Enantioselective Bioreduction of Prochiral Pyrimidine Base Derivatives by Boni Protect Fungicide Containing Live Cells of Aureobasidium pullulans

: The enzymatic enantioselective bioreduction of prochiral 1-substituted-5-methyl-3-(2-oxo-2-phenylethyl)pyrimidine-2,4(1 H ,3 H )-diones to corresponding chiral alcohols by Boni Protect fungicide containing live cells of Aureobasidium pullulans was studied. The microbe-catalyzed reduction of bulky-bulky ketones provides enantiomerically pure products (96–99% ee). In the presence of A. pullulans ( Aureobasidium pullulans ), one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to the si sides of the prochiral carbonyl group to give secondary alcohols with R conﬁguration. The reactions were performed under various conditions in order to optimize the procedure with respect to time, solvent, and temperature. The present methodology demonstrates an alternative green way for the synthesis of chiral alcohols in a simple, economical, and eco-friendly biotransformation.


Introduction
Biotransformation, i.e., enzyme-catalyzed reactions, have been used over millions of years in nature to carry out reactions that are complicated from a chemical point of view. Nowadays, biocatalysis has become an important method for the production of organic compounds. Biotransformations catalyzed by isolated enzymes and by whole cells of microorganisms, in terms of microorganisms growing or resting cells, are employed for the synthesis of chemicals such as pharmaceuticals, agrochemicals, and natural products [1][2][3].
Bioreductions of prochiral carbonyl compounds are mainly carried out using oxidoreductases, the most frequently used of which is the catalytic potential of NADH (Nicotinamide adenine dinucleotide)/NADPH (Nicotinamide adenine dinucleotide phosphate)-dependent dehydrogenases. Dehydrogenases in living organisms catalyze the reaction of the oxidation of alcohols to carbonyl compounds by mediating the transfer of a hydride ion from the cofactor on the substrate. They also have the ability to catalyze a reverse reaction in a reduction reaction. In the reduction reaction, dehydrogenases transfer the hydride ion (pro-S or pro-R) from the cofactor to one of the prochiral sides of the carbonyl group (face re or si) to give pure enantiomeric or diastereomeric products [1,2]. The main limitation to the use of dehydrogenases is the necessity of employing expensive cofactors. In contrast, biocatalysis in the presence of living organisms in the form of tissue cultures or whole microbial cells is a very promising and effective method because the dehydrogenase, cofactor, and its regenerating system are all located within the cell [1,[4][5][6].
Baker's yeast is the most popular whole-cell biocatalyst for the asymmetric reduction of prochiral ketones due to its unlimited availability, ease of growing, and low cost [7]. For asymmetric bioreduction of ketones, in addition to the popular Baker's yeast, most often the cells of bacterial [8], fungal [9][10][11][12][13], and plant tissue [14][15][16][17] are used. The application of bioreagents in enantioselective enzymatic desymmetrization of prochiral ketones leads to a broad spectrum of chiral alcohols used as intermediates in the syntheses of many pharmaceuticals and compounds presenting biological activity [6]. Not without significance is the fact that biocatalytic reactions are conducted under moderate conditions in aqueous solutions without the use of expensive and often toxic reagents. For this reason, they are environmentally friendly, which increases the scope of their applications [1,2].
In this work, we present the microbial biotransformation of the 1-substituted-5-methyl-3-(2-oxo-2phenylethyl)pyrimidine-2,4(1H,3H)-dione in a reduction catalyzed by Boni Protect fungicide containing live cells of Aureobasidium pullulans. Boni Protect is used in orchards because the microorganism contained in this antifungal preparation has an antagonistic activity against a number of phytopathogenic fungi (Botrytis cinerea, Penicillium expansum, Monilinia laxa, Pezicula malicorticis). It is used most often to combat white mold. The 3-substituted pyrimidine base derivatives exhibit biological activity, for e.g., N-3-substituted arabinofuranosyluracils and 4-thio analogues of N-3-substituted uridines possess hypnotic activity [18,19]. For this reason, the modification of these compounds in order to obtain enantiomerically pure derivatives is important from the point of view of using them as drugs.

