Non-natural amino acid as well as preparation method and application thereof

Abstract

The invention discloses a non-natural amino acid as well as a preparation method and application thereof. The novel non-natural amino acid is obtained by introducing long and soft side chain amino acid into a classical ASF side chain for the first time, the stability of the obtained non-natural amino acid is enhanced, the reaction radius of the non-natural amino acid is increased, the non-natural amino acid can touch amino acid residues with farther distance, and the reactivity is improved. In addition, compared with other covalent amino acids, the covalent amino acids have higher reaction rate and binding efficiency. The polypeptide and related drugs obtained based on the non-natural amino acid can be high in safety, can cross a blood brain barrier to realize brain targeting, and have better anti-tumor activity, so that the polypeptide and related drugs have extremely high medical value.

Description

Technical Field

[0001] The present invention relates to the technical field of drug development, and in particular to a non-natural amino acid and a preparation method and application thereof. Background Art

[0002] Traditional small molecule drugs mainly work by non-covalently binding drugs to enzyme or receptor target proteins. However, non-covalent binding can undergo reversible changes due to the activity of the protein or the surrounding environment, and may even be competed for by high concentrations of endogenous substrates, thus affecting the efficacy of the drug. Among the approximately 20,000 proteins in the human proteome, only about 3,000 proteins are considered to be useful for drug therapy, and in fact only some proteins have obvious druggable pockets that can be used for the development of conventional drugs, and currently there are only 700 drugs used to target these druggable pockets. Therefore, it is still necessary to explore a variety of drug design strategies to develop more effective therapeutic drugs.

[0003] With the development of biotechnology, drug design is not limited to traditional small molecule development, and scientists have shifted their attention to the design of covalent drugs. Covalent drug design is a strategy that uses covalent bonds to irreversibly bind drugs to target proteins to exert the biological effects of drugs. Covalent drugs not only have corresponding non-covalent interactions, but also react with specific amino acid residues of target proteins to form a variety of groups with covalent bonds, such as acrylamide, epoxide, α-halogenated ketone, methyl ester ketone, aziridine, vinyl sulfone, activated alkyne, etc. Covalent drugs have high efficacy, strong binding force and durability, which can significantly prolong the half-life of drugs in vivo, thereby reducing the dosage, reducing the toxic and side effects of drugs, avoiding certain drug resistance mechanisms, and thus enhancing patient compliance. Small molecule covalent drugs have attracted widespread attention due to their small molecular weight, high flexibility, and easier binding to target proteins in different pockets. However, they also have shortcomings such as lack of specificity and selectivity, and sometimes affect irrelevant biological targets to cause "off-target effects", which can easily cause adverse reactions. Covalent peptide drugs can combine the advantages of biocompatibility and covalent binding of peptides. Compared with small molecule covalent drugs, they are more specific and selective, providing a new approach for disease treatment. Unfortunately, covalent peptide drugs are obviously limited by the chemical reactivity of classical amino acids. Among the 20 classical amino acids, the disulfide bonds produced by cysteine-cysteine ​​cross-linking are weak, reversible, and sensitive to redox, which is not suitable for the development of covalent peptide and protein drugs. There is also a lack of typical electrophilic amino acids. Therefore, it is necessary to increase the diversity of covalent peptide drugs by introducing unnatural amino acids (Uaas).

[0004] In recent years, the Uaas with potential bioreactivity developed only react with specific adjacent natural amino acid residues. Among them, one of the representative applications of proximal bioreactive Uaas targeting various natural residues is the sulfur (VI) fluoride exchange (SuFEx) reaction, which is a representative of the new generation of click chemistry reactions after the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The core of the SuFEx reaction is to obtain a sulfate diester through the coupling between silicon-protected hydroxyl groups and arylsulfonyl fluoride, and then based on the sulfate diester as a sulfate precursor, further hydrolysis or hydrogenolysis is used to obtain the target sulfated product. With the development of SuFEx applications, arylsulfonyl fluoride (FSY) and arylsulfosulfate (ASF) are considered to be candidate warheads for targeting other nucleophilic residues in the protein environment. Among them, arylsulfosulfate (ASF) has attracted much attention due to its strong stability and high reaction rate. However, ASF has no flexible long side chain and its length is similar to that of classical amino acids, which limits its reaction radius and makes it unable to cross-link target residues farther away, thus hindering the universal applicability of covalent peptide drugs based on it and its potential to combat target mutations. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a non-natural amino acid and a preparation method and application thereof. The present invention prepares a non-natural amino acid based on aryl fluorosulfate modification, and finds that the active peptide obtained based on the non-natural amino acid has better binding rigidity and reaction rate, thereby enhancing the therapeutic activity and therapeutic effect of the active peptide, and has good application prospects and high economic value.

[0006] The first aspect of the present invention provides a modified amino acid, wherein the modified amino acid is selected from modified lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine.

[0007] In the present invention, the optional amino acids are required to have a certain length so that they can serve as side chains to improve the ASF reaction rigidity and reaction radius after being connected to ASF, thereby making the modified amino acids have better reaction rate and binding efficiency.

[0008] In some embodiments of the invention, the length of the optional amino acids is at least greater than

[0009] In some embodiments of the invention, the optional amino acid length is to

[0010] In some embodiments of the present invention, the modification is aryl fluorosulfate modification. In the present invention, the aryl fluorosulfate modification means that the -COOH on the side chain of aryl fluorosulfate (ASF) reacts with the side chain hydroxyl, amino or thiol of amino acid after activation.

