Thermal- and pH-Dependent Size Variable Radical Nanoparticles and Its Water Proton Relaxivity for Metal-Free MRI Functional Contrast Agents

Article abstract of DOI:10.1021/acs.joc.6b01509

For development of the metal-free MRI contrast agents, we prepared the supra-molecular organic radical, TEMPO-UBD, carrying TEMPO radical, as well as the urea, alkyl group, and phenyl ring, which demonstrate self-assembly behaviors using noncovalent bonds in an aqueous solution. In addition, TEMPO-UBD has the tertiary amine and the oligoethylene glycol chains (OEGs) for the function of pH and thermal responsiveness. By dynamic light scattering and transmission electron microscopy imaging, the resulting self-assembly was seen to form the spherical nanoparticles 10-150 nm in size. On heating, interestingly, the nanoparticles showed a lower critical solution temperature (LCST) behavior having two-step variation. This double-LCST behavior is the first such example among the supra-molecules. To evaluate of the ability as MRI contrast agents, the values of proton (¹H) longitudinal relaxivity (r₁) were determined using MRI apparatus. In conditions below and above CAC at pH 7.0, the distinguishable r₁ values were estimated to be 0.17 and 0.21 mM^-1 s¹, indicating the suppression of fast tumbling motion of TEMPO moiety in a nanoparticle. Furthermore, r₁ values became larger in the order of pH 7.0 > 9.0 > 5.0. Those thermal and pH dependencies indicated the possibility of metal-fee MRI functional contrast agents in the future.

Full text of DOI:10.1021/acs.joc.6b01509

Subscriber access provided by Northern Illinois University
Article
Thermal- and pH-Dependent Size Variable Radical Nano-particles and Its
Water Proton Relaxivity for Metal-free MRI Functional Contrast Agents
Kosuke Morishita, Shuhei Murayama, Takeru Araki, Ichio Aoki, and Satoru Karasawa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01509 • Publication Date (Web): 19 Aug 2016
Downloaded from http://pubs.acs.org on August 21, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Thermalꢀ and pHꢀDependent Size Variable Radical Nanoꢀparticles and
Its Water Proton Relaxivity for Metalꢀfree MRI Functional Contrast
Agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
1
3
1,2
Kosuke Morishita, Shuhei, Murayama, Takeru Araki, Ichio Aoki, and Satoru Karasawa *
1
Graduate School of Pharmaceutical Sciences, Kyushu University, 3ꢀ1ꢀ1 Maidashi, HigashiꢀKu,
2
Fukuoka, 812ꢀ8582 Japan. PRESTO, Japan Science and Technology Agency, Kawaguchi, 332ꢀ0012
3
Japan. Department of Molecular Imaging and Theranostics, National Institute of Radiological
Sciences (NIRS), QST, Anagawa 4ꢀ9ꢀ1, Inage, Chibaꢀcity, 263ꢀ8555 Japan.
Corresponding author: Satoru Karasawa.
Eꢀmail: karasawa@phar.kyushuꢀu.ac.jp
TEL: +81ꢀ92ꢀ642ꢀ6593, FAX: +81ꢀ92ꢀ642ꢀ6545
ABSTRACT
For development of the metalꢀfree MRI contrast agents, we prepared the supraꢀmolecular
organic radical, TEMPO-UBD, carrying TEMPO radical, as well as the urea, alkyl group,
and phenyl ring, which demonstrate selfꢀassembly behaviors using noncovalent bonds in an
aqueous solution. In addition, TEMPO-UBD has the tertiary amine and the
oligoethyleneglycol chains (OEGs) for the function of pH and thermal responsiveness. By
DLS and TEM imaging, the resulting selfꢀassembly was seen to form the spherical
nanoꢀparticles 10 – 150 nm in size. On heating, interestingly, the nanoꢀparticles showed a
lower critical solution temperature (LCST) behavior having twoꢀstep variation. This
doubleꢀLCST behavior is the first such example among the supraꢀmolecules. To evaluate of
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
the ability as MRI contrast agents, the values of proton ( H) longitudinal relaxivity (r
1
) were
determined using MRI apparatus. In conditions below and above CAC at pH 7.0, the
ꢀ1 1
distinguishable r
1
values were estimated to be 0.17 and 0.21 mM s , indicating the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
suppression of fast tumbling motion of TEMPO moiety in a nanoꢀparticle. Furthermore, r
1
values became larger in the order of pH 7.0 > 9.0 > 5.0. Those thermal and pH dependencies
indicated the possibility of metalꢀfee MRI functional contrast agents in the future.
INTRODUCTION
Magnetic resonance imaging (MRI) is widely used as a nonꢀinvasive diagnostic method
1
because of its properties of safety and deep penetration into the body. To obtain bioꢀimages
emphasizing for the specific tissues such as a tumor tissue, contrast agents (CAs) are often
used. Currently, Gadolinium (Gd ) complexes are widely used as CAs because Gd ions have a
2
largest spin quantum number of all the elements. However, Gd complex CAs have potential
3
sideꢀeffects such as renal disorder due to the free Gd ion and a lack of specificity to the
tissues. In addition, recently, accumulation in the brain, especially among children, was
4
reported, so the replacement of Gd complexes by new CAs is strongly desired. Stable organic
5
radicals such as TEMPO are widely used as probes for bioꢀESR imaging, spinꢀtrap against
6
reactive oxygen species (ROS), and so on. Such organic radicals can function as MRI CAs
1
due to their possession of electron spin. However, their water proton relaxivity values (r and
7
r
2
) are considerably smaller than those of Gd complexes. To increase the relaxivity value,
taking advantage of the suppression of fast tumbling of the TEMPO moiety rotational
R
correlation time (τ ) by enlarging the molecular size is a promising approach studied by many
8
groups. We previously reported that nanoꢀparticles with ~ 10 nm size consisting of
amphiphilic oligonucleotides carrying TEMPO exhibited unexpectedly large r
1
value
9
comparable with Gd complexes. The resulting behavior was based on a slower molecular
motion of the nanoꢀparticles and effective assembly of the water molecules against TEMPO
2
ACS Paragon Plus Environment

