Enantioselective Trans Dihydroxylation of Nonactivated C-C Double Bonds of Aliphatic Heterocycles with Sphingomonas sp. HXN-200

Article abstract of DOI:10.1021/jo034628e

The bacterial strain Sphingomonas sp. HXN-200 was used to catalyze the trans dihydroxylation of N-substituted 1,2,5,6-tetrahydropyridines 1 and 3-pyrrolines 4 giving the corresponding 3,4-dihydroxypiperidines 3 and 3,4-dihydroxypyrrolidines 6, respectivel

Full text of DOI:10.1021/jo034628e

En a n tioselective Tr a n s Dih yd r oxyla tion of Non a ctiva ted C-C
Dou ble Bon d s of Alip h a tic Heter ocycles w ith Sph in gom on a s sp .
HXN-200
Dongliang Chang, Maarten F. Heringa, Bernard Witholt, and Zhi Li*
Institute of Biotechnology, ETH-Ho¨nggerberg, CH-8093 Zurich, Switzerland
zhi@biotech.biol.ethz.ch
Received May 12, 2003
The bacterial strain Sphingomonas sp. HXN-200 was used to catalyze the trans dihydroxylation of
N-substituted 1,2,5,6-tetrahydropyridines 1 and 3-pyrrolines 4 giving the corresponding 3,4-
dihydroxypiperidines 3 and 3,4-dihydroxypyrrolidines 6, respectively, with high enantioselectivity
and high activity. The trans dihydroxylation was sequentially catalyzed by a monooxygenase and
an epoxide hydrolase in the strain with epoxide as intermediate. While both epoxidation and
hydrolysis steps contributed to the overall enantioselectivity in trans dihydroxylation of 1, the
enantioselectivity in trans dihydroxylation of the symmetric substrate 4 was generated only in the
hydrolysis of meso-epoxide 5. The absolute configuration for the bioproducts (+)-3 and (+)-6 was
established as (3R,4R) by chemical correlations. Preparative trans dihydroxylation of 1a and 4b
with frozen/thawed cells of Sphingomonas sp. HXN-200 afforded the corresponding (+)-(3R,4R)-
3,4-dihydroxypiperidine 3a and (+)-(3R,4R)-3,4-dihydroxy pyrrolidine 6b in 96% ee both and in
60% and 80% yield, respectively. These results represent first examples of enantioselective trans
dihydroxylation with nonterpene substrates and with bacterial catalyst, thus significantly extending
this methodology in practical synthesis of valuable and useful trans diols. Enantioselective hydrolysis
of racemic epoxide 2a with Sphingomonas sp. HXN-200 gave 34% of (-)-2a in >99% ee, which is
a versatile chiral building block. Further hydrolysis of (-)-2a with the same strain afforded (-)-
(3S,4S)-3a in 96% ee and 92% yield. Thus, both enantiomers of 3a can be prepared by
biotransformation with Sphingomonas sp. HXN-200.
In tr od u ction
tioselective trans dihydroxylation of a nonisoprene C-C
double bond has been synthetically unsuccessful: several
eukaryotic systems (plants, animal, and fungi) were
known to metabolize aromatic compounds transforming
the C-C double bond into the corresponding trans-
dihydrodiol with low yield;8 bioconversion of 1-benzyl-3-
methyl-1,2,5,6-tetrahydropyridine with Cunninghamella
verticillata VKPM F-430 gave a mixture of 3,4-trans-diol
and two monohydroxylated products.9 Moreover, trans
dihydroxylation catalysts reported so far are limited to
Enantiomerically pure vicinal diols are very useful
intermediates in the synthesis of a number of important
pharmaceuticals and biologically active molecules, and
asymmetric dihydroxylation of a C-C double bond pro-
vides a simple access to these diols. While asymmetric
cis dihydroxylation can be achieved with Sharpless
catalyst1 or a dioxygenase,2 epoxidation of a C-C double
bond followed by hydrolysis could give rise to the asym-
metric “trans dihydroxylation”. The latter can be realized
by use of a biological system containing a monooxygenase
and an epoxide hydrolase.3 Successful examples are the
fungus-catalyzed trans dihydroxylations of several acyclic
terpenes,4-6 limonene,7 and R-terpinene.7 However, enan-
(4) Imai, K.; Marumo, S.; Ohtaki, T. Tetrahedron Lett. 1976, 15,
1211-1214.
(5) (a) Fourneron, J . D.; Archelas, A.; Furstoss, R. J . Org. Chem.
1989, 54, 4686-4689. (b) Furstoss, R. Microbial Reagents In Organic
Synthesis, Servi, S., Ed. In NATO ASI Ser. 1992, 381, 333-346. (c)
Zhang, X. M.; Archelas, A.; Furstoss, R. J . Org. Chem. 1991, 56, 3814-
3817. (d) Zhang, X.; Archelas, A.; Meou, A.; Furstoss, R. Tetrahedron:
Asymmetry 1991, 2, 247-250. (e) Zhang, X. M.; Archelas, A.; Furstoss,
R. Tetrahedron: Asymmetry 1992, 3, 1373-1376.
* Author to whom correspondence should be addressed. Phone: +41
1 633 3811. Fax: +41 1 633 1051.
(1) J ohnson, R. A.; Sharpless, K. B. In Catalytic Asymmetric
Synthesis; Ojima, I., Ed.; VCH: New York, 2000; pp 357-398.
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17, 438. (b) Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep. 1998, 15,
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10.1021/jo034628e CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/03/2003
J . Org. Chem. 2003, 68, 8599-8606
8599

Chang et al.
SCHEME 1. Tr a n s Dih yd r oxyla tion of N-Su bstitu ted 1,2,5,6-Tetr a h yd r op yr id in es 1a ,b a n d 3-P yr r olin es
4a ,b w ith Sph in gom on a s sp . HXN-200
the eukaryotic systems. We are interested in expanding
the scope of enzymatic trans dihydroxylation in practical
syntheses by developing a highly active and easy to
handle bacterial catalyst with new substrate specificity.
Previously, we found that Sphingomonas sp. HXN-200,
an alkane-degrading bacterium, contains a soluble alkane
monooxygenase that catalyzes the regio- and stereose-
lective hydroxylation of the nonactivated carbon atom of
various aliphatic heterocycles.10 Recently we also found
a soluble epoxide hydrolase in this strain that catalyzes
the enantioselective hydrolysis of alicyclic meso-epox-
ides.11 Considering that the alkane monooxygenase
might catalyze the epoxidation of a C-C double bond,
the strain HXN-200 might be a promising catalyst for
trans dihydroxylation of the nonisoprene C-C double
bond of alicyclic compounds.
