Biotransformations of 2,3-epoxy-3-arylpropanenitriles by Debaryomyces hansenii and Mortierella isabellina cells

Article abstract of DOI:10.1016/j.tetasy.2008.06.004

Biotransformations of five substituted cis- and trans-oxiranecarbonitriles with Mortierella isabellina DSM 1414, a microbial whole-cell catalyst producing epoxide hydrolases, were investigated. The reactions were stopped when the conversion of the substrates reached 50%. They yielded the appropriate optically active dihydroxycarbonitriles and oxiranecarbonitriles in low enantiomeric purity. Kinetic resolution of rac-syn-2,3-dihydroxy-3-arylpropanenitriles by lipase catalyzed acetylation yielded almost enantiomerically pure (-)-dihydroxynitriles and mixtures of regioisomers of monoacetylated diols. Another microorganism, Debaryomyces hansenii DSM 3428, was used as a source of nitrile hydratases in the kinetic resolution of oxiranecarbonitriles. Only two trans-configured compounds were transformed into the corresponding oxiranecarboxamides.

Full text of DOI:10.1016/j.tetasy.2008.06.004

Tetrahedron: Asymmetry 19 (2008) 1455–1460
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Tetrahedron: Asymmetry
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Biotransformations of 2,3-epoxy-3-arylpropanenitriles by
Debaryomyces hansenii and Mortierella isabellina cells
*
Malgorzata Zagozda, Jan Plenkiewicz
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2008
Accepted 3 June 2008
Biotransformations of five substituted cis- and trans-oxiranecarbonitriles with Mortierella isabellina DSM
1414, a microbial whole-cell catalyst producing epoxide hydrolases, were investigated. The reactions
were stopped when the conversion of the substrates reached 50%. They yielded the appropriate optically
active dihydroxycarbonitriles and oxiranecarbonitriles in low enantiomeric purity. Kinetic resolution of
rac-syn-2,3-dihydroxy-3-arylpropanenitriles by lipase catalyzed acetylation yielded almost enantiomeri-
cally pure (ꢀ)-dihydroxynitriles and mixtures of regioisomers of monoacetylated diols. Another micro-
organism, Debaryomyces hansenii DSM 3428, was used as a source of nitrile hydratases in the kinetic
resolution of oxiranecarbonitriles. Only two trans-configured compounds were transformed into the
corresponding oxiranecarboxamides.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
A highly efficient and enantioselective hydrolysis of the cyano
group in 3-aryloxiranecarbonitrile and its 2-methyl- and 2,3-di-
The enzyme-catalyzed enantioselective ring opening or func-
tional group transformation of electrophilic oxiranes is still an
important and attractive reaction. In the case of phenylglycidonit-
rile derivatives, hydrolysis of the cyano group may give, depending
on the enzymes used to carry out the reaction, the corresponding
optically active oxiranecarboxamides or oxiranecarboxylic acids.
On the other hand, the oxirane ring may be hydrolyzed by epoxide
hydrolases to yield the appropriate diols. Optically active products
of such biotransformations are important substrates in the prepa-
ration of several biologically active compounds. For example, oxi-
ranecarboxamides and oxiranecarboxylic acid esters prepared
from the corresponding oxiranenitriles are known as useful build-
ing blocks in the synthesis of (+)-clausenamide,1 (+)-neoclausena-
mide,2 (ꢀ)-dehydroclausenamide,3 that is, compounds used in the
synthesis of TaxolÒ.4 The products of these biotransformations are
also important and versatile intermediates in the synthesis of sev-
eral pharmaceuticals such as the selective leukotriene antagonist
SK&F 1043535 or diltiazem,4b,6 a potent coronary vasodilating
agent. It is worth mentioning that some biologically active N-mon-
osubstituted and N,N-disubstituted phenylglycidamide derivatives
have been isolated7 from the Clausena indica and Clausena lansium
plants whose leaves and fruits are used in traditional medicine to
treat coughs, asthma, viral hepatitis as well as some dermatologi-
cal and gastrointestinal disorders.
methyl-derivatives have recently been described by Wang
et al.8,9 As the enzyme source, they employed Rhodococcus sp. AJ
270 cells producing nitrile hydratases and amidases. The biotrans-
formations of racemic trans-2,3-epoxy-3-arylpropanenitriles
afforded (2R,3S)-2-arylglycidamides in excellent yield and enantio-
meric purity as well as the unstable (2S,3R)-2-arylglycidacides
which were not isolated. In contrast to the racemic trans-2,3-
epoxy-3-arylpropanenitriles, the biotransformations of the race-
mic cis-counterparts proceeded with great difficulty. This indicates
that the steric factors control the reaction of nitrile hydratase with
nitrile substrates. Since the biotransformations of the racemic cis-
2,3-epoxy-3-arylpropanenitriles with the same microorganism
stopped at the stage of the corresponding racemic cis-amides, the
amidases produced by Rhodococcus sp. AJ270 were inactive against
the investigated cis-epoxyamides.
The cited authors8,9 confirmed the earlier results10 by conclud-
ing that the overall high enantioselectivity of the biotransforma-
tions arises from the combined effects of the highly (2S)-
enantioselective amidase (predominant effect) and the poorly
(2S) enantioselective nitrile hydratase involved in the reaction.
2. Results and discussion
Herein, we report the enzymatic hydrolysis of the oxiranecarbo-
nitrile derivatives catalyzed by Debaryomyces hansenii DSM 3428
and Mortierella isabellina DSM 1414 cells. Assuming that the fungi
produce nitrilases and/or nitrile hydratases we expected them to
be able to transform 2,3-epoxy-3-arylpropanecarbonitrile into
* Corresponding author. Tel.: +48 22 234 75 70; fax: +48 22 628 27 41.
