A method for preparing POCs using Fmoc solid-phase synthesis

Abstract

The application belongs to the technical field of preparing POCs using solid-phase synthesis method, and relates to a method for preparing POCs by using Fmoc solid-phase synthesis method. In view of the technical problem that the solid-phase synthesis method in the prior art cannot be used to prepare POCs containing arginine, histidine or tryptophan, small molecules C7NH2 are first coupled to a solid-phase carrier, then Fmoc-amino acids are used for solid-phase polypeptide synthesis, then solid-phase oligonucleotide synthesis is carried out, finally, the peptide-oligonucleotide conjugate POCs is released from the solid-phase carrier and precipitated, a borate solution at 90 DEG C (optimum condition) is used to remove the tert-butyloxycarbonyl (Boc) and tert-butyl (tBu) protecting groups, and the POCs are purified by using HPLC. The method is carried out on a solid support under mild conditions, can be manually carried out or carried out through a synthesizer, and is suitable for the synthesis of various peptide-oligonucleotide conjugates (POCs) containing arginine, histidine or tryptophan.

Description

Technical Field

[0001] This invention belongs to the technical field of POCs using solid-phase synthesis, specifically, it relates to a method for preparing POCs using the Fmoc solid-phase synthesis method. Background Technology

[0002] Oligonucleotides hold promise as effective therapeutic agents for treating microbial infections and human genetic diseases, including cancer. However, as antisense drugs, oligonucleotides suffer from drawbacks such as poor cellular uptake, poor intracellular transport, and sensitivity to enzymatic degradation, reducing their effectiveness in vivo. Covalent attachment of peptides to oligonucleotides (peptide-oligonucleotide conjugates) can significantly enhance their biological effects. In particular, binding to peptides effectively promotes the transmembrane transport of oligonucleotides and improves the stability of exonucleases.

[0003] For decades, scientists have been dedicated to developing various chemical methods for synthesizing peptide-oligonucleotide conjugates. Currently, there are two main methods for their synthesis: one is post-assembly conjugation, in which the peptide and oligonucleotide are synthesized and purified separately, and then linked in solution via a chemoselective reaction to form a peptide-oligonucleotide conjugate; the other is full-step synthesis, in which the peptide and oligonucleotide fragments are synthesized sequentially on the same solid support. Compared to the post-assembly conjugation strategy, full-step synthesis requires only one purification step to rapidly obtain the final product. However, a major problem with full-step synthesis is the incompatibility between peptides and oligonucleotides. For example, after solid-phase peptide synthesis, treatment with trifluoroacetic acid (TFA) is usually required to remove the protecting groups on the peptide side chains, but TFA causes the purine moieties on the oligonucleotides to be removed. To avoid this problem, Boc solid-phase synthesis has been applied to the synthesis of peptide-oligonucleotide conjugates, where protecting groups on the peptide side chains, such as Fmoc, Tfa, and For, are ultimately removed using a base. Although this method ultimately does not require strong acids to remove the protecting groups on the peptides, the α-Boc protecting group needs to be removed with strong acids each time amino acids are coupled, which is somewhat dangerous and less gentle than Fmoc solid-phase synthesis. Furthermore, overnight reaction in ammonia to remove side-chain protecting groups may lead to peptide bond breakage. Finally, the Fmoc protecting group on the arginine guanidine group is quickly removed by ammonia, resulting in deguanidinization and the formation of ornithine as a byproduct. When preparing POCs using Fmoc solid-phase synthesis, allyl oxycarbonyl (Alloc) and allyl (Allyl) groups can be used to protect the side chains of lysine, glutamic acid, and aspartic acid, but this method requires metal catalysts to remove Alloc and Allyl protecting groups, leading to heavy metal residues. Moreover, this method is only applicable to lysine, glutamic acid, and aspartic acid; POCs containing arginine, histidine, and tryptophan cannot be synthesized due to the incompatibility of Alloc and Allyl removal conditions with Fmoc.

[0004] The stepwise synthesis of peptide-oligonucleotide conjugates can also involve first synthesizing oligonucleotides and then synthesizing peptides using Fmoc-amino acids. However, the nitrile ethyl protecting group on the nucleotide phosphate triester during oligonucleotide synthesis is incompatible with the removal conditions of Fmoc (20% piperidine), leading to severe side reactions in the DNA chain. Highly acidic and unstable Bpoc and Dde have been used to protect the α-amino group of amino acids and have been successfully applied to the synthesis of peptide-oligonucleotide conjugates. Unfortunately, the compatibility of arginine and tryptophan with oligonucleotides remains problematic in this method. More seriously, due to the issue of side-chain protecting groups, classical Fmoc solid-phase peptide synthesis is still not well-suited for the synthesis of peptide-oligonucleotide conjugates; currently, the synthesis of peptide-oligonucleotide conjugates containing arginine, histidine, and tryptophan simultaneously using Fmoc solid-phase synthesis is still not feasible. Summary of the Invention

[0005] 1. The technical problem that the invention aims to solve

[0006] To address the technical problem that the Fmoc method in the prior art cannot be used for solid-phase synthesis of POCs containing arginine, histidine, or tryptophan, this application provides a method for preparing POCs using the Fmoc solid-phase synthesis method. The preparation process is carried out on a solid support under mild conditions and can be performed manually or by a synthesizer. It is suitable for the synthesis of various peptide-oligonucleotide conjugates containing arginine, histidine, or tryptophan.

