Synthesis of Knoevenagel Adducts Under Microwave Irradiation and Biocatalytic Ene-Reduction by the Marine-Derived Fungus Cladosporium sp. CBMAI 1237 for the Production of 2-Cyano-3-Phenylpropanamide Derivatives

Abstract

The organic synthesis has been driven by the need of sustainable processes, which also requires efficiency and cost-effectiveness. In this work, we described the synthesis of nine Knoevenagel adducts between cyanoacetamide and aromatic aldehydes ((E)-2-cyano-3-(phenyl)acrylamide derivatives), employing triethylamine as catalyst under microwave irradiation in 30 min with excellent yields (93–99% yield). Then, these adducts were employed in the C–C double bond bioreduction by the marine-derived fungus Cladosporium sp. CBMAI 1237 for obtention of 2-cyano-3-phenylpropanamide derivatives in mild conditions and short reaction time for a whole-cells reduction (phosphate buffer pH 7.0, 32 °C, 130 rpm, 8 h) with good yields (48–90%). It is important to emphasize that the experimental conditions, especially the reaction time, should be carefully evaluated for the obtention of high yields. Since a biodegradation process consumed the obtained product in extended periods, probably due to the use of the substrate as carbon and nitrogen source. This approach showed that the use of coupled and greener catalysis methods such as microwave irradiation and biocatalytic reduction, which employs unique biocatalysts like marine-derived fungi, can be an interesting tool for the obtention of organic molecules.

Introduction

The use of enzymes for reductive processes has been driven by the need of green and sustainable processes, which also requires efficiency and cost-effectiveness (González-Martínez et al. 2019; Toogood and Scrutton 2014). In this context, reductases have been employed for reduction of different substrates, including ketones, aldehydes, carboxylic acid derivatives, nitriles, and nitro compounds (Winkler et al. 2018). Moreover, whole-cells biocatalysis also presented interesting results, employing fungi, bacteria, plant, and algae for reduction reactions (Birolli et al. 2015; Garzon-Posse et al. 2018; Lin and Tao 2017).

Marine-derived fungi have been successfully employed for reduction of ketones (Liu et al. 2018) and alkenes (de Matos et al. 2019). For example, free and immobilized cells of Penicillium citrinum CBMAI 1186 reduced C–C double bonds of α,β-, di-α,β-, and mono-α,β,γ,δ-unsaturated ketones with good yields and selectivities (Ferreira et al. 2015; Ferreira et al. 2014), and also reduced in high yields benzylidene-malononitrile derivatives, which were produced by Knoevenagel condensation reactions (Jimenez et al. 2016).

The Knoevenagel condensation is a synthetic tool very useful for the increase of carbon chains, as well as, for functionalization (Khare et al. 2019; Nokami et al. 2001), being applied for the synthesis of polymers (Kwak and Fujiki 2004), intermediates (Zhang et al. 2014), and different bioactive organic compounds (Coulibaly et al. 2015) such as α-glucosidase inhibitors (Kashtoh et al. 2016), antidepressants (Mishra and Hajra 2015), cancer therapy (Ramesh and Lalitha 2015), vasodilators (Girgis et al. 2011), and antimicrobial substances (Al-Mousawi et al. 2016).

Microwave irradiation has been employed in the synthesis of Knoevenagel adducts in mild conditions, short times, easy handling, and using solvents with low toxicity (Zanin et al. 2018). The use of different methods that promote advances towards the green chemistry principles, as biocatalysis and low intensity irradiation, is an interesting approach for new methodologies (Erythropel et al. 2018).

Therefore, the aim of this work was the obtention of the Knoevenagel adducts from aromatic aldehydes and cyanoacetamide under microwave irradiation followed by a C–C double bond reduction by mycelia of marine-derived fungus.

Material and Methods

Reagents and Solvents

The 4-methoxybenzaldehyde (98%) 1a, 4-nitrobenzaldehyde (98%) 1b, 4-fluorobenzaldehyde (98%) 1c, 4-chlorobenzaldehyde (97%) 1d, 4-bromobenzaldehyde (99%) 1e, vanillin (99%) 1f, 3,4,5-trimetoxibenzaldehyde (99%) 1 g, syringaldehyde (98%) 1 h, 2-thiophenecarboxaldehyde (98%) 1i, cyanoacetamide (99%), and sodium borohydride (≥ 98%) were obtained from Sigma-Aldrich.

Anthracene (99%) was acquired from ACROS Organics. The salts employed for production of artificial seawater were purchased from Vetec and Synth. Deuterated chloroform (CDCl₃, 99.9%), methanol (MeOD, 99.9%), acetone-d₆ (99.9%), and DMSO-d₆ (99.9%) were acquired from the Cambridge Isotope Laboratories. The high-pressure liquid chromatography (HPLC) grade solvents; hexane, isopropanol and ethyl acetate were obtained from Panreac. The extraction and purification solvents, hexane and ethyl acetate, were obtained from Synth.

Knoevenagel Condensation of Cyanoacetamide and Aromatic Aldehydes 1a-i under Microwave Irradiation

In a round bottom flask of 25 mL was added cyanoacetamide (1.0 mmol), aromatic aldehyde 1a-i (1.0 mmol), H₂O (4 mL) and triethylamine (10 mol%). Then, the reaction was performed in a CEM Discover reflux open vessel of 300 W with infrared temperature control in 55 W at 85 °C for 30 min. The reaction progress was monitored by Thin Layer Chromatography (TLC) and revealed with iodine-impregnated silica gel.

Subsequently, the reaction was filtered and extracted three times with 10 mL of ethyl acetate. The obtained organic phases were combined, dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The products were purified in column chromatography with silica flash using hexane and ethyl acetate (7:3). All the obtained adducts 2a-I were characterized by melting point (m.p.), Fourier-Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS).

The m.p. was obtained employing a melting point apparatus Fisatom 431. The IR spectra were acquired from 400 to 400 cm⁻¹ with a Shimadzu IRAffinity spectrometer preparing the samples in KBr disks. The MS spectra were obtained in a Shimaduzu MS2010 spectrometer (70 eV). The NMR analyses were carried out on an Agilent 400/54 or Agilent 500/54 premium-shielded spectrometer operated at frequencies of 500 and 400 MHz (¹H NMR) or 125 and 100 MHz (¹³C NMR), respectively. All performed analyzes can be verified in the Electronic Supplementary Material-1 (ESM-1).

