Three D structures of chitosan

Crystal structures of two polymorphs of chitosan, tendon (hydrated) and annealed (anhydrous) polymorphs, have been reported. In both crystals, chitosan molecule takes up similar conformation (Type I form) to each other, an extended two-fold helix stabilized by intramolecular O3-O5 hydrogen bond, which is also similar to the conformation of chitin or cellulose. Three chitosan conformations other than Type I form have been found in the crystals of chitosan-acid salts. In the salts with acetic and some other acids, called Type II salts, chitosan molecule takes up a relaxed two-fold helix composed of asymmetric unit of tetrasaccharide. This conformation seems to be unstable because no strong intramolecular hydrogen bond like Type I form. Type II crystal changes to the annealed polymorph of chitosan by a spontaneous water-removing action of the acid. Chitosan molecule in its hydrogen iodide salt prepared at low temperature takes a 4/1 helix with asymmetric unit of disaccharide. The fourth chitosan conformation was found to be a 5/3 helix in chitosan salts with medical organic acids having phenyl group such as salicylic or gentisic acids. Similar conformation of chitosan molecule in the aspirin (acetylsalicylic acid) salt was suggested by a solid-sate NMR measurement.     

Fourth 3D structure of the chitosan molecule: conformation of chitosan in its salts with medical organic acids having a phenyl group

Chitosan salts with two medical organic acids having phenyl groups (salicylic and gentisic acids) exhibited fiber diffraction patterns of a new type of crystal which does not compare with known types I and II. The crystals, called type III salts, showed a fiber repeat of 2.550 nm and a meridional reflection at the 5th layer line. These results coupled with a conformational analysis indicate the chain conformation of chitosan with the salts to be a 5/3 helix, this helix differing from those of type I (an extended two-fold helix) and type II (a relaxed two-fold helix or a 4/1 helix). The fiber patterns of all the type III salts were similar. This observation has also been found with type II salts and is an indication that the acid ions are not arranged in regular positions in the crystals. A comparison of solid-state 13C-NMR spectra of the gentisic acid salt and the aspirin salt, which could not be crystallized, suggests that, in the latter salt, the chitosan molecules also formed a 5/3 helix.     

Crystalline Behavior of Chitosan Organic Acid Salts

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous “Annealed” polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated “Tendon” polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Crystalline behavior of chitosan organic acid salts.

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous "Annealed" polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated "Tendon" polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Plausible molecular and crystal structures of chitosan/HI type II salt

Chitosan/HI type II salt prepared from crab tendon was investigated by X-ray fiber diffraction. Two polymer chains and 16 iodide ions (I(-)) crystallized in a tetragonal unit cell with lattice parameters of a = b = 10.68(3), c (fiber axis) = 40.77(13) A, and a space group P4(1). Chitosan forms a fourfold helix with a 40.77 A fiber period having a disaccharide as the helical asymmetric unit. One of the O-3... O-5 intramolecular hydrogen bonds at the glycosidic linkage is weakened by interacting with iodide ions, which seems to cause the polymer to take the 4/1-helical symmetry rather than the extended 2/1-helix. The plausible orientations of two O-6 atoms in the helical asymmetric unit were found to be gt and gg. Two chains are running through at the corner and the center of the unit cell along the c-axis. They are linked by hydrogen bonds between N-21 and O-61 atoms. Two out of four independent iodide ions are packed between the corner chains while the other two are packed between the corner and center chains when viewing through the ab-plane. The crystal structure of the salt is stabilized by hydrogen bonds between these iodide ions and N-21, N-22, O-32, O-61, O-62 of the polymer chains.     

Molecular and crystal structures of chitosan/HI type I salt determined by X-ray fiber diffraction

The three-dimensional structure of chitosan/HI type I salt was determined by the X-ray fiber diffraction technique and linked-atom least-squares refinement method. Two polymer chains and four iodide ions (I(-)) crystallized in a monoclinic unit cell with dimensions a = 9.46(2), b = 9.79(2)], c (fiber axis)=10.33(2)A, beta = 105.2(2) degrees and a space group P2(1). Chitosan chains adopted an extended twofold helical conformation that was stabilized by O-3...O-5 hydrogen bonds, and the O-6 atom adopted nearly gt orientation. Polymer chains zigzag along the b-axis and directly connect to each other by N-2...O-6 hydrogen bonds. Two columns of iodide ions were shown to pack at the bending points of the zigzag sheets, and their locations are closely related to those of water columns in the hydrated chitosan. The iodide ions stabilized the salt structure by forming hydrogen bonds with the N-2 and O-6 atoms of the polymer chains together with an electrostatic interaction between N-2 and the iodide ions.     

