Keratin Protein-Catalyzed Nitroaldol (Henry)

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Keratin Protein-Catalyzed Nitroaldol (Henry) Reaction and Comparison with Other Biopolymers Marleen Häring 1,2 , Asja Pettignano 1,2 , Françoise Quignard 2 , Nathalie Tanchoux 2 and David Díaz Díaz 1,3, * 1 2

3

*

Institute of Organic Chemistry, University of Regensburg, Universitätsstr 31, Regensburg 93053, Germany; [email protected] (M.H.); [email protected] (A.P.) Institute Charles Gerhardt Montpellier-UMR 5253 CNRS/UM/ENSCM, Matériaux Avancés pour la Catalyse et la Santé, 8 rue de l'École Normale, Cedex 5, Montpellier 34296, France; [email protected] (F.Q.); [email protected] (N.T.) IQAC-CSIC, Jordi Girona 18–26, Barcelona 08034, Spain Correspondence: [email protected]; Tel.: +49-941-943-4373; Fax: +49-941-943-4121

Academic Editor: Christophe Len Received: 15 July 2016; Accepted: 2 August 2016; Published: 25 August 2016

Abstract: Here we describe a preliminary investigation on the ability of natural keratin to catalyze the nitroaldol (Henry) reaction between aldehydes and nitroalkanes. Both aromatic and heteroaromatic aldehydes bearing strong or moderate electron-withdrawing groups were converted into the corresponding β-nitroalcohol products in both DMSO and in water in the presence of tetrabutylammonium bromide (TBAB) as a phase transfer catalyst. Negligible background reactions (i.e., negative control experiment in the absence of keratin protein) were observed in these solvent systems. Aromatic aldehydes bearing electron-donating groups and aliphatic aldehydes showed poor or no conversion, respectively. In general, the reactions in water/TBAB required twice the amount of time than in DMSO to achieve similar conversions. Moreover, comparison of the kinetics of the keratin-mediated nitroaldol (Henry) reaction with other biopolymers revealed slower rates for the former and the possibility of fine-tuning the kinetics by appropriate selection of the biopolymer and solvent. Keywords: keratin; biopolymer; C-C bond formation; nitroaldol reaction; Henry reaction

1. Introduction Keratin is one of the most abundant non-food proteins and represents a group of insoluble, cysteine-rich and stable filament-forming materials derived primarily from wool, hair, feathers, beaks, hooves, horns and nails [1]. Depending on their sulfur content, keratins can be classified as soft or hard keratins. Soft keratins, with a cysteine content up to 2% and found in outer layers of the epidermis and hair core, have a weaker mechanical and chemical stability. Hard keratins with a cysteine content of 10%–14% in hair, wool and skin, exhibit a higher resistance to thermal and chemical influences due to higher content of disulfide bridges [2]. Keratins contain a central ~310 residue domain with four segments in α-helical conformation that are separated by three short linker segments in β-turn conformation [3]. The complete amino acid sequence of keratins obtained from different sources has been reported in several publications [4–7], making the protein a suitable candidate to be tested as a catalyst. Recently, keratinous materials have attracted increasing attention as an important source of renewable biomaterials, especially since keratin wastes have been estimated to be more than 5 million tons per year [8], being used after degradation for different applications including scaffolds for tissue engineering [9,10] and support matrices for catalytic metal nanoparticles [11], among others [12]. On the other hand, the nitroaldol (Henry) reaction is a highly versatile C-C bond-forming reaction between an aldehyde or ketone and a nitroalkane to yield β-nitroalcohols, which can be Molecules 2016, 21, 1122; doi:10.3390/molecules21091122

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2 of 9 hand, the nitroaldol (Henry) reaction is a highly versatile C-C bond-forming reaction between an aldehyde or ketone and a nitroalkane to yield β-nitroalcohols, which can be easily modifiedinto intovaluable valuable building blocks [13–17]. As of part our research programs easily modified building blocks [13–17]. As part oneofofone our of research programs devoted devoted to understanding the potential catalytic role of different biopolymers [18–21], we report to understanding the potential catalytic role of different biopolymers [18–21], we report here forhere the for the first time the catalytic properties of keratin protein towards the nitroaldol (Henry) reaction first time the catalytic properties of keratin protein towards the nitroaldol (Henry) reaction as well asasa well as comparison a critical comparison with the performance of otherpolymers natural polymers in process. the same process. critical with the performance of other natural in the same

