Isothiazolones; Thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF

Article abstract of DOI:10.1039/c0ob00464b

Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent cysteine protease cathepsin B and the histone acetylt

Full text of DOI:10.1039/c0ob00464b

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PAPER
Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and
histone acetyltransferase PCAF†
Rosalina Wisastra,a Massimo Ghizzoni,a Harm Maarsingh,b Adriaan J. Minnaard,c Hidde J. Haismaa and
Frank J. Dekker*a
Received 21st July 2010, Accepted 8th December 2010
DOI: 10.1039/c0ob00464b
Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak
nitrogen–sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent
cysteine protease cathepsin B and the histone acetyltransferase p300/CBP associated factor (PCAF)
based on their substitution pattern. Furthermore, enzyme kinetics and mass spectroscopy indicate
covalent binding of a 5-chloroisothiazolone to cathepsin B, which demonstrates their potential utility as
probes for activity-based protein profiling.
Introduction
Functional groups that react covalently with thiolates have been
used extensively for activity-based protein profiling of cysteine-
proteases.1 This technology depends on covalent labelling of
active site thiolates of enzymes in cell-lysates. Labelling is often
performed with biotinylated functionalities, which can be em-
ployed for purification with immobilized streptavidin. Cleavable
inhibitor-biotin linkers have been developed to improve the recov-
ery of the proteins of interest.2 Our current study indicates that
isothiazolones and 5-chloroisothiazolones are excellent candidates
for this purpose, because the disulfides that are formed upon
reaction with active site thiolates (Scheme 1) can be cleaved under
mild conditions.
Scheme 1 Reaction of isothiazolones and 5-chloroisothiazolones with
propane-1-thiolate results in cleavage of the N–S bond. (a) propane-1-thiol,
Et3N, CH2Cl2 (b) extended reaction times resulted in hydrolysis of 7.
The activity of isothiazolones and 5-chloroisothiazolones is
expected to originate mainly from the reactivity of these hete-
rocycles towards thiolates.3 In a careful study, Alvarez-Sa´nchez
et al.4 described that N-methyl-5-chloroisothiazolone reacts with
oxygen and nitrogen nucleophiles via an addition–elimination in
the 5-position, thereby substituting the chloride. Treatment of
N-methyl-5-chloroisothiazolone with thiol-nucleophiles, however,
provided ring-opened thioester and dithioester products that can
not be explained this way. This indicates that the thiol nucleophiles
react through cleavage of the N–S bond (Scheme 1).
Cathepsin B is a biologically relevant enzyme that is impli-
cated in tumor formation, angiogenesis, tumor invasion and
metastasis.5,6 Furthermore, cathepsin B has been considered
a potential biomarker for prognosis in cancer.7 Biotinylated
irreversible inhibitors can be used as a probe in activity-based
protein profiling of cathepsin B.8,9 In this study we identify
isothiazolones and 5-chloroisothiazolones as covalent inhibitors
of cathepsin B. Furthermore, we evaluated the selectivity of
cathepsin B inhibition compared to the histone acetyltransferase
(HAT) p300/CBP associated factor (PCAF). This enzyme plays
a role in the nuclear factor k B (NF kB) pathway, which is
an important regulator of several genes that are involved in
inflammation.10 Upon activation, NF kB binds to cofactors that
have intrinsic HAT activity, like PCAF, which in turn acetylate
lysines in core histone H4. This leads to a less compact chromatin
structure, which enables the transcription of proinflammatory
genes, including Interleukin 8 (IL-8).11,12 Isothiazolones and 5-
chloroisothiazolones (Scheme 1) are potent and cell-permeable
inhibitors of PCAF, and attempts to optimize their inhibitory
aDepartment of Pharmaceutical Gene Modulation, Groningen Research
Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1,
9713 AV, Groningen, The Netherlands. E-mail: f.j.dekker@rug.nl
bDepartment of Molecular Pharmacology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV,
Groningen, The Netherlands
cDepartment of Bio-organic Chemistry, Stratingh Institute for Chemistry,
University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Nether-
lands
† Electronic supplementary information (ESI) available. CCDC reference
numbers 763228 and 763229. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c0ob00464b
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activity have been made in several studies.13–17 However, their mode
of action and selectivity remain to be studied.
