Part 7 – Equilibrium reactions and how to achieve satisfactory yields.

Introduction

   Many reactions are truly reversible (see blog post Part 4) and, as a consequence, substrate(s) and product(s) are in equilibrium with each other. The position of this equilibrium depends on thermodynamics and process conditions. This blog post will deal with equilibriums and how to shift them more towards the product to allow you to isolate your product in an economic manner.

   There are two main ways of shifting an equilibrium towards the product side: push and pull. Let’s look at the example in Figure 1 (a) below. This is an ester hydrolysis in hexane as solvent where the desired molecule is the acid. Understandably, increasing the concentration of H₂O (b) will favor hydrolysis and the arrow in the forward reaction becomes bigger. I call that ‘pushing’ the equilibrium. We could also ‘pull’ the equilibrium towards the product by removing the methanol by evaporation (c). We could also do both (d). See how the arrows change?

Alternatively, we could pull the acid from the reaction medium by binding the acid at the right pH to an anion exchange resin[1], or make it precipitate as a salt with an appropriate counter ion, for instance.

Figure 1: Example of a lactic acid ester hydrolysis.

 Discussion

   I will now give a few examples of solving the equilibrium issues from enzyme classes that if you follow my blog should be familiar to you.

Ketoreductases

   Ketone reductions are fully reversible reactions that lead to a chemical equilibrium of ketone and alcohol (Figure 2).

Figure 2: The KRED catalyzed equilibrium reaction. [2]

   If we would use a large excess of NADH, we might be able to push the reaction to completion. However, the cost of NADH is too high to do that and, also, high concentration of NADH acts as an inhibitor of many KREDs. Economically, we can only use a little bit (a catalytic amount) and then only of the cheapest form: NAD⁺. Therefore, the cofactor needs to be recycled many times. This can be done by doing a similar reaction in reverse, using a sacrificial alcohol as a co-substrate. Isopropanol (iPA) is often used for this purpose. The scheme from Figure 2 changes to the one in Figure 3. Thus, in the first step, the KRED oxidizes iPA to acetone and the NAD⁺ cofactor is reduced to NADH. In the next step, the same KRED enzyme reduces the aryl ketone to the chiral alcohol (hopefully) or it reduces acetone back to iPA (L).

Clearly, the efficiency of this system depends on the binding affinities and turnover rates for iPA compared to aryl ketone. The aryl ketone must bind better, but not so much better that it cannot be displaced by iPA every now and then.

Figure 3: Formal scheme of KRED-catalyzed transfer hydrogenation.

   Most people only use the simplified scheme shown in Figure 4 which only becomes valid under a certain circumstance that I’ll get to in a moment. In the mean time, note that the scheme in Figure 3 really does not have any directional arrow. It is like the wheel of a car that can rotate in each direction but if we want to go somewhere, it better rotate in the same direction for a while! We need an external force that makes the wheel turn (clockwise in this example).

   That external force is created by adding a large excess of iPA: running the enzymatic reaction in 50/50 water/iPA, or sometimes even up to 90% iPA [3]! The large excess iPA pushes the equilibrium of the iPA oxidation all the way to the acetone side, de facto making that reaction uni-directional. Now, since that reaction now only goes in one direction, the coupled reaction can also only go in one direction: towards the chiral alcohol. We have created the scheme that is shown in Figure 4.

Figure 4: Simplied scheme for ketone reduction with excess iPA.

   In some cases, the excess of iPA is not enough to push the desired conversion above 99% yield [4, 5]. Removal of acetone by vacuum or a nitrogen gas sweep helps pull the lower reaction to completion and thus the coupled formation to chiral alcohol.[6] In some cases, precipitation of the chiral alcohol can be achieved by tailoring the reaction medium; this is another case of ‘pull’ [7].

   The iPA/Acetone system is not perfect for every case: sometimes the process conditions cannot be optimized to give adequate conversion, or the KRED might not accept iPA as substrate. In those cases, a selection of different coupled enzymatic reactions can be used [8]. Most popular are using glucose dehydrogenase (GDH, [9]), formate dehydrogenase (FDH, [10]) and phosphite dehydrogenase (PTDH, [11]) coupled systems. These reaction schemes are compiled in Figure 5.

Figure 5: Three popular enzymatic cofactor regeneration systems.

   What is important to remember in these multi-enzyme systems is that the cofactor needs to shuttle back and forth from one enzyme to the other. This shuttling is also something to keep in mind when KREDs are immobilized: the fixation to the carrier must not interfere with substrate and product transport as well as allow fast exchange of the cofactor which happens to be a rather large molecule and (may) requires conformational changes in the protein. Apparently it can be done since SPRIN in Italy offers a kit of 12 immobilized KREDs and also has an immobilized GDH.[12]

Transaminases

   Transaminases catalyze transfer of an amine group from a donor to a ketone and can use isopropyl amine (iPM) as amine donor, or use natural amino acids like alanine as donor. See Figure 6 and 7 for iPM and Alanine, respectively. Note that in these schemes, the recycle of the pyridoxal phosphate (PLP) cofactor is omitted for clarity.

Figure 6: ‘Chemical tricks’ to achieve better yield. Binding of the product-amine to a resin also alleviates the product inhibition that is often seen with TAs.

Figure 7: Two enzymatically coupled reactions to drive transaminations to completion. [15] And then I have not even shown another one using pyruvate decarboxylase [16].

