Regio- and stereoselective hydroxylation of N-substituted piperidin-2-ones with Sphingomonas sp. HXN-200

Article abstract of DOI:10.1016/S0957-4166(02)00534-7

High activity, excellent regioselectivity, and opposite enantioselectivity were achieved in the hydroxylation of N-benzyl- and N-tert-butoxycarbonylpiperidin-2-one with Sphingomonas sp. HXN-200. High yield preparations of 4-hydroxypiperidin-2-ones were demonstrated in a bioreactor and in a shaking flask by use of the frozen/thawed cells as biocatalyst. The absolute configuration for the bioproducts was established.

Full text of DOI:10.1016/S0957-4166(02)00534-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 2141–2147
Regio- and stereoselective hydroxylation of N-substituted
piperidin-2-ones with Sphingomonas sp. HXN-200
Dongliang Chang, Hans-J u¨ rgen Feiten, Bernard Witholt and Zhi Li*
Institute of Biotechnology, ETH Zurich, Hoenggerberg, CH-8093 Zurich, Switzerland
Received 9 August 2002; accepted 20 August 2002
Abstract—High activity, excellent regioselectivity, and opposite enantioselectivity were achieved in the hydroxylation of N-benzyl-
and N-tert-butoxycarbonylpiperidin-2-one with Sphingomonas sp. HXN-200. High yield preparations of 4-hydroxypiperidin-2-
ones were demonstrated in a bioreactor and in a shaking flask by use of the frozen/thawed cells as biocatalyst. The absolute
configuration for the bioproducts was established. © 2002 Elsevier Science Ltd. All rights reserved.
1
. Introduction
Optically active 4-hydroxypiperidin-2-one is a useful
synthon for preparing antibiotics such as Blasticidin
17a,b
17c
The regio- and stereoselective hydroxylation of non-
activated carbon atoms is a great challenge in organic
chemistry. Nature has found a general solution for this
reaction via oxygenases. Several biological systems have
been successfully applied in the hydroxylation of
steroids and several other alicyclic compounds.
S
and penem derivatives,
4-aminopiperidin-2-
17d
ones which are b-amino acid equivalents, and pepti-
domimetics with Homo-Freidinger lactam sub-
structure.
1
17e,f
The chemical preparation of 4-hydroxy-
piperidin-2-ones, including the oxidation of N-substi-
2
–11
Thus
18a
tuted 4-piperidinopiperidines,
pyridines,
1,2,3,6-tetrahydro-
far, soluble cytochrome P450 monooxygenases such as
18a
18b
or 4-hydroxypiperidines,
resulted in
1
2
13
P450 cam
and P450 B4M-3,
soluble methane
product mixtures. Preparation via biohydroxylation is
also difficult: hydroxylation of N-phenyl-, N-benzyl-,
or N-benzoyl-4-piperidin-2-ones with Beauveria
bassiana ATCC 7159 gave the corresponding 4-hydroxy-
1
monooxygenase (sMMO),
and membrane-bound
15
alkane hydroxylase (AlkB) of P. putida GPo1 have
been well investigated. In practice, the filamentous fun-
gus Beauveria bassiana ATCC 7159 has been widely
used in organic laboratories for the hydroxylation of a
6
piperidin-2-ones in low concentration with a yield of 7,
11e
11e
3,5–9,10c,11
10, and 27%, respectively. Herein, we report our
broad range of substrates,
although the activity
results on the hydroxylation of N-substituted piperidin-
and product concentration are rather low. We have
recently discovered that Sphingomonas sp. HXN-200,
an alkane-degrading bacterium containing a soluble
monooxygenase, is a highly active and regio- and
2
-ones with Sphingomonas sp. HXN-200, preparative
biotransformations and the stereochemistry of the
hydroxylation products.
