Reacciones- Red-ox Dr. Sinisterra Biotransformations Group Faculty of Pharmacy Universidad Complutense www.biotransformaciones.com Metodologías para reducir compuestos carbonílicos E3 E4 HO H HR HS O + N ADPR Alcohol S E1 Cara re E2 HS O HR H OH + N ADPR Cara si Cara si Alcohol R Método fotoquímico de regeneración de la coenzima LUZ Captura de la luz por la célula H2O Sistema de transferencia electrónica + NADPH O O2 NADP H OH X CO2 S 96-98% e.e. X Ciclo de Calvin Cianobacteria X= o-, m-, p- Clo-, m-, p-F; o-, m-, p-Me Nakamura et al. 2003, 14, 1659-2681 Comp. Orgánicos Los métodos experimentales para hacer la reducción se pueden clasificar en I)Búsqueda del biocatalizador. a.Screening b.Sobre expresión c.Evolución dirigida d.Anticuerpos catalíticos II)Modificación de sustratos III)Optimización de las condiciones de reacción a.Temperatura de reacción b.Ingeniería del disolvente. Fluidos supercríticos, líquidos iónicos, disolventes orgánicos, adsorbentes c.Tratamiento de la células: alteración de la permeabilidad con acetona o DMSO d.Empleo de Inhibidores enzimáticos Sustrato reducido Sustrato oxidado Reduction of C=O Using two enzymes NAD(P)H/H + NAD(P)+ Sustrato auxiliar reducido Sustrato auxiliar oxidado L S Reductase O Cl O Cl O NAD(P)H/H+ O HO H R NAD(P)+ CO2 + "H2" O H H OH H OH HO H H H+ O- Formiate dehydrogenase HO HO O OH O Glucose dehydrogenase HO HO HO H H OH H OH RS O cara Re RL RL Grupo grande Me Et Rs Grupo pequeño Me nBu Rs RL e.e.(%) S 67% S 82% CF3 CH3 S > 80% cara Si E3 E4 HO H HR HS O + N ADPR Alcohol S E1 Cara re E2 HS O HR H OH + N ADPR Cara si Cara si A lcohol R Esquema cinético de reducción cetonas proquirales NADH K2 Enz-A K1 K-1 K3 Enz-A-NADH K-2 NADH K-3 K'-1 NADH K'3 K'2 Enz-A' K'-2 NADH K4 K-4 Enz-NAD Enz + A K'1 Enz-R-NAD Enz-A'-NADH K' -3 K'-4 K'4 Enz-S-NAD R Enz + NAD S Tabla 6. Parámetros cinéticos de las oxido-reductasas aisladas.125 KM (mM) Kcat (s-1) pHopt. Tªopt. (ºC) D-Enzima-1 0.59 ----- 8.5 45 D-Enzima-2 4.54 303 6.5 45 L-Enzima-1 0.13 6.20 6.5 55 L-Enzima-2 0.15 4.66 5.5-6.0 40 Enzima HO H D-Enzima-2 O Cl OEt S O Cl O OEt L-Enzima-1 L-Enzima-2 H OH O Cl OEt R HO D-Enzima-2 Cl O O Cl O H Naftil S Naftil L-Enzima-1 L-Enzima-2 H OH Cl O R Naftil Tabla 5. Reducción enzimática de 4-cloro-3-oxo-butanoato de etilo. Pm (KDa) Act. total (U) Config. alcohol e.e. (%) D-Enzima-1 25 26 S > 99 % D-Enzima-2 1600 1442 S > 99 % L-Enzima-1 32 246 R > 99 % L-Enzima-2 32 3108 R > 99 % Enzima Nakamura y cols Figura 1.9. Alcohol deshidrogenasa de hígado de caballo. Protein Data Bank. www.pdb.org His 190 NH---- Figura 1.13. En el estado APO, en el centro activo de la enzima existen 3 moléculas de agua ligadas a aminoácidos de la maquinaria catalítica. HN O N pKa=6.7 2.7 A Ser 138 OH Tyr 151 H O H HO H O pKa= 9-9.5 2.7 A H 2.7 A +H3N H Lys 155 O H Protón cedido al medio Figura 1.12. Mecanismo de reacción de las alcohol deshidrogenasas Pro-47/His 47. Cesión al medio de un hidrógeno mediante formación de una molécula de agua. H N NAD+ N R HO His 47 H HO O O Tyr-48 N + H H2N O H O H H Zn+2 H N O N SH R2 CyS-46 HS R1 His67 Cys-178 His 190 NH---- HN Figura 1.14. Estado del NAD+ en el sitio activo unido a la enzima formando el complejo binario y unido a una molécula de agua O N pKa=6.7 Tyr 151 OH Ser 138 2. 