A method for preparing branched polypeptides based on stepwise functionalization by chemical-enzymatic method

Abstract

The application discloses a method for preparing branched polypeptides based on chemical-enzyme step-by-step functionalization, and belongs to the field of cross between biotechnology and synthetic chemistry. The method uses a peptide A containing an asparagine residue at a C terminal and a peptide B containing a cysteine residue at an N terminal as raw materials, first uses polypeptide amidase PAM15 to catalyze the reaction of the peptide A and hydrazine to generate a diacylhydrazine intermediate; then the diacylhydrazine intermediate is subjected to azidation by low-temperature sodium nitrite to obtain a diacyl azide intermediate; finally, the diacyl azide intermediate is selectively connected with the peptide B to obtain a branched polypeptide with an asparagine as a core branched node. The application has high site selectivity, requires less amount of a protecting group, has strong sequence compatibility, and has a clear product structure, breaks through the limitation of traditional lysine nodes, can efficiently construct diversified branched polypeptides, and is suitable for fields such as biomaterials, antibacterial peptides and vaccine research and development.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biotechnology and synthetic chemistry, and in particular to a method for preparing branched peptides based on chemical-enzymatic stepwise functionalization. Specifically, it involves a strategy that combines peptide amidase-specific catalysis with chemical coupling to construct branched peptides with asparagine residues as core nodes, which can be applied to the fields of biomaterials, antibacterial drugs and vaccine development. Background Technology

[0002] Branched-chain peptides, with their unique spatial topology, have significant application value in fields such as biomaterial construction, antimicrobial peptide development, multivalent display of vaccine antigens, and drug delivery system design. Compared to linear peptides, branched-chain peptides can significantly increase antigen loading, enhance immunogenicity, improve pharmacokinetic properties, and improve the stability of biological activity.

[0003] Currently, the mainstream methods for constructing branched peptides mainly include solid-phase polymerization and chemical linking of peptide fragments. Solid-phase polymerization typically uses lysine as the sole core branch node, utilizing the reaction differences between its α-amino and ε-amino groups, combined with the stepwise removal and coupling of multiple sets of orthogonal protecting groups to achieve branching. This method involves cumbersome synthesis steps, complex protecting group operations, numerous side reactions, and difficult purification; furthermore, its synthesis efficiency is easily affected by sequence.

[0004] While chemical linking of peptide fragments can avoid some defects of solid-phase synthesis, it still heavily relies on lysine as a branching node and requires strict control of the orthogonality of protecting groups to achieve selective linking. This method involves linking pre-synthesized linear peptide chains to the α- and γ-amino groups of lysine at the branching node via amide bonding reactions. Because this method does not introduce side-chain protecting groups, it often uses inert amino acids such as alanine, valine, and leucine in non-branching node regions, thus limiting the types of amino acids that can be introduced.

[0005] Existing technologies generally suffer from problems such as lysine dependence on core branch nodes, numerous non-specific linker side reactions, and limited amino acid types for non-branch nodes. Therefore, developing a branched peptide construction method that uses novel amino acids as branch nodes, has high linker selectivity, and has fewer restrictions on the types of amino acids for non-branch nodes has significant application value. Pini et al. (A.Pini, et al. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. The FASEB Journal 2010, 24(4): 1015-1022) reported a tetrabranched antimicrobial peptide that can neutralize bacterial lipopolysaccharide and effectively prevent septic shock in vivo, but its core branch node is still lysine, and the synthesis process depends on the orthogonal removal and coupling of multiple sets of protecting groups. Stach et al. (M. Stach, et al. Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug-Resistant Pseudomonas aeruginosa. Angewandte Chemie International Edition 2014, 53(47): 12827-12831) combined topology and sequence design to develop a dendritic polypeptide macromolecule targeting multidrug-resistant Pseudomonas aeruginosa. They also used lysine as the core of the branch, but the sequence compatibility was limited and it was difficult to adapt to complex peptides containing multiple active functional groups.

