CN106750262B - The synthesis of amphipathic block antibacterial peptide and its preparation method and application of assembly - Google Patents
The synthesis of amphipathic block antibacterial peptide and its preparation method and application of assembly Download PDFInfo
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Abstract
The present invention provides a kind of synthesis of amphipathic block antibacterial peptide and its preparation method and application of assembly.Amphipathic block antibacterial peptide is prepared by hydrophilic amino acid, hydrophobic amino acid, triphosgene, organic solvent, initiator, deprotection agent and precipitating reagent; amphiphilic diblock antibacterial peptide prepared by the present invention has wide spectrum and excellent anti-microbial property; it is a kind of economic, low toxicity and stable biomaterial, is with a wide range of applications and is worth；Its raw material is cheap and easily-available, low in cost；In addition, synthetic route of the invention is simple, condition is controllable.
Description
Technical field
The invention belongs to antibacterial biological material fields, and in particular to a kind of synthesis and its assembling of amphipathic block antibacterial peptide
The preparation method and application of body.
Background technique
Currently, leading to the generation and a wide range of propagation of super drug-resistant bacteria due to the long-term abuse of antibiotic, causing antibiosis
Element is reduced using effect or even some bacteriums have been difficult to be killed by antibiotic, such as carries the bacterium of MCR-1 gene.Therefore, it seeks
The antibacterial agent looked for novelty replaces conventional antibiotic very urgent.Although inorganic antiseptic such as Ag and its metal oxide etc. have compared with
Good antibiotic property, but its biocompatibility limits it and is widely used in human body；And organic antibacterial agent such as quaternary ammonium salt, halogenated
Amine, biguanides and chitosan class etc. are also limited its application because of the defects of poor biocompatibility or not satisfactory antibacterial effect.
Antibacterial peptide is a kind of peptide molecule for having and inhibiting, killing the microbial performances such as bacterium, fungi even virus, currently, antibacterial peptide
Preparation there are mainly two types of mode: first is that being obtained by biological Immune inducing in vivo separation, as Agricultural University Of South China Huang Ziran is taught
Research team obtains antibacterial peptide through artificial induction and extraction from tussah chrysalis；Second is that being synthesized by chemical synthesis, as liquid phase is closed
Cheng Fa, solid-phase synthesis and NCA ring-opening polymerisation method, Zhou Chuncai et al. cause NCA open loop with transition metal initiators and are prepared for one
Serial antibacterial peptide.
Summary of the invention
In view of the deficiencies of the prior art, primary and foremost purpose is to provide a kind of amphipathic block antibacterial peptide to the present invention.
Second object of the present invention is to provide a kind of synthesis of amphipathic block antibacterial peptide.
Third object of the present invention is to provide a kind of preparation method and application of assembly.
In order to achieve the above objectives, solution of the invention is:
A kind of amphipathic block antibacterial peptide, structural formula are as follows:
Wherein, the value range of X is the integer in 1-100, and the value range of Y is the integer in 1-100, the value model of Z
It encloses for the integer in 0-100, the value range of n is the integer in 1-5, R1And R3It is the hydrophilic radical of hydrophilic amino acid, R2It is
The hydrophobic grouping of hydrophobic amino acid；X, Y and Z respectively represents the degree of polymerization of the poly- polypeptide of each block, and n indicates R1And R2Representative
The repetition number of amphipathic copolymer, A indicate initiator.
A kind of preparation method of above-mentioned amphipathic block antibacterial peptide comprising following steps:
(1), the hydrophilic amino acid containing amido protecting group and triphosgene are reacted in organic solvent, is obtained hydrophilic
Acidic amino acid-N carboxy α amino acid anhydride monomer, hydrophobic amino acid and triphosgene are reacted in organic solvent, dredged
Aqueous amino acid-N carboxy α amino acid anhydride monomer；
(2), by 0.200-1.000mmol initiator and 0.200-100.000mmol hydrophilic amino acid-N- carboxyl-α-
Amino anhydride monomers mix in organic solvent, react under vacuum, obtain:
Wherein A indicates that initiator, Z ' indicate amido protecting group；
(3), it continuously adds hydrophobic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(4), step (2) and (3) n-1 times are repeated, are obtained after purification:
(5), it continuously adds hydrophilic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(6) by amphipathic block antibacterial peptide:
Blocking group Z ' is sloughed, is obtained:
Preferably, the deprotection reaction in step (6) includes the following steps:
(a), to amphipathic block antibacterial peptide:
Middle addition deprotection agent reaction, obtains mixed liquor；
(b), mixed liquor is added dropwise in precipitating reagent, takes precipitating as crude product；
(c), crude product is washed, is dialysed, it is dry, obtain amphipathic block antibacterial peptide:
Preferably, hydrophilic amino acid is one of arginine, lysine and histidine.
Preferably, hydrophobic amino acid is one of isoleucine, leucine, valine, tyrosine and phenylalanine.
Preferably, organic solvent is that tetrahydrofuran, n,N-Dimethylformamide, n,N-dimethylacetamide or dimethyl are sub-
One or more of sulfone.
Preferably, initiator is selected from one of n-hexylamine, diisopropylethylamine, n-butylamine, isobutyl amine and aniline.
Preferably, the molar ratio of hydrophilic amino acid and triphosgene is 2.3:1.
Preferably, the molar ratio of hydrophobic amino acid and triphosgene is 2.3:1.
Preferably, initiator and hydrophilic amino acid-N carboxy α amino acid anhydride monomer molar ratio are 1:1-100.
