2D Heteronuclear Single Quantum Correlation Research Articles

PagUNE12 encodes a basic helix-loop-helix transcription factor that regulates the development of secondary vascular tissue in poplar.

Secondary growth in woody plants generates new cells and tissues via the activity of the vascular cambium and drives the radial expansion of stems and roots. It is regulated by a series of endogenous factors, especially transcription factors. Here, we cloned the basic helix-loop-helix (bHLH) transcription factor gene UNFERTILIZED EMBRYO SAC12 (UNE12) from poplar (Populus alba × Populus glandulosa Uyeki) and used biochemical, molecular, and cytological assays to investigate the biological functions and regulatory mechanism of PagUNE12. PagUNE12 mainly localized in the nucleus and possessed transcriptional activation activity. It was widely expressed in vascular tissues, including primary phloem and xylem and secondary phloem and xylem. Poplar plants overexpressing PagUNE12 showed significantly reduced plant height, shorter internodes, and curled leaves compared with wild-type plants. Optical microscopy and transmission electron microscopy revealed that overexpressing PagUNE12 promoted secondary xylem development, with thicker secondary cell walls than wild-type poplar. Fourier transform infrared spectroscopy, confocal Raman microscopy, and 2D Heteronuclear Single Quantum Correlation analysis indicated that these plants also had increased lignin contents, with a lower relative abundance of syringyl lignin units and a higher relative abundance of guaiacyl lignin units. Therefore, overexpressing PagUNE12 promoted secondary xylem development and increased the lignin contents of secondary xylem in poplar, suggesting that this gene could be used to improve wood quality in the future.

NMR Titration Studies in Z-DNA Dynamics.

Chemical shift perturbation (CSP) is a simple NMR technique for studying the DNA binding of proteins. Titration of the unlabeled DNA into the 15N-labeled protein is monitored by acquiring a two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectrum at each step of the titration. CSP can also provide information on the DNA-binding dynamics of proteins, as well as protein-induced conformational changes in DNA. Here, we describe the titration of DNA for the 15N-labeled Z-DNA-binding protein, monitored via 2D HSQC spectra. NMR titration data can be analyzed with the active B-Z transition model to provide the protein-induced B-Z transition dynamics of DNA.

Alkylation modification for lignin color reduction and molecular weight adjustment

The application of industrial kraft lignin is limited by its low molecular weight, dark color, and low solubility. In this work, an efficient crosslinking reaction with N,N-Dimethylformamide (DMF) and 1,6-dibromohexane was proposed for adjusting the molecular weight and color of lignin. The chemical structure of alkylation lignin was systematically investigated by gel permeation chromatography (GPC), ultraviolet spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and 2D heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) spectra. After the alkylation modification, the molecular weights of the lignin were increased to 1643%. The resinol (β-β), β-aryl ether (β-O-4), and phenylcoumaran (β-5) linkages were still the main types of the linkages. The formation of β-β linkage would be inhibited at high temperatures. The color reduction of lignin can be attributed to the low content of chromophores and low packing density. This alkylation lignin will be a new and general approach for developing molecular weight-controlled and light-colored lignins, which can find more applications in cosmetics, packing, and other fields.

Speedy quantitative microstructure determination of Poly(ethylene-co-1-hexene) at triads by 1H–13C two-dimensional NMR

One of the most commonly utilized techniques for microstructure determination of polyolefins is quantitative 13C NMR, but it generally requires long experiment times and large amounts of samples to achieve reliable quantification for its low sensitivity. In this context, we report the use of two-dimensional (2D) 1H–13C heteronuclear single-quantum correlation (HSQC) spectroscopy to rapidly determine the microstructure of a typical linear low density polyethylene (LLDPE), poly(ethylene-co-1-hexene) at triads. Moreover, the 2D HSQC with non-uniform sampling (NUS) enable the reduction of experiment time from more than 23 h to several minutes with composition accuracy higher than 99.5%. The methodology is robust, and it can find wide-spread applications in areas where a quantitative analysis of polymers is necessary.

