Synthesis and evaluation of new NIR-fluorescent probes for cathepsin B: ICT versus FRET as a turn-ON mode-of-action

Article abstract of DOI:10.1016/j.bmcl.2014.04.022

Recent years have seen tremendous progress in the design and study of molecular imaging geared towards biological and biomedical applications. The expression or activity of specific enzymes including proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has been attracting significant attention in cancer research, and many imaging probes targeting its activity have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared (NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One probe is based on an ICT activation mechanism of a donor-two-acceptor π-electron dye system, while the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast.

Full text of DOI:10.1016/j.bmcl.2014.04.022

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2453–2458
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of new NIR-fluorescent probes for
cathepsin B: ICT versus FRET as a turn-ON mode-of-action
a,
Einat Kisin-Finfer a, Shiran Ferber b, Rachel Blau b, Ronit Satchi-Fainaro b, , Doron Shabat
⇑
⇑
a Department of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel
b Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2014
Revised 6 April 2014
Accepted 7 April 2014
Available online 16 April 2014
Recent years have seen tremendous progress in the design and study of molecular imaging geared
towards biological and biomedical applications. The expression or activity of specific enzymes including
proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in
tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has
been attracting significant attention in cancer research, and many imaging probes targeting its activity
have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared
(NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One
Keywords:
Fluorescent probes
Cathepsin B
probe is based on an ICT activation mechanism of a donor-two-acceptor
p-electron dye system, while
the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes
exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of
the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results
in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast.
Ó 2014 Elsevier Ltd. All rights reserved.
Near infrared fluorescence
Cyanine dyes
Protease activity is an important necessary factor for cell prolif-
eration. Numerous tumor types have been shown to contain ele-
vated levels of proteolytic enzymes, presumably in adaptation to
rapid cell cycling and for secretion in order to sustain invasion,
establish metastasis and promote angiogenesis.1,2 Cathepsin B is
a lysosomal cysteine protease involved in cellular protein turnover
and degradation, which is overexpressed in many tumors, as well
as by host cells at the tumor microenvironment. Its high expression
at tumor sites compared with that of its basal expression in healthy
tissues makes it an attractive target for tumor-selective treatment
and diagnosis.3 Recent years have seen tremendous progress in the
design and study of molecular imaging geared towards biological
and biomedical applications Expression and activity of specific
enzymes including proteases can now be monitored by various
molecular imaging techniques,4 including optical imaging in the
near-infrared (NIR) region.2 This technique enables detection of
molecular activity intravitally thanks to high penetration of NIR
photons through organic tissues and low autofluorescence back-
ground. Various NIR imaging probes were designed and applied
for diagnostics of a variety of diseases. Among them are enzyme-
sensitive probes, which can change their signal intensity in
response to specific enzymes at targeted sites. Cyanine dyes are
Figure 1. Illustration of the general design and mode of action of an ICT-based
fluorescent probe.
Figure 2. Illustration of the general design and mode of action of a FRET-based
turn-ON probe.
widely employed as fluorescence markers for NIR-imaging probes,
since they are compounds with large extinction coefficient, rela-
tively high quantum yield and photostability.5
⇑
Corresponding authors. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640 5761.
E-mail address: chdoron@post.tau.ac.il (D. Shabat).
http://dx.doi.org/10.1016/j.bmcl.2014.04.022
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2454
E. Kisin-Finfer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2453–2458
O
O
N
O
N
O
O
O
O
N
O
N
H
N
O
O
O
N
H
H2N
N
H
Cat B
O
N
N
Phe-lys
N
N
Probe 1
NH3
1a
O3S
O3S
SO3
SO3
Naphthol QCy7
N
O
HO
HN
O
N
O
N
N
N
N
N
NH
O3S
SO3
CO2
1b
O3S
SO3
Figure 3. Chemical structure and activation mechanism of the ICT-based fluorescent probe for cathepsin B with the cyanine dye naphthol-QCy7.
