Download pdf-download

Biotechnology
Enzymatic C-C coupling in the s
Dr Pascal Dünkelmann of Evocatal and Professor Michael Müller of the University of Freiburg
advocate biocatalytic C-C bond formation and cleavage to produce enantiopure hydroxy-ketones
effiiciently
Another example is the application of hydroxynitrile
lyases that catalyse the formation of hydrogen cyanide
in plants as a protection against herbivores. In synthetic chemistry, these catalysts can be used to form the
useful hydroxynitriles in an asymmetric manner, such
as in the formation of (R)-mandelic acid using an
enzyme derived from almonds.
A family of enzymes that has underestimated
potential, in terms of technical applications is the thiamine pyrophosphate (TPP)-dependant enzymes.2
These enzymes exhibit a highly interesting synthetic
potential They catalyse the formation or cleavage of CC bonds starting from carbonyl compounds. An activated aldehyde intermediate, generated by adding the
carbonyl compound to the TPP molecule, represents
the activated species in these conversions. (Figure 1)
any examples of where enzymes or whole
cells are applied efficiently to produce high
value chemicals have been introduced in
the past decade.1 Among these, hydrolases are the
most prominent. These enzymes cleave or form ester
or amide bonds by catalysing a saponification or the
corresponding back reaction. One example is the
racemic resolution of amino acids like methionine to
the desired enantiopure L-compounds.
Another class of enzymes that can be found regularly in commercialised processes are the so-called oxidoreductases, enzymes that perform oxidation or
reduction reactions. A well-known example is Eli Lilly’s
whole-cell transformation to generate (S)-(3,4-methylenedioxyphenyl)-2-propanol, the key intermediate in
the synthesis of 3,4-methylendioxy-N-methylamphetamine (Tenamfetamine).
Various industrial applications have also been established for other enzyme classes. In many examples,
stereoselectivity plays an outstanding role. However, in
most of these reactions, bonds between carbon and
hetero-atoms are formed, cleaved or modified.
Even though enzymes are already known to catalyse C-C bond formations, there are only a few examples of their industrial application. Enzymatic aldol
reactions catalysed by the enzyme 2-deoxyribose-5phosphate-aldolase (DERA) should be mentioned in
this context.
M
Formation of α-hydroxy-ketones
The usefulness of thiazolium-catalysts for C-C-bondforming reactions via ‘Umpolung’ was described by
Ugai et al. in a Japanese journal back in 1943.3 Since
then, a broad variety of different synthetic applications
of thiazolium- or derived catalysts have been shown,
in many cases as alternatives to known hydrogen
cyanide-catalysed ‘Umpolung’ reactions.
In 1966, Shehan and Hunneman introduced the
first asymmetric variant of the benzoin condensation.
Then, in the 1970s, Stetter et al. published intensive
OH
CH3
O
CH3
O
R1
R2
N+
(R)-Phenylacetylcarbinol (PAC)
O
Pyruvate
S
(Donor)
H
R1
CH3
Thiamine pyrophosphate
(TPP)
CH3
R1
R2
N+
R2
N+
S
S
HO
O-
H3C
HO
CH3
CH3
O-
O-
O
Ph
O
H
Benzaldehyde
R1
R2
N+
R1
R2
N
S
S
(Acceptor)
CO2
CH3
CH3
HO
HO C-
CH3
CH3
Activated aldehyde
Note: Shown as the formation of (R)-PAC starting from benzaldehyde & pyruvate
Figure 1 - Catalytic Cycle of thiamine pyrophosphate-dependant enzymes shown
16
studies on the condensation of aldehydes with α,βunsaturated carbonyl species, the so-called Stetter
reactions.
During the last two decades, interest in this kind of
transformation grew further. In this context, the transformations catalysed by triazolium compounds and
the investigations by Knight and Leeper who introduced novel bicyclic thiazolium salts for asymmetric CC bond formations, should be mentioned.
Many efforts have also been made to use such catalysts in the synthesis of pharmaceutical compounds.
One example is the application of a thiazolium derivative catalysing a Stetter reaction, leading to a key
intermediate in the synthesis of atorvastatin.4
What is mostly overlooked in this context is that all
these efforts only mimic what nature does very well,
after millions of years of evolution. Although the structure of TPP, the natural equivalent to the above mentioned heterocyclic catalysts, was not elucidated before
1937, a biotechnological process applying the synthetic principle, in which pyruvate derived from the central
metabolism of Saccharomyces cerevisiae and benzaldehyde are coupled to give (R)-phenyl-acetylcarbinol, a precursor in the synthesis of Ephedrine,
had already been established in 1921.
