Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction

European Polymer Journal xxx (2015) xxx–xxx

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction Ansgar Sehlinger a, Katrin Ochsenreither b, Nikolai Bartnick a, Michael A.R. Meier a,⇑ a

Laboratory of Applied Chemistry, Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany b

a r t i c l e

i n f o

Article history: Received 11 November 2014 Received in revised form 15 January 2015 Accepted 20 January 2015 Available online xxxx Keywords: Ugi four-component reaction Multi-component reaction Polyacrylamide Free radical polymerization Biocompatible

a b s t r a c t A novel class of acrylamide monomers was synthesized using the Ugi four-component reaction. The application of acrylic acid and a variety of amines, aldehydes, and isocyanides as reactants led to a set of diversely substituted acrylamides in a highly straightforward one-pot procedure. These acrylamides were subjected to free radical polymerization yielding amorphous polymers with various designable side-chains. Subsequently, the synthesized polyacrylamides were tested for their biological activity by a modified Japanese Industrial Standard Z 2801:2000 protocol. All tested polymers showed high tolerance toward Gram-negative Escherichia coli and Pseudomonas fluorescens as well as Gram-positive Bacillus subtilis bacteria, thus suggesting these ‘‘easy to tune’’ polymers for applications requiring biocompatibility. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Polymers evolved into indispensable materials in many areas of everyday life including clothing, packaging, or health care. While for most applications optical or mechanical properties are of main interest, medically applied polymers must fulfill further criteria, such as the possibility of being sterilized and particularly being biocompatible. Biocompatible materials are characterized by full acceptance of living organisms, e.g. by contact with biological fluid or tissue, and thus do not trigger immunological response [1]. Biocompatible polymers can be obtained synthetically or from natural resources. Natural biopolymers, mainly categorized into polysaccharides and proteins, are used for a long time in the food industry as thickeners, in health care or in the pharmaceutical industry as emulsifiers, or as drug carriers [2–5]. Typical representatives are starch,

⇑ Corresponding author. E-mail address: [email protected] (M.A.R. Meier).

chitosan, alginate and gelatin derivatives. Natural biopolymers generally stand out for their very low or non-existing toxicity and their good biodegradability, inherently implied by their biological origin. However, synthetic biocompatible polymers are often favorable due to their higher stability and mechanical strength, which can be required for specific biomedical applications [6]. Moreover, they usually show better performance in solubility, thus simplifying their processing and application [7]. In addition, synthetic polymers are nowadays thoroughly examined regarding their mechanical and thermal behavior or the kinetics of polymerization, providing the necessary knowledge to synthesize tailormade polymers for individual applications [8]. For example, polycaprolactone, often used in tissue engineering, is known for its excellent biocompatibility, but also for its poor thermal and mechanical properties [9]. Polymers, such as polyamides, overcome these drawbacks through blending or incorporation into copolymers, or by modification of their side-chains to introduce the desired physical properties [10–13]. Polyethylene glycol (PEG) has also

http://dx.doi.org/10.1016/j.eurpolymj.2015.01.032 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sehlinger A et al. Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction. Eur Polym J (2015), http://dx.doi.org/10.1016/j.eurpolymj.2015.01.032

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proven to be a synthetic polymer with excellent biocompatibility for use in many biomedical applications [14,15]. In drug delivery, conjugation of PEG to therapeutic agents (i.e. PEGylation) improved the drug’s pharmacological and biologic activity [16,17]. In comparison to PEG, which allows only a single-site modification, the use of polyoxazolines (POX) for POXylations provides greater versatility for incorporation of greater monomer variability and thus composition [18–20]. There still remains a synthetic challenge to synthesize biocompatible polymers with the desired high performance through controlled chemical modifications. Here, we present a novel synthetic methodology for the straightforward modification of acrylamide monomers that, when polymerized, represent another essential class of synthetic polymers [21–23]. The approach taken in this work was to take advantage of the Ugi four-component reaction (Ugi-4CR) [24], which is a multi-component reaction (MCR), to make a wide range of monomers with biocompatible side-groups. The Ugi reaction involves the coupling of four chemical functionalities, including carboxylic acids, amines, aldehydes, and isocyanides, and has been used extensively to produce synthetic peptides in drug discovery or for the total synthesis of drug candidates owing to the modular nature of the Ugi reaction [25–28]. However, there are only a few examples of this synthetic approach in polymer chemistry [29–31], as the formation of hydrogels through cross-linking of amine- or acid-containing polysaccharides (e.g. hyaluronic and alginic acid, carboxymethyl cellulose or 1(deoxylactit-1-yl)chitosan) [32–36]. The Ugi-4CR further provided a strategy for the efficient conjugation of two types of polymers to either a fluorescent molecule, chemical agent or protein [37,38]. With regard to the monomer synthesis, the Ugi-4CR allowed the preparation of polymers with polypeptide-like features [39], a-amide substituted polyamides [40], convertible polyamides [41], and polyurethanes [42]. Here, we report, for the first time, the synthesis of Ugi-derived acrylamide monomers and their polymers, allowing the simultaneous introduction of three different chemical moieties as part of the monomer side-group. The four components consisted of an amine, aldehyde and isocyanide, while acrylic acid was used throughout this work as the acid component. Due to the modular nature of this approach, each acrylamide can be customized for individual needs. To investigate their biocompatibility, these polyacrylamides were tested for their cell toxicity against Gram-negative and Gram-positive bacteria using a modified Japanese Industrial Standard Z 2801:2000 protocol [43,44]. In these tests, all employed polymers revealed biocompatibility, which makes this new class of polymers highly interesting candidates for biomedical applications.

2. Experimental section 2.1. Materials The following chemicals were used as received: cyclohexylamine 2a (99%, Aldrich), n-butylamine 2b (99.5%,

