CN116003565A - Preparation method of oligopeptide cyclic peptide - Google Patents

Preparation method of oligopeptide cyclic peptide Download PDF

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CN116003565A
CN116003565A CN202210895556.6A CN202210895556A CN116003565A CN 116003565 A CN116003565 A CN 116003565A CN 202210895556 A CN202210895556 A CN 202210895556A CN 116003565 A CN116003565 A CN 116003565A
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hst1
mad
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吴刚
卢云宇
吴丽勇
程柳行行
雷肖璇
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Hangzhou Huibo Technology Co ltd
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Abstract

The invention provides a preparation method of oligopeptide cyclopeptide, which comprises the steps of obtaining a shortest sequence capable of keeping Hst1 activity from a truncated linear Hst1 complete sequence, and preparing oligopeptide head-tail cyclopeptide with triazole ring by performing azide-alkyne cycloaddition reaction under specific conditions on the shortest sequence; the molecular weight of the oligopeptide head-tail cyclic peptide is only 1/3 of that of the linear histamine, and the biological activity is about 15 times that of the linear histamine; when the same activity is achieved, the cost of the oligopeptide head-tail cyclic peptide is only 1/100 of that of the linear histamine, the yield of the cyclic peptide directly cyclized relative to an amide bond is obviously improved, and the stability and the anti-biological metabolism degradation effect are obviously improved. The whole cyclization process of the oligopeptide cyclopeptide is mild, simple and efficient, can prepare the high-purity oligopeptide head-tail cyclopeptide, can be used for preparing anti-aging and repairing skin care products, health care products or medicines, and has a very good large-scale industrialized prospect.

Description

Preparation method of oligopeptide cyclic peptide
The present application claims priority from China, application number 202110851741.0, application day 2021, 7, 27; all of which are included as part of the present invention.
Technical Field
The invention relates to the fields of biomedical technology and personal care, in particular to a preparation method of oligopeptide cyclopeptides.
Background
The linear peptide has good biological activity and stability in vitro, but the activity disappears soon after entering the body. Because of the complex in vivo environment, a wide variety of enzymes exist. The linear peptide is easily degraded and metabolized under the action of enzymes, and then loses activity. In order to obtain polypeptides with good biological activity, long half-life and higher receptor selectivity, many methods for modification of polypeptides have been reported in the literature, including modification of linear peptides to cyclic peptides. The macrocyclic molecule has a definite fixed conformation, can better fit with a receptor, and has greatly reduced sensitivity to aminopeptidases and carboxypeptidases. The metabolic stability and bioavailability of cyclic peptides are thus far higher than those of linear peptides.
Cyclic peptides are classified according to the ring closure scheme into Head-to-tail, side-chain and side-chain-to-side, side-chain and end-chain-to-end, disulfide-bridge-containing cyclic peptides (disulid e-bridge), staple peptides (Stapled peptides), and cyclic peptides containing other bridging structures. In terms of the synthesis method, the synthesis difficulty of the cyclic peptide connected end to end is greatest. Because the peptide bond of the linear peptide has strong p bond characteristic, the molecule prefers to form a trans-conformation and is in a stretching state, so that carboxyl and amino groups of the end group belonging to a reaction center are far away in space, which is unfavorable for intramolecular condensation reaction, but is favorable for intermolecular condensation.
The end-to-end cyclic peptides are usually linear peptides free at the N-and C-termini in dilute solution (10 -3 ～10 -4 M) is synthesized by forming an amide bond from a carboxyl group and an amino group. The type and number of amino acids in the linear precursor plays a critical role in the ease of ring formation and the yield of cyclic peptides.
The diversity of cyclic peptide synthesis methods is due to the wide variety of numbers and types of amino acids contained in the cyclic peptide precursors, linear peptides. Reagents and methods that exhibit efficient, rapid condensation on certain linear peptides may become inefficient or ineffective on other peptide chains. Thus, finding a corresponding cyclic peptide synthesis method based on the sequence of the target cyclic peptide must go through long and serious exploration and effort.
End-to-end cyclization is an important method for improving the rigidity and stability of peptide structures. Since an exoprotease, such as aminopeptidase or carboxypeptidase, can recognize the N-terminal or C-terminal group of a linear peptide and hydrolyze it. Thus, cyclic peptides are more resistant to proteolytic degradation by enzymes than linear peptides. The cyclic structure limits the conformation of the cyclic peptide in advance, closes the amino groups exposed at the two ends, and makes the enzyme digestion effect poor, thereby reducing the entropy cost in the receptor binding process. This feature increases their binding affinity and specificity for receptor and protein targets. They are therefore suitable for detecting and modulating protein-protein interactions. By appropriate design, cyclic peptides can mimic the secondary structure of proteins (e.g., alpha helices and beta hairpins) that are key modules for receptor recognition. All of these advantageous pharmacological characteristics make cyclic peptides potential drug candidates.
The synthesis method of peptide chain mainly comprises liquid phase synthesis and solid phase synthesis. Liquid phase synthesis uses a fully protected linear precursor (except for the two cyclized ends) directly coupled in the presence of a coupling reagent. However, this strategy has several drawbacks, resulting in a low synthesis efficiency. First, cyclization must be performed in highly diluted solutions to prevent/reduce oligomerization due to intermolecular reactions, because peptide cyclization is an entropy-reducing process, resulting in a large waste of solvents, and cumbersome handling and post-treatment processes. Second, activated C-terminal carboxylic acids are prone to epimerization and thus a pair of stereoisomers, which may also be produced by the enolization of activated C-terminal carboxylic acids by deprotonation of the alpha protons under basic conditions, and thus liquid phase synthesis requires very high purification procedures, resulting in reduced synthesis yields. Finally, some fully protected linear peptide precursors have poor solubility in organic solvents, preventing cyclization. Thus, the solid-phase polypeptide synthesis (SPPS) method is more competitive.
The existing head-to-tail cyclization method mainly comprises the following steps: cyclization on resin (on-resin cyclization), chemical ligation (chemical ligation), azide-olefin cycloaddition (azide-alkyne cycloaddition), ring-closing metathesis (ring-closing metathesis), electrostatically controlled macrocyclization (electronically controlled macro-cyclizations), and the like.
Related reports (Sortase A as a tool for high-yield histatin cyclization, jan G.M.Bolscher, the FASEB journal. Research Communication) that a method for preparing cyclized Hst1 by adopting a Sortase A enzyme method is available, and high-activity Hst1 cyclic peptides are synthesized, but The prepared cyclic peptides have poor solubility due to complicated steps of The Sortase A enzyme method, and are low in price and belong to bacterial enzymes, and bacterial product residues are easily contained, so that The problems of process safety of immunogens, pathogenic microorganisms and The like are brought, and The method is difficult to prepare high-purity Hst1 cyclic peptides which can be produced in a large scale, in addition, large recognition units are needed, redundant amino acid sequences are introduced at The head end and The tail end of The method, so that The length and The sequence complexity of original polypeptides are unnecessarily increased, and The method is not particularly suitable for small-molecule oligopeptides. In addition, CN103249426a discloses a cyclic analogue of histidines prepared by disulfide bonding, but the cyclic peptides usually attached to side chains are prepared by disulfide bonding, it is difficult to prepare stable end-to-end cyclic peptides, and they are not suitable for polypeptide sequences containing no cysteine residues.
The complete sequence of The linear Hst1 is truncated, so that The shortest sequence Hst1-MAD capable of maintaining The activity of Hst1 can be obtained (see Structure-activity analysis of histatin, a potent wound healing peptide from human saliva: cyclization of histatin potentiates molar activity 1000-fold, the FASEB journal. Research Communication), and The required cost is obviously reduced when The same activity is achieved due to The obvious reduction of The molecular weight of The truncated sequence. If the head-tail cyclic peptide of the Hst1-MAD can be further prepared to improve the activity and stability, the unit activity production cost can be further reduced. Therefore, a preparation method of the head-to-tail cyclic peptide of the shortest sequence Hst1-MAD which is simpler, more efficient, less in side reaction, high in solubility of a cyclized product, safe, sterile and capable of realizing large-scale production and keeping the Hst1 activity is urgently needed, so that the unit activity of Hst1 series products is further improved, and the production cost is reduced.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method of oligopeptide cyclopeptide, which comprises the steps of obtaining a shortest sequence capable of keeping the activity of Hst1 after the whole sequence of linear HST1 is truncated, and preparing the oligopeptide head-tail cyclopeptide with triazole ring by performing azide-alkyne cycloaddition reaction under specific conditions on the shortest sequence; the molecular weight of the oligopeptide head-tail cyclic peptide is only 1/3 of that of the linear histamine, and the biological activity is about 15 times that of the linear histamine; when the same activity is achieved, the cost of the oligopeptide head-tail cyclic peptide is only 1/100 of that of the linear histamine, the yield of the cyclic peptide directly cyclized relative to an amide bond is obviously improved, and the stability and the anti-biological metabolism degradation effect are obviously improved. The whole cyclization process of the oligopeptide cyclopeptide is mild, simple and efficient, can prepare the high-purity oligopeptide head-tail cyclopeptide, can be used for preparing anti-aging and repairing skin care products, health care products or medicines, and has a very good large-scale industrialized prospect.
In one aspect, the invention provides an oligopeptide cyclopeptide, the structural formula of which is shown in formula 1 or formula 2:
Figure BDA0003766573290000031
wherein Xaa 1 、Xaa 2 、Xaa 3 、Xaa 4 Is any amino acid; the Hst1-MAD has an amino acid sequence shown as SEQ ID NO.1 in a sequence table; or a sequence having more than 80% homology with SEQ ID NO.1 and fragments thereof, or derivative modified peptide fragments of SEQ ID NO. 1.
In some aspects, the Xaa 1 、Xaa 2 、Xaa 3 、Xaa 4 The amino acids may be the same or different from each other.
Hst1-MAD is obtained by truncating the complete Hst1 (histamine 1) sequence, and the shortest sequence capable of maintaining the activity of Hst1 is Hst1 (20-32), namely the sequence of 20 th to 32 th amino acids in the histamine 1 sequence.
The head-tail connection cyclic peptide of Hst1-MAD is difficult to construct, has high requirements on reaction conditions, and is easy to cause side reaction. According to the invention, through azide-alkynyl cycloaddition reaction, screening of a large number of reactants and searching of process conditions, the Hst1-MAD oligopeptide cyclopeptide containing triazole rings is successfully prepared, and the activity of the oligopeptide cyclopeptide can reach about 15 times of that of linear histamine.
The Hst1-MAD oligopeptide cyclic peptide containing triazole rings provided by the invention can improve the biological activity and clinical efficacy of histamine, can obviously reduce the cost, has the molecular weight of only 1/3 of that of linear histamine, and has the biological activity improved by 1.5 times compared with that of linear histamine when the concentration is only 1/10 of that of linear histamine, so that the activity of the oligopeptide cyclic peptide can reach about 15 times that of linear histamine, the mass concentration is reduced by 4/5, and the cost of the oligopeptide head-tail cyclic peptide is only 1/100 of that of linear histamine when the same activity is achieved.
