Non-natural amino acid, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a non-natural amino acid, and a preparation method therefor and the use thereof. The present invention provides, for the first time, a new class of non-natural amino acids designed by introducing a long and flexible side-chain amino acid into a side chain of a canonical ASF. The design not only enhances the stability of the resulting non-natural amino acids, but also expands the reaction radius thereof, thus enabling same to access more distant amino acid residues and thereby improving the reactivity thereof. Moreover, compared with other covalent amino acids, the non-natural amino acid exhibits a higher reaction rate and binding efficiency. Polypeptides obtained on the basis of the non-natural amino acid and related drugs thereof exhibit high safety, can cross the blood-brain barrier to achieve brain targeting, and have a better anti-tumor activity, thereby demonstrating an extremely high medical value.

Description

A non-natural amino acid, its preparation method and application Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to a non-natural amino acid, its preparation method, and its application. Background Technology

[0002] Traditional small molecule drugs exert their effects primarily through non-covalent binding to target proteins (such as enzymes or receptors). However, non-covalent binding is susceptible to reversible changes due to protein activity or the surrounding environment, and can even be competitively bound by high concentrations of endogenous substrates, thus affecting drug efficacy. Of the approximately 20,000 proteins in the human proteome, only about 3,000 are considered therapeutically viable. In reality, only a portion of these proteins possess readily available druggable pockets for conventional drug development, and currently only about 700 drugs target these druggable pockets. Therefore, exploring diverse drug design strategies is still necessary to develop more effective therapeutic agents.

[0003] With the development of biotechnology, drug design is no longer limited to traditional small molecule development; scientists have turned 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 their biological effects. Covalent drugs not only have corresponding non-covalent interactions, but can also react with specific amino acid residues of the target protein to form various covalently bonded groups, such as acrylamide, epoxides, α-halogenated ketones, methyl ester ketones, azacyclopropane, vinyl sulfone, and activated alkynes. Covalent drugs are highly effective, have strong binding affinity, and are persistent, significantly prolonging the drug's half-life in vivo, thereby reducing dosage, minimizing drug toxicity and side effects, avoiding certain drug resistance mechanisms, and ultimately enhancing patient compliance. Small molecule covalent drugs have attracted widespread attention due to their small molecular weight, high flexibility, and ease of binding to target proteins in different pockets. However, they also have drawbacks such as a lack of specificity and selectivity, and sometimes affecting unrelated biological targets, causing "off-target effects," which can easily lead to adverse reactions. Covalent peptide drugs combine the advantages of peptide biocompatibility and covalent bonding, offering greater specificity and selectivity compared to small molecule covalent drugs, thus providing new avenues for disease treatment. However, covalent peptide drugs are unfortunately significantly limited by the chemical reactivity of classical amino acids. Among the 20 classical amino acids, the disulfide bonds formed by cysteine-cysteine ​​crosslinks are weak, reversible, and sensitive to redox reactions, making them unsuitable for the development of covalent peptide and protein drugs. Furthermore, typical electrophilic amino acids are currently lacking. Therefore, introducing non-natural amino acids (Uaas) to enhance the diversity of covalent peptide drugs is essential.

[0004] In recent years, bioreactive amino acids (UAAs) have been developed that react only with specific, adjacent natural amino acid residues. One representative application of proximal bioreactive UAs targeting various natural residues is the sulfur(VI)-fluoride exchange (SuFEx) reaction, a next-generation click chemistry reaction following the copper-catalyzed azide–alkyne cycloaddition (CuAAC). The core of the SuFEx reaction lies in the coupling between a silicon-protected hydroxyl group and an aryl sulfonyl fluoride to obtain a sulfate diester. Then, based on the sulfate diester as a sulfate precursor, further hydrolysis or hydrogenolysis yields the target sulfation product. With the continuous development of SuFEx applications, aryl sulfonyl fluorides (FSY) and aryl fluorosulfates (ASF) are considered potential covalent targets for other nucleophilic residues in the protein environment. ASF, in particular, has attracted significant attention due to its strong stability and high reaction rate. However, because ASF lacks flexible long side chains and its length is similar to that of classical amino acids, its reaction radius is limited, making it unable to crosslink with target residues further away. This hinders the general applicability of covalent peptide drugs based on ASF and their potential for combating target mutations. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide a non-natural amino acid, its preparation method, and its application. This invention prepares a non-natural amino acid based on aryl fluorosulfate modification, and discovers that the active peptide obtained based on this non-natural amino acid has better binding rigidity and reaction rate, thereby enhancing the therapeutic activity and efficacy of the active peptide, showing good application prospects and high economic value.

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

[0007] In this invention, the optional amino acid is required to have a certain length so that it can serve as a side chain to improve the reaction rigidity and reaction radius of ASF after being linked with ASF, thereby enabling the modified amino acid to have a better reaction rate and binding efficiency.

