enzimas redox 03.ppt [modo de compatibilidad]...lab scale: 103-104 thi l 10technical purposes: >...

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 aReduction reaction usually goes in hand with the generaton of astereogenic center, the desymmetrization of prochiral carbonylcompounds and C=C-bonds is predominant. In contrast, the

di ( Al h l id ticorresponding reverse process (e.g. Alcohol oxidation ordehydrogenation) leads to the destruction of a chiral center, whichis generally of limited use.


REDUCTION REACTIONS

- The major and crucial distinction between redox enzymesand hydrolases is that the former require redox cofactors,y q ,which donate or accept the chemical equivalents forreduction (or oxidation).


REDUCTION REACTIONS - For the majority of redox enzymes nicotinamideFor the majority of redox enzymes, nicotinamideadenine dinucleotide [NAD(H)] and its respectivephosphate [NADP(H)] are require by about 80 % and 10p p q y% of redox enzymes, respectively. Flavines (FMN, FAD)and pyrroloquinoline quinone (PQQ) are encountered more

lrarely.


REDUCTION REACTIONS

- The nicotinamide cofactors are relatively unstablemolecule and they are prohibitively expensive if used inmolecule and they are prohibitively expensive if used instoichiometric amounts.


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 105Technical purposes: > 105


OOH H

NICOTINAMIDE ADENINE DINUCLEOTIDE COFACTORS

NOOP-O O

NH2

NOOP-O O

NH2

H+ 2e-NAD(H) OH OHO

P

O

O-O

O

N

NN

N

NH2OH OHO

P

O

O-O

O

N

NN

N

NH2

H , 2eNAD(H)

OH OH

O

OH OH

O

NAD+NADH

O

NOOP-O O

NH2

O

NOOP-O O

NH2

OH H

OH OH

P

O

P

O

O O

O-O N

NN

N

NH2OH OHO

P

O

O-O N

NN

N

NH2

H+, 2e-

NADP(H)O

OH O

O NNO

OH O

O NN

NADP+NADPH

P OO-

O P OOO-O O-


RECYCLING OF REDUCED NICOTINAMIDE COFACTORS

Coupled-S b lSubstrate Process

SingleEnzyme

-In the couple-subtrate process the cofactor required for thet f ti f th i b t t i t tl t d btransformation of the main substrate is constantly regenerated byaddition of a second auxiliar substrate (DONOR) which is transformedby the same enzyme but into the opposite direction.-To shift the equilibrium of the reaction in the desired direction thedonor must be applied in excess leading to turnover numbers of up tp103.10 .



Coupled-Substrate SingleSubstrate Process

SingleEnzyme

Disadvantages:Th ll ffi i f th i li it d i th ´-The overall efficiency of the process is limited since the enzyme´s

activity is distributed between both the substrate and the hydrogendonor/acceptor- The producr has to be purified from large amounts of auxiliarsubstrate used in excessEnzyme deactivation when highly reactive carbonyl species are involved-Enzyme deactivation when highly reactive carbonyl species are involved

as auxiliar substrates- Enzyme inhibition caused by the high concentration of the auxiliarsubstrate.



Coupled-E i Enzime Process

TwoEnzymes

The use of two independent enzyme is more advantageous.The use of two independent enzyme is more advantageous.The two parallel redox reactions are catalyzed by two

different enzymes.yBoth enzymes should have sufficiently different

specificities for their respective substrates.


METHODS FOR RECYCLING NADHThe best and most The best and most widely used methodFDH commercially il blavailable

StableImmobilizedTNN 103-105

Another useful methos

GDH is highly GDH is highly stableExpensive


RECYCLING OF OXIDIZED NICOTINAMIDE COFACTRS

The best and most widely applied method for the regeneration ofNicotinamide Cofactore in their oxidized form involved the use ofGluDH.LDH is less expensive and exhibits a higher spcific activity than

GlcDH although the redox potential is smallerGlcDH, although the redox potential is smaller.



REDUCTION REACTIONSREDUCTION REACTIONS

1 REDUCTION OF ALDEHIDES AND1. REDUCTION OF ALDEHIDES ANDKETONES USING ISOLATED ENZYMES

2. REDUCTION OF ALDEHIDES ANDKETONES USING WHOLE CELLS

3. REDUCTION OF C=C-BONDS USINGWHOLE CELLSWHOLE CELLS


1 REDUCTION OF ALDEHIDES AND 1.REDUCTION OF ALDEHIDES AND KETONES USING ISOLATED

ENZYMES


REDUCTION OF ALDEHIDES AND KETONES USING ISOLATED ENZYMESUSING ISOLATED ENZYMES

A broad range of ketones can be reduced stereoselectivelyA broad range of ketones can be reduced stereoselectivelyusing DH to give chiral secondary alcohols.

During the course of the reaction the enzyme delivers thehydride preferentially from the si- or the re-side of thehy r pr f r nt a y from th s or th r s of thketone to give (R) or (S)-alcohols.

