Study on the (R)-oxynitrilase activity of Pouteria sapota

Article abstract of DOI:10.1016/j.tet.2004.07.102

Mamey (Pouteria sapota) defatted meal was used to catalyze the enantioselective addition of HCN to α,β-unsaturated aldehydes. Using a biphasic system of diisopropyl ether and citrate buffer (0.1 M, pH 5.0, 10% v/v), the (R)-cyanohydrins obtained showed go

Full text of DOI:10.1016/j.tet.2004.07.102

Tetrahedron 60 (2004) 10427–10431
Study on the (R)-oxynitrilase activity of Pouteria sapota
*
Aida Solıs, Hector Luna, Norberto Manjarrez and Herminia I. Perez
´
´
´
´ ´
Departamento Sistemas Biologicos, Universidad Autonoma Metropolitana, Unidad Xochimilco. Calz. del Hueso No. 1100,
´
Col Villa Quietud, Coyoacan, C.P. 04960 Mexico, D.F., Mexico
Received 21 May 2004; revised 12 July 2004; accepted 28 July 2004
Available online 28 August 2004
Abstract—Mamey (Pouteria sapota) defatted meal was used to catalyze the enantioselective addition of HCN to a,b-unsaturated aldehydes.
Using a biphasic system of diisopropyl ether and citrate buffer (0.1 M, pH 5.0, 10% v/v), the (R)-cyanohydrins obtained showed good
conversion (from 54 to 98%) and enantiomeric excess (from 74 to 99%).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
heteroaromatic, cinnamaldehyde and aliphatic aldehydes,
and except with cinnamaldehyde, the enantiomeric excesses
were good (77–98%) (Fig. 1).
Research on enzymatic processes to prepare optically active
compounds has been growing constantly in recent years, due
to the advantages enzymes have over metallic chiral
catalysts, mainly in the preparation of biologically active
compounds. Oxynitrilases or hydroxynitrile lyases are
enzymes that biocatalyze the enantioselective addition of
HCN to aldehydes or ketones to yield cyanohydrins. These
compounds are versatile building blocks that have been used
in the preparation of pharmaceuticals and agrochemicals.
Oxynitrilases have attracted the attention of many
researchers, in such way that actually there are very
interesting findings in this field.1–6
Hydroxynitrile lyases have been found in a wide variety of
plant sources, but the selectivity towards the biocatalyzed
addition of HCN to carbonyl compounds is not the same,
some accept better aromatic rather than aliphatic aldehydes,
some biocatalyze the addition of HCN to aldehydes and
ketones, others only to aldehydes. Due to this, it can be said
that the different sources of oxynitrilases are comple-
mentary.1–5,7,8 So, the study of alternative sources of
oxynitrilases becomes important.
Figure 1.
Due to the promising results with mamey, we extended our
study to find the reaction conditions to get the best
biocatalytic activity of the defatted meal of mamey seeds,
for the addition of HCN to a,b-unsaturated aldehydes. We
chose those aldehydes to prepare cyanohydrins which can
be used in the preparation of several interesting compounds9
such as insecticides.10
In previous work we found that seeds of Pouteria sapota
(mamey) are a source of (R)-oxynitrilase.7 We found that in
aqueous medium the defatted meal of mamey biocatalizes
the addition of HCN to aromatic aldehydes with conversions
from 24 to 73% and enantiomeric excesses from 34 to 77%.
We extended the study to determine the selectivity towards
different aldehydes in organic medium. We tested aromatic,
2. Results and discussion
It is well known that oxynitrilases need some water to
biocatalyze addition of HCN to aldehydes, but the
enantioselectivity of the reaction can be decreased by the
chemical reaction that is favored by the aqueous medium.
This last reaction can be diminished by lowering the pH and
Keywords: Oxynitrilases; Cyanohydrins; Mamey.
* Corresponding author. Tel.: C52-55-5483-7255; fax: C52-55-5483-
7237; e-mail: asolis@correo.xoc.uam.mx
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.07.102

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A. Solıs et al. / Tetrahedron 60 (2004) 10427–10431
10428
temperature of the reaction medium or using proper organic
solvents. The aim of this work is to improve the addition of
HCN to aldehydes biocatalyzed by mamey meal, the study
is divided into several parts to determine the solvent, the
acid used in the preparation of the HCN source, concen-
tration of buffer solution for the biphasic system, and the
water content of the biphasic system. The reactions for this
study were carried out with benzaldehyde and cinnamalde-
hyde, after analysis of the results the best reaction
conditions were applied to other a,b-unsaturated aldehydes.
the effect of this unwanted addition, a reaction under the
same conditions but without the meal was carried out, and
after 24 h no cyanohydrin was detected. We can conclude
that the enantiomeric excess of cyanohydrin 2a is not so
high because mamey meal is not very selective towards
aldehyde 1a, under the conditions of reaction tested.
