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Alginates consist of distinct blocks with different physical properties. This short review focuses on research carried out in Trondheim related to the early discovery of the block structure, their isolation, their different chemical and physical properties, and how they recently are utilized in diblock polysaccharides to obtain new nanostructuring properties.

Renewable resources can provide a range of different polysaccharide blocks that can be used to prepare new types of stimuli-responsive polysaccharide-based block copolymers. Alginates are natural polysaccharides widely used as biomaterials. Functional properties depend on the content and distribution of the two 4-linked monomers (β-D-mannuronate (M) and α-L-guluronate (G)). Blocks of L-guluronate (Gn) are responsible for cooperative binding of calcium ions and hydrogel formation. Incorporation of such blocks in block polysaccharide copolymers would represent a new class of engineered, Ca-sensitive biomacromolecules. Dioxyamines and dihydrazides have recently been shown to be well suited for preparation of block polysaccharide structures. Here we first show that when applied to alginate blocks (Gn and Mn) the two types are both very reactive, but the detailed distribution of acyclic (E)- and (Z)-forms and cyclic N-pyranosides, reaction kinetics, conjugate stability, and the rate of Schiff base reduction with α-picoline borane differ considerably, also compared to other polysaccharides. Hence, alginate specific protocols were developed. The linkers introduce a highly flexible joint in otherwise semiflexible Gn-based diblocks. This was demonstrated by SEC-MALS using a symmetrical Gn-b-Gn diblock, which in solution can best be described according to a broken rod model. Ca-Induced self-assembly of Gn-b-dextran diblocks was studied by dynamic light scattering, demonstrating that well defined nanoparticles could be prepared for certain combinations of chain lengths. Taken together, this approach provides a new class of engineered, stimuli-responsive block polysaccharide copolymers solely based on natural resources.

Schizophyllan, a triple helical polysaccharide, exhibits cooperative order-disorder transition (CODT) in aqueous solutions. The transition transforms the ordered structure (triple helix I) formed between the branched side chains and solvent molecules into the disordered structure (triple helix II) without dissociation of the triple helix. The CODT behaviors in H

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide 'click' reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

Sequential periodate-chlorite oxidation of sodium hyaluronate to 2,3-dicarboxylated hyaluronate (DCH), a novel biocompatible and highly functionalized derivative bearing additional pair of COOH groups at C2 and C3 carbons of oxidized ᴅ-glucuronic acid units, is investigated. The impact of various reaction parameters (time, oxidizer concentration, and molar amount) on DCH's composition, molecular weight, degree of oxidation, and cytotoxicity are investigated to guide the synthesis of DCH derivatives of desired properties. Subsequently, fully (99%) and partially (70%) oxidized DCH derivatives were compared to untreated sodium hyaluronate in terms of anticancer drug cisplatin loading efficacy, carrier capacity, drug release rates, and cytotoxicity towards healthy and cancerous cell lines. DCH derivatives were found to be superior in every aspect, having nearly twice the carrier capacity, significantly slower release rates, and higher efficacy. DCH is thus a highly interesting hyaluronate derivative with an adjustable degree of oxidation, molecular weight, and great potential for further modifications.

Injectable, self-setting calcium phosphate cements (CPCs) are synthetic bone substitutes considered favorable for the repair and regeneration of bone due to their osteocompatibility and unique handling properties. However, their clinical applicability can be compromised due to insufficient cohesion upon injection into the body coupled with poor degradation rates that restricts new bone formation. Consequently, carboxymethyl cellulose (CMC) was incorporated into CPC formulations to improve their cohesion and injectability while poly (D,L -lactic-co-glycolic acid) (PLGA) porogens were added to introduce macroporosity and improve their biodegradation rate. Like most biomaterials, CPCs are gamma irradiated before clinical use to ensure sufficient sterilization. However, it is well known that gamma irradiation also reduces the molecular weight of CMC and PLGA via chain scission, which affects their material properties. Therefore, the aim of this study is to measure the effect that gamma irradiation has on the molecular weight of CMC at varying doses of 15, 40, or 80 kGy and investigate how this affects the handling (i.e., injectability, cohesion, washout, and setting times) and in vitro degradation behavior of CPC formulations. Results reveal that the molecular weight of CMC decreases with increasing gamma irradiation dose, thereby reducing the viscosifying capabilities of CMC, which causes CPCs to deteriorate more readily. Further, the addition of CMC seems to inhibit the degree of phase transformation during cement setting while the subsequent reduction in molecular weight of PLGA after gamma irradiation improves the in vitro degradation rate of CPCs due to the faster degradation rate of low molecular weight PLGA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2216-2228, 2019.

