Substrate optimization for monitoring cathepsin C activity in live cells

Article abstract of DOI:10.1016/j.bmc.2008.02.002

A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH₂-aminobutyric-homophenylalanine)₂

Full text of DOI:10.1016/j.bmc.2008.02.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 1064–1070
Substrate optimization for monitoring cathepsin C activity
in live cells
Jun Li, H. Michael Petrassi, Christine Tumanut, Brian T. Masick,
Christopher Trussell and Jennifer L. Harris*
Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
Received 14 December 2007; revised 29 January 2008; accepted 4 February 2008
Available online 7 February 2008
Abstract—A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the
proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH2-aminobutyric-homophenylalanine)2-rhodamine
demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The
resulting FACS assay was validated through correlation of the IC50 values with a competitive radiolabeling assay against a series
of small molecule inhibitors of cathepsin C.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
hand, Cat C deficiency in mice renders resistance to
developing sepsis,7 arthritis,4,8 and abdominal aortic
aneurysms,9 indicating that Cat C may represent a
new opportunity for clinical intervention of inflamma-
tory diseases.
Cathepsin C (Cat C), also known as dipeptidyl peptidase
I (DPPI), is a widely expressed lysosomal cysteine prote-
ase of the papain-fold. It functions as a homotetramer
with each subunit containing an N-terminal exclusion
domain, a heavy (H) chain harboring the catalytic
Cys234 and a C-terminal light (L) chain. The exclusion
domain partially blocks the prime side of active cleft
making it only accessible to the N-terminus of a peptide
substrate.1 This unique structural arrangement makes
Cat C an efficient processing enzyme specialized in re-
moval of dipeptide from N-terminus of a protein
precursor.
To facilitate the in vivo characterization of Cat C activ-
ity and the development of a Cat C inhibitor with cellu-
lar efficacy, a sensitive and selective cell based assay is
crucial. A number of assay formats have been reported
to measure the intracellular activity of Cat C and in-
clude enzymatic assays of cell lysates,3 radiolabeled
activity-based probe in intact cells10 and fluorescently
quenched activity based probe in intact cells.11 However,
these methods either require cell lysate preparation and
sample separation, or involve covalent inactivation of
Cat C and thereby lack the catalytic amplification of
the signal. In this communication, we report the synthe-
sis and characterization of a series of rhodamine peptide
substrates designed to monitor Cat C activity in intact
cells. We identified one rhodamine substrate, 3a, that
is amenable to flow cytometry (FACS), allowing sensi-
tive and selective monitoring of Cat C proteolytic activ-
ity in live cells.
Cat C plays an important function in the activation of
hematopoietic serine proteases found in the granules
of cytotoxic lymphocytes, such as granzyme A and B,2
and inflammatory cells, such as chymase, cathepsin G,
and netrophil elastase.3,4 Consequently, loss of function
mutations in humans results in a decrease in host de-
fense and susceptibility to microbe infection, leading to
severe pre-pubertal peridontitis, Papillon–Lefevre syn-
drome and Haim–Munk syndrome.5,6 On the other
2. Substrate design and chemistry
Keywords: Cathepsin; Imaging; Rhodamine; Activity-based probe.
*
Corresponding author. Tel.: +1 858 812 1665; e-mail: harris@gnf.org
Rhodamine is an attractive fluorphore for cell based
enzymatic assays due to its high extinction coeffi-
cient, long excitation and emission wavelengths, high
Both J.L. and H.M.P. contributed equally to the work described in
this manuscript.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.002

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 1064–1070
1065
fluorescence quantum yield, photostability, and mem-
brane permeability.12 Rhodamine based peptide sub-
strates have been reported to measure cellular activity
of proteases of the papain fold.13 The synthesis of the
potential Cat C rhodamine substrates (Table 1) is out-
lined in Schemes 1a and 1b.
To generate the symmetrical compounds an excess of
amino acid (6 equiv) was coupled to rhodamine. To
generate a mono-peptidic substrate rhodamine was cou-
pled to morpholinecarbomyl chloride as previously de-
scribed. Ether symmetric of non-symmetric rhodamine
derivatives 2 or 4 was deprotected then coupled to a sec-
ond amino acid, or dipeptide in the case of 4. Deprotec-
tion of the terminal boc protecting group afforded the
desired dipeptidic substrates.
