A method for modifying cysteine residues and its use in the preparation of cyclic peptides

Abstract

The application provides a method for modifying a compound containing a cysteine residue by using aryl diazonium salt, and the reaction does not need heating, organic solvent and any catalyst, the reaction process is simple, and a complex reaction device is not needed, so that a very convenient new method is provided for modification based on a Cys site. The application further applies the reaction to construction and conformation regulation of a cyclic peptide, and realizes systematic regulation of a conformation constraint scale of the cyclic peptide by only changing a skeleton structure (a benzene ring, a biphenyl, a biphenyl ether) of a double-head aryl diazonium tetrafluoroborate crosslinking agent without changing a primary sequence of the polypeptide.

Description

Technical Field

[0001] This invention belongs to the field of chemical engineering, specifically relating to a method for modifying cysteine ​​residues, and the application of this modification method in the preparation of cyclic peptides and the regulation of cyclic peptide conformation. Background Technology

[0002] Peptide cyclization is an effective strategy for enhancing peptide metabolic stability, target affinity, and cell membrane permeability. However, existing classical cyclization methods share common limitations in conformational regulation: the ring size of lactamylation cyclization is entirely determined by the position of the Cys or terminal groups in the sequence; while disulfide bridging can form rings under mild conditions, its flexible backbone makes it difficult to lock the peptide into a single active conformation; and although olefin metathesis introduces non-natural cross-linking bridges, its ring size still depends on the position of the olefin side chain. None of these methods can achieve independent regulation of cyclic peptide conformation through cross-linking agent structural design without altering the peptide's primary sequence. Therefore, designing a two-site cross-linking agent that selectively couples with two specific amino acid residues in the peptide chain, and then adjusting the conformational constraint scale of the cyclic peptide by changing the structure of the cross-linking agent, provides a new approach to solving these problems.

[0003] Among 20 natural amino acids, cysteine ​​(Cys) is an ideal modification site for this method due to the high nucleophilicity of its side-chain thiol group, low natural abundance, and moderate pKa value. However, the application of aryl diazonium salts in cysteine ​​modification has been relatively limited, despite their being a class of highly reactive electrophilic reagents. Furthermore, existing studies rely on organic solvents or photo / metal catalysis, and the reactions all follow denitrification pathways, producing aryl thioether structures, thus enabling only single-site modification. To achieve peptide cyclization using aryl diazonium salts, two aryl diazonium reaction sites need to be designed to form a covalent link between two Cys residues on the same peptide chain.

[0004] There is an urgent need in this field to provide a method for regulating the conformation of cyclic peptides through the structural design of cross-linking agents without altering the primary sequence of the peptide. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the first aspect of the present invention provides a method for modifying a compound containing a cysteine ​​residue, the method comprising reacting an aryldiazotetrafluoroborate of Formula I with a compound containing a cysteine ​​residue to form an azo coupling product, wherein the compound containing a cysteine ​​residue has a free thiol group (-SH) and a free amino group (-NH2) or an amino group is present on an amino group adjacent to the cysteine ​​residue.

[0006] Formula I in, R is selected from -F, -Cl, -Br, -I, -H, -CF3, -CH3, or -OCH3; Ar is selected from phenyl, diphenyl, or diphenyl ether; n is an integer that is either 1 or 2.

[0007] In some embodiments, the aryldiazotetrafluoroborate has the structure shown in Formula II.

[0008] Formula II Wherein, R is selected from -F, -Cl, -Br, -I, -H, -CF3, -CH3, or -OCH3; In some implementations, R is -Br.

[0009] In some embodiments, aryldiazotetrafluoroborate comprises one or more of the following compounds: .

[0010] In some embodiments, the aryldiazotetrafluoroborate is 4-bromobenzenediazotetrafluoroborate.

[0011] In some embodiments, the aryldiazotetrafluoroborate and the compound containing cysteine ​​residues are processed under pure water conditions.

[0012] In some embodiments, the molar ratio of aryldiazotetrafluoroborate to the compound containing cysteine ​​residues is 1:1.

[0013] In some embodiments, aryldiazotetrafluoroborate and compounds containing cysteine ​​residues react under catalyst-free conditions.

[0014] In some embodiments, the reaction temperature of the aryldiazotetrafluoroborate and the compound containing cysteine ​​residues is room temperature.

[0015] In some embodiments, the reaction of the aryldiazotetrafluoroborate and the compound containing cysteine ​​residues takes 0-5 minutes.

[0016] In some embodiments, the general reaction formula for the aryldiazotetrafluoroborate and the compound containing cysteine ​​residues is: , Wherein, R is selected from -F, -Cl, -Br, -I, -H, -CF3, -CH3, -OCH3; R1 is selected from -OH, -OCH3, or -[AA1]. p R2 is selected from -NH2 or -[AA2] q ; AA1 and AA2 may be the same or different; AA1 and AA2 are independently selected from any natural amino acid residues; p, q≥1.

[0017] In some embodiments, compounds containing cysteine ​​residues include cysteine ​​or derivatives thereof, polypeptides containing cysteine ​​residues, and proteins.

[0018] In some embodiments, the cysteine ​​residue in the polypeptide is located in the middle of the polypeptide. In some embodiments, the cysteine ​​residue in the polypeptide is located at the N-terminus of the polypeptide. In some embodiments, the cysteine ​​residue in the polypeptide is located at the C-terminus of the polypeptide. In some embodiments, the cysteine ​​residue in the polypeptide is located at both the N-terminus and C-terminus of the polypeptide.

