Synthesis and microbial transformation of β-amino nitriles

Article abstract of DOI:10.1016/j.tet.2005.02.057

Rhodococcus equi A4, Rhodococcus erythropolis NCIMB 11540 and Rhodococcus sp. R312 were investigated towards their ability to produce β-amino amides and acids from β-amino nitriles. The microorganisms show comparable trends: five-membered alicyclic 2-amino nitriles were transformed significantly faster than the six-membered compounds and the products of trans-2-amino nitriles (amides and acids) were formed considerably faster than the cis-counterparts (amides). The trans-five membered nitriles gave the amides (1b, 5b) in excellent enantiomeric excess (94-99%), the biotransformation of trans-six membered substrates resulted in the formation of the acid (3c, 7c) in excellent ee (87-99%). The ee's of the cis-compounds were throughout lower. Fifteen new substances were synthesized and characterized in the course of this work.

Full text of DOI:10.1016/j.tet.2005.02.057

Tetrahedron 61 (2005) 4249–4260
Synthesis and microbial transformation of b-amino nitriles
a
Margit Winkler, Ludmila Martınkova, Astrid C. Knall, Stefan Krahulec
b
a
a
´
´
and Norbert Klempiera,
*
aInstitute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, A-8010 Graz, Austria
bInstitute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ 142 20 Prague 4, Czech Republic
Received 22 December 2004; accepted 21 February 2005
Available online 18 March 2005
Abstract—Rhodococcus equi A4, Rhodococcus erythropolis NCIMB 11540 and Rhodococcus sp. R312 were investigated towards their
ability to produce b-amino amides and acids from b-amino nitriles. The microorganisms show comparable trends: five-membered alicyclic 2-
amino nitriles were transformed significantly faster than the six-membered compounds and the products of trans-2-amino nitriles (amides
and acids) were formed considerably faster than the cis-counterparts (amides). The trans-five membered nitriles gave the amides (1b, 5b) in
excellent enantiomeric excess (94–99%), the biotransformation of trans-six membered substrates resulted in the formation of the acid (3c,
7c) in excellent ee (87–99%). The ee’s of the cis-compounds were throughout lower. Fifteen new substances were synthesized and
characterized in the course of this work.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
NCIMB 11540. These strains express nitrile hydratase and
amidase activity.
b-Amino acids are key structural components of a variety of
natural products and drugs.1,2 Recently, alicyclic b-amino
acids, for example, trans-aminocyclopentane carboxylic
acid, have been used for the synthesis of b-oligopeptides.
The folding properties of such synthetic oligomers with
unnatural backbones into defined three-dimensional struc-
tures have been explored.3–5 On the other hand (1R,2S)-cis-
aminocyclopentane carboxylic acid (cispentacin) itself is a
strong antifungal antibiotic,6 other alicyclic amino acids can
be used in heterocyclic chemistry.7 This interest in b-amino
acids and their synthesis is reflected in several recent
reviews.1,2,8
2. Results and discussion
In previously published work, we have demonstrated that
b-amino nitriles are readily converted to b-amino amides
and acids by nitrile hydratase/amidase containing micro-
organisms.12,13 In this work, we wish to report on the
stereoselective transformation of cis- and trans-amino-
cyclopentane-/hexane nitriles (1a–8a depicted in Fig. 1) in
dependence on ring size and relative configuration of the
In recent years, enzymatic methods have been established as
an alternative to the harsh reaction conditions of chemical
hydrolysis of nitriles.9 The respective biocatalysts—nitril-
ases,10 nitrile hydratases11 and amidases—are frequently
used as whole cell systems or, less frequently, in purified
form by several groups.
In this paper, we report the preparation of b-amino amides
and carboxylic acids by whole cells of Rhodococcus equi
A4, Rhodococcus sp. R312 and Rhodococcus erythropolis
Keywords: Microbial nitrile hydrolysis; Enantioselectivity; b-Amino acids.
*
Corresponding author. Tel.: C43 316 873 8245; fax: C43 316 873 8740;
e-mail: klempier@orgc.tu-graz.ac.at
Figure 1. Structures of racemic b-amino nitriles for whole cell
transformations (only one enantiomer is depicted).
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.02.057

4250
M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
Table 1. Biotransformations of racemic N-protected b-amino nitriles by whole cells—screening
Entry
Substrate
R. equi A4a
R. erythropolis NCIMB 11540b
R. sp. R 312c
Nitrile (%) Amide (%) Acid (%)
Nitrile (%) Amide (%) Acid (%)
Nitrile (%) Amide (%) Acid (%)
1
2
3
4
5
6
7
8
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
0
22
3d
20
1d
13
76
55
96
38
94
18
78
0d
50
0d
87
24
45
1
0
12
4
12
9
1
70
2
92
39
98
26
88
3
52
0
98
29
96
7
16
0
0
10
3
14
6
90
3
54
0
100
37
80
8
24
0
97d
31
99d
0
93
36
91
1
1
2
2
45
2
93
32
94
0
17
2
1
54
6
0
0
0
3
52
6
46
18
90
22
94
21
9
10
11
9
0
15
67
53
21
49
30
a Cell preparation OD 52.
b Cell preparation OD 68.
c Cell preparation OD 77.
d Cell preparation OD 12.
1,2-positions. The acyclic nitriles 9a–11a serve as pre-
cursors for b-phenylalanine, a-methylene-b-amino acids
and b-homo-phenylalanine, respectively, (Fig. 1).
influence of the ring-size on the transformation have been
observed for the analogous b-hydroxy cyclopentane/hexane
carbonitriles. This was previously reported for R. equi A4.14
As described later in this paper, the chemical route to the
single cis-diastereoisomeric nitriles 2a, 4a, 6a and 8a is
tedious and could be accomplished after all by preparative
chromatographic separation of a mixture of cis/trans-
diastereomers on silica gel. No synthetic approach to the
cis-compounds is reported up to now and our efforts to
synthesize the pure cis-isomers resulted in a 13:1 ratio at
best for 2a (Table 3) to nearly 1:1 for compound 6a (Scheme
3). The rate difference found in the microbial transformation
of cis- versus trans-isomers is, therefore, for the purpose of
a diastereospecific preparation, of great practical use.
2.1. Biotransformation
Initial screening experiments were performed using
Rhodococcus equi A4, Rhodococcus erythropolis NCIMB
11540 and Rhodococcus sp. R312. Different from our
previous work, the microorganisms were cultivated in an
inductive medium yielding less biomass, yet with better
activity. The results of these screening experiments are
depicted in Table 1. In summary they suggest the following
general trends: (i) the conversions by the microorganisms
with respect to a single substrate structure are comparable.
No significant differences in product formation were
observed; (ii) the biotransformations of trans-compounds
are in all cases faster than those of their cis-counterparts.
The cis-benzoates 2a and 4a are not accepted as substrates,
in contrast, trans-1a and trans-3a show rapid conversion;
(iii) the conversion of tosylated substrate 6a is resulting in a
significant accumulation of the amide 6b, while nitrile 5a is
converted to the acid 5c without accumulating detectable
amounts of amide; (iv) the transformation of five-membered
alicycles is significantly faster than for the six-membered
compounds.
This diastereodiscrimination was found to be more distinct
in case of the benzoates compared to the tosylates, thus
nitriles 2a and 4a are not converted at all. Unexpectedly,
the conversion rates in general were found to be faster for
the tosylated substrates.
To demonstrate the preparative value, larger scale biotrans-
formations were carried out with substrates chosen accord-
ing to the best screening results in Table 1. The larger scale
biotransformations were carried out with the tenfold
substrate concentration in contrast to the screening experi-
ments (10 mM versus 1 mM for the screening). In order to
provide comparable conversion results, each reaction was
Similar trends, that is, the faster conversion of trans-
substrates compared to the cis-counterparts and the distinct
Table 2. Biotransformations of racemic N-protected b-amino nitriles by whole cells—isolated yieldsa
Entry
Substrateb
R. equi A4
R. erythropolis NCIMB 11540
R. sp. R 312
Nitrile %
(ee %)c
Amide %
(ee %)c
Acid %
(ee %)c
Nitrile %
(ee %)c
Amide %
(ee %)c
Acid %
(ee %)c
Nitrile %
(ee %)c
Amide %
(ee %)c
Acid %
(ee %)c
1
2
3
4
5
6
7
1a
3a
5a
6a
7a
8a
10a
0
40 (94)
22 (56)
14 (O99)
14 (51)
54 (65)
48 (6)
55 (75)
36 (O95)d
44 (2)
0
30 (O99)
16 (67)
13 (O99)
49 (15)
56 (59)
41 (8)
63 (48)
15 (O95)d
86 (5)
0
15 (97)
0
7 (O99)
14 (38)
10 (O99)
75 (7)
42 (77)
43 (4)
25 (6)
87 (15)
7 (O95)d
34 (14)
0
16 (87)
38 (99)
40 (47)
71 (5)
26 (78)
47 (8)
84 (0)
59 (44)
0
50 (16)
24 (98)
50 (10)
91 (1)
61 (82)
46 (30)
11 (51)
33 (47)
44 (10)
73 (0)
0
13 (O99)
0
0
0
0
0
0
10 (11)
2 (32)
a Yields after chromatographic purification.
b Substrate amount given in the Section 4.
c Columns and chromatographic conditions for chiral separation are given in the Section 4.
d Since both enantiomers were not 100% baseline separated, the ee is given as O95%; the second enantiomer could not be actually detected.

M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
4251
stopped after a scheduled time (24 h), deliberately taking
into account a mixture of nitrile, amide and acid. The yields
of the remaining nitriles, product-amides and acids are
given after isolation and chromatographic purification in
Table 2.
AD-H, Chiralcel OJ, Chiral AGP, Chiral HSA and Chirobiotic
R) for the determination of the enantiomeric excess of all
products appears rather surprising. In the majority of cases a
simultaneous separation of nitrile, amide and/or acid was not
feasible. The respective results are included in Table 2. The
detailed conditions are given in the Section 4.
Transformation of the benzoates 1a and 3a resulted, in
consistency with the screening results, in a mixture of amide
and acid. However, the purification of isolated amide 3b
turned out to be troublesome due to its poor solubility even
in MeOH or DMSO. The tosylated nitriles 8a and 10a
were transformed exclusively to the amides. Nitrile 10a,
prepared by aza-Baylis-Hillman reaction, is a precursor for
a-methylene-b-amino acids. The related oxo-analogous
Baylis–Hillman carbonitrile was recently reported to
give exclusively the amide by incubation with cells of
Rhodococcus sp. AJ270.15 These results are in analogy to
our aza-compound 10a.
Despite this, 3c could not be resolved on any of the columns
mentioned, instead, it was finally resolved by gas chromato-
graphic separation of its methyl ester on Chirasil-Dex CB.
