PmHNL catalyzed synthesis of (R)-cyanohydrins derived from aliphatic aldehydes

Article abstract of DOI:10.1016/j.tetasy.2006.02.003

Hydroxynitrile lyase from the Japanese apricot (Prunus mume) catalyzes the formation of several aliphatic cyanohydrins in an asymmetric fashion. By employing a biphasic reaction system, aliphatic aldehydes with various structural features can be converted to the corresponding (R)-cyanohydrins with good overall yield and enantiomeric excess.

Full text of DOI:10.1016/j.tetasy.2006.02.003

Tetrahedron: Asymmetry 17 (2006) 735–741
PmHNL catalyzed synthesis of (R)-cyanohydrins derived
from aliphatic aldehydes
Samik Nanda, Yasuo Kato and Yasuhisa Asano*
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan
Received 8 September 2005; revised 3 February 2006; accepted 3 February 2006
Available online 15 March 2006
Abstract—Hydroxynitrile lyase from the Japanese apricot (Prunus mume) catalyzes the formation of several aliphatic cyanohydrins
in an asymmetric fashion. By employing a biphasic reaction system, aliphatic aldehydes with various structural features can be con-
verted to the corresponding (R)-cyanohydrins with good overall yield and enantiomeric excess.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Enantiomerically pure cyanohydrins have attracted the
attention of organic chemists, as well as enzymologists,
due to their immense potential as chiral building blocks
and interesting biological properties.1 Enantiopure cyano-
hydrins serve as immediate intermediates for several
industrially useful chemicals,2 and the use of chiral cyano-
hydrins as building blocks for the production of
important chemicals is likely to continue growing, as it
avoids problems associated with the optical resolution
or asymmetric synthesis of certain products. One of
the most promising and interesting ways to produce
enantiomerically pure cyanohydrins is the hydroxy-
nitrile lyase (HNL) catalyzed addition of cyanide to
the respective carbonyl compounds.3 Hydroxynitrile
lyase is a class of enzymes that catalyze the addition of
an appropriate cyanide source to carbonyl compounds
in a facial selective way, hence yielding the respective
cyanohydrins with excellent enantioselection. The chem-
istry and biology of HNLs have been extensively
explored over the past two decades,4 and active research
to find new HNLs with interesting catalytic properties is
still ongoing. In our search for new HNL species, we
previously reported interesting results with a new (R)-
HNL from Japanese apricot (PmHNL, Prunus mume).5
Herein, continuing on from this earlier report, we dis-
cuss PmHNL catalyzed asymmetric synthesis of cyano-
hydrins from aliphatic aldehydes with different
structural features.
In our previous report,5a we revealed that PmHNL
accepts a broad array of carbonyl compounds as its sub-
strate and produces the corresponding cyanohydrins
with excellent enantioselection. In this earlier study, it
was mainly aromatic aldehydes that were tested as pos-
sible PmHNL substrates with a few aliphatic aldehydes
and aliphatic methyl ketones. The aliphatic aldehydes
were shown to act as excellent substrates compared to
the aliphatic methyl ketones. This finding prompted us
to investigate a larger number of aliphatic aldehydes
with different structural features as possible PmHNL
substrates (Scheme 1). In general, a vigorously stirred
biphasic reaction media was employed,6 with acetone
cyanohydrin as the cyanating source (carbonyl com-
pounds: 5 g and 1.5 equiv of cyanating species; see
Experimental for details).
OH
O
PmHNL, Me2C(OH)CN
R
CN
R
H
DIPE, pH = 4.0
(R)-cyanohydrins 1a-34a
aldehydes 1-34
Scheme 1. PmHNL catalyzed (R)-cyanohydrin synthesis from ali-
phatic aldehydes.
2.1. Saturated aliphatic aldehydes
A series of aliphatic aldehydes were employed as the
PmHNL substrate. Aldehydes with 3 (propanal) to 10
carbon units (decanal) were tested for PmHNL cata-
lyzed cyanohydrin synthesis. By increasing chain length
*
Corresponding author. Tel.: +81 766 56 7500x530; fax: +81 766 56
2498; e-mail: asano@pu-toyama.ac.jp
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.02.003

736
S. Nanda et al. / Tetrahedron: Asymmetry 17 (2006) 735–741
R1
from propanal to decanal, the enzyme had a steady
effect on enantioselection. In agreement with our previ-
ous article,5a the short chain aldehydes propanal 1,
butyraldehyde 2, pentanal 6, and hexanal 7 were excel-
lent PmHNL substrates, and all provided excellent enan-
tioselection of the synthesized cyanohydrins with overall
good chemical yield (Table 1). In contrast, with the long
chain aldehydes heptanal 8 to decanal 10, a steady
decrease in enantioselectivity was observed, and decanal
provided an enantioselectivity of only 12% under similar
reaction conditions. By increasing the chain length, the
reaction time was largely enhanced to obtain an appre-
ciable chemical yield of the synthesized cyanohydrins.
