5, see SI for details). Under these conditions
the CalB-flow reactor was able to convert continuously 6.6
mmol of ECF in 120 minutes. Although a micro-aqueous
system improved the ECF hydrolysis, it is noteworthy that
in the absence of CalB only a small amount of ECF (≤5%) was
converted to HCN.
Figure 1 (a) CalB-catalyzed hydrolysis of ECF in a sample vial over time.
Reaction conditions: a mixture of Novozyme 435 (40 mg) and ECF (1
mmol) in 1 mL of micro-aqueous MTBE was stirred at room tempera-
ture (b) Study on the recyclability of a Novozyme 435-containing ‘tea-
bag’ over four consecutive cycles (see SI for details).
biocatalyst recycling, and solvent switching were problem-
atic, despite this being an attractive application of flow
chemistry. With the aim of improving this approach, we de-
cided to use the HCN-containing solution derived from the
CalB-catalyzed reaction in a second AtHNL-catalyzed addi-
tion of HCN to aldehydes in a linked multistep flow process.
Initial experiments were performed in a closed sample vial
equipped with 25 mg of AtHNL immobilized on celite
(celite-AtHNL, 6.2 mg of pure enzyme). A mixture of benz-
aldehyde (0.5 M) and HCN solution (1.5 M) in micro-aque-
ous MTBE was introduced into the vial and stirred at room
temperature, affording after 125 minutes (R)-mandeloni-
trile with high conversion (99%) and high ee (98%; see SI for
more details). Similar reaction conditions were applied in a
flow system equipped with a packed bed reactor (micro-
bore column 3 mm/50 mm) containing AtHNL immobilized
on celite (Scheme 2).
Table 1 Optimization of the reaction parameters for the ECF hydroly-
sis in the flow set-up
Entry
Flow rate (mL/min)
CalB (mg)
Conv. (%)a
1
2
3
4
5
0.15
0.05
0.04
0.04
0.04
34
34
55b
56b
84c
95d
97e
277
277
277
a The conversion was calculated by GC analysis checking for the ECF deple-
tion (see SI for details).
b A mixture of Novozyme 453 and celite was used to fill the reactor.
c Anhydrous MTBE was used.
d A mixture of MTBE/EtOH (3:1) was used.
e Micro-aqueous MTBE, prepared as described in SI, was used.
Celite-
OH
R
AtHNL
O
H
HCN
1 M
+
Ph
Pleasingly, impressive system stability was observed
when a 1 M solution of ECF in micro-aqueous MTBE was
passed (0.04 mL/min) through the CalB-reactor, showing
only minor inactivation of the biocatalyst from 97% after
one hour to 80% after eight hours of continuous operation
(see SI for details).
CN
Ph
5 bar
l (cm)
2a
1a
0.5 M
Scheme 2 Flow set-up of celite-AtHNL catalyzed synthesis of (R)-man-
delonitrile
When the same biotransformation was carried out in
batch mode in a screw-cap equipped glass vial (4 mL) a re-
action time of 120 minutes was necessary to hydrolyze 1
mmol of ECF quantitatively in the presence of 40 mg of No-
vozyme 453 (Figure 1, a), and a modest lifetime was ob-
served, with a 30% decrease in activity during the fourth cy-
cle (Figure 1, b).
This new in situ generation of HCN opens up further op-
portunities to use this hazardous reagent for reactions
which have often been neglected owing to safety reasons.
Recently, the flow preparation of protected mandelonitrile
derivatives by integrating an enzymatic step in aqueous
media with a chemical reaction in an organic solvent was
reported.23 However, the use of KCN as HCN source, lack of
The effect of the residence time and the amount of the
biocatalyst were considered, changing respectively the
length of the reactor (l) and the amount of biocatalyst.
When the residence time was increased a good ee and a sat-
isfactory conversion was observed (Table 2, entry 3). How-
ever, doubling the quantity of biocatalyst resulted in a ben-
eficial effect, achieving good conversion and very high ee
values (Table 2, entry 4).
With these very promising data in hand, we attempted
to develop a coupled process, assembling the two biotrans-
formations consecutively in a continuous flow set-up. A
solution of ECF (1 M in micro-aqueous MTBE) was pumped
(0.04 mL/min) into a 10 cm PBR containing 277 mg of CalB.