[0019] In some embodiments, compounds containing cysteine ​​residues include cysteine ​​methyl ester hydrochloride.

[0020] In some embodiments, compounds containing cysteine ​​residues include polypeptides or proteins containing one or more cysteine ​​residues.

[0021] In some embodiments, the compound containing cysteine ​​residues has the structure shown in Formula III.

[0022] Formula III in, R1 is selected from -OH, -OCH3, or -[AA1]. p R2 is selected from -NH2 or -[AA2] q ; AA1 and AA2 may be the same or different; AA1 and AA2 are independently selected from any natural amino acid residues; p, q≥1.

[0023] In some implementations, when p, q>1, each AA1 or AA2 may be the same or different.

[0024] In some implementations, -[AA1] p It must include at least one amino acid with an amino group in its side chain. In some embodiments, -[AA2] q It must contain at least one amino acid with an amino group in its side chain.

[0025] In some embodiments, the amino acids containing an amino group in their side chains include lysine, arginine, and histidine.

[0026] In some embodiments, the compound containing cysteine ​​residues also includes a free amino group.

[0027] In some implementations, R2 is -NH2.

[0028] In some implementations, p and q are selected from integers from 1 to 7.

[0029] In some implementations, -[AA1] p This includes the C-terminus of the polypeptide. In some embodiments, -[AA1] p Including the N-terminus of the polypeptide.

[0030] In some embodiments, the azo coupling product obtained by reacting the aryldiazotetrafluoroborate of Formula I with a compound containing a cysteine ​​residue is selected from any of the following structures. .

[0031] The second aspect of the present invention provides the application of the method described in the first aspect in the preparation of cyclic peptides and the regulation of cyclic peptide conformation.

[0032] A third aspect of the present invention provides a method for preparing a cyclic peptide, the method comprising reacting an aryldiazotetrafluoroborate as described in the first aspect with a linear polypeptide to undergo intramolecular cyclization to generate a cyclic peptide, wherein n is 2 in the aryldiazotetrafluoroborate.

[0033] In some embodiments, the aryl diazonium salt comprises one or more of the following compounds: .

[0034] In some embodiments, the linear polypeptide contains two cysteine ​​residues. In some embodiments, the linear polypeptide contains two free thiol groups.

[0035] In some embodiments, the linear polypeptide contains 1-7 natural amino acid residues.

[0036] In some embodiments, the N-terminus and C-terminus of the linear polypeptide are both cysteine ​​residues.

[0037] In some embodiments, at least one free amino group is present in the linear polypeptide at a position 0-7 amino acids away from the thiol group of the cysteine ​​residue.

[0038] In some embodiments, the linear polypeptide has a naked amino group at the N-terminus and a carboxylated C-terminus.

[0039] In some embodiments, the linear polypeptide has a naked amino group at the N-terminus and a naked carboxyl group at the C-terminus. In some embodiments, the thiol group of the cysteine ​​residue in the linear polypeptide is separated from the N-terminal amino group by 0-7 amino acids.

[0040] In some embodiments, aryldiazotetrafluoroborate is reacted with a linear polypeptide in pure water.

[0041] In some embodiments, the reaction time of aryldiazotetrafluoroborate with linear peptides is 0-5 minutes.

[0042] In some embodiments, the molar ratio of aryldiazotetrafluoroborate to the linear polypeptide is 1:1.

[0043] In some embodiments, the reaction temperature of the aryl diazonium salt and the linear polypeptide is room temperature.

[0044] In some embodiments, the reaction between the aryl diazonium salt and the linear polypeptide does not require a catalyst.

[0045] In some implementations, the linear peptide concentration is 50 mM.

[0046] In some embodiments, the structure of the linear peptide is as follows:

[0047] In some embodiments, the prepared cyclic peptide is selected from one of the following structures. .

[0048] A fourth aspect of the present invention provides a method for regulating the conformation of a cyclic peptide, the method comprising reacting an aryldiazotetrafluoroborate as described in the first aspect with a linear peptide to induce intramolecular cyclization, wherein n is 2.

[0049] In some embodiments, the sulfur atom spacing in the cyclic peptide is 5.801 Å–10.084 Å.

[0050] In some embodiments, the aryldiazotetrafluoroborate comprises one or more of the following compounds: .

[0051] In some embodiments, when the aryl diazonium salt is 10a, the distance between the two cysteine ​​sulfur atoms in the cyclic peptide is 5.801 Å.

[0052] In some embodiments, when the aryl diazonium salt is 11a, the distance between the two cysteine ​​sulfur atoms in the cyclic peptide is 9.306 Å.

[0053] In some embodiments, when the aryl diazonium salt is 12a, the distance between the two cysteine ​​sulfur atoms in the cyclic peptide is 10.084 Å.

