CN108070021B - Small molecular peptide capable of being assembled into highly ordered nanofiber and method for assembling and constructing highly ordered nanofiber - Google Patents

Small molecular peptide capable of being assembled into highly ordered nanofiber and method for assembling and constructing highly ordered nanofiber Download PDF

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CN108070021B
CN108070021B CN201711446630.1A CN201711446630A CN108070021B CN 108070021 B CN108070021 B CN 108070021B CN 201711446630 A CN201711446630 A CN 201711446630A CN 108070021 B CN108070021 B CN 108070021B
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highly ordered
resin
amino acid
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CN108070021A (en
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秦四勇
丁文强
张爱清
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South Central Minzu University
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South Central University for Nationalities
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
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Abstract

A small molecular peptide capable of being assembled into highly ordered nano-fibers and a method for assembling and constructing the highly ordered nano-fibers relate to the technical field of biological materials. The small molecular peptide capable of being assembled into the highly ordered nano fiber is mainly composed of a hydrophobic alkyl chain, beta-folded amino acid, hydrophilic amino acid and an N-end amino end capping end; the preparation method is characterized in that the polypeptide is synthesized by a polypeptide solid-phase synthesis method with an FMOC protection strategy, small-molecule peptide chain segments are sequentially prolonged from C end to N end on carrier resin, the reaction conditions are simple and mild, and the obtained small-molecule peptide has amphipathy and can be assembled into the highly-ordered nanofiber with good biocompatibility. The method for assembling the highly ordered nanofiber comprises the steps of dissolving the small molecular peptide in water until the concentration is 0.05wt% -12wt%, adjusting the pH value of the aqueous solution to 7-8, standing at room temperature, and obtaining the highly ordered nanofiber with good biocompatibility, wherein the reaction conditions are simple and mild.

Description

Small molecular peptide capable of being assembled into highly ordered nanofiber and method for assembling and constructing highly ordered nanofiber
Technical Field
The invention relates to the technical field of biological materials, in particular to small molecular peptides capable of being assembled into highly ordered nanofibers and a method for assembling and constructing the highly ordered nanofibers.
Background
In the nanometer technology, the construction of the highly ordered nanometer structure has wide application value. Recent studies have shown that highly ordered nanostructures have special biological functions in the biological field. Jiangxiangyu subject group pointed out in 2008 that: the stem cells can be differentiated according to a certain geometric direction to form functional cells such as neurons, muscle cells, vascular endothelial cells and the like, and the arrangement of the functional cells in the tissue has directionality, so that the functional cells can grow according to a certain direction to form a specific tissue. When the chemical structure and mechanical properties of the cell culture substrate are directional, the growth direction of the functional cells is selective, so that the control of the growth of the functional cells in a certain direction through the design of the culture substrate is an important subject of tissue engineering research. The ChungCY group in 2007 and the Stupp group in 2010 reported consecutively: a large number of highly ordered extracellular fibrils, which play an important role in the functioning of the respective organs, are also found in important organs such as the brain, heart, bone and spinal cord, but their formation process is still a mystery. In order to effectively explain the specific biological effects of the ordered structure, how to construct the ordered nano-structure with biocompatibility constitutes a primary problem.
Various techniques for constructing ordered nano-arrays have been researched and developed, including electrostatic spinning of polymers and vacuum deposition. The electrostatic spinning technology of polymer is a spinning method for obtaining micro-nano fibers by carrying out jet drawing on polymer solution or melt under the electrostatic action, and the method has harsh reaction conditions and needs high temperature and high pressure. The vacuum deposition technology is a technology for depositing a required coating on the surface of a base material by utilizing physical processes such as thermal evaporation, glow discharge, arc discharge and the like, and the technology needs high voltage, has poor biocompatibility of a product and is not beneficial to the application in a biological direction.
Therefore, there is a need for the construction of ordered fibers with simple and mild reaction conditions and good biocompatibility.
