REDUCTION REACTIONS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION REACTIONS Reduction reaction usually goes in hand with the generaton of a stereogenic center, the desymmetrization of prochiral carbonyl compounds and C=C-bonds is predominant. In contrast, the corresponding di reverse process (e.g. ( Al h l oxidation Alcohol id ti or dehydrogenation) leads to the destruction of a chiral center, which is generally of limited use. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION REACTIONS - The major and crucial distinction between redox enzymes y is that the former require q redox cofactors,, and hydrolases which donate or accept the chemical equivalents for reduction (or oxidation). Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION REACTIONS - For the majority of redox enzymes, enzymes nicotinamide adenine dinucleotide [NAD(H)] and its respective phosphate p p [NADP(H)] are require q by y about 80 % and 10 % of redox enzymes, respectively. Flavines (FMN, FAD) and pyrroloquinoline quinone (PQQ) are encountered more rarely. l Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION REACTIONS - The nicotinamide cofactors are relatively unstable molecule and they are prohibitively expensive if used in stoichiometric amounts. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Total Turnover Number Total number of moles of products formed per mole of cofactor during its entire life. Lab scale: 103-104 T h i l purposes: > 105 Technical Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM NICOTINAMIDE ADENINE DINUCLEOTIDE COFACTORS H H O O NH2 O O P O NAD(H) O - O - OH OH O P O N O N O H+, 2e- O O P O O NH2 - N N O N OH OH O P O N O N NADH OH OH NH2 N O OH OH H H O NH2 N N NAD+ O NH2 O O P O NADP(H) O - O OH OH O P O N O N O OH NH2 N O O P O O- O O P O H+, 2e - O NH2 N N NADPH O N - OH OH O P O N O N O OH NH2 N N NADP+ O O P O O- Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM RECYCLING OF REDUCED NICOTINAMIDE COFACTORS CoupledS b Substrate Process Single l Enzyme -In the couple-subtrate process the cofactor required for the t transformation f ti of f the th main i substrate b t t is i constantly t tl regenerated t d by b addition of a second auxiliar substrate (DONOR) which is transformed by the same enzyme but into the opposite direction. -To shift the equilibrium of the reaction in the desired direction the donor must be applied in excess leading to turnover numbers of up tp 103. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM RECYCLING OF REDUCED NICOTINAMIDE COFACTORS CoupledSubstrate Process Single Enzyme Disadvantages: -The Th overall ll efficiency ffi i of f the th process is i limited li it d since i th enzyme´s the ´ activity is distributed between both the substrate and the hydrogen donor/acceptor - The producr has to be purified from large amounts of auxiliar substrate used in excess -Enzyme deactivation when highly reactive carbonyl species are involved as auxiliar substrates - Enzyme inhibition caused by the high concentration of the auxiliar substrate. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM RECYCLING OF REDUCED NICOTINAMIDE COFACTORS CoupledE i Enzime Process Two Enzymes ¾The use of two independent enzyme is more advantageous. ¾The two parallel redox reactions are catalyzed by two y different enzymes. ¾Both enzymes should have sufficiently specificities for their respective substrates. different Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM METHODS FOR RECYCLING NADH The best and most widely used method FDH commercially available il bl Stable Immobilized TNN 103-105 Another useful methos GDH is highly stable Expensive Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM RECYCLING OF OXIDIZED NICOTINAMIDE COFACTRS ¾The best and most widely applied method for the regeneration of Nicotinamide Cofactore in their oxidized form involved the use of GluDH. ¾LDH is less expensive and exhibits a higher spcific activity than GlcDH although the redox potential is smaller. GlcDH, smaller Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION REACTIONS 1 REDUCTION 1. OF ALDEHIDES AND KETONES USING ISOLATED ENZYMES 2. REDUCTION OF ALDEHIDES KETONES USING WHOLE CELLS 3. REDUCTION WHOLE CELLS OF C=C-BONDS AND USING Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM 1 REDUCTION OF ALDEHIDES AND 1. KETONES USING ISOLATED ENZYMES Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF ALDEHIDES AND KETONES USING ISOLATED ENZYMES A broad range of ketones can be reduced stereoselectively using DH to give chiral secondary alcohols. During the course of the reaction the enzyme delivers the hydride hy r preferentially pr f r nt a y from th the ssi- or th the rre-side s of th the ketone to give (R) or (S)-alcohols. For most cases, the stereochemical course of the reaction, which is mainly dependent o the steric requirements of the substrate, may be predicted from a simple model which is generally referred to as Prelog´s Rule. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONES Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONES C sii Cara HR HS O HR HS O O NH2 N R S L NH2 N R Cara re Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONES Pro-R / cara re Pro-R / cara re Pro-R / cara re Pro R / cara si ProPro-S / cara si Pro-R / cara si Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM PREFERRED SUBSTRATE SIZE FOR DEHYDROGENASES Commercially available dehydrogenases: YADH = Yeast alcohol dehydrogenase HLADH = Horse liver alcohol dehydrogenase Microorganisms as Baker´s yeast TBADH = Thermoanaerobium brockii alcohol dehydrogenase Microbial dehydrogenases (e.g. Lactobacillus Kefir) Follow Prelog Prelog´ss Rule F ll Follow Anti-Prelog´s P l ´ Rule Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Horse Liver Alcohol Dehydrogenesas (HLADH) ¾ HLADH is a very y universal enzyme y with a broad susbtrates specificity and excelent stereoselectivity. ¾ The most useful applications of HLADH are found in the reduction of medium-ring monocyclic ketones (four to nine membered ring systems) and bicyclic ketones. Sterically demanding molecules which are larger than decalines are not readily accepted and acyclic ketones are usually ll reduced d d with ith low l wnantioselectivity. ti l ti it Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Alcohol Dehydrogenesas Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Forms,, Functions,, and a little fiction - structure and function of ADH and associated isoenzymes Humans have at least nine known forms of ADH ADH exists as a homo or heterodimer due to the fact there are two different types of monomer The two types are E and S for ethanol active and steroid active respectively. Although they have different p both are nearly y identical at 374 aa’s long g specificities, Therefore, possible types of ADH are: EE, SS, and ES hybrid ADH. EE is the most commonly found at 40-60% Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Characteristics of EE ADH EE ADH has a molecular weight of about 80 000 Th There are 8 chains, h i 60 helices, h li and d 74 beta b t strands in ADH Each monomer of the dimer has 2 subunits Each of the two subunits has a binding g site for one NAD+ and two Zn2+ (seen later) Activated by cyanate (NCO) and inhibited by heavy metals and chelating agents Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM For the Microbiologist in all of us Three distinct genes are responsible for the production of ADH However, gene products show a 93% h l homology Cross-species homology exists as well Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Homology Between Species Human EE ADH Equine EE ADH Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Interaction of Monomers Two residues are directly responsible for the monomer packing of ADH His-105 and Tyr-286 on each monomer interact with each other to seal the packing The ring side-chains of His-105 will stack on top of the Tyr-286 Tyr 286 side chain on the other monomer Th monomers are aligned The li d anti-paralell i l ll to each other Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Active Site Characteristics of ADH DH As mentioned A ti d earlier, li each h subunit b it of f one monomer contains one binding site for NAD+ and t two bi di sites binding it for f Zn Z 2+ Each Zinc ion is ligated directly between the side chains of Cys-46, His-67, Cys-174 and a water molecule which is hydrogen bonded to Ser-48. Between the two binding sites where the zinc is located,, there are two clefts. One which binds NAD+ and one which binds the substrate (ethanol) Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Zinc bound to Cys-46, His-67, Cys-174, and Ser-48 (Blue) and the coenzyme NAD (purple) NAD+ (pu pl ) attached tt ch d to t His-51 His 51 ((yellow) ll ) and nd Lys-228 L s 228 (cyan). (c n) Th The eight i ht zinc inc molecules are in red. The four zincs seen easily are not directly involved in the proton transfer chain. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Components and Interactions at th Bi the Binding di Sit Site of f ADH NAD+ is the coenzyme for ADH and is absolutely necessary for the i of f ethanol th l conversion f NAD+ is used to One m molecule of convert ethanol to acetaldehyde by proton transfer During hydrogen transfer, two h d hydrogens are stripped d off ff the h ethanol by y zinc Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Conformation Change g at the Active Site NAD+ binds at residues 293-298 and causes a 100 rotation This causes the catalytic domain to move closer to the coenzyme binding domain and closes the active site cleft S48 helps in the proton relay system Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM But I Must Know More! The two active sites are in clefts between th coenzyme binding the bi di core and d the th catalytic domains Eth Ethanol l binds bi d tto th the hydrophobic h d h bi core lined by nine amino acids, which surround the substrate After binding NAD+, the 100 rotation makes the protein go from its apo "open" open form to the halo "closed". This narrows the cleft brings the substrate binding site cleft, closer and excludes water from the active site which is vital for the activity y of ADH Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM •The hydrophobic pocket:pocket: Leu-57, Leu 57 Phe-93, Phe 93 Leu Leu-116, 116 Phe-110, Phe 110 Phe Phe-140, 140 Leu Leu-141, 141 Val-294, Pro-295 and Ile-318 (red). Zinc (orange), Cys-174 (purple), Cys-46 (yellow) and His-67 (green) Cxf (in this case) in blue and oxygen involved in the y g reaction shown in white dehydrogenation Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM •The zinc atom is held in place by cysteine 46 to the left, cysteine 174 to the right, and histidine 67 above. Ethanol binds to the zinc, and the g extends below the ethanol NAD analog Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM C Conclusions l i Alcohol Dehydrogenase is the Human Body’ss offensive line (colts) against Body alcoholic toxins being ingested ADH substrate specificity is broad, broad with most alcohols being potential targets (eg. Methanol Æ Formaldehyde) Once bound to zinc, however, a conformation change ensures tight binding. binding Homer Hypothesis is not feasible Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM SUBSTRATES RECONIZED BY HLADH Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM SUBSTRATES RECONIZED BY HLADH Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM SUBSTRATES RECONIZED BY HLADH Every kinetic resolution of bi bi- and polycyclic ketones suffers from one particular drawback becose the bridgehead carbon atoms make imposible to recycle the undesired enantiomer via racemization. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM SUBSTRATE MODEL FOR HLADH Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM DEHYDROGENASES FROM Thermoanaerobacter ethanolicus AND Thermoanaerobium brockii Useful for the asymmetric reduction of open-chain ketones Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM SUBSTRATES RECOGNIZED BY H dr x ster id DH (HSDH) Hydroxysteroid Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM 2.-REDUCTION OF ALDEHYDES AND KETONES USING WHOLE CELLS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM 2. REDUCTION OF ALDEHYDES AND KETONES USING WHOLE CELLS Ad Advantage: t 9They contain multiple dehydrogenases which are able to accept nonnatural substrates 9They contain all the necesary cofactors and the metab lic pathways for metabolic f r their regeneration re enerati n 9Cheap Ch carbon-sources b such h as saccharose h or glucose can be used as auxiliar substrates for ASYMMETRIC REDUCTION REACTIONS. REACTIONS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Disadvantage: 9The productivity of microbial conversions is usually low since the majority of nonnatural substrates are toxic to living organisms and are therefore only tolerated at low concentrations (0.1 (0 1-0 0.3 3 % per volume) 9The large amount of biomass present in the reaction medium causes l low overall ll yields i ld and d make k product d t recovery ttroublesome. bl 9Chiral transport phenomena into and out of the cell may influence the specificities p of the reaction,, particularly p y when racemic substrates are used. 9Different strains of microorganism can produce different specificities. specificities 9Low stereoselectivity by: 9Inherent p poor substrate recognition g 9Existence of two enzymes with opposite selectivities. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF ALDEHIDES AND KETONES BY BAKER´S BAKER S YEAST ¾Baker Baker´ss yeast (Saccharomyces cerevisiae cerev s ae) iss far the most w widely dely microorganism for the asymmetric reduction of ketones. ¾Reasonable R bl price. i ¾Not require sterile fermenters and can be handled using standard laboratory equipment. ¾A wide range of functional groups within the ketones are tolerated including heterocyclic, fluoro-, chloro-, bromo-, perfluoroalkyl-, cyano-, y , azido-,, nitro-,, hydroxyl-, y y , sulfur-, f , and dithianyl y g groups. p Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF ALIPHATIC KETONES USING BAKER´S YEAST Simple aliphatic and aromatic ketones are reduced to give the corresponding (S) (S)-alcohols alcohols in good optical purities. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF ACYCLIC β-KETOESTERS USING BAKER´S YEAST β-Hydroxyesters obtained serve as chiral starting materials for the synthesis of β-lactams, β-lactams insect pheromones and carotenoids Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM DIASTEREOSELECTIVE REDUCTION OF KETONS BY BAKER´S YEAST Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM MODEL FOR PREDICTING THE DIASTEREOSELECTIVITY IN YEAST-REDUCTIONS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM MICROBIAL REDUCTION OF α-SUBSTITUTED β KETOESTERS β-KETOESTERS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM YEAST-REDUCTION OF CYCLIC β-DIKETONES Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM YEAST-REDUCTION OF α-DIKETONES Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM DERACEMIZATION VIA MICROBIAL STEREOINVERSION OF SECONDARY ALCOHOLS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM 3 -REDUCTION 3. REDUCTION OF C C=C-BONDS C BONDS USING WHOLE CELLS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Difficult via chemical methods. Enzymes: enoate reductases (NADH dependant), involved in fatty acid biosynthesis, found in different microorganisms (even in baker’s yeast) Used generally as whole cells (although some of them have been isolated and characterized), because no regeneration of cofactor is needed and their extreme sensitivity to traces of oxygen. ElectronWithdrawing substituent Anti addition Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Only C=C bonds which are “activated” by electron-withdrawing substituents are reduced, while isolated double or triple bonds are not recognized. i d REDUCTION OF α,β α β-UNSATURATED UNSATURATED ESTERS/ACIDS Generally, G n ll the th ester is firstly hydrolyzed to the h acid. d Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF β-SUBSTITUED α,β-UNSATURATED LACTONES Very useful C5 chiral building block For terpenoid synthesis The sulfone is too polar = low chemical and optical yields Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF α,β-UNSATURATED CARBONYL COMPOUNDS Generally transformed in two steps: 1.- Reduction of the C=C bond by enoate reaductases. 2.- Reduction of the C=O bond to alcohol by ADHs. Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM REDUCTION OF NITROALKENES Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM