In-situ activation of biomimetic single-site bioorthogonal nanozyme for tumor-specific combination therapy.

Copper-catalyzed click chemistry offers creative strategies for activation of therapeutics without disrupting biological processes. Despite tremendous efforts, current copper catalysts face fundamental challenges in achieving high efficiency, atom economy, and tissue-specific selectivity. Herein, we develop a facile "mix-and-match synthetic strategy" to fabricate a biomimetic single-site copper-bipyridine-based cerium metal-organic framework (Cu/Ce-MOF@M) for efficient and tumor cell-specific bioorthogonal catalysis. This elegant methodology achieves isolated single-Cu-site within the MOF architecture, resulting in exceptionally high catalytic performance. Cu/Ce-MOF@M favors a 32.1-fold higher catalytic activity than the widely used MOF-supported copper nanoparticles at single-particle level, as first evidenced by single-molecule fluorescence microscopy. Furthermore, with cancer cell-membrane camouflage, Cu/Ce-MOF@M demonstrates preferential tropism for its parent cells. Simultaneously, the single-site CuII species within Cu/Ce-MOF@M are reduced by upregulated glutathione in cancerous cells to CuI for catalyzing the click reaction, enabling homotypic cancer cell-activated in situ drug synthesis. Additionally, Cu/Ce-MOF@M exhibits oxidase and peroxidase mimicking activities, further enhancing catalytic cancer therapy. This study guides the reasonable design of highly active heterogeneous transition-metal catalysts for targeted bioorthogonal reactions.

Click Chemistry in Detecting Protein Modification.

Click chemistry, also known as "link chemistry," is an important molecular connection method that can achieve simple and efficient connections between specific small molecular groups at the molecular level. Click chemistry offers several advantages, including high efficiency, good selectivity, mild conditions, and few side reactions. These features make it a valuable tool for in-depth analysis of various protein posttranslational modifications (PTMs) caused by changes in cell metabolism during viral infection. This chapter considers the palmitoylation, carbonylation, and alkylation of STING and presents detailed information and experimental procedures for measuring PTMs using click chemistry.

Facile fabrication of a thermal/pH responsive IPN hydrogel drug carrier based on cellulose and chitosan through simultaneous dual-click strategy.

Herein, an interpenetrating network hydrogel (IPN-Gel) based on cellulose and chitosan was synthesized via simultaneous amino-anhydride and azide-alkyne click reaction in water in one pot. The samples were characterized by various analytical methods including FTIR, SEM, XRD, XPS, 1H NMR and so forth. The fabrication conditions were optimized by single factor experiments with water uptake (WU) and gel mass fraction (GMF) as two indexes. The WU and GMF of the IPN-Gel prepared under optimized conditions were 1192.37 % and 74.00 %, respectively. Its WU descended with the ascension in temperature, and first descended and then gradually ascended with the ascension in pH, confirming that the IPN-Gel had thermal/pH dual responsiveness. Using 5-Fu as a model drug, the release behavior of 5-Fu in IPN-Gel was explored. Its release behavior could be regulated by changing temperature and pH values, and it followed the Korsmeyer Peppas model. The viability of 4 T1 cells and HUVEC cells exceeded 80 % after 48 h of incubation at a high concentration of 200 µg/mL IPN-Gel, and hemolytic percentage was below the allowed limit of 5 %. The study provides a new strategy for the preparation of the IPN-Gel with biocompatibility, swelling reversibility and controllable drug release.

Rapid preparation of injectable dual-network hydrogels for biomedical applications using UV-triggered sulfhydryl click reactions.

The use of hydrogels to mimic natural cartilage implantation can effectively solve the current problems of insufficient cartilage donors and low rate of injury healing. In particular, injectable hydrogels are less invasive in clinical applications and better able to fill uneven injury surfaces. Here, we prepared NorCS and CS-SH by modifying chitosan with 5-norbornene-2-carboxylic acid and N-Acetyl-L-cysteine, respectively. Dual-network hydrogels were prepared by using UV-triggered thiol-ene click reaction between NorCS and CS-SH and the metal coordination between SA and Ca2+. The prepared hydrogels can be cross-linked quickly and exhibit excellent degradability, self-healing and injectable properties. At the same time, the hydrogel also showed good cytocompatibility and could significantly restore the motor function of mice. This study provides an effective strategy for preparing injectable hydrogels capable of rapid cross-linking.

Stretchable Heat Transfer Eco-Materials: Mesogen Grafted NR-Based Nanocomposites with High Thermal Conductivity and Low Dielectric Constant.

