Functional monomer of chitosan oligosaccharide imine derivative as well as preparation method and application of functional monomer

Abstract

The invention discloses a functional monomer based on chitosan oligosaccharide imine derivatives as well as a preparation method and application thereof, chitosan oligosaccharide (COS) and 4-allyloxybenzaldehyde (4-AB) are taken as raw materials,-NH2 of COS and-CHO of 4-AB are subjected to condensation reaction through Schiff base reaction, and the chitosan oligosaccharide imine derivatives are successfully prepared. The structure of a target derivative is characterized by adopting an infrared spectrum, a high-resolution mass spectrum and a carbon nuclear magnetic resonance spectrum, and the target derivative is used as a novel functional monomer to be applied to preparation of molecularly imprinted polymers (MIPs); the synthesis method disclosed by the invention has the advantages of rapidness, simplicity, no need of filtration and dialysis, mild reaction conditions, low cost, high yield and the like, and the synthesized chitosan oligosaccharide imine derivative (COS / 4-AB = 1: 2, mmol / mmol) can be used as a novel molecular imprinting functional monomer and is expected to play an important application value in the aspects of MIPs preparation and the like.

Description

Technical Field

[0001] The invention relates to a functional monomer based on chitosan oligosaccharide imine derivatives and a preparation method and application thereof. Background Art

[0002] In recent years, molecular imprinting technology (MIT) has been used as an emerging molecular recognition technology. It combines template molecules and functional monomers together through non-covalent interactions (hydrogen bonds, van der Waals forces, ionic or hydrophobic interactions, and metal coordination), forms polymers under the action of porogens, crosslinkers, and initiators, and washes the template out of the polymer through solvents, leaving specific recognition sites that complement the template molecules in shape, size, and chemical function. At present, molecular imprinting technology has been widely used in medical diagnosis, chromatographic separation, antibody and receptor mimics, solid phase extraction, biosensors, biomedicine, drug delivery systems, and other fields.

[0003] MIPs have the characteristics of low cost, simple preparation, resistance to extreme environments, high reusability and physical stability. Good interaction between functional monomers and templates is the initial prerequisite for obtaining highly specific MIPs. Although molecular imprinting technology has been developed for decades, there are still few functional monomers that can be used to prepare MIPs. Typical functional monomers include carboxylic acids (acrylic acid, methacrylic acid, vinylbenzoic acid), sulfonic acids (2-acrylamide-2-methylpropane sulfonic acid, heteroaromatic bases (vinylpyridine, vinylimidazole), etc. Due to the limited types of interactions between functional monomers and template molecules and weak interaction forces, the recognition sites are easily deformed, and the prepared MIPs have poor selectivity and low specific recognition ability. Therefore, the development of functional monomers that can interact with multiple sites of template molecules has become a key link in the current research on highly specific molecular imprinting polymers.

[0004] Chitosan oligosaccharides (COS), also known as chitosan oligomers or chitosan oligomers, are highly valued in various industrial fields such as medicine, cosmetics, agriculture, food, and biomaterial engineering due to their abundant resources and high cost-effectiveness. The structure of chitosan oligosaccharides is composed of DN-acetylglucosamine and D-2 glucosamine connected in unequal amounts through β-1,4 glycosidic bonds. The degree of acetylation (DP) is less than 20 and the average molecular weight is less than 3.9kDa. It is a low-molecular-weight product with good water solubility, great functional effects, and high biological activity. In particular, the amino group at the C2 position and the hydroxyl groups at the C3 and C6 positions of chitosan oligosaccharides are highly reactive, and controllable chemical modification of their structure can be achieved through simple molecular design. The main modification methods at present are acylation reaction, etherification reaction, and Schiff base reaction.

[0005] Benzaldehyde derivative Schiff base is the most widely studied and important base compound. Schiff base and its metal complexes have excellent antiviral, anti-cell growth, antibacterial, and anti-cancer biological activities, and are widely used in medicine, catalysis, optical materials, sensors, and analysis. The azomethylene -N=CH- group in the Schiff base structure has good donor properties in coordination chemistry and can form very stable complexes with a variety of transition metal ions.