Preferably, hydrophilic amino acid-N carboxy α amino acid anhydride monomer and hydrophobic amino acid-N- carboxyl-alpha-amido
The molar ratio of anhydride monomers is 1:100-100:1.
Preferably, hydrophilic amino acid-N carboxy α amino acid anhydride monomer and hydrophobic amino acid-N- carboxyl-alpha-amido
The number for being alternately repeated addition of anhydride monomers is 0-4.
Preferably, the block number of amphipathic block antibacterial peptide is 2-11.
Preferably, in step (1), reaction temperature is 40-60 DEG C, reaction time 3-12h.
Preferably, in step (2), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C.
Preferably, in step (3), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C.
Preferably, in step (5), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C.
Preferably, deprotection agent is that trifluoroacetic acid, the ethanol solution containing 33wt%HBr and the acetic acid containing 33wt%HBr are molten
One of liquid.
Preferably, precipitating reagent be selected from one of water, n-hexane, hexamethylene, acetone, methanol, ethyl alcohol, propyl alcohol or butanol with
On.
Preferably, in step (a), reaction temperature is 35-55 DEG C, reaction time 3-5h.
A kind of assembly is self-assembly of by above-mentioned amphipathic block antibacterial peptide.
A kind of preparation method of above-mentioned assembly comprising following steps:
By amphipathic block antibacterial peptide:
It is dissolved in organic solvent, obtains suspension；Deionized water is added dropwise in suspension, dialyses, obtains assembly.
Preferably, organic solvent is one of tetrahydrofuran, dioxane and dimethyl sulfoxide.
Preferably, the time that deionized water is added dropwise is 5-30min, dialysis time 12-24h.
A kind of above-mentioned assembly is in the encapsulation and transport of drug, Targeting delivery, synthesizing nano-particle and the micro- reaction of chemistry
Device etc. application.
By adopting the above scheme, the beneficial effects of the present invention are:
The first, amphiphilic diblock antibacterial peptide prepared by the present invention has wide spectrum and excellent anti-microbial property.
The second, raw material of the invention is cheap and easily-available, low in cost；Synthetic route of the invention is simple, and condition is controllable.
Third, amphiphilic diblock antibacterial peptide prepared by the present invention are a kind of economic, low toxicity and stable biomaterial, tool
Have wide practical use and is worth.
Detailed description of the invention
Fig. 1 is the polydispersity index figure of assembly of the invention.
Fig. 2 is assembly (K of the invention30‐b‐F15) TEM figure.
Fig. 3 is assembly (K of the invention30‐b‐F30) TEM figure.
Fig. 4 is assembly (K of the invention30‐b‐F45) TEM figure.
Fig. 5 is assembly (K of the invention30‐b‐F45) cytotoxicity figure.
Specific embodiment
The present invention provides a kind of synthesis of amphipathic block antibacterial peptide and its preparation method and application of assembly.
<amphipathic block antibacterial peptide>
A kind of amphipathic block antibacterial peptide, structural formula are as follows:
Wherein, the value range of X is the integer in 1-100, and the value range of Y is the integer in 1-100, the value model of Z
It encloses for the integer in 0-100, the value range of n is the integer in 1-5, R1And R3It is the hydrophilic radical of hydrophilic amino acid, R2It is
The hydrophobic grouping of hydrophobic amino acid；X, Y and Z respectively represents the degree of polymerization of the poly- polypeptide of each block, and n indicates R1And R2Representative
The repetition number of amphipathic copolymer, A indicate initiator.
R1And R3One of hydrophilic radical selected from hydrophilic amino acid, such as: the ammonia of arginic guanidine radicals, lysine
The imidazole radicals of base and histidine.R1And R3It may be the same or different.
R2One of hydrophobic grouping selected from hydrophobic amino acid, such as: the first of the methyl of isoleucine, leucine
Base, the methyl of valine, the phenolic group of tyrosine and phenylalanine phenyl.
Work as n=1, when Z=0, amphipathic block antibacterial peptide is amphiphilic diblock antibacterial peptide.
Work as n=1, when Z=1, amphipathic block antibacterial peptide is amphipathic three block antibacterial peptide.
Work as n=2, when Z=0, amphipathic block antibacterial peptide is amphipathic four blocks antibacterial peptide.
Work as n=2, when Z=1, amphipathic block antibacterial peptide is amphipathic five blocks antibacterial peptide；And so on.
Antibacterial peptide is a kind of peptide molecule for having and inhibiting, killing the microbial performances such as bacterium and fungi even virus,
As one of nonspecific immunity system constituent in organism, there are the characteristics such as quick, broad-spectrum antiseptic.
Wherein, antibacterial peptide by N carboxy α amino acid anhydride (N-carboxy- α-anhydride, NCA) ring-opening polymerisation and
At.Antibacterial peptide is difficult to generate bacterial drug resistance, this is to have benefited from its mechanism of action to destroy bactericidal mechanism, i.e. antibacterial peptide molecule for film
Positive charge on chain is combined with the bacterial cell membrane with negative electrical charge by Electrostatic Absorption, and its hydrophobic segment can be inserted into bacterium
Inside destroys bacterial structure, causes the content in film to outflow, so as to cause bacterial death.