Haloalcohol dehalogenases

   The ring closure of 1-(4-nitrophenyl)-2-bromo ethan-1-ol (dubbed by a biochemist ‘PNSHH’ for para-nitrostyrene halo hydrin and the name caught on...) to 4-nitro-styrene oxide (PNSO) and bromide ion was scaled up. Reaction conditions: 15 gr substrate, 3 liters of 50% toluene/water biphasic mixture and 100 g of acetate-loaded ion exchange resin that bound the released halide. We achieved complete conversion with essentially absolute enantioselectivity .[17] Enzyme load was 50 mg total and was provided by Ms Lixia Tang.

Figure 8: Since it was his idea, Jeffrey lutje Spelberg got to hold the flask in this picture which was later simply put in a shaker at 30°C.

Conclusions

   Dealing with equilibrium reactions may seem daunting but as long as one of the reactions in a coupled system is or can be made uni-directional, the proof of concept has been made. Process optimization must follow and a rule of thumb is “less enzyme is cheaper”. However, if using multiple enzymes can lead to a 100% yield and a simple product isolation, it can still be a winner.

References

[1] Resins available at scale, for instance, from www.purolite.com. Also look at their Purolite LifeTech™ brand for biocatalysis and life sciences applications.

[2] Of course this discussion does not mention NADP(H) as a cofactor for simplicity.

[3] C Savile, JM Gruber, E Mundorff, GW Huisman, and SJ Collier. WO2010/025287 Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine. Patent granted to Codexis. Example 9 shows conversion of a ketone on preparative scale at 90% iPA.

[4] J Liang, E Mundorff, R Voladri, S Jenne, L Gilson, A Conway, A Krebber, J Wong, GW Huisman, S Truesdell, and J Lalonde. Highly Enantioselective Reduction of a Small Heterocyclic Ketone: Biocatalytic Reduction of Tetrahydrothiophene-3-one to the Corresponding (R)-Alcohol. Org. Process Res. Dev., 2010, 14 (1), pp 188–192.

[5] From an atom utilization standpoint, using ‘excess’ of something is less elegant and frowned upon according to the 12 principles of Green Chemistry: http://www.epa.gov/sciencematters/june2011/principles.htm . Use of excess reagents should be avoided where possible.

[6] SJ Calvin, D Mangan, I Miskelly, TS Moody, and PJ Stevenson. Overcoming Equilibrium Issues with Carbonyl Reductase Enzymes. Org. Process Res. Dev. 2012, 16, 82–86. http://www.almacgroup.com/wp-content/uploads/Overcoming-Equilibrium-Issues-with-Carbonyl-Reductase-Enzymes.pdf

[7] J Liang, J Lalonde, B Borup, V Mitchell, E Mundorff, N Trinh, DA Kochrekar, RN Cherat, and GG Pai. Development of a Biocatalytic Process as an Alternative to the (−)-DIP-Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast. Org. Process Res. Dev., 2010, 14 (1), pp 193–198.

[8] A very nice range of cofactor recycling enzymes can be acquired from Evocatal in Germany. http://www.evocatal.com/en/products/evozymes/cofactor-regeneration.html. Industrial scale quantities of GDH can be acquired from Codexis. FDH has decreased in popularity because of lack of robustness under process conditions although it was the basis for Degussa’s process for tert-Leucine. PTDH is still experimental.

[9] See reference 4, but there are many other examples.

[10] Pioneering work was done by my friend Vladimir Tishkov from Lomonosov University in Moscow to engineer NADP specificity in an NAD-dependent FDH and reviewed in a nice overview: http://www.enzyme.chem.msu.ru/~tishkov/Publications/Proteins_19_FDH.pdf

[11] TW Johannes, RD Woodyer, H Zhao. Efficient Regeneration of NADPH Using an Engineered Phosphite Dehydrogenase. Biotech. Bioeng. 2007, 96 (1), pp 18-26. http://scs.illinois.edu/~zhaogrp/publications/HZ53.pdf and also their nice review of cofactor recycle systems: http://www.scs.illinois.edu/~zhaogrp/publications/HZ21.pdf

[12] http://www.sprintechnologies.com or through their US distributor: http://www.itochu-purification.com/products/biocatalysis/new-immobilized-ketoreductases

[13] K Engelmark Cassimjee, C Branneby, V Abedi, A Wells, and P Berglund. Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration. Chem. Commun., 2010, 46, pp 5569-5571.

[14] MD Truppo, JD Rozzell, and NJ Turner. Efficient Production of Enantiomerically Pure Chiral Amines at Concentrations of 50 g/L Using Transaminases .Organic Process Research & Development 2010, 14(1), pp. 234-237.

[15] MD Truppo, JD Rozzell, JC Moore, and NJ Turner. Rapid screening and scale-up of transaminase catalysed reactions. Org. Biomol. Chem. 2009, 7, pp 395-398.

[16]M Höhne, S Kühl, K Robins, UT Bornscheuer. Efficient Asymmetric  Synthesis of Chiral Amines by Combining Transaminase and Pyruvate Decarboxylase. ChemBioChem 2008, 9(3), pp 363–365.

[17] JH lutje Spelberg, LX Tang, EJ de Vries, RM Kellogg, and DB Janssen. Biocatalytic preparation of optically pure epoxides and derivatives. Poster presentation at Biotrans 2003, Olomouc/CZ.