WO1994003498A1

WO1994003498A1 - Improved method for preparing soluble glucans

Info

Publication number: WO1994003498A1
Application number: PCT/US1993/007329
Authority: WO
Inventors: David L. Williams; Rose Brigid Mcnamee; Henry A. Pretus; Isaac William Browder
Original assignee: Bioglucans, L.P.
Priority date: 1992-08-05
Filing date: 1993-08-04
Publication date: 1994-02-17
Also published as: EP0654045A4; ZA935649B; SI9300415A; CN1091747A; AU4837693A; CA2141845A1; MX9304728A; EP0654045A1

Abstract

A highly efficient, rapid method for preparing aqueous soluble glucans is described.

Description

IMPROVED METHOD FOR PREPARING SOLUBLE GLUCANS

1. FIELD OF THE INVENTION This invention relates to an improved, highly efficient method for preparing soluble glucans. The method advantageously avoids the use of a highly polar solvent such as dimethyl sulfoxide (DMSO) . The soluble glucans prepared by the method of the invention are non-toxic and exert pronounced immunobiological responses when administered in vivo, most notably immunostimulation of macrophage activities and stimulation of hematopoietic bone marrow activity. The soluble glucans also exhibit significant effects against malignant neoplasms, including melanomas and sarcomas.

2. BACKGROUND OF THE INVENTION The term "glucan" refers generically to a variety of naturally occurring homopolysaccharides or polyglucoses, including polymers such as cellulose, amylose, glycogen, laminarians, starch, etc. Glucan encompasses branched and unbranched chains of glucose units linked by 1-3, 1-4, and 1-6 glucosidic bonds that may be of either the alpha or beta type. As defined herein, "particulate glucan" designates a water-insoluble particulate (about 1-3 μ) polyglucose such as that derived from the cell wall of the yeast Saccharomvces cerevisiae. Particulate glucan is macromolecular and comprises a closed chain of glucopyranose units united by a series of β-1-3 glucosidic linkages. (Hassid et al., 1941, J. A er. Chem. Soc. 63_.:295-298; DiLuzio et al., 1979, Int'l J. Cancer 2A_*.773-779) . X-ray diffraction studies have demonstrated that particulate glucans exist in the form of a triple-stranded helix. (Sarko et al., 1983, Biochem. Soc. Trans. 11:139-142) .

Particulate glucan is a potent activator of the macrophage/monocyte cell series, complement, as well as T and B cell lymphocytes. Thus, particulate glucan has profound effects on both the reticuloendothelial and immune systems. Particulate glucan has been shown to modify host resistance to a wide variety of infectious diseases (see review by DiLuzio, 1983, Trends in Pharmacol. Sci. 4_.:344-347 and references cited therein) . In addition particulate glucan has been shown to inhibit tumor growth and prolong survival in syngeneic murine tumor models (DiLuzio et al., 1979, Advances in Exp. Med. Biol. 12_1A:269-290) . In vitro studies using normal and tumor cells incubated with particulate glucan have demonstrated that glucan exerts a cytostatic effect on sarcoma and melanoma cells and a proliferative effect on normal spleen and bone marrow cells (Williams et al., 1985, Hepatology 5 :198-206) .

Notwithstanding the beneficial biological properties of particulate glucans, the adverse side effects of particulate glucans have made these compounds all but useless in clinical medicine. When particulate glucan is administered .in vivo to animals, a number of severe side effects have become apparent, the most notable being: (1) formation of granuloma (sarcoidosis) ; (2) development of hepatosplenomegaly; (3) increased susceptibility to gram-negative infections and endotoxins; (4) activation of complement (anaphylatoxin) ; (5) development of pulmonary granulomatous vasculitis; (6) development of hypotension following intravenous administration; and (7) development of microembolism when administered at high concentrations. Additionally, there is a relatively high degree of acute toxicity observed when particulate glucan is administered in vivo. For example, following a single intravenous injection of an aqueous suspension of particulate glucan, 20% and 100% mortality were observed in mice receiving glucan at 250 and 500 mg/kg body weight respectively.