Biomass-based functional polymers have received significant attention across various fields, in view of eco-friendly human society and sustainable growth. In this context, there are efforts to functionalize the biomass polymers for next-generation polymer materials. Here, stretchable heat transfer materials are focused on which are essential for stretchable electronics and future robotics. To achieve this goal, natural rubber (NR) is chemically modified with a thiol-terminated phenylnaphthalene (TTP), and then utilized as a thermally conductive NR (TCNR) matrix. Hexagonal boron nitride (h-BN), renowned for its high thermal conductivity and low electrical conductivity, is incorporated as a filler to develop stretchable heat transfer eco-materials. The optimized TCNR/h-BN composite elongates to 140% due to great elasticity of NR, and exhibits excellent dielectric properties (a low dielectric constant of 2.26 and a low dielectric loss of 0.006). Furthermore, synergetic phonon transfer of phenylnaphthalene crystallites and h-BN particles in the composite results in a high thermal conductivity of 0.87 W m-1 K-1. The outstanding thermal, mechanical, and dielectric properties of the newly developed TCNR/h-BN composite enable the successful demonstration as stretchable and shape-adaptable thermal management materials.

Visualization of lignification in flax stem cell walls with novel click-compatible monolignol analogs.

Introduction: As an essential part of plant cell walls, lignin provides mechanical support for plant growth, enhances water transport, and helps to defend against pathogens. As the most abundant natural aromatic-based renewable resource on earth, its biosynthesis has always been a research focus, and it is still currently under study. Methods: In this study, the p-coumaryl alcohol analog (HALK) and the coniferyl alcohol analog (GALK) containing an alkyne group at the ortho position were synthesized and applied to lignification in vivo and in vitro. The incorporation of these novel lignin monomers was observed via fluorescence imaging. Results and Discussion: It was found that the two monolignol analogs could be incorporated in dehydrogenated polymers (DHPs) in vitro and in flax cell walls in vivo. The results showed that as the cultivation time and precursor concentration varied, the deposition of H and G-type lignin exhibited differences in deposition mode. At the subcellular scale, the deposited lignin first appears in the cell corner and the middle lamella, and then gradually appears on the cell walls. Furthermore, lignin was also found in bast fiber. It was demonstrated that these new molecules could provide high-resolution localization of lignin during polymerization.

Thiol-chromene click reaction-triggered mitochondria-targeted ratiometric fluorescent probe for intracellular biothiol imaging.

Chromene as the efficient biothiol recognition site was widely used to develop fluorescent probes based on thiol-chromene click reaction. However, chromene-based fluorescent probes with the both properties of ratiometric measurement and mitochondria-targeted function have not been reported and remain challenging. In this paper, we skillfully designed and synthesized the first mitochondria-targeted ratiometric fluorescent probe (Probe 1) for biothiols based on chromene. Upon addition of biothiols (Cys, Hcy, and GSH), the absorption and fluorescence spectra of Probe 1 changed from 490 to 426 nm and from 567 to 498 nm respectively, accompanied by color changes from orange to pale yellow under natural light and from orange to blue under a 365-nm UV lamp, which can be attributed to the click reaction of biothiols with α,ß-unsaturated ketone of chromene moiety, subsequent pyran ring-opening, and phenol formation as well as 1,6-elimination of p-hydroxybenzyl moiety. Probe 1 not only exhibited high sensitivity (LODs of 149 nM, 133 nM, and 116 nM for Cys, GSH, and Hcy respectively), rapid response, and excellent selectivity for biothiols (Cys, Hcy, and GSH), but also could target in mitochondria and ratiometrically image the fluctuation of intracellular biothiols. Moreover, the novel design strategy of modifying chromene to the N atom of pyridine was proposed for the first time.

Macroporous PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates Prepared via Radical-Free Cross-Linking for Cartilage Tissue Engineering.

Trauma or repeated damage to joints can result in focal cartilage defects, significantly elevating the risk of osteoarthritis. Damaged cartilage has an inherently limited self-healing capacity and remains an urgent unmet clinical need. Consequently, there is growing interest in biodegradable hydrogels as potential scaffolds for the repair or reconstruction of cartilage defects. Here, we developed a biodegradable and macroporous hybrid double-network (DN) cryogel by combining two independently cross-linked networks of multiarm polyethylene glycol (PEG) acrylate and alginate.Hybrid DN cryogels are formed using highly biocompatible click reactions for the PEG network and ionic bonding for the alginate network. By judicious selection of various structurally similar cross-linkers to form the PEG network, we can generate hybrid DN cryogels with customizable degradation kinetics. The resulting PEG-alginate hybrid DN cryogels have an interconnected macroporous structure, high mechanical strength, and rapid swelling kinetics. The interconnected macropores in the cryogels support efficient mesenchymal stem cell infiltration at a high density. Finally, we demonstrate that PEG-alginate hybrid DN cryogels allow sustained release of chondrogenic growth factors and support chondrogenic differentiation of mouse mesenchymal stem cells. This study provides a novel method to generate macroporous hybrid DN cryogels with customizable degradation rates and a potential scaffold for cartilage tissue engineering.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst.

