Copper-mediated radioiodination reaction through aryl boronic acid or ester precursor and its application to direct radiolabeling of a cyclic peptide

Article abstract of DOI:10.1002/jlcr.3925

A copper-mediated radioiodination using aryl boronic precursors is attracting attention as a solution to oxidative iododestannylation and nickel-mediated radioiodination drawbacks. The copper-mediated radiolabeling method allows radioiodination at room te

Full text of DOI:10.1002/jlcr.3925

Kimura Hiroyuki (Orcid ID: 0000-0002-4291-3524)
RESEARCH ARTICLE
Title:
Copper-mediated radioiodination reaction through aryl boronic acid or ester precursor
and its application to direct radiolabeling of a cyclic peptide
Running head:
Direct labeling of a peptide by copper-mediated radioiodination
1
1
1
1,2
3
Yuto Kondo , Hiroyuki Kimura *, Chiaki Fukumoto , Yusuke Yagi , Yasunao Hattori ,
Hidekazu Kawashima , Hiroyuki Yasui1
4
1Department of Analytical and Bioinorganic Chemistry, Division of Analytical and Physical
Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto
6
07-8414, Japan
2Department of Radiological Technology, Faculty of medicinal science, Kyoto College of
Medical Science, 1-3 Imakita, Oyama-higashi, Sonobe, Nantan, Kyoto 622-0022, Japan
3Center for Instrumental Analysis, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi,
Yamashina-ku, Kyoto 607-8412, Japan
4Radioisotope Research Center, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi,
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3925
This article is protected by copyright. All rights reserved.

Yamashina-ku, Kyoto 607-8412, Japan
Correspondence
*
Hiroyuki Kimura, PhD, Phone: +81-75-595-4630; Fax: +81-75-595-4753; E-mail:
hkimura@mb.kyoto-phu.ac.jp
Funding information
This work was supported by a Private University Research Branding Project for Ministry of
Education, Culture, Sports, Science, and Technology, and JSPS KAKENHI with grant number
JP20K08061.
This article is protected by copyright. All rights reserved.

ABSTRACT
A copper-mediated radioiodination using aryl boronic precursors is attracting attention as a
solution to oxidative iododestannylation and nickel-mediated radioiodination drawbacks. The
copper-mediated radiolabeling method allows radioiodination at room temperature with stable
aryl boronic precursors without preparing complex starting materials or reagents, and can be
performed in a reaction vessel exposed to air. This method has good potential in
radiochemistry; however, studies on the scope of copper-mediated radioiodination through
boronic precursors are insufficient. In particular, few reports have demonstrated the effect of
protecting groups on radiolabeling efficiency. Therefore, the effect of the protecting group of
aryl boronic acids on the copper-mediated radioiodination was investigated. In addition, this
method, which does not require heating, is expected to be useful for direct radiolabeling of
peptides. Thus, we attempted direct radioiodination of c(RGDyk) as an example. The resulting
radioiodination method was well tolerated in various substrates and was unaffected by the
pinacol ester-type protecting group. Also, c(RGDyk) was labeled with 125I via copper-mediated
radioiodination using an aryl boronic acid precursor. The reaction time and yield were
improved, compared with the indirect method. Furthermore, the large difference in polarity
between the boronic acid precursor and the radiolabeled compound facilitated purification.
This article is protected by copyright. All rights reserved.

KEYWORDS
copper-mediated, radioiodination, aryl boronic precursor, 125I, cyclic peptide, c(RGDyk), αVβ3
integrin inhibitor.
1
. INTRODUCTION
An oxidative iododestannylation of aryl stannanes is widely used as a radioiodination method
1
,2
because of its high radiochemical purity. However, this method allows the presence of toxic
organotin residues in clinical preparations of radioiodinated imaging agents, and the instability
3
of some organotin precursors prevents long-term storage. In addition, hydrogen peroxide
(H
2
O
) and N-chlorosuccinimide, used as oxidizing agents, require careful handling.4,5
2
Alternative approaches have been developed to improve these issues. For example, in the
6
nickel-mediated direct radioiodination of aryl and heteroaryl bromides, this halogen-exchange
reaction carried out at high temperatures (e.g., 180C) employs Ni(cod) , an air-sensitive metal
2
complex requiring storage at 0C and inert atmosphere techniques for handling. Therefore, Ni-
based precursors are not trivial to prepare by non-chemist professionals performing
(pre)clinical studies.
An excellent radiolabeling method, copper-mediated radioiodination using aryl boronic
precursors through a Chen–Evans–Lam cross-coupling reaction, was reported in 2016.3,7
Makvandi and Mach group found that the copper complex [Cu(pyridine)
4
(OTf) ] (OTf =
2
This article is protected by copyright. All rights reserved.

