Part 6 – Multi-step enzymatic reactions

Introduction

Most chemists are inherently lazy and are always looking for short-cuts and ‘quick and dirty’ by doing as much as possible in one pot or by skipping product isolation after each step. This mindset is now part of what is called “process intensification”, a push toward higher efficiency in chemical processes.  I found a nice definition/explanation on the web[1]:

The objective of process intensification is to enable a process to achieve its maximal chemical or catalytic performance asymptote free of extraneous transport resistances and dispersive statistical phenomena, using minimal physical and financial resources. Crucial aspects of process intensification are well-defined operating conditions and its integrative approach, i.e. considering the overall process objectives rather than the isolated performance of individual unit operations. Due to tight operating windows, loss of degrees of freedom and nonlinear behaviour, integrated processes pose challenging control problems.

This blog issue will deal with sequential, multistep, reactions that require ‘action’, i.e. they do not happen spontaneously. This opposed to ‘cascade reactions’ or ‘domino reactions’ that require an (enzyme-catalyzed) initiating step that sets off a sequence of events. I once heard Nobel laureate Kyriacos Costa “KC” Nicolaou speak and I remember the most fantastic reaction schemes that I’m sure will impress you too and I am happy to include a reference to a review on cascades by his hand that also has some of his work.[2] Since most people use ‘cascades’ rather loosely, I will stick to that terminology as well.

Microorganisms are really good at cascades. Metabolic pathways tend to have high efficiency due to feedback controls that maintain a low concentration of irrelevant intermediates, all under relatively constant reaction conditions[3]. Also, if reactions are not compatible, the organism can perform them in different compartments (organelles). All in all pretty neat and worth trying to mimic in the lab!

I hope the following examples will give some  background and maybe some good ideas.

One enzyme, multiple substrates

Deoxyribose-phosphate aldolase (DERA)

This enzyme stitches together three molecules of aldehyde: chloroacetaldehyde to acetaldehyde to acetaldehyde. This cascade is the basis for DSM’s Atorvastatin-intermediate synthesis.[4]

Figure 1: Very chemo- and enantioselective coupling of several aldehydes by DERA. 

Polyketide synthases (PKS) and fatty acid biosynthesis (FAS)

The enzyme or enzyme complex stitches together a number of acetyl-CoA and malonyl-CoA molecules (for polyketides, [5]) (for fatty acids, [6]) to achieve the desired chain length.

Figure 2: The polyketide synthase enzyme complex couples an exact number of C2 fragments.

Haloalcohol dehalogenase (HHDH)

This enzyme can catalyze the reversible ring closure of a haloalcohol to give an epoxide. It can also perform ring-opening reactions with non-halogen nucleophiles. This is the basis for Codexis’ route to an Atorvastatin-intermediate.[7]

Figure 3: Ring closure and opening, catalyzed by HHDH. The epoxide is not isolated.

Cytochrome P450 oxidations

Cytochrome P450 enzymes can oxidize substrates leading to hydroxylation and other metabolism. In the case of multiple oxidations, it may happen that the product has not left the active site in-between reactions although in most cases it is assumed that it did. Measuring the time course of dihydroxylated product will show whether it follows the formation of mono-hydroxylated product or is simultaneous. In the metabolism of buspirone, changing the reaction conditions led to less di-hydroxylation so that would indicate a release of the product and a true two-step process.[8]

Figure 4: Cytochrome P450 oxidation of buspirone. Dihydroxylation can follow two pathways, each catalyzed by the same enzyme.

Multiple enzymes, one substrate

If you have a molecule with two functional groups that you both want to convert with enzymes, why not do all the enzymes in one pot and convert both groups? Although in principle this seems like a rather obvious thing to try, I’ll explain that this is deceptive. I think most people got it since I could not really find any good examples for this in half an hour. There is one report of reduction and hydrolysis of keto-esters but it does not specify whether ester hydrolysis is enzymatically catalyzed[9]. The resulting molecules are all reduced, leading me to believe that the sequence of events does not make a difference.

Figure 5: Two functional groups, two enzymes. What can be simpler?

Theoretically, this concept can be applied to any molecule with different functional groups for the respective enzymes to work on. However, there is a high risk of channeling the reactions towards a slow-reacting intermediate and this concept only works well when all four reactions (in the example above) proceed at the same rate. It is unlikely that the ester will have the same k_cat and K_m as the acid, for instance. Therefore there is a good chance of having a severe bottleneck and a two-step approach is almost always better.

