WO2008141452A1

WO2008141452A1 - Chitosan salts, methods of manufacture and uses thereof

Info

Publication number: WO2008141452A1
Application number: PCT/CA2008/000969
Authority: WO
Inventors: Jonathan Gagnon; Alain Binette
Original assignee: Rival S.E.C.
Priority date: 2007-05-18
Filing date: 2008-05-16
Publication date: 2008-11-27

Abstract

Organosoluble chitosan salts, their method of preparation and uses are described. The organosoluble chitosan salts may be prepared by reacting chitosan with an acid in a reaction solvent, wherein the acid comprises at least 3 fluorine atoms and is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid, and wherein the reaction solvent comprises an organic solvent, thereby to form the chitosan salt. The reaction solvent may further comprise water, which is removed without causing the solidification of the chitosan salt.

Description

TITLE OF THE INVENTION

[0001] CHITOSAN SALTS, METHODS OF MANUFACTURE AND USES

THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. provisional application Serial No. 60/938,743 filed on May 18, 2007, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to chitosan salts and their methods of manufacture and uses. More specifically, the present invention relates to chitosan salts amenable to organic conditions.

BACKGROUND OF THE INVENTION

[0004] Chitin is a component of the cell walls of fungi, the exoskeletons of insects and other arthropods, and some other animals. It is a polysaccharide constructed from units of acetylglucosamine (N-acetyl-2-amino-2-deoxy-D-glucopyranosyl) linked together in β-1 ,4 fashion as shown in the following formula.

[0005] Chitosan is produced by deacetylation of chitin. The degree of deacetylation (%DDA) of chitosan may vary. This degree of deacetylation can be determined by NMR spectroscopy and other techniques. Generally, the degree of deacetylation in commercial chitosans is in the range of 60 to 100 %. Totally deacetylated chitosan has the following formula:

[0006] The amino group in chitosan has a pKa value of about 6.5 in water, thus, chitosan is positively charged and soluble in acidic to neutral aqueous solution with a charge density dependent on pH and the degree of deacetylation.

[0007] Given its properties and availability, chitosan has been considered in the context of various applications such as in pharmaceutical delivery systems and chromatographic supports.

[0008] While polyanionic polymers, like polysaccharides including sulfate groups and/or glucuronic units, are abundant in nature (see Biopolymers, Volume 6, Polysaccharides II: Polysaccharides from Eukaryotes, Vandamme EJ. , De Baets S., Steinbϋchel A., Wiley-VCH, New York, 2002), protonated chitosan represents a rare example of polycationic biopolymer.

[0009] Chitosan salts are intensively studied because they are easily available, water-soluble and can form films or fibers. On the other hand, certain chemical transformations of chitosan prove difficult due to its insolubility in common organic solvents, thus limiting its application in certain fields.

[0010] Furthermore, group-specific modification of chitosan has proven difficult, resulting in unpredictable transformation of any and all of the chitosan functional groups, i.e., the amino group and the different alcohol groups. This generally leads to a mixture of products, in turn resulting in a low yield of the desired products and thus high costs. [0011] There have been some attempts in the literature to provide methods or chitosan-derived products for partial reaction of chitosan (or the derived product thereof) in organic solvents and/or allow the selective reaction of hydroxyl group by protecting the amino group.

[0012] Selective N-phthaloylation of chitosan provides N-phthaloyl-chitosan which has increased solubility in some organic solvents, such as N,N-dimethylformamide (DMF) and pyridine, and is N-protected. This N-phthaloyl-chitosan facilitates the chemical modifications of hydroxyl groups on chitosan (see Kurita K., lkeda H., Yoshida Y., Shimojoh M., Harata, M., Biomacromolecules 2002, 3: 1 ). However, upon subsequent reaction of the N-phthaloyl-chitosan, deprotection of amine groups may sometime occur (by hydrazinolysis) and lead to undesired reactions, like ester cleavage.

[0013] Protonation of chitosan amino groups to form a chitosan salt having the formula below is one approach to protect the amino group during reaction.

[0014] Indeed, protonation as a protection method has the advantage to provide easily removable protection without side reactions. Of course, to be useful for reactions in organic systems, the chitosan salts used would have to be soluble in organic solvents. However, inorganic and organic salts of chitosan are normally insoluble in such solvents and are only soluble in aqueous solutions.

[0015] Sashiwa et al. have reported the formation in water of some sulfonate chitosan salts, such as (1R)-(-)-10-camphorsulfonate (CSA), which are somewhat soluble in dimethylsulfoxide (DMSO) (Sashiwa H., Shigemasa Y., Roy R. Chem. Lett. 2000, 6: 596), but are not soluble in other organic solvents such as N,N-dimethylformamide (DMF). They have also reported the utilization of CSA chitosan salts for unselective N₁O- acylation by cyclic acid anhydrides (Sashiwa H., Shigemasa Y., Roy R. Chem. Lett. 2000, 10: 1186). [0016] However, the presence of water in these chitosan salts hinders their reactivity with water-sensitive reagents. Indeed, it is generally known that chitosan salts formed in an aqueous solution will contain a fair amount of water entrapped in the structure of the salt. The nucleophilicity of water and those of alcohol groups of chitosan are similar, thus both will react with the water-sensitive reagents. Generally, water content is higher than the quantity of alcohol groups and water has less steric hindrance than alcohol groups.

