Fluorogenic assay of alkaline phosphatase activity based on the modulation of excited-state intramolecular proton transfer

Article abstract of DOI:10.1016/j.bmcl.2012.07.021

A new fluorogenic substrate 1, which enables the fast and quantitative analysis of alkaline phosphatase activity, has been developed. Selective enzymatic hydrolysis of 1 instantly generated fluorescent compound 2 in aqueous media, which undergo an excited-state intramolecular proton transfer process, resulting in a remarkable fluorescence turn-on signal with an unusually large Stokes shift.

Full text of DOI:10.1016/j.bmcl.2012.07.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5541–5544
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Bioorganic & Medicinal Chemistry Letters
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Fluorogenic assay of alkaline phosphatase activity based on the modulation
of excited-state intramolecular proton transfer
Jihye Park a, Aasif Helal b, Hong-Seok Kim b, , Youngmi Kim a,
⇑
⇑
a Department of Chemistry, Institute of Nanosensor and Biotechnology, Dankook University, 126 Jukjeon-dong, Yongin-si, Gyeonggi-do 448-701, Republic of Korea
b Department of Applied Chemistry, Kyungpook National University, 80 University Road, Buk-gu, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new fluorogenic substrate 1, which enables the fast and quantitative analysis of alkaline phosphatase
activity, has been developed. Selective enzymatic hydrolysis of 1 instantly generated fluorescent com-
pound 2 in aqueous media, which undergo an excited-state intramolecular proton transfer process,
resulting in a remarkable fluorescence turn-on signal with an unusually large Stokes shift.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 April 2012
Revised 29 June 2012
Accepted 6 July 2012
Available online 14 July 2012
Keywords:
Alkaline phosphatase
Excited-state intramolecular proton transfer
Fluorogenic assay
Inhibitor assay
Probe
Introduction
substrates, in which an increase in fluorescence intensity occurs
upon the enzyme-catalyzed hydrolysis of a phosphate group.8–10
Alkaline phosphatases (ALPs), which catalyze hydrolysis or
transphosphorylation of a wide variety of phosphate compounds,
are a group of enzymes found in a variety of tissues (intestine, liver,
bone, kidney, and placenta) of nearly all living organisms.1–3 This
enzyme has been extensively used as a biomarker in enzyme
immunoassays and molecular biology.4 ALP is also one of the most
commonly assayed enzymes in routine clinical practice. Because of
its abnormal level of serum, ALP is an important diagnostic indica-
tor of many human diseases, such as bone diseases (osteoblastic
bone cancer, Paget’s disease, and osteomalacia) and liver diseases
(cancer, hepatitis, and obstructive jaundice).5–7 Therefore, a sensi-
tive and continuous assessment of ALP activity would be extremely
valuable for providing effective diagnostic approaches and/or ther-
apeutic targets in biomedical research as well as for offering the
possibility of studying the role of this enzyme in various physiolog-
ical and biological processes.
These probes offer high sensitivity and can be used in a continuous
assay of ALP activity; however, their water-soluble fluorescent
products often excret out of cells, limiting their use in detecting
endogenous ALP activities. In case of fluorescein-based substrates,
enzymatic products exhibit a fluorescence emission with small
Stokes shifts, resulting in high background signals and interfer-
ences (self-absorption, etc.).
Intramolecularly hydrogen-bonded molecules such as o-hydroxy-
benzoxazole, -benzimidazole, and -benzothiazole often undergo an
excited-state intramolecular proton transfer (ESIPT), in which rapid
photoinduced proton transfer results in the formation of two tauto-
meric forms.11,12 Such organic fluorescent compounds harnessing
an ESIPT process have attracted great attention due to their distinct
photophysical properties, which facilitate a wide variety of sensory
applications for ions, chemicals, and biomolecules.13–15
2-(20-Hydroxyphenyl)-4-phenylthiazole 2 is known to exhibit
an ESIPT process.16 In the ground state, compound 2 exists in the
enol form (2-E). Irradiation of chromophore 2 converts the enol
(2-E⁄) to the excited-state keto species (2-K⁄) in the subpicosecond
time scale (Scheme 1a). The resulting emission from 2-K⁄ exhibits
an unusually large Stokes shift. Since 2-K⁄ emission occurs in a re-
gion of the spectra that is clear of interferences (self-absorption,
etc.), we sought to explore the turn-on fluorescence probes that
rely on an ESIPT process. In this paper, we report an ESIPT-con-
trolled fluorescence turn-on probe for monitoring ALP activity in
aqueous media.
