Open research questions in Click Chemistry and Applications
46 unresolved questions extracted from the limitations and future-work sections of 115 Click Chemistry and Applications papers in our library. Each links back to the study that raised it.
What the literature leaves open
While the present study is computational in nature, it provides a robust foundation for subsequent experimental validation and further lead development. A key future step will be the chemical synthesis of the prioritized AREB analogs, which was deliberately considered throughout the optimization process. The majority of the selected R-groups are synthetically accessible using well-established organic transformations, including amide bond formation, urea or hydrazide coupling, heterocycle functionalization, reductive amination, cross-coupling reactions, and late-stage diversification of aromatic or heteroaromatic moieties. Importantly, the modular R1 - R4 design of the AREB scaffold lends itself naturally to a convergent combinatorial synthetic strategy, enabling rapid assembly of analog series and efficient structure–activity relationship (SAR) exploration. Crucially, PLpro represents an especially attractive antiviral target because its catalytic site and substrate-binding channel are highly conserved across coronaviruses, both at the sequence and structural levels. Large-scale genomic analyzes have shown that PLpro is relatively resistant to function- altering point mutations, with the few frequently observed mutations occurring predominantly outside the catalytic and inhibitor-binding regions.4,9,92 This structural and functional conservation is essential for maintaining viral viability. As a result, inhibitors that effectively target the ARTICLE IN PRESS ARTICLE IN PRESS ACCEPTED MANUSCRIPT ARTICLE IN PRESS catalytic site of this enzyme are less likely to lose efficacy against emerging viral variants. In this context, the newly identified AREB analogs may represent promising starting points for the development of pan-coronaviral therapeutics, though this proposition remains speculative in the absence of direct comparative analysis against PLpro homologs from other coronaviruses and awaits experimental validation against a broader panel of coronaviral variants. By targeting a conserved enzymatic function rather than highly variable viral surface proteins, PLpro inhibitors could complement existing antiviral strategies and provide a more mutation-resilient line of defense against coronavirus outbreaks. In summary, this study delivers a comprehensive computational framework for PLpro inhibitor discovery and provides a set of rationally designed AREB candidates that combine predicted high potency with improved drug-like characteristics. The integration of synthetic feasibility, pharmacokinetic considerations, and evolutionary conservation of the target underscores the translational relevance of the presented work. Experimental validation represents the logical next step toward the development of next- generation, broad-spectrum antiviral agents targeting coronaviral PLpro.
Computer-assisted design of arylethylbenzamides as predicted nanomolar inhibitors of papain-like cysteine protease of SARS-CoV-2 · 2026 · DOI9 Study Limitations Several limitations of this study should be acknowledged. Bioavailability enhancement strategies should be explored for curcumin and berberine, including nanoparticle formulations, liposomal encapsulation, phytosome complexes, and co-administration with piperine. First, molecular docking provides computational predictions of binding affinity but does not account for protein flexibility, solvent effects, or entropic contributions.
Molecular Docking Evaluation of Curcumin, Ursolic Acid, Quercetin, Berberine, and Andrographolide Against HIV-1 Reverse Transcriptase and Protease · 2026 · DOINo integrated pipeline exists for natural product-based antibiotic discovery that combines crude extract screening, active principle isolation, structure elucidation, and molecular docking-guided target validation. Current practice separates crude extract assays from pure compound characterization, and studies isolating secondary metabolites have limited bioactivity evaluations.