[0006] In recent years, research on the chemical modification of COS has gradually increased. For example, various types of chitosan oligosaccharide derivatives such as chitosan oligosaccharide nicotinic acid grafted derivatives (COS-N), amphiphilic chitosan oligosaccharide derivatives lauryl-succinylated chitosan oligosaccharide (LS-COS), and chitosan oligosaccharide Schiff base copper complex (5-I-SCOS-Cu) have been synthesized through grafting reaction, metal ion complexation reaction and acylation reaction using a variety of modification methods and reaction conditions (such as acetone precipitation, filtration, anhydrous ethanol washing and catalyst catalysis). However, the current large-scale preparation of molecular imprinting functional monomers of chitosan oligosaccharide derivatives is difficult, costly and low in purity. Summary of the invention

[0007] Purpose of the invention: The present invention aims to provide a functional monomer of chitosan oligosaccharide derivatives which is quick and simple, does not require filtration and dialysis, has mild reaction conditions, is low in cost and has high yield, and its preparation method and application.

[0008] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical scheme:

[0009] In the first aspect, the present invention provides a functional monomer of chitosan oligosaccharide imine derivatives, the general structural formula of which is:

[0010]

[0011] Where n=3-10.

[0012] In a second aspect, the present invention further provides a method for preparing the functional monomer of the above-mentioned chitosan oligosaccharide imine derivatives, the method comprising the following steps:

[0013] dissolving chitosan oligosaccharide in a solvent to obtain a chitosan oligosaccharide solution, wherein the solvent comprises methanol and water;

[0014] After adding 4-allyloxybenzaldehyde to the chitosan oligosaccharide solution, mixing and dissolving, centrifuging and placing in a water bath for heating reaction;

[0015] The target product is dried at a preset temperature.

[0016] In a preferred embodiment, the molar ratio of the chitosan oligosaccharide to the 4-allyloxybenzaldehyde is 1:1 to 1:7.

[0017] In a preferred embodiment, the volume of methanol in the solvent is 3-4 mL, and the volume of water in the solvent is 200-267 μL.

[0018] In a preferred embodiment, the mixing, dissolving, centrifuging, and then placing in a water bath for heating reaction comprises:

[0019] After ultrasonication for 5 to 10 minutes, shaking for 3 to 5 minutes is performed to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution;

[0020] After the reaction solution is transferred to a container, it is heated in a water bath at 60° C. to obtain a reaction product. The reaction time is 24 hours.

[0021] In a preferred embodiment, drying the reaction product at the preset temperature to obtain the target product includes: after the reaction is completed, drying the container containing the reaction product at 60° C. for 6 hours to obtain the target product, with a yield of 400 to 700 mg each time.

[0022] In a preferred embodiment, after the ultrasonic treatment for 5 to 10 minutes and the shaking for 3 to 5 minutes to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution, the method further comprises:

[0023] The reaction solution was centrifuged at 2000 r / min for 1 min.

[0024] In a third aspect, the present invention further provides a use of the functional monomer derived from chitosan oligosaccharide imine as described in the first aspect in biomolecular imprinting.

[0025] The advantages of the present invention are as follows: the present invention provides a functional monomer of a chitosan oligosaccharide imine derivative, a preparation method and application thereof; the derivative preparation method provided by the present invention is quick and simple, does not require filtration and dialysis, has mild reaction conditions, low cost and high yield, and the like; the chitosan oligosaccharide imine derivative synthesized by the present invention, COS / 4-AB=1:2 (mmol / mmol), greatly improves the specific adsorption capacity and imprinting effect of MIPs, can be used as a new type of molecular imprinting functional monomer, and is expected to play an important application value in the preparation of MIPs and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0027] Figure 1 The infrared spectra of chitosan oligosaccharide imine derivatives with different ratios (COS / 4-AB);

[0028] Figure 2This is the high-resolution mass spectrum of chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2);

[0029] Figure 3 It is a chitosan oligosaccharide imine derivative (COS / 4-AB=1:2). 13 C NMR spectrum;

[0030] Figure 4 The effect of different ratios (COS / 4-AB) of chitosan oligosaccharide imine derivatives on the adsorption performance of MIPs / NIPs;

[0031] Figure 5 This is the effect of chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2) on the adsorption properties of MIPs / NIPs.

[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] As described in the background art, the current molecular imprinting functional monomers of chitosan oligosaccharide derivatives are difficult to prepare on a large scale, with high costs and low purity. To solve the above problems, the present invention creatively proposes a functional monomer of chitosan oligosaccharide imine derivatives and its preparation method and application.

[0034] Specifically, the present invention provides a functional monomer of chitosan oligosaccharide imine derivatives, the general structural formula of which is:

[0035]

[0036] Wherein n=3-10. Exemplarily, n can be 3, 4, 5, 6, 7, 8, 9, 10.

