Notes
J . Org. Chem., Vol. 62, No. 15, 1997 5217
ratory (USA). Microorganism cultures were carried out in a
Setric 7 L fermentor. All reactions involving anhydrous condi-
tions were conducted in flame-dried glassware under a positive
atmosphere of oxygen-free nitrogen. All the dried solvents were
distilled under nitrogen immediately prior use. THF, Et2O, and
CH2Cl2 were dried by distillation, respectively, from sodium
benzophenone ketyl, on lithium aluminum hydride and on CaH.
Separation by flash chromatography were performed using silica
gel 60H and a pentane/ethyl acetate gradient. Melting points
are uncorrected. 1H NMR (250 MHz) and 13C NMR (62.5 MHz)
spectra were recorded in CDCl3 (unless otherwise specified) with
TMS as internal standard. Coupling constants (J ) are in hertz.
IR spectra were achieved in KBr film.
128.50, 129.12, 138.43; IR (ν) 1358, 1265, 1176, 945, 738, 704,
528 cm-1; MS EI (m/ z) 366 (1), 270 (32), 179 (13), 145 (64), 107
(56), 91 (100), 79 (65), 68 (63), 55 (43), 41 (32), 29 (19). Anal.
Calcd for C14H22O7S2 (366.4552): C, 45.88; H, 6.06. Found: C,
46.12; H, 6.31.
(S)-(+)-N-Ben zyl-3-[(ben zyloxy)m eth yl]p yr r olid in e (6).
Dimesylate (R)-(-)-5 (3.0 g, 8.2 mmol), benzylamine (1.8 g, 16.5
mmol), and triethylamine (3 mL) in dioxane (30 mL) were
refluxed for 12 h. The crude mixture was concentrated in vacuo,
and the residue was dissolved in CH2Cl2 (50 mL), washed with
5% HCl solution (2 × 10 mL), concentrated, and purified by flash
chromatography and bulb-to-bulb distillation (120 °C/0.1 mbar)
to give (S)-(+)-6 (2.0 g, 85%) as a colorless oil: [R]25 ) +0.43 (c
D
) 4.0, CH2Cl2); 1H-NMR δ 1.42-1.55 (m, 1H), 1.89-2.04 (m, 1H),
2.29 (dd, J ) 9.2, 5.9, 1H), 2.44-2.62 (m, 2H), 2.72 (dd, J ) 9.0,
7.8, 1H), 3.39 (d, J ) 7.2, 2H), 3.59 (d, J ) 2.8, 2H), 3.78-3.81
(m, 1H), 7.23-7.37 (m, 10H); 13C-NMR δ 28.25, 38.07, 54.44,
58.22, 61.15, 73.63, 74.72, 127.48, 128.13, 128.22, 128.82, 129.01,
129.43, 139.17, 139.85; IR (ν) 3062, 3028, 2956, 2924, 2854, 2789,
(R)-(-)-4-[(Ben zyloxy)m eth yl]-dih ydr ofu r an -2-on e (2).12,13
An adequate volume of suspension containing 2 × 107 spores
(in 0.5% Tween 80 solution) of C. echinulata NRRL 3655 was
used to inoculate a 5 L complex medium in a 7 L Setric fermentor
(complex medium composition: 100 g of corn steep liquor
(Roquette SA), 20 g of glucose, 5 g of KH2PO4, 10 g of K2HPO4,
10 g of NaNO3, 2.5 g of KCl, 2.5 g of MgSO4, 0.1 g of FeSO4, 1
mL of Pluronic PE 8100 (BASF), 0.25 mL of Antifoam Silicon
426R (Prolabo). Cells were grown for 60 h at 27 °C (450 rpm,
30 L/h air) and then harvested by filtration and washed with
water (5-7 g/L dry weight). They were suspended in 5 L of
phosphate buffer (5 g of KH2PO4, 10 g of K2HPO4, pH 7). To
this cell suspension was added an alcoholic solution of ketone 3
(5 g, 26 mmol, dissolved in 50 mL of EtOH) in the 7 L fermentor
(27 °C, 450 rpm, 30 L/h air). The reaction was stopped after
24-30 h, once the ketone disappeared (reaction monitored by
GC-BP 10 column). Biotransformation was quenched by addi-
tion of a HCl solution until pH 2. The medium was then
continuously extracted with CHCl3 for 48 h. After drying over
MgSO4, purification by flash chromatography afforded a mixture
of 3-[(benzyloxy)methyl]cyclobutanols (cis/trans: 12/1, 0.8 g,
16%), identified by comparison with literature data,11 and (R)-
1494, 1453, 1364, 1453, 1364, 1264, 1097, 1028, 737, 637 cm-1
;
MS EI (m/ z) 281 (3), 190 (24), 175 (65), 132 (10), 98 (17), 91
(100), 84 (48), 65 (19), 42 (19). Anal. Calcd for C19H23NO
(281.4013): C, 81.10; H, 8.24; N, 4.98. Found: C, 81.42; H, 8.33;
N, 4.75.
