Enzyme-catalyzed enantiomeric resolution of N-Boc-proline as the key-step in an expeditious route towards RAMP

Article abstract of DOI:10.1016/S0957-4166(03)00210-6

For the preparation of both enantiomers of N-carbamoyl-2-methoxymethylpyrrolidine, the precursors of Enders' chiral auxiliaries, SAMP and RAMP, enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, N-Boc, N-Cbz proline esters and prolinols were examined. B. licheniformis protease (subtilisin) preferentially hydrolyzed the (R)-carbamoylproline ester with an enantiomeric ratio (E) of 10. To a hydrophobic N-Cbz proline ester, subtilisin showed lower selectivity (E=2.8), and in contrast, a purified protease (isozyme A) from the earthworm showed the preference of (S)-enantiomer (E=13.6). In a practical sense, C. antarctica lipase B (Chirazyme L-2) was effective for the hydrolysis of both N-Boc and N-Cbz derivatives with E >100. The e.e. of (R)-N-carbamoyl-2-methoxymethylpyrrolidine was raised to be >99.9% by recrystallization at the N-Boc-prolinol stage, which was derived from the (R)-N-Boc-proline methyl ester (98.7% e.e.) through a preparative-scale enzyme-catalyzed resolution (49% yield) of the racemic substrate.

Full text of DOI:10.1016/S0957-4166(03)00210-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 1323–1333
Enzyme-catalyzed enantiomeric resolution of N-Boc-proline as
the key-step in an expeditious route towards RAMP
Masayuki Kurokawa,a Takeyuki Shindo,a Masumi Suzuki,a Nobuyoshi Nakajima,b Kohji Ishiharac
and Takeshi Sugaia,*
aDepartment of Chemistry, Keio University, Yokohama 223-8522, Japan
bDepartment of Nutritional Science, Okayama Prefectural University, Okayama 719-1197, Japan
cDepartment of Chemistry, Kyoto University of Education, Kyoto 612-8522, Japan
Received 23 January 2003; accepted 27 February 2003
Abstract—For the preparation of both enantiomers of N-carbamoyl-2-methoxymethylpyrrolidine, the precursors of Enders’ chiral
auxiliaries, SAMP and RAMP, enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, N-Boc, N-Cbz proline esters and
prolinols were examined. B. licheniformis protease (subtilisin) preferentially hydrolyzed the (R)-carbamoylproline ester with an
enantiomeric ratio (E) of 10. To a hydrophobic N-Cbz proline ester, subtilisin showed lower selectivity (E=2.8), and in contrast,
a purified protease (isozyme A) from the earthworm showed the preference of (S)-enantiomer (E=13.6). In a practical sense, C.
antarctica lipase B (Chirazyme L-2) was effective for the hydrolysis of both N-Boc and N-Cbz derivatives with E >100. The e.e.
of (R)-N-carbamoyl-2-methoxymethylpyrrolidine was raised to be >99.9% by recrystallization at the N-Boc-prolinol stage, which
was derived from the (R)-N-Boc-proline methyl ester (98.7% e.e.) through a preparative-scale enzyme-catalyzed resolution (49%
yield) of the racemic substrate. © 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
selective reactions as well as diastereofacially selective
reactions.2 SAMP and RAMP have so far been prepared
from the pure enantiomers of proline, by way of car-
bamoyl derivatives 1a as the key intermediates.3 Our
recent efforts on the study of the synthesis and reaction of
carbamoylamino compounds4 prompted an expeditious
way toward (R)- and (S)-1a, based on the enzyme-catal-
yzed resolution of the appropriate intermediates 1b or 2
from a racemic form of proline 3 (Scheme 1).
a-Substituted pyrrolidines have significant importance in
asymmetric synthesis in organic chemistry.1 Among
them, 1-amino-2-methoxymethylpyrrolidine in enan-
tiomerically enriched forms, SAMP and RAMP, devel-
oped by Enders, play important roles for the
derivatisation of prochiral carbonyl compounds to hydra-
zones, which allow a number of diastereotopic group-
Scheme 1.
* Corresponding author. Fax: +81-45-566-1697; e-mail: sugai@chem.keio.ac.jp
0957-4166/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00210-6

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M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
2. Attempted kinetic resolution of N-carbamoyl
derivatives
Our first approach was an attempted kinetic resolution
of derivatives with esters 2 and primary alcohols 1,
both of which bear an N-carbamoyl protecting group,
seldom used so far. Among commercially available
enzymes, protease from Bacillus licheniformis (subtil-
isin, Sigma)5 performed the hydrolysis of esters 2b–d
(Scheme 2) with preference for the (R)-enantiomers as
shown in Table 1 (entries 1–3). The enantioselectivity
(E value6) was 8–10, and did not significantly depend
upon the size or hydrophobicity of the ester moiety.
The closely related substrate, hydantoin 4 was not
hydrolyzed either by protease or hydantoinase.7
Scheme 3.
carbamoyl group into account (Fig. 1). It has been
suggested that the hydrogen atom on the acylamino
group of the substrate has a considerable importance
for the enantiomeric recognition through a hydrogen
bond formation between peptide residue of the enzyme
in the active site to fix the orientation of the side
chain.10 As the result, for example, Ghisalba reported
an excellent enantioselectivity (E >200)5g on the racemic
N-acetyl-tert-leucine chloroethyl ester, with the (S)-
enantiomer being preferentially hydrolyzed.
In our case, however, there is no hydrogen atom. If the
N-carbamoyl group acts as the bulky substituent,
instead of the side chain of the normal amino acid, the
inverted preference on the (R)-enantiomer is under-
standable. In this fitting model, it is possible that
hydrogen bonding between carbamoyl NH and the
peptide has another effect on the molecular recognition.
This hypothesis prompted us to introduce a hydropho-
bic Boc or Cbz group instead of the carbamoyl group.
Previously, an example demonstrating that the property
of the N-protecting group affected the selectivity has
been observed in pig liver esterase-catalyzed hydrolysis
of substrates related to b-amino acid structure.11
Scheme 2.
Next, the lipase-catalyzed kinetic resolution of hydroxy-
methyl derivatives (Scheme 3) was attempted, and the
results are shown in Table 2. In the case of Candida
antarctica lipase B-catalyzed reaction, the enantioselec-
tivity observed in the hydrolysis of the acetate was
higher (E=4.8, entry 2) than that in the acetylation of
alcohol in organic solvent (E=2.4, entry 1).8 The intro-
duction of benzoate affected the reaction rate, and the
hydrolysis became very slow. In contrast, Candida
rugosa lipase-catalyzed hydrolysis of the same substrate
1d proceeded; the enantioselectivity, however, was as
low as 3.0 compared with those of examples reported so
far.9
The enantioselectivity was nearly lost (E=1.3, entry 2,
Table 3) in the case of a bulky but hydrophobic group,
Boc derivative 5b (Scheme 4). Similarly, the Cbz group
also weakened the preference for the (R)-enantiomer
(E=2.8, entry 3) compared with the original carbamoyl
substrate (E=9.4, entry 1). At this juncture, we became
interested in another source of enzymes, from the earth-
worm,12 far from the microbial origin, and applied
some partially purified isozymes to the substrates
above. Although an enzyme (isozyme C) did not work
on the N-carbamoyl substrate 2b (entry 4), one of the
major isozymes13 (isozyme A) showed a preference for
3. Effect of the protecting group and source of enzymes
on selectivity
At this stage, we took the property of an N-protecting
Table 1. Enzymatic hydrolysis of N-carbamoylproline esters
Entry
Substrate
Enzyme
Product
% e.e.
