Photochemically-enabled, post-translational production of C-terminal amides

Abstract

C-terminal α-amidated peptides are attractive therapeutic targets, but preparative methods to access amidated pharmaceuticals are limited both on lab and manufacturing-scale. Here we report a straightforward and scalable approach to the C-terminal α-amidation of peptides and proteins from cysteine-extended polypeptide precursors. This amidation protocol consists of three highly efficient steps: 1) selective cysteine thiol substitution with a photolabel, 2) photoinduced decarboxylative elimination and 3) enamide cleavage by simple acidolysis or inverse electron demand Diels-Alder reaction. We provide a blueprint for applying this protocol to the semi-recombinant production of therapeutically relevant targets where gram scale C-terminal α-amidation is achieved in a photoflow reactor on a recombinantly prepared peptide YY analogue and a GLP-1/amylin co-agonist precursor peptide. Robust performance of this reaction cascade in flow highlights the potential of this chemistry to enable amidated drug leads to enter development that would not be viable on commercial scale using existing technology.

Fig. 1. C-terminal α-amidation reactions.

a Enzymatic (PAM) mediated α-amidation of peptides containing a C-terminal glycine residue. b Biomimetic C-terminal α-amidation of peptides containing a C-terminal cysteine residue (this work). The two reaction pathways provide complementary routes for accessing C-terminal α-amides that proceed through a common enamide intermediate, which can be converted to the amide under acidic conditions (Path A) via the biosynthetic carbinolamide intermediate, or under neutral conditions via an IEDDA reaction with a tetrazine (Path B).

Fig. 2. Evaluation of reaction conditions and scope of the α-amidation with respect to the penultimate position.

a Generalized reaction scheme. b Yield of C-terminal α-amides obtained by substitution with bromomaleimide (Route A) or NBD-Cl (Route B), followed by enamide cleavage with acid (route C) or by IEDDA (Route D). *All yields were determined by extracted ion chromatography (XIC).^≠ X = A, and the Trp residue at the 2-position is substituted to Phe (IFTKDHEEVYEA-NH₂).

Fig. 3. Demonstration of scope of the amidation on pharmaceutically relevant amidated peptides.

All reactions were performed at a C-terminal cysteine-extended peptide concentration of 250 µM in 25 mM BisTris + 50 mM glycine buffer at pH 6.4. The choice of photolabel and enamide cleavage conditions are indicated by the letters following yields and correspond to the routes described in Fig. 2. *All yields were determined by extracted ion chromatography (XIC).

Fig. 4. C-terminal α-amidation of a peptide containing a backbone disulfide bond.

^*Yield was determined by extracted ion chromatography (XIC).

Fig. 5. Demonstration of gram scale photo amidation using recombinant starting material.

a Multi-step synthesis of peptide YY analog 7 utilizing a photoflow reactor. b Streamlined multigram photoamidation process of a GLP1R-amylinR co-agonist precursor mimicking biopharmaceutical manufacturing conditions. Isolated recombinant disulfide backbone 8 was converted to amide 9 using an optimized photoamidation process protocol with both high chemical yield and high-volume yield.