A method for the synthesis of sequence-specific phosphorylated arginine polypeptides

Abstract

This invention discloses a method for synthesizing sequence-specific phosphorylated arginine peptides. The method first prepares Fmoc-Arg(N3)-OH and Boc-Arg(N3)-OH synthons; then, using a solid-phase peptide coupling synthesis method, azidoarginine is introduced at any designated site using the above synthons to prepare a precursor peptide containing ArgN3; next, the precursor peptide undergoes a Staudinger reaction with a phosphite compound to construct an N-P bond, yielding a phosphorylated arginine peptide containing a protecting group; finally, the protecting group is removed under mild conditions to obtain the sequence-specific phosphorylated arginine peptide. This invention realizes the chemical synthesis of azidoarginine synthons and the microwave-assisted SPPS preparation of ArgN3-containing precursor peptides. The entire route does not require a noble metal catalyst, is highly compatible with existing microwave SPPS processes, and is characterized by mild conditions and simple operation, applicable to the preparation of phosphorylated arginine peptides with arbitrary sequences.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide chemical synthesis technology, specifically relating to a method for synthesizing sequence-specific phosphorylated arginine polypeptides. Background Technology

[0002] Phosphorylation of arginine in proteins (pArg) is an important post-translational modification with well-defined physiological functions. Since the discovery of the first prokaryotic arginine protein kinase, McsB, in 2009, nearly two decades of research have shown that pArg is widely involved in bacterial signal transduction, bacterial stress response, and protein quality regulation. Recently, multiple research teams have identified numerous high-confidence pArg sites in Jurkat and HeLa cells, indicating the widespread existence of pArg modification in eukaryotic cells. Currently, research on the physiological functions of pArg is still in its early stages, and there is an urgent need to prepare sequence-specific pArg peptides for specific research purposes to further elucidate their biological effects and evaluate the performance of related antibodies.

[0003] Compared to the stable PO bond in traditional O-phosphorylation, the PN bond in pArg has a higher free energy, making it extremely prone to breakage under acidic or heating conditions. From a molecular structure analysis, the oxygen atom in the P=O bond of phosphoramide... 2 Hybrid orbitals and sp orbitals of phosphorus atoms 3 The orbitals form σ bonds, while the unhybridized p orbitals in the oxygen atom form d-pπ bonds with the 3d orbitals of the phosphorus atom. Because the d-pπ bonds are located in higher energy levels, their overlap with the lone pair electrons of the nitrogen atom is low. In physiological environments, the lone pair electrons of the nitrogen atom are easily captured by protons and protonated, thereby enhancing the leaving ability of the amino terminus and leading to a significant decrease in the stability of the PN bond (the specific mechanism is...). For example, the pArg model peptide has a half-life of only 1-2 hours at 25°C and pH < 4.0; when the temperature is increased to 60°C, the half-life is further shortened to less than 1 hour. This fully demonstrates the high sensitivity of pArg to acid and heat treatment.

[0004] However, existing solid-phase peptide synthesis (SPPS) strategies, whether using the Boc or Fmoc / tBu system, cannot avoid the use of strongly acidic reagents. The Boc system requires 20%-50% trifluoroacetic acid (TFA) during the removal of the main chain α-amino Boc protecting group, and the peptide is ultimately cleaved from the resin with hydrofluoric acid (HF) or trifluoromethanesulfonic acid (TFMSA). While the Fmoc / tBu system avoids strong acids during coupling cycles, the removal of side-chain protecting groups and resin cleavage still require 80%-95% TFA concentration. This makes it exceptionally difficult to introduce pArg groups containing unstable PN bonds at specific sites using conventional SPPS.

[0005] Currently, only two methods have been reported for preparing sequence-specific pArg peptides: one is an enzymatic synthesis method using McsB arginine kinase activity; the other is a chemical synthesis method using trichloroethyl (Tc) protected phosphorylated arginine as a synthon (Fmoc-Arg(PO3Tc2)-OH), which is coupled sequentially from the C-terminus to the N-terminus by conventional SPPS.

[0006] Enzymatic synthesis methods use ATP as a phosphate donor, utilizing McsB kinase to phosphorylate arginine residues in peptides or proteins in vitro. The McsB used in this method is typically a recombinant, heterologous protein, resulting in high costs. Furthermore, existing research shows that McsB exhibits a certain preference for substrate sequences, failing to explain its ability to phosphorylate hundreds of dynamically changing sites within bacteria. It is speculated that an unknown adapter protein may exist in vivo to guide McsB to recognize specific sites. Simultaneously, substrate structure, protein-protein interactions, and other factors further complicate the in vitro enzymatic reaction, making it difficult to prepare pArg peptides with arbitrary sequences, and even unable to reproduce certain identified pArg sites, resulting in low sequence universality. In addition, limited by enzyme reaction scale, kinetic equilibrium, and buffer composition, enzymatic methods suffer from low conversion rates and difficult product purification, hindering large-scale preparation. For example, when using a 13-peptide derived from CtsR as a substrate, the conversion rate of pArg-phosphorylated peptides is less than 40%.

[0007] The chemical synthesis method uses the Fmoc-Arg(PO3Tc2)-OH synthon, which is used to prepare peptides via SPPS coupling from the C-terminus to the N-terminus. The Tc protecting group can improve the acid tolerance of the PN bond. After side chain deprotection and cleavage, the Tc group is removed by Pd-catalyzed hydrogenolysis to obtain the target pArg peptide. Theoretically, this method has good sequence universality, but it has obvious limitations: the synthon Fmoc-Arg(PO3Tc2)-OH has no commercial source, the laboratory preparation steps are cumbersome, and the overall yield is low; both the synthon and Tc removal require a large amount of Pd / C noble metal catalysts, which is not only costly and uneconomical, but also may introduce the risk of heavy metal pollution. At the same time, the HBTU / DIPEA coupling system used in this method is water-sensitive and needs to be prepared and used immediately, and the reaction time is long. It has not been proven to be compatible with the current mainstream microwave-assisted SPPS (90℃ high temperature, DIC / Oxyma and other inexpensive coupling agents), and cannot make full use of efficient and rapid modern peptide synthesis technology.

