264
Vol. 56, No. 3
(neat) cmꢄ1: 3400—2800; ESI-MS (m/z): 200.13 (MꢅH)ꢅ.
catalysts were removed by filtration through Celite, dry ether was added to
precipitate crude peptide. The peptide was purified by HPLC (Inertsil
WP300C18 column, 20ꢂ250 mm, GL Sciences), eluted with a linear gradi-
ent of 15—30% acetonitrile in 0.1% aqueous trifluoroacetic acid in 130 min
at a flow rate of 10 ml/min. Fractions containing the peptide were collected,
and the solvent was removed by lyophilization to give 1 (6.0 mg, 62.3%) as a
tert-Butyl [(1S)-2-Cyano-1-(1H-indol-3-ylmethyl)ethyl]carbamate (21)
Sodium bicarbonate (282 mg, 3.35 mmol) in water (5.0 ml) and di-tert-butyl
dicarbonate (491.3 mg, 2.25 mmol) in THF (5.0 ml) were added to com-
pound 20 (335.1 mg, 1.68 mmol) in an ice bath. After stirring at room tem-
perature overnight, the solvent was concentrated and ethyl acetate was
added. The mixture was washed with 10% aqueous citric acid, then dried
and evaporated in vacuo. The residue was purified by silica gel chromatogra-
phy (chloroform : acetoneꢃ20 : 1) to afford 21 (278.8 mg, 55.4%) as a brown
1
brown solid. H-NMR (CD3OD) d (ppm): 2.20—3.30 (16H, m), 3.75 (1H,
m), 4.45 (2H, m), 6.98—7.10 (3H, m), 7.23—7.36 (7H, m), 7.56 (1H, d,
Jꢃ7.6 Hz), 8.65 (1H, s); ESI-MS (m/z): 602.42 (MꢅH)ꢅ.
1
H-His-Trp-Gly-Phe-OH (2) The protected peptidyl resin was con-
structed using an Fmoc-based solid phase methodology on a Wang resin.
Triethylsilane (60 ml), trifluoroacetic acid (4.9 ml), and distilled water (60 ml)
were added to the peptidyl-resin (104 mg), and the mixture was stirred at
room temperature for 2 h. After the resins were removed by filtration, dry
ether was added to precipitate a crude peptide. The peptide was purified by
HPLC (Inertsil WP300C18, 20ꢂ250 mm), eluted with a linear gradient of
15—22% acetonitrile in 0.1% aqueous trifluoroacetic acid in 40 min at a
flow rate of 16 ml/min. Fractions containing the peptide were collected, and
the solvent was removed by lyophilization to give 2 (8.8 mg) as a white
solid. 1H-NMR (CD3OD) d (ppm): 2.95—3.30 (4H, m), 3.62—3.99 (2H,
m), 4.09 (1H, m), 4.64—4.67 (2H, m), 6.99—7.34 (10H, m), 7.59 (1H, d,
Jꢃ8.0 Hz), 8.44 (1H, s); ESI-MS (m/z): 546.28 (MꢅH)ꢅ.
H-Phe-Gly-Trp-His-OH (3) Compound 3 was obtained as a white solid
according to the procedure described for 2. 1H-NMR (CD3OD) d (ppm):
2.97—3.27 (4H, m), 3.68—3.95 (2H, m), 4.06—4.10 (1H, m), 4.62—4.71
(2H, m), 6.97—7.10 (3H, m), 7.23—7.33 (7H, m), 7.56 (1H, d, Jꢃ7.6 Hz),
8.71 (1H, s); ESI-MS (m/z): 546.2 (MꢅH)ꢅ.
