Biotransformations in organic
chemistry
History of biotransformations
• wine and beer fermentation
• bread
6000 B.C. Summer, Babylon
4000 B. C. Egypt
Industrial production of fine chemicals:
L-Lactic acid
1880 USA
OH
COOH
Biotransformation in chiral separation
COOH
OH
COOH
HO
+
OH
COOH
(-)-tartaric acid
COOH
Penicilium glaucum
OH
COOH
HO
COOH
(+)-tartaric acid
Pasteur 1858
HO
- CO2
(-)-tartaric acid
Industrial production of efedrine
O
H
+
OH
pyruvate
decarboxylase
O
OH
O
H2/Pt
CH3NH2
OH
NHCH3
O
(-)-efedrine
1921
Industrial production of ascorbic acid
CH2OH
Acetobacter
suboxydans
HO
HO
CH2OH
O
HO
OH
ascorbic acid
OH
HO
HO
CH2OH
sorbitol
1924
CH2OH
sorbose
Biotransformations
• tissue cell cultures (plant cells)
• whole cells (bacteria, yeast)
• immobilized cells
• cell extracts
• isolated native enzymes
• recombinant enzymes
• modified/mutated enzymes
• stabilized enzymes (cross-linking)
• immobilized enzymes/multi-enzyme systems
Advantages of enzymatically catalyzed reactions
• high reaction specificity
• high regioselectivity
• high stereoselectivity (enantioselectivity, diastereoselectivity)
• good efficiency (high turnover)
• mild reaction conditions
• environmental friendly (green) processes
For most organic reactions there are some enzymes that
efficiently catalyze them; if not, artificial enzymes could be
developed by in vitro evolution.
Enzymes catalyze reverse reactions.
Disadvantages and problems of biotransformations
• sensitivity to harsh reaction conditions (low or high
temperatures, pressure, pH, reagents)
• high prices of many enzymes
• problematic co-factor regeneration (multi-enzyme systems)
• low conversions in some reactions (inhibition by the product)
• narrow substrate specificity of some enzymes
• limited use of non-aqueous solvents
• high dilutions (low volume efficiency)
Enzymes only lower activation barrier (accelerate reactions) –
they do not influence reaction balance!!!
Chirality
Enzymes in productions of enantiopure chiral compounds
STEREOSELECTIVE REACTION
enzyme
Substrate
Product
enantiopure chiral
achiral (prochiral)
KINETIC RESOLUTION
enzyme
(S)-
Substrate
Product
50%
chiral - racemate
(R)-
Substrate
enzyme
(R)-
Substrate
50%
racemization/reverse reaction
DYNAMIC KINETIC RESOLUTION
Enzymes
Oxidoreductases
OXIDATIONS
Substrate
oxidoreductase
Product
NAD regeneration
NAD+
+
FMNH2
FMN
H2O2
1/2 O2
H2O
+
NADH + H
catalase
Oxidoreductases
REDUCTIONS
Substrate
oxidoreductase
Product
NADH +H regeneration
NADH+ + H+
NAD+
NADH+ + H+
NAD+
glucose
CO2
HCOO-
gluconolacton
glucose dehydrogenase
formiate degydrogenase
OXIDATIONS
CH2OH
CHOH + O2
CH2OH
achiral
galactose oxidase
CH=O
HO
H
CH2OH
(S)-(-)-glyceraldehyde
galactose oxidase
CH2OH
CH2OH
CH=O
H
CHOH + O2
HO
H + HO
CH2Cl
CH2Cl
CH2Cl
racemic
(R)-aldehyde
REDUCTIONS
O
O
OH
alcohol degydrogenase
+
NADH
racemic
O
H
H
achiral
O alcohol degydrogenase
O
H
NADH
H
OH
Stereo- and regiospecific hydroxylation of non-activated CH
peroxidases, monoxygenases
O
O
Rhizopus nigrificans
O
cortison
O
11--hydroxyprogesteron
progesteron
HOOC
NH2
HO
HO
cytochrome
c-peroxidase
HO
HOOC
NH2
HO
tyrosine
L-DOPA
Oxidative deaminations/reductive aminations
CH3
HO
COOH
lactate
dehydrogenase
dextrane-NAD+
CH3
O
COOH
dextrane-NADH+
CH3
H2N
COOH
+
H2O
NH4
alanine
degydrogenase
TRANSFERASES OR LIGASES
used mostly for phosphorylations
Donor-P
Donor
Donors:
kinase 1
ADP + CH3COOP(O)(OH)2
ATP
ADP
ADP +
kinase 2
Substrate-P
Substrate
H2C C COOH
OP(O)(OH)2
acetate
kinase
pyruvate
kinase
ATP + CH3COOH
ATP + CH3COCOOH
Enzymatic phosphorylations
OH
HO
HO
O
hexokinase
OH OH
H
H2C C CH2
OH OH OH
O OP OO
ATP
glycerol kinase
ATP
O
HO
HO
HO
HO
OH OH
H
O O
O P O
Enzymatic sulfation of saccharides with the regeneration of the PAPS cofactor.