The reason for this trend remains unclear, but a similar
trend was also observed with the most widely known
HNL, PaHNL (Prunus amygdalus; almond).7 When
subjected to PmHNL catalyzed cyanohydrin synthesis,
isobutyraldehyde 3, pivalaldehyde 4, and isovaleralde-
hyde 5 revealed excellent enantioselection with good
chemical yield. The absolute configurations of the pro-
duct cyanohydrins were confirmed as (R) by comparing
their specific rotation values with the known compounds
reported in the literature.7,8
R
CHO
CHO
R2
R = Me; 24
R = Et; 25
R3
R1 = R2 = R3 = H; 12
R1 = R2 = H; R3 = Me; 13
R1 = R3 = H; R2 = Me; 14
R1 = H; R2 = R3 = Me; 15
R1 = R2 = Me; R3 = H; 16
R1 = H; R2 = Me; R3 = Et; 17
R1 = H; R2 = Et; R3 = Me; 18
R1 = H; R2 = n-Pr; R3 = Et; 19
R1 = H = R3; R2 = n-Pr; 20
R1 = H = R3; R2 = n-C4H9; 21
CHO
26
R1 = H = R3; R2 = n-C5H11; 22
R1 = H = R3; R2 = n-C6H13; 23
Scheme 2. Aliphatic unsaturated aldehydes as PmHNL substrates.
Table 2. PmHNL catalyzed (R)-cyanohydrin synthesis from unsatu-
rated aliphatic aldehydes
Substrate
Time (h)
Yield (%)
ee (%)a
Acrolein 12
2-Methyl-2-propenal 13
(E)-2-Butenal 14
(E)-2-Methyl-2-butenal 15
3-Methyl-2-butenal 16
(E)-2-Ethyl-2-butenal 17
(E)-2-Methyl-2-pentenal 18
(E)-2-Ethyl-2-hexenal 19
(E)-2-Hexenal 20
(E)-2-Heptenal 21b
(E)-2-Octenal 22b
(E)-2-Nonenal 23b
(E,E)-2,4-Hexadienal 24b
(E,E)-2,4-Heptadienal 25b
Geranial 26b
8
10
14
14
12
48
70
78
58
76
78
82
56
70
52
90
88
70
78
72
50
58
48
62
52
48
40
32
38
48
42
32
96
90
92
92
96
92
94
72
21
12
96
97
98
Table 1. PmHNL catalyzed (R)-cyanohydrin synthesis from aliphatic
aldehydes
Substratea
Time (h)
Yield (%)
ee (%)b
Propionaldehyde 1c
Butyraldehyde 2
Isobutyraldehyde 3c
Pivalaldehyde 4c
3-Methyl-1-butanal 5c
Pentanal 6
Hexanal 7
Heptanal 8
Octanal 9
Nonanal 10
11
14
10
52
13
16
60
28
36
54
72
68
58
62
6
65
57
22
56
58
42
38
94
90
94
96
95
88
90
82
52
20
12
Decanal 11
a Ee was determined by chiral GC analysis of the cyanohydrins as they
were clearly resolved under the GC conditions.
b These cyanohydrins were derivatized to the corresponding –TMS
ethers prior to GC analysis.
a Data for compounds 1–4, and 6–7 were taken from our previous
article.5a
b Ee was determined by chiral GC analysis by derivatizing the cyano-
hydrins to the corresponding –TMS ethers.
c These cyanohydrins were not derivatized as all were clearly resolved
under the GC conditions.
tor for enantioselectivity as 2-butenal 14 provided 96%
enantioselectivity, even though its hydrophobic para-
meters are similar to those of methacrolein 13. Thus,
hydrophobicity as well as the structural parameters of
the substrate both have a pertinent effect on enantio-
selection. This will be discussed later.
