A structurally diverse library of polypeptides and methods of construction and use thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a polypeptide library with diverse structures, a construction method and application thereof. Each polypeptide in the polypeptide library has a general formula shown in formula I: Acp-K-X1C1X2X3X4C2X5X6X7C3X8X9X 10 C4E (I); wherein, X1-X 10 are any amino acid residues which can be randomized; K is lysine; C1-C4 are cysteine; and E is glutamic acid. Through solid-phase synthesis technology under mild conditions, the polypeptide intramolecular disulfide bond and amide bond pairing are precisely controlled, and rapid cyclization is performed, so that a polypeptide library with diverse structures is constructed. The synthesized linear peptides, cyclic peptides and polycyclic peptides are subjected to high-throughput screening against different cell models, and a target cell-penetrating peptide with selectivity for penetrating cell membranes is obtained, which provides a solid foundation for active polypeptide molecule discovery and development of polypeptide innovative drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a structurally diverse polypeptide library, its construction method, and its applications. Background Technology

[0002] As is well known, peptide molecules combine the advantages of antibodies, small molecule drugs, and protein drugs, exhibiting affinity and precise targeting specificity similar to antibodies. Simultaneously, their small molecular weight (0.3–6 kilodaltons (Kda) allows them to rapidly and deeply penetrate tissues, enabling them to target lesions from within tissues and potentially avoid the immunogenicity issues that plague antibody development. Their peptide nature provides "tunable" pharmacokinetic half-life and renal clearance pathways, thus avoiding the liver and gastrointestinal toxicities common in other drug formulations, making them highly promising drugs. Furthermore, the basic building blocks of peptides include 20 different natural amino acids, resulting in a diverse and varied peptide library. Combined with their excellent biocompatibility, cell / tissue penetration ability, and chemical stability, peptides possess enormous potential and are widely used in research fields such as medicine and cosmetics.

[0003] With the continuous development of biotechnology, peptide drugs are playing an increasingly important role in the treatment of human diseases. In fact, peptide drugs have already been successfully used clinically, such as oxytocin, vancomycin, cyclosporine, plecanatide, and the anticancer drug octreotide. Like other new drug development, peptide drug development first requires identifying the active molecule. There are two main methods for discovering active peptide molecules: one is to search for active peptides in vivo, but peptide isolation, purification, and identification are time-consuming and difficult. To accelerate the discovery of active peptides, reduce the drug screening cycle, and speed up the development process of peptide drugs, high-throughput drug screening technology for screening active peptides from peptide libraries has become another, and increasingly important, pathway for peptide drug discovery.

[0004] Currently, the most advanced library construction methods for bioactive peptides include display techniques and chemical synthesis. Display techniques include phage display, yeast display, mRNA display, ribosome display, and deoxyribonucleic acid (DNA) display. These establish a link between phenotype (peptide) and genotype (DNA or RNA), generating a large peptide library that can be screened for therapeutic targets. After multiple rounds of screening, high-affinity peptide lead compounds are obtained, and then medicinal chemistry strategies are used to improve the druggability of the lead compounds. For example, O'Neil of Merck Pharmaceuticals first reported in 1992 the construction of phage-displayed cyclic peptide libraries, in the form of CX6C libraries (where X represents variable amino acids, C represents cysteine, and the subscript number indicates the number of variable amino acids). After the peptides are expressed on the phage surface, the cysteine ​​residues spontaneously oxidize to form rings, thus forming a cyclic peptide library. In 1997, Klaus Mosbach's group successfully constructed a bicyclic nonapeptide library for displaying M13 phage via oxidation to form disulfide bonds and screened for bicyclic peptide inhibitors of α-chymotrypsin. Bicycle Therapeutics provides a method for constructing and screening cyclic peptide libraries for displaying phage via chemical cyclization using phage display technology. These peptides have 16 amino acid residues and contain three fixed cysteine ​​residues at positions 2, 9, and 16. The cyclization structure is formed by bonds between the cysteine ​​residues and the chemical moiety, thus only forming structurally simple bicyclic peptides (see patent WO2009098450). MORPHOSYS AG's patent application WO2015166036A1, "Peptide Libraries," discloses libraries of linear and cyclic peptides of varying lengths constructed using phage display technology.

[0005] In the chemically synthesized peptide library method discovered by Sang Hoon Joo et al., due to cyclization failure, the final cyclic peptide exhibited only a single ring. (Sang Hoon Joo et al., High Throughput Sequence Determination of Cyclic Peptide Library Members by Partial Edman Degradation / MassSpectrometry, J.Am.Chem.Soc.(2006)128,13000-13009).