2.2. Effect of the acid used to prepare the KCN/acid
buffer
Another important condition in the addition of HCN to
aldehydes is the acid used in the preparation of the buffer
solution of KCN/acid (pH 5.0, 1 M), which is the source of
HCN.13 Acetic, citric, phosphoric, lactic and tartaric acids
were chosen to prepare KCN/acid buffer, the reaction of
addition of HCN was performed with benzaldehyde (1b)
using diisopropyl ether as solvent in microaqueous
medium12,14 and after 24 h of reaction at 4 8C the reaction
was analyzed. Although the enantiomeric excess of the
obtained mandelonitrile (2b) was O99%, with all the acids,
the conversion was not the same. From the results in
Figure 3 it can be stated that conversion was the highest for
citric and phosphoric (93%), than with the other acids. One
disadvantage for the use of phosphoric acid is that the
product becomes dark and it is difficult to purify.
2.1. Effect of solvent
It is reported that the biocatalytic activity of oxynitrilases is
better in biphasic systems, and that organic solvent has an
important effect on the yield and enantioselectivity.2,11,12 To
determine the most convenient solvent for the reaction of
addition of HCN to cinnamaldehyde (1a) to get the
corresponding cyanohydrin (2a) catalyzed by mamey
meal, different solvents were tested to extract the HCN
from a buffer solution (KCN/citric acid, pH 5.0, 1 M) and
use it as reaction solvent, then the reaction was carried out at
4 8C for 24 h, the results are in Figure 2.
Figure 2. Conversion (determined by 1H NMR) and enantiomeric excess
(determined by HPLC using an OD Chiracel column) of cyanohydrin 2a, in
different solvents. A: diethyether, B: diisopropyl ether, C: methyl tert-butyl
ether (MTBE), D: ethyl acetate, E: hexanes, F: heptane, G: octane, H:
toluene.
Figure 3. Effect of the acid used to prepare KCN/acid, in the conversion
(determined by 1H NMR) of benzaldehyde (1b) to mandelonitrile (2b).
Since cinnamaldehyde seems to be the less accepted
substrate for mamey oxynitrilase, we decided to do an
experiment similar to the one described above, to determine
the effect of the acid on the reactivity and enantioselectivity
with this aldehyde. Acetic, citric, ascorbic and lactic acids
were selected, the mixture was analyzed after 24 h of
reaction at 4 8C and the results are shown in Figure 3. The
enantiomeric excesses are similar in all the cases (approxi-
mately 50%), but the conversion is very low with acetic acid
(8%) and practically the same with citric, ascorbic and lactic
acids (approximately 25%). Due to the results obtained, we
decided to continue our studies with mamey meal using
diisopropyl ether and MTBE as solvents, and citric acid for
the KCN/acid buffer solution.
From the results in Figure 2, it can be observed that the
addition of HCN to cinnamaldehyde catalyzed by mamey
defatted meal is not favorable in hydrocarbons such as
hexanes (E), heptane (F), octane (G) and toluene (H), since
the conversion was less than 10%, and the enantiopurity was
also low. When ethyl acetate was used in this reaction the
enantioselectivity was the highest (72%), but as with
hydrocarbon solvents the conversion was also very low
(10%).
By using diethyl ether (A) as solvent, the enantiomeric
excess was also high (70%) and the conversion was a little
higher (17%) than with the previously mentioned solvents.
The best reactivities were obtained with diisopropyl ether
(B) and MTBE (C), the conversions were 26 and 25%
respectively, but with MTBE the enantiomeric excess was
superior (70%).
2.3. Effect of the concentration of the citrates buffer
solution
A decrease in enantiomeric excess can be due to the
formation of the racemic cyanohydrin because of the non-
biocatalized addition of HCN to the aldehyde. To determine
We evaluated the effect of the concentration of the citrate
buffer solution,13 on the preparation of cyanohydrin 2a
catalyzed by the defatted meal of mamey. The citrate buffer

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A. Solıs et al. / Tetrahedron 60 (2004) 10427–10431
10429
solution in concentrations of 0.02, 0.05, 0.1, 0.15 and 0.5 M
(pH 5.0) was added in a ratio of 1% (v/v) to the solution of
diisopropyl ether and HCN, then the meal and the aldehyde
1a were added, after 6 and 12 h at 4 8C the reaction mixture
was analyzed.
phenylpropanenitrile,17 and Lin reported that higher yields
and enantioselectivity in the preparation of several cyano-
hydrins are obtained in microaqueous medium (0.32%, v/v
of water content).12,14 All these studies were done using
almond meal.
