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Artificial enzymes

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Artificial enzyme made from natural enzyme

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Artificial enzymes

  1. 1. BY TATHAGATA PRADHAN M.PHARMA FIRST SEMESTER [PHARMACEUTICAL CHEMISTRY]
  2. 2. INTRODUCTION • Enzymes are bio macromolecules with three-dimensional structures composed of peptide polymers via supramolecular Interactions which include hydrophobic interactions , electro-static attractions, hydrogen bonding , van der waals interactions and metal-ligand coordination. • These interactions play important roles in both substrate recognition and the process of enzyme catalysis. Hence artificial enzymes have been designed and constructed on the basis of these supramolecular interactions.
  3. 3. • An artificial enzyme is a synthetic , organic molecule or ion that recreate some function of an enzyme. This promises to deliver catalysis at rate and selectivity observed similar to natural enzyme. • Enzyme catalysis of chemical reactions occur with high selectivity and rate by Artificial enzyme. • Substrate is activated in a small part of the enzyme’s macromolecule called the active site. • It is believed that a suitable microenvironment is required when an enzymatic reaction is carried out. An eligible pocket could not only segregate substrates and active sites of enzymes from the surroundings, but also provide the microenvironment for accomplishing the substrates recognition and enzymatic reaction. • Cavity containing molecules, such as cyclodextrins, calixarenes, container molecules, provide similar environment as present in the pocket of natural enzymes.
  4. 4. • Catalytic groups of Artificial enzyme bind with substrates, using backbone such as : - Cyclodextrins - calixarene - crown ether etc.
  5. 5. Applications of Artificial Enzyme Artificial enzymes are a class of catalysts that have been actively interest as potentially viable alternatives to natural enzymes. • Artificial enzymes have the desired advantages due to tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation . • Artificial enzymes have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels . • Pharmaceutical Industry - specifically the designing of synthetic enzymes that accelerates the formation of drugs and chemicals. • Medicine - use of synthetic enzymes as supplements for patients deficient in certain enzyme can be made instead of extracting natural enzymes from other organism. • Genetic Engineering – potentially designing synthetic enzymes that manipulate gene sequences to create genetically modified organisms or to help genology research.
  6. 6. • Tunable structures and catalytic efficiencies similar to natural enzyme • Excellent tolerance to experimental conditions • Purely synthetic routes for their preparation • High cost and low stability limit the application of natural enzymes • Speeds up the reaction at a relatively high rate
  7. 7. DESIGN APPROACH FOR ARTIFICIAL ENZYMES The traditional approach for constructing artrificial enzymes has been the designing of macromolecular receptors with appropriately placed functional groups (catalytic groups). These catalytic groups are usually chosen to mimic the amino acid residues known to be involved in the natural enzyme catalysed reaction. Generally used Macrocyclic molecule for constructing artificial enzymes: • Cyclodextrins as enzyme mimics • Cyclophane as enzyme mimics • Calixarene as enzyme mimics • Crown ethers as enzyme mimics
  8. 8. Macrocyclic molecules for the design of artificial enzymes  CYCLODEXTRINS • Made up of 6,7 or 8 units of α-1,4-linked D-glucopyranoses • It has a Hydrophobic cavity • Stable and water soluble • It is tunable (modify to change properties ) • The inner diameter of cavities is approx. 4.5Å - α-cyclodextrin 7.0Å - β-cyclodextrin 8.5Å - γ-cyclodextrin synthesis of cyclodextrin : Starch cyclodextrin (by the action of CTGase) i.e. cyclodextrin glucosyl transferase
  9. 9. • A complete artificial enzyme can be synthesised by modifying cyclodextrins to contain a catalytic site attached at an appropriate position. For example : The design of artificial redox enzyme consists of a cyclodextrin molecule acting as a binding site covalently attached to a flavin molecule as a catalytic site.
  10. 10. • The electrostatic environment in the binding site maintains the pKa balance required for various groups to participate in the catalytic fashion. • Histidine is often able to function both as acid and as base in catalysis ,influenced by this phenomenon Breslow and his Co –worker chose to mimic enzyme Ribonuclease A. • Ribonuclease A is a member of a group of enzymes that cleave RNA using general acid–base catalysis without a metal ion in the enzyme. • In ribonuclease A, such catalysis is performed by two imidazoles of histidine units, one as the free base (Im) and the other, protonated, as the acid (ImH+). To mimic this in an artificial enzyme, we prepared b-cyclodextrin bis-imidazoles.
  11. 11. A catalyst carrying only one imidazole showed only base catalysis, by the unprotonated imidazole group Im. Thus, in catalyst mixture , one imidazole was acting as a base – delivering a water molecule to the phosphate group of the bound substrate – while the imidazolium ion of the other catalytic group played a role as a general acid. It was thought that this imidazolium ion might be simply protonating the leaving group of the phosphate, as was normally assumed for the enzyme ribonuclease A.