2.2. Unsaturated aliphatic aldehydes
A series of unsaturated aliphatic aldehydes with different
structural features were also tested as PmHNL sub-
strates (Scheme 2). The simplest unsaturated aldehydes,
acrolein, and methacrolein 12 and 13 reacted rapidly
under the reaction conditions, with an almost quantita-
tive yield achieved at 8–10 h. However, the product cya-
nohydrin showed poor enantioselectivity (Table 2). The
reason for this was unclear, but a plausible explanation
is the high water solubility of these two compounds, the
chemical cyanation of which caused poor enantioselec-
tion. It had been established that during biphasic
HNL catalyzed cyanohydrin synthesis, the reaction
mainly occurs in the interphase and the hydrophobicity
of the substrates play an important role.8 However,
hydrophobicity alone could not be the determining fac-
3-Methyl-2-butenal and 2-methyl-2-butenal 16 and 15
were both shown to be excellent substrates of PmHNL,
producing corresponding cyanohydrins with very good
enantioselection and yield. 2-Ethyl-2-butenal, trans-2-
methyl-2-pentenal and (E)-2-ethyl-2-hexenal 17–19
required a longer reaction time for an appreciable chem-
ical yield (as indicated by TLC), but good enantioselec-
tion was observed with the synthesized cyanohydrins
(Table 3, 17a–19a). Under the reaction conditions, (E)-
2-hexenal 20 resulted in cyanohydrin formation with
excellent enantioselection; however, after 58-h reaction
time, only a 62% yield of respective cyanohydrin was

S. Nanda et al. / Tetrahedron: Asymmetry 17 (2006) 735–741
Table 3. PmHNL catalyzed cyanohydrin synthesis from cyclic aldehydes
737
Substratea
Time (h)
Yield (%)
ee (%)b
Cyclopropanecarbaldehyde 27
Cyclobutanecarbaldehyde 28b
Cyclopentanecarbaldehyde 29b
Cyclohexanecarbaldehyde 30b
Cyclohex-1-enecarbaldehyde 31b
Cyclohex-3-enecarbaldehyde 32c
Bicyclo[2.2.1]-hept-5-ene-2-carbaldehydec,d 33
(1R)-Myrtenal 34b
10
16
14
12
21
16
28
14
18
78
70
72
54
70
62
82
Nd
92
94
93
90
96, 94
89, 81
99
a Data for compounds 29 and 30 was taken from our previous article.5a
b Ee was determined by chiral GC analysis; these cyanohydrins were not derivatized as all were clearly resolved under the GC conditions.
c The ee for 32 and 33 (enantiomeric excess at the carbonyl carbon; molar percentage excess of 2R,3S over 2S,3S and 2R,3R over 2S,3R) was
determined with the help of chiral GC by derivatizing the cyanohydrins to the corresponding –TMS ethers. With the cyanohydrin–TMS ether of 32,
the composition of the four diastereomers was 49:1:48.5:1.5 and for 33 was 44.5:2.5:47.8:5.2.
d The starting material contained exo aldehyde (racemic) exclusively.
achieved. (E)-2-Heptenal 21 produced the correspond-
ing cyanohydrin with moderate enantioselection (72%),
whereas the higher chain aldehydes (E)-2-octenal and
(E)-2-nonenal 22 and 23 both provided very poor
enantioselectivity.
2.3. Cyclic aldehydes
We also tested several cyclic aldehydes with different
structural features as PmHNL substrates (Scheme 3).
The smallest cyclic aldehyde, cyclopropane carbalde-
hyde 27, was not accepted by PmHNL as a substrate,
and under the reaction conditions, yielded the corre-
sponding cyanohydrin with almost no enantioselection.
The chemical reactivity of cyclopropane carbaldehyde
was also very low: after 10 h incubation, a yield of only
18% of respective cyanohydrin was observed. Higher
aldehydes in the same series, cyclobutane carbaldehyde
28, cyclopentane carbaldehyde 29, and cyclohexane
carbaldehyde 30, were all excellent substrates of
PmHNL,5a and all provided cyanohydrins with excellent
enantioselection (Table 3) and good chemical yield. It
was also evident that cyclic aldehydes are better sub-
strates for PmHNL compared with their linear counter-
parts in terms of chemical yield as well as
enantioselectivity (Tables 1 and 3).
Increasing the chain length had a pronounced influence,
lowering the ee; this was also observed in the case of the
corresponding saturated aliphatic aldehydes. It was also
revealed that with an increase in chain length, the alde-
hydes required a longer reaction time to produce an
appreciable amount of products (although far below
the expected level). With an increase in chain length,
i.e. carbon number, the hydrophobic fragmental con-
stant value9 also increased. Therefore, if hydrophobicity
was the only factor responsible for enantioselection, we
should observe an enhanced ee value with an increase in
chain length, but in fact the opposite occurred. More-
over, surprisingly, (E)-geranial 26, even though it
contains 10 carbon atoms, also provided excellent
enantioselection, although low chemical yield of the cor-
responding cyanohydrin was observed. Thus, structural
parameters of the substrate also seem to play an impor-
tant role in governing the ee value.
