HSQC Spectra Research Articles

The disordered negatively charged C-terminus of the large HECT E3 ubiquitin ligase HERC2 provides structural and thermal stability to the HECT C-lobe.

Homologous to the C-terminus of E6AP (HECT) and RCC1-like domain (RLD)-containing protein 2 (HERC2) is a large, 528 kDa E3 ubiquitin ligase that is associated with cancer, oculocutaneous albanism type 2, Prader-Willi syndrome, and other neurological diseases. HERC2 has been found to contribute to double-stranded DNA break repairs, tumor suppression, maintaining centrosome architecture, and ubiquitylation. The C-terminal portion of the HECT domain (C-lobe) of HERC2 is responsible for transferring ubiquitin to a substrate but the precise function of the other eight domains in HERC2 are unknown. Interestingly, HERC2 contains a unique and negatively charged C-terminal tail adjoined to the C-lobe that is predicted to act as a linker to promote interactions between HERC2 and its binding partners. This study aims to better understand the function and relevance of HERC2 in disease by investigating the structural aspects of the HERC2 C-lobe and HERC2 C-terminal tail using AlphaFold followed by molecular dynamics (MD) simulations, multidimensional nuclear magnetic resonance (NMR), and circular dichroism (CD). Secondary structure content analysis from MD simulations and the fully resonance assigned 1H-15N HSQC spectra of the HERC2 C-lobe and the isolated C-terminal tail confirm that the C-lobe is well-folded but the C-terminal tail is disordered. CD melting curves indicate that the flexible C-terminal tail provides improved stability to the C-lobe. Additionally, MD simulations have identified that the interaction between residues D4829 and R4728 is prevalent among the non-bonded contacts between the tail and the C-lobe. Overall, our results demonstrate that the negatively charged C-terminal tail is disordered, provides stability to the C-lobe, and may act as a flexible scaffold for protein-protein interactions.

Ameloblastin binding to biomimetic models of cell membranes – A continuum of intrinsic disorder

ObjectiveA 37-residue amino acid sequence corresponding to the segment encoded by exon-5 of murine ameloblastin (Ambn), AB2 (Y67-Q103), has been implicated with membrane association, ameloblastin self-assembly, and amelogenin-binding. Our aim was to characterize, at the residue level, the structural behavior of AB2 bound to chemical mimics of biological membranes using NMR spectroscopy. DesignTo better define the structure of AB2 using NMR-based methods, recombinant 13C- and 15N-labelled AB2 (*AB2) was prepared and data collected free in solution and with deuterated dodecylphosphocholine (dPC) micelles, deuterated bicelles, and both small and large unilamellar vesicles. ResultsAmide chemical shift and intensity perturbations observed in 1H-15N HSQC spectra of *AB2 in the presence of bicelles and dPC micelles suggest that a region of *AB2, S6-E36 (murine Ambn S68 – E98), associates with the membrane biomimetics. A CSI-3 analysis of the NMR chemical shift assignments for *AB2 free in solution and bound to dPC micelles indicated the peptide remains disordered except for the adoption of a short, 12-residue α-helix, F10-G21 (murine Ambn F72-G83). In dPC micelles, the NOE NMR data was void of patterns characteristic of long-lived helical structure indicating this helix was transient in nature. ConclusionsA continuum of intrinsic disorder in the membrane-bound state may be responsible for ameloblastin’s ability to dynamically interact with multiple partners at the same site during amelogenesis.

(R)-3,3,6-Trimethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[c]furan

(R)-3,3,6-trimethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[c]furan was obtained by the acid cyclization of the alcohol (S)-(2-methyl-5-(prop-1-en-2-yl)cyclopent-1-en-1-yl)methanol, which, in turn, is obtained from (R)-limonene. The structure of the new compound was established using NMR spectroscopy (1H, 13C, COSY, HSQC, and HMBC spectra) and high-resolution mass spectrometry. The chemical shifts of the proton and carbon atoms were calculated at the DFT level with a high correlation between the calculated and experimental values.

Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid binding properties of AtGRP2-CSD. The 2D [1H,15N] HSQC spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of eleven residues at the C-terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a β-barrel composed of five antiparallel β-strands and a 310 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the β3-β4 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands β2 and β3, as well as the β3-β4 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the β3-β4 loop, evidenced by 15N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central β-sheet, completely abolished DNA binding, highlighting the significance of π-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.

Cumulative asparagine to aspartate deamidation fails to perturb γD-crystallin structure and stability.

Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1H-15N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation.

Identifying Anti-NSCLC Bioactive Compounds in Scutellaria via 2D NMR-Based Metabolomic Analysis of Pharmacologically Classified Crude Extracts.

We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.

1Hβ chemical shift-based phase modulated NMR methods for fast identification of amino acid types in proteins

Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use 1Hβ chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the 1Hβ spins during the experiments is allowed to evolve for a fixed evolution period that results in phase modulation (positive or negative) of the cross peaks corresponding to various amino acid residues on their 2D HN projections, resembling a typical 2D [1H–15N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their 1Hβ chemical shifts, resulting in unique phase patterns at different fixed evolution periods for 1Hβ, thus facilitating their identification. Remarkably, the PM-2D HN(HB) stands out among all amino acid type identification NMR techniques for its applicability with cost-effective and most routinely employed 15N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the 13Cβ chemical shift-based phase modulated NMR method (PM-2D HN(CACB)), these methods resolved the identification of large groups of amino acids into relatively smaller groups. Moreover, these techniques can accelerate the sequence-specific sequential resonance assignment (SSRA) process and would help in fast tracking of assigned NMR signals exhibiting chemical shift perturbation on the 2D [1H–15N]-HSQC spectrum of proteins during various experiments (e.g., temperature change, pH change, and protein or ligand or cofactor binding) as well as in site-directed mutagenesis.

COMPONENT COMPOSITIONS OF THE ESSENTIAL OIL AND PHENOLIC COMPOUNDS OF THE AERIAL PART OF LOPHANTHUS SCHTSCHUROVSKIANUS

The component composition of the essential oil (EO) obtained by hydrodistillation from the air dried aerial part of the Lophanthus schtschurowskianus (Regel) Lipsky, growing in the Surkhandarya region of the Republic of Uzbekistan, was studied. By the GC-MS method were identified 57 components in the composition of EO, which was 93.8% of the total amount of oil, of which 52 volatile compounds were identified. The main components of the essential oil were 1,8-cineol (13.4%), viridiflorol (8.5%), α-terpineol (4.3%), terpinen-4-ol (4.2%), τ-cadinol (4.1%), β-spatulenol (3.9%), α-p-dimethylstyrene (2.1%). Oxygenated monoterpenes (33.6%) and oxygenated sesquiterpenes (24.7%) predominate in the composition of the essential oil; compounds related to sesquiterpene hydrocarbons (8.6%) were also found. The composition of the EO components of L. schtschurowskianus significantly differs from the EOs of other studied plant species of the genus Lophanthus.
 The antibacterial and antifungal activity of the essential oil, as well as various extracts from the aerial parts of L. schtschurowskianus, was studied using a modified agar diffusion method. Gram-positive bacteria were found to be susceptible to all the studied samples. Among the studied samples, the essential oil showed the highest antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.
 Three individual phenolic compounds were isolated from various fractions of a 75% alcoholic extract of the aerial part of L. schtschurowskianus, which, based on the study of 1H, 13C NMR, HSQC and HMBC spectra, were identified as caffeic, rosmarinic acids and luteolin.

The effects of biological crowders on fibrillization, structure, diffusion, and conformational dynamics of α-synuclein.

