Chemical introduction of the green fluorescence: Imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore

Article abstract of DOI:10.1039/c3ob41341a

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

Full text of DOI:10.1039/c3ob41341a

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Chemical introduction of the green fluorescence:
imaging of cysteine cathepsins by an irreversibly
Cite this: DOI: 10.1039/c3ob41341a
locked GFP fluorophore†
Maxim Frizler,a Ilia V. Yampolsky,b Mikhail S. Baranov,b Marit Stirnberga and
Michael Gütschow*a
Received 30th June 2013,
Accepted 16th July 2013
An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evalu-
ated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable
for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.
DOI: 10.1039/c3ob41341a
www.rsc.org/obc
concept of ‘activity-based’ probes (ABPs) was applied to specifi-
cally address the active site of enzymes, using the example of
Introduction
Green fluorescent protein (GFP) from the jellyfish Aequorea the therapeutically relevant cysteine cathepsins.
victoria and related fluorescent proteins are widely used as
Human cysteine cathepsins (cat) belong to the subfamily of
universal genetically encoded fluorescent labels and have revo- papain-like cysteine proteases and are involved in a variety
lutionized the field of live cell imaging. Following protein of different (patho)physiological processes. In particular,
translation, the fluorophore, i.e. 4-(4-hydroxybenzylidene)-1H- cathepsins L, S, K and B are important subjects of several drug
imidazol-5(4H)-one (I, Fig. 1), is formed autocatalytically by discovery programs.15–18 Besides their importance in drug
cyclization of the protein backbone at positions 65–67 (Ser-Tyr- development, cysteine cathepsins are considered to be possible
Gly) and dehydrogenation of the Tyr side chain.1,2 Inspired by biomarkers for certain pathological conditions including
the GFP fluorophore, several studies have targeted its synthetic inflammation and cancer.19–24 Recently, remarkable scientific
analogues.3–10 The simple dimethyl compound II, identical to efforts have been made to develop sensitive methods for the
the native GFP core, does however not possess applicable detection of cysteine proteases using labeled inhibitors and
fluorescence properties. This is predominantly due to cis–trans fluorescently quenched substrates.24–33 Human cathepsin K,
isomerization and/or free rotation of the phenyl ring resulting playing a crucial role in bone remodeling and osteoporosis,
in a decay of the fluorophore’s excited state.11 However, when was chosen as a model enzyme in this study to demonstrate a
buried within the protective β-barrel structure of GFP, this useful procedure for the visualization of cathepsin activities by
internal conversion is suppressed. In the absence of the rigid the introduction of a green GFP-like label.
H-bonding protein environment, the radiationless decay
can be sufficiently prevented upon complexation with Zn2+ as
realized in III,12 or by introducing the BF2 entity leading to
irreversible locked analogues IV.13 Among the irreversibly
locked analogues, the phenol derivative V is structurally
closest to the native GFP fluorophore.14 Therefore, we have
selected the known fluorophore V to design a probe to exo-
genously label native proteins and to demonstrate the chemical
introduction of the green fluorescence. For this purpose, the
Results and discussion
The synthesis of the new fluorescent reporter 5, an irreversibly
locked analogue of the GFP fluorophore, is shown in Fig. 2.
The oxazolone derivative 14 was reacted with GABA under
basic conditions, followed by heating with Cs2CO3 in DMF.
The resulting compound 2 was treated with tert-butyldiphenyl-
chlorosilane (TBDPSCl) in the presence of N,N-diisopropylethyl-
amine (DIPEA) to protect both the phenolic and the carboxylic
group as tert-butyldiphenylsilyl ether/ester moieties. Com-
pound 3 was subsequently converted to the highly fluorescent
organoborane derivative 4 by consecutive reactions with BBr3,
EtOH and HF. The ethyl ester of 4 was finally saponified
leading to the green fluorophore 5, bearing a flexible linker
with a distal carboxyl group.
aPharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der
Immenburg 4, 53121 Bonn, Germany. E-mail: guetschow@uni-bonn.de;
Fax: +49 228 732567; Tel: +49 228 732317
bInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya
16/10, 117997 Moscow, Russia
†Electronic supplementary information (ESI) available: Inhibition assays for
cathepsins L, S, K, and B, spectral properties of 12, as well as 13C NMR spectra of
2, 4, 5, 7, 8, 9, 11, and 12. See DOI: 10.1039/c3ob41341a
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Fig. 1 The native GFP fluorophore and its synthetic analogues.
Fig. 2 Synthesis of the probe 12. (a) GABA, Et3N, EtOH, H2O, rt; (b) Cs2CO3, DMF, Δ; (c) TBDPSCl, DIPEA, imidazole, THF, rt; (d) BBr3, molecular sieves, CH2Cl2, rt;
(e) EtOH, THF, rt; (f) HF, THF, rt; (g) NaOH, H2O, rt; (h) P(OEt)3, 130 °C, sealed tube; (i) KMnO4, AcOH, H2O, rt; ( j) Boc-Gly-H, NaH, THF, rt; (k) AcCl, MeOH, AcOEt;
(l) i. NMM, ClCO2i-Bu, THF, −25 °C, ii. 9, NaOH, H2O, −25 °C to rt; (m) AcCl, MeOH, AcOEt, rt, basic extraction; (n) 5, NMM, ClCO2i-Bu, THF, −25 °C to rt.