Results and Discussion
Enantioselectivity and the efficiency of the microbial catalysis are mainly determined by the steric requirements of the substrate. For instance, Baker's yeast does not tolerate long-chain dialkyl ketones; however, one long alkyl chain is accepted if the methyl group has the other moiety. Highly stereoselective catalysis is achieved for the substrate with substituents of significantly different sizes [20].
The aim of the study was to use A. pullulans (Aureobasidium pullulans) to reduce the phenacyl of pyrimidine base derivatives with a prochiral carbon atom where there are two bulky substituents in the vicinity (Scheme 1). For this reason, they belong to the group of carbonyl compounds that are difficult to reduce by microbiological methods. dehydrogenases transfer the hydride ion (pro-S or pro-R) from the cofactor to one of the prochiral sides of the carbonyl group (face re or si) to give pure enantiomeric or diastereomeric products [1,2].
The main limitation to the use of dehydrogenases is the necessity of employing expensive cofactors. In contrast, biocatalysis in the presence of living organisms in the form of tissue cultures or whole microbial cells is a very promising and effective method because the dehydrogenase, cofactor, and its regenerating system are all located within the cell [1,[4][5][6].
Baker's yeast is the most popular whole-cell biocatalyst for the asymmetric reduction of prochiral ketones due to its unlimited availability, ease of growing, and low cost [7]. For asymmetric bioreduction of ketones, in addition to the popular Baker's yeast, most often the cells of bacterial [8], fungal [9][10][11][12][13], and plant tissue [14][15][16][17] are used. The application of bioreagents in enantioselective enzymatic desymmetrization of prochiral ketones leads to a broad spectrum of chiral alcohols used as intermediates in the syntheses of many pharmaceuticals and compounds presenting biological activity [6]. Not without significance is the fact that biocatalytic reactions are conducted under moderate conditions in aqueous solutions without the use of expensive and often toxic reagents. For this reason, they are environmentally friendly, which increases the scope of their applications [1,2].
In this work, we present the microbial biotransformation of the 1-substituted-5-methyl-3-(2-oxo-2-phenylethyl)pyrimidine-2,4(1H,3H)-dione in a reduction catalyzed by Boni Protect fungicide containing live cells of Aureobasidium pullulans. Boni Protect is used in orchards because the microorganism contained in this antifungal preparation has an antagonistic activity against a number of phytopathogenic fungi (Botrytis cinerea, Penicillium expansum, Monilinia laxa, Pezicula malicorticis). It is used most often to combat white mold. The 3-substituted pyrimidine base derivatives exhibit biological activity, for e.g., N-3-substituted arabinofuranosyluracils and 4-thio analogues of N-3substituted uridines possess hypnotic activity [18,19]. For this reason, the modification of these compounds in order to obtain enantiomerically pure derivatives is important from the point of view of using them as drugs.

Results and Discussion
Enantioselectivity and the efficiency of the microbial catalysis are mainly determined by the steric requirements of the substrate. For instance, Baker's yeast does not tolerate long-chain dialkyl ketones; however, one long alkyl chain is accepted if the methyl group has the other moiety. Highly stereoselective catalysis is achieved for the substrate with substituents of significantly different sizes [20].
We wanted to find out if the use of A. pullulans will enable the reduction of 1-3 and whether selectivity of bioreduction can be improved as a result.