Conformational preferences of the sequential fragments of the hinge region of the human IgA1 immunoglobulin molecule

The mean solution conformation of tetrapeptide fragments spanning the hinge region of human IgA1 was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences of IgA1 were found. In a series of tetrapeptides having the Thr-Pro-Pro-Thr sequence, the Pro-Pro fragment was ordered to the structure of a type II polyproline helix, but with unordered forms prevailing in the equilibria. In the case of the Pro-Pro-Thr-Pro sequence, a distinct preference for the beta-turn conformation was found. Acetylation of this tetrapeptide shifts the equilibrium towards unordered forms containing some elements of the type II polyproline helix. The peptide Thr-Pro-Ser-Pro exists predominantly in the beta-turn conformation whereas Pro-Ser-Pro-Ser-NH2 has, for the most part an unordered conformation.     

Effects of neutral salts on the conformational state of glycogen, interacting with iodine

The effects of salts on helix formation of linear (outer) chains of branched alpha-glucane, muscle glycogen, was judged upon by the state of polyiodine chromophore of the iodine reaction surrounded by a polysaccharide asymmetric helix and recorded by the circular dichroism method. It was demonstrated that apart from the known changes in the absorption of the iodine-glycogen complex the salts induce changes in the ellipticity by affecting the helix formation of the linear chains of the polysaccharide. The nature of these effects depends on the type and concentration of the salt used. Monovalent metal salts produce a more favourable effect on helix formation than the divalent metal salts. Among divalent metal salts transient metal salts produce a weaker effect. The action of neutral salts on helix formation is of dual nature: helix formation and helix destruction. The helix formation is largely impaired by high concentrations of LiCl (greater than 6 M) or CaCl2 (greater than 2M). It was shown that a certain role in the mechanism of neutral salt action on helix formation belongs both to electrostatic and lyotropic effects. The latter determine the effects of salts on helix formation at concentrations above the monomolar one, when the specific effect of a salt is especially well-pronounced. The salts which enhance the orderliness of H2O structure produce a favourable effect on helix formation of the linear polysaccharide chains and, consequently, on the iodine reaction. In terms of molar efficiency of their action on the iodine reaction the salts correspond to the Hofmeister lyotropic sequence.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.     

Alkaline chitosan solutions

Rigid and transparent hydrogels were obtained upon pouring chitosan salt solutions into saturated ammonium hydrogen carbonate. Incubation at 20 degrees C for 5 days yielded chitosan carbamate ammonium salt, Chit-NHCO(2)(-)NH(4)(+) a chemical species that either by hydrolysis or by thermal treatment decomposed to restore chitosan in free amine form. Chitosans of different degrees of acetylation, molecular sizes and origins (squid and crustaceans) were used as hydrochloride, acetate, glycolate, citrate and lactate salts. Their hydrogels obtained in ammonium hydrogen carbonate yielded chitosan solutions at pH values as high as 9.6, from which microspheres of regenerated chitosans were obtained upon spray-drying. These materials had a modest degree of crystallinity depending on the partial acylation that took place at the sprayer temperature (168 degrees C). Citrate could cross-link chitosan and impart insolubility to the microspheres. Chloride on the contrary permitted to prepare microspheres of chitosan in free amine form. By the NH(4)HCO(3) treatment, the cationicity of chitosan could be reversibly masked in view of mixing chitosan with alginate in equimolar ratio without coacervation. The clear and poorly viscous solutions of mixed chitosan carbamate and alginate were spray-dried at 115 degrees C to manufacture chitosan-alginate microspheres having prevailing diameter approx 2 micron.     

Conformational Behavior of Chitosan in the Acetate Salt: An X-Ray Study

A well-defined X-ray fiber pattern of chitosan acetate was obtained by immersing a tendon chitosan, prepared from a crab tendon chitin by a solid-state N-deacetylation, in an aqueous acetic acid-isopropanol solution at 110°C. This pattern was very similar to that of chitosan salts with some inorganic acids, such as HF, HCl, and H₂SO₄, in which chitosan chains form an 8/5 helix, indicating that chitosan acetate also take up this conformation. This information may give an influential clue to the chitosan conformation in the aqueous acetic acid solution, the most popular solvent for chitosan. However, after one month of storage of the chitosan acetate, the fiber pattern, the density and its IR spectrum changed to those of the anhydrous polymorph of chitosan, suggesting that the acetic acid was removed accompanied with water molecules from the crystal during storage and that the polymorph can be obtained not only by annealing chitosan, but also through the chitosan acetate.     