on phthalazine-piperizine hybrid. Nitrogen-containing heterocycles constitute a huge number of pharmaceuticals and bioactive natural products. Among them, diazaheterocycles such as phthalazines have been recognized as prominent motifs of numerous medicines and synthetic drug molecules. The development of these drug candidates is highly dependent on the sustainable synthetic methodologies and there remains a compelling need for a coordinated approach to discover novel therapeutics. This review presents a well-balanced overview of synthetic and medicinal chemistry aspects of phthalazine nucleus over the last decade. A reasonable degree of chemical complexity in the synthesis of these small molecules was achieved through various efficient and robust methods. High level of functional group tolerance was noticed in the structurally diversified phthalazine products. The use of newly developed approaches was investigated in the formation of drugs such as naproxen. Moreover, the halogenated phthalazine products were also synthetically modified using Buchwald-Hartwig amination protocol, thus enabling the formation of new carbon-nitrogen bonds. On the other hand, various biological activities of the phthalazine core have been reviewed. The careful and critical evaluation of the in vitro and in vivo bioactivity results revealed the significance of the substitution patterns. Structure-activity relationship analyses also provided insightful information leading to the generation of potent leads with enhanced inhibitory efficacy. The literature precedents (vatalanib, sunitinib, phenstatin, taladegib, vismodegib, sorafenib, Anta XV, MY 5445, PHR0007) were used to optimize and fine-tune the structures using seemingly simple exchange of bioactive pharmacophores providing a significant boost in potency. The possible binding modes of the most potent leads were visualized using molecular docking approach. Despite these remarkable achievements, there is still lack of an integrated strategy to overcome the challenges and deficit of potent leads in drug discovery arena. The green and sustainable syntheses need to be developed while applying the low-cost procedures and shortening the reaction steps. The careful selection of lead compounds considering their physical properties, pharmacological profiling, and 92 appropriate structure–activity relationships need to be developed. In vitro toxicity screening as well as in vivo testing need improvement at the earlier phase of drug discovery process, thus allowing medicinal chemists to safely design the drug candidate. Collectively, the developed synthetic methodologies and bioactive potential associated with the phthalazine nucleus will serve as a handy guide to design new strategies tightly linked to the discovery and development of effective therapeutic drugs.
Synthetic and medicinal chemistry of phthalazines: Recent developments, opportunities and challenges · 2020 · DOILate-stage [18F]fluorination of arenes and heteroarenes has been identified as an alternative to click chemistry approaches, but head-to-head comparisons of radiochemical yield, reaction time, and specific activity between [18F]fluorination methods and [18F]click chemistry routes for identical target molecules have not been systematically conducted.
The paper presents click chemistry applications for 18F-labeled peptides (RGD peptides, folate derivatives, apoptosis markers) and 99mTc-labeled compounds, but a systematic comparison of biological efficacy and in vivo targeting performance between click-generated radiopharmaceuticals and conventional fluorobenzoyl- or fluoropropionyl-labeled analogues across multiple tumor models is not comprehensively documented.
The application of copper-catalyzed azide-alkyne cycloaddition (CuAAC) for 11C click chemistry labeling has only been briefly demonstrated with in situ synthesis of [11C]methyl azide. Comprehensive evaluation of [11C]click chemistry regioselectivity, radiochemical purity, and specific activity across diverse 11C-labeled alkyne precursors in peptide and small molecule radiopharmaceuticals remains underdeveloped.
The inverse electron demand Diels-Alder (iEDDA) cycloaddition strategy using trans-cyclooctene dienophiles with tetrazine reagents has been referenced but not thoroughly evaluated in the context of 18F-radiopharmaceutical synthesis. Direct comparison of iEDDA reaction kinetics and labeling efficiency with traditional azide-alkyne click chemistry and SPAAC methods for 18F-labeled biomolecules needs systematic investigation.
While copper-free click chemistry with strain-promoted azide-alkyne cycloadditions (SPAAC) has been demonstrated for 18F-radiopharmaceutical labeling, the optimization of cyclooctyne and bicyclononyne reagents for rapid 18F-incorporation with the short half-life of fluorine-18 remains incompletely characterized. Systematic kinetic comparisons between different cycloalkyne scaffolds (monobenzocyclooctyne, difluorinated cyclooctynes, biarylazacyclooctynones) for 18F click chemistry efficiency and radiochemical yield are lacking.
Triazoles 1B30-37 were synthesized as derivatives for treating Alzheimer's disease using copper-catalyzed azide-alkyne cycloaddition with RF-azides, but their biological activity, target engagement, pharmacokinetics, and in vivo efficacy in Alzheimer's disease models have not been reported in this excerpt.
