Activation of polyethylene glycol(PEG)



The modification of proteins by polyethylene glycol is mainly achieved by the reaction between the terminal hydroxyl groups of polyethylene glycol and the amino acid residues of proteins. However, the terminal hydroxyl groups of polyethylene glycol have poor activity and are difficult to couple with proteins in a mild environment. , so an activator must be used to activate the hydroxyl group, so that the activated polyethylene glycol can covalently modify the protein in a mild environment. In recent years, the activation method of polyethylene glycol has become one of the research hotspots in this field.

Nonspecific activation method
(1) Cyanogen bromide method
One of the earliest activation methods for chromatographic or chromatographic solid-phase supports, that is, cyanogen bromide activation method. When the pH is high, cyanogen bromide reacts with the hydroxyl group inside the support and converts it into cyanate and iminocarbonate. Later, this method was also applied to the activation of macromolecular polymers with hydroxyl groups. The method is simple and easy to repeat and the reaction environment is mild, but the ligands (including protein R-NH2) after coupling are easy to fall off. It is caused by the formation of unstable isourea bonds between the activated carrier and the amino-containing ligand, and the toxic operation of cyanogen bromide must be carried out in a fume hood, and this method is gradually replaced by other methods.

(2) Carbonyl diimidazole method
This method was first used in the synthesis of polypeptides and proved to be a good reagent for forming amide bonds. Hydroxyl-containing carriers can react with carbonyldiimidazole (CDI) to obtain active acylimidazoles, and the primary amino groups of lysine residues in proteins can react with them. It reacts rapidly to form a stable amide bond, and this chemical bond is more stable than the coupled protein and is not easy to fall off.
CDI activated PEG should pay attention to:
(1) CDI generates CO2 and imidazole in contact with water, so the activation reaction should be carried out in an organic solvent and the solvent should not contain water
(2) The activated polyethylene glycol is preferably washed at low temperature to maintain high reactivity
(3) The activated polyethylene glycol should be freeze-dried to remove the residual organic solvent so as to avoid the failure of the coupled protein
(4) Do not contain amines (such as Tris or glycine) in the protein coupling buffer, otherwise it will compete with the protein molecules to be coupled for coupling.

(3) N-hydroxysuccinimide method
(a) Activation of N,N-succinimidyl carbonate This reaction also needs to be carried out under anhydrous conditions.
(b) Activation of succinic anhydride and N-hydroxysuccinimide
The polyethylene glycol activated by this method has high activity, and it is best to perform protein coupling in a non-aqueous environment, but for most biological macromolecules, aqueous systems are preferred, and at 4°C pH=8 The half-life of activated polyethylene glycol in water is about 20 minutes, so the coupling reaction is best completed within 20 minutes at low temperature.

(4) Cyanuryl chloride method
Cyanuryl chloride, also known as trichloride azine (TST), is a symmetrical heterocyclic compound containing three active acid chloride bonds, which is widely used in the dye industry. The three chlorine atoms on TST are prone to nucleophilic substitution reaction, and the substitution of one chlorine atom can stabilize other acid chloride bonds, so the first chlorine atom can react at 4°C, and the second at 25°C. The third reacted at 80°C. David et al. used the reaction between TST and the hydroxyl group on polyethylene glycol, only one chlorine atom was substituted, and the other chlorine atoms reacted with the amino group of the protein to inhibit the nucleophilic activity of the chlorine atom to control the speed and modification degree of the reaction, and aniline could be added first. Substituting one of the chlorine atoms, the remaining chlorine atoms are used for protein coupling. Although this reaction is simple and feasible, its application is limited due to the toxicity of TST and the strong nucleophilic activity of halogen atoms.

(5) The activation method that phosgene participates in Kurfuerst etc. mentioned some methods in its patent. Activated polyethylene glycol was prepared by reacting N-hydroxysuccinimide potassium salt nitrophenol and trichlorophenol with phosgene, respectively.

These activators should be reacted with polyethylene glycol in the organic phase. The operation method is similar to the activation effect and ligand coupling effect. However, the reaction has not been specifically evaluated, but the reaction is highly toxic due to the participation of phosgene, which increases the complexity of the operation.

(6) Change the structure of polyethylene glycol to improve the modification effect. Changing the structure of polyethylene glycol from linear to fork or comb can also play a better modification effect.

Its advantages are mainly reflected in:
(1) Forked polyethylene glycol can reduce the contact between protease and protein to weaken its hydrolysis
(2) The steric hindrance effect caused by the structural change of polyethylene glycol reduces the binding site of polyethylene glycol and protein and reduces protein inactivation
(3) The shielding effect of polyethylene glycol on proteins is more significant and better to reduce the antigenicity and immunogenicity of protein drugs

Modification of uricase with linear and forked polyethylene glycols, respectively, by Schiavon et al.
Forked polyethylene glycol-modified proteins can well retain the biological activity of the protein. In addition, by increasing the distance between the activated end and the polyethylene glycol chain, or adding branch chains on a-C, the activated polyethylene glycol can be extended in the The hydrolysis half-life in water is expected to improve the modification effect.