α-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, α-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. 15 N-1 H HSQC NMR results show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates over a timescale of 48 h. 15 N-edited diffusion measurements indicate that all crowders slow down the α-synuclein's diffusivity. Interestingly, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. 15 N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.

Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.

INCREASING THE EFFICIENCY OF USING WOOD BIOMASS BY PROVIDING SPECIAL PROPERTIES

Background. The search for ways to use logging waste is a distinct scientific direction, due to the large number of their permanent formation. The importance of such studies is associated with the need to reduce the loss of valuable wood raw materials in the form of logging waste. One of the possible ways of using waste from logging industries is the production of wood chips using a mobile complex of machines with its further recycling into a wood-fiber semi-finished product. Wood-fiber semi-finished product is, as it is known nowadays, one of the environmentally friendly types of insulation and finishing materials. A significant limitation in the use of wood fiber in various types of production is low fire and bio-resistance. As a consequence, imparting specific properties to a wood-fiber semi-finished product in the form of fire and bio-resistance is an acute problem of the modern wood processing industry.
 Purpose. Synthesis of 4-azofunctionalized pyrazoles with perfluoroalkyl substituent by cyclocondensation of polyfluorinated beta-diketones to confer special properties to the wood-fiber semi-finished product.
 Materials and methods. The 1H, 13C NMR and 1H-13C HSQC spectra were registered at the Krasnoyarsk Regional Center for Collective Use of the FITC KNC SB RAS on a Bruker Avance III Fourier spectrometer with a frequency of 600 MHz equipped with sensitive helium probes with a diameter of 5 mm and 1.7 mm. TMS is used as an internal standard. Chromato-mass spectra were obtained using ISQ 7610 Single Quadrupole GC-MS and Shimadzu LC/MS-2020 instruments. Methyl alcohol was used as the mobile phase. IR spectra were recorded on the FT-801 (FT-801) SIMEX IR Fourier spectrometer with a spectral range from 450 to 5700 cm-1. UV spectra were recorded on a HELIOS OMEGA spectrophotometer equipped with a tungsten-halogen lamp in quartz cuvettes with a diameter of 1 cm at a concentration of 1·10-4 mol/l for 200-400 nm and a concentration of 1·10-2 mol/l for 400-800 nm in ethanol. Melting temperatures were determined in open capillaries on a PTP device (TU 25-11-1144-76).
 Results. 3 new compounds were synthesized: 3(5)-(thiophene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole, 3(5)-phenyl-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole and 3(5)-(naphthalene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole.
 Conclusion. Thus, 3(5)-(thiophene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole, 3(5)-phenyl-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole and 3(5)-(naphthalene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole. Complex separable mixtures of substances containing pyrazoline, β-diketone and target nitrosopyrazoles were formed by a single-reactor method. The two-stage method of production proposed by us increases the purity and yield of the target products.

Stilbenes, phenanthrenes and antiproliferative activity of Cattleya intermedia

The phytochemical study of Cattleya intermedia (Orchidaceae) led to the isolation of two new stilbenoids and one new 9,10-dihydrophenanthrene, 4′,5-dihydroxy-2′,3-dimethoxy-dihydrostilbene (1), 3,6′-dihydroxy-4′-methoxy-dihydrostilbene (2) and 1,2,6-trihydroxy-3,8-dimethoxy-9,10-dihydrophenanthrene (3), named cattleymediol, cattleyol and phenanmediol, respectively, in addition to other five known compounds (4–8). The structural elucidations of the isolated compounds were carried out through the analyses of the one-dimensional 1H,1³C and NOE NMR spectra, and the 2D HSQC, HMBC, COSY and NOESY spectra, besides high-resolution mass spectrometry. In addition to this, the crude extract and its main fractions were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-QTOF-MS/MS), leading to the putative identification of several other compounds, including flavonoids and organic acids derivatives. Finally, the main fractions of the crude extract, and the pure compounds cattleymediol (1) and lusiantridine (7), had their antiproliferative activities evaluated against human cancerous HeLa and non-cancerous VERO cells.

