Commercial Landscape
Solid-Phase Peptide Synthesis and Coupling Control
Protecting Group Strategies and Stereochemical Control
Phosphoramidite Chemistry for Oligonucleotides
Managing Impurities and Side Products
Emerging Enzymatic and Flow-Based Approaches
Conclusion
References and Further Reading
Building sequence-defined medicines demands near-perfect chemistry. New advances in coupling control, purity management, and automated synthesis are making that precision achievable.
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Modern medicine depends on large, information-rich molecules such as peptides and oligonucleotides. These molecules carry biological instructions that can be directed toward specific disease pathways.
Over the past decade, their expansion into mainstream therapeutics has placed increasing demands on synthetic reliability and scalability. Their entry into the therapeutic landscape is enabled by precision chemistry - a framework of tightly controlled, sequential reactions that allow the stepwise assembly of complex sequences with high fidelity, yield, and reproducibility.
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This approach underpins the production of therapeutic peptides such as semaglutide and tirzepatide, as well as oligonucleotide drugs including inclisiran and various siRNA and antisense agents. In each case, the design of the synthetic pathway is as critical as the final molecular sequence.
Precision determines not only whether a molecule can be synthesized, but whether it can be produced consistently, at scale, and to regulatory standards.
Solid-Phase Peptide Synthesis and Coupling Control
Solid-phase peptide synthesis (SPPS) remains the primary method for peptide production at research and industrial scales. The growing peptide chain is anchored to an insoluble polymer support, simplifying purification and allowing excess reagents to drive each coupling step toward completion.
The method proceeds through iterative cycles of deprotection and coupling: the α-amino group of each incoming amino acid is protected (commonly with Fmoc or Boc), while side chains are masked with orthogonal protecting groups.
After loading the C-terminal amino acid onto the resin, the α-protecting group is removed, the next amino acid is activated and coupled, and unreacted sites are capped to prevent deletion sequences.1,2
This repetitive build-deprotect-couple-cap cycle forms the operational backbone of peptide manufacturing.
Recent refinements focus on maximizing coupling efficiency while suppressing side reactions. The choice of coupling reagents, such as phosphonium- or uronium-type activators, and additives, including oxyma derivatives, reduces epimerization and improves solvation, even for challenging sequences.
Resin and linker selection further influence swelling, chain accessibility, and cleavage conditions.
Together, these parameters increase crude purity, reduce truncated or misfolded species, and lower downstream purification demands - an important consideration for therapeutic-scale production.2,3
Protecting Group Strategies and Stereochemical Control
Protecting group design is central to synthetic precision. The widely adopted Fmoc/tBu strategy combines base-labile Fmoc removal with acid-labile side-chain deprotection, enabling controlled, orthogonal exposure of functional groups.
Amino acids such as lysine, glutamic acid, and cysteine can therefore be selectively manipulated. Careful control of deprotection kinetics and compatibility minimizes diketopiperazine formation, aspartimide rearrangements, and disulfide scrambling, preserving sequence integrity.1,2
In practice, protecting groups act as a molecular control system, dictating which functionalities are reactive at each stage.
Stereochemical integrity is maintained through the use of enantiomerically pure building blocks and minimized racemization during activation. Residues such as histidine and cysteine are particularly sensitive, requiring optimized activators, reduced temperatures, and short coupling times.
Modern automated SPPS platforms encode these parameters into standardized workflows, enabling reproducible synthesis of complex peptides, including semaglutide, with high crude purity and limited side products.4
Automation reduces operator variability and strengthens batch-to-batch consistency, which is critical for regulatory compliance.
Phosphoramidite Chemistry for Oligonucleotides
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For oligonucleotides, phosphoramidite chemistry provides an analogous framework for precision. Nucleotide monomers are functionalized with a 4,4'-dimethoxytrityl (DMT) group at the 5'-hydroxyl, base-protecting groups such as benzoyl or isobutyryl, and a 2-cyanoethyl phosphoramidite at the 3' position.
Each synthesis cycle comprises DMT deprotection, phosphoramidite coupling, capping of unreacted chains, and oxidation of the phosphite linkage to a phosphate or phosphorothioate.5,6
Repetition of this controlled four-step sequence enables incremental construction of defined DNA or RNA strands.
Coupling efficiencies typically exceed 98-99 % per step, permitting synthesis of oligonucleotides up to approximately 100 nucleotides in length with acceptable full-length yields. Advances in linker chemistry and monomer design have expanded compatibility with chemically modified nucleotides and non-nucleoside termini, broadening the structural diversity accessible through solid-phase synthesis.6
Even modest improvements in per-step efficiency produce exponential gains in full-length products as sequence length increases.
Managing Impurities and Side Products
Despite high efficiencies, both peptide and oligonucleotide synthesis generate side products. In oligonucleotides, typical impurities include truncated n-1 and n-2 sequences, depurinated strands, and phosphorothioate diastereomers. Peptide byproducts include deletion sequences, epimerized residues, and side-chain modifications such as aspartimide formation or cysteine oxidation.6,7
Accumulated across multiple cycles, even low-frequency reactions can compromise overall purity.
Impurity management begins with reaction optimization and extends to analytical characterization.
