SI9300415A - Improved method for preparing soluble glucans - Google Patents

Abstract

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

Description

1. FIELD OF THE INVENTION

The present invention relates to a sophisticated, highly efficient method for the preparation of soluble glucans. The method preferably avoids the use of highly polar solvents such as dimethylsulfoxide (DMSO). The soluble glucans prepared by the method of the present invention are non-toxic and exhibit a pronounced immune response when administered in vivo, most notably immunostimulating macrophage activity and stimulating bone marrow hamatopoietic activity. Soluble glucans also show significant effects against malignant neoplasms including melanoma and sarcoma.

2. BACKGROUND OF THE INVENTION

The term glucan generally refers to a variety of natural homopolysaccharides or polyglucose, including polymers such as: cellulose, amylose, glycogen, laminariates (a type of polysaccharide), starch, etc. Glucan comprises branched and unbranched chains of glucose units linked to 1-3, 14, and 1-6 glucoside bonds, which may be of type a or β.

Particular glucan (an aggregate of water-insoluble glucan particles) as named in the present invention is referred to as a water-insoluble particle (aggregate of glucan particles) (size 1-3 pm) of polyglucose obtained from the cell envelope of the yeast Saccharomyces cerevisiae. Particulate glucan is a macromolecule and comprises a closed chain of glucopyranose units linked to a series of β-1-3 glucoside bonds (Hassid et.al., 1941, J.Amer. Chem. Soc. 63: 295-298; DiLuzio et al., 1979 , Infl. J. Cancer 24: 773-779). X-ray diffraction studies have confirmed that particulate glucans form a three-fold helix. (Sarko et al., 1983, Biochem. Soc. Trans. 11: 139142).

Particulate glucan is a potent activator of macrophage / monocyte cell type, complement as well as T and B cell lymphocytes. Thus, particulate glucan has a pronounced effect on the reticuloendothelial and immune systems. Particulate glucan demonstrated the ability to modify host resistance to a wide range of infectious diseases (see review article DiLuzia, 1983, Trends and Pharmacol. Sci. 4: 344-347 and cited references). Additionally, particulate glucan also inhibits tumor growth and prolongs survival in congenital mouse tumor models (DiLuzio et.al., 1979; Advances in Exp. Med. Biol. 121A.-269-290). An in vitro study of normal and tumor cells incubated with particulate glucan showed that glucan exhibits a cytostatic effect on sarcoma and melanoma cells and a proliferative effect on normal liver and bone marrow cells (VVilliams et al. 1985, Hepatology 5: 198-206).

Despite the beneficial biological effects of particulate glucans, these various side effects have prevented their usefulness in clinical medicine. Addition of particulate glucan to animals in vivo has revealed serious side effects, the most notable of which are: (1) granuloma formation (sarcoidosis); (2) development of heptatosplenomegaly; (3) increasing susceptibility to gram-negative infections and endotoxins; (4) complement activation (anaphylatoxin); development of pulmonary granulomatous vasculitis; developing hypotension following intravenous administration; and (7) the growth of microembolism upon the addition of a highly concentrated reagent.

In addition, a relatively high level of acute toxicity was observed with in vivo addition of particulate glucan. In the case of a single intravenous injection of particulate glucan aqueous suspension, 20% and 100% mortality was observed in mice receiving 250 and 500 mg / kg glucan per body weight, respectively.

Furthermore, due to the particular nature of glucanan preparation (1-3 pm), glucan is very difficult to administer via the intravenous route. To illustrate, the patient needs constant monitoring of intravenous (IV) administration of particulate glucan, since continuous shaking of the IV droplet bottle is essential to maintain suspension and thus prevent embolism in the patient.

Even though weakly soluble neutral glucans are commercially available, these preparations are not suitable for intravenous administration because aqueous solutions have a very high viscosity and, more importantly, their use when administered to experimental animals is inevitably accompanied by considerable toxicity.

Due to all the disadvantages of β-1,3 glucan in in vivo administration, new pathways for the development of β-1,3 polyglucose, which is not toxic, would not induce characteristic pathology and still retain significant immunological activity, have been extensively investigated previously.