In addition, the cyclization can lock the spatial conformation of the polypeptide molecule to improve the stability, thereby improving the curative effect and the in vivo half-life. Two types of cyclic peptides shown in formula 1 and formula 2 can be prepared through azide-alkyne cycloaddition reaction by introducing azide and alkyne groups into the head and the tail of domain of Hst1 and simultaneously combining resin.
Due to the construction of triazole rings, the oligopeptide provided by the invention can resist biological metabolism degradation, so that the oligopeptide has excellent metabolic stability, can form hydrogen bond combination with a biological molecule target, can simulate a trans structure or a beta-sheet and corner secondary structure of a natural polypeptide peptide bond, improves solubility, can resist enzyme degradation, and has excellent stability to hydrolysis reaction and oxidation reaction.
Further, the derivatization modification includes one or more of alkylation, acylation, esterification, phosphorylation, sulfonation, glycosylation, PEGylation, biotin labeling, fluorescent labeling, isotopic labeling, D-form, linking linker, carrier protein coupling, or a specific amino acid.
In some embodiments, the specific amino acids include derived amino acids of the 20 basic amino acids that make up the natural protein, as well as specific amino acids that make up certain specific proteins, such as 4-hydroxyproline, 5-hydroxylysine, N-methyllysine, N-formylmethionine, gamma-carboxyglutamic acid, selenocysteine, pyrrolysine, desmin, isodesmin, and beta, gamma, delta-amino acids, and the like.
In some embodiments, the derivative modified peptide fragment of SEQ ID NO.1 includes, but is not limited to, methylation, alkylation, acylation, esterification, phosphorylation, sulfonation, glycosylation, PEGylation, biotin labeling, fluorescent or isotopic labeling, D-type, linking of various linker, carrier protein coupling, or specific amino acids, and the like.
In some embodiments, the derivative modified peptide fragment of SEQ ID NO.1 includes a peptide fragment obtained by adding/subtracting several amino acids at the beginning and end of SEQ ID NO.1, changing L-type amino acid to D-type amino acid, or phosphorylation, lipid acylation, glycosylation, methylation, hydroxylation, ubiquitination, lipidation, aliphatic hydrocarbon group, alicyclic hydrocarbon group, aromatic hydrocarbon group, heterocyclic group or halogenated derivative modification method of amino acid group.
In some embodiments, the derivative modified peptide fragment of SEQ ID No.1 comprises an N-terminal modification (e.g., ac acetylation, etc.) to SEQ ID No. 1; n-terminal fatty acid modification (Myr myristic acid, pal palmitic acid, ste stearic acid, lauric acid, capric acid, caprylic acid modification, etc.); c-terminal modification (-NH 2 amidation, -PNA, -AMC, -OME, -OET, etc.).
In some embodiments, the derivative modified peptide fragment of SEQ ID NO.1 includes modification of a fluorescent label of SEQ ID NO.1, such as Cy3, cy5, cy5.5, cy7, FAM, FITC, rhodamine B, TAMRA, and the like.
In some embodiments, the derivative modified peptide fragment of SEQ ID NO.1 comprises a BIOTIN-labeled modification (e.g., BIOTIN, etc.), or a PEGylation modification (the modified site is at the N-terminus, C-terminus, thiol group of Lys side chain and Cys, etc.), or a phosphorylation modification (e.g., p-Ser, p-Thr, p-Tyr, etc.), a methylation modification (e.g., lys (Me), arg (Me), etc.), of SEQ ID NO. 1.
Further, the structural formula is shown as formula 3 or formula 4:
Figure BDA0003766573290000041
wherein n=1 to 6, m=0 to 3, x=0 or 1, a=1 to 6, b=0 to 2, and R group is an R group of any amino acid.
The R group is the R group of any one of amino acids, and the amino acids comprise natural amino acids and non-natural amino acids, essential amino acids and non-essential amino acids, L-type amino acids, special amino acids (D-type amino acids, beta amino acids, homo amino acids), other various side chain modified amino acids and the like.
In some embodiments, the R group includes, but is not limited to, H, aliphatic hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, heterocyclic groups, halogenated groups, and the like.
In some embodiments, the R groups are 20 natural amino acids andthe fourth group, which is connected with the C atom, is different from the R group according to the amino acid, and mainly comprises: -H, -CH 3 、-CH 2 OH、-CH 2 SH、-CH(OH)CH 3 、-CH(CH 3 ) 2 、-CH 2 CH(CH 3 ) 2 、-CH(CH 3 )CH 2 CH 3 、-CH 2 CH 2 SCH 3 、-C 7 H 7 、
Figure BDA0003766573290000042
-CH 2 -benzene ring-OH, -CH 2 COOH、-CH 2 CONH 2 、-CH 2 CH 2 COOH、-CH 2 CH 2 CONH 2 、-CH 2 CH 2 CH 2 CH 2 NH 2 、-CH 2 CH 2 NHCNH 2 、
Figure BDA0003766573290000043
Etc.
Further, the R group includes a (substituted) aliphatic hydrocarbon group, alicyclic hydrocarbon group, aromatic hydrocarbon group, heterocyclic group, halogenated group or the like.
Further, the R group comprises-H, or any one of methyl, ethyl and phenyl.
Further, the amino group and/or the carboxyl group in formula 3 or formula 4 may be derivatized to obtain-NH 2 And/or histamine cyclic peptides from which-COOH was derivatized.
Further, derivatizing the amino group in formula 3 or formula 4 includes any one or more of acylating, alkylating, pegylating, biotin-labeling, or fluorescent-labeling the amino group; the derivatization of the carboxyl group in the formula 3 or the formula 4 comprises any one or more of amidation, esterification, glycosylation and PEGylation of the carboxyl group.
Such amino-derived modifications include, but are not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, and the like; carboxyl-derived modifications include, but are not limited to, amidation, esterification, glycosylation, PEGylation, and the like.
The amino group-NH 2 Derivatization, e.g. amidation, of the obtained modificationA derivative; the carboxyl-COOH derivatization is a modified derivative obtained by, for example, lipid acylation, palmitoylation, myristoylation, acetylation, etc.
Further, the R group is-H.
Further, the R group of the oligopeptide cyclopeptide is H, and the structural formula of the oligopeptide cyclopeptide is shown in formula 5 or formula 6:
Figure BDA0003766573290000051
where n=1 to 6, m=0 to 3, x=0 or 1, a=1 to 6, b=0 to 3.
Further, the structural formula is shown as formula 7 or formula 8:
Figure BDA0003766573290000052
where n=1 to 3, a=4, x=0 or 1.
Further, the Xaa 1 、Xaa 2 、Xaa 3 、Xaa 4 The carbon chain attached to the triazole ring may contain any one or more of a benzene ring, an alicyclic ring, an aromatic ring, a heterocyclic ring, or a halogenated ring.
Further, the oligopeptide cyclic peptide is obtained by connecting any one amino acid containing alkynyl at one end of Hst1-MAD, connecting any one amino acid containing azido at the other end, combining resin at any one end, cyclizing addition of the alkynyl and the azido, and removing the resin.
In another aspect, the invention provides a use of an oligopeptide cyclopeptide in promoting wound healing or skin care, wherein the oligopeptide cyclopeptide has a structural formula shown in formula 7 or formula 8:
Figure BDA0003766573290000061
where n=1 to 3, a=4, x=0 or 1.
In still another aspect, the present invention provides a method for preparing an oligopeptide cyclic peptide, wherein one end of a derivative modification product of Hst1-MAD and a fragment thereof is connected with any amino acid containing an alkynyl group, the other end is connected with any amino acid containing an azido group, and resin is combined at any end, and the alkynyl group and the azido group are cycloadded and removed from the resin to obtain the oligopeptide cyclic peptide; the Hst1-MAD has an amino acid sequence shown as SEQ ID NO.1 in a sequence table; or a sequence having more than 80% homology with SEQ ID NO.1 and fragments thereof, or derivative modified peptide fragments of SEQ ID NO. 1.
Further, xaa containing alkynyl groups is linked at the C-terminus of the derivative modification product of Hst1-MAD and fragments thereof 1 N-terminal connection of Xaa containing azido group 2 The Xaa 2 Upper bonding resin Xaa 1 Connected alkynyl and Xaa 2 Cyclizing addition of the connected azido groups, removing resin to obtain the histamine cyclic peptide, wherein the reaction formula is shown in the formula 9:
Figure BDA0003766573290000062
or by linking Xaa containing an azide group at the C-terminus of the derivative modification product of Hst1-MAD and fragments thereof 3 N-terminal connection of Xaa containing alkynyl 4 The Xaa 4 The azide of Xaa3 and the alkynyl of Xaa4 are cyclized and added to form a resin, and the resin is removed to obtain the oligopeptide cyclopeptide, wherein the reaction formula is shown in a formula 10:
Figure BDA0003766573290000063
wherein Xaa 1 、Xaa 2 、Xaa 3 、Xaa 4 Is any amino acid.
Such derivatization modifications include, but are not limited to, alkylation, acylation, esterification, phosphorylation, glycosylation, PEGylation, biotin labeling, fluorescent labeling, isotopic labeling, or specific amino acids, and the like.
Further, the alkynyl-containing Xaa 1 Or Xaa 4 The structural formula of (C) is shown as formula 11, or asAmino and/or carboxyl modified derivatives represented by formula 11:
Figure BDA0003766573290000071
xaa containing an azido group 2 Or Xaa 3 The structural formula of (a) is shown as a formula 12, or an amino and/or carboxyl modified derivative shown as a formula 12:
Figure BDA0003766573290000072
Wherein n=1 to 6, m=0 to 3, and the R group is an R group of any amino acid, and x=0 or 1.
In some embodiments, the R group includes, but is not limited to, H, aliphatic hydrocarbon, alicyclic hydrocarbon, aromatic hydrocarbon, heterocyclic, halogenated.
Further, the amino group and/or the carboxyl group in formula 11 or formula 12 may be further derivatized to obtain-NH 2 And/or histamine cyclic peptides from which-COOH was derivatized.
Derivatization of the amino groups and/or carboxyl groups, wherein the amino group derivatization modification includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, and the like, and the carboxyl group derivatization modification includes, but is not limited to, amidation, esterification, glycosylation, PEGylation, and the like
In some embodiments, the amino-NH 2 Derivatizing the modified derivative obtained by amidation or the like; the carboxyl-COOH derivatization is a modified derivative obtained by, for example, lipid acylation, palmitoylation, myristoylation, acetylation, etc.
Further, the R group is-H.
In some embodiments, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, and the like.
