Practical Syntheses of N-Substituted 3-Hydroxyazetidines and 4-Hydroxypiperidines by Hydroxylation with Sphingomonas sp. HXN-200

Article abstract of DOI:10.1021/ol025829s

(Equation Presented) Hydroxylation of N-substituted azetidines 11 and 12 and piperidines 15-19 with Sphingomonas sp. HXN-200 gave 91-98% of the corresponding 3-hydroxyazetidines 13 and 14 and 4-hydroxypiperidines 20-24, respectively, with high activity and excellent regioselectivity. High yields and high product concentrations (2 g/L) were achieved with frozen/thawed cells as biocatalyst. For the first time, rehydrated lyophilized cells were successfully used for the biohydroxylation.

Full text of DOI:10.1021/ol025829s

ORGANIC
LETTERS
2002
Vol. 4, No. 11
1859-1862
Practical Syntheses of N-Substituted
3-Hydroxyazetidines and
4-Hydroxypiperidines by Hydroxylation
with Sphingomonas sp. HXN-200
Dongliang Chang,† Hans-Ju1rgen Feiten,† Karl-H. Engesser,‡ Jan B. van Beilen,†
,†
Bernard Witholt,† and Zhi Li*
Institute of Biotechnology, ETH-Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland, and
Institut fu¨r Siedlungswasserbau, Wassergu¨te- und Abfallwirtschaft der UniVersita¨t
Stuttgart, Bandta¨le 2, D-70569 Stuttgart, Germany
zhi@biotech.biol.ethz.ch
Received March 6, 2002
ABSTRACT
Hydroxylation of N-substituted azetidines 11 and 12 and piperidines 15−19 with Sphingomonas sp. HXN-200 gave 91−98% of the corresponding
3-hydroxyazetidines 13 and 14 and 4-hydroxypiperidines 20−24, respectively, with high activity and excellent regioselectivity. High yields and
high product concentrations (2 g/L) were achieved with frozen/thawed cells as biocatalyst. For the first time, rehydrated lyophilized cells were
successfully used for the biohydroxylation.
3-Hydroxyazetidine and 4-hydroxypiperidine are useful
pharmaceutical intermediates. For example, 3-hydroxy-
azetidine is used in the synthesis of oral carbapenem
antibiotics L-036 1 and L-084 2, antiepileptic Dezinamide
3, and antihypertensive Azelnidipine 4;1 4-hydroxypiperidine
is used for the preparation of allergic rhinitis drugs Ebastine
5 and Betotastine besilate 6, antibacterial Nadifloxacin 7,
antiallergy/antiasthmatic Linazolast 8, and agents for anti-
platelet therapy Lamifiban 9 and Sibrafiban 102 (Scheme 1).
In practice it is often advantageous, if not required, to use
3-hydroxyazetidine and 4-hydroxypiperidine in their N-
protected form.
amination of epichlorohydrin is limited to primary hindered
amines;3a-e amination of 1,3-dichloro-2-propyl ether gives
low yield;3f halogenation of 2,3-epoxyamines has only one
example for tert-butylamine;3g the route via 1-azabicyclo-
[1.1.0]butane requires a special reagent;3h and reduction of
azetidinone requires difficult to obtain starting materials.3i
(1) (a) Abe, T.; Isoda, T.; Sato, C.; Mihira, A.; Tamai, S.; Kumagai T.
U.S. Patent 5,534,510, 1995. (b) Abe, T.; Kumagai, T. U.S. Patent 5,886,-
172, 1999. (c) Teng, L. C. (A. H. Robins Co., Inc.) EP 102194, 1984. (d)
Koike, H.; Yoshomoto, M.; Nishino, H. (Sankyo Co., Ltd.; Ube Ind., Ltd.)
EP 266922, 1988.