Herein we describe isothiazolones, 5-chloroisothiazolones and
5-chloroisothiazolone-1-oxides as new classes of cathepsin B
inhibitors. Selectivity between cathepsin B and PCAF inhi-
bition can be obtained by variation of the substitution. 5-
chloroisothiazolones are non-competitive inhibitors of cathepsin
B, and mass spectroscopy demonstrates covalent binding. The
reaction products of isothiazolones and 5-chloroisothiazolones
that are formed upon thiolate treatment proved to be disul-
fides, which could be advantageous in activity-based protein
profiling.
Results and discussion
Synthesis
Compounds 1, 5 and 11 were prepared by reaction of the corre-
sponding primary amines with 3,3¢-dithiobispropionyl chloride to
yield the 3,3¢-dithiobispropionic amides as described previously
for compounds 9, 10, 12–18 and 19–26.15,16 These amides were
treated with 3 equiv. of sulfuryl chloride in dichloromethane to
yield a mixture of the 5-chloroisothiazolone and isothiazolone,
which were separated by column chromatography.
Fig.
2 Crystal structure of (A) 2-phenylisothiazolone 1 and (B)
5-chloro-2-phenylisothiazolone 5. CCDC 763228 & 763229 contain the
supplementary crystallographic data for this paper†. These data can be
obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html.
Thiolate reactivity of (5-chloro)isothiazolones
The reaction products that were formed upon treatment with
thiolates were identified in order to study the thiol-reactivity of
isothiazolones and 5-chloroisothiazolones. Isothiazolones 1 and 2
were treated with 1.0 equiv. of propane-1-thiol and triethylamine in
dichloromethane (Scheme 1). The resulting products were isolated
and characterized by NMR and mass analysis. In substrate 1 the
numbering of the carbon atoms in the crystal structure deviates
from the IUPAC numbering in Scheme 1. The crystal structure
shows a planar isothiazolone ring for both 1 and 5. In comparison
with literature data (Table S1†) the C1–C2 bond length fits to a
double bond, C2–C3 fits to a single, conjugated bond and C3–O
to a double bond. This indicates that resonance structure I has a
major contribution to the structure and that the S–N bond length
has single bond character. Nevertheless, the C3–N bond is shorter
than expected for a single bond and the C1–S bond is also shorter
than observed for a single bond, which indicates that resonance
structures II and III also contribute to the structure.
The observation that thiol nucleophiles attack isothiazolones at
the sulfur atom can be explained by the strength of the formed
sulfur–sulfur bond and the weakness of the sulfur–nitrogen
bond. In contrast, isothiazolones are non-reactive towards oxy-
gen or nitrogen nucleophiles, whereas 5-chloroisothiazolones
react with these nucleophiles via addition–elimination at the
5-position.4
3
observed J(H4,H5) was 6.5 Hz, whereas in product 3 it amounted
3
to 14.1 Hz. Comparably, in 2 J(H4,H5) was 6.5 Hz, whereas it was
9.7 Hz in 4. This is a strong indication for an E-configuration of
the double bond in 3 and 4, which could be caused by a catalytic
amount of thiol that reacts via subsequent addition, rotation and
elimination. When 5-chloroisothiazolones 5 and 6 were subjected
to the same reaction conditions, 7 (Fig. S5†) was isolated upon
treatment of 5, however we were not able to isolate a comparable
product after treatment of 6. Upon extended reaction times for the
formation of 7, a new product 8 was isolated that was identified
1
as the hydrolysis product of 7. The H and 13C NMR spectra of
8 showed two isomers most likely due to keto–enol tautomerism
(Fig. S6†).
