1. Kinetic Resolution & Analysis of
(+/-) - 1- phenylethanol using a
DMAP Derivative and the
Subtilisin Carlsberg Enzyme
Kian Bagheri
Pomona College Organic Chemistry 110B
Laboratory

2. Man-Made vs. Natural?
SC (MW: 27,800
g/mol)
DMAP (MW: 660.6 g/mol)
DMAP
(Vasquez et. al. 2015)
Subtilisin DMAP

3. Comparing Kinetics by using the
same solvent
Tetrahydrofuran (THF)
(not ideal solvent)

4. Methods
● Kinetic Resolution: enantiomeric excess
● Silica Gel Column: remove catalyst
● Thin Layer Chromatography (TLC): identify gel column
fractions needed for the GC
● Gas chromatography (GC): quantify enantiomers

5. Kinetic Resolution: A Closer Look
Stopping a reaction before completion in order to
get close to 100% enantiomeric excess
%ee
Reaction Progress
100% ee

6. GC/MS: A closer look
Peak intensities correlate with major & minor
enantiomer products.
(teaching.shu.ac.uk)

7. Acyl Transfer Reaction of DMAP
(man-made catalyst)
Overall Reaction
Acetic
Anhydride
1-phenylethyl
acetate
(Vasquez et. al. 2015)

8. Proposed Mechanism of DMAP
1-phenylethyl acetate

9. Subtilisin Carlsberg Reaction (Naturally-
Occurring Enzyme)
Vinyl
buterate 1-phenylethyl butyrate
(Vasquez et. al. 2015)

10. Proposed Mechanism of Subtilisin
Enzyme

11. Subtilisin Mechanism (Continued)
(Vasquez et. al. 2015)
1-phenylethyl butyrate

12. Reagents (Comparable to MIT Lab)

13. GC
Results
for DMAP

14. GC
Results
for
Subtilisin

15. Data Analysis (% ee)
% ee= (major-minor)/(major + minor) [areas]
DMAP Catalyst: % ee = 80.7 %
● [(700,845 - 749,40)/(700,845 + 74,940)]
Subtilisin Enzyme: % ee = 50.0 %
● [(9,883,234 - 3,295,182)/(9,883,234 + 3,295,182)]

16. Data Analysis (% conversion)
% conversion= (products)/ (reactants + products)
DMAP Catalyst: % conversion = 11.8 %
● (700,845 + 749,40)/(700,845 + 749,40 +3,061,649+2,744,991)
Subtilisin Enzyme: % conversion = 78.4 %
● (9,883,234 + 3,295,182)/(9,883,234 + 3,295,182 +
3,630,602)
= starting material converted into product.

17. GC Analysis
SuDMAP
100% ee
50% ee

18. Conclusion
DMAP Subtilisin
%ee: 80.7 % %ee: 50.0 %
% c: 11.8 % % c: 78.4 %

19. Additional Literature/Considerations
● Bansal et al. concluded prolonged exposure causes active-site of Subtilisin to adopt different
binding conformation
● Enzyme activity decreased approximately 10-fold during 4-day incubation
● As a result, substrate binds in a less catalytically favorable conformation
● Relates back to Subtilisin experiment. Prolonged exposure yielded 78.4% conversion (want
closer to 50%)
Dioxane AcetonitrileGraphs courtesy of Bansal et al.

20. Future Experiments
● Reaction for less than 7 days
● Ideal solvents:
DMAP: Tert-Amyl alcohol Subtilisin: Dioxane

21. Acknowledgements
Bansal, V., Delgado Y., Fasoli, E., Ferrer, A., Griebenow, K., Secundo, F., and
Barletta, G., Effect of prolonged exposure to organic solvents on the active site
environment of subtilisin Carlsberg. Journal of Molecular Catalysis B. 2010.
Selassie, C., Sveinbjornsson, B., Vasquez, T. (2015). Organic Chemistry 110B
Laboratory Manual. 20-24. Print.
Solomons, T., Fryhle, C., & Snyder, S. (2013). Organic Chemistry (11th ed.). New
York: Wiley. 558-566. Print.