Inhibitor-Tethered Resin for Detection of ActiVe Matrix Metalloproteinases
6.42 (s, 1H), 7.07-7.10 (m, 2H), 7.12-7.19 (m, 3H), 7.62 (s, 1H),
Route A (Scheme 3). Commercial epoxy-activated Sepharose
8.56 (d, J ) 8.8 Hz, 1H), 11.5 (bs, 1H); 13C NMR (126 MHz,
CDCl3): δ 22.7, 22.8, 26.0, 26.6, 39.6, 41.9, 43.4, 54.7, 126.9,
127.4, 128.6, 129.5, 136.5, 139.2, 169.9, 173.2, 173.7; LC/MS m/z
345 (M - H)-; HRMS (FAB) for C19H26N2O4 [M + H]+ calcd
347.1971, found 347.1990.
6B resin (560 mg) was washed thoroughly with distilled water (500
mL) before use. The swollen resin was placed in a flask and purged
with argon. A solution of CH3COSH (0.5 mL) in 50% MeOH (5
mL) was added to the resin under argon. The resulting suspension
was swirled using a gyratory water-bath shaker under argon at 25
°C for 1 day. The thioacetyl-modified resin 19 was filtered and
washed with methanol (200 mL) and then with water (200 mL).
To generate the intermediate 18, a solution of compound 17 (180
mg, 0.4 mmol) and K2CO3 (56 mg, 0.4 mmol) in 50% MeOH (1
mL) was sonicated under argon for 5 min and then was transferred
via cannula to the swollen resin 19 followed by the addition of the
potassium carbonate solution pH 10.8 (15 mL). The suspension
was vigorously swirled for 15 min. Subsequently, oxygen was
bubbled into the reaction vessel to purge the residual inert gas from
the reaction mixture, and after overnight oxidative treatment the
modified Sepharose resin 2 was filtered, washed with methanol (5
× 40 mL), followed by 100 mM sodium acetate, 0.5 M NaCl, pH
4.0 (50 mL each, 5×). Finally, the resin was washed with 0.5 M
NaCl (100 mL) containing 1% sodium azide and was stored at 5
°C in the same solution.
Route B (Scheme 3). Commercial epoxy-activated Sepharose
6B (560 mg) resin was washed thoroughly with distilled water (500
mL) before use. The swollen resin was placed in a flask and purged
with argon. A solution of CH3COSH (0.5 mL) in 50% MeOH (5
mL) was added to the resin under argon. The resulting suspension
was swirled in a gyratory water-bath shaker under argon at 25 °C
for 1 day. The resulting resin 19 was filtered and washed with
methanol (200 mL) and then with water (200 mL), followed by
the addition of the potassium carbonate solution pH 10.8 (5 mL)
to the resin under argon. The resulting suspension was swirled under
argon at 25 °C for 1 h and washed with methanol (200 mL) and
then with CH2Cl2/MeOH (1:1, 200 mL). To generate the Sepha-
rose-supported disulfide 21, a solution of 2,2′-dithiobis(5-nitropy-
ridine) (DTNB; 310 mg, 1 mmol) in CH2Cl2 (1 mL) was transferred
via cannula to the swollen Sepharose 6B resin 20, and the mixture
was swirled in a gyratory water-bath shaker under argon at 25 °C
for 1 day. The resin was filtered, washed with CH2Cl2/MeOH (1:
1, 200 mL). To generate the intermediate 15, a solution of
compound 14 (174 mg, 0.4 mmol) and K2CO3 (56 mg, 0.4 mmol)
in 50% methanol (1 mL) was sonicated for 10 min, and the resultant
mixture was transferred via cannula to the modified resin 21
followed by the addition of TFA (40 µL, 0.5 mmol). The suspension
was swirled under argon at 25 °C for 1 day. The inhibitor-tethered
resin 3 was filtered and washed with methanol (5 × 40 mL),
followed by 100 mM sodium acetate, 0.5 M NaCl, pH 4.0 (50 mL
each, 5 times). Finally, the resin was washed with 0.5 M NaCl
(100 mL) containing 1% sodium azide and was stored at 5 °C in
the same solution.
