2.2. Enzymative Deracemization of Arylcarboxylic Acids
Enzymes act in solution, at ambient temperature and neutral pH. They demonstrate a high level of reaction stereoselectivity, as well as high chemo-, regio-, and enantioselectivity. Hydrolases are enzymes that catalyze hydrolysis reactions directly in water, through aqueous buffers, or even using two-phase organic solvent/water systems. The category of hydrolases is very broad, and there is much variation among hydrolases. For example, hydrolase activity typically requires dissolution of the substrate in the reaction medium, which does not apply to lipases. Indeed, the latter demonstrate low catalytic activity towards substrates dissolved in an aqueous solution. However, lipase activity increases significantly when the substrate reaches a critical micelle concentration, where it tends to form a second phase. This activation of the water/lipid interface is attributed to the presence of the oligopeptide. (helical “lid”), which blocks access to the active site of the enzyme. When interaction with a hydrophobic interface occurs, the enzyme rearranges itself into its active conformation. This phenomenon is specific to lipases and does not occur in other hydrolases. Hydrolysis uses esterases, proteases, and some lipases. The chiral or prochiral moiety of esters performs an acidic function. The substrates presented in this article are racemic esters. Cleavage of racemic esters by hydrolase-catalyzed enantioselective hydrolysis is an efficient and practical method for the preparation of pharmaceutical intermediates (
Figure 2). Transesterification in an organic medium is an equilibrium reaction. Therefore, it is important to shift the balance in the right direction to obtain the desired result. For example, during enzymatic digestion by
Burchholderia cepacia lipase, an aryl carboxylic acid ester can be transesterified in the presence of vinyl acetate. The vinyl alcohol thus formed tautomerizes into an aldehyde during the reaction, making it irreversible.
Enantioselective esterification of racemic acid with
Burkholderia cepacia lipase in hexane can be carried out, provided that the water formed during the reaction is removed. Enzymatic hydrolysis of the racemic aryl carboxylic acid ester was carried out in phosphate buffer pH 7.0 in the presence of Amano PS lipase. The mixture was stirred for 16 h at room temperature, then filtered; the filtrate was acidified with 2 M HCl to pH 2 and extracted with MTBE. Carboxylic acid esters
3a–
e were obtained in 50–80% yield and 93–100%
ee enantioselectivity. The aqueous solution after extraction with an organic solvent was acidified with 2M HCl to pH 2 and washed with MTBE. The resulting extract was washed with a solution of sodium chloride, dried with sodium sulfate, and evaporated in a vacuum. As a result, arylcarboxylic acids
3a–
e or
5a–
e were obtained in 52–60% yield and 77–99% enantiomeric purity. To increase the enantiomeric purity of arylcarboxylic acids, additional (double) enzymatic deracemization was used (
Figure 3).
To a solution of methyl aryl carboxylic acid in ethanol solution at 0 °C, 1.6 equiv of thionyl chloride was added dropwise over 10 min. The mixture was stirred for 16 h at room temperature, then evaporated in a vacuum. The residue was dissolved in water and neutralized with an aqueous solution of potash to pH > 9. The resulting ester was added to a suspension of Amano PS in a solution of MTBE with 5 eq. water. The reaction mixture was stirred for 16 h at 40 °C, then filtered, washed with MTBE, and the filtrate was evaporated in a vacuum. The residue was dissolved by heating in MTBE, then cooled and left in the refrigerator for 10 h. In total, several lipases and esterases were tested for the resolution of racemic 3-phenylbutanoic acid ester (±)-
5a. All hydrolases tested resulted in the hydrolysis of ethyl 3-phenylbutanoate (±)-
3a, and the screening results are summarized in
Table 1.
