The fungal strains Aspergillus terreus URM 3371 and A. terreus CCT 4083, isolated in Brazil, catalysed the deracemization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) and (RS)-1-[(4-ethylselanyl)phenyl]ethanol (2). Different mass of whole fungal cells (1–5 g), pH values (4 and 7), biotransformation temperature (20 and 32 °C) and additives (ethanol, butanol, propanol and cyclohexanol) were employed in attempt to improve product yield and selectivity. The A. terreus strain URM 3371 transformed (RS)-1 into (+)-(R)-1 with high enantiomeric excess (e.e.≥98 %), good conversion (≥98 %) and acceptable yield (53 %).Sojevi plijesni Aspergillus terreus URM 3371 i A. terreus CCT 4083, izolirani u Brazilu, katalizirali su deracemizaciju (RS)-1-[(4-metilselanil)fenil]etanola (1) i (RS)-1-[(4-etilselanil)fenil]etanola (2). Upotrijebljene su cijele stanice plijesni različite mase (1-5 g), pri različitoj pH-vrijednosti (4 i 7) i temperaturi biotransformacije (20 i 32 °C), uz dodatak raznih aditiva (etanol, butanol, propanol i cikloheksanol) radi poboljšanja prinosa i selektivnosti proizvoda. Soj A. terreus URM 3371 transformirao je (RS)-1 u (+)-(RS)-1 s velikim enantiomernim viškom (≥98 %), dobrom pretvorbom (≥98 %) i prihvatljivim prinosom (53 %)

