CN103880981A - Method for synthesizing phosphorylated chitin by taking methanesulfonic acid as solvent - Google Patents

Abstract

The invention discloses a method for synthesizing phosphorylated chitin by taking methanesulfonic acid as solvent, and belongs to the field of biochemical engineering. The method comprises: weighing phosphorus pentoxide in a dry state, mixed with methanesulfonic acid and dissolving for 1-2 h under the state of continuous stirring, so as to prepare Eaton reagent, wherein the mass ratio of methanesulfonic acid to phosphorus pentoxide is 8:1-14:1; uniformly mixing chitin with the Eaton reagent according to a chitin-to-Eaton reagent mass ratio of 1:6-1:18, continuing to stir and react at 0-5 DEG C for 2-3 h under protection of nitrogen; adding diethyl ether to perform precipitation, centrifuging, then successively washing the precipitate with acetone and methanol; dissolving the precipitate with deionized water, and performing turbid liquid dialysis, vacuum concentration and freeze drying to obtain the product. The preparation method for the chitin phosphorylation derivative is simple in operation, high in yield, small in environment pollution and wide in application. The obtained product is directly dissolvable in water and is widely applicable to fields such as agriculture, textile, medicines, foodstuff, cosmetic, environment protection and the like.

Description

A kind of method of synthesizing phosphorylation chitin take methylsulphonic acid as solvent

Technical field

The invention belongs to biological chemical field, be specifically related to a kind of chitin is carried out to phosphorylation modification make its soluble preparation method in water.

Background technology

Chitin (Chitin) has another name called chitin, chitin, chemistry by name (1,4)-2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-callose, with β-1, the linear biopolymer that 4 glycosidic links are connected, can from crustacean shell and fungal cell wall, extract, be to be only second to cellulosic second largest renewable natural biological polymer.But chitin due in molecule and intermolecular hydrogen bond action be insoluble in usual vehicle, this has limited further applying of chitin to a great extent.Water-soluble in order to increase it, conventionally chitin deacetylase is prepared to chitosan.Chitosan could dissolve under acidic conditions, has limited its range of application.And in the process of de-acetyl, remaining concentrated base can produce larger pollution to environment.

Phosphorus is the important element composition of human body, has extremely important effect in numerous vital movements.At present, in vital process, also do not find not phosphorous life entity.Why phosphorus is the important element in vital process, is because the catalyzed reaction of most enzymes is all to regulate and control with their phosphorylation and dephosphorylation.The required basic storage element ATP of organism has participated in almost each reaction in life chemistry process, and metabolic processes is often carried out take phosphorous derivant as intermediate.Between hereditary material DNA in organism and the nucleosides of RNA, connect by phosphodiester bond.Chitin is a kind of emerging Biofunctional materials, and it has been applied to, and sclerotin substitutes, organizational project and gene and pharmaceutical carrier etc. and life science and the closely bound up field of medical science.In recent years, researchist progressively recognizes phosphorus is attached to the derivative forming in chitin structure in some the special using value aspect life science and medical science.This may be also the distinctive feature that phosphorylation method of modifying is different from other modifying and decorating method.Especially the N,O-CMC after phosphorylation has been obtained breakthrough progress in the application of the aspects such as bone integration and organizational project, and this is that other water-solubility chitin derivative is too far behind to catch up.

Summary of the invention

For prior art exist deficiency, the invention provides a kind of simple to operate, productive rate is high, environmental pollution is little, the preparation method of widely used chitin phosphorylated derivative.In reaction, methylsulphonic acid is not only the good solvent of chitin, is also the effective catalyst of this phosphorylation reaction.The method not only efficiency is high, and can also obtain the product that substitution value is higher.

For achieving the above object, the technical solution used in the present invention is:

1) preparation of Eaton reagent: methylsulphonic acid is placed in the flask with calcium chloride tube.Under drying regime, take Vanadium Pentoxide in FLAKES, constantly stir lower and methylsulphonic acid mixed dissolution 1-2 hour.The mass ratio of methylsulphonic acid and Vanadium Pentoxide in FLAKES is 8:1-14:1.Reagent is as do not used immediately, and when being stored in tool plug bottle, life-time service is understood flavescence, but can obviously not affect the validity of reagent.

2) reaction: dried chitin is pulverized, crossed 60-100 mesh sieve.Under the protection of nitrogen, chitin dry powder is dissolved in Eaton reagent to 0-5 ℃ of lasting stirring reaction 2-3 hour.The mass ratio of chitin dry powder and Eaton reagent is 1:6-1:18.The substitution value that can improve by slowly add Vanadium Pentoxide in FLAKES in reaction system final product, Vanadium Pentoxide in FLAKES interpolation quality is 0.2-0.6 times of chitin dry powder.

3) separate: to reaction system add ether sedimentation, centrifugal after, more successively by acetone and methanol wash precipitation.Then use deionized water dissolution precipitation.Turbid liquid is that 3500-8000 dialysis tubing dialysis final vacuum is concentrated through molecular weight cut-off, and lyophilize obtains product.Organic solvent reclaims by rectifying.