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Separately, a vinyl sulfone ‘warhead’ for irreversible inter-
actions with the active-site cysteine of human cathepsins
was synthesized, since peptidyl vinyl sulfones have been
established as potent irreversible inhibitors of cysteine
cathepsins.34–39 Chloromethyl phenyl sulfide 6 was heated
with triethyl phosphite in a sealed tube, and the resulting
Arbuzov product was oxidized with KMnO4 leading to 7 which
was reacted with Boc-protected glycinal in the presence of NaH
to obtain the corresponding (E)-vinyl sulfone 8 via Wittig–
Horner reaction. Hydrogen chloride-promoted deprotection of
8 provided the Michael acceptor 9 as a hydrochloride salt.35
For the final synthetic steps of the convergent route to the
envisaged ABP 12, we chose phenylalanine as a suitable build-
ing block for the P2 position. Boc-protected phenylalanine 10
was activated via a mixed anhydride and coupled under basic
conditions with the vinyl sulfone 9 to obtain compound 11.
This was deprotected upon treatment with hydrogen chloride,
and the resulting product was coupled, after a previous basic
extraction, with the fluorescent reporter 5 leading to com-
pound 12.
Next, the probe 12 was evaluated in activity assays with
human cathepsins L, S, K and B in the presence of chromo-
genic or fluorogenic peptide substrates. The kinetic analyses
confirmed an irreversible, slow-binding mode of interaction
(Fig. 3). The progress curves were analyzed by non-linear
regression using the equation E/I = vi/kobs × (1 − exp(−kobs × t)) + d
where E/I is the extinction/fluorescence intensity, vi is the
initial rate, kobs is the observed first-order rate constant, and d
is the offset. The plots of the kobs values versus [12] proved to
Table 1 Kinetic parameters of 12. Data were calculated from duplicate experi-
ments with at least five different inhibitor concentrations
k2nd (M−1 s−1
)
kinac (s−1
)
Ki (µM)
Cat L
Cat S
Cat K
Cat B
2200 200
0.0031 0.0001
0.0020 0.0001
0.0011 0.0001
0.0029 0.0003
1.4 0.1
0.11 0.01
0.24 0.04
18 000 2000
4600 900
290 70
10
2
concentration, and the second-order rate constant k2nd was
calculated using the equation k2nd = kinac/Ki where Ki is the
true inhibition constant.
The kinetic parameters of 12 are listed in Table 1.
In general, the ABP 12 was active against all tested cathepsins.
The second-order rate constants (k2nd), representing the
main characteristic of irreversible inhibitors, were at least
290 M−1 s−1. Particularly high k2nd values were calculated for
cathepsin S (k2nd = 18 000 M−1 s−1) and cathepsin K (k2nd
4600 M−1 −1).
=
s
In the case of irreversible inhibition, the true inhibition
constants (Ki) describe the formation of non-covalent enzyme–
inhibitor complexes, prior to the subsequent inactivation step.
The first-order inactivation rate constants (kinac) were in the
same range. These findings are in agreement with the expected
common modification reaction, i.e. the nucleophilic attack of
the active-site cysteine at the Michael acceptor site of 12, gov-
erned by kinac. In contrast to this parameter, the corresponding
Ki values varied between 0.11 µM and 10 µM, reflecting the dis-
tinct affinity of 12 to the four target enzymes, determining its
inhibitory activity (k2nd = kinac/Ki) and slight preference for
cathepsins S and K.
be hyperbolic. Therefore, kinac and K′ constants were obtained
i
by non-linear regression of the data pairs (kobs, [I]) using the
The photochemical properties make compound 12 well
suited as an ABP. Due to the excited-state proton transfer, the
GFP-like fluorophore of 12 partly dissociates already at neutral
pH and thus exists as two different species in water.14 As
shown in Fig. 4, two emission maxima, i.e. at 486 nm (neutral
form) and at 528 nm (anionic form), were observed for 12 in
equation kobs = kinac[I]/(K′ + [I]) where kinac is the first-order
i
inactivation rate constant, K′ is the apparent inhibition
i
constant and [I] is the concentration of 12. The apparent
inhibition constant was corrected to the zero substrate
Fig. 3 Kinetic behavior of 12. Inset: monitoring of the human cathepsin L-cata-
lyzed hydrolysis of Z-Phe-Arg-pNA (100 µM) in the presence of 12 (from top to
bottom: 0 µM, 2 µM, 4 µM, 6 µM, 8 µM and 10 µM). Graph: plot of the kobs
values versus the increasing concentration of 12. Non-linear regression of the
resulting data pairs (kobs, [12]) gave an apparent inhibition constant K’ =
i
(1 + [S]/Km)Ki = 9.6 0.7 µM. The corresponding kinac value was calculated to be
Fig. 4 Representative normalized spectra of 12 (10 µM). CH2Cl2: λex = 423 nm,
λem = 480 nm; H2O: λex = 410 nm, λem = 486/528 nm.
0.18 0.01 min−1
.
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Fig. 5 Imaging and competition experiments. Top: fluorescence analysis.
Bottom: Coomassie staining. PS – protein standard; (I) 130 ng of 12, 280 ng cat
K, 930 ng of 13; (II) 130 ng of 12, 280 ng cat K; (III) 280 ng cat K; (IV) 130 ng of
12.
Fig. 6 Plot of calculated areas of the fluorescent bands versus increasing
amounts of the labeled cathepsin K. PS – protein standard; (I) 130 ng of 12,
280 ng cat K; (II) 66 ng of 12, 140 ng cat K; (III) 33 ng of 12, 69 ng cat K; (IV)
99 ng of 12, 35 ng cat K; (V) 50 ng of 12, 17 ng cat K. Slope = 50 1 square
pixels ng−1, correlation coefficient ∼1. For quantification, the ImageJ freeware
was used.
an aqueous environment. The corresponding absorption
maximum in water was at 410 nm allowing the excitation of 12
using a VIS transilluminator.
Next, it was intended to demonstrate the possibility of
direct in-gel fluorescence detection of cathepsins using
human cathepsin K, the fluorescent probe 12 and a common
gel documentation device. Moreover, the binding mode of 12
was investigated in
a competition experiment (Fig. 5).
