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Artificial enzymes
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.
Cyclodextrins as enzyme mimics
O
OHHO
OH
O
O
HO
HOOHO
OHO
OH
OH
O
O
HOOH
OH
OO
HO
OH
HOO
OOH
OHHO
O
OOH
HO
HO
O
-CD
Cyclophane 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 Diels–Alder reaction of thiophene dioxide 28 and benzoquinone 27
Reversibly self-assembled dimers as enzyme mimics
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.
Molecular imprinting
Non-covalent imprinting of 11--hydroxyprogesterone with methacrylic acid cross-linked with EGDMA to leave recognition sites used to screen binding of steroids
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 C–P reaction. The rate of conversion of the substrate d[S]/dt can be related to the rate constant of the catalysed reaction and the concentrations of substrate and catalyst by the Michaelis–Menten equation (Equation 2).
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 C–C bond formation and loss of water generated the , -unsaturated ketone (48).
Two views of the active site of bovine chymotrypsin, showing the relative positions of amino acids serine 195, histidine 57 and aspartic acid 102—the ‘catalytic triad’.
MIP chymotrypsin mimic prepared by Leonhardt and Mosbach for the hydrolysis of activated ester substrates.
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
Dynamic combinatorial libraries (DCLs)
Combinatorial chemistry vs.dynamic combinatorial chemistry
chemical libraries
large, static populations of discrete molecules
prepared by irreversible chemical reactions
prepared in absence of target
dynamic chemical libraries
virtual, dynamic set of molecules or supramolecules
prepared by reversible chemical reactions
prepared in presence of target
Reversibility
reversible covalent bondsor
noncovalent interactions (supramolecules)
Lehn J. M.: Chem. Eur. J. 1999, 5, 2455
Target-driven self-assembly
target
Casting for a substrate
Otto S., Furlan R. L. E., Sanders J. K. M.: Drug Discovery Today 2002, 7, 122
Target-driven self-assembly
Molding for a receptor
Otto S., Furlan R. L. E., Sanders J. K. M.: Drug Discovery Today 2002, 7, 122
Target-driven self-assembly
Target-driven self-assembly
Otto S., Furlan R. L. E., Sanders J. K. M.: Drug Discovery Today 2002, 7, 122
library is generated in situdynamic chemical libraries = virtual chemical libraries
relative concentration
abse
nce
of ta
rget
pres
ence
of t
arge
t
Target-driven self-assembly
Example: carbonic anhydrase inhibitor design
Huc I., Lehn J. M.: Proc. Natl. Acad. Sci 1997, 97, 2106-2110
Example: carbonic anhydrase inhibitor design
Huc I., Lehn J. M.: Proc. Natl. Acad. Sci 1997, 97, 2106-2110
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, 2455Huc I., Lehn J. M.: Proc. Natl. Acad. Sci 1997, 97, 2106-2110Otto S., Furlan R. L. E., Sanders J. K. M.: Drug Discovery Today 2002, 7, 122Hochgürtel M. et al.: Proc. Natl. Acad. Sci 2002, 99, 3382–3387
- formation of mutants - error prone replication/transcription
- mutagenesis
- combinatorial synthesis
- selection of active mutants
- use of the active mutants in another cycle
In vitro Evolution
In vitro selection of:
a) nucleic acids
b) proteins
Evolution of a ribozyme
Lipase evolution
- meranie absorbancie
Bacterial display
Phage display
Chemical Genetics
Forward Approach
muscular cells before compound treatment muscular cells after myoseverin treatment
Tubuline polymerization
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
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 protein’s 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.
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.
Reverse Approach
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.
normal metaphase after purvalanol treatment after myoseverin treatment
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.
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-compound with no activity, was applied.
