CN110499349A - First small-molecular peptides, the second small-molecular peptides that can produce active oxygen with oxygen carrying potential and preparation method thereof - Google Patents
First small-molecular peptides, the second small-molecular peptides that can produce active oxygen with oxygen carrying potential and preparation method thereof Download PDFInfo
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- CN110499349A CN110499349A CN201910716784.0A CN201910716784A CN110499349A CN 110499349 A CN110499349 A CN 110499349A CN 201910716784 A CN201910716784 A CN 201910716784A CN 110499349 A CN110499349 A CN 110499349A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Abstract
A kind of the first small-molecular peptides with oxygen carrying potential of the invention, second small-molecular peptides that can produce active oxygen and preparation method thereof.The first small-molecular peptides sequence with oxygen carrying potential of the invention is C17H31- CONH-VRGDS-COOH, the unsaturated double-bond functional group in structure can be formed conjugated double bond by fat oxidation oxydasis and side group generates hydroperoxides, and the method that the first small-molecular peptides construct the second small-molecular peptides is enzymatic method, and the second small-molecular peptides structure is C17H31O2-CONH-VRGDS-COOH.The method that second small-molecular peptides generate active oxygen is embryonic stem-like cells, C17H31O2- CONH-VRGDS-COOH is in Fe2+Catalysis under generate and have cytotoxic active oxygen.
Description
Technical field
The present invention relates to technical field of biological materials, more particularly, to a kind of the first small molecule with oxygen carrying potential
Peptide, second small-molecular peptides that can produce active oxygen and preparation method thereof.
Background technique
Tumour is to threaten the No.1 disease of whole mankind's health.In January, 2019, the national cancer of China national Cancer center publication
Disease statistical data shows that national de novo malignancy case load was about 392.9 ten thousand in 2015, and dead about 233.8 ten thousand people are average
Cancer is diagnosed as more than 10,000 people daily.The conventional means for the treatment of of cancer mainly have operation to cut off, radiotherapy and chemicals
Treatment etc..Personalized treatment is taken in order to effectively be directed to various cancers patient, reduces the side effect during conventional therapy, in recent years
To have emerged the therapeutic modality of various new, such as photodynamic therapy, photo-thermal therapy, ultrasonic therapy and immunization therapy etc..
Wherein, photodynamic therapy (PDT) is as a kind of emerging strategy of cancer treatment, since its distinctive operating performance effectively drops
The toxic side effect of low normal tissue, by the favor of researchers in the basic research of oncotherapy.PDT is predominantly specific
The laser irradiation of wavelength excites the photosensitizer of tumor tissues, and the photosensitizer of excitation state is by its energy transmission to the oxygen of surrounding
To generate the living radical (ROS) of cytotoxicity for treating malignant tumor.It is clear that the oncotherapy effect of PDT
Photosensitizer concentration, light intensity and the oxygen content of tumor region are depended on simultaneously.In order to realize the cancer target transport of photosensitizer,
It is usually medium by foreign vector.Carrier synthesis is complicated, and potential bio-toxicity cannot be guaranteed.Meanwhile tissue is to light
There is stronger absorption, the tissue penetration of general light source is low to cause the luminous intensity for reaching tumor region not high.In order to improve light
Penetration capacity, may be selected light source of the near infrared light as PDT.However, near infrared energy is lower, the efficiency of photosensitizer is activated
It is undesirable.In addition, the weary oxygen environment of tumor region also limits the therapeutic effect of PDT.
In view of the principle of PDT, the treating malignant tumor that is produced as of living radical provides new approaches.In order to overcome PDT
The problem of process, building are not necessarily to foreign vector and light source, while not depending on tumor region oxygen from delivery nanometer system
Active oxygen radical can be controllably generated under concentration, will effectively improve the effect for the treatment of malignant tumor.Therefore, building has biofacies
Capacitive is good, it is convieniently synthesized, can efficiently generate being of great significance from delivery nanometer treatment system for singlet oxygen again.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of the first small-molecular peptides with oxygen carrying potential, can
Generate the second small-molecular peptides and preparation method thereof of active oxygen.