Recovery
% e.e.
Ea
Yield (%)
Yield (%)
1
2
3
4
2b
2c
2d
4
Proteaseb
47.0
ND
ND
No reaction
64.0
ND
ND
24.0
41.3
40.5
86.6
81.1
87.0
9.4
8.8
9.8
c
c
Hydantoinased
a E(P) for entry 1, E(S) for entries 2 and 3. For the definition of E(P) and E(S), see Ref. 6.
b From B. licheniformis (subtilisin, Sigma, P5459).
c From B. licheniformis (subtilisin, Sigma, P5380).
d From Azuki bean (Sigma, H4028).

M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
1325
Table 2. Enzymatic hydrolysis and acetylation of N-carbamoylprolinol and esters
Entry
Substrate
Lipase
Conversiona (%)
Product (% e.e.)
Recovery (% e.e.)
Ea
1
2
3
4
1b
1c
1d
1d
C. antarctica
C. antarctica
C. antarctica
C. rugosa
86.4
20.3
No reaction
53.6
12.2
60.9
77.2
15.6
2.4
4.8
34.8
40.2
3.0
a Conversion and E value were calculated from the e.e.s of the product and the recovered substrate.
with further screening of substrates would be promising
as a new tool for biotransformation, as the diversity is
suggested through the entries shown in Table 3.
Scheme 4.
At the very outset of this study, many lipases were
dropped as candidates, by the screening of hydrolytic
enzymes on the N-carbamoyl substrate 2b (Scheme 5).
The diversity of stereoselectivity as well as the reactivity
observed in proteases led us to reexamine Candida
antarctica lipase B on the substrates with a hydropho-
bic protecting group. This was also suggested from the
successful results reported on structurally related com-
pounds.16 This lipase-catalyzed hydrolysis worked very
well with a preference for the (S)-enantiomer shown in
Table 4. In both N-Boc (5b, entry 2) and N-Cbz (6b,
entry 3) substrates, the E values exceeded 100, and the
enzyme activity was kept throughout the reaction to
achieve a nearly 50% conversion. Unnatural (R)-proline
derivatives became available in 98.7% e.e. in the prepar-
ative scale (see Section 6).
Figure 1.
the (S)-enantiomer with E=13.6 on substrate 6b (entry
5). Enhancement of the activity at a slightly raised pH
(8.5) was attempted, however, the background non-
enzymatic hydrolysis brought about an apparently low-
ered enantioselectivity (E=2.7, entry 6). To our
surprise, isozyme C (entry 7) showed the preference of
the (R)-enantiomer with a similar inclination with
Bacillus protease (entry 3). The structural similarities
and catalytic functions of enzyme proteins indicated
that isozyme A is classified as trypsin-type protease,
while isozyme C is attributed to elastase-type.14 This is
the first example of examination of substrate specificity
and enantioselectivity of the purified earthworm
proteases toward artificial compounds other than natu-
rally occurring peptide substrates. Although the specific
activity was low, overexpression of enzymes15 combined
4. Transformation to
N-carbamoyl-2-methoxymethylpyrrolidine
The final task was the transformation of enzymatically
Table 3. Protease-catalyzed hydrolysis of N-protected forms of proline
Entry
Substrate
Protease
Preferred enantiomer
Conversion (%) Product (% e.e.)
Recovery (% e.e.)
E
1
2
3
4
5
6
7
8
2b
5b
6b
2b
6b
6b
6b
6b
Subtilisin
Subtilisin
Subtilisin
R
S
R
53.2
23.3
23.4
64.0
11.2
42.9
86.6
3.4
13.1
9.4a
1.3
2.8
Isozyme Cb
Isozyme Ab
Isozyme Ac
Isozyme Cb
Isozyme Eb
No reaction
S
S
R
1.6
9.0
4.0
85.9
44.5
43.7
1.4
4.4
1.7
13.6
2.7
2.4
No reaction
a See Table 1, entry 1.
b Protease isozymes from Lumbricus rubellus.
c This reaction was under pH 8.5, while all other reactions were carried out at 7.0.

1326
M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
5. Conclusion
An expeditious route to both enantiomers of N-car-
bamoyl-2-methoxymethylpyrrolidine 1a was established
by means of the lipase-catalyzed enantioselective
hydrolysis of racemic N-Boc proline esters. The proper-
ties of the N-protecting group greatly affected the
enantioselectivity.
Scheme 5.
6. Experimental
6.1. General
resolved highly pure enantiomers to N-carbamoyl-2-
methoxymethylpyrrolidine 1a. The reduction of car-
boxylic esters in both 5b and 6b smoothly proceeded
with NaBH4–LiCl17 in 93 and 97% yield, respectively
(Scheme 6). Recrystallization of one of the resulting
alcohols 7a provided the pure (R)-enantiomer. In the
methylation step of the resulted hydroxymethyl group,
the advantage of N-Boc compound 7a was emphasized.
In the case of N-carbamoyl derivative 1b, which has the
closest relationship to 1a, however, N-methylated by-
product 1e also formed (15%) together with 1a (68%)
even under optimised conditions in terms of the equiva-
lent of the methylating reagent (NaH–MeI) as well as
the reaction temperature (−78°C to rt). In turn, an
oxazolidinone 9 was a by-product (33%) in the case of
N-Cbz precursor 8b. Finally, no problem of significant
by-product formation (9: <3%) was found for 7a giving
7b in 94% yield. The product was converted to 1a via
one-pot conversion18 involving deprotection of the Boc
group under acidic conditions and subsequent N-car-
bamoylation in 98% yield.
All melting points were uncorrected. IR spectra were
measured as thin films for oils or KBr disks of solids on
1
a JASCO FT/IR-410 spectrometer. H NMR spectra
were measured at 270 MHz on a JEOL JNM EX-270
spectrometer and at 400 MHz on a JEOL JNM GX-
400 spectrometer. 13C NMR spectra were measured at
100 MHz on a JEOL JNM GX-400 spectrometer. Mass
spectra were recorded on Hitachi M-80B spectrometer
at 70 eV. HPLC analyses were performed with a SSC-
5410 (Senshu Scientific Co., Ltd.) liquid chromato-
graphs and Chiralcel OJ (0.46×25 cm, Daicel Chemical
Ind.). Optical rotations were recorded on a JASCO DIP
360 polarimeter. Silica gel 60 (spherical, 100–210 mm,
37558-79) of Kanto Chemical Co. was used for column
chromatography.
6.2. ( )-N-Carbamoylproline methyl ester ( )-2b
A stirred solution of ( )-N-carbamoylproline19 2a, (1.50
g, 9.48 mmol) in MeOH (150 mL) was treated with
Table 4. C. antarctica lipase-catalyzed hydrolysis of N-protected forms of proline
Entry
Substrate
Preferred enantiomer
Conversion (%)
Product (% e.e.)