Aldrich), propylamine 2c (98%, Aldrich), benzylamine 2d (99%, Aldrich), aniline 2e (99%, Fluka), ethanolamine 2f (>99.5%, Aldrich), glycine methyl ester hydrochloride 2g (99%, Aldrich), isobutyraldehyde 3a (P98%, Aldrich), heptaldehyde 3b (95%, Aldrich), acetone 3c (>99.5%, VWR), paraformaldehyde 3d (P95%, Aldrich), benzaldehyde 3e (P99%, Aldrich), tert-butyl isocyanide 4a (98%, Aldrich), butyl isocyanide 4b (97%, Aldrich), benzyl isocyanide 4c (98%, Aldrich), phosphorous(V) oxychloride (phosphoryl chloride, 99%, Aldrich), 4-aminobutyric acid (>98%, Aldrich), thionyl chloride (>99%, Aldrich), trimethyl orthoformate (99%, Aldrich), diisopropylamine (>99%, Aldrich), 2,20 -azo-bis(2-methylpropionitrile) (AIBN, >98%, Aldrich), silica gel 60 (0.035–0.070, Aldrich), chloroform-d (CDCl3, 99.8 atom-% D, Euriso-Top), methanol-d4 (CD3OD, 99.8 atom-% D, Euriso-Top), polycaprolactone (average Mw = 14,000, average Mn = 10,000 by SEC, Aldrich), poly(D,L-lacide) (ester terminated, Mw = 10,000–18,000, Aldrich), Nylon 12 (pellets, 5 mm, Aldrich), CASO broth (for microbiology, Aldrich). Acrylic acid 1 (99%, Aldrich) was freshly distilled before use in Ugi-4CRs. Methyl 4-isocyanobutyrate 4d was synthesized according a procedure mentioned in several publications [40–45]. All solvents were used without any kind of purification. 2.2. Characterization NMR spectra were recorded on a Bruker AVANCE DPX spectrometer operating at 300 MHz for 1H- and at 75 MHz for 13C-measurements. CDCl3 was used as solvent and the resonance signal at 7.26 ppm and 77.16 ppm served as reference for the chemical shift d. In case of CD3OD the resonance signal at 3.31 ppm (1H) and 49.00 ppm (13C) was used. Polymers were characterized on a SEC System LC-20A (Shimadzu) equipped with a SIL-20A autosampler and RID-10A refractive index detector in THF (flow rate 1.0 mL/min) at 50 °C. The analysis was performed on the following column system: main-column PSS SDV analytical (5.0 lm, 300 mm  8.0 mm, 10,000 Å) with a PSS SDV analytical precolumn (5.0 lm, 50 mm  8.0 mm). For the calibration, narrow linear poly(methyl methacrylate) standards (Polymer Standards Service PPS, Germany) ranging from 1100 to 981,000 Da were used. The thermal properties of the prepared monomers and polymers were studied by differential scanning calorimetry (DSC) with a Mettler Toledo DSC stare system operating under nitrogen atmosphere using about 5 mg of the respective material for the analysis. The melting point of the monomers was determined by heating from 0 °C to 200 °C at 10 °C/min. The glass transition (Tg) of the polymers was recorded on the second heating scan by using the following method: heating from 20 °C to 100 °C at 20 °C/min, cooling from 100 °C to 20 °C at 20 °C/min and heating from 20 °C to 150 °C at 20 °C/min. Infrared spectra (IR) were recorded on a Bruker Alpha-p instrument in a frequency range from 3998 to 374 cm1 applying ATR technology. Fast atom bombardment (FAB) mass spectra were recorded on a Finnigan MAT 95 instrument. The protonated molecular ion is expressed by the term: [(M+H)]+.

Please cite this article in press as: Sehlinger A et al. Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction. Eur Polym J (2015), http://dx.doi.org/10.1016/j.eurpolymj.2015.01.032

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All thin layer chromatography experiments were performed on silica gel coated aluminum foil (silica gel 60 F254, Aldrich). Compounds were visualized by irradiation with a UV-lamp or by staining with Seebach-solution (mixture of phosphomolybdic acid hydrate, cerium(IV) sulfate, sulfuric acid and water). Bacteria tests according Japanese Industrial Standard Z 2801:2000 protocol were carried out at the Institute of Process Engineering in Life Sciences of the KIT. First, microscope slides were covered with 100 lL polymer solution (15 mg of the respective polymer per 1.0 mL chloroform), then shortly placed on a hot plate (180 °C) until a smooth and thin polymer film remained. The coated glass slides were exposed to UV light for sterilization overnight. Bacterial strains tested were Escherichia coli XL1-Blue (Stratagene), Pseudomonas fluorescens DSM 50,090, and Bacillus subtilis DSM 10T. For each strain, an overnight pre-culture (5.0 mL CASO broth in test tube, 30 °C, 150 rpm) was prepared from a single colony. A main-culture (10 mL CASO broth in 100 mL shaking flask, 30 °C, 150 rpm) was inoculated with 10 lL of pre-culture and cultivated to an OD600 of 0.6 (corresponds to approximately 5  108 cells/ mL). To obtain the working culture, the main culture was diluted 1:1000 with CASO broth to reach a final concentration of 5  105 cells/mL. The prepared glass slides were coated with 225 lL of the bacterial working culture and capped with a sterile cover glass. To prevent drying up of the cultures, the inoculated glass slides were placed on spacer inside a petri-dish filled with sterile water. After cultivation for 16 h at 30 °C, samples were transferred from the glass slide to an Eppendorf cup and diluted with fresh CASO broth in a 1:10 ratio and plated on CASO agar. After incubation for 24 h at 30 °C, colony-forming units (CFUs) were counted. Each polymer was tested for each bacterial strain in triplicates; the samples from each coated glass slide were diluted in duplicates. Therefore, presented CFUs are main values maintained from a sixfold determination. 2.3. Synthesis 2.3.1. General procedure for synthesis of acrylamides 5a–n via Ugi-4CR A mixture of amine 2a–g (12.0 mmol), aldehyde 3a–e (10.0 mmol), and 50 mL methanol was stirred at room temperature for 30 min. Then, acrylic acid 1 (865 mg, 816 lL, 12.0 mmol) and isocyanide 4a–d (10.0 mmol) were added. After 24 h reaction time, methanol was removed and the residue was purified by silica gel column chromatography to yield the respective acrylamides 5a–n. 2.3.1.1. N-(tert-butyl)-2-(N-cyclohexylacrylamido)-3-methylbutanamide 5a. Colorless solid (2.62 g, 85%); Rf = 0.46 (nhexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.78 (d, J = 6.3 Hz, 3 H, CHCH3), 0.99 (d, J = 6.4 Hz, 3 H, CHCH3), 1.06–1.94 (m, 10 H, 5 CH2), 1.30 (s, 9 H, t-Bu), 2.74–3.25 (m, 2 H, NCHCH), 3.61 (tt, J = 11.8, 3.5 Hz, 1 H, NCHCH2), 5.70 (dd, J = 10.4, 1.7 Hz, 1 H, CH2@CHCO), 6.26 (dd, J = 16.8, 1.7 Hz, 1 H, CH2@CHCO), 6.52 (dd, J = 16.8, 10.4 Hz, 1 H, CH2@CHCO), 8.41 (br, 1 H, NH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 19.87, 20.59, 25.08, 25.85, 26.25, 26.69, 28.66, 31.28, 31.57, 50.43,

