2. Description of the Previously Published Art Levan is a polymer of fructose, C6Hl2ogst and this polymer is a polysaccharide with P- (2->6) linkages between the fructose rings where the numbers describe the carbon atoms in the fructose ring which are linked and theP describes the stereochemical relationship. Levans have also been described as fructans in which the predominant glycosidic linkage between the D-fructofuranoside monomeric units is - (2->6). The levans are generally made by microorganisms and do not occur as high molecular weight compounds in plants. Some low molecular weight levans having a molecular weight of less than 100,000 Daltons can occur in grasses. Alternative spellings are laevan and levulosan. Alternative names include polyfructosan and polyfructose and polylevulan. Levan from some microbial sources is commercially available and sources include Sigma Chemical Co. and IGI Biotechnology.

The reactions in the present invention can be applied to levans made by the following exemplary microorganisms listed in Table 1, but they are not limited to only these microorganisms.

TABLE 1 Levan Forming Microorganisms A. aerogenes A. indicus A. versicolor Acetobacter suboxydans Achromobacter spp.

Actinomycenes sp.

Actinomyces viscosus Aerobacter aerogenes Aerobacterlevanicum Aspergillus sydowi Azotobacter chroococcum Bacillus polymyxa Bacillus licheniformis Bacillus macerans Bacillus megatherium Bacillus mesentericus Bacillus subtilis Bacillus vulgatus and other Bacillus spp.

Corynbacterium laevaniformans Erwinia herbicola Gluconobacter oxydans Leuconostoc mesenteroides Odontomyces viscosus Phytobacterium vitrosum Phytomonas pruni Ps. Fluorescens, and other Pseudomonads Ps. Syringe Pseudomonas prunicola Rothis dentocariosa Serratia kiliensis Steptococcus bovis Steptococcus mutans Steptococcus salivarius Xanthomonas campestris Xanthomonas pruni Zymomonas mobilis The literature dealing with the polymers of fructose or polyfructosans has some nomenclature problems because of the two types of polymers, each with a common name. The other polymer of fructose besides levan is inulin.

Inulin is a polymer of fructose with ß-(1->2) linkages between the fructose molecules. It is found in chicory, Jerusalem artichokes and in onions. It is now available as a commercial product in food processing. Inulins are naturally occurring in plants.

The following references disclose various uses of levan.

PCT Publication No. WO 86/04091 to IGI Biotechnology discloses the microbial production of polyfructose where a water soluble levan is made having a weight average molecular weight of 10, 000 to 40 million. There is no sulfate or other derivative mentioned in this publication.

The material is used as a colloidal stabilizer agent for foods, beverages, pharmaceuticals, dentifrices and cosmetics.

German Patent No. 3,725,554 and its corresponding U. S.

Patent No. 5,055,457 to Schrinner et al. at Hoechst discloses a combination of a polysaccharide sulfate and a xanthine derivative in tablets which shows an anti-AIDS activity by inhibiting reverse transcriptase. The examples given for formulations use a sodium salt of pentosan polysulfate. The formula of the pentosan polysulfate shows two sulfates per sugar unit, which are on a chain of xylose molecules (a pentosan) at the C-2 and C-3 carbons. The pentosan in this patent is a (1->4) linked polymer.

Pentosan is a polysaccharide of five-carbon sugar (xylose), not a six-carbon sugar (fructose). The sulfated polysaccharides are defined as having a molecular weight of 1,000 to 20,000 Daltons, with particularly preferred weight of about 5, 000 to 12,000 Daltons and especially of about 6,000 Daltons.

In the Efficacy Tests, in Table 1, levan sulfate is used either alone or mixed with pentoxifyllin according to their invention. The levan sulfate in Table 1 has a specific molecular weight of 8,000 Daltons and is 100%

sulfated. When used alone, the levan sulfate shows no activity at 10 bg/ml or 20 yg/ml toward the inhibition of syncytium (HIV) formation, whereas the product of the present invention shows 50% inhibition at 0.5 ßgtml and fully inhibits at 1.0 yg/ml.

The sulfated polysaccharides are prepared by a different procedure than the product of the present invention. This patent provides no structural details and thus comparison as to any similarity to the present product is not possible.