4) mensuration of phosphorylation chitin substitution value: use the digestion of sulfuric acid-nitrate mixture to destroy organic substance, and phosphate transfection is turned to orthophosphoric acid salt.Under acidic conditions, the ammonium phosphomolybdate that orthophosphoric acid salt and ammonium molybdate reaction generate, by reductive agent effect, forms " molybdenum blue ", the light absorption value by spectrophotometric determination blueness at 825nm wavelength.Read corresponding phosphorus content from typical curve.The substitution value (Degree of substitution, DS) of phosphate radical is defined as the number of phosphate radical contained in each acetylglucosamine unit, and calculation formula is as follows:

$\mathrm{DS}=\frac{203\·\left[P\right]}{31-80\·\left[P\right]}$

DS in formula---substitution value [P] of the sample---phosphorus content of sample

203---molecular weight 31 of chitin unit---molecular weight of phosphorus atom

80---the increasing amount of molecular weight after a phosphate group replaces

The main chemical reactions formula the present invention relates to is as follows:

Compared with prior art, the present invention has advantages of following:

1, the inventive method is simple to operation, productive rate is high, environmental friendliness, is applicable to suitability for industrialized production.

2, products therefrom substitution value is high.Modification group phosphate radical has certain surge capability, makes product keep dissolved state within the scope of very wide pH value.Phosphate radical also has very strong sequestering power simultaneously.

3, employing the inventive method can be prepared phosphorylation chitin dry powder that can be directly water-soluble, and products therefrom can be widely used in the fields such as agricultural, weaving, medicine, food, makeup, environment protection.

Accompanying drawing explanation

Fig. 1 is the infrared spectra comparison diagram of chitin and embodiment 1 gained phosphorylation chitin, and the infrared spectrogram that wherein (a) is chitin (b) is the infrared spectrogram of embodiment 1 gained phosphorylation chitin.For chitin, 3500-3400cm-1 is V _oH(stretching vibration of hydroxyl-OH) absorption band, 3265cm-1 is V _nH(stretching vibration of amino-NH) absorption band; 2890cm-1 is V _cH2(the upper methyne-CH2 of sugar ring-symmetrical stretching vibration) absorption band; 1661cm-1 is V _c=O(carbonyl in amido linkage) absorption band, 1558cm-1 is δ _nH(amino in amido linkage) absorption band.After chitin phosphorylation, disappear at the absorption peak at 1310cm-1 place.Occur strong peak at 524cm-1, this is the feature flexural vibration absorption band of P-O key.The Infrared spectroscopy of chitin and phosphorylation chitin shows that phosphate group closes with the acetylglucosamine loops of chitin.

Embodiment

Further illustrate the present invention below in conjunction with embodiment.

Embodiment 1:

2g chitin and 14ml Eaton reagent mix are even.Under the protection of nitrogen, 2 ℃ of lasting stirring reactions 2 hours.Add 70ml ether sedimentation, centrifugal.Use successively again 42ml acetone, 42ml methanol wash.Then by 50ml deionized water dissolving precipitation.Turbid liquid is that 3500 dialysis tubing dialysis final vacuums are concentrated through molecular weight cut-off, is dried to obtain product.Recording phosphorylation chitin substitution value is 0.71.

Embodiment 2:

3g chitin and 20ml Eaton reagent mix are even.Under the protection of nitrogen, 4 ℃ continue to stir, and slowly add Vanadium Pentoxide in FLAKES 1.5g, react 3 hours.Add 100ml ether sedimentation, centrifugal.Use successively again 60ml acetone, 60ml methanol wash.Again by 80ml deionized water dissolving precipitation.Turbid liquid is that 6000 dialysis tubing dialysis final vacuums are concentrated through molecular weight cut-off, is dried to obtain product.Recording phosphorylation chitin substitution value is 1.15.

Claims (6)

1. a method for synthetic phosphorylation chitin take methylsulphonic acid as solvent, is characterized in that comprising the following steps:

1) preparation of Eaton reagent: methylsulphonic acid is placed in flask; Under drying regime, take Vanadium Pentoxide in FLAKES and add in flask, constantly stir lower and methylsulphonic acid mixed dissolution 1-2 hour; The mass ratio of methylsulphonic acid and Vanadium Pentoxide in FLAKES is 8:1-14:1;

2) reaction: dried chitin is pulverized, crossed 60-100 mesh sieve, get the component by sieve aperture, under the protection of nitrogen, chitin dry powder is dissolved in Eaton reagent to 0-5 ℃ of lasting stirring reaction; The mass ratio of chitin dry powder and Eaton reagent is 1:6-1:18;

3) separate: to reaction system add ether sedimentation, centrifugal after, more successively by acetone and methanol wash precipitation; Then use deionized water dissolution precipitation; Turbid liquid dialysis final vacuum is concentrated, and concentrated solution lyophilize obtains product.

2. it is characterized in that in accordance with the method for claim 1:

Described in step 1), flask is the flask with calcium chloride tube.

3. it is characterized in that in accordance with the method for claim 1:

Step 2) under the protection of nitrogen, continuing the stirring reaction time is 2-3 hour.

4. it is characterized in that in accordance with the method for claim 1:

Step 2) can improve by add Vanadium Pentoxide in FLAKES in reaction system the substitution value of final product in reaction process, Vanadium Pentoxide in FLAKES add quality be chitin dry powder 0.2-0.6 doubly.

5. it is characterized in that in accordance with the method for claim 1:

It is 3500-8000 that step 3) dialysis procedure adopts the molecular weight cut-off of dialysis tubing.

6. it is characterized in that in accordance with the method for claim 1:

Adopt phosphorus molybdenum blue spectrophotometric method to measure the phosphorus content of product, be converted into the substitution value of phosphorylation chitin.