Polypeptide-manganese-carbonyl compound CO release molecule with phenylazo pyridine as ligand and synthetic method and application thereof
阅读说明：本技术 一种以苯偶氮基吡啶为配体的多肽-锰-羰基复合物co释放分子及其合成方法与应用 (Polypeptide-manganese-carbonyl compound CO release molecule with phenylazo pyridine as ligand and synthetic method and application thereof ) 是由 何春茂 周仪 于 2021-07-23 设计创作，主要内容包括：本发明提供了一种以苯偶氮基吡啶为配体的多肽-锰-羰基复合物CO释放分子及其合成方法与应用。本发明通过基于Fmoc保护基团的固相多肽合成法和带有正交保护基4-氨基苯丙氨酸的使用,得到带有反应位点的多肽氨基树脂,再利用固相米尔斯反应在多肽目标位点构建苯偶氮基吡啶类配位基团,最后利用五羰基溴化锰进行锰-羰基-配体的配位反应,乙醚萃取后即获得目标产物。本发明将苯偶氮基吡啶配体与多肽-CO释放分子结合起来,配体的使用能够使CO的释放光源能量明显降低,光源波长向近红外区域移动,有利于CO释放分子在生理生化研究中的实际应用,以多肽为载体有助与对特定部位的生理生化作用,为CO在相关疾病的治疗方面奠定良好基础。(The invention provides a polypeptide-manganese-carbonyl compound CO release molecule using phenylazo pyridine as a ligand, and a synthesis method and application thereof. The invention obtains polypeptide amino resin with reaction sites by a solid-phase polypeptide synthesis method based on Fmoc protective groups and the use of 4-aminophenylalanine with orthogonal protective groups, then constructs phenylazo pyridine coordination groups at polypeptide target sites by utilizing solid-phase Mills reaction, finally performs manganese-carbonyl-ligand coordination reaction by utilizing manganese pentacarbonyl bromide, and obtains target products after ether extraction. The invention combines the phenylazo pyridine ligand with the polypeptide-CO release molecule, the use of the ligand can obviously reduce the energy of the CO release light source, the wavelength of the light source moves to the near infrared region, the invention is beneficial to the practical application of the CO release molecule in the physiological and biochemical research, the polypeptide is taken as the carrier to help the physiological and biochemical action on the specific part, and the invention lays a good foundation for the treatment of the CO in the aspect of the relevant diseases.)
1. A solid phase synthesis method of polypeptide-manganese-carbonyl compound CO release molecules with phenylazo pyridine as a ligand is characterized in that: the method comprises the following steps:
(1) designing a target polypeptide sequence containing amino acid as a coordination group introduction site, adopting an Fmoc solid-phase polypeptide synthesis method, taking Fmoc amino resin as a carrier, sequentially condensing Fmoc protected amino acid from a C end to an N end according to the target polypeptide sequence, washing, and drying to obtain linear polypeptide resin; wherein:
amino acid as a coordination group introduction site is synthesized by using Fmoc-4-aminophenylalanine of which the amino protecting group of aniline is allyloxycarbonyl;
the rest amino acids are Fmoc amino acids without side chains, or the Fmoc amino acids of which the protecting groups of the side chains can be removed by trifluoroacetic acid are synthesized;
(2) adding the linear polypeptide resin prepared in the step (1) into a palladium catalytic system, reacting, washing and drying to obtain the linear polypeptide resin with the allyloxycarbonyl protecting group removed;
(3) taking the linear polypeptide resin prepared in the step (2), adding a nitrosopyridine compound, dichloromethane and glacial acetic acid, reacting, washing and drying to obtain the linear polypeptide resin containing the phenylazo pyridine coordination group;
(4) taking the linear polypeptide resin containing the phenylazo pyridine coordination group prepared in the step (3), adding a cutting reagent to remove the polypeptide chain from the Fmoc amino resin, and removing the residual side chain protecting group; filtering, spin-drying, extracting, centrifuging and freeze-drying to obtain a linear polypeptide crude product containing phenylazo pyridine coordination groups; further separating and purifying to obtain linear polypeptide containing phenylazo pyridine coordination groups;
(5) taking the linear polypeptide containing the phenylazo pyridine coordination group prepared in the step (4), adding manganese pentacarbonyl bromide, and performing coordination reaction by using a dichloromethane/methanol mixed solution as a reaction solvent; and (3) spin-drying the solvent, and extracting with diethyl ether to obtain the manganese-carbonyl-linear polypeptide compound containing the phenylazo pyridine coordination group.
2. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule using phenylazo pyridine as ligand according to claim 1, wherein:
the amino acid used as the site for introducing the coordination group in the step (1) is phenylalanine or tyrosine.
3. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule with phenylazopyridine as ligand according to claim 1 or 2, characterized in that:
the palladium catalysis system in the step (2) is a tetrakis (triphenylphosphine) palladium catalysis system, the dosage of the tetrakis (triphenylphosphine) palladium is 3-5 times equivalent of the polypeptide supported on the resin, and the reaction solvent is a chloroform-acetic acid-N-methylmorpholine mixed solution of chloroform, acetic acid and N-methylmorpholine according to the volume ratio of 37:2: 1;
the reaction conditions in the step (2) are that the temperature is 20-30 ℃ and the time is 2-3 h.
4. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule with phenylazopyridine as ligand according to claim 1 or 2, characterized in that:
the nitrosopyridine compound in the step (3) comprises nitrosopyridine and nitrosopyridine derivatives;
the chemical formula of the nitrosopyridine is C5H4N2O, the structural formula is as follows:
the dosage of the nitrosopyridine compound in the step (3) is 5-15 times of the equivalent of the polypeptide loaded on the resin;
the dosage of the dichloromethane in the step (3) is calculated according to the concentration of the nitrosopyridine compound in the system being more than 0.4 mol/L;
the dosage of the glacial acetic acid in the step (3) is 0.5 to 1.5 percent v/v of that of the dichloromethane;
the reaction condition in the step (3) is that the temperature is 20-30 ℃, and the reaction time is that the resin is colorless and transparent.
5. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule with phenylazopyridine as ligand according to claim 1 or 2, characterized in that:
the reagent for cutting in the step (4) is trifluoroacetic acid and water, and the ratio of the trifluoroacetic acid to the water is 95:5 by volume ratio;
the cutting time in the step (4) is 1-2 h;
the extractant extracted in the step (4) is ethyl acetate;
the extraction times in the step (4) are two times.
6. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule using phenylazo pyridine as ligand according to claim 1, wherein the solid phase synthesis method comprises the following steps:
the adding amount of the manganese pentacarbonyl bromide in the step (5) is 1-5 times of the equivalent of the polypeptide;
the volume ratio of the dichloromethane to the methanol in the dichloromethane/methanol mixed solution in the step (5) is 1:1, and the dosage of the dichloromethane/methanol mixed solution is calculated according to the concentration of manganese pentacarbonyl bromide in a system which is more than 0.4 mol/L;
the coordination reaction in the step (5) is carried out at the temperature of 20-30 ℃ for 4-6 h by oscillation reaction.
7. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule using phenylazo pyridine as ligand according to claim 1, wherein:
the dosage of the nitrosopyridine compound in the step (3) is 10 times of the equivalent of the polypeptide loaded on the resin;
the dosage of the dichloromethane in the step (3) is calculated according to the concentration of the nitrosopyridine compound in the system being 0.4-0.6 mol/L;
the dosage of the glacial acetic acid in the step (3) is 1% v/v of that of the dichloromethane;
the adding amount of the manganese pentacarbonyl bromide in the step (5) is 3 times of the equivalent of the polypeptide;
the volume ratio of the dichloromethane to the methanol in the dichloromethane/methanol mixed solution in the step (5) is 1:1, and the dosage of the dichloromethane/methanol mixed solution is calculated according to the concentration of the manganese pentacarbonyl bromide in a system being 0.4-0.6 mol/L.
8. The solid phase synthesis method of polypeptide-manganese-carbonyl complex CO releasing molecule using phenylazo pyridine as ligand according to claim 1, wherein:
the specific operation of sequentially condensing Fmoc protected amino acids from the C end to the N end in the step (1) is as follows: under the action of a coupling system, firstly, reacting the 1 st amino acid with Fmoc amino resin to generate amino acid-amino resin, and then coupling other Fmoc protected amino acids one by one to obtain linear polypeptide resin;
the Fmoc deprotection reagent in the coupling system is 20% piperidine/DMF;
the deprotection reaction time in the coupling system is 5-10 min;
the condensing agent in the coupling system is HOBT + DIC or TBTU + DIEA;
the amino resin in the step (1) is Rink Amide MBHA resin;
the loading amount of the amino resin in the step (1) is 0.2-0.8 mmoL/g;
the chemical formula of Fmoc-4-aminophenylalanine with the amino protecting group of the aniline being allyloxycarbonyl in the step (1) is Fmoc-L-Aph (alloc) -OH, and the structural formula is shown as follows:
the dosage of Fmoc-L-Aph (alloc) -OH is as follows: Fmoc-L-aph (alloc) -OH ═ 1: 3, calculating the molar ratio;
the coupling time of the Fmoc-L-Aph (alloc) -OH and the Fmoc amino resin is 4-12 h;
fmoc-protected amino acids other than Fmoc-L-Aph (alloc) -OH were used as Fmoc amino resin: fmoc protected amino acid ═ 1: 4, calculating the molar ratio;
the coupling time of the Fmoc protected amino acid except Fmoc-L-Aph (alloc) -OH and the Fmoc amino resin is 2-4 h.
9. A polypeptide-manganese-carbonyl compound CO release molecule using phenylazo pyridine as a ligand is characterized in that: obtained by the solid phase synthesis method according to any one of claims 1 to 8.
10. Use of the phenylazo pyridine based polypeptide-manganese-carbonyl complex CO releasing molecule of claim 9 for the preparation of a medicament and/or a medical material.