[0054] The beneficial effects of this invention are as follows: The method for modifying compounds containing cysteine ​​residues using aryldiazotetrafluoroborate provided by this invention requires no heating, no organic solvents, and no catalysts. The reaction process is simple and requires no complex reaction apparatus. Furthermore, this reaction exhibits excellent selectivity, reacting only with cysteine ​​in 20 natural amino acid methyl esters and specifically modifying only cysteine ​​sites in complex model peptides containing all nucleophilic residues. The second-order reaction rate constant is greater than 1.0 × 10⁻⁶. 4 M - ¹·s - ¹ This method is 2-4 orders of magnitude faster than traditional cysteine ​​modification methods, completing the reaction within seconds and achieving efficient conversion even at micromolar concentrations. The method provided by this invention, which uses a difunctional aryl diazonium tetrafluoroborate to prepare cyclic peptides via an azo coupling reaction, allows for systematic regulation of cyclic peptide conformation without altering the primary sequence of the peptide. The sulfur atom spacing varies from 5.801 Å to 10.084 Å, providing a new chemical tool for studying the structure-activity relationship between cyclic peptide conformation and biological function. Attached Figure Description

[0055] Figure 1 The aqueous reaction solution of compounds 1a and 2a 1 Rough H NMR spectrum.

[0056] Figure 2 High-resolution mass spectrometry of azo coupling product 3a.

[0057] Figure 3 The crystal structure of azo coupling product 3a is shown.

[0058] Figure 4 The figures are for reaction kinetic studies, where (a) is the UV absorption spectrum of compound 1a and azo coupling product 3a, (b) is the absorbance-concentration standard curve of azo coupling product 3a at 330 nm, (c) is the kinetic curve of the absorbance of azo coupling product 3a changing with time, and (d) is the curve of 1 / [1a] changing with time.

[0059] Figure 5 The stability of azo coupling product 3a was monitored under acidic and alkaline conditions. Among them, (a) is an aqueous solution at pH 2, with a monitoring wavelength of 330 nm; (b) is an aqueous solution at pH 2, with a monitoring wavelength of 280 nm; (c) is an aqueous solution at pH 10, with a monitoring wavelength of 330 nm; and (d) is an aqueous solution at pH 10, with a monitoring wavelength of 280 nm.

[0060] Figure 6The stability of azo-coupled product 3a was monitored under physiological and oxidative conditions. (a) was a PBS solution at pH 7.4, monitored at a wavelength of 330 nm; (b) was a PBS solution at pH 7.4, monitored at a wavelength of 280 nm; (c) was a PBS solution with 5 mM H₂O₂, monitored at a wavelength of 330 nm; and (d) was a PBS solution with 5 mM H₂O₂, monitored at a wavelength of 280 nm.

[0061] Figure 7 The images show the secondary mass spectrometry analysis of the peptide conjugate product 4b, where (a) is the MS / MS resolved spectrum and (b) is the structure of the peptide azo conjugate product 4b.

[0062] Figure 8 The results show the secondary mass spectrometry analysis of the peptide conjugate product 5b, where (a) is the MS / MS resolved spectrum and (b) is the structure of the peptide azo conjugate product 5b.

[0063] Figure 9 For the reaction solution of the single amino system 1 H NMR.

[0064] Figure 10 The reaction solution for the coexistence of monothiol and thiol-amino groups 1 ¹H NMR, where (a) is the reaction solution of compound 6a. 1 The crude 1H NMR spectrum, (b) is the reaction solution of compound 2a. 1 Rough H NMR spectrum.

[0065] Figure 11 LCMS analysis of the monothiol system and the thiol-amino coexistence system. (a) is the 254 nm chromatogram of the monothiol system (1a+6a), (b) is the 210 nm chromatogram of the monothiol system (1a+6a), (c) is the total ion chromatogram of the monothiol system (1a+6a), (d) is the 254 nm chromatogram of the thiol-amino coexistence system (1a+2a), (e) is the 210 nm chromatogram of the thiol-amino coexistence system (1a+2a), and (f) is the total ion chromatogram of the thiol-amino coexistence system (1a+2a).

[0066] Figure 12 The optimized conformation and sulfur atom spacing of linear peptide 13a after cyclization by aryldiazotetrafluoroborate crosslinking agent 10a-12a are shown, where (a) is the sulfur atom spacing of linear peptide 13a, (b) is the sulfur atom spacing of cyclic peptide 10b, (c) is the sulfur atom spacing of cyclic peptide 11b, and (d) is the sulfur atom spacing of cyclic peptide 12b. Detailed Implementation

[0067] The "natural amino acids" mentioned in this invention are selected from tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, or proline.

[0068] The "cysteine ​​derivative" mentioned in this invention refers to a compound containing a cysteine ​​sulfhydryl group and a free amino group, including but not limited to cysteine ​​salts or cysteine ​​esters.

[0069] The specific implementation of the present invention will be further described in detail below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0070] Information on the main experimental instruments and reagents used in this invention is shown in Tables 1 and 2: Table 1 Main Reagents

[0071] Table 2 Main Instruments

[0072] Example 1: Coupling reaction of L-cysteine ​​methyl ester hydrochloride with 4-bromobenzenediazotetrafluoroborate and confirmation of the product structure 0.1 mmol each of 4-bromobenzidine difluoroborate and L-cysteine ​​methyl ester hydrochloride were dissolved in pure water and mixed at room temperature. The reaction equation is shown below. The solution in the reaction system changed from colorless to yellow within seconds. Samples of the crude reaction solution were taken for further analysis. 1 H NMR analysis, results as follows Figure 1 As shown.

[0073]

[0074] Depend on Figure 1 It was observed that the β-CH2 signal of cysteine ​​methyl ester shifted from 3.10 ppm to 4.08 ppm, the α-CH signal from 4.40 ppm to 4.58 ppm, and the terminal CH3 signal from 3.83 ppm to 3.67 ppm; the aromatic ring protons of the aryl diazonium salt shifted from 8.40 ppm and 8.11 ppm to 7.46 ppm and 7.27 ppm. Comparison of characteristic peak integrals showed that the product conversion exceeded 90%.