[0006] The activity of a peptide is determined by both its sequence and structure. Clearly, existing peptide library construction techniques have the following drawbacks: ① Currently available peptide libraries are either all linear peptides or single-structure cyclic peptide libraries; ② When multiple pairs of bonded amino acid residues exist in the peptide sequence, spontaneous cyclization using phage display technology is difficult to control due to the lack of directionality in amino acid bond formation, making it hard to form a structurally defined target product; ③ Existing technologies have not yet disclosed structural peptide libraries integrating multiple structures. The structure of a peptide has a significant impact on its biological activity. Structural diversity allows for the screening of peptide libraries that yield more peptides with higher specificity, stronger binding affinity, and greater cell penetration. Therefore, constructing peptide libraries with rich structural diversity is extremely important.

[0007] In summary, to meet various research needs and screening objectives, there is an urgent need to construct a peptide library with multiple structural peptides. This would greatly enrich the library's form and screening capacity, allowing for the screening of more active peptides, expanding the scope of drug discovery and research, and providing a blueprint for the development of a wider range of peptide drugs. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a structurally diverse polypeptide library, its construction method, and its applications.

[0009] The present invention adopts the following technical solution:

[0010] A structurally diverse polypeptide library, wherein each polypeptide in the library has the general formula shown in Formula I:

[0011] Acp-K-X1C1X2X3X4C2X5X6X7C3X8X9X 10 C4E (Ⅰ);

[0012] Among them, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 It is any amino acid residue that can be randomized;

[0013] K stands for lysine;

[0014] C1, C2, C3, and C4 are cysteine;

[0015] E stands for glutamic acid.

[0016] The bonding modes between amino acid residues of peptides in the peptide library include: forming disulfide bonds between cysteine ​​residues at fixed positions, or forming amide bonds between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of the lysine side chain at the N-terminus of the peptide chain.

[0017] Furthermore, the polypeptide includes five structures: linear peptide, cyclic peptide, disulfide bond monocyclic peptide, bicyclic peptide, and tetracyclic peptide.

[0018] Furthermore, polypeptides with different structures are formed in the following ways;

[0019] 1) Linear peptide: A linear peptide with a fixed length of 17 amino acid residues;

[0020] 2) Cyclic peptides: An amide bond is formed between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain, thus forming a cyclic peptide.

[0021] 3) Disulfide bond monocyclic peptide: A disulfide bond is formed between C2 and C4 of cysteine, thus forming a disulfide bond monocyclic peptide;

[0022] 4) Bicyclic peptides are formed by the formation of disulfide bonds between cysteine ​​C1 and C3, and the formation of amide bonds between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain.

[0023] 5) Tetracyclic peptide: A disulfide bond is formed between cysteine ​​C1 and C3, a disulfide bond is formed between cysteine ​​C2 and C4, and an amide bond is formed between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain, thus forming a tetracyclic peptide.

[0024] Furthermore, the peptide library contains at least 5 × 10 20 One polypeptide member.

[0025] The method for constructing the peptide library includes the following steps:

[0026] S1. Using a fully automated microwave peptide synthesizer, the Fmoc solid-phase synthesis method was adopted. Wang resin, a solid-phase carrier, different amino acids, removal agents and peptide condensation reagents were added sequentially to synthesize the target peptide in a fully automated manner, resulting in a linear crude peptide of 17 amino acids.

[0027] S2. Remove the protecting groups of amino acid residues at the reaction sites from the linear crude peptide obtained in S1.

[0028] S3. Cyclic peptides are prepared by cyclizing the linear peptides obtained in S2 using chemical methods.

[0029] S4. Shrink dry the linear and cyclic peptides obtained in S1 and S2 and cleave the polypeptide resin.

[0030] S5. After separation HPLC purification and freeze drying, weigh and dispense into storage.

[0031] The application of the peptide library in screening for targeted membrane-penetrating peptides includes the following steps:

[0032] a. Culture cells in a system containing peptides from the aforementioned peptide library;

[0033] b. Remove peptides that do not have targeted membrane-penetrating ability; and

[0034] c. Recover peptides with targeted membrane-penetrating capabilities.

[0035] The present invention has the following beneficial effects:

[0036] 1. By using solid-phase synthesis and different side-chain protecting groups, precise pairing of intramolecular disulfide bonds and amide bonds can be achieved, resulting in target products with high purity and well-defined structures.

