Enzymatic Condensation Reactions in Ionic Liquids - American

Chapter 17

Enzymatic Condensation Reactions in Ionic Liquids 1

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Nicole Kaftzik , Sebastian Neumann , Maria-Regina Kula , and Udo Kragl * 1,

Downloaded by COLUMBIA UNIV on July 27, 2012 | http://pubs.acs.org Publication Date: August 26, 2003 | doi: 10.1021/bk-2003-0856.ch017

1

Department of Chemistry, Rostock University, 18051 Rostock, Germany Heinrich Heine University at Düsseldorf, Institute of Enzyme Technology, 52426 Jülich, Germany

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In an aqueous environment glycosidases and peptide amidases usually hydrolyse glycosidic bonds or amides, respectively. The reaction can be reversed by incubating the enzyme at lower water activity in the presence of ionic liquids, resulting in a higher yield of disaccharide or peptide amide, βGalactosidasefromBacillus circulans can be applied in nearly anhydrous ionic liquids for reverse hydrolysis with yields of lactose of up to 17%. Peptide amidase from Stenotrophomonas maltophilia is used for the direct C-terminal peptide amidation of H-Ala-Phe-OH.

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© 2003 American Chemical Society In Ionic Liquids as Green Solvents; Rogers, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


207 There is no question that biotransformations are an established method either in the lab or for industrial production of bulk and fine chemicals (1-3). Nevertheless, there are still problems with substrate solubility, yield or selectivity. Some progress has been made by the use of microemulsions (4), supercritical fluids (5) or use of organic solvents (6, 7). The use of hydrolytic enzymes in anhydrous organic media has become a valuable addition to the synthetic repertoire. Reactions may be performed that are impossible in water and the enzymes may even show enhanced thermostability (8, 9). During the past decade, ionic liquids have gained increasing attention for performing all types of reactions with sometimes remarkable results (10). Ionic liquids are salts with melting points below 100 °C; they have no measurable vapor pressure making them ideal tools for clean and sustainable processes (11). Most of the studies in this area have focused on transformations using transition-metal catalysts (10, 11). Recently the application of lipases (12-16), thermolysin or α-chymotrypsin (17, 18) in ionic liquids has been reported. We herein wish to report the results from our studies on the synthesis of disaccharides using the reverse hydrolysis activity of β-galactosidase from Bacillus circulons (19) with enhanced yield in the presence of ionic liquids. The second example is the use of a peptide amidase from Stenotrophomonas maltophilia (20) for the direct C-terminal peptide amidation of peptides using different ionic liquids instead of organic solvents.

β-Galactosidase Catalyzed Synthesis Of Disaccharides D-Galacto-oligosaccharides have been synthesized mainly by utilizing the transglycosylation activity of β-galactosidase of various origins (19, 21). However, it is difficult to separate the products from the starting disaccharides and to determine the optimum time to terminate the reaction in order to obtain high yields of the desired compounds. Nevertheless we have studied the behaviour of the β-galactosidasefromBacillus circulons in this type of reaction, in the presence of ionic liquids. These results are presented elsewhere (22,23). Here we studied the reverse hydrolytic activity of β-galactosidase from Bacillus circulons (Figure 1). The reaction was performed in both aqueous media containing different amounts of the ionic liquid and the pure ionic liquid 1,3dimethylimidazol methylsulfate ([MMIM] MeS0 ) 6. To favour the formation of disaccharides 4 and 5, high concentrations of the monosaccharides 1 and 2 are necessary. Ionic liquids offer the advantage of dissolving carbohydrates very well allowing higher concentrations than in water and especially mixtures of water and organic solvents as cosolvents. Furthermore, the ratio of substrates 1 and 3 must be as high as possible in order to minimize the formation of D-galactosyl-Dgalactoses. Therefore a solution containing 3 and 1 in a ratio of 1:5 was used together with β-galactosidasefromBacillus circulons (24). The reaction mixture was incubated for 24 h at 20,35 and 50 °C. After removal of the heat-denaturated enzyme the filtrate was analysed by HPLC (conditions: BioRad Aminex HPX87H, UV (208 nm) and RI detection, 0.006 M H S0 with 0.8 ml/min at 65 °C). The yield of disaccharide obtained is shown in Figure 2. 4

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In Ionic Liquids as Green Solvents; Rogers, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

