Preparation method of lysine derivative and polypeptide modified by lysine containing isocyano group or aldehyde group

Abstract

The invention discloses a preparation method of a lysine derivative and a polypeptide modified by isocyano-containing lysine or aldehyde lysine, and belongs to the technical field of polypeptide modification. The structure of the lysine derivative is shown as a formula 1, and PG is an amino protecting group. The preparation method comprises the following steps: introducing a lysine derivative with an isocyano-modified side chain, which has a structure as shown in a formula 1, into a polypeptide chain by virtue of an Fmoc solid-phase synthesis method, and carrying out solid-phase reaction on the basis of isocyano-tetrazine, so as to obtain aldehyde-containing lysine modified polypeptide; or converting the polypeptide modified by the lysine containing the isocyano group into the polypeptide modified by the lysine containing the aldehyde group based on the liquid phase reaction of the isocyano-tetrazine. The method has the advantages of simple steps, mild conditions and high conversion rate, is compatible with a liquid phase reaction and a conventional polypeptide solid phase synthesis reaction, and is widely applied. And the solvent is compatible with an organic solvent and a water phase environment, so that the expansibility is good. In addition, the polypeptide chain prepared by the method has the advantages of good tolerance and selectivity of amino acid side chain functional groups.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide modification, and in particular to a method for preparing a lysine derivative, and a polypeptide modified with isocyanolysine or aldehyde lysine. Background Art

[0002] Post-translational modifications play a crucial role in the structure and function of peptides and proteins. As biologically active molecules, the functions of peptides and proteins are often influenced not only by their amino acid sequences but also by post-translational modifications. Protein synthesis involves two main stages: transcription and translation. After the polypeptide chain is synthesized, a series of post-translational modifications chemically adjust it, conferring its specific structure and function.

[0003] Currently reported post-translational modifications include phosphorylation, glycosylation, ubiquitination, methylation, acetylation, and disulfide bond formation. These modifications can significantly influence a protein's structure, stability, activity, and interactions with other molecules. However, in biotechnology and drug development, synthetic peptides and proteins often require specific post-translational modifications to ensure their desired biological activity and stability. Therefore, in-depth research on the preparation and significance of peptides with post-translational modifications is of great value in promoting the development of biomedicine.

[0004] Aldehyde lysine modification is a product formed by the oxidative deamination of lysine residues under the catalysis of lysyl oxidase. Lysyl oxidase and aldehyde lysine modification are present at low levels in normal mammalian tissues. However, in pathological processes such as pulmonary fibrosis and hepatic fibrosis, the concentration of aldehyde lysine modification can increase significantly, reaching hundreds of micromolar levels. Therefore, it can serve as a potential biomarker for disease diagnosis (Essays in Biochemistry, 2019, EBC20180051). Furthermore, by regulating enzymes or pathways related to aldehyde lysine, new treatments for diseases such as fibrosis are expected. For example, studies have found that certain drugs can inhibit the activity of lysyl oxidase, thereby reducing the production of aldehyde lysine and slowing the progression of fibrosis. Furthermore, collagen with aldehyde lysine modification has potential applications in tissue engineering and regenerative medicine. By regulating the degree of collagen cross-linking, the performance of tissue repair materials can be improved, enhancing the effectiveness of tissue regeneration. Beyond medical research, research on aldehyde lysine modification can also be applied to fields such as materials science and chemistry. For example, in the research and development of biomaterials, the specific reaction of aldehyde lysine can be used to functionalize biomaterials and give them better biocompatibility, mechanical properties and biological activity; in addition, the special chemical structure of aldehyde lysine provides inspiration for the design and synthesis of new compounds, which can be used to develop bioactive molecules with specific functions.

[0005] Therefore, developing synthetic methods for peptides modified with aldehyde lysine is of great significance. Currently, few methods for modifying aldehyde lysine have been reported. Emenike et al. reported the first chemical synthesis of aldehyde lysine peptides, using Selectfluor as an oxidant to oxidize dimethyl lysine (Kme2) (Angew. Chem. Int. Ed. 2024, e202403215). However, this method has certain limitations. First, the oxidation of dimethyl lysine (Kme2) produces monomethyl lysine (Kme) and oxidized methionine as byproducts; second, the use of bioincompatible oxidants limits the application of this method in biological systems. Wang et al. also reported a biosynthetic method for aldehyde lysine-modified proteins based on the insertion of non-natural amino acids (Angew. Chem. Int. Ed. 2016, 55, 1-6). This method requires the construction of a specific plasmid for the insertion of non-natural amino acids and the synthesis of complex non-natural amino acids, resulting in a relatively cumbersome genetic manipulation process. Therefore, it is necessary to develop a simple, efficient, and biocompatible method for preparing aldehyde-lysine-modified polypeptides. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing lysine derivatives and polypeptides modified with isocyanolysine or aldehyde lysine. The preparation method has simple steps, mild conditions and high conversion rate.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a lysine derivative, the structure of which is shown in Formula 1:

[0009]

[0010] Wherein, PG is an amino protecting group.

[0011] Preferably, the amino protecting group is selected from 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz) or allyloxycarbonyl (Alloc).

[0012] The present invention also provides a method for preparing a polypeptide modified with isocyanolysine or aldehyde lysine, comprising the following steps:

[0013] (1) using the Fmoc solid-phase synthesis method, using a raw material system comprising the lysine derivative according to claim 1, to introduce a lysine derivative having an isocyanate-modified side chain into a polypeptide chain;

[0014] (2) reacting the resin connected to the polypeptide chain with a tetrazine compound in a solid phase, and cleaving the resin after the reaction to obtain a polypeptide modified with aldehyde-containing lysine; or, cleaving the resin connected to the polypeptide chain by an isocyanate-tolerant cleavage method to release a free polypeptide modified with isocyanate-containing lysine, and reacting the polypeptide with a tetrazine compound in a liquid phase to obtain a polypeptide modified with aldehyde-containing lysine.

[0015] The above preparation method introduces a lysine derivative having an isocyanate-modified side chain into a polypeptide chain (synthesizing an isocyanate-lysine-containing polypeptide) by the Fmoc solid-phase synthesis method, and obtains an aldehyde-lysine-modified polypeptide based on an isocyanate-tetrazine solid-phase reaction; alternatively, the isocyanate-lysine-modified polypeptide is converted into an aldehyde-lysine-modified polypeptide based on an isocyanate-tetrazine liquid-phase reaction.

[0016] The preparation method has simple steps, mild conditions, is compatible with liquid phase reactions and solid phase synthesis reactions, and has a wide range of applications.

[0017] Preferably, the resin used in the Fmoc solid phase synthesis method is selected from Wang resin, 2-Chlorotrityl resin, Rink Amide resin, Sieber Amide resin or TentaGel resin.

[0018] Preferably, the conditions of the Fmoc solid phase synthesis method in step (1) of the present invention are:

[0019] (1) Coupling conditions of the lysine derivative according to claim 1

[0020] The feed equivalent of the lysine derivative according to claim 1 is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method;

[0021] The amount of triethylamine used is 3-5 times the amount of resin loading used in the Fmoc solid phase synthesis method;

[0022] The reaction solvent is selected from N,N-dimethylformamide, and the reaction time is 2-4h;

[0023] (2) Other natural or non-natural Fmoc-amino acid coupling conditions

[0024] The feed equivalent of Fmoc-amino acid is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method;

[0025] The feeding amount of (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinyl uronium hexafluorophosphate (COMU) is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method;

[0026] The feed equivalent of 2,4,6-trimethylpyridine (TMP) is 6-10 times the resin loading amount used in the Fmoc solid phase synthesis method;

[0027] The reaction solvent is selected from N,N-dimethylformamide, and the reaction time is 0.5-2h;

[0028] The Fmoc deprotection conditions are: 1%-5% HOBT (w / v), 2%-20% DBU (v / v), 30-200 mM (calculated based on the resin loading), 1-10 min. Preferably, the tetrazine compound in step (2) is selected from 3,6-di-2-pyridyl-1,2,4,5-tetrazine.

