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CN-121992056-A - Chemoenzymatic synthesis method of high-sugar-content homogeneous mucin core domain and application thereof

CN121992056ACN 121992056 ACN121992056 ACN 121992056ACN-121992056-A

Abstract

The invention relates to a chemoenzymatic synthesis method of a high-sugar-content homogeneous mucin core domain. The method comprises the steps of firstly catalytically synthesizing key alpha-linked glycosylation amino acid building blocks Fmoc-GalNAc alpha-Ser/Thr-OH at low temperature (-20 ℃ to 0 ℃), then assembling the building blocks on repeated polypeptide skeletons rich in proline, serine and threonine at precise intervals (3-5 amino acid residues) through a solid-phase peptide synthesis technology to form a 'sugar chain growth bracket', then carrying out sequential and directional enzymatic extension of sugar chains on the bracket by utilizing a series of high-specificity glycosyltransferases to construct a uniform core structure (such as a core 3 type), and finally carrying out high-efficiency purification through three-step tandem methods of size exclusion chromatography, ion exchange chromatography and lectin affinity chromatography. The invention overcomes the technical bottleneck of non-uniformity (< 70%) of the traditional method, can prepare mucin core fragments with the uniformity of sugar equal to or more than 95% and the bioactivity highly similar to that of natural mucin in a large scale, and has wide application prospect in the biomedical fields such as mucous membrane protectants, drug delivery carriers and the like.

Inventors

  • Tang Aimei

Assignees

  • 乂乂智能科技(深圳)有限公司

Dates

Publication Date
20260508
Application Date
20260107

Claims (8)

  1. 1. A chemoenzymatic synthesis method of a high-sugar-content homogeneous mucin core domain, which is characterized by comprising the following steps in sequence: (1) Synthesizing Fmoc-protected N-acetylgalactosamine-serine or threonine building blocks connected by alpha-glycosidic bonds, namely Fmoc-GalNAc alpha-Ser-OH or Fmoc-GalNAc alpha-Thr-OH, by adopting a trichloroacetimidate method at a temperature of between-20 ℃ and 0 ℃ and using trifluoromethylsilyl triflate as a catalyst; (2) Preparing a polypeptide main chain on a solid phase carrier by Fmoc solid phase peptide synthesis technology by taking the building blocks obtained in the step (1) and conventional Fmoc-amino acids as raw materials, wherein the polypeptide main chain comprises 8 to 12 PTS repeating units with the general formula of (Pro-Xaa 1-Thr (GalNAc) -Xaa2-Xaa 3), xaa1 is selected from Ser or Ala, xaa2 is selected from Pro or Ala, xaa3 is selected from Ser, thr or Ala, and each building block is used as a glycosylation starting site to be separated by 3 to 5 amino acid residues in the main chain; (3) Enzymatically reacting the polypeptide backbone obtained in step (2), still attached to a solid support or cleaved and purified, with at least one glycosyltransferase and a corresponding sugar nucleotide donor in a buffer solution containing 5-10 mmgcl 2 , said reaction being performed in a dialysis device or a system comprising a nucleotide regenerating enzyme, to extend over GalNAc at the glycosylation initiation site to form a uniform O-sugar chain; (4) And (3) deprotecting the glycopeptide obtained in the step (3), and purifying sequentially by size exclusion chromatography, ion exchange chromatography and lectin affinity chromatography with concanavalin A as a ligand to obtain a mucin core domain with sugar uniformity of not less than 95%.
  2. 2. The method of claim 1, wherein in step (2), the solid support is RinkAmideMBHA resin and the condensing agent system is a combination of HOBt/HBTU and DIPEA.
  3. 3. The method according to claim 1, wherein in step (3), the glycosyltransferase is a β1, 3-galactosyltransferase and the corresponding sugar nucleotide donor is UDP-galactose for constructing a core type 3O-sugar chain structure.
  4. 4. A method according to claim 3, wherein in step (3) an alpha 2, 3-sialyltransferase and CMP-sialic acid are further added for sialylation modification on the core type 3 structure.
  5. 5. The method of claim 1, wherein in step (4), the size exclusion chromatography is performed using Sephacryls-200HR or equivalent medium, the ion exchange chromatography is performed using an anion exchange column MonoQ or equivalent medium, eluting with a gradient of 0-0.5MNaCl, and the lectin affinity chromatography is performed with a buffer containing 0.5M methyl alpha-D-mannoside.
  6. 6. A core domain of mucin having high glycoform uniformity, which is prepared by the method according to any one of claims 1 to 5, wherein the sugar chain structure is a core 3-type structure consisting of Galβ1-3 GalNAc. Alpha. -Ser/Thr, the glycoform uniformity is not less than 95%, and the molecular weight dispersity (Đ) is not more than 1.2.
  7. 7. Use of a high sugar type homogeneous mucin core domain according to claim 6 for the preparation of a pharmaceutical composition or medical device for preventing, alleviating or treating lesions of the digestive tract, respiratory tract or ocular mucosa.
  8. 8. Use of the high sugar type homogeneous mucin core domain according to claim 6 as a carrier for the preparation of a nano-pharmaceutical composition for delivery of small molecule chemicals, polypeptides, proteins or nucleic acid based therapeutic agents.

