Background and Overview
Peptides offer a significant structural advantage: new functional peptide sequences can be introduced at one or both ends of a peptide through solid-phase synthesis or biosynthesis, without affecting the original functional peptide fragment, to create multifunctional fusion peptides. The mechanism by which pH-sensitive liposomes with receptor/ligand binding ability enter cells often differs from the fusion mechanism of conventional liposomes. For example, binding mechanisms similar to RGD/αVβ3 or Transferrin/TfR induce active endocytosis of the liposomes by tumor cells, leading to drug/nucleic acid release. These peptide-modified pH-sensitive liposomes are known to release drugs/nucleic acids through drug/nucleic acid release. Common peptide modifications can be divided into four categories based on the modification site: C-terminal modifications (amidation, sulfation, etc.), N-terminal modifications (acetylation, fatty acidization, etc.), intermediate residue modifications (glycosylation for Ser-, Tyr-, Asn-, and Thr-binding; phosphorylation for Ser-, Tyr-, and Thr-binding), and cyclization. Peptide modification has become a hot topic of research.
Modification Methods
- Chemical Methods
Currently, the most mature and widely used modification methods for peptides, primarily encompassing liquid-phase and solid-phase methods.
1) Liquid-Phase Method
The liquid-phase method is a classic method with unique advantages in purity monitoring and scalable production. In peptide cyclization, to avoid intermolecular reactions that form linear or cyclic dimers and multimers, the reaction must be performed in a highly dilute solution (10-3 to 10-4 mol/L). However, these highly diluted conditions often result in extended reaction times and numerous side reactions, complicating post-processing. To address this issue, a sterically hindered dendritic silane was used as a substituted carbodiimide condensation agent, replacing the conventional reagent dicyclohexylcarbodiimide (DCC) for solution-based lactam cyclic peptide synthesis, yielding the target compound with high purity.
2) Solid-Phase Method
Solid-phase methods are widely used in peptide modification due to their ease of separation and purification. The yield and purity of peptide modifications synthesized using this method are significantly higher than those using liquid-phase methods. In solid-phase peptide synthesis, the C-terminal amino acid residue is attached to a support before the remaining amino acids are assembled, which inherently hinders the synthesis of C-terminally modified peptides. Using the relatively common 2-chlorotrityl chloride resin as a support, this paper used solid-phase synthesis of the fully protected peptide leuprorelin (Py r-His-Trp-Ser-Tyr-D-Leu-eu-Arg-Pro-NHEt). Following ethylamination at 25°C for 5 minutes, the side chain was directly cleaved. This method allows for the sequential coupling of ethylamination and side-chain cleavage steps, avoiding the need for intermediate separation and purification. This successful approach provides a reference for other C-terminal peptide modifications.
3) Enzymatic Method
Enzymes offer significant advantages in stereoselectivity at the modification site and mild, rapid, and efficient synthetic reaction conditions. In glycopeptide synthesis, glycosylamino acids are attached to oligopeptides via an enzymatic condensation reaction, followed by further oligosaccharide modification using glycosyltransferases. This enzymatic reaction can be performed in aqueous solution and requires minimal protection. However, enzymes have limitations, such as limited availability and limited access. Furthermore, the amino acid sequence of the peptide chain within the substrate may affect the efficiency of modification. When enzymatically attaching galactosamine to peptides to synthesize glycopeptides, the yield of the reaction is generally low, depending on both substrate structure and glycosylation. With the advancement of DNA technology and the continued advancement of research, enzymes will play an increasingly important role in peptide modification.
4) Chemoenzymatic Coupling
The use of a chemoenzymatic method, combining chemical and enzymatic methods, for peptide modification research can reduce contamination caused by excessive use of chemical reagents while ensuring high enzyme-catalyzed yields under mild conditions. Solid-phase synthesis of an N-terminally conjugated glycosylendomorphin-1 derivative was performed. The N-acetylglucosamine compound was then attached to a chemically synthesized glycopeptide under the catalysis of a glycosyltransferase to form the target N-terminal trisaccharide peptide endomorphin-1 analog. This synthesis process requires minimal protection. Using modified silica gel as a solid-phase support, the peptide bond was rapidly chemically synthesized and the glycosidic bond was enzymatically synthesized, resulting in the successful synthesis of the Sialyl lewis x antigen.
