A Bioluminogenic Probe for Monitoring Tyrosinase Activity

Article abstract of DOI:10.1002/asia.201601659

A bioluminogenic probe based on luciferin was designed and synthesized to monitor tyrosinase activity. This probe was efficient in assessing tyrosinase activity in a buffered aqueous solution and in measuring endogenous tyrosinase activity in melanoma cel

Full text of DOI:10.1002/asia.201601659

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: A bioluminogenic probe for monitoring tyrosinase activity
Authors: Jianguang Wang, Taesup Lee, Zhe Zhang, and Ching Tung
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Chemistry - An Asian Journal
10.1002/asia.201601659
COMMUNICATION
A bioluminogenic probe for monitoring tyrosinase activity
[
a][b] ‡
[a][c] ‡
[a]
[a]
Jianguang Wang,
Tae Sup Lee,
Zhe Zhang, and Ching-Hsuan Tung*
[
7]
Abstract: A novel bioluminogenic probe, based on luciferin, was
designed and synthesized to monitor tyrosinase activity. Its
applications were demonstrated in assessing tyrosinase activity
in a buffered aqueous solution, and in measuring endogenous
tyrosinase activity in melanoma cells.
the oxidative property of tyrosinase.
Aminoluciferin
(AminoLu), which is slightly different from the natural
luciferin structure, is known to undergo an oxidation
reaction with FLuc; however, studies have also shown that
FLuc is unable to recognize the amine derivatized
[
8]
AminoLu. We, thus, envisioned that a TYR/FLuc dual
detection could be achieved by employing a urea linkage to
conjugate the monophenol motif to AminoLu. The proposed
reaction mechanism is illustrated in Scheme 1. The
oxidation of the monophenol moiety to ortho-quinone by
Tyrosinase (TYR), a copper-containing monooxygenase
enzyme, is widespread in fungi, plants, and animals. TYR
has two oxidation enzyme activities: its monophenolase
property catalyzes the hydroxylation of monophenol to
ortho-diphenol, and its diphenolase property catalyzes the
oxidation of diphenol to ortho-quinone in the presence of
TYR would trigger
a
spontaneous self-immolative
elimination, freeing the caged AminoLu, which then could
be oxidized by FLuc to form oxyluciferin and generate
bioluminescence.
[
1]
oxygen. TYR plays a key role in the biosynthesis of
melanin – the pigment that generates skin color, protects
DNA in skin cells from ultraviolet, and scavenges reactive
COOH
COOH
COOH
[
2]
N
S
N
S
N
S
N
S
N
S
N
S
oxygen species. TYR has been reported to be over-
expressed in melanoma cells and a lack of TYR causes
type I oculocutaneous albinism. In addition, excessive TYR
can contribute to dopamine toxicity and neurodegeneration
HN
HN
HN
HN
O
Tyrosinase
HN
O
Tyrosinase
HN
O
O
O
2
O
2
OH
OH
LumiTYR
OH
O
[
3]
associated with Parkinson’s disease. TYR has been thus
identified as a major target for medical and industrial
applications, including cosmetic and agricultural industries.
The development of a sensitive and selective probe to
monitor TYR activity has been an active research field over
the past decade. A few TYR probes have been designed to
monitor enzymatic activity by measuring fluorescent or
COOH
N
N
COOH
N
N
S
COOH
S
S
CO
2
N
S
N
S
H
2
O
HN
H
2
N
S
HN
O
HN
AminoLu
HO
O
O
O
Fluc
O
ATP, O
2
, Mg2+
O
HN
O
N
S
N
O
O
H2
N
S
[
4]
2
H N
AMP, PP, CO
2
, light
Oxyluciferin
electrochemical changes. However, those methods are
more suitable for biochemical analysis because they require
external excitation or electricity. On the other hand,
bioluminescence technique has been widely employed in
biological assays, both in vitro and in vivo, due to its high
specificity, wide dynamic range, and relative inexpensive
Scheme 1. The structure of LumiTYR and proposed reaction
mechanism for the detection of tyrosinase activity.
The bioluminescent responsive probe, LumiTYR, was
synthesized by adapting several reported protocols with
modifications (detailed syntheses and characterizations
[
5]
instrument. Many bioluminescent probes based on the
luciferin-firefly luciferase system have exhibited excellent
[6d,9]
were described in the Supplementary Information),
The
[
6]
detection properties. The non-invasive optical imaging of
living cells or organs can be realized by detecting
bioluminescent signals generated deep under skin tissue
through a mechanism involving the reporter enzyme, firefly
luciferase (FLuc), the substrate, luciferin, Mg and ATP.
