PEG Polypeptides Chemical Modification

A polypeptide is a compound formed by multiple amino acids connected by peptide bonds. They are ubiquitous in organisms. So far, thousands of polypeptides have been found in organisms. Polypeptides play an important role in regulating the functional activities of various systems, organs, tissues, and cells in life activities. And it often used in functional analysis, antibody research, drug development, and other fields. With the maturity of biotechnology and peptide synthesis technology, more and more peptide drugs have been developed and applied in clinical practice.

There are many kinds of polypeptide modifications, which can be simply divided into post-modification and process modification(using derived amino acids). According to different modification sites, they have N-terminal modification, C-terminal modification, side-chain modification, amino acid modification, skeleton modification, etc. As an important means to change the main chain structure or side chain groups of the peptide chain, peptide modification can effectively change the physical and chemical properties of peptide compounds, increase water solubility, extend the time of action in the body, change their biological distribution, eliminate immunogenicity, reduce toxic side effects and so on. Mainly introduces several major peptide modification strategies and characteristics here.


Cyclic peptides have many applications in biomedicine, and many natural peptides with biological activity are cyclic peptides. Since cyclic peptides are more rigid than linear peptides, they are extremely resistant to the digestive system, survive in the digestive tract, and a stronger affinity for target receptors. Cyclization is the most direct way to synthesize cyclic polypeptides, especially for polypeptides with larger structural skeletons. According to the cyclization method, it can be divided into side chain-side chain type, terminal-side chain type, and terminal-terminal type.

(1) Sidechain-to-sidechain

The most common type of side chain-side chain cyclization is the disulfide bridge between cysteine ​​residues. The most common type of side-chain-side-chain cyclization is disulfide bridging between cysteine residues. Introducing the cyclization method is through deprotecting a pair of cysteine residues and then oxidizing to form a disulfide bond. It can remove the sulfhydryl protecting group to achieve form multiple rings by selectively. Cyclization can be done either in the solvent after dissociation, or on the resin before dissociation. Since the peptide on the resin is not easy to form a cyclization conformation, cyclization on the resin may be less efficient than cyclization in a solvent.

Another type of side chain-side chain cyclization is the formation of an amide structure between the aspartic acid or glutamic acid residues and the basic amino acid. It requires the side chain protection of the polypeptide whether it is on the resin or after dissociation The base must be able to be selectively removed. The third type of side chain-side chain cyclization is the formation of diphenyl ether through tyrosine or p-hydroxyphenylglycine. This type of cyclization in natural products only exists in microbial products, and cyclization products often have potential pharmaceutical value. The preparation of these compounds requires unique reaction conditions, so they are not often used in the synthesis of conventional polypeptides.

(2) Terminal-to-sidechain

Terminal-side chain cyclization usually involves the C-terminus and the amino group of the lysine or ornithine side chain, or the N-terminus and the aspartic acid or glutamic acid side chain. There are also some polypeptide cyclizations through the formation of an ether bond between terminal C and the side chain of serine or threonine.

(3)Terminal-terminal or head-to-tail

Chain polypeptides can be cyclized in a solvent or immobilized on a resin through side-chain cyclization. Cyclization in the solvent should use a low concentration of polypeptide to avoid oligomerization of the polypeptide. The yield of cyclic peptides synthesized head-to-tail depends on the sequence of the chain peptides. Therefore, before large-scale preparation of cyclic peptides, a library of possible chain-like lead peptides should be created first, and then cyclization should be performed to find the sequence that can achieve the best results.


N-methylation first appeared in natural peptides and was introduced into peptide synthesis to prevent the formation of hydrogen bonds, thereby making the peptides more resistant to biodegradation and clearance. Using N-methylated amino acid derivatives to synthesize peptides is the most important method. In addition, N-(2-nitrobenzenesulfonyl chloride) peptide-resin intermediates can also be used for the Mitsunobu reaction with methanol. This method has been used Prepare a library of cyclic peptides containing N-methylated amino acids.