[0017] There is therefore a need for chitosan derivatives, such as chitosan salts, which are more conducive to chemical modifications in an organic environment.

SUMMARY OF THE INVENTION

[0018] The present invention relates to chitosan salts and their methods of manufacture and uses. More specifically, the present invention relates to chitosan salts amenable to organic conditions.

[0019] In accordance with the present invention, there are provided organosoluble chitosan salts.

[0020] In an embodiment, the chitosan salts are soluble in at least one organic solvent other than dimethylsulfoxide (DMSO). "Soluble in at least one organic solvent other than DMSO" as used herein includes the situation wherein the salt is soluble in DMSO and is additionally soluble in at least another organic solvent other than DMSO.

[0021] The present inventors were the first to determine that a chitosan salt may be prepared in an organic solvent, with or without the additional presence of an aqueous solvent.

[0022] Without being bound to any particular theory, in cases where the chitosan salt is prepared in aqueous medium and subsequently isolated, the present inventors have determined that the degree of water removal has an impact on the form in which the salt is obtained (film-like, fiber-like or powder-like) and in turn has an impact on its solubility in organic solvents. The inventors further observed that when the chitosan salt changes from being film-like or fiber-like to being powder-like, it also becomes soluble in a plurality of organic solvents.

[0023] Interestingly, without being bound to any particular theory, the exact amount of water in the film is not by itself predictive of its solubility. For the same quantity of water, a salt may be powder-like (soluble) or film/fiber-like (insoluble) depending on the reagents and/or conditions of its preparation. As determined by the studies described herein, powder-like salts are soluble in organic solvents (in addition to the organic solvent DMSO), while fiber-like and film-like salts are not.

[0024] As used herein, a "film-like" salt refers to a salt that spontaneously forms small thin membranes, layers or sheets and a "fiber-like" salt refers to a salt that spontaneously forms fine elongated threads, strands or filaments. Finally, a "powder-like" salt refers to a salt that spontaneously forms finely divided loose particles. All these terms refer to the form spontaneously adopted by the salt when it is isolated from a reaction mixture, dried, washed, etc. It does not refer to the form of the salt after treatments are performed to change the form of the salt without changing its nature or water content. For example, a film-like salt that is ground until it becomes powdery is not considered a powder-like salt according to the description above because the powdery state is not spontaneously adopted by the salt, but forced by the grinding.

[0025] As used herein, a "soluble" chitosan salt refers to a chitosan salt which, when combined with a solvent, (1 ) dissolves or solubilizes to the point where no particulate matter is visible to the naked eye or (2) swells, that is incorporates the solvent into its structure, and achieves a gel-like, translucent state.

[0026] The instant inventors have thus developed organosoluble chitosan salts as well methods of producing such salts.

[0027] In an aspect, the salt can be prepared in a substantially anhydrous organic solvent. This allows production of a salt that contains very little water because there is very little water in the reaction mixture. The salt formed is therefore powder-like and soluble in a plurality of organic solvents. As noted above, because of their very well known incompatibility with an organic environment, prior to inventors' studies described herein, chitosan salts had only been prepared in aqueous conditions, not in organic environments.

[0028] In a further aspect, the salt can be prepared in a reaction solvent comprising an organic solvent and an aqueous solvent and then removing water from the reaction mixture via a method which does not cause solidification of the salt, before isolating or using the salt. As a result, the isolated salt is soluble in a plurality of organic solvents.

[0029] In a further aspect, the salt can be prepared in an aqueous solvent, following which an organic solvent is added and the water is removed from the reaction mixture via a method which does not cause solidification of the salt, before isolating or using the salt. Once again, the isolated salt is soluble in a plurality of organic solvents.

[0030] In a further aspect, the salt can be prepared in an aqueous solvent, and then the salt is isolated from this solvent. This produces a film-like or fiber-like salt that is not soluble in organic solvents other that DMSO. Then water is removed from this salt until it becomes powder-like salt and thus soluble in a plurality of organic solvents.

[0031] The present invention thus relates to organosoluble chitosan salts, their method of preparation and their uses. The inventors have developed chitosan salts which are compatible with organic solvents as well as methods of preparing these salts. Such salts can be used for example in reactions in solution for the derivatization or modification of chitosan, with derivatizing/modifying agents of interest.

[0032] In an aspect, the present invention relates to a method of preparing an organosoluble chitosan salt, the method comprising reacting chitosan with an acid in a reaction solvent, wherein the acid comprises at least 3 fluorine atoms and is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid and wherein the reaction solvent comprises an organic solvent and optionally further comprises water, thereby to form the chitosan salt ; and, if the reaction solvent comprises water, removing the water without causing the solidification of the salt.