8
trifluoromethanesulfonate) was suitable for the radioiodination reaction. This reaction process
does not require preparation of complex starting materials or reagents and can be performed in
a reaction vessel exposed to air. Recently, a reaction without a copper catalyst was also reported.
This report has enabled the development of a simple base-catalyzed halodeboronation suitable
for the preparation of 125I-labeled products for imaging applications.20
Copper-mediated transformations between aryl boronic precursors and various coupling
partners have already been investigated9,10 and their translations into radiochemistry have
gained considerable attention. Copper-mediated 18F-fluorination,11,12 77/76Br-bromination,13
123/125/131I-radioiodination,3,7 211
At-astatination, and C-cyanation methods via aryl boronic
8
11
14
precursors have been reported. Tracers labeled with various radioactive elements could be
synthesized through a common aryl boronic precursor. Notably, its applicability to 211At-
astatination is extremely valuable from the treatment viewpoint.
3
FDA-approved medicines containing boron have already been used in clinical practice, for
®
®
®
20
example, Kerydin (tavaborole), VELCADE (bortezomib), and Eucrisa (crisaborole). For
treatment, a high dose of the medicine is administered, compared with the medicine dose used
for single-photon emission computed tomography (SPECT). Therefore, the tolerability of
humans to medicines containing boronic acid has been proven; further, boronic acid precursors
overcome the issue of toxicity in clinical use.
This article is protected by copyright. All rights reserved.

Several protecting groups have been available for boronic acid, which are essential for the
synthesis of boronic precursors. Occasionally, the protecting groups may be unexpectedly
removed during the precursor synthesis. Conversely, the reaction to remove protecting groups
may not proceed efficiently. Therefore, in designing precursors, how the protecting groups of
boronic acid affect the radiolabeling efficiency should be determined; however, few studies
have been conducted on this data and they have not been investigated in an integrated
manner.3,7,8
In recent years, studies on the scope of the copper-mediated 18F-labeling method and
1
8
preparation of F-labeled PET radiotracers from a diverse range of boronic precursors have
been in progress.11,12,15 There are also studies on labeling method of small molecules,3,8
2
2
peptides, and antibodies via copper-mediated radioiodination using a boronic precursor. In
particular, its application to small molecule labeling using the copper-mediated radioiodination
reaction is increasing, but studies on the scope of copper-mediated radioiodination using
boronic precursors are insufficient so far. Notably, few studies have been conducted on the
direct radiolabeling of peptides via the copper-mediated radioiodination reaction using a
boronic precursor. First, the direct labeling method of peptides with high specificity was limited．
Direct labeling of c(RGDfK) with 125I or 211At using tin precursors has been reported, but the
issue of toxicity derived from residual tin is unavoidable. Vaidyanathan et al. reported that 131I-
iodinated MIBG-octreotate was yielded by heating a mixture of brominated precursor and
23
This article is protected by copyright. All rights reserved.

1
7
radioiodine in acetic acid at 100C [Radio chemical yield (RCY): 3%–36%, n = 10]. In
general, since the brominated peptide precursor and radioiodinated peptide have similar
polarities, it is difficult to separate them by high performance liquid chromatography (HPLC).
When this method is applied to longer-chain peptides, the isolation and purification are
expected to become more difficult. Also, a heating operation should be avoided for application
to a wide variety of peptides. On the contrary, copper-mediated radioiodination via boronic
precursors does not require oxidizing reagents such as chloramine-T and can be carried out at
8
room temperature. In addition, the precursor does not contain heavy metals, and the polarity
between the boronic acid precursor and the iodine compound is significantly different.
Therefore, this method would be a highly versatile direct labeling reaction for peptides (Figure
1
).
Based on previous reports illustrating the Chan–Evans–Lam reaction to access
radioiodination via boronic reagents, we focused on the most frequently used pinacol ester-
type protecting group and aimed at investigating the effect of the protecting group of an aryl
boronic acid on copper-mediated radioiodination. In addition, to evaluate the effectiveness of
copper-mediated radioiodination via boronic precursor in direct labeling peptides, we report
the direct radioiodination method of c(RGDyk), a selective αVβ3 integrin inhibitor.
This article is protected by copyright. All rights reserved.

2
. RESULTS AND DISCUSSION
Labeling the aromatic ring with radioiodine is a common in radiotracer synthesis from the
viewpoint of stability of the iodinated compound in vivo.2,16 Hence, the effect of substituents
on the aromatic ring and their positional isomers on the copper-mediated radioiodination
through the boronic acid or ester should be investigated. The precursors 1a–1j were selected to
complement those of previous reports.3,7,8 Precursors without protecting groups 1a–1j(i) or
with pinacol ester-type protecting groups 1a–1j(ii) were labeled with 125I by the method shown
in Figure 2. Cu(pyridine)
4
(OTf)
2
, which can be labeled efficiently without the presence of 1,10-
8
phenanthroline as a ligand, was used. As copper-catalyzed transformations of functional
1
8
groups from aryl boronic acids proceed under solvents containing water, an aqueous solution
1
25
of Na I was used for radiolabeling. Radiochemical conversions (RCCs) shown in Figure 2
were determined by radio-thin layer chromatography (radio-TLC).
As a result, all compounds tested (2a–2j) showed high RCCs (99.7%–84.6%), and no
significant difference was observed between the case of radiolabeling via boronic precursors
without protecting groups 2a–2j(i) and with protecting groups 2a–2j(ii). This labeling method
is well tolerated in both electron-rich (2a–c, 2h–2j) and electron-poor substrates (2d–2f, 2g),
including sterically crowded substrates (2c and 2f). There are studies on radioiodination of
boronic precursors containing nitrogen atoms using the copper-mediated radiolabeling
method,3,8 however, reports on radioiodination of precursors containing sulfur atoms, which
This article is protected by copyright. All rights reserved.