Recently the liquid chromatographic separation of unpro-
tected b-amino acids was reported employing a Chirobiotic
T column16 and a chiral crown ether (Crownpak CR(C)).17
The latter one proved to be more efficient in separating cis-
and trans-amino cyclohexane carboxylic acid, however, the
formation of an ammonium ion is a prerequisite for
successful separation. No separation of b-amino amides is
reported up to now.
We have investigated the hydrolysis of cis-2-NHTs-
cyclopentane nitrile (6a) over prolonged reaction time to
find out, whether the transformation can be brought to
completeness with regard to the acid (6c). These experi-
ments were carried out on a 3 mM level in order to obtain
sufficient material for the determination of the ee. In Figure
2, the conversion of 6a using Rhodococcus equi A4 is
plotted against the reaction time. On a 1 mM level, however,
the acid could be detected as the sole product after
prolonged reaction time. This is in agreement with results
we obtained from experiments, whereby the extend of
conversion of 6a was determined in dependence on its
concentration after a constant reaction time (20 h). In this
case, the highest conversions were achieved within a
concentration range of 0.9–1.5 mM of substrate. Rising
the concentration turned out to be accompanied by a
significant drop in conversion. As can be seen from Figure
2, 34% acid could be achieved after an incubation time of
206 h (3 mM). The application of extended reaction time to
other substrates, for example nitrile 10a resulted in 53%
isolated yield of the respective amide (10b) as the only
product after 120 h (70% conversion according to HPLC),
though no acid was formed after this time.
The enantioselectivity of the Rhodococci towards the trans-
isomers (1, 3, 5, 7) is significantly higher as compared to the
cis-counterparts (2, 4, 6, 8), providing the latter were
accepted as substrates at all. Thus, the trans-amino
cyclohexane carboxylic acids 3c and 7c could be prepared
by all three microorganisms with very high ee (95–99%),
instead, the intermediate amides were formed with moder-
ate ee regardless of their protecting group. Differently, the
five-membered trans-amides 1b and 5b were obtained
throughout in very high enantiopurity, but not so their acids.
The high enantiomeric purity of the remaining trans-nitriles
3a and 7a is remarkable, considering the extent of their
conversions(38 and24% resp.). Atleast for the transformation
3a to 3b, this can be attributed to the highly enantioselective
nitrile hydratase reported for Rhodococcus equi A4.18
In Figure 3, the conversion of cis-2-NHTs-cyclopentane
nitrile (6a), amide (6b) and acid (6c) is plotted versus their
enantiomeric excess. Again, the experiments were carried
out on a 3 mM level over a reaction time of 206 h. The curve
in Figure 3 suggests the enantiopurity-conversion depen-
dence expected from a kinetic resolution. Different from the
entries in Table 2, where the acid did not form within the
scheduled reaction time of 24 h, 33% acid could be obtained
in 34% ee after 206 h. Even at low conversion, the ee of the
acid did not exceed 56%. The reasons for that are unknown.
One explanation is the presence of an additional amidase
acting with opposite enantioselectivity.
Figure 2. Biotransformation of 6a by R. equi A4 in dependence on reaction
time. (C) nitrile; (&) amide; (:) acid.
Given the rather small structural variation within the cis-
and trans-series of substrates 1–8, the requirement of six
different chiral HPLC columns (Chiralcel OD-H, Chiralpak
Figure 3. Enantiomeric excess of 6a–c in dependence on conversion by
R. equi A4. (C) nitrile; (&) amide; (:) acid.

4252
M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
2.2. Synthesis of substrates
We have made several approaches to prepare 1a–4a, the
most preferred one for the trans-compounds being via the
aziridine ring opening. Generally, the synthetic availability
of benzoylated aziridines is limited.13 However, the
benzoylation of the corresponding tosylates with subsequent
tosyl-deprotection turned out to be a practicable synthetic
protocol for trans-compounds 1a and 3a, which has already
been used in this laboratory for the preparation of Boc-
protected amino nitriles.13 To obtain the cis-compounds 2a
and 4a, we developed a three step synthesis starting from
commercially available adiponitrile and heptanedinitrile,
respectively. Thorpe–Ziegler cyclization of the latter using
NaH yielded unsaturated aminonitriles 12 and 13.19
Scheme 2.
that applying other dehydrating agents than molecular
sieves partially results in formation of unwanted Baylis–
Hillman product, that is the alcohol. Use of quinuclidine
resulted in slightly better conversion rates versus DABCO.
In-triflate was superior to all the other Lewis acids with
respect to conversion and Baylis–Hillman product forma-
tion. Summarizing, the price of quinuclidine and In-triflate
compared to DABCO and Ti(i-OPr)4 rather suggests an
application of the latter reagents.
After benzoylation (BzCl/pyridine) to 14 and 15, the double
bond was catalytically hydrogenated using Pd/C in MeOH
at 50 bar and ambient temperature (Scheme 1). The cis-
selectivity in case of 2a was 90% after 4 h and 97%
conversion and could not be optimized further (Table 3). In
the case of the six-membered ring the desired cis-compound
4a could be isolated as a pure diastereoisomer after 3 days
under the same conditions at 33% conversion.
cis-Tosylates 6a and 8a could not be prepared in an
analogous way to Scheme 1, since the tosylation of 12 and
13 resulted in complex mixtures and the desired products
could only be isolated in yields lower than 5%. Compounds
6a and 8a were available through the three step synthesis
shown in Scheme 3, followed by silica gel separation of the
diastereomers.22
Scheme 1.
Scheme 3.
N-Ts-protected compounds 5a and 7a were prepared as
described in our previous paper.12 The same methodology
was used for 9a and 11a via 16 and 17, respectively,20
(Scheme 2). The known structure 10a was prepared by aza-
Baylis-Hillman reaction, although the reaction conditions
had to be optimized. We applied a number of modifications
of the procedure described in the literature,21 such as
Reference amides were prepared according to a standard
procedure,23 except 10b, which was available only through
biotransformation.
The chemical hydrolysis to reference acids was attempted
by refluxing the corresponding nitriles in NaOH concd.
Not surprisingly, the majority of the acids, that is, 1c–4c and
6c–8c could not be prepared by this way. Chemical
hydrolysis of 7a and 8a resulted in mixtures of nitrile,
˚
variation of the dehydrating agent (molecular sieve 4 A,
PPh3, DCC, CDI), variation of the base (DABCO,
quinuclidine) and the Lewis acid (Ti(i-OPr)4, Sc–, Y–,
Yb–, Gd–, La– and In-triflate). GC/MS evaluation revealed
Table 3. Optimization of reaction conditions for catalytic hydrogenation of 14
Entry
Catalyst
Solvent
Pressure (bar)
Time
14 (%)
1a (%)
2a (%)
1
2
3
4
5
6
7
8
9
Rh/Al2O3
10% Pt/C
10% Pd/C
10% Pd/C
10% Pd/C
Wilkinson
5% Pd/C
5% Pd/C
5% Pd/C
MeOH
MeOH
MeOH
Ethyl acetate
MeOH/H2O
MeOH
Ethyl acetate
MeOH
MeOH
1
1
1
1
2d
2d
7d
13d
2d
4 h
3d
100
100
4
!1
!1
100
9
0
0
0
0
16
12
26
0
17
21
7
80
84
74
0
74
79
90
1
50
50
50
50
20 h
4 h
0
3

M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
4253
amide and acid, even at reaction times longer than 14 days.
cis-Nitrile 6a epimerized under these drastic conditions to
the trans-acid 5c.
membered compounds. More specific, the products of the
trans-2-amino nitriles (amides and acids) are formed
considerably faster than the products of the cis-counterparts
(only amides). With exception of the a-methylene-b-amino
carbonitrile structure, which is exclusively transformed to
the amide, the other open chain nitriles show no clear trend
concerning the formation of a preferred product. These
product pattern mentioned in context with the alicyclic
substrates (1–8) is similar for the three microorganisms
investigated. In the same way, the enantioselectivities
achieved for the alicyclic compounds are strongly depen-
dent on the structure. Thus, the trans-five-membered nitriles
give exclusively the amides in excellent enantiopurity
(94–99% ee), in contrast, the biotransformation of trans-
six-membered substrates result in the formation of the acid
in excellent enantiopurity (87–99% ee). The ee’s of
the corresponding cis-compounds are throughout lower.
The concentration level of the biotransformation reaction
was found to exert some influence on the final distribution of
the products. As a consequence of the kinetic resolution,
improvement of product yield is gained at the expense of the
enantiomeric purity.
The benzoylated compounds were deprotected under these
conditions. Thus, 2c–4c, 7c and 8c were prepared by
protecting the corresponding commercially available carb-
oxylic acids using standard conditions. However, standard
tosylation of (G)-cispentacin 18 to (G)-6c yielded
compound 19, which was confirmed by NOESY-NMR
and single crystal structure (Fig. 4). A proposed mechanism
for this reaction path is depicted in Scheme 4, where the
amide 19 is generated by aminolysis of the intermediate
b-lactam with another molecule of cispentacin 18. The
same side reaction did not occur during the benzoylation of
(G)-cispentacin. Thus, the carboxylic acid 6c was solely
available by biotransformation.
In summary, the results suggest the application of the
investigated Rhodococci to the enantioselective hydrolysis
of five- and six-membered 2-amino substituted carbocyclic
nitriles on a preparative scale.
4. Experimental
Analytical thin layer chromatography was carried out on
Merck silica gel 60 F254 plates. Flash chromatography was
performed on Merck silica gel 60, 230–400 mesh.
Analytical HPLC was carried out with a Hewlett Packard
Series 1100 HPLC using a G1315A diode array detector or
MWD detector. For achiral analysis a LiChrospher 100
RP18e column (5 mm) was used. Chiral analysis was carried
out with an Astec Chirobiotic R column, a Chromtech
Chiral AGP 100.4 column (5 mm), a Chromtech Chiral HSA
100.4 column (5 mm), a Daicel Chiralpak AD-H (5 mm) and
a Chiralcel OD-H column (5 mm). For preparative HPLC a
Merck-Hitachi LC-6200 pump and L-4000 UV-detector
was used. Separations were performed on a 21.2!250 mm
Zorbax SB-C18 preparative HPLC column. CI-mass spectra
were recorded with an Agilent 5973N MSD and Agilent
6890 Series II GC. Chiral gas chromatographic analyses
were carried out on a Chrompack Chirasil-Dex CB (25 m!
0.32 mm; 0.25 mm film thickness; hydrogen carrier gas).