Activated cathepsin K was incubated with the probe 12. Simul-
taneously, the same amount of activated cathepsin K was
treated with the selective, active-site directed cathepsin K
inhibitor 13,40 prior to incubation with 12, in order to protect
cathepsin K from inactivation by 12. In the control experi-
ments, solutions of activated cathepsin K and 12 were separ-
ately prepared. Certain aliquots of each incubation mixture
were subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) under reducing conditions and analyzed using a gel
documentation device. No fluorescence was detectable at
∼26 kDa when cathepsin K was pre-incubated with compound
13 (lane I, Fig. 5, top). In contrast, a strong fluorescent signal
of the labeled cathepsin K was observed in the absence of the
competitor (lane II, Fig. 5, top). These findings indicated a
covalent interaction between 12 and the active-site cysteine of
cathepsin K, while the surface nucleophiles of the enzyme
were obviously not affected by the probe 12. To state the pres-
ence of cathepsin K in the labeling, competition and control
experiments, the gel was additionally stained with Coomassie
blue (Fig. 5, bottom).
Fig. 7 Selectivity of labeling. Top: fluorescence analysis. Bottom: western blot
using cathepsin K antibodies. PS – protein standard; (I) 260 ng of 12, 370 ng cat
K, 24 µg protein (HEK293 cell lysate); (II) 260 ng of 12, 370 ng cat K; (III) 260 ng
of 12, 24 µg protein (HEK293 cell lysate). Right: Coomassie staining of the gel,
which was performed with the same amounts of cathepsin K and proteins from
the HEK293 cell line as in the case of the fluorescence and western blot ana-
lyses, but in the absence of 12. PS – protein standard; (I) 370 ng cat K, 24 µg
protein (HEK293 cell lysate); (II) 370 ng cat K; (III) 24 µg protein (HEK293 cell
lysate).
In a further experiment, activated cathepsin K was incu-
bated with compound 12. Different aliquots of the incubation
mixtures were subjected to reducing SDS-PAGE, and the result-
ing gel was imaged and subsequently quantified. The calcu-
lated areas of the fluorescent bands were plotted against the
increasing amounts of cathepsin K showing a strong linear the conditions used, but not the proteins from the HEK293
dependence (Fig. 6).
cell lysate (lane I, Fig. 7, top). The sensitivity of the fluore-
The selectivity of labeling was investigated as follows. Acti- scence analysis was comparable with that of the corresponding
vated cathepsin K was mixed with a lysate from HEK293 cells western blot (lane I, Fig. 7, bottom), and the ABP-based detec-
and treated with 12. Simultaneously, activated cathepsin K
tion appeared to be even more selective. A further gel electro-
and the HEK293 lysate were separately incubated with 12 as phoresis was performed under the same conditions, but in the
positive and negative controls, respectively. Certain aliquots absence of 12. The following Coomassie staining indicated
of each incubation mixture were subjected to reducing
that the ratio between cathepsin K and the proteins from the
SDS-PAGE. The fluorescence analysis of the resulting gel HEK293 cell lysate, which had been used for the fluorescence
showed that the probe 12 selectively imaged cathepsin K under and western blot analyses, was appropriate to show the
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selectivity of labeling by 12. The cathepsin K band at ∼26 kDa performed using a Vario EL apparatus. LC-DAD chromato-
did not appear to be stronger than the bands of the proteins grams and ESI-MS spectra were recorded using an Agilent 1100
from the HEK293 cell lysate (Fig. 7, right).
HPLC system with an Applied Biosystems API-2000 mass
To summarize, the newly synthesized ABP 12 caused an spectrometer. For compound 12, the MS (ESI) spectra were
irreversible, slow-binding inhibition of human cathepsins with recorded using a Bruker Daltonics micrOTOF-Q spectrometer
a slight preference for cathepsins S and K. Using the example and the corresponding MS (EI) spectrum with an A.E.I. MS-50
of cathepsin K, we demonstrated the possibility of cathepsins’ spectrometer. To study the spectral properties of compound 12
direct in-gel fluorescence imaging by 12. The fluorescence pro- as well as its inhibitory activities on human cathepsins, a
perties of the GFP-like fluorophore of 12 turned out to be Varian Cary 50 Bio spectrophotometer and a Monaco Safas
optimal for a detection using a common gel documentation spectrofluorometer flx were used. Human cathepsins L and K
device, equipped with a commercially available VIS trans- were obtained from Enzo Life Science, Lörrach, Germany and
illuminator containing a light source for excitation between human cathepsins S and B from Calbiochem, Darmstadt,
about 420 and 500 nm. As an ABP, 12 allows for the detection Germany. The kinetic parameters were calculated using the
of only the active enzyme fraction. In contrast, other visualiz- software GraFit 5. SDS-PAGE (14%, w/v) was performed under
ation techniques such as Coomassie or silver staining as well reducing conditions using a Power Supply EV243 Consort.
as the western blot analysis provide, by a different degree of Direct in-gel fluorescence detection of cathepsin K was carried
selectivity, the imaging of the whole protein including both, out using a Gel iX Imager from Intas, equipped with a
active and inactive forms of cathepsins. We demonstrated the dark reader from MoBiTec (excitation wavelength about
strong selectivity, but also a notable sensitivity of the cathepsin 420–500 nm), a supersensitive ‘scientific grade’ CCD-camera
detection by ABP 12, comparable to that of the western blot and an ethidium bromide filter. For quantification, the ImageJ
protocol. ABP 12 proved to be appropriate for imaging and freeware was used. Coomassie staining was carried out with
quantification of cathepsin activities ex vivo. The suitability of the ‘Page Blue’ protein staining solution from Thermo Scienti-
the probe 12 to detect cysteine cathepsins in human materials fic, St. Leon-Rot, Germany. For western blotting, a polyclonal
should be investigated in further studies.
rabbit anti-cathepsin K antibody from Abcam, Cambridge, UK
was used.