(2) (a) Prieto, J.; Vega, A.; Moragues, J.; Spickett, R. G. W. (Fordonal
S.A.) U.S. Patent 4,550,116, 1985. (b) Koda, A.; Kuroki, Y.; Fujiwara, H.;
Takamura, S.; Yamano, K. (Ube Ind., Ltd.) EP 335586, 1989. (c) Ishikawa,
H.; Tabusa, F.; Miyamoto, H.; Kano, M.; Ueda, H.; Tamaoka, H.;
Nakagawa, K. Chem. Pharm. Bull. 1989, 37, 2103. (d) Tasaka, K. Drugs
Future 1988, 13, 1056. (e) Knopp, D.; Edenhofer, A.; Hadva´ry, P.; Weller,
T.; Hu¨rzeler, M.; Trzeciak, A.; Mu¨ller, M.; Steiner, B.; Alig, L. J. Med.
Chem. 1992, 35, 4393. (f) Graul, A.; Castan˜er, J.; Merlos, M. Drugs Future
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There are synthesis routes to 3-hydroxyazetidine and its
N-substituted derivatives, but each of them has drawbacks:
† Institute of Biotechnology.
‡ Institut fu¨r Siedlungswasserbau.
10.1021/ol025829s CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/30/2002

synthetic chemistry,5 biohydroxylation can be a useful tool
for this type of transformation.6-7 However, no successful
biohydroxylation of azetidine or N-substituted azetidines has
been reported thus far. Hydroxylations of N-substituted
piperidines with BeauVeria sulfurescens ATCC 71597i,8 or
Aspergillus niger VKM F-11199 are known, but the low
activity, yield, and product concentration (less than 0.1 g/L)
limit their synthetic applications.
Scheme 1
We have recently found that the bacterial strain Sphin-
gomonas sp. HXN-200 is an excellent biocatalyst for regio-
and stereoselective hydroxylations of pyrrolidines7a,c and
pyrrolidin-2-ones.7b Here, we report the hydroxylation with
this strain of N-substituted azetidines and piperidines, four-
and six-membered heterocycles, for the preparation of the
corresponding 3-hydroxyazetidines and 4-hydroxypiperidines
and the successful use of rehydrated lyophilized cell powder
as hydroxylation catalyst.
Sphingomonas sp. HXN-200 was grown on n-octane vapor
in 30 L of E2 medium10 at 30 °C and 1500 rpm for 90 h to
a cell density of 8.5 g/L. The cells were harvested, and the
cell pellets (2.5 kg wet cells consisting of 10% dry cells)
were stored at -80 °C. The frozen/thawed cells were used
(5) For reviews, see: (a) Reiser, O. Angew. Chem., Int. Ed. Engl. 1994,
33, 69. (b) Barton, D. H. R.; Doller, D. Acc. Chem. Res. 1992, 25, 504. (c)
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Watson, P. L.; Liebmann, J. F.; Greenberg, A. SelectiVe Hydrocarbon
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H. L. Organic Synthesis with OxidatiVe Enzymes; VCH: New York, 1992;
Chapter 3, p 55. (e) Holland, H. L.; Weber, H. Curr. Opin. Biotechnol.
2000, 11, 547. (f) Duetz, W. A.; van Beilen, J. B.; Witholt, B. Curr. Opin.
Biotechnol. 2001, 12, 419. (g) Lehman, L. R.; Stewart, J. D. Curr. Org.
Chem. 2001, 5, 439. (h) Li, Z.; van Beilen, J. B.; Duetz, W. A.; Schmid,
A.; de Raadt, A.; Griengl, H.; Witholt, B. Curr. Opin. Chem. Biol. 2002, 6,
136.
(7) For recent publications, see: (a) Li, Z.; Feiten, H.-J.; Chang, D.;
Duetz, W. A.; van Beilen, J. B.; Witholt, B. J. Org. Chem. 2001, 66, 8424.
(b) Chang, D.; Witholt, B.; Li, Z. Org. Lett. 2000, 2, 3949. (c) Li, Z.; Feiten,
H.-J.; van Beilen, J. B.; Duetz, W.; Witholt, B. Tetrahedron: Asymmetry
1999, 10, 1323. (d) de Raadt, A.; Griengl, H.; Weber, H. J. Chem. Eur. J.