Page 3 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
moiety. The exhibited high relaxivity indicated that using CAs with radicals is promising
10
method for development of metal free CAs. Separately, we reported that waterꢀsoluble
supraꢀmolecules consisting of urea benzene frameworks (UBDs) having oligoethyleneglycol
11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
chains (OEGs) showed thermal responsiveness in water solution (Figure 1). Upon heating
the solution, an abrupt selfꢀassemble behavior with low temperature critical temperature
12
(
LCST) took place due to the dehydration surrounding OEGs, to form microꢀsize particles.
13
This LCST property is rare for supraꢀmolecules. This time, UBDs carrying TEMPO
(TEMPO-UBD) as well as tertiary amino groups were prepared and thermal responsive
behaviors accompanied by LCST and structural changes in solution were revealed.
Furthermore, to confirm the driving force of the selfꢀassembly of UBDs in water solution,
UBD without TEMPO (H-UBD) as a hydrophobic moiety was prepared as reference
compound and carefully compared to TEMPO-UBD. Using TEMPO-UBD, the water proton
1 2
relaxivity values, r and r , were determined under the conditions between pH 5.0 – 9.0. We
herein describe the variations of physical properties and morphologies in response to various
stimuli and evaluate the candidacy for metalꢀfree MRI functional CAs. The molecular
structures of Tri-, H- and TEMPO-UBD are shown in Figure 1.
Figure 1. Molecular structures of Tri-, H- and TEMPO-UBD.
RESULTS and DISCUSSION
A. Syntheses of H- and TEMPO-UBD (Scheme 1).
14
A primary amine analogue having an amphiphilic side chain (Eg
3
NEg
3
C
6
NH
2
)
was
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
synthesized by three steps from 2ꢀaminoethanol as the starting materials via a tertiary amine
having bistriethyleneglycol at the amino group (TEG
Eg NEg Br), and a phtalimide having the amphiphilic chain (Eg
resulting Eg NEg NH was coupled with 1ꢀiodoꢀ3,5ꢀdiisocyanatobenzene, to afford the
2
EA), an amphiphilic compounds
(
3
3
C
6
3
NEg3C6Pht). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
3
3
6
2
diurea derivative having the amphiphilic chain (Iodo-UBD) as a colorless oil. The radical
analogue having the amphiphilic chain (TEMPO-UBD) was prepared by SuzukiꢀMiyaura
15
coupling between Iodo-UBD and the TEMPO analogue having the boronic acid pinacolato
16
ester. The analogue H-UBD without TEMPO radical as the reference compound was
prepared in a manner similar to TEMPO-UBD but using isophthalic acid in place of
5ꢀiodoisophthalic acid. The synthesis routes of H- and TEMPO-UBD are shown in Scheme
1.
Scheme 1. Synthesis routes of H- and TEMPO-UBD.
(a)
Eg3
N
(b)
Eg3
N
H N
2
OH
Eg3
OH
Eg3
O
Br
6
TEG EA (75%)
Eg NEg C Br (68%)
3 3 6
2
Eg3
N
O
(c)
(d)
Eg3
N
Eg3
O
N
6
Eg3
O
NH2
6
O
Eg NEg C NH (78%)
Eg NEg C Pht (80%)
3
3
6
2
3
3 6
o
(a) Eg -Ts, K CO , MeCN, reflux; (b) 1,6-dibromohexane, NaH, THF, 0 C;
3
2
3
o
(
c) potassium phthalimide, DMF, 110 C; (d) Hydrazine monohydrate, EtOH, reflux.
4
ACS Paragon Plus Environment

Page 5 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B. Self-assembly behaviors of H- and TEMPO-UBD in aqueous solutions.
To reveal the selfꢀassembly behavior of H- and TEMPO-UBD in aqueous solutions, the
1
concentration and the pH dependence of HꢀNMR for H-UBD and ESR for TEMPO-UBD
were examined using 0.1 – 10 mM solution at pH 9.0, 7.0, and 5.0 at 23 ºC.
1
B-1. H-NMR of H-UBD in a buffer solution.
Before investigation of the selfꢀassembly behavior, to determine the pKa value for H-UBD,
1
7, 18
pH titration of the chemical shift was performed (Figure S15 in S10).
As the pH value
decreased, the protons neighboring the N atom in the tertiary amino moiety were shifted
19
downfield due to the protonation of amines. The variation of the proton close to the N atom
g
(H in Figure S15 in S10) was plotted with pH (Figure S16 in S11) and the pKa value of
H-UBD was determined to be 7.6 according to eq. S1 in SI. This value indicates that the
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
protonation of amines took place completely and partially at pH 5.0 and 7.0, respectively, but
not at all at pH 9.0. TEMPO-UBD having the same framework as H-UBD may be expected
to show a similar pKa value.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
1
Figure 2. (a) HꢀNMR spectra of H-UBD in 0.1 mM buffer solution at pH 9.0. (b)
Concentration dependence of H-UBD in buffer solution at pH 9.0 in the expansions of
aromatic (left) and alkyl chain (right) regions. Hxs indicate the protons corresponding the
6
ACS Paragon Plus Environment

Page 7 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
molecular structure of H-UBD (upper). The dotted blue lines represent the fitting curves
according to the isodesmic model (See text). The asterisks denote the proton of DDS as
standard material.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the lowest concentration of 0.1 mM buffer solution at pH 9.0, protons corresponding to
the benzene ring (H
a b c d e g k
, H , and H ), oligoethyleneglycol chains (OEGs)(H , H , H , and H ) and
the alkyl chains (H , H , H , and H ) were observed at 7.3 ꢀ 7.0, 3.7 – 2.8, and 3.2 – 1.3 ppm,
f
h
i
j
respectively (Figure 2a). As the concentration increased from 0.1 to 10 mM (Figure 2b), the
protons at the aromatic region (H and H ), neighboring a tertiary nitrogen (H ), and alkyl
a
b
g
chains (Hhꢀj) clearly shifted with the broadening. In contrast, the peaks corresponding to the
OEGs showed little shifting. The observed upfield shifts of H , H , and H indicate the
b
g
hꢀj
typical behaviors of formation of the aggregate due to the shielding effect among the
20
molecules. In contrast, a downfield shift was observed in H
a
, which is the proton in ortho
position of two urea groups, revealing the formation of a pseudoꢀhydrogen bond between H
a
2
1
and O atoms in the carbonyl group of urea moiety (Figure S19(b) in S14). This interaction
might be led by the formation of the aggregate, and a similar deshielding of proton was
11
observed in the analogue of Tri-UBD, as previously reported.
(a)
(
b)
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Plots of chemical shifts of H (a) and H (b) vs concentration for H-UBD at pH 9.0
b
j
(blue), 7.0 (green), and 5.0 (red). The solid lines indicate the theoretical curves according to
the isodesmic model. See the text and Table 1.
The resulting chemical shifts of H , H , and H were plotted as functions of the concentration
b
g
j
(Figures 3 and S19 (a)). To evaluate the aggregation behavior, the shifting peaks were fitted
2
2, 23
according to the isodesmic model (eq. S2 in S13),
to give association constants (K) of
, H , and H , respectively (Table 1).
ꢀ
1
1
2.3 ± 0.1, 54.3 ± 11.8, and 16.2 ± 0.1 M in H
b
g
j
Furthermore, the inflection points in the curvature suggested that the value of critical
aggregation concentration (CAC) was 0.5 mM. Even though the concentration was over CAC
and the aggregate was forming, the peaks corresponding to OEGs showed little shifting even
at the highest concentration. This suggested that the OEGs were located outside the aggregate
so the OEGs could move and/or rotate faster than the benzene ring and alkyl group. In neutral
and acidic conditions at pH 7.0 and 5.0 of 0.1 mM, the obtained chemical shifts consisted of
peaks at pH 9.0 except for H
g
, which corresponded to the peak neighboring the tertiary N
atoms (Figures S15 and S19(a) in S10 and S14). Deshielding shifts of H
g
were observed in the
order pH 9.0 < 7.0 < 5.0, suggesting that the protonation took place at N atoms. In the plot of
chemical shift vs concentration, even though concentration dependences similar to those at pH
9.0 were observed, the degree of change in the chemical shifts was smaller than for those at
8
ACS Paragon Plus Environment

