PREPARED BY:

PHARMACY 2A-1
Albano, Creon Aoi
Bawalan, Hewlett Pearl
Cunanan, Phinehlopee
Pelayo, Ma. Kristiana
Tolentino, Suzzane

TOPIC: COMPLEXATION AND PROTEIN BINDING

Complexes or coordination compounds result from a donor-acceptor mechanism or Lewis acid-base
reaction between two or more different chemical constituents.

Nonmetallic atom or ion- electron donor

Metallic ion- electron acceptor

Intermolecular forces involved in the formation of complexes are

1- the van der Waals forces of dispersion.

2- dipolar, and induced dipolar types.
3- Hydrogen bonding provides a significant force in some molecular complexes.
4- coordinate covalence is important in metal complexes.

Classification of Complexes II. Organic molecular complexes

I. Metal ion complexes A. Quinhydrone type

A. Inorganic type B. Picric acid type

B. Chelates C. Caffeine and other drug complexes

C. Olefin type D. Polymer type

D. Aromatic type III. Inclusion/occlusion compounds

1. Pi (π) complexes A. Channel lattice type

2. Sigma (σ) complexes B. Layer type

3. “Sandwich” compounds C. Clathrates

D. Monomolecular type

E. Macromolecular Type
INORGANIC COMPLEXES

1. The ligand provides only one site for binding with metal.

Ligand- a molecule that interacts with another molecule, the substrate, to form a complex. It is an
ion or neutral molecule that bonds to a central metal atom or ion.

Simple ligands- Water, Ammonia, Chloride ions -> all have active lone pairs of electrons in
the outer energy level to form coordinate bonds with metal ion.

*ALL LIGANDS ARE LONE PAIR DONORS -> function as LEWIS BASES

* METALS in which ligands bond -> LEWIS ACIDS (electron acceptors)

COMPLEX ION- When the coordination complex carries a net charge

COORDINATION COMPOUNDS- These are compounds that contain a coordination complex

COORDINATION NUMBER- the number of donor atoms bonded to the central metal
atom/ion.

EX. Co 3+ + 6NH3 = [Co(NH3)6]3+ Coordination number

Acceptor/ Lewis acid Ligand/Lewis Base

CHELATES- refers to the special type of complex formed when a substance containing two or
more donor groups combine with a metal

MONODENTATE- When the ligand provides one group for attachment to the central
atom

BIDENTATE- Molecules with two donor groups

TRIDENTATE- molecules with three donor groups

 EDTA (Ethylebediaminetetraacetic acid) – a synthetic chelating agent that is used to tie

up or sequester iron and copper ions to that they cannot catalyze the oxidative
degradaration of ascorbic acid in fruit juices and in drug preparation.
o Also used as an antidote for heavy metal poisoning
o Anticoagulant

TWO GEOMETRIC FORMS

A. Cis isomer – 2 like ligands are adjacent
Example. Alcohol dehydrogenase enzymes (contains zinc)
-can undergo chelation, suggesting that the metal is bound in such a way as to leave
two cis positions available for chelation
B. Trans isomer- 2 like ligands are opposite to each other
Example. Vitamin B12 and hemeproteins
 -are incapable of reacting with chelating agents because their metal is already
coordinated in such a way that only the transcoordination positions of the metal are
available for complexation.
ORGANIC MOLECULAR COMPLEXES

- interaction between two organic molecules held together by hydrogen bonds, weak vander waal’s forces,
dipole-induced dipole interactions, and cannot be separated from solutions as definite compounds, with low
energy of attraction. In simple terms, these are organic coordination compounds or molecular complexes
consist of constituents held together by weak forces.

Hydrogen bonds- The compounds dimethyl aniline and 2,4,6- trinitroanisole react in the cold to give a
molecular complex. However, at elevated temperature, they react to yield a salt, in which molecules are
held together by primary valence bonds.

Donor-acceptor type- when bonds between uncharged species are formed and stabilized by dipole-dipole
interactions.

Charge transfer complex- one molecule polarizes the other, resulting in a type of ionic interaction or charge
transfer; stabilized by resonance

Example- benzene + trinitrobenzene

London dispersion forces and dipole–dipole interactions contribute more to the stability of the complex.

Drug and caffeine complex

- Dipole-dipole interaction or hydrogen bonding between acidic hydrogen atom and caffeine carboxyl group

- Interaction of non-polar part (insolubility with water)

Example-Acidic drugs + caffeine = complexes

Polymer complex

- Polymers with nucleophilic oxygen + drugs = complexes

Quinhydrone complex

- Formed by mixing alcoholic solutions of benzoquinone and hydroquinone forming green crystals

Picric acid complex

- Picric acid (strong acid) + strong base = salt

- Picric acid (strong acid) + weak base = complex

INCLUSION/OCCLUSION COMPOUNDS

Inclusion or Occlusion takes place when impurities or solvents are physically trapped within crystals. This
situation is different from the case of solid compounds. In inclusion or occlusion, impurities can be
occluded sporadically within crystals, whereas in solid compounds, impurities are distributed throughout the
crystal lattice.
 • Inclusion- is more applicable to cases where solvents or impurities are trapped within crystal cavities
during crystallization.

