Nitrogen compounds

Amines
Introduction
-

Amines are derivatives of ammonia in which one or all of its H atoms are
replaced by an alkyl or aryl group.
Amines may be classified as primary, secondary or tertiary depending on the
no. of alkyl or aryl groups attached to the N atom

Note: the primary, secondary and tertiary classification of amines is different

from that used in the classification of alcohols or alkyl halides.

Structure
-

Like the N atom in ammonia, the N atom of most amines is sp 3 hybridised

Amines are polar compounds.

No. of electron pairs around N atom = 4 electron pair geometry is

tetrahedral
There are 3 bond pairs and 1 lone pair around N. to minimise repulsion,
molecular geometry is trigonal pyramidal with respect to the N atom.
Net molecular dipole moment exists

Nomenclature
Aliphatic amines
-

Several different nomenclature exist for amines.

One common way to name aliphatic amines is to list the alkyl groups bonded
to the N atom in alphabetical order before the suffix amine.

Phenyl amines
-

Amines in which the N atom is attached directly to the benzene ring are
commonly named as derivatives of phenylamine.
For 2 and 3 phenylamines, substituent groups attached directly to the N
atom are identified by the prefix N- in the names of these compounds

Compounds with other functional groups of higher priority

-

In compounds with other functional groups of higher priority, the NH 2 group

is called the amino group. Such compounds are named by adding the prefix
amino- to the name of the parent compound.
E.g.

Amine salts
-

Salts of amines are generally named by replacing amine by ammonium and

adding the name of the anion (chloride, nitrate, sulfate etc.)
E.g.,

When an amine salt has four alkyl or aryl groups bonded to the N atom, it is
called a quaternary ammonium salt

Physical properties
Boiling point
-

Simple aliphatic amines which are gases possess the characteristic ammonia
smell, while higher liquid members have a distinctive fishy smell of decay.
Amines with very high Mr are solids

Amine vs. non-polar substance

- The boiling point of a amine is higher than those of non-polar substances of
similar Mr
- Only weak van der Waals forces (instantaneous dipole-induced dipole
interactions) exist between molecules of non-polar substances like alkanes.
- ON the other hand, polar N-H bonds in amine molecules result in stronger
intermolecular hydrogen bonding
Amine vs. corresponding alcohol
- The boiling point of an amine is lower than that of the corresponding alcohol
- While hydrogen bonds exist between molecules of both compounds,
hydrogen bonds between amine molecules are weaker than those between
alcohol molecules.
- This is due to the N-H bonding being less polar than the O-H bond, since
oxygen is more electronegative than nitrogen
Structurally isomeric amines
- The boiling points of isomeric amines (with the same number of atoms)
decrease in the following order.
2

1 amines > 2 amines > 3 amines

The 1 amine has the highest boiling point since its intermolecular hydrogen
bonding is the most extensive, with two H atoms available for hydrogen
bonding per molecule, as compared to one for the 2 amine and none for the
3 amine
The 3 amine has the lowest boiling point since only van der Waals forces
(permanent dipole-permanent dipole interactions) exist between its
molecules. 3 amine molecules are unable to form hydrogen bonds with each
other since there is no H atom attached to the electronegative N atom.

Effect of chain branching

- Chain branching reduces the boiling points of amines.
- A branched amine has a smaller surface area of contact as compared to its
straight chain isomer.
- Hydrogen bonding aside, this results in weaker van der Waals forces of
attraction between branched amine molecules.
- Therefore less energy is required to overcome these forces, resulting in a
comparatively lower boiling point

Solubility in water
-

Small 1, 2, and 3 amines are very soluble in water

All the amines (1, 2 and 3) can form hydrogen bonds with water
Although 3 amines do not have an H atom attached to the nitrogen and so
cannot form hydrogen bonds with themselves, they can form hydrogen bonds
with water molecules using the lone pair on the N atom.

