An improved fluorogenic substrate for the detection of alkaline phosphatase activity

Article abstract of DOI:10.1016/j.bmcl.2013.02.063

We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter 3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for the enzyme.

Full text of DOI:10.1016/j.bmcl.2013.02.063

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2332–2335
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An improved fluorogenic substrate for the detection of alkaline
phosphatase activity
⇑
Jeesook Park, Youngmi Kim
Department of Chemistry, Institute of Nanosensor and Biotechnology, Dankook University, 126 Jukjeon-dong, Yongin-si, Gyeonggi-do 448-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative
spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate
specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter
3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for
the enzyme.
Received 26 December 2012
Revised 31 January 2013
Accepted 13 February 2013
Available online 24 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Alkaline phosphatase
Fluorogenic substrate
Fluorometric assay
Inhibitor assay
Turn-on probe
Alkaline phosphatases (ALPs), which catalyze the hydrolysis of
phosphate esters, are widely distributed in mammalian tissues,
and are present in high concentration in the bones, intestines, kid-
neys, placenta, and liver.1,2 ALP is not only the most commonly
used conjugated enzyme for enzyme immunoassays in molecular
biology,3,4 but is also routinely used as a diagnostic marker for sev-
eral human diseases, including hepatobiliary and bone disorders.5–
7 Accordingly, the development of convenient and sensitive molec-
ular probes capable of reporting ALP expression and its dynamic
activity in various biological systems will be extremely valuable
for studying detailed mechanisms of ALP activity regulation during
pathogenesis, and for the development of efficient diagnostic and
therapeutic agents. Several optical assays in combination with
chemiluminescent,8 chromogenic,9 or fluorescent probes10 have
been employed for the detection of ALP activity. Among them,
fluorescence-based detection methods are generally superior in
terms of sensitivity, spatial and temporal resolution, and ease of
use. In that context, nonpeptidic ALP fluorogenic substrates such
as 4-methyl-7-hydroxycoumarinyl phosphate (MUP), 3,6-fluores-
cein diphosphate (FDP), and 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) are in widespread use.11
development of efficient ALP fluorogenic probes that have higher
enzyme affinity, as well as the modulation of fluorogenic sub-
strates for enzyme specificity, we newly designed probe 1 that
incorporates a reactive p-hydroxybenzyl linker, as a spacer unit be-
tween the phosphate moiety and profluorescent compound. The
presence of a spacer between the recognition unit and the bulky
profluorescent compound has the benefit of lower steric hindrance
that increases its accessibility to the enzyme’s active site, conse-
quently improving the substrate’s affinity for the enzyme (i.e., low-
er the Michaelis constant, KM) while maintaining a dye platform as
signal transducing element. Herein, we report the synthesis and
photophysical characterization of 1. The hydrolysis of 1 by ALP
was monitored in fluorescence-based assays to assess its utility
as an indicator of ALP activity. In addition, the kinetic parameters
of the enzymatic hydrolysis of 1 were determined, and its potential
for the screening of ALP inhibitors was investigated.
The fluorescence of probe 1 is effectively quenched through
internal free rotation about the vinylene linker. ALP-catalyzed
P–O cleavage in probe 1 causes a chemical breakdown of the linker
via 1,6-elimination of the p-hydroxybenzylether derivative, fol-
lowed by a spontaneous intramolecular cyclization, leading to
the release of benzothiazolyl iminocoumarin 3, which is water-
insoluble and highly emissive upon photoexcitation (Scheme 1).
Probe 1 was easily prepared in a five-step synthesis as shown in
Scheme 2, and the structural identification of 1 was confirmed by
1H NMR, 13C NMR, and HR-MS. It is also noted that probe 1 is
highly soluble in water. Dye 3, the expected product resulting from
the enzymatic reaction was also independently synthesized
according to a previously reported method.12
Recently, we reported a sensitive ALP probe (4) in which P–O
bond cleavage is triggered by ALP-catalyzed hydrolysis, and a
subsequent intramolecular nucleophilic reaction results in the
highly emissive cyclized product, the benzothiazolyl iminocouma-
rin (3).12 As a continuation of our research program aimed at the
⇑
Corresponding author. Tel.: +82 31 8005 3156; fax: +82 31 8005 3148.
E-mail address: youngmi@dankook.ac.kr (Y. Kim).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.063

