PEG的修饰作用和PEG修饰方法

聚乙二醇修饰即PEG化，是将活化的PEG通过化学方法以共价键偶联到蛋白质或多肽分子上。自Davies 1977年用PEG 修饰牛血清白蛋白以来， PEG修饰技术广泛应用于多种蛋白质和多肽的化学修饰，多个PEG修饰药物上市或在临床研究中。 PEG修饰具有半衰期延长、免疫原性降低或消失、毒副作用减少以及物理、化学和生物稳定性增强等。

高分子聚乙二醇（PEG）由于其毒性小、无抗原性、具有良好的两亲性，且生物相容性已获FDA认可，对蛋白质的改造具有无可取代的优势。聚乙二醇化修饰技术通过共价键，将聚乙二醇与被修饰药物耦联，改善药物的理化性质和生物学活性，这种技术现已广泛应用于蛋白质(肽类)、酶、抗体及小分子药物。

1 PEG化学结构及性质

1.1 PEG化学结构

结构式CH2（OH）-（CH2CH2O）n -CH2OH

1.2 PEG性质

聚乙二醇系列产品通常情况下溶于水和多种有机溶剂，不溶于脂肪烃、苯、乙二醇等，不会水解变质，有广泛的溶解范围和优良的相容性、很好的稳定性、润滑性、成膜性、增塑性、分散性等。系低毒物质，且无刺激性，属非离子型聚合物。

2 聚乙二醇修饰剂

根据化学修饰剂与蛋白质之间反应性质的不同，修饰反应主要分为酰化反应、烷基化反应、氧化还原反应、芳香环取代反应等类型，对蛋白质进行氨基、巯基和羧基等侧链基团进行化学修饰。根据被修饰化合物包括蛋白质、多肽、单克隆抗体分子片段、以及小分子化合物等的不同分子量大小、分子结构以及其理化特性，采取不同的聚乙二醇化技术方法对这些化合物进行修饰。

2.1随机修饰

随机修饰蛋白质多以赖氨酸的ε-NH2或α-NH2为修饰目标，由于赖氨酸在蛋白质内通常数量较多，这种修饰引起蛋白质中多个赖氨酸被修饰，得到的产物是聚乙二醇化修饰异构体的混合物，目前FDA批准的已经上市的聚乙二醇化新药多数为随机修饰的产物。

2.2定点修饰

聚乙二醇化定点修饰，是在随机修饰的基础上发展起来的第二代聚乙二醇修饰技术，通过对修饰方式、PEG修饰剂和反应pH等的优化选择，实现定点修饰，这种修饰所得的产物为均一产物，异构体少，活性保留较好，免疫原性大大降低。

2.2.1 N端氨基定点修饰

由于蛋白质、多肽中存在多个α-NH2 和ε-NH2基团，氨基的随机修饰可能导致药物活性的明显下降，这给蛋白质、多肽的修饰带来了障碍。目前我们采取对N端氨基定点修饰，特别是对于远离活性中心的 N端定点修饰可以有效地保持药物的原有生物活性。

2.2.2巯基定点修饰

结合基因工程技术或Mutagenesis等方法，将巯基或一些特异性基团引入到蛋白、多肽预设位点（如不影响活性的糖基化位点、抗原决定簇等）后，再进行对该基团的修饰，这样就可得到活性保留较高和免疫原性降低的定点修饰产物。

2.2.3羧基定点修饰

采用带酰肼活化基团的聚乙二醇衍生物对蛋白质、多肽进行羧基基团进行修饰， 同样可以获得定点的修饰产物。

2.2.4酶催化修饰

采用酶催化手段诱导聚乙二醇分子与蛋白质、多肽等分子上特定的位点进行定向修饰。此方法具有一下特点：

（1）不需要对原蛋白质、多肽进行结构改造，保持原化合物的理化特性不受改变；

（2）定点修饰；

（3）工艺简单，容易大规模化生产和质量控制。

PEG modification effect and PEG modification method

Polyethylene glycol modification, or PEGylation, is to chemically couple activated PEG to a protein or polypeptide molecule by a covalent bond. Since Davies modified bovine serum albumin with PEG in 1977, PEG modification technology has been widely used in chemical modification of various proteins and polypeptides, and many PEG modified drugs have been listed or in clinical research. PEG modification has the advantages of prolongation of half-life, reduction or disappearance of immunogenicity, reduction of toxic and side effects, and enhancement of physical, chemical and biological stability.