Non-phosphorylated glucan low molecular weight preparations prepared by hydrolysis of particulate glucan with formic acid have shown antitumor activity and efficacy against staphylococci (DiLuzio et al., Intemat'l J.Cancer 24: 773-779). Unfortunately, the low yields and the diversity of fractions obtained by this method prevented the use of these preparations for prophylactic and therapeutic purposes. (See DiLuzio, 1983, Trends in Pharmacological Sciences 4: 344-347).

Attempts to solubilize (solubility) the particulate gucana with the addition of dimethylsulfoxide (DMSO), a molecular relaxant, have also been unsuccessful. DMSO was assumed to loosen the configuration of the triple helix of the glucan molecule. Particulate glucan is indeed dissolved in the presence of DMSO. All attempts to isolate the particulate glucan from the DMSO solution, however, have proved unsuccessful. After diluting the DMSOglucan solution with various aqueous media, such as glucose solutions or salt solutions, the particulate glucan was transformed. After dilution of DMSO-soluble glucan solutions with brine, all animals that received injections of these solutions immediately died due to high concentrations of DMSO or rearrangement of particulate glucan. After the formation of particulate glucan with ethanol (100%), the precipitate was collected and lyophilized. The lyophilized glucan was transformed by the addition of water.

Early attempts to convert a neutral glucan preparation of particulate glucan into a polar-charged preparation with the addition of phosphate or sulfate groups, as well as acetylation, were also unsuccessful. All of these procedures were performed after solubilization of the particulate glucan with DMSO, and in all cases the particular glucan was transformed.

A neutral particulate glucan preparation was successfully converted to a stable form called soluble phosphorylated glucan (hereinafter referred to as glucan phosphate) by hydrolysis with phosphoric acid using the method briefly described below. As indicated herein, the terms glucan phosphate and soluble phosphorylated glucan refer to a series of giucans solubilized by the addition of phosphate groups via reaction with phosphoric acid. These are identical or substantially similar to the substances described in U.S. Pat. No. 4,739,064; 4,761,402; No. 4,818,752 and 4,833,131. This soluble phosphorylated glucan is non-toxic, non-immunogenic and substantially non-pyrogenic (see U.S. Patent Nos. 4,739,064; 4,761,402; 4,818,752 and 4,833,131).

According to the method described in U.S. Pat. patent no. No. 4,739,046, glucan phosphate was prepared as follows: Particulate glucan or a polyglucose protein complex were suspended in a strongly polar aprotic solvent: DMSO. A strong chaotropic reagent (a substance that causes protein denaturation) was added to the urea and the mixture was heated to 50-150 ° C under constant stirring, to which phosphoric acid was slowly added. The temperature is preferably kept at 100 ° C for 3-12 hours. This increases the yield of the bioactive product. The product is isolated and DMSO, urea, glucose and all unreacted phosphoric acid removed. The yield after reaction (6 hours, 100 ° C) is about 70-90%.

Another new type of soluble gyucan, where the polyglucopyranose chains have obtained a phosphor-free acid group, is described in patent application Serial Number 07 / 649,572. Soluble glucans with a charge-containing group, including such as sulfates and nitrates, are also able to show a pronounced immunobiological effect when administered in vivo. These soluble glucans immunostimulate the activity of macrophages through the corresponding activation of immunoactive cells in the reticuloendothelial and immune systems. These soluble glucans also increase the haematopoietic (hematopoietic) activity of the bone marrow.

In accordance with the methods of patent application serial number 07 / 649,527, soluble glucan was prepared as follows: particulate glucan was suspended in DMSO solution and urea; concentrated mixture heated to 50-150 ° C, concentrated acid added (sulfuric or nitric) alone or in DMSO and the reaction mixture heated while stirring at 50-150 ° C. The bioactive product was isolated and the DMSO, urea, glucose and all unreacted acid were removed. After about 6 hours at 100 ° C, the acid itself was used, the yield being given as 37.5%, but when the acid with DMSO was added, the yield was about 98%.