The levan sulfate of the present invention has a higher molecular weight of about 500,000 Daltons and is preferably not 100% sulfated. As seen in Formula 1 infra and in the description of the nmr results in Table 2, there is one sulfate on the C-1 of each sugar unit, and about half that number of sulfates distributed between the C-3 and C-4 carbons based on the stoichiometric data from the elemental analysis combined with the nmr spectral information on the position of the sulfates on the ring carbons. When one compares the small molecule of Schrinner with the much larger levan sulfate molecule of the present invention, one would expect that the smaller molecules of Schrinner with more of the toxic sulfur groups would be more effective for inhibition than the much larger molecule of the present invention with fewer sulfur groups per molecule. Unexpectedly, however, such is not the case. As indicated above, the 8,000 Dalton levan sulfate of Table 1 of Schrinner has no activity at 10 ßg/ml or 20 ßg/ml toward the inhibition of syncytium formation, whereas the product of the present invention shows 50% inhibition at 10 ßg/ml and fully inhibits at 20 yg/ml.

U. S. Patent No. 5,273,892 to Okutani discloses a hetropolysaccharide which contains a mixture of sugars and include components such as galactose, galaturonic acid, N- acetyl fucosamine and pyruvic acid in a ratio of 2: 3: 1: 1.

High molecular weight heteropolysaccharides are quite common in bacterial cell walls and in gum arabic. See D.

L. Cook et al,"Comparative Pharmacology and Chemistry of Synthetic Sulfated Polysaccharides,"Arch. int.

Pharmacodyn., 1963,144, No. 1-2,1963. High molecular weight homopolysaccharides, such as the present levan, are much less common. This polysaccharide in Okutani is sulfated at a low level of 10% or less sulfur and has antiviral activity including anti-AIDS activity. In Okutani's prior art section, he notes the action of sulfated polysaccharides against viruses and retroviruses, but concludes that novel sulfated polysaccharides are still desired. Nothing from this prior art reference renders obvious any modification for levan sulfate. The polysaccharides are so completely different. Okutani also does not state a molecular weight for the sulfated polysaccharide, since data is given only for the unsulfated.

Swiss Patent No. 329205 [in German] by K. Vogler at Hoffmann La Roche in 1958 discloses polysaccharide sulfates as blood anticoagulants which can be used as heparin substitutes. There are no activities shown and there are no molecular weights given. As will be seen from the description below of the present invention, Vogler has a different starting material, a different preparation and a different product. The preparation is different since Vogler uses a sulfating reagent in oleum (concentrated sulfuric acid) whereas chlorosulfonic acid is the preferred reactant used here. Vogler's starting materials were partially hydrolyzed polysaccharides with a low molecular weight, whereas the levan starting material here was not hydrolyzed and is of high molecular weight. Finally, the optical rotation of Vogler's polysulfate of hydrolyzed levan is-4.7° whereas the rotation of the levan sulfate here is-17.3°. This is a substantially significant

difference in this physical property and demonstrates the difference in these two materials.

French Patent Publication No. 2,163,622 (1973) to Lucius and Bruening of Hoechst which corresponds to German Patent No. 2,162,343, discloses using levan sulfates with. a molecular weight of 1, 000 to 5,000 to have a very pronounced inhibiting effect on the proteolytic activity of gastric juice, but only prolong blood coagulation time in a very reduced way. The sulfate is suggested for use for the treatment of ulcers in the digestive tract. The molecular weight range was significant since when the molecular weight compared was 45,000 instead of 2,000 the inhibition of gastric ulceration in Shay rats dropped from 74.7% to 49.5%. There is no suggestion of using levan sulfate having a high molecular weight of about 500,000 Daltons.

Additional references on the various syntheses and uses of levans are: G. Avigad, D. S. Feingold: Fructosides Formed from Sucrose by a Coryne Bacterium, Arch. Biochem. Biophys. 70 (1957) 178-184.

H. G. Pontis, E. Del Campilio: Biochemistry of Storage Carbohydrates in Green Plants (P. M. Dey, R. A. Dixon, Eds.), Academic Press, New York 1985, Chapter 5, pp 205-227.

E. Newbrun, S. Baker: Physicochemical Characteristics of the Levan Produced by Streptococcus salivarus, Carbohydr. Res. 6 (1968) 165-170.

B. Lindberg, J. Lonngren, J. L. Thompson: Methylation Studies on Levans, Acta Chem. Scand. 27 (1973) 1819.

J. R. Loewenberg, E. T. Reese: Microbial Fructosanes and Fructosananes, Can. J. Microbiol. 3 (1957) 643-650; Chem.

Abstr. 51 (1957) 13068b.