(Z)-4-(4-(4-Hydroxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-
1H-imidazol-1-yl)butanoic acid (2). Compound 14 (7.35 g,
30.0 mmol) was suspended in ethanol (150 mL), and a
Conclusion
The highly fluorescent probe 12, containing an irreversibly mixture of GABA (9.27 g, 89.9 mmol), Et3N (7.58 g, 74.9 mmol)
locked GFP-like fluorophore, was designed, synthesized and and water (10 mL) was added. The reaction mixture was
evaluated as an inhibitor of human cathepsins L, S, K and stirred for 3 h at room temperature and evaporated. The
B. While the biotechnologically introduced GFP domain residue was dissolved in dry DMF (80 mL), Cs2CO3 (3.26 g,
becomes an integral part of GFP-tagged fluorescent proteins 10.0 mmol) was added, and the mixture was refluxed for
ab initio, fluorescent labeling by 12 arises from a specific 15 min. The solvent was removed in vacuo, and the mixture
protein–ligand recognition process. Nevertheless, such probes was dissolved in water (100 mL), acidified to pH ∼ 5 and
also lead to a permanent labeling of the target protein, owing extracted with EtOAc (3 × 300 mL). The extract was washed
to the covalent, active-site directed modification. Thus, 12 can with water (50 mL) and brine (5 × 50 mL) and dried
be considered as a new tool for the chemical introduction of over Na2SO4 (in the case of the precipitate formation on this
green fluorescence.
or previous step, the precipitates should be collected by
filtration and combined with the residues of the subsequent
evaporation). The solution was removed in vacuo, the
product was washed with a cold EtOH–Et2O (1 : 2) mixture
and dry Et2O to obtain 2 as a yellowish solid (7.60 g, 88%
Experimental
General methods and materials
from 1). mp: 240–243 °C. 1H NMR (600 MHz, DMSO-d6)
3
Compounds 2–5: NMR spectra were recorded using Bruker δ 12.10 (bs, 1H), 10.10 (bs, 1H), 8.07 (d, J = 8.77 Hz, 2H), 6.88
Avance III-600 MHz and Bruker DRX-500 MHz instruments. (s, 1H), 6.83 (d, 3J = 8.77 Hz, 2H), 3.58 (t, 3J = 7.23 Hz, 2H), 2.35
HRMS spectra were recorded using a Bruker Daltonics micrO- (s, 3H), 2.26 (t, 3J = 7.02 Hz, 2H), 1.78 (m, 2H); 13C NMR
TOF II mass spectrometer using electrospray ionization. Thin (151 MHz, DMSO-d6) δ 15.14, 23.93, 30.67, 39.23, 115.71,
layer chromatography was performed using Merck aluminum 125.28, 125.60, 134.06, 159.57, 161.74, 169.82, 173.71; HRMS
sheets. Preparative column chromatography was performed (ESI) m/z = 289.1193 [M + H]+, calcd for C15H16N2O4 m/z =
using silica gel 60, 0.060–0.200 mm. Compounds 7–12: thin 289.1188 [M + H]+.
layer chromatography was performed using Merck aluminum
(Z)-tert-Butyldiphenylsilyl 4-(4-(4-((tert-butyldiphenylsilyl)-
sheets. Preparative column chromatography was performed oxy)benzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl)-
using silica gel 60, 0.060–0.200 mm. 13C NMR (125 MHz) and butanoate (3). A solution of compound 2 (7.20 g, 25.0 mmol),
1H NMR (500 MHz) spectra were recorded using a Bruker tert-butyl-diphenyl-silyl chloride (16.5 g, 60.0 mmol), diiso-
Avance DRX 500 spectrometer. Elemental analyses were propyl-ethylamine (9.68 g, 74.9 mmol) and imidazole (170 mg,
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2.50 mmol) was stirred for 30 h in dry THF (250 mL). The
Diethyl phenylsulfonylmethylphosphonate (7). A mixture of
solvent was evaporated, and EtOAc (500 mL) was added. The chloromethyl phenyl sulfide (6, 5.00 g, 31.5 mmol) and triethyl
solution was washed with aqueous HCl (5%, 150 mL), water phosphite (5.23 g, 31.5 mmol) was heated at 130 °C in a
(2 × 100 mL) and brine (2 × 100 mL) and dried over Na2SO4. sealed tube for 16 h. The resulting solution was distilled
The solvent was evaporated, and the product was purified under reduced pressure to remove the starting materials. The
by column chromatography using EtOAc–heptane (1 : 10) as an oily residue was diethyl phenylthiomethylphosphonate (7.69 g,
eluent to obtain 3 as a yellowish oil (15.3 g, 80%) which was 94%). Diethyl phenylthiomethylphosphonate (7.68 g,
used in the next step without further purification. HRMS (ESI) 29.5 mmol) was dissolved in ice acetic acid (20 mL). KMnO4
m/z = 765.3525 [M + H]+, calcd for C47H52N2O4Si2 m/z = (9.34 g, 59.1 mmol) was dissolved in H2O (50 mL) and slowly
765.3544 [M + H]+.
dropped into the reaction mixture. It was stirred for 1.5 h at
(Z)-Ethyl 4-(4-(2-(difluoroboryl)-4-hydroxybenzylidene)-2-methyl- room temperature, followed by the addition of a sat. KHSO3
5-oxo-4,5-dihydro-1H-imidazol-1-yl)butanoate (4). Compound solution until the reaction mixture became colorless. The
3 (15.3 g, 20.0 mmol) was dissolved in dry dichloromethane colorless aqueous suspension was extracted with ethyl acetate
(150 mL). A solution of boron tribromide in dichloromethane (3 × 30 mL), washed with brine (30 mL) and evaporated
(1 M, 100 mL) and molecular sieves (20 g. 4A, 20 g. 3A) was in vacuo to obtain 7 as an oily colorless product (6.80 g, 79%).
added. The mixture was stirred for 120 h at room temperature, 1H NMR (500 MHz, DMSO-d6) δ 1.14 (t, 3J = 7.1 Hz, 6H),
2
filtered, the sieves were washed with dry dichloromethane 3.93–4.02 (m, 4H), 4.41 (d, JPH = 17 Hz, 2H), 7.62–7.95 (m,
(3 × 100 mL), and the solvent was removed in vacuo. The 5H); 13C NMR (125 MHz, DMSO-d6) δ 16.07, 16.11, 51.68 (d,
residue was dissolved in dry THF (200 mL) and dry ethanol 1JPC = 131.3 Hz), 62.45, 62.50, 127.95, 129.16, 133.92, 140.65.