Page 9 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
pH 9.0. The resulting K values in H
b
, H
g j
, and, H , by fitting with the isodesmic model were
ꢀ1
4
±
.03 ± 0.01, 3.50 ± 0.01, and 4.31 ± 0.01 M at pH 7.0 and 1.35 ± 0.01, 1.28 ± 0.01, and1.37
ꢀ1
0.25 M at pH 5.0. Comparing to the K values between various pH conditions, the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
estimated K values increased in the order pH > 9.0 > 7.0 > 5.0 suggesting that protonated
H-UBD in acidic conditions was suppressed to form the aggregates owing to Coulomb
repulsion and/or increased hydrophilicity. The CAC values under conditions at pH 7.0 and 5.0
1
were 1 and 5 mM, respectively. The HꢀNMR spectrum of H-UBD in 0.1 m M and the
concentration dependence at pH 9.0 are shown in Figure 2 a) and b). Similar spectra at pH 7.0
b j g
and 5.0 are shown in Figure S17 (S12). The plots of H and H , and H as a function of
concentration at pH 9.0, 7.0, and 5.0 with the fitting curves are shown in Figure 3 and Figure
S19 (a) (S14). The obtained values of K and CAC are summarized in Table 1.
Table 1. Values of Association Constants (K) Estimated from Isodesmic Model and CAC
under Various Concentrations at pH 9.0, 7.0, and 5.0.
ꢀ1
pH
K values ( M )
CAC ( mM)
H
a
H
g
H
j
9
7
5
.0
.0
.0
12.3 ± 0.1
4.03 ± 0.01
1.35 ± 0.01
54.3 ± 11.8
3.50 ± 0.01
1.28 ± 0.01
16.2 ± 0.1
4.31 ± 0.01
1.37 ± 0.25
0.5
1
5
B-2. EPR spectra of TEMPO-UBD in buffer solution.
Xꢀband ( 0 =9.4 GHz) ESR measurements at various concentrations (0.1 – 2.0 mM) were
ν
performed at pH 9.0, 7.0, and 5.0. In the lowest concentration of 0.1 mM TEMPO-UBD
solution at pH 9.0, peaks with three wellꢀresolved lines due to splitting of the nucleus spin of
an N atom were observed at g = 2.0048 (Figure 4). The peak in the highest field showed the
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
weaker intensity and slight broadening compared to those typical TEMPO analogues,
R
indicating that the slow rotational correlation time (τ ) took place even at 0.1 mM due to the
large molecular size of TEMPO-UBD. As the concentration increased, the peak in the highest
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
field became slightly weaker, and broadened above 0.76 mM.
(a)
(b)
ꢀ10
.3x10
2
2
.2
.1
.0
.9
2
2
1
pH 5.0
pH 7.0
pH 9.0
1.8
2
3
4
5
6
7
8
9
2
0.1
1
Concentration / mM
Figure 4. (a) Normalized ESR spectra of TEMPO-UBD in 2.0 – 0.1 mM buffer solutions at
pH 9.0. Spectra were normalized by intensity at center peaks. Inset shows a magnified peaks
R
at highest field. (b) Plots of τ values estimated by Kivelson’s equation vs concentration at
given pHs. The colored solid line indicate the sigmoidal fitting as a guide.
To evaluate the global motions of molecules and local motion of TEMPO moiety below and
2
4
ꢀ10
R
above 0.76 mM, τ values using Kivelson’s equation were estimated as 2.05 x 10 and 2.34
ꢀ10
x 10 s, respectively (Figure 4, S20, and eq. S3 in S15). The resulting values above 0.76 mM
ꢀ11
were16 and 1.1 times larger than those of typical TEMPO (1.5 x 10 s) and the solution of
TEMPO-UBD below 0.76 mM, suggesting the formation of aggregate as seen for H-UBD,
and the inflection concentration implied the CAC value of TEMPO-UBD at pH 9.0. At pH
7.0 and 5.0, furthermore, the inflection points of τ values were both observed at the similar
R
concentration of 0.66 mM. Even though similar CAC values between pH 9.0, 7.0, and 5.0
were observed, the estimated τ values showed a large difference. Below and above CAC at
R
10
ACS Paragon Plus Environment