The reactivity of isothiazolones originates from the electronic
structure of the heterocycle, which is supposed to have considerable
aromatic character. This is illustrated by resonance structures
II and III in Fig. 1. To provide experimental evidence for the
aromaticity of isothiazolones, the crystal structures of 1 and 5
were resolved (Fig. 2, Table S1, CCDC 763228 and 763229†) The
Cathepsin B and PCAF inhibition
Cathepsin B inhibition was evaluated by the cleavage of 7-amino-
4-methyl coumarin (AMC) from the substrate Cbz-Arg-Arg-
AMC.18 Inhibitory concentration (IC50) values were derived after
15 min of pre-incubation with the inhibitors (Table 1). Since these
inhibitors are expected to interact covalently with the enzyme,
differences in potency reflect changes in enzyme binding as well as
differences in the reaction rate of covalent binding to the enzyme.
The assay for the HAT PCAF inhibition and the IC50 values of 9,
10, 12–18 and 19–26 have been reported previously.15,16
Disulfides 3, 4 and 7, isothiazolone 12 and 5-chloroisothia-
zolone 5 inhibited cathepsin B, whereas no inhibition of PCAF
Fig. 1 Three resonance structures of 1.
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Table 1 IC50 values for inhibition of cathepsin B and PCAF by isothia-
Table 2 Enzyme kinetics parameters for cathepsin B inhibition (n = 3)
zolones and 5-chloroisothiazolones and disulfides (Scheme 1)
[5] (mM)
Vmax (nmol s-1)
Km (mM)
KS (mM)
R2
R1 R2 R3
Cat Ba (mM) PCAFb (mM)
0
7
14
0.60 0.41
0.36 0.20
0.22 0.10
1.2 0.92
1.1 0.68
1.1 0.60
0.11 0.087
0.11 0.068
0.12 0.069
0.996
0.996
0.997
1
3
4
5
7
9
H H -phenyl
H H -phenyl
H H -(CH2)5CO2Me
Cl H -phenyl
Cl H -phenyl
>100
>10
>10
>10
>10
12
24
13
1
1
1
0.046 0.002 >10
>100 4.2 0.6
42
H H 3-Cl-4-F-phenyl
10 Cl H 3-Cl-4-F-phenyl
2
2.5 0.1
3.9 0.4
>10
2.9 0.3
1.8 0.2
2.6 0.6
>10
11 Cl H 2-Cl-4,5-dimethoxy phenethyl 4.1 0.2
12 H H -pentyl
13 Cl H -pentyl
14 Cl H -(CH2)2CO2Me
15 Cl Cl -(CH2)2CO2Me
16 Cl CH3 -(CH2)2CO2Me
17 Cl H -(CH2)2CO2Et
18 Cl H -(CH2)3CO2Me
13
7.3 0.5
12
8.5 0.7
1
1
11
14
11
1
2
1
2.0 0.2
2.5 0.3
a maximal concentration in the assay 100 mM. b maximal concentration in
the assays 10 mM.
Fig. 3 Conversion of the substrate Cbz-Arg-Arg-AMC by the enzyme
cathepsin B with no inhibitor present (᭡) showed substrate inhibition
at high substrate concentrations. The same was observed for the enzyme
was observed in the applied concentrations. Remarkably, disul-
fide 7 showed the highest potency for cathepsin B (46 nM).
Isothiazolone 9 inhibited HAT PCAF, whereas cathepsin B
was not inhibited, which demonstrated that selective PCAF
inhibition can be obtained with N-aryl substituents. This was
not observed for N-aliphatic substituted isothiazolones and 5-
chloroisothiazolones 12–18. Substitution in the isothiazolone
4-position (R2), in 14, 15 and 16, changed the cathepsin
B inhibition modestly. Remarkably, N-aliphatic substituted 5-
chloroisothiazolone 11 was more potent for cathepsin B than
the other N-aliphatic substituted 5-chloroisothiazolones. Further-
more, 5-chloroisothiazolone-1-oxides also inhibited cathepsin B
(Table S2†) and their potency also depended on their substi-
tution pattern. Taking these data together it is concluded that
isothiazolones, 5-chloroisothiazolones and 5-chloroisothiazolone-
1-oxides are inhibitors of cathepsin B and HAT PCAF and that
their potency and selectivity depends on the substitution at the 2-,
4- and 5-position.