[0008] In summary, existing enzymatic synthesis methods and Tc-protected chemical synthesis methods cannot meet the urgent need for preparing sequence-specific pArg peptides, as they suffer from high costs, difficulties in large-scale production, low sequence universality, environmental unfriendliness, or incompatibility with mainstream synthetic processes. Therefore, developing a novel, mild, noble metal catalyst-free, microwave SPPS compatible, sequence universality-highly universal, and environmentally friendly chemical synthesis method for sequence-specific phosphorylated arginine peptides has significant theoretical and practical value. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing sequence-specific phosphorylated arginine polypeptides.

[0010] The technical solution of the present invention is as follows:

[0011] A method for synthesizing a sequence-specific phosphorylated arginine polypeptide includes the following steps:

[0012] (1) A solid-phase polypeptide coupling synthesis method was adopted, using Fmoc-Arg(N3)-OH or Boc-Arg(N3)-OH as synthons to prepare precursor polypeptides containing azidoarginine.

[0013] The structural formula of Fmoc-Arg(N3)-OH is: ,

[0014] The structural formula of Boc-Arg(N3)-OH is: ;

[0015] (2) The precursor polypeptide obtained in step (1) is subjected to a Staudinger reaction with a phosphite compound to obtain a phosphorylated arginine polypeptide containing a protecting group.

[0016] (3) Remove the above protecting group to obtain sequence-specific phosphorylated arginine polypeptide.

[0017] In a preferred embodiment of the present invention, the Fmoc-Arg(N3)-OH or Boc-Arg(N3)-OH is prepared by reacting fluorosulfonyl azide with Fmoc-L-Arg or Boc-L-Arg in the presence of a catalyst, and the structural formula of the fluorosulfonyl azide is as follows: .

[0018] In a preferred embodiment of the present invention, step (1) employs the Fmoc / tBu solid-phase peptide synthesis strategy or the Boc solid-phase peptide synthesis strategy.

[0019] More preferably, the solid-phase polypeptide coupling synthesis method in step (1) adopts a non-microwave-assisted coupling method or a microwave-assisted coupling method.

[0020] More preferably, the reaction temperature of the solid-phase polypeptide coupling synthesis method is 15-120 °C.

[0021] In a preferred embodiment of the present invention, the phosphite compound in step (2) is photosensitive tris(4,5-dimethoxy-2-nitrobenzyl phosphite).

[0022] More preferably, the Staudinger reaction in step (2) is carried out under light-protected conditions at 0-60 °C.

[0023] More preferably, the solvent for the Staudinger reaction is selected from at least one of DMF, PBS buffer, and Tris buffer; and the Staudinger reaction is carried out under argon protection or without a protective atmosphere.

[0024] In a preferred embodiment of the present invention, the deprotection in step (3) is carried out under light irradiation of 280-420 nm; the solvent for the deprotection reaction is selected from at least one of PBS buffer, Tris buffer, ACN / DDW mixed solvent and ACN / DDW / lower alcohol mixed solvent, wherein the lower alcohol is selected from methanol, ethanol and propanol.

[0025] In a preferred embodiment of the present invention, the solid-phase polypeptide coupling synthesis method in step (1) uses MBHA Rink resin, Wang resin or CTC resin.

[0026] The beneficial effects of this invention are:

[0027] 1. The sequence of this invention has high universality, and can prepare sequence-specific pArg peptides at any specified site according to research needs, breaking through the bottleneck of enzymatic methods being limited by the specificity of kinase substrates;

[0028] 2. The synthetic route of this invention is novel. By first constructing a precursor polypeptide containing arginine (ArgN3) and then constructing a PN bond via the Staudinger reaction, the difficulty of directly coupling unstable pArg synthons is avoided, and the synthetic flexibility is significantly improved.

[0029] 3. This invention is highly compatible with existing microwave-assisted solid-phase peptide synthesis processes and can use inexpensive and efficient carbodiimide coupling agents, shortening coupling time and reducing reagent costs;

[0030] 4. The entire process of this invention does not require precious metal catalysts, the post-processing is simple, it is economical and environmentally friendly, and avoids the risk of heavy metal pollution.

[0031] 5. The reaction conditions of this invention are mild (the Staudinger reaction and subsequent deprotection are carried out under mild conditions), which effectively protects the stability of the PN bond. The resulting product has high purity and sufficient structural confirmation, which can meet the needs of downstream biological research and antibody evaluation.

[0032] 6. The overall technical solution of this invention provides a novel synthetic approach and practical tools for the field, promoting in-depth research on pArg-related post-translational modifications. It also provides new tools for studying N-phosphorylation modifications in eukaryotic organisms. Attached Figure Description

[0033] Figure 1 This is a route diagram for synthesizing pArg polypeptides of arbitrary sequences in this invention.

[0034] Figure 2 Showing different Cu in Embodiment 4 of the present invention 2+ The effect of concentration on reaction yield.

[0035] Figure 3 This invention demonstrates the use of semi-preparative chromatography to purify the ArgN3-containing precursor polypeptide (TRFTSER(N3)GYIVESKK-NH2) in Example 5. Chromatographic peak a: precursor polypeptide.

[0036] Figure 4 The chromatogram is of the purified precursor polypeptide (TRFTSER(N3)GYIVESKK-NH2) in Example 5 of this invention.

[0037] Figure 5 This is the MALDI-ToF-MS mass spectrum of the precursor polypeptide (TRFTSER(N3)GYIVESKK-NH2) in Example 5 of this invention. Theoretical mass-to-nuclear ratio: [M+H] + = 1825.967, Actual measurement: [M+H] + = 1825.795.