For most cases, the stereochemical course of the reaction,which is mainly dependent o the steric requirements of thesubstrate, may be predicted from a simple model which isgenerally referred to as Prelog´s Rule.


PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONESREDUCTION OF KETONES


PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONES

C i

REDUCTION OF KETONES

O OH H

Cara si

NH2

OHR HS O

NH2

OHR HS

S

N

R

2

N

R

LR R

Cara re


PRELOG´S RULE FOR THE ASYMMETRIC REDUCTION OF KETONES

Pro-R / cara rePro-R / cara re

Pro R / cara si

Pro-R / cara re

Pro-R / cara si

Pro-R / cara siPro-S / cara si


PREFERRED SUBSTRATE SIZE FOR DEHYDROGENASES

Commercially available dehydrogenases:Commercially available dehydrogenases:YADH = Yeast alcohol dehydrogenaseHLADH = Horse liver alcohol dehydrogenase Follow Prelog´s Microorganisms as Baker´s yeastTBADH = Thermoanaerobium brockii alcohol dehydrogenase

Follow Prelog s Rule

F ll P l ´ 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 susbtratesy y

specificity and excelent stereoselectivity.The most useful applications of HLADH are found in the reduction

of medium-ring monocyclic ketones (four to nine membered ringof medium-ring monocyclic ketones (four to nine membered ringsystems) and bicyclic ketones. Sterically demanding molecules whichare larger than decalines are not readily accepted and acyclic ketones

ll d d ith l ti l ti itare usually reduced with low wnantioselectivity.


Alcohol Dehydrogenesas


Forms, Functions, and a little fiction, ,- structure and function of ADH and associated isoenzymes

Humans have at least nine known forms of ADHADH exists as a homo or heterodimer due to the fact ADH exists as a homo or heterodimer due to the fact there are two different types of monomerThe two types are E and S for ethanol active and steroid 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 longp y gTherefore, possible types of ADH are: EE, SS, and ES hybrid ADH.EE is the most commonly found at 40-60%


Characteristics of EE ADHCharacteristics of EE ADHEE ADH has a molecular weight of about 80 EE ADH has a molecular weight of about 80 000Th 8 h i 60 h li d 74 b t There are 8 chains, 60 helices, and 74 beta strands in ADHEach monomer of the dimer has 2 subunitsEach of the two subunits has a binding site gfor one NAD+ and two Zn2+ (seen later)

Activated by cyanate (NCO) and inhibited Activated by cyanate (NCO) and inhibited by heavy metals and chelating agents


For the Microbiologist in all For the Microbiologist in all of usof us

Three distinct genes are responsible for the production of ADH

However, gene products show a 93% h lhomology

Cross-species homology exists as well


Homology Between SpeciesHomology Between Species

Human EE ADH Equine EE ADH


Interaction of Monomers

Two residues are directly responsible for Two residues are directly responsible for the monomer packing of ADHHis-105 and Tyr-286 on each monomer His-105 and Tyr-286 on each monomer interact with each other to seal the packingpackingThe ring side-chains of His-105 will stack on top of the Tyr 286 side chain on the on top of the Tyr-286 side chain on the other monomerTh li d i l ll The monomers are aligned anti-paralell to each other


Active Site Characteristics of DHADH

A ti d li h b it fAs mentioned earlier, each subunit of onemonomer contains one binding site for NAD+ andt bi di it f Z 2+two binding sites for Zn2+

Each Zinc ion is ligated directly between the sidechains of Cys-46, His-67, Cys-174 and a watermolecule which is hydrogen bonded to Ser-48.Between the two binding sites where the zinc islocated, there are two clefts. One 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 (pu pl ) tt ch d t His 51 ( ll ) nd L s 228 (c n) Th i ht inc NAD+ (purple) 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.


Components and Interactions at th Bi di Sit f ADHthe Binding Site of ADH

NAD+ is the coenzyme for ADH and is NAD+ is the coenzyme for ADH and is absolutely necessary for the

i f th lconversion of ethanolOne molecule of NAD+ is used to m fconvert ethanol to acetaldehyde by proton transferproton transferDuring hydrogen transfer, two h d d ff h hydrogens are stripped off the ethanol by zinc y


Conformation Change at the Active gSite

NAD+ binds at residues 293-298 and causes a 100 rotationThis causes the catalytic domain to move closer to the coenzyme binding domain and closes the active site cleft coenzyme binding domain and closes the active site cleft S48 helps in the proton relay system


But I Must Know More!The two active sites are in clefts between th bi di d th the coenzyme binding core and the catalytic domainsEth l bi d t th h d h bi Ethanol binds to the hydrophobic core lined by nine amino acids, which surround the substratethe substrateAfter binding NAD+, the 100 rotation makes the protein go from its apo "open" makes the protein go from its apo open form to the halo "closed". This narrows the cleft brings the substrate binding site cleft, brings the substrate binding site closer and excludes water from the active site which is vital for the activity of ADHy


•The hydrophobic pocket: Leu 57 Phe 93 Leu 116 Phe 110 Phe 140 Leu 141 •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 whitey g


