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 ^ H g g ^ O i o which corresponds to a 50% deacetylated anhydro-N-acetyl-D-

glucosamine -unit. L$wy (8) repeated the deacetylation experiment (8) b. Lowy, Biochcm. Z., 2£, hi (1910), by heating the purified chitin at 170-180° with k parts of dry caustic potash for 30 min., and studied the coi bitution of chitosan as its sulfate salt. The results also indicated that chitosan was a polymeric monoacetyldiglucosamine. In addition, he noted that chitosan could be split into D-glucosamine and acetic acid. Rigby (9) modified the deacetylation conditions by using $0%

(9) G. W. Rigby, U. S. Pat. 2,01*0,879, May 19, 1936. aqueous caustic and heating at 100° for hi hr. under a nitrogen atmosphere to prevent degradation of the chitosan molecule. The chitosan isolated was 90% deacetylated. However, an additional

72 hr. treatment with $0% aqueous sodium hydroxide did not alter the composition. If concentrated caustic and higher temperature were used, evolution of ammonia as well as destruction of the hexosamine molecule were frequently observed.

Enzymic study of chitosan with snail chitinase was reported by Karrer and Hoffman.(10) The product they obtained was neither

(10) P. Karrer and A. Hoffman, Helv. Chim. Acta, 12, 6l6 (1929).

N-acetyl-D-glucosamine nor D-glucosamine, but was a mixture of compounds that formed a colorless, amorphous hydrochloride. This could be separated into two fractions by extraction with hot - 5- alcoholj a soluble fraction, molecular weight 5hO, and an insoluble fraction, molecular weight 630-657* If chitosan was treated with acetic anhydride and sodium acetate (11) to form a monoacetyl

(11) F. Hoppe-Seyler, Ber., 27, 3329 (I89U). derivative first, then it would behave very similar to chitin with respect to snail chitinasej thus, after hydrolysis, 80%

N-acetyl-D-glucosamine was obtained.

The chitosan was completely dissolved by the action of nitrous acid, (12,13) resulting in a solution containing carbo-

(12) W. Areribrecht, Biochem. Z., £5, 108 (1919).

(13) K. rl. Meyer and H. Wehrli, Helv. Chim. Acta, 20, 353 (1937). hydrates of more than one type, but there was no crystalline sugar which could be isolated. However, when treating the solution with phenylhydrazine, a crystalline osazone could be isolated from the mixture. It had a melting point of 202° and was shown to be the osazone of chitose, a reducing anhydro sugar which has the structure shown below. CH2OH

OH OH

H H

Chitose -6-

Chitose also could be formed from the reaction of silver nitrate

or nitrogen tetroxide on D-glucosamine hydrochloride. (1U)

(1U) P. Schorigin and N. N. Makarowa-Semljanskaja, Ber., 68, 965 (193?).

When chitosan was reacted with barium hypobromite, (15)

(15) Y. Inouye and K. Onodera, J. Agr. Chem. Soc. Japan, 25, 553 (1952).

a product which subsequently yielded D-glucose phenylosazone with

phenylhydrazine could be isolated.

The periodate oxidation of chitosan was studied by Jeanloz

and Forchielli (16). They found that at 5° and pH U.1, l.U-1.5

(16) R. Jeanloz and E. Forchielli, Helv. Chim. Acta, 33, I69O (1950).

moles of periodate were consumed per D-glucosamine residue and

0 .6-0 .7 moles of ammonia and u.2 moles of formic acid were

liberated. This result applied very well to the formular proposed

by Meyer and Mark (17) as shown below.

(17) K. II. Meyer and H. Mark, Ber., 6l, 1936 (1928).

- ICif ^HCooH CHzOH CHiOH M 1 NHi — IOq. ' * HCOOH 'OH H O- o h k0 H M -0 .OH

^ H f chzoh CH2OH IO n Ucoo h 10 q j r\ NH, HC00H Structure of Chitosan and Its Oxidation by Periodate -7

Chitosan, in the presence of zinc chloride, exhibited a red violet color with iodine and a scarlet color with bromine#

However, the colors were easily destroyed by slight wanning or by treatment with alcohol. (8) If chitosan was treated with iodine in dilute acetic acid, a violet color was produced, and with the subsequent addition of a drop of 75% sulfuric acid, spherical crystals of chitosan sulfate separated; this was used for the detection of chitosan. (18)

(18) Van Wisselingh, Folia Microbiol., 2> 1^5 (191U).

The chitosan displayed crystalline areas as do chitin and cellulose. X-ray diffraction patterns showed that the unit cell in chitosan was orthorhombic with the dimensions a = 8.9, b = 10.25 and c ■ 17.0 %. (19) Its powder pattern was similar to that of

(19) G. L. Clark and A. F. Staith, J. Fhys. Chem., U0, 863 (I936). mercerized cotton. Molecular weight estimation of chitosan by means of copper number and iodine reduction methods gave a D.P.

(degree of polymerization) value of 20-30. (13,15) However, these end-group method values usually were grossly lower than viscosity values and since chitosan was capable of forming tough flexible films from the acid solution casts, Rigby (9) had proposed that the D.P. of chitosan must be over 50.

2. The Sulfation of Polysaccharides. -8-

The importance or tne -SO^H group for inhibiting blood clotting was rirst pointed out by Demole and Fischer (2u,21)

(20) Vo Demole and M. Reinert, Arch. Exptl. Pathol. Pharmakol,, l£8, 211 (1930).

(21) A. Fischer, Biochem. Z., 2U0, 36^ (I93I), when they noted that numerous anticoagulants, like heparin, hirudin and germanin all contained such a group. In 1935,

Bergstrom (22) treated a number of polysaccharides with chloro-

(22) S. Bergstrom, Z. physiol# Chem., 238, 163 (1936). sulfonic acid and pyridine (23 ; and discovered the anticoagulant

(23) R. Tamba, Biochem. Z., lltl, 2714- (1923). properties of these polysaccharide sulfuric acid esters. The highest activity, in term of Kerk's heparin, was 2 for a synthetic chondroitin-trisulfuric acid, the lowest was l/l2 for a glycogentrisulfuric acid, and sulfuric acid esters of chitin, cellulose, pectin and starch were intermediate in activity, whereas the sulfuric acid esters of the mono- and di-saccharides were inactive# Starting from that period, many investigators began to prepare the synthetic or se,.ii-synthetic anticoagulants from the sulfation of polysaccharide derivatives#

Chargaff and co-workers (2U) prepared cellulose sulfuric - 9-

(2h) E. Chargaff, F. W. Bancraft and Margaret Stanley-Brown, J. Biol. Chem., 115, 155 (1936). acid esters and studied the anticoagulant activity of these substances with high molecular weights. The results they obtained showed that no active anticoagulant could be found that was free from sulfur. However, a number of suhstances of high molecular weight containing sulfuric acid esters, such as sodium salt of cellulose monosulfuric acid, agar and the polysaccharide from cornea, were found to be inactive. The inactivity of penta- raethylene and decamethylene disulfuric acids supported the findings of Bergstrom regarding the sulfuric acid derivative of simple sugars which likewise were found to be inactive. Reuse (25) used Tamba's

(25) Jean Reuse, Compt. rend. soc. biol., 131, 83I1 (1939). method (23) for the sulfation of amylose. The product she isolated was the potassium salt of amylose disulfuric acid

*rhich would decrease the clotting time when injected into live rabbits at a dosage of 1 mg./kg. However, if 2 mg./kg. or more were used, the clotting time was increased, later, Karrer,

Koenig and IJsteri (26j studied the acid esters of polysaccharides.

(26; P. Karrer, H. Koenig and S. Usteri, Helv. Chim. Acta, 26, 1296 (19U3).

Phosphoric esters of starch, trihexosan, chitosan and gelatin -10- with a phosphorus content from 9-21;% were prepared which showed extremely small anticoagulant activity. The polysaccharide sulfuric acid esters were prepared by the action of chloro- sulfonic acid on various carbohydrates suspended in pyridine at o 0 . The ester of levoglucosan, containing 20.5% S, had no anti­ coagulant activity and thus it appeared that a high molecular weight was requisite in an anticoagulant, asters of chondroitin

(lh»l% S), cellulose (lb.9% S; and pectin had favorable activity but were from U-6 times less effective than heparin and were very toxic.

Experiments have been repeated on the sulfation of cellulose,

(27,28,29) starch, (27,28) chitin, (27; polyvinyl alcohols,

(27) T. Astrup, I. Galsman and M. Volkert, Acta Physiol. Scand., 8, 215 (19WO.

(28) E. Husemann, K. N. van Kaulla and R. Kappesser, Z.

Naturforschg., 1, 581; (19U6;.

(29) l/S. Solusol, Dan. Pat. 65,269, Dec. 30, 1956.

(28) glycogens, (28) xylans, (28) and alginic acid. (30,31) All

(30) E. G. Snyder, U. S. Pat. 2,508,533, Hay 23, 1950.

(31) D. Molko and J. Gotte, Bull. soc. chim. biol., 312 (1951).

of these substances were either too toxic or without significant

potency.

Since a large difference in anticoagulant properties has -11- been observed from different preparations, Meyer and associates (32)

(32; K. H. Meyer, R. P. Piroue and M. E. Odier, Helv. Chim. Acta, 35, 57U (1932;. had carried out a series of experiments to 3tudy the effect difference of the method of preparation on activity. They discovered

that polysaccharides, when treated with chlorosulfonic acid and pyridine, employed by most of the investigators would be degraded

considerably during the reaction, whereas a solution of sulfuric

anhydride in pyridine effected less degradation.

The utilization of sulfur dioxide as a solvent had been proposed by Burkhardt and Lapworth, (33; and also by Ross and co-

(33) U* N. Burkhardt and A. Lapworth, J. Chem. Soc., 68U (1926;. workers (3U; for the preparation of the arylsulfonic acid ester

(3U; J. Ross, J. H. Percy, R. L. Brandt, A. J, Gebhant, J. E. Mitchell and 3, Jolles, Ind. Eng. Chem., ^SU, 92b (19U2). and the sulfate of primary alcohols. However, it had not been used for the preparation of the polysaccharide sulfuric acid

esters. In view of the fact that it was an excellent solvent for

sulfur trioxide and was easily removable by evaporation, Meyer, / Hroue and Odier (32; used it to replace pyridine during the sulfation reaction. Their results are listed in Table I.

