A R T I C L E S
Wood et al.
N3-Nle-AMCA-Wang resin were added alkyne (0.02 M, 2.0 equiv),
i-Pr2EtN (1 M, 100 equiv), and CuI (0.0025 M, 0.25 equiv) in THF,
and the mixture was shaken for 20-48 h. The solution was removed,
the resin was washed with 20 mL of THF, and the process was repeated.
After removal of the solution, the resin was washed with three portions
(20 mL) each of THF, CH3OH, THF, and CH2Cl2, and then the product
was cleaved from the solid support [see General Procedure for Support
Cleavage and Purification of Product].
portion of the cleavage solution and three portions of CH2Cl2. The
combined washes were concentrated under reduced pressure. The crude
product mixture was purified by HPLC [preparatory reverse phase C18
column (24.1 × 250 mm), acetonitrile/H2O-0.1% CF3CO2H; 5-95%
over 50 min; 8 mL/min; 254 nm detection for 60 min] and either
lyophilized or extracted with ethyl acetate followed by solvent removal.
The identity and purity of each compound was confirmed by LCMS
analysis of the purified material.
General Procedure for Synthesis of Triazole Substrates Listed
in Entries 5-12 (Table 1). To the cartridge containing the pre-swollen
N3-Nle-AMCA-Wang resin (0.10-0.15 mmol) were added the HCl
salt of (S)-3-amino-3,4-dimethyl-1-pentyne (0.02 M, 1.5 equiv), i-Pr2-
EtN (1.3 M, 100 equiv), and CuI (0.04 M, 3.0 equiv) in THF, and the
mixture was shaken for 20-48 h. After removal of the solution, the
resin was washed with three portions (25 mL) each of THF, CH3OH,
acetonitrile, and THF. After solvent removal, i-Pr2EtN (8 equiv) was
added to the derivatized resin. To a THF solution of carboxylic acid
(0.1 M, 3.5 equiv) and triphosgene (0.03 M, 1.1 equiv) was added 2,4,6-
collidine (0.3 M, 10 equiv). The resulting slurry was stirred for 1 min
and then was added to the cartridge containing the derivatized resin.
The resulting mixture was shaken for 4-12 h. After removal of the
solution, the resin was washed with 25 mL of THF, and the coupling
was repeated two more times. After removal of the solution, the resin
was washed with three portions (25 mL) each of THF, CH3OH, THF,
and CH2Cl2, and then the product was cleaved from the solid support
[see General Procedure for Support Cleavage and Purification of
Product].
General Procedure for the Synthesis of Phenoxyacetyl Substrates
(Table 2). Loading of Chloroacetic Acid. A 20% solution of piperidine
(vol/vol) in DMF (17 mL) was added to a 40 mL cartridge containing
2.0 g (1.1 mmol, 0.553 mmol/g) of Fmoc-AMCA-Wang resin. The
mixture was shaken for 15 min, and the resin was washed with three
portions (20 mL) each of DMF, CH3OH, and CH2Cl2. After addition
of 20 mL of CH2Cl2-pyridine (20:1, vol/vol) and chloroacetic acid
anhydride (752 mg, 4.4 mmol), the reaction mixture was agitated for
2 h at room temperature, and then the resin was washed with two
portions (20 mL) each of DMF, water, DMF, and ethyl ether. The resin
was finally dried under reduced pressure to give the desired resin-bound
chloroacetamide-AMCA.
General Synthesis of 1,2,3-Triazole-aldehyde Inhibitors (Entries
1-5, Table 3). The HCl salt of (S)-3-amino-3,4-dimethyl-1-pentyne
was acylated with the appropriate benzoic anhydride derivative. A Cu-
(I)-catalyzed 1,3-dipolar cycloaddition between the terminal alkyne of
the benzamide product and N3-Nle-OH was next performed. The
carboxylic acid of the resulting 1,2,3-triazole derivative was then
methylated with diazomethane, and the resulting methyl ester was then
reduced with DIBAL to give the corresponding aldehyde. For the
synthesis of the aldehyde inhibitor listed in entry 5 (Table 3), it was
necessary to first protect (S)-3-amino-3,4-dimethyl-1-pentyne with Boc-
anhydride, followed by performing the copper-catalyzed 1,3-dipolar
cycloaddition with norleucine azide methyl ester (N3-Nle-OCH3). The
Boc group on the amine of the resulting triazole product was cleaved
with CF3CO2H, and the resulting amine was then acylated with the
acid chloride of thiophene-3-carboxylic acid. The methyl ester was
reduced with DIBAL to give the aldehyde inhibitor. Due to facile
aldehyde to enol tautomerization, significant epimerization of the
aldehyde inhibitors occurred upon reverse-phase HPLC purification and
isolation [detailed procedures and full analytical characterization are
provided in the Supporting Information].