Disclosure of Invention
The invention aims to provide the small molecular peptide capable of being assembled into the highly ordered nanofiber and the preparation method thereof, the reaction conditions are simple and mild, the obtained small molecular peptide has amphipathy, and the highly ordered nanofiber with good biocompatibility can be assembled.
The invention aims to provide the highly ordered nanofiber and the assembling method thereof, the reaction condition is simple and mild, and the obtained highly ordered nanofiber has good biocompatibility.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a small molecular peptide capable of being assembled into highly ordered nanofibers, wherein a small molecular peptide sequence is formed by sequentially sealing a hydrophobic alkyl chain, beta-folded amino acid, hydrophilic amino acid and an N-terminal amino group.
Further, in a preferred embodiment of the present invention, the hydrophobic alkyl chain includes one of stearic acid, palmitic acid, myristic acid, and lauric acid.
Further, in the preferred embodiment of the present invention, the β -sheet amino acid is a hydrophobic amino acid having β -sheet, and specifically includes one or more of alanine, valine, glycine, and phenylalanine.
Further, in the preferred embodiment of the present invention, the hydrophilic amino acid includes one or more of glutamic acid and lysine.
Further, in the preferred embodiment of the present invention, the small molecule peptide has the structural formula C15H31CO-AAAAAKK-CONH2Or C15H31CO-VVVVKKK-CONH2Wherein A is alanine, V is valine and K is lysine.
The invention provides a preparation method of the small molecular peptide capable of being assembled into the highly ordered nano fiber, which is synthesized by a polypeptide solid phase synthesis method adopting an FMOC protection strategy, wherein carrier Resin is Rink Amide-AM Resin, and small molecular peptide chain segments are sequentially prolonged from the C end to the N end on the carrier Resin.
Further, in the preferred embodiment of the present invention, it comprises the following steps:
s1, placing Rink Amide-AM Resin in a polypeptide solid phase synthesis column, washing with N, N-dimethylformamide, emptying the solvent, swelling with N, N-dimethylformamide, and emptying the solvent;
s2, adding a piperidine/N, N-dimethylformamide solution into the solid-phase synthesis column, stirring, washing with N, N-dimethylformamide, and emptying the solvent;
s3, adding beta-folded amino acid, benzotriazole-N, N, N ', N' -tetramethylurea fluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylethylamine/N, N-dimethylformamide solution into the solid-phase synthesis column, and stirring;
s4, sampling to check whether the beta-sheet amino acid is connected on the resin;
s5, if the beta-sheet amino acid is grafted on the resin, repeating the steps S2-S4 until the corresponding beta-sheet amino acid and the amount are grafted on the resin;
s6, adding a piperidine/N, N-dimethylformamide solution into the solid-phase synthesis column, stirring, washing with N, N-dimethylformamide, and emptying the solvent;
s7, adding hydrophilic amino acid, benzotriazole-N, N, N ', N' -tetramethylurea fluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylethylamine/N, N-dimethylformamide solution into the solid-phase synthesis column, and stirring for 1.5-4 h;
s8, sampling to check whether the hydrophilic amino acid is grafted on the resin;
s9, if the hydrophilic amino acid is grafted on the resin, repeating the steps S6-S8 until the corresponding hydrophilic amino acid and the corresponding amount are grafted on the resin;
s10, adding a hydrophobic alkyl chain, benzotriazole-N, N, N ', N' -tetramethylurea fluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylethylamine/N, N-dimethylformamide solution into the solid-phase synthesis column, and stirring;
s11, sampling to check whether the hydrophobic alkyl chain is connected on the resin;
s12, if the hydrophobic alkyl chain is connected to the resin, washing the resin with N, N-dimethylformamide, methanol and dichloromethane respectively, and drying in vacuum at normal temperature to obtain dried resin;
s13, adding a trifluoroacetic acid/water/triisopropylsilane mixed liquid into the dry resin, stirring at normal temperature, collecting a cutting liquid, carrying out rotary evaporation and concentration to obtain a viscous liquid, then dropwise adding the viscous liquid into cold ether for precipitation, centrifuging, removing a supernatant, and drying at normal temperature.