Figure 1 depicts the catalytic cycle of this reaction.
The first step is the addition of pyruvate, acting as the
donor, to the catalyst TPP. After the elimination of
CO2, an enamine complex, the so-called activated
aldehyde, is formed. To this, the second aldehyde, the
acceptor, is bound. Afterwards, the final 2-hydroxy
ketone is released. This reaction was established as a
whole-cell transformation using baker’s yeast. It is still
in use and represents the only commercialised process
applying a TPP-dependent enzyme.
Enzymes that have already been studied intensively because of their high synthetic potential include various pyruvate decarboxylases from bacteria or yeasts,
benzoylformate decarboxylase from the microorganism Pseudomonas putida and the branched-chain 2keto acid decarboxylase from Lactococcus lactis. These
proteins catalyse the non-oxidative decarboxylation of
2-keto acids to aldehydes as a main reaction.
The benzoin condensation-like C-C bond formation occurs as a side reaction. It is important to mention here that these enzymes are able to accept aldehydes as donors directly. This is, of course, beneficial,
as the aldehydes are much cheaper and more available than the corresponding 2-oxo-acids.
Typical products that result from these conversions
are aliphatic and aromatic 2-hydroxy-ketones. Among
these are 2-hydroxy-1-phenylpropanone (HPP) and
phenyl-acetylcarbinol (PAC). While the (S)-enantiomer
of HPP represents a key intermediate for the API
bupropione, the (R)-enantiomer of PAC can efficiently
be used in the synthesis of ephedrine, as described
above.
November 2011 Speciality Chemicals Magazine
www.specchemonline.com
Biotechnology
synthesis of fine chemicals
CH3
CH3
R
R
O
R'
O
OH
OH
CH3
CH3
H3C
OH
R
R'
Enzyme-catalysed Stetter reaction
O
CH3
OH
However, attempts to solve this problem by
changing the structure of the enzyme, as well as by
identifying homologues, are under investigation. In
this context, the elucidation of the three-dimensional structure of YerE, which is not yet known, will be
important.
O
O
OH
CH3
OH
R
O
O
R
O
O
R
CH3
H
+
H
O
R
+
O
TPP
CH3
OH
O
OH
OH
O
CH3
R
CH3
O
O
CH3
CH3
CH3
H3C
O
OH
OH
O
OH
CH3
R
Figure 2 - Chiral 2-hydroxy ketones accessible via TPP-catalysed syntheses
For other TPP-dependent enzymes the C-C bond
formation and cleavage represents the main reaction.
Enzymes that have been studied in this context are, for
example, transketolases which form D-seduheptulose7-phosphate by transferring 2-hydroxy-acetyl-residue
to D-ribose-5-phosphate and benzaldehyde lyase isolated from Pseudomomas fluorescens. The latter catalyses the reversible cleavage of one molecule of (R)benzoin into two molecules of benzaldehyde.
From a synthetic standpoint, it is more interesting
that this enzyme also catalyses the C-C bond formation highly efficiently. Not only (R)-benzoin but also a
vast variety of benzoin derivatives, including mixed
benzoins, plus (R)-HPP derivatives and aliphatic 2hydroxy ketones are accessible using this enzyme.
In addition to the (R)-enantiomer, the (S)-enantiomers are also available from these reactions, by resolution of the racemic compounds. In this case, the
enantiomerically pure (S)-compounds remain
untouched in the reaction while the (R)-enantiomer is
cleaved into the corresponding aldehydes.
Not surprisingly for enzymatic conversions the
described ligase and lyase reactions are, in most cases,
highly stereoselective. The number of compounds that
can be synthesised in such reactions is constantly
increasing. This is due to the fact that in many cases
the enzymes do not only catalyse the conversion of
their natural substrate but also that of a broad variety
of compounds with similar functionality.
In addition, scientists also take advantage of the fact
that more and more variants of these enzymes are
becoming available. Figure 2 shows various chiral
compounds that can be obtained by applying the
described TPP-dependant enzymes.
Synthetic route to tertiary alcohols
One TPP-dependent enzyme with a tremendous synthetic potential that has been studied in the recent
past, is a lyase that was isolated from Yersinia pseudotuberculosis.5 The application of this protein allows the
synthesis of tertiary alcohols in an asymmetric manner,
by catalysing the C-C bond formation between a
ketone and pyruvate or acetaldehyde, whereas the
synthesis of such compounds by classical organo-synthetic means is difficult to achieve.