[0037] The present invention also provides a method for preparing a functional monomer of chitosan oligosaccharide imine derivatives, the method comprising the following steps:

[0038] dissolving chitosan oligosaccharide in a solvent to obtain a chitosan oligosaccharide solution, wherein the solvent comprises methanol and water;

[0039] After adding 4-allyloxybenzaldehyde to the chitosan oligosaccharide solution, mixing and dissolving, centrifuging and placing in a water bath for heating reaction;

[0040] The target product is dried at a preset temperature.

[0041] In a preferred embodiment, the molar ratio of the chitosan oligosaccharide to the 4-allyloxybenzaldehyde is 1:1 to 1:7. Exemplarily, the molar ratio of the chitosan oligosaccharide to the 4-allyloxybenzaldehyde can be 1:1, 1:1.5, 1:2, 1:2.6, 1:3, 1:3.3, 1:4, 1:4.8, 1:5, 1:5.2, 1:6, 1:6.1, 1:7, or any ratio within the above ratio range.

[0042] In a preferred embodiment, the volume of methanol in the solvent is 3 to 4 mL, and the volume of water in the solvent is 200 to 267 μL. Exemplarily, the volume of methanol in the solvent can be 3 mL, 3.2 mL, 3.5 mL, 3.7 mL, 4 mL, or any value within the above range. The volume of water in the solvent can be 200 μL, 205 μL, 210 μL, 216 μL, 220 μL, 235 μL, 240 μL, 246 μL, 250 μL, 253 μL, 260 μL, 267 μL, or any value within the above range.

[0043] In a preferred embodiment, the mixing, dissolving, centrifuging, and then placing in a water bath for heating reaction comprises:

[0044] After ultrasonication for 5 to 10 minutes, shaking is performed for 3 to 5 minutes to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution. Exemplarily, a vortex shaker is used for shaking;

[0045] After the reaction solution is transferred to a container, it is heated in a water bath at 60° C. to obtain a reaction product, and the reaction time is 24 hours. For example, the container is a 30 mL vial.

[0046] In a preferred embodiment, drying the reaction product at the preset temperature to obtain the target product comprises: after the reaction is completed, drying the container containing the reaction product at 60° C. for 6 hours to obtain the target product, with a yield of 400 to 700 mg each time. The yield can be 400 mg, 470 mg, 530 mg, 600 mg, 610 mg, 670 mg, 700 mg, or any value within the above numerical range.

[0047] In a preferred embodiment, after the ultrasonic treatment for 5 to 10 minutes and the shaking for 3 to 5 minutes to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution, the method further comprises:

[0048] The reaction solution was centrifuged at 2000 r / min for 1 min to concentrate the reaction solution at the bottom of the centrifuge tube to improve the reaction efficiency.

[0049] In addition, the present invention also provides an application of the functional monomer of the chitosan oligosaccharide imine derivatives described above in biomolecular imprinting.

[0050] Example 1: Preparation of functional monomers of chitosan oligosaccharide imine derivatives:

[0051] Dissolve chitosan oligosaccharide in methanol and water, add 4-allyloxybenzaldehyde, mix and dissolve, centrifuge and place in a water bath for heating reaction;

[0052] In this embodiment, this step includes: accurately weighing 0.4 mmol of chitosan oligosaccharide and placing it in a 10 mL centrifuge tube, then adding 4 mL of methanol, ultrasonicating for 5 minutes, then adding 0.8 mmol of 4-AB and 220 μL of water, ultrasonicating for 10 minutes, shaking with a vortex shaker for 3 to 5 minutes until the raw materials are completely dissolved in the solution, placing in a centrifuge at 2000 r / min, centrifuging for 1 minute, transferring the reaction solution to a 30 mL vial, and reacting in a 60° C. water bath for 24 hours.

[0053] The target product is dried at a preset temperature. Specifically, after the reaction is completed, the vial is placed in a 60°C oven for drying for 6 hours to obtain the target derivative, and the product is ground into powder using a mortar and stored at room temperature for later use, with the yield of each bottle being about 700 mg each time.

[0054] Embodiment 2: The difference between this embodiment and embodiment 1 is that:

[0055] In this embodiment, 0.4 mmol chitosan oligosaccharide was accurately weighed and placed in a 10 mL centrifuge tube, followed by the addition of 3.5 mL methanol, ultrasonication for 5 min, and then 1.2 mmol 4-AB and 200 μL water were added. After ultrasonication for 10 min, the raw materials were shaken for 3 to 5 min with a vortex shaker until the raw materials were completely dissolved in the solution, placed in a centrifuge at 2000 r / min, centrifuged for 1 min, and the reaction solution was transferred to a 30 mL vial, and reacted in a 60 ° C water bath for 24 h. After the reaction was completed, the vial was placed in a 60 ° C oven for 6 h to obtain the target derivative, and the product was ground into powder with a mortar and stored at room temperature for standby use, with a yield of about 600 mg per bottle each time.