Due to unparalleled sensitivity, simplicity, rapid implementa-
tion, and applicability to high throughput screening, fluores-
cence-based probes are promising tools to detect the catalytic
activity of ALP. The most common fluorescent probes for monitor-
ing ALP activity are coumarin- and fluorescein-based fluorogenic
⇑
Corresponding authors. Tel.: +82 53 950 5588; fax: +82 53 950 6594 (H.-S.K.);
tel.: +82 31 8005 3156; fax: +82 31 8005 3148 (Y.K.)
E-mail addresses: kimhs@knu.ac.kr (H.-S. Kim), youngmi@dankook.ac.kr (Y.
Kim).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.021

5542
J. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5541–5544
(e
= 17380 Mꢀ1 cmꢀ1), and the emission spectrum showed a peak
maximum at 385 nm, which is characteristic of an enol-like emis-
sion (Fig. 1). To validate the proposed sensing mechanism, the
photophysical properties of 2-(20-hydroxyphenyl)-4-phenylthiaz-
ole 2—the expected product after reaction with ALP—were exam-
ined in water containing 2% EtOH as a cosolvent. The UV–visible
absorption spectrum of
2 displayed a maximum at 340 nm
(e
= 13750 Mꢀ1 cmꢀ1) and the emission spectrum of 2 showed a
strong keto tautomer emission (ꢁ522 nm) resulting from the
ESIPT. The emission quantum yields (UF) in the aqueous solution
were 0.03 for probe 1 and 0.12 for compound 2, relative to that
of quinine sulfate in 0.1 M H2SO4. Enhanced emission efficiency
and a large bathochromic shift in the emission maxima of 2 with
respect to those observed in probe 1 indicated that a higher sig-
nal-to-noise ratio can be obtained from the conversion of probe 1
to 2 upon reaction with ALP.
Upon incubation of probe 1 (20 lM) with ALP (50 nM) in a buf-
fered aqueous solution (10 mM Tris–HCl, pH 9.0, 37 °C), the
absorption spectrum showed a clear bathochromic shift from
316 nm to 339 nm, indicating the formation of compound 2. The
characteristic ESIPT-fluorescence signal of 2 at 521 nm immedi-
ately arose and increased significantly with time (Fig. 2) before
reaching a plateau within 10 min. A remarkable increase (ca.
455-fold) in fluorescence intensity at 521 nm was obtained upon
incubation with ALP (50 nM) for 10 min. The weak blue emissive
nature of probe 1 and the bright green fluorescence upon cleavage
of phosphate group of 1 were clearly visualized, as shown in
Figure 2.17
We further investigated the ALP-catalyzed hydrolysis of probe 1
as a function of incubation time using different enzyme concentra-
tions. Figure 3a illustrates the increase in fluorescence intensity at
525 nm for solutions with different ALP concentrations (0–50 nM),
in which fluorescence intensity was measured every 1 min. As ex-
pected, the fluorescence intensity increased with increasing [ALP]
within this concentration range. The increase in fluorescence
intensity at 525 nm was shown to be quantitatively related to
the increase of ESIPT-product 2, thus allowing real-time monitor-
ing of enzyme activity (Fig. 3B). We then found that ALP could be
assayed with a sensitivity limit as low as 0.25 nM of ALP.
We then conducted kinetic measurements for the enzymatic
hydrolysis reaction of fluorogenic substrate 1 and determined ki-
netic parameters using Lineweaver–Burk analysis, yielding values
Scheme 1. (a) General mechanism of the ESIPT process and chemical structures of
the 2-enol and 2-keto forms in the ESIPT process of compound 2. (b) Illustration of
the fluorometric assay for ALP using ESIPT-based probe 1.