<preparation method of amphipathic block antibacterial peptide>
A kind of preparation method of above-mentioned amphipathic block antibacterial peptide comprising following steps:
(1), the hydrophilic amino acid containing amido protecting group and triphosgene are reacted in organic solvent, is obtained hydrophilic
Acidic amino acid-N carboxy α amino acid anhydride monomer, hydrophobic amino acid and triphosgene are reacted in organic solvent, dredged
Aqueous amino acid-N carboxy α amino acid anhydride monomer；
(2), by 0.200-1.000mmol initiator and 0.200-100.000mmol hydrophilic amino acid-N- carboxyl-α-
Amino anhydride monomers mix in 20-50mL organic solvent, react under vacuum, obtain:
Wherein A indicates that initiator, Z ' indicate amido protecting group；
(3), it continuously adds hydrophobic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(4), step (2) and (3) n-1 times are repeated, are obtained after purification:
(5), it continuously adds hydrophilic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(6) by amphipathic block antibacterial peptide:
Blocking group Z ' is sloughed, is obtained:
Wherein, hydrophilic amino acid-N carboxy α amino acid anhydride monomer can be lysine-N carboxy α amino acid anhydride
Monomer (Lysine-N-carboxy- α-amino-anhydride；Lys-NCA), hydrophobic amino acid-N- carboxyl-a-amino acid
Anhydride monomer can be phenylalanine-N carboxy α amino acid anhydride monomer (Phenylalanine-N-carboxy- α-amino-
anhydride；Phe‐NCA).
If selected hydrophilic amino acid contains more than one amino group, such as lysine and arginine, then select
An amino in hydrophilic amino acid need to be protected with benzyloxycarbonyl protecting group or tertbutyloxycarbonyl protecting group.
(a), to amphipathic block antibacterial peptide:
Middle addition 50-100mL deprotection agent reaction, obtains mixed liquor；
(b), mixed liquor is added dropwise in 500-1000mL precipitating reagent, takes precipitating as crude product；
(c), crude product is washed, is dialysed, it is dry, obtain amphipathic block antibacterial peptide:
Wherein, hydrophilic amino acid can be one of arginine, lysine and histidine.
Hydrophobic amino acid can be one of isoleucine, leucine, valine, tyrosine and phenylalanine.
Organic solvent can for tetrahydrofuran (THF), N,N-dimethylformamide (DMF), DMAC N,N' dimethyl acetamide or
One or more of dimethyl sulfoxide.
Initiator can be selected from one of n-hexylamine, diisopropylethylamine, n-butylamine, isobutyl amine and aniline.
Deprotection agent can be in trifluoroacetic acid, the ethanol solution containing 33wt%HBr and the acetic acid solution containing 33wt%HBr
One kind.
Precipitating reagent can be selected from one or more of water, n-hexane, hexamethylene, acetone, methanol, ethyl alcohol, propyl alcohol or butanol.
In step (1), reaction temperature can be 40-60 DEG C, and preferably 40 DEG C, the reaction time can be 3-12h, preferably
For 3h.
In step (2), the vacuum reaction time can be 2-12h, and preferably 12h, temperature can be 40-60 DEG C, preferably
It is 50 DEG C.
In step (3), the vacuum reaction time can be 2-12h, and preferably 12h, temperature can be 40-60 DEG C, preferably
It is 50 DEG C.
In step (5), the vacuum reaction time can be 2-12h, and preferably 12h, temperature can be 40-60 DEG C, preferably
It is 50 DEG C.
Since hydrophilic amino acid-N carboxy α amino acid anhydride monomer ring-opening polymerisation initiator is amine initiator,
After the completion of hydrophilic amino acid-N carboxy α amino acid anhydride monomer polymerization reaction, the end N- of the entire poly- polypeptide of hydrophily energy again
Continue to cause hydrophobic amino acid-N carboxy α amino acid anhydride monomer and carry out polymerization reaction, it may be assumed that entire poly- polypeptide is in heating, true
Itself is a macromole evocating agents in empty reaction system, continue polymerization reaction.
In step (5), similarly, the poly- polypeptide that (4) step obtains can be used as a macromolecular again in the reaction system
Initiator causes R3Representative hydrophilic amino acid-N carboxy α amino acid anhydride monomer carries out ring-opening polymerisation.
In step (a), reaction temperature can be 35-55 DEG C, and preferably 40 DEG C, the reaction time can be 3-5h, preferably
For 4h.
The molar ratio of hydrophilic amino acid and triphosgene can be 2.3:1.
The molar ratio of hydrophobic amino acid and triphosgene can be 2.3:1.
Initiator and hydrophilic amino acid-N carboxy α amino acid anhydride monomer molar ratio can be 1:1-100, preferably
For 1:30.
Hydrophilic amino acid-N carboxy α amino acid anhydride monomer and hydrophobic amino acid-N- carboxyl-alpha-acid anhydride monomer
Molar ratio can be 1:100-100:1, preferably 2:1.
It is alternately repeated the hydrophilic amino acid-N carboxy α amino acid anhydride monomer and hydrophobic amino acid-N- carboxylic of addition
Base-a-amino acid anhydride monomer number can be 0-4.
The block number of amphipathic block antibacterial peptide can be 2-11, preferably 2.
<assembly>
A kind of assembly is self-assembly of by above-mentioned amphipathic block antibacterial peptide.
Self assembly refer to basic structural unit be based on non-covalent interaction, as hydrogen bond, hydrophobic effect, Van der Waals force and
Pi-pi bond accumulation etc., the stabilization or meta-stable that spontaneously form and the process with certain regular geometric structure.
Wherein, assembly can have a patterns such as rod-shaped micelle, vesica and vermiform, partial size 300-800nm, and more points of particle
Dissipating sex index (Polydispersity index, PDI) can be 0.005-0.3.
