A two-photon fluorescent probe for intracellular detection of tyrosinase activity

Article abstract of DOI:10.1002/asia.201200762

AAN effective sensor: The two-photon turn-on fluorescent probe NHU was synthesized to optically detect tyrosinase activity in vitro and in melanoma cells. NHU is composed of a 4-aminophenol moiety and a naphthylamine unit, both of which are connected through a urea linkage. Upon exposure to tyrosinase, the 4-aminophenol site is gradually oxidized to the corresponding orthoquinone, ultimately releasing the highly fluorescent product 6-acyl-N-methyl-2- naphthylamine (AAN). Copyright

Full text of DOI:10.1002/asia.201200762

COMMUNICATION
DOI: 10.1002/asia.201200762
A Two-photon Fluorescent Probe for Intracellular Detection of Tyrosinase
Activity
Shengyong Yan,[a] Rong Huang,[a] Changcheng Wang,[a] Yimin Zhou,[a] Jiaqi Wang,[a]
Boshi Fu,[a] Xiaocheng Weng,[a] and Xiang Zhou*[a, b]
Tyrosinase is a copper-containing enzyme and it can pro-
mote the hydroxylation of relevant catechol derivatives.
These products could be further synthesized to the orthoqui-
none oxidation products.[1] Tyrosinase is widespread in bio-
logical tissues and can be obtained from commercially avail-
able mushrooms, Agaricus bisporus.[2] Melanoma is a malig-
nant tumor originating in melanocytes, in which tyrosinase
mediates the biosynthesis of the pigment melanin.[3] Owing
to its overexpression level, tyrosinase is regarded as a bio-
marker for melanoma.[4] Consequently, the detection of tyro-
sinase with high sensitivity is of significant importance not
only for observing its activities in biological and pathological
processes but also for providing efficient diagnosis and treat-
ment in biomedical and clinical research.[5]
Recently, several methods for the detection of tyrosinase
have been developed on the basis of colorimetry, electro-
chemistry, and gold nanoparticles.[6] Nevertheless, fluorome-
try still attracts much attention due its accessibility and high
sensitivity. Until now, only a few fluorescent probes were
utilized for tyrosinase analysis, including quantum dots, con-
jugate polymers, and near-ultraviolet stimulated dyes.[7] Be-
sides these probes, however, the use of a two-photon fluo-
rescent group as signal output has been recognized because
of its lower excitation energy, increased penetration depth
(>500 nm), and greatly reduced autofluorescence in tissue.[8]
Previous studies illustrated that tyrosinase can be used to
mediate the release of a cytotoxic agent from urea prodrugs
activity in a turn-on mode. Herein, we design a novel tyrosi-
nase-detecting system that combines a phenol group as
a substrate for the enzyme and a naphthylamine group
(such a group is highly fluorescent due to its typical one-
photon and two-photon absorption properties) as activatable
signal reporter through a urea linkage.[10] In this approach,
compounds NHU (1-(6-acetylnaphthalen-2-yl)-3-(4-hydroxy-
phenyl)-1-methylurea) and NDU (1-(6-acetylnaphthalen-2-
yl)-3-(3,4-dihydroxyphenethyl)-1-methylurea) were newly
synthesized with satisfactory yield (Figure 1a; the synthetic
steps are described in the Supporting Information). As de-
tailed below, their fluorescent emission mainly depends on
the activity of the enzyme, which provides a method to se-
lectively monitor tyrosinase activity in vitro and in cancer
cells.
in
melanocyte-directed
enzyme
prodrug
therapy
(MDEPT).[9] This MDEPT mechanism can be employed to
release a latent fluorophore for visualization of tyrosinase
Figure 1. a) Structures of NHU and NDU. b,c) Dose-dependent one-
photon fluorescent emission spectra of NHU and NDU (5 mm) upon incu-
bation with tyrosinase for 24 h at 378C in 10 mm potassium phosphate
buffer (pH 6.4) containing 0.5% DMSO. The concentrations of tyrosi-
nase are 0 (control), 0.05, 0.1, 0.5, 1, 5, 10, 20, 40, 100 UmLÀ1, respective-
ly. The spectra were obtained after excitation at 382 nm.
[a] S. Yan,+ R. Huang,+ C. Wang, Y. Zhou, J. Wang, B. Fu, X. Weng,
Prof. Dr. X. Zhou
College of Chemistry and Molecular Sciences
Key Laboratory of Biomedical Polymers of Ministry of Education
State Key Laboratory of Virology
Wuhan University
Hubei, Wuhan, 430072 (P. R. China)
Fax : (+86)27-87336380
E-mail: xzhou@whu.edu.cn
First, to evaluate whether NHU and NDU can be used to
measure tyrosinase activity, one-photon fluorescent titration
experiments were conducted with these two compounds as
substrates (Figure 1b and 1c). Since the optimum pH value
for tyrosinase activity has been reported to lie between
pH 6–7, a potassium phosphate buffer of pH 6.4 was chosen
as simulated physiological conditions. In our experiments,
NHU and NDU (5 mm) were incubated in the absence and
[b] Prof. Dr. X. Zhou
State Key Laboratory of Natural and Biomimetic Drugs
Peking University
Beijing, 100871 (P. R. China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200762.
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Chem. Asian J. 2012, 7, 2782 – 2785

