METHOD FOR SYNTHESIS OF A BIOPOLYMER DERIVATIVE, A BIOPOLYMER DERIVATIVE AND ITS USE

Abstract

The invention relates to a method of synthesis of a biopolymer derivative, preferably a chitosan derivative, comprising the formation of a peptide bond. The invention also relates to the biopolymer derivative, and use of the biopolymer derivative, preferably a chitosan derivative. The biopolymer derivative has broad uses in the industry, environmental protection and can be used in pharmaceutical and cosmetic compositions. The invention also relates to a composition for prevention of symptoms of allergy caused by heavy metals, especially palladium, cobalt, chromium and gold, in particular nickel.

Claims

1. A derivative of an insoluble biopolymer, wherein said biopolymer contains reactive amine or carboxyl groups, characterized in that said derivative is modified with a modifier molecule with the formation of a peptide bond.

2. The derivative of claim 1, wherein the biopolymer is a poly/oligosaccharide, preferably chitosan.

3. The derivative of claim 1 or 2, wherein the peptide bonds are present between amine groups of units of poly(2-deoxy-2-aminoglucose), and available carboxyl groups of modifier molecules.

4. The derivative of any of claims 1-4, wherein the modifier molecule containing a carboxyl group is selected from carboxylic acids, amino acids, amino acid analogues, dipeptides, tripeptides, tetrapeptides, longer peptides, peptidomimetics, proteins and protein mimetics or any mixture thereof.

5. A method for synthesis of an insoluble biopolymer derivative comprising forming a peptide bond between an amine or carboxyl group of a unit in the biopolymer containing reactive amine or carboxyl groups, and one of the available carboxyl or amine groups of the modifier molecule being attached.

6. The method of claim 5, comprising: a) reacting a modifier molecule with Fmoc-Cl in a suitable solvent, preferably dioxane; b) dissolving Fmoc-modifier molecule in a solvent, preferably DMF, and reacting Fmoc-modifier molecule with the biopolymer in the presence of HBTU, HOBt and DIPEA; c) deprotecting the modifier molecule by removal of Fmoc group and d) suspending the biopolymer with the attached modifier molecule in distilled water and lyophilizing thereof.

7. The method of claim 5, comprising formation of the peptide bond between the amine groups of units of poly(2-deoxy-2-aminoglucose) and the modifier molecules with the field of microwaves.

8. The method of claim 5, wherein the step of obtaining the Fmoc protected molecule is dispensible.

9. The method of claim 7 or 8, comprising: a) processing the biopolymer with microwaves in a suitable solvent, b) adding the activators to activate the function group of the biopolymer, c) adding the unprotected modifier molecule and d) processing the reaction mixture with microwaves, e) washing the obtained modified biopolymer and lyophilizing thereof.

10. The method of claim 7-9, wherein said activators are either DCC and HOPfp or HBTU, HOBT and DIPEA.

11. The method of any of claims 7-9, wherein the microwave processing is continued for 1-30 minutes.

12. The method of any of claims 5-11, wherein the biopolymer is chitosan and/or the modifier molecule is at least one of carboxylic acids, amino acids, amino acid analogues, dipeptides, tripeptides, tetrapeptides, longer peptides, peptidomimetics, proteins or protein mimetics, or any mixture thereof, preferably the tripeptide molecule is glutathione.

13. A cosmetic or pharmaceutic composition containing a biopolymer derivative of any of claims 1-4.

14. The composition of claim 13, in form of an ointment, cream or lotion.

15. The composition of claim 13 or 14, for use in medicine.

16. The composition of claims 13-15, for use in preventing symptoms of allergy caused by heavy metals, preferably palladium, cobalt, chromium, gold and nickel, especially caused by the contact of skin with nickel.

17. The composition of claims 13-16, wherein the composition contains an additional biopolymer, preferably a biopolymer being a metal binding protein.

18. The chitosan derivative of claims 1-4, for use in preventing symptoms of skin allergy caused by the contact with metals, especially heavy metals, in particular nickel.

19. A use of biopolymer derivative of any of claims 1-4 in medicinal or cosmetic products.

20. The use of chitosan derivative of claim 1 or 2 and/or chitosan to prevent symptoms or alleviate heavy metal allergies.

21. The use of claim 20 to prevent or alleviate symptoms of nickel allergy.

22. The use of claims 18-21, wherein the composition contains an additional biopolymer, preferably a biopolymer being a metal binding protein.