[0033] The invention further provides a method of preparing an organosoluble chitosan salt, the method comprising reacting chitosan with an acid in aqueous solvent, wherein the acid comprises at least 3 fluorine atoms and is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid, thereby to form the chitosan salt; adding an organic solvent in which the salt is soluble; and removing water without causing the solidification of the salt.

[0034] "Removing water without causing the solidification of the salt" as used herein refers to methods of removing water which avoid solid formation. Examples of such methods which avoid solid formation include partial evaporation and treatment/washing/extraction with a water-removing agent or solvent. In contrast, water removal methods which would not avoid solid formation include precipitation, crystallization, and lyophilization.

[0035] The invention further provides a method of preparing an organosoluble chitosan salt, the method comprising reacting chitosan with an acid in an aqueous solvent; isolating the salt from the aqueous solvent, thereby producing a fiber- like or film-like chitosan salt; and removing water from the fiber-like or film-like chitosan salt until the fiber-like or film-like chitosan salt attains a powder-like form.

[0036] The chitosan salt may be prepared starting with commercially available chitosan, which may be ground before using, if desired.

[0037] In some embodiments, the chitosan salt may have a solubility in the organic solvent of at least about 0.1 mg/mL at ambient temperature (e.g., about 15^°C to about 30^°C, e.g., about 20^°C to about 25^°C, e.g., about 25^°C).

[0038] As used herein, an "organic" solvent refers to any organic solvent generally known in the art. Organosoluble" as used herein refers to a compound or material which is soluble in an organic solvent. For example, an organosoluble chitosan salt is soluble in an organic solvent.

[0039] In an embodiment, the organic solvent is an organic solvent other than

DMSO. Further, in embodiments, as used herein, a chitosan salt which is soluble in an organic solvent other than DMSO may additionally be soluble in DMSO.

[0040] In embodiments, the organic solvent in which the chitosan is soluble is

N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), ethylene glycol (1 ,2- ethanediol), formic acid, glycerol (1 ,2,3-propanetriol), methanol, or trifluoroacetic acid (TFA). In further embodiments, the above-mentioned solvent is DMF or DMA.

[0041] The reaction solvent is an organic solvent in which the chitosan salt to be prepared is soluble. This means, in this particular case, a solvent wherein the solubility of the formed chitosan salt is, in an embodiment, at least about 0.5 mg/mL, in a further embodiment at least about 1 mg/mL.

[0042] If the acid used to prepare the salt is liquid, in embodiments the acid can serve as the reaction solvent: the reaction will be carried out neat and the acid will be used in sufficient quantity to serve as the solvent for the reaction. This can be the case, for example, if trifluoroacetic acid is used.

[0043] In embodiments, the organic solvent may be an organic solvent having a dielectric constant higher than about 25.5 at a temperature between about 15 and about 30⁰C; a Gutmann's donor number higher than about 14.5 at a temperature between about 15 and about 3O⁰C; a value of E_τ(30) higher than about 41.0 kcal/mol at a temperature of about 25°C; and a pKa that does not permit the deprotonation of the salt in the organic solvent. These parameters may be found or determined by the skilled person using known sources and methods. For example, dielectric constant values can be obtained from CRC Handbook of Chemistry and Physics, Lide D. R., 87^th edition, CRC Press, 2006-2007, pp. 15-13 to 15-22; Gutmann's donor number values can be found in Hahn S., Miller W.M., Lichtenthaler R.N., Prausnitz J. M., J. Solution Chem., 1985, 14: 129-137; E_τ(30) values can be obtained from Marcus Y., J. Solution Chem., 1991 , 20: 929-94 and corresponding E_T ^N can be calculated from the equation and data reported in Adams DJ. , Dyson P.J., Tavener S.J., Chemistry in Alternative Reaction Media, John Wiley & Sons Ltd, West Sussex, England, 2004, pp. 18-21 (Equation 1.4 and Table 1.7). Furthermore, according to Kamlet M.J., Abboud J. -LM., Abraham M. H., Taft R.W., J. Org. Chem., 1983, 48: 2877-2887, " The β scale of HBA (hydrogen-bond acceptor) basicities provides a measure of the solvent's ability to accept a proton (donate an electron pair) in a solute-to-solvent hydrogen bond. "

[0044] The reaction solvent may advantageously be substantially anhydrous.

When such a solvent is used, the chitosan salts produced are relatively devoid of water, powder-like and soluble in organic solvents other that DMSO.

[0045] In embodiments, the substantially anhydrous reaction solvent may contain less that about 5% (w/w) water, preferably, less than about 3%, 2%, 1%, 0.5% water.

[0046] In an embodiment, the reaction solvent is a solvent in which chitosan is not soluble. This facilitates the removal of unreacted chitosan, if any and if desired, from the reaction mixture.

