www.asiachem.news December 2021 | 65 C4-carbonyl and C6-alkenyl or phenyl groups in 1-azatrienes enables reducing the energy gap between HOMO and LUMO to significantly accelerate the azaelectrocyclization and occur in a matter of a few minutes at room temperature.8 Then, We began to shift our interest to developing 6p-azaelectrocyclization for protein labeling. Althoughmany lysine conjugationmethodologies were developed at the time, most of them were too slow or not reactive enough. As a result, azaelectrocyclization for lysine-selective conjugation (later coinedas theRIKENclick reaction9-11) has become a standard technique heavily utilized in our research today. As shown in Figure 2B, we first began to prepare the aldehyde probe directly linkedwithmolecules of interest via amide linkage. 12-18 To simplify the operation of the RIKEN click reaction, we transitioned to using another reaction to link the molecules. This has led to the preparationof RIKENclick reagentsmodifiedwith groups such as a azide (for Staudinger ligation)19, dibenzocyclooctyne (for strain-promoted azidealkyne cycloaddition)20,21, and trans-cyclooctene (for tetrazine ligation)22-35. Numerous successfully applied molecules for protein modification clearly prove the versatility of the RIKEN click reaction. For instance, molecular imaging and radiotherapeutic applications have seen the usage of metal chelating agents, such as DOTA12,13,18,20,30, NOTA20,30, and closo-decaborate21. Moreover, the fluorescent imaging studies have utilized various fluorophores like coumarin12,14,15, NBD14,18, TAMRA12,13,17,23,30, Cy513,18,19, Hilyte Fluor 75016, fluorescein23, and FRET pairs24. Other molecules that have also found significant usage in our research include the conjugation of biotin17,19,22, andnumerous types of complex N-glycans19,22-34. In terms of conjugates done using the RIKEN click reagent, our studies have shownapplicability to various amine-containing scaffolds. A number of peptides and proteins have been conjugated under in vitro conditions; such as the cRGDyK peptide22,24,30, somatostatin12,14,22, albumin, orosomucoid12, and asialoorosomucoid12. This has also been extended todendrimer complexes18, aswell as a number of antibodies that include anti-GFP mAb12, anti-IGSF4mAb20, and trastuzumab21. For example, the rapid rate of theRIKENclick reaction has also been beneficial for the preparation of radiotherapeuticagents20,21. Toapproach it, aonepot reaction can be performed the RIKEN click reagent, a tetrazine-linked metal chelator, and a targeting antibody (Fig. 2C). These radiolabeled antibodies have been shown in mouse models to effectively accumulate to targeted tumors and suppress their growth. Intriguing, the RIKEN click reagent has also been shown to be applicable in labeling the surface proteins of live cells. For instance, to investigate and identify glycan-dependent mechanisms that could potentially influence in vivo lymphocyte trafficking in living animals, we labeled the lymphocyte that were extracted from nude mice with a(2,6)-sialic acid terminated complex N-glycan19. The glycosylated lymphocytes were then administered into DLD-1 tumor bearing mice. In the case of glycosylated lymphocytes, observations revealed that besides lymphocyte accumulation in spleen/lymph nodes, detection was also found in implanted tumor regions (Figure 2D). In a control setting, lymphocytes without glycan modifications naturally accumulated to the spleen and intestinal lymph nodes, while no detection was found in the tumor. Current literature has strongly implicated cancer cell glycosylation to be vital for mediating tumor metastasis and invasion35. On the basis of the concept, we first established four kinds of human cancer cells (two cancer cell metal-catalyzed uncaging reactions to release drugs have also utilized to cancer treatments due to bioorthogonal character and the outstanding catalytic activity of metals5. These approaches, however, still have some drawbacks. For example, the use of external stimuli requires expensive machinery, while click-to-release strategies use abiotic small molecules that need to be directly injectedat tumors sites. Since only fully developed tumors are acidic, pH triggereddrug release is not as effective for early-stage cancers. Encapsulation of metal nanoparticles can reduce toxicity of abiotic metals in vivo and are accumulated in cancer tissues by enhanced permeability and retention (EPR) effect, however, thousands of research papers gave a critical verdict, that is, the EPR effect works in rodents but not in humans6. And, the studies revealed that after treatment, nanomaterials were found to accumulate in the spleen, liver, brain, and lungs to cause oxidative stress via the production of reactive oxygen species (ROS), leading to significant toxicity7. Our group offers a vastly different approach by in vivo synthetic chemistry to achieve localized drug synthesis/release on cancers. By definition, in vivo synthetic chemistry is a term used by our group to describe the ability to perform non-natural chemical reactions within living biological systems. Because of the complexity of biological environments, however, a multitude of challenges need to be overcome to achieve the feat. Practically speaking, there are threemain areas to address. The first area of focus is related to the targetingmethodology.Without proper localizationof in vivo synthetic chemistry, this system would be incapable of applicability for biomedical research. The second is the need to develop an effective andbiocompatible catalyst for the implementation of in vivo synthetic chemistry. Our group felt that theadvantages affordedby abiotic transitionmetal catalysis, such as the potential for in vivo natural product synthesis, made it an attractive strategy. Lastly, development of biocompatible chemical reactions is also indispensable to the system. In vivo synthetic chemistry does not only requiremild and aqueous conditions, but also specific chemoselectively without interfering with biological metabolism. Although our group has only begun to challenge the immense feat, we have identified a path forward using a system that integrates different aspects of our past interesting research (Fig1E). The article is tohighlight the steps taken at each stage, and how they all ultimately fit together for therapeutic in vivo synthetic chemistry. RIKEN Click Reaction As shown in Fig. 2A, the thermal cyclization of 1-azatrienes to 1,2-dihydropyridines via 6p-azaelectrocyclization could be an attractive tool to be utilized for the modification of the lysine amino group on proteins through Schiff-base (imine) formation. However, the requirement for high temperatures and long reaction times for the strategy limited the application of the method in biological systems. Incorporating the interesting chemistry, we found that modification at the Figure 1. In vivo cancer therapeutic modalities based on strategies of localized drug delivery mediated by (A) external-stimuli responsive systems, (B) click-to-release chemistry, (C) the cancer microenvironment, or (D) the abiotic metal-mediated reactions. (E) Therapeutic in vivo synthetic chemistry via glycosylated artificial metalloenzymes.
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