Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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High Throughput Preparation of UV-Protective Polymers from Essential Oil Extracts via the Biginelli Reaction Tengfei Mao,†,‡ Guoqiang Liu,† Haibo Wu,† Yen Wei,† Yanzi Gou,‡ Jun Wang,‡ and Lei Tao*,† †
The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China ‡ Science and Technology on Advanced Ceramic Fibers and Composites Laboratory, National University of Defense Technology, Changsha, 410073, P. R. China S Supporting Information *
ABSTRACT: A high throughput (HTP) system has been developed to exploit new functional polymers. We synthesized 25 monomers in a mini-HTP manner through the tricomponent Biginelli reaction with high yields. The starting materials were five aldehydes extracted from essential oils. The 25 corresponding polymers were conveniently prepared via mini-HTP radical polymerization initially realizing the benefit of HTP methods to quickly fabricate sample libraries. The distinct radical scavenging ability of these Biginelli polymers was evaluated through a HTP measurement to choose the three best radical scavengers. This confirms the superiority of the HTP strategy to rapidly collect and analyze data. The selected polymers have been upgraded and screened according to different requirements for biomaterials and offer water-soluble and biocompatible copolymers that effectively protect cells from the fatal UV damage. This research is a straightforward way to establish new libraries of monomers with abundant diversity. It offers polymers with interesting functionalities. This suggests that a broader study of multicomponent reactions and HTP methods might be useful in many interdisciplinary fields. To the best of our knowledge, this is the first report of a HTP study of the Biginelli reaction to develop a promising polymeric biomaterial, which might have important implications for the organic chemistry and polymer communities.
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INTRODUCTION High throughput (HTP) methods have had huge successes in polymer science to exploit new functional polymers and polymeric materials.1−20 Many polymers with different main/ side chains have been synthesized in parallel and then characterized through HTP methods to study the subtle relationships between polymer structures and properties. This has led to the rapid preparation of polymers as potential gene/ drug carriers,3,8,13−17,21,22 sensors,23−25 tissue engineering scaffolds,12,26 antibacterial surfaces, 27,28 (stem)cell factories,29−31 etc. HTP strategies can accelerate the pace of research and the conversion of research results; thus, the development of new HTP methods to identify new polymers for interdisciplinary applications is important in fundamental research and practical applications. The preparation of new monomers for chain-growth polymerization is a direct route to new functional polymers. However, the tedious processes required during the syntheses of new monomers (chromatography separation, multistep reactions, protection/deprotection techniques, etc.) seriously limit large-scale preparation and further study/application of the products much less facilitate monomer/polymer libraries. Multicomponent reactions (MCRs) are a potential solution for several reasons: (1) MCRs use more than two reactants, and © XXXX American Chemical Society
thus the number and molecular diversity of the samples can be quickly increased through different combinations of reactants.32−34 (2) Some MCRs (Biginelli, Passerini, Ugi, Debus− Radziszewski, etc.) are highly efficient and can generate single compounds that are directly purified via simple precipitation or extraction.35,36 This avoids laborious column chromatography. (3) Many MCRs use functional groups (aldehyde, amine, carboxylic acid, etc.) that are common in many natural products and can create delicate compounds with potential bioactivity (antioxidant, antitumor, antibacterial, etc.).33,34,37 This offers many new choices to explore new functional polymers from easily available raw materials. The monomers prepared from MCRs have been studied by some pioneers in the field (Wright, Sun, Meier),38−42 but HTP methods are much less common. Herein we report a new HTP system to easily build a library of monomers via the Biginelli reaction for subsequent HTP radical polymerization (Scheme 1). We used five aromatic aldehydes that can be extracted from plant essential oils (EOs); 25 new monomers were quickly prepared through the Biginelli reaction in a mini-HTP manner. Received: February 13, 2018 Published: April 8, 2018 A
DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society Scheme 1. HTP Methods To Prepare Libraries of New Monomers and Polymers
These monomers were then used for radical polymerization to simultaneously obtain 25 polymers with different Biginelli structures as the side chains. This offers a HTP strategy to rapidly construct sample libraries. Although natural products such as chitosan, hyaluronic acid, oleic acids, etc., have been used to prepare new polymers via MCRs,33,43−45 the unique bioactivity of the resulting polymers is rarely studied in detail. Therefore, we tried HTP analyses to evaluate the specific bioactivity of the resulting polymers and obtained a biocompatible polymer that protects cells from UV damage. This demonstrates the utility of the HTP strategy to optimize and screen functional polymers for possible practical applications.