CHO
CHO
CHO
n
The geometry (orientation) of (E)-geranial, even though
it contains 10 carbon atoms, makes it sufficiently flexible
to fit in the PmHNL active site. Although no structural
data is currently available, as PmHNL is a relatively new
enzyme, it seems certain that one has to consider the
hydrophobic parameters as well as structural features
of a given set of substrates when considering the enantio-
selectivity of the reaction. Substrates with an addi-
tional conjugated double bond (e.g., 24 and 25) are
also accepted by PmHNL, but require a sufficiently
longer reaction time and result in a low chemical yield
of cyanohydrins. However, with both 24 and 25, excel-
lent enantioselection was observed (Table 2). The abso-
lute configurations of the product cyanohydrins were
assigned to (R) and confirmed by comparing their spe-
cific rotation values with known compounds reported
in the literature.6–8 For compounds 17a–19a and 25a,
the absolute configurations were assigned by analogy
since the specific rotation values of these compounds
are not reported elsewhere.
n = 1, 2, 3, 4
27-30
31
32
CHO
CHO
33
34
Scheme 3. Cyclic aldehydes as PmHNL substrates.
Cyclohex-1-enecarbaldehyde 31, when treated with
PmHNL under similar reaction conditions, showed
product cyanohydrins with good enantioselectivity, but
low chemical yield (54%) compared with its saturated
counterpart. Cyclohex-3-enecarbaldehyde 32 provided
excellent enantioselection and was our first experience
with a substrate with a preexisting stereogenic center
(adjacent to the –CHO group). Both enantiomers of
cyclohex-3-enecarbaldehyde (1R and 1S), when reacted
with PmHNL, resulted in product cyanohydrins with

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S. Nanda et al. / Tetrahedron: Asymmetry 17 (2006) 735–741
good chemical yield (70%). The enantioselectivity (enan-
tioselection at the prochiral carbonyl group) of the reac-
tion was measured with chiral-GC and compared with
racemic samples synthesized by chemical methods. In
the racemic cyanohydrin, the –TMS ether of 32 showed
four peaks with characteristic baseline separation in an
equimolar ratio, whereas the GC diagram of the enzy-
matic hydrocyanation reaction products obtained from
32 showed two major and two minor peaks (Table 3).
The two major peaks [corresponding to (2R,3S) and
(2R,3R)-isomers, but we are not sure, which peaks corre-
sponded to which isomer] existed in almost equimolar
amounts, indicating that the preexisting stereogenic cen-
ter in the starting aldehydes is not differentiated by
PmHNL as it accepts both enantiomers as a substrate,
yielding respective cyanohydrins (by Si-facial addition
of the CN source to both the 1R and 1S isomers of com-
pound 32).
whereas those of all other cyanohydrins was assigned
by analogy.
3. Conclusion
A careful literature survey of (R)-HNL catalyzed
aliphatic cyanohydrins synthesis revealed that PaHNL
is a widely explored HNL in biocatalytic asymmetric
synthesis.3 HNL from defatted apple meal and LuHNL
(Linum usitatissimum hydroxynitrile lyase from flax
seedlings) were also used in a few cases.13,14 However,
the reactivity and substrate specificity of HNL from
apple meal and LuHNL were restricted to very few ali-
phatic aldehydes, unlike PaHNL, which accepts various
aliphatic aldehydes, saturated, unsaturated, and cyclic,
and yields respective cyanohydrins with good to excel-
lent enantioselection and chemical yield.3b In our earlier
report,5a we revealed that PmHNL possesses a striking
similarity with PaHNL with regards to substrate speci-
ficity (chemical yield and enantioselectivity). Comparing
the results presented here with those obtained previously
with PaHNL catalyzed aliphatic cyanohydrin synthesis,
an excellent analogy was found with respect to both
chemical yield and enantioselection. It is important to
point out, however that a few substrates 17–19, 25, 28,
29, and 33 tested here with PmHNL were new and have
not been examined with PaHNL or any other HNL cata-
lyzed cyanohydrin synthesis.
In an earlier communication by Brussee et al.,10 the (R)-
PaHNL catalyzed the synthesis of cyanohydrins from
substrate 32 was examined by employing aqueous EtOH
as a cosolvent. However, the poor enantioselectivity of
the process made it inefficient and presented the scope
for us to examine our enzyme system using PmHNL,
which subsequently provided excellent enantioselection.