The output of this first PBR, consisting of a 1 M solution of
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A. Brahma et al.
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Table 2 Optimization of the reaction parameters for the celite-AtHNL-
Novozyme 435 catalyzed synthesis of (R)-mandelonitrile in the flow set-up
Novozyme 435
CalB
E. coli-
AtHNL
HCN
OH
(R)
5 bar
H
CN
Ph
10 cm
10 cm
2a
Entry
l (cm)
flow rate
(mL/min)
Celite-AtHNL (mg) Conv.a (%)
ee (%)
NCCOOEt
1.0 M
99% conv
95% ee
O
1
2
3
4
2.5
2.5
5.0
5.0
0.02
0.04
0.04
0.04
50b
50b
33
20
74
85
93
93
88
96
1a
0.5 M
Ph
50c
Scheme 4 Synthesis (R)-mandelonitrile by a two-step biotransforma-
tion (Novozyme 435-E. coli-AtHNL)
100b
a Determined by 1H NMR.
b Celite-AtHNL was used to fill the PBR.
c A mixture of celite-AtHNL (50 mg) and pure celite (50 mg) was used to fill
be addressed. The advantageous modularity of the flow sys-
tem allowed us to perform an in-line protection of the new-
ly formed cyanohydrins, as a three-step process. The new
flow configuration consisted of two consecutive PBRs, con-
taining Novozyme 435 and E. coli-AtHNL, respectively, a
back-pressure regulator (5 bar) and a 2.0 mL PTFE coil for
acetylation of the formed cyanohydrin (Scheme 5).
the PBR.
HCN, was mixed with a 0.5 M solution of benzaldehyde in
micro-aqueous MTBE (0.04 mL/min) by means of a tee
piece assembly. The resulting mixture was passed through a
second reactor (5 cm), containing 100 mg of celite-AtHNL
and equipped with a back pressure regulator to keep the
entire system pressurized at 5 bar (Scheme 3). As a proof of
concept, (R)-mandelonitrile 2a was successfully obtained in
very good conversion (97%) and excellent ee (99%).
Novozyme 435
CalB
E. coli-
AtHNL
HCN
2a-f
5 bar
A
C
10 cm
10 cm
Ac2O, Py
(1:1 v/v)
NCCOOEt
1.0 M
2.0 mL
Novozyme 435
Celite-
B
0.1 mL/min
OAc
∗
0.04 mL/min
CalB
HCN
AtHNL
O
OH
5 bar
Ph
1a–f
0.5 M
H
NCCOOEt
1.0 M
R
Ar
Ar
CN
CN
5 cm
10 cm
3a–f
0.04 mL/min
2a
97% conv
99% ee
O
OAc
R
OAc
R
1a
0.5 M
OAc
R
Ph
CN
MeO
CN
CN
Scheme 3 Synthesis of (R)-mandelonitrile by a two-step biotransfor-
mation (Novozyme-celite-AtHNL)
Br
3b
3c
3a
99% conv.
78% conv.
99% conv.
Although this two-step biocatalytic approach showed
persuasive advantages, the expensive and time-consuming
purification of isolated enzymes could become an issue, es-
pecially during scale-up. The application of recombinant
whole-cells as alternative biocatalyst appears more appeal-
ing in biotransformations, since no enzyme purification is
needed, and, typically, the microorganisms present a high
operational stability.24 The use of wild-type AtHNL-ex-
pressing E. coli BL21 (DE3) cells as whole-cell biocatalyst for
the synthesis of chiral cyanohydrins has been investigated
in detail by our group, demonstrating their efficiency and
extreme stability in the same micro-aqueous reaction sys-
tem as used in this study.12d We therefore studied AtHNL-
expressing E. coli cells as biocatalyst in our two-step cas-
cade synthesis of (R)-mandelonitrile, employing a PBR (10
cm) containing 250 mg of lyophilized cells (Scheme 4; see
SI for details). According to this protocol, the desired (R)-
mandelonitrile could be obtained in high conversion (99%)
and in very good ee (95%).
