CN102558388B - 6-amine-substituted-6-deoxidizing chitosan derivant and preparation method thereof - Google Patents
6-amine-substituted-6-deoxidizing chitosan derivant and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technology of ocean chemical engineering, in particular to a 6-amine-substituted-6-deoxidizing chitosan derivant and a preparation method thereof. As shown in the general formula I, an amino compound can be introduced at the 6 position of chitosan, so that the derivant is strong in area selectivity. By introducing toxic or non-toxic amino-compounds on the chitosan, remarkable effects for improving anti-bacteria effect of the chitosan can be achieved, and the production using process of the derivant is led to be low in toxicity and environment-friendly. The formula I is shown in the description, wherein m=0-20; and n=4-4000.
Description
Technical field
The invention belongs to marine chemical industry engineering, be specifically related to a kind of novel 6-amine replacement-6-deoxy-chitosan derivative and preparation method thereof.
Background technology
Along with social development, market, to antibacterials, comprises that the demand of veterinary drug and fodder additives, agricultural chemicals, anti-biotic material, food preservative etc. is increasing.Microbiotic is because the features such as cost is low, output large, good anti-bacterial effect are widely used.But antibiotic abuse causes the generation of drug-resistant microorganism, and cause drug residue contaminate environment, harm humans is healthy.Therefore developing green novel antibacterial medicine becomes study hotspot both domestic and external.
Chitosan (chitosan) also claim chitosan, is the product of de-acetyl chitin, and chemical name is poly-(Isosorbide-5-Nitrae glycosides)-2-amino-beta--D-Glucose.It is extensively present in the shell of the Crustacean such as shrimp, crab and insect, and the cell walls of fungi, is a kind of very abundant natural resources.Chitosan has nontoxic, and the characteristic such as biodegradable and good biocompatibility, is all having been widely used aspect chemical industry, agricultural, food, biotechnology, environment protection and makeup.Chitosan has certain restraining effect to bacterium, fungi etc. under acidic conditions, but because its solvability under the condition of pH neutrality is poor, and the bacteriostatic activity of itself is not strong, so that its be directly restricted as the use of antiseptic-germicide.Therefore according to different principle, chitosan is carried out to chemically modified and become the focus of Recent study to strengthen its anti-microbial activity.
It is many that to contain amino polymkeric substance positively charged under neutral and acid pH, can be by combining and make lysis with electronegative microorganism cells film, or with microorganism cells in electronegative material if DNA, RNA etc. are in conjunction with the metabolism of interference cell, thereby performance sterilization, press down mould and antiviral effect.Wherein, polyhexamethylene guanidine (PHMG) as commodity list marketing, has purposes extremely widely as environment-friendly type macromolecule polymkeric substance disinfection sanitizer in industrial, agriculture, medical and daily life.We are using chitosan as skeleton, and selectivity is substituted by 6 hydroxyls containing aminocompound, to obtaining efficient broad spectrum antibiotic.Yet there are no the report about 6-amine replacement-6-deoxy-chitosan derivative.
Summary of the invention
The object of the present invention is to provide a kind of 6-amine replacement-6-deoxy-chitosan derivative and preparation method thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of 6-amine replacement-6-deoxy-chitosan derivative, its general formula is suc as formula shown in I,
Formula I
In formula I, m=0-20; N=4-4000.
The preparation method of 6-amine replacement-6-deoxy-chitosan derivative, 1) the nucleophilic substitution reaction 12h-48h at 50-100 ℃ by bromo-6-6-'-deoxy-n-phthalyl chitosan and aminocompound, obtain suc as formula the intermediate shown in II, wherein, the mass volume ratio of the bromo-6-'-deoxy-n-phthalyl chitosan of 6-and solvent is 1: 10-80 is stand-by;
In formula II, m=0-20; N=4-4000;
2) by above-mentioned gained intermediate in N-Methyl pyrrolidone take 80-100 ℃ with concentration the hydrazine hydrate action-reaction 1h-4h as 80%, after reactant washing, obtain chitosan derivatives shown in general formula I; The mass volume ratio of described intermediate and solvent is 1: 20-80; The volume ratio of solvent and hydrazine hydrate is 1: 0.5-2.
Described step 1) in solvent be that aminocompound or aminocompound and water make mixed solvent, the wherein volume ratio 100 of aminocompound and water: 0-100.
