Biotransformation
DYNAMIC KINETIC RESOLUTION
Methods for Asymmetric Synthesis
Synthesis utilizing existing stereogenic centers (chiral substrates)
Catalytic enantioselective organic reactions
Chemocatalysis - metal-mediated, Lewis acid-mediated, organocatalysis
Biocatalysis - enzymes (hydrolases)
Resolution
Conventional separation procedures of two enantiomers in a racemic mixture.
i. Kinetic resolution: Resolution method based on kinetics of reaction
Problems associated with standard kinetic resolutions:
Theoretical yield can never exceed a limit of 50%.
Separation of the product from the remaining substrate may be laborious
In the majority of processes, only one stereoisomer is desired
Drop in enantiomeric purity as process nears 50% conversion
ii. Dynamic Kinetic resolution
If racemization can occur concurrently with kinetic resolution, then theoretically
100% of the racemic mixture can be converted to one enantiomer. This process is
known as dynamic kinetic resolution (DKR).
25
Biotransformation
Guidelines for Dynamic Kinetic Resolution
In order to design a successful DKR, both the inversion and resolution steps have to
be carefully tuned. Here are a few established general guidelines for an efficient
DKR:
i. The kinetic resolution should be irreversible in order to ensure high
enantioselectivity.
ii. The enantiomeric ratio (E = kR/kS) should be at least greater than ~20.
iii. To avoid depletion of SR, racemization (kinv) should be at least equal or
greater than the reaction rate of the fast enantiomer (kR).
Examples of Racemization
The racemization/inversion step is key to a successful DKR. Following are a number
of common techniques used for this step.
1. Acid or base catalyzed racemization
26
Biotransformation
5-oxazolones have been transformed into the corresponding enantiopure amino acid
derivatives by combining CALB as the biocatalyst and triethylamine as the base
2. Enzyme catalyzed racemization
One enantiomer of a racemic mixture is selectively oxidized to the corresponding
ketone under catalysis of a dehydrogenase, the ketone is reduced again in a
subsequent step by a different enzyme displaying opposite stereochemical
preferences
3. Schiff base-mediated racemization
The racemization proceeds via initial protonation of the imine, formed from reaction
of a primary amino group with an aldehyde or ketone, followed by proton abstraction
at the a-position of the acid.
27
Biotransformation
4. Racemization via sp2 intermediates (redox, addition/elimination)
5. Racemization via π-allyl intermediates
These are two general methods that have been used for the racemization using
transition metal catalyst: (i) racemization via hydrogen transfer and (ii) racemization
via π-allyl formation.
a) Enzymatic Methods – Hydrolysis
Amino acid synthesis
The racemization proceeds via initial protonation of the imine, formed from reaction
of a primary amino group with an aldehyde or ketone, followed by proton abstraction
at the a-position of the acid.
b) Enzymatic Methods – Esterifications
DKR of Hemiacetals, cyanohydrins, and derivatives. Achieved by combining lipase-
catalyzed transesterification with racemization via dissociation-recombination.
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Biotransformation
c) Other Enzymatic Methods via DKR
DKR applied to microbiological Baeyer-Villiger oxidation.
The dynamic resolution of racemic 2-benzyloxymethylcyclopentanone 1 upon
microbiologically mediated Baeyer–Villiger oxidation allowed the corresponding
(R)-lactone 2 to be prepared in 85% yield and 96% ee.
Combination of Enzymes and Transition Metals in DKR
Why use anything other than simple enzymes and nonmetallic racemization
methods? These DKR approaches are mainly limited to substrates that possess a
stereogenic center with an acidic proton
A Possible Solution: Transition Metal-Catalyzed Racemizations
29
Biotransformation
There are two general methods that have been used for the racemization using
transition metal catalyst: (i) racemization via hydrogen transfer and (ii) racemization
via π-allyl formation. Hydrogen transfer reaction mechanism involves metal
hydrides as key intermediates
a) Enzyme-Metal Catalysis - DKR of Alcohols /Diols
Efficient DKR of secondary alcohols was obtained by combining immobilized
CALB transesterification using p-chlorophenyl acetate as acyl donor and ruthenium-
catalyzed racemization
b) Enzyme-Metal Catalysis - Other Alcohol DKRs
The chemoenzymatic DKRs were carried out using ruthenium catalyst 1 as the
racemization the chemocatalyst, immobilized CALB as the biocatalyst, and
30
Biotransformation
p-chlorophenyl acetate 2 as the acyl donor in toluene
c) Enzyme-Metal Catalysis Utilizing Palladium
2-phenyl 2-cyclohexenyl acetate deracemized via lipase-catalyzed hydrolysis in
phosphate buffer and the unreactive enantiomer is racemized in situ with
PdCl2(MeCN)2
d) DKR of Amines
combination of immobilized CALB as biocatalysts and palladium on carbon as
racemization catalysts was used for the synthesis of (R)-N-(1-phenylethyl)acetamide
from 1-phenylethylamine in moderate yield (64%) and enantiomerically pure form
Challenges with Enzymatic DKR
There are currently a number of serious drawbacks towards applications of this
technology. Since enzymes and chemical catalysts usually work in different
environments, their combination in a one-pot transformation in far from
straightforward.
31
Biotransformation
For instance, with lipase catalyzed DKRs with transition-metal catalysts, solvents,
metal, acyl donor, and temperature all need to be optimized.
Potential Solution: Enzyme-free DKR
DKR Utilizing Organocatalysts
DKR of azlactones using urea-based bifunctional organocatalysts
Bifunctional organocatalysts of type D were used for the enantioselective Michael
addition of malonates to nitroolefins.
A substrate-catalyst interaction is expected to occur by hydrogen bonding of the
quasi Lewis acidic urea moiety to the azlactone carbonyl group.
N-benzoyl amino acid allyl esters (9 a–f) were obtained with greater than 70% ee
in the presence of 5 mol% of the urea catalyst
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