Biomolecular labeling based on lysine-clickable 6π-azaelectrocyclization
toward innovative cancer theranostics
Katsumasa Fujiki a,*
, Katsunori Tanaka b,c,d
a Isotope Science Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan b Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo
152-8552, Japan c Biofunctional Synthetic Chemistry Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan d Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia
ARTICLE INFO
Keywords:
6π-Azaelectrocyclization
Click reaction
Protein labeling
Cancer diagnosis
Cancer therapy
ABSTRACT
An amino group at side chain of lysine residue can be targeted for protein modification because of the conve￾nience for covalent bond formation. We have achieved an efficient protein modification by utilizing amine￾clickable 6π-azaelectrocyclization, termed RIKEN click reaction recently, which enabled direct click labeling
of protein without any introduction of specific functional groups such as alkynes and azides. On the basis of the
RIKEN click reaction, we established the double click labeling method. The double click methods composed of
copper-free strain-promoted [3 + 2] cyclization or tetrazine ligation and RIKEN click reaction were developed.
The double click method realized highly effective proteins including radiolabeling of bioactive peptides and anti￾tumor antibodies. In this personal review, the development of double click probes, practical radiolabeling of
biological active molecules such as cyclic RGDyK peptides, proteins, and antibodies with α-emission or
β-emission radionuclides, and their applications for PET imaging and α-emission cancer treatment are
summarized.
1. Introduction
The amine-reactive modification methods for biomolecule by iso￾thiocyanate,1,2 activated ester,3,4 and sulfonyl chloride5 derivatives
have been utilized.6–8 However, a disadvantage of these methods is the
low efficiency due to low reactivity and instability in aqueous solutions.
In recent years, amine-reactive reagents with high efficacy and aqueous￾stable property have been developed.9 The click reaction has been re￾ported as biorthogonal reaction10–12 and used for chemical modification
of biomolecules.13 A variety of click reactions have been developed,
such as photo-induced click reaction,14–18 Cu-free “azide–alkyne” liga￾tion,19 tetrazine ligation (the inverse electron-demand Diels-Alder re￾action) using trans-cyclooctene (TCO).20 These click reactions need
unnatural reactive groups. For efficient biomolecular labeling, click re￾action using natural functional groups, such as amine and thiol at the
side chain of lysine and cysteine, respectively, are ideal. There are re￾ports on amine-targeted labeling method for chroptical sensing of amino
acid.21,22 As amine-targeted click reaction, recently, Chen et al. reported
click labeling method for various α-amino acids via a three-component
coupling of primary amines with o-phthalaldehyde and p-toluenethiol
reagents.23 As for click-type reaction designed for amino group on
protein, Guo et al. developed a light-induced primary amines and o￾nitrobenzyl alcohols cyclization.24 We developed RIKEN click aldehyde
probes containing a series of functional molecules such as fluorophore,
metal chelator, and biotin (Fig. 1a). By using these aldehyde probes, we
accomplished efficient protein labeling via RIKEN click reaction (6π-
azaelectron cyclization that proceeds rapidly after imine formation with
the amino group of the lysine residue side chain of the protein).25–30
Recently, many researcher has devoted to synthesis of the RI thera￾peutic molecules such as radiolabeled antibody. However, there are few
examples of using the click reaction for radiolabeling, especially bio￾molecular labeling for radiotherapy.31 The development of radio￾labeling method based on click reaction could enable to accelerate the
development of radiotherapeutics. Therefore, we have developed an
efficient biomolecular radiolabeling method that can be applied to the
diagnosis and treatment of cancer using the RIKEN click reaction. As our
self-review, we summarize development and diagnostic and therapeutic
applications of efficient radiolabeled biomolecules, such as 64Cu-labeled
* Corresponding author.
E-mail address: [email protected] (K. Fujiki).
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc

https://doi.org/10.1016/j.bmc.2021.116238

Received 16 April 2021; Received in revised form 15 May 2021; Accepted 20 May 2021
Bioorganic & Medicinal Chemistry 42 (2021) 116238
2
cyclic RGDyK peptide32 and 67Cu-/211At-labeled antibody,33,34 via
RIKEN click reaction.
2. Double click labeling with βþ ray emission metal and its
application for PET
Summarized above, we developed the DOTA-introduced RIKEN click
aldehyde and succeeded in 68Ga labeling and PET imaging of the bio￾molecules.28 However, the synthesis of the aldehyde probe required
harsh conditions at high temperature for efficient metal chelation.
