Laboratory Experiment pubs.acs.org/jchemeduc
Lipase-Mediated Kinetic Resolution: An Introductory Approach to Practical Biocatalysis Pamela T. Bandeira, Juliana C. Thomas, Alfredo R. M. de Oliveira, and Leandro Piovan* Department of Chemistry, Universidade Federal do Paraná, Curitiba 81.531-991, Brazil S Supporting Information *
ABSTRACT: An experimental protocol that provides an excellent way to discuss concepts at the crossroads of organic chemistry and biochemistry employing biocatalysis is presented. By evaluating several reaction parameters (enzyme source, organic solvent, and acyl donor), it was possible to conduct an enzymatic kinetic resolution experiment using 1phenylethanol as a model compound. Students were then challenged to revisit and explore a variety of basic principles already addressed in previous organic chemistry or biochemistry courses to stimulate interdisciplinary research.
KEYWORDS: Upper-Division Undergraduate, Organic Chemistry, Laboratory Instruction, Biochemistry, Hands-On Learning/Manipulatives, Alcohols, Esters, Chirality/Optical Activity, Enzymes, Biotechnology
B
compounds in several contexts, undergraduate chemistry experiments that allow students to explore stereoselective biotransformations are rare. In this context, EKR is a powerful tool for reviewing basic concepts of stereochemistry and their applications in chiral catalysis. Moreover, this approach can be used to introduce enzyme-mediated reactions in organic classes starting from their basic concepts as ordinary organic reactions. For teachers, use of EKR as a tool is a great opportunity to discuss concepts that are at the crossroads of organic chemistry (i.e., stereochemistry, catalysis, enantioselective reactions, mechanisms. etc.) and biochemistry (amino acids, peptide bonds, proteins, enzymes, catalytic sites, etc.) and stimulate interdisciplinary research.7 To the best of our knowledge, few chemical-teaching laboratory experiments involving EKR processes are described in the scope of this Journal including the EKR of amino acids,8,9 1-phenylethyl acetate,3 secondary alcohols and derivatives,10 and 1-phenylethanol using fatty esters as acylating agents.11 Pertinent to this concept, here we describe a systematic discovery-based experiment intended to introduce the role of lipases as chiral biocatalysts in organic chemistry and highlight the influence of reaction parameters on enzymes’ catalytic properties through a practical biocatalysis course. The experiment is safe and simple to perform, its results are reproducible, and it can help students to develop scientific inquiry skills by aligning experimental data with several theoretical concepts
iocatalysis is a powerful biotechnological tool used to promote transformation of organic compounds using the high selectivity of enzymes. Enzymes are biodegradable catalysts, which can be isolated from renewable sources (yeast, fungi, bacteria, plants). Their use makes biocatalysis an environmentally friendly approach. 1 In this sense, biocatalysis is a well-established process used in both academic research and industry, especially in manufacturing of pharmaceuticals and fine chemicals. Sitagliptin (Merck and Co., Inc.), atorvastatin (Pfizer, Inc.), and rosuvastatin (AstraZeneca, PLC) are examples of pharmaceuticals produced employing at least one enzyme-mediated step.2 Notwithstanding the high relevance of biocatalysis in the field of biotechnology, this approach is commonly not well discussed in undergraduate chemistry curricula. As a consequence, there is a gap in the repertoire of lab experiments that explore the potential of enzymes in organic chemistry.3 Lipases (glycerol ester hydrolases EC 3.1.1.3) are among the enzymes that have been successfully applied in biocatalysis. These enzymes are widely distributed in nature,4 and their great success in synthetic organic chemistry is due to several reasons including their broad substrate specificities, relative activity and stability in nonaqueous media, and high selectivity, plus they do not require cofactors.5 The most widely used lipase-mediated reaction is the enzymatic kinetic resolution (EKR) of racemates. Kinetic resolution is a process based on unequal reaction rates of enantiomers in the presence of a chiral (bio) catalyst.6 EKR has special relevance in the preparation of optically active compounds because it allows access to both enantiomers separately. In spite of the importance of enantioenriched © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: August 10, 2016 Revised: March 9, 2017
A
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Each group performed a specific experiment for subsequent discussion of the results and aimed to establish the best system (enzyme, solvent, acyl donor). Therefore, all students received a protocol, with physicochemical properties of reagents and detailed experimental procedure (see Supporting Information). At this moment, stoichiometry calculation was performed, and the majority of students demonstrated mastery of the required calculations. Briefly, 20 mg of commercial lipases was added to a solution of (R,S)-1-phenylethanol (0.1 mmol), acyl donor (0.4 mmol), and organic solvent (2.0 mL). All reactions were carried out in 4 mL sealed vials equipped with magnetic stirrer and kept at 35 °C with the aid of a thermostatic bath for 1.5 h. After this interval, the lipases were filtered off, and the resulting sample was prepared through dilution of a 100 μL aliquot from the filtrate in 0.5 mL with hexanes for further chiral gas chromatography analyses (see Supporting Information for analytic conditions). Students’ preparation of their own GC samples is particularly valuable since this a very important step in most analytical techniques. All students were able to perform the experimental procedure within the allotted time (4 h).