[0047] The reaction solvent may advantageously be substantially anhydrous

N,N-dimethylformamide.

[0048] Alternatively, the reaction solvent may comprise water. For example, it may comprise up to 20% water. When such a solvent is used, the salt is formed and then the water is removed. This leaves the chitosan salt in solution in the anhydrous reaction solvent (without water). As above, this salt is thus relatively devoid of water, powder-like and soluble in organic solvents.

[0049] If water is present, it can be removed by a variety of means well known to the person of skill in the art. For example, if the reaction solvent has a boiling point higher than the boiling point of water, the water can be removed by a simple evaporation. [0050] The acid used in the above-method will be chosen depending on the chitosan salt that is desired. This acid may be a fluoroalkyl (e.g., a trifluoroalkyl) carboxylic acid or a fluoroalkyl (e.g., a trifluoroalkyl) sulfonic acid. More specifically, in certain embodiment, the alkyl group may have between 1 and 12, preferably, 1 and 6 and more preferably between 1 and 3 carbon atoms. In embodiments, the acid may be trifluoroacetic acid or trifluoromethanesulfonic acid.

[0051] The above-described method produces the chitosan salt in solution in the reaction solvent. From this solution, the salt can be easily isolated by methods well known to the person of skill in the art. This is however not always necessary because the chitosan salt solution may be directly used to perform a subsequent chemical reaction in the same solvent. That is further reactant(s) can be added to the solution in order to carry out a desired transformation of the salt. For example, where the chitosan salt is in solution in an organic solvent upon its preparation, further reactant(s) which are reactive in the same organic solvent may be added directly to the solution, and subsequent reaction(s) may proceed. As such, in an embodiment the chitosan salt may be prepared and then subsequently derivatized/modified in the same solvent in which it was prepared.

[0052] In embodiments, in cases where isolation of the chitosan salt is desired, the method may further comprise the step of isolating the salt from the reaction solvent. This can be easily done by any of a number of techniques known to the person of skill in the art. For example, the chitosan salts of the invention can be isolated by evaporation, distillation, lyophilization, precipitation, ultrafiltration, or combinations thereof.

[0053] Some of these isolation methods may involve the use of a non- solvent. A non-solvent is a solvent that causes a compound in a solution to precipitate out of solution, when it is added to that solution. As used herein, the expression "a non- solvent" means an organic solvent in which the chitosan salt of the invention is not significantly soluble. Such a non-solvent will cause the precipitation of the chitosan salt when it is added to a solution of this salt.

[0054] More specifically, the isolation may be done by adding a non-solvent in the reaction mixture, thereby precipitating the salt, and then separating (e.g., filtering) out the salt. If there was some chitosan left unreacted in the reaction solvent, and this unreacted chitosan is insoluble in the reaction solvent, the chitosan may be filtered out prior to the addition of the non-solvent.

[0055] In cases where an aqueous solvent is used, it may for example be water.

[0056] The removal of water until the salt becomes powder-like can also be easily performed by a number of techniques known to the person of skill in the art. For example, it can be one or more wash with appropriate solvents, vacuum drying, etc.

[0057] In the case where the chitosan salt is prepared in an aqueous solvent, the isolating step may comprise the evaporation or the lyophilization of the aqueous solvent and the water removing step may comprise washing the salt with at least one washing organic solvent.

[0058] The present invention also relates to chitosan salts produced by the above-mentioned methods.

[0059] Furthermore, the present invention relates to powder-like chitosan salts soluble in a least one organic solvent other than DMSO.

[0060] In embodiments, the chitosan salt may comprise less than about 6% water, less that about 5% or 4% (w/w) water, or in a further embodiment less that about 3.5% water.

[0061] In embodiments, the salt may be a fluoroalkyl carboxylate or a fluoroalkyl sulfonate and comprise at least 3 fluorine atoms. In further embodiments, the salt may be trifluoroacetate chitosan or trifluoromethanesulfonate chitosan.

[0062] These chitosan salts are useful intermediates for the transformation of chitosan in organic media. They allow, for example, the efficient and regioselectively development of a various chitosan derivatives. For example, the chitosan salt of the invention may be regioselectively O-6 silylated using, for example, tert- butylchlorodiphenylsilane (TBDPSCI) in DMF.

[0063] Therefore, the present invention further relates to methods/uses of these chitosan salts for producing a chitosan derivative in an organic solvent, for example for regioselectively modifying chitosan in an organic solvent (e.g., for regioselective O-6 silylation of chitosan in an organic solvent).

[0064] Furthermore, chitosan being a biodegradable, nontoxic and biocompatible material, many of its derivatives, such as those produced by the salts of the invention, may find application in various fields, such as pharmaceuticals.

[0065] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0066] The present invention is illustrated in further details by the following non-limiting examples.

[0067] All the water content values in these examples were obtained by Karl

Fisher analysis according to USP method <921>. All the water percentages are mass percentages (w/w). All the samples were vacuum dried before analysis.

