Bioorganic & Medicinal Chemistry Letters
A two-step strategy to radiolabel choline phospholipids with 99mTc
in S180 cell membranes via strain-promoted cyclooctyne–azide
cycloaddition reaction
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Qingxin Chen, Taiwei Chu
Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history:
As tumor markers, the radiolabeling of choline (Cho)-containing phospholipids in cellular membranes
with 99mTc is a challenge. The conventional strategy to combine the metallic radionuclide with Cho by
large ligand damages the bioactivity of Cho, resulting in low tumor-to-nontumor ratios. Pretargeting
strategy based on strain-promoted cyclooctyne–azide cycloaddition (SPAAC) reaction was applied to
solve this general problem. Functional click synthons were synthesized as pretargeting components:
azidoethyl-choline (AECho) serves as tumor marker and azadibenzocyclooctyne (ADIBO) conjugated to
bis(2-pieolyl) amine (BPA) ligand (ADIBO-BPA) as 99mTc(CO)3-labeling and azido-binding group. Both
in vitro cell experiment and in vivo biodistribution experiment indicate that it is versatile to radiolabel
Cho in cellular membranes via this two-step pretargeting strategy. We believe that this pretargeting
strategy can indeed enhance the target-specificity and also reduce background signals to optimize
imaging quality.
Received 18 July 2016
Revised 6 October 2016
Accepted 11 October 2016
Available online 12 October 2016
Keywords:
Tumor imaging
AECho
ADIBO-BPA
99mTc(CO)3-labeling
Pretargeting
SPAAC
Ó 2016 Elsevier Ltd. All rights reserved.
Phospholipids are important components of cellular mem-
branes. Choline (Cho) is a common precursor of phospholipid via
intrinsic biosynthesis route.1 Cho-containing phospholipids are
important structural components of membranes and play critical
roles in cell signaling, either as signaling molecules in their own
right or as precursors of secondary messengers.
Because tumor proliferates more rapidly than normal tissues,
the uptake of choline in the former is obviously higher than that
in the latter.2 So far, Cho derivatives have already been widely
researched as tumor markers in magnetic resonance imaging3
and radionuclide imaging of tumor.4,5 However, modifications of
the Cho molecule will probably decrease its bioactivity6, restrain-
ing its applications in nuclear medical imaging. In the case of
99mTc nuclide, the defect is especially obvious, as it requires large
ligands linked with Cho to coordinate with the 99mTc core.
Therefore, the pretargeting strategy is potentially able to overcome
this general problem.7,8
phospholipids involves the administration of a tagged unlabeled
choline and subsequent tumor targeting. The second step follows
with injection of a radiolabeled molecule with a high specificity
and bioorthogonal reactivity for the tag on the tumor localized
Cho-containing phospholipids. This strategy has been increasingly
used in radioimmunodetection and radio-immunotherapy of
tumors.9–11 The pretargeting strategy consists of two indispensable
components: click synthons used as reporter/effector and click
reaction as the link.12–14 Among all the click reactions, the strain-
promoted cyclooctyne–azide cycloaddition (SPAAC) represents a
rapid, efficient, and catalyst-free in vivo click reaction with high
specificity and versatility under mild conditions.15 SPAAC reaction
is being increasingly applied in microscopic optical visualization of
macromolecules such as glycoproteins16, proteins17, DNA18 and
RNA19, as well as in radiochemistry for molecular imaging.20–23
Labeling of Cho-containing phospholipid in vitro with
pretargeting strategy has been tried24–31; however, the in vivo trial
especially with radiochemical method has been rarely reported. In
this study, we attempted to use this strategy to radiolabel
Cho-containing phospholipid with the SPAAC reaction.
The pretargeting strategy is a multi-step process that utilizes
the high affinity of biomolecules, avoiding the drawbacks
associated with decreasing bioactivity and slow clearance.8 For
example, the first step of a pretargeting strategy in Cho-containing
Next, we investigated Cho and cyclooctyne derivatives suitable
as reporter/effector in the pretargeting strategy.
In 2013, Huang et al.25 biosynthesized and incorporated
choline (Cho) analogs such as azidoethyl-choline (AECho) and
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0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.