concerning chemistry and biochemistry while also contributing to extend biocatalysis experiments’ repertoire.
■
EXPERIMENT This experiment has been performed by 40 undergraduates over the past two years in a second-year organic chemistry introductory course. The complete experiment (including the postlab discussion) has been performed in 8 h at meetings twice a week for 4 h periods. The students worked in pairs, and individual understanding was performed based on the assessment of questions of teaching and learning in a postlab class, which involved the continuous monitoring of students’ understanding. The assessment of teaching goals was based on students’ answers, which were supported by practical results and analyzed by the teacher and PhD students. Students were presented with an outline about the practical biocatalysis course. Lipase-mediated transesterification of racemic 1-phenylethanol [(RS)-1] was chosen as the model reaction (Scheme 1) because1-phenylethanol is a standard substrate for lipases and consequently is well-described and reproducible.
■
HAZARDS All reactions should be performed in a proper fume hood. Students should wear gloves, lab coats, and splash-proof goggles during the experiment. 1-Phenylethanol and 1-phenylethyl acetate may slightly irritate skin, eyes, and mucous membranes. Hexanes should be carefully handled because they contain n-hexane, a neurotoxin. Toluene, if swallowed in high quantity, can cause central nervous system effects. Tertbutylmethyl ether and ethanol are extremely flammable (liquid and vapor). Acetonitrile, ethyl acetate, and vinyl acetate are harmful if ingested or if vapors are inhaled. Acetic anhydride is corrosive in case of skin contact. It is recommended that in future experiments hexanes be replaced by n-heptane, a greener solvent.
Scheme 1. Enzymatic Kinetic Resolution of (RS)-1Phenylethanol
Therefore, the use of three reaction parameters was investigated by the students: (i) lipase type based on structural aspects of selected enzymes; (ii) influence of organic solvent nature; and (iii) acyl donor structure. Three commercially available lipases obtained from different sources were selected: pancreatic pig lipase (PPL); Thermomyces lanuginosus lipase (TL-IM); and Candida antarctica lipase B (CAL-B). Hexanes, toluene, tert-butylmethyl ether (MTBE), ethanol (EtOH), and acetonitrile (ACN) were chosen as solvents. Finally, vinyl acetate, isopropenyl acetate, ethyl acetate, and acetic anhydride were chosen as acyl donors. To compare the results between reactions mediated and not mediated by lipases, an assay employing N,N-dimethylaminopyridine (DMAP) as catalyst was also performed. Each experimental parameter was planned with a teaching goal (Table 1) for postlab discussion supported by the students’ results.
■
RESULTS AND DISCUSSION To analyze the results and stimulate the postlab class discussion, each student received a printed copy of the standard chromatograms for 1-phenylethanol (1) and 1-phenylethyl acetate (1a) racemates (Figure 1) and another one corresponding to the reaction performed by them (Figure 2). Since the enantiopreference of commercial lipases tested in this experiment is well-known, it is possible to attribute the absolute configuration of peaks in gas chromatography (GC) analysis chromatograms. With the chromatograms in hand, the concepts of conversion rate (c), enantiomeric excess (e.e.), and enantiomeric ratio (E) and the theory behind it were introduced in a short lecture. Then students were requested to calculate the enantioselectivity parameters for their reactions according to Chen equations12 (see Supporting Information). Students’ data were presented in Table 2 through a PowerPoint presentation. After Table 2 was completed, the first topic of discussion was the lipase type. Students were able to observe significant changes in conversion rates for enzymatic assays mediated by different lipases (PPL, TL-IM, and CAL-B, Table 2, entries 1− 3, respectively). By using the results obtained, structural features of enzymes, such as amino acids, peptide bonds and three-dimensional structure (secondary, tertiary, and quaternary protein structures),13 and catalytic site could be discussed
Table 1. Teaching Goals To Be Assessed through Variation of Experimental Parameters in Enzymatic Kinetic Resolution of 1-Phenylethanol, (RS)-1 Parameter Lipases type Organic solvent effects Influence of acyl donor
Teaching Goals Discuss structural features of enzymes, catalytic site, mechanism of lipase-mediated reactions, chiral recognition, and enzymatic activity. Demonstrate that lipase-mediated kinetic resolution can be seen as any ordinary organic reactions; therefore, they are sensitive to the solvent’s nature. Correlate chemical reactivity of acyl donor to its influence on reactions mediated by lipases.