[0008] In some embodiments of the invention, the length of the optional amino acid is at least greater than 0.1 angstroms.

[0009] In some embodiments of the invention, the length of the optional amino acid is 0.1 angstroms. Up to 10 Angers

[0010] In some embodiments of the present invention, the modification is aryl fluorosulfate modification. In the present invention, aryl fluorosulfate modification refers to the reaction of the -COOH group on the side chain of aryl fluorosulfate (ASF) with the hydroxyl, amino, or sulfhydryl groups of the amino acid side chain after activation.

[0011] In some embodiments of the present invention, the modified amino acid and the aryl fluorosulfate are further linked 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] Among them, L 1 Including but not limited to carbonyl groups, amides, imines, and thioesters;

[0015] n is any integer from 0 to 4;

[0016] R 1 Including but not limited to alkoxycarbonyl groups (such as Cbz, Boc, Fmoc, Alloc, Teoc), acyl groups (such as Pht, Tos, Tfa), and alkyl groups (such as Trt, Dmb, Pmb, Bn);

[0017] R 2 This includes, but is not limited to, substituted or unsubstituted methyl esters, substituted or unsubstituted ethyl esters, tert-butyl esters, and benzyl esters.

[0018] In some embodiments of the present invention, the substituents include FMOC (9-fluorenylmethoxycarbonyl).

[0019] In some embodiments of the present invention, the substituted ethyl ester is OtBu (tert-butyl ester).

[0020] A second aspect of the present invention provides a method for preparing modified amino acids, comprising:

[0021] The activated aryl fluorosulfate is condensed with an amino acid to obtain the product.

[0022] In some embodiments of the present invention, the amino acid is selected from lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine.

[0023] In some embodiments of the present invention, the amino acid is attached with a protecting group.

[0024] In some embodiments of the present invention, the protecting groups include, but are not limited to, carbobenzyloxycarbonyl (CBZ), t-butyloxycarbonyl (BOC), and 9-fluorenylmethyloxycarbonyl (FMOC).

[0025] In some embodiments of the present invention, the amino acid may also be protected by acylation or benzylation.

[0026] In some embodiments of the present invention, the activation is to activate the carboxyl group of the aryl fluorosulfate.

[0027] In some embodiments of the present invention, the activation of the carboxyl group can be carried out using conventional methods in the art, including but not limited to: NCA synthesis methods, activated ester methods, carbodiimide methods, and mixed anhydride methods.

[0028] In some embodiments of the present invention, the activation of the carboxyl group specifically involves: dissolving the compound to be activated in a solvent, then adding N-methylmorpholine (NNM) and isobutyl chloroformate (IBCF) in one step, and reacting for 10-30 minutes.

[0029] In some embodiments of the present invention, the activation is carried out under ice-salt bath conditions.

[0030] In some embodiments of the present invention, the specific steps of the condensation reaction are as follows: after dissolving the protected amino acid in a solvent, NMM and carboxyl-activated aryl fluorosulfate are added sequentially, and the reaction is carried out in an ice bath for 20-40 min, followed by a further reaction at room temperature for 3-4 h.

[0031] In some embodiments of the present invention, the preparation method further includes: detection of the reaction process, extraction, and purification.

[0032] In some embodiments of the present invention, the reaction process is detected using thin-layer chromatography (TLC).

[0033] In some embodiments of the present invention, the extraction includes the following steps: rotary evaporation under reduced pressure to remove the solvent, followed by extraction with an extractant, and then washing with a detergent.

[0034] In some embodiments of the present invention, the extractant includes ethyl acetate, dichloromethane, chloroform, toluene, etc.

[0035] 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 solution.

[0036] In some embodiments of the present invention, the number of washing cycles is greater than or equal to 2.

[0037] In some embodiments of the present invention, the purification is performed by recrystallization purification.

[0038] In some embodiments of the present invention, the recrystallization purification includes the following steps: the washed solution is rotary evaporated under reduced pressure to remove the solvent, and then dried to obtain the final product.

[0039] In some embodiments of the present invention, the preparation method further includes: deprotecting the obtained product.

[0040] In some embodiments of the present invention, the preparation method is used to prepare the modified amino acids described above.

[0041] In some embodiments of the present invention, the reaction of the activated aryl fluorosulfate with amino acids includes, but is not limited to, a condensation reaction, and may or may not include a linker for the connection. Those skilled in the art may also use other methods to connect amino acids to the side chains of the activated aryl fluorosulfate to achieve the same or similar technical effects.

[0042] A third aspect of the present invention provides the use of the modified amino acids described above or the modified amino acids prepared by the preparation method described above in the synthesis of proteins or their active fragments.

