CN111040003B - Chitosan oligosaccharide derivative molecular imprinting functional monomer and preparation method thereof - Google Patents

Abstract

A chitosan oligosaccharide derivative molecularly imprinted functional monomer and a preparation method thereof relate to the field of chemical technology, in particular to the design and production process of the molecularly imprinted functional monomer. Dissolve asparagine, glutamine, methionine or threonine neutral amino acid and potassium carbonate in water, add pentenoyl chloride dropwise under ice-water bath conditions, and then react at room temperature to prepare N-pentenoyl- Amino acid solution; adjust the pH value of the N-pentenoyl-amino acid solution to neutral, then mix it with chitosan, EDC, and NHS for reaction, then use a dialysis bag for dialysis and freeze-drying to obtain N-(N'- pentenoyl-amino acid acyl)-chitooligosaccharide. The invention has simple synthesis reaction, mild reaction conditions, high yield, can save the preparation cost, and can obtain more effective recognition sites for the imprinted polymer.

Description

A kind of chitosan oligosaccharide derivative molecularly imprinted functional monomer and preparation method thereof

technical field

The invention relates to the technical field of chemical engineering, in particular to the design and production process of molecularly imprinted functional monomers.

Background technique

Molecular imprinting technology refers to the technology of preparing polymers with selective recognition ability for a specific molecule (also known as template molecule). The principle is: in a suitable medium, template molecules and functional monomers with appropriate functional groups form some kind of host-guest complex, add crosslinking agent, initiator, after polymerization is initiated, the template molecules will be imprinted to the polymerization in things. When the template molecule is removed, a hole with multiple action sites matching the spatial configuration of the template molecule is formed in the polymer. Such a hole has selective recognition properties for the template molecule and its analogues. Therefore, this polymer can also be called artificial antibody. In recent years, molecular imprinting technology has been widely used in many fields such as chromatographic separation, solid phase extraction, drug analysis, biosensor technology, and catalytic synthesis, and thus it has become one of the new fields intersecting chemistry and biology. The application prospect has been developed rapidly.

It is very important to select suitable functional monomers in molecularly imprinted polymers. On the one hand, the functional monomers must participate in the polymerization reaction of the crosslinking agent, that is, the grid structure formed by polymerization produces a certain spatial structure suitable for template molecules. On the other hand, the functional monomer has a sufficiently strong interaction with the template molecule to make the polymer generate a specific recognition site for the template molecule. However, because the polymer network structure is difficult to deform, only the polymer formed by the functional monomer with small steric hindrance of the recognition group can allow template molecules to enter the imprinted cavity. The strong molecular interactions between host and guest molecules are mainly ionic bonds and hydrogen bonds, so the current choice of functional monomers is mainly simple small molecule polar compounds containing alkenyl and polar groups, such as acrylic acid, Acrylamide, Vinylpyridine, etc.

Theoretically, the higher the specificity of the functional monomer to the template molecule, the stronger the specificity of the molecularly imprinted polymer to the template molecule recognition. Therefore, in order to improve the specificity of the functional monomer to the template molecule, it is necessary to use a functional monomer whose molecular structure matches the template molecule better. Obviously, this functional monomer is sterically more complex than a simple polar group, so the steric hindrance is greater. Therefore, to allow the template molecules to enter the imprinted cavity of the polymer smoothly, the grid space of the polymer must be enlarged.

Oligochitosan, also known as chitosan oligosaccharide and oligochitosan, is an oligosaccharide product composed of β-1,4 glycosidic bonds with a degree of polymerization between 2 and 20 obtained by degrading chitosan, with a molecular weight of ≤3200Da , the molecular structure is rich in hydroxyl and amino groups. Since its amino group is evenly distributed on both sides of the glucose structural unit, if a double bond can be introduced into the amino group, the polymer formed will have a larger grid structure.

Since chitosan molecules have a large number of polar groups, they can be used as functional monomer oligomers in molecularly imprinted materials. However, due to the rigidity of their molecular structure, it is obviously difficult to achieve specific structures. The template molecules form a good match and a stronger interaction occurs. In order to improve this situation, it is necessary to introduce some kind of flexible structure on the chitosan molecular structure.