diethyl ether rinse to remove acetic acid and other impurities (Table S1, Figures S1−S4, ∼7−18% isomers). In a typical example, the characteristic peaks of the Biginelli cyclization products (NH: 9.79−9.89 ppm; PhCH: 5.09−5.16 ppm) could be clearly identified from the 1H NMR spectra of M(X)(4) monomers (Figure 2). The integral ratio between the PhCH methine and the protons of vinyl group (I5.09−5.16/I5.66/I5.95 = 1/ 1/1) agreed well with the theoretical value (1/1/1) suggesting
RESULTS AND DISCUSSION HTP Synthesis of Monomers via the Biginelli Reaction. The Biginelli reaction uses three common components (aldehyde, β-ketoester, and (thio)urea or their derivatives) to effectively produce dihydropyrimidin-2(H)-ones (DHPMs).46,47 Recently, the Biginelli reaction has been used in polymer chemistry to prepare a few polymers with DHPMs in main/side chains.44,48,49 Here aromatic aldehydes (EOs extracts) were used to convert the commercially available monomer 2-(acetoacetoxy)ethyl methacrylate (AEMA) to new monomers via the Biginelli reaction (Figure 1a). The monomers were synthesized in a HTP manner by mixing 5 aromatic aldehydes (A(X)) and 5 (thio)urea compounds (B(Y)) through different combinations (5 × 5) to simultaneously create 25 Biginelli-monomers (M(X)(Y)) (Figure 1b). In a typical reaction, AEMA, A(X), and B(Y) were added to centrifuge tubes, and the molar ratio of each component was set as AEMA:A(X):B(Y) = 1:1:1.5. The excess urea/thiourea compounds were added to ensure complete reaction of the βketoester group in AEMA. Acetic acid and MgCl2 (20 mol % with respect to aldehydes) were used as the solvent and catalyst, respectively. The 25 tubes were charged with reactants, solvent, and catalyst and kept in a 100 °C isothermal shaker (Figure 1c) for 2 h. The 1H NMR analyses suggested that 25 M(X)(Y) monomers were efficiently generated with high conversion rates (∼79−99%) (Table S1). The target M(X)(Y) monomers were easily purified in 70− 87% yields after simple precipitation in cold water followed by a
Figure 2. (a) Reaction conditions: acetic acid as the solvent, 100 °C, and 2 h. AEMA: A(X):B(4):MgCl2 = 1:1:1.5:0.2. (b) 1H NMR spectra (DMSO-d6, 400 M) of M(X)(4) monomers.
Figure 1. (a) HTP preparation of monomers via the Biginelli reaction. (b) The 25 combinations of A(X) and B(Y). (c) Experimental setup for the HTP preparation of monomers.
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B
DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society facile preparation of different DHPM-type monomers via the HTP Biginelli reaction. HTP Polymerization of M(X)(Y) via Radical Polymerization. These new M(X)(Y) monomers were used for free radical polymerization. This polymerization was also performed in a HTP manner because of its convenience. Briefly, 2,2′azobis(isoheptonitrile) (ABVN) was used as the initiator, and 25 monomer solutions (1.0 M in DMF, in centrifuge tubes) were added with the ABVN (2 mol % to the monomer). These samples were purged with nitrogen for 15 min and then kept in a 70 °C isothermal shaker overnight (Figure S5). After 14 h, the polymerization was quenched in an ice−water bath, and the samples were analyzed by 1H NMR and gel-permeation chromatography (GPC). All monomers were polymerized with high conversions (∼90−99%, Figure S6 (M(X)(4) as a typical example), Table S2), and all Biginelli-polymers (P(X)(Y)) have satisfactory molecular weights (Mn(GPC): ∼31 000−128 600 g mol−1, Figure S7 (P(X)(4) as a typical example), Table S2), suggesting that the different DHPM groups in monomers are compatible with radical polymerization. The P(X)(Y) can also be purified through simple precipitation in diethyl ether. In one example, the 1H NMR spectra of P(X)(4) showed specific peaks of DHPM moieties (NH: 9.67−9.82 ppm; PhCH: 5.10−5.22 ppm) while the peaks of the vinyl group (5.95, 5.66 ppm) completely disappeared (Figure 3), suggesting successful preparation of a series of Biginelli-type polymers after