They found that degradation of the product was always accompanied by a drop in anticoagulant activity. The best product -12-

TABLE I

Methods Of Sulfation and Effects on Polysaccharides (32)

Moisture, Method of Time, Yield, Polysaccharide % Sulfation Tenp« hr. Degradation i SJ. chondroitin sulfuric acid very O 1 ro (sodium salt) 19 S02-S03 C 0 slight 86 13 same 19 SO2-CISO3H -20° 6 slight 9u 17 same 15 pyridine- 6u° 6 slight 91 9 503

same 15 pyridine- 20° 1/ very 75 8 503 slight

same 15 dioxane- 20° 25 very 90 6 SO slight j same 0 C1S03H -12° 17 very 55 Hi strong chondroitin sulfuric acid 0 pyridine- 60° 6 strong 70 Ik 503

galactomannan 7 SOo-SO -20° 6 strong 16 10 2 3 0 CM 0 lichenin 11 S02“S03 1 6 inter­ mediate 60 15 O 1 chitosan 11 SOp-SO^ ro O 6 slight Uo 15

chitosan 0 SC>2-S03 -20° 6 inter­ mediate 17 16

chitosan 18 C1S0-.H- 0 0 CM so2 * 1 6 slight 70 9 xylan 22 S02“S03 -20° 6 slight 25 10 - 13 - they obtained for chondroitin sulfuric acid possessed a molecular weight of the order of 26,000, which corresponds to a D.P, of 50 anhydrodisaccharide uints. The strongly degraded products were practically inactive. It also appeared that the content of -SO-jH group affected the anticoagulant activity. Only those products containing at least 11-12% S (which corresponds to nearly 2

-SO3H groups per period) exhibited a good anticoagulant activity.

However, it was not a general rule, because Marbet and Winterstein

(35) had isolated " fZ heparin", a polysaccharide similar to the

(35) Marbet and A. Winterstein, Helv. Chim. Acta, 3U, 2311 (1951). chondroitin sulfuric acid, containing only 6% S which had an activity about 25% that of heparin.

Many investigations have been carried out on dextran sulfate.

Ingelman and co-workers (36) had described three preparations

(36) A. Grdmwall, B. Ingelman and H. Mosimann, Upsala Lakareforen F8rh., 50, 397 (19U5); Chimie & industrie, 55, 206 (19U6). of dextran sulfate, The results they obtained showed that the degree of polymerization of the molecule played an important role in controlling the toxicity. Qnijy the preparation with molecular weight < 5u,000, which was about 3 times that of the molecular weight of heparin (17,000, as determined by using sedimentation and the dixfusion constant for a substantially pure preparation in the ultracentrixuge) was nontoxic. However, the high toxicity of irrulin sulfuric acid ester of molecular weight 1U,000 has been reported* (37) Therefore, Ingelman expressed the view that

(37) B. Ingelman, Arkiv, Kemi. Mineral, Geol,, 2 k , U (19U6).

"molecular weight" was not a principal factor in determining toxicity.

In order to determine the effect of molecular weight and sulfur content on toxicity and anticoagulant activity, Ricketts (38)

(38) G. R. Ricketts, Biochem. J., 5l, 129 (1952). had prepared and studied a series of sulfuric acid esters of dextran differing widely in molecular weight and sulfur content. He concluded that toxicity does increase with molecular weight. A dextran sulfate preparation of molecular weight 35,000 was nontoxic while those with a higher range were toxic. The anticoagulant activity appeared to be independent of molecular weight but depended on a certain minimum number of sulfate groups per D-glucose unit. Thus, dextran sulfate with 1.0-1.3 sulfate groups per D-glucose unit showed a sharp increase in activity. However, heparin has much greater activity with considerably less sulfate groups, 5 sulfate groups per anhydrotetrasaccharide unit being present in its molecule.

Berger and Lee (39) also had studied the relation between

(39) L, Berger and J. Lee, Abstracts Papers, XII th Intern. Congr. Pure and Appl. Chem., 3U3 (1951;• - 15-

the degree of sulfation, molecular size and shape, of various polysaccharides, and their anticoagulant activity and toxicity.

The results of Berger and Lee are presented in Table II.

TABLE II

Effect of Begree of Sulfation and Molecular Size and Shape on Anticoagulant Activity and Toxicity

Activity Activity Toxicity, in Vitro, in Vivo, (mouse, I.V., Sulfated Product S.ft U.S.P. Method (rabbits) g./kg.)

alginic acid 13.95 0.25 0.5-0.75 o.U

polymannuronic acid methyl ester methyl glycoside (type 1) 15.2 0.125 0.25 2.75

same (type 2) 1iw9 0.175 0.5 1.3

poly-D-galact- uronic acid methyl ester methyl glycoside 15.7 0.125 0.3-0 .5 6 .0

pectic acid 12.75 0.125 l.u 0 .1

heparin 13.0 1 .0 1 .0 2.0

The molecular weight of the polysulfuric ester of poly-D-

galacturonic acid methyl ester methyl glycoside, determined by

ultracentrifuge experiments, (39) was UOUO. It had a very low

toxicity and exhibited only about l/8 the activity of heparin

in vitro. Clinically, weight for weight, it showed about 1/3 the -16-

activity of heparin by the intravenous (I.V.; route and was

reported to be completely without side reactions.

Recently, after the present work was initiated and a prelimi­ nary report was published, (UO; the sulfation of chitosan was

(R0) M. L, Wolfrom, Tsung-men Shen and C. G. Summers, J. Amer* Chem. Soc., 75, 1519 (1953).

studied by several investigators. Ricketts (Ul; treated chitosan

(ljlj C. R. Ricketts, Research, 6, 17s (1953J. with chlorosulfonic acid in pyridine. The product he isolated had

an activity of 11 I.U./mg. Coleman and associates (u2; repeated

(U2 j L. L. Coleman, L. P. McCarty, D. E. Warner, R. F. Willy and J. F. Floksten, Abstracts Papers, Am. Chem. Soc., 123. 191 (1953;.

the sulfation experiment on chitosan by using SOp-SO^ at -10° for

10 hr. The resulting chitosan sulfate, containing 1.3-2 moles of

S per hexose unit, exhibited an activity l/6-l/u that of heparin;

its L.D.^q (the minimum dosage of a drug required to produce the

death of 50/ of the animals tested^ was duO mg./kg. (mouse, I.V.; which was about equal to the heparin, 75u mg./kg. - 17 -

TTT, DISCUSSION OF RESULTS

1, The Sulfation of Chitosan with Ryridine-Chlorosulfonic Acid.

The chitosan used for this experiment was a light-tan, flaky material (U2a) which was found to contain 0.11 acetyl group per

(li2a) This material was kindly supplied by Professor J. F. Haskins of The Ohio State University Chemistry Department. It had been prepared by the method of Rigby from the chitin obtained from shrimp shell. arihydro-D-glucosamine unit. It was thus 8 deacetylated. Owing to the insoluble nature of chitosan in pyridine, the surface condition of the suspended solid played an important role during the sulfation reaction. Failure to sulfate crude chitosan has been reported by previous workers. The activation process used here, in addition to any purification which it might render, produced a colloidal suspension readily susceptible to sulfation. Poorer results were obtained if the activated chitosan was dried, ground and resuspended in the reaction medium. A non-colloidal suspension was obtained in this case. In addition to the surface condition, the purity of the pyridine used seemed to be critical. In earlier

experiments, when three-degree range redistilled pyridine was used, not only the yield was lowered, but the product had a very low anticoagulant activity.

The sulfation of chitosan employing chiorosulfonic acid in dry pyridine, yielded a product which is isolated as an amorphous, water-soluble sodium salt. It contained two N-sulfate and two -18-

0-sulfate groups per anhydrodi sac charids unit and had a rotation of (X) 25 -23° (c 1.5, water;. The quantitative analysis of this D product is given in Table III.

TABLE HI

Analysis of the Sulfated Chitosan

Required for 1 (-12%806 ^ sQ3^a ^2^0^03N*,^2 J U.By + (0l2Hi8Q6(NCOCHj)g(OSO^NaUan Found

C 20.65 20.51* (HU)

H 2.6** 2.95 (HM;

N 3.92 3.1*1 (HM;

Na (H2 S01; ash; 12.uO 11.31 (HM;

S 16.?u 16.25 (HS;

N-acetyl (as COCH^; I.63 (HS;

-NH2 (^y ninhydrin), absent

The analysis, identified by (HMj , were conducted by the Hoffman Microanalytical Laboratories of Wheatridge, Colorado. Others, identified by (HS;, were made by the author herself in this laboratory.

* N-acetyl content was determined by the method described by Wolfrom and co-workers. (1*3;

(1*3; M. L. Wolfrom, D. I, Weisblat, J. V. Karabinos, W. H. McNeely, and J. McLean, J. Am. Chem. 5oc., £5. 208)4 (191*2;.______•19'

This product exhibited the behavior in the Van Slyke amino assay characteristic of the acid-labile N-sulfate group present in heparin (1,2,3,U) (see Fig. 1). Therefore, it is probable that both contained the same type of N-sulfate linkage. The anticoagulant activity of sulfated chitosan was £6 I.U./mg. Astrup (27,UH) has

(ijJU9 T. Astrup and I. Galsman, Acta Physiol. Scand., 13, 3^1 (191\h). sulfated chitin, without removing the N-acetyl function, to a degree of two sulfate groups per each anhydrodisaccharide unit, yet the product exhibited no significant activity. This was another evidence to support the findings of Wolfrom and his associates (2,U0) regarding the contribution of sulfamic acid linkages to the anticoagulant activity of polysaccharide poly-

sulfuric acid esters of the heaprin type. Doczi and co-workers (1*5)

(U5) J. Doczi, A. Fischerman and J. A. King, J. Am. Chem. Soc., 75, 1512 (1953). prepared a series of sulfated chitosans in which the amino and hydroxyl groups were sulfated to a varying degrees, and studied the

effect of the sulfamic acid groups on the anticoagulant activity.

Their results further confirmed the findings of this laboratory.