A. Fuchs, J. M. DeBruijn, C. L. Niedveld: Bacteria and Yeast as Possible Candidates for the Production of Inulinases as Levanases, Antonie Van Leewenhoek 51 (1985) 333-343, Chem. Abstr. 104 (1986) 107853y.

S. Hestrin, D. Avineri-Shapiro, M. Aschner: The Enzymic Production of Levan, Biochem. J. 37 (1943) 450-456.

T. H. Evans, H. Hibbert: Bacterial Polysaccharides, Adv. Carbohydr. Chem. 2 (1946) 204-233.

D. S. Feingold, M. Gehatia: The Structure and Properties of Levan, a Polymer of D-Fructose Produced by Cultures and Cell-free Extracts of Aerobacter levanicum, J. Polymer Sci. 23 (1957) 783-7900; Chem. Abstr. 51 (1957) 9797a.

T. D. Mays, E. L. Dally (IGI Biotechnology, Inc): Microbiological Production of Polyfructose, US Pat.

4,879,228 (1989); Chem. Abstr. 106 (1987) 48658e.

J. F. Kennedy, D. L. Stevenson, C. A. White, L. Viikari: The Chromatographic Behavior of a Series of Fructooligosaccharides Derived from Levan Produced by the Fermentation of Sucrose by Zymomnonas mobilis, Carbohydr.

Polym. 10 (1989) 103-113; Chem. Abstr. 111 (1989) 78521a.

3. Objects of the Invention It is an object of this invention to provide levan derivatives with unique properties.

It is a further object of this invention to provide high molecular weight levan sulfates, phosphates, and acetates.

It is a further object of this invention to provide a processes for producing high molecular weight levan derivatives.

It is a further object of this invention to provide a new use for levan derivatives as an inhibitor of smooth muscle cell proliferation.

It is a further object of this invention to provide a new use for levan derivatives as an anti-AIDS agent.

It is a further object of this invention to provide a new use for levan derivatives as an excipient for use in making tablets.

It is a further object of this invention to provide a new use for levan derivatives as an agent to transform water and organic solvents into a gel.

It is a further object of this invention to provide a new use for levan cross-linked by epichlorohydrin as a film-forming material and substrate for chiral separations.

It is a further object of this invention to provide a new use for levan acetate as a film former.

It is a further object of this invention to provide a new use for levan derivatives for enhancing wound healing in both human and veterinary applications.

These and further objects of the invention will become apparent as the description of the invention proceeds.

SUMMARY OF THE INVENTION Novel derivatives of levan which is the - (2- >6) polymer of fructose have been made with unique properties. The levan which is preferably used as the starting material has a much higher molecular weight than ordinary levan. It has a molecular weight of at least 1 x 106 Daltons and preferable an average molecular weight of about 2 x 106 Daltons, in a narrow distribution range in the natural state and it is produced in high yield from any available inexpensive sucrose. These novel levan derivatives, which are made from levan have high molecular weights, are on the order of about 500,000 Daltons. The derivatives are characterized as levan derivatives having predominant P- (2->6) glycosidic linkages between the D- fructofuranoside monomeric units. Among the preferred derivatives are sulfates, phosphates and acetates which can

be present in an amount of from 0.2 to 3 sulfates, phosphates or acetates per fructose ring.

These levan derivatives have many novel uses. Among them are as an inhibitor of smooth muscle cell proliferation, an anti-AIDS agent, as an excipient for use in making tablets, and as an agent to transform water and certain organic solvents into a gel. Other uses include wound healing for medical use, for dermatological use, for subcutaneous packing for medical or dental use, and for veterinary use in treatment of inflamed udders and mastitis in cattle. These uses are especially relevant if there are bleeding complications. The subcutaneous packing can be used, for example, for burns, surface wounds or skin infections. When the levan itself is cross-linked by epichlorohydrin it acts as does acetylated levan as a film former to form thin plastic films.

BRIEF DESCRIPTION OF THE DRAWING The Figure illustrates a comparison with heparin for use as an inhibitor of smooth muscle cell (smc) proliferation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The levan derivatives and levan cross-linked products are described below. The especially preferred levan sulfate begins the description.