To establish the mechanism of cathepsin B inhibition further, the
influence of inhibitors 5 and 14 on the Michaelis–Menten kinetics
for conversion of the substrate Cbz-Arg-Arg-AMC was deter-
mined. Cathepsin B conversion of Cbz-Arg-Arg-AMC showed
a strong substrate inhibition at higher concentrations (Fig. 3, S7
and S8†). Substrate inhibition of the enzyme activity was expected
to obey the model in Scheme 2 from which the kinetic parameters
could be derived by non-linear curve fitting of equation 1 (Fig. 4).
Inhibitor 5 caused a reduced Vmax and a constant Km and Ks
activity after pre-incubation with 7 mM inhibitor 5 ( ) and the enzyme
᭹
activity after pre-incubation with 14 mM inhibitor 5 (ꢀ). The data were
fitted to equation 1 (Fig. 4) and the parameters are shown in Table 2.
Fig. 4 Equation for non-linear curve fitting of enzyme kinetics that is
influenced by substrate inhibition according to the model in Scheme 219
v is the reaction velocity, Vmax is the maximal reaction velocity, [S] is
the substrate concentration and Km is the Michaelis–Menten constant.
Ks is the binding constant of a second substrate molecule to the enzyme
substrate complex resulting in inhibition of the enzyme activity.
(Table 2). The same was observed for 14 (Fig. S7, Table S3†). This
indicated non-competitive inhibition of the cathepsin B activity
by 5-chloroisothiazolones. Inhibition of cathepsin B activity by 5
proved to be time dependent (Table S4†) and dilution experiments
did not show reactivation of cathepsin B (Table S5†). Pre-steady
state kinetic analysis revealed a very fast inactivation of the enzyme
(50% inhibition after 20 s by 14 mM 5, Table 1). The IC50 value
proved to decrease slightly over time. This suggested a fast initial
binding event followed by a slow second binding event (Table
S4, S6†). Furthermore, mass-spectroscopy experiments showed an
increased mass after incubation of cathepsin B with the inhibitor
(Fig. S9, S10†). The mass spectrometry data showed that the
inhibitor can bind one or two times to the enzyme. The mass
spectrum of the non-reacted enzyme (Fig. S9†) gave m/z 27960
as the [M+H]+ peak and 13985 as the [M+2H]2+ peak. The mass
spectrum of the enzyme pre-incubated with the inhibitor (Figure
S10†) gave m/z 28111 and 28354 as the [M+H]+ and 14082 and
14177 as the [M+2H]2+ peaks. The difference between reacted
and non-reacted enzyme was 151 and 394 for the [M+H]+ peaks
Scheme 2 Enzyme kinetics influence by substrate inhibition.
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and 97 and 192 for the [M+2H]2+ peaks. This indicates that 5-
chloroisothiazolone 5 binds once or twice to the enzyme to give
a product corresponding to 8, which would mean a mass increase
of 194 or 388. Taking these experiments together, it is concluded
that the 5-chloroisothiazolone 5 binds covalently to the thiolate in
the enzyme active site and/or to another surface exposed cysteine
of cathepsin B.
SO2Cl2 was added dropwise to the solution. The mixture was
stirred at 0 ◦C for 2 h. The mixture was washed with water
(2 times) and brine (1 time), dried using Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography.
5-chloro-2-phenylisothiazol-3(2H)-one (5)
The product was obtained using synthetic procedure 1. Pu-
rification was performed using column chromatography with
hexane : EtOAc 10 : 1 (v/v) as eluent. Yield 43%. Off-white solid.
Crystals (colorless needles) for X-ray diffraction◦were obtained
by crystallization from EtOAc : hexanes 1 : 1 at 4 C over 2 days.