Technical Field
The invention relates to the technical field of synthesis and preparation of polypeptide-metal compounds, in particular to a polypeptide-manganese-carbonyl compound CO release molecule using phenylazo pyridine as a ligand, and a synthesis method and application thereof.
Background
Carbon monoxide (CO) has long been recognized as a toxic gas because of its strong binding to hemoglobin in the body. At the same time, however, CO, an important gas molecule, plays a variety of physiological roles such as signal transduction and cell protection in mammals (r. motterlini, nat. rev. drug. discov.,2010,9, 728-. In order to further explore the important role of CO in a life system and the application of CO in clinical medicine, one of the key problems to be solved is to adopt which mode to realize safe and controllable fixed-point transportation of CO.
Carbon monoxide releasing molecules (CORM) are one of the technical means commonly used for the controlled release of CO. Through the development over the years, according to the way in which CORMs are stimulated to release CO, CORMs can be divided into the following main 3 classes: 1. ligand exchange triggered CO-releasing CORMs; 2. enzyme-induced CO-releasing CORMs (ET-CORMs); 3. light controllable light induced CO release type corms (photocorms). PhotoCORMs are of interest to researchers because of their advantage of more controllable CO release patterns, typically manganese-carbonyl (Mn (CO))3) Phocorms, which induce the release of CO after irradiation with a high-energy light source (ultraviolet or violet light) (j. niesel, chem. commun.,2008, 1798-; pc, kunz, eur, j, inorg, chem, 2009, 5358-one 5366; G.chem.,2011,50, 4362-. However, the relatively high energy light source has poor tissue penetration and causes certain damage to normal cells, which limits the practical biological application of such PhotoCORMs requiring a high energy light source to release the light source.
According to the metal-to-ligand electron transfer (MLCT) theory, under the stimulation of a light source with enough energy, electrons are excited to jump from a metal d orbital to a pi orbital of an auxiliary ligand, so that the oxidation state of the central metal ion form is improved, and the synergistic effect between the metal and a carbonyl compound is destroyed. This process weakens the metal-carbonyl bond and promotes the release of CO. By introducing rails capable of facilitating participation in electron transferThe auxiliary ligand containing a super-conjugated system and a pi acceptor group with narrowed energy gap is expected to realize CO release of PhotoCORM under the stimulation of a low-energy light source with long wavelength. Ligands of this type include bipyridine, bipyridylmethylamine, pyridinolinamine, and phenylazopyridine, among others. Compared with bipyridyl and other alpha-diamine ligand systems, the 2-phenylazopyridine ligand (azpy) can stabilize central metal in a lower oxidation state, effectively promote electron transfer between the metal and the ligand in a visible light region, weaken sigma-pi bonds between metal-carbonyl groups, and further realize CO release by using visible light (MA. Gonzalez, Ino rg. chem.,2012,51, 601-. Construction of Mn (I) -CO Complex MnBr (azpy) (CO) containing Benzopyridinyland ligands by the investigator3And [ Mn (azpy) ((CO))3(PPh3)](ClO4) Can realize CO release (SJ. Carrington, chem. Commun.,2013,49,11254) under the stimulation of visible light (lambda is less than or equal to 520 nm).
The CORMs monomer generally has the defects of poor solubility in aqueous solution, poor air stability and poor biocompatibility, so that the defects are improved by introducing suitable carriers such as polypeptide, high molecular polymer, nano material and the like to construct corresponding compounds with the CORMs. Among them, the complex of polypeptides and CORMs is of great interest because of its better biocompatibility. Covalent coupling methods are commonly used to prepare the CORMs complexes of the polypeptides. The coupling of the metal-carbonyl structure and the polypeptide or the protein is realized by a chemical step-by-step synthesis method, namely, firstly connecting the structure with the metal-carbonyl with a functional group with reactivity through a linker, and then reacting the functional group with a reaction site originally or artificially introduced by the polypeptide or the protein through the reactive functional group. Common coupling reactions for metal-carbonyl structures with polypeptides or proteins include Sonogashira coupling, CuAAC-catalyzed click chemistry ligation, and oxime chemistry ligation (m.salaman, eur.j.inorg.chem.,2020, 21-35). Such coupling reactions typically require multi-step syntheses in a liquid phase environment, thus requiring multi-step purifications resulting in lower final yields.
The phenylazo pyridine ligand capable of realizing CO release by visible light is combined with the CORMs compound of the polypeptide, so that a novel polypeptide-PhotoCORMs which can release CO in response to visible light and has good biocompatibility can be expected to be constructed. However, no report on a related synthesis method of a CO release molecule of a polypeptide-manganese-carbonyl complex with phenylazo pyridine as a ligand exists at present. Therefore, it is necessary to develop a novel synthetic method with convenient synthesis and purification and modular synthesis for efficiently preparing the polypeptide-CORMs compound with the phenylazo pyridine as the ligand.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention mainly aims to provide a solid-phase synthesis method of polypeptide-manganese-carbonyl compound CO release molecules by using phenylazopyridine as a ligand.