A broad variety of carbonyl compounds were
accepted as acceptor in this conversion. Among these
acceptors are small cyclic aliphatic compounds like
cyclohexanone derivatives, open chain ketones, 1,2diketones as well as α- and β-keto esters (Figure 3).
The tertiary alcohols obtained in these reactions
represent valuable building blocks for the asymmetric synthesis of 1,2-diols vicinal amino alcohols or
two contiguous tertiary alcohols. Unfortunately, the
ee’s that can be achieved in these reactions are not
yet satisfactory.
O
O
Although the TPP-dependent enzymes catalyse the
stereoselective formation or cleavage of α-hydroxyketones, this class of enzymes is not limited to these
transformations. Recent studies have revealed that the
Stetter reaction also can be carried out biocatalytically
by using the appropriate enzyme-catalyst.
In the 1970s, Stetter and others showed that 1,4diketones are accessible by coupling an aldehyde to an
α,β-unsaturated ketone. Cyanide can be used in this
reaction as a catalyst, as can various thiazolium or triazolium salts.
Different studies have been published that dealt
with stereoselective variants of this reaction. However,
whilst some major improvements have been achieved
with regard to the conversion and yield, the enantioselectivity is still a difficult issue. Enzymes can be efficient alternatives in this respect.
It can be shown that various enzymatically catalysed
couplings, between α,β-unsaturated ketones and
mainly pyruvates, are catalysed with high enantioselectivity by applying the enzyme PigD (Figure 4).6 This
enzyme was isolated from the microorganism Serratia
marcescens and is involved in the biosynthesis of the
red pigment prodigiosin.
It is important to note that the formation of 1,2adducts, with ketones as acceptor substrates was not
observed, which nicely complements the aforementioned YerE-catalysed transformations. PigD catalyses
only the 1,4-addition. Interestingly, not only aliphatic
but also aromatic and some heterocyclic α,β-unsaturated ketones selectively reacted with pyruvate in the
presence of PigD.
Larger substituents, even on both sides of the carbonyl group, did not sterically affect the Stetter reaction. Pyruvate is the preferred donor substrate, but
2-oxobutanoate could also be employed. The yields
of isolated products are not yet satisfactory, but one
should be aware that all ketone compounds are
non-physiological substrates of the wild-type
enzyme PigD.
O
HO
+
CH3
H
CH3
CH3
O
YerE, TPP
CH3
O
Acceptor
Examples of additional acceptors:
O
Donor
Conversion after 25 hours: 20 %
ee = 96%
O
O
O
O
H3C
CH3
O
O
O
CH3
O
Figure 3 - Examples of the synthesis of chiral tertiary alcohols accessible via TPP-catalysed reactions
Speciality Chemicals Magazine November 2011
17
www.specchemonline.com
Biotechnology
O
O
H3C
CH3
+
H3C
O
O
-
- CO2
H3C
O
References:
O
PigD, TPP
H3C
1. C. Wandrey, A. Liese & D. Kihumbu, 2000 Org. Proc.
Res. Dev. 2000, 4, 286-290; M. Hall & A. S. Bommarius,
Chem. Rev. 2011, 111, 4088-4110
2. M. Müller, D. Gocke & M. Pohl, FEBS 2009, 276, 28942904; D. Gocke, T. Graf, H. Brosi, I. Frindi-Wosch, L.
Walter, M. Müller & M. Pohl, J. Mol. Catal. B: Enzymatic
2009, 61, 30-35; P. Domínguez de María, M. Pohl, D.
Gocke, H. Gröger, H. Trauthwein, T. Stillger, L. Walter &
M. Müller, Eur. J. Org. Chem. 2007, 2940-2944; P.
Hoyos, J.-V. Sinisterra, F. Molinari, A. Alcantara & P.
Dominguez de Maria, Acc. Chem. Res. 2010, 43, 288299
3. T. Ugai, R. Tanaka & T. Dokawa, J. Pharm. Soc. Jpn.
1943, 63, 296
4. K. L. Baumann, D.E. Butler, C. F. Deering, K.E. Mennen,
A. Millar, T.N. Nanninga, C.W. Palmer & B.D. Roth,
Tetrahedron Lett. 1992, 33, 2283-2284
5. P. Lehwald, M. Richter, C. Röhr, H.-W. Liu & M. Müller,
Angew. Chem. 2010, 122, 2439 -2442
6. C. Dresen, M. Richter, M. Pohl, S. Lüdeke & M. Müller,
Angew. Chem. 2010, 122, 6750 -6753
CH3
Yield: 38 %
ee > 99 %
Examples of additional acceptors:
O
O
H2C
O
CH3
O
CH3
CH3
O
OH
Figure 4 - Examples of asymmetric TPP-catalysed Stetter reactions
Catalyst optimisation
be optimised, for instance, for pH- and temperaturestability and solvent tolerance, and also for regio- or
stereoselectivity.