are important for drug development, are limited. In our study, this labeling method was applied
to precursors with sulfur atoms (2i and 2j) and showed high RCCs (97.2%99.0%). In addition,
approximately no difference in RCCs was observed between positional isomers, allowing
flexibly designing precursors. Boronic acids are used in well-known reactions, such as Suzuki–
1
9
Miyaura cross-coupling, and various types of boronic acid compounds can be purchased.
Therefore, the boronic acid precursor is easy to synthesize using commercial compounds. In
addition, it is highly stable and can be stored for a long time. If application data is accumulated,
the copper-mediated radioiodination method becomes an indispensable radiolabeling method.
Then, as an application example of this labeling method, c(RGDyk) was directly labeled with
radioiodine through the boronic precursor. The cyclic peptide c(RGDyk) is a selective αVβ3
integrin inhibitor. Zhang et al. indirectly labeled c(RGDyk) using 125I-labeled N-
125
hydroxysuccinimide ester of iodobenzoic acid ([ I]IB-NHS) synthesized by the copper-
mediated radioiodination from a boronic acid precursor, and the SPECT imaging results
7
confirmed the good targeting ability and in vivo stability of radiopharmaceuticals. In general,
the indirect radiolabeling method takes a long time to label and increases the exposure risk of
researchers; however, labeling methods that can directly label peptides with high specificity
are limited. The development of effective and versatile direct radiolabeling methods for
peptides is required. Therefore, we evaluated whether copper-mediated radioiodination via
boron precursors, which can react under mild conditions, can be applied to direct radiolabeling
This article is protected by copyright. All rights reserved.

of peptides such as c(RGDyk).
1
25
First, [ I]IB-c(RGDyk) was synthesized by the indirect radiolabeling method.
Simultaneously, we investigated the reaction condition of 125I-labeled N-hydroxysuccinimide
125
ester of iodobenzoic acid ([ I]IB-NHS, 4), commonly employed in indirect radiolabeling of
16
peptides, proteins, and antibodies. N-Hydroxysuccinimide esters of boronobenzoic acid (PB-
NHS, 3) can be easily handled because they have high crystallinity. In addition, boronic acid
precursors are highly polar and have significantly different retention times than the target tracer
labeled with radioiodine; therefore, they can be easily separated and removed by HPLC. Thus,
125
we decided to synthesize [ I]IB-NHS (4) using PB-NHS (3) as a precursor. The reaction was
carried out using a methanol solvent (Figure 3). The RCCs of 4 showed a high value after 10
min. However, RCCs decreased with time. Then, the reaction solvent was changed from
methanol (MeOH) to ethanol (EtOH), acetonitrile, or acetone and we briefly investigated RCCs
of the target compound and by-product with time using radio-TLC (Table S1). At 10 min after
the reaction, the RCCs of MeOH and EtOH showed high values (97.7%–98.8%). The increased
rate of by-products with time when EtOH was used as the reaction solvent was smaller than
that when MeOH was used. Therefore, EtOH was used as a reaction solvent for the synthesis
125
of [ I]IB-NHS (4) through PB-NHS (3). Finally, c(RGDyk) was indirectly labeled using
[125
I]IB-NHS as a radiotracer to give [ I]IB-c(RGDyk) [radioisolated yield: 36.5%–39.3% (n
125
=
3), working time: 140.5–157.3 min (n = 3), Scheme 1B]. Since the synthesis and purification
This article is protected by copyright. All rights reserved.

1
25
process of [ I]IB-NHS are required, this indirect radiolabeling method requires a long time
125
to yield [ I]IB-c(RGDyk). Thus, applying an indirect radiolabeling method to the labeling of
123I (t1/2 = 13.27 h), which has a half-life shorter than 125I (t1/2 = 59.40 day), is inappropriate.
Then, c(RGDyk) was directly labeled with radioiodine via copper-mediated radioiodination
using the boronic precursor (Scheme 1A). PB-NHS (3a) was reacted with c(RGDyk) in
triethylamine/dimethylformamide (DMF) to give PB-c(RGDyk) (Yield: 43.6%). We then
explored appropriate conditions for direct radioiodination of PB-c(RGDyk) as a precursor
1
25
(
Table S2). As a result, [ I]IB-c(RGDyk) was obtained in a methanol solution of
Cu(pyridine)
4
(OTf)
2
at 25C (RCC: >99%). When dimethyl sulfoxide (DMSO) or DMF was
used as the solvent, the radiolabeling efficiency was poor (RCC: 0%–15.3%). After optimizing
125
radiolabeling conditions, [ I]IB-c(RGDyk) was synthesized three times to determine the
radioisolated yield [64.4%–69.0% (n = 3)]. The working time was 27.7–32.1 min (n = 3), which
is faster than the indirect radiolabeling method. This direct radiolabeling method is particularly
effective when 123I is used as a tracer. Retention times of the boronic precursor PB-c(RGDyk)
1
25
and the tracer labeled with radioiodine [ I]IB-c(RGDyk) were significantly different, which
facilitates easy separation by HPLC (Figure 4). These results indicated that the copper-
mediated radiolabeling method via aryl boronic precursors was an effective method to achieve
direct radioiodination of c(RGDyk), a cyclic peptide.
This article is protected by copyright. All rights reserved.