<preparation method of assembly>
A kind of preparation method of above-mentioned assembly comprising following steps:
By amphipathic block antibacterial peptide:
It is dissolved in 2.0-5.0mL organic solvent, obtains suspension；5.0-10.0mL deionized water is added dropwise in suspension, dialyses,
Obtain assembly.
Wherein, organic solvent can be one of tetrahydrofuran, dioxane and dimethyl sulfoxide, preferably tetrahydro furan
It mutters.
The time that deionized water is added dropwise can be 5-30min, and preferably 10min, dialysis time can be 12-24h, preferably
For for 24 hours.
<application of assembly>
A kind of above-mentioned assembly can be micro- in the encapsulation and transport of drug, Targeting delivery, synthesizing nano-particle and chemistry
Reactor etc. application.
The present invention will be further described with reference to the accompanying drawings.
Embodiment 1:
Step 1: the preparation method of amphiphilic diblock antibacterial peptide
The preparation method of the amphiphilic diblock antibacterial peptide of the present embodiment comprising following steps:
(1), 30.000g (107.14mmol) benzyloxycarbonyl group-lysine and 13.790g (46.43mmol) triphosgene are existed
By necleophilic reaction in 40mLDMF, temperature is 40 DEG C, time 3h, and obtaining Z '-Lys-NCA monomer, (Z ' indicates that benzyloxycarbonyl group is protected
Protect base), 30.000g (181.18mmol) phenylalanine and 23.400g (78.78mmol) triphosgene are passed through in 40mL DMF
Necleophilic reaction, temperature are 40 DEG C, and time 3h obtains Phe-NCA monomer；
(2), by 0.022mL (0.217mmol) isobutyl amine (as initiator) and 2.000g (6.540mmol) Z '-Lys-
NCA monomer (Z ' indicates benzyloxycarbonyl protecting group), which is dissolved in 20mL (260mmol) DMF, carries out ring-opening polymerisation, reacts under vacuum
12h, temperature are 50 DEG C, obtain the hydrophilic amino acid block that the degree of polymerization is 30；
(3), continue addition 0.624g (3.27mmol) the Phe-NCA monomer in polyaminoacid solution to react under vacuum
12h, temperature are 50 DEG C, obtain the hydrophobic amino acid block that the degree of polymerization is 15；
(4), continue to be alternately repeated addition Z '-Lys-NCA monomer (Z ' expression benzyl according to the reaction condition of step (2) and (3)
Oxygen carbonyl protecting group) and Phe-NCA monomer 0 time, excessive DMF is evaporated off in vacuum backspin, and deionized water is added and is precipitated, obtains
To the first crude product, continues repeatedly to wash the first crude product with deionized water, further remove DMF, finally rotate under vacuum
Deionized water is removed, amphiphilic diblock antibacterial peptide (Z '-Lys) is obtained30‐b‐Phe15(Z ' indicates benzyloxycarbonyl protecting group)；
(5), to amphiphilic diblock antibacterial peptide (Z '-Lys)30‐b‐Phe15It is added in (Z ' indicates benzyloxycarbonyl protecting group)
Reaction 4h is sufficiently stirred in the acetic acid solution (as deprotection agent) containing 33wt%HBr of 50mL, and temperature is 40 DEG C, all dissolves
After obtain mixed liquor；Mixed liquor is added dropwise in 500mL acetone (as precipitating reagent), crude product is precipitated out, obtains second
Crude product；Second crude product is dissolved in deionized water, for 24 hours, is during which changed once every 2h with bag filter (Mn=3500) dialysis
Water, finally revolving removes deionized water under vacuum, obtains amphiphilic diblock antibacterial peptide K30‐b‐F15(wherein K is lysine,
F is phenylalanine).
Wherein, the structural formula of Z '-Lys-NCA monomer (Z ' indicates benzyloxycarbonyl protecting group) are as follows:
The structural formula of Phe-NCA monomer are as follows:
Hydrophilic amino acid can be one of arginine, lysine and histidine.
Hydrophobic amino acid can be one of isoleucine, leucine, valine, tyrosine and phenylalanine.
Organic solvent can for tetrahydrofuran (THF), N,N-dimethylformamide (DMF), DMAC N,N' dimethyl acetamide or
One or more of dimethyl sulfoxide.
Initiator can be selected from one of n-hexylamine, diisopropylethylamine, n-butylamine, isobutyl amine and aniline.
Deprotection agent can be in trifluoroacetic acid, the ethanol solution containing 33wt%HBr and the acetic acid solution containing 33wt%HBr
One kind.
Precipitating reagent can be selected from one or more of water, n-hexane, hexamethylene, acetone, methanol, ethyl alcohol, propyl alcohol or butanol.
In step (1), within 40-60 DEG C, the reaction time is possible reaction temperature within 3-12h.
In step (2), the vacuum reaction time, temperature was possible within 40-60 DEG C within 2-12h.
In step (3), the vacuum reaction time, temperature was possible within 40-60 DEG C within 2-12h.
In step (5), within 35-55 DEG C, the reaction time is all possible deprotection reaction temperature within 3-5h.
Initiator and hydrophilic amino acid-N carboxy α amino acid anhydride monomer molar ratio are within 1:1-100 can be with
's.
Hydrophilic amino acid-N carboxy α amino acid anhydride monomer and hydrophobic amino acid-N carboxy α amino acid anhydride list
The molar ratio of body is possible within 1:100-100:1.