[0037] 2. The constructed peptide libraries have diverse structures and large capacity, which have the advantage of high-throughput screening and can greatly shorten the time required for screening and validating active peptide molecules.

[0038] 3. The constructed peptide library has high structural diversity, which can screen out more peptide active molecules with better performance.

[0039] 4. The construction of structurally diverse peptide libraries has promoted the development of peptide library construction technology. Attached Figure Description

[0040] Figure 1 The diagram shows five structural representations of peptides in the peptide library: A is the linear peptide structure of LP, B is the cyclic peptide structure of CP, C is the monocyclic peptide structure of MP with disulfide bonds, D is the bicyclic peptide structure of BP, and E is the tetracyclic peptide structure of TP.

[0041] Figure 2 HPLC chromatograms of LP-10, CP-10, MP-10, BP-10, and TP-10 peptides;

[0042] Figure 3 MS spectra of LP-10, CP-10, MP-10, BP-10, and TP-10 protamines;

[0043] Figure 4 In the diagram, A is the fluorescence localization map of peptide LP-10 in cells (×200); B is the fluorescence localization map of peptide MP-10 in cells (×200); and C is the fluorescence localization map of peptide TP-10 in cells (×200). Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1: Solid-phase synthesis and purification of linear peptide LP-10

[0046] A linear peptide, LP-10, was synthesized using a microwave peptide synthesizer and the standard (fluorenemethyloxycarbonyl) Fmoc technique. The method includes the following steps:

[0047] 1) Weigh 0.1 mmol of Fmoc-Glu(OALL)-Wang resin into a solid-phase reactor, add 5 ml of DCM (dichloromethane), swell the resin for 30 min, and after the resin has fully swelled, vacuum dry the solvent.

[0048] 2) Place the Fmoc-Glu(OALL)-Wang resin obtained in step 1) into a microwave peptide synthesizer, add a 25% piperidine / DMF mixture with a volume ratio of 1:5, remove the Fmoc protecting group under microwave action, wash the resin three times with 4 ml DMF solution, and vacuum dry the solvent.

[0049] 3) Add 0.5 ml of condensing agent DIC solution, 2 ml of Oxyma solution and 1 ml of Fmoc-Cys(Mmt)-OH solution, and obtain Fmoc-Cys(Trt)-Glu(OALL)-Wang resin under microwave action.

[0050] 4) Repeat steps 2) and 3) sequentially, replacing the raw material amino acids sequentially with Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe(Boc)-OH, Fmoc-Phe(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Mtt)-OH, and Fmoc-ε-Acp-OH, finally obtaining a resin coupled sequentially with the above amino acids; the resulting sequence is: Acp-KRCFFWCKKKCPIQCE (SEQ ID Linear peptide (NO.1).

[0051] 5) Remove the side-chain protecting group OALL from the first amino acid Glu at the C-terminus.

[0052] The synthesized resin was swollen in 7 ml of freshly opened DCM (10 ml / g resin) for 30 min.

[0053] Then add 370 ml of benzyl silane, add a magnetic stirrer, dissolve 34.7 g of tetrakis(triphenylphosphine)palladium in 8 ml of DCM, and add it dropwise to the resin. React for 15–20 min. After the reaction is complete, wash with 5 ml of sodium diethyldithiocarbamate (trihydrate) for 15 min, then wash three times with 5 ml of DMF for 3 min each time. The dosage is calculated as follows: (n is 0.1 mmol)

[0054] V(phenylsilane) = n * 108.22 * 30eq / 0.877

[0055] m(tetraphenylphosphinepalladium) = n * 1155.56 * 0.3 eq

[0056] 6) Remove the protecting group Fmoc from the first amino acid Acp at the N-terminus.

[0057] Remove the solution twice with 5 ml of 20% piperidine / DMF solution, for 5 min the first time and 15 min the second time, and then wash five times with 5 ml of DMF.

[0058] 7) Kaiser method detection

[0059] The protective group in step 6) above is completely removed by indole testing; if the resin turns blue, it indicates that the Fmoc protective group has been completely eluted, and then proceed to the next step.

