CN114539432A - Spin-labeled starch and preparation method thereof - Google Patents
Spin-labeled starch and preparation method thereof Download PDFInfo
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- CN114539432A CN114539432A CN202210073775.6A CN202210073775A CN114539432A CN 114539432 A CN114539432 A CN 114539432A CN 202210073775 A CN202210073775 A CN 202210073775A CN 114539432 A CN114539432 A CN 114539432A
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- C08B31/00—Preparation of derivatives of starch
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- C08B33/00—Preparation of derivatives of amylose
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Abstract
The invention relates to the technical field of starch modification, in particular to a method for spin-labeling starch molecules and spin-labeled starch prepared by the method. The method is characterized in that a spinning marker with spinning characteristics is combined with starch molecules in a covalent combination method, so that the starch molecules have spinning characteristics; the spin label is 4-amino-2, 2,6, 6-tetramethylpiperidinyloxy free radical. The prepared spin-labeled starch has the following structural formula:
the invention labels the nitroxide free radicals with the spinning characteristic to the starch in a covalent binding mode, the labeled starch molecules have the spinning characteristic in a magnetic field, and the conformation and the structure of the starch molecules can be further analyzed under the condition of not damaging the structure of a starch chain.
Description
Technical Field
The invention relates to the technical field of starch modification, in particular to a method for spin-labeling starch molecules and spin-labeled starch prepared by the method.
Technical Field
Starch is a high molecular carbohydrate, not only a source of human energy intake, but also one of the main raw materials of renewable materials. Starch is one of the most common natural materials in life, and is widely applied to industries such as food, paper making, pharmacy and the like due to the advantages of high yield, low cost, natural degradation and the like. The research of starch molecules is closely related to the progress and development of human production and life.
The current research methods of starch mainly comprise differential scanning calorimetry, confocal laser scanning microscopy, small-angle x-ray scattering method and the like. These methods typically work only at the granularity level, with certain limitations. Starch molecules can only be studied at the molecular level by means of nuclear magnetic resonance of a nuclear magnetic microscope, an atomic force microscope, monomolecular force spectroscopy and the like, but the methods all process the starch molecules to destroy the original conformation of the molecules.
Disclosure of Invention
In order to solve the technical problems, the invention provides spin-labeled starch, wherein the starch molecules have spin characteristics in a magnetic field, and the characteristic spectrum of the starch molecules can be detected in electron paramagnetic resonance under the condition of not damaging the structure of a starch chain, so that the conformation and the structure of the starch molecules can be analyzed.
The invention adopts the following technical scheme:
a spin-labelled starch of the formula:
the invention also provides a preparation method of the spin-labeled starch, which combines a spin label with spin characteristic with starch molecules by a covalent combination method to make the starch molecules have the spin characteristic; the spin marker is 4-amino-2, 2,6, 6-tetramethylpiperidinyloxy free radical.
The method comprises the following steps:
s1, dissolving hexamethyl diisocyanate and 4-amino Tempo in anhydrous dimethyl sulfoxide DMSO, and continuously stirring for 2 hours;
s2, putting a certain amount of starch into a new anhydrous DMSO solution, and stirring until the starch is completely dissolved;
s3, mixing the DMSO solutions respectively obtained in the S1 and S2 steps together, and continuously stirring for 24 hours to obtain a mixed sample;
s4, putting the mixed sample into a dialysis bag, and dialyzing the mixed sample in an ethanol water solution until no signal is detected by electron paramagnetic resonance EPR of the mixed sample;
and S5, taking out the dialyzed mixed sample, and freeze-drying to obtain the required spin-labeled starch.
Preferably, in step S1, the hexamethyldiisocyanate, 4-amino Tempo and DMSO are mixed at a ratio of 1 mL: (1-100) mg: 10mL of the aqueous solution was used.
Preferably, in step S1, the starch and DMSO are dissolved in 20 to 40mL of DMSO per 2g of starch for use; the stirring time was 10 h.
Preferably, in step S4, the cut-off amount of the dialysis bag is 8000 to 16000 molecular weight, and the volume fraction of ethanol in the ethanol aqueous solution is 30%.
Preferably, in step S4, the dialysis is performed three times in total, and each dialysis time is 4 to 5 hours.
Preferably, the freeze-drying is: pre-freezing at-20 deg.C for 12 hr, taking out, and drying in-50 deg.C freeze-drying machine for 48 hr.
The invention has the beneficial effects that:
1. hexamethyl Diisocyanate (HDI) and 4-amino-2, 2,6, 6-tetramethyl piperidinyloxy free radical (4-amino Tempo) are dissolved in anhydrous dimethyl sulfoxide (DMSO), and a cyanate ester functional group on the HDI and an amino group on the 4-amino Tempo undergo an addition reaction to form an amide bond.
The reaction process is as follows:
the starch is added into DMSO, and is swelled and dissolved, so that starch molecules are opened and can easily participate in the reaction. After mixing, the hydroxyl on the starch molecule and the cyanate ester functional group at the other end of the HDI are subjected to esterification reaction to form an amido bond, so that the starch molecule has a spinning characteristic.
