Unveiling the Structural Characteristics of Lignin and Lignin-Carbohydrate Complexes in Fibers and Parenchyma Cells of Moso Bamboo during Different Growing Years.

Understanding and recognizing the structural characteristics of lignin-carbohydrate complexes (LCCs) and lignin in different growth stages and tissue types of bamboo will facilitate industrial processes and practical applications of bamboo biomass. Herein, the LCC and lignin samples were sequentially isolated from fibers and parenchyma cells of bamboo with different growth ages. The diverse yields of sequential fractions not only reflect the different biomass recalcitrance between bamboo fibers and parenchyma cells but also uncover the structural heterogeneity of these tissues at different growth stages. The molecular structures and structural inhomogeneities of the isolated lignin and LCC samples were comprehensively investigated. The results showed that the structural features of lignin and LCC linkages in parenchyma cells were abundant in ß-O-4 linkages but less with carbon-carbon linkages, suggesting that lignin and cross-linked LCC in parenchyma cells are simple in nature and easily to be tamed and tractable in the current biorefinery. Parallelly, the different ball-milled samples were directly characterized by high-resolution (800 M) solution-state 2D-HSQC NMR to analyze the whole lignocellulosic material. Overall, the scheme presented in this study will provide a comprehensive understanding of lignin and LCC linkages in fibers and parenchyma cells of bamboo and enable the utilization of bamboo biomass.

Water-soluble organic compounds (WSOC) from atmospheric aerosols in Bou-Ismail (Algeria).

In this contribution, we report the study of nuclear resonance magnetic spectroscopy techniques (1H-NMR, 13C-NMR, and 2D-NMR) efficiency in the characterisation of the functional composition of water-soluble organic compounds (WSOC) from atmospheric aerosols. The chosen site was our scientific and technical center of research (CRAPC) situated in Algerian Bou-Ismail city. where the concentrations of PM10 were found to be between 15.66 and 142.19 µg.m-3. As results, 1H-NMR analysis showed the coexistence of biological material and emissions from urban and biomass burning. The dominant source was identified by quantitative integration of each 1H NMR spectral region. By using the HSQC technique, many peaks are revealed in biogenic samples including biomass burning. On the other hand, the identification of the source of various organic compounds and their functional composition is possible through specific NMR spectra, which can also be used to adjust the surrounding organic aerosol sources.

The NOAH HSQC-COSY module revisited: A theoretical and practical comparison of pulse sequences.

NMR supersequences, as exemplified by the NOAH (NMR by Ordered Acquisition using 1H detection) technique, are a powerful way of acquiring multiple 2D data sets in much shorter durations. This is accomplished through targeted excitation and detection of the magnetisation belonging to specific isotopologues ('magnetisation pools'). Separately, the HSQC-COSY experiment has recently seen an increase in popularity due to the high signal dispersion in the indirect dimension and the removal of ambiguity traditionally associated with HSQC-TOCSY experiments. Here, we describe how the HSQC-COSY experiment can be integrated as a 'module' within NOAH supersequences. The benefits and drawbacks of several different pulse sequence implementations are discussed, with a particular focus on how sensitivities of other modules in the same supersequence are affected.

Discovery of anti-Mycobacterium tuberculosis desertomycins from Streptomyces flavofungini TRM90047 based on genome mining and HSQC-TOCSY.

Tuberculosis caused by Mycobacterium tuberculosis (M. tb) is a major public health problem with high morbidity and mortality worldwide. In our previous study, we found that a fermentation product of Streptomyces flavofungini TRM90047 exhibited anti-M. tb activity and decreased the expression level of several genes, including rpsL, Rplc and ClpC1. Guided by heteronuclear single quantum correlation-total correlation spectroscopy (HSQC-TOCSY) fingerprints and genome mining, we isolated two new 44-membered macrolides, desertomycin 44-1 (1) and desertomycin 44-2 (2), together with known desertomycin A (3) from S. flavofungini TRM90047. Three desertomycins showed anti-M. tb activity. The EC50 values of desertomycin A, desertomycin 44-1 and desertomycin 44-2 were 25 µg/mL, 25 µg/mL and 50 µg/mL, respectively. Molecular docking analyses revealed that the isolated desertomycins bound well to the RPSL, RPLC and CLPC1 proteins. In the present study, we describe the discovery of new anti-M. tb compounds guided by genome mining, HSQC-TOCSY and anti-M. tb bioassays.