Mp = 118.4 ◦C. Rf 0.52 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz,
CDCl3) d 6.35 (s, 1H), 7.27–7.55 (m, 5H). 13C NMR (50 MHz
CDCl3) d 114.9, 124.8, 127.8, 129.5, 135.7, 146.4, 165.4. MS
(ESI) m/z 211.9 [M+H]+. HRMS (m/z) 211.9931 [M+H]+, calcd
211.9931 C9H7ClNOS+.
Conclusion
In conclusion, isothiazolones and 5-chloroisothiazolones react
with thiolates by disulfide formation and cleavage of the nitrogen–
sulfur bond. The crystal structures illustrate the aromatic charac-
ter of isothiazolones. Whereas this aromaticity provides stability
to the compounds, the weak sulfur–nitrogen bond allows a fast
reaction with sulfur nucleophiles. Based on their reactivity, 5-
chloroisothiazolones and isothiazolones inhibit thiol containing
enzymes, such as the cysteine protease cathepsin B and HAT
PCAF. Their potency depends on the substitution pattern. Selec-
tivity for inhibition of HAT PCAF was observed for N-aryl sub-
stituted isothiazolone 9. Enzyme kinetics and mass spectroscopy
indicate covalent binding of 5-chloroisothiazolone 5 to the active
site thiol and/or to another surface exposed cysteine of cathepsin
B. The fact that isothiazolones and 5-chloroisothiazolones react
with thiolates to form disulfides makes them excellent candidates
for activity-based protein profiling of thiol-containing enzymes.
2-phenylisothiazol-3(2H)-one (1)
After elution of product 5 product 1 was eluted from the column
with hexane : EtOAc 1 : 1 (v/v) as eluent. The product was puri-
fied again using column chromatography using hexane : EtOAc
1 : 1 as eluent. Yield 29%. Off-white solid. Crystals (colorless
platelets) for X-ray diffraction were obtained by crystallization
from EtOAc : hexanes 3 : 4 at 4 ◦C over 2 days. Mp = 92.8 ◦C. Rf
0.16 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz, CDCl3) d 6.32 (d,
J = 6.2 Hz, 1H), 7.29–7.61 (m, 5H), 8.18 (d, J = 6.5 Hz). 13C NMR
(50 MHz CDCl3) d 114.8, 124.8, 127.5, 129.4, 136.5, 139.8, 167.6.
MS (ESI) m/z 178.1 [M+H]+. HRMS (m/z) 178.0321 [M+H]+,
calcd 178.0321 C9H8NOS+.
Experimental
General
All reagents and solvents were obtained from commercial suppliers
(Sigma-Aldrich, Acros Organics) and were used without further
purification. Dichloromethane was distilled over CaH2 before
use. Merck silica gel 60 F254 plates were used for analytical thin
layer chromatography (TLC) and spots were detected by UV
light, or stained using KMnO4 or ninhydrin solution. Column
5-chloro-2-(2-chloro-4,5-dimethoxyphenethyl)isothiazol-3(2H)-
one (11)
The product was obtained using synthetic procedure 1. Pu-
rification was performed using column chromatography with
hexane : EtOAc 1 : 1 (v/v) as eluent. Yield 6%. White-yellow solid.
Mp = 108.5 ◦C. Rf 0.20 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz,
CDCl3) d 3.04 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.86 (s, 3H), 3,95 (t,
J = 7.0 Hz, 2H), 6.25 (s, 1H), 6.68 (s, 1H), 6.86 (s, 1H). 13C NMR
(50 MHz CDCl3) d 32.8, 43.3, 56.0, 112.5, 113.2, 114.3, 124.9,
126.4, 145.8, 147.8, 148.5, 166.6. MS (ESI) m/z 334.3 [M+H]+.