EI-mass spectra were recorded with a Hewlett-Packard
5972 MSD and HP 6890 Series II GC equipped with a
HP5 column. 1H NMR (199.98 MHz) and 13C NMR
(50.29 MHz) spectra were recorded on a Varian GEMINI-
200BB. 1H (499.82 MHz) and 13C NMR (125.69 MHz)
Figure 4. Molecular structure of 19 with thermal ellipsoids at the 30%
probability level.
trans-2-Amino cyclopentane carboxylic acid 1c was
prepared by TFA-catalyzed deprotection of (G)-trans-2-
tert-butoxycarbonylamino cyclopentane carboxylic acid13
and subsequent benzoylation. (G)-2-[Phenyl-(toluene-4-
sulfonylamino)-methyl]-acrylic acid 10c could neither be
prepared by chemical nor enzymatic hydrolysis under the
above mentioned conditions.
To our knowledge, 1a–c, 2a–c, 4a, 6a–c, 8a–b, 10b, 15 and
19 have not been reported elsewhere up to now. Their
spectroscopic data as well as other physical data are given in
the Section 4.
3. Conclusion
The biotransformation of five-membered alicyclic 2-amino
nitriles proceeds significantly faster than in case of the six-
Scheme 4.

4254
M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
spectra were recorded on a Varian INOVA 500. 2D-tech-
niques (HSQC, HMBC) as well as DEPT, NOESY, TOCSY
and deuterium exchange were used to assist in structure
elucidation. Melting points were determined on a Electro-
thermal MEL-TEMP apparatus and are uncorrected. The
elemental analyses were performed on a Heraeus vario EL.
X-ray crystal structures were measured on a Bruker AXS
SMART APEX CCD diffractometer.
4.2.1. (G)-2-[Phenyl-(toluene-4-sulfonylamino)-methyl]-
acrylamide (10b). R. equi A4 (13.8 g wet cells, 150 ml
buffer, OD610Z48). Yield 79 mg (53%) at 70% conversion
after 120 h from 141 mg (G)-10a (3 mM); R. equi A4 (6.0 g
wet cells, 60 ml buffer, OD610Z60). Yield 20 mg (10%,
eeZ11%) from 187 mg (G)-10a (10 mM); R. ery. 11540
(0.8 g wet cells, 10 ml buffer, OD610Z46). Yield 3 mg (2%,
eeZ32%) from 125 mg (G)-10a (40 mM); R. sp R312
(5.0 g wet cells, 40 ml buffer, OD610Z77). Yield 33 mg
(25%, eeZ6%) from 124 mg (G)-10a (10 mM). White
solid, mp 199–200 8C; 1H NMR (DMSO-d6) d 2.32 (s, 3H),
5.42 (d, 1H, JZ9.8 Hz), 5.55 (s, 1H), 5.71 (s, 1H), 6.95 (s,
1H, NH2), 7.04–7.06 (m, 2H), 7.14–7.19 (3H, m), 7.25 (d,
2H, JZ8.3 Hz), 7.48 (s, 1H, NH2), 7.54 (d, 2H, JZ8.3 Hz),
8.32 (d, 1H, JZ9.8 Hz, NH); 13C NMR (DMSO-d6) d 21.63,
57.44, 118.98, 127.12, 127.75, 127.93, 128.71, 129.94,
139.33, 140.59, 142.96, 144.56, 168.84; (CI, methane) m/z
331 (MC1)C (3), 313 (42), 260 (6), 172 (91), 160 (100),
155 (24). Anal. Calcd for C17H18N2O3S: C, 61.80; H, 5.49;
N, 8.48. Found: C, 60.62; H, 5.48; N, 8.04. Chiral separation
on Chiralcel OD-H, n-heptane/i-propanol 50:50, 0.38 ml/
min, 15 8C.
4.1. Microorganisms and cultivation
4.1.1. Microorganisms. Rhodococcus equi A4 was isolated
ˇ
´
´
in Prague by the group of V. Kren and L. Martınkova and is
deposited in the Czech Collection of Microorganisms,
Masaryk University, Brno, Czech Republic. Rhodococcus
sp. R312 is commercially available (CBS 717.73).
Rhodococcus erythropolis NCIMB 11540 was obtained
from DSM Research, The Netherlands. R. equi A4 was
maintained on MPA agarplates. Merck Standard I nutrient
agar medium was used for maintainance of R. sp R312 and
R. erythropolis NCIMB 11540 on agarplates.
4.1.2. Cultivation. All strains were cultivated on BSB
medium24 using acetonitrile as the only source of nitrogen.
The microorganisms were cultured at 30 8C and 150 rpm in
250 ml shaking flasks, each containing 100 ml of the above
described medium. During the exponential phase of growth,
the cells were harvested by centrifugation (5500 rpm,
20 min, 4 8C). The cells were washed with phosphate buffer
(4.98 g/l Na2HPO4, 2.04 g/l KH2PO4, pH 7.5) and again
centrifuged.
4.2.2. (G)-cis-2-(Toluene-4-sulfonylamino)-cyclopentane
carboxylic acid (6c). R. sp R312 (27.0 g wet cells, 250 ml
buffer, OD610Z61). Yield 65 mg (92%) at 100% conversion
after 76 h from 66 mg (G)-6a (1 mM). White solid, mp
139–141 8C; 1H NMR (CDCl3) d 1.48–1.56 (m, 1H), 1.65–
1.82 (m, 3H), 1.88–2.00 (m, 2H), 2.43 (s, 3H), 2.91 (dt, 1H,
JZ5.7, 7.7 Hz, H-1), 3.78 (m, 1H, JZ8.1 Hz, H-2), 6.16 (d,
1H, JZ9.3 Hz, NH), 7.30 (d, 2H, JZ8.3 Hz), 7.76 (d, 2H,
JZ8.3 Hz), 5.70 (s, br, 1H, COOH); 13C NMR (CDCl3) d
21.78, 21.95, 28.06, 31.89, 46.55, 56.50, 127.31, 130.01,
137.77, 143.75, 178.46. Anal. Calcd for C13H17NO4S: C,
55.12; H, 6.05; N, 4.94. Found: C, 55.11; H, 5.99; N, 4.97.
Chiral separation on Chirobiotic R, polar organic mode
(MeOH/Et3N/AcOH 100:0.4:0.1), 0.80 ml/min, ambient
temperature.
4.1.3. Screening. For screening experiments, 0.5 ml of cell
suspension was put into Eppendorf vessels. The substrates
were added as 200 mM solutions in DMSO (2.5 ml) to give a
final concentration of 1 mM. The reactions were carried out
at 32 8C in an Eppendorf Thermomixer at 850 rpm. After
20 h, 50 ml of HCl (1 N) were added. Unreacted nitriles and
products were extracted twice with ethyl acetate. Conver-
sions were determined by RP18 HPLC analysis. In the case
of the benzoates 1–4 the ethyl acetate was removed and the
remaining mixture diluted with methanol.
4.3. General procedure for benzoylation and
detosylation
To a solution of 5a or 7a in anhydrous CH3CN 3.0 equiv of
benzoylchloride and DMAP were added. After refluxing for
4 h the solvent was removed under reduced pressure. The
remaining oil was diluted with CH2Cl2 and washed with
saturated NH4Cl. The aqueous phase was extracted with
CH2Cl2 three times. The combined organic layers were
dried with Na2SO4 and concentrated to give a crude oil,
which was used for detosylation without further purifi-
cation. Thus, the oil was dissolved in anhydrous MeOH.
After addition of 5.0 equiv of magnesium turnings, the
mixture was sonicated for 15 min. The mixture was allowed
to react for another 45 min. Then, it was filtered through a
plug of celite and washed with MeOH. The solvent was
evaporated and the remaining oil diluted with CH2Cl2. The
organic layer was washed with HCl (2 N), NaHCO3 satd and
brine. After drying with Na2SO4 and evaporation, the
product was purified using silica gel chromatography.
4.2. General procedure for large scale biotransformation
For preparative biotransformations, the cells were resus-
pended in the above given buffer, usually about 1 g wet cell
weight per 10 ml of buffer. The optical density of the cell
suspension was intended to be about 50, but as three
different strains were used, the OD-value was not repro-
ducible. The substrates were added as solutions in DMSO to
give a final concentration of 10 mM. The DMSO portion
was 2.5% (v/v) of the total volume. The reaction was
performed in a rotary shaker at 150 rpm and 30 8C for 24 h.
The biotransformations were stopped by addition of HCl
(2 N). After centrifugation, unreacted nitriles as well as
products were extracted from the aqueous phase using ethyl
acetate. To prevent from losses of precipitated nitrile, amide
and acid, the cells were also resuspended in ethyl acetate
and again centrifuged. The combined organic layers were
dried with Na2SO4 and concentrated in vacuo. Unreacted
nitrile and products were purified by silica gel
chromatography.
4.3.1. (G)-trans-N-(2-Cyano-cyclopentyl)-benzamide
(1a). White solid, mp 129–130 8C, H NMR (CDCl3) d
1.70–1.78 (m, 1H), 1.83–1.92 (m, 2H), 1.96–2.03 (m, 1H),
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M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
4255
2.15–2.21 (m, 1H), 2.22–2.29 (m, 1H), 2.83 (dt, 1H, JZ8.3,
7.1 Hz, H-1), 4.54 (m, 1H, JZ7.3 Hz, H-2), 6.46 (d, 1H, JZ
6.3 Hz, NH), 7.42 (m, 2H), 7.51 (t, 1H, JZ7.3 Hz), 7.75 (m,
2H); 13C NMR (CDCl3) d 23.20, 29.62, 31.77, 34.95, 56.40,
121.80, 127.23, 128.88, 132.10, 134.05, 167.80; m/z (EI)
213 (MK1)C (8), 161 (4), 105 (100), 77 (35). Anal. Calcd
for C13H14N2O: C, 72.87; H, 6.59; N, 13.07. Found: C,
71.19; H, 6.59; N, 12.43.
from 137 mg (G)-3a (10 mM). White solid, mp 217–
219 8C; H NMR (DMSO-d6) d 1.12–1.22 (m, 1H), 1.24–
1
1.35 (m, 2H), 1.42 (ddd, 1H, JZ12.7, 12.7, 3.4 Hz), 1.69
(m, 2H), 1.79–1.82 (m, 1H), 1.90 (m, 1H), 2.43 (dt, 1H, JZ
3.3, 11.6 Hz, H-1), 3.98 (m, 1H, H-2), 7.42–7.44 (m, 2H),
7.47–7.51 (m, 1H), 7.76–7.78 (m, 2H), 8.29 (d, 1H, JZ
8.8 Hz, NH), 12.01 (s, br, 1H, COOH); 13C NMR (DMSO-
d6) d 25.08, 25.30, 29.55, 32.67, 48.94, 50.21, 127.91,
128.83, 131.68, 135.55, 166.01, 176.08. Anal. Calcd for
C14H17NO3: C, 68.00; H, 6.93; N, 5.66. Found: C, 66.78; H,
6.96; N, 5.62. Chirasil-Dex CB after derivatization with
(trimethysilyl) diazomethane; 100 8C (2 min), 58/min
200 8C (H2, 1 bar).