The higher molecular weight levan sulfate of the present invention is a white granular solid which is very soluble in water and it is non-hygroscopic. In some reactions the color can be from cream to pale yellow. It stores well. The generic formula for levan sulfate can be written as:

SULFATED LEVAN Formula 1 which indicates that the C-1 position is fully sulfated and that the C-3 and C-4 positions are variously sulfated. The value of x varies from 0 to 2 and wherein the sulfate is present in an amount of 0.2 to 3 sulfates per fructose ring. The value of n ranges from about 1250 to 1550.

The levan polymer has about 8% branching and it is expected that the sulfated polymer has the same.

The sulfated levan compound has a molecular weight of about 500,000 Daltons as determined by gel permeation chromatography (GPC). This large size is unique and believed to be much larger than any other levan compound reported in the literature.

Further confirmation of the presence and position of sulfate moieties in levan sulfate is given by nuclear magnetic resonance spectroscopy (nmr) data. The 13C nmr assignments for highly sulfated levan and a reference levan are set forth in Table 2.

TABLE 2

C-1 C-2 C-3 C-4 C-5 C-6 Sulfate 55.39 103.66 80.20 79. 77 80. 75 67.01 Levan 103. 5 80.05 79. 67 80. 60 65.12 Ex. 1 79. 92 79. 40 80. 45 Levan 61. 4 105.0 77.0 76. 4 81. 1 64.2 See M. A. Clarke, E. J. Roberts, W. S. C. Tsang, et al, Proc.

Conf. Sugar Proc. Res, 1988, page 139 for information on the levan.

Shifts upfield for C-1 and downfield for C-3 and C-4 indicate addition of a sulfate (replacement of an OH with a charged group). Over the range of compounds the C-1 is preferentially sulfated with subsequent sulfate substitution occurring on C-3 and C-4 without preference.

In the highly sulfated compounds C-1 is fully sulfated, and C-3 and C-4 partially sulfated. Lack of splitting on C-1 indicates that the position is fully sulfated. Triplet splitting on C-3 and C-4 indicates that each position is partially sulfated (some sulfated, some not). The C-5 signal is split into a triplet by the three possible sulfated environments. For C-2 and C-6, the linkage carbons, they are shifted upfield and downfield, respectively, as expected when the molecule is substituted with a large negative group. The sulfate can be present in an amount of 0.2 to 3 sulfates per fructose ring.

Additional properties for the levan sulfate are an optical rotation of light of [a] 20 =-17. 3°.

The weight range of compositions for sulfated levans are preferably: C from 15% to 40% S from 2% to 20% O from 46% to 52%

As seen in Example 1 below, a preferred composition has C: 15%, S: 18.4%, O : 48.2%, H: 2.1% and Na: 14.18%.

A second preferred levan derivative is the phosphated levan which has a similar structure to levan sulfate with the phosphate groups substituted for the sulfate groups.

The generic formula for levan phosphate can be written as: PHOSPHATED LEVAN Formula 2 which indicates that the C-1 position is fully phosphated and that the C-3 and C-4 positions are variously sulfated.

The value of x varies from 0 to 2 and wherein the phosphate is present in an amount of 0.2 to 3 phosphates per fructose ring. The value of n ranges from about 1250 to 1550. The molecular formula for levan phosphate shows a minimum of one phosphate group per five fructose residues, as the sodium salt, with the phosphate groups acting to cross link chains of levan, with a range up to one phosphate group per fructose residue. Over the range of compounds the C-1 is preferentially phosphated with subsequent phosphate substitution occurring on C-3 and C-4 without preference.

In the highly phosphated compounds C-1 is fully phosphated, and C-3 and C-4 partially phosphated. The phosphate can be present in an amount of 0.2 to 3 phosphates per fructose ring.

The weight range of compositions for phosphated levans are preferably: C from 25% to 39% P from 14% to 2%.

The viscosity of a 1% aqueous levan phosphate solution was measured at 12 rpm for various temperatures and the results are given in Table 3.

TABLE 3 Temperature °C Viscosity Viscosity (SS) (centipoise) 20 2. 92 2. 6 40 3. 57 2. 97 60 4. 29 3. 89 80§ 0. 77 3. 56 * too solid to read at 12 rpm so 80 rpm was used.

An aqueous solution or gel of levan phosphate also maintains its gel integrity when frozen and thawed, and when heated to 100°C where it is still a gel and then cools back to room temperature. The gel retains it structure up to 100°C. The levan phosphate also gels certain organic solvents, including methanol, ethanol, 50% ethanol/water, n-propanol, iso-propanol, n-butanol, iso-butanol, tert- butanol, methyl ethyl ketone, dimethylformamide (DMF), dioxan, and dimethyl sulfoxide. This is very unusual since a polysaccharide does not normally gel organic solvents.