2. 2. Results Resultsand andDiscussion Discussion The The nitroaldol nitroaldol (Henry) (Henry) reaction reaction between between 4-nitrobenzaldehyde 4-nitrobenzaldehyde (1a, (1a, 0.1 0.1 mmol) mmol) and and nitromethane nitromethane (2a, 1.0 1.0mmol) mmol)was was chosen a model reaction to demonstrate the potential ability of to keratin to (2a, chosen as aasmodel reaction to demonstrate the potential ability of keratin promote promote C-C bond formation under mild conditions. Preliminary experiments using 10 mg of keratin C-C bond formation under mild conditions. Preliminary experiments using 10 mg of keratin in different in different solvents no of formation of the desired β-nitroalcohol 3a inTHF toluene, THF or CH3CN solvents showed no showed formation the desired β-nitroalcohol 3a in toluene, or CH 3 CN (Table 1, (Table entries yields 3a (ca. were in obtained CH2Cl 2, CHCl3 or H2O entries 1, 1–3), and 1–3), very and low very yieldslow of 3a (ca. of 10%) were10%) obtained CH2 Cl2in , CHCl 3 or H2 O (Table 1, (Table 1, entries 4, 5 and 7). However, DMSO and water containing tetrabutylammonium bromide entries 4, 5 and 7). However, DMSO and water containing tetrabutylammonium bromide (TBAB) as (TBAB) as phase-transfer catalyst were found to besolvents appropriate solvents allowing the selective phase-transfer catalyst were found to be appropriate allowing the selective formation of 3a formation 3a inatmodest yields at room temperature or very lowreaction background in modest of yields room temperature with no or verywith lowno background (Tablereaction 1, entry(Table 6 and 1, entry 6 and entry 8). Thus, formation of 3a could be unequivocally ascribed to the intrinsic catalytic entry 8). Thus, formation of 3a could be unequivocally ascribed to the intrinsic catalytic activity of the activity of the protein. These results are similar to those obtained with other[19]. biopolymers protein. These results are similar to those obtained with other biopolymers We found[19]. that We the found that the keratin loading could be reduced to 2 mg without detriment in the product yield (data keratin loading could be reduced to 2 mg without detriment in the product yield (data not shown). not shown). 10 mg were experiments used in mostfor experiments for practical reasons. addition, However, 10 However, mg were used in most practical reasons. In addition, theIn reaction canthe be reaction be1scaled (i.e.,without 1 mmoldetriment of 1a) without on the product yield. scaled upcan (i.e., mmolup of 1a) on thedetriment product yield. Table 1. Initial Initial solvent screening for for the the nitroaldol nitroaldol (Henry) (Henry) reaction reaction between between 1a 1a and and 2a 2a aa.. Table 1. solvent screening

Entry Solvent Yield c of 3a (%) Entry Solvent Yield c of 3a (%) 1 Toluene 0 1 Toluene 0 2 THF 0 2 THF 0 3 CH 3CN 0 3 CH CN 0 3 11 4 CH2Cl2 4 CH2 Cl2 11 10 5 CHCl3 5 CHCl3 10 6 DMSO 66 (0) d d 6 DMSO 66 (0) 7 H 7 H22O O 11 11 2O/TBAB bb 8 H d (3) d 8 H2 O/TBAB 57 (3) 57 a Reaction conditions: 1a (0.1 mmol), 2a (1.0 mmol), keratin (10 mg), solvent (0.5 mL), RT, orbital a Reaction conditions: 1a (0.1 mmol), 2a (1.0 mmol), keratin (10 mg), solvent (0.5 mL), RT, orbital shaking b TBAB (0.1 mmol). The addition of the phase transfer catalyst did not change shaking (15024rpm), 24 h.(0.1 (150 rpm), h; b TBAB mmol). The addition of the phase transfer catalyst did not change the pH of the c 1 H-NMR yieldscof solution; crude product obtained in the presenceobtained of dimethyl (DMA)ofasdimethyl internal yields of crude product inacetamide the presence the pH of the solution. 1H-NMR standard; d Background reaction without keratin. d acetamide (DMA) as internal standard. Background reaction without keratin.