[0029] Preferably, in the present invention, the solvent for the solid phase reaction and the liquid phase reaction with the tetrazine compound in step (2) is selected from N,N-dimethylformamide, water or a mixture of N,N-dimethylformamide and water;

[0030] The volume ratio of N,N-dimethylformamide to water in the mixture of N,N-dimethylformamide and water is 100:1-1:100; more preferably, the volume ratio of N,N-dimethylformamide to water in the mixture is 9:1.

[0031] The time of the solid phase reaction and the liquid phase reaction in (2) is independently 0.5-24h.

[0032] Preferably, the isocyanate-resistant cleavage method in step (2) is selected from photocleavage or weak acid cleavage;

[0033] The resin and polypeptide connecting chain fragment used in the photocleavage are selected from 3-amino-3-(2-nitrophenyl) propionic acid, and the cleavage conditions are that the resin reacts under 365nm light for 2-24h, and the solvent of the photoreaction is selected from methanol, acetonitrile or N,N-dimethylformamide;

[0034] The resin used in the weak acid cleavage and the connecting chain fragment of the polypeptide are selected from 2-chlorotriphenylphosphine. The cleavage conditions are that the resin is reacted in a mixed solution of trifluoroethanol and dichloromethane for 1-24 hours, and the volume ratio of trifluoroethanol to dichloromethane in the mixed solution is 2:1-1:4. The present invention also provides a polypeptide modified with isocyanolysine or aldehyde lysine, prepared by the above preparation method.

[0035] The present invention also provides the application of the above-mentioned polypeptide containing isocyanolysine or aldehyde lysine modification in protein post-translational modification research, biomedicine and material synthesis;

[0036] The polypeptide modified with isocyanolysine or aldehyde lysine is selected from fluorescent modified polypeptides, cyclic peptides, polypeptide-drug conjugates, nuclide-polypeptide conjugates, and bioactive polypeptides modified with aldehyde lysine after translation.

[0037] Compared with the prior art, the structure of the lysine derivative provided by the present invention is shown in Formula 1, wherein PG is an amino protecting group. The present invention introduces a lysine derivative having an isocyanate-modified side chain of the structure shown in Formula 1 into a polypeptide chain through the Fmoc solid-phase synthesis method, and obtains a polypeptide modified with aldehyde-containing lysine based on an isocyanate-tetrazine solid-phase reaction; alternatively, the polypeptide modified with isocyanate-tetrazine is converted into a polypeptide modified with aldehyde-containing lysine based on an isocyanate-tetrazine liquid-phase reaction. The method has simple steps, mild conditions, high conversion rate, is compatible with liquid-phase reactions and conventional polypeptide solid-phase synthesis reactions, and has a wide range of applications. The solvent used is compatible with organic solvents and aqueous environments and has good scalability. In addition, the polypeptide chain prepared by the method has the advantages of good tolerance and selectivity of amino acid side chain functional groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the chemical conversion route for the preparation of the polypeptide modified with isocyanolysine or aldehyde lysine according to the present invention;

[0039] Figure 2 This is the hydrogen spectrum of Fmoc-Lys(isocyano)-OPfp prepared in Example 1;

[0040] Figure 3 This is the carbon spectrum of Fmoc-Lys(isocyano)-OPfp prepared in Example 1;

[0041] Figure 4 This is the fluorine spectrum of Fmoc-Lys(isocyano)-OPfp prepared in Example 1;

[0042] Figure 5 From top to bottom are the LC-MS images of the solid-phase synthesized isonitrile-modified peptides Cpd.2, Cpd.3, and Cpd.4 in Example 4;

[0043] Figure 6 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.5 in Example 4;

[0044] Figure 7 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.6 in Example 4;

[0045] Figure 8 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.7 in Example 4;

[0046] Figure 9 This is the solid phase synthesis LC-MS chart of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.8 in Example 4; Figure 10 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified peptide Cpd.9 in Example 4;

[0047] Figure 11 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.12 in Example 4;

[0048] Figure 12 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified peptide Cpd.14 in Example 4;

[0049] Figure 13 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.18 in Example 4;

[0050] Figure 14 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified peptide Cpd.19 in Example 4;

[0051] Figure 15 This is the LC-MS graph of the solid phase synthesis of the isonitrile-modified polypeptide Cpd.21 in Example 4;

[0052] Figure 16 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.23 prepared in Example 5;

[0053] Figure 17 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.24 prepared in Example 5;

[0054] Figure 18 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.25 prepared in Example 5;

[0055] Figure 19 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.29 prepared in Example 5;

[0056] Figure 20 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.30 prepared in Example 5;

[0057] Figure 21 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.32 prepared in Example 5;

[0058] Figure 22 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.33 prepared in Example 5;

[0059] Figure 23 This is the LC-MS spectrum of the aldehyde lysine-modified polypeptide Cpd.36 prepared in liquid phase in Example 6;

[0060] Figure 24 This is the LC-MS spectrum of Cpd.42 after Pictet-splender reaction of the peptide modified with aldehyde lysine in Example 7;

[0061] Figure 25 This is the LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.45 prepared in Example 9 through reductive amination reaction;

[0062] Figure 26 LC-MS spectrum of the aldehyde lysine modified polypeptide Cpd.46 prepared in Example 10 by HWE reaction;

[0063] Figure 27 This is the LC-MS chart of the aldehyde lysine modified polypeptide Cpd.47 prepared in Example 10 by HWE reaction. DETAILED DESCRIPTION

[0064] To further illustrate the present invention, the preparation methods of the lysine derivatives and the polypeptides modified with isocyanolysine or aldehyde lysine provided by the present invention are described in detail below with reference to the examples.

[0065] Example 1

[0066] Preparation of Fmoc-Lys(isocyano)-OPfp

[0067]

[0068] (1) Preparation of Fmoc-Lys(For)-OH

[0069] The preparation was carried out according to the method reported in the literature (Journal of the American Chemical Society, 128(41), 13586-13591; 2006). Formic acid (1.5 mL, 39 mmol) was added dropwise to acetic anhydride (4.5 mL, 48 mmol) under ice-bath, heated to 60°C for 2 h, and then the mixture was cooled to 0°C. A dichloromethane solution of Fmoc-Lys-OH (2.5 g, 6.79 mmol) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the reaction solution was diluted with ethyl acetate, and the organic layer was washed with water and saturated sodium chloride solution in sequence. The organic phase was dried over Na2SO4 and concentrated. The compound Fmoc-Lys(For)-OH (1.61 g, 60% yield) was obtained by silica gel chromatography. 1H NMR(400MHz,DMSO-d6)δ12.59(s,1H),8.05-7.96(m,2H),7.90(d,J=7.6Hz,2H ),7.73(d,J=7.5Hz,2H),7.69-7.60(m,1H),7.42(t,J=7.4Hz,2H),7.33(td,J= 7.5,1.2Hz,2H),4.32-4.17(m,2H),3.91(ddd,J=9.6,7.9,4.6Hz,1H),3.35(s, 1H),3.07(q,J=6.5Hz,2H),1.69(s,1H),1.77-1.54(m,1H),1.47-1.27(m,3H). 13 C NMR (101MHz, DMSO) δ174.42,161.36,156.63,144.31,144.27,141.19,141.17,128.1 1,127.54,125.78,125.75,120.59,66.05,54.22,47.11,37.33,30.84,29.06,23.52.