Table 1. Characterization of rhodamine (Rd) substrates in FACS-
based assay
#
Structure
S/N
Relative signal
3a
3b
3c
3d
3e
3f
(H2N-Abu-Hph)2-Rd
(H2N-Leu-Hph)2-Rd
(H2N-Nva-Hph)2-Rd
(H2N-Pro-Hph)2-Rd
(H2N-Fpr-Hph)2-Rd
(H2N-Tze-Hph)2-Rd
(H2N-Thp-Hph)2-Rd
(H2N-Chn-Hph)2-Rd
(H2N-Leu-Leu)2-Rd
(H2N-Nva-Bip)2-Rd
H2N-Abu-Hph-Rd-Mor
H2N-Abu-Bip-Rd-Mor
H2N-Fpr-Bip-Rd-Mor
H2N-Leu-Mef-Rd-Mor
H2N-Nva-Mef-Rd-Mor
H2N-Fpr-Mef-Rd-Mor
7.9
1.7
4.1
12.2
4.3
3.3
6.0
16.9
1.7
6.9
1.0
3.2
2.9
1.0
2.2
1.3
1
0.06
0.44
0.14
0.02
0.05
0.00
0.02
0.08
0.00
0.22
0.03
0.01
0.03
0.01
0.00
3. Activity of rhodamines as intracellular substrates
3g
3h
3i
To evaluate these substrates, we identified 6 as a tool
inhibitor compound that was potent and selective for
Cat C with at least 75-fold selectivity over other cathep-
sin enzymes as well as known to have intracellular activ-
ity (Table 2).14 A known analogue of inhibitor 6
possessing a phenolic group and I125 radiolabel (com-
pound 9), readily enters B721 human B-lymphoblastoid
cells and covalently conjugates the lysosomal Cat C en-
3j
5a
5b
5c
5d
5e
5f
Scheme 1a. Synthesis of symmetric dipeptidic rhodamine substrates ((H2N-Xaa-Xaa)2-Rd).
Scheme 1b. Synthesis of non-symmetric mono-peptidic rhodamine substrates (H2N-Xaa-Xaa-Rd-Mor).

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J. Li et al. / Bioorg. Med. Chem. 17 (2009) 1064–1070
Table 2. Correlation between FACS-based and competitive irreversible radiolabeled inhibitor assays
Cat C Ki (nM)
Cat C IC50
(nM)
Cat K Ki (nM) Cat B Ki (nM) Cat S Ki (nM) Cat H Ki (nM)
FACS I125
0.2
70
100
120
1100
5.5
140
1800
2600
4000
1.2
64
<0.1
1400
1.5
6100
10,000 18,000
>105
>105
3700
zyme (Fig. 1). Unlabeled 6 blocks the conjugation of cel-
lular Cat C to the radiolabeled analogue with an IC50 of
69 nM. The reactivity and selectivity of the sixteen rho-
damine substrates were then analyzed in B721 cells by
flow cytometry in the presence of compound 6. Sub-
strate 3a generated the highest cellular fluorescent signal
and 5a, the monoamide analogue of 3a, produced inter-
mediate signal. Other substrates with significant cellular
turnover include 3c and 3d, both sharing Hph at P1.
Cleavage of 3a, 3c, and 3d is dependent on Cat C activ-
ity as the signal is sensitive to treatment with compound
6. Based on the relative velocity and signal-to-noise ra-
tio, substrate 3a, (NH2-aminobutyric-homophenylala-
nine)2-rhodamine ((H2N-Abu-Hph)2-Rd), was chosen
for further validation.
tested in both the flow cytometry assay with sub-
strate 3a and the competitive radiolabeled assay
with compound
9 (Fig. 1). A good correlation
was observed between these two assays (Table 2).
Compound 7 is a potent cathepsin S (Cat S) and
cathepsin K (Cat K) inhibitor with good intracellu-
lar permeability stability and potency as an inhibi-
tor of these proteases. The correlation between
the isolated biochemical inhibition, FACS, and
competitive radiolabeling support that Cat C activ-
ity is solely responsible for the FACS signal. The
known selective Cat C inhibitor 8 was also charac-
terized and displayed a good correlation between
the FACS and competitive radiolabeling assays.