The sensitivity of microorganisms to low pH can be utilized in food protection by preparing coatings based on macromolecular acids. Due to limited diffusivity of macromolecules low pH occurs primarily at the surface, while the interior parts of the food remain unaffected. This principle is demonstrated using food approved alginic acid in various types of coatings (aqueous, emulsions, dispersions, dry coating) on a wide range of foods including meat, fish, chicken, shrimp and boiled rice. Significant delay or inhibition of the natural flora is generally demonstrated, particularly when exposed to ‘temperature abuse’. Specifically, we show that the coatings reduce or inhibit regrowth of pathogens (Bacillus cereus, B. weihenstephanensis, Listeria monocytogenes serotype 1 and Staphylococcus aureus). In special cases like boiled rice, alginic acid may largely replace acetic acid for acidification and preservation, as demonstrated studying regrowth of added spores of B. cereus. Most formulations allow easy removal prior to further processing (cooking, frying). Temporary side effects such as ‘acid cooking’ obtained for high acid concentrations on sensitive surfaces (e.g. salmon) disappear during processing, recovering the normal taste and texture. The coating is hence suitable for a large variety of foods. © 2018. This is the authors’ accepted and refereed manuscript to the article. Locked until 16.8.2019 due to copyright restrictions. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide ‘click’ reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

Schizophyllan and scleroglucan are water-soluble polysaccharides having repeating units consisting of three β-1,3-linked glucose residues in the main chain and a single β-1,6-linked glucose residue as the side chain. This polysaccharide dissolves as a triple helix in an aqueous solution and shows a cooperative order-disorder transition between the side chain and solvent molecules while retaining the triple helical conformation. Periodate and subsequent chlorite oxidations selectively modify the side chain glucose to provide the corresponding dicarboxylate units. Optical rotation measurements and differential scanning calorimetry were performed on carboxylated schizophyllan/scleroglucan (‘sclerox’) samples to investigate the effects of the degree of carboxylation on the order-disorder transition in deuterium oxide with 0.1 M NaCl. The transition curves for the sclerox samples are strongly dependent on the degree of carboxylation. The modified side chains cannot take the ordered structure, resulting in a reduction of the transition enthalpy. The transition temperature for carboxylated schizophyllan becomes lowered and the transition curve broadens with increasing the degree of carboxylation. The permanent disordered units are included in a trimer by the carboxylation to inhibit a long sequence of the ordered units. © 2017. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Xanthan belongs to a special class of bacterial polysaccharides by having on one hand a oligosaccharide repeating unit and on the other higher order structures resulting in an unusually large chain rigidity. It is further characterized with an ionic strength, pH, and solvent-dependent cooperative conformational (order-disorder) transition. The ordered state is predominantly double-stranded, but partially denatured, fully or aggregated forms may coexist, depending on the sample history. In this chapter, we have reviewed the literature on chemical modifications of xanthan, and the resulting physical properties resulting from the modifications. It appears that research has been carried out without fully recognizing the complex conformational issues and possible degradations (depolymerizations) accompanying the modifications of xanthan. However, in recent years this issue has been more systematically dealt with, including studies on hydrophobization in both the ordered and disordered state.

