CN103087149A - Peptide fragment-containing cyanoacrylate derivative as well as preparation method and use thereof - Google Patents
Peptide fragment-containing cyanoacrylate derivative as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention relates to a peptide fragment-containing cyanoacrylate derivative, wherein the structure of the derivative is shown by a formula I, a formula II and a formula III, respectively. The ethylenic bond in the derivative can be initiated by anions to quickly carry out intermolecular polymerization and generate adhesive-glue adhesive capacity; the peptide fragment-containing cyanoacrylate derivative can be independently used or can be combined with other cyanoacrylate substances for use; moreover, the peptide fragment-containing cyanoacrylate derivative can be used as a degradable medical adhesive for adhering the wound, stopping bleeding for the large-area wound, as well as closing wounds of the internal organs and soft tissues; and besides, the peptide fragment-containing cyanoacrylate derivative also can be used as a universal modification way of peptide for slow release, and further also can be used for preparing tissue engineering materials.
Description
Technical field
The present invention relates to a kind of alpha-cyanoacrylate derivative, particularly relate to a kind of alpha-cyanoacrylate derivative that contains peptide fragment.Intermolecular polymerization can occur rapidly in the ethylene linkage in this derivative under the anionic initiation condition, and produce bonding stickiness, can share separately or with other cyanoacrylate material, as the biodegradable adhesive of medical, be used for wound bonding, the extensive wound hemostasis, and internal organ and soft tissue wound closure etc., also can be used as the general modification mode of peptide, be used for the slowly-releasing purpose, also can be used for preparing tissue engineering material.
Background technology
The α-cyanoacrylate quasi-molecule, owing to combining cyano group, ester group on alpha-carbon atom in its structure, so two bonding electron cloud density are very low, have very strong suction electrical, just can moment produce the intermolecular polymerization of negatively charged ion mediation under the negatively charged ion (the weak base base material of the amino that has as the biological tissue surface, hydroxyl and trace such as aqueous vapor etc.) of denier exists.
The more structure of this quasi-molecule research is mainly that the side chain ester group is less than the α-cyanoacrylatealkyl of 8 carbon.People have been developed its purposes as adhesive of medical.Usually free state be liquid, by sprinkling or the mode of smearing, after touching tissue, polymerization (less than 30 seconds) occurs rapidly and solidifies during use, and the polyreaction that occurs causes desired bonding or sealing intensity.Nineteen fifty-nine first cyanoacrylate sizing agent Eastman910(a-Methyl 2-cyanoacrylate that gone on the market), be used for binding skin and hemostasis.Various countries have synthesized a large amount of cyanoacrylate sizing agents subsequently, for example the AU-CRYLATE(main component of the U.S. be alpha-cyanoacrylate just/isobutyl ester), Germany HISTOACRYL BLUE(main component be α-cyanoacrylaten-butyl).China rose and begins one's study and produce medical adhesive in 1962, and principal item comprises good fortune philharmonic (the positive fourth/n-octyl of alpha-cyanoacrylate), alpha-cyanoacrylate butyl ester, isobutyl ester, n-octyl etc.
The appearance of adhesive of medical, changed the concept that only relies on needlework to sew up a wound on the traditional medicine, by bonding mode, substitute the stitching of needlework, both had simple to operate, wound is not caused the advantage of secondary damage, also have can use or assistance application in the advantage as positions such as the soft tissue of the more difficult stitching of the operation needlework such as liver, pancreas, internal organ, bones.In addition;, scratch hemorrhage for the big area shallow, burn, Battlefield Damage, field harm, intestines leakage etc.; the mode that this class adhesive of medical can also stop blooding by spreadability forms the protectiveness film at wound, thereby reaches hemostasis and the extraneous purpose that infects of isolation.
But there are some shortcomings in these α-cyanoacrylatealkyls, and for example polymkeric substance hardness is large, and snappiness is low, the skin poor compliance; And the linear high polymer that obtains due to monomer polymerization; its linear main chain is polymerized by carbon-carbon double bond; the carbon-carbon bond energy level is higher; be not easy destroyed; therefore this class polymer degradation is very slow, and this has also caused foreign body reaction, may cause new inflammation; even hinder wound and further heal, reduced them as the practical value of surgical adhesive/sealing agent.
For the structural modification of α-cyanoacrylate, can improve its performance, perhaps give this quasi-molecule with new purposes.
United States Patent (USP) discloses for No. 3995641 the ester chain portion has been modified, and is included in the method that this structure division is introduced more ester bond site such as alkoxycarbonyl alkyl ester, can accelerate polymer degradation.Chinese patent CN101180085A discloses and has contained the structure of a plurality of ester bonds and spacer groups in the side-chain structure with accelerated degradation.
Chinese patent CN1944399A partly changes into ethylene glycol to alkyl ester, utilize cyanoacrylate glycol ester to make the poly-cyanoacrylate glycol ester nano-micelle of medicine carrying, its particle diameter is little, has the ability of passing the target tissue endotheliocyte, can greatly improve curative effect of medication.PEG6000-PHDCA and for example, for the preparation of the nanoparticle medicine-carried system, its polymerization degree is controlled, toxicity is less, in addition, because the PEG chain has wetting ability, flexibility, given the long cycle characteristics of copolymer nano particle, thereby changed by pharmacokinetics behavior in the body of bag medicine carrying thing.
Two alpha-cyanoacrylate diol ester molecular structures are modified mode, generally believe the degree of crosslinking that can improve polymkeric substance, thereby increase polymer machinery intensity and hardness, and are perhaps more stable under wet gas environments.English Patent GB1048906A for example, US Patent No. 3142698, US Patent No. 6699940.Describe the intermediate segment structure in these patents and comprise polyethylene glycols (PEG), dimethyltrimethylene glycol, polyester, polycarbonate, polyethers etc., poly(silicon aether), polyene, carbene etc.
Structure to α-cyanoacrylate remains further to be optimized, and expectation obtains better biological effect and specific purpose, as improves its biocompatibility, improves degradation property etc.
Summary of the invention
The invention provides a kind of alpha-cyanoacrylate derivative that contains peptide fragment, its structure can represent with formula I, formula II or formula III:
Wherein, AA
1-AA
2-...-AAn represents peptide fragment, can be comprised of any natural amino acid or alpha-non-natural amino acid, and preferred side chain nonfunctional base and the amino acid that need not the Additional Protection base are as glycine, phenylalanine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine etc.;
N represents amino acid quantity, can be 1 to 50, preferred 2 to 30, further preferred 2 to 25,2 to 20 or 2 to 10; More preferably 2 to 8,2 to 7,2 to 6,2 to 5 or 2 to 4;
X is Sauerstoffatom or nitrogen-atoms, preferred Sauerstoffatom;
R
1The expression junction fragment plays peptide fragment C end or N is held the effect that derives hydroxyl and become ester with alpha-cyanoacrylate, can be alcohol acid class and alkamine molecule; Preferably, R
1Include, but are not limited to as lactic acid, oxyacetic acid, hydroxy-propionic acid, amino amylalcohol, amino-hexanol etc., when peptide fragment during with N-terminal amino linkage alpha-cyanoacrylate, perhaps by pendant hydroxyl group in its structure, amino when connecting alpha-cyanoacrylate, R
1Can be for default;
R
2Be peptide fragment end closure group, can be for ethanoyl, methyl, ethyl etc., when the AAn end is the PEPC terminal carboxyl(group), R
2Can be for default;
R
3The expression junction fragment, be used for connecting two or more peptide fragment, can be saturated/unsaturated alkyl chain, oxyalkyl chain, silylation chain, polyester, polyethers arbitrarily, preferred ethylene glycol, propylene glycol, Triethylene glycol, tetraethylene-glycol, PEG, amino PEG, oxalic acid, propanedioic acid, succinic acid etc., C end or N end with the peptide fragment at two ends, by mode bondings such as amido linkage, ester bonds, can also adopt the side chain sulfydryl of the halfcystine that belongs to respectively two peptide fragment between two peptide fragment, form disulfide linkage as middle junction fragment.
In said derivative, when containing one or two natural amino acid that pendant hydroxyl group, amino are arranged or alpha-non-natural amino acid in described peptide fragment, described peptide fragment can be connected with alpha-cyanoacrylate by hydroxyl, the amino of its side chain, described have pendant hydroxyl group, amino natural amino acid or alpha-non-natural amino acid to include, but are not limited to Serine, oxyproline etc.
As shown in formula III, the peptide fragment in said derivative can be to contain a peptide fragment, can be also to contain two or more peptide fragment, passes through junction fragment R between peptide fragment
3Connect junction fragment R
3Can be saturated/unsaturated alkyl chain, oxyalkyl chain, silylation chain, polyester, polyethers etc. arbitrarily, preferred ethylene glycol, propylene glycol, Triethylene glycol, tetraethylene-glycol, PEG, amino PEG, oxalic acid, propanedioic acid, succinic acid etc., C end or N end with the peptide fragment at two ends, by mode bondings such as amido linkage, ester bonds, can also adopt the side chain sulfydryl of the halfcystine that belongs to respectively two peptide fragment between two peptide fragment, form disulfide linkage as middle junction fragment.