[0043] In this invention, the term "protein or its active fragment" refers to any active or inactive protein and its fragments (peptides). Active proteins refer to all proteins that are active in life processes, including but not limited to enzymes, hormone proteins, transport and storage proteins, actin, receptor proteins, etc.; inactive proteins include proteins that are inactive but have biological protective and supportive functions, such as collagen, keratin, elastin, etc.

[0044] In this invention, the functionality of the synthesized protein or its active fragment is not limited.

[0045] In a fourth aspect, the present invention provides a protein or an active fragment thereof, wherein the protein or the active fragment thereof contains at least one of the modified amino acids described above or the modified amino acids prepared by the preparation method described above.

[0046] In some embodiments of the present invention, the modified amino acids described above or prepared by the preparation method account for 0.1%-100% of the total number of amino acid residues in the protein or its active fragment.

[0047] In some embodiments of the present invention, the modified amino acids described above or prepared by the preparation method account for 0.1%-20% of the total number of amino acid residues in the protein or its active fragment.

[0048] In some embodiments of the present invention, the protein or its active fragment contains 1 to 5 modified amino acids as described above or modified amino acids prepared by the preparation method.

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

[0050] In some embodiments of the present invention, the protein or its active fragment may contain both D-configuration and L-configuration amino acids.

[0051] In this invention, the ratio of D-configured and L-configured amino acid residues in the protein or its active fragment is not limited, and the above modifications can occur on either D-configured or L-configured amino acid residues.

[0052] In some embodiments of the present invention, the amino acids in the protein or its active fragment are all D-configuration amino acids.

[0053] A fifth aspect of the present invention provides a chimeric protein (fusion protein) or an active fragment thereof, said chimeric protein or active fragment thereof comprising one or more proteins or active fragments thereof from the same or different sources.

[0054] 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 (homologous) obtained by repeating the same homologous fragments or a chimeric protein composed of multiple non-homologous fragments (heterologous) in a certain connection mode or order.

[0055] In some embodiments of the present invention, at least one of the proteins or its active fragments contains at least one of the modified amino acids described above or the modified amino acids prepared by the preparation method.

[0056] 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 selectively add other elements, including but not limited to the elements described above, according to actual needs.

[0057] In some embodiments of the present invention, the chimeric protein or its active fragment contains a linker L. 2 .

[0058] In some embodiments of the present invention, the connector L 2 Including but not limited to -GS-, -(GGGS) n1 -、-(PEG m1 ) n2 -、-(Gly) n1 -and-(EAAK) n3 -. Where n1 is any integer from 0 to 8, n2 is any integer from 0 to 15, n3 is any integer from 0 to 3, and m1 is any integer from 1 to 24.

[0059] Of course, those skilled in the art can also use other connectors to connect multiple segments, including but not limited to the connectors described above.

[0060] In some embodiments of the present invention, the chimeric protein or its active fragment comprises:

[0061] ddfYVWWPNFpr

[0062] hrpyiah-L 2 -rvysf;

[0063] Wherein, at least one amino acid in the ddfYVWWPNFpr or rvysf fragment in the above sequence is a modified amino acid as described in the above aspects.

[0064] In some embodiments of the present invention, the rvysf fragment in the above sequence contains one, two, three, four or five of the modified amino acids described above.

[0065] 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 above.

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

[0067] A sixth aspect of the invention provides the use of the protein or its active fragment, or the chimeric protein or its active fragment, described above in at least one of the following (1)-(5):

[0068] (1) Preparation of drugs;

[0069] (2) Disease diagnosis or contrast agents;

[0070] (3) Enhance receptor-ligand binding activity;

[0071] (4) As a drug screening platform;

[0072] (5) Delivery carrier.

[0073] In this invention, the term "disease diagnostic or imaging reagent" refers to a class of reagents that can be used for imaging, tracing, qualitative and quantitative analysis of target proteins by labeling proteins or their active fragments with labeling substances such as fluorescein or radionuclides using conventional or feasible labeling or conjugation methods in the art.

[0074] In some embodiments of the present invention, the drugs described in (1) and (4) include: antitumor drugs, targeted protein binders or degraders.

[0075] In some embodiments of the present invention, the tumors include, but are not limited to, colon cancer, breast cancer, liver cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, bone cancer, head and neck cancer, glioma, melanoma, lymphoma, leukemia, etc.

[0076] In some embodiments of the present invention, the delivery carrier can cross the blood-brain barrier.

[0077] 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., obtained further based on the protein or its active fragment or the chimeric protein or its active fragment.

[0078] The beneficial effects of this invention are:

[0079] 1. This invention, for the first time, designs a new class of non-natural amino acids by introducing long and flexible side-chain amino acids into the classic ASF side chain. This design not only enhances the stability of the obtained non-natural amino acids but also increases their reaction radius, enabling them to reach amino acid residues at greater distances and improving their reactivity. Moreover, compared to other covalently modified FSY, the non-natural amino acids of this invention generally exhibit higher reaction rates and binding efficiencies.