André Luiz Meleiro Porto

Edna Kagohara

João Valdir Comasseto

Leandro Helgueira Andrade

Leonardo Fernandes Assis

Álvaro Takeo Omori

English

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ISSN 1330-9862 preliminary communication(FTB-1743)Deracemization of (RS)-1-[(4-Methylselanyl)Phenyl]Ethanoland (RS)-1-[(4-Ethylselanyl)Phenyl]Ethanol by Strains ofAspergillus terreusLeonardo Fernandes Assis1, Edna Kagohara1, Álvaro Takeo Omori1,João Valdir Comasseto1, Leandro Helgueira Andrade1 andAndré Luiz Meleiro Porto1,2*1Laboratório de Química Fina e Biocatálise, Instituto de Química, Universidade de São Paulo,Av. Prof. Lineu Prestes 748, CEP 05508-900, São Paulo, SP, Brazil2Laboratório de Química Orgânica e Biocatálise, Instituto de Química de São Carlos,Universidade de São Paulo, Av. Trabalhador São-Carlense 400, CEP 13566-590, São Carlos, SP, BrazilReceived: June 29, 2006Accepted: December 14, 2006SummaryThe fungal strains Aspergillus terreus URM 3371 and A. terreus CCT 4083, isolated inBrazil, catalysed the deracemization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) and(RS)-1-[(4-ethylselanyl)phenyl]ethanol (2). Different mass of whole fungal cells (1–5 g), pHvalues (4 and 7), biotransformation temperature (20 and 32 °C) and additives (ethanol,butanol, propanol and cyclohexanol) were employed in attempt to improve product yieldand selectivity. The A. terreus strain URM 3371 transformed (RS)-1 into (+)-(R)-1 with highenantiomeric excess (e.e.³98 %), good conversion (³98 %) and acceptable yield (53 %).Key words: Aspergillus terreus, biotransformation, chiral organoseleno alcohols, fungal cells,deracemizationIntroductionThe search for new enzymatic systems with applica-tion in the synthesis of chiral compounds continues toattract considerable interest (1). The screening of micro-organisms for novel biocatalytic activities may be facili-tated by using whole cells rather than isolated enzyma-tic systems (2).Organoselenium compounds have been widely em-ployed in synthetic transformations, and various enan-tioselective methodologies involving organoseleno inter-mediates have been developed (3,4). In view of this fact,we became interested in the application of biocatalysisin the synthesis of enantiopure chiral organoseleno com-pounds. Recently we have reported the biocatalytic pre-paration of chiral organoseleno alcohols by the asym-metric reduction of organoseleno acetophenones by vari-ous fungal strains isolated in Brazil (5) and by Daucuscarota roots (6). Moreover, the efficient enzymatic resolu-tion of (RS)-b-hydroxyselenides in organic media hasbeen promoted by several lipases (7,8).In previous work we employed whole cells of thefungus Aspergillus terreus to catalyse the deracemizationof several phenylethanol derivatives (9,10). In the pres-ent work we report the biotransformation of (RS)-1--[(4-methylselanyl)phenyl]ethanol (1), (RS)-1-[(4-ethylse-lanyl)phenyl]ethanol (2) and (RS)-1-[(4-phenylselanyl)phenyl]-ethanol (3) by whole cells of two strains of A.terreus under different conditions (pH, temperature, ad-ditives, fungal cell mass; Scheme 1).415L.F. ASSIS et al.: Deracemization of (RS)-Organoseleno Alcohols by A. terreus, Food Technol. Biotechnol. 45 (4) 415–419 (2007)*Corresponding author; Phone ++55 16 33 738 103; Fax ++55 16 33 739 952; E-mail: almporto@iqsc.usp.brMaterials and MethodsGeneral methods1H-NMR and 13C-NMR spectra were measured on aBruker model DPX300 spectrophotometer operating at300.1 and 75.5 MHz, respectively, with samples dis-solved in CDCl3 and with tetramethylsilane (TMS) as in-ternal standard.Chemical syntheses were monitored by TLC analy-sis using aluminium-backed silica gel 60 F254 layers (Merck)eluted with hexane and ethyl acetate; analytes were vi-sualized by spraying with p-anisaldehyde/sulphuric acidreagent and heating at approx. 