What Is Peptide Modification: An Overview
This chapter explores the various types of peptide modifications and their applications in pharmaceutical and research settings, covering:
-
- Natural and chemical modifications that enhance peptide stability and function
- Key modification sites including N-terminus, C-terminus, and side chains
- Specialized modifications like disulfide bridges, cyclization, and isotope labeling
- Advanced applications including fluorescent labeling and enzyme substrate development
- Techniques for increasing peptide half-life and metabolic stability
Peptide modification refers to permanent chemical alterations of the molecule, in contrast to protecting groups, which are removed after synthesis to obtain the final product (even in the case of so-called “permanent” protecting groups). Peptides can be modified in various ways, including functionalization of the N- and C-termini, derivatization of reactive groups in the 20 proteinogenic amino acids, and replacing specific amino acids with non-proteinogenic or unusual amino acids.
Types of peptide modifications
Natural peptide modifications
In nature, a diverse array of peptide/protein modifications occurs. Many of these are essential for the biological activity or function of the molecule. For example, the enzymatic oxidation of proline to hydroxyproline plays a crucial role in collagen’s structural stability, while the phosphorylation of serine, threonine, and tyrosine residues is central to biochemical signaling, effectively switching proteins “on” or “off”, depending on the phosphorylation state. Other modifications can indicate pathological processes and disease.
Modifications for stability
Certain modifications, such as N-terminal acetylation, N-terminal pyroglutamic acid (Pyr) formation, and C-terminal amidation, are used by nature to stabilize peptides. These “blocked” peptides are less rapidly broken down by enzymes, allowing them to persist longer. Additionally, natural peptide hormones, like gonadorelin and thyrotrophin-releasing hormone (TRH), are often modified at their end groups.
Chemical modifications for peptide stabilization
C-terminal amide groups are formed through the enzymatic degradation of glycine residues, while another enzyme catalyzes the cyclization of glutamine to Pyr:
H-Gln-Xaa-Yaa-…-Zaa-Gly-OH → Pyr-Xaa-Yaa-…-Zaa-NH2
Since oxidation of methionine in peptides often results in a loss of biological activity, methionine residues may be replaced with norleucine (Nle), a non-oxidizable, similarly structured amino acid. Chemical modifications also help stabilize or alter the spatial structure of a peptide.
Key N-terminal modifications
Peptide chemical synthesis offers numerous possibilities for modification, with the N-terminus being the easiest to modify, requiring only one additional step in Solid-Phase Peptide Synthesis (SPPS). Some important N-terminal modifications include:
- Acetylation
- Biotinylation — the attachment of biotin (vitamin B7) enables binding to the protein avidin.
- Fluorescent derivatization — attaching a fluorophore (e.g., 2-aminobenzoyl, coumarins, fluorescein) enables peptide detection in minute amounts.
Modifications to peptide side chains and backbone
The side chains of cysteine (Cys) and lysine (Lys) are also easily modified. Furthermore, the peptide backbone can be modified through the incorporation of special amino acids:
- D-amino acids
- N-methylated amino acids
- Non-proteinogenic amino acids (unusual amino acids)
- Replacement of the peptide bond with other bonds
- Ring closure, e.g., using disulfide bridges
C-terminal modifications
Peptidamides, the most important modification of the C-terminal carboxyl group, are readily accessible through SPPS. These derivatives are synthesized on specially developed resins, such as Ramage resin. More complex C-terminal modifications may require significant effort.
The diverse variety of peptide modifications available from Bachem is summarized in the infobox below.
