NMR Spectroscopy Research Articles

Unveiling the metabolomic profile of growth hormone deficiency children using NMR spectroscopy

IntroductionThe diagnosis of Growth Hormone Deficiency (GHD) during childhood has been the subject of much controversy over the last few years. Aiming to accurate medical treatment, there is a need for biomarker discovery.ObjectiveTo characterize the metabolic profile of GHD children, examine the effect of GH administration on the metabolic signature, and investigate the correlations between metabolites and IGF-1.MethodsNuclear Magnetic Resonance (NMR)-based untargeted and targeted metabolomic approach applied to study the metabolic profiles of children with GHD. Plasma, serum, and urine samples were collected from twenty-two children diagnosed with GHD and forty-eight age matched controls from the Pediatric Endocrinology Unit of the University Hospital of Patras. Experimental data were examined by both multivariate and univariate statistical analysis.ResultsThe results of this pilot study revealed a different metabolic fingerprint of children with GHD in comparison to age-matched healthy individuals. However, the detected alterations in the metabolite patterns before and after GH treatment were subtle and of minor discriminative statistical power.ConclusionsThis study provides evidence that metabolome plays a pivotal role in GHD, but large-scale multicenter studies are warranted to validate the results.

The Next Generation of Phosphorus Bisylide Superbases - Synthesis, Structures, Basicity and Proton Self Exchange.

Herein we present two phosphorus ylide superbases to enhance the basicity of MHPN - the first superbase with interacting carbon atoms as basicity centers. Its 1-pyrrolidinyl substituted analog MTPN and MHPN's theoretically predicted higher homologue P2-MHPN exhibit extreme pKaH values of 26.0 and 29.5 (experimental) in THF solution and 33.6 and 37.4 (estimated) in acetonitrile solution. The corresponding calculated gas phase basicity values are 281.4 and 284.6 kcal/mol, respectively. We prepared the neutral free bases together with the corresponding mono- and bisprotonated species which were characterized by NMR spectroscopy, ESI mass spectrometry, IR spectroscopy, elemental analysis and partly XRD analysis. The monoprotonated forms exhibit a rapid proton self-exchange between the two carbon atoms in peri-position and dynamic NMR spectroscopic methods revealed self-exchange rates of 2298 s-1 and 300 s-1 at 300 K for MTPN and P2-MHPN. However, computational studies reveal that the proton chelating effect, which typically considerably contributes to the basicity of proton sponges with basicity centers on nitrogen, is negligible in ylide proton sponges, as the 1,8-substitution pattern yields almost the same basicity as the corresponding 2,7- or 1,5-substituted analogues.

Novel Thiourea-Based Chemosensor for Ultrasensitive and Selective Detection of Hg2.

We have successfully developed a novel thiourea-based chemosensorN-((6-methylbenzo[d]thiazol-2-yl)carbamothioyl)-3,5-bis(trifluoromethyl)benzamide, TS, specifically designed for the detection of Hg2+. The structure of TS was comprehensively characterized using techniques such as 1H NMR, FT-IR, and fluorescence spectroscopy. TS exhibited exceptional fluorescence enhancement in the presence of Hg2+ ions when tested against a broad range of metal ions, including Cr3+, Co2+, Ca2+, Na+, Pb2+, Mn2+, K+, Ni2+, Mg2+, Zn2+, Cd2+, Cu2+, Ag+, and Al3+. The selectivity and sensitivity studies were performed in a MeOH/water (2:98, v/v) solvent system, where TS demonstrated a highly sensitive response to Hg2+ ions with a detection limit as low as 0.005µg/mL. Insights into the sensing mechanism were explained through fluorescence and Job's plot analysis, confirming a robust and efficient detection pathway with a 2:1 binding mode between TS and Hg2+. To assess its real-world applicability, recovery experiments were conducted on environmental and agricultural samples spiked with Hg2+ ions. The chemosensor achieved impressive recovery rates ranging from 90.0 ± 0.24% to 102.0 ± 0.58% across various concentration levels, demonstrating its accuracy and reliability. The unique attributes of TS, including its cost-effectiveness, simplicity, high selectivity, and ultra-sensitivity, make it an excellent candidate for the detection of trace levels of Hg2+ in diverse applications.

Kinetic Control and Trapping in the Supramolecular Polymerization of m-Terphenyl Bis-Urea Macrocycles.