Scheme 1b depicts our design rationale for ALP assays based on
an ESIPT fluorophore (2). Fluorogenic substrate 1 was expected to
display enol emission (E⁄) exclusively due to the inhibition of the
ESIPT process. In addition, the excited probe 1 was expected to
have less efficient emission because of the fast non-radiative decay
of the singlet excited state facilitated via internal bond rotations.
However, ALP-catalyzed cleavage of the phosphate group in probe
1 induces a fast generation of ESIPT-product 2, and increases the
quantum yields of the corresponding keto-tautomer emission
upon photoexcitation.
Probe 1, as a new substrate for ALP, was synthesized in a mod-
erate yield according to Scheme S1. 2-(20-Hydroxyphenyl)-4-phe-
nylthiazole 2 was phosphorylated with diethyl chlorophosphate
and NaH in THF to afford phosphate triester 3. Selective hydrolysis
of 3 was acheived with bromotrimethylsilane to give the water-
soluble probe 1 after neutralization with NaOH.
The UV–visible absorption spectrum of probe 1 in water
displayed
a strong electronic transition at kmax,abs = 315 nm
Figure 2. Emission spectra of probe 1 (20 lM) upon treatment with ALP (50 nM) for
Figure 1. Absorption (dashed lines) and emission spectra (solid lines) of probe 1
(black) and product 2 (red) in an aqueous solution at 25 °C. Probe 1 in water and 2
in 2% EtOH/H2O (v/v). Excited at 300 nm for probe 1 and at 340 nm for product 2.
different time periods (0–10 min) in Tris–HCl buffer (10 mM, pH 9.0, 37 °C). The
spectra were obtained every 1 min. Excited at 340 nm. Inset: Photographs of probe
1 (20
lM) in the absence (left) and presence (right) of ALP (50 nM) after incubation
[1] = [2] = 20 lM.
for 10 min at 37 °C under UV light (365 nm) illumination.

J. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5541–5544
5543
Figure 4. Inhibition assay of ALP activity by levamisole. Relative emission intensity
of probe 1 as a function of the concentration of the inhibitor. ALP (50 nM) was
incubated with the inhibitor for 10 min at 25 °C before the addition of probe 1
(20 lM). After 10 min incubation at 37 °C, emission intensity at 525 nm was
recorded (kex 340 nm). Each point was obtained from the average value of three
measurements.
In summary, we have developed a new ALP probe based on a
unique ESIPT turn-on mechanism. This probe exhibited a large
fluorescence response in addition to its excellent sensitivity to-
ward ALP. We anticipate that the probe will accelerate the devel-
opment and characterization of ALP inhibitors and activators
based on in vitro fluorogenic assays, as well as increase the under-
standing of its biological and pathological roles in living systems.
Acknowledgments
Figure 3. (A) Increase in fluorescence intensity at 525 nm, recorded every 1 min (0–
10 min) during real-time turn-on assay of probe (20 M) under varying
concentrations (0–50 nM) of ALP in Tris–HCl buffer (10 mM, pH 9.0) at 37 °C.
This research was supported by a Grant from the Fundamental
R&D Program for Core Technology of Materials funded by the Min-
istry of Knowledge Economy, Republic of Korea, and by a National
Research Foundation (NRF) Grant funded by the Korea government
(MEST) (No. 2010-0017220 and 2010-0010070).
1
l
Excited at 340 nm. (B) Relative fluorescence intensity at 525 nm as a function of
concentration of ALP. Incubation time = 10 min. F0 and
F
correspond to the
fluorescence intensity of probe
respectively.
1 in the absence and the presence of ALP,
Supplementary data
of the Michaelis constant Km = 1.90 lM and the pseudo-first-order
Supplementary data (detailed experimental procedures, addi-
tional UV–visible and emission spectra, enzyme assay studies)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.07.021.
catalytic constant kcat = 0.09 sꢀ1. Enzymatic efficiency for probe 1,
as estimated by a kcat/Km = 4.7 ꢂ 104 Mꢀ1 sꢀ1, was higher than that
of a commercially available reference substrate, 4-methylumbel-
liferyl phosphate (4-MUP, kcat/Km = 2.80 ꢂ 104 Mꢀ1 sꢀ1
) under
identical assay conditions.