With these results in in hand, the substrate scope of of the With these preliminary preliminary results hand, we we examined examined the substrate scope the reaction reaction using using different aldehydes in combination with nitromethane or nitroethane both in DMSO (Table 2) and different aldehydes in combination with nitromethane or nitroethane both in DMSO (Table 2) and H O/TBAB (Table 3) at room temperature. Aromatic aldehydes bearing strong electron-withdrawing 2 H2O/TBAB (Table 3) at room temperature. Aromatic aldehydes bearing strong electron-withdrawing substituents were were easily easily converted converted in DMSO into into the the corresponding corresponding β-nitroalcohols 3a–f in in excellent excellent substituents in DMSO β-nitroalcohols 3a–f yields regardless regardless the meta-, para-) para-) (Table (Table 2, Weaker yields the position position of of the the substituent substituent (ortho-, (ortho-, meta-, 2, entries entries 1–6). 1–6). Weaker acceptor substituents afforded the corresponding product 3 in moderate yields (Table 2, entries 7, 8, 8, acceptor substituents afforded the corresponding product 3 in moderate yields (Table 2, entries 7, 10, 12, 15). Interestingly, here the meta-position (Table 2, entry 9) of the substituent seemed to be more 10, 12, 15). Interestingly, here the meta-position (Table 2, entry 9) of the substituent seemed to be more favored, giving giving rise rise to to 3i 3i in in good good yield yield and and twice as much substrate (Table (Table 2, 2, favored, twice as much than than the the para-substituted para-substituted substrate entry 8). In contrast, benzaldehyde or aromatic aldehydes bearing electron-donating substituents entry 8). In contrast, benzaldehyde or aromatic aldehydes bearing electron-donating substituents yielded only only trace trace amounts amounts of of the the expected expected product product (Table (Table 2, Although we we did did not not yielded 2, entries entries 11, 11, 13,14). 13,14). Although

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perform these experiments under longer reaction times and/or higher temperatures, it is expected perform these experiments under longer reaction timesthe and/or higher temperatures, it is expected that that these factors could enhance to some extent yields as we have observed with other these factors could enhance to some extent the yields as we have observed with other biopolymers [19]. biopolymers [19]. Heteroaromatic aldehydes such as 2-pyridinecarboxaldehyde (Table 2, entry 16) Heteroaromatic aldehydes suchinasexcellent 2-pyridinecarboxaldehyde (Table 2,(Table entry 16) the desired lead to the desired product yield, whereas furfural 2, lead entryto18) and 1product in excellent yield, whereas furfural (Table 2, entry 18) and 1-naphthaldehyde (Table 2, entry 19) naphthaldehyde (Table 2, entry 19) proceeded only with low yields. proceeded onlythe with low yields. Regarding nature of the nucleophile, the use of nitroethane instead of nitromethane provided Regarding the nature ofdesired the nucleophile, the usewith of nitroethane of nitromethane provided slightly higher yields of the product albeit no or little instead diastereoselectivity favoring the slightly higher yields of the desired product albeit with no or little diastereoselectivity favoring the syn diastereomer (Table 2, entries 2, 6, 17 vs. 1, 5, 16, respectively). Slight anti-diastereoselectivy was syn diastereomer (Table 2, entries 2, 6, 17 (Table vs. 1, 5,2,16, respectively). anti-diastereoselectivy observed only with 3-nitrobenzaldehyde entry 4). RelativeSlight configurations were assignedwas by observed only with 3-nitrobenzaldehyde (Table 2, entry 4). Relative configurations were assigned by 1 comparison with H-NMR data reported in the literature. For instance, in the model reaction between 1 H-NMR data reported in the literature. For instance, in the model reaction between comparison with 1a and 2b, the anti diastereomer is characterized by a doublet at 4.85 ppm (J = 8.3 Hz), whereas the 1a and 2b, the anti shows diastereomer is characterized by a(Jdoublet at 4.85 ppmresults (J = 8.3 are Hz),similar whereas syn syn diastereomer the doublet at 5.41 ppm = 2.4 Hz). These tothe those diastereomer shows the doublet at 5.41 ppm (J = 2.4 Hz). These results are similar to those obtained obtained with other biopolymers such as gelatin [19] and suggest that the acidity of the nitroalkane with other biopolymers as gelatin pKa [19] and suggest that theinacidity the nitroalkane (nitroethane pKa = 8.6; such nitromethane = 10.2) constitutes most of cases here a more(nitroethane important pKa = 8.6; pKa[22]. = 10.2) constitutes in most cases herethat a more important factor than steric factor thannitromethane steric effects It is also worth mentioning aliphatic aldehydes such as effects [22]. It is also worth mentioning that aliphatic aldehydes such as isobutyraldehyde were not isobutyraldehyde were not converted (data not shown). Moreover, despite the α-helical chiral convertedof(data not shown). the α-helical chiral structure of the keratin, HPLC structure the keratin, HPLCMoreover, analysis ofdespite the reaction mixtures showed no enantioselection, as we analysis of the reaction mixtures showed no enantioselection, as we have previously observed with have previously observed with other biocatalysts in the same reaction [19–21]. other biocatalysts in the same reaction [19–21]. Table 2. Keratin-catalyzed nitroaldol (Henry) reaction in DMSO a. Table 2. Keratin-catalyzed nitroaldol (Henry) reaction in DMSO a .

