Enzymatic resolution of N-protected-β3-amino methyl esters, using lipase B from Candida antarctica

Article abstract of DOI:10.1016/j.tetasy.2004.11.082

Racemic β³-amino methyl esters bearing the amine function protected with Bz, Cbz, Boc, Fmoc and as aminobenzamide, were resolved by enantiospecific transesterifications catalyzed by lipase B from Candida antarctica. The reactions proceeded with

Full text of DOI:10.1016/j.tetasy.2004.11.082

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 629–634
Enzymatic resolution of N-protected-b3-amino methyl esters,
using lipase B from Candida antarctica
a
Patricia Flores-Sanchez, Jaime Escalante and Edmundo Castillo
a,
b
*
´
a
´
´
Centro de Investigaciones Quımicas, Universidad Autonoma del Estado de Morelos, Av. Universidad No. 1001,
´
´ ´ ´
Departamento de Bioingenierıa, Instituto de Biotecnologıa, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, Mexico
Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, Mexico
b
Received 24 September 2004; accepted 18 November 2004
Available online 21 January 2005
Abstract—Racemic b3-amino methyl esters bearing the amine function protected with Bz, Cbz, Boc, Fmoc and as aminobenzamide,
were resolved by enantiospecific transesterifications catalyzed by lipase B from Candida antarctica. The reactions proceeded with a
high conversion and yielded enantiomerically pure enantiomers.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
with groups such as Bz, Cbz, Boc, Fmoc and as amino-
benzamide (ABA) (Scheme 1). The resolutions were car-
ried out through transesterifications catalyzed by lipase
B from Candida antarctica (CALB).5
Due to the wide range of applications,1 the preparation
of optically active b3-amino acids is an area of cur-
rent interest.2 However, only few enzymatic routes to
these compounds have been described.3 Indeed, few
enzymes show activity and stereoselectivity in the pres-
ence of b3-amino acids, and it becomes even more diffi-
cult if the reactive site is the carboxy function, because
the stereocenter is remote from that.4
2. Results and discussion
2.1. Synthesis of N-protected methyl 3-aminobutanoate
( )-1a–e and methyl 3-amino-3-phenylbutanoate deriva-
tives ( )-2a–e
Here we report the enzymatic resolution of methyl 3-
aminobutanoate ( )-1 and methyl 3-amino-3-phenyl-
propanoate ( )-2 derivatives, with the amine protected
The N-protected-b3-amino methyl esters ( )-1a–d and
( )-2a–d were obtained from the corresponding racemic
PG
PG
Me
PG
NH
O
NH
O
NH
O
iBuOH
CALB
+
Me
OiBu
OiBu
OMe
Me
OMe
OMe
(+) - 1
1
3
PG
Ph
PG
Ph
PG
Ph
NH
O
NH
O
NH
O
iBuOH
CALB
+
OMe
(+) - 2
2
4
PG = Bz, Cbz, Boc, Fmoc, ABA
Scheme 1.
*
Corresponding author. Tel./fax: +52 777 3 29 79 97; e-mail: jaime@ciq.uaem.mx
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.082

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P. Flores-Sanchez et al. / Tetrahedron: Asymmetry 16 (2005) 629–634
630
NH2
O
Isatoic
anhydride
Et3N
NH
O
O
(+)-1a: R=Me, 86%
(+)-2a: R=Ph, 82%
OMe
R
Toluene-AcOEt
Bz
R
NH
BzCl
NaOH
(+)-1b: R=Me, 85%
(+)-2b: R=Ph, 88%
OMe
OMe
- +NH3
O
Cl
NH2
O
SOCl2
MeOH
R
OMe
Boc
R
OH
NH
O
(+)-1c: R=Me, 91%
(+)-2c: R=Ph, 90%
(+)-5: R=Me
(+)-6: R=Ph
(+)-7: R=Me, 94%
(+)-8: R=Ph, 97%
(Boc)2O
R
Et3N, MeOH
CbzCl
NaOH
Fmoc
NH
O
(+)-1d: R=Me, 91%
(+)-2d: R=Ph, 74%
FmocOSuc
NaHCO3
R
OMe
Cbz
Cbz
NH O
NH
O
SOCl2
MeOH
R
OH
R
OMe
(+)-9: R=Me, 92%
(+)-10: R=Ph, 88%
(+)-1e: R=Me, 94%
(+)-2e: R=Ph, 92%
Scheme 2.
b3-amino acids ( )-5 and ( )-6 (Scheme 2). For com-
pounds ( )-1e and ( )-2e, we first protected the amine
with CbzCl to obtain ( )-9 and ( )-10 and then pre-
pared the methyl esters.