[0060] 8) Fluorescence Coupling

[0061] Weigh out the appropriate amounts of the fluorescent substance 5-FAM and the condensing agent HATU, dissolve them completely in 3 ml of DMF, then add the condensing agent DIEA. Add the reaction solution to the resin and react for at least 4 hours. After the reaction, wash three times with 5 ml of DMF, 3 minutes each time. The completeness of the fluorescence coupling reaction is detected by indole detection. The solvent volume is calculated as follows: (n = 0.1 mmol)

[0062] m(5-FAM)=n*376*6eq

[0063] v(HATU) = n * 380.24 * 6eq * 0.95eq

[0064] V(DIEA)=n*129.24*6eq*1.5eq / o.782

[0065] 9) Shrink drying

[0066] Wash twice with 5ml DCM, once with 5ml methanol, once with 5ml DCM, and twice with 5ml methanol in alternating order, for 3 minutes each time. After shrinking and drying, promptly place the product in a vacuum drying oven to remove the solvent.

[0067] 10) Peptide resin cleavage

[0068] ① Prepare 8 ml of lysis buffer (the ratio of lysis buffer is: TFA:H2O:methyl phenyl sulfide:phenol:DODT = 33:2:2:2:1), cool to 0℃±1℃, and set aside for use; weigh 0.8 g of peptide resin, place it in the peptide lysis vessel, add the lysis buffer that has been pre-cooled to 0℃, start the lysis reaction program, and react at room temperature for 3 h; prepare methyl tert-butyl ether solution, cool to -10℃, and set aside for use.

[0069] ② Add the reaction solution to 80 ml of methyl tert-butyl ether solution. A yellow, sludge-like solid will appear. Turn on the centrifuge at 4500 rpm for 3 minutes. Centrifuge the precipitate, discard the supernatant into a waste container, and collect the yellow, sludge-like solid. Then add fresh methyl tert-butyl ether solution to the yellow, sludge-like solid and centrifuge again. Repeat the above process twice more, i.e., washing and centrifuging. After the last washing and centrifugation, collect the yellow, sludge-like substance into a centrifuge tube and vacuum dry it at 20°C to constant weight. After the yellow, sludge-like substance is completely dry, weigh it and place it in a low-density polyethylene bag for further purification.

[0070] 11) The product obtained in step 10) is purified and freeze-dried to obtain the linear polypeptide LP-10. The polypeptide contains the following amino acid sequence: [5-FAM]-(Acp-KRCFFWCKKKCPIQCE) (hereinafter referred to as LP-10).

[0071] Experimental Results: The crude peptide was purified by HPLC and lyophilized to obtain LP-10 linear peptide. Spectral data obtained by HPLC and mass spectrometry are shown below. Figure 2 and Figure 3 As shown, the purity of LP-10 linear peptide is 98.48%, and the molecular weight M+H obtained by mass spectrometry is 2518.096 (the theoretical molecular weight is 2517.142).

[0072] 12) Peptide purity detection and quality control:

[0073] ① Quality control during the linear crude peptide synthesis process employs the sensitive Kaiser assay to detect free amino groups on the resin. This is the primary method for determining the complete elution of the Fmoc protecting group, the incompleteness of the fluorescence coupling reaction, and the completeness of the condensation reaction. Specifically: A small amount of thoroughly washed and dried resin is placed in a small glass test tube. Indene test reagents are added sequentially: 1 drop of 5% ninhydrin anhydrous ethanol solution (w / v), 1 drop of freshly distilled phenol anhydrous ethanol solution (4:1, w / v), and 2 drops of pyridine. The mixture is heated to boiling for 3 minutes, and the colors of the resin and solution are observed. A blue or light red resin and a blue or brown solution indicate exposed amino groups; a colorless resin and a light yellow solution indicate no exposed amino groups, thus indicating whether the reaction is complete.

[0074] ② HPLC-MS was used for purity determination and quality control of the LP-10 linear peptide, as follows: After drying the obtained crude peptide under reduced pressure, the purity of the LP-10 linear peptide was detected by HPLC and confirmed by MS. Detection conditions: The chromatogram used octadecylsilane-bonded silica gel as the stationary phase, with 0.1% TFA and 80% acetonitrile aqueous solution as mobile phase A, and 0.1% TFA aqueous solution as mobile phase B, using gradient elution; wavelengths were 220 nm and 254 nm.

[0075] ③ High-performance liquid chromatography (HPLC) was used to perform quality control during sample purification using gradient elution. Analytical conditions: Mobile phase 1: 0.1% TFA 80% acetonitrile / water; Mobile phase 2: 0.1% TFA water; Flow rate: 10 ml / min; Detection wavelengths: 220 nm, 254 nm.

[0076] Column temperature: 35℃.

