A journey in unraveling the enantiorecognition mechanism of 3,5-dinitrobenzoyl-amino acids with two Cinchona alkaloid-based chiral stationary phases: The power of molecular dynamic simulations.

BACKGROUND: Innovations in computer hardware and software capabilities have paved the way for advances in molecular modelling techniques and methods, leading to an unprecedented expansion of their potential applications. In contrast to the docking technique, which usually identifies the most stable selector-selectand (SO-SA) complex for each enantiomer, the molecular dynamics (MD) technique enables the consideration of a distribution of the SO-SA complexes based on their energy profile. This approach provides a more truthful representation of the processes occurring within the column. However, benchmark procedures and focused guidelines for computational treatment of enantioselectivity at the molecular level are still missing. RESULTS: Twenty-eight molecular dynamics simulations were performed to study the enantiorecognition mechanisms of seven N-3,5-dinitrobenzoylated α- and ß-amino acids (DNB-AAs), occurring with the two quinine- and quinidine-based (QN-AX and QD-AX) chiral stationary phases (CSPs), under polar-ionic conditions. The MD protocol was optimized in terms of box size, simulation run time, and frame recording frequency. Subsequently, all the trajectories were analyzed by calculating both the type and amount of the interactions engaged by the selectands (SAs) with the two chiral selectors (SOs), as well as the conformational and interaction energy profiles of the formed SA-SO associates. All the MDs were in strict agreement with the experimental enantiomeric elution order and allowed to establish (i) that salt-bridge and H-bond interactions play a pivotal role in the enantiorecognition mechanisms, and (ii) that the π-cation and π-π interactions are the discriminant chemical features between the two SOs in ruling the chiral recognition mechanism. SIGNIFICANCE: The results of this work clearly demonstrate the high contribution given by MD simulations in the comprehension of the enantiorecognition mechanism with Cinchona alkaloid-based CSPs. However, from this research endeavor it clearly emerged that the MD protocol optimization is crucial for the quality of the produced results.

Oxidized reducing ends in celluloses: Quantitative profiling relative to molar mass distribution by fluorescence labeling.

Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone. Furthermore, we show that to achieve high selectivity for carboxyl groups at the C1 position, keto and aldehyde groups need to be selectively reduced (e.g., by NaBH4), and carboxyl groups other than at C1 need to be blocked (e.g., by methylation with (trimethylsilyl)diazomethane) prior to fluorescence labeling of carboxyl groups at C1 position. Finally, we demonstrate the practical value of the analytical method by measuring the content of the C1-oxidized REGs in cellulose samples after chemical (by Pinnick oxidation) or enzymatic (by treatment with C1-oxidizing LPMO enzymes) oxidation of various pulp samples.

From liquid to solid-state, solvent-free oxidative ammonolysis of lignins - an easy, alternative approach to generate "N-lignins".

A new chemical modification protocol to generate N-lignins is presented, based on Indulin AT and Mg2+-lignosulfonate. The already known ammonoxidation reaction in liquid phase was used as a starting point and stepwise optimised towards a full solid-state approach. The "classical" liquid ammonoxidation products, the transition products from the optimization trials, as well as the "solid-state" products were comprehensively analysed and compared to the literature. The N-lignins obtained with the conventional ammonoxidation protocol showed the same properties as reported. Their molar mass distributions and the hydroxy group contents, hitherto not accessible due to solubility problems, were measured according to a recently reported protocol. N-Indulin showed an N-content up to 11 wt% and N-lignosulfonate up to 16 wt%. The transition experiments from liquid to solid-state gave insights into the influence of chemical components and reaction conditions. The use of a single chemical, the urea-hydrogen peroxide complex (UHP, "carbamide peroxide"), was sufficient to generate N-lignins with satisfying N-content. This chemical acts both as an N-source and as the oxidant. Following the optimization, a series of solid-state ammonoxidation tests were carried out. High N-contents of 10% in the case of Indulin and 11% in the case of lignosulfonate were obtained. By varying the ratio of UHP to lignin, the N-content can be controlled. Structural analysis showed that the N is organically bound to the lignin, similar to the "classical" ammonoxidation products obtained under homogeneous conditions. Overall, a new ammonoxidation protocol was developed which does not require an external gas supply nor liquids or dissolved reactants. This opens the possibility for carrying out the lignin modification in closed continuous reactor systems, such as extruders. The new, facile solid-state protocol will hopefully help N-lignins to find more consideration as a fertilizing material and in soil-improving materials.

