Synthesis of Tryptophan–Folate Conjugates

Abstract

A mild protocol for the synthesis of folate–peptide/protein conjugates targeting tryptophan residues is described. This synthetic protocol is advantageous for homogeneous conjugation of chemically sensitive folates to biomolecules, with potential applications in cancer therapy and diagnostics.

Key words

bioconjugation - folate - tryptophan - proteins - peptides

• References and Notes

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• 20 Trp-Targeted Conjugation of an ABNO-PEG–Folate to a Peptide; Typical ProcedureLeuprorelin acetate (25.4 μg, 0.02 μmol, 2.54 μL of 10 mg/mL stock solution), ABNO-PEG₄-folate (34.0 μg, 0.04 μmol, 1.70 μL of 20 mg/mL stock solution), NaNO₂ (1.66 μg, 0.024 μmol, 1.66 μL of 1 mg/mL stock solution), and AcOH (0.02 μL, 2.00 μL of 0.01 μL/μL stock solution) were dissolved in H₂O (12.10 μL; final volume 20 μL), and the resulting mixture was stirred at rt for 90 min. The reaction was quenched by the addition of 20 mM phosphate-buffered saline (PBS; pH 7.4) and the mixture was analyzed by HPLC and LC/MS. The yield was calculated to be 41% from the HPLC peak area of the product at 254 nm. HPLC: [YMC-Triart C18 (4.6 × 150 mm); linear gradient B/A 0–100% (A = 0.1% aq TFA, B: MeCN) during 2–42 min at a flow rate of 1 mL/min]; t _R = 21.6 min. ESI-MS: m/z calcd [M + 2H]²⁺ for C₉₇H₁₃₅N₂₉O₂₂: 1029.5; found: 1029.6 Trp-Targeted Conjugation of an ABNO-PEG–Folate to a Protein; Typical ExampleLysozyme (358 μg, 0.02 μmol, 7.15 μL of 50 mg/mL stock solution), ABNO-PEG₄-folate (85.0 μg, 0.1 μmol, 4.25 μL of 20 mg/mL stock solution), NaNO₂ (4.14 μg, 0.06 μmol, 4.14 μL of 1 mg/mL stock solution), and AcOH (0.1 μL, 1.00 μL of 1 mg/mL stock solution) were dissolved in H₂O (3.46 μL, final volume 20 μL), and the resulting mixture was stirred at rt for 15 min. The mixture was then transferred into 20 mM PBS (pH 7.4, 200 μL) to quench the reaction. Small molecules were removed by ultrafiltration (Amicon Ultra, 10 K) (10×; 200 μL of PBS was added each time). The final volume was adjusted to ~ 250 μL, and the UV/Vis spectrum was recorded. The folate-to-protein ratio (N) was calculated to be 0.72 from the absorbances at 347 and 278 nm, based on the Beer–Lambert law. The solution (80 μL) was exchanged with H₂O by ultrafiltration (Amicon Ultra, 10 K), and the final volume was ~ 40 μL. The resulting solution was analyzed by LC-MS. ESI-Q-TOF MS: m/z [M]⁺ calcd for C₆₅₁H₁₀₀₈N₂₀₆O₁₉₅S₁₀: 15151.2; found: 15151.6.