The animal (mouse, I.V.) toxicity test (by Dr. H. L. Dickison,

Bristol Laboratories, Syracuse, N. Y.) showed that sulfated

chitosan was approximately twice as toxic as heparin. It was as

effective as heparin in the anticoagulation of freshly drawn f^3r Corrt of N itr o g e n 0 3P IP 0 Figure /. Jo Variation o / Amino- N ( Van Van ( N Amino- / o Variation Nt Ti e im T \Nith Tm, Minutes 'T/me, 6o 0 C 0 r y s t a l l i n e 0

h e pan n o t e suffafmd 90 5 hitoson ch yke) e k ly bar/om

acid

J20

02“ -21- dog’s "blood but was less effective than heparin in prolonging the clotting time of the circulating blood of rabbits. However, there was indication that there may be a potentiation of the heparin effect when sulfated chitosan was given later,

2. The Molecular Weight Study on Sulfated Chitosan,

From the published results, it was found that toxicity, in the form of a greatly increased tendency to haemorrhage, was associated with high molecular weight, such as the larger dextran sulfate molecules. Therefore, a thorough study on the adjustment of the molecular weight of sulfated chitosan was considered desirable.

Experiments have been carried out by means of the light scattering technique.

Although the theory of light scattering was established in

1871 by Lord Rayleigh, (]*.6,1|.7jU8) it has not been widely applied

(1*6) Lord Rayleigh, Phil Mag., 1*1, 107, 27U (1871),

(1*7) Lord Rayleigh, Proc. Roy. Soc., 8i*A, (1910).

(1*8) Lord Rayleigh, Phil. Mag., 373 (1918), until in the past few years when Debye (1*9*50) introduced the

(1*9) P. Debye, J. Applied Phys., 15, 338 (191*1*;.

(£0) P. Debye, J. Phys. Colloid Chem., j?l, 18 (19l*7). light scattering technique to determine the absolute molecular weight of polymers in solution and developed equations that could -22- be used for that purpose. A complete review of this subject is by

Edsall and Danliker, (51) therefore, only a brief description of

(51) J. T. Edsall and W. Danliker, Fortshr. Chem. Forsch. Ed., 2, S.l (1951).

the method will be given here.

A. Theory of Light Scattering.

li/hen a beam of light falls upon a nonabsorbing medium, the

electric field associated with the light induces periodic

oscillations of the electrons in the material. The material then

derves as a secondary source of light and radiates light in the form of scattered radiation with a wavelength equal to the incident

light. The above consideration, however, excludes the relatively

small amount of light which is re-emitted with an altered wavelength when the molecules are raised to higher energy states by the incident light, i.e., the Raman scattering. The intensity of the scattering light has been shown to depend on the microscopic heterogeneity

of the solution. In the case of high polymer solutions, the principal

cause of heterogeneity is usually the fluctuations in density and

concentration. The extent of these fluctuations is a function of

the work necessary to produce such a local variation in concentration in a volume element, and is related to the free energy of dilution and hence to the osmotic pressure and molecular size. On this basis, Debye (50) has derived an equation as follows; -23-

Hc 1 rY a M 4 2Bc where,

H - 32 ttW u n/°)2 3 \ k N

and is called the proportionality constant which has the dimension

cm2/g 2.

N ■ Avogadro's number

c ■ concentration, in g. per 100 c.c.

7\ - wavelength of light incident upon the system

n0 » the refractive index of solvent

A n ■ difference between refractive index of solution and

refractive index of solvent; it can be evaluated from

the differential refractometer

M ■ molecular weight of the solute

s y b the turbidity, caused by concentration fluctuations and

B, which measures the deviation from Van't Hoff's law, is

coefficient of the square term in concentration in the expression

for the osmotic pressure. In all practical applications of the

light scattering technique to the determination of molecular weights, it has now become customary to determine H by refraction measurements, and T for various concentrations and then plot Hc/|-

as a function of c. A straight line drawn through the observed point will cut the vertical axis (c » 0 ) of the plot at an

ordinate which is equal to l/M. -21*-

The derivation of the equation was based on the assumption that the scattering particles were small compared to the light wavelength used as the source, the polarizability, o( , is therefore independent of the direction in the particle. The direction of the electric field associated with the incident light always coincides with the direction of the induced moment, and the scattered light o is perfectly plane-polarized in the direction of 9 « 90 . If, how­ ever, particles are greater than l/20th to l/lOth the wavelength of the light, an important correction must be made for the dis­ symmetry which arises due to the interference between light waves scattered from the same particle. This internal interference causes a diminution of the intensity of the scattered light which, at concentrations sufficiently low to eliminate external inter­ ference, is given by the expression, (52 )

(52) G. Oster, Chem. Revs., h3, 317 (191*8).

P (©, -ZLZL sin ksrjj 1 3 ksrij where the double summation extends all pairs of scattering elements i and j; k ■ 2 TT/a !; X,’ is the wavelength of light in solution; s = 2 sin 9/2; and is the distance between the ith and jth scattering element. This general expression was derived originally by Debye (53) to treat the case of internal interference arising

(53) P. Debye, Physik. Zeits, 28, 135 (1927) -85- in x-ray diffraction by atoms. There, it is known as the square of the atomic scattering factor; in the present case, it is called the particle scattering factor.

Expressions have been obtained for the particle scattering factor for the most important particle shapes, ($h) i.e., sphere,

(5UJ B. Zimn, R. S. Stein and P. Debye, Polymer Bull., 1, pO (19U5)• rod and random coil. These expressions are: sphere

P (©) (sin x ” x cos x ^) » x ■ rod (2x p (SJ = i ) do). toL X /o ^ x J x " — random coil

P (Q) mrn - | - [ ee_A “x - (1 - x)jx ) I ;: X ■ - 2. P-2 -?.2 X where, D is the diameter of sphere, L is the length of the rod and

R is the root square of the distance between ends of the random coil.

The particle scattering factors serve two purposes in the most common method of interpretation of light scattering data, these two purposes are: (a) the measurement of the ratio of the intensities at any two angles permits the evaluation of the -26-

dimension (D, L or RJ if the particle shape is known; (b) the same measurement make possible the correction of the observed intensity

at 90° for the loss due to internal interference. It is this

corrected intensity, or the turbidity calculated therefrom which must be employed in equations.

It has become fairly standard practice to choose the angle

-h $ ° and -135° for measurement and the ratio of these intensities

is known as the dissymmetry, 2 . A table of precise values of z

and l/P (90° ) for selected values of p/a 1 is given as follows,

(#)

($5) P. Doty and R. S. Stein, J. Chem. Phys., 18, 1211 (19^0). ■27-

TABLE IV

Dissymmetry and Correction Factors for Random Coil as a Function of Dimension/A1

P (0; s -L. [e"x - (1 - x)J ; x -

s s 2 sin 9/2; k * 2 Tf / a 'S A' • 'Vn

R is the root mean square distance between ends of the random coil

= P(-a5°)/P(-1350,); A « U37 rap ; x1/2 = 1.115 x 10'

Dimension/ A 1 i/p ( 9 o ° ; z

0 .0 5 1.016 1.012

0 .1 0 1.066 1.09a

oai5 1.1U8 1.200

0.2 0 1.263 i.3 a o

0 .2 5 1.3 9 1 1.519

0.3 0 1.5 2 1.715

0 .3 5 1 .7 6 1. 92a

o .a o 2.051 2.151

o .a5 2.330 2.360

o .5 o 2.6U2 2.569

o .5 5 2.987 2.779

0.60 3.365 2.980

0.6 5 3.776 3.169

0.70 a . 218 3.35a

0 .7 5 a. 695 3.523

0 .8 0 . 5.205 3.681 -28-

Figure 2 also shews the relation, between z and l/P(9 0°j.

The molecular weight is corrected by multiplying the value calculated from the observed turbidity by the factor l/P(90°).

Since P(90°) is a function of dimension/X 1, a measurement of z will define 1/P(9o7» This value may become very large for large molecules. The molecular weight obtained by this method is a weighted molecular weight. (56)

(56) B. H. Zimm and P. Doty, J. Chem. Phys.. 12, 203 (l?hU)*

B. The Effect or Polymerelectrolytes.

Polymerelectrolytes are capable of ionizing in aqueous solution, thus becoming electrically charged by an amount depending on the concentration* The electrostatic repulsions of the charges cause the distance of closest approach of two molecules to be much greater than the actual diameter, thus effecting the molecular size and giving rise to particular light scattering curves.

However, Doty and Stein (57) have found that all of these molecule

(57) P* Doty and R. S. Stein, J. Chem. Phys., 1Y, 7I4.3 (19U9;. effects may be eliminated by using high salt concentrations

^ 0 . 0 1 M) and extropolating the Hc/7 - through low values of concentration.

G. The Effect of Extraneous Particles.

Since the presence of foreign particles, particularly those of relatively large size, increases the light scattering properties -29- -30- of a solution, it is essential to eliminate these particles beforehand in order to secure reasonably accurate results. Using filtration through an ultra-fine fritted glass filter is a convenient means of removing the extraneous particles; however, in some cases, this method was found unsatisfactory, principally because of the high viscosity of the solution. Consequently, for this class of solutions, ultracentrifuge (,5 8J was used in lieu of

(58) F, Foster and E. F. Paschall, J. Am. Chem. Soc., 75, 1182 U 953J. direct filtration.

The molecular weight of sulfated chitosan prepared by the chlorosulfonic acid and dry pyridine method has been determined in a 0.2 M sodium chloride solution. It had an observed molecular weight of Ii56,000, i.e., a weight average D.P. of 638 anhydro- disaccharide units. Apparently, this might be the main factor which cause a high toxicity of the sulfated chitosan. In order to reduce such undesirable effect, other sulfation methods also have been investigated.

3. The Traube Sulfation. (I4.J

The chitosan was sulfated by means of sulfur trioxide in sulfur dioxide solvent and by sulfur trioxide in dry, ethanol-free chloroform solvent. The material isolated from the reaction in the latter media was found to have a sulfur content of 15 .U5, corresponding to about 3.5 sulfate groups per anhydrodisaccharide unit. However, -31- this water soluble material was found to have an anticoagulant activity of approximately 12 I.U./mg., which was much lower than the sulfated chitosan prepared from the chiorosulfonic acid and dry pyridine. No more work was done with this sulfation procedure.

U. The Sulfation of Chitosan with Sulfur Trioxide-N.I-I, -Dimethyl-

formamide Complex.