The in vitro synthesis of cellulose - A mini-review.

The implementation of cellulose as a green alternative to classical polymers sparks research on the synthesis of defined derivatives of this biopolymer for various high-tech applications. Apart from the scientific challenge, the in vitro synthesis of cellulose using a bottom-up approach provides specimens with absolutely accurate substituent patterns and degrees of polymerization, not accessible from native cellulose. Synthetic cellulose exhibiting a comparably high degree of polymerization (DP) was obtained starting from cellobiose by biocatalytic synthesis implementing cellulase. Cationic ring-opening polymerization has been established in the last two decades, representing an excellent means of precise modification with regards to regio- and stereoselective substitution. This method rendered isotopically enriched cellulose as well as enantiomers of native cellulose ("l-cellulose", "d,l-cellulose") accessible. In this review, techniques for in vitro cellulose synthesis are summarized and critically compared - with a special focus on more recent developments. This is complemented by a brief overview of alternative enzymatic approaches.

Synthesis of Polyanionic Cellulose Carbamates by Homogeneous Aminolysis in an Ionic Liquid/DMF Medium.

Polyanionic cellulose carbamates were synthesized by rapid and efficient homogeneous aminolysis of cellulose carbonate half-esters in an ionic liquid/DMF medium. Cellulose bis-2,3-O-(3,5-dimethylphenyl carbamate), as a model compound, reacted with different chloroformates to cellulose carbonates. These intermediates were subjected to aminolysis, for which both the reactivity of different chloroformates towards C6-OH and the reactivity/suitability of the respective carbonate half-ester in the aminolysis were comprehensively studied. Phenyl chloroformate and 4-chlorophenyl chloroformate readily reacted with C6-OH of the model cellulose derivative, while 4-nitrophenyl chloroformate did not. The intermediate 4-chlorophenyl carbonate derivative with the highest DS (1.05) was then used to evaluate different aminolysis pathways, applying three different amines (propargyl amine, ß-alanine, and taurine) as reactants. The latter two zwitterionic compounds are only sparingly soluble in pure DMF as the typical reaction medium for aminolysis; therefore, several alternative procedures were suggested, carefully evaluated, and critically compared. Solubility problems with ß-alanine and taurine were overcome by the binary solvent system DMF/[EMIM]OAc (1:1, v/v), which was shown to be a promising medium for rapid and efficient homogeneous aminolysis and for the preparation of the corresponding cellulose carbamate derivatives or other compounds that are not accessible by conventional isocyanate chemistry. The zwitterionic cellulose carbamate derivatives presented in this work could be promising chiral cation exchangers for HPLC enantiomer separations.

A general solvent system for the analysis of lignosulfonates by 31P NMR.

31P nuclear magnetic resonance (NMR) spectroscopy is the most common and most accurate analytical method to quantitatively determine the hydroxy group contents of technical lignins. However, for lignosulfonates, liquid-state NMR analysis is often limited due to solubility problems in commonly used solvent systems, which may arise from the broad range of lignosulfonates from different wood sources, pulping conditions, and purification procedures used in biorefineries. Finding a suitable solvent system is even more difficult for chemically modified or fractionated lignosulfonates. In this study, a novel and fast approach for the solubilization of genuine, modified, and fractionated lignosulfonates and subsequent quantitative analysis of hydroxy groups by 31P NMR after derivatization with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane is presented. The implementation of the ionic liquid 1-ethyl-3-methylimidazolium chloride [emim]Cl to the already validated and commonly used DMF/pyridine solvent system caused complete solubility of previously insoluble samples, especially in the case of hard-to-dissolve ammonoxidized lignosulfonates. The applicability, accuracy, and robustness of the novel solvent system for 31P NMR analysis were comprehensively investigated with lignin model compounds and commercial lignosulfonates, including otherwise insoluble, real-world lignosulfonate specimens. The results were compared to the conventional DMF/pyridine solvent system. With the novel solvent system in hand, a much larger number of different lignosulfonates can be analyzed. In particular, the hydroxy group contents of ammonoxidized lignosulfonates were determined for the first time directly by 31P liquid-state NMR.