Time-Resolved Evaluation of L-Dopa Metabolism in Bacteria-Host Symbiotic System and the Effect on Parkinson's Molecular Pathology.

The gut microbiome influences drug metabolism and therapeutic efficacy. Still, the lack of a general label-free approach for monitoring bacterial or host metabolic contribution hampers deeper insights. Here, a 2D nuclear magnetic resonance (NMR) approach is introduced that enables real-time monitoring of the metabolism of Levodopa (L-dopa), an anti-Parkinson drug, in both live bacteria and bacteria-host (Caenorhabditis elegans) symbiotic systems. The quantitative method reveals that discrete Enterococcus faecalis substrains produce different amounts of dopamine in live hosts, even though they are a single species and all have the Tyrosine decarboxylase (TyrDC) gene involved in L-dopa metabolism. The differential bacterial metabolic activity correlates with differing Parkinson's molecular pathology concerning alpha-synuclein aggregation as well as behavioral phenotypes. The gene's existence or expression is not an indicator of metabolic activity is also shown, underscoring the significance of quantitative metabolic estimation in vivo. This simple approach is widely adaptable to any chemical drug to elucidate pharmacomicrobiomic relationships and may help rapidly screen bacterial metabolic effects in drug development.

Multianalytical Approach to Understand Polyphenol-Mal d 1 Interactions to Predict Their Impact on the Allergenic Potential of Apples.

Interactions between phenolic compounds and the allergen Mal d 1 are discussed to be the reason for better tolerance of apple cultivars, which are rich in polyphenols. Because Mal d 1 is susceptible to proteolytic digestion and allergenic symptoms are usually restricted to the mouth and throat area, the release of native Mal d 1 during the oral phase is of particular interest. Therefore, we studied the release of Mal d 1 under different in vitro oral digestion conditions and revealed that only 6-15% of the total Mal d 1 present in apples is released. To investigate proposed polyphenol-Mal d 1 interactions, various analytical methods, e.g., isothermal titration calorimetry, 1H-15N-HSQC NMR, and untargeted mass spectrometry, were applied. For monomeric polyphenols, only limited noncovalent interactions were observed, whereas oligomeric polyphenols and browning products caused aggregation. While covalent modifications were not detectable in apple samples, a Michael addition of epicatechin at cysteine 107 in r-Mal d 1.01 was observed.

Synthesis of novel indol-3-acetamido analogues as potent anticancer agents, biological evaluation and molecular modeling studies.

Cannabinoids bind to cannabinoid receptors CB1 and CB2 and their antitumoral activity has been reported against some various cancer cell lines. Some synthetic cannabinoids possessing indole rings such as JWH-015 and JWH-133 particularly bind to the cannabinoid CB2 receptor and it was reported that they inhibit the proliferation and growth of various cancer cells without their psychoactive effects. However, the pharmacological action mechanisms of the cannabinoids are completely unknown. In this study, we report the synthesis of some new cannabinoidic novel indoles and evaluate their anticancer activity on various cancerous and normal cell lines (U87, RPMI 8226, HL60 and L929) using several cellular and molecular assays including MTT assay, real-time q-PCR, scratch assay, DAPI assay, Annexin V-PE/7AAD staining, caspase3/7 activity tests. Our findings indicated that compounds 7, 10, 13, 16, and 17 could reduce cell viability effectively. Compound 17 markedly increased proapoptotic genes (BAX, BAD, and BIM), tumor suppressor gene (p53) expression levels as well as the BAX/BCL-2 ratio in U87 cells. In addition, 17 inhibited cell migration. Based on these results, 17 was chosen for determining the mechanism of cell death in U87 cells. DAPI and Annexin V-7AAD staining results showed that 17 induced apoptosis, moreover activated caspase 3/7 significantly. Hence, compound 17, was selected as a lead compound for further pharmacomodulation. To rationalize the observed biological activities of 17, our study also included a comprehensive analysis using molecular docking and MD simulations. This integrative approach revealed that 17 fits tightly into the active site of the CB2 receptor and is involved in key interactions that may be responsible for its anti-proliferative effects.