In the process of antibody recognition of antigen, the antibody effectively recognizes the corresponding part of the antigen by using the amino acid residues of specific parts. Therefore, if a specific amino acid unit can be introduced into molecularly imprinted polymers, and the flexibility of its residues can be used to assist in the recognition of template molecules, the recognition performance of molecularly imprinted polymers for template molecules will definitely be improved. After searching, the construction of this kind of functional monomer has not been reported yet.

Contents of the invention

Aiming at the deficiency that the molecular structure of the functional monomer cannot match well with the template molecule in the current molecular imprinting technology, the present invention proposes a chitosan oligosaccharide derivative molecular imprinting functional monomer that has a strong effect on the curcumin molecule.

The chitosan oligosaccharide derivative molecularly imprinted functional monomer referred to in the present invention is N-(N'-pentenoyl-amino acid acyl)-chitosan oligosaccharide.

Its molecular structure general formula is as follows:

Where n=3-10, R is a side chain group of a neutral amino acid, and the neutral amino acid is asparagine, glutamine, methionine or threonine.

The invention uses curcumin molecules as template molecules, chitosan oligosaccharides as carriers, and basic structural units of functional monomer oligomers as recognition molecules.

Since the formed small peptide chain structure (-CONH-CH(R)-CONH-) also presents a certain degree of rigidity, it is spatially intersected with the rigid chitosan molecular chain, which is beneficial to Maintain the shape of the grid structure (it will not be deformed due to the increase of the grid), which is conducive to maintaining the permeability of the imprinted material, allowing more template molecules to enter, so that the imprinted polymer can be more effectively recognized site.

Another object of the present invention is to propose a method for preparing the molecularly imprinted functional monomer of the chitosan oligosaccharide derivative.

The preparation method of the present invention comprises the following steps:

1) Dissolving neutral amino acid and potassium carbonate in water, adding pentenoyl chloride dropwise in an ice-water bath, and reacting at room temperature after the dropwise addition, to obtain N-pentenoyl-amino acid solution; the neutral amino acid is asparagine, glutamine, methionine or threonine;

2) Adjust the pH of the N-pentenoyl-amino acid solution to neutral and mix with chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) Mix and react with N-hydroxysuccinimide (NHS) to generate N-(N'-pentenoyl-aminoacyl)-chitooligosaccharide solution;

3) The reaction solution obtained in step 2) is dialyzed with a dialysis bag, and the dialysate is freeze-dried to obtain N-(N'-pentenoyl-amino acid acyl)-chitooligosaccharide.

Reaction formula of the present invention is as follows:

Wherein n=3～6, R is the side chain group of neutral amino acid.

The advantages of the present invention are:

(1) The pentenoyl group is introduced into the amino group of a neutral amino acid, and when the pentenoyl amino acid is coupled to the amino group of the chitosan molecule, a double bond and a flexible amino acid side chain can be simultaneously introduced into the chitosan molecule group, and will not greatly reduce the water solubility of chitosan oligosaccharides due to the pentenoyl group.

(2) The synthesis reaction is simple, the reaction conditions are mild, the yield is high, and the reaction can be quantified.

(3) The use of pentenoyl chloride as the reaction raw material only requires two steps of reaction, and chitosan oligosaccharide can be coupled without separation after the first step of reaction, which can greatly save the preparation cost.

Further, the molar ratio of neutral amino acid and pentenoyl chloride in the present invention is 1:1.05-1.10. Due to the higher activity of the acid chloride (can react with water), in order to ensure that the amino acid can all participate in the reaction, the molar ratio of the pentenoyl chloride in the present invention is appropriately excessive.

Description of drawings

Figure 1 is the infrared spectrum of N-(N'-pentenoyl-glutaminyl)-chitooligosaccharide.

Fig. 2 is a comparative graph of cyclic voltammetry of a clean glassy carbon electrode, a molecularly imprinted electrode before curcumin elution, a molecularly imprinted electrode after curcumin elution, and a molecularly imprinted electrode after re-adsorbing curcumin.

Fig. 3 is a comparison chart of the decrease value of the cyclic voltammetry current response of the molecularly imprinted electrode to curcumin, tetrahydrocurcumin, ferulic acid, carotene and quercetin after curcumin was eluted.