[0007] 2. Technical Solution

[0008] To achieve the above objectives, the provided technical solution is as follows:

[0009] The present invention discloses a method for preparing POCs using the Fmoc solid-phase synthesis method, comprising the following steps: releasing and precipitating POCs from a solid support, removing the protecting groups of the polypeptide side chains using a borate solution and purifying the POCs; wherein the POCs comprise one or more of glycine, alanine, valine, leucine, isoleucine, oroleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or amino acid analogs.

[0010] Preferably, the amino acid analogue is selenocysteine ​​or pyrrolidone.

[0011] Furthermore, it also includes the following steps:

[0012] S1, coupling a small molecule to a solid support, wherein the small molecule has an amino functional group and the amino group is protected by Fmoc;

[0013] S2, peptide synthesis was performed using Fmoc-amino acids. After completion, a hydroxyl-containing linker arm was added to the peptide, the hydroxyl group was protected with DMTr, and then solid-phase oligonucleotide synthesis was performed.

[0014] S3, POCs are released from the solid support and precipitated, and the peptide side chain protecting groups are removed and the POCs are purified using borate solution.

[0015] Furthermore,

[0016] S1, a small molecule is coupled to a solid support, wherein the small molecule has both amino and hydroxyl functional groups, the amino group is protected by Fmoc and the hydroxyl group is protected by DMTr.

[0017] S2, uses Fmoc-amino acids for peptide synthesis, followed by oligonucleotide synthesis;

[0018] S3, POCs are released from the solid support and precipitated, and the peptide side chain protecting groups are removed and the POCs are purified using borate solution.

[0019] Further, the borate solution is a sodium borate buffer solution with a pH of 5.0–9.0 and a concentration of 50–300 mM.

[0020] Furthermore, the sodium borate buffer solution has a pH of 8.5 and a concentration of 250 mM.

[0021] Furthermore, the small molecule is C7NH2.

[0022] Furthermore, the solid support is a controllable microporous glass bead or resin.

[0023] Furthermore, the amino acid side chain protecting groups are tert-butyloxycarbonyl (Boc) and tert-butyl (tBu).

[0024] Preferably, the side-chain protecting group of histidine can be replaced with Dmbz, but the protecting group needs to be removed by reacting in ammonia water for 12 hours. The conditions are very harsh and may cause peptide bond breakage.

[0025] Furthermore, a condensing agent is used, wherein the condensing agent is HCTU.

[0026] Further purification was performed using high-performance liquid chromatography (HPLC).

[0027] Furthermore, the temperature for removing tert-butyloxycarbonyl (Boc) and tert-butyl (tBu) using borate solution is 75–95 °C, and the time is 1–8 h.

[0028] Furthermore, the POCs comprise one or more of tryptophan, aspartic acid, glutamic acid, arginine, or histidine.

[0029] 3. Beneficial effects

[0030] Compared with existing known technologies, the technical solution provided by this invention has the following beneficial effects:

[0031] This invention discloses a method for preparing peptide-oligonucleotide conjugates (POCs) using Fmoc solid-phase synthesis. First, a small molecule possessing both amino and / or hydroxyl functional groups, such as C7NH2, is coupled to a solid support. The amino group is protected with Fmoc. Then, Fmoc-amino acids protected with side chains such as Boc or tBu are used to synthesize peptides and oligonucleotides. Finally, the POCs are released from the solid support and precipitated. The protecting groups are removed using a borate solution, and the POCs are purified by HPLC. Fmoc solid-phase synthesis provides efficient and rapid acquisition of peptide-oligonucleotide conjugates (POCs), requiring only one HPLC purification step. Using sodium borate buffer at 90°C (optimal conditions) instead of trifluoroacetic acid to remove protecting groups from peptides avoids damage to oligonucleotides from strong acids, making it possible to synthesize peptide-oligonucleotide conjugates containing arginine, histidine, and tryptophan using Fmoc solid-phase synthesis. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process flow for a method for preparing POCs using the Fmoc solid-phase synthesis method of this application.

[0033] Figure 2 This is a schematic diagram of the process and structure of the product prepared in Example 1.

[0034] Figure 3 The images show the mass spectrum and HPLC analysis of the product prepared in Example 1 before the side-chain protecting groups were removed.

[0035] Figure 4 The mass spectrometry and HPLC chromatograms of the product prepared in Example 1 after the side-chain protecting groups were removed are shown.