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127.83, 130.15, 168.24; FAB of C18H32N2O2 (M+H+ = 309.3); HRMS (FAB) of C18H32N2O2 [M+H]+ calc. 309.2537, found 309.2537; IR (ATR) m = 3263.3, 2962.7, 2929.7, 2856.7, 1667.4, 1637.6, 1601.7, 1546.1, 1436.6, 1389.1, 1361.2, 1302.6, 1288.7, 1225.6, 1160.9, 1144.1, 1110.6, 1054.5, 993.7, 977.9, 894.1, 798.7, 692.9, 609.5, 486.5 cm1; Tm = 75 °C. 2.3.1.2. N-(tert-butyl)-2-(N-butylacrylamido)-3-methylbutanamide 5b. Colorless solid (2.31 g, 82%); Rf = 0.34 (n-hexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.80 (d, J = 6.6 Hz, 3 H, CHCH3), 0.90 (t, J = 7.3 Hz, 3 H, CH2CH3), 0.96 (d, J = 6.4 Hz, 3 H, CHCH3), 1.20–1.34 (m, 2 H, CH2CH3), 1.30 (s, 9 H, t-Bu), 1.41–1.68 (m, 2 H, NCH2CH2), 2.28–2.51 (m, 1 H, CH(CH3)2), 3.21– 3.47 (m, 2 H, NCH2), 4.09–4.36 (m, 1 H, NCHCO), 5.74 (dd, J = 10.1, 2.2 Hz, 1 H, CH2@CHCO), 6.35–6.49 (br, 1 H, NH), 6.39 (dd, J = 16.6, 2.2 Hz, 1 H, CH2@CHCO), 6.56 (dd, J = 16.6, 10.1 Hz, 1 H, CH2@CHCO); 13C NMR (CDCl3, 75 MHz): d (ppm) = 13.72, 18.92, 19.86, 20.32, 26.60, 28.65, 32.41, 51.11, 128.00, 128.70, 167.58, 169.99; FAB of C16H30N2O2 (M+H+ = 283.3); HRMS (FAB) of C16H30N2O2 [M+H]+ calc. 283.2380, found 283.2382; IR (ATR) m = 3295.3, 3073.2, 2959.9, 2869.8, 1670.9, 1642.4, 1605.2, 1554.7, 1479.2, 1437.5, 1359.9, 1342.5, 1282.2, 1249.1, 1221.0, 1183.7, 1142.3, 1125.4, 1062.5, 983.8, 964.4, 850.4, 797.0, 752.0, 670.6, 628.8, 600.6, 451.5 cm1; Tm = 113 °C. 2.3.1.3. N-(tert-butyl)-3-methyl-2-(N-propylacrylamido) butanamide 5c. Colorless solid (1.98 g, 74%); Rf = 0.30 (nhexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.80 (d, J = 6.6 Hz, 3 H, CHCH3), 0.86 (t, J = 7.4 Hz, 3 H, CH2CH3), 0.96 (d, J = 6.4 Hz, 3 H, CHCH3), 1.30 (s, 9 H, t-Bu), 1.43–1.74 (m, 2 H, CH2CH3), 2.28–2.51 (m, 1 H, CH(CH3)2), 3.20–3.41 (m, 2 H, NCH2), 4.10–4.35 (m, 1 H, NCHCO), 5.74 (dd, J = 10.1, 2.2 Hz, 1 H, CH2@CHCO), 6.35–6.60 (br, 1 H, NH), 6.39 (dd, J = 16.6, 2.2 Hz, 1 H, CH2@CHCO), 6.55 (dd, J = 16.6, 10.1 Hz, 1 H, CH2@CHCO); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.41, 18.91, 19.86, 23.58, 26.59, 28.65, 51.09, 127.99, 128.70, 167.60, 170.04; FAB of C15H28N2O2 (M+H+ = 269.2); HRMS (FAB) of C15H28N2O2 [M+H]+ calc. 269.2224, found 269.2222; IR (ATR) m = 3294.2, 3076.4, 2959.0, 2872.0, 1670.8, 1643.4, 1604.5, 1554.7, 1478.4, 1438.4, 1361.9, 1342.5, 1306.9, 1282.6, 1241.1, 1225.1, 1193.5, 1146.2, 1123.2, 1061.5, 986.0, 965.8, 901.7, 832.6, 797.1, 759.1, 743.1, 630.9, 600.7, 474.8, 439.5, 408.7 cm1; Tm = 127 °C. 2.3.1.4. 2-(N-benzylacrylamido)-N-(tert-butyl)-3-methylbutanamide 5d. Colorless solid (2.42 g, 77%); Rf = 0.39 (nhexane/ethyl acetate = 4:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.82 (d, J = 6.6 Hz, 3 H, CHCH3), 0.96 (d, J = 6.5 Hz, 3 H, CHCH3), 1.25 (s, 9 H, t-Bu), 2.29–2.50 (m, 1 H, CH(CH3)2), 4.45 (d, J = 10.8 Hz, 1 H, NCHCO), 4.60– 4.88 (m, 2 H, ArACH2), 5.58–5.67 (m, 1 H, CH2@CHCO), 6.27 (br, 1 H, NH), 6.37–6.46 (m, 2 H, CH2@CHCO, CH2@CHCO), 7.10–7.36 (m, 5 H, 5 ArAH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 19.06, 19.72, 27.51, 28.63, 51.36, 126.49, 127.25, 128.25, 128.57, 129.21, 137.84, 168.29, 169.17; FAB of C19H28N2O2 (M+H+ = 317.2); HRMS (FAB)

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of C19H28N2O2 [M+H]+ calc. 317.2229, found 317.2226; IR (ATR) m = 3321.5, 2962.3, 1673.7, 1637.3, 1599.3, 1537.2, 1496.8, 1446.7, 1418.9, 1393.6, 1367.9, 1329.1, 1297.8, 1279.2, 1223.4, 1164.7, 1128.1, 1060.4, 1020.3, 992.9, 971.4, 941.7, 798.0, 735.8, 695.0, 656.6, 606.1, 534.5, 495.0, 455.1 cm1; Tm = 111 °C. 2.3.1.5. N-(tert-butyl)-3-methyl-2-(N-phenylacrylamido) butanamide 5e. Colorless solid (1.60 g, 53%); Rf = 0.43 (nhexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.96 (d, J = 6.5 Hz, 3 H, CHCH3), 0.97 (d, J = 6.4 Hz, 3 H, CHCH3), 1.35 (s, 9 H, t-Bu), 2.24–2.44 (m, 1 H, CH(CH3)2), 4.25 (d, J = 11.1 Hz, 1 H, NCHCO), 5.52 (dd, J = 10.3, 1.9 Hz, 1 H, CH2@CHCO), 5.93 (dd, J = 16.8, 10.3 Hz, 1 H, CH2@CHCO), 6.34 (dd, J = 16.8, 1.9 Hz, 1 H, CH2@CHCO), 6.87 (s, 1 H, NH), 7.17–7.46 (m, 5 H, 5 ArAH); 13 C NMR (CDCl3, 75 MHz): d (ppm) = 19.73, 20.05, 27.03, 28.71, 51.07, 70.97, 127.94, 128.28, 129.02, 129.29, 129.33, 140.50, 167.09, 169.53; FAB of C18H26N2O2 (M+H+ = 303.2); HRMS (FAB) of C18H26N2O2 [M+H]+ calc. 303.2067, found 303.2065; IR (ATR) m = 3317.1, 3068.1, 2962.7, 1676.4, 1642.1, 1607.3, 1590.3, 1552.6, 1488.4, 1451.3, 1405.4, 1359.6, 1336.5, 1309.7, 1251.8, 1174.2, 1044.4, 979.3, 963.4, 927.5, 810.8, 790.0, 765.8, 728.1, 700.6, 658.8, 630.9, 604.9, 517.1, 487.4, 437.9, 418.9 cm1; Tm = 112 °C. 2.3.1.6. N-(tert-butyl)-2-(N-(2-hydroxyethyl)acrylamido)-3methylbutanamide 5f. Colorless solid (1.06 g, 39%); Rf = 0.04 (n-hexane/ethyl acetate = 5:1); 1H NMR (CD3OD, 300 MHz, 2:3 mixture of cis/trans-amide bonds): d (ppm) = 0.77–0.87 (m, 3 H, CHCH3), 0.92–1.01 (m, 3 H, CHCH3), 1.32 (s, 9 H, t-Bu), 2.23–2.43 (m, 1 H, CH(CH3)2), 3.52–3.77 (m, 4 H, NCH2CH2), 3.86, 4.33 (2 d, J = 10.5 and 10.9 Hz, 1 H, NCHCO), 5.69–5.83 (m, 1 H, CH2@CHCO), 6.18–6.35 (m, 1 H, CH2@CHCO), 6.77–6.92 (m, 1 H, CH2@CHCO); 13C NMR (CDCl3, 75 MHz, 2:3 mixture of cis/trans-amide bonds): d (ppm) = 19.12, 20.09, 28.44, 28.69, 29.87, 47.94, 52.16, 52.34, 60.95, 61.79, 66.29, 128.97, 129.33, 129.57, 129.87, 169.79, 170.09, 171.23, 171.81; FAB of C14H26N2O3 (M+H+ = 271.3); HRMS (FAB) of C14H26N2O3 [M+H]+ calc. 271.2016, found 271.2018; IR (ATR) m = 3394.9, 3292.1, 3082.0, 2962.5, 1635.5, 1603.8, 1558.4, 1455.2, 1429.7, 1391.1, 1363.3, 1347.7, 1270.6, 1257.2, 1215.1, 1175.9, 1061.6, 981.1, 930.0, 851.9, 798.8, 635.2, 599.8, 491.3, 440.0, 413.4 cm1; Tm = 117 °C. 2.3.1.7. Methyl 2-(N-(1-(tert-butylamino)-3-methyl-1oxobutan-2-yl)acrylamido)acetate 5g. Colorless solid (2.71 g, 91%); Rf = 0.05 (n-hexane/ethyl acetate = 5:1); 1H NMR (CD3OD, 300 MHz, 1:1 mixture of cis/trans-amide bonds): d (ppm) = 0.75–0.87 (m, 3 H, CHCH3), 0.88–0.97 (m, 3 H, CHCH3), 1.27, 1.30 (2 s, 9 H, t-Bu), 1.98–2.21 (m, 1 H, CH(CH3)2), 3.65, 3.67 (2 s, 3 H, OMe), 4.17–4.50 (m, 2 H, NCH2), 4.02, 4.63 (2 d, J = 10.5 and 11.0 Hz, 1 H, NCHCO), 5.72, 5.78 (2 dd, J = 10.6, 1.9 Hz, 1 H, CH2@CHCO), 6.19–6.32 (m, 1 H, CH2@CHCO), 6.53, 6.90 (2 dd, J = 16.6, 10.6 Hz, 1 H, CH2@CHCO), 7.68, 7.88 (2 br, 1 H, NH); 13C NMR (CD3OD, 75 MHz, 1:1 mixture of cis/trans-amide bonds): d (ppm) = 18.86, 19.20, 19.48, 28.67, 28.78, 29.99,