Described peptide fragment can also be the peptide fragment with pharmaceutical activity, includes but not limited to antibacterial peptide, phosphopeptide caseinate, gsh, immune-active peptides etc.
In a preferred embodiment of the present invention, the derivative shown in formula I, formula II or formula III, wherein:
X is O;
R
1Be hydroxyacetyl, hydroxy propionyl group;
R
2Be default;
R
3For-O-CH2CH2-O-;
Described peptide fragment is Ac-Ser-Gly-Gly, Gly-Gly, Phe-Phe, Phe-Phe-Phe-Ser or Ser-Phe-Gly-Ser.
In a preferred embodiment of the present invention, the derivative shown in formula I provided by the invention, formula II or formula III, it is selected from:
Alpha-cyano acryloyl (Ac-Ser-Gly-Gly-OH) ester;
Alpha-cyano acryloyl (Gly-Gly-OH) amine;
Two alpha-cyanoacrylates (hydroxyl acetyl Phe-Phe-O-CH2CH2-O-Phe-Phe hydroxyl acetyl) ester;
Two alpha-cyanoacrylates (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) ester;
Alpha-cyanoacrylate (CH
3CONHSer (OH)-Phe-Phe-PHe-Ser (OH) CONHCH
2CH
3) ester;
Alpha-cyanoacrylate (CH
3CONHSer (OH)-Phe-Gly-Ser (OH) CONHCH
2CH
3) ester.
A second aspect of the present invention relates to the preparation method of the alpha-cyanoacrylate derivative that contains peptide fragment shown in formula I or formula II or formula III, and the method comprises the following steps:
(1) peptide fragment is synthetic: adopt general peptide solid phase synthesis process in industry, the synthetic peptide fragment AA that contains arbitrary amino acid
1-AA
2-...-AAn; Perhaps from middle fragment R
3(for example ethylene glycol) beginning is extended to both sides, adopts the synthetic peptide fragment AA that contains arbitrary amino acid of method of liquid phase synthetic peptide
1-AA
2-... R
3-AAn;
(2) anthracene closes the alpha-cyanoacrylate derivatize: as required, connect anthracene in the one or both ends of described peptide fragment and close alpha-cyanoacrylate, when X is the N atomic time, the N end of the peptide fragment that obtains by step (1) is amino to be closed alpha-cyanoacrylate with anthracene and directly becomes amido linkage, perhaps working as X is the O atomic time, the peptide fragment end that obtains in step (1) is derived hydroxyl, then close alpha-cyanoacrylate with anthracene and is connected, the anthracene of acquisition peptide fragment closes the alpha-cyanoacrylate derivative;
Perhaps, when the natural amino acid in the peptide fragment that step (1) obtains or alpha-non-natural amino acid contain pendant hydroxyl group or amino (for example Serine), close alpha-cyanoacrylate by described pendant hydroxyl group or the amino anthracene that connects, the anthracene that obtains peptide fragment closes the alpha-cyanoacrylate derivative; Perhaps, after first containing pendant hydroxyl group or amino natural amino acid or alpha-non-natural amino acid and connecting anthracene and close alpha-cyanoacrylate, then with its amino acid synthesis unit as a peptide fragment, adopt general peptide synthetic method to be synthesized in peptide fragment, the anthracene that obtains peptide fragment closes the alpha-cyanoacrylate derivative;
(3) remove anthracene protection: the anthracene of the peptide fragment that step (2) is obtained closes the alpha-cyanoacrylate derivative and removes the anthracene protecting group, namely obtains finally suc as formula compound shown in I, formula II or formula III.
In above-mentioned preparation method, formula II is to contain in peptide fragment two hydroxyls or amino in order to be connected alpha-cyanoacrylate with the difference of formula I.The specific embodiment Isosorbide-5-Nitrae, how the particular compound of 5,6 correspondences obtains target formula I or formula II compound in order to illustrate, but is not restricted to ad hoc structure.The preparation method of formula III adopts from middle fragment (as ethylene glycol) beginning to extend to both sides, the method for liquid phase synthetic peptide, then become ester or become acid amides by closing alpha-cyanoacrylate with anthracene, remove the anthracene protection, obtain end product.How the particular compound of specific embodiment 2,3 correspondences obtains target formula III compound in order to illustrate, but is not restricted to ad hoc structure.
For example described alpha-cyano acryloyl (Ac-Ser-Gly-Gly-OH) ester (Compound I-1) is by solid phase synthesis Gly-Gly-resin; anthracene after introducing on solid phase is end modified closes cyano group acryloyl (Fmoc-Ser-OH) ester again; be intermediate (4); take off again Fmoc; peptide resin; obtain intermediate (5), then remove the anthracene protecting group and obtain the finished product (Compound I-1).And two alpha-cyanoacrylates (hydroxyl acetyl Phe-Phe-O-CH2CH2-O-Phe-Phe hydroxyl acetyl) ester and two these two kinds of molecules of alpha-cyanoacrylate (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) ester are all to adopt to begin to both sides to extend from middle ethylene glycol, the method of liquid phase synthetic peptide finally obtains end product.
a third aspect of the present invention relates to a kind of composition, comprises one or more formulas I, formula II, the alpha-cyanoacrylate derivative that contains peptide fragment shown in formula III, the preferred alpha-cyanoacrylate derivative that comprises simultaneously a kind of formula I or formula II or formula III, randomly, other α-cyanoacrylate quasi-molecules, described other α-cyanoacrylate quasi-molecules comprise but are not restricted to: ethyl α-cyanoacrylate, α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate, isobutyl alpha-cyanoacrylate, the different monooctyl ester of alpha-cyanoacrylate, the alpha-cyanoacrylate glycol ester, the alpha-cyanoacrylate macrogol ester, alpha-cyanoacrylate poly glycol monomethyl ether (mPEG) ester, two alpha-cyanoacrylate PEG diester, two alpha-cyanoacrylate alkyl diol esters, two alpha-cyanoacrylates (poly(lactic acid), PLA) diester, two alpha-cyanoacrylates (polyglycolic acid, PGA) diester, two alpha-cyanoacrylates (poly lactic coglycolic acid, PLGA) diester, two alpha-cyanoacrylates (PLA-PEG-PLA segmented copolymer) diester, two alpha-cyanoacrylates (PGA-PEG-PGA segmented copolymer) diester etc., preferred α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate.
In a specific embodiment, the formula of described composition is:
Two alpha-cyanoacrylates (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) Zhi ︰ α-cyanoacrylaten-butyl is 50% ︰ 50%(weight ratio);
In another specific embodiment, the formula of described composition is:
Two alpha-cyanoacrylates (hydroxyl acetyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxyl acetyl) Zhi ︰ α-cyanoacrylaten-butyl is 50% ︰ 50%(weight ratio).
The alpha-cyanoacrylate derivative that contains peptide fragment shown in the formula I of first aspect present invention, formula II, formula III, intermolecular polymerization can occur rapidly in the ethylene linkage in this derivative under the anionic initiation condition, and produce bonding stickiness, and obtain superpolymer, this derivative can share separately or with other cyanoacrylate material, as the biodegradable adhesive of medical, is used for wound bonding, extensive wound hemostasis, and internal organ and soft tissue wound closure etc.Described negatively charged ion blood, body fluid, tissue juice or skin-OH ,-NH2.
Therefore, a fourth aspect of the present invention relates to the purposes of composition of formula I, formula II, the alpha-cyanoacrylate derivative that contains peptide fragment shown in formula III or the third aspect present invention of first aspect present invention, described purposes is the purposes as adhesive of medical, or for the preparation of the purposes in adhesive of medical.Described adhesive of medical is used for hemostasis, bonding, covering, leak stopping, sclerous tissues to be fixed, or internal organ and the soft tissue wound closed.Can be used in the human body Medicine and Surgery operations such as outer in ambition, uropoiesis, neural outer, tumour, general outer liver and gall pancreas stomach and intestine, bone are outer, oral cavity, otorhinolaryngology, or extensive wound is when impaired, hemostasis, bonding, covering, leak stopping, sclerous tissues are fixed, or internal organ and soft tissue wound closure etc.This derivative can also be for the preparation of the purposes of biological degradability tissue engineering material.
a fifth aspect of the present invention relates to a kind of pharmaceutical composition, comprise formula I, formula II, the alpha-cyanoacrylate derivative that has the peptide fragment of pharmaceutical activity containing shown in formula III, when described peptide fragment does not have pharmaceutical activity, this pharmaceutical composition can also comprise one or more and be selected from small molecules, peptide class or nucleic acid drug, for example include but not limited to anti-inflammation analgesic, tranquilizer, local anesthetic, NSAID (non-steroidal anti-inflammatory drug), anti-allergy agent, anti-ulcerative drug, microbiotic, antimicrobial drug, antiviral drug, antifungal drug, immunosuppressor, nature derived protein or engineered protein, polysaccharide, glycoprotein or ester gp, oligonucleotide, polypeptide drugs, antibody, antigen, chemotherapeutics, Procoagulants and hemostatic agent, as thrombogen, zymoplasm, Fibrinogen, scleroproein, Fibronectin, thrombin, tissue factor, collagen protein, gelatin, vassopressin, Type 1 plasminogen activator inhibitor, inducer of platelet activation and have a synthetic peptide of styptic activity.Biocompatible agent includes but not limited to as sodium bisulfite.This pharmaceutical composition can be made Nano microsphere or nano-microcapsule.
