Heavy metal-free 19F NMR probes for quantitative measurements of glutathione reductase activity using silica nanoparticles as a signal quencher

Article abstract of DOI:10.1016/j.bmc.2011.11.026

For the quantitative assessment of the glutathione reductase (GR) activity with a ¹⁹F NMR spectroscopy, we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers is highly restricted, and the magnitude of ¹⁹F NMR signals from the perfluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from the surfaces of the nanoparticles. Consequently, the ¹⁹F NMR signals of perfluorinated dendrimers were recovered. The enzymatic activity of GR was determined from the increase of the magnitude of ¹⁹F NMR signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value determined from our method showed a good agreement with that from the conventional method.

Full text of DOI:10.1016/j.bmc.2011.11.026

Bioorganic & Medicinal Chemistry 20 (2012) 96–100
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Heavy metal-free 19F NMR probes for quantitative measurements
of glutathione reductase activity using silica nanoparticles as a signal quencher
⇑
Kazuo Tanaka, Narufumi Kitamura, Yoshiki Chujo
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e i n f o
a b s t r a c t
For the quantitative assessment of the glutathione reductase (GR) activity with a 19F NMR spectroscopy,
we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble
perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation
of the perfluorinated dendrimers is highly restricted, and the magnitude of 19F NMR signals from the per-
fluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the
reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from
the surfaces of the nanoparticles. Consequently, the 19F NMR signals of perfluorinated dendrimers were
recovered. The enzymatic activity of GR was determined from the increase of the magnitude of 19F NMR
signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules
under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value
determined from our method showed a good agreement with that from the conventional method.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 26 September 2011
Revised 14 November 2011
Accepted 15 November 2011
Available online 23 November 2011
Keywords:
Glutathione reductase
19F NMR
Silica nanoparticle
Molecular probe
Quantitative assay
1. Introduction
advance, the quantitative evaluation of the enzymatic activity
has been reported.8 The stimuli-responsive MR probes can be clas-
Glutathione reductase (GR) is the NADPH-dependent enzyme
which catalyzes the reduction of oxidized glutathione (GSSG) to re-
duced glutathione (GSH). The GSH production plays a central role
in buffering an intercellular redox condition and in activating the
anti-oxidation system.1 In addition, it has been reported that a sig-
nificant enhancement of the GR activity was observed in malignant
tumor cells.2 Therefore, the quantification of the GR activity at the
deep spot inside vital organs is of great significance for further
understanding of the stress responses and carcinogenesis. On the
other hand, the standard assay for measuring the GR activity is
to follow the decrease in absorbance of NADPH at 340 nm.3
Because of the low light permeability through a body, it is far
difficult to apply the conventional methods for the in vivo assay.
Hence, the new methodology is desired for the quantitative analy-
sis of the GR activity.
Magnetic resonance imaging (MRI) contrast agents provide us
the information from the deep spots inside vital organisms. In
the recent years, a variety of fluorinated compounds have been
developed as a versatile probe with a 19F MRI or a 19F NMR spec-
troscopy for monitoring the biological molecules and events such
as gene repression,4 protein existence,5,6 enzymatic activity,7–10
environmental alteration,11–16 and biological reactions.17 In
sified in two groups. One is based on the metal complexes in which
the center metal ion such as a gadolinium or a manganese ion can
show the different contrast ability by recognizing the target.18
However, GR itself would be activated in the presence of these
heavy metal species in the cells.1 Therefore, it should be difficult
to eliminate errors on the detection using the metal complex-
based MR probes. Another group is categorized into the surface-
modified nanoparticles (NPs) such as superparamagnetic iron
oxide.19–21 By changing the aggregation/dispersion states of the
NPs, the contrast ability can be greatly controlled.21 However, be-
cause of difficulties to maintain the uniform morphology and par-
ticularly assemble the NPs in the cells, there are many problems to
be overcome for realizing the quantitative assay with the NP-based
probes. We have previously reported the silica NP can work as a
quencher for the 19F NMR signals.7,8 Although the probe is com-
posed of NPs, the interparticle interaction was not necessary for
the target detection. Consequently, the quantitative evaluation of
enzymatic reactions can be accomplished with this system.8 In
addition, silica is known to be a biocompatible material.22 It can
be expected to avoid the perturbation originated from the antiox-
idant activity. Because of these advantages, we aimed to construct
the assay system for the GR activity using the modified silica NPs.
