Flow photochemistry unlocks scalable peptide amidation

Scientists at Novo Nordisk have demonstrated a photochemically enabled strategy for the functionalisation of peptides and proteins, employing Vapourtec’s R-Series flow chemistry system and UV-150 photochemical reactor. Their work [1], published in Nature Communications, introduces a method for post-translational C-terminal α-amination of peptides, presenting a scalable alternative to traditional approaches.

The study, ‘Photochemically-enabled, post-translational production of C-terminal amides’, describes a three-step process involving cysteine thiol substitution with a photolabel, photoinduced decarboxylative elimination, and cleavage of the enamide. This strategy was successfully applied to a recombinantly produced peptide YY analogue, achieving gram-scale synthesis. The findings highlight how flow photochemistry facilitates the development of amidated peptide drug candidates that would otherwise be impractical using conventional synthetic methods.

Peptide-based therapeutics have seen renewed interest due to advancements in chemical and structural biology. Overcoming key limitations such as short half-life and poor oral bioavailability has driven innovation in the field [2]. While solid-phase peptide synthesis (SPPS) remains a standard method, large-scale production is often challenging. Recombinant expression provides an alternative, but peptides containing a C-terminal α-amide are notoriously difficult to produce this way.

Building on prior work by the Baker group [3], the researchers employed a photolabile reagent, 4-chloro-7-nitrobenzofurazan (NBD-Cl), to conjugate C-terminal cysteine residues. Upon irradiation, this intermediate underwent a decarboxylation–fragmentation sequence, yielding a C-terminal N-vinyl amide. Subsequent hydrolysis or inverse electron demand Diels-Alder (IEDDA) reaction enabled selective conversion to the desired C-terminal α-amide while preserving acid-sensitive structures such as glycopeptides.

Initial small-scale experiments demonstrated the method’s utility in synthesizing GLP-1(7-36), a GLP-1R agonist relevant to marketed drugs like exenatide and lixisenatide. The approach was also applied to biologically significant peptides, including gastrin-releasing peptide, osteocrin, and bulevertide.

Scalability was validated using Vapourtec’s R-Series system in conjunction with the UV-150 photochemical reactor. A model reaction converting a PYY analogue was performed under flow conditions (430 nm, 24 W, 99% lamp power, 2 mL coil, 20 mL/min, residence time = 6 sec). The final amidated peptide was obtained in 20% overall yield after a single purification. Another large-scale conversion of a recombinant 81-amino acid GLP1R-amylinR co-agonist precursor resulted in a 78% yield with high efficiency.

The researchers demonstrated that this process allows C-terminal amidation in a single day, significantly improving upon existing methodologies. The broad substrate scope, tolerance for synthetic and recombinant peptides, and scalability make this an important advancement. Without the photochemical flow approach, commercial viability of this transformation would not be feasible, underscoring the impact of enabling technologies in modern peptide science.

More information online

1. Photochemically-enabled, post-translational production of C-terminal amides. (D. Hymel, F. Wokcik, K. S. Halskov et al., Nature Comm., 2024, 15, 7162). https://doi.org/10.1038/s41467-024-51005-5 

2. The current state of peptide drug discovery: back to the future? (A. Henninot, J. C. Collins, J. M. Nuss, J. Med. Chem., 2018, 61, 1382). https://doi.org/10.1021/acs.jmedchem.7b00318 

3. Photochemically re-bridging disulfide bonds and the discovery of a thiomaleimide mediated photodecarboxylation of C-terminal cysteines. (D. A. Richards, S. A. Fletcher, M. Nobles et al., Org. Biomol. Chem., 2016, 14, 455). https://doi.org/10.1039/C5OB02120K