(E)-2-Cyano-3-(4-Methoxyphenyl)Acrylamide (2a)

C₁₁H₁₀N₂O₂, 202.21 g.mol⁻¹; (99% yield); white crystal; m.p. = 117–118 °C; FTIR ν_max (cm⁻¹) = 3445, 3305, 3175, 2209, 1698, 1582, 1508; ¹H NMR (500 MHz, CDCl₃) δ (ppm) = 8.3 (s, 1H), 8.0 (m, 2H), 7.0 (m, 2H), 3.9 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ (ppm) = 164, 163, 153, 133, 124, 115, 99, 56; MS (70 eV) m/z = 202 (100%); 201 (100%); 158 (33%); 89 (28%); 186 (24%); 77 (18%); 115 (18%); 114 (18%).

(E)-2-Cyano-3-(4-Nitrophenyl)Acrylamide (2b)

C₁₂H₇N₃O₃, 217.18 g.mol⁻¹; (95% yield); red solid; m.p. = 110–112 °C; FTIR ν_max (cm⁻¹) = 3435, 3344, 2223, 1691, 1601, 1516, 1345; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) = 8.4 (m, 2H), 8.3 (s, 1H), 8.1 (d, J = 8.8 Hz, 2H), 8.1 (s, 1H), 7.9 (s, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ (ppm) = 163, 149, 148, 138, 131, 131, 125, 124, 116, 111; MS (70 eV) m/z = 216 (100%); 200 (92%); 170 (81%); 217 (63%); 89 (49%); 44 (46%); 101 (42%); 75 (41%).

(E)-2-Cyano-3-(4-Fluorophenyl)Acrylamide (2c)

C₁₀H₇N₂OF, 190.18 g.mol⁻¹; (94% yield); white crystal; m.p. = 89–91 °C; FTIR ν_max (cm⁻¹) = 3336, 3192, 2924, 2211, 1691, 1613, 1588, 1487; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) = 8.2 (s, 1H), 8.0 (m, 2H), 7.8 (d, J = 8.0 Hz, 2H), 7.4 (m, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ (ppm) = 165 (d, J = 252 Hz, 1C), 163, 163, 150, 133 (d, J = 9 Hz, 1C), 129 (d, J = 3 Hz, 1C), 117 (d, J = 22 Hz, 1C), 117,107; MS (70 eV) m/z = 189 (100%); 120 (60%); 190 (59%); 146 (41%); 126 (35%); 44 (30%); 75 (28%); 147 (21%).

(E)-2-Cyano-3-(4-Chlorophenyl)Acrylamide (2d)

C₁₀H₇N₂OCl, 206.63 g.mol⁻¹; (92% yield); pale yellow solid; m.p. = 91–93 °C; FTIR ν_max (cm⁻¹) = 3336, 3192, 2215, 1681, 1597, 1380; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) = 8.2 (s, 1H), 8.0 (m, 3H), 7.8 (s, 1H), 7.7 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm) = 166, 163, 150, 137, 132, 132, 131, 131, 130, 129, 117, 108; MS (70 eV) m/z = 205 (100.00%); 206 (62%); 207 (39%); 136 (33%); 127 (28%); 75 (26%); 162 (25%); 208 (22%).

(E)-2-Cyano-3-(4-Bromophenyl)Acrylamide (2e)

C₁₀H₇N₂OBr, 249.08 g.mol⁻¹; (91% yield); yellow solid; m.p. = 95–97 °C; FTIR ν_max (cm⁻¹) = 3336, 3192, 2217, 1683, 1597, 1465, 1370; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) = δ 8.2 (s, 1H), 7.9 (m, 2H), 7.8 (m, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ (ppm) = 163, 150, 133, 132, 132, 126, 117, 108; MS (70 eV) m/z = 251 (100%); 249 (99%); 127 (79%); 250 (62%); 252 (61%); 75 (42%); 128 (40%); 171 (39%).

(E)-2-Cyano-3-(4-Hydroxy-3-Methoxyphenyl)Acrylamide (2f)

C₁₁H₁₀N₂O₃, 218.21 g.mol⁻¹; (96% yield); black solid; m.p. = 134–135 °C; FTIR ν_max(cm⁻¹) = 3335, 3198, 2924, 2213, 1667, 1570, 1514, 1290; ¹H NMR (500 MHz, MeOD) δ (ppm) = 8.1 (s, 1H), 7.8 (d, J = 2.1 Hz, 1H), 7.5 (d, J = 2.3 Hz, 1H), 7.4 (d, J = 2.3 Hz, 1H), 6.9 (d, J = 8.3 Hz, 1H), 3.9 (s, 3H); ¹³C NMR (126 MHz, MeOD) δ (ppm) = 165, 152, 151, 148, 137, 127, 124, 117, 115, 114, 112, 100, 55; MS (70 eV) m/z = 218 (100%); 217 (82%); 44 (59%); 77 (42%); 76 (38%); 137 (35%); 51 (29%); 114 (29%).

(E)-2-Cyano-3-(3,4,5-Trimethoxyphenyl)Acrylamide (2g)

C₁₃H₁₄N₂O₄, 262.27 g mol⁻¹; (95% yield); yellow crystal; m.p. = 125–127 °C; FTIR ν_max(cm⁻¹) = 3346, 3208, 2924, 2213, 1633, 1588, 1510, 1320; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) = 8.1 (s, 1H), 7.8 (m, 2H), 7.4 (s, 2H), 3.8 (s, 6H), 3.7 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ (ppm) = 163, 153, 151, 141, 127, 117, 108, 105, 61, 56; MS (70 eV) m/z = 262 (100%); 247 (50%); 202 (27%); 188 (25%); 187 (24%); 44 (19%); 161 (18%); 144 (17%).