The invention also aims to provide the polypeptide-manganese-carbonyl compound CO release molecule which is obtained by the solid-phase synthesis method and takes the phenylazopyridine as the ligand.
The invention also aims to provide application of the polypeptide-manganese-carbonyl compound CO release molecule with phenylazo pyridine as a ligand.
The purpose of the invention is realized by the following technical scheme:
a solid phase synthesis method of polypeptide-manganese-carbonyl compound CO release molecules with phenylazo pyridine as a ligand comprises the following steps:
(1) designing a target polypeptide sequence containing amino acid as a coordination group introduction site, adopting an Fmoc solid-phase polypeptide synthesis method, taking Fmoc amino resin as a carrier, sequentially condensing Fmoc protected amino acid from a C end to an N end according to the target polypeptide sequence, washing, and drying to obtain linear polypeptide resin; wherein:
amino acid at the site of introduction of the coordinating group was synthesized using Fmoc-4-aminophenylalanine (Fmoc-L-Aph (Alloc) -OH) in which the amino-protecting group of aniline was Alloc (allyloxycarbonyl);
the rest amino acids are Fmoc amino acids without side chains, or the Fmoc amino acids of which the protecting groups of the side chains can be removed by trifluoroacetic acid are synthesized;
(2) adding the linear polypeptide resin prepared in the step (1) into a palladium catalytic system, reacting, washing and drying to obtain linear polypeptide resin with the Alloc protecting group removed;
(3) adding a nitrosopyridine compound, Dichloromethane (DCM) and glacial acetic acid (AcOH) into the linear polypeptide resin prepared in the step (2), reacting, washing and drying to obtain the linear polypeptide resin containing the phenylazo pyridine coordination group;
(4) taking the linear polypeptide resin containing the phenylazo pyridine coordination group prepared in the step (3), adding a cutting reagent to remove the polypeptide chain from the Fmoc amino resin, and removing the residual side chain protecting group; filtering, spin-drying, extracting, centrifuging and freeze-drying to obtain a linear polypeptide crude product containing phenylazo pyridine coordination groups; further separating and purifying to obtain linear polypeptide containing phenylazo pyridine coordination groups;
(5) taking the linear polypeptide containing the phenylazo pyridine coordination group prepared in the step (4), adding manganese pentacarbonyl bromide, and performing coordination reaction by using a dichloromethane/methanol mixed solution as a reaction solvent; and (3) spin-drying the solvent, and extracting with diethyl ether to obtain the manganese-carbonyl-linear polypeptide compound containing the phenylazo pyridine coordination group.
The amino acid to be introduced as a coordinating group in the step (1) is preferably phenylalanine or tyrosine. The side chain of the modification site is changed from a natural aliphatic chain structure to a structure with an aromatic ring conjugated system, and the structure change is large, so that certain influence on the property of the polypeptide can be caused. The phenylalanine side chain has a benzene ring, the tyrosine side chain group has phenol, and the original structure of the polypeptide can be kept to the maximum extent without excessive change by taking the phenylalanine side chain group as a site for constructing the ligand group.
The percentage of the amino acid as the site for introducing a coordinating group in step (1) to the total number of amino acids is 20% or less, preferably 15% or less. In view of the effect of steric hindrance of the modifying group on the potential synthetic difficulties associated with solid phase modification, there should be at least 3 amino acid separations, preferably at least 5 amino acid separations, between two adjacent amino acids designated as side chain modification sites.
The specific operation of sequentially condensing Fmoc protected amino acids from the C end to the N end in the step (1) is as follows: under the action of a coupling system, the 1 st amino acid reacts with Fmoc amino resin to generate amino acid-amino resin, and then other Fmoc protected amino acids are coupled one by one to obtain linear polypeptide resin.
The Fmoc deprotection reagent in the coupling system is preferably 20% piperidine/DMF.
The deprotection reaction time in the coupling system is preferably 5-10 min.
The condensing agent in the coupling system is preferably HOBT + DIC or TBTU + DIEA.
The amino resin in the step (1) is preferably Rink Amide MBHA resin.
The loading amount of the amino resin in the step (1) is preferably 0.2-0.8 mmoL/g.
The chemical formula of Fmoc-4-aminophenylalanine with the aniline amino protecting group of Alloc (allyloxycarbonyl) in the step (1) is Fmoc-L-Aph (Alloc) -OH, and the structural formula is shown as follows:
the Fmoc-L-Aph (alloc) -OH is preferably used in an amount of Fmoc amino resin: Fmoc-L-aph (alloc) -OH ═ 1: 3, calculated as a molar ratio.
The coupling time of the Fmoc-L-Aph (alloc) -OH and the Fmoc amino resin is preferably 4-12 h; more preferably 12 h.
The Fmoc-protected amino acids other than Fmoc-L-Aph (alloc) -OH are preferably used in an amount such that the Fmoc amino resin: fmoc protected amino acid ═ 1: 4 in terms of molar ratio.