[0070] (2) Preparation of Fmoc-Lys(For)-OPfp

[0071] Fmoc-Lys(For)-OH (1.5 g, 3.78 mmol) and EDCI (725.38 mg, 3.78 mL) were dissolved in a dry DCM / DMF mixture (23 mL:1 mL) at room temperature. After stirring for 15 minutes, pentafluorophenol (1.082 mL, 10.33 mmol) was added. After the reaction was complete, the reaction solution was washed with saturated NaHCO₃ solution ×3 and saturated NaCl solution ×3. The organic phase was dried over Na₂SO₄ and concentrated to yield 1.86 g of Fmoc-Lys(For)-OPfp as a solid (87.7% yield). This product was used directly in the next step without further purification.

[0072] (3) Preparation of Fmoc-Lys(isocyano)-OPfp

[0073] Fmoc-Lys(For)-OPfp (1.8 g, 3.32 mmol) and Et3N (2.466 mL, 17.79 mmol) were dissolved in dry DCM and stirred at -20°C for 15 min. POCl3 (0.515 mL, 5.61 mmol) was slowly added. TLC monitoring indicated the reaction was complete. The organic solution was washed three times with saturated NaHCO3 solution. The organic phase was dried over Na2SO4 and concentrated. Purification by silica gel column chromatography (EtOAc / DCM = 1:30) afforded 1.11 g of Fmoc-Lys(isocyano)-OPfp as a solid (61.5% yield). 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.6Hz,2H),7.60(d,J=7.6Hz,2H),7.41(t,J=7.5Hz,2H),7.32(t,J=7.5Hz,2H),5.33(d,J=8.5Hz,1H),4.80-4.70(m,1 H),4.49(d,J=6.8Hz,2H),4.24(t,J=6.8Hz,1H),3.42(d,J=6.8Hz,2H),2.0 8(dd,J=15.2,7.7Hz,1H),1.90(dq,J=15.0,7.9Hz,1H),1.83-1.69(m,2H). 13 C NMR (101MHz, CDCl3) δ168.60,156.56,155.76,143.64,141.34,140.08-136.64(fluoro-conjugating carbon),127.79,127.10,124.97,120.05,120.03,67.27,53.38,47.10,41.24,31.58,28.42,22.05. 19 F NMR (376MHz, CDCl3) δ-152.32 (d, J=17.6Hz, 2F), -156.72 (t, J=21.7Hz, 1F), -161.51 (dd, J=22.0, 17.3Hz, 2F).

[0074] Example 2

[0075] Preparation of Cbz-Lys(isocyano)-OPfp

[0076]

[0077] The preparation route of Cbz-Lys(isocyano)-Opfp is the same as that in Example 1. The Cbz-Lys-OH raw material is subjected to formylation, esterification with pentafluorophenol, and dehydration to obtain the target product. Cbz-Lys(isocyano)-Opfp is a white solid. 1 H NMR(400MHz,Chloroform-d)δ7.32–7.25(m,5H),5.26(d,J=8.3Hz,1H),5.08(s,2H),4.70(td,J=8.1,5.1Hz,1H),3.3 4(d,J=6.8Hz,2H),2.02(q,J=7.6,6.9Hz,1H),1.84(dq,J=14.6,7.6Hz,1H),1.70(d,J=9.5Hz,2H),1.62–1.52(m,2H).

[0078] Example 3

[0079] Solid-Phase Synthesis of Aldehyde-Lysine-Modified Amino Acids

[0080]

[0081] The fatty aldehyde group of aldehyde lysine is relatively unstable and easily changes during the separation and purification process. Therefore, in this example, after the solid-phase synthesis reaction of aldehyde lysine-modified amino acids was carried out on Rink Amide resin, a reductive amination reaction was carried out in situ on the solid phase of the generated fatty aldehyde group, and purification and structure confirmation were performed.

[0082] The reaction scheme, as shown above, begins by coupling Cbz-Lys(isocyano)-OPfp (the final product of Example 2) to Rink Amide resin (0.3 mmol loading). After reaction with the tetrazine compound 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine, the blue-fluorescent compound Carbazol-NH2 is attached to the resin via reductive amination. After the reaction, the resin emits a strong blue fluorescence under UV light. The compound is then cleaved with 95% trifluoroacetic acid and purified by silica gel column chromatography to yield compound Cpd.1.

[0083] Cpd.1, white solid. 1 H NMR (400MHz, DMSO-d6) δ8.00 (d, J=7.6Hz, 1H),

[0084] 7.45(d,J=8.4Hz,1H),7.38–7.29(m,10H),7.24(d,J=2.2Hz,1H),7.07(t, J=7.4Hz,1H),7.03–6.98(m,1H),6.86(dd,J=8.7,2.3Hz,1H),5.03(s,2H), 4.31(q,J=7.2Hz,2H),3.95(dd,J=8.7,4.9Hz,1H),3.11–3.03(m,2H),1.75 –1.65(m,2H),1.65–1.55(m,2H),1.49–1.39(m,2H),1.25(t,J=7.1Hz,3H). 13 C NMR(101MHz,DMSO)δ174.69,156.50,137.49,128.82,128.26,128.13,125.65,122.44,1 20.61,118.20,110.02,109.25,65.85,55.30,55.00,37.31,32.19,28.59,23.75,14.18.

[0085] Example 4

[0086] The general method for Fmoc solid phase synthesis includes the following steps:

[0087] a. Fmoc Deprotection: Add Fmoc-Ala-2-Chlorotrityl Resin (Gill Biochemical) to a solid-phase synthesis tube and allow to swell in DMF for 30 minutes at room temperature. Drain the reaction solvent and add a mixture of 1% HOBT (w / v) and 2% DBU (v / v) in DMF to the tube (TentaGel resin does not have an Fmoc protecting group, so this step is omitted). Vortex for 2 minutes, drain the reaction solvent, and repeat three times. Wash the resin nine times with DMF, DCM, and DMF, alternating between the two.

[0088] b.1) Amino acid coupling

[0089] Coupling of isocyanate-modified lysine: Dissolve Fmoc-Lys(isocyano)-OPfp (3.0 eq) and Et3N (5.0 eq) in DMF, vortex for 2 minutes, transfer to the peptide reaction tube, and react on a shaker at room temperature for 120 minutes. Drain the reaction solvent and wash the resin nine times with DMF, DCM, and DMF.

[0090] For coupling of other natural or unnatural Fmoc amino acids: Dissolve Fmoc-AA-OH (5.0 eq), COMU (5.0 eq), and TMP (7.5 eq) in DMF. Vortex for 2 minutes, transfer to the peptide reaction tube, and react on a rocking platform at room temperature for 30 minutes. Drain the reaction solvent and wash the resin nine times with DMF, DCM, and DMF.

[0091] 2) Fmoc deprotection: Add a mixed solution of 1% HOBT (w / v) and 2% DBU (v / v) in DMF to a solid phase synthesis tube, vortex for 2 minutes, drain the reaction solvent, and repeat three times. Wash the resin nine times with DMF, DCM, and DMF alternately.

[0092] Repeat the above two steps to extend the peptide segment. AA represents amino acid, and the above general formula does not limit the length of the polypeptide but is only used to illustrate the structure of the polypeptide. AA can be any amino acid independently.

[0093] (1) This section demonstrates the compatibility of Fmoc-Lys(isocyano)-OPfp (the final product synthesized in Example 1) for Fmoc solid phase synthesis using TentaGel resin.