The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

Cobalt (II) imprinted chitosan for selective removal of cobalt during nuclear reactor decontamination

The selectivity of chitosan has been modified through metal ion imprinting technique for its potential application in nuclear industry. Considerable reduction in radioactive waste volume, generated during the chemical decontamination of nuclear power plants, can be achieved through the selective removal of the radionuclides. In this context, a Co(II) imprinted chitosan was synthesized using epichlorohydrin as the crosslinker. The selective removal of Co(II) in presence of Fe(II), which is the major non-radioactive ion present in excess during decontamination, was studied. The imprinted chitosan showed selective sorption of Co(II) over Fe(II), while the raw chitosan was selective to Fe(II) over Co(II). The imprinted chitosan was found to retain the enhanced selectivity towards Co(II) under various solution conditions, including typical nuclear reactor decontamination formulations containing strong complexants. The highest uptake by the imprinted chitosan, with maximum selectivity for Co(II) over Fe(II), was obtained in citrate medium at pH 4.8.     

X‐ray crystal structure of anhydrous chitosan at atomic resolution

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P2₁2₁2₁] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016.     

Hydration of the left spiral of the poly-L-proline type. Study by the Monte Carlo method]

The paper exhibits results of hydration shell Monte Carlo calculations in poly-L-proline II and extended helix conformation and in alpha-helical and beta-structural conformations for comparison. It was found that left-handed helix of poly-L-proline II type as well as epsilon-helix are characterized by very favorable hydration. Therefore this conformation has preference as compared to other standard conformations of the main polypeptide chain. This determined inevitability of cold denaturation of protein.     

A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.     

Interaction between chitosan and uranyl ions: Part 1. Role of physicochemical parameters

This work is devoted to the comprehension of the sorption mechanism of uranyl ions on chitosan particle dispersions. The uranyl concentration measurements were obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES) and we considered the role of various physicochemical parameters (pH; nature and concentration of added salts; degree of acetylation, DA). The use of appropriate calculation software allowed us to determine the chemical nature of uranyl species in solution in relation to these different parameters. The optimal pH of fixation has been found to be within 6.5–7.5 and can be related to the necessity of having both deprotonated amino groups and no carbonate ions, which are a strong complexant of uranyl ions, thus inhibiting their interaction with chitosan. The decrease of metal uptake with an increase of DA and the lack of influence of ionic strength, confirm the results obtained with pH and allowed us to suppose the formation of a complex with chitosan amino groups rather than interactions of an electrostatic nature.     

Optical properties of the polyglycine II helix

The circular dichroism spectrum obtained from a dilute aqueous solution of poly (ala-gly-gly) resembles that described for charged polypeptides such as the salt form of poly glutamic acid. A similar spectrum is found for films cast from aqueous solution where x-ray studies reported elsewhere have indicated a poly-glycinc II conformation. Evidence is presented for a heat induced poly-glycine II to unordered state transition similar to that described for collagen. The interpretation of this, the first observation of the optical properties of a poly-amino acid in the poly glycine II conformation, is further rationalized on the basis of spectra obtained from a number of polypeptides whose conformation approaches that of a 3₁ helix.     

Spectral studies of beta-melanocyte-stimulating hormone and beta-lipotropic hormone fragment]

Secondary structure of beta-melanocyte-stimulating hormone was studied by circular dichoism and infra-red spectroscopy. Left-helical conformation of poly-L-proline II type which was stabilized by temperature reduction was found in aqueous solution; in 60% ethanole the quota of this structure sharply decreased. The investigation of hormone films at different values of relative humidity (in the course of H--D metabolism) made it possible to discover a twisted beta-form and an elongated helix of poly-L-proline II type. Temperature induced changes of circular dichroism spectra specify the peculiarities of poly-L-proline II conformation in C-end fragment of beta-lipotropic hormone.     

Evaluation of modified chitosan as matrix for hydrophobic interaction chromatography

Chitosan beads were modified with glutaraldehyde and modified chitosan was investigated as matrix for hydrophobic interaction chromatography. The influence of temperature, type of salt and its ionic strength on the adsorption of -galactosidase was studied. -Galactosidase was found to bind in presence of high concentration of ammonium sulphate (3 M, w/v) and 90% of the bound enzyme was eluted with decreasing salt concentration in presence of 10% ethylene glycol. Attempt was made to purify -galactosidase from modified chitosan, -galactosidase showed 1.7-fold purification with 43.96% recovery of enzyme activity. The SDS–PAGE analysis of enzyme showed considerable purification and its molecular weight was found to be 63–64 kDa. Unlike other chromatographic matrices, the prepared chitosan beads were used five times. The results showed that purification and recovery of the enzyme did not change even when column size was increased.