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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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]).

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.