The reaction process is as follows:
during dialysis, ethanol molecules and water molecules continuously exchange unreacted 4-amino Tempo (molecular weight 171.260), HDI (molecular weight 168.22) and DMSO (molecular weight 78.13) molecules in the mixture, and the self-selected labeled starch is purified.
2. The invention labels nitroxide free radical with spin characteristic, namely 4-amino Tempo, on starch in a covalent bonding chemical mode, and the labeled starch molecule has spin characteristic in a magnetic field and has stable chemical structure. The characteristic spectrum can be detected in electron paramagnetic resonance under the condition of not destroying the structure of the starch chain, thereby analyzing the conformation and the structure of the starch molecule.
3. The method has the advantages of simple chemical reaction principle, simple and easy process route and easy operation. The method can be widely applied to spin labeling synthesis of other types of polysaccharide molecules such as dextran, cellulose and the like besides being applied to starch molecules, and provides a new thought and method for analyzing the structure and conformation of the polysaccharide molecules.
Drawings
FIG. 1 shows the electron paramagnetic resonance (ESR) detection results of the spin-labeled corn starch prepared in example 1;
FIG. 2 shows the electron paramagnetic resonance (NMR) detection results of spin-labeled corn starch prepared in example 1 dissolved in DMSO;
FIG. 3 shows the electron paramagnetic resonance detection of the spin-labeled amylose prepared in example 2;
FIG. 4 shows the Electron Paramagnetic Resonance (EPR) detection results of spin-labeled amylose prepared in example 2 dissolved in DMSO;
FIG. 5 shows the electron paramagnetic resonance detection of the spin-labeled wheat starch prepared in example 3;
FIG. 6 shows the Electron Paramagnetic Resonance (EPR) detection of spin-labeled wheat starch prepared in example 3 dissolved in DMSO.
Detailed Description
For the convenience of understanding, the technical solution of the present invention will be described in more detail with reference to the following embodiments and the accompanying drawings.
Example 1
2.0g of corn starch is added into 25mL of anhydrous DMSO, and dissolved for 10 hours at 25 ℃ to prepare starch solution for later use. After 1g of hexamethyldiisocyanate and 50mg of 4-aminoTempo were dissolved in 10mL of anhydrous DMSO and continuously stirred for 2 hours, it was mixed with the above starch solution and continuously stirred for 24 hours to obtain a mixed sample.
And (3) filling the mixed sample in a dialysis bag with the molecular weight of 8000-16000, sealing, immersing the dialysis bag in an ethanol solution with the volume fraction of 30%, and dialyzing for three times for 4-5 h each time to remove unreacted 4-amino Tempo, HDI and DMSO molecules. And (3) taking out the mixed sample after dialysis, freeze-drying, pre-freezing in a refrigerator at-20 ℃ for 12 hours, taking out, and drying in a freeze dryer for 48 hours to finally obtain the required spin-labeled corn starch.
50mg of the spin-labeled corn starch was put in a quartz tube for Electron Paramagnetic Resonance (EPR) detection, and the detection results are shown in FIG. 1. And 2mg of the spin-labeled corn starch is taken again to be dissolved in 10mL of anhydrous DMSO, and then the mixture is placed in a quartz tube for electron paramagnetic resonance detection, and the detection result is shown in figure 2.
As can be seen in FIG. 1, the spin-labeled corn starch has strong spin signal and accumulation of signals in an anisotropic state in a solid powder state. As can be seen in FIG. 2, the spin-labeled corn starch in the solution state has three approximately equal-height and equal-width peaks, which are isotropic, and the peaks are characteristic peaks of the spin label, indicating that the spin-labeled corn starch exhibits good spin characteristics.
Example 2
2.0g of amylose is added into 35mL of anhydrous DMSO and dissolved for 10 hours at 25 ℃ to prepare a starch solution for later use. After 1g of hexamethyldiisocyanate and 10mg of 4-aminoTempo were dissolved in 10mL of DMSO and continuously stirred for 2 hours, it was mixed with the above starch solution and continuously stirred for 24 hours to obtain a mixed sample.
And (3) filling the mixed sample in a dialysis bag with the molecular weight of 8000-16000, sealing, immersing the dialysis bag in an ethanol solution with the volume fraction of 30%, and dialyzing for three times for 4-5 h each time to remove unreacted 4-amino Tempo, HDI and DMSO molecules. And (3) taking out the mixed sample after dialysis is finished, freezing and drying the mixed sample, pre-freezing the mixed sample in a refrigerator at the temperature of-20 ℃ for 12 hours, taking out the mixed sample, and drying the mixed sample in a freeze dryer for 48 hours to finally obtain the required spin-labeled amylose.
50mg of the spin-labeled amylose was put in a quartz tube for Electron Paramagnetic Resonance (EPR) detection, and the detection results are shown in FIG. 3. And dissolving 2mg of the spin-labeled corn starch in 10mL of DMSO again, and placing the obtained solution in a quartz tube for electron paramagnetic resonance detection, wherein the detection result is shown in figure 4.
