Design and synthesis of an ER-specific fluorescent probe based on carboxylesterase activity with quinone methide cleavage process

Article abstract of DOI:10.1016/j.bmcl.2011.04.066

CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subsequent hydrolytic activation by CEs causes spontaneou

Full text of DOI:10.1016/j.bmcl.2011.04.066

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3206–3209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of an ER-specific fluorescent probe based
on carboxylesterase activity with quinone methide cleavage process
⇑
Wataru Hakamata , Aki Machida, Tadatake Oku, Toshiyuki Nishio
Department of Chemistry and Life Sciences, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 February 2011
Revised 8 April 2011
Accepted 13 April 2011
Available online 22 April 2011
CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new
mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subse-
quent hydrolytic activation by CEs causes spontaneously quinone methide cleavage, resulting in bright
red fluorescence in ER with high specificity. Probe 1 was developed for CE activity imaging and inhibitor
screening at the cellular level.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Carboxylesterase
Fluorescent probe
Quinone methide cleavage
Resorufin
In mammals, CEs (EC 3.1.1.1) are expressed in numerous tis-
sues.1 CEs hydrolyze various prodrugs, including the anti-influenza
prodrug oseltamivir, the anti-thrombogenic agent clopidogrel, the
cholesterol reduction drug lovastatin, and the b-blocker esmolol.2
The hydrolytic activity of CEs has important roles in drug protec-
tion and metabolism,3 yet fluoresces probes for these enzymes to
study these important processes at the cellular level are insuffi-
cient. In addition, the development of inhibitors of CEs has many
potential uses, including increasing the drug lifetime and improv-
ing, delaying, controlling, and specifically expressing the action of
the parent drug using a prodrug.
CE-activated fluorescent probes that are commercially avail-
able, mainly ester-protected fluorescein derivatives such as fluo-
rescein diacetate, carboxy fluorescein diacetate, and calcein-AM,
Figure 1. Schematic illustration of the fluorescence revealing by quinone methide-type cleavage.
Abbreviations: CE, carboxylesterase; ER, endoplasmic reticulum; TBSCl, tert-butyldimethylchlorosilane; DMF, N,N-dimethylformamide; TBAF, tetrabutyl ammonium
fluoride; THF, tetrahydrofuran; TBP, tributylphosphine; ADDP, 1,10-(azodicarbonyl)dipiperidine.
⇑
Corresponding author.
E-mail address: hakamata.wataru@nihon-u.ac.jp (W. Hakamata).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.066

