Applied Biochemistry and Biotechnology

, Volume 185, Issue 3, pp 578–592 | Cite as

CALB-Catalyzed Two-Step Alcoholytic Desymmetrization of 3-Methylglutaric Diazolides in MTBE

  • Ting-Yi Wu
  • Yuan-Rong Lai
  • Shau-Wei Tsai


Optically pure 3-substituted glutarates can be prepared from the alcoholic ring-opening of cyclic anhydride derivatives, esterification of 3-substituted glutaric acid, and hydrolysis, alcoholysis, aminolysis, and ammonolysis of the diester derivatives via hydrolases or organocatalysts. Unfortunately, most of them mainly focus on the first-step desymmetrization, leading to the difficulty on producing optically pure enantiomers. As a general trend in lipase-catalyzed desymmetrization of 3-methylglutarates, poorer enantiomeric excesses with lower chemical yields were found, as the methyl substituent is relatively small to induce a high enzyme stereodiscrimination. The two-step desymmetrization for CALB-catalyzed alcoholysis of 3-methylglutaric di-1,2,4-triazolide 1a in anhydrous MTBE is first developed to increase the enzyme activity in each reaction step. The enantioselectivity for the second-step kinetic resolution is furthermore improved by using 3-methylglutaric dipyrazolide 1b as the substrate. The kinetic and thermodynamic analysis is, moreover, addressed for shedding insights into the desymmetrization process.


Two-step desymmetrization Kinetic and thermodynamic analysis CALB 3-Methylglutaric diazolide 



enzyme concentration (mg mL−1)


selectivity defined as k10k20−1

E2, E3

dimensionless parameters defined as k30(k10 + k20)−1and k40(k10 + k20)−1, respectively


enantiomeric excess defined as (X2S − X2R)(X2S + X2R)−1

Km1, Km2, Km3, Km4

Michaelis-Menten constant for each reaction step in Scheme 1 (mM)


inhibition constant from alcohol (mM)

k1, k2, k3, k4

kinetic constants in Scheme 1 (h−1)


initial alcohol concentration (mM)

(S)0, (S)

diazolide concentration initially and at a specific time (mM)


absolute temperature (K)


time (h)


dimensionless time defined as (k1 + k2)t

Vmax,1, Vmax,2, Vmax,3, Vmax,4

maximum velocity for each reaction step in Scheme 1 (mM h−1)

X1, X2R,X2S, X3

molar fractions based on (S)0 for 1, (R)-2, (S)-2, and 3, respectively


free energy difference for two transition states (kJ mol−1)


enthalpy difference for two transition states (kJ mol−1)


entropy differences for two transition states (J mol−1 K−1)



for dimensionless group



initial state


Funding Information

Financial supports of MOST 104-2221-E-182-058-MY2 from the Ministry of Science and Technology (Taiwan, Republic of China) are appreciated.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12010_2017_2675_MOESM1_ESM.doc (3 mb)
ESM 1 (DOC 3086 kb)