N-Substituted 1,2,5,6-tetrahydropyridine and 3-pyrro-
line are interesting substrates, since trans dihydroxyla-
tion of these compounds could give the corresponding
optically active trans-3,4-diols. While (3R,4R)-3,4-dihy-
droxypiperidine is a potent inhibitor of â-D-glucuronidase12a
and an intermediate for the preparation of xylanase
inhibitor isofagomine,12b (3R,4R)-3,4-dihydroxypyrroli-
dine is a useful intermediate for the preparation of
biologically active Sialyl Lewis X mimetics,13a new
carbapenem13b and cephalosporin13c antibiotics, and novel
carbohydrate mimics.13d The chemical synthesis of (3R,4R)-
3,4-dihydroxypiperidine requires 10 steps from either
D-arabinose12a,14 or D-tartaric acid12b,15 with low overall
yield, whereas the preparation of (3R,4R)-3,4-dihydroxy-
pyrrolidine involves a difficult reduction step from D-
tartaric acid.16,13b Here, we report the highly enantiose-
lective trans dihydroxylation of N-substituted 1,2,5,6-
tetrahydropyridine and 3-pyrroline with Sphingomonas
sp. HXN-200 as biocatalyst and the high yield prepara-
tion of (3R,4R)-3,4-dihydroxypiperidine and (3R,4R)-3,4-
dihydroxypyrrolidine. In addition, we report the practical
synthesis of enantiomerically pure 3,4-epoxypiperidine
and (3S,4S)-3,4-dihydroxypiperidine by enantioselective
hydrolysis with the same strain.
Resu lt a n d Discu ssion
E n a n t ioselect ive Tr a n s Dih yd r oxyla t ion w it h
Sph in gom on a s sp . HXN-200. Sphingomonas sp. HXN-
20010c was grown on n-octane in E2 medium as de-
scribed,10b the cells were harvested, and the cell pellets
were stored at -80 °C. The easy to handle frozen/thawed
cells were subsequently used for trans dihydroxylation
studies. Since 1,2,5,6-tetrahydropyridine and 3-pyrroline
themselves were not substrates for the monooxy-
genase of Sphingomonas sp. HXN-200, docking/protecting
groups17,10 were introduced. N-Phenoxycarbonyl and N-
benzyloxycarbonyl groups were shown to be excellent
docking/protecting groups for the hydroxylation of pyrroli-
dine,10b thus being selected as the docking/protecting
groups of 1,2,5,6-tetrahydropyridines and 3-pyrrolines for
the trans dihydroxylation (Scheme 1).
(9) Terent’ev, P. B.; Parshikov, I. A.; Grishina, G. V.; Piskunkova,
N. F.; Chumakov, T. I.; Bulakhov, G. A. Chem. Heterocycl. Compds.
1997, 33, 619-620.
Substrate 1a 18 was synthesized in 70% yield by treat-
ment of 1,2,5,6-tetrahydropyridine with phenyl chloro-
formate, and 1b and 4a were prepared by similar
procedures.19,20 To facilitate the analysis of the biocon-
(10) (a) Li, Z.; Feiten, H.-J .; van Beilen, J . B.; Duetz, W.; Witholt,
B. Tetrahedron: Asymmetry 1999, 10, 1323-1333. (b) Li, Z.; Feiten,
H.-J .; Chang, D.; Duetz, W. A.; van Beilen, J . B.; Witholt, B. J . Org.
Chem. 2001, 66, 8424-8430. (c) Chang, D.; Witholt, B.; Li, Z. Org. Lett.
2000, 2, 3949-3952. (d) Chang, D.; Feiten, H.-J .; Engesser, K.-H.; van
Beilen, J . B.; Witholt, B.; Li, Z. Org. Lett. 2002, 4, 1859-1862. (e)
Chang, D.; Feiten, H.-J .; Witholt, B.; Li, Z. Tetrahedron: Asymmetry
2002, 13, 2141-2147.
(14) Legler, G.; Stuetz, A. E.; Immich, H. Carbohydr. Res. 1995, 272,
17-30.
(15) Lucas, H. J .; Baumgarten, W. J . Am. Chem. Soc. 1941, 63,
1653-1657.
(11) Chang, D.; Wang, Z.; Heringa, M. F.; Wirthner, R.; Witholt, B.;
Li, Z. Chem. Commun. 2003, 960-961.
(16) (a) Nagel, U.; Kinzel, E.; Andrade, J .; Prescher, G. Chem. Ber.
1986, 119, 3326-3343. (b) Skarzewski, J .; Anil, G. Tetrahedron:
Asymmetry 1997, 8, 1861-1868.
(12) (a) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J . Am.
Chem. Soc. 1998, 120, 3007-3018. (b) Williams, S. J .; Hoos, R.;
Withers, S. G. J . Am. Chem. Soc. 2000, 122, 2223-2235.
(13) (a) Huang, H.; Wong, C.-H. J . Org. Chem. 1995, 60, 3100-3106.
(b) Kang, Y. K.; Shin, K. J .; Yoo, K. H.; Seo, K. J .; Park, S. Y.; Kim, D.
J .; Park, S. W. Bioorg. Med. Chem. Lett. 1999, 9, 2385-2390. (c) Lee,
Y. S.; Lee, J . Y.; J ung, S. H.; Woo, E.-R.; Suk, D. H.; Seo, S. H.; Park,
H. J . Antibiot. 1994, 47, 609-612. (d) Davis, B. G.; Maughan, M. A.
T.; Chapman, T. M.; Villard, R.; Courtney, S. Org. Lett. 2002, 4, 103-
106.
(17) (a) de Raadt, A.; Griengl, H.; Weber, H. J . Chem. Eur. J . 2001,
7, 27. (b) de Raadt, A.; Griengl, H. Curr. Opin. Biotechnol. 2002, 13,
537-542.
(18) Oediger, H.; J oop, N. Liebigs Ann. Chem. 1972, 764, 21-27.
(19) D’Andrea, S. V.; Michalson, E. T.; Freeman, J . P.; Chidester,
C. G.; Szmuszkovicz, J . J . Org. Chem. 1991, 56, 3133-3137.
(20) Punniyamurthy, T.; Katsuki, T. Tetrahedron 1999, 55, 9439-
9454.
8600 J . Org. Chem., Vol. 68, No. 22, 2003

Enantioselective Trans Dihydroxylation of Nonactivated CdC
F IGURE 1. Trans dihydroxylation of 1a ,b and 4a ,b with frozen/thawed cells (10 g cdw/L) of Sphingomonas sp. HXN-200: (a) 1a
(10 mM); (b) 1b (8 mM); (c) 4a (5 mM); and (d) 4b (3 mM).
version, epoxides 2a ,b and 5a ,b and diols 3a ,b and 6a ,b
were also prepared chemically: epoxidation of 1a and 4a
with m-CBPA gave 79% and 67% of 2a and 5a , respec-
tively; hydrolysis of 2a and 5a with TFA afforded the
corresponding trans-diol 3a and 6a in 52% and 82% yield,
respectively; similar reactions gave the epoxides 2b19 and
5b 13d and diols 3b12b and 6b .13a,d The structures of all
synthesized compounds were confirmed by the MS, IR,
1H, and 13C NMR spectra.
Small-scale biotransformations were performed with
frozen/thawed cells (cell concentration: 10 g cdw/L,
cdw ) cell dry weight; 1 g cdw corresponds to about 4 g
frozen cell weight) of Sphingomonas sp. HXN-200 in 10
mL of 50 mM K-phosphate buffer (pH 8.0). Glucose (2%,
w/v) was added to increase the yield via regeneration of
the cofactor for the epoxidation, and the mixture was
shaken at 200 rpm and 30 °C. Aliquots (0.1-0.2 mL) were
taken from the bioconversion mixture at predetermined
time points, diluted in methanol, and the cells were
removed by centrifugation. The samples were analyzed
by reverse-phase HPLC to follow directly the reaction in
aqueous phase. The conversion was quantitated by
comparing the integrated peak areas at 210 nm of the
substrate and product in an analytic sample with the
corresponding standards.
cells that were boiled for 20 min; therefore, the hydrolysis
during dihydroxylation must be catalyzed by the epoxide
hydrolase from the strain.