In another example, the exo-isomer of bicyclo[2.2.1]-
hept-5-ene-2-carbaldehyde (racemic mixture, 33), when
reacted with PmHNL, resulted in good overall enantiose-
lection (at the prochiral carbonyl group) (Table 3). It was
previously reported that the two enantiomers of racemic
aldehydes substituted at the a-position reacted differ-
ently with PaHNL catalyzed asymmetric hydrocyana-
tion, but that the enzyme cannot distinguish between
the enantiomers of racemic aldehydes devoid of any a-
substitution.11 Moreover, substitution at the b-position
had absolutely no effect on the reactivity of the two enan-
tiomers.11 A substrate structurally related to 32 (1,2,3,4-
tetrahydronaphthalene-2-carbaldehyde) was previously
subjected to PaHNL catalyzed hydrocyanation by Riva
et al.,12 who revealed that both enantiomers were active
and that the enantioselectivity was not effected by the
existing chirality of the molecule. It can, therefore, be
concluded that, in general, both enantiomers of a race-
mic aldehyde (without any a-substitution) are accepted
by PaHNL. As our PmHNL system poses striking simi-
larities in reactivity as well as enantioselectivity, PmHNL
is expected to exhibit a similar trend, and indeed did so in
the case of aldehydes 32 and 33. Further experimental
work is required to confirm the above concept and is cur-
rently under progress in our laboratory.
We can conclude that the compatibility of PmHNL with
the novel substrates presented herein will provide the
opportunity for further research focusing on its catalytic
behavior with customized substrates. Further studies
directed toward the enzymological properties of PmHNL
will also allow the prediction of an efficient model for
determining the structure–activity relationship of these
kinds of enzymes.
4. Experimental
4.1. General
Unless otherwise stated, materials were obtained from
commercial suppliers and used without further purifica-
tion. Ripen Ume fruit (P. mume) was obtained from a
local fruit market and stored at 4 ꢁC. All aldehydes were
freshly distilled or washed with aq NaHCO3 solution to
minimize the amount of free acid, which is thought to
inhibit HNL activity. Mandelonitrile and acetone cyano-
hydrins were freshly distilled prior to use. Reactions
were monitored by TLC carried out on 0.25 mm thick
silica gel plates (Merck) with UV light, ethanolic vanil-
lin, and phosphomolybdic acid/heat as developing
agents. Silica gel 100–200 mesh was used for column
chromatography. Chemical yield (isolated yield) refers
to chromatographically and spectroscopically homo-
geneous materials unless otherwise stated. NMR spectra
were recorded on a 400 MHz spectrometer (JEOL
JNM-CA 400) at 25 ꢁC in CDCl3 using TMS as the
internal standard. Chemical shifts are shown in d. 13C
NMR spectra were recorded in a complete proton
The last example in this series was 1(R)-myrtenal 34,
which has a similar bicyclic framework as norbornene
aldehydes 33, with two extra geminal –Me groups in
the bridge. Under similar reaction conditions, 1(R)-
myrtenal provided the corresponding cyanohydrins with
excellent chemical and optical yield (Table 3). Thus,
bicyclic substrates also seem to be accepted by the
PmHNL active site. The absolute configurations of the
product cyanohydrins of 29, 30, 31, and 34 were con-
firmed as (R) by comparing their specific rotation values
with known compounds reported in the literature,1f,7,8

S. Nanda et al. / Tetrahedron: Asymmetry 17 (2006) 735–741
739
decoupling environment. The chemical shift value is
dissolved in anhydrous DCM, then treated with excess
imidazole and TMS-Cl at room temperature. After com-
plete derivatization, the products were purified by pass-
ing through a small pad of silica gel and eluting with
hexane/ETOAc (9:1). Method b, synthesis of racemic
cyanohydrin–TMS ethers as the GC Standard: Aldehydes
(1 equiv) were dissolved in anhydrous DCM, followed
by the addition of a catalytic amount of anhydrous
ZnCl2 (0.03 equiv). The solution was stirred for 5 min
at room temperature then trimethylsilylnitrile
(1.5 equiv) was added to the reaction mixture. After
complete conversion by TLC, the organic solvent was
removed under vacuum and the product was purified
as described earlier.
1
listed as dH and dC for H and 13C, respectively. Chiral
GC analysis was performed in a Schimadzu autosampler
with cyclodextrin columns as the chiral stationary phase
(Fused silica capillary column, 30 m · 0.25 mm ·
0.25 lm thick: b-Dex-120 and b-Dex-325 from SUPE-
LCO, USA) using He as a carrier gas (detector temper-
ature: 230 ꢁC and injection temperature: 220 ꢁC).