The phosphated levans can be used to cause water to gel or become a solid phase. Applications of this gel include use as topical applications of pharmaceuticals and for cosmetic applications. The gels can be used to extinguish fires, by holding water in place at the site of the fire. They can also be used to hold water in place either in or on soil and earth. The phosphated levans can

be used as a food ingredient, including foods to be frozen or heated, as a thickening agent or a fat substitute.

A third preferred derivative is levan acetate which is insoluble in water, methanol and ethanol. It is partly soluble in chloroform, and acetonitrile, and very soluble in dimethylsulfoxide.

The generic formula for levan acetate can be described as ACETYLATED LEVAN Formula 3 which indicates that the C-1 position is fully acetylated and that the C-3 and C-4 positions are variously acetylated, with the total degree of acetylation on C-3 and C-4 being X, where X varies from 0 to 2 and wherein the acetate can be present in an amount of 0.2 to 3 acetates per fructose ring. The value of n varies from about 1325 to 1650. Over the range of compounds the C-1 is preferentially acetylated with subsequent acetylate substitution occurring on C-3 and C-4 without preference.

In the highly acetylated compounds C-1 is fully acetylated, and C-3 and C-4 partially acetylated. The acetate can be present in an amount of 0.2 to 3 acetates per fructose ring. The 13C nmr assignments for acetylated levan and a reference levan are set forth in Table 4.

TABLE 4

See M. A. Clarke, E. J. Roberts, W. S. C. Tsang, et al, Proc.

Conf. Sugar Proc. Res, 1988, page 139 for information on the'levan.

The nuclear magnetic resonance (nmr) shows the structure with the acetate preferentially fully substituted on C-1 and subsequently partially substituted on C-3 and C-4. This is about the same substitution pattern as seen on the levan sulfate.

The weight range of components for levan acetates are preferably: C from 43% to 47% H from 5.0% to 8.0% O from 48% to 51.0% with about 28-29% weight in acetyl groups. It is a primary acetyl compound with approximately 0.3 acetyl groups per fructose in secondary positions.

Levan acetate has an insolubility in water and a chiral nature. One potential use for levan acetate is as a chromatographic support for chiral separations. Levan acetate can also be used to make thin films with potential use in foods and packaging. To prepare a thin film, levan acetate (2 g) is dissolved in one of several organic solvents (20 to 30 ml) and the solution is spread on a surface. As the solvent is evaporated (mild heat and forced air) a transparent or semitransparent film forms.

In another embodiment of the invention levan is crosslinked with epichlorohydrin. This material can be used to separate racemic mixtures of chiral compounds

including sugars and amino acids. The compound exhibits a very low pressure drop across a column. Thus, the compound can also be used as a substrate for capillary electrophoresis.

The preparation of the levan derivatives begins with an original fructose polymer which has an average molecular weight of about 2 million Daltons where the molecular weight is a distribution around this average value. One such levan, made by B. polymyxa, can be made from sucrose cheaply and in large quantities. See U. S. Patent No.

5,547,863 (U. S. Patent Application Serial No. 07/393,604, filed August 14,1989, issuing August 20,1996) by Han and Clarke. A further description of this higher molecular weight levan is also given in"Production and Characterization of Microbial Levan,"by Y. W. Han and M.

A. Clarke, J. Agriculture and Food Chemistry, 1990,38,393 and also in"Polyfructose: a New Microbial Polysaccharide" by M. A. Clarke, A. V. Bailey, E. J. Roberts and W. S.

Tsang in Carbohydrates as Organic Raw Materials, Ed. F. W.

Lichtenthaler. VCH N. Y. 1990.367 pp. These levans or any other levans having a molecular weight over 0.5 x 106 Daltons can be used as the material to prepare the levan derivatives.

Having described the basic aspects of the invention, the following examples are given to illustrate specific embodiments thereof.

Examples 1-5 In these examples, a series of runs were conducted to make the sulfated levan.

The procedure involves starting with a high molecular weight form of levan produced by the procedure described by Y. W. Han and M. A. Clarke in"Production and Characterization of Microbial Levan,"J. Agriculture and Food Chemistry, 1990,38,393. The levan was sulfated by the procedure generally described in Biochem. J. 58,532-

536 (1954) using 5.8 ml of chlorosulfonic acid in 36 ml of pyridine and 2.65 g of levan. The procedure described in the article was followed except the aqueous layer was dialyzed and no ethanol was added because when reacting in pyridine the levan sulfate derivative was insoluble in pyridine and separated as a semisolid. The product was dialyzed for 48 hours and concentrated. It weighed 5.2 g.