The block number of amphipathic block antibacterial peptide is possible within 2-11.
Step 2: being self-assembly of antibacterial peptide rod-shaped micelle assembly
The preparation method of the antibacterial peptide rod-shaped micelle assembly of the present embodiment comprising following steps:
By 5.0mg (7.294 × 10‐4mmol)K30‐b‐F15It is dissolved in 2.0mL (24.7mmol) THF, 2h is sufficiently stirred, obtains
To suspension；Slowly add 5.0mL deionized water dropwise in suspension, time 10min is stirred overnight after being added dropwise, stirs
After the completion of mixing, for 24 hours with bag filter (Mn=3500) dialysis, a water during which is changed every 2h, THF is removed, obtains K30‐b‐F15It is anti-
Bacterium peptide rod-shaped micelle assembly.
Wherein, assembly can be the patterns such as rod-shaped micelle, vesica and vermiform vesica.
Organic solvent is that one of tetrahydrofuran, dioxane and dimethyl sulfoxide are possible.
The time of deionized water is added dropwise within 5-30min, dialysis time is possible within 12-24h.
Embodiment 2:
Step 1: the preparation method of amphiphilic diblock antibacterial peptide
The preparation method of the amphiphilic diblock antibacterial peptide of the present embodiment comprising following steps:
(1), 30.000g (107.14mmol) benzyloxycarbonyl group-lysine and 13.790g (46.43mmol) triphosgene are existed
By necleophilic reaction in 40mL DMF, temperature is 40 DEG C, time 3h, and obtaining Z '-Lys-NCA monomer, (Z ' indicates benzyloxycarbonyl group
Protecting group), 30.000g (181.18mmol) phenylalanine and 23.400g (78.78mmol) triphosgene are led in 40mL DMF
Necleophilic reaction is crossed, temperature is 40 DEG C, and time 3h obtains Phe-NCA monomer；
(2), by 0.022mL (0.217mmol) isobutyl amine (as initiator) and 2.000g (6.540mmol) Z '-Lys-
NCA monomer (Z ' indicates benzyloxycarbonyl protecting group), which is dissolved in 20mLDMF, carries out ring-opening polymerisation, reacts 12h, temperature under vacuum
It is 50 DEG C, obtains the hydrophilic amino acid block that the degree of polymerization is 30；
(3), continue addition 1.25g (6.55mmol) the Phe-NCA monomer in polyaminoacid solution to react under vacuum
12h, temperature are 50 DEG C, obtain the hydrophobic amino acid block that the degree of polymerization is 30；
(4), continue to be alternately repeated addition Z '-Lys-NCA monomer (Z ' expression benzyl according to the reaction condition of step (2) and (3)
Oxygen carbonyl protecting group) and Phe-NCA monomer 0 time, excessive DMF is evaporated off in vacuum backspin, and deionized water is added and is precipitated, obtains
To the first crude product, continues repeatedly to wash the first crude product with deionized water, further remove DMF, finally rotate under vacuum
Deionized water is removed, amphiphilic diblock antibacterial peptide (Z '-Lys) is obtained30‐b‐Phe30(Z ' indicates benzyloxycarbonyl protecting group)；
(5), to amphiphilic diblock antibacterial peptide (Z '-Lys)30‐b‐Phe30It is added in (Z ' indicates benzyloxycarbonyl protecting group)
Reaction 4h is sufficiently stirred in the acetic acid solution (as deprotection agent) containing 33wt%HBr of 50mL, and temperature is 40 DEG C, all dissolves
After obtain mixed liquor；Mixed liquor is added dropwise in 500mL acetone (as precipitating reagent), crude product is precipitated out, obtains second
Crude product；Second crude product is dissolved in deionized water, for 24 hours, is during which changed once every 2h with bag filter (Mn=3500) dialysis
Water, finally revolving removes deionized water under vacuum, obtains amphiphilic diblock antibacterial peptide K30‐b‐F30(wherein K is lysine,
F is phenylalanine).
Step 2: being self-assembly of antibacterial peptide vesica assembly
The preparation method of the antibacterial peptide vesica assembly of the present embodiment comprising following steps:
By 5.0mg (5.3590 × 10‐4mmol)K30‐b‐F30It is dissolved in 2.0mL THF, 2h is sufficiently stirred, obtain suspended
Liquid；Slowly add 5.0mL deionized water dropwise in suspension, time 10min is stirred overnight after being added dropwise, and stirring is completed
Afterwards, for 24 hours with bag filter (Mn=3500) dialysis, a water during which is changed every 2h, THF is removed, obtains K30‐b‐F30Antibacterial peptide capsule
Steep assembly.
