阅读说明：本技术 一种基于多肽-锰-羰基复合物的co释放分子的固相合成方法及其应用 (Solid-phase synthesis method and application of polypeptide-manganese-carbonyl compound-based CO release molecule ) 是由 何春茂 周仪 陈永渌 于 2021-02-05 设计创作，主要内容包括：本发明提供了一种基于多肽-锰-羰基复合物的CO释放分子的固相合成方法及其应用。本方法采用Fmoc固相多肽合成法,在需引入侧链修饰的位点使用侧链Alloc保护的赖氨酸,从C端到N端依次缩合Fmoc保护氨基酸,得到侧链带有Alloc保护的多肽氨基树脂；进行树脂上的Alloc基团脱除,暴露可用于修饰反应的自由氨基；在树脂上采用还原胺化反应,将树脂上多肽链上的自由氨基修饰为可进行化学配位的配体基团；加入五羰基溴化锰进行树脂上的化学配位；最后将目标多肽从树脂裂解下来并纯化,即可得到带有锰-羰基配位的多肽。本发明有助于研究一氧化碳对特定部位的生理生化作用,同时为一氧化碳在相关疾病的治疗方面奠定良好基础。(The invention provides a solid-phase synthesis method of a CO release molecule based on a polypeptide-manganese-carbonyl compound and application thereof. The method adopts an Fmoc solid-phase polypeptide synthesis method, lysine with side chain Alloc protection is used at a site needing side chain modification, Fmoc protected amino acids are condensed in sequence from a C end to an N end to obtain polypeptide amino resin with the side chain with Alloc protection; removing Alloc groups on the resin to expose free amino groups which can be used for modification reaction; modifying free amino groups on polypeptide chains on the resin into ligand groups capable of chemical coordination by adopting reductive amination reaction on the resin; adding manganese pentacarbonyl bromide to perform chemical coordination on the resin; finally, the target polypeptide is cracked from the resin and purified, and the polypeptide with manganese-carbonyl coordination can be obtained. The invention is helpful to research the physiological and biochemical effects of carbon monoxide on specific parts, and lays a good foundation for the treatment of carbon monoxide in related diseases.)

1. A solid phase synthesis method of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex, characterized in that: the method comprises the following steps:

(1) adopting an Fmoc solid-phase polypeptide synthesis method, taking Fmoc amino resin as a carrier, sequentially condensing Fmoc protected amino acids from a C end to an N end according to a target polypeptide sequence, washing, and drying to obtain linear polypeptide resin; wherein:

the target polypeptide sequence contains lysine with any number and any position as a side chain modification site, the lysine is used as the side chain modification site, and Fmoc lysine with side chain amino protecting group of Alloc is used for synthesis;

when the target polypeptide sequence also contains lysine which is not used as a side chain modification site, the lysine which is not used as the side chain modification site is synthesized by Fmoc lysine of which the side chain amino protecting group is Boc;

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) taking the linear polypeptide resin prepared in the step (2), adding an azacyclic compound with aldehyde group, sodium triacetoxyborohydride and dichloroethane, reacting, washing and drying to obtain the linear polypeptide resin with lysine side chains containing coordination groups;

(4) taking the linear polypeptide resin prepared in the step (3), adding manganese pentacarbonyl bromide and dichloromethane, performing coordination reaction, washing and drying to obtain a manganese-carbonyl-linear polypeptide resin compound;

(5) taking the manganese-carbonyl-linear polypeptide resin compound prepared in the step (4), 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 crude product of the polypeptide-manganese-carbonyl compound; further separating and purifying to obtain the polypeptide-manganese-carbonyl compound.

2. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, 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 prepared by mixing chloroform, acetic acid and N-methylmorpholine according to a volume ratio of 37:2: 1.

3. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

the nitrogen heterocyclic compound with aldehyde group in the step (3) is at least one of 2-formaldehyde pyridine and 2-formaldehyde quinoline;

the dosage of the nitrogen heterocyclic compound with aldehyde group and the sodium triacetoxyborohydride in the step (3) is 5-15 times of the equivalent of the polypeptide loaded on the resin;

the dosage of the dichloroethane in the step (3) is calculated according to the concentration of the nitrogen heterocyclic compound with aldehyde group and sodium triacetoxyborohydride in the system being more than 0.4 mol/L.

4. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

the adding amount of the manganese pentacarbonyl bromide in the step (4) is 1-5 times of the equivalent of the polypeptide loaded on the resin;

the dosage of the dichloromethane in the step (4) is as follows: 5-10 mL of Fmoc amino resin: and 1g is calculated.

5. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

the reaction conditions in the step (2) are that the temperature is 20-30 ℃ and the time is 2-4 h;

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;

the coordination reaction in the step (4) is carried out at the temperature of 20-30 ℃ for 4-6 h by oscillation reaction.

6. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

the reagent for cleavage in step (5) is trifluoroacetic acid, water and 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid according to a ratio of 95:2.5:2.5 volume ratio of the obtained solution;

and (5) cutting for 2-4 h.

7. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

the extractant extracted in the step (5) is ethyl acetate;

the extraction times in the step (5) are two times;

the separation and purification in the step (5) are realized by reverse liquid chromatography;

the mobile phase of the reverse phase liquid chromatography is acetonitrile/water mixed solution containing 0.1 percent of trifluoroacetic acid.

8. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

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.

9. The method for the solid phase synthesis of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to claim 1, characterized in that:

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 Fmoc lysine with the side chain amino protecting group of Alloc in the step (1) has the chemical formula of Fmoc-L-Lys (Alloc) -OH, and the structural formula is shown as follows:

the Fmoc-L-Lys (alloc) -OH is used according to the weight ratio of Fmoc amino resin: Fmoc-L-lys (alloc) -OH ═ 1: 3, calculating the molar ratio;

the coupling time of the Fmoc-L-Lys (alloc) -OH and the Fmoc amino resin is 4-12 h;

fmoc-protected amino acids other than Fmoc-L-Lys (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-Lys (alloc) -OH and the Fmoc amino resin is 2-4 h.

10. Use of the solid phase synthesis method of a CO releasing molecule based on a polypeptide-manganese-carbonyl complex according to any one of claims 1 to 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 solid-phase synthesis method of a CO release molecule based on a polypeptide-manganese-carbonyl compound 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.

The use of carbon monoxide releasing molecules (CORMs) is one of the possible approaches to the controlled delivery of CO. Most CORMs are metal-carbonyl complexes, CO coordinates with transition metals in low oxidation states, and can release CO in the molecular structure after a certain stimulus (r. alberto, dalton. trans.,2007, 1651-. After years of development, CORMs can be classified into the following main 3 classes according to the way they are triggered to release CO: 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) Basic PhotoCORMs. However, the CORMs monomers generally have the disadvantages of poor solubility in aqueous solution, poor air stability and poor biocompatibility, which limit the practical application of the CORMs monomers (r.d. rimmer, coord.chem.rev.2012, 256, 1509-.

The above problems of the CORMs can be solved by constructing the resulting complex with a suitable carrier. The carriers used for constructing the CORMs compounds generally include polypeptides, high molecular polymers, nanomaterials, and the like. Among them, the complex of polypeptides and CORMs is of great interest because of its better biocompatibility. In addition, due to the specific targeting of certain specific types of polypeptides, controllable and fixed-point CO local release is expected to be realized.

The preparation of the CORMs complexes of the polypeptides described above generally employs covalent coupling methods. 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.