• Occlusion- is more applicable to cases where surface liquid is trapped within crystal clusters or
agglomerates during drying.

CHANNEL LATTICE TYPE

Channels are formed by crystallization of the host molecules, the guest component is usually limited to
long, unbranched straight chain compounds. It provides a mean of separation to optical isomers.

LAYER TYPE

• Type of inclusion compound in which the guest molecule is diffused between the layers of carbon
atom, to form alternate layers of guest and host molecules.

• Compounds such as clays, montomorillorite (constituent of bentonite), can entrap hydrocarbons,

alcohols, and glycols.

• They form alternate monomolecular (monoatomic) layers of guest and host.

CLATHRATES

Clathrate is a structure in which water molecules under certain conditions bond to form complex
networks of molecules forming cage-like structures that encapsulate a guest molecule, which is a gas.

Example. Warfarin sodium – a form of crystalline clathrate containing water and Isopropyl alcohol.

MONOMOLECULAR TYPE

These interact generally on a 1:1 basis with the guest molecule, which is enclosed within a cavity in the
host molecule. The cyclodextrins antibiotics and certain proteins are included in this category.

MACROMOLECULAR TYPE

The host framework in macromolecular clathrate is completely constructed using covalent bonds; it is
impossible to distinguish an individual host molecule and the framework is a macromolecule as a
whole. Examples of this category include zeolites, which act as molecular sieves. These compounds
have been investigated extensively and have wide use in industrial and laboratory processes.

METHODS OF ANALYSIS

ESTIMATION OF 2 PARAMETERS:
1. Stoichiometric Ratio of Ligand: Metal (Donor: Acceptor)
2. Stability Constant of Complex “formation constant / binding constant” – a measure of strength of
the interaction between the reagents that come together to form the complex

METHODS:

1. Methods of Continuous Variation – measurement of additive properties (Dielectric constant,

Refractive Index, Spectrophotmetric extinction coefficient)
2. Spectroscopy methods

-The UV spectroscopy is used extensively in determining rate constants, equilibrium constants, acid
base dissociation constants for chemical reaction.

-Used to investigate electron donor-acceptor or change transfer complexation

3. Distribution Method

-Method of distributing a solute between two immiscible solvents for determining stability constant

4. Solubility Method

-Excess quantities of drugs are placed in a well-stoppered containers with a solution of the
complexing agent. Bottles are agitated to reach equilibrium.

5. pH titration Method

-Suitable if complexation produces change in pH

Ex. Chelation of cupric ion in glycine (results in decrease in pH because product results to addition
of two H+)

6. OTHER METHODS
a. NMR (Nuclear Magnetic Resonance)
b. Infrared spectroscopy
c. Polarography
d. Circular dichroism
e. Kinetics
f. X-ray diffraction
g. Electron diffraction
PROTEIN BINDING

Protein binding is important for many drug substances. It could influence different drug actions. Also,
methods such as the equilibrium dialysis and ultrafiltration are important for determining protein binding.

Binding Equilibria

• Klotz reciprocal plot (If v independent binding sites are available):

1/r = 1/νK 1/[Df] + 1/ν

• Scatchard plot:

r/[Df] = νK – rK

• If the nature and the amount of protein in the experimental system are unknown:

[Db]/[Df] = −K[Db] + νK[Pt]

Equilibrium Dialysis (ED)

• Equilibrium dialysis procedure is for studying the complexation between metal ions or small
molecules and macromolecules that cannot pass through a semipermeable membrane.

• Classic technique for protein binding.

• If binding occurs, the drug concentration in the sac containing the protein is greater at equilibrium
than the concentration of drug in the vessel outside the sac.

• Potential errors: possible binding of drug to the membrane, transfer of substantial amounts of drug
from the plasma to the buffer side of the membrane, and osmotic volume shifts of fluid to the
plasma side.

Ultrafiltration (UF)

• Ultrafiltration methods are more convenient for routine determination (less time consuming &
similar to Equilibrium Dialysis).

• Hydraulic pressure or centrifugation is used to force the solvent and the small molecules, unbound
drug, through the membrane while preventing the passage of the drug bound to the protein.

Dynamic Dialysis

• Kinetic method for determining the concentrations of bound drug in a protein solution.

• Relatively rapid, economical in terms of the amount of protein required, and readily applied to the
study of competitive inhibition of protein binding.

Apparatus:

• 200mL of buffer solution into a 400mL jacketed (temperature-controlled) beaker.

• 7mL cellophane dialysis bag with drug or drug–protein solution suspended in the buffer solution.
Hydrophobic Interaction

• Attraction of hydrophobic species, resulting from their unwelcome reception in water.

• Avoids water because they are not readily accommodated in the hydrogen-bonding structure of
water.

• Favored thermodynamically.

Self-Association

• Self-associates to form dimers, trimers, or aggregates of larger sizes.

• May affect solubility, diffusion, transport through membranes, and therapeutic action.

• Insulin

Factors Affecting Complexation and Protein Binding

• Significant correlation between stability constant of the complexes and the hydrophobicity of the
ligands.

• Ligand hydrophobicity is the main contribution to the formation of water-soluble complexes.

• Warfarin