The product gave a positive test for sulfate and was dialyzed again.

The following larger scale runs were conducted and the reactants and analysis are set forth in Table 5. Example 1 has the best yield and exemplifies preferred conditions.

In Examples 2,3 and 4, when the amount of chlorosulfonic acid is reduced, the yield also is reduced. In Example 3, the amount of pyridine dilutes further and results in low yields. In Example 5, there is sufficient chlorosulfonic acid, but with the low amount of pyridine, the yield is not as large as in Example 1.

TABLE 5 Example 1 2 3 4-5 pyridine ml 72 5 75 5 5 chlorosulfonic 11 7 5 3 15 acid ml levan g. 10 10 10 10 10 yield g. 20 11. 5 6 10. 0 12.8 C wt. % 15. 10 31. 55 29.82 39.22 17.8 14.93 H wt. % 2.03 3.36 4.73 6.30 2.41 2.18 S wt. % 18.31 10.72 8.13 1.88 17.61 18.44 Na wt. % 14.13 7.82 0.37 1.33 12.38 14.24 0 wt. % 48.29 45.30 51.95 52.01 50.03 47.95

Notes: 4a carried out the same as Ex. 2 except the temperature never raised above 60°C The data from Table 5 can be used to derive molar formulas for the products. Consider first Example 1.

After obtaining the elemental weight from the two runs, the number of moles of each element is calculated. Since a fructose ring has 6 carbon atoms, the ratio of molar amounts are normalized to C equal 6. The factor used was 4.8. The average molar amounts for the two runs are determined and the results are set forth in Table 6 below.

TABLE 6

wt. % atomic wt. moles moles per Avg Mole C as 6 ratio C15.10 12. 01 1. 26 6. 03 6.00 C14.93 12. 01 1. 24 5.97 H2.03 1. 01 2. 01 9. 65 10.00 H2.18 1. 01 2. 16 10.36 S18.31 32. 06 0. 57 2. 74 2.75 S18.44 32. 06 0. 58 2.76 Na14.13 22. 96 0. 62 2. 95 2.97 Na14.24 22. 96 0. 62 2.98 O48.29 16. 00 3. 02 14. 49 14.44 O47.95 16. 00 3. 00 14.39

To fit the sulfur data of 2.75 moles to a formula with whole integers, a chain of 4 fructoses can be taken where there would be 3 sugars substituted with 3 sulfates and 1 sugar with 2 sulfates, making the average sulfur content 2.75. The sulfur and oxygen balance for these 4 fructose chains are set forth in Table 7.

TABLE 7

Chain S O-sulfate O-fructose Total O 1 3 6 15 2 3 9 6 15 3 3 9 6 15 4 2 6 6 12 Average 2. 75 14. 25

The sodium is slightly higher than the sulfur, indicating 3 positive charges/monomer, rather than the 2.75 positive charges from the varied substitution. The higher sodium could be from an incompletely dialyzed salt.

The proposed formula is C6H7.25S2. 75014. 2. 5Na2. 75 or in a whole integer form (C6H7S3O15Na3)3 (C6H8S2O12Na2) An analysis can be made for the material in the single run in Example 5. The factor was 4.05 and the results are set forth in Table 8.

TABLE 8

wt. % atomic wt. moles moles per C as 6 C17.80 12. 01 1. 48 6.00 H2.41 1. 01 2. 39 9.66 S17.61 32. 06 0. 55 2.22 Na12.38 22. 96 0. 54 2.18 O50. 03 16. 00 3. 13 12. 66

To accommodate the S value of about 2 and 1/4 moles, a 5 fructose chain can be considered where four fructose chains have 2 sulfates and one fructose chain has 3 sulfates.

The O balance is given in Table 9.

TABLE 9

Chain S 0-0-Total O Total sulfate fructose per O sugar 4 fructoses 2 6 6 12 48 with 2 sulfates 1 fructoses 3 9 6 15 15 with 3 sulfates Total 63 Average 12. 6 Proposed formula C6H7. 8S2. 2°12 6Na2. 2 or in a whole integer form (C6H8S2O12Na2)4 (C6H7S3O15Na3) Examples 6-9 These examples illustrate the production of levan phosphate by Method 1.

