Modification Of Ring Research Articles

Dynamic features of virus protein 1 and substitutions in the 3-phenyl ring determine the potency and broad-spectrum activity of capsid-binding pyrazolo[3,4-d]pyrimidines against rhinoviruses

Pyrazolo[3,4-d]pyrimidines represent one potent class of well tolerated and highly active rhinovirus (RV) inhibitors that act as capsid binders. The lead compound OBR-5-340 inhibits a broad-spectrum of RVs. Aiming to improve lead activity, we evaluated the impact of structural modifications in the 3-phenyl ring of OBR-5-340 on its potency and spectrum of anti-RV activity vitro. Our results demonstrate the crucial role of substitution at position 4 for strong, broad-spectrum anti-RV activity. The 4-methyl (RCB23137) and 4-chloro (RCB23138) derivatives outperformed OBR-5-340 in terms of potency and anti-RV activity spectrum. Based on these findings, the compounds were selected for computational binding studies. Molecular dynamic simulations with six RVs differing in OBR-5-340, RCB23137, and RCB23138 sensitivity proved the impact of dynamic features of two VP1 loops enveloping these inhibitors on antiviral potency.

In silico Approaches for Exploring the Pharmacological Activities of Benzimidazole Derivatives: A Comprehensive Review.

This article reviews computational research on benzimidazole derivatives. Cytotoxicity for all compounds against cancer cell lines was measured and the results revealed that many compounds exhibited high inhibitions. This research examines the varied pharmacological properties like anticancer, antibacterial, antioxidant, anti-inflammatory and anticonvulsant activities of benzimidazole derivatives. The suggested method summarises In silico research for each activity. This review examines benzimidazole derivative structure-activity relationships and pharmacological effects. In silico investigations can anticipate structural alterations and their effects on these derivative's pharmacological characteristics and efficacy through many computational methods. Molecular docking, molecular dynamics simulations and virtual screening help anticipate pharmacological effects and optimize chemical design. These trials will improve lead optimization, target selection, and ADMET property prediction in drug development. In silico benzimidazole derivative studies will be assessed for gaps and future research. Prospective studies might include empirical verification, pharmacodynamic analysis, and computational methodology improvement. This review discusses benzimidazole derivative In silico research to understand their specific pharmacological effects. This will help scientists design new drugs and guide future research. Latest, authentic and published reports on various benzimidazole derivatives and their activities are being thoroughly studied and analyzed. The overview of benzimidazole derivatives is more comprehensive, highlighting their structural diversity, synthetic strategies, mechanisms of action, and the computational tools used to study them. In silico studies help to understand the structure-activity relationship (SAR) of benzimidazole derivatives. Through meticulous alterations of substituents, ring modifications, and linker groups, this study identified the structural factors influencing the pharmacological activity of benzimidazole derivatives. These findings enable the rational design and optimization of more potent and selective compounds.

Sequential Modification of Pyrrole Ring with up to Three Different Nucleophiles.

An umpolung strategy was used for the preparation of highly functionalized 3-pyrrolin-2-ones. This approach involves dearomative double chlorination of 1H-pyrroles to form highly reactive dichloro-substituted 2H-pyrroles. The resulting intermediate reacts selectively with wet alcohols to form the corresponding alkoxy-substituted 3-pyrrolin-2-ones via double nucleophilic substitution in up to 99% yield. The subsequent reaction with different N-, O-, and S-nucleophiles opens access to highly functionalized pyrrolinones bearing additional functionality. The overall outcome of the reported sequence is step-by-step nucleophilic modification of pyrroles with three different nucleophiles. All steps were found to be highly efficient and 100% regioselective. This transformation proceeds under mild conditions and does not require any catalyst to give final products in very high yields. The obtained experimental results are in perfect agreement with the data obtained by theoretical investigation of these reactions.

Hyperthermia Intensifies α-Mangostin and Synthetic Xanthones' Antimalignancy Properties.

