Chemo-Enzymatic Synthesis of (R)- and (S)-2-Hydroxy-4-phenylbutanoic Acid via Enantio-Complementary Deracemization of (±)-2-Hydroxy-4-phenyl-3-butenoic Acid Using a Racemase-Lipase Two-Enzyme System

Abstract

Deracemization of (±)-2-hydroxy-4-phenylbut-3-enoic acid was accomplished by lipase-catalyzed kinetic resolution ­coupled to mandelate racemase-mediated racemization of the non-reacting substrate enantiomer. Stepwise cyclic repetition of this ­sequence led to a single enantiomeric product, the stereochemical outcome of which could be controlled by switching between lipase-catalyzed acyl-transfer/ester hydrolysis reactions. Both enantio­meric products were easily transformed into (R)- and (S)-2-­hydroxy-4-phenylbutanoic acid, important building blocks for ACE-inhibitors.

deracemization - lipase - mandelate racemase - biocatalysis - enzyme catalysis

References

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For the concept of deracemization see:

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8

Although several processes producing enantiomeric products were reported depending on the choice of biocatalyst, the search for e.g. ‘anti-Kazlauskas’ carboxyl ester hydrolyses and ‘anti-Prelog’ alcohol dehydrogenases still represents a major challenge in biocatalysis.

9 Strauss UT. Faber K. Tetrahedron: Asymmetry  1999,  10:  4079 

10 For a review on enzyme-catalyzed racemization see: Schnell B. Faber K. Kroutil W. Adv. Synth. Catal.  2003,  345:  653 

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13 rac-2-Hydroxy-4-phenyl-3-butenoic acid (rac-1) was prepared according to: Nerdel F. Rachel H. Chem. Ber.  1956,  89:  671; mp 137-139 °C, lit. mp 137 °C 

14 For a large-scale preparation of mandelate racemase see: Stecher H. Felfer U. Faber K. J. Biotechnol.  1997,  56:  33 

15

( S )-2-Acetoxy-4-phenyl-3-butenoic Acid [( S )-2] via Deracemization of [ rac -1].
Kinetic resolution step (a): to a solution of rac-1 (0.25 g, 1.4 mmol) in diisopropyl ether (25 mL), vinyl acetate (2.5 mL) and lipase PS-C ‘Amano’ II (0.25 g) were added and the mixture was shaken for 48 h at 25 °C and 150 rpm. The enzyme was filtered and dried for reuse; the filtrate was evaporated to dryness. HPLC analysis showed a conversion of 50% [Chiralpak AD column, Daicel, heptane-2-PrOH-CF₃COOH, 90:10:0.1; 0.4 mL/min, 18 °C, (S)-2: τ = 29.3 min, (R)-1: τ = 42.6 min].
Racemization step (b): to a solution of (S)-2 and (R)-1 obtained from step (a) in Hepes buffer (10 mL, 50 mmol, pH 7.5, 10 mM MgCl₂), mandelate racemase [EC 5.1.2.2] (1.5 g, prepared as described in ref. 14) rehydrated in 15 mL Hepes buffer was added. The mixture was shaken for 24 h at 30 °C and 150 rpm. After centrifugation the solution was acidified to pH 1-2 and extracted with EtOAc, dried (Na₂SO₄) and evaporated. HPLC analyses showed complete racemization of (R)-1; (S)-1: τ = 36.2 min. After repeating step (a) for three times and step (b) for two times, the residue was purified by flash chromatography to yield (S)-2 as the sole product (0.21g, 68% overall yield from rac-1); mp 80-82 °C; mp lit. 82 °C; [α]_D ²⁰ +100.3 (c 0.47, EtOH, >99% ee); lit. [α]_D ²⁵ +108.0 (c 0.36, EtOH).
( S )-2-Hydroxy-4-phenyl-3-butenoic Acid [( S )-1].
A mixture of (S)-2 (110 mg, 0.5 mmol), MeOH (4 mL) and K₂CO₃ (0.5 g) was stirred at 0 °C. After 3-4 h the mixture was acidified with HCl (3 M) to pH 1-2 and then extracted three times with EtOAc. The organic layer was dried (Na₂SO₄), evaporated and the residue was purified by flash chromatography to yield (S)-1 (56 mg; 63%); mp 132-133 °C; lit. mp 104 °C; [α]_D ²⁰ +96.5 (c 0.27, MeOH, >99% ee); lit. [α]_D ²⁵ +85.2 (c 0.55, MeOH, 94% ee).
( S )-2-Hydroxy-4-phenylbutanoic Acid [( S )-3].
(S)-1 (50 mg, 0.28 mmol) was hydrogenated employing a rubber balloon using a catalytic amount of Pd on C (10%, 5 mg) in MeOH for 10 min. Then the catalyst was filtered off and the solvent was evaporated to yield (S)-3 (42 mg, 83%); mp 115-117 °C; lit. mp 114 °C; [α]_D ²⁰ +8.1 (c 1.0, EtOH, >99% ee); lit. [α]_D ²⁵ +7.5 (c 0.5, EtOH, 84% ee); chiral HPLC analysis using the method described above showed a single peak at τ = 26.8 min.