2001, 7, 27. (e) de Raadt, A.; Fetz, B.; Griengl, H.; Klingler, M. F.; Krenn,
B.; Mereiter, K.; Mu¨nzer, D. F.; Plachota, P.; Weber, H. J., Saf, R.
Tetrahedron 2001, 57, 8151. (f) de Raadt, A.; Fetz, B.; Griengl, H.; Klingler,
M. F.; Kopper, I.; Krenn, B.; Mu¨nzer, D. F.; Ott, R. G.; Plachota, P.; Weber,
H. J.; Braunegg, G.; Mosler, W.; Saf, R. Eur. J. Org. Chem. 2000, 3835.
(g) Aranda, G.; Moreno, L.; Cortes, M.; Prange, T.; Maurs, M.; Azerad, R.
Tetrahedron 2001, 57, 6051. (h) Hemenway, M. S.; Olivo, H. F. J. Org.
Chem. 1999, 64, 6312. (i) Holland, H. L.; Morris, T. A.; Nava, P. J.; Zabic,
M. Tetrahedron 1999, 55, 7441. (j) Flitsch, S. L.; Aitken, S. J.; Chow, C.
S.-Y.; Grogan, G.; Staines, A. Bioorg. Chem. 1999, 27, 81. (k) Shibasaki,
T.; Sakurai, W.; Hasegawa, A.; Uosaki, Y.; Mori, H.; Yoshida M.; Ozaki,
A. Tetrahedron Lett. 1999, 40, 5227.
Syntheses of 4-hydroxypiperidine and N-substituted 4-hy-
droxypiperidines are also not straightforward: preparations
involving reduction of N-substituted 4-piperidones,4a-e hy-
drogenation of 4-hydroxypyridine or N-substituted 1H-
pyridin-4-one,4f-h or hydrogenation and cyclization of 3-hy-
droxy-glutaronitrile4i give low overall yields in multisteps;
syntheses via Mannich-type cyclization of formaldehyde with
benzylbut-3-enyl-amine4j or with N-benzylammonium trif-
luoroacetate and allyl-trimethyl-silane4k are not practical;
hydroboration of N-trimethylsilanyl- or N-benzyloxylcarbonyl-
1,2,5,6-tetrahydropyridine4l-n gives a mixture of 3- and
4-hydroxy piperidines.
Regioselective hydroxylation of azetidine and piperidine
represents one of the simplest ways for preparing the
hydroxylated derivatives. While selective hydroxylation on
a nonactivated carbon atom remains still a challenge in
(3) (a) Gaertner, V. R. J. Org. Chem. 1967, 32, 2972. (b) Laguerre, M.;
Boyer, C.; Leger, J.-M.; Carpy, A. Can. J. Chem. 1989, 67, 1514. (c)
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Karikomi, M.; Arai, K.; Toda, T. Tetrahedron Lett. 1997, 38, 6059. (h)
Hayashi, K.; Sato, C.; Hiki, S.; Kumagai, T.; Tamai, S.; Abe, T.; Nagao,
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Polym. Sci. 1959,40, 377. (g) Hall, H. K., Jr. J. Am. Chem. Soc. 1958, 80,
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S. F.; Overman, L. E. J. Am. Chem. Soc. 1987, 109, 6107. (k) Larsen, S.
D.; Grieso, P. A.; Fobare, W. F. J. Am. Chem. Soc. 1986, 108, 3512. (l)
Dicko, A.; Montury, M.; Baboulene, M. Tetrahedron Lett. 1987, 28, 6041.
(m) Brown, H. C.; Prasad, J. V. N. V. J. Am. Chem. Soc. 1986, 108, 2049.
(n) Brown, H. C.; Prasad, J. V. N. V.; Zee, S.-H. J. Org. Chem. 1985, 50,
1582.
(8) (a) Aitken, S. J.; Grogan, G.; Chow, C.; Turner, N. J.; Flitsch, S. L.