According to an aspect of the invention, there is provided a kind of sequence of the first small-molecular peptides with oxygen carrying potential successively by
Hydrophobic alkyl chain, hydrophobic amino acid, hydrophilic amino acid and N- Amino End Group sealing end are constituted.
On the basis of above scheme preferably, the hydrophobic alkyl chain contains cis- unsaturated double-bond
Functional group, the cis- unsaturated double-bond functional group is oxidized to form conjugated double bond under the catalysis of lipoxidase and side group produces
Raw hydroperoxides (- OOH), i.e.,
On the basis of above scheme preferably, the hydrophobic amino acid is one or more of valine, glycine.
On the basis of above scheme preferably, the hydrophilic amino acid is one of aspartic acid, serine, arginine
Or it is several.
On the basis of above scheme preferably, the structural formula of first small-molecular peptides with oxygen carrying potential is C17H31-
CONH-VRGDS-COOH, wherein V is valine, and R is arginine, and G is glycine, and D is aspartic acid, and S is serine.
On the basis of above scheme preferably, the preparation of first small-molecular peptides with oxygen carrying potential uses dichloro triphen
Methyl chloride resin as carrier, by FMOC polypeptide solid-state reaction method by small-molecular peptides segment on carrier from C-terminal to N-terminal successively
Extend.The synthetic method of the first small-molecular peptides with oxygen carrying potential is mature and simple, and reaction condition is mild, obtained small molecule
Peptide has amphipathic and tumour cell targeting, the cis- unsaturated double-bond containedFunctional group is in rouge
It is oxidized to form conjugated double bond under the catalysis of fat oxidizing ferment and side group generates hydroperoxides (- OOH), has potential oxygen carrying function
Can, and it can be self-assembly of the nanometer spiral fiber with good biocompatibility in aqueous solution.
The present invention also provides a kind of the second small-molecular peptides that can produce active oxygen, second small-molecular peptides are using as above
First small-molecular peptides with oxygen carrying potential are obtained as the method for raw material enzymatically, described to can produce the of active oxygen
The structural formula of two small-molecular peptides is C17H31O2- CONH-VRGDS-COOH, and V is valine, R is arginine, and G is glycine, D
For aspartic acid, S is serine.This small-molecular peptides prepares that reaction condition is simply mild, and obtained small-molecular peptides have amphiphilic
Property, tumour cell targeting and from function of carrying oxygen, can be efficiently from carrying active oxygen oxygen source-hydroperoxides (- OOH) and generate tool
Cytotoxic active oxygen.
A kind of method for the second small-molecular peptides that can produce active oxygen as described above is prepared the present invention also provides a kind of,
The following steps are included:
S1, the first small-molecular peptides (C is weighed17H31- CONH-VRGDS-COOH) solid powder in container, be added 0.1M and
The PBS buffer solution that pH is 9.0 dissolves, and under uniform stirring, injects air bubble 20 minutes；
S2, lipoxygenase is weighed in container, the PBS buffer solution that 0.1M and pH are 9.0 is added and dissolves；
S3, the solution in above-mentioned S2 is added in solution described in S1, is kept for 5 DEG C of temperature, react 2h, acquired solution is close
Seal cryo-conservation.
The present invention is prepared the anti-of the second small-molecular peptides that can produce active oxygen by the first small-molecular peptides with oxygen carrying potential
Answer condition simple, mild, obtained the second small-molecular peptides that can produce active oxygen are with amphipathic, tumour cell targeting and certainly
Function of carrying oxygen efficiently from carrying active oxygen oxygen source-hydroperoxides (- OOH) and can generate the cytotoxic active oxygen of tool.
The present invention also provides a kind of methods that the second small-molecular peptides that can produce active oxygen generate active oxygen, in institute as above
FeCl is added in the second small-molecular peptides stated2Solution, second small-molecular peptides are in FeCl2Fe in solution2+Effect is lower to be generated
Has cytotoxic active oxygen.