Moreover, due to the particulate nature of the glucan preparation (1-3 μ) , it is difficult to administer via an intravenous route. By way of illustration, one patient receiving particulate glucan required constant supervision during intravenous (IV) administration, continuous shaking of the IV drip bottle being essential to maintain the particulate glucan in suspension to avoid formation of emboli in the patient.

Although slightly soluble neutral glucans are commercially available, these preparations are not suitable for intravenous administration because the aqueous solutions have very high viscosity and, more importantly, because their use when administered to experimental animals has inevitably been accompanied by considerable toxicity.

In view of the disadvantages of particulate S-,1,3 glucans for in vivo administration, extensive studies were previously undertaken to develop a soluble 0-1,3 polyglucose which might be non-toxic, induce no significant pathology, and yet retain significant immunobiological activity. A low molecular weight non-phosphorylated soluble glucan preparation prepared by formic acid hydrolysis of particulate glucan has been shown to have anti-tumor and anti-sraphylococcal activity (DiLuzio et al., 1979, Internat'l J. Cancer 24:773- 779) . Unfortunately, the low yield and diversity of fractions obtained by this method made this preparation non-useful for prophylactic and therapeutic applications. (See DiLuzio, 1983, Trends in Pharmacological Sciences 4.:344-347). Similarly, attempts to solubilize particulate glucan by the addition of dimethylsulfoxide (DMSO) a "molecular relaxant" were also unsuccessful. It was thought the DMSO would "relax" the triple helical configuration of the glucan molecule. Indeed, particulate glucan dissolves in the presence of DMSO. All attempts to isolate a soluble glucan from the DMSO solution, however, resulted in failure. Upon dilution of the DMSO-glucan solution with various aqueous media such as glucose or saline solutions, the particulate glucan was reformed.

Following dilution of the DMSO-soluble glucan solution with saline, all animals receiving injections of these solutions died immediately upon injection due to high concentration of DMSO or the reformation of the particulate glucan. Upon precipitation of the glucan in DMSO solution by the addition of ethanol (100%) , the precipitate was collected and lyophilized. When this lyophilized glucan was added to water, the particulate glucan reformed. Early attempts to convert the neutral glucan preparation of particulate glucan to a polar-charged preparation by the addition of phosphate or sulfate groups as well as by acetylation were also unsuccessful. Each of these procedures was conducted following the solubilization of particulate glucan by DMSO and in each instance the particulate glucan was reformed.

A neutral preparation of particulate glucan was successfully converted into a stable solubilized form termed "soluble phosphorylated glucan" (hereinafter termed "glucan phosphate") through phosphoric acid hydrolysis using the method described briefly below. As defined herein, the term "glucan. phosphate" or "soluble phosphorylated glucan" refers to the class of glucans solubilized by the addition of charged phosphate groups through reaction with phosphoric acid. These are the same or substantially similar to those substances as described in U.S. Patents Nos. 4,739,046; 4,761,402; 4,818,752 and 4,833,131. This soluble phosphorylated glucan is non- toxic, non-i munogenic, and substantially non- pyrogenic (see U.S. Patent Nos. 4,739,046; 4,761,402; 4,818,752 and 4,833,131).

According to the method of U.S. Patent No. 4,739,046, glucan phosphate was prepared as follows: particulate glucan or a polyglucose-protein complex was suspended in the highly polar aprotic solvent DMSO. A strong chaotropic agent, urea, was added, the mixture heated and maintained at 50-150°C with constant stirring while phosphoric acid was slowly added. Preferably, the reaction mixture was maintained at about 100°C for about 3-12 hours to increase the yield of the bioactive product. The product was isolated and the DMSO, urea, glucose, and any unreacted phosphoric acid were removed. The yield, after reaction for about 6 hours at 100°C, is stated to be about 70-90%.

Another new class of soluble glucans, in which the polyglαcopyranouse chains have acquired a charged group from a non-phosphorous containing hydrolytic acid, are described in copending application Serial No. 07/649,527. The soluble glucans having a charged group, including such as a sulfate or nitrate group, are also capable of exerting a pronounced immunobiological effect when administered in vivo. These soluble glucans immunostimulate macrophage activity with resulting activation of the immunoactive cells in the reticuloendothelial and immune systems. In addition, these soluble glucans enhance hematopoietic bone marrow activity.