[0038] Figure 6 This is a high-resolution mass spectrometry characterization (first-order mass spectrometry) of ArgN3 Peptide in Example 5 of the present invention. Theoretical mass: [M+2H] 2+ = 913.487, [M+3H] 3+ = 609.3271, [M+4H] 4+ = 457.2471, Actual observation: [M+2H] 2+ = 913.4872, [M+3H] 3+ = 609.3272, [M+4H] 4+ = 457.2471. δ=0.2 ppm

[0039] Figure 7 This is a high-resolution secondary mass spectrum of the ArgN3-containing precursor polypeptide in Example 5 of the present invention.

[0040] Figure 8 This illustrates the chromatographic purification of the TRFTSER(P(OR3))GYIVESKK-NH2 polypeptide in Example 7 of the present invention. Chromatographic peak a: precursor polypeptide TRFTSER(N3)GYIVESKK-NH2; chromatographic peak b: target polypeptide TRFTSER(P(OR3))GYIVESKK-NH2; chromatographic peak c: excess phosphite (tris(4,5-dimethoxy-2-nitrobenzyl) phosphite). Chromatographic conditions: TC C18(2) 5 µm, 250 x 4.6 mm, 170 Å pore size column; mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient: 0–5 min 5% → 16.25% B, 5–35 min 16.25% → 38.75% B, 35–37 min 38.75% → 100% B, 37–47 min 100% B.

[0041] Figure 9 The chromatogram is of the purified pArg polypeptide TRFTSER(P(OR3))GYIVESKK-NH2 containing a photocage protecting group in Example 7 of this invention.

[0042] Figure 10This demonstrates the reversed-phase high-performance liquid chromatography (RP-HPLC) purification process of the arginine phosphorylated polypeptide (TRFTSEpRGYIVESKK-NH2) in Example 8 of this invention. Chromatographic peak a: dephosphorylated polypeptide TRFTSERGYIVESKK-NH2; chromatographic peak b: target pArg polypeptide TRFTSEpRGYIVESKK-NH2; chromatographic peak c: photocage-protected polypeptide TRFTSER(P(OR3))GYIVESKK-NH2. Chromatographic conditions: TC C18(2) 5 µm, 250 x 4.6 mm, 170 Å pore size column; mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient: 0–5 min 5% → 16.25% B, 5–35 min 16.25% → 38.75% B, 35–37 min 38.75% → 100% B, 37–47 min 100% B.

[0043] Figure 11 This is the chromatogram of the purified pArg polypeptide TRFTSEpRGYIVESKK-NH2 in Example 8 of the present invention.

[0044] Figure 12 This is a high-resolution first-order mass spectrum (ESI-Orbitrap) of the pArg polypeptide (H-TRFTSEpRGYIVESKK-NH2) synthesized in Example 8 of this invention. Theoretical value [M+3H] 3+ =627.3190, actual measured value [M+3H] 3+ =627.3216, δ=4.1 ppm

[0045] Figure 13 This is a secondary mass spectrum of the pArg polypeptide (H-TRFTSEpRGYIVESKK-NH2) synthesized in Example 8 of this invention.

[0046] Figure 14 The image shows the MALDI-ToF-MS mass spectrum of (TRFTSER(N3)GYIVESKK-NH2) synthesized in Example 9 of this invention. The SPPS compatible temperature can reach 110℃.

[0047] Figure 15 The MALDI-ToF-MS mass spectrum of (TRFTSER(N3)GYIVESKK-NH2) synthesized in Example 9 of this invention can be reduced to 50°C with SPPS compatibility.

[0048] Figure 16The MALDI-ToF-MS mass spectrum of (TRFTSER(N3)GYIVESKK-NH2) synthesized in Example 9 of this invention can be reduced to 15℃ for SPPS compatibility.

[0049] Figure 17 The image shows the MALDI-ToF-MS mass spectrum of (TRFTSER(N3)GYIVESKK-NH2) synthesized in Example 9 of this invention. The SPPS compatible temperature cannot exceed 120°C.

[0050] Figure 18 In Example 10 of this invention, DMF was replaced with PBS buffer and no argon protection was used. The temperature was 45-48°C. In the figure, a represents the Staudinger conjugate product ArgPOR3 peptide.

[0051] Figure 19 In Example 10 of this invention, DMF was replaced with Tris Buffer and no argon protection was used. The temperature was 45-48°C. In the figure, a represents the Staudinger conjugate product ArgPOR3 peptide.

[0052] Figure 20 In Example 10 of this invention, DMF was used under argon protection and the temperature was lowered to room temperature. In the figure, a represents the Staudinger conjugate product ArgPOR3 peptide.

[0053] Figure 21 In Example 10 of this invention, DMF was used under argon protection and the temperature was lowered to 0°C. In the figure, a represents the Staudinger conjugate product ArgPOR3 peptide.

[0054] Figure 22 In Example 10 of this invention, DMF was used under argon protection and the temperature was raised to 60°C. In the figure, a represents the Staudinger conjugate product ArgPOR3 peptide.

[0055] Figure 23 In Example 11 of this invention, photolysis was performed using light with a wavelength of 280-300 nm, with Tris Buffer as the solvent. (Figure a)

[0056] This represents the deprotected product pArg peptide.

[0057] Figure 24 In Example 11 of this invention, photolysis was performed using light with a wavelength of 400-420 nm, with Tris Buffer as the solvent. (Figure a)

[0058] b represents the deprotected product pArg peptide; b represents the unprotected ArgPOR3 peptide.

[0059] Figure 25 In Example 11 of this invention, 365 nm wavelength light was used for photolysis, and ACN / DDW / lower alcohol was used as the solvent.

[0060] In the figure, a represents the deprotected product pArg peptide; b represents the unprotected ArgPOR3 peptide.