SO3
N
NH2
OH
NH2
O
Cat B
H
N
Cy5
SO3H
O
N
H
H2N
Quencher
O
H
N
H
N
N
O
O
N
H
H
N
N
H
O
O
O
O
NH
Quencher
Cy5
CO2
N
N
Probe 2
N
O3S
Figure 4. Chemical structure and activation mechanism of a FRET-based cathepsin B fluorescent probe with the cyanine dye Cy5.
H2N
COOH
O
1.
O
, NMM
NH2
O
O
Cl
O
O
OH
H
N
H
N
COOH
Et3N
O
O
N
H
O
N
H
O
N
H
N
H
DMF
65%
O
HN
O
O
O
NO2
2.
HO
O
HN
O
THF, -20oC
35%
HN
O
O
O
1e
1f
1c
1d
NO2
N
O
O
N
Boc
H
N
1h
DMF, 75%
O
1. TFA : DCM
O
O
O
O
N
O
Boc
O
O
H
N
H
N
2. Triphosgene,
THF
O
PNP-Cl, Et3N, DMAP
THF, 82%
N
H
N
H
N
H
N
H
O
O
56%
HN
HN
1g
1i
O
O
O
O
N
OH
O
CHO
1k
1m
1. 2eq
O
Cl
O
O3S
N
O
O
O
O
N
O
CHO
O
N
H
N
H
N
N
Probe1
O
O
N
H
N
H
N
N
NaOAc, Ac2O
H
H
Pyridine
70%
O
O
O
O
2. 2% Hydrazine, DMF
15%
CHO
HN
1j
HN
1l
CHO
O
O
O
O
Figure 5. Chemical synthesis of probe 1.

E. Kisin-Finfer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2453–2458
2455
There are several known approaches to turn-ON a fluorescent
probe such as Förster Resonance Energy transfer (FRET), Photo–
induced Energy Transfer (PET), and Internal Charge Transfer
(ICT).6–8 Here, we report the synthesis and activity evaluation of
two new NIR-fluorescent cyanine-probes for detection of cathepsin
B; one is based on an ICT turn-ON mode and the other on a FRET
turn-ON mechanism.
A fluorescent ICT-based probe is usually composed of a dye,
masked by a substrate that acts as a trigger (Fig. 1). The substrate
is attached to a functional group of the dye and thus, masks the
emission in the NIR region.13 One dye, naphthol-QCy7, composed
of naphthol donor and two indolium acceptors, was found to have
superb optical characteristics in terms of NIR fluorescence effi-
ciency.14 Thus, we have selected naphthol-QCy7 as a fluorescent
dye for preparation of a turn-ON probe for cathepsin B. The chem-
ical structure and activation mechanism of the probe are presented
in Figure 3. Probe 1 is composed of the dipeptide Z-Phe-Lys (red) as
a substrate for cathepsin B, attached to naphthol-QCy7 (pink)
through a self-immolative linker15 (blue). Cleavage of the amide
linkage between the dipeptide and the linker by cathepsin B,
results in the formation of aniline 1a. The latter undergoes 1,6-
elimination followed by decarboxylation to release amine 1b. This
amine spontaneously undergoes cyclization reaction to form a cyc-
lic urea and to release the free naphthol-QCy7.
optical signal by decrease of
p-electrons conjugation. Removal of
the triggering substrate by an analyte or an enzyme of interest,
results in release of the free dye and activation of an optical signal,
such as NIR fluorescence.
FRET-based probes can be composed of a fluorescent dye
attached through a specific linker to a quencher (Fig. 2).9,10 Under
such circumstances, the excited fluorophore transfers its excitation
energy to the nearby quencher-chromophore in a non-radiative
manner through long rang dipole–dipole interactions. Cleavage of
the linker moiety by the analyte or enzyme of interest, results in
diffusion of the fluorescent dye away from the quencher and
thereby, generation of a measurable fluorescent signal.