Embodiment 3:
Step 1: the preparation method of amphiphilic diblock antibacterial peptide
The preparation method of the amphiphilic diblock antibacterial peptide of the present embodiment comprising following steps:
(1), 30.000g (107.14mmol) benzyloxycarbonyl group-lysine and 13.790g (46.43mmol) triphosgene are existed
By necleophilic reaction in 40mL DMF, temperature is 40 DEG C, time 3h, and obtaining Z '-Lys-NCA monomer, (Z ' indicates benzyloxycarbonyl group
Protecting group), 30.000g (181.18mmol) phenylalanine and 23.400g (78.78mmol) triphosgene are led in 40mL DMF
Necleophilic reaction is crossed, temperature is 40 DEG C, and time 3h obtains Phe-NCA monomer；
(2), by 0.022mL (0.217mmol) isobutyl amine (as initiator) and 2.000g (6.540mmol) Z '-Lys-
NCA monomer (Z ' indicates benzyloxycarbonyl protecting group), which is dissolved in 20mL DMF, carries out ring-opening polymerisation, reacts 12h under vacuum, temperature
Degree is 50 DEG C, obtains the hydrophilic amino acid block that the degree of polymerization is 30；
(3), continue addition 1.87g (9.80mmol) the Phe-NCA monomer in polyaminoacid solution to react under vacuum
12h, temperature are 50 DEG C, obtain the hydrophobic amino acid block that the degree of polymerization is 45；
(4), continue to be alternately repeated addition Z '-Lys-NCA monomer (Z ' expression benzyl according to the reaction condition of step (2) and (3)
Oxygen carbonyl protecting group) and Phe-NCA monomer 0 time, excessive DMF is evaporated off in vacuum backspin, and deionized water is added and is precipitated, obtains
To the first crude product, continues repeatedly to wash the first crude product with deionized water, further remove DMF, finally rotate under vacuum
Deionized water is removed, amphiphilic diblock antibacterial peptide (Z '-Lys) is obtained30‐b‐Phe45(Z ' indicates benzyloxycarbonyl protecting group)；
(5), to amphiphilic diblock antibacterial peptide (Z '-Lys)30‐b‐Phe45It is added in (Z ' indicates benzyloxycarbonyl protecting group)
Reaction 4h is sufficiently stirred in the acetic acid solution (as deprotection agent) containing 33wt%HBr of 50mL, and temperature is 40 DEG C, all dissolves
After obtain mixed liquor；Mixed liquor is added dropwise in 500mL acetone (as precipitating reagent), crude product is precipitated out, obtains second
Crude product；Second crude product is dissolved in deionized water, for 24 hours, is during which changed once every 2h with bag filter (Mn=3500) dialysis
Water, finally revolving removes deionized water under vacuum, obtains amphiphilic diblock antibacterial peptide K30‐b‐F45(wherein K is lysine,
F is phenylalanine).
Step 2: being self-assembly of antibacterial peptide vermiform vesica assembly
The preparation method of the antibacterial peptide vermiform vesica assembly of the present embodiment comprising following steps:
By 5.0mg (4.235 × 10‐4mmol)K30‐b‐F45It is dissolved in 2.0mL THF, 2h is sufficiently stirred, obtain suspension；
Slowly adding 5.0mL deionized water dropwise in suspension, time 10min is stirred overnight after being added dropwise, after the completion of stirring,
For 24 hours with bag filter (Mn=3500) dialysis, a water during which is changed every 2h, THF is removed, obtains K30‐b‐F45Antibacterial peptide vermiform
Assembly.
Embodiment 4:
The preparation method of the amphipathic three block antibacterial peptide of the present embodiment comprising following steps:
(1), 30.000g (107.14mmol) benzyloxycarbonyl group-lysine and 13.790g (46.43mmol) triphosgene are existed
By necleophilic reaction in 40mL DMF, temperature is 40 DEG C, time 3h, and obtaining Z '-Lys-NCA monomer, (Z ' indicates benzyloxycarbonyl group
Protecting group), 30.000g (181.18mmol) phenylalanine and 23.400g (78.78mmol) triphosgene are led in 40mL DMF
Necleophilic reaction is crossed, temperature is 40 DEG C, and time 3h obtains Phe-NCA monomer；
(2), by 0.022mL (0.217mmol) isobutyl amine (as initiator) and 2.000g (6.540mmol) Z '-Lys-
NCA monomer (Z ' indicates benzyloxycarbonyl protecting group), which is dissolved in 20mL (260mmol) DMF, carries out ring-opening polymerisation, reacts under vacuum
12h, temperature are 50 DEG C, obtain the hydrophilic amino acid block that the degree of polymerization is 30；
(3), continue addition 0.624g (3.27mmol) the Phe-NCA monomer in polyaminoacid solution to react under vacuum
12h, temperature are 50 DEG C, obtain the hydrophobic amino acid block that the degree of polymerization is 15；
(4), continue to be alternately repeated addition Z '-Lys-NCA monomer (Z ' expression benzyl according to the reaction condition of step (2) and (3)
Oxygen carbonyl protecting group) and Phe-NCA monomer 0 time, and 2.168g (6.510mmol) Z '-is continuously added in the reaction system
Arg-NCA monomer (Z ' indicates benzyloxycarbonyl protecting group) reacts 12h under vacuum, and temperature is 50 DEG C, and obtaining the degree of polymerization is 30
Hydrophily arginine block；
(5) excessive DMF is evaporated off in vacuum backspin, and deionized water is added and is precipitated, obtains the first crude product, continues to use
Deionized water repeatedly washs the first crude product, further removes DMF, and finally revolving removes deionized water under vacuum, obtains two
Parent's property three block antibacterial peptide (Z '-Lys)30‐b‐Phe15‐b‐(Z’‐Arg)30(Z ' indicates benzyloxycarbonyl protecting group)；
(6) to amphipathic three block antibacterial peptide (Z '-Lys)30‐b‐Phe15‐b‐(Z’‐Arg)30(Z ' indicates that benzyloxycarbonyl group is protected
Protect base) in be added 50mL the acetic acid solution (as deprotection agent) containing 33wt%HBr, be sufficiently stirred reaction 4h, temperature 40
DEG C, all mixed liquor is obtained after dissolution；Mixed liquor is added dropwise in 500mL acetone (as precipitating reagent), crude product is settled out
Come, obtains the second crude product；Second crude product is dissolved in deionized water, it is for 24 hours, during which every with bag filter (Mn=3500) dialysis
A water is changed every 2h, finally revolving removes deionized water under vacuum, obtains amphipathic three block antibacterial peptide K30‐b‐F15‐b‐A30
(wherein K is lysine, and F is phenylalanine, and A is arginine).