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Figure 2. Synthetic scheme of probe 1.
reduced fluorescence of the background obtained from cells. Probe
1 is used as a fluorescent dye in cells as it has appropriate mem-
brane permeability. Waring reported that a log D value of >1.7 is
required for compounds of molecule weight 350–400 for good
membrane permeation.4 The designed probe 1, which has a molec-
ular weight of 389 and a c log D value of 4.87 at pH 7–8,5 will have
good membrane permeability.
Details of the synthetic scheme of 1 are outlined in Figure 2. In
the final step, Mitsunobu reaction conditions, using compound 56
and resorufin, yielded the respective product 1.7 The overall four-
step synthesis had a 50.0% yield from starting material. To be a
CE activity imaging tool in cells, probe 1 should not have emission
spectra under resorufin excitation conditions at 571 nm. To ob-
serve the emission spectra of resorufin and 1 in PBS(-) at 571 nm,
these emission spectra were measured and indicated no emission
spectra from 1.8
We investigated the release of resorufin from 1 activated by the
porcine liver esterase in vitro and observed clearly the release of
the fluorescence signal of resorufin when probe 1 was co-incu-
bated with the esterase (Fig. 3). In that reaction, the enzyme show
appropriate activity, 0.05 unit/mg protein, against 1.9 These results
show that the enzyme transformation of 1 to release resorufin is
through a tandem reaction, ester hydrolyzation, and spontaneous
quinone methide cleavage, at 37 °C in phosphate buffer at pH 8.0.
The most important application for 1 is as a CE activity imaging
tool in cells. To test this capability, we tried to transfer 1 into cells
across membranes, and the imaging potential of 1 based on CE
activity was then evaluated in cell culture using a human fibrosar-
coma cell line, HT-1080, and kidney cells of the African green mon-
Figure 3. Esterase-assisted probe activation. Hydrolysis of probe
without porcine liver esterase at 37 °C.
1 with and
key, COS-1. Following incubation of both cell lines with 1 (50 lM)
for 120 min and Hoechst 33258 as the nuclear fluorescence dye,
phase-contrast microscopy images and fluorescence images were
taken by a fluorescence microscope.10 The fluorescence signal of
1 indicated intracellular accumulation of resorufin around the nu-
cleus which was dyed by Hoechst 33258, as shown in Figure 4. To
specify the fluorescence dyeing area of 1, the ERs of both cell lines
were dyed with 1 and ER-Tracker Blue–White DPX, a highly selec-
tive dye for the ER. The results show that the fluorescence dyeing
areas of 1 and ER-Tracker complementally overlapped, as shown
in Figure 5. These images indicated that 1 had good selectivity
against ERs of both cell lines with bright red fluorescence.
Clearly, probe 1 is feasible as an ER-specific fluorescent probe
based on CE activity: in particular, the selectivity for ER dyeing
was high, whereas ester-protected fluorescein derivatives, which
were activated by CEs, showed dyeing ability for cytoplasm. Previ-
ous research on the location of CEs in human cells show that CEs
are located in ER11 and cytoplasm.12 These results showed that
probe 1 can enhance the importance and usefulness of CE activity
have been applied to studies of living cell labeling, cell viability,
and cytotoxicity for a variety of cells. After permeation of protected
fluorescein derivatives into cells, the ester bonds can be hydro-
lyzed by CEs. Hydrolysis often results in direct conversion of the
protected fluorescein derivatives into fluorescent forms that are re-
tained and dye the cell cytoplasm green.
Here we describe the design, synthesis, photochemical, and
biological properties, and cell imaging of resorufin releasing
fluorescence probe 1 in Figure 1. The fluorogenic chemical trans-
formation of 1 triggered by the CEs in cells is through a tandem
reaction, ester hydrolyzation, and quinone methide-type cleavage
reaction, which are spontaneous and irreversible at physiological
temperature in aqueous media (also see Fig. 1). Resorufin is the
parent fluorescent used widely in cell imaging, and has an emission
spectrum at 585 nm to long wavelength excitation at 571 nm. The
major benefits of long wavelength excitation include much

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W. Hakamata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3206–3209
Figure 4. Images of HT1080 cells (a–d) and COS-1 cells (e–h). The phase-contrast images are a and e. The images of 1, in red, are b and f. The images of Hoechst 33258, in blue,
are c and g. The overlaid images of 1 and Hoechst 33258 are d and h.
Figure 5. Images of HT1080 cells (a–c) and COS-1 cells (d–f). The phase-contrast images are a and d. The images of 1, in red, are b and e. The images of ER-Tracker Blue–White,
in blue, are c and f.
imaging, functional analysis of CEs, and inhibitor screening at the
cellular level.
References and notes
1. (a) Satoh, T.; Hosokawa, M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 257; (b)
Satoh, T.; Hosokawa, M. Chem. Biol. Interact. 2006, 162, 195.
2. Wheelock, C. E.; Nakagawa, Y. J. Pestic. Sci. 2010, 35, 215.
3. Imai, T.; Hosokawa, M. J. Pestic. Sci. 2010, 35, 229.
Acknowledgments
4. Waring, M. J. Expert Opin. Drug Disc. 2010, 5, 235.
This research was partly supported by the Ministry of
Education, Science, Sports and Culture, a Grant-in-Aid for Young
Scientists (B)(No. 17790097) to W.H. and Health and Labour
Sciences Research Grants for Research on HIV/AIDS to W.H. from
the Ministry of Health, Labour and Welfare, Japan.
5. Advanced Chemistry Development Inc., Structure Design Suite Ver. 12.
6. Briggs, A. D.; Camplo, M.; Freeman, S.; Lundströmb, J.; Brian, G. P. Tetrahedron
1996, 52, 14937.
7. Synthesis of 1. To a solution of benzyl alcohol derivative 5 (107 mg, 0.55 mmol)
in dry THF 200 mL and the mixture was added ADDP (831 mg, 3.3 mmol), TBP
(814 lL, 3.3 mmol), and resorufin (141 mg, 0.66 mmol). After stirring the