The second small-molecular peptides proposed by the present invention that can produce active oxygen, in Fe2+In the presence of, can produce has carefully
Cellular toxicity singlet oxygen (1O2).In order to solve the problems, such as PDT process, the present invention is using hydroperoxides as active oxygen
Generating source is bonded building with Functional Polypeptides from the nanometer system for delivering hydroperoxides (- OOH), is not necessarily to foreign vector and light source, together
When active oxygen radical can be controllably generated in the case where not depending on tumor region oxygen concentration, new think of can be provided for the treatment of malignant tumour
Road.The the second small-molecular peptides generation method proposed by the present invention that can produce active oxygen is easy and has good biocompatibility.
Present invention effectively prevents traditional inorganic nano-particle subcarriers may occur what aggregation caused in use
Biosafety issues.Meanwhile in order to overcome the generation of active oxygen during PDT to tumor region oxygen and H2O2The dependence of concentration
Problem, the first small-molecular peptides with oxygen carrying potential designed by the present invention can produce the second small-molecular peptides of active oxygen, in nothing
Foreign carrier and light source are needed, while not depending on and controllably generating active oxygen radical under tumor region oxygen concentration.
Detailed description of the invention
Fig. 1 is the structure chart for the first small-molecular peptides with oxygen carrying potential that the embodiment of the present invention 1 provides；
Fig. 2 is the mass spectrogram for the first small-molecular peptides with oxygen carrying potential that the embodiment of the present invention 1 provides；
Fig. 3 is transmission electron microscope (TEM) figure for the nanometer spiral fiber that the embodiment of the present invention 1 provides；
Fig. 4 is atomic force microscope (AFM) figure for the nanometer spiral fiber that the embodiment of the present invention 1 provides；
Fig. 5 is the structure chart for the second small-molecular peptides that can produce active oxygen that the embodiment of the present invention 2 provides；
Fig. 6 is the mass spectrogram for the second small-molecular peptides that can produce active oxygen that the embodiment of the present invention 2 provides；
Fig. 7 is that the second small-molecular peptides that can produce active oxygen that the embodiment of the present invention 3 provides generate active oxygen for Fe2+Oxidation
For Fe3+Color change figure；
Fig. 8 is that the second small-molecular peptides that can produce active oxygen that the embodiment of the present invention 3 provides and corresponding control group exist
The fluorescence intensity change tendency chart of active oxygen is generated in 140min；
Fig. 9 is the second small-molecular peptides that can produce active oxygen that provide of the embodiment of the present invention 3 and corresponding control group the
The fluorescence detection figure of active oxygen is generated at 12 hours；
Figure 10 is that the second small-molecular peptides that can produce active oxygen that the embodiment of the present invention 3 provides and corresponding control group generate
Have cytotoxic singlet oxygen (1O2) fluorescence detection figure；
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Each raw material sources approach in following embodiment: dichloro trityl chloride resin (resin degree of substitution 0.97mmol/g),
Valine (FMOC-Val-OH) that amino is protected by N- fluorenes -9- methoxycarbonyl group, amino are protected sweet by N- fluorenes -9- methoxycarbonyl group
The serine that the carboxyl of propylhomoserin (FMOC-Gly-OH), amino and side group is protected by N- fluorenes -9- methoxycarbonyl group and tert-butyl respectively
The amino of (FMOC-Ser (tBu)-OH), amino and side group are respectively by N- fluorenes -9- methoxycarbonyl group and 2,2,4,6,7- pentamethyls
The arginine (FMOC-Arg (Pbf)-OH) of Dihydrobenzofuranes -5- sulfonyl protection, amino and side group carboxyl respectively by
Aspartic acid (FMOC-Asp (OtBu)-OH), the benzotriazole-N, N, N of N- fluorenes -9- methoxycarbonyl group and tert-butoxy protection ',
N '-tetramethylurea fluorophosphate (HBTU) and 1- hydroxy benzo triazole (HOBT) are bought in biochemical (Shanghai) the limited public affairs of gill
Department.