A 10 g sample of levan of high molecular weight of at least 1 x 106 Daltons was dissolved in 200 ml pyridine, cooled to 0°C, and treated with 10 ml PC13, added dropwise.

The mixture was stirred until it reached room temperature.

Then, 200 ml of saturated sodium carbonate was added, and the aqueous layer was separated from the pyridine layer.

After dialysis, the aqueous fraction was concentrated and freeze-dried. The product was a white powder. Three runs using PC13 are given as Examples 6-8 in Table 10 and a fourth run using POC13 as the phosphorylating agent is given as Example 9.

TABLE 10

Example6789 pyridine ml 200 200 200 200 phosphorylating PCl3 PC1, PCJL POCl. agent amount of 10 ml 10 ml 10 ml 10 g phosphorylating agent levan g. 10 10 10 10 yield g. 11.3 11.3 5. 0 6.0 C wt. % 30.22 37.65 17.23 38.33 H wt. % 5.37 5.87 4.03 6.08 O wt. % 47.28 P wt. % 8.58 4.28 11.86 2.26 Na wt. % 6.25 2.60 11. 91 0.42 In Example 6 of the levan used was finely ground to pass a 24 mesh screen and, therefore, had a much larger surface area for reaction than did the levan in Example 8.

It is believed that this is the reason why Example 6 has a higher yield of product with a lower phosphorous content per weight.

Example 10 This example illustrates another method to produce levan phosphate by Method 2.

A 10 g sample of levan obtained by the procedure as used in Example 1 was dissolved in 200 ml pyridine and the solution was cooled to 0°C. Then, 10 g of POCl3 was added slowly. A condenser was then connected to the flask and the solution was heated to 80°C for four hours. The

solution was cooled and 200 ml of saturated sodium carbonate was added. The aqueous layer was separated and dialyzed for 100 hours. Some of the material remaining in the bag was suspended in water and freeze dried. The effluent from the centrifuge was concentrated and freeze- dried, yielding 6.0 g of white powder.

Example 11 This example illustrates a method to produce levan acetate.

A 10 g sample of levan obtained by the procedure as used in Example 1 was-suspended in 150 ml of dry pyridine and 25 ml of acetic anhydride was added. The mixture was heated to 100°C and held at 100°C for two hours. The mixture was poured into 700 ml of absolute ethanol with stirring. The solid precipitate that formed was filtered off, washed with ethanol and air dried. The yield was 13 g and the determination of the degree of acetylation showed 28.17 % acetyl groups.

Example 12 This example illustrates the production of levan crosslinked with epichlorohydrin.

A 10 g sample of levan, obtained by the procedure as used in Example 1, was dissolved in 150 ml dimethyl sulfoxide and 10 g powdered sodium hydroxide and 20 g of epichlorohydrin were added. The mixture was heated at 70°C for two hours. Then 100 ml of water was added, and the solution was neutralized with acetic acid. The solid material was filtered off and washed well with water. The yield was 11.8 g. The cross-linked material is a pale yellow granular solid. When added to water, the grains swell, but do not dissolve and form a suspension (pH 5.5).

The composition data for the levan cross-linked with epichlorohydrin are set forth in Table 11.

TABLE 11 Product Average General Range Composition Value wt % wt % C 40. 7 33-46 H 7. 22 4-11 Cl <0. 5 0.3-0.8 O 41. 76 28-44 This crosslinked product can be used to form thin plastic films as well as being used as a support medium for separation of chiral compounds. A column of cross-linked epichlorohydrin was found to separate a mixture of D (+) and L (-) fucose. The epichlorohydrin cross-linked levan may serve as a substrate to separate racemic mixtures of stereoisomers. The cross-linked material may serve as a substrate for gel permeation chromatography because of its swelling characteristics.

The higher molecular weight levan derivatives, such as levan sulfates, phosphates, and acetates, according to the present invention have many unique properties. The following are some of the applications for which they can be used.

The levan derivatives can be used for the inhibition of the proliferation of smooth muscle cells (smc). The materials have an anti-clotting factor activity or smooth muscle cell inhibition. They can be used for heart patients and in all surgery and for treatment to reduce, delay or prevent restenosis after angioplasty. Compounds which inhibit the proliferation of smooth muscle cells are a goal for many drug companies and the compound tested below exhibited better activity than heparin at low doses in the range of less than 1.0 ßg/ml and at medium doses in

the range of between 1 and 10 ßg/ml. The activity of this material is given in Example 13.