W. Hakamata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3206–3209
3209
mixture at rt for 30 min, the resulting mixture was concentrated and then the
resulting residue was purified by column chromatography on silica gel (1:1
pH 8.0 (100
lL) followed by incubation at 37 °C. The assays were followed by
the monitoring fluorescence intensity change of resorufin. One unit of enzyme
hexane–ethyl acetate). Obtained fractions containing probe
1
were
activity was defined as the amount of enzyme required to liberate 1
resorufin/min.
lmol of
concentrated and then the resulting residue was purified by column
chromatography on silica gel (20:1 dichloromethane–methanol) to afford
191 mg (89.0%) of probe 1 and was recrystallized from CH2Cl2/hexane to give a
light orange solid: mp 266–267 °C; 1H NMR (500 MHz, CDCl3): d 1.04 (t, 3H,
J = 7.5, –CH3), 1.78 (qt, 2H, J = 7.5, –CH2–CH3), 2.55 (t, 2H, –CO–CH2–), 5.15 (s,
2H, benzyl position), 6.32 (d, 1H, J = 2.3), 6.83 (dd, 1H, J = 2.3, 9.7), 6.87 (d, 1H,
J = 2.9), 7.00 (dd, 1H, J = 2.6, 8.9), 7.13 (d, 2H, J = 8.0), 7.41 (d, 1H, J = 9.8), 7.45
(d, 2H, J = 8.6), 7.71 (d, 1H, J = 9.2), 13C NMR (125 MHz, CDCl3): d 13.64 (–CH2–
CH3), 18.43 (–CH2–CH2–), 36.19 (–CO–CH2–), 70.25 (benzyl position), 101.03,
106.78, 114.22, 122.07, 128.51, 128.69, 131.63, 132.82, 134.28, 134.70, 145.58,
145.74, 149.77, 150.79, 162.46, 172.09 (–C@O, butyl ester), 186.33 (–C@O,
resorufin), ESI-MS (Positive): m/z = 390 [M+H]+. Anal. Calcd for C23H19NO5: C,
70.94; H, 4.92; N, 3.60. Found: C, 70.92; H, 4.91; N, 3.54.
10. Fluorescence microscopy studies. The human fibrosarcoma cell line, HT-1080
(RCB1956), and kidney cells of the African green monkey, COS-1 (RCB0143)
were provided by the RIKEN BRC through the National Bio-Resource Project of
the MEXT, Japan. HT1080 and COS-1 cells were cultured, medium was
exchanged for fresh medium, and then incubated at 37 °C in the presence of
50 lM of 1 for 120 min. After incubation, both cells were removed from the
medium and under went formalin fixation. In this culture condition, probe 1
showed no significant cytotoxicity composed to the trypan blue method. The
fluorescence signal of the cells was recorded using an BIOREVO BZ-9000
fluorescence microscope (KEYENCE Inc.) equipped with TRITC (exciter; 540/
25 nm, emitter; 605/55 nm) and DAPI (exciter; 360/40, emitter; 460/50 nm)
filter sets.
8. Photochemical characterization. The solutions of resorufin and probe 1 (10
l
M)
11. (a) Hosokawa, M. Molecules 2008, 13, 412; (b) Fukami, T.; Nakajima, M.;
Maruichi, T.; Takahashi, S.; Takamiya, M.; Aoki, Y.; McLeod, H. L.; Yokoi, T.
Pharmacogenet. Genomics 2008, 18, 911.
12. (a) Hennebelle, I.; Terret, C.; Chatelut, E.; Bugat, R.; Canal, P.; Guichard, S.
Anticancer Drugs 2000, 11, 465; (b) Humerickhouse, R.; Lohrbach, K.; Li, L.;
Bosron, W. F.; Dolan, M. E. Cancer Res. 2000, 60, 1189; (c) Takahashi, S.; Katoh,
M.; Saitoh, T.; Nakajima, M.; Yokoi, T. J. Pharm. Sci. 2008, 97, 5434.
in PBS(-) containing 0.1% DMSO, respectively. The fluorescence emission
spectra of these solutions were recorded with an excitation wavelength of
571 nm.
9. Enzyme assays. Probe 1 was prepared as a 1 mM solution in DMSO. Esterase
(from porcine liver, purchased from SIGMA) was prepared as a 16.5
solution in 200 mM Na phosphate buffer (pH 8.0). The assay were conducted by
adding esterase solution (85 L) to 1 (5 L) and 200 mM Na phosphate buffer,
lg/mL
l
l