U.S. patent no. No. 4,707,471 describes a water-soluble aminated β-1-3 bound D-glucan mixture and a process for preparing such a mixture. According to the inclusion of one of the methods, β 1,3-D-glucan, preferably cheesy or flaky, was hydrolyzed in 90% formic acid. The acid was removed by evaporation, water was added and the mixture heated to reflux for one hour. The mixture was then separated on a Sephadex G-50 column and the fraction with the highest molar mass separated, which was further processed as follows. The hydrolyzed glucan was dissolved in water containing bromine at pH 7 where it stood in this solution until complete consumption of bromine (24-48 hours). The pH was then adjusted to 5.0 and the mixture dialyzed into water and lyophilized. Oxidized glucan was added to a solution of ammonium, acetate or 1,6-diaminohexane in water at pH adjusted to 7.0 using acetic acid together with sodium cyanoborohydride and the solution allowed to stand with stirring for 7 days. Aminated glucan was obtained after dialysis and lyophilized. In another embodiment, the hydrolyzed scales were dissolved in DMSO prior to acetylation and then aminated as described above.

VVO91 / 03495 Jamasa describes soluble preparations of neutral glucan polymers by a process comprising treating glucan particles with a unique sequence of acid and base action. The whole glucan particles were suspended in an acidic solution, usually around pH = 1-5 at 20-100 ° C, preferably using organic acids such as acetic and formic acid; the acid-insoluble glucan was removed and the pH was adjusted to 7-14. The whey was resuspended in hot base (NaOH or KOH) (0.1-1 ON) at 4-120 ° C. The soluble glucan was then recovered and further purified.

U.S. patent no. No. 3,883,505 describes a process for improving the 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 lower N-alkyl derivatives.

U.S. patent no. 3,987,166; and 3,943,247 describe the treatment of animal tumors and the prevention and treatment of bacterial infections. Completely unlike phosphorylated glucan prepared by this process which is non-viscous, glucan prepared according to these patents are highly viscous and difficult to prepare in aqueous solutions of higher concentrations than 0.5%.

Despite the above procedures, highly effective, faster processes are still required to obtain bioactive soluble glucans that can be used as biological response modifiers. The process presented meets this long-imagined need.

3. SUMMARY OF THE INVENTION

The present invention provides an improved highly efficient method for the preparation of water-soluble glucans. The process avoids the use of DMSO.

The process of the present invention for the preparation of soluble glucans comprises the following steps:

(a) mixing neutral polyglucose or glycoprotein polyglucose with a strong chaotropic reagent and grinding the mixture to a fine powder;

* there is no relevant Slovenian expression (b) react the fine powder mixture with a strong solution of concentrated phosphoric acid to form soluble glucan and to obtain soluble glucan from the mixture.

4. BRIEF DESCRIPTION OF THE DIAGRAMS AND FIGURES

The present invention can be better understood by the following detailed description, examples of specific embodiments of the invention and additional drawings in which:

Figure 1. (AB) presents an analysis of the spiral roll transition. Dextran (70kD) (Δ-Δ) serves as a linear control. Congo red in sodium hydroxide (oo) serves as a negative control. Figure 1A is an analysis of the helix transition for soluble phosphorylated glucan prepared according to the process of the present invention. FIG. 1B is an analysis of the transition state of a helix for soluble, phosphorylated glucan prepared by known methods. Figure 2. (AB) shows the ¹³ C-NMR spectra of soluble phosphorylated glucan. Figure 2A shows the NMR spectrum of soluble phosphorylated glucan prepared by a method known in the art. Figure 2B shows the NMR spectrum of soluble phosphorylated glucan prepared according to the method described in the present invention.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. PROCEDURE FOR PREPARATION OF SOLUBLE GLUCANES