Polysaccharide- and ß-Cyclodextrin-Based Chiral Selectors for Enantiomer Resolution: Recent Developments and Applications.

Polysaccharides, oligosaccharides, and their derivatives, particularly of amylose, cellulose, chitosan, and ß-cyclodextrin, are well-known chiral selectors (CSs) of chiral stationary phases (CSPs) in chromatography, because they can separate a wide range of enantiomers. Typically, such CSPs are prepared by physically coating, or chemically immobilizing the polysaccharide and ß-cyclodextrin derivatives onto inert silica gel carriers as chromatographic support. Over the past few years, new chiral selectors have been introduced, and progressive methods to prepare CSPs have been exploited. Also, chiral recognition mechanisms, which play a crucial role in the investigation of chiral separations, have been better elucidated. Further insights into the broad functional performance of commercially available chiral column materials and/or the respective newly developed chiral phase materials on enantiomeric separation (ES) have been gained. This review summarizes the recent developments in CSs, CSP preparation, chiral recognition mechanisms, and enantiomeric separation methods, based on polysaccharides and ß-cyclodextrins as CSs, with a focus on the years 2019-2020 of this rapidly developing field.

On nitrogen fixation and "residual nitrogen content" in cellulosic pulps.

Cellulosic material is capable of permanently retaining nitrogen compounds (mostly having amino functions), which is reflected in a residual nitrogen content (in the low per mille range to the low percent range) of some pulps and certain lab samples. Merely adsorptively bound compounds can be removed by mild acidic washing, but part of the nitrogen seems to be resistant and very tightly bound, and thus not accessible for removal by washing. Tertiary and aromatic amines are not retained in this way, but only primary and secondary amines. There is only a weak correlation between the "firmly bound nitrogen" and the carbonyl content in cellulosics (because of oxidative damage), so that possible aminal, Schiff base and enamine structures can hardly be relevant as major nitrogen sources. However, there is a very good linear correlation between the ISO brightness (chromophore content) in aged pulps and the residual nitrogen content. In particular the concentration of the cellulosic key chromophore 2,5-dihydroxy-[1,4]-benzoquinone (DHBQ) determines the permanent N-binding capacity of the pulp. DHBQ reacts very readily with primary and secondary amines under ambient conditions to 2,5-diamino-substituted [1,4]-benzoquinones, which have very low solubility (because of zwitterionic resonance contributions) and thus remain on/in the pulp. Examples of nitrogen fixation in pulps are the binding of piperidine (a common amine catalyst in derivatization reactions), amine degradation products of the cellulose solvent NMMO, dimethylamine in materials processed from the cellulose solvent DMAc/LiCl, imidazole (a degradation product of 1-alkyl-3-methylimidazolium ionic liquids), and of amino groups in proteins after enzymatic treatment. The nature of the respective DHBQ-amine addition compound has been verified by complete structure determination.

Reaction of 2,5-dihydroxy-[1,4]-benzoquinone with nucleophiles - ipso-substitution vs. addition/elimination.