23. A matrix for attaching modifier molecules, containing the biopolymer derivative of any of claims 1-4.

24. A method for purification of industrial and domestic waste wherein the biopolymer derivative of any of claims 1-4 is contacted with said waste to entrap the pollutants and said biopolymer derivative with entrapped pollutant is resolved.

25. A method for recovery of metals wherein the material containing metal ion is contacted with the biopolymer derivative of any of claims 1-4.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] The method according to the invention is explained on the basis of the specific embodiments in more detail on Figures wherein:

[0057] FIG. 1 presents the scheme of reaction of chitosan modification with a modifier molecule under the conditions of peptide bond formation.

[0058] FIG. 2 presents the ESI-MS spectrum of 9-fluorenylmetoxycarbonyl-glutathione obtained in the reaction of glutathione coupling with Fmoc-Cl.

[0059] FIG. 3 presents a comparison of behavior of commercially available chitosan (A) and glutathione-modified chitosan (A[GSH]) in contact with a 50 mM solution of nickel(II) chloride. The precipitate in test tube marked (A) is brown and the precipitate in test tube marked (A[GSH]) is green. Nickel complexes with unmodified chitosan are green, while nickel complexes with glutathione-modified chitosan are brown.

[0060] FIG. 4 presents a graph illustrating a comparison of nickel binding potency of commercial chitosan (A) and glutathione-modified chitosan (A[GSH]).

EXAMPLES

[0061] Examples are provided herein below. However, the disclosed and claimed invention is to be understood to not be limited in its application to the specific experimentation, results and laboratory procedures. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

Example 1

Synthesis of 9-fluorenylmetoxycarbonyl-glutathione

[0062] In a 200 ml three-necked flask 3 g of glutathione (10 mmoles) was dissolved in a mixture of 26 ml of dioxane and 68 ml of 10% NaCO.sub.3 under anaerobic conditions. The flask fitted with a dropping funnel, stirring magnet, argon balloon and a bubbler was mounted over a magnetic stirrer. 2.71 g of Fmoc-Cl (10.5 mmoles) was dissolved in 26 ml of dioxane and added dropwise slowly over 15 minutes. The reaction was kept in the ice bath during addition. Then, the ice bath was removed. The reaction was allowed to proceed for 10 hours under argon, while monitoring its progress by ESI-MS. Next, the solution was acidified to pH3. The precipitate formed was separated on a Schott funnel. The remaining solution was evaporated until a significant amount of precipitate formed. This precipitate was separated on a Schott funnel and washed with distilled water. Fmoc-glutathione was obtained, having molecular mass 529.17 g/mole (FIG. 2).

Example 2

Attaching of Glutathione to Chitosan

[0063] 0.68 g of chitosan was placed in a reaction vessel for solid state peptide synthesis. 3 g of Fmoc-glutathione (3 mol equivalents) was dissolved in 20 ml of DMF. To this solution 2.14 g (3 mol equivalents) of HBTU, 1.29 g (3 mol equivalents) of HOBt and 1.98 ml (6 mol equivalents) of DIPEA were added. The reagents were mixed together and added to the reaction vessel containing chitosan. The mixture was allowed to react for 2.5 hours on a laboratory shaker. This procedure was repeated three times. Next, the solution was filtered off and the remaining biopolymer was washed three times with DMF. In order to remove the Fmoc protecting group from glutathione, a 20% solution of piperidine in DMF was added twice, followed by shaking for 20 minutes. Following the Fmoc group removal, the biopolymer was washed three times with DMF. The DMF solution was sucked up and the biopolymer with glutathione was suspended in distilled water and lyophilized. The reaction yield determined by elemental analysis Y<0.3%.