E-mail address: plenki@ch.pw.edu.pl (J. Plenkiewicz).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.06.004

1456
M. Zagozda, J. Plenkiewicz / Tetrahedron: Asymmetry 19 (2008) 1455–1460
O
O
O
CN
Whole cells
CN
CONH2
+
D. hansenii DSM 3428
R
R
R
trans-1a-e
2a,d
3a,d
a: R= H, b: R= m-CH3, c: R= p-CH3
d: R= p-Cl, e: R= p-Br
Scheme 1.
the corresponding optically active 3-arylglycidamides or the
appropriate acids. Moreover, if the investigated microorganisms
proved to be able to produce epoxide hydrolases, it would also
be possible to convert the oxiranecarbonitrile substrates into the
appropriate optically active 2,3-dihydroxy-3-arylpropanenitriles
or 2,3-dihydroxy-3-arylpropaneamides.
The reactions were carried out with whole cells of the microor-
ganisms harvested on the third day of cultivation. To the wet cells
suspended in a 2% glucose solution in distilled water the oxirane-
carbonitrile substrate was added as a solution in a small volume
of ethanol. The mixtures were incubated at 30 °C until the sub-
strate conversion was approximately 50% as estimated by TLC
monitoring in a hexane/ethyl acetate mixture.
In the experiments with D. hansenii DSM 3428 cells, only the
trans-isomers of two of the five investigated 2,3-epoxy-3-arylpro-
panecarbonitriles, namely, 1a and 1d, were accepted by the nitrile
hydratases produced by this microorganism (see Scheme 1).
As the products of kinetic resolution of racemates of trans-1a
and 1d, we obtained (2R,3S)-3-phenylglycidamide 3a, or (2R,3S)
3-(p-chlorophenyl)glycidamide 3d and the unreacted (2R,3R) 2,3-
epoxypropanecarbonitriles 2a and 2d in good yields, but of low
enantiomeric purity. The results of the biotransformation are pre-
sented in Table 1.
unchanged, even after extending the reaction time up to seven
days.
Using the same substrates 1a–e and the standard biotransfor-
mation procedure, we investigated M. isabellina DSM 1414 cells
as the catalyst. Unlike D. hansenii DSM 3428, that strain was found
to produce epoxide hydrolases able to open the oxirane ring with
no effect on the cyano substituent. With racemic trans-2,3-
epoxy-3-arylpropanecarbonitriles, a 50% conversion was achieved
in the smoothly proceeding reactions. The products contained the
unchanged (2R,3R)-oxiranecarbonitrile and the corresponding
syn-2,3-dihydroxy-3-arylpropanenitrile formed by hydrolysis of
the oxirane ring (see Scheme 2).
Unfortunately, only the reactions with three of the five investi-
gated substrates 1c–e proceeded, yielding optically active products
although with poor stereoselectivity. The phenyl and m-methyl-
phenyl derivatives 1a and 1b yielded racemic 2,3-dihydroxy-3-
arylpropanenitriles 4a and 4b and racemic oxiranecarbonitriles
2a and 2b. These results are similar to those reported by Wang9
for the Rhodococcus sp. AJ270-catalyzed hydrolysis of the cyano
group in cis-2-methyl-3-phenyloxiranecarbonitrile. In this reac-
tion, carried out under the kinetic resolution conditions, both the
product and substrate were racemates. The results obtained are
summarized in Table 2.
Attempts at biotransformation of the cis-isomers of 1a–e by D.
hansenii DSM 3428 cells failed and the substrates were isolated
Since it was not known which position of the oxirane ring
was the target of the attack, we could not forecast the absolute
Table 1
Results of 2,3-epoxy-3-arylpropanenitrile hydrolysis by Debaryomyces hansenii DSM 3428
Substrate 1
Time (h)
Ca (%)
trans-2
3
E
Yb (%)
eec (%)
½
a 2D5
ꢁ
Yb (%)
eec (%)
Mp (°C)
149–151d
187–188e
½ ꢁ
a 2D5
a
d
3
6
45
43
43
46
29
19
+24.3
+16.0
40
36
36
25
ꢀ33.2d
2.8
2.0
ꢀ11.8e
a
Conversions were calculated from the enantiomeric excesses of substrates trans-2 (ees) and products 3 using the formula: c = ees/(ees + eep).
Yield after purification on a chromatography column.
b
c
Enantiomeric excess determined by HPLC.
Lit.8 mp = 157–158 °C, ½a 2D5
¼ ꢀ160:0.
ꢁ
d
e
Lit.8 mp = 190–191 °C.
Table 2
Results of trans-1a-e hydrolysis by Mortierella isabellina DSM 1414 cells
Substrate trans-1
Time (h)
C (%)
2
4
E
Yc (%)
eed (%)
½
a 2D4
ꢁ
Yc (%)
eed (%)
Mp (°C)
½ ꢁ
a 2D4
a
b
c
d
e
5.5
4
5
24
96
ꢂ50a
ꢂ50a
44b
49
34
38
46
25
rac
rac
13
44
18
—
—
+15.8
+25.4
+14.5
32
41
43
34
19
rac
rac
17
55
34
Oil
Oil
Oil
99–101
92–94
—
—
ꢀ4.6
ꢀ11.4
ꢀ9.1
—
—
1.6
5.3
2.4
45b
35b
a
Approximate conversion on the basis of spot area on TLC plates.
