Systematic study of protein detection mechanism of self-assembling 19F NMR/MRI nanoprobes toward rational design and improved sensitivity

Article abstract of DOI:10.1021/ja203996c

¹⁹F NMR/MRI probe is expected to be a powerful tool for selective sensing of biologically active agents owing to its high sensitivity and no background signals in live bodies. We have recently reported a unique supramolecular strategy for specific protein detection using a protein ligand-tethered self-assembling ¹⁹F probe. This method is based on a recognition-driven disassembly of the nanoprobes, which induced a clear turn-on signal of ¹⁹F NMR/MRI. In the present study, we conducted a systematic investigation of the relationship between structure and properties of the probe to elucidate the mechanism of this turn-on ¹⁹F NMR sensing in detail. Newly synthesized ¹⁹F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes. We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe aggregates and that "moderate stability" of the aggregates produced an ideal turn-on response in protein detection. We also successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed understanding of the detection mechanism allowed us to rationally design a turn-on ¹⁹F NMR probe with improved sensitivity, giving a higher image intensity for the target protein in ¹⁹F MRI.

Full text of DOI:10.1021/ja203996c

ARTICLE
pubs.acs.org/JACS
Systematic Study of Protein Detection Mechanism of Self-Assembling
19F NMR/MRI Nanoprobes toward Rational Design and Improved
Sensitivity
Yousuke Takaoka,† Keishi Kiminami,† Keigo Mizusawa,† Kazuya Matsuo,† Michiko Narazaki,‡
Tetsuya Matsuda,‡ and Itaru Hamachi*,†,§
†Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan
‡Department of Systems Science, Graduate School of Informatics, Kyoto University, 36-1 Yoshida-Honmachi, Sakyo-ku,
Kyoto 606-8501, Japan
§Japan Science and Technology Agency, CREST, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
S Supporting Information
b
ABSTRACT: 19F NMR/MRI probe is expected to be a power-
ful tool for selective sensing of biologically active agents owing
to its high sensitivity and no background signals in live bodies.
We have recently reported a unique supramolecular strategy for
specific protein detection using a protein ligand-tethered self-
assembling 19F probe. This method is based on a recognition-
driven disassembly of the nanoprobes, which induced a clear
turn-on signal of 19F NMR/MRI. In the present study, we
conducted a systematic investigation of the relationship be-
tween structure and properties of the probe to elucidate the mechanism of this turn-on 19F NMR sensing in detail. Newly
synthesized 19F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes.
We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe
aggregates and that “moderate stability” of the aggregates produced an ideal turn-on response in protein detection. We also
successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed
understanding of the detection mechanism allowed us to rationally design a turn-on 19F NMR probe with improved sensitivity,
giving a higher image intensity for the target protein in 19F MRI.
’ INTRODUCTION
is advantageous for deep-tissue and noninvasive imaging in vivo.9
19F MRI is anticipated to be a promising alternative to conven-
tional 1H MRI because of its high sensitivity (83% relative to 1H)
and no background signals in animal bodies.10 However, success-
ful examples of the application of 19F-based chemical probes for
specific protein detection have been very limited,11 and thus the
development of functional 19F probes is now highly desirable.
Here we describe a systematic structureꢀproperty relation-
ship study for elucidating the mechanism of turn-on 19F NMR
sensing using the newly synthesized 19F probe derivatives with
varying self-assembling properties. Interestingly, in addition to
the turn-on probes, we found that some probes gave the 19F
NMR signal both in the absence and in the presence of the target
protein, indicating that they were always-on probes. Further-
more, some probes produced no signals regardless of the target
was present; therefore, these were classified as always-off probes.
We clearly demonstrated that the response was strongly con-
trolled by the stability of the self-aggregates in aqueous solution,
Chemical probes for selective sensing and detection of
biologically active molecules have attracted a great deal of
attention, not only in basic biological research but also in medical
diagnosis and pharmaceutical applications.1ꢀ4 Protein is one of
the most important targets among a variety of biomarkers, and
chemical probes for versatile and convenient detection or
imaging of proteins are in high demand. However, development
of protein-specific chemical probes is generally difficult due to
the complicated 3D structures of proteins with diverse functions.
