Evaluation of guanabana (Annona muricata) seed meal as a source of (S)-oxynitrilase

Article abstract of DOI:10.1016/S0957-4166(03)00500-7

Guanabana seed meal is a source of (S)-oxynitrilase which biocatalyzes the enantioselective addition of HCN to aromatic, heteroaromatic and α,β-unsaturated aldehydes to produce cyanohydrins.

Full text of DOI:10.1016/S0957-4166(03)00500-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2351–2353
Evaluation of guanabana (Annona muricata) seed meal as a
source of (S)-oxynitrilase
Aida Sol´ıs,* He´ctor Luna, Herminia I. Pe´rez and Norberto Manjarrez
Departamento Sistemas Biolo´gicos, Universidad Auto´noma Metropolitana, Unidad Xochimilco, Calz. del Hueso No. 1100,
Col. Villa Quietud, Coyoaca´n, C.P. 04960, Mexico, D.F., Mexico
Received 31 March 2003; accepted 17 June 2003
Abstract—Guanabana seed meal is a source of (S)-oxynitrilase which biocatalyzes the enantioselective addition of HCN to
aromatic, heteroaromatic and a,b-unsaturated aldehydes to produce cyanohydrins.
© 2003 Elsevier Ltd. All rights reserved.
Nowadays the use of biocatalysts for the preparation of
optically active compounds is increasing constantly, due
to the advantages of biocatalysts over chemical chiral
catalysts. It is well documented that enzymes accept a
wide array of substrates, and catalyze enantio-, chemo-
and regioselective reactions.1,2 In the processes for the
formation of carbonꢀcarbon bonds, lyases are very
attractive options and among them are the (R)- and
(S)-oxynitrilases, that catalyze the addition of HCN to
aldehydes or ketones to give (R)- or (S)-cyanohydrins
respectively. Enzymatic procedures to obtain chiral
cyanohydrins are very important, because these sub-
stances are very valuable starting materials for the
synthesis of agrochemicals and drugs.3–6
worked at different pH values, the HCN generated in a
KCN/citric acid buffer (pH 5.5, 5.0, 4.5, 4.0, 3.6) was
extracted with isopropylether; second, the temperatures
of the reaction were 4 and 25°C, the reaction mixture
was stirred for 48 h, before the crude product was
analyzed.
From the results in Table 1 it can be concluded that
there is a relation between pH and the enantioselectiv-
We are currently engaged in the search for new and
accessible sources of oxynitrilases,7 and therefore we
tested the capability of guanabana (Annona muricata)
seed meal to biocatalyze the addition of HCN to 2-fur-
furaldehyde to produce the corresponding cyanohydrin.
Scheme 1.
Table 1. Enantiomeric excess conversion of the biocata-
lyzed addition of HCN to 2-furfuraldehyde
Entry
pH
T (°C)
Conversion (%)a
Ee (%)b
It is well known that the enzymatic preparation of
enantiopure cyanohydrins can be accompanied by the
nonenzymatic formation of the unwanted racemic
product. This disadvantage can be diminished using an
aqueous–organic biphasic system, reducing the temper-
ature and/or reducing the pH.8,9 Since we were dealing
with a new oxynitrilase source, we tested different
reaction conditions for the biocatalyzed addition of
HCN to 2-furfuraldehyde 1a (Scheme 1). First, we
1
2
3
4
5
6
7
8
9
3.6
3.6
4.0
4.0
4.5
4.5
5.0
5.0
5.5
5.5
25
4
25
4
25
4
25
4
87
95
87
90
87
91
84
82
93
97
73
87
66
84
65
81
62
69
49
51
25
4
10
a Determined by GC as the naproxenate derivatives.10
b Determined by HPLC using Chiracel OD column.
* Corresponding author. E-mail: asolis@cueyatl.uam.mx
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00500-7

