Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.

Computational Organic Chemistry: The Frontier for Understanding and Designing Bioorthogonal Cycloadditions.

Computational organic chemistry has become a valuable tool in the field of bioorthogonal chemistry, offering insights and aiding in the progression of this branch of chemistry. In this review, I present an overview of computational work in this field, including an exploration of both the primary computational analysis methods used and their application in the main areas of bioorthogonal chemistry: (3 + 2) and [4 + 2] cycloadditions. In the context of (3 + 2) cycloadditions, detailed studies of electronic effects have informed the evolution of cycloalkyne/1,3-dipole cycloadditions. Through computational techniques, researchers have found ways to adjust the electronic structure via hyperconjugation to enhance reactions without compromising stability. For [4 + 2] cycloadditions, methods such as distortion/interaction analysis and energy decomposition analysis have been beneficial, leading to the development of bioorthogonal reactants with improved reactivity and the creation of orthogonal reaction pairs. To conclude, I touch upon the emerging fields of cheminformatics and machine learning, which promise to play a role in future reaction discovery and optimization.

Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.

Indole as a Versatile Building Block in Cycloaddition Reactions: Synthesis of Diverse Heterocyclic Frameworks.

Indole, a ubiquitous and structurally versatile aromatic compound, has emerged as a key player in the synthesis of diverse heterocyclic frameworks via cycloaddition reactions. These reactions are completely atom-economical and, hence, are considered as green reactions. This review article provides a comprehensive overview of the pivotal role played by indole in the construction of complex and biologically relevant heterocyclic compounds. Here we explore the chemistry of indole-based cycloadditions, highlighting their synthetic utility in accessing a wide array of heterocyclic architectures, including cyclohepta[b]indoles, tetrahydrocarbazoles, tetrahydroindolo[3,2-c]quinoline, and indolines, among others. Additionally, we discuss the mechanistic insights that underpin these transformations, emphasizing the strategic importance of indole as a building block. The content of this article will certainly encourage the readers to explore more work in this area.

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

[Fe(NCMe)6](BF4)2 is a bifunctional catalyst for styrene aziridination by nitrene transfer and heterocycle expansion by subsequent dipolar insertion.

The solvated iron(II) salt [Fe(NCMe)6](BF4)2 (Me = methyl) is shown to be a bifunctional catalyst with respect to aziridination of styrene. The salt serves as an active catalyst for nitrene transfer from PhINTs to styrene to form 2-phenyl-N-tosylaziridine (Ph = phenyl; Ts = tosyl, -S{O}2-p-C6H4Me). The iron(II) salt also acts as a Lewis acid in non-coordinating CH2Cl2 solution, to catalyze heterolytic CN bond cleavage of the aziridine and insertion of dipolarophiles. The 1,3-zwitterionic intermediate is presumably supported by interaction of the metal dication with the anion, and by resonance stabilization of the carbocation. Nucleophilic dipolarophiles then insert to give a five-membered heterocyclic ring. The result is a two-step cycloaddition, formally [2 + 1 + 2], that is typically regiospecific, but not stereospecific. This reaction mechanism was confirmed by conducting a series of one-step, [3 + 2] additions of unsaturated molecules into pre-formed 2-phenyl-N-tosylaziridine, also catalyzed by [Fe(NCMe)6](BF4)2. Relevant substrates include styrenes, carbonyl compounds and alkynes. These yield five-membered heterocylic rings, including pyrrolidines, oxazolidines and dihydropyrroles, respectively. The reaction scope appears limited only by the barrier to formation of the dipolar intermediate, and by the nucleophilicity of the captured dipolarophile. The bifunctionality of an inexpensive, earth-abundant and non-toxic catalyst suggests a general strategy for one-pot construction of heterocyclic rings, as demonstrated specifically for pyrrolidine ring formation.

Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

A Synthetic Strategy toward S-, N-, and O-Heterocyclooctynes Facilitates Bioconjugation Using Multifunctional Handles.