Both activities were approximately 4000- to 7000-
fold less potent than the isolated biochemical
inhibition. The poor stability of this compound in
cellular media and lack of cell permeability could
explain the difference in the observed activities.
These three compounds demonstrate the utility of
the FACS-based activity assay in identifying potent
cellular Cat C inhibitors.
4. Assessment of cathepsin C inhibitors intracellular
activity
To validate the (H2N-Abu-Hph)2-Rd substrate, the
C inhibitors was
cellular potency of three Cat
Figure 1. Competitive labeling of intracellular cathepsin C enzyme.

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 1064–1070
1067
5. Conclusions
6.4. N,N0-NH2-Leu-Hph-rhodamine110 (3b)
In conclusion, we identified 3a as a selective reagent
for monitoring Cat C activity in live cells. The cleav-
age of 3a is dependent on Cat C activity and readily
detected by FACS. This assay will aid in the
investigation of cellular Cat C activity in physiologi-
cal and pathological conditions. It also has sufficient
throughput necessary to support the development of
Compound 3b was prepared as described for the synthe-
sis of 3a. HPLC. 1H NMR (400 MHz, MeOHꢀd4) d 8.04
(d, J = 8.1, 1H), 7.70–7.80 (m, 4H), 7.15–7.28 (m, 12H),
6.72 (d, J = 8.1 Hz, 2H), 4.57 (dd, J = 0.8, 1.4 Hz, 2H),
4.01 (dd, J = 2.1, 1.4 Hz, 2H), 2.66–2.86 (m, 4H),
2.03–2.22 (m, 4H), 1.68–1.85 (m, 6H), 1.02 (t,
J = 1.6 Hz, 11H). 13C NMR (100 MHz, MeOH-d4) d
172.31, 171.35, 170.63, 154.41, 152.91, 142.13 136.88,
131.39, 129.56, 129.46, 127.58, 127.26, 126.02, 125.15,
84.22, 55.68, 54.25, 35.13, 34.89, 33.21, 19.16, 14.07.
Cat C inhibitors for therapeutic and biomarker
development.
6. Experimental
6.5. N,N0-NH2-Nva-Hph-rhodamine110 (3c)
6.1. General materials and methods
Compound 3c was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.06 (d,
J = 8.0 Hz, 1H), 7.70–7.80 (m, 4H), 7.11–7.27 (m,
12H), 6.72 (d, J = 2.2 Hz, 2H), 4.57 (dd, J = 0.8,
8.0 Hz, 2H), 4.01 (dd, J = 2.1, 1.4 Hz, 2H), 2.66–2.86
(m, 4H), 1.68–2.24 (m, 8H), 1.41 (m, 4H), 1.02 (t,
J = 1.6 Hz, 6H). 13C NMR (100 MHz, MeOH-d4) d
174.36, 171.04, 167.89, 163.14, 160.11, 151.25, 150.92,
141.11, 140.01, 136.88, 131.41, 129.49, 127.27, 116.44,
116.12, 114.91, 106.22, 84.24, 55.68, 51.57, 40.01,
38.21, 33.24, 29.11, 25.38, 21.11, 20.21.
All reagents were purchased from Aldrich, Novabio-
1
chem, and standard commercial sources. All H NMR
were recorded at 400 MHz. All 13C NMR were recorded
at 100 MHz. Flash chromatography was performed on
an Isco using pre-packed silica gel cartridges shielded
from light with foil.
6.2. N,N0-Boc-Hph-rhodamine110 (1a)
To an amber vial were charged a Boc-Hpe-OH (744 mg,
2.66 mmol, 6 equiv), HATU (163 mg, 0.44 mmol, 6
equiv) and DMF (6 mL). After stirring for 5 min DIEA
(1.4 mL, 8 mmol, 18 equiv) was added. To the solution
was added rhodamine110 and the reaction was allowed
to stir for 72 h. A standard aqueous EtOAc workup and
purification via silica gel chromatography afforded the
desired product.
