Sensitive luciferin derived probes for selective carboxypeptidase activity

Article abstract of DOI:10.1016/j.bmcl.2011.05.023

Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzym

Full text of DOI:10.1016/j.bmcl.2011.05.023

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3931–3934
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sensitive luciferin derived probes for selective carboxypeptidase activity
⇑
Yu-Cheng Chang, Pei-Wen Chao, Ching-Hsuan Tung
Department of Radiology, The Methodist Hospital Research Institute, Weill Cornell Medical College, 6565 Fannin Street, #B5-009, Houston, TX 77030, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 February 2011
Revised 6 May 2011
Accepted 9 May 2011
Available online 14 May 2011
Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity
were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then
they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypep-
tidases can be determined using this technology.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Luciferin
Carboxypeptidase A
Carboxypeptidase G
Luciferase
Glutamic acid
Tyrosine
Luciferase has been widely used as an image reporter to study
numerous biological events in vivo.1–3 This light-producing system
upregulated in pancreatitis and pancreatic cancer; therefore, it
has been proposed as a biomarker for early detection.16,17 In addi-
tion to CPA and CPB, carboxypeptidase G, a bacterial enzyme that
hydrolyzes folic acid, has been proposed as a potential drug activa-
tor to convert an anticancer prodrug for enhanced potency.18,19
These observations provide the motivation for developing new
protease-specific agents that can provide important information
of biological processes.
The current assay for CPA activity is based on the hydrolysis of
N-(4-methoxyphenylazoformyl)-Phe-OH by monitoring the
absorption decrease at 350 nm.20 This method is light-sensitive21
and not suitable for cell assay. Since the light-emitting luciferase
assay is more sensitive and potentially useful for cell or in vivo
imaging, new luciferin derivatives with tyrosine (QLUC-TYR) and
glutamate (LUC-GLU) residues were designed to detect carboxy-
peptidase activities for CPA and CPG, respectively (Fig. 1).22
Aiming to distinguish different carboxypeptidases, the sub-
strates were designed to report enzyme selectivity by lumines-
cence of the specific wavelength. Quinolylluciferin (QLUC), a
luciferase substrate, was selected because of its known property
of producing light at long wavelength.23 QLUC was synthesized
from 6-methoxy-2-quinoline-carbonitrile, which was demethylat-
ed by pyridine hydrochloride and followed by cycloaddition with
gives high signal with low background in the presence of O2, Mg2+
,
ATP, and D-luciferin. Luciferase specifically oxidizes luciferin; how-
ever, this substrate selectivity limits its application to a small num-
ber of suitable light-emitting compounds and its utility as a read-
out of this biochemical reaction. Modifications at the carboxyl or
hydroxyl groups of luciferin often prohibit its conversion to the
key intermediate, oxyluciferin, and abolish the luminescence.4 Re-
cently, few modified luciferins, whose carboxyl or hydroxyl groups
were blocked by various chemical moieties, have been reported to
yield additional biological information.5–9 Removing blocking moi-
eties through reactions either enzymatically catalyzed or photo-
activated released free luciferin for subsequent bioluminescence.
Such strategies have extended the luciferase platform to study
other enzymes. In this study, we reported a new type of ‘caged’
luciferins to investigate carboxypeptidase activities.
Carboxypeptidases are a family of proteases that cleave at spe-
cific C-terminal amino acid residues in polypeptides and proteins
with critical roles in normal biological processes and in diseases.10
For example, carboxypeptidase A (CPA) and B (CPB), mainly pro-
duced by the pancreas, are metalloenzymes which are similar in
primary amino acid structure and substrate specificity.11,12 It has
been shown that CPA in mast cells can enhance the resistance to
snake and honeybee venoms.13 In plasma, the precursor proteins,
procarboxypeptidase A, exhibit only low catalytic activity before
activation by trypsin cleavage.14,15 CPA activity was found to be
D-cysteine to give the desired compound, using a previously
reported procedure.23 QLUC-TYR was prepared by solid-phase syn-
thesis24 using Wang resin. Fmoc-Tyr(2-ClTrt)-OH was activated by
1-methylimidazole and 1-(2-mesitylenesulfonyl)-3-nitro-1H-
1,2,4-triazole and linked to the resin (Scheme 1). The Fmoc group
was removed by piperidine/DMF (1:3) and then reacted with quin-
olylluciferin catalyzed by HOBt/HBTU/DIPEA. The resin was treated
⇑
Corresponding author.
E-mail address: ctung@tmhs.org (C.-H. Tung).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.023

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Y.-C. Chang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3931–3934
Figure 1. The structures of QLUC-TYR (a) and LUC-GLU (b) and their enzymatic degradation are shown.
Scheme 1. Reagents and synthetic conditions: (i) 2a or 2b, Wang resin, 1-methylimidazole, 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole, CH2Cl2, rt, 2.5 h; (ii) 25%
piperidine in DMF, rt, 25 min, 2 times; (iii) quinolylluciferin or -luciferin, HOBt, HBTU, DIPEA, CH2Cl2, rt, 2 h; (iv) TFA/H2O/TIS (38:1:1), rt, 2 h.
D
with H2O/TRIS/TFA (1:1:38) solution to give QLUC-TYR (6a). Same
procedure was utilized to prepare LUC-GLU (6b) using Fmoc-
Glu(OtBu)-OH (Scheme 1). Although both derivatives can be pre-
pared by solution phase synthesis, the solid phase synthesis pro-
vides high efficiency without multiple purification steps.
The normalized fluorescence emission spectra of QLUC-TYR and
LUC-GLU were recorded in water with excitation at 340 nm, and
the observed kmax value was 524 and 540 nm for QLUC-TYR and
LUC-GLU, respectively (Fig. 2). It shifted slightly from the parent
molecules, the kmax of QLUC and LUC was 518 and 531 nm,