HRMS (m/z) 334.0065 [M+H]+, calcd 334.0066 C13H14Cl2NO3S+;
chromatography was performed with MP Ecochrom silica gel 32–
1
˚
63, 60 A using the flash chromatography technique. H (200 MHz)
and 13C (50 MHz) NMR spectra were recorded on a Varian
Gemini 200 spectrometer in CDCl3 unless otherwise specified. 1H
chemical shifts are reported in ppm (d) downfield from internal
tetramethylsilane (TMS). 13C NMR spectra were recorded using
the attached proton test (APT) and chemical shifts are relative to
the solvent. Melting points were determined using an Electrocome
digital melting point measurement apparatus. High-resolution
mass spectra (HR-MS) were recorded using a flow injection
method on a LTQ-Orbitrap XL mass spectrometer (Thermo
Electron, Bremen, Germany) with a resolution of 60,000 at m/z
400. Protonated testosterone (lock mass m/z = 289.2162) was used
for internal recalibration in real time.
+
(m/z) 199.0521 [M-C3HClNOS]+, calcd 199.0526 C10H12ClO2 .
Synthetic procedure 2
The 5-chloroisothiazolone (1 mmol) was dissolved in 2 mL CH2Cl2
and cooled to 0 ◦C. A 0.5 M propane-1-thiol solution in CH2Cl2
(1 mL) was added, and the reaction was followed by TLC. A 0.5 M
triethylamine (TEA) solution in CH2Cl2 (1 mL) was added to the
mixture and reacted for another 30 min. The mixture was diluted
with 20 mL of CH2Cl2 and washed with H2O (2 times 50 mL),
brine (1 times 50 mL) and dried using Na2SO4.
The enzyme cathepsin B (human liver) and cathepsin Substrate
III, Fluorogenic (Z-RR-AMC) was obtained from Calbiochem.
7-amino 4-methyl coumarin was obtained from Sigma.
Synthetic procedure 1
N-phenyl-3-(propyldisulfanyl)acrylamide (3)
Isothiazolones and 5-chloroisothiazolones were synthesized ac-
cording to the procedure described by Dekker et al.15,16 The 3,3¢-
dithiobis-propionamide was dissolved in dry CH2Cl2 at 0 ◦C and
The product was obtained using synthetic procedure 2. Pu-
rification was performed using column chromatography with
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hexane : EtOAc 15 : 1 (v/v) as eluent. Yield 21%. White solid. Rf
0.70 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz, CD3CN) d 1.03
(t, J = 7.4 Hz, 3H), 1.72 (m, 2H), 2.80 (t, J = 7.2 Hz, 2H), 6.50 (d,
J = 14.1 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H)
7.51–7.64 (m, 3H), 8.50 (br. 1H). 13C NMR (50 MHz CD3CN)
d 13.2, 23.2, 40.4, 120.4, 121.2, 124.7, 129.8, 139.9, 142.9, 163.0.
MS (ESI) m/z 254.1 [M+H]+. HRMS (m/z) 254.0668 [M+H]+,
The cathepsin B enzyme (0.5 mg ml-1) was diluted 1 : 4000 with
cathepsin B assay buffer (100 mM Na2HPO4·2H2O; 1.25 mM
EDTA.2Na; pH = 6.8) containing 1 mM DTT. The inhibitor
(10 mM in CH3CN) was diluted with cathepsin B assay buffer
containing 0.1% (v/v) Triton-X to the desired concentration.
Per assay 50 mL inhibitor solution was mixed with 50 mL
enzyme solution and after 15 minutes incubation 50 mL substrate
solution was added. Calculations were performed with Excel
2003 and non-linear curve fitting was performed with Origin 7
software.
