Nitrilase-catalysed hydrolysis of cyanomethyl p-tolyl sulfoxide: stereochemistry and mechanism

Article abstract of DOI:10.1016/j.tetasy.2008.01.021

Several commercially available nitrilases have been used for the enantioselective hydrolysis of cyanomethyl p-tolyl sulfoxide into the corresponding amide and acid, which are formed in different proportions and with varying stereoselectivities, depending on the nitrilase involved. It was shown that the externally added amide is not transformed into the acid, which can be explained by assuming that both products must be produced in concurrent reactions. It was also demonstrated that the absolute configuration of the substrate exerts substantial influence on the product ratio. Two alternative explanations of the stereochemical course are presented.

Full text of DOI:10.1016/j.tetasy.2008.01.021

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 562–567
Nitrilase-catalysed hydrolysis of cyanomethyl p-tolyl sulfoxide:
stereochemistry and mechanism
a,
a
a
b,
*
*
´
Piotr Kiełbasinski, Michał Rachwalski, Marian Mikołajczyk and Floris P. J. T. Rutjes
aCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Heteroorganic Chemistry,
Sienkiewicza 112, 90-363 Ło´dz´, Poland
bRadboud University Nijmegen, Institute for Molecules and Materials, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
Received 14 December 2007; accepted 16 January 2008
Available online 7 February 2008
Abstract—Several commercially available nitrilases have been used for the enantioselective hydrolysis of cyanomethyl p-tolyl sulfoxide
into the corresponding amide and acid, which are formed in different proportions and with varying stereoselectivities, depending on the
nitrilase involved. It was shown that the externally added amide is not transformed into the acid, which can be explained by assuming
that both products must be produced in concurrent reactions. It was also demonstrated that the absolute configuration of the substrate
exerts substantial influence on the product ratio. Two alternative explanations of the stereochemical course are presented.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ence of two different enzymes, which can be achieved using
microorganisms or whole cell techniques.
Nitriles constitute as an important and versatile class of
intermediates for synthetic chemistry.1 They are syntheti-
cally particularly useful and, hence, widely used for the
preparation of various carboxylic acids and amine deriva-
tives. However, their chemical transformation into the cor-
responding carboxamides and carboxylic acids or esters
usually requires the use of strong bases, acids or heavy me-
tal salts. Therefore, an alternative approach to achieve this,
which rests upon the use of nitrile hydrolysing enzymes,
has been a fast developing area of research in recent
years.2–7
O
nitrilase(NL)
R
C N
R
OH
amidase
nitrile
hydratase
(NHase)
O
R
NH2
Scheme 1. Pathways of enzyme-catalysed nitrile hydrolysis.
This simple picture has recently turned out to be insuffi-
cient for explaining new findings. For example, it was
reported that certain nitrilases, besides their anticipated
behaviour, exhibited NHase activity, which resulted in
the formation of both the corresponding amides and
acids.9–15 In principle, such results could be explained by
invoking the second pathway. However, on the basis of
common observations that nitrilases do not hydrolyse
carboxamides,13 the assumption was made that both prod-
ucts of the hydrolysis were formed in parallel reactions. A
plausible mechanism, which could account for such results,
was proposed by Sheldon et al.13
It is generally accepted that the enzyme-catalysed hydrolysis
of nitriles follows one of the two pathways (Scheme 1).8 In
the first pathway, a nitrilase (NL) converts the nitrile di-
rectly into the carboxylic acid, via addition of two molecules
of water. In the second one, a nitrile hydratase (NHase)
catalyses single hydration of the nitrile to give the corre-
sponding amide, which is followed by amidase-catalysed
hydrolysis to the appropriate acid. Thus, according to this
scheme, the latter pathway requires the simultaneous pres-
Our previous work on the deracemisation of racemic and
desymmetrisation of prochiral heteroorganic compounds
led to efficient approaches for the synthesis of a variety
*
Corresponding authors. Tel.: +48 42 680 32 34; fax: +48 42 6847126
(P.K.); tel.: +31 24 3653202; fax: +31 24 3653393 (F.P.J.T.); e-mail
addresses: piokiel@bilbo. cbmm.lodz.pl; f.rutjes@science.ru.nl
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.01.021

P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 19 (2008) 562–567
563
of optically active heteroatom derivatives.14–18 In particu-
lar, our results on the enzymatic desymmetrisation of
bis(cyanomethyl) sulfoxide14 and bis(cyanomethyl)phenyl-
phosphine oxide15 represented the first examples of a
nitrilase-catalysed stereoselective hydrolysis of dinitriles
containing prostereogenic centres located on a hetero-
atom—the sulfinyl sulfur and phosphinyl phosphorus,
respectively. In these cases, the hydrolysis also led to two
out of five possible products, namely the corresponding
monoamide and monoacid, formed in different proportions
and with varying enantiomeric excesses, depending on the
kind of the nitrilase. Interestingly, in certain cases the abso-
lute configurations of the amide and acid produced in the
same reaction were identical, while in others they were
opposite. The former would substantiate the assumption
that both products were formed in a concurrent reaction
(as proposed by Sheldon et al.),13 while the latter would
suggest that the amide was first formed non-stereoselective-
ly and then stereoselectively hydrolysed to the acid under
kinetically controlled resolution conditions.