Because of its low toxicity, no antigenicity, good amphiphilicity, and biocompatibility, macromolecular polyethylene glycol (PEG) has an irreplaceable advantage in protein modification. The PEGylation modification technology couples polyethylene glycol with the modified drug through covalent bonds to improve the physicochemical properties and biological activity of the drug. This technology has been widely used in proteins (peptides), enzymes, antibodies and small molecule drugs.

1 PEG chemical structure and properties

1.1 Chemical structure of PEG

Structural formula CH2(OH)-(CH2CH2O)n-CH2OH

1.2 Properties of PEG

Polyethylene glycol series products are usually soluble in water and various organic solvents, insoluble in aliphatic hydrocarbons, benzene, ethylene glycol, etc., and will not be hydrolyzed and deteriorated. They have a wide range of dissolution and excellent compatibility. Stability, lubricity, film formation, plasticity, dispersibility, etc. Department of low toxic substances, and non-irritating, is a non-ionic polymer.

2 polyethylene glycol modifier

According to the different nature of the reaction between chemical modifiers and proteins, the modification reactions are mainly divided into acylation reactions, alkylation reactions, redox reactions, aromatic ring substitution reactions, etc. group for chemical modification. According to the different molecular weights, molecular structures and physicochemical properties of the compounds to be modified, including proteins, polypeptides, monoclonal antibody fragments, and small molecular compounds, different PEGylation techniques are used to modify these compounds.

2.1 Random modification

Randomly modified proteins mostly target ε-NH2 or α-NH2 of lysine. Since lysine is usually in large quantity in the protein, this modification causes multiple lysines in the protein to be modified, and the resulting product is The mixture of PEGylated isomers, most of the new PEGylated drugs approved by the FDA are currently randomly modified products.

2.2 Fixed-point modification

PEGylation site-directed modification is a second-generation polyethylene glycol modification technology developed on the basis of random modification. By optimizing the selection of modification methods, PEG modifiers and reaction pH, site-directed modification is realized. This modification The obtained product is a homogeneous product with less isomers, better activity retention and greatly reduced immunogenicity.

2.2.1 N-terminal amino group site-directed modification

Due to the existence of multiple α-NH2 and ε-NH2 groups in proteins and polypeptides, random modification of amino groups may lead to a significant decrease in drug activity, which brings obstacles to the modification of proteins and polypeptides. At present, we adopt the site-directed modification of the N-terminal amino group, especially for the N-terminal site-directed modification far from the active center, which can effectively maintain the original biological activity of the drug.

2.2.2 Thiol site-directed modification

Combined with genetic engineering technology or Mutagenesis and other methods, thiol groups or some specific groups are introduced into the preset sites of proteins and polypeptides (such as glycosylation sites that do not affect activity, antigenic determinants, etc.) In this way, site-directed modification products with higher activity retention and reduced immunogenicity can be obtained.

2.2.3 Carboxyl site-directed modification

Using polyethylene glycol derivatives with hydrazide activating groups to modify the carboxyl groups of proteins and polypeptides, a site-specific modification product can also be obtained.

2.2.4 Enzymatic modification

Enzyme catalysis is used to induce targeted modification of polyethylene glycol molecules and specific sites on proteins, polypeptides and other molecules. This method has the following characteristics:

(1) No structural modification of the original protein and polypeptide is required, and the physical and chemical properties of the original compound are kept unchanged;

(2) Fixed-point modification;

(3) The process is simple, and it is easy for large-scale production and quality control.