[0006] In summary, existing technologies generally suffer from problems such as single branch nodes, high dependence on protecting groups, poor site selectivity, weak sequence compatibility, and numerous byproducts. There is an urgent need to develop a novel, efficient, highly selective, and sequence-compatible method for preparing branched peptides. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing branched-chain peptide preparation technologies, such as lysine dependence on core nodes, excessive use of protecting groups, low site selectivity, poor sequence compatibility, numerous side reactions, and complex synthesis processes. It provides a method for preparing branched-chain peptides based on a chemical-enzymatic stepwise functionalization process. This invention uses asparagine residues as novel core branching nodes, combining specific catalysis by peptide amidase with efficient chemical coupling to achieve precise single-residue dual-site modification and selective linkage. This results in the construction of branched-chain peptides with well-defined structures, strong sequence compatibility, and simple synthesis, meeting the application needs of biomaterials, antimicrobial peptides, and vaccine development.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one embodiment, the present invention provides a method for preparing branched polypeptides based on chemical-enzymatic stepwise functionalization, comprising the following steps: (1) Preparation of peptide A and peptide B: linear polypeptide peptide A with asparagine residues at the C-terminus and linear polypeptide peptide B with cysteine ​​residues at the N-terminus are synthesized by solid-phase synthesis; (2) Enzymatic catalytic dihydrazideization: peptide A is reacted with hydrazine hydrate at pH 8.0-12.0 and temperature 20-37℃ for 15-90 min using peptide amidase PAM15, and the amide group at the C-terminus of peptide A main chain and the β-amide group of the asparagine side chain are simultaneously converted into hydrazide to obtain a dihydrazide intermediate; (3) Low-temperature azidation: the dihydrazide intermediate is reacted with sodium nitrite at 0-4℃ and pH 2.0-6.0 for 5-30 min to generate a diacyl azide intermediate; (4) Selective coupling: the diacyl azide intermediate is reacted with peptide B at pH 4.0-8.0 and 0-4℃ for 15-90 min. min, forming branched polypeptides with asparagine residues as branch nodes.

[0010] In another embodiment, the C-terminal backbone of peptide A of the present invention is an amide group or a methyl ester group.

[0011] In another embodiment, the asparagine side chain of peptide A of the present invention has a β-amide group, providing two-site reactivity.

[0012] In another embodiment, the N-terminus of peptide B of the present invention is a free α-amino group, and the cysteine ​​side chain thiol group is in a free state.

[0013] In another embodiment, the amino acid sequence of the polypeptide amidase PAM15 of the present invention is shown in SEQ ID NO.1, and the nucleotide sequence encoding it is shown in SEQ ID NO.2.

[0014] In another embodiment, the reaction buffer in step (2) of the present invention is one of phosphate buffer, acetate buffer or borate buffer.

[0015] In another embodiment, 4-mercaptophenylacetic acid is added in step (4) of the present invention to promote the coupling reaction between acyl azide and peptide B.

[0016] In another embodiment, the branched polypeptide obtained by the present invention has a Y-shaped bibranched structure, with two branches respectively connected to the C-terminus of the asparagine residue main chain and the β-position of the side chain.

[0017] In another embodiment, the non-branched node region sequence of the branched polypeptide prepared according to the present invention may contain at least one of acidic amino acids, basic amino acids, polar amino acids, or nonpolar amino acids.

[0018] In one embodiment, the present invention provides a branched polypeptide prepared by the method according to the present invention, with asparagine residues as the only core branched nodes, for use in the preparation of biomaterials, antimicrobial peptides or vaccine formulations.

[0019] The present invention has the following outstanding beneficial effects:

[0020] (1) This invention is the first to replace traditional lysine with asparagine residues as the core branch node. By utilizing the C-terminal amide group of its main chain and the β-amide group of the side chain to form a natural double reaction site, a Y-type double branch structure is constructed, which breaks away from the topological limitation of the α-amino / ε-amino reaction mode of lysine and significantly expands the structural diversity and design freedom of branched peptides.