Next, we sought to prepare a FRET-activated probe for cathepsin
B with a cyanine dye. The chemical structure and the activation
mechanism of such a probe based on a FRET technique are pre-
sented in Figure 4. The probe is composed of Cy5 fluorescent dye
attached through a cathepsin B substrate to a quencher dye. Simi-
larly, as was described for the ICT-probe, the selected cathepsin B
substrate was the dipeptide Phe-Lys. The NH2-terminus of the
dipeptide was linked to Cy5 and the COOH-terminus was linked
We have recently reported a novel strategy for the design of
through a
short self-immolative spacer16,17 to the quencher
long-wavelength fluorogenic probes with a turn-ON option.11,12
(Fig. 4). Cleavage of the amide linkage after the lysine, followed
by 1,6-elimination of the self-immolative spacer, results in separa-
tion between the Cy5 and the quencher. Consequently, a turn-ON
NIR fluorescent signal should be obtained from the emission of
the Cy5 dye.
Their design is based on a donor-two-acceptor
dye that can undergo an internal charge transfer to form a new
fluorochrome with an extended -conjugated system. This strategy
p-electron cyanine
p
was translated into synthesis of a library of dyes with fluorescence
O
O
NO2
O
HO
NO2
O
COOH
NH2
H
N
, DCC
H
N
OH
COOH
Et3N
O
EtOH, reflux
quant.
EtOAc, 0oC
83%
O
O
O
2a
2b
O
O
HN
O
HN
O
O
PNP-Cl, Et3N, DMAP
THF, 83%
1.
, NMM
COOH
O
O
Cl
O
O
O
H
N
H
N
O
, Et3N
H2N
N
H
O
H
N
N
H
OH
N
H
2.
HO
O
O
OH
NH2
DMF
93%
O
O
THF, -15oC
43%
2d
2c
O
O
O
O
NH
O
NH
Quencher
NHBoc
O
2f
O
O
1. TFA:DCM
H
N
H
N
O
1.Hydrazine 2%, DMF, 51%
2.
H
N
H
N
Probe 2
2. Et3N, DMF
73%
N
H
H
N
H
, 12%
O
O
N
NHS
2h
3. TFA: DCM
Quencher
NHBoc
Cy5
O
O
O
OPNP
O
O
2g
2e
Quencher
Cy5
NHS
SO3H
O3S
N
N
N
SO3
N
2f
N
2h
O
HN
NH
O
O
O
N
O
O
O
Figure 6. Chemical synthesis of probe 2.

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E. Kisin-Finfer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2453–2458
The synthesis of probe 1 was achieved as presented in Figure 5.
With probes 1 and 2 in hand, we initially sought to evaluate
their turn-ON response upon reaction with cathepsin B. Probes 1
and 2 were incubated with cathepsin B in activity buffer solution
(pH = 6.0) and the NIR fluorescence emission was monitored using
a spectrofluorometer. Figure 7A shows the increase of the emitted
fluorescence at wavelength of 740 nm indicating the release of free
naphthol-QCy7 fluorophore as a function of time upon addition of
cathepsin B. No change in the NIR fluorescence emission was
observed in the background reaction over more than 1 h where
no enzyme was added. Figure 7B shows the increase of the emitted
fluorescence at wavelength of 670 nm upon incubation of probe 2
with cathepsin B, whereas hardly, no change in fluorescence was
observed in the absence of the enzyme.
To evaluate the capability of the probes to serve as imaging
agents, we assessed their turn-ON response upon reaction with
cathepsin B by the CRI Maestro™ imaging system (Fig. 8). Figure 8A
and C show images of probes 1 and 2 in the presence and the
absence of cathepsin B versus the free naphthol-QCy7 and Cy5
fluorophores, respectively. The images were acquired 1 min fol-
lowing addition of cathepsin B. Figure 8B and D show the increase
of the emitted fluorescence of probe 1 and 2, 4 h following the
addition of cathepsin B.
Under these conditions both probes exhibit no emission of NIR
fluorescence in their OFF state. Probe 1 shows strong emission of
NIR fluorescence, 4 h after exposure to cathepsin B, while no fluo-
rescence is observed in the absence of the enzyme at this time
point. Probe 2 also exhibits strong emission of NIR fluorescence,
4 h. after exposure to cathepsin B, however, in the absence of the
enzyme, the background signal of the probe clearly reveals certain
fluorescence emission.