As can be seen in FIG. 3, the spin-labeled amylose has strong spin signal and the signal is stacked and anisotropic in the solid powder state. As can be seen in FIG. 4, the spin-labeled amylose in the solution state has three approximately equal-height and equal-width peaks, which are isotropic, and the peaks are characteristic peaks of the spin label, which indicates that the spin-labeled maize starch has good spin characteristics.
Example 3
2.0g of wheat starch is added into 35mL of anhydrous DMSO and dissolved for 10 hours at 25 ℃ to prepare a starch solution for later use. After 1g of hexamethyldiisocyanate and 80mg of 4-aminoTempo were dissolved in 10mL of anhydrous DMSO and continuously stirred for 2 hours, it was mixed with the above starch solution and continuously stirred for 24 hours to obtain a mixed sample.
And (3) filling the mixed sample in a dialysis bag with the molecular weight of 8000-16000, sealing, immersing the dialysis bag in an ethanol solution with the volume fraction of 30%, and dialyzing for three times for 4-5 h each time to remove unreacted 4-amino Tempo, HDI and DMSO molecules. And (3) taking out the mixed sample after dialysis, freeze-drying, pre-freezing in a refrigerator at-20 ℃ for 12h, taking out, and drying in a freeze dryer for 48 h to finally obtain the required spin-labeled wheat starch.
50mg of the spin-labeled wheat starch was put in a quartz tube for Electron Paramagnetic Resonance (EPR) detection, and the detection results are shown in FIG. 5. And then 2mg of the spin-labeled wheat starch is taken again to be dissolved in 10mL of DMSO, and then the mixture is placed in a quartz tube for electron paramagnetic resonance detection, and the detection result is shown in figure 6.
As can be seen in FIG. 5, the spin-labeled wheat starch has strong spin signal and the signal is accumulated and is anisotropic in the solid powder state. As can be seen in FIG. 6, in the solution state, three peaks, which are characteristic peaks of the spin label, were still detected due to the low concentration of the spin label, indicating that the spin-labeled wheat starch exhibits good spin characteristics.
The above embodiments are only used to illustrate the technical solutions of the present invention, and do not limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A spin-labeled starch having the structural formula:
2. a method for preparing a spin-labeled starch molecule according to claim 1, wherein the method comprises the steps of binding a spin label having spin characteristics to the starch molecule by covalent binding to impart spin characteristics to the starch molecule; the spin label is 4-amino-2, 2,6, 6-tetramethyl piperidinyloxy radical.
3. The method of claim 2, comprising the steps of:
s1, dissolving hexamethyl diisocyanate HDI and 4-amino Tempo in anhydrous DMSO, and continuously stirring for 2 h;
s2, putting a certain amount of starch into new anhydrous DMSO, and stirring until the starch is completely dissolved;
s3, mixing the DMSO solutions respectively obtained in the S1 and S2 steps together, and continuously stirring for 24 hours to obtain a mixed sample;
s4, putting the mixed sample into a dialysis bag, and dialyzing the mixed sample in an ethanol water solution until no signal is detected by electron paramagnetic resonance EPR of the mixed sample;
and S5, taking out the dialyzed mixed sample, and freeze-drying to obtain the required spin-labeled starch.
4. A method of spin labeling starch molecules as claimed in claim 3, wherein in step S1, the hexamethyldiisocyanate, 4-amino Tempo and DMSO are mixed at a ratio of 1 mL: (1-100) mg: 10mL of the aqueous solution was used.
5. The method according to claim 3, wherein in step S1, the starch and DMSO are dissolved in 20-40 mL of anhydrous DMSO per 2g of starch; the stirring time was 10 h.
6. The method according to claim 3, wherein in step S4, the cut-off amount of the dialysis bag is 8000-16000 molecular weight, and the volume fraction of ethanol in the ethanol aqueous solution is 30%.
7. A method according to claim 6, wherein in step S4, the dialysis is performed three times for 4-5 h.
8. A method of spin labeling starch molecules as claimed in claim 3, wherein in step S5, the freeze drying is: pre-freezing at-20 deg.C for 12 hr, taking out, and drying in-50 deg.C freeze-drying machine for 48 hr.
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| CN202210073775.6A CN114539432A (en) | 2022-01-21 | 2022-01-21 | Spin-labeled starch and preparation method thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5250672A (en) * | 1985-09-11 | 1993-10-05 | Guerbet S.A. | Contrast agent for NMR imaging |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5250672A (en) * | 1985-09-11 | 1993-10-05 | Guerbet S.A. | Contrast agent for NMR imaging |
Non-Patent Citations (3)
| Title |
|---|
| COSTAS G. BILIADERIS等: ""Electron Spin Resonance Studies of Starch-Water-Probe Interactions"", 《CARBOHYDRATE POLYMERS》 * |
| SHOJI TAKIGAMI等: ""E.s.r. study of the conformational transition of spin-labelled xanthan gum in aqueous solution"", 《INT.J.BIOL.MACROMOL.,》 * |
| YOSHIMASA YAMANO等: ""Relationship Between Macroscopic and Microscopic Viscosities in Starch Gels"", 《J. DISPERSION SCIENCE AND TECHNOLOGY》 * |
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