Variation pattern in the macromolecular (cellulose, hemicelluloses, lignin) composition of cell walls in Pinus tabulaeformis tree trunks at different ages as revealed using multiple techniques.

The plant cell wall is a complex, heterogeneous structure primarily composed of cellulose, hemicelluloses, and lignin. Exploring the variations in these three macromolecules over time is crucial for understanding wood formation to enhance chemical processing and utilization. Here, we comprehensively analyzed the chemical composition of cell walls in the trunks of Pinus tabulaeformis using multiple techniques. In situ analysis showed that macromolecules accumulated gradually in the cell wall as the plant aged, and the distribution pattern of lignin was opposite that of polysaccharides, and both showed heterogenous distribution patterns. In addition, gel permeation chromatography (GPC) results revealed that the molecular weights of hemicelluloses decreased while that of lignin increased with age. Two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) analysis indicated that hemicelluloses mainly comprised galactoglucomannan and arabinoglucuronoxylan, and the lignin types were mainly comprised guaiacyl (G) and p-hydroxyphenyl (H) units with three main linkage types: ß-O-4, ß-ß, and ß-5. Furthermore, the C-O bond (ß-O-4) signals of lignin decreased while the C-C bonds (ß-ß and ß-5) signals increased over time. Taken together, these findings shed light on wood formation in P. tabulaeformis and lay the foundation for enhancing the processing and use of wood and timber products.

Clean PDI-1 SQ: Suppression of HSQC artifacts in 2D proton-detected INADEQUATE spectra by pulse sequence redesign.

Proton-detected INADEQUATE NMR experiments are widely used for structure elucidation of small molecules, in particular the implementations that display 13C single-quantum rather than double-quantum frequencies in the indirect dimension of 2D spectra. But unfortunately, such spectra in addition to the desired 1H-13C two-bond correlations also contain HSQC artifacts of comparable magnitude. The redesigned versatile experiment presented in this paper requires no compromise based on different 13C multiplicities and suppresses the HSQC artifacts that are a source of possible spectral misinterpretation. Demonstration of the new method is shown by applications to typical small molecules of different complexity.

1D and 2D NMR for KRAS:Ligand Binding.

Fragment-based screening by ligand-observed 1D NMR and binding interface mapping by protein-observed 2D NMR are popular methods used in drug discovery. These methods allow researchers to detect compound binding over a wide range of affinities and offer a simultaneous assessment of solubility, purity, and chemical formula accuracy of the target compounds and the 15N-labeled protein when examined by 1D and 2D NMR, respectively. These methods can be applied for screening fragment binding to the active (GMPPNP-bound) and inactive (GDP-bound) states of oncogenic KRAS mutants.

DFT investigation of coupling constant anomalies in substituted ß-lactams.

ß-lactams are a chemically diverse group of molecules with a wide range of biological activities. Having recently observed curious trends in 2JHH coupling values in studies on this structural class, we sought to obtain a more comprehensive understanding of these diagnostic NMR parameters, specifically interrogating 1JCH, 2JCH, and 2JHH, to differentiate 3- and 4-monosubstituted ß-lactams. Further investigation using computational chemistry methods was employed to explore the geometric and electronic origins for the observed and calculated differences between the two substitution patterns.