In accordance with the process of the present invention, an aqueous soluble glucan is prepared from neutral polyglucose or glycoprotein, which is obtained from several microbial sources as follows: the neutral polyglucose or glycoprotein is mixed with a strong chaotropic reagent such as urea, and the dry mixture is stirred vigorously and finely powdered . Any grinding method is suitable. To prepare small quantities, we can use a mortar and pestle. In practice, about 1-4 g of neutral polyglucan or glycoprotein is mixed with about 10-20 g of urea. Concentrated phosphoric acid (5-50 ml, conc. 20-43%) was added to form a mud mixture and the reaction mixture was heated to 60-80 ° C with constant stirring. The reaction mixture was kept at 60-80 ° C for 1-6 hours until a precipitate of soluble phosphorylated glucan formed. Add 10 ml of distilled water to transform the sludge, keeping the reaction mixture at 60-80 ° C with constant stirring; Repeat the addition of water (preferably three times) to keep the sludge and continue heating. It is best to keep the temperature at 60-80 ° C for 1-2 hours. In practice, after a two-hour reaction, 97% yield is obtained. During the reaction, ammonia is released from urea, which is most noticeable 1-2 hours after the start of heating.

In one case, mix 1-4 g of parti-culate glucan with 10-20 g of urea and 20-25 ml of concentrated phosphoric acid to prepare the sludge treated as above.

Soluble phosphorylated glucan is isolated from the reaction mixture as follows: The reaction mixture is removed from the heat bath and dissolved in a large volume of distilled water by resuspending the precipitate. The appropriate solution is filtered through a coarse, medium and fine sinter filter to remove any residue of the precipitate. Molecular sieves are then added to the solution, removing all components smaller than 10,000 moles. Urea, glucose and unreacted phosphoric acid are appropriately removed from the solution. Any known method that uses molecular sieves can be depicted. As an example, the use of membrane dialysis sieves via Spectrapor membrane tubes can be demonstrated and the mixture dialyzed against liquid distilled water. In the second case, we use a Millipor dialyzer / concentrator with a membrane filter of 10,000 daltons of molar mass and a large volume of dialysis fluid.

The process described in the present invention is faster and more efficient than previously known processes for the preparation of soluble phosphate glucan. We reduced the required time to less than 2 hours compared to 6 hours. The new process does not require as intense heating as the previously known method.

The neutral polyglucose used in the present process for the preparation of soluble phosphorylated glucan may be a particulate glucan isolated from S. cerevisiae cell envelopes by known methods (see DiLuzio et al. 1979, lnt'l J. Cancer 224: 773-779: Hassid et al., 1941, J.Amer. Chem. Soc., 63: 295-298.

Additionally, soluble phosphorylated glucan from neutral polyglucose or glycoproteins obtained from various microbial sources can be prepared. In Table 1, we present a non-exhaustive list of these U.S. sources. patent no. No. 4,739,046, which is incorporated herein by reference.

5.2 SOLUBLE GLUCAN PRODUCTS AND THEIR

APPLICATION

The soluble glucan products prepared by the corresponding process of the present invention are non-toxic, pyrogenic and non-immunogenic when tested by interfacial ring test. As shown in Example 7, soluble phosphorylated glucan prepared by these improved processes are substantially identical in composition to those prepared by previously known methods.

The soluble glucans prepared by the methods presented show a thoroughly expressed biological response when administered in vivo. More specifically, because they are effective as biological modifiers of biological response, the products obtained by the following methods are useful for the prophylaxis and therapy of infectious diseases caused by a variety of microorganisms, including but not limited to bacteria, viruses, fungi and protozoan parasites. Additionally, soluble glucans can be used to prevent and / or treat opportunistic infections in animals and humans that are immunosuppressive as a result of prenatal or acquired immunodeficiency.

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

Administration is, but is not limited to, the following: oral, injectable, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular and topical application. Soluble glucans can be added in combination with water, with an aqueous solution or with any biologically acceptable carrier.

As an illustration, we will present the following series of examples, but not as limiting to the scope of the present invention.