Described aminocompound is quadrol, diethylenetriamine, triethylene tetramine or polyethylene polyamine.Step 1) gained intermediate, solution after nucleophilic substitution reaction is poured into ethanol, Virahol or acetone, 4 ℃ staticly settle, decompress filter gained solid precipitates with ethanol, centrifugal, after supernatant discarded, add washing with alcohol, more centrifugal, repeatedly 3-5 all over after, the lyophilize of gained solid obtains target compound.
Principle: 6-amine replaces in-6 deoxy-chitosan structures has increased amino content, easily protonated under acidity or neutrallty condition, can improve the water-soluble of derivative, and contribute to improve the ability to function of derivative and microorganism negative charge, significantly improve the biological activity of derivative; Derivative of the present invention will be under the protected condition of 2 bit amino, and 6 of Selective activations, then carry out amido replacement by nucleophilic substitution reaction to 6, finally slough 2 blocking groups, obtain target compound.In the present invention, first synthesize the bromo-6-deoxidation-chitosan derivatives of 6-take NBS as bromide reagent selectivity; 6 carbon that amino on aminocompound replaces chitosan bromine carry out nucleophilic substitution; finally with hydrazine hydrate, 2 blocking groups of chitosan are sloughed, obtained 6-amine and replace-6 deoxy-chitosans.The derivative of gained shows that through Infrared spectroscopy chitosan is combined effectively with the group of access, and original blocking group removes completely, generates 6-amine and replaces-6 deoxy-chitosans.
Advantage of the present invention:
1. the present invention can introduce containing aminocompound on 6 of chitosans, and regioselectivity is strong.
2. the present invention, by introduce the aminocompound of nontoxic or low toxicity on chitosan, reaches the effect of remarkable enhancing chitosan fungistatic effect, makes the equal low toxicity of production and application process, the environmental protection of derivative.
3. chitosan derivatives of the present invention has good solvability, has potential using value in fields such as agricultural chemicals, veterinary drug, makeup, sterilizing agents.
Accompanying drawing explanation
The infrared spectrogram of the chitosan that Fig. 1 provides for the embodiment of the present invention, its infrared signature absorbs (cm
-1): 3370.71,2877.24,1654.21,1596.40,1423.54,1379.86,1319.02,1154.84,1075.22,895.68,568.81.
The infrared spectrogram of preparing the bromo-6-'-deoxy-n-phthalyl of important intermediate 6-chitosan in chitosan derivatives that Fig. 2 provides for the embodiment of the present invention, its infrared signature absorbs (cm
-1): 3444.84,2889.92,1777.59,1711.21,1468.04,1417.30,1390.64,1062.27,872.35,796.39,721.09,663.79,528.0.
The infrared spectrogram of the chitosan derivatives 1 that Fig. 3 provides for the embodiment of the present invention, its infrared signature absorbs (cm
-1): 3366.36,2868.27,1652.11,1583.70,1460.73,1421.66,1377.64,1324.29,1074.68,665.38.
The infrared spectrogram of the chitosan derivatives 2 that Fig. 4 provides for the embodiment of the present invention, its infrared signature absorbs (cm-1): 3425.93,2872.43,1653.678,1455.87,1310.54,1064.27.
The infrared spectrogram of the chitosan derivatives 3 that Fig. 5 provides for the embodiment of the present invention, its infrared signature absorbs (cm-1): 3370.93,2868.65,1656.37,1460.75,1374.99,1063.56.
Embodiment
Below in conjunction with Figure of description, the invention will be further described, and protection scope of the present invention is not only confined to following examples.
The preparation method of derivative: chitosan is being protected to 2 NH with Tetra hydro Phthalic anhydride in dimethyl formamide (DMF)
2prerequisite under carry out 6 bromination reactions, temperature of reaction is 100-130 ℃, the reaction times is 5-10 hour; React after product and poured in frozen water, filtered, by enough water and washing with alcohol filter cake post-drying, obtained N-phthalyl chitosan.Wherein the mass volume ratio of chitosan and DMF is 1: 20-100, chitosan is 1 with the mol ratio of reacting of Tetra hydro Phthalic anhydride: 3-5.Above-mentioned N-phthalyl chitosan derivatives is dispersed in N-Methyl pyrrolidone (NMP); under ice-water bath and nitrogen protection, add respectively N-bromo-succinimide (NBS) and triphenylphosphine (TPP), then 50-100 ℃ of reaction 1-4 hour.Products therefrom precipitates with ethanol, and centrifugal supernatant discarded is also used ethanol repetitive scrubbing, finally dries and obtains suc as formula the bromo-6-'-deoxy-n-phthalyl of 6-shown in chitosan.Wherein the mass volume ratio of N-phthalyl chitosan and NMP is 1: 100-300, N-phthalyl chitosan is 1 with the mol ratio of reacting of NBS: 5-15, N-phthalyl chitosan is 1 with the mol ratio of reacting of TPP: 5-15.(the bromo-6-'-deoxy-n-phthalyl of 6-chitosan also can be referring to preparing with Publication about Document
Satoh，T.，et al.，6-Amino-6-deoxy-chitosan.Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier.Carbohydrate Research，2006.341(14)：p.2406-2413.)