+
calcd 254.0668 C12H16NOS2 ; (m/z) 178.0323 [M-C3H7S]+, calcd
178.0321 C9H8NOS+.
Methyl 6-(3-(propyldisulfanyl)acrylamido)hexanoate (4)
Michaelis–Menten enzyme kinetics
The product was obtained using synthetic procedure 2. Pu-
rification was performed using column chromatography with
hexane : EtOAc 3 : 1 (v/v) as eluent. Yield 24%. White solid. Rf
0.36 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz, CD3CN) d 0.98 (t,
J = 7.2 Hz, 3H), 1.25–1.70 (m, 10H), 2.77 (t, J = 7.2 Hz, 2H), 3.10–
3.21 (m, 2H), 3.60 (s, 3H), 5.86 (d, J = 9.7 Hz, 1H), 6.59 (br. 1H),
6.99 (d, J = 9.7 Hz, 1H). 13C NMR (50 MHz CD3CN) d 12.2, 22.1,
24.3, 26.0, 28.9, 33.4, 38.6, 41.0, 50.9, 118.0, 152.1, 165.9, 173.8.
MS (ESI) m/z 306.0 [M+H]+. HRMS (m/z) 328.1009 [M+Na]+,
The enzyme kinetic experiments were performed using the same
assay setup as used for the IC50 determinations. Various con-
centrations of fluorogenic substrate Cbz-Arg-Arg-AMC (7.81–
1000 mM) were added to the enzyme. The fluorescence values were
converted to concentrations using a 7-amino 4-methyl coumarin
calibration curve (0.78–25 mM). The concentration change over
time is the reaction velocity (v) that is plotted against the substrate
concentration ([S]) in Fig. 3, S7, S8†. The experiments were
performed in triplicate and the average triplicate values and their
standard deviations are plotted. Calculations were performed with
Excel 2003 and non-linear curve fitting with equation 1 (Fig. 4)
was performed with Origin 7 software. The results are shown in
Table 2 and Table S3.†
+
calcd 328.1012 C13H23NNaO3S2 ; (m/z) 306.1191 [M+H]+, calcd
+
306.1192 C13H24NO3S2 ; (m/z) 230.0844 [M-C3H7S]+, calcd
230.0845 C10H16NO3S+.
N-phenyl-3-(propyldisulfanyl)-3-chloro-acrylamide (7)
The product was obtained using synthetic procedure 2. Pu-
rification was performed using column chromatography with
hexane : EtOAc 20 : 1 (v/v) as eluent. Yield 60%. White solid. Rf
0.67 (hexane : EtOAc 1 : 1). 1H NMR (200 MHz, CD3CN) d 0.99
(t, J = 7.4 Hz, 3H), 1.72 (m, 2H), 2.79 (t, J = 7.2 Hz, 2H), 6.56
(s, 1H), 7.10 (t, J = 7.4 Hz, 1H), 7.33 (m, 2H) 7.58 (d, J = 7.6 Hz,
2H), 8.50 (br. 1H). 13C NMR (50 MHz CD3CN) d 13.2, 22.7,
42.8, 120.3, 122.2, 125.0, 129.8, 139.4, 151.4, 163.3. MS (ESI) m/z
288.1 [M+H]+. HRMS (m/z) 288.0277 [M+H]+, calcd 288.0278
Mass spectrometry
Inhibitor 5 (50 mM) and cathepsin B (0.25 mg mL-1) were incu-
bated in phosphate buffer (100 mM Na2HPO4·2H2O; 1.25 mM
EDTA.2Na; pH = 6.8) for 20 min at room temperature. This
mixture was mixed 1 to 1 with the sinapinic acid matrix and
subjected to MALDI TOF mass spectroscopy.
+
C12H15ClNOS2 ; (m/z) 211.9931 [M-C3H7S]+, calcd 211.9931
C9H7ClNOS+.
Notes and references
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N-phenyl-3-(propyldisulfanyl)-3-hydroxy-acrylamide (8)
The product was obtained using synthetic procedure 2 with
overnight reaction time. Purification was performed using colomn
chromatography with hexane : EtOAc 5 : 1 (v/v) as eluent. Yield
30%. Yellow solid. Rf 0.57 (hexane : EtOAc 1 : 1). 1H NMR
(200 MHz, CDCl3) d 0.97 (m, 3H), 1.63 (m, 2H), 2.67 (m, 2H),
7.22 (m, 1H), 7.25 (m, 2H), 7.50 (m, 3H), 8.73 (br. 1H). 13C NMR
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