2.1.2. Synthesis of racemic p-toluenesulfinylacetamide 2.
Racemic 2 was synthesised via a two-step method from
bromoacetamide, which upon treatment with p-toluene-
thiol in the presence of triethylamine in Et2O, provided
the corresponding sulfide in quantitative yield. The latter
was oxidised with sodium periodate in a 1:1 mixture of
water and ethanol to give the desired racemic amide 2 in
85% yield after purification (Scheme 3).
O
p-TolSH
O
O
S
O
Et3N
NaIO4
S
NH2
p-Tol
NH2
p-Tol
NH2
Et2O
EtOH/H2O
Br
100%
2 (85%)
Scheme 3. Synthesis of racemic amide 2.
2.2. Kinetic resolution of racemic cyanomethyl p-tolyl
sulfoxide 1
The racemic sulfoxide 1 was subjected to hydrolysis in a
phosphate buffer and a co-solvent to dissolve the substrate,
by a variety of nitrilases under kinetic resolution condi-
tions. The hydrolysis should in principle give rise to two
products: p-toluenesulfinylacetamide 2 and p-toluenesulfin-
ylacetic acid 3, together with (probably) enantiomerically
enriched starting material 1 (Scheme 4, absolute configura-
tions of 2 and 3 are arbitrarily chosen). The products were
purified by column chromatography and analysed spectro-
scopically. The results are shown in Table 1.
The use of prochiral substrates did not allow us to distin-
guish the two possible pathways employed by the enzymes.
First of all, it was not possible to determine whether the
first step leading to the amide was stereoselective, since
the unreacted substrate was not chiral. Additionally, it ap-
peared rather difficult to synthesise a racemic monoamide
for subjection to hydrolysis by a nitrilase. In order to gain
better insight into the hydrolysis mechanism, we decided to
investigate the nitrilase-catalysed kinetic resolution of a
racemic cyanomethyl sulfoxide since this would provide
more information about the stereochemistry of the reac-
tion. As a model substrate, the easily available racemic
cyanomethyl p-tolyl sulfoxide 1 was chosen.
O
S
O
S
Nitrilase
O
S
+
NH2
p-Tol
p-Tol
CN
phosphate
buffer
OH
p-Tol
pH 7.2
O
O
1
2
3
solvent
2. Results and discussion
2.1. Synthesis of substrates
Scheme 4. Enzymatic hydrolysis of sulfoxide 1.
2.1.1. Synthesis of racemic and enantiomeric cyanomethyl
p-tolyl sulfoxides. The starting material, racemic cyano-
methyl p-tolyl sulfoxide, 1, was synthesised in a two-step
procedure starting from chloroacetonitrile, which on sub-
jection to p-toluenethiol in the presence of sodium meth-
oxide in methanol provided cyanomethyl p-tolyl sulfide
in quantitative yield. The latter was oxidised with sodium
periodate in a 1:1 mixture of water and ethanol to give
the desired sulfoxide 1 in 80% yield after purification
(Scheme 2).
The enantiomeric excesses of acid 3 were calculated by
comparing the [a]D value of a given sample with that
reported in the chemical literature,20 while the enantio-
meric excesses of unreacted nitrile 1 and amide 2 were
determined by chiral HPLC. The absolute configurations
of 1 and 3 are known from the literature to be (+)-
(R).19,20 To determine the absolute configuration of amide
2, CD spectra of (+)-(R)-1, (+)-(R)-3 and of (+)- and (À)-2
were measured as shown in Figure 1.
