Coherence Nuclear Magnetic Resonance Spectroscopy Research Articles

Folds from fold: Exploring topological isoforms of a single-domain protein

Expanding the protein fold space beyond linear chains is of fundamental significance, yet remains largely unexplored. Herein, we report the creation of seven topological isoforms (i.e., linear, cyclic, knot, lasso, pseudorotaxane, and catenane) from a single protein fold precursor by rewiring the connectivity of secondary structure elements of the SpyTag-SpyCatcher complex and mutating the reactive residue on SpyTag to abolish the isopeptide bonding. These topological isoforms can be directly expressed in cells. Their topologies were confirmed by combined techniques of proteolytic digestion, fluorescence correlation spectroscopy (FCS), size-exclusion chromatography (SEC), and topological transformation. To study the effects of topology on their structures and properties, their biophysical properties were characterized by differential scanning calorimetry (DSC), heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC-NMR), and circular dichroism (CD) spectroscopy. Molecular dynamics (MD) simulations were further performed to reveal the atomic details of structural changes upon unfolding. Both experimental and simulation results suggest that they share a similar, well-folded hydrophobic core but exhibit distinct folding/unfolding dynamic behaviors. These results shed light onto the folding landscape of topological isoforms derived from the same protein fold. As a model system, this work improves our understanding of protein structure and dynamics beyond linear chains and suggests that protein folds are highly amenable to topological variation.

Hydrogen Transfer Hydrogenolysis of Organosolv Chinese Fir Lignin to Monophenols over NiZnAlOx Catalyst

Lignin is the only naturally renewable aromatized polymer consisting of several phenyl propane structures linked by C–O and C–C bonds, so lignin can be depolymerized into value-added chemicals or liquid fuels. In this study, M5Zn5AlOx (M = Co, Ni and Cu) catalysts were obtained by the co-precipitation method and then were used in organosolv lignin depolymerization. Among these catalysts, the Ni5Zn5AlOx catalyst possessed the largest surface area and abundant surface oxygen vacancies as well as strong acidic sites on the surface, giving the highest yield of monophenols (about 14.49 wt %). The effect of Ni/Zn ratios on the lignin depolymerization was also investigated, and it was found that the surface area and the proportion of surface oxygen vacancies and strong acidic sites of the NiZnAlOx catalysts increased and then decreased with the Ni/Zn ratios increasing. Similarly, the yield of monomeric compounds increased and then decreased with the Ni/Zn ratios increasing. The highest yield of monophenols was 17.18 wt % obtained over the Ni3Zn7AlOx catalyst, a remarkable monomer yield from organosolv lignin. The two-dimensional 1H-13C heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of bio-oil revealed that the linkage bonds in lignin could be effectively broken over the Ni3Zn7AlOx catalyst. This study provided an effective route to obtain high-value chemicals from organosolv lignin under nickel-based catalysts.

Suppression Effect of Gamma-Valerolactone on the Mild Alkaline Pretreatment of Hybrid Pennisetum

Gamma-valerolactone (GVL) assisted NaOH/water pretreatment for hybrid pennisetum delignification was first investigated. Contrary to the hypothesized results, the addition of GVL significantly suppressed the delignification and hemicellulose removal by mild alkaline pretreatment (37 °C for 24 h). An addition of GVL to GVL/NaOH molar ratio of 1/1 resulted in lignin removal at ∼23% compared with that at ∼75% without GVL. This suppression effect was further confirmed by scanning electron microscopy and Fourier-transform infrared spectroscopy. The extracted lignin was further characterized by two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy, suggesting that the addition of GVL suppressed the decomposition of large lignin molecules by NaOH/water and the release of hemicellulose, resulting in extracted lignin with lower molecular weights and less hemicellulose. The decrease in the partial alkalinity (PA) of alkaline solutions caused by GVL addition was likely the main reason for the suppressed delignification, suggested by the good correlation between PA and delignification in both GVL/NaOH/water and NaOH/water (R2 = 0.967). This study suggested that solvent-assisted alkaline pretreatment can also result in an unwanted suppression effect, and the changes in PA could be referred to as an indicator while selecting organic solvents.

Solvothermal-Based Lignin Fractionation From Corn Stover: Process Optimization and Product Characteristics.

