The Mechanochemical Release of Naphthalimide Fluorophores from ?-Carbonate and ?-Carbamate Disulfide-Centered Polymers

Open AccessCCS ChemistryRESEARCH ARTICLE1 Nov 2021The Mechanochemical Release of Naphthalimide Fluorophores from ?-Carbonate and ?-Carbamate Disulfide-Centered Polymers Zhiyuan Shi, Qingchuan Song, Robert Göstl Andreas Herrmann Shi DWI—Leibniz Institute for Interactive Materials, Aachen 52056 Technical Macromolecular Chemistry, RWTH University, 52074 Department I Internal Medicine, University Hospital Cologne, Cologne 50937 Google Scholar More articles by this author , Song *Corresponding authors: E-mail Address: [email protected] Zernike Advanced Groningen, 9747 AG Groningen https://doi.org/10.31635/ccschem.021.202101147 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail The covalent attachment cargo molecules (e.g., drugs fluorophores) in ?-position a disulfide moiety through carbamate carbonate bonds finds many applications responsiverelease systems. Recently, we showed that the combination release process with polymer mechanochemistry-induced scission enabled remote-controlled small molecule fluorophores their inactive parent macromolecules using ultrasound. nature linker bond largely governed subsequent kinetics, an aspect has not been investigated so far. To compare differences, here employ disulfide-centered polymers releasing either hydroxyl- or amino-naphthalimides respective ?-carbonate -carbamate linkers force-induced intramolecular 5-exo-trig cyclization. We present synthesis, characterization, cell imaging evaluation three naphthalimides featuring colorimetric green fluorescence turn-on upon release, allowing monitoring process. believe insights gained these experiments would advance tailoring rates pharmacotherapy. Download figure PowerPoint Introduction Small fluorescent biosensors feature high resolution, low cost, as well real-time monitoring, are used explore disease-related cellular events biological systems.1 Besides sensitivity, operational simplicity, biocompatibility, fluorogenic probes offer excellent spatial resolution tissue contrast. Thus, number such coumarin,2 pyrene,3 xanthenes,4 benzobisimidazolium,5 cyanine,6 borondipyrromethene difluoride,7 were track prodrugs.1,8 Typically, prodrugs comprise fluorophore covalently connected linker, which could be cleaved selectively intracellular thiols,1,9 enzymes,10–13 ions,14–16 changes pH17,18 followed alteration dyes’ optical properties. Among physicochemical stimuli, exploitation redox potential differences is one most approaches. Using led exploration multifunctional theranostic conjugates, providing therapeutic effect while drug distribution.19,20 Within disulfide-based delivery systems, often employed due comparably stability option fine-tuning associated rates.2,6,7,21–25 cleavage chemical between ultimate step before can achieved, hence, its significantly influences rate. tune precisely, motifs on kinetics was previous reports.21,26 In field mechanochemistry, mechanical force induces transformations mechanoresponsive (mechanophores) embedded structures.27–29 Mechanophores were, example, force-activate -modulate properties,30–33 catalysis,34–36 molecules.37–40 However, employing mechanophores controlled activation ultrasound area research only recently established.41–43 Previously, released consisting active pharmaceutical ingredients scission44–49 cyclization.42,43 principle, system allows both amino-bearing via ?-carbamate linkers, respectively. thus Since different known21 but relative interplay apparent rate mechanochemical scission, deeper investigation consecutively coupled desirable. Here, show design, linked mechanophore unit motif (Scheme 1a). By this, effects identity absorption spectroscopy shown have greater sensitivity purpose compared liquid chromatographic methods.42,43 incorporation within center water-soluble poly(oligo(ethylene glycol)methyl ether acrylate) (POEGMEA) chain ultrasonication, amino-terminated solution. visible color alterations caused modulation charge transfer (ICT) processes provided simple readout.16 Finally, polymer-dye conjugates examined capability HeLa cells. Scheme 1 | (a) Reaction sequence naphthalimide 1–3 P1–P3 including synthesis I1–I3. (b) Chemical structures terminally substituted control polymers, P4 P5, work; syntheses I4 I5 listed. Experimental Methods Materials Commercial starting reagents solvents without further purification unless otherwise stated. 