Conversions were calculated from the enantiomeric excesses of substrates trans-2 (ees) and products 4 using the formula: c = ees/(ees + eep).
Yield after purification on chromatography column.
b
c
d
Enantiomeric excess determined by HPLC.

M. Zagozda, J. Plenkiewicz / Tetrahedron: Asymmetry 19 (2008) 1455–1460
1457
OH
O
O
OH
CN
Whole cells
CN
+
M. isabellina DSM 1414
CN
R
R
R
rac-2a,b
(2R,3R)-2c-e
rac-4a,b
(-)-4c-e
trans-1a-e
a: R= H, b: R= m-CH3, c: R= p-CH3
d: R= p-Cl, e: R= p-Br
Scheme 2.
Table 3
Results of hydrolysis of cis-1a-e by Mortierella isabellina DSM 1414 cells
Substrate cis-1
Time (h)
C (%)
5
6
E
Yc (%)
eed (%)
½
a 2D5
ꢁ
Yc (%)
eed (%)
Mp (°C)
½ ꢁ
a 2D5
a
b
c
d
e
24
24
4
24
72
ꢂ50a
48b
39b
59b
51b
43
39
29
47
47
rac
23
23
36
20
—
39
32
24
29
26
rac
25
37
25
19
Oil
Oil
91–92
98–99
96–97
—
—
+23.8
+22.4
ꢀ19.5
ꢀ15.1
ꢀ3.1
ꢀ15.2
+8.9
2.1
2.7
2.3
1.8
+3.2
a
Approximate conversion on the basis of spot area on TLC plates.
Conversions were calculated from the enantiomeric excesses of substrates 5 (ees) and products 6 using the formula c = ees/(ees + eep).
Yield after purification on a chromatography column.
b
c
d
Enantiomeric excess determined by HPLC.
OH
O
O
OH
CN
Whole cells
CN
+
M. isabellina DSM 1414
CN
R
R
R
rac-5a
rac-6a
cis-1a-e
(+)-5b,c
(-)-5d,e
(-)-6b,c
(+)-6d,e
a: R= H, b: R= m-CH3, c: R= p-CH3
d: R= p-Cl, e: R= p-Br
Scheme 3.
configuration of the optically active 2,3-dihydroxy-3-arylpropane-
nitriles obtained.
products of the trans-oxiranecarbonitrile isomers (see Table 3,
see Scheme 3).
Unlike with the D. hansenii DSM 3428 cells, the cultures of Mor-
tierella isabellina DSM 1414 produced epoxide hydrolases active
against cis-2,3-epoxy-3-aryloxiranecarbonitriles.
Poor enantiomeric purities of syn- and anti-2,3-dihydroxy-3-
arylpropanenitriles prompted us to attempt their purification by
kinetic resolution using the lipase-catalyzed esterification. The
solutions of racemic syn-2,3-dihydroxy-3-phenyl- and 2,3-dihy-
droxy-3-(p-methylphenyl)propanenitrile 4a and 4c in a non-polar
organic solvent were treated with vinyl acetate in the presence
of Novozym 435 (Candida Antarctica) or Amano AK (Pseudomonas
fluorescens) lipase and the reactions were carried on until the
conversion was approximately 50%. The regioselectivity of the
reaction was rather low and two isomeric monoacetylated diols
7a and 7c and 8a and 8c were formed (see Scheme 4).
The reactions, which were carried out under the same condi-
tions, yielded optically active products of the kinetic resolution.
Only the unsubstituted compound 1a gave racemic mixtures.
The hydrolytic opening of the oxirane ring of cis-2,3-epoxy-3-
arylpropanecarbonitriles 1a–e proceeded much slower than that
of the trans-isomers, while the enantiomeric purities of the anti-
2,3-dihydroxy-3-arylpropanenitriles 6a–e as well as of the
unchanged cis-carbonitriles 5a–e were even lower than in the
Scheme 4.

1458
M. Zagozda, J. Plenkiewicz / Tetrahedron: Asymmetry 19 (2008) 1455–1460
Table 4
Results of lipase catalyzed acetylation of syn-rac-4a,c
4
Lipase
Molar excess of vinyl acetate
Temp. (°C)
Time (days)
Solvent
syn-(ꢀ)-4a,c
Monoester ratio 7/8c
Ya (%)
eeb (%)
½ ꢁ (EtOH)
a 2D4
a
a
c
c
a
a
c
c
Novozym 435
Amano AK
Novozym 435
Amano AK
Novozym 435
Amano AK
Novozym 435
Amano AK
10
10
10
10
5
5
5
5
35
35
35
35
22
22
22
22
18
9
21
6
18
11
30
21
Toluene
Toluene
Toluene
Toluene
TBME
TBME
TBME
TBME
41
44
34
38
39
43
35
38
34
68
77
99
59
89
94
92
ꢀ11.0 (c 0.71)
ꢀ22.0 (c 0.76)
ꢀ23.8 (c 0.80)
ꢀ37.9 (c 0.66)
ꢀ13.6 (c 0.81)
ꢀ28.7 (c 0.84)
ꢀ36.1 (c 0.69)
ꢀ31.7 (c 0.76)
2/1
3/1
7/1
9/1
4/1
5/1
7/1
2/1
a
b
c
Yield after purification on a chromatography column.
Enantiomeric excess determined by HPLC.
Monoester ratio determined on the basis of 1H NMR spectra.
The reaction conditions were optimized by changing the sol-
vents and other parameters as shown in Table 4.