It is known that sulfur trioxide has commonly been brought into reaction with those organic compounds that are labile to sulfuric acid, as its co-ordinate complex with dioxane (5 9) or

(59) C. IT. Suter, P. B. Evans and J. IT. Kiefer, J. Am. Chem. Soc., 60, 538 (1938). with pyridine. (60) The former is an unstable solid used as a

(60) P. Baumgarten, Ber., 59, 1166 (1926). suspension in ethylene dichloride and therefore can not be added in a measured quantity. The latter is an easily prepared solid but would probably yield an acylpyridinium salt rather than the desired ester sulfate under mild conditions.

As a base of intermediate strength, N,N-dimethyIformamide is more suitable for preliminary combination with the sulfur trioxide.

The resulting complex salt is a white crystalline substance, soluble in N,N-dimethylf ormamide to a concentration of ca. 2.5 N. ^0 -503" S 03 ♦ h g o n ( c h3 j 2 ------h - c - n ( c h 3)2

(i; -32-

Since (I; reacts readily with the sodiura salt of amino acids

(61; in N,N-dime thy Iformamide to form a mixed anhydride and has

(6lj G. W. Kenner and R. J. Stedman, J. Chem. Soc,, 2069 (19^2). also been used for the sulfation of leuco dyes, (62) it appeared

(6 2 ) S. Coffey, G. W. Driver, D. A* Fairweather and F. Irving, British Patents 610,117, Oct. 12, I9U85 6^2,206, Aug. 30, 19^0.

that (I) may be used to sulfate those polysaccharides which are

soluble in IT, N-dime thy If ormamide, such as mucopolysaccharides, in the following manner.

-COO -o so 2 ^o-so3 - C Ox ^ ° " ♦ H-C-N(GH3 ; 2 -HH, -NHSOgCH

-CH - o s o 2 o h

•COO

- n h s o 2 o h

-o s o 2oh

Experiments have been carried out on chitosan (ca. 90£ N-

deacetylated). Although this polysaccharide is not quite soluble

in N,N-dimethyIformamide, it dissolves when S0^*HC0N(CH3J2 complex -33-

is added, to form a homogeneous solution* The product obtained

from this process is an amorphous, water-soluble sodium salt,

containing essentially two N-sulfate and two O-sulfate groups per 27 anhydrodisaccharide unit; f°0 p -17 (c 1 *67, water)*

Anal* Calcd, for jc-j^H^O^/NSOyia^COSO^Na^] 0*89 4

[ci2 H1 806(NC°GH3)2 (0S03Na)2 ] 0 .1 1 : S» l6*?0* S, 16.33

(HS), -NHg (by ninhydrin) absent; NAc (by p-toluene sulfonic

acid method) as Ac, 1.63*

Molecular weight determination with light scattering

technique, shows that it has a molecular weight of 188,600 or a

weight average D.P. of 263 anhydrodisaccharide units which is much

smaller than that obtained (1*36,000 or a weight average D.P. of

638) from the sulfated chitosan prepared by the chlorosulfonic

acid and pyridine method. Its anticoagulant activity was approxi­

mately 30 I.U./mg* and a marked decrease in toxicity for this

preparation was reported. Its L.D.^q (mouse, I.V.) = 773 mg,/kg.

which was about equal to that of heparin (730 mg./kg.). This

result further confirmed the point that toxicity was effected by

the molecular weight of polysaccharide polysulfuric acid esters.

The new sulfation method established here has several

advantages. Since a solution of (I) is stable on storage, it

can be handled with ease and accuracy. Besides, judging from o the sensitivity of (I), reactions can be achieved below 5 for -3U- sensitive compounds , thus avoiding the degradation of the poly­ saccharide chain. Also, compounds such as mucopolysaccharides which are insoluble in other solvents can be thus sulfated in a homogeneous medium and probably to a higher extent.

5. The Anticoagulant Assay by the Sheep Plasma Method.

The sheep plasma method used here for the estimation of the anticoagulant activity was originally described by Swoap and

Kuizenga and is a modification of that previously reported by

Kuizenga, (63) Nelson and Cartland. (6UJ The Method is based on

(63J F. Swoap and M. H. Kuizenga, J. Am, Fharm. Assoc., 38, 563 (19U9).

(6I4J M. H. Kuizenga, J. Nelson and G. F. Cartland, Am, J. Physiol., 612 {19k3)» the simultaneous comparison of the minimum amounts of standard and unknown sample which, when contained in a volume of 0J 4 ml., will keep 1 ml. of decalcified sheep plasma more than $0% fluid for 1 hr. at room temperature. The method is short, accurate and easy to use.

It has definite advantages over other methods as follows.

A. The amount of calcium chloride necessary for recalcification of the plasma need be determined only once for each lot of frozen plasma.

B. The calculation of the results is rapid and simple. The dosage response curve is steeper than those found with other methods or with other plasma. c* No water bath is required. The tubes stand for one hour at

room temperature.

D. The end point is easy to determine. Reailltd can be determined

with sufficient accuracy without plotting the data obtained*

Foster's (65) method, as well as others, are much more time-

(65;, R. H. K. Foster, J. Lab. Clin. Med., 27, 820 (19U2). consuming and complicated and are not suitable for routine testing of a large number of samples.

5. Model Compound Preparation.

The fundamental chemistry of simple model compounds were v e r y important for the proper manipulation of the heparin-like anti­ coagulant being studied. Certain derivatives of D-glucosamine hydrochloride were very essential in any study of 0 -sulfate and

N-sulfate groups in carbohydrate compounds. Considerable work has been done in attempts to prepare the barium salt of the fully sulfated methyl glycoside of D-glucosamine hydrochloride which in turn had been prepared from methyl tri-O-acetyl- -D-glucosaminide hydrobromide. The latter compound has been reported by Irvine (6 6;

(6 6; J. C. Irvine, D. McNicoll and A. Hynd, J. Chem. Soc., 99 250 (1911). and his co-workers. Their experiments were carried out to prepare the tri-O-acetyl compound but the results were unsatisfactory.

After considerable efforts, the procedure has been modified so as to give a fair yield of the desired product in a reproducible -36- manner.

A. The Preparation of Tri-O-acetyl-2-amino-(hydrobromide;-2-

deoxy- d -D-glucopyranosyl Bromide.

The modified procedure for obtaining the tri-0-acetyl-2-

amino-(hydrobromide )-2 -deoxy- d -D-glucopyranosyl bromide was to react

D-glucosamine hydrochloride with acetyl bromide until the mixture

suddenly solidified. At this point, the flask was immediately

removed and connected to a series of drying tubes which contained

anhydrous calcium chloride and soda lime. Any hydrogen bromide was drawn from the flask into the drying tubes by means of a vacuum applied to this closed system. It has been found very

important to remove all of the hydrogan bromide immediately because

it readily absorbed moisture and thus decomposed the desired

product. A very slightly excess of acetyl bromide should be used

in the reaction as otherwise solidification does not occur at the

end of the reaction. An over extended rraction gradually destroys

the product, only tri-oacetyl-D-glucosamine hydrobromide being

obtained. The yield of the bromo compound by this procedure was

85%. It formed colorless needle-luke crystals, m.p. lUy-l50°

(recorded (6 6;, l5u°;.

B. The Preparation of Methyl 2-Amino-2-deoxy-N-sulfo-tri-O-

sulf o- -D-glucopyranoside Dibarium Salt.

Methyl 2-amino-2-deoxy- ^ -D-glucopyranoside hydrochloride

(6 6; was sulfated by chlorosulfonic acid and dry pyridine at 60°.

The product was isolated as an amorphous, water-soluble barium -37- salt of methyl 2-amino-2-deoxy-N-sulfo-tri-O-sulfo- (3 -D-gluco- 25 pyranosidej m.p. 22> dec., (U) D + U° {c j.U, water).

Anal. Calcd. for CyH-QO-^S^Bag* 2%0: S, 15.635 Ba, 33.1*8.

Found: S, 15.61* (HM); Ba, 32.98 (HM).

Information concerning the relative stability of the amino and hydroxyl sulfate linkages was investigated by means of dilute acid hydrolysis. It was found that a 3 x 10 ^ M solution of this substance in 0.01* iK hydrochloric acid at 95° lost 3*6% of S, which was equivalent to approximately 1 .0 mole of sulfate in 6 20 minutes with the concomitant release of the free amino group (see

Figs. 3 and- U). The 0-sulfate was removed relatively more slowly and only completely so after twelve hours. Mild acid hydrolysis has also been investigated previously by Wolfrom and McNeely (1) in experiments on the deactivation of heparin. The result showed a loss of only of the total sulfur content. However, on the basis of present knowledge of the heparin molecule, this sulfate loss is about equivalent to the amino group released so that a sulfate group shift is not a required postulation* (2) It is also possible that the barium sulfate was peptized by the nearly intact heparin molecule, thus leading to a low value for sulfate \ release.

From the above results, it appeared that the sulfamic acid group is a potent contributor to anticoagulant activity, and it is less stable than the sulfate ester group toward mild acid hydrolysis. Per Cent of So/fur H ydrolyzed 0 2 2 A < A 2 O iue. aito fte ufrCreto the.of Variation £ -Qmino of Sulfur -Corient 2 Figure3. - -deoxy N-hri-O guoyaoie toim I Wt Tn o HdlSS 9^11 HO. ^ 1/1 t 95^ o o t a HydmlySiS of Tine With o It S thorium D -gtucopyranoside d Time Time Hours j 0 2 (A (2 10 8 - Sulfo-Q- /6 O 4 3 /2 2o 24 28 32 4o 44 43 42 Time, Minutes Figure if- . Variation ot Amino- N ( Ninhydnn) with Time. IV. EXPERIMENTAL

1. The Activation of Chitosan.

Ten grams of chitosan (U2a) was suspended in one liter of 2% acetic acid with efficient stirring until most of the solid dissolved. The insoluble residue was removed by centrifugation and the clear gelatinous solution was then neutralized with 2 .5

N sodium hydroxide using the universal indicator strip as a guide.

The white precipitate formed was collected by centrifugation and washed successively with distilled water (four times), alcohol, absolute alcohol, ether and dry pyridine. It was finally suspended in 80 ml. of dry pyridine as a pale tan colloid.

2. The Sulfation of Chitosan with Chlorosulfonic Acid and Dry

Pyridine.

An amount of 60 ml. of freshly distilled dry pyridine was placed in a three-necked flask previously cooled in an ice bath.