Pseudomonas cepacia and
Burcholderia cepacia showed excellent enantioselectivity for hydrolysis of the substrate (±)-
3a. It was previously reported that hydrolysis of
Burholderia cepacia (±)-
3a methyl ester (E > 50) provides access to (
S)-
3a-acid with 89%
ee. Here, these enzymes produce (
S)-3a-acid with a high enantioselectivity of 97%. Unreacted (
R)-
3a was isolated at 98%
ee, allowing access to both enantiomeric series in a single resolution. The presence of a bulky trifluoromethyl group in ethyl (
S)-4,4,4-trifluoro-3-phenylbutanoate
4f significantly increased the reaction time, which made it difficult to bring the reaction to the optimal 50% hydrolysis (
Figure 4). Therefore, we resorted to initiating the reaction using microwave radiation, which allowed the reaction to be completed within an optimal term. The reaction in toluene at 40 °C proceeded at a constantly high
ee. Kinetic resolution occurred under the influence of microwave irradiation, and the conversion and
ee remained stable throughout the entire resolution process (
Table 1). In recent years, the trend towards the use of chiral pharmaceuticals has been steadily increasing, although requirements on this issue were published by the US Food and Drug Administration (FDA) back in 1992 in a document entitled “Development of new stereoisomeric drugs” [
1,
2].
General procedure for the resolution of fluorinated arylcarboxylic acids by lipase. A solution of 0.25 mol of racemic aryl carboxylic acid ester 2(a–e) in 1500 mL of phosphate buffer pH 7.0. Add 20 g of Amano PS lipase. The mixture was stirred for 16 h at room temperature, and then filtered, the filtrate was acidified with 2 M HCl to pH 2 and extracted with MTBE (3 × 300 mL). The organic extract was washed with 0.2 M aqueous potassium carbonate solution (2 × 300 mL), dried with sodium sulfate, and evaporated in a vacuum. Carboxylic acid esters 2(a–e) or 4(a–e) were obtained. Yield 50–80%; 93–100% ee. After extraction, the aqueous solution was acidified with 2 M HCl to pH 2 with an organic solvent and extracted with MTBE (3 × 500 mL). The extract was washed with sodium chloride solution, dried with sodium sulfate, and evaporated in a vacuum. Arylcarboxylic acids 3(a–e) or 5(a–e) were obtained. Yield 52–60%, 77–99% ee. To an enantiomerically enriched solution of 55 g (0.335 mol; 1 eq.) of aryl carboxylic acid in 500 mL of ethanol at 0 °C, 29.1 mL (0.4 mol; 1.2 eq.), thionyl chloride was added dropwise over a period of 10 min. The mixture was stirred for 16 h at room temperature, then concentrated in vacuo. The residue was dissolved in 500 mL of MTBE and washed twice with a 5% solution of sodium bicarbonate in water (2 × 300 mL). The organic layer was dried over Na2SO4 and evaporated in a vacuum. The resulting ester was added to a suspension of 5.5 g Amano PS (10 weight percent) in 550 mL phosphate buffer pH 7.0 and stirred for 16 h at room temperature. The mixture was filtered and washed with MTBE (2 × 200 mL); the filtrate was acidified to pH 2 with 2M HCl and extracted three times with MTBE (3 × 200 mL). The organic layer was washed twice with 0.5 M (2 × 300 mL) potassium carbonate solution and disposed of. The aqueous layer was acidified to pH 2 with 2M HCl and extracted three times with MTBE (3 × 500 mL). The organic layer was washed twice with saturated sodium chloride solution (2 × 500 mL), dried over sulfate, and evaporated in a vacuum. We obtained 41.5 g (252.7 mmol; ee = 99; yield 75.5%) of substance 4a in the form of a pale yellow liquid.
(R)-3-(2-Fluorophenyl)-2-methylpropanoic acid ((R)-3a). Pale yellow oil (97% yield). [α]D20 = −25.22 (C = 0.5, CH3OH), ee 95%. 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.18 (d, J = 6.8 Hz, 3H, CH3); 2.75 m (2H, CH2); 3.1 (1H, CHCH3); 6.96–7.21 m (4H, C6H4). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −118.34; LCMS: M 182.19 (M 182.09) (R); HPLC: 10.177 min (R); 9.674 min (S); Column: Chiralpak AD-H (250 × 4.6 mm, 5 mkm)—ADH0CE-WG064—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 90:10; Flow Rate: 0.6 mL/min.