There are few reports on direct peptide labeling via copper-mediated radioiodination using
boronic precursors. Therefore, an accumulation of further application examples is very
important. Our results facilitate the application of this synthetic method to direct labeling of
peptides.
3
. CONCLUSION
This study explored the effect of pinacol ester-type protecting group of aryl boronic acids on
copper-mediated radioiodination and found that the radiolabeling method is well tolerated in
both electron-rich and electron-poor substrates, including sterically crowded substrates,
unaffected by protecting groups. In addition, we adapted the copper-mediated radiolabeling
methodology to achieve direct radioiodination of c(RGDyk). The reaction time and yield were
improved, compared with the indirect method. Also, the large difference in polarity between
the boronic acid precursor and the radiolabeled target tracer facilitated purification. These
results indicate that the copper-mediated radiolabeling reaction via aryl boronic precursors is
an effective radiolabeling method for direct radioiodination of peptides. We hope that our report
promotes development of peptide tracers.
This article is protected by copyright. All rights reserved.

4
4
. EXPERIMENTAL
.1 General
All reagents were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Nakalai
Tesque Inc. (Kyoto, Japan), Fujifilm Wako Chemical Corp. (Osaka, Japan), and Fluorochem
Ltd. (Hadfield, Derbyshire, UK), and were used without further purification unless otherwise
indicated. The cyclic peptide, c(RGDyk), was provided by Nard Institute, Ltd. (Hyogo, Japan).
125
125
Sodium [ I]iodide ([ I]NaI) (carrier-free) solution was purchased from PerkinElmer, Inc.
(Waltham, MA, USA).
1H NMR and 13C NMR spectra were measured on AscendTM 500 (Bruker, Billerica,
Massachusetts, USA) with CDCl
3
or DMSO-d as a solvent. Data were reported as follows:
6
chemical shifts are reported as  in units of parts per million (ppm) relative to an internal
standard (tetramethylsilane); multiplicities are reported as follows: s (singlet), d (doublet), t
(triplet), q (quintet), br. s (broad singlet), dd (doublet of doublets), or m (multiplet); coupling
constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given
resonance is indicated as nH, based on spectral integration values.
An autoradiograph of the reagents on the TLC sheet (5.0 × 2.0 cm; TLC silica gel 60 F254
aluminum plate; Merck, Kenilworth, NJ, USA) was acquired using an image analyzer
(Typhoon 9410; GE Healthcare, Waukesha, WI, USA). The radioactive signal intensities of
each TLC were evaluated using ImageQuant TL software (GE Healthcare) from the respective
This article is protected by copyright. All rights reserved.

images.
An LD-20AD (Shimadzu, Kyoto, Japan) was used for HPLC, along with an SPD-20A
(
Shimadzu) ultraviolet (UV) detector (k = 254 nm) and γ-survey meter TCS-172 (ALOKA,
Mitaka, Japan) or Gavi Nova (M&S Instruments Inc., Osaka, Japan) RI detector. A Cosmosil
18-AR-II column (4.6ID × 150 mm or 10.0ID × 150 mm; Nacalai Tesque Inc.) was used
5
C
for reverse phase HPLC.
Low-resolution mass spectra were obtained by Agilent LC/MS 6130B (Agilent Technologies,
California, USA). High-resolution mass spectra were obtained using LCMS-IT-TOF (ESI;
Shimadzu, Kyoto, Japan), GC mate II (EI, JEOL, Tokyo, Japan), or SX-102A (FAB; JEOL,
Tokyo, Japan).
4
.2 Synthesis of precursor 3a
-Carboxyphenylboronic acid (p-PB, 745.0 mg, 4.5 mmol) and N-hydroxy succinimide (NHS,
59.2 mg, 4.9 mmol) were dissolved in tetrahydrofuran (THF, 22.0 mL), and then
4
5
dicyclohexylcarbodiimide (DCC, 1.0 g, 4.9 mmol) was added into the solution. The reaction
was stirred at room temperature under nitrogen for 3 h. The reaction solution was concentrated
by the rotary evaporator and dissolved in ethyl acetate (200 mL). The organic layer was washed
with sat. NH
4
Cl aq. (100 mL × 2), sat. NaHCO aq. (100 mL × 2), and brine (50 mL × 1). The
3
obtained organic solution was dried over MgSO and concentrated using a rotary evaporator.
4
Residual water was removed by the freeze-drying to yield p-PB-NHS (682.0 mg, 2.6 mmol).
This article is protected by copyright. All rights reserved.

