Integrating Metabolic Oligosaccharide Engineering and SPAAC Click Chemistry for Constructing Fibrinolytic Cell Surfaces.

To effectively solve the problem of significant loss of transplanted cells caused by thrombosis during cell transplantation, this study simulates the human fibrinolytic system and combines metabolic oligosaccharide engineering with strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry to construct a cell surface with fibrinolytic activity. First, a copolymer (POL) of oligoethylene glycol methacrylate (OEGMA) and 6-amino-2-(2-methylamido)hexanoic acid (Lys) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization, and the dibenzocyclooctyne (DBCO) functional group was introduced into the side chain of the copolymer through an active ester reaction, resulting in a functionalized copolymer DBCO-PEG4-POL with ε-lysine ligands. Then, azide functional groups were introduced onto the surface of HeLa model cells through metabolic oligosaccharide engineering, and DBCO-PEG4-POL was further specifically modified onto the surface of HeLa cells via the SPAAC "click" reaction. In vitro investigations revealed that compared with unmodified HeLa cells, modified cells not only resist the adsorption of nonspecific proteins such as fibrinogen and human serum albumin but also selectively bind to plasminogen in plasma while maintaining good cell viability and proliferative activity. More importantly, upon the activation of adsorbed plasminogen into plasmin, the modified cells exhibited remarkable fibrinolytic activity and were capable of promptly dissolving the primary thrombus formed on their surfaces. This research not only provides a novel approach for constructing transplantable cells with fibrinolytic activity but also offers a new perspective for effectively addressing the significant loss of transplanted cells caused by thrombosis.

Synthesis and Antiproliferative Effect of New Alkyne-Tethered Vindoline Hybrids Containing Pharmacophoric Fragments.

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.

Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation.

Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).

Are Terminal Alkynes Necessary for MAO-A/MAO-B Inhibition? A New Scaffold Is Revealed.

A versatile family of quaternary propargylamines was synthesized employing the KA2 multicomponent reaction, through the single-step coupling of a number of amines, ketones, and terminal alkynes. Sustainable synthetic procedures using transition metal catalysts were employed in all cases. The inhibitory activity of these molecules was evaluated against human monoaminoxidase (hMAO)-A and hMAO-B enzymes and was found to be significant. The IC50 values for hMAO-B range from 152.1 to 164.7 nM while the IC50 values for hMAO-A range from 765.6 to 861.6 nM. Furthermore, these compounds comply with Lipinski's rule of five and exhibit no predicted toxicity. To understand their binding properties with the two target enzymes, key interactions were studied using molecular docking, all-atom molecular dynamics (MD) simulations, and MM/GBSA binding free energy calculations. Overall, herein, the reported family of propargylamines exhibits promise as potential treatments for neurodegenerative disorders, such as Parkinson's disease. Interestingly, this is the first time a propargylamine scaffold bearing an internal alkyne has been reported to show activity against monoaminoxidases.

Utilization of Spontaneous Alkyne-Gold Self-Assembly Chemistry as an Alternative Method for Fabricating Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a promising class of biosensors which use structure-switching redox-labeled oligonucleotides (aptamers) codeposited with passivating alkanethiol monolayers on electrode surfaces to specifically bind and detect target analytes. Signaling in E-AB sensors is an outcome of aptamer conformational changes upon target binding, with the sequence of the aptamer imparting specificity toward the analyte of interest. The change in conformation translates to a change in electron transfer between the redox label attached to the aptamer and the underlying electrode and is related to analyte concentration, allowing specific electrochemical detection of nonelectroactive analytes. E-AB sensor measurements are reagentless with time resolutions of seconds or less and may be miniaturized into the submicron range. Traditionally these sensors are fabricated using thiol-on-gold chemistry. Here we present an alternate immobilization chemistry, gold-alkyne binding, which results in an increase in sensor lifetimes under ideal conditions by up to ∼100%. We find that gold-alkyne binding is spontaneous and supports efficient E-AB sensor signaling with analytical performance characteristics similar to those of thiol generated monolayers. The surface modification differs from gold-thiol binding only in the time and aptamer concentration required to achieve similar aptamer surface coverages. In addition, alkynated aptamers differ from their thiolated analogues only by their chemical handle for surface attachment, so any existing aptamers can be easily adapted to utilize this attachment strategy.