In order to improve naturally occurring xanthones' anticancer properties, chemical synthesis is proposed. In this study, from eight novel xanthone derivatives coupled to morpholine or aminoalkyl morpholine, only the two most active ones were chosen. For additional enhancement of the anticancer activity of our tested compounds, we combined chemotherapy with hyperthermia in the range of 39-41 °C, from which the mild conditions of 39 °C were the most influencing. This approach had a profound impact on the anticancer properties of the tested compounds. TOV-21G and SC-OV-3 ovarian cell line motility and metastasis behavior were tested in native and hyperthermia conditions, indicating decreased wound healing properties and clonogenic activity. Similarly, the expression of genes involved in metastasis was hampered. The expression of heat shock proteins involved in cancer progression (Hsc70, HSP90A, and HSP90B) was significantly influenced by xanthone derivatives. Chemotherapy in mild hyperthermia conditions had also an impact on decreasing mitochondria potential, visualized with JC-1. Synthetic xanthone ring modifications may increase the anticancer activity of the obtained substances. Additional improvement of their activity can be achieved by applying mild hyperthermia conditions. Further development of a combined anticancer therapy approach may result in increasing currently known chemotherapeutics, resulting in a greater recovery rate and diminishment of the cytotoxicity of drugs.

Investigation Into Novel Mukanadin B, Mukanadin D and Mukanadin F Derivatives as Antagonists of 5-HT1A Signalling.

Marine bromopyrrole alkaloids are a diverse family of natural products with a large array of biological applications. The mukanadin family is a group of molecules consisting of seven members (mukanadin A-G) that possess a range of biological activities. Inhibition of serotonergic signaling has been demonstrated by mukanadin B derivatives, presenting this chemical scaffold as a candidate for further SAR exploration. A library of thirteen novel mukanadin B and D derivatives with structural variation targeted at the pyrrole ring, central linker and hydantoin ring, were synthesized. These analogues were subsequently assessed for serotonergic antagonism, in addition to natural products, mukanadin B, D, F and 9-hydroxy mukanadin B. A collection of compounds exhibited significant 5-HT1A signaling, including five of the novel derivatives and two of the naturally occurring bromopyrroles, mukanadin B and F. Particular SAR information could be determined from these results, such as modification of the pyrrole ring being a well-tolerated strategy for improving serotonergic inhibition. Other changes to the pharmacophore led to significant reduction in activity such as saturation of the linker region, or no conclusive improvement in inhibitory activity such as a 9-OH group or replacement of the hydantoin ring with a triazole moiety.

Comparative effectiveness of nitro dihydrocapsaicin, new synthetic derivative capsaicinoid, and capsaicin in alleviating oxidative stress and inflammation on lipopolysaccharide-stimulated corneal epithelial cells

Loss of tear homeostasis, characterized by hyperosmolarity of the ocular surface, induces cell damage through inflammation and oxidation. Transient receptor potential vanilloid 1 (TRPV1), a sensor for osmotic changes, plays a crucial role as a calcium ion channel in the pathogenesis of hypertonic-related eye diseases. Capsaicin (CAP), a potent phytochemical, alleviates inflammation during oxidative stress events by activating TRPV1. However, the pharmacological use of CAP for eye treatment is limited by its pungency. Nitro dihydrocapsaicin (NDHC) was synthesized with aromatic ring modification of CAP structure to overcome the pungent effect. We compared the molecular features of NDHC and CAP, along with their biological activities in human corneal epithelial (HCE) cells, focusing on antioxidant and anti-inflammatory activities. The results demonstrated that NDHC maintained cell viability, cell shape, and exhibited lower cytotoxicity compared to CAP-treated cells. Moreover, NDHC prevented oxidative stress and inflammation in HCE cells following lipopolysaccharide (LPS) administration. These findings underscore the beneficial effect of NDHC in alleviating ocular surface inflammation, suggesting that NDHC may serve as an alternative anti-inflammatory agent targeting TRPV1 for improving hyperosmotic stress-induced ocular surface damage.