[0094] The feasibility of solid-phase synthesis of isonitrile-modified peptides was verified using TentaGel resin, a solid-phase synthesis support compatible with bioactivity screening systems. Using the previously synthesized Fmoc-Lys(isocyano)-OPfp and the conventional Fmoc solid-phase synthesis process, a linker containing an ANP photocleavage site, aminocaproic acid, and 4-bromophenylalanine was synthesized. Lys(isocyano), Met, Pro, Trp(Boc), and Val amino acids were then coupled sequentially, and finally, three different N-terminal amino acids were coupled. After nine coupling-deprotection cycles, a portion of the resin was placed in a 96-well plate, 200 μL of acetonitrile was added, and cleavage was performed under 365 nm light for 2 hours. The cleavage solution was collected to obtain the target peptide Cpd.2-4.

[0095]

[0096] Cpd.2: Boc-Ala-Val-Trp(Boc)-Pro-Met(O)-Lys(isocyano)-Phe(4-Br)

[0097] -Ahx-NH2

[0098] C 61 H 89 BrN 11 O 13 S + [M+H] +Exact Mass: 1294.5545; Measured Mass: 1294.5506; Liquid phase UV peak retention time: 8.1min, LC purity> 80% (254nm). (Attached Figure 5 )

[0099]

[0100] Cpd.3: Fmoc-Trp-Val-Trp(Boc)-Pro-Met(O)-Lys(isocyano)-Phe(4-Br)

[0101] -Ahx-NH2

[0102] C 79 H 97 BrN 12 O 13 S ++ [M+2H] 2+ Exact Mass: 766.3101; Measured Mass: 766.3130; Liquid phase UV peak retention time: 9.5min, LC purity> 75% (254nm). (Attached Figure 5 )

[0103]

[0104] Cpd.4: Bza(4-Ethynyl)-Trp-Val-Trp(Boc)-Pro-Met(O)-Lys(isocyano)-Phe(4-Br)-Ahx-NH2

[0105] C 62 H 81 BrN 10 O 11 S ++ [M+2H] 2+ :626.2495; Measured Mass:626.2497; Liquid phase UV peak retention time: 8.2min, LC purity>85% (254nm). Note: The oxidation of Met in the Cpd.2-4 structure is caused by oxidation during resin photocleavage. (Attached Figure 5 )

[0106] After the TentaGel resin was swollen in DMF for 30 minutes, the Fmoc solid-phase synthesis process was used to sequentially couple Phe (4-Br), Lys (isocyano), Phe, Pro, Ala, and Trp (Boc) amino acids. After five coupling-deprotection operations, 2 mg of the resin was placed in a photoreactor, 500 μL of acetonitrile was added, and the resin was cleaved for 1 hour. The cleavage liquid was collected to obtain the target peptide Cpd.5.

[0107]

[0108] Cpd.5:Fmoc-Trp(Boc)-Ala-Pro-Phe-Lys(isocyano)-Phe(4-Br)-NH2

[0109] C 63 H 73 N9O 10 [M+H] + :1204.4429; Measured Mass:1204.4570; Liquid phase UV peak retention time: 11.33 min; LC purity = 67.72% (254 nm). Note: The UV peak at 11.78 minutes corresponds to the methyl ester product generated by alcoholysis of the amide product in methanol solvent during the photolysis process. (Appendix Figure 6 )

[0110] (2) This section of the examples verifies the compatibility of Fmoc-Lys(isocyano)-OPfp (the final product synthesized in Example 1) for Fmoc solid phase synthesis on RinkAmide resin.

[0111] Using the previously synthesized Fmoc-Lys(isocyano)-OPfp and the conventional Fmoc solid-phase synthesis process, a peptide with an ANP photocleavage site was synthesized, and then Phe(4-Br), Lys(isocyano), Phe, Pro, Ala and Trp amino acids were coupled in sequence. After seven coupling-deprotection operations, 2 mg of resin was placed in a photoreactor, 500 μL of acetonitrile was added, and cleavage was carried out under 365 nm light for 1 hour. The cleavage liquid was collected to obtain the target peptide Cpd.6.

[0112]

[0113] Cpd.6: Fmoc-Trp-Ala-Pro-Phe-Lys(isocyano)-Phe(4-Br)-NH2

[0114] C 59 H 62 BrN9O8 + [M+H]+ :1104.3483; Measured Mass:1104.3950; Liquid phase UV peak retention time: 8.9min, LC purity = 84.51% (254nm). (Attached Figure 7 )

[0115] In this example, Rink Amide resin was swollen in DMF for 30 minutes, followed by Fmoc deprotection. ANP with a photocleavage site was synthesized using a conventional Fmoc solid-phase synthesis process. Phe(4-Br), Lys(isocyano), Trp(Boc), Glu(OBzl), and Ac-Ala amino acid coupling were then performed sequentially. After five coupling-deprotection cycles, 2 mg of the resin was placed in a photoreactor, 500 μL of DMF was added, and cleavage was performed under 365 nm light for 1 hour. The cleavage solution was collected to obtain the target peptide Cpd.7.

[0116]

[0117] Cpd.7:Ac-Ala-Glu(OBzl)-Trp(Boc)-Lys(isocyano)-Phe(4-Br)-NH2

[0118] C 49 H 59 BrN8O 10 + [M+H] + :999.3616; Measured Mass:999.3452; Liquid phase UV peak retention time: 8.4min, LC purity = 82.7% (254nm). (Attached Figure 8 )

[0119] In this example, Rink Amide resin was swollen in DMF for 30 minutes, deprotected with Fmoc, and then synthesized using the Fmoc solid-phase synthesis process with an ANP photocleavage site. Phe (4-Br), Lys (isocyano), Trp, Val, Cys (StBu), and Ac-Phe amino acids were then coupled in sequence. After seven coupling-deprotection operations, 2 mg of the resin was placed in a photoreactor, 500 μL of DMF was added, and cleavage was carried out under 365 nm light for 1 hour. The cleavage solution was collected to obtain the target peptide Cpd.8.

[0120]

[0121] Cpd.8: Ac-Phe-Cys(StBu)-Val-Trp-Lys(isocyano)-Phe(4-Br)-NH2

[0122] C 50 H 64 BrN9O7S2 + [M+H] + :1046.3554; Measured Mass:1046.3459; Liquid phase UV peak retention time: 8.4min, LC purity = 77.6% (254nm). (Attached Figure 9 )

[0123] Using the previously synthesized Fmoc-Lys(isocyano)-OPfp and a conventional Fmoc solid-phase synthesis process, a peptide with an ANP photocleavage site was synthesized. Gly, Lys(isocyano), Gly, Abz, and HaloTag ligands were then coupled sequentially. After six coupling-deprotection cycles, a portion of the resin was placed in a photoreactor, 500 μL of acetonitrile was added, and cleavage was performed under 365 nm light for one hour. The cleavage solution was collected to obtain the target peptide Cpd.9. This compound can be used as a tool probe for biological studies of post-translational modification of aldehyde lysines based on the HaloTag ligand.

[0124]

[0125] Cpd.9: HaloTAG-Abz-Gly-Lys(isocyano)-Gly-NH2

[0126] C 34 H 54 ClN6O 10 + [M+H] + :741.3584; Measured Mass:741.3535; Liquid phase UV peak retention time: 4.1min; LC purity = 84.7% (254nm). (Attached Figure 10 )

[0127] Rink Amide resin was swollen in DMF for 30 minutes and then deprotected with Fmoc. The Fmoc solid-phase synthesis process was then used to sequentially couple amino acids: Phe(4-Br), Trp(Boc), His(Trt), Gly, Lys(isocyano), Pro, and Ac-Ala. After seven coupling and deprotection cycles, a portion of the resin was placed in a photoreactor and cleaved with 200 μL of a 95:5 mixture of TFA and H₂O. The resin was then allowed to stand for 15 minutes and the cleavage solution was collected to yield the target peptide Cpd.10. (The isocyanate group on the Lys(isocyano) reacts with the acid cleavage solution to convert it to a formyl group.)