  12. 12. Cleavage of uridyluridine (39, UpU) by artificial enzyme was used to study the cleavage of this dimeric piece of RNA . We saw that high concentrations of imidazole buffer could catalyze this cleavage, mimicking the high effective local concentrations of imidazole in the enzyme, and concluded that with this buffer there was sequential base, then acid catalysis. Hence imidazole attached with cyclodextrin can be utilised for making artificial enzyme.
  13. 13.  CYCLOPHANE • Also known as Diedrich’s pyruvate oxidase mimic • Pyruvate oxidase employs two co factors ThDP(Thiamine di phosphate) and Flavin to water or alcohol to carboxylic acids or esters by simple thiazolinium ions.
  14. 14.  CALIXARENE • Calixarene is a cyclic oligomer based on the hydroxyalkylation product of a phenol and formaldehyde • The word calixarene is derived from calix because this type of molecule resembles a vase and the word arene refers to the aromatic building block • Calixarene are efficient Na+ ionophores and are potentially used in chemical sensors. They also form complexes with Cadmium, lead , lanthanides and actinides.
  15. 15. • The p-sulfonatocalix[n]arenes 1 were developed by Shinkai et al. in the 1980s as water-soluble calixarenes for catalytic studies in water solution. p-sulfonatocalix[n]arenes 1 with the protonated form of basic amino acid derivatives, as shown in 2 for the His-1 (n=4) combination
  16. 16. CALIXARENE 3-D STRUCTURE
  17. 17. APPLICATIONS OF CALIXARENES • Calixarenes have been extensively used as molecular platform to build up supramolecular catalysts. • The design of this catalyst is the functionalizing the upper rim and lower rim of calixarene with ligands able to bind metal cations notably Cu2+ or Zn2+ • Calixarenes accelerates reactions taking place inside the cavity. Different type of mimics shown by calixarene : As Acyltransferase enzyme As Ribonuclease enzyme As ATPase enzyme As Aldolase enzyme As Carbonic anhydrase enzyme
  18. 18. CALIXARENE AS ACYLTRANSFERASE ENZYME MIMIC • Co operativity of functional groups is important for the catalytic properties of the supra molecular enzyme mimic. • Imidazole moieties were often used as an acid /base couple or nucleophile which can enhance hydrolytic processes or Aldol type condensation reaction. • Calix[4]arenes which bear imidazole groups at different positions were reported recently by schatz et al. as metal free enzyme mimics with trans – acyltransferase activity. • The macrocyclic skeleton improve the hydrolysis by 13% compared with the non macrocyclic catalysed and by 52% toward the blank hydrolysis. • Diimidazole calixarene bearing the catalytic group in a distal arrangement double the initial reaction rate indicating some kind of co operativity of the catalytic site.
  19. 19.  CALIXARENE AS ATPase ENZYME MIMIC • Nucleotide polyphosphate , particularly ATP, ADP, AMP are internal parts of the energy cycle for a vast range of biological processes such as oxidative phosphorylation , muscle contraction, photosynthetic phosphorylation etc. • The function of ATPase is simply to hydrolyse the terminal phosphate residue of triphosphate tail of ATP to yield ADP and iP. • This releases about 35KJ/mol of energy and results in temporary photo phosphorylation of the enzyme • An example of supramolecular catalysis show a remarkable effect of supramolecular interaction of the catalysis calixarene on hydrolysis of ATP • The hydroxides at the lower rim were found to cause strong molecular H-Bonding with guest molecule, using Laser photolysis and pulse radiolysis. • This electrostatic interaction between calixarene and substrate was suggested as essentiality for the catalysis. • The hydrolysis of ATP in pure aqueous solution was found to be slow and acceleration in the speed is noted after the addition of water soluble calixarene into the solution.
  20. 20.  CROWN ETHER • Crown ethers are cyclic chemical compounds that consist of a ring containing several ether groups. The most common crown ethers are cyclic oligomers of ethylene oxide, the repeating unit being ethyleneoxy, i.e., –CH2CH2O–. • Important members of this series are the tetramer (n = 4), the pentamer (n = 5), and the hexamer (n = 6). The term "crown" refers to the resemblance between the structure of a crown ether bound to a cation, and a crown sitting on a person's head. • The first number in a crown ether's name refers to the number of atoms in the cycle, and the second number refers to the number of those atoms that are oxygen. Crown ethers are much broader than the oligomers of ethylene oxide; an important group are derived from catechol. • Crown ethers strongly bind certain cations, forming complexes. The oxygen atoms are well situated to coordinate with a cation located at the interior of the ring, whereas the exterior of the ring is hydrophobic. • 18-crown-6 has high affinity for potassium cation, 15-crown-5 for sodium cation, and 12-crown-4 for lithium cation.
  21. 21. APPLICATION OF CROWN ETHER • 18-Crown-6 binds to a variety of small cations, using all six oxygens as donor atoms. Crown ethers can be used in the laboratory as phase transfer catalysts. Salts which are normally insoluble in organic solvents are made soluble by crown ether. For example, potassium permanganate dissolves in benzene In the presence of 18-crown-6, giving the so-called "purple benzene", which can be used to oxidize diverse organic compounds.
  22. 22. • Crown ether ammonium ion binding occurs by hydrogen bonding between oxygen atoms (or nitrogen, sulfur or other free electron pair in hetero crown ethers) and N+–H bonds.

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