[0080] 2. This invention also constructs various targeted binding peptides based on the design concept of this non-natural amino acid. These peptides can be used for targeted binding to target sites, and can even achieve functional blocking, internalization, and degradation of target sites through dual-target binding of chimeric peptides, thereby realizing the construction of a peptide-based targeted degradation platform (Pep-TACs). Furthermore, through the design of a series of linkers, different binding modes to different targets can be explored, optimizing the design of peptides modified with non-natural amino acids.

[0081] 3. The targeted binding platform based on modified peptides constructed in this invention can be effectively applied to drug design and screening. This platform exhibits higher affinity and stronger binding force to the target, significantly improving both the target binding rate and degradation rate. Furthermore, this invention reveals that the platform demonstrates even higher affinity and stronger target degradation ability in acidic environments, thus enabling it to effectively block target degradation in acidic environments such as the tumor microenvironment. Moreover, the resulting drug is simple to design, easy to synthesize, and low in cost.

[0082] The chimeric protein constructed based on the design concept of non-natural amino acids in this invention can effectively improve the therapeutic environment, such as degrading the immunosuppressive target PD-L1 to a greater extent, thereby reversing the immunosuppressive environment in the tumor microenvironment and enhancing anti-tumor immunity. Moreover, it has high safety and can cross the blood-brain barrier to achieve brain targeting, thus being effective for brain diseases. Attached Figure Description

[0083] Figure 1 shows the mass spectrum of Fmoc-k-ASF-OtBu.

[0084] Figures 2A-2C show the comparison of the covalent reaction rates of ASF and k-ASF.

[0085] Figure 3 shows the sequence design of covalently modified peptides.

[0086] Figure 4 shows the Western blot (WB) results of the covalent binding of f3ASF to PD-1.

[0087] Figure 5 shows the design process of the dual-target chimeric peptide.

[0088] Figures 6A and 6B show representative flow cytometry images and data comparison results of peptide blocking PD-1 / PD-L1 in humans and mice in the experimental group.

[0089] Figures 7A-7C show the effects of the experimental group peptides on the expression of PD-L1 and TFRC on the surface of B16 and MC38 cells. Figures 7A-7B show the results of flow cytometry, and Figure 7C shows the results of Western blot analysis.

[0090] Figures 8A and 8B show the flow cytometry results of the effect of the experimental group peptides on the expression of PD-L1 on the surface of B16 and MC38 cells under pH 6.0 conditions.

[0091] Figure 9 is a flowchart of the in vivo antitumor experiment of covalently modified drugs.

[0092] Figures 10A and 10B show the changes in tumor volume and blood routine test results of mice in each group.

[0093] Figure 11 shows the experimental procedure for administering covalently modified drugs to the brain in an antitumor experiment.

[0094] Figures 12A and 12B show the survival status of mice in each group and IVIS images of brain tumors. Detailed Implementation

[0095] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0096] In this invention, the terms "amino acid" and "amino acid residue" include naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids (e.g., the α-carbon bound to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds whose structure differs from that of ordinary amino acids, but which function similarly to naturally occurring amino acids.

[0097] In this invention, the synthesis of peptide chains is not limited, and includes, but is not limited to, chemical synthesis and biosynthesis. Examples of chemical synthesis include solid-phase synthesis (such as the Fmoc method), liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis. During synthesis, if conventional modifications are made at the C-terminus or N-terminus, the peptide chain may contain the conventionally modified groups.

[0098] The natural amino acids and their various expression forms described in this article are well known to those skilled in the art, and their specific correspondences are shown in the table below:

[0099] In this invention, when an amino acid residue is represented by a single letter, unless otherwise stated, an uppercase letter indicates that it is in the L configuration and a lowercase letter indicates that it is in the D configuration.

[0100] In this invention, the aryl fluorosulfate (ASF) used to construct non-natural amino acids is as shown in Formula II, with a relative molecular weight of 204.18.

[0101] Example 1

[0102] This embodiment provides a method for preparing a covalently non-natural amino acid—D-lysine-arylfluorosulfate (k-ASF). The synthetic route is shown below:

[0103] The specific preparation steps are as follows:

[0104] (1) Carboxyl activation: Weigh 100 mg ASF (0.5 mmol) and dissolve it in 5 mL of tetrahydrofuran (THF) in a 25 mL flask. Under ice-salt bath conditions (ice-salt mass ratio of 3:1, salt is NaCl, temperature is -18℃ to -22℃), stir with a magnetic stir bar, and then slowly add 56 μL of N-methylmorpholine (NNM, 1.2 equivalents, 0.6 mmol). Add 67 μL of isobutyl chloroformate (IBCF, 0.5 mmol). After reacting for 15 min, remove the ice-salt bath and replace it with an ice bath.