B
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 1. Chromatogram of 1-phenylethanol (1) and 1-phenylethyl acetate (1a) racemates.
Figure 2. Chromatogram of direct injection of a reaction sample (1.5 h). Reaction conditions: CAL-B, hexanes, acetic anhydride. Peak top numbers represent chromatogram area.
Table 2. Enzymatic Kinetic Resolution of (RS)-1-Phenylethanol, (RS)-1a Enantiomeric Excess, % Group/Entry
Solvent
Acyl Donor
Catalyst
Conversion Rate, %
(R)-1a
(S)-1
Enantiomeric Ratio
1 2 3 4 5 6 7 8 9 10 11
Hexanes Hexanes Hexanes ACN MTBEb Toluene Ethanol Hexanes Hexanes Hexanes Hexanes
Vinyl acetate Vinyl acetate Vinyl acetate Vinyl acetate Vinyl acetate Vinyl acetate Vinyl acetate Acetic anhydride Isopropenyl acetate Ethyl acetate Acetic anhydride
PPLc TL IMd CAL-Be CAL-Be CAL-Be CAL-Be CAL-Be CAL-Be CAL-Be CAL-Be DMAPf
99 >99 >99 >99 g 86 >99 >99 0
g 20 >99 10 84 >99 g 32 >99 34 h
g >200 >200 >200 >200 >200 g 18 >200 >200 1
Reaction conditions: (RS)-1 (0.01 mmol, 12 mg); lipase (20 mg); acyl donor (0.04 mmol); organic solvent (2 mL); 35 °C for 1.5 h. bMTBE: tertbutylmethyl ether. cPPL: Porcine pancreas lipase. dTL-IM: Thermomyces lanuginosus lipase. eCAL-B: Candida antarctica lipase-B. fDMAP: N,Ndimethylaminopyridine. gNot measured due to low conversion. hNot applied.
a
in the classroom. General mechanistic aspects of enzyme catalysis were also introduced including Fischer’s “lock-andkey”,13 Koshland’s “induced fit” mechanisms,14 and Dewar’s “desolvation and solvation-substitution” theory.15 Because of chirality recognition by lipases and their specificity toward substrate (supported by (R)-1a preferential formation or by (R)-1 consumption), concepts of stereochemistry, chiral recognition, and the mechanism of lipase transesterification catalysis were also introduced16 (Figure 3) based on Ogston’s “three point attachment” rule17 and Kazlaukas’ rule,18 which predicts the enantioselectivity of lipases in accordance with the fit of the substituent in the catalytic site (Figure 4).
From these results, the students were able to deduce that enzymes/lipases are not “all equal”. In other words, although all lipases have the same amino acid residues in their catalytic sites, particular enzymes’ catalytic properties/activity can be influenced according to their three-dimensional structure, which results in different conversion rates for the same reaction,19 as observed in assays 1−3 (Table 2). Moreover, the students were able to deduce after discussion that TL-IMmediated EKR of (RS)-1 can reach 50% conversion in a longer time since TL-IM activity was lower than CAL-B activity. The second topic of discussion was the organic solvent’s effects on the reactions. For reactions employing solvents such as hexanes, MTBE, and toluene (Table 2, entries 3, 5, and 6), C
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 3. Mechanism of lipase-mediated transesterification reaction of (RS)-1-phenylethanol, (RS)-1.
acetate) were employed in the EKR reactions using CAL-B as catalyst and hexanes as organic solvent (Table 2, entries 8−10). The conversion rates for the reactions employing vinyl acetate and isopropenyl acetate (Table 2, entries 3 and 9, respectively) were higher than those employing acetic anhydride and ethyl acetate (Table 2, entries 8 and 10). When acetic anhydride was employed as an acyl donor, a decrease of enantioselectivity was also observed compared to other acyl donors. As part of a postlaboratory test (see Supporting Information), the students were requested to draw the mechanism of nucleophilic substitution to carbonyl compounds26 by relating it to the enzymatic catalysis mechanism (Figure 3). The key step to explain the changes in conversion rates is the identification of the leaving group in the second step of the enzymatic mechanism (Figure 5). Nucleophilic addition to vinyl acetate leads to release of a vinyl alcohol moiety, which is converted
Figure 4. Preferential fitting of (R)-1-phenylethanol according to Kazlauskas rule prediction.