Recovery (% e.e.)
E
1
2
3
2b
5b
6b
No reaction
S
S
49.7
49.5
\99.9
99.8
98.7
97.8
\100
\100
Scheme 6.

M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
1327
Amberlyst-15 (1.50 g) and refluxed for 2 h. After cooling
to room temperature, the reaction mixture was filtered.
The filtrate was concentrated in vacuo and the residue
was purified by column chromatography (silica gel, 38
g). Elution with CHCl3–EtOH (10:1) afforded ( )-2b
(98%) as a colorless fine powder. Mp 170.0–170.5°C; 1H
NMR (270 MHz, D2O) l 4.23 (dd, 1H, J=3.7, 6.6 Hz),
3.58 (s, 3H), 3.25 (m, 2H) 2.12 (m, 1H), 1.85 (m, 3H);
13C NMR (100 MHz, D2O; acetonitrile l 1.7 as internal
standard) l 176.5, 159.9, 59.8, 53.6, 47.3, 30.3, 24.9; IR
(KBr) 3400, 3179, 1739, 1651, 1610, 1600, 1470, 1456,
1213, 1200, 1179, 1104, 996, 778, 609 cm−1. Anal. calcd
for C7H12N2O3: C, 48.83; H, 7.03; N, 16.27. Found: C,
48.80; H, 6.99; N, 16.30.
s, 2H), 4.51 (m, 1H), 3.50 (m, 2H), 2.30–1.95 (m, 4H);
13C NMR (100 MHz, CDCl3) l 191.1, 172.2, 157.2,
132.6, 132.2, 129.2, 66.1, 58.9, 46.6, 30.3, 24.5; IR (KBr)
3398, 3185, 2949, 2882, 1746, 1700, 1649, 1609, 1596,
1472, 1455, 1399, 1366, 1236, 1179, 1103, 1071, 1010,
971, 809, 776, 612 cm−1; MS (70 eV) m/e (rel. int.,%):
355 (M+1, 2), 183 (30), 171 (4), 113 (35), 90 (4), 70 (base
peak), 41 (17); HR-MS (70 eV): M+ 354.0238 (calcd.
354.0215). Anal. calcd for C14H15BrN2O4: C, 47.34; H,
4.26; N, 7.89. Found: C, 47.14; H, 4.05; N, 7.57.
6.5. Subtilisin-catalyzed hydrolysis of N-carbamoylpro-
line esters. (R)-N-Carbamoylproline (R)-2a and (S)-N-
carbamoylproline methyl ester (S)-2b
6.3. ( )-N-Carbamoylproline benzyl ester ( )-2c
Subtilisin (Sigma, P5459, 300 mL, 199 units) was added
to a solution of the methyl ester 2b (1.00 g, 5.81 mmol)
in 0.1 M phosphate buffer (pH 6.5, 60 mL), and the
mixture was stirred at 24°C for 25 h, while keeping its
pH to 6.5 by a pH controller. The reaction was moni-
tored by TLC analysis (AcOEt–EtOH=1:1, Rf=0.50
for 2b; n-BuOH–AcOH–H2O=3:1:1, Rf=0.45 for 2a).
A small portion of the residue was dissolved in D2O and
KCNO (0.757 g, 9.33 mmol) was added to a solution of
( )-proline (1.02 g, 8.88 mmol) in water (5.0 mL), and
the mixture was stirred at 70°C for 1 h. After cooling to
room temperature, THF (11.0 mL), BnBr (1.0 mL, 8.44
mmol), KI (0.153 g, 0.92 mmol) and DMF (15.0 mL)
were added and the mixture was stirred at room temper-
ature for 161 h. The remaining BnBr was quenched with
aq. NH3 soln (total 54.0 mL, 0.88 mmol) for 1 h at room
temperature and the mixture was concentrated in vacuo.
The residue was dissolved in a mixture of water and
AcOEt, and then the organic layer was separated. The
aqueous layer was extracted with AcOEt, and the com-
bined organic layer was washed with 10% aq. Na2S2O3
soln and brine, dried over Na2SO4, and concentrated in
vacuo. The solid residue was further washed with Et2O
and the precipitate was filtered and dried in vacuo to
give ( )-2c (two steps, 71%) as a white solid. Mp
1
its H NMR spectrum of the mixture was measured.
The ratio of the expecting product (R)-2a to the starting
material (S)-2b was estimated by comparing the H-2
signal of the substrate (l 4.23) with that of the product
(l 3.94). The reaction mixture was filtered through
Amicon YM 10 ultrafiltration membrane (62 mm f).
Purification was performed by filtration through an
−
anion-exchange resin (HCO3 form). The column was
eluted with H2O and a gradient from 0 to 1.0 M aq.
NH4HCO3 soln. Fractions containing (R)-2a were com-
bined and concentrated in vacuo to give a white solid.
The acid (R)-2a was further derived to the benzyl ester
(R)-2c (two steps, 47%) for the evaluation of enan-
tiomeric excess. Unreacted (S)-2b was recorved in 24%
yield.
1
147.5–148.3°C; H NMR (400 MHz, CDCl3) l 7.34 (m,
5H), 5.18 (s, 2H), 4.62 (br s, 2H), 4.40 (m, 1H), 3.46 (m,
2H), 2.28–1.90 (m, 4H); 13C NMR (100 MHz, CDCl3)
l 172.6, 157.1, 135.6, 128.5, 128.1, 127.9, 66.8, 59.1,
46.4, 29.9, 24.5; IR (KBr) 3404, 3176, 2963, 2879, 1739,
1650, 1609, 1598, 1494, 1473, 1455, 1378, 1212, 1170,
1102, 970, 754, 699, 597 cm−1. Anal. calcd for
C13H16N2O3: C, 62.89; H, 6.50; N, 11.28. Found: C,
62.80; H, 6.43; N, 11.33.
(R)-2c: Solidified upon standing: mp 75.0–76.0°C; [h]D18
+41.7 (c 1.02, CHCl3). This was estimated to be 64.0%
e.e. based on specific rotation which was compared with
that of an authentic (R)-enantiomer, prepared from the
commercial (R)-proline (Aldrich Co. 85,891-9) in the
same manner as before [mp 78.5–79.3°C, [h]1D9 +65.7 (c
1.00, CHCl3)]. Its NMR spectrum was identical with
that of this authentic specimen.
6.4. ( )-N-Carbamoylproline p-bromophenacyl ester ( )-
2d
( )-N-Carbamoylproline19 2a (0.159 g, 0.950 mmol) was
dissolved in 5% aq. NaOH soln (0.8 mL). After the
solution was stirred at room temperature for 30 min,
EtOH (1.5 mL) and p-bromophenacyl bromide (0.279 g,
1.01 mmol) were added to the mixture and stirred at
reflux for 5 h. The mixture was concentrated in vacuo.
The residue was dissolved in a mixture of water and
AcOEt, and then the organic layer was separated. The
aqueous layer was extracted with AcOEt, and the com-
bined organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 12 g).