[0105] (2) Condensation reaction: Weigh 277 mg of protected hydrochloric acid D configuration Lys (Fmoc-D-Lys-OtBu·HCl, 1.2 equivalents, 0.6 mmol), dissolve it in 1 mL of N,N-dimethylformamide (DMF), add 56 μL of NMM, mix well, and slowly add it 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.

[0106] The reaction progress was monitored using thin-layer chromatography (TLC). The specific method was as follows: small amounts of ASF and Fmoc-D-Lys-OtBu·HCl were dissolved in dichloromethane (DCM) to obtain ASF and Fmoc-D-Lys-OtBu·HCl control solutions. The two control solutions and the product obtained in step (2) were spotted onto the TLC plate using a capillary glass tube. The TLC plate was then placed in a developing tank containing a DCM:methanol mixture of 10:1. After the solvent (DCM) had expanded upwards to approximately 90% of the plate length, the plate was removed, the solvent was dried, and the reaction was observed under ultraviolet light to determine if it was complete.

[0107] (3) Product extraction: After the reaction was detected by TLC, the product obtained in step (2) was subjected to rotary evaporation under reduced pressure to remove the solvent. 6 mL of ethyl acetate (EtOAc) was added. After the ethyl acetate dissolved, it was transferred to a 60 mL separatory funnel and washed with saturated NaHCO3, ultrapure water, 5% citric acid and saturated saline solution in sequence, twice.

[0108] (4) Recrystallization purification: Add the washed EtOAc solution to a rotary evaporation flask, evaporate under reduced pressure to remove the solvent, add a small amount of EtOAc to dissolve the product by sonication, and then slowly add petroleum ether (PE). Stop adding PE (about 10-20 times the volume of EtOAc) when a large amount of white precipitate is produced. Let the flask stand at 4°C overnight for recrystallization.

[0109] (5) Mass spectrometry verification: The solvent of the product obtained after overnight recrystallization was removed by rotary evaporation under reduced pressure. Then, the oily substance was dried using an oil pump to obtain product powder. The product powder was collected and the yield was calculated. A small amount of powder was taken, dissolved in methanol, and filtered through a 0.22 μm filter membrane. The correctness of Fmoc-k-ASF-OtBu (compound 3, MW:633) in the filtrate was detected by mass spectrometry.

[0110] The mass spectrum of Fmoc-k-ASF-OtBu is shown in Figure 1.

[0111] (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 min, then add a large amount (excess) of diethyl ether for precipitation. After precipitation is complete, remove the solvent by rotary evaporation under reduced pressure. Wash with diethyl ether and evaporate to dryness, repeating 2-3 times. The product obtained by rotary evaporation is dried by pumping oil to obtain product powder (final product), which is used for subsequent peptide synthesis steps. Among them, reactant 4 can be deprotected by treatment with 20% piperidine at room temperature for 10 min to obtain D-lysine-arylfluorosulfate (k-ASF, i.e., the covalent non-natural amino acid in this invention).

[0112] The covalent reaction rate of the k-ASF obtained in this example was determined. Specifically, the k-ASF obtained in this example was dissolved in a reaction solvent (MeCN:H₂O:DMSO = 1:1:1.1). Then, under shaking conditions at room temperature, 20 equivalents of N,N-diisopropylethylamine (DIPEA) 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 collected at 5 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, filtered through a 0.22 μm filter membrane, and the filtrate was analyzed by HPLC. The peak area of ​​the target product at each reaction time point was calculated to determine the covalent reaction rate. Untreated ASF was used as a control.

[0113] The results are shown in Figures 2A-2C.

[0114] It can be observed that the k-ASF obtained in this embodiment has a significantly higher covalent reaction rate compared to ASF without any treatment.

[0115] Following the same method as in this embodiment, D-tyrosine-aryl fluorosulfate and D-histidine-aryl fluorosulfate can also be obtained.

[0116] Example 2

[0117] In this embodiment, peptide construction is performed based on the k-ASF obtained in the above embodiments. In this embodiment, a PD-1 affinity peptide is used as an example to demonstrate the effect of peptides constructed using k-ASF. It should be understood that this embodiment does not limit the range of peptides that k-ASF can be used to construct; peptides that can be constructed using k-ASF include, but are not limited to, the PD-1 affinity peptide used as an example in this embodiment.