The reactions were monitored by GC and the results
are described in Table 1.
At 60 ꢁC we observed slow reactions for compounds ( )-
2a–e where R = Ph (entries 6–10), and after time >192 h,
conversion did not reach 50%. This might be due to the
steric effect exerted by the Ph group. However, we found
that the enzyme could present selectivity against only
one of the enantiomers, since, after isolation, we deter-
mined the specific rotations of products (+)-4a–e. In
the case of compounds ( )-1a and b,where R = Me
2.2. Transesterification of methyl 3-aminobutanoate ( )-
1a–e and methyl 3-amino-3-phenylbutanoate ( )-2a–e
derivatives, catalyzed by CALB
The enzymatic transesterifications were performed in
toluene, using isobutyl alcohol, in presence of CALB.
Table 1. Transesterification of ( )-1a–e and ( )-2a–e, catalyzed by CALB
PG
PG
PG
NH
O
NH
O
NH O
iBuOH, Toluene
CALB, T ºC
+
R
OiBu
R
OMe
R
OMe
1a-e: R=Me
+
+
(
(
)-1a-e: R=Me
)-2a-e: R=Ph
3a-e: R=Me
4a-e: R=Ph
2a-e: R=Ph
Conversiona (%)
Eeb (%)
Ec
[a]D
Absolute configuratione
d
Entry
Product
R
PG
T (ꢁC)
Time (h)
1
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
Me
Me
Me
Me
Me
Ph
ABA
Bz
Boc
60
60
45
45
45
60
60
60
45
60
7
50
50
50
50
50
32
30
43
14
28
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>458
>458
>458
>458
>458
>14
+53.4, c = 1.10
+34.9, c = 0.75
+19.7, c = 0.60
+14.9, c = 1.01
+14.6, c = 1.03
+18.3, c = 0.71
+22.8, c = 0.70
ꢀ21.1, c = 1.02
ꢀ8.2, c = 0.8
R
R
R
R
R
S
S
S
S
S
9
3
0.75
0.75
0.75
4
Fmoc
Cbz
ABA
Bz
5
6
240
7
Ph
Ph
200
192
240
240
>13
>37
8
Boc
Fmoc
Cbz
9
10
Ph
Ph
>8
>12
ꢀ9.6, c = 1.00
a Determined by GC, using a Megabore column, HP 1, 5 m; for PG = ABA, Bz, Boc, Cbz, 140 ꢁC for 1 min ! 180 ꢁC (T rise 5 ꢁC/min); for
PG = Fmoc, 240 ꢁC for 1 min ! 280 ꢁC (T rise 5 ꢁC/min).
b Determined by 1H NMR using europium tris[3-(heptafluropropylhydroximethylen)-(+)-camphorate as chiral shift reagent.
c The E values were calculated from E = ln[(1 ꢀ c)(1 ꢀ ee)]/ln[(1 ꢀ c)(1 + ee)].
d Specific rotations were measured with a Perkin–Elmer 341 polarimeter, using CHCl3 as solvent.
e Assigned by chemical correlation.

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P. Flores-Sanchez et al. / Tetrahedron: Asymmetry 16 (2005) 629–634
631
Ph
Ph
Ph
N
Li
O
Ph
N
O
NH2 O
H2, PdOH/C
R
OR'
R
OR'
R
OR'
MeOH, AcOH 60lb
THF
13: R= R'=Me
11: R= R'=Me
12: R= R'=Me
16: R=Ph, R'=tBu
14: R=Ph, R'=tBu
15: R=Ph, R'=tBu
NH2
O
Isatoic
Bz
anhydride
AcOEt
BzCl
NaOH
NH
O
NH
O
13
Me
OR
Me
OR
(-)-1a: R=Me
[α]D24 = -49.9, (c 1.0, CHCl3)
(-)-1b: R=Me
iBuOLi
THF
iBuOLi
THF
[α]D24 = -39.8, (c 0.77, CHCl3)
(-)-3b, R=iBu
(-)-3a: R=iBu
[α]D24 = -34.9, (c 0.75, CHCl3)
[α]D24 = -53.4, (c 1.1, CHCl3)
1. CF3CO2H,
NH2
O
1.-BzCl, NaOH
CH2Cl2
Bz
Ph
2.- CF3CO2H,
2. SOCl2, MeOH
NH
O
NH
O
16
CH2Cl2
3. Isatoic anhydride
Et3N, Toluene
OR
Ph
OR
3.- SOCl2, MeOH
(-)-2a: R=Me
(-)-2b: R=Me
[α]D = -31.0, (c 0.70, CHCl3)
24
iBuOLi
THF
iBuOLi
THF
[α]D24 = -36.3, (c 0.80, CHCl3)
(-)-4b, R=iBu
(-)-4a, R=iBu
[α]D24 = -18.2, (c 0.71, CHCl3)
24
[α]D = -22.6, (c 0.70, CHCl3)
Scheme 3.