Elution with AcOEt–EtOH (1:0 to 8:1) afforded ( )-2d
(S)-2b: Solidified upon standing: mp 136.0°C; [h]D18
−53.5 (c 1.00, MeOH). This was estimated to be 86.6%
e.e. based on specific rotation which was compared with
that of an authentic (S)-enantiomer, prepared from the
commercial (S)-proline (Aldrich Co. 13,154-7) in the
same manner as before [mp 135.0–136.0°C, [h]2D0 −61.8
(c 1.00, MeOH)]. Its NMR spectrum was identical with
that of this authentic specimen.
6.6. (S)-N-Carbamoylproline benzyl ester (S)-2c
Subtilisin (Sigma, P5380, 25.0 mg, 240 units) was added
to a suspension of the benzyl ester (2c, 400 mg, 1.61
mmol) in 0.1 M phosphate buffer (pH 7.0, 5.6 mL) and
acetone (2.4 mL), and the mixture was stirred at 25°C
1
(78%) as a white solid. Mp 163.3–164.0°C; H NMR
(270 MHz, CDCl3) l 7.76 (m, 2H), 7.64 (m, 2H), 5.47
(d, 1H, J=16.3 Hz), 5.26 (d, 1H, J=16.3 Hz), 4.72 (br

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M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
for 6 h, while keeping its pH to 6.5 by pH controller.
The reaction mixture was extracted with AcOEt, and
the combined organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The
residue was purified by column chromatography (silica
gel, 15 g). Elution with CHCl3–MeOH (20:1) afforded
(S)-2c (41%) as a white solid. In this case, another
product (R)-2a stayed in the aqueous phase and was
not recovered.
6.9. ( )-N-Carbamoylprolinol ( )-1b
KCNO (1.02 g, 12.5 mmol) and AcOH (1.16 mL) were
added to a solution of ( )-prolinol (0.510 g, 5.00 mmol)
in DMF (3.0 mL), and the mixture was stirred
overnight at room temperature. The reaction mixture
was concentrated in vacuo, and the residual solid was
thoroughly extracted with CHCl3. The extract was con-
centrated in vacuo. On the other hand, the residual
solid at the stage of CHCl3 extraction was dissolved in
water, and the mixture was desalted by AC-110-10 on
Asahi Chemical Micro Acylyzer S1. At the initial stage,
the conductivity was 62 mS (4.3 V) and after the
desaltation at the 3.4 V, it reached 619 mS (26 h). The
recovered solution was frozen and lyophilized. This
lyophilizate was combined with the CHCl3 extract, and
further purified by column chromatography (silica gel,
20 g). Elution with AcOEt–EtOH (3:1) afforded ( )-1b
(S)-2c: [h]2D2 −53.3 (c 1.00, CHCl3). This was estimated
to be 81.1% e.e. based on specific rotation which was
compared with that of an authentic (R)-2c as before. Its
NMR spectrum was identical with this authentic
specimen.
6.7. (S)-N-Carbamoylproline p-bromophenacyl ester
(S)-2d
1
(84%) as a white solid. Mp 115.2–116.0°C; H NMR
(270 MHz, CDCl3) l 4.64 (br s, 3H), 4.01 (m, 1H), 3.58
(m, 2H), 3.40 (m, 2H), 2.07–1.86 (m, 3H), 1.60 (m, 1H);
IR (KBr) 3358, 3253, 3195, 2975, 2877, 1646, 1593,
1484, 1456, 1353, 1096, 1040, 903, 770, 612, 584 cm−1.
Its NMR spectrum was identical with that reported
previously.20
Subtilisin (Sigma, P5380, 74.7 mg, 710 units) was added
to a suspension of the p-bromophenacyl ester (2d, 399
mg, 1.12 mmol) in 0.1 M phosphate buffer (pH 7.0,
10.6 mL) and DMSO (4.4 mL), and the mixture was
stirred at 30°C for 44 h, while keeping its pH to 6.5 by
pH controller. The reaction mixture was extracted with
AcOEt, and the combined organic layer was washed
with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy (silica gel, 22 g). Elution with AcOEt–EtOH (1:0
to 8:1) afforded (S)-2d (41%) as a white solid. In this
case also, the product (R)-2a remained in the aqueous
phase.
6.10. ( )-N-Carbamoylprolinol O-acetate ( )-1c
Ac2O (0.340 g, 3.33 mmol) was added to the solution of
the alcohol ( )-1b (0.309 g, 2.14 mmol) in pyridine (3.5
mL), and the mixture was stirred overnight at room
temperature. Water was added to the reaction mixture
and was stirred for 30 min to decompose excess Ac2O.
After the mixture was concentrated in vacuo, then
toluene and EtOH were added and further evaporated
until pyridine and AcOH were removed. The residue
was purified by column chromatography (silica gel, 20
g). Elution with AcOEt–EtOH (8:1) afforded ( )-1c
(S)-2d: [h]2D1 −57.9 (c 0.96, EtOH). This was estimated
to be 85.8% e.e. based on the specific rotation which
was compared with that of an authentic (S)-2d, pre-
pared from (S)-proline in the same manner as described
for racemic form [mp 71.6–72.5°C, [h]2D0 −67.5 (c 0.98,
EtOH)]. Its NMR spectrum was identical with that of
this authentic specimen.
1
(84%) as a white solid. Mp 139.5–140.2°C; H NMR
(270 MHz, CDCl3) l 4.62 (s, 2H), 4.18 (m, 1H), 3.95
(m, 1H), 3.81 (m, 1H), 3.30 (m, 2H), 2.01 (s, 3H), 1.89
(m, 4H); 13C NMR (100 MHz, CDCl3) l 171.0, 157.7,
65.4, 55.6, 46.4, 28.7, 23.4, 21.2; IR (KBr) 3395, 3194,
2957, 2880, 1740, 1650, 1604, 1454, 1384, 1366, 1252,
1240, 1097, 1039, 778, 580 cm−1. Anal. calcd for
C8H14N2O3: C, 51.61; H, 7.58; N, 15.04. Found: C,
51.21; H, 7.59; N, 15.07.
6.8. Attempt for
D-hydantoinase-catalyzed hydrolysis of
(R)-proline-hydantoin (R)-4
D
-Hydantoinase (Sigma, H4028, 30.0 mg, 9.07 units)
was added to a solution of (R)-proline-hydantoin19
4
(224 mg, 1.60 mmol) in 0.16 M borate buffer (pH 9.0,
10 mL), and the mixture was stirred at 40°C for 6 days.
The acid 2a was estimated to be 1.28 mmol, which
indicated that the progress of the reaction was 80%
6.11. ( )-N-Carbamoylprolinol O-benzoate ( )-1d
Bz2O (1.71 g, 7.54 mmol) and DMAP (0.049 g, 0.40
mmol) were added to a solution of the alcohol ( )-1b
(0.543 g, 3.77 mmol) in pyridine (6.0 mL), and the
mixture was stirred overnight at room temperature.
3-(Dimethylamino)propylamine (0.5 mL, 4.01 mmol)
was added to destroy the excess Bz2O, and the mixture
was poured into ice-cooled 2 M HCl. The mixture was
extracted with AcOEt, and the combined organic layer
was washed with aq. NaHCO3 soln and brine, dried
over Na2SO4, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 20
g). Elution with AcOEt–EtOH (8:1) afforded ( )-1d
yield. In the control experiment without
D-hydan-
toinase at pH 9.0, however, the spontaneous consump-
tion (72%) of substrate was observed.