The coupling time of the Fmoc-protected amino acid except Fmoc-L-Aph (alloc) -OH and the Fmoc amino resin is preferably 2-4 h; more preferably 2 h.
The palladium catalysis system in the step (2) is preferably a tetrakis (triphenylphosphine) palladium catalysis system, the dosage of the tetrakis (triphenylphosphine) palladium is preferably 3-5 times equivalent of polypeptide supported on resin, and the reaction solvent is a chloroform-acetic acid-N-methylmorpholine mixed solution of chloroform, acetic acid and N-methylmorpholine according to the volume ratio of 37:2: 1.
The reaction conditions in the step (2) are 20-30 ℃ (room temperature) and 2-3 h.
The nitrosopyridine compound in the step (3) comprises nitrosopyridine and nitrosopyridine derivatives. The nitrosopyridine derivatives include, but are not limited to, nitrosoquinoline.
The chemical formula of the nitrosopyridine is C5H4N2O, the structural formula is as follows:
the dosage of the nitrosopyridine compound in the step (3) is preferably 5-15 times of the equivalent of the polypeptide supported on the resin, and more preferably 10 times of the equivalent.
The amount of the dichloromethane in the step (3) is preferably more than 0.4mol/L of the concentration of the nitrosopyridine compound in the system; more preferably 0.4 to 0.6 mol/L.
The dosage of the glacial acetic acid in the step (3) is preferably 0.5-1.5% (v/v) of that of the dichloromethane; more preferably 1% (v/v).
And (3) reacting at 20-30 ℃ (room temperature) for a period of time until the resin is colorless and transparent.
The cleavage reagent described in step (4) is preferably trifluoroacetic acid (TFA) and water at a ratio of 95:5 volume ratio of the resulting solution.
The cutting time in the step (4) is preferably 1-2 h.
The extractant for the extraction in the step (4) is preferably ethyl glacial ether.
The number of extractions described in step (4) is preferably two.
The adding amount of the manganese pentacarbonyl bromide in the step (5) is preferably 1-5 times of the equivalent of the polypeptide, and more preferably 3 times of the equivalent.
The volume ratio of the dichloromethane to the methanol in the dichloromethane/methanol mixed solution in the step (5) is preferably 1:1, and the usage amount is preferably more than 0.4mol/L, and more preferably 0.4-0.6 mol/L of the manganese pentacarbonyl bromide in the system.
The coordination reaction in the step (5) is preferably carried out at the temperature of 20-30 ℃ (room temperature) for 4-6 h by oscillation reaction.
A polypeptide-manganese-carbonyl compound CO release molecule using phenylazo pyridine as ligand is prepared through solid-phase synthesis.
The polypeptide-manganese-carbonyl compound CO release molecule taking phenylazo pyridine as a ligand is applied to preparation of medicines and/or medical materials.
The principle of the invention is as follows: synthesis of unnatural amino acid Fmoc-4-aminophenylalanine (Fmoc-L-Aph (Alloc) -OH) with side chain anilino-amino group temporarily protected with Alloc protecting group. Adopting Fmoc solid-phase polypeptide synthesis method, using Fmoc amino resin as carrier, according to the sequence of target polypeptide, using Fmoc-L-aph (Alloc) -OH at the site needing introducing side chain modification, and sequentially condensing Fmoc protected amino acids from C end to N end to obtain polypeptide amino resin with side chain having Alloc protection; wherein, the removing mode of the Alloc protecting group is different from the side chain protecting groups of other amino acids; secondly, removing the Alloc group on the resin by adopting the removing condition of the Alloc protecting group, and exposing free amino of aniline for modification reaction; then synthesizing nitrosopyridine compounds, and modifying aniline free amino groups on polypeptide chains on resin into phenylazo pyridine ligand groups capable of carrying out chemical coordination by adopting a Mills reaction on the resin; cracking and purifying target polypeptide from resin, and then adding manganese pentacarbonyl bromide for chemical coordination; extracting with diethyl ether to obtain the polypeptide with manganese-carbonyl coordination.
Compared with the prior art, the invention has the following advantages and effects:
the method provides a brand-new synthesis means for introducing the phenylazo pyridine coordination functional group to the polypeptide resin and carrying out manganese-carbonyl coordination, and provides a corresponding example. The synthesis route only needs one-step purification by reversed phase liquid chromatography, greatly simplifies the number of the synthesis steps of the existing polypeptide/protein metal compound synthesis, and shortens the time required by synthesis.
In the method of the invention, the polypeptide is introduced in solid phase synthesisAdding unnatural amino acid Fmoc-4-aminophenylalanine (Fmoc-L-Aph (Alloc)) -OH with side chain aniline amino protecting group of Alloc through Pd (PPh)3)4After removal of (a), the free amino groups are exposed. The free amino group has the ability to undergo a variety of different types of reactions, and thus, a variety of functional group modifications can be made by different reaction types.