•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 ethanolg


C l iConclusions

Alcohol Dehydrogenase is the Human Body’s offensive line (colts) against Body s offensive line (colts) against alcoholic toxins being ingestedADH substrate specificity is broad with ADH substrate specificity is broad, with most alcohols being potential targets (eg. Methanol Formaldehyde)Methanol Formaldehyde)Once bound to zinc, however, a conformation change ensures tight bindingconformation change ensures tight binding.Homer Hypothesis is not feasible


SUBSTRATES RECONIZED BY HLADH


SUBSTRATES RECONIZED BY HLADH


SUBSTRATES RECONIZED BY HLADHEvery kinetic resolution of bi- and polycyclic ketones suffers from one particularEvery kinetic resolution of bi and polycyclic ketones suffers from one particulardrawback becose the bridgehead carbon atoms make imposible to recycle theundesired enantiomer via racemization.


SUBSTRATE MODEL FOR HLADH


DEHYDROGENASES FROM Thermoanaerobacter ethanolicus AND Thermoanaerobium brockii

Useful for the asymmetric reduction of open-chain ketones


SUBSTRATES RECOGNIZED BY H dr x ster id DH (HSDH)Hydroxysteroid DH (HSDH)


2.-REDUCTION OF ALDEHYDES AND KETONES USING WHOLE CELLSKETONES USING WHOLE CELLS


2. REDUCTION OF ALDEHYDES AND KETONES USING WHOLE CELLSKETONES USING WHOLE CELLS

Ad tAdvantage:

They contain multiple dehydrogenases which areable to accept nonnatural substrates

They contain all the necesary cofactors and themetab lic pathways f r their re enerati nmetabolic pathways for their regeneration

Ch b h hCheap carbon-sources such as saccharose orglucose can be used as auxiliar substrates forASYMMETRIC REDUCTION REACTIONSASYMMETRIC REDUCTION REACTIONS.


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 (0 1-0 3 % per are therefore only tolerated at low concentrations (0.1 0.3 % per volume)The large amount of biomass present in the reaction medium causes

l ll i ld d k d t t bllow 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 p , p yare used.Different strains of microorganism can produce different

specificitiesspecificities.Low stereoselectivity by:

Inherent poor substrate recognitionp gExistence of two enzymes with opposite selectivities.


REDUCTION OF ALDEHIDES AND KETONES BY BAKER´S YEASTBAKER S YEAST

Baker´s yeast (Saccharomyces cerevisiae) is far the most widelyBaker s yeast (Saccharomyces cerev s ae) s far the most w delymicroorganism for the asymmetric reduction of ketones.

R bl iReasonable price.

Not require sterile fermenters and can be handled using standardNot require sterile fermenters and can be handled using standardlaboratory equipment.

A wide range of functional groups within the ketones are toleratedincluding heterocyclic, fluoro-, chloro-, bromo-, perfluoroalkyl-,cyano-, azido-, nitro-, hydroxyl-, sulfur-, and dithianyl groups.y , , , y y , f , y g p


REDUCTION OF ALIPHATIC KETONES USING BAKER´S YEAST

Simple aliphatic and aromatic ketones are reduced to give thecorresponding (S)-alcohols in good optical purities.corresponding (S) alcohols in good optical purities.


REDUCTION OF ACYCLIC β-KETOESTERS USING BAKER´S YEAST

β-Hydroxyesters obtained serve as chiral starting materials for thesynthesis of β-lactams insect pheromones and carotenoidssynthesis of β-lactams, insect pheromones and carotenoids


DIASTEREOSELECTIVE REDUCTION OF KETONS BY BAKER´S YEAST


MODEL FOR PREDICTING THE DIASTEREOSELECTIVITYIN YEAST-REDUCTIONS


MICROBIAL REDUCTION OF α-SUBSTITUTED β-KETOESTERSβ KETOESTERS


YEAST-REDUCTION OF CYCLIC β-DIKETONES


YEAST-REDUCTION OF α-DIKETONES


DERACEMIZATION VIA MICROBIAL STEREO-INVERSION OF SECONDARY ALCOHOLS



3 -REDUCTION OF C=C-BONDS 3. REDUCTION OF C C BONDS USING WHOLE CELLS


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.

Antiaddition

Electron-Withdrawingsubstituent


Only C=C bonds which are “activated” by electron-withdrawingsubstituents are reduced, while isolated double or triple bonds are not

i drecognized.

REDUCTION OF α β-UNSATURATED ESTERS/ACIDSREDUCTION OF α,β UNSATURATED ESTERS/ACIDS

G n ll thGenerally, theester is firstlyhydrolyzed toh dthe acid.


REDUCTION OF β-SUBSTITUED α,β-UNSATURATED LACTONES

Very usefulVery usefulC5 chiral building blockFor terpenoid synthesis

The sulfone is too polar = low chemical and optical yields


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.



REDUCTION OF NITROALKENES


enzimas redox 03.ppt [modo de compatibilidad]...lab scale: 103-104 thi l 10technical purposes: >...

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