Recently, Rotello and co-workers5 and Thayumanavan and co-
workers6 have independently proposed supramolecular ap-
proaches to protein detection using amphiphilic polymers or
dendrons, showing that such nanomaterials have the potential for
use in protein sensing and profiling applications. We also recently
reported a unique supramolecular strategy for protein detection
using protein ligand-tethered self-assembling nanoprobes.7,8
This technique is based on a protein-recognition-driven
disassembly of the nanoprobes, which produced a clear turn-on
signal of 19F NMR/MRI or fluorescence in the detection
modality (Figure 1a). Among many detection modalities, MRI
Received: April 30, 2011
Published: June 23, 2011
r
2011 American Chemical Society
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Figure 1. (a) Schematic of self-assembling 19F NMR off/on probe for protein detection. (b) Chemical structures of probes used in this study: 1, 3, and 4
for human carbonic anhydrase I (hCAI), 5ꢀ7 for avidin, 8ꢀ10 for dihydrofolate reductase (DHFR), and 2 for pH-sensitive probe.
signal/noise ratio, to afford enhanced sensitivity and brighter 19F
MRI in the turn-on detection mode.
’ RESULTS AND DISCUSSION
19F NMR Signal Intensity Influenced by Hydrophilicity of
the Head Group. We initially noticed that the signal-off or -on of
19F NMR is highly dependent on the hydrophilicity of the 19F-
tethered molecule when we measured the 19F NMR of a pH-
sensitive amphiphilic molecule 2 (Figure 1b), with a structure
similar to the ideal off/on probe 1. The benzoic acid head group
of 2 is neutral at acidic pH, whereas it is negatively charged as
benzoate at basic pH. When 2 was dissolved in acidic buffer
(pH 3), no 19F signal was observed. In contrast, a sharp signal
appeared at ꢀ62.9 ppm in basic buffer (pH 8) (Figure 2a,b).12 It
was clear from the pH titration experiment that the 19F NMR
signal turned on at approximately pH 5.3, which corresponds to
the apparent pKa of benzoic acid (Figure 2c). The visible
absorption spectrum of 2 in acidic buffer showed a broad band
around 500ꢀ700 nm owing to the visible-light scattering, whereas
the solution was not turbid in basic solution (Figure S1a,
Supporting Information; the scattering intensity at 600 nm
showed an 80-fold decrease relative to that in the acidic buffer).
These findings suggested that there are aggregates of 2 in the
acidic solution but not in the basic solution. Indeed, atomic force
microscopy (AFM) revealed the formation of spherical or oval
aggregates of 2 with diameters ranging from 10 to 100 nm
(Figure S1b, Supporting Information) in the acidic buffer. Con-
sistently, dynamic light scattering (DLS) measurements in acidic
buffer containing 2 showed aggregates with a mean diameter of
13 nm (Figure S1c, Supporting Information), whereas negligible
Figure 2. (a, b) 19F NMR spectra of pH-sensitive off/on probe 2
(100 μM) at (a) pH 3.0 or (b) pH 8.0 in a citrate/phosphate buffer
system of constant ionic strength12 [0.5 ion strength, 0.2 mM TFA, 10%
D2O (v/v), 500 μL]. (c) pH profile of signal intensity change of probe 2
was assessed in the same system as shown in panels a and b
(trifluoroacetic acid, TFA, was used for an internal standard for signal
intensity). Experiments were performed in triplicate to obtain mean and
standard deviation values (shown as error bars).
which was modulated by the hydrophilicity/hydrophobicity
balance of the probe molecules. The present mechanistic under-
standing enabled us to design self-assembling probes with improved
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Figure 3. 19F NMR spectra of hCAI-type probes (a) 1, (b) 3, and (c) 4 (25 μM), (top) with or (bottom) without 25 μM hCAI, in 50 mM HEPES buffer
[pH 7.2, 0.2 mM TFA, 10% D2O (v/v), 500 μL].