208

PH

0

u

OH

OH

OH

H0 2

R

OH

H

^

R

4R: OH 5 R: NHAc

1 R: OH 2 R: NHAc

[MMIM]MeS0 [BMIM]MeS0 [EMIM]BF [BMIMJBTA

6 R: Me A: MeS0 7 R:Bu A":MeS0 8 R: Et A":BF 9 R:Bu A: [CF S0 ) N 10[Et NH]MeSO 11 [Et NMe]MeS0

4

4

4

4

4

4

3


R HO HO" R

3

2 2

4

3

4

Figure 1. β-Galactosidase catalyzed synthesis of disaccharides and ionic liquids used. The highest yield of 4 was obtained at 35 °C and in almost pure ionic liquid containing only 0.6% v/v water. There was no further increase in the product concentration after 24 h. Compared to data published previously, where the maximum yield was reached after 5 d, the reaction velocity is much faster in the presence of ionic liquids (19). Kren and coworkers reported similary results using high salt concentrations in a thermodynamically controlled reaction, but only after a reaction time of 6 days (25).

0

10

20

30

40

50

60

70

80

90

100

amount of ionic liquid [% v/v]

Figure 2. Yield of lactose 4 as function of amount of the ionic liquid IMMIM] MeS0 6; conditions: 100 mmol/l 1, 20 mmol/l 3, 2 mg/ml galactosidase. 4


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Peptide Amidase Catalyzed Amidation Peptides and their synthetic analogues represent an interesting class of biologically active compounds, which find application in all areas of immunology, pharmaceutical research and therapy. Many bioactive peptides and more than half of all peptides hormones are amidated at their C-terminal carboxyl group to exhibite full biological activity (26, 27). A peptide amidase that hydrolyses exclusively C-terminal amide groups in peptides, without affecting the internal peptide bonds, wasfirstdescribed by Steinke and Kula (28). This enzyme is also able to catalyze the reverse reaction, the direct C-terminal peptide amidation (29) (Figure 3). The best results of amide syntheses using model substrates of a large series of I^-protected di-, tri-, tetra- and penta-peptide were achieved in a medium consisting of acetonitrile with 25 % v/v of dimethylformamide and 3 % v/v of water (30). The aim of this work was to investigate the synthetic possibilities of peptide amidase-catalyzed peptide amidation with a microbial peptide amidasefromStenotrophomonas maltophilia (20) using H-Ala-Phe-OH 12 as model substrate in the presence of NH4HCO313 as the ammonium source in ionic liquids as reaction media.

NH2 H

/ - ^

Ν

O

Ν γ Χ

Ο

NHg Ο

Η

+

NH HCQ3 4

Ph

12

•

^ γ

Ν

Η

γ ^ Ν Η 2

Ο

13

Ο

+ Η0 2

Ph

14

Figure 3. Peptide amidation using peptide amidase from Stenotrophomonas maltophilia. A series of different ionic liquids 6 - 1 1 (Scheme 1) from total water miscible to water insoluble ionic liquids was investigated. Solubility properties and amidation rates were compared with the results obtained in oganic solvents like acetonitrile, glycerine or ethylenglycol. Amongst the ionic liquids tested best results were obtained with 7. Reactions were carried out in sealed 2 ml screw-cap plastic tubes incubated in a rotatory shaker. The peptide (0.025 mmol) was dissolved in a mixture of ionic liquid (125 μΐ, 250 μΐ, 375 μΐ, 450 μΐ) and aqueous buffer-solution (325 μΐ, 200 μΐ, 75 μΐ, 50 μΐ) and in pure ionic liquid. After the addition of 1 mg 13 (0.035 mmol of N H 4 * ) and lyophilized amidase (6 mg) respectively 50 μΐ enzyme solution (10 mM in buffer-solution) the reactionmixture was shaken at 37 °C for 12 h. Both substrates and the enzyme were apparently soluble in 6, 7,10 and 11 as well as in glycerine and in ethylenglycol. The influence of ionic liquid and water concentration on the peptide amidasecatalyzed amidation of 12 was analyzed by HPLC (conditions: RP 18 Hypersil OPS 5, UV detection 220 nm, 90% water, 10% acetonitrile, 0.1% trifluoracetic acid with 1 ml/min at room temperature).


210 Lowering the total amount of water to almost 0 % v/v results in a maximum yield of 15% in 7. This result is in the same order of magnitude obtained in acetonitrile containing 25 % v/v DMF and 4 % v/v of water (31). Further studies have to show whether the ionic liquids will improve the enzyme stability as well.


Conclusions We have shown that ionic liquids can serve as sole solvents for enzymes others than lipases and proteases reported so far. They may be especially useful in thermodynamically controlled reactions where proper control of the water activity in the medium is of great importance. For the β-galactosidase-catalyzed formation of disaccharides we have found an improved enzyme stability also at higher temperatures and an increased formation of disaccharide by lowering the total water content in the reaction media. Reaction rates of peptide amidasecatalyzed amidation were comparable with those observed in conventional organic reaction media. In both cases ionic liquids show good or even better solubility properties for both substrates and enzymes. To represent a real alternative to synthesis in organic solvent for example, it is important to demonstrate that the ionic liquid can be recycled after the reaction. This aspect is subject to further studies. Additionally, other influencing factors such as the nature of the medium, diffusion rates, the water content and the question which of the ions are responsible for the effects have to be investigated as well.