4.3.2. (G)-trans-N-(2-Cyano-cyclohexyl)-benzamide
(3a). White solid, mp 167–168 8C; H NMR (CDCl3) d
1
1.31–1.39 (m, 1H), 1.47–1.55 (m, 2H), 1.72–1.84 (m, 3H),
2.13–2.16 (m, 2H), 2.82 (dt, 1H, JZ3.4, 10.0 Hz, H-1), 4.20
(m, 1H, H-2), 6.23 (d, 1H, JZ7.8 Hz, NH), 7.44 (m, 2H),
7.52 (m, 1H), 7.78 (m, 2H); 13C NMR (CDCl3) d 24.00,
24.04, 28.85, 31.66, 34.49, 50.27, 120.73, 127.23, 128.90,
132.02, 134.45, 167.47; m/z (EI) 228 MC (10), 160 (4), 123
(5), 105 (100), 77 (43). Anal. Calcd for C14H16N2O: C,
73.66; H, 7.06; N, 12.27. Found: C, 74.16; H, 7.27; N, 12.08.
Chiral separation on Chiralcel OD-H, n-heptane/i-propanol
50:50, 0.38 ml/min, 15 8C.
4.4.4. (G)-cis-2-Benzoylamino-cyclohexane carboxylic
acid (4c). White solid, mp 175–176 8C; H NMR (DMSO-
1
d6) d 1.32–1.41 (m, 2H), 1.49–1.65 (m, 4H), 1.77–1.83 (m,
1H), 1.96–2.02 (m, 1H), 2.74 (m, 1H, H-1), 4.30 (m, 1H,
H-2), 7.43 (m, 2H), 7.50 (m, 1H), 7.74–7.76 (m, 2H), 8.00
(d, 1H, JZ8.3 Hz, NH), 12.22 (s, br, 1H, COOH); 13C NMR
(DMSO-d6) d 22.78, 23.55, 25.49, 29.95, 44.41, 48.19,
128.07, 128.83, 131.72, 135.60, 166.71, 175.52. Anal. Calcd
for C14H17NO3: C, 68.00; H, 6.93; N, 5.66. Found: C, 66.72;
H, 6.86; N, 5.60.
4.4. General procedure for benzoylation
Analogous to Section 4.3 but pyridine was used as the base.
4.4.5. N-(2-Cyano-cyclopent-1-enyl)-benzamide (14).
White solid; mp 90–92 8C; H NMR (200 MHz, CDCl3) d
1
4.4.1. (G)-trans-2-Benzoylamino-cyclopentane carb-
oxylic acid (1c). R. equi A4 (7.1 g wet cells, 70 ml buffer,
OD610Z40): Yield 90 mg (55%, eeZ75%) from 150 mg
(G)-1a (10 mM); R. ery. 11540 (8.2 g wet cells, 80 ml
buffer, OD610Z32). Yield 118 mg (63%, eeZ48%) from
171 mg (G)-1a (10 mM); R. sp R312 (7.9 g wet cells, 70 ml
buffer, OD610Z46). Yield 192 mg (87%, eeZ15%) from
150 mg (G)-1a (10 mM). White solid, mp 184–186 8C; 1H
NMR (DMSO-d6) d 1.53–1.65 (m, 2H), 1.67–1.77 (m, 2H),
1.94–2.00 (m, 2H), 2.76 (dt, 1H, JZ8.8, 7.3 Hz, H-1), 4.44
(m, 1H, JZ7.4 Hz, H-2), 7.44 (m, 2H), 7.50 (m, 1H), 7.81–
7.83 (m, 2H), 8.47 (d, 1H, JZ7.8 Hz, NH), 12.20 (s, br, 1H,
COOH); 13C NMR (DMSO-d6) d 23.93, 29.35, 33.27, 49.98,
54.87, 127.96, 128.88, 131.80, 135.17, 166.63, 176.72.
Anal. Calcd for C13H15NO3: C, 66.94; H, 6.48; N, 6.00.
Found: C, 65.86; H, 6.40; N, 5.96. Chiral separation on
Chirobiotic R, polar organic mode (MeOH/Et3N/AcOH
100:0.4:0.1), 0.80 ml/min, ambient temperature.
2.03 (m, 2H), 2.55 (t, 2H, JZ6.8 Hz), 3.22 (t, 2H, JZ
7.7 Hz), 7.42–7.61 (m, 3H), 7.92 (m, 2H), 8.39 (s, 1H, NH);
13C NMR (50 MHz, CDCl3) d 22.44, 30.33, 33.84, 89.90,
116.62, 127.72, 129.16, 133.03, 133.24, 156.67, 164.90; m/z
(EI) 211 (MK1)C (31), 105 (100), 77 (50), 51 (11).
4.4.6. N-(2-Cyano-cyclohex-1-enyl)-benzamide (15).
White solid; mp 102–104 8C; H NMR (200 MHz, CDCl3)
1
d 1.71–1.77 (m, 4H), 2.33 (m, 2H), 3.00 (m, 2H), 7.45–7.58
(m, 3H), 7.83 (m, 2H), 8.20 (s, 1H, NH); 13C NMR
(50 MHz, CDCl3) d 21.24, 21.72, 25.69, 27.96, 91.38,
118.30, 127.48, 129.23, 132.82, 133.93, 152.30, 165.25; m/z
(EI) 226 (M)C (9), 206 (1), 105 (100), 77 (39), 51 (9).
4.5. General procedure for aziridine formation and ring
opening
See Ref. 20.
4.4.2. (G)-cis-2-Benzoylamino-cyclopentane carboxylic
acid (2c). White solid, mp 187–189 8C; H NMR (DMSO-
1
4.5.1. (G)-2-Phenyl-1-(toluene-4-sulfonyl)-aziridine
(16). White solid; H NMR (200 MHz, CDCl3) d 2.39 (d,
d6) d 1.47–1.55 (m, 1H), 1.73–1.86 (m, 3H), 1.87–2.00 (m,
2H), 2.93 (dt, 1H, JZ7.3, 7.8 Hz, H-1), 4.55 (m, 1H, JZ
7.5 Hz, H-2), 7.42 (m, 2H), 7.49 (m, 1H), 7.76–7.78 (m,
2H), 8.17 (d, 1H, JZ8.3 Hz, NH), 11.96 (s, br, 1H, COOH);
13C NMR (DMSO-d6) d 22.90, 27.81, 31.50, 47.91, 52.98,
128.09, 128.74, 131.65, 135.58, 166.84, 175.30. Anal. Calcd
for C13H15NO3: C, 66.94; H, 6.48; N, 6.00. Found: C, 66.59;
H, 6.47; N, 6.42.
1
1H, JZ4.5 Hz), 2.43 (s, 3H), 2.98 (d, 1H, JZ7.3 Hz), 3.78
(dd, 1H, JZ7.3, 4.5 Hz), 7.20–7.37 (m, 7H), 7.87 (d, 2H,
JZ7.9 Hz); 13C NMR (50 MHz, CDCl3) d 21.89, 36.18,
41.28, 126.81, 128.19, 128.55, 128.81, 130.14, 135.20,
135.29, 144.92; m/z (EI) 155 (1), 118 (32), 91 (100), 77 (5),
65 (44).
4.4.3. (G)-trans-2-Benzoylamino-cyclohexane carb-
oxylic acid (3c). R. equi A4 (4.1 g wet cells, 40 ml buffer,
OD610Z40). Yield 36 mg (36%, eeO95%) from 93 mg
(G)-3a (10 mM); R. ery. 11540 (9.0 g wet cells, 90 ml
buffer, OD610Z38). Yield 33 mg (15%, eeO95%) from
205 mg (G)-3a (10 mM); R. sp. R312 (7.0 g wet cells,
60 ml buffer, OD610Z54). Yield 11 mg (7%, eeO95%)
4.5.2. (G)-2-Benzyl-1-(toluene-4-sulfonyl)-aziridine (17).
White solid; H NMR (CDCl3) d 2.18 (d, 1H, JZ4.9 Hz),
2.43 (s, 3H), 2.69 (dd, 1H, JZ14.5, 7.1 Hz), 2.72 (d, 1H,
JZ7.1 Hz), 2.82 (dd, 1H, JZ14.5, 4.9 Hz), 2.96 (tt, 1H, JZ
7.1, 4.9 Hz), 7.03–7.06 (m, 2H), 7.15–7.18 (m, 3H), 7.22 (d,
2H, JZ8.6 Hz), 7.69 (d, 2H, JZ8.6 Hz); 13C NMR (CDCl3)
d 21.87, 33.07, 37.73, 41.43, 126.74, 128.11, 128.69,
1

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M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
128.96, 129.83, 135.06, 137.25, 144.56; m/z (EI) 287 MC
(5), 172 (4), 155 (6), 132 (100), 105 (57), 91 (64).
temperature, the solvent was removed under reduced
pressure. The crude residue was diluted with NaOH (1 N).
Solid NaCl was added to give a 10% solution. The aqueous
phase was extracted with CH2Cl2. The product was removed
from the organic phase by extraction with HCl (2 N)
(CAUTION! Possible formation of HCN). Subsequently,
the aqueous phase was made alkaline with NaOH concd and
extracted four times with CH2Cl2. After drying with
Na2SO4, the solvent was removed to give a brown oil
which was used for standard tosylation of nitriles (see
Section 4.7).
4.5.3. (G)-trans-N-(2-Cyano-cyclopentyl)-4-methyl ben-
zene sulfonamide (5a). White solid, mp 109–110 8C; H
1
NMR (CDCl3) d 1.44–1.51 (m, 1H), 1.65–1.80 (m, 2H),
1.83–1.90 (m, 1H), 1.93–2.00 (m, 1H), 2.06–2.13 (m, 1H),
2.44 (s, 3H), 2.83 (dt, 1H, JZ8.6, 6.2 Hz, H-1), 3.73 (m, 1H,
JZ6.6 Hz, H-2), 5.72 (d, 1H, JZ7.2 Hz, NH), 7.35 (d, 2H,
JZ8.3 Hz), 7.81 (2H, d, JZ8.3 Hz); 13C NMR (CDCl3) d
21.84, 22.88, 29.22, 32.85, 35.89, 58.92, 121.41, 127.49,
130.26, 136.76, 144.36; m/z (EI) 264 MC (10), 210 (27),
155 (38), 109 (26), 91 (100). Anal. Calcd for C13H16N2O2S:
C, 59.07; H, 6.10; N, 10.60. Found: C, 59.63; H, 6.13; N,
10.63. Chiral separation on Chiralcel AD-H, n-heptane/
i-propanol 50:50, 0.50 ml/min, 15 8C.
4.7. General procedure for tosylation of amino nitriles
The unprotected amino nitriles were suspended in CH2Cl2.