HTP radical polymerization. HTP Analysis of the Radical Scavenging Ability of P(X)(Y). Many DHPM derivatives have been studied as potential hypotensors, calcium antagonists, anticarcinogens, antioxidants, etc.50 EOs have been widely used as antioxidants, antibacterial agents, repellents, anti-inflammatory agents, etc., in cosmetic, pharmaceutical, and food industries.51 Therefore, some value-added polymers might be obtained via the combination of the Biginelli reaction, polymer chemistry, and EOs extracts. To verify that hypothesis, the radical scavenging ability of resulting polymers was evaluated through a HTP measurement. The resulting polymers (water insoluble) were dissolved in DMSO (200 μL, 5 mg/mL) and put into a 96-well plate, and DMSO was used as the control. The 1,1-diphenyl-2picrylhydrazyl (DPPH) radical was used as a model radical. After addition of the solution of DPPH radical (20 μL, 5 mg/ mL in DMSO) to the plate, the dark blue color of the DPPH radical quickly faded with P(1)(2), P(1)(4), and P(2)(4) (Figure 4a, 4b, 4c). This indicates that these three polymers can quench the DPPH radical much faster than the others. Direct visual observation showed that the order of radical scavenging ability is P(1)(2) > P(2)(4) > P(1)(4) ≫ P(1)(1) ≈ DMSO. This is consistent with the quantitative data analysis (Figure 4d). Thus, the HTP method is a simple but practical method to quickly detect polymers’ ability to quench free radicals. The M(X)(Y) monomers were also tested via the same HTP approach to identify some radical scavenging monomers (Figure S8). All monomers could be polymerized via radical polymerization, suggesting that the radical scavenging process is slower than polymerization. M(4)(2) and M(4)(4) had the best radical scavenging ability of all M(X)(2) and M(X)(4), respectively. The inconsistent results between monomers and polymers might be due to the steric effects of giant polymer chains that block the interactions between the side groups and radicals. Therefore, monomers and corresponding polymers
Figure 3. (a) Reaction conditions: DMF as the solvent, 70 °C, and 14 h. M(X)(4) monomer: ABVN = 1:0.02. (b) 1H NMR spectra (DMSO-d6, 400 M) of P(X)(4).
Figure 4. HTP analysis of polymers: polymer solutions (200 μL and 5 mg/mL in DMSO); DMSO served as the control. (a) Samples after adding the DPPH radical solution (20 μL, 5 mg/mL in DMSO), t = 0 min; (b) 10 min; (c) 20 min; (d) DPPH radical concentration vs time in the presence of different polymers with absorbance at 520 nm. These mixtures (20 μL) were diluted in DMSO (100 μL), and the data are presented as means ± SD, n = 3.
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DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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cell-counting kit-8 (CCK-8) assay. The murine fibroblast cell line L929 was chosen as the model cell. L929 cells (∼ 5 × 104/ mL) were cultured (37 °C, 5% CO 2 ) with different concentrations of copolymers (0.5, 1, 2, 5, and 10 mg/mL) for 24 h. P(PEGMA) (Figure S12, Mn(GPC) ∼ 73 800 g mol−1) was prepared by radical polymerization and served as the control. Cell viability in culture medium only was defined as 100% viability (Figure 6a).
should be studied separately to ensure the accuracy of results; the features of the monomers are merely references. Preparation of Water-Soluble Copolymers. P(1)(2), P(1)(4), and P(2)(4) had particularly strong radical scavenging activity, but they still require further refinement to achieve biomaterials for applications in personal care, medicine, cosmetics, and food. The M(1)(2), M(1)(4), and M(2)(4) monomers were copolymerized with poly(ethylene glycol methyl ether) methacrylate (PEGMA, Mn ∼ 950 g mol−1) via radical polymerization to generate water-soluble copolymers (Figure 5a).
Figure 5. (a) Reaction conditions: DMF as the solvent, 70 °C, 12 h. M(X)(Y) monomer:PEGMA:ABVN = 0.5:0.5:0.02. (b) 1H NMR spectra (DMSO-d6, 400 M) of the copolymers.