Example 13 This example illustrates the property of the inhibition of the proliferation of smooth muscle cells by levan sulfate.

A sample of levan sulfate as made by the procedure in Example 1 was tested for antiproliferative activity against smooth muscle cells in culture by using the standard assay for smc proliferation in which cell count is assayed. The comparison material was commercial heparin. The results of the test are set forth in the Figure. The activity of the levan sulfate, code named Spriex-1, was approximately one log greater than the commercial heparin when expressed in % inhibition in heparin equivalents for smc growth inhibition.

The levan derivatives can be used for their anti-AIDS activity. The compounds have a positive effect against AIDS in high doses and they are relatively non-toxic to the patient. The evaluation of the material for its anti-AIDS activity is set forth in Example 14.

Example 14 This example illustrates the anti-AIDS activity of levan sulfate.

Levan sulfate made according to Example 1 from a levan with a high molecular weight was tested for inhibition of HIV replication in a human T-cell line (MT4). Effective dose giving 50% reduction of virus growth (ED50) was 0.5 ßg/ml. The levan sulfate shows 50% inhibition of syncytium formation at 0.5 Ug/ml and fully inhibits at 1.0 ßg/ml which results are totally unexpected.

This is to be compared with the results from the Schrinner et al. U. S. Patent No. 5,055,457, where the control levan sulfate, having a specific low molecular weight of 8,000 Daltons and 100% sulfated, shows no

activity at either 10 ßg/ml or 20 Ug/ml toward the inhibition of syncytium formation as set forth in their Efficacy Tests in Table 1.

The levan derivatives can be used as a carrier compound or excipient when making drug formulations with other drugs. Formulas for tableting are known and there are standard recipes. These levan derivatives serve this function because the original polyfructose also serves this function.

The levan derivatives can be used as a gel former to transform water into a gel and certain organic solvents into gel form. An example is described in Example 15.

Example 15 This example illustrates the use of levan phosphate as a gel former.

When levan phosphate as made in Examples 6 to 9 is added to water in an amount of from about 0.1-10%, or when it is added to a dilute salt solution, the water becomes a gel. The gel has a viscosity in the ranges set forth in Table 3 which depend on the temperature.

Similarly, when a very small amount of levan phosphate is sprinkled into a small dish of water, it will immediately turn the water into a gel.

Among the uses for these gels are in applications where water can be used to exclude air such as in fire extinguishing. Additional medical applications include using the gel as a vehicle to apply medicine to body skin such as in the treatment of burns and wound healing.

Additional uses in food manufacture and processing are as fat substitutes, to add stability, texture and mouthfeel to reduced fat foods.

Example 16 This example illustrates the use of levan phosphate as a gel former with certain organic solvents.

When levan phosphate, as made in Examples 6-9, is added to any of, by not only, ethanol, methanol, propanol, butanol and their mixtures with water, or to dimethyl sulfoxide, or to formamide, or other polar organic solvents, the liquid becomes a gel. Levan phosphate is added in an amount of 0.5% to 10t. Ethanol/water mixtures include potable ethanol liquors, liquerers, cordials and distilled liquors.

Among the uses for these gels are in food processing, to carry flavor and color compounds; in frozen foods as stabilizers; and in medicine as carriers for pharmaceuticals.

Example 17 This example illustrates the use of levan acetate in film formation.

A sample of levan acetate as made by the procedure in Example 11 in a white powder form is dissolved in an organic solvent such as dimethyl sulfoxide and the solution is applied to a surface. When the solvent has evaporated with heat the levan acetate remains as a thin transparent film. It is anticipated that this film is edible.

The levan which was crossed-linked by epichlorohydrin as made in Example 7 can use used for physical separation of chiral mixtures such as a racemic mixture of D-and L- forms of amino acids and sugars. Example 17 illustrates the separation possible with D (+) and L (-) fucose.

Example 18 This example illustrate the use of cross-linked levan as a separation agent.

A separatory column was filled with the granular, pale yellow cross-linked levan as prepared in Example 7, which was crossed-linked by epichlorohydrin and which was insoluble in water, but which swells in water, holding water within the granules. An aqueous solution of a chiral mixture of D (+) and L (-) fucose was passed over the column

and the components were separated on the column into their optically active components.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of this invention.