Latacz, Gniewomir

Pękala, Elżbieta

Wójcik, Tomasz

BioTechnologia

English

Jagiellonian Univeristy Repository

PRACE EKSPERYMENTALNEAdres do korespoadoK^Elżbieta Pękala, Department of Technology and Biotechnology of Drugs,Medical College of jagiellonian Unherslty, ul. Medyczna 9,30-688 Kraków; e-mail:mipekala@cyf-kr.edu.plbiotechnologia2 C77) 174-180 2007Enantioselective bioreduction of propentofylline using yeast in water and organic solventsElżbieta Pękala, Gniewomir Latacz, Tomasz WójcikDepartment of Technology and Biotechnology of Drugs,Medical College of jagiellonian University, KrakówEnantioselective bioreduction of propentofylline using yeast in water and organic solventsSummaryPropentofylline (PPT) is a drug used in the treatment of both vascular de­mentia and Alzheimer type dementia. Hydroxy-metabolites of propentofylline (OHPPT) also demonstrate the same biological activity as the parent compound. As steroisomers of HOPPT are not commercially available, we had to produce them for pharmacological and pharmacokinetic studies. The aim of this study was to find the strains of Saccharomyces cerevisiae yielding enantiomerically pure (R)- or (S)-l-(5-hydroxyhexyl)-3-methyl-7-propylxanthin (OHPPT) from PPT. In this paper, we present the results of stereoselective reduction of PPT into OHPPT when catalysed by whole cells of baker’s and a few strains of wine yeast in water and organic solvents.Key words:propentofylline, metabolites of propentofylline, Saccharomyces cerevisiae, enantiomeric excess.1. IntroductionPropentofylline (PPT, 3-methyl-l-(5-oxohexyl)-7-propyl-xan- thine) has been reported to be beneficial in the treatment of both vascular dementia and Alzheimer type dementia (1-4). PPT increases the solubility of lipids by substituting a methyl group for a propyl one in position 7 of the purine backbone of pentoxi­fylline. The pharmacological effects of PPT may be observed inEnantioselective bioreduction of propentofylline using yeast in water and organic solventsSaccharomyces coravisiaeFig. Bioreduction pathway of propentofylline by yeast.Stimulation of the nerve growth factor, increased cerebral blood flow and inhibition of adenosine uptake. PPT also enhances extracellular adenosine concentrations and decreases extracellular levels of glutamate in vivo during ischemia (5,6).To date, despite the known pharmacological effects, few clinical pharmacokinetic or metabolism studies of PPT have been reported in the literature. The short half-life of PPT at the terminal elimination phase and poor bioavailability, after oral administration to rabbits, suggest that this drug undergoes extensive first-pass me­tabolism in liver. PPT is metabolised in vitro to the racemic compound - (±)-l-{5-hydroxyhexyl)-3-methyl-7-propyl-xanthine (HOPPT) (7-10).Studies have shown that the racemic mixture and the steroisomers of HOPPT demonstrate biological activity. Hydroxy-metabolites of PPT inhibited [^HJnitro- benzylthioinosyne binding in rat brains with a similar affinity to propentofylline, and also inhibited [^HJadenosine uptake by es transport as effectively as propento­fylline. Since inhibition of adenosine transport appears to be important for the neuroprotective effects of PPT, the hydroxy-metabolites may also provide neuro­protection. As steroisomers of HOPPT are not commercially available, we had to produce them for pharmacological and pharmacokinetic studies.In this paper, we present the results of stereoselective biotransformation of PPT to HOPPT when catalysed by whole cells of baker’s and a few strains of wine yeast in both water and organic solvents. Alcohol dehydrogenases (YADH’s) in yeast {Saccharomyces