Biomimetic Dehydrogenation of Non-Conventional Lignin Monomers on Cellulose Ferulate Interfaces.

Cellulose ferulate, synthesized by Mitsunobu reaction, is shaped into thin films and also used as an aqueous dispersion to perform artificial lignin polymerization on anchor groups. This biomimetic approach is carried out in a Quartz crystal microbalance with a dissipation monitoring (QCM-D) device to enable online monitoring of the dehydrogenation, applying H2O2 and adsorbed horseradish peroxidase (HRP). The systematic use of phenylpropanoids with different oxidation states, i.e., ferulic acid, coniferyl aldehyde, coniferyl alcohol, and eugenol allowed to conclude structure-property relationships. Both the deposited material, as well as the surface roughness increased with the hydrophobicity of the monomers. Beyond surface characterizations, py-GC-MS, HSQC NMR spectroscopy and Size exclusion chromatography (SEC) measurements revealed the linkage types ß-ß, ß-5, 5-5, and ß-O-4, as well as the oligomeric character of the dehydrogenation products. All samples possessed an antibacterial activity against B. subtilis and can be used in the field of antimicrobial biomaterials.

Multinuclear 1H/13C/15N chemical shift assignment of therapeutic octreotide acetate performed at natural abundance.

Octreotide acetate, the active pharmaceutical ingredient in the long-acting release (LAR) drug product Sandostatin®, is a cyclic octapeptide that mimics the naturally occurring somatostatin peptide hormone. Modern NMR can be a robust analytical method to identify and quantify octreotide molecules. Previous 1H chemical shift assignments were mostly performed in organic solvents, and no assignments for heteronuclear 13C, 15N, and aromatic 1H nuclei are available. Here, using state-of-the-art 1D and 2D homo- and heteronuclear NMR experiments, octreotide was fully assigned, including water exchangeable amide protons, in aqueous buffer except for 13CO and 15NH of F1, 15NH of C2, and 15NζHζ of K5 that were not observed because of water exchange or conformational exchange. The solution NMR spectra were then directly compared with 1D 1H/13C/15N solid-state NMR (SSNMR) spectra showing the potential applicability of 13C/15N SSNMR for octreotide drug product characterization.

Sensitivity-enhanced NMR 15N R1 and R1ρ relaxation experiments for the investigation of intrinsically disordered proteins at high magnetic fields.

NMR relaxation experiments provide residue-specific insights into the structural dynamics of proteins. Here, we present an optimized set of sensitivity-enhanced 15N R1 and R1ρ relaxation experiments applicable to fully protonated proteins. The NMR pulse sequences are conceptually similar to the set of TROSY-based sequences and their HSQC counterpart (Lakomek et al., J. Biomol. NMR 2012). Instead of the TROSY read-out scheme, a sensitivity-enhanced HSQC read-out scheme is used, with improved and easier optimized water suppression. The presented pulse sequences are applied on the cytoplasmic domain of the SNARE protein Synpatobrevin-2 (Syb-2), which is intrinsically disordered in its monomeric pre-fusion state. A two-fold increase in the obtained signal-to-noise ratio is observed for this intrinsically disordered protein, therefore offering a four-fold reduction of measurement time compared to the TROSY-detected version. The inter-scan recovery delay can be shortened to two seconds. Pulse sequences were tested at 600 MHz and 1200 MHz 1H Larmor frequency, thus applicable over a wide magnetic field range. A comparison between protonated and deuterated protein samples reveals high agreement, indicating that reliable 15N R1 and R1ρ rate constants can be extracted for fully protonated and deuterated samples. The presented pulse sequences will benefit not only for IDPs but also for an entire range of low and medium-sized proteins.

Best method to determine DNA G-quadruplex folding: The 1H-13C HSQC NMR experiment.