120 °C for 1 min. Con-version yields and enantiomeric excesses (e.e.) of enzyme--catalysed reactions were determined using a ShimadzuGC-17A (FID) gas chromatograph equipped with a Chi-rasil-Dex CB b-cyclodextrin (25 m×0.25 mm i.d.) fusedsilica capillary column.Optical rotations were determined using a JascoDIP-378 polarimeter with a 1-dm cuvette and were mea-sured with reference to the sodium D-line. The absoluteconfiguration of (+)-(R)-1 was determined by compari-son of its optical rotation ([a]D20=+44.4° (c 1.4, CHCl3;e.e.>98 %) with that reported in the literature for(–)-(S)-1 ([a]D20=–56.8°; c 0.47, CHCl3; e.e.=96 %) (5). Theabsolute configuration of 2 was determined by compari-son of the chiral chromatograms with the literature data(5).GC-MS analyses were performed on a Shimadzu QP5050A instrument equipped with a J&W Scientific DB-5capillary column (30 m´0.25 mm i.d.; 0.25 mm) employ-ing helium as the carrier gas.Synthesis of the racemic alcohols 1–3The racemic organoseleno alcohols 1–3 were ob-tained by reduction of the corresponding organoselenoacetophenones with sodium borohydride in methanol,as previously described by Andrade et al. (5). The orga-noseleno acetophenones were prepared by methods de-scribed by Omori et al. (8).Fungal culturesThe fungal strains employed were A. terreus CCT4083 (isolated by L. Pfenning in August 1994 from pas-ture soil in Belém, PA, Brazil) and A. terreus URM 3371(isolated in 1993 from the rhizosphere soil of Vernoniaherbaceae in São Paulo, SP, Brazil). A slant culture wasused to inoculate 1 L of sterilised malt extract medium(Oxoid, 20 g/L) contained in a 2-litre Erlenmeyer flask,which was incubated on an orbital shaker (Tecnal TE-421or Superohm G-25; rotational speed 160 rpm) for 96 h at32 °C. Cells were harvested by vacuum filtration andwashed under sterile conditions in laminar flow cabinet.Small scale biotransformation reactionsSamples (5 mg) of 1–3, dissolved in 200 mL of an ad-ditive (ethanol, propanol, butanol or cyclohexanol), wereintroduced under sterile conditions into the Erlenmeyerflasks (125 mL) containing suspensions of washed, wetcells of A. terreus (1–5 g, see Tables 1–3) in 50 mL of 0.1M phosphate buffer (pH=4 or 7) (11). The mixtures wereincubated on an orbital shaker (rotational speed 160 rpm)at 32 °C, and the progress of the reaction was monitoredat 3, 5 and 10 days by collecting samples of 1 or 2 mL.These samples were extracted by stirring with ethyl ace-tate (0.5 mL), and aliquots (4 mL) of the organic phasewere analysed by gas chromatography-flame ionizationdetector (GC-FID) using a fused silica chiral capillarycolumn. The products of the deracemization were com-pared with the racemic mixture of organoseleno alcoholsobtained by chemical reduction of the respective orga-noseleno ketones.Semi-preparative scale deracemization reactionsAn aliquot (100 mL) of (RS)-1-[(4-methylselanyl)phe-nyl]ethanol (1), dissolved in 600 mL of additive (ethanol),was introduced under sterile conditions into an Erlen-meyer flask (500 mL) containing a suspension of washed,wet cells of A. terreus URM 3371 (15 g) in 200 mL ofphosphate buffer (pH=4). The mixture was incubated onan orbital shaker (rotational speed 160 rpm) at 32 °C un-til the substrate had been completely consumed (18 days).Following incubation, the cell suspension was filteredand the cells were washed with ethyl acetate (4×100 mL).The organic phases were combined, dried over MgSO4,and the solvent removed under vacuum. The residue waspurified by column chromatography on silica