Phosphorylation is one of the most common post-translational modifications in nature. In human cells, more than 30% of proteins are phosphorylated. Phosphorylation, especially reversible phosphorylation, plays an important role in the control of many cellular processes, such as signal transduction, gene expression, cell cycle and cytoskeletal regulation, and apoptosis.

Phosphorylation can be observed on various amino acid residues, but the most common phosphorylation targets are serine, threonine and tyrosine residues. Phosphotyrosine, phosphothreonine and phosphoserine derivatives can either be introduced into the polypeptide during synthesis or can be formed after the synthesis of the polypeptide. Selective phosphorylation can be achieved by using serine, threonine, and tyrosine residues that can selectively remove protective groups. Some phosphorylation reagents can also be post-modified to introduce phosphate groups into the polypeptide. In recent years, some scholars have used the chemoselective Staudinger-phosphite reaction to achieve site-specific phosphorylation of lysine.

4.Myristoylation and palmitoylation

Acylation of the N-terminus with fatty acids allows the peptide or protein to bind to the cell membrane. The myristoylated sequence on the N-terminus can target the protein kinases of the Src family and the reverse transcriptase Gaq protein to bind to the cell membrane. The myristic acid can be connected to the N-terminus of the resin-polypeptide using standard coupling reactions, and the resulting lipopeptides can be dissociated under standard conditions and purified by RP-HPLC.


Glycopeptides such as vancomycin and teicoplanin are important antibiotics for the treatment of resistant bacterial infections, and other glycopeptides are often used to stimulate the immune system. In addition, because many microbial antigens are glycosylated, the study of glycopeptides is of great significance for improving the therapeutic effect of infections. On the other hand, some studies have found that the proteins on the cell membrane of tumor cells exhibit abnormal glycosylation, which makes glycopeptides also play an important role in cancer and tumor immune defense research. The preparation of glycopeptides generally uses the Fmoc/t-Bu method. Glycosylated residues, such as threonine and serine, are often introduced into polypeptides via Fmoc protected glycosylated amino acids activated by pentafluorophenol ester.


Prenylation occurs at cysteine ​​residues on the side chain near the C-terminus. The prenylation of proteins can increase the affinity of cell membranes and form protein-protein interactions. Prenylated proteins include tyrosine phosphatase, small GTPases, co-chaperone molecules, lamina, and centromere binding proteins. Prenylated polypeptides can be prepared by prenylation on resin or by introducing cysteine ​​derivatives.

7.Polyethylene glycol (PEG) modification

PEG modification can be used to improve proteolytic stability, biodistribution, and peptide solubility. The introduction of PEG chains on polypeptides can improve their pharmacological properties and can also inhibit the hydrolysis of polypeptides by proteolytic enzymes. PEG polypeptides are easier to pass through the cross-section of glomerular capillaries than ordinary polypeptides, greatly reducing renal clearance.

Since the effective half-life of PEG polypeptides in the body is prolonged, the normal therapeutic level can be maintained by using lower doses and lower frequency of polypeptide drugs. But PEG modification also has negative effects. A large amount of PEG prevents the enzyme from degrading the polypeptide but also reduces the binding of the polypeptide to the target receptor. However, the low affinity of PEG polypeptides is usually offset by its longer pharmacokinetic half-life. By staying longer in the body, PEG polypeptides are more likely to be absorbed by target tissues. Therefore, the specifications of PEG polymers should be optimized for the best results. On the other hand, due to reduced renal clearance, PEG polypeptides can accumulate in the liver and cause the macromolecular syndrome. Therefore, PEG modifications need to be designed more carefully when peptides are used in drug testing.