International audience; Reducing end activation of poly-and oligosaccharides by bifunctional dioxyamines and dihydrazides enables aniline-free and cyanoborohydride-free conjugation to aldehyde-containing molecules, particles and surfaces without compromising the chain structure. Chitosans are due to their polycationic character, biodegradability, and bioactivity important candidates for conjugation. Here, we present a kinetic and structural study of the conjugation of a dioxyamine and a dihydrazide to enzymatically produced chitooligosaccharides ranging from N,N'-diacetylchitobiose to a decamer, all having N-acetyl D-glucosamine at the reducing end. Conjugation of the dioxyamine resulted in mixtures of (E)-and (Z)-oximes and β-N-pyranoside, whereas the dihydrazide yielded cyclic N-glycosides. Reaction kinetics was essentially independent of DP. Stable secondary amines were in both cases obtained by reduction with α-picoline borane, but higher temperatures were needed to obtain acceptable reduction rate. Comparison to dextran oligomers shows that the nature of the reducing end strongly influences the kinetics of both the conjugation and reduction.

β‐1,3‐d‐glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water‐soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi‐angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith‐degraded β‐1,3‐d‐glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide–hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order‐disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation. Locked until 12.06.2020 due to copyright restrictions. This is the peer reviewed version of an article, which has been published in final form at [DOI:10.1002/bip.23315 ]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Injectable, self-setting calcium phosphate cements (CPCs) are synthetic bone substitutes considered favorable for the repair and regeneration of bone due to their osteocompatibility and unique handling properties. However, their clinical applicability can be compromised due to insufficient cohesion upon injection into the body coupled with poor degradation rates that restricts new bone formation. Consequently, carboxymethyl cellulose (CMC) was incorporated into CPC formulations to improve their cohesion and injectability while poly (

Alginate-based biomaterials and medical devices are commonly subjected to γ-irradiation as a means of sterilization, either in the dry state or the gel (hydrated) state. In this process the alginate chains degrade randomly in a dose-dependent manner, altering alginates’ material properties. The addition of free radical scavenging amino acids such as histidine and phenylalanine protects the alginate significantly against degradation, as shown by monitoring changes in the molecular weight distributions using SEC-MALLS and determining the pseudo first order rate constants of degradation. Tris buffer (0.5 M), but not acetate, citrate, or phosphate buffers had a similar effect on the degradation rate. Changes in pH itself had only marginal effects on the rate of alginate degradation and on the protective effect of amino acids. Contrary to previous reports, the chemical composition (M/G profile) of the alginates, including homopolymeric mannuronan, was unaltered following irradiation up to 10 kGy. © American Chemical Society 2014. This is the authors accepted and refereed manuscript to the article.

Degradation of alginate remains a critical issue to allow predictable biological performance upon implantation of alginate-based materials. Therefore, the objective of the current study is to compare the effects of gamma-irradiation (dry state, 20-80 kGy), partial (1 and 4%) periodate oxidation (aqueous solution), and autoclaving (dry state) on the molecular weight of alginate, as well as the degradation behavior of alginate-based composites. The results show that gamma-irradiation is by far the most destructive technique characterized by strongly reduced molecular weights and rapid loss of composite integrity upon soaking in simulated body fluid. Partial periodate oxidation is less destructive as characterized by more moderate decreases in molecular weight, but the production of hydrolytically labile bonds compromises the integrity of the resulting composites. Autoclaving is shown to be a powerful tool to reduce the molecular weight of alginate in a controllable and mild manner without compromising the integrity of the resulting alginate-hydroxyapatite composites, simply by increasing the number of repetitive autoclaving cycles.

The molecular weight distributions of artificially aged transformer papers were studied using SEC–MALLS with 0.5 % DMAc/LiCl as solvent, providing chain length distributions and averages. The slow dissolution and presence of non-cellulosic contaminants tend in some cases to introduce errors in the DPw estimates, and a modified data processing procedure was developed to correct for extraneous scattering. Data were compared to the intrinsic viscosities obtained in 0.5 M Cuen and the ‘viscosimetric DP’ calculated thereof according to ISO 5351. DPvisc was 2–3 times lower than the weight average DP (DPw), but closer to or slightly above the number average DP (DPn) obtained by SEC–MALLS in the case of pure cellulose. Ageing of transformer papers were in some cases associated with changes in the polydispersity (DPw/DPn). The apparent degradation rate (defined as δ(1/DP)/δt) gradually decreased with time, resulting in a tendency for a ‘level-off’ DP in the range 200–300 for DPvisc and DPn, and roughly 1,000 for DPw.