[0077] Example 2: Solid-phase synthesis and purification of the proto- and posterior cyclic peptide CP-10

[0078] 1) The resin peptide obtained according to steps 1)-5) in Example 1 shall be used for the next step;

[0079] 2) Remove the Mtt side-chain protecting group from the second N-terminal amino acid Lys;

[0080] First, wash the conversion system 5 times with 5 ml of DCM, 3 min each time; then wash with 5 ml of the prepared 0.8% TFA and 5% TIS / DCM reaction solution to remove Mtt, 5 times; wash with 5 ml of DMF 4 times; and wash with 5 ml of 0.25% DIEA / DMF solution once (volume ratio), for 3 min each time; check whether the removal is complete by indene test.

[0081] 3) Looping of the beginning and end loops

[0082] Weigh an appropriate amount of PyBop, dissolve it in 3 ml of a mixed solution of DMF and DCM (volume ratio: DMF:DCM = 1:1), add DIEA, then add the reaction solution to the resin and react overnight. Wash three times with 5 ml of DMF, and check whether the first and last ring condensation of the side chains is complete using an indole test. The solvent volume is calculated as follows: (n is 0.1 mmol).

[0083] m(PyBop) = n * 520 * 4eq

[0084] v(DIEA) = n * 129.24 * 8eq / 0.782

[0085] 4) Repeat steps 6)-12) in Example 1 to obtain the CP-10 cyclic peptide, which contains the following amino acid sequence: [5-FAM]-(Acp-KRCFFWCKKKCPIQCE) (hereinafter referred to as CP-10).

[0086] Experimental Results: The crude peptide was purified by HPLC and lyophilized to obtain CP-10 peptide. Spectral data obtained by HPLC and mass spectrometry are shown below. Figure 2 and Figure 3 As shown, the purity of CP-10 peptide is 99.83%, and the molecular weight M+H obtained by mass spectrometry is 2500.0788 (the theoretical molecular weight is 2499.1036).

[0087] Example 3: Solid-phase synthesis and purification of disulfide bond monocyclic peptide MP-10

[0088] 1) The resin peptide obtained according to steps 1)-5) in Example 1 shall be used for the next step;

[0089] 2) Remove C2 and C4 side chain protecting groups Mmt

[0090] First, wash the conversion system three times with 5 ml of DCM, 3 min each time; then wash with 5 ml of the prepared 0.8% TFA and 5% TIS / DCM reaction solution to remove Mmt, elute 5 times, then wash with 5 ml of DMF 4 times, and then wash with 5 ml of 0.25% DIEA / DMF solution once, for 3 min each time; check whether the removal is complete by indole detection.

[0091] 3) C2 and C4 condense to form a disulfide ring.

[0092] Weigh an appropriate amount of N-chlorosuccinimide (NCS), dissolve it in DMF, add it to the resin for cyclization, react for 15 min, and repeat the above cyclization step; then wash 5 times with 5 ml of DMF; the dosage is calculated as follows:

[0093] m(NCS) = n * 133.53 * 2eq (n is 0.1 mmol)

[0094] 4) Repeat steps 6)-12) in Example 1 to obtain the MP-10 cyclic peptide, which contains the following amino acid sequence: [5-FAM]-(Acp-KRCFFWCKKKCPIQCE) (here referred to as MP-10).

[0095] Experimental Results: The crude peptide was purified by HPLC and lyophilized to obtain MP-10 peptide. Spectral data obtained by HPLC and mass spectrometry are shown below. Figure 2 and Figure 3As shown, the purity of MP-10 peptide is 94.065%, and the molecular weight M+H obtained by mass spectrometry is 2516.0719 (the theoretical molecular weight is 2515.0986).

[0096] Example 4: Solid-phase synthesis and purification of bicyclic peptide BP-10

[0097] 1) The resin peptide obtained according to steps 1)-5) in Example 1 shall be used for the next step;

[0098] 2) Remove the C1 and C3 side chain protecting groups Mmt and the Lys side chain protecting group Mtt

[0099] First, wash the conversion system three times with 5 ml of DCM, 3 min each time; then wash with 5 ml of the prepared 0.8% TFA and 5% TIS / DCM reaction solution to remove Mtt, 5 times, then wash with 5 ml of DMF four times, and then wash with 5 ml of 0.25% DIEA / DMF solution once, for 3 min each time; check whether the removal is complete by indole detection.