Linoleic acid, piperidines (Piperiding), ninhydrin, n,N-Dimethylformamide (DMF), methanol, methylene chloride
(DCM) purchase is in Chinese medicines group.
Trifluoroacetic acid (TFA), n,N-diisopropylethylamine (DIEA) are bought in Aladdin (Aladdin).
Tri isopropyl silane (TIS) is bought in Sa En chemical technology (Shanghai) Co., Ltd..
Lipoxidase is bought in Sigma (sigma).
For ease of description, the first small-molecular peptides with oxygen carrying potential are named as OPA1 below, can produce active oxygen
The second small-molecular peptides be named as OPA2, phosphate buffer is referred to as PBS, and n,N-Dimethylformamide is referred to as DMF.
Embodiment 1
The present embodiment provides a kind of the first small-molecular peptides with oxygen carrying potential, structural formula C17H31-CONH-
VRGDS-COOH, specific synthesis step are as follows:
1) it weighs a certain amount of resin to be placed in Solid-phase synthesis peptides column, is washed three times with DMF, empty solvent, then with suitable
Solvent is emptied after the DMF swelling 1h of amount.
2) DMF solution that FMOC-Ser (tBu)-OH, 6 times of equivalent DIEA of 3 times of molar equivalents is added in Xiang Hecheng column is suitable
Amount, is slowly stirred 2h.
3) the FMOC blocking group in first amino acid serine is removed.20% is added into synthesis in solid state column
Piperiding/DMF (V/V) is deprotected liquid, after stirring 30min, is washed three times with DMF, empties solvent.
4) FMOC-Asp (OtBu)-OH, HBTU of 2.4 times of molar equivalents of 2 times of molar equivalents is added in Xiang Hecheng column,
The DMF solution of HOBT and 6 times of equivalent DIEA is appropriate, is slowly stirred 2h.
5) a small amount of resin in 4) is taken, ebuillition of heated 3- in the methanol solution (concentration 0.1mg/mL) of ninhydrin is put into
5min tests color, if non-discolouring, shows that amino acid couples successfully.
6) the FMOC blocking group in FMOC-Asp (OtBu)-OH is removed.20% is added into synthesis in solid state column
The deprotection liquid of Piperiding/DMF (V/V) after stirring 30min, is washed three times with DMF, empties solvent.
7) FMOC-Gly-OH, the HBTU of 2.4 times of molar equivalents, HOBT and 6 of 2 times of molar equivalents are added in Xiang Hecheng column
The DMF solution of times equivalent DIEA is appropriate, is slowly stirred 2h.
8) a small amount of resin in 7) is taken, ebuillition of heated 3-5min in the methanol solution (0.1mg/mL) of ninhydrin is put into and tests
Color shows that amino acid has coupled success if non-discolouring.
9) the FMOC blocking group in FMOC-Gly-OH is removed.20%Piperiding/ is added into synthesis in solid state column
The deprotection liquid of DMF (V/V) after stirring 30min, is washed three times with DMF, empties solvent.
10) FMOC-Arg (Pbf)-OH, HBTU of 2.4 times of molar equivalents of 2 times of molar equivalents is added in Xiang Hecheng column,
The DMF solution of HOBT and 6 times of equivalent DIEA is appropriate, is slowly stirred 2h.
11) a small amount of peptide resin in 10) is taken, ebuillition of heated 3-5min in the methanol solution (0.1mg/mL) of ninhydrin is put into
Color is tested, if non-discolouring, shows that amino acid has coupled success.
12) the FMOC blocking group in Fmoc-Val-OH is removed.20%Piperiding/ is added into synthesis in solid state column
The deprotection liquid of DMF (V/V) after stirring 30min, is washed three times with DMF, empties solvent.