J. Park, Y. Kim / Bioorg. Med. Chem. Lett. 23 (2013) 2332–2335
2333
Scheme 1. Chemical structures of fluorogenic substrates 1 and 4, and the proposed sensing mechanism.
Scheme 2. Synthesis of probe 1: Reagents and conditions: (a) (EtO)2POCl, NaH, THF, room temperature, 24 h, 81%; (b) CBr4, PPh3, CH2Cl2, room temperature, 1 h, 88%; (c)
4-(diethylamino)salicylaldehyde, K2CO3, DMF, 60 °C, overnight 80%; (d) 2-benzothiazoleacetonitrile, iperidine, EtOH, room temperature, 6 h, 84%; (e) (i) TMSBr, CH2Cl2, 24 h,
rt (ii) aq NaOH, 27%.
The photophysical properties of probe 1 and dye 3 were initially
investigated to confirm their suitability as substrate for fluorogenic
reaction in aqueous buffer solution (10 mM Tris–HCl, pH 8). Probe
1 exhibited a maximum absorbance band centered at 470 nm
fluorescence change under the same conditions, implying insignif-
icant nonenzymatic hydrolysis of probe 1 (Fig. 1B). The nonemis-
sive nature of probe 1 and the significant fluorescence turn-on of
ALP-treated probe 1 are clearly visualized (Fig. 1A, inset).
In order to confirm the origin of the fluorescence increase
during the enzymatic reaction, we monitored the assay solution
(e
= 3.3 Â 104 MÀ1 cmÀ1) and a fluorescence maximum at kmax
=
530 nm with weak green emission (Ufl = 0.0035). The absorption
and fluorescence emission maxima of
3
were displayed at
by HPLC–MS. Incubation of probe 1 (10 lM) with ALP (100 nM)
470 nm (
e
= 2.8 Â 104 MÀ1 cmÀ1) and 530 nm, respectively, with a
gave rise to complete 1 ? 3 conversion in 30 min. The formation
of intermediates (2a and/or 2b) was not detected in our HPLCÀMS
experiments throughout the duration of the 1 ? 3 conversion
process (Fig. 2). This reveals that the fluorescence increase is attrib-
uted to the formation of expected product 3 through the sequence
of rapid self-immolative reaction after catalytic cleavage of the P–O
bond upon treatment of probe 1 with ALP, with a concomitant
increase in fluorescence.
Encouraged by these results, we further investigated the
ALP-catalyzed hydrolysis of probe 1 as a function of incubation
time at different ALP concentrations. Figure 3A shows the increase
in fluorescence intensity at 530 nm for solutions of probe 1 (5 lM)
incubated with ALP concentrations ranging from 0 to 30 nM, in
fluorescence quantum yield (Ufl) of 0.10, which was about 30 times
greater than that of fluorogenic substrate 1.
The hydrolysis of probe 1 by ALP was monitored by fluorimetry
(Fig. 1). In these experiments, probe 1 (5 lM) was incubated with
ALP (30 nM) in Tris–HCl buffer (10 mM, pH 8) at 37 °C, and the
emission spectra of the resulting solution were measured at
4 min intervals over 40 min with excitation at kex = 440 nm. The
fluorescence emission intensity at 530 nm gradually increased
with the incubation time from 0 to 40 min and reached a plateau
after 30 min ( Fig. 1A). An approximately 30-fold fluorescence
enhancement was obtained upon incubation with ALP (30 nM)
for 30 min. In contrast, it was observed that the fluorescence inten-
sity of probe 1 without addition of ALP displayed no noticeable
which fluorescence intensity was measured every 2 min. As