[0061] Figure 26 In Example 11 of this invention, photolysis was performed using 365 nm wavelength light, with ACN / DDW as the solvent. In the figure, a represents the deprotected product pArg peptide; b represents the unprotected ArgPOR3 peptide.

[0062] Figure 27 In Example 11 of this invention, photolysis was performed using 365 nm wavelength light, with PBS Buffer as the solvent. In the figure, a represents the deprotected product pArg peptide; b represents the unprotected ArgPOR3 peptide. Detailed Implementation

[0063] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0064] Figure 1 This is a roadmap for synthesizing pArg peptides of arbitrary sequences in this invention. First, solid-phase peptide synthons containing guanidinium azide structures (Fmoc-Arg(N3)-OH, Boc-Arg(N3)-OH) were synthesized. Precursor peptides containing ArgN3 were then synthesized using the conventional SPPS method. Subsequently, under mild conditions, the precursor peptides and phosphites were reacted with a Staudinger reaction to form a PN bond. The protecting group was then removed using conditions compatible with PN bonds, ultimately yielding a target sequence-specific phosphorylated arginine peptide. Examples 1-8 below follow... Figure 1 The shown route proceeds through each step of the reaction sequentially, and Examples 9-11 further demonstrate the compatibility and scope of the technical features.

[0065] Example 1 Synthesis of fluorosulfonyl azide (FSO2N3)

[0066] The structural formula of the fluorosulfonyl azide synthesized in this embodiment is as follows: The specific method is as follows:

[0067] In a 25 mL plastic reaction flask equipped with a magnetic stirrer, 5 mL of a 0.5 mol / L sodium azide aqueous solution (containing 165 mg of sodium azide, 2.52 mmol) was mixed with an equal volume of methyl tert-butyl ether (MTBE). Subsequently, 1 g (3 mmol) of 1-(fluorothio)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate (SuFEx-IT) was dissolved in 256 μL of acetonitrile, and the resulting viscous solution was rapidly added to the reaction mixture, which was cooled in an ice-water bath and vigorously stirred. The container holding SuFEx-IT was rinsed once with 256 μL of acetonitrile, and the rinse solution was incorporated into the reaction system to ensure complete transfer of materials. The reaction flask was sealed with a loose stopper and placed in an ice-water bath, where the mixture was vigorously stirred at 600 r / min for 10 min.

[0068] After the reaction was complete, the system was allowed to stand for 30 seconds to allow for phase separation. The upper organic phase containing the target product FSO₂N₃ was carefully aspirated using a pipette. The concentration of the FSO₂N₃ solution was determined using 19F NMR with a known concentration of benzyl sulfonyl fluoride (PMSF) as an internal standard, and the final concentration was adjusted to 200 mmol / L by adding DMF. Based on the determined concentration and solution volume, the yield of FSO₂N₃ was calculated to be 86%. This solution was used directly for subsequent reactions without further purification.

[0069] The NMR data of FSO2N3 obtained in this embodiment are as follows: 19 F NMR (375 MHz, methyl tert-butyl ether as solvent): δ = 62.4 (δ = 53.4 with PMSF as standard).

[0070] Example 2 Synthesis of Boc-Arg(N3)-OH

[0071] The structural formula of the Boc-Arg(N3)-OH synthesized in this embodiment is: The specific method is as follows:

[0072] In a 10 mL reaction flask, Boc-L-Arg (172.8 mg, 0.63 mmol, 1.0 eq) was dissolved in 700 μL of N,N-dimethylformamide (DMF) and 300 μL of dimethyl sulfoxide (DMSO), followed by the addition of 188.3 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 191.8 mg, 1.26 mmol, 2.0 eq), and the mixture was stirred at room temperature for 5 min. Then, 100 μL of copper sulfate solution (10 mg / mL, 0.02% eq) was added, and stirring continued until the solution turned a stable bright blue. The reaction flask was transferred to an ice bath, and 1 mL of FSO2N3 (78.8 mg, 0.63 mmol, 1.0 eq) prepared in Example 1 was slowly added dropwise, and stirring continued for 8 h after the addition was complete.

[0073] After the reaction was complete, 10 mL of water was added for dilution, and the solution was acidified to pH 1–2 with 1 mol / L hydrochloric acid. Extraction was performed with ethyl acetate (EA, 20 mL × 3), maintaining the pH of the aqueous phase at 1–2 throughout the extraction process. The organic phases were combined and washed successively with saturated sodium chloride (20 mL, containing 0.1 mol / L hydrochloric acid) and saturated lithium chloride (20 mL, containing both 0.1 mol / L hydrochloric acid and 0.1 mol / L lithium chloride), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation under reduced pressure at no more than 35 °C. The crude product was purified by MP-200 reversed-phase medium-pressure chromatography (mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; flow rate: 12 mL / min; detection wavelength: 214 nm, 254 nm; gradient: 0–5 min 5% B, 5–40 min 5%→95% B, 40–45 min 100% B), and concentrated under reduced pressure to obtain a white solid Boc-Arg(N3)-OH (28 mg, yield 15%). Besides 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), other usable organic bases include triethylamine (TEA), diisopropylethylamine (DIPEA) and its derivatives, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and its derivatives, which can also successfully prepare this synthon. Since the final product is the same, redundant characterization is not performed again.

[0074] The NMR data of Boc-Arg(N3)-OH obtained in this embodiment are as follows:

[0075] 1H NMR (400 MHz, CDCl3) δ 3.47 (dd, J = 7.7, 6.5 Hz, 2H), 3.39 (d, J =9.9 Hz, 1H), 3.39 (s, 1H), 3.35 (t, J = 6.8 Hz, 2H), 2.59 – 2.52 (m, 2H), 1.84 (q, J = 7.0 Hz, 2H), 1.82 – 1.73 (m, 1H), 1.76 – 1.62 (m, 3H), 1.28 (s, 1H).

[0076] 13 C NMR (101 MHz, CDCl3) δ 176.29, 50.15, 49.21, 46.15, 37.02, 29.91, 28.62, 27.59, 23.34.