A sixth aspect of the present invention relates to a kind of polymkeric substance, described polymkeric substance is polymerized under the negatively charged ion effect by formula I, formula II, the alpha-cyanoacrylate derivative that contains peptide fragment shown in formula III or the composition of third aspect present invention of first aspect present invention.Preferably, described negatively charged ion is the negatively charged ion of denier.More preferably, the negatively charged ion of described denier be blood, body fluid, tissue juice or skin-OH ,-NH
2
Detailed Description Of The Invention
The alpha-cyanoacrylate derivative molecular that contains peptide fragment shown in formula I, formula II, formula III, under the anionic initiation condition of denier, polymerization can occur rapidly in the ethylene linkage in the monomer whose molecule, produces bonding stickiness and obtains superpolymer.These molecules can be independent or several as the polymeric matrix composition, also can jointly form polymeric matrix with other α-cyanoacrylate quasi-molecules.other α-cyanoacrylate quasi-molecules can comprise but not be restricted to: ethyl α-cyanoacrylate, α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate, isobutyl alpha-cyanoacrylate, the different monooctyl ester of alpha-cyanoacrylate, the alpha-cyanoacrylate glycol ester, the alpha-cyanoacrylate macrogol ester, alpha-cyanoacrylate poly glycol monomethyl ether (mPEG) ester, two alpha-cyanoacrylate PEG diester, two alpha-cyanoacrylate alkyl diol esters, two alpha-cyanoacrylates (poly(lactic acid) (PLA)) diester, two alpha-cyanoacrylates (polyglycolic acid (PGA)) diester, two alpha-cyanoacrylates (poly lactic coglycolic acid (PLGA)) diester, two alpha-cyanoacrylates (PLA-PEG-PLA segmented copolymer) diester, two alpha-cyanoacrylates (PGA-PEG-PGA segmented copolymer) diester etc., preferred α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate.
Peptide matters has good biocompatibility and multiple biological activity, adopt the alpha-cyanoacrylate derivative molecular of peptide fragment structural modification, can binding peptide and the characteristic of cyanoacrylate two quasi-molecules, except the character that general α-cyanoacrylate quasi-molecule has, this quasi-molecule that the present invention relates to also has following special performance.
One, alpha-cyanoacrylate peptide derivant molecule polymerizable forms superpolymer, produces corresponding bonding force and intensity.Simultaneously, if contain two alpha-cyanoacrylate peptide derivant molecules in polymer matrix, can give superpolymer good biological degradability.Reason is:
1. be different from the linear high polymer (shown in Fig. 1-A) that single α-cyanoacrylate linear polymerization obtains; Contain two alpha-cyanoacrylate peptide derivants and divide the period of the day from 11 p.m. to 1 a.m in polymeric matrix, due to two ethylene linkages that polymerization can occur being arranged in the monomer whose molecule, the functionalized molecule degree is 4, and it can be used as linking agent, makes polymeric matrix generation copolymerzation with cross-linking, obtains 3D build superpolymer.Be subjected to the mode confinement of its structure and crosslinking polymerization, polymeric matrix carries out chainpropagation with three-dimensional multiple spot polymerization methods, and gelation rate is fast, and the linear polymerization degree of double-bond polymerization is low.The prolongation carbochain that double-bond polymerization produces, and the peptide fragment structure division of two alpha-cyanoacrylate peptide derivants have consisted of the grid main chain (shown in Fig. 1-B) on the space jointly.
2. be different from the carbon carbon backbone chain non-degradable of poly-single α-cyanoacrylate; Peptide fragment in two alpha-cyanoacrylate peptide derivant molecules, participate in to form the grid main chain, the cross-linking set of grid, i.e. peptide fragment and carbon carbochain junction, comprise ester bond, amido linkage, these key bond energys are lower, easily are degraded in vivo, therefore, the grid main chain also just can be destroyed from these sites, be that these sites are main chain degradation site of superpolymer, in addition, the sites such as ester bond, amido linkage that in like manner also can design other in peptide fragment reach the purpose of destroying grid main chain, degraded superpolymer.When the grid structure main chain of superpolymer be degraded destroy after, in fact superpolymer is decomposed into the carbon carbon backbone chain of polybutylcyanoacrylate, and the peptide fragment main chain, and some other degraded fragment as the side chain ester.Peptide fragment has good biocompatibility, can further bring into play active function, perhaps is degraded to the little peptide fragment of more short-movie section and amino acid and absorbs to excrete; Simultaneously, as previously mentioned, due to the restriction that the structure steric hindrance of dibasic acid esters is brought, the polymerization degree of two key linear polymeric, i.e. the degree of carbochain prolongation is less, and the carbon carbochain molecular weight of the polybutylcyanoacrylate that decomposites is also less, thereby also can be absorbed discharge.
3. by the amino acid arrangement mode in the specific peptide fragment of design, can also introduce the specificity degradation site of enzyme in this structure, promote polymer degradation.For example, the peptide bond that forms between Methionin, arginine is easily by trypsin hydrolyzing, the peptide bond that forms by die aromatischen Aminosaeuren or with the amino acid carboxyl of larger non-polar sidechain is easily by the chymotrypsin protein enzymic hydrolysis, and the peptide bond that the amino acid whose carboxyl of L-Ala, glycine and short aliphatic chain forms is easily by elastin enzymic hydrolysis etc.In addition, if design the peptide fragment that contains polyglycine in the two alpha-cyanoacrylate diol ester structures that the present invention relates to, it is hydrolyzed the glycine that obtains, and itself also has the function that promotes wound healing.
4. different two alpha-cyanoacrylate peptide derivant molecular structures and the polymer matrix formula space conformation that can change superpolymer, by regulating length and the kind of peptide fragment in two alpha-cyanoacrylate peptide derivants, and the dibasic acid esters in polymeric matrix/monoesters proportioning, just can the aggregation degree, polymerization time, degradation property etc.
Two, this class alpha-cyanoacrylate peptide derivant molecule, can realize the purpose to the peptide medicament slowly-releasing.
With some activated peptide fragment structures, design is connected with the alpha-cyanoacrylate structure, the list that obtains the present invention relates to/pair alpha-cyanoacrylate peptide derivant molecule.After the polymer matrix generation polymerization that contains these monomer molecules obtains superpolymer, peptide fragment wherein, owing to becoming ester bond, amido linkage to be connected with the acrylic acid carbon carbochain of paracyanogen base, therefore, be all can be from superpolymer these connection site, mode by chemical bond rupture, hydrolysis dissociates out, and further brings into play its active function.The mode that this chemistry dissociates is different from the release of dissociating on physics meanings such as capsule commonly used on pharmaceutics, micella, thereby can keeps relatively slow level and speed, thereby reaches the purpose of slowly-releasing.By the Combinatorial Optimization to polymer matrix and additive, can regulate slow release speed.
In addition, in recent years, Polyalkylcyanoacrylanano class colloidal nanoparticles, in good body, biocompatibility is widely used in controlled release drug delivery system owing to having, and according to preparation technology's difference, can form Nano microsphere and nano-microcapsule.The list that the present invention relates to/pair alpha-cyanoacrylate peptide derivant molecule can apply to prepare Nano microsphere and nano-microcapsule equally, and after entering blood circulation, the mode by chemical bond is degraded discharges peptide medicament, reaches the effect of slow controlled release.Nano microsphere and the nano-microcapsule of this molecule preparation also can be used for other small molecules of physical load and peptide class, nucleic acid drug.
Three, this class alpha-cyanoacrylate peptide derivant molecule, can satisfy the requirement of tissue engineering material.
The purpose of tissue engineering bracket material is to provide a three-dimensional rack for building histocyte, be conducive to adhesion, propagation and even the differentiation of cell, for Growth of Cells provides suitable external environment, its condition that need to satisfy comprises: good biocompatibility, without obvious toxicity, inflammatory reaction and immunological rejection; Degradability and suitable degradation rate; Suitable hole dimension, high porosity and the pore morphology that is connected; The structural strength that is complementary with the mechanical property of implant site tissue; Be convenient to be processed into desirable two dimension or three-dimensional structure etc.This class alpha-cyanoacrylate peptide derivant molecule that the present invention relates to, combine the good biocompatibility of peptide matters, as described in characteristics one, can have good degradation property, 3D grid space structure, and can utilize polymerization property, forming specific two dimension or three-dimensional structure, therefore, is the requirement that can satisfy these tissue engineering materials.