1
6
stereoselective catalyst for hydroxylations. The easily
handled frozen/thawed cells of Sphingomonas sp. HXN-
2
. Results and discussion
200 have been successfully used in the high yield prepa-
ration of several pharmaceutical intermediates such as
N-substituted (S)- and (R)-3-hydroxypyrrolidines, (S)-
For hydroxylation studies, cells of Sphingomonas sp.
HXN-200 were grown on n-octane vapor in 30 L E2
4
4
-hydroxypyrrolidin-2-ones, 3-hydroxyazetidines, and
-hydroxypiperidines. We wanted to explore the syn-
16a,b
16
medium, as previously described.
The cells were
thetic application of this catalytic system further in the
hydroxylation of other alicyclic compounds.
harvested and the cell pellets were stored at −80°C.
Frozen/thawed cells were used for biohydroxylation.
2
.1. Small-scale biohydroxylation of 1–2
Based on the previous results from hydroxylation with
Sphingomonas sp. HXN-200, the hydrophobic N-ben-
*
0
Corresponding author. Tel.: +41 1 633 3811; fax: +41 1 633 1051;
e-mail: zhi@biotech.biol.ethz.ch
16
957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00534-7

2
142
D. Chang et al. / Tetrahedron: Asymmetry 13 (2002) 2141–2147
zyl- and N-tert-butoxycarbonyl-piperidin-2-one 1 and 2
were chosen as substrates. Compounds 1 and 2 were
rather clean. Obviously, Sphingomonas sp. HXN-200 is
significantly better than Beauveria sulfurescens ATCC
7159 in hydroxylation of N-substituted piperidin-2-
19,20
prepared according to established procedures.
6,11e
Small-scale hydroxylations were performed with frozen/
thawed cells of Sphingomonas sp. HXN-200 in 10 ml of
one.
5
0 mM K-phosphate buffer (pH 7.0–8.0) at a cell
2
.2. Determination of the ee for bioproduct 3 and 4
density of 4.0 g cdw/L. Glucose (2%, w/v) was added to
improve the conversion via regeneration of the cofac-
tor, and the mixture was shaken at 200 rpm and 30°C.
Aliquots (0.1–0.2 ml) were taken from the bioconver-
sion mixture at predetermined time points, diluted with
MeOH, and the cells were removed by centrifugation.
The samples were analyzed by reverse phase HPLC to
follow the reaction in the aqueous phase. No extraction
was needed, which is advantageous in comparison with
GC analysis. Biohydroxylation of 1 and 2 afforded the
desired products 3 and 4, respectively. The conversion
was quantified by comparing the integrated peak areas
at 210 nm of the samples with the substrate and
product standards. The results are given in Table 1.
For the determination of ee, a racemic standard of 3
was prepared chemically: oxidation of the bioproduct 3
with Jones reagent gave the corresponding ketone 5 in
3
4% yield; reduction of 5 with NaBH afforded the
4
racemic 3 in 95% yield (Scheme 1). The two enan-
tiomers were separated by HPLC on a chiralpak AS
column with retention times of 8.7 and 10.4 min. Simi-
larly, a pure sample of the bioproduct 4 was obtained
by HPLC on a chiralcel OB-H column with retention
times of 28.3 and 30.8 min for the two enantiomers.
As shown in Table 1, the ee of the bioproducts 3 and 4
are about 30–36 and 65–71%, respectively. No signifi-
cant influence of pH on the ee was observed. While the
ee of bioproduct 4 remained nearly unchanged during
the biotransformation, there was a slight decrease in the
ee of 3 (Fig. 1). This is probably due to trace alcohol
dehydrogenase activity in the whole cells. While excel-
High activity (12–15 U/g cdw) was observed in the
hydroxylation of N-benzyl-piperidin-2-one, 1. Biotrans-
formation of a 10 mM solution of 1 gave 90% of
N-benzyl-4-hydroxypiperidin-2-one
3
at pH 8.0.