Y.-C. Chang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3931–3934
3933
Figure 4. The luminescent wavelength spectra of (a) QLUC-TYR system with CPA
and (b) LUC-GLU with CPG are compared.
by luciferase to yield luminescence (Fig. 3). Without carboxypep-
tidases, luciferase alone was not able to generate luminescence, be-
cause the caged luciferins containing amino acid groups were not
substrates for luciferase. To further evaluate the enzyme specificity
in this process, a reported luciferase inhibitor, methyl ether of
luciferin,25 was added (22.8 mM, 2
lL) to the assay under the same
reaction conditions. It resulted in a significant suppression of the
luminescence signal of QLUC-TYR and LUC-GLU (Fig. 3), supporting
the specificity of the luminescence to luciferase and luciferin. Titra-
tion experiments showed that these probes are detectable even at
sub-nM concentration (Fig. S1). CPA and CPG could detect their
corresponding substrates as low as 0.9 and 0.09 nM, respectively.
Further quantitative measurement of CPA and CPG both showed
a dose dependent activation (Figs. S2 and S3).
While examining the luminescent intensity, the luminescence
of the QLUC-TYR system was found significantly lower than that
of the LUC-GLU system. Several experimental conditions, such as
addition of Zn2+, reaction temperature (to 37 °C), enzyme concen-
tration, and incubation time were all varied; however, the relative
intensity of luminescence was not significantly improved (data not
shown). It has been reported that addition of FBS or BSA could
facilitate the hydrolysis19; however, their inclusion in our reaction
Figure 2. The fluorescent emission spectra of QLUC-TYR & QLUC (a) and LUC-GLU &
LUC (b) with excitation at 340 nm in water are compared.
respectively, suggesting that the attachment of amino acids has a
small effect on the fluorescent wavelength.
To investigate the enzyme activation systems, QLUC-TYR and
LUC-GLU (22.8 mM, 2
30 °C for 4 h, respectively. The reaction mixture was then treated
with luciferase (39,800 units, 10 L), and luminescence was mea-
lL) were first treated with CPA and CPG at
l
sured with a luminometer. This observation indicated that the
enzymatic hydrolysis of QLUC-TYR and LUC-GLU released quin-
olylluciferin and luciferin, which were subsequently hydrolyzed
Figure 3. The luminescent emission spectra of QLUC-TYR (a) and LUC-GLU (b) are
compared. These two derivatives were treated with CPA in (a) and CPG in (b) that
gave the luminescent emission (gray dashed line). It showed no luminescence
without enzyme activation (black hard line) and low emission with addition of
luciferase essay inhibitor (gray dashed line with triangles).
Figure 5. The luminescent emission spectra of (a) QLUC-TYR system with CPA
(dashed line), CPB (dashed line with square), and CPG (solid line with triangle); (b)
LUC-GLU system with CPG (dashed line), CPA (dashed line with square), and CPB
(solid line with triangle) are compared.

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Y.-C. Chang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3931–3934
mixture did not alter the luminescent signal (data not shown).
These observations may suggest that the low intensity of lumines-
cence is due to the nature of the quinolylluciferin but not the en-
zyme activity. For efficient luminescence, the structure of
luciferin appears to be critical.26
was not successful in our experimental system, this concept is
technically feasible.
Acknowledgment
In contrast to fluorescent characteristics, the luminescent
wavelength emitted by QLUC is different compared to that pro-
duced by luciferin. This distinction provides a practical way to
monitor enzyme activities using a single compound or a mixture
of these two derivatives. When QLUC-TYR was incubated with
CPA and treated with luciferase, the emission kmax was 603 nm
(Fig. 4). Conversely, when LUC-GLU was underwent the same
condition with CPG, the emission kmax was 556 nm (Fig. 4). These
readings were similar to the direct measurement using QLUC and
LUC with luciferase, their luminescence kmax were 600 and
554 nm, respectively, which are consistent with reported find-
ings.23 This data suggests that the emission wavelength was not
affected by the neighboring amino acid residues, and a specific en-
zyme could be detected using a mixture of substrates. However,
we have not been able to achieve this yet since only luciferin-spe-
cific wavelength at 556 nm was clearly observed, probably due to
the dramatic difference in the luminescent intensity and substrate
specificity of the luciferase.
Since the enzyme-substrate specificity is crucial for the system,
the correlation between the analogs and three carboxypeptidases
was further investigated. QLUC-TYR was treated with CPA, CPB,
and CPG separately under the same condition (Figs. 5a and S4).
The luminescence of QLUC-TYR/CPA combination was significantly
increased as described previously. In contrast, the results in QLUC-
TYR/CPB (<10% compared to QLUC-TYR/CPA) and QLUC-TYR/CPG
both showed low signal. When the LUC-GLU system was evaluated,
LUC-GLU/CPG gave the most intensive signal relative to LUC-GLU/
CPA (2% compared to LUC-GLU/CPG) and LUC-GLU/CPB (Fig. 5b).
The result further confirmed the high selectivity between the en-
zymes and substrates in our design.
This work was supported in part by NIH CA135312.
Supplementary data
Supplementary data (experimental details and characteriza-
tion) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2011.05.023.
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Luciferin has been extensively used to measure luciferase activ-
ity both in vitro and in vivo. Our design has broadened the applica-
tion of the luciferase assay to carboxypeptidases. Since the reaction
required two co-existing enzymes, in vivo imaging might not be
practical. However, the in vitro detection of carboxypeptidases
would be effective. High selectivity between the enzyme and sub-
strate makes this system potentially useful for monitoring different
enzyme activities. Although the attempt of using bioluminescence
at specific wavelengths to measure the different enzyme activity
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