[0075] The structure of the reaction products was further confirmed using high-resolution mass spectrometry, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that [M+H] + The peak mass-to-charge ratios were 317.9901 and 319.9880 (theoretical values ​​of 317.9904 and 319.9883), confirming that the azo group of compound 1a was completely retained in the product molecule and that no azo group was lost during the reaction. This reaction is an azo coupling that retains the diazo group.

[0076] To obtain direct structural information about the product, the purified coupling product 3a was subjected to X-ray single-crystal diffraction analysis. The crystal analysis results confirmed the formation of an azo bond, as shown below. Figure 3 As shown in Table 3.

[0077] Table 3 Crystal data and structure refinement parameters

[0078] Example 2 Kinetic determination of the coupling reaction between cysteine ​​residues and aryl diazonium salts Kinetic experiments were conducted using a stationary flow spectrometer (Applied Photophysics SX20), and the reaction rate was measured at room temperature. Because the azo coupling product 3a exhibits characteristic absorption at 330 nm, while compound 1a has extremely low absorption at this wavelength, and compound 2a shows no significant UV absorption, ... Figure 4 As shown in (a), the product formation process can be accurately tracked by monitoring the change in absorbance at 330 nm over time. Compounds 1a and 2a were prepared into aqueous solutions of the same concentration (initial concentrations of 10, 15, 20, and 25 μM). They were rapidly mixed in equal volumes (resulting in concentrations of approximately 5.0, 7.5, 10, and 12.5 μM), and the absorbance at 330 nm, a characteristic absorption of the azo product, was monitored over time. A standard curve was established by measuring the absorbance of azo coupling product 3a at different known concentrations at 330 nm to obtain a quantitative relationship between absorbance and concentration, as shown in (a). Figure 4 As shown in (b). The absorbance data measured during the reaction were then substituted into the standard curve to calculate the product concentration at different time points. Since the stoichiometric ratio of the reaction is 1:1, the amount of reactant 1a consumed can be calculated based on the amount of product 3a produced, thus obtaining the concentration change curve of compound 1a over time. The above concentration data were processed according to a second-order reaction kinetic model, and its linear form is... , where [1a] is the lower concentration (μM) of compound 1a at different times, and [1a]0 is the initial concentration (μM) of compound 1a. k 2 is the second-order reaction rate constant (M). -1 ·s -1 ), where t is the reaction time (s).

[0079] Experimental results show that the absorbance of azo coupling product 3a at 330 nm is as follows: Figure 4 As shown in (c), by Figure 4 It can be seen that the absorbance of the azo coupling product 3a at 330 nm increases rapidly with time and then plateaus within a few seconds. A plot of 1 / [1a] against time t shows good linearity, as shown in the figure. Figure 4 As shown in (d), the second-order reaction rate constant was calculated based on the slope of the straight line fitted at different initial concentrations. k 2. As shown in Table 4.

[0080] Table 4. Second-order rate constants of the reaction at different initial concentrations.

[0081] As shown in the table above, compared to traditional cysteine ​​modification methods (such as iodoacetamide alkylation), the second-order reaction rate constant is typically around 10. 0 -10² M - ¹·s - ¹ The reaction rate of the cysteine ​​residue modification method provided in this invention is 2-4 orders of magnitude faster ([9] F.-J. Chen and J. Gao, “Fast CysteineBioconjugation Chemistry,” Chemistry – A European Journal 28 (2022):e202201843, https: / / doi.org / 10.1002 / chem.202201843).

[0082] Example 3: Stability verification of azo coupling products This embodiment investigates the stability of azo-coupled product 3a under acidic conditions (aqueous solution at pH 2), alkaline conditions (aqueous solution at pH 10), physiological conditions (PBS buffer at pH 7.4), and oxidative environments (PBS buffer containing 5 mM H2O2).

[0083] Azo coupling product 3a was dissolved in the four solutions mentioned above and incubated at room temperature. Samples were taken every 1.5 hours, and LC-MS analysis was used to monitor the appearance of new degradation products for a total monitoring time of 24 hours. Chromatographic detection was performed at wavelengths of 280 nm and 330 nm. 330 nm is the characteristic absorption wavelength of the azo product, and 280 nm is the characteristic absorption wavelength of the aryl compound, used to simultaneously monitor changes in the aryl structure. Under these chromatographic conditions, the retention time of product 3a was 2.75 min. The stability of the product under different conditions was qualitatively assessed by comparing the weakening or disappearance of the starting peak at 2.75 min and the appearance of new peaks in the LC-MS chromatograms at different time points. Chromatograms of different solutions at different time points are shown below. Figure 5 and Figure 6 As shown.