(2S,3R)-3-[1-(Methylcarbamoyl)-(2S)-2-phenylethylcarbam-
oyl]-2-(2-oxopropyl)hexanoic Acid (14). Compound 13 (0.69 g,
2 mmol) in THF (5 mL) was treated with thioacetic acid (210 µL,
3 mmol) and two drops of a 1:1 solution of triethylamine in THF.
The mixture was stirred at room temperature overnight under an
atmosphere of nitrogen, followed by the addition of hexane (25
mL) to yield a white solid 14 (0.75 g, 89%): 1H NMR (compound
14, 500 MHz, DMSO-d6) δ 0.69 (d, J ) 6.4 Hz, 3H), 0.75 (d, J )
6.4 Hz, 3H), 0.81-0.87 (m, 1H), 1.21-1.27 (m, 1H), 1.40-1.46
(m, 1H), 2.21 (s, 3H), 2.25-2.31 (m, 2H), 2.38-2.43 (m, 1H),
2.45-2.49 (m, 1H), 2.52 (d, J ) 4.5 Hz, 3H), 2.74 (dd, J ) 13.5,
10.2 Hz, 1H), 2.90 (dd, J ) 13.6, 4.8 Hz, 1H), 4.48-4.53 (m, 1H),
7.06 (t, J ) 7.2 Hz, 1H), 7.14 (t, J ) 7.4 Hz, 2H), 7.18-7.22 (m,
2H), 7.82 (d, J ) 4.6 Hz, 1H), 8.31 (d, J ) 8.5 Hz, 1H), 12.2 (s,
1H); 13C NMR (126 MHz, DMSO-d6) δ 22.2, 24.5, 25.8, 26.2,
29.1, 31.0, 38.2, 47.0, 49.2, 54.7, 126.8, 128.6, 129.7, 138.5, 172.0,
172.4, 174.6, 194.5; LC/MS m/z 423 [M + H]+; HRMS (FAB) for
C21H30N2O5S [M + H]+ calcd 423.1954, found 423.1938.
(2R)-N1-[1-(Methylcarbamoyl)-(2S)-2-phenylethyl)-N4-hydroxy-
2-isobutyl-3-methylenesuccinamide (16). To an ice-cold solution
of 13 (3.4 g, 10 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, 1.6 g, 10 mmol) in dry DMF (40 mL) was added 2,4,6-
trichloro[1,3,5]triazine (TCT, 0.76 g, 4.2 mmol), and the mixture
was stirred under N2 for 1 h. The resulting mixture was added by
syringe to a flask containing a suspension of hydroxylamine
hydrochloride (3.0 g, 40 mmol) and Et3N (5.6 mL, 40 mmol) in
DMF. The mixture was then stirred for 2 h at room temperature.
Removal of solvent in vacuo gave an oil that was copiously washed
with water to give a solid material, which was then extracted into
CH2Cl2. The extract was dried over MgSO4 and concentrated on a
rotary evaporator to give 16 (2.9 g, 82%): 1H NMR (compound
16, 600 MHz, 10% CD3OD in CDCl3) δ 0.65 (d, J ) 5.3 Hz, 3H),
0.66-0.73 (m, 3H), 1.24 (d, J ) 2.9 Hz, 2H), 1.37 (m, 1H), 2.52-
2.59 (m, 4H), 2.76 (s, 1H), 2.96 (d, J ) 7.8 Hz, 1H), 3.15-3.23
(m, 1H), 4.38 (s, 1H), 5.18 (s, 1H), 5.48 (s, 1H), 6.98-7.05 (m,
3H), 7.09 (t, J ) 6.8 Hz, 2H), 7.17 (d, J ) 6.8 Hz, 1H); 13C NMR
(75 MHz, MeOD-d4) δ 22.8, 22.9, 26.4, 27.0, 39.0, 41.5, 46.5, 56.2,
126.8, 127.7, 129.4, 130.3, 138.4, 141.3, 170.4, 174.0, 175.2; LC/
MS m/z 362 [M + H]+; HRMS (FAB) for C19H27N3O4 [M + H]+
calcd 362.2080, found 362.2101.
