Enantiopure Synthesis of (R)-Mandelonitrile Using Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.) [ParsHNL] in Aqueous/Organic Biphasic System

Article abstract of DOI:10.1007/s10562-017-2025-5

Abstract: Hydroxynitrile lyases (HNLs) are increasingly finding application in the synthesis of enantiomerically pure cyanohydrins which are important intermediates in the production of pharmaceuticals and agrochemicals. Synthesis of enantiopure mandelonitrile was carried out using HNL of wild apricot (Prunus armeniaca L.) [ParsHNL] in aqueous/organic biphasic system. The optimum pH and temperature of the reaction were 4.0 and 15 °C respectively, which are important parameters to suppress the non-enzymatic catalysis. ParsHNL catalyses synthesis of (R)-mandelonitrile in methyl-tbutyl ether (MTBE)/citrate buffer biphasic system with >99% ee. Synthesis of mandelonitrile was carried out in batch reaction at 40?ml scale and finally 2.7 mmoles of (R)-mandelonitrile was recovered which corresponded to 90% molar conversion in 46?h reaction. In fed batch reaction 6.37 mmoles of (R)-mandelonitrile could be produced which corresponds to 91% molar conversion in 46?h. In both reactions, enzyme produces (R)-mandelonitrile with > 99% ee which showed enhanced selectivity as compared to aqueous reaction (96% ee) by ParsHNL. The results showed potential of ParsHNL to synthesize (R)-mandelonitrile in both, batch reaction and fed-batch reaction and can be effectively used in the synthesis of (R)-mandelonitrile. Graphical Abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s10562-017-2025-5

Catal Lett
DOI 10.1007/s10562-017-2025-5
Enantiopure Synthesis of (R)-Mandelonitrile Using
Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.)
[ParsHNL] in Aqueous/Organic Biphasic System
Mohammad Asif1 · Tek Chand Bhalla1
Received: 9 January 2017 / Accepted: 5 March 2017
© Springer Science+Business Media New York 2017
Abstract Hydroxynitrile lyases (HNLs) are increasingly
finding application in the synthesis of enantiomerically
pure cyanohydrins which are important intermediates in
the production of pharmaceuticals and agrochemicals. Syn-
thesis of enantiopure mandelonitrile was carried out using
HNL of wild apricot (Prunus armeniaca L.) [ParsHNL]
in aqueous/organic biphasic system. The optimum pH and
temperature of the reaction were 4.0 and 15°C respectively,
which are important parameters to suppress the non-enzy-
matic catalysis. ParsHNL catalyses synthesis of (R)-man-
delonitrile in methyl-tbutyl ether (MTBE)/citrate buffer
biphasic system with >99% ee. Synthesis of mandelonitrile
was carried out in batch reaction at 40 ml scale and finally
2.7 mmoles of (R)-mandelonitrile was recovered which
corresponded to 90% molar conversion in 46 h reaction. In
fed batch reaction 6.37 mmoles of (R)-mandelonitrile could
be produced which corresponds to 91% molar conversion in
46 h. In both reactions, enzyme produces (R)-mandeloni-
trile with >99% ee which showed enhanced selectivity
as compared to aqueous reaction (96% ee) by ParsHNL.
The results showed potential of ParsHNL to synthesize
(R)-mandelonitrile in both, batch reaction and fed-batch
reaction and can be effectively used in the synthesis of
(R)-mandelonitrile.
Graphical Abstract
3000
Product Formed (µmole)
3000
2700
2640
2598
Substrate le (µmole)
2500
2088
1948
2000
1500
1000
500
0
1052
912
402
360
300
0
0
3
6
24
28
46
Time (h)
HCN
ParsHNL
(R)-MANDELONITRILE
CHIRAL SYNTHESIS
BENZALDEHYDE
6370
91
6067
5952
85
100
6000
4800
3600
2400
1200
0
4708
64.9
80
86.7
Substrate
Product
60
3000
54.6
2252
2292
40
20
0
% conversion
1948
1908
1052
1048
933
630
0
6
12
18
24
30
36
42
48
Time (h)
Keywords Prunus armeniaca L. · Wild apricot · (R)-
mandelonitrile · Biphasic reaction · Hydroxynitrile lyase ·
ParsHNL · Cyanohydrin · Batch-fed batch reaction
1 Introduction
* Tek Chand Bhalla
bhallatc@rediffmail.com
Hydroxynitrile lyases (HNL, EC 4.1.2.x) are the enzymes
which mediate the release of hydrogen cyanide (HCN) and
1
Department of Biotechnology, Himachal Pradesh University,
Summer Hill, Shimla, Himachal Pradesh 171005, India
Vol.:(0123456789)
1 3

M. Asif, T. C. Bhalla
aldehyde or ketone from their corresponding cyanohydrins
[1] and catalyses enantioselective synthesis of cyanohy-
drins through reverse reaction [2]. The cyanohydrins and
their derivatives are finding wide applications in indus-
tries for production of pharmaceuticals, agrochemicals
and cosmetics, as both functional groups of cyanohydrins,
hydroxyl and cyanide are attached to same carbon atom,
which can be readily converted into other synthetically rel-
evant building blocks like α-hydroxy ketones, α-hydroxy
carboxylic acids and β-amino acids [3].