Water-soluble (1→3)-β- d -glucans with 1,6-linked branches (SBG), originally isolated from the cell walls of Saccharomyces cerevisiae and partially depolymerised to a weight average degree of polymerisation (DP w ) in the range 120–160 for optimal performance in wound healing applications, were studied by dynamic light scattering (DLS), SEC MALLS and AFM. Results indicate that dilute aqueous SBG solutions (1 μg/ml to 3 mg/ml) contain higher order structures with a very wide size distribution in water (10–500 nm), corresponding to a mixture of single chains, multi-chain aggregates including triple-stranded motifs, and particulate materials. The latter were enriched in longer chains compared to non-particulate fractions. The size distribution of SBG aggregates shifted to slightly lower values upon heating, but showed hysteresis upon cooling. AFM images prepared from very dilute aqueous solution (1–5 μg/ml) analysis showed by comparison to other (1→3)-β- d -glucans that some of the structures were the triple helical species coexisting with larger aggregates and single chains, in contrast to carboxymethylated SBG, which contained predominantly single chains. The ability to control the aggregation behaviour of SBG enables tailoring of the physical, and possibly bioactive, properties of SBG preparations.

Alginates in the format of scaffolds provide important functions as materials for cell encapsulation, drug delivery, tissue engineering and wound healing among others. The method for preparation of alginate-based foams presented here is based on homogeneous, ionotropic gelation of aerated alginate solutions, followed by air drying. The method allows higher flexibility and better control of the pore structure, hydration properties and mechanical integrity compared to foams prepared by other techniques. The main variables for tailoring hydrogel properties include operational parameters such as degree of aeration and mixing times and concentration of alginate, as well as macromolecular properties such as the type of alginate (chemical composition and molecular weight distribution). Exposure of foams to γ-irradiation resulted in a dose-dependent (0-30 kGy) reduction in molecular weight of the alginate and a corresponding reduction in tensile strength of the foams.

Water-soluble (1→3)-β-D-glucans with 1,6-linked branches (SBG), originally isolated from the cell walls of Saccharomyces cerevisiae and partially depolymerised for optimal performance in wound healing applications, were studied by size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) detector and a viscosity detector at both high and ambient column temperatures. The strongly aggregating materials could be dispersed as single chains in water following partial carboxymethylation (degree of substitution (DS) 0.51 or higher). Lower DS (0.23) also dispersed as single chains provided a column temperature of 80 °C was applied. Reduction of reducing ends prior to carboxymethylation was required to avoid alkaline peeling and hence to obtain correct molecular weight distributions of the native material. DS was determined using (13)C NMR and potentiometric titration (range 0.23-0.91). Further analysis of CM-SBG in the single chain state suggested a randomly coiled behaviour with marginal influence of the branches in terms of macromolecular dimensions, which were close to those of CM-curdlan. The result of the investigation is a simple and reliable protocol for preparing undegraded and un-aggregated SBG derivatives, which are well suited as a standard analysis of the molecular weight distribution of SBG-like molecules.

The localisation of pectin in Sphagnum moss leaves and its role in preservation has been investigated. Light microscopy using ruthenium red to detect pectin in whole and sectioned Sphagnum papillosum leaves revealed it is abundant in hyaline cell walls, fibrils, papillae, chlorophyllous cell walls and thickenings around hyaline cell pores. Transmission electron microscopy of ultrathin cell walls labelled with poly- l -lysine colloidal gold revealed pectin was distributed throughout the cell wall. The preservative/microbiocidal properties of these pectins are explained by the acid-dissociation properties of galacturonic acid carboxyls and their incorporation in the unique cell arrangement of the Sphagnum leaf. Liquid from a salmon fillet absorbed into S. papillosum leaves and incubated at room temperature for 22 h had a pH around 4.85, was dominated by Lactobacillus sp. and smelled fresh compared to experimental controls. Chlorite-treated Sphagnum leaves could have a potential as a food tray pad that absorbs liquid and prevents the growth of spoilage bacteria inside it.