According to the methods of Application Serial No. 07/649,527, the soluble glucans were prepared as follows: particulate glucan was suspended in a solution of DMSO and urea. The concentrated mixture was heated to about 50-150°C, a concentrated hydrolytic acid, such as sulfuric or nitric acid alone or in the presence of DMSO was added and the reaction mixture was maintained at about 50-150°C with stirring. The bioactive product was isolated and the DMSO, urea, glucose and any unreacted acid were removed. After about 6 hours at 100°C, the hydrolytic acid was used alone, the yield is stated to be about 37.5%, when the hydrolytic acid was added with additional DMSO, the yield is stated to be about 98%. U.S. Patent No. 4,707,471 describes a water- soluble aminated /S-l-3 bound D-glucan composition and a method for preparing such composition. According to one embodiment of the method, β 1,3-D-glucan, preferably curdlan or laminarian was hydrolyzed in 90% formic acid. The acid was removed by evaporation, water was added and the mixture was refluxed for an hour. The mixture was then separated on a Sephadex G- 50 column and the highest molecular weight fraction recovered and further reacted as follows. ^,he hydrolyzed glucan was dissolved in water containing bromine at pH 7 and allowed to stand until all the bromine was consumed (24-48 hours) . The pH was adjusted to 5.0 and the mixture dialyzed against water and freeze dried. The oxidized glucan was added to a solution of ammonium acetate or 1,6-diaminohexane in -1-

water adjusted to pH 7.0 with acetic acid together with sodium cyanoborohydride and allowed to stand for 7 days with stirring. The aminated glucan was obtained after dialysis and freeze drying. In another embodiment of the method, the hydrolyzed laminarian was dissolved in DMSO prior to acetylation and then aminated as described above.

WO91/03495 by Jamas describes soluble preparations of neutral glucan polymers by a method involving treatment of glucan particles with a "unique sequence of acid and alkaline treatments." Whole glucan particles were suspended in acid solution, generally about pH 1-5 at 20-100°C, preferably using an organic acid such as acetic or formic acid. The acid insoluble glucan was removed and the pH adjusted to pH 7-14. The slurry was resuspended in hot alkali, such as NaOH or KOH at 0.1-10 N at 4-120°C. The soluble glucan was recovered and further purified.

U.S. Patent No. 3,883,505 describes a method for improving solubility of poorly soluble or water- insoluble polysaccharides such as pachyman, explain, lentinan, etc. , using strong, hot aqueous solutions of urea, thiourea, guanidine and their N-lower alkyl derivatives. U.S. Patent Nos. 3,987,166 and 3,943,247 describe, respectively, treatment of animal tumors and prevention and treatment of bacterial infections. As indicated, completely unlike the soluble phosphorylated glucans prepared by the present method which are non-viscous, the glucans of these patents are highly viscous and difficult to prepare in aqueous solution of higher concentrations than 0.5% aqueous solution.

Notwithstanding the above methods, there still remains a need for high efficiency, faster ethods for obtaining bioactive soluble glucans which can be used as biological response modifiers. The present method meets this long felt need.

3. SUMMARY OF THE PRESENT INVENTION

The present invention provides an improved, highly efficient method for preparing aqueous soluble glucans. The method avoids the use of DMSO.

The method of the invention for preparing a soluble glucan, comprises the steps of:

(a) mixing a neutral polyglucose or a polyglucose protein complex with a strong chaotropic reagent and grinding the mixture to form a fine powder; (b) reacting the fine powder mixture with a strong solution of concentrated phosphoric acid to form a soluble glucan and recovering the resultant soluble glucan from the mixture.

4. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more fully understood by reference to the following detailed description of the invention, examples of specific embodiments of the invention and the appended figures in which:

FIG. 1 (A-B) illustrates helical coil transition analyses. Dextran (70kD) (Δ-Δ) served as the linear control. Congo Red in sodium hydroxide (o- o) served as the negative control. FIG. 1A is the helical coil transition analysis for soluble phosphorylated glucan prepared according to the method of the present invention. FIG. IB is the helical coil transition analysis for soluble, phosphorylated ylucan prepared according to the prior art method. FIG. 2 (A-B) illustrates ¹³C-NMR spectra of soluble phosphorylated glucan. FIG. 2A shows the NMR spectrum of soluble phosphorylated glucan prepared according to the prior art method. FIG. 2B shows^" the NMR spectrum of soluble phosphorylated glucan prepared according to the method of the present invention.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 PROCESS FOR PREPARING SOLUBLE GLUCANS According to the method of the present invention, aqueous soluble glucan is prepared from a neutral polyglucose or polyglucose-protein complex obtained from a variety of microbial sources as follows: a neutral polyglucose or a polyglucose- protein complex is mixed with a strong chaotropic agent, such as urea, and the dry mixture is thoroughly mixed and ground into a fine powder. Any means of grinding to form a fine powder is suitable. For preparation of small batches, a mortar and pestle is satisfactory. In practice, about 1-4 gm of neutral polyglucan or a polyglucose-protein-complex is mixed with about 10-20 gm of urea. Concentrated phosphoric acid, about 5-50 ml (concentrated, about 20-43%) is added to form a slurry and the reaction mixture is heated to about 60-80°C with constant stirring. The reaction mixture is maintained at about 60-80°C for 1- 6 hours until a precipitate comprising the soluble phosphorylated glucan forms. About 10 ml of distilled water is added to reform a slurry and the reaction mixture maintained at about 60-80°C with stirring. The addition of water is repeated several times, preferably about three times, to maintain a slurry and heating is continued. It is preferable to maintain the reaction mixture at about 60-80°C for about 1-2 hours. In practice, after reaction for about two hours, the yield is about 97%. During the reaction, ammonia is released from the urea and the smell of . ammonia is most noticeable between about 1-2 hours after heating is begun.

In one embodiment, about 1-4 gm of particulate glucan is mixed with about 10-20 gm of urea and about 20-25 ml of concentrated phosphoric acid is used to form the slurry which is treated as above.

The soluble phosphorylated glucan is isolated from the reaction mixture as follows: the mixture is removed from the heat and dissolved in a large volume of distilled water so that the precipitate is resuspended. The resulting solution is filtered through coarse, medium and fine sintered filters to remove any remaining precipitate. The solution is then molecularly sieved to remove all components of less than 10,000 MW. Accordingly, any urea, glucose and unreacted phosphoric acid are removed from the solution. Any suitable method known in the art for molecular sieving can be used. For example, the solution can be sieved using Spectrapor membrane dialysis tubing dialyzed against running distilled water. In another example, the solution can be sieved using a Millipore dialyzer/concentrator with a 10,000 dalton MW membrane filter and a larger volume of dialyzing solution.

The method of the present invention is more rapid and more efficient than the prior art method for preparing soluble glucan phosphate. The time required for solubilization is reduced from 6 hours to less than two hours. The new method does not require as intense heating as the prior art method. The neutral polyglucose used in the present method for preparing the soluble phosphorylated glucan can be particulate glucan isolated from the cell wall of ∑ . cerevisiae by known methods (see e.g., DiLuzio et al., 1979, Int'l J. Cancer 224:773-779: Hassid et al., 1941, J. Amer. Chem. Soc, 6J3:295-298) . Additionally, soluble phosphorylated glucan can be prepared from neutral polyglucose or polyglucose- protein products derived from a variety of microbial sources. A non-exhaustive list of such sources is presented in Table 1 of U.S. Patent No. 4,739,046 incorporated herein by reference.

5.2 SOLUBLE GLUCAN PRODUCTS AND THEIR USES The aqueous soluble glucan products prepared according to the method of the present invention are non-toxic, non-pyrogenic and non-immunogenic when evaluated using the interfacial ring test. As demonstrated in Example 7 infra, the soluble phosphorylated glucan prepared using the improved method of the present invention is substantially the same in composition as that formed by the prior art method.

The soluble glucans prepared using the present method exert profound immunobiological responses when administered in vivo. More particularly, because they are active as biological response modifiers, the products obtained using the present method are useful for prophylaxis and therapy of infectious diseases induced by a variety of microorganisms including, but not limited to, bacteria, virus, fungi and protozoal parasites. Additionally, the soluble glucans may be used for the prevention and/or treatment of opportunistic infections in animals and man which are immunosuppressed as a result of congenital or acquired immunodeficiency.