2.2 Preparation of Crude Enzyme Extract
Crude hydroxynitrile lyase was extracted from the kernel
of P. armeniaca seeds according to method outlined by
Han et al. [17] with slight modification [16]. The crude
enzyme obtained was concentrated using vacuumed-
freeze drier. The protein concentration in the enzyme
preparation was determined following the method of
Bradford [18].
The enantiomeric purity of the synthesized caynohy-
drins is highly dependent on the spontaneous non-enzy-
matic catalysis which is major drawback of aqueous mono-
phasic reaction. To overcome this problem and to maintain
the substrate and product concentration to avoid catalytic
inhibition, immiscible biphasic reactions are introduced.
Biphasic reactions are also preferred due to easy recovery
of product formed from the organic phase. HNL medi-
ated synthesis of cyanohydrins involves two substrates
and therefore it is a complex reaction that involves several
parameters [1, 4]. The role of organic solvent has been well
studied on PaHNL activity and stability [5]. Biphasic reac-
tion is known for its suppression of non-enzymatic catalysis
and hence enhancing the enantiopurity of product formed.
Previous studies on the syntheses of chiral cyanohydrins in
biphasic systems were carried out on the HNLs from Mani-
hot esculenta (MeHNL), Hevea brasiliensis (HbHNL),
Sorghum bicolour (SbHNL), Prunus amygdalus (PaHNL),
Passiflora edulis (PeHNL) and Prunus dulcis (PdHNL)
[6–15], but no further information on cyanohydrins synthe-
sis is available for recently characterized HNLs.
2.3 Preparation of HCN Solution
Preparation of cyanide solution was carried out following
the method used by Alagöz et al. [15] with slight modifi-
cation. In brief potassium cyanide (13.0 g) was added into
100 ml of citrate buffer (50 mM, pH 5.0) on ice and then
25 ml of methyl-tbutyl ether (MTBE) was added onto cya-
nide solution. The mixture was continuously stirred for
25 min followed by addition of HCl (35%) until the final
pH was dropped to 3.0. MTBE layer containing HCN was
separated and further MTBE was added repeatedly onto
the citrate buffer so that HCN formed was extracted fur-
ther. The combined MTBE layers were collected together
and stored over citrate buffer (50 mM, pH 4.0) in a dark
bottle at 4°C. Concentration of HCN was determined as
described in the literature [19].
2.4 Effect of pH
Recently purification and characterization of HNL from
wild apricot (Prunus armeniaca L.) has been reported [16].
The HNL from P. armeniaca (ParsHNL) catalyzed the syn-
thesis of (R)-mandelonitrile from benzaldehyde in a citrate
buffer system employing potassium cyanide as a cyanide
source [16], but the yield and enantiomeric excess of the
(R)-mandelonitrile was low in this aqueous medium.
The effect of pH on the synthesis of mandelonitrile was
studied. For this purpose reaction mixture (20 ml) was pre-
pared with aqueous/organic phase (MTBE) [1:3 ratio (v/v)]
containing 1500 µmole of benzaldehyde dissolve in DMSO
(0.5 ml), and HCN was used in excess amount with citrate
buffer of molarity 400 mM was used and pH was varied
from 4.0 to 6.0. The reaction was carried out at 15°C at
shaking condition and the percentage conversion and % ee
was recorded.
Therefore, in the present studies, an attempt has been
made to synthesize (R)-mandelonitrile using ParsHNL in
aqueous/organic biphasic system and study productivity in
batch and fed batch conditions with aim to enhance both
volumetric yield and enantiomeric excess of the product.