It is justifiable to assume that the comparison of the Cotton
effects exhibited by each sample allows to ascribe an abso-
lute configuration to amide 2, since the stereogenic sulfur
atom in all the compounds is bound to three identical
substituents, and the fourth is only slightly different at a
distant position. As can be seen in Figure 1, the Cotton
effects exhibited by the laevorotatory amide (À)-2 and the
laevorotatory acid (À)-(S)-3 are almost identical. Further-
more, the Cotton effects exhibited by the dextrorotatory
amide (+)-2 and the dextrorotatory nitrile (+)-(R)-1 are
identical as well. Hence, it seems reasonable to assign the
absolute configuration of amide 2 as being (+)-(R).
p-TolSH
O
S
NaIO4
MeONa
MeOH
S
CN
Cl
CN
p-Tol
100%
Scheme 2. Synthesis of racemic sulfoxide 1.
CN
p-Tol
EtOH/H2O
1
(80%)
The enantiopure compounds (+)-(R)-1 and (À)-(S)-1 were
synthesised using a method described by Hiroi and
Umemura.19

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P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 19 (2008) 562–567
Table 1. Nitrilase-promoted hydrolysis of cyanomethyl p-tolyl sulfoxide
Entry
1
Nitrilase
NL 103
Solvent
CHCl3
Time (h)
120
Product; yield (%)
[a]D
+42.5a
ee (%)
Absolute configuration
1; 40.0
2; 30.0
3; 30.0
1; 46.0
2; 27.0
3; 19.0
1; 10.2
2; 32.1
3; 57.1
1; 26.0
2; 33.0
3; 36.4
1; 72.4
3; 25.3
1; 80.0
3; 9.1
2; 10.0
3; 90.0
2; 36.0
3; 60.1
1; 62.8
3; 30.5
1; 100
1; 100
2; 62.0
3; 38.0
2; 34.5
3; 58.2
37c
21c
35d
33c
20c
31d
44c
71c
27d
26c
62c
24d
0
(R)
(S)
(S)
(R)
(S)
(S)
(R)
(R)
(S)
(R)
(R)
(S)
—
À51.5b
À63.8b
+38.0a
À47.0b
À56.0b
+50.1a
+171.2b
À48.8b
+29.8a
+150.0b
À43.0b
0a
2
3
4
NL 103
NL 104
NL 104
Acetone
CHCl3
120
72
Acetone
72
5
6
7
8
9
NL 105
NL 105
NL 106
NL 107
NL 108
CHCl3
Acetone
CHCl3
CHCl3
CHCl3
120
120
72
À2.0b
1d
(S)
—
0a
0
À1.6b
+90.0b
À13.4b
+31.5b
À30.2b
0a
1d
(S)
(R)
(S)
(R)
(S)
—
(S)
—
—
(R)
(S)
(R)
(S)
37c
7d
72
13c
17d
0
120
À3.8b
2d
0
10
11
12
NL 110
NL 111
NL 112
CHCl3
CHCl3
CHCl3
120
120
120
0a
0a
0
+109.0b
À149.1b
+100.0b
À137.5b
45c
83d
42c
76d
13
NL 112
Acetone
120
a In CHCl3 (c 1).
b In MeOH (c 1).
c Determined by chiral HPLC.
d Enantiomeric excess, as determined by comparison of [a]D values.
both nitrile hydrolysis products, that is, amide 2 and acid
3. No substantial differences were observed with the change
of a co-solvent from chloroform (a biphasic system) to ace-
tone (homogeneous solution). Interestingly, nitrilases NL
110 and NL 111 (entries 10 and 11) turned out to be com-
pletely unreactive under the conditions applied. In two
cases, it was possible to recover the enantiomerically en-
riched unreacted substrate 1 (entries 1 and 4), while in three
other cases, the isolated unreacted substrate was racemic
(entries 5, 6 and 9). The stereoselectivity of the hydrolysis
was generally lower as compared to the desymmetrisations
that we previously described.14,15 Nevertheless, the stereo-
chemical outcome of particular experiments allowed us to
draw some conclusions concerning the reaction mecha-
nism. To start with, the reaction in which the absolute con-
figuration of both products, amide 2 and acid 3, were the
same (entries 1 and 2) may be taken as proof for the bidi-
rectional mechanism in which both products are formed
concurrently.13 On the other hand, the cases in which the
absolute configurations of the resulting amide and acid
are opposite (entries 7, 8, 12 and 13) point to the pathway
in which the amide is formed first and then subsequently
hydrolysed. However, the assumption that the real stereo-
recognition is the result of a kinetic resolution of the ini-
tially formed racemic amide must be discarded since the
isolated unreacted substrate was enantiomerically enriched
(entries 1–4). In this context, it should be stressed that the
three examples in which the recovered substrate is racemic
Figure 1. The CD spectra of compounds 1, 2 and 3.