Detailed ways

1. Taking N-(N′-pentenoyl-glutaminyl)-chitooligosaccharide as an example, the synthesis steps are as follows.

1. Dissolve 0.125g (1.05mmol) of pentenoyl chloride in 2mL of anhydrous N,N-dimethylformamide (DMF) for later use. Weigh 0.146g (1.0mmol) of glutamine and 0.138g (1.0mmol) of potassium carbonate, add an appropriate amount of water to dissolve, cool in an ice-water bath to 0°C, then add the DMF solution of pentenoyl chloride drop by drop, and continue the reaction for 1h. A solution of N-pentenoyl glutamine was prepared.

2. Adjust the pH of the N-pentenoyl glutamine solution to neutral with 3N hydrochloric acid, add 0.17g chitosan oligosaccharide (can be dissolved in a small amount of water in advance), 0.201g (1.05mmol) 1-(3-dimethylamino Propyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.121g (1.05mmol) N-hydroxysuccinimide (NHS), reacted at room temperature for 24h, and obtained enoylglutaminyl)-chitooligosaccharide reaction solution.

3. Transfer the reaction solution to a dialysis bag with a cut-off molecular weight of 1000-2000 for dialysis, change the water every 6 hours, and complete the dialysis in 2 days. The dialysate was taken and freeze-dried to obtain N-(N'-pentenoylglutaminyl)-chitooligosaccharide.

2. Characterization of the prepared N-(N'-pentenoyl-glutaminyl)-chitooligosaccharides:

The infrared spectrum of N-(N′-pentenoyl-glutaminyl)-chitooligosaccharide obtained by characterization is shown in Figure 1.

In Figure 1, the broad peaks around 3387 cm ^-1 are mainly the OH on the hydroxyl groups on the chitosan oligosaccharides, and the NH stretching vibration peaks on the amido groups, and the absorptions near 2928 cm ^-1 and 2856 cm ^-1 are the methyl and methylene groups. The stretching vibration peaks of CH bonds; 1655 cm ^-1 and 1561 cm ^-1 are mainly the stretching vibration peaks of amide groups. 1072 cm ^-1 is mainly the absorption vibration peak of primary alcohol, secondary alcohol and tertiary alcohol hydroxyl group.

3. Elemental analysis and characterization of target compounds.

After analysis and characterization, the mass percentage content of each element is:

C: 49.1%; H: 6.96%; N: 10.54%; O: 33.44%. The content of each element is basically consistent with the theoretical results.

It can be seen from the results of infrared spectrum and elemental analysis that N-(N'-pentenoyl-glutaminyl)-chitooligosaccharide is obtained by this method.

4. Application examples.

1. The preparation method of curcumin electrochemical molecular imprinting sensor is as follows:

Add 3.16mg of curcumin and 8.79mg of N-(N′-pentenoyl-glutaminyl)-oligochitosan to 3mL of a mixed solvent composed of equal volume ratio of DMF and H ₂ O, ultrasonically dissolve at room temperature, and then add 50 mg crosslinker ethylene glycol dimethacrylate (EGDMA), 1 mg initiator ammonium persulfate. After standing for 5 hours, purify with nitrogen for at least 10 minutes to remove dissolved oxygen and obtain a mixed solution.

Pipette 3 μl of the mixed solution onto the surface of a clean glassy carbon electrode, and then cover it with a clean cover slip. Then heated in an oven at 60°C for 10 h, and after removing the cover glass, a layer of transparent polymer film was formed on the surface of the glassy carbon electrode to obtain the molecularly imprinted electrode before curcumin elution.

The molecularly imprinted electrode before curcumin elution was eluted with a mixed solution composed of methanol and acetic acid in equal volume ratio for 50 minutes, and the molecularly imprinted electrode after curcumin elution was obtained, that is, the curcumin electrochemical molecularly imprinted sensor.

2. Cyclic voltammetry curves of curcumin molecularly imprinted membrane-modified electrodes in different states:

Figure 2 shows the clean glassy carbon electrode (control), the molecularly imprinted electrode before curcumin elution, the molecularly imprinted electrode after curcumin elution, and the molecularly imprinted electrode after curcumin was re-adsorbed in the presence of 1.0 mM K ₃ [Fe(CN ) ₆ ] The cyclic voltammetry curve in 10mL0.25MNaAc/HAc (pH6.5) solution.