(E)-2-Cyano-3-(4-Hydroxy-3,5-Dimethoxyphenyl)Acrylamide (2h)

C₁₂H₁₂N₂O₄, 248.24 g.mol⁻¹; (93% yield); yellow solid; m.p. = 123–124 °C; FTIR ν_max (cm⁻¹) = 3346, 3204, 2930, 2211, 1675, 1576, 1510, 1314; ¹H NMR (400 MHz, MeOD) δ (ppm) = 8.1 (s, 1H), 7.4 (s, 2H), 3.9 (s, 6H).; ¹³C NMR (100 MHz, MeOD) δ (ppm) = 165, 152, 148, 141, 122, 117, 108, 106, 100, 56, 55; MS (70 eV) m/z = 248 (100%); 247 (54%); 217 (24%); 55 (22%); 73 (21%); 44 (20%); 43 (19%); 117 (18%).

(E)-2-Cyano-3-(Thiophen-2-Yl)Acrylamide (2i)

C₈H₆N₂OS, 178.21 g.mol⁻¹; (94% yield); brown crystal; m.p. = 115–117 °C; FTIR ν_max (cm⁻¹) = 3334, 3194, 2211, 1675, 1578, 1386; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.4 (s, 1H), 8.1 (dt, J = 5.0 and 0.9 Hz, 1H), 7.8 (dd, J = 3.7 and 0.7 Hz, 1H), 7.3 (dd, J = 5.0 and 3.7 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ (ppm) = 163, 144, 138, 136, 135, 129, 117, 102; MS (70 eV) m/z = 178 (100%); 177 (67%); 134 (43%); 108 (41%); 162 (39%); 90 (22%); 44 (22%); 135 (21%).

General Procedure for Reducing the Knoevenagel Adducts 2a-i Employing NaBH₄

The Knoevenagel adduct 2a-i (0.2 mmol) was placed into a 25-mL round flask and dissolved with 5 mL of MeOH. After that, the solution was cooled to 0 °C, and sodium borohydride (NaBH₄, 0.2 mmol) was added. The reaction was carried out for 20 min and monitored by TLC (in a mixture containing hexane and ethyl acetate 7:3) and revealed with sublimated iodine impregnated on silica gel. The reaction was extracted with ethyl acetate (3 × 10 mL), and the organic phase was combined, dried with anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The obtained product was purified by column chromatography with silica flash using hexane and ethyl acetate (7:3). All the obtained adducts 3a-i were characterized by m.p., FTIR, NMR and MS, as described in the previous section. All performed analyzes can be verified in the ESM-2.

2-Cyano-3-(4-Methoxyphenyl)Propanamide (3a)

C₁₁H₁₂N₂O₂, 204.09 g mol⁻¹ (70% yield); white solid; m.p. = 169 °C; FTIR ν_max (cm⁻¹) = 3407, 3308, 3208, 2949, 2251, 1663, 1613, 1514, 1404, 1315, 1244, 1165, 1035, 826, 597; ¹H NMR (500 MHz, CDCl₃) δ 7.2 (d, J = 8.7 Hz, 2H), 6.9 (d, J = 8.7 Hz, 2H), 3.8 (s, 3H), 3.6 (dd, J = 7.8 and 5.2 Hz, 1H), 3.2 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 166, 159, 130, 127, 118, 114, 55, 41, 35; MS (70 eV) m/z = 121 (100%); 77 (10%); 204 (10%); 122 (9%); 91 (7%); 78 (6%); 89 (4%); 44 (3.1%).

2-Cyano-3-(4-Nitrophenyl)Propanamide (3b)

C₁₂H₉N₃O₃, 219.06 g.mol⁻¹ (79% yield); yellow solid; m.p. = 157 °C; FTIR ν_max (cm⁻¹) = 3412, 3311, 3200, 2929, 2241, 1604, 1514, 1345, 1214, 1115, 846, 706, 617; ¹H NMR (500 MHz, DMSO-d₆) δ 8.2 (d, J = 8.8 Hz, 2H), 7.6 (d, J = 8.8 Hz, 2H), 4.1 (dd, J = 8.6, 6.8 Hz, 1H), 3.2 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166, 147, 145, 131, 124, 118, 39, 35; MS (70 eV) m/z = 175 (100%); 129 (72%); 77 (50%); 89 (44%); 44 (41%); 78 (39%); 106 (38%); 128 (37%).

2-Cyano-3-(4-Fluorophenyl)Propanamide (3c)

C₁₀H₉N₂OF, 192.07 g.mol⁻¹ (83% yield); white solid; m.p. = 140 °C; FTIR ν_max (cm⁻¹) = 3468, 3308, 3188, 2929, 2241, 1683, 1604, 1514, 1404, 1315, 1274, 1205, 1165, 1035, 816, 607; ¹H NMR (400 MHz, acetone-d₆) δ 7.4 (ddd, J = 8.5, 5.4 and 2.6 Hz, 2H), 7.1 (m, 2H), 3.9 (dd, J = 8.5 and 6.7 Hz, 1H), 3.2 (m, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 167, 163 (d, J = 243.70 Hz, 1C), 134 (d, J = 3.40 Hz, 1C), 132 (d, J = 8.10 Hz, 1C), 118, 116 (d, J = 21.40 Hz, 1C), 40, 36; MS (70 eV) m/z = 109 (100%); 148 (22%); 83 (15%); 192 (13%); 44 (12%); 101 (11%); 122 (9%); 75 (9%).

2-Cyano-3-(4-Chlorophenyl)Propanamide (3d)

C₁₀H₉N₂OCl, 208.04 g.mol⁻¹; (89% yield); white solid; m.p. = 169 °C; FTIR ν_max (cm⁻¹) = 3428, 3198,, 2909, 2241, 1663, 1604, 1503, 1404, 1096, 1016, 906, 806, 727, 577; ¹H NMR (400 MHz, acetone-d₆) δ 7.4 (s, 5H), 4.0 (dd, J = 8.4 and 6.7 Hz, 1H), 3.2 (m, 2H). ¹³C NMR (101 MHz, acetone-d₆) δ 167, 137, 133, 132, 129, 118, 40, 36; MS (70 eV) m/z = 125 (100%); 127 (34%); 164 (20%); 208 (19%); 89 (14%); 128 (12%); 101 (10%); 75 (10%).