- Peptide Modification Strategies
In the peptide modification process, general condensation strategies, unit-building strategies, and native chemical ligation are commonly employed. Different strategies are often employed to address the interplay between peptide chain condensation and the structures of different modifying groups during the modification process.
1) General Condensation Strategy
The general condensation strategy involves catalytically conjugating a pre-prepared polypeptide chain with the target modifier to minimize the impact of reagents used during peptide chain elongation on the efficiency of the modifier-peptide linkage. Taking glycopeptides as an example, during the condensation process, the peptide and oligosaccharide are first synthesized separately, and then the peptide acceptor and oligosaccharide donor are condensed to form an N- or O-glycopeptide. This strategy avoids the effects of acidic deprotection conditions on the O-glycosidic bond during peptide chain elongation. However, direct glycosylation of serine or threonine in the peptide chain remains challenging. A solid-phase glycosylation reaction on a resin was attempted. Acetyl-protected glucose with an allylcarbamate terminal end group was reacted with a tripeptide attached to a threonine residue on the resin. After Pd-catalyzed cleavage, the raw material was recovered in a 64% yield, while the overall product yield was only 4%. Currently, this strategy is primarily used for N-linked glycopeptides where the sugar moiety is linked to asparagine.
2) Unit-Building Strategy
The unit-building strategy involves first preparing units with different modification groups and then catalytically coupling them one by one. During glycosylation, the glycosidic bond between the amino acid and the sugar is formed before peptide chain elongation, making site and stereoselectivity relatively easy to control. This strategy has become a common synthetic method, particularly suitable for solid-phase synthesis and the synthesis of glycopeptides containing multiple glycosyl groups. This strategy requires the pre-synthesis of the glycosylated amino acid in liquid phase. Building blocks linked to asparagine have been developed, including glucose, galactose, N-acetylglucosamine, GlcβGlc, Man, glycomimetic compounds, and ribose. The introduction of microwave technology to synthesize Fmoc-L-Asn(GlcAc4) has shortened the condensation time from 16–24 h to 5 min (100W, 70°C), greatly facilitating the synthesis of N-glycopeptides. Regarding the strategy of choice, the Fmoc solid-phase synthesis strategy was employed to study the synthesis of phosphorylated angiotensin II, using N,N-diethyl-di-tert-butyloxyphosphoramidite as the phosphorylation reagent. The global phosphorylation method uses tyrosine with unprotected side chain hydroxyl groups as a monomer for solid-phase synthesis of peptides. Peptide synthesis was performed using the phosphorylated monomer Fmoc-Tyr(PO₃But₂)-OH. Both the bulk phosphorylation and phosphorylated monomer methods yielded the desired phosphorylated angiotensin II. The bulk method yielded both phosphopeptides and unphosphorylated peptides, but the phosphorylation efficiency was low, with a yield of approximately 17%, and the crude peptide was difficult to isolate. The monomer method, on the other hand, offered a more direct synthesis step, higher efficiency, fewer byproducts, and a yield of up to 38%. Therefore, the phosphorylated monomer method is considered preferable for synthesizing peptides containing phosphotyrosine.
- Native Chemical Ligation
Solid-phase synthesis often yields peptide modifications of fewer than 50 amino acids. Furthermore, when there are multiple modification sites on the peptide chain, conventional strategies often present significant challenges. To address these limitations, an efficient fragment ligation method, native chemical ligation (NCL), has been developed. This involves the reaction of a peptide thioester at the C-terminus with an unprotected cysteine residue at the N-terminus to form a native cysteine residue that links the two fragments. Alkanesulfonamides were used as linkers to create C-terminal peptide thioesters. Two glycopeptide fragments were linked using the NCL strategy to form the 82-residue antibacterial glycoprotein volycin. Solid-phase peptide synthesis was employed in the synthesis of the cyclic peptide, using the Boc/Bzl strategy to gradually extend the peptide chain from the C-terminus to the N-terminus, yielding a peptide resin containing a linear peptide thioester. The cleaved and purified linear peptide thioester was then subjected to spontaneous acyl migration from the S atom to the N atom in aqueous NaHCO₃. While there are numerous types of peptide modifications, this article focuses on the latest research progress on several of the most important peptide modifications.