1.1 equiv of tosylchloride and 1.5 equiv of Et3N were added
and the mixture was allowed to react at reflux. The reaction
was monitored by TLC. After disappearance of starting
material, the organic phase was washed with HCl (2 N),
NaHCO3 satd and NaCl satd. After drying with Na2SO4 the
organic solvent was removed and the products were
separated by silica gel chromatography.
4.5.4. (G)-trans-N-(2-Cyano-cyclohexyl)-4-methyl-ben-
zene sulfonamide (7a). White solid, mp 106–108 8C; H
1
NMR (CDCl3) d 1.25–1.39 (m, 3H), 1.58–1.68 (m, 3H),
1.93–1.97 (m, 1H), 2.01–2.06 (m, 1H), 2.44 (s, 3H), 2.62–
2.68 (m, 1H, H-1), 3.35–3.41 (ddt, 1H, JZ4.1, 8.3, 8.3 Hz,
H-2), 5.23 (d, 1H, JZ8.3 Hz, NH), 7.34 (d, 2H, JZ8.5 Hz),
7.82 (d, 2H, JZ8.5 Hz); 13C NMR (CDCl3) d 21.83, 22.78,
23.14, 27.43, 31.66, 34.69, 52.91, 120.46, 127.46, 130.11,
137.31, 144.19; m/z (EI) 278 MC (4), 210 (33), 155 (32),
123 (16), 91 (100). Anal. Calcd for C14H18N2O2S: C, 60.41;
H, 6.52; N, 10.06. Found: C, 59.72; H, 6.42; N, 9.51. Chiral
separation on Chiralcel OD-H, n-heptane/i-propanol 50:50,
0.38 ml/min, 15 8C.
4.7.1. (G)-cis-N-(2-Cyano-cyclopentyl)-4-methyl-ben-
zene sulfonamide (6a). White solid, mp 117–119 8C; H
1
NMR (CDCl3) d 1.57–1.65 (m, 2H), 1.82–1.96 (m, 3H),
1.98–2.04 (m, 1H), 2.44 (s, 3H), 2.83 (ddd, 1H, JZ7.3, 6.8,
3.4 Hz, H-1), 3.77 (m, 1H, H-2), 5.29 (d, 1H, JZ8.8 Hz,
NH), 7.33 (d, 2H, JZ8.3 Hz), 7.81 (d, 2H, JZ8.3 Hz); 13C
NMR (CDCl3) d 20.85, 21.82, 28.28, 30.77, 35.23, 56.18,
119.91, 127.41, 130.15, 137.51, 144.15; m/z (EI) 264 MC
(16), 210 (31), 155 (40), 109 (28), 91 (100). Anal. Calcd for
C13H16N2O2S: C, 59.07; H, 6.10; N, 10.60. Found: C, 59.30;
H, 5.88; N, 10.75. Chiral separation on Chiralpak AD-H,
EtOH, 0.55 ml/min, 40 8C.
4.5.5. (G)-N-(2-Cyano-1-phenyl-ethyl)-4-methyl-ben-
zene sulfonamide (9a). White solid, mp 141–142 8C; H
1
NMR (CDCl3) d 2.41 (s, 3H), 2.90 (dd, 1H, JZ16.5,
7.0 Hz), 2.95 (dd, 1H, JZ16.5, 5.5 Hz), 4.57 (dt, 1H, JZ
5.5, 7.0 Hz), 5.40 (d, 1H, JZ7.0 Hz, NH), 7.11–7.13 (m,
2H), 7.24 (d, 2H, JZ8.6 Hz), 7.27–7.29 (m, 3H), 7.67 (d,
2H, JZ8.6 Hz); 13C NMR (CDCl3) d 21.80, 26.55, 54.38,
116.69, 126.48, 127.38, 129.22, 129.44, 130.05, 136.70,
137.38, 144.29; m/z (EI) 260 (60), 155 (52), 145 (5), 91
(100), 77 (20). Anal. Calcd for C16H16N2O2S: C, 64.98; H,
5.37; N, 9.33. Found: C, 64.19; H, 5.33; N, 9.26.
4.7.2. (G)-cis-N-(2-Cyano-cyclohexyl)-4-methyl-benzene
sulfonamide (8a). White solid, mp 133–134 8C; H NMR
1
(CDCl3) d 1.12–1.21 (m, 1H), 1.35–1.52 (m, 3H), 1.53–1.61
(m, 2H), 1.68–1.72 (m, 1H), 1.88–1.91 (m, 1H), 2.37 (s,
3H), 3.01 (m, 1H, H-1), 3.24 (m, 1H, JZ4.1 Hz, H-2), 4.91
(d, 1H, JZ8.3 Hz, NH), 7.25 (d, 2H, JZ8.3 Hz), 7.71 (d,
2H, JZ8.3 Hz); 13C NMR (CDCl3) d 21.13, 21.82, 24.80,
27.91, 29.89, 35.76, 52.72, 119.52, 127.11, 130.18, 138.10,
144.08; m/z (EI) 278 MC (0.1), 210 (1), 155 (2), 123 (4), 91
(100). Anal. Calcd for C14H18N2O2S: C, 60.41; H, 6.52; N,
10.06. Found: C, 60.60; H, 6.63; N, 10.01. Chiral separation
on Chiralpak AD-H, n-heptane/i-propanol 50:50, 0.5 ml/
min, 15 8C.
4.5.6. (G)-N-(1-Benzyl-2-cyano-ethyl)-4-methyl-benzene
sulfonamide (11a). White solid, mp 106–107 8C; 1H NMR
(CDCl3) d 2.43 (s, 3H), 2.57 (dd, 1H, JZ16.6, 3.9 Hz), 2.66
(dd, 1H, JZ16.6, 6.3 Hz), 2.78 (dd, 1H, JZ14.0, 7.6 Hz),
2.90 (dd, 1H, JZ14.0, 6.8 Hz), 3.64 (m, 1H), 4.79 (d, 1H,
JZ7.3 Hz, NH), 6.98–7.00 (m, 2H), 7.19–7.23 (m, 5H),
7.55 (d, 2H, JZ8.3 Hz); 13C NMR (CDCl3) d 21.81, 24.39,
40.04, 51.49, 116.95, 127.19, 127.67, 129.19, 129.31,
130.11, 135.20, 136.50, 144.11; m/z (EI) 223 (57), 155
(100), 91 (100). Anal. Calcd for C17H18N2O2S: C, 64.94; H,
5.77; N, 8.91. Found: C, 65.33; H, 5.79; N, 8.80.
4.8. General procedure for tosylation of amino acids
The unprotected amino acids were suspended in CH3CN.
1.1 equiv of tosylchloride and 1.5 equiv of Et3N were added
and the mixture was allowed to react at reflux. The reaction
was monitored by TLC. After disappearance of starting
material, HCl (2 N) was added and the organic solvent was
removed. The aqueous phase was extracted three times
with CH2Cl2. The carboxylic acids were removed from the
aqueous phase by extraction with NaOH (2 N) which was
subsequently acidified with HCl (2 N). The product was
extracted three times with CH2Cl2 and three times with
ethyl acetate. After drying with Na2SO4, the solvent was
4.6. General procedure for cyanoborohydride reduction
and protection
To a solution of compound 12 or 13 in MeOH bromocresole
green was added and the pH of the solution was adjusted
with methanolic HCl till the indicator turned to yellow.
NaCNBH3 was added in portions while the pH was kept
acidic using methanolic HCl. After stirring 30 min at room

M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
4257
removed to give pure white solids (7c and 8c) or a crude
product (19), which was purified by silica gel chroma-
tography.
and acetic acid were added slowly. The organic layer was
separated and the aqueous phase extracted three times with
ethyl acetate. The combined organic layers were dried with
Na2SO4 and the solvent was evaporated. The product was
purified by recrystallization.
4.8.1. (G)-trans-2-(Toluene-4-sulfonylamino)-cyclo-
hexane carboxylic acid (7c). R. equi A4 (7 g wet cells,
80 ml buffer, OD610Z53). Yield 31 mg (13%, ee O 99%)
from 223 mg (G)-7a (10 mM); R. ery. 11540 (7.5 g wet
cells, 80 ml buffer, OD610Z39). Yield 35 mg (15%, eeZ
97%) from 223 mg (G)-7a (10 mM); R. sp. R312 (8.1 g wet
cells, 80 ml buffer, OD610Z63). Yield 39 mg (16%, eeZ
87%) from 223 mg (G)-7a (10 mM). White solid, mp 175–
4.9.1. 2-Amino-cyclopent-1-ene carbonitrile (12). Pale
1
brown solid; H NMR (200 MHz, CDCl3) d 1.90 (m, 2H),
2.47 (m, 4H), 4.50 (s, br, 2H, NH2); 13C NMR (50 MHz,
CDCl3) d 22.19, 31.45, 34.48, 74.61, 119.26, 162.65; m/z
(EI) 107 (M-1)C (100), 93 (1), 80 (20), 67 (3), 53 (10).
1
176 8C; H NMR (CDCl3) d 1.16–1.30 (m, 3H), 1.47–1.55
4.9.2. 2-Amino-cyclohex-1-ene carbonitrile (13). Pale
1
(m, 1H), 1.67 (m, 2H), 1.96–1.99 (m, 2H), 2.32 (dt, 1H, JZ
3.6, 10.5 Hz, H-1), 2.42 (s, 3H), 3.36 (ddt, 1H, JZ3.7, 7.6,
10.5 Hz, H-2), 5.31 (d, 1H, JZ7.6 Hz, NH), 7.29 (d, 2H,
JZ8.3 Hz), 7.76 (d, 2H, JZ8.3 Hz), 8.25 (s, br, 1H,
COOH); 13C NMR (CDCl3) d 21.83, 24.29, 24.52, 28.86,
33.38, 49.68, 54.03, 127.41, 129.86, 137.96, 143.63, 178.87.
Anal. Calcd for C14H19NO4S: C, 56.55; H, 6.44; N, 4.71.
Found: C, 55.98; H, 6.44; N, 4.52. Chiral separation on
Chiralpak AD-H n-heptane/ethanol 70:30, 0.80 ml/min,
15 8C.
brown solid; H NMR (200 MHz, CDCl3) d 1.65 (m, 4H),
2.12–2.21 (m, 4H), 4.22 (s, br, 2H, NH2); 13C NMR
(50 MHz, CDCl3) d 21.84, 22.21, 24.48, 28.41, 74.33,
121.17, 156.40; m/z (EI) 121 (MK1)C (61), 93 (100), 81
(7), 66 (25).
4.10. General procedure for catalytic hydrogenation
The catalyst (Pd/5% on charcoal) was added to a solution of
the olefin 14 or 15 in MeOH. 1 bar (balloon) or 50 bar
(autoclave) of hydrogen were applied after evaporating and
purging the vessels three times. The reactions were carried
out at room temperature and monitored by GC/MS.