(100 mL) was added. The mixture was stirred for 20 min, and
(E)-tert-Butyl 3-(phenylsulfonyl)allylcarbamate (8). Com-
saturated aqueous HF (20 mL) was added. After 30 min, the pound 7 (3.20 g, 10.9 mmol) was dissolved in dry THF (20 mL),
solvent was evaporated to 1/3 of the initial volume, and the treated with 60% NaH in mineral oil (0.52 g, 13.1 mmol) and
residue was dissolved in EtOAc (400 mL), washed with water stirred for 30 min at room temperature. N-Boc-glycinal (1.74 g,
(5 × 100 mL), aqueous K2CO3 (5%, 2 × 100 mL) and brine 10.9 mmol) was added, and the resulting reaction mixture
(2 × 100 mL) and dried over Na2SO4. The solvent was evapor- was stirred for
2 h at rt. THF was evaporated under
ated, and the product was purified by column chromatography reduced pressure, and the oily residue was suspended in
using CHCl3–EtOH (20 : 1) as an eluent to obtain 4 as a yellow H2O. The aqueous suspension was extracted with ethyl acetate
solid (2.62 g, 36% from 3). mp 185–187 °C; 1H NMR (600 MHz, (3 × 30 mL), washed with brine (30 mL), dried over Na2SO4 and
3
DMSO-d6) δ 10.22 (bs, 1H), 7.55 (s, 1H), 7.49 (d, J = 8.33 Hz, evaporated in vacuo. The crude product was purified by
1H), 7.01 (d, 4J = 2.19 Hz, 1H), 6.74 (dd, 3J = 8.33 Hz, 4J = column chromatography using ethyl acetate–petroleum ether
3
3
2.19 Hz, 1H), 4.02 (q, J = 7.02 Hz, 2H), 3.75 (t, J = 7.23 Hz, (1 : 1) to obtain 8 as a white solid (1.68 g, 52%). 1H NMR
3
3
2H), 2.74 (s, 3H), 2.40 (t, J = 7.02 Hz, 2H), 1.89 (m, 2H), 1.16 (500 MHz, DMSO-d6) δ 1.35 (s, 9H), 3.78 (bs, 2H), 6.65 (d, J =
(t, 3J = 7.02 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ 12.61, 15.4 Hz, 1H), 6.82 (dt, J = 15.1 Hz, J = 4.6 Hz, 1H), 7.15 (bs,
13.97, 23.02, 30.56, 40.42, 59.91, 115.14, 118.50, 123.87, 1H), 7.62–7.85 (m, 5H); 13C NMR (125 MHz, DMSO-d6) δ 28.24,
124.84, 129.41, 134.33, 161.47, 162.86, 164.11, 172.19; HRMS 78.36, 127.25, 129.71, 130.00, 133.76, 140.47, 144.80, 155.57;
(ESI) m/z = 403.0984 [M + K]+, calcd for C17H19BF2N2O4 m/z = MS (ESI) m/z = 315.3 ([M + NH4]+); Anal. C14H19NO4S (297.37 g
3
3
403.1041 [M + K]+.
mol−1) calcd C 56.55, H 6.44, N 4.71; found C 56.47, H 6.38,
(Z)-4-(4-(2-(Difluoroboryl)-4-hydroxybenzylidene)-2-methyl- N 4.75.
5-oxo-4,5-dihydro-1H-imid-azol-1-yl)butanoic acid (5). Com-
(E)-3-(Phenylsulfonyl)prop-2-en-1-amine hydrochloride (9).
pound 4 (1.82 g, 5.00 mmol) was dissolved in water (200 mL), Compound 8 (0.90 g, 3.03 mmol) was dissolved in ethyl acetate
and ethanol (50 mL) and NaOH (400 mg, 10.0 mmol) were (10 mL). AcCl (11.0 g, 140 mmol) was slowly dropped into
added. The mixture was stirred for 120 min at room temp- MeOH (10 mL) under ice-cooling and stirred for 10 min. The
erature, acidified by aqueous HF, extracted with EtOAc solutions were combined and stirred for 10 min at rt. The pre-
(3 × 100 mL), washed with water (50 mL), brine (2 × 50 mL) cipitated white solid was filtered off, and the filtrate was
and dried over Na2SO4. The solvent was evaporated, and the stirred additionally for 30 min at room temperature. The preci-
product was purified by column chromatography using pitated white solid was again filtered off. The precipitates were
CHCl3–EtOH (5 : 1) as an eluent to obtain 5 as a yellow solid combined, washed with n-hexane and dried in vacuo to obtain
1
(1.29 g, 77%). mp: >200 °C under decomposition. 1H NMR 9 as a white solid (0.55 g, 78%). H NMR (500 MHz, DMSO-d6)
3
4
3
(600 MHz, DMSO-d6) δ 11.88 (bs, 1H), 10.30 (bs, 1H), 7.55 (s, δ 3.71 (dd, J = 5.4 Hz, J = 1.6 Hz, 2H), 6.90 (dt, J = 15.4 Hz,
1H), 7.49 (d, 3J = 8.2 Hz, 1H), 7.00 (d, 4J = 2.2 Hz, 1H), 6.74 (dd, 3J = 5.4 Hz, 1H), 7.10 (dt, 3J = 15.4 Hz, 4J = 1.6 Hz, 1H),
4
3
3J = 8.2 Hz, J = 2.2 Hz, 1H), 3.75 (t, J = 7.23 Hz, 2H), 2.754 (s, 7.65–7.88 (m, 5H), 8.50 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
3H), 2.32 (t, 3J = 7.02 Hz, 2H), 1.85 (m, 2H); 13C NMR δ 38.61, 127.42, 129.84, 133.15, 134.08, 138.88, 139.86.