Formula one
Bromo-gained 6-6-'-deoxy-n-phthalyl chitosan is distributed in the mixing solutions of aminocompound or aminocompound and water, and the mass volume ratio of the bromo-6-'-deoxy-n-phthalyl chitosan of 6-and mixing solutions is 1: 10-80; When mixing solutions is aminocompound and water, between aminocompound and water, volume ratio is 100: 0-100; Temperature of reaction is 50-100 ℃; Reaction times is 12h-48h.After having reacted, solution is poured into ethanol, Virahol or acetone, 4 ℃ staticly settle, and decompress filter gained solid fully rinses post-drying with ethanol, obtains intermediate.
Intermediate is removed 2 blocking groups through hydrazine hydrate effect in N-Methyl pyrrolidone.The mass volume ratio of intermediate and N-Methyl pyrrolidone is 1: 20-80; N-Methyl pyrrolidone and concentration are that the volume ratio of 80% hydrazine hydrate is 1: 0.5-2; Temperature of reaction is 80-100 ℃; Reaction times is 1h-4h.Reacted rear by ethanol precipitation, centrifugal, after supernatant discarded, added washing with alcohol, more centrifugal, repeatedly 3-5 all over after, the lyophilize of gained solid, obtains target compound.Described target compound molecular weight is 1000-700kDa.Oven temperature is 50 ℃, and lyophilize temperature is-50 ℃.
The preparation of embodiment 1 derivative 1
1.0 grams of bromo-6-'-deoxy-n-phthalyl of 6-chitosans, 26ml quadrols are put into there-necked flask, and 80 ℃ are reacted 24 hours, and reactant is poured in 50ml Virahol, and gained precipitation is used ethanol repetitive scrubbing, and centrifugal postlyophilization, obtains intermediate.Get 0.5g intermediate and add in there-necked flask, then add 25mlNMP and 25ml hydrazine hydrate, 100 ℃ are reacted 4 hours, and reaction product precipitates with ethanol, centrifuge washing repeatedly, and lyophilize obtains derivative 1, and structural formula is referring to table 1.
Infrared spectra shows: the infrared spectrum (Fig. 3) of chitosan derivatives 1 is compared with the infrared spectrum (Fig. 1) of chitosan, and primary amine groups N-H stretching vibration peak broadening, makes 2889.92cm
-1the C-H stretching vibration peak at place becomes acromion; 1583.70cm simultaneously
-1the N-H of place in-plane bending vibration intensity enhancing, illustrates on chitosan skeleton and has extra amino to increase.1468.04cm
-1the absorption peak of the medium tenacity at place is-CH
2-characteristic peak.Above-mentioned infrared spectrum changes proves that target derivative 1 synthesizes successfully.
The preparation of embodiment 2 derivatives 2
By the 1.0 grams of bromo-6-'-deoxy-n-phthalyl of 6-chitosans, 26ml diethylenetriamine, 10ml distilled water is put into there-necked flask, and 80 ℃ are reacted 48 hours, reactant is poured in 50ml Virahol, gained precipitation is used ethanol repetitive scrubbing, and centrifugal postlyophilization, obtains intermediate.Get 0.5g intermediate and add in there-necked flask, then add 25mlNMP and 25ml hydrazine hydrate, 100 ℃ are reacted 4 hours, and reaction product precipitates with ethanol, centrifuge washing repeatedly, and lyophilize obtains derivative 2, and structural formula is referring to table 1.
Infrared spectra shows: the infrared spectrum (Fig. 4) of chitosan derivatives 2 compared with the infrared spectrum (Fig. 1) of chitosan, 3425.93 and 2872.43cm
-1place's primary amine groups N-H stretching vibration peak and hydrocarbon stretching vibration peak-to-peak signal further strengthen, 1455cm
-1place occurs that the absorption peak of medium tenacity is CH
2absorption peak prove derivative 2 synthesize successfully.