| Available peptide modifications |
|---|
| Acetylation, acylation (e.g., lipopeptides) |
| Amidation |
| Backbone modifications, e.g.:
• hydroxyethylene isosteres |
| Biotinylation |
| C-terminal esters and thioesters |
| Conjugation to carrier proteins (e.g., KLH, BSA, OVA) |
| Conjugation to polymers |
| Conjugation to small molecules (including imaging agents) |
| Cyclization
• Head-to-tail |
| Depsipeptides (replacement of amide groups with ester groups) |
| Hydrocarbon-stapled peptides |
| Introduction/incorporation of:
• α- and N-methylated amino acids |
| Labeling with stable isotopes (e.g., 2H, 13C, 15N) |
| PEGylation |
| Peptide alcohols and aldehydes |
| Phosphorylation and sulfation |
Selected examples of peptide modifications
Peptides with disulfide bridges
A peptide may contain several disulfide bridges. For instance: One disulfide bridge can be found in various natural peptide hormones (e.g., calcitonin, somatostatin, vasopressin, oxytocin) and hormone analogs/APIs (e.g., octreotide, desmopressin). Several disulfide bridges are present in various peptide hormones and toxins (e.g., endothelin-1 (two bridges), conotoxin (three bridges), GaTx1 and hepcidin-25 (four bridges each)). Some peptide hormones contain two peptide chains linked by interchain disulfide bridges (e.g., insulin, relaxins).
Disulfide bridging and the correct linkage of the Cys pairs (if there are multiple bridges) are extremely important for the biological activity of a peptide. In general, peptides, especially longer peptides, do not exist as elongated chains but instead form three-dimensional structures that are stabilized by various interactions between the individual amino acids. Disulfide bridges are one such type of interaction that plays a crucial role in stabilizing the “correct” structure.
Disulfide bonds in peptides are formed by the oxidation of the thiol groups of two Cys residues in the peptide chain. In the case of Fmoc-SPPS, this oxidation step is carried out after cleavage from the resin, during which the Trt protecting groups are removed from Cys residues. Acm groups are cleaved with iodine and simultaneously oxidized to yield disulfide bonds.
The synthesis of peptides containing three or more disulfide bridges requires special care to ensure the homogeneity and expected biological activity of the purified product. Two strategies are commonly employed for this purpose: oxidative folding and “directed” disulfide formation. Oxidative folding is essentially a “biomimetic” process in which the reduced precursor sequence is folded under near-physiological conditions to yield the thermodynamically stable disulfide-bridged product. Directed disulfide formation, on the other hand, uses unambiguous chemical methods in a stepwise manner to prepare the product with the desired disulfide connectivity, which is “programmed” into the synthetic route. Both methods have advantages and disadvantages. They have been implemented at Bachem on milligram to kilogram scales for the synthesis of a wide range of targets.
Cyclization via amide bond formation
Besides disulfide bridging, peptide cyclization can be achieved by other methods. In so-called “head-to-tail cyclization”, an amide bond is formed between the N- and C-termini of the peptide after SPPS. Stabilization of a desired conformation can also be accomplished by the cyclization of side-chain amino and carboxy groups, which requires an additional level of protecting group orthogonality.
These bridges can be introduced between a variety of carboxy-containing amino acids (e.g., Asp, Glu, Aad, Asu) and amino-containing amino acids (e.g., Dap, Dab, Orn, Lys), which allows the ring size, flexibility, and direction of bond formation to be adjusted. Additionally, these amide bridges are more stable than disulfide bridges. If required, other modes of cyclization (e.g., thioether formation, metathesis) can also be employed.
Phosphopeptides and sulfopeptides
O-Phosphorylation and O-sulfation are very common post-translational modifications of proteins, and they are commonly requested for synthetic peptides. However, the limited chemical stability of phosphorylated and sulfated peptides necessitates the use of specialized procedures during synthesis and purification.
At Bachem, several synthetic methodologies are commonly utilized for these targets, which are largely based on the use of pre-derivatized building blocks to minimize the incidence of side reactions and ensure site-specific modification. Nonetheless, these products require meticulous attention during manufacturing and careful handling upon receipt by our customers, and we are always happy to provide guidance on stability optimization and recommendations to ensure the integrity of the target molecule during testing.