b d 0 (2) 33 dr Entry R (1) R′ R (2) Yield Entry R (1) Yield bofof3 3(%) (%) dr d(anti/syn) (anti/syn) 1 (4-NO2)-C6H4 H 84 3a 1 (4-NO2 )-C6 H4 H 84 3a 2 (4-NO2)-C6H4 CH3 96 3b 1:1 2 (4-NO2 )-C6 H4 CH3 96 3b 1:1 33 (3-NO 2)-C6H4 HH 9393 3c3c -(3-NO 2 )-C6 H4 2)-C6H4 CH 3 98 3d 1:0.8 44 (3-NO (3-NO2 )-C6 H4 CH3 98 3d 1:0.8 2)-C)-C 6H 4 H HH 9494 3e3e -55 (2-NO (2-NO 2 6 4 66 (2-NO 2)-C 6 H 4 CH 3 96 3f 1:1.3 (2-NO )-C H CH 96 3f 1:1.3 2 6 4 3 (4-F)-C 3g 6H6 4 H4 HH 1010 3g -77 (4-F)-C (4-Cl)-C 3h 6H6 4 H4 HH 2525 3h -88 (4-Cl)-C (3-Cl)-C 99 (3-Cl)-C 6H6 4 H4 HH 4848 3i3i -(4-Br)-C H4 6H6 4 HH 2525 3j3j -1010 (4-Br)-C 11 (4-OH)-C6 H4 H 5 3k H 5 3k 11 (4-OH)-C6H4 12 (4-CN)-C6 H4 H 30 3l 12 (4-CN)-C6H4 H 30 3l 13 (4-Me)-C6 H4 H 7 3m 6H 4 HH 79 3m -1314 (4-Me)-C C6 H 3n 4 c 1415 C 6H 4 H 9 3n (4-CHO)-C6 H4 H 65 3o c 6H 4 HH 6590 3o -1516 (4-CHO)-C Pyrid-2-yl 3p 1617 Pyrid-2-yl HCH3 9096 3p Pyrid-2-yl 3q 1:1.7 Furfur-2-yl 1718 Pyrid-2-yl CH3H 963 3q3r 1:1.7 Naphtha-1-yl 1819 Furfur-2-yl HH 313 3r3s -a Reaction 19conditions: Naphtha-1-yl H 1 (0.1 mmol), 2 (1.0 mmol), keratin (10 13 mg), DMSO (0.5 3s mL), RT, orbital shaking (150 rpm),

b

1

c

24 h; Yield determined by H-NMR the presence of DMA(10 as internal standard; Yield RT, of monosubstituted Reaction conditions: 1 (0.1 mmol), in 2 (1.0 mmol), keratin mg), DMSO (0.5 mL), orbital shaking 1 H-NMR analyses. “Not applicable” is represented β-nitroalcohol; dbDiastereomeric ratio anti/syn determined by 1H-NMR in the presence of DMA as internal standard. c Yield (150 rpm), 24 h. Yield determined by by a dash (-). of monosubstituted β-nitroalcohol. d Diastereomeric ratio anti/syn determined by 1H-NMR analyses. “Not applicable” is represented by a dash (-). Although the desired β-nitroalcohols 3 were also obtained in H2 O/TBAB, the reactions took a

two times longer than in DMSO to achieve similar conversions. In H2 O/TBAB the substituent Although the desired β-nitroalcohols 3 were also obtained in H2O/TBAB, the reactions took two times longer than in DMSO to achieve similar conversions. In H2O/TBAB the substituent position in

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the aldehyde 1 was found to have higher influence than in DMSO. For example, strong electronwithdrawing orthoandtopara-position the reaction (TableFor 3, entries 1 and 5), position in thesubstituents aldehyde 1in was found have higher favored influence than in DMSO. example, strong whereas a significant substituents decrease of in theorthoyield was observed with thethe same substituent electron-withdrawing and para-position favored reaction (Table 3,placed entriesin1 meta-position (Table 3, entry 3). The reasons for the differences observed in H 2 O/TBAB vs. DMSO and 5), whereas a significant decrease of the yield was observed with the same substituent placed in may be related to the solubilization of the reactants and/or involve important variations in the meta-position (Table 3, entry 3). The reasons for the differences observed in H2 O/TBAB vs. DMSO mechanistic pathways, which remain unclear and and/or will beinvolve investigated in variations future research. may be related to the solubilization of the reactants important in the Furthermore, the expected products were obtained in good yields also with 4-cyanobenzaldehyde mechanistic pathways, which remain unclear and will be investigated in future research. Furthermore, (Table 3, entry 12) and 3, entry 17). Other (Table aldehydes were the expected products were2-pyridine obtained incarboxaldehyde good yields also(Table with 4-cyanobenzaldehyde 3, entry 12) converted less efficiently. Furthermore, the use of nitroethane instead of nitromethane provided and 2-pyridine carboxaldehyde (Table 3, entry 17). Other aldehydes were converted less efficiently. significant higher yields of the desired product (Table 3, entries 2, 6, 13, significant 18 vs. 1, 5, 12, 17, respectively) Furthermore, the use of nitroethane instead of nitromethane provided higher yields of the albeit with almost negligible diastereoselectivity (Table 3, respectively) entries 6, 18).albeit It is worth to mention that desired product (Table 3, entries 2, 6, 13, 18 vs. 1, 5, 12, 17, with almost negligible the replacement of TBAB by other ionic materials such as 1-butyl-3-methylimidazolium diastereoselectivity (Table 3, entries 6, 18). It is worth to mention that the replacement of TBAB by hexaflorophosphate 6) afforded the desired compound albeit in lower(BMIM-PF yield even6 )at longer other ionic materials(BMIM-PF such as 1-butyl-3-methylimidazolium hexaflorophosphate afforded reaction time (Table 3, entry the desired compound albeit1). in lower yield even at longer reaction time (Table 3, entry 1). a a. Table 3. Keratin-catalyzed nitroaldol (Henry) reaction 2O/ tetrabutylammonium bromide (TBAB) nitroaldol (Henry) reactionininHH Table 3. Keratin-catalyzed 2 O/tetrabutylammonium bromide (TBAB) .