[0056] 1. The structure of the target derivative was characterized by infrared spectroscopy, high-resolution mass spectrometry and carbon nuclear magnetic resonance spectroscopy.

[0057] Figure 1 The spectral analysis of 4-AB, COS and chitosan oligosaccharide imine derivatives in different ratios (COS / 4-AB) was carried out by Fourier transform infrared spectrometer (FTIR). Figure 1 As shown in a, the functional group characteristic peaks of chitosan oligosaccharide imine derivatives with different ratios are basically the same, but the absorption intensity is slightly different. Figure 1As shown in b, COS is at 1561.57cm -1 There is a strong -NH2 characteristic absorption peak at 1690cm -1 There is a strong -C=O characteristic absorption peak at 3371cm -1 There is a broad and strong absorption peak at 1689-1600cm, which is the -OH stretching vibration absorption peak of the coordinated water and crystal water in the product, or the broad and strong hydrogen bond absorption peak formed by the ionic association of -OH and -NH2 of COS. -1 In the area (1639.97cm -1 ) The sharp and strong absorption peak is the stretching vibration absorption peak of the imine C=N, which is the characteristic peak of the ligand, indicating that the chitosan oligosaccharide Schiff base ligand has been formed. -1 The sharp and strong absorption peak at 1579.94cm is the stretching vibration absorption peak of the benzene ring skeleton. Since the derivative molecule contains aromatic functional groups, it is affected by the strong vibration peak of the benzene ring skeleton. -1 The bending vibration absorption peak at 1561.57cm -1 Compared with the absorption peak at 832.60cm, it becomes smaller and sharper. -1 There is a primary amino group NH swing vibration absorption peak at 1309.78cm -1 and 1254.99cm -1 The medium-strong absorption peak at is the stretching vibration absorption peak of CN. The synthesis of the target product can be preliminarily judged from the infrared spectrum.

[0058] like Figure 2 As shown, the chitosan oligosaccharide imine derivatives (COS / 4-AB = 1:2) were subjected to ESI-MS molecular detection. The m / z [M+Na + ]The theoretical value is 1457.8205; the measured value is 1457.8198, the error is -0.5ppm, and the molecular formula is C65H122N6O28Na.

[0059] Figure 3 pass 13C NMR was used to further characterize the structure of chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2). 55.37(C2), 60.08(C6), 70.23(C3), 74.47(C5), 75.27(C4), 102.18ppm(C1) were the chemical shifts of the pyranose ring of chitosan oligosaccharide unit. The chemical shifts of the carbon chain of 4-AB were located at 69.04(C12), 115.17(C10), 118.16(C14), 129.44(C8), 130.18(C9), 133.89(C13), 160.69(C11). 163.61ppm was the chemical shift of the characteristic functional group C=N carbon of Schiff base, which indicated that the -NH2 of COS had undergone condensation reaction with the -CHO on 4-AB. Based on the above analysis, it can be determined that the chitosan oligosaccharide imine derivatives have been successfully synthesized.

[0060] 2. Application of functional monomers of chitosan oligosaccharide imine derivatives in biomolecular imprinting.

[0061] 1. Preparation of MMP-9 carboxyl-terminal nonapeptide molecularly imprinted polymer:

[0062] 0.012mmol template molecule YDILQCPED, 0.024mmol zinc acrylate, 0-0.048mmol chitosan oligosaccharide derivative were accurately weighed and placed in a 30mL vial, followed by the addition of 5mL methanol, ultrasonic mixing, and the addition of 0.192mmol ethylene glycol dimethacrylate. After ultrasonication for 5min, 0.04g dimethyl azobisisobutyrate was added and reacted in a 65℃ water bath for 10h. After the reaction, the polymer was washed thoroughly with methanol / acetic acid (1:1, v / v) and methanol / acetic acid (9:1, v / v) in turn, and the template molecule content in the eluent was detected by HPLC until the template molecule could not be detected. Finally, the polymer was placed in a 60℃ oven and dried for 3h to obtain MIPs. In the preparation of NIPs, except that the template molecule nonapeptide was not added, the other operation steps were prepared and eluted in the same way as MIPs.