Due to the inability to stimulate macrophage activity and proliferation, the soluble glucans can be used, alone or in combination for therapy of neoplasms.

Routes of administration include, but are not limited to: oral, injection, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, and topical routes. The soluble glucans can be administered in combination with water, an aqueous solution or any physiologically acceptable carrier.

The following series of examples are presented for purposes of illustration and not by way of limitation on the scope of the invention.

6. PREPARATION OF SOLUBLE PHOSPHORYLATED GLUCAN Particulate glucan was prepared from Saccharomyces cerevisiae according to the method of DiLuzio et al., 1979, Int'l J. Cancer 24:773-779. Briefly, using a 6 1 flask, 540 gm of dry yeast (Universal Foods Corp., Milwaukee, WI) was suspended in 3% aqueous sodium hydroxide solution to a total volume of 5 1. The suspension was placed in boiling water bath for 4 hours, cooled overnight and the supernatant decanted. This procedure was repeated three times. The residue was then acidified with 5.75% hydrochloric acid to a total volume of 5 1 and placed in a boiling water bath for 4 hours. The suspension was allowed to stand overnight and the supernatant decanted. The residue was further digested twice with of 3% hydrochloric acid to a total volume of 5 1 at 100°C. The residue was washed with boiling water and decanted numerous times until the residue became floceulent. One 1 of ethyl alcohol was added to the residue, mixed thoroughly and allowed to stand a minimum of 24 hours for maximum extraction. The dark reddish-brown alcohol supernatant was aspirated from the residue and discarded. The alcohol extraction procedure was repeated until the alcohol supernatant was essentially colorless. The alcohol was removed by washing the residue four times with hot water; the particulate glucan preparation was then collected by centrifugation, frozen and lyophilized. Soluble phosphorylated glucan was prepared according to the present invention by solubilization and phosphorylation of the particulate glucan as follows: 18 gm of urea was mixed with 1 gm of particulate glucan and ground with a pestle in a mortar to form a finely ground powder mixture. Twenty-five ml of phosphoric acid (43%) was added slowly to the powder mixture to form a slurry. The mixture was heated to about 60-80°C and maintained at that temperature for about 1-2 hours with stirring. A precipitate was formed which became visible after about 1-1.5 hours and increased in amounts thereafter. About 10 ml of distilled water (Milli-Q water) was added to the mixture and heating continued with stirring. The addition of about 10 ml of distilled water was repeated three times and heating continued for 1-2 hours. The mixture was then removed from the heat, cooled and diluted with about 1 1 of distilled water to resuspend the precipitate. The mixture was filtered using a series of filters to remove any remaining precipitate.

The resulting solution containing the soluble phosphorylated glucan was then molecularly sieved to remove low molecular weight fractions, including glucose and urea. In one series of experiments, the mixture was filtered through coarse (1-3 μ) , medium (0.8 μ, 0.65 μ) and fine (0.45 μ) sintered Millipore filters to remove the precipitate. The solution was then molecularly sieved using a Millipore dialyzer/concentrator (Millipore Corp. , Bedford, MA) with a 10,000 MW membrane filter. Dialysis against about 24-100 L of distilled water (Milli-Q grade water) was used to remove low MW compounds.

Following molecular sieving, the solution containing the soluble phosphorylate glucan was concentrated and lyophilized. This yield was about 97%.

7. CHARACTERIZATION OF SOLUBLE PHOSPHORYLATED GLUCAN PREPARED BY THE PRESENT METHOD

A series of experiments were conducted to compare the soluble phosphorylated glucan (designated

Glucan Phosphate-no DMSO) prepared using the method of the invention as described in Section 5 above with the soluble phosphorylated glucan (designated Glucan

Phosphate) prepared using the prior art method, i.e., the method of U.S. Patent No. 4,739,046.

