3492 J . Org. Chem., Vol. 62, No. 11, 1997
Wang et al.
activity and was without loss of enantioselectivity.20 In
contrast, crude alcalase21 lost more than 50% of its
original activity after two cycles.
HP LC Con d it ion s. (a) Microsorb C18 column (4.6 × 50
mm, 5 µm, 300A); mobile phase: gradient, ratio (v/v) of water
(with 0.1% trifluoroacetic acid) and acetonitrile (with 0.1%
trifluoroacetic acid) was 90:10 at 0 min, 50:50 at 8 min, 90:10
at 10 min; flow rate: 1 mL/min; UV detection at 254 nm;
Retention times: N-cbz-L-Phe-OBzl, 8.61 min; N-cbz-L-Phe-L-
Leu-NH2, 5.75 min; N-Boc-L-Tyr-OMe, 6.89 min; N-Boc-L-Tyr-
L-Leu-NH2, 6.36 min; N-Boc-3-(2-naphthyl)alanine methyl
ester, 7.77 min; N-Boc-L-Nap-L-Leu-NH2, 6.49 min; N-cbz-L-
Val-L-Phe-OMe, 9.70 min; N-cbz-L-Val-L-Phe-L-Leu-NH2, 8.95
min; N-cbz-L-Val-L-Phe-L-Phe-NH2, 9.25 min. (b) Microsorb
C18 column (4.6 × 150 mm, 5 µm, 100A); mobile phase:
gradient, ratio (v/v) of water (with 0.1% trifluoroacetic acid)
and acetonitrile (with 0.1% trifluoroacetic acid) was 90:10 at
0 min, 20:80 at 10 min, 20:80 at 12 min, 90:10 at 15 min; flow
rate: 1 mL/min; UV detection at 254 nm; retention times:
N-cbz-L-Phe-OBzl, 12.59 min; Phe-NH2, 3.84 min; N-cbz-L-Phe-
L-Phe-NH2, 9.81 min; N-cbz-L-Phe-L-Leu-OtBu, 12.99 min;
N-cbz-L-Phe-L-Ala-OtBu, 11.68 min; N-cbz-L-Ser-L-Leu-NH2,
7.45 min; N-cbz-L-Glu-DiOMe, 7.99 min; N-cbz-L-Glu(OMe)-
L-Leu-NH2, 7.55 min.
N-cbz-L-P h e-L-Leu -NH2 (CD3OD/TMS): 0.88 (3H, d, J )
6.5 Hz), 0.92 (3H, d, J ) 6.5 Hz), 1.57 (2H, dd, J ) 6.5, 7.5
Hz), 1.60 (1H, m), 2.89 (1H, dd, J ) 9.0, 13.5 Hz), 3.11 (1H,
dd, J ) 6.0, 13.5 Hz), 4.36 (2H, m), 5.03 (2H, s), 7.15-7.35
(10H, m).
N-cbz-L-P h e-D-Leu -NH2 (CD3OD/TMS): 0.69 (3H, d, J )
6.5 Hz), 0.76 (3H, d, J ) 6.5 Hz), 0.82-0.92 (1H, m), 1.36 (1H,
ddd, J ) 4.0, 11.5, 14.0 Hz), 1.51 (1H, ddd, J ) 4.0, 10.5, 14.0
Hz), 2.96 (2H, d, J ) 8.0 Hz), 4.17 (1H, dd, J ) 3.5, 11.5 Hz),
4.30 (1H, t, J ) 8.0 Hz), 5.06 (2H, s), 7.15-7.35 (10H, m).
Con clu sion s
Subtilisin-CLEC is a versatile catalyst that combines
high activity and stability in different reaction media. It
is useful for the syntheses of peptides and peptidomi-
metics, mild hydrolyses of amino acid and peptide
amides, enantio- and regioselective reactions, and trans-
esterifications. The combination of these features makes
subtilisin-CLEC a useful catalyst for organic synthesis
both in the laboratory and in large-scale applications.
Exp er im en ta l Section
NMR spectra were recorded on a Bruker 500 spectrometer.
1H-chemical shifts are reported relative to TMS at δ 0. HPLC
analyses were conducted with a Hewlett-Packard 1050 chro-
matograph. GC analyses were conducted with a Hewlett-
Packard 5890 chromatograph. CLEC-subtilisin and crude
subtilisin activities were assayed by hydrolysis of the substrate
N-R-p-tosyl-L-arginine methyl ester. Unit definition: 1 unit
converts 1 µmol of tosyl-L-arginine methyl ester per minute
at 25 °C, pH 8.0.