[0084] Depend on Figure 5 As shown in (a) and (b), the chromatogram of product 3a showed no significant change within 24 hours in an acidic aqueous solution at pH 2, and no new degradation product signals were detected. Figure 5 As shown in (c) and (d), the stability of product 3a is significantly reduced in alkaline aqueous solution at pH 10. After approximately 3 hours, a significant weakening of the starting material peak and the formation of a new peak were observed in both the 330 nm and 280 nm channels, indicating that product 3a underwent degradation. Figure 6 As shown in (a) and (b), product 3a remained stable in the initial stage of the experiment (0-9 hours) in PBS buffer at pH 7.4, with no significant changes observed in the chromatogram. After approximately 9 hours, the starting peak weakened and a new peak formed, indicating that the azo thioether bond has a stability window of approximately 9 hours at near physiological pH. Figure 6 As shown in (c) and (d), in an oxidizing environment, significant degradation signals were detected in the 330 nm and 280 nm channels after approximately 4.5 hours. In summary, the degradation characteristics of product 3a are closely related to the pH and oxidizing properties of the medium. Acidic or neutral conditions are more conducive to product stability.

[0085] Example 4 Synthesis of aryldiazotetrafluoroborate This embodiment synthesized aryldiazotetrafluoroborates 1b-1j with different electronically affected substituents, as shown in Table 5. The synthesized aryldiazotetrafluoroborates encompass a variety of substituent types, including electron-donating groups such as methoxy and methyl, weakly electron-withdrawing halogen substituents such as fluorine, chlorine, bromine, and iodine, as well as the strongly electron-withdrawing substituent trifluoromethyl, and also included unsubstituted benzenediazotetrafluoroborate. All products were synthesized by... 1 H NMR and 13 Its structure was confirmed by C NMR characterization, and its purity met the requirements for subsequent reaction studies.

[0086] The specific synthetic method is as follows: Aniline derivatives with different substituents (50 mmol, gram-scale reaction) were dissolved in a homogeneous mixture of 20 mL water and 50 wt% fluoroboric acid (96.5 mmol, 1.92 equivalents), and the mixture was cooled to 0°C in an ice-water bath. Then, sodium nitrite solution (50 mmol, dissolved in 7.5 mL water) was slowly added, and the mixture was stirred at 0°C for 30 minutes, ensuring the temperature did not exceed 5°C throughout the reaction. After the reaction was complete, the precipitate was collected by filtration, washed with a small amount of cold water, and dried under reduced pressure to obtain aryldiazotetrafluoroborates with different substituents, which were then stored at -20°C for later use. The yields shown in the table are the separation yields.

[0087] Table 5. Aryldiazotetrafluoroborate 1b-1j

[0088] Example 5: Expansion of substrates for azo coupling of aryl diazonium tetrafluoroborates with different substituents The diazonium salt (0.1 mmol, 100 mM) prepared in Example 4 was reacted with compound 2a (0.1 mmol, 100 mM) in pure water at room temperature for 5 minutes. After the reaction, an equimolar amount of sodium bicarbonate (0.1 mmol) was added to the aqueous phase to neutralize some of the acid generated during the reaction. The mixture was then extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was then dissolved in a small amount of dichloromethane, and an appropriate amount of silica gel was added to the organic phase. The solvent was removed under reduced pressure. Column chromatography purification was performed using 300-400 mesh silica gel as the stationary phase and petroleum ether and ethyl acetate as eluents. Gradient elution was performed from ethyl acetate:petroleum ether = 2:3 (v / v) to ethyl acetate:petroleum ether = 1:1 (v / v). The target fractions were collected, and the solvent was removed under reduced pressure to obtain the azo coupling products (3a-3j), as shown in Table 6. The yields shown in the table are NMR quantification yields. Maleic acid was used as an internal standard. 1 Yield was determined by H NMR.

[0089] Table 6. Azo coupling products of aryl diazo tetrafluoroborates with different substituents

[0090] Experimental results show that the electronic effects of different substituents have a significant impact on the reaction yield. When the para-position is occupied by an electron-donating group such as methoxy or methyl, the azo coupling yield is relatively low; when the para-position is occupied by an electron-withdrawing group, the yield is generally higher.

[0091] Example 6: Verification of Selective Modification of 20 Amino Acid Monomers by Aryl Diazonium Salts Compound 1a (0.1 mmol) was reacted with methyl ester hydrochloride of 20 natural amino acids (0.1 mmol) in pure water at room temperature. 1 The reaction process was monitored by ¹H NMR. Only the reaction system of cysteine ​​methyl ester showed a new characteristic peak of azo coupling product, while the other 19 amino acids did not react, as shown in Table 7, including lysine (ε-amino), arginine (guanidinyl), histidine (imidazolyl), serine (hydroxyl), tyrosine (phenolic hydroxyl), etc., which contain nucleophilic side chains.

[0092] Table 7. Reaction selectivity at the monomer level of 20 amino acids

[0093] As shown in the table above, only the reaction system of cysteine ​​methyl ester hydrochloride showed a new characteristic peak of azo coupling product; no new peaks were detected in the other 19 amino acid systems. This is consistent with the control spectrum of the blank starting material. Figure 1 Even amino acids containing reactive nucleophilic side chains, such as lysine (ε-amino), arginine (guanidinyl), histidine (imidazolyl), serine and threonine (hydroxyl), tyrosine (phenolic hydroxyl), etc., in 1 No new products were observed during H NMR monitoring. This result indicates that the reaction exhibits excellent chemoselectivity for cysteine ​​among 20 natural amino acids, while other nucleophilic amino acid residues do not participate in coupling under these reaction conditions.

[0094] Example 7: Verification of the selective modification of peptides by aryl diazonium salts This embodiment further extends the system to peptide substrates to evaluate the chemoselectivity of the azo coupling reaction on more complex peptide substrates. Both model peptides (4a) and (5a) were synthesized via Fmoc solid-phase peptide synthesis (SPPS).