[0077] HRMS (ESI / Q-ToF): [M+H]⁺ Calcd for C 11 H 20 N6O4 301.1616; Found 301.1619; δ: 1.0 ppm.

[0078] Example 3 Synthesis of Fmoc-Arg(N3)-OH

[0079] The structural formula of the Fmoc-Arg(N3)-OH synthesized in this embodiment is: The specific method is as follows:

[0080] In a 10 mL reaction flask, Fmoc-L-Arg (249.8 mg, 0.63 mmol, 1.0 eq) was dissolved in 700 μL of N,N-dimethylformamide (DMF) and 300 μL of dimethyl sulfoxide (DMSO) solution, and stirred at room temperature for 5 min. Then, 100 μL of copper sulfate solution (10 mg / mL, 0.02% eq) was added, and stirring continued until the system turned a stable bright blue color. The reaction flask was transferred to an ice bath, and FSO2N3 (78.8 mg, 0.63 mmol, 1.0 eq) prepared in Example 1 was slowly added dropwise. After the addition was complete, stirring was continued for 6 h.

[0081] After the reaction was complete, 10 mL of water was added for dilution, and the solution was acidified to pH 1–2 with 1 mol / L hydrochloric acid. Extraction was performed with ethyl acetate (EA, 20 mL × 3), ensuring the pH of the aqueous phase remained between 1 and 2 during extraction. The organic phases were combined and washed successively with saturated sodium chloride (20 mL, containing 0.1 mol / L hydrochloric acid) and saturated lithium chloride (20 mL, containing both 0.1 mol / L hydrochloric acid and 0.1 mol / L lithium chloride). The solutions were dried over anhydrous sodium sulfate and concentrated by rotary evaporation under reduced pressure at no more than 35 °C. The crude product was purified by an MP-200 reversed-phase preparative chromatography system (mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; flow rate: 12 mL / min; gradient: 0–5 min 5% B, 5–40 min 5%→95% B, 40–45 min 100% B), and concentrated under reduced pressure to give a white solid compound Fmoc-Arg(N3)-OH (58 mg, yield 22%).

[0082] The NMR data of Fmoc-Arg(N3)-OH obtained in this embodiment are as follows:

[0083] 1 H NMR (400 MHz, CD3OD) δ 7.81 (dd, J = 7.5, 3.4 Hz, 2H), 7.69 (t, J =7.4 Hz, 1H), 7.44 – 7.27 (m, 3H), 4.44 – 4.38 (m, 1H), 4.22 (dt, J = 26.0, 6.8 Hz, 3H), 1.92 (dt, J = 12.2, 7.2 Hz, 2H), 1.74 (q, J = 9.9, 9.1 Hz, 1H).

[0084] 13 C NMR (101 MHz, DMSO-d6) δ 178.35, 173.98, 156.63, 155.78, 141.18,128.10, 127.53, 125.73, 120.57, 66.13, 53.84, 47.13, 44.45, 28.19, 25.71.

[0085] HRMS (ESI / Q-ToF): [M+H]⁺ Calcd for C 21 H 22 N6O4 423.1775; Found 423.1776; δ: 0.3 ppm.

[0086] Example 4 Different Cu2+ Concentration range and its donor

[0087] Cu used in this embodiment 2+ The donor salts included copper sulfate (CuSO4) and copper trifluoromethanesulfonate (Cu(OTf)2). Both successfully synthesized the target synthon, and the type of anion had no effect on the yield.

[0088] In this embodiment, Cu 2+ Concentration affects the reaction yield; a range of 0.01% to 5% allows for the successful synthesis of Boc- or Fmoc-azidoarginine, with yields similar to Cu. 2+ The relationship between concentrations is shown in the figure. Figure 2 .

[0089] Example 5: Synthesis of the ArgN3-containing precursor polypeptide TRFTSER(N3)GYIVESKK-NH2 using Fmoc-ArgN3-OH

[0090] The precursor polypeptide TRFTSER(N3)GYIVESKK-NH2 (SEQ ID NO.01) synthesized in this embodiment has the following structural formula: The specific method is as follows:

[0091] The Fmoc / tBu solid-phase peptide synthesis strategy was employed, and the synthesis was carried out on MBHA Rink resin (0.312 mmol / g) at a synthesis scale of 25 μmol. Fmoc protected amino acids: Thr(tBu), Arg(Pbf), Phe, Thr(tBu), Ser(tBu), Glu(OtBu), Gly, Tyr(tBu), Ile, Val, Glu(OtBu), Ser(tBu), Lys(Boc), Lys(Boc), and Fmoc-Arg(N3)-OH prepared in Example 3 were used for peptide synthesis in a CEM microwave reactor.

[0092] Deprotection was performed using a 20% piperidine DMF solution under microwave conditions of 90 °C, 60 W, with a heating time of 60 s, a holding time of 50 s, and low-speed stirring. For the coupling reaction of conventional amino acids, a 5-fold excess of Fmoc was used to protect the amino acid, using the CarboMAX coupling formulation, under microwave conditions of 90 °C, 70 W, with a heating time of 60 s, a holding time of 100 s, and low-speed stirring.

[0093] Coupling of the first amino acid on the Rink resin: Add 2 mL of a DMF / DCM mixture (DMF / DCM = 1:1) to 25 μmol MBHA Rink resin, and allow it to swell for 1 h at room temperature with shaking. Transfer the resin to a peptide synthesis tube, wash the resin with 2 mL of DMF, and repeat once. Add 3 mL of a 20% piperidine DMF solution, heat at 90 °C and 60 W for 60 s, maintain the temperature for 50 s, and stir at low speed to deprotect the resin. After deprotection, wash four times with 2 mL of DMF each time. Subsequently, 250 μL of a 5-fold excess of Fmoc-L-Lys(Boc)-OH (0.125 mmol, 58.6 mg) in DMF, 500 μL of 0.5 M DIC in DMF, 250 μL of 1 M Oxyma in DMF, and 8.6 μL of DIPEA were added to the peptide reaction tube. The mixture was coupled in a microwave reactor at 90 °C, 70 W, for 60 s, held for 100 s, and stirred at low speed. After coupling, the sample was washed three times with 2 mL of DMF each time. The coupling process was repeated once until the resin showed no color change when detected by Kaiser assay.

