R. M. Rodr´ıguez Sarmiento et al. / Tetrahedron: Asymmetry 14 (2003) 1547–1551
1549
glycine was added portionwise during 1 h to a solution
of 21.7 g (126 mmol) (E)-butenedioic acid diethyl ester
in 1000 ml toluene at 105°C. The reaction mixture was
refluxed overnight and the water collected in a Deam-
Stark. After evaporation of the organic solvent, the
remaining crude product was dissolved in 500 ml hex-
ane, filtrated and the organic solvent evaporated. The
excess of paraformaldehyde was removed by distillation
at 175°C. The remaining product 34 g (89%), a brown
potassium phosphate buffer pH 7.5. 200 mg of Lipase
OF was added and the pH kept constant by the con-
trolled addition (pH-stat) of 1.0N NaOH solution.
After termination of the reaction the mixture was aci-
dified to pH 2.0 with 25% hydrochloric acid and
extracted twice with 300 ml ethyl acetate. The com-
bined organic phases were dried over Na2SO4, evapo-
rated and dried on a HV to give 4.60 g crude product.
After chromatography on silica gel (hexane/ethyl ace-
tate 1/1) 3.45 g (75.7%) of the racemic monoacid 2 was
obtained. Analysis: purity: 99.6% GLC (methylated).
1
oil, was identified by NMR. Analysis: >95% pure by H
NMR. 1H NMR (400 MHz; CDCl3): 1.27 (t, 6H, -CH3),
2.80–2.77 (m, 2H, -NCH2-), 2.92–2.88 (m, 2H, -NCH2-),
3.44–3.42 (m, 2H, -CH-CH-), 3.60 (s, 2H, N-CH2-Ph),
4.16 (q, 4H, OCH2-), 7.30–7.29 (m, 5H, Ph). ISP MS:
306.3 (M+H+). IR (neat): 1727 (ester), 1171 (ester).
4.2.6. Diethyl (R,R)-N-Boc-pyrrolidine-3,4-dicarboxylate
1b. 13.00 g (40.98 mmol) chiral diester 1 (99.4%) was
emulsified under vigorous stirring in 650 ml 0.1 M
NaCl, 3 mM potassium phosphate buffer pH 7.0. 130
mg of Hydrolase ESP-ESL 1199 was added and the pH
kept constant by the controlled addition (pH-stat) of
1.0N NaOH solution. After termination of the reaction
(ꢀ50% conversion; 20 h) the reaction mixture was
extracted thrice with 500 ml CH2Cl2. The combined
organic phases were washed with 400 ml 2% NaHCO3
and 500 ml water (phase separation after filtration on 75
g dicalite prewashed with CH2Cl2 and water). The
organic phase was dried over Na2SO4, evaporated and
dried on a HV to give 6.00 g (46.4%) of the chiral diester
1b as a yellowish oil. Analysis: purity: 100% GLC
(methylated); >99% ee. Specific rotation [h]D
4.2.2. Diethyl trans-N-Boc-pyrrolidine-3,4-dicarboxylate
rac-1. 24.5 g (80 mmol) of 1-benzyl-pyrrolidine-3,4-
dicarboxylic acid diethyl ester and 19.2 g (88 mmol)
di-tert-butyldicarbonate were dissolved in 500 ml etha-
nol, and 2.55 g (2.4 mmol) Pd–C 10% was added. The
reaction was stirred under hydrogen for 2 h and then the
balloon with hydrogen was replaced because of CO2
formation. After 3.5 h the reaction was finished. The
Pd/C was removed by filtration and rinsed with ethanol.
The filtrate was concentrated and the residue purified by
column chromatography (silica gel, EtOAc/hexane 2/3)
to give 24.6 g (98%) of a colorless oil. Analysis: >95%
pure by 1H NMR. 1H NMR (400 MHz; CDCl3): 1.27 (t,
6H, -CH3), 1.46 (s, 9H, Boc), 3.30–3.44 (m, 2H, -NCH2-
), 3.46–3.58 (m, 2H, -NCH2-), 3.68–3.83 (m, 2H, -CH-
CH-), 4.18 (q, 4H, OCH2-). ISP MS: 338.2 (M+Na+),
1
+27.8 [c 0.92; CHCl3]. H NMR (400 MHz; CDCl3):
1.27 (t, 6H, -CH3), 1.46 (s, 9H, Boc), 3.30–3.44 (m, 2H,
-NCH2-), 3.46–3.58 (m, 2H, -NCH2-), 3.68–3.83 (m, 2H,
-CH-CH-), 4.18 (q, 4H, OCH2-). EI MS: 316.2 (M+H+),
300.2 (M−Me), 258.0 (M−tBu), 242.1 (M−COOEt). IR
(neat): 1732 (ester), 1695 (carbamate), 1161 (ester).