The biotransformation a reaction catalyzed by A. pullulans was carried out in aqueous solution (phosphate buffer pH = 7.0) at 30 • C, in the presence of glucose as the energy source and with the addition of ethanol. Under these conditions, regeneration of the cofactor takes place in situ. First, compound 1 as a model substrate was subjected to bioreduction and the reaction was completed after 3, 5, and 6 days ( Table 1, entry 1-3). We found that the process of biotransformation of the compound is very slow and in order to achieve an effective performance of greater than 50%, the reaction should be carried out for at least 144 h. The enantiomerically pure product of R configuration ((R)-1 ) was obtained, regardless of at which stage the reaction was finished. In order to improve the reaction yield, it was decided to reduce 1 with the addition of organic solvents: In a two-phase system phosphate buffer (pH = 7.0): Hexane (1:1 v/v), phosphate buffer (pH = 7.0):Hexane (4:1 v/v), and with organic solvents as cosolvents (Table 1, entry [4][5][6][7][8][9][10][11][12]. Surprisingly, regardless of the polarity of organic solvents, the performance of reduction decreased. The highest yield (11.6%) was obtained in a solution of phosphate buffer (pH = 7.0) with the addition of 2% ionic Further optimization of the reaction conditions concerned the change in pH of phosphate buffer solution and the introduction of sucrose as a carbon source in the pre-incubation stage. According to the literature, the optimal pH for the microorganism A. pullulans is slightly acidic, therefore, the reduction of 1 in an environment of pH = 6.5 was carried out [20]. The obtained results are given in Table 1 (entry  13,15).
The highest yield was obtained in a solution with pH = 7.0 with sucrose (75.6%; see HPLC analysis of reduction of 1 in this condition- Figure 1). Bioreduction in aqueous solution with A. pullulans as a catalyst was also carried out for 2 and 3. For compound 2, the highest efficiency and high enantioselectivity (99% ee) were obtained in a phosphate buffer solution with pH = 7.0 in the presence of glucose, which was higher than the efficiency in the solution with pH = 6.5. However, the addition of sucrose to the reaction mixture, independent of the pH of the solution, resulted in a reduction in the reaction yield ( Table 2). As in the case of 1, the microorganism A. pullulans ensures excellent selectivity of biotransformation giving chiral R-alcohol with 96-99% ee. The most difficult object to reduce was compound 3. For this compound, as for 2, the conditions such as phosphate buffer, pH = 7.0, and glucose are the most optimal. After 144 h, the best performance was obtained, which, however, did not exceed 30%. Interestingly, lowering pH to 6.5 resulted in a decrease in performance. Under these conditions, 3a accounted for only 10% of the reaction mixture. As a consequence, (R)-3a was obtained with low yield and high enantiomeric excess ( Table 2).
Compounds 1-3 are structural analogues, differing in substituents in the N-1 position. Despite the similarity in the chemical structure, they are reduced in the same conditions at different rates. Therefore, for each of the prochiral reagents, the conditions of biocatalysis should be individually optimized. Based on the results obtained, the rate of the reaction catalyzed by A. pullulans and thus the efficiency is influenced by the size and chemical nature of the substituent on the first nitrogen in the pyrimidine ring. The presence of the allyl substituent increases the reaction rate.
In the next stage, biotransformation was performed at two additional temperatures to improve the efficiency of the reaction: 33 °C and 36 °C. The reaction rate increases with the increasing temperature, the enzymatic catalysis is also regulated by this dependence, but only within a certain temperature range. The optimal temperature that is characteristic of the enzyme ensures the highest activity of the biocatalyst, which results in the maximum degree of conversion. The inverse relationship applies to the selectivity of the process, in most cases the optical purity of a product decreases along with an increase in temperature [28]. We expected that as the temperature increased, the yield of chiral alcohols would increase without adversely affecting the enantioselectivity of the catalyzed reaction. Bioreduction in aqueous solution with A. pullulans as a catalyst was also carried out for 2 and 3. For compound 2, the highest efficiency and high enantioselectivity (99% ee) were obtained in a phosphate buffer solution with pH = 7.0 in the presence of glucose, which was higher than the efficiency in the solution with pH = 6.5. However, the addition of sucrose to the reaction mixture, independent of the pH of the solution, resulted in a reduction in the reaction yield ( Table 2). As in the case of 1, the microorganism A. pullulans ensures excellent selectivity of biotransformation giving chiral R-alcohol with 96-99% ee. The most difficult object to reduce was compound 3. For this compound, as for 2, the conditions such as phosphate buffer, pH = 7.0, and glucose are the most optimal. After 144 h, the best performance was obtained, which, however, did not exceed 30%. Interestingly, lowering pH to 6.5 resulted in a decrease in performance. Under these conditions, 3a accounted for only 10% of the reaction mixture. As a consequence, (R)-3a was obtained with low yield and high enantiomeric excess (Table 2). Compounds 1-3 are structural analogues, differing in substituents in the N-1 position. Despite the similarity in the chemical structure, they are reduced in the same conditions at different rates. Therefore, for each of the prochiral reagents, the conditions of biocatalysis should be individually optimized. Based on the results obtained, the rate of the reaction catalyzed by A. pullulans and thus the efficiency is influenced by the size and chemical nature of the substituent on the first nitrogen in the pyrimidine ring. The presence of the allyl substituent increases the reaction rate.