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide-alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min-1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Hoechst modification by strain-promoted azide-alkyne cycloaddition for transport of functional molecules into the cell nucleus.

The delivery of functional molecules to the cell nucleus enables the visualization of nuclear function and the development of effective medical treatments. In this study, we successfully modified the Hoechst molecule, which is a well-documented nuclear-staining agent, using the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We prepared Hoechst derivatives bearing an azide group (Hoe-N3) and characterized their SPAAC reactions in the presence of corresponding molecules with a dibenzylcyclooctyne unit (DBCO). The SPAAC reaction of Hoe-N3 with alkylamine bearing DBCO, fluorescent TAMRA, or Cy5 molecules bearing DBCO led to the formation of the coupling products Hoe-Amine, Hoe-TAMRA, and Hoe-Cy5, respectively. These Hoechst derivatives retained their DNA-binding properties. In addition, Hoe-TAMRA and Hoe-Cy5 exhibited properties of dual accumulation in the cell nucleus and mitochondria. Initial incubation of these molecules in living cells resulted in its accumulation in mitochondria, while after mitochondrial depolarization, it was smoothly released from mitochondria and translocated into the cell nucleus. Thus, mitochondrial depolarization could be monitored by measuring the emission of Hoe-TAMRA and Hoe-Cy5 at the cell nucleus.

Antibody-Directing Antibody Conjugates (ADACs) Enabled by Orthogonal Click Chemistry for Targeted Intracellular Delivery.

Using orthogonal click chemistries for efficient nanoscale self-assembly, a new antibody-directing antibody conjugate (ADAC) nanogel is generated. In this system, one of the antibodies is displayed on the nanogel surface to specifically recognize cell-surface epitopes while the other antibody is encapsulated inside the nanogel core. The system is programmed to release the latter antibody in its functional form in the cytosolic environment of a specific cell to engage intracellular targets. ADACs offer a potential solution to harness the advantages seen with antibody-drug conjugates (ADCs) to deliver therapeutic cargos to specific tissues, but with the added capability of carrying biologics as the cargo. In this manuscript, this potential is demonstrated through delivery of antibodies against intracellular targets in specific cells. This platform offers new avenues for precise therapeutic interventions and the potential to address previously "undruggable" cellular targets.

Surface Passivation of Norland Optical Adhesive Improves the Guiding Efficiency of Gliding Microtubules in Microfluidic Devices.

The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system's application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.

Structurally Defined Amphiphilic AAO Membranes Using UV-Assisted Thiol-Yne Chemistry: Applications in Anti-Counterfeiting and Electronics.

In this study, we fabricate and characterize amphiphilic anodic aluminum oxide (AAO) membranes using UV-triggered thiol-yne click reactions and photomasks for various innovative applications, including driven polymer nanopatterns, anti-counterfeiting, and conductive pathways. Specifically, we synthesize 10-undecynyl-terminated-AAO membranes and subsequently prepare amphiphilic AAO membranes with superhydrophilic and superhydrophobic regions. Various analytical methods, including grazing angle X-ray photoelectron spectroscopy (GIXPS), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), nanofocused synchrotron X-ray techniques (nano-XRD and nano-XRF), and water contact angle measurements, confirm the modifications and distinct properties of the modified areas. This work achieves a series of applications, such as driven polymer nanopatterns, solvent- and light-triggered anti-counterfeiting, and region-selective conductive pathways using silver paint with lower resistivity. Besides, the amphiphilic AAO membrane exhibits successful stability, durability, and reusability. To sum up, this study highlights the versatility and potential of amphiphilic AAO membranes in advanced material design and smart applications.

Synthesis and Characterization of Thiol-Ene Networks Derived from Levoglucosenone.

Levoglucosenone (LGO), a renewable compound obtained from cellulose biomass, has been utilized to prepare novel monomers bearing alkene functional groups. These monomer derivatives of LGO were subsequently cured via ultraviolet (UV)-initiated radical thiol-ene "click" chemistry with commercially available multifunctional thiols to obtain colourless, optically transparent cross-linked thermosets. The monomers prepared in this work are unique due to utilising the internal double bond of the LGO ring during polymerization as part of the cross-linked network. The thermal and mechanical properties along with the degradation of thermosets containing both ether and ester linkages within the LGO monomers were studied. These thermosets had tensile strengths of 1.3-3.3 MPa, glass transition temperatures between 23.2 and 27.2 °C, and good thermal stability of up to 300 °C.