2,5-Dihydroxy-[1,4]-benzoquinone (DHBQ) reacts readily with secondary amines to 2,5-diamino compounds and with thiols to the corresponding 2,5-dithioether derivatives. The literature has been inconclusive about the detailed mechanism, as both ipso-substitution at C-2/C-5 and the sequence of C-3/C-6 addition and C-2/C-5 elimination would give exactly the same products. By means of selectively 13C isotopically labelled DHBQ it was demonstrated that the reaction with morpholine is an ipso-substitution, while the reaction with benzenethiol and 1-hexanethiol proceeds according to the addition/elimination mechanism, and the reaction with the respective thiolates according to both mechanisms in parallel. Strongly acidic media cause a peculiar "OH-fluxuational" effect in DHBQ with the two quinone oxygens and the two hydroxy groups changing positions relative to the carbon skeleton while maintaining the 2,5-dihydroxy-[1,4]-diketo pattern. With this study, the mechanism of the unwanted nitrogen and sulfur fixation in cellulose fiber processing and during degradation could be better understood.

Aerogels from Cellulose Phosphates of Low Degree of Substitution: A TBAF·H2O/DMSO Based Approach.

Biopolymer aerogels of appropriate open-porous morphology, nanotopology, surface chemistry, and mechanical properties can be promising cell scaffolding materials. Here, we report a facile approach towards the preparation of cellulose phosphate aerogels from two types of cellulosic source materials. Since high degrees of phosphorylation would afford water-soluble products inappropriate for cell scaffolding, products of low DSP (ca. 0.2) were prepared by a heterogeneous approach. Aiming at both i) full preservation of chemical integrity of cellulose during dissolution and ii) utilization of specific phase separation mechanisms upon coagulation of cellulose, TBAF·H2O/DMSO was employed as a non-derivatizing solvent. Sequential dissolution of cellulose phosphates, casting, coagulation, solvent exchange, and scCO2 drying afforded lightweight, nano-porous aerogels. Compared to their non-derivatized counterparts, cellulose phosphate aerogels are less sensitive towards shrinking during solvent exchange. This is presumably due to electrostatic repulsion and translates into faster scCO2 drying. The low DSP values have no negative impact on pore size distribution, specific surface (SBET ≤ 310 m2 g-1), porosity (Π 95.5-97 vol.%), or stiffness (Eρ ≤ 211 MPa cm3 g-1). Considering the sterilization capabilities of scCO2, existing templating opportunities to afford dual-porous scaffolds and the good hemocompatibility of phosphorylated cellulose, TBAF·H2O/DMSO can be regarded a promising solvent system for the manufacture of cell scaffolding materials.

Fabrication of bacterial cellulose-based wound dressings with improved performance by impregnation with alginate.

Bacterial cellulose (BC) hydrogels are among the most efficient materials already being used for the treatment of complex wounds. The moist environment provided by the BC dressing is a key feature assuring efficient wound recovery. Improving the dressings´ moisture-holding ability facilitates its application and leads to an economically preferable extended wear time. To produce materials with reduced moisture loss, BC dressings were impregnated with a secondary hydrophilic component: alginate. The feasibility of an industrial fabrication of this composite was evaluated on pilot scale equipment. It was shown that the procedure can easily be scaled up without significantly increasing the manufacturing time. The resultant composite possessed improved water-retention properties, providing a smooth dressing exchange as demonstrated by a wound-imitating model. The new materials were moreover shown to be compatible with an antimicrobially active compound, which assures their efficiency in the treatment of highly colonized wounds.

Electrostatic attraction-repulsion model with Cinchona alkaloid-based zwitterionic chiral stationary phases exemplified for zwitterionic analytes.

In the present paper, we demonstrated that Cinchona alkaloid cyclohexyl sulfonic acid-based zwitterionic chiral selectors (SOs) and the respective chiral stationary phases (CSPs) can be successfully employed for the enantioseparation of underivatized thus zwitterionic amino acids (the selectands, SAs) even in the absence of ionic additives in the eluent, generally used as displacer counter-ions in ion exchange chromatography. Therefore, we provided evidence that cooperative "intramolecular and intermolecular counter-ion effects" of the zwitterionic SO moiety and the zwitterionic SAs can be sufficient to modulate alone the retention characteristics without a loss of stereoselectivity. Four fully constrained ß-amino acids were used as target compounds for this study. The analyses were carried out with either neat methanol, acetonitrile, water or their binary hydro-organic mixtures. A U-shaped retention profile was observed both with methanol- and acetonitrile-based eluents. Except a few cases, enantioselectivity experienced a remarkable amelioration at the "balanced region" of a buffer free hydroorganic mobile phase composition. At "the bottom" of the U-shaped curve, high α- and resolution values could be reached with most of the screened mobile phases. An electrostatically driven "attraction-repulsion model" was postulated to explain the very favourable characteristic of the two studied zwitterion-type CSPs for the retention and enantiomer separation of zwitterionic analytes.