6.6. N,N0-NH2-Pro-Hph-rhodamine110 (3d)
Compound 3d was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.04 (d,
J = 8.2 Hz, 1H), 7.70–7.83 (m, 4H), 7.14–7.29 (m,
12H), 6.74 (d, J = 3 Hz, 2H), 4.53 (dd, J = 8.2, 1.3 Hz,
2H), 4.38 (dd, J = 2.1, 1.7 Hz, 2H), 3.33–3.45 (m, 4H),
2.66–2.85 (m, 4H), 2.45–2.54 (m, 2H), 2.01–2.21 (m,
10H). 13C NMR (100 MHz, MeOH-d4) 172.38, 171.24,
169.98, 154.39, 152.91, 142.05, 136.84, 131.40, 129.59,
129.47, 127.62, 127.29, 126.02, 125.12, 116.81, 116.79,
115.53, 108.65, 84.06, 60.97, 55.86, 47.51, 35.05, 33.24,
31.12, 25.0.
6.3. N,N0-NH2-Abu-Hph-rhodamine110 (3a)
To an amber vial were charged N,N0-Boc-Hph-rhoda-
mine110 (1a) (45 mg, 0.053 mmol, 1 equiv), dichloro-
ethane/TFA 4/1 (v/v) and water. The reaction was
allowed to stir for 2 h at room temperature and then
all volatile reagents were removed under reduced pres-
sure to afford N,N0-NH2-Hph-rhodamine110 as a TFA
6.7. N,N0-NH2-Fpr-Hph-rhodamine110 (3e)
salt. Boc-Abu-OH (67 mg, 0.317 mmol,
6
equiv),
Compound 3e was prepared as described for the synthesis
of 3a 1H NMR (400 MHz, MeOH-d4) d 8.04 (d,
J = 8.0 Hz, 1H), 7.70–7.83 (m, 4H), 7.14–7.29, 6.74 (d,
J = 2.1 Hz, 2H), 5.48 (t, J = 0.9 Hz, 1H), 5.35 (t,
J = 1.0 Hz, 1H), 4.53–4.59 (m, 4H), 3.77 (q, J = 4.6,
0.4 Hz, 2H), 3.53 (q, J = 8.9, 0.9 Hz, 2H), 2.56–2.89 (m,
9H), 2.04–2.24 (m, 5H). 13C NMR (100 MHz, MeOH-
d4) d 172.16, 171.29, 169.37, 154.45, 152.91, 142.09,
141.96, 136.87, 131.41, 129.62, 129.50, 127.31, 126.02,
125.18, 116.92, 115.58, 108.78, 93.70, 91.94, 84.10,
59.91, 55.91, 53.49, 53.25, 38.19, 37.97, 35.16, 33.22.
HATU (120 mg, 0.317 mmol, 6 equiv) and DMF
(mL) were all charged to an amber vial. After stirring
for 5 min DIEA (165 lL, 0.951 mmol, 18 equiv) was
added. To the solution was added N,N0-NH2-Hph-rho-
damine110 and the reaction was allowed to stir for 4 h.
A standard aqueous EtOAc workup and purification
via silica gel chromatography afforded N,N0-Boc-Abu-
Hph-rhodamine110. This material was TFA deprotec-
ted as described above and the desired product was
purified from the reaction mixture via HPLC. 1H
NMR (400 MHz, MeOH-d4) d 8.01 (d, J = 8.2 Hz,
1H), 7.67–7.75(m, 4H), 7.12–7.26 (m, 12H), 6.68 (d,
J = 8.4 Hz, 2H), 4.53–4.56 (m 2H), 3.96 (t, J = 6.4 Hz,
2H), 2.65–2.83 (m, 4H), 1.86–2.20 (m, 8H), 1.06 (t,
J = 7.2 Hz, 6H), 13C NMR (100 MHz, MeOH-d4) d
172.31, 171.35, 170.44, 154.36, 152.87, 142.12, 152.87,
142.12, 141.97, 136.87, 129.57, 129.45, 127.55, 127.24,
126.00, 125.13, 116.83, 115.42, 108.69, 84.20, 55.69,
35.10, 33.12, 26.07, 9.45.
