Cell-Penetrating Dynamic-Covalent Benzopolysulfane Networks

Abstract

Cyclic oligochalcogenides (COCs) are emerging as promising systems to penetrate cells. Clearly better than and different to the reported diselenolanes and epidithiodiketopiperazines, we introduce the benzopolysulfanes (BPS), which show efficient delivery, insensitivity to inhibitors of endocytosis, and compatibility with substrates as large as proteins. This high activity coincides with high reactivity, selectively toward thiols, exceeding exchange rates of disulfides under tension. The result is a dynamic-covalent network of extreme sulfur species, including cyclic oligomers, from dimers to heptamers, with up to nineteen sulfurs in the ring. Selection from this unfolding adaptive network then yields the reactivities and selectivities needed to access new uptake pathways. Contrary to other COCs, BPS show high retention on thiol affinity columns. The identification of new modes of cell penetration is important because they promise new solutions to challenges in delivery and beyond.

Keywords: adaptive networks; cellular uptake; cyclic oligochalcogenides; dynamic-covalent chemistry; polysulfanes.

Figure 1

Cellular uptake of BPS₅  1 into HeLa Kyoto cells compared to 4–8. a) Selected original flow cytometry data (20 μm, 45 min, Leibovitz's medium). The position of the compound numbers reflects the approximate fluorescence intensities after correction using quenching factors (QFs) of reduced COCs (note the log scale). b–g) Uncorrected CLSM images (10 μm, scale bar: 10 μm; laser power (LP): 15 %, with QFs of reduced COCs for correction if desired). Previously reported COCs are in gray.10, 11

Scheme 1

a) 6 steps, Scheme S1;3 b) S₂Cl₂, 0 °C to rt, 24 h, 20 %;3 c) FL–NH₂, DMF, rt, 12 h, 14 %; d) 6 steps, Scheme S5;11 e) 1. NH₃, MeOH, rt, 30 min, 2. S₂Cl₂, CH₂Cl₂, 0 °C to rt, 2 h, 58 %; f) 3 steps; g) 10 steps, Scheme S6. PNP=p‐nitrophenyl.

Figure 2

a) Possible ring expansion and contraction of BPS₅, interconvertible ring‐opened intermediates RI, and selected products from intermolecular exchange. b–d) Diagnostic region of ¹H NMR spectra of 3 in deuterated PBS buffer, 2 weeks (b), and with GSH, 5 min, pD 8.0 (c), and pD 5.5 (d). e–g) HPLC traces of BPS₅  1 in PBS buffer, pH 7.4, 30 min (e), and with 2 equiv GSH (f), followed by 100 equiv GSSG (g). h) Signal cluster of 23 ₄ in UHPLC‐TOF HRMS of 1 with 22 (2 equiv), with i) a zoom and a simulated spectrum for 23 ₄ (Σn=2), and j) signal clusters of 23 ₂, 23 ₅, and 23 ₇ in LC‐MS of 1 with 22 (2 equiv). R′′: See 1, 3, Figure 1; R′: See 22.

Figure 3

Thiol‐exchange affinity column chromatograms of a) 7,12 b) 4, and c) 1 in 10 mm Tris, 0.1 m NaCl, 1 mm EDTA, pH 7.5 with a 0–50 mm DTT gradient at t=60–70 min (solid) and constant 50 mm DTT from t=0 (dashed), R′′: See 1, 4, and 7, Figure 1.

Figure 4

a) Fluorescence intensities in HeLa Kyoto cells treated with 31 (2.5 μm) or 28 (0.5–10 μm), then, after washing, with 27 (10 μm). CLSM images of HeLa Kyoto cells incubated with b) 32 and c) 34 (10 μm, 8 h; scale bars: 10 μm; LP: 30 %).