In recent years, the development and applications of protein drugs have attracted extensive attention from researchers. However, the shortcomings of protein drugs also limit their further development. Therefore, bioactive peptides isolated or simulated from protein polymers have broad application prospects in food, medicine, biotechnology, and other industries. Such peptides have a molecular weight distribution between 180 and 1000 Da. As a small molecule substance, bioactive peptide is usually degraded by various enzymes in the organism and have a short half-life. At the same time, such substances have poor stability and are difficult to produce and store. Therefore, these active peptides may be modified through phosphorylation, glycosylation, and acylation. Compared with other protein drugs, the modified active peptides are more easily absorbed by the body, have longer half-life, stronger targeting, and fewer side effects in addition to higher bioavailability. In the light of their functions, bioactive peptide can be divided into antimicrobial, anti-tumour, anti-angiogenic, antioxidant, anti-fatigue, and anti-hypertensive peptides.

1. peptide cyclization

The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation. These approaches range from the direct linkage of N- and C- termini to the connection of amino acid side chains, which can be applied both in reaction vessels and in living systems. Cyclisation can be categorised into four general classes: side chain-to-side chain, head-to-tail (also known as backbone cyclisation), head-to-side chain and side chain-to-tail (Fig. 1).

(1). Side chain-to-side chain cyclisation occurs when a bond is formed between the side chain functionalities of two amino acid residues (Fig. 1a). One prominent example is intramolecular disulphide bond formation between the thiol functionalities of two cysteine residues, leading to a type of cyclic structure, commonly found in peptides and proteins such as insulin and antibodies. It is estimated that about 50% of cysteine residues in polypeptides are found in the form of disulphide bonds. Other types of side chain cyclisation, including non-native linkages, are also possible and will be discussed throughout this review.

(2). Head-to-tail terminus cyclisation is another commonly observed form of cyclisation. As the first residue in a chain of amino acids has an amino functionality (i.e. N-terminus), and the last residue has a carboxylate functionality (i.e. C-terminus), polypeptides are typically directional. Subsequently, cyclisation can be achieved by joining the N- and C-termini through an amide bond (Fig. 1b).

(3). Head-to-tail peptide cyclisation has been observed in microorganisms and plants, such as kalata B1 from the plant Oldenlandia affinis and bacteriocin AS-48 produced by the bacterium Enterococcus faecalis. Furthermore, a recent report shows that head-to-tail cyclic peptides are prevalent in normal flora such as those in the human gut. Meanwhile, the formation of a lactam, lactone or thiolactone between either terminus with an appropriate side chain functional group (Fig. 1c and d) results in side chain-to-terminus cyclisation. For example, bacitracin is an antibiotic side chain-to-tail cyclic peptide produced by Bacillus subtilis, in which a bond is formed between a lysine side chain and the C-terminus.

2. N-Methylation peptide

N-Methylation is one of the simplest chemical modifications often occurring in peptides and proteins of prokaryotes and higher eukaryotes. Over years of evolution, nature has employed N-methylation of peptides as an ingenious technique to modulate biological function, often as a mode of survival through the production of antibiotics. This small structural change can not only mobilize large protein complexes (as in the histone methylation), but also inhibits the action of enzymes by selective recognition of protein–protein interaction surfaces. In recent years through the advancement in synthetic approaches, the potential of N-methylation has begun to be revealed, not only in modulating biological activity and selectivity as well as pharmacokinetic properties of peptides, but also in delivering novel drugs. N-methylated amino acids are the mostly used to synthesize N-methylated peptides, furthermore, 2-Nitrobenzenesulfonyl chloride-peptide-resin can be reacted with Methanol using Mitsunobu reaction to synthesize cyclic peptide liabary containing N-methylated amino acids.

3. Phosphorylation peptide

Protein phosphorylation is a posttranslational modification of proteins in which a serine, a threonine or a tyrosine residue is phospohorylated by a protein kinase by the addition of a covalently bound phosphate group.Protein kinases share a conserved catalytic domain, which catalyses the transfer of the gamma-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. Phosphorylation occurs mainly on the hydroxyl side chain of serine and threonine residues and, to a lesser extent, on the phenolic side chain of tyrosine residues. Regulation of proteins by phosphorylation is one of the most common modes of regulation of protein function. Protein phosphorylation plays essential roles in nearly every aspect of cell life.

In order to study phosphorylation events, site-specific synthesis of phosphopeptides is crucial. Phosphorylated peptides are generally produced by the specific incorporation of protected phospho-amino acids into the sequence. Alternative approaches, such as “post-assembly” phosphorylation of Ser/Thr/Tyr-containing peptide resins can also be applied.

4. Myristoylation and Palmitoylation

Myristoylation is a lipidation modification where a myristoyl group, derived from myristic acid, is covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue. Myristic acid is a 14-carbon saturated fatty acid (14:0) with the systematic name of n-Tetradecanoic acid. This modification can be added either co-translationally or post-translationally. N-myristoyltransferase (NMT) catalyzes the myristic acid addition reaction in the cytoplasm of cells. This lipidation event is the most found type of fatty acylation and is common among many organisms including animals, plants, fungi, protozoans and viruses. Myristoylation allows for weak protein–protein and protein–lipid interactions and plays an essential role in membrane targeting, protein–protein interactions and functions widely in a variety of signal transduction pathways.

Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine (S-palmitoylation) and less frequently to serine and threonine (O-palmitoylation) residues of proteins, which are typically membrane proteins. The precise function of palmitoylation depends on the particular protein being considered. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. Palmitoylation also appears to play a significant role in subcellular trafficking of proteins between membrane compartments, as well as in modulating protein–protein interactions. In contrast to prenylation and myristoylation, palmitoylation is usually reversible (because the bond between palmitic acid and protein is often a thioester bond). The reverse reaction in mammalian cells is catalyzed by acyl-protein thioesterases (APTs) in the cytosol and palmitoyl protein thioesterases in lysosomes. Because palmitoylation is a dynamic, post-translational process, it is believed to be employed by the cell to alter the subcellular localization, protein–protein interactions, or binding capacities of a protein.

An example of a protein that undergoes palmitoylation is hemagglutinin, a membrane glycoprotein used by influenza to attach to host cell receptors. The palmitoylation cycles of a wide array of enzymes have been characterized in the past few years, including H-Ras, Gsα, the β2-adrenergic receptor, and endothelial nitric oxide synthase (eNOS). In signal transduction via G protein, palmitoylation of the α subunit, prenylation of the γ subunit, and myristoylation is involved in tethering the G protein to the inner surface of the plasma membrane so that the G protein can interact with its receptor.

5. Peptide Glycosylation

Glycosylation is the most abundant polypeptide chain modification in nature. Glycosylation of peptides is a promising strategy for modulating the physicochemical properties of peptide drugs and for improving their absorption through biological membranes. The glycosylated peptide can target specific organs, enhance the biodistribution in tissues, improve penetration through biological membranes, increase metabolic stability and lower the clearance rate, receptor-binding, protect amino acid’s side chain from oxidation, and maintain and stabilize the physical properties of peptides, such as precipitation, aggregation and thermal and kinetic denaturation. Glycans can be covalently attached to the amide nitrogen of Asn residues (N-glycosylation), to the hydroxyl oxygen of Ser or Thr residues (O-glycosylation), and the indole C2 carbon of Trp through a C–C linkage (C-mannosylation).

Usually, We need to synthesize the glycosylated amino acids, which are compatible with standard protocols in Fmoc solid phase peptide synthesis first. The pre-synthesised glycosylated amino acid is coupled to the elongating peptide using solid phase peptide synthesis (SPPS) in a stepwise fashion. Typical synthesis is for the peptide of 10-20 amino acids. The integration of long peptides with more than 50 residues is difficult by stepwise synthesis, due to the incomplete couplings and epimerization.

6. Peptide Prenylation

Farnesylation is a type of prenylation, a post-translational modification of proteins by which an isoprenyl group is added to a cysteine residue. It is an important process to mediate protein–protein interactions and protein–membrane interactions. Peptide Prenylation can be made by using prenylated cysteine

7. Peptide PEGylation

EGylation (or pegylation) is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, in pharmacy called macrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated. PEGylation affects the resulting derivatives or aggregates interactions, which typically slows down their coalescence and degradation as well as elimination in vivo.

PEGylation is routinely achieved by the incubation of a reactive derivative of PEG with the target molecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host’s immune system (reducing immunogenicity and antigenicity), and increase its hydrodynamic size (size in solution), which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. Having proven its pharmacological advantages and acceptability, PEGylation technology is the foundation of a growing multibillion-dollar industry.

8. Peptide Biotinylation

Because of its especially high affinity and binding specificity, the biotin-avidin (or biotin-streptavidin) interaction has been used for several decades as the basis of many laboratory research techniques to label, detect and purify peptide, proteins and other biomolecules. The biotin moiety has a very strong affinity for streptavidin (Kd<10-10 M) and biotinylation of peptides is therefore an efficient method to specifically bind peptides to streptavidin coated surfaces.

Peptide biotinylation can be performed either at the N- or C-terminus. Biotinylation at the N-terminus can be performed directly to the primary-terminal amino group, whereas biotinylation is usually performed at the ε-amino group of an (extra) C-terminal lysine. An important consideration when making a biotinylated peptide is to ensure there is a sufficient spacer arm between the biotin group and the amino acids in the peptide which are expected to interact with a macromolecule (such as an antibody). To avoid sterical hindrance, a linker can be inserted between biotin and the peptide sequence. A hydrophobic straight chain spacer such as the 6-carbon ε-aminohexanoic (Ahx) is frequently applied or a hydrophilic tetrapeptide such as -SGSG- can be inserted.

One limitation of the biotin-(strept)avidin interaction in purification applications is that it is essentially irreversible under physiological conditions. Binding is resistant to pH and/or temperature changes, organic solutions and denaturing reagents. Therefore in case a reversible interaction to (strept)avidin is desired, the use of biotin analogs is recommended.

One limitation of the biotin-(strept)avidin interaction in purification applications is that it is essentially irreversible under physiological conditions. Binding is resistant to pH and/or temperature changes, organic solutions and denaturing reagents. Therefore in case a reversible interaction to (strept)avidin is desired, the use of biotin analogs is recommended.

Similarly desthiobiotin, a non-sulfur containing biotin analog, can be used. Desthiobiotin binds less tightly to avidin and streptavidin than biotin but nonetheless provides a high level of specificity. Moreover it is less prone to oxidation.