333.3 (M+NH4 ), 316.2 (M+H+). IR (neat): 1732 (ester),
+
1695 (carbamate), 1161 (ester).
4.2.3. pH-indicator assay. Into pre-loaded 96 well plates
containing 0.5 mg enzyme/well a buffered indicator
solution (190 ml, 7.5 mM Tris–HCl, pH 8.0, 0.02%
NaN3, 50 mg/l cresol red) and the substrate solution (0.5
mg 1 in 10 ml EtOH) were added with a liquid handler
(Lissy; Zinsser Analytics). For up to 2 days the color
change of the indicator from red to yellow was moni-
tored at 410 nm with a well plate reader (Tecan Sunrise)
and a well plate autosampler (Twister).
4.2.7. Ethyl (S,S)-N-Boc-pyrrolidine-3,4-dicarboxylate
2a. The aqueous phase of the above experiment was
washed twice with 500 ml EtOAc, acidified to pH 2.5
with 25% HCl and extracted thrice with 500 ml CH2Cl2.
The combined organic phases were dried over Na2SO4,
evaporated and dried on a HV to give 4.50 g (38.1%) of
the chiral monoacid 2a as a yellowish viscous oil.
Analysis: purity: 99.7% GLC (methylated; 0.3% diacid);
1
98% ee. Specific rotation [h]D −23.4 [c 1.09; CHCl3]. H
NMR (400 MHz; CDCl3): 1.28 (t, 3H, -CH3), 1.46 (s,
9H, Boc), 3.32–3.64 (2×m, 4H, NCH2-), 3.68–3.86 (m,
2H, -CH-CH-), 4.20 (m, 2H, OCH2), ꢀ9.6 (bs, ꢀ1H,
COOH). EI MS: 288.2 (M+H+), 230.1 (M−tBu), 214.1
(M−COOEt). IR (neat): 2400–3200 (acid), 1733 (ester),
1698 (carbamate), 1673 (acid).
4.2.4. Automated pH-stat (Metrohm) screening
procedure9. 15–20 mg (47–63 mmol) chiral diester 1
(99.4%) was added as an ethanol solution (0.3 ml) into
a freshly prepared reaction solution (19.5 ml 0.1 M
NaCl, 3 mM potassium phosphate buffer pH 7.0, 0.02%
NaN3) containing an enzyme aliquot (ꢀ3–10 mg
lyophilisate). Under vigorous stirring the pH was kept
constant by the controlled addition (pH-stat) of 0.05N
NaOH solution. After termination of the reaction
(ꢀ10–50% conversion; maximal 3–5 h) the reaction
mixture was acidified to pH 2.5 with 0.5N HCl and
extracted with 10–20 ml CH2Cl2. The organic phase was
subjected to GLC analysis for ee-determination of the
reaction products.
4.2.8. Ethyl (R,R)-N-Boc-pyrrolidine-3,4-dicarboxylate
2b. 5.70 g (18.07 mmol) chiral diester 1b was emulsified
under vigorous stirring in 430 ml 0.1 M NaCl, 3 mM
potassium phosphate buffer pH 7.5. 40 mg of Lipase
OF was added and the pH kept constant by the con-
trolled addition (pH-stat) of 1.0N NaOH solution.
After termination of the reaction (40 h) the reaction
mixture was extracted twice with 400 ml CH2Cl2 and
once with 400 ml EtOAc. The aqueous phase was
acidified to pH 2.5 with 25% hydrochloric acid and
extracted thrice with 400 ml CH2Cl2. The combined
organic phases were dried over Na2SO4, evaporated
4.2.5. Ethyl trans-N-Boc-pyrrolidine-3,4-dicarboxylate
rac-2. 5.0 g (15.85 mmol) chiral diester 1 was emulsified
under vigorous stirring in 300 ml 0.1 M NaCl, 3 mM