Part 5 - Chirality, enantiomers, and enzyme enantioselectivity

Dear readers,

In the first month I had more than 500 page views from all over the World. I thank you all for your interest and hope you'll come back every now and then to enjoy some more.

Introduction

People seem to like my car analogies, so here comes another one. Enantiomers are mirror images of each other. In Figure 1A, you can see two cars. One is the left-hand drive (LHD) version and the other one is the opposite, right-hand drive (RHD). So, as long as we can tell which is the front of the car, by looking at where the steering wheel is placed we can say whether it is the English version (RHD) or the continental (LHD). Aerodynamic design conventions make it rather easy to determine what the front end of a car is, except for weird and failed exercises like in Figure 1B. This Zündapp model aptly names Janus shown here is an (almost) symmetrical car and consumers could not make heads or tails out of it. 

Figure 1: Cars as enantiomers[1].

How an enzyme treats enantiomers is like how a Brit would drive a continental rental car: the stick shift is on the wrong side and if feels uncomfortable enough to have to drive slower than your regular mainland European citizen. Of course in this analogy, the Brit is the non-preferred substrate, the stick is the catalytically active amino acid and the speed is the catalytic rate. Changing the enantioselectivity of an enzyme by protein engineering is like converting a car from LHD to RHD.

Are there gradations in chirality; is one molecule more chiral than another?[2]

A carbon atom with four different substituents is (at least somewhat) tetrahedral[3] and chiral since the groups can be arranged on the central atom in two mirror-image fashions.[4] In Figure 2A, I show three different chiral molecules; they all have four different substituents and I think most people will be inclined to say that molecule 1 is the ‘most chiral’ since the substituents differ the most, close to the central C-atom. In Figure 2B, an example where the discriminator (chlorine) is far away, yet the carbon atom has four different groups and the molecule is chiral. Valiant attempts at developing mathematical tools to quantify chirality have been made. The jury is still out as far as I’m concerned and I have listed two references to get you started [2]. Two thoughts occur to me: how do you make them and how do you analyze them? This is beyond the scope of this story, though.

Figure 2: A1, chloroethylbenzene; A2, deuterium (²H) and tritium (³H) labeled groups leading to chirality; A3, tetra-alkyl methane; B, (S)-4-chloro-diphenylmethanol.

Enzymes and enantioselectivity

Traditionally, an enzyme has been regarded as a lock to which only one of the enantiomeric keys would fit. Later, this model was modified to incorporate enzyme flexibility and became a glove that only fits one hand. I will use a few examples to show that enantioselectivity is even more subtle than that.

Example 1: Ketoreductase (KRED) reduction of ketothiolane (Figure 3A) and 4-chloro-benzophenone (Figure 3B).

A KRED enzyme uses so-called ‘three point interaction’ to orient the substrate in the active site. In this case, the ketone oxygen forms a hydrogen bond to a group in the enzyme that always locks it into place. Then, there are two sub-sites in the active site that need to accommodate the two groups flanking the ketone. Once the substrate molecule is bound in this preferred way (Figure 3A/B), the enzyme makes sure that the reducing hydride always comes from, say, above the plane of the paper/screen. In this way, only one enantiomer of the alcohol product is formed.

There are two 'flipped' alternative binding modes that can cause imperfect enantioselectivity[5] and they are shown in Figure 3C. Since the reduction of ketothiolane is rather temperature dependent, it is likely that enzyme flexibility is a factor that allows the substrate to occupy the alternative binding mode. At higher temperatures, the enzyme cannot distinguish what is ‘left’ and ‘right’. In the case of 4-chloro-benzophenone, it is more likely that the hydrogen bond is broken and that the ketone group exposes the other side (or ‘face’), leading to the opposite enantiomer.

Should one want to improve the selectivity of the KRED enzyme, in the case of ketothiolane one would want to focus on improving rigidity of the enzyme active site, whereas in the benzophenone case it seems more rewarding to strengthen the hydrogen bond to keep the ketone properly oriented. Thus, a detailed understanding of the root cause of lack of selectivity can guide a mutagenesis approach.

            By the way, there are KREDs that achieve >99% e.e. of these alcohols so it can be done! [6]

Figure 3: Some very basic active site models (A and B) and two alternative binding modes that lead to the opposite product-enantiomer (C).

Example 2: Epoxide hydrolysis by epoxide hydrolases.

Enantioselective hydrolysis of styrene oxide to phenylethane diol and its catalytic mechanism has been described by several groups. [7] A crude active site model is shown in Figure 4. A simplistic explanation for enantioselectivity in this case can be that with the non-preferred enantiomer, the catalytically active aspartate ‘cannot reach’ to the epoxide ring (indicated by the dotted line in Figure 4B) and no reaction takes place.