1
Yield: 57.8%; mp 262.5°C–266.1°C; H NMR (500 MHz, DMSO-d ) δ: 2.90 (br. s, 4H), 8.01
6
1
3
(
d, J = 8.2 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 8.46 (br. s., 2H); C NMR (126 MHz, DMSO-
d
6
) δ: 24.93, 124.91, 128.12, 134.19, 161.33, 169.73; LRESIMS: m/z 286.0510 [M + Na]+;
+
HRESIMS: calcd for C11
H
10NO
6
B [M + Na] 286.0495, found 286.0497.
4
.3 Synthesis of precursor 3b
-Carboxyphenylboronic acid (m-PB, 401.2 mg, 2.4 mmol) was dissolved in dichloromethane
DCM, 5.0 mL) and added with N-hydroxy succinimide (NHS, 231.9 mg, 2.0 mmol), 1-(3-
3
(
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCHCl, 386.3 mg, 2.0 mmol),
and triethylamine (279.3 μL, 2.0 mmol). The reaction was stirred at room temperature for 4 h.
The solvent was removed under reduced pressure and dissolved in ethyl acetate (100 mL). The
organic layer was washed with water (50 mL × 2), sat. NH
4
Cl aq. (50 mL × 2), sat. NaHCO
3
aq. (50 mL × 2), and brine (50 mL × 1). The obtained organic solution was dried over MgSO
4
and removed under reduced pressure. The residual water was removed by the freeze-drying to
yield m-PB-NHS (453.8 mg, 1.7 mmol) as a white powder. Yield: 71.4%; mp 252.6°C–
1
2
54.5°C; H NMR (500 MHz, DMSO-d
6
) δ: 2.90 (br. s., 4H), 7.63 (t, J = 7.6 Hz, 1H), 8.11 (d,
13
J = 7.8 Hz, 1H), 8.20 (d, J = 7.3 Hz, 1H), 8.44 (br. s., 2H), 8.52 (s, 1H); C NMR (126 MHz,
DMSO-d ) δ: 25.56, 123.79, 128.66, 131.48, 135.59, 140.99, 162.09, 170.41; LRESIMS: m/z
6
+
+
2
86.0503 [M + Na] ; HRESIMS: calcd for C11
H
10NO B [M + Na] 286.0495, found 286.0492.
6
This article is protected by copyright. All rights reserved.

4
.4 Synthesis of standard compound 4a
-Iodobenzoic acid (p-IB, 103.5 mg, 0.42 mmol) was dissolved in tetrahydrofuran (THF, 4.1
4
mL), and N-hydroxy succinimide (NHS, 61.5 mg, 0.53 mmol) and N,N’-
dicyclohexylcarbodiimide (DCC, 111.9 mg, 0.54 mmol) were then added into the solution. The
reaction was stirred at room temperature under nitrogen for 4 h. The reaction solution was
concentrated using a rotary evaporator and dissolved in ethyl acetate (50 mL). The organic
layer was washed with sat. NH
4
Cl aq. (30 mL × 2), sat. NaHCO aq. (30 mL × 2), and brine
3
(
20 mL × 1). The obtained organic solution was dried over MgSO and concentrated using a
4
rotary evaporator. The residual water was removed by freeze-drying to yield 2,5-
dioxopyrrolidin-1-yl 4-iodobenzoate (p-IB-NHS, 142.5 mg, 0.41 mmol). Yield: 98.4%; mp
1
2
7
1
4
25.6°C–227.7°C; H NMR (500 MHz, CDCl
3
) δ: 2.92 (br. s., 4H), 7.84 (d, J = 8.5 Hz, 2H),
1
3
.90 (d, J = 8.5 Hz, 2 H); C NMR (126 MHz, CDCl
3
) δ: 25.65, 103.39, 124.55, 131.67,
+
38.30, 161.58, 169.05; HREIMS: calcd for C11
H
8
INO
4
[M] 344.9498, found 344.9498.
.5 Synthesis of standard compound 4b
3
-Iodobenzoic acid (m-IB, 1.9 g, 7.8 mmol) was dissolved in tetrahydrofuran (THF, 20 mL),
and then with N-hydroxysuccinimide (NHS, 1.0 g, 8.6 mmol) and N,N'-
dicyclohexylcarbodiimide (DCC, 1.8 g, 8.6 mmol). The reaction was stirred at room
temperature for 22 h. The solution was filtered, and then the mother liquid was concentrated
using a rotary evaporator and purified using medium pressure liquid chromatography (SiO ,
2
This article is protected by copyright. All rights reserved.

hexane:ethyl acetate = 1:1) to yield 2,5-dioxopyrrolidin-1-yl 3-iodobenzoate (m-IB-NHS, 2.6
1
g, 7.6 mmol) as white solid. Yield: 96.8%; mp 141.2°C–143.1°C; H NMR (500 MHz, DMSO-
d
1
1
6
) δ: 2.90 (br. s., 4H), 7.46 (t, J = 7.9 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.1 Hz,
1
3
H), 8.35 (s, 1H); C NMR (126 MHz, DMSO-d
6
) δ: 25.56, 95.53, 126.40, 129.29, 131.61,
+
37.85, 144.10, 160.58, 170.22; LRFABMS: m/z 345.7 [M + H] ; HRFABMS: calcd for
+
C
11
H
8
INO
4
[M + H] 345.9578, found 345.9577.
4
.6 Synthesis of IB-c(RGDyk), the standard sample
c(RGDyk) (20.0 mg, 32.3 mol) and 4a (10.6 mg, 32.3 mol) were dissolved in DMF (2.0
mL), and then triethylamine (100 L) was added into the solution and reacted at room
temperature for 16 h. The reaction solvent was removed using a rotary evaporator and purified
by HPLC [method F; Cosmosil 5C18-AR-II (10.0 ID × 150 mm); Flow: 4.0 mL/min; Temp.:
4
0°C; t : 16.8 min] to yield IB-c(RGDyk) (16.7 mg) as white powder; Yield: 60.8%; Purity:
R
1
>
99%; H NMR (DMSO-d , 500 MHz) δ: 1.10 (m, 2H), 1.27-1.51 (m, 6H), 1.58 (m, 1H), 1.65-
6
1
3
.74 (m, 1H), 2.34-2.41 (m, 1H), 2.62-2.74 (m, 2H), 2.74-2.81 (m, 1H), 3.02-3.13 (m, 2H),
.15- 3.27 (m, 3H), 3.88-3.95 (m, 1H), 3.99-4.06 (m, 1H), 4.11-4.18 (m, 1H), 4.35 (q, J = 7.2
Hz, 1H), 4.59-4.66 (m, 1H), 6.63 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 7.49 (t, J = 5.7
Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.95 (d,
J = 7.2 Hz, 1H), 8.06 (d, J = 7.0 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H), 8.43 (dd, J
1
= 6.8, J = 4.8
2
13
Hz, 1H), 8.55 (t, J = 5.5 Hz, 1H), 9.19 (br. s., 1H); C NMR (DMSO-d , 126 MHz) δ: 1.63,
6
This article is protected by copyright. All rights reserved.