To the cooled pyridine was added slowly, through a dropping funnel, 10 ml. of technical chlorosulfcnic acid over a period of

3QJi0 min. This addition must be carried out cautiously, as the reaction was violent and much heat was evolved. On cooling, a pyridine-sulfur trioxide complex crystallized out beautifully.

To this mixture, U0 ml. of the above described suspension of chitosan in pyridine (containing 3*5 g. of chitosan) was added and the whole mixture was heated on a boiling water bath for 1 hr.

It was observed that a sticky reddish brown mass separated out from the pyridine suspension after reaction had proceeded for 20 -ia- min.

After cooling to room temperature, the reaction mixture was poured into 2 0 0 ml. of water to give a clear brown solution, and

75 ml. of 2.5 N sodium hydroxide was added. The sodium salt of crude sulfated chitosan was then precipitated with 5 0 0 ml. of ethanol. The precipitate was redissolved in 200 ml. of distilled water and subjected to dialysis in a seamless tubing (Visking Co*,

Chicago, 111., wall thickness, 0.0023 in.; for 3 days against distilled water. After the solution was concentrated under reduced pressure to 1 0 0 ml., 1 0 ml, of saturated sodium chloride solution was added to the concentrate and the product was precipitated as its sodium salt with 150 ml. of ethanol. The salt was washed with ethanol, ether and then dried over phosphorus pentoxide in vacuo. Yield of the amorphous pale tan solid was 3.2 g. It was readily dissolved in water to form a viscous clear solution. No free inorganic sulfate was detectable.

3. The Molecular Weight Determination of the Sulfated Chitosan.

A. Procedure.

The molecular weight measurements were conducted on the light scattering photometer and the differential refractometer which were designed by Brice, Halwer and Speiser (,6 7) and have been

(67; A. B. Brice, M. Halwer and R. Speiser, J. Opt. Soc. Amer., l£, 768 (1950). described in the literature. The following was a description of these measurements. -U2-

Sulfated chitosan was dissolved in 0.2 M sodium chloride

solution (25 to 50 mg,/ml.) and the solution was placed in several

small lustroid tubes, each containing approximately 9 ml. (tube maximum was 10 ml.) and centrifuged in the Spinco ultracentrifuge for 2 hr. at 3u,000 RFM. The resultant supernatant liquid was

carefully filtered through an ultra-fine fritted glass filter

and the clear filtrate was kept in a clean glass container. Next*

50 ml. of the 0.2 M sodium chloride solution was clarified by

filtering through another clean ultra-fine fritted glass filter

and allowed to fill the dissymetry cell directly. The turbidity

of the solvent was then measured. The photometer with the shutter

closed was allowed to warm up for several minutes with the gal­ vanometer sensitivity turned to maximum. This setting was not

changed during the measurements.

The cell containing the solvent was placed on the platform o and the box was closed. Then the disc was set to read 0

scattering, All of the four neutral filters were inserted into

the filter holder and the shutter was opened. The photomultiplier

sensitivity was slowly turned clockwise until a full scale

deflection was obtained on the galvanometer, The shutter was re­

opened and the scale reading recorded. Readings at -U5 and -90°

and at -135° were also taken using the p r o p e r filter combinations

to bring the deflection on the scale. This procedure was followed

for the wavelength U37 rap.

The photomultiplier was turned to zero and the cell was -113- reinoved from the box. One ml* of the sulfated chitosan solution was added to the solvent by means of a pipet and the solution was mixed thoroughly by swirling. The .cell was replaced in the box and identical measurements were made as for the solvent. This was repeated for as many concentrations as desiredj at least five were recommended since the data must be extropolated to zero concentration.

The refractive increment of the sulfated chitosan solution was determined by pipeting 2 ml. of the solution into one side of the cell and the solvent in the other. Then the handle, which rotates the cell, was placed at the most foward position and the deflection was read through the eyepiece. It was necessary to adjust the slit to a fine line and to focus the microscope before setting the marked line over the slit image and reading the deflection. On turning the handle through 180°, the image was refocused and the deflection was again read. It was necessary to take readings for the solvent in both sides in order to correct the result. The difference in refractive index could then be determined and the concentration of the sample solution was evaluated from the expression:

m a n (RnO - Rtrq0) - 0*075 x O.OOO96 K " K -uu- where,

0 , 0 7 0 * cell correction

0 , 0 0 0 9 6 » correction factor for blue light

For sulfated chitosan,

K ® 1 . 0 9 9 X 1 0 "3, at "A r ^ 3 7 mjL

This was determined from the above equation by measuring in the differential refractometer a series samples of known concentrations,

B, Calculations,

The light scattering measurements on sulfated chitosan prepared from two different methods were shown in Tables V and'

VI.

TABLE V

light Scattering Measurements on Sulfated Chitosan (Prepared by the Fyridine-ClSOcjH MethodJ

Ml. of Sample in 00 mi. of

1 1 0.2 M NaCl 1 F_ cP 1 I O 1 Kh 1 O -130° e ! |vO 1° £ 3 L

0 3U 2 bX 0 01 2 9k 1,2,3,U

1 01 3 5k 2 80 3 93 l,2j3,i

2 37 1,3 U2 3 62 1,3 93 1,2,3,U

3 H9 1,3 07 3 82 1,3 9U 1,2,3,k

h 3k U 72 3 08 h 93 1,2,3jU

0 kO k 80 3 68 k 96 1,2,3,k

# F are the filter factors. Their values are listed in Table VII, -k $ -

TABLE VI

Light Scattering Measurements on Sulfated Chitosan (Prepared by the SO^»HCON(CHt Jq Method)

Ml. of Sample

in 5>0 ml. of 0 Os 0 * 1 F* 0.2 M NaCl -135° F* -1*5° F_ £ F

0 3H.5 2 39 0 1*0 2 97 1 ,2 ,3,1*

1 1*2 3 95.5 1 53 3 95 1,2 ,3,U

2 69 3 83 2 81* 3 99 1 ,2 ,3,1*

3 Uh 1,3 53 3 52 1,3 98 1 ,2 ,3,U

h 52 1,3 65 3 61 1,3 93 1 ,2 ,3,U

5 62 1,3 79 3 72 1,3 95 1,2,3,U

For the B-S Light Scattering Photometer, the theoretical soundness of the method xdLth respect to measurements and calculations has been thoroughly reported,(6 7) The expression used for calculation the turbidity is given as;

_ l6TDan2 Rw Gs 1 - ^ x F where,

T ■ transmittance of the reference standard = 0,288

D ■ correction for standard diffusor ■ 0,8U

a * constant relating the working opal glass standard to the

reference standard = 0*28

1.0U5 is a correction for the reflection of the emerging

transmitted beam -14,6 -

h « the width of diaphragm near cell, cm., = 1 . 2

— is a refraction correction that as almost independent Rc of wavelength, but is related to refractive index. For

water, » 1.025

n ■ refractive index of water ■ 1.33L

s —2s ■ the measured ratio of intensity of light scattered at

90° to the intensity of the incident beam

F is the filter factor. The value is listed in the following

table.

TABLE VII

The Value for the Filter Factors

Filter Number Filter Factor

1 0.1;71

2 0.220

3 0.1OU

it 0.027/

Combining these constants, the turbidity for blue light, i.e., at it37 mjx, may be found from the following expression,

'Y - 0.5256 F 2S 0* where,

F (GsA*wi * the filter factor times the ratio of the 90° -U7-

galvanometer reading to the 0° deflection.

The next step is to evaluate the constant H. Since,

H » 32 It3 rip lAn/cJ2 3 A.4 N where,

noa 1.3J4

X » 437 x 10"7

N a 6.023 X 1023

^n/c a K ■ 1.099 x IcT"3 therefore,

H a 3.23h x 10“6

The calculations from the experimental data in Table V and

Table VI are shown in Table VIII and Table IX respectively.

Plotting Hc/"']“ as a function of concentration, c, and extropolating to zero concentration, gives the value which is equal to l/M. Here M is the "uncorrected molecular weight"

(see Figs. t>,7).

From these data, one obtains an uncorrected molecular weight M2 , for the sulfated chitosan prepared from the pyridine- chlorosulfonic acid method, as 2y0,00o and M^, for the sulfated chitosan prepared from the sulfur trioxide-N,N-dimethyIformamide method, as l6i|.,000.

In order to correct the molecular weight, it is necessary to assume a molecular shape. For sulfated chitosan, a random -U8-

TABLE VIII

Calculations from the Light Scattering Data in Table V

Ml. o f Gs ^ _ c■ 1n He 7 Sample K«xlO~ TxlQ3 T c o r r . ______c 7 ^ x 1 0 z

0 0 .U362 156.9 0 .068U ------—------

1 0.5807 713.3 O.I4IU2 0.3U58 0.03365 0.9736 3.1U9 1.7U5

2 O.U515 1509 0.6813 0 .6 1 2 9 0 .0 6 6 0 1 1.077 3.U82 1.721

3 0 .606U 1509 0.5150 0.8U66 0.0971U 1.1U7 3.71 1.705

U 0.7U21 1509 1.12 1.0516 0.1271 1.209 3.908 1.739

5 0.885k 1509 1.336 1.2676 0.1560 1.230 3.978 1.720 where,

K> » 0.5256 x F and the value z could be evaluated by plotting z as a function of

concentration, c, and extropolating to the zero concentration* It was equal to 1.739 (see Figure $ )*

TABLE IX

Calculations from the Light Scattering Data in Table VI

Ml. of %

-6 H O ^ c l O 7 Sample 0[j K'xlO Txlo3 '1 corr. c z

0 0.U021 156.9 0 .0 6 3 1 ------

1 1.0055 333.2 0.33U9 0.2717 0.0523 1.925 6.226 1.327

0 0.8385 713. k 0.5982 0.5351 0 .1 0 2 6 1.918 6.203 1.235

3 0.5U09 1509 0.816 0.7529 0 .1 5 1 0 2 .0 0 6 6.k87 1.186

k 0.6 9 8 8 1509 1.05U 0.9909 0.1976 1.99k 6.k50 1.176 r* > 0.810-5 1509 1.255 1.1919 O.2 I4.26 2 .03k 6.577 1 .1 6 2 where the average z was equal to 1.22. 2 0

19

/■8 N Z = /7382 - 0-1281 C

• ‘l H,

1-5 i

...... C 0.OZ 0.0*f- O.oio O.oQ o.!o 012. o./F <3/6 a/8 C j p m per loo cc Figure S ■ f^igsgmmeby Z >/&rius Concerrtrzrkon C. — X /O 7 ( C3 5 u n 'h ) 3.6 3 2 0 3 0 gore r o ig E . lclr iQgt f uPhd hk*n n 2 . 0 In Ch,k>*on SuJPahcd VilQigbt of olecular M &. •02 rpae by Te i ne oodfni Aci Me+hod) d o h + e M c.id A ic — lorosd-fon e h in C rid y P The y b Prepxaned ( f • o cy cy ==-xio =2.987 + 6.86 0 + 6.86 ==-xio=2.987 08 g m per per m g loo ■to cc 72 — Solution. d a N 4 / •

16 ■18 ( i. y i ur,tsj 5"9 0 . 6 (o-l - 2 o& h o 4 0 o2 O ------F igure . M o lecu la r W eighf eighf W r la lecu o M . igure F ( P repared by by repared P (

/ 1 1- h 8 ■2o 18 lh 14- 12 ./o g™ P c 50 - P d) ePm M ^- ^ of- i°° cc i°° SulPaied Ch,ioSan >n 0 2 - N a d Soluf Soluf d a N - 2 >n 0 Ch,ioSan SulPaied 22 4 6 8 o 3 28 26 24 on

-52-

coil model is chosen. Then in making correction in shape factor,

the following equation is used.