Details of the analysis for reaction progress were as
follows: a 400 mL aliquot of the reaction mixture was
taken and the reaction was terminated by the addition
of 700 mL of 12% (w/v) trichloroacetic acid, followed by
100 mL of 10% (w/v) p-dimethylaminobenzaldehyde in
6 M HCl. After the cells and debris were removed by
centrifugation, the absorbance at 450 nm was
measured.
1
(90%) as a white solid. Mp 162.7–163.5°C; H NMR

M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
1329
(270 MHz, CDCl3) l 7.96 (m, 2H), 7.51 (m, 1H), 7.38
(m, 2H), 4.72 (s, 2H), 4.44 (m, 1H), 4.09 (m, 2H), 3.37
(m, 2H), 1.92 (m, 4H); 13C NMR (100 MHz, CDCl3) l
166.6, 157.6, 133.1, 129.6, 129.5, 128.3, 65.6, 55.7, 46.5,
28.8, 23.5; IR (KBr) 3396, 3195, 2970, 2886, 1718, 1650,
1605, 1449, 1385, 1316, 1279, 1177, 1117, 1025, 776,
709, 586 cm−1. Anal. calcd for C13H16N2O3: C, 62.89;
H, 6.50; N, 11.28. Found: C, 62.66; H, 6.44; N, 11.15.
6.14. (R)-N-Carbamoylprolinol [(R)-1b] and (S)-N-carb-
amoylprolinol O-benzoate (S)-1d: lipase-catalyzed
hydrolysis
Candida rugosa lipase (Meito OF, 504 mg, 181,400
units) was added to a suspension of the benzoate 1d
(505 mg, 2.03 mmol) in 0.02 M phosphate buffer (pH
7.0, 100 mL), and the mixture was stirred at 35°C for
24 h, while keeping its pH to 7.0 by pH controller. The
mixture was diluted with AcOEt and filtered through
Celite pad. The filtrate was extracted with Et2O and
AcOEt, and the combined organic layer was washed
with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy (silica gel, 10 g). Elution with AcOEt–EtOH (8:1)
afforded (S)-1d (32%) as a white solid. After the extrac-
tion of (S)-1d, the aqueous layer was desalted and
lyophilized. The residue was purified by column chro-
matography (silica gel, 20g). Elution with AcOEt–
EtOH (3:1) afforded (R)-1b (52%) as a white solid.
6.12. Lipase-catalyzed resolution of N-carbamoylprolin-
ol and esters. (S)-N-Carbamoylprolinol O-acetate (S)-
1c and (R)-N-carbamoylprolinol (R)-1b:
lipase-catalyzed transesterification
Candida antarctica lipase (Chirazyme L-2, 25.0 mg) was
added to a suspension of the alcohol 1b (50.0 mg, 0.347
mmol), vinyl acetate (1.0 mL), tert-butylamine (0.10
mL), and small portion of BHT in THF (0.75 mL), and
the mixture was stirred at 20°C for 14 h. The reaction
mixture was filtered and the filtrate was evaporated.
The residue was purified by column chromatography
(silica gel, 10 g). Elution with AcOEt–EtOH (8:1 to 3:1)
afforded (S)-1c (72%) as a white solid and (R)-1b (12%)
as a white solid. The e.e.s of both (S)-1c and (R)-1b
were estimated to be 12.2 and 77.2%, respectively, by
the HPLC analysis of 1d. The conditions were as
follows: eluent, hexane/i-PrOH=6/1; flow rate, 0.5 mL/
min; retention time, 36.1 and 44.8 min. The absolute
configurations of present 1b and 1c were confirmed by
comparing its retention time of the resulted 1d that of
the authentic (R)-1d (44.6 min), prepared from the
commercial (R)-prolinol (Tokyo Kasei Kogyo Co., Ltd.
P1274) in the same manner as before.
(R)-1b: [h]2D0 +18.7 (c 0.54, EtOH), 34.8% e.e. by HPLC
analysis as above. Its NMR spectrum was identical
with that reported previously.20
(S)-1d: [h]2D4 −24.8 (c 1.01, EtOH) [an authentic speci-
men: mp 94.3–95.0°C, [h]2D5 −56.0 (c 1.00, EtOH) for
(S)-1d, which was prepared from (S)-prolinol in the
same manner as described for racemic form], 40.2% e.e.
by HPLC analysis as above. Its NMR spectrum was
identical with that of this authentic specimen.
6.15. ( )-N-tert-Butoxycarbonylproline methyl ester
( )-5b
6.13. (S)-N-Carbamoylprolinol (S)-1b and (R)-N-carb-
amoylprolinol O-acetate (R)-1c: lipase-catalyzed
hydrolysis
According to the reported procedure,21c free NH group
of ( )-proline (1.40 g, 12.2 mmol) was protected with
Boc group. K2CO3 (5.03 g, 36.4 mmol) was added to a
solution of the crude 5a in DMF (35.0 mL), and the
mixture was stirred at room temperature for 30 min.
MeI (2.40 mL, 38.5 mmol) was then added and the
reaction mixture was stirred for 10 h at room tempera-
ture. The solvent was evaporated and the residue was
diluted with water and AcOEt, then the organic layer
was separated. The aqueous layer was extracted with
AcOEt, and the combined organic layer was washed
with water, 10% aq. Na2S2O3 soln and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 105 g).
Elution with hexane–AcOEt (7:1 to 5:1) afforded ( )-5b
Candida antarctica lipase (Chirazyme L-2, 201 mg) was
added to a solution of the acetate (1c, 502 mg, 2.69
mmol) in 0.02 M phosphate buffer (pH 7.0, 20 mL),
and the mixture was stirred at 24°C for 51 h, while
keeping its pH to 7.0 by pH controller. The reaction
mixture was filtered and the filtrate was lyophilized.
The residue was purified by column chromatography
(silica gel, 30 g). Elution with AcOEt–EtOH (8:1 to 3:1)
afforded (R)-1c (74%) as a white solid and (S)-1b (24%)
as a white solid.
(S)-1b: [h]1D9 −33.6 (c 0.50, EtOH), [an authentic speci-
men: mp 92.7–93.5°C, [h]2D2 +57.2 (c 0.54, EtOH) for
(R)-1b, which was prepared from (R)-prolinol in the
same manner as described for racemic form], 60.9% e.e.
by the HPLC analysis of 1d. Its NMR spectrum was
identical with that reported previously.20
1
(two steps, 91%) as a colorless oil. H NMR (400 MHz,
CDCl3) l 4.25 (m, 1H), 3.70 (s, 3H), 3.50 (m, 2H), 2.20
(m, 1H), 2.00–1.80 (m, 3H), 1.40 (s, 9H); IR (neat)
2977, 2881, 1748, 1698, 1395, 1162, 1121, 1089, 1034,
999, 974, 888, 858, 772 cm−1. Its NMR and IR spectra
were identical with those reported previously.21b
(R)-1c: [h]2D0 +8.2 (c 1.00, EtOH) [an authentic speci-
men: mp 58.5–59.3°C, [h]1D8 −50.0 (c 0.97, EtOH) for
(S)-1c, which was prepared from the commercial (S)-
prolinol (Tokyo Kasei Kogyo Co., Ltd. P1087) in the
same manner as described for racemic form], 15.6% e.e.
by the HPLC analysis of 1d. Its NMR spectrum was
identical with that of this authentic specimen.