Based on the important characteristic of above three classes, the purposes of this class alpha-cyanoacrylate peptide derivant molecule that proposition the present invention relates to:
1, this class alpha-cyanoacrylate peptide derivant molecule that the present invention relates to can be used for forming the polymer matrix moiety of medical glue paste, especially two alpha-cyanoacrylate peptide derivants, by the rational structure of design optimization, composition and ratio etc., can reach the purpose of effective raising colloid degradation property, and degraded product is nontoxic absorbs.The medical adhesive that comprises this class alpha-cyanoacrylate peptide derivant molecule, applicable to human body Medicine and Surgery operation, the hemostasis in the operations such as, oral cavity outer such as outer, neural outer, the tumour in: ambition, uropoiesis, general outer liver and gall pancreas stomach and intestine, bone, otorhinolaryngology, bonding, covering, leak stopping, sclerous tissues are fixing etc.
2, this class alpha-cyanoacrylate peptide derivant molecular structures mode that the present invention relates to can with it as a kind of general peptide medicament modifying method, be used for slow controlled release purpose.By connecting alpha-cyanoacrylate at the peptide fragment end, polymerization obtains high-polymer molecular, and in superpolymer, the ester bond of connection peptides fragment can be hydrolyzed gradually, thereby release peptide fragment again reaches slow release effect.This modification mode can be used for peptide medicament in the sustained-release administration purpose at the positions such as local patholoic change tissue or tumour.Simultaneously, the list that the present invention relates to/pair alpha-cyanoacrylate peptide derivant molecule, also can be used for preparing Nano microsphere and nano-microcapsule, peptide medicament in Chemical bond, release monomer, or other small-molecule drugs of physical load, peptide medicament, nucleic acid drug etc., reach through sanguimotor slow controlled release drug administration.
3, the biocompatibility that utilizes this class alpha-cyanoacrylate peptide derivant molecule to have, mechanical characteristic, the performance adjustability, the easy characteristics of processing forming also can be used for preparing the purpose of biodegradability tissue engineering material.Can form the solid form with some strength and supporting degree by polymerization, satisfy the requirement of tissue engineering material, and can be along with tissue growth, self degrades gradually, and degraded product is nontoxic to be absorbed.
Advantage of the present invention is: the molecule that a novel list of class/pair alpha-cyanoacrylate peptide derivant is provided.Comprise the medical adhesive that this quasi-molecule is the polymeric matrix composition, outside the effect of the conventional medical adhesive of decapacitation performance, also can have better biocompatibility, the polymer biological degradability, degraded product is nontoxic to be absorbed; On the other hand, the structure of this quasi-molecule provides a kind of modifying method of general peptide medicament, and be used for the chemical load peptide medicament, or form the mode of microcapsules and microsphere, at local organization, tumor locus, or sanguimotor sustained-release administration purpose; Moreover the present invention also provides the new texture molecule of a class for the preparation of the biodegradability tissue engineering material.
Description of drawings
Fig. 1-A: the linear high polymer schematic diagram that single α-cyanoacrylate polymerization obtains.
Fig. 1-B: contain the build superpolymer schematic diagram that two alpha-cyanoacrylate diol ester polymerizations obtain.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1:
Alpha-cyano acryloyl (Ac-Ser-Gly-Gly-OH) esterSynthesizing of (Compound I-1):
This compound is a specific embodiment of formula I compound, and wherein X is O, R
1Default, in this compound, the AAn end is the PEPC terminal carboxyl(group), R
2For default.
Synthetic route is as follows: first prepare the alpha-non-natural amino acid 4 that the alpha-cyanoacrylate of anthracene protection is connected with the Serine side chain, alpha-non-natural amino acid 4 as last amino acid introducing peptide solid phase synthesis, is finally removed the anthracene protecting group and obtains product I-1.
Intermediate (1): H-Ser-OBzl TosOH
With H-Ser-OH10.50g(0.1mol), benzylalcohol 40ml(0.4mol), TosOH22.80g(0.12mol), 100ml benzene joins in the round-bottomed flask of 250ml, and the oil bath reflux is divided water, until till not having water to be told, approximately 5h is carried out in reaction again.Stopped heating, cooling, evaporate to dryness benzene adds sherwood oil to wash residual liquid, until the benzylalcohol in reaction solution is washed away fully, obtains approximately 40g of oily liquids product (intermediate 1).
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 8.10(s, 3H), δ 7.65(d, 2H) and, δ 7.17-7.26(m, 5H), δ 6.98(d, 2H), δ 4.96-5.05(q, 2H), δ 3.88-4.09(m, 2H) and, δ 3.85-3.87(m, 1H), δ 2.23(s, 3H) (JNM-ECA-400, Tokyo, Japan
Mass spectrum: 196.2(M+1)
Intermediate (2): Fmoc-Ser-OBzl
With Na
2CO
342.40g(0.4mol) be dissolved in 500ml water, ice bath is cooling, add approximately 40g(0.1mol of the thick product of H-Ser-OBzl TosOH) stirring and dissolving, keep the ice bath state, slowly drip FmocOSu33.71g(0.1mol) 200ml tetrahydrofuran (THF) (tetrahydrofuran (THF), THF) solution, drip off in 3h, ice bath 0.5h rises to room temperature reaction 5h, decompression steams THF, and make up water is to Na
2CO
3Dissolving fully, dichloromethane extraction are to the aqueous phase product-free, and the saturated NaCl aqueous solution is washed methylene dichloride mutually to neutral, Na
2SO
4Drying, the evaporate to dryness methylene dichloride obtains the pale yellow oily liquid body, PE-EtOAc(3:1) crosses post and separates, and obtains white solid product 24.94g(intermediate 2), two step overall yields 59.8%.
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 7.76(m, 2H), δ 7.60(m, 2H) and, δ 7.25-7.40(m, 9H), δ 5.74(d, 1H) and, δ 5.22(s, 2H), δ 4.41-4.49(m, 3H) and, δ 4.21(t, 1H), δ 3.92-4.04(m, 2H)
Mass spectrum: 418.4(M+1)
Intermediate (3): anthracene closes cyano group acryloyl (Fmoc-Ser-OBzl) ester
Fmoc-Ser-OBzl23.40g(56mmol), anthracene closes alpha-cyanoacrylate 30.8g(112mmol), EDC(1-ethyl-(3-dimethylaminopropyl) carbodiimide, the 21.40g(112mmol of N-(3-Dimethylaminopropyl)-N '-ethylcarbodiimide)), the DMAP(4-Dimethylamino pyridine, 4-Dimethylaminopyridine) 1.30g(10.7mmol) be added in the round-bottomed flask of 1L, the methylene dichloride that adds the 800ml drying, stirring and dissolving, room temperature reaction 5h, saturated sodium bicarbonate aqueous solution is washed, saturated sodium-chloride water solution is washed, anhydrous sodium sulfate drying, concentrated pale yellow oily liquid body, PE-EtOAc(8:1) crossing post separates, get white products (intermediate 3) 19.9g productive rate 52.70%
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 7.76(m, 4H), δ 7.60(m, 2H), δ 7.25-7.40(m, 32H), δ 6.89-7.10(m, 4H), δ 5.59(d, 1H) δ 5.26(s, 2H), δ 4.64-4.49(m, 3H), δ 4.25-4.49(m, 12H), δ 2.69(d, 1H), δ 2.50(d, 1H), δ 2.09(d, 1H), δ 2.05(d, 1H)
Mass spectrum: 585.4(M+1)
Intermediate (4): anthracene closes cyano group acryloyl (Fmoc-Ser-OH) ester
Anthracene closes cyano group acryloyl (Fmoc-Ser-OBzl) ester 14.48g(21.50mmol), 10%Pd/C2.32g (2.19mmol), ammonium formiate 4.06g(64.4mmol), join in reaction flask, add 500ml methyl alcohol to make it dissolving, reflux 1h, stopped heating reacts completely, let cool filtration, evaporate to dryness DCM-MeOH(40:1) cross post and obtain white solid product (intermediate 4) 5.5g productive rate 43.8%
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 7.76(m, 2H), δ 7.60(m, 1H) and, δ 7.25-7.40(m, 13H), δ 6.15(s, 1H), δ 5.70(d, 1H), δ 5.29(s, 2H) and, δ 4.78-4.84(d, 2H), δ 4.20-4.49(m, 6H), δ 2.69(dd, 2H) and, δ 2.18(dd, 2H)
Mass spectrum: 692.5(M+18)
Intermediate (5): anthracene closes cyano group acryloyl (Ac-Ser-Gly-Gly-OH) ester
Adopting the wang resin is carrier (carrying capacity is 1.3mmol/g), with the tactful solid phase synthesis of peptide of Fmoc amino acid protection.