Figure 4. Stabilities of the self-assembling aggregates of probes 3, 1, and 4. (aꢀc) 19F NMR spectra of probes 3 (a, 25 μM), 1 (b, 25 μM), and 4
(c, 25 μM) in aqueous solution [pH 7.2, 50 mM HEPES buffer (0.2 mM TFA as an internal standard for peak intensity and chemical shift), 500 μL] with
various DMSO-d6 contents. (d) Relative peak intensity changes of probes 3 (O), 1 (0), and 4 (]) in aqueous solution with various DMSO contents.
(e) Concentration-dependent change of scattering intensities of hCAI probes 3 (Δ), 1 (ꢁ), and 4 (b) in aqueous solution (pH 7.2, 50 mM HEPES
buffer). These intensities were collected from DLS analyses with the same laser intensity and sensitivity.
DLS intensity was obtained in the basic buffer. These results
indicate that aggregates of 2 formed in the acidic buffer with
protonation of the benzoic acid moiety but they collapsed
upon deprotonation in the basic buffer. Aggregation in acidic
solution resulted in the apparent molecular mass (Mr) of 2
dramatically increasing from 8 ꢁ 102 (monomer) to 5 ꢁ 105 Da.
It may be reasonable that such an increase in Mr caused a
significant increase in the 19F relaxation rate, relative to that in
the monomeric form of 2, affording no NMR signals.7a It should
be noted that the aggregation/disaggregation was modulated by
a change in the hydrophilicity of the head group, which directly
led to the disappearance/appearance of the 19F NMR signal.
Three Distinct Response Patterns in Protein Detection via
Human Carbonic Anhydrase I 19F Probes. The aggregation
property of 19F probes is reasonably considered to be control-
lable by other modules, as well as the head group, which may
influence the turn-on response to a target protein. To examine
the effects of the linker module on human carbonic anhydrase I
(hCAI) response, we prepared two 19F probes, 3 and 4, with
different linkers based on the structure of the ideal off/on probe 1
for hCAI detection. Our probes consisted of three modules: (i) a
hydrophilic ligand specific to a protein of interest (head group,
benzenesulfonamide for hCAI),13 (ii) a hydrophobic 3,5-bis-
(trifluoromethyl)benzene for the 19F NMR detection modality
(tail group), and (iii) a relatively hydrophobic linker group to
connect these two modules (Figure 1b). Interestingly, three
different responses to hCAI were found for these probes in the
absence and presence of hCAI: off/on, always-on ,and always-off
responses. Probe 1 alone was silent in 19F NMR spectroscopy but
produced a sharp signal at ꢀ62.6 ppm in response to the addition
of hCAI (Figure 3a). In contrast, when probe 3, which has a
hydrophilic hepta(ethylene glycol) unit as a linker group, was
dissolved in aqueous solution, a sharp 19F signal was observed at
ꢀ62.9 ppm even without hCAI (“always-on” as shown in
Figure 3b).14 Conversely, probe 4, which had a hydrophobic
alkyl chain linker, gave no 19F signal in the absence or presence
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Table 1. Stability Parameters and Response Patterns of Target
aggregates of the always-off type probe 4 were too stable to be
disassembled by hCAI recognition. Indeed, we confirmed that
the mean diameter of the aggregates 4 did not change substan-
tially in response to the hCAI addition from DLS measurements
[97 nm for a buffer solution containing 4 alone and 114 nm after
hCAI addition (Figure S2c, Supporting Information)],15 unlike
in the case of the probe 1. The optical density measurements
showed that the scattering of 4 alone in buffer solution was
0.02 at 600 nm, and this value increased slightly in response to
the addition of hCAI (Figure S2e, Supporting Information).
These findings were in sharp contrast to the behavior of the
off/on probe 1, which showed a 10-fold decrease in response to
hCAI addition.7a We also confirmed that the enzymatic activity
of hCAI was partially inhibited by 4 (Figure S3, Supporting
Information), implying that an interaction between 4 and hCAI
occurred. These results suggest that the aggregates of 4 did not
collapse regardless of the recognition by hCAI; this can be
attributed to the high stability of the probes, and therefore, the
19F NMR signals were not observed.