Isomerization of the olefin structure of RIKEN click aldehyde occurred
under these conditions. Therefore, by utilizing the click reaction, effi￾cient preparation of unsaturated aldehyde probes modified with various
functional groups has been developed.32 The aldehyde 1 having the
cyclooctyne DIBO (dibenzoannulated cyclooctyne), reported by Boons
et al.,35 was designed and synthesized. It was thought that introduction
of DOTA (1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid) and
NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) to 1 by using the
strain-promoted [3 + 2] cyclization reaction can be carried out effi￾ciently. To develop this synthetic strategy, DOTA-azide probe 2 was
designed and synthesized. Moreover, given the half-life of 68Ga (68
min), we sought to complete the labeling process within 5 h from the
elution of 68Ga from the 68Ge/68Ga generator to the purification of 68Ga￾labeled molecule (Scheme 1).
The 68Ga labeling of the cyclic RGDyK peptide as ligand of αVβ3
integrin was investigated. The 212 MBq 68Ga solution was eluted from
the 68Ge/68Ga generator, and the solution was concentrated to 100 μL
(<pH 1.0). DOTA-azide 2 (10 nmol) in 4-(2-hydroxyethyl)-1-piper￾azineethanesulfonic acid (HEPES) buffer (pH 3.5) was reacted at 120 ◦C
under microwave irradiation for 10 min. HPLC purification was carried
out, followed by evaporation of the solvent under heating conditions to
give 108 MBq of 68Ga-chelated DOTA-azide 3 in 81% radiochemical
yield (RCY). The obtained 3 was reacted with a DMSO/MeCN solution of
Fig. 1. (a) RIKEN click aldehyde that enables efficient amine-clickable reac￾tion.25–30 (b) Double click reaction by combination of RIKEN click with strain￾promoted [3 + 2] cyclization/tetrazine ligation.32–34.
Scheme 1. Double click labeling process of cyclic RGDyK peptide with 68Ga.32.
Fig. 2. PET imaging of 68Ga-labeled RGDyK peptide 5 in U87MG cancer
xenograft mouse.32.
K. Fujiki and K. Tanaka
Bioorganic & Medicinal Chemistry 42 (2021) 116238
3
5 nmol of aldehyde 1 at 75 ◦C for 15 min. HPLC analysis of the reaction
mixture showed that the production of 61 MBq of 68Ga-chelated DOTA￾labeled aldehyde 4 in 76% RCY, calculated by 68Ga radioactivity elution
from the generator. Finally, the resulting 4 was reacted with a 5 nmol
cyclic RGDyK peptide DMF solution at 37 ◦C. The RIKEN click on the
amino group on the side chain of the lysine residue was completed after
25 min. After HPLC purification, 18 MBq of 68Ga-chelated DOTA-labeled
RGDyK 5 was successfully obtained. This 68Ga labeling process was
carried out in 2 h from the elution of 68Ga from the generator to HPLC
purification of 5 in practical RCY. The results demonstrated that this 68Ga labeling strategy with a double click reaction was efficient.
Furthermore, with the obtained 5 through the double click reaction,
the PET imaging was performed. The 5 MBq of 5 was intravenously
injected to BALB nude mouse xenografted with U-87MG cells derived
from brain cancer. It was shown that the accumulation of 5 to the tumor
was observed from 60 to 110 min after injection (Fig. 2).
3. Development of one-pot three-component double-click and
highly efficient 67Cu labeling
The strain-promoted [3 + 2] cyclization needed heating conditions
at 75 ◦C to complete the reaction rapidly. To address this, we focused on
tetrazine ligation reported by Fox et al., which can be carried out under
mild conditions.36 Instead of the strain-promoted [3 + 2] cyclization, by
combining tetrazine ligation with RIKEN click, we established highly
efficient “one-pot three-component double-click”, which enabled to
prepare functional group-introduced aldehyde probe and modification
of biomolecule by RIKEN click simply by mixing tetrazine, aldehyde,
and biomolecule under mild conditions.33
As tetrazine probes, DOTA-tetrazine 6 and NOTA-tetrazine 7 were
developed. With these tetrazine probes and TCO-aldehyde 8, one-pot
three-component double click labeling of human serum albumin
(HSA) was examined (Table 1). The 1.0 × 10− 5 M aqueous solution of
HSA was reacted with 5.0 × 10− 5 M DMSO solution of 6 and 1.5 × 10− 4
M DMSO solution of 8 at 37 ◦C for 1 h. The Mass spectrometry of the
product by using Matrix-assisted laser desorption/ionization-time-of￾flight (MALDI-TOF) showed the DOTA-introduced HSA 9, which was
attached with two molecules of DOTA (entry 1). Furthermore, when 1.0
× 10− 4 M DMSO solution of 6 and 3.0 × 10− 4 M solution of 8 were
reacted with 1.0 × 10− 5 M aqueous solution of HSA, four molecules of
DOTA could be introduced to HSA (entry 2). From these results, the
introduced number of DOTA could be controlled by concentrations of
the probes. Similarly, under the same conditions as entry 2, three mol￾ecules of NOTA could be introduced to HSA (entry 3).