References and notes
Next, we investigated the utility of probe 1 for the evaluation of
the ALP inhibitor. The inhibition of ALP activity was performed
with a well-known ALP-inhibitor,18 levamisole, at pH 9.0. ALP
was pre-incubated with different concentrations of the inhibitor
for 10 min at 25 °C. The inhibitor-pretreated ALP-solution was then
added to probe 1 in an aqueous solution, and the enzyme-
catalyzed hydrolysis reaction was monitored by measuring the
fluorescence intensity at 525 nm. As shown in Figure 4, the
enhancement of the fluorescence intensity of probe 1 was inhibited
1. Herz, F. Experientia 1985, 41, 1357.
2. Fernley, H. N. Enzyme 1971, 4, 417.
3. McComb, R. B.; Bowers, G. N.; Posen, S., Jr. Alkaline Phosphatase; Plenum Press:
New York, 1979.
4. Paragas, V. B.; Kramer, J. A.; Fox, C.; Haugland, R. P.; Singer, V. L. J. Microsc. 2002,
206, 106.
5. Goltzman, D.; Miao, D. Encyclopedia Endocr. Dis. 2004, 1, 164.
6. Ooi, K.; Shiraki, K.; Morishita, Y.; Nobori, T. J. Clin. Lab. Anal. 2007, 21, 133.
7. Wolf, P. L. J. Clin. Lab. Anal. 1994, 8, 172.
8. Berg, S.; Luneberg, E.; Frosch, M. Mol. Cell. Probes 1996, 10, 7.
9. Hasegawa, T.; Sugita, M.; Takatani, K.; Matsuura, H.; Umemura, T.; Haraguchi,
H. Bull. Chem. Soc. Jpn. 2006, 79, 1211.
10. Kamiya, M.; Urano, Y.; Ebata, N.; Yamamoto, M.; Kosuge, J.; Nagano, T. Angew.
Chem., Int. Ed. 2005, 44, 5439.
11. Pla-Dalmau, A. J. Org. Chem. 1995, 60, 5468.
12. Qian, Y.; Li, S.; Zhang, G.; Wang, Q.; Wang, S.; Xu, H.; Li, C.; Li, Y.; Yang, G. J.
Phys. Chem. B 2007, 111, 5861.
in a dose-dependent fashion. In particular, the addition of 75 lM
levamisole entirely blocked the increase of fluorescence intensity,
confirming that the enzymatic activity of ALP was required to
hydrolyze probe 1 and result in fluorescent product 2. The IC50
was determined to be 13.3 lM for levamisole, which is in good
agreement with the reported literature value determined by other
methods.19 These results highlight the utilities of probe 1 for both
ALP activity assays and the evaluation of inhibitor.
13. Kwon, J. E.; Park, S. Y. Adv. Mater. 2011, 23, 3615.
14. Tanaka, K.; Kumagai, T.; Aoki, H.; Deguchi, M.; Iwata, S. J. Org. Chem. 2001, 66,
7328.

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15. Baranov, M. S.; Lukyanov, K. A.; Borissova, A. O.; Shamir, J.; Kosenkov, D.;
Slipchenko, L. V.; Tolbert, L. M.; Yampolsky, I. V.; Solntsev, K. M. J. Am. Chem.
Soc. 2012, 134, 6025.
17. Under the assay conditions, compound 2 remains fully homogeneous, as
evidenced by the lack of aggregation features in its spectra and picture (Fig. 2).
18. Felix, R.; Fleisch, H. Biochem. J. 1979, 183, 73.
16. Helal, A.; Thao, N.; Lee, S.; Kim, H.-S. J. Inclusion Phenom. Macrocyclic Chem.
2010, 66, 87.
19. Sergienko, E. A.; Milan, J. L. Nat. Protoc. 2010, 5, 1431.