Solubility falls off as the hydrocarbon chains get longer, with borderline
solubility being reached at about six C atoms (amines are soluble in less polar
or non-polar solvents like ether, alcohol, benzene etc.)

Aromatic amines are insoluble in water.

For higher aliphatic amines and aromatic amines, vdW interactions between
long hydrocarbon chains and between benzene rings dominate
these interfere with hydrogen bonding between the water and amine
molecules
The energy released when hydrogen bonds are formed between these amines
and water molecules is insufficient to compensate for the energy required to
overcome strong hydrogen bonding between water molecules.

Relative basicity
-

An amine, like ammonia is a Bronsted-Lowry base i.e. proton acceptor

Reason: it has a lone pair of electrons on the N atom which can form a dative
covalent bond with an H+ ion
In water, an amine is a relatively weak base as it dissociates only partially in
solution

Factors affecting base strength

1. Availability of the lone pair on the N atom for forming a dative bond with H +.
The greater the availability of this lone pair, the stronger the base strength
2. Ease by which the conjugate acid i.e. the protonated amine, is solvated by
water molecules and so becomes stabilised

Relative basicity of phenylamine, ammonia & ethylamine

-

Relative basicity increases in the following order: phenylamine<

ammonia<ethylamine

Ethyl amine is a stronger base than ammonia

Reason: the ethyl group in ethyl amine is electron-donating. It increases the
electron density at the N atom, making the lone pair more available for forming a
dative bond with H+ as compared to that of NH3

Phenyl amine is a weaker base than ammonia

Reason:
-

In phenylamine, the lone pair of electrons on the N atom is delocalised into

the electron cloud of the benzene ring.
This decreases the availability of the lone pair on N atom for forming a dative
bond with H+ as compared to that of NH3

Relative basicity of methylamine, dimethylamine and trimethylamine

-

Relative basicity increases in the following order: 3 amine < 1 amine < 2
amine
2 amine is more basic than 1 amine
In 1 amine, there is one electron-donating alkyl group bonded to the N atom
In 2 amine, there are two electron-donating alkyl groups bonded to the N
atom

With more electron-donating alkyl groups, the electron density at the N atom
in 2 amine is increased to a greater extent, making its lone pair more readily
available for forming a dative bond with H + than that of 1 amine
The base strength of 3 amine is expected to be greater than that of 1
amine or 2 amine since there are 3 electron-donating alkyl groups bonded to
the N atom.
However 3 amine is actually less basic than 1 amine or 2 amine
The three bulky alkyl groups in the conjugate acid, reduce available space
around the N atom for water molecules. This poses a hindrance to effective
solution (and hence stabilisation) of the conjugate acid
In general, the base strength of aliphatic amines increases in the following
order: ammonia < 1 aliphatic amine < 2 aliphatic amine

Effect of substituent groups on base strength of phenylamines

-

Relative basicity: 2-chlorophenylamine < phenylamine < 2methylphenylamine

2-chlorophenylamine is a weaker base than phenylamine
In 2-chlorophenylamine, the Cl atom is electron-withdrawing.
It enhances the delocalisation of the lone pair on N atom with the electrons
in the benzene ring
This decreases electron density at the N atom of the NH 2 group, making the
lone pair of electrons less available for forming a dative bond with H + as
compared to that of the phenylamine

2-methylphenylamine is a stronger base than phenylamine

In 2-methylphenylamine, the CH3 group is electron-donating. It increases
electron density at the N atom of the NH2 group, making the lone pair of
electrons more available for forming a dative bond with H + as compared to
that of phenyl amine
In general, electron-withdrawing substituent groups decreases the base
strength of phenylamines, while electron-donating substituent groups
increase the base strength of phenylamines.

Preparation
1.
2.
3.
4.
5.