Eco-friendly electrosynthesis of Cu-PANI-pencil composite: Enhanced their current value, acid sensing, click chemistry and turn on-off sunlight photo-catalytical H2O2 activation.

In this contribution, we report a straightforwardly and easily one-step synthesis of a small family of composites based in polyaniline grafted on HB2 graphite (PANI@UG) and their copper-doped derivatives (Cu50PANI@UG5-6). The PANI@UG composites were synthesized through electrochemical polymerization using cyclic voltammetry (CV) in three different acidic media: i) acetic acid (AcOH) at high and low concentration (12 and 1 M, using KCl as electrolytic support); ii) a mixture of AcOH and sulfuric acid (H2SO4, which have two roles: as electrolytic support and proton source) and iii) a mixture of acetonitrile (NCCH3) and H2SO4, under atmospheric conditions. Once the best conditions were achieved, our next step was focused on obtaining the Cu50PANI@UG5-6 composites using a solution of aniline and CuSO4 (50 mM) in AcOH:H2SO4 and NCCH3:H2SO4 solutions, respectively. All composites were characterized by CV, FT-IR, SEM and MALDI-TOF experiments. So, the current value was enhanced for the Cu50PANI@UG6 composite, which have three potential catalytical applications in: i) HClO4 acid sensing, ii) click chemistry and iii) sunlight drive photo-activation of H2O2.

Investigation of relationships between metabolic chemical reporter structures and S-glyco-modification effects.

The approach of metabolic chemical reporters (MCRs) for labeling proteins has been widely used in the past several decades. Nevertheless, artificial side reaction generated with fully protected MCRs, termed S-glyco-modification, occurs with cysteine residues through base-promoted ß-elimination and Michael addition, leading to false positives in the proteomic identification. Therefore, next generation of MCRs, including partially protected strategy and modifications on the backbone of monosaccharides, have emerged to improve the labeling efficiency. In this paper, we prepared fifteen kinds of unnatural monosaccharides to investigate the relationships of structures and S-glyco-modification labeling. Our results demonstrated that Ac4GlcNAz and Ac4GalNAz exhibited the most remarkable labeling effects among the detected compounds. Of note, Ac4ManNAz, Ac46AzGlucose and Ac46AzGalactose containing similar structures but did not show similar robust signals as them. Moreover, other modifications on the 1-, 2-, 3-, 4- and 6-site indicated minimal side reactions of S-glyco-modification, raising a possibility that subtle modifications of monosaccharide substrate may alter its role in the process of biosynthesis, for example, by change of electronegativity or enhancement of steric hindrance effects. In conclusion, our discoveries provide a new avenue to choose appropriate probe for selective label proteins in vitro and in vivo without undesired S-glyco-modification.

Facile synthesis of a new chiral polyimine macrocycle and its application for enantioseparation in high-performance liquid chromatography.

Macrocyclic compounds such as crown ethers and cyclodextrins play an important role in the field of chromatography and show excellent separation performance. The design of simple and convenient methods for the efficient synthesis of novel chiral macrocycles for chromatographic separation is of great significance. In this work, a novel chiral polyimine macrocycle (PIMC) was designed and synthesized by the simply one-step reaction of 2,6-diformyl-4-tert-butylphenol with (S)-(-)-1,2-propanediamine. Then, it was bonded onto silica by the thiol-ene click reaction to construct a new chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC). The chiral separation performance of the proposed CSP was examined by separating various racemates in the normal-phase (NP) and reversed-phase (RP) HPLC. In total, twelve and nine racemates, including ethers, esters, amines, alcohols, organic acids, ketones, and epoxides, were separated to varying degrees via NP-HPLC and RP-HPLC, respectively, Moreover, the CSP offered good chiral separation complementarity to Chiralcel OD-H and Chiralpak AD-H columns for resolution of these test racemates, and it can separate several racemic compounds that either cannot be separated or cannot be separated well be separated by the two commercially available columns. After the column was used for hundreds of injections, the relative standard deviations of the retention time and resolution were below 0.56 % and 0.45 %, respectively, showing the good reproducibility and stability of the CSP. This study provides a simple and convenient approach to synthesize a novel chiral macrocycle and CSP and also indicates the broad application prospects of such chiral PIMCs in HPLC chiral separation.