P r ep a r a tion of Su btilisin -CLEC. Alcalase (Novo Nord-
isk) from Bacillus licheniformis was crystallized and cross-
linked with glutaraldehyde according to the procedure reported
for subtilisin Carlsberg by Tu¨chsen and Ottesen22 with slight
modifications. Cross-linked enzyme crystals of subtilisin are
sold under the trade names Chir°CLEC-BL (for chiral resolu-
tions) and PeptiCLEC-BL (for peptide synthesis), respectively,
and are commercial products of Altus Biologics, Inc. (Cam-
bridge, MA).
N-Boc-L-Tyr -L-Leu -NH2 (CD3OD/TMS): 0.89 (3H, d, J )
6.0 Hz), 0.93 (3H, d, J ) 6.0 Hz), 1.39 (9H, s), 1.58 (2H, dd, J
) 6.5, 7.2 Hz), 1.61 (1H, m), 2.77 (1H, dd, J ) 8.5, 14.0 Hz),
2.98 (1H, dd, J ) 6.0, 14.0 Hz), 4.22 (1H, dd, J ) 6.0, 8.5 Hz),
4.36 (1H, t, J ) 7.2 Hz), 6.70 (2H, d, J ) 8.5 Hz), 7.06 (2H, d,
J ) 8.5 Hz).
N-cbz-L-P h e-L-P h e-NH2 (CD3OD/TMS): 2.76 (1H, dd, J )
9.2, 13.8 Hz), 2.91 (1H, dd, J ) 8.6, 13.8 Hz), 2.99 (1H, dd, J
) 5.8, 13.8 Hz), 3.14 (1H, dd, J ) 5.6, 13.8 Hz), 4.31 (1H, dd,
J ) 5.8, 9.2 Hz), 4.58 (1H, dd, J ) 5.6, 8.6 Hz), 4.99 (1H, d, J
) 12.5 Hz), 5.03 (1H, d, J ) 12.5 Hz), 7.15-7.35 (15H, m).
N-cbz-L-P h e-D-P h e-NH2 (CD3OD/TMS): 2.68 (1H, dd, J )
9.0, 13.8 Hz), 2.84 (1H, dd, J ) 8.8, 13.8 Hz), 2.85 (1H, dd, J
) 5.6, 14.0 Hz), 3.12 (1H, dd, J ) 5.1, 14.0 Hz), 4.27 (1H, dd,
J ) 5.6, 8.8 Hz), 4.60 (1H, dd, J ) 5.1, 9.0 Hz), 4.99 (1H, d, J
) 12.6 Hz), 5.02 (1H, d, J ) 12.6 Hz), 7.15-7.35 (15H, m).
Activity Assa y. Subtilisin-CLEC activity was assayed by
hydrolysis of the substrate N-R-p-tosyl-L-arginine methyl ester
(TAME). Assay conditions: 2 mmol of TAME (80 mM) in 25
mL of 0.3 M phosphate buffer (pH 7.5) was incubated with 2
mg of subtilisin-CLEC at room temperature. One unit was
definited as hydrolysis of 1 µmol substrate per minute per mg
of CLEC at room temperature. The activity of subtilisin-CLEC
used in here was 33-36 U. This catalyst was very stable in
storage. It did not lose activity after 6 months of storage at
room temperature.
P r ep a r a tion of Su r fa cta n t-Tr ea ted Su btilisin -CLEC.
Subtilisin-CLEC (20 g protein) suspended in 10 mM Tris, 10
mM CaCl2, pH 7.0 was transferred to a sintered glass funnel
(porosity ∼5 µm). The buffer above the CLECs was decanted
or removed by suction. The equal volume of 2-butanone
containing 30 g of the detergent Tergitol Type 15-S-3 (poly-
(glycol ether)) was added to the CLECs cake. The solvent and
surfactant were removed by gentle suction. The mixture was
transferred to a fritted pressure filter funnel after breaking
up any lumps and dried in a stream of nitrogen to a water
content of about 2-3% as determined by Karl Fisher titration.