[0118] (1) Peptide synthesis:

[0119] The parent PD-1 affinity peptide, R4-Y ddf-pr Its amino acid sequence is Asp D -Asp D -Phe D -Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D In this configuration, the superscript 'D' indicates the D configuration, and the absence of a superscript indicates the L configuration. The single-letter abbreviation (uppercase letters for L configuration, lowercase letters for D configuration) is: ddfYVWWPNFpr (SEQ ID NO: 1), MW: 1808.95. R4-Y ddf-prMolecular docking and molecular dynamics simulations were performed with PD-1 protein (PDB database ID: 4ZQK) to observe R4-Y. ddf-pr The binding surface of peptides to proteins. Based on observations, amino acids adjacent to lysine, tyrosine, or histidine on the protein were selected and replaced with the k-ASF-modified amino acids described in the above examples. The resulting peptides were synthesized using the standard solid-phase Fmoc synthesis method. The peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC), and their relative molecular weight (MW) was verified using electrospray ionization mass spectrometry (ESI-MS). The synthesized k-ASF-modified peptide was named f3ASF, and its amino acid sequence is Asp. D -Asp D -x-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D The corresponding sequence information is shown in Figure 3.

[0120] Meanwhile, CPBP-12, a covalently modified polypeptide with amino acid substitutions of isotope arylsulfonyl fluoride (FSY), i.e., fluorosulfate-modified tyrosine Tyr(O-SO2F), was used as a control. Its amino acid sequence is Asp. D -Asp D -Phe D -Tyr(O-SO2F)-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D The preparation of fluorosulfate-modified tyrosine in CPBP-12 was described in Wang N, Yang B, Fu C, et al. Genetically Encoding Fluorosulfate-l-tyrosine To React with 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 synthesized using the standard solid-phase Fmoc synthesis method.

[0121] (2) Covalent labeling: Equal amounts of k3ASF and CPBP-12 were labeled with biotin and then co-incubated with equal amounts of hPD-1 protein (purchased from Sino Biological). SDS-PAGE gel electrophoresis was then performed. After protein transfer, the protein was co-incubated with HRP-labeled Streptavidin (purchased from Abcam, ab7403). Western blotting (WB) was used to detect whether the peptide and protein successfully formed a covalent bond.

[0122] The results are shown in Figure 4.

[0123] It can be observed that k3ASF modified with non-natural covalent amino acid k-ASF can covalently bind to PD-1 protein and has a stronger binding force than other covalent modifications.

[0124] Furthermore, the inventors also discovered that, in the case of R4-Y ddf-pr The enhanced binding affinity can also be achieved by substituting at least one or two amino acids at positions 3, 5, and 7 (e.g., substitution at position 3, position 5, position 7, or substitution at positions 5 and 7) using the aforementioned FSY modification. This indicates that the binding affinity will be stronger after k-ASF modification at the above sites.

[0125] Example 3

[0126] In this embodiment, the use of the non-natural covalent amino acid modifications and the resulting peptides from the above embodiments as targeted blocking drugs is provided, and their use in drug screening is also provided.

[0127] In this embodiment, the covalently modified polypeptide drug used for example is a dual-targeting chimeric peptide composed of a lysozyme endocytosis receptor-targeting peptide and a protein of interest (POI)-targeting peptide. This chimeric peptide can target and bind to both the lysozyme endocytosis receptor and the POI protein, thereby simultaneously blocking the binding of the lysozyme endocytosis receptor to its endogenous ligand and the POI protein receptor and ligand. In addition to its blocking function, this invention also reveals that the chimeric peptide has a targeted degradation function; that is, after the lysozyme endocytosis receptor-targeting peptide binds to the lysozyme endocytosis receptor, it draws the ternary complex formed by the lysozyme endocytosis receptor-chimeric peptide-POI protein into the lysosome for degradation based on the endocytosis action of the lysozyme endocytosis receptor.

[0128] In this embodiment, the lysozyme endocytosis receptor is the transferrin receptor (TFRC), and the POI protein is the programmed death ligand 1 (PD-L1), an immune checkpoint molecule. The lysozyme endocytosis receptor targeting peptide is the TFRC targeting peptide. D T7 (amino acid sequence: hrpyiah (SEQ ID NO: 2)) has all its amino acid residues in the D configuration. The specific POI protein targeting peptide selected is the PD-L1 targeting peptide OPBP1 (8-12), with the amino acid sequence: rvysf (SEQ ID NO: 3), also having all its amino acid residues in the 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. D After T7 ligation, various dual-targeting chimeric peptides with different flexibility and length were obtained. D The T7-linker-OPBP1 uses the following connectors: -GS- and -(GGGS). n1 -、-(PEG m1 ) n2 - where n1 is any integer from 0 to 8, n2 is any integer from 0 to 15, and m1 is any integer from 1 to 24.

[0129] Table 1 Dual-targeting chimeric peptides D T7-linker(L 2 )-OPBP1(8-12)

[0130] The following verification was performed using Pep-1 as an example. In Pep-1, the f, s, y, and v amino acids were replaced at substitution sites, and the resulting sequences were named f12x (hrpyiah-rvysx, MW:1713), s11x (hrpyiah-rvyxf, MW:1773), y10x (hrpyiah-rvxsf, MW:1697), and v9x (hrpyiah-rxysf, MW:1761), respectively. In this embodiment, the peptides were synthesized using the standard solid-phase Fmoc synthesis method. The synthesized peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC), and their relative molecular masses were verified using electrospray ionization mass spectrometry (ESI-MS). The specific process is shown in Figure 5.