With the DOTA-, NOTA-introduced HSA 9 and 10 synthesized by
using one-pot three-component double-click, the 67Cu labeling of 9 and
10 was examined (Scheme 2). The 67Cu is known as a theranostics
radionuclide, which enables therapy with β-ray emission and diagnosis
with γ-ray emission.37 In addition, the large amount of 67Cu can be
produced by using the AVF cyclotron at RIKEN Nishina Accelerator
Science Research Center (RIKEN Nishina Center).38 The labeling of HSA
9 and 10 with 67Cu was successful simply by treatment with 11 MBq of 67Cu at 40 ◦C for 1 h, the 67Cu-labeled HSA 11 in 72% RCY and the 67Cu￾labeled HSA 12 in 19% RCY, respectively. The DOTA-introduced anti￾body 13 could be labeled with 67Cu in 51% RCY under same conditions
as the labeling of 9. Furthermore, the labeling of NOTA-introduced
antibody 14 with 67Cu resulted in 7% RCY. Therefore, we successfully
established the efficient radiolabeling process of biomolecule based on
our one-pot three-component double click and the collaboration with
RIKEN Nishina center.
4. Practical synthesis of α-ray nuclide 211At-labeled antibody
and cancer treatment
The α-emission nuclide 211At with half-life of 7.2 h has much
attention for the development of new cancer-targeted radiotherapeutic
molecule because of the potent effect by α-emission.39,40 Wilbur et al.
have reported the synthesis of various 211At-labeled molecules.41–43
However, establishment of efficient labeling method for protein with 211At is still challenging, thereby the cancer therapeutic effect of 211At
has not been investigated in detail.44 By using one-pot three-component
double click, we established an efficient synthetic protocol of 211At￾labeled antibody.34
To develop an efficient synthetic method for 211At-labeled molecules
and evaluate the therapeutic effects, tetrazine 15 modified with the
closo-decaborate as prothestic group of 211At was designed and synthe￾sized (Scheme 3). With 15 and TCO-aldehyde 8, introduction of closo￾decaborate to HSA and antibody by one-pot three-component double￾click was examined. As summarized, 1.0 × 10− 4 M DMSO solution of
15 and 1.0 × 10− 4 M DMSO solution of 8, and 1.0 × 10− 5 M aqueous
solution of HSA were reacted at 37 ◦C for 1 h and it was successful that
closo-decaborate-introduced HSA 16 was obtained (Scheme 3a). The
MALDI-TOF analysis demonstrated that attachment of two molecules of
closo-decaborates to HSA. Under the same conditions, introduction of
closo-decaborate to trastuzumab as anti-HER2 antibody was investigated
and closo-decaborate-introduced trastuzumab 17 was efficiently syn￾thesized (Scheme 3b).
Next, the synthesis of 211At-labeled trastuzumab was investigated
(Table 2). The 211At labeling was performed by reacting 5.8 MBq of 211At
with 1.0 × 10− 5 M of 17 in the presence of chloramine T as oxidant at
room temperature for 5 min (entry 1). In our synthesis of the 211At￾labeled trastuzumab, purification was carried out by ultrafiltration for 5
min, which was performed 2 times. Through this simple synthetic pro￾cedure, the 211At-labeled trastuzumab 18 was obtained in 78% RCY with
specific activity of 0.050 MBq µg− 1
. To investigate whether non-specific
adsorption on trastuzumab occurred, intact trastuzumab was reacted
with 211At (entry 2). It resulted in the RCY after ultrafiltration was 5% or
less. It was anticipated that radioactivity from the residual 211At,
therefore non-specific adsorption was not observed. When the 211At￾labeled trastuzumab 18 was prepared by using 1.0 × 10− 6 M of 17 with
75 MBq of 211At in the presence of chloramine T at room temperature for
5 min, the dissociation constant Kd of the obtained with a specific
Table 1
One-pot three-component double clicks for introduction of DOTA/NOTA to HSA.33
Entry Tetazine (concentration [10− 4 M]) 8 [10− 4 M] Introduced number
1 6 (0.5) 1.5 2
2 6 (1.0) 3.0 4
3 7 (1.0) 3.0 3
K. Fujiki and K. Tanaka
Bioorganic & Medicinal Chemistry 42 (2021) 116238
4
activity of 1.7 MBq μg− 1 was examined by quartz crystal microbalance
(QCM) method and showed 1.0 nM (entry 3). It demonstrated that the
recognition activity of 18 prepared was completely retained and our
double click method enabled invasive labeling with 211At.