* W . - ’Wcrr. * •where,

P"^(90°) can be obtained from Figure 2.

Since at z equal to l,7h, P~^-(90°) ■ 1.57

therefore,

Mg n Mg x 1.57 » U56,000 corr. uncorr.

Similarly, at z equal to 1.22, P”^(90°) » 1.15

and

Mo - M9 x 1.15 = 188,600 ■^corr. -uincorr.

The Traube Sulfation.

The chitosan was activated as described above and the ether was replaced with chloroform (ethanol-free). This was added to

liquid sulfur dioxide containing sulfur trioxide (U per Nj

freshly distilled monomer) and maintained at -20° for 30

The solvent was then removed under reduced pressure. All operations were carried out in a dry, closed pressure system. The solid

product was neutralized with aqueous sodium hydroxide and

precipitated as an amorphous water-soluble solid -with ethanol. It

was then dissolved in 200 ml. of distilled water and treated as

described in the previous sulfation procedures.

5. The Sulfation of Chitosan with Sulfur Trioxide-N,N-Dimethyl-

formamide Comolex, -53-

A. The Purification of N,N-Dimethylformamide.

The commercial N ,N-dimethylformarriide was redistilled through a heated Vigreux column (U”). The foreruns were discarded and the fraction of b.p. 152° was collected. Care was taken to protect the distillation vessel from moisture. Distillation can also be carried out under reduced pressure.

B. The Preparation of Sulfur Trioxide-N,N-Dimethylformamide

Complex in N, N-Dimethylf ormamide.

Sulfur trioxide was generated by heating 30p oleum over phosphoric anhydride in a Claisen flask, and was conducted into a receiver containing N,N-dimethyIformamide so that the sulfur trioxide was absorbed immediately. Vilien the solution became saturated with the complex, a white deposit began to appear and the distillation was stopped. Enough N,N-dimethyIformamide was added to the solution to dissolve the excess SO^'HCOI^CII^)^ the final concentration (2.5 Nj was obtained by titrating 2 ml.

of the solution in water with saturated sodium hydroxide (0.1 N)«

The solution was kept in a glass-stoppered (silicon grease) bottle, preferably at 15°« It gradually developed a yellow color on standing.

C. The Solubility of the Compound to be Sulfated in N,N-

Dime thy If ormamide.

The solubility of the canpound to be sulfated in N,N-

dimethylformamide should be determined in advance. Since the -in­ solubility of a salt generally increases according to the series

Ba, Ca jd. K, Na KHj^ < H, the starting material may be converted into a more soluble salt with the aid of ion-exchange resins.

D. The Sulfation Procedure.

An amount of 2 g. of the purified chitosan, previously swollen in N,M-dimethy Iformamide, was placed in a three-necked flask, fitted with a drying tube, mercury-sealed mechanical stirrer, and a dropping funnel. A solution of sulfur trioxide-N,N-dimethyIformamide complex was added (in 20% excess) at room temperature. After the addition was completed, the solution was stirred for 12 hr.

The crude sulfated chitosan could be isolated as the sodium salt by the addition of solid sodium bicarbonate to the reaction mixture, followed by the filtration of the insoluble inorganic salts, and addition of ethanol to the filtrate to complete the precipitation of the product. The precipitate was redissolved in

£0u ml. of x-rater and subjected to dialysis for 3 days. The purified product was obtained by precipitation with ethanol, p. The Anti coagulant Assay by the Sheep Plasma Method.

A. The Determination of Calcium Chloride for Recalcification

of the Plasma.

The optimum amount of calcium chloride necessary to bring about comolete coagulation of the plasma in the shortest possible time was determined by placing 1 ml. samples in each of U clean, dry tubes and adding the necessary quantities of a 2p calcium chloride solution as shown in Table X.

TABLE X

The Determination of Calcium Chloride for Hecalcification of the Plasma

Tube Plasma, ml, ILL. NaCl, 0,9% Ml. CaClp, 20 mg./ml.

1 1 0.3 0.0^

2 1 0.3 0.10

3 1 0.3 0.15

h l 0.3 0.20

The optimum amount of calcium chloride was that which, in the shortest time, produces a firm clot that cannot be removed from the tube upon inverting it. This optimum amount of calcium chloride was then used per ml, of that particular lot of plasma.

B. Plasma.

Fresh frozen sheep plasnja was obtained from the Wilson laboratories in Chicago. The frozen plasma was thawed before use in a water bath not exceeding 37° until fluid and then strained through glass wool to remove any suspended matter.

C. The Preparation of the Sample Solution.

All of the sample solutions, as well as the heparin standard, were made with isotonic sodium chloride. The solution of the provisional International Heparin Reference standard in 0.9%

sodium chloride had a concentration of exactly 50 f of heparin per ml. The solution was kept in a glass-stoppered container and was -56- stored in the refrigerator at 0° when not in use* This solution was stable for at least three months*

D. The Anticoagulant Test*

An individual series of test tubes (size 11 x 100 mm. previously cleaned in chromic acid solution) was set up for the standard and for each unknown to be tested. Varying amounts of the heparin solutions were accurately measured into these sets of tubes* The amounts were selected so that the minimum amount necessary to maintain fluidity of the plasma, as determined by a preliminary trial, was approximately at the center of the series of tubes* The volume of the heparin solution in each test tube was then made up to 0 *U ml, with 0 *9% sodium chloride solution*

TABLE XI

The Anticoagulant Test

Heparin Standard, CaCl2 , ml* Tube Plasma, ml* 50 r/ml. NaCl, 0.9% 20 mg./ml.

1 1 0.16 0 .2 U 0 .2

2 1 0 .1 8 0 .2 2 0 .2

3 1 0*20 O.2 0 0 .2

h 1 0.22 0.18 0.2

5 1 0 .2 u 0.16 0 .2

6 1 0 .2 6 o.iu 0 .2

A similar series of tubes was used for the unknown solution, -57- using the same amount of calcium chloride per ml. of plasma. The amount of calcium chloride was added last and each tube, upon making up to O.U ml. was immediately stoppered with a paraffined

cork and the contents were mixed by inverting the tube three

times. The tubes thus prepared were kept at room temperature.

Exactly 1 hr. after the recalcification, each tube was examined for the extent of coagulation. As in all biological work, the

dosage response curve was S shaped, with the steepest part giving

the most accurate end-point for the comparison of the standard

and unknowns. Experience has shown that sheep plasma gives a much steeper curve than does beef or horse plasma and thus the

end-point is sharper. Complete clotting was determined easily by

inverting the tube. Those tubes falling on the steep part of the

curve were evaluated by estimating visually the amount of clot

and fluid present. Because of the steepness of the dosage

response curve, very few questionable tubes were encountered.

Any tube showing partial clotting was shaken sufficiently to

estimate i-rtiether the clot was greater than $0% of the total

contents of the tube. If the clot was greater than 50n', the tube

was considered clotted; if less than $0%, the tube was considered

as fluid.

E. The Evaluation of the Results.

The end point was taken as the minimum amount of heparin

necessary to maintain fluidity of the plasma (less than 50$ clot;

and this value was recorded for both the standard and unknown. -58-

From these values the potency of the unknown was determined in term of the standard.

If, for example, tube No. 2 of the unknown series containing y IT of sample, matched the No. U of the standard series containing

11 T of standard heparin, then the activity of the unknown sample was:

110 y -1-1. - 13U.5 I.U./mg.

■ 673 Roche A.C.U./mg.

(5 Roche A.C.U. = 1 I.U.).

Standard heparin has an activity of 110 I.U./mg. which was equivalent to 55>0 Roche A.C.U./mg.

7. Model Compound Preparation.

A. The Preparation of Tri-0-acetyl-2-amino-(hydrobromide;-2-

deoxy- -D-glucopyranosyl Bromide.

An amount of lu g. of D-glucosamine ftydrochloride (1.2 moles; was introduced into a dry three-nocked flask, which was fitted with a drying tube and mercury sealed mechanical stirrer. An amount of 25 g. (? moles) of acetyl bromide was added to the vigorously stirred mixture with cautious heating to 60-70° at which temperature range the reaction began. This temperature was maintained by means of an oil bath for about 2 hr. or until the mixture suddenly solidified. Hydrogan bromide was constantly- evolved throughout the reaction. At this point, the flask was -59-

immediately removed from the oil hath and connected to a series

of drying tubes which contained calcium chloride and soda lime.

All of the hydrogen bromide thus evolved could be drawn out immediately through the system by means of the vacuum.

The crude, slightly colored, product was extracted with hot

ethanol-free chloroform (prepared by washing four times with

concentrated sulfuric acid, then water, sodium bicarbonate, water, and finally dried over sodium sulfate for 30 min.) from the remaining D-glucosamine salt, A small amount of absolute ether

(previously dried over anhydrous calcium chloride for 2 days, to remove any of the ethanol) was then added. Upon stirring vigorously for a few sec., the desired purified product (tri-O- acetyl-2 -amino-(hydrobromide)-2 -deoxy- -D-glucopyranosyl brOmide) was precipitated,

B, The Preparation of Methyl Tri-0-acetyl-2-amino-2-deoxy-

-B-glucopyranoside Hydrobromide,

An amount of 5 g* of the tri-O-acety1-2-amino-(hydrobromide)-

2 -deoxy- oi -D-glucopyranosyl bromide was dissolved in 100 ml, of

absolutely dry methanol containing 11 of anhydrous pyridine. The reaction was completed after 6 hr, at room temperature and is reported to be quantitative. The solvent was removed under

reduced pressure and the product was recrystallized from a methanol-ether solvent.