[0131] Peptide blocking: Pep-1 and its variants were diluted to 100 μM with PBS at pH 7.4. The peptides were then incubated with 50 ng of hPD-L1-Fc (purchased from Sino Biological) or mPD-L1-Fc (purchased from Sino Biological) at 37°C for 2 or 6 hours. CHO-K1 cells (Chinese hamster ovary cells K1) overexpressing PD-1 (CHO-K1-PD-1) were collected, and the above peptide and protein mixture was added to the CHO-K1-PD-1 cells and incubated on ice for 30 min. After adding PBS, the cells were centrifuged at 3000 rpm for 5 min to remove unbound protein. Then, fluorescently labeled anti-Fc antibody (eBioscience, USA) was added, and the cells were stained on ice for 30 min. After adding PBS, the cells were centrifuged at 3000 rpm for 5 min to remove unbound Fc antibody. The mean fluorescence intensity of the cells was measured using flow cytometry. Cells without Fc-tagged protein and peptide, but only incubated with fluorescently labeled Fc antibody, were used as the negative control group. Cells without peptide, but only incubated with Fc-tagged protein and fluorescently labeled Fc antibody, were used as the positive control group. The blocking rate of the above peptides was calculated using the following formula.

[0132] The results are shown in Figures 6A and 6B.

[0133] It can be observed that f12x and v9x modified with k-ASF according to the embodiments of the present invention have significantly improved blocking rates, with v9x exhibiting the highest blocking rate. The blocking rate also increases significantly over time.

[0134] Further testing was conducted to determine the target degradation effect of the aforementioned peptides:

[0135] The above-mentioned peptides (Pep-1 and its variants) were incubated at 25 μM for 2 × 10⁻⁶ days. 5 B16 and MC38 cells / mL were incubated for 0h, 2h, 6h, 24h, or 48h. The pH was maintained at 7.4 or 6.0. After incubation, cells were collected and washed with PBS by centrifugation. Cells were stained at 4°C for 30 min with anti-mouse PD-L1PE (clone: ​​MIH5, eBioscience), anti-mouse TFRC APC (clone: ​​R17217, eBioscience), or isotype control antibodies. Fluorescence intensity was assessed by flow cytometry.

[0136] Simultaneously, PD-L1 expression in cells incubated with 25 μM v9x for 24 h was detected by Western blotting. The specific method was as follows: After cell collection, cells were lysed using Radioimmunoprecipitation Assay (RIPA) buffer (Thermo Scientific), and then the protein lysates were continuously purified by 10% SDS-PAGE and a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After blocking with 5% skim milk (room temperature 1 h), the membrane was incubated overnight at 4 °C with primary antibodies against mPD-L1 (D363307, BBI) and GAPDH (D110016, BBI). Subsequently, the membrane was treated with a secondary antibody conjugated with horseradish peroxidase (HRP). Enhanced chemiluminescence reagent (ECL, GBCBIO, China) was added, and protein bands were imaged.

[0137] Furthermore, flow cytometry was used to detect changes in PD-L1 expression in cells under acidic pH 6.0 conditions.

[0138] The PBS-treated group served as a negative control. Unmodified PBS was also tested. D T7's target degradation effect.

[0139] The results are shown in Figures 7A-7C and 8A-B.

[0140] The results show that the covalently modified drug obtained based on the embodiments of the present invention can significantly degrade the PD-L1 target with a degradation efficiency of over 80%. Moreover, the covalent binding force is stronger under acidic conditions, which can further reduce the expression of PD-L1.

[0141] Example 4

[0142] In this embodiment, a mouse subcutaneous tumor model was constructed to test the in vivo antitumor effect of the above-mentioned covalently modified drug. The specific experimental steps are as follows:

[0143] Construction of a mouse subcutaneous tumor model: C57BL / 6 mice were used as experimental mice. 2×10⁻⁶ tumor cells were subcutaneously injected into the right side of each C57BL / 6 mouse. 6 MC38 cells. Monitor tumor volume; when the tumor volume reaches 40-70 mm. 3 Subsequent drug administration was then carried out. The long diameter (a), wide diameter (b), and high diameter (c) of the tumor were measured using digital calipers, and the results were analyzed using the formula... Calculate the volume of the tumor.