45.71, 46.30, 52.20, 52.36, 52.41, 52.71, 63.54, 67.30, 128.91, 129.22, 129.29, 129.58, 169.59, 170.10, 170.21, 170.66, 171.05, 171.33; FAB of C15H26N2O4 (M+H+ = 299.2); HRMS (FAB) of C15H26N2O4 [M+H]+ calc. 299.1965, found 299.1976; IR (ATR) m = 3342.7, 2958.9, 1752.2, 1675.3, 1643.2, 1603.1, 1542.6, 1447.3, 1420.7, 1394.2, 1363.5, 1303.6, 1208.2, 1187.1, 1055.7, 1024.8, 973.3, 836.0, 795.3, 759.8, 739.5, 716.2, 601.2, 554.0, 488.3, 438.2, 417.5 cm1; Tm = 142 °C. 2.3.1.8. N-(tert-butyl)-2-(N-propylacrylamido)octanamide 5h. Yellowish oil (1.35 g, 44%); Rf = 0.29 (n-hexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.80– 0.94 (m, 6 H, 2 CH3), 1.15–1.36 (m, 8 H, 4 CH2), 1.29 (s, 9 H, t-Bu), 1.42–1.74 (m, 3 H, NCHCH2a, NCH2CH2), 1.84– 2.02 (m, 1 H, NCHCH2b), 3.14–3.36 (m, 2 H, NCH2), 4.77 (t, J = 7.6 Hz, 1 H, NCHCO), 5.75 (dd, J = 10.1, 2.1 Hz, 1 H, CH2@CHCO), 6.37–6.59 (br, 1 H, NH), 6.40 (dd, J = 16.5, 2.1 Hz, 1 H, CH2@CHCO), 6.55 (dd, J = 16.6, 10.1 Hz, 1 H, CH2@CHCO); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.23, 13.93, 22.44, 23.80, 26.02, 28.02, 28.50, 29.01, 31.55, 46.30, 50.80, 58.01, 127.80, 128.58, 167.42, 170.34; FAB of C18H34N2O2 (M+H+ = 311.3); HRMS (FAB) of C18H34N2O2 [M+H]+ calc. 311.2626, found 311.2625; IR (ATR) m = 3312.9, 2959.2, 2925.6, 2857.0, 1679.3, 1641.8, 1604.5, 1536.3, 1452.2, 1428.3, 1390.7, 1362.2, 1225.1, 1134.7, 1058.3, 977.0, 956.3, 899.5, 795.1, 725.0, 628.3 cm1. 2.3.1.9. N-(1-(tert-butylamino)-2-methyl-1-oxopropan-2-yl)N-propylacrylamide 5i. Colorless solid (0.87 g, 34%); Rf = 0.32 (n-hexane/ethyl acetate = 1:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.92 (t, J = 7.4 Hz, 3 H, CH2CH3), 1.30 (s, 9 H, t-Bu), 1.49 (s, 6 H, C(CH3)2), 1.57–1.74 (m, 2 H, CH2CH3), 3.32–3.38 (m, 2 H, NCH2), 5.56 (br, 1 H, NH), 5.64 (dd, J = 10.2, 2.2 Hz, 1 H, CH2@CHCO), 6.30 (dd, J = 16.6, 2.2 Hz, 1 H, CH2@CHCO), 6.49 (dd, J = 16.6, 10.2 Hz, 1 H, CH2@CHCO); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.22, 25.03, 28.53, 46.08, 50.75, 62.64, 127.99, 129.19, 166.86, 174.14; FAB of C14H26N2O2 (M+H+ = 255.3); HRMS (FAB) of C14H26N2O2 [M+H]+ calc. 255.2067, found 255.2065; IR (ATR) m = 3338.2, 2959.9, 1647.4, 1614.6, 1519.8, 1449.0, 1417.3, 1358.1, 1280.0, 1247.0, 1215.9, 1175.8, 1139.4, 1117.2, 987.3, 949.4, 896.3, 799.3, 775.6, 745.2, 629.5, 558.3, 468.2, 401.0 cm1; Tm = 81 °C. 2.3.1.10. N-(2-(tert-butylamino)-2-oxoethyl)-N-propylacrylamide 5j. Colorless solid (0.73 g, 32%); Rf = 0.32 (n-hexane/ethyl acetate = 1:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.91 (t, J = 7.4 Hz, 3 H, CH3), 1.31 (s, 9 H, t-Bu), 1.54–1.73 (m, 2 H, CH2CH3), 3.34–3.44 (m, 2 H, NCH2), 3.93 (s, 2 H, CONCH2CO), 5.75 (dd, J = 10.2, 2.1 Hz, 1 H, CH2@CHCO), 6.39 (dd, J = 16.5, 1.8 Hz, 1 H, CH2@CHCO), 6.47 (br, 1 H, NH), 6.58 (dd, J = 16.7, 10.3 Hz, 1 H, CH2@CHCO); 13 C NMR (CDCl3, 75 MHz): d (ppm) = 11.08, 22.30, 28.64, 51.03, 51.44, 52.85, 126.97, 129.03, 167.05, 168.71; FAB of C12H22N2O2 (M+H+ = 227.3); HRMS (FAB) of C12H22N2O2 [M+H]+ calc. 227.1754, found 227.1752; IR (ATR) m = 3304.6, 3075.1, 2964.1, 2931.8, 2874.5, 1642.7, 1606.0, 1545.3, 1451.0, 1391.5, 1361.8, 1263.4, 1222.3,

