Synthesis of (R)-cyanohydrins by crude (R)-oxynitrilase-catalyzed reactions in micro-aqueous medium

Article abstract of DOI:10.1016/S0957-4166(98)00177-3

In diisopropyl ether or ethyl acetate under micro-aqueous conditions, the enantioselective synthesis of (R)-cyanohydrins from aldehydes and methyl ketones was studied using crude (R)-oxynitrilase prepared from almonds. This reaction system performed well

Full text of DOI:10.1016/S0957-4166(98)00177-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1835–1838
Synthesis of (R)-cyanohydrins by crude (R)-oxynitrilase-
catalyzed reactions in micro-aqueous medium
Shiqing Han, Guoqiang Lin ∗ and Zuyi Li
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, PR China
Received 30 March 1998; accepted 30 April 1998
Abstract
In diisopropyl ether or ethyl acetate under micro-aqueous conditions, the enantioselective synthesis of (R)-
cyanohydrins from aldehydes and methyl ketones was studied using crude (R)-oxynitrilase prepared from almonds.
This reaction system performed well over the temperature range of 4°C to 30°C. © 1998 Elsevier Science Ltd. All
rights reserved.
Asymmetric cyanohydrination is an important process in organic synthesis because cyanohydrins can
be easily converted into a variety of valuable synthetic intermediates such as α-hydroxyketones, α-
hydroxyacids, β-aminoalcohols, amino-nitriles and aziridines. Due to the availability and the excellent
stereoselectivities, the biocatalytic formation of (R)-cyanohydrins using almond enzymes has been
thoroughly investigated and is considered to be a reliable method.1,2
The (R)-oxynitrilase [EC 4.1.2.10] catalytic reaction has been reported so far to be successful with
both crude enzyme preparations3–8 and purified enzyme.1,9,10 In the case of isolated oxynitrilase, it is
necessary to establish certain conditions (essential water, pH, temperature, solvent, etc.) to retain the
catalytic activity of the enzyme. In general, aqueous buffer media was chosen for its favorable reaction
conditions,3,11 however, due to a competing nonenzymatic reaction of the substrate with hydrogen
cyanide in aqueous media, it is most likely to lead to a decrease in the enantiomeric purity of the
products. Moreover, the enantiomeric purity of the enzymatic products could be further compromised
by their racemization in the aqueous buffer during the course of the reaction. Many substrates have been
investigated by Effenberger et al. in detail with the purified oxynitrilase both in water/ethanol and in
water/water-immiscible organic solvent systems.1,9,10 They found that higher yields and enantiomeric
excesses could be achieved in water/water-immiscible organic biphases. Recently it was reported that
2–10% (v/v) aqueous buffers were employed in the reaction with the crude enzyme.4–8
The crude enzyme is swollen with aqueous buffer to congeal to a pulp (System I in Scheme 1),
so efficient stirring is essential for ensuring the contact of enzymes with substrates. Nonetheless, the
∗
Corresponding author. Email: lingq@pub.sioc.ac.cn
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00177-3