[0011] In some embodiments of the present invention, in the modified amino acid, the amino acid and the aryl fluorosulfate are further connected with a linker.

[0012] In some embodiments of the present invention, the amino acids include D-configuration amino acids and L-configuration amino acids.

[0013] In some embodiments of the present invention, the modified amino acid has a structure as shown in Formula I:

[0014]

[0015] Among them, L 1 Including but not limited to carbonyl, amide, imine, thioester;

[0016] n Any integer between 0 and 4;

[0017] R 1 Including but not limited to alkoxycarbonyl (such as Cbz, Boc, Fmoc, Alloc, Teoc), acyl (such as Pht, Tos, Tfa) and alkyl (such as Trt, Dmb, Pmb, Bn);

[0018] R 2 These include, but are not limited to, substituted or unsubstituted methyl esters, substituted or unsubstituted ethyl esters, tert-butyl esters, and benzyl esters.

[0019] In some embodiments of the invention, the substituent includes FMOC (9-fluorenylmethoxycarbonyl).

[0020] In some embodiments of the present invention, the substituted ethyl ester is OtBu (tert-butyl ester).

[0021] The second aspect of the present invention provides a method for preparing a modified amino acid, comprising:

[0022] The activated aryl fluorosulfate is subjected to condensation reaction with amino acid to obtain the product.

[0023] In some embodiments of the present invention, the amino acid is selected from lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine.

[0024] In some embodiments of the invention, the amino acid is linked to a protecting group.

[0025] In some embodiments of the present invention, the protecting group includes, but is not limited to, CBZ (carboxybenzyloxycarbonyl), BOC (tert-butyloxycarbonyl) and FMOC (9-fluorenylmethyloxycarbonyl).

[0026] In some embodiments of the present invention, the amino acid may also be protected by acylation or benzylation.

[0027] In some embodiments of the present invention, the activation is to activate the carboxyl group of the aryl fluorosulfate.

[0028] In some embodiments of the present invention, activation of the carboxyl group can be performed by conventional methods in the art, including but not limited to: NCA synthesis method, activated ester method, carbodiimide method and mixed anhydride method.

[0029] In some embodiments of the present invention, the activation of the carboxyl group is specifically as follows: after dissolving the compound to be activated in a solvent, N-methylmorpholine (NNM) and isobutyl chloroformate (IBCF) are added at once, and the reaction is carried out for 10-30 minutes.

[0030] In some embodiments of the present invention, the activation is performed under ice-salt bath conditions.

[0031] In some embodiments of the present invention, the specific steps of the condensation reaction are: after dissolving the protected amino acid in a solvent, NMM and carboxyl-activated aryl fluorosulfate are added in sequence, reacting in an ice bath for 20-40 minutes and then continuing the reaction at room temperature for 3-4 hours.

[0032] In some embodiments of the present invention, the preparation method further comprises: detection of reaction progress, extraction and purification.

[0033] In some embodiments of the present invention, the reaction progress is detected by thin layer chromatography TLC.

[0034] In some embodiments of the present invention, the extraction comprises the following steps: vacuum rotary evaporation to remove the solvent, adding an extractant to perform extraction, and then washing with a detergent.

[0035] In some embodiments of the present invention, the extractant includes ethyl acetate, dichloromethane, chloroform, toluene, and the like.

[0036] In some embodiments of the present invention, the detergent comprises NaHCO3, water, citric acid and NaCl; in some embodiments of the present invention, the detergent is saturated NaHCO3, ultrapure water, 5% citric acid and saturated saline.

[0037] In some embodiments of the present invention, the number of washing times is greater than or equal to 2.

[0038] In some embodiments of the present invention, the purification is performed by recrystallization.

[0039] In some embodiments of the present invention, the recrystallization purification comprises the following steps: removing the solvent from the washed solution by rotary evaporation under reduced pressure, and then drying the solution to obtain the product.

[0040] In some embodiments of the present invention, the preparation method further comprises: deprotecting the obtained product.

[0041] In some embodiments of the present invention, the preparation method is used to prepare the modified amino acid described in the above aspects.

[0042] In some embodiments of the present invention, the reaction of connecting the activated aryl fluorosulfate with the amino acid includes but is not limited to a condensation reaction, and the connection may or may not include a linker. Those skilled in the art may also use other methods to connect the amino acid to the side chain of the activated aryl fluorosulfate to achieve the same or similar technical effects.

[0043] The third aspect of the present invention provides the use of the modified amino acid described in the above aspects or the modified amino acid prepared by the preparation method in the synthesis of proteins or active fragments thereof.

[0044] In the present invention, the term "protein or its active fragment" refers to any active or inactive protein and its fragment (peptide segment). Among them, active protein refers to all active proteins in the process of life activities, including but not limited to enzymes, hormone proteins, transport and storage proteins, actin, receptor proteins, etc.; inactive protein includes inactive proteins with biological protection and support functions, such as collagen, keratin, elastin, etc.

[0045] In the present invention, the functionality of the synthesized protein or its active fragment is not limited.

[0046] The fourth aspect of the present invention provides a protein or an active fragment thereof, wherein the protein or the active fragment thereof contains at least one modified amino acid described in the above aspects or a modified amino acid prepared by the preparation method.

[0047] In some embodiments of the present invention, in the protein or its active fragment, the modified amino acids described in the above aspects or the modified amino acids prepared by the preparation method account for 0.1%-100% of the total amino acid residues.

[0048] In some embodiments of the present invention, in the protein or its active fragment, the modified amino acids described in the above aspects or the modified amino acids prepared by the preparation method account for 0.1%-20% of the total amino acid residues.

[0049] In some embodiments of the present invention, the protein or its active fragment contains 1 to 5 modified amino acids described in the above aspects or modified amino acids prepared by the preparation method.

[0050] In some embodiments of the present invention, the protein or its active fragment contains one modified amino acid described in the above aspects or a modified amino acid prepared by the preparation method.

[0051] In some embodiments of the present invention, amino acids in both D and L configurations may coexist in the protein or its active fragment.

[0052] In the present invention, the ratio of amino acid residues in D configuration and L configuration in the protein or its active fragment is not limited, and the above-mentioned modification can occur in amino acid residues in D configuration or in L configuration.

[0053] In some embodiments of the present invention, the amino acids in the protein or its active fragment are all D-configuration amino acids.

[0054] The fifth aspect of the present invention provides a chimeric protein (fusion protein) or an active fragment thereof, wherein the chimeric protein or the active fragment thereof comprises multiple segments of one or more proteins or active fragments thereof of the same or different origins.

[0055] In some embodiments of the present invention, the chimeric protein or its active fragment may be composed of one or more homologous or heterologous proteins or their active fragments, such as a chimeric protein obtained by repeating homologous identical fragments or a chimeric protein composed of multiple non-identical fragments (homologous or heterologous) in a certain connection mode or order.

[0056] In some embodiments of the present invention, at least one segment of the protein or its active fragment contains at least one modified amino acid described in the above aspects or a modified amino acid prepared by the preparation method.

[0057] In some embodiments of the present invention, the chimeric protein or its active fragment further contains at least one of a linker, a tag protein, and a selection marker. Of course, those skilled in the art can also choose to add other elements according to actual needs, including but not limited to the above elements.

[0058] In some embodiments of the present invention, the chimeric protein or its active fragment contains a linker L 2 .

[0059] In some embodiments of the present invention, the linker L 2Including but not limited to -GS-, -(GGGS) n1 -、-(PEG m1 ) n2 -、-(Gly) n1 -and-(EAAK) n3 -.in n1 Any integer between 0 and 8. n2 Any integer between 0 and 15. n3 Any integer between 0 and 3. m1 Any integer from 1 to 24.

[0060] Of course, those skilled in the art can also use other linkers to connect multiple fragments, including but not limited to the linkers mentioned above.

[0061] In some embodiments of the present invention, the chimeric protein or active fragment thereof comprises:

[0062] ddfYVWWPNFpr

[0063] hrpyiah-L 2 -rvysf;

[0064] Wherein, at least one amino acid in the ddfYVWWPNFpr or rvysf fragment in the above sequence is the modified amino acid described in the above aspect.

[0065] In some embodiments of the present invention, the rvysf fragment in the above sequence contains 1, 2, 3, 4 or 5 of the modified amino acids described in the above aspects.

[0066] In some embodiments of the present invention, at least one of the amino acids f, s, y or v in the rvysf fragment is a modified amino acid as described in the above aspects.

[0067] In some embodiments of the present invention, f in the ddfYVWWPNFpr fragment is the modified amino acid described in the above aspects, and one of f, s, y or v in the rvysf fragment is the modified amino acid described in the above aspects.

[0068] The sixth aspect of the present invention provides the use of the protein or its active fragment, or the chimeric protein or its active fragment described in the above aspects in at least one of the following (1)-(5):

[0069] (1) Preparation of drugs;

[0070] (2) Disease diagnosis or imaging agents;

[0071] (3) Improve receptor-ligand binding activity;

[0072] (4) As a drug screening platform;

[0073] (5) Delivery vehicle.

[0074] In the present invention, the term "disease diagnosis or imaging agent" refers to an agent that labels a protein or its active fragment with fluorescence or radionuclide by a labeling or coupling method that can be inferred in the art, so as to achieve imaging, tracing, qualitative and quantitative analysis of the target protein.

[0075] In some embodiments of the present invention, the drugs in (1) and (3) include: anti-tumor drugs, targeted protein binding agents or degradation agents.

[0076] In some embodiments of the present invention, the tumor includes but is not limited to colon cancer, breast cancer, liver cancer, lung cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, bone cancer, head and neck cancer, brain glioma, melanoma, lymphoma, leukemia, etc.

[0077] In some embodiments of the invention, the delivery vehicle is capable of penetrating the blood-brain barrier.

[0078] In some embodiments of the present invention, the protein or its active fragment, or the chimeric protein or its active fragment can also be used as a probe, including but not limited to fluorescent molecular imaging probes, radionuclide probes, etc. further obtained based on the protein or its active fragment, or the chimeric protein or its active fragment.

[0079] The beneficial effects of the present invention are:

[0080] 1. The present invention introduces a long and flexible side chain amino acid design into the classic ASF side chain for the first time to obtain a new type of non-natural amino acid. This design not only enhances the stability of the obtained non-natural amino acid, but also increases its reaction radius, enabling it to reach amino acid residues farther away and improve reactivity. Moreover, compared with other covalently modified FSY, the non-natural amino acids in the present invention have generally higher reaction rates and binding efficiencies.

