Artificial enzymes

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Artificial enzymes
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In general these different approaches can be
divided into three categories The design
approach. A host molecule is designed with
salient functionality (often also present in the
natural enzyme counterpart) which is expected to
be involved in catalysis of the chosen reaction.
Catalytic cyclodextrins are one such
example. The transition state
analogue-selection approach. A library of hosts
is generated in the presence of a transition
state analogue (TSA) and the best host is then
selected from the library. This latter approach
has been employed with considerable success in
the field of catalytic antibodies and has more
recently inspired the process of molecular
imprinting (vide infra). The catalytic
activity-selection approach. This takes
advantage of the combinatorial chemistry
revolution wherein a library of possible
catalysts is generated and screened directly for
enzyme-like activity.
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Cyclodextrins as enzyme mimics
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Cyclophane enzyme mimics
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Reversibly self-assembled dimers as enzyme mimics
Rebek and co-workers have carried out much
research into the synthesis of reversibly
self-assembled dimers. The extended polycyclic
system in 26 exists as a hydrogen-bonded dimer in
organic solvents and adopts a pseudo spherical
structure (described as a hydroxy-softball)
which is able to form and dissipate on a
timescale of milliseconds. This dynamic
behaviour, coupled with the microenvironment
provided by the softball led Rebek et al. to
investigate the catalytic potential of 26 towards
the DielsAlder reaction of thiophene dioxide 28
and benzoquinone 27
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Molecular imprinting
Schematic diagram of the molecular imprinting
process (i) the template is mixed with vinyl
monomers, selected to interact with specific
functionality of the template, (ii) the
template-monomer complex may be formed by
covalent or non-covalent associations (or a
mixture of both), (iii) the complex is
co-polymerised with an excess of cross-linking
monomer ethylene glycol dimethacrylate (EGDMA)
or divinylbenzene (DVB) typically being used
whilst the inclusion of a small amount of solvent
ensures that the polymer structure is porous,
allowing access to the sites within the polymer
monolith, and (iv) the polymer is usually ground
to a powder for ease of handling and the template
removed by solvent extraction or chemical
treatment. The sites created in the polymer are
complementary in shape to the template and bear
the functionality originally involved in complex
formation, precisely arranged to interact with
the template on rebinding.
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                   Non-covalent imprinting of
11-a-hydroxyprogesterone with methacrylic acid
cross-linked with EGDMA to leave recognition
sites used to screen binding of steroids
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     Figure 2. Schematic of pathways involved in
enzyme-analogous catalysis. A substrate S
associates with catalyst C leading to the
products P. Stabilisation of the transition state
T.S. by the catalyst lowers the activation energy
of the CP reaction. The rate of conversion of
the substrate dS/dt can be related to the rate
constant of the catalysed reaction and the
concentrations of substrate and catalyst by the
MichaelisMenten equation (Equation 2).
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                                      Class II
aldolase dibenzoylmethane imprinted as the
cobalt-bis(4-vinylpyridine) complex (47) to leave
a metal coordination site. Subsequent rebinding
of acetophenone and benzaldehyde in the site,
followed by catalysed CC bond formation and loss
of water generated the a,b -unsaturated ketone
(48).
10
  Two views of the active site of bovine
chymotrypsin, showing the relative positions of
amino acids serine 195, histidine 57 and aspartic
acid 102the catalytic triad.
MIP chymotrypsin mimic prepared by Leonhardt and
Mosbach for the hydrolysis of activated ester
substrates.   
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The catalytic activity-selection approach
Combinatorial polymers as enzyme mimics
In a highly original approach to artificial
enzymes, Menger et al. have developed the
combinatorial derivatisation of pollyallylamine.
The basic idea was to attach various combinations
of carboxylic acids to polyallylamine backbones
and then screen for catalysis in the presence of
a metal ion. The idea that a vast number of
molecules can be generated from a very restricted
number of initial partners is of particular in
interest in terms of evolutionary chemistry.
Phosphatase activity and reduction of
benzoylformate to mandelate
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Dynamic combinatorial libraries (DCLs)
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Combinatorial chemistry vs. dynamic combinatorial
chemistry
dynamic chemical libraries virtual, dynamic set
of molecules or supramolecules prepared by
reversible chemical reactions prepared in
presence of target

• chemical libraries
• large, static populations of discrete molecules
• prepared by irreversible chemical reactions
• prepared in absence of target

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Reversibility
reversible covalent bonds or noncovalent
interactions (supramolecules)
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Lehn J. M. Chem. Eur. J. 1999, 5, 2455
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Target-driven self-assembly
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Target-driven self-assembly
Casting for a substrate
Otto S., Furlan R. L. E., Sanders J. K. M. Drug
Discovery Today 2002, 7, 122
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Target-driven self-assembly
Molding for a receptor
Otto S., Furlan R. L. E., Sanders J. K. M. Drug
Discovery Today 2002, 7, 122
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Target-driven self-assembly
Otto S., Furlan R. L. E., Sanders J. K. M. Drug
Discovery Today 2002, 7, 122
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Target-driven self-assembly
relative concentration
library is generated in situ dynamic chemical
libraries virtual chemical libraries
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Example carbonic anhydrase inhibitor design
Huc I., Lehn J. M. Proc. Natl. Acad. Sci 1997,
97, 2106-2110
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Example carbonic anhydrase inhibitor design
Huc I., Lehn J. M. Proc. Natl. Acad. Sci 1997,
97, 2106-2110
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Advantages of dynamic combinatorial chemistry
high speed of a process single step is
used only active compounds are formed in
detectable quantities and further
processed rapid generation of broad structural
diversity
low cost
Lehn J. M. Chem. Eur. J. 1999, 5, 2455 Huc I.,
Lehn J. M. Proc. Natl. Acad. Sci 1997, 97,
2106-2110 Otto S., Furlan R. L. E., Sanders J. K.
M. Drug Discovery Today 2002, 7, 122 Hochgürtel
M. et al. Proc. Natl. Acad. Sci 2002, 99,
33823387
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In vitro Evolution