Page 11 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
ꢀ10
pH 7.0, and 5.0, the τ
R
values were 1.85 and 2.09 x 10 s, for pH 7.0 and 1.77 and 2.04 x
ꢀ10
10
s for pH 5.0, respectively. Below and above CAC, the obtained τ
R
values increased in
the order pH 9.0 > 7.0 > 5.0. Below CAC, TEMPO-UBD in pH 5.0 solution exists as cationic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
monomer protonated at the tertiary N atoms, to give the fast molecular motion and smallest τ
R
values due to Coulomb repulsion, while in pH 9.0 solution, the monomer of TEMPO-UBD
exists as a neutral form and showed the largest τ
Above CAC, the resulting aggregate including Coulomb repulsion in an acidic condition also
gave faster molecular motion and smaller τ values. In addition, the neutral aggregate gave
values. Comparing the CAC values estimated from
R
values compared to those at pH 7.0 and 5.0.
R
the slower molecular motion and larger τ
R
1
HꢀNMR for H-UBD and ESR for TEMPO-UBD, the values of TEMPO-UBD were smaller
than those of H-UBD, indicating that the association constant (K) of TEMPO-UBD might be
higher than that for H-UBD. The difference of the selfꢀassembly behaviors among UBDs is
responsible for the hydrophobicity of TEMPO moiety. ESR spectra of TEMPO-UBD in 2.0 –
0
.1 mM buffer solutions at pH 9.0, 7.0, and 5.0 are shown in Figure 4(a) and S21(S15). Plots
of τ vs concentration at pH 9.0, 7.0, and 5.0 are shown in Figure 4 (b). The τ values
obtained under various conditions are summarized in Table 2.
R
R
R
Table 2. Values of τ (s) Estimated from Kivelson’s Equation and CAC under Various
Concentration Conditions at pH 9.0, 7.0, and 5.0.
ꢀ10
pH
τR (10 s)
CAC (mM)
Below CAC
Above CAC
2.34
9
.0
7.0
.0
2.05
1.85
1.77
0.76
0.66
0.66
2.09
5
2.04
C. Thermal behaviors of H- and TEMPO-UBD in water and buffer solution.
C-1) Transmittance change of H- and TEMPO-UBDs by heating process.
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We previously reported that the urea benzene derivatives (UBDs) carrying the OEGs showed
thermal responsiveness in an aqueous solution, to give a cloudy solution with lower critical
11
solution temperature (LCST) by heating. This behavior is based on the dehydration
surrounding OEGs in response to temperature, such that the selfꢀassembly is accelerated by
the increase of hydrophobicity of the molecules as well as the increasing size of the aggregate.
To understand the thermal behavior of H- and TEMPO-UBD, the transmittance at 800 nm
was monitored in the range 20 – 70 ºC at various pHs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the case of 5 mM H-UBD solution at pH 9.0, which is above CAC, the transparent
solution turned abruptly cloudy at 48 ºC, which corresponds to the LCST value, and the
resulting cloudy solution was maintained over 70 ºC (Figure 5(a)). In contrast, the solution at
0.1 mM, which is below CAC, showed no LCST behavior until 70 ºC, indicating a
dependency on concentration. Similarly, the thermal behaviors in 5 mM buffer solution at
various pH conditions were tested: LCST behavior at 63 ºC and no LCST behavior until 70 ºC,
the same as for the dilute solution, were observed at pH 7.0 and 5.0, respectively. As the pH
values decreased, the LCST became higher in the order pH 9.0 < 7.0 < 5.0, indicating that
aggregates including the cation moiety in acidic conditions can’t easily form a large size
aggregate and/or the amount of the aggregate is maintained. This is because Coulomb
repulsion between cationic species took place by itself and/or the cationic species showed a
large hydrophilicity than under basic conditions.
In the case of TEMPO-UBD in phosphate buffer solutions, interestingly, twoꢀstep decreases
of the transmittance were observed (Figure 5(b)). At pH 9.0, the first and second steps began
at 31 and 37 ºC, respectively. The former step showed gradual change of the transmittance
from 98 to 83%, and the latter step changed abruptly from 83 to 0%. Similar twoꢀstep LCST
behaviors were observed at pH 8.0, and 7.0. This twoꢀstep behavior has been reported in the
25
case of polymer compounds and was classified as “double –LCST behavior”. It was noted
that this thermal doubleꢀLCST behavior is first such example among the supraꢀmolecule
12
ACS Paragon Plus Environment

Page 13 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
category. Furthermore, it was found that missing doubleꢀLCST behavior took place in the
conditions using a pure water solution, and in the case of H-UBD even at a high
concentration.
(aꢀ1)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
aꢀ2)
bꢀ1)
(
1
00
8
6
4
2
0
0
0
0
pH 5.0
pH 6.0
pH 7.0
pH 7.5
pH 8.0
pH 9.0
H2O
0
20
30
40
50
60
70
Temperature / ∞C
(bꢀ2)
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 5. Thermal responsiveness monitored by changes of the transmittance at 800 nm in 5
mM aqueous solutions of (a) H-UBD and (b) TEMPO-UBD at given pHs and in pure water
condition (black). The pictures indicate the change between transparent and turbid solutions in
vials by thermal stimulus (5 mM at pH 7.0).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nd
The abrupt decrease of the transmittance observed in the 2 step corresponds to the typical
LCST behavior due to selfꢀassembly by the dehydration surrounding OEGs. The gradual
st
decrease of the transmittance observed in the 1 step might be led by the demetalation and
consequently the selfꢀassembly process in buffer solution including the salts of NaCl and KCl.
Above 50 ºC, in addition, a gradual increase of the transmittance occurred until 70 ºC,
suggesting the formation of precipitation due to a stronger dehydration effect. Actually, small
precipitations in a cuvette were observed during the measurement at 70 ºC. Comparing the
LCST behavior between H- and TEMPO- UBD, the LCST value of TEMPO-UBD shifted to
a lower temperature in same conditions, suggesting that the hydrophobicity in the aggregates
consisting of TEMPO- UBD was higher than in those of H-UBD. The reason for no
doubleꢀLCST behavior in the H-UBD might be the differences in hydrophobicity and stability
for the aggregate in buffer solution. The thermal responsiveness monitored by changes of the
transmittance at 800 nm in 5 mM aqueous solutions of H- and TEMPO-UBD at given pHs,
in addition in a pure water are shown in Figure 5 and Figure S22 (S16). The thermal variation
between transparent and turbid solutions into vials were photographed and are represented in
Figure 5.
Table 3. LCST Values of H- and TEMPO-UBD under Various pH Conditions.
LCST value (ºC)
pH
.0
H-UBD
TEMPO-UBD
*
9
45
31
#
37
14
ACS Paragon Plus Environment