As shown in Table 1, the average epoxidation activity
during the first 30 min is quite high for 1a (10 mM) and
1b (10 mM): 16 and 18 U/g cdw (U ) µmol/min),
respectively. While the diol 3a was formed in 94% at 5
h, only 65% of the diol 3b was observed at the same time.
Nevertheless, biotransformation of 1b at 8 mM for 5 h
afforded 82% of 3b. The epoxidation activity for 4a (5-8
mM) was also quite high (9-12 U/g cdw), but the diol 6a
was formed in only 45-48% at 5 h. In contrast, biotrans-
formation of 4b (2-3 mM) showed lower epoxidation
activity (4.4-5.0 U/g cdw), but gave 64-97% of the diol
6b. In fact, the diol formation depends on both epoxida-
tion and hydrolysis. As shown in Figure 1, the concentra-
tion of the epoxides 2a ,b and 5a ,b reached maxima at
0.5-1.0 h. The epoxides 2a ,b and 5b were rapidly
hydrolyzed afterward, while the epoxide 5a was hydro-
lyzed rather slowly resulting in low conversion to 6a . It
was also clearly shown in Figure 1 that the epoxidation
rate became much slower after 0.5 h in all the cases,
possibly due to the inhibition of the monooxygenase by
the formed epoxides.
The alkane monooxygenase of HXN-200 showed higher
epoxidation activity for the six-member ring substrate
1a ,b than for the five-member ring substrates 4a ,b.
Epoxidation of 1,2,5,6-tetrahydropyridine 1a and 3-pyr-
roline 4a with a N-phenoxycarbonyl docking/protecting
group is slightly less active than the hydroxylation of the
corresponding saturated substrate piperidine and pyr-
rolidine,10b,d respectively. Epoxidation of N-benzyloxy-
carbonyl-1,2,5,6-tetrahydropyridines 1b is more active
than the hydroxylation of the corresponding piperidine,10d
During the biotransformation of 1a ,b and 4a ,b, the
epoxides 2a ,b and 5a ,b were formed and then hydrolyzed
to the corresponding diols 3a ,b and 6a ,b (Figure 1). The
formed epoxides and diols were extracted from the
reaction mixture and purified, and their structures were
1
identified by H and 13C NMR analyses. These results
suggested that the alkane monooxygenase of HXN-200
is able to catalyze the epoxidation of the nonactivated
C-C double bond. No hydrolysis of the chemically
prepared epoxide 2a ,b and 5a ,b were observed with the
J . Org. Chem, Vol. 68, No. 22, 2003 8601

Chang et al.
TABLE 1. En a n tioselective Tr a n s Dih yd r oxyla tion of N-Su bstitu ted 1,2,5,6-Tetr a h yd r op yr id in es 1a ,b a n d 3-P yr r olin es
4a ,b w ith F r ozen /Th a w ed Cells (10 g cd w /L) of Sph in gom on a s sp . HXN-200
conversion and eeb (%)c
substrate
(mM)
activitya
(U/g cdw)
entry
1
product
0.5 h
1 h
2 h
3 h
4 h
5 h
1a (10)
1a (15)
1b (8.0)
1b (10)
4a (5.0)
4a (8.0)
4b (2.0)
4b (3.0)
16
14
17
18
9.0
12
4.4
5.0
(+)-2a
(+)-3a
(+)-2a
(+)-3a
2b
(+)-3b
2b
(+)-3b
5a
(+)-6a
5a
(+)-6a
5b
(+)-6b
5b
25 (48)
24 (98)
14 (49)
14 (98)
37
27 (51)
32
23 (51)
38
16 (79)
33
14 (78)
40
26 (95)
35
25 (31)
41 (94)
18 (37)
27 (98)
33
41 (49)
30
35 (51)
39
22 (76)
35
23 (77)
28
56
27
14 (-22)
72 (93)
21 (13)
54 (94)
25
52 (52)
24
39 (49)
35
31 (80)
31
30 (76)
5.1
90
12
5.8 (-84)
93 (88)
10 (-29)
74 (91)
16
67 (49)
16
56 (51)
28
43 (78)
26
37 (75)
0
97 (95)
3.3
4.0 (>-99)
96 (83)
7.2 (-68)
82 (93)
11
73 (48)
11
61 (48)
25
1.7 (>-99)
94 (77)
2
3
4
5
6
7
8
5.3
82 (49)
8.4
65 (50)
22
48 (77)
21
45 (78)
45 (77)
23
39 (75)
1.6
1.7
(+)-6b
15 (95)
28 (95)
48 (95)
59 (95)
62 (95)
64 (95)
a
Biotransformation was performed in a 10 mL cell suspension in 50 mM K-phosphate buffer (pH 8.0) containing glucose (2%), and
activity is the epoxidation activity based on the total product formation over the first 30 min. Ee is given in parentheses. c Conversion
b
and ee were determined by HPLC analysis; error limit is 2% of the stated values; “-” for ee means the opposite enantiomer is in excess.
whereas epoxidation of 3-pyrroline 4b is less active than
the hydroxylation of N-benzyloxycarbonyl-pyrrolidine.10b
In the case of trans dihydroxylation of the symmetric
substrate 4a ,b, the enantioselectivity was generated only
in the hydrolysis step, since epoxidation gave meso-
epoxide 5a ,b. While biotransformation of 4a with a
phenoxycarbonyl docking/protecting group gave diol 6a
in 78% ee, much higher enantioselectivity was observed
in dihydroxylation of 3b with a benzyloxycarbonyl dock-
ing/protecting group: the diol 6b was formed in 95% ee.
To our knowledge, these are the first examples of
enantioselective trans dihydroxylation with a bacterial
catalyst. These are also the first enantioselective dihy-
droxylations of 1,2,5,6-tetrahydropyridines and 3-pyrro-
lines, since Osmium-catalyzed dihydroxylations of 1,2,5,6-
tetrahydro pyridines and 3-prrolines resulted in racemic
and meso-cis-diols, respectively.22,23
En a n tioselective Hyd r olysis of Ra cem ic Ep oxid e
2a ,b a n d m eso-Ep oxid es 5a ,b. To investigate the
second reaction step of the trans dihydroxylation, enzy-
matic hydrolysis was performed with racemic epoxides
2a ,b and meso-epoxides 5a ,b as substrates, respectively.
The biotransformation was carried out with frozen/
thawed cells in 10 mL of 50 mM K-phosphate buffer (pH
8.0) and followed by HPLC analysis as described above.