Herein, we examine pathway complexity in the supramolecular polymerization of a novelm-terphenyl bis-urea macrocycle. Designed to induce kinetically metastable states, the macrocycle's concentration-dependent aggregation was studied via ¹H NMR and IR spectroscopy in THF and CHCl₃. Temperature-dependent UV-Vis spectroscopy in water/THF revealed a cooperative nucleation-growth mechanism, indicated by a shift in λmax to longer wavelengths upon cooling. Morphological studies using DLS, AFM, and SEM demonstrated fibrous aggregate formation. Thermal hysteresis observed in assembly-disassembly cycles indicated kinetically trapped species, with cooling governed by kinetic control and heating by thermodynamic processes. Deviations in ΔH values during cooling, compared to van't Hoff analysis and alignment of heating ΔH values with thermodynamic predictions, reinforced this distinction. Spontaneous nucleation retardation, resulting from monomer trapping, led to lag times of up to 50 minutes under specific conditions. Computational studies revealed parallel urea conformation as the more stable monomer configuration, whereas the antiparallel conformation is more stable in dimers. By probing pathway complexity of the macrocycle, we demonstrate a distinct ability to control and stabilize kinetically trapped states, broadening the scope for designing macrocyclic supramolecular polymers with tailored properties. This work deepens our understanding of supramolecular dynamics, exploring ON-pathway mechanisms and advancing tunable supramolecular materials.

Defect Identification in Zeolitic Imidazolate Framework Glasses by Solid-State NMR Spectroscopy Using 13CO2 as Probe

Defect Identification in Zeolitic Imidazolate Framework Glasses by Solid-State NMR Spectroscopy Using ¹³CO₂ as Probe

Uranium(III) and Uranium(IV) meta-Terphenylthiolate Complexes.

We report the synthesis and characterization of crystalline m-terphenylthiolate uranium complexes supported by the bulky ligand system, SAriPr6 (SAriPr6 = {SC6H3-2,6-(Tripp)2}; Tripp = 2,4,6-iPr-C6H2). Treatment of UIVCl4 with 2 equiv of KSAriPr6 in Et2O afforded both [UIV(SAriPr6)2(Cl)2] (1) and the Et2O adduct, [UIV(SAriPr6)2(Cl)2(Et2O)2] (1·Et2O) in poor yield. The reaction between [UIV(BH4)4] and 1 equiv of KSAriPr6 in toluene gave several crystals of the double salt, [UIV(μ-SAriPr6)(BH4)2(μ-BH4)(μ3-BH4)K]2 (2), and exposing the crude reaction mixture to Et2O gave the disulfide dimer, (SAriPr6)2. The reaction between [UIV(BH4)4] and 1 equiv of HSAriPr6 in hot toluene gave [UIII(H3B·SAriPr6 κS,H,H)(BH4)2] (3) which proved resistant to further substitution using either HSAriPr6 or KSAriPr6. Two U(III) mono-terphenylthiolates, [UIII(SAriPr6)(BH4)2] (4a) and [{UIII(SAriPr6)(BH4)}2{μ-B2H6}] (4b), were isolated as a mixture from the reaction between [UIII(BH3)3(toluene)] and 1 equiv of KSAriPr6, while using 2 equiv of KSAriPr6 gave the bis-terphenylthiolate complex [UIII(SAriPr6)2(BH4)] (5). Complex 4b is a rare example of a nido-metalloborane. Complexes 1-5 have been characterized variously by single-crystal and powder X-ray diffraction, multinuclear NMR spectroscopy, infrared spectroscopy, UV-Vis-NIR spectroscopy, SQUID magnetometry, and elemental analyses as appropriate. Quantum chemical calculations have been employed to interpret the nature of the U-S bonding interactions across these complexes.

Effects of Chelators on Fluoride Removal by AlCl3 and Al13 Coagulation: Performance and Mechanism.