[0100] 3) C1 and C3 cyclization into disulfide rings

[0101] Weigh an appropriate amount of N-chlorosuccinimide (NCS), dissolve it in DMF, add it to the resin for cyclization, react for 15 min, and repeat the above cyclization steps; then wash five times with 5 ml of DMF each time; the dosage is calculated as follows: (n is 0.1 mmol)

[0102] m(NCS) = n * 133.53 * 2eq

[0103] 4) Cycling of the head and tail rings in the solid phase

[0104] Weigh an appropriate amount of PyBop, dissolve it in 3 ml of a mixed solution of DMF and DCM (volume ratio DMF:DCM = 1:1), add DIEA, then add the reaction solution to the resin and react overnight. Wash three times with 5 ml of DMF each time, and check the indole test to see if the first and last ring condensation is complete. The dosage is calculated as follows: (n is 0.1 mmol).

[0105] m(PyBop) = n * 520 * 3eq

[0106] v(DIEA) = n * 129.24 * 6eq / 0.782

[0107] 5) Repeat steps 6)-12) in Example 1 to obtain the BP-10 cyclic peptide, which contains the following amino acid sequence: [5-FAM]-(Acp-KRCFFWCKKKCPIQCE) (hereinafter referred to as BP-10).

[0108] Experimental Results: The crude peptide was purified by HPLC and lyophilized to obtain BP-10 peptide. Spectral data obtained by HPLC and mass spectrometry are shown below. Figure 2 and 3 As shown, the purity of BP-10 peptide is 97.275%, and the molecular weight M+H obtained by mass spectrometry is 2498.0670 (the theoretical molecular weight is 2497.0880).

[0109] Example 5: Solid-phase synthesis and purification of tetracyclic peptide TP-10

[0110] 1) The resin peptide obtained according to steps 1)-5) in Example 1 shall be used for the next step;

[0111] 2) Remove the C2 and C4 side chain protecting groups Mmt and the Lys side chain protecting group Mtt

[0112] First, wash the conversion system three times with 5 ml of DCM, 3 min each time; then wash with 5 ml of the prepared 0.8% TFA and 5% TIS / DCM reaction solution to remove Mtt, eluting five times for 15 min, 15 min, 20 min, 20 min, and 20 min respectively. Then wash with 5 ml of DMF four times and 5 ml of 0.25% DIEA / DMF solution once for 3 min. The complete removal is then checked by indole detection.

[0113] 3) C2 and C4 form a disulfide ring.

[0114] Weigh an appropriate amount of N-chlorosuccinimide (NCS), dissolve it in DMF, add it to the resin for cyclization, react for 15 min, and repeat the above cyclization step; then wash five times with 5 ml of DMF each time; the dosage is calculated as follows:

[0115] m(NCS) = n * 133.53 * 2eq (n is 0.1 mmol)

[0116] 4) Solid-phase iodine oxidation of C1 and C3 to form disulfide rings

[0117] Weigh an appropriate amount of I2, dissolve it in DMF, add the dilute I2 solution to the resin obtained above, react for 4 hours, wash twice with 5 ml L-ascorbic acid solution for 5 min and 3 min respectively, and wash twice with 5 ml DMF for 3 min each time; the dosage is calculated as follows:

[0118] m(I2) = n * 253 * 10 eq (n is 0.1 mmol)

[0119] 5) Cycling of the head and tail rings in the solid phase

[0120] Weigh an appropriate amount of PyBop, dissolve it in 3 ml of a mixed solution of DMF and DCM (volume ratio DMF:DCM = 1:1), add DIEA, then add the reaction solution to the resin and react overnight. Wash three times with 5 ml of DMF each time, and check the indole test to see if the first and last ring condensation is complete. The solvent volume is calculated as follows: (n is 0.1 mmol).

[0121] m(PyBop) = n * 520 * 3eq

[0122] v(DIEA) = n * 129.24 * 6eq / 0.782

[0123] 6) Repeat steps 6)-12) in Example 1 to obtain the TP-10 cyclic peptide, which contains the following amino acid sequence: [5-FAM]-(Acp-KRCFFWCKKKCPIQCE) (hereinafter referred to as TP-10).

[0124] Experimental Results: The crude peptide was purified by HPLC and lyophilized to obtain TP-10 peptide. Spectral data obtained by HPLC and mass spectrometry are shown below. Figure 2 and Figure 3 As shown, the purity of TP-10 peptide is 96.064%, and the molecular weight obtained by mass spectrometry is 2496.0472 (the theoretical molecular weight is 2495.0723).

