Peptides are biomolecules composed of multiple amino acids connected by amide bonds (peptide bonds). They are the basic building blocks of proteins and the core structures of numerous drugs, hormones, antibacterial agents, and signaling molecules. With the development of biomedicine, new materials, and chemical biology, artificial synthesis of peptides has become an important technology in both scientific research and industrial fields. This article will systematically introduce the basic principles, mainstream methods, key steps, and applications of peptide synthesis.

1、 What is peptide synthesis?

Peptide synthesis refers to the process of chemically connecting amino acids one by one according to a predetermined amino acid sequence under laboratory or industrial conditions, forming peptide chains of specific length and structure. According to the different reaction systems, it can be divided into liquid-phase synthesis and solid-phase synthesis. Among them, Solid Phase Peptide Synthesis (SPPS) was studied by American chemist R. Bruce Merrifield in 1963 and won the Nobel Prize in Chemistry in 1984. It has now become a mainstream technology.

2、 Basic principles of solid-phase peptide synthesis

The core idea of SPPS is to covalently connect the C-terminus (carboxyl terminus) of a polypeptide chain to an insoluble polymer resin, and then add amino acids one by one starting from the N-terminus (amino terminus). Each amino acid added completes a cycle of "deprotection coupling washing". The entire process is carried out on a solid-phase carrier, and unreacted reagents and by-products can be removed through simple filtration and washing, greatly simplifying the purification steps.

3、 Key steps in peptide synthesis

1. Resin selection and fixation of first amino acid

The synthesis begins with the selection of suitable solid-phase carriers (such as cross-linked polystyrene, PEG-PS resin, etc.) with reactive functional groups on their surfaces (such as hydroxyl groups in Wang resin and amino groups in Rink Amide resin). The carboxyl group of the first amino acid is connected to the resin through ester or amide bonds, and its alpha amino group needs to be protected in advance.

2. Protection and deprotection of amino acids

To prevent side reactions, the amino acids involved in the reaction must be selectively protected:

Alpha amino: commonly protected with Fmoc (9-fluorenylmethoxycarbonyl) or Boc (tert butoxycarbonyl);

Side chain active groups (such as Lys's ε - amino group, Cys' thiol group, Asp/Glu's carboxyl group, etc.): acid sensitive protecting groups (such as tBu, Trt, etc.) are used;

Carboxyl group: It is usually not protected in solid-phase synthesis as it has been used for coupling.

Before each round of coupling, the protective group at the N-terminus of the polypeptide chain on the current resin needs to be removed, exposing the free amino group for the next reaction step.

3. Peptide formation reaction (coupling)

Activate the carboxyl group of the next protected amino acid (commonly used condensation systems such as HBTU/HOBt/DIPEA, PyBOP, DIC/Oxyma, etc.) and react with the free amino group of the peptide on the resin to form a new peptide bond. The reaction should be efficient and rapid, avoiding racemization or the formation of by-products.

4. Cracking and deprotection of side chains

After the synthesis of the target sequence is completed, a strong acid reagent (such as TFA) is needed to cleave the peptide from the resin and remove all side chain protecting groups. Fmoc strategy commonly uses TFA/water/scavenger (such as TIS) mixture; The Boc strategy requires more stringent conditions (such as HF or TFMSA).

5. Purification and identification

The crude peptide obtained from cracking contains missing sequences, by-products, and impurities, which need to be purified by reverse phase high performance liquid chromatography (RP-HPLC). Subsequently, molecular weight was confirmed using mass spectrometry (MS), purity was evaluated using analytical HPLC, and amino acid composition analysis or circular dichroism (CD) structural characterization was performed if necessary.

4、 Two mainstream synthesis strategies: Fmoc vs Boc

project	Fmoclaw	Boclaw
α -Amino protecting group	Fmoc(Alkali removal)	Boc(Acid removal)
Deprotection reagent	20%piperidine/DMF	TFA
Side chain protection base	Acid sensitive type (such astBu）	Strong acid stable type (such asBzl）
Cracking conditions	TFAaccomplish in one step	needHFOr strong acid, dangerous operation
Safety and popularity	High, mainstream laboratory	Low, special sequence usage

At present, the Fmoc/tBu strategy has become the ideal choice for the synthesis of the vast majority of peptides due to its safe operation, mild conditions, and strong compatibility.

5、 Application of Peptide Synthesis

Drug development: such as insulin analogues, GLP-1 receptor agonists (semaglutide), antimicrobial peptides, etc;

Vaccine development: synthesis of antigen epitope peptides for immunological research;

Basic scientific research: preparation of labeled peptides, enzyme substrates, receptor ligands, etc;

Cosmetics and health products: such as signal peptides (promoting collagen production), glutathione, etc;

Materials Science: Self assembling peptide nanomaterials, biosensors, etc.

6、 Challenges and Future Directions

Although peptide synthesis technology is quite mature, it still faces challenges such as low efficiency in long peptide synthesis, racemization risk, and difficulty in dissolving hydrophobic peptide aggregates. Current research hotspots include:

Automation and high-throughput synthesis platform;

Microwave assisted accelerated coupling;

Synthesis of ultra long peptides or proteins using Native Chemical Ligation method;

Replace DMF with green solvents;

Artificial intelligence assisted sequence design and synthesis optimization.

Peptide synthesis is a precision engineering that combines organic chemistry, polymer science, and biotechnology. From Merrifield's pioneering work to today's automated synthesis platform, this technology has not only driven the development of life sciences, but also demonstrated enormous potential in the field of medicine and health.