Structure and property variations of mixed hardwood kraft lignins

Kraft lignin as an emerging renewable feedstock can be used to produce fuels, chemicals, and materials. Hardwood kraft lignin bears intrinsic variation due to wood species and the isolation process. The structure and property variation of hardwood kraft lignin could introduce new challenges and opportunities for its application. To better understand such variation, seven kraft lignin samples, originated from southern mixed hardwood (North America), northern mixed hardwood (North America), and Asian mixed hardwood, were isolated from commercial kraft pulping black liquor using both LignoBoost and LignoForce processes. Modern analytical techniques were used to elucidate the characteristics of mixed hardwood kraft lignins, including chemical composition, molecular weight, functional groups, and thermal properties. All lignin samples had a lignin content over 90% (92% to 96%) with one exception, which was northern mixed hardwood kraft lignin with 86% of lignin content and 6% polysaccharides. The elemental and methoxy analyses revealed the expected variation of hardwood kraft lignins with the methoxy content ranging from 0.85 to 1.20 per C9 unit. The weight average molecular weight exhibited a higher variation (from 4800 to 1895 Da) with a descending order of southern mixed hardwood kraft lignins, northern mixed hardwood lignins, and Asian mixed hardwood lignins. The aliphatic hydroxy groups ranged from 14 to 25 per 100 C9 units, and phenolic hydroxy groups ranged from 65 to 112 per 100 C9 units. The catecholic group content ranged from 12 to 34 per 100 C9 units, which is higher than softwood kraft lignin. The lignins isolated from the rapid displacement heating (RDH) pulping process were more condensed than from the regular kraft pulping process. 2D HSQC and quantitative 13C NMR revealed the drastic structure change upon kraft pulp with the low abundance of native lignin linkages and formation of new interunit linkages, such as stilbene, enol ethers, and 1-1’/5’. The S/G ratio was calculated using 2D HSQC spectra correcting for signal shift caused by the catecholic groups.

Multipronged Approach to Profiling Metabolites in Beta vulgaris L. Dried Pulp Extracts Using Chromatography, NMR and Other Spectroscopy Methods.

Beetroot (Beta vulgaris L.) is known for being a rich source of phytochemicals, minerals and vitamins. This study aims to show how the combination of extraction/chromatography/mass spectrometry and NMR offers an efficient way to profile metabolites in the extracts of beetroot. Such combination may lead to the identification of more nutritional or medicinal compounds in natural products, and it is essential for our ongoing investigation to study the selective adsorption/desorption of these metabolites' on/off nanoparticles. The aqueous and organic extracts underwent analyses using UV-vis spectroscopy; GC-MS; LC-MS; 1H, 13C, 31P, TOCSY, HSQC, and selective TOCSY NMR experiments. Polar Extract: The two forms of betalain pigment were identified by UV-vis and LC MS. Fourteen amino acids, sucrose, and other compounds, among which is riboflavin, were identified by LC-MS. Two-dimensional TOCSY showed the spin coupling correlations corresponding to some of these compounds. The HSQC spectrum showed 1H/13C spin correlation in sucrose, confirming its high abundance in beetroot. Organic Extract: GC-MS data enabled the identification of several compounds including six fatty acid methyl esters (FAME) with higher than, on average, 90% similarity score. Selective TOCSY NMR data showed the spin coupling pattern corresponding to oleic, linoleic, and linolenic fatty acids. 31P NMR spectra indicate that phospholipids exist in both the organic and aqueous phase.

Evolution of Cd2+ and Cu+ binding in Helix pomatia metallothioneins.