Example 3

Attaching of Glutathione to Chitosan in the Field of Microwaves According to Method 1 with the Use of DCC and HOPfp

[0064] To a reaction vessel for solid state peptide synthesis 2.27 g of chitosan suspended in 5 ml of 2:1 DMF:H.sub.2O mixture was added and processed with microwaves (t=5 minutes, P=25 W, T=75 C.). 0.613 g of HOPfp and 0.687 g of DCC were dissolved in 5 ml of 2:1 DMF:H.sub.2O mixture and added to the reaction vessel. The resulting mixture of chitosan with the activators was processed with microwaves (t=5 minutes, P=25 W, T=75 C.), which activated the function groups of the polymer. Next, 0.568 g of glutathione (free molecule, not protected with Fmoc) in 5 ml of DMF was added to the vessel and subjected twice to the microwaves (t=5 minutes, P=12 W, T<50 C.). The suspension was added to a centrifugation vessel and centrifuged. The supernatant was decanted. The obtained modified biopolymer was suspended three times in fresh portions of DMF, and then centrifuged and decanted. This procedure was repeated with the use of methylene chloride. After these three washes with methylene chloride the precipitation was frozen in liquid nitrogen and lyophilized. The lyophilized precipitate was washed three times with distilled water and lyophilized again. Elemental analysis revealed the presence of sulfur, and therefore the presence of glutathione attached to the polymer. The reaction yield determined by elemental analysis Y=23%.

Example 4

Attaching of Glutathione to Chitosan in the Field of Microwaves According to Method 2 with the Use of HBTU, HOBT and DIPEA

[0065] To a reaction vessel for solid state peptide synthesis 2.27 g of chitosan suspended in 5 ml of DMF was added and subjected to the action of microwaves (t=5 minutes, P=25 W, T=75 C.). 2.14 g HBTU, 1.29 g HOBT and 1.98 ml DIPEA in 5 ml of DMF were added to the reaction vessel. The resulting mixture of chitosan with the activators was subjected to the action of microwaves (t=5 minutes, P=25 W, T=75 C.), which activated the function groups of the polymer. Next, 0.568 g of glutathione (free molecule, not protected with Fmoc) in 5 ml of DMF was added to the vessel and subjected twice to the action of microwaves (t=5 minutes, P=12 W, T<50 C.). The suspension was added to a centrifugation vessel and centrifuged. The supernatant was decanted. The obtained modified biopolymer was suspended three times in fresh portions of DMF, and then centrifuged and decanted. This procedure was repeated with the use of methylene chloride. After these three washes with methylene chloride the precipitation was frozen in liquid nitrogen and lyophilized. The lyophilized precipitate was washed three times with distilled water and lyophilized again. The reaction yield determined by elemental analysis Y=30%.

Example 5

Attaching of Bacitracin to Chitosan in the Field of Microwaves. According to Method 2 with the Use of HBTU, HOBT and DIPEA

[0066] 2.27 g of chitosan suspended in 5 ml of DMF was placed in a reaction vessel for solid state peptide synthesis and subjected to the action of microwaves (t=5 minutes, P=25 W, T=75 C.). 2.14 g HBTU, 1.29 g HOBT and 1.98 ml DIPEA in 5 ml of DMF was added to the reaction vessel. The resulting mixture of chitosan with the activators was processed with microwaves (t=5 minutes, P=25 W, T=75 C.), thus activating the function groups of the polymer. Next, 2.630 g of bacitracin dissolved in 5 ml of 1:1 DMF:H.sub.2O mixture was added to the vessel and subjected twice to the action of microwaves (t=5 minutes, P=12 W, T<50 C.). The suspension was transferred to a centrifugation vessel and centrifuged. The supernatant was decanted. The obtained modified biopolymer was suspended three times in fresh portions of DMF, and then centrifuged and decanted. This procedure was repeated with the use of methylene chloride. After these three washes with methylene chloride the precipitation was frozen in liquid nitrogen and lyophilized. The lyophilized precipitate was washed three times with distilled water and lyophilized again. The reaction yield determined by elemental analysis Y=44%.

Example 6

Attaching of Ticarcillin to Chitosan in the Field of Microwaves According to Method 2 with the Use of HBTU, HOBT and DIPEA

[0067] 2.27 g of chitosan suspended in 5 ml of DMF was placed in a reaction vessel for solid state peptide synthesis and subjected to the action of microwaves (t=5 minutes, P=25 W, T=75 C.). 2.14 g HBTU, 1.29 g HOBT and 1.98 ml DIPEA in 5 ml of DMF was added to the reaction vessel. The resulting mixture of chitosan with the activators was processed with microwaves (t=5 minutes, P=25 W, T=75 C.), thus activating the function groups of the polymer. Next, 0.792 g of ticarcillin dissolved in 5 ml of 1:1 DMF:H.sub.2O mixture was added to the vessel and subjected twice to the action of microwaves (t=5 minutes, P=12 W, T<50 C.). The suspension was transferred to a centrifugation vessel and centrifuged. The supernatant was decanted. The obtained modified biopolymer was suspended three times in fresh portions of DMF, and then centrifuged and decanted. This procedure was repeated with the use of methylene chloride. After these three washes with methylene chloride the precipitation was frozen in liquid nitrogen and lyophilized. The lyophilized precipitate was washed three times with distilled water and lyophilized again. The reaction yield determined by elemental analysis Y=18%.