The enantiomeric excess of the product obtained in all the
cases was very high (99%), so the enantioselectivity of the
reaction was not affected by the concentration of the buffer
solution, but the degree of conversion showed a dependence
of the concentration. From Figure 4 it is clear that there is an
increase in the conversion as the solution becomes more
concentrated until a maximum of activity of the mamey
meal is reached, at a concentration of 0.1 M of the buffer
solution, then the reactivity is decreased with more
concentrated solutions (Fig. 5).
Since there are no data about the effect of water content on
the oxynitrilase activity of mamey meal, we carried out the
following experiment. To a solution of HCN in diisopropyl
ether or MTBE were added different quantities of citrate
buffer solution, then the mamey meal, followed by
cinnamaldehyde (1a), the reaction was monitored at 24,
48, 72, and 96 h, at 4 8C.
From the results in Figure 6 it can be stated that the
conversion is higher when the reaction is carried out in
diisopropyl ether than in MTBE. It is also observed that the
conversion increases proportionally with the increase in
water content, in such way that after 48 h the maximum
conversion (54%) is obtained with 90% of aqueous phase in
diisopropyl ether (f), but it is noticeable that after that time
the cyanohydrin began to transform into the aldehyde.
Waiting longer than 48 h for a higher conversion degree will
decrease cyanohydrin concentration, due to the reversibility
of the reaction.
Figure 4. Effect of the acid used to prepare KCN/acid, in the conversion
1
(determined by H NMR) and enantiomeric excess (determined by HPLC
using an OD Chiracel column) of aldehyde (1a) to cyanohydrin (2a).
Figure 6. Effect of water content on conversion (determined by 1 H NMR)
% of aldehyde (1a) to cyanohydrin (2a) catalyzed by mamey meal. Water
content in diisopropyl ether, a: microaqueous medium, b: 1%, c: 5%, d:
10%, e: 50%, f: 90%. Water content in methyl tert-butyl ether, MTBa:
microaqueous medium, MTBb: 1%, MTBc: 5%.
In the rest of the reactions, an important increase in
conversion from the beginning of the reaction until
approximately 48 h is observed, but from 72 to 96 h the
change in the degree of conversion is minimal, and in some
cases there is no change at all (a and b). It is possible that
around this time the biocatalyzed reaction has reached an
equilibrium. From Figure 7 some interesting facts can be
observed: with the highest water content (f, 90% of water in
diisopropyl ether) and the lowest (a, microaqueous medium
in diisopropyl ether and MTBa, microaqueous medium in
methyl tert-butyl ether), the enantiomeric excess have the
lowest values. In Figure 7 it is noticeable that in almost all
the cases the enantiomeric excess reaches a maximum at
48 h and after that time the enantiomeric excess begins to
decrease. This can be explained by an important compe-
tition between the biocatalyzed and chemical addition of
HCN to the aldehyde. In addition, the cyanohydrin began to
decompose and racemize, this behavior is similar to that
observed for conversion (Fig. 6).
Figure 5. Effect of buffer concentration (M) on the conversion (determined
by 1H NMR) of benzaldehyde (1b) to mandelonitrile (2b), biocatalyzed by
mamey meal.
2.4. Effect of the water content of the biphasic system
It is well known that some quantity of water is necessary for
the optimum activity of oxynitrilases, but there is not an
agreement on the minimum and maximum water content
needed for these enzymes to work. It is also known that at
high water content the undesirable chemical addition of
HCN to aldehydes is a favored reaction, resulting in a lower
enantiomeric excess. Some examples are the following:
Straathof15 found that a 50% aqueous phase is the optimum
for (R)-mandelonitrile production; the same author deter-
mined that a 15% aqueous phase is the optimum for (R)-4-
hydroxymandelonitrile production,16 Kanerva determined
13.5% as optimum for the resolution of 2-hydroxy-2-

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A. Solıs et al. / Tetrahedron 60 (2004) 10427–10431
10430
From the results in Table 1, it can be observed that the
reactivity and enantioselectivity of the addition of HCN to
aldehydes 1c, 1d, 1e catalyzed by mamey meal, increases
constantly with the increase in water content in the
reaction medium. These facts are in agreement with that
shown in Figures 6 and 7. The best results were obtained
with 50% water content in the reaction, however, the
disadvantage of employing 50% of water is that the
recovery of the product becomes difficult, so it is better to
use 10% of water since the results obtained were similar to
those with 50%.