5 A H H O H O -O pKa= 7.6 3.7 A 4.5 A H2N +N H NAD + O (R) O +H2N (R) O R H Lys 155 His 190 NH---- HN Figura 1.15. Complejo ternario Enzima-NAD+-Alcohol O N pKa=6.7 Tyr 151 OH Ser 138 R1 O R2 Alcohol R1>R2 O H 2N H -O H pKa= 7.6 H (E) (Z) N H NAD + O (S) O +H2N (R) O R H Lys 155 NH---- HN His 190 Figure 1.1.6- Complejo ternario Enzima -NAD + - Cetona O N pKa=6.7 Al exterior del centro activo R1 R2 Tyr 151 OH Ser 138 2.5 A O 2.6 A O H2N H H O pKa= 10 H (E) (Z) N H NAD + O (S) O +H2N (R) O R H Lys 155 His 190 Figura 1.17- Centro activo después de liberar la cetona, quedando en su lugar una molécula de agua NH---- HN O N pKa=6.7 Tyr 151 OH Ser 138 H H O HO O H2 N H pKa= 10 H (E) (Z) N H NAD H O (S) O +H2N (R) O R H Lys 155 Figura 1.20. Disposición estérica de los oxígenos del Asp-150 unido al Zn(II). HO OH NADH O N Cys 37 NH2 His 59 HS Hs O N N Zn(II) HR H O O- O -O Asp 150 Glu 60 HO OH (S) Figura 1.22. Se aprecian 2 moléculas de agua unidas al Zn (II). (S) NADH O N NH2 (E) (Z) Hs H O H Hr O H O O H His 67 H Ser 48 Zn(II) N S H Cys 46 N H Tabla 3.5. Microorganismos seleccionados como potenciales biocatalizadores para la reducción de cetonas. Conversiones medias reducción de ciclohexanona. Colección & código Nº Ref. Bibliográficas[1 ] especie (género) Origen Gongronella butleri (Absidia butleri) CBS 157.25 8 (176) Indonesia H. filamentoso 95 Monascus kaoliang CBS 302.78 1 (139) Taiwan H. filamentoso 86 Diplogelasinospora grovesii IMI 171018 1 (1) Japón H. filamentoso 85 Schizosaccharomyces octosporus NCYC 427 4 (4090) India Levadura 84 Absidia glauca CBS 100.48 27 (176) Alemania H. filamentoso 79 Pyrenochaeta oryzae IMI 195679 0 (33) Swazilandia H. filamentoso 70 Neosartorya hiratsukae CBS 294.93 1 (48) Japón H. filamentoso 66 Echinosporangium transversale CBS 357.67 1 (1) Nevada USA. H. filamentoso 55 Actinoplanes sp. DSMZ 43031 (144) - Actinomicet os 53 Nocardia uniformis ATCC 21806 3 (1106) - Actinomicet os 48 Cepa a Media de las conversiones obtenidas en los tres ensayos. (-) Procedencia desconocida J.D. Carballeira- Tesis Doctora. Fac. Farmacia . UCM 2003 Grupo % conv. a CoMFA model for reduction of ketones CARBALLEIRA et al. High throughput screening and QSAR-3D/CoMFA : Useful tools to design predictive models of substrate specificity for biocatalysts Molecules 2004, 9 pp 673-693 A) B) The CoMFA models obtained for depict the substrate requirements common to G. candidum (A) and S. octosporus (B) During the evaluation of the results obtained in the reduction reactions and the CoMFA models we concluded that the pattern of substrate affinity was similar between the ADHs from different strains. This fact was astonishing as far as ADHs present in microbial strains – distant from the taxonomic point of view - were showing considerable similarities in substrate scope. In the bibliography different genetic and biochemical publications pointed out the structural relationships between the different ADHs, with special homology at the active site and even the hypothesis about the existence of a common ancestor based in structural studies of different proteins of the same family. The building of 3D-QSAR/CoMFA models