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nonenzymatic addition still exists in these aqueous phases. Therefore, on most occasions in System I
it is necessary to carry out the enzymatic reaction in a lower range of pH values (3.5–5.5) and lower
temperatures (0–4°C) to supress the competitive chemical addition.4,6–8
Scheme 1. Comparison of a water–organic solvent biphase system with a micro-aqueous organic phase system
Herein, we wish to report our results where almond meal is used as an (R)-oxynitrilase source for the
enantioselective synthesis of (R)-cyanohydrins (Scheme 2) in a micro-aqueous organic phase (System II
in Scheme 1). In this case the organic phase serves as a big reservoir of substrates and products, where
the catalytic enzyme powder can mix well and act as a highly effective mode of transfer, therefore it is
easier for the substrates to interact with the enzymes to give the products. Furthermore, it is very easy to
recover the enzyme for re-use from System II.
Scheme 2.
The results of the enzymatic cyanohydrinations are presented in Table 1 (System I) and Table 2
(System II) for comparison. At a lower temperature (4°C), the synthesis of (R)-mandelonitrile 2a was
accomplished in quantitative yield and excellent enantiomeric purity (>99% ee) both in System I (Entry
1 and 2) and in System II (Entry 8). With an increase in temperature in System I, Table 1 shows that at
12°C the yields and enantiomeric excesses of 2a decreased to 92% and 97.5% respectively in IPE/buffer
(Entry 4), and at 30°C decreased to 65% and 92% in EA/buffer (Entry 5) as well as to 77% and 93.9%
in IPE/buffer (Entry 6), respectively. This means that a lower temperature was necessary to have better
results for the reaction in a water–organic biphase (System I). At higher temperatures (30°C) the rates of
chemical addition and decomposition were enhanced, resulting in a decrease of the chemical yields and
ees. While in System II (see Table 2) higher yields and enantioselectivities were achieved, ie, at 12°C
100% yield and >99% ee of 2a (Entry 9 and 10), at 30°C 98% yield and 98.2% ee in EA (Entry 11) as well
as 98% yield and 97% ee in IPE (Entry 12) of 2a, respectively. As shown in Fig. 1, the nonenzymatic
addition and decomposition that occurred in the aqueous phase (A) were suppressed deeply in micro-
aqueous media (B and C).
Furthermore, in System II (S)-(+)-2-(2-furyl)-2-hydroxyacetonitrile 2b was obtained not only with
100% yield and 99% ee at 4°C (Entry 13) but also with 90% yield and 95% ee at 30°C (Entry 14), while
in System I it was only obtained with 70% yield and 73% ee at 30°C (Entry 7). In contrast to the reported
result that (E)-cinnamaldehyde 1c was not accepted by the enzyme in aqueous medium,3 it could be

S. Han et al. / Tetrahedron: Asymmetry 9 (1998) 1835–1838
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Table 1
Investigations for the enantioselective syntheses of (R)-mandelonitrile in System I
Table 2
Investigations for the enantioselective syntheses of (R)-cyanohydrins in System II
converted into the corresponding (R)-cyanohydrin with moderate enantiomeric purity by using System
II (Entry 15, 16 and 17), the yield of 2c which was only 9.6% at 4°C (Entry 15) reached 58% at 30°C
(Entry 17). The reactions of methyl ketones under the conditions of System II gave the results of 78% ee
of 2d (Entry 18) and 98.6% ee of 2e (Entry 19).
In conclusion, (R)-cyanohydrins can be synthesized with high enantiomeric excess by the application
of almond meal to a micro-aqueous organic phase in the temperature range of 4°C to 30°C. The procedure
for System I: In a typical experiment, the enzyme preparation (500 mg)12 was swollen with 0.53 ml of
a 0.02 M citrate buffer pH 5.5 for l0 min and then mixed with freshly distilled aldehyde (2.5 mmol)
and 1.5 equiv. HCN in 10 ml diisopropyl ether.13 The resulting aqueous pulp of almond meal in organic
solvent was stirred at 30°C for the times indicated in Table 1. The procedure for System II: In a typical
experiment, the enzyme preparation (500 mg), freshly distilled aldehyde (2.5 mmol) and 1.5 equiv. HCN
in 10 ml diisopropyl ether (the solution was dried over Na2SO4 before use, water content 0.32% v/v)

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S. Han et al. / Tetrahedron: Asymmetry 9 (1998) 1835–1838
Fig. 1. Comparison of the rate of decomposition of (ꢀ)-mandelonitrile (420 µM) to that of benzaldehyde in pH=5.5 citrate
buffer (A) and in micro-aqueous medium (EA: B; IPE: C) at 25°C, by determining the intensity of the absorbance of PhCHO at
249.6 nm on a Beckman DU-70 spectrometer
were mixed. The fine enzyme powder mixed homogeneously in the organic solvent when the mixture
was stirred at 30°C for the times indicated in Table 2. Upon removal of the crude enzyme by filtration,
the filtrate was concentrated under reduced pressure, and the crude cyanohydrin was purified by column
chromatography on silica gel. The enantiomeric purity of the cyanohydrins were determined with the
chiral HPLC resolution on CHIRALPAK AD column as the corresponding O-protected derivatives.
Acknowledgements
The authors thank the National Natural Science Foundation of China for financial support and
professor Lijun Xia, Ms. Minhua Tang for performing the HPLC analysis.
References
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12. Almond was collected from mature garden fruits, after being granulated in a homogenizer, the preparation was defatted
four times by treatment with ethyl acetate, then sealed and stored in a refrigerator at 4°C (water content in the meal was
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13. Hydrogen cyanide in diisopropyl ether or ethyl acetate solution was prepared according to the literature4 method.