Aluminum (Al) coagulation is one of the most widely used techniques for the defluoridation of industrial effluents. This study examined the influence of two common chelating agents, i.e., ethylenediaminetetraacetic acid (EDTA) and citric acid (CA), on fluoride removal during coagulation using monomeric Al (AlCl3) and polymeric aluminum species [Al13O4(OH)24(H2O)127+, Al13]. The results revealed that EDTA reduced the fluoride removal efficiency by up to 95.4% with AlCl3 and by 28.3% with Al13. In contrast, CA inhibited fluoride removal by up to 100% with AlCl3 and 90.6% with Al13. Both chelators impaired fluoride removal primarily by disrupting floc formation. Advanced analytical techniques, including 19F/27Al NMR spectroscopy combined with DFT calculations, demonstrated that this interference was due to the formation of EDTA-Al-F ternary complexes and Al-CA binary complexes. Additionally, the formation of a bridging fluoride (μ-F) in the Al13 structure enhanced its thermodynamic stability against chelators. Specifically, Al13 maintained structural stability even in the presence of up to 1.50 mmol/L EDTA, whereas CA progressively destabilized the Al13 structure by forming soluble Al-CA complexes. These findings are believed to help guide the rational design of Al-based coagulants and optimize water treatment strategies aimed at defluoridation of industrial effluents.

Protein structure and interactions elucidated with in-cell NMR for different cell cycle phases and in 3D human tissue models

Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.

Amidated and Aminated PMSSO-Hydrogels as a Promising Enzyme-Sensitive Vehicle for Antianemic Drugs

In this study, we report the synthesis and characterization of aminated poly(methyl silsesquioxane)-based hydrogels ((AP/MS)SO-hydrogels) as potential enzyme-sensitive vehicles for antianemic drugs. The hydrogels were synthesized via sol–gel polymerization and functionalized with amine groups. Characterization techniques included Congo red assay, Brunauer–Emmett–Teller (BET) surface area analysis, scanning electron microscopy, elemental analysis, 13C NMR, 29Si NMR, and ATR-FTIR spectroscopy and microscopy of hydrogels. The sorption of ferric chloride and ferrous D-gluconate, as well as complexes of ferrous D-gluconate with HPCD, was evaluated. Crosslinking of the gel with bifunctional agents was performed to create a new amide enzyme-sensitive bond, followed by infrared characterization of the crosslinked product. Trypsin-mediated degradation studies demonstrated the sensitivity of the hydrogel to enzymatic cleavage under model conditions. Iron release experiments in gastric and intestine-simulating media confirmed prolonged release. Overall, our findings suggest that aminated PMSSO-hydrogels hold promise as versatile and biocompatible carriers for targeted delivery of antianemic agents, warranting further exploration in preclinical and clinical applications.

Proline cis/trans Conformational Selection Controls 14-3-3 Binding.

Intrinsically disordered protein regions (IDRs) are structurally dynamic yet functional, often interacting with other proteins through short linear motifs (SLiMs). Proline residues in IDRs introduce conformational heterogeneity on a uniquely slow time scale arising from cis/trans isomerization of the Xaa-Pro peptide bond. Here, we explore the role of proline isomerization in the interaction between the prolactin receptor (PRLR) and 14-3-3. Using NMR spectroscopy, thermodynamic profiling, and molecular dynamics (MD) simulations, we uncover a unique proline isomer-dependent binding, with a cis conformation affinity 3 orders of magnitude higher than the trans. MD simulations identify structural constraints in the narrow 14-3-3 binding groove that provide an explanation for the observed isomer selectivity. The cis preference of WT PRLR introduces a slow kinetic component relevant to signal propagation and a steric component that impacts chain direction. Proline isomerization constitutes a previously unrecognized selective component relevant to the ubiquitous 14-3-3 interactome. Given the prevalence of prolines in IDRs and SLiMs, our study highlights the importance of considering the distinct properties of proline isomers in experimental design and data interpretation to fully comprehend IDR functionality.

HSQCid: A Powerful Tool for Paving the Way to High-Throughput Structural Dereplication of Natural Products Based on Fast NMR Experiments.