6.16. ( )-N-Benzyloxycarbonylproline methyl ester ( )-
6b
In a similar manner as above, racemic N-Cbz-proline22
( )-6a (1.17 g, 4.69 mmol) was treated with K2CO3
(1.96 g, 14.2 mmol) and MeI (1.46 mL, 23.5 mmol) in

1330
M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
DMF (22.0 mL) to give crude ( )-6b. This was purified
by column chromatography (silica gel, 30 g). Elution
with hexane–AcOEt (4:1) afforded ( )-6b (98%) as a
a solution of (R)-2b (2.0 mg, 0.012 mmol) in 0.05 M
Tris–HCl buffer (pH 7.0, 1.0 mL), and the mixture was
stirred at 37°C for 72 h. To (S)-enantiomer, same
reaction was performed. The reaction was monitored
by TLC analysis, however, any trace of 2a was not
observed in either case.
1
colorless oil. H NMR (270 MHz, CDCl3) l 7.34 (m,
5H), 5.15 (m, 2H), 4.35 (m, 1H), 3.86–3.40 (m, 5H),
2.30–1.80 (m, 4H); IR (neat) 3033, 2953, 2882, 1747,
1713, 1416, 1353, 1281, 1201, 1179, 1119, 1089, 1002,
919, 769, 699, 612, 554 cm−1. Its NMR and IR spectra
were identical with those reported previously.22
6.20. Isozyme A-catalyzed hydrolysis of ( )-N-benzyl-
oxycarbonylproline methyl ester ( )-6b
6.17. Subtilisin-catalyzed hydrolysis of N-substituted
proline esters. (S)-N-tert-Butoxycarbonylproline (S)-5a
and (R)-N-tert-butoxycarbonylproline methyl ester (R)-
5b
Earthworm protease (isozyme A, 30.0 mg) was added
to an emulsion of the methyl ester 6b (28.0 mg, 0.106
mmol) in 0.05 M Tris–HCl buffer (pH 7.0, 0.5 mL),
and the mixture was stirred at 37°C for 72 h. The
reaction mixture was acidified by 2 M HCl to pH 2.0
and was extracted with AcOEt. The combined organic
layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by
column chromatography (silica gel, 11 g). Elution with
hexane–AcOEt (3:1) afforded (R)-6b (93%) as a color-
less oil and further elution with pure AcOEt gave
(S)-6a (5%) as a colorless oil. The e.e.s of both (S)-6a
and (R)-6b were estimated to be 1.4 and 85.9%, respec-
tively, by the HPLC analysis of 6b as described before.
The same reaction was performed at pH 8.5 at 37°C for
120 h. The ester (R)-6b (89%, 4.4% e.e.) and the acid
(R)-6a (6%, 44.5% e.e.) were obtained.
Subtilisin (Sigma, P5380, 15.1 mg, 140 units) was added
to an emulsion of the methyl ester 5b (150 mg, 0.653
mmol) in 0.1 M phosphate buffer (pH 7.0, 3.0 mL), and
the mixture was stirred at 24°C for 23 h. The reaction
mixture was acidified by 2 M HCl to pH 2.5 and was
extracted with AcOEt. The combined organic layer was
washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by column
chromatography (silica gel, 10 g). Elution with hexane–
AcOEt (5:1 to 4:1) afforded (R)-5b (70%) as a colorless
oil, and further elution with hexane–AcOEt (1:1) gave
(S)-5a (25%) as a white solid. The acid (S)-5a was
further derived to (S)-5b by the treatment with
TMSCHN2 for analysis.
6.21. Isozyme C-catalyzed hydrolysis of ( )-N-benzyl-
oxycarbonylproline methyl ester ( )-6b
(S)-5b: [h]2D3 −7.3 (c 1.40, MeOH). This was estimated
to be 11.2% e.e. based on specific rotation which was
compared with the reported value [lit.21d [h]D23 −65.0 (c
2.00, MeOH)].
Isozyme C (10.0 mg) was added to an emulsion of the
methyl ester 6b (20.0 mg, 0.076 mmol) in 0.05 M
Tris–HCl buffer (pH 7.0, 1.0 mL), and the mixture was
stirred at 37°C for 72 h. The ester (S)-6b (83%, 1.7%
e.e.) and (R)-6a (7%, 43.7% e.e.) were obtained.
(R)-5b: [h]2D4 +2.2 (c 3.20, MeOH), 3.4% e.e. in a similar
way as above.
6.18. (R)-N-Benzyloxycarbonylproline (R)-6a and (S)-
N-benzyloxycarbonylproline methyl ester (S)-6b
6.22. Attempt for isozyme E-catalyzed hydrolsis of ( )-
N-benzyloxycarbonylproline methyl ester ( )-6b
Subtilisin (Sigma, P5380, 11.5 mg, 106 units) was added
to an emulsion of the methyl ester 6b (107 mg, 0.407
mmol) in 0.1 M phosphate buffer (pH 7.0, 2.1 mL), and
the mixture was stirred at 30°C for 48 h. The reaction
was monitored by TLC analysis (hexane–AcOEt=1:1,
Rf=0.75 for 6b; Rf=0.10 for 6a). The ester (S)-6b
(63%) and the acid (R)-6a (22%) were obtained. The
acid (R)-6a was further derived to (R)-6b by the treat-
ment with TMSCHN2 for analysis.
In a similar manner as above, isozyme E (28 mg) was
incubated with 6b (28 mg, 0.106 mmol) for 72 h,
however, any trace of 6a was not observed by TLC
analysis.
6.23. Lipase-catalyzed hydrolysis of N-protected forms
of proline. Attempt for lipase-catalyzed hydrolysis of
N-carbamoylproline methyl ester 2b
Candida antarctica lipase (Chirazyme L-2) was incu-
bated with both enantiomers of 2b by applying enzyme
(7.7 mg) to substrate (21.7 mg, 0.13 mmol) in 0.1 M
phosphate buffer (pH 7.0, 1.0 mL), and the mixture was
stirred at 30°C for 72 h. The reaction was monitored by
TLC analysis, but any trace of 2a was not observed.
(R)-6b: This was estimated to be 42.9% e.e. based on
HPLC analysis. The conditions were as follows: eluent,
hexane/i-PrOH=9/1; flow rate, 0.5 mL/min; retention
time, 41.7 (R) and 50.0 (S) min.
(S)-6b: This was estimated to be 12.6% e.e. by HPLC
analysis as above.
6.24. (S)-N-tert-Butoxycarbonylproline (S)-5a and (R)-
N-tert-butoxycarbonylproline methyl ester (R)-5b
6.19. Earthworm protease isozyme-catalyzed hydrolysis
of N-substituted proline esters. Attempt for hydrolysis
of N-carbamoylproline methyl ester 2b
Candida antarctica lipase (Chirazyme L-2, 0.882 g) was
added to an emulsion of the methyl ester 5b (2.19 g,
9.55 mmol) in 0.02 M phosphate buffer (pH 7.0, 110
Earthworm protease (isozyme C, 10.0 mg) was added to

M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
1331
mL), and the mixture was stirred at 30°C for 25 h,
while keeping its pH to 6.8 by pH controller. The
reaction mixture was acidified by 2 M HCl to pH 2.5.