(S)-(-)-N-Ben zyl-3-(h yd r oxym eth yl)p yr r olid in e (7).7
A
suspension of (S)-(+)-6 (0.5 g, 1.8 mmol) and 10% Pd/C (200 mg)
was stirred 24 h in MeOH under H2 (1 atm). The palladium
carbon catalyst was filtered off, the solution concentrated, and
the residue distilled (110 °C/0.1 mbar) to give (S)-(-)-7 (0.32 g,
95%) as a colorless oil: [R]25D ) -3.1 (c ) 4.1, EtOH) (lit.7 [R]20
D
) -2.55 (c ) 3.98, EtOH)); 1H-NMR δ 1.25-1.42 (m, 1H), 1.70-
1.90 (m, 1H), 1.95 (s/br, 1H), 2.22-2.40 (m, 1H), 2.61 (dd, J )
11.1, 5.7, 1H), 2.71-2.90 (m, 2H), 2.90-2.99 (m, 1H), 3.20-3.40
(m, 2H), 4.44 (s, 2H), 7.25-7.35 (m, 5H); 13C-NMR δ 30.07, 39.78,
47.55, 51.28, 73.62, 74.08, 128.09, 128.13, 128.96, 139.05; IR (ν)
3461, 3030, 2939, 2866, 1635, 1351, 1171, 1100, 940, 840, 736,
701, 529 cm-1; MS CI (m/ z) 192 (M+ + 1, 100), 107 (35), 100
(35), 91 (84), 85 (96), 68 (35), 43 (96), 28 (80), 18 (50). Anal.
Calcd for C12H17NO (191.2754): C, 75.35; H, 8.96; N, 7.32.
Found: C, 75.48; H, 9.14; N, 7.18.
(-)-2 (3.2 g, 60%), as a colorless oil: [R]19 ) -36.8 (c ) 1.4,
D
CHCl3, ee g 97%18) (lit.13 [R]23 for (S)-(+)-2 ) +32.5 (c ) 0.9,
D
CHCl3, ee ) 95%)); 1H NMR δ 2.45 (dd, J ) 17.6, 6.3, 1H), 2.69
(dd, J ) 17.6, 8.9, 1H), 2.85-3.00 (m, 1H), 3.52-3.60 (m, 2H),
4.26 (dd, J ) 9.2, 5.5, 1H), 4.48 (dd, J ) 9.2, 7.5, 1H), 4.60 (s,
2H), 7.33-7.48 (m, 5H); 13C NMR δ 31.06, 35.28, 70.41, 70.73,
73.19, 127.60, 127.80, 128.44, 137.70, 176.92; IR (ν) 2862, 1774,
1365, 1172, 1101, 1023, 741, 700 cm-1; MS EI (m/ z) 206 (4),
177 (5), 160 (3), 120 (27), 105 (11), 91 (100), 65 (21), 39 (13), 27
(2). Anal. Calcd for C12H14O3 (206.2436): C, 69.88; H, 6.84.
Found: C, 69.63; H, 6.97.
(S)-(-)-3-(Hyd r oxym eth yl)p yr r olid in e (8).7 A suspension
of (S)-(+)-6 (1.9 g, 6.7 mmol) and 10% Pd/C (200 mg), in EtOH
was stirred 24 h under H2 (1 atm). The palladium carbon
catalyst was filtered off, the solution concentrated in vacuo, and
the residue distilled (100 °C/0.1 mbar) to give (S)-(-)-8 (0.64 g,
95%) as a colorless oil: [R]25 ) -26 (c ) 6.0, EtOH) (lit.7 [R]20
D
D
) -19.1 (c ) 4.6, EtOH)); 1H-NMR δ 1.32-1.46 (m, 1H), 1.74-
1.88 (m, 1H), 2.13-2.32 (m, 1H), 2.62-2.71 (m, 1H), 2.74-2.98
(m, 3H), 4;35 (s/br, 2H), 3.34-3.54 (m, 2H); 13C-NMR δ 29.40,
(S)-(-)-2-[(Ben zyloxy)m eth yl]bu ta n e-1,4-d iol (4). To a
suspension of lithium aluminum hydride (1.9 g, 50 mmol) in 10
mL of dry THF, at 0 °C, was added dropwise a solution of (R)-
(-)-2 (2.06 g, 10 mmol) in 20 mL of dry THF was added dropwise.