Herein, we report the quantitative assay in 19F NMR for the GR
activity. Perfluorinated dendrimers were anchored on silica NPs via
the disulfide linkers (Scheme 1). On silica NPs, the molecular rota-
tion of the perfluorinated dendrimers should be highly restricted,
⇑
Corresponding author. Tel.: +81 75 383 2604; fax: +81 75 383 2605.
E-mail address: chujo@chujo.synchem.kyoto-u.ac.jp (Y. Chujo).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.026

K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 96–100
97
2.3. Compound 225
To the solution of 3,30-dithioldipropionic acid (1 g, 4.75 mmol)
in dichloromethane (20 mL), 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (WSC) (2.16 g, 10.5 mmol) and
N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added. The mix-
ture was stirred for 2 h at room temperature. The solution was con-
centrated in vacuo, and the obtained residue was subjected to silica
column chromatography (ethyl acetate/hexane = 1:5) to give the
NHS ester 2 (1.63 g, 85%). 1H NMR (DMSO-d6, 400 MHz) d 3.16
(m, 8H), 2.82 (s, 8H). FAB (DMSO-d6) [(M+H)+] calcd 405, found
405.
Scheme 1. Schematic mechanism for GR activity using the NP probe.
2.4. Preparation of the amino-presenting silica NPs7,26
and the NMR signals from the perfluorinated dendrimers can be
suppressed. Corresponded to progress the enzymatic reaction with
GR in the presence of GSH and b-NADPH as a cofactor, the perfluo-
rinated dendrimers were released from the surfaces, and the NMR
signals from perfluorinated dendrimers were enhanced due to the
recovery of the molecular rotation. Consequently, the enzymatic
activity of GR can be evaluated as the increases of the magnitude
of 19F NMR signals. Furthermore, the values of the GR activities
determined from the conventional and our method were compared
using the cell lysate to demonstrate the feasibility of the probe in
the presence of miscellaneous molecules under bio-mimetic
conditions.
A solution containing 2 mL of tetraethoxysilane, 1 mL of 3-ami-
nopropyltriethoxysilane, 7 mL of water, and 2 mL of ammonium
hydroxide in 50 mL of ethanol was stirred at ambient temperature
for 16 h, and then the white precipitate was separated by centrifu-
gation. After washing with ethanol three times, the particles
(152 11 nm diameter) were obtained as a white powder. To
determine the amounts of the reactive amino groups at the surface
of the particles, we treated the silica nanoparticles with ethyl tri-
fluoroacetate. After washing with methanol, the particles were dis-
solved in 1 N aqueous sodium hydroxide, and the peak height was
compared to the standard samples. Consequently, it was found that
960 nmol of trifluoroacetyl groups were attached to the particles.
2. Experimental section
2.1. General
2.5. Preparation of the NP probes
To the solution of the NHS ester 2 (404 mg. 1 mmol) and trieth-
ylamine (1 mL) in dichloromethane, 100 mg of amino-presented
silica NPs were added. After 1 h stirring at room temperature,
NPs were centrifuged and washed with dichloromethane. After
drying in vacuo, the solution of 1 and triethylamine in methanol
was added. The mixture was stirred at room temperature under
ultrasound irradiation. After 1 h stirring, the NPs were centrifuged
and washed with methanol and dichloromethane. The white pow-
der was obtained after drying (167 10 nm diameter from TEM).
To estimate the amount of 1 on the NP (18 nmol/mg, F atom:
216 nmol/mg), the 19F NMR spectrum of the solution containing
the NP probe dissolved in 1 N aqueous sodium hydroxide was
compared to the spectra of the standard samples with various
concentrations of trifluoroacetic acid.
1H and 13C NMR spectra were measured with a JEOL EX-400
spectrometer operating at 400 MHz for 1H and 100 MHz for 13C.
19F and 29Si NMR spectra were measured with a JEOL JNM-A400
spectrometer operating at 370 MHz for 19F and 80 MHz for 29Si.
Coupling constants (J value) are reported in Hertz. The chemical
shifts in 19F NMR are expressed in ppm downfield from trifluoro-
acetic acid as an external reference. Masses were determined with
a MALDI-TOF mass spectroscopy (acceleration voltage 21 kV, neg-
ative mode) with 2,5-dihydroxybenzoic acid (DHB) as a matrix.