Although all the aldehydes investigated were a,b-unsatu-
rated (1a, 1c, 1d, 1e), except for benzaldehyde (1b), the
reactivity towards addition of HCN catalyzed by mamey
was very different. With cinnamaldehyde (1a), the maxi-
mum conversion and enantioselectivity were not so high, 54
and 75%, respectively (Figs. 6 and 7). Something similar
was observed with 1,4-hexadienal (1e), the highest
conversion was 69% (Table 1) and enantioselectivity 79%
(Table 1).
Figure 7. Effect of water content on enantiomeric excess % (determined by
HPLC using an OD Chiracel column) of cyanohydrin (2a) catalyzed by
mamey meal. Water content in diisopropyl ether, a: microaqueous medium,
b: 1%, c: 5%, d: 10%, e: 50%, f: 90%. Water content in methyl tert-butyl
ether, MTBa: microaqueous medium, MTBb: 1%, MTBc: 5%.
The highest enantiopurity is reached after 48 h with MTBE
(water content of 5 and 10%, v/v) and diisopropyl ether at
water content of 5 and 10%. In the case of diisopropyl ether
it is very interesting that with 1, 5, 10 and 50% of water
content, enantiomeric excess are very similar at 48 h, but
after that time the decrease is much more significant with 10
and 50% of water. This could be because of the competition
between biocatalyzed and chemical reaction, which results
in racemization of the reaction.
2-Methyl-2-pentenal (1c) and trans-2-hexenal (1d) were
found to be more reactive under the reaction conditions
tested and had better enantiopurities, for 2c 84 and 90%,
respectively (Table 1), for 2d 88 and 98%, respectively
(Table 1). For some reason it seems that substrates having
more extended conjugated systems become less reactive to
the addition of HCN catalyzed by mamey meal, this can
explain the less favorable results with substrates 1a and 1e.
It is worth mentioning that 1,4-addition was not observed in
these experiments.
From these results the best conditions for the addition of
HCN to cinnamaldehyde biocatalyzed by mamey meal
were: the use of diisopropyl ether in a biphasic system, with
a water content of 5%, with reaction times not longer than
48 h, at 4 8C.
2.5. Preparation of cyanohydrins from a,b-unsaturated
aldehydes
3. Conclusions
From the results obtained we determined that mamey meal
is an (R)-oxynitrilase source which displays its best
biocatalytic activity in the addition of HCN to aldehydes
in the following reaction conditions, diisopropyl ether as
the solvent, citric acid for the preparation of KCN/acid
buffer as HCN source, and a water content of 10% in a
biphasic system. Benzaldehyde (1b), 2-methyl-2-pentenal
(1c) and trans-2-hexenal (1d) proved to be more reactive
under the mentioned reaction conditions, whereas cinna-
maldehyde (1a) and 1,4-hexadienal (1e) showed less
reactivity.
Due to their synthetic utility and because they are precursors
of insecticides, we explored the effect of water content on
the addition of HCN to a,b-unsaturated aldehydes (1c, 1d,
1e), catalyzed by mamey meal. According to results from
previous experiments, the solvent selected was diisopropyl
ether, KCN/citric acid buffer (pH 5.0, 1 N) as HCN source,
for the aqueous phase citrate buffer (pH 5.0, 0.1 M), the
amounts of the last one were: microaqueous medium
(approximately 0.32%), 1, 5, 10 and 50%, v/v. After 48 h
at 4 8C the reaction mixture was analyzed, the results are
shown in Table 1.
Table 1. Effect of water content on conversion and enantiomeric excess of cyanohydrins 2c, 2d and 2e of the reaction catalyzed by mamey meal
Cyanohydrin 2c
Conv. %a
Cyanohydrin 2d
Conv. %a
Cyanohydrin 2e
Conv. %a
% of water content
(v/v)
ee %b
70
ee %b
87
ee %b
52
Microaqueous
medium
68
37
27
1
5
10
50
74
77
84
83
77
88
89
90
56
67
62
88
94
98
92
95
51
50
63
69
79
72
70
75
a
1
Determined by H NMR.
Determined by HPLC using an OD Chiracel column.
b

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10431
4. Experimental
2.08 (m, 2H), 3.72 (br, 1H), 4.94 (d, JZ6.4 Hz, 1H), 5.59
(dd, JZ15.5, 6.4 Hz, 1H), 6.03 (m, 1H).