Let’s explain this by a simplified example. Assume we have two pathways from S --> P, with intermediates A and B. Further, assume that the rates are as listed in the figure, e.g. S --> A proceeds at 100/hr and the conversion of A --> P is much slower, at 5/hr. The upper route proceeds at an overall rate of 5/hr and thus intermediate A accumulates. Similarly, the lower route is at 25/hr and because the second step is so much faster, B has only a very low transient concentration.

When we measure the formation of P through both routes, it occurs at 30/hr. In the beginning of the process everything seems alright: the combination of enzymes gives a higher rate than one could get with the individual enzyme (ignoring inhibition of A or P on the conversion of S --> B in this simplification). Now, let us assume we have 500 molecules of S. In 4 hours, all the S is gone and we have sent 400 molecules through the upper route to A and 100 to B. The B is immediately converted to P as soon as it is formed, but A --> P needs 76 more hrs to complete (380/5).

Now consider using one enzyme first. S --> B can be done in 20 hours, and B --> P takes another 10 hrs, giving an overall process duration of 30 hrs. This is almost three times faster! As an added bonus, it now makes sense to use even more enzyme to convert B --> P, decreasing overall process time even further.

Convinced that there is no efficiency gain when using two enzymes simultaneously?

Figure 6: Simplified numerical example for two pathways. In practice, the rates are not constant and will slow down once the concentration approaches the K_m. Also, depending on the k_cat/K_m for A and S, the rate will not be 25+5, but lower than that.

Multiple enzymes, reaction sequence/pathway

For this concept to be elegant, I would limit this to cases where all the enzymes are present from the start and do not interfere with each other. Just a few examples here and many more in literature if you look for the work of Wolfgang Kroutil and Kurt Faber, both at Graz University, who have been at this for a while.

Nitrilase --> Penicillin acylase

My good friends from Delft have demonstrated the use of a nitrilase and penicillin acylase in antibiotic synthesis[10].

Figure 7: Some nitrilases are stuck in the amide stage and act more like a nitrile hydratase. This property is used in this strategy.

Phytase --> GPO/Catalase --> Aldolase --> Phytase

Conversion of glycerol and an aldehyde (R-CHO) to non-natural carbohydrates, also from Delft [11].

Figure 8: The phytase in step 1 is the same as in step 4 but requires a pH-shift.

KRED --> FDH, or KRED --> GHD, or KRED --> KRED

Technically, all the redox cofactor recycle systems would fall in this category. There are de-symmetrization oxidation-reduction cascades (cycles) that are very useful, as reviewed by folks from the University of Graz. [12]

Figure 9: KRED-FDH redox cycle.

KRED --> HNL --> NR

Jörg Schrittwieser is currently working on developing this sequence that includes a hydroxynitrile lyase and a new nitrile reductase enzyme while in Delft.[13] For the moment, chemical aerobic oxidation is considered but an alcohol oxidase (flavin dependent, with O₂) or a ketoreductase (NAD or NADP dependent) could be used too.

Figure 10: Oxidation – Reduction should use the same cofactor, in this case NADP⁺. There will be an imbalance, though, since the nitrile reduction requires 2 equivalents of hydride.

The challenge

Below is a partly hypothetical sequence that involves nine reaction steps that should not interfere with each other. Parts of this have been proven as shown above and in literature[14] and some of this idea was already posed a while back [15]. I challenge you to email me (devries.erikjan@gmail.com) a longer sequence (but don’t come with “repeat steps 6-9”) and I’ll compile them and will do a follow-up posting with your ideas. Let’s not worry about economic feasibility or technicalities like enzyme selectivity or optimal reaction conditions etcetera since we can change that easily by mutagenesis.

Figure 11: Now, this starts to look like a real pathway.

References

[1] http://www.bci.tu-dortmund.de/en/research/process-intensification

[2] KC Nicolaou, DJ Edmonds, and PG Bulger. Cascade reactions in total synthesis. Angew. Chem. Int. Ed. 2006, 45, pp 7134-7186. (http://medchem.rutgers.edu/mc504/pdfs/cascade_reactions.pdf) and for a review on enzyme-triggered cascades: SF Mayer, W Kroutil, and K Faber. Enzyme-initiated domino (cascade) reactions. Chem. Soc. Rev., 2001, 30, pp 332-339.