In the next stage, biotransformation was performed at two additional temperatures to improve the efficiency of the reaction: 33 • C and 36 • C. The reaction rate increases with the increasing temperature, the enzymatic catalysis is also regulated by this dependence, but only within a certain temperature range. The optimal temperature that is characteristic of the enzyme ensures the highest activity of the biocatalyst, which results in the maximum degree of conversion. The inverse relationship applies to the selectivity of the process, in most cases the optical purity of a product decreases along with an increase in temperature [28]. We expected that as the temperature increased, the yield of chiral alcohols would increase without adversely affecting the enantioselectivity of the catalyzed reaction.
For each reagent, the two optimal conditions in which the reductions were performed at elevated temperatures were selected ( Table 3). The best results were obtained for 3 at 33 • C. In a phosphate For each reagent, the two optimal conditions in which the reductions were performed at elevated temperatures were selected ( Table 3). The best results were obtained for 3 at 33 °C. In a phosphate buffer solution with pH = 7.0 with glucose (Figure 2), the yield of the reaction increased 2-fold and even 3-fold under the same conditions with sucrose added as a carbon source.  A slight increase in yield was observed for 2 at 33 °C, and over 60% yield was obtained in the buffer solution with pH = 7.0 with glucose ( Figure 3). In the case of 1, a 3 °C increase in temperature resulted in a decrease in performance. A further increase in temperature to 36 °C adversely affected the conversion rate of each reagent, but did not have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield of 1a-3a alcohols at different temperatures.  A slight increase in yield was observed for 2 at 33 • C, and over 60% yield was obtained in the buffer solution with pH = 7.0 with glucose ( Figure 3). For each reagent, the two optimal conditions in which the reductions were performed at elevated temperatures were selected ( Table 3). The best results were obtained for 3 at 33 °C. In a phosphate buffer solution with pH = 7.0 with glucose (Figure 2), the yield of the reaction increased 2-fold and even 3-fold under the same conditions with sucrose added as a carbon source.  A slight increase in yield was observed for 2 at 33 °C, and over 60% yield was obtained in the buffer solution with pH = 7.0 with glucose ( Figure 3). In the case of 1, a 3 °C increase in temperature resulted in a decrease in performance. A further increase in temperature to 36 °C adversely affected the conversion rate of each reagent, but did not have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield of 1a-3a alcohols at different temperatures. In the case of 1, a 3 • C increase in temperature resulted in a decrease in performance. A further increase in temperature to 36 • C adversely affected the conversion rate of each reagent, but did not have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield of 1a-3a alcohols at different temperatures.  The advantage of the microbiological method we use is a unique enantioselectivity, which combined with the ecological and economic aspect can be an interesting alternative to organocatalysis. Dehydrogenases contained in the microorganism A. pullulans selectively transfer one of the enantiotopic hydrogen ions of the dihydropyridine ring coenzyme to the si sides of the prochiral carbonyl group and provide secondary alcohols with the R configuration. Therefore, the Boni Protect could be considered as an efficient bioreagent for preparation of optically pure alcohols of derivatives of N-1 substituted thymine.