2334
J. Park, Y. Kim / Bioorg. Med. Chem. Lett. 23 (2013) 2332–2335
Figure 1. (A) Time-dependent emission spectra (kex = 440 nm) of probe 1 (5
lM) upon treatment with ALP (30 nM) in Tris–HCl buffer (10 mM, pH 8.0) at 37 °C. The spectra
were obtained every 4 min (0–40 min). Inset: Photographs of probe 1 (10 M) in the absence (left) and presence (right) of ALP (30 nM) after incubation for 40 min at 37 °C
l
under UV light (365 nm) illumination. (B) Relative fluorescence intensity (F/F0) at 530 nm with and without ALP (30 nM) as a function of incubation time.
intensity was obtained with the ALP incubation time of 30 min.
These observations indicate that 1 can be utilized as a probe to
assay ALP concentration quantitatively, thus achieving real-time
monitoring of enzyme activity. The detection limit for ALP using
probe 1 under optimized conditions was determined to be 1 Â 10
À10 M on the basis of the signal-to-noise ratio (S/N = 3), which is
impressively sensitive compared with existing fluorescent probes
for ALP detection.11
To directly compare enzyme specificity and kinetics between
probe 1 and the previously described 4, kinetic parameters for
the enzymatic hydrolysis reaction of 1 and 4 were determined un-
der the same assay conditions (see the Supplementary data). By
using Lineweaver–Burk analysis, we obtained the kinetic value of
KM = 0.90
(KM = 20.3
1. Thus, even the lower turnover number of 1 (kcat = 0.06 sÀ1
l
l
M for probe 1, which is smaller compared to probe 4
M), indicating that ALP has a higher affinity for probe
Figure 2. HPLC chromatograms of probe 1 without ALP treatment (top); with ALP
treatment for 30 min at 37 °C (middle); and 3 only (bottom). The samples were
analyzed by HPLC–MS (eluent A/B = 50:50, (A) deionized water with 1% acetic acid,
(B) acetonitrile, flow rate 0.3 mL/min, UV: 340 nm). The MW at the retention time of
8.5 min is 536.1, which corresponds to [M+H]+ for probe 1 and the MW at the
retention time of 2.2 min is 350.1, which corresponds to [M+H]+ for 3.
)
translated to the higher kcat/KM value of 6.2 Â 104 MÀ1 sÀ1 relative
to the kcat/KM of 4.5 Â 104 MÀ1 sÀ1 for 4. These results confirmed
that the incorporation of a spacer linker not only maintained, but
actually enhanced efficiency and specificity of fluorogenic sub-
strate 1 by the targeted enzyme ALP compared to that of 4, which
has a direct linkage between the profluorescent compound and
phosphate moiety. Better affinity of ALP for probe 1 is advanta-
geous for in vivo imaging as it allows the use of lower overall doses
of probe, thus preventing extended circulation that can possibly
cause cross-reactivity with other biomolecules.
[1] = [3] = 10 lM, [ALP] = 100 nM.
expected, the fluorescence intensity increased with increasing ALP
concentration within the measured range ( Fig. 3B). Subsequent
data analysis revealed a good linear relationship (R2 = 0.98) be-
tween the normalized fluorescence signal at 530 nm and the ALP
concentrations from 0 to 5 nM (Fig. 3B, inset), where the emission
Figure 3. (A) Increase in fluorescence intensity at 530 nm recorded every 2 min (0–40 min) during real-time turn-on assay of probe 1 (5 lM) under varying ALP
concentrations (0–30 nM) in Tris–HCl buffer (10 mM, pH 8.0) at 37 °C. kex = 440 nm. (B) Plot of relative fluorescence intensity (F/F0) at 530 nm versus ALP concentration
(0–30 nM). Inset shows the linear relationship between fluorescence intensity and ALP concentration (0–5 nM). Incubation time = 30 min. F0 and F correspond to the
fluorescence intensity of probe 1 in the absence and the presence of ALP, respectively. Data points are averages of three independent measurements.