4.8.2. (G)-cis-2-(Toluene-4-sulfonylamino)-cyclohexane
carboxylic acid (8c). White solid, mp 155 158 8C; 1H NMR
(CDCl3) d 1.25–1.29 (m, 2H), 1.45–1.49 (m, 2H), 1.54–1.60
(m, 1H), 1.66 (m, 1H), 1.76–1.84 (m, 1H), 2.07 (m, 1H),
2.43 (s, 3H), 2.78 (dt, 1H, JZ4.4, 4.6 Hz, H-1), 3.42 (m, 1H,
H-2), 6.01 (d, 1H, JZ9.8 Hz, NH), 7.30 (d, 2H, JZ8.3 Hz),
7.76 (d, 2H, JZ8.3 Hz), 9.58 (s, br, 1H, COOH); 13C NMR
(CDCl3) d 21.80, 22.21, 24.42, 27.74, 29.89, 45.19, 52.68,
127.12, 129.97, 138.53, 143.60, 178.33. Anal. Calcd for
C14H19NO4S: C, 56.55; H, 6.44; N, 4.71. Found: C, 57.82;
H, 6.99; N, 4.97.
4.10.1. (G)-cis-N-(2-Cyano-cyclopentyl)-benzamide
(2a). White solid, mp 107–109 8C, H NMR (CDCl3) d
1
1.72–1.82 (m, 2H), 1.95–2.04 (m, 1H), 2.06–2.15 (m, 2H),
2.17–2.22 (m, 1H), 3.39 (dt, 1H, JZ3.9, 7.3 Hz, H-1), 4.56–
4.63 (m, 1H, H-2), 6.45 (d, 1H, JZ6.5 Hz, NH), 7.44 (m,
2H), 7.52 (m, 1H), 7.80 (m, 2H); 13C NMR (CDCl3) d 21.85,
29.02, 30.50, 34.72, 52.63, 120.61, 127.30, 128.93, 132.14,
134.07, 168.03; m/z (EI) 214 MC (10), 161 (3), 105 (100),
77 (36). Anal. Calcd for C13H14N2O: C, 72.87; H, 6.59; N,
13.07. Found: C, 72.56; H, 6.55; N, 12.75.
4.8.3. (G)-cis-2-{[cis-2-(Toluene-4-sulfonylamino) cyclo-
pentanecarbonyl]-amino}-cyclopentane carboxylic acid
1
(19). White solid, mp 176–177 8C; H NMR (DMSO-d6) d
4.10.2. (G)-cis-N-(2-Cyano-cyclohexyl)-benzamide (4a).
White solid, mp 177–178 8C; 1H NMR (CDCl3) d 1.43 (tq,
1H, JZ3.7, 13.2 Hz), 1.53–1.63 (m, 1H), 1.67–1.75 (m,
3H), 1.92 (m, 2H), 2.06 (m, 1H), 3.48 (m, 1H, H-1), 4.14 (m,
1H, JZ4.0 Hz, H-2), 6.38 (d, 1H, JZ6.8 Hz, NH), 7.43–
7.47 (m, 2H), 7.52 (m, 1H), 7.78 (d, 2H, JZ7.8 Hz); 13C
NMR (CDCl3) d 21.48, 24.85, 27.68, 28.84, 34.21, 48.99,
120.30, 127.30, 128.91, 132.14, 134.08, 167.44; m/z (EI)
228 MC (16), 160 (7), 123 (6), 105 (100), 77 (38). Anal.
Calcd for C14H16N2O: C, 73.66; H, 7.06; N, 12.27. Found:
C, 73.69; H, 7.13; N, 12.08.
1.30–1.44 (m, 2H), 1.45–1.56 (m, 3H), 1.57–1.65 (m, 2H),
1.71–1.78 (m, 4H), 1.83–1.90 (m, 1H), 2.36 (s, 3H), 2.59 (q,
1H, JZ7.4 Hz), 2.8 (q, 1H, JZ7.6 Hz), 3.47 (m, 1H,), 4.27
(m, 1H), 7.32 (d, 1H, JZ6.7 Hz, NH), 7.35 (d, 2H, JZ
8.0 Hz), 7.57 (d, 1H, JZ8.7 Hz, NH), 7.64 (d, 2H, JZ
8.0 Hz), 11.95 (s, br, 1H, COOH); 13C NMR (DMSO-d6) d
21.67, 22.11, 22.37, 27.20, 28.04, 31.98, 32.47, 47.22,
47.78, 52.42, 57.07, 127.31, 130.25, 138.68, 143.20, 172.95,
175.01. Crystal data25 for C19H26N2O5S: orthorhombic,
˚
space group P212121(19), aZ6.3805(13) A, bZ
˚
˚
16.940(3) A, cZ17.430(4) A, aZbZgZ908, VZ
1883.9(7) A , ZZ4, dcZ1.391 g cmK1, mZ0.206 mmK1
,
3
˚
4.11. General procedure for Aza-Baylis-Hillman
reaction
˚
(Mo Ka, lZ0.71073 A) TZ100 K, the structure was solved
by direct methods and refined by full matrix least squares
procedures (SHELXL97): R1Z0.0691 and 0.0756 (wR2Z
0.1321 and 0.1345) for 3326 unique measured reflections.
Goodness of fit: 1.277.
Tosylamide was suspended in i-PrOH together with
˚
molecular sieve (4 A) and 0.15 equiv of DABCO.
1.00 equiv of benzaldehyde, 1.10 equiv of acrylonitrile
and 0.75 equiv of Ti(i-OPr)4 were added subsequently. The
reaction was stirred at room temperature and monitored by
TLC. After 36 h, another 0.75 equiv of Ti(i-OPr)4 were
added and the reaction was stirred for another 24 h.
Subsequently, the mixture was filtered over celite and
washed with i-PrOH three times. The solvent was removed
and the residue was treated with MeOH and H2SO4 (2 N) for
4.9. General procedure for Thorpe–Ziegler cyclization
1.05 equiv of NaH was stirred in dry toluene and a solution
of adipodinitrile or heptanedinitrile in toluene was added
dropwise. The mixture was then refluxed for 3 h. EtOH was
added in order to destroy excess NaH. Subsequently, water

4258
M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
90 min. MeOH was removed under reduced pressure and
the aqueous layer was extracted with CH2Cl2. The organic
layer was washed with NaHCO3 satd, water and brine. After
drying with Na2SO4 the product was purified by silica gel
chromatography.
4.12.3. (G)-trans-N-(2-Carbamoyl-cyclohexyl)-benz-
amide (3b). R. equi A4 (4.1 g wet cells, 40 ml buffer,
OD610Z40): Yield 22 mg (22%, eeZ56%) from 93 mg
(G)-3a (10 mM); R. ery. 11540 (9.0 g wet cells, 90 ml
buffer, OD610Z38). Yield 35 mg (16%, eeZ67%) from
205 mg (G)-3a (10 mM); R. sp. R312 (7.0 g wet cells,
60 ml buffer, OD610Z54). Yield 20 mg (14%, eeZ38%)
from 137 mg (G)-3a (10 mM). White solid, mp 290–
4.11.1. (G)-N-(2-Cyano-1-phenyl-2-propenyl)-4-methyl-
benzenesulfonamide (10a). White solid, mp 126–127 8C;
1H NMR (CDCl3) d 2.44 (s, 3H), 5.06 (d, 1H, JZ7.3 Hz),
5.23 (d, 1H, JZ7.3 Hz, NH), 6.00 (d, 1H, JZ1.0 Hz), 6.06
(d, 1H, JZ1.0 Hz), 7.11–7.13 (m, 2H), 7.28 (d, 2H, JZ
8.5 Hz), 7.30–7.32 (m, 3H), 7.71 (d, 2H, JZ8.5 Hz); 13C
NMR (CDCl3) d 21.82, 60.03, 116.77, 123.60, 127.08,
127.56, 129.35, 129.52, 130.02, 132.09, 136.35, 137.00,
144.33; m/z (EI) 260 (24), 157 (100), 155 (38), 91 (89), 77
(20). Anal. Calcd for C17H16N2O2S: C, 65.36; H, 5.16; N,
8.97. Found: C, 65.57; H, 5.05; N, 8.95. Chiral separation on
AGP, 10 mM phosphatebuffer pH 7.02/acetonitrile 85:15,
0.80 ml/min, 15 8C.
1
291 8C; H NMR (DMSO-d6) d 0.87–1.12 (m, 3H), 1.24
(ddt, 1H, JZ3.4, 12.9, 12.9 Hz), 1.47 (m, 2H), 1.56 (m, 1H),
1.66 (m, 1H), 2.15 (dt, 1H, JZ3.4, 11.6 Hz, H-1), 3.69 (m,
1H, H-2), 6.51 (s, br, 1H, NH2), 6.90 (s, br, 1H, NH2), 7.20–
7.23 (m, 2H), 7.28 (m, 1H), 7.58 (m, 2H), 7.94 (d, 1H, JZ
8.8 Hz, NH); 13C NMR (DMSO-d6) d 25.31, 25.41, 29.73,
33.18, 49.40, 50.46, 127.93, 128.82, 131.66, 135.58, 165.97,
176.13; m/z (EI) 246 MC (1), 201 (4), 141 (14), 105 (100),
77 (67). Anal. Calcd for C14H18N2O2: C, 68.27; H, 7.37; N,
11.37. Found: C, 63.97; H, 7.13; N, 9.89. Chiral separation
on HSA, 20 mM phosphate buffer (pH 7.00)/2-propanol
97:3, 0.90 ml/min, ambient temperature.
4.12. General procedure for amide formation
4.12.4. (G)-cis-N-(2-Carbamoyl-cyclohexyl)-benzamide
(4b). White solid, mp 173–174 8C; H NMR (DMSO-d6) d
1
100 mg of nitrile was dissolved in 5 ml of MeOH. 5.0 equiv
of K2CO3 and 1 ml of aqueous H2O2 (35%) were added. The
reaction was stirred at room temperature and monitored by
TLC. After completion, the MeOH was removed under
reduced pressure, the aqueous phase diluted with water and
the product extracted three times with CH2Cl2. After drying
with Na2SO4 and evaporation of the solvent, the product-
amides crystallized as pure white substances.
1.29–1.38 (m, 2H), 1.44–1.62 (m, 4H), 1.92–1.99 (m, 2H),
2.57–2.60 (m, 1H, H-1), 4.19 (m, 1H, H-2), 6.86 (s, br, 1H,
NH2), 7.35 (s, br, 1H, NH2), 7.44 (m, 2H), 7.50 (m, 1H),
7.73–7.76 (m, 2H), 7.91 (d, 1H, JZ7.3 Hz, NH); 13C NMR
(DMSO-d6) d 22.50, 23.72, 25.88, 30.00, 44.51, 48.74,
127.84, 128.96, 131.78, 135.59, 166.56, 176.41; m/z (EI)
245 (MK1)C (7), 201 (11), 141 (28), 105 (100), 77 (52).