Fractionation of lignocellulosic is a fundamental step in the production of value-added biobased products. This work proposes an initiative to efficiently extract lignin from the corn stover using a single-step solvothermal fractionation in the presence of an acid promoter (H2SO4). The organic solvent mixture used consists of ethyl acetate, ethanol, and water at a ratio of 30: 25:45 (v/v), respectively. H2SO4 was utilized as a promoter to improve the performance and selectivity of lignin removal from the solid phase and to increase the amount of recovered lignin in the organic phase. The optimal conditions for this extraction, based on response surface methodology (RSM), are a temperature of 180°C maintained for 49.1 min at an H2SO4 concentration of 0.08 M. The optimal conditions show an efficient reaction with 98.0% cellulose yield and 75.0% lignin removal corresponding to 72.9% lignin recovery. In addition, the extracted lignin fractions, chemical composition, and structural features were investigated using Fourier transform infrared spectroscopy, thermogravimetric analysis, elemental analysis, and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D-HSQC NMR). The results indicate that the recovered lignin primarily contains a β-O-4 linking motif based on 2D-HSQC spectra. In addition, new C–C inter-unit linkages (i.e., β-β, and β-5) are not formed in the recovered lignin during H2SO4-catalyzed solvothermal pretreatment. This work facilitates effective valorization of lignin into value-added chemicals and fuels.

Synthesis and characterization of an isocyanate-terminated hyperbranched polymer and its waterborne study

An isocyanate-terminated hyperbranched polymer (HBI) was successfully synthesized by reacting hyperbranched polyester (BoltornTM HB-20) with isophorone diisocyanate (IPDI). Gel permeation chromatography (GPC) was used to monitor the molecular weight and its distribution changes during the synthesis. The structure and composition of IPDI were characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The structure of HBI was confirmed by one-dimensional 1H, 13C NMR, and distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy as well as two-dimensional heteronuclear multiple bond coherence (2D-HMBC) and heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Furthermore, HBI was reacted with methoxypolyethylene glycols (MPEG) to obtain a water-dispersible polyisocyanate with hyperbranched structure.

Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis.

The dosing of peptide and protein therapeutics is complicated by rapid clearance from the blood pool and poor cellular membrane permeability. Encapsulation into nanocarriers such as liposomes or polymersomes has long been explored to overcome these limitations, but manufacturing challenges have limited clinical translation by these approaches. Recently, inverse Flash NanoPrecipitation (iFNP) has been developed to produce highly loaded polymeric nanocarriers with the peptide or protein contained within a hydrophilic core, stabilized by a hydrophobic polymer shell. Encapsulation of proteins with higher-order structure requires understanding how processing may affect their conformational state. We demonstrate a combined experimental/simulation approach to characterize protein behavior during iFNP processing steps using the Trp-cage protein TC5b as a model. Explicit-solvent fully atomistic molecular dynamics simulations with enhanced sampling techniques are coupled with two-dimensional heteronuclear multiple-quantum coherence nuclear magnetic resonance spectroscopy (2D-HMQC NMR) and circular dichroism to determine the structure of TC5b during mixed-solvent exposure encountered in iFNP processing. The simulations involve atomistic models of mixed solvents and protein to capture the complexity of the hydrogen bonding and hydrophobic interactions between water, dimethylsulfoxide (DMSO), and the protein. The combined analyses reveal structural unfolding of the protein in 11 M DMSO but confirm complete refolding after release from the polymeric nanocarrier back into an aqueous phase. These results highlight the insights that simulations and NMR provide for the formulation of proteins in nanocarriers.

High-quality and phenolic monomer-rich bio-oil production from lignin in supercritical ethanol over synergistic Ru and Mg-Zr-oxide catalysts

The efficient depolymerization of technical lignin to produce high-quality and phenolic monomer-rich bio-oil is challenging owing to the inherent recalcitrant structure of lignin. In addition, the phenolic fragments formed during lignin depolymerization are highly reactive and easily undergo rapid repolymerization to yield undesired heavy oils or char. In the present work, we report a new hybrid catalyst of Ru/C combined with MgO/ZrO2 that is highly effective at depolymerizing lignin to high-quality bio-oil by a cooperative catalysis of Ru-catalyzed reductive depolymerization and MgO/ZrO2-catalyzed stabilization of the reactive phenolic monomers through in-situ capping of the phenol repolymerization initiator, i.e., formaldehyde, by reaction with ethanol. A screening study of various catalyst combinations, including transition metal catalysts such as Pd, Ni, and Ru supported on carbon or Al2O3 as well as basic oxide catalysts such as MgO, MgO/ZrO2, ZrO2, and Mg-Al mixed oxides, revealed that Ru/C–MgO/ZrO2 was the only combination that had a significant positive synergistic catalytic effect on lignin depolymerization, thereby highlighting its unique and exceptional activity. To elucidate the synergistic roles of Ru and MgO/ZrO2 in lignin depolymerization, their individual and integrated catalysis as well as the effect of the Mg/Zr ratio were systematically investigated through a comprehensive analysis of the bio-oil products using several techniques, including 2D heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC-NMR). A high bio-oil yield of 76.2 wt% with a low molecular weight of 401 g/mol, large HHV of 34.47 MJ/kg, and high phenolic monomer yield of 31.44 wt% was successfully obtained over the integrated catalyst of Ru/C and MgO/ZrO2 (Mg/Zr = 3) at 350 °C and 30 bar-H2 after 4 h.