Retention time was denoted as tR and tS for (R)- and
(S)-enantiomers, respectively; values in parenthesis indi-
cate the column temperature. Optical rotations were
measured in a digital polarimeter HORIBA Sepa-3000
instrument. Mass spectra were measured in a QSTAR-
XL (LC/MS) spectrometer (Applied Biosystems).
4.2. PmHNL isolation and assay
4.6. NMR values and GC retention times of aliphatic
cyanohydrins
Details of the enzyme isolation and assay procedures are
reported in our previous article.5a The protein content of
PmHNL was measured using the Bradford method with
a Bio-Rad protein assay kit using BSA as the stan-
dard.15 Accordingly, the protein content of a crude ex-
tract of PmHNL was found to be roughly 10 mg/mL
and activity was 120 U/mL. The enzyme is a flavopro-
tein (FAD containing), as evident by its yellow color
and confirmed by absorption spectroscopy.
Spectral data (1H NMR, 13C NMR, and MS) for most
compounds were in full agreement with the previously
published data.3,6–8,14,17,18 Except for compounds 17a,
23a, and 25a, all cyanohydrins reported here have been
reported elsewhere. The following referencing index will
help the reader locate those compounds:
Compounds 1a–11a, 12a–16a, 20a, 22a, 24a, 26a, 30a–
32a can be found in Ref. 3b (and cross references cited
there in); 18a,18 19a,18c 21a,18b 27a,18d 28a,18e 29a,18d
33a,18f 34a.1f
4.3. Preparative scale biphasic synthesis of cyanohydrins
using PmHNL
Carbonyl compounds (5 g) were dissolved in diisoprop-
ylether (DIPE, 100 mL), saturated with 5 mL of
400 mM Na-citrate buffer (pH = 4.0). HNL solution
was prepared by dissolving concentrated crude extract
of PmHNL (100 mg of protein) in 10 mL of the above
buffer. The enzyme solution was added to the reaction
vessel followed by the addition of an appropriate cya-
nide source (acetonecyanohydrin: 1.5 equiv) then vigor-
ously stirred until the desired conversion was achieved
(by TLC at 25 ꢁC). The reaction mixture was extracted
with ether (3 · 50 mL) and dried over Na2SO4. The
product cyanohydrins were purified by silica gel chro-
matography (hexane/ethyl acetate 8:2).
The GC retention times for cyanohydrins 1a–4a, 6a–7a,
and 29a–30a were reported in our previous article5a and
hence are not given here.
4.6.1. (2R)-Hydroxy-4-methylpentanenitrile 5a. tR =
26.1; tS = 27.2 (125).
4.6.2. (2R)-Hydroxyoctanenitrile 8a. tR = 46.8; tS =
47.9 (100).
4.6.3. (2R)-Hydroxynonanenitrile 9a. tR = 53.0; tS =
54.3 (110).
4.6.4. (2R)-Hydroxydecanenitrile 10a. tR = 57.3; tS =
4.4. Preparation of racemic cyanohydrins as the GC
standard
58.6 (120).
4.6.5. (2R)-Hydroxyundecanenitrile 11a. tR = 59.8;
tS = 60.9 (130).
Following a common protocol, an aqueous solution of
KCN (3 equiv) in water was added to a solution of car-
bonyl compounds (1 equiv) in acetic acid.16 After com-
pletion of the reaction (by TLC), the reaction mixture
was neutralized with aq NaHCO3 solution then
extracted with ether and dried over anhydrous Na2SO4.
Evaporation and purification by column chromatogra-
phy on silica resulted in a good yield of cyanohydrins.
Racemic cyanohydrins obtained by this method were
fully characterized by NMR spectroscopy and used as
the GC standard.
4.6.6. (2R)-2-Hydroxy-3-butenenitrile 12a. tR = 45.2;
tS = 46.0 (90).
4.6.7. (2R)-2-Hydroxy-3-methyl-3-butenenitrile 13a.
tR = 32.2; tS = 33.1 (110).
4.6.8. (2R,3E)-2-Hydroxy-3-pentenenitrile 14a. tR =
61.7; tS = 63.4 (100).
4.6.9. (2R,3E)-2-Hydroxy-3-methyl-3-pentenenitrile 15a.
tR = 34.1; tS = 32.3 (125).
4.5. Derivatization of cyanohydrins as their TMS ethers
Method a, for determination of enantioselectivity by chi-
ral GC: Cyanohydrins synthesized enzymatically were
4.6.10. (2R)-2-Hydroxy-4-methyl-3-pentenenitrile 16a.
tR = 29.3; tS = 26.7 (130).