J. Chem. Soc., Perkin Trans. 1 1998, 3365. (b) Archelas, A.; Furstoss, R.;
Srairi, D.; Maury, G. Bull. Soc. Chem. Fr. 1986, 234. (c) Johnson, R. A.;
Herr, M. E.; Murray, H. C.; Fonken, G. S. J. Org. Chem. 1968, 3187. (d)
Floyd, N.; Munyemana, F.; Roberts, S. M.; Willetts, A. J. J. Chem. Soc.,
Perkin Trans. 1 1993, 881.
(9) Parshikov, I. A.; Modyanova, L. V.; Dovgilivich, E. V.; Terent’ev,
P. B.; Vorob’eva, L. I.; Grishina, G. V. Chem. Heterocycl. Compd. (Engl.
Transl.) 1992, 28, 159.
(10) Lageveen, R. G.; Huisman, G. W.; Preusting, H.; Ketelaar, P.;
Eggink, G.; Witholt, B. Appl. EnViron. Microbiol. 1988, 54, 2924.
1860
Org. Lett., Vol. 4, No. 11, 2002

for hydroxylation of N-substituted azetidines 11 and 12 and
piperidines 15-19. Substrates 11 and 1211 were synthesized
by reaction of azetidine with phenyl chloroformate and di-
tert-butyldicarbonate in 67% and 83% yield, respectively.12
Piperidines 15,13 16,8a and 1814 were prepared according to
established procedures, and 1715 was prepared in an improved
yield of 82%.
min, cdw ) cell dry weight) and high conversions. No
byproducts were detected, indicating the excellent regiose-
lectivity and clean biotransformation. Moreover, higher
product concentrations could be achieved as follows: hy-
droxylation of 10 mM solutions of 11 and 12 gave 82% and
89% of 3-hydroxyazetidine 13 and 14, respectively. This
demonstrates the first successful biohydroxylation of N-
substituted azetidines.
Hydroxylations were performed with frozen/thawed cells
of Sphingomonas sp. HXN-200 on a 10-mL scale in the
exploratory stage,16 and the bioconversions were followed
by HPLC analyses.17 As shown in Table 1, hydroxylation
Similarly, hydroxylation of piperidines 15-19 afforded
the desired 4-hydroxypiperidines 20-24. As shown in Table
2, high activity was obtained in hydroxylations of 15-18
Table 2. Hydroxylation of N-Substituted Piperidines 15-19
with Frozen/Thawed Cells of Sphingomonas sp. HXN-200 (4.0
g cdw/L)
Table 1. Hydroxylation of N-Substituted Azetidines 11 and 12
with Frozen/Thawed Cells of Sphingomonas sp. HXN-200 (4.0
g cdw/L)
conversionb (%)
substrate
(mM)
activitya
product (U/g cdw) 0.5 h 1 h 2 h 3 h 5 h
conversionb (%)
11 (5.0)
11 (7.0)
11 (10.0)
12 (5.0)
12 (10.0)
13
13
13
14
14
15
16
15
17
17
33
27
18
39
20
61
44
30
60
35
96
71
49
93
60
substrate
(mM)
activitya
89
64
98
82
produdct (U/g cdw) 0.5 h 1 h 2 h 3 h
5 hc
15 (5.0)
16 (2.0)
16 (3.0)
17 (7.0)
17 (8.0)
18 (5.0)
18 (6.0)
19 (2.0)
19 (3.0)
20
21
21
22
22
23
23
24
24
20
12
13
19
18
29
27
49
71
49
31
26
69
54
27
16
77 94 98 98
83 87 87 91 (1.4)
61 65 68 69
53 84 91 94 (5.1)
51 76 88 94 (4.1)
91 94 94 94 (6.3)
79 86 93 93 (6.6)
50 78 91 97 (1.8)
32 57 74 90 (1.7)
74
89
a Activity was determined over the first 30 min. b Conversion was
determined by HPLC analysis; error limit, 2% of the stated values.
of azetidine 11 and 12 gave the desired 3-hydroxyazetidine
13 and 14 with high activities (15-17 U/g cdw, U ) µmol/
4.5
4.0
a Activity was determined over the first 30 min. b Conversion was
determined by HPLC analysis; error limit, 2% of the stated values. c Number
in bracket is the conversion to the corresponding 4-ketones at 5 h; no bracket
indicates no ketones formed.