[0021] (2) This invention utilizes the strict substrate specificity of the polypeptide amidase PAM15 to accurately identify and catalyze the simultaneous acylhydrazideation of the diamide group of asparagine residues, avoiding non-specific modification; the subsequent azidation and coupling reaction conditions are mild and highly selective, requiring no large amount of orthogonal protecting groups, significantly reducing byproducts, and the product has a single structure, high purity, and controllable molecular weight.

[0022] (3) The reaction system of the present invention is highly compatible with common functional groups such as amino, carboxyl, hydroxyl and thiol groups on polypeptide chains. Acidic, basic, polar or nonpolar amino acids can be freely introduced into the non-branched node region, no longer limited to inert amino acids, which can meet the design requirements of diverse functional peptide sequences and adapt to different application scenarios such as biomaterials, antimicrobial peptides, and vaccines.

[0023] (4) The present invention adopts a stepwise functionalization strategy of enzyme catalysis and chemical coupling, with fewer synthesis steps, mild reaction conditions, and very little amount of protecting group. It avoids the cumbersome deprotection-coupling cycle of traditional methods, greatly reduces the difficulty of operation and time cost, and has high yield of intermediates and final products, and has the potential for large-scale preparation.

[0024] (5) The branched peptides prepared by this method have well-defined structures, good stability, and controllable biological activity. They can be directly used in the fields of antimicrobial peptide development, multivalent antigen display of vaccines, and construction of tissue engineering scaffold materials, providing efficient and reliable preparation technology support for related biomedical and biomaterial industries. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the overall reaction process for preparing branched peptides based on the chemical-enzymatic stepwise functionalization method of the present invention.

[0026] Figure 2 This is a mass spectrometry chromatogram for identifying the dihydrazide functionalized intermediate and the final branched polypeptide product of this invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the experimental conditions are conventional conditions in the art.

[0028] Example 1: Synthesis of branched-chain polypeptides

[0029] I. Synthesis of raw material peptides:

[0030] Peptide A: Ac-DFSKN-CONH2 (N-terminal acetylation protection, C-terminal amidation, amino acid sequence: aspartic acid-phenylalanine-serine-lysine-asparagine).

[0031] Peptide B: H2N-CLKKA-CONH2 (N-terminal free amino group, C-terminal amidation, amino acid sequence: cysteine-leucine-lysine-lysine-alanine).

[0032] A Fmoc solid-phase synthesis strategy was employed, sequentially coupling Fmoc-protected aspartic acid, phenylalanine, serine, lysine, and asparagine onto Rink amide resin. N-terminal acetylation was achieved using acetic anhydride blocking, and C-terminal amidation was performed to form -CONH2. After synthesis, the peptide chains were cleaved from the resin and the side-chain protecting groups were removed using a cleavage reagent (TFA / H2O / TIS = 95:2.5:2.5). The crude product was precipitated with cold diethyl ether and purified by preparative HPLC (C18 column, acetonitrile / water gradient elution), followed by lyophilization to obtain a white powder. HPLC purity >95%, and mass spectrometry-determined molecular weight: 650.3 Da (theoretical value 650.3 Da).

[0033] Using the same Fmoc solid-phase synthesis strategy, cysteine, leucine, lysine, lysine, and alanine were sequentially coupled onto Rink amide resin. The N-terminus retained a free amino group, while the C-terminus was amidated. The cleavage and purification steps were the same as peptide A. HPLC purity >95%, and mass spectrometry measured molecular weight: 560.3 Da (theoretical value 560.3 Da).

[0034] Peptide A: Purity > 95%, mass spectrometry measured 650.3 Da (theoretical value 650.3 Da);

[0035] Peptide B: Purity > 95%, mass spectrometry measured 560.3 Da (theoretical value 560.3 Da).