a

Entry R (1) Entry R (1) 1 (4-NO2)-C6H4 (4-NO 21 (4-NO 2)-C 6H6 4 H4 2 )-C (4-NO 2 )-C 32 (3-NO 2)-C 6H6 4 H4 (3-NO H4 2 )-C 2)-C 6H6 4 53 (2-NO (2-NO H4 2 )-C 2)-C 6H6 4 65 (2-NO 6 (2-NO2 )-C6 H4 7 (4-F)-C6H4 7 (4-F)-C6 H4 6H 4 88 (4-Cl)-C (4-Cl)-C 6 H4 6H 4 99 (3-Cl)-C (3-Cl)-C 6 H4 1010 (4-Br)-C 6H 4 H (4-Br)-C 6 4 6H 4 H 1111 (4-OH)-C (4-OH)-C 6 4 (4-CN)-C 1212 (4-CN)-C 6H64 H4 (4-CN)-C 6H64 H4 1313 (4-CN)-C 14 (4-Me)-C 14 (4-Me)-C6H64 H4 15 C64 H4 15 C6H 16 (4-CHO)-C 16 (4-CHO)-C6H64 H4 17 Pyrid-2-yl 17 Pyrid-2-yl 18 Pyrid-2-yl 18 Pyrid-2-yl 19 Furfur-2-yl 1920 Furfur-2-yl Naphtha-1-yl 20 Naphtha-1-yl

R’ (2) R’ (2) H CH3H HCH3 HH CH3H CH H 3 H HH HH HH HH HH CHCH 3 3 HH HH HH H H CH3 CH3 H HH H

Yield b of 3 (%) Yield b of c3 (%) 73 (50) 7394 (50) c 2194 7021 9870 98 9 9 1818 3434 1414 11 6666 8989 44 88 d 19 19 d 72 72 85 85 11 110 0

3 3 3a 3a 3b 3c3b 3e3c 3f3e 3f 3g 3g 3h 3h 3i3i 3j3j 3k 3k 3l3l 3m 3m 3n 3n 3o 3o 3p 3p 3q 3q 3r 3r 3s 3s3t 3t

dr e e(anti/syn) dr (anti/syn) n.d. n.d. -1:0.8 1:0.8 -----n.d. n.d. ---1:0.9 1:0.9 --

Reaction conditions: 1 (0.1 mmol), 2 (1.0 mmol), TBAB (0.1 mmol), keratin (10 mg), H2 O (0.5 mL), RT, orbital

1 H-NMR c Yield Reaction conditions: mmol), 2 (1.0bymmol), TBAB mmol), keratin (10 mg), H2O (0.5 mL), shaking (150 rpm), 48 h;1 b(0.1 Yield determined in the(0.1 presence of DMA as internal standard; b 1 of the reaction carried out in the presence of BMIM-PF (0.1 mmol) instead of TBAB. Reaction time = 72 h; RT, orbital shaking (150 rpm), 48 h. Yield determined 6 by H-NMR in the presence of DMA as internal d Yield of monosubstituted β-nitroalcohol; e Diastereomeric ratio anti/syn determined by 1 H-NMR analyses. c standard. Yield of the reaction carried out in the presence of BMIM-PF6 (0.1 mmol) instead of TBAB. Abbreviation: n.d. = not determined. “Not applicable” is represented by a dash (-). Reaction time = 72 h. d Yield of monosubstituted β-nitroalcohol. e Diastereomeric ratio anti/syn determined by 1H-NMR analyses. Abbreviation: n.d. = not determined. “Not applicable” is The heterogeneous nature of the protein catalyst allowed its recovery from the reaction mixture represented by a dash (-). a