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H–15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.

Comparison of the structural characterization of lignophenols and alkaline lignins before and after methylolation

This study reports on the isolation of bamboo lignophenols (LPs) and alkaline lignins (ALs) followed by their subsequent modification by methylolation. Methylolated lignins are quite useful and in high demand in wood–plastics composites or phenolic resin. The structural characterizations of the two lignin samples both before and after methylolation were compared by gel permeation chromatography, Fourier transform infrared spectroscopy, and 1H–13C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D HSQC NMR). It was found that after methylolation, the weight average molecular weight (Mw) and polydispersity (D = Mw/Mn) of the LPs varied to a greater extent than those of the ALs (c.f., Mw: from 1167 (D = 1.96) to 8267 (D = 5.47) for the LPs and Mw: from 917 (D = 1.45) to 1580 (D = 1.62) for the ALs). These results indicated that the LPs have more functional groups prone to methylolation compared to ALs. Quantitative 13C and 2D-HSQC NMR analysis demonstrated that both LPs and ALs were hydroxyl phenol, guaiacyl, syringyl-type (HGS-type), and that the esterified p-coumarate groups in the native bamboo lignins were cleaved under alkaline cooking conditions but were not cleaved in the phase separation system. In addition, the 1,1-bis(aryl)propane-2-O-aryl ether unit was confirmed to be the basic unit of the LPs, and both the introduced and unreacted p-cresol moieties were observed by 2D HSQC NMR spectroscopy. Finally, the higher ratio of syringyl unit to guaiacyl unit (S/G ratio) and methoxy group content of the ALs compared to the LPs explained the poorer reactivity of the ALs toward methylolation.

Fractionating Wheat Straw via Phosphoric Acid with Hydrogen Peroxide Pretreatment and Structural Elucidation of the Derived Lignin

As a newly-developed method for pretreating lignocellulosic biomass, phosphoric acid plus hydrogen peroxide (PHP) was employed as a pretreatment solvent to fractionate wheat straw. The structural properties of the derived lignin were elucidated in particular. Results indicated that 100.0 g of wheat straw (dry basis) yielded 39.7 g of cellulose-rich fraction, 4.7 g of oligosaccharides, and 4.5 g of lignin at mild conditions of 50 °C within 1.0 h. The resultant cellulose-rich fraction was highly accessible to hydrolytic enzymes with 88–96% cellulose–glucose conversion in 24 h, suggesting a great potential for producing biofuels. The derived lignin was characterized as having high purity (≤1.0% residual carbohydrates) and relatively low molecular weight (Mw < 1436 g mol–1) and being abundant in carboxylic acid functional groups. According to the 31P, 13C, and 2D heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy results on the derived lignin, the degree of condensation was quite l...

Adhesion/growth-regulatory galectins tested in combination: evidence for formation of hybrids as heterodimers.

The delineation of the physiological significance of protein (lectin)-glycan recognition and the structural analysis of individual lectins have directed our attention to studying them in combination. In this report, we tested the hypothesis of hybrid formation by using binary mixtures of homodimeric galectin-1 and -7 as well as a proteolytically truncated version of chimera-type galectin-3. Initial supportive evidence is provided by affinity chromatography using resin-presented galectin-7. Intriguingly, the extent of cell binding by cross-linking of surface counter-receptor increased significantly for monomeric galectin-3 form by the presence of galectin-1 or -7. Pulsed-field gradient NMR (nuclear magnetic resonance) diffusion measurements on these galectin mixtures indicated formation of heterodimers as opposed to larger oligomers. 15N-1H heteronuclear single quantum coherence NMR spectroscopy and molecular dynamics simulations allowed us to delineate how different galectins interact in the heterodimer. The possibility of domain exchange between galectins introduces a new concept for understanding the spectrum of their functionality, particularly when these effector molecules are spatially and temporally co-expressed as found in vivo.