2
1
1
3.39, 25.69, 29.00, 31.29, 35.45, 37.15, 43.69, 49.33, 52.31, 55.07, 55.15, 99.21, 115.38,
27.68, 129.61, 130.47, 134.42, 137.60, 156.26, 157.00, 158.57, 165.91, 170.03, 170.38,
+
71.36, 171.67, 172.16, 172.64; LRESIMS m/z 850.0 [M + H] ; HRESIMS: calcd for
+
C
34
H
44
N
9
O
9
I [M + H] 850.2379 found 850.2359.
4
.7 Synthesis of PB-c(RGDyk)
c(RGDyk) (20.0 mg, 32.3 mol) and 3a (8.0 mg, 32.3 mol) were dissolved in DMF (2.0
mL), and then triethylamine (100 L) was added into the solution and reacted at room
temperature for 16 h. The reaction solvent was removed using a rotary evaporator and purified
by HPLC [method F; Cosmosil 5C18-AR-II (10.0 ID × 150 mm); Flow: 4.0 mL/min; Temp.:
4
4
6
8
0°C; t
R
: 11.4 min] to yield PB-c(RGDyk) (10.8 mg, 14.1 mol) as white powder. Yield:
1
3.6%; Purity: 98.5%; H NMR (500 MHz, DMSO-d
6
) δ: 1.07-1.17 (m, 1H), 1.29-1.53 (m,
H) 1.54-1.62 (m, 1H) 1.65-1.75 (m, 1H), 2.34-2.42 (m, 1H), 2.62-2.74 (m, 2H), 2.79 (m, J =
.9 Hz, 1H), 3.03-3.13 (m, 2H), 3.16-3.28 (m, 3H), 3.92 (t, J = 9.4 Hz, 1H), 4.03 (m, J = 14.9,
1
2
J = 7.7 Hz, 1H), 4.16 (q, J = 7.5 Hz, 1H), 4.36 (q, J = 7.3 Hz, 1 H), 4.58-4.66 (m, 1H), 6.63
(
d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 7.49 (br. s., 1H), 7.60 (d, J = 6.7 Hz, 1H), 7.79
d, J = 7.9 Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 7.0 Hz, 1H), 8.07 (d, J = 7.2 Hz,
(
1
H), 8.12 (d, J = 8.4 Hz, 1H), 8.23 (s, 2H), 8.42 (dd, J
1
= 6.4, J = 4.6 Hz, 1H) 8.48 (t, J = 5.6
2
1
3
Hz, 1H), 9.19 (s, 1H), 12.29 (br. s., 1H); C NMR (126 MHz, DMSO-d ) δ: 23.42, 25.69,
6
2
8.99, 29.13, 31.33, 37.16, 43.69, 49.34, 52.30, 55.13, 115.39, 126.50, 127.69, 130.48, 134.39,
This article is protected by copyright. All rights reserved.

1
36.32, 156.27, 157.03, 166.77, 170.01, 170.41, 171.37, 171.69, 172.19, 172.67; LRESIMS:
+
+
m/z 790.1 [M + Na] ; HRESIMS: calcd for C34
H
46
N O11B [M + Na] 790.3308 found 790.3309.
9
4
3
.8 General procedure of 125I-radiolabeling using boronic acid or ester precursors (1a–1j,
a–b)
Boronic acid or ester precursor (0.5 mg) and Cu(pyridine)
mg/mL, 100 L) were added into a 1.5-mL microtube. About 10-μM NaOH aq. solution of 125I
0.5 L, 1–4 MBq) was added to the mixture in the microtube. Then, the tube was gently
4
(OTf)
2
in MeOH or EtOH (2.2
(
vortexed for 5 s. The reaction was left at room temperature for 10 min. RCCs were determined
by radio-TLC (Supplementary information). The reaction mixture was analyzed by radio-
HPLC (Supplementary information).
.9 Indirect 125I-labeling of c(RGDyk) using [ I]IB-NHS
125
4
p-PB-NHS (3a, 0.2 mg) and Cu(pyridine)
4
(OTf) in EtOH (2.2 mg/mL, 50 μL) were added to
2
1
25
a 1.5-mL microtube. About 10-M NaOH aq. solution of I (1.0 L, 2.4–2.9 MBq) was added
to the mixture in the microtube. Then, the tube was gently vortexed for 5 s. The reaction was
left to set at room temperature for 10 min. The obtained solution was dried under a flow of
nitrogen for 2 min and dissolved in acetonitrile (40 μL). 4a was isolated using HPLC [method
B, t : 7.42 min]. The solution of 4a was diluted with water (30 mL), passed through a C18 Sep-
R
Pak column, and washed with water (4 mL). Finally, 4a was eluted with acetonitrile (0.5 mL)
and dried under flow of nitrogen for 10–20 min. c(RGDyk) (10 mg/mL, 20 L), triethylamine
This article is protected by copyright. All rights reserved.

