biotransformations in organic chemistry. history of biotransformations wine and beer...
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Biotransformations in organic chemistry
History of biotransformations
• wine and beer fermentation 6000 B.C. Summer, Babylon• bread 4000 B. C. Egypt
Industrial production of fine chemicals:L-Lactic acid 1880 USA
COOH
OH
COOH
COOH
HO
OH
COOH
COOH
OH
HO
(-)-tartaric acid (+)-tartaric acid
+Penicilium glaucum
- CO2
COOH
COOH
HO
OH
(-)-tartaric acid
Biotransformation in chiral separation
Pasteur 1858
Industrial production of efedrine
1921
O
HO
O
OH
OH
O
OH
NHCH3
pyruvate decarboxylase
+
H2/PtCH3NH2
(-)-efedrine
Industrial production of ascorbic acid
1924
CH2OH
HO
OH
HO
CH2OH
HO
CH2OH
HO
OH
O
CH2OH
HO
Acetobacter suboxydans
sorbitol sorbose
ascorbic acid
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
Substrate
achiral (prochiral)
Product
enantiopure chiral
STEREOSELECTIVE REACTION
enzyme
Substrate
chiral - racemate
Product
KINETIC RESOLUTION
enzyme(S)-
Substrate(R)- enzyme Substrate(R)-
50%
50%
racemization/reverse reaction
DYNAMIC KINETIC RESOLUTION
Enzymes in productions of enantiopure chiral compounds
Enzymes
Oxidoreductases
NADH+ + H+
H2O2
FMN
NAD+
FMNH2
H2O
OXIDATIONS
Substrate Product
1/2 O2
catalase
NAD regeneration
oxidoreductase
Oxidoreductases
NADH+ + H+
glucose
NAD+
gluconolacton
REDUCTIONS
NADH+ + H+
HCOO-
NAD+
CO2
Substrate Product
NADH +H regeneration
glucose dehydrogenase formiate degydrogenase
oxidoreductase
CH2OH
CHOH
CH2OH
galactose oxidase CH=O
CH2OH
achiral
HO H
(S)-(-)-glyceraldehyde
CH2OH
CHOH
CH2Cl
galactose oxidase CH=O
CH2Cl
racemic
HO H
(R)-aldehyde
CH2OH
CH2Cl
HO H+
+ O2
+ O2
OXIDATIONS
REDUCTIONS
O
NADH
H
H
O O
NADH
OH
H
H
O OH
Oalcohol degydrogenase
racemic
+
achiral
alcohol degydrogenase
O
O
progesteron
Rhizopus nigrificans
O
O
HO
11--hydroxyprogesteron
cortison
Stereo- and regiospecific hydroxylation of non-activated CH
peroxidases, monoxygenases
HONH2
HOOC
HO
HONH2
HOOCcytochrome c-peroxidase
tyrosine L-DOPA
Oxidative deaminations/reductive aminations
CH3
HO COOH
CH3
O COOH
dextrane-NAD+ dextrane-NADH+
CH3
H2N COOHNH4
+H2O alanine
degydrogenase
lactate dehydrogenase
TRANSFERASES OR LIGASES
used mostly for phosphorylations
SubstrateSubstrate-P
ADP ATP
kinase 2
kinase 1Donor-P Donor Donors:
ADP + CH3COOP(O)(OH)2 ATP + CH3COOH
acetate kinase
ADP + ATP + CH3COCOOH
pyruvate kinaseH2C C
OP(O)(OH)2
COOH
OHO
HO OHOH
OHhexokinase
ATPO
HOHO OHOH
OP
O O-
O-
H2CHC CH2
OH OH OH
glycerol kinase
ATPHO O
HO HP
O O-
O-
Enzymatic phosphorylations
Enzymatic sulfation of saccharides with the regeneration of the PAPS cofactor. left: proposed transition state of the reaction.
HYDROLASES – hydrolyses or condensations
R
O
HN
R'R
O
H2NR'
OH
proteasespeptidasesamidohydrolasesaminoacylases
R
O
OR'
R
O
HOR'
OHlipasesesterases
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
SS PS
retentionSR
inversion
O
racemate O
O
OH
OH
OH
OH
OH
OH
+
+
89% ee
Aspergillus niger
Beauveria sulfurescens
Aspergillus niger +Beauveria sulfurescens
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.
Catalytic antibodies
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.
HAPTEN HAPTEN BSA
covalent chemical binding to BSA
BSA = bovine serum albumine
.ANTIBODIES
isolation
Immunization
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.
Transesterification
a) Acyl transfer from the ester 2 to the alcohol 1 catalyzed by antibody 13D6.1, which was generated against the phosphonate diester 5;
b) NMR structure of the Michaelis complex, with 1 shown in blue and 2 in orange.
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.
Oxy-Cope rearrangement
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.
Aldolization
Generation of an aldolase antibody by reactive immunization with the 2-diketone hapten 13.