Transmission electron microscopy (TEM) was performed using a
JEOL JEM-100SX microscope operating at 100 kV. The sizes of the
particles were determined as an average of 100 particles in the
TEM images. Emission spectra of the samples were monitored by
a Perkin Elmer LS50B fluorometer at 25 °C using a 1 cm path length
cell. The excitation bandwidth was 0.1 nm. The emission band-
width was 0.1 nm. GR, GSH, GSSG, and b-NADPH were purchased
from Wako Pure Chemicals Industries, Ltd (Osaka, Japan).
2.6. Reaction conditions with the NP probe
The 500 lL of samples containing the NP probe (2.5 mg,
1.08 mM F atom) with 500 mM NaBH4, 500 mM DTT, 1 mM b-
NADPH, 1 mM GSH, or 1 mM GSSG in PBS were incubated at
37 °C for 30 min. In the case of enzymatic reactions with GR, the
2.2. Perfluorinated dendrimer, F-POSS, 17
To a suspension of octaammonium polyhedral oligomeric sils-
esquioxane (POSS)23,24 (1 g, 0.852 mmol) and ethyl trifluoroacetate
(406 lL, 3.41 mmol) in methanol (20 mL), triethylamine (2 mL,
NP probe (2.5 mg) was incubated in 500 lL of the reaction solu-
tions containing 1 mM b-NADPH and 0.5 mM GSSG with various
concentrations of GR in PBS at 37 °C. The reaction was terminated
by centrifuging the reaction mixture, and then the supernatants
were analyzed by a 19F NMR spectroscopy.
14.4 mmol) was added, and the reaction mixture was stirred at
room temperature for 3 h. The resulting mixture was evaporated,
and the crude 1 was directly used in the next step. The analyzed
sample as a clear oil was obtained after dialysis (895 mg, 83%).
1H NMR (D2O, 400 MHz) d 3.26 (t, 8H, J = 7.0 Hz), 2.94 (t, 8H,
J = 7.0 Hz), 1.70 (br s, 8H), 1.61 (br s, 8H), 0.64 (m, 16H). 13C NMR
(D2O, 100 MHz) d 155.9, 117.4, 41.52, 40.92, 21.76, 20.05, 8.77,
8.72. 29Si NMR (D2O, 80 MHz) d ꢀ68.7, ꢀ68.4. 19F NMR (D2O,
2.7. Preparation for HeLa cell lysate
HeLa cells (1.0 ꢁ 106 cells) were cultured in a dish (90% conflu-
ent in
u
100 mm dishes) and washed twice with ice-cold PBS(ꢀ).
The cell lysate was then harvested with 2 mL of ice-cold CelLytic
M Cell Lysis Reagent (SIGMA-ALDRICH, Inc., St. Louis, MO), kept at
ambient temperature for 15 min, and centrifuged at 12,000 rpm
for 5 min to remove the cell debris. The enzymatic volume activity
(U/mL) of the supernatant was measured from the conversion rate
373 MHz)
d
ꢀ75.4. MALDI-TOF [(M+H)+], [POSS-TFA2] calcd
1074.52, found 1073.29, [POSS-TFA3] calcd 1170.53, found
1170.00, [POSS-TFA4] calcd 1266.53, found 1266.16, [POSS-TFA5]
calcd 1362.54, found 1361.76.

98
K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 96–100
of NADPH. The solutions (100
l
L) containing 1
l
L of the cell lysate
the standard line. Initial velocities (vi) were determined from the
and 1 mM NADPH in PBS were incubated at 37 °C, and the absorp-
tion changes at 340 nm were monitored. The volume activity was
estimated from fitting of the standard curve with GR.
slopes of the increases of the 19F NMR signal intensities from 0 to
15 min in Figure 3. Figure 4 represents the plots of these vi values
to the enzymatic concentrations and the approximate line with the
plots in lower concentration region. The vi value determined from
the lysate sample was fitted on the approximate line, and the enzy-
matic activity was estimated as an x value.