Synchrotron x-ray spectra characterisation for radiation therapy applications at the ESRF - ID17 biomedical beamline

Objective. Radiation therapy requires reliable dosimetry protocols to deliver successful treatments with high accuracy and precision. In this context, accurate knowledge of the beam’s energy spectra is mandatory. The goal of this study was to validate the synchrotron x-ray spectrum of the ID17 beamline at the European Synchrotron Radiation Facility (ESRF). The modification of the synchrotron storage ring and beamline in recent years necessitates a new characterisation of the radiation spectra of the ID17 beamline. The validated spectra will be a starting point for possible future clinical applications. Approach. The half value layer method was used to measure the attenuation of the x-ray spectrum in Al and Cu. Experimental data was validated against theoretical data produced using OASYS; an in-house developed software for calculating beamline spectra. Two different spectral configurations, ‘conventional’ and ‘clinical’, were investigated. The characterised spectra were used to perform dosimetric validation of depth dose profiles measured in a water-equivalent phantom. The dose profile was measured using two different detectors and compared with calculations generated using two different Monte Carlo algorithms. Main results. The results showed good agreement between measured and predicted half value layers, with differences of less than 1% in most cases. Excellent dosimetric agreement to within 3% was obtained, an agreement that satisfies the requirements in conventional radiotherapy for approvable treatment planning. Significance. Accurate spectra have been defined and validated for the ESRF—ID17 Biomedical beamline. The validated spectra can be used as input for future dosimetric studies and treatment planning systems in the context of preclinical studies and possible future clinical trials.

Enhancing Fast RISC in Hot‐Exciton Thermally Activated Delayed Fluorescence Emitter Through Fused Ring Modification: A Theoretical Insights

AbstractRecent progress has shown promising advancements in enhancing the Reverse intersystem crossing (RISC) process between high‐energy triplet states (Tn, n ≥ 1) and radiative singlet states (Sm) through the utilization of the classic D‐A‐D' strategy. In this study, 12 molecules employing phenyl carbazole and a peripheral phenanthroimidazole as the donors and fused cores as acceptors are theoretically designed. Additionally, electron‐withdrawing groups such as fluorine and cyano, as well as electron‐donating group like methoxy, are incorporated into anthracene, napthaoxadiazole, and napthothiadiazole derivatives, acting as fused core components. Theoretical analyses reveal that among the molecules examined, eight adhere to the three golden principle rules, demonstrating a large energy gap between S1‐T1, the minimal ΔES1‐T2, and large T2‐T1. Additionally, these molecules exhibit significant Spin‐orbit coupling (SOC) and maintain a strong Hybridized local and charge transfer (HLCT) character, facilitating the fast hRISC from T2 to S1. The auxiliary inclusion of heteroatoms, such as oxygen and sulfur, on the anthracene core, aligns with the prerequisite strategy, ultimately reinforcing the SOC. Furthermore, replacing the methoxy group on the Nz bridge significantly enhances the SOC, resulting in a substantial increase in the hRISC rate (10 +08 s−1). Overall, the findings substantially elevate the photophysical attributes of the designed molecules through the utilization of the hRISC channel.

Dehydrogenative synthesis of N-functionalized 2-aminophenols from cyclohexanones and amines: Molecular complexities via one-shot assembly.