[0063] 2. Effects of functional monomers of chitosan oligosaccharide imine derivatives in different ratios (COS / 4-AB) on the adsorption properties of MIPs / NIPs:

[0064] From the infrared results, we can see that the characteristic peak structures of the derivatives of COS / 4-AB=1:4 and COS / 4-AB=1:5 are basically the same, and the characteristic peak structures of the derivatives of COS / 4-AB=1:6 and COS / 4-AB=1:7 are also basically the same. Therefore, this study only selected chitosan oligosaccharide imine derivatives with COS / 4-AB of 1:1, 1:2, 1:3, 1:4, and 1:6 as new molecular imprinting functional monomers, zinc acrylate as a co-monomer, and the ratio of chitosan oligosaccharide imine derivatives / zinc acrylate was 2:2 (mmol / mmol). MMP-9 carboxyl terminal nonapeptide MIPs / NIPs were prepared, and the adsorption performance of MIPs / NIPs was investigated. Figure 4 As shown in the figure, when COS / 4-AB is 1:1, the adsorption amount of MIPs / NIPs is the largest and IF is the smallest. As the amount of 4-AB increases gradually, IF first increases and then decreases. When COS / 4-AB is 1:2, IF reaches the maximum (IF=2.59). Therefore, chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2) have the greatest influence on the adsorption performance of MIPs / NIPs, so they can be used as the best new functional monomer.

[0065] 3. Effect of chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2) on the adsorption properties of MIPs / NIPs:

[0066] In this study, chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2) were selected as a new functional monomer and zinc acrylate as a co-monomer. When the amount of zinc acrylate was unchanged, the effect of the amount of chitosan oligosaccharide imine derivatives (COS / 4-AB=1:2) on the adsorption performance of MMP-9 carboxyl terminal nonapeptide MIPs / NIPs was investigated. Figure 5 As shown in the figure, when the ratio of chitosan oligosaccharide imine derivatives / zinc acrylate is 4:2 (mmol / mmol), the specific adsorption capacity of MIPs is the strongest and the imprinting effect is the best (adsorption amount = 48.7 mg / g, IF = 4.14). Moreover, the adsorption performance of MIPs will also weaken with the reduction of the amount of chitosan oligosaccharide imine derivatives. The above results show that in the synergistic effect of the two functional monomers, chitosan oligosaccharide imine derivatives play a leading role in the specific adsorption efficiency of MIPs, and their appropriate dosage will also significantly improve the adsorption efficiency of MIPs.

[0067] Of course, the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any modifications made according to the spirit of the main technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A functional monomer of a chitosan oligosaccharide imine derivative, the general structural formula of which is: Where n=3-10.

2. A method for preparing a functional monomer of chitosan oligosaccharide imine derivatives as claimed in claim 1, characterized in that: The method comprises the following steps: dissolving chitosan oligosaccharide in a solvent to obtain a chitosan oligosaccharide solution, wherein the solvent comprises methanol and water; After adding 4-allyloxybenzaldehyde to the chitosan oligosaccharide solution, mixing and dissolving, centrifuging and placing in a water bath for heating reaction to obtain a reaction product; The reaction product is dried at a preset temperature to obtain a target product.

3. The method for preparing the functional monomer of chitosan oligosaccharide imine derivatives according to claim 2, characterized in that: The feeding molar ratio of the chitosan oligosaccharide to the 4-allyloxybenzaldehyde is 1:1-1:

7.

4. The method for preparing the functional monomer of chitosan oligosaccharide imine derivatives according to claim 2, characterized in that: The volume of methanol in the solvent is 3-4 mL, and the volume of water in the solvent is 200-267 μL.

5. The method for preparing the functional monomer of chitosan oligosaccharide imine derivatives according to claim 2, characterized in that: The mixing, dissolving, and heating reaction after centrifugation comprises: After ultrasonication for 5 to 10 minutes, shaking for 3 to 5 minutes is performed to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution; After the reaction solution is transferred to a container, it is heated in a water bath at 60° C. to obtain a reaction product. The reaction time is 24 hours.

6. The method for preparing the functional monomer of chitosan oligosaccharide imine derivatives according to claim 5, characterized in that: The step of drying the reaction product at the preset temperature to obtain the target product comprises: after the reaction is completed, drying the container containing the reaction product at 60° C. for 6 hours to obtain the target product, with a yield of about 400 to 700 mg each time.

7. The method for preparing the functional monomer of chitosan oligosaccharide imine derivatives according to claim 5, characterized in that: After the ultrasonic treatment for 5 to 10 minutes and the shaking for 3 to 5 minutes to completely dissolve the chitosan oligosaccharide and 4-allyloxybenzaldehyde to obtain a reaction solution, the method further comprises: The reaction solution was centrifuged at 2000 r / min for 1 min.

8. Use of the functional monomer of the chitosan oligosaccharide imine derivatives as claimed in claim 1 in biomolecular imprinting.

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