Correctness of this assignment was ultimately confirmed by
chemical, acid-catalysed hydrolysis of (+)-amide 2, which
gave acid (+)-(R)-3. Since the transformation took place
beyond the stereogenic sulfur atom, the absolute configura-
tion of the amide must be (+)-(R)-2.
Inspection of Table 1 clearly shows that in most cases, the
reactions were not selective and result in the formation of

P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 19 (2008) 562–567
565
(entries 5, 6 and 9) show the exclusive formation of the acid
in low yield and with very low enantiomeric excess.
subjected to hydrolysis by selected nitrilases. The results
are shown in Table 2.
Inspection of Table 2 reveals a tendency towards the pref-
erential formation of amide 2 from (+)-(R)-1 and of acid 3
from (À)-(S)-1 for NL 103 (entries 1 and 2) and NL 112
(entries 3 and 4). Although the relationship is not so
clear-cut, probably due to the relatively low stereoselectiv-
ity of the hydrolysis (cf. Table 1, entries 1 and 12), it shows
that the absolute configuration of the substrate has a sub-
stantial influence on the reaction course.
2.3. Attempted nitrilase-catalysed hydrolysis of the racemic
amide 2
To finally clarify the issue of the involvement of amide 2 as
an intermediate in the formation of acid 3, the racemic
amide was synthesised and subjected to hydrolysis by
selected nitrilases in order to investigate the formation of
all possible reaction products (Scheme 5).
O
S
O
Nitrilases 103 , 105 and 112
3. Mechanism of action
no reaction
p-Tol
NH2
phosphate buffer
pH 7.2/acetone
There is no X-ray analysis of nitrilases available so that the
actual structure of their active sites remain unknown. How-
ever, it is generally accepted that cysteine, which is part of
the catalytic triad Cys-Glu-Lys, plays the role of the
reactive amino acid. The sulfhydryl group of cysteine is
believed to attack the nitrile carbon atom in the first step
to form a thioimidate intermediate. The bidirectional
mechanism of nitrile hydrolysis proposed by Sheldon
et al.13 assumes that the thioimidate is the common
primary intermediate for both hydrolysis pathways. Once
formed, it can be transformed in two ways: it either reacts
with a single water molecule to form an amide, or it reacts
with two water molecules to provide an acid. This mecha-
nism also predicts that transformation of the amide into
the corresponding acid is energetically unfavourable.
2
Scheme 5. Attempt to perform a nitrilase-catalysed hydrolysis of amide 2.
Surprisingly, in none of these cases was the formation of
acid 3 observed. This seems obvious for NL 103, which
catalyses the nitrile hydrolysis to form both products 2
and 3 having the same absolute configuration (S), and to
leave the non-racemic unreacted substrate 1 of opposite
absolute (R)-configuration (entries 1 and 2). Hence, it is
logical to assume that this nitrilase must employ the bidi-
rectional mechanistic pathway. However, in the case of
NL 112, which leads to the formation of both products
having opposite absolute configurations (entries 12 and
13), this additional experiment suggests that the amide is
not involved in the formation of the acid. Finally, in the
case of NL 105 (entries 5 and 6), the result shown in
Scheme 5 led us to discard the possible explanation that
the amide is non-stereoselectively formed in a rate-deter-
mining step and then fully hydrolysed to the acid. On the
other hand, the observed lack of reactivity of amides may
only prove that the externally added amide cannot enter
the active site. However, as suggested by a Referee,21 if
the amide was generated within the active site by hydrolysis
of the nitrile, it could still react and this would be compat-
ible with our findings (see the discussion in Section 3).