An isothermal microcalorimeter has been used to study ageing of oil impregnated kraft paper. Under the assumption that the heat flow is proportional to the ageing rate of paper it is found that activation energy seems to be lower for oxidation than for hydrolysis. This observation corresponds well with results obtained by traditional ageing methods. The results have also been confirmed by measuring the changes in DP of the samples used in the calorimetric measurements. Comparing degradation of aged and unaged oil impregnated paper in air gave a higher heat flow for the unaged paper, but the activation energy for the processes remained the same. The process was also studied with another cellulosic material and in addition varying size of the sample holder of the calorimeter, giving similar results. Hence, since the microcalorimeter appears to give the same qualitative ageing characteristics for oxidation and hydrolysis as more time consuming methods, calorimeter could be a useful tool for quick ageing investigations.

Limited periodate oxidation (0–8%) of alginates results in enhanced degradability, extending the range of applications of alginates as biomaterials, e.g. in tissue engineering. Oxidation produces dialdehydes that are highly sensitive to alkaline β-elimination, even under physiological conditions (pH 7.4, 37 °C). Although all periodate is consumed during limited oxidation, not all of the resulting dialdehydes are equally degradable, leading to a characteristic level off in molecular weight upon prolonged degradation. A significant fraction (20–50%) of the oxidized residues were resistant even at pH 10.4. We tentatively assign this finding to the presence of particularly stable intramolecular hemiacetals known to form in periodate oxidized alginates (POA). The enhanced degradation of POA persists into the gel state, allowing tailoring of more biodegradable alginate gels. The activation energy for β-elimination of dialdehydes was 88 kJ/mol compared to 113 kJ/mol for unoxidized residues, presumably reflecting the absence of strain in the non-cyclic transition state of the former.

Fully de-N-acetylated chito-oligomers were prepared by de-amination of chitosan using nitrous acid (HNO2). The dimer to hexamer fractions of chitosan oligomers were fractionated with preparative size-exclusion chromatography (SEC) and characterized using 1H NMR spectroscopy. The purified oligomers were polymerized by reductive N-alkylation by the action of NaCNBH3, in a reaction between the terminal 2,5-anhydro- d -mannofuranose (M-unit) and the primary amines of the repeating units (1 → 4) bound β- d -glucosamine (D-unit) resulting in a covalent bond and formation of secondary amines. The polymerized products were studied by SEC-RI and SEC-MALLS-RI and were confirmed to be branched reaching molecular weights of up to 10 000 g/mol. Analysis by 1H NMR spectroscopy confirmed that the chitosan oligomers had been branched by formation of secondary amines. Fully de-N-acetylated chitosan (FA

We studied the effect of the mole fraction ( F M ) of β- d -mannuronate residues in alginate chains and the weight-average molecular weight ( M w ) of alginates on intestinal immunological activity through Peyer's patch cells of C3H/HeJ mice. Over most of the range of M w (30,000–690,000), alginates with high mannuronate content (high-M alginates, F M 0.69–0.86) showed immunological activity, but alginates with F M lower than 0.31 did not. For high-M alginates with F M = 0.78 extracted from Laminaria japonica , those with M w lower than 200,000 showed the highest activity.

The paper presents a new investigation of a native and a hydrophobically modified hyaluronan (HMHA) C8NH-HA100 interactions with surfactants SDS, DTAB, and a dye polarity probe Coomassie Brilliant Blue (CBB), and their macromolecular properties in aqueous environment using isothermal titration calorimetry (ITC) and SEC-MALLS. A novel alkylated HA derivative was prepared by the introduction of octyl chains onto –OH groups of the HA d-glucuronic acid unit via carbamate bond at a high degree of substitution preserving the HA carboxylic groups. Large and abrupt endothermic enthalpy changes (ΔH) during the titration of HA by DTAB evidence a strong electrostatic interaction and a formation of complex aggregates between HA and DTAB micelles. Heat effects of HA–SDS titration are moderate and reveal a lowering of the surfactant CMC in the presence of HA. The C8NH-HA100 displays binding with CBB in terms of exothermic enthalpy changes, which is ascribed to hydrophobic interactions, while such binding is not observed for HA. The assessment of the polysaccharides backbone stiffness and the coefficient of the Mark–Houwink–Sakurada plot (MHS), a, by SEC-MALLS shows a little alternation of the HA backbone stiffness after its modification.