2-Cyano-3-(4-Bromophenyl)Propanamide (3e)

C₁₀H₉N₂OBr, 251.99 g.mol⁻¹; (80% yield); white solid; m.p. = 174 °C; FTIR ν_max (cm⁻¹) = 3407, 3198, 2939, 2251, 1683, 1604, 1494, 1395, 1075, 995, 906, 786, 607, 477; ¹H NMR (400 MHz, acetone-d₆) δ 7.5 (d, J = 8.4 Hz, 2H), 7.3 (d, J = 8.3 Hz, 2H), 4.0 (dd, J = 8.3 and 6.8 Hz, 1H), 3.2 (m, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 167, 137, 132, 121, 118, 40, 36, 1; MS (70 eV) m/z = 169 (100%); 171 (95%); 90 (33%); 129 (32%); 89 (26%); 102 (21%); 44 (20%); 252 (19%).

2-Cyano-3-(4-Hydroxy-3-Methoxyphenyl)Propanamide (3f)

C₁₁H₁₂N₂O₃, 220.08 g.mol⁻¹; (88% yield); yellow oil; FTIR ν_max (cm⁻¹) = 3407, 3308, 3188, 2919, 2251, 1673, 1613, 1524, 1404, 1254, 1214, 1115, 1025, 786, 626;¹H NMR (500 MHz, acetone-d₆) δ 6.9 (d, J = 8.2 Hz, 1H), 6.8 (d, J = 2.2 Hz, 1H), 6.7 (dd, J = 8.2 and 2.2 Hz, 1H), 3.9 (dd, J = 8.6 and 6.6 Hz, 1H), 3.8 (s, 3H), 3.1 (m, 2H). ¹³C NMR (126 MHz, acetone-d₆) δ 167, 148, 147, 131, 121, 119, 117, 112, 56, 41, 36; MS (70 eV) m/z = 137 (100%) 44 (24%); 77 (18%); 94 (17%); 122 (16%); 40 (13%); 220 (12%); 43 (12%).

2-Cyano-3-(3,4,5-Trimethoxyphenyl)Propanamideacrilamida (3g)

C₁₃H₁₆N₂O₄, 264.11 g.mol⁻¹; (83% yield); white oil; FTIR ν_max (cm⁻¹) = 3447, 3358, 3219, 2949, 2829 2241, 1713, 1604, 1503, 1463, 1384, 1235, 1115, 995, 796, 527; ¹H NMR (500 MHz, acetone-d₆) δ 6.7 (s, 2H), 4.0 (dd, J = 8.5 and 6.7 Hz, 1H), 3.8 (s, 3H), 3.7 (s, 3H), 3.1 (m, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 167, 154, 138, 133, 119, 107, 60, 56, 41, 37; MS (70 eV) m/z = 181 (100%); 264 (18%); 148 (13%); 182 (11%); 77 (6%); 91 (5%); 220 (5%); 137 (4%).

2-Cyano-3-(4-Hydroxy-3,5-Dimethoxyphenyl)Propanamide (3h)

C₁₂H₁₄N₂O₄, 250.10 g.mol⁻¹; (78% yield); dark yellow solid; m.p. = 173 °C; FTIR ν_max (cm⁻¹) = 3392, 3275, 3206, 2950, 2846, 2253, 1683, 1604, 1521, 1452, 1335, 1243, 1115, 894, 801, 661; ¹H NMR (400 MHz, DMSO-d₆) δ 6.6 (s, 2H), 3.9 (dd, J = 9.0 and 6.6 Hz, 1H), 3.7 (s, 3H), 3.0 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167, 148, 135, 127, 119, 106, 56, 40, 35. MS (70 eV) m/z = 167 (100%); 250 (21%); 168 (10%); 123 (7%); 77 (4%); 174 (4%); 106 (4%); 78 (4%).

2-Cyano-3-(Thiophen-2-yl)Propanamide (3i)

C₈H₈N₂OS, 180.04 g.mol⁻¹ (61% yield); yellow solid; m.p. = 123 °C; FTIR ν_max (cm⁻¹) = 3367, 3180, 2919, 2800, 2260, 1673, 1604, 1434, 1404, 1284, 1205, 1137, 859, 661; ¹H NMR (400 MHz, acetone-d₆) δ (ppm) 7.3 (dd, J = 5.1 and 1.2 Hz, 1H), 7.0 (dd, J = 3.5 and 1.1 Hz, 1H), 7.0 (dd, J = 5.1 and 3.5 Hz, 1H), 4.0 (dd, J = 8.0 and 6.5 Hz, 1H), 3.4 (m, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 167, 139, 128, 127, 126, 118, 41, 31; MS (70 eV) m/z = 97 (100%); 180 (20%); 136 (15%); 45 (10%); 53 (7%); 109 (7%); 98 (6%); 44 (6%).

Isolation and Cultivation of Marine-Derived Fungi

The marine-derived strains employed in this study were collected by Roberto G. S. Berlinck (professor of the Chemistry Institute of São Carlos, University of São Paulo, IQSC-USP, Brazil) from the South Atlantic Ocean at São Sebastião, São Paulo, Brazil. The marine-derived fungi Aspergillus sydowii CBMAI 935 was isolated from the sponge Chelonaplysilla erecta. Whereas Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, and Westerdykella sp. CBMAI 1679 were isolated from Dragmacidon reticulatum (Birolli et al. 2016).

The strains Penicillium citrinum CBMAI 1186 and Penicillium oxalicum CBMAI 1185 were obtained from the marine alga Caulerpa sp. (Ferreira et al. 2014; Rocha et al. 2012). Whereas the strain Trichoderma harzianum CBMAI 1677 was isolated from the sponge Didemnum ligulum (Vacondio 2015). All the strains were deposited and are available at the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI–http://webdrm.cpqba.unicamp.br/cbmai/, WDCM 823).

These fungi were cultivated in malt 2% medium composed of agar (15 g L⁻¹) for solid medium and malt (20 g L⁻¹) in artificial seawater, which was composed of: CaCl₂.2H₂O (1.30 g L⁻¹), MgCl₂.6H₂O (9.68 g L⁻¹), KCl (0.61 g L⁻¹), NaCl (30.00 g L⁻¹), Na₂HPO₄ (0.014 g L⁻¹), Na₂SO₄ (3.47 g L⁻¹), NaHCO₃ (0.17 g L⁻¹), KBr (0.10 g L⁻¹), SrCl₂.6H₂O (0.04 g L⁻¹), and H₃BO₃ (0.03 g L⁻¹) (Vacondio et al. 2015). The pH was adjusted to 7.0 with a KOH aqueous solution (0.1 mol L⁻¹).