6.8. N,N0-NH2-Tze -Hph-rhodamine110 (3f)
Compound 3f was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.04 (d,
J = 8.0 Hz, 1H), 7.70–7.80 (m, 4H), 7.15–7.28 (m,
12H), 6.72 (d, J = 8.0 Hz, 2H), 4.57 (dd, J = 0.8,
1.4 Hz, 2H), 4.01 (dd, J = 2.1, 1.4 Hz, 2H), 2.66–2.86
(m, 4H), 2.03–2.22 (m, 4H), 1.68–1.85 (m, 6H), 1.02 (t,

1068
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 1064–1070
J = 1.6 Hz, 11H). 13C NMR (100 MHz, MeOH-d4)
172.18, 171.27, 169.52, 156.13, 154.43, 152.95, 151.52,
142.15, 136.89, 131.45, 129.62, 129.49, 127.29, 126.07,
125.15, 118.74, 117.03, 116.99, 115.66, 108.86, 84.08,
55.55, 54.20, 35.14, 33.31, 33.16.
charged to an amber vial. After stirring for 5 min DIEA
(313 lL, 1.8 mmol, 18 equiv) was added. To the solution
was added N-Mor-rhodamine110 (4) (44 mg, 0.1 mmol,
1 equiv) and the reaction was allowed to stir for 4 h. A
standard aqueous EtOAc workup and purification via
silica gel chromatography afforded N-Boc-Abu-Hph-
N0-Mor-rhodamine110. This material was TFA depro-
tected as described above and the desired product was
purified from the reaction mixture via HPLC 1H
NMR (400 MHz, MeOHꢀd4) d 8.02 (d, J = 7.6, 1H),
7.71–7.82 (m, 3H), 7.57 (d, J = 2.4 Hz, 1H) 7.05–7.28
(m, 7H), 6.72 (d, J = 8.8 Hz, 1H), 6.66 (d, J = 8.8 Hz,
1H), 4.56–4.60 (m, 1H), 3.91 (m, 1H), 3.71 (t,
J = 4.4 Hz, 4H), 3.52 (t, J = 4.4 Hz, 4H), 2.61–2.37 (m,
2H), 1.79–2.23(m, 5H), 1.07 (t, J = 7.6 Hz, 3H). 13C
NMR (100 MHz, MeOH-d4) d 173.15, 171.70, 170.94,
165.20, 160.32, 160.11, 159.15, 158.89, 145.32, 140.96,
139.87, 130.98, 127.09, 124.21, 123.07, 120.96, 120.04,
115.06, 113.87, 109.68, 85.32, 81.36, 55.36, 44.36,
43.21, 41.11, 40.39, 36.25, 32.78, 31.01, 30.68, 25.46,
22.98, 20.02.
6.9. N,N0-NH2-Thp-Hph-rhodamine110 (3g)
Compound 3g was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.04 (d,
J = 7.9 Hz, 1H), 7.70–7.83 (m, 4H), 7.14–7.30 (m,
12H), 6.73 (d, J = 2.1 Hz, 2H), 4.55 (dd, J = 2.1,
1.5 Hz, 2H), 3.90–3.96 (m, 4H), 3.74–3.79 (m, 4H),
2.66–2.86 (m, 4H), 2.44–2.58 (m, 5H) 2.13–2.24 (m,
4H), 1.82–1.87 (m, 4H). 13C NMR (100 MHz,
MeOHꢀd4) d 172.73, 171.91, 171.27, 163.42, 163.08,
162.73, 162.35, 162.32, 154.38, 152.93, 142.07, 136.87,
131.43, 129.64, 129.54, 127.32, 126.05, 125.13, 116.81,
115.54, 108.67, 108.65, 84.09, 63.54, 59.15, 56.30,
34.52, 33.55, 32.68, 32.55.
6.10. N,N0-NH2-Chn-Hph-rhodamine110 (3h)
6.14. N-NH2-Abu-Bip-N0-Mor-rhodamine110 (5b)
Compound 3h was prepared as described for the synthe-
sis of 3a1H NMR (400 MHz, MeOH-d4) d 8.03 (d,
J = 7.9 Hz, 1H), 7.69–7.81 (m, 4H), 7.14–7.28 (m,
12H), 4.53 (dd, J = 7.9, 1.5 Hz, 2H), 2.65–2.84 (m,
4H), 2.14–2.26 (m, 9H), 1.79–1.93 (m, 10H), 1.50–1.65
(m, 6H). 13C NMR (100 MHz, MeOH-d4) d 173.37,
172.80, 171.31, 163.20, 162.85, 162.50, 162.15, 154.32,
152.90, 142.06, 136.87, 131.42, 129.61, 129.52, 127.27,
126.03, 125.11, 116.86, 115.46, 108.68, 84.16, 61.81,
56.10, 34.55, 33.47, 32.52, 32.34, 25.03, 21.58, 21.53.