The mixture was diluted with AcOEt and filtered
through Celite pad. The filtrate was extracted with
AcOEt and the combined organic layer was washed
with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy (silica gel, 105 g). Elution with hexane–AcOEt
(5:1 to 4:1) afforded (R)-5b (49%) as a colorless oil, and
further elution with hexane–AcOEt (1:1) gave (S)-5a
(50%) as a white solid.
(S)-6b: [h]2D0 −55.2 (c 1.23, MeOH), 99.8% e.e. by
HPLC analysis as above. Anal. calcd for C14H17NO4:
C, 63.87; H, 6.51; N, 5.32. Found: C, 63.74; H, 6.69; N,
5.00.
6.26. (R)-N-tert-Butoxycarbonylprolinol (R)-7a
According to the reported procedure,17b the methyl
ester (R)-5b (0.941 g, 4.10 mmol) was treated with
NaBH4 (0.381 g, 10.3 mmol) and LiCl (0.386 g, 9.11
mmol) to give (R)-7a (quant.). The product was recrys-
tallized from hexane–Et2O to afford colorless prisms
(93%, >99.9% e.e. by the HPLC analysis of 7c). Mp
60.5–60.8°C (lit.17b 58.5–59.4°C); [h]2D0 +54.4 (c 1.02,
MeOH) [lit.17b [h]2D5 −53.9 (c 1.04, MeOH) for (S)-7a];
1H NMR (400 MHz, CDCl3) l 3.99 (m, 1H), 3.70 (br s,
1H), 3.58 (m, 2H), 3.40 (m, 1H), 3.25 (m, 1H), 2.05–
1.70 (m, 4H), 1.42 (s, 9H). Its NMR spectrum was
identical with that reported previously.17b Anal. calcd
for C10H19NO3: C, 59.68; H, 9.52; N, 6.96. Found: C,
59.87; H, 9.54; N, 7.07.
(S)-5a: Mp 135.0–135.7°C (lit.21c 133.5–135.0°C); [h]2D0
−60.3 (c 2.02, AcOH) [lit.21a [h]2D5 −60.2 (c 2.01, AcOH)
1
for (S)-5a]; H NMR (400 MHz, CDCl3) l 8.94 (br s,
1H), 4.30 (m, 1H), 3.50 (m, 2H), 2.35–1.90 (m, 4H),
1.43 (s, 9H); IR (KBr) 3463, 2977, 2897, 1739, 1639,
1480, 1432, 1367, 1218, 1188, 1164, 1131, 902, 775
cm−1. Its NMR and IR spectra were identical with
those reported previously.21c Anal. calcd for
C10H17NO4: C, 55.80; H, 7.96; N, 6.51. Found: C,
55.79; H, 7.97; N, 6.32. A small portion of 5a was
treated with TMSCHN2 to afford 5b. Then the methyl
ester 5b was reduced (see below) and further derived to
a benzoate 7c for the HPLC analysis, in a conventional
6.27. (R)-N-Benzyloxycarbonylprolinol (R)-8a
In a similar manner as above,17a the methyl ester (R)-6b
(1.23 g, 4.67 mmol) was treated with NaBH4 (0.345 g,
9.12 mmol) and LiCl (0.398 g, 9.39 mmol) to give crude
(R)-8a. This was purified by column chromatography
(silica gel, 30 g). Elution with hexane–AcOEt (5:1 to
2:1) afforded (R)-8a (97%) as a colorless oil. [h]2D0 +45.4
1
manner. H NMR (270 MHz, CDCl3) l 8.00 (m, 2H),
7.55 (m, 1H), 7.43 (m, 2H), 4.40 (m, 1H), 4.20 (m, 2H),
3.40 (m, 2H), 2.10–1.90 (m, 4H), 1.45 (s, 9H); 13C
NMR (100 MHz, CDCl3) l 166.2, 154.3, 132.9, 132.8,
129.9, 129.5, 128.3, 79.7, 79.5, 65.2, 65.0, 55.6, 46.7,
46.5, 28.9, 28.5, 27.9, 23.9, 23.1; IR (neat) 2975, 2879,
1722, 1695, 1452, 1393, 1366, 1314, 1272, 1172, 1108,
1027, 908, 865, 773, 712 cm−1; HR-MS (70 eV): M++H
306.1659 (calcd. 306.1704). Anal. calcd for C17H23NO4:
C, 66.86; H, 7.59; N, 4.59. Found: C, 66.97; H, 7.59; N,
4.56. The e.e. of (S)-5a was estimated to be 99.9% by
the HPLC analysis of 7c. The conditions were as fol-
lows: eluent, hexane/i-PrOH=39/1; flow rate, 0.5 mL/
min; retention time, 15.4 (S) and 17.0 (R) min.
1
(c 1.05, MeOH); H NMR (270 MHz, CDCl3) l 7.36
(m, 5H), 5.16 (s, 2H), 4.03 (m, 1H), 3.65 (m, 2H), 3.55
(m, 1H), 3.40 (m, 1H), 2.24 (s, 1H), 2.10–1.79 (m, 4H);
IR (neat) 3429, 2953, 2879, 1699, 1417, 1359, 1193,
1104, 913, 769, 698 cm−1. Its NMR and IR spectra were
identical with those reported previously.23
6.28. ( )-N-Carbamoyl-2-methoxymethylpyrrolidine ( )-
1a
NaH (10.6 mg, 0.443 mmol) and MeI (30.0 ml, 0.482
mmol) were added to a suspension of the alcohol 1b
(49.5 mg, 0.343 mmol) in THF (1.0 mL) at −78°C, and
the cooling bath was removed. The mixture was stirred
at room temperature for 4 h and saturated aq. NH4Cl
soln was then added. The mixture was extracted with
CHCl3 to ensure the complete recovery, and the com-
bined organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 10 g).
Elution with AcOEt–EtOH (8:1) afforded ( )-1a (68%)
as a white solid and further N-methylated form [( )-1e,
15%] as a white solid.
(R)-5b: [h]D20 +63.6 (c 1.55, MeOH) [lit.21d [h]2D3 −65.0 (c
2.00, MeOH) for (S)-5b], 98.7% e.e. by the HPLC
analysis of 7c as above. Anal. calcd for C11H19NO4: C,
57.63; H, 8.35; N, 6.11. Found: C, 57.55; H, 8.39; N,
5.99.
6.25. (S)-N-Benzyloxycarbonylproline (S)-6a and (R)-
N-benzyloxycarbonylproline methyl ester (R)-6b
Candida antarctica lipase (Chirazyme L-2, 96.5 mg) was
added to an emulsion of the methyl ester 6b (241 mg,
0.917 mmol) in 0.1 M phosphate buffer (pH 7.0, 12.0
mL), and the mixture was stirred at 30°C for 38 h. The
ester (R)-6b (46%) and the acid (S)-6a (46%) were
obtained. The acid (S)-6a was further derived to (S)-6b
by the treatment with TMSCHN2 for analysis.