[0081] 2. The present invention also constructs a variety of targeted binding peptides based on the design concept of the non-natural amino acid, which can be used as the targeted binding of the target, and can even rely on the dual-target binding of the chimeric peptide to achieve the functional blocking, endocytosis, degradation, etc. of the target, thereby realizing the construction of a peptide-based targeted degradation platform (Pep-TACs). And through the design of a series of linkers, it is possible to explore different binding modes of different targets and optimize the design of non-natural amino acid modified peptides.

[0082] 3. The modified peptide-based targeted binding platform constructed in the present invention can be effectively used for drug design and screening. It has higher affinity for the target, stronger binding force, and can significantly improve the target binding rate and target degradation rate. It is also found that it has higher affinity and stronger target degradation ability in an acidic environment, so it can be used to exert target blocking degradation ability in an acidic environment such as a tumor microenvironment. Moreover, the drug is simple in design, easy to synthesize, and low in cost.

[0083] 4. The chimeric protein constructed based on the design concept of the non-natural amino acid in the present invention can effectively improve the treatment environment, such as degrading the immunosuppressive target PD-L1 to a greater extent, thereby reversing the immunosuppressive environment in the tumor microenvironment and improving anti-tumor immunity. Moreover, it has high safety and can cross the blood-brain barrier to achieve brain targeting, so it is effectively used for brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is the mass spectrum of Fmoc-k-ASF-OtBu.

[0085] Figure 2 Comparison results of the covalent reaction rates of ASF and k-ASF.

[0086] Figure 3 Designed for covalently modifying peptide sequences.

[0087] Figure 4 This is the WB detection result of the covalent binding of f3ASF and PD-1.

[0088] Figure 5 Design process for dual-targeting chimeric peptides.

[0089] Figure 6 Representative flow cytometry images and data comparison results of the experimental group peptides blocking human and mouse PD-1 / PD-L1.

[0090] Figure 7 The effect of the experimental group peptides on the expression of PD-L1 and TFRC on the surface of B16 and MC38 cells, where A is the result of flow cytometry detection and B is the result of WB detection.

[0091] Figure 8 This is the flow cytometry result of the effect of the peptides in the experimental group on the expression of PD-L1 on the surface of B16 and MC38 cells under pH 6.0 conditions.

[0092] Fig. 9 Flow chart of in vivo anti-tumor experiments of covalently modified drugs.

[0093] Fig.10The changes in tumor volume of mice in each group (A) and the results of blood routine test (B).

[0094] Fig.11 This is an experimental procedure for the anti-tumor delivery of covalently modified drugs to the brain.

[0095] Fig.12 Shown are the survival status of mice in each group (A) and IVIS images of brain tumors (B). DETAILED DESCRIPTION

[0096] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0097] In the present invention, the terms "amino acid", "amino acid residue" include naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure (e.g., α carbon bound to hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but function similarly to naturally occurring amino acids.

[0098] In the present invention, the synthesis of peptide chains is not limited, including but not limited to chemical synthesis and biosynthesis. Examples of chemical synthesis include solid phase synthesis (such as Fmoc method), liquid phase synthesis and solid-liquid phase synthesis. Synthesis strategies include C-terminal synthesis, N-terminal synthesis and segmented synthesis. If conventional modification is performed at the C-terminal or N-terminal during synthesis, the peptide chain may contain the conventional modification group.

[0099] The natural amino acids and their various expressions described herein are well known to those skilled in the art, and their specific corresponding situations are shown in the following table:

[0100] name Single letter Three letters name Single letter Three letters Alanine A Ala Leucine L Leu Arginine R Arg Lysine K Lys Asparagine N Asn Methionine M Met Aspartic acid D Asp Phenylalanine F Phe Cysteine C Cys Proline P Pro Glutamine Q Gln Serine S Ser Glutamate E Glu Threonine T Thr Glycine G Gly Tryptophan W Trp Histidine H His Tyrosine Y Tyr Isoleucine I Ile Valine V Val

[0101] In the present invention, when amino acid residues are represented by single letters, unless otherwise specified, capital letters represent the L configuration and lowercase letters represent the D configuration.

[0102] In the present invention, the aryl fluorosulfate (ASF) used to construct the non-natural amino acid is shown in Formula II, and its relative molecular weight is 204.18.

[0103]

[0104] Example 1

[0105] This example provides a method for preparing a covalent non-natural amino acid, D-lysine-aryl fluorosulfate (k-ASF). The synthetic route is as follows:

[0106]

[0107] The specific preparation steps are as follows:

[0108] (1) Activation of carboxyl groups: Weigh 100 mg ASF (0.5 mmol), dissolve it in 5 mL tetrahydrofuran (THF) in a 25 mL flask, stir it with a magnetic stirrer in an ice-salt bath (ice-salt mass ratio 3:1, salt is NaCl, temperature is -18°C to -22°C), and then slowly drop 56 μL N-methylmorpholine (NNM, 1.2 equivalents, 0.6 mmol). Add 67 μL isobutyl chloroformate (IBCF, 0.5 mmol). After reacting for 15 min, remove the ice-salt bath and replace it with an ice bath.