[0144] According to the grouping, MC38 tumor-bearing mice were intraperitoneally injected with physiological saline, OPBP1 (8-12) (0.5 mg / kg), and... DAn equimolar mixture of T7 and OPBP1(8-12) (1.3 mg / kg), Pep-1 (1.3 mg / kg), and equimolar concentrations of low-dose (1.3 mg / kg) and high-dose (3.9 mg / kg) v9x at 0.75 μmol / kg, and 10 mg / kg anti-PD-L1 (clone: ​​10F.9G2, Bioxcell) as a positive control were administered. Except for the PD-L1 antibody, all other groups were administered once daily for 2 weeks. The PD-L1 antibody was administered once every 3 days for 2 weeks. Tumor volume changes in each group of mice were continuously monitored. After the experiment, blood was collected from each group of mice, and the levels of alanine aminotransferase, aspartate aminotransferase, and creatinine were measured using standard methods in the art.

[0145] The flowchart is shown in Figure 9.

[0146] The results are shown in Figures 10A and 10B.

[0147] It was found that, compared with other groups, v9x based on k-ASF covalent modification significantly inhibited tumor growth in mice. Furthermore, after blood routine tests on mice following drug administration, the levels of alanine aminotransferase (ALT, normal range 10.06–96.47 U / L), aspartate aminotransferase (AST, normal range 36.31–235.48 U / L), and creatinine (CR, normal range 10.91–85.09 μmol / L) in the covalently modified drug group were all within the normal range, and there were no significant differences between the groups, indicating that the covalently modified drug obtained based on the embodiments of this invention has good safety.

[0148] Example 5

[0149] In this embodiment, it was verified that the covalently modified drug (covalently modified peptide) obtained in the embodiments of the present invention can be administered to the brain across the blood-brain barrier.

[0150] Construction of a mouse brain tumor model: C57BL / 6 mice were used as experimental mice. After anesthetizing, C57BL / 6 mice were fixed on a stereotaxic apparatus, and GL261-Luc cells (luciferase-labeled mouse glioma cells, 1×10⁻⁶) were injected into the right ventricle of the mouse at a depth of 3 mm using a 10 μL Hamilton syringe. 5 (Cells / 5 μL PBS). Successful model construction can be confirmed by detecting bioluminescence in tumor cells expressing luciferase using an in vivo imaging system.

[0151] The tumor-bearing mice were randomly assigned to groups. According to the group, the mice were intraperitoneally injected with saline, Pep-1 (3.4 mg / kg), V9x (3.9 mg / kg), or anti-PD-L1 (10 mg / kg), respectively. The administration frequency was the same as in the previous example. In situ tumor chemiluminescence was measured weekly using IVIS after successful modeling to determine the therapeutic effect.

[0152] The flowchart is shown in Figure 11.

[0153] The results are shown in Figures 12A and 12B.

[0154] It can be observed that, compared with other groups, v9x based on k-ASF covalent modification can cross the blood-brain barrier to achieve brain targeting, inhibit brain tumor growth, and prolong the survival of mice.

[0155] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A modified amino acid, characterized in that, The modified amino acids are selected from modified lysine, tyrosine, histidine, arginine, serine, cysteine, glutamine, threonine, and asparagine. The modification is aryl fluorosulfate modification; preferably, in the modified amino acid, the amino acid and the aryl fluorosulfate are also connected to 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 groups, amides, imines, and thioesters; n is any integer from 0 to 4; R 1 Including alkoxycarbonyl, acyl, and alkyl groups; R 2 This includes substituted or unsubstituted methyl esters, substituted or unsubstituted ethyl esters, tert-butyl esters, and benzyl esters.

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

4. The use of the modified amino acid as described in claim 1 or 2, or the modified amino acid prepared by the preparation method described in 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 as described in claim 1 or 2, or a modified amino acid prepared by the preparation method as described in claim 3.

6. A chimeric protein or its active fragment, characterized in that, The chimeric protein or its active fragment includes 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 as described in claim 1 or 2, or a modified amino acid prepared by the preparation method as described in claim 3.

7. The chimeric protein or its active fragment 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 connector L 2 Including -GS- and -(GGGS) n1 -、-(PEG m1 ) n2 -、-(Gly) n1 -and-(EAAK) n3 -; Where n1 is any integer from 0 to 8, n2 is any integer from 0 to 15, n3 is any integer from 0 to 3, and m1 is any integer from 1 to 24.

8. The chimeric protein or its active fragment according to claim 7, characterized in that, The chimeric protein or its active fragment includes: 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 a modified amino acid as described in claim 1 or 2, and at least one amino acid among f, s, y or v in the rvysf fragment is a modified amino acid as described in claim 1 or 2.

9. The use of the protein of claim 5 or its active fragment, or the chimeric protein of any one of claims 6-8 or its active fragment, in at least one of the following (1)-(5): (1) Preparation of drugs; (2) Disease diagnosis or contrast agents; (3) Enhance receptor-ligand binding activity; (4) As a drug screening platform; (5) Delivery carrier.

10. The application according to claim 9, characterized in that, The drugs mentioned in (1) and (4) include: antitumor drugs, targeted protein binding agents or degrading agents.

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