(S)-3-(2-Fluorophenyl)-2-methylpropanoic acid ((S)-3a). Pale yellow oil (97% yield), [α]D20 = +25.34 (C = 0.5, CH3OH), 97.5% ee. 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.18 (d, J = 6.8 Hz, 3H, CH3); 2.75 m (2H, CH2) 3.1 (1H, CHCH3); 6.96–7.21 m (4H, C6H4). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −118.34; LCMS: M 182.19 (M 182.09) (S); HPLC: 10.081 min (R); 9.656 min (S), Column: Chiralpak AD-H (250 × 4.6 mm, 5 mkm)—ADH0CE-WG064—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 90:10; Flow Rate: 0.6 mL/min.
(R)-3-(4-fluorophenyl)-2-methylpropanoic acid ((R)-3b). Pale yellow oil (97% yield). [α]D20 =−27.1 (C = 0.5, CH3OH) ee 99%. 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.19 (d, J = 6.7 Hz, 3H, CH3); 2.73 m (2H, CH2) 3.02 (1H, CHCH3); 6.97–7.14 m (4H, C6H4). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −117.24; LCMS: M 182.19 (M 182.09) (R); HPLC 7.944 min (R); 8.958 min (S), Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—3; Mobile Phase: Hexane/IPA, 95:5; Flow Rate: 1.0 mL/min.
(S)-3-(4-fluorophenyl)-2-methylpropanoic acid ((S)-3b). Pale yellow oil (97% yield). [α]D20 = +27.2 (C = 0.5, CH3OH), ee 94% (S). 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.19 (d, J = 6.7 Hz, 3H, CH3); 2.73 m (2H, CH2) 3.02 (1H, CHCH3); 6.97–7.14 m (4H, C6H4). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −117.67. LCMS: M 182.19 (M 182.09);HPLC 8.125 min (R); 9.148 min (S), Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—3; Mobile Phase: Hexane/IPA, 95:5; Flow Rate: 1.0 mL/min.
(R)-2-methyl-3-(3-(trifluoromethyl)phenyl)propanoic acid ((R)-3c). Oil; [α]D20 = −25.04 (C = 0.5, CH3OH), (S); 97% ee. 1H NMR (500 MHz, ppm, J, Hz, DMSO) δ 1.04 (d, J = 6.8 Hz, CH3); 2.70 m (2H, CH2) 2.90 (1H, CHCH3); 7.5 d (4H, C6H4); 12.19 br (1H, OH). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 16.5 s; 38.95 s; 41.1 s; 123.42 q; 125.7 q; 128.87 s; 131 q; 132.4 s; 139.9 s; 182.2 s; J 120.08; 122.78; 125.49; 128.19. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.47; LCMS: M 232.2 (M 232.09); HPLC 7.24 min (R); 7.67 min (S), Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 97:3.
(S)-2-methyl-3-(3-(trifluoro-methyl)phenyl) propanoic acid ((S)-3c). Oil; [α]D20 = +25.27 (C = 0.5, CH3OH)(S); 99% ee 1H NMR (500 MHz, ppm, J, Hz, DMSO) δ 1.04 (d, J = 6.8 Hz, CH3); 2.70 m (2H, CH2) 2.90 (1H, CHCH3); 7.5 d (4H, C6H4); 12.19 br (1H, OH), 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 16.5 s; 38.95 s; 41.1 s; 123.42 q; 125.7 q; 128.87 s; 131 q; 132.4 s; 139.9 s; 182.2 s; JCF 120.08; 122.78; 125.49; 128.19. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.47; LCMS: M 232.2 (M 232.09); HPLC 7.24 min (R); 7.67 min (S), Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 97:3.