  1. 1.
    Bornscheuer, U. T., & Kazlauskas, R. J. (2006). Hydrolases in organic synthesis: regio- and stereoselective biotransformations (2nd ed.). Weinheim: Wiley-VCH.Google Scholar
  2. 2.
    Ghanem, A. (2007). Trends in lipase-catalyzed asymmetric access to enantiomerically pure/enriched compounds. Tetrahedron, 63(8), 1721–1754. Scholar
  3. 3.
    Faber, K. (2011). Biotransformations in organic chemistry: a textbook (6th ed.). New York: Springer. Scholar
  4. 4.
    Paravidino, M., Bohm, P., Groger, H., & Hanefeld, U. (2012). Hydrolysis and formation of carboxylic acid esters. In K. Drauz, H. Gröger, & M. May (Eds.), Enzyme catalysis in organic synthesis (Vol. 1, 3rd ed.). Weinheim: Wiley-VCH.Google Scholar
  5. 5.
    Schoffers, E., Golebiowski, A., & Johnson, C. R. (1996). Enantioselective synthesis through enzymatic asymmetrization. Tetrahedron, 52(11), 3769–3826. Scholar
  6. 6.
    García-Urdiales, E., Alfonso, I., & Gotor, V. (2011). Update 1 of: enantioselective enzymatic desymmetrizations in organic synthesis. Chemical Reviews, 111(5), PR110–PR180. Scholar
  7. 7.
    Palomo, J. M., & Cabrera, Z. (2012). Enzymatic desymmetrization of prochiral molecules. Current Organic Synthesis, 9(6), 791–805. Scholar
  8. 8.
    Atodiresei, I., Schiffers, I., & Bolm, C. (2007). Stereoselective anhydride openings. Chemical Reviews, 107(12), 5683–5712. Scholar
  9. 9.
    Sajisha, V. S., Anas, S., John, J., & Radhakrishnan, K. V. (2009). Desymmetrization of meso-bicyclic hydrazines: an efficient strategy towards the synthesis of functionalized cyclopentenes. Synlett, 18, 2885–2895.Google Scholar
  10. 10.
    Díaz-De-Villegas, M. D., Gálvez, J. A., Etayo, P., Badorrey, R., & Lopez-Ram-De-Viu, P. (2011). Recent advances in enantioselective organocatalyzed anhydride desymmetrization and its application to the synthesis of valuable enantiopure compounds. Chemical Society Reviews, 10, 5564–5587.CrossRefGoogle Scholar
  11. 11.
    Suzuki, T. (2011). Desymmetrization of meso diols. In E. M. Carreira & H. Yamamoto (Eds.), Comprehensive chirality (Vol. 5). Amsterdam: Elsevier.Google Scholar
  12. 12.
    Furuta, T., & Kawabata, T. (2011). C-O bond formation: acylation of meso diols. In E. M. Carreira & H. Yamamoto (Eds.), Comprehensive chirality (Vol. 6). Amsterdam: Elsevier.Google Scholar
  13. 13.
    Moteki, S. A. (2011). C-O bond formation: desymmetrization of acid anhydride. In E. M. Carreira & H. Yamamoto (Eds.), Comprehensive chirality (Vol. 6). Amsterdam: Elsevier.Google Scholar
  14. 14.
    De, C. K., & Seidel, D. (2011). Catalytic enantioselective desymmetrization of meso-diamines: a dual small-molecule catalysis approach. Journal of the American Chemical Society, 133(37), 14538–14541. Scholar
  15. 15.
    Muller, C. E., & Schreiner, P. R. (2011). Organocatalytic enantioselective acyl transfer onto racemic as well as meso alcohols. Angewandte Chemie International Edition, 50(27), 6012–6042. Scholar
  16. 16.
    Fryszkowska, A., Komar, M., Koszelewski, D., & Ostaszewski, R. (2005). Enzymatic desymmetrization of 3-arylglutaric acid anhydrides. Tetrahedron: Asymmetry, 16(14), 2475–2485. Scholar
  17. 17.
    Gopinath, P., Watanabe, T., & Shibasaki, M. (2012). Studies on catalytic enantioselective total synthesis of caprazamycin B: construction of the western zone. Journal of Organic Chemistry, 77, 92605–99267.CrossRefGoogle Scholar
  18. 18.
    Jung, J.-H., Yoon, D.-H., Kang, P., Lee, W. K., Eum, H., & Ha, H.-J. (2013). CAL-B catalyzed desymmetrization of 3-alkylglutarate: “olefin effect” and asymmetric synthesis of pregabalin. Organic & Biomolecular Chemistry, 11, 3635–3641, 22, doi: Scholar
  19. 19.
    Yamamoto, Y., Yamamoto, K., Nishioka, T., & Oda, J. (1988). Asymmetric synthesis of optically active lactones from cyclic acid anhydrides using lipases in organic solvents. Agricultural Biological Chemistry, 52, 3087–3092.Google Scholar
  20. 20.
    Fryszkowska, A., Komar, M., Koszelewski, D., & Ostaszewski, R. (2006). Studies on enzymatic synthesis of chiral non-racemic 3-arylglutaric acid monoesters. Tetrahedron: Asymmetry, 17(6), 961–965. Scholar
  21. 21.
    Chaubey, N., Date, S. M., & Ghosh, S. K. (2008). An efficient asymmetric desymmetrization of prochiral glutaric anhydrides with SuperQuat chiral oxazolidin-2-ones. Tetrahedron: Asymmetry, 19(23), 2721–2730. Scholar
  22. 22.
    Park, S. E., Nam, E. H., Jang, H. B., Oh, J. S., Some, S., Lee, Y. S., & Song, C. E. (2010). Enantioselective alcoholysis of meso-glutaric anhydrides catalyzed by Cinchona-based sulfonamide catalysts. Advanced Synthesis & Catalysis, 352(13), 2211–2217. Scholar
  23. 23.
    Liu, W., Hu, Y., Zhang, Y., Ma, Y., & Huang, H. (2014). Enzymatic desymmetrization of 3-(4-fluorophenyl)glutaric anhydride through enantioselective alcoholysis in organic solvents. Biotechnology and Bioprocess Engineering, 19(3), 449–455. Scholar
  24. 24.
    Roy, S., Chen, K. F., Gurubrahamam, R., & Chen, K. (2014). Organocatalytic kinetic resolution of racemic secondary nitroallylic alcohols combined with simultaneous desymmetrization of prochiral cyclic anhydrides. Journal of Organic Chemistry, 79(19), 8955–8959. Scholar
  25. 25.
    Lam, L. K. P., & Hui, R. A. H. F. (Jones, J. B. (1986). Enzymes in organic synthesis. 35. Stereoselective pig liver esterase catalyzed hydrolyses of 3-substituted glutarate diesters. Optimization of enantiomeric excess via reaction conditions control. Journal of Organic Chemistry, 51, 2074–2050.Google Scholar
  26. 26.
    Yu, M. S., Lantos, I., Peng, Z.-Q., Yu, J., & Cacchio, T. (2000). Asymmetric synthesis of (−)-paroxetine using PLE hydrolysis. Tetrahedron Letters, 41(30), 5647–5651. Scholar
  27. 27.
    Homann, M. J., Vail, R., Morgan, B., Sabesan, V., Levy, C., Dodds, D. R. C., & Zaks, A. (2001). Enzymatic hydrolysis of a prochiral 3-substituted glutarate ester, an intermediate in the synthesis of an NK1/NK2 dual antagonist. Advanced Synthesis & Catalysis, 343(6–7), 744–749.<744::AID-ADSC744>3.0.CO;2-E.CrossRefGoogle Scholar
  28. 28.
    Lopez-Garcia, M., Alfonso, I., & Gotor, V. (2003). Desymmetrization of dimethyl 3-substituted glutarates through enzymatic ammonolysis and aminolysis reactions. Tetrahedron: Asymmetry, 14(5), 603–609. Scholar
  29. 29.
    Cabrera, Z., Fernandez-Lorente, G., Fernandez-Lafuente, R., Palomo, J. M., & Guisan, J. M. (2009). Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports. Journal of Molecular Catalysis B: Enzymatic, 57(1-4), 171–176. Scholar
  30. 30.
    Dong, H.-P., Wang, Y.-J., & Zheng, Y.-G. (2010). Enantioselective hydrolysis of diethyl 3-hydroxyglutarate to ethyl (S)-3-hydroxyglutarate by immobilized Candida antarctica lipase B. Journal of Molecular Catalysis B: Enzymatic, 66(1–2), 90–94. Scholar
  31. 31.
    Wang, B., Liu, J., Tang, X., Cheng, C., Gu, J., Dai, L., & Yu, H. (2010). Enzymatic synthesis of (S)-glutaric acid monoesters aided by molecular docking. Tetrahedron Letters, 51(2), 309–312. Scholar
  32. 32.
    Cabrera, Z., & Palomo, J. (2011). Enantioselective desymmetrization of prochiral diesters catalyzed by immobilized Rhizopus oryzae lipase. Tetrahedron: Asymmetry, 22(24), 2080–2084. Scholar
  33. 33.
    Liu, W., Hu, Y., Jiang, L., Zou, B., & Huang, H. (2012). Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester. Process Biochemistry, 47(7), 1037–1041. Scholar
  34. 34.
    Nojiri, M., Uekita, K., Ohnuki, M., Taoka, N., & Yasohara, Y. (2013). Microbial asymmetric hydrolysis of 3-substituted glutaric acid diamides. Journal of Applied Microbiology, 115(5), 1127–1133. Scholar
  35. 35.
    Chan, P.-H., & Tsai, S. W. (2016). Two-step desymmetrization of dipyrazolidyl 3-phenylglutarate via lipase-catalyzed hydrolysis in organic solvents. Chemical Engineering Science, 139, 41–48. Scholar
  36. 36.
    Hsiao, H.-T., Lin, S.-Y., & Tsai, S. W. (2016). Quantitative insights and improvements of enzyme activity and stereoselectivity for CALB-catalyzed alcoholysis in two-step desymmetrization. Journal of Molecular Catalysis B: Enzymatic, 127, 82–88. Scholar
  37. 37.
    Tsai, S. W. (2016). Enantiopreference of Candida antarctica lipase B toward carboxylic acids: Substrate models and enantioselectivity thereof. Journal of Molecular Catalysis B: Enzymatic, 127, 98–116. Scholar
  38. 38.
    Yang, H., Henke, E., & Bornscheuer, U. T. (1999). The use of vinyl esters significantly enhanced enantioselectivities and reaction rates in lipase-catalyzed resolutions of arylaliphatic carboxylic acids. Journal of Organic Chemistry, 64(5), 1709–1712. Scholar
  39. 39.
    Chen, R., Wu, C. H., Wang, P. Y., & Tsai, S. W. (2012). Kinetic and thermodynamic investigation of lipase-catalyzed hydrolysis of (R,S)-3-phenylbutyl azolides. Industrial & Engineering Chemistry Research, 51(9), 3580–3586. Scholar
  40. 40.
    Staab, H. A., Bauer, H., & Schneider, K. M. (1998). Azolides in organic synthesis and biochemistry. Weinheim: Wiley-VCH. Scholar
  41. 41.
    Wang, P. Y., Chen, Y. J., Wu, A. C., Lin, Y. S., Kao, M. F., Chen, J. R., Ciou, J. F., & Tsai, S. W. (2009). (R,S)-Azolides as novel substrates for lipase-catalyzed hydrolytic resolution in organic solvents. Advanced Synthesis & Catalysis, 351(14–15), 2333–2341. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018
corrected publication January/2018

Authors and Affiliations

  1. 1.Department of Chemical and Materials EngineeringChang Gung UniversityTaoyuan CityTaiwan
  2. 2.Graduate Institute of Biochemical and Biomedical EngineeringChang Gung UniversityTaoyuan CityTaiwan

Personalised recommendations