[0094] Coupling of arginine azide (ArgN3) in peptide sequences: 3 mL of 20% piperidine DMF solution was added to the peptide synthesis tube. The mixture was deprotected in a microwave reactor at 90 °C and 60 W for 60 s, held for 50 s, and stirred at low speed. After deprotection, the peptide was washed four times with 2 mL of DMF each time. Experiments verified that ArgN3 is resistant to microwave fields and high temperatures up to 120 °C. The following two methods can be used to couple Fmoc-Arg(N3)-OH:

[0095] Method 1 (Low-Temperature Coupling Method): Add DMF (840 μL), a DMF solution of 0.5 M HATU (90 μL), a DMF solution of 1 M HOBt (48 μL), and 17.2 μL of DIPEA to a 1.5 mL centrifuge tube containing a 2-fold excess of Fmoc-Arg(N3)-OH synthon (50 μmol, 21.2 mg). Stir at low speed overnight at room temperature. Use a small amount of resin to test the coupling efficiency using the Kaiser method. If coupling is incomplete, continue the reaction at room temperature for 12-24 h, or microwave at 50 °C for 10 min.

[0096] Method 2 (High-Temperature Coupling Method): Add DMF (250 μL), 0.5 M DIC DMF solution (500 μL), 1 M Oxyma DMF solution (250 μL), and DIPEA 8.6 μL to a 1.5 mL centrifuge tube containing 2 times excess Fmoc-Arg(N3)-OH synthon (50 μmol, 21.2 mg). React at 90 °C for 100 s in a microwave reactor. If Kaiser assay indicates insufficient coupling, the coupling can be repeated once.

[0097] Subsequent deprotection was performed using a 20% piperidine DMF solution under microwave conditions of 90 °C, 60 W, with a heating time of 60 s, a holding time of 50 s, and low-speed stirring. The amino acid coupling reaction used a 5-fold excess of Fmoc to protect the amino acid, employing the CarboMAX coupling formulation, under microwave conditions of 90 °C, 70 W, with a heating time of 60 s, a holding time of 100 s, and low-speed stirring.

[0098] Peptide cleavage and purification: Add 1-2 mL of cleavage solution (TFA:H2O:TIS = 95:2.5:2.5) to the DCM-washed and dried resin, and microwave at 38 °C for 35 min. Add 30 mL of ice-cold diethyl ether to the cleavage solution to precipitate the crude peptide, and then centrifuge at 5000 rpm for 5 min at 4 °C to separate the crude peptide. The crude peptide was purified by semi-preparative chromatography (Unisil 10-120-C18 column, 10 µm, 250 × 21.2 mm, 120 Å; mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient: 0–5 min 5% B, 5–30 min 5%→52% B, 30–32 min 52%→100% B, 32–37 min 100% B). The fraction containing the target product was lyophilized to obtain the precursor peptide TRFTSER(N3)GYIVESKK-NH2 (28.7 mg, 63%, see...). Figure 3 ).

[0099] The purified peptide was analyzed by analytical HPLC and the purity was >95%. Figure 4 The [M+H]⁺ value determined by low-resolution mass spectrometry (MALDI-ToF-MS) was 1825.795, which is close to the theoretical value of 1825.967. Figure 5 ). By high-resolution mass spectrometry ( Figure 6 ) and secondary mass spectrometry ( Figure 7 Further confirmation showed that the composition and sequence of the polypeptide were consistent with the theory, proving that the precursor polypeptide containing the ArgN3 structure was successfully prepared in this embodiment.

[0100] Example 6 Synthesis of the ArgN3-containing precursor polypeptide TRFTSER(N3)GYIVESKK-NH2 using Boc-ArgN3-OH

[0101] The precursor polypeptide TRFTSER(N3)GYIVESKK-NH2 (SEQ ID NO.01) synthesized in this embodiment has the following structural formula: ,

[0102] The specific method is as follows:

[0103] A mixed solid-phase peptide synthesis strategy using Fmoc / tBu and Boc was employed, with a synthesis scale of 25 μmol on MBHA Rink resin (0.312 mmol / g). Fmoc was used to protect the following amino acids: Thr(tBu), Arg(Pbf), Phe, Thr(tBu), Ser(tBu), Glu(OtBu), Gly, Tyr(tBu), Ile, Val, Glu(OtBu), Ser(tBu), Lys(Boc), Lys(Boc), and Boc-Arg(N3)-OH prepared in Example 2. Peptide synthesis was carried out in a CEM microwave reactor.

[0104] Deprotection was performed using a 20% piperidine DMF solution under microwave conditions of 90 °C, 60 W, with a heating time of 60 s, a holding time of 50 s, and low-speed stirring. For the coupling reaction of conventional amino acids, a 5-fold excess of Fmoc was used to protect the amino acid, using the CarboMAX coupling formulation, under microwave conditions of 90 °C, 70 W, with a heating time of 60 s, a holding time of 100 s, and low-speed stirring.