Changing the ‘docking/protecting’ group to a tert-
butoxycarbonyl group resulted in a slightly lower
hydroxylation activity (7.7–9.0 U/g cdw). Nevertheless,
hydroxylation of a 10 mM solution of 2 produced
6
2
9–75% of N-tert-butoxycarbonyl-4-hydroxypiperidin-
-one 4. While higher activity and conversion were
achieved at pH 8.0 for the reaction of 1 to 3, no
significant pH effect was observed for the transforma-
tion of 2 to 4. In both cases no other hydroxylation
products were formed, indicating the excellent regiose-
lectivity of the alkane monooxygenase of Sphingomonas
sp. HXN-200. Moreover, no overoxidation products
were detected, suggesting that the bioconversion is
Scheme 1.
Table 1. Hydroxylation of N-substituted piperidin-2-ones 1–2 with frozen/thawed cells of Sphingomonas sp. HXN-200 (4.0 g
cdw/L)
pH Activitya (U/g cdw)
Conv.b (ee ) (%)
c
Entry
Substrate (mM) Prod.
0
.5 h
1 h
2 h
3 h
4 h
5 h
1
2
3
4
5
6
1
(8.0)
3
3
3
4
4
4
7.5
7.5
8.0
7.0
7.5
8.0
12
12
15
9.0
8.6
7.7
18 (36)
14 (35)
18 (36)
11 (71)
10 (66)
9.2 (68)
36 (33)
27 (33)
34 (34)
25 (66)
24 (66)
21 (68)
55 (31)
40 (31)
58 (33)
45 (66)
47 (65)
40 (67)
68 (31)
47 (33)
77 (33)
59 (65)
64 (65)
55 (68)
77 (31)
51 (31)
83 (31)
68 (64)
74 (63)
66 (68)
79 (30)
52 (31)
90 (30)
69 (69)
75 (65)
71 (68)
1 (10.0)
1 (10.0)
2 (10.0)
2 (10.0)
2 (10.0)
a
Activity was determined over the first 30 min.
Conversion was determined by HPLC analysis; error limit: 2% of the stated values.
Number in brackets is the ee determined by HPLC analysis with a chiral column.
b
c

D. Chang et al. / Tetrahedron: Asymmetry 13 (2002) 2141–2147
2143
1
H
6
-C-OH at 4.64 and 4.21 ppm in the H NMR spectra,
and the signal for C-OH at 65.23 and 65.17 ppm in the
C NMR spectra, respectively. The existence of the
signals of the NCH and NCOCH2 for both 3 and 4 in
the H NMR spectra exclude the possibility of OH
groups at positions 6 or 3. The couplings of H-C-OH
6
13
6
6
2
1
6
1
with NCOCH2 and not with NCH6 2 in the H and
H,H) COSY NMR spectra suggest clearly that the OH
group in 3–4 is at the 4-position.
6
(
2
.4. Stereochemistry of the bioproducts 3–4
For determination of the absolute configuration of 3–4,
(
(
R)- and (S)-N-benzyl-4-hydroxypyrrolidin-2-one 6 and
R)- and (S)-N-tert-butoxycarbonyl-4-hydroxypyrro-
lidin-2-one 7 were prepared chemically according to our
16c
previously published procedures. In the CD spectrum
shown in Fig. 2(c), a negative Cotton effect for (−)-3 at
2
45 nm and a negative Cotton effect at 240 nm for
(
R)-6 was observed, suggesting that (−)-3 has the same
configuration at the C4-position as (R)-6 (Scheme 2).
According to the CIP nomenclature, this configuration
is (S) for (−)-3. Similarly, a negative Cotton effect was
observed for bioproduct (+)-4 and (S)-7 at 260 and 266
nm, respectively, in Fig. 2(d). This indicates the same
configuration at the C4-position for (+)-4 and (S)-7.
Thus, according to the CIP principle, the configuration
of (+)-4 is established as (R) (Scheme 2).
Figure 1. Biotransformation of 1 (10mM) to 3 and of 2 (10
mM) to 4 with frozen/thawed cells of Sphingomonas sp.
