Synlett 2020; 31(08): 784-787
DOI: 10.1055/s-0039-1691735
letter
© Georg Thieme Verlag Stuttgart · New York

Synthesis of Tryptophan–Folate Conjugates

Katsuya Maruyama
,
Katarzyna Joanna Malawska
,
Natsuki Konoue
,
,
Motomu Kanai
This work was supported in part by JSPS KAKENHI Grant Number JP17H01522 (for M.K.); JSPS KAKENHI Grant Number JP18H04239 (Precisely Designed Catalysts with Customized Scaffolding), JP18K06545 (Scientific Research C); research grants from the Noguchi Institute and the Kobayashi Foundation for Cancer Research (for K.O.); and a MEXT Scholarship (for K.J.M.).
Further Information

Publication History

Received: 24 January 2020

Accepted after revision: 04 February 2020

Publication Date:
26 February 2020 (online)


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.

Supporting Information

 
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  • 20 Trp-Targeted Conjugation of an ABNO-PEGFolate 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-PEGFolate 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.