BY TATHAGATA PRADHAN
• 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
• 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
• Catalytic groups of Artificial enzyme bind with substrates, using
backbone such as :
- crown ether etc.
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.
• 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
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
Generally used Macrocyclic molecule for constructing artificial
• Cyclodextrins as enzyme mimics
• Cyclophane as enzyme mimics
• Calixarene as enzyme mimics
• Crown ethers as enzyme mimics
Macrocyclic molecules for the design of artificial enzymes
• 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
• A complete artificial enzyme can be synthesised by modifying
cyclodextrins to contain a catalytic site attached at an appropriate
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.
• 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.
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
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.
• 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
• Calixarene is a cyclic oligomer based on the hydroxyalkylation product of a phenol
• 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
• The p-sulfonatocalix[n]arenes 1 were developed by Shinkai et al. in
the 1980s as water-soluble calixarenes for catalytic studies in water
p-sulfonatocalix[n]arenes 1 with the
protonated form of basic amino acid
derivatives, as shown in 2 for the His-1 (n=4)
APPLICATIONS OF CALIXARENES
• Calixarenes have been extensively used as molecular platform to build up
• 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
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
• Calixarenes which bear imidazole groups at different positions were
reported recently by schatz et al. as metal free enzyme mimics with trans –
• 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
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.
• 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.
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.
• 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.