A Biocatalytic One‐Pot Approach for the Preparation of Lignin Oligomers Using an Oxidase/Peroxidase Cascade Enzyme System

AbstractSynthetic lignin was prepared biocatalytically in a one‐pot, two‐step reaction using an oxidase/peroxidase cascade enzyme system. Using eugenol in combination with eugenol oxidase and a peroxidase, lignin‐like material was produced. The cascade reaction takes advantage of the ability of the oxidase to produce coniferyl alcohol and hydrogen peroxide from eugenol and molecular oxygen. The hydrogen peroxide is used by the peroxidase for the formation of crosslinks that typify lignin. As eugenol oxidase has a broad substrate acceptance profile, also 4‐allylphenol (chavicol) and 4‐allyl‐2,6‐dimethoxyphenol could be used as precursors of the synthetic lignin. As a result, all three naturally occurring monolignols could be prepared and incorporated in the synthetic lignin. The reaction was optimized in order to achieve the highest possible yield of insoluble lignin oligomers and scaled up to 1 gram. Analysis of the water‐insoluble product by gel permeation chromatography revealed the formation of relatively small lignin oligomers (≈1000 dalton). By using two‐dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D HSQC NMR) analysis it could be demonstrated that the material contained α‐O‐4/β‐O‐4, β‐O‐4, β‐β, β‐5 linkages and dibenzodioxocin units. All these features indicate that the biocatalytically produced material closely resembles natural lignin. While 54% of eugenol was converted into water‐insoluble oligomers, the remaining substrate was converted into water‐soluble dimers and tetramers which are important lignin model compounds. Therefore, the presented method represents a valuable and facile biocatalytic approach for the preparation of lignin‐like material and potentially valuable chemicals.magnified image

(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.

BackgroundThe aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies (13C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP).MethodsPorcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope 13C ([U-13C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D 1H and 2D 1H,13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing 13C in both perfusate and tissue samples.ResultsThere was significant enrichment of 13C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples.ConclusionsThis study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the 13C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.

Characterization of Endocarp Biomass and Extracted Lignin Using Pyrolysis and Spectroscopic Methods

Pyrolysis-GC/mass spectrometry (Py-GC/MS) and thermogravimetric analysis (TGA) was used to analyze the thermal decomposition of several endocarp sources, namely, coconut shells, walnut shells, peach pits, and olive pits, as well as their respective lignin fractions. To determine whether extraction procedures influenced pyrolysate composition and thermal decomposition processes, lignin was extracted from these feedstocks using two different procedures based on the use of formic acid and sulfuric acid (National Renewable Energy Laboratory (NREL) laboratory analytical procedure), after which the lignin-derived pyrolysates and TGA profiles were compared. Qualitative analysis of the distribution of pyrolysates provided predictive information about the structure and composition of the lignin in each sample. Results suggest that the lignin extract pyrolysates contained a different distribution of linkages and monomers in comparison to the non-extracted biomass, suggesting that lignin processing can influence bio-oil composition. Moreover, we identify the types of products obtainable by pyrolysis of these feedstocks and their lignin extracts. Heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC NMR) and Fourier transform infrared spectroscopy (FTIR) were also used to elucidate the structures of the extracted lignin samples.

O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

NMR Studies of the Q5A, G6S Unmyristylated Feline Immunodeficiency Virus Matrix Protein

Feline Immunodeficiency Virus (FIV) has been identified as an animal model for studying Human Immunodeficiency Virus. The Gag polyprotein is targeted to the plasma membrane for viral budding via the matrix protein. A myristate group is covalently bound to the N-terminus of the matrix protein by the enzyme N-Myristyltransferase (NMT). Inhibitors targeted to the matrix protein have been proposed as possible drug treatments. The structure of myristylated FIV matrix protein is being solved using Nuclear Magnetic Resonance Spectroscopy (NMR).In order to myristylate FIV matrix with yeast NMT, mutations (Q5A, G6S) were introduced into the matrix gene. There is an interest, however, in comparing the unmyristylated mutant form of matrix with the unmyristylated wildtype form in order to evaluate the physiological relevance of the mutations. It is hypothesized that because only two residues of the protein were mutated, the overall three-dimensional fold of the mutant would be similar to the wildtype.To test this, FIV unmyristylated Q5A, G6S matrix protein was expressed, labeled with a 15N source, and purified. A Heteronuclear Single Quantum Coherence (HSQC) NMR experiment was performed and compared to HSQC spectra from the wildtype matrix protein. The comparison showed strong similarities between wildtype and mutant unmyristyalated FIV MA. The structures of FIV matrix and HIV matrix can be compared to determine the effectiveness of FIV as an animal model for drug trials.