Incorporation of 5-Nitroisatin for Tailored Hydroxyapatite Nanorods and its Effect on Cervical Cancer Cells: A Nanoarchitectonics Approach

Hydroxyapatite (HAP) have been widely used as bone implants for its biocompatibility, and bioactivity. Other approaches like scaffold development, drug delivery and nanomaterials preparation are explored recently. Herein, hydroxyapatite incorporated with 5-nitroisatin (HAP-5 N) were prepared and tested for its anticancer potential. The prepared HAP-5 N were found to have rod like structures as seen using scanning electron microscopic analysis. The structural elucidation of 5-nitroisatin were confirmed by performing 2D heteronuclear single quantum correlation experiment. The synthesized HAP-5 N were assessed for its possible role as an anticancer agent against cervical cancer (SiHa) cells. From the study, it was found that HAP-5 N is efficient in inhibiting the growth of cervical cancer cell lines.

Synthesis and Characterization of Lignin-grafted-poly(ε-caprolactone) from Different Biomass Sources

Modification of lignin with poly(ε-caprolactone) is a promising approach to valorize industrial low-value lignins and to advance the bioeconomy. We have synthesized lignin grafted poly(ε-caprolactone) (lignin-g-PCL) copolymers via ring-opening polymerization of ε-caprolactone with different types of lignins of varying botanical sources (G-type pine lignin, S/G-type poplar lignin, and C-type Vanilla seeds lignin) and lignin extraction methods (Kraft and ethanol organosolv pulping). The lignin-g-PCL copolymer showed remarkably improved compatibility and dispersion in acetone, chloroform, and toluene in comparison to non-modified lignins. The structure and thermal properties of the lignin-g-PCL were investigated using Fourier-transform infrared spectroscopy (FTIR), 31P nuclear magnetic resonance (NMR), 2D heteronuclear single quantum correlation (HSQC) NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). We have found that all the technical lignins were reactive to the copolymerization reaction regardless of their plant source and isolation methods. The molecular weights of the synthesized lignin-g-PCL copolymers were positively correlated with the content of aliphatic lignin hydroxyls, suggesting that the copolymerization reaction tends to occur preferentially at the aliphatic hydroxyls rather than the phenolic hydroxyls of lignin. Thermal analyses of the lignin-g-PCL copolymers were studied, and in general, a reduction of melting temperature and crystallinity percentage in comparison to the neat PCL was observed. However, the thermal behavior of lignin-g-PCL copolymers varied depending on the lignin feedstocks employed in the copolymerization reaction.

Unveiling TMP Process Water Potential As an Industrial Sourcing of Valuable Lignin–Carbohydrate Complexes toward Zero-Waste Biorefineries

The development of zero-waste biorefineries is a promising yet challenging pathway toward a sustainable bioeconomy. Deep insights on the composition of biorefineries side streams along with the development of green technologies are mandatory to achieve such a goal. In this Article, a sustainable pilot-scale membrane process was used to recover valuable organic matter dissolved in the main side stream of a thermomechanical pulping (TMP) plant. A great number of analytical tools were combined to get an extensive characterization of the generated fraction, including inductively coupled plasma atomic emission spectroscopy (ICP-AES), high-performance ion chromatography coupled with pulse amperometric detection (HPIC-PAD), Fourier transform infrared (FTIR), size-exclusion chromatography (SEC), and C-13 and 2D heteronuclear single quantum correlation (HSQC) NMR. The results revealed the presence of a considerable amount of native lignin-carbohydrate complexes (LCCs) in the fraction representing 40% of the dry weight. The LCCs are composed of partially acetylated galactoglucomannans (GGMs) covalently linked to low condensed and ) beta-O-4-rich lignin through phenyl glycosidic bonds (25.5 bonds/100 aromatic rings (ar)) and characterized by a relatively low molecular weight around 7 kDa. Such complexes present an interesting potential for valorization with unique properties resulting from the combination of their aromatic and sugar moieties. Different applications are discussed in regards to the generated data in this work as an industrial sourcing of LCC is reported.

2-[(2,6-Dimethylmorpholin-4-yl)methyl]-4-[(E)-2-{3-[(E)-2-{3-[(2,6-dimethylmorpholin-4-yl)methyl]-4-hydroxy-5-methoxyphenyl}ethenyl]-1H-pyrazol-5-yl}ethenyl]-6-methoxyphenol

A novel di-Mannich derivative of curcumin pyrazole, 2-[(2,6-dimethyl morpholin-4-yl)methyl]-4-[(E)-2-{3-[(E)-2-{3-[(2,6-dimethylmorpholin-4-yl)methyl]-4-hydroxy-5-methoxyphenyl}ethenyl]-1H-pyrazol-5-yl}ethenyl]-6-methoxyphenol (2), has been synthesized through a Mannich reaction of curcumin pyrazole (1), formaldehyde, and 2,6-dimethylmorpholine. The structure of the synthesized compound was confirmed on the basis of FTIR, 1H-NMR, 13C-NMR, 2D Heteronuclear Single-Quantum Correlation (HSQC) and 2D Heteronuclear Multiple Bond Correlation (HMBC), and mass spectral data. The water solubility was evaluated and the result showed that compound 2 was three times more soluble than that of curcumin pyrazole (1) and curcumin.