The method of the invention innovates a manganese-carbonyl coordination mode which takes manganese pentacarbonyl bromide as a reaction substrate. The manganese pentacarbonyl bromide is dissolved in the organic solvent, so that the problem of poor solubility of the manganese pentacarbonyl bromide when liquid phase coordination is carried out in a buffer solution is solved. After the coordination is finished, the residual non-coordinated manganese pentacarbonyl bromide can be easily and conveniently removed by spin-drying the solvent and adding sufficient ether for extraction, so that the liquid phase purification step after the coordination is saved, and the synthesis time and cost are greatly shortened.
The solid phase construction of the polypeptide-manganese-carbonyl complex with phenylazo pyridine ligand according to the invention is to prepare the polypeptide-manganese-carbonyl (Mn (CO))3) One innovation of the compound. The invention obtains polypeptide amino resin with reaction sites by a solid-phase polypeptide synthesis method based on Fmoc protective groups and the use of 4-aminophenylalanine with orthogonal protective groups, and constructs phenylazo pyridine coordination groups at polypeptide target sites by utilizing solid-phase Mills reaction on the resin. The constructed product with the phenylazo pyridine coordination group can be kept stable in the reaction process of resin cutting, and no shedding condition is found. Then utilizing manganese pentacarbonyl bromide to carry out coordination reaction of manganese-carbonyl-ligand, extracting with diethyl ether, and successfully preparing polypeptide-manganese-carbonyl (Mn (CO)) using phenylazo pyridine as coordination group3) And (c) a complex.
The polypeptide-manganese-carbonyl (Mn (CO)) which is prepared by the method and takes phenylazo pyridine as a coordination group3) The targeting property of the polypeptide part and the light controllability of the carbon monoxide release of the manganese carbonyl group are combined to construct the carbon monoxide release molecule which has high biocompatibility and stable physiological environment, realizes the targeted transportation and the controllable release of the carbon monoxide and takes the polypeptide as a bracket. Phenylazo pyridine ligand is helpful to add oxygenThe release wavelength of the carbon monoxide is prolonged to the red light range or even the near infrared range, which is beneficial to researching the physiological and biochemical effects of the carbon monoxide on specific parts and lays a good foundation for the carbon monoxide in the aspect of treating related diseases.
Drawings
FIG. 1 is a schematic HPLC diagram of each stage of the solid phase synthesis of TAT (Y11Aph (azpy-Mn-CO)) containing phenylazopyridine and manganese-carbonyl coordination prepared in example 1.
FIG. 2 shows the structure and theoretical molecular weight of a TAT (Y11Aph (azpy-Mn-CO)) complexed with a phenylazopyridine and a manganese-carbonyl group prepared in example 1.
FIG. 3 is a graph of ESI-MS characterization of TAT (Y11Aph (azpy-Mn-CO)) complexed with phenylazopyridine and manganese-carbonyl prepared in example 1.
FIG. 4 is a Fourier transform infrared (FT-IR) spectrum of TAT (Y11Aph (azpy-Mn-CO)) complexed to a phenylazopyridine and manganese-carbonyl complex obtained in example 1.
FIG. 5 is a graph of the results of the experiment for the optically controlled CO release of red (. about.700 nm, LED,5W) light from TAT (Y11Aph (azpy-Mn-CO) prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Fmoc-L-Aph (alloc) -OH used in the following examples, which has the following structural formula, was prepared according to the method described in references "L.Leelavastanakij, J.peptide Res.,2000,56, 80-87":
the remaining Fmoc-protected amino acids are Fmoc-L-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser-tBu) -OH, (Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Val-OH.
Nitrosopyridines used in the following examples have the following structural formula:
the following examples were HPLC using an Agilent 1260, a Phenomenex C18 column as column and water and acetonitrile as mobile phase (0.1% (v/v) TFA).
The sequences of the polypeptides with ligand functional groups synthesized in the following examples are as follows, and the C-terminal of the synthesized polypeptides is amidated:
TAT(Y11Aph(azpy)):Aph(azpy)GRKKRRQRRR；
names and abbreviations of reagents used in the following examples:
DMF: n, N-dimethylformamide;
DCM: dichloromethane;
HOBT: 1-hydroxybenzotriazole;
DIC: n, N-diisopropylcarbodiimide;
TBTU: benzotriazole tetramethyltetrafluoroboric acid;
DIEA: n, N-diisopropylethylamine;
NMM: n-methylmorpholine;
TFA: trifluoroacetic acid;
MeCN: acetonitrile
MeOH: methanol.
Example 1: the preparation method of the TAT-manganese carbonyl compound with the side chain coordination group of phenylazo pyridine (azpy) comprises the following steps:
(1) preparation of Fmoc-Arg (Pbf) -MBHA resin: taking 500mg Rink Amide MBHA resin into a polypeptide synthesis tube, adding 15mL of DMF, oscillating and swelling twice at room temperature for 15min each time, draining, adding 10mL of 20% piperidine/DMF into the resin, oscillating and reacting for 5min at room temperature, washing twice with DMF, adding 10mL of 20% piperidine/DMF, oscillating and reacting for 5min at room temperature, washing twice with DMF, DCM and DMF in sequence, draining the solvent to obtain the resin with the Fmoc protection removed, weighing Fmoc-Arg (Pbf) -OH (1mmol), TBTU (2mmol) and DIEA (0.98mmol), dissolving Fmoc-Arg (Pbf) -OH and TBTU with a small amount of DMF, adding DIEA, oscillating and reacting for 2min at room temperature, adding the activated amino acid into the resin, oscillating and reacting for 2h at room temperature, washing three times respectively with DMF and DCM, drying the resin by nitrogen to obtain dry resin, detecting the resin load of the resin by ultraviolet to finally obtain Fmoc-Arg (Pbf) -MBHA resin with the load of about 0.44 mmol/g.