J. Park, Y. Kim / Bioorg. Med. Chem. Lett. 23 (2013) 2332–2335
2335
Figure 4. (A) Inhibition assay of ALP activity by levamisole showing emission intensity of probe 1 as a function of inhibitor concentration (0–5 mM). ALP (30 nM) was
incubated with the inhibitor for 120 min at 25 °C before addition of probe 1 (5 M). After 30 min of incubation at 37 °C, emission intensity at 530 nm was recorded (kex
440 nm). (B) Relative activity of ALP versus concentration of levamisole at 38 min time point. IC50 value was determined as 71 M for levamisole.
l
l
Next, the potential utility of probe 1 for the screening of ALP
inhibitor was investigated. The inhibition of ALP activity was
tested with levamisole—a known ALP inhibitor—in Tris–HCl buffer
(10 mM, pH 8).13 ALP was preincubated with different concentra-
tions of inhibitor for 120 min at 25 °C. The solution of ALP pre-
treated with the inhibitor was then added to probe 1 in aqueous
solution and the enzyme reaction was monitored by measuring
the fluorescence intensity at 530 nm. As shown in Figure 4, the
enhancement of fluorescence intensity of probe 1 was inhibited
istry of Knowledge Economy, Republic of Korea, and by a National
Research Foundation (NRF) grant funded by the Korea government
(MEST) (No. 2012R1A1A2038694).
Supplementary data
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.2013.02.063.
in a dose-dependent manner. In particular, addition of 5000 lM
of levamisole entirely blocked the fluorescence increase, providing
confirmation that the enzymatic activity of ALP was required for
hydrolysis of probe 1. The IC50 was calculated to be 71 lM for lev-
References and notes
[1]. Millan, J. L. Mammalian Alkaline Phosphatases. From Biology to Applications in
Medicine and Biotechnology; Wiley-VCH: Weinheim, Germany, 2006. pp 1–
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[2]. McComb, R. B.; Bowers, G. N.; Posen, S. Alkaline Phosphatase; Plenum: New
York, 1979.
[3]. Paragas, V. B.; Kramer, J. A.; Fox, C.; Haugland, R. P.; Singer, V. L. J. Microsc.
2002, 206, 106.
[4]. Yam, L. T.; Khansur, T.; Tavassoli, M. Pathol. Immunopathol. Res. 1988, 7, 169.
[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.
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M.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. ChemBioChem 2008, 9, 714.
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[10]. Kamiya, M.; Urano, Y.; Ebata, N.; Yamamoto, M.; Kosuge, J.; Nagano, T. Angew.
Chem. 2005, 117, 5575.
[11]. Haughland, R. P. In Handbook of Fluorescent Probes and Research Products;
Vani, S., Ed., 11th ed.; Molecular Probes, Inc.: Eugene, OR, 2010; pp 427–441.
[12]. Kim, T.-I.; Kim, H.; Choi, Y.; Kim, Y. Chem. Commun. 2011, 47, 9825.
[13]. Chang, L.; Duy, D. L.; Médarek, S.; Popowycz, F.; Pellet-Rostaing, S.; Lemaire,
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amisole, which is in good agreement with the reported literature
value determined by other methods.12 These results emphasize
that probe 1 can be a useful tool for ALP activity assays, as well
as the screening of inhibitors.
In summary, we have designed and synthesized an efficient
fluorogenic probe 1, suitable for real-time dynamic monitoring of
ALP activity. In this probe, a self-immolative spacer was employed
to improve enzyme’s affinity for the substrate. Fluorogenic assays
proved that probe 1 exhibited high enzyme affinity with the ability
to detect ALP at concentrations as low as 0.1 nM. In addition, the
potential utility of 1 in the screening of ALP inhibitors was demon-
strated with levamisole. This work demonstrates the utility of
self-immolative linkers in the design of fluorogenic substrates for
enzymes possessing a comparatively difficult to access active cata-
lytic site and opens the possibility for the improvement of existing
fluorogenic substrates for various enzymes.
Acknowledgments
This research was supported by a grant from the Fundamental
R&D Program for Core Technology of Materials funded by the Min-