Figure 4: Side view (A) and top view (B). Note that only the enzyme-substrate alkylation step is shown because it is this step that determines the stereochemical outcome. In the following step, the covalent enzyme-substrate intermediate is simply hydrolyzed to retrieve the enzyme and release the diol.

This model can also explain a rather nice phenomenon that is often observed in epoxide hydrolysis: enantioconvergence. This happens when one substrate-enantiomer reacts on the terminal β-position with retention of configuration and the other substrate-enantiomer moves out of the binding pocket just a bit to allow reaction at the benzylic α-position with inversion of configuration. The result is going from a racemic epoxide to 100% enantiomeric excess (e.e.) of the diol in 100% yield.

Figure 5: Enantioconvergence rationalized.

X-Ray crystallography and computational chemistry can certainly aid in finding favorable binding modes. An example that I am familiar with: crystal structure elucidation of haloalcohol dehalogenases enzymes with substrate enantiomers in the active site showed how non-productive binding of the ‘wrong enantiomer’ occurred in the crystal. [8] I would also like to point to the impressive body of quantum chemical computational work done by Kathrin Hopmann on epoxide hydrolases and haloalcohol dehalogenases that present compelling evidence for the origins of the regioselectivity. [9]

Now, these simple cartoon models are exactly that: simple. They ignore kinetic parameters (rates and affinity constants) so the next two examples will show how kinetics can determine enantioselectivity.

Example 3: Epoxide hydrolysis by epoxide hydrolases (yes, again).

In Figure 6, two reactions catalyzed by EH from Agrobacterium radiobacter are shown. [10] In panel A, we see a ‘normal’ kinetic behavior: the most reactive enantiomer (R)-Cl-SO's concentration decreases rapidly. After 20 minutes, the remaining (S)-enantiomer is hydrolyzed at a slower pace. In panel B, we see that a slight modification to the substrate structure gives a completely different picture. In this case, the enantiomer that binds best is hydrolyzed preferentially but at a slow pace. After 50 minutes, when (R)-SO is gone, the second ‘gets its chance to bind’ and is hydrolyzed very rapidly and follows suit in just 10 minutes more. Well, you all know how I like comics and cartoons, but here a cartoon model is clearly inadequate at explaining this phenomenon.

Figure 6: Hydrolysis of 4-chlorostyrene oxide (A, Cl-SO) and styrene oxide (B, SO).

Example 4: Switch of enantioselectivity of Subtilisin Carlsberg catalyzed transesterifications in organic solvent due to additives.

The effect of co-solvents and additives on the reaction rates of (L) and (D) phenylalanine derivatives in cyclohexane was studied (Figures 7 and 8, [11]). It turned out that the effect on the reaction rates of the (L)-enantiomer was different than on the (D)-enantiomer. This spurred a few years of debate between Alex Klibanov and Jaap Broos about water activity, enzyme flexibility and conformational change. Anyway, the sum of the effects is that in some cases the enzyme is S-selective and in some cases R-selective for the same reaction, without mutagenesis. I remember a Friday afternoon discussion I had with Jaap about the rate limiting step in this reaction. It could be that with some additives the acylation is rate limiting whereas in the presence of other additives de-acylation is rate limiting. If these two reaction steps proceed with different enantioselectivity, this could give an opposite stereochemical outcome. A detailed study into the kinetics of each individual reaction step should give the answer but I don’t think that was ever done.

Figure 7: Subtilisin catalyzed transesterification.

Figure 8: Effect of additives on the reaction rates (V₀) of the individual enantiomers (V₀(L) and V₀(D)). Additives used were entry 1: ethanol; 2: acetonitrile; 3: 1-propanol; 4: propionitrile; 5: butyronitrile; 6: no additive; 7: tert-butanol; 8: 2-methyl-2-pentanol; 9: 3-methyl-3-pentanol; 10: 2-methyl-2-butanol.

Conclusions

Successfully predicting whether an enzyme will be R-selective or S-selective in a certain reaction is very hard to do. Even the best crystal structure with the highest resolution, into which substrate or products are soaked or computer docked will only give an indication. Granted, computational chemistry has come a long way from the days of “you tell me the answer and I’ll calculate it for you”[12] but I challenge this readership to show me a case where a computational prediction of enantioselectivity was accurate (sign and magnitude). I think I have shown that even a rather simple extrapolation of results from one molecule to an analog or from one reaction condition to another is tricky.

Fortunately, it is not difficult to just measure the enantioselectivity. (Semi-) random mutagenesis is very powerful and rather agnostic of kinetic parameters and has been used quite often to improve or even really switch enantioselectivity.

