REDUCTION REACTIONS

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
ƒ FDH commercially
available
il bl
ƒ Stable
ƒ Immobilized
ƒ TNN 103-105
Another useful
methos
ƒ GDH is highly
stable
ƒ 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