Rhodium-Promoted C-H Activation/Annulation between DNA-Linked Terminal Alkyne and Aromatic Acid: A Finding from the Selection Outcomes.

Inspired by previous selection outcomes, we investigated and developed a rhodium-promoted C-H activation/annulation reaction of DNA-linked terminal alkynes and aromatic acids. This reaction exhibits excellent efficiency with high conversions and a broad substrate scope. Most importantly, the unique DEL-compatible conditions provide a better scenario for yielding an isocoumarin scaffold compared to conventional organic reaction conditions, and this newly developed on-DNA method has confirmed its feasibility in preparing DNA-encoded libraries.

A CuSO4/Bicinchoninic acid/Reducing sugar based stable and non-ROS catalyst system for the CuAAC reaction in bioanalysis.

The limitations of commonly used sodium ascorbate-based catalyst system for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction include excess production of reactive oxygen species and rapid catalyst deactivation. In this study instead of using a highly active reducing agent, such as, sodium ascorbate, we chose reducing sugar as a mild reducing agent to build up the catalyst system for CuAAC reaction. Interestingly, the bicinchoninic acid (BCA) assay system containing reducing sugar satisfies the essential elements of the catalyst system for CuAAC reaction. We found that CuSO4/BCA/Reducing sugar system can catalyze the CuAAC reaction but with low yield. Rational analyses of various parameters in CuSO4/BCA/Glucose catalyst system suggested storage at room temperature might enhance the catalytic activity, which was proven to be the case. Importantly, the system remains stable at room temperature and minimal H2O2 was detected. Notably, our study showed that the coordination between the slow reduction of Cu(I) by reducing sugar and the selective chelation of Cu(I) by BCA is key to developing this system. The CuSO4/BCA/Reducing sugar catalyst system was successfully applied to various CuAAC reaction based bioanalyses, and it is suitable for the CuAAC reaction based bioanalyses that are sensitive to ROS or request long reaction time.

Thiol-yne click post-synthesis of phenylboronic acid-functionalized magnetic cyclodextrin microporous organic network for selective and efficient extraction of antiepileptic drugs.

Owing to their incomplete digestion in the human body and inadequate removal by sewage treatment plants, antiepileptic drugs (AEDs) accumulate in water bodies, potentially affecting the exposed humans and aquatic organisms. Therefore, sensitive and reliable detection methods must be urgently developed for monitoring trace AEDs in environmental water samples. Herein, a novel phenylboronic acid-functionalized magnetic cyclodextrin microporous organic network (Fe3O4@CD-MON-PBA) was designed and synthesized via the thiol-yne click post-modification strategy for selective and efficient magnetic solid-phase extraction (MSPE) of trace AEDs from complex sample matrices through the specific B-N coordination, π-π, hydrogen bonding, electrostatic, and host-guest interactions. Fe3O4@CD-MON-PBA exhibited a large surface area (118.5 m2 g-1), rapid magnetic responsiveness (38.6 emu g-1, 15 s), good stability and reusability (at least 8 times), and abundant binding sites for AEDs. Under optimal extraction conditions, the proposed Fe3O4@CD-MON-PBA-MSPE-HPLC-UV method exhibited a wide linear range (0.5-1000 µg L-1), low limits of detection (0.1-0.5 µg L-1) and quantitation (0.3-2 µg L-1), good anti-interference ability, and large enrichment factors (92.2-104.3 to 92.3-98.0) for four typical AEDs. This work confirmed the feasibility of the thiol-yne click post-synthesis strategy for constructing novel and efficient multifunctional magnetic CD-MONs for sample pretreatment and elucidated the significance of B-N coordination between PBA and N-containing AEDs.