Compound 5b was prepared as described for the synthe-
sis of 5a 1H NMR (400 MHz, MeOH-d4) d 7.95–8.04 (m,
4H), 7.63–7.76 (m, 3H), 7.44–7.55 (m, 5H), 7.31–7.38
(m, 4H), 7.24–7.28 (m, 1H), 7.05–7.12 (m, 3H), 6.60–
6.68 (m, 2H), 3.81–3.85 (m, 1H), 3.71 (t, J = 4.4 Hz,
4H), 3.52 (t, J = 4.4 Hz, 4H), 3.31–3.35 (m, 2H), 1.89–
1.95 (m, 3H), 1.31–1.38 (m, 5H), 1.01–1.05 (m, 3H).
13C NMR (100 MHz, MeOH-d4) d 171.80, 171.37,
170.26, 164.91, 157.33, 153.00, 143.89, 141.93, 141.90,
136.97, 131.32, 130.87, 129.89, 129.14, 128.15, 127.75,
12.02, 125.25, 117.38, 116.80, 115.68, 115.68, 114.00,
08.70, 108.60, 67.68, 57.26, 55.81, 55.27, 45.66, 37.02,
31.71, 26.08, 18.72, 7.28, 13.18, 9.41.
6.11. N,N0-NH2-Leu-Leu-rhodamine110 (3i)
Compound 3i was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.02 (d,
J = 7.6, 1H), 7.68–7.80 (m, 4H), 7.01–7.06 (m, 3H),
6.71 (d, J = 7.6 Hz, 2H), 4.58–4.62 (m, 2H), 3.90–3.97
(m, 2H), 1.45–1.80 (m, 12H), 0.87–0.96 (m, 24H), 13C
NMR (100 MHz, MeOH-d4) d 173.37, 172.65, 170.65,
154.69, 152.36, 140.58, 136.55, 130.25, 128.65, 125.63,
127.63, 125.21, 116.95, 115.32, 108.54, 82.65, 56.96,
54.71, 44.28, 28.63, 27.36, 25.63, 24.15, 23.65.
6.15. N-NH2-Fpr-Bip-N0-Mor-rhodamine110 (5c)
Compound 5c was prepared as described for the synthe-
1
sis of 5a H NMR (400 MHz, MeOH-d4) d 7.97–7.99
(m, 1H), 7.69–7.74 (m, 3H), 7.22–7.51 (m, 11H), 7.02–
7.13 (m, 3H), 6.58–6.61 (m, 2H), 4.98 (m, 1H), 4.55
(m, 1 H) 3.71 (t, J = 4.4 Hz, 4H), 3.52 (t, J = 4.4 Hz,
4H), 3.31–3.35 (m, 2H), 1.80–1.97 (m, 6H), 1.31–1.38
(m, 5H), 1.01–1.05 (m, 3H).13C NMR (100 MHz,
MeOH-d4) d 171.55, 171.38, 169.13, 159.18, 158.77,
157.32, 153.96, 153.05, 143.89, 141.81, 136.89, 136.74,
130.88, 129.88, 128.13, 127.83, 126.07, 125.28, 120.39,
117.45, 116.49, 115.74, 114.63, 114.01, 108.87, 93.69,
91.93, 67.66, 59.77, 57.48, 55.16, 45.64, 38.91, 37.05.
