Hanno Baumann

Concepts for improved regioselective placement of O-sulfo, N-sulfo, N-acetyl, and N-carboxymethyl groups in chitosan derivatives☆

In the present paper a new strategy has been studied to introduce solely or in combination N-sulfo, O-sulfo, N-acetyl, and N-carboxymethyl groups into chitosan with highest possible regioselectivity and completeness and defined distribution along the polymer chain. The aim was to generate compounds having lowest toxicity for determining the pharmacological structure function relationships among different backbone structures and differently arranged functional groups compared to those of heparin and heparan sulfate. The water-soluble starting material, chitosan, with a degree of acetylation (DA) of 0.14 and a molecular weight of 29 kD, allows one to apply most of the known reactions of chitosan as well as some reactions of heparin chemistry successfully and with improved regioselectivity and completeness. On the other hand, a number of these reactions were not successful by application to water-soluble high-molecular-weight chitosan (DA 0.45 and 150 kD). The starting material showed statistical N-acetyl (N-Ac) distribution along the polymer chain according to the rules of Bernoulli, with highest abundance of the GlcNAc-GlcNAc diad along with a lower abundance of triads, tetrads, and pentads. The space between the N-Ac groups was filled up in homogeneous reactions by N-sulfo and/or N-carboxymethyl groups, which also resulted in a Bernoulli statistical distribution. The N-substitution reaction showed highest regioselectivity and completeness with up to three combined different functional groups. The regioselectivity of the 3-O-sulfo groups was improved by regioselective 6-desulfation of nearly completely sulfated 3,6-di-O-sulfochitosan. By means of desulfation reactions, all of the possible intermediate sulfated products are possible. 6-O-Sulfo groups can also be introduced with highest regioselectivity and completeness, and a number of partially 6-desulfated products are possible.

Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.

Photochemical immobilization of heparin, dermatan sulphate, dextran sulphate and endothelial cell surface heparan sulphate onto cellulose membranes for the preparation of athrombogenic and antithrombogenic polymers.

Heparin (HE), dextran sulphate (DX) of molecular weight 40000 and 500000, dermatan sulphate (DS) and endothelial cell surface heparan sulphate (ES-HS) were immobilized covalently onto cellulose membranes (Visking dialysis tubes) using the photochemical heterobifunctional reagent 4-azido-1-fluoro-2-nitrobenzene (AFNB); 120 pmol HE/cm2 and 40 pmol DS/cm2, 3.4 pmol DX 500,000/cm2, 50 pmol DX 40,000/cm2 and 3.6 pmol ES-HS/cm2 were immobilized. The platelet adhesion of the modified membranes was measured in a modified Baumgartner perfusion chamber with citrated human blood at a defined shear rate. Membranes modified with DX 40,000 and DX 500,000 showed 80% and 30% platelet adhesion, respectively, heparinized and DS coated membranes showed 50% and 60% platelet adhesion, respectively, compared with a subendothelial matrix (100% platelet adhesion). ES-HS modified membranes showed no platelet adhesion.

Novel regio- and stereoselective O-6-desulfation of the glucosamine moiety of heparin with N-methylpyrrolidinone–water or N,N-dimethylformamide–water mixtures

The degree of completeness and selectivity of the solvolytic O-6-desulfation reactions of the glucosamine moiety adjacent to the 2-O-sulfoiduronic acid group of heparin was systematically studied. Using solutions of various ammonium salts of heparin (salts of tributylamine, quinoline and pyridine) in mixtures of 9:1 aprotic solvents and water (solvents of medium polarity, in order of decreasing polarity: Me2SO > Me2NCHO > Me2NAc > N-methylpyrrolidinone), the influence of different reaction conditions were studied. The ammonium salt of heparin with a strong base (e.g., tributylamine) in Me2SO showed almost no desulfation, while in Me2NCHO a relatively low degree of completeness of O-6-desulfation (30%) with moderate selectivity (15% [I-2(OS)]-desulfation) was observed. Weak bases like quinoline or pyridine in Me2SO-water resulted in nearly complete [A-6(OS)]-desulfation (95 and 94%, respectively) with low selectivity [I-2(OS)]-desulfation (49 and 35%, respectively). The heparin pyridinium salt in Me2NCHO-water showed both a relatively high degree of completeness and high selectivity (72% [A-6(OS)]- and 8% [I-2(OS)]-desulfation). The highest regioselectivity (i.e., a high degree of completeness accompanied by high selectivity) was achieved using an N-methylpyrrolidinone-water mixture (88% [A-6(OS)]-desulfation and 10% [I-2(OS)]-desulfation). A nearly complete O-6-desulfation (95%), accompanied by a lower selectivity (18% [I-2(OS)]-desulfation), was achieved when the reaction was carried out twice. Lower temperature improved selectivity (5% [I-2(OS)]-desulfation) but reduced the completeness of [A-6(OS)]-desulfation (72%). In comparison with the variety of O-6-desulfations reported to date, the novel reactions presented in this article led to remarkable increase in completeness and regioselectivity of the reactions that were investigated.