2.5 Effect of Temperature
Temperature plays an important role being a reversible cat-
alytic property of HNLs. Effect of temperature was studied
and the synthesis of mandelonitrile was carried out at dif-
ferent temperatures (10–20°C). For this purpose reaction
mixture (20 ml) was prepared with aqueous/organic phase
(MTBE) [1:3 ratio (v/v)] containing 1500 µmole of benza-
ldehyde dissolve in DMSO (0.5 ml), and HCN was used in
excess amount with citrate buffer of molarity 400 mM at
pH 4.0 was used.
2 Materials and Methods
2.1 Plant Material
Seeds of wild apricot (P. armeniaca) used as source of
HNL were obtained from the Department of Food Science
and Technology, Dr. Y S Parmar University of Horticulture
and Forestry, Solan, India.
1 3

Enantiopure Synthesis of (R)-Mandelonitrile Using Hydroxynitrile Lyase of Wild Apricot…
2.6 Effect of Time
2.9 Analysis of Mandelonitrile Produced
Effect of time on % molar conversion and %ee was studied.
The reaction was continued for 46 h and % molar conver-
sion and %ee was determined. For this purpose reaction
mixture (40 ml) was prepared with aqueous/organic phase
(MTBE) [1:3 ratio (v/v)] containing 3000 µmole of ben-
zaldehyde dissolve in DMSO (1 ml), and HCN was used
in excess amount with citrate buffer of molarity 400 mM
at pH 4.0 was used. The reaction was carried out at 15°C
at shaking condition, samples were withdrawn (3, 6, 24,
28 and 46 h) and the percentage conversion and % ee was
recorded.
The mandelonitrile synthesized in reactions was quantified
by HPLC using 65% acetonitrile in water as mobile phase
using Shimadzu Prominence System with auto sampler
equipped with C18 5 µm (4.6×250 mm) column and dual
wavelength detector with flow rate of 60 ml/h. Absorbance
was measured at 210 nm wavelength and retention time
(RT) of 4.0 and 5.0 min were observed for mandelonitrile
and benzaldehyde respectively.
2.10 Determination of Enantioselectivity
To determine the enantiomeric excess (%ee), chiral chro-
matography was performed using Chiralcel-OJH 5 µm
(4.6×250 mm) column (Daicel) and SPD-20A vp UV
detector, with mobile phase of n-hexane:isopropanol in
ratio of 80:20 respectively with flow rate of 60 ml/h. Sam-
ple prepared in isopropanol was injected using auto sam-
pler and the absorbance was recorded at 210 nm and data
2.7 Synthesis of Chiral Mandelonitrile in Batch
Reaction
Batch reaction was performed using 3000 µmole of benza-
ldehyde dissolve in DMSO (1 ml), and HCN was used in
excess amount to ensure the availability throughout the car-
boligation reaction. Reaction mixture (40 ml) was prepared
with aqueous/organic phase (MTBE) [1:3 ratios (v/v)] con-
taining 75 mg crude ParsHNL, citrate buffer of pH 4.0,
molarity 400 mM. The reaction was carried out at 15°C in
shaking condition at 160 rpm.
analyzed using Shimadzo LC solution software. The %ee
R−S
was calculated as
× 100, where R and S are the amount
R+S
of mandelonitrile formed as (R)-mandelonitrile and (S)-
mandelonitrile respectively. A blank reaction was also per-
formed without enzyme and amount of mandelonitrile
formed in blank was subtracted from enzyme catalyzed
reaction.
2.8 Fed Batch Reaction
A fed batch reaction was carried out starting with
3000 µmole of benzaldehyde dissolved in DMSO (1 ml).
Reaction mixture (40 ml) was prepared with aqueous/
organic phase (MTBE) [1:3 ratios (v/v)] containing
75 mg crude ParsHNL, citrate buffer of pH 4.0, molarity
400 mM. The reaction was carried out at 15°C in shak-
ing condition at 160 rpm. Feeding of substrates were done
at 3 h (1200 µmole) and 6 h (2800 µmole), hence total of
7000 µmole of substrate was used. Samples were with-
drawn and amount of product formed, substrate left and
%ee was determined at regular interval of time.