In each case, the expected trans-diol was formed without
any byproduct. Hydrolysis of (()-2a was examined with
three different concentrations (Table 2), and the enzy-
matic activity reached 15-18 U/g cdw, which is nearly
the same as the epoxidation activity for 1a . As expected,
(+)-2a was hydrolyzed faster than (-)-2a , giving (-)-2a
in high ee at 3-5 h. The enantioselectivity factor E was
calculated as 12.8 (for 20 mM at 5 h) from the equation
E ) ln(1 - c)(1 - ees)/ln(1 - c)(1 + ees).24
To investigate the enantioselectivity for the trans
dihydroxylation, analytic samples were prepared by
taking 0.5-mL aliquots from biotransformation mixture
and extracting with equal volume of ethyl acetate. HPLC
analysis with a chiral column (Chiralcel OB-H, OD-H,
or Chiralpak AS) gave the ee values of the epoxide 2a
and diols 3a ,b and 6a ,b. During dihydroxylation of 1a
(10 mM), the ee of the diol (+)-3a was 98% at 30 min
and then decreased to 77% at 5 h (Figure 1a). The ee of
the epoxide (+)-2a was 48% at 30 min and changed to
>-99% at 5 h (Table 1). These results suggested the
following: (a) the enantioselective epoxidation of 1a
initially gave (+)-2a in about 73% ee; (b) (+)-2a was
preferably hydrolyzed affording (+)-3a in very high ee
at the beginning; (c) (-)-2a was also hydrolyzed at a
slower rate resulting in a decrease of the ee of (+)-3a
with time; and (d) pure (-)-2a remained at 5 h as a result
of resolution. Similar results for the trans dihydroxyla-
tion of a 15 mM solution of 1a were obtained (Table 1).
Reaction for 4 h gave 82% of (+)-3a in 93% ee. Changing
the docking/protecting group of 1 from the phenoxycar-
bonyl to the benzyloxycarbonyl group resulted in lower
enantioselectivity for the trans dihydroxylation. As shown
in Figure 1b, the ee of the diol 3b was 51% at 30 min
and remained nearly unchanged during the reaction.
Although an attempt to determine the ee of the epoxide
2b by HPLC analysis with a chiral column (Chiralcel OB-
H, OD-H, OJ or Chiralpak AS) failed, the constant low
ee value of 3b during the reaction suggested that the
enantioselectivity for hydrolysis of 2b is very poor. It can
be thus assumed that epoxidation of 1b gave the epoxide
2b in about 50% ee. The fact that HXN-200 catalyzes the
enantioselective epoxidation of 1a ,b is remarkable, since
enantioselective epoxidation of the aliphatic heterocyclic
C-C double bond was proven to be difficult with mi-
crobial enzyme: only Rhodococcus rhodochrous 9703
(ATCC19067) was reported to oxidize 1b giving the
corresponding epoxide as the major product with no
information about the ee.21
As shown in Figure 2, 2b was hydrolyzed as fast as
2a . Although the ee of 2b could not be determined, the
ee of the formed diol 3b was found to be very low. The
enantioselectivity factor was calculated as 2.7 (for 10 mM
(21) Flitsch, S. L.; Aitken, S. J .; Chow, C. S.-Y.; Grogan, G.; Staines,
A. Bioorg. Chem. 1999, 27, 81-90.
(22) (a) Dupau, P.; Epple, R.; Thomas, A. A.; Fokin, V. V.; Sharpless,
K. B. Adv. Synth. Catal. 2002, 344, 421-433. (b) J ensen, H. H.;
Lyngbye, L.; J ensen, A.; Bols, M. Chem. Eur. J . 2002, 8, 1218-1226.
8602 J . Org. Chem., Vol. 68, No. 22, 2003

Enantioselective Trans Dihydroxylation of Nonactivated CdC
TABLE 2. Hyd r olysis of N-Su bstitu ted 3,4-Ep oxy-p ip er id in es 2a ,b a n d 3,4-Ep oxy-p yr r olid in es 5a ,b w ith F r ozen /Th a w ed
Cells (10 g cd w /L) of Sph in gom on a s sp . HXN-200
conversion and eeb (%)c
substrate
(mM)
activitya
(U/g cdw)
entry
1
product
0.5 h
1 h
2 h
3 h
4 h
5 h
2a (10)
2a (15)
2a (20)
2b (10)
2b (20)
15
15
18
15
17
(-)-2a
(+)-3a
(-)-2a
(+)-3a
(-)-2a
(+)-3a
2b
56 (30)
44 (63)
71 (17)
29 (64)
73 (13)
27 (66)
56
46 (49)
54 (60)
61 (28)
39 (69)
63 (20)
37 (68)
50
32 (92)
68 (39)
46 (58)
54 (64)
53 (45)
47 (66)
33
27 (>99)
73 (23)
39 (87)
61 (57)
45 (71)
55 (62)
23
2
3
4
5
32 (>99)
68 (36)
41 (89)
59 (56)
11
35 (97)
65 (50)
8.9
91 (7)
39
(+)-3b
2b
44 (10)
75
50 (11)
68
67 (14)
58
77 (16)
48
89 (11)
45
(+)-3b
(+)-6a
(+)-6a
(+)-6a
(+)-6b
(+)-6b
(+)-6b
25 (12)
37 (68)
31 (68)
28 (67)
52 (95)
37 (95)
27 (95)
32 (16)
49
36
36
72
42 (19)
83
63
57
88
52 (17)
93
77
70
98
55 (19)
98
83
61 (15)
>99 (68)
95 (66)
91 (67)
>99 (95)
98 (95)
85 (95)
6
7
8
9
10
11
5a (5.0)
5a (8.0)
5a (10)
5b (10)d
5b (15)d
5b (20)d
6.2
8.1
9.2
17
18
18
76
>99
96
79
53
43
70
56
86
70
a
Biotransformation was performed in a 10 mL cell suspension in 50 mM K-phosphate buffer (pH 8.0) containing glucose (2%), and the
activity was determined over the first 30 min. Ee is given in parentheses. c Conversion and ee were determined by HPLC analysis;
error limit is 2% of the stated values. Data from ref 11.
b
d
grams suggested the same enantiomer in excess for diols
3a ,b and 6a ,b obtained from dihydroxylation of 1a ,b and
4a ,b and from hydrolysis of 2a ,b and 5a ,b, respectively.
P r ep a r a tive Tr a n s Dih yd r oxyla tion of 1a a n d 4b
a n d Absolu te Ster eoch em istr y. To demonstrate the
synthetic applicability of the enantioselective trans di-
hydroxylation, preparative biotransformations were per-
formed with frozen/thawed cells as biocatalyst in a
shaking flask (Scheme 2). Bioconversion of 1a (10 mM)
on a 100-mL scale at a cell density of 10 g cdw/L gave
91% of (+)-3a at 3 h. Extraction of the product with
n-butanol/ethyl acetate (1:1) and purification by flash
chromatography on silica gel afforded 77% of (+)-3a in
90% ee (Table 3). Reaction of 1a at an increased concen-
tration (15 mM) for 3 h gave (+)-3a in 96% ee with 83%
conversion, and the pure product was isolated in 60%
yield (106.9 mg) with an [R]25D of +4.24 (c 1.44 in CHCl3).
Similarly, biotransformation of 4b (2.0 mM) on a 100-
mL scale for 3 h gave 95% of (+)-6b. Workup and
chromatographic purification afforded 80% (37.8 mg) of
the pure product in 96% ee with an [R]25D of +7.56 (c 1.80
in CHCl3).