4.2.1. 2,3-Epoxy-3-phenylpropanenitrile 1a
Yield 73%; colorless oil; bp = 123–125 °C/15 Torr (lit.13
bp = 119–121 °C/6 Torr). Mixture of diastereoisomers.
Separation of the monoacetylated diols failed, therefore the iso-
mer ratio was established by analysis of the 1H NMR spectra. The
particular signals were attributed to the isomers on the basis of
the literature11 data. As it can be seen the 2-acetoxy compound
is predominant. The enantiomeric purities ee of the separated levo-
rotatory diols were above 90%. A similar purification procedure was
applied to the racemic mixture of anti-2,3-dihydroxy-3-(p-bromo-
phenyl)propanenitrile 6e. In the resulting acetylation catalyzed by
the Amano AK lipase we obtained a 3:1 mixture of 2- and 3-mono-
acetoxy derivatives and the dextrarotatory diol of 95% enantiomeric
purity and in 35% yield.
4.2.2. 2,3-Epoxy-3-(m-methylphenyl)propanenitrile 1b
Yield 62%; colorless oil; bp = 74–75 °C/0.1 Torr. Anal. Calcd for
C10H9NO (159.18): C, 75.45; H, 5.70; N, 8.80. Found: C, 75.28; H,
5.65; N, 8.73. trans-1b: 1H NMR (400 MHz, CDCl3): d 2.37 (s, 3H,
CH3-Ar); 3.41 (d, J = 2.0 Hz, 1H, Ar-CH–); 4.25 (d, J = 2.0 Hz, 1H,
–CH–CN); 7.26–7.41 (m, 4H, Ar-H). 13C NMR (100 MHz, CDCl3): d
21.28; 44.50; 58.48; 116.02; 122.76; 126.08; 128.81; 130.54;
132.61; 138.83. cis-1b: 1H NMR (400 MHz, CDCl3): d 2.40 (s, 3H,
CH3-Ar); 3.76 (d, J = 3.6 Hz, 1H, Ar-CH–); 4.21 (d, J = 3.6 Hz, 1H,
–CH–CN); 7.21–7.35 (m, 4H, Ar-H). 13C NMR (100 MHz, CDCl3): d
21.35; 44.98; 57.66; 115.01; 123.29; 126.77; 128.51; 130.43;
131.23; 138.42.
3. Conclusion
Our results indicate that whole cells of D. hansenii DSM 3428
can selectively catalyze the hydrolysis of the cyano group in some
trans-2,3-epoxy-3-aryl-propanenitriles to give the corresponding
amides in high yields but with low enantioselectivity. On the other
hand, M. isabellina DSM 1414 cells catalyze the oxirane ring
opening of both cis- and trans-2,3-epoxy-3-aryl-propanenitriles
yielding the corresponding optically active 2,3-hydroxy-3-
arylpropanenitriles.
4.2.3. 2,3-Epoxy-3-(p-methylphenyl)propanenitrile 1c
Yield 80%; colorless oil; bp = 87–88 °C/0.4 Torr (lit.14 bp = 140–
145 °C/17 Torr). Mixture of diastereoisomers.
4.2.4. 2,3-Epoxy-3-(p-chlorophenyl)propanenitrile 1d
Yield 73%; colorless crystals; trans-1d: mp = 66–67 °C
(lit.14 mp = 68–69 °C), cis-1d: mp = 78–80 °C (lit.14 mp = 79–
81 °C).
4. Experimental
4.1. General
4.2.5. 2,3-Epoxy-3-(p-bromophenyl)propanenitrile 1e
Yield 78%; colorless crystals. Anal. Calcd for C9H6BrNO (224.05):
C, 48.25; H, 2.70; N, 6.25; Br, 35.66. Found: C, 48.29; H, 2.89; N,
6.22; Br, 35.61. trans-1e: mp = 90–92 °C. 1H NMR (400 MHz,
CDCl3): d 3.39 (d, J = 2.0 Hz, 1H, Ar-CH–); 4.26 (d, J = 2.0 Hz, 1H,
–CH–CN); 7.13–7.17 (m, 2H, Ar-H); 7.51–7.54 (m, 2H, Ar-H). 13C
NMR (100 MHz, CDCl3): d 44.54; 57.88; 115.65; 123.93; 127.20;
131.73; 132.15. cis-1e: mp = 96–97 °C. 1H NMR (400 MHz, CDCl3):
d 3.79 (d, J = 3.6 Hz, 1H, Ar-CH–); 4.22 (d, J = 3.6 Hz, 1H, –CH–CN);
7.27–7.31 (m, 2H, Ar-H); 7.56–7.59 (m, 2H, Ar-H). 13C NMR
1H (400 MHz) and 13C NMR (100 MHz) spectra were recorded
with a Varian Mercury 400 MHz spectrometer in CDCl3 solution,
and chemical shifts (d) are reported in parts per million. Optical
rotations were measured in EtOH using a PolAAr 32 polarimeter.
Elemental analyses were performed with a CHNS/O Perkin–Elmer
type 2400 instrument. Enantiomeric excesses (ee%) were deter-
mined by HPLC analysis on a Thermo-Separation Products P-100
instrument with Chiracel OD-H column in n-hexane/iso-propanol
as the eluent in comparison with racemates. The biotransformation
reactions were monitored by TLC on Silica Gel 60 F254 plates. Ama-
no AK was kindly granted by Amano and Novozym SP 435 by
Novozymes.
(100 MHz, CDCl3):
130.40; 131.90.
d 44.97; 57.14; 114.70; 123.93; 127.89;
4.3. Microorganisms
The DSM 1414 strain of M. isabellina and DSM 3428 strain of
D. hansenii were purchased from the German Collection of Microor-
ganisms and Cell Cultures (DSMZ).