Further, before the reaction, the Xaa 1 、Xaa 3 、Xaa 3 Or Xaa 4 Of (2) NH 2 The Fmoc of the protecting group is required to be connected, and after the reaction is finished, the Fmoc of the protecting group is removed to form Is naked-NH 2 Or bare-NH 2 Is further derivatized.
In some embodiments, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, and the like.
Further, before the reaction, the Xaa 1 Or Xaa 3 Of (2) NH 2 The Fmoc of the protecting group is required to be connected, and after the reaction is finished, the Fmoc of the protecting group is removed to become naked-NH 2 Or bare-NH 2 Is further derivatized.
In some embodiments, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, and the like.
The amino group-NH 2 Derivatization such as amidation and the like.
Further, the alkynyl-containing Xaa 1 The structural formula of (2) is shown as formula 13:
Figure BDA0003766573290000073
Figure BDA0003766573290000081
xaa containing an azido group 2 The structural formula of (2) is shown as formula 14:
Figure BDA0003766573290000082
xaa containing an azido group 3 The structural formula of (2) is shown as formula 15:
Figure BDA0003766573290000083
xaa containing alkynyl 4 The structural formula of (2) is shown as formula 16:
Figure BDA0003766573290000084
where n=1 to 4, a=4, x=0 or 1.
Further, the Xaa 1 And/or Xaa 4 Any one or more of benzene ring, alicyclic ring, aromatic ring or heterocyclic ring can be contained in the carbon chain connected with alkynyl; xaa 2 And/or Xaa 3 The carbon chain attached to the azide group may contain any one or more of a benzene ring, an alicyclic ring, an aromatic ring, a heterocyclic ring, or a halogenated group.
In some embodiments, the reaction scheme of the preparation method is shown in formula 17.
Figure BDA0003766573290000085
Further, the method comprises the steps of:
1) Placing the linear peptide prepared by the solid-phase polypeptide synthesis process into a container, and adding a solvent; the linear peptide is formed by connecting Xaa1 containing alkynyl at the C end of linear histamine and Xaa2 containing azido at the N end, and resin is bonded on Xaa 2; or Xaa3 containing azido is connected to the C end of the linear histamine, xaa4 containing alkynyl is connected to the N end of the linear histamine, and resin is bonded to Xaa 4;
2) Adding a catalyst;
3) Adding a ligand;
4) Bubbling with nitrogen;
5) The container is tightly covered and closed, and the mixture is stood and stirred;
6) Washing with disodium ethylenediamine tetraacetate, and removing the washing liquid;
7) Washing with water, washing with an organic solvent, and removing the washing liquid;
8) Vacuum drying;
9) Adding a cutting mixed solution to cut the polypeptide from the resin;
10 Purified polypeptide.
In some embodiments, the solvent may be a protic solvent such as selected from methanol, ethanol, t-butanol, polyethylene glycol PEG, trifluoroethanol TFE, hexafluoroisopropanol HFIP, water, a dipolar aprotic solvent such as acetonitrile MeCN, dimethyl sulfoxide DMSO, acetone, N-dimethylformamide DMF, N-diisopropylethylamine DIPEA, N-methylpyrrolidone NMP, pyridine, piperidine, a nonpolar solvent such as dichloromethane DCM, chloroform, tetrahydrofuran THF, ethyl acetate, diethyl ether, benzene, toluene, carbon tetrachloride, dioxane, N-hexane, cyclohexane, and the like, and may be any one or more of two or three thereof in a certain ratio such as t-butanol/water, methanol/dichloromethane, dichloromethane/acetonitrile, dichloromethane/water, acetonitrile/dimethyl sulfoxide, acetone/dimethyl sulfoxide, ethanol/water, N-methylpyrrolidone/acetonitrile, N-methylpyrrolidone/dichloromethane/water, N-hexane/dioxane, N-dimethylformamide/trifluoroethanol, N-dimethylformamide/hexafluoroisopropanol, and the like.
Steric hindrance, electrical properties, polarity, protonicity, hydrogen bonding and ability and proportion of the solvent affect the reaction speed, the formation proportion of side reaction byproducts such as dimers, trimer multimers and decomposition products, and the like.
Further, the invention adopts a mixed solution of acetonitrile and dimethyl sulfoxide as a solvent.
Further, the solvent in the step 1) is acetonitrile and dimethyl sulfoxide (MeCN: DMSO), and the solution is prepared according to the ratio of 4:1 as the solvent.
It was found that the concentration of domain of Hst1 oligopeptide in the solution influences the reaction speed, resulting in an increase of the proportion of side reaction byproducts such as dimers and trimer multimers, but in general, SPPS solid phase synthesis is more favorable to side reactions such as intramolecular cyclization rather than intermolecular cyclization compared with liquid phase synthesis, which is one of the reasons for selecting SPPS in the invention.
In some embodiments, the invention preferably uses 750mg domain of Hst1 oligopeptides, with the addition of 20ml of acetonitrile and solvent dimethyl sulfoxide (MeCN: dmso=4:1).
Further, the Resin is selected from any one of Wang Resin, rink Amide AM Resin, rink Amide MBHA Resin, 2-Chlorotrityl Resin, PEGylated Rink-modified TentaGel (TGR) Resin, aminomethyl Resin, sieber Amide Resin, PAM Resin and the like.
Different resins affect the reaction rate and lead to increased side reaction byproducts.
In some embodiments, rink Amide MBHA Resin is employed to increase the reaction rate and significantly reduce the occurrence of side reaction byproducts.
Further, the catalyst of the present invention may be a copper or rhodium reagent.
In some embodiments, the catalyst of the present invention is a copper reagent selected from the group consisting of monovalent copper salts and complexes thereof such as copper iodide CuI, copper chloride CuCl, copper iodide/triethyl phosphite CuI.P (OEt) 3 Cuprous bromide, cuprous bromide dimethyl sulfide, cuprous bromide tris (triphenylphosphine) [ CuBr (PPh) 3 ) 3 ]Cuprous tris (triphenylphosphine) fluoride [ CuF (PPh) 3 ) 3 ]Tetraethyl cyanocopper tetrafluoroborate [ Cu (MeCN) 4 ]BF 4 Tetraethyl cyanogen copper hexafluorophosphate [ Cu (MeCN) 4 ]PF 6 Tetraethyl cyanocopper triflate [ Cu (MeCN) 4 ]OTf, various Cu (I) -carbene complexes; can also be selected from cupric salts and their complexes such as copper sulfate CuSO 4 Copper chloride CuCl 2 Copper acetate Cu (OAc) 2 Copper nitrate Cu (NO) 3 ) 2 Copper acetylacetonate Cu (acac) 2 ) A composition of the copper nanoparticles and a reducing agent such as sodium ascorbate, or a composition of the copper nanoparticles.
In addition, the loading of the copper/rhodium reagent also affects the reaction completion to some extent, ranging from 0.05eq to 4eq, and may be non-copper/rhodium reagents such as HBTU/HOBt, but the effect varies significantly.
Further, the copper catalyst of the invention is cuprous iodide.
Further, the addition amount of the cuprous iodide is 20mM.
Further, the ligand of the present invention is generally a nitrogen-containing ligand, and may be selected from any one of 2,6-lutidine (2, 6-lutidine), diethylamine, triethylamine, n-propylamine, diisopropylamine, tributylamine, diisopropylethylamine DIPEA, dimethylaminopyridine DMAP, 1, 8-diazabicyclo [5.4.0] undec-7-ene DBU, pentamethyldiethylenetriamine PMDETA, hexamethyltriethylenetetramine HMTETA, tris (2-dimethylaminoethyl) amine Me6tre, piperidine, pyridine, bipyridine, 2':6',2 "-terpyridyl tpy, tris (2-pyridylmethyl) amine TPMA, bipyridine, t-butyltrichloroacetyliminoester TBTA, tris (3-hydroxypropyl triazolylmethyl) amine THPTA, TTTA, BTTAA, TABTA, BTTE, BTTP; the phosphorus-containing ligand can be any one selected from triphenylphosphine, tricyclohexylphosphine PCy3, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine BINAP, 1, 2-bis (diphenylphosphine) ethane DPPE, 1, 3-bis (diphenylphosphine) propane DPPP, 1, 4-bis (diphenylphosphine) butane DPPB and 1, 5-bis (diphenylphosphine) pentane DPPPe; the sulfur-containing ligand may be any one selected from 4,4' -thiobis (6-tert-butyl-3-methylphenol BPS, BTTES, BTTPS).
When the catalyst is a copper reagent, the alkaline, donor performance and steric hindrance of the ligand stabilize monovalent copper ions, so that the monovalent copper ions are prevented from being oxidized or disproportionated, meanwhile, the formation of a cuprous acetylene complex is promoted, the loading capacity of the copper reagent is reduced, the cyclization reaction efficiency is improved, the ratio of the ligand to copper is varied from 0.5/1 to 4/1, and the excess is different, depending on the relative activity, polarity and donor capability of the ligand, the copper reagent and the solvent.
Further, the ligand of the present invention is 2,6-lutidine (2, 6-lutidine).
Further, the addition amount of 2,6-lutidine of the present invention was 30. Mu.L.
Further, the reaction conditions for the azide-alkynyl cycloaddition reaction of the present invention are also severely limited.
In some embodiments, the reaction system of the reactions described herein can tolerate a wide range of acidity of 4-12.
Further, the reaction process of the present invention must be deoxygenated;
oxygen oxidizes the copper reagent, which results in failure of the cyclization process, and thus deoxygenation is important, while humidity affects the reaction efficiency.
Further, the reaction process of the present invention requires bubbling with nitrogen for 2 to 5 minutes. Nitrogen bubbling can be used for 2 to 5 minutes to ensure deoxygenation of the reaction process.
Further, air insulation is also required: and (5) tightly covering and sealing to isolate oxygen.
Further, the reaction temperature of the present invention is room temperature to 100 ℃. The reaction temperature affects the reaction rate and the relative proportions of product/by-product and decomposition products and their stability.
Further, the reaction time of the invention is 10min to 100h.
In some ways, the reaction of the present invention may also be carried out under microwave (room temperature to 100 ℃) conditions, which may accelerate the reaction, but our studies have demonstrated that microwave conditions are prone to by-products and decomposition products.
Further, after the completion of the reaction, the reaction mixture was washed with saturated disodium ethylenediamine tetraacetate solution (saturated of disodium EDTA) by shaking, and chelating agent EDTA was used for removing copper ions.
Further, the washing was performed with EDTA shaking for 2 times each for 10 minutes.
Further, the washing solution is removed by washing with water 1 time, acetonitrile 1 time, dichloromethane 1 time, and diethyl ether once.
Further, vacuum drying is required for 1 hour after the washing is completed.
Further, after drying is completed, cleavage is performed: according to the standard flow: 20ml of the mixture (95.5% trifluoroacetic acid, 2% phenol aqueous solution, 2% phenylsulfide, 0.5% triisopropylsilane) was shaken for 3 hours to shear the polypeptide from the resin. The polypeptide was filtered through cold diethyl ether, washed 3 times and dried.