Protein Detection of 19F NMR Probes 1 and 3 ꢀ 10
probe
target protein
HMDC, %
CAC,a μM
response patterns
1
hCAI
15
0
5
50
1
off/on
3
hCAI
always-on
always-off
off/on
partial always-onb
always-off
off/on
4
hCAI
65
35
2
5
avidin
avidin
avidin
DHFR
DHFR
DHFR
5
6
20
1
7
60
39
0
8
2.5
500
1
9
always-on
partial always-offc
10
59
a CAC values were determined from concentration-dependent DLS
measurements, shown in Figure 4e and Figure S6, Supporting Informa-
tion. b Peak intensity of probe 6 alone was observed no more than 40%
relative to the theoretical value in 50 mM HEPES buffer [pH 7.2,
0.2 mM TFA, 10% D2O (v/v) without NaCl]. c Peak intensity of probe
10 recovered only up to 5% by addition of DHFR relative to the
theoretical value.
Similar Correlation Observed in Other Self-Assembling
Probes. Such a relationship was also observed for other self-
assembling probes that detect different proteins in the turn-
on mode. The modular design of our probes enables the
detection of various target proteins by use of appropriate probes
with a hydrophilic head group that has been replaced with a
corresponding ligand moiety. We developed two other classes of
probes displaying a different ligand group instead of benzene-
sulfonamide, that is, biotin-tethered probes for avidin16 (probes
5ꢀ7) and probes containing methotrexate (MTX) as a specific
inhibitor of dihydrofolate reductase (DHFR)17 (probes 8ꢀ10).
As can be inferred from Table 1 and Figure S4 (Supporting
Information), always-on and always-off probes were found in
both classes of probes, in addition to the turn-on probes. Because
the hydrophilicity of the ligand parts differed among probes, the
linker structure of the probes that showed the three different
responses was varied slightly. For example, the C5 linker gave the
turn-on response for the hCAI and DHFR probes (1 and 8,
respectively), whereas the C8 linker was needed for the turn-on
response for avidin (5). In the case of avidin probes, probe 6 with
the C5 linker exhibited the 19F signal to some extent, indicating
that 6 was not a perfect “off/on” probe but rather a partial always-
on probe.18 It should be noted that such a response pattern was
closely related to the aggregate stability, which was evaluated on
the basis of the HMDC value as in the case of hCAI probes. That
is, the HMDC values were 35% and 39% for 5 and 8, respectively
(Figure S5, Supporting Information, and Table 1), which are
similar to that of the turn-on probe 1 (15%). On the other hand,
the HMDC values were 60% and 59% for always-off probes 7 and
10, respectively, which are in the same range as that of the always-
off probe 4 for hCAI (65%).
of hCAI (“always-off” as shown in Figure 3c). Undoubtedly,
both always-on and always-off responses are not suitable for
accurate protein detection. These differences in hCAI response
may be explained as follows: probe 3 was too hydrophilic to form
stable self-assembling aggregates that are essential for the signal
off state, whereas probe 4 formed aggregates that were too robust
to undergo the recognition-driven disassembly.
Correlation between Stabilities of Self-Assembling Aggre-
gates and Off/On Response Patterns of 19F Probes. Next, we
evaluated the stability of self-assembling aggregates of 19F probes
1, 3, and 4 with various measurements. As mentioned above, 19F
NMR signals of these probes cannot be observed when they are
stably aggregated in aqueous solution, and the signals appear
when they are homogeneously dispersed. We attempted to
monitor the collapse of the 19F probe aggregates in response
to the addition of dimethyl sulfoxide (DMSO) on the basis of
disappearance and appearance of 19F NMR signals (Figure 4). As
an index of the aggregate stability, the critical DMSO content
in aqueous solution that gave the 19F signal appearance at half of
the maximum intensity (HMDC) was determined (Figure 4d).
The collapse of the aggregates was also confirmed by the decrease
in optical density at 600 nm (Figure S2a,b, Supporting In-
formation). The HDMC values for the probes 3, 1, and 4 were
determined to be 0%, 15%, and 65%, respectively, indicating that
the order of the stability of the aggregates is 3 < 1 < 4 (Table 1).