(11) Billotte, S. Synlett 1998, 4, 379.
(12) Data for 11: Rf 0.15 (silica gel, ethyl acetate/hexane 2:8); mp 41.1-
1
42.3 °C; H NMR (300 MHz, 243 K) δ 7.41-7.34 (m, 2 H), 7.24-7.19
(m, 1 H), 7.14-7.10 (m, 2 H), 4.22 (t, 2 H, J ) 7.8), 4.12 (t, 2 H, J ) 7.8),
2.32 (quin, 2 H, J ) 7.8); 13C NMR (75 MHz) δ 154.19(s), 150.58 (s),
129.30 (d), 125.29 (d), 121.66 (d), 50.04 (t), 48.94 (t), 15.48 (t); MS m/z
178 (100%, M + 1); IR (CHCl3) ν 1716, 1595 cm-1. Two sets of signals
for NCH2 in the 1H NMR spectrum at 243 K indicates the existence of two
rotamers due to the restricted rotation of the N-CO bond.
(12-29 U/g cdw), while moderate activity (4.5 U/g cdw)
was observed for the hydroxylation of 19, probably as a result
of the steric hindrance of the N-benzoyl group in the
substrate. No other regioisomers were detected during
hydroxylation, indicating excellent regioselectivity. Hydroxy-
lation of 15 was a clean reaction, while hydroxylation of
16-19 gave a small amount of the corresponding 4-ketones.
Nevertheless, over 91% of 4-hydroxypiperidines 20-24 were
formed in hydroxylation of 15 (5 mM), 16 (2 mM), 17 (8
mM), 18 (6 mM), and 19 (5 mM), respectively.
(13) Ferrer, M.; Sanchez-Baeza, F.; Messeguer, A. Tetrahedron 1997,
53, 15877.
(14) Beak, P.; Lee, W. K. J. Org. Chem. 1993, 58, 1109.
(15) Patonay, T.; Patonay-Peli, E.; Mogyorodi, F. Synth. Commun. 1990,
20, 2865.
(16) General Procedure. Substrate was added to a 10-mL suspension
of the frozen/thawed cells (0.40 g with 40 mg cdw) in 50 mM K-phosphate
buffer (pH ) 8.0) containing glucose (2%, w/v). The mixture was shaken
at 200 rpm and 30 °C for 5 h. Aliquots (0.1-0.2 mL) were taken out at
predetermined time points and diluted in MeOH, and the cells were removed
by centrifugation. The samples were analyzed by HPLC.
(17) HPLC Analysis. Column, Hypersil BDS-C18 (5 µm, 125 mm ×
4 mm); eluent, a mixture of A (10 mM K-phosphate buffer, pH 7.0) and B
(acetonitrile); flow, 1.0 mL/min.; detection, UV at 210, 225, and 254 nm;
retention time, 2.1 min for 13, 5.4 min for 11 (A/B 70:30), 2.7 min for 14,
9.2 min for 12 (A/B 75:25), 3.0 min for 20, 5.2 min for 15 (A/B 85:15),
2.0 min for 21, 8.8 min for 16 (A/B 55:44), 1.7 min for 22, 6.6 min for 17
(A/B 55:45), 1.5 min for 23, 5.6 min for 18 (A/B 50:50), 1.5 min for 24,
5.4 min for 19 (A/B 70:30); the conversion was quantified by comparing
the integrated peak areas at 210 nm of the samples with the substrate and
product standards.
Preparative hydroxylations were carried out on scales of
60 mL to 2 L with frozen/thawed cells, as shown in Table
3. Hydroxylation of azetidine 12 (15.8 mM) on a 1-L scale
gave 2.140 g (79%) of 3-hydroxyazetidine 14.18 Hydroxy-
lation of 11 on a 60-mL scale gave 1319 in 81% yield.
