Unnatural Amino Acids for Peptide Synthesis

Unnatural amino acids are amino acids that do not exist in nature or are not produced by natural synthetic pathways. These amino acids can be prepared by chemical synthesis or enzymatic reactions, and they have more structural diversity and functionality than natural amino acids. Unnatural amino acids have a wide range of applications in peptide synthesis. By introducing these special amino acids, scientists can regulate the physical, chemical and biological properties of peptides. Specific applications include enhancing the stability of peptides, increasing their functions and specificity, and improving the pharmacokinetic properties of drugs. For example, peptides containing unnatural amino acids can better resist degradation by enzymes in the body and provide longer biological activity; they can also achieve specific labeling and identification of biological molecules by introducing fluorescent groups or targeting molecules. These applications have greatly expanded the potential of peptides in drug development, diagnostic tools and biomaterials.

What are Peptides?

Peptide is a compound formed by α-amino acids connected together by peptide bonds. It is also an intermediate product of protein hydrolysis. Compounds usually formed by dehydration and condensation of 10 to 100 amino acid molecules are called peptides. Peptides are widely present in organisms, and most of them have certain physiological activities. At present, peptides are mainly used in peptide functional foods, dietary supplements, cosmetics, peptide drugs, peptide drug carriers, etc. Humans have used peptides as drugs for more than 70 years. There are nearly 100 peptide drugs on the market worldwide, mainly focusing on the treatment of chronic diseases such as diabetes, anti-tumor, immunomodulation, and cardiovascular diseases.

Peptides Benefits

The molecular size of peptide drugs is between small molecule drugs and protein drugs, which cleverly fills the gap between small molecule drugs and protein drugs and forms its unique ecological niche. Compared with small molecule chemical drugs, peptide drugs have the advantages of safety, high efficiency, high selectivity, and not easy to accumulate in the body. The disadvantages are also very obvious. The chemical properties of peptide drugs are unstable, prone to oxidation and hydrolysis, short half-life, fast metabolic rate, poor cell membrane penetration, and cannot be taken orally. Therefore, as a special chemical reagent that plays an important role in constructing amide bonds in the synthesis of peptide drugs and small molecule chemical drugs, the development of the peptide synthesis reagent industry is closely related to the development of downstream peptide drugs and small molecule chemical drugs.

Peptide Synthesis

Peptide synthesis refers to the process of connecting amino acids in a certain order to form a peptide chain. It is one of the important technologies in the fields of biochemistry, organic chemistry and biotechnology, and is often used to synthesize proteins and peptide compounds. The basic principle of peptide synthesis is to condense the carboxyl group and amino group of amino acids by chemical means to form a peptide bond. This reaction is usually called a peptide bond formation reaction or a peptide bond synthesis reaction. The formation of peptide bonds is achieved by the formation of amide bonds between carboxyl groups and amino groups.

• Enzymatic Synthesis of Peptides

Enzymatic hydrolysis is the use of biological enzymes to degrade plant proteins and animal proteins to obtain small molecule peptides. Enzymatic hydrolysis has not been able to achieve industrial production due to its low peptide yield, large investment, long cycle and serious pollution. The peptides obtained by enzymatic hydrolysis can retain the original nutritional value of protein and can obtain more functions than the original protein, which is greener and healthier.

• Genetic Engineering Synthesis of Peptides

Genetic engineering is mainly based on DNA recombination technology, and controls the sequence synthesis of peptides through appropriate DNA templates. Some researchers have obtained quasi-elastin-polyvaline-proline-glycine-valine-glycine peptide (VPGVG) through genetic engineering. Active peptides produced by genetic engineering technology include peptide antibiotics, interferons, interleukins, growth factors, tumor necrosis factor, human growth hormone, blood coagulation factors, erythropoietin, tissue non-protein plasminogen, etc. The genetic engineering method for synthesizing peptides has the advantages of strong expression orientation, safety and hygiene, wide source of raw materials and low cost, but it is difficult to achieve large-scale production due to the problems of efficient expression, difficulty in separation and low yield.

• Fermentation of Peptides

The fermentation method is a method for obtaining peptides from microbial metabolites. Although the fermentation method is low-cost, its application range is relatively narrow, because the only polyamino acids that microorganisms can independently synthesize are ε-polylysine (ε-PL), γ-polyglutamic acid (γ-PGA) and cyanobacterial peptides.

• Chemical Synthesis of Peptides

Chemical synthesis is mainly divided into liquid phase synthesis and solid phase synthesis. Liquid phase synthesis is to condense amino acids and peptides in a solution to form peptide bonds, followed by purification. It is mainly used in the synthesis of oligopeptides. The disadvantage is that each step requires purification and the synthesis efficiency is low. In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS), which is to link amino acids to a solid phase carrier. After the amino acids are linked through a condensation reaction, the product is purified by rapid filtration and washing. Because solid phase synthesis is convenient and rapid, it is currently the preferred and main method for peptide synthesis.