13) linoleic acid of 3 times of molar equivalents, HBTU, the DMF solution of HOBT and 6 times of equivalent DIEA are added in Xiang Hecheng column
In right amount, it is slowly stirred 8h.
14) a small amount of resin in 13) is taken, ebuillition of heated 3-5min in the methanol solution (0.1mg/mL) of ninhydrin is put into and tests
Color shows that linoleic acid has connect on a upper amino acid if non-discolouring.
15) DMF is used respectively, and methanol and DCM wash resin, and after normal-temperature vacuum is dry, the dry resin obtained is stand-by.
16) it cuts and falls polypeptide.The TFA/H of certain volume is added into the dry resin synthesis column 15) obtained2O/TIS(V/V/
V=95%/2.5%/2.5%) mixing liquid, stirring at normal temperature 2h, collection, which is cut, falls liquid concentrated by rotary evaporation to thick liquid, then will
It is added dropwise in cold ether and precipitates, and supernatant is removed after centrifugation, air drying 12h obtains peptide powder, and final products are low
Temperature freeze-drying is stand-by.
The first small-molecular peptides (OPA1) with oxygen carrying potential resulting to the present embodiment detect, and Fig. 1 is this implementation
The structure chart of the resulting OPA1 of example；Fig. 2 is the mass spectrogram of the resulting OPA1 of the present embodiment, and abscissa m/z indicates mass-to-charge ratio in figure,
Ordinate indicates that abscissa corresponds to the relative intensity of peak value.The result shows that: the actual molecular weight of OPA1 is consistent with theoretical molecular weight.
Fig. 3 is transmission electron microscope (TEM) figure of the nanometer spiral fiber of OPA1 self assembly provided in this embodiment；Fig. 4 is this reality
Atomic force microscope (AFM) figure of the nanometer spiral fiber of the OPA1 self assembly of example offer is provided；The result shows that: the self assembly of OPA1
Pattern be helical form nanofiber.
Embodiment 2
The present embodiment provides a kind of the second small-molecular peptides (OPA2) production methods that can produce active oxygen, and specific steps are such as
Under:
1) appropriate OPA1 (C is weighed17H31- CONH-VRGDS-COOH) in container, appropriate PBS is added in solid powder
(0.1M, pH 9.0) buffer solution injects air bubble 20 minutes under stiring；
2) appropriate lipoxygenase is weighed in container, and appropriate PBS (0.1M, pH 9.0) buffer solution is added；
3) by it is above-mentioned 2) in solution be added 1) described in solution in, kept for 5 DEG C of temperature, react 2h, acquired solution is close
Seal cryo-conservation.
The second small-molecular peptides (OPA2) that can produce active oxygen resulting to the present embodiment detect, and Fig. 5 is the present invention
The structure chart for the second small-molecular peptides that embodiment 2 provides；Fig. 6 is the mass spectrum for the second small-molecular peptides that the embodiment of the present invention 2 provides
Scheme, abscissa m/z indicates mass-to-charge ratio in figure, and ordinate indicates that abscissa corresponds to the relative intensity of peak value；The result shows that: second is small
The actual molecular weight of molecular peptide is consistent with theoretical molecular weight, and the second small-molecular peptides are C17H31O2-CONH-VRGDS-COOH。
Embodiment 3
The method that the second small-molecular peptides that active oxygen be can produce the present embodiment provides a kind of generate active oxygen, be specifically according to
According to embryonic stem-like cells, i.e., in Fe2+In the presence of, can produce in the second small-molecular peptides structure of active oxygen hydroperoxides (-
OOH it) generates and has cytotoxic active oxygen (ROS).
The active oxygen that the second small-molecular peptides that can produce active oxygen resulting to the present embodiment generate detects, and Fig. 7 is
By Fe after the second small-molecular peptides generation active oxygen that can produce active oxygen of the offer of the embodiment of the present invention 32+(concentration used is 100 μ
M) it is oxidized to Fe3+Color change figure；The result shows that: in OPA2 (C17H31O2- CONH-VRGDS-COOH) in the presence of, Fe2+
Fe can be oxidized to by it3+, FeCl2Solution becomes yellow clear from achromaticity and clarification is transparent.