Incorporation of Intracellular NanoSIMS Tracers to Oligonucleotide Conjugates via Strain Promoted Sydnone-Alkyne Cycloaddition.

Extensive efforts have been dedicated to developing cell-specific targeting ligands that can be conjugated to therapeutic cargo, offering a promising yet still challenging strategy to deliver oligonucleotide therapeutics beyond the liver. Indeed, while the cargo and the ligand are crucial, the third component, the linker, is integral but is often overlooked. Here, we present strain-promoted sydnone-alkyne cycloaddition as a versatile linker chemistry for oligonucleotide synthesis, expanding the choices for bioconjugation of therapeutics while enabling subcellular detection of the linker and payload using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging. This strategy was successfully applied to peptide and lipid ligands and profiled using the well characterized N-acetylgalactosamine (GalNAc) targeting ligand. The linker did not affect the expected activity of the conjugate and was detectable and distinguishable from the labeled cargo. Finally, this work not only offers a practical bioconjugation method but also enables the assessment of the linker's subcellular behavior, facilitating NanoSIMS imaging to monitor the three key components of therapeutic conjugates.

3D printed dispersible efavirenz tablets: A strategy for nasogastric administration in children.

Enteral feeding tubes (EFTs) can be placed in children diagnosed with HIV which need nutritional support due to malnutrition. EFTs are the main route for medication administration in these patients, bringing up concerns about off label use of medicines, dose inaccuracy and tube clogging. Here we report for the first time the use of selective laser sintering (SLS) 3D printing to develop efavirenz (EFZ) dispersible printlets for patients with HIV that require EFT administration. Water soluble polymers Parteck® MXP and Kollidon® VA64 were used to obtain both 500 mg (P500 and K500) and 1000 mg printlets (P1000 and K1000) containing 200 mg of EFZ each. The use of SLS 3D printing obtained porous dosage forms with high drug content (20 % and 40 % w/w) and drug amorphization using both polymers. P500, K500 and K1000 printlets reached disintegration in under 230 s in 20 mL of water (25 ± 1 °C), whilst P1000 only partially disintegrated, possibly due to saturation of the polymer in the medium. As a result, the development of dispersible EFZ printlets using hydrophilic polymers can be explored as a potential strategy for drug delivery through EFTs in paediatrics with HIV, paving the way towards the exploration of more rapidly disintegrating polymers and excipients for SLS 3D printing.

Efficient conversion of hemoglobin to a non-vasoactive oxygen carrier by site-specific cross-linking with azido acyl methyl phosphates followed by bio-orthogonal CuAAC with a bis-alkyne.

While cross-linked hemoglobin tetramers are functional acellular oxygen carriers, their ability to scavenge endogenous nitric oxide (NO) by endothelial pore penetration results in adverse cardiovascular effects. Animal studies established that cross-linked human hemoglobins, chemically joined into a double protein, avoid NO scavenging, presumably due to their larger size preventing penetration into endothelial regions that produce NO. In the present report, we utilize azide-containing acyl phosphate reagents to form cross-linked hemoglobins then bio-orthogonally click-couple them with a bis-alkyne (CuAAC). The production of these larger oxygen-carrying hemoglobin conjugates is obtained in high yields through subunit-specific cross-linking between each ßLys82 ε-amino group. The methyl phosphate leaving groups provide electrostatically induced ß-subunit site-selectivity, producing azido-cross-linked hemoglobin that undergoes highly efficient CuAAC compared with previous cross-linkers. The acyl phosphates also efficiently cross-link both T-state and R-state hemoglobin. The resulting bis- and tris-tetrameric hemoglobin conjugates exhibit oxygen affinity and cooperativity that are comparable to those of the native protein. The hemoglobin derivatives from the process we describe can function as sources of oxygen in biomedical applications, such as in ex-vivo donor organ perfusion.