[0109] (2) Condensation reaction: Weigh 277 mg of protected D-lysine hydrochloride (Fmoc-D-Lys-OtBu·HCl, 1.2 equivalents, 0.6 mmol) and dissolve it in 1 mL of N,N-dimethylformamide (DMF). Add 56 μL of NMM and mix well. Then slowly add dropwise to the reaction system of step (1). After reacting for 0.5 h, remove the ice bath and continue the reaction for 3-4 h.

[0110] Wherein, thin layer chromatography TLC is used to detect the progress of the reaction. The specific method is: use dichloromethane (DCM) to dissolve a small amount of ASF and Fmoc-D-Lys-OtBu·HCl respectively to obtain ASF and Fmoc-D-Lys-OtBu·HCl control solutions, use a capillary glass tube to absorb the two control solutions and the product obtained in step (2) respectively for spotting, and then place the thin plate in a developing tank if there is a developing agent, and the developing agent is a mixed solution of DCM: methanol = 10:1. After the solvent (DCM) is expanded upward to about 90% of the length of the thin plate, take out the thin plate to dry the solvent, and observe whether the reaction is complete under ultraviolet light.

[0111] (3) Product extraction: After the reaction is complete as determined by TLC, the product obtained in step (2) is subjected to reduced pressure rotary evaporation, and after removing the solvent, 6 mL of ethyl acetate (EtOAc) is added to dissolve it and then transferred to a 60 mL separatory funnel. The product is washed twice with saturated NaHCO3, ultrapure water, 5% citric acid and saturated brine in sequence.

[0112] (4) Recrystallization purification: Pour the washed EtOAc solution into a rotary evaporation flask, perform rotary evaporation under reduced pressure, remove the solvent, add a small amount of EtOAc and ultrasonically dissolve the product, then slowly add petroleum ether (PE) dropwise until a large amount of white precipitate is produced, stop adding PE (about 10-20 times the volume of EtOAc), and place the flask at 4°C overnight for recrystallization.

[0113] (5) Mass spectrometry verification: The product obtained after overnight recrystallization was subjected to vacuum rotary evaporation to remove the solvent, and then the obtained oil was pumped dry using an oil pump to obtain a product powder, which was collected to calculate the yield. A small amount of the powder was taken and dissolved in methanol, and then filtered through a 0.22 μm filter membrane, and the correctness of Fmoc-k-ASF-OtBu (compound 3, MW: 633) in the filtrate was detected by mass spectrometry.

[0114] Among them, the mass spectrum of Fmoc-k-ASF-OtBu is as follows Figure 1 shown.

[0115] (6) Deprotection: Before the final peptide synthesis, take an appropriate amount of Fmoc-k-ASF-OtBu and dissolve it in a flask with a small amount of 90% trifluoroacetic acid (TFA, prepared with dichloromethane), stir the reaction for 30 minutes, then add a large amount (excessive) of ether for precipitation. After precipitation is complete, vacuum evaporation is used to remove the solvent. Add ether for washing and spin dry, and repeat 2-3 times. The product obtained by spin drying is pumped dry by an oil pump to obtain a product powder (final product) for subsequent peptide synthesis steps. Among them, reactant 4 can be treated with 20% piperidine at room temperature for 10 minutes to remove the protecting group protection to obtain D-lysine-aryl fluorosulfate (k-ASF, i.e., the covalent non-natural amino acid in the present invention).

[0116] The k-ASF obtained in this example was subjected to a covalent reaction rate test. The specific method is as follows: the k-ASF obtained in this example was dissolved in a reaction solvent (MeCN:H2O:DMSO=1:1:1.1). Then, under room temperature oscillation conditions, 20 equivalents of DIPEA (N,N-diisopropylethylamine) were added dropwise for base correction. Then, 10 equivalents of Fmoc-D-Lys-OtBu·HCl were added dropwise. After the reaction started, the reaction solution was filtered through a 0.22 μm filter membrane at 5 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h, and the obtained filtrate was detected using HPLC, and then the peak area of ​​the target product at each reaction time point was calculated to determine the covalent reaction rate. ASF without any treatment was used as a control.

[0117] The results are as follows Figure 2 shown.

[0118] It can be found that the k-ASF obtained in this example has a significantly higher covalent reaction rate than the ASF without any treatment.

[0119] According to the same method as in this example, D-tyrosine-aryl fluorosulfate and D-histidine-aryl fluorosulfate can also be obtained.

[0120] Example 2

[0121] In this example, polypeptides were constructed based on the k-ASF obtained in the above examples. In this example, the PD-1 affinity peptide was used as an example to demonstrate the effect of the polypeptides constructed using k-ASF. Of course, it should be understood that this example does not limit the scope of polypeptides that can be constructed using k-ASF, and the polypeptides that can be constructed include but are not limited to the PD-1 affinity peptide used as an example in this example.