and its reuse for further cycles. However, a gradual deactivation of the catalyst could be observed in both organic and aqueous media, although the reduction of the catalytic activity was clearly more The heterogeneous nature of the protein catalyst allowed its recovery from the reaction mixture pronounced in DMSO (Figure 1). Such behavior has also been observed with other biopolymers [19–21] and its reuse for further cycles. However, a gradual deactivation of the catalyst could be observed in and could be associated to loss of catalyst loading (e.g., inefficient isolation by filtration) after each both organic and aqueous media, although the reduction of the catalytic activity was clearly more cycle and/or formation of intermediate linear or cyclic aminals that could block catalytic sites of the pronounced in DMSO (Figure 1). Such behavior has also been observed with other biopolymers [19–21] and could be associated to loss of catalyst loading (e.g., inefficient isolation by filtration) after each

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cycle and/or formation of intermediate linear or cyclic aminals that could block catalytic sites of the protein. These potential competitive processes are somehow more more critical critical in in DMSO DMSO than than in in aqueous aqueous protein. however, the the reasons reasonsbehind behindthese thesedifferences differencesremain remainunclear. unclear. solution; however,

Figure 1. Typical recycling experiments for keratin-catalyzed nitroaldol (Henry) reaction in DMSO Figure 1. Typical recycling experiments for keratin-catalyzed nitroaldol (Henry) reaction in DMSO and H2O/TBAB. Reaction conditions: 4-Nitrobenzaldehyde (1a, 15.1 mg, 0.1 mmol), nitromethane (2a, and H2 O/TBAB. Reaction conditions: 4-Nitrobenzaldehyde (1a, 15.1 mg, 0.1 mmol), nitromethane 54 μL, solvent (0.5 mL), keratin powder (10 mg), 24–48 h. Yields (2a, 54 1.0 µL, mmol), 1.0 mmol), solvent (0.5 mL), keratin powder (10 room mg), temperature, room temperature, 24–48 h. 1H-NMR values obtained from at least two experiments. The error bars represent the correspond to 1 Yields correspond to H-NMR values obtained from at least two experiments. The error bars represent O/TBAB, addition of newofTBAB standard deviation of the For the in H2in the standard deviation of measurements. the measurements. Forexperiments the experiments H2 O/TBAB, addition new after each cycle (i.e., 32.2 mg, 0.1 mmol) was necessary in order to ensure a constant concentration TBAB after each cycle (i.e., 32.2 mg, 0.1 mmol) was necessary in order to ensure a constant concentration during the the reactions reactions as as confirmed confirmed by by NMR NMR analyses analyses of of the thereaction reactionmixtures. mixtures. during

At this this point, point, we we carried carried out out kinetic kinetic analyses analyses of of the the model model reaction reaction between between 1a 1a and and 2a 2a catalyzed catalyzed At O/TBAB under by keratin keratin in in both both DMSO DMSO and and H H2O/TBAB by under the the described described conditions. conditions. The The results results were were compared compared 2 with the performance of other biopolymers as biocatalysts for the same reaction that we we have have with the performance of other biopolymers as biocatalysts for the same reaction that previously studied in our group and others (i.e., chitosan [18], gelatin [19], collagen [19], bovine serum previously studied in our group and others (i.e., chitosan [18], gelatin [19], collagen [19], bovine albumin (BSA) [19,23], silk fibroin and[20] alginate [21]) under optimized conditions. Figure 2 shows serum albumin (BSA) [19,23], silk[20] fibroin and alginate [21]) under optimized conditions. Figurethe 2 first-order kinetics corresponding to each biopolymer within the first hours of reaction. In general, shows the first-order kinetics corresponding to each biopolymer within the first hours of reaction. In powdered keratin keratin displayed rate constants in the of of aerogel calcium and general, powdered displayed rate constants in range the range aerogel calciumalginate alginate [21] [21] and significantly below below the the other other biopolymers biopolymers demonstrating demonstrating the the possibility possibility of of fine-tuning fine-tuning the the kinetics kinetics of of significantly the nitroaldol (Henry) reaction by appropriate selection of the biopolymer and solvent system. It is the nitroaldol (Henry) reaction by appropriate selection of the biopolymer and solvent system. It is worth mentioning that although these biopolymers are not superior to standard base catalysts such worth mentioning that although these biopolymers are not superior to standard base catalysts such as tetramethylethylenediamine tetramethylethylenediamine [16], byproducts and allowed to as [16],the theformer formeravoided avoidedthe theformation formationofof byproducts and allowed work under ecofriendly and heterogeneous conditions. However, far beyond the interest of these to work under ecofriendly and heterogeneous conditions. However, far beyond the interest of these materials as standard catalyst, their mechanism of action to promote C-C bond formation reactions materials as standard catalyst, their mechanism of action to promote C-C bond formation reactions may be more relevant within the context of biological evolution. may be more relevant within the context of biological evolution.