(R)-2-methyl-3-(4-(trifluoromethyl)phenyl)propanoic acid ((R)-3d). Mp 61 °C; [α]D20 = −24.16 (C = 0.5, CH3OH)(R); ee 98%, 1H NMR (500 MHz, ppm, J, Hz, DMSO); δ 1.04 (d, J = 6.7 Hz, 3H, CH3); 2.70 m (2H, CH2); 2.93 (1H, CHCH3); 7.40–7.63 m (4H, C6H4); 12.19 br (1H, OH). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 16.07s; 38.41 s; 40.45 s; 122.62 s; 124.8 5q, JCF 128.7; 142.57 s; 181.58 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.23; LCMS: M 232.2 (M 232.09); HPLC 9.89 min (R); 9.275 min (S), Column: Chiralpak AD-H (250 × 4.6 mm, 5 mkm)-3; Mobile Phase: Hexane (0.1% TFA)/IPA, 97:3 Flow Rate: 1.0 mL/min.
(S)-2-methyl-3-(4-(trifluoromethyl)phenyl)propanoic acid ((S)-3d). Mp 61 °C; [α]D20 = +22.5 (C = 0.5, CH3OH)(S); ee 98%. 1H NMR (500 MHz, ppm, J, Hz, DMSO) δ 1.04 (d, J = 6.7 Hz, 3H, CH3); 2.70 m (2H, CH2); 2.93 (1H, CHCH3); 7.40–7.63 m (4H, C6H4); 12.19 br (1H, OH). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 16.07 s; 38.41 s; 40.45 s; 122.62 s; 124.8 5q, JCF 128.7; 142.57 s; 181.58 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.23; LCMS: M 232.2 (M 232.09); HPLC 9.89 min (R); 9.275 min (S); Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)-3; Mobile Phase: Hexane/IPA, 98:2 Flow Rate: 0.6 mL/min.
(S)-2-methyl-3-(perfluorophenyl)propanoic acid ((S)-3e). [α]D20 = +16 (C = 0.5, CH3OH)(S); ee 99%. 1H NMR (400 MHz, ppm, J, Hz, CDCl3) δ 1.21 (d, J = 7 Hz); 2.81 m (2H, CH2); 3.1 m (1H, CHCH3). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −143 dd; −156.62 t; −162.8 dt. LCMS: M 254.04 (M 233.0 Neg [-HF]); HPLC 8.65 min (R); 9.14 min (S); Column: Chiralpak IB (250 × 4.6 mm, 5 mkm)—IB00CE-BN025—14; Mobile Phase: Hexane (0.1% TFA)/IPA, 97:3; Flow Rate: 0.6 mL/min.
(R)-2-methyl-3-(perfluorophenyl)propanoic acid ((R)-3e). [α]D20 = −16 (C = 0.5, CH3OH)(S); ee 49%. 1H NMR (400 MHz, ppm, J, Hz, CDCl3) δ 1.21 (d, J = 7 Hz); 2.81 m (2H, CH2); 3.1 m (1H, CHCH3). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −143 dd; −156.62 t; −162.8 dt. LCMS: M 254.04 (M 233.0 Neg [-HF]); HPLC 8.75 min (R); 9.25 min (S); Column: Chiralpak IB (250 × 4.6 mm, 5 mkm)—IB00CE-BN025—14; Mobile Phase: Hexane (0.1% TFA)/IPA, 97:3; Flow Rate: 0.6 mL/min.
(S)-3-(2-fluorophenyl)butanoic acid ((S)-5a). Mp 36 °C; [α]D20 = +29 (C = 0.5, CH2Cl2), ee 99%. 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.3 d (CH3); 2.55–2.8 m (2H, CH2) 3.5 (1H, CHCH3); 6.9–7.25 m (4H, C6H4)). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 19.9 s; 29.6 s; 40.4 s; 115.1 d; 123.7 d, 124.45 d; 124.51 d; 131.5 d; 159.2–161.5 d, 178.35s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −118.6; LCMS: M 182.19 (M 182.09) (S); HPLC: 12.07 min (R); 10.45 min (S), Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 90:10; Flow Rate: 0.6 mL/min.