(2) Preparation of TAT (Y11Aph (Alloc)) -MBHA resin: adding the obtained Fmoc-Arg (Pbf) -MBHA resin into 15mL of DMF, oscillating and swelling twice at room temperature for 15min each time, draining, adding 10mL of 20% acetic anhydride/DMF into the resin, oscillating and reacting for 20min at room temperature to seal off the amino group of the resin without amino acid coupling, preventing the next reaction, washing twice with DMF, DCM and DMF in turn, adding 10mL of 20% piperidine/DMF into the resin, oscillating and reacting for 5min at room temperature, washing twice with DMF, adding 10mL of 20% piperidine/DMF again, oscillating and reacting for 5min at room temperature, washing twice with DMF, DCM and DMF in turn, draining the solvent to obtain the resin without Fmoc protection, weighing Fmoc-Arg (Pbf) -OH (2mmol), TBTU (4mmol) and DIEA (1.96mmol), dissolving Fmoc amino acid and HOBT with a small amount of DMF, adding DIC, oscillating and reacting for activating carboxyl group for 2min at room temperature, the activated amino acid was then added to the resin, shaken for 2h at room temperature, monitored with Kaiser's reagent, washed twice with DMF, DCM, DMF, and the above experimental procedure was repeated (amino acid content was calculated as 4-fold molar equivalent of resin, shaken for 2h), followed by sequential coupling of Fmoc-L-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-L-Aph (alloc) -OH. After the synthesis, the resin was washed with DMF and DCM for three times, and dried with nitrogen to obtain 1.2g of dried resin. The loading of the resin at this point was about 0.18mmol/g due to the increased mass of the resin.
(3) Removal of Alloc protecting group: taking 1g of the resin obtained in the step (2), and adding 3 times of equivalent Pd (PPh) of the polypeptide supported on the resin3)4Then, 15mL of a mixed solution of chloroform, acetic acid and NMM at a volume ratio of 37:2:1 was added, and the mixture was reacted at room temperature for 2 to 3 hours.
(4) Preparation of nitrosopyridine: in a method described in "EC.Taylor, J.org.chem.,1982,47, 552-555", 0.94g (0.01mol) of 2-aminopyridine and 0.68g (0.8mL,0.011mL) of dimethylsulfide were dissolved in 10mL of DCM and stirred at-20 ℃. While stirring at-20 ℃, 1.33g (0.01mol) of N-chlorosuccimide (N-chlorosuccinimide, CAS #:128-09-6) was dissolved in 25mL of DCM, and the solution was added dropwise to the reaction flask, after the addition, the reaction solution was stirred at-20 ℃ for 1 hour, and then the temperature was returned to room temperature and stirred for 1 hour. 0.405g of sodium methoxide was dissolved in 7.5mL of methanol, and the resulting solution was added to the reaction system and stirred for 10min, followed by addition of 15mL of water and stirring at room temperature for 4 hours. The organic layer was separated, the aqueous layer was extracted twice with 5mL DCM, all organic layers were pooled and the liquid was washed with water, dried over anhydrous sodium sulfate and the solvent was spin dried. 10mL of DCM were added, stirring at 0 ℃ and 0.201g (0.0119mol) of m-chloroperbenzoic acid (m-chloroperoxybenzoic acid, CAS #:937-14-4) (80% -90%) dissolved in ultra-dry DCM were added. After stirring and reacting for 90min at 0-5 ℃ (bright yellow solution), 0.3mL dimethyl sulfide is added, and stirring is carried out for 30min without changing the reaction temperature. And adding 50mL of saturated sodium bicarbonate solution into the reaction bottle, separating, keeping an organic phase, fully washing the separated liquid with water, drying the separated liquid with anhydrous sodium sulfate, and spin-drying the solvent. And recrystallizing with ethanol to obtain bright yellow solid which is nitrosopyridine.
(5) Preparation of TAT (Y11Aph (azpy)) -MBHA resin: 500mg of the resin obtained in step (3) was added with nitrosopyridine (0.9mmol), 2.25mL of DCM and 22.5. mu.L of AcOH were added, the reaction was shaken at room temperature and monitored with Kaiser's reagent until the resin was bright orange and no longer discolored. After the reaction is finished, DMF and DCM are adopted to wash the mixture for three times respectively, and the mixture is dried by nitrogen to obtain dry resin.