High-performance liquid chromatography (HPLC) and mass spectrometry identify dominant side reactions, enabling targeted adjustments in stoichiometry, coupling conditions, or protecting group strategies. Downstream purification - often via reversed-phase or ion-exchange chromatography - removes truncated or misfolded species while preserving yield.7,8
In a stringent regulatory environment, impurity profiling is integral to process validation rather than a secondary analytical step.
Emerging Enzymatic and Flow-Based Approaches
Complementing traditional chemical methods, enzymatic and flow-based strategies are expanding the synthetic toolkit. Enzymatic peptide synthesis employs proteases or ligases to form peptide bonds under aqueous conditions with high chemoselectivity. Such systems facilitate access to glycopeptides and other post-translationally modified structures that are challenging to assemble chemically.9
In oligonucleotide synthesis, template-independent enzymatic or chemoenzymatic methods use polymerases and transferases to build RNA or DNA strands in aqueous media, reducing reliance on organic solvents.
Flow-based and membrane-assisted liquid-phase systems integrate coupling, sulfurization, and purification within continuous processes, improving reaction control and lowering process-mass intensity.10,11
Continuous formats also enhance control over mixing, residence time, and reagent exposure, supporting more consistent outcomes at scale.
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Conclusion
The convergence of solid-phase, enzymatic, and flow-based methods reflects a maturing field in which synthetic precision underpins therapeutic viability. Integrated platforms combining automated SPPS with chemical modifications such as cyclization or stapling enable efficient access to conformationally constrained, drug-like peptides within controlled workflows.
Precision chemistry in peptide and oligonucleotide synthesis is not a single technique but a coordinated framework encompassing protecting group design, optimized coupling, stereochemical control, and impurity management.
As these modalities transition from specialized applications to widely prescribed therapies, reproducible control over each synthetic step will influence scalability, cost, and global access. Continued progress in sustainable reagents, real-time analytical monitoring, and hybrid chemoenzymatic systems will determine how far sequence complexity can advance without compromising manufacturability.
References and Further Reading
- Guzmán, F. et al. (2023). Peptides, solid-phase synthesis and characterization: Tailor-made methodologies. Electronic Journal of Biotechnology, 64, 27-33. DOI:10.1016/j.ejbt.2023.01.005, https://www.sciencedirect.com/science/article/pii/S0717345823000143
- Albericio, F. (2022). Practical Protocols for Solid-Phase Peptide Synthesis 4.0. Methods and Protocols, 5(6). DOI:10.3390/mps5060085, https://www.mdpi.com/2409-9279/5/6/85
- Phungula, A. et al. (2025). Aqueous Solid-Phase Peptide Synthesis (ASPPS) using Standard Fmoc/tBu-Protected Amino Acids. ACS Sustainable Chem. Eng, 13, 45, 19833–19848. DOI:10.1021/acssuschemeng.5c09191, https://pubs.acs.org/doi/10.1021/acssuschemeng.5c09191
- Zero, J. et al. (2025). Universal peptide synthesis via solid-phase methods fused with chemputation. Nature Communications, 16(1), 7322. DOI:10.1038/s41467-025-62344-2, https://www.nature.com/articles/s41467-025-62344-2
- Mohammed, A.A. et al. (2024). Oligonucleotides: evolution and innovation. Med Chem Res 33, 2204–2220. DOI:10.1007/s00044-024-03352-7, https://link.springer.com/article/10.1007/s00044-024-03352-7
- Yamamoto, K. et al. (2023). Expansion of Phosphoramidite Chemistry in Solid-Phase Oligonucleotide Synthesis: Rapid 3′-Dephosphorylation and Strand Cleavage. J. Org. Chem, 88, 5, 2726–2734. DOI:10.1021/acs.joc.2c02195, https://pubs.acs.org/doi/10.1021/acs.joc.2c02195
- Kelly, R., Parga, C., & Ferguson, S. (2025). Scalable Membrane Enabled One-Pot Liquid-Phase Oligonucleotide Synthesis. Organic Process Research & Development. DOI:10.1021/acs.oprd.5c00117, https://pubs.acs.org/doi/10.1021/acs.oprd.5c00117
- Abbasi Somehsaraie, M. H. et al. (2022). Chemical Wastes in the Peptide Synthesis Process and Ways to Reduce Them. Iranian Journal of Pharmaceutical Research: IJPR, 21(1), e123879. DOI:10.5812/ijpr-123879, https://brieflands.com/journals/ijpr/articles/123879
- Bizat, P. N., Sabat, N., & Hollenstein, M. (2025). Recent advances in biocatalytic and chemoenzymatic synthesis of oligonucleotides. ChemBioChem, Volume 26, Issue 9. DOI:10.1002/cbic.202400987, https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400987
- Wiegand, D. J. et al. (2024). Template-independent enzymatic synthesis of RNA oligonucleotides. Nature Biotechnology, 43(5), 762. DOI:10.1038/s41587-024-02244-w, https://www.nature.com/articles/s41587-024-02244-w
- Pichon, M., & Hollenstein, M. (2024). Controlled enzymatic synthesis of oligonucleotides. Communications Chemistry, 7, 138. DOI:10.1038/s42004-024-01216-0, https://www.nature.com/articles/s42004-024-01216-0
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