(entries 1 and 2), at 60 ꢁC the reactions occurred faster
than ( )-2a–e, and 50% conversion was detected. For
compounds ( )-1c–e (entries 3–5), transesterifications
were performed at 45 ꢁC, for 0.75 h at 50% conversion.
Products (+)-3a–e were also optically active.
(ꢀ)-1b and (ꢀ)-2b. So, for (+)-3c–e the absolute config-
uration is R, with ee >98%, and for (ꢀ)-4c–e the abso-
lute configurations is S, with ee >98%. As we can see
Bz
R
Boc
R
NH
O
NH
O
1.-TFA, CH2Cl2
2.- NaOH, BzCl
In order to determine the enantiomeric excesses and the
absolute configurations of (+)-3a and b and (+)-4a and
b, we prepared compounds (ꢀ)-3a and b, and (ꢀ)-4a,
b of absolute configuration S and R, respectively,
through the asymmetric synthesis described by Davies
and co-workers (Scheme 3).6 We compared the specific
rotations values and in all cases we observed that they
were in agreement. On the other hand, the absolute con-
figuration of these compounds was assigned by compar-
ing the sign of their specific rotations; so for (+)-3a and
b is R and for (+)-4a and b is S.
OiBu
OMe
3.- SOCl2, MeOH
(+)-3c: R= Me
(-)-4c: R= Ph
(+)-3b: R= Me
[α]D24 = +39.8, (c 0.77, CHCl3)
(+)-4b: R= Ph
[α]D24 = +31.2, (c 0.70, CHCl3)
Fmoc
R
Bz
R
NH
O
NH
O
OiBu
OMe
1.- NaOH, BzCl
2.- SOCl2, MeOH
(+)-3d: R=Me
(-)-4d: R= Ph
(+)-3b: R= Me
1
The enantiomeric excesses were also determined by H
[α]D24 = +39.7, (c 0.77, CHCl3)
(+)-4b R= Ph
NMR, using europium tris[3-(heptafluropropylhydroxi-
methylen)-(+)-camphorate as chiral shift reagent.7 After
the addition of the chiral shift reagent to a solution of
(+)-3a,b and (+)-4a,b only one diastereoisomeric com-
plex was detected, for each case, confirming the ee to
be >98%.
[α]D24 = +31.1, (c 0.71, CHCl3)
Cbz
R
Bz
R
NH
O
1.-H2, Pd/C
NH
O
MeOH
OiBu
2.- NaOH, BzCl
3.- SOCl2, MeOH
OMe
(+)-3b: R= Me
The enantiomeric excesses and the absolute configura-
tions of compounds (+)-3c–e and (ꢀ)-4c–e were deter-
mined by transforming them into N-benzoyl-b3-amino
methyl esters (Scheme 4), and then comparing their spe-
cific rotations with the ones from enantiomerically pure
(+)-3e: R =Me
(-)-4e: R= Ph
[α]D24 = +39.6, (c 0.77, CHCl3)
(+)-4b: R= Ph
[α]D24 = +31.0, (c 0.70, CHCl3)
Scheme 4.

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P. Flores-Sanchez et al. / Tetrahedron: Asymmetry 16 (2005) 629–634
632
in Scheme 4, CALB was selective for only those enantio-
mers where the N-protected group is pointing toward
the observer.8
2.62 (2H, d, J = 5.2 Hz), 3.70 (3H, s), 4.40–4.60 (1H,
m), 5.50 (2H, b), 6.59–6.66, (2H, m) 6.76 (1H, b), 7.18
(1H, Jmeta = 1.4 Hz, Jortho = 8.1 Hz), 7.31 (1H, d,
Jortho = 8.4 Hz); 13C NMR (50 MHz, CDCl3) d 20.5,
40.1, 42.2, 52.0, 116.1, 116.7, 117.3, 127.2, 132.2,
148.7, 168.3, 172.2. Anal. Calcd for C12H16NO3: C,
61.00; H, 6.83; N, 11.86. Found: C, 61.73; H, 7.06; N,
10.86.