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn2+, Cd2+, and Cu+, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd2+ and Cu+ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [15N,1H]-HSQC spectra and T2 relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd2+ complexes and closely resembles the Cd2+-specific HpCdMT isoform, suggesting a role in Cd2+ detoxification for the ancestral protein. All Cu+-MT complexes, including the Cu+-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu+-specific HpCuMT isoforms form Cu+ complexes with comparable characteristics. It is possible to follow how Cd2+ and Cu+ binding changed throughout evolution. Interestingly, Cu+ binding improved independently in the lineages leading to the unspecific and the Cu+-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.

Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3.

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, 1H-NMR, 2D 1H-1H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

DeepSAT: Learning Molecular Structures from Nuclear Magnetic Resonance Data

The identification of molecular structure is essential for understanding chemical diversity and for developing drug leads from small molecules. Nevertheless, the structure elucidation of small molecules by Nuclear Magnetic Resonance (NMR) experiments is often a long and non-trivial process that relies on years of training. To achieve this process efficiently, several spectral databases have been established to retrieve reference NMR spectra. However, the number of reference NMR spectra available is limited and has mostly facilitated annotation of commercially available derivatives. Here, we introduce DeepSAT, a neural network-based structure annotation and scaffold prediction system that directly extracts the chemical features associated with molecular structures from their NMR spectra. Using only the 1H-13C HSQC spectrum, DeepSAT identifies related known compounds and thus efficiently assists in the identification of molecular structures. DeepSAT is expected to accelerate chemical and biomedical research by accelerating the identification of molecular structures.

NMR spectroscopy based configurational and compositional analysis of isobornyl methacrylate—Acrylonitrile copolymers

The authors conducted free radical polymerization using an initiator to synthesize a copolymer of isobornyl methacrylate—Acrylonitrile (I/A). The reactivity ratios of I (r1) and A (r2) monomers were determined as r1 = 1.63 ± 0.14, r2 = 0.61 ± 0.06 for linear KT (Kelen–Tudos) method and r1 = 1.58, r2 = 0.60 for the EVM (Error-in-Variable Method). We interpreted 1H and 13C{1H} NMR spectra of the I/A copolymers using DEPT-135 and 2D HSQC spectra. The α-CH3 carbon in the I-unit was identified and confirmed using a 2D HSQC NMR spectrum, up to the level of triad of compositional and configurational sequences. The CH (C14) and β-CH2 carbon peaks were also identified up to the triad level and higher, respectively. A 2D TOCSY spectrum revealed geminal and vicinal interactions within various CH and β-CH2 protons. A 2D HMBC NMR spectrum provided a complete assignment of the coupling between nitrile, carbonyl, and quaternary carbons with CH3 and CH2 protons.

Quantification of sesquiterpene pyridine alkaloids from genus Tripterygium by band-selective HSQC NMR

Sesquiterpene pyridine alkaloids (SPAs) are bioactive analogues derived from the genus Tripterygium and have anti-inflammatory and anti-rheumatic properties. Attributed to the similar sesquiterpene structures, the total SPAs showed severe peak overlap in 1D NMR and HPLC, leading to difficulties in identification and quantification. Interestingly, the application of band-selective HSQC NMR that specifically excited the region corresponding to the H-3 of SPAs prompted a signal separation of the total SPAs. Based on the high resolution, 23 SPAs were identified from the band-selective HSQC spectrum. The coupling constants (JCH, JHH) and relaxation times (T1, T2) of SPAs were measured, and it was found that they caused less than 1% attenuation of the HSQC signals, so the HSQC signals of SPAs had almost uniform responses. The concentrations of 23 SPAs were determined by standard curve method, using wilforgine as the calibration. In addition, we extended the pulse length-based concentration determination (PULCON) as a more efficient external standard method to the band-selective HSQC spectrum, and the results showed that the concentrations of alkaloids determined by PULCON were consistent with those measured by standard curve method. The developed quantification approach was validated according to the <761> of United States Pharmacopoeia (USP), demonstrating that the established band-selective HSQC approach is reliable for the rapid quantification of analogues in botanical extracts.

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.