Reduction of nitriles
Reaction of ammonia with halogenoalkanes
hydrolysis of amides
Reduction of amides
Reduction of nitrobenzene

Reduction of nitriles
1. Reagent and conditions: LiAlH4 in dry ether or H2 with Ni catalyst, high
temperature and pressure
2. Type of reaction: reduction
3. General equation:

Reaction of ammonia with halogenoalkanes

Aliphatic amines may be prepared by nucleophilic substitution of halogenoalkanes
(R-X) with ammonia
In the presence of excess NH3, the product is mainly the 1 amine.
1.
2.
3.
4.

Reagent: excess conc. NH3

Conditions: (dissolve) in alcohol, heat in a sealed tube
Type of reaction: nucleophilic substitution
General equation:

This method is limited to the preparation of aliphatic amines because aryl halides do
not undergo nucleophilic substitution reactions with ammonia under such conditions
A mixture of products is obtained due to polyalkylation.

An excess of NH3 is used to discourage polyalkylation

Hydrolysis of amides
1.
2.
3.
4.

Reagent: HCl, NaOH

Condition: prolonged heating under reflux
Type of reaction: nucleophilic acyl substitution
Equation:

Reduction of nitrobenzene
1.
2.
3.

Used to prepare phenylamine or substituted phenylamines

Reagent and conditions: Sn/ conc. HCl, heat under reflux or NaOH(aq)
Type of reaction: reduction
Equation:

Reactions
With acids
Like ammonia, amines can act as Bronsted-Lowry bases i.e. H + acceptors
because of the availability of a lone pair of electrons on the N atom for
forming a dative covalent bond with an H + ion.
1. Reagent & conditions: Acid, room temperature
2. Type of reaction: neutralisation
3. Equation:
-

The amine can be liberated from the amine salt by reacting the salt with an alkali
e.g. NaOH (aq)
Properties
amines

Amine salts

Insoluble in water except for amines

with low Mr
Soluble in organic solvents
Smaller amines are volatile
Generally has a fishy odour, with
smaller amines smelling like ammonia

Ionic salts
Relatively more soluble in water
Generally soluble in organic solvents
Non-volatile solids with high melting
points
No odour

With halogenoalkanes (aminolysis of halogenoalkanes)

1.
2.
3.
4.

An amine can act as a nucleophile in the nucleophilic substitution of a

halogenoalkane
Reagent: excess conc. RNH2
Conditions: (dissolved) in alcohol, heat in sealed tube
Type of reaction: nucleophilic substitution
General equation:

Heating is done in a sealed tube when either a lower amine or halogenoalkane is

used as these are either gaseous or have low boiling points at r.t.p.
Polyalkylation can occur, especially if the halogenoalkane is in excess, until a
quaternary ammonium salt is obtained. This is because the 2 and 3 amine
produced during the reaction can also act as nucleophiles

With acyl chlorides (acylation of amines)

-

Acyl chlorides undergo condensation or nucleophilic acyl substitution with

amines to produce amides
1. Reagent & conditions: acyl chloride in anhydrous conditions
2. Type of reaction: nucleophilic acyl substitution/ condensation
3. Equation:

Ring reactions of phenyl amine

-

The NH2 group strongly activates the benzene ring towards electrophilic
substitution
As such, the bromination of phenylamine using Br 2 (aq) occurs readily at
room temperature without the need for a halogen carrier catalyst like FeBr 3
1. Reagent and conditions: halide (e.g. Br2 (aq)), room temperature
2. Type of reaction: electrophilic substitution
3. Equation:

Observations:
-

Immediate decolourisation of reddish-brown Br 2 (aq)

Formation of a white ppt of 2,4,6-tribromophenylamine
Cl2 (aq) reacts similarly to form 2,4,6-trichlorophenylamine

Amides
-

Amides are carboxylic acid derivatives in which the hydroxyl group (-OH) of
the carboxylic acid has been replaced by the NR 2 group where R=H, alkyl or
aryl group
Amide functional group:
Like amines, amides are classified as primary (1), secondary (2) or tertiary
(3) according to the number of alkyl groups bonded to the N atom of the
amide group