6.12. N,N0-NH2-Nva-Bip-rhodamine110 (3j)
Compound 3j was prepared as described for the synthe-
sis of 3a 1H NMR (400 MHz, MeOH-d4) d 8.01–8.03 (m,
1H), 7.21–7.39 (m, 27 H), 6.90–7.14 (m, 3H),6.58–6.62
(m, 2H), 4.53 (dd, J = 2.2, 1.5 Hz, 2H), 2.65–2.84 (m,
4H), 2.14–2.26 (m, 9H), 1.79–1.93 (m, 10H), 1.50–1.65
(m, 6H). 13C NMR (100 MHz, MeOH-d4) d 173.37,
172.80, 171.31, 163.20, 162.85, 162.50, 162.15, 154.32,
152.90, 142.06, 136.87, 131.42, 129.61, 129.52, 127.27,
126.03, 125.11, 116.86, 115.46, 108.68, 84.16, 61.81,
56.10, 34.55, 33.47, 32.52, 32.34, 25.03, 21.58, 21.53.
6.16. N-NH2-Leu-Mef-N0-Mor-rhodamine110 (5d)
Compound 5d was prepared as described for the synthe-
sis of 5a 1H NMR (400 MHz, MeOH-d4) d 7.95–8.01 (m,
3H), 7.53–7.96 (m, 6H), 6.97–7.25 (m, 7H), 6.45–6.81
(m, 7H), 3.81–3.85 (m, 1H), 3.71 (t, J = 4.4 Hz, 4H),
3.52 (t, J = 4.4 Hz, 4H), 3.31–3.35 (m, 2H), 1.89–1.95
(m, 2H), 1.31–1.38 (m, 6H), 1.01–1.05 (m, 3H). 13C
NMR (100 MHz, MeOH-d4) d 171.90, 171.49, 170.45,
161.18, 158.45, 155.98, 154.89, 153.15, 142.18, 140.67,
6.13. N-NH2-Abu-Hph-N0-Mor-rhodamine110 (5a)
Boc-Abu-Hph-OH (218 mg, 0.6 mmol, 6 equiv), HATU
(228 mg, 0.6 mmol, 6 equiv) and DMF (1 mL) were all

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 1064–1070
1069
139.70, 129.94, 129.52, 128.07, 126.32, 126.00, 123.27,
118.52, 117.01, 115.21, 114.19, 113.00, 112.10, 110.65,
107.54, 107.03, 68.56, 58.21, 50.47, 44.16, 37.29, 35.62,
33.74, 18.62, 14.96.
About 500 lL of 1 · 106 cells/mL was plated in a 24-well
flat bottom DMSO-resistant plate. Compounds and
DMSO were immediately added to a final concentration
of 50, 10, 1, 0.1, and 0 lM. The cell-compound mixtures
were incubated for 30 min at 37 ꢁC in a 5% CO2 humid-
ified incubator. After incubation, 2 nM of 125I-labeled
compound 9 was added to each sample and incubated
for another 30 min at 37 ꢁC. Cells were pelleted by cen-
trifugation at 1200 rpm for 10 min, lysed immediately
and boiled for 20 min. Samples were resolved on SDS–
PAGE gels and the resulting gels were vacuum dried
for 2 h. Dried gels were exposed to a phosphor screen
(Amersham, cat# 63-0034-89) overnight and bands were
detected using Storm 860 phosphorimaging machine
and quantified with ImageQuant TL software program
(Amersham).
6.17. N-NH2-Nva-Mef-N0-Mor-rhodamine110 (5e)
Compound 5e was prepared as described for the synthe-
1
sis of 5a H NMR (400 MHz, MeOH-d4) d 8.00 (d,
J = 7.8 Hz, 1H), 7.67–7.76 (m, 3H), 7.53 (s, 1H), 7.11–
7.21 (m, 3H), 7.04–7.08 (m, 2H), 6.75–6.81 (m, 2H),
6.61–6.68 (m, 2H), 4.85–4.87 (m, 1H), 3.89–3.95 (m,
1H) 3.71 (t, J = 4.4 Hz, 4H), 3.52 (t, J = 4.4 Hz, 4H),
3.31–3.35 (m, 6H), 1.89–1.95 (m, 4H), 1.31–1.38 (m,
3H), 1.01–1.05 (m, 3H).13C NMR (100 MHz, MeOH-
d4) d 171.92, 171.39, 170.38, 160.18, 157.35, 154.13,
153.06, 153.02, 143.88, 141.87, 136.76, 131.34, 129.67,
129.17, 127.76, 1236.05, 125.27, 117.48, 117.42, 116.85,
116.85, 115.68, 114.99, 114.00, 108.76, 108.63, 67.68,
57.50, 55.67, 45.66, 38.29, 34.88, 19.13, 14.07.