(6) Preparation of TAT (Y11Aph (azpy)) polypeptide: 100mg of the resin obtained in step (5) was taken, and 40mL of a cleavage reagent (TFA: H) was added2O is 95:5 in volume ratio), shaking for 1-2h, filtering to obtain yellow brown transparent liquid, and rotating the liquid in a rotary evaporatorAfter drying, extraction was performed twice by adding about 15mL of glacial ethyl ether, the precipitate was collected after centrifugation, and the sample was lyophilized to obtain about 30mg of TAT (Y11Aph (azpy)) polypeptide, after which the crude polypeptide was isolated and purified by HPLC if necessary.
(7)TAT(Y11Aph(azpy)-Mn(CO)3) The preparation of (1): the TAT (Y11Aph (azpy)) polypeptide obtained in step (6) was taken, manganese pentacarbonyl bromide (0.36mmol) was added, DCM/MeOH ═ 1/1(v/v) was added in 0.7mL, and the reaction was stirred at room temperature for 4 hours. The unreacted manganese pentacarbonyl bromide was removed by extraction with frozen diethyl ether. The obtained dark blue solid is the target product TAT (Y11Aph (azpy) -Mn (CO)3) A polypeptide.
At TAT (Y11Aph (azpy) -Mn (CO)3) The solid phase synthesis process of (1) is carried out by carrying out a small amount of cleavage test on the resin obtained in each stage and detecting the products in different stages by using HPLC, and the results are shown in FIG. 1. ESI-MS identification of TAT (Y11Aph (azpy) -Mn (CO) obtained3) The results are shown in FIG. 3. The Fourier transform Infrared Spectroscopy (FT-IR) characterization results are shown in FIG. 4.
Example 2: TAT (Y11Aph (azpy) -Mn (CO)3) Light-controlled CO release experiment
Experiment raw materials: TAT (Y11Aph (azpy) -Mn (CO)3) PBS buffer, visible red torch (-700 nm, LED, 5W).
The experimental steps are as follows:
the PBS buffer was deoxygenated by bubbling nitrogen for 30 min.
② TAT (Y11Aph (azpy) -Mn (CO) is added3) Polypeptide-manganese complex (final concentration 0.16 mg/mL).
Fourthly, illuminating the cuvette by using a visible light red flashlight, and recording spectrograms of different accumulated illumination time by using an ultraviolet visible spectrum. The results are shown in FIG. 5.
From the change of characteristic peaks of the ultraviolet spectrum, the following conclusion was drawn that TAT-Mn (CO) was not irradiated with light3CO is not released; ② TAT (Y11Aph (azpy) -Mn (CO) with 546.5nm in ultraviolet visible spectrum after being illuminated by visible light red light3) The metal-to-ligand electron transfer (MLCT) in (a) decreased, demonstrating the rapid release of CO therein.
Comparative example 1: construction of phenylazo pyridine coordination group by using tyrosine as amino acid substrate
When tyrosine is chosen as the amino acid substrate for the construction of the phenylazo pyridine coordinating group, a diazo coupling reaction can be used. Under proper conditions, the diazonium salt can generate electrophilic substitution reaction with some aromatic compounds, such as phenol, amine, etc., with strong electron donating group on the aromatic ring to generate azo (-N ═ N-) compounds. When the coupling component is a phenolic compound, diazo coupling is often carried out in a weakly basic aqueous environment. In order to verify the feasibility of constructing phenylazo pyridine coordination on tyrosine by diazo coupling reaction:
(1) route exploration for diazo coupling of aniline and p-cresol with a simple substrate
Firstly, aniline reacts with sodium nitrite in an aqueous solution of concentrated hydrochloric acid at 0-5 ℃ to generate aniline diazonium salt; then, the aniline diazonium salt solution is directly dropped into an aqueous solution of p-cresol at 0 ℃, and a saturated sodium carbonate solution is used to control the pH to be 8-9 weak alkaline.
The reaction system is kept at 0 ℃ for 3 hours, and the reaction system turns deep red. Extraction with diethyl ether gave a dark brown oil after spin drying. The product was subjected to APCI-MS and1H-NMR characterization and final comprehensive analysis to determine that the target product is 4-methyl-2-phenylazo phenol.
(2) Exploration of diazo coupling on Fmoc-protected amino acids was performed using Fmoc-protected tyrosine (Fmoc-L-Tyr-OH) as substrate for the N-terminal amino group used in the polypeptide synthesis
As diazo coupling is mainly carried out in aqueous solution such as buffer solution, and the solubility of Fmoc-L-Tyr-OH in aqueous solution is low, the reaction sites can not be exposed in the aqueous solution. Under the condition, the coupling efficiency of aniline diazonium salt and Fmoc-L-Tyr-OH is extremely low, and a target product is not generated.
It is presumed that the reaction system of the resin reaction in the conventional solid phase polypeptide synthesis is an organic solvent system, and the aqueous phase reaction system required for diazo coupling limits the application of the reaction to the solid phase one-pot synthesis of a polypeptide-metal carbonyl complex having phenylazopyridine as a coordinating group.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.