Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus in Racemization of Secondary Alcohols Musa M. Musa * Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261 KSA *musam@kfupm.edu.sa Introduction Dynamic Kinetic resolution (DKR) has gained considerable interest in the last two decades to produce optically active compounds like alcohols. Racemization is the key step successful DKR. New progresses in biotechnology have made biocatalysis a practical and environmentally benign alternative to conventional organic and organometallic catalysis in a wide variety of reactions [1]. A pair of alcohol dehydrogenases (ADHs) with opposite stereopreferences has been employed in racemization of enantiopure alcohols [2]. We have reported that phenylacetone was reduced to (S)-1-phenyl-2-propanol ((S)-1a) by W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TeSADH) with low enantioselectivity (37% ee), which can be explained by the reverse fit of that substrate in the active site of the enzyme that leads to delivering the hydride from the undesired face of the ketone, i.e. Si face instead of Re face, leading to what is called “selectivity mistake” [3]. This low enantioselectivity drew our interest to study the W110A TeSADH-catalyzed racemization of secondary alcohols. Herein, W110A TeSADH was employed in racemization of phenyl-ring-containing secondary alcohols in an attempt to recycle the slow reacting alcohol enantiomer in KR either in situ to achieve DKR or separately by sequential KR and racemization cycles. Materials and Methods General procedure for W110A TeSADH-catalyzed recemization: A mixture of enantiopure alcohol (3 μL), NADP + (0.5 mg), NADPH (1.0 mg), W110A TeSADH (0.3 mg), Tris-HCl buffer solution (800 μL, pH adjusted to 8.0 at 25 ◦ C), and acetonitrile (200 μL) were placed in a 1.5 mL plastic tube. The reaction mixture was shaken at 50 ◦ C at 200 rpm for 48 h then it was extracted with ethyl acetate (2×500 μL). The combined organic layer was dried with sodium sulfate and concentrated to dryness. The acetate derivatives were analyzed by GC equipped with a chiral column to determine their ee. Results and Discussion We describe W110A TeSADH-catalyzed racemization of enantiopure secondary alcohols. This racemization was achieved by controlling the reversible interconversion between ketone and alcohol substrates in W110A TeSADH-catalyzed redox transformations. It was conducted in Tris-HCl buffer containing 20% (v/v) acetonitrile as a cosolvent to improve the solubility of the substrate. Both NADPH and NADP + (2:1, wt/wt) were included in the reaction medium to allow for the racemization to take place by facilitating the equilibrium in the redox reaction. The ee of (S)-1a was reduced from >99% to 6.3% in 48 h at 50  C employing this racemization method, as shown in Table 1. The same procedure was employed to racemize (R)-1a, and its ee was reduced from >99% to 10.3%. (S)-4-(4ʹ-Methoxyphenyl)-2-butanol ((S)-2a) was exposed to the same racemization conditions, and its ee was reduced from 91% to 44%. As expected, (S)-2a was racemized to a lesser extent than (S)-1a, which is simply explained by the difference in enantiomeric ratio, i.e. E-value, for the W110A TeSADH-catalyzed enantiospecific KR for their racemates. Table 1. W110A TeSADH-catalyzed racemisation of enantiopure phenyl-ring-containing secondary alcohols. R 1 R 2 OH (S)- or (R)-alcohol R 1 R 2 OH W110A TeSADH, NADPH, NADP +, 50 o C (rac)-alcohol Entry R Substrate ee (%) before after 1 PhCH 2 (S)-1a >99 6.3 (S) 2 PhCH 2 (R)-1a >99 10.3 (R) 3 p-MeOC 6 H 4 (CH 2 ) 2 (S)-2a 91 44.0 (S ) 4 PhCH 2 CH 2 (R)-3a >99 34.5 (R) 5 PhCH 2 CH 2 (S)-3a 99 82.4 (S ) 6 PhCH 2 CH 2 (S)-3a 72.7 51.8 (S ) Using the same racemization procedure, we were able to decrease the ee of ( R)-3a from >99% to 34.5% in 48 h (Table 1). However, the ee of (S)-3a was reduced from 99% to 82.4% in 48 h. In a separate experiment, we were able to reduce the ee of (S)-3a from 72.7% to 51.5% in 48 h. This indicates that (S)-3a can be racemized by W110A TeSADH to a lesser extent than (R)-3a, but that it could be racemized if sufficient time is allowed. These results indicate that even alcohols accepted by W110A TeSADH with high enantiomeric discrimination can still be racemized by this method. Significance We demonstrated that W110A TeSADH-catalyzed racemization is valid for both enantiomers of alcohols accepted by this enzyme with not only low, but also high enantiomeric discriminations. The capability of TeSADH to perform racemization under mild conditions in addition to its high thermal stability and high tolerance to organic solvents is of great interest. It represents an attracting approach especially if it proves successful in situ with other interesting reactions such as KR. Acknowledgment The author acknowledges the support provided by King Abdulaziz City for Science and Technology (KACST) through the Science and Technology Unit at KFUPM through project No. 11-BIO1666-04 as part of the National Science, Technology and Innovation plan. References 1. Bornscheuer, U. T., Huisman, G. W., Kazlauskas, R. J., Lutz, S., Moore, J. C., Robins, K. Nature, 485, 185-194 (2012). 2. Gruber, C. C., Nestl, B. M., Gross, J., Hildebrant, P., Bornscheuer, U. T., Faber, K., Kroutil, W. Chem. Eur. J. , 13, 8271-8276 (2007). 3. Musa, M. M., Ziegelmann-Fjeld, K. I., Vieille, C., Zeikus, J. G., Phillips, R. S. J. Org. Chem., 72, 30-34 (2007).