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Figure 2. First-order kinetics plots for the nitroaldol (Henry) reaction between 1a and 2a catalyzed by Figure 2. First-order kinetics plots for the nitroaldol (Henry) reaction between 1a and 2a catalyzed different biopolymers in powder form. Unless otherwise indicated, reactions were made in DMSO as by different biopolymers in powder form. Unless otherwise indicated, reactions were made in described in the text and experimental section. Apparent rate constants are given in units of h−1. Each DMSO as described in the text and experimental section. Apparent rate constants are given in data point represents the average of at least two independent measurements. Cinfi = final units of h−1 . Each data point represents the average of at least two independent measurements. concentration, at infinite time; Ct = concentration at given time t; C0 = initial concentration at t = zero C infi = final concentration, at infinite time; Ct = concentration at given time t; C0 = initial concentration time. at t = zero time.

3. Materials 3. Materials and and Methods Methods 3.1. Materials Materials 3.1. Unless otherwise otherwise indicated, indicated, analytical analytical grade grade solvents solvents and and reactants reactants were were commercially commercially available available Unless and used used as as received received without without further further purification. purification. Deionized Deionized water was was used used for for experiments experiments in in and aqueous solutions. Aldehydes (purity by GC > 98%) were purchased from TCI Europe (TCI Europe, aqueous solutions. Aldehydes > Zwijndrecht, Belgium). Keratin extracted from wool Cat. Nr. AB 250197) purchased Zwijndrecht, Belgium). Keratin extracted from(CAS wool69430-36-0, (CAS 69430-36-0, Cat. Nr.was AB 250197) frompurchased ABCR. Tetrabutylammonium bromide (CAS 1643-19-2, Cat Nr. 86860) was purchased was from ABCR. Tetrabutylammonium bromide (CAS 1643-19-2, Cat Nr. 86860) from was Fluka (Fluka Chemical WI, USA). 1-Buthyl-3-methylimidazolium hexafluorophosphate purchased from Fluka Corp., (FlukaMilwaukee, Chemical Corp., Milwaukee, WI, USA). 1-Buthyl-3-methylimidazolium (BMIM-PF6) was purchased from TCI Europe (CAS 174501-64-5, Cat. Nr. B2320). hexafluorophosphate (BMIM-PF ) was purchased from TCI Europe (CAS 174501-64-5, Cat. Nr. B2320). 6 3.2. 3.2. Methods Methods 11H-NMR

H-NMR spectra were recorded on Avance 300 or Avance Avance 400 400 spectrometers spectrometers (Bruker, (Bruker, Billerica, Billerica, ◦ C. Chemical shifts for 11H-NMR were reported as δ, parts per million, relative to MA, USA) at 25 MA, USA) at 25 °C. Chemical shifts for reported as δ, parts per million, relative to external externalstandards. standards.Yields Yieldswere weredetermined determinedby by11H-NMR H-NMRanalyses analysesof ofthe thecrude crudeproduct productin inCDCl CDCl33 using using dimethyl dimethyl acetamide acetamide (0.1 (0.1 mmol, mmol, 9.2 µL) μL) as as internal internal standard standard after after complete complete work-up of the reaction. Enantiomeric Enantiomeric excess excess was was evaluated evaluated by by chiral chiral HPLC HPLC (Agilent (Agilent Technologies, Technologies, Santa Santa Clara, Clara, CA, CA, USA) USA) (column: (column: Phenomenex Phenomenex (Phenomenex, (Phenomenex, Aschaffenburg, Aschaffenburg,Germany) Germany)Lux LuxCellulose-1, Cellulose-1,4.6 4.6mm mm×× 250 mm, 55 µm, μm,eluents: eluents: n-heptane, n-heptane,i-propanol i-propanol70:30, 70:30,flow: flow:0.5 0.5mL/min). mL/min). Relative configurations configurations were were assigned assigned 1 1 by by comparison comparison with with H-NMR H-NMR data data reported reported in in the the literature. literature. For example, example, in in the the model model reaction reaction between and2b, 2b,the theanti anti diastereomer identified a doublet 4.85(Jppm (J = whereas 8.3 Hz), between 1a and diastereomer waswas identified by aby doublet at 4.85atppm = 8.3 Hz), whereas the syn diastereomer the doublet at 5.41 (J = 2.4 For kinetics calculations, the syn diastereomer showed showed the doublet at 5.41 ppm (J =ppm 2.4 Hz). ForHz). kinetics calculations, the 1H1 the H-NMR analyses the reaction mixtures performed in presence the presence of an internal standard NMR analyses of the of reaction mixtures werewere performed in the of an internal standard as as above indicated. general,given givenyield yieldvalues valuescorrespond correspondto tothe theaverage average of of at at least two independent above indicated. InIn general, independent measurements measurementswith withSTDV STDV± ± 2%–4%. Among various kinetics models, the straight straight lines lines shown shown in in the the kinetics kinetics plots plots correspond correspondto tothe thebest bestfit fitof ofthe thefirst-order first-ordermodel model(e.g., (e.g.,(nitromethane) (nitromethane)≥≥ (aldehyde)). (aldehyde).

