Synthesis of Asparagusic Acid Modified Lysine and its Application in Solid-Phase Synthesis of Peptides with Enhanced Cellular Uptake

Published as part of the Special Section 9^th EuCheMS Organic Division Young Investigator Workshop

Abstract

Cyclic disulfides, such as asparagusic acid, enhance the uptake of a variety of cargoes into live cells. Here, we report a robust and scalable synthesis of an asparagusic acid modified lysine. This amino acid can be used in solid-phase peptide synthesis. We confirmed that incorporation of this building block into the sequence of a peptide increases its cellular uptake substantially.

Key words

asparagusic acid - modified amino acid - cellular uptake - solid-phase peptide synthesis - cell-penetrating peptides

• References and Notes

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• 16 N ²-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-N ⁶-(1,2-dithiolane-4-carbonyl)-l-lysine (Fmoc-Lys(AspA)-OH, 6) Crude NHS ester 5 (328 mg, 1.32 mmol) was dissolved in dioxane-PBS, 3:1 (28 mL) at room temperature (25 °C) and Fmoc-Lys-OH (538 mg, 1.46 mmol, 1.1 equiv) was added in one portion. After 4 h, the solution was acidified by slow addition of 1 M HCl (ca. 2 mL) and CH₂Cl₂ (ca. 25 mL) until phase separation occurred. The aqueous phase was extracted again with CH₂Cl₂ (2 × 25 mL). A white emulsion formed, which could be separated by centrifugation (3 min at 1750 × G). The combined organic phases were dried over MgSO₄, filtered, and concentrated under reduced pressure to give a very viscous light-yellow oil. The product was precipitated from pentane. The obtained solid was purified by flash column chromatography (SiO₂; CH₂Cl₂–MeOH, 100 to 90:10) to give a white solid (198 mg, 30% yield). ¹H NMR (400 MHz, DMSO-d ₆): δ = 1.24–1.46 (m, 4 H, H-5 and H-6), 1.54–1.76 (m, 2 H, H-7), 2.98–3.22 (m, 5 H, H-4, H-2, two of H-1), 3.37 (m, 2 H, two of H-1), 3.86–3.95 (m, 1 H, H-8), 4.19–4.26 (m, 1 H, H-11), 4.25–4.30 (m, 2 H, H-10), 7.33 (td, J = 7.4, 1.1 Hz, 2 H, H-13), 7.42 (td, J = 7.5, 1.6 Hz, 2 H, H-14), 7.59 (d, J = 8.0 Hz, 1 H, H-9), 7.73 (d, J = 7.5 Hz, 2 H, H-12), 7.89 (d, J = 7.5 Hz, 2 H, H-15), 8.11 (t, J = 5.6 Hz, 1 H, H-3) ppm. ¹³C NMR (101 MHz, DMSO-d ₆): δ = 23.5, 28.9, 30.8, 39.0, 42.5, 42.6, 47.1, 52.0, 54.1, 66.0, 120.5, 120.6, 125.7, 125.8, 127.5 (2 C), 128.1 (2 C), 141.1, 141.2, 144.2, 144.3, 156.6, 170.6, 174.4 ppm. ESI-HRMS: m/z calcd for [C₂₅H₂₈N₂O₅S₂Na]⁺: 523.1332; found: 523.1340.