benzole soln sealing polypeptide reactor with trimethylchlorosilane spends the night, hydroxyl on the closed glass vessel is to reduce glass to the absorption of resin, reclaim trimethylchlorosilane, with each wash bottle of methyl alcohol-sherwood oil 3 times, add wang resin 0.308g(0.40mmol), with methylene dichloride swelling resin 20min, drain, add 0.47g(1.6mmol in the polypeptide reactor) Fmoc-Gly-OH, 0.33g(1.6mmol) DCC (1, the 3-dicyclohexylcarbodiimide, N, N'-dicyclohexylcarbodiimide), 0.22g(1.6mmol) the HOBt(1-hydroxy benzo triazole, Hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazine), each 5ml of DMF/DCM begins reaction, slowly stir (preventing that resin from being stirred brokenly), react and add 0.015g(10% after one hour) DMAP, react again 12h, after completing, drain reaction solution, resin is with the DMF(dimethyl formamide, N, N-Dimethylformamide), methyl alcohol, methylene dichloride is respectively washed three times, washed two minutes at every turn, again add 0.47g(1.6mmol in reactor) Fmoc-Gly-OH, 0.33g(1.6mmol) DCC, 0.22g(1.6mmol) HOBt, each 5ml of DMF/DCM begins reaction, slowly stir (preventing that resin from being stirred brokenly), react and add 0.015g(10% after one hour) DMAP, react again 12h, drain reaction solution, resin is with DMF, methyl alcohol, methylene dichloride is respectively washed three times, washed two minutes at every turn, add the 2ml aceticanhydride in resin, 2mlDIEA, add the 10ml methylene dichloride, reaction 0.5h.Unreacted resin is sealed.DMF, methylene dichloride, methyl alcohol respectively wash three times.25% piperidines/DMF solution takes off Fmoc, 5min once, 25min for the second time, after deprotection, with DMF, methylene dichloride, methyl alcohol are respectively washed three times.Whether after completing, detect Fmoc and be removed, three kinds of indicator, 110 ℃ of heating 5min detect if respectively adding one.
Drop into 0.47g(1.6mmol) Fmoc-Gly-OH, 0.33g(1.6mmol) DCC, 0.22g(1.6mmol) HOBt, each 5ml of DMF/DCM begins reaction, and slowly stirring reaction 4h, drain reaction solution, and DMF, methylene dichloride, methyl alcohol respectively wash three times.After completing, whether the amino acid that detects on resin reacts completely, and three kinds of indicator respectively add one, and 110 ℃ of heating 5min detect.25% piperidines/DMF solution takes off Fmoc, 5min once, 25min for the second time, after deprotection, with DMF, methylene dichloride, methyl alcohol are respectively washed three times.Whether after completing, detect Fmoc and be removed, three kinds of indicator, 110 ℃ of heating 5min detect if respectively adding one.
Input 0.72g(1.2mmol) anthracene closes cyano group acryloyl (Fmoc-Ser-OH) ester, 0.26g(1.2mmol) DCC, 0.18g(1.2mmol) HOBt, each 5ml of DMF/DCM begins reaction, and slowly stirring reaction 4h, drain reaction solution, DMF, methylene dichloride, methyl alcohol respectively wash three times.After completing, whether the amino acid that detects on resin reacts completely, and three kinds of indicator respectively add one, and 110 ℃ of heating 5min detect.25% piperidines/DMF solution takes off the Fmoc(fluorenylmethyloxycarbonyl, Fluorenylmethyloxycarbonyl), 5min once, 25min for the second time, after deprotection, with DMF, methylene dichloride, methyl alcohol are respectively washed three times.Whether after completing, detect Fmoc and be removed, three kinds of indicator, 110 ℃ of heating 5min detect if respectively adding one.
Add the 2ml aceticanhydride in resin, 2ml DIEA(N, the N-diisopropylethylamine, 2-(1H-Benzotriazole-1-yl)-1,1,3 3-tetramethyluroniumtetrafluoroborate), adds the 10ml methylene dichloride, reaction 0.5h.Unreacted resin is sealed.DMF, methylene dichloride, methyl alcohol respectively wash three times.Drain the peptide resin 0.59g that weighs.
Under ice bath, the lysate 10ml peptide resin 1h with TFA-meta-cresol-methyl-phenoxide-water (95-2-1.5-1.5) then rises to room temperature reaction 1h, and decompression steams TFA, DCM-MeOH(15:1) cross post and obtain product (intermediate 5)
The 1H spectrum:: 1HNMR(DMSO-D6, δ ppm): δ 9.04(t, 1H), δ 7.62(s, 1H), δ 7.46-7.03(m, 8H), δ 5.20(s, 1H), δ 4.53(s, 1H), δ 3.68(m, 2H), δ 3.63(m, 1H) δ 3.58(s, 2H), δ 3.17(s, 2H), δ 2.77(dd, 1H), δ 2.04(dd, 1H), δ 1.23(s, 3H).
Mass spectrum: 519.4(M+1)
The finished product (I-1): alpha-cyano acryloyl (Ac-Ser-Gly-Gly-OH) ester
Anthracene closes cyano group acryloyl (Ac-Ser-Gly-Gly-OH) ester 0.10g(0.19mmol), maleic anhydride 0.117g(1.2mmol) dimethylbenzene 10ml adds stirring and dissolving in the 25ml flask, add Vanadium Pentoxide in FLAKES 10mg, quinhydrones 5mg, reflux 6h, 140 ℃ of reflux temperatures, stopped reaction, be cooled to room temperature, pressure reducing and steaming dimethylbenzene can obtain approximately 0.08 gram (Compound I-1) of yellow solid product.H-composes evaluation, at δ 7.04(s, 1H, the trans hydrogen of CH2=C CN), δ 6.61(s, 1H, CH2=CCN cis hydrogen) two key hydrogen characteristic peaks of cyanoacrylate are arranged.
With the product (Compound I-1) that obtains, use DMF(N, N-Dimethylformamide, DMF, nitrogen dimethylformamide) after the dissolving, drip in 1 centimetre of diameter (Compound I-1 with DMF mass ratio 1:1), and long 5 centimetres of silicagel column tops are very fast, silicagel column top silica gel is bonded, illustrates that product has certain adhesiveproperties.Product silica gel piece after polymerization is solid state, and is water insoluble.This polyreaction is the alpha-cyanoacrylate ester cpds that the hydroxyl isopolarity group on silica gel causes, i.e. ethylene linkage polyreaction between each monomer of I-1.
Embodiment 2: the synthetic of two alpha-cyano propylene (Phe-Phe-O-CH2CH2-O-Phe-Phe) peptide fragment given an example
Synthesizing of two alpha-cyanoacrylates (hydroxyl acetyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxyl acetyl) ester (compound III-1)
This compound is a specific embodiment of formula III, and wherein X is O, and R1 is the oxyacetic acid part, and R3 is-O-CH2CH2-O-.
Synthetic route is as follows:
Intermediate (6): Boc-Phe-OCH
2CH
2O-Phe-Boc
Boc-Phe – OH19.14g(0.072mol), ethylene glycol 1.49g(24mmol), EDCHCl 13.85g(0.072mol), DMAP0.84g(7.2mmol) be added in the reaction flask of 500ml, add the 350ml methylene dichloride to make it to dissolve fully, reaction 6hTLC detects, and saturated sodium bicarbonate is washed, anhydrous sodium sulfate drying concentrates and crosses post with sherwood oil-methylene dichloride-ethyl acetate 20:20:1.Obtain white solid (intermediate 6) 12.5g.Productive rate 93%.
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 7.1-7.3(m, 10H), δ 4.99(d, 2H) and, δ 4.58(m, 2H), δ 4.26(s, 4H) and, δ 3.0-3.2(m, 4H), δ 1.41(s, 18H)
Mass spectrum: 557.6(M+1)
Intermediate (7): Phe-O CH
2CH
2O-Phe
Boc-Phe-OCH2CH2O-Phe-Boc10.5g(18.9mmol), add the 25mlTFA(0.33mol under ice bath in batches) in, until completely dissolved, temperature of reaction rises to room temperature, reaction 1h, decompression steams TFA, with the 100mlDCM dissolution residual substance, adds 30ml water, add the sodium carbonate solid and regulate PH to 7-8, saturated nacl aqueous solution is washed dichloromethane layer, and anhydrous sodium sulfate drying, concentrating under reduced pressure obtain yellow liquid product (intermediate 7) 6.0g.Productive rate 89.2%.
1The H spectrum:
1HNMR(DMSO-d
6, δ ppm): δ 7.1-7.3(m, 10H), δ 4.15(m, 4H) and, δ 3.58(m, 2H), δ 2.7-2.9(m, 4H) and, δ 1.74(s, 4H)
Mass spectrum: 357.4(M+1)
Intermediate (8): Boc-Phe-Phe-O CH
2CH
2O-Phe-Phe-Boc
add Boc-Phe-OH10.81g(0.040mol in the there-necked flask of 250ml), Phe-OCH2CH2O-Phe6.70g(0.0188mol), HOBt5.41g(0.040mol), add 10mlDMF dissolving raw material, add the 350ml methylene dichloride, stir under ice bath, after being down to 0 ℃, slowly add DIC4.1ml(0.040mol), N-methylmorpholine 4.1ml(0.40mol), react 5h under ice bath, and then spend the night at room temperature reaction, TLC detects, the evaporate to dryness reaction solution, with the acetic acid ethyl dissolution residue, filter, successively with water, 10% citric acid, saturated sodium bicarbonate solution, saturated nacl aqueous solution is washed ethyl acetate layer, anhydrous sodium sulfate drying, 160:1 crosses post with methylene chloride-methanol, obtain white solid (intermediate 8) 14.7g productive rate 91.8%.