Similarly, hydroxylation of piperidine 15 (5.0 mM) at a cell
concentration of 4.0 g cdw/L gave 4-hydroxypiperidine 2020
Org. Lett., Vol. 4, No. 11, 2002
1861

Table 3. Preparation of N-Substituted 3-Hydroxyazetidines 13
and 14 and 4-Hydroxypiperidines 20-24 by Hydroxylation with
Frozen/Thawed Cells of Sphingomonas sp. HXN-200
yieldb
substrate
(mM)
scale
cells
time
(h)
conva
(%)
(mL) (g cdw/L)
%
mg
11 (4.0)
12c (15.8)
15 (5.0)
15 (15.0)
16 (2.0)
17 (7.0)
18 (5.0)
19 (2.0)
60
1000
2000
1000
100
100
100
100
4.0
10.2
4.0
10.2
4.0
4.0
4.0
4.0
1.5
5.0
4.0
5.2
3.0
4.0
2.0
5.0
98
83
98
98
96
91
96
83
81.0
79.0
82.9
76.2
70.2
83.2
69.5
71.5
37.5
2140
1501
2072
33.0
43.6
69.3
29.3
a Conversion was determined by HPLC analysis; error limit, 2% of the
stated values. b Yield of the isolated pure product. c Substrate was added
at different time points.18
Figure 1. Biohydroxylation of N-benzylpiperidine 15 (5.0 mM)
to 20 with rehydrated lyophilized cells and frozen/thawed cells of
Sphingomonas sp. HXN-200 (4.0 g cdw/L) in 20 mL of 50 mM
K-phosphate buffer (pH 8.0) containing glucose (2%).
in 83% yield. The product concentration was easily increased
to 2.072 g/L by use of a higher cell density (10.2 g cdw/L)
and higher substrate concentration (15 mM). Compounds
21,21 22,22 23,23 and 249 were also prepared in good yields
by hydroxylation of 16-19, respectively. These results are
clearly superior to those obtained with other hydroxylation
systems.7i,8,9
200 as a practical catalyst for use in organic synthesis.
Hydroxylation of piperidine 15 (5 mM) with the rehydrated
catalyst powder24 at a density of 4.0 g cdw/L afforded 80%
of 4-hydroxypiperidine 20. Comparing with a similar hy-
droxylation with frozen/thawed cells, shown in Figure 1, 85%
of the activity was achieved with the lyophilized powder. It
has been shown that hydroxylation with Sphingomonas sp.
HXN-200 is catalyzed by a NADH-dependent enzyme.7a The
fact that rehydrated lyophilized cells are able to carry out
such a NADH-dependent hydroxylation indicates that these
cells are capable of retaining and regenerating NADH at rates
equal to or exceeding the rate of hydroxylation. Although it
is known that lyophilized microbial cells retain activities for
hydrolytic reactions after rehydration,25 our result is the first
example of the use of lyophilized cells for a cofactor-
dependent hydroxylation.
To facilitate the application of this interesting biohydroxy-
lation system in organic synthesis, we developed a lyophi-
lized cell powder preparation of Sphingomonas sp. HXN-
(18) Preparation of 14. A 1-L suspension of the frozen/thawed cells
(102 g with 10.2 g cdw) in 50 mM K-phosphate buffer (pH 8.0) containing
glucose (2%, w/v, for the intracellular regeneration of cofactors) was stirred
at 1500 rpm and at 30 °C under the introduction of air at 1 L/min in a 3-L
bioreactor. Substrate 12 was added at different time points: 10.1 mmol at
the beginning, 2.0 mmol at 30 min, 2.0 mmol at 147 min, and 1.7 mmol at
180 min. The biotransformation was followed by analytical HPLC and
stopped at 5 h by centrifugation. The pH of the supernatant was adjusted
to 11-12 by addition of KOH followed by extraction with ethyl acetate.