[0122] (1) Peptide synthesis:

[0123] The parent PD-1 affinity peptide, R4-Y ddf-pr , whose amino acid sequence is Asp D -Asp D -Phe D -Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D , wherein the superscript D indicates the D configuration, and no superscript indicates the L configuration, and the single-letter abbreviation (capital letters indicate the L configuration, lowercase letters indicate the D configuration) is: ddfYVWWPNFpr (SEQ ID NO: 1), MW: 1808.95. R4-Y ddf-pr Molecular docking and molecular dynamics simulation were performed with PD-1 protein (PDB database number: 4ZQK) to observe R4-Yddf-pr Based on the observation results, the amino acids adjacent to lysine, tyrosine or histidine on the protein were selected for replacement with the amino acids modified by k-ASF in the above examples. The peptides after amino acid replacement were synthesized by standard solid phase Fmoc synthesis. The peptides were purified by reverse phase high performance liquid chromatography (RP-HPLC), and then their relative molecular mass was verified by electrospray ionization mass spectrometry (ESI-MS). The synthesized k-ASF modified peptide was named f3ASF, and its amino acid sequence was Asp D -Asp D -x-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D The corresponding sequence information is as follows Figure 3 shown.

[0124] At the same time, the covalently modified peptide CPBP-12 with amino acid substitution by isotopic arylsulfonyl fluoride (FSY), i.e., fluorosulfate-modified tyrosine Tyr (O-SO2F), was used as a control, and its amino acid sequence is Asp D -Asp D -Phe D -Tyr(O-SO2F)-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D Among them, the preparation of fluorosulfate-modified tyrosine in CPBP-12 was based on Wang N, Yang B, Fu C, et al. Genetically Encoding Fluorosulfate-l-tyrosine To Reactwith Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140, 4995–4999. After obtaining fluorosulfate-modified tyrosine, CPBP-12 was obtained by standard solid phase Fmoc synthesis.

[0125] (2) Covalent labeling: Equal amounts of k3ASF and CPBP-12 were labeled with biotin, and then incubated with the same amount of hPD-1 protein (purchased from Sino Biological) and subjected to SDS-PAGE gel electrophoresis. After protein transfer, HRP-labeled Streptavidin (purchased from Abcam, ab7403) was used for co-incubation. Western Blot was used to detect whether the peptide and protein were successfully covalently bound.

[0126] The results are as follows Figure 4 shown.

[0127] It can be found that k3ASF modified based on the non-natural covalent amino acid k-ASF can covalently bind to the PD-1 protein and has stronger binding force than other covalent modifications.

[0128] In addition, the inventors also found that in the case of R4-Y ddf-pr After at least 1-2 amino acids in the 3rd, 5th, and 7th amino acids (such as 3rd substitution, 5th substitution, 7th substitution, 5th and 7th substitution) are modified with the above FSY, the binding force can also be enhanced. This shows that after the above sites are modified with k-ASF, the binding force will be stronger.

[0129] Example 3

[0130] In this embodiment, the use of the non-natural covalent amino acid modifications and the resulting polypeptides in the above embodiments as targeted blocking drugs is provided, and their use in drug screening is also provided.

[0131] In this embodiment, the covalently modified polypeptide drug used for the example is a dual-targeted chimeric peptide composed of a lysosomal endocytic receptor targeting peptide and a protein of interest (POI) targeting peptide. Among them, the chimeric peptide can target and bind to the lysosomal endocytic receptor and the POI protein, thereby simultaneously blocking the binding of the lysosomal endocytic receptor to its endogenous ligand and the POI protein ligand. In addition to the blocking function, the present invention also found that the chimeric peptide has a targeted degradation function, that is, after the lysosomal endocytic receptor targeting peptide binds to the lysosomal endocytic receptor, it will pull the ternary complex formed by the lysosomal endocytic receptor-chimeric peptide-POI protein into the lysosome for degradation based on the endocytosis of the lysosomal endocytic receptor.

[0132] In this embodiment, the endocytic receptor is transferrin receptor (TFRC), and the POI protein is the immune checkpoint molecule programmed death receptor ligand 1 (PD-L1). The endocytic receptor targeting peptide is the TFRC targeting peptide. D T7 (amino acid sequence is hrpyiah (SEQ ID NO: 2)), whose amino acid residues are all in D configuration. The POI protein targeting peptide is specifically selected as the PD-L1 targeting peptide OPBP1 (8-12), whose amino acid sequence is: rvysf (SEQ ID NO: 3), whose amino acid residues are also all in D configuration. As shown in Table 1, the PD-L1 targeting peptide OPBP1 (8-12) and the TFRC targeting peptide are linked by different linkers. DAfter T7 connection, a variety of dual-targeting chimeric peptides with different softness and length were obtained D T7-linker-OPBP1, where the linker used is -GS-, -(GGGS) n1 -、-(PEG m1 ) n2 -,in n1 Any integer between 0 and 8. n2 Any integer between 0 and 15. m1 Any integer from 1 to 24.

[0133] Table 1 Dual targeting chimeric peptides D T7-linker(L 2 )-OPBP1(8-12)

[0134] serial number Amino acid sequence Relative molecular weight (MW) Pep-1 hrpyiah-rvysf 1546 Pep-2 hrpyiah-GS-rvysf 1690 Pep-3 hrpyiah-GGGS-rvysf 1804 Pep-4 hrpyiah-GGGSGGGS-rvysf 2063 Pep-5 <![CDATA[hrpyiah-PEG4-rvysf]]> 1793 Pep-6 <![CDATA[hrpyiah-(PEG4)2-rvysf]]> 2039

[0135] Among them, Pep-1 is used as an example for the following verification. Among them, f, s, y, and v in Pep-1 are used as replacement sites for amino acid replacement, and the replaced sequences are named f12x (hrpyiah-rvysx, MW: 1713), s11x (hrpyiah-rvyxf, MW: 1773), y10x (hrpyiah-rvxsf, MW: 1697), and v9x (hrpyiah-rxysf, MW: 1761). In this embodiment, the synthesis of the polypeptides adopts the standard solid phase Fmoc synthesis method, and the synthesized polypeptides are purified by reverse phase high performance liquid chromatography (RP-HPLC), and the relative molecular mass is verified by electrospray ionization mass spectrometry (ESI-MS). The specific process is as follows Figure 5 shown.