gel (230–400 mesh) eluted with mixtures of hexane and ethyl ace-tate (9:1 and 8:2) to afford (+)-(R)-1.Chromatographic conditions employed in theGC separation of the enantiomers of the chiralorganoseleno phenylethanols 1–3Chromatographic conditions were: injector tempera-ture 220 °C; split ratio 1:20; detector temperature 220 °C;hydrogen carrier gas at 100 kPa and oven temperatureprogram: (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) –GC conditions: 150 °C for 30 min, then rising to 180 °Cat 1 °C/min; retention time: R-isomer 17 min, S-isomer18 min; (RS)-1-[(4-ethylselanyl)phenyl]ethanol (2) – GCconditions: 150 °C for 30 min, then rising to 180 °C at1 °C/min; retention time: R-isomer 13 min, S-isomer 14min; and (RS)-1-[(4-phenylselanyl)phenyl]ethanol (3) – GCconditions: 150 to 180 °C at 2 °C/min, retention time:R-isomer 34 min, S-isomer 35 min.416 L.F. ASSIS et al.: Deracemization of (RS)-Organoseleno Alcohols by A. terreus, Food Technol. Biotechnol. 45 (4) 415–419 (2007)OHROROHR(RS)-1-3 1a-2a (R)-1, (R)-2Fungal strainconditions+R = SeMe (1)SeEt (2)SePh (3)Fungal strains: A. terreus URM 3371 and A. terreus CCT 4083Scheme 1. Deracemization of (RS)-organoseleno alcohols bywhole cells of Aspergillus terreusResults and DiscussionMicrobial deracemizations of the organoseleno phe-nyl alcohols (RS)-1, (RS)-2, and (RS)-3 (Scheme 1) werestudied using buffer solutions containing whole cells ofA. terreus URM 3371 and A. terreus CCT 4083. The reac-tions were performed varying the experimental condi-tions (pH, temperature, mass of fungal cells and addi-tives). Initially, the reactions were carried out by incubatingthe (RS)-organoseleno alcohols 1–3 with 1–5 g of wholecells of A. terreus URM 3371 at pH=4 and at 32 °C in thepresence of ethanol as an additive for a reaction periodof up to 10 days. For all experimental conditions investi-gated, the compound (RS)-3 was inactive when in con-tact with the whole cells of A. terreus. The most efficientderacemization was achieved with compound (RS)-1.When 2–5 g of whole fungal cells were employed, (+)--(R)-1 was obtained in high conversion rate and selectiv-ity (entries 6–15 for compound 1 in Table 1 and Fig. 1).In contrast, (RS)-2 was not efficiently deracemized by A.terreus URM 3371 under the employed conditions, theorganoseleno acetophenone 2a being the main product(entries 6, 8–9, 11–12, 14 and 15 for compound 2 in Table1), while (S)-2 was obtained only with modest selectiv-ity.In the light of these preliminary results, the bio-transformation of (RS)-1 and (RS)-2 was performed at 32°C using the two fungal strains, A. terreus URM 3371and A. terreus CCT 4083, in buffer solutions at pH=4 and7 (Table 2). The most efficient conversions were obtainedwith A. terreus URM 3371 using (RS)-1 as substrate (en-tries 1–6 for substrate 1 in Table 2). Under these condi-tions, (RS)-1 was deracemized to yield (+)-(R)-1 in highoptical purity (e.e.³98 %) at both pH, 4 and 7. Apprecia-bly lower deracemization of (RS)-1 was obtained usingA. terreus CCT 4083, particularly under neutral condi-tions (entries 10–12 for substrate 1 in Table 2), the majorproduct being the p-(organoseleno)acetophenone 1a. Themicrobial deracemization of (RS)-2 by A. terreus URM3371 led to the formation of (S)-2 with low selectivity417L.F. ASSIS et al.: Deracemization of (RS)-Organoseleno Alcohols by A. terreus, Food Technol. Biotechnol. 