Monofluoroalkyl-substituted 1,2,3-triazoles 1B44 with remote fluorine atoms were synthesized and evaluated for dopamine receptor D2long, D2short, D3, and D4 binding, but the structure-activity relationships between fluoroalkyl chain length, position, and receptor selectivity across these dopamine receptor subtypes remain incompletely characterized.
Bu- and Ph-substituted 1-RF-triazoles decompose with nitrogen elimination at 160–170 °C producing specific products (122, 123), but the thermal decomposition mechanism and temperature-dependent fragmentation pathways for these fluoroalkyl-substituted triazole variants have not been characterized in detail.
The proposed explanation for high stability of the =N–CF2–R moiety in 1-RF-1,2,3-triazoles (nitrogen lone electron pair incorporation into aromatic system) requires experimental validation through computational modeling or spectroscopic analysis to confirm the mechanistic basis of this stabilization.
Unexpected variability in reactivity of copper-catalyzed azide-alkyne cycloaddition was observed for [18F]2-fluoroethyl azide forming 1,4-disubstituted 1-alkyl-1,2,3-triazoles (1B42) across different alkyne analogues; the structural or electronic factors responsible for this variability in 18F-labeled triazole synthesis require identification.
RF-vinylic azides reacting with dimethyl acetylenedicarboxylate (DMAD) via 1,3-dipolar cycloaddition consistently yielded triazoles 1B41 at maximum 60% despite multiple optimization attempts varying reaction parameters; the specific conditions limiting yields above 60% remain unidentified and require systematic investigation.
The thiol-parafluoro, thiol-halogeno, and thiol-ene/yne photochemical reactions on thiolfunctionalized glass using visible light are described generically without specifying the exact wavelength ranges tested, substrate material variations, or quantitative coupling efficiencies; systematic comparison of these three reaction pathways under identical visible light conditions is absent.
The visible/near-infrared light-controlled singlet oxygen-mediated activation of multifunctional photo-unclickable prodrugs for tumor removal is mentioned as a concept by You and coworkers, but the paper truncates before providing implementation details; specific wavelengths, singlet oxygen generation efficiency, and in vivo tissue penetration depth for this photo-unclick chemistry approach require complete characterization.
The photodegradable nanogel synthesis using methoxynitrobenzyl ether derivatives showed size increases and hydrodynamic changes upon UV exposure (λ = 365 nm, 10 mW/cm²) for 5 minutes; however, the paper does not address the reproducibility of crosslinking efficiency, degradation kinetics under varying light intensities, or the stability of encapsulated cargo during the photocleavage process.
The photoinduced bio-orthogonal 1,3-dipolar cycloaddition reactions employing o-allyltyrosine and homoallylglycine were performed at λ = 302 nm for 10 minutes; however, the paper lacks systematic investigation of photon dosage effects, wavelength optimization, and potential phototoxicity thresholds for sustained spatiotemporal protein synthesis detection in mammalian cells.
While photoclick modification of oligonucleotides using diaryltetrazole photoactivatable groups has been demonstrated with LEDs at λ = 365 nm for chemical biology applications, the paper does not specify the quantum yield or photon efficiency of this reaction, nor does it provide comparative data on reaction efficiency across different LED wavelengths or intensities relevant for intracellular DNA modification.
The photochemical fixation of DNA nanoconstructs using UV irradiation (λ = 365 nm) has only been demonstrated with 80-mer cyclic DNA and 10 base pair duplexes; the scalability and efficacy of this metal-free strain-promoted click chemistry approach on larger DNA oligomeric structures (>200 nucleotides) and varying sequence complexities remain unexplored.
Fragment-based drug discovery using click chemistry assumes that azide and alkyne components can reach the enzyme active site in sufficient concentration and orientation; however, the paper does not address how library size, fragment molecular weight distribution, or cellular permeability constraints affect screening success rates across diverse protein targets.
The in situ click chemistry concept relies on enzyme-catalyzed triazole formation at room temperature through proper fragment orientation in the active site, but the paper does not specify quantitative kinetic parameters (Km, kcat, enzyme specificity constants) comparing in situ-generated triazoles to conventionally synthesized analogs on the same enzyme targets.