740
S. Nanda et al. / Tetrahedron: Asymmetry 17 (2006) 735–741
4.6.11. (2R,3E)-3-Ethyl-2-hydroxy-3-pentenenitrile 17a.
dH: 5.8 (q, J = 7.3 Hz, 1H), 4.82 (s, 1H), 3.85 (br s, 1H),
2.2 (m, 2H), 1.72 (d, J = 7.2 Hz, 3H), 1.0 (t, J = 7.2 Hz,
4.6.20. (2R,3E)-2-Hydroxy-4,8-dimethyl-3,7-nonadiene-
nitrile 26a. tR = 59.8; tS = 59.3 (120).
3H). dC: 136.2, 125.1, 118.9, 56.8, 29.8, 19.6, 11.8.
tR = 41.4; tS = 39.0 (130). ½aꢀD ¼ ꢁ32:1 (c 1.0, CHCl3).
4.6.21. (2R)-2-Cyclobutyl-2-hydroxyacetonitrile 28a.
dH: 4.28 (d, J = 7.0 Hz, 1H), 1.75–2.1 (m, 7H). dC:
25
25
119.2, 67.7, 35.2, 21.2, 19.0. ½aꢀD ¼ þ3:8 (c 1.1, CHCl3)
ESIMS for C7H11ON calcd 125.0913, obsd m/z 125.0916
tR = 25.5; tS = 24.8 (120).
(M+).
4.6.22. (2R)-2-Cyclopentyl-2-hydroxyacetonitrile 29a.
dH: 4.38 (d, J = 7.2 Hz, 1H), 2.8 (br s, 1H), 1.4–1.72
4.6.12. (2R,3E)-2-Hydroxy-3-methyl-3-hexenenitrile 18a.
dH: 5.68 (t, J = 8.2 Hz, 1H), 4.82 (s, 1H), 3.76 (br s, 1H),
25
(m, 9H). dC: 118.5, 65.2, 35.9, 29.2, 27.3. ½aꢀD ¼ þ11:2
2.08 (m, 2H), 1.8 (s, 3H), 1.0 (t, J = 7.2 Hz, 3H). dC:
(c 1.0, CHCl3).
25
134.2, 128.9, 118.6, 67.2, 21.2, 13.3, 11.8. ½aꢀD ¼ ꢁ26:8
(c 1.2, CHCl3). tR = 30.7; tS = 29.4 (135).
4.6.23. (2R)-2-Cyclohexenyl-2-hydroxyacetonitrile 31a.
dH: 6.0 (m, 1H), 4.9 (s, 1H), 3.48 (br s, 1H), 2.1 (m,
2H), 1.5–1.8 (m, 6H). dC: 138.7, 129.4, 118.6, 65.3,
4.6.13. (2R,3E)-3-Ethyl-2-hydroxy-3-heptenenitrile 19a.
dH: 5.7 (m, 1H), 4.82 (s, 1H), 3.85 (br s, 1H), 2.2–1.98
(m, 4H), 1.52 (m, 2H), 1.0 (t, J = 7.2 Hz, 3H), 0.96 (t,
J = 7.2 Hz, 3H). dC: 142.4, 122.5, 118.4, 68.5, 29.3,
25.1, 24.7, 18.7, 18.0. ESIMS for C8H11ON calcd
25
137.0913, obsd m/z 137.0906 (M+). ½aꢀD ¼ ꢁ16:7 (c
1.5, CHCl3). tR = 35.9; tS = 37.6 (160).
23.4, 17.6, 14.2, 12.8. tR = 30.8; tS = 30.0 (150).
25
½aꢀD ¼ ꢁ36:8 (c 1.0, CHCl3).
4.6.24.
(2R)-2-(3-Cyclohexenyl)-2-hydroxyacetonitrile
32a. dH: 5.7 (m, 2H), 4.4 (d, J = 7.1 Hz, 1H), 2.1–
1.91 (m, 4H), 1.7–1.48 (m, 3H). dC: 126.1, 125.8, 118.9,
4.6.14. (2R,3E)-2-Hydroxy-3-heptenenitrile 20a. dH:
6.0 (m, 1H), 5.6 (dd, J = 14.8, 6.8 Hz, 1H), 4.92 (d,
J = 6.8 Hz, 1H), 4.0 (br s, 1H), 2.08 (m, 2H), 1.45 (m,
2H), 0.92 (t, J = 7.2 Hz, 3H). dC: 137.7, 123.6, 118.6,
61.6, 33.9, 21.7, 13.4 tR = 42.9; tS = 42.1 (130).
