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SULFATION of CHITOSAN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Tfiilosophy In

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SULFATION of CHITOSAN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Tfiilosophy In SULFATION OF CHITOSAN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of tfiilosophy in the Graduate School of The Ohio State University 3jr (.Mrs.) Tsung-men Shen Han, B. Sc., M. Sc. The Ohio State University 19$U Approved by: ____ Advisor Department of TABLE OF CONTENTS Page I. Introduction and Statement of Problem. ......1 II. historical ..... 3 1. The Chemistry of Chitosan,......... ...3 2. The Sulfation of Polysaccharides................ ....7 III. Discussion of Results ........... 17 1. The Sulfation of Chitosan with Fyridine- Chlorosulfonic Acid..................... ....17 2. The Molecular Weight Study on Sulfated Chitosan 21 A. The Theory of Light Scattering........ 22 B. The Effect of Polymerelectrolytes............... 28 C. The Effect of Extraneous Particles............. .28 3. The Traube Sulfation............. 30 U. The Sulfation of Chitosan with Sulfur Trioxide- N,N-Dimethylf ormamide Complex..................... 31 3. The Anticoagulant Assay by the Sheep Plasma Method. .......... ........... ......3U 6. Model Compound Preparation....... 35 A. The Preparation of Tri-0-acetyl-2-amino- (hydrobromide)-2-deoxy- o£. -D-glucopyranosyl Bromide............ 36 i Page B. The Preparation of Methyl 2-Amino- 2-deoxy-N-sulfo-tri-O-sulfo--D- glucopyranoside Dibarium Salt................ 36 IV. Experimental............. ..UO 1. The Activation of Chitosan........ ..UO 2. The Sulfation of Chitosan with Chlorosulfonic Acid and Dry Pyridine.......................... !+0 3. The Molecular Weight Determination of the Sulfated Chitosan.......................... ip. A. Procedure ............... *Ul B. Calculations...... hh U. The Traube Sulfation...................... .. 32 3. The Sulfation of Chitosan with Sulfur Trioxide- N, N-Diinethylf ormamide Complex, ............... • 52 A. The Purification of N, N-Dimethylf ormamide.... .53 B. The Preparation of Sulfur Trioxide- NjN-DimethyIformamide Complex in N,N-Dimethylf ormamide........... ...53 C. The Solubility of the Compound to be Sulfated in N, N-Dimethylf ormamide............ 53 D. The Sulfation Procedure............ 5U 6. The Anticoagulant Assay by the Sheep Plasma Method................................. *5h ii Page A. The Determination of Calcium. Chloride for Recalcification of the Plasma.......... «.5H B. Plasma* ............ 55 C. The Preparation of Sarrqple Solution*.......... 55 D. The Anticoagulant Test. ......... ....56 E. The Evaluation of the Results................ 57 7 • Model Compound Preparation. ..... 58 A. The Preparation of Tri-O-acetyl-2-amino- (hydrobromide J-2-deoxy-^ -D-gluco- pyranosyl Bromide............... ••....••58 B. The Preparation of Methyl Tri-Q-acetyl- 2-amino-2-deoxy- ^ -D-glucopyranoside Hydrobromide. ............... 5 9 C. The Preparation of Methyl 2-Amino-2- deoxy- (3 -D-glucopyranoside Hydrochloride...... 60 D. The Preparation of Methyl 2-amino-2- deoxy-H-sulfo-tri-O-sulfo-^ -D- glucopyranoside Dibarium Salt................ 60 8. Calibration of the Hellige Turbidimeter for Sulfate Assay............................... ...61 A. Ins trumentation...... 61 5. Reagents. ................ 62 C. Procedure....................................62 iii Page 9, The Quantitative Estimation of Sulfur Content after the Hydrolysis of Methyl 2-Amino-2 -deoxy-N-sulf o-tri-O-sulfo- ^ -D-glucopyranoside Dibarium Salt with O.Olj. N Hydrochloric Acid, at 65 10, The Quantitative Estimation of Amino Nitrogen after the Hydrolysis of Methyl 2-Amino-2-deoxy-N-sulfo-tri-O- sulf 0- ^-D-glucopyranoside Dibarium Salt with 0.0U N hydrochloric Acid, at 95°.........,.6 6 V, Summary .................. 6 9 VI, Acknowledgment............... 71 VII, Collected Bibliography........................... ,72 VIII, Autobiography,........ 76 iv LIST OF FIGURES AND TABLES (in order of appearances) Page Table I* Methods of Sulfation and Effects on Polysaccharides. .............. ..12 Table II. Effect of Degree of Sulfation and Molecular Size and Shape on Anti­ coagulant Activity and Toxicity............. 15 Table III# Analysis of the Sulfated Chitosan... ........Id Figure 1. Variation of Amino Nitrogen (Van Slyke) with Time ...........». 2u Table IV, Dissymmetry and Correction Factors for Random Coil as a Function of Dimension/ ..............................27 Figure 2, Relationship Between Dissymmetry and Correction Factors.............. .29 Figure 3» Variation of the Sulfur Content of 2-Amino-2 -deoxy-N-sulfo-tri-O-sulfo- ^3 -D-glucopyranoside Dibarium Salt with Time of Hydrolysis at 93°, in 0.01+ N Hydrochloric Acid................ 3 8 Figure 1+. Variation of the Amino Nitrogen (ninhydrin) with Time............ ...........39 v Page Table V. Light Scattering Measurements on Sulfated Chitosan (Rrepared by the Pyridine-Chlorosulfonic Acid Method)....... «Uli Table VI. Light Scattering Measurements on Sulfated Chitosan (Prepared by the SCy HCON (CH3) 2 Method) ..................... h$ Table VII. The Value for the Filter Factors............ lj.6 Table VIII. Calculation from the Light Scattering Data in Table V. ................ ......U8 Table IX. Calculations from the Light Scattering data in Tabl^ VI.................. U8 Figure 5» Dissymmetry z Versus Concentration ........ h9 Figure 6. Molecular ./eight of Sulfated Chitosan in 0.2 M Sodium Chloride Solution (Prepared by the iyridine-Chlorosulfonic Acid Method).................................... 50 Figure 7. Molecular ./eight of Sulfated Chitosan in 0.2 M Sodium Chloride Solution (Prepared by the SO^.HCOI'KC/^ Method)...............51 Table X. The Determination of Calcium Chloride for Recalcification of the Plasma,,...... ...55 Table XI. The Anticoagulant Test...................... 56 Figure 0. Diagrammatic View of Hellige Turbidimeter. ..6 3 vi Page Table XII. Turbidity Measurements (BaSO^J with Tube of 10 min. Viewing Depth ...... 6£ Table XIII. The Hydrolysis of 2-Amino-2-deoxy- N-sulf o-tri-O-sulf o - ^ -D-gluco- pyranoside Dibarium Salt with 0.0U N Hydrochloric Acid, at 95°».. ....... .66 Table XIV. Estimation of the Amino Nitrogen with D-Qlucosamine as Standard. .... 67 Table XV. Estimation of the Amino Nitrogen after the Hydrolysis of 2-Amino-2-deoxy-N- Sulfo-tri-O-sulfo-f t-D-glucopyranoside Dibarium Salt with O.Oli N Hydrochloric Acid, at 95 .................. .68 vii -1- I. INTRODUCTION AND STATEMENT OF PROBLEM For years heparin, a natural occuring potent anticoagulant with low toxicity, has been used clinically to prevent blood clotting. It has however, the great disadvantages of being difficult to obtain free from pyrogens and being very expensive. Consequently, efforts have been made to find a satisfactory substitute for heparin which would be less toxic, cheaper and readily available. Many investigations have been carried out on such compounds as .sulfates of cellulose, pectin, chi tin, chondroitin and the sulfates of derivatives of such polysaccharides. All of these materials investigated, containing the free hydroxyl groups esterified with sulfuric acid, were proved to have some anticoagulant activity; yet the high toxicity and sufficiently low potency caused them to be still impractical for use as a heparin substitute. The work of Wolfrom (1,2) and other investigators (3,U) has (1) M. L. Wolfrom and W. H. McNeely, J. Am. Chem. Soc., 67, 7I4.8 (19U5). (2) M. L. Wolfrom, R. Montgomery, J. V. Karabinos and P. Rathgeb, J. Am. Chem. Soc., 72, 5796 (1950). (39 U. E. Jorpes, H. Borstrom and V. Mutt, J. Biol. Chem., 183, 607 (1950). (U) K. H. Meyer and D. E. Schwartz, Helv. Chira. Acta, 33, 1651 (1950). demonstrated the presence of a linkage between the amino group of D-glucosamlne and sulfuric acid in tne heparin molecule. Other polysaccharides contain D-glucosamine combined with acetic acid. -2- In view of the fact that the anticoagulant activity of heparin, about 110 I.U. (International Anticoagulant Units)/mg., is substantially greater than that of the other polysaccharide sulfuric acid esters, usually between 10-30 I.U./mg., it is of interest to attempt to prepare a polysaccharide sulfuric acid ester containing such N-sulfate groups and to determine its anticoagulant activity. To this end, the sulfation of chitosan, a polymer which is formed from the naturally occuring carbohydrate chitin, by de- acetylation, appears attractive, because this would permit the introduction of sulfamate groups as well as sulfate ester groups into a carbohydrate skeleton for the purpose of producing a heparin-like anticoagulant. - 3 - II. HISTORICAL 1. The Chemistry of Chitosan. The most abundant polysaccharide which contains amino sugars is chitin, a long uribranched molecule constituted entirely of N-acetyl-D-glucosamine units linked by (3 -1,U' bonds. It occurs widely as the major organic skeletal substance in the animal kingdom and is also a frequent constituent of the nycelia and spores of fungi. Chitin was discovered by Odier (5) in 1823 from (5) A. Odier, Mem. Soc. Hist. Nat. Paris, 1, 29 (1823). the elytra of the cockchafer, as the material which was insoluble in caustic potash. Since then, many investigations have been carried out on this substance. In 1859, Rouget (6) first reported the isolation of chitosan (6) M. Rouget, Compt. rend., U8, 792 (1859)• from the deacetylation reaction of chitin with caustic potash. The product he obtained was soluble in dilute acid; however, it formed an insoluble salt in cold concentrated acid. Later, Araki (7) carried out an analysis on a very similar product and (7) T. Araki, Z. Physiol. Chem., 20, 1#8 (1895). found that chitosan has an elementary forraular of G ^
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