[0136] Peptide blocking: Pep-1 and its variants were diluted to a concentration of 100 μM with PBS at pH 7.4. Then 50 ng of hPD-L1-Fc (purchased from Sino Biological) or mPD-L1-Fc (purchased from Sino Biological) was incubated with the polypeptide at 37°C for 2 hours or 6 hours. CHO-K1 cells (Chinese hamster ovary cells K1) (CHO-K1-PD-1) overexpressing PD-1 were collected, and the above-mentioned peptide and protein mixture was added to CHO-K1-PD-1 cells and incubated on ice for 30 minutes. After adding PBS, centrifugation at 3000 rpm for 5 minutes was performed to remove unbound proteins. Then a fluorescently labeled anti-Fc antibody (eBioscience, USA) was added and stained on ice for 30 minutes. After adding PBS, centrifugation at 3000 rpm for 5 minutes was performed to remove unbound Fc antibodies. The mean fluorescence intensity of the cells was measured using flow cytometry. The cells without Fc tag protein and peptide and only incubated with fluorescently labeled Fc antibody were used as the negative control group, and the cells without incubation with peptide but only incubated with Fc tag protein and fluorescently labeled Fc antibody were used as the positive control group. The blocking rate of the above polypeptide was calculated using the following formula.

[0137]

[0138] The results are as follows Figure 6 shown.

[0139] It can be found that f12x and v9x modified with k-ASF in the embodiment of the present invention have significantly improved blocking rates, and v9x has the highest blocking rate. The blocking rate also increases significantly with the extension of time.

[0140] Further testing of the target degradation effect of the above peptides:

[0141] 2×10 5 B16 and MC38 cells were incubated at 0h, 2h, 6h, 24h or 48h at 400 μg / mL. The pH was maintained at 7.4 or 6.0. After incubation, the cells were collected and washed by centrifugation in PBS. Anti-mouse PD-L1 PE (clone: ​​MIH5, eBioscience), anti-mouse TFRC APC (clone: ​​R17217, eBioscience) or isotype control antibodies were used for staining at 4°C for 30min. Fluorescence intensity was evaluated by flow cytometry.

[0142] At the same time, the expression of PD-L1 in cells incubated with 25 μM v9x for 24 h was detected by WB. The specific method is as follows: after collecting the cells, they were lysed with immunoprecipitation analysis (RIPA) lysis buffer (Thermo Scientific), and then the protein lysates were continuously separated and purified by 10% SDS-PAGE and polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). After sealing the membrane with 5% skim milk (room temperature for 1 h), it was incubated overnight at 4 ° C with primary antibodies for mPD-L1 (D363307, BBI) and GAPDH (D110016, BBI). Subsequently, the membrane was treated with a secondary antibody coupled to horseradish peroxidase (HRP). Enhanced chemiluminescence reagent (ECL, GBCBIO, China) was added and the protein bands were imaged.

[0143] In addition, flow cytometry was used to detect changes in cell PD-L1 expression under acidic pH 6.0 conditions.

[0144] The PBS-treated group was used as a negative control. D Target degradation effect of T7.

[0145] The results are as follows Figure 7-8 shown.

[0146] The results show that the covalently modified drug obtained based on the embodiment of the present invention can significantly degrade the PD-L1 target, and the degradation efficiency can reach more than 80%. Moreover, the covalent binding force is stronger under acidic conditions, and the expression of PD-L1 can be further reduced.

[0147] Example 4

[0148] In this example, a mouse subcutaneous tumor model was constructed to test the in vivo anti-tumor effect of the above covalently modified drug. The specific experimental steps are as follows:

[0149] Construction of mouse subcutaneous tumor model: C57BL / 6 mice were used as experimental mice. 2×10 6 MC38 cells. Monitor tumor volume. When the tumor volume reaches 40-70mm 3 The tumor length (a), width (b), and height (c) were measured with a digital caliper, and the formula Tumor volume was calculated.

[0150] MC38 tumor-bearing mice were intraperitoneally injected with normal saline, OPBP1(8-12) (0.5 mg / kg), DAn equimolar mixture of T7 and OPBP1 (8-12) (1.3 mg / kg), Pep-1 (1.3 mg / kg), and an equimolar concentration of 0.75 μmol / kg low-dose (1.3 mg / kg) and high-dose (3.9 mg / kg) v9x, and 10 mg / kg anti-PD-L1 (clone: ​​10F.9G2, Bioxcell) as positive controls. Except for the PD-L1 antibody, the other groups were given once a day for 2 weeks. The PD-L1 antibody was administered once every 3 days for the same 2 weeks. The changes in tumor volume of mice in each group were continuously monitored. After the experiment, blood was drawn from each group of mice and serum was collected, and the levels of alanine aminotransferase, aspartate aminotransferase and creatinine were detected by conventional methods in the art.