The preparation of embodiment 3 derivatives 3
By the 1.0 grams of bromo-6-'-deoxy-n-phthalyl of 6-chitosans, 26ml triethylene tetramine, 10ml water is put into there-necked flask, and 80 ℃ are reacted 48 hours, reactant is poured in 50ml Virahol, gained precipitation is used ethanol repetitive scrubbing, and centrifugal postlyophilization, obtains intermediate.Get 0.5g intermediate and add in there-necked flask, then add 25ml NMP and 25ml hydrazine hydrate, 100 ℃ are reacted 4 hours, and reaction product precipitates with ethanol, centrifuge washing repeatedly, and lyophilize obtains derivative 3, and structural formula is referring to table 1.
Infrared spectra shows: the infrared spectrum (Fig. 5) of chitosan derivatives 3 compared with the infrared spectrum (Fig. 1) of chitosan, 3370.93 and 2868.65cm
-1place's primary amine groups N-H stretching vibration peak and hydrocarbon stretching vibration peak-to-peak signal further strengthen, 1460cm
-1place occurs that the absorption peak of medium tenacity is CH
2absorption peak prove derivative 3 synthesize successfully.
The structure of table 1 general formula I derivative
Formula I
| | m | |
| 1 | 0 | |
| 2 | 1 | |
| 3 | 2 |
Antibacterial Activity: adopt the minimal inhibitory concentration (MIC) of micro-broth dilution method sample to intestinal bacteria and streptococcus aureus.
Above-described embodiment of getting respectively 1024 μ g/mL is prepared the each 50 μ l of gained derivative storing solution and is put into the 96 front holes of orifice plate, and doubly rare to the 11st hole with MH broth culture etc. since the second hole, the 12nd hole adds 100 μ l substratum as blank.The 1st hole to the 12 hole liquor strengths are respectively 1024,512, and 256,128,64,32,16,8,4,2,1 μ g/mL and blank 0 μ g/mL.To be diluted to about 1-2 × 10 at the bacteria suspension of logarithmic phase with MH broth culture
6cFU/mL.Above-mentioned bacteria suspension is got 50 μ l and is joined in the liquid of 96 orifice plates, and the final concentration that makes 1 to 12 hole liquid is 512,256,128,64,32,16,8,4,2,1,0.5,0 μ g/mL.With molecular weight 230,000, the chitosan of deacetylation 80% and polyhexamethylene guanidine are as positive control.
Experimental result is in table 2
| Sample | MIC Intestinal bacteria(μg/mL) | MIC Streptococcus aureus(μg/mL) |
| |
16 | 32 |
| |
8 | 16 |
| |
16 | 16 |
| |
8 | 4 |
| Chitosan | ＞1000 | ＞1000 |
Claims (4)
1. a 6-amine replacement-6-deoxy-chitosan derivative, is characterized in that, its general formula is suc as formula shown in I,
In formula I, m=0-20; N=4-4000.
2. a preparation method for 6-amine replacement-6-deoxy-chitosan derivative claimed in claim 1, is characterized in that,
1) the nucleophilic substitution reaction 12h-48h at 50-100 ℃ by bromo-6-6-'-deoxy-n-phthalyl chitosan and aminocompound, obtain suc as formula the intermediate shown in II, wherein, the mass volume ratio of the bromo-6-'-deoxy-n-phthalyl chitosan of 6-and solvent is that 1:10-80 is stand-by;
In formula II, m=0-20; N=4-4000;
2) by above-mentioned gained intermediate in N-Methyl pyrrolidone with 80-100 ℃ with hydrazine hydrate action-reaction 1h-4h, after reactant washing, obtain chitosan derivatives shown in general formula I; The mass volume ratio of described intermediate and solvent is 1:20-80; The volume ratio of solvent and hydrazine hydrate is 1:0.5-2.
3. by the preparation method of 6-amine replacement-6-deoxy-chitosan derivative claimed in claim 2, it is characterized in that, in described step 1), solvent is that aminocompound or aminocompound and water make mixed solvent, the wherein volume ratio 100:0-100 of aminocompound and water.
4. by the preparation method of 6-amine replacement-6-deoxy-chitosan derivative claimed in claim 3, it is characterized in that, described aminocompound is quadrol or polyethylene polyamine.
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