Example 1 TFA-Chitosan Salt Prepared in DMF

[0068] Preparation

[0069] Chitosan trifluoroacetate salt (TFA-chitosan) was synthesized by direct reaction between completely deacetylated high molecular weight chitosan and trifluoroacetic acid in DMF. [0070] Completely deacetylated chitosan from shrimp shells with a molecular weight of 546 kDa determined by gel permeation chromatography (GPC) was purchased from Marinard Biotech inc. (Riviere-au-renard, Canada). Chitosan was ground to 0.6 mm prior to utilization. DMF was dried over calcium hydride prior to distillation under vacuum.

[0071] A suspension of chitosan (1.00 g, 6.21 mmol of pyranosyl units) in anhydrous DMF (200 ml.) was slowly treated with trifluoroacetic acid (2.10 ml_, 27.3 mmol). The suspension was heated to 80⁰C for 45 min and then allowed to reach room temperature. After 24h, the suspension was filtered to remove the unreacted chitosan. The filtrate was concentrated to 100 mL and the chitosan salt was precipitated with 300 mL diethyl ether. The resulting suspension was filtered to isolate TFA-chitosan. The isolated salt was then washed with three portions of diethyl ether (15 mL) and dried under vacuum. A pale yellow solid was obtained with 85 % yield. IR: y (OH), 3418 cm^"1; y (C=O), 1678 cm^"1; y (NH3⁺), 1529 cm^'1; y (COO^"), 1432 cm^"1; Y (C-F), 1205 cm^"1; y (C-O), 1134 cm^"1 and 1073 cm^'1. ¹³C NMR: C2, 56.3 ppm; C6, 60.4 ppm; C3, 70.60 ppm; C5, 75.2 ppm; C4, 78.1 ppm; C1 , 98.1 ppm; CF₃, 116.8 ppm, (q), ¹J_C-_F = 290 Hz; C=O, 161.8 ppm, (q), ²Jc_-F = 36 Hz.

[0072] This salt was in the form of a powder.

[0073] The solubility of the TFA-chitosan salt obtained in different organic solvents and water was assessed and is shown in Table 1 below. 10 mg of TFA-chitosan salt was added to 10 mL of solvent, the suspension was stirred and incubated at room temperature for 24 h. Thereafter, if the suspension persisted, it was heated at 9O⁰C during one additional hour. A positive test of solubility was determined by a homogeneous solution at room temperature.

[0074] As can be seen in Table I, the chitosan salt obtained is highly soluble in several organic solvents such as DMF, DMA, DMSO, methanol, formic acid, trifluoroacetic acid (TFA), glycerol and ethylene glycol.

Table I. Solubility of the Obtained TFA-Chitosan Salt in Different Solvents

N/A: not available

[0075] Dielectric constant values at ambient temperature values were obtained from CRC Handbook of Chemistry and Physics, Lide D. R., 87^th edition, CRC Press, 2006-2007, pp. 15-13 to 15-22.

[0076] Donor number values were obtained from Marcus Y., Chem. Soc.

Rev., 1993, 22: 409-416 and Hahn S., Miller W.M., Lichtenthaler R.N., Prausnitz J. M., J. Solution Chem., 1985, 14: 129-137.

[0077] Kamlet-Taft parameter β values were obtained from Kamlet MJ. ,

Abboud J.-L.M., Abraham M.H., Taft R.W., J. Org. Chem., 1983, 48: 2877-2887; Marcus Y., J. Solution Chem., 1991 , 20: 929-944; and Marcus Y., Chem. Soc. Rev., 1993, 22: 409-416. This value for acetonitrile is not the same in each of these references. Kamlet et al. report a value of 0.31 , while Marcus reports a value of 0.40. This latter value is originally based on the article of Boeck J., Gritzner G., Z. Phys. Chem. (Neue Folge) 1982, 130: 181-192.

[0078] E_T(30) values were obtained from Marcus Y., J. Solution Chem., 1991 , 20: 929-944; and Marcus Y., Chem. Soc. Rev., 1993, 22: 409-416.

[0079] This TFA-chitosan salt produced contained 3.4 % of water.

Example 2 TFA-Chitosan Salt Prepared Using Chitosan of Different Molecular Weights

[0080] In Example 1 , TFA-chitosan salts have been successfully synthesized using completely deacetylated chitosan of 546 kDa.

[0081] Complete deacetylation of chitosan DDA 88% of 261 kDa (Kurita K.,

Hirakawa M., Kikuchi S., Yamanaka H., Yang J., Carbohydr. Polym., 2004, 56: 333-337) was performed to obtain completely deacetylated chitosan. TFA-chitosan salts have been successfully synthesized using this chitosan according to the method described in Example 1. This salt, a pale yellow solid, is a powder that is soluble in DMSO, DMF and DMA.