Mitochondrial Function in Diabetes: Novel Methodology and New Insight

Interpreting mitochondrial function as affected by comparative physiologic conditions is confounding because individual functional parameters are interdependent. Here, we studied muscle mitochondrial function in insulin-deficient diabetes using a novel, highly sensitive, and specific method to quantify ATP production simultaneously with reactive oxygen species (ROS) at clamped levels of inner mitochondrial membrane potential (ΔΨ), enabling more detailed study. We used a 2-deoxyglucose (2DOG) energy clamp to set ΔΨ at fixed levels and to quantify ATP production as 2DOG conversion to 2DOG-phosphate measured by one-dimensional 1H and two-dimensional 1H/13C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional 31P nuclear magnetic resonance and allowed high-throughput study of small mitochondrial isolates. Over conditions ranging from state 4 to state 3 respiration, ATP production was lower and ROS per unit of ATP generated was greater in mitochondria isolated from diabetic muscle. Moreover, ROS began to increase at a lower threshold for inner membrane potential in diabetic mitochondria. Further, ATP production in diabetic mitochondria is limited not only by respiration but also by limited capacity to use ΔΨ for ATP synthesis. In summary, we describe novel methodology for measuring ATP and provide new mechanistic insight into the dysregulation of ATP production and ROS in mitochondria of insulin-deficient rodents.

Dissolution and acetylation of ball-milled birch (Betula platyphylla) and bamboo (Phyllostachys nigra) in the ionic liquid [Bmim]Cl for HSQC NMR analysis

Abstract A method for nuclear magnetic resonance (NMR) characterization of whole cell wall components, including lignin, cellulose and hemicelluloses, was recently developed in our laboratory. The method described for fir (Abies sachalinensis) as a softwood consists of ball-milling of cell wall, dissolution in an ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), in situ acetylation, recovery of the material from the solution, and characterization of the product by 1H-13C correlation heteronuclear single quantum coherence (HSQC) NMR spectroscopy in dimethyl sulfoxide (DMSO)-d6. In the present paper, the performance of the method should be tested for a hardwood and a bamboo. Thus, Japanese white birch (Betula platyphylla) and hachiku bamboo (Phyllostachys nigra) have been investigated. Finely ball-milled birch and bamboo materials were completely dissolved in [Bmim]Cl at 100°C without severe chemical modification of the cell wall components. The dissolved cell walls were then subjected to in situ acetylation, and the ball-milled and fully acetylated cell walls were recovered from [Bmim]Cl. Longer ball-milling time was required for birch and bamboo cell walls, because of the lower solubility of acetylated birch and bamboo materials in DMSO-d6compared to the acetylated fir material. However, HSQC NMR experiments were successfully conducted, and the acetylated whole cell wall components in the birch and bamboo could be fully characterized. This method is applicable for the analysis of cell wall components of various plant biomasses without previous isolation. Further studies are necessary to improve the method.

Structural Characterization of Switchgrass Lignin after Ethanol Organosolv Pretreatment

Kanlow switchgrass was subjected to aqueous ethanol organosolv pretreatment, and ethanol organosolv lignin (EOL) was recovered through precipitation of pretreatment effluents. Ball-milled switchgrass lignin was also isolated from unpretreated and pretreated switchgrass. The obtained switchgrass lignins were characterized using Fourier transform infrared (FTIR) spectroscopy, 31P nuclear magnetic resonance (NMR), two-dimensional (2D) heteronuclear single-quantum coherence (HSQC) NMR spectroscopy, and gel permeation chromatography (GPC). The results showed that the molecular weight of switchgrass EOL lignin decreased while polydispersity increased. The pretreatment resulted in an increase of free phenolic guaiacyl and syringyl units and a decrease of the β-O-4 linkage content.

Structural features for α-galactomannan binding to galectin-1.

Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-D-galacto-β(1 → 4)-D-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with (15)N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-D-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [6(1)-α(1 → 6)-D-galactosyl-β-D-mannotriaose and 6(3),6(4)-di-α(1 → 6)-D-galactosyl-β-D-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.