 [3] This is probably the best use of enzyme product inhibition that you can imagine. Other feedback controls are regulated cofactor regeneration, or energy/ATP use. No big pH swings are allowed, obviously all reactions occur at the same temperature. http://en.wikipedia.org/wiki/Biochemical_cascade

[4] S Jennewein, M Schürmann, M Wolberg, I Hilker, R Luiten, M Wubbolts, D Mink. Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase. Biotechnol. J. 2006, 1 (5), pp 537-548. WA Greenberg, A Varvak, SR Hanson, K Wong, H Huang, P Chen, and MJ Burk. Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. PNAS 2004, 101 (16), pp 5788-5793. WO2007137816 Process for the preparation of epoxide intermediates for pharmaceutical compounds such as statins. D Mink, JH lutje Spelberg, EJ de Vries, 2007. WO2007128469 Process for the preparation of enantiomerically enriched nitriles. D Mink, JH lutje Spelberg, EJ de Vries, 2007.

And while I was researching for this blog post, I stumbled across the nice thesis by Angela Kinnell from Leeds University: Development of an Organo- and Enzyme-Catalysed Cascade Reaction, 2010. (http://etheses.whiterose.ac.uk/1132/1/Angela_Kinnell_Thesis_Final.pdf).

[5] Although technically this is sometimes one enzyme and sometimes a rather a complex multi-enzyme system that is forming a cluster: http://en.wikipedia.org/wiki/Polyketide_synthase

[6] The FAS principle is very similar to PKS: http://en.wikipedia.org/wiki/Fatty_acid_synthesis

[7] SK Ma, J Gruber, SC Davis, L Newman, D Gray, A Wang, J Grate,  GW Huisman  and RA Sheldon. A green-by-design biocatalytic process for atorvastatin intermediate. Green Chem., 2010, 12, pp 81-86.

[8] M Zhu, W Zhao, H Jimenez, D Zhang, S Yeola, R Dai, N Vachharajani, and J Mitroka. Cytochrome P450 3A- mediated metabolism of buspirone in human liver microsomes. Drug Metabolism and Disposition 2005, 33 (4), pp 500-507. (http://dmd.aspetjournals.org/content/33/4/500.full)

[9] NA Salvi, S Chattopadhyay. Asymmetric reduction of 3-aryl-3-keto esters using Rhizopus species. Bioorganic & Medicinal Chemistry 2006, 14 (14), pp 4918–4922.

[10] MA Wegman, LM van Langen, F van Rantwijk, RA Sheldon. A two-step, one-pot enzymatic synthesis of cephalexin from D-phenylglycine nitrile. Biotechnology and Bioengineering 2002, 79 (3), pp 356–361.

[11] R Schoevaart, F van Rantwijk, RA Sheldon. A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol. J Org Chem. 2000, 65 (21), pp 6940-3.

[12] JH Schrittwieser, J Sattler, V Resch, FG Mutti, and W Kroutil. Recent biocatalytic oxidation–reduction cascades. Curr. Opin. Chem. Biol., 2011, 15 (2), pp 249–256. (http://www.sciencedirect.com/science/article/pii/S1367593110001869)

[13] JH Schrittwieser. Asymmetric Cascade Reactions Incorporating Hydroxynitrile-Lyase Catalysed C–C Bond Formation. Go to http://www.tnw.tudelft.nl/en/ and search for ‘cascade schrittwieser’.

[14] JH Schrittwieser, I Lavandera, B Seisser, B Mautner, and W Kroutil. Biocatalytic Cascade for the Synthesis of Enantiopure β-Azidoalcohols and β-Hydroxynitriles. Eur. J. Org. Chem., 2009, 14, pp 2293–2298. And: B Seisser, I Lavandera, K Faber, JH lutje Spelberg, and W Kroutil. Stereo-Complementary Two-Step Cascades Using a Two-Enzyme System Leading to Enantiopure Epoxides. Adv. Synth. & Catal. 2007, 349 (8-9), pp 1399–1404.

 [15] JH lutje Spelberg, and EJ de Vries. Multi-step biocatalytic synthesis of epoxides & derivatives.  Specialty chemicals online News Article ID: 11764, 23 October 2006. I can email you a copy since it is not available online anymore.