Structural dereplication is an essential step in the study of natural products (NPs). The number of discovered NPs is so large that efficient dereplication is highly desirable. NMR spectroscopy is still the gold standard for structural identification. 13C NMR spectrum is an effective molecular fingerprint, but their acquisition is time-consuming, especially for mass-limited NPs. Several alternative methods or tools have been proposed but have never reached general use for some reasons. Here, a new artificial intelligence tool, HSQCid, using contrastive learning between 1H-13C HSQC spectra and structures, is proposed for effective structural identification. Two structure encoders are compared, and the graph neural network is preferred over the Transformer. In this way, 80% and 20% of about 400,000 predicted data could be used for training and testing, respectively. Besides, with 17,971 experimental data as external test data, the top-1 and top-10 accuracies reach 74.5% and 94.8%, respectively. Top-1 accuracy increases by at least 12% when combined with other easily obtainable structure features, such as the total number of hydrogens connected to carbons from 1H NMR spectra. Further data analysis shows that the filters by structure features nearly eliminate the influence (>10%) of the difference between predicted and experimental data. Surprisingly, the influence of the number or the ratio of nonprotonated carbons on the identification accuracy is only significant in specific and rare cases (2.65%). Furthermore, the benchmark method, which matches 13C peaks, is compared and is markedly inferior to the proposed method, with or without structural features. The HSQCid code is available online. It is believed that HSQCid contributes to paving the way for high-throughput or highly effective structural dereplication of NPs.

A New Combined Computational and Experimental Approach to Characterize Photoactive Conjugated 3D Polymers.

A tractable new computational protocol is proposed to elucidate oligomeric-scale detail from experimental spectra, providing insight into the local and longer-range electronic and molecular structures of amorphous materials. The protocol uses an in-house code Ambuild to grow kinetically-controlled representative oligomeric clusters of an amorphous polymeric material. Generating many clusters, the statistical prevalence of different structural motifs is identified, and used to develop a 'subset' of structures that capture a broad range of important morphologies. Subsequent electronic structure calculations allow the prediction of IR, NMR, and UV-vis spectra of the bulk materials, providing significant insight into oligomeric scale topologies and helping develop structure-property relationships by identifying the underlying structural origins of different spectral features observed experimentally. Two known, and two novel, pyrene-based conjugated microporous polymers (CMPs) are synthesized and characterized as a test bed for this newly-proposed protocol. Meaningful IR, NMR, and UV-vis absorption spectral data, and experimentally comparable computationally derived spectra are obtained. Whilst IR and NMR reliably probe the local environment, UV-vis absorption spectroscopy is found to be particularly sensitive to the longer-range structural motifs observed on an oligomeric scale, providing significant structural insight into the synthesized materials with reasonable computational cost.

Examining Terpyridine Ligand Binding Interactions with Cobalt and Aluminium: Synthesis, ADME Assessment, Reversibility, and DFT Analysis.

This article emphasizes the influence of pyridine ring substitutions in 2,2':6',2″-terpyridine unit, allowing for precise adjustment of ligand properties. This study explores terpyridine-based molecular systems, with its unique coordination properties assessed against a range of competing metal ions, highlighting the specific affinity of the terpyridine ligand towards aluminium (Al) and cobalt (Co) detection in Acetonitrile-water (ACN-H2O) mixed solvent (80:20). The terpyridine-based probe's structural component is meticulously designed and detailed investigated to understand their influence on sensing performance. Synthesised probes are characterised using NMR, IR, UV-Vis spectroscopy, and mass spectrometry. Such modifications present ample opportunities to customise the attributes and applications of resultant metal complexes. The interaction between the terpyridine-based probes and target metal ions is investigated through various experimental methods, including fluorescence spectroscopy and UV studies along with unassisted discernment of Cobalt through Naked-Eye. Observation turns out that the limit of detection is 4.4 × 10-8mol/L for cobalt and 4 × 10-7mol/L for aluminium and coordination features Showing the binding stoichiometry to be 1:1 for R1. ADME Studies have been performed to analyse pharmacokinetic and biological actions. DFT calculations were performed to investigate the molecular probe's coordination features and its corresponding metal complex.

Synthesis, Characterization, Cytotoxicity, Biological Evaluation, DFT Calculations, and Molecular Docking of a Novel Schiff Base and Its Pt(IV) Complex

ABSTRACTA novel Schiff base ligand derived from 1,2,4‐triazole‐3‐thione was synthesized and complexed with platinum (IV) ion to enhance its bioactivity. Characterization techniques, including FTIR, NMR, UV‐Vis, and mass spectrometry, confirmed the formation of Schiff base molecule and revealed that its Pt(IV) complex has an octahedral geometry. The antibacterial and antifungal activity of the synthesized compounds was evaluated against Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus flavus, with the metal complex outperforming the ligand. The antioxidant properties were evaluated using a DPPH assay, with the Pt(IV) complex demonstrating superior radical‐scavenging ability. Cytotoxicity studies via MTT assays on MCF‐7 breast cancer cells showed dose‐dependent reductions in cell viability, with the Pt(IV) complex inducing enhanced antiproliferative effects compared with the ligand. DFT calculations were performed to investigate the electronic properties of the compounds, and molecular docking studies were conducted against the alpha estrogen receptor (PDB ID: 3ERT) to predict their binding affinities. The results demonstrated that the Pt(IV) complex exhibited higher biological activity than the free ligand, suggesting its potential as an effective therapeutic agent.