[0125] Example 6 Cell permeabilization experiment of the polypeptides LP-10, CP-10, MP-10, BP-10, and TP-10 prepared in the above examples.

[0126] 1. Introduction to Experimental Methods

[0127] 1.1 Reagents and Instruments

[0128] (1) Reagents:

[0129] 1) 96-well cell culture plates (purchased from Corning);

[0130] 2) RPMI 1640 medium, DMEM medium and MEM medium (purchased from Gibco);

[0131] 3) FBS, dual antibodies (purchased from Gibco);

[0132] (2) Instruments:

[0133] 1) 10μL pipettes, 20μL pipettes, 200μL pipettes, 1mL pipettes, 100μL multipipes (purchased from Eppendoff).

[0134] 2) 10μL Pipette Tip (purchased from QSP), 200μL Pipette Tip (purchased from Axygen), 1mL Pipette Tip (purchased from Axygen)

[0135] 3) 1.5ml, 15ml, and 50ml EP tubes (purchased from Axygen)

[0136] 4) Genespeed centrifuge (purchased from Gene Company)

[0137] 5) Vortex-Genie2 vortex mixer (purchased from Scientific Industries)

[0138] 6) Leica THUNDER Imager Fluorescence Microscope

[0139] 7) CO2 cell incubator (Esco)

[0140] 8) Biosafety cabinet (ESCO)

[0141] 1.2 Selected cell lines:

[0142] HCT-116 (colorectal cancer), NCI-H226 (lung squamous cell carcinoma), AGS (gastric cancer cells), A2780 (human ovarian cancer cells), MDA-MB-231 (breast cancer cells), U251 (human glioblastoma cells), A172 (human glioblastoma cells), 293T (human embryonic kidney cells), HL-60 (human promyelocytic leukemia cell line), SK-MEL-2 (melanoma cells), A375 (melanoma cells), HEK-a (human epidermal keratinocytes).

[0143] 1.3 Cell Culture Experiment

[0144] SK-MEL-2 (melanoma cells) were cultured in MEM + 20% FBS + 1% P / S complete medium; HCT-116 (colorectal cancer cells), NCI-H226 (lung squamous cell carcinoma cells), AGS (gastric cancer cells), and A2780 (human ovarian cancer cells) were cultured in RPMI medium. MDA-MB-231 (breast cancer cells), 293T (human embryonic kidney cells), A375 (melanoma cells), and HEK-a (human epidermal keratinocytes) were cultured in DMEM + 10% FBS + 1% P / S complete medium. U251 (human glioblastoma cells) and A172 (human glioblastoma cells) were cultured in DMEM (047) + 10% FBS + 1% P / S complete medium. HL-60 (human promyelocytic leukemia cell line) was cultured in IMDM + 20% FBS + 1% P / S complete medium. All cells were cultured in a 37°C, 5% CO2 incubator, and passaged when the cell density reached 80%.

[0145] 2. Cell plating

[0146] Cell counting was performed based on the cell numbers determined in previous preliminary experiments with different cell types for plate formation. The required cell numbers for different cell types are as follows:

[0147] Table 1 Cell Density

[0148]

[0149]

[0150] Dilute the cell count, calculated by the cell counter, to the appropriate concentration in the culture medium. Take 100 μL and seed it into a 96-well plate.

[0151] 3. Cell-based drug delivery

[0152] The polypeptide samples LP-10, CP-10, MP-10, BP-10, and TP-10 obtained in Examples 1, 2, 3, 4, and 5 were prepared into sample solutions with a stock solution concentration of 100 μM. The old culture medium in the 96-well plate was discarded, and 50 μL of serum-free culture medium was added to wash the cells. After discarding the washing medium, 95 μL of serum-free culture medium was added, and then 5 μL of the stock solution was added directly to the 96-well plate to ensure a final sample concentration of 5 μM. The plates were then incubated at 37°C in a 5% CO2 incubator for 4 hours.

[0153] 4. Fluorescence photography: Take the PBS and culture medium out of the 4°C freezer and let them come to room temperature in advance.

[0154] (1) Washing cells: Remove the 96 cell plate and discard the cell culture medium; wash once with PBS buffer; if it is HL-60 cells, since they are suspension cells, they need to be centrifuged (1000 rpm, 3 min) before washing the cells.

[0155] (2) Add fresh culture medium: Since the photography time is long, adding PBS will make the cells adhere poorly. Therefore, after washing, add the corresponding fresh culture medium for photography.