HXN-200 (4 g cdw/L) at pH 8.0.
lent enantioselectivity was observed in hydroxylations
of N-benzyl- and N-tert-butoxycarbonyl piperidin-2-
1
6c
one, hydroxylation of 1–2 occurred with only moder-
ate to good enantioselectivity.
2.3. Preparative biohydroxylation
As an example of preparation on a gram scale, biocon-
version of N-benzyl-piperidin-2-one 1 was carried out
in a 3 L bioreactor. Biotransformation of substrate 1
(
1.52 g, 8.04 mmol) in a 1 L suspension of the frozen/
thawed cells in 50 mM of K-phosphate buffer (pH 8.0)
containing glucose (2%, w/v) at a cell density of 10 g
cdw/L for 5 h gave 75% conversion to 3. Work-up and
chromatographic purification afforded 1.168 g (71%) of
Scheme 2.
2
5
the pure 3 as white powder with an [h] of −6.5 (c 1.10,
D
CHCl ) and an ee of 31% (Fig. 2a).
The opposite enantioselectivity in the hydroxylation of
N-benzyl- and N-tert-butoxycarbonyl-piperidin-2-one
is very interesting. This provides the possibility of
preparing both enantiomers of 4-hydroxypyrrolidin-2-
ones by biohydroxylation with Sphingomonas sp. HXN-
200. The enantioselectivity might be further improved
by changing the docking/protecting groups or modify-
ing the enzyme.
3
The preparation of 4 was demonstrated by biotransfor-
mation with a shaking flask. Biohydroxylation of 2 (199
mg) in 100 ml of 50mM K-phosphate buffer (pH 8.0)
containing glucose (2%, w/v) at a cell density of 4.0 g
cdw/L gave 71% of conversion to 4 at 5 h. Extraction
of the product with n-butanol/ethyl acetate (1:1) and
purification by flash chromatography afforded 96.2 mg
2
5
(
45%) of 4 in 68% ee (Fig. 2b) with an [h]D of +6.7 (c
.50, CHCl ). The low isolated yield is probably caused
1
3. Conclusion
3
by the incomplete extraction of the product 4 due to its
hydrophilic nature. Continuous extraction would
increase the yield.
The hydroxylations of N-substituted pyrrolidin-2-ones
1–2 with Sphingomonas sp. HXN-200 occur with high
activity and regioselectivity. Preparative transforma-
tions of 1 and 2 with the frozen/thawed cells afforded
the corresponding 4-hydroxypyrrolidin-2-ones 3 and 4
in high concentration and good yield. Opposite enan-
tioselectivity was observed for hydroxylation of 1 and
2, giving the corresponding product 3 and 4 in 31% ee
(S) and 68% ee (R), respectively.
The existence of an OH group in products 3 and 4 was
−
1
evidenced by the absorption at 3382 and 3446 cm in
the IR spectra and the signal at 2.64 and 2.60–2.30 ppm
1
in the H NMR spectra, respectively. This was further
confirmed by the MS signal at 206 and 215 (16 higher
than the corresponding substrate), the resonance for

2
144
D. Chang et al. / Tetrahedron: Asymmetry 13 (2002) 2141–2147
Figure 2. (a) HPLC analysis of ee for bioproduct (−)-3. (b) HPLC analysis of ee for bioproduct (+)-4. (c) CD spectra of
bioproduct (−)-3 and (R)- and (S)-N-benzyl-4-hydroxypyrrolidin-2-one 6 (in acetonitrile). (d) CD spectra of bioproduct (+)-4 and
(
R)- and (S)-N-tert-butoxycarbonyl-4-hydroxypyrrolidin-2-one 7 (in acetonitrile).
4
. Experimental
LC-MS apparatus. Melting points are uncorrected.