Description

Chemoenzymatic synthesis method of high-sugar-content homogeneous mucin core domain and application thereof Technical Field The invention belongs to the field of biotechnology and synthetic chemistry intersection, and particularly relates to a chemoenzymatic synthesis method of Mucin (Mucin) core functional domains with precisely controllable structures and high uniformity of sugar types. More specifically, it relates to the large-scale preparation of mucin mimics with defined biological activity by designing and synthesizing specific glycosylated polypeptide backbones and performing precise sugar chain extension using glycosyltransferases. Background Mucins are a class of high molecular weight, highly glycosylated glycoproteins that coat all mucosal surfaces of the human body. They undergo intensive O-glycosylation through their tandem repeats (PTS domains) rich in serine (Ser), threonine (Thr) and proline (Pro), forming a gel layer with viscoelastic and lubricating effects, playing a key role in mucosal physical barrier, lubrication, cell signaling and microbial interactions. The mucin or the core functional fragment thereof with uniform structure is obtained and is important for the deep research of the structure-activity relationship and the development of novel mucous membrane protective agents, drug delivery systems and biological materials based on mucin. Currently, there are three main approaches to mucin acquisition: 1. Natural extraction and recombinant expression are extracted from animal tissue (such as pig stomach and ox submaxillary gland) or recombinant expression by using mammalian cells and yeast (such as Pichia pastoris system disclosed in CN 118109499A). The products obtained by the methods are highly heterogeneous mixtures of sugar chains, the sugar chain structure is complex and uncontrollable, and basic research and high-end application with strict requirements on the uniformity of materials cannot be met. 2. Full chemical synthesis, although the solid phase peptide synthesis technology is mature, for mucins containing tens or even hundreds of glycosylation sites, oligosaccharide chains with complex structures are synthesized in advance and coupled with peptide chains, the steps are extremely complicated, the total yield is extremely low (usually < 1%), the cost is high, and the practicability and the scale potential are not realized. 3. Conventional chemoenzymatic methods generally employ strategies of "first synthesizing sugar chains followed by linking peptide chains" or "random glycosylation on simple peptide chains". The former is still limited by the inefficiency of complex sugar block synthesis, while the latter results in a mixture of multiple glycoforms with poor uniformity (typically < 70%) and difficult prediction and reproducibility of biological activity due to lack of precise control over glycosylation sites and sequence. In view of the above, a long-standing technical bottleneck in the art is the lack of a method capable of efficiently and controllably synthesizing a high molecular weight mucin fragment having a predetermined, uniform sugar chain structure from a relatively simple starting material. This bottleneck severely restricts the application of mucin in precise medical and high-end biomaterials. Disclosure of Invention Object of the Invention The primary aim of the invention is to overcome the defects of the prior art and provide a chemical enzymatic synthesis method of mucin core domain, which has high sugar uniformity and strong structure controllability and is suitable for large-scale preparation. It is a further object of the present invention to provide a high sugar type homogeneous mucin core domain having a definite sugar chain structure and excellent biological activity, which is produced by the method. It is still a further object of the present invention to provide the use of the above mucin core domain in the fields of mucosal protection, drug delivery, etc. Technical proposal To achieve the above object, the present inventors have made intensive studies and creatively proposed a novel synthesis strategy of "in situ, sequential enzymatic sugar chain assembly on a polypeptide skeleton designed and synthesized in advance" starting from a minimum glycosyl unit (GalNAc). The core of the strategy is to perfectly combine the sequence precise control capability of solid phase peptide synthesis with the catalytic specificity of glycosyltransferase, and the strategy is realized by the following steps of synergistic effect: First step, low-temperature and high-selectivity preparation of key glycosylated amino acid building blocks To ensure the singleness and correct configuration of the starting points for the subsequent sugar chain extension, the present invention first requires a high purity, high stereoselectivity starting block. The reaction of Fmoc-Ser-OH or Fmoc-Thr-OH with the trichloroacetimidate donor of GalNAc was catalyzed by a catalytic amount (0.05-