7.1 ELEMENTAL COMPOSITION The elemental composition of the Glucan Phosphate-no DMSO was determined by Galbraith Laboratories, Knoxville, TN. A comparison of the elemental composition with that of Glucan Phosphate is shown in Table 1. Table 1

Chemical Composition of Soluble Glucans

The elemental composition of Glucan Phosphate prepared according to the method of the present invention indicates that it is essentially identical to the art Glucan Phosphate. As shown in Table 1, the only difference seen between the product of the present method i.e. Glucan Phosphate-no DMSO and the art Glucan Phosphate is the degree of phosphorylation. Glucan Phosphate-no DMSO has approximately one phosphate substitution for every 3 glucose units, whereas Glucan Phosphate has approximately one phosphate substitution for every 7 glucose units.

7.2 MOLECULAR WEIGHT DISTRIBUTION The molecular weight (polymer) distribution of the two glucans was determined by aqueous gel permeation chromatography (GPC) . The basic GPC system consisted of a Waters 600E solvent delivery system, a U6K manual injector and a column heating chamber (Waters Chromatography Division, Millipore Corp. , Milford, MA). The mobile phase, 0.05 M sodium nitrite, was stored in a sterile reservoir (Kontes, Vineland NJ) , and was thoroughly degassed by sparging and blanketing with helium prior to use. Mobile phase was delivered at a flow rate of 0.5 ml/min. Three Ultrahydrogel (Waters Chromatography Division, Milford, MA) aqueous GPC columns having exclusion limits of 2 x 10⁶ D, 5 x 10^s D and 1.2 x 10⁵ D were connected in series along with an Ultrahydrogel guard column. The columns were maintained at 30°C. Flow rate, column temperature, and pump operating conditions were controlled by Maxima 820 GPC software (Dynamic Solutions, Ventura CA) .

The system was calibrated using narrow-band pullulan standards and dextran standards. For analysis, the glucans were dissolved in mobile phase at a concentration of 2-3 mg/ml by gentle rocking until completely hydrated (about 2-3 hrs) . A 200 μl injection volume was used for all analyses.

Absolute molecular weights of the glucan were determined by on-line multi-angle laser light scattering (MALLS) photometry employing a Dawn-F MALLS photometer fitted with a K5 flow cell (Wyatt Technology Corp., Santa Barbara, CA) . Absolute MW distribution, molecular weight moments (number-average MW, Z-average MW, weight-average MW) , peak MW, polydispersity and root mean square (rms) radius moments were established with ASTRA software (v. 2.0). A differential index of refraction (dn/dc) of 0.146 cm³/g was assumed. Reported MWs of pullulan and dextran standards used to check column calibration showed good agreement with MALLS data.

Intrinsic viscosity ([η]) of the polymeric glucans were determined by on-line differential viscometry (d.v.). For determination of [ η ] the column eluent was passed through a Viscotek Model 200 differential refractometer/viscometer and data were analyzed with Unical software (Viscotek, Porter, TX) . Molecular weight determinations of standards using . this technique showed good agreement with MALLS data. Intrinsic viscosity of pullulan standards was determined to be in close agreement with previous data. The molecular weight averages, polydispersity, and intrinsic viscosity of glucan phosphate without DMSO are shown in Table 2. For comparison, analogous data for glucan phosphate prepared according to the method of Patent No. 4,739,046 are also presented.

Table2

Molecular Weight Characteristics

a M_n represents: number-average MW

M_w represents: weight-average MW

M_z represents: z-average MW

M_w RMS Radius: weight-average root-mean square radius (nm)

All MW are expressed as g/mol. η represents Intrinsic Viscosity b The RMS Radius could not be determined for this portion of the sample, c Due to the low concentration of polymers in peak 1 , it was not possible to determine intrinsic viscosity in this portion of the sample. As demonstrated in Table 2, the molecular weight characteristics of glucan phosphate-no DMSO and the prior art glucan phosphate are strikingly similar, in fact, substantially identical. Using the presently described techniques, two peaks were noted in each of the glucan phosphate preparations. The greater majority of the polymers of both preparations, however were found in peak #2; in both preparations, peak #1 comprises < 2% of the total polymers. As clearly shown in Table 2, M„, indicative of the proportion of low MW polymers, K^, indicative of the average molecular weight, and M_z, indicative of the proportion of high MW polymers were substantially identical for peak #2 in the two glucan phosphate preparations. Additionally, the index of polydispersity was substantially identical showing that compositions had identical polymer homogeneity. Finally, as shown in Table 2, the intrinsic viscosity of the two compositions was virtually identical.