65.7, 38.0, 26.6, 24.8, 23.4. tmajor = 48.4, 49.6; tminor
=
47.7, 49.3 (105).
4.6.25. (2R)-2-(Bicyclo[2.2.1]-hept-5-en-2-yl)-2-hydroxy-
acetonitrile 33a. dH: 5.65 (m, 2H), 4.24 (m, 1H), 2.26
(m, 2H), 1.94 (m, 1H), 1.72–1.58 (m, 3H), 1.34 (m,
1H). dC: 136.2, 135.8, 118.5, 63.4, 51.2, 42.7, 42.3,
32.8, 26.3. ESIMS for C9H11ON calcd 149.0913, obsd
m/z 149.0915 (M+). tmajor = 43.9, 49.4; tminor = 45.1,
50.5 (160).
4.6.15. (2R,3E)-2-Hydroxy-3-octenenitrile 21a. dH: 6.0
(m, 1H), 5.64 (dd, J = 14.8, 6.8 Hz, 1H), 4.92 (d, J =
6.8 Hz, 1H), 4.0 (br s, 1H), 2.08 (m, 2H), 1.45–1.6 (m,
4H), 0.92 (t, J = 7.2 Hz, 3H). dC: 134.5, 126.4, 118.9,
66.6, 32.7, 32.2, 22.9, 14.2. tR = 38.9; tS = 37.7 (110).
4.6.16. (2R,3E)-2-Hydroxy-3-nonenenitrile 22a. dH: 6.1
(m, 1H), 5.46 (dd, J = 15.2, 6.9 Hz, 1H), 4.88 (d, J =
6.9 Hz, 1H), 3.5 (br s, 1H), 2.1 (m, 2H), 1.2–1.5 (m,
6H), 0.95 (br t, 3H). dC: 137.8, 123.5, 118.6, 61.6, 32.2,
31.1, 28.0, 22.6, 13.8. tR = 42.5; tS = 41.2 (120).
4.6.26. (2R)-2-Hydroxy-2-(6,6-dimethylbicyclo[3.1.1]hept-
2-en-3-yl)acetonitrile 34a. dH: 5.9 (s, 1H), 4.85 (s, 1H),
2.65–2.1 (m, 6H), 1.6 (s, 3H), 1.3 (s, 3H). dC: 124.8,
123.6, 118.3, 63.4, 43.2, 40.0, 38.3, 34.1, 29.0, 25.8,
25
24.1. ½aꢀD ¼ ꢁ21:1 (c 1.4, CHCl3). ESIMS for
C11H15ON calcd 177.0913, obsd m/z 177.0916 (M+).
tR = 36.3; tS = 37.6 (170).
4.6.17. (2R,3E)-2-Hydroxy-3-decenenitrile 23a. dH:
6.07 (m, 1H), 5.48 (dd, J = 15.0, 6.8 Hz, 1H), 4.87 (d,
J = 6.8 Hz, 1H), 3.5 (br s, 1H), 2.1 (m, 2H), 1.2–1.5
(m, 8H), 0.92 (br t, 3H). dC: 137.8, 123.6, 118.6, 62.0,
31.8, 31.3, 28.9, 28.7, 22.4, 13.9. tR = 44.8; tS = 43.7
(130). ESIMS for C10H17ON calcd 167.0913, obsd m/z
167.0918 (M+).
Acknowledgments
A postdoctoral fellowship from the Japan Society for
Promotion of Science (JSPS) to S.N. is gratefully
acknowledged. This research was supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan. We are also thankful to Ms. Ai Kayou of To-
yama Prefectural University for her excellent technical
help.
4.6.18. (2R,3E,5E)-2-Hydroxy-3,5-heptadienenitrile 24a.
tR = 32.1; tS = 32.8 (110).
4.6.19. (2R,3E,5E)-2-Hydroxy-3,5-octadienenitrile 25a.
dH: 6.55 (dd, J = 15.0, 10.5 Hz, 1H), 6.08 (dd,
J = 15.0, 10.5 Hz, 1H), 5.93 (dq, J = 15.0, 7.0 Hz, 1H),
5.65 (dd, J = 15.0, 7.0 Hz, 1H), 5.0 (d, J = 7.0 Hz,
1H), 2.88 (br s, 1H), 2.1 (q, J = 7.0 Hz, 2H), 1.02 (t,
J = 7.0 Hz, 3H). dC: 141.6, 135.9, 126.8, 122.7, 118.3,
61.7, 25.7, 13.0.
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