Artificial Transfer Hydrogenase

The asymmetric transfer hydrogenation of ketones is an effective way to prepare enan-tiopure alcohols. We were attracted to this reaction as we anticipated that one could exploit the reversibility of the reaction to perform either for the enantioselective reduction or for the kinetic resolution of racemic alcohols via oxidation. This behaviour is reminiscent of alcohol dehydrogenases which can operate either as oxidases or reductases.  

In the context of transfer-hydrogenation, dialkylketones are challenging substrates as 

Having developed an efficient artificial transfer hydrogenase, we attempted to apply the same methodology to the reverse reaction the kinetic resolution of racemic alcohols. To our disappointment, we were forced to use strong oxidizing agents (eg. f-BuOOH rather than acetone, in the spirit of an Oppenauer-type mechanism) to drive the reaction to completion. We speculate that, in the presence of water, the ruthenium is unable to abstract the j8-hydrogen on the prochiral alcohol. 

Despite the excitanent generated by the X-ray structure, the localization of the biotinylated metal complex within the protein environment was a source of disappointment. Indeed, we anticipated that the active catalyst would be encapsulated within the biotin-binding pocket of Sav rather than on the surface of the protein. To overcome this problem we tested whether the polar residues present within the biotin-binding pocket itself may be capable of binding a polar coordination complex. With this goal in mind, we tested the catalytic potential of vanadyl as a catalyst.  

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Letondor, C., Humbert, N. and Ward, TR. (2005) Artificial metaUoenzymes based on biotin-avidin technology for the enantioselective reduction of ketones by transfer hydrogenation. Proc. Natl. Acad. Sci. U.S.A., 102, 4683-4687 Letondor, C., Pordea, A., Humbert, N., Ivanova, A., Mazurek, S., Novic, M. and Ward, TR. (2006) Artificial transfer hydrogenases based on the biotin-(strept)avidin technology Fine tuning the selectivity by saturation mutagenesis of the host protein. J. Am. Chem. Soc., 128, 8320-8328. 

Letondor C, Pordea A, Humbert N, Ivanova A, Mazurek S, Novic M, Ward TR. Artificial transfer hydrogenases based on the biotin-(strept)avidin technology fine tuning the selectivity by saturation mutagenesis of the host protein. J. Am. Chem. Soc. 2006 128 8320-8328. 

After many unsuccessful attempts, we were fortunate to crystallize the most promising (5)-selective artificial transfer hydrogenase [RuCl(Ti -benzene)(Biot-Vipara-3)](-sii2K Sav (Fig. 7) [57]. Several noteworthy features are apparent from this X-ray stracture ... 

The result of the screening of the immobilized artificial transfer hydrogenases is displayed as a fingerprint in Fig. 9. The most promising results of artificial transfer hydrogenases were subsequently reproduced using the purified non-immobilized... 

Fig. 9 Fingerprint display of the results for the chemogenetic optimization of the reduction of 4-bromo acetophenone and 4-phenyl-2-butanone in the presence of biotin-sepharose-immobihzed artificial transfer hydrogenases [RuH(r -arene)(Biot-VP -3)]cstreptavidin mutant [57]...

A further exciting development in the field of chemoenzymatic one-pot synthesis is the integration of artificial metalloproteins (which can then be regarded as the chemocatalytic component ) in such processes. Such a concept was successfully realized by HoUmann, Turner, and Ward et al. in the combination of an artificial transfer hydrogenase with various redox biocatalysts, comprising NADH-, FAD, and heme-dependent enzymes [47]. A selected example is shown in Scheme 19.18. Therein, readily available L-lysine is oxidized by an L-amino acid oxidase toward Al-piperidine<arboxylic acid (52), which is then reduced by the iridium complex-containing metalloprotein to racemic pipecohc acid (rac-53). 

Cellular processes require orthogonal catalysts, that is, ones that can function unaffected by all the other cell components. Organometallic catalysts often fail to act in concert with enzymes because of mutual inactivation. A Cp Ir(chelate)Cl transfer hydrogenation catalyst has now been successfully incorporated into the protein, streptavidin, as an artificial transfer hydrogenase in order to protect it from deactivation in cooperative catalysis with monoamine oxidases. ... 

Flafner et al first reported an oxidase-catalyzed deracemization method using amino acids as the substrate and pkDAAOx or LAAOx from Crotalus adamanteus together with sodium borohydride as the chemical reductant in 1971 [42]. A procedure for the successful deracemization of amino acids was previously reported by Soda et al. [43]. They focused on proline and pipecolic acid as substrates for the production of L-enantiomer by deracemization because these substrates formed stable imines rather than unfavorable keto acids in water by DAAOx. However, the enzyme was denatured by the chemical reaction with sodium borohydride. Turner et al developed an effective production method for (R)- or (S)-amino acids and (S)-amines by a deracemization method using milder chemical reducing reagents such as sodium cyanoborohydride and artificial transfer hydrogenase [44,45]. 

A second approach created a fusion protein from a PSI subunit (PsaE) and a nickel-iron [NiFe] hydrogenase [12], This new protein was then assembled into a PSI mutant lacking the PsaE subunit. The fused enzymatic system was attached to a gold surface in the same way as the PSn electrode described above using a His-tag on PSI, Ni(n) and NTA functionalities on the surface (Fig. 4a, right side). A soluble electron shuttle was used to transfer electrons from the electrode to PSI. From these two approaches the fusion protein is to date the most effective artificial enzymatic system for photo-driven hydrogen production and the activity is comparable to the electrocatalytic activity of the hydrogenase alone immobilized directly on an electrode. 

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