1The H spectrum:
1HNMR(DMSO-d
6, δ ppm): δ 8.38(d, 2H), δ 7.1-7.3(m, 20H) and, δ 6.89(d, 2H), δ 4.53(m, 2H) and, δ 4.18(m, 4H), δ 2.6-2.9(m, 8H) and, δ 1.27(s, 18H)
Mass spectrum 851.5(M+1)
Intermediate (9): Phe-Phe-O CH
2CH
2O-Phe-Phe
Boc-Phe-Phe-OCH2CH2O-Phe-Phe-Boc11.5g(13.5mmol), add the 27mlTFA(0.35mol under ice bath in batches) in, until completely dissolved, temperature of reaction rises to room temperature, reaction 1h, decompression steams TFA, with the 100mlDCM dissolution residual substance, adds 30ml water, add the sodium carbonate solid and regulate PH to 7-8, saturated nacl aqueous solution is washed dichloromethane layer, and anhydrous sodium sulfate drying, concentrating under reduced pressure obtain yellow liquid product (intermediate 9) 7.3g.Productive rate 83.2%.
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 7.80(d, 2H), δ 7.1-7.3(m, 20H) and, δ 4.89(m, 2H), δ 4.28(m, 4H) and, δ 3.57(m, 2H), δ 3.11(m, 8H) and, δ 1.67(s, 4H).
Mass spectrum: 651.3(M+1)
Intermediate (10): dihydroxy acetyl Phe-Phe-O CH
2CH
2O-Phe-Phe hydroxyl acetyl
With Phe-Phe-O CH
2CH
2O-Phe-Phe0.225g(0.346mmol), oxyacetic acid 0.052g(0.684mmol), BOP0,306g (0.694mmol), DIEA0.184g (1.42mmol) drops in reaction flask, be dissolved in the 15ml methylene dichloride, room temperature reaction 3h reacts completely, with successively with water, 10% citric acid, saturated sodium bicarbonate solution, saturated nacl aqueous solution is washed, anhydrous sodium sulfate drying, cross post with petroleum ether-ethyl acetate-methyl alcohol (10:10:1), obtain 0.12g product (intermediate 10), productive rate 45.2%.
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 8.64(d, 2H) δ 7.60(d, 2H), δ 7.1-7.3(m, 20H) and, δ 5.52(m, 2H), δ 4.5-4.6(m, 4H) and, δ 4.18(m, 4H), δ 3.71(d, 4H) and, δ 3.0(m, 8H) δ δ δ
Mass spectrum: 767.6(M+1)
Intermediate (11): two anthracenes close alpha-cyanoacrylate hydroxyl acetyl (Phe-Phe-OCH
2CH
2O-Phe-Phe) ester
Anthracene closes alpha-cyanoacrylate 1.65g(5.0mmol), hydroxyl acetyl Phe-Phe-OCH2CH2O-Phe-Phe hydroxyl acetyl 1.38g(1.96mmol), EDCI0.96g(5.0mmol), DMAP0.12g(1mmol), join in the 10ml methylene dichloride, room temperature reaction 6h, saturated sodium bicarbonate solution is washed, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, concentrated, methylene chloride-methanol (200:1) is crossed post, obtain 0.92g product (intermediate 11), productive rate 36%.
1The H spectrum:
1HNMR(CDCl
3, δ ppm): δ 8.44(s, 2H) δ 8.01(m, 2H), δ 7.0-7.5(m, 36H) and, δ 4.82(s, 2H), δ 4.90(s, 2H), δ 4.65(m, 4H) and, δ 4.44(m, 8H), δ 4.13(m, 4H), δ 3.94(m, 8H) δ 2.71(dd, 2H), δ 2.20(dd, 2H)
Mass spectrum: 1282.2(M+2)
The finished product (compound III-1): two alpha-cyanoacrylates (hydroxyl acetyl Phe-Phe-OCH 2 CH 2 O-Phe-Phe hydroxyl acetyl) ester(compound III-1)
Two anthracenes close alpha-cyanoacrylate hydroxyl acetyl (Phe-Phe-OCH2CH2O-Phe-Phe) ester 0.299g(0.23mmol), maleic anhydride 0.145g(1.44mmol), dimethylbenzene 10ml adds stirring and dissolving in the 25ml flask, add Vanadium Pentoxide in FLAKES 17mg, reflux 6h, 140 ℃ of reflux temperatures, stopped reaction, be cooled to room temperature, pressure reducing and steaming dimethylbenzene can obtain yellow solid product (compound III-1).H-composes evaluation, at δ 7.04(s, 1H, the trans hydrogen of CH2=C CN), δ 6.61(s, 1H, CH2=CCN cis hydrogen) two key hydrogen characteristic peaks of cyanoacrylate are arranged.
With the product (compound III-1) that obtains, use and be applied in the pigskin surface after micro-DMF dissolving (compound III-1 is 1:1 with the mass ratio of DMF), at this moment, (two pigskins are smeared one side and got final product) overlaps another piece pigskin rapidly, after 15 seconds, by intensely bonding together, illustrate that product has good adhesiveproperties.
With the product (compound III-1) that obtains, after using the DMF dissolving, (the mass ratio 1:1 of compound III-1 and DMF) drips in 1 centimetre of diameter, and the silicagel column of long 5 centimetres is very fast, the loose silica gel powder in silicagel column top is bonded into bulk, illustrates that product has certain adhesiveproperties.Product after polymerization is solid state, and is water insoluble.
Embodiment 3: the two alpha-cyanoacrylate diol esters that contain peptide fragment are for example synthetic
Synthesizing of two alpha-cyanoacrylates (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) ester (compound III-2)
This compound is a specific embodiment of formula III compound, wherein, and R
1Be hydroxy propionyl group, R
3For-O-CH2CH2-O-.
Adopt synthetic method similar to Example 2, obtain yellow solid product (compound III-2).
With the product (compound III-2) that obtains, use the mass ratio 1:1 of micro-DMF(compound III-2 and DMF) be applied in the pigskin surface after dissolving, at this moment, pigskin is overlapped rapidly, after 15 seconds, by intensely bonding together, illustrate that product has good adhesiveproperties.
With the product (compound III-2) that obtains, use the mass ratio 1:1 of DMF(compound III-2 and DMF) drip in 1 centimetre of diameter after dissolving, the silicagel column of long 5 centimetres, very fast, silicagel column top silica gel is bonded, illustrates that product has certain adhesiveproperties.Product after polymerization is solid state, and is water insoluble.
Embodiment 4:
Alpha-cyanoacrylate (CH 3 CONHSer (OH)-Phe-Phe-PHe-Ser (OH) CONHCH 2 CH 3 ) ester (Compound I I-1)Synthetic
This compound
II-1Be a specific embodiment of formula II compound, wherein, X is O, R
1Default, peptide fragment can but be not limited to peptide fragment in the present embodiment.
Synthetic method: adopt peptide solid phase synthesis universal method similar to Example 1; select Merrifield resin (chloromethyl resin) to carry out the solid phase synthesis of peptide fragment; N holds acetylize; peptide fragment uses ethamine/DMF solution cutting; re-use palladium-carbon as catalyzer; catalytic hydrogenation 20h obtains the peptide fragment intermediate:
CH
3CONHSer(OH)PhePhePheSer(OH)CONH
2CH
2CH
3
Wherein, contain two exposed hydroxyls, the C end becomes respectively acid amides with the N end, and peptide fragment is adopted the universal synthesis method described in embodiment 2,3, can connect alpha-cyanoacrylate from hydroxyl and become ester, finally obtain the finished product (Compound I I-1) of peptide fragment two ends alpha-cyanoacrylate esterification.
With the product (Compound I I-1) that obtains, use mass ratio 1 ﹕ 1 of micro-DMF(Compound I I-1 and DMF) be applied in the pigskin surface after dissolving, at this moment, pigskin is overlapped rapidly, after 15 seconds, by intensely bonding together, illustrate that product has good adhesiveproperties.
With the product that obtains
(Compound I I-1), use mass ratio 1 ﹕ 1 of DMF(Compound I I-1 and DMF) drip in 1 centimetre of diameter after dissolving, the silicagel column that length is 5 centimetres, very fast, silicagel column top silica gel is bonded, illustrates that product has certain adhesiveproperties.Product after polymerization is solid state, and is water insoluble.
H-composes evaluation, at δ 7.04(s, 1H, the trans hydrogen of CH2=C CN), δ 6.61(s, 1H, CH2=CCN cis hydrogen) two key hydrogen characteristic peaks of cyanoacrylate are arranged.