Further, in a preferred embodiment of the present invention, the method of checking whether the group is attached to the resin is: and (3) taking the resin, putting the resin into a methanol solution of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the group is connected on the resin.
The invention provides an assembly method of highly ordered nano-fibers, which is to dissolve the small molecular peptide in water to the concentration of 0.05wt% -12wt%, adjust the pH of the water solution to 7-8, and place at room temperature.
The invention provides a highly ordered nanofiber, which is obtained by adopting the assembly method of the highly ordered nanofiber.
The small molecular peptide capable of being assembled into the highly ordered nanofiber and the method for assembling and constructing the highly ordered nanofiber have the advantages that: the small molecule peptide sequence capable of being assembled into the highly ordered nano fiber is formed by a hydrophobic alkyl chain, beta-folded amino acid, hydrophilic amino acid and an N-terminal amino end capping end in sequence; the preparation method is characterized in that the polypeptide is synthesized by a polypeptide solid-phase synthesis method with an FMOC protection strategy, carrier Resin is Rink Amide-AM Resin, small-molecule peptide chain segments are sequentially prolonged from the C end to the N end on the carrier Resin, the reaction conditions are simple and mild, and the obtained small-molecule peptide has amphipathy and can be assembled into the highly-ordered nano fiber with good biocompatibility. The method for assembling the highly ordered nanofibers comprises the steps of dissolving the small molecular peptide in water to a concentration of 0.05wt% -12wt%, adjusting the pH value of an aqueous solution to 7-8, standing at room temperature, and obtaining the highly ordered nanofibers with good biocompatibility, wherein the reaction conditions are simple and mild.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a structural diagram of a small molecule peptide provided in example 1 of the present invention;
FIG. 2 is a mass spectrum of a small molecule peptide provided in example 1 of the present invention;
FIG. 3 is an SEM image of highly ordered nanofibers provided in example 2 of the present invention;
FIG. 4 is a polarization microscope photograph of highly ordered nanofibers provided in example 3 of the present invention;
FIG. 5 is a structural diagram of a small molecule peptide provided in example 4 of the present invention;
FIG. 6 is a mass spectrum of a small molecule peptide provided in example 4 of the present invention;
fig. 7 is an SEM image of highly ordered nanofibers provided in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following is a detailed description of the small molecule peptide capable of assembling into the highly ordered nanofiber and the method for assembling and constructing the highly ordered nanofiber according to the embodiment of the present invention.
The embodiment of the invention provides the micromolecule peptide which can be assembled into the highly ordered nano-fiber, and meanwhile, the micromolecule peptide has good water solubility and excellent biocompatibility. The small molecule peptide sequence is composed of hydrophobic alkyl chain, beta-folded amino acid, hydrophilic amino acid and N-end amino end capping in sequence, and the general formula of the sequence is hydrophobic alkyl chain + beta-folded amino acid + hydrophilic amino acid-CONH2. Wherein the hydrophobic alkyl chain comprises one of stearic acid (C18), palmitic acid (C16), myristic acid (C14) and lauric acid (C12), and the structural general formula of the hydrophobic alkyl chain is shown in the specification
Figure 689495DEST_PATH_IMAGE002
(ii) a The beta-sheet amino acid is hydrophobic amino acid with beta sheet, and specifically comprises one or more of alanine (A), valine (V), glycine (G) and phenylalanine (F); the hydrophilic amino acid comprises one or more of glutamic acid (E) and lysine (K).
In this example, the small moleculeThe peptide may have the formula C15H31CO-AAAAAKK-CONH2Or C15H31CO-VVVVKKK-CONH2Wherein A is alanine, V is valine and K is lysine.