[0128]

[0129] Cpd.10:Ac-Ala-Pro-Lys(For)-Gly-His-Trp-Phe(4-Br)-NH2

[0130] C 45 H 58 BrN 12 O9 + [M+H] + :989.3555; Measured Mass:989.3605; Liquid phase UV peak retention time: 2.8min; LC purity = 74.2% (254nm)

[0131] RinkAmide resin was swollen in DMF for 30 minutes and then deprotected with Fmoc. The Fmoc solid-phase synthesis process was then used to sequentially couple amino acids: Phe(4-Br), Val, Gly, Lys(isocyano), Leu, Pro, Tyr(tBu), Ser(Trt), and Ac-Phe. After eight coupling-deprotection cycles, a portion of the resin was placed in a photoreactor and cleaved with 200 μL of a 95:5 mixture of TFA and H₂O. The resin was then allowed to stand for 15 minutes and the cleavage solution was collected to yield the target peptide Cpd.11. The isocyanate group on the Lys(isocyano) reacted with the acid cleavage solution to convert it to a formyl group.

[0132]

[0133] Cpd.11:Ac-Phe-Ser-Tyr-Leu-Lys(For)-Gly-Val-Phe(4-Br)-NH2

[0134] C 52 H 72 BrN 10 O 12 + [M+H] + :1107.4436; Measured Mass:1107.4422; Liquid phase UV peak retention time: 4.2 min; LC purity = 58.9% (254 nm).

[0135] (3) This section demonstrates the compatibility of Fmoc-Lys(isocyano)-OPfp (the final product synthesized in Example 1) for Fmoc solid phase synthesis on 2-Chorotrityl resin.

[0136] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Val, Phe, Ala, Lys (isocyano), Gly, Ile, Trp, and Pro amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.12.

[0137]

[0138] Cpd.12: Fmoc-Pro-Trp-Ile-Gly-Lys(isocyano)-Ala-Phe-Val-Ala-OH

[0139] C 66 H 82 N 11 O 12 + [M+H] + :1220.6139; Measured Mass:1220.6084; Liquid phase UV peak retention time:10.201min; LC purity=81.4%(254nm).(Attached Figure 11 )

[0140] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Val, Phe, Ala, Lys (isocyano), Gly, Ile, Trp, Pro, and Boc-Trp amino acids. After nine coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.13.

[0141]

[0142] Cpd.13: Boc-Trp-Pro-Trp-Ile-Gly-Lys(isocyano)-Ala-Phe-Val-Ala-OH

[0143] C 67 H 90 N 13 O 13 + [M+H] +:1284.6776; Measured Mass:1284.6927; Liquid phase UV peak retention time: 7.86 min; LC purity = 82.15% (254 nm).

[0144] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes, and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Lys (Alloc), Val, Cys (Stmp), Ala, Lys (isocyano), Phe, Leu, and Gly amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL TFE and DCM (1:1) was added. The resin was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.14.

[0145]

[0146] Cpd.14: Fmoc-Gly-Leu-Phe-Lys(isocyano)-Ala-Cys(Stmp)-Val-Lys(Alloc)-Ala-OH

[0147] C 67 H 90 N 13 O 13 + [M+H] + :1340.6418; Measured Mass:1340.6469; Liquid phase UV peak retention time:11.08min; LC purity=84.68%(254nm).(Attached Figure 12 )

[0148] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Lys (Alloc), Cys (Stmp), Ala, Lys (isocyano), Phe, Leu, Gly, and Boc-Trp amino acids. After nine coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.15.

[0149]

[0150] Cpd.15: Boc-Trp-Gly-Leu-Phe-Lys(isocyano)-Ala-Cys(Stmp)-Val-Lys(Alloc)-Ala-OH

[0151] C 68 H 102 N 13 O 15 S2 + [M+H] + :1404.7454; Measured Mass:1404.7250; Liquid phase UV peak retention time:11.6min; LC purity =78.82% (254nm).

[0152] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Glu (OAll), Pro, Lys (isocyano), Ala, Gly, Trp, Arg (Pbf), and Phe amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.16.

[0153]

[0154] Cpd.16: Fmoc-Phe-Arg(Pbf)-Trp-Gly-Ala-Lys(isocyano)-Pro-Glu(OAll)

[0155] -Ala-OH

[0156] C 82 H 101 N 14 O 17 S + [M+H] + :1585.7481; Measured Mass:1585.7446; Liquid phase UV peak retention time:10.22min; LC purity =67.47% (254nm).

[0157] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Glu (OAll), Pro, Lys (isocyano), Ala, Gly, Trp, Arg (Pbf), Phe, and Boc-Trp amino acids. After nine coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.17.

[0158]

[0159] Cpd.17: Boc-Trp-Phe-Arg(Pbf)-Trp-Gly-Ala-Lys(isocyano)-Pro-Glu(OAll)-Ala-OH

[0160] C 83 H 109 N 16 O 18 S + [M+H] + :1649.7821; Measured Mass:1649.7887; Liquid phase UV peak retention time:9.04min; LC purity =70.46% (254nm).

[0161] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Ile, Ala, Lys (isocyano), Phe, Tyr (All), Trp, Pro, and Val amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.18.

[0162]

[0163] Cpd.18:Fmoc-Val-Pro-Tyr(All)-Phe-Lys(isocyano)-Ala-Ile-Ala-OH

[0164] C 76 H 92 N 16 O 12 + [M+H]+ :1366.6871; Measured Mass:1366.7116; Liquid phase UV peak retention time:11.07min; LC purity=84.23%(254nm).(Attached Figure 13 )

[0165] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes, and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Ile, Ala, Lys (isocyano), Phe, Tyr (All), Trp, Pro, Val, and Boc-Trp amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL TFE and DCM (1:1) was added. The resin was allowed to stand for 1 hour and the cleavage liquid was collected to obtain the target peptide Cpd.19.

[0166]

[0167] Cpd.19:Boc-Trp-Val-Pro-Trp-Tyr(All)-Phe-Lys(isocyano)-Ala-Ile-Ala-OH

[0168] C 76 H 92 N 16 O 12 + [M+H] + :1430.7507; Measured Mass:1430.7542; Liquid phase UV peak retention time:9.60min; LC purity=75.60% (254nm). (Attached Figure 14 )

[0169] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes, and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Trp, Lys (isocyano), Phe, Pro, Cys (Stmp), and Leu amino acids. After six coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.20.

[0170]

[0171] Cpd.20:Fmoc-Leu-Cys(Stmp)-Pro-Phe-Lys(isocyano)-Trp-Ala-OH

[0172] C 68 H 81 N9O 13 S2 + [M+H] + :1294.5312; Measured Mass:1294.5384; Liquid phase UV peak retention time:11.33min; LC purity =40.79% (254nm).

[0173] The Fmoc-Ala-2-Chorotrityl resin was swollen in DMF for 30 minutes, and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Tyr (OAll), Lys (isocyano), Val, Ile, Lys (Alloc), Leu, Gly, Ala, and Phe amino acids. After eight coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.21.

[0174]

[0175] Cpd.21:Fmoc-Phe-Ala-Gly-Lys(Alloc)-Ile-Val-Lys(isocyano)-Tyr(OAll)-Ala-OH

[0176] C 72 H 94 N 11 O 15 + [M+H] + :1352.6925; Measured Mass:1352.6873; Liquid phase UV peak retention time:10.37min; LC purity=75.34%(254nm).(Attached Figure 15 )

[0177] (3) The examples in this section verify the compatibility of Fmoc-Lys(isocyano)-OPfp (the final product synthesized in Example 1) for Fmoc solid phase synthesis on Sieber resin.