MMP-2 Inhibition Assay Gelatin (from bovine bone, Wako) was dis-
solved in phosphate buffer (0.3 M, pH 7.4) to a concentration of 1 mg/ml. To
this solution (100 ml) was added [125I]NaI (6.1 MBq, 3 ml, GE Healthcare)
and a chloramine T solution (1 mg/ml, 10 ml). After a 20-min reaction, the
solution was removed and eluted through a PD-10 column (GE Healthcare)
to separate non-reacted [125I]NaI, according to the manufacturer’s instruc-
tions. The radiochemical purity was determined to be ꢀ96% by TLC devel-
oped with acetone/water/n-butanol/ammoniaꢃ13 : 1 : 4 : 2.
solid, mp 48—50 °C. H-NMR (CD3OD) d (ppm): 1.45 (9H, s), 2.44—2.71
(2H, m), 3.02—3.20 (2H, m), 4.21 (1H, m), 4.77 (1H, bs), 7.13—7.25 (3H,
m), 7.39 (1H, d, Jꢃ8.4 Hz), 7.65 (1H, d, Jꢃ7.6 Hz), 8.10 (1H, s); IR (KBr)
cmꢄ1: 3350, 3100—2800, 1693; ESI-MS (m/z): 300.19 (MꢅH)ꢅ; [a]D27
ꢄ8.9° (cꢃ1.35, CH2Cl2).
Boc-b3-Trp-OH (22) To a solution of 21 (18 mg, 0.06 mmol) in
ethanol/H2Oꢃ5 : 1 (1.3 ml) was added potassium hydroxide (33.7 mg,
0.6 mmol). The mixture was heated under reflux for 9 h and concentrated in
vacuo. The reaction mixture, after addition of water (2 ml) and neutralization
with 10% aqueous citric acid, was extracted with ethyl acetate. The organic
layer was dried, concentrated in vacuo, and purified by silica gel chromatog-
raphy (chloroform : methanolꢃ20 : 1) to afford 22 (5.6 mg, 29.3%) as a pale
brown solid, mp 137—138 °C. 1H-NMR (CD3OD) d (ppm): 1.37 (9H, s),
2.39—2.59 (2H, m), 2.95 (2H, d, Jꢃ6.4 Hz), 4.19—4.22 (1H, m), 6.97—
7.09 (3H, m), 7.13 (1H, d, Jꢃ8.0 Hz), 7.60 (1H, d, Jꢃ8.0 Hz); IR (neat)
cmꢄ1: 3338, 3100—2800, 1683; ESI-MS (m/z): 319.2 (MꢅH)ꢅ; [a]D27 ꢄ8.9°
(cꢃ0.45, CH2Cl2).
Z-b3-Phe-b-Ala-OMe (23) Under argon, to a solution of b-alanine
methyl ester (22.3 mg, 0.16 mmol) in dry dichloromethane (0.5 ml) was
added triethylamine (0.12 ml, 0.8 mmol), 15 (50 mg, 0.16 mmol) in dry DMF
(0.5 ml), HOBt (29.4 mg, 0.19 mmol), and EDC (36.4 mg, 0.19 mmol) in an
ice bath. After stirring for 10 h at room temperature, chloroform (10 ml) was
added. The mixture was washed with a 1 M hydrogen chloride solution, a sat-
urated sodium hydrogencarbonate solution and brine, and then dried and
evaporated in vacuo. The residue was purified by chromatography on a silica
gel column (chloroform : methanolꢃ30 : 1) to afford 23 (35.1 mg, 55.1%) as
a white solid, mp 108—110 °C. 1H-NMR (CD3OD) d (ppm): 2.25—2.43
(2H, m), 2.52 (2H, t, Jꢃ6.0 Hz), 2.78—3.01 (2H, m), 3.46—3.51 (2H, q,
Jꢃ6.0 Hz), 3.68 (3H, s), 4.09—4.18 (1H, m), 5.07 (2H, s), 5.75 (1H, br s),
6.13 (1H, br s), 7.15—7.36 (10H, m); IR (KBr) cmꢄ1: 3312, 3032, 1693,
1636; FAB-MS (m/z): 399.2 (MꢅH)ꢅ; [a]D27 ꢅ10.5° (cꢃ0.21, CHCl3).