3 Results and Discussion
3.1 Effect of pH
The effect of pH on asymmetric synthesis of (R)-man-
delonitrile in the biphasic system (citrate buffer-MTBE) is
shown in Fig. 1. It was observed that at pH 4.0, 65% molar
conversion of substrate and ee (>99%) were obtained at the
end of 3 h reaction time. On increasing the pH above 4.0,
upto 6.0 caused increase in the molar conversion (85%) but
99
% molar conversion
Fig. 1 The effect of pH on
100
92
%ee
molar conversion (in percent)
85
and %ee value of (R)-man-
80
72
delonitrile synthesized by
65
ParsHNL in biphasic condition
57
60
40
20
0
4
5
6
pH
1 3

M. Asif, T. C. Bhalla
a drastic loss in the %ee of (R)-mandelonitrile (57%) was
observed.
avoided. The molar conversion and %ee were obtained as
60% and >99 respectively at 10°C, pH 4.0 at the end of
3 h. On increasing the temperature, the % molar conversion
and %ee values of (R)-mandelonitrile were 65 and >99% at
15°C. When the temperature was increased to 20°C, 64%
molar conversion was recorded whereas the %ee observed
for (R)-mandelonitrile was 95%. This observation can be
explained that the increase in temperature slightly accel-
erates the non-enzymatic carboligation, thus leading to
decrease in %ee of (R)-mandelonitrile and similar trends
have been reported earlier [14, 21].
This result indicated that the rate of non-enzymatic car-
boligation of cyanide molecule to benzaldehyde increased
at pH 5.0 and 6.0 and this decreased the %ee value of (R)-
mandelonitrile in the reaction. pH 4.0 has been earlier
reported to be the optimum pH for synthesis of (R)-man-
delonitrile catalysed by HNL from P. amygdalus turcoman-
ica [20], P. dulcis [15] and Passiflora edulis [14].
3.2 Effect of Temperature
The effect of the temperature on the enantioselectivity of
ParsHNL was examined at 10–20°C and the molar con-
version and %ee change of synthesized (R)-mandelonitrile
depending on temperature were shown in Fig. 2.
3.3 Time Course Reaction
As demonstrated in Fig. 3, the % molar conversion
increases with respect to reaction time while the %ee of
(R)-mandelonitrile was constant. The % molar conversion
of (R)-mandelonitrile increased from 64.9 to 90% when
the reaction time was increased from 3 to 46 h at pH 4.0
ParsHNL catalysed the reaction effectively in the tem-
perature range varying from 10 to 20°C. Increasing the
temperature above 20°C, favours the reverse reaction i.e.
conversion of mandelonitrile to benzaldehyde and hence
120
Fig. 2 The effect of tempera-
% molar conversion
% ee
ture on molar conversion (in
99
99
95
100
percent) and % ee value of (R)-
mandelonitrile synthesized by
80
ParsHNL in biphasic condition
65
64
60
60
40
20
0
10
15
20
Temperature (°C)
3500
100
80
60
40
20
0
3000
90
88
86.6
3000
Product
Formed
(µmole)
Substrate
le (µmole)
2700
2640
2500
2000
1500
1000
500
69.6
2598
64.9
2088
%
1948
1052
conversion
%ee
912
402
360
300
0
0
0
0
3
6
24
28
46
Time (h)
Fig. 3 The effect of time course on molar conversion (in percent) and % ee value of (R)-mandelonitrile, product formation and substrate left in
batch reaction synthesized by ParsHNL in biphasic condition
1 3

Enantiopure Synthesis of (R)-Mandelonitrile Using Hydroxynitrile Lyase of Wild Apricot…
6370
91
6067
86.7
5952
85
100
80
60
40
20
0
6000
4800
3600
2400
1200
0
4708
64.9
Substrate
Product
3000
54.6
2292
2252
1948
% conversion
1908
1052
1048
933
630
0
6
12
18
24
30
36
42
48
Time (h)
Fig. 4 Effect of time on product formation (filled triangle), substrate left (multiply symbol) and percentage conversion (filled circle) in fed-batch
reaction synthesized by ParsHNL in biphasic condition
and 15°C and no increament in % molar conversion was
observed on increasing the reaction time further.
of 1200 µmole substrate at this point, total 2292 µmole of
product was synthesized after 6 h which corresponded to
54.6% molar conversion. At 6 h finally 2800 µmole of sub-
strate was added and reaction was continued up to 46 h. At
24, 28 and 46 h, the % molar conversion observed was 85,
86.7 and 91 respectively. Hence a total of 91% molar con-
version was achieved in 46 h reaction which corresponded
to 6370 µmole of mandelonitrile produced (Fig. 4). The
%ee was >99% though out the reaction. As compared with
batch reaction, the fed batch reaction was observed to be
more efficient in terms of % molar conversion and the
amount of product formed.