Further, purification by high-performance liquid chromatography (HPLC) was also required, and the detection wavelength was 210nm by gradient elution using an acetonitrile aqueous solution containing 0.1% (v/v) trifluoroacetic acid as a mobile phase.
Further, the preparation method comprises the following steps:
1) 750mg of linear peptide prepared by the solid-phase polypeptide synthesis process is placed in a container, and 20ml of mixed solution prepared by acetonitrile and dimethyl sulfoxide according to the proportion of 1:1-10:1 is added; the linear peptide is formed by connecting Xaa1 containing alkynyl at the C end of linear histamine and Xaa2 containing azido at the N end, and resin is bonded on Xaa 2; or Xaa3 containing azido is connected to the C end of the linear histamine, xaa4 containing alkynyl is connected to the N end of the linear histamine, and resin is bonded to Xaa 4;
2) 750mg of resin was added;
3) Adding 20mM cuprous iodide;
4) 30. Mu.L of 2, 6-lutidine was added;
5) Bubbling with nitrogen for 2-5 min;
6) Closing the container and closing;
7) Stirring and reacting for 40-80 minutes at room temperature;
8) Shaking and cleaning for 2 times by using saturated disodium ethylenediamine tetraacetate solution for 10 minutes each time, and removing the cleaning solution;
9) Washing with water for 1 time, acetonitrile for 1 time, dichloromethane for 1 time, diethyl ether for one time, and removing washing liquid;
10 Vacuum drying for 1 hour;
11 Cutting: preparing a cutting mixed solution comprising trifluoroacetic acid, phenol, phenylsulfide and triisopropylsilane=95.5:2:2:0.5, v/v), adding 20ml of the cutting mixed solution into the sample obtained in the step 10), shaking for 3 hours, and cutting the polypeptide from the resin. Filtering the polypeptide with cold diethyl ether, washing for 3 times, and drying;
12 High performance liquid chromatography purification.
Researches prove that the cyclization method provided by the invention can be used for performing end-to-end cyclization on a long peptide chain, and can successfully cyclize any histamine Hst 1-Hst 12, so that the histamine cyclic peptide containing the triazole ring is prepared, and the cyclic peptide is simple and convenient in preparation process, high in purity and activity and easy for large-scale industrial production.
The invention prepares histamine cyclic peptide containing triazole ring through copper catalyzed azido-alkyne cycloaddition reaction, which only generates 1, 4-disubstituted [1,2,3] triazole products, and has similarities with natural peptide bonds in many aspects, such as hydrogen bond forming capacity, planarity, 1, 4-substituent distance, conformational restriction of peptide skeleton and the like. Thus, the triazole in the modified peptide can be used to replace peptide bonds, and can display a secondary structure similar to that of the natural polypeptide.
The beneficial effects of the invention are as follows:
1. creatively adopts copper to catalyze the azide-alkyne cycloaddition reaction to prepare a series of Hst1-MAD oligopeptide head-tail cyclic peptides with triazole rings;
2. The molecular weight of the prepared Hst1-MAD oligopeptide head-tail cyclic peptide is only 1/3 of that of the linear histamine, and the biological activity is 15 times that of the linear histamine;
3. the prepared Hst1-MAD oligopeptide head-tail cyclic peptide has excellent stability and biological metabolism degradation resistance, can improve the solubility, has unique conformation, and remarkably improves the biological activity and clinical efficacy of histamine;
4. when the same activity is achieved, the cost of the oligopeptide head-tail cyclic peptide is only 1/100 of that of the linear histamine;
5. the cyclization process adopted by the invention is mild, simple and convenient, the efficiency is high, the purity of the prepared oligopeptide head-tail cyclic peptide is higher, and the method has very good large-scale and industrialized application prospects.
Drawings
FIG. 1 is a mass spectrum MS detection pattern of the cyclization reaction product of example 1;
FIG. 2 is a high performance liquid chromatography HPLC detection chart of the cyclization reaction product of example 1;
FIG. 3 is an infrared IR detection spectrum of the cyclization reaction product of example 1;
FIG. 4 is a photograph showing the healing of the wound surface of four groups of mice in example 10 on days 0, 3 and 5;
FIG. 5 is a graph showing the comparison of wound healing rates of the four groups of mice in example 10 on days 3, 5 and 10;
FIG. 6 is a photograph showing the detection of wound epidermis neogenesis on day 10 of HE staining of four groups of wound tissues in example 10;
FIG. 7 is a comparison of the thickness of the epidermis of the wound tissue of the four groups in example 10;
FIG. 8 is a photograph of a Masson stain on day 10 of four groups of wound tissue from example 10 to detect collagen neogenesis;
FIG. 9 is a comparative schematic diagram of wound collagen regeneration on day 10 of the four groups of wound tissues in example 10;
FIG. 10 is a graph showing the results of the number of new CD31 positive blood vessels generated on days 5 and 10 of the four groups of wound tissues in example 10;
FIG. 11 is a graph showing quantitative analysis and comparison of the numbers of new CD31 positive blood vessels on days 5 and 10 of the four groups of wound tissues in example 10;
FIG. 12 is a graph showing VEGF expression level at days 5 and 10 of the four groups of wound tissues in example 10;
FIG. 13 is a graph showing quantitative analysis and comparison of VEGF expression levels on days 5 and 10 of the four groups of wound tissues in example 10;
FIG. 14 is a photograph showing the detection of dendritic cells (MHC II+ and CD11c+) of four groups of wound tissues at days 3 and 5 by immunofluorescence double staining in example 10;
FIG. 15 is a graph showing the comparison of the effect of the four drugs of example 10 on the modulation of wound dendritic cells (MHC II+ and CD11c+);
FIG. 16 is a photograph showing the detection of M1 macrophages (CD68+ and CD80+) of four groups of wound tissue at days 3 and 5 by immunofluorescence double staining in example 10;
FIG. 17 is a graph showing the comparison of the effect of the four drugs in example 10 on the modulation of wound M1 macrophages (CD68+ and CD80+);
FIG. 18 is a photograph showing the detection of M2 macrophages (CD68+ and CD206+) of four groups of wound tissue at days 3 and 5 by immunofluorescence double staining in example 10;
FIG. 19 is a graph showing the comparison of the effect of the four drugs in example 10 on the modulation of wound M2 macrophages (CD68+ and CD206+);
FIG. 20 is a graph showing analysis of M1/M2 macrophage results in example 10;
FIG. 21 is a photograph showing the immunofluorescence double-staining test of four sets of wound surface connective protein (Claudin 1) expression in example 10;
FIG. 22 is a graph showing the comparison of the effects of four drugs in example 10 on the regulation of wound connexin (Claudin 1);
FIG. 23 is a photograph showing the immunofluorescence double-staining test of four sets of wound surface connective protein (Claudin 2) expression in example 10;
FIG. 24 is a graph showing the comparison of the effects of four drugs of example 10 on the regulation of wound connexin (Claudin 2);
FIG. 25 is a photograph showing the detection of Nrf2/HO-1/NQO1 signal pathway expression of four groups of wounds by Western blotting (Western Blot) in example 10;
FIG. 26 is a graph showing comparison of the expression of antioxidant stress Nrf2/HO-1/NQO1 signal pathways on days 3 and 5 of the wound surface for the four groups of drugs in example 10;
FIG. 27 is a photograph showing the detection of inflammatory factors IL-6, TNF-. Alpha., iNOS and MIP-1β expressed in four groups of wounds by Western blotting (Western Blot) in example 10;
FIG. 28 is a graph showing the comparison of the expression of inflammatory factors IL-6, TNF- α, iNOS and MIP-1β on days 3 and 5 of the wound surface by the four drugs of example 10.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The reagents used in the invention are all commercial reagents.
EXAMPLE 1 preparation of propynylglycine-Hst 1-MAD cyclopeptides
The preparation method of the embodiment comprises the following steps:
1. alkynyl and protecting group Fmoc were introduced into glycine, azido and protecting group Fmoc were introduced into lysine to obtain Fmoc-propynylglycine (formula 18) for connection of C-terminal (Fmoc-propynylglycine from Merck KGaA was directly used in this example) and Fmoc-azido lysine (formula 19) for connection of N-terminal (Fmoc-azido lysine from Merck KGaA was directly used in this example), the structural formula was as follows:
Figure BDA0003766573290000131
2. on the basis of Fmoc-azido lysine, adopting a polypeptide solid phase synthesis method (SPPS) from right to left to synthesize linear amino acid of Hst1-MAD (SEQ ID NO. 1) in one step, and connecting Fmoc-propynylglycine at the C end of Hst1-MAD (SEQ ID NO. 1);
3. Preparing cyclic peptide (Click-Hst 1-MAD) of Hst1-MAD (SEQ ID NO. 1) by copper-catalyzed azido-alkyne cycloaddition reaction by using the Hst1-MAD linear polypeptide prepared in the step 2, wherein the preparation method comprises the following steps:
1) 750mg domain of Hst1 linear polypeptide is placed in a container, and 20ml of mixed solution prepared by acetonitrile and dimethyl sulfoxide according to the ratio of 4:1 is added;
2) 750mg of resin Rink Amide MBHA Resin (available from Merck KGaA) was added;
3) Adding 20mM cuprous iodide;
4) 30 μl of 2, 6-lutidine (lutidine) was added;
5) Bubbling with nitrogen for 5 minutes;
6) Closing the container and closing;
7) Stirring and reacting for 40 minutes at room temperature;
8) Shaking and cleaning for 2 times by using saturated disodium ethylenediamine tetraacetate solution for 10 minutes each time, and removing the cleaning solution;
9) Washing with water for 1 time, acetonitrile for 1 time, dichloromethane for 1 time, diethyl ether for one time, and removing washing liquid;
10 Vacuum drying for 1 hour;
11 Shearing: preparing a shearing mixed solution comprising 95.5% of trifluoroacetic acid, 2% of phenol water solution, 2% of phenylsulfide and 0.5% of triisopropylsilane, adding 20ml of the shearing mixed solution into the sample obtained in the step 10), and shaking for 3 hours to shear the polypeptide from the template; filtering the polypeptide with cold diethyl ether, washing for 3 times, and drying;
12 High-performance liquid chromatography (HPLC) purification, gradient elution with acetonitrile aqueous solution containing 0.1% (v/v) trifluoroacetic acid as mobile phase, detection wavelength 210nm.
707mg of propynylglycine-Hst 1-MAD cyclopeptide was obtained.
The structural formula of the propynylglycine-Hst 1-MAD cyclic peptide is shown in formula 20:
Figure BDA0003766573290000141
the mass spectrum detection shows that the purity of the detection spectrum is more than 95% and the recovery rate is 94% as shown in figure 1. The HPLC detection shows the detection pattern as shown in figure 2; the infrared IR detection shows that the detection spectrum is shown in figure 3, and has characteristic peak of triazole (3100 cm) -1 、1400cm -1 、1100cm -1 、700cm -1 ) No azide (2100 cm) -1 ) And alkyne (2200 cm) -1 ) Is a characteristic peak of (2).