In order to examine OPA2 to generate the effect of active oxygen, tested with the material in following table, to examine the present invention
The second small-molecular peptides that can produce active oxygen the fluorescence intensity of active oxygen is generated in 140min.
OPA2 (C is detected using ROS fluorescent index agent DCFH-DA17H31O2- CONH-VRGDS-COOH) in Fe2+Deposit
In the active oxygen (ROS) of lower generation, vitamin C (VC) is as active oxygen killer, and experimental results are shown in figure 8, and Fig. 8 is this hair
The fluorescence intensity change that the second small-molecular peptides and corresponding control group that bright embodiment 3 provides generate active oxygen in 140min becomes
Gesture figure.Abscissa Time represents meaning as the time in Fig. 8, and ordinate DCF.IF. represents activity oxygen indicator DCFH by active oxygen
(ROS) oxidation generates the fluorescence intensity of DCF, the generation of indirect proof active oxygen (ROS)；Experimental group 5 represents embodiment 3 and mentions in figure
The second small-molecular peptides supplied are in Fe2+In the presence of in 140min generate active oxygen (ROS) fluorescence intensity change trend, experiment
Group 1 represents activity oxygen indicator DCFH fluorescence intensity change trend in 140min.The result shows that: in Fe2+Catalytic action under,
OPA2 can quickly and efficiently generate ROS within the initial short time, significantly increase the fluorescence intensity of active oxygen indicator, with experiment
Group 1 compares, its active oxygen (ROS) generated makes the fluorescence intensity of active oxygen indicator increase about 250 times in 140min,
Illustrate the second small-molecular peptides (OPA2) of the offer of the embodiment of the present invention 3 in Fe2+Active oxygen is quickly generated under catalytic action.
In order to further examine the second small-molecular peptides to generate the effect of active oxygen, carried out with the material in following table real
It tests, it is strong with the fluorescence after examining the second small-molecular peptides that can produce active oxygen of the invention to generate active oxygen at the 12nd hour
Degree.
Fig. 9 is the second small-molecular peptides that can produce active oxygen that provide of the embodiment of the present invention 3 and corresponding control group the
The fluorescence detection figure that active oxygen is generated at 12 hours, abscissa Wavelength represents meaning as wavelength in figure, ordinate
DCF.IF. it represents activity oxygen indicator DCFH and is aoxidized the fluorescence intensity for generating DCF, indirect proof active oxygen by active oxygen (ROS)
(ROS) generation.
OPA2 (C is detected using ROS fluorescent index agent DCFH-DA17H31O2- CONH-VRGDS-COOH) by Fe2+Catalysis
The active oxygen (ROS) generated afterwards, vitamin C (VC) are used as active oxygen killer, and 5 curve of experimental group shows OPA2 in Fe in figure2+
In the presence of generate active oxygen (ROS), and the fluorescence intensity of active oxygen indicator increases to when being reacted to 12 hours
About 325, when to being added (i.e. experimental group 4) after active oxygen killer vitamin C (VC) co-cultures in experimental group 5, experimental group 4 it is glimmering
Luminous intensity is substantially reduced, it is suppressed that the generation of active oxygen；The result shows that: with the extension of time and in Fe2+In the presence of,
OPA2 can be more lasting generation have cytotoxic active oxygen (ROS).
In order to prove the active oxygen that the second small-molecular peptides generate for have cytotoxic singlet oxygen (1O2), with following tables
Material in lattice is tested, with the singlet oxygen for examining the second small-molecular peptides provided by the invention that can produce active oxygen to generate
(1O2)。
As shown in Figure 10, Figure 10 is the second small-molecular peptides that can produce active oxygen that provide of the embodiment of the present invention 3 and corresponding
Control group generate have cytotoxic singlet oxygen (1O2) fluorescence detection figure, abscissa Wavelength (nm) generation in figure
Table meaning is wavelength, ordinate1O2.IF. represent generate have cytotoxic singlet oxygen (1O2) fluorescence intensity.
Using with single selective singlet oxygen (1O2) green fluorescence index agent SOSG detects OPA2 (C17H31O2-
CONH-VRGDS-COOH) in Fe2+In the presence of generate singlet oxygen (1O2), vitamin C (VC) is as active oxygen killer.
The results showed that in Fe2+In the presence of, the second small-molecular peptides and the singlet oxygen with single selective
(1O2) after green fluorescence index agent SOSG is co-cultured (experimental group 5 i.e. in figure) showed that apparent fluorescence increases at 0.05 hour
By force, fluorescence intensity has lasting enhancing (experimental group 9 in such as figure) and in 12 hours, when to addition activity oxygen inhibition in experimental group 5
After agent vitamin C (VC) co-cultures (i.e. experimental group 4), in 0.05 hour and 12 hours, compared to experimental group 5 and experimental group 9,
Correspondingly, the fluorescence intensity of experimental group 4 and experimental group 8 is substantially reduced, and generated singlet oxygen is obviously suppressed, it was demonstrated that OPA2
By Fe2+Efficient catalytic generate the cytotoxic singlet oxygen of tool (1O2)。
In conclusion the embodiment of the present invention constructs a kind of the second small-molecular peptides that can produce active oxygen, in Fe2+Presence
Under, the cytotoxic active oxygen of tool can efficiently and be constantly generated in 12 hours, and this active oxygen is with cell
Toxicity singlet oxygen (1O2)。
In order to solve the problems, such as PDT process, the embodiment of the present invention using hydroperoxides as the generating source of active oxygen,
It is bonded building with Functional Polypeptides and is not necessarily to foreign vector and light source from the nanometer system for delivering hydroperoxides (- OOH), while not
Active oxygen radical can controllably be generated under the oxygen concentration of tumor region by relying on, and will effectively improve the effect for the treatment of malignant tumor.This
Inventive embodiments not only avoid inorganic nano-particle, and the biological safety that aggregation causes may occur in use process in vivo
Problem also effectively overcomes the generation of active oxygen to tumor region oxygen and H2O2The Dependence Problem of concentration.
Finally, method of the invention is only preferable embodiment, it is not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention
Within the scope of.
Claims (10)
1. a kind of the first small-molecular peptides with oxygen carrying potential, which is characterized in that first small-molecular peptides are successively by hydrophobic alkane
Base chain, hydrophobic amino acid, hydrophilic amino acid and N- Amino End Group sealing end are constituted.
2. a kind of the first small-molecular peptides with oxygen carrying potential as described in claim 1, which is characterized in that the hydrophobic alkyl
Chain contains cis- unsaturated double-bond functional group, and the cis- unsaturated double-bond functional group is oxidized under the catalysis of lipoxidase
It forms conjugated double bond and side group generates hydroperoxides.
3. a kind of the first small-molecular peptides with oxygen carrying potential as described in claim 1, which is characterized in that the hydrophobic amino
Acid is one or more of valine, glycine.
4. a kind of the first small-molecular peptides with oxygen carrying potential as described in claim 1, which is characterized in that the hydrophilic ammonia
Base acid is one or more of aspartic acid, serine, arginine.
5. a kind of the first small-molecular peptides with oxygen carrying potential as described in claim 1, which is characterized in that described first small point
The structural formula of sub- peptide is C17H31- CONH-VRGDS-COOH, wherein V is valine, and R is arginine, and G is glycine, and D is day
Aspartic acid, S are serine.
6. a kind of the first small-molecular peptides with oxygen carrying potential as claimed in claim 5, which is characterized in that described first small point
In the sequence of sub- peptide-RGD- sequence can hydrophilic and hydrophobic in balance peptide structure, and can also targets neoplastic cells.