In addition, the method of obtaining the molecularly imprinted electrode after re-absorbing curcumin: soak the molecularly imprinted electrode after curcumin has been eluted in a curcumin ethanol solution with a concentration of 0.2M to absorb curcumin to saturation.

Curve a is the cyclic voltammetry curve of clean glassy carbon electrode in 10mL0.25M NaAc/HAc (pH6.5) solution containing 1.0 mM K ₃ [Fe(CN) ₆ ].

Curve b is the cyclic voltammetry curve of the molecularly imprinted electrode after elution of curcumin in 10mL of 0.25M NaAc/HAc (pH6.5) solution containing 1.0 mM K ₃ [Fe(CN) ₆ ].

Curve c is the cyclic voltammetry curve of the molecularly imprinted electrode of curcumin in 10mL of 0.25M NaAc/HAc (pH6.5) solution containing 1.0 mM K ₃ [Fe(CN) ₆ ] before elution.

Curve d is the cyclic voltammetry curve of the molecularly imprinted electrode after re-adsorbed curcumin in 10mL of 0.25M NaAc/HAc (pH6.5) solution containing 1.0 mM K ₃ [Fe(CN) ₆ ].

It can be seen from Figure 2 that the cyclic voltammetry current curve response of the clean glassy carbon electrode is relatively large (curve a), and when the surface is covered with a molecularly imprinted film (that is, contains a template molecularly imprinted film), the current response curve of the cyclic voltammetry drops sharply (curve c), and when the molecular imprinted membrane containing the template was eluted, the current response of cyclic voltammetry increased significantly (curve b). down (curve d).

3. Analysis of the cyclic voltammetry current response of curcumin electrochemical molecular imprinting sensor to curcumin and its analogues:

Figure 3 is the cyclic voltammetry of curcumin and its analogues (tetrahydrocurcumin, ferulic acid, carotene, quercetin) on the molecularly imprinted electrode (that is, curcumin electrochemical molecularly imprinted sensor) after curcumin elution Comparison graph of the drop value of the current response.

It can be seen from Figure 3 that the peak current drop value of the electrode for cyclic voltammetry of curcumin is the largest (that is, it has a large adsorption of curcumin), and the response to other analogues is small (and the adsorption of other analogues is relatively small). Smaller), it can be seen that the molecularly imprinted membrane has better selectivity.

In summary, N-(N′-pentenoyl-glutaminyl)-oligochitosan can be used as a functional monomer oligomer for curcumin recognition, and can be used for the determination of curcumin content in sample solutions.

Claims (3)

1. A chitosan oligosaccharide derivative molecular imprinting functional monomer is N- (N' -pentenoyl-amino acid acyl) -chitosan oligosaccharide, and has a molecular structure general formula as follows:

wherein n = 3-7,R is a side chain group of a neutral amino acid molecule, and the neutral amino acid is glutamine.

2. The method for preparing the molecularly imprinted functional monomer of the chitosan oligosaccharide derivative as claimed in claim 1, which comprises the steps of:

1) Dissolving neutral amino acid and potassium carbonate in water, dropwise adding pentenoyl chloride under the condition of ice-water bath, and then placing at room temperature for reaction to prepare an N-pentenoyl-amino acid solution; the neutral amino acid is glutamine;

2) Adjusting the pH value of the N-pentenoyl-amino acid solution to be neutral, and then mixing the solution with chitosan oligosaccharide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for reaction to generate an N- (N' -pentenoyl-amino acid acyl) -chitosan oligosaccharide solution;

3) Dialyzing the reaction solution obtained in the step 2) by using a dialysis bag, and freeze-drying the dialyzate to obtain the N- (N' -pentenoyl-amino acid acyl) -chitosan oligosaccharide.

3. The method for preparing the chitosan oligosaccharide derivative molecular imprinting functional monomer according to claim 2, wherein the feeding molar ratio of the neutral amino acid to the pentenoyl chloride is 1: 1.05-1.10.

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