(2.5 L), and DMF (20 L) were added into the microtube containing 4a. The reaction was left
125
at room temperature for 1 h. [ I]IB-c(RGDyk) (0.9–1.1 MBq) was isolated by HPLC (method
F; radioisolated yield: 36.5%–39.3%; working time: 140.5–157.3 min, n = 3). The mixture of
purified and cold samples was analyzed by UV- and radio-HPLC, showing a single peak (t :
R
1
6.92 min) corresponding to cold sample (t
R
: 16.85 min).
4
.10 Direct 125I-radiolabeling through PB-c(RGDyk)
PB-c(RGDyk) (0.1 mg) and Cu(pyridine)
4
(OTf) in MeOH (2.2 mg/mL, 50.0 μL) were added
2
to a microtube. About 10-μM NaOH aq. solution of 125I (1.0 L, 2.6-2.8 MBq) was added to
the mixture. Then, the tube was gently vortexed for 5 s. The reaction was left at room
1
25
temperature for 10 min. [ I]IB-c(RGDyk) (1.8-1.9 MBq) was isolated by HPLC [method F].
RCCs were determined by radio-HPLC analysis; RCC: 82.4%–99.9% (n = 3); radioisolated
yield: 64.4%–69.0% (n = 3); and working time: 27.7–32.1 min (n = 3). The mixture of purified
and cold samples was analyzed using UV- and radio-HPLC, which showed a single peak (t :
R
1
6.91 min) corresponding to cold sample (t : 16.83 min).
R
CONFLICT OF INTEREST
The authors declare no competing financial interests.
This article is protected by copyright. All rights reserved.

REFERENCES
1
2
3
4
5
6
. Zhuang ZP, Kung MP, Wilson A, Lee CW, Ploessl K, Hou C, Holtzman David M, Kung
Hank F. Structure-activity relationship of imidazo[1,2-a]pyridines as ligands for detecting
β-amyloid plaques in the brain. J Med Chem 2003;46:237-243.
. Kil KE, Zhu A, Zhang Z., Choi JK, Kura S, Gong C, Brownell A. Development of
[123
I]IPEB and [ I]IMPEB as SPECT radioligands for metabotropic glutamate receptor
123
subtype 5. ACS Med Chem Lett 2014;5:652-656.
. Wilson TC, McSweeney G, Preshlock S, Verhoog S, Tredwell M, Cailly T, Gouverneur V.
Radiosynthesis of SPECT tracers via a copper mediated 123I iodination of (hetero)aryl
boron reagents. Chem Commun 2016;52:13277-13280.
. Chitneni SK, Reitman ZJ, Spicehandler R, Gooden DM, Yan H, Zalutsky MR. Synthesis
and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging
mutant IDH1 expression in tumors. Bioorg Med Chem Lett 2018;28:694-699.
. Rajerison H, Faye D, Roumesy A, Louaisil N, Boeda F, Faivre-Chauvet A, Gestin JF,
Legoupy S. Ionic liquid supported organotin reagents to prepare molecular imaging and
therapy agents. Org Biomol Chem 2016;14:2121-2126.
. Cant AA, Champion S, Bhalla R, Pimlott SL, Sutherland A. Nickel-mediated
radioiodination of aryl and heteroaryl bromides: rapid synthesis of tracers for SPECT
imaging. Angew Chem Int Ed 2013;52:7829-7832.
This article is protected by copyright. All rights reserved.

7
8
9
1
1
. Zhang P, Zhuang R, Guo Z, Su X, Chen X, Zhang X. A highly efficient copper-mediated
radioiodination approach using aryl boronic acids. Chem -Eur J 2016;22:16783-16786.
2
11
. Reilly SW, Makvandi M, Xu K, Mach RH. Rapid Cu-catalyzed [ At]astatination and
[125
I]iodination of boronic esters at room temperature. Org Lett 2018;20:1752-1755.
. Zhang G, Zhang L, Hu M, Cheng J. Copper(I)-mediated cyanation of boronic acids. Adv
Synth Catal 2011;353:291-294.
0. Sanjeeva Rao K, Wu TS. Chan-Lam coupling reactions: synthesis of heterocycles.
Tetrahedron 2012;68:7735-7754.
1. Tredwell M, Preshlock Sean M, Taylor Nicholas J, Gruber S, Huiban M, Passchier J,
Mercier J, Genicot C, Gouverneur V. A general copper-mediated nucleophilic 18F
fluorination of arenes. Angew Chem Int Ed 2014;53:7751-7755.
1
1
1
2. Mossine AV, Brooks AF, Makaravage KJ, Miller JM, Ichiishi N, Sanford MS, Scott PJ.
1
8
18
Synthesis of [ F]arenes via the copper-mediated [ F]fluorination of boronic acids. Org
Lett 2015;17:5780-5783.
3. Zhou D, Chu W, Voller T, Katzenellenbogen JA. Copper-mediated nucleophilic
radiobromination of aryl boron precursors: convenient preparation of a radiobrominated
PARP-1 inhibitor. Tetrahedron Lett 2018;59:1963-1967.
11
4. Ma L, Placzek Michael S, Hooker Jacob M, Vasdev N, Liang Steven H. [ C]Cyanation of
arylboronic acids in aqueous solutions. Chem Commun 2017;53:6597-6600.
This article is protected by copyright. All rights reserved.