Nomenclature
-

1 amide: name carboxylic acid, replace oic acid suffix with amide
20 and 30 amide: treat the alkyl groups on N as substituents, specifying
their positions by the prefix N-

Physical properties
Melting and boiling points
-

In general, amides have high melting and boiling points

E.g. all 1 amides are crystalline solids with the exception of methanamide,
which is a liquid
Each 1 amide has two partially positive H atoms and two lone pairs of
electrons on the O atom which allows for numerous hydrogen bonding
possibilities between the H and O atoms in the amide functional group
A lot of energy is required to break these hydrogen bonds, thereby increasing
the melting and boiling points of 1 amides significantly

The 1 amide has the highest melting and boiling points since its
intermolecular hydrogen bonding is the most extensive, with two H atoms
available for hydrogen bonding per molecule as compared to one for the
secondary amide and none for the 3 amide
The 3 amide has the lowest melting and boiling points since only vdW forces
(permanent dipole-dipole interactions exist between its molecules
3 amide molecules are unable to form H bonds with each other since there is
no H atom attached to the electronegative N atom.

Solubility in water
-

Lower members of aliphatic amides (up to 5 or 6 C atoms) are soluble in

water because amide molecules (1, 2 or 3) can form hydrogen bonding
with water molecules
However solubility decreases as the length of hydrocarbon chain increases
vdW interactions between long hydrocarbon chains interfere with H bonding
between water and amide molecules
The energy released when hydrogen bonds are formed between these amides
and water molecules is insufficient to compensate for the energy required to
overcome strong hydrogen bonding between water molecules

Lack of basicity
-

Both amine and amides have the NH2 group. Unlike amines, amides are
neutral
The lack of basicity in amides arises because the lone pair of electrons on the
N atom is delocalised with the -electron cloud of the adjacent C=O bond
This decreases the availability of the lone pair on N atom to form a dative
covalent bond with an H+ ion.

Preparation
Reaction of ammonia or amines with acyl chloride (acylation of ammonia or
amines)
1. Reagent & conditions: acyl chlorides
2. Type of reaction: nucleophilic acyl substitution/condensation
3. Equation:

3 amines do not react with acyl chlorides

Dehydration of ammonium carboxylate salts RCO2-NH4+

Amides cannot be prepared from the reaction between carboxylic acids and
amines at room temperature.
- The carboxylic acid has to be converted into an ammonium carboxylate salt,
which then produces an amide on heating
1. Type of reaction: acid-base , elimination
2. Equations:
-

10

Yield of amide is usually low as the ammonium carboxylate salt tends to

dissociate to form ammonia and the parent acid during heating
As this dissociation is reversible the use of excess carboxylic acid in step 1
helps to prevent this from happening by moving the position of equilibrium to
the right

Reactions
-

Amides are the least reactive towards nucleophilic acyl substitution amongst
all carboxylic acid derivatives
The lone pair of electrons on the N atom is delocalised with the electron
cloud of the adjacent C=O bond

This reduces the + charge on the carbonyl carbon, making it less

susceptible to nucleophilic attack
The amide group is resonance stabilised. This creates a partial double bond
between the C and N atoms and reduces the ability of the NH 2 group to
leave in a substitution reaction.

Hydrolysis
1. Reagent and conditions: acid/ alkaline, prolonged heating under reflux
2. Type of reaction: hydrolysis/ nucleophilic acyl substitution)
3. Equation:
In the presence of acid, hydrolysis of an amide yields its parent carboxylic acid
and the corresponding ammonium salt.

In the presence of an alkali, hydrolysis of an amide gives the salt of its parent
carboxylic acid with the evolution of ammonia or the liberation of a 1 or 2
amine

11

To predict the parent acid and amine from the given amide, just add OH to
the acid part and a H to the amine part

Reduction
1.
2.
3.