[0178] The Sieber resin was swollen in DMF for 30 minutes and then deprotected by Fmoc. The Fmoc solid-phase synthesis process was used to sequentially couple Tyr (2-ClTrt), Trp, Pro, Ser (Trt), Val, Thr (Trt), Leu, Gly, and Lys (isocyano) -Cbz amino acids. After nine coupling-deprotection operations, 2 mg of the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFA and DCM (1:99) was added. The mixture was allowed to stand for 1 hour for cleavage, and the cleavage liquid was collected to obtain the target peptide Cpd.22.

[0179]

[0180] Cpd.22:Cbz-Lys(For)-Gly-Leu-Thr(Trt)-Val-Ser(Trt)-Pro-Trp-Tyr(2-ClTrt)-N H2

[0181] C 60 H 83 N 12 O 15 + [M+H] + :1211.6095; Measured Mass:1211.6011; Liquid phase UV peak retention time: 4.32 min; LC purity = 20.36% (254 nm).

[0182] Example 5

[0183] Solid-Phase Synthesis of Aldehyde-Lysine Modified Peptides

[0184] In this example, RinkAmide resin was used to sequentially couple Phe (4-Br), Lys (isocyano), Trp, Val, Cys (StBu), and Ac-Phe amino acids. 5 mg of the dried resin was then reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a photoreactor, 500 μL of DMF was added, and cleavage was performed under 365 nm light for 1 h. The cleavage liquid was collected to obtain Cpd.23.

[0185]

[0186] C 49 H 63 BrN8O8S2 + [M+H] + :1035.3472; Measured Mass:1035.3336; Liquid phase UV peak retention time:8.0min, LC conversion rate>90%. (Attached Figure 16 ).

[0187] In this example, RinkAmide resin was used to sequentially couple Phe(4-Br), Lys(isocyano), Trp(Boc), Glu(OBzl), and Ac-Ala amino acids. 10 mg of the dried resin was then reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After the reaction, the resin was placed in a photoreactor, 500 μL of DMF was added, and cleavage was performed under 365 nm light for 1 h. The cleavage solution was collected to obtain Cpd.24.

[0188]

[0189] C 48 H 58 BrN7O 11 + [M+H] + :988.3456; Measured Mass:988.3324; Liquid phase UV peak retention time: 7.8min, LC conversion rate>99% (254nm). (Attached Figure 17 ).

[0190] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Val, Phe, Ala, Lys (isocyano), Gly, Ile, Trp, and Pro amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.25.

[0191]

[0192] C 65 H 81 N 10 O 13 + [M+H] + :1209.5979; Measured Mass:1209.5982; Liquid phase UV peak retention time: 8.88min, LC conversion rate>99% (254nm). (Attached Figure 18 )

[0193] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Val, Phe, Ala, Lys (isocyano), Gly, Ile, Trp, Pro, and Boc-Trp amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.26.

[0194]

[0195] C 66 H 90 N 12 O 14 + [M+H] + :1273.6616; Measured Mass:1273.6584; Liquid UV peak retention time: 7.66 min, LC conversion rate>90% (254 nm).

[0196] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys (Alloc), Val, Cys (Stmp), Ala, Lys (isocyano), Phe, Leu, and Gly amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.27.

[0197]

[0198] C 66 H 92 N 10 O 15 S2 + [M+H] + :1329.6258; Measured Mass:1329.6184; Liquid UV peak retention time:11.128min, LC conversion>90% (254nm).

[0199] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys (Alloc), Val, Cys (Stmp), Ala, Lys (isocyano), Phe, Leu, Gly, and Boc-Trp amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.28.

[0200]

[0201] C 67 H 101 N 12 O 16 S2 + [M+H] + :1393.6894; Measured Mass:1393.6928; Liquid UV peak retention time:10.48min, LC conversion>90% (254nm).

[0202] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Glu (OAll), Pro, Lys (isocyano), Ala, Gly, Trp, Arg (Pbf), and Phe amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.29.

[0203]

[0204] C 81 H 101 N 13 O 18 S + [M+H] + :1574.7025; Measured Mass:1574.6931; Liquid phase UV peak retention time: 9.77min; 10.13min, LC conversion rate>90% (254nm). (Attached Figure 19 )

[0205] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Glu (OAll), Pro, Lys (isocyano), Ala, Gly, Trp, Arg (Pbf), Phe, and Boc-Trp amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.30.

[0206]

[0207] C 82 H 108 N 15 O 19 S + [M+H] + :1638.7661; Measured Mass:1638.7713; Liquid phase UV peak retention time: 8.54min, LC conversion rate>90% (254nm). (Attached Figure 20 )

[0208] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Ile, Ala, Lys (isocyano), Phe, Tyr (All), Trp, Pro, and Val amino acids. 2 mg of the dried resin was then reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.31.

[0209]

[0210] C 75 H 92 N 10 O 14 + [M+H] + :1355.6711; Measured Mass:1355.6623; Liquid UV peak retention time:10.54min, LC conversion>90% (254nm).

[0211] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Ile, Ala, Lys (isocyano), Phe, Tyr (All), Trp, Pro, Val, and Boc-Trp amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and a mixed solution of 500 μL TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.32.

[0212]

[0213] C 76 H 99 N 12 O 15 + [M+H] + :1419.1347; Measured Mass:1419.7368; Liquid phase UV peak retention time: 9.05min, LC conversion rate>90% (254nm). (Attached Figure 21 )

[0214] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Gly, Val, His (Trt), Pro, Ile, Ala, Lys (isocyano), and Asn (Trt) amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.33.

[0215]

[0216] C 93 H 103 N 12 O 14 + [M+H] + :1611.7711; Measured Mass:1611.7623; Liquid phase UV peak retention time: 8.42min, LC conversion rate>90% (254nm). (Attached Figure 22 )

[0217] In this example, TentaGel resin was used to sequentially couple AHX (6), Lys (isocyano), Gly, Pro, Pro, Trp, Val, and Trp amino acids. 2 mg of dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a photoreactor, 500 μL of a mixed solution of ACN was added, and the reaction was cleaved for 1 h. The cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.34.

[0218]

[0219] C 75 H 88 BrN 12 O 12 + [M+H] + :1427.5823; Measured Mass:1427.5744; Liquid UV peak retention time:8.04min, LC conversion>90% (254nm).

[0220] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Tyr (OAll), Lys (isocyano), Val, Ile, Lys (Alloc), Gly, Ala, and Phe amino acids. 2 mg of the dried resin was reacted with 2 eq of tetrazine (DMF: H2O = 9:1) for 2 h, and the reaction was repeated once. After the reaction, the resin was placed in a 1.5 mL EP tube, and 500 μL of a mixed solution of TFE and DCM (1:1) was added. The tube was allowed to stand for 1 h and the cleavage liquid was collected to obtain the target peptide. The cleavage liquid was collected to obtain Cpd.35.

[0221]

[0222] C 73 H 91 N 10 O 16 + [M+H] + :1341.6766; Measured Mass:1341.6805; Liquid phase UV peak retention time: 9.85 min, LC conversion rate>90% (254 nm).

[0223] Example 6

[0224] Liquid Phase Synthesis of Aldehyde-Lysine Modified Peptides

[0225] In this example, Rink Amide resin was used to sequentially couple Phe (4-Br), Lys (isocyano), Trp, Val, Cys (StBu), and Ac-Phe amino acids. 10 mg of the resin was placed in a photoreactor, 500 μL of DMF was added, and cleavage was carried out under 365 nm light for 1 h. The cleavage solution was collected and reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h. The reaction was repeated once to obtain Cpd.36.

