Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

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.

Advances in the Synthesis of Bioorthogonal Reagents: s-Tetrazines, 1,2,4-Triazines, Cyclooctynes, Heterocycloheptynes, and trans-Cyclooctenes.

Aligned with the increasing importance of bioorthogonal chemistry has been an increasing demand for more potent, affordable, multifunctional, and programmable bioorthogonal reagents. More advanced synthetic chemistry techniques, including transition-metal-catalyzed cross-coupling reactions, C-H activation, photoinduced chemistry, and continuous flow chemistry, have been employed in synthesizing novel bioorthogonal reagents for universal purposes. We discuss herein recent developments regarding the synthesis of popular bioorthogonal reagents, with a focus on s-tetrazines, 1,2,4-triazines, trans-cyclooctenes, cyclooctynes, hetero-cycloheptynes, and -trans-cycloheptenes. This review aims to summarize and discuss the most representative synthetic approaches of these reagents and their derivatives that are useful in bioorthogonal chemistry. The preparation of these molecules and their derivatives utilizes both classical approaches as well as the latest organic chemistry methodologies.

Fluorescent coumarin-alkynes for labeling of amino acids and peptides via manganese(I)-catalyzed C-H alkenylation.

The late-stage fluorescent labeling of structurally complex peptides bears immense potential for molecular imaging. Herein, we report on a manganese(I)-catalyzed peptide C-H alkenylation under exceedingly mild conditions with natural fluorophores as coumarin- and chromone-derivatives. The robustness and efficiency of the manganese(I) catalysis regime was reflected by a broad functional group tolerance and low catalyst loading in a resource- and atom-economical fashion.

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.

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.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

Transfer Hydrogenation of Vinyl Arenes and Aryl Acetylenes with Ammonia Borane Catalyzed by Schiff Base Cobalt(II) Complexes.

A series of bench-stable Co(II) complexes containing hydrazone Schiff base ligands were evaluated in terms of their activity and selectivity in carbon-carbon multiple bond transfer hydrogenation. These cobalt complexes, especially a Co(II) precatalyst bearing pyridine-2-yl-N(Me)N=C-(1-methyl)imidazole-2-yl ligand, activated by LiHBEt3, were successfully used in the transfer hydrogenation of substituted styrenes and phenylacetylenes with ammonia borane as a hydrogen source. Key advantages of the reported catalytic system include mild reaction conditions, high selectivity and tolerance to functional groups of substrates.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Reflections on a Copenhagen-Minneapolis Axis in Bioorganic Chemistry.

The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

Asymmetric Carbene-Alkyne Metathesis-Mediated Cascade: Synthesis of Benzoxazine Polychiral Polyheterocycles and Discovery of a Novel Pain Blocker.

This study introduces a novel approach for synthesizing Benzoxazine-centered Polychiral Polyheterocycles (BPCPHCs) via an innovative asymmetric carbene-alkyne metathesis-triggered cascade. Overcoming challenges associated with intricate stereochemistry and multiple chiral centers, the catalytic asymmetric Carbene Alkyne Metathesis-mediated Cascade (CAMC) is employed using dirhodium catalyst/Brønsted acid co-catalysis, ensuring precise stereo control as validated by X-ray crystallography. Systematic substrate scope evaluation establishes exceptional diastereo- and enantioselectivities, creating a unique library of BPCPHCs. Pharmacological exploration identifies twelve BPCPHCs as potent Nav ion channel blockers, notably compound 8 g. In vivo studies demonstrate that intrathecal injection of 8 g effectively reverses mechanical hyperalgesia associated with chemotherapy-induced peripheral neuropathy (CIPN), suggesting a promising therapeutic avenue. Electrophysiological investigations unveil the inhibitory effects of 8 g on Nav1.7 currents. Molecular docking, dynamics simulations and surface plasmon resonance (SPR) assay provide insights into the stable complex formation and favorable binding free energy of 8 g with C5aR1. This research represents a significant advancement in asymmetric CAMC for BPCPHCs and unveils BPCPHC 8 g as a promising, uniquely acting pain blocker, establishing a C5aR1-Nav1.7 connection in the context of CIPN.

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.

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.

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.

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.

3-Acyl-4-Pyranone as a Lysine Residue-Selective Bioconjugation Reagent for Peptide and Protein Modification.

Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide-alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.

Bioinspired Selenium-Nitrogen Exchange (SeNEx) Click Chemistry Suitable for Nanomole-Scale Medicinal Chemistry and Bioconjugation.

Click chemistry is a powerful molecular assembly strategy for rapid functional discovery. The development of click reactions with new connecting linkage is of great importance for expanding the click chemistry toolbox. We report the first selenium-nitrogen exchange (SeNEx) click reaction between benzoselenazolones and terminal alkynes (Se-N to Se-C), which is inspired by the biochemical SeNEx between Ebselen and cysteine (Cys) residue (Se-N to Se-S). The formed selenoalkyne connection is readily elaborated, thus endowing this chemistry with multidimensional molecular diversity. Besides, this reaction is modular, predictable, and high-yielding, features fast kinetics (k2≥14.43 M-1 s-1), excellent functional group compatibility, and works well at miniaturization (nanomole-scale), opening up many interesting opportunities for organo-Se synthesis and bioconjugation, as exemplified by sequential click chemistry (coupled with ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) and sulfur-fluoride exchange (SuFEx)), selenomacrocycle synthesis, nanomole-scale synthesis of Se-containing natural product library and DNA-encoded library (DEL), late-stage peptide modification and ligation, and multiple functionalization of proteins. These results indicated that SeNEx is a useful strategy for new click chemistry developments, and the established SeNEx chemistry will serve as a transformative platform in multidisciplinary fields such as synthetic chemistry, material science, chemical biology, medical chemistry, and drug discovery.

1,3,4,5-Tetrasubstituted Poly(1,2,3-triazolium) Obtained through Metal-Free AA+BB Polyaddition of a Diazide and an Activated Internal Dialkyne.

A tetra(ethylene glycol)-based 1,3,4,5-tetrasubstituted poly(1,2,3-triazolium) is synthesized in two steps including: i) the catalyst-free polyaddition of a diazide and an activated internal dialkyne and ii) the N-alkylation of the resulting 1,2,3-triazole groups. In order to provide detailed structure/properties correlations different analogs are also synthesized. First, parent poly(1,2,3-triazole)s are obtained via AA+BB polyaddition using copper(I)-catalyzed alkyne-azide cycloaddition or metal-free thermal alkyne-azide cycloaddition (TAAC). Poly(1,2,3-triazole)s with higher molar masses are obtained in higher yields by TAAC polyaddition. A 1,3,4-trisubstituted poly(1,2,3-triazolium) structural analog obtained by TAAC polyaddition using a terminal activated dialkyne and subsequent N-alkylation of the 1,2,3-triazole groups enables discussing the influence of the methyl group in the C-4 or C-5 position on thermal and ion conducting properties. Obtained polymers are characterized by 1H, 13C, and 19F NMR spectroscopy, differential scanning calorimetry, thermogravimetric analysis, size exclusion chromatography, and broadband dielectric spectroscopy. The targeted 1,3,4,5-tetrasubstituted poly(1,2,3-triazolium) exhibits a glass transition temperature of -23 °C and a direct current ionic conductivity of 2.0 × 10-6 S cm-1 at 30 °C under anhydrous conditions. The developed strategy offers opportunities to further tune the electron delocalization of the 1,2,3-triazolium cation and the properties of poly(1,2,3-triazolium)s using this additional substituent as structural handle.