7.3 CONFORMATIONAL STRUCTURE ANALYSIS

Conformational structure was assessed using the technique of Ogawa and co-workers (Ogawa and Hatana, 1978, Carbohyd. Res. 62:527-535; Ogawa and Tsurugi, 1973, Carbohyd. Res. .29:397-403). This technique determines the absorption maxima of polymer solutions complexed with Congo Red in the presence of various concentrations of hydroxide ion. The presence of a triple-helical compound, for example, would be indicated by a shift in the absorption maxima of the solution as sodium hydroxide concentration increases. Disruption of hydrogen bonds occurs with the relaxation of the polymer helix and subsequently, the Congo Red complexes with the carbohydrate. It has been previously shown that an ordered (e.g. helical) conformation is essential for carbohydrates such as glucan to form complexes with the dye Congo Red. Aqueous solutions of Congo Red (44 μM) were prepared at various concentrations of NaOH (1 mM to 1000 mM) . Results are presented in FIG. 1 (A and B) for Glucan Phosphate-no-DMSO and Glucan Phosphate, respectively.

As shown in FIG. 1 (A and B) , analysis of the conformational structure of both the Glucan

Phosphate and Glucan Phosphate-no DMSO indicates an ordered or triple-helical conformation. In both cases, a shift in the absorption maxima at sodium hydroxide concentration of 0.1 to about 0.4 M were observed.

7.4 NUCLEAR RESONANCE SPECTROSCOPY Carbon-13 nuclear magnetic resonance spectroscopy (¹³C-NMR) using a Bruker 260 MHz NMR spectrometer (Bruker Instruments, Inc., Billerica, MA) was performed to determine the nature of interchain linkages in Glucan Phosphate and Glucan Phosphate-no- DMSO. Soluble Glucan Phosphate or Glucan Phosphate-no DMSO was dissolved in D₂0 at 50 mg/ml. Conditions were as follows:

FIELD STRENGTH: 50 MH; RELAXATION DELAY: 1 second, PULSE WINDOW: 15°-20°, NUMBER OF SCANS: Glucan phosphate 694 scans

Glucan phosphate-no DMSO, 14,900 scans.

Results are shown in FIG. 2 (A-B) and Table

3. Table 3

"C-NMR Chemical Shifts of Glucans*

Chemical Shifts in ppm

The only difference between Glucan Phosphate and Glucan Phosphate-no DMSO is that Glucan Phosphate- no DMSO shows peaks C4 and C5^b, which do not appear in Glucan Phosphate. It is apparent, however, that the compounds show ^I3C-NMR spectra which agree well with a jβ-1,3-linkage (Colson, Carbohydrate Research 71: 265 , 1979) .

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. A number of references are cited, the disclosure of each of which is incorporated herein in its entirety by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a soluble phosphorylated glucan, comprising:

(a) mixing a neutral polyglucose or a polyglucose-protein complex with a strong chaotropic reagent and grinding the mixture to form a fine powder; (b) reacting the fine powder with a strong solution of concentrated phosphoric acid to form a reaction mixture containing soluble phosphorylated glucan; and

(c) recovering the resultant soluble phosphorylated glucan from the reaction mixture.

2. The method according to claim 1 in which the chaotropic reagent comprises urea.

3. The method according to claim 1, which further comprises heating the reaction mixture of step (b) to about 60-80°C for about 1-6 hours.

4. The method according to claim 3, in which the reaction mixture is heated to about 60-80°C for about 1-2 hours.

5. The method according to claim 1, in which the soluble phosphorylated glucan is recovered by:

(a) allowing the soluble phosphorylated glucan to precipitate;

(L>) adding a sufficient amount of water to resuspend the precipitated soluble phosphorylated glucan; and (c) removing all components of less than about 10,000 daltons molecular weight.

6. The method according to claim 1, in which the neutral polyglucose comprises particulate glucan obtained from a microbial organism.

7. The method according to claim 6, in which the particulate glucan was obtained from Saccharomyces cerevisiae.