[0226]

[0227] C 49 H 63 BrN8O8S2 + [M+H] + :1035.3472; Measured Mass:1035.3335; Liquid phase UV peak retention time: 8.0min, LC purity>99% (254nm). (Attached Figure 23 ).

[0228] Example 7

[0229] Derivatization of aldehyde-lysine-modified peptides based on hydrazide compounds

[0230] After coupling ANP, Phe(4-Br), Lys(isocyano), Trp, Val, Cys(StBu), and Ac-Phe amino acids using Rink Amide resin, 10 mg of dried resin was reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After completion of the reaction, the resin was washed three times with DMF, DCM, and DMF alternately. Then, 2 eq of biotin hydrazide was added to the resin. After reacting for 6 h, the resin was placed in a photoreactor and cleaved under 365 nm light for 1 h. The cleavage solution was collected to obtain Cpd.37.

[0231]

[0232] C 59 H 79 BrN 12 O9S3 + [M+H] + :1275.4517; Measured Mass:1275.4505; Liquid phase UV peak retention time: 6.4 min, LC conversion = 67.2% (254 nm).

[0233] After coupling ANP, Ahx(6), Lys(isocyano), Gly, Pro, Pro, Trp, Val, and Trp amino acids using Rink Amide resin, 2 mg of dried resin was reacted with 2 eq of tetrazine (DMF:H2O=9:1) for 2 h. This reaction was repeated once. After the reaction, the resin was washed three times with DMF, DCM, and DMF alternately. Then, 2 eq of biotin hydrazide was added to the resin. After reacting for 1 h, the resin was placed in a photoreactor and cleaved under 365 nm light for 1 h. The cleavage solution was collected to obtain Cpd.38.

[0234]

[0235] C 59 H 79 BrN 12 O9S3 + [M+2H] 2+ :1667.6867; Measured Mass:1667.6765; Liquid phase UV peak retention time: 7.21 min, LC conversion = 67.75% (254 nm).

[0236] After coupling Phe(4-Br), Gly, Gln, Trp, Lys(isocyano), Lys(Boc), and Ac-Ala amino acids using Rink Amide resin, 5 mg of dried resin was reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After completion of the reaction, the resin was washed three times with DMF, DCM, and DMF, followed by the addition of 2 eq of biotin hydrazide. After 6 h of reaction, the resin was cleaved in 500 μL of a 95:5 TFA:H2O mixture for 15 min. The cleavage solution was collected to yield Cpd.39.

[0237]

[0238] C 54 H 77 BrN 15 O 11 S + [M+H] + :1222.4753; Measured Mass:1222.4752; Liquid phase UV peak retention time:2.8min.

[0239] Example 8

[0240] Derivatization of Aldehyde-Lysine-Modified Peptides Based on the Pictet-splender Reaction

[0241] In this example, Rink Amide resin was used to sequentially couple Phe(4-Br), Gly, Gln, Trp, Lys(isocyano), Lys(Boc), and Ac-Ala amino acids. 5 mg of dried resin was then reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After completion of the reaction, the resin was washed three times with DMF, DCM, and DMF. The resin was then added to a 1:1 mixture of acetonitrile and sodium acetate solution (0.1 M, pH = 5), followed by 10 eq of 2-(1H-pyrrol-1-yl)ethanamine. After reacting at room temperature for 1 h, the resin was washed three times with DMF, DCM, and MeOH. Finally, 500 μL of a 95:5 mixture of TFA:H2O was added to the resin. The resin was allowed to stand for 15 min and cleaved. The cleavage solution was collected to obtain Cpd.40.

[0242]

[0243] C 50 H 69 BrN 13 O9 + [M+H] + :1074.4446; Measured Mass:1074.4483; Liquid phase UV peak retention time:2.7min.

[0244] In this example, Rink Amide resin was used to sequentially couple Phe (4-Br), Leu, Trp, Lys (isocyano), Pro, Arg (Pbf), and Ac-Phe amino acids. 5 mg of dried resin was reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h; the reaction was repeated once. After the reaction, the resin was washed three times with DMF, DCM, and DMF. The resin was then added to a 1:1 mixture of acetonitrile and sodium acetate solution (0.1 M, pH = 5). 10 eq of 2-(1H-pyrrol-1-yl)ethylamine was added. After reacting at room temperature for 1 h, the resin was washed three times with DMF, DCM, and MeOH. Finally, 500 μL of a 95:5 mixture of TFA:H2O was added to the resin. The resin was allowed to stand for 15 min and cleaved. The cleavage solution was collected to obtain Cpd.41.

[0245]

[0246] C 62 H 86 BrN 12 O 10 S + [M+H] +:1269.5416; Measured Mass:1269.5291; Liquid phase UV peak retention time:5.4min. In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys(isocyano), Pro, Tyr(OAll), Phe, Val, and Leu amino acids. 5mg of the dried resin was then reacted with 2eq of tetrazine (DMF:H2O=9:1) for 2h. The reaction was repeated once. After the reaction, the resin was washed three times with DMF, DCM, and DMF alternately. The resin was added to a 1:1 mixed solvent of acetonitrile and sodium acetate solution (0.1 M, PH = 5), and 10 eq of 2-(1H-pyrrol-1-yl)ethylamine was added. After reacting at room temperature for 1 hour, the resin was washed three times with DMF, DCM, and MeOH alternately. Finally, 500 μL of a 1:1 mixed solution of TFE:DCM was added to the resin, and the resin was allowed to stand for 15 minutes for cutting. The cutting liquid was collected to obtain Cpd.42.

[0247]

[0248] C 67 H 84 N9O 11 + [M+H] + :1190.6212; Measured Mass:1190.6257; Liquid phase UV peak retention time:8.67min. (Attached Figure 24 In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys(isocyano), Leu, Trp, Ile, Ala, and Val amino acids. 5 mg of dried resin was then reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After the reaction, the resin was washed three times with DMF, DCM, and DMF alternately. The resin was then added to a 1:1 mixture of acetonitrile and sodium acetate solution (0.1 M, pH = 5), followed by 10 eq of 2-(1H-pyrrol-1-yl)ethanamine. After reacting at room temperature for 1 h, the resin was washed three times with DMF, DCM, and MeOH alternately. Finally, 500 μL of a 1:1 mixture of TFE:DCM was added to the resin, and the mixture was allowed to stand for 15 min for cleavage. The cleavage solution was collected to obtain Cpd.43.

[0249]

[0250] C 61 H 81 N 10 O 10 + [M+H] +:1113.6059; Measured Mass:1113.6012; Liquid phase UV peak retention time:8.06min.

[0251] Example 9

[0252] Derivatization of Aldehyde-Lysine-Modified Peptides via Reductive Amination

[0253] In this example, Rink Amide resin was used in the Fmoc solid-phase synthesis process to sequentially couple Phe (4-Br), Trp (Boc), His (Trt), Gly, Lys (isocyano), Pro, and Ac-Ala amino acids. After seven coupling and deprotection cycles, the resin was dried in a vacuum oven. 5 mg of the dried resin was reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. The reaction was repeated once. After the reaction, 2 eq of aminocarbazole was dissolved in TMOF and allowed to react for 1 h. Then, 5 eq of NaBH3CN was dissolved in a 2:1 DCM:MeOH mixture. Acetic acid was added to 1 / 30 of the total solution volume and allowed to react overnight. After the reaction, the resin was washed three times with alternating DMF, DCM, and MeOH. Finally, 500 μL of a 95:2.5:2.5 TFA:TIPS:H2O mixture was added to the resin, and the resin was allowed to cleave for 15 min. The cleavage solution was collected to obtain Cpd.44.