[0151] Flowchart as Fig. 9 shown.

[0152] The results are as follows Fig.10 shown.

[0153] It can be found that compared with other groups, v9x covalently modified with k-ASF can significantly inhibit the growth of mouse tumors. And after the blood routine test of mice after administration, the alanine aminotransferase (ALT, normal range 10.06-96.47U / L), aspartate aminotransferase (AST, normal range 36.31-235.48U / L), and creatinine (CR, normal range 10.91-85.09μmol / L) of the covalently modified drug group after administration were all within the normal range, and there was no significant difference between the groups, indicating that the covalently modified drug obtained based on the embodiment of the present invention has good safety.

[0154] Example 5

[0155] In this example, it was verified that the covalently modified drug (covalently modified peptide) obtained in the example of the present invention can be administered to the brain across the blood-brain barrier.

[0156] Construction of mouse brain tumor model: C57BL / 6 mice were used as experimental mice. C57BL / 6J mice were anesthetized and fixed on a brain stereotaxic instrument. GL261-Luc cells (luciferase-labeled mouse glioma cells, 1×10 5 cells / 5μL PBS), and the bioluminescence of tumor cells expressing luciferase was detected using an in vivo imaging system to confirm that the model was successfully constructed.

[0157] The constructed tumor-bearing mice were randomly divided into groups. According to the grouping, the experimental mice were intraperitoneally injected with normal saline, Pep-1 (3.4 mg / kg), v9x (3.9 mg / kg) or anti-PD-L1 (10 mg / kg). The frequency of administration was the same as in the above example. After the modeling was successful, in situ tumor luminescence measurement was performed once a week by IVIS to determine its therapeutic effect.

[0158] Flowchart as Fig.11 shown.

[0159] The results are as follows Fig.12 shown.

[0160] It can be found that compared with other groups, v9x covalently modified with k-ASF can cross the blood-brain barrier to achieve brain targeting, inhibit the growth of brain tumors, and prolong the survival of mice.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A modified amino acid, characterized in that The modified amino acid is selected from modified lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine; The modification is an aryl fluorosulfate modification, Preferably, in the modified amino acid, the amino acid and the aryl fluorosulfate are further connected with a linker.

2. The modified amino acid according to claim 1, characterized in that The modified amino acid has a structure as shown in Formula I: Among them, L 1 Including carbonyl, amide, imine, thioester; n Any integer between 0 and 4; R 1 including alkoxycarbonyl, acyl and alkyl groups; R 2 Including substituted or unsubstituted methyl esters, substituted or unsubstituted ethyl esters, tert-butyl esters, benzyl esters.

3. A method for preparing a modified amino acid, comprising: The activated aryl fluorosulfate is subjected to a condensation reaction with an amino acid to obtain; Wherein, the amino acid is selected from lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine; preferably, the amino acid is connected with a protecting group.

4. Use of the modified amino acid according to claim 1 or 2 or the modified amino acid prepared by the preparation method according to claim 3 in the synthesis of proteins or their active fragments.

5. A protein or an active fragment thereof, characterized in that: The protein or its active fragment contains at least one modified amino acid according to claim 1 or 2 or a modified amino acid prepared by the preparation method according to claim 3.

6. A chimeric protein or an active fragment thereof, characterized in that: The chimeric protein or its active fragment comprises multiple segments of one or more proteins or their active fragments from the same or different sources; Wherein, at least one segment of the protein or its active fragment contains at least one modified amino acid according to claim 1 or 2 or a modified amino acid prepared by the preparation method according to claim 3.

7. The chimeric protein or active fragment thereof according to claim 6, characterized in that: The chimeric protein or its active fragment further contains at least one of a linker, a tag protein, and a selection marker; Preferably, the chimeric protein or its active fragment contains a linker L 2 ; Preferably, the linker L 2 Including -GS-, -(GGGS) n1 -、-(PEG m1 ) n2 -、-(Gly) n1 -and-(EAAK) n3 -; in n1 Any integer between 0 and 8. n2 Any integer between 0 and 15. n3 Any integer between 0 and 3. m1 Any integer from 1 to 24.

8. The chimeric protein or active fragment thereof according to claim 7, characterized in that: The chimeric protein or its active fragment comprises: DDFYVWWPNFPR HRPYIAH-L 2 -RVYSF; Preferably, the chimeric protein or its active fragment comprises: ddfYVWWPNFpr hrpyiah-L 2 -rvysf; Wherein, at least one amino acid in the chimeric protein or its active fragment is a modified amino acid as described in claim 1 or 2; Preferably, f in the ddfYVWWPNFpr fragment is the modified amino acid described in claim 1 or 2, and at least one of f, s, y or v in the rvysf fragment is the modified amino acid described in claim 1 or 2.

9. Use of the protein or active fragment thereof according to claim 5 or the chimeric protein or active fragment thereof according to any one of claims 6 to 8 in at least one of the following (1) to (5): (1) Preparation of drugs; (2) Disease diagnosis or imaging agents; (3) Improve receptor-ligand binding activity; (4) As a drug screening platform; (5) Delivery vehicle.

10. The use according to claim 9, characterized in that: The drugs described in (1) and (3) include: anti-tumor drugs, targeted protein binders or degradation agents.

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