(151 MHz, DMSO-d6) δ 12.61, 23.13, 30.67, 40.42, 115.12,
(S,E)-tert-Butyl 1-oxo-3-phenyl-1-(3-(phenylsulfonyl)allylamino)-
118.48, 123.86, 124.83, 129.38, 134.29, 161.47, 162.85, 164.14, propan-2-ylcarbamate (11). N-Boc-phenylalanine (10, 1.21 g,
173.71; HRMS (ESI) m/z = 359.0977 [M + Na]+, calcd for 4.56 mmol) was dissolved in dry THF (20 mL) and cooled to
C15H15BF2N2O4 m/z = 359.0988 [M + Na]+.
−25 °C. N-Methylmorpholine (0.51 g, 5.04 mmol) and isobutyl
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4
chloroformate (0.69 g, 5.05 mmol) were added consecutively. 3J = 4.2 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 7.12–7.22 (m, 5H),
Compound 9 (1.07 g, 4.58 mmol) was dissolved in H2O (2 mL), 7.48 (d, 3J = 8.5 Hz, 1H), 7.55 (s, 1H), 7.61–7.82 (m, 5H), 8.27 (t,
treated with 2 N NaOH (3 mL) and added to the reaction 3J = 7.9 Hz, 1H), (t, 3J = 5.7 Hz, 1H); 13C NMR (125 MHz,
mixture when the precipitation of N-methylmorpholinium DMSO-d6) δ 12.76, 14.21, 23.87, 31.97, 37.47, 39.00, 54.42,
chloride occurred. It was allowed to warm to room temperature 115.34, 118.68, 124.00, 125.02, 126.43, 127.19, 128.19, 129.17,
within 30 min, and stirred for an additional 90 min. The 129.67, 129.79, 133.70, 134.56, 137.89, 140.50, 144.37, 161.71,
solvent was evaporated, and the resulting white solid was 162.93, 164.18, 171.45; LC-DAD (90% H2O to 100% MeOH
extracted with ethyl acetate (3 × 30 mL). The combined organic in 20 min, then 100% MeOH to 30 min, DAD 220.0–500.0 nm)
layers were washed with 10% KHSO4 (30 mL) and brine tr = 8.59, 94% purity; MS (ESI) (pos.) m/z = 685.2 ([M + Na]+);
(30 mL). The solvent was dried (Na2SO4) and evaporated. The MS (ESI) (neg.) 661.2 ([M − H]−); MS (EI) m/z = 662.4 (M˙+).
oily product was purified by column chromatography on silica
gel using ethyl acetate–petroleum ether (1 : 1) as an eluent to Enzyme kinetics
obtain 11 as a white solid (1.05 g, 52%). 1H NMR (500 MHz,
Compound 12 was tested on human cathepsins L, S, K and B
DMSO-d6) δ 1.28 (s, 9H), 2.74 (dd, 2J = 13.6 Hz, 3J = 9.5 Hz, 2H),
using the conditions described,41 at five different inhibitor
2.90 (dd, 2J = 13.7 Hz, 3J = 5.2 Hz, 2H) 3.82–4.02 (m, 2H),
concentrations and in duplicate experiments. The enzymatic
4.06–411 (m, 1H), 6.59 (d, 3J = 15.1 Hz, 1H), 6.85 (dt, 3J =
reactions were followed for 30 min, see also S2 of the ESI.†
3
3
15.1 Hz, J = 4.1 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H), 7.14–7.24
(m, 5H), 7.62–7.83 (m, 5H), 8.22 (t, J = 5.7 Hz, 1H); 13C NMR
3
Spectral properties
(125 MHz, DMSO-d6) δ 28.27, 37.24, 56.15, 78.26, 126.36,
127.19, 128.16, 129.25, 129.69, 129.72, 133.70, 138.15, 140.56,
144.65, 155.48, 171.97.
To study the spectral properties of the probe 12, 10 μL of the
compound stock solution in DMSO (1 mM) were added to
990 μL of the corresponding solvent (dichloromethane, metha-
nol and water). The absorption spectra were recorded using a
Cary 50 Bio spectrophotometer in a range between 800 nm
and 200 nm after baseline correction. The emission spectra
were performed using a Monaco Safas spektrofluorometer flx
in a range between 800 nm and 200 nm after baseline correc-
tion, see also S2 of the ESI.†
4-((Z)-4-(2-(Difluoroboryl)-4-hydroxybenzylidene)-2-methyl-
5-oxo-4,5-dihydro-1H-imidazol-1-yl)-N-((S)-1-oxo-3-phenyl-1-((E)-
3-(phenylsulfonyl)allylamino)propan-2-yl)butanamide (12).
Compound 11 (1.00 g, 2.25 mmol) was dissolved in ethyl
acetate (20 mL). Acetyl chloride (10 mL) was slowly added to
MeOH (9 mL) under ice-cooling. The solutions were combined
and stirred for 30 min at room temperature. The solvent was
evaporated under reduced pressure, and the resulting residue
was dissolved in H2O and adjusted to a pH of ∼9 with sat.