4.2. Synthesis of 2,3-epoxy-3-arylpropanenitriles 1a–e
2,3-Epoxy-3-arylpropanenitriles were prepared from the appro-
priate aldehyde and chloroacetonitrile in a Darzens reaction
according to the described12 procedure. The cis-/trans-isomers sep-
aration was accomplished by column chromatography.
4.4. Cultivation of microorganisms
The microorganism was grown on a plate with an agar medium
for 3 days at 27 °C until well sporulated. Spores from the surface

M. Zagozda, J. Plenkiewicz / Tetrahedron: Asymmetry 19 (2008) 1455–1460
1459
culture were used to inoculate flasks containing sterile liquid
medium (sterilized at 121 °C for 15 min), next incubated at 30 °C
for 3 days on a rotary shaker at 140 rpm.
Growing medium for both microorganisms investigated con-
tains malt extract (30 g), soya peptone (3 g), dissolved in 1.0 L of
distilled water, and adjusted pH to 5.6.
The biomass of fungal cells of M. isabellina was separated by fil-
tration under reduced pressure, while D. hansenii was separated by
centrifugation of the medium.
4.6.2. rac-syn-2,3-Dihydroxy-3-phenylpropanenitrile 4a
Yield 32%; colorless oil. 1H NMR (400 MHz, CDCl3): d 2.83 (d,
J = 3.2 Hz, 1H, –OH); 3.32 (d, J = 8.8 Hz, 1H, –OH); 4.55 (dd,
J = 8.8 Hz, J = 4.0 Hz, 1H, –CH); 5.00 (dd, J = 4.0 Hz, J = 3.2 Hz, 1H,
–CH); 7.37–7.47 (m, 5H, Ar-H). 13C NMR (100 MHz, CDCl3): d
67.27; 74.73; 117.15; 126.22; 128.92; 129.24; 136.74. Anal. Calcd
for C9H9NO2 (163.17): C, 66.25; H, 5.56; N, 8.58. Found: C, 66.32;
H, 5.59; N, 8.60.
4.6.3. anti-(ꢀ)-2,3-Dihydroxy-3-(m-methylphenyl)-
propanenitrile 6b
4.5. Hydrolysis of trans-2,3-epoxy-3-phenylpropanenitrile 1a
and trans-2,3-epoxy-3-(p-chlorophenyl)-propanenitrile 1d by
D. hansenii DSM 3428
Yield 32%; colorless oil. 1H NMR (400 MHz, CDCl3): d 2.38 (s, 3H,
CH3-Ar); 2.88 (br s, 1H, –OH); 3.21 (br s, 1H, –OH); 4.51 (d,
J = 6.4 Hz, 1H, –CH); 4.88 (d, J = 6.4 Hz, 1H, –CH); 7.19–7.32 (m,
4H, Ar-H).13C NMR (100 MHz, CDCl3): d 21.38; 66.16; 74.72;
117.96; 123.82; 127.35; 128.71; 130.05; 136.77; 138.64. Anal.
Calcd for C10H11NO2 (177.20): C, 67.78; H, 6.26; N, 7.90. Found:
C, 67.81; H, 6.28; N, 7.93.
Wet cells of the microorganism (58 g) were suspended in
116 mL of 2% glucose solution in sterile water, and a solution of
0.24 g (1.5 mmol) of 1a in 1.5 mL of ethanol or 0.3 g (1.5 mmol)
of 1d in 1.5 mL of DMF was added. The reaction was carried at
30 °C using an orbital shaker (140 rpm). The reaction monitored
by TLC was quenched after about 50% of the substrate reacted.
Ethyl acetate (90 mL) was added to the reaction flask, shaken for
20 min, and 25 g of CeliteÒ was added next. After filtration, the
supernatant was extracted three times with ethyl acetate. The
extract was dried with sodium sulfate and evaporated to dryness.
The residue was separated on silica gel column with hexane–ethyl
acetate (gradient of concentration) yielding unreacted nitriles
(2R,3R)-2a or 2d and the amides (2R,3S) 3a or 3d. The enantiomeric
excesses were determined by HPLC analysis using a Chiracel OD-H
column (in n-hexane–iso-propanol, 7/3 v/v, f = 0.6 mL/min). The 1H
NMR spectra of the amides were consistent with those presented
in the literature.8
4.6.4. rac-syn-2,3-Dihydroxy-3-(m-methylphenyl)-
propanenitrile 4b
Yield 41%; colorless oil. 1H NMR (400 MHz, CDCl3): d 2.38 (s, 3H,
CH3-Ar); 2.82 (br s, 1H, –OH); 3.35 (br s, 1H, –OH); 4.54 (d,
J = 4.0 Hz, 1H, –CH); 4.96 (d, J = 4.0 Hz, 1H, –CH); 7.18–7.33 (m,
4H, Ar-H).13C NMR (100 MHz, CDCl3): d 21.41; 67.31; 74.68;
117.48; 123.24; 126.82; 128.75; 129.86; 136.77; 138.65. Anal.
Calcd for C10H11NO2 (177.20): C, 67.78; H, 6.26; N, 7.90. Found:
C, 67.89; H, 6.29; N, 7.95.