A limited degree (typically 1-20%) of periodate oxidation of polysaccharides may give rise to derivatives with entirely altered chemical and physical properties. Notably, the ring opening caused by periodate leads to the formation of highly flexible 'hinges' in otherwise rather semiflexible or rigid structures. This review highlights selected examples with the main focus on periodate oxidation of alginates, chitosans, hyaluronan, scleroglucan/schizophyllan, and cellulose.

Novel alginates prepared by independent control of chain stiffness and distribution of G-residues : Structure and gelling properties

The carbonyl content of a pectic polysaccharide from Sphagnum papillosum (sphagnan) and periodate oxidised alginates was investigated using three different carbonyl labelling strategies combined with size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and on-line fluorescence or off-line tritium detection. The labelling strategies were tritium incorporation via NaB3H4 reduction, and fluorescent labelling with carbazole carbonyl oxyamine (CCOA), or 2-aminobenzamide (2-AB), respectively. Carbonyl quantification was based on labelled pullulan, dextran and alginate standards possessing only the reducing end carbonyl group. As a result the carbonyl distribution in the polysaccharides could be determined. In sphagnan it was found that the carbonyl content increased with increasing molecular weight, whereas in periodate oxidised alginate the carbonyl content was as expected independent of the molecular weight. The methods proved useful for carbonyl detection in water soluble polysaccharides in general. The tritium incorporation method was preferred for alkali stable polysaccharides, while the CCOA method was most suitable for acid stable polysaccharides with low carbonyl content. The 2-AB method is applicable for all polysaccharides tested with varying carbonyl content; however, it lacks the ability to detect ketone functionalities.

Chitosans with different chemical compositions ( F A = 0.05–0.65) and different molecular weights ( M w = 36.000–460.000) were studied by size-exclusion chromatography combined with multi-angle laser light scattering (MALS) and viscosity detectors to provide R G – M and [ η ]– M data as basis for further analysis using the wormlike chain model. In both cases intrinsic persistence lengths ( q 0 ) in the range 5.1–7.6 nm were obtained, with little or no detectable dependence on F A . These values are significantly lower than values obtained for alginates (12 nm), including homopolymeric mannuronan, using the same approach. This finding is also corroborated by differences in the Smidsrod B -parameter, confirming that chitosans are less extended than alginates, despite the similarity in basic chain geometry (cellulose type) and linear charge density. Partial periodate oxidation of chitosans led to a pronounced increase in the chain flexibility as shown by a gradual decrease in persistence length, approaching 2 nm for the most oxidised samples.

Summary The industrially widely used polysaccharide alginate is a co-polymer of β-d-mannuronic acid and α-l-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1–7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1–4 and algE6–7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.

The purpose of this research was to evaluate if pectin-like polysaccharides, collectively known as sphagnan, extracted by acid hydrolysis from the leaves of Sphagnum moss have a unique ability to react with phenylhydrazine, o-phenylenediamine or collagen. A previous assay for determination of carbonyl groups in sphagnan by reaction with phenylhydrazine was disproved due to spectrophotometric interference from furans. The actual carbonyl content of sphagnan is estimated to be much less than previously thought. NMR spectroscopy showed that small amounts of o-phenylenediamine and/or its oxidation products bind to sphagnan probably via imine formation, but evidence of quinoxaline formation was inconclusive. Sphagnan-gelatin mixtures formed complex coacervates at pH 2.0-4.8 at low ionic strength, which is typical of electrostatic polyelectrolyte interactions, rather than covalent carbonyl-amine reactions. Measurements of hydrothermal stability and collagenase-degradation of sphagnan-treated hide powder collagen suggest that sphagnan is a poor tanning agent. The results indicate the suggested preservative properties of sphagnan are not related to tanning.