[0036] II. Preparation of Peptidase PAM15

[0037] The nucleotide sequence encoding PAM15 (SEQ ID NO.2) was cloned into pET-28a and transformed into BL21 (DE3); it was induced at 30℃ with 1 mM IPTG for 12 h, sonicated, and purified with Ni-NTA; SDS-PAGE showed approximately 55 kDa and purity >90%.

[0038] The amino acid sequence of SEQ ID NO. 1: Peptidase PAM15 is: MAEPASRNVPFPYAETDVADLQARMTAGELDSTTLTQAYLQRIAALDRTGPRLHAVIELNPDALKEAAERDRERRDGRLRGPLHGIPLLLKDGINAAPMATSAGSLALQDFRPDDAYLVRRLRDAGAVVLGKTNLSEWGNFRSNNSISGWSARGGQTRNPYRPSHSPCGSSSGSAVAVAANLASVAIGTETDGSIVCPAAINGVVGLKPTVGLVSRDGGIPGSFSQDTPGPMARSVADAAAVLTA IAGRDPADPATATMPGRAVYDYTARLDPQGLRGKRIGLLQVPLLKYRGMPPLIEQAATELRRAGAVVVPVELPNYGAWAEAERTLLLYEFKAGLERYFNTHRAPLRSLADLIAFNQAHSKQELALFGQELLV EADATAGLADPAYIRARSDARRLAGPEGIDAALAAHQLDALVAPTTGVAWPIRPEGDDFPGESYSAAAVAGYPSLTVPMGQIDGLPVGLLFMGTAWSEPKLIEMAYAYEQRTRARRPPHFDTTDALIDAGEP.

[0039]

[0040] III. Enzyme-catalyzed diacylhydrazide

[0041] 10 mg of peptide A was dissolved in 2 mL of 100 mM phosphate buffer (pH 9.0), and 0.1 mL of hydrazine hydrate (50 mM) and 0.2 mg of PAM15 were added. The reaction was carried out at 25 °C for 60 min, and HPLC monitoring was performed until the starting peak disappeared and a new peak was formed (12.1 min). The overall process is shown in Figure 1, which is a schematic diagram of the overall reaction process of this invention, showing the entire process of enzyme-catalyzed dihydrazidation, azidation, and coupling.

[0042] IV. Enzymatic preparation of dihydrazide-modified peptide A (intermediate II)

[0043] 10 mg of peptide A was dissolved in 2 mL of 100 mM phosphate buffer (pH 9.0), and 0.1 mL of hydrazine hydrate (final concentration 50 mM) and 0.2 mg of PAM15 enzyme were added. The reaction was carried out with gentle shaking at 25 °C for 60 minutes. HPLC monitoring showed the disappearance of the main peak and the formation of a new peak (retention time 12.1 min). After preparative HPLC purification, intermediate II was obtained by lyophilization. The yield was calculated to be 92% by weighing the lyophilized powder. The molecular weight determined by mass spectrometry was 680.3 Da (theoretical value 680.3 Da, corresponding to a weight gain of 30 Da from dihydrazide modification), as shown in the upper part of Figure 2.

[0044] V. Azide Conversion

[0045] Dissolve 5 mg of intermediate II in 1 mL of 100 mM HCl (pH 3.0), place in an ice bath at 0°C, add 0.5 mL of pre-cooled 0.5 M NaNO2, and react for 15 min to obtain a diazid intermediate solution.

[0046] VI. Construction of Branched Peptides

[0047] Add 1 mL of peptide B solution (peptide A: peptide B = 1:2, pH 7.0 phosphate buffer, containing 50 mM 4-mercaptophenylacetic acid) to the diazytidyl azide intermediate solution and react at 0℃ for 60 min; HPLC showed a new peak (18.3 min), with a conversion rate of 76%; mass spectrometry measured 1736.7 Da (theoretical value 1736.9 Da), confirming it as the target Y-type branched polypeptide, as shown in the lower half of Figure 2. Figure 2 shows the mass spectra of intermediate II and the product, with the measured / theoretical molecular weight and structure labeled to verify the correctness of the structure.