2352
A. Sol´ıs et al. / Tetrahedron: Asymmetry 14 (2003) 2351–2353
Scheme 2.
ity of the biocatalyzed reaction; at lower pH (3.6, 4.0,
4.5, entries 2, 4, 6) the enantiomeric excess is higher
than 80%, whereas at pH 5.0 and 5.5 the enantiomeric
excess was drastically reduced to 69 and 51% respec-
tively (entries 8 and 10). The reaction temperature also
played an important role over the enantioselectivity
because at lower temperature the enantiomeric excess
was higher, for example at pH 4.0 and 4°C (entry 4) the
enantiomeric excess was 84%, in contrast to 66% at
25°C (entry 3).
From all these facts it can be stated that the defatted
meal of guanabana seeds is a new source of (S)-oxyni-
trilase, a class of enzymes, which are much less com-
mon than (R)-oxynitrilases. The advantage of this
source of (S)-oxynitrilase is that it is very cheap and
accessible since guanabana can be purchased from local
markets and the seeds are very abundant in the fruit. It
is worth mentioning that the common sources of (S)-
oxynitrilases, Hevea brasiliens12 and Manihot escu-
lenta,13 have been overexpressed in several
microorganisms in order to have sufficient amounts of
the enzyme for synthetic applications.
Very interesting was the fact that the optical rotation of
the cyanohydrin synthesized in this way was negative,
meaning that the cyanohydrin of 2-furfuraldehyde pre-
pared using defatted meal of guanabana has the (R)-
configuration (assigned according Cahn–Ingold–Prelog)
and the meal contains an (S)-oxynitrilase.11
1. Experimental
1H NMR spectra were recorded on a Varian of 400
MHz instrument in CDCl3 using tetramethylsilane as
an internal reference. Optical rotations were measured
using a Perkin Elmer 341 polarimeter and methylene
chloride as solvent. Analysis of naproxenates was
accomplished by gas chromatography on a Hewlett
Packard 6890 series instrument using a HP-5 capillary
column, and analysis of underivatized cyanohydrins by
HPLC on a Hewlett Packard 1050 instrument, employ-
ing a Chiracel OD column with n-hexane/isopropanol
mixtures.
Due to the fact that guanabana seeds are a new source
of (S)-oxynitrilase, we decided to explore the scope and
limitations of this biocatalytic material in the reaction
of HCN addition to aromatic, heteroaromatic, a,b-
unsaturated and aliphatic aldehydes (Scheme 2), at pH
4.0, 4°C.
From the results in Tables 1 and 2, it can be stated that
the defatted meal of guanabana seeds biocatalyzed the
addition of HCN to heteroaromatic 1a, aromatic 1b–d
and a,b-unsaturated 1e aldehydes, but did not work
with aliphatic aldehydes 1f and 1g.
The best conversion and enantiomeric excess were
obtained with the aldehyde 1a (Table 1). The reaction
with cinnamaldehyde 1e proceeded in high enan-
tiomeric excess (82%) but with low conversion; in the
case of the reaction with aromatic aldehydes 1b–d, the
enantiomeric excess and conversions are not very high.
Table 2. Cyanohydrins synthesized by the addition of
HCN to aldehydes catalyzed by guanabana seed meal
Product
Conversion (%)a
Ee (%)b
2b
2c
2d
2e
2f
10
24
20
11
nc
nc
50
46
58
82
–
It is possible that the reaction conditions are adequate
for the aldehyde 1a, but not for aromatic or a,b-unsat-
urated aldehydes. All the cyanohydrins prepared in this
way showed negative optical rotations, which corre-
sponds to (S)-cyanohydrins, except for 2a which
configuration is ‘R’. In the reaction mixtures, only the
corresponding cyanohydrin and the unreacted aldehyde
2g
–
a Determined by 1H NMR.
b Determined by HPLC using a Chiracel OD column.
nc, no conversion.
1
were detected by H NMR.

A. Sol´ıs et al. / Tetrahedron: Asymmetry 14 (2003) 2351–2353
2353
1.1. Guanabana seed meal
Ciencia y Tecnolog´ıa (CONACyT), Me´xico, Grant No.
37272-N.
Guanabana fruit was purchased in local markets, the
seeds from the fruit were peeled, then the meal was
prepared by grinding the seeds and defatting three
times with ethyl acetate, the meal was filtered, dried
and stored at 4°C.
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hyde (0.98 mmol) were added, the mixture was magnet-
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1
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We thank the financial support of Consejo Nacional de