<experiment>
It is tested as follows using the amphiphilic diblock antibacterial peptide of above-described embodiment and assembly as product.
<experiment 1>
This experiment is to verify amphiphilic diblock antibacterial peptide in gram-positive bacteria (staphylococcus aureus) and leather
The anti-microbial property of Lan Shi negative bacterium (Escherichia coli).
Minimal inhibitory concentration (MIC) is to assess the important parameter of antibacterial antiplaque agent performance.This experiment uses gram respectively
Negative bacterium (Escherichia coli) and gram-positive bacteria (staphylococcus aureus) measure the antibacterial of amphiphilic diblock antibacterial peptide
Performance.Experimental procedure is as follows:
(1) the LB bone broth of 10mL is added in culture dish；
(2) the LB bone broth of 160 μ L is added in the first lattice of the first row of 96 orifice plates, 100 μ L are added in subsequent every lattice
LB bone broth.Then the amphiphilic diblock antibacterial peptide aqueous solution that 40 μ L concentration are 5mg/mL is added in the first lattice, sufficiently
Mixing takes the 100 μ L mixed liquors to be added in 96 the second lattice of orifice plate the first row, is uniformly mixed, and then takes the 100 μ L mixed liquors again
To the row third lattice, and so on；
(3) by 10 μ L, activated bacterium is added in the 10mL LB bone broth in step (1), is then therefrom respectively taken
100 μ L are added in the grid of mixed liquor of each of step (2).
It is added (staphylococcus aureus or Escherichia coli) after 100 μ L bacterium liquid, amphipathic two in the mixed liquor of 96 orifice plates
Block antibacterial peptide concentration is respectively 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL, 31.25 μ g/mL, 15.63 μ g/
ML, 7.81 μ g/mL, 3.90 μ g/mL, 1.95 μ g/mL and 0.98 μ g/mL, and read after 18 hours.Amphiphilic diblock
The test result of antibacterial peptide and natural antibacterial peptide is as shown in table 1.
Table 1: the MIC value of amphiphilic diblock antibacterial peptide and natural antibacterial peptide
Wherein, MIC (Minimum inhibitory concentration) is minimum inhibitory concentration, i.e. MIC value is smaller,
Its anti-microbial property is better.
As can be seen from the above table, the MIC value (2-16) of amphiphilic diblock antibacterial peptide prepared by the present invention compares natural antibacterial
The MIC value of peptide is small, and all has to Escherichia coli (Gram-negative bacteria) and staphylococcus aureus (gram-positive bacteria)
Therefore good bactericidal activity illustrates that amphiphilic diblock antibacterial peptide prepared by the present invention has wide spectrum and excellent antibiotic property
Energy.
<experiment 2>
The purpose of this experiment is to study the assembling situation of assembly.
Particle polydispersity index PDI (Polydispersed index) expression of assembly particle diameter distribution situation, when
When PDI is less than 0.3, then it represents that the particle size dispersion of assembly is uniform.Researcher is first each by assembled assembly difference
It takes 1.5mL to be put into glass dish, is then placed in Zetasizer Nano serial nano granularity and zeta potential instrument and carries out dynamic
Light scattering test.
Assembly Kn‐b‐Fm(n=30, m=15,30 and particle polydispersity index PDI value 45) as shown in Figure 1, its
In, (a) n:m=2:1, (b) n:m=1:1；(c) n:m=2:3.
As shown in Figure 1, the partial size of assembly is between 450-700nm, and PDI value is respectively less than 0.3, illustrates system of the present invention
The uniform particle sizes of standby assembly particle, it is well dispersed, can preliminary judgement assemble successfully.
The transmission electron microscope shape appearance figure of assembly is as shown in figs 2-4:
The good amphiphilic diblock antibacterial peptide aqueous solution of self assembly is diluted to 200 μ g/mL by researcher at room temperature, is taken
10 μ L drops are dyed after being dried at room temperature for phosphotungstic acid (PTA, 1.0wt%) solution on copper mesh.Take a drop PTA solution
Drop is placed on PTA drop top in hydrophobic membrane, by what copper mesh had loaded sample on one side, infiltrates 1min, extra with Adsorption of Filter Paper
PTA solution after, copper mesh is dried at room temperature for a night, transmission electron microscope test can be carried out.
Assembly Kn‐b‐FmThe TEM figure of (n=10-50, m=10-100) is as shown in figs 2-4, wherein (a) n:m=2:
1；(b) n:m=1:1；(c) n:m=2:3.
It can be seen from Fig.2-Fig.4 that amphiphilic diblock antibacterial peptide K30‐b‐F15, K30‐b‐F30And K30‐b‐F45It is received being assembled into
Its pattern is respectively rod-shaped micelle, vesica and vermiform vesica after rice grain.
<experiment 3>
The purpose of this experiment is to study amphiphilic diblock antibacterial peptide K30‐b‐F45Vermiform vesica assembly to L02
The toxicity of cell.
