Synthesis of liraglutide

Liraglutide is a GLP-1 (glucagon-like peptide-1) analog composed of 31 amino acids, characterized by C-terminal amide modification and palmitate esterification of the Lys20 (lysine at position 20) side chain. Due to its large molecular weight, easy accumulation of long chains, and presence of hydrophobic fat chains, its synthesis mainly adopts a "solid-and-liquid combination" aggregation synthesis strategy or a biotechnological approach of "recombinant protein fermentation combined with enzyme modification." The specific synthesis plan is as follows:

1. Chemical Synthesis Method (Mainstream Industrial Route) The chemical synthesis method mainly uses solid-phase peptide synthesis (SPPS) combined with fragment condensation and side chain modification to overcome the difficulties of long peptide chain coupling and peptide chain aggregation.

1. Fragment Splitting and Solid-Phase Synthesis (SPPS)

1.  Fragment Resolution: Break down 31 amino acids into several short peptide fragments (such as 1-8, 9-16, 17-31), with fragment length controlled within 10 amino acids to improve coupling efficiency and solubility.

2. Solid-phase synthesis: Using resins (such as Wang resin, 2-CTC resin, or HMB-CM resin) as carriers, and employing Fmoc (fluoremethoxycarbonyl) or Boc (tert-butoxycarbonyl) protection strategies, amino acids are gradually coupled to synthesize various short peptide fragments.

2. Fragment Condensation (Aggregation Synthesis)

1. Purified short peptide fragments are condensed in liquid or solid phase and spliced into complete long peptide main chains (such as 1-8 and 9-31 docking). Coupling agents (such as HCTU, DIC/HOBt) are commonly used during condensation, along with additives that prevent peptide chain aggregation (such as LiCl or pseudoproline) to improve conversion rates.

3. Side chain modification (palmitotic acid)

1. Palmitot acid modification was performed at the Lys20 site of the long peptide backbone. Pal-glutamyl-tert-butyl ester (Pal-Glu-OBu) active esters are typically synthesized first, and under alkaline conditions (pH 10-12), they are coupled with the Lys20 side-chain amino group to achieve long-chain modification.

4. Cutting and removing protection

1. The peptide resin with side chain modification is deprotected with a cutting solution (such as TFA/TIS/H2O mixture), and the peptide chain is cleaved from the resin with an alkaline solution (such as NaOH) to obtain crude peptides.

5. Purification and refining

1. The crude peptide is purified by reversed-phase high-performance liquid chromatography (RP-HPLC) or hydrophobic interaction chromatography (HI-LPLC), and finally freeze-dried to obtain high-purity (>98%) liraglutide API.

2. Biosynthesis (Recombinant Fermentation Route) 

For some industrial production, recombinant DNA technology can also be used, combining engineering microbial fermentation with enzymatic modification for synthesis. 

1. Recombinant fusion protein expression

1. The liraglutide gene (containing C-terminal glycine) is fused with the GST (glutathione S-transferase) tag, inducing expression in E. coli (such as BL21) to form inclusions.

2. Inclusion of body complexity

1. After denatured and dissolved inclusions, reformation is carried out through a redox system (GSH/GSSG) to restore the correct disulfide bonds.

3. Enzyme digestion and modification

1. GST tags are removed with thrombin, releasing liraglutide precursors.

2. The C-terminal amide modification was performed using PAM (peptidylglycine α-amimatated monooxygenase), and the palmitotto acidation of the Lys20 side chain was modified with Bacillus subtilis protease to finally obtain mature liraglutide.

4. Purification and validation

1. Purified by high-performance liquid chromatography (HPLC), and verified for bioactivity and purity.

Each of the two synthetic routes has its own advantages and disadvantages: the chemical synthesis method is highly purified and easy to control, suitable for precise synthesis with complex modifications; Biosynthesis is suitable for large-scale fermentation production but requires solving process issues such as short peptide degradation, inclusion body reconstruction, and enzymatic modification.

The intermediates for liraglutide

Fmoc-Lys(Pal-Glu-OtBu)-OH  Cas:1491158-62-3

Pal-Glu(OSU)-OH  Cas:294855-91-7

Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH  Cas:1418291-58-3

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