The embodiment of the invention provides a preparation method of the small molecular peptide capable of being assembled into the highly ordered nanofiber, which is characterized in that the small molecular peptide is synthesized by a polypeptide solid phase synthesis method adopting an FMOC protection strategy, carrier Resin is Rink Amide-AM Resin (the Resin substitution degree is 0.6-0.65 mmol/g), small molecular peptide chain segments are sequentially prolonged from the C end to the N end on the carrier Resin, and the specific synthesis steps are as follows:
s1, placing 0.5-1.5g Rink Amide-AM Resin in a polypeptide solid phase synthesis column, washing at least three times by using N, N-Dimethylformamide (DMF), emptying the solvent, swelling for 0.5-2h by using 15-40ml of N, N-dimethylformamide, and emptying the solvent.
S2, removing alpha-amino protected FMOC group in Rink Amide-AM Resin, specifically adding the FMOC group into a solid phase synthesis column according to the volume ratio of 15% -40%: 60% -85% piperidine (piperidine)/N, N-dimethylformamide solution, stirring for 20-60min, washing with N, N-dimethylformamide at least three times, and draining off the solvent.
S3, adding 2-4 times of molar equivalent of beta-folded amino acid (such as Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH), benzotriazole-N, N, N ', N' -tetramethyluronium fluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBT) and 5-20% by volume into a solid phase synthesis column: 80 to 95 percent of N, N-Diisopropylethylamine (DIEA)/N, N-dimethylformamide solution, and slowly stirring for 1.5 to 4 hours.
S4, sampling to check whether the beta-folded amino acid is connected on the resin, specifically, taking a small amount of resin, putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the beta-folded amino acid is connected on the resin;
s5, if the beta-sheet amino acid is grafted on the resin, repeating the steps S2-S4 until the corresponding beta-sheet amino acid and the amount are grafted on the resin;
s6, removing alpha-amino protected FMOC group in Rink Amide-AM Resin, specifically adding the FMOC group into a solid phase synthesis column according to the volume ratio of 15% -40%: stirring 60-85% piperidine/N, N-dimethylformamide solution for 20-60min, washing with N, N-dimethylformamide for at least three times, and draining off solvent;
s7, adding 1.5-4 times of molar equivalent of hydrophilic amino acid and HBTU, 2-3 times of molar equivalent of HOBT and 5-20% of volume ratio into a solid phase synthesis column: 80 to 95 percent of N, N-diisopropylethylamine/N, N-dimethylformamide solution is stirred for 1.5 to 4 hours;
s8, sampling to check whether the hydrophilic amino acid is connected on the resin, specifically, taking a small amount of resin, putting the small amount of resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the hydrophilic group is connected on the resin;
s9, if the hydrophilic amino acid is grafted on the resin, repeating the steps S6-S8 until the corresponding hydrophilic amino acid and the corresponding amount are grafted on the resin;
s10, adding 2.5-4 times of molar equivalent of hydrophobic alkyl chain, HBTU, HOBT and DIEA/DMF solution into the solid phase synthesis column, and stirring;
s11, sampling to check whether a hydrophobic alkyl chain is connected to the resin, specifically, taking a small amount of resin, putting the small amount of resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the hydrophobic alkyl chain is connected to the resin;
s12, if the hydrophobic alkyl chain is connected to the resin, washing the resin with N, N-dimethylformamide, methanol and Dichloromethane (DCM) respectively, and drying in vacuum at normal temperature to obtain dried resin;
s13, cutting off polypeptide, specifically, adding 94-96% volume ratio of the polypeptide into dry resin in a solid phase synthesis column: 2% -3%: 2% -3% trifluoroacetic acid (TFA)/H2And stirring the O/Triisopropylsilane (TIS) mixed liquid at normal temperature for 1.5-4h, collecting the cut liquid, concentrating by rotary evaporation to obtain a viscous liquid, then dropwise adding the viscous liquid into cold ether for precipitation, centrifuging, removing the supernatant, and drying at normal temperature for more than 12h to obtain the small molecular peptide.