Solid Phase Peptide Synthesis

The basic principle of solid phase peptide synthesis is to first connect the hydroxyl group of the hydroxyl terminal amino acid of the peptide chain to be synthesized with an insoluble polymer resin by a covalent bond structure. Then, the amino acid bound to the solid phase carrier is used as the amino component, and the amino protecting group is removed and reacted with an excess of activated carboxyl components to extend the peptide chain. Then repeat the operation (condensation → washing → deprotection → neutralization and washing → next round of condensation) to achieve the desired peptide chain length, and finally cleave the peptide chain from the resin, and after purification and other treatments, the desired peptide is obtained. Among them, the α-amino group is protected by Boc (tert-butyloxycarbonyl) and is called the Boc solid phase synthesis method, and the α-amino group is protected by Fmoc (9-fluorenylmethyloxycarbonyl) and is called the Fmoc solid phase synthesis method.

• Boc Solid Phase Synthesis

Boc peptide synthesis is a classic peptide solid phase synthesis method, using Boc as the protecting group of amino acid α-amino group and benzyl alcohol as the protecting group of side chain. When Boc peptide solid phase synthesis is performed by trifluoroacetic acid, the N-α-amino acid protected by Boc is covalently cross-linked to the resin, TFA removes the Boc protecting group, and the N-terminus is neutralized with a weak base. The peptide chain is extended by dicyclohexylcarbodiimide activation and coupling, and finally the synthesized target peptide is dissociated from the resin by strong acid hydrofluoric acid (HF) method or trifluoromethanesulfonic acid (TFMSA).

* Boc-Amino Acids:

• Fmoc Solid Phase Synthesis

The Fmoc peptide synthesis method uses Fmoc as the protecting group of the α-amino group of amino acids. Its advantage is that it is stable under acidic conditions, is not affected by reagents such as TFA, and can be deprotected by mild alkali treatment, so the side chain can be protected with a Boc protecting group that is easily removed by acid. The reaction conditions are mild, the side reactions are few, the yield is high, and the Fmoc group itself has characteristic ultraviolet absorption, which makes it easy to monitor and control the progress of the reaction.

* Fmoc-Amino Acids:

What are Peptides Used For?

The application of unnatural amino acids in peptide therapy has brought exciting prospects for drug development. Compared with natural amino acids, unnatural amino acids have unique chemical structures that provide enhanced stability, specificity and functional diversity. Their introduction into peptide chains can improve the resistance of peptide drugs to enzymatic hydrolysis, thereby extending the half-life and enhancing in vivo stability. In addition, unnatural amino acids can also affect the conformation of peptides, and also enhance their specific binding to target molecules and improve therapeutic effects. By optimizing the structure and physicochemical properties of peptide chains, scientists can design more effective drugs with fewer side effects.

• Drug Development

Unnatural amino acids have great potential in drug development. By introducing unnatural amino acids, new biological activities can be given to peptides and protein drugs, their stability can be improved, their half-life can be increased, and their pharmacokinetic properties can be improved. For example, certain unnatural amino acids such as fluorinated amino acids and sulfur-containing amino acids can significantly improve the enzymatic stability of peptides, thereby extending the half-life of drugs in the body and improving their efficacy. In addition, cyclic peptide drugs can enhance their specificity and binding ability by introducing unnatural amino acids on the ring, which has important applications in the development of anti-tumor drugs and antiviral drugs.

• Materials Sciences

Unnatural amino acids are also widely used in materials science, especially in the development of new functional materials. The introduction of specific unnatural amino acids in the synthesis of peptide materials can regulate the physical and chemical properties of the materials. For example, by introducing unnatural amino acids with UV absorption properties into peptide materials, materials with UV light shielding functions can be developed. In addition, some unnatural amino acids can form cross-links through substrate reactions, thereby enhancing the mechanical strength and stability of the material.

• Biotechnology Applications

In biotechnology, the use of unnatural amino acids can increase the diversity and function of biomolecules. For example, new biomolecular sensors or separation media can be developed by introducing unnatural amino acids that can specifically bind to specific ligands or other small molecules into proteins. In addition, peptide active factors based on unnatural amino acids can play a key role in cell culture and regenerative medicine.

• Synthetic Biology

The goal of synthetic biology is to design and construct new biological systems or functions. By integrating unnatural amino acids into the gene coding system, the genetic code of life can be expanded, thereby creating biological systems with new functions. For example, scientists have successfully incorporated some unnatural amino acids into the protein synthesis system of Escherichia coli and yeast, which provides a new path for the development of new functional proteins.

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