Biocatalyzed transformations

This chapter describes, to the best of author s knowledge, the state of the art in the enzymatic chemo- and regioselective acylaton of polyfunchonalized compounds at the end of2006. Twenty years after the first reports by Klibanov s group, these peculiar biocatalyzed transformations have shown an unexpected efficiency and versatility. 

Fig. 35. Light-induced biocatalyzed transformations utilizing photochemically regenerated NAD(P)H cofactors...

FIG U RE 17.6 Nitrilase biocatalyzed transformation of racemic (i /5)-mandelonitrile for the production of (i )-mandelic acid. 

Nitrile Hydratase/Amidase Biocatalyzed Transformation of 3-Hydroxy-4-Aryloxybutanenitriles and 3-Hydroxy-3-Arylpropanenitriles... 

The photochemical carboxylation of pyruvic acid by this process is endergonic by about AG° = 11.5 kcal mol and represents a true uphill photosynthetic pathway. The carbon dioxide fixation product can then act as the source substrate for subsequent biocatalyzed transformations. For example, photogenerated malic acid can act as the source substrate for aspartic acid (Figure 35). In this case, malic acid is dehydrated by fumarase (Fum) and the intermediate fumaric acid is aminated in the presence of aspartase (Asp) to give aspartic acid. 

For practical photoinduced synthetic biocatalyzed transformations, it is important to integrate biocatalysts in immobilized matrices that allow the recycling of the photosystems. The fact that bipyridinium sites act as electron mediators for various redox enzymes was used to develop two paradigms for the electrical contacting and photoactivation of the biocatalyst (Figure 39). By one approach, the bipyridinium electron relays are tethered by covalent bonds to the protein backbone (Figure 39A). These electron relays act as oxidative quenchers of the excited state of the dye and, upon photoreduction of the electron acceptor units, they act as electron carriers that activate the reductive functions of the enzyme. As an example, the... 

Figure 39. Electrical communication between an enzyme redox center and a photoexcited species attaining light-induced biocatalyzed transformations (A) direct electrical wiring of the protein by its chemical modification with tethered electron-relay units (B) electrical communication by the immobilization of the protein into a redox-functionalized polymer matrix.

By the second approach, the enzyme is immobilized in a redox polymer assembly (Figure 39B). Electron-transfer quenching of the photosensitizer by the polymer matrix generates an electron pool for the activation of the enzyme. Photoreduction of nitrate to nitrite was accomplished by the physical encapsulation of NitraR in a redox-functionalized 4,4 -bipyridinium acrylamide copolymer [234]. In this photosystem, Ru(bpy)3 + was used as a photosensitizer and EDTA as a sacrificial electron donor. Oxidation of the excited photosensitizer results in electron transfer to the redox polymer, and the redox sites on the polymer mediate further electron transfer to the enzyme redox center, where the biocatalyzed transformation occurs. The rate constant for the MET from the redox polymer functionalities to the enzyme active site is — (9 + 3) x 10 s. Similarly, the enzyme glutathione reductase was electrically wired by interacting the enzyme with a redox polymer composed of polylysine modified with A-methyl-A -carboxyalkyl-4,4 -bipyridinium. The photosensitized reduction of oxidized glutathione (GSSG) (Eq. 21) ... 

Xiao, Y., Pavlov, V., Levine, S., Niazov, T., Markovich, G., and Willner, I., Catalytic growth of Au-nanoparticles by NAD(P)H cofactors optical sensors for NAD(P) -dependent biocatalyzed transformations, Angew. Chem. Int. Ed., 43, 4519M 522, 2004. 

Figure 19. Photochemical-biocatalyzed C02-fixation processes and secondary biocatalyzed transformations using the carboxylation products by primary light-induced regeneration of NADPH.

Figure 24. Photosensitized biocatalyzed transformations in electrically communicated redox functionalized polymers (a) reduction of nitrate in a bipyridinium-acrylamide-cross-linked polymer (b) reduction of oxidized glutathione using a redox functionalized bipyridinium-polylysine polymer.

Figure 4 Schematic amplified analysis of a target DNA using (A) An oligonucleotide-enzyme conjugate and a biocatalyzed transformation as amplification route. (B) An oligonucleotide-functionaUzed particle (liposome or nanoparticle) as an amplifying unit.

Various biocatalyzed transformations induce the polymerization of a thin film [37], or the precipitation of an insoluble product [38,39] on the transducer, leading to the electrode support insulation (increase of electrode resistance and decrease of interfacial electron-transfer) or to an increase in the mass associated with a... 

Figure 8 Biocatalyzed transformations leading to the precipitation of insoluble products on the transducer interfaces.

V. BIOCATALYZED TRANSFORMATIONS ON NUCLEIC ACID-FUNCTIONALIZED SURFACES... 

Different biocatalyzed transformations proceed with single-stranded or double-stranded DNA assanbhes. The Ugation of nucleic acids, replication of double-stranded assemblies, or the sequence-specified scission of the double-stranded system by specific endonucleases are a few representative examples. Bioelectro-catalytic transformations occurring on surfaces provide additional nanometric tools to manipulate and nanoengineer the nucleic acid surfaces and to amplify the sensing processes by the replication route. The different electronic transduction means that follow the surface functionalization by DNA may then be applied to follow the biocatalyzed transformations occurring on the nucleic acid-functionalized supports. 

A series of biocatalyzed transformations involving nucleic acids that include the surface-stimulated ligation, replication, and specific scission of nucleic acids by a restriction enzyme were transduced electronically using faradaic impedance and QCM transduction tools [47]. 

Fig. 20 Bioelectrocatalytic reduction systems using (a) AlcDH and FNR for the biocatalyzed transformation and the regeneration of NADPH, respectively, and (b) using AlcDH for both processes, where the biocatalytic cycle Substrate 1/Product 1 performs the mediating function for the NADPH regeneration and the second biocatalytic cycle results in the formation of the aim product 2.

Recently, this strategy was applied to the deracemization of propargylic alcohols that are important synthons for the preparation of biologically active compounds such as mifepristone, efavirenz, or petrosynol [63]. A one-pot two-step process employing whole cells from Candida parapsilosis ATCC 7330 was carried out in aqueous medium using short reaction times of 1—4h (Scheme 4.15). Biocatalyzed transformations afforded excellent enantiomeric excess (up to 99%) and isolated yields were from 60-81%. 

This book is intended to describe the state of the art of these efforts for the advantage of both the academic and the industrial audience. The book will focus on the current available toolbox of biocatalyzed reductions of C=0, C=C, and formal C=N double bonds, in order to show (i) which are the reliable biocatalyzed transformations to be used by organic chemists involved in the development of manufacturing processes, and (ii) which are the biotransformations still requiring improvements and investigations. Bioreductions have been chosen as the main topic of the book, because of their widespread applications in organic synthesis and their versatility in the creation of stereogenic centers in chiral molecules. 

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