REDUCTION REACTIONS Prof. A. R. Alcántara, Grupo de Biotransformaciones, Facultad de Farmacia, UCM

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1 REDUCTIN REACTINS

2 REDUCTIN REACTINS 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 reverse process (e.g. Alcohol l oxidation or dehydrogenation) leads to the destruction of a chiral center, which is generally of limited use. REDUCTIN REACTINS - The major and crucial distinction between redox enzymes and hydrolases is that the former require redox cofactors, which donate or accept the chemical equivalents for reduction (or oxidation).

3 REDUCTIN REACTINS - For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)] and its respective phosphate p [NADP(H)] are require by about 80 % and 10 % of redox enzymes, respectively. Flavines (FMN, FAD) and pyrroloquinoline quinone (PQQ) are encountered more rarely. REDUCTIN REACTINS - The nicotinamide cofactors are relatively unstable molecule and they are prohibitively expensive if used in stoichiometric amounts.

4 Total Turnover Number Total number of moles of products formed per mole of cofactor during its entire life. Lab scale: Technical purposes: > 10 5 NICTINAMIDE ADENINE DINUCLETIDE CFACTRS H H - P N NH 2 - P H +, 2e NAD(H) - H H NH H H 2 - P N N - P N N N N N NH 2 NH 2 N N H H NADH H H NAD + H H NADP(H) - P N NH 2 H H NH 2 H +, 2e - - P N N N N NH 2 N P H H NH 2 - P N N N N H NADPH H P P NADP +

5 RECYCLING F REDUCED NICTINAMIDE CFACTRS Single Enzyme -In the couple-subtrate process the cofactor required for the transformation ti of the main substrate bt t is constantly tl regenerated by addition of a second auxiliar substrate (DNR) 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 RECYCLING F REDUCED NICTINAMIDE CFACTRS Coupled- Substrate l Process Coupled- Substrate Process Single Enzyme Disadvantages: -The overall efficiency i of the process is limitedit since the enzyme s 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.

6 RECYCLING F REDUCED NICTINAMIDE CFACTRS Coupled- Enzime Process Two Enzymes The use of two independent enzyme is more advantageous. The two parallel redox reactions are catalyzed by two different enzymes. Both enzymes should have sufficiently different specificities for their respective substrates. The best and most widely used method FDH commercially available Stable Immobilized TNN Another useful methos METHDS FR RECYCLING NADH GDH is highly stable Expensive

7 RECYCLING F XIDIZED NICTINAMIDE CFACTRS The best and most widely applied method for the regeneration of Nicotinamide Cofactore in their oxidized form involved the use of GluDH. LDH is less expensive and exhibits a higher spcific activity than GlcDH, although the redox potential is smaller.

8 REDUCTIN REACTINS 1. REDUCTIN F ALDEHIDES AND KETNES USING ISLATED ENZYMES 2. REDUCTIN F ALDEHIDES AND KETNES USING WHLE CELLS 3. REDUCTIN F C=C-BNDS USING WHLE CELLS 1.REDUCTIN F ALDEHIDES AND KETNES USING ISLATED ENZYMES

9 REDUCTIN F ALDEHIDES AND KETNES USING ISLATED ENZYMES A broad range of ketones can be reduced stereoselectively using DH to give chiral secondary alcohols. Duringthecourseofthereactiontheenzymedeliversthe hydride preferentially r from the si- or the re-sider of 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. PRELG S RULE FR THE ASYMMETRIC REDUCTIN F KETNES

10 PRELG S RULE FR THE ASYMMETRIC REDUCTIN F KETNES Cara si H R H S H R H S N R NH 2 Cara re S L N R NH 2 PRELG S RULE FR THE ASYMMETRIC REDUCTIN F KETNES Pro-R / cara re Pro-R / cara re Pro-R / cara re Pro-R R /carasi Pro-S / cara si Pro-R / cara si

11 PREFERRED SUBSTRATE SIZE FR DEHYDRGENASES Commercially available dehydrogenases: YADH = Yeast alcohol dehydrogenase HLADH = Horse liver alcohol dehydrogenase Microorganisms as Baker s yeast TBADH = Thermoanaerobium brockii alcohol dehydrogenase Follow Prelog s Rule Microbial dehydrogenases (e.g. Lactobacillus Kefir) Follow Anti-Prelog s Rule Horse Liver Alcohol Dehydrogenesas (HLADH) HLADH is a very universal enzyme with a broad susbtrates specificity and excelent stereoselectivity. 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 reduced d with low wnantioselectivity. it

12 Alcohol Dehydrogenesas 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 specificities, both are nearly identical at 374 aa s long Therefore, possible types of ADH are: EE, SS, and ES hybrid ADH. EE is the most commonly found at 40-60%

13 Characteristics of EE ADH EE ADH has a molecular weight of about There are 8 chains, 60 helices, and 74 beta strands in ADH Each monomer of the dimer has 2 subunits Each of the two subunits has a binding site for one NAD + and two Zn 2+ (seen later) Activated by cyanate (NC) and inhibited by heavy metals and chelating agents For the Microbiologist in all of us Three distinct genes are responsible for the production of ADH However, gene products show a 93% homology Cross-species homology exists as well

14 Homology Between Species Human EE ADH Equine EE ADH 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 side chain on the other monomer The monomers are aligned anti-paralell ll to each other

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16 Active Site Characteristics of ADH As mentioned earlier, each subunit of one monomer contains one binding site for NAD + and two binding sites for Zn 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. ne which binds NAD + and one which binds the substrate (ethanol) Zinc bound to Cys-46, His-67, Cys-174, and Ser-48 (Blue) and the coenzyme NAD+ (purple) pl attached to His-51 (yellow) and Lys-228 (cyan). The eight zinc molecules are in red. The four zincs seen easily are not directly involved in the proton transfer chain.