The embodiment of the invention provides an assembly method of highly ordered nano fibers, which comprises the following steps:
dissolving small molecular peptides capable of being assembled into highly ordered nanofibers in water to form an aqueous solution with the concentration of 0.05-12 wt%;
adjusting the pH value of the aqueous solution to 7-8 to obtain an assembly;
and standing the assembly at room temperature for a period of time to obtain the highly ordered nanofiber.
In the above process, the small molecule peptide can be dissolved in water to form an aqueous solution with a concentration of 0.05wt% to 0.1wt% (low concentration), and then the pH of the aqueous solution is adjusted to 7 to 8 to obtain an assembly, and finally the highly ordered nanofiber is obtained. The small molecular peptide can also be dissolved in water to form a water solution with the concentration of 7wt% -12wt% (high concentration), the pH value of the water solution is adjusted to 7-8, the water solution is heated for 1-2h at 70-90 ℃ to obtain an assembly, and finally the highly ordered nano-fiber arranged in parallel is obtained.
The embodiment of the invention provides a highly ordered nanofiber, which is obtained by adopting the assembly method of the highly ordered nanofiber.
The features and properties of the present invention are described in further detail below with reference to examples.
The following examples illustrate the source routes of the various feedstocks: rink Amide-AM Resin (Resin substitution degree 0.625 mmol/g), alanine with alpha-amino acid protected by 9-fluorenylmethyloxycarbonyl (Fmoc-Ala-OH), alpha-amino group and lysine with pendant amino group protected by 9-fluorenylmethyloxycarbonyl and tert-butylcarbonyl (Fmoc-Lys (Boc) -OH), benzotriazole-N, N, N ', N' -tetramethyluronium fluorophosphate (HBTU) and 1-Hydroxybenzotriazole (HOBT) were purchased from Gill Biochemical (Shanghai) Co., Ltd.
Palmitic acid, piperidine (Piperiding), ninhydrin, N-Dimethylformamide (DMF), methanol, Dichloromethane (DCM) were purchased from the national pharmaceutical group.
Trifluoroacetic acid (TFA), N-Diisopropylethylamine (DIEA) were purchased from aladin (aladin).
Triisopropylsilane (TIS) was purchased from Sahn chemical technology (Shanghai) Inc.
Example 1
This example provides a small molecule peptide with the structural formula C15H31CO-AAAAAKK-CONH2The specific synthesis steps are as follows:
1) weighing 1g of the resin, placing the resin in a polypeptide solid phase synthesis column, washing the resin with DMF three times, emptying the solvent, swelling the resin with 20ml of DMF for 1 hour, and emptying the solvent.
2) Removing the alpha-amino protected FMOC group in the RinkAmide-AMResin resin. To the solid phase synthesis column was added 20% Piperiding/DMF (V/V), stirred for 30min, washed three times with DMF and the solvent was drained.
3) 20ml of 3-fold molar equivalent of Fmoc-Lys (Boc) -OH, HBTU, HOBT and 2ml of DIEA in DMF was added to the synthesis column and stirred slowly for 2 h.
4) Taking a small amount of the resin in the step 3), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the amino acid is connected on the resin.
5) Repeating the steps 2), 3) and 4) until the corresponding amino acid number is completed.
6) And removing the alpha-amino protected FMOC group in Fmoc-Ala-OH. To the solid phase synthesis column was added 20% Piperiding/DMF (V/V), stirred for 30min, washed three times with DMF and the solvent was drained.
7) 20ml of 2-fold molar equivalent of Fmoc-Lys (Boc) -OH, 2.4-fold molar equivalent of HBTU, HOBT and 2ml of DIEA in DMF was added to the synthesis column and stirred slowly for 2 h.
8) Taking a small amount of the resin in the step 7), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the amino acid is attached to the resin.
9) Repeating the steps 6), 7) and 8) until the corresponding amino acid number is completed.
10) 20ml of a 3-fold molar equivalent solution of palmitic acid, HBTU, HOBT and 2ml of DIEA in DMF was added to the synthesis column and stirred slowly for 8 h.