[0156] (3) Fluorescence imaging: The fluorescence microscope used was a Leica THUNDER Imager, and the imaging was performed under the condition of green fluorescence wavelength of 475nm.

[0157] Results of cell membrane penetration experiments for 5.5 peptide samples

[0158] The observation indicator was the presence of visible green fluorescence aggregation within cells under a fluorescence microscope. This indicated that the polypeptide sample had cell-penetrating ability and was a cell-penetrating peptide. The cell-penetrating results are shown in Table 2. Three images were taken for each sample and each cell type: a visible light image, a fluorescence image, and a merged image of the fluorescence and visible light.

[0159] Table 2. Cell membrane penetration results of five structural peptides

[0160]

[0161] Note: In the table, items marked with the symbol "+" indicate that they have a targeted membrane penetration effect; items marked with the symbol "-" indicate that they do not have a targeted membrane penetration effect.

[0162] Experimental results: The polypeptide LP-10 was able to penetrate the cell membrane of NCI-H226 cells and enter the cell, as shown in the following results. Figure 4 As shown in Figure A; MP-10 can penetrate the cell membranes of NCI-H226 and SK-MEL-2 cells and enter the cell, as shown in Figure A. Figure 4 As shown in B; TP-10 can penetrate the cell membranes of NCI-H226 and SK-MEL-2 cells and enter the cell, as shown in the results. Figure 4 As shown in Figure C. Therefore, peptides LP-10, MP-10, and TP-10 have targeted membrane-penetrating effects.

[0163] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0164] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A structurally diverse polypeptide library, characterized in that, Each polypeptide in the polypeptide library has the general formula shown in Formula I: Acp-K-X1C1X2X3X4C2X5X6X7C3X8X9X 10 C4E(Ⅰ); Among them, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 It consists of randomized arbitrary amino acid residues; K stands for lysine; C1, C2, C3, and C4 are cysteine; E stands for glutamic acid; The bonding modes between amino acid residues of peptides in the peptide library include: forming disulfide bonds between cysteine ​​residues at fixed positions, or forming amide bonds between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of the lysine side chain at the N-terminus of the peptide chain. The polypeptide has five structures: linear peptide, cyclic peptide with an initial and final ring, monocyclic peptide with disulfide bond, bicyclic peptide, and tricyclic peptide. Different peptide structures are formed in the following ways; 1) Linear peptide: A linear peptide with a fixed length of 17 amino acid residues; 2) Cyclic peptides: An amide bond is formed between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain, thus forming a cyclic peptide. 3) Disulfide bond monocyclic peptide: A disulfide bond is formed between C2 and C4 of cysteine, thus forming a disulfide bond monocyclic peptide; 4) Bicyclic peptide: A disulfide bond is formed between cysteine ​​C1 and C3, and an amide bond is formed between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain, thus forming a bicyclic peptide. 5) Tricyclic peptide: A disulfide bond is formed between cysteine ​​C1 and C3, a disulfide bond is formed between cysteine ​​C2 and C4, and an amide bond is formed between the carboxyl group of glutamic acid at the C-terminus of the peptide chain and the amino group of lysine side chain at the N-terminus of the peptide chain, thus forming a tricyclic peptide.

2. The polypeptide library according to claim 1, characterized in that, The peptide library contains at least 5 × 20 10 One polypeptide member.

3. The method for constructing the polypeptide library according to claim 2, characterized in that, Includes the following steps: S1. Using a fully automated microwave peptide synthesizer, the Fmoc solid-phase synthesis method was adopted. Wang resin, a solid-phase carrier, different amino acids, removal agents and peptide condensation reagents were added sequentially to synthesize the target peptide in a fully automated manner, resulting in a linear crude peptide of 17 amino acids. S2. Remove the protecting groups of the amino acid residues at the reaction sites from the linear crude peptide obtained in S1 to obtain a linear peptide. S3. Cyclic peptides are prepared by cyclizing the linear peptides obtained in S2 using chemical methods. S4. Shrink dry the linear and cyclic peptides obtained in S2 and S3 and cleave the polypeptide resin. S5. After separation HPLC purification and freeze drying, weigh and dispense into storage.

4. The application of the polypeptide library of claim 1 in screening targeted membrane-penetrating peptides, characterized in that, Includes the following steps: a. Culturing cells in a system containing peptides from the peptide library of claim 1; b. Remove peptides that do not have targeted membrane-penetrating ability; and c. Recover peptides with targeted membrane-penetrating capabilities.

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