Optimization of Structure‐Guided Development of Chemical Probes for the Pseudoknot RNA of the Frameshift Element in SARS‐CoV‐2

AbstractTargeting the RNA genome of SARS‐CoV‐2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS‐CoV‐2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites. Available experimental 3D structures of the pseudoknot element were compared to SAXS and NMR data to validate its dominant folding state in solution. In order to experimentally map in silico predicted binding sites, NMR assignments of the majority of nucleobases were achieved by segmental labeling of the pseudoknot RNA and isotope‐filtered NMR experiments at 1.2 GHz, demonstrating the value of NMR spectroscopy to supplement modelling and docking data. Optimized ligands with enhanced affinity were shown to specifically inhibit frameshifting without affecting 0‐frame translation in cell‐free translation assays, establishing the frameshift element as target for drug‐like ligands of low molecular weight.

Detection of Exchangeable Protons in NMR Metabolomic Analysis Using AI-Designed Water Irradiation Devoid Pulses.

1H NMR spectroscopy has enabled the quantitative profiling of metabolites in various biofluids, emerging as a possible diagnostic tool for metabolic disorders and other diseases. To boost the signal-to-noise ratio and detect proton resonances near the water signal, current 1H NMR experiments require solvent suppression schemes (e.g., presaturation, jump-and-return, WATERGATE, excitation sculpting, etc.). Unfortunately, these techniques affect the quantitative assessment of analytes containing exchangeable protons. To address this issue, we introduce two new one-dimensional (1D) 1H NMR techniques that eliminate the water signal, preserving the intensities of exchangeable protons. Using GENETICS-AI, a software that combines an evolutionary algorithm and artificial intelligence, we tailored new water irradiation devoid (WADE) pulses and optimized the 1D 1H NOESY sequence for metabolomic analysis. When applied to human urine samples, kidney tissue extract, and plasma, the WADE technique allowed for accurate measurement of typical metabolites and direct quantification of urea, which is usually challenging to measure using standard NMR experiments. We anticipate that these new NMR techniques will significantly improve the accuracy and reliability of metabolite quantitative assessment for a wide range of biological fluids.

Water-Soluble Curcumin Derivatives Including Aza-Crown Ether Macrocycles as Enhancers of Their Cytotoxic Activity.

The synthesis of three novel curcumin derivative compounds, featuring aza-crown ether macrocycles of various sizes (aza-12-crown-4, aza-15-crown-5, and aza-18-crown-6), is described. The incorporation of these aza-crown macrocycles significantly enhances their water solubility, positioning them as groundbreaking instances of curcumin derivatives that are fully soluble in aqueous environments. These curcumin ligands (L1, L2, and L3) were then reacted with zinc acetate to afford the coordination metal complexes (L1-Zn, L2-Zn, and L3-Zn). Comprehensive characterization of all compounds was achieved using various analytical techniques, including 1D and 2D NMR spectroscopy, ATR-FTIR spectroscopy, mass spectrometry (ESI+), elemental analysis and UV-Vis spectroscopy. The in vitro cytotoxic activity of both, ligands and complexes were evaluated on three human cancer cell lines (U-251, MCF-7, and SK-LU-1). Compared to conventional curcumin, these compounds demonstrated improved antiproliferative potential. Additionally, a wound healing assay was conducted to assess their antimigration properties. The obtained results suggest that these modifications to the curcumin structure represent a promising approach for developing therapeutic agents with enhanced cytotoxic properties.