The dopamine D3 receptor ligands (compounds 96-99) generated via solid-phase click chemistry demonstrated single-digit nanomolar Ki values, but selectivity against off-target dopamine receptor subtypes and potential metabolic stability or toxicity concerns are not addressed, limiting preclinical candidate assessment for neuropsychiatric indications.
The human histamine H3 receptor (hH3R) antagonists showed nanosubnanomolar binding affinity in vitro, but the paper does not report blood-brain barrier penetration data, in vivo pharmacokinetics, or efficacy in cognitive impairment or sleep/wake disorder animal models, limiting translation to CNS therapeutic applications.
Most-cited papers in Click Chemistry and Applications
- Dual-Locked Enzyme-Activatable Bioorthogonal Fluorescence Turn-On Imaging of Senescent Cancer Cells · Journal of the American Chemical Society · 2024 · 93 citations
- DrugMap: A quantitative pan-cancer analysis of cysteine ligandability · Cell · 2024 · 92 citations
- Photochemical tuning of materials: A click chemistry perspective · Materials Today Chemistry · 2018 · 60 citations
- Synthetic and medicinal chemistry of phthalazines: Recent developments, opportunities and challenges · Bioorganic Chemistry · 2020 · 52 citations
- Bioorthogonal Activation of Deep Red Photoredox Catalysis Inducing Pyroptosis · Journal of the American Chemical Society · 2024 · 52 citations
- Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells · Nature Chemistry · 2024 · 51 citations
- The 2022 Nobel Prize in Chemistry for the development of click chemistry and bioorthogonal chemistry · Analytical and Bioanalytical Chemistry · 2023 · 28 citations
- Chemistry of fluoroalkyl-substituted 1,2,3-triazoles · Journal of Fluorine Chemistry · 2018 · 23 citations
- Recent Perspectives on Anticancer Potential of Coumarin Against Different Human Malignancies: An Updated Review · Food Science & Nutrition · 2024 · 23 citations
- An in silico investigation of 1,2,4-triazole derivatives as potential antioxidant agents using molecular docking, MD simulations, MM-PBSA free energy calculations and ADME predictions · Pharmacia · 2023 · 15 citations
Most recent work
- Synthetic developments and biological insights of 1,2,3-triazoles: Unravelling scope for drug discovery · European Journal of Medicinal Chemistry Reports · 2026
- A Catalyst-Free, Tyrosine-Selective Bioconjugation Strategy via Triazine-Pyridine Chemistry for Antibodies · Organic Letters · 2026
- Nicotine-Inspired, De Novo-Designed SARS-CoV-2 Main Protease Inhibitors Reveal Unique Chemistry for Covalently Conjugating Both Cysteine and Histidine Residues in the Catalytic Dyad · Journal of the American Chemical Society · 2026
- Case studies I: Clickable chemistry in analytical chemistry · Comprehensive Analytical Chemistry · 2026
- Exploration of Multicomponent Triazole-Oxadiazole Clubbed Imidazole Hybrids: Design, Synthesis, In Silico Docking, and DFT Analysis as Potent Pharmacological Agents · Russian Journal of General Chemistry · 2026
- Should I Stay or Should I Go? - Leveraging an Overlooked Feature of Click-to-Release Chemistry for Affinity Labeling · ChemRxiv · 2026
- Modular Synthesis of Bis(triazole)s by Sequential Triazole Formation and Michael Addition · ChemRxiv · 2026
- Ambient Temperature Synthesis of Triazole-4-carbaldehyde Reagent by Dimroth Rearrangement Enabling Facile N-Terminal-specific Modification and Immobilization of Proteins · Bioconjugate Chemistry · 2026
- Biophysical Modeling Elucidates Mechanistic Principles for Rational Molecular Glue Design · Journal of Chemical Information and Modeling · 2026
- Synthesis of Reversible Sequence-Defined Oligourethane Macrocycles through Click and Declick Thiol-Amine Conjugation with a Meldrum’s Acid-Derived Conjugate Acceptor · The Journal of Organic Chemistry · 2026
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