Deciphering the cell surface sugar-coating via Biochemical Pathways.

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i.e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol-anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and the advancement of glycan-targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid-based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications.

A Comparative Analysis of Click ABR and Multi-ASSR in Assessing Infant Hearing: A Cross-Sectional Study.

Introduction: This study investigates the comparative effectiveness of Click Auditory Brainstem Response (Click ABR) and Multiple Auditory Steady-State Response (Multi-ASSR) in identifying hearing impairments in infants. Recognizing auditory issues early is crucial for a child's cognitive and language development, as emphasized by the Joint Committee on Infant Hearing (JCIH) and the American Academy of Audiology (AAA). While Click ABR is widely utilized, Multi-ASSR offers a modern technique for detailed hearing assessment. Methods: A comparative analysis was conducted on 111 infants aged 1-6 months, previously screened for hearing at a tertiary care centre. The study employed both Click ABR and Multi-ASSR to evaluate their respective efficacy in assessing infant hearing. Results: Click ABR detected normal hearing in 87.4% of the infants, slightly higher than Multi-ASSR's 84.7%. A noteworthy finding was the higher incidence of bilateral versus unilateral hearing loss, with Click ABR identifying bilateral loss in 10 infants and unilateral loss in 4, compared to Multi-ASSR, which found bilateral loss in 12 infants and unilateral loss in 5. There was a minor but significant difference in auditory thresholds between the methods, with a mean discrepancy of 1.2 dB and a significant statistical variance (t-value of 15; p < 0.001), indicating variations in sensitivity. Conclusion: Both Click ABR and Multi-ASSR are indispensable tools in paediatric audiology, each with unique advantages. Click ABR excels in efficiency, suitable for rapid assessments and early detection. In contrast, Multi-ASSR offers comprehensive frequency-specific data, facilitating thorough evaluations. Healthcare professionals must grasp these methods' strengths to optimize infant hearing screenings and enhance early intervention strategies, aligning with JCIH and AAA guidelines. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-024-04639-2.

Preparation and evaluation of a 1,1'-bi-2-naphthol-based chiral macrocycle bonded silica chiral stationary phase for high performance liquid chromatography.

Macrocycles play vital roles in supramolecular chemistry and chromatography. 1,1'-Bi-2-naphthol (BINOL)-based chiral polyimine macrocycles are an emerging class of chiral macrocycles that can be constructed by one-step aldehyde-amine condensation of BINOL derivatives with other building blocks. These macrocycles exhibit good characteristics, such as facile preparation, rigid cyclic structures, multiple chiral centers, and defined molecular cavities, that make them good candidates as new chiral recognition materials for chromatographic enantioseparations. In this study, a BINOL-based [2+2] chiral polyimine macrocycle was synthesized by one-step condensation of enantiopure (S)-2,2'-dihydroxy-[1,1'-binaphthalene]-3,3'-dicarboxaldehyde with (1R,2R)-1,2-diaminocyclohexane. The product was modified with 5-bromo-1-pentene and then attached to thiolated silica using click chemistry to construct a new chiral stationary phase (CSP). The enantioselectivity of the new CSP was explored by separating various racemates under normal phase (NP) and reversed phase (RP) high performance liquid chromatography (HPLC). Thirteen racemates and eight racemates were enantioseparated under the two separation modes, respectively, including chiral alcohols, phenols, esters, ketones, amines, and organic acids. Among them, nine racemates achieved baseline separation under NP-HPLC and seven racemates achieved baseline separation under RP-HPLC. High resolution separation was observed with benzoin (Rs = 5.10), epinephrine (Rs = 4.98), 3-benzyloxy-1,2-propanediol (Rs = 4.42), and 4,4'-dimethylbenzoin (Rs = 4.52) in NP-HPLC, and with 4-methylbenzhydrol (Rs = 4.72), benzoin ethyl ether (Rs = 3.79), 1-phenyl-1-pentanol (Rs = 3.68), and 1-(3-bromophenyl)ethanol (Rs = 3.60) in RP-HPLC. Interestingly, the CSP complemented Chiralcel OD-H, Chiralpak AD-H, and CYCLOBOND I 2000 RSP columns for resolution of these test racemates, separating several racemic compounds that could not be well separated by the three commercially available columns. The influences of injected sample amount on separation were also evaluated. It was found that the column exhibited excellent stability and reproducibility after hundreds of injections, and the relative standard deviations (n = 5) of the retention time and resolution were less than 0.49% and 0.69%, respectively. This study indicates that the BINOL-based chiral macrocycle has great potential for HPLC enantioseparation.