Candida tropicalis CE 017 : a New Brazilian Enzymatic Source for the Bioreduction of Aromatic Prochiral Ketones

A reatividade e estereosseletividade apresentadas por uma nova cepa de Candida tropicalis na redução de cetonas pró-quirais foram comparadas com cepas de microrganismos, estudadas em nosso laboratório, provenientes da biodiversidade brasileira. Neste caso, Candida tropicalis demonstrou-se como um agente redutor versátil e estereosseletivo na biorredução de uma série de cetonas aromáticas. Estes compostos pró-quirais foram convertidos aos correspondentes álcoois opticamente ativos com moderada a excelente estereopreferência dependendo da estrutura do substrato. Entre as cetonas utilizadas, as nitroacetofenonas foram enzimaticamente reduzidas aos correspondentes (S)-álcoois enantiopuros com completa conversão.


Introduction
Chiral alcohols are an important class of organic substrates due to their properties as bioactive compounds and as starting materials for the synthesis of various biologically active materials. 1For example, enantiopure 1-phenylethanol is a chiral building block used in the fine chemical and pharmaceutical industries as an ophthalmic preservative, a solvatochromic dye, an inhibitor of cholesterol intestinal absorption and a mild floral fragrance. 2In the synthesis of enantiopure chiral secondary alcohols, prochiral ketones are commonly used as starting materials using stereoselective chemical transformations or biocatalytic methods.Asymmetric reduction by chemical methods usually involves the use of expensive reagents or heavy metal catalysts. 3By contrast, biocatalysis applied to industrial processes has been shown as a very advantageous alternative to conventional chemical methods, and is widely used for the preparation of enantiomerically pure pharmaceuticals and other high added value compounds. 4nzymatic reduction of carbonyl groups represents one of the most important reactions employed in the synthesis of chiral alcohols.Enzymes that can be used for this transformation are oxidoreductases, which require the presence of a coenzyme, such as NADH or NADPH, which transfers the hydride anion to the carbonyl compound, being formed NAD + or NADP + .Whole cells of microorganisms can be also used in the enzymatic reduction of the carbonyl group.Each cell represents a small factory fully equipped for the reduction of the substrate bearing the carbonyl moiety.In order to find optimal conditions for the action of cell factories, the process designer has only to plan in which way the ketonic substrate should be added, and which medium should be used for the designed reaction.Nowadays, a wide variety of cultured cells from microorganisms or vegetables are available for enzymemediated reduction of carbonyl compounds. 5t is well known that the screening of a wide variety of microorganisms living in the environment is an efficient method to obtain the desired enzyme towards an unnatural substrate.Acetophenone has been used as a model substrate for the isolation of microorganisms having ketone reductase activity. 6Recently, a new strain of Candida tropicalis PBR-2 MTCC 5158 has been reported as an efficient reducing agent to acetophenone and its derivatives. 7he use of microorganism new strains as biocatalysts may offer an alternative opportunity to investigate the local resources for the effective conduct of key synthetic transformations with significant economic and ecological implications. 8ur research interest is based on the development of chemoenzymatic methodologies to obtain chiral alcohols using Brazilian local sources of low cost.Herein, we report our current investigations in the study of the Candida tropicalis CE017 strain behavior as a novel stereoselective reducing agent of aromatic prochiral ketones to the corresponding chiral alcohols.The experimental results obtained will be compared to the one previously obtained by our research group using different Brazilian enzymatic sources. 9,10

Bioreduction of acetophenone using C. tropicalis CE017
Acetophenone 1a was tested in the bioreduction using growing cells of the yeast as biocatalyst in a potato dextrose broth medium.Enzymatic reductions have been followed by analyzing the product 1b formed after 1, 3, 6, 9, 12 and 15 days of reaction, being summarized the data in Table 1.(S)-1-phenylethanol 1b was obtained with high optical purities (97-99% ee) in all cases, observing the maximum conversion (62%, entry 4) after 9 days of reaction.Longer reaction times led to the formation of acetophenone due to an oxidizing activity showed by Candida tropicalis.
In our efforts to improve the yield of (S)-1-phenylethanol 1b, some other reaction parameters were analyzed as culture medium, pH, temperature and substrate concentration.
These results revealed that all the new culture media have similar effect in the enzymatic activity (11-13%  conversion), allowing the recovery of (S)-1-phenylethanol in enantiomerically pure form.All these media led to notable lower isolated yields in comparison with potato dextrose medium (62% yield at pH 7.0).

O OH 1a (S)-1b
Growing cells of Candida tropicalis PD medium 125 rpm, 28 ºC Scheme 1. Bioreduction of acetophenone 1a by C. tropicalis.Reactions were analyzed after 9 days using potato dextrose broth as culture medium in the following pHs: 4.0, 5.5, 7.0 and 8.0.The medium was adjusted to pH 8.0 and 7.0 with a sterile solution of NaOH 1.0 mol L -1 , and pH 4.0 was adjusted by using a sterilized 10% tartaric acid solution.The results were summarized in Table 3.
The optimal pH for this biocatalytic system varied from 5.5 to 7.0 (entries 2 and 3), observing a decrease of conversion when more acidic or basic media were employed (entries 1 and 4).