Imaging and competition experiments
NaHCO3. It was extracted with ethyl acetate (3 × 30 mL). The Thirty µL of a human recombinant cathepsin K (Enzo Life
combined organic layers were washed with brine (30 mL). Sciences) stock solution (23 μg mL−1 in 50 mM sodium
The solvent was dried (Na2SO4) and evaporated to obtain (S,E)- acetate, pH 5.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM DTT) were
2-amino-3-phenyl-N-(3-(phenylsulfonyl)allyl)propanamide as a treated with 19 µL of the cathepsin K assay buffer (100 mM
colourless oil (0.63 g, 81%). Compound 5 (0.10 g, 0.30 mmol) sodium citrate, pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01%
was dissolved in dry THF (20 mL) and cooled to −25 °C. CHAPS) containing 5 mM DTT and incubated for 30 min at
N-Methylmorpholine (30.0 mg, 0.30 mmol) and isobutyl 37 °C. Activated cathepsin K was treated with 0.5 µL of the
chloroformate (41.0 mg, 0.30 mmol) were added consecutively. probe 12 (1 mM) and 0.5 µL of DMSO, and the resulting solu-
(S,E)-2-Amino-3-phenyl-N-(3-(phenylsulfonyl)allyl)propanamide tion (13.8 µg mL−1 of cathepsin K, 10 µM of 12, 2% DMSO)
(0.19 g, 0.55 mmol) in dry THF (10 mL) was added to the reac- was incubated for 40 min at 37 °C. Simultaneously, the same
tion mixture when the precipitation of N-methylmorpholinium amount of activated cathepsin K was preincubated for 10 min
chloride occurred. It was allowed to warm to room temperature with 0.5 µL of the selective, active-site directed cathepsin K
within 30 min, and stirred for an additional 3.5 h. The solvent inhibitor 13 (10 mM)40 treated subsequently with 0.5 µL of 12
was evaporated, and the resulting white solid was extracted (1 mM) and incubated for 40 min at 37 °C. The final con-
with ethyl acetate (3 × 30 mL). The combined organic layers centrations were 13.8 µg mL−1 of cathepsin K, 100 µM of 13,
were washed with 10% KHSO4 (30 mL), sat. NaHCO3 (fast with 10 µM of 12, and 2% DMSO. In the control experiments, acti-
30 mL) and brine (30 mL). The solvent was dried (Na2SO4) and vated cathepsin K (13.8 µg mL−1) and 12 (10 µM) were separ-
evaporated. The brown solid was purified by column chro- ately shaken in the presence of 2% DMSO for 40 min at 37 °C
matography on silica gel using ethyl acetate (twelve 100 mL frac- in the assay buffer containing 5 mM DTT. Certain amounts
tions) and then ethyl acetate–MeOH (5 : 1) as eluents to obtain (20 µL) of each incubation medium were treated with the redu-
12 as a yellow green solid (0.10 g, 50% from 5). 1H NMR cing running buffer, heated for 4 min at 98 °C and filled into
(500 MHz, DMSO-d6) δ 1.67–1.77 (m, 2H), 2.08–2.15 (m, 2H), the sample wells of a 14% (w/v) polyacrylamide gel. After separ-
2
3
2.67 (s, 3H), 2.74 (dd, J = 13.7 Hz, J = 9.3 Hz, 2H), 2.94 (dd, ation, the resulting gel was analyzed using a Gel iX Imager
3
2J = 13.9 Hz, J = 5.7 Hz, 2H), 3.55–3.67 (m, 2H), 3.84–3.95 (m, from Intas equipped with a transilluminator from MoBiTec
3
4
2H), 4.40–4.44 (m, 1H), 6.53 (dt, J = 15.1 Hz, J = 1.9 H, 1H), (excitation wavelength about 420–500 nm). Subsequently,
3
3
6.73 (dd, J = 8.2 Hz, 4J = 2.5 Hz, 1H), 6.82 (dt, J = 15.3 Hz, Coomassie staining was performed.
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Quantification experiment
37 °C. Incubation medium (b): A volume of 20 µL of a human
recombinant cathepsin K (Enzo Life Sciences) stock solution
(23 μg mL−1 in 50 mM sodium acetate, pH 5.5, 50 mM NaCl,
0.5 mM EDTA, 5 mM DTT) was treated with 29 µL of the cath-
epsin K assay buffer (100 mM sodium citrate, pH 5.0, 100 mM
NaCl, 1 mM EDTA, 0.01% CHAPS) containing 5 mM DTT, and
incubated for 30 min at 37 °C. A volume of 0.5 µL of the probe
12 (1 mM) and 0.5 µL of DMSO were added, and the resulting
solution (9.2 µg mL−1 of cathepsin K, 10 µM of 12, 2% DMSO)
was incubated for 40 min at 37 °C. Incubation medium (c):
A volume of 10 µL of HEK293 cell lysate (3 mg mL−1) was treated
with 39 µL of the cathepsin K assay buffer (100 mM sodium
citrate, pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01% CHAPS) con-
taining 5 mM DTT and incubated for 30 min at 37 °C.
A volume of 0.5 µL of the probe 12 (1 mM) and 0.5 µL of DMSO
were added, and the resulting solution (600 µg mL−1 of the
HEK293 cell lysate, 10 µM of 12, 2% DMSO) was incubated for
40 min at 37 °C. Certain volumes (40 µL) of the incubation
media (a), (b) and (c) were treated with the reducing loading
buffer, heated for 4 min at 98 °C and filled into the sample
wells of a 14% (w/v) polyacrylamide gel. Electrophoresis was
performed as described above.