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3.3. General Procedure for Keratin-Catalyzed Nitroaldol (Henry) Reaction Keratin (10 mg) was added in one portion to a mixture of 4-nitrobenzaldehyde (1a, 0.1 mmol, 15.1 mg), nitromethane (2a, 1.0 mmol, 54 µL) and solvent (0.5 mL) placed into a 4-mL screw-capped vial. The resulting reaction mixture was gently shaked in an orbital shaker (150 rpm) for the appropriate time at room temperature. After completion, water (1 mL) was added. The reaction mixture was extracted with EtOAc (4 × 1.5 mL), dried over anhydrous sodium sulfate, filtrated and evaporated under reduced pressure. Yield was determined by 1 H-NMR of the crude product in CDCl3 using dimethylacetamide (9.2 µL, 0.1 mmol) as internal standard. All β-nitroalcohol products are known and the spectroscopic data obtained from NMR analysis of the reaction mixtures were in agreement with those reported in the literature. 3.4. Typical Recycling Procedure After reaction and extraction with EtOAc, the aqueous phase with remaining catalyst was freeze-dried prior addition of the reaction substrates and solvent for the next run. 3.5. Kinetic Studies Reaction conversions were unequivocally calculated by 1 H-NMR analysis of the reaction mixtures according to the integration of characteristic signals of the species in the reaction mixture in the presence of a suitable internal standard. Each experimental point represents the average of at least two experiments. Among various kinetics models, lines presented in the kinetic plots show best-fits of the first-order model for each case (i.e., (NO2 R) ≥ (aldehyde)). Due to the fact that not all reactions reached 100% yield, data fitting was made according to the variation of ln((Ct − C∞ )/(C∞ − C0 )) with time, where Ct is the concentration at a given time t; C∞ the final concentration (at infinite time) and C0 the initial concentration (at t = zero time). For reaction conversions close to 100%, plots of ln(Ct /C0 ) versus time provided consistent results (C∞ = 0). Under these considerations, minor differences were observed between the exponential and linear fits. All errors reported for the rate constants k were calculated by graphical analysis. Kinetics data for other biopolymers have been previously reported by us and were used for comparison: Chitosan [18], gelatin [19], collagen [19], BSA [19], freeze-dried silk fibroin [20] and calcium alginate aerogel [21]. Sample preparation and details of the reactions can be found in the corresponding references. 4. Conclusions In conclusion, we have demonstrated that keratin proteins are able to promote C-C bond formation via the nitroaldol (Henry) reaction between various aldehydes and nitroalkanes. Appropriate control experiments demonstrated the intrinsic catalytic activity of the keratin. Both aromatic and heteroaromatic aldehydes having strong or moderate electron-withdrawing groups were converted exclusively into the corresponding β-nitroalcohol products in both DMSO and in water in the presence of TBAB as phase transfer catalyst. In contrast, aromatic aldehydes bearing electron-donating groups and aliphatic aldehydes showed poor or no conversion, respectively. In general, the reactions in water/TBAB required twice the amount of reaction time than in DMSO to achieve similar conversions. Although the heterogeneous nature of the reaction allowed for the recovery and reuse of the keratin, a gradual deactivation of the catalyst was observed after each cycle. Comparative kinetic studies with other biopolymers revealed that the rate of the nitroaldol (Henry) reaction strongly depends on the nature of the biopolymer. The effect of different forms of keratins on the ability to catalyze this and other C-C bond forming reactions, as well as detailed mechanistic studies in both organic and aqueous medium, including different ionic liquids, are currently under study in our laboratories and the results will be reported in due course.

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Acknowledgments: D.D.D. thanks the Deutsche Forschungsgemeinschaft (DFG) for the Heisenberg Professorship Award. A.P. thanks the SINCHEM program for her PhD Grant. SINCHEM is a Joint Doctorate program selected under the Erasmus Mundus Action 1 Program—FPA 2013-0037. Author Contributions: M.H. and A.P. performed the experiments. F.Q. and N.T. contributed to results discussion. D.D.D. designed the experiments. M.H. and D.D.D. wrote the paper. All authors took part in data analysis and discussion. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).