Gen er a l P r oced u r es for th e Syn th esis of P ep tid es. To
a solution of 0.3 mmol of N-protected amino acid ester (acyl
donor) and 0.9 mmol of amino acid amide (or other nucleo-
philes) in 15 mL of acetonitrile was added 100 mg of dry
subtilisin-CLECs. The resulting mixture was incubated on a
rotary shaker at 40 °C. The reaction was monitored by HPLC.
After the acyl donor had disappeared, the catalyst was
removed by filtration. The excess nucleophile was removed
by washing with 1 N HCl, and the products were then
separated by silica gel column chromatography or by recrys-
tallization.
N-cbz-L-Va l-L-P h e-L-Leu -NH2 (CD3OD/TMS): 0.85 (6H, d,
J ) 7.0 Hz), 0.87 (3H, d, J ) 6.0 Hz), 0.91 (3H, d, J ) 6.0 Hz),
1.57 (2H, dd, J ) 6.0, 7.5 Hz), 1.61 (1H, m), 1.96 (1H, m), 2.97
(1H, dd, J ) 8.0, 13.5 Hz), 3.13 (1H, dd, J ) 6.5, 13.5 Hz),
3.87 (1H, d, J ) 7.0 Hz), 4.33 (1H, t, J ) 7.5 Hz), 4.63 (1H, dd,
J ) 6.5, 8.0 Hz), 5.04 (1H, d, J ) 12.0 Hz), 5.10 (1H, d, J )
12.0 Hz), 7.15-7.35 (10H, m).
N-cbz-L-Va l-L-P h e-L-P h e-NH2 (CD3OD/TMS): 0.85 (6H, d,
J ) 7.0 Hz), 1.95 (1H, m), 2.75 (1H, dd, J ) 9.2, 13.8 Hz), 2.91
(1H, dd, J ) 8.6, 13.8 Hz), 2.99 (1H, dd, J ) 5.8, 13.8 Hz),
3.14 (1H, dd, J ) 5.6, 13.8 Hz), 3.87 (1H, d, J ) 7.0 Hz), 4.31
(1H, dd, J ) 5.8, 9.2 Hz), 4.58 (1H, dd, J ) 5.6, 8.6 Hz), 5.04
(1H, d, J ) 12.0 Hz), 5.10 (1H, d, J ) 12.0 Hz), 7.15-7.35
(15H, m).
N-cbz-L-P h e-L-P h e-L-Leu -NH2 (CD3OD/TMS): 0.88 (3H,
d, J ) 6.5 Hz), 0.92 (3H, d, J ) 6.5 Hz), 1.57 (2H, dd, J ) 6.0,
7.5 Hz), 1.60 (1H, m), 2.75 (1H, dd, J ) 9.2, 13.8 Hz), 2.90
(1H, dd, J ) 8.8, 13.8 Hz), 2.99 (1H, dd, J ) 5.8, 13.8 Hz),
3.12 (1H, dd, J ) 5.8, 13.8 Hz), 4.33 (1H, t, J ) 7.5 Hz), 4.36
(1H, dd, J ) 5.8, 9.2 Hz), 4.63 (1H, dd, J ) 5.8, 8.8 Hz), 4.99
(1H, d, J ) 12.5 Hz), 5.03 (1H, d, J ) 12.5 Hz), 7.15-7.35
(15H, m).
N-Boc-L-Na p -L-Leu -NH2 (CD3OD/TMS): 0.87 (3H, d, J )
5.2 Hz), 0.89 (3H, d, J ) 5.2 Hz), 1.32 (9H, s), 1.58 (3H, m),
3.03 (1H, dd, J ) 9.0, 13.5 Hz), 3.28 (1H, dd, J ) 5.5, 13.5
Hz), 4.39 (1H, m), 4.41 (1H, dd, J ) 5.5, 9.0 Hz), 7.38-7.48
(3H, m), 7.71 (1H, s), 7.76-7.83 (3H, m).
(20) After each reaction cycle, the catalysts were filtered off and
washed with 3-methyl-3-pentanol. The washed catalysts were directly
used in the next cycle.
(21) The dry crude alcalase was prepared according to the procedure
described in ref 12b.
(22) Tu¨chsen, E.; Ottesen, M. Carlsberg Res. Commun. 1977, 42,
407.
N-cbz-L-P h e-L-Leu -OtBu (CD3OD/TMS): 0.89 (3H, d, J )
6.5 Hz), 0.93 (3H, d, J ) 6.5 Hz), 1.40 (9H, s), 1.58-1.62 (3H,