the conversion rates were considerably higher than those in polar ones such as ACN and ethanol (Table 2, entries 4 and 7, respectively). These results prompted the students to conclude that, as in all ordinary organic reactions, lipase-mediated reactions are not inert to the solvent and their course can be altered by solvent properties.20 Several aspects concerning solvent influence on reaction components and on lipases were discussed: polarity, based on log P21 and permittivity;22 substrate solubility; effect on lipase structure;23 and models to explain enzyme behavior in nonaqueous media,24 including enzymes’ essential water layer, which can be removed by polar solvents, lowering the lipase catalytic activity.25 After analysis of all theoretical explanations and results presented in Table 2, the students were able to answer why hexanes and toluene (hydrophobic organic solvents) were better for lipase-mediated reactions than ethanol and acetonitrile (hydrophilic ones). Students’ answers were supported by the practical results and this allowed them to correlate experimental data with theoretical concepts providing a more effective learning. The last reaction parameter evaluated in this experiment was the nature of the acyl donor agent involved in the first step of the enzymatic transesterification of 1-phenylethanol (Figure 3). In addition to vinyl acetate (Table 2, entry 3), three other acyl donors (acetic anhydride, isopropenyl acetate, and ethyl
Figure 5. Mechanism-step of intermediate acyl-enzyme formation using different acyl donors. D
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
quickly to acetaldehyde (a volatile substance, bp 20 °C). This is easily removed from the medium and thus shifts the reaction equilibrium (Figure 5[A]). When isopropenyl acetate was used as acyl donor, the leaving group (enol) was tautomerized to propanone, another volatile species (Figure 5[B]). For the reaction employing acetic anhydride as acyl donor (Table 2, entry 10), the leaving group is acetic acid (Figure 5[C]), while for the reaction with ethyl acetate the leaving group is ethanol (Figure 5[D]). About 90% of students were able to understand that, due to reversibility of enzymatic reactions, in cases when the leaving group is tautomerized, the equilibrium is shifted to form an intermediate acyl-enzyme. For reactions with ethyl acetate and acetic anhydride, students were also able to observe that the reaction rate was directly related to basicity of the leaving group. The loss of selectivity when acetic anhydride was employed as an acyl donor was explained based on results of a control reaction (without the biocatalyst), when a low spontaneous reaction between (RS)-1 and acetic anhydride was observed. At this point, comparison between the control reaction (blank), which happened without catalysis, and the reaction with a chemical catalyst (DMAP) allowed highlighting the effect of catalysts on reaction rates (see Supporting Information). Finally, the nonbiocatalyzed reaction (Table 2, entry 11) resulted in 100% conversion and product 1a was obtained as a racemate. With this result, the differences between a nonchiral and a chiral catalyst were also discussed, and the students could understand the importance of the presence of a chiral catalyst in the reaction medium.
ORCID
Leandro Piovan: 0000-0002-2835-5001 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank the Brazilian National Council for Scientific and Technological Development (CNPq, Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico, Brazil) for financial support (Proc. 456834/2014) and Office to Improve University Personnel, Ministry of Education (CAPES, Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı ́vel Superior) for fellowships.