4.6.5. anti-(ꢀ)-2,3-Dihydroxy-3-(p-methylphenyl)-
propanenitrile 6c
Yield 24%; colorless crystals. 1H NMR (400 MHz, CDCl3): d 2.37
(s, 3H, CH3-Ar); 2.70 (br s, 1H, –OH); 2.97 (br s, 1H, –OH); 4.51
(d, J = 6.4 Hz, 1H, –CH); 4.90 (d, J = 6.4 Hz, 1H, –CH); 7.22–7.24
(m, 2H, Ar-H); 7.32–7.34 (m, 2H, Ar-H).13C NMR (100 MHz, CDCl3):
d 21.21; 66.33; 74.67; 117.66; 126.62; 129.58; 133.87; 139.38.
Anal. Calcd for C10H11NO2 (177.20): C, 67.78; H, 6.26; N, 7.90.
Found: C, 67.69; H, 6.21; N, 7.94.
4.6. Hydrolysis of cis- or trans-2,3-epoxy-3-aryl-propanenitriles
by M. isabellina DSM 1414
To an Erlenmeyer flask (300 mL) covered by cotton wool,
110 mL of 2% glucose solution in sterilized water and M. isabellina
DSM 1414 cells (55 g wet weight) were added. The appropriate iso-
mer of the substrate (1.1 mmol) in 2 mL of ethanol was added in
one portion to the flask, and the mixture incubated at 30 °C using
an orbital shaker (140 rpm). The reaction monitored by TLC was
quenched after a specified period of time (see Tables 2 and 3) by
the addition of ethyl acetate and shaking for an additional
20 min. The biomass was filtered off, and washed with a small vol-
ume of ethyl acetate. The organic phase of the filtrate was sepa-
rated, and the water phase extracted with ethyl acetate. The
combined extracts after drying with sodium sulfate and solvent
evaporation were chromatographed on silica gel column with gra-
dient of hexane–ethyl acetate mixture, yielding the appropriate
unreacted 2,3-epoxy-3-arylpropanenitrile and 2,3-dihydroxy-3-
arylpropanenitrile as the product of kinetic resolution. The enan-
tiomeric excesses were determined by HPLC analysis using a Chira-
cel OD-H column (in n-hexane–i-propanol, 7/3 v/v for unreacted
nitriles and 9/1 v/v for cyanohydrins, f = 0.6 mL/min). 1H, 13C
NMR spectra and elemental analyses of the isolated products are
reported below.
4.6.6. syn-(ꢀ)-2,3-Dihydroxy-3-(p-methylphenyl)-
propanenitrile 4c
Yield 43%; colorless oil. 1H NMR (400 MHz, CDCl3): d 2.34 (s, 3H,
CH3-Ar); 3.42 (br s, 1H, –OH); 4.03 (d, J = 8.0 Hz, 1H, –OH); 4.49
(dd, J = 8.0 Hz, J = 3.6 Hz, 1H, –CH); 4.92 (d, J = 3.6 Hz, 1H, –CH);
7.18–7.20 (m, 2H, Ar-H); 7.28–7.30 (m, 2H, Ar-H).13
C NMR
(100 MHz, CDCl3): d 21.15; 67.29; 74.50; 117.57; 126.13; 129.49;
133.79; 138. 96. Anal. Calcd for C10H11NO2 (177.20): C, 67.78; H,
6.26; N, 7.90. Found: C, 67.62; H, 6.29; N, 7.71.
4.6.7. anti-(+)-2,3-Dihydroxy-3-(p-chlorophenyl)-
propanenitrile 6d
Yield 29%; colorless crystals. 1H NMR (400 MHz, CDCl3): d 2.78
(d, J = 3.2 Hz, 1H, –OH); 2.94 (d, J = 6.8 Hz, 1H, –OH); 4.49 (dd,
J = 6.8 Hz, J = 6.4 Hz, 1H, –CH); 4.93 (dd, J = 6.4 Hz, J = 3.2 Hz, 1H,
–CH); 7.21–7.37 (m, 4H, Ar-H).13C NMR (100 MHz, CDCl3): d
67.23; 73.88; 117.23; 127.56; 129.08; 135.08; 135.14. Anal. Calcd
for C9H8ClNO2 (197.62): C, 54.70; H, 4.08; Cl, 17.94; N, 7.09. Found:
C, 54.74; H, 4.10; Cl, 17.99; N, 7.14.
4.6.1. rac-anti-2,3-Dihydroxy-3-phenylpropanenitrile 6a
Yield 39%; colorless oil. 1H NMR (400 MHz, CDCl3): d 3.09
(s, 1H, –OH); 3.46 (d, J = 6.0 Hz, 1H, –OH); 4.50 (dd, J = 6.4 Hz,
J = 6.0 Hz, 1H, –CH); 4.90 (d, J = 6.4 Hz, 1H, –CH); 7.38–7.45 (m,
5H, Ar-H). 13C NMR (100 MHz, CDCl3): d 66.42; 74.94; 118.27;
127.06; 129.07; 129.52; 137.11. Anal. Calcd for C9H9NO2
(163.17): C, 66.25; H, 5.56; N, 8.58. Found: C, 66.18; H, 5.53; N,
8.55.