Thermal stability of low and high Mw fractions of bio-oil derived from lignin conversion in subcritical water

The thermal stability of bio-oil influences its application in industry and is, therefore, a very important factor that must be taken into consideration. In this study, the stability of low and high molecular weight (Mw) fractions of bio-oil obtained from the hydrothermal liquefaction (HTL) of lignin in subcritical water was studied at an elevated temperature (80 °C) for a period of 1 h, 1 day and 1 week. The changes in molecular weight (gel permeation chromatography (GPC)) and chemical composition (gas chromatography–mass spectrometry (GC–MS) and 2D heteronuclear single quantum correlation (HSQC) NMR (18.8 T, DMSO-d6)) of low and high Mw fractions of the HTL bio-oil (i.e. light oil (LO) and heavy oil (HO)) were evaluated before and after ageing. It was found that only a slight formation of high Mw insoluble structures was obtained during ageing at elevated temperature for 1 week: 0.5% for the LO and 3.1% for the HO. These higher Mw moieties might be formed from different polymerisation/condensation reactions of the reactive compounds (i.e. anisoles, guaiacols, phenols, methylene (–CH2–) groups in phenolic dimers and xanthene). The high Mw insolubles in both the LO and the HO were analysed for structural composition using 2D HSQC NMR to obtain a better understanding of the changes in the composition of bio-oil fractions during the accelerated ageing process. In addition, a chemical shift database in DMSO-d6 was analysed for a subset of phenolic model compounds to simplify the interpretation of the 2D HSQC NMR spectra.

Synthesis and Characterization of Cellulose-graft-poly(p-dioxanone) Copolymers via Homogeneous Ring-Opening Graft Polymerization in Ionic Liquids

Cellulose-graft-poly(p-dioxanone) copolymers (cellulose-g-PPDO) were homogeneously prepared via ring-opening graft polymerization (ROP) between p-dioxanone (PDO) and hydroxyl groups of cellulose using the catalyst 4-dimethylaminoptridine (DMAP) in the ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl). Chemical structures and physical properties of the cellulose-g-PPDO copolymers were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H, 13C, 1H-13C 2D heteronuclear single quantum correlation (HSQC)-NMR)), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) techniques. By adjusting the reaction conditions, including the molar ratio of PDO to the anhydroglucose unit (AGU), amount of DMAP, and reaction temperature and time, the structure of the graft copolymers could be altered, and a series of copolymers with molar substitutions (MSs) in the range of 1.09 to 6.97 and polymerization degrees (DPs) varying from 1.85 to 2.88 were obtained. The graft copolymers exhibited a noticeably lower thermal stability than cellulose. After the attachment of PPDO groups, the crystalline structure of cellulose was disrupted because of the elimination of the inter- and intra-molecular hydrogen bonds of cellulose chains.

Influence of alkaline hydrothermal pretreatment on shrub wood Tamarix ramosissima: Characteristics of degraded lignin

The objective of this work was to evaluate the influence of alkaline hydrothermal (AH) pretreatment on the physicochemical properties of the degraded lignins, attempt to upgrade the potential of lignin for value-added chemicals and fuel production. For this purpose, shrub wood Tamarix ramosissima lignin was fractionated using a two-stage process based on an AH pretreatment followed by an alkaline ethanol post-treatment. The recovered lignin fractions were investigated by comparison with milled wood lignin (MWL) in terms of fractionation yield, carbohydrate composition, gel permeation chromatography, Fourier transform infrared spectroscopy, 13C and 2D heteronuclear single quantum correlation nuclear magnetic resonance, as well as pyrolysis-gas chromatography/mass spectrometry. The result showed that AH pretreatment led to the degradation of β-O-4 linkages and consequently the increased severity caused a release of more S-units lignin fractions with molecular weights between 1300 and 2500 g/mol in the liquid but higher molecular weights (3000–4400 g/mol) in the residues. Moreover, it was found that the lignin syringyl-to-guaiacyl (S/G) ratios from analytical pyrolysis slightly changed after AH pretreatment (S/G, 1.8–2.3) but higher than those of MWL (S/G, 1.7). Overall, the present study demonstrates that these lignins dissolved during AH pretreatment and those recovered from the solid residues isolated with alkaline ethanol post-treatment could be profitably exploited as feedstock in integrated forest biorefineries, rather than traditional use as low-value energy sources.