This experiment measures the toxicity for L02 (normal human liver cell) by CCK-8 kit.Researcher uses
100 μ L cell suspensions (4000) and culture medium is added in 37 DEG C, the incubator of 5% relative humidity in 96 hole bed boards, every hole
It cultivates together for 24 hours, CO is full of in incubator2；Then 31.25 μ g/mL, 62.5 μ are separately added into the cell suspension in each hole
G/mL, 125 μ g/mL, 250 μ g/mL, 500 μ g/mL and 1000 μ g/mL amphiphilic diblock antibacterial peptide vesicle solution be further continued for
Culture is for 24 hours.Use not the cell with the processing of antibacterial peptide solution as blank control group.Experimental group and control group culture complete it
Afterwards, CCK-8 coloring agent is added in each aperture, cultivates 1h at 37 DEG C.Researcher uses double wave by microplate reader
Absorbance of the sample of regular way measurement experiment group and control group at 450nm and 630nm.Each sample of experimental group and control group
Duplicate measurements four times, cell survival rate is obtained according to the ratio calculation of normal liver cell survival volume and control group liver cell total amount,
Test results are shown in figure 5, wherein abscissa indicates vermiform vesica concentration (Vesicle concentration), ordinate
It indicates cell survival rate (Relative cell viability).
As shown in Figure 5, when the concentration of amphiphilic diblock antibacterial peptide solution is 31.25 μ g/mL, cell survival rate still has
85% or more, it may be assumed that when minimum inhibitory concentration is 2 μ g/mL (gram-positive bacteria) and 8 μ g/mL (Gram-negative bacteria), human body
Hepatocyte viability can reach 85% or more, therefore, amphiphilic diblock antibacterial peptide K prepared by the present invention30‐b‐F45Worm
Shape vesica is very low to the toxicity of cell.
The above-mentioned description to embodiment is that this hair can be understood and used for the ease of those skilled in the art
It is bright.Those skilled in the art obviously readily can make various modifications to these embodiments, and described herein one
As principle be applied in other embodiments, without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments.
Those skilled in the art's principle according to the present invention, not departing from improvement that scope of the invention is made and modification all should be at this
Within the protection scope of invention.
Claims (7)
1. a kind of amphipathic three block antibacterial peptide, it is characterised in that: structural formula is as follows:
Wherein, the value range of X is the integer in 1-100, and the value range of Y is the integer in 1-100, and the value range of Z is
Integer in 1-100, R1And R3It is the hydrophilic radical of hydrophilic amino acid, R2It is the hydrophobic grouping of hydrophobic amino acid；X, Y and Z
The degree of polymerization of the poly- polypeptide of each block is respectively represented, A indicates initiator.
2. a kind of preparation method of amphipathic three block antibacterial peptide as described in claim 1, it is characterised in that: including walking as follows
It is rapid:
(1), the hydrophilic amino acid containing amido protecting group and triphosgene are reacted in organic solvent, obtains hydrophilic ammonia
Base acid-N carboxy α amino acid anhydride monomer, hydrophobic amino acid and triphosgene are reacted in organic solvent, obtain hydrophobicity
Amino acid-N carboxy α amino acid anhydride monomer；
(2), by hydrophilic amino acid-N- carboxyl-α-described in 0.200-1.000mmol initiator and 0.200-100.000mmol
Amino anhydride monomers mix in organic solvent, react under vacuum, obtain:
Wherein A indicates that initiator, Z ' indicate amido protecting group；
(3), it continuously adds the hydrophobic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(4), it continuously adds hydrophilic amino acid-N carboxy α amino acid anhydride monomer and reacts under vacuum, obtain:
(5), by the amphipathic block antibacterial peptide:
Blocking group Z ' is sloughed, is obtained:
3. preparation method according to claim 2, it is characterised in that: the hydrophilic amino acid is arginine, lysine
One of with histidine；Alternatively,
The hydrophobic amino acid is one of isoleucine, leucine, valine, tyrosine and phenylalanine；Alternatively,
The initiator is selected from one of n-hexylamine, diisopropylethylamine, n-butylamine, isobutyl amine and aniline；Alternatively,
The molar ratio of the hydrophilic amino acid and the triphosgene is 2.3:1；Alternatively,
The molar ratio of the hydrophobic amino acid and the triphosgene is 2.3:1；Alternatively,
The initiator and the hydrophilic amino acid-N carboxy α amino acid anhydride monomer molar ratio are 1:1-100；Alternatively,
The hydrophilic amino acid-N carboxy α amino acid anhydride monomer and the hydrophobic amino acid-N- carboxyl-a-amino acid
The molar ratio of anhydride monomer is 1:100-100:1.
4. preparation method according to claim 2, it is characterised in that: in the step (1), reaction temperature is 40-60 DEG C,
Reaction time is 3-12h；Alternatively,
In the step (2), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C；Alternatively,
In the step (3), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C；Alternatively,
In the step (4), the vacuum reaction time is 2-12h, and temperature is 40-60 DEG C.
5. a kind of assembly, it is characterised in that: be self-assembly of by amphipathic three block antibacterial peptide as described in claim 1.
6. a kind of preparation method of assembly as claimed in claim 5, characterized by the following steps:
By amphipathic three block antibacterial peptide:
It is dissolved in organic solvent, obtains suspension；Deionized water is added dropwise in suspension, dialyses, obtains assembly.
7. the preparation method of assembly according to claim 6, it is characterised in that:
The organic solvent is one of tetrahydrofuran, dioxane and dimethyl sulfoxide；Alternatively,
The time that deionized water is added dropwise is 10-30min；Alternatively,
The dialysis time is 12-24h.
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