For instance, the M(1)(2) monomer and PEGMA were mixed in DMF (0.5/0.5, 1.0 M) using ABVN as the initiator (2 mol % to monomers). After removal of oxygen by nitrogen flow, the mixture was kept in a 70 °C oil bath for 12 h and then decanted into diethyl ether to precipitate the copolymer P(1) (2)-co-P(PEGMA). Other copolymers were similarly prepared. The 1H NMR spectra of the copolymers (Figure 5b) showed specific peaks of the DHPM structures (PhCH: 5.05−5.20 ppm). The methyl group in the PEG moieties (CH3O: 3.23 ppm) is obvious. All copolymers can easily dissolve in water (>150 mg/mL, Table S3) and have high molecular weights (Mn(GPC): ∼84400−91300 g mol−1; Figure S9) and DHPM/ PEG ratios (∼0.90−0.93) (Table S3), indicating smooth preparation of the desired copolymers. M(4)(2) and M(4) (4) were also copolymerized with PEGMA to obtain copolymers, respectively (Figure S10). The five copolymers were tested in parallel by a DPPH assay (Figure S11), and the order of radical scavenging ability is P(1)(2)-co-P(PEGMA) > P(2)(4)-co-P(PEGMA) > P(1)(4)-co-P(PEGMA) > P(4)(2)co-P(PEGMA) > P(4)(4)-co-P(PEGMA). Thus, the first three copolymers were used for the next study. Cytotoxicity Evaluation and Cell-Protection Study. Polymers should be biocompatible for use with living systems. Thus, we evaluated the cytotoxicity of the copolymers with a
Figure 6. (a) Cytotoxicity of copolymers to L929 cells. Twenty-four hour culture, P(PEGMA) as the control, cell viability in culture medium is 100% viability. (b) Cell viability with different copolymers after exposure to UV light (∼254 nm, 40 w). Fifteen minutes of UV irradiation (300 ± 20 μw/cm2) + 24 h culture, P(PEGMA) as the control; cell viability in culture medium without UV irradiation is 100% viability. The data are presented as means ± SD, n = 5.
All polymers have low cytotoxicity at low concentrations (≤2 mg/mL). With increased concentrations, P(2)(4)-co-P(PEGMA) demonstrated moderate cytotoxicity: 76% and 61% viability at 5 and 10 mg/mL, respectively. On the contrary, ∼88% and ∼100% cells remained viable with 10 mg/ mL of P(1)(2)-co-P(PEGMA) and P(1)(4)-co-P(PEGMA), respectively, suggesting the good and excellent cellular safety of P(1)(2)-co-P(PEGMA) and P(1)(4)-co-P(PEGMA), respectively (p < 0.01, contrast with the cells in the presence of P(2) (4)-co-P(PEGMA), ≥5 mg/mL). D
DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society UV light is known to destroy DNA through radical processes;52 thus, the ability of copolymers to protect cells from UV damage was also evaluated because of the excellent radical-fighting DHPM groups on their chains. To L929 cells were added different concentrations of copolymers, and then they were exposed to a UV sterilamp (∼254 nm, 40 W) for 15 min (300 ± 20 μw/cm2) prior to 24 h culture. The samples were analyzed by a CCK-8 assay using P(PEGMA) (Figure S12) as the control; the viability of the cells in culture medium without UV irradiation was defined as 100% (Figure 6b). When incubated with P(PEGMA), most cells (86−98%) lost viability after UV irradiation, but there was 47−74% viability with 2 mg/mL of copolymers and exposure to UV light. This indicates that the DHPM side groups in the copolymers protect cells from the UV light. At increasing concentrations (≥5 mg/ mL), P(1)(4)-co-P(PEGMA) retained excellent cytoprotection, but the cytotoxicity of P(1)(2)-co-P(PEGMA) and P(2)(4)-coP(PEGMA) could not be neglected (p < 0.01, contrast with the cells in the presence of P(1)(4)-co-P(PEGMA), ≥ 5 mg/mL). This counterbalanced the cell-protective effect and led to obvious apoptosis. Thus, P(1)(4)-co-P(PEGMA) was selected for further study as a potential sunscreen; it has excellent safety and protects cells from UV damage. Cell-Protection Ability of Selected Copolymer in Comparison with Superoxide Dismutase (SOD). We designed an experiment to test the protection ability of P(1) (4)-co-P(PEGMA) to act as a sunscreen in comparison with superoxide dismutase (SOD), a well-known antioxidase which has protective and beneficial roles in many diseases53,54 and is also safe to cells (Figure S13). After adding culture medium containing P(1)(4)-co-P(PEGMA) (10 mg/mL), the L929 cells (∼5 × 104/mL) were exposed to a UV sterilamp for 15 min and then cultured for 24 h prior to further analyses. The cells in the culture medium containing SOD (10 mg/mL, from bovine erythrocytes, ≥3000 units/mg) were tested as the