Anal. Calcd for C14H18N2O2: C, 68.27; H, 7.37; N, 11.37.
Found: C, 64.00; H, 7.02; N, 10.28.
4.12.1. (G)-trans-N-(2-Carbamoyl-cyclopentyl)-benz-
amide (1b). R. equi A4 (7.1 g wet cells, 70 ml buffer,
OD610Z40): Yield 66 mg (40%, eeZ94%) from 150 mg
(G)-1a (10 mM); R. ery.11540 (8.2 g wet cells, 80 ml
buffer, OD610Z32). Yield 55 mg (30%, ee O 99%) from
171 mg (G)-1a (10 mM); R. sp R312 (7.9 g wet cells, 70 ml
buffer, OD610Z46). Yield 13 mg (7%, ee O 99%) from
150 mg (G)-1a (10 mM). White solid, mp 237–239 8C; 1H
NMR (DMSO-d6) d 1.52–1.65 (m, 2H), 1.67–1.74 (m, 2H),
1.85–1.91 (m, 1H), 1.92–1.98 (m, 1H), 2.65 (dt, 1H, JZ8.3,
7.6 Hz, H-1), 4.34 (m, 1H, JZ7.3 Hz, H-2), 6.77 (s, br, 1H,
NH2), 7.27 (s, br, 1H, NH2), 7.44 (m, 2H), 7.51 (m, 1H),
7.82–7.83 (m, 2H), 8.33 (d, 1H, JZ7.8 Hz, NH); 13C NMR
(DMSO-d6) d 24.24, 29.53, 33.41, 50.90, 55.08, 128.00,
128.84, 131.75, 135.33, 166.70, 176.41; m/z (EI) 232 MC
(0.5), 188 (3), 187 (13), 127 (20), 105 (100), 77 (58). Anal.
Calcd for C13H16N2O2: C, 67.22; H, 6.94; N, 12.06. Found:
C, 65.83; H, 6.62; N, 11.49. Chiral separation on AGP,
10 mM phosphate buffer pH 7.00 (with 1 mM dimethyl-
octylamine), 0.90 ml/min, ambient temperature.
4.12.5. (G)-trans-2-(Toluene-4-sulfonylamino)-cyclopen-
tane carboxamide (5b). R. equi A4 (7 g wet cells, 80 ml
buffer, OD610Z56). Yield 32 mg (14%, ee O 99%) from
211 mg (G)-5a (10 mM); R. ery. 11540 (8.0 g wet cells,
80 ml buffer, OD610Z32). Yield 30 mg (13%, ee O 99%)
from 211 mg (G)-5a (10 mM); R. sp. R312 (8.0 g wet cells,
80 ml buffer, OD610Z62). Yield 23 mg (10%, ee O 99%)
from 211 mg (G)-5a (10 mM). White solid, mp 180–
1
181 8C; H NMR (DMSO-d6) d 1.15–1.22 (m, 1H), 1.38–
1.53 (m, 4H), 1.78–1.82 (m, 1H), 2.37 (s, 3H), 2.44 (dt, 1H,
JZ8.8, 6.8 Hz, H-1), 3.66 (m, 1H, JZ7.1 Hz, H-2), 6.72 (s,
br, 1H, NH2), 7.14 (s, br, 1H, NH2), 7.35 (d, 2H, JZ8.1 Hz),
7.61 (d, 1H, JZ7.8 Hz, NH), 7.65 (2H, d, JZ8.1 Hz); 13C
NMR (DMSO-d6) d 21.68, 24.03, 29.97, 33.47, 51.60,
57.58, 127.23, 130.20, 139.36, 143.06, 176.09; m/z (EI) 281
(MK1)C (0.02), 238 (0.1), 155 (7), 127 (100), 110 (27), 91
(89). Anal. Calcd for C13H18N2O3S: C, 55.30; H, 6.43; N,
9.92. Found: C, 55.21; H, 6.12; N, 9.96. Chiral separation on
Chiralcel OD-H, n-heptane/i-propanol 50:50, 0.38 ml/min,
15 8C.
4.12.2. (G)-cis-N-(2-Carbamoyl-cyclopentyl)-benzamide
(2b). White solid, mp 198–199 8C; H NMR (DMSO-d6) d
1
1.45–1.55 (m, 1H), 1.73–1.86 (m, 1H), 1.88–1.97 (m, 4H),
2.85 (dt, 1H, JZ7.3, 8.3 Hz, H-1), 4.43 (m, 1H, JZ7.1 Hz,
H-2), 6.87 (s, br, 1H, NH2), 7.33 (s, br, 1H, NH2), 7.44 (m,
2H), 7.48–7.51 (m, 1H), 7.74–7.76 (m, 2H), 8.12 (d, 1H, JZ
7.3 Hz, NH); 13C NMR (DMSO-d6) d 23.25, 28.66, 32.75,
47.24, 53.17, 127.77, 128.93, 131.73, 135.52, 166.47,
175.96; m/z (EI) 232 MC (0.1), 231 (2), 187 (6), 127 (10),
105 (100), 77 (55). Anal. Calcd for C13H16N2O2: C, 67.22;
H, 6.94; N, 12.06. Found: C, 66.47; H, 6.75; N, 11.53.
4.12.6. (G)-cis-2-(Toluene-4-sulfonylamino)-cyclopen-
tane carboxamide (6b). R. equi A4 (2.9 g wet cells,
30 ml buffer, OD610Z38). Yield 13 mg (14%, eeZ51%)
from 88 mg (G)-6a (10 mM); R. ery. 11540 (7.5 g wet cells,
70 ml buffer, OD610Z29). Yield 96 mg (49%, eeZ15%)
from 185 mg (G)-6a (10 mM); R. sp. R312 (5.3 g wet cells,
50 ml buffer, OD610Z50). Yield 106 mg (75%, eeZ7%)
from 132 mg (G)-6a (10 mM). White solid, mp
1
167–168 8C; H NMR (DMSO-d6) d 1.29–1.39 (m, 2H),

M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
4259
1.43–1.51 (m, 1H), 1.57–1.78 (m, 3H), 2.36 (s, 3H), 2.59
(dt, 1H, JZ6.8, 8.1 Hz, H-1), 3.53 (m, 1H, JZ6.3 Hz, H-2),
6.92 (s, br, 1H, NH2), 7.26 (s, br, 1H, NH2), 7.35 (d, 2H, JZ
8.1 Hz), 7.40 (d, 1H, JZ6.8 Hz, NH), 7.67 (2H, d, JZ
8.1 Hz); 13C NMR (DMSO-d6) d 21.66, 22.10, 27.72, 32.20,
47.56, 57.11, 127.33, 130.23, 138.92, 143.18, 175.55; m/z
(EI) 281 (MK1)C (1), 238 (1), 155 (16), 127 (51), 110 (20),
91 (100). Anal. Calcd for C13H18N2O3S: C, 55.30; H, 6.43;
N, 9.92. Found: C, 54.76; H, 6.42; N, 9.23. Chiral separation
on Chiralpak AD-H, EtOH, 0.55 ml/min, 40 8C.
4.12.10. (G)-4-Phenyl-3-(toluene-4-sulfonylamino)-
butyramide (11b). White solid, mp 183–184 8C; H NMR
1
(DMSO-d6) d 2.10 (d, 2H, JZ6.8 Hz), 2.33 (s, 3H), 2.48
(dd, 1H, JZ13.7, 7.8 Hz), 2.64 (dd, 1H, JZ13.7, 5.4 Hz),
3.65 (m, 1H), 6.84 (s, br, 1H, NH2), 7.02–7.03 (m, 2H),
7.12–7.18 (m, 3H), 7.22 (d, 2H, JZ8.1 Hz), 7.30 (s, br, 1H,
NH2), 7.46 (d, 2H, JZ8.1 Hz), 7.59 (d, 1H, JZ8.3 Hz, NH);
13C NMR (acetone-d6) d 20.73, 38.55, 40.81, 53.05, 126.42,
127.06, 128.44, 129.60, 129.66, 138.36, 138.97, 142.89,
172.69. Anal. Calcd for C17H20N2O3S: C, 61.43; H, 6.06; N,
8.43. Found: C, 60.69; H, 5.94; N, 8.06.
4.12.7. (G)-trans-2-(Toluene-4-sulfonylamino)-cyclohex-
ane carboxamide (7b). R. equi A4 (7 g wet cells, 80 ml
buffer, OD610Z53). Yield 129 mg (54%, eeZ65%) from
223 mg (G)-7a (10 mM); R. ery. 11540 (7.5 g wet cells,
80 ml buffer, OD610Z39). Yield 133 mg (56%, eeZ59%)
from 223 mg (G)-7a (10 mM); R. sp. R312 (8.1 g wet cells,
80 ml buffer, OD610Z62). Yield 100 mg (42%, eeZ77%)
from 223 mg (G)-7a (10 mM). White solid, mp 212–
4.13. General procedure for chemical hydratation of
nitriles to carboxylic acids
100 mg of nitrile was suspended in 5 ml of NaOH concd and
refluxed overnight. Except for the acids 7c and 8c, the
saponification was complete. The acid was released by
addition of HCl and dilution of the aqueous phase with
water. Extraction with CH2Cl2 or/and ethyl acetate and
drying with Na2SO4 generally yielded pure crystals. In some
cases, the product had to be purified by recrystallization or
silica gel chromatography.
1
213 8C; H NMR (DMSO-d6) d 0.93–1.09 (m, 3H), 1.32–
1.39 (m, 1H), 1.44–1.51 (m, 3H), 1.69–1.71 (m, 1H), 2.03
(dt, 1H, JZ3.9, 10.8 Hz, H-1), 2.35 (s, 3H), 3.26 (ddt, 1H,
JZ3.7, 9.3, 10.5 Hz, H-2), 6.70 (s, br, 1H, NH2), 7.01 (s, br,
1H, NH2), 7.32 (d, 2H, JZ8.0 Hz), 7.42 (d, 1H, JZ9.3 Hz,
NH), 7.65 (d, 2H, JZ8.0 Hz); 13C NMR (DMSO-d6) d
21.66, 24.81, 24.84, 29.79, 33.12, 50.44, 53.92, 126.96,
130.04, 140.95, 142.69, 175.55; m/z (EI) 252 (1), 155 (10),
141 (100), 124 (55), 91 (83). Anal. Calcd for C14H20N2O3S:
C, 56.74; H, 6.80; N, 9.45. Found: C, 56.77; H, 6.60; N,
9.46. Chiral separation on Chiralcel AD-H, n-heptane/
ethanol 70:30, 0.80 ml/min, 15 8C.