Incubation medium (a): A volume of 30 µL of a human recom-
binant cathepsin K (Enzo Life Sciences) stock solution (23 μg
mL−1 in 50 mM sodium acetate, pH 5.5, 50 mM NaCl, 0.5 mM
EDTA, 5 mM DTT) was treated with 19 µL of the cathepsin K
assay buffer (100 mM sodium citrate, pH 5.0, 100 mM NaCl,
1 mM EDTA, 0.01% CHAPS) containing 5 mM DTT and incu-
bated for 30 min at 37 °C. 0.5 µL of the probe 12 (1 mM) and
0.5 µL of DMSO were added, and the resulting solution
(13.8 µg mL−1 of cathepsin K, 10 µM of 12, 2% DMSO) was
incubated for 40 min at 37 °C. Incubation medium (b):
A volume of 5 µL of a human recombinant cathepsin K (Enzo
Life Sciences) stock solution (23 μg mL−1 in 50 mM sodium
acetate, pH 5.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM DTT) was
treated with 44 µL of the cathepsin K assay buffer (100 mM
sodium citrate, pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01%
CHAPS) containing 5 mM DTT and incubated for 30 min at
37 °C. A volume of 0.5 µL of the probe 12 (1 mM) and 0.5 µL of
DMSO were added, and the resulting solution (2.3 µg mL−1 of
cathepsin K, 10 µM of 12, 2% DMSO) was incubated for
40 min at 37 °C. Volumes of 20 µL, 10 µL, and 5 µL of the incu-
bation medium (a) and 15 µL and 7.5 µL of the incubation
medium (b) were treated with the reducing running buffer,
heated for 4 min at 98 °C and filled into the sample wells of a
14% (w/v) polyacrylamide gel. Electrophoresis was performed
as described above.
Western blotting
After SDS-PAGE, separated proteins (see above) were trans-
ferred onto the nitrocellulose membrane (Schleicher & Schuell,
Dassel, Germany). Nitrocellulose membranes were blocked by
incubation with 5% (w/v) non-fat dried milk (Carl Roth) in
PBST (PBS with 0.2% (v/v) Tween-20) for 1 h at room temp-
erature. Antigens were detected by incubation with polyclonal
rabbit anti-cathepsin K antibody (Abcam ab19027; 1 : 66) for
1 h at room temperature. After three washing steps with PBST,
primary antibody was incubated with species specific alkaline
phosphatase-conjugated secondary antibody diluted to 1 : 5000
in PBST (goat anti-rabbit IgG; Calbiochem) followed by three
washing steps with PBST and one washing step with PBS. The
resulting antigen–antibody complexes were detected with NBT/
BCIP (nitro tetrazolium blue chloride/5-bromo-4-chloro-
3-indolyl phosphate, Sigma-Aldrich, Saint Louis, USA).
Preparation of the cell lysate
Human embryonic kidney cells (HEK) 293 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM), supplemented
with penicillin (100 U mL−1), streptomycin (100 μg mL−1),
glutamine (2 mM; all substances from Invitrogen, Karlsruhe,
Germany) and fetal bovine serum (10% (v/v); PAA, Linz,
Austria) under a humidified atmosphere of 5% CO2 at 37 °C.
For the preparation of the cell lysate, confluent HEK293 cells
seeded on a 5 cm2 growth area were washed three times with
PBS and scraped off with 1 mL of ice-cold PBS buffer followed
by homogenization via ten aspirations through a 23-gauge
needle. Intact cells and nuclei were removed by centrifugation
(2000g,
5 min, 4 °C). The resulting supernatants were Coomassie staining
subjected to protein quantification with a Roti-Nanoquant
(Carl Roth, Karlsruhe, Germany).
Solution (a): A volume of 20 µL of a human recombinant cath-
epsin K (Enzo Life Sciences) stock solution (23 μg mL−1 in
50 mM sodium acetate, pH 5.5, 50 mM NaCl, 0.5 mM EDTA,
5 mM DTT) was treated with 10 µL of a cell lysate from the
SDS-PAGE and fluorescence analysis
Incubation medium (a): A volume of 20 µL of a human recom- HEK293 cell line (3 mg mL−1) and 20 µL of demineralized
binant cathepsin K (Enzo Life Sciences) stock solution (23 μg mL−1 water to obtain a solution containing 9.2 µg mL−1 of cathepsin
in 50 mM sodium acetate, pH 5.5, 50 mM NaCl, 0.5 mM K and 600 µg mL−1 of the HEK293 cell lysate. Solution (b):
EDTA, 5 mM DTT) were treated with 10 µL of HEK293 cell A volume of 20 µL of a human recombinant cathepsin K (Enzo
lysate (3 mg mL−1) and 19 µL of the cathepsin K assay buffer Life Sciences) stock solution (23 μg mL−1 in 50 mM sodium
(100 mM sodium citrate, pH 5.0, 100 mM NaCl, 1 mM EDTA, acetate, pH 5.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM DTT) was
0.01% CHAPS) containing 5 mM DTT and incubated for treated with 30 µL of demineralized water to obtain a solution
30 min at 37 °C. A volume of 0.5 µL of the probe 12 (1 mM) containing 9.2 µg mL−1 of cathepsin K. Solution (c): A volume
and 0.5 µL of DMSO were added, and the resulting solution of 10 µL of a lysate from the HEK293 cell line (3 mg mL−1) was
(9.2 µg mL−1 of cathepsin K, 600 µg mL−1 of the HEK293 cell treated with 40 µL of demineralized water to obtain a solution
lysate, 10 µM of 12, 2% DMSO) was incubated for 40 min at containing 600 µg mL−1 of the HEK293 cell lysate. Certain
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volumes (40 µL) of solutions (a), (b) and (c) were treated with 20 Y. Liu, X. Li, D. Peng, Z. Tan, H. Liu, Y. Qing, Y. Xue and
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Electrophoresis was performed, and the resulting gel was
stained using the protein staining solution ‘Page Blue’ from
Thermo Scientific.
R. E. Burden, H. L. Barrett, C. J. Scott, J. A. Johnston,
R. H. Wilson, E. W. Kay, P. G. Johnston and S. A. Olwill,
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