Herein, we report the first bioluminogenic probe to monitor
TYR activity.
absorption, fluorescent and bioluminescent spectra of the
prepared LumiTYR in PBS and in the presence of TYR
were shown in Fig. 1 and Fig. S1. It is expected that in the
presence of TYR, LumiTYR would be converted to AminoLu,
which could be further activated by FLuc. As shown in Fig.
S1A & B, LumiTYR showed two characteristic maxima
absorption peaks at 337 nm and 250 nm. Upon incubation
with TYR for 12 h, the absorption peak at 337 nm
broadened. It is known that the urea group could increase
the solubility of the hydrocarbons through the hydrogen
bond with water. It was thus postulated that the broadening
2
+
To detect tyrosinase activity, a probe was designed based on
[
a]
Dr. J. Wang, Dr. T.S. Lee, Dr. Z. Zhang, Prof. C.-H. Tung
Molecular Imaging Innovations Institute, Department of Radiology,
[
10]
of the peak could be due to the decrease in its solubility.
After incubation with TYR, the FLuc-induced
Weill Cornell Medicine,
th
4
13 East 69 Street, New York, NY 10065, USA.
E-mail: cht2018@med.cornell.edu
Current address: J. Wang
bioluminescence (Fig. 1A) and fluorescence (Fig. S1C)
were significantly enhanced. It is shown that the maximal
fluorescence peak is around 517 nm and the maxima
bioluminescence peak is around 600 nm. As displayed by
the insert in Fig. S1C, LumiTYR alone did not exhibit any
fluorescence; however, after a 12 h incubation with TYR,
the mixture exhibited green fluorescence. Similarly,
[
[
b]
c]
School of Chemical and Environmental Engineering,
Anyang Institute of Technology,
West of HuangHe Road, Anyang, 455000, PR China.
Current address: T.S. Lee
Division of RI-Convergence Research,
Korea Institute of Radiological and Medical Sciences
7
5 Nowon-ro, Nowon-gu, Seoul, 01812, Republic of Korea
‡
These authors contributed equally to this work
LumiTYR
alone
was
unable
to
generate
any
Supporting information for this article is available: Experimental
details, additional characterization studies, and additional
information about experiments performed in cells.
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Chemistry - An Asian Journal
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bioluminescence unless it coexisted with both TYR and
FLuc (Fig. 1A).
to the mixture after a 24 h pre-incubation with TYR. The control
sample, without the presence of TYR, did not produce a
detectable bioluminescence even after incubation with FLuc for
24 h. On the other hand, samples of LumiTYR, which were
incubated with different concentrations of TYR in advance and
followed by the addition of FLuc, showed enhanced
bioluminescence with increasing concentrations of TYR. The
assay is found to be very sensitive. The bioluminescence could
be detected even with only 0.057U of TYR. The
bioluminescence was enhanced 25-fold when 114U/mL of TYR
was used (Fig. 2B).
To validate the formation of the ortho-quinone
intermediate by TYR triggered oxidation, an established
MBTH (3-methyl-2-benzothiazolinone hydrazone) quinone-
trapping assay was used (Fig. 1B, Supplementary
[
11]
information).
TYR and LumiTYR is in agreement
The pink color from the mixture of MBTH,
Fig.2. Fluorescent (A) and bioluminescent (B) spectra of LumiTYR (12 µM in
sodium phosphate, pH=6.8) upon incubation with different concentration of
o
tyrosinase for 24
h
at 37 C. For bioluminescence measurement, fresh
prepared FLuc solution (0.43 µg/mL) was added prior to the measurement.