F IGURE 2. Hydrolysis of (()-2a ,b and 5a ,b (all 10 mM) to
the corresponding 3a ,b and 6a ,b with frozen/thawed cells (10
g cdw/L) of Sphingomonas sp. HXN-200.
at 4 h) according to the equation E ) ln[1 - c(1 + eep)]/
ln[1 - c(1 - eep)].24 Hydrolysis of the meso-epoxide 5b is
the fastest one among all hydrolyses, and the trans-diol
6b was obtained in 95% ee and >99% conversion.11 For
this asymmetric reaction, E was calculated as 39 accord-
ing to the equation E ) (1 + eep)/(1 - eep).24 Hydrolysis
of 5a showed the lowest activity (6-9 U/g cdw), which
explains why a significant amount of 5a remained in the
trans dihydroxylation of 4a (Figure 1c). The diol 6a was
formed in 67% ee, corresponding to an E of 5.1. Obviously,
the epoxide hydrolase of HXN-200 prefers substrates
with special size: N-phenoxycarbonyl epoxypiperidine 2a
and N-benzyloxycarbonyl epoxypyrrolidine 5b have simi-
lar sizes, and both of them are excellent substrates for
the hydrolysis with high activity and enantioselectivity;
hydrolysis of 2b with one carbon more or 5a with one
carbon less is significantly less enantioselective. Com-
parison of the retention times in chiral HPLC chromato-
To establish the absolute configuration, bioproduct (+)-
3a was transformed into 3,4-dihydroxy piperidine hy-
drochloride 7 in 72% yield by treatment with 6 N HCl
(Scheme 2). The obtained compound 7 has an [R]25 of
D
-14.0 (c 0.50 in MeOH), while the chemically synthesized
(3R,4R)-725 has an [R]25 of -15.0 (c 0.24 in MeOH) that
D
was determined in this study. Therefore, the absolute
configuration of bioproduct (+)-3a can be deduced as
(3R,4R). Treatment of (-)-7 with benzyl chloroformate
gave (+)-(3R,4R)-3b in 71% yield. Comparison of the
retention times of (+)-(3R,4R)-3b and bioproduct 3b in
chiral HPLC chromatograms suggested the (+)-(3R,4R)
for the major enantiomer of bioproduct 3b. Deprotection
of bioproduct (+)-6b by hydrogenation gave 94% of (-)-
(3R,4R)-8 with an [R]25D of -18.6 (c 0.80, in MeOH), thus
establishing the absolute configuration of bioproduct (+)-
6b as (3R,4R).11,26 Reaction of (-)-8 with phenyl chloro-
(23) (a) Punniyamurthy, T.; Katsuki, T. Tetrahedron 1999, 55,
9439-9454. (b) Rosenberg, S. H.; Woods, K. W.; Sham, H. L.; Kleinert,
H. D.; Martin, D. L.; Stein, H.; Cohen, J .; Egan, D. A.; Bopp, B.; Merits,
I.; Garren, K. W.; Hoffman, D. J .; Plattner, J . J . J . Med. Chem. 1990,
33, 1962-1969.
(24) Straathof, A. J . J .; J ongejan, J . A. Enzyme Microb. Technol.
1997, 21, 559-571.
(25) An authentic sample of (3R,4R)-7 prepared according to ref 12a
was given by Prof. Y. Ichikawa at J ohns Hopkins University.
J . Org. Chem, Vol. 68, No. 22, 2003 8603

Chang et al.
TABLE 3. P r ep a r a tion of (+)-3a , (+)-6b, (-)-2a , a n d (-)-3a by Biotr a n sfor m a tion w ith F r ozen /Th a w ed Cells (10 g cd w /L)
of Sph in gom on a s sp . HXN-200
substrate
(mM)
scale
(mL)
activity
(U/g cdw)
entry
product
time (h)
conversiona (%)
yieldb (%)
eea (%)
1
2
3
4
5
1a (10.0)
1a (15.0)
4b (2.0)
(()-2a (15.0)
(-)-2a (5.0)
100
50
100
50
27c
37c
6.0c
13d
6.7d
(+)-3a
(+)-3a
(+)-6b
(-)-2a
(-)-3a
3.0
3.0
3.0
3.0
6.0
91
83
95
66
>99
77.4
60.0
79.8
33.5
91.6
90
96
96
>99
96
20
a
b
Conversion and ee were determined by HPLC analysis; error limit is 2% of the stated values. Yield of the isolated pure product.
c Epoxidation activity was determined form the total product formation over the first 30 min. Hydrolysis activity was determined over
d
the first 30 min.
SCHEME 2. P r ep a r a tion of (+)-3a a n d (+)-6b
by En a n tioselective Tr a n s Dih yd r oxyla tion of 1a
a n d 4b w ith Sph in gom on a s sp . HXN-200 a n d
Ster eoch em istr y Cor r ela tion of Biop r od u cts 3a ,b
a n d 6a ,b
F IGURE 3. Hydrolysis of (()-2a (15 mM) to 3a with frozen/
thawed cells (10 g cdw/L) of Sphingomonas sp. HXN-200.
or enzymatic methods and it can be a versatile chiral
building block. Following the highly regioselective ring
opening of racemic 3,4-epoxypiperidine with HBr,27 (-)-
2a could be transformed into enantiopure trans-4-bromo-
3-hydroxy-piperidine, which can be further transformed
into other enantiopure compounds such as cis-4-amino-
3-hydroxy- and cis-3,4-dihydroxy-piperidine. Moreover,
(-)-2a is a useful intermediate for the preparation of
single enantiomer of antidepressant Ifoxetine sulfate28
and prokinetic agent Cisapride hydrate.29,30
While (+)-3a was prepared from 1a by enantioselective
trans dihydroxylation, (-)-3a was also successfully pre-
pared by hydrolysis of (-)-2a (5 mM) with HXN-200
(Scheme 3). The hydrolysis activity for (-)-2a is 6.6 U/g
cdw, which is half of that for the racemic 2a . Neverthe-
less, (-)-2a was totally converted to the corresponding
trans-diol 3a after 6 h of biotransformation (Table 3).
Workup and chromatographic purification afforded (-)-
3a in 92% yield and 96% ee with an [R]25 of - 4.05 (c
formate afforded (+)-(3R,4R)-6a in 65% yield, and com-
parison of the retention times in chiral HPLC analysis
established the absolute configuration as (+)-(3R,4R) for
the major enantiomer of bioproduct 6a .
D
0.65 in CHCl3). The high ee of the product indicates that
the ring opening of (-)-2a is highly regioselective.
P r ep a r a tion of (-)-2a a n d (-)-3a by Hyd r olysis
w ith Sph in gom on a s sp . HXN-200. The epoxide hydro-
lase of HXN-200 was shown to be enantioselective in
hydrolysis of racemic 2a , thus enabling the preparation
of enantiomerically pure epoxide 2a via resolution (Fig-
ure 3). Hydrolysis of racemic 2a (15 mM) for 3 h gave
(-)-2a in >99.9% ee. Workup and purification afforded
Con clu sion
Sphingomonas sp. HXN-200 containing a monooxyge-
nase and an epoxide hydrolase catalyzes the trans
dihydroxylation of N-phenoxycarbonyl-1,2,5,6-tetrahy-
(27) Imanishi, T.; Shin, H.; Hanaoka, M.; Momose, T.; Imanishi, I.
Chem. Pharm. Bull. 1982, 30, 3617-3623.
(28) Waldmeier, P. C.; Delini-Stula, A.; Paioni, R. Drugs Future
1987, 12, 126.
(29) Serradell, M. N.; Castaner, J .; Neuman, M. Drugs Future 1984,
9, 497.
34% of (-)-2a with an [R]25 of - 18.05 (c 1.23 in CHCl3)
D
(Table 3). This synthesis is important, since enantiopure
epoxide 2a is very difficult to prepare by either chemical
(30) Kim, B. J .; Pyun, D. K.; J ung, H. J .; Kwak, H. J .; Kim, J . H.;
Kim, E. J .; J eong, W. J .; Lee, C. H. Synth. Commun. 2001, 31, 1081-
1089.
(26) Cardona, F.; Goti, A.; Picasso, S.; Vogel, P.; Brandi, A. J .
Carbohydr. Chem. 2000, 19, 585-601.
8604 J . Org. Chem., Vol. 68, No. 22, 2003

Enantioselective Trans Dihydroxylation of Nonactivated CdC
4.15 (s, 1 H), 4.04 (s, 1 H), 3.74 (s, 1 H), 3.65 (s, 1 H), 2.25 (s,
2 H); 13C NMR (CDCl3, 100 MHz) δ 155.2, 152.5, 130.3, 126.3,
122.9, 126.7, 126.2, 125.4, 124.8, 44.9, 42.4, 41.6, 26.4, 25.9;
MS m/z 204 (100%, M + 1); IR (CHCl3) ν 1713, 1425, 1238,
SCHEME 3. P r ep a r a tion of (-)-2a a n d (-)-3a by
Hyd r olysis w ith Sph in gom on a s sp . HXN-200
1204 cm-1
.
N-P h en oxyca r bon yl-3,4-ep oxyp ip er id in e (2a ). m-CPBA
(1.43 g, 8.34 mmol) was added to a solution of 1a (740.6 mg,
3.64 mmol) in CH2Cl2 (20 mL) and the mixture was stirred at
room temperature for 24 h. NaOH (1 N, 15 mL) was added
and the mixture was extracted with CH2Cl2 (3 × 20 mL). The
organic phase was separated and dried over Na2SO4, and the
solvent was removed. Column chromatography on a silica gel
gave 631.2 mg (79.0%) of 2a with 99.7% purity (GC, tR ) 9.02
min). The spectroscopic data are the same as those for (-)-2a
reported below.
N-P h en oxycar bon yl-3,4-dih ydr oxypiper idin e (3a). TFA
(0.30 mL) was added to a solution of 2a (30.0 mg, 0.137 mmol)
in CHCl3 (2 mL) and the mixture was stirred at reflux for 6 h.
pH was adjusted to 8.0 by addition of 10% Na2CO3 and the
mixture was extracted with CHCl3 (3 × 20 mL). The organic
phase was dried over Na2SO4 and the solvent was removed.
Column chromatography on silica gel afforded 16.7 mg (51.5%)
of 3a with a purity of 99.3% (GC, tR ) 10.30 min). The
spectroscopic data are the same as those for (+)-3a reported
below.
N-Ben zyloxyca r bon yl-3,4-d ih yd r oxyp ip er id in e (3b ).
Similar to the preparation of 3a , hydrolysis of 2b (30.0 mg,
0.129 mmol) gave 19.9 mg (61.6%) of 3b with 98.7% purity
(GC, tR ) 9.53 min). The spectroscopic data agree with those
reported in ref 12b.
dropyridine 1a and N-benzyloxycarbonyl-3-pyrroline 4b,
respectively, with high activity and high enantioselec-
tivity. While enantioselectivity for the trans dihydroxyl-
ation of 1a is a combination of those for the epoxidation
of 1a and hydrolysis of 2a , the enantioselectivity for the
trans dihydroxylation of symmetric substrate 4b is due
only to the hydrolysis of meso-epoxide 5b. The absolute
configuration was established as (3R,4R) for both (+)-3a
and (+)-6b by chemical correlations. Preparative trans
dihydroxylations of 1a and 4b with frozen/thawed cells
of Sphingomonas sp. HXN-200 as biocatalyst afforded the
corresponding (+)-3,4-dihydroxypiperidine 3a and (+)-
3,4-dihydroxypyrrolidine 6b in 60% and 80% yield,
respectively, and both in 96% ee. These highly yielding
and highly enantioselective trans dihydroxylations pro-
vide the simplest syntheses of the trans-diols which are
useful pharmaceutical intermediates. These are the first
enantioselective trans dihydroxylations of a nonisoprene
C-C double bond of an aliphatic substrate and the first
bacterium-catalyzed enantioselective trans dihydroxyla-
tions, thus significantly extending this useful methodol-
ogy in organic synthesis.
N-P h en oxyca r bon yl-3,4-ep oxy-p yr r olid in e (5a ). Simi-
lar to the preparation of 2a , reaction of m-CPBA (0.52 g, 3.0
mmol) with 4a (284 mg, 1.50 mmol) gave 205.0 mg (66.7%) of
5a as solid. Mp 72.1-73.0 °C; Rf 0.42 (n-hexane/ethyl acetate
1
1/1); purity 99.0% (GC, tR ) 9.05 min); H NMR (CDCl3, 400
MHz) δ 7.35 (t, 2 H, J ) 7.41 Hz), 7.20 (t, 1 H, J ) 7.2 Hz),
7.11 (d, 2 H, J ) 7.2 Hz), 4.04 (d, 1 H, J ) 13.2 Hz), 3.95 (d,
1 H, J ) 13.2 Hz), 3.77 (s, 2 H), 3.58 (d, 1 H, J ) 13.2 Hz),
3.48 (d, 1 H, J ) 13.2 Hz); 13C NMR (CDCl3, 100 MHz) δ 154.7,
152.1, 130.4, 126.5, 122.8, 56.6, 56.1, 48.7; MS m/z 206 (100%,
M + 1); IR (CHCl3) ν 1721, 1420, 1392, 1204 cm-1
.
N-P h en oxyca r b on yl-3,4-d ih yd r oxy-p yr r olid in e (6a ).
Similar to the preparation of 3a , reaction of 5a (30.0 mg, 0.15
mmol) with TFA (0.30 mL) afforded 26.9 mg (82.4%) of 6a with
97.6% purity (GC, tR ) 9.30 min). The spectroscopic data are
the same as those for (+)-6a reported below.
Enantioselective hydrolysis of the racemic epoxide 2a
with the epoxide hydrolase of HXN-200 afforded (-)-2a ,
a versatile chiral building block, in >99% ee and 34%
yield. Further hydrolysis of (-)-2a with HXN-200 showed
excellent regioselectivity and gave (-)-(3S,4S)-3a in 96%
ee and 92% yield. Thus, both enantiomers of 3a can be
prepared in high ee by biotransformation with Sphin-
gomonas sp. HXN-200.
Gen er a l P r oced u r e for Biocon ver sion on
a Sm a ll
Sca le. Frozen cells of Sphingomonas sp. HXN-200 were
thawed and suspended to a cell density of 10 g cdw/L in 10
mL of 50 mM K-phosphate buffer containing glucose (2% w/v)
at pH 8.0 in a 100-mL Erlenmeyer flask. Substrates were
added either directly (1a , 2b, 4a , 5a ) or as a MeOH solution
(1b, 2a , 4b, 5b) to a final concentration of 2-20 mM. The
mixture was shaken at 200 rpm at 30 °C for 3-5 h and the
reaction was followed by HPLC analysis of samples that were
prepared by taking aliquots (0.1-0.2 mL) from a bioconversion
mixture at predetermined time points and mixing with an
equal volume of MeOH followed by removal of the cells by
centrifugation. The ee of the products was determined by
HPLC analysis on a chiral column, and the samples were
prepared by taking aliquots (0.5 mL) from a biotransformation
mixture, mixing with an equal volume of ethyl acetate,
removing the cells, separating the organic phase after cen-
trifugation, drying over sodium sulfate, and filtering. The
results are listed in Tables 1 and 2.