Anhydrous conditions must be maintained at all times during -60-

the reaction because the pyridine salt in the reaction has deliquescent properties and produces sirupy products. The yield was about 50-5 7/> of the theoretical; m.p. 230 -231;° dec.

C. The Preparation of Methyl 2-Amino-2-deoxy- (3 -D-gluco-

pyranoside Hydrochloride.

An amount of 3*3 g. of methyl tri-0-acetyl-2-amino-2-deoxy-

(3 -D-glucopyranoside hydrobromide was dissolved in 100 ml. of

aqueous barium hydroxide containing 7.81; g. of Ba(0H)2 * 8H2 O and the solution was boiled under reflux for 5U min. The solution was neutralized with N sulfuric acid and the precipitate filtered.

The supernatant liquid was evaporated to dryness under reduced pressure and the solid residue was extracted with hot alcohol.

Evaporation of the ethanol extract yielded a sirup which gave a positive Fehling test. This sirup was dissolved in methanol

containing 0 .25 /' hydrogen chloride and heated to 90° for 12 hr.

The acid was removed by precipitation with powdered lead

carbonate and the filtered solution was evaporated to a sirup which reacted as a strong base. On dissolving in cold concentrated hydrochloric acid and precipitating with excess acetone, a

crystalline product was obtained; yield 1 .5 g. of methyl 2 -

amino-2 -deoxy- (3-D-glucopyranoside hydrochloride.

D. The Preparation of Methyl 2-Amino-2-deoxy-N-sulfo-tri-

0-sulfo- -D-glucopyranoside Dibarium Salt.

An amount of 1 g. of methyl 2-amino-2-deoxy- 16 -D-gluco- -61-

pyranoside hydrochloride was sulfated by the pyridine-chloro-

sulfonic acid method, using 25 ml, of anhydrous pyridine and 1 ml,

of chlorosulfonic acid, and maintaining the reaction at 60° for

1 hr. After the reaction was completed, the material was

dissolved in water and neutralized with saturated sodium

carbonate solution. The solution was evaporated to dryness under

reduced pressure and was redissolved in lOu ml, of water. Barium

acetate solution was added until no more precipitate was formed

upon further addition. The barium sulfate precipitate was filtered by means of Filter Cel and alcohol was added to the supernatant

liquid until precipitation occurred. The precipitate was washed with hot absolute alcohol in order to remove any trace of

sodium acetate and barium acetate. Finally, the product was precipitated from water by the addition of ethanolj m,p,225 ° dec,,

l°0 ^ * h° (c_ 3>,h, water). The yield was about 175 of the

theoretical,

8. Calibration of the Hellige Turbidimeter for Sulfate Assay,

A, Instrumentation.

The principle of the Hellige Turbidimeter is based upon the

comparison of a beam of transmitted light with the scattered

light (Tyndall effect) produced by a lateral illumination of

the specimen by the same light source. Since transmitted light was compared against scattered light and the method only

involved the sample itself, the use of standard suspensions and -62- their tedious preparation were entirely eliminated. The apparatus measured 7" x 5" x 16" and was finished in dull crystal black. An opal glass bulb, a vitreous enameled reflector, and a precision slit were mounted in the housing. The slit was operated by a knob with dial. At the front of the apparatus was a door which exposed a platform supporting the specimen tube, a special mirror with aperture, and a filter frame. An opal glass reflector was located under the platform (see Fig. 8j.

B. Reagents.

(a) BaClg* 2 H2 U crystals, 2 U-30 mesh*

(b; Acid salt solution:

2u0 g. of sodium chloride was dissolved in 200 ml. of

distilled water; 20 ml. of concentrated hydrochloric

acid was added and then diluted to luuO ml. with water.

The solution was filtered in order to obtain a solution

of zero turbidity,

(c) Sample solution:

1.0365 g. of sodium sulfate was dissolved in luO ml.

of water.

C. Procedure.

The sample solution was pipetted accurately into a graduated cylinder and 10 ml. of the acid salt solution was added.

Approximately 0,2y g. of the BaCl2* 2H2O crystals were added, as measured bj filling the measuring cup level full, and the time -63-

P - Plunger R - Reflector T - Tube M - M irror E> - Bulb OR - Opal Glass Reflector S - S//f

/“ / > fi. Z ) iogrxamrnatic View of

h le llic g e Turb,

repeated inversion of the cylinder until the crystal were

completely dissolved. The mixing was repeated occasionally for 5

min# period following the BaCl2 * 2 H20 addition.

The sample was poured into a 20 mm. viewing depth tube, if

the sulfate content was expected to be low (below £0 P.P.II.). In

the case of higher concentrations (£0-100 P.P.M.), a 10 ml.

viewing depth tube was used. The plunger was inserted into the

tube and the assembly was placed on the platform. The light

source was turned on and the door was closed. The light rays

emanating from the opal glass bulb were reflected and laterally

illuminated the suspension in the tube. Observed through the

ocular, the light scattered by the suspension was seen as the

outer portion of an annular split field. light rays from the opal

glass bulb also passed through a precision slit and illuminated

an opal glass reflector. Observed from the ocular through the

suspension and through the aperture of a mirror, these light rays

formed the inner portion of the annular field. The outer and

inner portions of the field were seen lighter or darker, depending

on the quantity of light which passed through the slit. By

revolving the knob and adjusting the opening of the slit, it was

possible to regulate the light until the two portions of the

field appear alike. In this knob position, a scale reading was

taken. By using a series of different concentrations of the sodium -65- sulfate solution, a standard graph could be plotted. The data thus obtained are given in Table XII.

TABLE XII

Turbidity Measurements (BaSCV) With Tube of lu urn. Viewing Depth

(bulb B, no filter)

Distilled Acid Salt Sulfur Content Na2S0i. Water, ml. Solution, ml. (micrograms)

0.1 49.9 10 233.5

0.2 49.8 lu 466.5

0.3 b 9 .7 lo 70u.5

0.4 U9.6 10 934.6

0.5 49.5 10 1168.0

9. The Quantitative Estimation of Sulfur Content after the

Hydrolysis of Methyl 2-Amino-2-deaxy-N-sulfo-tri-0-sulfo-

(8 -D-glncopyranoside Dibarium Salt With O.Qli N Hydrochloric

Acid, at 95°.

An accurately weighed sample was dissolved in 50 ml. of distilled water, then 10 ml. of the acid salt solution and 0.9 g. of barium chloride crystals were added to it. The solution was placed immediately into a water bath at 95° and the time was noted.

After that, the solution was cooled at once. The solution was poured into a 10 ml. viewing depth tube, the plunger was inserted and read in the turbidimeter. From the recorded scale, the amount -66- of sulfur liberated could be estimated by means of the standard graph previously. Table XIII gives the data obtained.

TABLE XIII

The Hydrolysis of Methyl 2-Amino-2 deoxy- N-sulfo-tri-Q-sulfo- f3 -D-glucopyranoside Dibarium Salt With O.Oh N Hydrochloric Acid, ______at 95°______

Weight of Scale Time, Sulfur Content, Sample, g. Reading min. micrograms Sulfur, %

0.0153 3U.5 20 550 3.6

0.0137 U7 6o 778 5.6/

0.0128 U8 80 798 6.12

0.0103 67.5 150 1158 11. 2b

0.012U 87 330 1513 12.20

0.0101 91.5 720 1580 15.6u

The total sulfur content of this compound was 15.62^,

Therefore, the above data indicated that after 12 hr., the sulfate groups were completely hydrolyzed with O.Oli N hydrochloric acid at 95°»

10. The ^Quantitative Estimation of Amino Nitrogen after the

Hydrolysis of Methyl 2-Amino-2-deoxy-N-sulfo-tri-0-sulfo-

-D-glucopyranodide Dibarium Salt With 0.0U N hydrochloric

Acid, at 95°

The samples were hydrolysed by 0.0U N hydrochloric acid at

95° for different time intervals as described above. They were -67- then cooled and neutralized to pH 7 by means of 0.1 N sodium hydroxide. An amount of 0.5 ml* (10$ by volume) of aqueous pyridine and 0,5 ml* of aqueous ninhydrin (17) solution were added, and placed in the boiling water bath for 2u min. The solution was cooled in an ice bath and diluted to 50 ml. with water. The amount of amino nitrogen was estimated in a photoelectric colorimeter using a 5h00 S. filter. A blank was carried out in the same manner except that a 5 ml, sample was replaced by 5 ml. of water. The standard curve was obtained with D-glucosamine hydrochloride. The data obtained are listed in the following

Tables (XIV and XV).

TABLE XIV

Estimation of the Amino Nitrogen With D-glucosamine as Standard

Weight of Nitrogen, Scale R<

0.5 3. h

lb. 3 8

27.5 13

3U.U 17

L3.75 21.0

52. u 25.1

62.2 5 2y.h

65.06 32.0 -68-

TABLE XV

Estimation of the Amino Nitrogen After the Hydrolysis of Methyl 2-Amino-2-deoxy-N-sulfo-tri-O-sulfo- /3 -D-gluco- pyranoside Dibarium Salt With 0.0U N Hydrochloric Acid, ______at ?5° ______

Time, Scale Weight of Nitrogen min. Reading Nitrogen, hydrolyzed,

2 iu3 8.75 16.73

7 1U.3 29.U 56.21

15 23.U 1+8,25 92.25

20 25.2 51.8 99.0U

ho 25 51.5 98.1+7

5o 25.3 52.6 100.00 -69-

V. SUMMARY

1. A modified, technique for the sulfation of heparin-like polysaccharides with pyridine and chlorosulfonic acid x^as developed.

2. The sulfated chitosan was found to have an anticoagulant activity of 56 I.U./mg. which was much higher than any other previously obtained by sulfating any polysaccharide.

3. The molecular weight of this preparation was determined by means of light scattering technique. It had a molecular xjeight of 1|5>6,000 or a weight average D.P. of 638 anhydro- disaccharide units.