Analytical high-performance liquid chromatography
(HPLC) was carried out with a Hewlett Packard 1050
4
.1. General methods
instrument. IR spectra were measured in CHCl . Opti-
3
1
13
H and C NMR spectra were determined at 300 K in
CDCl , chemical shifts are reported in ppm relative to
TMS, coupling constants (J) are reported in Hz. Mass
spectra were obtained by Atmospheric Pressure Chemi-
cal Ionization at 40 eV with a Hewlett Packard 1100
cal rotations were determined using a Perkin Elmer 241
polarimeter. CD spectra were measured in acetonitrile
at 20°C on a JASCO J-710 spectropolarimeter. Biocon-
version was assayed by HPLC. The purity of the prod-
ucts was established by GC analyses with a Chrompack
3

D. Chang et al. / Tetrahedron: Asymmetry 13 (2002) 2141–2147
2145
CP–Sil-5CB column (25 m×0.32 mm, temperature pro-
gram: 60°C for 2 min, increase to 280°C at a rate of
HPLC analysis. Column: Hypersil BDS-C18 (5 mm,
125×4 mm); eluent: a mixture of A (10 mM K-phos-
phate buffer, pH 7.0) and B (acetonitrile); flow: 1.0
25°C/min, then 280°C for 1 min).
ml/min; detection: UV at 210, 225, and 254 nm; t of 3:
R
4
.2. Materials
1.8 min, t of 1: 4.4 min (A/B 70:30); t of 4: 1.8 min,
R
R
tR of 2: 4.3 min (A/B 70:30).
1
9
20
Compounds 1, and 2 were prepared according to
published procedures. Cells of Sphingomonas sp. HXN-
Determination of ee by HPLC. Chiral column (250×4.6
mm), UV detection at 210 and 254 nm, eluent A:
n-hexane, and eluent B: isopropanol. For 3: chiralpak
2
00 were produced by growth on n-octane in 30 L E2
21 16a,b
medium, as previously reported.
AS column; flow rate 1.0 ml/min, A/B (75/25); t : 8.7
R
4.3. N-Benzyl-piperidine-2,4-dione 5
and 10.4 min. For 4: chiralcel OB-H column; flow rate
0
.5 ml/min, A/B (95/5); t : 28.3 and 30.8 min.
R
A solution of chromium(VI) oxide (30 mg, 0.30 mmol)
in sulfuric acid (30 ml) and water (90 ml) was added
dropwise at 0°C to a stirred solution of N-benzyl-4-
hydroxypiperidin-2-one 3 (61 mg, 0.30 mmol) in ace-
tone (5 ml). The mixture was stirred at rt for 5 min and
the reaction was stopped by addition of isopropanol (2
ml). After evaporation, the residue was treated with
water (2 ml) and the product was extracted into CHCl3
4.6. Preparation of N-benzyl-4-hydroxypiperidin-2-one 3
by biohydroxylation in a bioreactor
N-Benzyl-piperidin-2-one 1 (1.52 g, 8.04 mmol) was
added to a 1 L suspension (10 g cdw/g) of frozen/
thawed cells of Sphingomonas sp. HXN-200 in 50 mM
of K-phosphate buffer (pH 8.0) containing glucose (2%,
w/v) in a 3 L bioreactor, the mixture was stirred at 1500
rpm at 30°C under the introduction of air at 1 L/min.
The biotransformation was followed by analytical
HPLC and stopped at 5 h. After removing the cells, the
supernatant pH was adjusted to 11–12 by addition of
KOH followed by extraction with ethyl acetate. The
organic phase was separated, dried over Na SO , and
(
100 ml) and ethyl acetate (50 ml). Drying over
Na SO , filtration, and evaporation afforded the crude
2
4
product, which was purified by column chromatogra-
22
phy on silica gel giving 5 (20.7 mg, 34%). Purity:
1
9
5.7% (GC); R 0.32 (ethyl acetate), H NMR (400 Hz)
f
l 7.36–7.25 (5H, m), 4.68 (2H, s), 3.48 (2H, t, J=6.0),
1
3
3
.42 (2H, s), 2.53 (2H, t, J=6.0); C NMR (100 MHz)
l 204.60 (s), 167.42 (s), 137.32 (s), 129.97 (d), 129.20
d), 129.00 (d), 51.10 (t), 49.99 (t), 43.38 (t), 39.75 (t);
MS (m/z): 204 (100%, M+1).
