Biocatalysis 2017; 3: 27–36 Communication Open Access Juliana Christina Thomas, Martha Daniela Burich, Pamela Taisline Bandeira, Alfredo Ricardo Marques de Oliveira, Leandro Piovan* Biocatalysis in continuous-flow mode: A case- study in the enzymatic kinetic resolution of secondary alcohols via acylation and deacylation reactions mediated by Novozym 435® DOI 10.1515/boca-2017-0003 Received December 4, 2016; accepted February 7, 2017 Abstract: Enzymatic kinetic resolution reactions are a well- established way to achieve optically active compounds. When enzymatic reactions are combined to continuous- flow methodologies, other benefits are added, including reproducibility, optimized energy use, minimized waste generation, among others. In this context, we herein report a case study involving lipase-mediated transesterification by acylation and deacylation reactions of secondary alcohols/esters in batch and continuous-flow modes. 1 Introduction Acylation reactions were performed with high values of Enzymatic kinetic resolution (EKR) is a well-established enantiomeric excess (72 up to >99%) and enantioselectivity approach in the preparation of optically active compounds. (E > 200) for both batch and continuous-flow modes. On Nowadays, enzyme-mediated transformations, such as the other hand, for deacylation reactions using n-butanol EKR by lipases, are present in the toolbox of synthetic as nucleophile, enatiomeric excess ranged between 38 to chemists [1,2]. Enzymes are exceptional catalysts >99% and E from 6 to >200 were observed for batch mode. presenting enormous stereoselectivity for a number of For deacylation reactions in continuous-flow mode, natural and non-natural substrates, which allows the results were disappointing, as in some cases, very low or synthesis of valuable chiral intermediates, building- no conversion was observed. Enantiomeric excess ranged blocks and products with high enantiopurity. Currently, from 16 to >99% and enantioselectivity from 5 to >200 were there is an upward trend in the use and performance observed. In terms of productivity, continuous-flow mode of biocatalytic transformations in continuous-flow reactions were superior in both strategies (acylation: r mode [3]. Application of continuous-flow systems in from 1.1 up to 18.1-fold higher, deacylation: 2.8 up to 7.4- organic reactions offers several advantages, such as fold higher in continuous-flow than in batch mode). reproducibility, efficient control of reaction parameters, fast/homogeneous heating and lower costs in the Keywords: enzymatic kinetic resolution, acylation/ optimization of reaction conditions.[4,5] They are in deacylation, continuous-flow chemistry, lipases, harmony with several principles of green chemistry, such secondary alcohols as optimized energy use, increased processes safety, minimized use of solvents and waste generation.[6] When *Corresponding author: Leandro Piovan, Department of Chemistry, continuous-flow systems are combined with biocatalysis, Universidade Federal do Paraná, Paraná, Brazil, other benefits are added, including no enzyme lixiviation E-mail: [email protected] from support, removing the product almost immediately Juliana Christina Thomas, Martha Daniela Burich, Pamela Taisline from the contact with biocatalyst, especially in cases Bandeira, Alfredo Ricardo Marques de Oliveira, Department of Chemistry, Universidade Federal do Paraná, Paraná, Brazil where they can act as enzyme inhibitors [7], in addition © 2017 Juliana Christina Thomas et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. 28 J.Ch. Thomas, et al. to the three major types of well-known chemo-, regio- (3 x 10 mL), dried over anhydrous MgSO4, filtered off and and stereoselectivity inherent to enzymes [8,9]. Aldolases dichloromethane was evaporated under reduced pressure [10], amidases [11], transaminases [12], oxidases [13,14], (alcohols 1-3) or distilled (alcohols 4-6). All compounds peroxidases [15] are some enzymes that have been were obtained with high purity and no additional steps of successfully applied to a continuous-flow approach. purification were required. Additionally, lipase-mediated esterification [16-21], Phenylethan-1-ol (1). Yield: 92% (5.620 g). GC-MS (70 interesterification [22,23], transesterification [24-26] and eV), m/z (relative intensity): 122 (M•+, 34%), 107 (90%), 79 enzymatic kinetic resolution (EKR) of racemic compounds (100%), 77 (54%), 51 (21%), 43 (24%). 1H NMR (200 MHz, [27-32], have demonstrated the potential of combining CDCl3, TMS), δ (ppm): 1.42 (d, J = 6.5 Hz, 3H), 4.79 (q, J = 13 continuous-flow systems and biocatalysis. 