17 Components and Interactions at the Binding Site of ADH NAD+ is the coenzyme for ADH and is absolutely necessary for the conversion of ethanol ne molecule of NAD+ is used to convert ethanol to acetaldehyde by proton transfer During hydrogen transfer, two hydrogens are stripped off the ethanol by zinc Conformation Change at the Active Site NAD+ binds at residues and causes a 10 0 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

18 But I Must Know More! The two active sites are in clefts between the coenzyme binding core and the catalytic domains Ethanol binds to the hydrophobic h core lined by nine amino acids, which surround the substrate After binding NAD+, the 10 0 rotation makes the protein go from its apo "open" form to the halo "closed". This narrows the cleft, brings the substrate binding site closer and excludes water from the active site which is vital for the activity of ADH The hydrophobic pocket:- Leu-57, Phe-93, Leu-116, Phe-110, Phe-140, Leu-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 dehydrogenation reaction shown in white

19 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 NAD analog extends below the ethanol Conclusions Alcohol Dehydrogenase is the Human Body s offensive line (colts) against alcoholic toxins being ingested ADH substrate specificity is broad, with most alcohols being potential targets (eg. Methanol Formaldehyde) nce bound to zinc, however, a conformation change ensures tight binding. Homer Hypothesis is not feasible

20 SUBSTRATES RECNIZED BY HLADH SUBSTRATES RECNIZED BY HLADH

21 SUBSTRATES RECNIZED BY HLADH Every kinetic resolution of bi- and polycyclic ketones suffers from one particular drawback becose the bridgehead carbon atoms make imposible to recycle the undesired enantiomer via racemization. SUBSTRATE MDEL FR HLADH

22 DEHYDRGENASES FRM Thermoanaerobacter ethanolicus AND Thermoanaerobium brockii Useful for the asymmetric reduction of open-chain ketones SUBSTRATES RECGNIZED BY Hydroxysteroid DH (HSDH)

23 2.-REDUCTIN F ALDEHYDES AND KETNES USING WHLE CELLS 2. REDUCTIN F ALDEHYDES AND KETNES USING WHLE CELLS Advantage: They contain multiple dehydrogenases which are able to accept nonnatural substrates They contain all the necesary cofactors and the metabolic pathways for their regenerationenerati n Cheap carbon-sources such as saccharose or glucose can be used as auxiliar substrates for ASYMMETRIC REDUCTIN REACTINS.

24 Disadvantage: The 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 ( % per volume) The large amount of biomass present in the reaction medium causes low overall yields and make product recovery troublesome. Chiral transport phenomena into and out of the cell may influence the specificities of the reaction, particularly when racemic substrates are used. Different strains of microorganism can produce different specificities. Low stereoselectivity by: Inherent poor substrate recognition Existence of two enzymes with opposite selectivities. REDUCTIN F ALDEHIDES AND KETNES BY BAKER S YEAST Baker s s yeast (Saccharomyces cerevisiae) is far the most widely wdely microorganism for the asymmetric reduction of ketones. ReasonableR price. Not require sterile fermenters and can be handled using standard laboratory equipment. A wide range of functional groups within the ketones are tolerated including heterocyclic, fluoro-, chloro-, bromo-, perfluoroalkyl-, cyano-, azido-, nitro-, hydroxyl-, y sulfur-, and dithianyl groups.

25 REDUCTIN F ALIPHATIC KETNES USING BAKER S YEAST Simple aliphatic and aromatic ketones are reduced to give the corresponding (S)-alcohols in good optical purities. REDUCTIN F ACYCLIC β-ketesters USING BAKER S YEAST β-hydroxyesters obtained serve as chiral starting materials for the synthesis of β-lactams, insect pheromones and carotenoids

26 DIASTERESELECTIVE REDUCTIN F KETNS BY BAKER S YEAST MDEL FR PREDICTING THE DIASTERESELECTIVITY IN YEAST-REDUCTINS

27 MICRBIAL REDUCTIN F α-substituted β-ketesters YEAST-REDUCTIN F CYCLIC β-diketnes

28 YEAST-REDUCTIN F α-diketnes DERACEMIZATIN VIA MICRBIAL STERE- INVERSIN F SECNDARY ALCHLS

29 3. -REDUCTIN F C=C-BNDS C USING WHLE CELLS

30 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. Electron- Withdrawing substituent Anti addition nly C=C bonds which are activated by electron-withdrawing substituents are reduced, while isolated double or triple bonds are not recognized. REDUCTIN F α,β-unsaturated ESTERS/ACIDS Generally, the ester is firstly hydrolyzed to the acid.

31 REDUCTIN F β-substitued α,β-unsaturated LACTNES Very useful C5 chiral building block For terpenoid synthesis The sulfone is too polar = low chemical and optical yields REDUCTIN F α,β-unsaturated CARBNYL CMPUNDS Generally transformed in two steps: 1.- Reduction of the C=C bond by enoate reaductases. 2.- Reduction of the C= bond to alcohol by ADHs.

32 REDUCTIN F NITRALKENES