• formation of mutants - error prone
  replication/transcription
• - mutagenesis
• - combinatorial
  synthesis
• selection of active mutants
• use of the active mutants in another cycle

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In vitro selection of a) nucleic acids
b) proteins
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Evolution of a ribozyme
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Lipase evolution
- meranie absorbancie
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Bacterial display
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Phage display
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Chemical Genetics
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Forward Approach
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muscular cells before compound treatment   muscula
r cells after myoseverin treatment
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Tubuline polymerization
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Tubulin has GTP binding site and also is a sort
of GTPase which make GTP to GDP through
hydrolysis in making microtubules. Microtubule
has growing end and reducing end. In cell
division, formation and destruction of well
controlled microtubule is required for exact
chromosome transfer. Natural substances (vinca
alkaloids, cholchicine), destructing microtubules
or preventing synthesis from tubulin, interrupt
normal cell division. Cholchicine is a substance
that was used to make seedless watermelon. On the
other hand, taxol, which excessively stables
microtubule and prevents its dynamic change, is
also used as anti-cancer medicine because it
stops normal cell division. For the microtubule
to work properly, MAP (microtubule associate
proteins) is also important. Hence, it is not
certain if myoseverin function on tubulin
directly or on other MAP. To verify it, purified
tubulin was bought from Cytoskeleton and it makes
microtubule in a certain solvent condition. When
myoseverin was inserted, tube structures were
clearly disappeared. Therefore, it was confirmed
that myoseverin directly works on tubulin or
microtubule.                                    
                                                 
                                                
                          

 microtubules before compound
treatment              microtubules after
myoseverin treatment
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                                      modified
affinity molecules of myoseverin
                                                  
                                              
                                                  
                     in vivo tubulin fishing
(1 affinity molecule, Ms myoseverin) In case
of myoseverin, instead of using linkers to bind
to resins, biotin, which binds very tightly to
Streptavidin, and nucleophile with strongly
active functional group an affinity molecule were
used. The advantage of this method is that the
affinity molecule can be induced to bind to
object proteins simply by insert it into a living
cell, instead of making protein mixture by
grinding cells. If the object protein binds to
the molecule, chemical active group will bind to
the proteins nucleophile by covalent bonding so
that one can catch the object protein with
streptavidin column by using biotin. It was
proved after the experiment that in vivo tubulin
binds to the affinity molecule.
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Reverse Approach
Selection of object protein Cell division is
like a harmonious orchestra of various
well-functioning proteins. CDK (cyclin dependant
kinases) are control-switch-proteins in each cell
division step, and among them, CDK2 takes part in
G1 to S step and CDK does in G2 to M step. Very
active researches are going on to find out their
specific functions. Therefore, in this research
we decided to research on chemical compounds that
inhibit functions of CDK1 or CDK2.
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Development of CDK inhibitor Purine library from
Forward Approach was used in screening enzyme
inhibitor compounds on purified the selected CDK1
and CDK2. Because purine compounds were expected
to bind competitively to ATP binding sites using
coenzymes, purine was employed. To accelerate the
screening process, emzyme activation was achieved
by using radioactive labeled ATP and histone
protein in 96 well plates and measured through
radioactivity of phosphate group transferred to
histone from proteins sieved with nitro cellulose
paper. Starting from olomocine (IC50 7mM), we
could achieve approximately 1000 times more
activated purvalanol series compounds at the end
of several steps of repetition. These compounds
inhibited both CDK1 and CDK2 at the similar
activities. It is because of both the enzymes are
built up through very similar pathways and the
similarities of their ATP binding sites.
                                                  
                                                  
                                                  

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In the regular state, DNA folds to form
chromosomes and these chromosomes aligned. Then
microtubules attach to them and drag them to two
sides. However, if purvalanol is added to this
state, DNA does not fold completely and
microtubules do not find their attaching sites.
It seems like G2 to M step was attacked. So to
speak, the inhibition is toward CDK1 than CDK2.
In addition, when myoseverin was added to the
same system, DNA folding did not affected at all,
but microtubule structures were completely
disappeared. It can be attack on microtubules at
M state right after G2.
                                                
                                                 
                                                 
                              normal
metaphase              after purvalanol
treatment        after myoseverin treatment
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Verification of Purvalanol binding protein To
verify which protein binds to purvalanol,
affinity column of agarose resins was used to
fish up the unknown protein. Generally, in
affinity column, even some proteins without any
selectivity are also obtained with object
proteins due to the other basic materials in the
column. To separate these unnecessary proteins,
comparison affinity column, of purvalanol-like-com
pound with no activity, was applied.