Synthetic peptides are a kind of special drug, which can be regarded as a kind of drug between small organic molecules and protein macromolecules. Liquid phase synthesis and solid phase synthesis are the main methods of peptide drug synthesis. Compared with the classical liquid phase synthesis of peptides, solid phase peptide synthesis has become a conventional method of peptide synthesis and extended to other organic fields such as nucleotide synthesis because of its outstanding advantages of time-saving, labor-saving, and material-saving.

The birth of solid-phase peptide synthesis

The research of peptide synthesis has gone through a brilliant course of more than one hundred years. Emil Fischer, a German chemist who won the Nobel Prize in chemistry for successfully solving the structure of sugar and his research achievements in purine derivatives and peptides in 1902, first began to pay attention to peptide synthesis. Because the knowledge of peptide synthesis was too little at that time, the progress was quite slow. It was not until 1932 that peptide synthesis began to have a certain development.

In the 1950s, organic chemists synthesized a large number of bioactive peptides, including oxytocin and insulin, and made a lot of achievements in peptide synthesis and amino acid protective groups. This provides an experimental and theoretical basis for the emergence of solid-phase synthesis methods.

In 1963, Merrifield first proposed solid-phase peptide synthesis (SPPS), which is a milestone in peptide chemistry. As soon as it appeared, it became the preferred method of peptide synthesis because of its convenience and rapidness. It also brought a revolution in peptide organic synthesis and became an independent subject-solid phase organic synthesis (SPOS). For this reason, Merrifield won the Nobel Prize in chemistry in 1984. Merrifield invented the first peptide synthesizer in the late 1960s and synthesized biological protease, ribonuclease (124 amino acids) for the first time.   

Detection of solid-phase synthetic peptides

Even the efficient coupling technology cannot guarantee that the acylation reaction can be carried out at 100%. Moreover, the efficiency of the coupling reaction is greatly reduced when the sequences such as stereo barrier or bilayer are encountered. There are always missing or truncated peptide chains on the polymer carrier, and when they are released, they also enter into the product, which brings great difficulties to separation. Therefore, when solid-phase synthesis of peptides, especially longer peptides, the condensation rate of each amino acid should reach 99.9%, otherwise the product will be very impure. Therefore, it is particularly important to monitor the progress of each reaction.

• Qualitative color reaction

Ninhydrin chromogenic method (Kaiser method) is a rapid determination of amino groups on the resin by ninhydrin color reaction, to determine whether the acylation reaction is complete. The sensitivity of the ninhydrin method for the determination of amino groups of polystyrene resin can reach 5μmol/g. This sensitivity can detect whether the condensation reaction has been carried out by more than 99%. In the detection of ninhydrin, the color intensity is different due to the difference in terminal amino acid residues and sequences. Aspartic acid (Asp) and asparagine (Asn) produce very weak blue or light brown. The reaction of the chromogenic reagent 2, 4, 4, 6-trinitrobenzenesulfonic acid with the amino group on the resin showed orange-red, and the sensitivity was 5μmol/g resin.

• Quantitative free amino group detection

The salicylaldehyde method can be used to determine the amount of residual amino groups on the resin after receiving peptides and the total amino content after the removal of protective groups, which can quantitatively detect whether the condensation reaction and the removal of protective groups are complete. If not completely, it can be dealt with repeatedly in time. The amino group on the resin was reacted with 2% salicylaldehyde + 6% pyridine ethanol solution (60℃, 30min). After washing, the salicylaldehyde was replaced by a 5% benzylamine ethanol solution (60℃, 30 min). After the ethanol solution with benzylamine was diluted, the light absorption value of 315nm was read, and the amount of amino group was calculated.

• HPLC detection

The partially protected intermediate peptides were in the middle of peptide synthesis. A small amount of peptide resin (3~10mg) was cleaved, precipitated with ether, dissolved in appropriate solvents, and directly analyzed by HPLC. During cleavage, the N-terminal protective group of the peptide was retained or removed as needed. When the synthesized peptide is a short polar peptide, the protective group can be retained, otherwise, the retention time in HPLC is too short, which is not conducive to analysis. Although this method is troublesome, it takes a short time and has high accuracy.

Advantage of solid phase peptide synthesis

Solid phase synthesis has obvious advantages for peptide synthesis: it simplifies and accelerates multi-step synthesis, and because the reaction can be carried out in a simple reaction vessel, the loss caused by manual operation and repeated material transfer can be avoided.

The covalently linked peptide chain of the solid phase carrier is in a suitable physical state, and the intermediate purification can be quickly filtered and washed to avoid the lengthy recrystallization or column separation steps in the synthesis of liquid phase peptides. A large number of losses in the separation and purification of intermediates can be avoided.

Excessive reactants are used to force individual reactions to be complete so that the final product can get a high yield.

Increase solvation and reduce the focus of intermediate products; the peptide chain on the solid phase carrier and the slightly cross-linked polymer chain are closely mixed, resulting in a mutual solvent effect, which is disadvantageous to the thermodynamic of peptide self-aggregation but suitable for the reaction.

Conclusion

Due to the increasing demand for peptide products in the market, especially in medical and health, the peptide synthesis industry is developing rapidly. With the continuous improvement of peptide synthesis technology and instruments, rare amino acids can be introduced into the solid-phase synthesis of peptides to study the structure and function of new proteins. Solid-phase synthesis of peptides has incomparable advantages in drug research, protein structure research, immunology research, and so on.

References

1. Amblard, M., Fehrentz, J. A., Martinez, J., & Subra, G. (2006). Methods and protocols of modern solid phase peptide synthesis. Molecular biotechnology, 33(3), 239-254.
2. Behrendt, R., White, P., & Offer, J. (2016). Advances in Fmoc solid‐phase peptide synthesis. Journal of Peptide Science, 22(1), 4-27.
3. Varnava, K. G., & Sarojini, V. (2019). Making solid‐phase peptide synthesis greener: a review of the literature. Chemistry–An Asian Journal, 14(8), 1088-1097.
4. Palomo, J. M. (2014). Solid-phase peptide synthesis: an overview focused on the preparation of biologically relevant peptides. RSC Advances, 4(62), 32658-32672.