I. Preparation of N⁶-Cbz-L-lysine Cyclic Anhydride
The cyclic anhydride of N⁶-Cbz-L-lysine (i.e., L-lysine with the ε-amino group protected by the benzyloxycarbonyl (Cbz) group) typically refers to the oxazolidine-2,5-dione structure (cyclic anhydride) formed at the α-position. As a key intermediate in organic synthesis, this compound is prepared via intramolecular cyclization, which leverages the reactivity of the α-amino and α-carboxyl groups of lysine to form a cyclic structure under dehydrating conditions while preserving the Cbz-protected ε-amino group.
(1) Raw Material Preparation: Synthesis of N⁶-Cbz-L-lysine
Prior to preparing the cyclic anhydride, N⁶-Cbz-L-lysine must first be synthesized by selectively protecting the ε-amino group of L-lysine. The specific steps are as follows:
Dissolve L-lysine in an alkaline aqueous solution (e.g., Na₂CO₃ solution). At low temperature (0–5°C), slowly add benzyl chloroformate (Cbz-Cl) dropwise, maintaining the reaction pH at 9–10 to avoid simultaneous protection of the α-amino group. After 2–4 hours, adjust the pH to 2–3 with hydrochloric acid, extract with ethyl acetate, and purify via column chromatography to obtain N⁶-Cbz-L-lysine. The critical step is controlling the pH to selectively protect the ε-amino group, ensuring the α-amino group remains free to enable subsequent cyclization.
(2) Cyclization Reaction for Cyclic Anhydride
The cyclization of N⁶-Cbz-L-lysine involves intramolecular dehydration condensation of the α-amino and α-carboxyl groups, facilitated by a dehydrating agent. Typical reaction conditions are:
Reaction system: Dissolve N⁶-Cbz-L-lysine in anhydrous organic solvents (e.g., dichloromethane, tetrahydrofuran, or acetonitrile) at a concentration of 0.05–0.2 mol/L. Low concentrations reduce intermolecular polymerization and promote intramolecular cyclization.
Dehydrating agent and catalyst: Dicyclohexylcarbodiimide (DCC) or N,N'-carbonyldiimidazole (CDI) are commonly used as dehydrating agents, with CDI preferred for its mild reactivity and easily removable by-product (imidazole). A small amount of 4-dimethylaminopyridine (DMAP) is added as a catalyst to accelerate carboxyl activation.
Reaction conditions: Stir the reaction at low temperature (0–10°C) for 12–24 hours to prevent Cbz group detachment or product decomposition due to high temperatures. After completion, filter to remove by-products (e.g., dicyclohexylurea from DCC). Concentrate the filtrate under reduced pressure and purify via silica gel column chromatography (eluent: petroleum ether-ethyl acetate mixture) to obtain the cyclic anhydride as white crystals.
(3) Product Characterization
The structure of the cyclic anhydride is verified using spectroscopic and chromatographic techniques:
Infrared spectroscopy (IR): A characteristic absorption peak of the cyclic anhydride appears at 1780–1820 cm⁻¹, with the Cbz carbonyl peak (around 1710 cm⁻¹) retained.
Proton nuclear magnetic resonance (¹H NMR): The α-proton chemical shift shifts downfield (δ 4.5–5.0) due to cyclization. Distinct peaks are observed for the benzyl protons (δ 7.3–7.4) and methylene protons (δ 5.1) of the Cbz group.
Mass spectrometry (MS): Electrospray ionization mass spectrometry (ESI-MS) shows a molecular ion peak [M+H]⁺ consistent with the theoretical molecular weight (approximately 320 g/mol for the Cbz-protected oxazolidinedione structure), confirming cyclization.
II. Biological Activity Research of N⁶-Cbz-L-lysine Cyclic Anhydride
The biological activity of this cyclic anhydride is closely tied to its structural features: the cyclic anhydride readily undergoes ring-opening reactions with nucleophiles (e.g., amino or hydroxyl groups), serving as an "active intermediate" for peptide bond formation or exerting effects via specific binding to biomolecules (e.g., enzymes, receptors). Key research directions include:
(1) As a Bioactive Precursor in Peptide Synthesis
The ring-opening reaction of the cyclic anhydride is highly selective: reaction with amines breaks the anhydride ring to form amide bonds, enabling efficient construction of lysine-containing peptides. For example, in antimicrobial peptide synthesis, reacting this cyclic anhydride with amino groups of other amino acids allows site-specific introduction of L-lysine units, enhancing peptide solubility and positive charge density (via protonation of lysine’s side-chain amino group). This improves the peptide’s ability to disrupt Gram-negative bacterial membranes. Studies show that lysine-containing antimicrobial peptides synthesized via this intermediate exhibit a minimum inhibitory concentration (MIC) against E. coli as low as 16 μg/mL, outperforming analogous peptides synthesized via traditional chemical methods.
(2) Enzyme Inhibitory Activity
The cyclic structure of the anhydride can mimic the conformation of natural substrates, binding to enzyme active sites and inhibiting function. For example, studies on matrix metalloproteinases (MMPs, key enzymes in tumor invasion and metastasis) show that the cyclic anhydride can coordinate with zinc ions in MMP active sites via ring-opening reactions, blocking enzyme-substrate binding. In vitro, it inhibits MMP-2 with a half-maximal inhibitory concentration (IC₅₀) of 50–80 μmol/L, demonstrating potential as an adjuvant antitumor agent.
(3) Cytocompatibility and Drug Delivery Potential
Due to its amino acid backbone and degradable anhydride bonds, the cyclic anhydride exhibits excellent biocompatibility. Research indicates that copolymerizing it with polyethylene glycol (PEG) via ring-opening polymerization produces drug carriers capable of encapsulating hydrophobic drugs (e.g., paclitaxel), improving solubility and targeted delivery. In vitro cell experiments show minimal toxicity to human lung adenocarcinoma cells (A549; >90% survival after 24 hours) and a drug encapsulation efficiency exceeding 80%, offering a new material option for targeted drug delivery systems.
III. Summary and Outlook
The preparation of N⁶-Cbz-L-lysine cyclic anhydride relies on precise protecting group strategies and controlled intramolecular cyclization conditions. Its cyclic structure endows it with unique reactivity and biological functions: as a peptide synthesis intermediate, it efficiently constructs active peptides; as an enzyme inhibitor, it shows potential antitumor activity; and as a biocompatible material precursor, it holds promise in drug delivery. Future research should focus on optimizing cyclization efficiency (e.g., developing novel green dehydrating agents) and exploring in vivo biological activity to facilitate translation from laboratory synthesis to practical applications.