2
4
the solvent was removed by evaporation. The crude
product was purified by flash chromatography on silica
(
gel (methanol/ethyl acetate, 5:95, R 0.05) giving pure 3
f
as a white powder (1.168 g, 71% yield). ee: 31% (S);
2
5
purity: 98.4% (GC), 99.2% (HPLC); [h] =−6.5 (c 1.10,
D
4.4. Racemic N-benzyl 4-hydroxypiperidin-2-one, 3
11e
1
CHCl ); mp 98.1–99.7°C, lit: 92–94°C; H NMR (400
3
MHz) l 7.33–7.22 (5H, m), 4.64 (1H, d, J=14.4), 4.55
A mixture of N-benzyl-piperidin-2,4-dione 5 (6.5 mg,
(
1H, d, J=14.4), 4.18 (1H, m), 3.41 (1H, m), 3.14 (1H,
0
.032 mmol) and NaBH4 (2.4 mg, 0.064 mmol) in
quin, J=6.0), 2.73 (1H, dd, J=16.0, 4.4), 2.64 (1H, s,
br), 2.50 (1H, dd, J=16.0, 4.4), 1.95 (1H, m), 1.84 (1H,
m); C NMR (100 MHz) l 169.87 (s), 137.85 (s),
CH Cl /acetic acid (9:1, 2 ml) was stirred at rt for 10 h.
2
2
The product was extracted into CHCl (50 ml). Drying
3
13
over Na SO , filtration, and evaporation afforded the
2
4
1
4
29.68 (d), 128.93 (d), 128.45 (d), 65.23 (d), 51.03 (t),
crude product that was purified by column chromatog-
raphy on silica gel giving 3 (6.2 mg, 95%). Purity: 95.9%
4.15 (t), 42.01 (t), 31.33 (t); MS (m/z): 206 (100%,
−
1
M+1), 188 (8%); IR (CHCl ) w 3382, 1632 cm .
3
(
GC); R 0.34 (ethyl acetate/MeOH, 9:1).
f
4
.7. Preparation of N-tert-butoxycarbonyl-4-hydroxy-
4.5. General procedures for small-scale hydroxylation
piperidin-2-one 4 by biohydroxylation in a shaking flask
The frozen cells of Sphingomonas sp. HXN-200 were
thawed and suspended to a cell density of 4.0 g cdw/L
in 10 ml of 50 mM K-phosphate buffer containing
glucose (2% w/v) at pH 7.0–8.0 in a 100 ml Erlenmeyer
flask. Substrates were added directly to a final concen-
tration of 2–10 mM. The mixture was shaken at 200
rpm at 30°C for 5 h. Aliquots (0.1–0.2 ml) were taken
from the bioconversion mixture at predetermined time
points and mixed with an equal volume of MeOH
followed by centrifugation to remove the cells. The
supernatant portion of the samples were analyzed by
HPLC to quantify the conversion. At the same time,
aliquots (0.5 ml) were taken from the bioconversion
mixture, the cells were removed, and the product was
extracted with ethyl acetate (0.5 ml). These samples
were analysed by HPLC with a chiral column to deter-
mine the ee. All results are summarized in Table 1.