General Synthesis of Phenoxyacetyl Aldehyde Inhibitors (Entries
1-4, Table 4). The appropriate 2-hydroxybiphenyl derivative, which
for the inhibitors listed in entries 1-4 (Table 4) was prepared by Suzuki
reaction between 2-bromophenol and the appropriate aryl boronic acid,
was coupled with ethyl bromoacetate under basic conditions followed
by reduction with LiAlH4. Dess-Martin oxidation of the resulting
alcohol provided the desired aldehyde inhibitor, which was purified
either by alumina or by silica gel chromatography [detailed procedures
and full analytical characterization are provided in the Supporting
Information].
Loading of Phenol Derivatives. To resin-bound chloroacetamide-
AMCA (0.1 mmol) was added a solution of the appropriate phenol
derivative (5 equiv) and 2-tert-butylimino-2-diethylamino-1,3-dimethyl-
perhydro-1,3,2-diazaphosphorine (3 equiv) in THF (2 mL). After being
stirred gently at 70 °C for 15-18 h, the resin was transferred into a 10
mL polypropylene cartridge and subsequently rinsed with two portions
(2 mL) each of DMF, CH3OH, and CH2Cl2 to give the desired resin.
Suzuki Coupling (Table 2, Entries 5-8). To the resin obtained
above using 2-bromophenol (0.1 mmol) was added either DME (2 mL),
a 2 M aqueous solution of Na2CO3 (0.25 mL, 0.5 mmol), arylboronic
acid (0.5 mmol, 5 equiv), and tetrakis(triphenylphosphine)palladium
(11 mg, 0.01 mmol) or potassium phosphate (106 mg, 0.5 mmol),
arylboronic acid (5 equiv), tris(dibenzylideneacetone)dipalladium(0)
chloroform adduct (10 mg, 0.01 mmol), 2-(dicyclohexylphosphino)-
biphenyl (14 mg, 0.04 mmol), and dioxane (2 mL). After being purged
with N2, the capped reaction vessel was placed in a 80-90 °C oil bath,
and the mixture was gently stirred for 15-18 h. The resin was
transferred into a 10 mL polypropylene cartridge and subsequently
rinsed with two portions (2 mL) each of DMF, water, CH3OH, and
CH2Cl2 to give resin-bound biaryloxyacetamide-AMCA. The product
was then cleaved from support according to the general cleavage and
purification conditions.
General Assay Procedure. The proteolytic cleavage of N-acyl
aminocoumarins by cathepsin S was conducted in DYNATECH
Microfluor fluorescence 96-well microtiter plates (black plates), and
readings were taken on a Molecular Devices Spectra Max Gemini XS
instrument. The excitation wavelength was 370 nm, and the emission
wavelength was 455 nm with a cutoff of 435 nm. For CbzLeuArg-
AMC, an excitation wavelength of 355 nm and an emission wavelength
of 450 were used. The assay buffer consisted of a 100 mM solution of
pH 6.1 sodium phosphate buffer with 100 mM sodium chloride, 1 mM
of DTT, 1 mM of EDTA, and 0.001% Tween-20.
Assay Procedure for N-Acyl Aminocoumarin Substrates. Assays
were conducted at 37 °C in duplicate with and without the enzyme. In
each well were placed 38 µL of enzyme solution and 2 µL of a DMSO
solution of an N-acyl aminocoumarin. For assays longer than 10 min,
SealPlate (adhesive sealing films for microplates) was used to seal the
plate between readings, to prevent evaporation. Relative fluorescent
units (RFU) were measured at regular intervals over a period of time
(maximum 4 h). A plot of RFU versus time was made for each library
member with and without cathepsin S.
To determine the Km and kcat of selected substrates, assays were
conducted at 37 °C in duplicate with and without the enzyme at six
substrate concentrations above and below the Km of each substrate. In
each well were placed 38 µL of enzyme solution and 2 µL of a DMSO
substrate solution. RFU were measured at regular intervals over a period
of time (maximum 1 h). A plot of RFU/s versus substrate concentration,
General Procedure for Support Cleavage and Purification of
Product. The resin was swollen in CH2Cl2. To the swollen resin was
then added a 5 mL solution of 9:1 CH2Cl2:(95% CF3CO2H, 2.5% H2O,
2.5% triisopropylsilane). The mixture was shaken or allowed to sit for
1-2 h. Upon removal of the solution, the resin was washed with one
analyzed using KaleidaGraph, was used to determine Km and Vmax
.
9
15526 J. AM. CHEM. SOC. VOL. 127, NO. 44, 2005