6.21. Enzymatic assays
Recombinant human cathepsin enzymes were used in all
enzyme inhibition assays. The standard assay format
was 30 lL in a 384-well plate that contained 50 lM
fluorogenic peptide substrate (Ac-His-Pro-Val-Lys-
6.18. N-NH2-Fpr-Mef-N0-Mor-rhodamine110 (5f)
Compound 5f was prepared as described for the synthe-
sis of 5a 1H NMR (400 MHz, MeOH-d4) d 7.95–8.05 (m,
2H), 7.68–7.76 (m, 3H), 7.51 (s, 1H), 7.0–7.20 (m, 3H),
7.04–7.08 (m, 2H), 6.75–6.81 (m, 2H), 6.61–6.68 (m,
2H), 5.36 (d, JF = 52 Hz, 1H), 4.76 (t, J = 7.6 Hz, 1H),
4.47 (dd, J = 4, 10.8 Hz, 1H), 3.97 (s, 3H) 3.71 (t,
J = 4.4 Hz, 4H), 3.56 (t, J = 4.4 Hz, 4H), 3.35 (s, 4H),
1.31–1.38 (m, 3H), 1.01–1.05 (m, 3H).13C NMR
(100 MHz, MeOH-d4) d 171.68, 171.36, 169.04, 164.92,
160.16, 159.18, 157.33, 154.05, 153.04, 153.00, 143.88,
141.81, 136.75, 131.38, 129.61, 1289.45, 127.74, 126.06,
125.28, 117.45, 115.72, 114.97, 114.63, 114.66, 114.02,
108.84, 96.68, 91.95 67.66, 59.74, 57.69, 55.65,
55.17,53.42, 45.64, 37.02, 31.7.
7-amino-3-carbamoylmethyl-4-methyl-coumarin
cathepsin S and cathepsin B, Ac-Lys-His-Pro-Lys-
7-amino-3-carbamoylmethyl-4-methyl-coumarin for
for
cathepsin K; Ac-His-Lys-Phe-Lys-7-amino-3-carbamoyl-
methyl-4-methyl-coumarin for cathepsin L; H-Arg-7-
amino-4-methyl-coumarin for cathepsin H, NH2-Gly-
Phe-7-amino-4-methyl-coumarin for cathepsin C) in
100 mM NaOAc, 1 mM EDTA, 0.01% Brij-35 and
5 mM DTT, pH 5.5 at 37 ꢁC. The recombinant human
cathepsin enzyme was preincubated with inhibitor for
20 min prior to addition of substrate. The liberation of
the coumarin group was monitored by the increase in
fluorescence at an excitation wavelength of 380 nm
and an emission wavelength of 450 nm on a Gemini
EM fluorometer. The reaction progress curve was
fitted to the Morrison equation using PlateKi
(BioKin) and the apparent inhibition constant was
converted to the inhibition constant (Ki) for competitive
inhibitors.
6.19. Cathepsin C FACS assay
B721 EBV-immortalized B cells were washed in serum-
free and phenol red-free RPMI 1640 media. Cells were
resuspended in the same media and 150 lL of 1 · 106
cells/mL was dispensed into a 96-well plate. The cells were
then pre-treated with compounds or DMSO to final con-
centrations of 10, 3.3, 1.1, 0.37, 0.12, and 0 lM. The cell-
compound mixture was incubated for 30 min at 37 ꢁC in a
5% CO2 humidified incubator. Following incubation,
(H2N-Abu-Hph)2-Rd substrate (3a) and Propidium Io-
dide were added to the cells to a final concentration of
2 lM and 4 lg/mL, respectively. Samples were immedi-
ately read using BD Biosciences FACSCalibur flow
cytometer (excitation = 488 nm; emission = 530 nm).
Three time points at 15-min intervals were taken to mea-
sure the increase of cleaved rhodamine over time. Data
were analyzed using FlowJo 5.7.2 software and the EC50
for each compound was calculated and plotted using Pla-
teKi software version 4.07.019.
References and notes
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´
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