CHONDROITIN SULFATE MODIFICATIONS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By BIENVENIDO OCHOA JULIANO, B. S., M. Sc. The Ohio State University 1959 Approved by; Ju. Adviser Department of Chemistry ACKNOWLEDGMENTS The candidate expresses appreciation to Professor M. L. Wolfrom for his advice, encouragement and interest in this work. The research group has been very accomodating and its pleasant company provided stimulating discussions and criticisms which were invaluable. This investigation was made possible through a National Science Foundation Predoctoral Fellowship from November, 1957, to June, 1958, under Grant NSF-GVf9^ to The Ohio State University, and through a C. F. Kettering Research Foundation Fellowship from July, 1958, to August, 1959* TABLE OF CONTENTS Page 1. INTRODUCTION AND STATEMENT OF PROBLEM 1 II. HISTORICAL Chemistry of Chondroitin Sulfates Early developments 2 Methods of isolation and purification 5 Isomeric chondroitin sulfates, distribution and properties 7 Nature of chondrosine l^f Sulfate ester group attachment 18 Hexuronidic linkage 21 Galactosaminidic linkage 21 Chondroitin Zb Keratosulfate Zb If-Deacetylation and Sulfation of Polysaccharides N-Deacetylation of mucopolysaccharides 25 Sulfated polysaccharides 27 III. DISCUSSION OF RESULTS Purification and Characterization of Chondroitin Sulfate A Sodium chondroitin sulfate A 33 Paper chromatographic analysis 35 Calcium chondroitin sulfate A 36 Keratosulfate isolation and desulfation 36 Barium chondroitin sulfate A 38 Potassium chondroitin sulfate A 39 Carboxyl-reduced Chondroitin_______ _______ Polymeric reduced chondroitin 39 Carboxyl-reduced chondrosine bZ Degraded, reduced chondroitin 50 iii xv Page N-Deacetylation Studies N-Deacetylation of Barium Chondroitin Sulfate A Alkaline reagents 52 Acidic reagents 56 Hydrolytic studies on N-deacetylated chondroitin sulfate A 59 Hydrazinolysis of Chondroitin Sulfate Modifications 6l Sulfated Chondroitin Sulfate Modifications 63 IV. EXPERIMENTAL i Purification and Characterization of Chondroitin Sulfate A Sodium chondroitin sulfate A 66 Paper chromatographic analysis 67 Calcium chondroitin sulfate A 68 Keratosulfate isolation and desulfation 69 Barium chondroitin sulfate A 70 Potassium chondroitin sulfate A 72 Carboxyl-reduced Chondroitin 90% Reduced Polymeric Chondroitin 72 96% Reduced Polymeric Chondroitin 75 Carboxyl-reduced Chondrosine Fractionation of hydrolyzate 79 P-D-Glucose pentaacetate 80 N-Acetyl-oc-D-galactosamine monohydrate 8l p-D-Galactosamine pentaacetate 8l Carboxyl-reduced N-acetylchondrosine dihydrate 82 Carboxyl-reduced N-acetylchondrosinol 83 83% Reduced, Degraded Chondroitin 85 N-Deacetylation Studies N-Deacetylation of Barium Chondroitin Sulfate A Hydrazinolysis 89 V Page Hydrolytic studies on N-deacetylated chondroitin sulfate A 90 Deacetylation with 5 N sodium hydroxide 91 Deacetylation with 5 N hydrochloric acid 91 Deacetylation with methanolic hydrogen chloride 91 Deacetylation with benzyl alcohol- hydrogen chloride 93 Hydrazinolysis of Chondroitin Sulfate Modifications N-Deacetylated carbonyl-reduced sodium chondroitin sulfate A 9k IJ-Deace tylated sodium chondroitin sulfate A 9^ N-Deacetylated Sodium chondroitin 9*+ N-Deacetylated carboxyl-reduced chondroitin 95 Sulfated Chondroitin Sulfate Modifications Sulfated Jtf-deacetylated chondroitin sulfate, sodium salt 95 Sulfated N-deacetylated carbonyl-reduced chondroitin sulfate, sodium salt 98 Sulfated chondroitin sulfate, sodium salt 98 Sulfated N-deacetylated chondroitin, sodium salt 99 Sulfated N-deacetylated carboxyl-reduced chondroitin, sodium salt 99 V. SUGGESTIONS FOR FURTHER RESEARCH 108 VI. SUMMARY AND CONCLUSIONS 109 CHRONOLOGICAL BIBLIOGRAPHY 111 AUTOBIOGRAPHY 120 LIST OF TABLES Comparison of Properties of Chondroitin Sulfates A, B and C Deacetylation of Sodium Chondroitin Sulfate A Characterization of Silicate-purified Sodium Chondroitin Sulfate A rf-Deacetylation of Barium Chondroitin Sulfate A Hydrazinolysis of Chondroitin Sulfate A and Its Modifications S t Values of Component Sugars in Three Solvent glucose ° Systems LIST OF FIGURES Figure Page 1. Reaction of 5 W Hydrochloric Acid with Polymeric Chondroitin Sulfate A at 37-^0° 57 2. Infrared Spectrum of Sodium Chondroitin Sulfate A 100 3. Infrared Spectrum of Crude Keratosulfate 100 h. Infrared Spectrum of Partially Desulfated Crude Keratosulfate 101 5* Infrared Spectrum of Chondroitin Acetate 101 6. Infrared Spectrum of Chondroitin Methyl Ester 102 7. Infrared Spectrum of Carboxyl-reduced Chondroitin 102 8. Infrared Spectrum of Carboxyl-reduced N- Acetylchondrosine Dihydrate 103 9. Infrared Spectrum of N-Acetyl-D-galactosamine Monohydrate 103 10. Infrared Spectrum of P-D-Galactosamine