6. PREPARATION OF SOLUBLE Phosphorylated

GLUKANA

Particulate glucan was prepared from Saccharomyces cerevisiae according to the method of DiLuzia et al., 1979, Infl J. Cancer 24: 773-779. 540 g of dry yeast (Universal Foods Corp., Milvvaukee, Wl) was suspended in 3% aqueous sodium hydroxide solution in a 6 I container until a final volume of 5 I. The suspension was placed in a boiling water bath for 4 hours, cooled overnight and decanted. fluid. We repeated this process three times. The residue was then acidified with 5.75% hydrochloric acid to a final volume of 5 I and placed in a boiling water bath for 4 hours. The suspension was then allowed to stand overnight and the liquid was decanted again. The residue was diluted twice with 3% hydrochloric acid to a final volume of 5 I at 100 ° C. The residue was washed with boiling water and decanted several times until the residue became colloidal. 1 I of ethanol was added to the residue, the mixture was carefully mixed and allowed to stand for a minimum of 24 hours to maximize extraction. The dark red liquor mother liquor was discarded from the rest and discarded. The alcohol extraction process was repeated until a colorless supernatant was obtained. The alcohol was removed by washing the residue four times with hot water; the particulate glucan preparation was then decided by centrifugation, frozen and lyophilized.

Soluble phosphorylated glucan was prepared according to the present invention by solubilizing and phosphorylating the particulate glucan as follows:

g of particulate glucan was mixed with 18 g of urea, the mixture was ground with a pestle in a mortar and thus a finely ground powder mixture was prepared. Slowly added 25 ml of phosphoric acid (43%) and converted the powder mixture to sludge. The mixture was then heated to 60-80 ° C and held at this temperature for a further 1-2 hours with constant stirring. A precipitate formed, which became visible within 1-1.5 hours and subsequently increased. About 10 ml of distilled water (Milli-Q- water) was added and heating and stirring was continued. The addition of distilled water (10 ml) was repeated three times and continued to warm (1-2 hours). The mixture was then stopped warming, cooled and diluted with about 1 I of distilled water to resuspend the precipitate. A series of filters filtered the mixture to remove residual precipitate.

The corresponding solution containing phosphorylated glucan was then treated with molecular sieves to remove low molecular weight fractions, including urea and glucose. In this type of experiment, the mixture was filtered through a coarse (1-3 pm), a mean 0.8 pm, 0.65 pm and a fine (0.45 pm) sinter Millipore filter to remove the precipitate. The solution was then - re-treated with molecular sieves via a dialyser / concentrator (Millipore Corp. Bedford, MA) with a membrane filter (10,000 MW). Then, dialysis against 24-100 I distilled water (Milli-Q water) eliminated niche molecular weight compounds.

After treatment of the solution containing soluble phosphorylated glucan with molecular sieves, the solution was concentrated and lyophilized. The yield was 97%.

7. CHARACTERIZATION OF SOLUBLE PHOSPHORILATED

GLUCANS PREPARED AFTER PROPOSED

IZUMU.

A series of experiments were performed to compare soluble phosphorylated glucan (labeled glucan phosphate DMSO) prepared by the process of the present invention described in section 5 above with soluble phosphorylated glucan (labeled glucan phosphate) prepared by a prior known method, by the method of US patent no. 4.739.046.

7.1 ELEMENTAL COMPOSITION

The elemental composition of glucan phosphate-no DMSO was determined at Galbright Laboratoires in Knoxville, TN. Comparison of elemental composition with glucan phosphate composition is given in Table 1.

Table 1

Chemical composition of soluble glucans

element glucan phosphate Mol% glucan phosphate (No DMSO) Mol% Carbon 34.66 32.72 Hydrogen 6.29 6.32 Oxygen 42.83 48.67 Phosphorus 2.23 4.37

The elemental composition of glucan phosphate prepared according to the process of the present invention characterizes it as substantially identical to that of known gucan phosphate. Table 1 shows that the only visible difference between the products is the degree of phosphorylation. Glucan phosphate DMSO has one phosphate substitution for every 3 glucose units, whereas for glucan phosphate one phosphate substitution per 7 glucose units is observed.

7. 2 MOLECULAR WEIGHT DISTRIBUTION

The molecular weight (polymers) distribution of both glucan was determined by aqueous gel permeable chromatography (GPC). The basic GPC system consists of a Waters 600E solvent drain system, a U6K manual injector and a column heating chamber (Waters Chromatography Division, Millipore Corp., Milford, MA). The mobile phase is 0.05 M sodium nitrite stored in a sterile tank (Kontess, Vineland, NJ). Carefully remove the helium blowing air before use. The mobile phase flow was 0.5 ml / min. Three Ultrahydrogel aqueous GPC columns (Waters Chromatography Division, Milford, Ma) with exclusion limits of 2 χ 10 ⁶ D, 5 χ 10 ⁵ D and 1.2 x 10 ⁵ D were connected in series with an additional Ultrahydrogel protection column. The columns were kept at 30 ° C. Flow rate, column temperature and pump performance were controlled with Maxima 820 GPC software (Dynamic Solutions, Ventura CA).