(R)-3-(2-fluorophenyl)butanoic acid ((R)-5a). Mp 36 °C; [α]D20 = −25.95 (C = 0.5, CH2Cl2) ee 99% 1H NMR (CDCl3, 500 MHz, 25 °C): δH 1.3 d (CH3); 2.55–2.8 m (2H, CH2) 3.5 (1H, CHCH3); 6.9–7.25 m (4H, C6H4). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 19.9 s; 29.6 s; 40.4 s; 115.1 d; 123.7 d, 124.45 d; 124.51 d; 131.5 d; 159.2–161.5 d, 178.35 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −118.6; LCMS: M 182.19 (M 182.09) (R); HPLC: 11.94 min (R); 10.34 min (S), Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 90:10; Flow Rate: 0.6 mL/min.
(S)-3-(4-fluorophenyl)butanoic acid ((S)-5b). Oil; [α]D20 = +43.09 (C = 0.5, CHCl3) ee 99%. 1H NMR (DMSO, 500 MHz, 25 °C): δH 1.17 d (CH3); 2.45 d (2H, CH2) 3.13 m (1H, CHCH3); 7.09 t, 7.27 d (4H, C6H4), 12.04 br (1H, OH). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −117.7; LCMS: M 182.19 (M 182.09) (S); HPLC: 10.12 min (R); 8.88 min (S), Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 80:20; Flow Rate: 0.6 mL/min.
(R)-3-(4-fluorophenyl)butanoic acid ((R)-5b). Oil; [α]D20 = −39 (C = 0.5, CHCl3) ee 99%. 1H NMR (DMSO, 500 MHz, 25 °C): δH 1.17 d (CH3); 2.45 d (2H, CH2) 3.13 m (1H, CHCH3); 7.09 t, 7.27 d (4H, C6H4), 12.04 br (1H, OH). 19F NMR (CDCl3, 376 MHz, 25 °C): δF −117.7; LCMS: M 182.19 (M 182.09) (R); HPLC: 14.9 min (R); 11.95 min (S), Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—OJH0CE-UF008—3; Mobile Phase: Hexane/IPA, 90:10; Flow Rate: 0.6 mL/min.
(S)-3-(3-(trifluoromethyl)phenyl)butanoic acid ((S)-5c). Oil; [α]D20 = +22.5 (C = 0.5, MeOH) ee 99%. 1H NMR (DMSO, 500 MHz, 25 °C): δH 1.14 d (CH3); 2.55 d (2H, CH2); 3.16 m (1H, CHCH3); 7.4–7.6m (4H, C6H4), 12.04 br (1H, OH). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 21.13 s; 35.48; 41.8 s; JF 120.4, 122.56, 124.73, 125.9; 122.92 d; 123.05 d; 128.5 s; 129.67 s; 130.33 q; 145.79; 177.72. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.38; LCMS: M 232.2 (M 232.09) (S); HPLC: 9.45 min (R); 8.48 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 90:10; Flow Rate: 0.6 mL/min.
(R)-3-(3-(trifluoromethyl)phenyl)butanoic acid ((R)-5c). Oil; [α]D20 = −22.5 (C = 0.5, MeOH) ee 98%. 1H NMR (DMSO, 500 MHz, 25 °C): δH 1.14 d (CH3); 2.55 d (2H, CH2); 3.16 m (1H, CHCH3); 7.4–7.6 m (4H, C6H4), 12.04 br (1H, OH). 13C NMR (CDCl3, 273.8 MHz, 25 °C): δC = 21.13 s; 35.48; 41.8 s; JF 120.4, 122.56, 124.73, 125.9; 122.92 d; 123.05 d; 128.5 s; 129.67 s; 130.33 q; 145.79; 177.72. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −61.38;LCMS: M 232.2 (M 232.09) (S); HPLC: 9.12 min (R); 8.22 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 90:10; Flow Rate: 0.6 mL/min.
