Nuclear Magnetic Resonance (NMR) spectroscopy is a core technique for identifying the chemical structure of N⁶-Cbz-L-lysine. By analyzing differences in the magnetic environments of various atomic nuclei in the molecule, it provides rich information on chemical bond connections, spatial configurations, and functional groups. Its specific applications are as follows:
I. Application of Proton Nuclear Magnetic Resonance (¹H NMR)
¹H NMR can parse the types, quantities, and adjacent group connections of hydrogen atoms in the molecule through parameters such as chemical shifts, integral areas, and coupling constants. In N⁶-Cbz-L-lysine, hydrogen atoms at different positions exhibit characteristic signals:
The benzene ring hydrogens in the benzyloxycarbonyl (Cbz) group typically appear as a set of multiplets at δ7.3-7.4 ppm. Their integral area corresponds to 5 hydrogen atoms on the benzene ring, serving as direct evidence for the presence of the Cbz group.
The methylene hydrogens in the ester group (-O-CH₂-) (the -CH₂- linked to the benzene ring) generally show a chemical shift around δ5.1 ppm (as a singlet or doublet) due to the magnetic anisotropy of the benzene ring, with weak coupling to the benzene ring hydrogens.
The amino (-NH-) hydrogens in the lysine main chain have a chemical shift highly dependent on solvent and temperature, usually appearing as a broad peak in the δ6.0-8.0 ppm range.
Methylene (-CH₂-) and methine (-CH-) hydrogens in the side chain and main chain exhibit chemical shifts distributed between δ1.2-4.0 ppm due to varying chemical environments. Coupling splitting (e.g., triplets, quintets) can infer the carbon chain connectivity. For example, the methine hydrogen adjacent to the carboxyl group (-CH-COOH) shows a relatively high chemical shift (δ3.5-4.0 ppm) due to the electron-withdrawing effect of the carboxyl group, while the methylene hydrogen at the end of the side chain linked to Cbz (-CH₂-NH-Cbz) appears at δ3.0-3.5 ppm due to the influence of the amino group.
By calculating integral areas, the quantity ratio of each type of hydrogen atom can be verified against the molecular formula of N⁶-Cbz-L-lysine (e.g., total methylene hydrogens in the main chain and side chain, number of benzene ring hydrogens), confirming the basic molecular skeleton.
II. Application of Carbon-13 Nuclear Magnetic Resonance (¹³C NMR)
¹³C NMR primarily distinguishes carbon atoms in different chemical environments via chemical shifts, clarifying the types of functional groups and carbon chain structures. In N⁶-Cbz-L-lysine:
The benzene ring carbon signals of the Cbz group typically appear at δ128-137 ppm. The methylene carbon linked to the oxygen atom (-O-CH₂-) shows a chemical shift of δ67-68 ppm due to the electronegativity of oxygen.
Carbonyl carbons (-COO- and -COOH) exhibit significantly different chemical shifts: the ester carbonyl in Cbz (-COO-) appears at δ155-157 ppm due to conjugation with oxygen, while the carboxyl (-COOH) carbonyl carbon appears at δ170-175 ppm.
Saturated carbons (-CH₂-, -CH-) in the lysine main chain and side chain show chemical shifts varying with the electronegativity of substituents. For example, the methylene carbon linked to the amino group (-CH₂-NH-) appears at δ40-45 ppm due to the electron-withdrawing effect of the amino group, while methylene carbons distant from functional groups appear at δ20-30 ppm.
By comparing the number and chemical shifts of carbon signals in the ¹³C NMR spectrum, the integrity of the carbon skeleton and correct connectivity of functional groups can be confirmed—for instance, verifying that the Cbz group is attached only to the ε-amino group (N⁶ position) of lysine rather than the α-amino group, thus excluding isomeric impurities.
III. Application of Two-Dimensional Nuclear Magnetic Resonance (2D NMR)
2D spectra further clarify spatial connectivity between atoms, resolving signal overlap in 1D spectra:
¹H-¹H Correlation Spectroscopy (COSY) reveals coupling between adjacent hydrogen atoms, constructing a network of hydrogen connections in the carbon chain. For example, coupling signals between -CH- and adjacent -CH₂- in the lysine main chain clearly indicate the continuous structure of the carbon chain.
Heteronuclear Single Quantum Coherence (HSQC) correlates each carbon atom with its directly bonded hydrogen atoms, confirming the hydrogen environment corresponding to each carbon signal—e.g., distinguishing the carbon-hydrogen correspondence of -O-CH₂- in the Cbz group.
Heteronuclear Multiple Bond Correlation (HMBC) observes long-range (2-3 bonds) carbon-hydrogen correlations, such as between the ester carbonyl carbon of Cbz and benzene ring hydrogens, or between the methylene carbon linked to the side chain amino group and the methine hydrogen in the main chain. This verifies the connection site of functional groups with the main chain, confirming the correctness of N⁶ substitution.
IV. Verification of Chiral Configuration
The chiral center (C2 position) of L-lysine can be confirmed using ¹H NMR combined with chiral shift reagents or spatial effect analysis in 2D spectra. For example, the chemical shift and coupling pattern of the methine hydrogen linked to the chiral carbon (-CH-) are influenced by the spatial configuration of surrounding groups, differing from those in the D-isomer. Combined with characteristic chiral signals of amino acids, this verifies that the product is in the L configuration.
Through multi-dimensional spectral analysis—from the chemical environments of hydrogen and carbon atoms to atomic connectivity and chiral configuration—NMR spectroscopy comprehensively verifies the molecular structure of N⁶-Cbz-L-lysine, ensuring the accuracy of functional group substitution sites, carbon chain skeletons, and stereoconfiguration. It remains the authoritative method for confirming the structure of synthetic products.