2.8. 19F NMR measurements for determining reaction yields and
velocities in enzymatic reactions
Relaxation times of 1 and trifluoroacetic acid in 19F NMR were
3. Results and discussion
taken with the following parameter sets; relaxation delay, 15 s;
pulse width (90°), 51 ls; acquisition time, 88 ms; scan time, 4
Scheme 1 illustrates the detection mechanism using the NP-
based probe for enzymatic activity in this study. The probes consist
of three significant components, the signal unit, the silica NP as a
quencher for NMR signals, and the linker to release the signal unit
after the target recognition. As a 19F NMR signal unit, we used
water-soluble perfluorinated POSS (F-POSS) which can be a good
scaffold to construct functional 19F MR probes by the modification
with the signal regulation modulus because of high water-solubil-
ity and stability under biological conditions.7,16,17 The silica NPs
work as a quencher for 19F NMR signals of the surface-tethered
F-POSS molecules.7,8 On the surface of the NP, the molecular rota-
tion of F-POSS should be restricted like in the solid phase, which
suppresses the NMR signals. Indeed, the T1 and T2 relaxation times
of fluorine atoms were determined to be 2.90 and 1.94 s in trifluo-
roacetic acid and 1.067 and 0.992 s in F-POSS, respectively. On the
other hand, those values in the NP probes were not determined be-
cause of shorter transverse relaxation times and the low signal
intensity of the 19F resonance. These results clearly indicate that
the silica particles can accelerate the relaxation for 19F NMR signals
times. 19F longitudinal (T1) relaxation data were collected with
10 delay times (1, 5, 10, 20, 50, 100, 250, 500, 1000, and
3000 ms) using a standard 1D inverse recovery pulse sequence.
19F transverse (T2) relaxation data were collected with nine delay
times (400, 500, 600, 700, 800, 900, 1000, 2000, and 8000 ms)
using a standard 1D Carr–Purcel–Meiboom–Gill pulse sequence.
Resonance intensities in relaxation experiments were measured
and fit to an exponential function. The reaction yields were deter-
mined as below: 19F NMR spectra for the analysis of synthetic com-
pounds were taken at 25 °C with the following parameter sets;
relaxation delay, 6 s; pulse width (45°), 12 ls; acquisition time,
88 ms; scan time, 8 times; receiver gain 30. Totally 1 min was re-
quired for 1D NMR acquisition. Standard solutions containing 1
with the concentration region 100 lM to 1 mM were prepared,
and the peak height was determined. The linear relationship was
observed between the concentration and the peak height from tri-
fluoromethyl groups in 1 in the 19F NMR spectra. The reaction
yields were determined by fitting the observed peak heights to
Scheme 2. Synthetic scheme of the NP probe. Reagents and conditions: (a) Ethyl trifluoroacetate, triethylamine, methanol, rt, 3 h, 83%; (b) N-hydroxysuccinimide, WSC,
dichloromethane, rt, 24 h, 81%; (c) amino-modified silica NPs, 2, DMF, rt, 2 h; (d) F-POSS, triethylamine, methanol, rt, 2 h.

K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 96–100
99
and work as a quencher for NMR signals. By the cleavage of the
linker, F-POSS can be released, and the 19F NMR signals of F-POSS
are promised to be enhanced. The disulfide-containing compound
was used as the linker between F-POSS and the surface of NP.
Hence, the enzymatic reaction can be monitored by the increase
of the signal molecule, F-POSS in 19F NMR measurements.
F-POSS was synthesized using octaamino-POSS as a starting
material by the introduction of trifluoroacetyl groups according
to the previous reports.7,16,17 We prepared amino-presenting silica
NPs averaging 160 nm in diameter with the Stöber method,7,26 and
then the amino groups were modified with the disulfide linker
(Scheme 2). Subsequently, F-POSS was reacted with another end
of the linker on the surface of NPs. After washing with water and
methanol thoroughly, the monodispersed modified NPs were ob-
tained (Fig. 1). From the measurements of the particle radii with
TEM, the slight increases were observed. In addition, less signifi-
cant increases were observed from DLS measurements
(287.5 45.6 nm). These data indicate that the inter-particle cross-
linking should hardly occur in the product. Precipitation or aggre-
gation hardly occurred during all reactions.
Figure 1. TEM image of the F-POSS-coated silica NPs. The scale bar represents
200 nm length.
To estimate the amount of F-POSS on the NP, the 19F NMR signal
intensities of the solution containing the NP probe dissolved in 1 N
aqueous sodium hydroxide were compared to those of the stan-
dard samples with various concentrations of trifluoroacetic acid.