left: proposed transition state of the reaction.
HYDROLASES – hydrolyses or condensations
H
N
R
R'
proteases
peptidases
amidohydrolases
aminoacylases
R
O
R
R'
O
O
O
OH H2N
R'
lipases
esterases
R
OH
O
HO
R'
Fig. 2. Typical biotransformations with enantioselective amidohydrolases in whole cells
of R. equi, A. aurescens and R. globerulus.
Dynamic kinetic resolution – enzyme + racemization catalyst
Dynamic kinetic resolution – enzyme + racemization reagent
Enantioconvergent synthesis
inversion
retention
SS
PS
SR
O
OH
OH
Aspergillus niger
+
O
OH
Aspergillus niger +
Beauveria sulfurescens
OH
89% ee
racemate
Beauveria sulfurescens
O
+
OH
OH
Catalytic antibodies
If one accepts the basic principle that catalytic function results from the
selective use of binding energy to stabilize transition states or to
destabilize ground states preferentially, then the problem is simplified to
one of synthesizing highly selective molecular receptors. While this remains a
major challenge for synthetic chemistry, there does exist a biological solution
to the problem of molecular recognition. It is a well-known fact in
immunochemistry that the immune response can generate an antibody
that is complementary to virtually any foreign molecular structure
presented to it. The process whereby these selective, high-affinity receptors
are generated resembles in many ways the natural evolution of enzymes.
R. Lerner, K. Janda and P. Schultz – Scripps
Table 1. A comparison of the evolution of enzymes and
antibodies.
Enzymes
Antibodies
exon shuffling
V-D-J rearrangement
gene duplication
batteries of V, D, and J gene elements
accumulation of point
somatic hypermutation
mutations
natural selection
clonal selection
timescale: 101-108 years
timescale: weeks
The generation of immunological diversity by genetic recombination and somatic mutation.
Immunization
covalent chemical
binding to BSA
HAPTEN
HAPTEN
BSA
BSA = bovine serum albumine
ANTIBODIES
isolation
.
Transesterification
a) Acyl transfer from the ester 6 to the alcohol 7, catalyzed by antibody 21H3, which was
generated against the hapten 9; b) modeled structure of the acyl-antibody intermediate
based on the X-ray crystal structure of the antibody-hapten 9 complex.
a)
b)
Acyl transfer from the
ester 2 to the alcohol 1
catalyzed by antibody
13D6.1, which was
generated against the
phosphonate diester 5;
NMR structure of the
Michaelis complex,
with 1 shown in blue
and 2 in orange.
Oxy-Cope rearrangement
Transition-state analogue 19 and the oxy-Cope
rearrangement catalyzed by antibody AZ28.
Overlay of the active sites for the germline
antibody structures of AZ28 with the hapten 19
(blue) and without hapten (green). The hapten
is shown in yellow.
Aldolization
a) Broad substrate scope of antibody-catalyzed aldol reactions. The two antibodies have antipodal activities; b) substrate binding pockets for the antibodies 33F12 (left) and
93F3 (right). The light chain is shown in pink and the heavy chain in blue. The active-site lysine residue is also shown.
Generation of an aldolase
antibody by reactive
immunization with the 2diketone hapten 13.