Each fungus strain was grown for 5 days in a Petri dish (90 mm × 15 mm) on malt 2% solid medium sterilized in autoclave (20 min, 121 °C, 1.5 kPa). Then, seven small disks with 5-mm diameter from the mycelial colony were transferred to five 250 mL-Erlenmeyer flasks containing 100 mL of 2% malt liquid medium. The flasks were incubated in an orbital shaker (130 rpm, 32 °C) for 5 days and then employed for the reduction reaction (Birolli et al. 2017).

Reduction of Knoevenagel Adducts 2a-i by Marine-Derived Fungi

The bioreduction reaction was carried out employing whole-cells of marine-derived fungi. The mycelial mass was filtered using a Buchner apparatus, and 5.0 g of wet-cells were transferred to a 250-mL Erlenmeyer flask containing 100 mL of phosphate buffer (Na₂HPO₄/KH₂PO₄, pH = 7.0, 0.1 mol L⁻¹) and a cotton plug that enables air exchange.

Then, 50 mg of Knoevenagel adduct 2a-i was dissolved in 400 μL of DMSO and added to the reactional media. All the manipulations were carried out with sterile instruments in a laminar flow cabinet (Veco), and the reactions were carried out in an orbital shaker (Tecnal TE-421) at 32 °C and 130 rpm for 0.1 to 120 h. These experiments were performed to verify biodegradation products.

Biodegradation of 2-Cyano-3-(4-Methoxyphenyl)Propanamide 3a

The biodegradation of the reduced adduct 3a was performed as described for the reduction reaction in the “Reduction of Knoevenagel Adducts by Marine-Derived Fungi” section at 32 °C and 130 rpm for 1 to 15 days.

Product Extraction

For extraction of the obtained product by marine-derived fungus, 100 mL of ethyl acetate was added to the reactional flask, and the sample was vigorous stirred magnetically for 30 min for cell lysis and product extraction. The obtained material was filtered using a Buchner apparatus, and a liquid-liquid extraction was carried out with the supernatant. A second step of liquid–liquid extraction was performed with the addition of 50 mL of ethyl acetate to the aqueous phase. Subsequently, the organic phases were combined, dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure.

Determination of Yield and Conversion

The obtained extract was resuspended in a 10-mL volumetric flask, and 800 μL of the sample was placed in a 1-mL volumetric flask with 200 μL of an anthracene solution of 2000 mg L⁻¹ employed as internal standard. Resulting in a sample with a maximum concentration of 4000 mg L⁻¹ of product 2a with 400 mg L⁻¹ of anthracene, which was employed for GC-MS analyses.

The GC-MS analyses were carried out in a Shimadzu GC2010plus coupled to a mass selective detector (Shimadzu MS2010plus) in electron ionization (EI, 70 eV) mode. The GC-MS (equipped with a 30 m × 0.25 mm × 0.25 μm J&W Scientific DB5 column) conditions were oven temperature started at 90 °C for 2 min, increased to 280 °C at 6 °C min⁻¹, and held for 7.3 min in this temperature. The injector and interface temperature were maintained at 250 °C. The injection volume was 1 μL with split ratio of 5. Helium was used as the carrier gas at a constant column flow of 0.75 mL min⁻¹ with a total run time of 41 min. The MS was employed in scan mode of m/z 40–500. This method was modified from a previous work and was also employed for metabolites identification (Birolli et al. 2016).

A calibration curve was obtained for the product 3a employing solutions of 500, 1500, 2500, 3500, and 4500 mg L⁻¹, c_{product 3a} = ((A_{product 3a}/A_anthracene) − 0.3093)/0.0009) with R² of 0.99, where c_product = product 3a concentration, A_{product 3a} = product 3a area and A_anthracene = area of the internal standard anthracene. The chromatographic yield (y) was determined by the equation y = (c_{product 3a}/4040) × 100.

A calibration curve was constructed for the reagent 2a also employing solutions of 500, 1500, 2500, 3500, and 4500 mg L⁻¹, c_{reagent 2a} = ((A_{reagent 2a}/A_anthracene) − 0.5674)/0.0007) with R² of 0.98, where c_reagent = reagent 2a concentration, A_{reagent 2a} = reagent 2a area, and A_anthracene = area of the internal standard anthracene. The chromatographic conversion (conv.) was defined as the reagent consumption determined by conv. = 100 − (c_{reagent 2a}/4000) × 100.

For the isolated yield determination, the products 3a-i were purified by column chromatography with silica flash employing hexane and ethyl acetate (7:3) as eluent. The obtained product was dried, weighted, and the isolated yield was determined. The remaining reagent was weighted, and the conversion was defined as the consumed percentage of the substrate.

Determination of the Enantiomeric Excess

The enantiomeric excess was determined in a Shimadzu HPLC system composed of: LC-20AT pump, DGU-20A5 degasser, SIL-20AHT sampler, SPD-M20A UV-VIS detector, CTO-20A column oven and CBM-20A controller. A chiralpak AD-H column (25.0 cm × 4.6 mm × 5 μm of particle size) was employed in normal isocratic mode composed of 30% ethanol in hexane at a flow rate of 1 mL min⁻¹ with detection at 225 nm. Chromatograms can be verified in ESM-4.

Results and Discussion

Synthesis of the Knoevenagel Adducts 2a-i Under Microwave Irradiation

The synthesis of Knoevenagel adducts 2a-i was carried out using triethylamine and water under microwave irradiation (Scheme 1). A total of nine adducts were obtained and employed for bioreduction by marine-derived fungi. The obtained yields were high (y > 93%) regardless of the substituent groups present in the aromatic aldehyde employed as reagent. The compound 2a, which presented the highest yield, was employed as model for maximization of the bioreduction reaction.

Scheme 1

Synthesis of Knoevenagel adducts 2a-i under microwave irradiation

Reduction of the Knoevenagel Adducts 3a-I for Obtention of Racemic Standards

A reduction of the Knoevenagel adducts 2a-i with NaBH₄ was carried out for the obtention of racemic standards of 3a-i for analyses of enantiomeric excess. All the employed adducts resulted in excellent yields, > 70% (Scheme 2).