[0048] The high-performance liquid chromatography-mass spectrometry (HPLC-MS) analytical method used in the examples is as follows:

[0049] Instrumentation: Agilent 1200 high-performance liquid chromatography system + G1946D single quadrupole mass spectrometer

[0050] Instrument setup: A C18 reversed-phase column (4.6 mm × 250 mm, pore size 120 Å, particle size 5 μm) was used for analysis. Chromatographic conditions were as follows: mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile (CH3CN); flow rate was 1.0 mL / min. -1 The column temperature was 30℃, the UV detection wavelength was 220 nm, and the run time was 15 min. The elution program was a linear gradient elution: the initial mobile phase contained 5% B, which linearly increased to 45% B within 24 min, and then held at 5% B for 30 min. The mass spectrometry was performed in positive ion mode, with a scanned mass range of m / z 250–1250. The measured molecular weights were compared with the theoretical molecular weights to confirm the structural correctness of each intermediate and the final product.

[0051] Therefore, this invention is compatible with acidic amino acid sequences and polar / nonpolar amino acid sequences. It uses asparagine as the core node and efficiently prepares well-defined Y-type branched polypeptides through enzymatic-chemical stepwise modification. It does not require complex protecting groups, has good selectivity, and high yield, which fully demonstrates the versatility and practicality of this invention.

[0052] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for preparing branched peptides based on chemical-enzymatic stepwise functionalization, characterized in that, Includes the following steps: (1) Preparation of peptide A and peptide B: linear polypeptides containing asparagine residues at the C-terminus, namely peptide A and linear polypeptides containing cysteine ​​residues at the N-terminus, namely peptide B, were synthesized by solid-phase synthesis. (2) Enzymatic dihydrazideation: Peptide A reacts with hydrazine hydrate at pH 8.0-12.0 and temperature 20-37℃ for 15-90 min using peptide amidase PAM15 to simultaneously convert the C-terminal amide group of peptide A main chain and the β-amide group of asparagine side chain into hydrazide to obtain dihydrazide intermediate; (3) Low-temperature azidation: Under the conditions of 0-4℃ and pH 2.0-6.0, the diacylhydrazine intermediate reacts with sodium nitrite for 5-30 min to generate the diacyl azid intermediate; (4) Selective coupling: Under pH 4.0-8.0 and 0-4℃ conditions, the diacyl azide intermediate reacts with peptide B for 15-90 min to form a branched polypeptide with asparagine residues as branch nodes.

2. The method according to claim 1, characterized in that, The C-terminal backbone of peptide A is an amide group or a methyl ester group.

3. The method according to claim 1, characterized in that, The asparagine side chain of peptide A has a β-amide group, providing two-site reactivity.

4. The method according to claim 1, characterized in that, The N-terminus of peptide B is a free α-amino group, and the cysteine ​​side chain thiol group is in a free state.

5. The method according to claim 1, characterized in that, The amino acid sequence of the polypeptide amidase PAM15 is shown in SEQ ID NO.1, and the nucleotide sequence encoding it is shown in SEQ ID NO.

2.

6. The method according to claim 1, characterized in that, In step (2), the reaction buffer is one of phosphate buffer, acetate buffer or borate buffer.

7. The method according to claim 1, characterized in that, In step (4), 4-mercaptophenylacetic acid is added to promote the coupling reaction between acyl azide and peptide B.

8. The method according to claim 1, characterized in that, The resulting branched polypeptide has a Y-shaped bibranched structure, with two branches connected to the C-terminus of the asparagine main chain and the β-position of the side chain, respectively.

9. The method according to claim 1, characterized in that, The unbranched node region sequence of the prepared branched polypeptide may contain at least one of acidic amino acids, basic amino acids, polar amino acids, or nonpolar amino acids.

10. The branched polypeptide prepared by the method according to any one of claims 1-9, characterized in that, Using asparagine residues as the sole core branch node, it is used to prepare biomaterials, antimicrobial peptides, or vaccine formulations.

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