We determine the E values for the enzymatic transeste-
rifications finding excellent values (E >458) for (+)-3a–
e, while for (+)-4a–b, (ꢀ)-3c–e, they were low (E <14).
3. Conclusions
4.2.1.2. (rac)-Methyl 3-(2-aminobenzamido)-3-phenyl-
propanoate, ( )-2a. Yield: 82%. Yellow oil, H NMR
1
We have reported a new enzymatic method to resolve N-
protected-b3-amino methyl ester through transesterifica-
tion catalyzed by CALB. Despite the stereocenter being
remote from the reactive site of the molecules, we ob-
serve that CALB presents selectivity for only one enan-
tiomer, to yield enantiomerically pure compounds with
a high conversion. The esters resolved can be suitable
for b-peptide synthesis in solid phase,9 or solution tech-
niques;10 furthermore, after hydrolysis, they would yield
to the corresponding b3-amino acids in enantiomerically
pure form.
(200 MHz, CDCl3) 2.97 (2H, t, J = 5.4 Hz), 3.63 (3H,
s), 5.51–5.61 (3H, m), 6.62–6.70 (4H, m), 7.06–7.44,
(6H, m); 13C NMR (50 MHz, CDCl3) d 40.2, 49.8,
52.2, 115.4, 116.7, 117.4, 126.1, 127.3,127.6, 128.7,
132.5, 140.6, 149.0, 168.3, 171.6. Anal. Calcd for
C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C,
68.00; H, 6.23; N, 8.86.
4.2.2. (rac)-Methyl 3-[(9H-fluren-9-ylmethoxycarbonyl)-
3-phenylpropanoate, ( )-2d. Yield: 74%. Methyl ester
hydrochloride 8 (5 mmol) was dissolved in 20 ml of dis-
tilled H2O and cool down to 0 ꢁC then NaHCO3 1.0 M
was added (20 mmol, 4 equiv) and the reaction was stir-
red for 0.3 h. Then, FmocSuc (6 mmol, 1.2 equiv) in ace-
tone. The reaction continued for 3 h. After this time the
acetone was evaporated, and reaction mixture extracted
with CH2Cl2 (3 · 50 ml). The organic layer was dried
over MgSO4 and concentrated under reduced pressure.
The crude product was purified by FC (hexane:ethyl
acetate 90:10 ! 70:30) to produce ( )-2d as a white so-
lid, mp 128 ꢁC, 1H NMR (200 MHz, CDCl3) d 2.95 (2H,
b), 3.88 (3H, s), 4.26 (1H, t, J = 6.5 Hz), 4.46 (2H, d,
J = 7.4 Hz), 5.22 (1H, b), 5.83 (1H, b), 7.30–7.84 (13H,
m); 13C NMR (50 MHz, CDCl3) d 47.6, 50.7, 52.2,
65.4, 67.1, 120.0, 124.8, 125.1, 126.1, 127.1, 127.6,
127.7, 128.8, 141.3, 146.8, 155.6, 171.5. Anal. Calcd
for C25H23NO4: C, 74.79; H, 5.77; N, 3.49. Found: C,
74.51; H, 6.00; N, 3.32.
4. Experimental section
4.1. General
All reagents were purchased from commercial suppliers
and used without further purification. Toluene and THF
1
were distilled from sodium. H and 13C NMR spectra
were obtained on a Varian Gemini 200 or Inova 400
spectrometer and were recorded relative to TMS as
internal standard. Microanalyses were performed in Ele-
mentar Vario EL III. Optical rotations: 10 cm, 1 mL
cell, Perkin–Elmer-341 polarimeter. Melting points were
measured in open glass capillaries with a Buchi appara-
¨
tus and are uncorrected. Conversion was determined by
GC, using a Megabore column, HP 1, 5 m; for PG = A-
BA, Bz, Boc, Cbz, 140 ꢁC for 1 min ! 180 ꢁC (tempera-
ture rise 5 ꢁC/min); for PG = Fmoc, 240 ꢁC for 1 min !
280 ꢁC (temperature rise 5 ꢁC/min).
