Reduction Reaction

The reduction of ketones, aldehydes, and olefins has been extensively explored using chemical and biological methods. As the latter method, reduction by heterotrophic microbes has been widely used for the synthesis of chiral alcohols. On the contrary, the use of autotrophic photosynthetic organisms such as plant cell and algae is relatively rare and has not been explored because the method for cultivation is different from that of heterotrophic microbes. Therefore, the investigation using photosynthetic organisms may lead to novel biotransformations.

Indeed, recent research on the use of a cyanobacterium as a biocatalyst has opened up this area: asymmetric reduction of ketones by a cyanobacteria, Syne-chococcus elongates PCC 7942, with the aid of light energy proceeded smoothly

Figure 1: Reduction of ketones by cyanobacteria.

and afforded the corresponding (S)-alcohols in excellent enantioselectivities1-3 (Fig. 1). Characteristics of the reduction reaction using cyanobacteria are as follows:

(a) Biocatalyst/substrate (bis) ratios are low:

A large amount of biocatalyst is usually required to reduce a considerable amount of substrate (the bis for baker's yeast is about 50-350). On the contrary, a low bis ratio (2.6-0.5) could be achieved using the cyanobacteria. The improvement in the bis ratio is caused by the fact that the cyanobacterium can utilize the power of light effectively to reduce the substrate.

(b) The reaction afforded high selectivity and wide substrate specificity: Ketones used in this report are reduced by the cyanobacterium with excellent enan-tioselectivities (> 96% ee). An enzyme exhibiting high enantioselectivity usually shows a relatively strict substrate specificity; hence, there scarcely is a catalyst that reacts with many kinds of substrates and also shows high selectivities. This alga can reduce a wide variety of aryl methyl ketones and afford the corresponding alcohols with high enantioselectivities.

(c) Light energy can be used for reduction:

Reduction of substrates usually requires a large input of energy, and in microbial reductions, carbohydrates such as sugars have been used as the energy source. These carbohydrates are generated through photosynthesis with sunlight energy. In other words, we have been indirectly using light energy for asymmetric reduction in the heterotrophic organism-catalyzed reduction. However, in the bioreduction using microalgae, the reaction was promoted by the direct use of light energy.

For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)] and its respective phosphate [NADP(H)] are required. These cofactors are prohibitively expensive if used in stoichiometric amounts. Since it is only the oxidation state of the cofactor that changes during the reaction, it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Usually in the heterotrophic organism-catalyzed reduction, formate, glucose, and simple alcohols such as ethanol and 2-propanol are used to transform the oxidized form of the coenzyme to the reduced form. These reductants originally stem from bio-products of CO2 with phototrophs with the aid of sunlight.

Phototrophs such as algae and plants capture light energy to generate NADPH from NADP+ through photosynthetic electron-transfer reactions. Subsequently, CO2 is converted into sugar, generally using NADPH. The reducing power of NADPH generated through photosynthesis can also be used in the reduction of exogenous substrates such as unnatural ketones to yield useful optically active alcohols. Thus, cofactor-recycling is no problem when photosynthetic living cells are used as biocatalysts for reduction. Accordingly, we can use solar energy directly for bioconversion of artificial substrates.2

(d) The stereochemical course of the reduction is possibly changed by light conditions:

The reduction of a,a-difluoroacetophenone by a cyanobacterium proceeded both under light and in the dark, and the poor enantioselectivities (20-30% ee) observed in the dark were improved by irradiation. Thus, the enantioselectivities increased according to the lightness (70% ee under light (1000 lux)). The use of DCMU, a photosynthetic inhibitor, decreased the enantioselectivity of the reduction even under light conditions. The stereochemical course of the reduction is controlled by illumination or by adding DCMU.3

(e) The microalgae is easily manipulated and has high growth rate: Photosynthetic plant cell cultures typically grow quite slowly. However, cyanobac-teria grow much faster than plant cell cultures in spite of the fact that the algae are types of phototrophs. Thus, contamination with microbes during the cultivation of algae can be prevented, and sufficient amount for biotransformation can be obtained easily.

(f) Ecological system:

Algae have the ability to directly utilize sunlight and carbon dioxide for photosynthesis. Due to this activity, cyanobacteria may help to solve a global environmental problem, the greenhouse effect, which increasingly threatens mankind at the beginning of the 21st century.

Other examples of microalgae-catalyzed reductions of carbonyl groups are summarized below and shown in Fig. 2:

• Reduction of carbonyl groups: Terpene4 and aromatic56 aldehydes (100 ppm) were reduced by microalgae. In a series of chlorinated benzaldehyde, m- or p-chlorobenzaldehyde reacted faster than the o-derivative.5 Due to toxicity, the substrate concentrations are difficult to increase. Asymmetric reductions of ketones by microalgae were reported. Thus, aliphatic7-9 and aromatic1-31011 ketones were reduced.

Figure 2: Examples of reduction and oxidation reactions.

• Reduction of carbon-carbon double bond: Microalgae easily reduce carboncarbon double bonds in enone.12 Usually, the reduction of carbonyl group and carbon-carbon double bond proceeds concomitantly to afford the mixture of corresponding saturated ketone, saturated alcohol, and unsaturated alcohol because a whole cell of microalgae has two types of reductases to reduce carbonyl and olefinic groups. The use of isolated reductase, which reduces carbon-carbon double bond chemoselectively, can produce saturated ketones selectively.

The future direction of reduction of carbonyl compounds is as follows:

1. Development of new reduction systems that reduce sterically hindered compounds: The reported examples of reduction of carbonyl compounds are usually for the substrates that can be easily reduced such as methyl ketones. Since the demand for reduction of various types of compounds is increasing, investigation of new biocatalytic reductions is required. Photosynthetic organisms are not investigated yet, and they may have new type of enzymes, which can reduce sterically hindered compounds.

2. Development of a new system for biocatalysis by photosynthetic organisms: Since most of the present reactors of biocatalysis are for heterotrophic microorganisms, their systems are not usable for the reaction by photosyn-thetic organisms. Since they require light for growing, it is necessary to develop a new type of photobioreactor to utilize the light energy as reduction power efficiently.

3. Development of new methods for controlling reactions: Although many methods are already known in microbial reactions using heterotroph to control the reaction,13 little is known about the methods using photosynthetic organisms. In usual microbial reactions, three methods, screening of suitable microbes, screening of suitable substrates, and modifying the reaction conditions, have been used for controlling the reaction. These methods have to be modified to use for the reaction by photosynthetic organisms. For example, for the efficient screening of photosynthetic organisms, the formation of large culture stock is necessary.

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