Ionizing

groups in Biology and their ionization tendencies

Anionic groups
Anionic groups in biomolecules contain O and S.
The unionized form of an anionic group loses H+ to water producing H3O+, and thereby
becoming negatively (-) charged.
According to the Bronsted definition, they are also termed acidic groups.

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1. The hydroxyl (-OH) group

Phosphoric acid ester

(amide)

2. The thiol (-SH) group

Cationic groups
! Cationic groups in biomolecules contain N.
! The unionized form of a cationic group gains H+ from water producing OH, and thereby
becoming positively (+) charged.
! According to the Bronsted definition, they are also referred to as the basic groups.
1. Amino (-NH2) or imino (=NH) groups

2. Non-aromatic N-containing rings

3. Aromatic N-containing rings

4. Important non-ionizing groups in Biochemistry

Some N-containing groups do not ionize to any appreciable degree, even though it might
seem like they should. Their dissociation equilibrium is far, far on the side of the neutral
structure.

Explanations on ionization tendencies in the next pages.

Ionization tendencies
! Kinetic tendency of a reaction is given by the rate for the reaction. However, aqueous H+ transfer
reactions are the fastest in biochemistry. There is no appreciable kinetic barrier to these reactions.
Therefore, ionization tendency does not refer to how fast the ionization reaction happens. Ionization
tendency is not used to refer to kinetics.
! Ionization tendency concerns reaction thermodynamics. Aqueous ionization reactions are controlled
by thermodynamic factors (not by kinetic factors).
! The ionization tendency of a functional group depends on the energy difference between the ionized
products and the unionized reactants, and therefore dependent on the difference in thermodynamic
stability between the ionized products and unionized reactants (and not by how fast the unionized
reactants are converted to ionized products).
! Thermodynamic tendency is quantified by the Keq (Ka or Kb) for the reaction.
! The more thermodynamically stable the products are relative to the reactants, the higher the Keq value,
the more complete the reaction will be at (dynamic) equilibrium.
1. Why do some groups have more of a tendency to ionize in water than others?
! Different functional groups have different ionization tendencies. They have different Ka or Kb values.
! The differences of Ka or Kb values are due to different stabilization of the reactants relative to the
products, or of the products relative to the reactants.
! If the reactants are particularly stabilized, then the ionization tendency will be relatively low and the Ka
or Kb value will be low. If the products are particularly stabilized, then the ionization tendency will be
relatively high and the Ka or Kb value will be high.
! One of the factors involved is resonance stabilization of reactants or products.
! Other factors involved are hybridization state (sp, sp2, sp3) and electronegativity.
2. Amino-N and amido-N atoms as examples:
An amide-N has a lesser tendency to ionize than an amine-N. WHY???
To answer this question, lets compare the apparently similar molecules ethylamine and acetamide,
shown below:

! Because of the proximity of the carbonyl C=O within an amido group, the lone pair of electrons of the N-
atom are delocalized. This resonance stabilizes the entire group, and therefore the undissociated
reactants.

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! Similar resonance stabilization does not occur for the product of amide protonation. Therefore, the
equilibrium of the ionization reaction is far to the side of the reactants. The amide groups shows
negligible ionization tendency.
! The lone pair of electrons on the amido-N behaves as if it was not available for accepting a proton.
! Amido-N is not a base under biological conditions.
! In the case of ethylammonium, however, no such resonance stabilization is possible on either the
reactant or the product side.

! Therefore, the equilibrium mixture will contain some reactants and some products. The amino group
shows some ionization tendency.
! The lone pair of electrons on the amino-N is available for accepting a proton.
! In summary, amino-N has some ionization tendency, amido-N does not.

3. How about guanidino N-atoms?

The guanidino N-atoms show a greater tendency to ionize than the N-atoms of amines. WHY??? To answer
this question, lets compare the molecules ethylguanidine and ethylamine (pKb ~ 4).

! Because of the proximity of the other N atoms and their lone pairs of electrons within a guanidino
group, the positive charge of the protonated product is delocalized. This resonance stabilizes the
charged product.
! The lone pair of electrons on the imino-N of the guanidine group behaves as if it was more available
for accepting a proton.
! Guanidino-N is not a relatively strong base.
! In the case of ethylammonium, however, no such resonance stabilization of the positive charge is
possible on the product side.

! Thus guanidino N-atoms show a greater tendency to ionize than amino-N atoms.
4. How about the indole N-atom?
Like the amido group, lone pair electrons apparently associated with the N-atom of the indole group are
resonance delocalized (in this case into a ring contributing to its aromaticity) and are therefore
unavailable to be protonated. Thus, the indole-N atom shows a negligible tendency to ionize.

5. How about the imidazole N-atoms?

Note that the imidazole ring contains two N atoms with different bonding. One of them ionizes (acts as a
base), the other one doesnt.
! The single-bonded N-atom within the imidazole group behaves like the one in the indole group. Its
lone pair is delocalized into the ring (contributing to its aromaticity) and is therefore unavailable to
be protonated.
! The lone pair electrons of the other N-atom can be protonated, resulting in ionization.
Methylimidazole

Methylimidazolium

Ionization tendency

Kb ~ 10-7

Kb ~ 10-4

Ethylamine

Ethylammonium

WHY DOES THE IMIDAZOLE-N HAVE LOWER IONIZATION TENDENCY THAN AMINO-N???
To answer this question, consider several factors stabilizing reactants and/or products:
! The positive charge carried by the ionized form can be spread out (stabilized) by resonance. This reason
alone suggests that the imidazole group should have higher ionization tendency than the amino group.

! However, the unshared pair of electrons functioning as the H+ acceptor in imidazole lies in an sp2 hybrid
orbital, while the unshared H+-accepting pair of electrons in a simple amine lie in an sp3 hybrid orbital.
The higher the s character of an orbital, the more stable (= at lower energy) it will be. Thus, its electrons
will be less available for accepting a proton and the less basic the atom will be (recall from CHEM
123/233). This reason suggests that the imidazole group should have lower ionization tendency than
the amino group.
! The hybridization effect in this case is stronger than the resonance stabilization effect. Therefore, the
imidazole-N is less basic than the amino-N.