4.1. Chemicals and sources of enzymes
4.2.5. (R)-(K)-2-Hydroxy-3E,5E-heptadienenitrile (2e).
Conversion 79%, [a]2D2ZK52 (c 3.85, CH2Cl2) ee 69%;
lit.:18 [a]2D3ZK24.9 (c 1, CHCl3), ee 96%. 1H NMR
(CDCl3): dZ1.8 (d, JZ6.45 Hz, 3H), 3.87 (br, 1H), 4.99 (d,
JZ6.0 Hz, 1H), 5.62 (dd, JZ15.2, 6.0 Hz, 1H), 5.91 (m,
1H), 6.06 (dd, JZ15.0, 10.4 Hz, 1H), 6.46 (dd, JZ15.0,
10.4 Hz, 1H).
Ripe mamey (Pouteria sapota) seeds were obtained from a
fresh fruit purchased in a local grocery store. The mamey
seeds were ground and washed three times with acetone and
once with diisopropyl ether, after filtration by suction the
powder was air dried and stored at 4 8C. Commercial
aldehydes were purchased from Aldrich.
Optical rotations were measured in a Perkin–Elmer
polarimeter model 341. Enantiomeric excesses were
determined by HPLC analyses, using a Chiracel OD column
using hexanes–isopropanol as eluent in a Hewlett–Packard
1050 series, equipped with a diode array detector. Conver-
sion percentages were determined by NMR. 1H NMR
spectra were recorded on a Varian at 400 MHz using CDCl3
as a solvent and TMS as internal reference.
Acknowledgements
We thank the financial support of Consejo Nacional de
´ ´
Ciencia y Tecnologıa (CONACyT), Mexico, Grant Num.
37272-N.
4.2. General procedure for enzymatic reactions
References and notes
HCN (1.5 equiv) was extracted from a buffer solution
(KCN/citric acid, 1 M, pH 5.0) with the proper solvent (2!
2.5 mL). To this solution was added the citrate buffer
solution (0.1 M, pH 5.0); the quantity added to the reaction
medium depended on the experiment, then 200 mg of
mamey defatted meal were added and stirred for 10 min at
room temperature, after this 1 mmol of the aldehyde was
added. The reaction mixture was stirred for the necessary
time at 4 8C, filtered, dried over sodium sulfate and the
solvent was evaporated to dryness. Enantiomeric excess was
determined by HPLC using a Chiracel OD column with
hexanes–isopropyl alcohol as eluent and conversion per-
centage was determined by 1H NMR, of the crude product.
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4.2.1. (R)-(C)-2-Hydroxy-4-phenyl-3E-butenenitrile
(2a). Conversion 54%, [a]2D2ZC17 (c 1, CH2Cl2), ee
1
72%; lit.:12 [a]D13ZC26.1 (c 0.78, CHCl3), ee 69.3%. H
´
´
NMR (CDCl3): dZ5.18 (d, JZ6.0 Hz, 1H), 6.26 (dd, JZ
16.0, 6.0 Hz, 1H), 6.9 (d, JZ6.0 Hz, 1H), 7.35 (m, 5H).
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version 95%, [a]D22ZC46 (c 1.9, CH2Cl2); lit.:12 [a]D20Z
C47.5 (c 1.89, CHCl3), ee O99%. H NMR (CDCl3):
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1
dZ5.52 (s, 1H), 7.49 (m, 5H).
4.2.3. (R)-(K)-2-Hydroxy-3-methyl-3E-hexanenitrile
(2c). Conversion 84%, [a]2D2ZK72 (c 3.9, CH2Cl2), ee
89%. 1H NMR (CDCl3): dZ1.0 (t, JZ7.6 Hz, 3H), 1.78 (s,
3H), 2.09 (m, 2H), 3.42 (br, 1H), 4.83 (s, 1H), 5.72 (t, JZ
7.6 Hz, 1H). Configuration was assigned on the basis of
mamey meal selectivity, and of elution order of the
enantiomers on chiracel OD column using hexanes–
ispropanol (95:5) as eluent, (R)-enantiomer trZ11.37 min,
(S)-enantiomer trZ11.036 min.
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¨
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4.2.4. (R)-(K)-2-Hydroxy-3E-heptanenitrile (2d). Con-
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1
[a]2D0ZC20.3 (c 0.3, CHCl3), (S)-enantiomer, ee 95%. H
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8649–8698.
NMR (CDCl3): dZ0.92 (t, JZ7.6 Hz, 3H), 1.43 (m, 2H),