( )-1a: Mp 153.9–154.6°C; 1H NMR (270 MHz,
CDCl3) l 5.10 (br s, 2H), 3.96 (m, 1H), 3.56–3.17 (m,
4H), 3.30 (s, 3H), 2.10–1.75 (m, 4H); 13C NMR (100
MHz, CDCl3) l 159.2, 76.8, 59.1, 57.4, 46.8, 29.1, 23.8;
MS (70 eV) m/e (rel. int.,%): 159 (M+1, 4), 126 (13),
113 (base peak), 70 (61), 45 (9); IR (KBr) 3386, 3185,
2982, 2873, 1653, 1603, 1460, 1115, 974, 782, 678 cm−1.
Its NMR and IR spectra were identical with the
reported data.18a
(R)-6b: [h]2D0 +54.9 (c 1.92, MeOH) [lit.22 [h]2D0 −58.1 (c
1.80, MeOH) for (S)-6b], 97.8% e.e. by HPLC analysis
as above. Anal. calcd for C14H17NO4: C, 63.87; H, 6.51;
N, 5.32. Found: C, 64.10; H, 6.54; N, 5.20.

1332
M. Kurokawa et al. / Tetrahedron: Asymmetry 14 (2003) 1323–1333
( )-1e: Mp 138.8–139.6°C; 1H NMR (270 MHz,
CDCl3) l 5.52 (br s, 1H), 3.90 (m, 1H), 3.60 (m, 1H),
3.42–3.35 (m, 5H), 2.90 (m, 1H), 2.75 (s, 3H), 2.10–1.79
(m, 4H); HR-MS (70 eV): M+ 172.1192 (calcd.
172.1211).
conc. HCl (6.0 mL), and the mixture was stirred for 2
h at room temperature. After saturated aq. KOH soln
was added to the reaction mixture under 15°C until the
pH of the mixture reached 2.8, KCNO (0.435 g, 5.37
mmol) was added and the resulting mixture was stirred
at room temperature for 48 h. As mentioned before,
due to the hydrophilicity of the product, the mixture
was extracted several times with CHCl3. The crude
material was purified by column chromatography (silica
gel, 20 g). Elution with AcOEt–EtOH (9:1 to 6:1)
afforded (R)-1a (98%) as a white solid. Mp 61.7–62.5°C
(lit.18a 60.5–61.5°C); [h]D22 +65.1 (c 2.00, EtOH) [lit.18a
6.29. (R)-N-tert-Butoxycarbonyl-2-methoxymethyl-
pyrrolidine (R)-7b
MeI (0.35 mL, 5.62 mmol) and NaH (0.132 g, 5.50
mmol) were added to a solution of the alcohol (R)-7a
(0.658 g, 3.27 mmol) in THF (13.0 mL) at −78°C. Then
the temperature was slowly raised to room temperture,
and the mixture was stirred overnight. The reaction
mixture was cooled to 0°C and saturated aq. NH4Cl
soln was then added. The mixture was extracted with
AcOEt and the combined organic layer was dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 20 g).
Elution with hexane–AcOEt (9:1 to 6:1) afforded (R)-
7b (94%) as a colorless oil. [h]1D9 +68.1 (c 1.35, MeOH);
1H NMR (400 MHz, CDCl3) l 3.90 (m, 1H), 3.50 (m,
1H), 3.40–3.20 (m, 6H), 2.00–1.76 (m, 4H), 1.45 (s, 9H);
13C NMR (100 MHz, CDCl3) l 154.4, 79.7, 73.5, 72.9,
59.0, 56.2, 46.8, 46.4, 28.7, 28.5, 27.9, 23.7, 22.9; IR
(neat) 2976, 2878, 1695, 1478, 1455, 1393, 1255, 1173,
1102, 975, 907, 867, 773 cm−1. Anal. calcd for
C11H21NO3: C, 61.37; H, 9.83; N, 6.51. Found: C,
61.49; H, 9.66; N, 6.31.
1
[h]2D0 −6.7 (c 2.00, EtOH) for (S)-1a]; H NMR (270
MHz, CDCl3) l 5.20 (br s, 2H), 3.96 (m, 1H), 3.58–3.20
(m, 4H), 3.30 (s, 3H), 2.10–1.79 (m, 4H); 13C NMR
(100 MHz, CDCl3) l 159.3, 76.7, 59.0, 57.4, 46.7, 29.1,
23.7; IR (KBr) 3389, 3200, 2968, 2881, 1663, 1614,
1451, 1361, 1197, 1110, 905, 778, 679 cm−1. Anal. calcd
for C7H14N2O2: C, 53.15; H, 8.92; N, 17.71. Found: C,
52.76; H, 8.93; N, 17.41. Its NMR and IR spectra were
identical with those reported previously.18a
Acknowledgements
The authors thank Meito Co. for the generous gift of
C. rugosa lipase OF. We express our deep thanks to
Professors Romas Kazlauskas of McGill University
and Tadashi Ema of Okayama University for their
discussion on enantiomeric recognition of protease-cat-
alyzed hydrolysis, on the occasion of the 5th Japanese
Symposium on the Chemistry of Biocatalysis. The
authors also thank Dr. Shuji Akai of Osaka University
for information on lipase-catalyzed hydrolysis of ben-
zoates. This work was also supported by a Grant-in-
Aid for Scientific Research (No.14560084) and the 21st
Century COE program (KEIO LCC) from the Ministry
of Education, Sports, Culture, Science, and Technol-
ogy, Japan, the Monbu-Kagakusho, and is acknowl-
edged with thanks.
6.30. (R)-N-Benzyloxycarbonyl-2-methoxymethylpyrro-
lidine (R)-8b
In a similar way as above, the alcohol (R)-8a (0.728 g,
3.09 mmol) was treated with MeI (1.17 mL, 18.8 mmol)
and NaH (0.156 g, 6.5 mmol) in THF (7.3 mL) under
−25°C for 15 h and for overnight at room temperature
to give crude (R)-8b. This was purified by column
chromatography (silica gel, 10 g). Elution with hexane–
AcOEt (8:1 to 1:1) afforded (R)-8b (58%) as a colorless
oil and an oxazolidinone 9 (33%) as a colorless oil.
(R)-8b: [h]2D2 +57.5 (c 1.44, MeOH); 1H NMR (400
MHz, CDCl3) l 7.35 (m, 5H), 5.15 (s, 2H), 4.00 (m,
1H), 3.55–3.20 (m, 7H), 2.00–1.75 (m, 4H); 13C NMR
(100 MHz, CDCl3) l 154.8, 136.8, 128.3, 127.7, 127.6,
73.4, 72.7, 66.6, 66.5, 59.0, 56.9, 56.3, 46.9, 46.6, 28.7,
27.9, 23.8, 22.9; IR (neat) 2976, 2879, 1704, 1453, 1412,
1358, 1198, 1098, 974, 914, 769, 748, 698 cm−1. Anal.
calcd for C14H19NO3: C, 67.45; H, 7.68; N, 5.62.
Found: C, 67.30; H, 7.45; N, 5.48.
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