Interestingly, these findings are in good agreement with the
decrease in hydrophilicity of the linker module of these probes.
We also conducted concentration-dependent DLS measure-
ments to determine the critical aggregation concentration
(CAC) of these probes, yielding 50, 5, and 1 μM for 3, 1, and
4, respectively (Figure 4e). Clearly, the order of CAC values was
well consistent with that of the aforementioned HMDC. Given
the stability data among the three probes, we may ascribe the
three different responses of the 19F NMR signal to the distinct
stability of the self-assembling aggregates. That is, the 19F signal
of the always-on type probe 3 appeared even before the addition
of hCAI, because the aggregates of 3 were less stable. In contrast,
the perfect off/on type probe 1 self-assembled without hCAI and
then disassembled in response to hCAI, owing to the moderate
stability of the aggregates. Conversely, it was conceivable that
Given all data regarding the three off/on probes toward the
different target proteins, it is generally accepted that the 19F
NMR signal response to a target protein is closely related to the
stability of the self-assembling aggregates of the probe. To achieve
an ideal turn-on response, moderate stability is crucial, and it can
be designed by modulating the hydrophobicity/hydrophilicity
balance of 19F probe molecules.
Rational Design of 19F Probe for Enhanced Sensitivity
without Loss of Turn-on Mode. Our understanding of the
detection mechanism enabled us to design a turn-on 19F NMR/
MRI probe with increased sensitivity. It might be simply con-
sidered that the sensitivity in 19F NMR is easily improved by
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Figure 5. (a) Chemical structures of 19F probes 11ꢀ14 for hCAI detection. (bꢀe) 19F NMR spectra of probes (b) 13, (c) 14, (d) 1, and (e) 12 (each
concentration was 100 μM) with hCAI (100 μM) in 50 mM HEPES buffer [pH 7.2, 0.2 mM TFA, 10% D2O (v/v), 500 μL]. (f) Relative integral
intensity change with increasing number of 19F atoms in each probes [normalized in probe 13 (19F = 1) in 50 mM HEPES buffer [pH 7.2, 0.2 mM TFA as
an internal standard for peak height and chemical shift, 10% D2O (v/v)]. Experiments were performed in triplicate to obtain mean and standard
deviation values (shown as error bars). (g) 19F MR images of probes 13 (F = 1), 14 (F = 3), 1 (F = 6) and 12 (F = 12) (each concentration was 100 μM),
(top) with or (bottom) without hCAI (100 μM) in 50 mM HEPES buffer (pH 7.2, 0.2 mM TFA, 2 mL). The signal-to-noise ratios (SNR) were 1.51,
3.42, 5.55, and 9.96 for 13, 14, 1, and 12, respectively.
increasing the number of 19F nuclei in the probe. However, the
stability of the probe aggregates is inevitably enhanced due to the
strong hydrophobicity of the additionally incorporated 19F atom,
which yields the always-off probe instead of the off/on probe. We
thus needed to carefully adjust the aggregates stability by
controlling the hydrophilicity of the linker group. In fact, we
prepared two new 19F probes (11 and 12, Figure 5a) containing
twelve 19F nuclei as the dimeric 3,5-bis(trifluoromethyl)benzene unit
with tetra- or nona(ethylene glycol) (oligo-EG) linker and exam-
ined the off/on response. In both cases, 19F NMR signals were
not observed when the probes were dissolved alone in aqueous
solution, whereas sharp signals appeared after the addition of
hCAI. However, for 11 (4-EG), the 19F signal recovered only up
to 50% of its intensity in response to hCAI, probably owing to the
rather robust aggregates that were formed. In contrast, we found
that almost 100% of the 19F signal was recovered in response to
hCAI for probe 12 (9-EG), indicating that the aggregate stability
was suitably tuned by the elongated ethylene glycol linker. These
response patterns were again explained by the stability of the
aggregates. That is, the HMDC value of the off/on probe 12 was
27%, which was similar to that of the ideal off/on probe 1 (15%).
On the other hand, the HMDC value of the partial off/on probe
11 was found to be 45%, which was rather close to that of the
always-off probe 4 (65%) (Figure S7, Supporting Information).