[0105] Coupling of the first amino acid on Rink resin: 2 mL of a DMF / DCM mixture (DMF / DCM = 1:1) was added to 25 μmol MBHA resin, and the mixture was shaken up and down at room temperature for 1 h to allow swelling. The resin was transferred to a peptide synthesis tube, and washed with 2 mL of DMF, repeated once. 3 mL of a 20% piperidine DMF solution was added, and the mixture was heated at 90 °C and 60 W for 60 s, held at that temperature for 50 s, and stirred at low speed for deprotection. After deprotection, the mixture was washed 4 times with 2 mL of DMF each time. Subsequently, 250 μL of a 5-fold excess of Fmoc-L-Lys(Boc)-OH (0.125 mmol, 58.6 mg) in DMF solution, 0.0125 mM HOBT, 0.0125 mM HBTU, and 8.6 μL of DIPEA were added to the peptide reaction tube, and the mixture was incubated in an ice-water bath at 0 °C. The resin was coupled at 50 °C and 45 W in a microwave reactor for 60 seconds, held for 10 minutes, and stirred at low speed. After coupling, the resin was washed three times with 2 mL of DMF each time. The coupling process was repeated once until the resin did not change color when tested with Kaiser.

[0106] Coupling of arginine azide (ArgN3) in the polypeptide sequence: 3 mL of 20% piperidine DMF solution was added to the polypeptide synthesis tube. The mixture was deprotected in a microwave reactor at 90 °C and 60 W for 60 s, held for 50 s, and stirred at low speed. After deprotection, the sample was washed four times with 2 mL of DMF each time. Experiments verified that ArgN3 is resistant to microwave field and 120 °C high temperature, and can be coupled to Boc-Arg(N3)-OH using the following two methods:

[0107] Method 1 (Low-Temperature Coupling Method): Add DMF (840 μL), a DMF solution of 0.5 M HATU (90 μL), a DMF solution of 1 M HOBt (48 μL), and 17.2 μL of DIPEA to a 1.5 mL centrifuge tube containing 2 times excess Boc-Arg(N3)-OH synthon (50 μmol, 14.9 mg). Stir at low speed overnight at room temperature. Use a small amount of resin to test the coupling efficiency using the Kaiser method. If coupling is incomplete, continue the reaction at room temperature for 12-24 h, or microwave at 50 °C for 10 min.

[0108] Method 2 (High-Temperature Coupling Method): Add DMF (250 μL), 0.5 M DIC DMF solution (500 μL), 1 M Oxyma DMF solution (250 μL), and DIPEA 8.6 μL to a 1.5 mL centrifuge tube containing 2 times excess Boc-Arg(N3)-OH synthon (50 μmol, 14.9 mg). React at 90 °C for 100 s in a microwave reactor. If Kaiser analysis indicates insufficient coupling, the coupling can be repeated once.

[0109] Deprotection of the Boc-Arg(N3)-OH synthon was performed using a 30% EA / HCl solution with stirring at room temperature for 2 h. Subsequent amino acid coupling reactions used a 5-fold excess of Fmoc to protect the amino acids, employing the CarboMAX coupling formulation under microwave conditions of 90 °C, 70 W, with a heating time of 60 s, a holding time of 100 s, and low-speed stirring. Deprotection of amino acids other than the Boc-Arg(N3)-OH synthon was performed using a 20% piperidine DMF solution under microwave conditions of 90 °C, 60 W, with a heating time of 60 s, a holding time of 50 s, and low-speed stirring.

[0110] Peptide cleavage and purification: 1-2 mL of pre-cleavage solution (TFA:DCM = 1:1) was added to the resin (without DCM washing) and stirred at room temperature for 5 min. After filtration, the peptide was resuspended in 1-2 mL of pre-cleavage solution (TFA:DCM = 1:1) and reacted for 30 min. After washing and drying with DCM, 200 μL of anisole and 100 μL of ethylenedithiol were added per 100 mg of resin. The flask was cooled in an ice bath, and 2 mL of TFA was added per 100 mg of resin. The mixture was stirred for 5 min. 200 μL of trifluoromethanesulfonic acid was slowly added dropwise in an ice bath and the reaction was allowed to proceed for 1 h. After the reaction was complete, the resin was washed with 2 mL of TFA, and 30 mL of ice-cold ether was added to the cleavage buffer to precipitate the crude peptide. The crude peptide was then separated by centrifugation at 5000 rpm for 5 min at 4 °C. The crude peptide was purified by semi-preparative chromatography (Unisil 10-120-C18 column, 10 µm, 250 × 21.2 mm, 120 Å; mobile phase A: water + 0.1% trifluoroacetic acid; mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient: 0–5 min 5% B, 5–30 min 5%→52% B, 30–32 min 52%→100% B, 32–37 min 100% B). The final product was consistent with that in Example 5, and its characterization was not repeated here.

[0111] Example 7: Constructing PN bonds on precursor peptides

[0112] The precursor peptide TRFTSER(N3)GYIVESKK-NH2 (5 mg, 2.74 μmol) obtained in Example 5 or Example 6 was dissolved in 150 μL of dry DMF. Tris(4,5-dimethoxy-2-nitrobenzyl ester) (8.3 mg, 13.7 μmol) was dissolved in 30 μL of dry DMF and then added to the precursor peptide. The reaction was carried out under argon protection at 45-48 °C in the dark for 1 day. The reaction solution was purified by reversed-phase chromatography (TC C18(2) 5 µm, 250 × 4.6 mm, 170 Å column; mobile phase A: water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient: 0–5 min 5% → 16.25% B, 5–35 min 16.25% → 38.75% B, 35–37 min 38.75% → 100% B, 37–47 min 100% B). Figure 8 The fraction containing the target product was lyophilized to obtain the pArg polypeptide TRFTSER(P(OR3))GYIVESKK-NH2 (4.46 mg, 72%) containing a photocage protecting group, with the following structural formula: .

[0113] The product was analyzed by HPLC and its purity was >98%. Figure 9 ). Detected by high-resolution mass spectrometry (HRMS), [M+3H] 3+ The measured value of 758.0159 is consistent with the theoretical value of 758.0184 (δ=3.3 ppm), proving that this embodiment successfully prepared a photocage-protected pArg polypeptide containing PN bonds.