The aforementioned results strongly favour this mecha-
nism, particularly if one takes into account our findings
that the externally added amide was in none of these cases
transformed into the acid. However, this mechanism can-
not account for all the results obtained. First of all, it is
not applicable to all nitrilases used in these investigations,
since the hydrolysis results were in some cases entirely dif-
ferent. Secondly, it does not explain the stereochemistry of
the reaction. The case in which both the amide and the acid
formed have the same and the recovered unreacted sub-
strate the opposite absolute configurations may be taken
as proof that both reactions take place within the same chi-
ral environment influencing stereorecognition. On the
other hand, the results in which the amide and the acid
formed have opposite absolute configurations lead to an
assumption that each reaction may employ different pock-
ets of the enzyme active site at the enantioselective water
2.4. Nitrilase-catalysed hydrolysis of the enantiomers of 1
To determine the relationship between the absolute config-
uration of substrate 1 and the ratio of the particular hydro-
lysis products 2 and 3, each enantiomer of 1 was separately
Table 2. Nitrilase-promoted hydrolysis of enantiomers of cyanomethyl p-tolyl sulfoxide 1
Entry
1
Nitrilase
NL 103
Substrate
Time (h)
120
Product; yield (%)
[a]D
ee (%)
Absolute configuration
(+)-1 ee >95%
1; 78.0
2; 14.0
3; 8.0
2; 11.0
3; 86.0
2; 86.0
3; 13.0
2; 20.0
3; 75.0
+115
+180
+7.2
À230
À120
+201
+18
>99
75
4
95
66
83
10
66
55
(R)
(R)
(R)
(S)
(S)
(R)
(R)
(S)
(S)
2
3
4
NL 103
NL 112
NL 112
(À)-1 ee = 75%
(+)-1 ee >95%
(À)-1 ee = 75%
120
72
72
À160
À100

566
P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 19 (2008) 562–567
attack on the preliminary thioimidate intermediate. The
fact that each enantiomer of the substrate produces prefer-
entially one of the hydrolysis products may support this
assumption.
5.2. Synthesis of racemic cyanomethyl p-tolyl sulfoxide 1
To a solution of sodium methoxide, prepared from metha-
nol (30 mL) and sodium (1.86 g, 0.081 mol) a solution of
p-toluenethiol (10 g, 0.081 mol) in methanol (100 mL) was
added, followed by chloroacetonitrile (6.12 g, 0.081 mol).
The mixture was stirred at room temperature for 1 h. After
this time TLC revealed only one spot. Methanol was evap-
orated, the residue was treated with water and extracted
with chloroform. After drying over anhydrous MgSO4
and evaporation of CHCl3 cyanomethyl p-tolyl sulfide
was obtained as yellow crystals (13.14 g, 100%); 1H
NMR (CDCl3): d = 2.42 (s, 3H), 3.55 (s, 2H), 7.38–7.62
(m, 4H).
For the sake of the completeness of the discussion, an-
other possible explanation of the stereochemical course
of the reaction investigated can be taken into account.21
Thus, it can be assumed that the nitrile is first converted
into the amide. The active site may be large enough to
enable the amide to rotate within it. For the nitrile, the
sulfoxide oxygen is likely to H-bond to a residue in the
active site, but for the amide it may be the carbonyl which
for some enzymes selectively H-bonds to that residue,
thus inverting the enantioselectivity of the hydrolysis to
the acid. For other enzyme/substrate combinations, such
a rotation may not be possible and so both hydrolyses
occur with the same enantioselectivity. In both cases,
the amide may be expelled from the active site at a rate
commensurate with its subsequent hydrolysis and, if elec-
trostatic effects prevent its re-entry to the active site, this
would explain the fact that both the amide and the acid
are obtained and the external amide does not undergo
hydrolysis.
The sulfide (13.1 g, 0.081 mol) was dissolved in an ethanol/
water mixture (1:1, 200 mL), then NaIO4 (34 g, 0.162 mol)
was added and the mixture was stirred at room tempera-
ture until TLC revealed completion of the reaction (ca.
12 h). A white precipitate was filtered off, ethanol was
evaporated and the aqueous residue was extracted with
chloroform. After drying of the combined organic layers
over anhydrous MgSO4 and evaporation of chloroform,
the residue was purified by column chromatography
(CHCl3) to afford 1 as a white powder (11.54 g, 80%); mp
1
60–62 °C; H NMR (CDCl3): d = 2.42 (s, 3H), 3.74 (AB
system, 2H), 7.38–7.62 (m, 4H); 13C NMR (CDCl3):
d = 21.36, 44.53, 111.19, 123.97, 129.94, 130.18, 138.03,
143.31; MS (CI): m/z 180 (M+H). Anal. Calcd for
C9H9NOS: C, 60.31; H, 5.06; N, 7.81. Found: C, 60.15;
H, 4.91; N, 7.75.