11) Taking a small amount of the resin in the step 10), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the palmitic acid is connected on the resin.
12) The resin was washed with DMF, methanol and DCM, respectively, and dried under vacuum at ambient temperature to give a dry resin for use.
13) And (4) cutting off the polypeptide. To the dry resin synthesis column obtained in 12) was added 30ml of TFA/H2And stirring the mixed liquid of O/TIS (V/V/V =95%/2.5%/2.5%) for 2h at normal temperature, collecting the cut liquid, performing rotary evaporation and concentration to obtain viscous liquid, then dropwise adding the viscous liquid into cold ether for precipitation, centrifuging, removing the supernatant, and drying at normal temperature for 12h to obtain the small molecular peptide.
Detecting the small molecule peptide obtained in this example, wherein FIG. 1 is a structural diagram of the small molecule peptide obtained in this example; FIG. 2 is a mass spectrum of the small molecule peptide obtained in this example. The results show that: the actual molecular weight of the small molecular peptide is consistent with the theoretical molecular weight, and the small molecular peptide is C15H31CO-AAAAAKK-CONH2。
Example 2
This example provides a highly ordered nanofiber, which is prepared according to the following method:
the small molecule peptide C prepared in example 115H31CO-AAAAAKK-CONH2Dissolving in water to a concentration of 0.1wt% to obtain an aqueous solution.
The pH of the aqueous solution was adjusted to 7.4 to obtain an assembly.
And placing the assembly at room temperature for a period of time to obtain the highly ordered nanofiber.
The highly ordered nanofibers obtained in this example were examined, and fig. 3 is an SEM image of the highly ordered nanofibers obtained in this example. The results show that: small molecule peptide C15H31CO-AAAAAKK-CONH2Indeed, highly ordered nanofibers can be assembled.
Example 3
This example provides a nanofiber with orientation, which is prepared according to the following method:
the small molecule peptide C prepared in example 115H31CO-AAAAAKK-CONH2Dissolving in water to a concentration of 10wt% to obtain an aqueous solution.
Adjusting the pH value of the aqueous solution to 7.4, and heating the aqueous solution at the temperature of 70-90 ℃ for 1-2h to obtain an assembly.
And standing the assembly at room temperature for a period of time to obtain the oriented nanofiber.
The oriented nanofibers obtained in this example were examined, and FIG. 4 is a polarization microscope image of the highly ordered nanofibers obtained in this example. The results show that: small molecule peptide C15H31CO-AAAAAKK-CONH2Indeed, it was possible to assemble nanofibers with orientation, and small molecule peptide C was laterally described15H31CO-AAAAAKK-CONH2The assembled nano-fibers have the property of being arranged in parallel.
Example 4
This example provides a small molecule peptide with the structural formula C15H31CO-VVVVKKK-CONH2The specific synthesis steps are as follows:
1) weighing 1g of the resin, placing the resin in a polypeptide solid phase synthesis column, washing the resin with DMF three times, emptying the solvent, swelling the resin with 20ml of DMF for 1 hour, and emptying the solvent.
2) Removing alpha-amino protected FMOC group in Rinkamide-AMResin resin, adding 20% of pipeciding/DMF (V/V) into a solid phase synthesis column, stirring for 30min, washing with DMF for three times, and emptying the solvent.
3) To the synthesis column was added 3 times molar equivalent of Fmoc-Lys (Boc) -OH, HBTU, HOBT and 2ml DIEA in 20ml DMF and stirred slowly for 2 h.
4) Taking a small amount of the resin in the step 3), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the amino acid is connected on the resin.
5) Repeating the steps 2), 3) and 4) until the corresponding amino acid number is completed.
6) And removing the alpha-amino protected FMOC group in Fmoc-Val-OH. To the solid phase synthesis column was added 20% Piperiding/DMF (V/V), stirred for 30min, washed three times with DMF and the solvent was drained.