EXAMPLE 2 preparation of homopropynylglycine-Hst 1-MAD cyclopeptides
The preparation method of the embodiment comprises the following steps:
1. alkynyl and protecting group Fmoc were introduced into glycine, azido and protecting group Fmoc were introduced into lysine to prepare Fmoc-propynylglycine for connection to C-terminal (formula 21) (Fmoc-propynylglycine from Merck KGaA was directly used in this example), and Fmoc-azido lysine for connection to N-terminal (formula 19) (Fmoc-azido lysine from Merck KGaA was directly used in this example), respectively, with the following structural formula:
Figure BDA0003766573290000151
2. on the basis of Fmoc-azido lysine, synthesizing linear amino acid of Hst1-MAD (SEQ ID NO. 1) from right to left by adopting a polypeptide solid phase synthesis method (SPPS), and connecting Fmoc-propynylglycine at the C end of Hst 1-MAD;
3. Hst1-MAD cyclic peptide was prepared by copper-catalyzed azido-alkyne cycloaddition using the Hst1-MAD linear polypeptide prepared in step 2, and the preparation method was prepared according to the method provided in example 1.
698mg of propynylglycine-Hst 1-MAD cyclopeptide was obtained.
The structural formula of the propynylglycine-Hst 1-MAD cyclic peptide is shown in formula 22:
Figure BDA0003766573290000152
the purity of the product is over 95 percent and the recovery rate is 93 percent through mass spectrum detection.The characteristic peak of triazole (3100 cm) is shown by infrared IR detection -1 、1400cm -1 、1100cm -1 、700cm -1 ) No azide (2100 cm) -1 ) And alkyne (2200 cm) -1 ) Is a characteristic peak of (2).
EXAMPLE 3 preparation of Bispyric glycine-Hst 1-MAD cyclopeptides
The preparation method of the embodiment comprises the following steps:
1. alkynyl and protecting group Fmoc were introduced into glycine, azido and protecting group Fmoc were introduced into lysine to prepare Fmoc-propynylglycine (formula 23) for connection to C-terminal (Fmoc-propynylglycine available from Merck KGaA was directly used in this example) and Fmoc-azido lysine (formula 19) for connection to N-terminal (Fmoc-azido lysine available from Merck KGaA was directly used in this example), respectively, and the structural formulae were as follows:
Figure BDA0003766573290000153
2. on the basis of Fmoc-azido lysine, synthesizing linear amino acid of Hst1-MAD (SEQ ID NO. 1) from right to left by adopting a polypeptide solid phase synthesis method (SPPS), and connecting Fmoc-propynylglycine at the C end of Hst 1-MAD;
3. Hst1-MAD cyclic peptide was prepared by copper-catalyzed azido-alkyne cycloaddition using the Hst1-MAD linear polypeptide prepared in step 2, and the preparation method was prepared according to the method provided in example 1.
699mg of propynylglycine-Hst 1-MAD cyclopeptide was obtained.
The structural formula of the propynylglycine-Hst 1 cyclic peptide is shown in formula 24:
Figure BDA0003766573290000161
the purity of the product is over 95 percent and the recovery rate is 93 percent through mass spectrum detection. The characteristic peak of triazole (3100 cm) is shown by infrared IR detection -1 、1400cm -1 、1100cm -1 、700cm -1 ) No azide (2100 cm) -1 ) Andalkyne (2200 cm) -1 ) Is a characteristic peak of (2).
EXAMPLE 4 Effect of Linear oligopeptide concentration on cyclization reaction
In this example, propynylglycine-Hst 1-MAD cyclic peptide was prepared according to the method provided in example 1, 250, 500, 750 and 1000mg of linear oligopeptide was added to a mixed solution of acetonitrile and dimethyl sulfoxide prepared in a ratio of 4:1, the reaction was performed, the required reaction time was recorded, the yield of the reaction product was detected by detecting HPLC, the proportion of side reactions (including by-products such as diploid, triploid and tetraploid of intermolecular condensation) in the reaction product was calculated, and the proportion of the by-products to the oligopeptides Hst1-MAD head-tail cyclic peptide was calculated by mass spectrometry molecular weight confirmation, so that the influence of different linear Hst1-MAD oligopeptide concentrations on the cyclization reaction was examined, and the results are shown in table 1.
TABLE 1 Effect of different linear Hst1-MAD oligopeptide additions on cyclization reactions
Linear oligopeptide dosage (mg) Reaction time (min) Yield (%) Side reaction occurrence ratio (%)
250 40 82 15
500 40 91 7
750 40 94 3
1000 60 80 16
As can be seen from Table 1, the cyclization reaction was significantly affected by different amounts of the linear Hst1-MAD oligopeptide, and the reaction rate was decreased as the amount of the linear oligopeptide was increased, and the yield and the side reaction occurrence ratio were also changed accordingly, wherein the reaction rate was still faster and the yield reached 94% with the linear oligopeptide addition of 750mg, and the side reaction occurrence ratio was very low, so that the most preferable linear oligopeptide addition was 750mg in 20ml of the solvent.
EXAMPLE 5 Effect of different solvents on cyclization reactions
In this example, a propynylglycine-Hst 1-MAD cyclic peptide was prepared according to the method provided in example 1, and the different solvents shown in table 2 were used for the reaction, the required reaction time was recorded, and the yield of the reaction product was measured by detecting HPLC, and the proportion of side reactions occurring in the reaction product was calculated, thereby examining the influence of the different solvents on the cyclization reaction, and the results are shown in table 2.
TABLE 2 influence of different solvents on the cyclization reaction
Solvent(s) Reaction time (min) Yield (%) Side reaction occurrence ratio (%)
Methanol 120 44 51
Tert-butanol 90 54 41
Dimethyl sulfoxide 60 67 29
Acetonitrile 90 57 40
Acetonitrile: dimethyl sulfoxide (1:1) 60 84 11
Acetonitrile: dimethyl sulfoxide (2:1) 40 94 3
Acetonitrile: dimethyl sulfoxide (4:1) 40 91 7
Acetonitrile: dimethyl sulfoxide (8:1) 60 89 9
As can be seen from Table 2, there is a significant effect on the cyclization reaction by different solvents, mainly because steric hindrance, electric property, polarity, protonic property, hydrogen bonding and ability and ratio are different from each other due to different solvents, and the reaction rate, side reaction by-products such as dimer, trimer polymer and decomposition products, etc. are affected. Acetonitrile is therefore preferred: dimethyl sulfoxide (2:1) was used as a solvent.
EXAMPLE 6 Effect of different resins on cyclization reactions
In this example, a propynylglycine-Hst 1-MAD cyclic peptide was prepared as provided in example 1, and the reaction was carried out using Wang Resin, rink Amide AM Resin, rink Amide MBHA Resin, 2-Chlorotrityl Resin, PEGylated Rink-modified TentaGel (TGR) Resin, aminomethyl Resin, sieber Amide Resin, and PAM Resin, respectively, for a total of 8 kinds of resins, the reaction time was recorded, and the yield of the reaction product was measured by detecting HPLC, and the ratio of occurrence of side reaction in the reaction product was calculated to examine the effect of different resins on the cyclization reaction, and the results are shown in Table 3.
TABLE 3 influence of different resins on cyclization reactions
Figure BDA0003766573290000171
As can be seen from Table 3, the different resins are selected to have obvious influence on the cyclization reaction, the reaction speed, the yield and the side reaction occurrence ratio are all obviously changed, and Rink Amide MBHA Resin resin is most preferably adopted, so that the product yield can reach more than 94%, the side reaction ratio is reduced to 3%, and the side reaction can be prevented by combining with Rink Amide MBHA Resin resin, and the efficiency of the cyclization reaction is improved.
EXAMPLE 7 Effect of different catalysts on cyclization reactions
In this example, propynylglycine-Hst 1-MAD cyclopeptides were prepared as provided in example 1, using CuI, cuCl, cuI/triethyl phosphite (OEt) 3 Cuprous bromide, cuprous bromide dimethyl sulfide, cuprous fluoride tris (triphenylphosphine) [ CuF (PPh) 3 ) 3 ]Tetraethyl cyanocopper tetrafluoroborate [ Cu (MeCN) 4 ]BF 4 Tetraethyl cyanogen copper hexafluorophosphate [ Cu (MeCN) 4 ]PF 6 As a catalyst, the reaction was performed, the required reaction time was recorded, and the yield of the reaction product was measured by detecting HPLC, and the proportion of occurrence of side reactions in the reaction product was calculated, thereby examining the influence of different catalysts on the cyclization reaction, and the results are shown in table 4.
TABLE 4 influence of different catalysts on cyclization reactions
Catalyst Reaction time (min) Yield (%) Side reaction occurrence ratio (%)
Cuprous iodide 40 94 3
Cuprous chloride 60 89 9
Cuprous iodide/triethyl phosphite 40 56 41
Cuprous bromide 70 77 20
Cuprous bromide dimethyl sulfide 60 69 25
Cuprous tris (triphenylphosphine) fluoride 80 70 27
Tetraethylcyanocopper tetrafluoroborate 60 61 36
Tetraethyl cyanogen copper hexafluorophosphate 60 55 43
As can be seen from Table 4, the choice of different catalysts has a significant effect on the cyclization reaction, and the reaction rate, yield and the occurrence ratio of side reactions all vary significantly, but only when cuprous iodide is used as the catalyst, the yield of the product can be 94%, and the occurrence ratio of side reactions is the lowest, so that cuprous iodide is most preferable as the catalyst.
EXAMPLE 8 Effect of different ligands on cyclization reactions
In this example, propynylglycine-Hst 1-MAD cyclic peptide was prepared according to the method provided in example 1, and 2,6-lutidine (2, 6-lutidine), diethylamine, triethylamine, n-propylamine, diisopropylamine, tributylamine, diisopropylethylamine DIPEA, dimethylaminopyridine DMAP were used as ligands for reaction, the reaction time was recorded, and the yield of the reaction product was measured by detecting HPLC, and the proportion of side reaction occurring in the reaction product was calculated to examine the effect of different ligands on cyclization reaction, and the results are shown in Table 5.
TABLE 5 influence of different ligands on cyclization reactions
Ligand Reaction time (min) Yield (%) Side reaction occurrence ratio (%)
2, 6-lutidine 40 94 3
Diethylamine 60 87 11
Triethylamine 50 66 31
N-propylamine 70 78 20
Diisopropylamine 60 70 26
Tributylamine 80 64 30
Diisopropylethylamine DIPEA 60 79 20
Dimethylaminopyridine DMAP 60 81 15
As can be seen from Table 5, the choice of different ligands has a significant effect on the cyclization reaction, and the reaction rate, yield and ratio of side reactions are all significantly varied, so that 2, 6-lutidine is most preferred as the ligand.