The organic phase was separated and dried over Na2SO4, and the solvent
was removed by evaporation. The product was purified by column
chromatography on silica gel (Rf 0.27, n-hexane/ethyl acetate 1:1) to give
14 in 79% yield (2.140 g): mp 44.8-46.8 °C; 1H NMR (400 MHz) δ 4.53
(m, 1 H); 4.11-4.07 (dd, 2 H, J ) 10.4, 7.2 Hz), 3.78-3.74 (dd, 2 H, J )
8.8, 4.4 Hz), 3.60 (s, 1 H); 1.38 (s, 9 H); 13C NMR (100 MHz) δ 157.54
(s), 80.83 (s), 62.33 (d), 60.06 (t), 29.42 (q); MS m/z 173 (11%, M), 130
Acknowledgment. Financial support by ETH Zu¨rich is
gratefully acknowledged.
Supporting Information Available: Growth curve of
Sphingomonas sp. HXN-200 on a 30-L scale; experimental
details for the chemical preparation of 11, 12, and 17 and
(100%), 118 (40%); IR (CHCl3) ν 3400, 1683 cm-1
.
(19) Data for 13: Rf 0.13 (silica gel, n-hexane/ethyl acetate 1:1); mp
100.8-102.6 °C; 1H NMR (400 MHz) δ 7.38-7.08 (m, 5 H), 4.57 (m, br,
1 H), 4.30 (s, br, 2 H), 3.97 (s, br, 2 H), 3.14 (s, br, 1 H); 13C NMR (100
MHz) δ 155.63 (s), 151.96 (s), 130.41 (d), 126.52 (d), 122.65 (d), 62.56
(d), 60.85 (t), 59.95 (t); MS m/z 194 (100%, M + 1), 113 (12%); IR (CHCl3)
1
biocatalytic preparation of 13, 14, and 20-24; H and 13C
NMR spectra of bioproducts 13, 14, and 20-24 and
substrates 11, 12, and 17. This material is available free of
charge via the Internet at http://pubs.acs.org.
ν 3401, 1720, 1595 cm-1
.
(20) Jucker, E.; Schenker, E. (Sandoz) CH 463504, 1965).
(21) Alig, L.; Edenhofer, A.; Hadvary, P.; Huerzeler, M.; Knopp, D.;
Mueller, M.; Steiner, B.; Trzeciak, A.; Weller, T. J. Med. Chem. 1992, 35,
4393.
OL025829S
(24) Hydroxylation of 15 with Rehydrated Lyophilized Cell Powder.
Frozen/thawed cells of Sphingomonas sp. HXN-200 were lyophilized at
low temperature for 3 days. For the experiment of Figure 1, the dry powder
(80 mg), which was stored at 4 °C for 2 weeks, was suspended in 20 mL
of 50 mM K-phosphate buffer (pH ) 8.0) containing glucose (2% w/v) in
a 100 mL Erlenmeyer flask. Piperidine 15 (17.5 mg) was added to the
suspension. The flask was shaken at 200 rpm at 30 °C for 4 h, and the
formation of 20 was followed by HPLC analysis.17
(22) Data for 22: Rf 0.11 (silica gel, n-hexane/ethyl acetate 1:1); mp
1
116.5-118.3 °C; H NMR (200 MHz) δ 7.40-7.06 (m, 5 H), 4.10-3.85
(m, 3 H), 3.28 (s, br., 2 H), 1.98-1.85 (m, 2 H), 1.81 (s, 1 H), 1.66-1.48
(ddt, 2 H, J ) 13.0, 8.8, and 4.1); 13C NMR (50 MHz) δ 153.75 (s), 151.42
(s), 129.26 (d), 125.25 (d), 121.73 (d), 67.08 (d), 41.62 (t), 33.97 (t); MS
m/z 222 (100%, M + 1), 206 (24%); IR (CHCl3) ν 3452, 1710, 1594 cm-1
.
(23) Kucznierz, R.; Grams, F.; Leinert, H.; Marzenell, K.; Engh, R.; von
der Saal, W. J. Med. Chem. 1998, 41, 4983.
(25) Mischitz M.; Faber K.; Willetts, A. Biotechnol. Lett. 1995, 17, 893.
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