Dimethylene nitrogen heterocycles, such as dimethylene pyridine (dpa) and dimethylene quinoline (dqa), have been used in recent work as coordinating groups for the construction of PhotoCORMs (M.A. Gonzalez, Inorg. chem.,2012,51, 601-608). By changing the type of nitrogen atom heterocyclic compound or adding substituent groups on the heterocyclic ring, the wavelength of a light source for light-induced release can be adjusted. Nitrogen heterocycles allow a wide variety of chemical complexation, and so have been constructed by researchers by click-coupling followed by ligand-to-metal coordination or by ligand-to-metal coordination followed by click-coupling (m.salaman, n.fischer-Durand b.rudolf, eur.j.inorg.chem.,2020, 21-35). In addition, synthetic Single Amino Acid Chelates (SAACs) with coordinating groups based on Fmoc-protected Lysine (Fmoc-L-Lysine) have also been used to construct polypeptide-metal complexes. For example, a dimethylene pyridine ligand (dpa) group or a dimethylene quinoline ligand (dqa) is introduced into the side chain of Fmoc-L-Lysine by chemical synthesis, SAAC-dpa or SAACQ-dqa with a coordinating group can be synthesized, respectively, and a polypeptide-metal complex (M) with a dpa or dqa group can be obtained by standard polypeptide solid phase synthesis and coordination procedures.Commun, 2009, 493-. The above-mentioned polypeptide-metal complex is generally constructed by: firstly, obtaining target SCCA through organic synthesis and column chromatography purification; secondly, introducing SAAC into the target position of the polypeptide sequence through solid-phase polypeptide synthesis; finally, by resin crackingAnd (4) obtaining a crude product, and further purifying to obtain the target polypeptide-metal compound.

The method combines the abundant chemical coordination capability of nitrogen atom heterocyclic compounds with the synthesis scheme of the existing polypeptide-metal compound, and can be used for constructing the PhotoCORMs based on the polypeptide-metal carbonyl compound. However, the SAAC preparation process in the existing polypeptide-metal complex synthesis scheme is complicated and time-consuming (requires organic reaction in liquid phase and purification by column chromatography). Furthermore, if it is desired to adjust the wavelength of the light source by changing the type of the heterocyclic compound of the nitrogen atom or adding a substituent group to the heterocyclic ring, new SAAC needs to be synthesized again and then the step-by-step polypeptide synthesis is performed, so that the whole synthetic process is more tedious and time-consuming. Therefore, there is a need to develop a novel synthesis method which is convenient for synthesis and purification and can be synthesized in a modularized manner, so as to efficiently prepare the PhotoCORMs based on the polypeptide-metal carbonyl compound.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a solid-phase synthesis method of a CO release molecule based on a polypeptide-manganese-carbonyl compound.

Another object of the present invention is to provide the use of the above-mentioned solid phase synthesis method of CO releasing molecules based on polypeptide-manganese-carbonyl complexes.

The purpose of the invention is realized by the following technical scheme:

a method for solid phase synthesis of a polypeptide-manganese-carbonyl complex-based CO releasing molecule comprising the steps of:

(1) adopting an Fmoc solid-phase polypeptide synthesis method, taking Fmoc amino resin as a carrier, sequentially condensing Fmoc protected amino acids from a C end to an N end according to a target polypeptide sequence, washing, and drying to obtain linear polypeptide resin; wherein:

the target polypeptide sequence contains lysine with any number and any position as a side chain modification site, and the lysine as the side chain modification site is synthesized by using Fmoc lysine (Fmoc-L-Lys (Alloc) -OH) with side chain amino protecting group of Alloc (allyloxycarbonyl);

when the target polypeptide sequence further contains lysine which is not used as a side chain modification site, the lysine which is not used as a side chain modification site is synthesized using Fmoc lysine (Fmoc-L-Lys (Boc) -OH) whose side chain amino protecting group is Boc (t-butyloxycarbonyl);

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) taking the linear polypeptide resin prepared in the step (2), adding an azacyclic compound with aldehyde group and sodium triacetoxyborohydride (Na (CH)₃CO₂)₃BH) and Dichloroethane (DCE), reacting, washing and drying to obtain the linear polypeptide resin with lysine side chains containing coordination groups;

(4) taking the linear polypeptide resin prepared in the step (3), adding manganese pentacarbonyl bromide and dichloromethane, performing coordination reaction, washing and drying to obtain a manganese-carbonyl-linear polypeptide resin compound;

(5) taking the manganese-carbonyl-linear polypeptide resin compound prepared in the step (4), 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 crude product of the polypeptide-manganese-carbonyl compound; further separating and purifying to obtain the polypeptide-manganese-carbonyl compound. 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 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 Fmoc lysine with the side chain amino protecting group of Alloc in the step (1) has the chemical formula of Fmoc-L-Lys (Alloc) -OH, and the structural formula is shown as follows:

said Fmoc-L-Lys (alloc) -OH is preferably used in an amount such that Fmoc amino resin: Fmoc-L-lys (alloc) -OH ═ 1: 3, calculated as a molar ratio.