cerevisiae) play an important role in the reduction of carbonyl func­tions in aldehydes and ketones (11). YADH has very narrow substrate specificity and generally accepts only aldehydes and methyl ketones. It is therefore of only limited use in the preparation of the chiral secondary alcohols.Microbiological reduction with yeast was carried out under non-fermenting con­ditions. The stereoselectivity of this biotransformation process was determined us­ing the HPLC technique with a chiral column. It was established that different yeast strains favoured the biotransformation of PPT into different enantiomers.BIOTECHNOLOGIA 2 (77) 174^180 2007 175Elżbieta Pękala, Gniewomir Latacz, Tomasz Wójcik2. Materials and methods2.1. ChemicalsPropentofylline was obtained from Intervet GmbH, Germany; n-hexane and 2-propanol of HPLC grade were purchased from Merck KgaA Darmstadt, Germany. Other reagents were all of analytical grade.2.2. MicroorganismsFive strains of Saccharomyces cerevisiae: KKP13, KKP35, KKP82, KKP295, KKPU were obtained from the Collection of Productive Microorganisms, Department of Technical Microbiology and Biochemistry in Warsaw, Poland; another two strains: L’hirondelle and Wołczyn were taken from Lesaffre Biocorporation in Warsaw, Po­land; and Krakowskie was obtained from Baker’s Company in Cracow, Poland.2.3. Chemical synthesisl-(5-R,S-hydroxyhexyl)-3-methyl-7-propylxanthine (HOPPT) - racemic standard.This compound was synthesised from PPT using sodium borohydride reduction. Briefly, 140 mg (0.5 mmol) of PPT was dissolved in 10 ml of methanol and 160 mg (2.5 mmol) of sodium borohydride was added. The mixture was stirred overnight at room temperature. The progress of the reaction was followed using thin layer chro­matography (benzene:acetone 1:1). After solvent evaporation, the reaction product was extracted with methylene chloride. The organic layer was washed three times with saturated sodium chloride solution and concentrated under reduced pressure. The residue was recrystallized from ethyl ether. M.p. 77-79°C; 90% yield; HPLC: tr = 14.50; 17.25 min;IR(KBr) [cm-i]:3372 (OH), 1699 (CO), 1645 (CO); ’HNMRBOOMHz [DMSOde): 6 [ppm]: 0.83 (t, J = 7.2 Hz, 3H, CH^-CH), 1.03 (d, J=6.3 Hz, 3H, CH-CH3), 1.30 (m, 4H, CH2-CH2-CH2-CH2), 1.50 (m, 2H, CH2-CH2-CH2), 1.78 (q, J=7.4, 4H, CH- CH2- CH2), 3.43 (s, 3H, N-CH3), 3.56 (q, J = 5.5 Hz, IH, CH), 3.85 (t,J=7.2 Hz, 2H, N-CH2-CH2), 4.21 (t, J=7.1 Hz, 2H, N-CH2-CH2), 4.33 (d, J=7.2 Hz, IH, OH), 8.09 (s, IH, lm”-H).176 PRACE EKSPERYMENTALNEEnantioselective bioreduction of propentofylline using yeast in water and organic solvents2.4. Microbiological methods2.4.1. Biotransformation of PPT in an aqueous mediumIn a typical experiment, 2 g of wine yeast (KKP) or 5 g of baker’s yeast were sus­pended in 150 mL of phosphate buffer. After stirring for 30 min at 30°C, 5 g of glu­cose and 235 mg (1 mmol) of PPT were added. After stirring for 7 days at 30°C, the yeast was filtered off. The filtrate was extracted three times with 100 mL of methy­lene chloride and dried over Na2S04. The residues obtained from the evaporation of the solvent were either dissolved in 2 mL 2-propanol for HPLC analysis or purified using silica gel chromatography.2.4.2. Biotransformation of PPT in organic mediaDry baker’s or wine yeast (2 g) was suspended in 25 mL of the organic solvent and 2 mL of tridestilled water was added. After the addition of 58 mg (0.25 mmol) of PPT the suspension was stirred for 3 days at 27®C. The yeast was then filtered off, the solution concentrated in vacuo, and the residue was dissolved in 2.0 mL of 2-propanol for analytical purposes.2.5. Methods of analysisPolarimetry: Measurement