Regioselectively Modified Cellulose and Chitosan Derivatives for Mono- and Multilayer Surface Coatings of Hemocompatible Biomaterials

In this study different synthetic strategies were developed and applied to introduce solely or in combination heparin/heparansulfate-like functional groups such as N-sulfo, O-sulfo, N-acetyl, and N-carboxymethyl groups into chitosan and cellulose with highest possible regioselectivity and completeness and defined distribution along the polymer chain. Completely substituted 6-amino-6-deoxycellulose and related derivatives were prepared from tosylcellulose (DS 2.02; C6 1.0) by nucleophilic substitution with azido groups only in the 6-position at 50 °C with subsequent reduction to amino groups and completely removing tosyl groups in the 2,3-position. 2,6-Di-O-sulfocellulose was prepared using the reactivity difference between C-2, C-6 and C-3 of cellulose. The reactivity difference between amino groups and hydroxyl groups was used to prepare various N-substituted derivatives. Partially 2,6-di-O-sulfated cellulose was obtained from trimethylsilylcellulose by the insertion of sulfurtrioxide into the Si–O ether linkage. Partially 3-O-sulfocellulose was synthesized by protecting C-2 and C-6 with trifluoroacetyl groups. A copper–chitosan complex was used to synthesize 6-O-sulfochitosan with a DS of 1.0 at C-6 and various partially 6-O-desulfonated products are possible. Using the phthalimido group to increase the solubility of chitosan in DMF, the regioselectivity of 3-O-sulfo groups was improved by regioselective 6-O-desulfonation of nearly complete 3,6-O-disulfochitosan. The platelet adhesion properties of immobilized regioselectively modified water-soluble derivatives on membranes have been tested in vitro. Some regioselectively modified chitosan and cellulose derivatives are potential candidates for the surface coatings of biomaterials if the regioselective reactions are somewhat further optimized.

Surface modification of the polymers present in a polysulfone hollow fiber hemodialyser by covalent binding of heparin or endothelial cell surface heparan sulfate: Flow characteristics and platelet adhesion

The present study addresses the problem of simultaneous surface modification of various polymers, i.e. polysulfone (PSU), polycarbonate (PC), and polyurethane (PU), which constitute the Ultraflux AV 600 S® hollow fibre hemodialyser. An investigation was first made into six different chemical routes aimed at introducing carboxyl groups onto the surface of PSU, PC, and PU model polymers to which heparin (HE) or endothelial cell surface heparan sulfate (ESHS) was covalently bound via the reaction of residual amino groups and a coupling reagent. Carboxyl groups were introduced using three specific reactions based on their nucleophilic or electrophilic introduction into aromatic repeating units of the polymers and three non-specific carboxylation reactions, i.e. UV, heat or redoxactivation via nitrene or radical species. Concentrations of 1-20 nmol COOH groups per cm-2 led to HE or ESHS surface concentrations corresponding to one or several layers. Two nonspecific carboxylation reactions followed by HE- or ESHS-coupling provided the lowest change in membrane pore structure according to cut off, clearance (urea, phosphate, maltose), ultrafiltration, and diafiltration assessments. In some cases the introduction of excess negatively-charged carboxyl groups and HE improved the flux properties of the modified membranes. The various methods were applied to the dialysis module. Platelet adhesion was not observed in the case of the ESHS-coating of PSU membrane at shear rates of 1050 s-1, whereas HE and subendothelial matrix showed 56 and 100% coverage, respectively, under similar conditions. The coating of PSU or of other highflux membranes by ESHS appears a promising method for improving membrane properties and to generate biocompatibility characteristics similar to those of natural blood vessels, i.e. inertness to platelet adhesion and no level effects for complement and intrinsic coagulation cascade activation. The ESHS coating may be used without anticoagulants.