Please cite this article in press as: Sehlinger A et al. Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction. Eur Polym J (2015), http://dx.doi.org/10.1016/j.eurpolymj.2015.01.032

A. Sehlinger et al. / European Polymer Journal xxx (2015) xxx–xxx

1126.3, 1061.4, 976.5, 954.4, 888.8, 794.6, 665.4, 560.3, 469.8 cm1; Tm = 77 °C. 2.3.1.11. N-(2-(tert-butylamino)-2-oxo-1-phenylethyl)-Npropylacrylamide 5k. Colorless solid (2.54 g, 84%); Rf = 0.08 (n-hexane/ethyl acetate = 5:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.65 (t, J = 7.3 Hz, 3 H, CH3), 0.83– 1.03 (m, 1 H, NCH2CH2a), 1.34 (s, 9 H, t-Bu), 1.40–1.51 (m, 1 H, NCH2CH2b), 3.24–3.42 (m, 2 H, NCH2), 5.74 (dd, J = 10.1, 2.1 Hz, 1 H, CH2@CHCO), 5.78 (br, 1 H, NH), 5.97 (s, 1 H, NCHCO), 6.43 (dd, J = 16.5, 1.6 Hz, 1 H, CH2@CHCO), 6.57 (dd, J = 16.6, 10.0 Hz, 1 H, CH2@CHCO), 7.30–7.47 (m, 5 H, 5 ArAH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.16, 23.74, 28.66, 48.17, 51.56, 62.25, 127.89, 128.32, 128.70, 128.80, 129.34, 135.94, 166.88, 169.09; FAB of C18H26N2O2 (M+H+ = 303.3); HRMS (FAB) of C18H26N2O2 [M+H]+ calc. 303.2067, found 303.2068; IR (ATR) m = 3302.6, 2965.8, 1675.9, 1636.9, 1590.9, 1551.9, 1479.8, 1433.7, 1391.1, 1358.9, 1276.0, 1250.1, 1223.3, 1198.1, 1129.3, 1059.1, 1032.4, 978.9, 960.1, 900.9, 807.7, 761.7, 737.9, 701.2, 655.7, 614.0, 556.3, 501.9, 454.3, 413.5 cm1; Tm = 101 °C. 2.3.1.12. N-butyl-3-methyl-2-(N-propylacrylamido)butanamide 5l. Colorless oil (2.29 g, 85%); Rf = 0.38 (n-hexane/ethyl acetate = 2:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.83 (d, J = 6.7 Hz, 3 H, CHCH3), 0.85–0.94 (m, 6 H, 2 CH2CH3), 0.97 (d, J = 6.4 Hz, 3 H, CHCH3), 1.22–1.37 (m, 2 H, NCH2CH2CH2), 1.38–1.50 (m, 2 H, NCH2CH2CH2), 1.52–1.67 (m, 2 H, NCH2CH2CH3), 2.38–2.58 (m, 1 H, CH(CH3)2), 3.12–3.45 (m, 4 H, 2 NCH2), 4.08–4.34 (m, 1 H, NCHCO), 5.75 (dd, J = 10.1, 2.1 Hz, 1 H, CH2@CHCO), 6.39 (dd, J = 16.6, 2.1 Hz, 1 H, CH2@CHCO), 6.54 (dd, J = 16.6, 10.1 Hz, 1 H, CH2@CHCO), 6.75 (br, 1 H, NH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.19, 13.60, 18.90, 19.75, 19.90, 23.36, 26.39, 31.33, 38.86, 47.74, 65.80, 127.86, 128.60, 167.50, 170.70; FAB of C15H28N2O2 (M+H+ = 269.3); HRMS (FAB) of C15H28N2O2 [M+H]+ calc. 269.2224, found 269.2222; IR (ATR) m = 3315.0, 3077.3, 2962.9, 2874.5, 1644.4, 1607.1, 1548.1, 1433.7, 1369.9, 1280.7, 1237.4, 1195.4, 1145.8, 1060.1, 978.9, 901.7, 796.3, 745.8, 633.7 cm1. 2.3.1.13. N-benzyl-3-methyl-2-(N-propylacrylamido)butanamide 5m. Colorless oil (2.20 g, 73%); Rf = 0.43 (n-hexane/ethyl acetate = 2:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.76–0.90 (m, 6 H, CHCH3, CH2CH3), 0.99 (d, J = 6.4 Hz, 3 H, CHCH3), 1.38–1.63 (m, 2 H, CH2CH3), 2.40–2.62 (m, 1 H, CH(CH3)2), 3.15–3.45 (m, 2 H, NCH2CH2), 4.20–4.50 (m, 3 H, NCHCO, CH2Ar), 5.74 (dd, J = 10.1, 2.1 Hz, 1 H, CH2@CHCO), 6.37 (dd, J = 16.6, 2.1 Hz, 1 H, CH2@CHCO), 6.53 (dd, J = 16.6, 10.1 Hz, 1 H, CH2@CHCO), 7.04–7.40 (m, 6 H, 5 ArAH, NH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.25, 18.97, 19.83, 23.38, 26.46, 43.14, 47.70, 65.58, 127.15, 127.54, 127.78, 128.46, 128.80, 138.33, 167.63, 170.74; FAB of C18H26N2O2 (M+H+ = 303.3); HRMS (FAB) of C18H26N2O2 [M+H]+ calc. 303.2067, found 303.2069; IR (ATR) m = 3296.1, 2961.6, 2872.3, 1673.1, 1640.5, 1603.2, 1535.9, 1453.4, 1428.0, 1364.8, 1278.8, 1234.9, 1191.0, 1143.2, 1123.5, 1080.7, 1059.4, 1029.1, 977.3, 901.1, 794.7, 750.2, 698.2, 604.3, 499.1, 454.2 cm1.