Acknowledgments We thank P. Wasserscheid and V. Cerovsky for fruitful discussions, Solvent Innovation, Cologne, for the gift of ionic liquids and the "Fonds der Chemischen Industrie" for partial financial support.

References 1. 2. 3. 4. 5. 6. 7. 8.

Faber, K. Biotransformation in Organic Chemistry, Springer, Berlin 2000. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis, WileyVCH, Weinheim 1999. Liese, Α.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, WileyVCH, Weinheim 2000. Orlich, B.; Schomäcker, R. Biotech. Bioeng. 1999, 65, 357-362. Hartmann, T.; Schwabe, E.; Scheper, T. "Enzyme catalysis in supercritical fluids" in R. Pathel, Stereoselective Biocatalysis, Marcel Decker 2000, pp. 799838. Carrea, G.; Riva, S. Angew. Chem. Int. Ed. 2000, 39, 2226-2254. Cabrai, J. M. S.; Aires-Barros, M. R.; Pinheiro, H.; Prazeres, D. M. F. J. Biotechnol. 1997, 59, 133. Dai, L.; Klibanov, A. M. Proc. Natl. Acad.Sci.USA 1999, 96, 9475-9478.


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9. 10. 11. 12. 13. 14.


15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Sellek, G. Α.; Chaudhuri, J. B. Enzyme Microb. Technol.1999,25, 471-482. Welton, T. Chem. Rev. 1999, 99, 2071-. Wasserscheid, P.; Keim, W. Angew. Chem. Int. Ed. 2000, 39, 3773-3789. Lau, R. M.; Van Rantwijk, F.; Seddon, K. R.; Sheldon, R. A. Org. Lett. 2000, 2, 4189-4191. Schöfer, S.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001, 425-426. Kwang-Wook Kim, Boyoung Song, Min-Young Choi, Mahn-Joo Kim Org. Lett. 2001, 3, 1507-1509. Itoh, T.; Akasaki, E.; Kudo, K.; Shirakami, S. Chem. Lett. 2001, 3, 262-263. Lozano, P., DeDiego, T.; Carrie, D.; Vaultier, M., Iborra, J. L. Biotechnol. Lett. 2001, 23, 1529-1533. Erbeldinger, M.; Mesiano, A. J.; Russel, A. Biotechnol. Prog. 2000, 16, 1131-. Laszlo, J. Α.; Compton, D. L.; Biotechnol. Bioeng. 2001, 75, 181-186. Ajisaka, K.; Fujimoto, H.; Nishida, H. Carbohydr. Res. 1988, 180, 35-42. Cerovsky, V.; Kula, M.-R. Biotechnol. Appl. Biochem. 2001, 33, 183-187. Crout, D. H. G.; Vic, G.; Curr. Opin. Chem. Biol. 1998, 2, 98-111. Kragl, U.; Kaftzik, N.; Schöfer, S.H.;Eckstein, M.; Wasserscheid, P.; Hilgers, C.; Chim. Oggi 2001, 19, 22-24. Kaftzik, N.; Wasserscheid,P.;Kragl, U., Org. Process Res. Dev. 2002, 6, 553557. β-Galactosidase from Bacillus circulans is commercial available from Daiwa Kasei, Osaka, Japan. Rajnochova, E.; Dvorakova, J.; Hunkova, Z.; Kren, V.; Biotechnol. Lett. 1997, 19, 869-872. Merkler, D.J. J. Enzyme Microb. Technol. 1999, 16, 450-456. Pigge, S. T.; Mains, R. E.; Eipper, Β. Α.; Amzel, L. M. Cell. Mol. Life. Sci. 2000, 57, 1236-1259. Steinke, D.; Kula, M.-R. Angew. Chem. Int. Ed. Engl. 1990, 29, 1139-1140. Stelkes-Ritter, U.; Wyzgol, K.; Kula, M.-R. Appl. Microb. Biotechnol. 1995, 44, 393-398. Cerovsky, V.; Kula, M.-R. Angew. Chem. Int. Ed. Engl. 1998, 37, 1885-1887. Neumann, S., Kula, M. R. Appl Microbiol Biotechnol. 2002, 58, 772-780.


Enzymatic Condensation Reactions in Ionic Liquids - American

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