Embodiment 5:
Alpha-cyanoacrylate (CH 3 CONHSer (OH)-Phe-Gly-Ser (OH) CONHCH 2 CH 3 ) ester(Compound I I-2's) is synthetic
Compound I I-2 is another specific embodiment of formula II compound, wherein, peptide fragment can but be not limited to peptide fragment in the present embodiment.
Synthetic method: adopt universal method similar to Example 4, obtain peptide fragment intermediate and the finished product that obtains two ends alpha-cyanoacrylate esterification.
With the product (Compound I I-2) that obtains, use mass ratio 1 ﹕ 1 of micro-DMF(Compound I I-12 and DMF) be applied in the pigskin surface after dissolving, at this moment, pigskin is overlapped rapidly, after 15 seconds, by intensely bonding together, illustrate that product has good adhesiveproperties.
With the product (Compound I I-2) that obtains, use mass ratio 1 ﹕ 1 of DMF(Compound I I-2 and DMF) drip in 1 centimetre of diameter after dissolving, the silicagel column that length is 5 centimetres, very fast, silicagel column top silica gel is bonded, illustrates that product has certain adhesiveproperties.Product after polymerization is solid state, and is water insoluble.
H-composes evaluation, at δ 7.04(s, 1H, the trans hydrogen of CH2=C CN), δ 6.61(s, 1H, CH2=CCN cis hydrogen) two key hydrogen characteristic peaks of cyanoacrylate are arranged.
Embodiment 6:
Alpha-cyano acryloyl (Gly-Gly-OH) amine (compoundI-2)
Compound shown in formula I-2 is another embodiment of formula I compound, and wherein, peptide fragment Gly-Gly can substitute with the peptide fragment that arbitrary amino acid forms.
Synthetic method: adopt general peptide solid phase synthesis strategy, adopting the wang resin is carrier (carrying capacity is 1.3mmol/g), with the tactful solid phase synthesis of peptide of Fmoc amino acid protection.Synthetic Gly-Gly dipeptides in peptide solid phase synthesis device is after the N end is amino exposed, with embodiment 2; the anthracene that uses in 3 closes alpha-cyanoacrylate and adds reactor as last amino acid, after normal condensation reaction, and the cleavage of peptide sequence; adopt identical method to remove the anthracene protecting group, obtain finished product I-2.
Mass spectrum: 212.5 (+H).
Peptide fragment in the compound of above-described embodiment 1-5 preparation can but be not limited to described peptide fragment in embodiment 1-6.
Composition and the preparation of 7: two kinds of formula glue of embodiment
Formula glue 1: two alpha-cyanoacrylates (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) ester: α-cyanoacrylaten-butyl (weight percent 50%:50%)
Formula glue 2: two alpha-cyanoacrylates (hydroxyl acetyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxyl acetyl) ester: α-cyanoacrylaten-butyl (weight percent 50%:50%)
Concrete preparation method is as follows:
1. sterilization all uses acid (0.5M dilute sulphuric acid) to soak afterwash, high-temperature sterilization in container used, and raw material is organic system millipore filtration (solarbio company) filtration sterilization of 0.22um with the aperture.
2. under drying conditions, add the various raw materials in above-mentioned formula glue 1 or formula glue 2 in the container, use the spiral shaker to shake fast 10min, mixture is colourless transparent liquid, namely gets medical adhesive.
3. preserve, the medical adhesive that step 2 is obtained passes into the protection of inert gas such as nitrogen, argon gas after packing under dry environment, and sealed vessel is placed in refrigerator and preserves (4 ℃ or lower), can stablize and preserve over 3 months.
4. use, the medical adhesive that the medical adhesive that preparation process 2 is obtained directly takes out or step 3 is preserved takes out from low temperature environment, return back to room temperature, medical adhesive is in a liquid state, this moment, wherein institute's cyanoacrylate-containing molecule was still the free state that polymerization does not occur, after liquid medical adhesive is contacted wound or tissue and uses the position by modes such as smearing, spray, splash into, medical adhesive occur rapidly polymerization form solid high-polymer and and tissue between produce bonding force.Specific embodiment 8,9 will further describe its set time and degradation property in 10.
As the formula in embodiment 7, can change the heavy class of raw material and proportioning, concrete preparation method is identical with preparation method in embodiment 7, and the medical adhesive that obtains all is the colourless or light yellow transparent liquid of even shape at room temperature condition.After touching skin and bio-tissue, polymerization occurs rapidly form solid high-polymer, produce bonding force.
Embodiment 8: measure the set time of the medical adhesive that obtains in embodiment 7
Testing method: with the medical adhesive of the different ingredients of preparation in embodiment 7, at ambient temperature, take a morsel and drip in the pigskin surface, another piece pigskin is overlapped thereon rapidly, by pullling respectively pigskin to two side directions, the bonding force between the check pigskin.To begin to produce the time point of bonding force between two pigskins, the completely crued time point of liquid glue after the overlap joint certain hour between pigskin is designated as set time.Two kinds of formula glue polymerization times in embodiment 7 are measured and are 5-15 second.
Embodiment 9: the degradation property of the medical adhesive that obtains in embodiment 7 is measured
Testing method: with medical adhesive (formula glue 1 and formula glue 2) and the control sample (BCA) that obtains in specific embodiment 7, each 100mg, drip respectively on the sheet glass of 2.5 * 3cm, after it solidifies fully, its initial weight of vacuum-drying and weighing, sheet glass is placed in 0.1M phosphate buffered saline buffer (PBS damping fluid, the K that pH7.2-7.4 is housed
2HPO
4.3H
2O1.85g/L, KH
2PO
40.24g/L, NaCl 8.00g/L, KCl 0.20g/L), perhaps bovine serum (prosperous Golden Horse biotechnology development corporation, Ltd. of Beijing unit, new-born calf serum) in Erlenmeyer flask, Erlenmeyer flask is put in 37.5 ℃ of constant-temperature tables degrades, weigh after 14 days and measure the remaining solid amount, calculate degradation rate, with this judge as its degradation property, be that remaining solid is fewer, the amount of being degraded is larger, and the degradation capability of expression colloid is stronger.3 parts of replicate(determination)s, data are averaged.
Degradation rate=(initial solid weight-remaining solid weight)/initial solid weight
Formula glue 1 in embodiment 7 and formula glue 2, its degradation rate is respectively 37.68% and 34.37%, and its degradation capability is greater than the BCA (degradation rate of BCA is 4.18%) of contrast.
Embodiment 10: rat/guinea pig skin otch damage bond test
Get 5 of adult rats, 5 of cavys (available from Military Medical Science Institute's animal center), back depilation, intraperitoneal injection of anesthesia agent (vetanarcol, dosage is 45mg/kg), after the sterilization of back, do at the place at the other approximately 1cm of dorsomeson and be about 2cm stringer otch, deeply reach the flesh layer, after hemostasis, skin incision is involutory tight, the formula glue 1 and 2 for preparing in the specific embodiment of the invention 7 is evenly spread upon rapidly respectively the wound skin surface, fixing approximately 30 seconds, wound well was bonded together.Approximately after 7 days, wound healing is all right, without cracking phenomena.
Embodiment 11: tissue engineering material moulding test
The formula glue 1 and 2 of preparation in the specific embodiment of the invention 7 is injected respectively the common plastics centrifuge tube, place, after it is polymerized to solid, can take out from plastics tubing, two kinds of solid colloids that formula glue obtains having definite shape.Can change the shape of mould, thereby obtain required tissue engineering material shape.
Claims (11)
1. alpha-cyanoacrylate derivative that contains peptide fragment, its structure can represent with formula I, formula II or formula III:
Wherein, AA
1-AA
2-...-AAn represents peptide fragment, can be comprised of any natural amino acid or alpha-non-natural amino acid, and preferred side chain nonfunctional base and the amino acid that need not the Additional Protection base are as glycine, phenylalanine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine;
N represents amino acid quantity, can be 1 to 50, preferred 2 to 30; Further preferred 2 to 25,2 to 20 or 2 to 10; More preferably 2 to 8,2 to 7,2 to 6,2 to 5 or 2 to 4;
X is Sauerstoffatom or nitrogen-atoms, preferred Sauerstoffatom;
R
1The expression junction fragment plays peptide fragment C end or N is held the effect that derives hydroxyl and become ester with alpha-cyanoacrylate, can be alcohol acid class and alkamine molecule, R
1Preferably include but be not restricted to as lactic acid, oxyacetic acid, hydroxy-propionic acid, amino amylalcohol, amino-hexanol, when peptide fragment connects alpha-cyanoacrylate so that N-terminal is amino, when perhaps passing through pendant hydroxyl group in its structure, amino connection alpha-cyanoacrylate, R1 can be for default;
R
2Be peptide fragment end closure group, can be for ethanoyl, methyl, ethyl etc., when the AAn end is the PEPC terminal carboxyl(group), R
2Can be default;
R
3The expression junction fragment, be used for connecting two or more peptide fragment, can be saturated/unsaturated alkyl chain, oxyalkyl chain, silylation chain, polyester, polyethers arbitrarily, preferred ethylene glycol, propylene glycol, Triethylene glycol, tetraethylene-glycol, PEG, amino PEG, oxalic acid, propanedioic acid, succinic acid etc., C end or N end with the peptide fragment at two ends, by mode bondings such as amido linkage, ester bonds, can also adopt the side chain sulfydryl of the halfcystine that belongs to respectively two peptide fragment between two peptide fragment, form disulfide linkage as middle junction fragment.