In the case of previously reduced unsymmetrical ketones (methyl ketones or bromomethyl ketones), in order to improve enantioselectivity, it was necessary to add appropriate inhibitors, i.e., additives which inhibit oxidoreductases with a specific stereopreference. Without the additives, reduction with A. pullulans proceeded with an enantiomeric excess ranging from 0% to 65% ee [26].

Analytical Methods
Nuclear magnetic resonance (NMR) spectra were performed with Bruker spectrometers (Billerica, MA, USA, 400/700 MHz). Chemical shifts are reported in δ ppm from tetramethylsilane (TMS) as an internal standard.
The samples were incubated in an orbital shaker (VORTEMP 1550 S2050; Equimed, Cracov, Poland). The advantage of the microbiological method we use is a unique enantioselectivity, which combined with the ecological and economic aspect can be an interesting alternative to organocatalysis. Dehydrogenases contained in the microorganism A. pullulans selectively transfer one of the enantiotopic hydrogen ions of the dihydropyridine ring coenzyme to the si sides of the prochiral carbonyl group and provide secondary alcohols with the R configuration. Therefore, the Boni Protect could be considered as an efficient bioreagent for preparation of optically pure alcohols of derivatives of N-1 substituted thymine.
In the case of previously reduced unsymmetrical ketones (methyl ketones or bromomethyl ketones), in order to improve enantioselectivity, it was necessary to add appropriate inhibitors, i.e., additives which inhibit oxidoreductases with a specific stereopreference. Without the additives, reduction with A. pullulans proceeded with an enantiomeric excess ranging from 0% to 65% ee [26].

Analytical Methods
Nuclear magnetic resonance (NMR) spectra were performed with Bruker spectrometers (Billerica, MA, USA, 400/700 MHz). Chemical shifts are reported in δ ppm from tetramethylsilane (TMS) as an internal standard.
The structure of 1a-3a was verified by 1 H NMR and spectra date was compared with the literature [27].
The absolute configurations of the chiral molecules were determined by various chiroptical methods (electronic circular dichroism (ECD) and vibrational circular dichroism (VCD)) [27].

General Procedure of Asymmetric Reduction by Aureobasidium pullulans
For a typical experiment, to a suspension of Boni Protect (0.5 g) in 7.5 mL of potassium phosphate buffer (pH 7.0) was added 2 × 10 −4 mol glucose/sucrose, and the resulting suspension was incubated in an orbital shaker (350 rpm) at specific temperature (30 • C, 33 • C, 36 • C) for 30 min. After pre-incubation, the appropriate ketone (2.5 × 10 −5 mol in 0.5 mL EtOH) was added, and stirring was continued at the same temperature. The reaction progress was monitored by Thin Layer Chromatography-TLC (the solvent system used was n-hexane:ethyl acetate 1:3 v/v). After the reaction, hyflo-super cel and ethyl acetate were added and the mixture was filtered. The celit was washed with ethyl acetate, and combined filtrates were extracted with ethyl acetate (3 × 20 mL). The organic phase was dried over anhydrous MgSO 4 , and the solvent was evaporated under vacuum. After that, each reaction mixture was purified by column chromatography using n-hexane:ethyl acetate 1:3 v/v to afford the product. The enantiomeric ratios were determined on the HPLC system using a chiral column.
NMR and HPLC spectra of 1-3 and 1a-3a were attached in the Supplementary Materials.

Conclusions
In summary, we have described an eco-friendly and environmentally benign asymmetric reduction system employing easily available Boni Protect fungicide as a biocatalyst. This reduction method is simple, economical (the need of costly cofactor is eliminated), and does not require the cultivation of the bioreagent. The bioreduction of different phenacyls of pyrimidine base derivatives to corresponding optically chiral alcohols has shown an exclusively (R) configuration. Thus, this study demonstrates an inexpensive approach in the synthesis of optically pure (R)-heterocyclic compounds of biological importance.