5

2.3.1.14. Methyl 4-(3-methyl-2-(N-propylacrylamido) butanamido)butanoate 5n. Colorless oil (2.77 g, 89%); Rf = 0.30 (n-hexane/ethyl acetate = 1:1); 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.83 (d, J = 6.7 Hz, 3 H, CHCH3), 0.84–0.91 (m, 3 H, CH2CH3), 0.97 (d, J = 6.5 Hz, 3 H, CHCH3), 1.46–1.65 (m, 2 H, CH2CH3), 1.74–1.87 (m, 2 H, COCH2CH2), 2.30 (t, J = 7.5 Hz, 2 H, COCH2), 2.40–2.58 (m, 1 H, CH(CH3)2), 3.14–3.41 (m, 4 H, 2 NCH2), 3.66 (s, 3 H, COOMe), 4.07–4.28 (m, 1 H, NCHCO), 5.76 (dd, J = 10.0, 2.2 Hz, 1 H, CH2@CHCO), 6.40 (dd, J = 16.6, 2.2 Hz, 1 H, CH2@CHCO), 6.54 (dd, J = 16.6, 10.0 Hz, 1 H, CH2@CHCO), 6.77–7.04 (br, 1 H, NH); 13C NMR (CDCl3, 75 MHz): d (ppm) = 11.17, 18.88, 19.72, 23.35, 24.58, 26.38, 31.12, 38.37, 47.75, 51.49, 65.83, 127.77, 128.75, 167.51, 170.92, 173.29; FAB of C16H28N2O4 (M+H+ = 313.3); HRMS (FAB) of C16H28N2O4 [M+H]+ calc. 313.2122, found 313.2121; IR (ATR) m = 3306.1, 2961.2, 2873.2, 1735.9, 1673.7, 1641.2, 1604.3, 1538.8, 1431.3, 1366.9, 1278.7, 1235.1, 1193.2, 1169.3, 1144.5, 1058.5, 978.8, 901.4, 879.8, 795.4, 745.8, 631.9, 440.6 cm1.

2.3.2. General polymerization procedure Acrylamides 5a–n (1.00 mmol) and 1.64 mg AIBN (0.01 mmol, 1 mol%) were placed in a round-bottom flask and dissolved in 1.5 mL ethyl acetate. The reaction mixture was degassed with argon for ten minutes and afterwards heated to 70 °C for seven hours. After this period, the solvent was evaporated and the residue was taken up in 1.0 mL dichloromethane. This solution was slowly dropped into cold hexane. The precipitate was filtered off and dried in vacuum to yield polyacrylamides P1–14. Polymer 1: Polyacrylamide derived from monomer 5a. Colorless solid (254 mg, 82%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.61–2.00 (m, 27 H, 2 CHCH3, 5 CH2, t-Bu, CH2 backbone), 2.05–2.40 (br, 1 H, CH backbone), 2.65–3.20 (br, 2 H, NCHCH), 3.40–4.20 (br, 1 H, NCHCH2), 8.15 (br, 1 H, NH); Tg = 59 °C. Polymer 2: Polyacrylamide derived from monomer 5b. Colorless solid (222 mg, 79%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.60–1.10 (m, 9 H, 2 CHCH3, CH2CH3), 1.10– 1.68 (m, 13 H, NCH2CH2, t-Bu, CH2CH3), 1.79–2.66 (m, 4 H, CH(CH3)2, CH and CH2 backbone), 2.82–3.71 (br, 2 H, NCH2), 4.11–4.97 (br, 1 H, NCHCO), 8.16 (br, 1 H, NH); Tg = 62 °C. Polymer 3: Polyacrylamide derived from monomer 5c. Colorless solid (218 mg, 81%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.60–1.06 (m, 6 H, 2 CHCH3), 1.27 (br, 9 H, tBu), 1.62–2.62 (m, 6 H, CH(CH3)2, CH and CH2 backbone, CH2CH3), 2.76–3.66 (br, 2 H, NCH2), 4.07–4.76 (br, 1 H, NCHCO), 7.95 (br, 1 H, NH); Tg = 68 °C. Polymer 6: Polyacrylamide derived from monomer 5f. Colorless solid (246 mg, 91%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.58–1.08 (m, 6 H, 2 CHCH3), 1.30 (br, 9 H, tBu), 1.71–2.64 (m, 3 H, CH(CH3)2, CH2 backbone), 2.68– 5.15 (m, 6 H, NCHCO, NCH2CH2, CH backbone), 8.11 (br, 1 H, NH); Tg = 42 °C. Polymer 7: Polyacrylamide derived from monomer 5g. Colorless solid (251 mg, 84%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.59–1.08 (m, 6 H, 2 CHCH3), 1.27 (br, 9 H, tBu), 1.61–2.61 (m, 4 H, CH(CH3)2, CH and CH2 backbone),

Please cite this article in press as: Sehlinger A et al. Potentially biocompatible polyacrylamides derived by the Ugi four-component reaction. Eur Polym J (2015), http://dx.doi.org/10.1016/j.eurpolymj.2015.01.032

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3.62 (br, 3 H, OMe), 3.95–4.83 (m, 3 H, NCH2, NCHCO), 8.24 (br, 1 H, NH); Tg = 62 °C. Polymer 8: Polyacrylamide derived from monomer 5h. Colorless solid (233 mg, 75%).1H NMR (CDCl3, 300 MHz): d (ppm) = 0.69–1.36 (m, 14 H, 2 CH3, 4 CH2), 1.26 (s, 9 H, t-Bu), 1.55–2.74 (m, 7 H, NCHCH2, NCH2CH2, CH and CH2 backbone), 2.81–3.71 (br, 2 H, NCH2), 4.56 (br, 1 H, NCHCO), 6.50 (br, 1 H, NH); Tg = 56 °C. Polymer 9: Polyacrylamide derived from monomer 5i. Colorless solid (212 mg, 83%).1H NMR (CDCl3, 300 MHz): d (ppm) = 0.62–1.65 (m, 18 H, CH2CH3, t-Bu, C(CH3)2), 1.66–1.96 (m, 4 H, CH2CH3, CH2 backbone), 2.11–3.63 (m, 3 H, NCH2, CH backbone), 5.38 (br, 1 H, NH); Tg = 49 °C. Polymer 10: Polyacrylamide derived from monomer 5j. Colorless solid (217 mg, 96%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.83 (br, 3 H, CH3), 1.32 (br, 9 H, t-Bu), 1.69– 2.10 (m, 4 H, CH2CH3, CH2 backbone), 2.20–4.59 (m, 5 H, NCH2, CONCH2CO, CH backbone), 6.67 (br, 1 H, NH); Tg = 41 °C. Polymer 12: Polyacrylamide derived from monomer 5l. Colorless solid (229 mg, 85%).1H NMR (CDCl3, 300 MHz): d (ppm) = 0.57–1.10 (m, 12 H, 2 CHCH3, 2 CH2CH3), 1.11– 1.77 (m, 8 H, NCH2CH2CH3, NCH2CH2CH2, CH2 backbone), 1.90–3.99 (m, 6 H, 2 NCH2, CH(CH3)2, CH backbone), 4.11–4.83 (br, 1 H, NCHCO), 8.06 (br, 1 H, NH); Tg = 52 °C. Polymer 13: Polyacrylamide derived from monomer 5m. Colorless solid (239 mg, 79%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.51–1.06 (m, 9 H, 2 CHCH3, CH2CH3), 1.13– 1.88 (m, 4 H, CH2CH3, CH2 backbone), 1.89–2.57 (m, 2 H, CH(CH3)2, CH backbone), 2.63–3.41 (m, 2 H, NCH2CH2), 3.44–5.12 (m, 3 H, NCHCO, CH2Ar), 6.29–7.35 (m, 6 H, 5 ArAH, NH); Tg = 61 °C. Polymer 14: Polyacrylamide derived from monomer 5n. Colorless solid (267 mg, 86%). 1H NMR (CDCl3, 300 MHz): d (ppm) = 0.53–1.18 (m, 9 H, 2 CHCH3, CH2CH3), 1.25– 2.00 (m, 6 H, CH2CH3, COCH2CH2, CH2 backbone), 2.02– 2.51 (m, 3 H, COCH2, CH backbone), 2.61–3.56 (m, 5 H, 2 NCH2, CH(CH3)2), 3.64 (s, 3 H, COOMe), 4.50 (br, 1 H, NCHCO), 7.04 (br, 1 H, NH); Tg = 68 °C. 3. Results and discussion 3.1. Monomer synthesis In order to obtain acrylamide monomers, acrylic acid 1 was used as key substrate in the Ugi-4CR along with a variety of amines 2a–g, aldehydes 3a–e, and isocyanides 4a–d. Performing the reaction, first the aldehyde and amine component were mixed to pre-form the corresponding imine. Then, acrylic acid and the isocyanide were added and the reaction was performed at room temperature for one day (Scheme 1). To increase the yield, acrylic acid and the amine component were used in slight access of 1.2 equivalents. Their remainder was easily removed by subsequent silica gel column chromatography. First, the amine component was varied bearing aliphatic 2a–c, aromatic 2d–e, or functional group containing moieties 2f–g, while isobutyraldehyde 3a and tert-butyl isocyanide 4a were used in the respective Ugi-4CRs (Table 1, entry 5a–g). Most of the resulting acrylamides were obtained in very good yields of 74–91%, except for

