Biohydroxylation of pyrrolidines
J . Org. Chem., Vol. 66, No. 25, 2001 8429
Gen er a l P r oced u r e for Hyd r oxyla tion of P yr r olid in es
8-12 on a Sm a ll Sca le. The cells of Sphingomonas sp. HXN-
20043 were resuspended into 20 mL of 50 mM K-phosphate
buffer containing glucose (0 or 2% w/v) at pH 7.5 to a cell
density of 3-6 g/L in a 100 mL Erlenmeyer flask. Pure
substrates 8-10 and 12 and a solution of 10% 11 in MeOH
were added to the mixture to a final concentration of 2-10
mM, respectively. The mixtures were shaken at 200 rpm at
30 °C for 1-24 h, and the reactions were followed by HPLC
analysis of samples that were prepared by taking aliquots
(0.1-0.2 mL) from the bioconversion mixtures at predeter-
mined time points and mixing with equal volume of MeOH
followed by removal of cells by centrifugation.
HP LC An a lysis for Bioh yd r oxyla tion of P yr r olid in es
8-12. The following conditions were used to analyze samples:
Hypersil BDS-C18 (5 µm) column (125 mm × 4 mm), UV
detection at 210 nm, flow rate of 1 mL/min; eluent A,
acetonitrile, and eluent B, 10 mM K-phosphate buffer (pH 7.0).
Retention times (min): tR of 15, 2.7; tR of 8, 4.6, A/B 20/80; tR
of 16, 1.5; tR of 9, 3.2, A/B 30/70; tR of 17, 1.8; tR of 10, 4.2, A/B
50/50; tR of 18, 2.1; tR of 11, 5.9, A/B 35/65; tR of 19, 1.7; tR of
12, 5.5, A/B 40/60.
pyrrolidines 16-19 in an ee of 53% (S) to 75% (R) with
high activity, high conversion, and excellent regioselec-
tivity. Improvement of the enantioselectivity and the
enantiocomplementary formation of 3-hydroxypyrro-
lidines are achieved by changing the docking/protecting
group in pyrrolidines. The enantioselectivity could be
further improved by use of an appropriate docking/
protecting group. Simple crystallizations increased the
ee of bioproducts 16-18 to 95% (R), 98% (R), and 96%
(S), respectively. The alkane monooxygenase of Sphin-
gomonas sp. HXN-200 is a soluble enzyme, different from
the well-known membrane-bound alkane hydroxylase of
P. putida GPo1. Cells of Sphingomonas sp. HXN-200 can
be easily prepared in large amounts, stored at -80 °C
for 2 years without significant loss of activity, and the
frozen cells can be thawed and resuspended for bio-
hydroxylation. With high activity and broad substrate
range, the easy-handling frozen/thawed cells of Sphin-
gomonas sp. HXN-200 can be used as a routine biocata-
lyst for regio- and stereoselective hydroxylation in organic
synthesis. Finally, we have developed the first practical
biohydroxylation for preparing (S)- and (R)-3-hydroxy-
pyrrolidines by use of either frozen/thawed or growing
cells of Sphingomonas sp. HXN-200 as biocatalysts.
Sta n d a r d Wor k u p P r oced u r e for Bioh yd r oxyla tion of
P yr r olid in es 8-12. The cells were removed from the biocon-
version mixture by centrifugation, and the supernatant was
adjusted to pH 11-12 by addition of KOH followed by
extraction with ethyl acetate. The organic phase was separated
and dried over MgSO4, and the solvent was removed by
evaporation. The product was purified by column chromatog-
raphy on aluminum oxide or silica gel.
Exp er im en ta l Section
Deter m in a tion of ee of Biop r od u cts 16-19 by HP LC
An a lysis. The following conditions were used to analyze
samples: Chiral column (250 mm × 4.6 mm), UV detection at
210 and 254 nm, flow rate at 1.0 mL/min; eluent A, n-hexane,
and eluent B, 2-propanol. Retention times (min): tR of (S)-16,
19.5; tR of (R)-16, 22.9, column, chiralcel OD-H, A/B 9/1; tR of
(S)-17, 13.5; tR of (R)-17, 15.4, column, chiralpak AS, A/B 9/1;
tR of (S)-18, 15.1; tR of (R)-18, 12.0, column, chiralcel OJ , A/B
8/2; tR of (S)-19, 34.8; tR of (R)-19, 39.0, column, chiralpak AS,
A/B (99/1). ee of 16-19 from exploratory hydroxylation: 52%
(R) for 16; 75% (R) for 17; 39% (S) for 18; and 23% (R) for 19.
Gen er a l P r oced u r e for Hyd r oxyla tion of P yr r olid in es
8-12 w ith Solu ble Cell-F r ee Extr a cts. The cells of Sphin-
gomonas sp. HXN-200 were suspended in 60 mL of Tris-HCl
buffer (pH 7.5) to a density of 20 g/L. After three passages
through the French press, the cell debris was removed by
centrifugation at 245 000g for 45 min yielding soluble cell-free
extracts containing no membrane proteins. To each 10 mL of
extract was added the substrate 8-12 and equimolar NADH,
respectively. The mixture was shaken at 200 rpm and at 30
°C for 1 h and the products 15-19 were isolated. The
conversion and the ee of 15-19 were analyzed by HPLC and
listed in Table 2.
Gen er a l P r oced u r e for P r ep a r a tive Bioh yd r oxyla -
tion s w ith F r ozen /Th a w ed Cells. Frozen cells of Sphingo-
monas sp. HXN-20043 were thawed and suspended in 0.9-2 L
of 50 mM of K-phosphate buffer (pH 8.0) containing glucose
(2%, w/v) in a 3 L bioreactor, the substrate was added, and
the mixture was stirred at 1500 rpm and at 30 °C under the
introduction of air at 1 L/min. The biotransformation was
followed by analytical HPLC. Workup according to the same
procedure described above and purification by column chro-
matography on silica gel afforded the pure products.
Gen er a l Meth od s. 1H and 13C NMR spectra were deter-
mined at 300 (1H) and 75 (13C) MHz, all in CDCl3, with
chemical shifts in ppm relative to TMS and coupling constants
J in Hz. Mass spectra were obtained by atmospheric pressure
chemical ionization (APCI) with LC-MS. IR spectra were
measured in CHCl3. Optical rotations were determined using
a Perkin Elmer 241 polarimeter. Melting points are uncor-
rected. Bioconversion was analyzed by HPLC. The purity of
the products was established by GC analyses with a Chrompack
CP-Sil-5CB column (25 m × 0.32 mm, temperature program
60 °C for 2 min, increase to 280 °C at a rate of 25 °C/min,
then 280 °C for 1 min). The ee of the products was determined
by HPLC analysis with a chiral column.
Ma ter ia ls. Pyrrolidine (>99.5%) and (R)-3-hydroxypyrro-
lidine (>99%) were purchased from Fluka, (S)-3-hydroxypyr-
rolidine (97%) from Synthon Co., and N-tert-butoxycarbonyl-
pyrrolidine 12 (97%), (R)- and (S)-N-benzyl-3-hydroxypyrrolidine
15 (98% and 99%, respectively) from Aldrich. Pyrrolidines
8,5a 9,34 1035, 11,36 13,37 and 1438 and the standard (R)- and
(S)-3-hydroxypyrrolidines 16, 17,31a and 1939 were prepared
according to the published procedures.
(R)-N-P h en oxycar bon yl-3-h ydr oxypyr r olidin e 18. Phen-
yl chloroformate (0.37 mL, 2.15 mmol) was added dropwise at
room temperature to a mixture of (R)-3-hydroxypyrrolidine
(260 mg, 2.98 mmol) and NaHCO3 (350 mg, 4.17 mmol) in THF
(3 mL) and H2O (3 mL), and the mixture was stirred for 2 h.
CH2Cl2 (10 mL) was added; the organic phase was separated,
washed with 5% Na2CO3 (5 mL), dried over Na2SO4, and
filtered; and the solvent removed by evaporation. Column
chromatography on silica gel gave 148 mg (24%) of (R)-18: Rf
0.12 (ethyl acetate/n-hexane 1:1); mp 74.3-75.7 °C; [R]20
D
1
-23.8 (c 1.04, CHCl3); H NMR (CDCl3) δ 7.35 (2 H, tt, J )
7.8, 2.1 Hz), 7.18 (1 H, tt, J ) 7.4, 1.1 Hz), 7.12 (2 H, d, J )
8.3 Hz), 4.43 (1 H, br, s), 3.76-3.45 (4 H, m), 2.48 (0.5 H, br,
s), 2.46 (0.5 H, br, s), 2.09-1.82 (2 H, m); 13CNMR (CDCl3) δ
153.47 (s), 153.38 (s), 151.32 (s), 129.27 (d), 125.26 (d), 121.76
(d), 70.91 (d), 70.04 (d), 54.83 (t), 54.62 (t), 44.37 (t), 34.09 (t),
33.52 (t); MS (40 eV) m/z 208 (M + 1, 100%); IR (CHCl3) ν
N-Ben zoyl-3-h yd r oxyp yr r olid in e 16. Biohydroxylation of
N-benzoylpyrrolidine 9 (380 mg, 2.17 mmol) was started in 1
L of cell suspension (7.3 g/L). Additional 9 were added to the
mixture at 1 h (352 mg, 2.01 mmol), 3.5 h (357 mg, 2.04 mmol),
7.5 h (581 mg, 3.32 mmol), and 10.6 h (520 mg, 2.97 mmol).
New cells suspension (130 mL) and glucose (30 mL, 50% w/v)
were added at 5.5 h to a cell density of 13.1 g/L. Biotransfor-
mation for 24 h gave 75% of 16. Workup with CHCl3 as
3425, 1712 cm-1
.
(S)-N-P h en oxyca r bon yl-3-h yd r oxyp yr r olid in e 18. In
the same procedure, 150 mg (34%) of (S)-18 was prepared from
(S)-3-hydroxypyrrolidine (189 mg, 2.17 mmol), NaHCO3 (302
mg, 3.59 mmol), and phenyl chloroformate (0.27 mL, 2.94
(43) For preparation of cells of Sphingomonas sp. HXN-200 see
Supporting Information.
mmol): mp 74.0-74.7 °C; [R]20 23.2 (c 1.01, CHCl3).
D