Polyfunctionalized arenes are privileged structural motifs in both academic and industrial chemistry. Conventional methods for accessing this class of chemicals usually involve stepwise modification of phenyl rings, often necessitating expensive noble metal catalysts and suffering from low reactivity and selectivity when introducing multiple functionalities. We herein report dehydrogenative synthesis of N-functionalized 2-aminophenols from cyclohexanones and amines. The developed reaction system enables incorporating amino and hydroxyl groups into aromatic rings in a one-shot fashion, which simplifies polyfunctionalized 2-aminophenol synthesis by circumventing issues associated with traditional arene modifications. The wide substrate scope and excellent functional group tolerance are exemplified by late-stage modification of complex natural products and pharmaceuticals that are unattainable by existing methods. This dehydrogenative protocol benefits from using 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) as oxidant that offers interesting chemo- and regio-selective oxidation processes. More notably, the essential role of in situ generated water is disclosed, which protects aliphatic amine moieties from overoxidation via hydrogen bond-enabled interaction.

Environmental risk mitigation of lambda-cyhalothrin using tryptophan-modified magnetic nanoparticles: A sustainable emergency response strategy

Lambda-cyhalothrin, a synthetic pyrethroid pesticide extensively used in agriculture, poses significant environmental challenges due to its extensive and long-term application. To remediate pollution caused by such organic contaminants, our study introduces a rapid and convenient one-pot synthesis method for creating amino acid-modified magnetic nano-adsorbents (AA-MNPs). Research found that after modification with tryptophan (Trp), the adsorption effectiveness of MNPs on Lambda-cyhalothrin significantly increases. This modification enhances the carbon content of the magnetic nanoparticles, while minimally affecting the magnetic saturation of the MNPs. Trp-MNP exhibited an approximate particle size of 450 nm and a specific surface area of 52.36 m2 g-1. The isotherm and kinetic experiments were well-fitted to the Liu isotherm model and the pseudo-second-order model, respectively. Thermodynamic assessments suggested that the adsorption process is spontaneous and exothermic. Results indicated that the maximum adsorption capacity 144.34 mg g-1 for Trp-MNPs at 298 K, outperforming adsorbents reported in the literature. The half-adsorption time was reduced to 8 min at 298 K with an initial concentration of 60 mg L-1. This enhancement was likely influenced by the aromatic ring modification and hydrophobicity of Trp-MNPs. The study explored potential removal mechanisms from a tri-phase perspective, investigating the interactions between the adsorbent, adsorbate, and solvent.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1–3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, β-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

“An Overview On Biological Behavior Of Benzotriazole: Synthesis And Docking Study On Its Versatile Biological Activities”

The practice of medicinal chemistry is devoted to the discovery and development of new chemical agents for treating diseases. Triazoles are obtained by a slight modification of azole ring and similar or improved activities as well as fewer adverse effects are reported for triazole derivatives several advantages are the notable attraction for using benzotriazole moiety-dependent methodologies, in my research work I synthesized various derivatives of n-(1h-benzotriazol-6-yl)-benzamide from Benzene 1,2,4-triamine and benzoic acid with high yield and the synthesized derivatives characterized by FT-IR spectrum, H1 NMR spectrum, and mass spectrum data of synthesized derivatives compounds of benzotriazole Scheme analysis proves that resultant compounds n-(1h-benzotriazol-6-yl)-benzamide derivatives. The molecular docking studies validated the outcome results from the anti-inflammatory and antiarthritic agents and signifies the potential of these derivatives as crystal structure of C-terminus of voltage-gated sodium channel in complex (PDB ID:4DCK) and COX 2 Inhibitor (PDB ID:1CX2) inhibitors. So, these compounds can be modified further for the development of new anti-inflammatory and antiarthritic agents. This study strongly suggests that most of molecules synthesized in this study may indeed be promising drug candidates with interesting pharmacological profile and most of these derivatives could be a fruitful for further development of better anti-inflammatory and antiarthritic activity.