Next, the biodistribution of the obtained 18 was evaluated. The 5 µL
of 0.05% tween 20-contained PBS solution (PBS-T) of 1.4 MBq of 18
containing 6.3 μg was administered by intratumor injection to A431
cells-xenografted mice. HER2 was highly expressed on A431 cells. The
radioactivity of each organ of the dissected mice was measured at one or
two days after administration (Fig. 3a). At one day after administration,
strong radioactivity was observed in the tumor. Furthermore, sufficient
radioactivity was observed in the tumor after 2 days. On the other hand,
no radioactivity was observed in the tumor, when 5 µL of PBS solution
containing 1.4 MBq of Na211At was injected. In addition, low radioac￾tivity was observed in the thyroid area and most of the injected Na211At
was excreted from the body after one day (Fig. 3b).
Furthermore, the α-emission therapeutic effect of the obtained 18
was evaluated. The 18 was injected by intratumor injection under the
same conditions as in the biodistribution analysis, and the tumor size
was measured for 37 days based on the Battelle Columbus laboratories
protocol method (Fig. 4a).45 The tumor growth was clearly enhanced at
7 days post injection. Afterwards, the slow tumor growth was observed.
In addition, all the mice that was injected with 18 could survive without
death (Fig. 4b). The therapeutic effect by α-emission was clearly
observed. By contrast, intratumor injection of PBS solution of trastuzu￾mab or PBS to A431 cells-xenografted mice clearly increased the tumor
size (Fig. 4a). Most mice did not survive at the 37 day post injection
(Fig. 4b). In addition, the 18 prepared via our double click reaction
showed no toxicity from the result of body weight after intratumor in￾jection of 18, although 9% loss of the body weight was observed at 4 day
post injection (Fig. 4c).
5. Perspective on RIKEN click labeling for in vivo synthesis
As a prospective application of RIKEN click labeling, in vivo RIKEN
click radiolabeling would be examined. Our RIKEN click labeling
method has been applied for invasive introduction of various function￾alized molecules to proteins, antibodies, and cyclic peptides so far. The
sizes of these biomolecules range from middle to large molecules and the
biological activity of these molecules is interfered with modifications
only in few cases. However, it is challenging to label small molecules
with retention of the biological activity. To address this, we anticipate
that in vivo RIKEN labeling of the molecule at the cancer tissue would be
effective. For example, masking an aldehyde with acid-labile protective
group enables in vivo specific labeling of cancer cells by RIKEN click
reaction around weakly acidic tumor tissue. This strategy enables direct
injection of the intact molecule with the masked RIKEN click probe to
the body and labeling in the body, thereby eliminating the risk to
diminish the biological activity. This in vivo synthesis by RIKEN click
labeling is currently underway in our group and would be one of the
innovative prodrug pharmaceuticals in the future.
6. Conclusion
We described the development of efficient labeling methods for
Scheme 2. Radiolabeling of DOTA-/NOTA-introduced HSA/antibody
with 67Cu.33.
Scheme 3. Introduction of closo-decaborate to (a) HSA and (b) trastuzumab by
one-pot three-component double click reaction.34.
Table 2
The 211At labeling and the evaluation of the binding affinity of closo-decaborate￾introduced
trastuzumab.34￾Entry 17 [M]) X
[MBq]
RCY
[%]
Specific activity
[MBq/μg]
Kd
[nM]
1 1.0 × 10− 5 5.8 78 0.050 –
2 Trastuzumab 1.0 ×
10− 5
5.2 5 – –
3 1.0 × 10− 6 75 49 1.7 1.0
K. Fujiki and K. Tanaka
Bioorganic & Medicinal Chemistry 42 (2021) 116238
5
biomolecules by a double click reaction based on RIKEN click that tar￾gets the amino group at the side chain of lysine residue. Our double click
labeling enabled PET imaging and cancer therapy. By using the one-pot
three-component double click reaction, the labeling with 67Cu as a
theranostic radionuclide, which could be produced in large scale at
RIKEN Nishina center, was successful. Furthermore, an efficient syn￾thesis of 211At-labeled antibody, which has a potent therapeutic effect by
α-emission, was achieved, thereby enabling α-emission therapy in
tumor-xenografted mouse. Our RIKEN click labeling strategy would be
compatible with in vivo labeling in the future and innovate effective
small molecular labeling. On the basis of RIKEN click labeling, the
development of new in vivo synthetic cancer diagnostic and therapeutic
methods will be accomplished in our prospective research.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the JSPS KAKENHI Grant Numbers
JP16H03287, JP18K19154, JP15H05843 (to K.T.), and 20 K15419 (to
K.F.), and by RIKEN Incentive Research Projects 2016 and 2018. This
work was also funded by the subsidy allocated to Kazan Federal Uni￾versity for the state assignment in the sphere of scientific activities
(0671-2020-0063), with the support of the Kazan Federal University
Strategic Academic Leadership Program. We are grateful to Dr. Hir￾omitsu Haba, Dr. Yasuyoshi Watanabe, Dr. Yousuke Kanayama, Dr.
Takuya Yokokita, and Dr. Yukiko Komori (RIKEN) for help in producing
the radiotracer.
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