Exp er im en ta l Section
N-P h en oxyca r bon yl-1,2,5,6-tetr a h yd r op yr id in e (1a ). A
solution of phenyl chloroformate (1.50 mL, 12.0 mmol) in THF
(5 mL) was added dropwise to a stirred mixture of 1,2,5,6-
tetrahydropyridine (0.996 g, 12.0 mmol) and NaHCO3 (1.30 g,
15.6 mmol) in THF-water (1:1, 12 mL) at 0 °C, and the
mixture was stirred at room temperature for 3 h. CHCl3 (20
mL) and 5% aqueous Na2CO3 (10 mL) were added, the organic
phase was separated, and the aqueous phase was extracted
with CHCl3 (50 mL). The combined organic phase was dried
over Na2SO4 and filtered, and the solvent was removed by
evaporation. Column chromatography on silica gel gave 1.6953
g (69.6%) of 1a 18 as solid. Mp 55.9-56.7 °C; Rf 0.38, (n-hexane/
ethyl acetate 8/2); purity 99.0% (GC, tR ) 8.20 min); 1H NMR
(CDCl3, 400 MHz) δ 7.36 (t, 2 H, J ) 7.4 Hz), 7.20 (t, 1 H, J )
7.2 Hz), 7.12 (d, 2 H, J ) 7.2 Hz), 5.90 (s, 1 H), 5.73 (s, 1 H),
(a ) HP LC a n a lysis of con ver sion : Hypersil BDS-C18
column (5 µm, 125 mm × 4 mm); eluent, a mixture of A (10
mM K-phosphate buffer, pH 7.0) and B (acetonitrile); flow rate,
1.0 mL/min; detection, UV at 210, 225, and 254 nm; tR of 3a )
1.6 min, tR of 2a ) 3.4 min, tR of 1a ) 9.1 min (A/B 65:35);
tR of 3b ) 1.8 min, tR of 2b ) 4.1 min, tR of 1b ) 10.8 min
(A/B 70:30); tR of 6a ) 1.8 min, tR of 5a ) 3.3 min, tR of 4a )
J . Org. Chem, Vol. 68, No. 22, 2003 8605

Chang et al.
7.5 min (A/B 70:30); tR of 6b ) 2.0 min, tR of 5b ) 4.2 min, tR
of 4b ) 9.2 min (A/B 70:30).
product (+)-3a (50.0 mg, 0.21 mmol) in MeOH (1 mL) and 6 N
HCl (5 mL) was stirred at reflux for 2 h. pH was adjusted to
10 by addition of 12 N NaOH and the solvent was evaporated
to dryness. The residue was treated with MeOH and purified
by column chromatography on silica gel (Rf 0.18, CHCl3/MeOH/
25%aq NH3H2O 8:3:1) to afford 20.2 mg (81.8%) of 3,4-
dihydroxypiperidine. Part of this product (10.0 mg, 0.085
mmol) was dissolved in MeOH (2 mL), HCl (32%, 50 µL) was
added, and the resulting mixture was evaporated to dryness
giving 11.5 mg (87.7%) of (-)-7 as a solid: [R]25D -14.0 (c 0.50,
MeOH). The spectroscopic data agree with those for (3R,4R)-7
(b) HP LC a n a lysis of ee: chiral column (250 mm × 4.6
mm); UV detection at 210 nm; a mixture of A (n-hexane) and
B (2-propanol). For 2a : Chiralcel OB-H column; flow rate, 0.5
mL/min, A/B (60/40); tR ) 49.4 and 55.4 min. For 3a : Chiralcel
OB-H column; flow rate, 0.5 mL/min, A/B (95/5); tR ) 92.7 and
110.4 min. For 3b: Chiralpak AS column; flow rate, 1.0 mL/
min, A/B (93/7); tR ) 25.9 and 29.0 min. For 6a : Chiralcel
OD-H column; flow rate, 0.5 mL/min, A/B (85/15); tR ) 18.1
and 25.5 min. For 6b: Chiralpak AS column; flow rate, 1.0
mL/min, A/B (97/3); tR ) 114.1 and 130.4 min.
reported in ref 12a. [R]25 of a synthetic sample of (3R,4R)-725
D
Gen er a l P r oced u r e for Biotr a n sfor m a tion on a P r e-
p a r a tive Sca le. Substrate was added to a 20-100 mL
suspension of frozen/thawed cells of Sphingomonas sp. HXN-
200 (10.0 g cdw/L) in 50 mM K-phosphate buffer (pH 8.0)
containing glucose (2%) in a 100-500 mL shaking flask. The
mixture was shaken at 200 rpm and 30 °C for 3-6 h. The cells
were removed by centrifugation and the supernatant was
extracted with n-butanol/ethyl acetate (1:1) (for 3a , 6b) or ethyl
acetate (for 2a ). 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
and the results are summarized in Table 3.
was determined as -15.0 (c 0.24, MeOH).
Tr a n sfor m a tion of (-)-7 to (+)-(3R,4R)-N-Ben zyloxy-
ca r bon yl-3,4-d ih yd r oxy-p ip er id in e (3b). Phenyl chlorofor-
mate (0.050 mL, 0.397 mmol) in THF (2 mL) was added
dropwise to a mixture of (-)-7 (10.0 mg, 0.085 mmol) and
NaHCO3 (160 mg, 1.905 mmol) in THF/H2O (1:1, 4 mL) at room
temperature, the mixture was stirred for 20 h, and ethyl
acetate (30 mL) was added. The organic phase was separated,
dried over Na2SO4, and filtered, and the solvent was removed
by evaporation. Column chromatography on silica gel gave 15.2
mg (71.2%; 0.061 mmol) of (3R,4R)-3b: [R]25 +3.51 (c 0.76,
D
CHCl3); Rf 0.38 (ethyl acetate); purity 97.9% (GC, tR ) 9.53
min). The spectroscopic data agree with those reported in ref
12b. The major enantiomer of bioproduct 3b has the same
retention time as the chemically prepared (+)-(3R,4R)-3b.
Tr a n sfor m a tion of Biop r od u ct (+)-6b to (-)-(3R,4R)-
3,4-Dih yd r oxyp yr r olid in e (8). A mixture of bioproduct (+)-
6b (100 mg, 0.42 mmol) and 20% Pd(OH)2/C (55 mg) in MeOH
(3.0 mL) was stirred under hydrogen for 24 h and filtered
through Celite. Column chromatography on silica gel afforded
8 in 93.9% (40.8 mg) as waxy solid: Rf 0.17, (CH2Cl2/MeOH/
(+)-(3R,4R)-N-P h en oxyca r bon yl-3,4-d ih yd r oxy-p ip er i-
d in e (3a ). Trans dihydroxylation of 1a (152.3 mg, 0.75 mmol)
on a 50-mL scale gave 106.9 mg (60.0%) of (+)-3a as solid. Mp
136.8-137.5 °C; ee 96% (3R,4R); [R]25 +4.24 (c 1.44, CHCl3);
D
Rf 0.32 (ethyl acetate); purity 99.3% (GC, tR ) 10.30 min); 1H
NMR (CDCl3, 400 MHz) δ 7.36 (t, 2 H, J ) 7.8 Hz), 7.20 (t, 1
H, J ) 7.4 Hz), 7.08 (d, 2 H, J ) 8.4 Hz), 4.34-4.15 (m, 2 H);
3.54-3.45 (m, 2 H), 3.12-2.77 (m, 3 H), 2.04-1.96 (m, 1 H),
1.90 (s, 1 H), 1.59-1.56 (m, 1 H); 13C NMR (CDCl3, 100 MHz)
δ 155.2, 152.2, 130.4, 126.6, 122.8, 74.5, 73.9, 72.9, 72.5, 49.2,
49.0, 43.8, 43.5, 32.4; MS m/z 238 (9%, M + 1), 118 (100%); IR
25% aq NH3H2O 8:3:1); [R]D -18.6 (c 0.80, MeOH); 1H NMR
25
(D2O, 400 MHz) δ 4.03-4.01 (m, 2 H), 3.08 (dd, 1 H, J ) 12.8,
(CHCl3) ν 3614, 3425, 1713, 1428, 1204 cm-1
.