4.6.8. syn-(ꢀ)-2,3-Dihydroxy-3-(p-chlorophenyl)-
propanenitrile 4d
Yield 34%; colorless crystals. 1H NMR (400 MHz, CDCl3): d 3.41
(br s, 1H, –OH), 3.95 (br s, 1H, –OH), 4.52 (d, J = 4.0 Hz, 1H, –CH);
4.96 (d, J = 4.0 Hz, 1H, –CH); 7.28–7.39 (m, 4H, Ar-H). 13C NMR
(100 MHz, CDCl3):
d 67.10; 73.99; 117.19; 127.65; 129.06;

1460
M. Zagozda, J. Plenkiewicz / Tetrahedron: Asymmetry 19 (2008) 1455–1460
135.04; 135.26. Anal. Calcd for C9H8ClNO2 (197.62): C, 54.70; H,
4.08; Cl, 17.94; N, 7.09. Found: C, 54.86; H, 4.12; Cl, 17.98; N, 7.16.
was monitored by TLC. After the appropriate time, the reaction
was stopped by filtering off the enzyme and the solvent was evap-
orated under reduced pressure. The crude mixture was separated
by column chromatography on silica gel with a hexane–ethyl ace-
tate (3:1 v/v) mixture as the eluent, yielding the appropriate unre-
acted 2,3-dihydroxy-3-(p-methylphenyl)propanenitrile 4c and the
mixture of two isomeric monoacetylated diols 7c and 8c as the
products of kinetic resolution (see Table 4).
4.6.9. anti-(+)-2,3-Dihydroxy-3-(p-bromophenyl)-
propanenitrile 6e
Yield 26%; colorless crystals. 1H NMR (400 MHz, CDCl3): d 2.71
(br s, 1H, –OH); 3.01 (br s, 1H, –OH); 4.79 (d, J = 5.6 Hz, 1H,
–CH); 5.01 (d, J = 5.6 Hz, 1H, –CH); 7.28–7.39 (m, 2H, Ar-H);
7.56–7.59 (m, 2H, Ar-H). 13C NMR (100 MHz, CDCl3): d 66.32;
74.61; 116.87; 123.59; 127.89; 131.93; 135.42. Anal. Calcd for
C9H8BrNO2 (242.07): C, 44.66; H, 3.33; Br, 33.01; N, 5.79. Found:
C, 44.70; H, 3.40; Br, 33.12; N, 5.77.
The 1H and 13C NMR spectra spectra of enantiomerically en-
riched cyanohydrin (ꢀ)-4c were identical with those obtained by
hydrolysis of trans-1c. The spectra and elemental analyses of the
obtained monoacetylated diols are reported below.
Mixture of 3-acetoxy-2-hydroxy-3-(p-methylphenyl)propane-
nitrile 7c and 2-acetoxy-3-hydroxy-3-(p-methylphenyl)propaneni-
trile 8c in ratio 9/1. Colorless liquid. Anal. Calcd for C12H13NO3
(219.24): C, 65.74; H, 5.98; N, 6.39. Found: C, 65.55; H, 5.88; N,
6.29.
4.6.10. syn-(ꢀ)-2,3-Dihydroxy-3-(p-bromophenyl)-
propanenitrile 4e
Yield 19%; colorless crystals. 1H NMR (400 MHz, CDCl3): d 3.52
(br s, 1H, –OH); 4.07 (br s, 1H, –OH); 4.51 (d, J = 4.0 Hz, 1H,
–CH); 4.92 (d, J = 4.0 Hz, 1H, –CH); 7.27–7.31 (m, 2H, Ar-H);
7.50–7.54 (m, 2H, Ar-H).13C NMR (100 MHz, CDCl3): d 67.01;
73.99; 117.25; 123.18; 127.94; 131.99; 135.76. Anal. Calcd for
C9H8BrNO2 (242.07): C, 44.66; H, 3.33; Br, 33.01; N, 5.79. Found:
C, 44.85; H, 3.51; Br, 33.14; N, 5.61.
4.8.1. 3-Acetoxy-2-hydroxy-3-(p-methylphenyl)-propanenitrile
7c
1H NMR (400 MHz, CDCl3): d 2.20 (s, 3H, –CH3); 2.35 (s, 3H,
–CH3); 3.66 (br s, 1H, –OH); 4.63 (d, J = 4.8 Hz, 1H, –CH); 5.93 (d,
J = 4.8 Hz, 1H, –CH); 7.20–7.34 (m, 4H, Ar-H). 13C NMR (100 MHz,
CDCl3): d 20.91; 21.26; 66.45; 75.63; 116.39; 126.67; 129.61;
130.86; 139.63; 170.51.
4.7. Transesterification procedure of syn-4a
syn-2,3-Dihydroxy-3-phenylpropanenitrile 4a 0.1 g (0.63
mmol) was dissolved in 12 mL of TBME (tert-butyl methyl ether),
and vinyl acetate 0.29 mL (3.2 mmol) as well as 0.13 g of Amano
AK lipase was added. The mixture was stirred at room temperature
and the conversion was monitored by TLC. After the appropriate
time, the reaction was stopped by filtering off the enzyme and
the solvent was evaporated under reduced pressure. The crude
mixture was separated by column chromatography on silica gel
with a hexane–ethyl acetate (3:1 v/v) mixture as the eluent, yield-
ing the appropriate unreacted 2,3-dihydroxy-3-phenylpropanenit-
rile 4a and the mixture of two isomeric monoacetylated diols 7a
and 8a as the products of kinetic resolution (see Table 4). 1H and
13C NMR spectra of enantiomerically enriched cyanohydrin
(ꢀ)-4a were identical with those obtained by hydrolysis of trans-
1a. The spectra and elemental analyses of the obtained mono-
acetylated diols are reported below.
4.8.2. 2-Acetoxy-3-hydroxy-3-(p-methylphenyl)-propanenitrile
8c
1H NMR (400 MHz, CDCl3): d 2.13 (s, 0.3H, –CH3); 2.36 (s, 0.3H,
–CH3); 2.98 (br s, 0.1H, –OH); 5.01 (d, J = 4.8 Hz, 0.1H, –CH); 5.43
(d, J = 4.8 Hz, 0.1H, –CH); 7.20–7.34 (m, 0.4H, Ar-H). 13C NMR
(100 MHz, CDCl3): d 20.29; 21.19; 65.58; 74.16; 115.08; 126.58;
129.54; 130.77; 139.50; 169.43.