4.3. General procedure for the enzymatic transesterifica-
tions of ( )-1a–e and ( )-2a–e
4.2. Synthesis of N-protected-b3-amino methyl ester
The enzymatic transesterifications were performed in
closed, screw capped 8 ml reaction vessels containing
0.25 mmol of the corresponding N-protected-b3-amino
methyl esters, 0.5 ml (0.5 mmol) of anhydrous isobutyl
alcohol, 160–200 mg of CALB (Novozym 435) and
7 ml of anhydrous toluene. The reactions were incu-
bated in a thermostated water bath at 45 or 60 ꢁC,
and slowly stirred. In order to monitor the reaction pro-
gress, samples of 200 ll were withdrawn at different
intervals, centrifuged to separate enzyme and analyzed
by GC. To finish the reactions, the enzyme was recov-
ered and washed with acetone. Solvent was removed un-
der reduced pressure and the residues were purified by
column chromatography on silica gel with hexane–ethyl
acetate as eluent (90:10 ! 70:30), to obtain N-protected-
b3-amino isobutyl esters 3a–e and 4a–e.
N-protected-b3-amino methyl esters ( )-1b–e and ( )-
2b–e, were prepared by standard methods.11 Only prep-
aration and properties of new compounds are described
afterwards.
4.2.1. (rac)-Methyl 3-(2-aminobenzamido)butanoate, ( )-
1a and (rac)-methyl 3-(2-aminobenzamido)-phenylpropan-
oate, ( )-2a. A suspension of methyl esters hydrochlo-
rides 7 and 8 (8 mmol) was treated with triethylamine
(9.6 mmol, 1.2 equiv) and isatoic anhydride (8.8 mmol,
1.1 equiv), in a mixture of toluene/ethyl acetate (1:1).
The reaction mixture was warmed to 50 ꢁC for 2 h.
The triethylamine hydrochloride was removed by filtra-
tion. The solvent from the filtrate was removed under re-
duced pressure and the crude product purified by FC.
4.2.1.1. (rac)-Methyl 3-(2-aminobenzamido)butanoate
( )-1a. Yield: 86%. White solid, mp 92–93 ꢁC, 1H
NMR (200 MHz, CDCl3) d 1.31 (3H, d, J = 6.9 Hz),
4.3.1. (R)-Isobutyl 3-(2-aminobenzamido)butanoate, (+)-
3a. Yield after isolation 49%. Yellow solid, mp 68 ꢁC,
24
½aꢁ ¼ þ53:1 (c 1.10, CHCl3), 1H NMR (200 MHz,
D

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P. Flores-Sanchez et al. / Tetrahedron: Asymmetry 16 (2005) 629–634
633
CDCl3) d 0.93 (6H, d, J = 6.9 Hz), 1.32 (3H, d,
J = 6.6 Hz), 1.94 (1H, m, J = 6.6 Hz), 2.63 (2H, d,
J = 5.2 Hz), 3.89 (2H, d, 6.6 MHz), 4.41–4.61 (1H, m),
5.52 (2H, b), 6.58–6.66, (2H, m) 6.81 (1H, b), 7.18
(1H, Jmeta = 1.4 Hz, Jortho = 8.1 Hz), 7.31 (1H, d,
Jortho = 8.4 Hz); 13C NMR (50 MHz, CDCl3) d 19.5,
20.5, 28.0, 40.2, 42.2, 71.1, 116.1, 116.7, 117.3, 127.2,
132.2, 148.7, 168.3, 171.9. Anal. Calcd for C15H22NO3:
C, 64.73; H, 7.97; N, 10.06. Found: C, 64.56; H, 7.97;
N, 9.51.
4.3.6. (S)-Isobutyl 3-(2-aminobenzamido)-3-phenylpro-
panoate, (+)-4a. Yield after isolation 31%. Yellow oil,
24
D
½aꢁ ¼ þ18:3 (c 0.71, CHCl3), 1H NMR (200 MHz,
CDCl3) d 0.81 (6H, d, J = 6.8 Hz), 1.82 (1H, m,
J = 6.6 Hz), 2.97 (2H, dd, J = 5.2 Hz, J = 14.5 Hz),
3.80 (2H, d, 6.8 Hz), 5.51-5.61 (3H, m), 6.61–6.69 (4H,
m), 7.15–7.45, (6H, m); 13C NMR (50 MHz, CDCl3)
d 19.3, 27.91, 40.4, 49.9, 71.3, 115.4, 116.7, 117.4,
126.1, 127.3,127.6, 128.7, 132.5, 140.6, 149.0,
168.3, 171.6. Anal. Calcd for C20H24N2O3: C,
70.56; H, 7.11; N, 8.23. Found: C, 70.15; H, 7.06; N,
9.29.