Studies on Some Wool Components : Skin Flakes, Cuticle, and Cell Membrane Material

When wool is vigorously shaken in formic acid, initially, the skin flakes and; later on, cuticle cells are liberated with a dissolution of some wool protein. The rate of release of skin flakes and dissolution of protein is increased with increasing periods of shaking wool in formic acid. The cuticle is isolated by ultrasonication of wool in formic acid. Amino acid compositions of wool and its components are determined and discussed in terms of helix and nonhelix indices, a degree of polarity, and cystine content. Skin flakes contain only cysteic acid but no cystine, double the amount of aspartic acid, half the ambunt of proline and tyrosine, and more glycine than the respective amino acids present in cuticle. Electron microscope studies revealed that the skin flakes and cuticle differ from each other in their structural features.

Endothelial Cell Surface Heparan Sulfate (ESHS) and Synthetic Heparin Derivatives as Hemocompatible Coating for Biomaterials

In the present overview a coating procedure, that has been developed in our working group for medical devices e. g. implants, which are exposed to permanent blood contact and therefore have to fulfill the highest standard of hemocompatibility is described. For this purpose an endothelial cell surface heparansulfate, which belongs to the class of glycosaminoglycans is used as coating substance. This substance can be isolated from endothelial cell culture, tissue extracts or organ perfusates. Alternatively chemical regio- and stereoselective modified derivatives of the structurally related anticoagulant heparin were brought to action. These substances are anchored covalently or ionically by application of a wide spectrum of immobilization techniques on many different material surfaces. Polymer materials modified as described here have been tested for hemocompatibility in invitro and in invivo experiments with Austrian sheep. The results show, that the described method is an advanced solution for the creation of long term hemocompatible artificial material surfaces.

Biocompatibility testing of new polymers in a moving implant bed.

Total reconstruction of the auricle requires a skilful surgical technique and an appropriate material for the shape-supporting frame. Up to now, there is no such material apart from autologous rib cartilage. The combination of chronic microtraumatisation of adjacent tissue caused by the mobility of an implant bed such as the auricle and the foreign-body reaction to currently available artificial polymers frequently results in extrusion. In our animal model (rats), polymers of different elasticity were implanted in a moving implant bed to analyse differences in foreign-body reaction related to implant elasticity. The results were significantly better for a rather stiff control material (porous polyethylene). A contributing factor may be better fixation of the implant material by tissue ingrowth into its micropores.

Isolation and Characterisation of Endothelial Cell Surface Heparan Sulphate from Whole Bovine Lung for Coating of Biomaterials to Improve Haemocompatibility

Heparan-sulphate was isolated for the first time as a byproduct of commercial heparin isolation in 1948 and described as a low sulphated heparin by Jorpes and Gardell. During early stage some researchers thought heparan-sulphate to represent a new class of glycosaminoglycans. Others supposed it to be an intermediate of the heparin-biosynthesis. In contrast to heparin, which is found in the granules of mastocytes, heparan-sulphate occurs ubiquitous on cell surfaces . This fact clarifies former distinguishing problems among the two glycosaminoglycans who nowadays represent the so called type 2 class of GAGs. Heparin as well as heparan sulphate obtain the same backbone structure which is composed from an alternating copolymer of uronic acid and glucosamine. Apart from the different occurrence in mammalians the type 2 GAGs also differ significantly e.g. according to their degree of sulphation, acetylation and especially according their structural heterogeneity which is exposed in the distinct domain structure of the heparan sulphates. The first researcher who described heparane sulphate localized on the luminal surface of endothelial