Anhydrous acetone

All the solvents were purchased from Sigma-Aldrich (Burlington, MA, USA). The SHFE films were prepared by dissolving 5.1 g of PVDF-HFP (3M™ Dyneon™ Fluoroelastomer FC 1650, 3M Co., Ltd., St. Paul, MN, USA) and 900 mg of DBP (Sigma-Aldrich, USA) in 30 mL of anhydrous acetone (Sigma-Aldrich, USA) and stirring at room temperature for 3 h. The mold for film formation employed polyethylene. Initially, a square glass of 10 cm $\times$ 10 cm was prepared. Double-sided tape was applied to one side of the glass entirely. A polyethylene bag was cut into a size of 12 cm $\times$ 12 cm. The cut polyethylene bag was aligned to the center of the glass. The corners were folded and secured with tape to form the mold. The inside of the mold was cleaned with ethanol. Next, the solution was poured into a polyethylene mold and dried at room temperature overnight to remove the solvent residue. Upon drying and the evaporation of the solvents, the SHFE film was delicately peeled off and placed into a polyethylene bag. Any air bubbles that may have formed during the transfer were carefully pushed out and removed.
In the case of colored films, which were produced to clearly illustrate the self-healing aspect, marker pens were dissolved in the solvent to create the desired colors (red and blue). This involved a process of dissolving the non-permanent marker in the solvent and allowing the dye in the ink to mix thoroughly with the solvent, thereby producing a colored film when the solution was applied and dried.

Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Gallic acid (98%), quercetin (>95%), methanol (≥99.9%), tert-butyl methyl ether (≥99.9%), hydrochloric acid (37%), ethyl acetate (≥99.5%), petroleum ether, sodium hydroxide (97%), phenolphthalein, sodium nitrite (≥97.0%), aluminum chloride, 2,2-diphenyl-1-picrylhydrazyl, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, sodium citrate (≥99.0%), trisodium citrate, silver nitrate (≥99.0%), hexane reagent (>99%), boric acid (≥99.5%), hydrochloric acid (37%), KjTabs VST, sulfuric acid (95.0–98.0%), potassium hydroxide (≥85%), n-octanol (≥99%), anhydrous acetone (≥99.5%) were obtained from Sigma (Darmstadt, Germany; Tokyo, Japan; Shanghai, China). Lutein and β-carotene were purchased from Extrasynthese (Lyon, France). A Specord 200 Plus spectrophotometer (Jena, Germany) was used for spectrophotometric measurements.

Carnosic acid was obtained from Combi-Blocks (San Diego, CA.) or Sigma-Aldrich (St. Louis, MO). Pisiferic acid was obtained from Combi-Blocks. Carnosol was commercially available from Sigma-Aldrich or prepared from Carnosic acid using the method of Han et al.^{48 (link)}. Carnosic acid diacetate, methyl carnosate and Carnosic acid γ-lactone were prepared using the method of Han, et al.^{48 (link)}. Conc. hydrochloric acid (35–38% in water) was from Fisher. Methanol (MeOH), ethyl acetate (EtOAc), toluene, dichloromethane (CH₂Cl₂) and hexanes were HPLC or LC/MS grade from Fisher or VWR International. Anhydrous acetone was supplied by Sigma-Aldrich. Water was 18.2 mΩ-cm from a Barnstead NANOpure Diamond system. Trifluoroacetic acid was obtained from EMD Millipore. Melting points (mp) were determined on an Electrothermal MEL-TEMP 3.0 apparatus (Barnstead International, Dubuque, IA) and are not corrected.
Preparative HPLC separations were carried out using a Shimadzu system consisting of two LC-8A pumps, a fraction collector (FRC-10A), a SIL-10AP auto sampler, a diode array detector (CPD-M20A) and a CBM-20A communication module. The separations employed a Waters PREP Nova-Pak® HR C18 6 µM 60 Å 40 ×100 mm reversed phase column with a 40 ×10 mm Guard-Pak insert and a Waters PrepLC Universal Base. The solvent system employed was MeOH/water gradients both containing 0.1 % TFA. Fractions were collected based on their response at 254 nm. Flash chromatography was carried out on 230–400 mesh silica gel (Silica gel 60, Geduran) obtained from Fisher Scientific using the method of Still et al.^{49 (link)}. Columns were eluted with EtOAc/hexanes mixtures or CH₂Cl₂. Thin layer chromatography (TLC) employed Analtech GHLF UV254 Uniplate silica gel plates from Miles Scientific. Plates were visualized under UV light or with I₂ vapor.