has several potential utilities that justify the use of this theoretical method for biotransformations and directed evolution, especially when there is no reference structure of the enzyme available (Carballeira et al 2006). Indeed, when an enzyme or library of enzymes is screened against a collection of substrates it is not easy to have a 3D overview of the capabilities of the enzyme. CoMFA is a good option to display a comprehensive model from the large amount of data generated that could serve as reference for the rational interpretation of the results and furthermore for the selection a priori of new potential substrates. Nowadays, new biocatalysts could be created by directed evolution enhancing the activity and selectivity of a known enzyme structure (Reetz and Carballeira 2007) and overcoming some of the catalytic restrictions of the natural protein. In this way CoMFA models are especially interesting when the x-ray structure of the enzyme is not available with the substrate docked at the active site because CoMFA models generated from different docking ways of the ligands may help in the proper location of the substrate in the binding site. CoMFA models are predictive. Indeed, to test this predictiveness of the model, we tried to hypothesize the activity of the biocatalysts against two new cyclic substrates: pulegone and menthone (Carballeira et al 2006). The substrates must be aligned according to the fitting model. After the fitting we observe the CoMFA zones occupied by these substrates. We can see that menthone fit into the model and so, it can be reduced by the ADHs. Indeed 52% yield of menthol – the only product - is achieved. According to the model, pulegone would not be reduced by the strains, due to the fitting of the double bond in an electrostatic zone where the presence of negative charge is not allowed (blue zone) (previous slide). Using the same model we can predict that acetophenone or α- bromo-acetophenone would be reduced by Rhodotorula sp AS 2241 giving (S)-1-phenylethanol (34.7% yield, >99.5% e.e) and (R )-2-bromo-1-phenylethanol (20% yield, > 99% e.e.) as predicted by our CoMFA model. This result indicates the evolutive conservation of active site of ADHs. O Pulegone O Menthone Table 5- Effect of the reaction conditions on the selectivity of reduction of the 1-acetoxy-3-phenoxy`ropan-2-one by Baker’s yeast. Substrate: 500mg; sodium phosphate buffer (0.15M), 200ml; yeast wet 12g o 2.5 g lyophilized. Co-substrate glucose: 5g. . Ar Baker's yeast OAc O Ar O Yeast Aryl group Wet caked O OAc HO H pH Time (h) Yield(%) e.e(%) S>R Ph Non controlled 3 n.r. 52 Wet caked Ph 7 1 84 80 Wet caked Ph 8 1 n.r 78 Lyophilized Ph 7 1.5 n.r 64 Wet caked 2-iPrphenyl 7 1 36 28 3-Cl-phenyl 7 2 38 93 4-Cl-phenyl 7 2 65 61 2-Me-phenyl 7 2 32 65 3-Me-phenyl 7 2 66 77 4-Me-phenyl 7 4 36 52 2,6-diMe-phenyl 7 1.5 60 >95 2,4,6-dri-Mephenyl 7 6 58 95 •Egri et al Baker’s yeast mediated steoselective biotransformation of 1-acetoxy-3-aryloxypropan-2-ones. Tetrahedron: Asymmetry 9, 271-283.(1998) Table 6.