7) 20ml of 2-fold molar equivalent of Fmoc-Lys (Boc) -OH, 2.4-fold molar equivalent of HBTU, HOBT and 2ml of DIEA in DMF was added to the synthesis column and stirred slowly for 2 h.
8) Taking a small amount of the resin in the step 7), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the amino acid is attached to the resin.
9) Repeating the steps 6), 7) and 8) until the corresponding amino acid number is completed.
10) To the synthesis column was added 3 times molar equivalent of palmitic acid, HBTU, HOBT and 2ml of DIEA in 20ml of DMF and stirred slowly for 8 h.
11) Taking a small amount of the resin in the step 10), putting the resin into a methanol solution (0.01 mg/ml) of ninhydrin, heating and boiling for 3-5 minutes, and checking color, wherein if the color is not changed, the palmitic acid is connected on the resin.
12) The resin was washed with DMF, methanol and DCM, respectively, and dried under vacuum at ambient temperature to give a dry resin for use.
13) Cleaving the polypeptide, adding 30ml of TFA/H to the dried resin synthesis column obtained in 12)2And stirring the mixed liquid of O/TIS (V/V/V =95%/2.5%/2.5%) for 2h at normal temperature, collecting the cut liquid, performing rotary evaporation and concentration to obtain viscous liquid, then dropwise adding the viscous liquid into cold ether for precipitation, centrifuging, removing the supernatant, and drying at normal temperature for 12h to obtain the small molecular peptide.
Detecting the small molecule peptide obtained in this example, wherein FIG. 5 is a structural diagram of the small molecule peptide obtained in this example; FIG. 6 is a mass spectrum of the small molecule peptide obtained in this example. The results show that: the actual molecular weight of the small molecular peptide is consistent with the theoretical molecular weight, and the small molecular peptide is C15H31CO-VVVVKKK-CONH2。
Example 5
This example provides a highly ordered nanofiber, which is prepared according to the following method:
the small molecule peptide C prepared in example 315H31CO-VVVVKKK-CONH2Dissolving in water to a concentration of 0.1wt% to obtain an aqueous solution.
The pH of the aqueous solution was adjusted to 7.4 to obtain an assembly.
And placing the assembly at room temperature for a period of time to obtain the highly ordered nanofiber.
The highly ordered nanofibers obtained in this example were examined, and fig. 7 is an SEM image of the highly ordered nanofibers obtained in this example. The results show that: small molecule peptide C15H31CO-VVVVKKK-CONH2Indeed, highly ordered nanofibers can be assembled.
In conclusion, the small molecular peptide capable of being assembled into the highly ordered nanofiber and the preparation method thereof provided by the embodiment of the invention have the advantages that the reaction conditions are simple and mild, the obtained small molecular peptide has amphipathy, and the highly ordered nanofiber with good biocompatibility can be assembled. The highly ordered nanofiber and the assembling method thereof provided by the embodiment of the invention have the advantages that the reaction conditions are simple and mild, and the obtained highly ordered nanofiber has good biocompatibility.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (3)

1. The small molecule peptide capable of being assembled into the highly ordered nanofiber is characterized in that a small molecule peptide sequence is formed by blocking a hydrophobic alkyl chain, a beta-folded amino acid, a hydrophilic amino acid and a C-terminal amino group in sequence, wherein the hydrophobic alkyl chain is palmitic acid, the beta-folded amino acid is alanine or valine, and the hydrophilic amino acid is lysine;
the structural formula of the small molecular peptide is C15H31CO-AAAAAKK-CONH2Or C15H31CO-VVVVKKK-CONH2Wherein A is alanine, V is valine and K is lysine.
2. A method for assembling highly ordered nanofibers by dissolving the small molecule peptide of claim 1 in water to a concentration of 0.1wt%, adjusting the pH of the aqueous solution to 7.4, and standing at room temperature.
3. A highly ordered nanofiber obtained by the method for assembling a highly ordered nanofiber as claimed in claim 2.
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