Effect of different immobilization strategies on chiral recognition properties of Cinchona-based anion exchangers.

In the enantiomeric separation of highly polar compounds, a traditionally challenging task for high-performance liquid chromatography, ion-exchange chiral stationary phases have found the main field of application. In this contribution, we present a series of novel anion-exchange-type chiral stationary phases for enantiomer separation of protected amino phosphonates and N-protected amino acids. Two of the prepared selectors possessed a double and triple bond within a single molecule. Thus, they were immobilized onto silica support employing either a thiol-ene (radical) or an azide-yne (copper(I)-catalyzed) click reaction. We evaluated the selectivity and the effect of immobilization proceeding either by the double bond of the Cinchona alkaloid or a triple bond of the carbamoyl moiety on the chromatographic performance of the chiral stationary phases using analytes with protecting groups of different size, flexibility, and π-acidity. The previously observed preference toward protecting groups possessing π-acidic units, which is a typical feature of Cinchona-based chiral stationary phases, was preserved. In addition, increasing the bulkiness of the selectors' carbamoyl units leads to significantly reduced retention times, while very high selectivity toward the tested analytes is retained.

Mechanism of ascaridole activation in Leishmania.

Endoperoxides (EP) are an emerging class of drugs which have potential in antiparasitic therapy, but also in other fields. For malaria therapy the EP artemisinin (Art) and its derivatives are successfully used. We have shown in the past that the EP ascaridole (Asc) is useful for the treatment of cutaneous leishmaniasis in a mouse model. Biomimetic experiments suggested that these drugs need activation in the respective target pathogens to exert their function. In spite of this idea, direct activation of EP to radicals inside cells has never been demonstrated. Therefore, this study was initiated to explore the activation of Asc in biomimetic systems and inside Leishmania in comparison to Art. Using electron paramagnetic resonance spectroscopy (EPR) in combination with spin-trapping we identified the secondary alkyl radical intermediates arising from reduction by Fe2+ in cell-free systems. Combined GC/NMR analysis confirmed the loss of isopropyl residues from Asc during this process as intermediates. This activation of Asc was stimulated by low molecular Fe2+ complexes or alternatively by hemin in conjunction with thiol reductants, such as cysteine (Cys). In Leishmania tarentolae promastigotes (LtP) as model for pathogenic forms of Leishmania carbon-centered radicals were identified in the presence of Asc by EPR spin-trapping. Both Asc and Art inhibited the viability in LtP with IC50 values in the low micromolar range while IC50 values for J774 macrophages were considerably higher. A similar structure without EP bridge (1,4-cineole) resulted in no detectable radicals and possessed much less cytotoxicity in LtP and no selectivity for LtP compared to J774 cells. The Asc-derived radical formation in LtP was inhibited by the iron chelator deferoxamine (DFO), and stimulated by Cys (a suitable reductant for hemin). The IC50 values for LtP viability in the presence of Asc or Art were increased significantly by the spin trap DMPO, while Cys and DFO increased only IC50 values for Art. In a heme association assay Asc demonstrated a lower binding affinity to heme than Art. ICP-OES measurements revealed that in LtP the total iron concentrations were twice as high as values in J774 macrophages. Since low molecular iron was important in Asc activation we studied the influence of Asc on the labile iron pool (LIP) in LtP. Low temperature EPR experiments demonstrated that Asc shifts the redox balance of iron in the LIP to its oxidized state. These data demonstrate that univalent cleavage of Asc/Art in LtP is an essential part of their pharmacological mechanism. The structure of the EP determines whether activation by low molecular iron or heme is favored and the availability of these intracellular activators modulates their cytotoxicity. These findings may be helpful for synthesis of new Asc derivatives and understanding the action of EP in other cell types.