Scheme 2

Reduction of the Knoevenagel adducts with NaBH₄

Fungi Screening for Reduction of the Knoevenagel Adducts 2a-i

An initial screening was performed for selection of an efficient biocatalyst for the reduction of the Knoevenagel adducts. Therefore, 8 strains of marine-derived fungi were screened for the ene-reduction of the compound 2a (Table 1).

Table 1 Screening of marine-derived fungi for ene-reduction of the Knoevenagel adduct 2a by marine-derived fungi^a

Acremonium sp. CBMAI 1676 presented 14, 27, and 41% yield for 24, 48, and 120 h (Entries 1–3, Table 1), showing that the mycelia remain active for bioreduction for at least 120 h in the employed conditions. In addition, increasing values of conversion (conv.) were also obtained, 16, 30, and 45% for 24, 48, and 120 h, respectively (Entries 1–3, Table 1).

A. sydowii CBMAI 935 presented 56% conv. with 38% yield for 24 h, but for 48 h, 98% conv. was determined (Entries 4 and 5, Table 1). After 120 h, 97% conv. with 44% yield was obtained (Entry 6, Table 1). This conversion of about 100% showed that this yield value cannot be maximized with extended reaction times.

Therefore, a secondary process of biodegradation occurs at the same time of the bioreduction, since about 100% yield was not observed, although about 100% conv. for 3a was determined. Additionally, the secondary product 3-(4-methoxyphenyl)propanenitrile 4a considered a biodegradation metabolite was identified by GC-MS analysis (96% of similarity). Additional experiments exploring the process of product biodegradation responsible for the reduced yields for prolonged periods of reaction were described in the “Biodegradation of 2-Cyano-3-(4-Methoxyphenyl)Propanamide 3a” section.

The same patterns for these bioreduction and biodegradation processes was observed for Cladosporium sp. CBMAI 1237, but with a high 3a yield of 86% with 96% conv. 2a for 24 h (Entry 7, Table 1), showing that the reaction can achieve about 90% yield in just 1 day. Additionally, 59 and 6% yield with complete conversion (100%) were determined for 48 and 120 h, respectively (Entries 8 and 9, Table 1).

T. harzianum CBMAI 1677 showed a reduced yield for the 24-h reaction, presenting 28% yield 3a with 35% conv. 2a (Entry 10, Table 1). Whereas for 48 h, 98% yield with 98% conv. were determined for 3a (Entry 11, Table 1), showing that the contribution of biodegradation in this reaction was small for this strain in these reactional conditions, since yield and conversion presented the same value. However, for 120 h, a biodegradation process occurred, and a yield of 60% was determined with 100% conversion (Entry 12, Table 1).

Another strain, Westerdykella sp. CBMAI 1679 presented a high 3a yield of 84% with 99% conv. 2a for only 24 h (Entry 13, Table 1). Whereas for 48 h, a yield of 29% with 99% conv. was determined (Entry 14, Table 1), showing that a greater biodegradation process was observed for longer reaction times. For 120 h, 100% conv. (Entry 15, Table 1) was observed without detected product, showing that the substrate and product were completely biodegraded.

The same accelerated biodegradation was observed for P. oxalicum CBMAI 1996, in which for 24 h, only 16% yield was observed, although 100% conv. was determined for 3a (Entry 16, Table 1). The about 100% conv. for 48 and 120 h of reaction without yield observation (Entries 17 and 18, Table 1) was due to the fast consumption of both substrate 2a and product 3a.

For P. citrinum CBMAI1186, 74% yield 3a with 88% conv. 2a was determined for 24 h, showing a relatively fast reduction of the substrate (Entry 19, Table 1). However, no yield was observed for 48 and 120 h (Entries 20 and 21, Table 1), showing that the rate of consumption of the reduced product was similar or higher than the rate of reduction of the Knoevenagel adduct, since no product was detected with 100% conv.

The strain Microsphaeropsis sp. CBMAI 1675 presented about 80% yield 3a for 24 and 48 h with about 90% conv. 2a, showing that for this strain the biodegradation process was slower than for the others evaluated fungi (Entries 22 and 23, Table 1). However, for 120 h, a complete consumption of the substrate and product was observed; therefore, no yield with 100% conv. was determined (Entry 24, Table 1).

The strain Trichoderma harzianum CBMAI 1677, which presented 98% yield for 48 h with reduced contribution of the biodegradation process in the reduction reaction, was selected for further studies of experimental conditions aiming the obtention of higher yields with shorter reaction times.

Different reactional media for the bioreduction of 2a in 16 h were evaluated for the biocatalyst T. harzianum CBMAI 1677, including the addition of glucose, the employment of a biphasic system, and the use of different co-solvents as methanol, isopropanol, and DMSO (Table 2).

Table 2 Exploration of reactional conditions for the reduction of the Knoevenagel adduct 2a^a

The reaction in phosphate buffer (0.1 M, pH 7.0) presented 16% yield 3a with 29% conv. 2a (Entry 1, Table 2), whereas the addition of 5% (v/v) of methanol to the reaction media aiming increased solubilization of the substrate that promoted an increase in the 3a yield to 22% with 28% conv. 2a (Entry 2, Table 2), as observed in the literature (Velankar and Heble 2003). It is notewhorthy that the biodegradation product 4a was present in this analysis in trace concentration. Since the employment of a co-solvent increased the obtained yield, the use of isopropanol and DMSO as co-solvents was also evaluated.

The reaction medium composed of phosphate buffer (0.1 M pH 7.0) with 5% of isopropanol presented 17% yield 3a with 33% conv. 2a (Entry 3, Table 2), a very similar value to the reaction in the absence of a co-solvent (16% yield, Entry 1, Table 2) and inferior to the use of methanol (22% yield, Entry 2, Table 2). The use of DMSO in the reactional medium promoted a yield decrease, 14% yield 3a with about 30% conv. (Entry 4, Table 2).

The supplementation of the reactional medium with glucose aiming the acceleration of the cell metabolism for the obtention of higher yields in shorter reaction times was not effective, since the 3a yield slightly increased to 19% with a conversion of 33% 2a (Entry 5, Table 2) in comparison with the reaction without an energy source (16% yield, Entry 1, Table 2). The use of both glucose and the co-solvent methanol resulted in 20% yield with 46% conv. (Entry 6, Table 2), showing that these conditions favored the biodegradation process, since the difference between yield and conv. increased significantly.

