Since Emil Fischer pioneered peptide synthesis in 1901, the techniques for synthesizing peptides have significantly advanced. Innovations such as solid-phase peptide synthesis (SPPS), various protection and deprotection strategies, efficient coupling agents, automation, and high-yielding cyclization methods have all contributed to today’s ability to produce peptides. The modular nature of peptides and the effective coupling of amine to carboxylic acid groups facilitate the synthesis of diverse peptide sequences using hundreds of available amino acid building blocks through fully automated processes. Although synthesizing simple peptides is generally straightforward, challenges persist with longer sequences, those containing complex building blocks such as N-methylated amino acids, or peptides incorporating non-standard peptide bonds.
Nature utilizes sophisticated systems to synthesize polymers like peptides, DNA, RNA, proteins, and polyketides. It employs both ribosomal synthesis, which assembles the 20 standard amino acids via a DNA template, and non-ribosomal synthesis, which assembles non-canonical amino acids using enzyme complexes. The laboratory emulation of this process generally proceeds in the opposite direction, from the C-terminus to the N-terminus, proving more efficient for chemical peptide synthesis.
Modern SPPS allows for the rapid, parallel production of diverse peptides. Commercial synthesizers can concurrently synthesize about 50 different peptides, and even larger numbers can be handled using 96-well plates. Subsequent purification of these peptides is performed using automated liquid chromatography-mass spectrometry systems. At the largest scales, peptides are synthesized on membrane arrays for applications like rapid protein epitope mapping.
Peptide synthesis has expanded beyond natural peptides thanks to the extensive availability of amino acid building blocks. The sheer number of accessible Fmoc amino acids, which surpass a thousand in commercial catalogs, demonstrates the potential for exploring new peptide-based innovations in research and development at affordable costs.
Peptoids, a class of peptide mimetics, exemplify this innovation. They are synthesized using a two-step process that sequentially attaches sub-monomers to a solid support, allowing for the incorporation of a vast array of primary amine building blocks. This diversity enables the rapid creation of peptide variants, optimizing properties such as binding affinity, specificity, stability, solubility, and membrane permeability.
Naturally occurring cyclic peptides are often cyclized through disulfide bridges or through head-to-tail connections involving the amino group of Lys, Glu, or Asp. Examples include hormones like oxytocin and vasopressin and drugs like the anticancer agent romidepsin. Various natural and synthetic cyclization strategies have been explored to achieve efficient ring closure.
Chemically, synthetic peptides are cyclized using strategies such as disulfide formation, macrolactamization, thiol alkylation, and copper-catalyzed azide-alkyne cycloaddition (CuAAC). However, achieving successful cyclization can be challenging due to the need for peptides to adopt specific conformations conducive to ring closure. This process can be hindered by the peptides’ tendencies to form intermolecular bonds at high concentrations. To avoid this, cyclization reactions are usually conducted at low concentrations.
The success of cyclization reactions often depends on the specific chemistry employed and can vary with the peptide’s length and sequence. Techniques like thiol-based cyclization and macrolactamization have shown high efficiency, although the latter’s effectiveness can vary. Other methods, such as CuAAC and ring-closing metathesis, while versatile, generally yield lower efficiency but can accommodate various functional groups.