Example 3 Triflate-Chitosan Salt Prepared in DMF

[0082] Moreover, triflate-chitosan salt has been synthesized from completely deacetylated chitosan using the method described in Example 1. More specifically, a suspension of chitosan (0.50 g, 3.10 mmol of pyranosyl units) in anhydrous DMF (90 ml_) was slowly treated with trifluoromethanesulfonic acid (10.0 g, 66.6 mmol). The suspension was heated to 80°C for 90 min and then allowed to reach room temperature. The suspension was filtered to remove the small fraction of unreacted chitosan. The filtrate was evaporated to dryness and the chitosan salt was washed with diethyl ether (500 ml_). The resulting solid was filtered. The isolated salt was then washed with 250 mL of diethyl ether and dried under vacuum. A pale yellow solid was obtained with 65 % yield.

[0083] The obtained salt was in the form of a powder.

Example 4

TFA-Chitosan Salt Prepared in DMF Containing 20% Water

[0084] A suspension of chitosan (0.50 g) in DMF (grade ACS) containing 20

% (v/v) of added distilled water (100 mL of solvent) was treated with trifluoroacetic acid (5.0 mL, 20 eq.). The mixture was stirred at 6O⁰C for 1 hour. The solution was allowed to reach room temperature. The solution was concentrated to a volume of 50 mL. Since DMF has a higher boiling point than water, substantially all the water was evaporated in this step, leaving DMF only as a solvent.

[0085] The solution was then precipitated with diethyl ether (500 mL) and filtered. The resulting solid was washed with acetone (250 mL) overnight. The solid was filtered and washed with diethyl ether (250 mL) overnight and finally dried under vacuum

[0086] This salt was in the form a powder.

[0087] This TFA-chitosan salt was soluble in DMSO and swelled in DMF and

DMA.

[0088] This TFA-chitosan salt contained 3.2% of water. Example 5 TFA-Chitosan Salt Prepared in Water

[0089] A suspension of chitosan (0.50 g) in distilled water (90 mL) was treated with trifluoroacetic acid (5.0 mL, 20 eq.). The suspension was heated at 60⁰C during 1 hour and was allowed to reach room temperature. The solution was concentrated to 20 mL. Ethanol (250 mL) was added to the solution and the mixture was evaporated to dryness. The obtained solid was then washed with acetone (250 mL) and was filtered. The solid was also washed with diethyl ether (100 mL) and was dried under vacuum.

[0090] This salt was in the form a powder.

[0091] The TFA-chitosan salt obtained was soluble in DMSO and swelled in DMA and DMF.

[0092] This TFA-chitosan salt contained 5.5% of water.

Example 6 TFA-Chitosan Salts Produced in Other Conditions

[0093] Using the method described in Example 1 , TFA-chitosan salts were successfully synthesized in methanol and in neat conditions (in the absence of an additional solvent) directly in trifluoroacetic acid.

[0094] The TFA-chitosan salt synthesized in methanol was obtained by evaporating the solution to dryness and subsequently washing the salt with diethyl ether. The salt was a powder. It was soluble in DMSO and swelled in DMF and DMA.

Comparative Example 1 TFA-Chitosan salts in the form of film or fibers

[0095] TFA-chitosan salts were synthesized directly in water and isolated either by evaporation or lyophilization (also called freeze-drying). These procedures are similar to that described by Sashiwa H., Shigemasa Y., Roy R. Chem. Lett. 2000, 6: 596. With both these methods of isolation, the water/chitosan salt mixture is dried (solidified) in the presence of water.

[0096] These TFA-chitosan salts obtained were soluble in DMSO (10 mg /1 ml.) but not soluble in DMF (10 mg /70 mL) and DMA (10 mg /70 ml_).

[0097] The TFA-chitosan salt obtained by evaporation formed a film and contained 4.5 % of water. The TFA-chitosan salt obtained by lyophilization formed fibers and contained 6.3 % of water.

Comparative Example 2 p-toluenesulfonic -Chitosan salts in the form of film or fibers

[0098] Also, p-toluenesulfonic chitosan salts were obtained using he procedure reported by Sashiwa et al. (see comparative Example 1). These chitosan salts, which are obtained by evaporation or lyophilization in the presence of water, formed films and fibers, respectively.

[0099] These salts were soluble in DMSO (10 mg /1 mL) as reported by

Sashiwa et al., but were not soluble in DMF (10 mg /70 mL) or in DMA (10 mg /70 mL).

[00100] The p-toluenesulfonate chitosan salt, which was in the form of fibers, contained 6.7 % of water.

Example 7 Regioselective O-6 silylation of TFA-chitosan

[00101] The chitosan salt of Example 1 was found to be a useful reagent for regioselective modifications of chitosan in homogeneous conditions. Indeed, O-6 positions of TFA-chitosan were selectively protected using ferf-butyldiphenylsilyl (TBDPS) groups with a degree of substitution near 1.00 followed by deprotonation of amine groups in a "one-pot" (i.e., all reagents combined in the same reaction vessel) procedure. [00102] Chitosan protonation during all the course of the reaction avoids substitution of the amino groups. A "one-pot" procedure was developed for the regioselective protection of O-6 positions and therefore for the formation of chitosan etiolating N,O-polyligand. This polyligand may be utilized, for example, in transition metals coordination chemistry.