[0036] Figure 5 This is a schematic diagram of the process and structure of the product prepared in Example 2.

[0037] Figure 6 The images show the mass spectrum and HPLC analysis of the product prepared in Example 2 before the side chain protecting groups were removed.

[0038] Figure 7 The images show the mass spectra and HPLC analyses of the product prepared in Example 2 after the side-chain protecting groups were removed.

[0039] Figure 8 This is a thin-layer chromatography chromatogram of Fmoc-His(Boc)-OH after being soaked in TCA in Example 2.

[0040] Figure 9 This is a schematic diagram of the process and structure of the product prepared in Example 3.

[0041] Figure 10 The images show the mass spectrum and HPLC analysis of the product prepared in Example 3 before the removal of the side chain protecting groups.

[0042] Figure 11 The mass spectrometry and HPLC analysis results of the product prepared in Example 3 after the side-chain protecting groups were removed are shown.

[0043] Figure 12 This is a schematic diagram of the process and structure of the product prepared in Example 4.

[0044] Figure 13 The images show the mass spectrum and HPLC analysis of the product prepared in Example 4 before the removal of the side-chain protecting groups.

[0045] Figure 14 The images show the mass spectra and HPLC analyses of the product prepared in Example 4 after the side-chain protecting groups were removed.

[0046] Figure 15 This is a schematic diagram of the process and structure of the product prepared in Example 5.

[0047] Figure 16 The images show the mass spectrometry and HPLC analysis of the product prepared in Example 5 before Boc removal.

[0048] Figure 17 The mass spectrometry and HPLC chromatograms of the product prepared in Example 5 after Boc removal are shown.

[0049] Figure 18 This is a schematic diagram of the process and structure of the product prepared in Example 6.

[0050] Figure 19 The mass spectrometry and HPLC analysis results of the product prepared in Example 6 before Boc removal are shown.

[0051] Figure 20 The mass spectrometry and HPLC analysis results of the product prepared in Example 6 after Boc removal are shown.

[0052] Figure 21 This is a schematic diagram of the process and structure of the product prepared in Example 7.

[0053] Figure 22 The mass spectrum and HPLC analysis chromatograms of the product prepared in Example 7 are shown.

[0054] Figure 23 This is a schematic diagram of the process and structure of the product prepared in Example 8.

[0055] Figure 24The mass spectrum and HPLC analysis chromatograms of the product prepared in Example 8 are shown.

[0056] Figure 25 This is a schematic diagram of the process and structure of the product prepared in Example 9.

[0057] Figure 26 The mass spectrum and HPLC analysis chromatograms of the product prepared in Example 9 are shown.

[0058] Figure 27 This is a schematic diagram of the process and structure of the product prepared in Example 10.

[0059] Figure 28 The mass spectrum and HPLC analysis chromatograms of the product prepared in Example 10 are shown.

[0060] Figure 29 This is a schematic diagram of the process flow for preparing POCs by solid-phase synthesis of Boc in Comparative Example 1.

[0061] Figure 30 This is a schematic diagram of the process flow for preparing POCs by first synthesizing oligonucleotides and then peptides in Comparative Example 2. Detailed Implementation

[0062] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] Explanation of abbreviations or nouns

[0064] POCs or POC (Peptide-Oligonucleotide Conjugates) are peptide-oligonucleotide conjugates.

[0065] CPG (Controlled Pore Glass) refers to glass beads with controllable micropores or glass spheres with controllable pore size.

[0066] Fmoc, nonafluorenemethoxycarbonyl;

[0067] Boc(t-Butyloxy carbonyl), tert-Butyloxy carbonyl;

[0068] DMTr, 4,4'-dimethoxytriphenylmethyl;

[0069] Dmbz (DiMethyl BenzimidaZole), dimethylbenzimidazole;

[0070] DIEA [N-Ethyl-N-(1-methylethyl)-2-propanamine], N,N-diisopropylethylamine;

[0071] HPLC (High Performance Liquid Chromatography);

[0072] MS (Mass Spectrum)

[0073] HUTU, Chlorobenzotriazole-1,1,3,3-Tetramethylurea hexafluorophosphate

[0074] DMF, N,N-dimethylformamide;

[0075] TFA, trifluoroacetic acid;

[0076] Protecting group: As used herein, the term “protecting group” is well known in the art, and suitable amino protecting groups include nonafluorene methoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), and tert-butyl (tBu).

[0077] Condensation reagent: In condensation reactions, the term "condensation reagent" refers to a reagent that activates less reactive sites and makes them more susceptible to attack by another reagent.

[0078] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group can then be demasked by removing the blocking group.

[0079] Solid support: The term "solid support" refers to any support that enables the synthesis of POCs. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used to perform the synthetic reaction steps and is derived to include reactive groups. In some embodiments, the solid support is a resin or CPG.