1) PEG-peptide conjugates: Currently, monomethoxypolyethylene glycol (mPEG: CH₃O-(CH₂-CH₂O)n-H) is the most commonly used PEG-modified peptide. This modification involves introducing active groups such as carboxyl or amino groups at the termini of mPEG, or preparing mPEG-modified amino acid derivatives, which are then coupled to peptide sequences using solid-phase or liquid-phase methods. This allows for PEGylation of the N-terminus, C-terminus, and certain amino acid side chains of the peptide. The effects of different PEG molecular weights and different attachment methods on the biological activity and thermal stability of PEGylated insulin were investigated. Three mPEGs with molecular weights of 1100, 2000, and 5000 g/mol were selected and activated with succinic anhydride (SA), cyanuric chloride (CC), and p-toluenesulfonyl chloride (TC), respectively. The results showed that the modified insulin exhibited reduced biological activity. However, insulin modified with the higher molecular weight mPEG exhibited greater affinity for the enzyme-substrate binding site and significantly improved thermal stability. Furthermore, insulin modified with cyanuric chloride-activated CC-mPEG (5000 g/mol) exhibited a significantly improved thermal stability. In recent years, with the increasing advancement of PEGylation chemistry, heterodifunctional PEG derivatives have also found significant applications in peptide chemistry. PEG is protected at both ends with DMT and Fmoc, respectively, with the DMT terminus attached to the oligonucleotide chain and the Fmoc terminus attached to the peptide. Oligonucleotide-PEG-peptide conjugates are obtained using a liquid-phase method. A carboxyl group was successfully introduced at the terminus of PEG using a solid-phase synthesis strategy to synthesize octreotide-PEG-DSPE (octadecanoylphosphatidylethanolamine (phosphatidylceramide)). While PEG modification of synthetic peptides has achieved some methodological progress, practical challenges remain. Because PEG is a polymer, its relative molecular mass cannot be determined. Since the molecular mass of peptide-modified PEG is typically between 5,000 and 20,000, solid-phase synthesis is challenging, resulting in slow reaction rates and low yields. Therefore, modification in liquid phase can be considered, but this inevitably increases the number of post-processing steps and negatively impacts the biological activity of the product.
2) Glycopeptides
Glycopeptides, the products of peptide glycosylation, serve as model tools for scientific research on the structure and function of glycoproteins. Therefore, the synthesis of glycopeptides is particularly important. Currently, the main linkages between oligosaccharides and polypeptide chains are C-, N-, O-, and S-glycosidic bonds, with N- and O-glycosidic bonds being the most commonly used. The chemical instability of glycosidic bonds greatly complicates peptide synthesis. Glycosidic bonds typically hydrolyze under acidic conditions, and all glycosyl serine and threonine derivatives are susceptible to β-elimination reactions, even under very weak alkaline conditions. N-linked glycosylation introduces a glycosylamine, which is then protected by the amide-carboxyl group of the asparagine residue. This method does not require special protection of the sugar or peptide, and the glycosylamine is bound to the asparagine residue in the polypeptide chain to produce an N-linked glycopeptide. In the synthesis of O-linked glycopeptides, the hydroxyl groups of serine and threonine serve as glycosyl acceptors, necessitating the protection of both the sugar and the peptide. Selective removal of protecting groups from glycosylamino acids or glycopeptides remains a long-standing problem, despite being proposed as early as the late 1970s. The Z group of β-glycosylamino acid derivatives cannot be removed using HBr/AcOH without disrupting the glycosidic bond. Although removal of Boc residues is possible under certain conditions, deprotection reactions under acidic conditions in glycopeptide synthesis require careful consideration. As mentioned above, the base-sensitive nature of glycosylserine or threonine derivatives (β-elimination reactions) further limits the scope of deprotection reactions. Therefore, protecting group removal can only be performed under mild or neutral reaction conditions.