NMR spectroscopy is the major method for G-quadruplex structure determination under physiologically relevant solution conditions. Unlike duplex B-DNA, in which all nucleotides adopt an anti glycosidic conformation, the core tetrad-guanines in a G-quadruplex can adopt anti or syn glycosidic conformation depending on the folding structure. An experimental method that can clearly and unambiguously determine syn and anti tetrad-Gs in a G-quadruplex is highly desirable and necessary. In the present study, we exploit the advantages of the 1H-13C HSQC experiment to determine tetrad-G's glycosidic conformation and thus folding topology of G-quadruplexes. We use several examples to demonstrate the clear and straightforward determination of the guanine glycosidic conformations and G-quadruplex folding structures. Moreover, 1H-13C HSQC data can readily identify adenine H2 resonances as well as determine unusual syn conformation in loop and flanking sequences, a challenging task by standard 2D NOESY.

Non-uniform sampling to enhance the performance of compact NMR for characterizing new psychoactive substances.

Efficient and robust analytical methods are needed to improve the identification and subsequent regulation of new psychoactive substances (NPS). NMR spectroscopy is a unique method able to determine the structure of small molecules such as NPS even in mixtures. However, high-field NMR analysis is associated with expensive purchase and maintenance costs. For more than a decade, compact NMR spectrometers have changed this paradigm. It was recently shown that a dedicated analytical workflow combining compact NMR and databases could identify the molecular structure of NPS, in spite of the lower spectral dispersion and sensitivity of compact spectrometers. This approach relies on 1H-13C HSQC to both recognize NPS and elucidate the structure of unknown substances. Still, its performance is limited by the need to compromise between resolution and experiment time. Here, we show that this strategy can be significantly improved by implementing non-uniform sampling (NUS) to improve spectral resolution in the 13C dimension of HSQC at no cost in terms of experiment time. Gains in the range of 3 to 4 in resolution are achieved for pure NPS and for a mixture. Finally, 2D HSQC with NUS was applied to improve the identification of NPS with the assistance of databases. The resulting method appears as a useful tool for the characterization of NPS in mixtures, which is essential for forensic laboratories.

Development of a specific and potent IGF2BP1 inhibitor: A promising therapeutic agent for IGF2BP1-expressing cancers.

IGF2BP1 is a protein that controls the stability, localization, and translation of various mRNA targets. Poor clinical outcomes in numerous cancer types have been associated with its overexpression. As it has been demonstrated to impede tumor growth and metastasis in animal models, inhibiting IGF2BP1 function is a promising strategy for combating cancer. A lead chemical, 7773, which specifically decreased IGF2BP1 RNA binding and cellular activities, was previously identified in a high-throughput screen for effective IGF2BP1 inhibitors. Additional optimization of 7773 described in this manuscript led to the discovery of six compounds that performed equally well or better than 7773. In cell lines with high levels of endogenous IGF2BP1, one of 7773 derivatives, AVJ16, was found to be most efficient at preventing cell migration. Further, AVJ16 was found to be IGF2BP1-specific because it had no effect on cell lines that expressed little or no IGF2BP1 protein. The direct binding of AVJ16 to IGF2BP1 was validated by binding tests, with a 12-fold increase in binding efficiency over the lead compound. AVJ16 was shown to bind to a hydrophobic region at the protein's KH34 di-domain interface between the KH3 and KH4 domains. Overall, the findings imply that AVJ16 is a potent and specific inhibitor of IGF2BP1 activity.

Synthesis and Tyrosinase Inhibitory Activity of Novel Benzimidazole/Thiazolidin-4-one Hybrid Derivatives.