The employment of a biphasic system composed of phosphate buffer (0.1 M, pH 7.0) and hexane (2:1) was also evaluated for reduction of possible inhibition effects of the substrate and product during the reaction (Ou et al. 2019). An excellent reaction yield of 40% with 45% conv. was obtained (Entry 7, Table 2), showing that this approach was interesting for a faster bioreduction with decreased biodegradation.

However, the addition of glucose (5% yield with 49% conv., Entry 8, Table 2), methanol (5% yield with 50% conv., Entry 9, Table 2), and both glucose and methanol (6% yield with 48% conv., Entry 10, Table 2) in the biphasic reactional medium failed in the promotion of synergistic effects that would promote better results.

The reduced yields occurred due to increased biodegradation with the formation of 4a, 4-methoxybenzaldehyde, 4-methoxyphenol, 1,4-dimethoxybenzene, and 2-cyanoacetamide (Fig. 1). Additionally, the product composition was estimated employing the GC-MS analysis. Mass spectra were presented in ESM-3.

Fig. 1

Biotransformation and biodegradation of the Knoevenagel adduct 2a by T. harzianum CBMAI 1677 in biphasic system (0.1 M phosphate buffer pH 7.0 and hexane (1:1) with 5% (v/v) methanol and 0.5% (w/v) glucose, 32 °C, 130 rpm, 16 h). ^a Conv. = conversion; ^b Percentage of product composition estimated by GC-MS.

The enantiomeric excess was determined employing liquid chromatography. However, none of the samples presented significant enantioselectivity (see ESM-4). A racemization process for this class of compounds already was described in the literature for reduced Knoevenagel adducts of malononitrile and aromatic aldehydes (Jimenez et al. 2019).

A tautomeric equilibrium based in the formation of a prototropic ketenimine form was proposed for (±)-2-cyano-3-(furan-2-yl)propanamide, justifying the production of a racemic mixture. This phenomenon is common in compounds containing cyano groups with acidic hydrogens on α-C positions with the nitrile form being the predominant structure (Jimenez et al. 2019; Kasturi et al. 1962; Kasturi et al. 1973).

After the evaluation of different experimental media, a second screening of marine-derived fungi was carried out for 16 h in phosphate buffer and in biphasic system (0.1 M phosphate buffer pH 7.0 and hexane (1:1), 32 °C, 130 rpm, 16 h). The strains P. oxalicum CBMAI 1996, Microsphaeropsis sp. CBMAI 1675, and Cladosporium sp. CBMAI 1237, which presented a faster biotransformation in the screening with about 90% conv. 2a in 24 h (Entries 16, 22 and 7, Table 1), were reevaluated and compared to T. harzianum CBMAI 1677 (Fig. 2).

Fig. 2

Screening of marine fungi for the reduction of the Knoevenagel condensation product 2a in phosphate buffer and in a biphasic system (reactional conditions: 50 mg of 2a, 5.0 g of humid whole-cells of marine-derived fungi, 100 mL of 0.1 M phosphate buffer pH 7.0 with or without 50 mL of hexane for biphasic system (2:1), 32 °C, 130 rpm, 16 h).

P. oxalicum CBMAI 1996 presented 20% yield 3a with 97% conv. 2a in phosphate buffer for a 16-h reaction, showing that the biodegradation process was also present for a shorter reaction time (Entry 1, Fig. 2). The employment of the biphasic system resulted in 16% yield (Entry 2, Fig. 2), differently from the reduction by T. harzianum CBMAI 1677, which presented a yield increase in this reactional condition (from 16 to 40% yield, Entries 7 and 8, Fig. 2).

The same pattern was observed for the fungus Microsphaeropsis sp. CBMAI 1675 that presented 45% yield with 84% conv. in phosphate buffer (Entry 3, Fig. 2) and 11% yield with 42% conv. for the biphasic system (Entry 4, Fig. 2), showing that this approach was not successful for this strain either.

The strain Cladosporium sp. CBMAI 1237 presented an excellent yield of 95% with 96% conv. for 16 h in phosphate buffer (Entry 5, Fig. 2), whereas in the biphasic system 82% yield with 91% conv. was determined (Entry 6, Fig. 2). The presence of solvents in the reaction medium may promote toxic effects on the employed strains, reducing their performance as biocatalyst, and promoting reduced yields and conversions in the biphasic system.

It was observed that the increased catalysis observed for T. harzianum CBMAI 1677 in biphasic system was specific for the enzymes from this fungus strain during the reaction process. But the employment of the strain Cladosporium sp. CBMAI 1237 in phosphate buffer constitute a greener methodology, since it avoids the use of hexane and promote increased yields.

Reduction of Knoevenagel Adducts by Cladosporium sp. CBMAI 1237

The kinetic of the reduction reaction of 2a by Cladosporium sp. CBMAI 1237 was evaluated aiming the obtention of high yields with reduction of the reactional time. The reaction was evaluated from 0 to 24 h with independent samples, showing that the reaction reaches a maximum yield of 97% after 8 h (Fig. 3).

Fig. 3

Reduction of the Knoevenagel adduct 2a by Cladosporium sp. CBMAI 1237 over time. Reactional conditions: 50 mg of adduct 2a, 5.0 g of humid whole cells of Cladosporium sp. 1237, 100 mL of 0.1 M phosphate buffer pH 7.0, 32 °C, 130 rpm, 0.2–24 h.

A reaction kinetic of zero-order (c = c_initial + kt, where c = concentration (mol L⁻¹), c_initial = initial concentration (mol L⁻¹), k = zero-order rate constant (mol L⁻¹ h⁻¹), and t = time (h)) was determined for the production of the reduced product, c = 0.23 + 0.42 t with R² = 0.98, showing a productivity from time 0.1 to 5.0 h of 0.42 mol L⁻¹ h⁻¹.

This zero-order kinetic is common in enzyme-catalyzed reactions and occurs when a bottleneck is limiting the reaction. Therefore, the observed constant reaction rate can be promoted by reduced contact of the substrate with the enzymes due to limited transportation of the reagent thought the cellular membrane, or by enzyme saturation due to excess of reagent (Atkins and De Paula 2006; Tinoco Jr and Wang 1995).