[0080] According to specific embodiments of this application, such as Figure 1 As shown, the specific steps include the following:

[0081] (1) Couple the C7NH2 small molecule to CPG or resin. This small molecule contains an amino group and a hydroxyl group: the amino group is protected with Fmoc, and peptide synthesis can be carried out on this amino group; the hydroxyl group is protected with DMTr, and oligonucleotide synthesis can be carried out on this hydroxyl group.

[0082] (2) Peptide solid-phase synthesis was performed using Boc,tBu protected Fmoc-amino acids, followed by oligonucleotide solid-phase synthesis.

[0083] (3) The peptide-oligonucleotide conjugate is released from the solid support using ammonia water, and then precipitated from the ammonia water using anhydrous ethanol. At this point, the protecting groups on the oligonucleotides have been removed from the ammonia water, but the protecting groups on the polypeptides still remain.

[0084] (4) The above peptide-oligonucleotide conjugate was placed in sodium borate buffer (pH 8.5; concentration 250mM) at 90℃ and reacted for 1-8 hours, and then separated and purified by HPLC.

[0085] Example 1

[0086] This embodiment describes the removal of Boc from the model peptide LWFA, including the following steps:

[0087] (1) Synthesis of model peptide LWFA on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid alanine does not need to be added with condensing agent, and amino acid and DIEA are added directly, with dichloromethane as solvent, and the reaction is carried out overnight; after washing, it is blocked with methanol for half an hour; the remaining three amino acids need to be added with condensing agent, and each amino acid is reacted for 40 minutes, with DMF as solvent; the Fmoc protecting group is removed with 20% piperidine during the peptide synthesis process.

[0088] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0089] (3) A portion of the fully protected peptide was analyzed by HPLC. The mass spectrometry results showed that the Boc on the model peptide LWFA was still on tryptophan, and its molecular weight (636.35) was 100 greater than that of the normal peptide (536.30).

[0090] (4) Take a portion of the fully protected peptide and place it in sodium borate buffer at 90°C for 1 hour. Perform HPLC and MS analysis on the reaction solution. The Boc on the model peptide LWFA was successfully removed, and its molecular weight changed from 636.35 to 536.30.

[0091] Example 2

[0092] This embodiment describes the removal of Boc from the model peptide HLWFA, including the following steps:

[0093] (1) Synthesis of model peptide HLWFA on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid alanine does not need to be added with condensing agent, and amino acid and DIEA are added directly, with dichloromethane as solvent, and the reaction is carried out overnight; and blocked with methanol for half an hour; the remaining 4 amino acids need to be added with condensing agent, and each amino acid is reacted for 40 minutes, with DMF as solvent; histidine is Fmoc-His(Boc)-OH protected by side chain Boc.

[0094] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0095] (3) A portion of the fully protected peptide was analyzed by HPLC. There were two peaks on the chromatogram. The second peak showed two Bocs on MS (873.4), while the first peak showed only one Boc on MS (773.4). It is possible that hexafluoroisopropanol caused some Bocs on histidine to be removed, but this did not affect the subsequent Boc removal experiment.

[0096] (4) The Boc-containing model peptide HLWFA was placed in sodium borate buffer at 90°C and reacted for 1 hour. The reaction solution was analyzed by HPLC and MS. The HPLC and MS results showed that all Boc on the model peptide HLWFA was successfully removed, and there was only one peak in the chromatogram. The molecular weight changed from the original 873.4 and 773.4 to 673.3.

[0097] (5) The weak acid TCA is required to remove the DMTr group at each step of oligonucleotide synthesis, so it is necessary to verify whether TCA removes Boc from histidine. By adding Fmoc-His(Boc)-OH to the TCA solution, we found that Boc remained stable on histidine even after 2 hours.

[0098] Example 3

[0099] This embodiment describes the removal of Boc from the model peptide FARL, including the following steps:

[0100] (1) Synthesis of model peptide FARL on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid leucine does not require the addition of condensing agent, and amino acid and DIEA are added directly, with dichloromethane as solvent, and the reaction is carried out overnight; methanol is used as a blocking agent for half an hour; the remaining amino acids require the addition of condensing agents, and each amino acid is reacted for 40 minutes; histidine uses Fmoc-His(Boc)-OH with Boc protection on the side chain; arginine uses Fmoc-Arg(Boc2)-OH with double Boc protection on the side chain. This arginine needs to be condensed with PyAop and Hoat and reacted overnight.

[0101] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0102] (3) A portion of the fully protected peptide was analyzed by HPLC. There were two peaks on the chromatogram. The second peak showed two Bocs on MS (873.4), while the first peak showed only one Boc on MS (773.4). It is possible that hexafluoroisopropanol caused some Bocs on arginine to be removed, but this did not affect the subsequent Boc removal experiment.

[0103] (4) The Boc-containing model peptide FARL was placed in sodium borate buffer at 90°C and reacted for 1 hour. The reaction solution was analyzed by HPLC and MS. The HPLC and MS results showed that all Boc on the model peptide FARL was successfully removed, and there was only one peak in the chromatogram. The molecular weight changed from the original 706.4 and 606.35 to 506.35.

[0104] Example 4

[0105] This embodiment describes the removal of Boc from the model peptide WHFARL, including the following steps:

[0106] (1) Synthesis of model peptide WHFARL on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid leucine does not require the addition of condensing agent, and amino acid and DIEA are added directly, with dichloromethane as solvent, and the reaction is carried out overnight and blocked with methanol for half an hour; the remaining amino acids require the addition of condensing agent, and each amino acid is reacted for 40 minutes; histidine uses Fmoc-His(Boc)-OH with Boc side chain protection; arginine uses Fmoc-Arg(Boc)-OH with double Boc side chain protection, and this arginine needs to be condensed with PyAop and Hoat and reacted overnight.

[0107] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0108] (3) A portion of the fully protected peptide was analyzed by HPLC. There were three peaks on the chromatogram. The three peaks had the same molecular weight and contained three Bocs. It is possible that hexafluoroisopropanol caused some of the Bocs on arginine or histidine to be removed, but this did not affect the subsequent Boc removal experiment.

[0109] (4) The Boc-containing model peptide WHFARL was placed in sodium borate buffer at 90°C and reacted for 1 hour. The reaction solution was analyzed by HPLC and MS. The HPLC and MS results showed that all Boc on the model peptide FARL was successfully removed, and there was only one peak in the chromatogram. The molecular weight changed from the original 1129.7 to 829.4.

[0110] Example 5

[0111] This embodiment describes the removal of Boc and tBu from the model peptide WDGFAL, including the following steps:

[0112] (1) Synthesis of model peptide WDGFAL on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid leucine does not need to be added with condensing agent, but directly added with amino acid and DIEA, with dichloromethane as solvent, reacted overnight, and blocked with methanol for half an hour; the remaining amino acids need to be added with condensing agent, and each amino acid reacted for 40 minutes.

[0113] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0114] (3) A portion of the fully protected peptide was analyzed by HPLC. There was one peak on the chromatogram and the mass spectrometry result was also correct (864.50), indicating that the Boc of tryptophan and the tBu of aspartic acid were still on the model peptide.

[0115] (4) The model peptide WDGFAL containing Boc and tBu was placed in sodium borate buffer at 90℃ and reacted for 2 hours. The reaction solution was analyzed by HPLC and MS. The results of HPLC and MS showed that Boc and tBu on the model peptide FARL were successfully removed. There was only one peak in the chromatogram and the molecular weight changed from 864.5 to 708.35.

[0116] Example 6

[0117] This embodiment describes the removal of Boc and tBu from the model peptide WEGFAL, including the following steps:

[0118] (1) Synthesis of model peptide WEGFAL on chlorinated resin: condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq); the first amino acid leucine does not need to be added with condensing agent, but directly added with amino acid and DIEA, solvent is dichloromethane, reacted overnight, and blocked with methanol for half an hour; the remaining amino acids need to be added with condensing agent, and each amino acid reacted for 40 minutes.

[0119] (2) Prepare a 20% hexafluoroisopropanol solution with dichloromethane as the solvent; after the polypeptide synthesis is completed, perform full protection cleavage with the above cleavage reagent, react for 1 hour, and finally precipitate the polypeptide with anhydrous diethyl ether.

[0120] (3) A portion of the fully protected peptide was analyzed by HPLC. There was one peak on the chromatogram and the mass spectrometry result was also correct (878.50), indicating that the Boc of tryptophan and the tBu of glutamate were still on the model peptide.

[0121] (4) The model peptide WDGFAL containing Boc and tBu was placed in sodium borate buffer at 90℃ and reacted for 2 hours. The reaction solution was analyzed by HPLC and MS. The HPLC and MS results showed that Boc and tBu on the model peptide FARL was successfully removed. There was only one peak in the chromatogram, and the molecular weight changed from the original 878.5 to 722.4.

[0122] Example 7

[0123] This embodiment describes an application of a method for preparing POCs using the Fmoc solid-phase synthesis method, specifically for preparing the peptide-oligonucleotide conjugate POC-1, comprising the following steps:

[0124] (1) Take 250 mg CPG into a polypeptide synthesis tube, take 20.65 mg HCTU and 38.9 mg C7-NH2 small molecules into a 10 mL centrifuge tube, dissolve them with 2 mL DMF, add 16.5 μL DIEA, put them into the polypeptide synthesis tube, and react for 1.5 hours.

[0125] (2) Add 1 mL of acetic anhydride and 1 mL of DIEA, and then bring the volume to 10 mL with DMF. Drain the reaction solution in the polypeptide synthesis tube and wash it with DMF 6 times. Finally, block the unreacted amino groups with a blocking reagent. Repeat the reaction twice, each time for 5 minutes.