4-Bromo-1,8-naphthalic anhydride (95%), butylamine (99.5%), hydrogen iodide (HI; 57 wt % H2O, distilled, stabilized, 99.95%), 4-amino-1,8-naphthalic t-butyl 3-butenoate (?98.0%), 2-bromoisobutyric (98%), 1-thioglycerol (?97%), H2O2 (30 H2O), CuBr2 (99%), N,N-diisopropylethylamine N,N?-disuccinimidyl (?95%), oligo(ethylene acrylate (OEGMEA, Mn ? 300) purchased Sigma-Aldrich (Taufkirchen, Germany). Triphosgene (98%) tris(2-(dimethylamino)ethyl)amine (Me6TREN, >98.0%) obtained TCI Chemicals (Eschborn, OEGMEA purified passing over short column inhibitor remover. Cu-wire (d = 0.25 mm) washed concentrated aqueous HCl 15 min, rinsed MiliQ H2O (Merck, Darmstadt, Germany) acetone, dried under N2, immediately. Analytical instrumentation Proton carbon-13 nuclear magnetic resonance (1H- 13C NMR) spectra recorded at room temperature dimethyl sulfoxide (DMSO)-d6, CDCl3, CD3OD 300 MHz DPX spectrometer (13C: 75 MHz), 400 Avance 101 600 DPX-600 FT-NMR 131 MHz) (Bruker, Berlin, shifts reported ? units residual protonated solvent signals internal standard. Coupling constants (J) given Hz refer H,H-couplings. Thin-layer chromatography (TLC) performed TLC Silica gel 60 F254 plates indicator 254 365 nm UV-lamp visualization. size range 40–63 ?m flash chromatography. Gel permeation (GPC) tetrahydrofuran (THF) [high-performance (HPLC) grade, VWR International GmbH, Germany] HPLC pump (PU-2080plus; Jasco, Pfungstadt, equipped refractive index (RI) detector (RI-2031plus; Jasco). sample contained 250 mg·mL?1 3,5-di-tert-4-butylhydroxytoluene (BHT, ?99%, Fluka Chemicals, Erkrath, One precolumn (8×50 four SDplus columns (8 × mm, SDplus, MZ Analysentechnik Mainz, applied flow 1.0 mL·min?1 20 °C. diameter particles 5 ?m, nominal pore widths 50, 102, 103, 104 Å. Calibration achieved narrowly distributed poly(methyl methacrylate) standards (Polymer Standards Service, Molar masses (Mn Mw) molar mass distributions (ÐM) calculated WinGPC UniChrom software Version 8.1.1; https://www.pss-polymer.com/en/products/chromatography-software/wingpc-unichrom-mcds.html). UV–vis spectroscopies Thermo Evolution (Thermo Fisher Scientific, Waltham, MA, USA) Fluoromax-4P (Horiba, Leichlingen, Germany), respectively, temperature. acquired background correction function. For measurements hydroxyl-naphthalimide, samples excited ?exc 450 nm. kinetic measurements, emission increase measured time, ?em 550 amino-naphthalimide, 430 time 530 spectral bandwidths set 3 excitation emission. data interval integration 0.1 s. All spectroscopic carried out quartz cuvettes Hellma Analytics (Müllheim, Sonication VCX 500 ultrasonic processor (Sonics & Newtown, CT, 13 mm probe, f kHz, 30% maximum amplitude 125 ?m. Pulsed sonication (2 s on, off) used. (100 ?M) dissolved MeCN:H2O (v:v 2:3) injected into Suslick immersed ice-water bath constant N2. Then mixture sonicated (3–4 h), taken during Cell cells cultured Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) supplemented 100 U·mL?1 penicillin ?g·mL?1 streptomycin 37 °C humidified atmosphere 5% CO2. seeded 24-well plate density 105 per culture media. After 24 h, they incubated varying concentrations non-sonicated 1, P1, 3, P3 phosphate-buffered saline (PBS) 30 min PBS 3×. Then, confocal laser scanning microscope (CLSM; TCS STP8, Leica, Wetzlar, (?exc 458 pass cut-off (>505 nm) filter) analyzed ImageJ ( https://imagej.nih.gov/ij/index.html). Flow cytometry T25 flasks 1·106 flask 4 mL growth 48 h min. incubation 3× rinsing buffer, cytometer (BD Accuri C6, BD Biosciences, San Jose, CA, 500,000 events’ collection. MTS proliferation assays evaluate cytotoxicity P3, P3by culturing basal DMEM, FBS 1% antibiotics/antimycotics. CO2 