4.3.2. (R)-Isobutyl 3-[(benzoyl)amino]butanoate, (+)-
3b. Yield after isolation 49%. White crystals, mp 71–
24
D
72 ꢁC, ½aꢁ ¼ þ34:9 (c 0.75, CHCl3), 1H NMR
4.3.7. (S)-Isobutyl 3-[(benzoyl)amino]-3-phenylpropano-
ate, (+)-4b. Yield after isolation 29%. White crystals,
(200 MHz, CDCl3) d 0.93 (6H, d, J = 6.9 Hz), 1.34
(3H, d, J = 6.6 Hz), 1.94 (1H, m, J = 6.6 Hz), 2.65
(2H, dd, J = 5.2 Hz, J = 15.0 Hz), 3.90 (2H, d, 6.8 Hz),
4.49–4.66 (1H, m), 7.02 (1H, b), 7.35–7.54, (3H, m)
7.74–7.78 (2H, m); 13C NMR (50 MHz, CDCl3) d
19.5, 20.4, 28.0, 40.0, 42.6, 71.0, 166.3, 172.0. Anal.
Calcd for C15H21NO3: C, 68.42; H, 8.04; N, 5.32.
Found: C, 68.51; H, 7.99; N, 5.23.
24
D
mp 70 ꢁC, ½aꢁ ¼ þ22:8 (c 0.70, CHCl3), 1H NMR
(200 MHz, CDCl3) d 0.82 (6H, d, J = 6.9 Hz), 1.82
(1H, m, J = 6.6 Hz), 3.00 (2H, dd, J = 5.4 Hz, J = 15.0
Hz), 3.80 (2H, d, 6.6 Hz), 5.58–5.67 (1H, m), 7.02 (1H,
b), 7.20–7.53 (8H, m), 7.58 (1H, b), 7.84–7.91 (2H, m);
13C NMR (50 MHz, CDCl3) d 19.3 27.9, 40.1, 50.1,
71.3, 126.2, 127.1, 127.6, 128.6, 128.7, 131.6, 134.2,
140.5, 166.3, 171.7. Anal. Calcd for C15H21NO3: C,
73.82; H, 7.12; N, 4.30. Found: C, 71.71; H, 7.53; N,
3.68.
4.3.3. (R)-Isobutyl 3-[(tert-butoxycarbonyl)amino]butano-
ate, (+)-3c. Yield after isolation 47%. Colorless oil,
24
D
½aꢁ ¼ þ19:7 (c 0.60, CHCl3), 1H NMR (200 MHz,
CDCl3) d 0.93 (6H, d, J = 7.0 Hz), 1.21 (3H, d,
J = 6.8 Hz), 1.43 (9H, s), 1.92 (1H, m, J = 6.8 Hz),
2.51 (2H, d, J = 6.6 Hz), 3.88 (2H, d, J = 6.8 Hz),
3.92–4.18 (1H, m), 4.95 (1H, b); 13C NMR (50
MHz, CDCl3) d 19.5, 20.9, 28.0, 41.1, 43.8, 70.9,
79.5, 154.9, 171.4. Anal. Calcd for C13H25NO4: C,
60.21; H, 9.72; N, 5.40. Found: C, 59.72; H, 9.49; N,
5.21.
4.3.8. (S)-Isobutyl 3-[(tert-butoxycarbonyl)amino]-3-phen-
ylpropanoate, (ꢀ)-4c. Yield after isolation 42%. Color-
24
D
less oil, ½aꢁ ¼ ꢀ21:1 (c 1.02, CHCl3), 1H NMR
(200 MHz, CDCl3) d 0.83 (6H, d, J = 6.6 Hz), 1.41
(9H, s), 1.43 (9H, s), 1.82 (1H, m, J = 6.8 Hz), 2.84
(2H, t, J = 4.4 Hz), 3.78 (2H, d, J = 6.6 Hz), 5.08 (1H,
b), 5.47 (1H, b), 7.21–7.31 (5H, m); 13C NMR (50
MHz, CDCl3) d 19.4, 27.9, 28.7, 41.3, 51.6, 71.0, 79.8,
126.1, 127.5, 128.6, 141.2, 154.9, 170.8. Anal. Calcd
for C18H27NO4: C, 67.26; H, 8.47; N, 4.36. Found: C,
66.98; H, 9.04; N, 4.02.
4.3.4. (R)-Isobutyl 3-[(9H-fluren-9-ylmethoxycarbonyl)-
amino]butanoate, (+)-3d. Yield after isolation 49%.