Background A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution. Methods Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage. Results P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues. Conclusions A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.

Mild acid hydrolysis was used to release polysaccharides from the chlorite-treated leaves of Sphagnum papillosum . These polysaccharides, collectively known as sphagnan, were physically and chemically characterised. No evidence was found of the presence of a previously described 5-keto- d -mannuronic acid (5-KMA) monomer in sphagnan, or in the chlorite-treated leaves from which sphagnan was extracted. After fractionation by anion-exchange chromatography the majority of polysaccharides were similar to rhamnogalacturonan I-type pectins. They were highly branched and had a weight average molecular weight of 3.9 × 10 g/mol.

The aim of this study was to improve the gene delivery efficacy of chitosan oligomer polyplexes by introducing a trisaccharide branch that targets cell-surface lectins. For this purpose, chitosan oligomers were substituted by a trisaccharide with the N-acetylglucosamine residue at the free end, and the ability of the trisaccharide-substituted chitosan oligomers (TCO) polyplexes to transfect various cell lines in vitro and lung tissue after in vivo administration to mice was investigated. Live-cell confocal microscopy showed improved cellular uptake in HEK 293 cells (11-fold, p

Alginate is a linear copolymer of β- d -mannuronic acid and its C-5-epimer, α- l -guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system ( eexDEF ). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (≤2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.

A series of alginates isolated from the stem and leaf of a brown algae (Laminaria hyperborea), bacterial mannuronan, in vitro epimerized mannuronans, and periodate oxidized alginates were analyzed by size-exclusion chromatography (SEC) combined with online multiangle laser light scattering (MALS) and viscometry (collectively abbreviated SMV). Selected samples were also analyzed off-line using low-angle laser light scattering and capillary viscometry. Excellent agreement between the two methods was obtained for properly purified samples. In contrast, abnormal results were obtained for some industrial samples due to the presence of particulate material. Naturally occurring alginates and in vitro epimerized mannuronans were found to obey essentially the same RG-M and [eta]-M relations, and hence, the same Mark-Houwink-Sakurada (MHS) equations (valid for I = 0.10 M): 20 000 g/molM100 000 g/mol, [eta] = 0.0054 .M(1.00); 100 000 g/molM1 000 000 g/mol, [eta] = 0.071 .M(0.89). Application of the wormlike chain model to the [eta]-M data obtained by SMV yielded persistence lengths (q) of 15 nm for all alginates at an ionic strength of 0.17 M. Intrinsic viscosities corresponding to infinite ionic strength were estimated on the basis of Smidsrød's B-parameter, and the wormlike chain model then yielded q = 12 nm. Periodate oxidized alginates showed, in contrast, a pronounced decrease in persistence length with increasing degree of oxidation, reaching values below 4 nm at 44% oxidation. Periodate oxidation also resulted in some depolymerization, even in the presence of a free-radical scavenger.

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter . The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

Fungi cause serious problems in wood utilization, and environmentally benign wood protection is required as an alternative to traditional chemicals. Chitosan has shown promising antimicrobial properties against several microorganisms. In this study, we present the characterization of and antifungal properties of a commercial chitosan formulation developed for impregnation of wood. A broad range of chemical and mycological methods were used to evaluate the uptake, fixation, and antifungal properties of chitosan for wood preservation. The results show that the higher the uptake of chitosan the lower the relative recovery of chitosan in wood after leaching, and the higher the molecular weight of chitosan the higher the recovery. Chitosan with high molecular weight proved to be more efficient against decay fungi than chitosan with low molecular weight. The fungi tested on chitosan-amended nutrient agar medium were totally inhibited at 1% (w/v) concentration. In decay studies using small wood blocks, 4.8% (w/v) chitosan concentration gave the best protection against brown rot fungi.