Mass spectroscopy employed a Thermo Scientific TSQ Quantum Ultra triple-stage quadrupole mass spectrometer. Heated-electrospray ionization (H-ESI) was used in negative or positive ionization mode depending on the structure of the analyte. Automatic methods for the optimization of instrument parameters were used to maximize sensitivity. Samples were analyzed by direct injection in MeOH or MeOH/water (TFA conc kept at 0.01% or less) using a syringe pump.
For Carnosic acid diacetate synthesis, Carnosic acid (1.02 g, 3.05 mmol) in CH₂Cl₂ (13 mL) was treated with neat acetic anhydride (1.6 mL) and 4-dimethylaminopyridine (DMAP, 443 mg). The reaction was warmed after the DMAP was added. The resulting yellow solution was stirred at rt for 2 days. The reaction was then diluted with CHCl₃ (40 mL) and extracted with a 1 M aq. HCl solution (25 mL). The organic layer was separated and washed with brine, dried (MgSO₄), filtered, and conc to dryness. The crude product was purified by flash chromatography with a gradient from 4:1 to 3:1 hexanes/EtOAc. The product was isolated as an oil (480 mg) that solidified on standing. The resulting solid had mp 94–95 ^oC (lit mp 158–159 ^oC^{50 (link)}. MS ESI⁻ m/z 415 (M – H⁺).
For methyl carnosate synthesis, Carnosic acid (329 mg, 0.99 mmol) was dissolved in 5:1 toluene/MeOH (6 mL) under N₂ and cooled in an ice-water bath. The reaction was treated with a 1.8 to 2.4 M solution of TMSCN₂ in hexanes (Thermo-Fisher; 1.1 mL) added dropwise via syringe. The reaction was stirred cold and then allowed to warm to rt and stir overnight. The reaction was cooled in an ice-water bath and a 2 M aq. HOAc solution was added (10 mL). The two-phase mixture was partitioned between water and EtOAc. The organic layer was separated, washed with brine, dried (MgSO₄), filtered, and conc. in vacuo. Flash chromatography with 9:1 hexanes/EtOAc gave 191 mg of the methyl ester as a white solid with mp softens at 118 ^oC, melts 126–127 ^oC (lit mp 118–119 ^oC^{51 (link)}. MS ESI^- m/z 345 (M – H⁺).
For Carnosic acid γ-lactone synthesis, Carnosic acid (125 mg, 0.30 mmol) in 3 mL of CH₂Cl₂ was treated with solid DCC (86 mg). A ppt formed almost immediately. Sold DMAP (7.7 mg) was then added. The mixture was stirred at rt under N₂ for 5 h and then filtered. The mother liquor was conc to dryness and purified by flash chromatography (100% CH₂Cl₂). The lactone (40 mg) was isolated as a yellow foam. Lit mp for the lactone is 106–109 ^oC^{52 (link)}. MS ESI⁻ m/z 313 (M – H⁺).
The dimethyl ether of Carnosol was prepared using the method of Luis et al. (Luis, J. G. et al.⁵³ . except that the compound was purified by reverse-phase HPLC.
For di-d₃-methyl Carnosol synthesis, Carnosol (100 mg, 0.30 mmol) in acetone (16 mL) in an ice-water bath was treated with neat d₃-MeI (500 µL, 1.16 g, 8.00 mmol) and 2 eq. of K₂CO₃ (84 mg, 0.60 mmol). The resulting mixture was stirred cold and then allowed to warm to rt and stir overnight. The reaction was diluted with cold water and extracted with EtOAc (3 × 40 mL). The pooled organic layer (yellow) washed with brine, dried (MgSO₄), filtered and conc to dryness, tare 130.835 g, weight 138 mg. Crude product was purified by RPHPLC (20 to 100% MeOH/water containing 0.1% TFA). Fractions with RT 6.6 min were collected and concentrated to 13 mg of di-d₃-methyl Carnosol as an oil; lit mp 155–157 ^oC^{50 (link)}. MS (ESI⁺) m/z 365 (M + H⁺) and 387 (M + Na⁺).