Anagostic Axial Interactions Inhibit Cross‐Coupling Catalytic Activity in Square Planar Pyridinophane Nickel Complexes

AbstractHerein, we report for the first time the use of the nitrogen‐based bidentate molecule [2.2]pyridinophane (N2) as a ligand for metal complexes. Additionally, its improved synthesis allows for electronic modification of the pyridine rings to access the new para‐dimethylamino‐[2.2]pyridinophane ligand (p‐NMe2N2). These ligands bind nickel in an analogous fashion to other pyridinophane ligands, completing the series of tetra‐, tri‐, and bidentate pyridinophane‐nickel complexes. The new compounds exhibit geometrically enforced C−H anagostic interactions between the ethylene bridge protons and the nickel center that are not present in other pyridinophane systems. These ethylene bridge groups also act as an unusual form of steric encumbrance, enforcing square planar geometries in ligand fields that would otherwise adopt tetrahedral structures. In addition, these anagostic interactions inhibit the catalytic performance in Csp3–Csp3 Kumada cross coupling reactions relative to other common bidentate N‐ligand platforms, possibly by preventing the formation of the 5‐coordinate oxidative addition intermediates.

Jugiones A-D: Antibacterial Xanthone-Anthraquinone Heterodimers from Australian Soil-Derived Penicillium shearii CMB-STF067.

The Australian roadside soil-derived fungus Penicillium shearii CMB-STF067 was prioritized for chemical investigation based on an SDA cultivation extract exhibiting both antibacterial properties and natural products with unprecedented molecular formulae (GNPS). Subsequent miniaturized 24-well plate cultivation profiling (MATRIX) identified red rice as optimal for the production of the target chemistry, with scaled-up cultivation, extraction and fractionation yielding four new xanthone-anthraquinone heterodimers, jugiones A-D (1-4), whose structures were assigned by detailed spectroscopic analysis and biosynthetic considerations. Of note, where 1-2 and 4 were active against the Gram-positive bacteria vancomycin-resistant Enterococcus faecalis (IC50 2.6-3.9 μM) and multiple-drug-resistant clinical isolates of Staphylococcus aureus (IC50 1.8-6.4 μM), and inactive against the Gram-negative bacteria Escherichia coli (IC50 > 30 μM), the closely related analog 3 exhibited no antibacterial properties (IC50 > 30 μM). Furthermore, where 1 was cytotoxic to human carcinoma (IC50 9.0-9.8 μM) and fungal (IC50 4.1 μM) cells, 2 and 4 displayed no such cytotoxicity (IC50 > 30 μM), revealing an informative structure activity relationship (SAR). We also extended the SAR study to other known compounds of this heterodimer class, which showed that the modification of ring G can reduce or eliminate the cytotoxicity while retaining the antibacterial activity.

Synthesis and Reactivity of Fluorenyl-Based Germanium(II) Compounds.

We present the synthesis of alkyl-substituted germylenes GeFlu2, Ge(FluTMS)2, and FluTMSGeCl (Flu = 9H-fluorenyl, FluTMS = 9-trimethylsilyl-9H-fluorenyl) using bulky fluorenyl ring systems and modifications of that. GeFlu2 can only be crystallized as its three-membered ring trimer, whereby the reaction is accompanied by the formation of several byproducts, such as [Li(THF)4][Ge(Ge3Flu7H)]. These results led to the modification of the fluorenyl framework by substitution the one H atom in the 9-position by a TMS group. With the synthesis of the corresponding Li salt LiFluTMS, Ge(FluTMS)2 could be isolated in good yields in a further reaction. The homoleptic Ge(FluTMS)2 is found in its crystalline form as a monomer and thus belongs to the series of monomeric alkyl-substituted germylenes. Also, the corresponding monoalkyl-substituted halogenido germylene was isolated as a four-membered ring tetramer [FluTMSGeCl]4 during an unselective reaction. However, FluTMSGeCl undergoes significant stabilization through the formation of the monomeric phosphane adduct FluTMSGeCl·PEt3, which greatly increases the selectivity of the reaction. During further reactions of Ge(FluTMS)2 with a GeBr solution (toluol/nPr3P), more impressions of the reactivity of Ge(I)X solutions with germylenes were achieved, showing that those germylenes take part in the disproportionation reaction of metastable Ge(I) solutions to give oxidized Ge(IV) compounds like (FluTMS)2GeBr2.

