N⁶-Cbz-L-lysine is a benzyloxycarbonyl-protected product of the amino group of lysine, appearing as a white to off-white solid powder. The following is an introduction to its preparation method and structural characterization techniques:
I. Preparation Method
Raw Materials and Principle: It is usually synthesized starting from lysine. Utilizing the nucleophilicity of the amino group in the lysine molecule, a reaction with benzyl chloroformate is carried out to introduce the benzyloxycarbonyl (Cbz) group onto the N⁶-position amino group of lysine, thereby obtaining N⁶-Cbz-L-lysine.
Specific Steps: Add a dichloromethane solution of lysine to a clean, dry round-bottom flask and cool to -5–5°C. Add an appropriate amount of triethylamine and stir for half an hour. Triethylamine acts as a base to neutralize the hydrogen chloride generated during the reaction, promoting the reaction progress. Dropwise add a prepared dichloromethane solution of benzyl chloroformate into the reaction system, maintain the temperature at -5–5°C, and stir for 40 minutes. After the reaction is complete, add 3N HCl solution dropwise, control the reaction temperature at 0–10°C, stir for half an hour, and then let stand to separate the layers. Wash the organic phase with water and evaporate dichloromethane under reduced pressure. Add petroleum ether, stir, and cool to 0°C; N⁶-Cbz-L-lysine will precipitate. Continue stirring for 40 minutes, then filter, wash the filter cake with petroleum ether, and dry under vacuum to obtain white N⁶-Cbz-L-lysine solid, with a typical yield of 60–80%.
II. Structural Characterization
Infrared Spectroscopy (IR): In the infrared spectrum of N⁶-Cbz-L-lysine, characteristic absorption peaks of the amino group appear around 3300 cm⁻¹, presenting as a double peak (primary amine). Due to the presence of a carboxyl group, a strong absorption peak of the carbonyl group is observed at 1700–1750 cm⁻¹, and a broad peak of the hydroxyl group in the carboxyl group appears at 3400–3300 cm⁻¹. Additionally, characteristic absorption peaks of the benzene ring can be observed around 1600 cm⁻¹ and in the fingerprint region below, which is used to verify the benzene ring structure in the benzyloxycarbonyl group.
Proton Nuclear Magnetic Resonance (¹H NMR): ¹H NMR can identify hydrogen atoms in different chemical environments within the molecule. For example, hydrogen atoms on the benzene ring of the benzyloxycarbonyl group show characteristic peaks at a chemical shift of δ = 7.2–7.5 ppm. Hydrogen atoms on the lysine main chain, as well as those on the amino and carboxyl groups, also appear at corresponding chemical shifts. The molecular structure can be inferred by analyzing the positions, integral areas, and splitting patterns of these peaks.
Carbon-13 Nuclear Magnetic Resonance (¹³C NMR): ¹³C NMR is used to determine the chemical environment of carbon atoms in the molecule. Carbon atoms in the benzyloxycarbonyl group, those on the lysine main chain, and others in N⁶-Cbz-L-lysine exhibit peaks at different chemical shifts. For instance, carbonyl carbons generally appear around δ = 160–180 ppm, and benzene ring carbons around δ = 110–160 ppm. The molecular structure can be verified by comparing with standard spectra or analyzing according to chemical shift rules.
Mass Spectrometry (MS): Mass spectrometry provides information about the molecular weight of the compound. The molecular ion peak of N⁶-Cbz-L-lysine appears in the mass spectrum; by comparing with the theoretical molecular weight, the molecular formula of the compound can be preliminarily confirmed. Meanwhile, the structural fragments of the molecule and the cleavage patterns of chemical bonds can be further inferred from its fragment ion peaks, assisting in structural identification.
