Caroline E. Paul

Mimicking nature: synthetic nicotinamide cofactors for C═C bioreduction using enoate reductases.

A series of synthetic nicotinamide cofactors were synthesized to replace natural nicotinamide cofactors and promote enoate reductase (ER) catalyzed reactions without compromising the activity or stereoselectivity of the bioreduction process. Conversions and enantioselectivities of >99% were obtained for C═C bioreductions, and the process was successfully upscaled. Furthermore, high chemoselectivity was observed when employing these nicotinamide cofactor mimics (mNADs) with crude extracts in ER-catalyzed reactions.

Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes

The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the “ene” reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. “Better-than-Nature” biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost.

Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong alpha-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M(-1)) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 degrees C), were determined. The half-lives of the conjugates varied between 3h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly.

In situ formation of H2O2 for P450 peroxygenases

An in situ H2O2 generation approach to promote P450 peroxygenases catalysis was developed through the use of the nicotinamide cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) and flavin mononucleotide (FMN). Final productivity could be enhanced due to higher enzyme stability at low H2O2 concentrations. The H2O2 generation represented the rate-limiting step, however it could be easily controlled by varying both FMN and BNAH concentrations. Further characterization can result in an optimized ratio of FMN/BNAH/O2/biocatalyst enabling high reaction rates while minimizing H2O2-related inactivation of the enzyme.

Recombinant Cyanobacteria for the Asymmetric Reduction of C=C Bonds Fueled by the Biocatalytic Oxidation of Water

A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves the atom efficiency and avoids the formation of undesired side products. Crucial factors for product formation are the availability of NADPH and the amount of active enzyme in the cells. The efficiency of the reaction is comparable to typical whole-cell biotransformations in E. coli. Under optimized conditions, a solution of 100 mg prochiral 2-methylmaleimide was reduced to optically pure 2-methylsuccinimide (99 % ee, 80 % yield of isolated product). High product yields and excellent optical purities demonstrate the synthetic usefulness of light-catalyzed whole-cell biotransformations using recombinant cyanobacteria.

Escherichia coli/ADH‐A: An All‐Inclusive Catalyst for the Selective Biooxidation and Deracemisation of Secondary Alcohols

The nicotinamide adenine dinucleotide regeneration system present in Escherichia coli cells was exploited for the oxidation and deracemisation of secondary alcohols with the overexpressed alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (E. coli/ADH‐A). Thus, various racemic alcohols were selectively oxidised with lyophilised or resting E. coli/ADH‐A cells without need for an external cofactor or co‐substrate. The addition of these substrates to the E. coli/ADH‐A cells in buffer afforded the corresponding ketones and the remaining enantioenriched (R)‐alcohols. This methodology was used for the desymmetrisation of a meso‐diol and for the synthesis of the highly valuable raspberry ketone. Moreover, a biocatalytic concurrent process was developed with the resting cells of E. coli/ADH‐A, ADH from Lactobacillus brevis, and glucose dehydrogenase for the deracemisation of various secondary alcohols, which afforded the desired enantiopure alcohols in more than 99 % ee starting from the racemic mixture. The reaction time of deracemisation of 1‐phenylethanol was estimated to be less than 30 min. The stereoinversion of (S)‐1‐phenylethanol to its pure (R)‐enantiomer was also achieved, which provided a biocatalytic alternative to the chemical Mitsunobu inversion reaction.

Cofactor-Free, Direct Photoactivation of Enoate Reductases for the Asymmetric Reduction of C=C Bonds

Abstract Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans‐hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light‐driven activation of OYEs through NAD(P)H‐free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light‐emitting‐diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2‐methylcyclohexenone resulted in enantiopure (ee>99 %) (R)‐2‐methylcyclohexanone with conversion yields as high as 80–90 %. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.

A survey of synthetic nicotinamide cofactors in enzymatic processes

Synthetic nicotinamide cofactors are analogues of the natural cofactors used by oxidoreductases as redox intermediates. Their ability to be fine-tuned makes these biomimetics an attractive alternative to the natural cofactors in terms of stability, reactivity, and cost. The following mini-review focuses on the current state of the art of those biomimetics in enzymatic processes.

Functional characterization and stability improvement of a 'thermophilic-like' ene-reductase from Rhodococcus opacus 1CP.

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

Chemoenzymatic preparation of optically active 3-(1H-imidazol-1-yl)cyclohexanol-based ionic liquids: application in organocatalysis and toxicity studies

A straightforward and robust modular synthetic approach was developed for the asymmetric synthesis of imidazolium salts, in which several engineered molecular vectors were considered to evaluate their toxicological profile. The diastereoselective synthesis of cis-3-(1H-imidazol-1-yl)cyclohexanol was achieved by the Michael addition of imidazole to cyclohex-2-en-1-one under microwave conditions followed by reduction of the ketone. The racemic cis-alcohol obtained was successfully resolved through a lipase-catalysed kinetic resolution, Pseudomonas cepacia lipase proved to be a good biocatalyst for the exclusive acetylation of the (1R,3S)-cis enantiomer. Using the remaining (1S,3R)-cis alcohol enantiomer as a synthetic precursor, the optically active (1R,3R)-trans alcohol enantiomer was also prepared. The corresponding chiral salts and ionic liquids were obtained via quaternisation with alkyl halides, followed by anion exchange with inorganic salts. In this manner, a family of novel imidazolium-based ionic liquids was prepared, and their properties as phase-transfer catalysts in the Michael addition of diethyl malonate to trans-chalcone were analysed. The toxicity of these compounds against E. coli cells was also evaluated to understand their structural implications through the presented systematic synthetic approach.