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6 protocols using 2 2 azobis

1

Recrystallization of Organic Compounds

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DDA (TCI) was recrystallized from chloroform/hexane.
2,2′-Azobis(isobutyronitrile) (AIBN; Fujifilm Wako Pure Chemical
Corp.) was recrystallized from ethanol. TMSPA (TCI), toluene (super
dehydrated, Fujifilm Wako Pure Chemical Corp.), n-octyltrichlorosilane (Sigma-Aldrich), acetone (Nacalai Tesque Inc.),
isopropyl alcohol (Nacalai Tesque Inc.), acetonitrile (Nakalai Tesque
Inc.), chloroform (Kanto Chemical Co., Inc.), and tetrahydrofuran
(THF; Kanto Chemical Co., Inc.) were used as received.
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2

Synthesis and Characterization of Thermoresponsive Hydrogels

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NIPAAm
was purchased from
KJ Chemicals (Tokyo, Japan) and was purified by recrystallization
from toluene and n-hexane. PEG (Mn: 10 kDa), oxalyl chloride, and chloroform-d were purchased from Sigma-Aldrich (St Louis, MO) and were used without
further purification. DDMAT was purchased from Trylead Chemical Technology
(Hangzhou, China). NAS, 2,2′-azobis(2,4-dimethylvaleronitrile)
(V-65), 2,2′-azobis(isobutyronitrile) (AIBN), dichloromethane
(super dehydrated), acetone, toluene, n-hexane, ethanol,
1,4-dioxane, diethyl ether, DEEA, triethylamine, N,N-dimethylformamide, hydrochloric acid, sodium
carbonate, acetic acid, and sodium acetate trihydrate were purchased
from Wako Pure Chemical (Osaka, Japan) and were used without purification.
PAA (Mn: 150 kDa, 40 wt % solution) was
kindly provided by Nittobo Medical (Tokyo, Japan).
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3

Synthesis of Photochromic Spectacle Lens

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Example 3

A total of 84 g of Compound 1-1, 16 g of polyethylene glycol dimethacrylate (trade name: NK ESTER 4G, manufactured by Shin-Nakamura Chemical Co., Ltd., average number of moles added of ethylene glycol: 4), 0.1 g of 2,2′-azobis (2,4-dimethylvaleronitrile) (trade name: V-65 manufactured by Wako Pure Chemical Industries, Ltd.) as a radical initiator, 0.07 g of the above-described Compound 3-1 as a photochromic compound, and 0.15 g of a mixture of butoxyethyl acid phosphate and dibutoxyethyl acid phosphate (mass ratio 5:5) as a release agent were placed into a 300 mL eggplant-shaped flask and dissolution was performed under stirring. Then, stirring was performed for 30 min under reduced pressure at 0.5 kPa to prepare a composition. This composition was filtered using a 1.0 μm PTFE⋅membrane filter. The filtrate was poured into a mold made of a glass mold and a tape. Casting polymerization was carried out for 24 h at a temperature program from 40° C. to a final temperature of 100° C. to obtain a spectacle lens substrate having a thickness of 2.0 mm. The spectacle lens substrate was further annealed for 2 h at 100° C.

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4

Synthesis and Characterization of PNVF and PAAm Networks

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The reagents for PNVF and PAAm networks are as follows [29 (link)]. N-vinyl formamide (NVF; Sigma-Aldrich, St. Louis, MO, USA: 98%) monomer was purified by distillation under vacuum at 80 °C and stored at −10 °C before polymerization. A novel cross-linker, 2-(N-vinyl formamide) ethyl ether (NVEE; liquid of density of 1.3 g/mL), was synthesized and characterized by our previously reported procedure [10 (link),29 (link),44 (link)]. The monomer, acrylamide (AAm; Sigma-Aldrich: 99+%) and the cross-linker, N,N’-methylenebisacrylamide (BIS; Sigma-Aldrich, St. Louis, MO: 99+%) are electrophoresis grade and were used as received. Hydrolysis was carried out using 0.1 M NaOH (Fisher Scientific, Pittsburgh, PA, USA). The initiator, 2,2′-Azobis[2 -(2-imidazolin-2-yl) propane] dihydrochloride (VA-044; Wako Pure Chemical Industries, Ltd., Osaka, Japan), was used as received. The different pH solutions were made by dilution of stock solutions of HCl (Fisher Scientific, Pittsburgh, PA) and of NaOH.
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5

Synthesis of 2-Cyano-2-Propyl Benzodithioate

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Unless otherwise stated, all commercial reagents were used as received. N-Ethylmaleimide (EtMI; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan, 98%) and 2,2′-azobis(isobutyronitrile) (AIBN; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, 98%) were used as received. Diethylene glycol mono vinyl ether (DEGV; Maruzen Petrochemical Co., Ltd., Tokyo, Japan, 99.4%) was dried overnight over KOH pellets, and distilled twice over CaH2. 2-Cyano-2-propyl benzodithioate (CPDB) was prepared according to the literature [23 (link)].
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6

Fluorescent Protein Conjugation Protocol

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2,2′-Azobis(4-methoxy-2,4-dimethylvaleronitrile)
(V-70), fluorescein-4-isothiocyanate, dimethyl sulfoxide (DMSO), N,N′-methylenebis(acrylamide) (MBAAm),
and HEA were purchased from FUJIFILM Wako Pure Chemical Corporation.
(Osaka, Japan). DMSO was distilled under reduced pressure before use
(0.5 kPa, 95.0 °C). Poly(ethylene glycol) monomethacrylate (PEGMA)
(Mw 2000), bovine serum albumin-fluorescein
isothiocyanate conjugate (FITC–BSA), and fibrinogen fraction
I type I-S were purchased from Sigma-Aldrich (MO, USA). 2-Methylene-1,3-dioxepane
(MDO) was prepared by a two-step reaction according to the previous
reports.13 (link)−16 (link) FITC–fibrinogen was synthesized from fibrinogen and FITC
in carbonate buffer (pH 8.5) for 20 h at 25 °C.
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