Acknowledgments
We acknowledge the evaluation of this manuscript by Professor
Jerzy Lange and suggestions provided by him. This work was finan-
cially supported by Warsaw University of Technology.
References
Mixture of 3-acetoxy-2-hydroxy-3-phenylpropanenitrile 7a
and 2-acetoxy-3-hydroxy-3-phenylpropanenitrile 8a in a ratio of
5/1. Colorless liquid. Anal. Calcd for C11H11NO3 (205.21): C,
64.38; H, 5.40; N, 6.83. Found: C, 64.42; H, 5.46; N, 6.89.
1. Cappi, M. W.; Chen, W.-P.; Flood, R. W.; Liao, Y. W.; Roberts, S. M.; Skidmore, J.;
Smith, J. A.; Williamson, N. M. Chem. Commun. 1998, 1159.
2. Wang, J.; Tian, W. J. Chem. Soc., Perkin Trans. 1 1996, 209.
3. Huang, D.-F.; Huang, L. Tetrahedron 1990, 46, 3135.
4. (a) Deng, L.; Jacobsen, E. N. J. Org. Chem. 1992, 57, 4320; (b) Jacobsen, E. N.;
Deng, L.; Furukawa, Y.; Martinez, L. E. Tetrahedron 1994, 50, 4323; (c) Righi, G.;
Rumboldt, G.; Bonini, C. J. Org. Chem. 1996, 61, 3557.
5. (a) Gleasson, J. G.; Hall, R. F.; Perchenock, C. D.; Erhard, K. F.; Frazee, J. S.; Ku, T.
W.; Kondrad, K.; McCarthy, M. E.; Mong, S.; Crooke, S. T.; Chi-Rosso, G.;
Wasserman, M. A.; Torphy, T. J.; Muccitelli, R. M.; Hay, D. W.; Tucker, S. S.;
Vickery-Clark, L. J. Med. Chem. 1987, 30, 959; (b) Kolb, H. C.; Sharpless, K. B.
Tetrahedron 1992, 48, 10515; (c) Flisak, J. R.; Gombatz, K. J.; Holmes, M. M.;
Jarmas, A. A.; Lantos, I.; Mendelson, W. L.; Novack, V. J.; Remich, J. J.; Snyder, L. J.
J. Org. Chem. 1993, 58, 6247.
6. Hashiyama, T.; Inoue, H.; Konda, M.; Takeda, M. J. Chem. Soc., Perkin Trans. 1
1984, 1725.
7. (a) Riemer, B.; Hofer, O.; Greger, H. Phytochemistry 1997, 45, 337; (b) Milner, P.
H.; Coates, N. J.; Gilpin, N. J.; Spear, S. R.; Eggleston, D. S. J. Nat. Prod. 1996, 59,
400.
8. Wang, M.-X.; Lin, S.-J.; Liu, C.-S.; Zheng, Q.-Y.; Li, J.-S. J. Org. Chem. 2003, 68,
4570.
9. Wang, M.-X.; Deng, G.; Wang, D.-X.; Zheng, Q.-Y. J. Org. Chem. 2005, 70,
2439.
4.7.1. 3-Acetoxy-2-hydroxy-3-phenylpropanenitrile 7a
1H NMR (400 MHz, CDCl3): d 2.20 (s, 3H, –CH3); 3.77 (br s, 1H,
–OH); 4.65 (d, J = 4.4 Hz, 1H, –CH); 5.96 (d, J = 4.4 Hz, 1H, –CH);
7.38–7.44 (m, 5H, Ar-H). 13C NMR (100 MHz, CDCl3): d 20.89;
66.24; 75.79; 116.81; 126.75; 128.92; 129.45; 136.42; 170.40.
4.7.2. 2-Acetoxy-3-hydroxy-3-phenylpropanenitrile 8a
1H NMR (400 MHz, CDCl3): d 2.12 (s, 0.6H, –CH3); 3.10 (br s,
0.2H, –OH); 5.04 (d, J = 4.4 Hz, 0.2H, –CH); 5.45 (d, J = 4.4 Hz,
0.2H, –CH); 7.38–7.44 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3):
d 20.27; 65.53; 72.91; 114.72; 126.38; 128.81; 129.29; 133.82;
168.81.
4.8. Transesterification procedure of syn-4c
10. Faber, K. Biotransformations in Organic Chemistry, 3rd ed.; Springer: Berlin
Heidelberg New York, 1997.
11. Althoff, W.; Karsdorf, R.; Tinapp, P. Arch. Pharm. 1981, 314, 518.
12. Jon´ czyk, A.; Gadaj, A. Pol. J. Chem. 2007, 81, 1595.
syn-2,3-Dihydroxy-3-(p-methylphenyl)propanenitrile 4c 0.08 g
(0.48 mmol) was dissolved in 8 mL of toluene and vinyl acetate
0.44 mL (4.8 mmol) as well as 0.08 g of Amano AK lipase was
added. The mixture was stirred at 35 °C and the conversion
´
´
13. Jonczyk, A.; Fedorynski, M.; Ma˛kosza, M. Tetrahedron Lett. 1972, 23, 2395.
ˇ
14. Svoboda, J.; Kocfeldová, Z.; Palecek, J. Coll. Czech. Chem. Commun. 1988, 53,
822.