Temperature effect on Candida tropicalis
Reactions were performed using potato dextrose broth at higher temperatures (45 and 55 ºC), as shown in Table 4.
These results revealed that C. tropicalis does not present a good thermostabilitity, as a decrease of the ketone reductase activity was observed by working at temperatures above 28 ºC (entries 1 and 2), leading to a dramatic loss of yield and also of the enantiomeric excess especially over 45 ºC (entry 3).
A slight improvement was observed in the conversion when less quantity of substrate (6-12 μL) was used, but in these conditions was observed a 85% ee in (S)-1phenylethanol (entries 1-4).The conversion decreased when more quantity of substrate was used (entries 5-9), however higher concentrations of ketone led to better stereopreference values, being in this case 15-20 μL the appropriate quantity of ketone (entries 6-7).
Thus, a temperature of 28 ºC, 9 days of reaction time, pH of 5.5, potato-dextrose broth as culture medium and 20 μL of acetophenone proved to be the optimum conditions to obtain (S)-1-phenylethanol (1b) in good yield (62%) and high enantiomeric excess (97% ee).

Bioreduction of acetophenone derivatives
After these encouraging results, we decided to extend our studies toward the analysis of the bioreduction of substituted acetophenones.Thus, ketones 2a-10a (Scheme 2) were reduced by growing cells of C. tropicalis CE017 using the optimal reaction conditions attained in the bioreduction of acetophenone.The results are summarized in Table 6.
The influence of an electron-donor (OMe) group and an electron-withdrawing (NO 2 ) group in ortho, meta-and para-position of the aromatic moiety was analyzed.Reduction of nitroacetophenones occurred with complete conversion yielding the corresponding alcohols (S)-2b-4b with excellent enantioselectivities (entries 1-3).The presence of the nitro group strongly increased the enzymatic activity when compared with acetophenone.Ketone 5a having the methoxy group in the ortho position yielded the corresponding alcohol (S)-5b with moderate conversion and selectivity (entry 4).When the methoxy group was located at meta position, the conversion was lower when compared with ketone 5a, being achieved a higher selectivity (entry 5).A strongly decrease in the activity and selectivity was observed for the reduction of 4-methoxyacetophenone (entry 6).The presence of the methoxy group in the para position seemed to be worst for enzymatic activity and selectivity.4-Chloroacetophenone 8a yielded the (S)-8b with moderate conversion and selectivity (entry 7).The presence of the chlorine atom in the α-position to the carbonyl group produced a strong decrease in the product conversion with a good enantioselectivity, as shown in entry 8 for the preparation of alcohol (S)-9b, yielding in this case the (R)-alcohol due to CIP rules (entry 8).
Bioreduction of 10a, compound presenting two chlorine atoms in the aromatic ring and one chlorine atom in the α-position to the carbonyl group, occurred with a similar conversion to acetophenone 8a and a similar stereoselectivity compared to ketone 9a (entry 9).Again, (R)-10b was obtained due to a change of priority in the CIP rules.
Our research group has been working to select several microorganisms from Brazilian biodiversity in order to perform selective bioreduction reactions using whole cells.We have found some interesting suppliers of alcohol dehydrogenases such Lasiodiplodia theobromae 9 and Lentinus strigellus 10 and now we can compare the results previously obtained in these manuscripts with Candida tropicalis.Each one is worth of attention due to their particularities.Candida tropicalis has not a good affinity for acetophenones bearing methoxy group moiety.Opposite, fungal cells of L. theobromae and L. strigellus catalyze the bioreduction of these methoxyacetophenones with higher enantioselectivities (87-99%).On the other hand, L. theobromae had poor selectivity for nitroacetophenones, 3a (only 45% of ee) and 4a (42% of ee).Lentinus strigellus had good enantioselectivities (98-99%) but moderate conversion of 2a (48%), that could not be improved after longer reaction times.Candida tropicalis was the most successful among them to reduce nitroacetophenones with high to excellent conversion and enantioselectivity values.It must be emphasize that nitrocompounds are important building blocks in organic synthesis 13 as well as precursors Scheme 2. Bioreduction of prochiral aromatic ketones 2a-10a using C. tropicalis of the corresponding amines after a reduction process. 14In addition, alcohol 6b is a relevant compound in the synthesis of (S)-Rivastigmine 15 and the alcohol 10b can be used in the preparation of the pharmacological active Sertraline. 16n conclusion, all this family of chiral alcohols can be obtained by an enzymatic strategy, just selecting the best local microorganism in each specific case.