Glycopeptides
N-Glycosylation and O-glycosylation are also important post-translational modifications of peptides and proteins. Glycosylated peptides may act to stimulate the immune system. The demand for such compounds is constantly increasing, despite the considerable challenges associated with their synthesis. These synthetic problems are caused by a variety of factors, such as the limited choice of protecting groups for the glycoside moiety and the high lability of the O-glycosidic bond
Lipopeptides
Substantial difficulties have to be expected during the purification of lipopeptides owing to their increased hydrophobicity. Before starting to synthesize a lipopeptide, the sequence to be lipidated is studied carefully to determine the most suitable position for the introduction of the lipid moiety. A considerable number of palmitoylated peptides have been successfully synthesized at Bachem.. These compounds, which resemble the N-terminus of the lipoprotein from the outer membrane of Escherichia coli, have been used at Bachem for synthesizing immunogenic conjugates such as peptide mitogens or vaccines.
Peptides labeled with stable isotopes
The production of peptides labeled with stable isotopes, such as 13C, 15N, or 2H, is limited only by the commercial availability of the correspondingly labeled amino acids. Isotope labels are especially useful in NMR studies of peptides. A range of protected 15N-labeled amino acids is available from stock at Bachem. Further labeled amino acids can be acquired, if they are commercially available, for the synthesis of suitably protected derivatives.
Fluorophores and chromophores
Bachem has at its disposal a broad range of fluorophore and chromophore derivatives suitable for labeling peptides. In most cases, the dyes are introduced at either the N- or C-terminus. The synthesis of a C-terminally labeled peptide is usually more complex because N-terminal incorporation simply means an additional step in the SPPS protocol, even though more elaborate coupling procedures are available if necessary. The expense of many fluorescent dyes requires the use of specialized coupling protocols to maximize reaction yields with fewer stoichiometric equivalents of these costly raw materials.
Additionally, dyes can be linked to a peptide by selective reaction with a Cys thiol moiety or the less hindered ε-amino group of a Lys residue. The insertion of a spacer moiety between the dye and peptide helps to avoid interactions between the two components, which is beneficial for retaining the peptide conformation and biological activity. Additional effects may be attained by varying the length, flexibility, and hydrophilicity of the spacer.
When devising FRET substrates, the Förster distance, i.e., the distance between the fluorophore and quencher that affords an energy transfer efficiency of 50% (typically 20–90 Å), must be achieved at a minimum to obtain a good quenching effect. This depends on the choice of fluorophore/quencher pair. Only a limited number of amino acids can be inserted between the dye and quencher moieties; otherwise, the background fluorescence may reach unacceptable levels. The incorporation of a flexible spacer may also disturb the energy transfer.
Enzyme substrates and inhibitors
C-terminal chromophores and fluorophores such as 4-nitroanilide (pNA) and 7-amido-4-methylcoumarin (AMC) are commonly incorporated into peptides to obtain substrates for the detection and quantification of enzymatic activity. A different type of C-terminal residue is required to turn a substrate interacting with the active center of the enzyme into an inhibitor that binds reversibly or even irreversibly to this site.
Aldehyde, hydroxamate, fluoromethyl ketone, and chloromethyl ketone moieties are among the most common C-terminal modifications of peptides for generating effective inhibitors. The incorporation of such highly reactive moieties requires the adaptation of the synthetic strategy to each case, but our specialists can rely on their vast experience in SPPS and solution chemistry.
O-Acylated peptides
The peptide hormone ghrelin containing an O-acylated Ser residue and its analogs have found widespread application in obesity research. Bachem has developed considerable experience in performing this modification during the synthesis of numerous ghrelin analogs.
Acid-sensitive modifications
Our chemists are able to fine-tune their synthetic approaches so precisely that even highly acid-sensitive peptides containing two or more sulfated Tyr residues can be obtained.
Stabilizing modifications
A range of modifications for prolonging the half-life and increasing the metabolic stability of biologically active peptides can be performed, including selective PEGylation, incorporation of N-methylated amino acids, and the generation of pseudopeptide bonds that resist enzymatic cleavage (e.g., reduced peptide bonds, psi-[CH2-NH]).
Peptides containing chelating groups
Complexes formed between peptides bearing a chelating moiety such as DOTA or DTPA and radionuclides are increasingly used as imaging agents or for radionuclide delivery. The derivatives of the chelators required for coupling with peptides are synthesized in-house.