The system was calibrated using narrow-band half-wave and dextran standards. For analysis, glucans were dissolved in the mobile phase at a concentration of 2-3 mg / ml with gentle shaking until complete hydration (2-3 hours) had elapsed. 200 ml injection volumes were used in all analyzes.

Absolute molecular weights of glucan were determined by on-line multilayer laser light scattering photometry (MALLS), using a Dawn-F MALLS photometer equipped with a K5 flow cell (Wyatt Technology Corp, Santa Barbara CA). The absolute distribution of molecular masses (MW), MW moments (numerical average MW, Z-average MW, weighted average MW), MW peak, polydispersity, and root mean square radius of the torque were determined by ASTRA software (v.2,0). A differential refractive index (dn / dc) of 0.146 cm3 / g was assumed. The published molecular weights of the pollulan and dextran standards used to verify column calibrations showed good agreement with the MALLS results.

The dynamic viscosity ([η]) of polymer glucan was determined by on-line differential viscometry (d.v.). For the determination of the column eluent, it was used to flow through the Viscotek Model 200, differential refractometer / viscometer, and analyze the data with Unical software (Viscotek, Porter, TX). Molecular weight determination of standards using the above techniques showed good agreement with MALLS results. The dynamic viscosity of the Polulan standards closely matched the previous data. The transverse molar masses, polydispersity and dynamic viscosity of glucan phosphate without DMSO are given in Table 2. For comparison, analogous data for glucan phosphate prepared according to the process of U.S. Pat. No. No. 4,739,046.

Table 2

Molecular mass characteristics

parameter ³ glucan phosphate glucan phosphate no DMSO top 1 top 2 top 1 top 2 M _a 1.28 χ 10 ⁶ 0.25x10 ⁵ 2.77x10 ⁶ 0.18 χ 10 ⁵ M _w 3.57 χ 10 ⁶ 1.10Χ10 ⁵ 5.45x10 ⁶ 1.10x 10 ⁵ M _z 1.22 χ 10 ⁷ 3.04x10 ⁵ 1.47 χ 10 ⁷ 2.75 x 10 ⁵ M _w 31.7 25.4 40.3 b RMSR (nm) Polydispersity (l) 3.2 6.2 1.97 6.1 [η] c 0.29 c 0.30

^a M _n represents: numerically transverse MW

M _w represents: mostly transverse MW M _z represents: z-transverse MW

M _w RMS radius: weight transverse square root of the radius (nm)

All MW are expressed in g / mol. η represents the dynamic viscosity ^{b The} RMS radius could not be determined for this part of the sample ^c Due to the low concentration of polymers at the top 1, it was not possible to determine the dynamic viscosity on this part of the sample.

Table 2 shows that the molecular weight characteristics of glucan phosphate DMSO and glucan phosphate prepared according to the previous methodology are extremely similar, in fact essentially identical. Using these techniques, two peaks were observed for both prepared phosphate glucan. Most of the polymers in both preparations are in the top 2; in both preparations the peak contains 1 <2% of all polymers. Table 2 clearly shows that M _n characteristic of the fraction of polymers with low MW and M _w characteristic of the fraction of transverse MW and Ml, the fraction characteristic of the proportion of polymers with high MW, are essentially identical at the top 2, in of both prepared glucan phosphates. The polydispersity index further confirms the identity, as it indicates the identical homogeneity of the polymers. Table 2 also finally shows that the dynamic viscosity of the two components is actually identical.