presence of tyrosinase (from 0.05 to 40 UmLÀ1) for 24 hours
at 378C. At the excitation wavelength of 382 nm, the emis-
sion peak of NHU was centered at 503 nm; it was weak ini-
tially and then gradually increased with increasing concen-
tration of tyrosinase, displaying a nearly 12-fold enhance-
ment when 40 UmLÀ1 tyrosinase were used. By contrast,
NDU exhibited a small change in fluorescence, with only
a about two-fold increase in the presence of 40 UmLÀ1 tyro-
sinase.
Hence, these results suggest dose-dependent changes in
emission spectra of NHU and NDU upon the addition of ty-
rosinase. With regard to sensitivity, NHU is more suitable
for tyrosinase detection in comparison with NDU. Further-
more, the nonfluorescent nature of NHU and the distinctive
fluorescence turn-on response of NHU can clearly be visual-
ized by the naked eye (Scheme 1 and Scheme S1 in the Sup-
porting Information). A similar increase in fluorescence in-
tensity was observed in the two-photon fluorescent spectrum
of NHU upon addition of tyrosinase (Figure S1 in the Sup-
porting Information, lEx =770 nm).
tion). Next, we evaluated the selectivity of the probe NHU
with other oxidizing enzymes. Alcohol dehydrogenase
(ADH), which is widely distributed in human and animal
liver, and known to facilitate the interconversion between
alcohols and aldehydes or ketones, was chosen for the com-
parison (Figure S5 in the Supporting Information). As ex-
pected, NHU responded only slightly toward ADH at the
concentration of 40 UmLÀ1, thereby confirming that this
probe is highly selective toward the target enzyme tyrosi-
nase rather than other oxidizing enzymes.
The mechanism by which the tyrosinase-catalyzed reac-
tion is postulated to occur is illustrated in Scheme 1. The 4-
aminophenol group that was attached to the fluorescent
group 6-acyl-N-methyl-2-naphthylamine (AAN) through
a urea linkage supposedly serves as the substate for tyrosi-
nase. It is hypothesized that tyrosinase-triggered two-step
oxidation produces an orthoquinone intermediate that is un-
stable under aqueous conditions and, consequently, under-
goes a rapid intramolecular cyclization to release the fluo-
rescent AAN. In support of the proposed mechanism, the 3-
methyl-2-
benzothiazolinone
hydrazone (MBTH) color test
was performed to trap the
formed orthoquinone inter-
mediate in our current study
(Figure 2a).[11] Upon exposure
to tyrosinase, the 4-aminophe-
nol group was oxidized to or-
thoquinone which reacted with
MBTH through a Michael reac-
tion to form a dark-pink prod-
uct. Furthermore, complemen-
tary HPLC analysis of a sample
of NHU incubated with tyrosi-
Scheme 1. Proposed mechanism for turn-on fluorescent detection of tyrosinase by NHU.
To confirm that the enzyme-catalyzed oxidation reaction
is time-dependent, kinetic analysis was undertaken within
a fixed time of 12 hours. As shown in Figure S2 in the Sup-
porting Information, the change in one-photon fluorescent
intensity at 503 nm, which was recorded once every hour, is
directly proportional to the amount of enzyme added. Con-
trol experiments revealed a small increase in the emission of
NHU in the absence of tyrosinase during the same period of
time. These results indicate that the oxidation of NHU can
be accelerated by tyrosinase catalysis, through which the flu-
orescent response was significantly enhanced.
To determine the optimal pH conditions for the detecting
system, tyrosinase-mediated oxidation of NHU was investi-
gated in 10 mm potassium phosphate buffer at pH values of
6.0, 6.4, and 7.4. The most rapid fluorescent response was
observed at pH 6.4 (Figure S3 in the Supporting Informa-
tion), in agreement with the optimal pH value reported in
the literature.[3] Moreover, any interference of the biologi-
cally essential metal ions (e.g., Na+, K+, Mg2+, Fe3+, and
Ca2+) with the tyrosinase-catalyzed oxidation should be ex-
cluded. The results demonstrate that NHU has high selectiv-
ity toward tyrosinase even in the presence of these poten-
tially interfering ions (Figure S4 in the Supporting Informa-
nase validated the generation of the final fluorescent prod-
uct AAN. As shown in Figure 2b, the peak at the retention
time of 5.87 min, which corresponded to NHU, disappeared
after incubation with tyrosinase. Instead, a new peak with
a retention time identical to ANN (t=7.37 min) was ob-
served. Taken together, the results confirm that tyrosinase
triggers the structural conversion of the 4-aminophenol
group in NHU into orthoquinone and ultimately releases
the product AAN.
Next, we assessed the feasibility of NHU as a two-photon