All reactions were run under dry N₂. Any reactions involving the generation or use of either Grignard reagents or LDA were run in glassware previously flame-dried under vacuum. Anhydrous acetone, TEOS, dimethylsulfide, anhydrous diethyl ether, and 2,2,2-trifluoroethyl trifluoroacetate were obtained from Aldrich and were used as received. LDA was obtained in the form of a commercial solution from Aldrich (1.5 M solution in cyclohexane) and was used as received. Anhydrous THF was prepared by distillation from a solvent still containing Na/benzophenone ketal immediately prior to use. Vacuum distillations were carried out using a Kugelrohr apparatus with all boiling points being reported at 0.20 mmHg. A PCI ozonizer (1 lb/day generation capacity) was used for the ozonolysis reactions with dry O₂ being used as the feed gas source. FT-IR spectra were obtained using a Nicolet Avatar 360 spectrophotometer with NaCl plates being used for all thin films. ¹H-NMR and ¹³C-NMR spectra were obtained using a Varian Unity Plus 300 MHz spectrometer and were measured at 300 and 75 MHz respectively, with all chemical shifts reported in ppm using TMS as the internal standard and all determined J values reported in Hz.
2-(4-(Triethoxysilyl)phenyl)propene (6). Into a 250-mL round-bottom three-neck flask were placed magnesium turnings (875 mg, 36.0 mmol) and a magnetic stirring bar and the resulting assembly was flame-dried under vacuum. The flask was then fitted with a reflux condensor and pressure-equalized dropping funnel, was flame-dried under a stream of dry N₂ and allowed to cool to RT. The dropping funnel was charged with a solution of 2-(4-bromophenyl)propene (5) (5.91 g, 30.0 mmol) in anhydrous THF (60 mL). Approximately 15 mL of the bromide-THF solution was discharged from the dropping funnel, 1,2-dibromoethane (four drops) was added to the solution in the flask and the resulting mixture was heated to reflux and stirred until the formation of the Grignard reagent had commenced (circa 10 min). The remainder of the bromide-THF solution was then added dropwise with continued heating and stirring over the course of 1 h and the reaction mixture was refluxed with stirring for an additional 90 min. The reaction mixture was allowed to cool to RT, a solution of tetraethoxysilane (6.87 g, 7.35 mL, 33.0 mmol) in anhydrous THF (10 mL) was added rapidly with stirring over the course of 2 min, and the resulting mixture was refluxed with continued stirring for an additional 16 h. The reaction mixture was then allowed to cool to RT, was cooled to 0-5^°C by immersion in an icebath, sat. NH₄Cl(aq) (15 mL) was added dropwise with vigorous stirring over the course of 5 min and the resulting mixture was stirred for an additional 10 min with continued cooling. The mixture was filtered through Celite^® and the collected magnesium salts were washed with three portions of ether (50 mL each). The combined organic extracts were washed with water and saturated aqueous NaCl, dried (MgSO₄) and concentrated in vacuo to afford the crude silane product as a thick, golden-yellow oil that was purified by vacuum distillation: 6.78 g (24.2 mmol, 81%); isolated as a clear, colorless thick oil; bp 92-95^°C (Kugelrohr, 0.20 mmHg); FT IR (thin film) 1628 (mod.), 1616 (w) cm^-1; ¹H-NMR (CDCl₃) δ 7.69 (2H, d, J = 8.1 Hz), 7.52 (2H, d, J = 8.1 Hz), 5.45 (1H, d, J_geminal = 1.5 Hz), 5.15 (1H, d, J_geminal = 1.5 Hz), 3.92 (6H, q, J = 6.3 Hz), 2.19 (3H, s), 1.29 (9H, t, J = 6.3 Hz); ¹³C- NMR (CDCl₃) δ 143.4, 143.3, 135.1, 130.1, 125.2, 113.3, 59.0, 21.9, 18.50.