References

[1] A: The air intake on the rear fender indicates that this is a rear-engine car (think Porsche or VW bugs) so the placement of the engine is not indicative of where the front is. Likewise with which wheels are driven by the transmission (FWD vs. RWD). The best indicator is looking at which wheels move when you turn the steering wheel (unless you’re looking at a fork lift) and where the mirrors are positioned. B: The Janus’s fenders have a cut-out to allow wheels to turn corners, so the right hand side of the car in the picture is the front. And while we’re at it, the Janus has an internal mirror plane and could be called a ‘micro car’ as well as a ‘meso car’.           http://en.wikipedia.org/wiki/Zündapp_Janus

[2] PW Fowler. Quantification of chirality: attempting the impossible. Symmetry: Culture and Science 2005, 16(4), pp 321-334 (http://symmetry.hu/content/fowler-05-4.pdf). And then have a look at this nice presentation from N Duncan-Gould. Recent advances in quantifying chirality. 2008. http://www.scs.illinois.edu/denmark/presentations/2008/gm-2008-12_9.pdf

[3] Winner of the first-ever Nobel prize for Chemistry, oddly enough not for the tetrahedral carbon configuration. http://en.wikipedia.org/wiki/Jacobus_Henricus_van_'t_Hoff and look at http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1901/

And look this paper full of definitions: K Mislow, and J Siegel. Stereoisomerism and Local Chirality. J. Am. Chem. Soc. 1984, 106 (11) pp 3319-3328 (https://www.uzh.ch/oci/efiles/OCVII/ja00323a043.pdf)

[4] I’d like to take this opportunity to highlight the work of Dr. Wolter ten Hoeve who in his thesis with the best title ever (“The long and winding road to planar carbon”) describes the synthesis of molecule 3 from Figure 2A in 80% enantiomeric excess, as well as efforts at making a carbon atom with four substituents that is mostly flat. Check out the (Dutch) summary: http://dissertations.ub.rug.nl/FILES/faculties/science/1979/w.ten.hove/Planar_carbon0001.PDF.

Ten Hoeve worked in the group of stereochemistry pioneer Prof Hans Wynberg, an extraordinary man that I had the pleasure to meet frequently. You have to check out these links to get a glimmer: http://en.wikipedia.org/wiki/Frederick_Mayer_(spy)#Hans_Wynberg and the obituary that does him justice: http://www.grinnell.edu/offices/communications/magazine/extras/hans-wynberg

[5] I know the term enantioselectivity is wrong, but most people call it like this.

[6] J Liang, E Mundorff, R Voladri, S Jenne, L Gilson, A Conway, A Krebber , J Wong, G 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; MD Truppo, D Pollard, and P Devine. Enzyme-Catalyzed Enantioselective Diaryl Ketone Reductions. Org. Lett., 2007, 9 (2), pp 335–338.

[7] A few come to mind from the EPOX project: Dick Janssen, Roland Furstoss, Michael (Ernie) Arand, Manfred Reetz, but there are many, many more. For the main catalytic mechanisms, just Google “catalytic mechanism epoxide hydrolase”.

[8] RM de Jong (LI: nl.linkedin.com/pub/rené-de-jong/29/297/3b2), JJW Tiesinga, A Villa, LX Tang, DB Janssen, and BW Dijkstra. Structural Basis for the Enantioselectivity of an Epoxide Ring Opening Reaction Catalyzed by Halo Alcohol Dehalogenase HheC. J. Am. Chem. Soc., 2005, 127 (38), pp 13338-13343. http://gbb.eldoc.ub.rug.nl/FILES/root/2005/JAmChemSocdeJong/2005JAmChemSocdeJong.pdf

[9] KH Hopmann (LI: www.linkedin.com/pub/kathrin-hopmann/9/966/a12). Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanism and Selectivities. KTH, Stockholm, Doctoral thesis, 2008. 

http://kth.diva-portal.org/smash/record.jsf?pid=diva2:13342

[10] JH lutje Spelberg, R Rink, RM Kellogg, and DB Janssen. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter. Tetrahedron: Asymmetry 1998, 9 (3), pp 459–466

[11] J Broos, JFJ Engbersen, IK Sakodinskaya, W Verboom, and DN Reinhoudt. Activity and enantioselectivity of serine proteases in transesterification reactions in organic media. J. Chem. Soc., Perkin Trans. 1, 1995, pp 2899-2905. (http://pac.iupac.org/publications/pac/pdf/1996/pdf/6811x2171.pdf ) and some of the follow-up discussions are summarized here: J Broos. Impact of the Enzyme Flexibility on the Enzyme Enantioselectivity in Organic Media Towards Specific and Non-specific Substrates. Biocatalysis and Biotransformation, 2002, 20 (4), pp. 291–295. (http://gbb.eldoc.ub.rug.nl/FILES/root/2002/BiocatBiotrBroos/2002BiocatalBiotransfBroos.pdf)

[12] I had to follow a course on thermodynamics given by Herman Berendsen while he and his group were writing the GROMOS software and I seemed to hear that joke a lot from them.