Investigation of the Influence of the Extraction System and Seasonality on the Pharmacological Potential of Eugenia punicifolia Leaves

The chemical complexity of natural products, such as Eugenia punicifolia (Kunth) DC. plant, presents a challenge when extracting and identifying bioactive compounds. This study investigates the impact of different extraction systems and seasonal variations on the chemical profile and pharmacological potential of E. punicifolia leaves using NMR spectroscopy for chemical analysis and canonical correlation analysis (CCA) for bioactivity correlation. Extracts obtained with methanol (M), ethanol (E), methanol/ethanol (1:1, ME), and methanol/ethanol/water (3:1:1, MEW) were analyzed for antioxidant, antiglycation, and antiviral activities. Quantitative ¹H NMR, combined with the PULCON method, was used to quantify phenolic compounds such as quercetin, myricetin, catechin, and gallic acid. The results showed that the MEW extract obtained in the rainy season exhibited the highest antioxidant and antiglycation activities, with a greater than 93% of advanced-glycation end-products (AGEs) inhibition capacity. Furthermore, our results showed that all the extracts were able to inhibit over 94% of the Zika virus (ZIKV) infection in Vero E6 cells. The CCA established strong correlations between the phenolic compounds and bioactivities, identifying gallic acid, catechin, quercetin, and myricetin as key chemical markers. This study demonstrates the importance of selecting appropriate extraction systems and considering seasonality to optimize the pharmacological potential of E. punicifolia leaves and highlights the efficacy of NMR in linking chemical composition with bioactivities.

Mechanistic Insights into the Photocatalytic Indigo Carmine Dye Decolorization by Co3O4/TiO2.

TiO2 is widely studied as an efficient UV-light photocatalyst for organic compound degradation through reactive oxygen species (ROS) generation. TiO2 can be modified to show photocatalytic activity under visible light illumination by combining with visible-light absorbing metal oxides. Here, we investigated Co3O4/TiO2 composite materials as visible-light absorbing photocatalysts, with various weight loadings of Co3O4, for the decolorization of wastewater pollutant indigo carmine. Under green LED light, 1.4 wt% Co3O4/TiO2 showed the highest decolorization rate compared to other weight loadings and bare TiO2. While UV-Vis spectroscopy indicated that Co3O4/TiO2 composite materials and bare TiO2 cause similar dye decolorization behavior, NMR spectroscopy showed that after 24 h, reaction products were present in the reaction mixture for 1.4 wt% Co3O4/TiO2, while TiO2 showed no reaction products. The lack of photocatalytic activity of Co3O4/zeolite and other Co3O4/oxide composite materials suggests a synergistic effect between Co3O4 and TiO2, where a small amount of Co3O4 enables TiO2 to utilize visible light without compromising the surface area available for ROS creation. Lastly, we emphasize the need to be cautious when drawing conclusions regarding the dye degradation, since we showed that decolorization does not necessarily equate to full degradation, using a unique combination of UV-Vis and NMR spectroscopy.

Discovery, Isolation, and Characterization of 23-Spirocholestane Derivatives as Novel Plant Root Growth Inhibitors from Ypsilandra thibetica.

In the present study, we first discovered that the 70% ethanol extract of Ypsilandra thibetica significantly inhibited Arabidopsis root growth. Through a bioactivity-guided strategy, 18 rare 23-spirocholestane derivatives (1-18), including 12 new ones (1-12), featuring a dioxane ring between C-24/C-23 of the aglycone moiety and C-1'/C-2' of the d-fucose ligand, were obtained from the strongest active elution fraction. Their structures were elucidated via a comprehensive method of MS and NMR spectroscopy, single-crystal X-ray diffraction, and chemical methods. Evaluation of their inhibitory effects of all isolates on Arabidopsis root growth revealed that 5/6, 3/4, 1, and 2 exhibited pronounced activity, with EC50 values of 6.89, 9.13, 18.01, and 26.83 μM, respectively. Then, a preliminary structure-activity relationship (SAR) analysis of these compounds was conducted. Furthermore, transcriptome data indicated that Arabidopsis responded to the stress caused by these bioactive compounds through the modulation of ethylene and auxin signaling pathways and the activation of various detoxification-related genes. Finally, the herbicidal evaluation showed that 3/4 and 5/6 significantly inhibited the root growth of Echinochloa crusgalli, while 3/4 remarkably suppressed the root growth of Lolium multiflorum. These findings highlight that 23-spirocholestane derivatives represent promising candidates for the development of new bioherbicides.