Page 15 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
*
#
*
#
8.0
7.0
6.0
47
63
36
40
42
51
N. D.
56
64
39
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
.0
ꢀ
Water
48
#
st
nd
*
and indicate the values at starting the 1 and the 2 LCST steps, respectively.
C-2. Variable Temperature DLS measurements of H- and TEMPO-UBD in a buffer
solution.
To understand the size of aggregate as well as the thermal selfꢀassembly accompanying
LCST, variable temperature dynamic light scattering (VTꢀDLS) measurements in 5 mM of
aqueous solutions were performed for H- and TEMPO-UBD, respectively. Each
H
hydrodynamic diameter (D ) was estimated as the average of three time measurements. The 5
mM solutions was selected as the concentration forms the aggregate in both UBDs
derivatives.
In a buffer solution at pH 9.0 for H-UBD (Figure 6a), single broadening peaks
H
corresponding to D values of 10~80 and over 1000 nm were observed at 20 and 58 ºC,
respectively, indicating that the aggregate below LCST was nanoꢀparticles 10~80 nm in size
and the grown aggregate above LCST was microꢀparticles over 1000 nm in size. In the neutral
H
condition (Figure 6a), similar D values of 20~100 and ~1000 nm at pH 7.0 were observed
below (20 ºC) and above LCST (70 ºC), indicating that nanoꢀparticles and microꢀparticles
were formed below and above LCST. In addition, the pH dependence was negligible (Table 4).
Even in pure water, the resulting D
Figure S22 in S16). In contrast, in TEMPO-UBD with pH 9.0 solution (Figure 6b and c),
which had the doubleꢀLCST behavior at 31 and 37 ºC (Figure 5 and Table 3), the D values at
4 and 36 ºC showed 30~150 nm as a single broadening peak, and two broadening peaks at
H
values were consistent with those in buffer solution
(
H
2
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
~
300 and over 1000 nm, respectively, suggesting that nanoꢀparticles 30~150 nm in size, as for
st
H-UBD, and grown particles over 100 nm in size were formed below and above the 1 LCST
nd
step. Above the 2 LCST of 40 ºC, all particles exhibited over 1000 nm in size and the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resulting thermal behavior was consistent with those of H-UBD.
(a)
(b)
(c)
(d)
Figure 6. Variable temperature DLS measurement in 5 mM buffer solutions. D
H-UBD and (b) TEMPO-UBD for given conditions of temperatures and pHs. (c) Detailed
changse of D values at various temperature at pH 9.0 for TEMPO-UBD. (d) Plots of the
average D value (right axis and blue mark) and transmittance (left axis and red mark) change
vs temperature at pH 9.0 for TEMPO-UBD. Dotted circle in (d) represents the steady step
H
values of (a)
H
H
st
nd
between the 1 and 2 LCST behaviors.
H H
To reveal the relationship between the transmittance and D value, the D values changed by
the heating process were plotted as a function of the temperature accompanied by the
16
ACS Paragon Plus Environment

Page 17 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
transmittance change (Figure 6d). As the temperature increased from 20 to 48 ºC, two
H
nonꢀcontinuous increase of D values were observed at 32 and 36 ºC, suggesting that changes
in particle size took place at the same temperature as the doubleꢀLCST behavior obtained
from the thermal transmittance change (left axis in Figure 6d ). As the temperature increased
above 48 ºC, a decrease of the microꢀparticle size was observed. This behavior occurred due
to the formation of precipitations by a stronger dehydration effect (vide supra). At pH 7.0 and
in pure water for TEMPO-UBD (Figures 6(b) and S23 in S17), comparable size changes with
pH 9.0 were observed below and above LCST. Comparing the size of the particles between
H- and TEMPO-UBD, even though TEMPO-UBD introduced a TEMPO moiety into
H-UBD, no considerable difference in the particle size below and above LCST was seen. The
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
obtained D
Figure 6a and b and are summarized in Table 4. The detailed changes of D
temperatures at pH 9.0 for TEMPO-UBD are shown in Figure 6c. Plots of D
H
values of H- and TEMPO-UBD under variable temperature and pH are shown in
values at various
value (right
H
H
axis) and transmittance (left axis) change vs temperature at pH 9.0 for TEMPO-UBD are
given in Figure 6d.
Table 4. Temperature and pH Dependencies of the Size Obtained from DLS for H- and
TEMPO-UBD with 5 mM Buffer Conditions.
Temperatu
re ranges
Size / nm (ºC)
H-UBD
pH 7.0
TEMPO-UBD
(ºC)
pH 9.0
10~80
Pure
water
20~100 7~60
pH 9.0
pH 7.0
Pure
water
2
0
10~150
10~150
20~150
2
1‒30
30~150 (24)
100~ (36),
31 ‒40
3000~ (40)
41‒50
~1000 (50)
100 (60)
5
1 ‒ 69
70
~1000 (58)
~1000 (68)
~1000
~700
~1000
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The parentheses indicate the temperature recorded DLS data.
C-3) TEM and SEM images of H- and TEMPO UBD.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To identify the morphologies of the solution samples, transmission electron microscopy
(TEM) were carried out and stained images for H- and TEMPO-UBD were obtained. In
addition, scanning electron microscopy (SEM) for TEMPO-UBD was performed. In TEM,
the 5 mM water solution samples were mounted as 5 ꢀl on a carbon grid with hydrophilic
treatment and the residual solution was sucked up by a filter paper at 23 ºC for the sample
below LCST. In contrast, the residual solution was evaporated at 70 ºC for the sample above
LCST. Each sample was stained with 5% uranyl acetate solution for the negative stained
images. The salts often prevented observation of the morphology for organic materials even
after staining so the samples prepared in pure waters solution were used. In SEM, the samples
were prepared from freezingꢀdry for the below LCST, while heated at 100 ºC on hot plate for
the sample above LCST. Each sample was coated with Pt moisture on a carbon tape onto a
stage.
c-3-1) TEM.
In H-UBD, spherical nanoꢀparticles 20 ~ 60 nm in size were obtained below LCST, while
amorphousꢀlike assembly of over 100 nm in size were observed in the samples above LCST
(Figure 7a and S25 in S18). The sizes obtained below and above LCST were slightly smaller
than those obtained from DLS measurements. This suggests that the samples in solution
included water molecules, making the sizes of samples obtained by DLS larger size compared
to those on TEM images. In the case of TEMPO-UBD (Figure 7b and S25 in S18), the
sample below LCST showed spherical nanoꢀparticles with 10ꢀ 150 nm sizes same as H-UBD.
In contrast, the samples above LCST exhibited formation of assemblies consisting of
nanoꢀparticles of size 300ꢀ 500 nm. The resulting sizes below and above LCST were smaller
than those estimated from DLS measurement for the same reason as with H-UBD. Above
18
ACS Paragon Plus Environment

Page 19 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
LCST, interestingly, a distinguishable morphology difference between H- and TEMPO-UBD,
being amorphousꢀlike particles and assemblies of the nanoꢀparticles, was clearly observed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(aꢀ1)
(aꢀ2)
(bꢀ1)
(bꢀ2)
Figure 7. TEM images of (a) H- and (b) TEMPO-UBD prepared at (1) 23 and (2) 70 ºC.
Scale bars indicate (aꢀ1)100, (aꢀ2) 200, (bꢀ1) 200, and (bꢀ2) 500 nm, respectively.
c-3-2) SEM.
In the sample after freezingꢀdry for TEMPO-UBD, many spherical nanoꢀparticle ~ 60 nm in
size were clearly observed. In contrast, the image of sample heated at 100 ºC showed the
grown particles with 100 ~ 500 nm in size and looks like the disordered amorphous shape,
even though the TEM image gave the ordered assemblies consisting of nanoꢀparticles. This
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
discrepancy might resulted from melting of the particles on a hot plate under preparing the
SEM sample. TEM mages and plots of count vs diameter for H- and TEMPO-UBD are
shown in Figure 7 (a) and (b), and S25 (in S18). SEM images for TEMPO-UBD are shown in
Figure S26 in S19.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C-4) Structured differences of self-assembly between H- and TEMPO-UBD.
Comparing the thermal properties and the morphologies between H- and TEMPO-UBD,
interestingly, we observed distinguishable differences in the LCST behavior and the
morphology above LCST were observed. With respect to the LCST behaviors, doubleꢀLCST
behavior was observed only in the case of TEMPO-UBD. Furthermore, the morphology of
the microꢀparticles obtained above LCST was different with microꢀparticle being disordered
amorphous or ordered assemblies of nanoꢀparticles in H- and TEMPO-UBD, respectively.
These results suggest that different thermal selfꢀassembly behaviors took place by different
mechanisms at the molecular level. Below LCST, the nanoꢀparticles of both H- and
TEMPO-UBD were hydrated with many water molecules and metal ions at OEGs in the
buffer solution. As the temperature increased, initially demetallation and then dehydration
surrounding OEGs took place, creating more hydrophobicity at the molecule level and in
grown microꢀparticles. In the case of the H-UBD sample (Figure 8a), upon heating the
spherical nanoꢀparticles sized ~100 nm collapse and the resulting amorphous microꢀparticles
might form a disordered random monomer. Although UBDs are supraꢀmolecules, the
microꢀparticles formed a globuleꢀlike structure, as also reported in thermoꢀresponsive
2
6
waterꢀsoluble polymers such as PNIPAM. In the case of the TEMPO-UBD (Figure 8b), in
contrast, the spherical nanoꢀparticles 20~ 150 nm in size obtained below LCST accumulated
by themselves and maintained their morphology and size even at 70 ºC, to give new particles
of micrometer order. Since the hydrophobicity of TEMPO-UBD is higher than that of
H-UBD, the stability of the nanoꢀparticles composing TEMPO-UBD in aqueous solution is
20
ACS Paragon Plus Environment

Page 21 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
also higher than that of H-UBD, so a difference in morphology change took place in both
UBDs over LCST. With respect to the doubleꢀLCST behavior in TEMPO-UBD, we consider
st
nd
that the 1 and the 2 steps of LCST correspond to the demetallation and dehydration
processes, respectively (Figure 8b). Even though the conditions were similar, the absence of
doubleꢀLCST behavior in H-UBD is likely to be led by the weaker association constant of the
nanoꢀparticles and so disordered microꢀparticles (globules) were observed. The plausible
structures and thermal mechanism of H- and TEMPO-UBD in buffer solution are shown in
Figure 8.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(a)
(b)
Figure 8. Schematic drawing of plausible structures of (a) H- and (b) TEMPO-UBD in buffer
solution.
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
D) Water-proton longitudinal and transverse relaxivity (
TEMPO-UBD in aqueous conditions at pH 9.0, 7.0, and 5.0.
To reveal the potential of TEMPO-UBD as a MRI contrast agent, waterꢀproton relaxivites,
and r , were determined from relaxation times, T and T obtained using 7 T MRI apparatus
at various concentrations and at pH 9.0, 7.0, and 5.0, respectively. In addition, T ꢀ and T
weighted images were acquired (Figure 10). Considering bioꢀimaging applications, the T and
values were obtained at 23 ºC maintained using a gradient coil cooling system and air
conditioners. Samples at 10.0, 7.5, 5.0, 3.3, 2.5, 1.0, 0.5, 0.25, and 0.125 mM were prepared
in PCR tubes and used. The values of r and r were estimated from the slopes in the plots of
vs concentration. To evaluate the r and r values of TEMPO-UBD, those values
were compared with simple TEMPO derivatives of Oxo-TEMPO.
1 2
r and r ) and MR imaging of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
r
1
2
1
2
1
2
–
1
T
2
1
2
ꢀ1
ꢀ1
T
1
or T
2
1
2
(a)
r
1
= 0.21
r
2
.0
.5
1
1
= 0.15
1.0
0.5
r
1
= 0.17
0
2
4
6
8
10
Concentration / mM
(b)
22
ACS Paragon Plus Environment

Page 23 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
6
.0
r = 0.33
2
5.0
4
.0
.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
r2 =0.19
3
0
2
4
6
8
10
Concentration / mM
ꢀ
1
ꢀ1
Figure 9. Plots of (a) T
1
and (b) T
2
vs concentration for TEMPO-UBD (blue and red) and
oxo-TEMPO (black) as reference at pH 7.0. The ranges for higher (10.0 – 1.0 mM) and lower
(0.5 – 0 mM) concentrations of TEMPO-UBD are shown blue and red filled circles,
respectively. The solid lines indicate the least squares fitting of each slope.
ꢀ
1
ꢀ1
The plots of relaxation rate, T
There were two different slopes, below and above 1.0 mM. From the τ
ESR at pH 9.0, nanoꢀparticles formed above CAC value of 0.76 mM, which is consistent with
the inflection values of 1 mM of r and r . The values below and above CAC of r were 0.14
1
or T
2
at pH 7.0 vs concentration are shown in Figure 9.
R
value estimated from
1
2
1
ꢀ1 ꢀ1
and 0.18 mM s , respectively. In addition, Oxo-TEMPO showed no inflection point from 10
ꢀ1 ꢀ1
–
1
0.1 mM and an r value of 0.15 mM s , suggesting that above CAC TEMPO-UBD
exhibited a 1.2 times larger value than below CAC and Oxo-TEMPO, respectively. This
result indicates that nanoꢀparticles exhibited larger r values due to the suppression of fast
molecular motion, as expected. Similarly, at pH 7.0 and 5.0, inflection concentration of r
1
1
1
value was observed and was consistent with the value obtained from
R
τ . The resulting r
ꢀ1 ꢀ1
values below and above CAC were 0.17 and 0.21 mM s at pH 7.0, and 0.15 and 0.19
ꢀ1 ꢀ1
2 2 2
mM s at pH 5.0, respectively. In the r values estimated from T ꢀweighted images, r values
ꢀ
1 ꢀ1
above CAC were 0.28, 0.33, and 0.39 mM s , at pH 5.0, 7.0, and 9.0, respectively,
suggesting a strong pH dependency. The reference of Oxo-TEMPO showed a smaller value
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ1 ꢀ1
of 0.19 mM s . As pH increased, the r value increased in the order pH 9.0 > 7.0 > 5.0 and
2
2 2
the resulting r values were larger than that of the reference. Since the r value is directly
affected by the spin quantum number and the amount of spin number compared to r , the
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aggregate number of nanoꢀparticles at pH 9.0 might be the largest, and in the order pH 9.0 >
ꢀ1
ꢀ1
7
.0 > 5.0. Plots of T1 and T vs concentration for TEMPO-UBD with Oxo-TEMPO at pH
2
9
1 2
.0, 7.0, and 5.0 are shown in Figure 9, Figure S27(a) and (b) in S20. T ꢀ and T ꢀweighted
images at pH 7.0 are shown in Figure 10. The values of r and r at pH 9.0, 7.0, and 5.0 are
1
2
summarized in Table 5.
(a)
(b)
1 2
Figure 10. (a)T ꢀ and (b)T ꢀweighted images of TEMPO-UBD at pH 7.0 and at various
concentrations with standard materials. The numbers next to each image indicate the samples
for 1) 10.0, 2) 7.5, 3) 5.0, 4) 3.3, 5) 2.5, 6) 1.0, 7) 0.5, 8) 0.25, and 9) 0.125 mM of
2
TEMPO-UBD. 10) 0.25, 11) 0.125 mM of MnCl . 12) pure water, respectively.
Table 5. Values of r
1 2
and r for TEMPO-UBD in Buffer Solutions under Various Conditions
and a Blood Solution at pH 7.0 with Results of Oxo-TEMPO as Reference.
pH
TEMPO-UBD
ꢀ1 ꢀ1
ꢀ1 ꢀ1
r (mM s ) r (mM s )
1
2
24
ACS Paragon Plus Environment

Page 25 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
9
.0
Above CAC 0.18
Below CAC 0.14
Above CAC 0.21
0.39
0.21
0.33
0.56*
7.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.24*
#
#
0
.24
0.38
Below CAC 0.17
N. D.
0.88*
0
0
.20*
#
#
.17
0.25
5.0
Above CAC 0.19
Below CAC 0.15
0.28
0.22
Oxo-TEMPO
.15
0
0.19
#
*
measured under blood solution. after annealed at 70 ºC and measured 25 ºC.
Comparing the r
neutral condition at pH 7.0 in spite of the highest
discrepancy between r and values can be explained as follows. In the acidic condition at
pH 5.0, the nanoꢀparticles showed smaller values in ESR because the nanoꢀparticles were
1
values above CAC at various pHs, the highest value was obtained in the
τ
R
value being seen at pH 9.0. This
1
τ
R
τ
R
carrying a cationic moiety, to lead a fast local motion of TEMPO moiety and/or fast global
motion of the nanoꢀparticles due to Coulomb repulsion. While in the basic condition at pH 9.0,
even though the nanoꢀparticles showed a larger
R
τ value in ESR, the number of water
molecules surrounding the TEMPO moiety is smaller owing to deprotonated tertiary N atoms
and stronger hydrophobicity in the molecule. In the neutral condition at pH 7.0, even though
the nanoꢀparticle showed a smaller
τ
R
value rather than those at pH 9.0, many water
1
molecules exist surrounding TEMPO moiety so the largest r values were observed due to
25
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
optimized conditions. Plausible molecular motions and environments of water molecules in
nanoꢀparticles comprising TEMPO-UBD at pH 9.0, 7.0, and 5.0 are shown in Figure 11.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 11. Schematic drawing of plausible molecular motions and environments of water
molecules in nanoꢀparticles comprising TEMO-UBD at pH 9.0, 7.0, and 5.0.
For bioꢀimaging, furthermore, the stability of TEMPO-UBD in blood was confirmed by the
1 2 1 2
variation of values of relaxivities (r and r ). The resulting values of r and r were 1.2 and 1.8
times larger than those in buffer solution at pH 7.0, suggesting that TEMPO-UBD was not
2
7
labile in the blood and showed resistance against reductants such as ascorbic acid and
28
glutathione . in blood. The reason for the increase of relaxivities might be the interaction
29
between proteins such as albumin and increase size. Surprisingly, increased values of r
1
and
r
2
were also observed even with annealing treatment of the sample until 70 ºC. This result
suggested that the heating process creates on the optimized formation of nanoꢀparticles for the
increase of relaxivities and/or the water molecules reꢀoriented into TEMPO-UBD. Plots of
ꢀ1
ꢀ1
T
1
2
and T vs concentration for TEMPO-UBD in blood in Figure S28 in S21 and values are
summarized in Table 5.
CONCLUSION.
26
ACS Paragon Plus Environment

Page 27 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
In conclusion, we prepared a supraꢀmolecular organic radical, TEMPO-UBD, carrying
TEMPO radical as a candidate metalꢀfree MRI contrast agent. TEMPO-UBD showed thermal
and pH responsiveness in addition to MR function. In buffer solutions above CAC,
TEMPO-UBD formed spherical nanoꢀparticle 20 ꢀ 150 nm in size. These nanoꢀparticles
exhibited a two steps “doubleꢀLCST” thermosꢀresponsive behavior and turned into
microꢀparticles above LCST. This doubleꢀLCST is the first example among supraꢀmolecules.
As pH decreased, LCST values increased due to the formation of cationic TEMPO-UBD. To
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
evaluate its potential as an MRI contrast agent, waterꢀproton relaxivites, r
estimated under various pH conditions. The resulting r and r at pH 7.0 were 1.2 and 3 times
larger than those of Oxo-TEMPO, indicating that an effective molecular size effect took
place. In a blood sample, the relaxivities of r and r were 1.2 and 1.8 times larger than those
1 2
and r were
1
2
1
2
in buffer solutions, indicating that TEMPO-UBD is not labile in a blood and thus is useful for
bioꢀimaging. In vivo imaging using TEMPO-UBD is under investigation. In the present stage,
no extremely large r
oligonucleotideꢀTEMPO system were observed. Because two insufficient factors to increase
the r and r values were raised in the TEMPO-UBD. One is insufficient suppression of
tumbling of the TEMPO moiety, another one is few number of water molecular surrounding
the TEMPO. To prepare candidate MRI contrast agents having larger r and r values using
1
and r
2
values such as the nanoꢀparticles consisted of
9)
1
2
1
2
the nanoꢀparticles, thus, the design and preparation of radicals introducing the polar moiety
such as OEGs, hydroxyl and carboxylic acid neighboring the radical center are in progress.
EXPERIMENTAL SECTION.
General Information
1
13
Infrared and UVꢀVis spectra were recorded. H and CNMR spectra were measured using
CDCl or DMSOꢀd6, or D O including TMS or DDS as standard material. HRMS using ESI
3
2
mass spectra (ESI MS) were recorded. ESR spectra were recorded on Xꢀband (9.4 GHz)
27
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
spectrometer equipped with a microwave frequency counter. Sample solutions in phosphate
buffer were placed in capillary tubes and were measured at 25 °C. DLS measurements were
performed. The images of transmission electron microscopy (TEM) images were obtained.
The sample mounted on a carbon grid with hydrophilic treatment was stained with 5% uranyl
acetate aq.. Scanning electron microscopy (SEM) was carried out. The samples were coated
by Pt moisture by an ion coater and immobilized on a carbon tape onto a stage.
Relaxivity Measurements
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1 2
The longitudinal (spinꢀlattice) and transverse (spinꢀspin) relaxation times (T and T ,
respectively) were obtained on 25 MHz (0.59 T). The sample solutions (ca. 0.1 − 0.7 mM) in
phosphate buffer were placed in 10 mm o.d. glass tubes and were measured at 25 °C. The
1 2
values of relaxivity, r and r , were calculated with equations (1) and (2).
1
/T
1
2
= 1/T
0
0
+ r
1
2
C
C
(1)
(2)
1
/T
= 1/T
+ r
,
where T
concentration of the paramagnetic species, respectively.
- and -weighted MRI for samples. MRI acquisitions of contrast agents were performed
0
and C are the relaxation time in the absence of the paramagnetic species and the
T
1
T
2
on a 7.0 TꢀMRI scanner with a volume coil (35 mm innerꢀdiameter, transmission and
reception). Aqueous solution of contrast agents (150 ꢀl) was put into a polymerization chain
reaction (PCR) tube cluster plate. The PCR tube cluster plate was set in the center of the
volume coil. Sample temperature was maintained at 23.0 ± 0.5 °C throughout all experiments
using a gradient coil cooling system and air conditioners. MRI scanner, horizontal singleꢀslice
1
T ꢀweighted MR images were acquired with the following parameters: spin echo, TR/TE
=400/9.6 ms, slice thickness = 2.0 mm, matrix = 256 × 256, field of view (FOV) =38.4 × 38.4
2
mm , number of averages (NA) = 1, number of slices = 1. For longitudinal relaxation time
1 1
(T ) and longitudinal relaxivity (r ) calculations, horizontal singleꢀslice inversionꢀrecovery
MRI was obtained using RARE (rapid acquisition with relaxation enhancement) acquisition
28
ACS Paragon Plus Environment

Page 29 of 38
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
with the following parameters: TR = 10,000 ms, TE = 20 ms, inversion time = 52, 100, 200,
2
4
00, 800, 1600, 3200, 6400 ms, matrix size = 128 × 128, FOV = 38.4 × 38.4mm , slice
thickness = 2.0 mm, RARE factor = 4, and NA = 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Materials
Unless otherwise stated, the solvent and reagents were used without the
purification. 2ꢀAminoethanol, 1,6ꢀdibromohexane, 5ꢀaminoꢀ1,3ꢀisophthalicacid, potassium
phthalimide, isophthaloyl chloride, and Oxo-TEMPO were purchased and used without
purification. 2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethyl 4ꢀmethylbenzenesulfonate (Eg
3
Ts) and
tetramethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ3,6ꢀdihydropyridinꢀ1(2H)ꢀyloxyl
1
4, 16
radical were prepared according to the literatures.
60 F254 (Merck).
TLC was performed on silica gel plates
1
1ꢀ(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethyl)ꢀ2,5,8ꢀtrioxaꢀ11ꢀazatridecanꢀ13ꢀol (TEG
solution of Eg Ts 33 g (0.10 mol) and 2ꢀaminoethanol (2.5 g, 40 mmol) in 50 mL CHCN
added K CO (25g, 0.18 mol) and refluxed for 6 h. The solution was cooled to r.t. and the
residual was removed by suction. The resulting solution was evaporated under reduced
pressure. The crude residual was chromatographed on silica gel using with CHCl : MeOH
(100 : 1 – 50 : 1) as eluent to afford a colorless oil (10.8g, 30.6 mmol) in 75% yield. IR (NaCl,
2
EA). To a
3
3
was
2
3
3
ꢀ1
1
cm ) 3472, 2873, 1456, 1352, 1294, 1245, 1200, 1108, 1045; HꢀNMR (CDCl
3
, 500 MHz) δ
3.67ꢀ3.60 (m, 12H), 3.59ꢀ3.51 (m, 10H), 3.38 (s, 6H), 2.79 (t, J = 5.7 Hz, 4H), 2.72 (t, J = 5.0
1
3
Hz, 2H); CꢀNMR (CDCl
3
) δ 71.9, 70.6, 70.5, 70.4, 69.8, 59.5, 59.0, 56.8, 54.2; ESIꢀMS m/z
+
+
354.25 [M+H] ; HRMS (ESI) Calcd for C16
H36NO
7
[M+H] : 354.2486, Found: 354.2519.
2ꢀ((6ꢀbromohexyl)oxy)ꢀN,Nꢀbis(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethyl)ethanꢀ1ꢀamine
Eg NEg Br). To a solution of 1,6ꢀdibromohexane (14.5 g, 59.4 mmol) in dist. THF (25
mL) was added a solution of NaH (1.5 g, 64 mmol) in dist. THF in an ice bath and stirred for
several minutes. To the solution was added dropwise TEG EA (7.3 g, 21 mmol) in dist. THF
10 mL) and stirred overnight. To the reaction mixture was added sat. NH Cl aqueous and the
(
3
3 6
C
2
(
4
mixture was extracted with Et
2
O three times. The combined organic layer was dried over
29
ACS Paragon Plus Environment