The coupling time of Fmoc-L-Lys (alloc) -OH and Fmoc amino resin is preferably 4-12 h; more preferably 12 h.

The Fmoc-protected amino acids other than Fmoc-L-Lys (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-Lys (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 nitrogen heterocyclic compound with aldehyde group in the step (3) is preferably at least one of 2-formaldehyde pyridine and 2-formaldehyde quinoline.

The dosage of the nitrogen heterocyclic compound with aldehyde group and the sodium triacetoxyborohydride in the step (3) is preferably 5-15 times equivalent, and more preferably 10 times equivalent of the polypeptide supported on the resin.

The dosage of the dichloroethane in the step (3) is preferably calculated according to the concentration of the nitrogen heterocyclic compound with aldehyde group and sodium triacetoxyborohydride in the system being more than 0.4 mol/L; more preferably 0.4 to 0.6 mol/L.

And (3) reacting at 20-30 ℃ (room temperature) for a period of time until the resin is colorless and transparent.

The adding amount of the manganese pentacarbonyl bromide in the step (4) is preferably 1-5 times of the equivalent of the polypeptide supported on the resin, and more preferably 3 times of the equivalent.

The amount of the dichloromethane in the step (4) is preferably determined according to the following ratio of dichloromethane: 5-10 mL of Fmoc amino resin: and 1g is calculated.

The coordination reaction in the step (4) is preferably carried out at the temperature of 20-30 ℃ (room temperature) for 4-6 h by oscillation reaction.

The cleavage reagent described in step (5) is preferably trifluoroacetic acid (TFA), water and 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DODT) in a ratio of 95:2.5: the resulting solution was mixed at a volume ratio of 2.5.

The cutting time in the step (5) is preferably 2-4 h.

The extractant for the extraction in step (5) is preferably ethyl glacial ether.

The number of extractions described in step (5) is preferably two.

The separation and purification in step (5) is preferably carried out by reverse phase liquid chromatography.

The mobile phase of the reverse phase liquid chromatography is acetonitrile/water mixed solution containing 0.1 percent of trifluoroacetic acid.

The solid-phase synthesis method of the CO release molecule based on the polypeptide-manganese-carbonyl compound is applied to the preparation of medicines and/or medical materials.

The principle of the method of the invention is as follows: adopting an Fmoc solid-phase polypeptide synthesis method, using Fmoc amino resin as a carrier, using lysine with side chain Alloc protection at a site needing side chain modification according to a sequence of target polypeptide, and sequentially condensing Fmoc protected amino acids from a C end to an N end to obtain polypeptide amino resin with the side chain with 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 group which can be used for modification reaction; then, carrying out reductive amination reaction on the resin to modify free amino groups on polypeptide chains on the resin into ligand groups capable of carrying out chemical coordination; then adding manganese pentacarbonyl bromide to carry out chemical coordination on the resin; finally, the target polypeptide is cracked from the resin and purified, and the polypeptide with manganese-carbonyl coordination can be obtained.

Compared with the prior art, the invention has the following advantages and effects:

the method provides a whole-process solid-phase synthesis example for introducing a coordination functional group into the polypeptide and carrying out manganese-carbonyl coordination, and the target product can be obtained only by the purification of a final reaction liquid chromatography, so that the number of the synthesis steps for synthesizing the polypeptide/protein metal compound is greatly simplified, and the time required by synthesis is shortened.

In the method, lysine with side chain protecting group of Alloc is introduced in polypeptide solid phase synthesis 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 an organic solvent used in the solid phase synthesis of the polypeptide, 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. By using the solid-phase coordination method on the resin, the residual uncomplexed manganese pentacarbonyl bromide can be easily and conveniently removed by washing the resin after coordination is finished, the liquid-phase purification step after coordination is saved, and the synthesis time and cost are greatly shortened.