An efficient method for the production of isotopically enriched cholesterol for NMR

(13)C-Cholesterol was produced with high efficiency by a genetically engineered yeast strain. The method produces ∼ 1 mg of cholesterol per gram of glucose using 100 ml of culture medium. Uniform 94% enrichment where the most abundant product is the fully enriched isotopomer (u-(13)C(27)) is obtained using (u-(13)C(6), 99%) glucose medium. High enrichment is very important for relaxation experiments, but for NMR applications where carbon-carbon couplings are measured, this is problematic. A good compromise between sensitivity and cost consists in diluting (u-(13)C(6), 25%) with natural-abundance glucose. With a 2:3 ratio, the maximal amount of singlets can be obtained in 1 dimensional (D) carbon and 2D heteronuclear single-quantum correlation (HSQC) spectra with 6× intensity increase relative to natural-abundance samples. The use of (1-(13)C(1)-glucose, 99%) or (2-(13)C(1)-glucose, 99%) as isotope sources allows the labeling of the cholesterol in multiple mostly nonvicinal positions and reach 45× intensity increase. As an alternative, the dilution of (u-(13)C(6), 99%) glucose can be used to simultaneously enrich eleven pairs of (13)C up to ∼ 1,000× natural-abundance probability, which should be very beneficial to double-quantum NMR experiments including the INADEQUATE and related pulse sequences. The flexibility of the method and the potential to adapt the culture protocol to specific needs should find many applications in chemistry and biology and in different fields of NMR and MS.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Quantification of Metabolites from Two-Dimensional Nuclear Magnetic Resonance Spectroscopy: Application to Human Urine Samples

We present a general scheme for metabolite quantification from a two-dimensional (2D) (1)H-(13)C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance (NMR) experiment of body fluids observed in natural abundance. The scheme of quantification from 2D HSQC spectra consists of measurement of relaxation parameters of proton resonances, such as T(1) and T(2) of the metabolites and (1)H-(13)C heteronuclear J-coupling for accurate quantification. The measured cross-peak volume from 2D HSQC NMR spectra is multiplied by a calculated correction factor (which depends upon two-dimensional NMR experimental parameters and relaxation parameters) to measure accurate quantification of the metabolite. The correction factor is theoretically derived from the solution of the Bloch equation and product operator formalism. The accuracy of the scheme is tested on a solution containing a mixture of amino acids of known concentration. For human urine samples, the accuracy of the method for measuring the concentration of various metabolites was tested with spike-in experiments. The scheme is general in nature and can be applied to any other body fluid samples for metabonomic studies. We also test the measured cross-peak volume of various metabolites from 2D (1)H-(13)C HSQC NMR spectra of human urine samples for clustering analysis with scatter plots, making the scheme complete for metabolic profiling.

One- and two-dimensional NMR studies of methyl acrylate/vinyl acetate/ N-vinyl carbazole terpolymers

Terpolymers of methyl acrylate/vinyl acetate/ N-vinyl carbazole (M/A/C) with different compositions were synthesized by solution polymerization using AIBN as an initiator. Composition of terpolymers was determined from quantitative 13C{ 1H} NMR spectrum. Two-dimensional heteronuclear single quantum correlation (HSQC) and total correlated spectroscopy (TOCSY) were used to assign the methylene and methine carbon resonances by analyzing two and three bond order couplings. Various resonance signals were assigned to different compositional and configurational sequences with the help of one- and two-dimensional NMR spectra. Three and four bond order coupling between carbonyl carbon and other neighboring protons have been investigated with the help of 2D heteronuclear multiple bond correlation (HMBC) spectra. The complex and overlapped 1H NMR spectrum of terpolymer was analyzed completely with the help of 2D HSQC and TOCSY spectra.