7. a kind of the first small-molecular peptides with oxygen carrying potential as claimed in claim 5, which is characterized in that described first small point
Sub- peptide, as vector resin, is existed small-molecular peptides segment by FMOC polypeptide solid-state reaction method using dichloro trityl chloride resin
Successively extend from C-terminal to N-terminal to acquire on vector resin.
8. a kind of the second small-molecular peptides that can produce active oxygen, which is characterized in that second small-molecular peptides are used as right is wanted
Described in any item first small-molecular peptides of 1-7 are asked to obtain as the method for raw material enzymatically, the structure of second small-molecular peptides
Formula is C17H31O2- CONH-VRGDS-COOH, and V is valine, R is arginine, and G is glycine, and D is aspartic acid, and S is silk
Propylhomoserin.
9. a kind of prepare the method that can produce the second small-molecular peptides of active oxygen as claimed in claim 8, which is characterized in that packet
Include following steps:
S1, C is weighed17H31The PBS buffer solution that 0.1M and pH are 9.0 is added in container in-CONH-VRGDS-COOH solid powder
Dissolution is injected air bubble 20 minutes under uniform stirring；
S2, lipoxygenase is weighed in container, the PBS buffer solution that 0.1M and pH are 9.0 is added and dissolves；
S3, the solution in above-mentioned S2 is added in solution described in S1, is kept for 5 DEG C of temperature, react 2h, acquired solution sealing is low
Temperature saves.
10. a kind of method that second small-molecular peptides generate active oxygen, which is characterized in that small as claimed in claim 8 second
FeCl is added in molecular peptide2Solution, second small-molecular peptides are in Fe2+Lower generate of effect has cytotoxic active oxygen.
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| CN201910716784.0A CN110499349B (en) | 2019-08-05 | 2019-08-05 | First small molecule peptide with oxygen carrying potential, second small molecule peptide capable of generating active oxygen and preparation method thereof |
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Citations (4)
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| WO2001010450A1 (en) * | 1999-08-10 | 2001-02-15 | Imarx Therapeutics, Inc. | Targeted thrombolytic agents |
| WO2003090255A2 (en) * | 2002-04-18 | 2003-10-30 | Northwestern University | Encapsulation of nanotubes via self-assembled nanostructures |
| WO2004018628A2 (en) * | 2002-08-21 | 2004-03-04 | Northwestern University | Charged peptide-amphiphile solutions & self-assembled peptide nanofiber networks formed therefrom |
| WO2008067145A2 (en) * | 2006-11-09 | 2008-06-05 | Northwestern University | Self-assembling peptide amphiphiles |
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Patent Citations (4)
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| WO2001010450A1 (en) * | 1999-08-10 | 2001-02-15 | Imarx Therapeutics, Inc. | Targeted thrombolytic agents |
| WO2003090255A2 (en) * | 2002-04-18 | 2003-10-30 | Northwestern University | Encapsulation of nanotubes via self-assembled nanostructures |
| WO2004018628A2 (en) * | 2002-08-21 | 2004-03-04 | Northwestern University | Charged peptide-amphiphile solutions & self-assembled peptide nanofiber networks formed therefrom |
| WO2008067145A2 (en) * | 2006-11-09 | 2008-06-05 | Northwestern University | Self-assembling peptide amphiphiles |
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| GULER MO.等: "Presentation of RGDS Epitopes on Self-Assembled Nanofibers of Branched Peptide Amphiphiles", 《BIOMACROMOLECULES》 * |
| HARTGERINK JD.等: "Self-Assembly and Mineralization of Peptide-Amphiphile Nanofiber", 《SCIENCE》 * |
| LI QL.等: "A novel self-assembled oligopeptide amphiphile for biomimetic mineralization of enamel", 《BMC BIOTECHNOLOGY》 * |
| 胡廷章等: "植物脂肪氧化酶的研究进展 ", 《生物工程学报》 * |
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