control. The cells in the culture medium only without UV irradiation were used as the blank. The fluorescein diacetate (FDA)/propidium iodide (PI) assay is a simple and rapid approach to simultaneously observe living and dead cells (FDA can only enter and accumulate in living cells, and PI only stains the nucleus of dead cells).55 We used FDA/PI double staining to qualitatively evaluate cell viability after UV irradiation (Figure 7). The cells cultured with SOD were almost completely stained by PI as red spots after exposure to UV light for 15 min (Figure 7b). This indicates the poor protection of SOD to cells, which might be caused by the deactivation of SOD under this experimental condition or the poor transmembrane efficiency of SOD or both. Cells cultured with P(1)(4)-co-P(PEGMA) survived the same process (Figure 7c) with viability similar to that of the blank (Figure 7a). Cells were healthy even after 30 min UV irradiation (Figure S14a); cell necrosis was observed after 45 min UV irradiation (Figure S14b). These results agreed well with the quantitative data obtained by a CCK-8 assay (Figure S14c), confirming that P(1)(4)-co-P(PEGMA) is a better sunscreen than SOD in this condition to protect cells from fatal UV damage. Mechanism of the UV-Protection. A possible UVprotection mechanism was studied. Here L929 cells (∼5 × 104/mL) with P(1)(4)-co-P(PEGMA) (10 mg/mL) were exposed to UV light for 15 min and then cultured for 24 h. The cells were fixed and stained with Phospho-histone H2A.X (Ser139) rabbit monoclonal antibody followed by Alexa Fluor
Figure 7. FDA/PI double staining of L929 cells (a) without UV irradiation, (b) with UV irradiation (15 min) in the presence of SOD (10 mg/mL), and (c) with UV irradiation (15 min) in the presence of P(1)(4)-co-P(PEGMA) (10 mg/mL), bar = 100 μm.
555-labeled donkey antirabbit IgG prior to observation under green light (515−560 nm band-pass excitation filters). L929 cells cultured in medium without copolymer served as the control. In the control group, almost all cells died after UV irradiation. The red-stained damaged DNA was obvious (Figure 8a, Figure S15a, green light), indicating that the DNA in the dead cells had been destroyed. However, the cells cultured with the copolymer maintained normal growth; only a few spots of damaged DNA were identified (Figure 8b, Figure S15b, green light). Moreover, flow cytometry analysis data indicated damaged DNA in ∼3.8% and ∼85.1% of cells before and after UV irradiation, respectively (Figure 8c, left and middle). In contrast, in ∼3.7% of cells cultured with P(1)(4)-coP(PEGMA) damaged DNA was detected after exposure to UV light (Figure 8c, right), verifying that the copolymer played an important role in protecting the DNA despite a vague molecular mechanism. Possible Extension and Challenges. This was an initial attempt to seek new synthetic methods that combine MCRs and HTP to prepare monomers/polymers; however, the current research still has room for improvement. Besides aromatic aldehydes, many other EOs extracts (terpenes, flavonoids, vitamins, etc.) are bioactive and contain carboxylic acids, aldehydes, ketones, and amine groups. These natural products might also be used directly as reactants for different MCRs and can prepare new polymers with interesting E
DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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quickly synthesized via single-electron transfer−atom transfer radical polymerization (SET-ATRP), sulfur-free reversible addition−fragmentation chain transfer (RAFT) emulsion polymerization, ultrafast RAFT polymerization, photoinduced control radical polymerizations (CRPs), etc.75−84 Some oxygen-tolerant CRPs make the polymerization easier in open tubes or well-plates without the degassing process. Examples are photoinduced electron/energy transfer-RAFT (PETRAFT), ultrafast RAFT, etc.79,85−90 More recently, the HTP strategy has been employed for oxygen-tolerant CRPs to easily synthesize well-defined (co)polymers and study the interesting structure−activity relationships.18,85,86,88,90 Thus, the future combination of methods in the current research with these modern CRP techniques might further simplify polymerization and offer more choices (main chain sequences, molecular weights, topology structures, etc.) to tune the properties/ functions of polymers, leading to further amplified number and diversity of polymer library.