[0126] (3) Condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq), each amino acid reacted for 1 hour; leucine, tryptophan, and histidine were sequentially added to CPG; histidine was protected with Fmoc-His(Boc)-OH with side chain Boc.

[0127] (4) After the polypeptide synthesis is completed, solid-phase oligonucleotides are synthesized with the sequence GTGAGAGAGTTCCATTCCAC.

[0128] (5) Remove the DMTr protecting group at the 5' end of the oligonucleotide with TCA, then put CPG into ammonia water and react for 1 hour. Finally, precipitate the peptide-oligonucleotide conjugate (white solid) with anhydrous ethanol. At this time, the protecting group on the peptide is still present.

[0129] (6) The above white solid was placed in sodium borate buffer at 90°C and reacted for 1 hour, and then purified by HPLC to obtain the final POC-1; mass spectrometry confirmed that POC-1 was correct. This shows that sodium borate buffer at 90°C can not only successfully remove Boc from POC, but also does not cause any damage to oligonucleotides.

[0130] Example 8

[0131] This embodiment describes an application of a method for preparing POCs using the Fmoc solid-phase synthesis method, specifically for preparing the peptide-oligonucleotide conjugate POC-2, comprising the following steps:

[0132] (1) Take 250 mg CPG into a polypeptide synthesis tube, take 20.65 mg HCTU and 38.9 mg C7-NH2 small molecules into a 10 mL centrifuge tube, dissolve them with 2 mL DMF, add 16.5 μL DIEA, put them into the polypeptide synthesis tube, and react for 1.5 hours.

[0133] (2) Add 1 mL of acetic anhydride and 1 mL of DIEA, and then bring the volume to 10 mL with DMF. Drain the reaction solution in the polypeptide synthesis tube and wash it with DMF 6 times. Finally, block the unreacted amino groups with a blocking reagent. Repeat the reaction twice, each time for 5 minutes.

[0134] (3) Condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq), react each amino acid for 1 hour; arginine and alanine are sequentially added to CPG; arginine uses Fmoc-Arg(Boc2)-OH with double Boc protected side chain, this arginine needs to be condensed with PyAop and Hoat as condensing agents and reacted overnight.

[0135] (4) After the polypeptide synthesis is completed, solid-phase oligonucleotide synthesis is carried out, with the sequence being TTTTTTTTTT.

[0136] (5) Remove the DMTr protecting group at the 5' end of the oligonucleotide with TCA, then put CPG into ammonia water and react for 1 hour. Finally, precipitate the peptide-oligonucleotide conjugate (white solid) with anhydrous ethanol. At this time, the protecting group on the peptide is still present.

[0137] (6) The above white solid was placed in sodium borate buffer at 90°C and reacted for 1 hour. Then, it was purified by HPLC to obtain the final POC-2. Mass spectrometry confirmed that POC-2 was correct. This shows that sodium borate buffer at 90°C can not only successfully remove the Boc of arginine on POC, but also does not cause any damage to the oligonucleotide. At the same time, when ammonolyzed with ammonia, ammonia does not have any effect on the guanidine group of arginine.

[0138] Example 9

[0139] This embodiment describes an application of a method for preparing POCs using the Fmoc solid-phase synthesis method, specifically for preparing the peptide-oligonucleotide conjugate POC-3, comprising the following steps:

[0140] (1) Take 250 mg CPG into a peptide synthesis tube, take 20.65 mg HCTU and 38.9 mg C7-NH2 small molecules into a 10 mL centrifuge tube, dissolve them with 2 mL DMF, add 16.5 μL DIEA, put them into the peptide synthesis tube, and react for 1.5 hours. (2) Add 1 mL acetic anhydride and 1 mL DIEA, and then make up to 10 mL with DMF; drain the reaction solution in the peptide synthesis tube, wash it 6 times with DMF, and finally block the unreacted amino groups with a blocking reagent. React twice, 5 minutes each time.

[0141] (3) Condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq), each amino acid reacted for 1 hour; phenylalanine, alanine, leucine, tryptophan, alanine, arginine, histidine were sequentially added to CPG; arginine was treated with Fmoc-Arg(Boc2)-OH with a double Boc protected side chain, and this arginine required PyAop and Hoat as condensing agents and reacted overnight; histidine was treated with Fmoc-His(Boc)-OH with a Boc protected side chain.

[0142] (4) After the polypeptide synthesis is completed, solid-phase oligonucleotides are synthesized with the sequence GCTAGTCTATTGCTGACGTCACTCACCTGTCAGTATAC.

[0143] (5) Remove the DMTr protecting group at the 5' end of the oligonucleotide with TCA, then put CPG into ammonia water and react for 1 hour. Finally, precipitate the peptide-oligonucleotide conjugate (white solid) with anhydrous ethanol. At this time, the protecting group on the peptide is still present.

[0144] (6) The above white solid was placed in sodium borate buffer at 90°C and reacted for 1 hour. Then, it was purified by HPLC to obtain the final POC-3. Mass spectrometry confirmed that POC-3 was correct. This shows that sodium borate buffer at 90°C can not only successfully remove Boc from the peptide, but also does not cause any damage to the oligonucleotide.