probe for tyrosinase detection in living cells. To this end,
imaging of tyrosinase activity by confocal microscopy was
carried out in the murine melanoma cell line B16-F1 (which
highly expresses tyrosinase) and in HeLa cells (a tyrosinase-
deficient cell line). As shown in Figure 3, under irradiation
at 770 nm, B16-F1 cells that were incubated with NHU
(10 mm) for 12 hours displayed a bright green fluorescence in
the cytoplasm, whereas analogously treated HeLa cells eli-
cited a relatively weak response. These data imply that the
oxidation reaction of NHU can be activiated by the overex-
pressed tyrosinase in the melanoma cells. Brightfield imag-
ing confirmed cell viability during the imaging experiment.
Chem. Asian J. 2012, 7, 2782 – 2785
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www.chemasianj.org
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Xiang Zhou et al.
COMMUNICATION
Experimental Section
Optical Properties Study
One-photon fluorescent emission spectra were collected in the range
420–650 nm on a PerkinElmer LS 55 fluorescence spectrometer with an
excitation wavelength of 382 nm using excitation and emission slit widths
of 10 and 12 nm, respectively. A quartz cuvette with a capacity of 600 mL
was used for emission measurements. For two-photon excitation experi-
ments, all samples were excited at 770 nm by a mode-locked Ti:Sapphire
femtosecond pulsed laser (Chameleon Ultra I, Coherent Inc.) with
a pulse width of 140 fs at a repetition rate of 80 MHz. Photoluminescence
was recorded on a DCS200PC Photon Counting with single-photon sensi-
tivity through an Omni-l5008 monochromator (Beijing Zolix Instruments
Co., Ltd). Unless otherwise specified, all spectra were taken at 378C in
10 mm sodium phosphate buffer.
Cell Culture and Confocal Imaging
B16-F1 and HeLa cell lines were purchased from the China Center for
Type Culture Collection (CCTCC) and cultured in Modified Eagle
Medium (MEM) supplemented
with 10% FBS, penicillin
Figure 2. a) MBTH color test based to trap the formed intermediate or-
thoquinone during the tyrosinase oxidation. The concentrations of NHU,
MBTH, and tyrosinase were fixed at 500 mm, 1 mm, and 40 UmLÀ1, re-
spectively. a) Images of vials containing NHU and MBTH (1), NHU and
tyrosinase (2), MBTH and tyrosinase (3), and NHU, tyrosinase, and
MBTH (4). b) HPLC analysis of NHU alone (top), NHU incubated for
24 h at 378C with tyrosinase (middle), and fluorescent AAN only
(bottom). The retention time at 5.87 min corresponds to NHU, while that
at 7.37 min corresponds to the fluorescent product AAN. The signals
were monitored at 503 nm under irradiation at 382 nm.
(100 unitsmLÀ1), and streptomycin (100 mgmLÀ1). Cells were maintained
in a humidified atmosphere of 5% CO2 at 378C. One day before imag-
ing, the cells were harvested using trypsin and plated on glassbottomed
dishes (Hyclone). Subsequently, both kinds of cells were incubated with
10 mm NHU for 12 h at 378C. Two-photon fluorescence microscopy
images of NHU-labeled cells were obtained with spectral confocal and
multiphoton microscopes with aꢁ40 objective lens. Two-photon micro-
scopic images were collected at 460–550 nm upon excitation at 770 nm.
Cells are shown representative images from replicate experiments (n=
4).
MBTH Assay
Compound NHU (500 mm) was incubated with 40 UmLÀ1 of mushroom
tyrosinase (this enzyme and alcohol dehydrogenase (ADH) were ob-
tained from Sigma–Aldrich) and 1 mm MBTH for 1 h in 10 mm sodium
phosphate buffer. MBTH solutions were freshly prepared before use.
Acknowledgements
This work was supported by the National Basic Research Program of
China (973 Program) (2012CB720600, 2012CB720603), the National Sci-
ence Foundation of China (No. 90813031, 21072115, 30973605, 20921062),
the National Grand Program on Key Infectious Disease
(2012ZX10003002-014), the Program for Changjiang Scholars and Inno-
vative Research Team in University (IRT1030), and by the Fundamental
Research Funds for the Central Universities.
Figure 3. Imaging of B16-F1 cells (a–c) and HeLa cells (d–f) after treat-
ment with NHU (10 mm) for 24 h. a,d) Two-photon microscopic images
collected at 460–550 nm upon excitation at 770 nm. b,e) Brightfield
images. c,f) Merged images. Cells shown are representative images from
replicate experiments (n=4).
Keywords: cell imaging · enzymes · fluorescent probes ·
two-photon · tyrosinase
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Received: August 18, 2012
Published online: September 28, 2012
Chem. Asian J. 2012, 7, 2782 – 2785
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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