[0114] Example 8 Synthesis of phosphorylated arginine polypeptide TRFTSEpRGYIVESKK-NH2

[0115] The photocage-protected pArg peptide TRFTSER(P(OR3))GYIVESKK-NH2 (4.46 mg, 1.96 μmol) obtained in Example 7 was dissolved in 50 μL of a mixed solvent (ACN / DDW / MeOH / NH4OH = 10 / 7 / 2 / 1 v / v) and photolyzed at 365 nm for 1-2 h in an ice bath (average power 3 mW). The reaction solution was purified by reversed-phase chromatography (under the same chromatographic conditions as in Example 7). Figure 10 The fraction containing the target product was lyophilized to obtain pArg peptide TRFTSEpRGYIVESKK-NH2 (3.32 mg, 90%).

[0116] The product was analyzed by HPLC and its purity was >98%. Figure 11High-resolution mass spectrometry (HRMS) analysis showed that the measured value of [M+3H]³⁺ was 627.3216, consistent with the theoretical value of 627.3190 (δ=4.1 ppm). Figure 12 Further secondary mass spectrometry sequencing showed that, via y9 2+ (530.814) fragments and y8 + The quality difference of (922.534), and b7 2+ (430.725) and b6 + The mass difference of (722.349) confirmed that position 7 of the peptide was pArg, proving that the pArg peptide was successfully synthesized and that the synthesized sequence was consistent with the target sequence. Figure 13 The structural formula is .

[0117] Example 9: Temperature compatibility range of ArgN3 synthon for SPPS

[0118] The specific steps for polypeptide synthesis are the same as in Example 5, only the reaction temperature is changed. Figures 14 to 17 As shown, the target polypeptide TRFTSER(N3)GYIVESKK-NH2 can be synthesized within the temperature range of 15℃ to 120℃.

[0119] Example 10 Staudinger Reaction Compatibility Range

[0120] The raw materials for constructing the PN bond are the same as in Example 7. Changing the solvent and temperature also allows for the construction of the pArg polypeptide TRFTSER(P(OR3))GYIVESKK-NH2 containing a photocage protecting group. Figures 18 to 22 As shown, PBS buffer and Tris buffer can be used instead of DMF as solvent; the reaction temperature is compatible with a range of 0℃-60℃.

[0121] Example 11: Photodegradation protection wavelength and solvent compatibility range

[0122] The deprotection method is the same as in Example 8; changing the wavelength, temperature, and solvent also yields the pArg peptide TRFTSEpRGYIVESKK-NH2. Figures 23 to 27 As shown, PBS buffer and Tris buffer can be used as photolysis solvents, and ultraviolet or visible light in the wavelength range of 280-420 nm can be used as photolysis wavelengths. The addition of base and lower alcohol helps to improve the purity and yield of photolysis. The aforementioned lower alcohol is selected from methanol, ethanol, and propanol.

[0123] In the above embodiments, all characterization data (HPLC, MALDI-ToF-MS, HRMS, MS / MS, etc.) were obtained using conventional and mature methods in the art, which fully demonstrates the correctness of the structure of the reaction products in each step of the present invention and the successful construction of PN bonds.

[0124] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for synthesizing a sequence-specific phosphorylated arginine polypeptide, characterized in that: Includes the following steps: (1) A solid-phase polypeptide coupling synthesis method was adopted, using Fmoc-Arg(N3)-OH or Boc-Arg(N3)-OH as synthons to prepare precursor polypeptides containing azidoarginine. The structural formula of Fmoc-Arg(N3)-OH is: , The structural formula of Boc-Arg(N3)-OH is: ; (2) The precursor polypeptide obtained in step (1) is subjected to a Staudinger reaction with a phosphite compound to obtain a phosphorylated arginine polypeptide containing a protecting group. (3) Remove the above protecting group to obtain sequence-specific phosphorylated arginine polypeptide.

2. The synthesis method according to claim 1, characterized in that: The Fmoc-Arg(N3)-OH or Boc-Arg(N3)-OH is prepared by reacting fluorosulfonyl azide with Fmoc-L-Arg or Boc-L-Arg in the presence of a catalyst. The structural formula of the fluorosulfonyl azide is as follows: This catalyst provides Cu 2+ The donor salt, and the addition of a catalyst, makes the Cu in the reaction system 2+ The molar percentage concentration ranges from 0.01% to 5%.

3. The synthesis method as described in claim 1, characterized in that: Step (1) employs either the Fmoc / tBu solid-phase peptide synthesis strategy or the Boc solid-phase peptide synthesis strategy.

4. The synthesis method as described in claim 3, characterized in that: The solid-phase polypeptide coupling synthesis method in step (1) adopts either non-microwave-assisted coupling or microwave-assisted coupling.

5. The synthesis method as described in claim 3 or 4, characterized in that: The reaction temperature of the solid-phase polypeptide coupling synthesis method is 15-120 ℃.

6. The synthesis method according to claim 1, characterized in that: The phosphite compound in step (2) is photosensitive tris(4,5-dimethoxy-2-nitrobenzyl phosphite).

7. The synthesis method according to claim 6, characterized in that: The Staudinger reaction in step (2) is carried out under light-protected conditions at 0-60°C.

8. The synthesis method according to claim 7, characterized in that: The solvent for the Staudinger reaction is selected from at least one of DMF, PBS buffer, and Tris buffer; and the Staudinger reaction is carried out under argon protection or without a protective atmosphere.

9. The synthesis method according to claim 1, characterized in that: The deprotection in step (3) is carried out under light with a wavelength of 280-420 nm; the solvent for the deprotection reaction is selected from at least one of PBS buffer, Tris buffer, ACN / DDW mixed solvent and ACN / DDW / lower alcohol mixed solvent, wherein the lower alcohol is selected from methanol, ethanol and propanol.

10. The synthesis method according to claim 1, characterized in that: The solid-phase polypeptide coupling synthesis method in step (1) uses MBHA Rink resin, Wang resin or CTC resin.

Images