■
(1) Watson, W. J. W. How do the fine chemical, pharmaceutical, and related industries approach green chemistry and sustainability? Green Chem. 2012, 14, 251−259. (2) Ciriminna, R.; Pagliaro, M. Green chemistry in the fine chemicals and pharmaceutical industries. Org. Process Res. Dev. 2013, 17, 1479− 1484. (3) Stetca, D.; Arends, I. W. C. E.; Hanefeld, H. Synthesis of a racemic ester and its lipase-catalyzed kinetic resolution. J. Chem. Educ. 2002, 79 (11), 1351−1352. (4) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis, 2nd ed.; Wiley-VCH: Weinheim, 2006; pp 61−183. (5) Roberts, S. M. Using microorganisms in synthetic organic chemistry. J. Chem. Educ. 2000, 77 (3), 344−348. (6) Moss, G. P. Basic terminology of stereochemistry. Pure Appl. Chem. 1996, 68 (12), 2193−2222. (7) Beers, M.; Archer, C.; Feske, B. D.; Mateer, S. C. Using Biocatalysis to Integrate Organic Chemistry into a Molecular Biology Laboratory Course. Biochem. Mol. Biol. Educ. 2012, 40 (2), 130−137. (8) Mohrig, J. R.; Shapiro, S. M. An enzyme-catalyzed resolution of aminoacids. J. Chem. Educ. 1976, 53 (9), 586−589. (9) Clement, G. E.; Potter, R. Enzymatic resolution. An organicbiochemical laboratory experiment. J. Chem. Educ. 1971, 48 (10), 695−696. (10) Rebolledo, F.; Liz, R. Multi-choice enzymatic resolutions of racemic secondary alcohols using Candida antarctica lipase B. A collaborative experiment for advanced undergraduates. J. Chem. Educ. 2005, 82 (6), 930−933. (11) Monteiro, C. M.; Afonso, C. A. M.; Lourenco, N. M. T. Enzymatic resolution and separation of secondary alcohols based on fatty esters as acylating agents. J. Chem. Educ. 2010, 87 (4), 423−425. (12) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. Quantitative analyses of biochemical kinetic resolutions of enantiomers. J. Am. Chem. Soc. 1982, 104 (25), 7294−7299. (13) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 4th ed.; Wiley: New York, 2005; pp 75−190. (14) Koshland, D. E. Application of a Theory of Enzyme Specificity to Protein Synthesis. Proc. Natl. Acad. Sci. U. S. A. 1958, 44 (2), 98− 104. (15) Dewar, M. J. S. New ideas about enzyme reactions. Enzyme 1986, 36, 8−20. (16) Faber, K. Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin, 2011; p 89. (17) Ogston, A. G. Interpretation of Experiments on Metabolic Processes, using Isotopic Tracer Elements. Nature 1948, 162 (4129), 963−963. (18) Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. A Rule To Predict Which Enantiomer of a Secondary Alcohol Reacts Faster in Reactions Catalyzed by Cholesterol Esterase, Lipase from Pseudomonas cepacia, and Lipase from Candida rugosa. J. Org. Chem. 1991, 56 (8), 2656−2665. (19) Benkovic, S. J.; Hammes-Schiffer, S. A Perspective on Enzyme Catalysis. Science 2003, 301 (5637), 1196−1202.
■
CONCLUSION A useful laboratory experiment was successfully implemented to introduce practical biocatalysis in undergraduate chemistry courses. The experimental procedure is simple and easy to follow, performed under very mild reaction conditions, and employs heterogeneous biocatalysts, which are easily removed from the medium. Through a process of guided exploration, the students were able to correlate experimental data with theoretical organic chemistry and biochemistry subjects and make a significant contribution to the development of scientific inquiry skills. Furthermore, this experimental approach offers an opportunity for students to learn about the use of enzymes in organic synthesis. Thus, the students were presented with a powerful tool of the current technological demand since enzymes are highly efficient natural catalysts widely applied in scientific research and industry.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00606. Instructor notes, experimental procedure record, list of chemicals, postlaboratory quiz, and GC analysis conditions (PDF, DOC)
■
REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. E
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
(20) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry, 3rd ed.; Wiley: Weinheim, 2003. (21) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rules for optimization of Biocatalysis in Organic Solvents. Biotechnol. Bioeng. 1987, 30, 81− 87. (22) Kitamoto, Y.; Kuruma, Y.; Suzuki, K.; Hattori, T. Effect of solvent polarity in enantioselectivity in Candida Antarctica Lipase B catalyzed kinetic resolution of primary and secondary alcohols. J. Org. Chem. 2015, 80 (1), 521−527. (23) Trodler, P.; Pleiss, J. Modeling structure and flexibility of Candida antartica lipase B in organic solvents. BMC Struct. Biol. 2008, 8 (1), 9. (24) Klibanov, A. M. Improving enzymes by using them in organic solvents. Nature 2001, 409 (6817), 241−246. (25) Zaks, A.; Klibanov, A. M. The Effect of Water on Enzyme Action in Organic Media. J. Biol. Chem. 1988, 263 (17), 8017−8021. (26) Clayden, J.; Greeves, N.; Warren, S.; Wothers, P. Organic Chemistry; 1st ed.; Oxford University Press: New York, 2001; p 279.
F
DOI: 10.1021/acs.jchemed.6b00606 J. Chem. Educ. XXXX, XXX, XXX−XXX