EXAMPLE 9 Effect of reaction conditions on cyclization reactions
In this example, a propynylglycine-Hst 1-MAD cyclic peptide was prepared according to the method provided in example 1, and the reaction was carried out under different reaction conditions as shown in table 6, the reaction time was recorded, and the yield of the reaction product was measured by detecting HPLC, and the proportion of side reaction occurring in the reaction product was calculated, thereby examining the influence of different reaction conditions on the cyclization reaction, and the results are shown in table 6.
TABLE 6 influence of different reaction conditions on the cyclization reaction
Ligand Reaction time (min) Yield (%) Side reaction occurrence ratio (%)
Nitrogen bubbling for 5 min at 25 deg.c 40 94 3
Nitrogen bubbling for 5 min at 50 deg.c 60 87 10
Nitrogen bubbling for 5 min at 100 deg.c 50 54 44
Nitrogen bubbling for 5 min, micro Wave-guide 60 77 20
No nitrogen bubbling at 25 DEG C 40 11 84
As can be seen from Table 6, the choice of different reaction conditions has a significant effect on the cyclization reaction, and the reaction rate, yield and side reaction occurrence ratio all vary significantly, so that the most preferable reaction conditions are nitrogen bubbling for 5 minutes at 25 ℃.
EXAMPLE 10 Effect of Hst1-MAD cyclopeptides on wound repair
In this example, animal experiments were carried out using the propynylglycine-Hst 1-MAD cyclic peptide (Click-Hst 1-MAD), the linear Hst1-MAD, and the linear peptide Hst1 (linear histamine 1) provided in example 1, while setting a Control (blank) group, and observing the effect of the Click-Hst1-MAD on the wound healing rate of mice, wherein the concentration of the linear peptide Hst1 was 10. Mu.M, the concentration of the linear Hst1-MAD was 1. Mu.M, and the concentration of the Click-Hst1-MAD was 1. Mu.M.
1 materials and methods
The study was approved by the medical animal center and ethics committee of the general hospital in the southern war zone of the liberation army of Chinese people.
1.1 animals and major reagents and instrumental sources
36 healthy male specific pathogen-free grade C57 BL/6 mice, 6-8 weeks old, with a mass of 22-28g, purchased from the laboratory animal center in Guangdong province. Diaminobenzidine (DAB) was purchased from Bodhisattva bioengineering, inc., rabbit anti-CD 31 antibody, mouse anti-VEGF antibody, goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Servicebio, U.S. and Ethylene Diamine Tetraacetic Acid (EDTA) antigen buffer, immunohistochemical pen, bovine serum albumin, collagenase A, hematoxylin were all purchased from Bodhisattva biotechnology, inc., inverted fluorescence microscope was purchased from Nikon, japan, dehydrator was purchased from Bodhisattva electronics, inc., paraffin microtome was purchased from Shanghai Leika instruments, inc.
1.2 grouping and treatment of animals
36 healthy male specific pathogen-free C57 BL/6 mice (purchased from Experimental animal center, guangdong province), 6-8 weeks old, and 22-28g in mass. 5ml/kg of 1% pentobarbital sodium was intraperitoneally injected for anesthesia, and after the mice had no corneal reflex, the hair on the backs of the mice was carefully removed and sterilized with alcohol. The back of each mouse was prepared with a circular skin punch to prepare 2 1cm x 1cm full-thickness skin defect wounds. 36 mice were divided into 9 Control (blank) groups, 9 10. Mu.M linear peptide Hst1 (Histatin 1) groups, 9 1. Mu.M Hst1-MAD linear peptide groups, and 9 1. Mu.M propynylglycine-Hst 1-MAD cyclic peptide (Click-Hst 1-MAD) groups according to the random number table method. Four medicines of 0.5ml are respectively added into each wound surface, the medicines are administered every day until the wound surface of an experimental group heals, photographing is respectively carried out on days 0, 3, 5 and 10 after the operation, and the materials of the wound surface tissues are obtained on days 3, 5 and 10, so that the subsequent pathological staining is carried out.
1.3 Observation index
1.3.1 wound healing Rate
The general conditions of wound healing of mice were observed 0, 3, 5 and 10 days after injury, and photographs were taken with a digital camera (see fig. 4) and wound healing rates were calculated (see fig. 5). Wound healing rate = (immediate wound area after wound-non-healing wound area at each time point)/(immediate wound area after wound x 100%).
1.3.2 histomorphometric assay
The wound specimens were fixed in 4% paraformaldehyde for 48 hours, rinsed with tap water for 10 minutes, dehydrated with gradient ethanol, transparent with xylene, paraffin-embedded, and 4 μm thick sections were prepared. Part of the sections were taken for HE staining to detect epidermis thickness and Masson staining to detect collagen neogenesis. The effect of the four groups of drugs on wound epidermal growth was assessed on day 10 by hematoxylin-eosin staining (HE) (fig. 6 and 7). The effect of four groups of drugs on wound collagen regeneration on day 5 and day 10 was assessed by Masson staining (Masson) (fig. 8 and 9). Each mouse was sectioned in 5 sections, each section having 5 fields of view. Data analysis was performed with software Image-pro Plus.
1.3.3 immunohistochemical staining
Paraffin sections prepared in 1.3.2 after injury for 10 days were dewaxed with xylene, rehydrated, and antigen thermally repaired in 0.1mol/L citric acid buffer (pH 6.0) for 20 minutes, 10 minutes with 10% volume fraction hydrogen peroxide inactivated endogenous peroxidase, and 50g/L bovine serum albumin blocked for 2 hours. Rabbit anti-murine CD31 primary antibody (dilution ratio 1:300) and murine anti-VEGF (dilution ratio 1:100) were added, incubated overnight at 4℃and washed with PBS. Rewarming for 1 hour, washing with PBS, dripping the instant biotinylated goat anti-mouse and goat anti-rabbit IgG secondary antibodies, incubating for 2 hours, and washing with PBS. DAB color development, hematoxylin counterstain, gradient ethanol dehydration, xylene transparency and resin sealing. Each mouse was sectioned in 5 pieces, each section was sectioned in 5 fields, and observed under a 400-fold inverted fluorescent microscope and analyzed for CD31 positive blood vessel number (see fig. 10) and VEGF expression (see fig. 11) using Image-pro Plus software.
1.3.4 immunofluorescent double-staining detection of dendritic cells, M1 macrophages, claudin1 and Claudin2
Immunofluorescence double-staining detection of dendritic cells (MHC ii+ and cd11c+): the effect of 1. Mu.M Hst1-MAD on wound dendritic cell (MHC II+ and CD11c+) regulation was assessed by immunofluorescence double staining on day 3 and day 5.
Immunofluorescent double staining method for detecting M1 macrophages (cd68+and cd80+) and M2 (cd68+and cd206+) macrophages: the effect of 1. Mu.M Hst1-MAD on wound macrophage regulation was assessed by immunofluorescence double staining on day 3 and day 5.
The immunofluorescence double-staining method for detecting Claudin1 comprises the following steps: the effect of 1. Mu.M Hst1-MAD on wound surface connexin (Claudin 1) on day 10 was assessed by immunofluorescence counterstaining.
The immunofluorescence double-staining method for detecting Claudin2 comprises the following steps: the effect of 1. Mu.M Hst1-MAD on wound surface connexin (Claudin 2) on day 10 was assessed by immunofluorescence counterstaining.
The method comprises the following specific steps: the previous steps were the same as 1.3.3, antibodies MHC II (orb 101661;1:500;Biorbyt Biotechnology Co., ltd., wuhan, CN), CD11c (97585;1:200;Cell Signaling Technology Inc., boston, mass.), CD68 (GB 11067;1:300;Servicebio Inc), CD80 (GB 11034;1:300;Servicebio Inc), CD206 (GB 11062;1:500;Servicebio Inc), claudin1 (37-4900;1:100;Thermo Fisher Scientific Co, ltd, shanghai, CN), claudin2 (32-5600;1:200;Thermo Fisher Scientific Co, ltd.) overnight at 4 ℃. The fluorescently labeled secondary antibody was incubated at room temperature for 1h, then 4',6-diamidino-2' -phenylindole (DAPI) (G1012; google Biotechnology Co., ltd., wuhan, CN). The stained sections were photographed with a fluorescence microscope (Nikon Eclipse TI-SR, tokyo, japan). Positive cells were analyzed by automatic counting using Image-Pro Plus software. Expression levels of claudin1 and claudin2 were quantified with MOD values.
1.3.5 Western blotting
Western Blot (Western Blot) detection of Nrf2/HO-1/NQO1 Signal pathway method: the effect of 1. Mu.M Hst1-MAD and Click-Hst1-MAD on the oxidative stress Nrf2/HO-1/NQO1 signaling pathway was evaluated by WB experiments on days 3 and 5.
Western Blot (Western Blot) method for detecting inflammatory factor expression: the effect of 1 mu M Hst1-MAD and Click-Hst1-MAD on the expression regulation of wound inflammatory factors on the 3 rd and 5 th days was evaluated by WB experiments.
The method comprises the following specific steps: in order to detect the expression of inflammatory factors and the regulation of oxidative stress, a wound tissue is taken for western blot detection. The wound tissue was washed with PBS, lysis buffer was added, homogenized 12000g and centrifuged for 10min, and the supernatant was collected. Bicinchoninic acid (BCA) kit (p 0010; beyotime Biotechnology co., ltd., shanghai, CN) was used to mix with a sample buffer of reduced Sodium Dodecyl Sulfate (SDS) and then boiled for 5min. Samples were loaded on SDS-PAGE gels, followed by electrophoresis at 80 volts for 30min and 120 volts for 1h. After transfer to a polyvinylidene fluoride (PVDF) membrane, incubation with 5% skim milk powder for 1h, primary antibodies Nrf2 (ab 137550;1:1000;Abcam Trade Co, ltd.), NQO1 (ab 28947;1:1000;Abcam Trade Co, ltd.), TNF- α (ab 6671;1:1000;Abcam Trade Co, ltd.), IL-6 (ab 9324;1:1000;Abcam Trade Co, ltd.), macrophage inflammatory protein-1β (MIP-1β) (C04131; 1:1000;Signalway Antibody Co, ltd, nanjin, CN) were incubated overnight at 4 ℃. Subsequently, goat anti-mouse secondary antibody (SA 00001-2;1:3000;Proteintech Group Inc, wuhan, CN) and HRP-labeled goat anti-mouse secondary antibody (GB 23301;1:3000;Proteintech Group Inc) were incubated at room temperature for 1 hour, and then displayed by chemiluminescence. To detect the expression of the above proteins, western blot bands were analyzed using Image J software.
1.4 statistical treatment
The metering data are expressed by M+/-SD, and SPSS 20.0 software is used for carrying out single-factor analysis of variance and independent sample t test on the data to detect whether the differences among all groups have statistical significance, and the two-by-two comparison is carried out by Bonferroni test. * p <0.05, < p <0.01, < p <0.001 > is statistically significant, and p >0.05 is not statistically significant.