90% ee
89% ee
96% ee
OAc
R
OAc
R
OAc
S
CN
NO2
CN
CN
O
F3C
3d
3e
3f
99% conv.
40% ee
86% conv.
75% ee
75% conv.
70% ee
Scheme 5 Three-step chemo-biocatalytic cascade towards O-protect-
ed chiral cyanohydrins
This flow system was shown to be robust and applicable
to a range of O-acetylcyanohydrins,25 both electron-rich
(3c) and electron-poor (3f), with high conversions over
three steps (75–99%, Scheme 5, conversion has been deter-
mined by 1H NMR).
In conclusion, the advantages of the continuous flow
technology have been explored with regard to a novel or-
thogonal biocatalytic approach. CalB and AtHNL were em-
ployed in a robust continuous telescoped process, involving
an in situ HCN generation followed by addition to alde-
Although this new biocatalytic tactic in flow is very
promising, the well-known instability of cyanohydrins at
neutral to alkaline pH and at room temperature needed to
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265
A. Brahma et al.
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Synlett
hydes. High stereocontrol was observed in the subsequent
hydrocyanation reaction. An in-line chemical acetylation
enabled stabilization of newly formed cyanohydrins, giving
access to a class of O-acetylcyanohydrins with very good
conversions and ee values over three steps (75–99% conver-
sion; 40–98% ee). The remarkable efficiency of whole re-
combinant cells as biocatalysts opens up further opportuni-
ties to more complex transformations, involving a combina-
tion of enzymes in flow reactor systems.
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Acknowledgement
We gratefully acknowledge the Deutsche Forschungsgemeinschaft
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Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560644.
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carried in a well ventilated fumehood and a gas monitor for
HCN was used during all the stages of the experiments. The
excess of HCN (1 eq) was treated with an aqueous solution of
sodium hypochlorite (10–15%) until neutralization.
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resis 2004, 25, 1327
(25) General procedure for the three-step cascade synthesis of
chiral O-acetylcyanohydrins in flow: A 1 M solution of ECF in
microaqueous MTBE was pumped (0.04 mL/min) into a 10 cm
packed-bed reactor containing CalB (277 mg). This first output,
consisting of 1 M solution of HCN, was mixed with a 0.5 M solu-
tion of aldehyde 1a–f in micro-aqueous MTBE (0.04 mL/min–1
)
by means of a tee piece assembly. The resulting mixture was
passed through a second bioreactor (Kinesis, Benchmark micro-
bore column 3 mm/100 mm 2 × F), containing lyophilized
E. coli-AtHNL- (250 mg), prepared as described in the SI (section
S5). A back pressure regulator (5 Bar) was introduced after the
packed bed reactor. The out coming solution was mixed with a
mixture of pyridine/acetic anhydride (1:1) pumped by
a
(R)-2-O-Acetyl-2-(2-nitrophenyl)acetonitrile (3d): 1H NMR
(400 MHz, CDCl3) δ 2.21 (s, 3 H), 7.09 (s, 1 H), 7.68 (dt, J = 8.2,
1.4 Hz, 1 H), 7.80 (dt, J = 7.7, 1.3 Hz, 1 H), 7.93 (dd, J = 7.8, 1.2 Hz,
1 H), 8.20 (dd, J = 8.2, 1.2 Hz, 1 H). 13C NMR (150 MHz, CDCl3):
δ 20.1, 59.6, 115.0, 125.8, 129.3, 131.3, 134.5, 147.0, 168.3. IR
(Neat): 3123, 3036, 2333, 1756, 1399, 1116, 1064 cm–1. HRMS:
m/z [M + H]+ calcd for C10H9O4N2: 221.0557; found: 221.0547.
compact HPLC pump (Knauer) and the resulting solution was
passed through a PTFE coil (2 mL). The layers were collected and
a sample (50 μL in 500 μL of CDCl3) was analyzed by NMR to
check the formation of (R)-3a, (R)-3b, (R)-3c, (R)-3d, (S)-3e and
(R)-3f (75–99%). The optical purity of the products (40–98%)
was assessed by GC analysis. CAUTION: special precautions
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 262–266