[0145] Example 10

[0146] This embodiment describes an application of a method for preparing POCs using the Fmoc solid-phase synthesis method, specifically for preparing the peptide-oligonucleotide conjugate POC-4, which includes the following steps:

[0147] (1) Take 250 mg CPG into a polypeptide synthesis tube, take 20.65 mg HCTU and 38.9 mg C7-NH2 small molecules into a 10 mL centrifuge tube, dissolve them with 2 mL DMF, add 16.5 μL DIEA, put them into the polypeptide synthesis tube, and react for 1.5 hours.

[0148] (2) Add 1 mL of acetic anhydride and 1 mL of DIEA, and then bring the volume to 10 mL with DMF. Drain the reaction solution in the polypeptide synthesis tube and wash it with DMF 6 times. Finally, block the unreacted amino groups with a blocking reagent. Repeat the reaction twice, each time for 5 minutes.

[0149] (3) Condensing agent HCTU (3.8 eq), amino acids (4 eq), DIEA (8 eq), reacted for 1 hour for each amino acid; phenylalanine, bingans, tryptophan, aspartic acid, histidine, and proline were sequentially added to CPG; histidine was prepared using Fmoc-His(Boc)-OH with Boc-protected side chain.

[0150] (4) After the polypeptide synthesis is completed, solid-phase oligonucleotides are synthesized with the sequence GTGAGAGAGTTCCATTCCAC.

[0151] (5) Remove the DMTr protecting group at the 5' end of the oligonucleotide with TCA, then put CPG into ammonia water and react for 1 hour. Finally, precipitate the peptide-oligonucleotide conjugate (white solid) with anhydrous ethanol. At this time, the protecting group on the peptide is still present.

[0152] (6) The above white solid was placed in sodium borate buffer at 90°C and reacted for 2 hours. Then, it was purified by HPLC to obtain the final POC-4. Mass spectrometry confirmed that POC-4 was correct. This shows that sodium borate buffer at 90°C can not only successfully remove Boc and tBu from POC, but also does not cause any damage to the oligonucleotide.

[0153] Example 11

[0154] This embodiment describes an application of a method for preparing POCs using the Fmoc solid-phase synthesis method, specifically for preparing the peptide-oligonucleotide conjugate POC-4, which includes the following steps:

[0155] (1) Weigh CPG into a polypeptide synthesis tube, HCTU, and small molecules into a 10 mL centrifuge tube, dissolve them with 2 mL DMF, add DIEA, and place them into a polypeptide synthesis tube. React for 1.5 hours. This small molecule contains only one amino group, and the amino group is protected with Fmoc.

[0156] (2) Add 1 mL of acetic anhydride and 1 mL of DIEA, and then bring the volume to 10 mL with DMF. Drain the reaction solution in the polypeptide synthesis tube and wash it with DMF 6 times. Finally, block the unreacted amino groups with a blocking reagent. Repeat the reaction twice, each time for 5 minutes.

[0157] (3) Condensing agent HCTU (3.8 eq), amino acid (4 eq), DIEA (8 eq), react each amino acid for 1 hour; add each amino acid sequentially to CPG.

[0158] (4) After the polypeptide synthesis is completed, a linker arm (Fmoc-Ser(DMTr)-OH) is attached to the amino terminus of the polypeptide. This linker arm contains a hydroxyl group, which is protected by DMTr.

[0159] (4) After the linker arm is attached, solid-phase oligonucleotides are synthesized, with the sequence being GTGAGAGAGTTCCATTCCAC.

[0160] (5) Place CPG in ammonia water and react for 1 hour. Finally, precipitate the peptide-oligonucleotide conjugate (white solid) with anhydrous ethanol. At this time, the protecting group on the peptide is still present.

[0161] (6) The above white solid was placed in sodium borate buffer at 90°C and reacted for 2 hours. The final polypeptide-oligonucleotide conjugate POC was then purified by HPLC.

[0162] Comparative Example 1

[0163] This comparative example is basically the same as the example. The Boc solid-phase synthesis method can realize the preparation of POC containing arginine, histidine, and tryptophan. The difference is:

[0164] (1) The amino acids used in the Boc solid-phase synthesis method are Boc-Arg(Fmoc)2-OH, Boc-His(Dnp)-OH, and Boc-Trp(For)-OH; the amino acids used in this scheme for Fmoc solid-phase synthesis are Fmoc-Arg(Boc)2-OH, Fmoc-His(Boc)-OH, and Fmoc-Trp(Boc)-OH; the two methods use different amino acids.

[0165] (2) When synthesizing peptides using the Boc solid-phase synthesis method, trifluoroacetic acid is required to remove the Boc on the α-amino group in each step, which is relatively dangerous and not as gentle as the Fmoc method. In this scheme, the Fmoc solid-phase synthesis only requires a weak base to remove the Fmoc on the α-amino group, followed by the next amino acid.