4.13.1. (G)-trans-2-(Toluene-4-sulfonylamino)-cyclopen-
tane carboxylic acid (5c). R. equi A4 (7 g wet cells,
80 ml buffer, OD610Z56). Yield 100 mg (44%, eeZ2%)
from 211 mg (G)-5a (10 mM); R. ery. 11540 (8.0 g wet
cells, 80 ml buffer, OD610Z32). Yield 195 mg (86%, eeZ
5%) from 211 mg (G)-5a (10 mM); R. sp. R312 (8.0 g wet
cells, 80 ml buffer, OD610Z62). Yield 76 mg (34%, eeZ
14%) from 211 mg (G)-5a (10 mM). White solid, mp 124–
1
125 8C; H NMR (CDCl3) d 1.44–1.52 (m, 1H), 1.60–1.76
4.12.8. (G)-cis-2-(Toluene-4-sulfonylamino)-cyclohexane
carboxamide (8b). R. equi A4 (6.9 g wet cells, 60 ml
buffer, OD610Z76): Yield 85 mg (48%, eeZ6%) from
167 mg (G)-8a (10 mM); R. ery. 11540 (8.5 g wet cells,
80 ml buffer, OD610Z38). Yield 98 mg (41%, eeZ8%)
from 223 mg (G)-8a (10 mM); R. sp. R312 (8.3 g wet cells,
80 ml buffer, OD610Z52). Yield 103 mg (43%, eeZ4%)
from 223 mg (G)-8a (10 mM). White solid, mp 161–
(m, 2H), 1.78–1.85 (m, 1H), 1.95–2.09 (m, 2H), 2.43 (s,
3H), 2.73 (dt, 1H, JZ8.8, 7.5 Hz, H-1), 3.80 (m, 1H, JZ
7.0 Hz, H-2), 5.19 (d, 1H, JZ6.4 Hz, NH), 7.30 (d, 2H, JZ
8.2 Hz), 7.77 (d, 2H, JZ8.2 Hz), 9.20 (s, br, 1H, COOH);
13C NMR (CDCl3) d 21.80, 23.18, 28.47, 33.71, 50.87,
57.73, 127.53, 129.98, 137.13, 143.92, 179.74. Anal. Calcd
for C13H17NO4S: C, 55.11; H, 6.05; N, 4.94. Found: C,
55.27; H, 6.05; N, 4.92. Chiral separation on Chirobiotic R,
polar organic mode (MeOH/Et3N/AcOH 100:0.4:0.1),
0.8 ml/min, ambient temperature.
1
162 8C; H NMR (DMSO-d6) d 1.07–1.19 (m, 3H), 1.39–
1.48 (m, 3H), 1.66–1.74 (m, 2H), 2.32–2.35 (m, 1H, H-1),
2.36 (s, 3H), 3.33 (m, 1H, H-2), 6.82 (s, br, 1H, NH2), 7.24
(s, br, 1H, NH2), 7.34 (m, 3H), 7.81 (d, 2H, JZ7.8 Hz); 13
C
4.13.2. (G)-3-Phenyl-3-(toluene-4-sulfonylamino)-pro-
pionic acid (9c). White solid, mp 149–151 8C; H NMR
1
NMR (DMSO-d6) d 21.53, 21.66, 23.67, 25.64, 29.65,
45.44, 52.49, 127.22, 130.18, 139.25, 143.11, 176.08; m/z
(EI) 252 (0.6), 155 (20), 141 (100), 124 (63), 91 (77). Anal.
Calcd for C14H20N2O3S: C, 56.74; H, 6.80; N, 9.45. Found:
C, 56.69; H, 6.87; N, 9.23. Chiral separation on Chiralcel
OD-H, n-heptane/i-propanol 50:50, 0.38 ml/min, 15 8C.
(CDCl3) d 2.37 (s, 3H), 2.83 (dd, 1H, JZ16.5, 6.3 Hz), 2.93
(dd, 1H, JZ16.5, 6.1 Hz), 4.71–4.75 (m, 1H), 5.74 (d, 1H,
JZ7.8 Hz, NH), 7.10–7.12 (m, 2H), 7.17 (d, 2H, JZ
8.3 Hz), 7.19–7.21 (m, 3H), 7.60 (d, 2H, JZ8.3 Hz); 13C
NMR (CDCl3) d 21.74, 40.89, 54.20, 126.68, 127.35,
128.16, 128.89, 129.75, 137.33, 139.15, 143.68, 174.90.
Anal. Calcd for C16H17NO4S: C, 60.17; H, 5.36; N, 4.39.
Found: C, 59.72; H, 5.20; N, 4.42.
4.12.9. (G)-3-Phenyl-3-(toluene-4-sulfonylamino)-pro-
pionamide (9b). White solid, mp 213–214 8C; H NMR
1
(DMSO-d6) d 2.30 (s, 3H), 2.28–2.46 (m, 2H), 4.62 (dt, 1H,
JZ6.3, 8.8 Hz), 6.72 (s, br, 1H, NH2), 7.08–7.14 (m, 5H),
7.18 (d, 2H, JZ8.1 Hz), 7.23 (s, br, 1H, NH2), 7.46 (d, 2H,
JZ8.1 Hz), 8.18 (d, 1H, JZ8.8 Hz, NH); 13C NMR
(DMSO-d6) d 21.60, 43.44, 55.17, 127.04, 127.43, 127.51,
128.56, 129.86, 139.26, 141.80, 142.77, 171.32; m/z (CI,
methane) 319 (MC1)C (2), 260 (100), 172 (69), 155 (17),
148 (41). Anal. Calcd for C16H18N2O3S: C, 60.36; H, 5.70;
N, 8.80. Found: C, 57.97; H, 5.48; N, 8.00.
4.13.3. (G)-4-Phenyl-3-(toluene-4-sulfonylamino)-buty-
ric acid (11c). White solid, mp 101–103 8C; 1H NMR
(CDCl3) d 2.41 (s, 3H), 2.55 (d, 2H, JZ4.9 Hz), 2.79 (dd,
1H, JZ13.7, 6.9 Hz), 2.86 (dd, 1H, JZ13.7, 7.8 Hz), 3.76
(m, 1H), 5.41 (d, 1H, JZ8.3 Hz, NH), 7.01–7.03 (m, 2H),
7.20–7.23 (m, 5H), 9.72 (s, br, 1H, COOH), 7.62 (d, 2H, JZ
8.3 Hz); 13C NMR (CDCl3) d 21.78, 37.92, 40.75, 51.91,
127.14, 127.22, 128.97, 129.46, 129.94, 136.87, 137.37,

4260
M. Winkler et al. / Tetrahedron 61 (2005) 4249–4260
Chem. Rev. 2001, 101, 2181–2204. (d) Sewald, N. Angew.
Chem., Int. Ed. 2003, 42, 5794–5795.
143.69, 176.47. Anal. Calcd for C17H19NO4S: C, 61.24; H,
5.74; N, 4.20. Found: C, 62.47; H, 5.93; N, 4.16.
´
´
ˇ
9. Martınkova, L.; Kren, V. Biocatal. Biotrans. 2002, 20, 73–93.
10. Brenner, C. Curr. Opin. Struct. Biol. 2002, 12, 775–782.
11. Kobayashi, M.; Shimizu, S. Curr. Opin. Chem. Biol. 2000, 4,
95–102.
Acknowledgements
We are grateful for financial support of this work by the
Austrian Science Fond (project No. P-15810), COST
(project No. D25/0002/02), the Czech Science Foundation
(project No. 203/05/3367) and the Ministry of Education of
the Czech Republic (project No. OC D25/001). We also
12. Preiml, M.; Hillmayer, K.; Klempier, N. Tetrahedron Lett.
2003, 44, 5057–5059.
¨
13. Preiml, M.; Honig, H.; Klempier, N. J. Mol. Catal. B: Enzym.
2004, 29, 115–121.
´ ´ ´
14. Martınkova, L.; Klempier, N.; Preiml, M.; Ovesna, M.;
´ ˇ
Kuzma, M.; Mylerova, V.; Kren, V. Can. J. Chem. 2002, 80,
¨
thank Prof. Helmut Honig and Prof. Kurt Faber for valuable
discussions, Astrid Preisz for proof-reading and Prof. Jorg
¨
724–727.
15. Wang, M.-X.; Wu, Y. Org. Biomol. Chem. 2003, 1, 535–540.
´
¨ ¨
16. Peter, A.; Torok, G.; Armstrong, D. W. J. Chromatogr. 1998,
793, 283–296.
Weber for special NMR experiments. We are grateful
to DSM, the Netherlands, for providing Rhodococcus
erythropolis NCIMB 11540.
´
´
´
´
¨ ¨
17. Peter, A.; Torok, G.; Csomos, P.; Peter, M.; Bernath, G.;
¨
Fu¨lop, F. J. Chromatogr. 1997, 761, 103–113.
´
´ ´
18. Prepechalova, I.; Martınkova, L.; Stolz, A.; Ovesna, M.;
´
´
Bezouska, K.; Kopecky, J.; Kren, V. Appl. Microbiol.
References and notes
Biotechnol. 2001, 55, 150–156.
19. Williams, J. K. J. Org. Chem. 1963, 28, 1054–1059.
20. Wu, J.; Hou, X.-L.; Dai, L.-X. J. Org. Chem. 2000, 65,
1344–1348.
1. Enantioselective Synthesis of b-Amino Acids; Juaristi, E., Ed.;
Wiley-VCH: London, 1997.
2. Cardillo, G.; Tomasini, C. Chem. Soc. Rev. 1996, 25, 117–128.
3. Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.;
Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V.
Helv. Chim. Acta 1998, 81, 932–982.
21. Balan, D.; Adolfsson, H. J. Org. Chem. 2002, 67, 2329–2334.
22. Atay, E.; Blagoeva, I. B.; Chubb, F. L.; Edward, J. T.;
Pojarlieff, I. G.; Toteva, M. M. Can. J. Chem. 2000, 78, 84–94.
23. Berrien, J. F.; Royer, J.; Husson, H. P. J. Org. Chem. 1994, 59,
3769–3774.
4. Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev.
2001, 101, 3219–3232.
24. DiGeronimo, M. J.; Antoine, A. D. Appl. Environ. Microbiol.
1976, 31, 900–906.
¨
5. Martinek, T. A.; Fu¨lop, F. Eur. J. Biochem. 2003, 270,
3657–3666.
25. CCDC 246741 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB2 1EZ, UK; fax: C44 1223 336033.
6. Konishi, M.; Nishio, M.; Saitoh, K.; Miyaki, T.; Oki, T.;
Kawaguchi, H. J. Antibiot. 1989, 42, 1749–1755.
´ ´
7. Szakonyi, Z.; Fu¨lop, F.; Bernath, G.; Torok, G.; Peter, A.
¨
¨ ¨
Tetrahedron: Asymmetry 1998, 9, 993–999.
8. (a) Liu, M.; Sibi, M. P. Tetrahedron 2002, 58, 7991–8035.
¨
(b) Cole, D. C. Tetrahedron 1994, 50, 9517–9582. (c) Fu¨lop, F.