products 5e and 5f. Product 5e was obtained in a moderate yield of 53%, caused by the lower nucleophilicity of the amine component due to the mesomeric effect of the aromatic ring. In case of 5f, the lower yield of 39% might result from the alcohol moiety of the amine, which interferes in the reaction mechanism. Compared to the amine component, variation of the aldehyde component is more restricted. Here, it is crucial to use aldehydes with blocked a-positions since otherwise undesired aldol reactions occur [46]. This side reaction can be observed when employing heptaldehyde 3b, propylamine 2c, and tert-butyl isocyanide 4a in the Ugi-4CR, consequently resulting in a moderate isolated yield of 44% (5h). Furthermore, acetone 3c and paraformaldehyde 3d were used, which yielded highly polar acrylamides 5i and 5j. Nevertheless, low yields were obtained since ketones are less reactive and paraformaldehyde is known for its low reactivity in isocyanide-based MCRs. Only benzaldehyde 3e (besides isobutyraldehyde 3a) demonstrates an excellent substrate for Ugi-4CRs giving 84% yield of acrylamide 5k. Finally, the isocyanide moiety R3 was altered while keeping propylamine 2c and isobutyraldehyde 3a fixed. Isocyanides are highly tolerated in isocyanide-based MCRs, and thus, regardless of the substitution, aliphatic, aromatic or functionalized isocyanides were incorporated into the acrylamide products 5l–n in good yields ranging from 73% to 89%. The successful synthesis of Ugi-derived monomers 5a– n was evidenced by mass spectrometry, IR, and NMR spectroscopy. Beside the detection of the expected mass, IR analysis verified the formation of amide linkages that all monomers have in common (Fig. 1). Two characteristic bands appear in the area between 1600 and 1680 cm1 corresponding to the C@O stretching of the secondary and tertiary amide. The absorption of the NAH stretching of the secondary amide is observed at around 3300 cm1. Furthermore, distinguishable signals referring to the ‘‘finger-print’’ area of amides are present at 1200–1330 cm1 and 1520–1580 cm1. In the 13C NMR spectrum, the carbonyl carbons appear in the very low field at 167 ppm (tertiary amide) and 170 ppm (secondary amide). Another characteristic functional group all monomers have in common is the conjugated double bond. The respective CAH resonance frequency of the valence vibrations occurs at 3000 cm1, whereas the deformation vibrations were observed between 900 and 1000 cm1. In the 13C NMR spectrum, the two carbons of the double bond appear at approximately 130 ppm. Both discussed functional groups (amide and acrylic system) show specific chemical shifts in the 1H NMR spectrum as well. For example, acrylamide 5n gives three distinguishable signals (dd) at 5.76, 6.40 and 6.54 ppm according to the acrylic protons (k), (k0 ) and (l) (Fig. 2). Moreover, the broad signal (m), corresponding to the amide proton, is observed at 6.93 ppm. All other signals are assigned in Fig. 2. In most cases, the Ugi products were obtained as colorless solids having a melting point around 100 °C, except for acrylamide monomers 5h and 5l–n, which are highly viscous substances. Interestingly, the modification of the isocyanide moiety seems to have the highest influence on the melting point.

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Scheme 1. Ugi-4CR of acrylic acid 1 and various amines (2a–g), aldehydes (3a–e) and isocyanides (4a–d) to yield diversely substituted acrylamide monomers 5a–n.

Table 1 Summary of all synthesized Ugi-4CR derived acrylamides 5a–n.

a

Producta

R1

R2

R3

Isolated yield (%)

Tm (°C)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n

-c-Hx (2a) -n-Bu (2b) -n-Pr (2c) -Bn (2d) -Ph (2e) -(CH2)2OH (2f) -Gly-Me (2g) -n-Pr (2c) -n-Pr (2c) -n-Pr (2c) -n-Pr (2c) -n-Pr (2c) -n-Pr (2c) -n-Pr (2c)

-i-Pr (3a) -i-Pr (3a) -i-Pr (3a) -i-Pr (3a) -i-Pr (3a) -i-Pr (3a) -i-Pr (3a) -n-Hx (3b) -(CH3)2 (3c) -H (3d) -Ph (3e) -i-Pr (3a) -i-Pr (3a) -i-Pr (3a)

-t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -t-Bu (4a) -n-Bu (4b) -Bn (4c) -(CH2)3CO2Me (4d)

85 82 74 77 53 39 91 44 34 32 84 85 73 89

75 113 127 111 112 117 142 Oil 81 77 101 Oil Oil Oil

Reaction conditions: room temperature, 24 h reaction time, 0.2 M solution in methanol, 1.2 eq excess of amine and carboxylic acid.

3.2. Polymerization Monomers 5a–n were then subjected to free radical polymerization. The reaction conditions were adopted from our previous study polymerizing diversely substituted acrylate monomers [45]. In short, the polymerization was

conducted in ethyl acetate at 70 °C with 1.0 mol% AIBN as thermal initiator (Scheme 2). After seven hours, the polymerization process was stopped (90% conversion) and the crude reaction mixture was purified by precipitation into cold hexane. In this way, high molecular weight polymers have been synthesized in yields of around 80% (Table 2).

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Scheme 2. Free radical polymerization of acrylamides 5a–n.

Fig. 1. IR spectra (ATR) of monomer 5n and respective polymer P14. While most absorptions remain unchanged, the vinyl band is absent in the polymer.