24
White crystals, mp 84 ꢁC, ½aꢁ ¼ þ14:9 (c 1.01, CHCl3),
D
1H NMR (200 MHz, CDCl3) d 0.92 (6H, d, J = 6.6 Hz),
1.25 (3H, d, J = 6.6 Hz), 1.92 (1H, m, J = 6.6 Hz), 2.54
(2H, d, J = 5.6 Hz), 3.87 (2H, d, 6.6 Hz), 4.1 (1H, m),
4.19 (1H, t, J = 6.6 Hz), 4.36 (2H, d, J = 6.9 Hz), 7.28
(2H, td, Jmeta = 1.6 Hz, Jortho = 7.1 Hz); 7.37 (2H, td,
4.3.9. (S)-Isobutyl 3-[(9H-fluren-9-ylmethoxycarbonyl)-3-
phenylpropanoate, (ꢀ)-4d. Yield after isolation 12%.
24
D
Colorless oil, ½aꢁ ¼ ꢀ8:2 (c 0.8, CHCl3), 1H NMR
(200 MHz, CDCl3) d 0.83 (6H, d, J = 7.0 Hz), 1.82
(1H, m, J = 6.6 Hz), 2.89 (2H, d, J = 5.2 Hz), 3.79
(2H, d, 6.6 Hz), 4.19 (1H, t, J = 7.0 Hz), 4.39 (2H, d,
J = 7.2 Hz), 5.16 (1H, b), 5.80 (1H, b), 7.24–7.41
(9H, m), 7.56 (2H, d, Jortho = 7.4 Hz), 7.73 (2H, d,
Jmeta = 1.2 Hz, Jortho = 7.3 Hz), 7.57 (2H, dd, Jmeta
=
0.8 Hz, Jortho = 7.4 Hz) 7.73 (2H, d, Jortho = 7.1 Hz);
13C NMR (50 MHz, CDCl3) d 19.5, 20.8, 28.0, 40.8,
44.5, 47.5, 66.9, 70.9, 120.0, 125.1, 127.0, 127.7, 141.3,
143.9, 155.5, 171.4. Anal. Calcd for C23H27NO3: C,
72.42; H, 7.13; N, 3.67. Found: C, 72.49; H, 7.18; N,
3.65.
Jortho = 7.2 Hz); 13C NMR (50 MHz, CDCl3)
d
19.4, 27.9, 40.9, 47.6, 51.9, 67.1, 71.2, 120.0,
125.1, 126.1, 127.1, 127.7, 128.8, 141.3, 143.9, 155.5,
172.1.
4.3.10. (S)-Isobutyl 3-[(benzyloxycarbonyl)amino]-3-phen-
ylpropanoate, (ꢀ)-4e. Yield after isolation 28%. Yellow
4.3.5. (R)-Isobutyl 3-[(benzyloxycarbonyl)amino]butano-
ate, (+)-3e. Yield after isolation 48%. Colorless oil,
24
D
1
24
D
½aꢁ ¼ þ14:6 (c 1.03, CHCl3), 1H NMR (200 MHz,
oil, ½aꢁ ¼ ꢀ9:6 (c 1.00, CHCl3), H NMR (200 MHz,
CDCl3) d 0.1 (6H, d, J = 7.0 Hz), 1.80 (1H, m,
J = 6.6 Hz), 2.87 (2H, d, J = 5.4 Hz), 3.77 (2H, d,
6.6 Hz), 5.10–5.21 (1H, m), 5.08 (2H, s), 7.27–7.31,
(5H, m), 5.78 (1H, b); 13C NMR (50 MHz, CDCl3) d
19.3, 20.8, 27.9, 41.0, 52.0, 67.0, 71.1, 126.1, 127.7,
128.1, 128.5, 128.7, 136.3, 140.7, 155.5, 170.7. Anal.
Calcd for C21H25NO4: C, 70.96; H, 7.09; N, 3.94.
Found: C, 70.58; H, 6.97; N, 3.06.
CDCl3) d 0.92 (6H, d, J = 7.0 Hz), 1.24 (3H, d,
J = 6.6 Hz), 1.91 (1H, m, J = 6.6 Hz), 2.53 (2H, d,
J = 6.6), 3.85 (2H, d, 6.6 Hz), 4.04–4.17 (1H, m), 5.08
(2H, s), 5.20 (1H, b), 7.31–7.35, (5H, m); 13C NMR
(50 MHz, CDCl3) d 19.5, 20.8, 28.0, 40.8, 44.4, 66.9,
70.9, 128.1, 128.5, 136.5, 155.4, 171.8. Anal. Calcd for
C16H23NO4: C, 65.51; H, 7.90; N, 4.77. Found: C,
65.08; H, 8.02; N, 4.66.