2. the derivative of claim 1, wherein said peptide fragment can contain a peptide fragment, can be also to contain two or more peptide fragment, between peptide fragment by junction fragment R
3Connect.
3. the derivative of claim 1, wherein,
X is O;
R
1Be hydroxyacetyl, hydroxy propionyl group;
R
2Be default;
R
3For-O-CH2CH2-O-;
Described peptide fragment is Ac-Ser-Gly-Gly, Gly-Gly, Phe-Phe, Phe-Phe-Phe-Ser or Ser-Phe-Gly-Ser.
4. the derivative of claims 1 to 3 any one, it is selected from:
Alpha-cyano acryloyl (Ac-Ser-Gly-Gly-OH) ester;
Alpha-cyano acryloyl (Gly-Gly-OH) amine;
Two alpha-cyanoacrylates (hydroxyl acetyl Phe-Phe-O CH
2CH
2O-Phe-Phe hydroxyl acetyl) ester;
Two alpha-cyanoacrylates (hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) ester;
Alpha-cyanoacrylate (CH
3CONHSer (OH)-Phe-Phe-PHe-Ser (OH) CONHCH
2CH
3) ester;
Alpha-cyanoacrylate (CH
3CONHSer (OH)-Phe-Gly-Ser (OH) CONHCH
2CH
3) ester.
5. the preparation method of the derivative of claim 1-4 any one comprises the following steps:
(1) peptide fragment is synthetic: adopt general peptide solid phase synthesis process in industry, the synthetic peptide fragment AA that contains arbitrary amino acid
1-AA
2-...-AAn; Perhaps from middle fragment R
3Beginning is extended to both sides, adopts the synthetic peptide fragment AA that contains arbitrary amino acid of method of liquid phase synthetic peptide
1-AA
2-... R3 ...-AAn;
(2) anthracene closes the alpha-cyanoacrylate derivatize: as required, connect anthracene in the one or both ends of described peptide fragment and close alpha-cyanoacrylate, when X is the N atomic time, the N end of the peptide fragment that obtains by step (1) is amino to be closed alpha-cyanoacrylate with anthracene and directly becomes amido linkage, perhaps working as X is the O atomic time, the peptide fragment end that obtains in step (1) is derived hydroxyl, then close alpha-cyanoacrylate with anthracene and is connected, the anthracene of acquisition peptide fragment closes the alpha-cyanoacrylate derivative;
Perhaps, when the natural amino acid in the peptide fragment that step (1) obtains or alpha-non-natural amino acid contain pendant hydroxyl group or amino (for example Serine), close alpha-cyanoacrylate by described pendant hydroxyl group or the amino anthracene that connects, the anthracene that obtains peptide fragment closes the alpha-cyanoacrylate derivative; Perhaps, after first containing pendant hydroxyl group or amino natural amino acid or alpha-non-natural amino acid and connecting anthracene and close alpha-cyanoacrylate, then with its amino acid synthesis unit as a peptide fragment, adopt general peptide synthetic method to be synthesized in peptide fragment, the anthracene that obtains peptide fragment closes the alpha-cyanoacrylate derivative;
(3) remove anthracene protection: the anthracene of the peptide fragment that step (2) is obtained closes the alpha-cyanoacrylate derivative and removes the anthracene protecting group, namely obtains finally suc as formula compound shown in I, formula II or formula III.
6. composition, at least comprise the described alpha-cyanoacrylate derivative that contains peptide fragment of one or more claims 1-4 any one, the preferred alpha-cyanoacrylate derivative that comprises simultaneously a kind of formula I or formula II or formula III, randomly, other α-cyanoacrylate quasi-molecules, described other α-cyanoacrylate quasi-molecules comprise but are not restricted to: ethyl α-cyanoacrylate, α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate, isobutyl alpha-cyanoacrylate, the different monooctyl ester of alpha-cyanoacrylate, the alpha-cyanoacrylate glycol ester, the alpha-cyanoacrylate macrogol ester, alpha-cyanoacrylate poly glycol monomethyl ether ester, two alpha-cyanoacrylate PEG diester, two alpha-cyanoacrylate alkyl diol esters, two alpha-cyanoacrylates (poly(lactic acid), PLA) diester, two alpha-cyanoacrylates (polyglycolic acid, PGA) diester, two alpha-cyanoacrylates (poly lactic coglycolic acid, PLGA) diester, two alpha-cyanoacrylates (PLA-PEG-PLA segmented copolymer) diester, two alpha-cyanoacrylates (PGA-PEG-PGA segmented copolymer) diester etc., preferred α-cyanoacrylaten-butyl, α-n-octylcyanoacrylate,
Preferably, the formula of described composition is:
(hydroxypropyl acyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxypropyl acyl) Zhi ︰ α-cyanoacrylaten-butyl is 50% ︰ 50% weight ratio to two alpha-cyanoacrylates;
Preferably, the formula of described composition is:
(hydroxyl acetyl-Phe-Phe-O-CH2CH2-O-Phe-Phe-hydroxyl acetyl) Zhi ︰ α-cyanoacrylaten-butyl is 50% ︰ 50% weight ratio to two alpha-cyanoacrylates.
7. the composition of the derivative of claim 1-4 any one or claim 6, as the purposes of adhesive of medical, or for the preparation of the purposes in adhesive of medical.
8. the purposes of claim 6, described adhesive of medical are used for hemostasis, bonding, covering, leak stopping, sclerous tissues fixes, or internal organ and the soft tissue wound closed;
Preferably, be used for hemostasis when impaired of the human body Medicine and Surgery operation such as outer, neural outer, the tumour in ambition, uropoiesis, general outer liver and gall pancreas stomach and intestine, bone are outer, oral cavity, otorhinolaryngology or extensive wound, bonding, covering, leak stopping, sclerous tissues and fix, or internal organ and the soft tissue wound closed.
9. the composition of the derivative of claim 1-3 any one or claim 5 is for the preparation of the purposes of biological degradability tissue engineering material.
10. pharmaceutical composition comprises the derivative of claim 1-3 any one and one or more are selected from small molecules, peptide class or nucleic acid drug;
Preferably, described pharmaceutical composition, its formulation can be prepared into Nano microsphere or nano-microcapsule.
11. a polymkeric substance, the composition by the described derivative of claim 1 to 4 any one or claim 6 is polymerized under the negatively charged ion effect;
Preferably, described negatively charged ion is the negatively charged ion of denier; More preferably, described negatively charged ion be blood, body fluid, tissue juice or skin-OH ,-NH
2
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106699841A (en) * | 2017-01-05 | 2017-05-24 | 北京大学深圳研究生院 | Self-assembled polypeptide nanorod and preparation method thereof |
| CN114773196A (en) * | 2022-05-19 | 2022-07-22 | 辽宁华星日化产业技术研究院有限公司 | Method for purifying methacrylic acid high-carbon ester |
| CN115093497A (en) * | 2019-09-05 | 2022-09-23 | 大连合元医疗器械有限公司 | Poly (2-cyanoacrylate) and application thereof in embolization microspheres |
| CN115216000A (en) * | 2022-06-29 | 2022-10-21 | 山西医科大学 | Disulfide bond-containing dicyano acrylate, preparation method and application thereof, biological material, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101178A1 (en) * | 2009-03-03 | 2010-09-10 | 公立大学法人横浜市立大学 | Amino acid-conjugated cyanoacrylate polymer particles |
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2012
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101178A1 (en) * | 2009-03-03 | 2010-09-10 | 公立大学法人横浜市立大学 | Amino acid-conjugated cyanoacrylate polymer particles |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106699841A (en) * | 2017-01-05 | 2017-05-24 | 北京大学深圳研究生院 | Self-assembled polypeptide nanorod and preparation method thereof |
| CN115093497A (en) * | 2019-09-05 | 2022-09-23 | 大连合元医疗器械有限公司 | Poly (2-cyanoacrylate) and application thereof in embolization microspheres |
| CN114773196A (en) * | 2022-05-19 | 2022-07-22 | 辽宁华星日化产业技术研究院有限公司 | Method for purifying methacrylic acid high-carbon ester |
| CN114773196B (en) * | 2022-05-19 | 2024-02-13 | 辽宁华星日化产业技术研究院有限公司 | Purification method of high-carbon methacrylate |
| CN115216000A (en) * | 2022-06-29 | 2022-10-21 | 山西医科大学 | Disulfide bond-containing dicyano acrylate, preparation method and application thereof, biological material, preparation method and application thereof |
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