(S)-3-(4-(trifluoromethyl)phenyl)butanoic acid ((S)-5d). Mp 56 °C; [α]D20 = +35.0 (C = 1.1, CHCl3); ee 99%. 1H NMR (500 MHz, ppm, J, Hz, CDCl3) δ 1.3 d (CH3); 2.63 m (2H, CH2); 3.32 m (1H, CHCH3); 7.32 d, 7.54d, 4H (C6H4). 13C NMR (237.8 MHz, CDCl3) δC = 21.7 s; 35.98 s; 42.08 s; 122.5 s, 125.5 q; 127.1 s; 130 q; 149.3 s; 177.96 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −62.94; LCMS Mol 232.2 (M 232.09) (S). HPLC: 13.057 min (R); 10.79 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3.
(R)-3-(4-(trifluoromethyl)phenyl)butanoic acid ((R)-5d). Mp 56 °C; [α]D20 = −23.7 (C = 0.5, MeOH); ee 99%. 1H NMR (500 MHz, ppm, J, Hz, CDCl3) δ 1.3 d (CH3); 2.63 m (2H, CH2); 3.32 m (1H, CHCH3); 7.32d, 7.54d, 4H (C6H4). 13C NMR (237.8 MHz, CDCl3) δC = 21.7 s; 35.98 s; 42.08 s; 122.5 s, 125.5 q; 127.1 s; 130 q; 149.3 s; 177.96 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −62.94; LCMS Mol 232.2 (M 232.09) (S). HPLC: 13.057 min (R); 10.76 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 95:5; Flow Rate: 0.6 mL/min.
(S)-3-(3-(trifluoromethoxy)phenyl)butanoic acid ((S)-5e). Oil; [α]D20 = +32.0 (C = 0.5, CHCl3); ee 99%. 1H NMR (500 MHz, ppm, J, Hz, CDCl3) δH 1.31 d (J 10, 3H, CH3); 2.64 m (2H, CH2) 3.32 (1H, CHCH3); 7.05–7.35 m, 4H (C6H4). 13C NMR (237.8 MHz, CDCl3) δC = 21.1 s; 35.38 s; 41.77 s; 118.4 s, 118.9 s; 121 s; 124.6 s; 129.3 s; 147.2 s; 148.9 s; 177.49 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −62.94; LCMS Mol 248.2 (M 248.08) (S) HPLC: 22.87 min (R); 20.95 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 99:1; Flow Rate: 0.6 mL/min.
(R)-3-(3-(trifluoromethoxy) phenyl)butanoic acid ((R)-5e). Oil; [α]D20 = −20 (C = 0.5, CHCl3); ee 96%. 1H NMR (500 MHz, ppm, J, Hz, CDCl3) δH 1.31 d (J 10, 3H, CH3); 2.64 m (2H, CH2) 3.32 (1H, CHCH3); 7.05–7.35 m, 4H (C6H4). 13C NMR (237.8 MHz, CDCl3) δC = 21.1 s; 35.38 s; 41.77 s; 118.4 s, 118.9 s; 121 s; 124.6 s; 129.3 s; 147.2 s; 148.9 s; 177.49 s. 19F NMR (CDCl3, 376 MHz, 25 °C): δF −62.94; LCMS Mol 248.2 (M 248.08) (S) HPLC: 23.05 min (R); 20.99 min (S), Column: Chiralpak AS-H (250 × 4.6 mm, 5 mkm)—203VB002-DB181—3; Mobile Phase: Hexane (0.1% TFA)/IPA, 99:1; Flow Rate: 0.6 mL/min.