The whole course solid phase synthesis method of 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 Fmoc protective group-based solid-phase polypeptide synthesis method and orthogonal protective group lysine, and utilizes reductive amination reaction on the resin to proceed at polypeptide target sitesFunctional modifications for coordination are available, followed by coordination reactions of manganese-carbonyl-ligands on the resin using manganese pentacarbonyl bromide. The coordinated product can be kept stable in the reaction process of resin cutting, and no shedding is found, so that only one-step purification is needed to successfully prepare the polypeptide-manganese-carbonyl (Mn (CO))₃) And (c) a complex.

The polypeptide-manganese-carbonyl (Mn (CO)) prepared by the method of the invention₃) The compound constructs a carbon monoxide release molecule which has high biocompatibility and stable physiological environment, realizes targeted transportation and controllable release of carbon monoxide and takes the polypeptide as a bracket by targeting the polypeptide part and the light controllability of carbon monoxide release of manganese carbonyl, is favorable for researching the physiological and biochemical effects of the carbon monoxide on a specific part, and lays a good foundation for the carbon monoxide in the aspect of treatment of related diseases.

Drawings

FIG. 1 is a schematic HPLC of each stage of the solid phase synthesis of TATK7K (dpa-Mn-CO) containing a bis-methylene pyridine and a manganese-carbonyl complex prepared in example 1.

FIG. 2 is a representation of ESI-MS of TATK7K (dpa-Mn-CO) containing a bis-methylene pyridine and a manganese-carbonyl complex prepared in example 1.

FIG. 3 is a schematic HPLC of each stage of the solid phase synthesis of TATK7K (dqa-Mn-CO) containing the coordination of bismethylene quinoline and manganese-carbonyl as prepared in example 2.

FIG. 4 is a representation of ESI-MS of TATK7K (dqa-Mn-CO) containing a bis-methylene quinoline and a manganese-carbonyl complex prepared in example 2.

FIG. 5 is a schematic representation of preparation of TATK7K (dpa-Mn (CO))₃) Light-operated CO release experiment result chart.

FIG. 6 is a scheme showing preparation of TATK7K (dqa-Mn (CO))₃) Light-operated CO release experiment result chart.

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.

The Fmoc-L-Lys (alloc) -OH used in the following examples has the following structural formula:

the remaining Fmoc protected amino acids are Fmoc-L-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH.

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 polypeptides with two different ligand functions synthesized in the following examples are as follows, and the C-termini of the synthesized polypeptides are amidated:

TAT(K7K(dpa)):YGRKK(dpa)RRQRRR；

TAT(K7K(dqa)):YGRKK(dqa)RRQRRR。

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;

DEC: ethylene dichloride;

TFA: trifluoroacetic acid;

DODT: 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid;

ACN: and (3) acetonitrile.

Example 1: the preparation method of the compound with the side chain coordination group of the bis-methylene pyridine (dpa) TAT-manganese carbonyl 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 TATK7K (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 the 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, shaking for 2h), followed by sequential coupling of Fmoc-L-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -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 TATK7K (dpa) -MBHA resin: and (3) taking 500mg of the resin obtained in the step (3), adding 2-formaldehyde pyridine (0.9mmol) and sodium triacetoxyborohydride (0.9mmol), adding 2.25mL of DCE, shaking for reaction at room temperature, and monitoring the reaction by using a Kaiser reagent until the resin is colorless and transparent. 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.

(5)TATK7K(dpa-Mn(CO)₃) Preparation of MBHA resin: 500mg of the resin obtained in step (4) was added with manganese pentacarbonyl bromide (0.36mmol), 2.5mL of DCM was added, and the reaction was stirred at room temperature for 4 h. 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)TATK7K(dpa-Mn(CO)₃) Preparation of the polypeptide: 250mg of the resin from step (5) was taken and 5mL of a cleavage reagent (TFA: DODT: H) was added₂O is 95:2.5:2.5) in volume ratio, shaking for 2-4h, filtering to obtain a yellow-brown transparent liquid, spin-drying the liquid by a rotary evaporator, adding about 15mL of ethyl glacial ether for extraction twice, centrifuging, collecting precipitates, and freeze-drying the sample to obtain about 80mg of TATK7K (dpa-Mn (CO)₃) The polypeptide is then separated and purified by HPLC to obtain 68mg of TATK7K (dpa-Mn (CO))₃) A polypeptide.