45 (4) 415–419 (2007)Table 1. Deracemization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) and (RS)-1-[(4-ethylselanyl)phenyl]ethanol (2) by whole cellsof Aspergillus terreus URM 3371 at pH=4 and 32 °C in the presence of 200 mL of ethanolEntrym(fungalcell)/gt/dayReaction products with 5 mg of compound 1 Reaction products with 5 mg of compound 21a 1 2a 2ca/% ca/% e.e./% A.C. ca/% ca/% e.e./% A.C.113 – – – – 12 88 17 S2 5 15 85 41 R 48 52 20 S3 10 19 81 56 R 26 74 10 S423 42 58 47 R 63 37 19 S5 5 72 28 98 R 44 56 18 S6 10 17 83 98 R 89 11 30 S733 11 89 30 R 40 60 2 S8 5 11 89 99 R 74 26 42 S9 10 0 98 99 R 79 21 60 S1043 0 98 98 R 55 45 14 S11 5 0 98 98 R 72 28 9 S12 10 0 98 98 R 81 19 53 S1353 0 98 99 R 57 43 28 S14 5 0 98 99 R 70 30 9 S15 10 0 98 99 R 98 0 0 –aconcentration determined by GC analysise.e.=enantiomeric excessA.C.=absolute configurationFig 1. GC chromatograms showing the progress of the derace-mization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) by wholecells of Aspergillus terreus URM 3371 (2 g) at pH=4 and 32 °C;e.e. enantiomeric excessand was accompanied, under both acidic and neutralconditions, by high levels of conversion to the ketone 2a(Table 2). With A. terreus CCT 4083, a low degree ofderacemization of (RS)-2 was observed at both pH (4and 7), and 2a was the major product (entries 7–12 forsubstrate 2 in Table 2).The deracemization of (RS)-1–2 was performed usingA. terreus URM 3371 in the presence of other additives,namely propanol, butanol, and cyclohexanol, and at dif-ferent temperatures (20 and 32 °C). Under these condi-tions the deracemization of alcohol (RS)-2 was not effi-cient, the organoseleno acetophenone 2a being observedin high concentration (Table 2). Under the same condi-tions (RS)-1 gave (+)-(R)-1, but with low enantioselecti-vity in most cases (Table 3). It is concluded that the pre-sence of the additives promoted preferentially the oxi-dation of the organoseleno phenylethanols to the cor-responding acetophenones.Since the most efficient deracemization of (RS)-orga-noseleno alcohols was obtained when whole cells of A.terreus URM 3371 were used with (RS)-1 as substrate,the reaction was carried out on a preparative scale inwhich 15 g of wet fungal cells were incubated at 32 °C,pH=4.0, with 100 mL of 1 and 600 mL of ethanol. After 18days, (+)-(R)-1-[(4-methylselanyl)phenyl]ethanol could beisolated in 53 % yield (0.0163 mmol/g of biomass) andthe product exhibited an e.e.>98 %.418 L.F. ASSIS et al.: Deracemization of (RS)-Organoseleno Alcohols by A. terreus, Food Technol. Biotechnol. 45 (4) 415–419 (2007)Table 2. Deracemization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) and (RS)-1-[(4-ethylselanyl)phenyl]ethanol (2) by whole cells(2 g) of Aspergillus terreus at 32 °C in the presence of 200 mL of ethanolEntry pHt/dayReaction products with 5 mg of compound 1 Reaction products with 5 mg of compound 21a 1 2a 2ca/% ca/% e.e./% A.C. ca/% ca/% e.e./% A.C.Aspergillus terreus URM 3371143 0 98 99 R 43 57 13 S2 5 8 92 99 R 62 38 38 S3 10 0 99 99 R 82 18 18 S473 40 60 56 R 65 35 44 S5 5 0 98 99 R 77 23 24 S6 10 0 98 99 R 99 0 0 –Aspergillus terreus CCT 4083743 45 55 56 R 33 67 2 R8 5 60 40 98 R 70 30 46 S9 10 81 19 98 R 88 12 99 S1073 75 25 99 R 99 0 0 –11 5 80 20 65 R 90 10 12 R12 10 82 28 49 R 98 0 0 –aconcentration determined by GC analysise.e.=enantiomeric excessA.C.=absolute configurationTable 3. Deracemization of (RS)-1-[(4-methylselanyl)phenyl]ethanol (1) by whole cells (2 g) of Aspergillus terreus URM 3371 at pH=4in the presence of 200 mL of butanol, propanol or cyclohexanolEntry Additive t/dayProducts of the reaction at 32 °Ca Products of the reaction at 20 °Ca1a 1 1a 1cb/% cb/% e.e./% A.C. cb/% cb/% e.e./% A.C.1Butanol3 37 67 47 R 14 86 12 R2 5 46 54 25 R 28 72 23 R3 10 45 55 99 R 59 41 56 R4Propanol3 43 57 17 R – – – –5 5 27 73 32 R – 98 54 R6 10 – – – – 17 83 51 R7Cyclo-hexanol3 – 98 4 R – 98 8 R8 5 – 98 17 R – 98 6 R9 10 – – – – – 98 55 RaThe mass of the substrate 1 employed was 5 mgbconcentration determined by GC analysise.e.