16 ( R )-2-Acetoxy-4-phenyl-3-butenoic Acid [( R )-2] via Deracemization of rac -1. Acylation step (c): a solution of rac-1 (0.5g, 2.8 mmol) and acetic anhydride (5 mL) in pyridine (0.2 mL) was kept at 0-5 °C. After 6 h the solution was poured into ice-water (100 mL), which was acidified with HCl (3 M) to pH 1-2 and extracted three times with EtOAc. The combined organic layers were washed with H₂O and brine, dried (Na₂SO₄) and evaporated to yield rac-2 (0.45 g, 72%); mp 74-77 °C.Kinetic resolution step (f): to a solution of rac-2 (0.45 g, 2.0 mmol) in acetone (4.5 mL) and phosphate buffer (45 mL, 50 mmol, pH 7.5), lipase from Candida antarctica B (Novozyme 435, 1.8 g) were added and the mixture was shaken for 24 h at 30 °C and 130 rpm. The reaction mixture was filtered and the recovered lipase was dried for reuse. The filtrate was evaporated from acetone, the residue was acidified with HCl (3 M) to pH 1-2, extracted three times with EtOAc, dried (Na₂SO₄) und evaporated. HPLC analysis as described above showed a conversion of 50%; (S)-1: τ = 36.2 min, (R)-2: τ = 31.9 min. For the racemization step (b) see above. After repeating (e) and (f) for two times and (b) once, the residue was purified by flash chromatography to yield (R)-2 as the sole product (0.33g, 53%); [α]_D ²⁰ -114.3 (c 0.49, EtOH, >99% ee). (R)-2-Hydroxy-4-phenyl-3-butenoic acid [(R)-1] was prepared as described for (S)-1. Yield 61%; [α]_D ²⁰ -71.9 (c 0.29, MeOH, ee >99%; lit. [α]_D ²⁵ -90.6 (c 1.9, MeOH). (R)-2-Hydroxy-4-phenylbutanoic acid [(R)-3] was prepared as described for (S)-3. Yield 85%; [α]_D ²⁰ -8.5 (c 1.0, EtOH, ee >99%); lit. [α]_D ²⁵ -9.0 (c 1.0, EtOH). Chiral HPLC analysis as described above showed a single peak at τ = 24.8 min. For spectroscopic and physical data of (R)-1-3 and (S)-1-3 see: Chadha A. Manohar M. Tetrahedron: Asymmetry  1995,  6:  651 

17 Although mandelate racemase is not deactivated in various organic solvents, it is catalytically inactive at low water activity; see: Pogorevc M. Stecher H. Faber K. Biotechnol. Lett.  2002,  24:  857 

18 Strauss UT. Kandelbauer A. Faber K. Biotechnol. Lett.  2000,  22:  515