Super-Resolution Imaging of Clickable Lipids With Lipid Expansion Microscopy (LExM).

Fluorescent imaging of cellular membranes is challenged by the size of lipid bilayers, which are smaller than the diffraction limit of light. Recently, expansion microscopy (ExM) has emerged as an approachable super-resolution method that requires only widely accessible confocal microscopes. In this method, biomolecules of interest are anchored to hydrogel-based, polymeric networks that are expanded through osmosis to physically separate and resolve features smaller than the diffraction limit of light. Whereas ExM has been employed for super-resolution imaging of proteins, DNA, RNA, and glycans, the application of this method to the study of lipids is challenged by the requirement of permeabilization procedures that remove lipids and compromise the integrity of the membrane. Here, we describe our recently developed protocols for lipid expansion microscopy (LExM), a method that enables ExM of membranes without permeabilization. These detailed protocols and accompanying commentary sections aim to make LExM accessible to any experimentalist interested in imaging membranes with super-resolution. © 2024 Wiley Periodicals LLC. Basic Protocol 1: LExM of alkyne-choline lipids Basic Protocol 2: LExM of IMPACT-labeled lipids Basic Protocol 3: LExM of clickable cholesterol Basic Protocol 4: Determining the expansion factor.

Multivalent dendritic DNA aptamer molecules for the enhancement of therapeutic effects.

Dendritic DNA molecules, referred to as DNA dendrons, consist of multiple covalently linked strands and are expected to improve the cellular uptake and potency of therapeutic oligonucleotides because of their multivalency. In this study, we developed an efficient synthetic method for producing DNA dendrons using strain-promoted azide-alkyne cycloaddition. Integration of the antitumor aptamer AS1411 into DNA dendrons enhanced cellular uptake and antiproliferative activity in cancer cells. These findings demonstrate that the incorporation of multivalent aptamers into DNA dendrons can effectively boost their therapeutic effects.

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.

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.

Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson-Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann-Ohira reaction for the generation of the alkyne moiety.

Functionalization of Siloxanes with Arynes Generated from o-Triazenylarylboronic Acids.

Herein, we report the functionalization of polyhedral oligosilsesquioxanes (POSS) and related siloxanes with arynes. Using o-triazenylarylboronic acids as aryne precursors and silica gel as the activator, the transformation of siloxane bearing various arynophilic moieties on the side chains was achieved with high yields without touching the siloxane core. This method was applied to the conjugation of POSS and pharmaceutical cores using an aryne derived from the synthetic intermediate of cabozantinib. Furthermore, orthogonal dual functionalization of POSS was realized by combining the aryne reaction with Huisgen cyclization.

Introducing terminal alkyne groups at the reducing end of cellulose nanocrystals by aldimine condensation for further click reaction.

In recent years, click reactions with cellulose nanocrystals (CNC) participation have gradually become a research hotspot. Carboxylamine condensation is the most used method to introduce terminal alkyne groups at the reducing end of CNC as reaction sites for click reactions. However, hydroxyl groups on CNC surface would be slightly oxidized during the carboxyamine condensation process, inducing the potential positions of introduced alkynes would be not only at the reducing end but also on CNC surface. Here, aldimine condensation was proposed to introduce terminal alkyne groups just at the reducing end of CNC, and a systematic comparison analysis was conducted with carboxylamine condensation. Firstly, the selectivity and extent of alkynylation were characterized by XPS and EA. Secondly, the end aldehyde content in these CNC samples was measured by the BCA method, which quantitatively explained the grafting efficiency of aldimine condensation and further verified its feasibility. Thirdly, the clickability of the modified CNC samples was confirmed through XPS analysis of the products after a pre-designed click reaction. In sum, aldimine condensation was proven to be a simple and effective strategy for introducing terminal alkyne groups at the reducing end of CNC, which could be used as reaction sites for further click reactions.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

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.