[0254]

[0255] C 59 H 73 BrN 13 O8 + [M+H] +:1154.4570; Measured Mass:1154.4292; Liquid phase UV peak retention time:3.3min. This example uses RinkAmide resin, a solid-phase synthesis support compatible with biological activity screening systems, and utilizes the Fmoc solid-phase synthesis process to sequentially couple Phe(4-Br), Val, Gly, Lys(isocyano), Leu, Pro, Tyr(tBu), Ser(Trt), and Ac-Ala amino acids. After nine coupling-deprotection cycles, the resin is dried in a vacuum oven. 5mg of the dried resin is reacted with 2eq of tetrazine (DMF:H2O=9:1) for 2h; the reaction is repeated once. After the reaction, 2 eq of aminocarbazole was dissolved in TMOF. After 1 hour, 5 eq of NaBH3CN was dissolved in a 2:1 DCM:MeOH mixture. Acetic acid was added, representing 1 / 30 of the total solution volume, and the reaction was allowed to proceed overnight. The next day, 5 mg of the resin was reacted with 2 eq of tetrazine in a 9:1 DMF:H2O mixture for 2 hours. This reaction was repeated once. After the reaction, the resin was washed three times with alternating DMF, DCM, and MeOH. Finally, 500 μL of a 95:2.5:2.5 TFA:TIPS:H2O mixture was added to the resin. The resin was allowed to stand for 15 minutes for cleavage. The cleavage solution was collected to obtain Cpd.45.

[0256]

[0257] C 65 H 83 BrN 11 O 11 + [M+H] + :1272.5379; Measured Mass:1272.5312; Liquid phase UV peak retention time: 5.4min; LC conversion rate = 51.18% (254nm). (Attached Figure 25 )

[0258] Example 10

[0259] Derivatization of Aldehyde-Lysine-Modified Peptides Based on HWE Reaction

[0260] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys (isocyano), Pro, Tyr (OAll), Phe, Val, and Leu amino acids. 5 mg of dried resin was then reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After completion of the reaction, the resin was washed three times with DMF, DCM, and DMF. After drying, 10 eq of tert-butyl diethylphosphonate, 10 eq of LiBr, and 10 eq of Et3N were added to the resin in anhydrous THF and allowed to react for 24 h. After completion of the reaction, the resin was washed three times with DMF, DCM, and DMF. Finally, 500 μL of a 1:1 TFE:DCM mixture was added to the resin, and the resin was allowed to cleave for 15 min. The cleavage solution was collected to obtain Cpd.46.

[0261]

[0262] C 67 H 86 N7O 13 + [M+H] + :1196.6205; Measured Mass:1196.6192; Liquid phase UV peak retention time:12.12min. (Attached Figure 26 )

[0263] In this example, Fmoc-Ala-2-Chorotrityl resin was used to sequentially couple Lys (isocyano), Leu, Trp, Ile, Ala, and Val amino acids. 5 mg of dried resin was then reacted with 2 eq of tetrazine (DMF:H2O = 9:1) for 2 h. This reaction was repeated once. After the reaction, the resin was washed three times with DMF, DCM, and DMF. After drying, 10 eq of tert-butyl diethylphosphonate, 10 eq of LiBr, and 10 eq of Et3N were added to the resin in anhydrous THF and allowed to react for 24 h. After the reaction, the resin was washed three times with DMF, DCM, and then washed three times with DMF. Finally, 500 μL of a 95:5 mixture of TFA and H2O was added to the resin, which was allowed to cleave for 15 min. The cleavage solution was collected to obtain Cpd.47.

[0264]

[0265] C 61 H 83 N8O 12 + [M+H] + :1119.6052; Measured Mass:1119.6057; Liquid phase UV peak retention time:11.91min. (Attached Figure 27 )

[0266] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A lysine derivative, characterized in that The structure is shown in formula 1: Wherein, PG is an amino protecting group.

2. The lysine derivative according to claim 1, characterized in that The amino protecting group is selected from 9-fluorenylmethyloxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl.

3. A method for preparing a polypeptide modified with isocyanolysine or aldehyde lysine, characterized in that: The following steps are involved: (1) using the Fmoc solid-phase synthesis method, using a raw material system comprising the lysine derivative according to claim 1, to introduce a lysine derivative having an isocyanate-modified side chain into a polypeptide chain; (2) reacting the resin connected to the polypeptide chain with a tetrazine compound in a solid phase, and cleaving the resin after the reaction to obtain a polypeptide modified with aldehyde-containing lysine; or, cleaving the resin connected to the polypeptide chain by an isocyanate-tolerant cleavage method to release a free polypeptide modified with isocyanate-containing lysine, and reacting the polypeptide with a tetrazine compound in a liquid phase to obtain a polypeptide modified with aldehyde-containing lysine.

4. The preparation method according to claim 3, characterized in that The resin used in the Fmoc solid phase synthesis method is selected from Wang resin, 2-Chlorotrityl resin, Rink Amide resin, Sieber Amide resin or TentaGel resin.

5. The preparation method according to claim 3, characterized in that The conditions of the Fmoc solid phase synthesis method in step (1) are: (1) Coupling conditions of the lysine derivative according to claim 1 The feed equivalent of the lysine derivative according to claim 1 is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method; The amount of triethylamine used is 3-5 times the amount of resin loading used in the Fmoc solid phase synthesis method; The reaction solvent is selected from N,N-dimethylformamide, and the reaction time is 2-4h; (2) Other natural or non-natural Fmoc-amino acid coupling conditions The feed equivalent of Fmoc-amino acid is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method; The feeding amount of (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinyl uronium hexafluorophosphate (COMU) is 3-5 times the resin loading amount used in the Fmoc solid phase synthesis method; The feed equivalent of 2,4,6-trimethylpyridine (TMP) is 6-10 times the resin loading amount used in the Fmoc solid phase synthesis method; The reaction solvent is selected from N,N-dimethylformamide, and the reaction time is 0.5-2h; The Fmoc deprotection conditions are: 1%-5% HOBT (w / v), 2%-20% DBU (v / v), 30-200 mM (calculated based on the resin loading amount), 1-10 min.

6. The preparation method according to claim 3, characterized in that The tetrazine compound in step (2) is selected from 3,6-di-2-pyridyl-1,2,4,5-tetrazine.

7. The preparation method according to claim 3, characterized in that The solvent for the solid phase reaction and liquid phase reaction with the tetrazine compound in step (2) is selected from N,N-dimethylformamide, water or a mixture of N,N-dimethylformamide and water; The volume ratio of N,N-dimethylformamide to water in the mixture of N,N-dimethylformamide and water is 100:1-1:

100.

8. The preparation method according to claim 3, characterized in that The isocyanate-tolerant cleavage method in step (2) is selected from photocleavage or weak acid cleavage; The resin and polypeptide connecting chain fragment used in the photocleavage are selected from 3-amino-3-(2-nitrophenyl) propionic acid, and the cleavage conditions are that the resin reacts under 365nm light for 2-24h, and the solvent of the photoreaction is selected from methanol, acetonitrile or N,N-dimethylformamide; The resin used in the weak acid cleavage and the connecting chain fragment of the polypeptide are selected from 2-chlorotriphenyl, and the cleavage conditions are that the resin reacts in a mixed solution of trifluoroethanol and dichloromethane for 1-24 hours, and the volume ratio of trifluoroethanol to dichloromethane in the mixed solution of trifluoroethanol and dichloromethane is 2:1-1:

4.

9. A polypeptide modified with isocyanolysine or aldehyde lysine, characterized in that: The invention is prepared by the preparation method according to any one of claims 3 to 8.

10. Use of the polypeptide modified with isocyanolysine or aldehyde lysine according to claim 9 in protein post-translational modification research, biomedicine and material synthesis.

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