4. Conclusions
We have shown that a variety of nitrilases convert cyano-
methyl p-tolyl sulfoxide 1 into the corresponding amide
and acid, which are formed in various proportions and
with various stereoselectivities, depending on the nitrilase
involved. We have proven that the externally added amide
is not transformed into the acid, which suggests that both
products may be produced in concurrent reactions. We
have found that the absolute configuration of the substrate
exerts substantial influence on the ratio of products. On
this basis, two alternative explanations of the stereochemi-
cal course of the reaction have been presented. Although
most of the results obtained by us are in agreement with
the bidirectional mechanism proposed by Sheldon et al.,13
some of them clearly demonstrate that the Sheldon
mechanism is incapable of accommodating all reactions
outcomes. Possibly, better explanations have to await
determination of crystal structures of nitrilases, which so
far do not exist.
5.3. Synthesis of racemic p-toluenesulfinylacetamide 2
p-Toluenethiol (1.86 g, 0.015 mol) and bromoacetamide
2.07 g (0.015 mol) were dissolved in Et2O (40 mL), and tri-
ethylamine (2.09 mL, 0.015 mol) in Et2O (10 mL) was
added slowly to the solution. The mixture was stirred for
1 h at room temperature and monitored by TLC. After
completion of the reaction, Et2O was evaporated, the resi-
due was treated with water and extracted with CH2Cl2.
After drying of the organic solution over anhydrous
MgSO4 and evaporation of dichloromethane, the corre-
sponding sulfide was obtained as a white powder (2.72 g,
100%). The crude sulfide (TLC pure) was oxidised as above
and purified by column chromatography (CHCl3/MeOH
from 100:1 to 25:1) to afford racemic 2 as a white powder
(2.52 g, 85%); mp 141–143 °C; 1H NMR (CD3CN):
d = 2.40 (s, 3H), 3.58 (AB system, 2H), 5.88 (br s, 1H),
6.50 (br s, 1H), 7.38–7.55 (m, 4H); 13C NMR (CD3CN):
d = 21.40, 47.50, 125.58, 131.21, 140.39, 143.92, 170.29;
MS (CI): m/z 198 (M+H). Anal. Calcd for C9H11NO2S:
C, 54.80; H, 5.62; N, 7.10. Found: C, 54.62; H, 5.70; N,
7.03.
5. Experimental
5.1. General
The enzymes were purchased from BioCatalytic Europe
GmbH, Grambach, Austria. NMR spectra were recorded
on Bruker instruments at 200 MHz with CDCl3, CD3CN
and CD3OD as solvents. Optical rotations were measured
on a Perkin–Elmer 241 MC polarimeter (c 1). Column
chromatography was carried out using Merck 60 silica
gel. TLC was performed on Merck 60 F254 silica gel plates.
The enantiomeric excess (ee) values were determined by
chiral HPLC (Varian Pro Star 210, Chiralpak AS).
5.4. Nitrilase-catalysed kinetic resolution of 1—general
procedure
Cyanomethyl p-tolyl sulfoxide 1 (racemic or optically ac-
tive) (0.100 g, 0.56 mmol) was suspended in a phosphate
buffer solution (pH 7.2) and a co-solvent (see Tables) was
added. After addition of an enzyme (10 mg) the mixture

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567
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was shaken at 30 °C for 72–120 h (see Tables) and moni-
tored by TLC. Then, water was evaporated and the residue
was separated by column chromatography (CHCl3/metha-
nol in gradient) or preparative TLC (CHCl3/methanol 1:1)
to yield the corresponding products 2 and 3 and the unre-
acted substrate 1. The yields and specific rotations are
shown in Tables 1 and 2.
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1
}
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64.1, 125.69, 131.18, 140.38, 143.56, 172.17; MS (CI): m/z
199 (M+H), 154 (MÀCO2). Anal. Calcd for C9H10O3S:
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Acknowledgements
This work was carried out under a Polish-Dutch Joint
Research Project: ‘Dynamic kinetic resolution and desym-
metrisation of sulfur and phosphorus compounds using
enzymes’. Financial support from the Ministry of Science
and Information Society Technologies (Poland), Grant
No. 3 T09A 166 27 for P.K., is gratefully acknowledged.
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21. We thank a Referee for this suggestion.