2 results
2.1 general observations and wound healing Rate
Four groups of wound surfaces gradually shrink along with the time growth after 3 days, 5 days and 10 days after operation, and the wound surface area of a 1 mu M propynylglycine-Hst 1-MAD cyclic peptide (Click-Hst 1-MAD) group is smaller than that of the other three groups at 3 days and 5 days (figure 4). As can be seen from FIG. 5, on days 3 and 5, the wound healing rate of 1 mu M Click-Hst1-MAD group is significantly higher than that of Control, histatin group and Hst1-MAD linear peptide group (P < 0.05), and on day 10, the wound of the mice is basically repaired without reference significance; wherein ∈3: the 1. Mu.M Click-Hst1-MAD group, the 1. Mu.M Hst1-MAD group, the 10. Mu.M Hst group and the Control group have significant differences (P < 0.05). The 1 mu M Click-Hst1-MAD group can better promote wound healing, the effect is better than that of the 1 mu M Hst1-MAD linear peptide group, even better than that of the 10 mu M Histatin group, and meanwhile, the effect of the 1 mu M Hst1-MAD linear peptide group is equivalent to that of the 10 mu M Histatin linear peptide group.
2.2 histomorphology observations
To assess the effect of four groups on epidermis thickness and dermis collagen expression, wound tissues were HE stained (fig. 6). On day 10, the 1. Mu.M Click-Hst1-MAD group had the thickest wound surface epidermis, the 10. Mu.M Histatin1 group next, the Control group and the 1. Mu.M Hst1-MAD group had the thinnest epidermis thickness (P < 0.05) (FIG. 7).
The results of Masson staining detection of collagen neogenesis are shown in FIG. 8, and the collagen neogenesis expression levels of 1 mu M Click-Hst1-MAD group are significantly higher than those of the other three groups (FIG. 9) on days 5 and 10, wherein the 1 mu M Hst1-MAD group is equivalent to the dermis collagen expression level of 10 mu M Histatin1 group on day 5. Wherein,, 3,: the 1 mu M Click-Hst1-MAD group, the 1 mu M Hst1-MAD group, the 10 mu M Histatin1 group and the Control group have significant differences (P < 0.05). The 1 mu M Click-Hst1-MAD group can better promote the collagen neogenesis expression.
2.3 immunohistochemical staining
CD31 and VEGF are angiogenesis markers, and the effect of 1 mu M Click-Hst1-MAD group, 1 mu M Hst1-MAD group, 10 mu M Histatin1 and Control group on wound angiogenesis was evaluated by immunohistochemical staining.
The results of immunohistochemical staining evaluation of the number of blood vessels positive for 4 groups of wound surface newly generated CD31 are shown in FIGS. 10 and 11. As can be seen from fig. 10, the number of CD31 positive blood vessels per field of 1 μm Click-Hst1-MAD group, 1 μm Hst1-MAD group and 10 μm Histatin1 group was significantly higher than Control (arrow) scale=50 μm 5 days and 10 days after surgery; as can be seen from FIG. 11, the CD31 positive blood vessel number was quantitatively analyzed, and the 1. Mu.M Click-Hst1-MAD group had more neovascular numbers than the other three groups. 3,: the 1 mu M Click-Hst1-MAD group, the 10 mu M Histatin1 group, the 1 mu M Hst1-MAD group and the Control group have significant differences (P < 0.05).
The results of immunohistochemical staining evaluation of the 4-group wound VEGF expression level are shown in FIGS. 12 and 13. From fig. 12, 10 days post-operation, 1 μm Click-Hst1-MAD group and 10 μm Histatin1 group showed higher positive expression levels of VEGF (indicated by arrows) scale = 50 μm; from FIG. 13, wherein (1) is the result of the analysis of the integrated density of VEGF and (2) is the result of the analysis of the expression area of VEGF, the quantitative analysis of VEGF according to FIGS. 13 (1) and (2) shows that three groups of 1. Mu.M of Click-Hst1-MAD group, 10. Mu.M of Histatin1 group and 1. Mu.M of Hst1-MAD group have equivalent expression amount of VEGF at 5 days; 10 days later, the VEGF expression level of 1 mu M Click-Hst1-MAD group is obviously higher than that of the other three groups. The 1. Mu.M Click-Hst1-MAD group, the 10. Mu.M Histatin1 group, the 1. Mu.M Hst1-MAD group and the Control group are significantly different (P < 0.05).
2.4 immunofluorescence double staining
The photograph of immunofluorescence double-staining detection of dendritic cells (MHCII+ and CD11c+) is shown in FIG. 14, and the analysis of the effect of four groups of drugs on wound dendritic cell (MHCII+ and CD11c+) regulation is shown in FIG. 15. As can be seen from FIG. 15, on the 3 rd day and 5 th day of the evaluation, on the 3 rd day after the operation, the numbers of dendritic cells in the 10. Mu.M Hst1 group and the 1. Mu.M Click-Hst1-MAD group were 1.75 times and 1.95 times that of the Control group, respectively, and the numbers of dendritic cells in the 1. Mu.M Hst1-MAD group were slightly lower than those in the 10. Mu.M Histatin1 group, but were significantly higher than those in the Control group, and the numbers of dendritic cells in the Click-Hst1-MAD group were significantly higher than those in the other three groups, so that the 1. Mu.M Click-Hst1-MAD group (1. Mu.M promethazine-Hst 1-MAD cyclic peptide) significantly promoted the growth of dendritic cells (MHC II+ and CD11 c+).
The photograph of immunofluorescence double-staining assay M1 macrophages (CD68+ and CD80+) is shown in FIG. 16, and the analysis of the effect of four groups of drugs on wound surface M1 macrophages (CD68+ and CD80+) regulation is shown in FIG. 17. From FIG. 17, the conditions on days 3 and 5 were evaluated, and the number of M1 macrophages (CD68+ and CD80+) in the 1. Mu.M Hst1-MAD group was slightly higher than that in the 10. Mu.M Histatin1 group, but lower than that in the Control group, and it was seen that the Hst1-MAD group and the Histatin1 group reduced the number of M1 macrophages (CD68+ and CD80+) in the wound surface; on day 3, the number of M1 macrophages (CD68+ and CD80+) of the Click-Hst1-MAD group was comparable to that of the Control group, while on day 5, the number of M1 macrophages (CD68+ and CD80+) of the Click-Hst1-MAD group was significantly reduced compared to the other three groups, and it was seen that 1. Mu.M of the propioglycine-Hst 1-MAD cyclic peptide significantly reduced the number of M1 macrophages (CD68+ and CD80+) after day 5, indicating that 1. Mu.M of the propioglycine-Hst 1-MAD cyclic peptide was able to better reduce the inflammatory response at the late stage of inflammation.
The photograph of immunofluorescent double-staining assay for M2 macrophages (CD68+ and CD206+) is shown in FIG. 18, and the analysis of the effect of four groups of drugs on wound surface M2 macrophages (CD68+ and CD206+) regulation is shown in FIG. 19. From FIG. 19, on day 3, the M2 macrophages (CD68+ and CD206+) of the Click-Hst1-MAD group were significantly higher than those of the other three groups, the 1 μM Click-Hst1-MAD group (16.40+ -5.86) and the 10 μM Hst1 group (12.67+ -7.34) were higher than those of the control group (12.17+ -4.44); on day 5, the number of M2 macrophages (CD68+ and CD206+) in the Click-Hst1-MAD group is slightly higher than that in the other three groups, and M2 cells secrete inflammatory factors to promote wound healing, so that 1 mu M propynylglycine-Hst 1-MAD cyclic peptide can more effectively promote wound healing.
As can be seen, the M2/M1 macrophage ratio was significantly higher in the Click-Hst1-MAD group than in the control group (2.61-fold and 2.17-fold, respectively), the 10. Mu.M Histatin1 group and the 1. Mu.M Hst1-MAD group. Demonstrating that the Click-Hst1-MAD group better promoted M1 pro-inflammatory macrophages to convert M2 pro-healing macrophages (FIG. 20).
The photograph of immunofluorescence double-staining detection wound surface connexin (Claudin 1) is shown in figure 21, and the analysis of the influence of four groups of medicines on the regulation and control of wound surface connexin (Claudin 1) is shown in figure 22. From FIG. 22, on day 10, the Claudin1 expression intensity of the Click-Hst1-MAD group was significantly higher than that of the other three groups, and the levels of Claudin1 expression promoted by the 10. Mu.M Histatin1 group and the 1. Mu.M Hst1-MAD group were equivalent, and the 1. Mu.M Click-Hst1-MAD group was able to further effectively promote Claudin1 expression.
The photograph of immunofluorescence double-staining detection wound surface connexin (Claudin 2) is shown in figure 23, and the analysis of the influence of four groups of medicines on the regulation and control of wound surface connexin (Claudin 2) is shown in figure 24. From FIG. 24, on day 10, the Claudin2 expression intensity of the Click-Hst1-MAD group was significantly higher than that of the other three groups, and the next 1. Mu.M Hst1-MAD group, and the effect of promoting Claudin2 expression of the 10. Mu.M Histatin1 group was not significant, unlike the Control group. It can be seen that the Hst1-MAD linear peptide and the Click-Hst1-MAD cyclic peptide can more effectively promote the expression of Claudin2, and particularly the effect of the Click-Hst1-MAD cyclic peptide (1 mu M propynylglycine-Hst 1-MAD cyclic peptide) is particularly remarkable compared with the 10 mu M Histatin1 linear peptide.
2.5 Western blotting
Western Blot (Western Blot) assay results photographs of Nrf2/HO-1/NQO1 signal pathways are shown in FIG. 25, and analysis of the effect of four groups of drugs on antioxidant stress Nrf2/HO-1/NQO1 signal pathways on day 3 and day 5 is shown in FIG. 26. As can be seen from fig. 25 and 26, on the 3 rd day after operation, the expression levels of Nrf2 and HO-1 in the 10 μm Histatin1 group are 1.27 and 1.80 times that of the Control group, the expression level of NQO1 in the 10 μm Hst1 group is significantly higher than that in the Control group (2.08 times), and the expression levels of Nrf2 and HO-1 in the Click-Hst1-MAD group and the Hst1-MAD group are also improved relative to the Control group, which is equivalent to that in the Histatin1 group; on the 5 th day after operation, the expression level of the 1 mu M Click-Hst1-MAD group on Nrf2, HO-1 and NQO1 signal paths is obviously improved compared with that of the control group and the Histatin1 group.