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CONCLUSIONS In summary, we used five aromatic aldehydes that can be extracted from EOs to prepare 25 monomers via a mini-HTP Biginelli reaction. These monomers were used to prepare 25 corresponding polymers via HTP radical polymerization. This confirmed the superiority of MCRs in quickly amplifying molecular diversity. This improved the HTP methods to rapidly establish sample libraries. The HTP analysis can then screen polymers according to different criteria and finally achieve a water-soluble copolymer with low cytotoxicity to protect cells against UV damage. This highlights the value of MCRs and HTP methods to achieve new functional polymers with useful properties. This is the first facile preparation of monomer/polymer libraries by the combination of MCRs and the HTP strategy. These results might prompt a broader study of MCRs in polymer science and lead to the development of other HTP systems through different MCRs that can achieve various libraries of new monomers/polymers.
Figure 8. Staining of damaged DNA in L929 cells after UV irradiation (15 min) (a) without or (b) with P(1)(4)-co-P(PEGMA) (10 mg/ mL). Green light: 515−560 nm band-pass excitation filters, bar = 100 μm; (c) flow cytometry analyses of damaged DNA in L929 cells (left: cells prior to UV irradiation; middle: cells after 15 min UV irradiation; right: cells after 15 min UV irradiation in the presence of P(1)(4)-coP(PEGMA)). DsRed-A channel: excitation wavelength 561 nm.
physicochemical and/or biological properties. Meanwhile, some natural polyphenols (catechin, quercetin, etc.) were used to prepare bioactive polymers.56,57 These natural products and their derivatives will also be good resources to prepare new functional polymers via suitable MCRs. The Biginelli reaction is powerful but does have some limitations. In the current research, heating is necessary for the Biginelli reaction, which might limit the preparation of thermosensitive monomers/polymers. The preparation of polymers via MCRs has been a growing area of research in polymer chemistry since Meier and co-workers reported the preparation of polymers via the tricomponent Passerini reaction.40 Many MCRs have been used to prepare many elegant polymers. They offer additional choices when the reactants or conditions or both are not suitable for the Biginelli reaction. Examples include Passerini, Ugi, Kabachnik−Fields, Hantzsch, Mannich, and Debus−Radziszewski reactions40,42,58−67 as well as the mecaptoacetic acid locking imine (MALI), thiolactone-based, and metal-catalyzed multicomponent reactions.49,68−74 The HTP methods in the current research are very simple (small sample pool, simple polymer structures, and semiquantitative analysis), but this work still resulted in a polymer with potential UV-protective applications. This highlights the utility of HTP methods in developing new functional polymers. Nowadays, polymers with controlled molecular weights, narrow polydispersity indices, and complex topologic structures can be
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EXPERIMENTAL SECTION
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ASSOCIATED CONTENT
HTP Preparation of P(X)(Y) Polymers. The polymers (P(X)(Y)) were prepared through HTP radical polymerization of M(X)(Y). Typically, M(1)(1) (0.69 g, 2.0 mmol) and ABVN (0.01 g, 0.04 mmol) were charged into a 15 mL centrifuge tube along with DMF (2.0 mL). The tube was sealed with a rubber septum and purged by nitrogen flow for 15 min and then put into an isothermal shaker (70 °C) for 14 h. The polymerization was quenched by putting the test tube into an ice−water bath. A sample (∼20−40 μL) was taken for 1H NMR and GPC analyses. The polymer was purified by precipitation in diethyl ether three times and then dried under vacuum to a white powder (P(1)(1)) (0.62 g, ∼90%). The other polymers were simultaneously prepared through the same procedure. Cell Culture. L929 cells are a fibroblast cell line from mice. They were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were incubated at 37 °C, 5% CO2. Culture medium was changed every 2 days to maintain the exponential growth of the cells.
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b01576. F
DOI: 10.1021/jacs.8b01576 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Detailed HTP methods to synthesize/analyze monomers and polymers, cell experiments, etc. (PDF)
AUTHOR INFORMATION
Corresponding Author
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[email protected] ORCID
Lei Tao: 0000-0002-1735-6586 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Science Foundation of China (21574073, 21534006) and the Postdoctoral Science Foundation of China (2014M552685).
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