1

H NMR analysis confirmed the successful polymerization by the disappearance of the acrylic protons and broadening of all other signals (Fig. 3, representative P14). Only in case of acrylamides 5d, 5e, and 5k almost pure starting material was isolated. This result can either be explained by steric reasons (all three monomers contain stiff and bulky aromatic moieties) or by electronic reasons, such as the formation of a highly stable radical in benzylic position of 5d or 5k by chain transfer to monomer. However, since monomer 5m, having a benzylic position as well, was almost fully converted after seven hours reaction time (95% conversion, Mn = 22,650 g/mol), the latter possibility is less likely, whereas the former reason is confirmed comparing the molecular weight of other bulky monomers such as in

P1, P7 and P13 with polymers bearing flexible moieties. Here, SEC analysis clearly revealed lower Mn values for these polymers ranging between 20 and 30 kDa. Thus, the bulkiness of the substituents clearly limits the propagation rate or even prevents the polymerization completely (compare monomers 5d, 5e and 5k). The other way around is demonstrated with polyacrylamide P10. Accordingly, the formaldehyde derived monomer 5j shows the highest polymerization rate indicated by the high molecular weight (Mn) of 270,100 g/mol and dispersity of 3.05 (Fig. 4). This is certainly caused by the moiety R2 being only a proton. All other polymers reached Mn values of 50 kDa or more and dispersities around two (see Table 2). DSC measurements of the synthesized polymers revealed only glass and no melting transitions, which is typical for acrylamides. Compared to N,N-dimethylacrylamide (Tg = 89 °C) or N,N-diethylacrylamide (Tg = 80 °C), all Ugi-derived polymers exhibit lower glass transitions in the range of 41–68 °C. Some trends can be observed, for instance, for P12–14, the variation of R3 from flexible butyl to bulky benzyl or polar methyl butyrate moiety increases the Tg in an expected manner from 52 °C to 61 °C, or 68 °C, respectively.

Fig. 2. Representative 1H NMR (CDCl3) spectrum of acrylamide 5n with assigned signals.

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Table 2 Results of free radical polymerization of acrylamides 5a–n. Polymera

Monomer

Mn (g/mol)

Ð

Yield (%)

Tg (°C)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n

29,350 58,600 52,550 – – 52,950 30,050 54,400 68,750 270,100 – 56,600 22,650 77,900

2.00 1.96 1.99 – – 2.25 2.19 1.94 2.26 3.05 – 1.91 2.09 2.40

82 79 81 – – 91 84 75 83 96 – 85 79 86

59 62 68 – – 42 62 56 49 41 – 52 61 68

a Reaction conditions: 70 °C, 7 h reaction time, 1.0 mol% AIBN, 0.67 M solution in ethyl acetate, argon atmosphere.

Fig. 4. SEC traces of P1, P10 and P14 demonstrating the strong influence of the substituents on the polymerization rate expressed by the obtained molecular weights.

3.3. Testing of biological activity Polyacrylamides are valuable polymers for biomedical applications like hydrogel formation or tissue engineering [21–23,47]. Here, N-isopropylacrylamide and N,N-dimethylacrylamide are the most widely used monomers. The Ugi-derived monomers exhibit a related structure, which suggests similar applications for them. Thus, the synthesized polymers were tested for their biological compatibility by a modified Japanese Industrial Standard Z 2801:2000 protocol [43,44,48]. This test consists in the exposition of different bacterial strains to polymer coated glass slides for 16 h at 30 °C. After this period of cultivation, the bacterial suspension was diluted and transferred on CASO agar, incubated for 24 h at 30 °C, and subsequently the

CFUs were determined. Referred to the CFUs of coated glass slides of well-known biocompatible polymers or even non-coated glass slides, the tested polymers can be distinguished in microbicidal (decrease of CFU log value >2), bacteriostatic (decrease of CFU log value <2) or bio-tolerated (no change in CFU log value) materials [43,44]. For the antimicrobial activity tests, three bacterial strains were investigated, being Gram-negative E. coli and P. fluorescens, and Gram-positive B. subtilis. For this test, five representative polyacrylamides were selected exhibiting aliphatic (P9 and P12), aromatic (P13), alcohol or ester functionalized (P6 and P14) moieties. As reference, well-known synthetic biocompatible polymers, namely polyamide 12 (PA 12), polycaprolactone (PCL) and

Fig. 3. Representative 1H NMR (CDCl3) spectrum of polyacrylamide P14 with assigned signals.

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Fig. 5. Test results of the antimicrobial testing with Escherichia coli.

Fig. 6. Test results of the antimicrobial testing with Pseudomonas fluorescens.

Fig. 7. Test results of the antimicrobial testing with Bacillus subtilis.

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poly(D,L-lactide) (PL) were used. Regarding E. coli, the exposition to the three reference polymers resulted in an average CFU log value of 7.69 cells/mL. Interestingly, all other tested polymers formed slightly more CFUs in the range of log value 8.00 cells/mL (Fig. 5), which unambiguously implies full tolerance of the synthesized polyacrylamides against E. coli bacteria. In case of P. fluorescens, the situation was inversed. The average CFU log level of the three known biocompatible polymers was determined to 9.18 cells/mL (Fig. 6). This value is only exceeded by P12, which was already the best tolerated polymer by E. coli. All other evaluated polyacrylamides gave only slightly lower values, which is still in the range of biological variability. The lowest CFU log level compared to the average reference level was observed with polymer P9 (D = 0.56), anyhow still not being bacteriostatic. Finally, as representative for Gram-positive bacteria, the polymer effect on B. subtilis was examined. Both kinds of polymers, the reference samples and the different polyacrylamides, revealed log values of CFUs of around 8.50 cells/mL (Fig. 7). Summarizing, all tested polyacrylamides showed full tolerance against the three different bacterial strains suggesting them for applications requiring biocompatibility. However, these results give only an indication for their biocompatibility and conclusive statements can only be given after further evaluation and testing. 4. Conclusions The Ugi-4CR was successfully used for the synthesis of diverse acrylamide monomers in reasonable to good yields. It was shown that the modular nature of this MCR approach allows the independent alteration of three different moieties enabling individually designed monomers. Free radical polymerization of these acrylamides yielded amorphous polymers of high molecular weights. The only limitation observed was that monomers exhibiting bulky substituents did not polymerize due to their steric hindrance. Finally, a testing of the synthesized polyacrylamides for their biological activity revealed that this new class of polymers shows potential biocompatible behavior, which makes them highly interesting for biomedical applications. For example, simple introduction of chemical agents, fluorescent markers or protein reactive functionalities is facilitated. Acknowledgement A. S. is gratefully thankful for a scholarship from the Carl-Zeiss-Stiftung. References [1] Williams DF. Biomaterials 2008;29(20):2941–53. [2] Anwunobi AP, Emeje MO. J Nanomed Nanotechnol 2011:S4–002. [3] Aravamudhan A, Ramos DM, Nada AA, Kumbar SG. Natural polymers: polysaccharides and their derivatives for biomedical applications. In: Kumbar SG, Laurencin CT, Deng M, editors. Natural and synthetic biomedical polymers. Oxford: Elsevier; 2014. p. 67–89 [chapter 4].

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