Photographs of experimental results of detecting the expression level of inflammatory factors by Western Blot (Western Blot) are shown in FIG. 27, and analysis of the effect of the four groups of drugs on the regulation of wound inflammatory factor expression on the 3 rd and 5 th days is shown in FIG. 27. As can be seen from fig. 27 and 28, the three groups of drugs were effective in inhibiting the expression levels of inflammatory factors IL-6, TNF- α, iNOS and MIP-1β relative to the Control group, wherein the 1 μm Click-Hst1-MAD group inhibited significantly better than the other three groups for inflammatory factors TNF- α, whether 3 days or 5 days after surgery; for inflammatory factors iNOS, the inhibition effect of 1 mu M of Click-Hst1-MAD group is obviously better than that of the other three groups in 3 days after operation, and the inhibition effect of 1 mu M of Click-Hst1-MAD group is similar to that of 1 mu M of Hst1-MAD group in 5 days after operation, and is obviously better than that of a control group and a 10 mu M of Histatin1 group; the inhibition effect of the inflammatory factors IL-6 and MIP-1 beta in the 1 mu M Click-Hst1-MAD group and the 1 mu M Hst1-MAD group is also obviously better than that of the control group and the 10 mu M Histatin1 group.
EXAMPLE 11 comparison of the Activity of cyclic peptides
In this example, the propynylglycine-Hst 1-MAD cyclic peptide, the homopropynylglycine-Hst 1-MAD cyclic peptide, the dihomopropynylglycine-Hst 1-MAD cyclic peptide and The linear Hst1 prepared in examples 1, 2 and 3, and The Hst1-MAD head-to-tail cyclic peptide prepared by The method of amide bond ring formation as provided in Sortase A as a tool for high-yield histatin cyclization, jan g.m. bolscher, the FASEB journal. Research Communication literature were used, respectively, the reaction time was recorded, the yield of The reaction product was detected by detecting HPLC, the ratio of occurrence of side reaction in The reaction product was calculated, and The activity comparison was performed, the activity comparison method was a scratch test, and The dose required for achieving The same efficacy was calculated, and The specific method was: at the same time, fibroblasts (NIH/3T3;GNM 6;Chinese Academy of Sciences,China) were prepared according to 2X 10 6 The number of wells/wells was inoculated into 6-well plates and cultured to a density of 70-80%. Cells were starved for 8-12 hours before experiments were performed. Mitomycin C (Sigma Aldrich, USA) was added to the medium to a concentration of 15. Mu.g/ml in the medium, and the medium was changed after 3 hours of treatment. Scratches were made with the same force using a 200. Mu.l gun head perpendicular to the bottom of the well plate, and after the scratch was completed, the wells were rinsed 2 times with PBS. DEME medium (10% FBS,1% diabody) was added to each well, and 10 μΜ linear Hst1, 1 μΜ propynyl glycerol were added separately to the blank The experimental groups were set up of an amino acid-Hst 1-MAD cyclic peptide, a 1. Mu.m propioglycine-Hst 1-MAD cyclic peptide, a 1. Mu.m linear Hst1-MAD, and a Hst1-MAD head-to-tail cyclic peptide prepared by a 1. Mu.m amide bond cyclization method. The cells were then placed at 37℃in 5% CO 2 The incubation was performed, and after 12 hours and 24 hours from the incubation, the scratch areas of each group were calculated using imageJ (Rawak Software, inc. Germany) Software, and the scratch area of 0 hour was the initial scratch area. The scratch healing rate was calculated by: w% (scratch healing rate) = (W0-Wt)/w0×100%, where w0=initial scratch area, wt=remaining scratch area; the results are shown in Table 7.
TABLE 7 comparison of Activity
Figure BDA0003766573290000231
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Figure BDA0003766573290000241
As can be seen from Table 7, the Hst1-MAD oligopeptide prepared by the method provided by the invention has the advantages that the activity is improved by more than 15 times compared with that of the linear peptide Hst1, the yield is higher, the side reaction is very few, the activity of Hst1-MAD directly cyclized by adopting the traditional amide bond is lower, even lower than that of the linear peptide Hst1-MAD, the yield is only 10%, the side reaction occurrence ratio is up to 89%, the operation process is complicated, the reaction time is long, and the Hst1-MAD oligopeptide is difficult to prepare.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Sequence listing
SEQ ID NO.1
Hst1-MAD：SHREFPFYGDYGS

Claims (14)

1. A preparation method of oligopeptide cyclic peptide is characterized in that one end of derivative modification products of Hst1-MAD and fragments thereof is connected with any amino acid containing alkynyl, the other end is connected with any amino acid containing azido, resin is combined with any end, the alkynyl and azido are cycloadded, and resin is removed to obtain oligopeptide cyclic peptide; the Hst1-MAD has an amino acid sequence shown as SEQ ID NO.1 in a sequence table; or a sequence having more than 80% homology with SEQ ID NO.1 and fragments thereof, or derivative modified peptide fragments of SEQ ID NO. 1.
2. The method of claim 1, wherein Xaa containing alkynyl is attached to the C-terminus of the derivative modification product of Hst1-MAD and fragments thereof 1 N-terminal connection of Xaa containing azido group 2 The Xaa 2 Upper bonding resin Xaa 1 Connected alkynyl and Xaa 2 Cyclizing addition of the connected azido groups, removing resin to obtain the histamine cyclic peptide, wherein the reaction formula is shown in the formula 9:
Figure FDA0003766573280000011
or by linking Xaa containing an azide group at the C-terminus of the derivative modification product of Hst1-MAD and fragments thereof 3 N-terminal connection of Xaa containing alkynyl 4 The Xaa 4 The azide of Xaa3 and the alkynyl of Xaa4 are cyclized and added to form a resin, and the resin is removed to obtain the oligopeptide cyclopeptide, wherein the reaction formula is shown in a formula 10:
Figure FDA0003766573280000012
Wherein Xaa 1 、Xaa 2 、Xaa 3 、Xaa 4 Is any amino acid.
3. The method of claim 2, wherein Xaa containing alkynyl 1 Or Xaa 4 The structural formula of (C) is shown as formula 11, or amino and amino shown as formula 11And/or a carboxyl modified derivative:
Figure FDA0003766573280000013
xaa containing an azido group 2 Or Xaa 3 The structural formula of (a) is shown as a formula 12, or an amino and/or carboxyl modified derivative shown as a formula 12:
Figure FDA0003766573280000014
wherein n=1 to 6, m=0 to 3, and the R group is an R group of any amino acid, and x=0 or 1.
4. A process according to claim 3, wherein the amino and/or carboxyl groups of formula 11 or formula 12 are derivatised.
5. The method of claim 4, wherein R is-H.
6. The method of claim 5, wherein before the reacting, xaa 1 、Xaa 3 、Xaa 3 Or Xaa 4 Of (2) NH 2 The Fmoc of the protecting group is required to be connected, and after the reaction is finished, the Fmoc of the protecting group is removed to become naked-NH 2 Or bare-NH 2 Is further derivatized.
7. The method of claim 6, wherein before the reacting, xaa 1 Or Xaa 3 Of (2) NH 2 The Fmoc of the protecting group is required to be connected, and after the reaction is finished, the Fmoc of the protecting group is removed to become naked-NH 2 Or bare-NH 2 Is further derivatized.
8. The method of claim 7, whereinXaa containing alkynyl 1 The structural formula of (2) is shown as formula 13:
Figure FDA0003766573280000021
xaa containing an azido group 2 The structural formula of (2) is shown as formula 14:
Figure FDA0003766573280000022
xaa containing an azido group 3 The structural formula of (2) is shown as formula 15:
Figure FDA0003766573280000023
xaa containing alkynyl 4 The structural formula of (2) is shown as formula 16:
Figure FDA0003766573280000024
where n=1 to 4, a=4, x=0 or 1.
9. The method of preparation of claim 8, wherein Xaa 1 And/or Xaa 4 Any one or more of benzene ring, alicyclic ring, aromatic ring or heterocyclic ring can be contained in the carbon chain connected with alkynyl; xaa 2 And/or Xaa 3 The carbon chain attached to the azide group may contain any one or more of a benzene ring, an alicyclic ring, an aromatic ring, a heterocyclic ring, or a halogenated group.
10. The method of preparing as claimed in claim 9, comprising the steps of:
1) Placing the linear peptide prepared by the solid-phase polypeptide synthesis process into a container, and adding a solvent; the linear peptide is formed by connecting Xaa1 containing alkynyl at the C end of linear histamine and Xaa2 containing azido at the N end, and resin is bonded on Xaa 2; or Xaa3 containing azido is connected to the C end of the linear histamine, xaa4 containing alkynyl is connected to the N end of the linear histamine, and resin is bonded to Xaa 4;
2) Adding a catalyst;
3) Adding a ligand;
4) Bubbling with nitrogen;
5) The container is tightly covered and closed, and the mixture is stood and stirred;
6) Washing with disodium ethylenediamine tetraacetate, and removing the washing liquid;
7) Washing with water, washing with an organic solvent, and removing the washing liquid;
8) Vacuum drying;
9) Adding a cutting mixed solution to cut the polypeptide from the resin;
10 Purified polypeptide.
11. The method of claim 10, wherein the solvent of step 1) is a mixture of acetonitrile and dimethyl sulfoxide; the resin was Rink Amide MBHA Resin.
12. The method of claim 11, wherein the catalyst of step 2) is cuprous iodide; the ligand in the step 3) is 2, 6-lutidine.
13. The method according to claim 12, wherein the steps 4) to 5) are reaction processes, the pH of the reaction process is 4 to 12, the reaction temperature is room temperature to 100, and the reaction time is 10min to 100h.
14. The method of manufacturing of claim 13, comprising the steps of:
1) 750mg of linear peptide prepared by the solid-phase polypeptide synthesis process is placed in a container, and 20ml of mixed solution prepared by acetonitrile and dimethyl sulfoxide according to the proportion of 1:1-10:1 is added;
2) 750mg of resin was added;
3) Adding 20mM cuprous iodide;
4) 30. Mu.L of 2, 6-lutidine was added;
5) Bubbling with nitrogen for 2-5 min;
6) Closing the container and closing;
7) Stirring and reacting for 40-80 minutes at room temperature;
8) Shaking and cleaning for 2 times by using saturated disodium ethylenediamine tetraacetate solution for 10 minutes each time, and removing the cleaning solution;
9) Washing with water for 1 time, acetonitrile for 1 time, dichloromethane for 1 time, diethyl ether for one time, and removing washing liquid;
10 Vacuum drying for 1 hour;
11 Cutting: preparing a cutting mixed solution comprising trifluoroacetic acid, phenol, phenylsulfide and triisopropylsilane=95.5:2:2:0.5, v/v), adding 20ml of the cutting mixed solution into the sample obtained in the step 10), shaking for 3 hours, and cutting the polypeptide from the resin. Filtering the polypeptide with cold diethyl ether, washing for 3 times, and drying;
12 High performance liquid chromatography purification.
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CN202210888704.1A Pending CN116333086A (en) 2021-07-27 2022-07-27 Oligopeptide cyclic peptide and application thereof
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