Fig. 1. (A) Bioluminescence response of LumiTYR towards TYR. The insert is
a snapshot of bioluminescence. Black line and left image: 30 µM LumiTYR,
Red line and right image: 30 µM LumiTYR incubated with TYR for 12h. Fresh
prepared FLuc solution (0.43 µg/mL) was added prior the measurement. (B)
MBTH color test based on TYR’s oxidizing activity towards LumiTYR (from left
The time dependent changes of the fluorescent and
luminescent spectra of LumiTYR were also tested in the
absence of TYR. As shown in Fig. 3, no fluorescence or
luminescence was detected at 0 h, immediately after mixing the
LumiTYR and TYR. Additionally, no spectrum was detected for
the sample of LumiTYR in the absence of TYR after 9 h
incubation, indicating that LumiTYR remained intact during
incubation. When LumiTYR was incubated with 22.8 U/mL TYR
for 1 to 9 h, the fluorescence and luminescence spectra were
enhanced gradually. The luminescence signal intensity of the
LumiTYR sample incubated with 22.8 U/mL TYR has exceeded
the detection limit. The turn-on fluorescent and especially the
turn-on luminescent properties of LumiTYR towards TYR
suggested that LumiTYR is an excellent probe for TYR activity
detection.
to right: LumiTYR and MBTH; MBTH and TYR; LumiTYR and TYR; LumiTYR,
o
TYR and MBTH), The mixture was incubated at 37 C for 3h. (C) HPLC
retention time that generated by using an analytical HPLC column. The
retention time of AminoLu, LumiTYR incubated with TYR for 12h, and
LumiTYR is 3.80 min, 3.83 min, and 12.78 min, respectively.
with the literature, supporting the formation of conversion of
[
4f]
the phenyl group to ortho-quinone intermediate. The LC-
MS analysis of the reaction mixture also showed the correct
molecular mass of the ortho-quinone intermediate (Fig. S2).
Subsequently, the unstable ortho-quinone intermediate
underwent
AminoLu, which could be utilized by FLuc to generate
a rapid self-immolative elimination to free
2
+
bioluminescence in the presence of ATP and Mg . To
further confirm the proposed mechanism, AminoLu was
synthesized as the standard. As shown in Fig. 1C,
LumiTYR has
a retention time of 12.7 min, which
disappeared after its incubation with TYR; a new peak,
corresponding to the formation of AminoLu, was observed
at 3.8 min. The LC-MS analysis of the mixture also
confirmed the formation of AminoLu (Fig. S2).
To examine the dose dependent reactivity, LumiTYR was
incubated with different concentrations of TYR (0 - 114U) for 24
Fig. 3. Fluorescent (A) and Luminescent (B) spectra of LumiTYR (12 µM in
sodium phosphate, pH=6.8) upon treatment with 22.8 U/mL tyrosinase for
different time periods. For bioluminescence measurement, fresh prepared
FLuc solution (0.43 µg/mL) was added prior to the measurement.
o
h at 37 C. Fig. 2A shows the fluorescence change of LumiTYR
on titration of TYR. Without TYR, the fluorescence is at a
background level. An increased concentration of TYR led to a
higher fluorescence. Specifically, when LumiTYR was incubated
with 114U/mL of TYR, the fluorescence was enhanced 27-fold.
Under an optimized condition, a near linear correlation of
fluorescence vs TYR activity was observed with 2 to 10 Units of
[
12]
Since TYR is
a
copper-containing enzyme,
the
interference of common metal ions other than copper, including
2
+
3+
+
+
2+
2+
Zn , Fe , Na , K , Ca , and Mg , was examined. The
fluorescence and luminescence intensity of LumiTYR in the
presence of different metal ions showed that the other metal
TYR and LumiTYR (12 µM) in sodium phosphate buffer (Fig. S3). ions could not interfere with the detecting property (Fig. S4). We
2
+
To generate bioluminescence, ATP, Mg and FLuc was added
then tested the fluorescent and luminescent signal of LumiTYR
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at different pH environments. The reported optimal pH of TYR is
LumiTYR to AminoLu, but was unable to generate
bioluminescence. These results indicated that LumiTYR could
monitor and image TYR activity in mammalian cells in the
presence of FLuc. This tyrosinase specific bioluminescence
assay using FLuc expressing B16-F10 cells and LumiTYR could
be applied to screen various tyrosinase inhibitors.
[
4]
between 6 - 7, and the functional pH of FLuc is 7.44 - 8.14 with
[
13]
maximal activity at pH of 7.8. The pH effect of the LumiTYR
property in a 0.1M sodium phosphate buffer at pH values 5.8
and 8.0 were shown in Fig. S5. The excellent probing ability of
LumiTYR was observed at pH 6.8, which is in agreement with
[
6]
the previous report. This finding supports the potential usage of
LumiTYR in a physiological condition.