The % molar conversion of (R)-mandelonitrile was
90% whereas its %ee value was still >99%. These results
showed that ParsHNL had a unique enantio-specificity and
the non-emzymatic spontaneous addition of HCN and ben-
zaldehyde has completely suppressed at pH 4.0 and 15°C
during long reaction time.
3.4 Synthesis of (R)-Mandelonitrile in Batch Reaction
A batch reaction was carried out using 3000 µmole of
benzaldehyde dissolved in DMSO, at 15°C, pH 4.0 and it
was observed that a total of 90% molar conversion of sub-
strate added was achieved in 46 h reaction and no further
increase in conversion was observed on extending the reac-
tion time. The %ee was >99% throughout the reaction. A
total of 2700 µmole of (R)-mandelonitrile was produced
which corresponds to 90% molar conversion of substrate
used (Fig. 3). In earlier studies Alagöz et al. have observed
91% yield with 93%ee for (R)-mandelonitrile after 3 days
of reaction with PatHNL [20]. Cabirol et al. have reported
98% yield with 97%ee for (R)-mandelonitrile with crude
PaHNL extract after 4 h of incubation in batch scale [22]
while Nanda et al. obtained 65% yield with 95% ee for
(R)-mandelonitrile using crude P. mume HNL after 24 h of
incubation in 50 ml batch reaction [23].
The productivity of mandelonitrile produced (µmol/h)
was calculated for both batch and fed batch reactions and
it was observed that fed batch reaction seemed better than
batch reaction (Fig. 5). At the end of 46 h the productivity
for (R)-mandelonitrile (µmol/h) was 2.4 times for fed-batch
reaction as compare with batch reaction. These results sug-
gest that the fed batch mode of reaction can be employed
for achieving higher volumetric yield and % enantio-
meric excess of (R)-mandelonitrile in a biphasic reaction
condition.
4 Conclusion
The potential of the ParsHNL for enantioselective synthe-
sis of (R)-mandelonitrile has been explored. The results
showed that the catalytic performance of ParsHNL was
significantly influenced by reaction conditions like pH
and temperature. The fed-batch reaction seemed more effi-
cient as compared to batch reaction in the term of percent-
age conversion as well as amount of product synthesized.
These results demonsitrate that ParsHNL is a powerful and
3.5 Fed Batch Reaction
A fed batch reaction was performed starting with
3000 µmole of benzaldehyde dissolved in DMSO,
pH 4.0, at 15°C. In 3 h 64.9% molar conversion was
observed yielding 1948 µmole of product. After feeding
1 3

M. Asif, T. C. Bhalla
Fig. 5 Comparison of produc-
tivity of (R)-mandelonitrile syn-
thesized in batch and fed-batch
conditions with time catalysed
by ParsHNL in biphasic condi-
tion
700
600
500
400
300
200
100
0
649.3649.3
Product Formed Fed
Batch
Product Formed Batch
382
348
248
216.7
138.5
58.7
108.3
94.3
3
6
24
Time (h)
28
46
8. Costes D, Wehtje E, Adlercreutz P (1999) Enzyme Microb Tech-
nol 25:384
cheap biocatalyst for the synthesis of (R)-mandelonitrile
in biphasic conditions. Moreover, the appropriate biphasic
system for synthesis of (R)-mandelonitrile indicated that
ParsHNL has high potential for enantiopure synthesis of
(R)-mandelonitrile.
9. Loos WT, Geluk HW, Ruijken MMA, Kruse CG, Brussee J, Van
der Gen A (1995) Biocatal Biotransform 12:255
10. Hickel A, Radke CJ, Blanch HW (2001) Biotechnol Bioeng
74:18
11. Persson M, Costes D, Wehtje E, Adlercreutz P (2002) Enzyme
Microb Technol 30:916
Acknowledgements Authors are highly grateful to University
Grants Commission (UGC), New Delhi, India for providing financial
assistance to Mr. Mohammad Asif.
12. Cascão-Pereira LG, Hickel A, Radke CJ, Blanch HW (2003)
Biotechnol Bioeng 83:498
13. Lin G, Han S, Li Z (1999) Tetrahedron 55:3531
14. Techawaree U, Tamura K, Ohmiya T, Kittikun AH, Asano Y
(2010) Enzyme Microb Technol 46:456
15. Alagöz D, Tükel SS, Yildirim D (2015) Appl Biochem Biotech-
nol 177:1348
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