Cyclic peptides are more stable cyclic peptides with various physiological functions and medicinal values than linear peptides. Since the discovery of the first antibacterial cyclic peptide GramicidinS in the 1940s, people have continuously isolated cyclic peptides with special structures from plants, fungi, bacteria and marine organisms. For example, melanotan 2 is a mature cyclic peptide that can be synthesized.

Analytical identification of the structures of cyclic peptides requires a combination of methods and a great deal of work and experience. Two commonly used analytical methods are described below: mass spectrometry and nuclear magnetic resonance.

Mass Spectrometry (MS)
Biological mass spectrometry is mainly used to solve two analytical problems: accurate determination of the molecular weight of biological macromolecules and provide molecular structure information; qualitative or quantitative analysis of trace or trace small molecular biologically active substances in complex systems of life. The sequence of cyclic peptides determined by mass spectrometry is deduced from the fragment ions of mass spectrometry, and the fragments of sequence information appearing in mass spectrometry are mainly formed by the cleavage of amide bonds. Making a "peptide map" is an important sequencing method. Continuous flow fast atom bombardment mass spectrometry (cf-FAB) and electrospray ionization mass spectrometry (EIS) are new methods developed in recent years. cf-FAB is often used in combination with HPLC, CEZ and other methods to achieve separation and analysis purposes. For example, H ideaki et al. used this method to study the series of tetrapeptide compounds of L-Pro and L-Ala, which proved that L-Pro can maintain the structural stability of small peptides. , is of great significance in terms of linking molecules. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) is currently the method for accurate determination of relative molecular mass in the identification of cyclic peptides.

Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance can be used to determine amino acid sequences, quantify the composition of each component in mixtures, etc., especially with the application of two-dimensional, three-dimensional and four-dimensional NMR, it has gradually become the main method for analyzing peptide substances. At present, the use of chemical shifts, cleavage constants, etc. to obtain structural information of cyclic peptides has become a routine test method. 2D-NMR technology can provide a variety of correlation information between various nuclei in cyclic peptide molecules, such as the spin coupling correlation between nuclei through chemical bonds, the space dipole coupling (NOE) correlation, and the same kind of nuclei. Coupling correlation between different nuclei, coupling correlation between heterogeneous nuclei, direct correlation and long-distance correlation between nuclei and nuclei. Based on this relevant information, the atoms in the cyclic peptide can be connected to each other through chemical bonds or spatial relationships. This not only greatly improves the separation ability of a large number of resonance signals, but also reduces the overlap between resonance signals, and can provide many structural information that 1D-NMR spectroscopy cannot provide, such as the finely divided morphology of each group of signals in the overlapping resonance signals. , the exact coupling constant, determine the sign of the coupling constant and distinguish between direct and remote coupling, etc. Therefore, nuclear magnetic resonance provides important information in the structural identification of cyclic peptides and is an indispensable and powerful tool.