To investigate the capability of LumiTYR for screening a
TYR inhibitor, TYR was pre-incubated with a commonly used
[
14]
inhibitor , benzaldehyde, for 15 min at room temperature
before its incubation with LumiTYR. As demonstrated in Fig. 4,
the catalysing activity of TYR was significantly inhibited with 5
µM of benzaldehyde. When the sample of LumiTYR and TYR
was pre-incubated with 10 µM benzaldehyde, an almost
complete inhibition effect was observed. These results further
verified that LumiTYR is a potential lumionogenic probe for TYR
detection.
Fig. 5. Tyrosinase specific bioluminescence of various cancer cells using
LumiTYR. (A) Bioluminescent signal intensity of MDA-MB-231 (TYR-/Fluc-),
MDA-MB-231-Luc (TYR-/FLuc+), B16-F10 (TYR+/FLuc-), and B16-F10-RF
(
TYR+/FLuc+) cells. (B) Bioluminescence image of B16-F10 (TYR+/FLuc-),
and B16-F10-RF (TYR+/FLuc+) cells in triplicate.
In conclusion, a novel bioluminogenic probe LumiTYR towards
TYR detection was developed for the first time. The detecting
activity of LumiTYR was characterized in vitro with mushroom
tyrosinase and mammalian tyrosinase based assays. The
detection mechanism of TYR activity is through TYR mediated
oxidative reactions followed by FLuc catalyzed bioluminescence
generation. In addition, LumiTYR is also fluorogenic. After
reacting with TYR, the enzyme activity could be conveniently
quantitated by measuring the fluorescence increase at 517 nm.
Similar to the other reported TYR fluorescence probes,
LumiTYR would be useful in cell free assays. However, the
fluorescence probes have limited applications in biological
systems, such as cells and animals, due to background,
penetration and diffusion issues. The unique bioluminogenic
capability of LumiTYR becomes handy. Because of this dual
fluorogenic/bioluminogenic property, LumiTYR can be applied to
detect the activity of TYR in cell free assay, cells, and,
potentially, in animals. It could provide an efficient screening
methodology of a tyrosinase inhibitor in biomedical and industrial
fields.
Fig. 4. Inhibition efficiency response of LumiTYR (12 µM in sodium phosphate,
pH=6.8) upon treatment with 57U/mL tyrosinase and different concentrations
(
control, 0 µM) of inhibitor(benzaldehyde) for 12h.
To evaluate the applicability of detecting endogenous TYR
activity in a living cell, LumiTYR was validated with B16-F10
melanoma cells that are known to overexpress TYR. Two
commercially available MDA-MB-231 breast cancer cells (TYR-
/
FLuc-) and MDA-MB-231-Luc (TYR-/FLuc+) cells, which have
no TYR activity, were used as the negative controls. The parent
B16-F10 cells (TYR+/FLuc-) were first transduced with
luciferase/RFP genes, and a stable clone was selected for the
study. As shown in Fig. S6, the parental B16-F10 (TYR+/FLuc-)
cells displayed no red fluorescence signal nor bioluminescence,
while the transfected B16-F10-RF (TYR+/FLuc+) cells
expressed red fluorescent protein and firefly luciferase which
were verified by fluorescence imaging and a bioluminescence
assay using D-luciferin as the substrate. An excellent correlation
2
(
R = 0.993) between the bioluminescent signal intensity and the
cell number was observed. LumiTYR was then tested with MDA-
MB-231 (TYR-/FLuc-) MDA-MB-231-Luc (TYR-/FLuc+), B16-F10
(
TYR+/FLuc-) and B16-F10-RF (TYR+/FLuc+). As demonstrated
in the tyrosinase specific bioluminescence assay using LumiTYR,
MDA-MB-231 (TYR-/FLuc-) and B16-F10 (TYR+/FLuc-) cells
only showed a background level bioluminescent signal. MDA-
Acknowledgements
MB-231-Luc bearing only FLuc showed
a
low level
This work was supported in part by NIH GM094880. We thank
the Nuclear Magnetic Resonance Core facility at Weill Cornell
Medical College
bioluminescent signal, but B16-F10-RF cells bearing both TYR
and FLuc activity showed a greater bioluminescent signal, as
compared to other cells using a luminometer (Fig. 5A). In
bioluminescence imaging using LumiTYR, B16-F10-RF cells
were successfully visualized by the tyrosinase triggered and
firefly luciferase mediated bioluminescence process (Fig. 5B).
The endogenous TYR alone in B16-F10 cells could convert
Keywords: Tyrosinase • Bioluminescence • Fluorescence,
Probe • Luciferin
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