The common modifying groups of PEG modifiers can be summarized as follows: Amino (-Amine)-NH2, aminomethyl-CH2-NH2, hydroxyl-OH, carboxyl-COOH, sulfhydryl (-Thiol)-SH, maleimide -MAL, succinimide carbonate-SC, succinimide acetate-SCM, succinimide propionate-SPA, N-hydroxysuccinimide-NHS, propionic acid group-CH2CH2COOH, aldehyde group -CHO (such as propionaldehyde-ALD, butyraldehyde-butyrALD), acrylic (-Acrylate)-ACRL, azido-Azide, biotin-Biotin, fluorescein-Fluorescein, glutaric acid-GA, acyl Hydrazide-Hydrazide, alkynyl-Alkyne, p-toluenesulfonate-OTs, succinimidyl succinate-SS, etc. The PEG derivatives with carboxylic acid can be coupled to the N-terminal amine or lysine side chain. Amino-activated PEG can be coupled with aspartic acid or glutamic acid side chains. MAL-activated PEG can be coupled with the thiol of the fully deprotected cysteine ​​side chain [11]. Common classifications of PEG modifiers are as follows (Note: mPEG is methoxy-PEG, CH3O-(CH2CH2O)n-CH2CH2-OH):

(1)Linear PEG modifier


(2)Bifunctional PEG modifier


(3)Branched PEG modifier

(mPEG)2-NHS, (mPEG)2-ALD, (mPEG)2-NH2, (mPEG)2-MA7


Biotin can bind strongly to avidin or streptavidin, and the binding strength is even close to the covalent bond. Biotin-labeled peptides are commonly used in immunoassays, histocytochemistry, and fluorescence-based flow cytometry. Labeled anti-biotin antibodies can also be used to bind biotinylated polypeptides. The biotin tag is often attached to the side chain of lysine or the N-terminus. Usually 6-aminocaproic acid is used as a link between peptides and biotin. The link can flexibly bind to the substrate, and it can bind better under steric hindrance.

9.Fluorescent labeling

Fluorescent labels can be used to track peptides in living cells, as well as to study enzymes and mechanisms of action. Tryptophan (Trp) is fluorescent, so it can be used for internal labeling. The emission spectrum of tryptophan depends on the surrounding environment and decreases as the solvent polarity decreases. This property is very useful for detecting peptide structure and receptor binding. Tryptophan fluorescence can be quenched by protonated aspartic acid and glutamic acid, which may limit its use. Dansyl chloride group (Dansyl) is highly fluorescent when combined with amino groups, and is also often used for fluorescent labeling of amino acids or proteins.

Fluorescence resonance energy conversion (FRET) is very useful for enzyme research. When FRET is used, the substrate peptide often contains a fluorescent labeling group and a fluorescence quenching group. The labeled fluorophore will be quenched by the quencher through non-photon energy transfer. When the peptide dissociates from the enzyme under study, the labeling group emits fluorescence.

10.Cladding peptides

The clathrate polypeptide has an optically removable protective group, which can shield the binding of the polypeptide to the receptor. When irradiated by UV, the peptide will be activated, restoring its affinity with the receptor. Since this optical activation can be controlled based on time, amplitude, or location, clathrate polypeptides can be used to study reactions that occur in cells. The most commonly used protecting groups for clathrate polypeptides are 2-nitrobenzyl and its derivatives, which can be introduced through protected amino acid derivatives during polypeptide synthesis. The amino acid derivatives that have been developed include lysine, cysteine, serine and tyrosine. However, aspartic acid and glutamic acid derivatives are not commonly used because they are easy to cyclize during peptide synthesis and dissociation.

11.Multimeric antigen peptide (MAP)

Short peptides are usually not immune and must be coupled with carrier proteins to produce antibodies. Multimeric antigen peptide (MAP) is composed of multiple identical polypeptides connected to the lysine core, which can specifically express high-efficiency immunogens and can be used to prepare peptide-carrier protein couplings. MAP polypeptides can be synthesized stepwise by solid-phase synthesis on MAP resin. However, the incomplete coupling will result in missing or truncated peptide chains on some branches, thus failing to show the properties of the original MAP polypeptide. As an alternative method, the polypeptide can be separately prepared and purified and then coupled to the MAP. The sequence of the peptide linked to the core of the peptide is clear and can be easily characterized by mass spectrometry.


Peptide modification is an important method for designing peptides. Chemically modified peptides can not only maintain high biological activity but also can effectively avoid the disadvantages of immunogenicity and toxicity. At the same time, chemical modification can give peptides some new excellent properties. . In recent years, methods for late-stage modification of peptides by means of C-H activation have also been rapidly developed, and many important results have been achieved.