[00103] Tert-butyldiphenylsilyl (TBDPS) group is a well-known protecting group selective to primary alcohols notably with saccharides due to its stability in varying reaction conditions (steric hindrance). TBDPS group is easily introduced and can be removed by numerous methods as mild reaction with fluoride anion (see Greene T. W., Wuts P. G. M. Protective Groups in Organic Synthesis, third edition, John Wiley and Sons, 1999.).

[00104] Regioselective silylation of primary hydroxyl groups in the chitosan salt of Example 1 was attempted with te/t-butylchlorodiphenylsilane (TBDPSCI) in DMF. Instantaneously after addition of this silylating reagent, a white chitosan precipitate was observed. This precipitation is attributable to an anion exchange between trifluoroacetate and chloride ions that are formed by nucleophilic substitution reaction of trifluoroacetate on TBDPSCI. Chitosan chloride salt is insoluble in organic solvents. Based on these results, preferred leaving groups on the silylating reagent are trifluoroacetate or a weaker conjugate base to minimize precipitation before silylation begins.

[00105] Selective silylation of the chitosan salt of Example 1 at room temperature during 48 h was performed with ferf-butyldiphenylsilyl trifluoroacetate, which was prepared by reacting TBDPSCI and sodium trifluoroacetate in DMF. The reaction scheme was:

CF₃COO^"

[00106] Sodium trifluoroacetate 5.02 g (36.9 mmol) was dissolved and stirred in dry DMF (25 ml.) at room temperature, then 8.0 ml_ (31 mmol) of TBDPSCI was added to the mixture. The appearance of a fine white crystalline precipitation was observed. The mixture was used without purification nor characterization.

[00107] To a suspension of chitosan 0.502 g (3.12 mmol) in 40 mL of DMF,

0.30 mL (3.9mmol) of TFA was added. The temperature was raised to 70⁰C, after 30 min. of stirring, 0.10 mL (13 mmol) of TFA was added. Then a flow of N₂ bubbled in the mixture for 30 min. The heating was stopped and the mixture was allowed to cool to room temperature. A solution of TBDPS-TFA in DMF 25 mL (1.25 M) was added to the mixture using a cannula and stirred for 48 h at room temperature. The reactional mixture was quenched with 100 mL of EtOH-H₂O (1 :1 (v/v)). The mixture was neutralized by dripping a solution of NaOH (0.656 g in 125 mL of EtOH-water (1 :1 (v/v)) till the pH reached 8. The precipitate was filtered and washed with acetone (3 x 15 mL), a drab powder was collected and dried under vacuum. Yield 83 %, 1.02 g (0.259 mmol).

[00108] The IR spectrum of product obtained from precipitation in a

NaOH/H₂O, EtOH mixture (1 :1 v/v) shows a weaker and sharper hydroxyl band at 3455 cm^"1 compared to chitosan and a NH₂ deformation at 1674 cm^"1. The presence of silyl groups was also confirmed by a band attributable to phenyl groups at 3068 cm^"1 and a strong absorption band at 1113 cm^"1 assigned to Si-O elongations superimposed to C-O elongations of pyranosyl units.

[00109] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

[00110] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing an organosoluble chitosan salt, the method comprising: a) reacting chitosan with an acid in a reaction solvent, wherein said acid comprises at least 3 fluorine atoms and is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid, and wherein said reaction solvent comprises an organic solvent and optionally further comprises water, thereby to form said chitosan salt; and b) if said reaction solvent comprises water, removing said water without causing the solidification of the salt.

2. The method of claim 1 , wherein said acid is trifluoroacetic acid or trifluoromethane sulfonic acid.

3. The method of claim 1 or 2, wherein said chitosan is not soluble in said reaction solvent.

4. The method of any one of claims 1 to 3, wherein said organic solvent has: a) a dielectric constant higher than about 25.5 at a temperature between about

15°C and about 3O⁰C; b) a Gutmann's donor number higher than about 14.5 at a temperature between about 15°C and about 3O⁰C; c) a value of E_τ(30) higher than about 41.0 kcal/mol at a temperature of about 25°C; and d) a pKa that does not permit the deprotonation of the salt in said organic solvent.

5. The method of any one of claims 1 to 4, wherein said reaction solvent is substantially anhydrous.

6. The method any one of claims 1 to 4, wherein said reaction solvent comprises up to 20% water.

7. The method of any one of claims 1 to 6, wherein said organic solvent is N₁N- dimethylformamide.

8. The method of any one of claims 1 to 3, wherein said acid is a liquid, and said organic solvent consists essentially of said acid.

9. The method of any one of claims 1 to 8, wherein said organic solvent has a boiling point higher than the boiling point of said water and said water is removed by evaporation.

10. The method of any one of claims 1 to 9, further comprising the step of isolating said salt from the reaction solvent.