Boc-b3-Trp-b3-His-OBn (25) Compound 25 was obtained as a brown
solid in 57.6% yield, mp 92—94 °C, from 22 and 9 according to the proce-
Compound 1, 2, and 3 and cCTT were dissolved in DMF and diluted in
the assay buffer (pH 7.5, 50 mM Tris–HCl, 0.2 M NaCl, 5 mM CaCl2, 0.1%
Triton X-100) to the appropriate concentrations (0.001—1 mM final concen-
tration). These compounds at the desired concentrations were added to the
enzyme solution (active MMP-2, Calbiochem, 3 nM final concentration) and
incubated for 30 min at 37 °C. Finally, 125I-labeled gelatin (30 MBq) was
added to each assay and incubated for 1 h at 37 °C. The degradation of 125I-
labeled gelatin was determined by counting the radioactivity in the super-
natant after precipitation of the undegraded gelatin with 20% trichloroacetic
acid.
1
dure described for 23. H-NMR (CDCl3) d (ppm): 1.41 (9H, s), 2.22—2.36
(2H, m), 2.43—2.62 (2H, m), 2.78—3.04 (4H, m), 4.21 (1H, m), 4.52—4.57
(1H, m), 5.11 (2H, s), 5.52 (1H, br s), 6.67 (1H, bs), 6.74 (1H, s), 6.97 (1H,
d, Jꢃ2.0 Hz), 7.09 (1H, t, Jꢃ7.4 Hz), 7.17 (1H, t, Jꢃ7.6 Hz), 7.26—7.35
(5H, m), 7.51 (1H, s), 7.63 (1H, d, Jꢃ8.0 Hz), 8.16 (1H, s); IR (KBr) cmꢄ1
:
Acknowledgments This work was supported in part by the research
fund of Kyushu University Foundation.
3361, 2900, 1680; ESI-MS (m/z): 560.2 (MꢅH)ꢅ; [a]D27 ꢅ47.7° (cꢃ0.13,
CHCl3).
Z-b3-Phe-b-Ala-b3-Trp-b3-His-OBn (27) To a solution of 23 (285 mg,
0.72 mmol) in dry methanol (5.0 ml) was added a 1 M sodium hydroxide so-
lution (1.44 ml) in an ice bath. The mixture was stirred at room temperature
for 30 min, neutralized by a 1 M hydrogen chloride solution and then concen-
trated in vacuo. The residue was diluted with a saturated sodium hydrogen-
carbonate solution and washed with ether. After acidification by the addition
of a 1 M hydrogen chloride solution, the mixture was extracted by ethyl ac-
etate. The organic layer was dried, and evaporated in vacuo to obtain crude
dipeptide (Z-b3-Phe-b-Ala-OH, 24) (260.7 mg) as a white solid.
References
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A mixture of 25 (17 mg, 0.03 mmol) and trifluoroacetic acid (0.3 ml) was
stirred at room temperature for 30 min, and concentrated in vacuo. The
residue was dissolved in distilled water, and the solution was lyophilized to
give a crude dipeptide (H-b3-Trp-b3-His-OBn, 26) (9.9 mg) as a white solid.
Under argon, to a solution of 26 (15.0 mg, 0.033 mmol) in dry dichloro-
methane (0.5 ml) was added triethylamine (23 ml, 0.165 mmol), 24 (12.7 mg,
0.033 mmol) in dry DMF (0.5 ml), HOBt (7.7 mg, 0.05 mmol), and EDC
(9.6 mg, 0.05 mmol) in an ice bath. After stirring for 10 h at room tempera-
ture, chloroform (10 ml) was added. The mixture was washed with water,
and then dried and evaporated in vacuo. The residue was purified by chro-
matography on a silica gel column (chloroform : methanolꢃ30 : 1) to afford
27 (8.8 mg, 32.3%) as a white solid. ESI-MS (m/z): 826.50 (MꢅH)ꢅ; [a]D27
ꢄ8.0° (cꢃ0.20, CH3OH).
H-b3-Phe-b-Ala-b3-Trp-b3-His-OH (1) A mixture of 27 (13.6 mg,
0.016 mmol) and Pd–C (10%, ca. 15 mg) in dry DMF (0.7 ml) was stirred
at room temperature for 2.5 h under atmospheric pressure of H2. After the