MetaboMiner – semi-automated identification of metabolites from 2D NMR spectra of complex biofluids

BackgroundOne-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomic studies involving biofluids and tissue extracts. There are several software packages that support compound identification and quantification via 1D 1H NMR by spectral fitting techniques. Because 1D 1H NMR spectra are characterized by extensive peak overlap or spectral congestion, two-dimensional (2D) NMR, with its increased spectral resolution, could potentially improve and even automate compound identification or quantification. However, the lack of dedicated software for this purpose significantly restricts the application of 2D NMR methods to most metabolomic studies.ResultsWe describe a standalone graphics software tool, called MetaboMiner, which can be used to automatically or semi-automatically identify metabolites in complex biofluids from 2D NMR spectra. MetaboMiner is able to handle both 1H-1H total correlation spectroscopy (TOCSY) and 1H-13C heteronuclear single quantum correlation (HSQC) data. It identifies compounds by comparing 2D spectral patterns in the NMR spectrum of the biofluid mixture with specially constructed libraries containing reference spectra of ~500 pure compounds. Tests using a variety of synthetic and real spectra of compound mixtures showed that MetaboMiner is able to identify >80% of detectable metabolites from good quality NMR spectra.ConclusionMetaboMiner is a freely available, easy-to-use, NMR-based metabolomics tool that facilitates automatic peak processing, rapid compound identification, and facile spectrum annotation from either 2D TOCSY or HSQC spectra. Using comprehensive reference libraries coupled with robust algorithms for peak matching and compound identification, the program greatly simplifies the process of metabolite identification in complex 2D NMR spectra.

Urinary Metabolite Quantification Employing 2D NMR Spectroscopy

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a fairly novel method for the quantification of metabolites in biological fluids and tissue extracts. We show in this contribution that, compared to 1D 1H spectra, superior quantification of human urinary metabolites is obtained from 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra measured at natural abundance. This was accomplished by the generation of separate calibration curves for the different 2D HSQC signals of each metabolite. Lower limits of detection were in the low to mid micromolar range and were generally the lower the greater the number of methyl groups contained in an analyte. The quantitative 2D NMR data obtained for a selected set of 17 urinary metabolites were compared to those obtained independently by means of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry of amino acids and hippurate as well as enzymatic and colorimetric measurements of creatinine. As a typical application, 2D-NMR was used for the investigation of urine from patients with inborn errors of metabolism.

Structure and stereochemistry of products of hydroxylation of human steroid hormones by a housefly cytochrome P450 (CYP6A1)

The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronuclear multiple bond correlation (HMBC) using broadband adiabatic 13C pulses and phase-sensitive data presentation. Testosterone, androstenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the beta-face of the steroids, at positions 2, 12, and 15.

Microstructure analysis of methyl acrylate/methyl methacrylate copolymers by two‐dimensional NMR spectroscopy

AbstractMethyl acrylate (A)/methyl methacrylate (B) copolymers of different compositions were synthesized in bulk at 50°C and the compositions were determined from 1H NMR spectra. Reactivity ratios were optimized using the least square methodology. Compositional and configurational assignments were done using two‐dimensional (2D) Heteronuclear Single Quantum Correlation (HSQC) and Total Correlation Spectroscopy (TOCSY) experiments. Methylene proton and carbon resonances were assigned for compositional and configurational sensitivity at tetrad level. Carbon resonances of methine group of methyl acrylate were assigned for compositional sensitivity up to triad level with the help of 2D HSQC spectra. α‐Methyl group of methyl methacrylate was assigned up to triad level of compositional and configurational placements for carbon and proton resonances by 2D HSQC spectroscopy. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 1437–1445, 2006

2D NMR studies of acrylonitrile–methyl acrylate copolymers

The microstructure of acrylonitrile–methyl acrylate copolymers prepared by the solution polymerization using 2,2′-azobisisobutyronitrile (AIBN) as free radical initiator was investigated by two-dimensional NMR techniques. 2D-heteronuclear single quantum correlation (HSQC) and the total correlation spectroscopy (TOCSY) have been utilized to resolve the complex 1H NMR spectrum and to establish the compositional and configurational sequences of acrylonitrile–methyl acrylate copolymers. 2D HSQC and TOCSY showed compositional and configurational sensitivity of methine protons of A and M units upto the triad level. Heteronuclear multiple-bond correlation (HMBC) spectroscopy has been used to study carbon (carbonyl/nitrile)–proton coupling. The carbonyl and nitrile carbons showed compositional sensitivity upto the triad level. The values of reactivity ratios were determined by Kelen–Tudos (KT) and non-linear error in variable method (RREVM).