Enhancing stability and bioavailability through peptide modifications 

 

 

Peptides, short sequences of amino acids, play important roles in medical diagnostics, immunology, and drug discovery due to their high selectivity, low toxicity, and significant biological activity. However, whether used as active pharmacological components, transport vectors, or diagnostic imaging agents, their applications are often hindered by low metabolic stability, short half-life and poor bioavailability. 

Peptides are highly susceptible to enzymatic degradation, undergo rapid renal clearance, and often struggle with membrane permeability, all of which significantly limit their clinical potential. To address these limitations, a variety of chemical modification strategies have been developed to improve peptides’ pharmacological and pharmacokinetic profiles. The conversion of a native peptide into an effective drug delivery tool typically begins with a subtle chemical modification to its natural structure that preserves the peptide backbone, resulting in a modified peptide. This is followed by the creation of a pseudopeptide and ultimately the development of a fully synthetic peptide mimetic. 

Experimental strategies for assessing peptide stability 

Identifying vulnerable enzymatic cleavage sites is critical for the rational design and chemical modification of peptides to enhance their in vivo stability. While computational modeling and database analyses offer valuable preliminary insights, experimental validation remains essential. 

Typically, peptides are incubated with biological matrices such as tissue homogenates, plasma, or serum to simulate physiological conditions. Their degradation kinetics and fragmentation patterns are monitored using high-performance liquid chromatography (HPLC), followed by detailed characterization through sequencing and mass spectrometry, particularly liquid chromatography–mass spectrometry (LC-MS). 

LC-MS, a highly sensitive analytical technique, enables the detection of low-abundance metabolites without requiring radio labeling. Ultimately, in vivo studies are vital to verify and contextualize the metabolic pathways identified through in vitro experiments. 

Peptide modification strategies 

To increase peptide drug stability and bioavailability, researchers employ both chemical and structural modification techniques. These enhancements are often enabled by advanced protein expression systems such as bacterial, yeast, or mammalian platforms, which facilitate scalable production of engineered peptides and fusion proteins. 

Below are key strategies used to improve peptide performance: 

Cyclization 

Cyclization, either head-to-tail or side-chain-to-side-chain, reduces the conformational flexibility of peptides, making them less recognizable to proteolytic enzymes. This modification not only increases resistance to degradation but also often potency, selectivity, stability and bioavailability, as well as membrane barrier permeability. For instance, cyclic peptides like cyclosporine A exhibit significantly improved stability and bioactivity compared to their linear counterparts. 

Backbone modification 

Replacing L-amino acids with their D-enantiomers, inserting N-alkylated amino acids, and incorporating β-amino acids or α/β-substituted αamino acids are the most utilized techniques as backbone modification approaches. These modifications of the peptide sequence can dramatically improve resistance to enzymatic cleavage, thus enhancing the half-life and stability of peptides. For example, introducing d-amino acids into lanthipeptides shows the potency of backbone modifications to overcome proteolytic instability. 

Further strategies involve isosteric substitutions, where peptide bonds are replaced with structurally similar but non-cleavable alternatives: 

• Azapeptides: Contain a nitrogen atom in place of the α-carbon, mimicking natural peptides with improved resistance. A recent azapeptide-based SARS-CoV-2 main protease (Mpro) inhibitor effectively targets the viral enzyme, showcasing clinical promise. 

• Retro-inverso peptides: Feature reversed sequences and D-amino acids, offering excellent resistance to degradation and acting as anti-inflammatory and immunomodulatory agents. 

• Peptoids: Composed of N-alkylated glycine, they offer structural flexibility. They show potent applications in oncology, neurology, and autoimmune diseases. 

N-terminal and C-terminal modifications 

Blocking the N- and C-termini of peptides through acetylation or amidation (N-acetylation, C-amidation) can protect against exopeptidase activity. These modifications prevent the enzymes from recognizing and cleaving peptide termini, thereby extending their half-life in vivo. For instance, The N-terminal acetylated somatostatin analog has a longer in vivo half-life than the native peptide, reaching over 400 minutes. 

Conjugation strategies 

Conjugation of peptides with larger molecules like lipids or polymers improves both bioavailability and metabolic stability. For example, the attachment of polyethylene glycol (PEGylation) increases the molecular weight of peptides, reducing renal filtration and providing a steric shield against proteases. 

Conjugating lipid moieties to peptides (lipidation) enhances their binding to serum albumin, reducing renal clearance and degradation. Semaglutide, a lipidated GLP-1 analog, demonstrates a 100-fold improvement in half-life (up to 168 hours) compared to its unmodified version. 

Side chain modifications 

Targeted modification of amino acid side chains, especially those at enzymatic recognition sites, is another effective method for improving the stability and bioavailability of peptides. The ideas underlying this strategy involve modifying the amino acids of the recognition site of the enzyme with analogs during peptide synthesis to prevent the cleavage while staying as close as possible to the original sequence to preserve the activity of the peptide. These types of alterations help to increase the binding affinity and target selectivity of peptides, thus increasing the metabolic stability. 

Research peptides represent a transformative tool in drug development, offering a foundation for innovative chemical modifications that improve their therapeutic potential. By enhancing enzymatic resistance, bioavailability, and pharmacokinetics, these strategies extend peptide half-life, reduce dosing frequency, and improve patient adherence. 

The combination of peptide engineering, protein expression systems, and cutting-edge bioanalytical techniques continues to drive the evolution of next-generation peptide therapies. As these tools advance, they pave the way for more effective, stable, and accessible peptide-based treatments across a wide range of clinical applications. 

 

References  

 

1. Adessi, C., & Soto, C. Converting a peptide into a drug: strategies to improve stability and bioavailability. Current medicinal chemistry. 9(9), 963-978 (2002).  

https://pubmed.ncbi.nlm.nih.gov/11966456/ 

 

2. Xiao, W., Jiang, W., Chen, Z., et al. Advance in peptide-based drug development: delivery platforms, therapeutics and vaccines. Signal Transduction and Targeted Therapy. 10(1), 74 (2025). 

https://www.nature.com/articles/s41392-024-02107-5 

 

3. Menacho-Melgar, R., Decker, J. S., Hennigan, J. N., et al. A review of lipidation in the development of advanced protein and peptide therapeutics. Journal of Controlled Release. 295, 1-12 (2019). 

https://pmc.ncbi.nlm.nih.gov/articles/PMC7520907/