In this study, novel 3-(phenylamino)thiazolidin-4-one 2 a-d and 3-(phenyl)thiazolidin-4-one 3 a-g derivatives which are having benzimidazole moiety were synthesized and their tyrosinase inhibitory activity were investigated. The structures of the target compounds were elucidated using 1 H/13 C-NMR, IR and MS. The structure of 2 b was also characterized using HSQC NMR technique. Among the target compounds, 3 b-g demonstrated stronger tyrosinase inhibitory activity (IC50 values for 3 b-g ranged from 80.93 to 119.20 µM), compared to the positive control kojic acid (IC50 : 125.08 µM). With IC50 value of 80.93 µM, 5-(2-(4-(1H-benzimidazol-1-yl)phenyl)-4-oxothiazolidin-3-yl)-2-methylbenzenesulfonamide 3 g was found to be the most active derivative of the series. Molecular docking studies were conducted to elucidate the binding interactions between compounds and tyrosinase. The MTT assay studies used to determine the cytotoxicity of 3 b-g showed that 3 c, 3 d, 3 f and 3 g were not cytotoxic in the range of 0-200 µM. Considering its tyrosinase inhibitory activity and cytotoxic effect, 3 g exhibits promising potential for further research and development as a novel tyrosinase inhibitor.

A New Dawn for Targeted Cancer Therapy: Small Molecule Covalent Binding Inhibitor Targeting K-Ras (G12C).

K-Ras is a frequently mutated oncogene in human malignancies, and the development of inhibitors targeting various oncogenic K-Ras mutant proteins is a major challenge in targeted cancer therapy, especially K-Ras(G12C) is the most common mutant, which occurs in pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and other highly prevalent malignancies. In recent years, significant progress has been made in developing small molecule covalent inhibitors targeting K-Ras(G12C), thanks to the production of nucleophilic cysteine by the G12C mutant, breaking the "spell" that K-Ras protein cannot be used as a drug target. With the successful launch of sotorasib and adagrasib, the development of small molecule inhibitors targeting various K-Ras mutants has continued to gain momentum. In recent years, with the popularization of highly sensitive surface plasmon resonance (SPR) technology, fragment-based drug design strategies have shown great potential in the development of small molecule inhibitors targeting K-Ras(G12C), but with the increasing number of clinically reported acquired drug resistance, addressing inhibitor resistance has gradually become the focus of this field, indirectly indicating that such small molecule inhibitors still the potential for the development of these small molecule inhibitors are also indirectly indicated. This paper traces the development of small molecule covalent inhibitors targeting K-Ras(G12C), highlighting and analyzing the structural evolution and optimization process of each series of inhibitors and the previous inhibitor design methods and strategies, as well as their common problems and general solutions, in order to provide inspiration and help to the subsequent researchers.

Low-temperature pretreatment by AlCl3-catalyzed 1,4-butanediol solution for producing 'ideal' lignin with super-high content of ß-O-4 linkages.

High contents of internal ß-O-4 linkages in lignin are critical for high-yield production of high-value aromatic monomers by depolymerization. However, it remains great challenge due to lack of suitable protection strategy. In this work, a very effective lignin-first strategy was developed to produce ideal lignin with a super high content of ß-O-4 linkages (up to 72 %) from poplar, in which the pretreatment was undertaken at low temperatures of 90-130 °C with the use of AlCl3-catalyzed 1, 4-butanediol solution. 2D-HSQC NMR spectra revealed that lignin ß-O-4 linkages were protected from etherification of the OH group by 1, 4-butanediol at the α position of lignin aliphatic chains. Besides, the OH groups at the γ position of lignin was also etherified, leading the formation of a structure of Ph-CH=CHCH2O(CH2)4OH. Interestingly, structure protection facilitated the formation of lignin nanoparticles via self-assembly (<100 nm). In addition, it was observed from pyrolysis results that addition of 1, 4-butanediol remarkably protected the structure of lignin by avoiding condensation, promoting the production of aromatics. The cellulose-rich fraction possessed a high cellulose digestibility of 91.64 % by enzymatic hydrolysis at a cellulase dosage of 15 FPU/g cellulose, approximately 6-fold untreated poplar (15.91 %). This low-temperature lignin-first strategy was of great importance for multi-products biorefining lignocellulose because it leads to the production of both lignin with super high content of ß-O-4 linkages for depolymerization and highly digestible cellulose for sugar production.