1
1
1
5. Taylor NJ, Emer E, Preshlock S, Schedler M, Tredwell M, Verhoog S, Mercier J, Genicot
1
8
C, Gouverneur V. Derisking the Cu-mediated F-fluorination of heterocyclic positron
emission tomography radioligands. J Am Chem Soc 2017;139:8267-8276.
6. Guerard F, Navarro L, Lee YS, Roumesy A, Alliot C, Cherel M, Brechbiel MW, Gestin JF.
Bifunctional aryliodonium salts for highly efficient radioiodination and astatination of
antibodies. Bioorg Med Chem 2017;25:5975-5980.
7. Vaidyanathan G, Affleck DJ, Norman J, O'Dorisio S, Zalutsky MR. A radioiodinated
MIBG-octreotate conjugate exhibiting enhanced uptake and retention in SSTR2-
expressing tumor cells. Bioconjugate Chem 2007;18:2122-2130.
1
1
2
8. Yang H, Li Y, Jiang M, Wang J, Fu H. General copper-catalyzed transformations of
functional groups from arylboronic acids in water. Chem Eur J 2011;17:5652-5660.
9. Altman RA, Buchwald SL. Pd-catalyzed Suzuki–Miyaura reactions of aryl halides using
bulky biarylmonophosphine ligands. Nature Protocols 2007;2:3115-3121.
0. Molloy JJ, O'Rourke KM, Frias CP, Sloan NL, West MJ, Pimlott SL, Sutherland A, Watson
AJB. Mechanism of Cu-catalyzed aryl boronic acid halodeboronation using electrophilic
halogen: development of a base-catalyzed iododeboronation for radiolabeling applications.
Org Lett 2019;21:2488-2492.
2
1. Fernandes GFS, Denny WA, Dos Santos JL. Boron in drug design: recent advances in the
development of new therapeutic agents. Eur J Med Chem 2019;179:791-804.
This article is protected by copyright. All rights reserved.

2
2. Berdal M, Gouard S, Eychenne R, Marionneau-Lambot S, Croyal M, Faivre-Chauvet A,
Cherel M, Gaschet J, Gestin JF, Guerard F. Investigation on the reactivity of nucleophilic
radiohalogens with arylboronic acids in water: access to an efficient single-step method
for the radioiodination and astatination of antibodies. Chem Sci 2021;12:1458-1468.
3. Ogawa K, Takeda T, Mishiro K, ToyoshimaA, Shiba K, Yoshimura T, ShinoharaA, Kinuya
S, Odani A. Radiotheranostics coupled between an At-211-labeled RGD Peptide and the
corresponding radioiodine-labeled RGD peptide. ACS Omega 2019;4:4584-4591.
2
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section
at the end of this article.
This article is protected by copyright. All rights reserved.

FIGURE 1 Application of the copper-mediated radioiodination through the boronic precursor
for direct radiolabeling of peptides.
This article is protected by copyright. All rights reserved.

FIGURE 2 The scope of copper-mediated 125I-iodination via aryl boronic acid or pinacol ester.
RCCs of radiolabeled compounds were determined by radio-TLC (n = 3). The reaction solution
was analyzed using the radio-HPLC to confirm identity of 125I-radiolabeled compounds.
*
pinacol boronic ester group = Bpin.
This article is protected by copyright. All rights reserved.

FIGURE 3 Copper-mediated 125I-radiolabeling of the active ester of carboxyphenylboronic
acid (n = 3).
This article is protected by copyright. All rights reserved.

FIGURE 4 (A) UV- and (B) radio-HPLC chromatogram of the reaction solution after the
copper-mediated direct radiolabeling of c(RGDyk) via PB-c(RGDyk). PB-c(RGDyk) t
R
=
125
1
1.39 min; [ I]IB-c(RGDyk) t
R
= 16.98 min. The first peak detected at 1.50–2.00 min is a
1
25
peak derived from the copper catalyst or [ I]NaI.
This article is protected by copyright. All rights reserved.

SCHEME 1 (A) Direct radioiodination of c(RGDyk) through the boronic precursor, PB-
c(RGDyk), (n = 3). (B) Indirect radioiodination of c(RGDyk) using 125I-labeled N-
hydroxysuccinimide ester of iodobenzoic acid (n = 3).
This article is protected by copyright. All rights reserved.

Graphical Abstract
This article is protected by copyright. All rights reserved.