4.4 Hz); 2.73 (d, 1 H, J ) 12.8 Hz); 13C NMR (D2O, 100 MHz)
δ 79.6, 54.6; MS (m/z) 104 (100%, M + 1); [R]26 of (3S,4S)-8
(+)-(3R,4R)-N-Ben zyloxyca r bon yl-3,4-d ih yd r oxy-p yr -
r olid in e (6b). Trans dihydroxylation of 4b (40.6 mg, 0.20
mmol) on a 100-mL scale gave 37.8 mg (79.8%) of (+)-6b as a
D
was reported to be +20.7 (c 0.3, MeOH).26
Tr a n sfor m a tion of (-)-8 to (+)-(3R,4R)-N-P h en oxyca r -
bon yl-3,4-d ih yd r oxy-p yr r olid in e (6a ). Similar to the trans-
formation of (-)-7 to (+)-3b, reaction of (-)-8 (10.0 mg, 0.097
mmol) with phenyl chloroformate (0.050 mL, 0.397 mmol) in
THF (2 mL) gave 14.0 mg (64.9%, 0.063 mmol) of (3R,4R)-6a
syrup: ee 96% (3R,4R); [R]25 +7.56 (c 1.80, CHCl3); Rf 0.27
D
(ethyl acetate); purity 98.9% (GC, tR ) 10.61 min). The
spectroscopic data agree with those for (3R,4R)-6b reported
in ref 13a.
(-)-N-P h en oxyca r bon yl-3,4-ep oxy-p ip er id in e (2a ). Hy-
drolysis of racemic 2a (164.3 mg, 0.75 mmol) on a 50-mL scale
afforded 54.9 mg (33.5%) of (-)-2a as a syrup: ee >99.9%;
[R]25D -18.05 (c 1.23, CHCl3); Rf 0.24 (hexane/ethyl acetate 2/1);
purity 99.7% (GC, tR ) 9.02 min); 1H NMR (CDCl3, 400 MHz)
δ 7.36 (t, 2 H, J ) 7.8 Hz), 7.20 (t, 1 H, J ) 7.2 Hz), 7.10 (dd,
2 H, J ) 7.2, 6.8 Hz), 4.08-3.97 (m, 2 H), 3.80 (d, 0.5 H, J )
17.2 Hz), 3.67 (dt, 0.5 H, J ) 13.2, 5.2 Hz), 3.55 (dt, 0.5 H,
J ) 13.2, 5.2 Hz), 3.39-3.27 (m, 2.5 H), 2.16 (dt, 1 H, J )
14.8, 4.4 Hz), 2.08-1.98 (m, 1 H); 13C NMR (CDCl3, 100 MHz)
δ 155.1, 154.9, 152.3, 130.4, 126.5, 122.8, 51.8, 51.6, 51.4, 51.1,
44.0, 43.8, 39.2, 38.7, 25.8, 25.2; MS m/z 220 (90%, M + 1),
as solid: mp 125.4-126.1 °C; [R]25 +17.71 (c 0.70, MeOH);
D
1
Rf 0.22 (ethyl acetate); purity 97.8% (GC, tR ) 9.30 min); H
NMR (CDCl3, 300 MHz) δ 7.36 (t, 2 H, J ) 7.4 Hz), 7.20 (t, 1
H, J ) 7.2 Hz), 7.12 (d, 2 H, J ) 7.2 Hz), 4.22 (m, 1 H), 4.18
(m, 1 H), 3.86 (dd, 1 H, J ) 12.1, 4.4 Hz), 3.76 (dd, 1 H, J )
12.1, 4.4 Hz), 3.58 (d, 1 H, J ) 12.1 Hz), 3.50 (d, 1 H, J ) 12.1
Hz), 2.13 (s, 2 H); 13C NMR (CD3OD, 75 MHz) δ 155.3, 152.5,
130.2, 126.4, 122.7, 76.2, 75.4, 53.3; MS (m/z) 224 (100%, M +
1); IR (CHCl3) ν 3620, 3444, 1714, 1408 cm-1. The major
enantiomer of bioproduct 6a has the same retention time as
the chemically prepared (+)-(3R,4R)-6a .
118 (100%); IR (CHCl3) ν 1716, 1428, 1205 cm-1
.
Ack n ow led gm en t. We thank Prof. Y. Ichikawa at
J ohns Hopkins University for providing an authentic
sample of (3R,4R)-7 and Prof. K. Engesser at the
University of Stuttgart for supplying us with the HXN-
200 strain.
(-)-(3S,4S)-N-P h en oxyca r bon yl-3,4-d ih yd r oxy-p ip er i-
d in e (3a ). Hydrolysis of (-)-2a (21.9 mg, 0.10 mmol) on a 20-
mL scale gave 21.7 mg (91.6%) of (-)-3a : purity 97.9% (GC,
tR ) 10.30 min); ee 96% (3S,4S); [R]25 -4.05 (c 0.65, CHCl3);
D
1H NMR (CDCl3, 300 MHz) δ 7.36 (t, 2 H, J ) 7.8 Hz), 7.20 (t,
1 H, J ) 7.5 Hz), 7.08 (d, 2 H, J ) 7.8 Hz), 4.38-4.15 (m, 2
H), 3.56-3.47 (m, 2 H), 3.18-2.75 (m, 2 H), 2.52 (s, 2 H), 2.05-
1.95 (m, 1 H), 1.60-1.55 (m, 1 H); 13C NMR (CDCl3, 75 MHz)
δ 153.6, 151.0, 129.2, 125.4, 121.6, 73.4, 72.9, 71.9, 71.5, 48.2,
48.1, 42.7, 42.4, 31.4; MS m/z 238 (100%, M + 1); IR (CHCl3)
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal details; 1H and 13C NMR spectra of the bioproducts (-)-
2a , (-)-3a , (+)-3a , and (+)-6b and the chemically prepared
compounds 1a , 2a , 3a , (+)-3b, 5a , 6a , (+)-6a , (-)-7, and (-)-
8. This material is available free of charge via the Internet at
http://pubs.acs.org.
ν 3621, 3457, 1713, 1428, 1209 cm-1
.
Absolu te Ster eoch em istr y of Biop r od u cts. Tr a n sfor -
m a tion of Biop r od u ct (+)-3a to (-)-(3R,4R)-3,4-Dih y-
d r oxyp ip er id in e Hyd r och lor id e (7). A suspension of bio-
J O034628E
8606 J . Org. Chem., Vol. 68, No. 22, 2003