Part 1 - How many catalysts for ketone reductions do you need?

Introduction

Let’s start off with a simple question. If you already have a bicycle and you want to go faster on it, wouldn’t it be easier to just switch out some gears and maybe get better handle bars instead of finding all the new and improved parts that you need to build an entire bike?

It is exactly like that in current-day biocatalysis: it is much easier to modify an existing enzyme scaffold to enable a reaction to the exact specifications than it is to find a new enzyme in Nature.

Results

In the figure 1 below, I show six examples of products of ketone reduction with mutants of one and the same ketoreductase enzyme (KRED). This particular KRED has been studied for years now and all the relevant issues have been dealt with: manufacturability at scale, inherent enzyme stability, screening data is available, etc. In other words “we have a pretty good bicycle”. To achieve the results that are summarized in the table, protein engineering was applied (‘directed evolution’) to modify the active site for substrate-fit and selectivity, much like in changing out the gears on a bicycle.

Figure 1: One KRED catalyst adapted for six processes.[1-5]

Table 1: Summary process descriptions. The alcohols had an e.e.>99% (or d.e. for penem’s) in all cases and the yields were >95% of theoretical in less than 24 hrs.

Discussion

I hope you will agree that it is pretty impressive that this enzyme’s active site is so flexible that mutagenesis allows it to accept a large molecule like the Montelukast intermediate but also a tiny, almost symmetrical molecule like the Sulopenem intermediate.

Some more conclusions:

• Substrate loading is always higher than 100 g/L (10%), even up to 36% without loss of productivity.
• Temperature is relatively mild (but can be high if required), as is the pH.
• Use of co-solvents is fine, even up to 90% 2-propanol (iPA).

It is results like the above that, already in 2007 led Merck researchers to write “Isolated enzymes have clearly supplanted whole cell bioreductions and in most instances chemo-catalytic ketones reductions at Merck.”[6]

Conclusion

Now, the big question that remains is “is this one catalyst, or are these 6 different ones?”. Of course, every enzyme variant is a specific unique chemical entity and as such it is a different catalyst. However, keep in mind that these catalysts are more than 90% identical, so humor me, and agree that you only need one catalyst :-). Let me know what you think!

References

[1] From my webinar hosted together with process chemists from Merck, Oct 2010. 21st Century Biocatalysis: an easy-to-use precision tool for every Process Chemists’ toolbox. http://www.icis.com/Webcast.htm

[2] For a nice general paper on KREDs: Gjalt W Huisman, Jack Liang, and Anke Krebber

Practical chiral alcohol manufacture using ketoreductases
Current Opinion in Chemical Biology, Volume 14, Issue 2, April 2010, Pages 122-129

[3] For Sulopenem: Jack Liang , Emily Mundorff, Rama Voladri, Stephan Jenne, Lynne Gilson, Aaron Conway , Anke Krebber , John Wong, Gjalt Huisman, Susan Truesdell, and James 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

[4] For Montelukast: Jack Liang, James Lalonde, Birthe Borup, Vesna Mitchell, Emily Mundorff, Na Trinh, D. A. Kochrekar, Ramachandran Nair Cherat, and G. Ganesh 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

[5] Patent numbers are shown in the figure.

[6] Jeffrey C. Moore, David J. Pollard, Birgit Kosjek, and Paul N. Devine

Advances in the Enzymatic Reduction of Ketones

Acc. Chem. Res. 2007, 40, 1412–1419

This is from their conclusions:” The substrate range and enantioselectivity for ketone reductions are excellent, providing high ee of either alcohol on the majority of ketone substrates. The enzymes also demonstrate valuable chemoselectivity and diastereoselectivity as described on the para-diketone 10 and several other substrates. The enzymes can be screened and scaled-up as rapidly as their chemocatalytic counterparts, and their cost to use at large scale and the environmental impact of their use is less. They have been used at Merck to economically deliver kilogram quantities of chiral intermediates with excellent yields and ee values. As a result, ketoreductases are the preferred catalyst for ketone reductions at Merck.”.