´
P. Flores-Sanchez et al. / Tetrahedron: Asymmetry 16 (2005) 629–634
634
M.; Brieva, R. Tetrahedron: Asymmetry 1997, 8, 995; (d)
¨
Orrenius, C.; Ohrner, N.; Rottici, D.; Mattson, A.; Hult,
K.; Norin, T. Tetrahedron: Asymmetry 1995, 6, 1217; (e)
Johnson, C. R.; Xu, Y.; Nicolau, K. C.; Yang, Z.; Guy,
R.; Dong, J. G.; Berova, N. Tetrahedron Lett. 1995, 36,
4.4. Procedure for the determination of the enantiomeric
excess by NMR, using europium tris[3-(heptafluropropyl-
hydroximethylen)-(+)-camphorate Eu(hfc)3
In a typical experiment, racemic mixtures and resolved
compounds (0.015 mmol) were weighed into a NMR
tube and dissolved in 0.2 ml of CDCl3. Then 0.1 ml of
a solution of Eu(hfc)3 (5 mM in CDCl3) was added.
The solutions were mixed and then 1H NMR spectra re-
corded. More solution of Eu(hfc)3 was added and the
process repeated until the detection of the diastereoiso-
meric complexes.
´
3291; (f) Ferrero, M.; Fernandez, S.; Gotor, V. J. Org.
Chem. 1997, 62, 4358; (g) Daniell, B.; Luisetti, M.;
Sampagnaro, G.; Carrea, G.; Riva, S. J. Mol. Catal B:
Enzym. 1997, 3, 193; (h) Bertirotti, A.; Carrea, G.;
Ottolina, G.; Riva, S. Tetrahedron 1994, 50, 13165; (i)
´
Cordova, A.; Iversen, T.; Hult, K.; Martinelle, M.
Polymer 1998, 39, 6519.
6. (a) Davies, S. G.; Ichihara, O. Tetrahedron: Asymmetry
1991, 2, 183; (b) Bunnage, M. E.; Davies, S. G.; Goodwin,
C. J. J. Chem. Soc., Perkin Trans. 1 1993, 1375; (c)
Bunnage, M. E.; Chernega, A. N.; Davies, S. G.; Good-
win, C. J. J. Chem. Soc., Perkin Trans. 1 1994, 2373.
7. (a) Thunhorst, M.; Holzgrabe, U. Magn. Reson. Chem.
1998, 36, 211; (b) McCReary, M. D.; Lewis, D. W.;
Wernick, D. L.; Whitesides, G. M. J. Am. Chem. Soc.
1974, 96, 1038; (c) Goering, H. L.; Eikenberry, J. N.;
Koerner, G. S.; Lattimer, C. J. J. Am. Chem. Soc. 1974,
96, 1493.
8. Several empirical and computational models have been
proposed as attempts to rationalize the selectivity of
CALB in the resolution of secondary alcohols (see
references). However, they do not apply to the results
presented here; so we must consider an analysis to explain
them. (a) Haeffner, F.; Norin, T.; Hult, K. Biophys. J.
1998, 74, 1251; (b) Orrenius, C.; Haeffner, F.; Rotticci, D.;
Acknowledgements
We are grateful to Consejo Nacional de Ciencia y Tec-
´
nologıa (CONACyT) for economical support under
grant 38187-E. We also thank to Dr. Isamel Leon-Riv-
´
´
era for technical support in the use of GC and to Gaston
Contreras and Carlos F. Ban˜os for experimental contri-
butions. PF thanks the CONACyT for scholarship
´
142044. We thank ÔNovozymes MexicoÕ for their gener-
ous donation of enzyme CALB.
References and notes
¨
Ohrner, N.; Norin, T.; Hult, K. Biocatal. Biotransform.
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¨
9. Atherton, E.; Sheppard, R. C. Solid Phase Peptide
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4. Estermann, H.; Seebach, D. Helv. Chim. Acta 1988, 71,
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5. (a) Lipase B from Candida antarctica, has shown utility in
enantiospecific opening of b-lactams, resolution of cyclic
and acyclic alcohols, in natural products synthesis, regio-
selective esterification of sugar and esteroids and poly-
´
merizations. Forro, E.; Fulop, F. Org. Lett. 2003, 5, 1209;
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¨
´
¨
´
573; (c) Gotor, V.; Limeres, F. L.; Garcıa, M. J.; Bayond,