Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> A strain-induced copper-free click reaction mediated by a new and easily prepared cyclooctyne derivative was used to efficiently assemble a DOTA−biotin adduct capable of radionuclide (68Ga) uptake. This synthetic strategy offers a potentially general and convenient means of preparing targeted radiolabeling and radiotherapeutic agents. <s> BIB001 </s> Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> The copper-free strain-promoted click chemistry between (18)F-labeled aza-dibenzocyclooctyne [(18)F]FB-DBCO and various azides is described. [(18)F]FB-DBCO was prepared in 85% isolated radiochemical yield (decay-corrected) through acylation of amino aza-dibenzocyclooctyne 1 with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB). [(18)F]FB-DBCO showed promising radiopharmacological profil with fast blood clearance as assessed with dynamic small animal PET studies. Metabolic stability of [(18)F]FB-DBCO was 60% of intact compound after 60 min post injection in normal Balb/C mice and blood clearance half-life was determined to be 53 s based on the time-activity-curve (TAC). Copper-free click chemistry was performed with various azides at low concentrations (1-2 μM) which differed in their structural complexity in different solvents (methanol, water, phosphate buffer and in bovine serum albumin (BSA) solution). Reaction proceeded best in methanol (>95% yield after 15 min at room temperature), whereas reaction in BSA required longer reaction times of 60 min and 40 °C upon completion. <s> BIB002 </s> Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> Bombesin is a 14 amino acid (Pyr-Gln-Arg-Leu-Gly-AsnGln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) neuropeptide, which binds with high affinity to the gastrin-releasing peptide receptor (GRPR). Bombesin has received much attention in the field of nuclear imaging because the GRPR is massively overexpressed on a variety of tumor cells, including breast and prostate tumor cells, thus making bombesin a promising radioligand for the diagnosis and imaging of cancer. Much effort has been invested in the development of labeled bombesin analogues. Bombesin is often modified in the form of Lys[3]-bombesin, which allows for site-selective introduction of the radionuclide at the terminal amino <s> BIB003 </s> Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> We introduce the high-throughput synthesis of various (18)F-labeled peptide tracers by a straightforward (18)F-labeling protocol based on a chemo-orthogonal strain-promoted alkyne azide cycloaddition (SPAAC) using aza-dibenzocyclootyne-substituted peptides as precursors with (18)F-azide synthon to develop peptide based positron emission tomography (PET) molecular imaging probes. The SPAAC reaction and subsequent chemo-orthogonal purification reaction with azide resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided four (18)F-labeled tumor targetable bioactive peptides such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP), and apoptosis targeting peptide (ApoPep) in high radiochemical yields as direct injectable solutions without any HPLC purification and/or formulation processes. In vitro binding assay and in vivo PET molecular imaging study using the (18)F-labeled cRGD peptide also demonstrated a successful application of our (18)F-labeling protocol. <s> BIB004 </s> Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile bioorthogonal conjugation techniques especially for the radiolabeling of biologically active high molecular weight compounds like peptides, proteins or antibodies. Taking into consideration that the introduction of fluorine-18 (t(1/2) = 109.8 min) proceeds under harsh conditions, radiolabeling of these biologically active molecules represents an outstanding challenge and is of enormous interest. Special attention has to be paid to the method of 18F-introduction. It should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. For these reasons and due to the high number of functional groups found in these compounds, a specific labeling procedure has to be developed for every bioactive macromolecule. Bioorthogonal strategies including the Cu-assisted Huisgen cycloaddition and its copper-free click variant, both Staudinger Ligations or the tetrazine-click reaction have been successfully applied and represent valuable alternatives for the selective introduction of fluorine-18 to overcome the afore mentioned obstacles. This comprehensive review deals with the progress and illustrates the latest developments in the field of bioorthogonal labeling with the focus on the preparation of radiofluorinated building blocks and tracers for molecular imaging. <s> BIB005 </s> Small Molecule Radiopharmaceuticals – A Review of Current Approaches <s> Strain-Promoted Click Chemistry/Strain-Promoted Azide Alkyne Cycloaddition <s> An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles. <s> BIB006
Largely driven by the requirement of copper-free click chemistry due to copper linked toxicity (cytotoxicity, non-compatibility with oligonucleotides, hepatitis, and implications in Alzheimer's disease and neurological diseases), strain-promoted, and copper-free variants of click chemistry are being validated in radiopharmacy BIB005 . Apart from being copper-free, the reaction proceeds at a faster rate and can be used for short-lived radioisotopes like 64 Cu (67); it is efficient, has high specificity, and requires mild reaction conditions BIB006 . These were first reported in 2011 BIB002 . Since then, the reaction has been used for radiolabeling of peptides [BBN BIB003 , RGD  BIB004 , c-Met-binding peptide BIB004 , apoptosis-targeting peptide (ApoPep) (72) , somatostatin analogs (72), DOTA-biotin conjugate BIB001 , and NPs (68, 74)]. However, the concern for strain-promoted azide alkyne cycloaddition (SPAAC) include (a) effect of bulky moieties such as DBCO and ADIBO on lipophilicity, binding affinity with the target and the variation on pharmacokinetic behavior, and (b) non-regioselective product formation consisting both 1,4 and 1,5 regioisomers (72). 