We subsequently compared the off/on 19F signal intensity
among four different probes, 13, 14, 1, and 12, which had one,
three, six, and twelve magnetically equivalent 19F nuclei, respectively
(Figure 5a). We separately confirmed that all of these were
off/on probes for hCAI. As shown in Figure 5bꢀf, the signal
intensity increased linearly as the number of 19F nuclei in these
probes increased.19
These turn-on 19F NMR probes can visualize the target protein
in a 19F MRI phantom, and the above-mentioned sensitivity
improvements of our self-assembling 19F probes resulted in
clearer MR images. As shown in Figure 5g, the signal intensities
of 19F MRI were greatly enhanced and thus the higher image
intensity was obtained as the number of 19F nuclei increased for
probes 13, 14, 1, and 12, in the presence of the same concentra-
tion of hCAI in each solution. In phantom experiments con-
ducted on our 19F MRI instrument, the minimum times required
to obtain clear images (signal-to-noise ratios greater than 2) were
3 h, 1 h, and 10 min for 14, 1, and 12, respectively.20 These results
clearly indicated that the designed probe containing twelve 19F nuclei
is indeed able to improve the sensitivity of 19F MRI.
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’ CONCLUSION
with TFA as an internal standard (ꢀ75.6 ppm). All the experi-
ments were acquired at 25 °C.
In the present systematic structureꢀproperty relationship
study of our self-assembling 19F NMR/MRI probes, we char-
acterized probes that showed three different behaviors in re-
sponse to the target protein: off/on, always-on, and always-off types.
Through optical density measurements and 19F NMR spectroscopy
following the addition of DMSO, these differences in protein
responses were found to be closely related to the stability of the
probe aggregates. It was clear that the “moderate stability” of the
self-assembling aggregates was critical to an ideal turn-on re-
sponse for protein detection and that the stability could be
controlled by altering the hydrophobicity/hydrophilicity balance
of the probes. Importance of this balance for efficient protein
detection using self-assembling systems was also pointed out by
Thayumanavan and co-workers.6b,c On the basis of our under-
standing of the detection mechanism, we successfully designed
a turn-on 19F NMR/MRI probe with improved sensitivity.
We believe that the present mechanism-based probe design is
an important first step toward imaging biologically significant
target proteins by use of self-assembling 19F MRI probes in
cells or in vivo.
Evaluation of Stabilities of Self-Assembling Aggregates
by 19F NMR Spectroscopy. The concentration of 19F probe was
fixed at 25 μM in each experiment. The stock solution of each 19F
probe was added to the mixed solvent of DMSO-d6 and 50 mM
HEPES buffer (pH 7.2, 0.2 mM TFA). These samples dissolved
in arbitrary DMSO contents (0ꢀ95%) were analyzed by 19F
NMR spectroscopy, and the relative signal intensities of
probe itself were compared with TFA (as an internal standard,
ꢀ75.6 ppm) for calculating the peak recovery ratio against the
theoretical value of which whole aggregates dispersed in the
solution.
MRI Experiments of 19F Probes 12ꢀ14 and 1. The samples
were prepared as described in a preceding section, with hCAI
(200 μM) and probes (200 μM) (2 mL, depth of sample tube
20 mm). 19F magnetic resonance images of samples were
obtained by fast spin echo with repetition time/echo time
1000/5.5 ms, echo train length 32, field of view 24 ꢁ 6 cm
without slice selection, matrix size 128 ꢁ 32, and the number of
accumulations 10 800. The excitation pulse width was 1370 Hz.
The sine window function was applied to the 19F magnetic
resonance images. For determination of the signal-to-noise ratios
(SNR), background signal intensity was used as the noise
intensity. All the images were acquired at 20 °C.