Ultimately, we have performed intravital imaging evaluation of
probes 1 and 2 turn-ON response. We have studied the probes’
fluorescence dependency on endogenous cathepsin B activity upon
intra-tumoral injection to cathepsin B-overexpressing 4T1 mam-
mary adenocarcinoma cells. Probes 1 and 2 were injected intratu-
morally and their NIR fluorescence emission was monitored over
4 h. Intravital non-invasive imaging and quantification of time-
dependent fluorescence signal within tumors are presented in
Figure 9.
4-Nitrophenyl-ester 1c was reacted with lysine derivative 1d to
afford dipeptide 1e. The latter was treated with 4-amino-benzylal-
cohol in the presence of N-methyl-morpholine and isobutyl-chlo-
roformate to generate benzylalcohol 1f. The obtained
benzylalcohol was reacted with 4-nitrophenyl chloroformate to
give carbonate 1g, which was then treated with amine derivative
1h to afford carbamate 1i. Compound 1i was first treated with tri-
fluoreoacetic acid to remove the Boc-protecting group, and then
with triphosgene to produce carbamoyl chloride 1j. The latter
was reacted with naphthol dialdehyde 1k to afford compound 1l,
which was then condensed with two equivalents of indolium-
propane-sulfonate 1m. The obtained crude product was subjected
to 2% of hydrazine solution in DMF for removal of amino-lysine
protecting group to afford probe 1.
The chemical synthesis of probe 2 is presented in Figure 6. The
phenylalanine amino acid was initially reacted with 1-(4,4-
dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl
(ivDde)
protecting group to afford compound 2a. The latter was coupled
with 4-nitrophenol using N,N0-dicyclohexylcarbodiimide coupling
reagent to obtain ester 2b. Compound 2b was reacted with H-lys
(Boc)-OH to give dipeptide 2c. The latter was treated with 4-
amino-benzylalcohol in the presence of N-methyl morpholine and
isobutyl-chloroformate to give benzylalcohol 2d. The alcohol was
activated with 4-nitrophenyl chloroformate to afford carbonate
2e. The Quencher moiety 2f was deprotected with trifluoroacetic
acid and then reacted with carbonate 2e to afford carbamate 2g.
Compound 2g was first deprotected using 2% of hydrazine in solu-
tion of DMF and then reacted with Cy5-NHS derivative 2h. The
obtained crude product was treated with trifluoroacetic acid to
remove the Boc-protecting group and to afford probe 2.
Probes 1 and 2 present a distinguishable increase of their NIR
fluorescence over 4 h. after the injection into the tumors. The
time-dependent fluorescence signal and the images of the tumors
following injection of the probes clearly show considerable higher
initial background signal emitted from FRET-based probe 2 in com-
parison with ICT-based probe 1. This observation is in agreement
with the in vitro images of the probes shown in Figure 8.
Both probes described in this study are composed of cyanine
molecule as the fluorogenic dye. Cyanine dyes are widely
employed as fluorescence labels for NIR imaging, because they
are compounds with large extinction coefficient and relatively high
quantum yield. These qualities allow their fluorescence to pene-
trate deep tissues and thus, cyanines serve as attractive probes
for intravital non-invasive imaging.18,19 In order to generate a
turn-ON system for a cyanine molecule, a FRET approach is usually
applied. To incorporate a cyanine molecule in a FRET-based probe,
an additional dye must be included in the molecular system, sim-
ilarly as was applied in probe 2. An alternative approach, to turn
ON a fluorophore, could be achieved by applying changes in the
pull–push conjugated p-electron system of the dye. This approach
is demonstrated in the chemical design of probe 1. The naphthol-
QCy7 fluorophore belongs to the recently developed donor-
two-acceptor dye family that is activated through an ICT. When
the phenol donor is masked by the cathepsin B-cleavable substrate,
the NIR fluorescence emission of the dye is almost completely
turned OFF. This outcome is responsible for a low background
signal of the probe and therefore, high signal-to-noise ratio is
Figure 7. (A) NIR fluorescence (kex = 680 nm, kem = 740 nm) emitted upon incu-
bation of probe 1 [50
[1.4 U/ml] in activity buffer (pH = 6.0) solution. (B) NIR fluorescence (kex = 620 nm,
kem = 670 nm) emitted upon incubation of probe 2 [25 M, 10% DMSO] in the
[1.4 U/ml] in activity buffer
lM] in the presence (blue) or absence (red) of cathepsin B
l
presence (red) or absence (blue) of cathepsin
(pH = 6.0) solution.