[0095] Peptide Synthesis: Rink-Amide resin was used as the solid-phase support, and the resin was first fully swollen. The α-amino group of the amino acid was protected with a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, which was then removed by piperidine and N,N-dimethylformamide solution. The carboxyl group of the amino acid was activated by the condensing reagents N,N′-diisopropylcarbodiimide and ethyl cyanohydroxyimino acetate, and amidation occurred with the free amino group on the resin, thereby achieving stepwise linkage of amino acids. After each condensation step, the presence of free amino groups on the resin was detected by ninhydrin colorimetry to determine whether the reaction was complete. By repeatedly performing deprotection, amino acid condensation, and reaction detection steps, the peptide chain was gradually extended, and the peptide with the target sequence was finally obtained. After synthesis, the peptide was cleaved from the resin using trifluoroacetic acid, and the side chain protecting groups were removed. The crude product was precipitated with cold diethyl ether, centrifuged, and washed, and then further purified by semi-preparative high-performance liquid chromatography (HPLC). The structure of the final product was confirmed by HRMS and ¹H NMR. The purified peptides 4a and 5a both achieved a purity of over 95%, meeting the requirements for subsequent reaction studies.

[0096] The structures of the two polypeptides are shown below:

[0097] .

[0098] The peptide coupling reaction was carried out in a pure water system. Specifically, a 50 mM aqueous solution of the peptide was reacted with an equimolar amount of compound 1a in pure water at room temperature for 5 minutes. The product was then analyzed by first-stage mass spectrometry using Q-Exactive liquid chromatography-quadrupole orbital trap high-resolution mass spectrometry. The determined molecular weight of product 4b after the reaction of peptide 4a was [M+H]. + 1289.4643 (theoretical value 1289.4629), [M+2H]2 + 645.2351 (theoretical value for unit site modification: 645.2350); the measured molecular weight of product 5b after the reaction of peptide 5a is [M+H]. + 1289.4623 (theoretical value 1289.4629), [M+2H]2 + 645.2353 (theoretical value of unit point modification is 645.2350).

[0099] The results showed that both model peptides produced product molecular weights consistent with single-site coupling modification, and high-resolution mass spectrometry did not detect multiple modification byproducts. This demonstrates that even in complex peptide environments containing multiple nucleophilic residues, the reaction maintains good single-site modification. Thanks to the ultrafast kinetics of this azo coupling reaction, when the coupling reaction between the target site and the diazonium salt is fast enough, the main reaction can be completed before competing side reactions (such as non-specific modification or intermolecular crosslinking) occur, thus effectively suppressing the formation of byproducts.

[0100] Next, the precursor ions (4b: m / z 1289.4643, 5b: m / z 645.2353) detected in the primary mass spectrometry were analyzed by high-energy C-trap dissociation (HCD) secondary mass spectrometry. In HCD mode, the charged precursor ions collide multiple times with inert gas molecules in the collision cell, easily breaking the amide bonds of the peptide backbone, thus generating a series of characteristic fragment ions. For peptides, the breakage usually occurs at the peptide bonds, forming b-type ion fragments starting from the N-terminus and y-type ion fragments starting from the C-terminus. By analyzing the mass changes of these continuous fragment ions, the specific amino acid of the peptide can be accurately identified as the site of modification. The secondary mass spectrometry and fragment ion resolution results are shown in Tables 8 and 9. Figure 7 and Figure 8 As shown.

[0101] Table 8. Secondary mass spectrometry fragment ion analysis of the modified peptide 4b

[0102] Table 9. Secondary mass spectrometry fragment ion analysis of the modified peptide 5b

[0103] The results in the table above show that ion fragments containing Cys residues all exhibited mass shifts consistent with modification, while y-series ion fragments ruled out the possibility of modifications at other sites, such as... Figure 7 and Figure 8 As shown, this confirms that the modification only occurs at the Cys site.

[0104] Example 8: Verification of the synergistic effect of thiol-amino groups Using S-methyl-L-cysteine ​​methyl ester hydrochloride 7a (thiol group protected by methyl group) as a substrate, it reacts with compound 1a. 1 No change in H NMR, such as Figure 9 As shown; using N-acetyl-L-cysteine ​​methyl ester 6a (amino group protected by acetyl group) as a substrate, it reacts with compound 1a. 1The aromatic region of ¹H NMR was disordered, and byproducts such as disulfide bonds and phenyl sulfides were detected by LC-MS. Azo products disappeared. Figure 10 As shown in (a); using L-cysteine ​​methyl ester hydrochloride 2a (with both thiol and amino groups present) as a substrate, the reaction proceeds efficiently with a yield ≥ 95%, as shown in (a). Figure 10 As shown in (b), the chaotic spectrum of the monothiol system contrasts sharply with the clear spectrum of the system with coexisting thiol and amino groups, indicating that when the amino group is protected, the reaction pathway becomes uncontrolled and cannot proceed along a single coupling pathway.

