The synthesis of semaglutide is an extremely complex process because it is a large, chemically modified peptide class (consisting of 31 amino acids). It is not easily done by pure chemical synthesis like small molecule drugs. Its synthesis can relies on a combination of solid-phase peptide synthesis(SPPS) and selective liquid-phase chemical modification. Here's an overview of the key steps of its synthesis route:

1. Solid-phase peptide synthesis Construct the main peptide chain backbone (GLP-1 analogue part):

◦ Step-by-step assembly of GLP-1 from the C-terminal (carboxyl terminus) to the N-terminus (amino terminus) using solid-phase peptide synthesis techniques (7-37) Key sequences of the skeleton (sequence: his-aib-glu-gly-thr-phe-thr-ser-asp-val-ser-ser-tyr-leu-glu-gly-gln-ala-ala-lys-glu-phe-ile-ala-trp-leu-val-arg-g-arg-gly).

◦ Key Points:

▪ Use the FMOC-protection strategy: Each amino terminal is protected by FMOC (methoxycarbonyl group). Before each step of coupling, reagents such as piperidine are used to remove the FMOC protection group of the previous amino acid to expose the free amino group.

▪ Side-chain protective groups: The side-chain active groups of key amino acids (e.g., Lys, Glu, Asp, Arg, etc.) must be protected with other protective groups (e.g., Boc, Trt, Pbf, OtBu, etc.) to prevent side reactions during synthesis.

▪ Conjugation Reagents: Promote peptide bond formation using highly efficient conjugation reagent systems such as HBTU/HOBt/DIPEA or similar.

▪ Carrier resins: Synthesis is performed on specific polymer resins such as Wang resins or Rink Amide resins, and the end product can be C-terminal carboxylic acids or C-terminal amides. Semaglutide is a C-terminal amide.

▪ AIB: Position 8 is not a natural ALA, but an unnatural amino acid 2-aminoisobutyric acid, which enhances the peptide's resistance to enzyme degradation.

2. Introduce the Glutamate Connection Arm:

◦ In the SPPPS process, introduce a Lys with orthogonal protection bases (such as Dde or ivDde) at a specific location (Lys26).

◦ After backbone synthesis is completed and the Dde/ivDde protective group of the Lys is removed, the ε-amino groups of the Lys are exposed.

◦ Subsequently, in the liquid phase (or on resins, but it is often more convenient to transfer to the liquid phase), the space arm modifier (Nε-[(2S)-2-(16-(octadecyde)-4,9,12,15-tetraoxa-17-azoctadecane-1-acyl)γ-glutamic acid]) is amide-coupled to the ε-amino group of this Lys by its α-carboxyl group.

◦ Key Points:

▪ This structure is very complex: it contains a glutamate backbone (its α-carboxyl group is used to connect Lys26) and the γ-carboxyl group is attached to a very long, branched lipophilic chain. This lipophilic chain is composed of an 18-carbon fatty acid (octadecanoic acid) and a polyethylene glycol (PEG) linker (here 4 ethylene glycol units, OEG, [2-(2-aminoethoxy)ethoxy]acetic acid]).

▪ Dual Action: 18-carbon fatty acids provide strong albumin binding ability; OEG linkers increase hydrophilicity, solubility, and molecular flexibility.

▪ Typically, this complex side-chain modification is pre-synthesized separately in the liquid phase with appropriate protective groups for selective ligation to the ε-amino group of Lys26.

3. Polyethylene Glycylation (PEGylation):

◦ The OEG linker sub-end introduced in the above step is an amino group (requiring pre-protection, such as Boc).

◦ After the connection is completed (usually also in the liquid phase), the protective group (such as Boc) at the end of the OEG is removed, exposing the amino group.

◦ This amino group is amide-coupled with an activated PEG molecule (usually a methoxypolyethylene glycol propionic acid or its active ester with a suitable molecular weight, such as 2000 Da or 3000 Da).

◦ Key Points:

▪ This step further increases hydrophilicity and molecular weight, greatly extending the half-life of the drug, which is at the heart of long-acting GLP-1 receptor agonists.

▪ Activated PEGs usually carry N-hydroxysuccinimide (NHS) esters or other activating esters to facilitate reaction with amino groups.

4. Total Side Chain Protection and Resin Cracking:

◦ Once all amino acids have been assembled and critical side chain modifications (steps 2 and 3) have been completed on the resin or after transfer to the liquid phase, global deprotection is required.

◦ Use a strong acid reagent mixture (such as trifluoroacetic acid as a main component, combined with water, triisopropylsilane, ethyl dimercaptan, etc.) to remove the protective groups of all amino acid side chains.

◦ Simultaneous cleavage: This strong acid treatment also separates the peptide chains from the solid phase resin (cleavage).

5. Purification:

◦ The resulting crude product contains target products, unreacted raw materials, short peptide fragments, reagent impurities, etc., which is very complex.

◦ Multistep purification using high-performance preparative reversed-phase HPLC. Select an optimized column (e.g., C18) and mobile phase conditions (gradient elution) to separate the target semaglutide single peak.

◦ This is often one of the most challenging and time-consuming steps in the entire synthesis.

6. Freeze Drying:

◦ The purified semaglutide-containing eluent is freeze-dried into a solid powder after desalting and buffer replacement, which is convenient for storage, transportation and preparation.

Summarize the key points and difficulties:

• Solid-liquid combination: The backbone is primarily synthesized in solid phase, and key chemical modifications (large side chains and PEGs) are usually performed in the liquid phase after selective deprotection for greater efficiency.

• Complex modifications: The synthesis and precise coupling of large chemical modifications (including fatty acids, connecting arms, and PEGs) on Lys26 are core challenges and technical keys.

• Orthogonal Protection Strategy: It is crucial to manage numerous protection bases (main chain FMOC, side chain various protection groups, protection groups on modifiers) to ensure that the reaction is in the correct position.

• Difficult purification: Preparing high-purity target products is very difficult due to their large molecular weight, coexistence of hydrophobic and hydrophilic moieties, and complex impurities.

• Process scale-up: Process scale-up from small-scale synthesis in the laboratory to industrial production is a significant engineering challenge.

Simple schematic diagram:

  1. Solid-phase synthesis of His-Aib-Glu...-Lys (ε-protection) -...- Arg-Gly resin -> backbone
  2. Remove the ε-protective group of Lys26 -> Expose Lys26 ε-NH2
  3. Liquid phase conjugated ε-NH2 + HOOC-Glu(α)-[Connecting Arm (OEG)-NHBoc]-O-Fatty Acid Sidechain Modifier --> Connecting Fatty Acid/OEG Fraction
  4. De-Boc protection --> exposes NH2 at the end of the OEG
  5. Liquid phase conjugated OEG end - NH2 + mPEG-SPA (or similar activated PEG) --> ligated PEG
  6. Global deprotection with resin cleavage (TFA, etc.) --> crude peptides
  7. Reversed-phase preparation of HPLC purification -> high-purity semaglutide
  8. Freeze drying

It should be emphasized that the specific synthesis route details, the types of protective groups used, the selection of conjugation reagents, and the purification conditions are all highly confidential intellectual property rights of Novo Nordisk. The above is a general description of the principles of chemical modification of peptide drugs based on the comprehensive published literature. The actual operation is extremely complex and requires a high degree of expertise and precise equipment control.

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标签: SPPS

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