Pentaacetate 104 11. Infrared Spectrum of p-D-Glucose Pentaacetate 104 12. Infrared Spectrum of Sodium Chondroitin 105 13. Infrared Spectrum of Partially N-Deacetylated Sodium Chondroitin 105 1^+. Infrared Spectrum of Partially N-Deacetylated Chondroitin Sulfate A, Sodium Salt 106 15* Infrared Spectrum of Sulfated Partially N-Deacetylated Chondroitin Sulfate, Sodium Salt 106 16. Infrared Spectrum of Sulfated Chondroitin Sulfate, Sodium Salt 107 17. Infrared Spectrum of Heparin, Sodium Salt 107 vii I. INTRODUCTION AND STATEMENT OF PROBLEM Chondroitin sulfate A, an acidic mucopolysaccharide readily available from cartilage, is closely related structurally to heparin, a known blood anticoagulant. An important difference is that the amino group of their anhydrohexosamine units is acetylated in chondroitin sulfate and sulfated in heparin. In attempts in this laboratory to prepare polymeric sulfated N-deacetylated chondroitin sulfate, the serious drawbacks had been the absence of a suitable jN-deacetylating reagent and the sensitivity of chondroitin sulfate A toward alkaline degradation, alkaline reagents having been used for deacetylation. This work concerns itself with the preparation and characterization of chondroitin sulfate modifications and the search for a fairly mild and efficient N-deacetylation technique. 1 II. HISTORICAL Chemistry of Chondroitin Sulfates Early developments. Although Krukenburg (1) was not the first (1) C. F. W. Krukenburg, Z. Biol., 20, 30? (1884). to isolate chondroitin sulfuric acid from cartilage, his preparation was of relatively pure form, whereas previous workers (2) obtained (2) G. Fischer and C. Boedeker, Ann., 117, 111 (l86l). mixtures. Among the nineteenth century researchers, Schmiedeberg (3) (3) 0. Schmiedeberg, Arch, exptl. Pathol. Pharmakol., 2£3, 335 (1891). contributed most toward the elucidation of the structure of chondroitin sulfate. He was the first to isolate the disaccharide, chondrosine, on acid hydrolysis of the polymer. To him was also given the credit of first noting the presence of sulfate and acetic acid in the hydrolyzate. He claimed to have obtained, by the action of dilute hydrochloric acid on chondroitin sulfate, the desulfated polymer which he termed chondroitin. However, his proposed formulas for these compounds were later proven to be inadequate (*0. (*0 P. A. Levene, ’'Hexosamines and Mucoproteins," Longmans, Green and Co., London, 1925* The preparation by Hebting (5) of a crystalline derivative (5) J* Hebting, Biochem. Z., 63, 353 (191*0 • of chondrosine was of great importance since hitherto all 3 preparations and all degradation products characterized were amorphous and thus conclusions deduced from these materials were not very reliable. Hebting obtained the crystalline ethyl ester hydrochloride of chondrosine by treating oxalic acid-hydrolyzed chondroitin sulfate with 3% ethanolic hydrogen chloride. Oxidative hydrolysis of chondroitin sulfate from cartilage with acidic bromine gave D-glucaric acid as a principal product (6). (6) M. L. SiVolfrom and W. B. Neely, J. Am. Chera. Soc., 7 31 2778 (1953); P* A. Levene and W. A. Jacobs, J. Exptl. Med., 10, 537 (1908). This acid was identified as the potassium acid salt by Wolfrom and Neely (6) and as the silver salt, by analysis, by Levene and Jacobs (6). The glycaric acid thus obtained may be accounted for by the oxidation of the anomeric group of either D-glucuronic or L-guluronic acid. The isolation of the first crystalline derivative of the uronic acid from chondroitin sulfate was made by Stacey and coworkers (7). They isolated the crystalline (7) H. G. Bray, J. E. Gregory and M. Stacey, Biochem. J., 38, 142 (1944). methyl glycoside of 2,3»4-tri-0-methyl-a-D-glucuronamide from the methanolysis of the methylated, degraded polysaccharide. This ether was identical to that derived from authentic D-glucuronic acid, thus proving that the uronic acid of chondroitin sulfate from cartilage is D-glucuronic acid. Levene and LaForge (8) isolated the hexosamine component of (8) P. A. Levene and F. B. LaForge, J. Biol. Chem., 18, 123 (1914). k chondroitin sulfate, which they termed chondrosamine. They showed that it was different from D-glucosamine (2-amino-2- deoxy-D-glucose or chitosamine). Preparation was effected by hydrolysis of the barium salt of chondroitin sulfate in refluxing hydrochloric acid in the presence of stannous chloride. This amino sugar was observed to give the same phenylosazone as D-galactose, signifying that the amino group was attached at C2, and that chondrosamine was either of the epimeric hexosamines, D-galactosamine (2-amino-2-deoxy-D-galactose) or D-talosamine (2-amino-2-deoxy-D-talose) (9)- These findings were confirmed (9) P. A. Levene, J.