Lignosulfonates obtained from spruce (Picea abies), aspen (Populus sp.) and two species of Eucalyptus (E. globulus and E. grandis) were characterized by aqueous size exclusion chromatography (SEC) combined with in-line multi-angle laser light scattering (MALLS). In general, the hardwood lignosulfonates were shifted to lower molecular weights (M w = 5.700–12.000 g/mol) as compared to softwood lignosulfonates (M w = 36.000–61.000 g/mol). Lignosulfonates from E. grandis were further fractionated to obtain fractions of different molecular weights (3.500–30.000 g/mol). The degree of sulfonation increased with decreasing M w for the fractions as previously found for fractions of spruce lignosulfonate (Fredheim, G.; Braaten S.M.; Christensen, B.E. Molecular weight determination of lignosulfonates by size exclusion chromatography and multi-angle laser lightscattering. J. Chromatogr. 2002, 942, 191–199). The relationship between the intrinsic viscosity (in 0.1 M NaCl) and molecular weight was essentially ...

The trimer 2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1--4)-2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1--4)-2,5-anhydro-D-mannofuranose (A-A-M) was reductively N-alkylated onto a fully de-N-acetylated chitosan (F(A)0.001, DP(n)=25) to obtain branched chitosans with degree of substitution (DS) of 0.070, 0.23 and 0.40, as determined by 1H NMR spectroscopy. The apparent pK(a) values of the primary and secondary amines of the chitosans substituted with the trimer A-A-M were determined by monitoring the chemical shift of the H-2 of GlcN, and were determined as 6.5-6.9 for the primary (unsubstituted) amines and as 5.0-5.2 for the secondary (substituted) amines. The intrinsic pK(a) values (pK(int)) were found to be 7.3-7.4 for the substituted and 8.7 for the unsubstituted amines. The chitosan branched with A-A-M (DS 0.40) was found to be soluble in aqueous solution over the entire pH range. SEC-MALLS (size-exclusion chromatography with a multi-angle laser light scattering detector) further showed that addition of branches did not affect the molar hydrodynamic volume of the chitosan.

Oat β-glucan was partially degraded with (1→4)-β- d -glucan 4-glucanohydrolase for different periods of time. Weight average molecular weight and weight average intrinsic viscosity were obtained from SEC-RI-RALLS-Visc and ranged from 2200 to 213,900 g/mol and 7 to 316 ml/g, respectively. The viscosity equation determined [ η ] w =1.06×10 −2 M w 0.86 indicated an extended random coil conformation. When the coil was modelled as a worm-like chain a persistent length of 3.65 nm was obtained. The hydrolysis products identified after extensive degradation were glucose, cellobiose, laminaribiose, 4-O-β-laminaribiosyl d -glucose, 4-O-β-laminaribiosyl d -cellobiose and 3-O-β-cellobiosyl d -cellobiose showing that other enzyme activities were present. The depolymerization kinetics suggested that longer sequences of consecutive (1→4)-linkages represent fast hydrolysable sites and the presence of higher relative proportions of (1→3)-linkages restricts the affinity of the enzyme.

Polysaccharides such as scleroglucan and schizophyllan contain periodate-resistant β-1,3-linked d -glucan backbones with regularly distributed side chains consisting primarily of single β-1,6-linked d -glucose residues that are susceptible to periodate oxidation. Side chain oxidation results in the formation of dialdehyde groups and the production of formic acid. During the initial periodate oxidation an increase in the storage modulus (G′) associated with the formation of gels, was observed when the reduced concentration, Cp [η] was above 3. These gels appeared to be physically stable, whereas aldehyde reduction (e.g. with NaBH4) or oxidation to carboxyl (e.g. with NaClO2 in acetic acid) dissolved the gels. The mechanism of cross-linking can be explained by the formation of intermolecular hemiacetal linkages. The gels were further characterised rheologically in terms of the initial increase in the storage modulus and the stability when stored in seawater at 55 and 90°C, respectively.

Two chitosans with widely different chemical composition (fraction of N-acetylated units (F(A))