[0166] (3) After the Boc solid-phase synthesis method for POC is completed, all side chain protecting groups need to be removed with ammonia water. The Fmoc on the guanidine group of arginine will be removed quickly, and then the guanidine group will undergo a deguanidinization reaction in ammonia water to form ornithine byproduct. In this scheme, when the Fmoc solid-phase synthesis method for POC is used, the side chain protecting group of arginine is Boc. Ammonia water will not remove Boc, thus avoiding the deguanidinization reaction to form ornithine byproduct.

[0167] Comparative Example 2

[0168] This comparative example is basically the same as the previous example. The method of first synthesizing oligonucleotides and then synthesizing peptides to prepare POCs can also achieve the preparation of POCs containing histidine. The difference is:

[0169] (1) The method of first synthesizing oligonucleotides and then synthesizing polypeptides requires the pre-preparation of various amino acids with α-amino groups protected by Bpoc. The side chain protecting groups can be removed under alkaline conditions, such as Bpoc-His(Dmbz)-OH. The amino acids used in the solid-phase synthesis of Fmoc in this scheme are all available and do not need to be prepared in advance.

[0170] (2) The method of first synthesizing oligonucleotides and then synthesizing peptides to prepare POCs requires the pre-prepared α-amino group to be a Bpoc-protected amino acid. Currently, only glycine, alanine, phenylalanine, histidine, and leucine are available. Bpoc-protected arginine and tryptophan have not been synthesized and cannot be used for POC preparation. In addition to the above-mentioned amino acids, the amino acids used in the Fmoc solid-phase synthesis of POCs in this scheme also include arginine, tryptophan, aspartic acid, glutamic acid, lysine, etc., which is a much wider range of amino acids.

[0171] As can be seen from the examples and comparative examples, the method for preparing POC by solid-phase synthesis of Fmoc in this scheme is simple and mild, can be used with a sufficient number of amino acids, and will not produce side reactions similar to arginine deguanidinization.

[0172] This application provides a novel and practical strategy for the synthesis of peptide-oligonucleotide conjugates (POCs), which involves peptide synthesis using Fmoc-amino acids, followed by oligonucleotide synthesis on the same solid-phase support. After synthesis, the peptide is released from the (CPG or resin) support using ammonia, and finally, Boc and tBu are removed using a borate buffer at 75–95°C instead of trifluoroacetic acid, thus avoiding the degradation of oligonucleotides by strong acids. At both the peptide and POC levels, it has been demonstrated that the Boc protecting groups on histidine, tryptophan, and arginine, and the tBu protecting groups on glutamic acid and aspartic acid, can be rapidly removed in an optimal 90°C borate buffer without any damage to the peptide or oligonucleotide.

Claims

1. A method for preparing POCs using the Fmoc solid-phase synthesis method, characterized in that: Includes the following steps: S1, coupling the small molecule to the solid support, wherein the structural formula of the small molecule is as follows: ， The amino group of the small molecule is protected with Fmoc, and the hydroxyl group of the small molecule is protected with DMTr. The solid support is a controllable microporous glass bead or resin. S2, removing the Fmoc protecting group from the amino group of the small molecule, using Fmoc-amino acids to perform solid-phase polypeptide synthesis on the amino group of the small molecule, and then performing solid-phase oligonucleotide synthesis on the hydroxyl group of the small molecule; wherein, the polypeptide is composed of one or more of glycine, alanine, leucine, proline, phenylalanine, tryptophan, aspartic acid, glutamic acid, arginine and histidine, and contains at least one of tryptophan, aspartic acid, glutamic acid, arginine or histidine; the side chains of tryptophan, arginine and histidine in the polypeptide are protected with tert-butyloxycarbonyl (Boc), and the side chains of aspartic acid and glutamic acid in the polypeptide are protected with tert-butyl (tBu); S3. The POCs are released from the solid support with ammonia water and then precipitated with anhydrous ethanol. The precipitated POCs are placed in a sodium borate buffer solution with a concentration of 250 mM and a pH of 8.5 and reacted at 90 °C for 1-8 hours to remove the Boc and / or tBu protecting groups of the peptide side chains. The POCs are then purified by high performance liquid chromatography (HPLC).

2. The method for preparing POCs using the Fmoc solid-phase synthesis method according to claim 1, characterized in that: When the side chain protecting group of the polypeptide is only Boc, the reaction time in the sodium borate buffer in step S3 is 1 hour.

3. The method for preparing POCs using the Fmoc solid-state synthesis method according to claim 1, characterized in that: When the polypeptide contains at least one of aspartic acid or glutamic acid, the reaction time in the sodium borate buffer in step S3 is 2 hours.

4. The method for preparing POCs using the Fmoc solid-phase synthesis method according to claim 1, characterized in that: The solid support is a controllable microporous glass bead.

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