At TATK7K (dpa-Mn (CO))₃) The whole solid phase synthesis process of (2) is carried out by carrying out a small amount of cutting tests on the resin obtained in each stage and detecting the products in different stages by using HPLC, and the results are shown in figure 1. ESI-MS identification of the resulting TATK7K (dpa-Mn (CO))₃) The results are shown in FIG. 2.

Example 2: the preparation method of the compound with the side chain coordination group of dimethylene quinoline (dqa) TAT-manganese carbonyl comprises the following steps:

(1) 500mg of Fmoc-Arg (Pbf) -MBHA resin was obtained at a loading of about 0.44mmol/g according to the procedure of example 1.

(2) Preparation of TATK7K (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 the 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, shaking for 2h), followed by sequential coupling of Fmoc-L-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -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: 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 TATK7K (dqa) -MBHA resin: 500mg of the resin was added with 2-carboxaldehyde quinoline (0.9mmol), 2.25mL of DCE and reacted at room temperature with shaking for 2 h. Sodium triacetoxyborohydride (0.9mmol) was then added and the reaction was continued with shaking at room temperature and monitored with Kaiser's reagent until the resin was colorless and transparent. 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.

(5)TATK7K(dqa-Mn(CO)₃) Preparation of MBHA resin: 500mg of the resulting resin (3) was added with manganese pentacarbonyl bromide (0.36mmol), 2.5mL of DCM was added, and the mixture was reacted at room temperature with shaking for 4 h. 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.

(5)TATK7K(dqa-Mn(CO)₃) Preparation of the polypeptide: 250mg of resin was taken and 5mL of cleavage reagent (TFA: DODT: H) was added₂O95: 2.5:2.5), shaking for 2-4h, filtering to obtain a yellow-brown transparent liquid, spin-drying the liquid in a rotary evaporator, adding about 15mL of ethyl glacial ether for extraction twice, centrifuging, collecting precipitates, and freeze-drying the sample to obtain about 75mg of TATK7K (dqa-mn (co)₃) The crude polypeptide is then separated and purified by HPLC to obtain about 62mg of TATK7K (dqa-Mn (CO))₃) A polypeptide.

At TATK7K (dqa-Mn (CO))₃) The whole solid phase synthesis process of (2) is carried out by carrying out a small amount of cutting test on the resin obtained in each stage and detecting the products in different stages by using HPLC, and the result is shown in FIG. 3. ESI-MS identification of the resulting TATK7K (dqa-Mn (CO))₃) The results are shown in FIG. 4.

Example 3: light-controlled CO release experiment

Experiment raw materials: myoglobin (Mb) (sigma aldrich), sodium dithionite, TATK7K (dpa-Mn (CO))₃)，TATK7K(dqa-Mn(CO)₃) PBS buffer solution, and a visible light purple flashlight (380-415 nm, LED, 5W).

The experimental steps are as follows:

the PBS buffer was deoxygenated by bubbling nitrogen for 30 min.

② Mb is prepared to a final concentration of 40 mu mol/L by using the PBS after oxygen removal, and added into a quartz cuvette. Mb was reduced by adding sodium dithionite (final concentration 3.2 mmol/L).

③ adding TAT-Mn (CO)₃Polypeptide-manganese complex (final concentration 10. mu. mol/L).

Fourthly, illuminating the cuvette by using a visible light purple flashlight, and recording spectrograms of different accumulated illumination time by using an ultraviolet visible spectrum. The results are shown in FIGS. 5 and 6.

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 after lighting, TAT-Mn (CO)₃CO in the material is released rapidly, and 540nm and 570nm characteristic absorption double peaks of Mb-CO appear in ultraviolet.

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.

Sequence listing

<110> university of southern China's science

<120> solid-phase synthesis method of CO release molecule based on polypeptide-manganese-carbonyl compound and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> lysine (K) at position 5 having a coordinating group of bismethylenepyridine (dpa)

<400> 1

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 2

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> lysine (K) at position 5 having a coordinating group of bismethylene quinoline (dqa)

<400> 2

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10