of the optical rotation of the products of the stereoselective reduction PTX was carried out on a Digital Polarimeter - DIP 2000 (Jasco Inc., Japan). The optical rotations R- and S-1-(5-hydroxyhexyl)-3-methyl- -7-propylxanthine were in accordance with the literature values (11).Spectroscopy: NMR spectra for the racemic standard and each enantiomerwere recorded using a Varian VM 300 Hz instrument using tetramethylsilane (TMS) as an internal standard. The IR spectra were recorded in KBr pellets on a jasco Spec­trometer FT/lR-410.High performance liquid chromatography: The identification of the starting ma­terials and reduction products as well as the yields of transformation and enantiomeric excess were determined by HPLC analysis on a Daicel ChiralPack AD Column. The high-performance liquid chromatograph (Dionex Corporation, USA) consisted of an isocratic solvent delivery system (Dionex HPLC Pump Series P580), an inlet equipped with a 20 pL loop and a variable wavelength UV detector (model Dionex UVWIS detector UVD 170S/340S), set at 275 nm. The analytical chiral column was 250 X 4.6 mm i.d. Daicel ChiralPack AD (Chemical Industries, France). The tem­perature was set at 25°C. The mobile phase was n-hexane:2-propanol 780:220 perBIOTECHNOLOGIA 2 (77) 174-180 2007 177Elżbieta Pękala, Gniewomir Latacz, Tomasz WójcikIL phase, vacuum-degassed before use, with a flow rate of ImL The analytes were disolved in 2-propanol (at a concentration of Img/mL).Chromatographic characteristics: Chromatographic data are given in Table 1.Table 1Chromatc^raphic dataki k2 a Rs -A(AG°) 0- mol-i)3.02 4.02 1.33 2.25 630.53The chromatognęhlc conditions: n-hexane/2-propanol 780/220 (v/?);flow rate 1.0 mL/min (isocratic); column temperature 25°C; UV detection at 275 nm; injection 20 The separation factor (a) was expressed as a = k2 /ki, where kj, kj are the retention factors for the first and second elu­ting enantiotner.The retention factors ki and k2 were calculated as follows:hi = (t*i - to) / to and k2 = (tn2-to) / to, where to, tRi and t*2 are the dead elution times of enantiomers.The resolution (lO of the first and second eluting enantiomers was calculated by the ratio of the difference between the elu­tion times tm and tR2 to the arithmetic mean of the two peaks’ widhts wi and W2.R, = 2(tR2 - tRi) / (wi + W2)The difference in the free energy (-A(AG°)) was calculated from the separation factor according to the following equ- ation:-A(AG“) = RT Ina3. Results and discussionl-(5-Oxohexyl)-3-methyl-7-propylxanthine (PPT) was reduced with NaBH4 to R,S-l-(5-hydroxyhexyl)-3-methyl-7-propylxanthine (HOPPT), which was used as the reference standard in further analytical studies.The aim of this study was to find the strains of Saccharomyces cerevisiae yielding enantiomerically pure (R)- or (S)-l-(5-hydroxyhexyl)-3-methyl-7-propylxanthine from PPT. Microbiological reduction using yeast was carried out under non-fermenting conditions. In Table 2, a summary of the results obtained using aqueous and organic media is given. PPT was reduced using baker’s yeast (Saccharomyces cerevisiae: strains - L’hirondelle, Wołczyn and Krakowskie), in an aqueous medium according to Prelog’s rule (12) to give an (S)-alcohol with a good optical purity only in the case of the L’hirondelle strain, with an ee of 78% for the (S) enantiomer. Bioreductions using baker’s yeast were also performed in organic solvents such as ethyl acetate and n-hexane. Table 2 shows that in several cases such reductions can be carried out quite successfully. However, in organic media yields of the reduction of PPT to HOPPT were quite low (from 1 to 23%). In ethyl acetate, all the baker’s