S-(2S,3R)-3-[1-(Methylcarbamoyl)-(2S)-2-phenylethylcarbam-
oyl]-2-(hydroxycarbamoyl)-5-methylhexyl ethanethioate (17).
The derivative 16 (0.72 g, 2 mmol) in THF (5 mL) was treated
with thioacetic acid (420 µL, 6 mmol) and two drops of a 1:1
solution of triethylamine in THF. The mixture was stirred at room
temperature for 1 day under an atmosphere of nitrogen, followed
by removal of solvents to yield a white solid 17 (0.76 g, 87%),
which was purified by HPLC. Reversed-phase semipreparative
HPLC purification was performed using DeltaPak C-18 column
(300 mm × 19 mm) and monitored with a Waters 2996 photodiode
array detector: 1H NMR (compound 17, 500 MHz, 3% CD3OD in
CDCl3) δ 0.74-0.82 (m, 6H), 1.03 (ddd, J ) 13.3, 9.9, 3.6 Hz,
1H), 1.34 (td, J ) 6.5, 3.1 Hz, 1H), 1.59-1.68 (m, 1H), 2.23-
2.28 (s, 3H), 2.70-2.79 (m, 6H), 2.98-3.07 (m, 1H), 3.09-3.18
(m, 1H), 4.84-4.90 (m, 1H), 7.01 (d, J ) 4.8 Hz, 1H), 7.15 (dt, J
) 8.6, 4.3 Hz, 1H), 7.19-7.25 (m, 4H), 7.71 (d, J ) 8.6 Hz, 1H);
13C NMR (126 MHz, 3% MeOD-d4 in CDCl3) δ 21.2, 23.3, 25.6,
25.9, 28.2, 30.3, 31.2, 39.5, 46.5, 47.3, 54.3, 126.6, 128.3, 129.0,
136.8, 171.6, 172.4, 173.3, 194.5; LC/MS m/z 438 [M + H]+;
HRMS (FAB) for C21H31N3O5S [M + H]+ calcd 438.2063, found
438.2083.
Binding of MMP-2 to the Inhibitor-Tethered Resin. Recom-
binant pro-MMP-2 was activated by incubation with 1 mM
p-aminophenylmercuric acetate (APMA), followed by dialysis
against CB buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 5 mM
CaCl2 and 0.02% Brij 35) to remove APMA as previously
described.28 Active MMP-2 (100 nM in CB buffer) was incubated
at room temperature without or with 10 µL of the inhibitor-tethered
resin (0.5 mL final volume). At various times (5, 30, and 60 min),
the samples were centrifuged at 15000g for 1 min. Aliquots of the
supernatants (unbound fractions) were collected, and enzyme
activity was measured using the fluorescence-quenching peptide
substrate (MOCAc-Pro-Leu-Gly-Leu-A2pr (Dnp)-Ala-Arg-NH2).
Substrate hydrolysis was monitored for 15 min by fluorescence
spectrophotometer at excitation and emission wavelengths of 328
and 393 nm, respectively, and the percentages of remaining activity
were compared. The total of MMP-2 activity (100%) was attributed
Preparation of the Inhibitor-Tethered Resins 2 and 3. Two
different synthetic approaches were attempted, as follows:
(28) Fridman, R.; Toth, M.; Pena, D.; Mobashery, S. Cancer Res. 1995,
55, 2548-2555.
J. Org. Chem, Vol. 71, No. 16, 2006 5853