From 8 to 18 h, the reaction yield and conversion remained constant at about 95% of 3a, and, after this, a biodegradation process became significant affecting the reaction yield that decreased to 86% from 18 to 24 h. Then the consumption of the product 3a for energy obtention by the biocatalyst increased and yields of 59% and 6% were determined for 48 h and 120 h (Entries 8 and 9, Table 1).

A first-order kinetic (c = c_initial.e^−kt, where c = concentration (mol L⁻¹), c_initial = initial concentration (mol L⁻¹), k = first-order rate constant (h⁻¹), and t = time (h)) of biodegradation was obtained for the reduced product considering the data for 18 (initial time of the biodegradation process, thus 0 h), 21, 24, and 48 h, c = 2.40.e^0.0168t with R² = 0.99, and a half-life of 41.3 h.

The reaction product obtained from the 8-h reaction was purified, and an isolated yield of 88% was obtained for 3a (Entry 1, Table 3), showing that the reduction reaction of nitrogenated compounds can be performed in high reaction yields. However, it is necessary to emphasize that the reaction time is an important variable of this process, since biodegradation decreases the yield.

Table 3 Reduction of different Knoevenagel adducts by Cladosporium sp. CBMAI 1237.^a,b,c

Then, different substrates were employed for the evaluation of the reaction scope employing Cladosporium sp. CBMAI 1237 as catalyst. The strong electron withdrawing group p-NO₂ was employed, and 61% yield was determined for 3b (Entry 2, Table 3), showing that the employed method can be applied with this type of group in the aromatic ring.

Halogenated substrates were also reduced; the product 3c with the substituent p-F was obtained with 64% yield (Entry 3, Table 3), whereas 3d with p-Cl and 3e with p-Br presented 58% and 65% yield (Entries 4 and 5, Table 3) respectively, showing that electronic effects did not affect significantly the reaction.

Larger aromatic aldehydes were also employed, and the product 3f with p-OH and m-OCH₃ was obtained with 48% yield (Entry 6, Table 3); a similar yield of 49% for 3g with 3,4,5-OCH₃ was also determined (Entry 7, Table 3). However, 3i with 3-OCH₃, 5-OCH₃, and 4-OH was not obtained; therefore, 0% of yield was determined with a conversion of 79% 2i (Entry 9, Table 3). Probably, the biodegradation process was faster than the reduction of the substrate, consuming all the obtained product.

Moreover, 3h was obtained in 90% yield (highest production of this study) in the employed conditions (Entry 8, Table 3), showing that compounds with heteroatoms in the aromatic ring can also be reduced by the marine-derived fungus Clasporium sp. CBMAI 1237.

Each compound probably presents an optimal experimental condition for the obtention of a maximum yield. For example, the time in our study strongly influenced the quantity of obtained product; thus, the reaction period should be determined for each employed substrate for obtention of more interesting results. Especially for those that presented 100% of conversion, because shorter reaction times would probably promote increased yields due to reduced biodegradation (Entries 2–5 and 8, Table 3).

The reduction of α,β-unsaturations of malononitrile derivatives was performed by the marine fungus Penicillium citrinum CBMAI 1186 in high yields (up to 98%) in reactions of 6 days (32 °C, 130 rpm) without a significant presence of a biodegradation process, as also observed for E-2-cyano-3-(furan-2-yl)acrylamide reduction with yields up to 99% for 3 days reactions (32 °C, 130 rpm) by P. citrinum CBMAI 1186, A. sydowii CBMAI 935, Trichoderma sp. CBMAI 932, Aspergillus sp. FPZSP152, and Aspergillus sp. FPZSP 146 (Jimenez et al. 2019; Jimenez et al. 2016).

Probably, small changes in the substrate structure promoted a faster attack of degradative enzymes, effect that may be enhanced by the employment of nitrogenated compounds that can be used by the strain as carbon and nitrogen source. The biodegradation of substrate in biocatalytic reactions by whole cells already was described for Knoevenagel adducts and isatin, which presented low yields (up to 37%) due to a biodegradation process and moderate enantioselectivities (up to 66%) (Birolli et al. 2017).

The enantiomeric excesses were determined by HPLC. However, all the obtained products were racemic. Chromatograms are available in ESM-4.

It is important to note that biodegradation metabolites were observed for all the employed substrates, except for the product 3i. For example, the 2a reaction had 94% conversion with 88% of 3a isolated yield, and 6.6% was determined as the biodegradation metabolite 4a (percentage determined by CG-MS analysis).

Some substrates presented about 10% of biodegradation metabolites such as 4a, 4c, 4d, and 4 h, whereas some compounds were about 20% of the obtained reaction material, including 4 g and 4e. Other compounds presented about 30% in secondary products, 4b and 4 f. These findings showed that the biodegradation process is present in the bioreduction of all the employed substrates (Fig. 4). Mass spectra were presented in ESM-3.

Fig. 4

Biodegradation metabolites identified by GC-MS.

Biodegradation of 2-Cyano-3-(4-Methoxyphenyl)Propanamide 3a

The biodegradation of 3a was carried out for 1, 2, 5, and 14 days, and conversions of 10, 66, 97, and 100% were determined, respectively. The only produced compound was 4a in small concentrations, thus the substrate 3a of this reaction was probably employed as carbon and nitrogen source by the biocatalyst Cladosporium sp. CBMAI 1237.

Conclusion

A bioreduction methodology of the Knoevenagel adducts 2a-h between cyanoacetamide and the aromatic aldehydes was carried out in good yields (48–90%) with whole-cells of the marine-derived fungus Cladosporium sp. CBMAI 1237, showing the presence of ene-reductases. However, enantioenriched products were not obtained due to the ketenimine equilibrium which racemized the products.

In addition, it is important to emphasize that the experimental conditions, especially the time, should be carefully evaluated for the obtention of high yields. Since a biodegradation process consume the obtained product in extended periods of reaction, probably due to the use of the substrate as carbon and nitrogen source. This study presented a coupled green approach of microwave irradiation and bioreduction for production of organic molecules in a more sustainable process.