- Reduction of 1-chloro-3(1-naphthyloxy)propan-2-one using different microorganisms . Growing or resting cells conditions (Martinez-Lagos et al 2004). Resting cells b Fermentor conditions a Microorganism Yield. c (%) Enantiopref.d e.e.c (%) M. kaoliang 55 R>S 1 5 R>S 10 N. hiratsukae 39 R>S 6 10 R>S 90 Y. lipolytica CECT 1240 20 S>>R 99 87 S>>>R 99 P. mexicana CECT 11015 19 R>>S 85 85 R>>>S 95 S. bayanus CECT 1969 15 R>S 52 40 R>>S 87 S. cerevisiae (Type II) 12 S>R 70 30 S>>R 70 S. cerevisiae CECT 1317 13 R>S 70 27 R>S 71 a Reductions Yield. Enantiopr e.e.c c (%) ef.d (%) were carried out with growing cells (48 hrs.). were carried out using 0.19 mmol of substrate and 4 g of sucrose in 25 mL of reaction media (0.1M sodium phosphate buffer, pH 8.0). Incubations were performed at 30 ºC for 48 h. c Molar Conversion and e.e. determined by chiral HPLC . d Absolute configuration of the halohydrin was assigned by specific rotation value. b Reductions The synthesis of (R)-3-Fluoro-4-[[hydroxyl(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl – 2 –naphthalenyl)acetyl]amino] benzoic acid (1), a retinoic acid receptor gamma -specific agonist with dermatological and anti-tumour activities can be performed by stereoselective reduction of the ketoester 2 or of the ketoacid 3 or of the ketoamide 4. These processes have been performed in Bristol Myers Squibb Research Institute (USA) O OR O HO microbial reduction OR O 2 O OH microbial reduction HO OH O HO O H N 3 F OR O 1 microbial reduction O H N R= H, F OR O 4 O O •Patel et al. Enantioselective microbial reduction of 2-(1’,2’,3’,4’-tetrahydro-1’,1’,4’,4’-tetramethyl-6’-naphtalenyl)acetic acid and its ethyl ester. Tetrahedron: Asymmetry 13, 349-355 (2002). Looking for anti-Prelog’s rule specificity in order to obtain the (R)-alcohol, precursor of 1, several interesting microorganisms were selected by BMS The reductions were performed using glucose as second substrate (25mg/ml) in order to regenerate the coenzyme NADPH/H+. Aureobasidium pullutans SC13894 can reduce the keto-ester 2 and the keto-amide 4. The keto-acid is efficiently reduced by Candida maltosa SC 16112 and Candida utilis SC 13983 that show specificity for the keto-acid. Finally, Hunsemula anamola SC 16158 shows a good selectivity versus 4 The cell extract of A. pullutans shows better activity than the whole cells, especially if NADPH is added as coenzyme This positive effect can be associated to the diminution of the mass transfer through the cell wall and/or membrane that is the rate controlling step in the whole cell catalyzed reactions. pH=7.0, 20% w/v wet cells, 1mg/ml ketone in MeOH 25mg ml-1 of glucose. T=28ºC. Reaction time = 48hr. . Substrate Product yield(%) e.e.(%) 2 98 98 -R 4 12 96-R Candida maltosa SC 16112 3 56 99-R Candida utilis SC 13983 3 64 98-R Hunsemula anamola SC 16158 4 20 85-R Aureobasidium pullutans SC13894a 3b 98 98-R Aureobasidium pullutans SC13894a 3c 12 98-R Aureobasidium pullutans SC13894a 4b 60 96-R Aureobasidium pullutans SC13894a 4c 10 97-R Microorganism Aureobasidium pullutans SC13894 Syntheses of the key intermediate of 34- squalene synthase inhibitor Synthesis of β-adrenergic receptor -137The synthesis is performed in a 8L fermentor Pro-R / cara re Pro-R / cara re Pro-R / cara re Pro-R / cara si Pro-S / cara si Pro-R / cara si Cara si H H R S O H O N H N R Cara re 2 S L H R S O N H N R 2