Lipase-catalyzed microwave-assisted enzymatic separation of (S)-4,4,4-trifluoro-3-phenylbutanoic acid (S)-5(f). Lipase-catalyzed hydrolysis of ethyl 4,4,4-trifluoro-3-phenylbutanoate under microwave irradiation was carried out in SEM Discover. Model Explorer SP 48. Power 100 W (CEM Corporation, Matthews, North Carolina, USA). Phosphate buffer (10 mL pH 7.0), lipase (160 mg Amano Lipase PS), and aryl carboxylic acid ester 2f, (0.5 g, 0,2 mmol). The entire reaction solution was placed in a microwave apparatus and irradiated at a frequency of 2.45 GHz, and at 200 m. The formation of the hydrolyzed product was monitored by TLC and NMR. The reaction mixture was filtered, and evaporated in a vacuum, the remaining ether was purified by chromatography on columns with silica gel and hexane-ethyl acetate eluent (95:53:1). The separated 4,4,4-trifluoro-3-phenylbutanoic acid was recrystallized from toluene. The reaction mixture was analyzed by GC-FLD, after irradiation, then the reaction flask was moved and the temperature of the reaction mixture was 45, °C.Mp 45 °C; [α]D20 = +42 (C = 0.5, CHCl3)(S); ee 92%. 1H NMR (400 MHz, ppm, J, Hz, CDCl3) δ 2.9–3.1 m (2H, CH2); 3.85 m (1H, CHCH3), 7.3 m (4H, C6H4). HPLC 15.85 min (R); 13.5 min (S); Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—173WE001-DC191—7; Mobile Phase: Hexane/IPA/MeOH, 90:5:5; Flow Rate: 0.6 mL/min which corresponds to the compound 5f previously described in the literature.
(R)-4,4,4-trifluoro-3-phenylbutanoic acid ((R)-3f). Mp 45 °C; [α]D20 = −43 (C = 0.5, CHCl3)(S); ee 47%. 1H NMR (400 MHz, ppm, J, Hz, CDCl3) δ 2.9–3.1 m (2H, CH2); 3.85 m (1H, CHCH3), 7.3 m (4H, C6H4). HPLC 15.851 min (R); 13.5 min (S); Column: Chiralcel OJ-H (250 × 4.6 mm, 5 mkm)—173WE001-DC191—7; Mobile Phase: Hexane/IPA/MeOH, 90:5:5; Flow Rate: 0.6 mL/min. which corresponds to the compound 3f previously described in the literature.
2.2.1. Docking
As a model structure, the crystal structure with the record number 1HQD in the Protein Data Bank was chosen [
https://www.rcsb.org/structure/1HQD] (accessed on 22 August 2001). The structure had a good resolution of −2.30 Å and already contained a covalently bound ligand, which best suited our purposes.
For the best imitation of the transition state, the covalent docking method was chosen. Calculations were conducted using the Schrödinger software package [Schrödinger Release 2024-1: CovDock, Schrödinger, LLC, New York, NY, USA 2024]. Ester forms of acids were considered as ligands. The compounds were protonated and energy-minimized using the LigPrep module under the force field of OPLS4, and the remaining parameters were set as default. The protein did not undergo mutation treatment during covalent virtual screening, and the default parameters of the Protein Preparation Wizard module were used to preprocess the protein structure. The structures were processed to remove water (since the compounds we are studying are larger in volume and have a different chemical structure) and calcium ions, as they were not involved in the reaction and could interfere with accurate calculations; also, hydrogen atoms were optimized, and energy was minimized directly.
We used the CovDock module to specify the receptor-active amino acid residues (Ser87) and reaction type (Nucleophhilic Addition to a double bond), specified the interfaced pocket based on the original ligand position of the crystal structure, and performeded covalent virtual screening in Pose Prediction (Thorough) mode.
2.2.2. Molecular Dynamics Simulation
Calculations were conducted using the Schrödinger software package [Schrödinger Release 2024-1: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, USA, 2024. Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, USA 2024]. The complexes obtained after molecular docking were used for the MD simulation. Each system was placed into the center of a periodic orthorhombic box, which was then filled with TIP3P water molecules. A minimum 1 nm distance was maintained between the nearest atom of the complex and the edge of the simulation box, so that the complex could fully immerse with water and rotate freely. Then, to neutralize the system and mimic the cellular environment (pH = 7), Na+ and Cl− ions were added to bring the ionic concentration to 150 mM. Here, the solvent molecules are replaced with monoatomic ions, randomly. Next, the obtained complex was energy-minimized, which also relieved any steric clashes. For each complex, a molecular dynamics simulation in water was carried out for 20 ns using an NPT ensemble, OPLS4 force field at a temperature of 300 K. To accurately reproduce the transitional state complex, the carbonyl oxygen of the ligand and His286 were additionally charged.