strains pre­ferred to form an (S) isomer, whereas in the n-hexane L’hirondelle strain an (R) iso­mer was preferred. Drastic solvent dependent ee variations were found for the re­178 PRACE EKSPERYMENTALNEduction of PPT using the L’hirondelle strain: 78% ee in favour of the (S) enantiomer in water and 100% ee favouring the (R) enantiomer in n-hexane.Enantioselective bioreduction of propentofylline using yeast in water and organic solventsTable 2Bloconversion of PPT to OHPPT In water and in organic solventsStrainsYields in water Yields in AcOEt Yields in n-hexane[%] ee[%] l%] ee[%] [%] ee[%]KKP 13 32 9 (R) nd ndKKP 35 48 50 (R) nd ndKKP 82 94 67 (R) nd ndKKP 295 75 31 (S) nd ndKKPU 59 14 (R) nd ndL’hirondelle 67 78 (S) 10 100 (S) 1 100 (R)Wołczyn 37 42 (S) 23 100 (S) 2 100 (S)Krakowskie 41 44 (S) 1 100 (S) 1 100 (S)nd: not determinedPPT was also bioreduced in the presence of wine yeast in an aqueous medium contrary to Prelog’s rule to give the (R)-alcohol, as is the case with reduction using LKADH (alcohol dehydrogenase from Lactobacillus kefiri) (13,14). The bio­transformation of PPT in water using wine yeast gave (R) HOPPT with variable enantiomeric excesses (ees from 14 for KKPU to 67% for KKP82), and yields ranging from 32 to 94%. Only in the case of biotransformation with KKP295, an (S) isomer (ee=31%) was obtained. The KKP82 strain was found to most favour the production of (R) HOPPT.4. ConclusionsThe above results demonstrate that the reduction of PPT in ethyl acetate with the yeast strain Wołczyn offers a new highly stereoselective method of (S)-l-(5-hydroxyhexyl)-3-methyl-7-propylxanthine preparation. Other reduction con­ditions employed were less effective.BIOTECHNOLOGIA 2 (77) 174-180 2007 179Elżbieta Pękala, Gniewomir Latacz, Tomasz WójcikAcknowledgementsThe research was supported by a scholarship from the Rector’s Fund of the Jagiellonian University. The authors are grateful to Intervet Company for a free sample of PPT and P. Zamojski for wine yeast supply.Literature1. Plaschke K., Grant M., Weigand M. A., Ziichner J., Martin E., Bardenheuer H. j, (2001), Br. J. Pharma­col.. 133, 107-116.Koriyama Y., Chiba K., Mohri., (2003), Eur. J. Pharm., 458, 235-241.Chauhan N. B., Siegel G. j., Feinstein D. L., (2005), Neuropharmacology, 48, 93-104.Ringheim G. E., (2000), Ann. N. Y. Acad. Sci., 903, 529-534.Raghavendra V., Tanga F., Rutkowski M. D., DeLeo j. A., (2003), Pain, 104, 655-664. Raghavendra V., Tanga F., DeLeo J. A., (2004), Nuuropsychopharmacology, 29, 327-334. Wirtz-Brugger F., Giovanni A., (2000), Neuroscience, 99 (4), 737-750.Kuroda N., Hamachi Y., Aoki N., Wada M., Tanigawa M., Nakashima K., (1999), Biomed. Chromat., 13(5), 340-343.Minami M., Arai H., Takahashi T., Kimura M., Noguchi 1., Suzuki T., Inoue R., (1995), Prog. Neu- ro-Psychopharmacol. and Biol. Psychiat., 19, 59-64.Kwon 0., Ryu j-C., (2000), Arch. Pharm. Res., 23 (4), 374-380.Furrer H., Gebert U., Rudolph! K., (Hoechst, Frankfurt am Main, Germany), (1995), US Patent 5, 407, 815.12. Faber K,, (2000), Biotransformations in Organic Chemistry '♦*ed., 2,160-184, Springer Verlag, Berlin.13. Amidjojo M., Franco-Lara E., Nowak A., Link H., Weuster-Botz D., (2005), Appl. Microb. Biotechnol., 69, 9-15.14. Ernst M., Kaup B., Miiller M., Bringer-Meyer S., Sahm H., (2005), Appl. Microbiol. Biotechnol., 66, 629-634.2.3.4.5.6.7.8.9.10.11.180 PRACE EKSPERYMENTALNE

Enantioselective bioreduction of propentofylline using yeast in water and organic solvents

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Enantioselective bioreduction of propentofylline using yeast in water and organic solvents

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