Conclusions
Growing cells of a new yeast strain, C. tropicalis CE017 in potato dextrose have been shown as an excellent and inexpensive biocatalyst for the introduction of chirality in bioreduction processes.When reducing substituted aromatic ketones, the presence of a nitro group in the aromatic moiety has a positive effect in the biocatalytic properties of C. tropicalis CE017, yielding the corresponding alcohols with complete conversion and ee > 95%.On the other hand, methoxyacetophenones seemed to be poorer substrates for this yeast.In all cases the biocatalyst has presented Prelog selectivity for the production of optically active alcohols.Comparison with two microorganisms previously studied in our group (L.theobromae and L. strigellus) revealed its particularities.In this case, Candida tropicalis was the most successful among them to reduce nitroacetophenones with great conversion and enantioselectivity.The evaluation of locally available microorganisms for a selection of standard organic chemical reactions of commercial significance could prove to be a very valuable economic endeavor.It may well offer new opportunities to expand the local resources as sustainable enzymatic systems where highcost, unstable coenzymes are presently used.

General
All reagents were purchased from Aldrich and used without further purification.Dry solvents were distilled over an adequate desiccant under nitrogen.Flash chromatographies were performed using silica gel 60 (230-240 mesh).High performance liquid chromatography (HPLC) analysis were carried out in a Hewlett Packard 1100 chromatograph UV detector at 210 nm using a Daicel Chiralcel OB-H, Chiralpak AS or Chiralpak IA column (25 cm × 4.6 mm I.D.) varying the conditions depending on the specific substrate, and using mixtures of hexane and 2-propanol (IPA).Gas chromatography (GC) analysis were performed on a Hewlett Packard 6890 Series II chromatograph equipped with a CP-Chiralsil DEX CB or a HP-1 column (30 m × 0.25 mm × 0.25 μm, 1.0 bar N 2 ) from Varian for determining the optical purities. 1H, 13 C NMR, DEPT, and 1 H- 13 C heteronuclear experiments were obtained using AC-200 ( 1 H, 200.13 MHz and 13 C, 50.3 MHz), AC-300 ( 1 H, 300.13 MHz and 13 C, 75.5 MHz), DPX-300 ( 1 H, 300.13 MHz and 13 C, 75.5 MHz) or AV-400 ( 1 H, 400.13 MHz and 13 C, 100.6 MHz) Bruker spectrometers.The chemical shifts are given in delta (d) values and the coupling constants (J) in Hertz (Hz).Optical rotations were performed in a Perkin-Elmer 241 polarimeter.

Candida tropicalis strain CE017
The strain CE017 was previously isolated from an oil refinery wastewater located in Fortaleza city (Ceara state, Brazil) by enrichment cultivation. 17This strain was identified as Candida tropicalis according to micromorphological and biochemical characteristics. 18It is noteworthy that C. tropicalis CE017 showed a particular ability to degrade high phenol concentration and might be useful in bioremediation activities. 17This strain is deposited in the Culture Collection of the Microbial Ecology and Biotechnology Laboratory at Biology Department of the Federal University of Ceará, Brazil.

Culture conditions
For inoculums preparation, stock cultures were cultivated in potato broth (Potato 4 g L -1 , Dextrose 20 g L -1 , HIMEDIA) pH 5.5 for 24 h at 28 ºC under shaking conditions (125 rpm).After this, the absorbance of the culture was adjusted with 0.15 mol L -1 NaCl solution to 0.5 at 600 nm to obtain a suspension of 32 × 10 6 CFU mL -1 determined by counting of colony forming units (CFU) on Potato Agar Plates.An aliquot of 1.0 mL of this suspension was inoculated in 250 mL Erlenmeyer flask containing 100 mL of Potato broth to produce biomass.When Peptone/ Dextrose was used as medium the counting of CFU was 15 × 10 6 CFU mL -1 .In the case of Czapeck medium the CFU value was 57 × 10 3 CFU mL -1 .

Procedure for the bioreduction of acetophenone and its derivatives using Candida tropicalis
The growing cells of C. tropicalis CE017 were used for bioreduction reactions according literature procedure. 19Then, 20 mg of substrate were added into erlenmeyer flask and the reactions were shaken for 9 days of reaction.The content of each flask was saturated with sodium chloride, and then the aqueous phase was extracted with EtOAc (3 × 80 mL).The organic phase was dried over with Na 2 SO 4 and then the solvent was evaporated under reduced pressure.The reaction crude was analyzed by the appropriate condition and purified by flash chromatography.All the bioreduction experiments were done in triplicate.

Table 1 .
Effect of incubation time on the reduction of acetophenone by

Table 2 .
Effect a Conversion and enantiomeric excesses were determined by GC.Vol.21, No. 8, 2010 pH Influence

Table 3 .
Effect of pH on the bioreduction of acetophenone by C. tropicalis CE017 a Conversion and enantiomeric excesses (ee) were determined by GC.

Table 4 .
Effect of temperature on the bioreduction of acetophenone by a Conversion and enantiomeric excesses (ee) were determined by GC.

Table 5 .
Effect of the quantity of substrate on the bioreduction of acetophenone (1a) by C. tropicalis CE017 a Conversion and enantiomeric excesses (ee) were determined by GC.

Table 6 .
Candida tropicalis CE017 biocatalyzed reduction of ketones 2a-10a in potato-dextrose medium at 28 °C, 125 rpm and 9 days a Conversion and enantiomeric excesses (ee) were determined by HPLC or GC (see Experimental section).Absolute configuration of the optically alcohol in brackets.