=enantiomeric excessA.C.=absolute configurationConclusionsThis study demonstrated that fungal strains of A.terreus, isolated from different provenances in Brazil, of-fer a potential for deracemization of (RS)-organoselenoalcohols. Under specific conditions, whole cells of Asper-gillus terreus catalysed the deracemization of (RS)-1-[(4--methylselanyl)phenyl]ethanol (1) to give an acceptableyield of (+)-(R)-1 with high enantiomeric excess (>98 %).The deracemization reactions were sensitive to pH, aci-dic medium providing the best conversion. The presenceof additives promoted mainly the oxidation of the sec-ondary alcohols.AcknowledgementsThe authors thank the Brazilian Culture Collectionsof the Botanical Institute of São Paulo, SP, Brazil (http://www.ibot.sp.gov.br) and to the Federal University of Per-nambuco (Recife, PE, Brazil) for donations of the fungalstrains A. terreus CCT 4083 and A. terreus URM 3371. A.L. M. Porto, L. H. Andrade and J. V. Comasseto thankFAPESP and CNPq (Proc. 472663/2004-6) for support.References1. K. Faber, W. Kroutil, New enzymes for biotransformations,Curr. Opin. Chem. Biol. 9 (2005) 181–187.2. N.J. Turner, Enzyme catalysed deracemization and dyna-mic kinetic resolution reactions, Curr. Opin. Chem. Biol. 8(2004) 114–119.3. T. Back: Organoselenium Chemistry, Oxford University Press,Oxford, UK (1999).4. M. Tiecco: Organoselenium Chemistry: Modern Develop-ments in Organic Synthesis (Topics in Current Chemistry).In: Electrophilic Selenium, Selenocyclizations, T. Wirth (Ed.),Springer-Verlag, Heidelberg, Germany (2000).5. L.H. Andrade, A.T. Omori, A.L.M. Porto, J.V. Comasseto,Asymmetric synthesis of arylseleno alcohols by means ofthe reduction of organoseleno acetophenones by whole fun-gal cells, J. Mol. Catal. B-Enzym. 29 (2004) 47–54.6. J.V. Comasseto, A.T. Omori, A.L.M. Porto, L.H. Andrade,Preparation of chiral organochalcogeno-a-methylbenzyl al-cohols via biocatalysis. The role of Daucus carota root, Tet-rahedron Lett. 45 (2004) 473–476.7. C.E. Da Costa, G.C. Clososki, H.B. Barchesi, S.P. Zanotto,M.G. Nascimento, J.V. Comasseto, Enzymatic resolution of(RS)-b-hydroxy selenides in organic media, Tetrahedron: Asym-metry, 15 (2004) 3945–3954.8. A.T. Omori, L.F. Assis, L.H. Andrade, J.V. Comasseto, A.L.M.Porto, Enantiomerically pure organoseleno-1-arylethanolsby enzymatic resolution with Candida antarctica lipase – No-vozyme 435, Tetrahedron: Asymmetry, 18 (2007) 1048–1053.9. J.V. Comasseto, L.H. Andrade, A.T. Omori, L.F. Assis, A.L.M.Porto, Deracemization of aryl ethanols and reduction of ace-tophenones by whole fungal cells of Aspergillus terreus CCT4083, A. terreus CCT 3320 and Rhizopus oryzae CCT 4964, J.Mol. Catal. B-Enzym. 29 (2004) 55–61.10. J.V. Comasseto, L.F. Assis, L.H. Andrade, I.H. Schoenlein--Crusius, A.L.M. Porto, Biotransformations of ortho-, meta-and para-aromatic nitrocompounds by strains of Aspergil-lus terreus: Reduction of ketones and deracemization of al-cohols, J. Mol. Catal. B-Enzym. 39 (2006) 24–30.11. R.M.V. Assumpção, T. Morita: Manual of Solutions, Reagentsand Solvents, University of São Paulo, São Paulo, Brazil(1968).419L.F. ASSIS et al.: Deracemization of (RS)-Organoseleno Alcohols by A. terreus, Food Technol. Biotechnol. 45 (4) 415–419 (2007)

Deracemizacija (RS)-1-[(4-metilselanil)fenil]etanola i (RS)-1-[(4-etilselanil)fenil]etanola s pomoću sojeva Aspergillus terreus

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Deracemizacija (RS)-1-[(4-metilselanil)fenil]etanola i (RS)-1-[(4-etilselanil)fenil]etanola s pomoću sojeva Aspergillus terreus

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