Consequently, the amount of F-POSS was determined as 18 nmol
on 1 mg of the NPs (F atom, 216 nmol/mg). The signals were not
observed after 24 h incubation at 37 °C at pH 7.0 in the presence
of proteinase K. In addition, the significant degradation following
the undesired signal output or the aggregation was not observed
by adding to BSA (1 mg/mL) which is known as an extracellular
reducing material, the serum or the pH alteration between pH 5
and 9. These results suggest that the probe could provide clear sig-
nals without loss of sensitivity caused by unexpected interactions.
Initially, to examine the reactivity of the probes, we carried out
the reactions with the conventional reducing agents, GSH, GSSG,
and NADPH. The sample mixtures (0.5 mL) containing 5 mg/mL
of the modified NPs (1.08 mM fluorine atoms) and the series of
the reagents described on each spectrum in PBS were incubated
at 37 °C for 30 min, and 19F NMR signals from the supernatants
after the centrifugation with the samples were monitored. The sig-
nals increased after incubation in the presence of NaBH4, dithio-
threitol (DTT), and GSH (Fig. 2). These results indicate that F-
POSS can be released by reductive cleavage of disulfide bonds at
the linker. On the other hand, both signals hardly increased after
the incubation in the presence of GSSG and NADPH. This means
that coexisting molecules should less affect the linker cleavage,
and therefore only the GR activity can be evaluated using our probe
as the increase of 19F NMR signals.
Figure 2. 19F NMR spectra of the supernatants after the incubation. The reaction
mixtures containing 5 mg/mL of the NPs were incubated in the presence of the
reagents in PBS at 37 °C for 30 min.
We carried out the reactions with various concentrations of GR,
and the signal intensities from the reaction mixtures were moni-
tored with 19F NMR measurements (Fig. 3). The signal intensities
were compared with the standards and converted as the reaction
yields. The initial velocity (vi) was estimated from the slope of
the increases of 19F NMR signals. In the presence of 5 U/mL of
GR, the reactions reached a plateau after the 20 min incubation.
In contrast, less significant increase of the NMR signal intensity
was observed in the absence of GR. The detection limits were cor-
respondingly 0.5 U/mL of GR. These data present that the NP
probes can provide the 19F NMR signal via the enzymatic reaction
with GR. Moreover, the reaction rates are seemed to be enhanced
corresponded to the increase of the amount of GR in the reaction
samples. In particular, the linear relationship was obtained be-
tween the GR concentration and the vi values in the lower concen-
tration region (Fig. 4). These results satisfy the requirements for
quantitatively evaluating the GR activity from the degree of the
reaction rates.
Figure 3. Time-courses of the intensity changes of 19F NMR signals form the
supernatants after enzymatic reactions. The NP probe (5 mg) was incubated in
500
lL of the reaction solutions containing various concentration of GR, 1 mM b-
NADPH, 0.5 mM GSSG in PBS at 37 °C. The reaction yields were monitored with 19
F
NMR and calculated by fitting on the standards. The errors represent the standard
deviation calculated from the three data sets.

100
K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 96–100
the particles, for instance in the vessels and extracellular or inter-
cellular matrices. Hence, this system promises to be a valid strat-
egy for gathering the variety of quantitative information with
MRI at various sites.
Acknowledgment
This research was partly supported by Magnetic Health Science
Foundation (for K.T.).
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Figure 4. Determination of GR activity in the HeLa cell lysate. The vertical axis
represents the initial velocities (vi) determined from the increases of the 19F NMR
signal intensities of the samples. Fitting of the obtained value (clear triangular dot)
from the 19F NMR measurements to the standard line according to the results of
Figure 3. The plots represent the averages calculated from the three data sets. The
error bars represent the standard deviations.
To demonstrate feasibility of the analysis of the probe in the
presence of the miscellaneous molecules under bio-mimetic condi-
tions, the reactions were carried out with the HeLa cell lysate. Ini-
tially, the GR activity was determined from the evaluation of the
absorption change of b-NADPH, and it was determined as
0.61 0.05 U/mL. In contrast, the enzymatic activity of the cell ly-
sate was evaluated to be 0.65 0.14 U/mL by fitting the vi value to
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4. Conclusion
We present here the quantitative detection system in 19F NMR
for GR activity. Perfluorinated dendrimers tethered with silica
NPs via disulfide bonds provided signal increase in the enzymatic
reactions in the presence of GR. In addition, the quantitative detec-
tion was achieved by using our probe. Furthermore, the localiza-
tion of the probes was regulated by modulating the diameter of
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