The common way to identify the appropriate
enzyme for a conversion, carried out by enzyme optimisation specialists is to screen enzyme libraries consisting of variants generated by molecular biology
mutation techniques. The enzyme variant showing the
best performance might be used as the catalyst or
might undergo additional mutation rounds until sufficient performance is obtained.
Various enzyme classes, especially those mentioned
in the introduction, are also commercially available as
ready-to-use test kits allowing every scientist to study
the viability of enzyme catalysis for the conversion of
interest. The TPP-dependant enzymes have not been
commercially available so far. Evocatal recently
launched a screening kit with a selection of these
enzymes so that they are now easily accessible for academic and industrial research.
The increasing acceptance of biocatalysis as a tool in
organic synthesis which is, of course, growing with
every commercialised process, has also resulted in a
strong stimulation of different kinds of investigation
into how to make enzymes available for any application that goes beyond proof-of-principle.
It is, nowadays, state of the art to produce biocatalysts above in special recombinant production
organisms like Eschericha, Bacillus or Pichia strains.
Such organisms in combination with special microbiological protein-production tools, allow the production of the desired protein at an almost unlimited scale.
As a result, the costs for these catalysts can be
reduced below those of the classical non-enzymatic
chiral catalysts. Moreover, due to the increasing number and improving capabilities of evolution techniques,
enzymes can be adapted perfectly to the requirements of the needed conversion. The catalyst might
OH OHOHO
OH
OH
OH O
OH O
OH
OH O
OH
OH
OH
OH O
OH
OH
OH O
OH
OH
O
OH O
OH
OH O O
OH O
OH
OH O
OH
Cl
OH
O
OH O
O
OH
OH
O
OH
OH OH
O
OH O
OH
O
OH O
OH
O
OH
OH
OH
OH OOH
OH OH
O
OH O
O
OH
OH O OH
Cl
OH
O
OH
OH
OH
OH
OH
OH O
OH O O
O
OH
OH O
O
O
OH
OH O
OH OH
O O
O
OH
OH O
OH
Cl
OH
Cl
O
O
OH
OH
O
OH
OH
OH O
OH
OH
OH O
OH
OHOHO
OH
O O
OH
O OH
OH O
Cl
OH
OHOH O
OH
OHO O
OH
Cl
OH
OH
O
OH O
O
OH
OH O
OH
Cl O
OH
O
OH
OH
O
OH OH
OH
OH
OH O
Cl O
OH
OH
Cl
O
O
OH
O
O
OH
OH
O
OH
O
Cl
O
OH O
OH
O
OH O
OH
OH O
OH
O
OH
O
OH
O
OH O
OH OH
OH O
OH
O
O
OH
O
OH
Cl
OH
OH
O
OH O
OH
O
OH
OH
OH
OH
O
Cl
OH
Cl OH
OH
OH
OH
OH
OH
OH O
OH
OH O
OH O
O
OH O
Cl
O
evocatal – the art of chiral purity
evocatal develops and produces high performance biocatalysts
for the chemical and pharmaceutical industries. Our customers
gZ\VgYjhVhVegZb^jbegdk^YZgd[]^\]fjVa^in!ÂcZX]^gVaWj^aY"
ing blocks: from a few grams to several tons. If you are interested
in our products, feel free to visit our website and download our
catalogue.
g
t
www.evocatal.com · +49 211 1576095-0
O
OH
OH O
O
OH
OH
OH O
O
OH
OH
OH
OH
OH
O O
OH
O
OOH
OH
OH
OH O OH
OH
Cl
OH O
OH O
OH
OH O
OH
OH O
O
OH O
OH
OH
OH
OH OO
O
OH
OH O
OH O
OH O
OH
OH OO
OH
Cl
OH O
OH
OH
OH O
OH
OH
O
OH O
OH
OH O
OH
OH
O
OH O
OH
OH O
OH O
OH
O
O
OH
O
OH
Cl
For more information, please contact:
Dr Pascal Dünkelmann
Director of Commercialisation
Evocatal GmbH
Merowingerplatz 1a
D-40225 Düsseldorf
Germany
Tel: +49 211 157 6095-0
E-mail: [email protected]
Website: www.evocatal.com