N-tert-Butoxycarbonyl-piperidin-2-one 2 (199 mg, 1.00
mol) was added to a 100 ml suspension (4.0 g cdw/L) of
frozen/thawed cells of Sphingomonas sp. HXN-200 in
50 mM K-phosphate buffer (pH 8.0) containing glucose
(2%) in a 500 ml shaking flask. The mixture was shaken
at 200 rpm and 30°C for 5 h and the bioconversion was
followed by analytical HPLC. The cells were removed
from the bioconversion mixture by centrifugation and
the pH of the supernatant was adjusted to 11–12 by
addition of KOH followed by extraction with ethyl
acetate/n-butanol (1:1). The organic phase was col-
lected, water was removed by azeotropic distillation
with MeOH, and the solvent was dried over Na SO
2
4
and removed by evaporation. The product was purified
by column chromatography on silica gel (ethyl acetate,
R 0.21), yielding 4 as a white powder (96.2 mg, 45%).
f
ee: 68% (R); purity: 97.7% (GC), 99.9% (HPLC);

2
146
D. Chang et al. / Tetrahedron: Asymmetry 13 (2002) 2141–2147
2
5
1
[
(
3
(
h] =+6.7 (c 1.50, CHCl ); mp 61.9–63.6°C; H NMR
Saf, R. Eur. J. Org. Chem. 2000, 3835; (d) Braunegg, G.;
de Raadt, A.; Feichtenhofer, S.; Griengl, H.; Kopper, I.;
Lehmann, A.; Weber, H. J. Angew. Chem., Int. Ed. Engl.
1999, 38, 2763.
D
3
400 MHz) l 4.21 (1H, s), 3.83 (1H, dd, J=12.0, 4.4),
.57 (1H, dd, J=12.0, 4.4), 2.74 (1H, d, J=15.6), 2.53
1H, d, J=15.6), 2.60–2.30 (1H, s, br), 2.02 (1H, d,
J=3.2), 1.88 (1H, m), 1.50 (9H, s); C NMR (100
MHz) l 170.89 (s), 153.39 (s), 84.25 (s), 65.17 (d), 44.75
t), 43.12 (t), 31.81 (t), 29.03 (q); MS (m/z): 215 (14%,
M), 116 (100%), 100 (39%); IR (CHCl ) w 3446, 1766,
13
4. Aranda, G.; Moreno, L.; Cortes, M.; Prange, T.; Maurs,
M.; Azerad, R. Tetrahedron 2001, 57, 6051.
5. Hemenway, M. S.; Olivo, H. F. J. Org. Chem. 1999, 64,
6312.
6. Holland, H. L.; Morris, T. A.; Nava, P. J.; Zabic, M.
Tetrahedron 1999, 55, 7441.
(
3
−
1
1
715 cm .
7
. (a) Flitsch, S. L.; Aitken, S. J.; Chow, C. S.-Y.; Grogan,
G.; Staines, A. Bioorg. Chem. 1999, 27, 81; (b) Aitken, S.
J.; Grogan, G.; Chow, C.; Turner, N. J.; Flitsch, S. L. J.
Chem. Soc., Perkin Trans. 1 1998, 3365.
Acknowledgements
We thank Dr. J. B. van Beilen (ETH Zurich) for a
helpful discussion and Professor K. Engesser (Univer-
sity of Stuttgart) for supplying us with the HXN-200
strain.
8. (a) Haufe, G.; W o¨ lker, D.; Fr o¨ hlich, R. J. Org. Chem.
2002, 67, 3022; (b) Pietz, S.; Fr o¨ hlich, R.; Haufe, G.
Tetrahedron 1997, 53, 17055; (c) Pietz, S.; W o¨ lker, D.;
Haufe, G. Tetrahedron 1997, 53, 17067.
9
. (a) Davis, C. R.; Johnson, R. A.; Cialdella, J. I.; Liggett,
W. F.; Mizsak, S. A.; Marshall, V. P. J. Org. Chem. 1997,
62, 2244; (b) Davis, C. R.; Johnson, R. A.; Cialdella, J.
I.; Liggett, W. F.; Mizsak, S. A.; Han, F.; Marshall, V. P.
J. Org. Chem. 1997, 62, 2252.
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