Example 7

Comparison of Capabilities of Chitosan and Glutathione Modified Chitosan to Bind Nickel(II) Ions

[0068] A 35 mg portion of unmodified commercially available chitosan and a 35 mg portion of glutathione modified chitosan obtained according to the invention were dispensed separately into two test tubes, followed by the addition of 1 ml of 50 mM nickel(II) chloride solution. A discoloration of pale green nickel(II) chloride solution was observed, accompanied by a change of the polymer color, to green for the unmodified chitosan, and to brown for the glutathione modified chitosan (FIG. 3). The precipitate settled at the bottom of the test tube, leaving a clear colorless supernatant above. The Ni(II) content in the supernatant was determined by spectrophotometry, using its colored DTT complexes. The change of Ni(II) concentration in solution is illustrated on FIG. 4.

Example 8

Barrier Activity of Glutathione Modified Chitosan Against Metal Ions, in Particular Nickel(II) Ions

[0069] In a vessel composed of three elements, designed for the purpose of this experiment and made with a 3D printer, two layers of dialysis membrane were mounted. The vessel was placed in a 25 ml beaker containing 10 ml of deionized water and a magnetic stirrer. The setup was placed on a magnetic stirrer and used as control experiment. In two further identical vessels 81 mg of commercially available chitosan or 81 mg of glutathione modified chitosan according to the invention was placed between the membrane layers. 0.5 ml of a 100 mM solution of nickel(II) chloride was placed in the inner cylinder of each vessel, and the vessels were placed in 25 ml beakers containing 10 ml of deionized water each. All setups were stirred for 24 hours, thus allowing for diffusion of Ni.sup.2+ ions across the dialysis membranes and across the layer of chitosan or modified chitosan present between the membranes, respectively. Then, the Ni.sup.2+concentrations present in water solutions in each of the beakers were determined. Both polymers demonstrated barrier action with the metal ion concentration detected nearly 8 times lower than that in the control. The results are presented in Table 1. The results in the last column of Table 1 were calculated on the basis of reaction yield Y=30% given in Example 4.

TABLE-US-00001 TABLE 1 Results of spectrophotometric assay for concentrations of Ni.sup.2+ ions in solution. Ni.sup.2+ Ni.sup.2+ in Ni.sup.2+ per Test Absorption concentration in solution vs. polymer unit no. at 465 nm solution [mM] control [mol/mol] Control 1 0.62 4.8 100% 0 2 0.62 4.8 100% 0 Biopolymer 1 0.03 0.6 13% 0.092 (0.455 mmol) 2 0.03 0.6 13% 0.092 Modified 1 0.07 0.8 17% 0.131 biopolymer 2 0.07 0.8 17% 0.131 (0.306 mmol) Blank 1 0.00 0.00 0% 0
The above Table 1 clearly shows that the presence of the chitosan derivative causes a significant reduction of diffusion of Ni.sup.2+ ions to solution and the chitosan derivative is over 40% more potent than chitosan itself in capturing Ni.sup.2+. A cosmetic composition containing the chitosan derivative according to the invention as active component has analogous properties, limiting the access of sensitizing ions after placing the composition on the skin.

Examples of Industrial Applications of the Invention

[0070] A use of a new agent (glutathione modified chitosan) provides for effective and simple recovery of metals from water solutions. The use of any desired modifier molecule of chitosan provides an opportunity for controlling the metal chelation properties of the biopolymer, while preserving its biocompatibility and nontoxicity.

[0071] The use of chitosan or other biocompatible biopolymer susceptible for attaching modifier molecules, such as of antibiotics used for treatment of dermatitis provides a basis for obtaining new materials for dermatological use.

[0072] Peptide LL-37 used broadly in the cosmetic industry as antimicrobial agent, lactobionic acid helpful in wound healing and combating juvenile acne, and p-aminobenzoic acid used for UVB photoprotection can be listed as such modifiers.