In the Laboratory
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Synthesis of a Racemic Ester and Its Lipase-Catalyzed Kinetic Resolution Delia Stetca, Isabel W. C. E. Arends, and Ulf Hanefeld* Gebouw voor Scheikunde, Technische Universiteit Delft, Delft, The Netherlands; *
[email protected] The application of enzymes as enantioselective catalysts in organic synthesis has attracted considerable interest over the last decades (1–4). However, there is still a great lack of experiments for the chemical-teaching laboratory that demonstrate the potential of enzyme chemistry. For an integrated biochemistry and chemistry first-year course, we recently devised a reaction sequence to familiarize the students with the use of enzymes in organic chemistry. The experiments are set up exactly in the same way as they would be performed in a research laboratory. Moreover, special attention has been paid to the application of environmentally-friendly conditions; thus, no chlorinated solvents were used. 1-Phenylethanol, 1, was chosen as substrate for the reaction sequence. This substrate is a ‘golden standard’ in research directed towards the development of enantioselective catalysts for alcohol conversion. A catalyst that cannot distinguish between the bulky phenyl and the small methyl group will not be suitable for the enantioselective conversion of more demanding substrates. The overall experiment consists of two separate steps: the synthesis of a racemic ester and the kinetic resolution thereof, catalyzed by an enzyme. In the first step, racemic 1 was converted by standard methodology into ␣-methylbenzyl acetate, 2 (Scheme I). The addition of two equivalents of pyridine and 1.5 equivalents of acetic anhydride at room temperature in ether led to excellent conversion within two hours. After a standard workup (extraction with 1 M HCl and saturated aqueous NaHCO3, drying with MgSO4, removing solvents with a rotary evaporator) a vacuum distillation was performed (yield up to 80 %). While performing this experiment the students will learn all those techniques that are needed for standard organic reactions: how to build a straightforward reaction setup, how to perform an extraction followed by drying and filtration, as well as how to use a rotary evaporator, and how to perform a vacuum distillation. In the second step, a kinetic resolution of the rac-2 was performed. For this kinetic resolution Candida antarctica lipase B (CAL-B) was utilized (5, 6). The enzyme is available in an immobilized form, which eases its handling and recycling. This lipase is an ideal catalyst for the hydrolysis of the (R) enantiomer of 2 while (S)-2 remains almost unconverted (Scheme II). In a kinetic resolution the difference of the speed
OH
O
+
O
of reaction of the two enantiomers is utilized (5). While the reaction (in this case a hydrolysis) of one of the two enantiomers is catalyzed, the same reaction is virtually not catalyzed for the other enantiomer. This unreactive enantiomer therefore remains almost unchanged so that the products at the end of the reaction are two different compounds, here an alcohol and an ester.1 These can be separated readily. To perform the reaction, the racemic ester 2 and a phosphate buffer (pH = 7) were stirred at room temperature and the enzyme was added. The pH value of the reaction mixture dropped, since acetic acid was released during the reaction. This was monitored with a pH meter. Whenever the pH dropped below 7 dilute NaOH was added. This task can be performed either with an automatic burette or by hand. The reaction was stopped once the pH remained stable (after 3 to 4 h). From the amount of NaOH added the students can calculate the degree of conversion (typically around 50%). The mixture was extracted with ether and TLC analysis showed two compounds, 1 and 2. These compounds were separated by column chromatography. The optical rotation of both compounds was determined to establish the optical purity. The enzyme can be recycled. However, some loss of activity was observed. This loss might be due to leaching or deactivation during the reaction and workup. By performing this reaction the students get to experience an enzyme in action and see the great ease with which these biocatalysts can be applied in enantioselective conversions. If desired, the kinetics of the reaction can be followed by monitoring the addition of the base. TLC analysis and column chromatography are introduced as standard techniques in today’s laboratory. The first-year students performed these two steps within four days and obtained yields of up to 80% for rac-2 in the first reaction and 30% (ee up to 93%) for (S)-2 and 14% (ee up to 83%) for (R)-1 in the enzyme-catalyzed reaction. Hazards All experiments should be performed in fumehoods, and suitable eye protection and a laboratory coat should be worn. Compounds 1 [98-85-1] and 2 [93-92-5] are irritants and any eye and skin contact, inhalation, or ingestion should be avoided. Acetic anhydride [108-24-7] causes burns and reacts
O
O
room temperature
rac-2
Scheme I. Synthesis of the ester rac-2.
CAL-B pH = 7 buffer
rac-2
OH
O
+
room temperature
O rac-1
O
O
O pyridine, ether
S-2
R-1
Scheme II. Enzyme-catalyzed kinetic resolution of rac-2.
JChemEd.chem.wisc.edu • Vol. 79 No. 11 November 2002 • Journal of Chemical Education
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In the Laboratory
violently with water: any eye and skin contact, inhalation, or ingestion should be avoided. Pyridine [110-86-1] is harmful and any eye and skin contact, inhalation, or ingestion should be avoided. Skin contact or inhalation of the enzyme should be avoided, as should the inhalation of silica gel [112926-00-8]. Ether [60-29-7] can form explosive peroxides and its inhalation or ingestion is harmful. Methanol [6556-1] and petroleum ether [8032-32-4] are highly flammable, toxic by inhalation or skin contact, and they are irritants. W
Supplemental Material
Lab handouts for the students and instructor notes are available in this issue of JCE Online. Acknowledgment We would like to thank Roche Diagnostics Penzberg (W. Tischer, U. Hill) for the generous donation of the enzyme (CAL-B, Chirazyme L-2, c.-f., C2, Lyo). U. H. and I. W. C. E. A. thank the Royal Netherlands Academy of Arts and Sci-
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ences for fellowships. We thank D. Klomp, P. P. Pescarmona and A. de Winter for valuable discussions and contributions. Note 1. If one does wait long enough (several days), both enantiomers of this ester will eventually be hydrolyzed and the racemic alcohol will be obtained.
Literature Cited 1. Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409, 258–268. 2. Sime, J. T. J. Chem. Educ. 1999, 76, 1658–1661. 3. Roberts, S. M. J. Chem. Educ. 2000, 77, 344–348. 4. Lee, M. J. Chem. Educ. 1998, 75, 217–219. 5. Bornscheuer, U. T.; Kaslauskas R. J. Hydrolases in Organic Synthesis; Wiley-VCH: Weinheim, 1999. 6. Roberts, S. M. Preparative Biotransformations; John Wiley & Sons: Chichester, New York, Brisbane, Toronto, Singapore, 1993, 1:6.41-1:6.48.
Journal of Chemical Education • Vol. 79 No. 11 November 2002 • JChemEd.chem.wisc.edu