Imidazole, a New Tunable Reagent for Producing Nanocellulose, Part I: Xylan-Coated CNCs and CNFs.

Imidazole is reported to be an effective reactant for the production of nanocellulose from hardwood pulp. The morphologies and surface properties of the nanocellulose can be simply tailored according to the water content in the imidazole system: with pure imidazole, cellulose nanofibrils (CNFs) in a yield of 10 wt % can be produced. With 25 wt % of water in imidazole, cellulose nanocrystals (CNCs) are obtained in 20 wt % yield. Both nanocelluloses exhibit crystallinity indices in the order of 70%. Interestingly, they retain the original xylan from the pulp with ca. 9⁻10 wt % of residual xylan content.

Aqueous Modification of Nano- and Microfibrillar Cellulose with a Click Synthon.

The modification of cellulose as a renewable resource has received wide attention in research and industry. A major problem regarding chemical modification, including heating and drying, is related to hornification that causes pore-system collapse and results in decreased reactivity and changes in the 3D structure of the material. A mild and green approach for the modification of different never-dried and thus wet cellulose substrates (pulp, nanostructured celluloses, and viscose fibers) by an alkoxysilane-azide in water is presented. A kinetic study of the silanization reaction demonstrates that alkoxy-trans-silanization of the cellulose surface is accomplished in water as a suspension medium within a few hours at room temperature. The resulting, azido-equipped celluloses are widely applicable precursor materials for subsequent functionalization by so-called click chemistry, for example, with a fluorescent Rhodamine derivative as a representative reagent. Successful covalent bonding was shown by GPC and a model reaction. The 3D structure of the materials remained intact, as was inter alia visualized by optical and fluorescence microscopy.

Silane meets click chemistry: towards the functionalization of wet bacterial cellulose sheets.

The modification of cellulosic materials is of great interest in materials research. Wet bacterial cellulose sheets were modified by an alkoxysilane under mild conditions to make them accessible to click chemistry derivatization. For this purpose (3-azidopropyl)triethoxysilane was grafted covalently onto the cellulosic surface. The silanized bacterial cellulose sheets were characterized comprehensively by attenuated total reflectance FTIR spectroscopy, solid-state NMR spectroscopy, thermogravimetric analysis, SEM with energy-dispersive X-ray spectroscopy, and elemental analysis. To demonstrate subsequent click chemistry functionalization, a new fluorophore based on fluorescein was synthesized and clicked to the silane-modified bacterial cellulose. The new method renders bacterial cellulose and other never-dried cellulosic materials susceptible to direct and facile functionalization in an aqueous medium without the need to work in water-free organic phases or to employ extensive protecting group chemistry and functional group interconversion.

Novel carbamoyl type quinine and quinidine based chiral anion exchangers implementing alkyne-azide cycloaddition immobilization chemistry.

The synthesis and chromatographic evaluation of a series of new Cinchona derived chiral weak anion exchangers is presented. Huisgen Cu(I) mediated alkyne-azide cycloaddition, so-called click chemistry, was used as an immobilization strategy. In this way it was possible to immobilize about 90% of offered selector via 1,2,3-triazole linker, which displays a more efficient way of binding the selector to modified silica compared to common radical mediated thiol-ene addition. Problems associated with potential radical scavenging properties of chiral selectors thereby could be circumvented. The evaluation of the synthesized chiral stationary phases regarding chromatographic behavior was carried out using polar organic mode mobile phase composition and a set of representative chiral organic acids. Different loading densities revealed an optimum selector density of about 310µmol/g chiral stationary phase with respect to resolution and selectivity. A decrease of performance was observed for higher loading, indicating mutual spatial influence of selector units leading to sterical hindrance. In addition, we observed that the effect of free azide groups on retention is negligible and the overall chromatographic behavior is comparable to other Cinchona derived chiral stationary phases.