Cp*Co(III)-catalyzed synthesis of isoquinolones via controlled annulation of primary arylamides with internal alkynes.

In this study, we present the first cobalt(III)-catalyzed direct synthesis of isoquinolones from readily available primary arylamides and internal alkynes through a controlled oxidative C-H/N-H annulation reaction. This innovative protocol eliminates the need for expensive transition metal salts and external auxiliaries, producing the desired mono-annulated product exclusively while accommodating a wide range of substrates. Preliminary mechanistic studies highlight the critical role of copper oxide in facilitating the transformation. Additionally, peripheral modifications of the core isoquinolone rings have been performed to synthesize complex heterocyclic systems.

Nitrogen monoxide and calix[4]pyrrolato aluminate: structural constraint enabled NO dimerization.

The dimerization of nitrogen monoxide (NO) is highly relevant in homo- and heterogeneous biochemical and environmental redox processes, but a broader understanding is challenged by the endergonic nature of this equilibrium. The present work describes NO-dimerization leveraged by structurally constrained aluminum and metal-ligand cooperativity at the anionic calix[4]pyrrolato aluminate(III). Quantum chemical calculations reveal the driving force for N-N bond formation, while reactivity tests shed light on subsequent redox chemistry and NO decomposition at metal surfaces. Inhibiting the dimerization pathway by saturating NO's unpaired electron with a phenyl group (nitrosobenzene) allows trapping the 1,2-adduct as a key intermediate. Elevated temperatures result in an unprecedented and high-yielding rearrangement of the calix[4]pyrrolato ligand scaffold. Kinetic and theoretical studies provide a comprehensive picture of the rearrangement mechanism and delineate systematics for ring modification of the prominent calix[4]pyrrole macrocycle.

Expanding Diterpene Complexity and Diversity via Photoinduced Ring Distortions.

Natural products and their semi-synthetic derivatives undoubtedly constitute an important source of therapeutic agents. Their importance lies in their own origin and evolution, since they have great chemical diversity, biochemical specificity, and pharmacological properties. Currently, there is a renewed interest in the development of methodologies capable of efficiently modifying the chemical structure of these bioactive platforms. In this work, the photoderivatization of the diterpene solidagenone was performed using a complexity-to-diversity-oriented approach. By exploring [2+2]-photocycloaddition, photoinduced-hydrogen abstraction, and photoxygenation reactions, a set of solidagenone derivatives was obtained, showing different ring fusions, side chain rearrangements, and modifications of the original furan ring's substitution pattern. The derivatives obtained were characterised by NMR methodologies. To evaluate the structural diversity of the labdane-derived compounds, their physicochemical properties, structural similarity, and chemical space were analysed. These results suggest that photochemical reactions are a useful tool for performing ring distortion transformations, generating derivatives of natural compounds with wide diversity, structural complexity, and with potential biological properties.

4-Hydroxy-2-pyrones: Synthesis, Natural Products, and Application

4-Hydroxy-2-pyrones are of interest as potential biorenewable molecules for a sustainable transition from biomass feedstock to valuable chemical products. This review focuses on the methodologies for the synthesis of 4-hydroxy-2-pyrones published over the last 20 years. These pyrones as polyketides are widespread in Nature and possess versatile bioactivity that makes them an attractive target for synthesis and modification. Biosynthetic paths of the pyrones are actively developed and used as biotechnological approaches for the construction of natural and unnatural polysubstituted 4-hydroxy-2-pyrones. The major synthetical methods are biomimetic and are based on the cyclization of tricarbonyl compounds. Novel chemical methods of de novo synthesis based on alkyne cyclizations using transition metal complexes and ketene transformations allow for straightforward access to 4-hydroxy-2-pyrones and have been applied for the construction of natural products. Possible directions for further pyrone ring modification are discussed.

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes.

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.