Over the past two decades, the introduction of bioorthogonal reactions has transformed the ways in which chemoselective labeling, isolation, imaging, and drug delivery are carried out in a complex biological milieu. A key feature of a good bioorthogonal probe is the ease with which it can be attached to a target compound through bioconjugation. This paper describes the expansion of the utility of a class of unique S-, N-, and O-containing heterocyclooctynes (SNO-OCTs), which show chemoselective reactivity with type I and type II dipoles and divergent reactivities in response to electronic tuning of the alkyne. Currently, bioconjugation of SNO-OCTs to a desired target is achieved through an inconvenient aryl or amide linker at the sulfamate nitrogen. Herein, a new synthetic approach toward general SNO-OCT scaffolds is demonstrated that enables the installation of functional handles at both propargylic carbons of the heterocycloalkyne. This capability increases the utility of SNO-OCTs as labeling reagents through the design of bifunctional bioorthogonal probes with expanded capabilities. NMR kinetics also revealed up to sixfold improvement in cycloaddition rates of new analogues compared to first-generation SNO-OCTs.

Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation.

Site-selective modification of complex peptides and the functionalization of their C-H bonds hold great promise for expanding their use in therapeutics and biomedical research. Herein, we leverage the power of late-stage chemoenzymatic catalysis using an indole prenyltransferase (IPT) enzyme and alkyl diphosphates to specifically modify the indole ring of tryptophan in clinically relevant peptides. Furthermore, the installed handle enables bioorthogonal click chemistry through an inverse electron-demand Diels-Alder (IEDDA) reaction with a biotin-conjugated tetrazine probe.

Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

The evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba.

Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.

Antimicrobial properties of triazolato terpyridine Pd(II) and Pt(II) complexes formed by [3+2] cycloaddition coupling reaction.

Modern classes of antimicrobials are crucial because most drugs in development today are basically antibiotic derivatives. Even though a large number of metal-based compounds have been studied as antimicrobial agents, relatively few studies have examined the antimicrobial properties of Pd(II) and Pt(II) compounds. The [3+2] cycloaddition reactions of [M(N3)L]PF6 (M = Pd(II) and Pt(II); L = 4'-(2-pyridyl)-2,2':6',2″-terpyridine) with 4,4,4-trifluoro-2-butynoic acid ethyl ester gave the corresponding triazolate complexes. The reaction products were fully characterized with a variety of analytical and spectroscopic tools including X-ray crystallographic analysis. The crystal structure of [Pd(triazolatoCF3,COOCH2CH3)L]PF6 provided cut-off evidence that the kinetically formed N1-triazolato isomer favoured the isomerization to the thermodynamically stable N2-analogue. The experimental work was complemented with computational work to get an insight into the nature of the predominant triazolate isomer. The lysozyme binding affinity of the triazolate complexes was examined by mass spectrometry. An analysis of the lysozyme Pd(II) adducts suggests a coordinative covalent mode of binding via the loss of the triazolato ligand. The free ligand and its triazolate complexes displayed selective toxicity against Candida albicans and Cryptococcus neoformans, while no cytotoxicity was observed against the normal human embryonic kidney cell line.

Semisynthesis and characterization of versatile azide intermediates using sodium alginate and its homopolymeric derivatives as starting material.

Alginate, a polyuronic biopolymer composed of mannuronic and guluronic acid units, contain hydroxyl and carboxyl groups as targeting modification sites to obtain structures with new and/or improved biological properties. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a versatile click reaction for polymer functionalization, but it typically requires a "pre-click" modification to introduce azide or alkyne groups. Here, we described a straightforward chemical path to selectively modify alginate carboxyl groups producing versatile azido derivatives through N-acylation using 3-azydopropylamine. The resulting azide-functionalized polysaccharides underwent click chemistry to yield amino derivatives, confirmed by NMR and FTIR analyses. The 1H NMR spectrum reveals a characteristic triazole group signal at 8.15 ppm. The absence of the azide FTIR band for all amino derivatives, previously observed for the N-acylation products, indicated reaction success. Antibacterial and antioxidant assessments revealed that the initial polysaccharide lacks E. coli inhibition, while the click chemistry-derived amine products exhibit growth inhibition at 5.0 mg/mL. Lower molecular weight derivatives demonstrate superior DPPH scavenging ability, particularly amino-derivatives (24-33 % at 1.2 mg/mL). This innovative chemical pathway offers a promising strategy for developing polysaccharide structures with enhanced properties, demonstrating potential applications in various fields.

Enantioselective total synthesis of (‒)-lucidumone enabled by tandem prins cyclization/cycloetherification sequence.