B

E. Kisin-Finfer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2453–2458
2457
A
- 1 minute following Cat. B addition
B
- 4h following Cat. B addition
C - 1 minute following Cat. B addition
D - 4h following Cat. B addition
Figure 8. NIR fluorescence turn-ON response of probes 1 and 2 upon reaction with cathepsin B (solutions in activity buffer pH 6.0). (A) Images of probe 1 [0.01 mM] and
naphthol-QCy7 [0.01 mM] in the presence (1 min after addition) and in the absence of cathepsin B [10 U/ml]. (B) Images of probe 1 in the presence (4 h after addition) and in
the absence of cathepsin B [10 U/ml]. (C) Images of probe 2 [0.01 mM] and Cy5 [0.01 mM] in the presence (1 min after addition) and in the absence of cathepsin B [10 U/ml].
(D) Images of probe 2 [0.01 mM] in the presence (4 h after addition) and in the absence of cathepsin B [10 U/ml]. Images were taken by CRI Maestro™ Imaging system. Filter
set: naphthol-QCy7 and Probe 1-excitation at 661 nm, emission cut-off filter of 750 nm, Cy5 and Probe 2- excitation at 635 nm, emission cut-off filter of 675 nm.
Figure 9. Intravital non-invasive imaging of probes 1 and 2 upon activation with cathepsin B. (A) Representative images of Probes 1 and 2 fluorescence dependency on
endogenous cathepsin B activity upon intratumoral injection into cathepsin B-overexpressing 4T1 mammary adenocarcinoma [50 ll; 0.01 mM]. (B) Quantification of
time-dependent fluorescence signal within tumors. Images were acquired and quantified using non-invasive intravital CRI Maestro™ imaging system. Filter set: ICT Based
probe 1-excitation at 661 nm, emission cut-off filter of 750 nm; FRET Based probe 2-excitation at 635 nm, emission cut-off filter of 675 nm.

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E. Kisin-Finfer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2453–2458
obtained from probe 1 after activation by the enzyme. On the other
hand in probe 2, the NIR fluorescence emission in the OFF state is
noticeably higher than that of probe 1. The limited ability of the
quencher dye in probe 2 to absorb the emission of Cy5, generates
certain background fluorescence, which results in lower signal-
to-noise ratio. This outcome is also reflected in the in vitro and
in vivo images of probes 1 and 2 activated by cathepsin B (see
Figs. 8 and 9).
In summary, we have described the design, synthesis and
evaluation of two novel NIR fluorescent probes for detection of
cathepsin B with different turn-ON mechanisms. One probe is
based on an ICT activation mechanism of a donor-two-acceptor
Initiative (INNI), Focal Technology Area (FTA) program:
Nanomedicine for Personalized Theranostics, and by The Leona
M. and Harry B. Helmsley Nanotechnology Research Fund.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
022.
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p-electron dye system, while the other is based on FRET mecha-
nism obtained by a fluorescent dye and a quencher. The two probes
exhibit significant fluorescence turn-ON response upon reaction
with cathepsin B, whereas almost no fluorescence change is
observed in the absence of the target enzyme. The probes demon-
strated potential to image cathepsin B activity in vivo, when
evaluated with cathepsin B overexpressing 4T1 mammary adeno-
carcinoma subcutaneously-inoculated tumors. The NIR fluores-
cence of the ICT probe in its OFF state was significantly lower
than that of the FRET-based probe. This effect leads to a higher
signal-to-noise ratio and consequently increased sensitivity and
better contrast of images. The obtained in vitro and in vivo results
indicate that both probes could be suitable for detection of cathep-
sin B activity and potential imaging, detection and progression of
tumors.
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Acknowledgments
D.S. and R.S.-F. thanks the Israel Science Foundation (ISF), the
Binational Science Foundation (BSF) and the German Israeli
Foundation (GIF) for financial support. This work was partially
supported by grants from the Israeli National Nanotechnology