[0105] like Figure 11 As shown, further LC-MS analysis of the monothiol system (1a+6a) revealed that although a few UV absorption peaks were present in the chromatograms at 254 nm and 210 nm ( Figure 11 a, 11b), but the total ion chromatogram (TIC, Figure 11 The presence of numerous signal peaks in c) indicates a complex reaction product with weak UV absorption. Among them, disulfide crosslinking products (t) were detected. R ≈ 2.8 min), phenyl sulfide byproducts (t R ≈ 3.9 min) and several unidentified aryl byproducts. (The asterisks in the figure indicate...) The marked areas represent inherent impurity signals from the instrument. In contrast, LC-MS analysis of the thiol-amino coexistence system (1a+2a) yielded drastically different results: the product peaks in the chromatograms at 254 nm and 210 nm were single (…). Figure 11 d, 11e), total ion chromatogram ( Figure 11 The main signal in f) is the azo coupling product 3a(t) R (≈ 3.45 min), only trace amounts of denitroaryl sulfide byproducts were detected.

[0106] LC-MS analysis showed that the products of the single-thiol system were complex and diverse, while the thiol-amino coexistence system produced only a single azo coupling product, confirming the key regulatory role of the amino group in guiding the reaction along the selective azo coupling pathway.

[0107] The control experiments described above demonstrate that although the amino group does not participate in the final bond formation, it plays a crucial regulatory role in the selection of the reaction pathway. When the amino and thiol groups coexist, the reaction proceeds efficiently along the azo coupling pathway; the absence of the amino group leads to an uncontrolled reaction pathway, generating various byproducts; while the absence of the thiol group prevents the reaction from occurring at all. This result confirms that the synergistic effect of the thiol and amino groups is the core mechanism for achieving high selectivity in this reaction: the amino group activates the thiol group through internal base catalysis, enhancing the competitiveness of the coupling pathway; simultaneously, in conjunction with the electronic effects of the diazonium salt itself, they jointly guide the reaction to directionally generate the target azo coupling product.

[0108] Example 9: Effect of thiol-amino sequence distance on the reaction To systematically investigate the effect of the sequence distance between the thiol and amino groups on the reaction efficiency, this embodiment designed and synthesized a series of model peptides using glycine (Gly) as the spacer unit. This series of peptides used Cys as the thiol donor and the N-terminal free amino group as the cooperating site. Model systems 8a-h with spacers of 0 to 7 amino acids were constructed by introducing different numbers of glycine residues, specifically the sequences CG, GC, GGC, GGGC (SEQ ID NO.1), GGGGC (SEQ ID NO.2), GGGGGC (SEQ ID NO.3), GGGGGGC (SEQ ID NO.4), and GGGGGGGC (SEQ ID NO.5). All model peptides were prepared using the standard Fmoc solid-phase peptide synthesis strategy. Each model peptide (50 mM) was reacted with 1a (50 mM) in pure water at room temperature for 5 minutes. The general reaction formula is as follows:

[0109] After the reaction, an equimolar sodium bicarbonate aqueous solution (50 mM) was added, and the azo coupling product precipitated. The product was centrifuged at 10000 r / min for 2 min, the supernatant was discarded, and the product was washed three times with pure water, centrifuged at 10000 r / min for 2 min after each wash, and the supernatant was discarded. The precipitate was concentrated under reduced pressure using an oil pump and dried to obtain the azo coupling product of the model peptide. The yields of the azo coupling reactions of each model peptide with compound 1a are shown in Table 10: the yield was 93% when there was no glycine between the thiol and amino groups, 50-65% when there were 1-4 glycine groups, 45-57% when there were 5-7 glycine groups, and still reached 45% when there was a 7 glycine group. This is in stark contrast to the single-thiol system (completely unreacted). The yields shown in the table are NMR quantification yields, with maleic acid as an internal standard. 1 Yield was determined by H NMR.

[0110] Table 10. Azo coupling yield of GC model peptides with different interval lengths

[0111] The results above show that the number of spacer amino acids has a significant impact on the azo coupling yield. While azo coupling can still occur with increasing spacer number, the overall yield decreases. However, the model peptide with the largest spacer (8h) can still undergo azo coupling, indicating that amino synergistic effects are effective over a relatively wide sequence range.

[0112] Example 10 Synthesis of a two-headed diazonium salt crosslinking agent and construction of cyclic peptides 1. Synthesis of Bifunctional Diazonium Salt Crosslinking Agents: Three bifunctional aryl diazonium tetrafluoroborate crosslinking agents 10a-12a were synthesized using 1,4-phenylenediamine, benzidine, or 4,4'-diaminodiphenyl ether as raw materials, as shown in Table 11. The synthesis method is as follows: The corresponding aromatic diamine (15 mmol) was dissolved in a mixture of 120 mL water and 12 mL of 50 wt% fluoroboric acid to form a homogeneous solution. The reaction system was cooled to 0°C in an ice-water bath, and saturated sodium nitrite aqueous solution (55 mmol) was slowly added dropwise, controlling the reaction temperature not to exceed 5°C. After the addition was complete, stirring was continued for 30 min. After the reaction was complete, the diazonium salt product precipitated. The precipitate was collected by vacuum filtration, washed successively with ice water (20 mL) and diethyl ether (20 mL), dried under vacuum to obtain the target product, and stored in a -20°C refrigerator for later use. The structures of the obtained diazonium salts were confirmed to be correct by ¹H NMR and ¹³C NMR, and the purity of the products met the requirements for subsequent reactions. The yields shown in the table are the isolated yields.