7.3 CONFORMITY STRUCTURE ANALYSIS

The conformational structure was evaluated using the technique of Ogawe et al. (Ogawa and Hatana, 1978; Carbohydrate Res. 6Z: 527535; Ogawa and Tsurugi, 1973; Carbohydrate Res. 29: 397-403). This technique determines the absorption maxima of polymer solutions complexed with Congo red in the presence of different concentrations of hydroxyl ion. For example, the presence of compounds with a triple helix would be indicated by a shift in the absorption maximum of the solution with increasing sodium hydroxide concentration. Hydrogen bonds are broken by polymer helix relaxation, resulting in the complexation of Congo red with carbohydrate.

Earlier, they showed that an orderly (helical) conformation is essential for carbohydrates such as glucan to form a complex with Congo dye red. Aqueous solutions of Congo red (44 pm) were prepared at various NaOH concentrations (1 mM to 1000 mM). The results are presented in Figure 1. (A and B) for glucan phosphate DMSO and glucan phosphate, respectively.

As can be seen from Figure 1. (A and B), the conformational structure analysis of the two giucans, glucan phosphate and glucan phosphate DMSO, show an ordered conformation of the triple helix. In both cases, a shift of the absorption maximum was observed when the sodium hydroxide concentration changed from 0.1 to about 0.4 M.

7.4 NUCLEAR MAGNETIC RESONANCE

SPECTROSCOPY

13c Nuclear Magnetic Resonance Spectroscopy ( ¹³ CNMR) on a Bruker 260 MHz NMR spectrometer (Bruker Instruments, INc., Billerica, MA) was used to determine the position of the interconnections in glucan phosphate and glucan phosphate DMSO. Soluble glucan phosphate or glucan phosphate DMSO was dissolved in D ₂ O (50 mg / ml) and measurements were made under the following conditions:

FIELD POWER: 50 MHz

RELAXATION LOCK: 1 second,

PULSE SIZE: 15 ° - 20 °,

REPEAT NUMBER: Glucan Phosphate 694 Mon.

Glucan Phosphate DMSO, 14,900 Mon.

The results are shown in Figure 2. (A-B) and Table 3.

Table 3

13C-NMR chemical shifts of glucans *

carbon atom glucan phosphate glucan phosphate (No DMSO) C-1 102.58 105.02 C-2 73.23 75.88 C-3 84.45 86.76 C-4a 68.21 70.79 C-4b 70.37 C-5a 75.66 78.26 C-5b 77.15 C-6 60.81 63.40

* Chemical shifts in ppm

Peaks C4 ^ and C5 ¹³ observed with glucan phosphate DMSO are not observed with glucan phosphate, which is the only difference between glucan phosphate and glucan phosphate DMSO. It is clear that the compounds exhibit ¹³ C-NMR spectra that are characteristic of the β-1,3-coupling (Colson, Carbohydrate Res., 71/265, 1979).

The invention claimed and explained herein is not limited in scope by the specific presentations described, since these representations are intended to illustrate several aspects of the invention. All equivalent representations are limited within the scope of the present invention. Various modifications of the present invention will be truly evident, in addition to those already described herein, for all experts. These modifications should also coincide with the scope of the additional claims. A number of references are cited, the content of which is comprehensively included.

Claims (7)

PATENT APPLICATIONS A process for the preparation of soluble phosphorylated glucan, characterized in that (a) the neutral polyglucose or glycoprotein is mixed with a strongly chaotropic reagent and the mixture is ground to a fine powder; (b) react the fine powder with concentrated phosphoric acid to form a reaction mixture containing soluble phosphorylated glucan; and (c) obtain the corresponding soluble phosphorylated glucan from the reaction mixture. A process according to claim 1, characterized in that the urea is a chaotropic reagent. The process of claim 1, further comprising heating the reaction mixture according to step (b) to 60-80 ° C for about 6 hours. Process according to claim 3, characterized in that the reaction mixture is heated to 60-80 ° C for 1-2 hours. Process according to claim 1, characterized in that soluble phosphorylated glucan is isolated by: (a) precipitation of soluble phosphorylated glucan; (b) addition of a sufficient amount of water to resuspend the precipitated soluble phosphorylated glucan; (c) removal of all components with a molecular weight less than 10,000 daltons. Method according to claim 1, characterized in that the neutral polyglucose contains particulate glucan obtained from microorganisms. A method according to claim 6, characterized in that the particulate glucan is derived from Saccharomyces cerevisiae.