U. A new homogeneous sulfation method for chitosan, using sulfur trioxide-N,N-dimethylformamide complex in N,K-dimethyl-

formamide was established.

5. Sulfated chitosan, prepared from the sulfur trioxide-N,N- dimethylformamide complex, had an anticoagulant activity of approximately 50 I.U./mg. and its molecular weight was 188,600 or a weight average D.P. of 263 anhydrodisaecharide units.

6. Sulfated chitosan with the high molecular weight of

1x56,000 was found to be twice as toxic as heparin. However, the lower molecular weight one (188,600) was found to be non-toxic.

Its L.D.50 (mouse, I.V.) = 775 mg./kg. was about equal to that of heparin (L.D.^0 ■ 750 mg./kg.). -70-

7. Methyl 2-amino-2-deoxy-N-suKo-tri-0-sulfo-|g -D-gluco- pyranoside dibarium salt was prepared as a model compound for studying the relative stability of the amino and hydroxyl sulfate linkages toward dilute acid hydrolysis.

8. It was found that a 3 x 1 0 M solution of this substance in 0.0U N hydrochloric acid solution , at 9^°» lost 1 mole of sulfate in — 20 minutes with the concomitant release of the free amino group. The 0-sulfate was removed relatively slowly and only completely so after 12 hr.

9. Evidence is presented that the sulfamic acid group is a potent contributor to anticoagulant activity. -71-

VI. ACKNOWLEDGMENT

The author wishes to express her thanks to Professor

M. L. Wolfrom for his interest, guidance and encouragement throughout this work.

The author would also like to thank Dr. Quentin Van Winkle for his advice and help in using the light scattering equipment for the molecular weight determinations.

The sulfation of mucopolysaccharides by using sulfur trioxide-II,N-dimethylformamide complex was suggested by Dr.

T. Y. Shen and preliminary experiments were carried out by him in this laboratory during 1 9 5 2-1 9 5 3.

This work was supported in part by the Dristol Laboratories,

Syracuse, N. Y., under contract with The Ohio State University

Research Fondation (Project h32)» -72-

VII. COLLECTED BIBLIOGRAPHY

(arranged alphabet!cally according to the surnames of the principal authors)

(7) Araki, T., Z. Physiol. Chem., 20, U98 (1895).

(12) Armbrecht, W., Biochem. Z., 95, 108 (1919).

{kU) Astrup, T., and Galsman, I., Acta Physiol. Scand., 8, 36I (19UU).

(27) Astrup, T*, Galsman, I., and Volkert, M., Acta Physiol. Scand., 8, 215 (19UU).

(60) Baumgarten, P., Ber., 5£., 1166 (1926).

(39) Berger, L., and Lee, J., Abstracts Papers, Xllth Intern. Congr. Pure and Appl. Chem., 3U3 (1951).

(22) Bergstrom, S., Z. Physiol. Chem., 238, I63 (1938).

(67) Brice, A. B., Halwer, M., and Speiser, R., J. Opt. Soc. Amer., U0, 768 (1950).

(33) Burkhardt, G. N., and Lapworth, A., J. Chem. Soc., 68U (1926).

(2L) Chargaff, E., Bancraft, F. W., and Stanley-Brown, Margaret, J. Biol. Chem., 115, 155 (1936).

(19) Clark, G. L., and Smith, A. F., J. Phys. Chem., U0, 863 (1936).

(62) Coffey, S., Driver, G. W. , Fairweather, D. A., and Irving, F., British Patents 610,117, Oct. 12, 19U8; 6U2,206, Aug. 30, 1950.

(U2) Coleman, L. L., McCarty, L. P., Warner, D. E., Willy, R. F., and Floksten, J. F., Abstracts Papers, Am. Chem. Soc., 123. 19L (1953).

(U9) Debye, P., J. Applied Phys., 1£, 338 (19Uii).

(50) Debye, P., J. Phys. Colloid Chem., 51, 18 (19U7)•

(53) Debye, P., Physik. Zeits, 28, 135 (1927). -73-

(20; Demole, V., and Reinert, M., Arch. Exptl. Pathol. Pharmakol., 158, 211 (1930).

OS) Doczi, J., Fischerman, A., and King, J. A., J. Am. Chem. Soc., 75, 1512 (1953).

(57) Doty, P., and Stein, R. S., J. Chem. Phys., 17, 7U3 (19U9). (55; Doty, P., and Stein, R. S., J. Chem. Phys., 18, 1211 (1950).

(51) Edsall, J. T., and Danliker, W., Fortshr. Chem. Forsch. Ed., 2, S.l (1951).

(21) Fischer, A., Biochem. Z., 2 k 0 , 36i* (1931).

(65; Foster, R. H. K., J. Lab. Clin. Med., 27, 820 (19U2).

(58; Foster, F., and Paschall, E. F., J. Am. Chem. Soc., 75,, 1182 (1953;.

(36; Gr&mwall, A., Ingelman, B., and Mosimann, H., Upsala Lakarefyren Forh., 50, 397 0~9hS)', Chimie & industrie, 55, 206 (19R6).

(ii; Hoppe-Seyler, F., Ber., 27, 3329 (189R;.

(2 8 ; Husemann, E., von Kaulla, K. N., and Kappesser, R., Z. Naturforschg., 1, 58U (19R6;.

(37; Ingelman, B., Arkiv. Kemi. Mineral. Geol., 21j_, U (19U6). (15) Inouye, Y., and Onodera, K., J. Agr. Chem. Soc. Japan, 25, 553 (1952).

(66; Irvine, J. C., McNicoll, D., and Hynd, A., J. Chem. Soc., 99, 250 (1911).

(16) Jeanloz, R., and Forchielli, E., Helv. Chim. Acta, 33 1690 (1950).

(3) Jorpes, J. E., Borstrom, H., and Mutt, V., J. Biol. Chem., 183. 607 (1950).

(1 0 ) Karrer, P., and Hoffman, A., Helv. Chim. Acta, 12_, 616 (1929;. -7U-

(26) Karrer, P., Koenig, H., and Usteri, E., Helv. Chim. Acta, 26, 1296 (19U3).

(61) Kenner, G. W., and Stedraan, R. J., J. Chem. Soc., 2u69 (1952).

(61+) Kuizenga, M. H., Nelson, J. W., and Cartland, G. F., Am. J. Physiol., 612 (19U3).

(1+6) Lord Rayleigh, Phil Mag., Ljl, 107, 27b (1871).

(U7) Lord RayLeigh, Proc. Roy. Soc., 81+A, 25 (1910).

(1+8) Lord Rayleigh, Phil. Mag., ^5, 373 (1918).

(8) Lowy, E., Biochem. Z., 2^, k7 (1910).

(35) Marbet, R., and Winterstein, A., Helv. Chim. Acta, 3U, 2311 (1951).

(17) Meyer, K. H., and Mark, H., Ber., 61, 1936 (1928).

(32) Meyer, K. II., Piroue, R. P., and Odier, M. E., Helv. Chim, Acta, 571+ (1952).

(1+; Meyer, K. H., and Schwartz, D. E., Helv. Chim. Acta, 33, 1651 (1950).

(13) Meyer, K. H., and Uehrli, H., Helv. Chim. Acta, 20, 353 (1937). (31) Molko, D., and Cotte, J., P ill. soc. chim. b iol., ^2, 312 (1951).

(5) Odder, A., Mem. Soc. Hist. Nat. Paris, 1, 29 (1823).

(52) Oster, G., Chem. Revs., 1+2, 317 (191+8).

(25) Reuse, Jean, Compt. rend. soc. biol., 131, 83I+ (1939).

(38) Ricketts, C. R,, Biochem. J., 51, 129 (1952).

(LpL) Ricketts, C. R., Research, 6, 17s (1953).

(9) Rigby, G. w., U. S. Pat. 2,Oi|0,8?9, May 19, 1936. -75-

(3U) Ross, J,, Percy, J. H., Brandt, R. L., Gebhant, A. J., Mitchell, J. E., and Jolles, S., Ind. Eng. Chem., ^ k , 9 2 k (19U2).

(6; Rouget, M., Compt. rend., U8, 792 (1859)•

(1U) Schorigin, P., and Makarowa-Semljanskaja, N. N,, Ber., 68, 965 (1935).

(30) Snyder, E. G., U. S. Pat. 2,5u8,U33, May 23, 1950.

(29) Solusol, I/S., Dan. Pat. 65,269, Dec. 30, 19U6.

(59) Suter, C. M., Evans, P. B., and Kiefer, J. M., J. Am. Chem. Soc., So, 538 (1938).

(6 3; Swoap, 0. F., and Kuizenga, M. H., J. Am. Pharm. Assoc., 2 8, 563 (19U9).

(23) Tamba, R., Biochem. Z., lltl. 2'fh (1923).

(18) Van Wisselingh, Folia Microbiol., 3, 165 (191U).

(1) Wolfrom, M. L., and McNeely, W, H., J. Am. Chem. Soc., 67, 7U8'(19U5).

(2) Wolfrom, M. L., Montgomery, R., Karabinos, J. V., and Rathgeb, P., J. Am. Chem. Soc., 72. 5796 (1950).

(UO) Wolfrom, M. L., Shen, Tsung-men, and Summers, C. G., J. Am. C .em. Soc., 75., 1519 (1953).

(kj>) Wolfrom, M. L., Weisblat, D. I., Karabinos, J. V., McNeely, W. H., and Mclean, J., J. Am. Chem. Soc., 65, 208U (19U2). -76-

VIII, AUTOBIOGRAPHI

I, Mrs, Tsung-men Shen Han, was born in Peking, China, on September 8, 1927, I attended primary schools in Shanghai and graduated from the Nanki High School, Chungking, China, in

19l4u After that, I enrolled in the National Medical College of

Shanghai, School of Pharmacy, specializing in pharmaceutical chemistry and subsequently graduated therefrom in 19l|8 with the degree Bachelor of Science,

For a period of six months after graduation, I was employed as a research chemist by the Shanghai Pharmaceutical Company.

In l9U9j I came to the United States and studied biochemistry at Emory University, Emory University, Georgia, I received the degree

Master of Science from Emory University in June, 1950, I briefly- attended the University of Maryland and then came to the Ohio

State University in March, 1951, I was appointed as Research

Fellow in the Department of Chemistry, The appointment was terminated June, 195h,