6.5 Hz, 1H), 7.20-7.32 (m, 5H). C NMR (50 MHz, CDCl3), δ Recently, we described the EKR of cyanohydrins in (ppm): 25.1, 70.3, 125.4, 127.4, 128.5, 145.9. IR (cm-1): 3352, continuous-flow system [33] and observed a contrasting 2973, 1449, 1074, 698. behaviour between transesterification by acylation (4-Methoxyphenyl)ethan-1-ol (2). Yield: 82% (6.240 g). and deacylation reactions. While a deacylation GC-MS (70 eV), m/z (relative intensity): 152 (M•+, 30%), 137 employing n-butanol led to cyanohydrin derivatives in (100%), 109 (64%), 94 (35%), 77 (32%), 43 (21%). 1H NMR high productivity and enantioselectivity, for acylation (200 MHz, CDCl3, TMS), δ (ppm): 1.45 (d, J = 6.5 Hz, 3H), reaction, in continuous-flow mode, a very low conversion 3.79 (s, 3H), 4.82 (q, J = 6.5 Hz, 1H), 6.86 (d, J = 8.6 Hz, 13 rate was observed, which made this acylation strategy 2H), 7.28 (d, J = 8.7 Hz, 2H). C NMR (50 MHz, CDCl3), δ unfeasible. Due to this observation, we asked ourselves (ppm): 25.0, 55.3, 69.9, 113.8, 126.7, 138.0, 158.9. IR (cm-1): if this behaviour was a particular case to cyanohydrins 3377, 2968, 2056, 1889, 1611, 1458, 1244, 831. or would other organic compounds be susceptible to (4-Nitrophenyl)ethan-1-ol (3). Yield: 88% (7.355 g). the same particular behaviour, according to acylation or GC-MS (70 eV), m/z (relative intensity): 166 (M•+, 1%), deacylation conditions. In this way, secondary alcohols 152 (100%), 122 (25%), 107 (75%), 94 (41%), 77 (89%), 51 1 are an appropriate choice, especially due to high numbers (30%), 43 (58%). H NMR (200 MHz, CDCl3, TMS), δ (ppm): of EKR studies involving acylation and deacylation of this 1.50 (d, J = 6.5 Hz, 3H), 5.00 (q, J = 6.5 Hz, 1H), 7.52 (d, J = 8.6 13 class of organic compounds. Hz, 2H), 8.15 (d, J = 8.6 Hz, 2H). C NMR (50 MHz, CDCl3), In this context, in order to evaluate the differences δ (ppm): 25.3, 22.2, 71.2, 123.8, 126.7, 147.4, 148.9, 170.0. IR between transesterification by acylation and deacylation (cm-1): 3376, 1932, 1803, 1680, 1516, 1343, 1108, 855. reactions under continuous-flow conditions, a series of Octan-2-ol (4). Yield: 89% (5.795 g). 1H NMR (200 MHz, well-known Novozym 435® substrates [34-43], composed CDCl3, TMS), δ (ppm): 0.89 (m, 3H), 1.19 (d, J = 6.2 Hz, 3H), by benzylic and aliphatic alcohols and their respective 1.24-1.50 (m, 10H), 3.78 (sext, J = 6.2 Hz, 1H). 13C NMR (50 esters were evaluated both in batch and continuous-flow MHz, CDCl3), δ (ppm): 14.0, 22.6, 23.4, 25.7, 29.3, 31.8, 39.3, modes. Finally, a multigram scale reaction was performed 68.1. IR (cm-1): 3341, 2973, 2929, 2856, 1459, 1378, 1327. to validate our analytical investigation. 4-Methyl-pentan-2-ol (5). Yield: 94% (4.802 g). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 0.92 (d, J = 6.6 Hz, 6H), 1.19 (d, J = 3.2 Hz, 3H), 1.35-1.48 (m, 1H), 1.63-1.84 (m, 1H), 2 Materials and Methods 13 3.80-3.96 (m, 1H). C NMR (50 MHz, CDCl3), δ (ppm): 22.3, 23.1, 23.9, 24.8, 48.6, 66.1. IR (cm-1): 3348, 2922, 2966, 2863, 2.1 General Experimental Procedure for 1473, 1371, 1327. Syntheses of Racemic Alcohols 1-6 Hexan-3-ol (6). Yield: 92% (4.700 g). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 0,93 (t, J = 7.2 Hz, 3H), 0.94 (t, Corresponding ketone (50 mmol) was solubilized in J = 6.9 Hz, 3H), 1.33-1.54 (m, 6H), 3.48-3.59 (m, 1H). 13C NMR methanol (50 mL) in an ice bath and NaBH4 (51 mmol) (50 MHz, CDCl3), δ (ppm): 9.8, 14.1, 18.8, 30.1, 39.1, 73.0. IR was added. After gas evolution stopped, the ice bath was (cm-1): 3348, 2966, 2944, 1459, 1319. removed and the reaction was carried out under magnetic stirring at room temperature. The reaction was monitored 2.2 General Experimental Procedure for by TLC until the consumption of ketone. Then, solvent was Syntheses of Racemic Esters 1a-6a removed under reduced pressure (alcohols 1-3) or distilled under atmospheric pressure (alcohols 4-6). Water and To a solution of corresponding alcohol (25 mmol) in aqueous HCl solution (1 mol L-1) were added until pH 6. dichloromethane (25 mL), acetic anhydride (50 mmol) The reaction media was extracted with dichloromethane and DMAP (1 crystal) were added and the reaction was Biocatalysis in continuous-flow mode 29 maintained under magnetic stirring at room temperature 2.3 General Procedure for Enzymatic Kinetic overnight. Then, the mixture was filtered through silica Resolution in Batch Mode via Acylation and the solvent was evaporated under reduced pressure. 1-Phenylethyl acetate (1a). Yield: 75% (3.079 g). To a solution of substrate (0.1 mmol) in n-hexane (2 mL), GC-MS (70 eV), m/z (relative intensity): 164 (M•+, 23%), vinyl acetate (0.4 mmol) and 20 mg of Novozym 435® 122 (100%), 105 (67%), 104 (89%), 107 (36%), 51 (15%), 43 were added and the reaction was carried out at 50°C 1 (55%).