3) Phosphopeptides
Protein phosphorylation and dephosphorylation regulate nearly all biological processes, including cell proliferation, development, and differentiation, neural activity, muscle contraction, metabolism, and tumorigenesis. Phosphopeptides are the best models for simulating the structural changes in their parent proteins during phosphorylation. Phospho-peptides can be divided into four categories based on the phosphorylated amino acid residues: N-phospho-peptides, O-phospho-peptides, acylphosphopeptides, and S-phospho-peptides. O-phospho-peptides are formed by phosphorylation of hydroxyamino acids, such as serine, threonine, tyrosine, hydroxyproline, or hydroxylysine; N-phospho-peptides are formed by phosphorylation of arginine, lysine, or histidine; acylphosphopeptides are formed by phosphorylation of aspartic acid or glutamic acid; and S-phospho-peptides are formed by phosphorylation of cysteine. A common method for phosphopeptide synthesis involves enzymatic phosphorylation of a synthesized, unphosphorylated peptide with the action of a kinase. However, this method is not applicable to peptides that cannot be phosphorylated by kinases. To date, the chemical synthesis of phosphopeptides and their analogs remains challenging. L-leucine is activated with phosphorus oxychloride for 72 hours using a one-pot method. The mixture is then cooled with 1,4-dioxane, and diisopropylphosphinate (DIPPH) and sodium hypochlorite (NaClO) are added and allowed to react. This yields an N-phosphorylated homologous peptide. Heterologous peptides and their phosphorylation are also under investigation.
4) Cyclic Peptides
Shorter linear peptides are readily degraded by various enzymes in vivo. Cyclic peptides can enhance enzymatic and chemical stability. Since cyclic peptides lack C- and N-termini, they can eliminate or reduce degradation by aminopeptidases and carboxypeptidases, thereby increasing their resistance to enzymatic degradation. Furthermore, the cyclic structure increases conformational constraints, potentially increasing peptide receptor affinity and selectivity, enhancing activity, and reducing side effects. These cyclic peptides have become a new direction in drug development in recent years. Cyclic peptides can be divided into two categories: homocyclic peptides, in which amino acids are linked by amide bonds; and heterocyclic peptides, in which, in addition to amide bonds, they also contain ester, ether, thioester, and disulfide bonds.
A: Homocyclic Peptides
Hypercyclic peptides essentially form intramolecular peptide bonds. The factor influencing cyclization is the size of the ring: peptides containing seven or more amino acid residues can cyclize smoothly, while the synthesis of hexapeptides, pentapeptides, and even smaller cyclic peptides is difficult. A cyclic pentapeptide (Gly-Ala-Tyr-Leu-Ala) isolated from Chickweed was used as a model peptide. Two different linear peptide precursors were synthesized using the developed organophosphorus compound DEPBT as a condensation reagent using liquid and solid phase methods, respectively. Cyclization reactions were completed using DEPBT as the condensation reagent, yielding the desired cyclic pentapeptides. However, the yields were only 6% and 6.5%, which are very low. Analysis indicates that the key to the synthesis of cyclic pentapeptides lies in inhibiting the formation of cyclic decapeptides.
B: Heterocyclic Peptides
The non-amide bonds between amino acid residues in heterocyclic peptides are primarily ester bonds and disulfide bonds. The formation process of the former is similar to that of amide bonds, while the synthesis of the latter differs significantly from that of homocyclic peptides, essentially involving the oxidation of two Cys sulfhydryl groups to -S-S- bonds using an oxidant. Currently, the synthesis of peptide disulfide bonds typically involves cleaving the reduced peptide containing sulfhydryl groups from the resin, followed by oxidative cyclization in the liquid phase. The main cyclization methods include DMSO, potassium ferricyanide, iodine oxidation, air oxidation, and glutathione oxidation. However, direct oxidation to disulfide bonds on the resin has rarely been reported. Using Rink Amide-MBHA resin as a support, an Fmoc/tBu orthogonally protected solid-phase synthesis strategy and microwave irradiation, a resin loaded with reduced oxytocin or lysine vasopressin peptides was rapidly and efficiently synthesized. The reduced peptides attached to the resin were then cyclized to form disulfide bonds using iodine oxidation under microwave irradiation and room temperature, respectively, to produce the cyclic peptides. This method for synthesizing such cyclic peptides has the advantages of being fast, efficient, and easy to purify. The entire cyclic peptide synthesis process only takes a few hours, laying the foundation for the large-scale production of such cyclic peptides.