[0113] Table 11 Dual-headed diazonium salt crosslinking agents

[0114] 2. Cyclization reaction study of the model cyclic peptide CRGDKC (SEQ ID NO.6) 1) Construction of cyclic peptides Linear peptide CRGDKC (13a) was prepared using the standard Fmoc solid-phase synthesis method, with a purity >95%. Linear peptide 13a (50 mM) was reacted with each cross-linking agent (50 mM) in pure aqueous phase at room temperature for 5 minutes. The reaction solution was directly purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The target components were collected and lyophilized. The semi-preparative RP-HPLC model was Shimadzu LC-16P, with a C18 reversed-phase column (250 mm × 20 mm, 5 μm). Mobile phase A was an aqueous phase containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile phase containing 0.1% trifluoroacetic acid. The detection wavelengths were 214 nm and 254 nm. Before sample loading, the sample was filtered through a 0.22 μm filter membrane. The liquid phase separation method is shown in Table 12. The corresponding chromatographic peak components were collected according to the retention time of the target product, combined, and lyophilized to obtain purified products 10b-12b. Maleic acid was used as an internal standard. 1 The yield of the cyclized product was determined by 1H NMR.

[0115] Table 12 Liquid phase separation methods

[0116] Table 13 Cyclic Peptide Structures

[0117] 2) Conformation regulation This embodiment quantifies the conformational constraints imposed on cyclic peptides by different crosslinking agents by calculating the spatial distance between the sulfur atoms of the two cysteine ​​side chains. This allows for structural verification of whether the conformation of cyclic peptides can be regulated by changing only the structure of the crosslinking agent without altering the primary sequence of the peptide.

[0118] The conformations of the cyclic peptides were evaluated using density functional theory (DFT). All calculations were performed using the Gaussian 16 (Revision A.03) software package. Geometric optimization was performed at the M06-2X / 6-31G(d) theoretical level to locate all stationary points. Grimme's D3 dispersion correction was incorporated during optimization. Frequency analysis confirmed that the optimized geometry was a minimum point, and thermal corrections to the Gibbs free energy were obtained. Single-point energies were calculated at the M06-2X-D3 / 6-311+G(d,p) level. The sulfur atom spacings of the linear peptide 13a and the cyclized cyclic peptides 10b, 11b, and 12b are shown in Figure 12.

[0119] Calculations show that the spatial distance between the sulfur atoms in the two cysteine ​​side chains of a linear peptide is 11.227 Å. When different bi-headed diazonium salt crosslinking agents are introduced, the distance between the two sulfur atoms changes significantly: in cyclic peptides crosslinked with 1,4-didiazobenzenetetrafluoroborate (10a), the distance between the two sulfur atoms decreases to 5.801 Å; in cyclic peptides crosslinked with 4,4'-diphenyl ether diazonium tetrafluoroborate (11a), the distance is 9.306 Å; and in cyclic peptides crosslinked with 4,4'-diphenyl ether diazonium tetrafluoroborate (12a), the distance is the largest, reaching 10.084 Å. This change in distance directly reflects the significant influence of the crosslinking agent backbone length on the geometric constraint scale of cyclic peptides. Therefore, when the primary sequence of the peptide is completely identical, the conformational constraint scale of cyclic peptides can be systematically regulated simply by changing the structure of the crosslinking agent. This strategy breaks through the limitations of traditional cyclization methods where the ring size is restricted by the linear precursor sequence itself, providing an important structural basis for subsequent research on the structure-activity relationship between conformational changes and peptide function within the same linear precursor sequence context.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for modifying a compound containing cysteine ​​residues, characterized in that, The aryl diazonium tetrafluoroborate of Formula I is reacted with a compound containing a cysteine ​​residue to form an azo coupling product, wherein the compound containing the cysteine ​​residue has a free thiol group (-SH) and a free amino group (-NH2), or an amino group is present on an amino group adjacent to the cysteine ​​residue; the structure of Formula I is as follows: Formula I Wherein, R is selected from -F, -Cl, -Br, -I, -H, -CF3, -CH3 or -OCH3; Ar is selected from phenyl, diphenyl, or diphenyl ether; n is an integer that is either 1 or 2.

2. The method according to claim 1, characterized in that, The aryldiazotetrafluoroborate includes one or more of the following compounds: 。 3. The method according to claim 1, characterized in that, The reaction of the aryldiazotetrafluoroborate and the compound containing cysteine ​​residues is carried out in pure water.

4. The method according to claim 1, characterized in that, The compounds containing cysteine ​​residues include cysteine ​​or its derivatives, polypeptides or proteins containing cysteine ​​residues.

5. The application of the method of claim 1 in the preparation of cyclic peptides or in regulating the conformation of cyclic peptides.

6. A method for preparing a cyclic peptide, characterized in that, The preparation method includes reacting the aryldiazotetrafluoroborate of claim 1 with a linear polypeptide to undergo intramolecular cyclization, generating a cyclic peptide, wherein n is 2 in the aryldiazotetrafluoroborate.

7. The preparation method according to claim 6, characterized in that, The aryldiazotetrafluoroborate includes any one of the following compounds: 。 8. The preparation method according to claim 6, characterized in that, The linear polypeptide contains two cysteine ​​residues.

9. The preparation method according to claim 6, characterized in that, The linear polypeptide contains free amino groups at positions 0-7 amino acids apart from the thiol groups in cysteine ​​residues.

10. The preparation method according to claim 6, characterized in that, The intramolecular cyclization reaction between the aryldiazotetrafluoroborate and the linear polypeptide was carried out in pure water.

Images