The Ganoderma meroterpenoids are a growing class of natural products with architectural complexity, and exhibit a wide range of biological activities. Here, we report an enantioselective total synthesis of the Ganoderma meroterpenoid (‒)-lucidumone. The synthetic route features several key transformations, including a) a Cu-catalyzed enantioselective silicon-tethered intramolecular Diels-Alder cycloaddition to construct the highly functionalized bicyclo[2.2.2]octane moiety; b) Brønsted acid promoted tandem O-deprotection/Prins cyclization/Cycloetherification sequence followed by oxidation to install concurrently the tetrahydrofuran and the fused indanone framework; c) Fleming-Tamao oxidation to generate the secondary hydroxyl; d) an iron-catalyzed Wacker-type oxidation of hindered vinyl group to methyl ketone.

Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Trisarcglaboids A and B, two cytotoxic lindenane sesquiterpenoid trimers with a unique polymerization mode isolated from Sarcandra glabra.

Trisarcglaboids A and B (1 and 2), representing the first example of lindenane sesquiterpenoid trimers repolymerized based on the classical [4 + 2] type dimer, together with known biogenic precursors chlorahololide D (3) and sarcandrolide A (4), were identified as chemical components of the root of Sarcandra glabra. The novel trimeric lindenane sesquiterpenoid skeletons, including their absolute configurations, were characterized using MS, NMR, ECD, and X-ray single crystal diffraction. The proposed Diels-Alder cycloaddition between Δ2(3) of the tiglic acyl group of the classical [4 + 2] type dimer and Δ15(4),5(6) of the third lindenane may serve as the key biogenic step. In addition, compound 1 exerted significant cytotoxicity against five human cancer cell lines with IC50 values ranging from 1 to 7 µM, potentially through blocking Akt phosphorylation and activating the endogenous apoptosis pathway.

Cyclopropenes as Chemical Reporters for Dual Bioorthogonal and Orthogonal Metabolic Labeling of DNA.

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross-reactivity. We developed a pair of differently modified 2'-deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse-electron demand Diels-Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1- and 3-methylcyclopropenyl-modified 2'-deoxynucleosides, two 2'-deoxyuridines were identified to be the fastest-reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2'-deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2'-deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine-styryl tetrazine and a fluorogenic pyrene-tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.

Innovative Arylimidazole-Fused Phytovirucides via Carbene-Catalyzed [3+4] Cycloaddition: Locking Viral Cell-To-Cell Movement by Out-Competing Virus Capsid-Host Interactions.

The control of potato virus Y (PVY) induced crop failure is a challengeable issue in agricultural chemistry. Although many anti-PVY agents are designed to focus on the functionally important coat protein (CP) of virus, how these drugs act on CP to inactivate viral pathogenicity, remains largely unknown. Herein, a PVY CP inhibitor -3j (S) is disclosed, which is accessed by developing unusually efficient (up to 99% yield) and chemo-selective (> 99:1 er in most cases) carbene-catalyzed [3+4] cycloaddition reactions. Compound -3j bears a unique arylimidazole-fused diazepine skeleton and shows chirality-preferred performance against PVY. In addition, -3j (S) as a mediator allows ARG191 (R191) of CP to be identified as a key amino acid site responsible for intercellular movement of virions. R191 is further demonstrated to be critical for the interaction between PVY CP and the plant functional protein NtCPIP, enabling virions to cross plasmodesmata. This key step can be significantly inhibited through bonding with the -3j (S) to further impair pathogenic behaviors involving systemic infection and particle assembly. The study reveals the in-depth mechanism of action of antiviral agents targeting PVY CP, and contributes to new drug structures and synthetic strategies for PVY management.

Halo-1,2,3-triazoles: Valuable Compounds to Access Biologically Relevant Molecules.

1,2,3-triazole is an important building block in organic chemistry. It is now well known as a bioisostere for various functions, such as the amide or the ester bond, positioning it as a key pharmacophore in medicinal chemistry and it has found applications in various fields including life sciences. Attention was first focused on the synthesis of 1,4-disubstituted 1,2,3-triazole molecules however 1,4,5-trisubstituted 1,2,3-triazoles have now emerged as valuable molecules due to the possibility to expand the structural modularity. In the last decade, methods mainly derived from the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction have been developed to access halo-triazole compounds and have been applied to nucleosides, carbohydrates, peptides and proteins. In addition, late-stage modification of halo-triazole derivatives by metal-mediated cross-coupling or halo-exchange reactions offer the possibility to access highly functionalized molecules that can be used as tools for chemical biology. This review summarizes the synthesis, the functionalization, and the applications of 1,4,5-trisubstituted halo-1,2,3-triazoles in biologically relevant molecules.