11. The method of claim 10, wherein said isolating step comprises: a) adding a non-solvent to said reaction solvent, thereby precipitating the salt; and b) separating the salt from the solvent.

12. A method of preparing an organosoluble chitosan salt, the method comprising: a) reacting chitosan with an acid in aqueous solvent, wherein said acid comprises at least 3 fluorine atoms and is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid, thereby to form said chitosan salt; and b) adding an organic solvent in which the salt is soluble; and c) removing water without causing the solidification of the salt.

13. The method of claim 12, wherein said aqueous solvent is water.

14. The method of claim 12 or 13, wherein said acid is trifluoroacetic acid or trifluoromethanesulfonic acid.

15. The method of any one of claims 12 to 14, wherein said organic solvent has a boiling point higher than the boiling point of water and water is removed by evaporation.

16. The method of any one of claims 12 to 15, further comprising the step of isolating the salt from the organic solvent.

17. The method of claim 16 wherein said isolating step comprises: a) adding a non-solvent to said reaction solvent, thereby precipitating the salt; and b) separating the precipitated salt from the solvent.

18. The method of any one of claims 12 to 17, wherein said organic solvent has: a) a dielectric constant higher than about 25.5 at a temperature between about 15°C and about 3O⁰C; b) a Gutmann's donor number higher than about 14.5 at a temperature between about 15⁰C and about 30⁰C; c) a value of E_τ(30) higher than about 41.0 kcal/mol at a temperature of about 25°C; and d) a pKa that does not permit the deprotonation of the salt in said organic solvent.

19. A method of preparing an organosoluble chitosan salt, the method comprising: a) reacting chitosan with an acid in an aqueous solvent; b) isolating the salt from said aqueous solvent, thereby producing a fiber-like or film-like chitosan salt; c) removing water from the fiber-like or film-like chitosan salt until the fiber-like or film-like chitosan salt attains a powder-like form.

20. The method of claim 19, wherein said aqueous solvent is water.

21. The method of claim 19 or 20, wherein said acid is a fluoroalkyl carboxylic acid or a fluoroalkyl sulfonic acid and wherein said acid comprises at least 3 fluorine atoms.

22. The method of claim 21 , wherein said acid is trifluoroacetic acid or trifluoromethanesulfonic acid.

23. The method of any one of claims 19 to 22, wherein said isolating step comprises evaporating or lyophilizing said aqueous solvent.

24. The method of any one of claims 19 to 23, wherein said water removing step comprise washing the salt with at least one washing organic solvent.

25. The method of any one of claims 1 to 24, wherein said organosoluble chitosan salt is soluble in N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), ethylene glycol (1 ,2-ethanediol), formic acid, glycerol (1 ,2,3-propanetriol), methanol, or trifluoroacetic acid.

26. The method of claim 25, wherein said organosoluble chitosan salt is soluble in N₁N- dimethylacetamide (DMA) or N,N-dimethylformamide (DMF).

27. A chitosan salt produced by the method of any one of claims 1 to 26.

28. A powder-like chitosan salt soluble in an organic solvent other than dimethylsulfoxide (DMSO).

29. The salt of claim 27 or 28, comprising less than 6% (w/w) of water.

30. The salt of claim 29, comprising less than 5% (w/w) of water.

31. The salt of claim 30, comprising less than 4% (w/w) of water.

32. The salt of claim 31 , comprising less than 3.5% (w/w) of water.

33. The salt of any one of claims 28 to 32, wherein said salt is a fluoroalkyl carboxylate or a fluoroalkyl sulfonate and wherein said salt comprises at least 3 fluorine atoms.

34. The salt of claim 33, wherein said salt is trifluoroacetate chitosan or trifluoromethanesulfonate chitosan.

35. The salt of any one of claims 28 to 34, wherein said organic solvent other than DMSO is N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), ethylene glycol (1 ,2-ethanediol), formic acid, glycerol (1 ,2,3-propanetriol), methanol, or trifluoroacetic acid.

36. The salt of claim 35, wherein said organic solvent other than DMSO is N, N- dimethylacetamide (DMA) or N,N-dimethylformamide (DMF).

37. Use of the salt of any one of claims 27 to 36 for producing a chitosan derivative in an organic solvent.

38. Use of the salt of any one of claims 27 to 36 for regioselectively modifying chitosan in an organic solvent.

39. Use of the salt of any one of claims 27 to 36 for regioselective 0-6 silylation of chitosan in an organic solvent.

40. A method for producing a chitosan derivative in an organic solvent, said method comprising reacting the salt of any one of claims 27 to 36 with a derivatizing agent in said organic solvent.

41. A method for regioselectively modifying chitosan in an organic solvent, said method comprising reacting the salt of any one of claims 27 to 36 with a modifying agent in said organic solvent.

42. A method for regioselective 0-6 silylation of chitosan in an organic solvent, said method comprising reacting the salt of any one of claims 27 to 36 with a silylating agent in said organic solvent.