The mixture was stirred 30 min at rt, refluxed for 1 h, cooled at
0 °C, diluted with Et2O (50 mL), and quenched with water (5
mL). The solid was filtered off and washed again with water
(20 mL). The organic phase was dried on MgSO4 and concen-
trated to give, after flash chromatography, (S)-(-)-4 (2.0 g, 95%)
as an undistillable colorless oil: [R]25D ) -8.4 (c ) 2.1, CH2Cl2);
1H-NMR δ 1.63 (dd, J ) 11.9, 6.5, 2H), 1.94-2.08 (m, 1H), 2.99
(s/br, 2H), 3.50 (m, 1H), 3.53 (m, 1H), 3.63-3.74 (m, 4H), 4.52
(s, 2H), 7.29-7.39 (m, 5H); 13C-NMR δ 32.99, 39.85, 61.26, 65.31,
73.29, 73.98, 128.09, 128.31, 129.09, 138.56; IR (ν) 3342, 2867,
1454, 1364, 1266, 1206, 1062, 737, 699, 612 cm-1; MS EI (m/ z)
210 (0.5), 192 (2), 107 (25), 91 (100), 65 (23), 31 (22), 29 (10).
41.93, 47.15, 50.51, 65.11; IR (ν) 3394, 1522, 1435, 762 cm-1
;
MS EI (m/ z) 101 (12), 82 (10), 68 (13), 55 (9), 43 (100), 28 (30),
15 (3). Anal. Calcd for C5H11NO (101.1495): C, 59.36; H, 10.98;
N, 13.85. Found: C, 59.52; H, 10.63; N, 13.67.
(S)-(-)-N-[(Ben zyloxy)ca r bon yl]-3-(h yd r oxym eth yl)p yr -
r olid in e (9). Amino alcohol (S)-(-)-8 (0.55 g, 5.4 mmol) and
K2CO3 (3.7 g, 27 mmol) were dissolved in THF/H2O (3/1, 100
mL) and treated with (benzyloxy)carbonyl chloride (1.9 g, 11
mmol) at 0 °C under stirring for 4 h. The reaction mixture was
concentrated, and the residue was dissolved in CH2Cl2 (200 mL),
washed with water (20 mL) and brine (10 mL), and concentrated
in vacuo. After flash chromatography, (S)-(-)-9 (1.14 g, 90%)
was obtained as an undistillable colorless oil: [R]25D ) -15 (c )
11.5, CHCl3); 1H-NMR δ 1.44-1.64 (m, 1H), 1.70-1.92 (m, 1H),
2.15-2.38 (m, 1H), 3.05-3.12 (m, 1H), 3.15-3.32 (m, 1H), 3.32-
3.50 (m, 4H), 3.79 (s/br, 1H), 4.99 (s, 2H), 7.08-7.35 (m, 5H);
13C-NMR δ 27.75, 28.48, 40.84, 41.67, 45.75, 46.13, 49.01, 49.34,
64.19, 67.16, 128.15, 128.34, 128.86, 137.25, 155.49; IR (ν) 3422,
2949, 2879, 1686, 1426, 1360, 1128, 910, 733 cm-1; MS EI (m/
z) 235 (48), 190 (18), 160 (3), 144 (16), 128 (41), 114 (12), 100
(22), 91 (100), 65 (38), 55 (15), 41 (17), 28 (20). Anal. Calcd for
Anal. Calcd for
C12H18O3 (210.2755): C, 68.53; H, 8.64.
Found: C, 68.35; H, 8.86.
(R )-(-)-2-[(B e n zy lo x y )m e t h y l]-1,4-b is(m e t h a n su lfo -
n yloxy)bu ta n e (5). To a solution of (S)-(-)-4 (1.9 g, 9.0 mmol),
(dimethylamino)pyridine (120 mg, 1 mmol), and Et3N (2.7 g, 27
mmol) in dry CH2Cl2 (10 mL) was added mesyl chloride (2.6 g,
22.5 mmol) at 0 °C, and the mixture was stirred for 30 min and
quenched with ice (2 g). The crude mixture was washed with
water (10 mL) and 5% NaHCO3 aqueous solution (10 mL). The
organic layer was dried on MgSO4, filtered off, and concentrated
in vacuo, to give, after flash chromatography (R)-(-)-5 (3.13 g,
C
13H17NO3 (235.2853): C, 66.35; H, 7.29; N, 5.95. Found: C,
66.13; H, 7.51; N, 5.72.
(S)-(+)-N-[(Ben zyloxy)ca r b on yl]-3-ca r b oxyp yr r olid in e
(10). Alcohol (S)-(-)-9 (1.04 g, 4.4 mmol) dissolved in acetone
(50 mL) was treated dropwise with freshly prepared J ones
reagent under vigorous stirring at 0 °C until a lasting orange
color was obtained. 2-Propanol (0.5 mL) was added and the
mixture filtered through Celite and washed with acetone/acetic
95%) as an undistillable colorless oil: [R]25 ) -4.9 (c ) 3.1,
D
1
CH2Cl2); H-NMR δ 1.90 (dd, J ) 12.9, 6.4, 2H), 2.22-2.32 (m,
1H), 2.99 (s, 6H), 3.50 (d, J ) 1.9, 1H), 3.52 (d, J ) 1.2, 1H),
4.27-4.34 (m, 4H), 4.51 (s, 2H), 7.29-7.42 (m, 5H); 13C-NMR δ
28.37, 36.19, 37.70, 37.94, 68.14, 69.33, 70.26, 73.93, 128.31,