4-(Triethoxysilyl)acetophenone (7). Into a 200-mL round-bottom three-neck reaction flask equipped with a magnetic stirring bar, a fire-polished bubbler tube, a glass stopper and a gas outlet portal was placed a solution of 6 (3.00 g, 10.7 mmol) dissolved in a 1:4 (v/v) mixture of dry ethanol in dichloromethane (60 mL) and the stirred solution was cooled to -78^°C. With continued cooling and stirring, the solution was treated with O₃ for 5 min [16 ]. Following the purgement of excess ozone gas from the solution, dimethyl sulfide (3.5 mL, 3.0 g, 48.3 mmol) was added to the cooled and stirred solution over the course of 2 min and the resulting mixture was allowed to warm to and stir at RT for an additional 12 h. The reaction mixture was concentrated in vacuo and the isolated residue was dissolved into ether (50 mL). The resulting ethereal solution was washed with two portions of water (50 mL each) and saturated aqueous NaCl, dried (MgSO₄) and concentrated in vacuo to afford the crude ketone product as a thick, light-yellow oil that was purified by vacuum distillation: 2.12 g (7.51 mmol, 70%); isolated as a clear, colorless thick oil; bp 128-131^°C (Kugelrohr, 0.20 mmHg); FT IR (thin film) 1689 cm^-1 (vs, C=O); ¹H-NMR (CDCl₃) δ 7.95 (2H, d, J = 8.1 Hz), 7.79 (2H, d, J = 8.1 Hz), 3.89 (6H, q, J = 7.1 Hz), 2.63 (3H, s), 1.26 (9H, t, J = 7.1 Hz); ¹³C-NMR (CDCl₃) δ 198.7, 138.6, 137.5, 135.3, 127.5, 59.1, 26.9, 18.4.
1-(4-(Triethoxysilyl)phenyl)-4,4,4-trifluoro-1,3-butanedione (2). The general approach described by Zayia [15 (link)] was followed with several modifications. A 50-mL round-bottom three-neck reaction flask was flame-dried under vacuum and was equipped with a magnetic stirring bar, three rubber septa and a positive-pressure N₂ inlet line. The flask was charged with a solution of 7 (1.00 g, 3.55 mmol) dissolved in anhydrous diethyl ether (15 mL). The flask was cooled to -78^°C, and with stirring, a 1.5 M solution of LDA in cyclohexane (2.61 mL, 3.91 mmol of LDA) was added dropwise with a syringe over the course of 2 min and the resulting mixture was stirred at -78^°C for an additional 90 min. With continued cooling and stirring, neat 2,2,2-trifluoroethyl trifluoroacetate (2.38 mL, 3.48 g, 17.75 mmol) was quickly added over the course of 1 min with a syringe and the resulting mixture was stirred at -78^°C for 4 h. Neat H₂SO₄ (0.40 mL, 0.732 g, 7.46 mmol) was then added in one portion with vigorous stirring using a microsyringe and the reaction mixture was allowed to warm to RT. The mixture was poured into a 125-mL separatory funnel containing ether (25 mL) and water (25 mL), the organic layer was isolated, and was washed with water, 10% aqueous NaHCO₃ and saturated aqueous NaCl, dried (MgSO₄) and concentrated in vacuo to afford the title diketone 2 as a thick, clear oil that was used without further purification: 0.98 g (2.59 mmol, 73%); FT IR (thin film) 3520-3280 (mod , O-H), 1605 (vs and broad, enolic form), and 1162 cm-1 (vs and broad, C-O); ¹H-NMR (CDCl₃) d 14.9 (1H, bs), 7.95 (2H, d, J = 7.8 Hz), 7.80 (2H, d, J = 7.8 Hz), 6.62 (1H, s), 3.87 (6H, q, J = 7.2 Hz), 1.31 (9H, t, J = 7.2 Hz); ¹³C-NMR (CDCl₃) d 185.8, 177.8 (q, ²J_CF = 35.9 Hz), 138.9, 135.3, 130.3, 126.5, 117.1 (q, , ¹J_CF = 284.3 Hz), 92.5, 59.0, 18.2. The product as isolated was found to be of sufficient purity for use in the subsequent sol-gel studies. Attempts to obtain an analytical sample of 2via vacuum distillation failed due to the labile nature of the product though it was possible to obtain an enriched fraction of >95% purity (bp 142-146^°C, Kugelrohr, 0.20 mmHg).