’ EXPERIMENTAL SECTION
General Materials and Methods. All proteins and chemicals
were obtained from commercial suppliers (Sigma, Aldrich, TCI,
Wako, or Watanabe Chemical Industries) and used without
further purification. 19F NMR spectra were recorded on a JEOL
ECX-400P (376.5 MHz) spectrometer and calibrated with TFA
(ꢀ75.6 ppm). Standard parameters were used with a 36 kHz
spectral width, 8 μs pulse length, 0.46 s acquisition time, and
0.50 s relaxation delay. A 0.1 Hz line broadening was applied. The
number of accumulations was 1024. UVꢀvisible spectra were
recorded on a Shimadzu UVꢀvisible 2550 spectrometer. AFM
measurements were performed with a Shimadzu SPM-9600.
DLS measurements were performed with a NICOMP 380zls.
The scattering angle was 108° and the wavelength of the laser was
520 nm. 19F MR images were obtained on a 7 T Bruker-Biospec
70/20 USR system (282 MHz for 19F) with 72 mm i.d. 1H/19F
radio frequency (RF) volume coil (Bruker Biospin, Germany).
1H NMR spectra were recorded on a Varian Mercury-400
(400 MHz). High-resolution fast atomic bombardment mass
spectrometry (HR-FAB MS) spectra and high-resolution elec-
trospray ionization quadrupole Fourier transform mass spectro-
metry (HR-ESI-MS) spectra were performed on a JEOL JMS-
HX110A with 3-nitrobenzyl alcohol (NBA) as the matrix and
on a Bruker apex-ultra (7 T) mass spectrometer, respectively, by
Dr. Keiko Kuwata (Department of Synthetic Chemistry and
Biological Chemistry, Graduate School of Engineering, Kyoto
University).
’ ASSOCIATED CONTENT
S
Supporting Information. Seven figures as described in the
b
text, two tables, and additional text and 12 schemes describing
experimental details and synthesis procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
ihamachi@sbchem.kyoto-u.ac.jp
’ ACKNOWLEDGMENT
We thank Dr. S. Tsukiji (Nagaoka University of Technology)
for helpful discussion during the early stages of this project. We
thank Dr. T. Sera (Kyoto University, Umeda Lab) for help with
DLS measurements. We thank Dr. M. Ikeda (Kyoto University,
Hamachi Lab) for help with AFM measurements. Y.T.,
K. Mizusawa, and K. Matsuo acknowledge JSPS Research Fellow-
ships for Young Scientists. This work was partly supported by the
CK Integrated Medical Bioimaging Project (MEXT) and
CREST (Japan Science and Technology Agency).
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Evaluation of hCAI Detection Properties of 19F Probes 1,
3, and 4. hCAI (Sigma, C4396) was dissolved in 50 mM N-
(2-hydroxyethylpiperazine)-N0-2-ethanesulfonic acid (HEPES)
buffer [pH 7.2, 10% D2O (v/v), 0.2 mM TFA, 500 μL]. The
concentration of hCAI was determined by measuring the absor-
bance at 280 nm with the molar extinction coefficient (49 000
M
ꢀ1cmꢀ1),21 and the stock solution was prepared. 19F probe 1,
3, or 4 was dissolved in dimethyl sulfoxide (DMSO) as the stock
solution and slowly added to the hCAI solution [0.6% DMSO
(v/v)]. These samples were analyzed by 19F NMR spectroscopy
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(14) By addition of hCAI, the original signal at ꢀ62.9 ppm
disappeared and a distinct broad signal appeared at ꢀ62.6 ppm
(Figure 3b). This chemical shift change clearly indicated that the probe
3 was bound to hCAI, similar to probe 1. These were supported by the
transverse relaxation times (T2) of probe 3 with or without hCAI
(shown in Table S1, Supporting Information).
(15) AFM consistently revealed the formation of spherical or oval
aggregates of 4 with diameters ranging from 80 to 100 nm, as in the case
of probe 1. Moreover, aggregates of 4 were also observed after addition
of hCAI, and the diameters slightly increased (ranging from 100 to
150 nm) in the AFM experiments (Figure S2d, Supporting In-
formation). These results were consisted in the DLS measurements.
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degree of aggregation.
(19) The detection limit of hCAI with probe 12 was less than
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(20) We could not obtain any clear images from the sample contain-
ing probe 13 (F = 1) and hCAI, despite accumulation for 3 h.
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