Vapourtec’s R-Series flow chemistry system, equipped with a UV-150 photochemical reactor, has been cited in research into the C-terminal α-amination of peptides and proteins from cysteine-extended polypeptide precursors by scientists at Novo Nordisk.
Published in a paper in Nature Communications entitled ‘Photochemically-enabled, post-translational production of C-terminal amides’, the process involved cysteine thiol substitution with a photolabel, photoinduced decarboxylative elimination and cleavage of the enamide. Gram-scale synthesis was achieved on a recombinantly prepared peptide YY analogue. The flow reaction demonstrated potential for amidated drug leads to enter development that would not be viable on commercial scale using existing technology.
Progress in chemical and structural biology, along with improvements to short half-life and poor oral bioavailability, have increased popularity of peptide-based therapies. Recombinant production was used in place of more traditional SPPS to synthesise RecomPeptides, although peptides containing a C-terminal α-amide are particularly challenging to prepare using this method.
C-terminal cysteine residues were conjugated to the photolabile reagent 4-chloro-7-nitrobenzofurazan (NBD-Cl). Upon irradiation, this intermediately underwent a decarboxylation–fragmentation sequence, to provide a C-terminal N-vinyl amide.
The vinyl group could be removed to reveal the C-terminal α-amide either through acid hydrolysis or an inverse electron demand Diels-Alder (IEDDA) reaction, which allows compatibility with peptides and proteins sensitive to acid (e.g. glycopeptides).
On small scale (250 μM), the protocol was successfully used in batch for the synthesis of the GLP-1R agonist, GLP-1(7-36), one example of an important class of therapeutic peptides that includes the marketed drugs exenatide and lixisenatide, as well as in the other biologically-relevant targets including gastrin-releasing peptide, osteocrin and bulevertide.
A scaled-up process, using Vapourtec’s R-Series system in conjunction with the UV-150 photochemical reactor module, was proven to be efficient. One example demonstrated the preparation of an analogue of PYY, an amidated gastrointestinal hormone that is involved in the regulation of appetite [4, 5].
Using 4g of the starting material, the C-terminal cysteine was reacted with NBD-Cl then subjected to photo-initiated cleavage under flow conditions (430 nm, 24 W, 99% lamp power, 2 mL coil, 20 mL/min, tres = 6 secs). Enzymatic digestion of the N-terminus and conversion of the enamide, using phosphoric acid, to the intended amidated peptide was accomplished in 20% overall yield after a single final purification.
A second example showed conversion of 12g of a recombinant 81 amino acid GLP1R-amylinR co-agonist precursor peptide to the target peptide amide in 78% yield, rapidly and cleanly.
In summary, the work demonstrated an efficient approach to convert peptides and proteins to their corresponding C-terminal amides in good yield. It was noted that the work enabled C-terminal amidation (photolabeling and photochemical conversion) to be undertaken in one day, which significantly improves current capabilities. The substrate scope is broad, and both synthetic and recombinant peptides are tolerated. The authors noted that the reaction would not be viable on a commercial scale without the photochemical flow process.
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