95% humidity. viability assay monitored tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt, reagent) electron acceptor dye (phenazine ethosulfate; PES) (Promega, according manufacturer’s instructions. Briefly, approximately 5000 suspended ?L 96-well microtiter plates. overnight incubation, removed exchanged fresh ?L) test compounds. Control cultures treated DMSO alone. final concentration did exceed 0.5%. media removed, then reagent added served negative control. resulting suspension mixed thoroughly, absorbance 490 microplate spectrophotometer (SpectraMax M3 reader, Molecular Devices, USA). after 2 survival determination. triplicates. Results Discussion Syntheses derivatives 1– prepared previously procedures Supporting Information S1).16,50 amino-naphthalimide-containing P2, 1a), P5 1b) carrying end, opposed center, routes established us earlier Schemes S2 S3).42,43 While N-butylated bore hydroxyl amino functionality 6-position, ester N-alkyl terminus group 6-position. identities all molecular compounds confirmed 1H NMR, electrospray ionization spectrometry (ESI-MS) analyses Figures S1–S26). dye-containing POEGMEA chains received average dispersities P1 113.9 kDa, ?M 1.62), P 84.2 1.35), 80.3 117.5 1.68), 101.8 determined GPC RI S27–S29). hydroxyl-naphthalimide gain insight first properties initiator I1 bound free Free exhibited strong ?max located (Figure When (?max 340 shifted hypsochromically enabling easy spectroscopy. Figure GSH-induced I1. (solid lines; 12.5 (dashed I1: 6.25 ?M; 1: 5.00 spectra. Excitation (I1) (1) Emission intensity nm; progressing reaction (I1: GSH: ?M.). (c) GSH reduction ?M). comparison. 2:3 (v/v) verified species chemically reducing disulfides bifunctional glutathione (GSH), whereby solution excess GSH, 1c) recorded. emergence new band indicated successful reduction. analogous polymer-centered derivative reduced tris(2-carboxyethyl)phosphine (TCEP), evolution 2). Comparable I1, initial vanished together position (Figures 2a 2b). Concomitantly, wavelength observed 2c). Note correlation stronger than regime possible nonlinear attenuation beam, quenching, emission-reabsorption phenomena. Together, results centered successfully TCEP, 1. TCEP-induced P1. line) (P1: 2.5 mg·mL?1; TCEP: 150 emission, Absorption Hereafter, irradiating immersion probe sonicator (f kHz). During P1sonication, specific points (0–4 reductions decrease S30a). fraction ca. 50% calibration curve 3a Importantly, subjecting same conditions nonspecific 11% underlining contribution S30b). Ultrasound-induced (3 (v/v)). Calculated time. each withdrawal, allowed proceed h. Visual (top) (bottom) course reasoned rooted instability undergoing hydrolysis. Notably, accompanied distinguishable visually indicating 3b). Henceforth, application mechanism staining CLSM fluorescence-activated sorting (FACS). pristine slight CLSM, clearly green-fluorescent stained cells, indicative uptake 4a). Subsequent FACS corroborated observations. negligible revealed considerably increased 4b). remarkably, cargo, highlighting effective shielding random coil backbone rendering it resistant thiol exchange physiological conditions. Additional evaluations toxicity experimental S31a). micrographs preincubated °C, replaced (50 Imaging Scale bar: analysis identical (a). Further, propensity dyes investigated. analogy agents I3 3. nm, 5a). 370 470 blueshifted respect I3. line; 3: 50 I3: 25 5.0 (I3: 14 days characteristics react TCEP 18 5b). gradual reacted days, corresponding occurred, also 5c). Similar I2, 2, comparable observations S32). Most notably, carbonate-linked efficiently reduction, I2 remained almost unaltered. Only prolonging notable yet non-quantitative amino-substituted observed, poor leaving amines carbamates. similar 6). I3, decreased along 6a 6b). 6c). 6 P3. (P3: 200 similarly slower addition, P2 gave S33). sonication, fractions 1.8% 2.8% 7a 7c S34 S35). 7 diminished here. inertness expressed lower parallel P5. Next, force-activated FACS. ultrasonicated CLSM; application, fluorescence, 8a). considerable 8b). b