Amides can undergo reduction to form amines

The C=O group in the amide is reduced to a CH 2- group
Reagent & conditions: LiAlH4 in dry ether, room temperature
Type of reaction: reduction
Equation:

12

Amino acids
-

Organic compounds that contain at least one amine group (-NH 2) and one
carboxylic acid group (-COOH)
Amino acids can be classified as , , and so on, according to the location
of the NH2 group relative to the C atom that bears the COOH group

Protein molecules in all organisms are made from the same set of 20 amino
acids
General formula:

Where the side-chain R may be a hydrocarbon group or a group that contains

other reactive groups like OH, -SH, other NH 2 or COOH groups

Nomenclature and classification

-

Amino acids are systematically named as amino derivatives of carboxylic

acids. However they are more commonly referred to by their common names
Amino acids are classified according to the characteristics of their side-chain
R groups:
o (Neutral) non-polar, if R group is a hydrocarbon alkyl or aromatic group
o (neutral) polar, if R contains a polar group like an amide, alcohol,
phenol or thiol (-SH)
o Acidic, if R has a COOH group
o Basic, if R has a NH2 group or other basic groups like the imidazole or
guanidine group.

Physical properties
Optical activity
-

With the exception of glycine, all amino acids contain at least one chiral
carbon atom and consequently display optical isomerism
amino acids isolated from natural sources consist purely of one enantiomer
and are optically active
On the other hand, amino acids which are synthesised in the laboratory are
usually racemic mixtures containing both enantiomers and are thus optically
inactive.
13

Formation of Zwitterions
-

the amino acid molecule undergoes an intramolecular acid-base reaction in

which
the COOH group loses a H+ ion resulting in a carboxylate (-COO-) group
the NH2 group is protonated to form an ammonium (-NH 3+) group
the resulting molecule, which has no overall electrical charge but which
contains separate parts which are positively and negatively charged, is
known as a zwitterion

Although zwitterions are often referred to as a dipolar ion, it is, strictly speaking, not
an ion but an electrically neutral molecule with oppositely charged ends.

Properties of zwitterions
-

in contrast to amines and carboxylic acids, amino acids are non-volatile

crystalline solids which melt with decomposition at fairly high temperatures
in solid state, amino acid molecules exist as zwitterions, held together by
strong ionic bonds
a large amount of energy is needed to overcome strong electrostatic forces of
attraction between oppositely charged ends of neighbouring zwitterions
hence the melting or decomposition points of amino acids are fairly high

amino acids are insoluble in non-polar solvents like benzene or ether but are
appreciably soluble in aqueous solutions

Reason:
-

due to their charged ends, zwitterions are unable to form energetically

favourable solute-solvent interactions with non-polar molecules
the energy evolved from these interactions is insufficient to overcome the
strong ionic bond between zwitterions
thus amino acids are insoluble in non-polar solvents
Though amino acids show higher solubility in aqueous solutions, their
solubility in aqueous solutions is dependent on pH.

An Amino acid shows lowest solubility in an aqueous solution at isoelectric

point, pI
In more acidic or alkaline solutions, the amino acid becomes more soluble.
14

Reason:
-

At pH= pI, amino acids exist as zwitterions, the oppositely charged NH 3+ and
CO2- ends on zwitterions prefer to form strong ionic bonds with one another
than to form weaker ion-dipole interactions with water molecules
Thus amino acids show the lowest solubility at pI
Precipitation is often observed at pI, in particular for amino acids with large
hydrocarbon R groups
At low or high pH, amino acids exist as cations or anions respectively. These
charged ions dissolve readily by forming energetically favourable ion-dipole
interactions with water molecules

The Ka and Kb values are very low for the COOH and NH 2 groups in amino
acid molecules

Reason:
-

The measured Ka value refers to the acidity of the ammonium ion, RNH 3+
which is much weaker than a typical carboxylic acid RCO 2H

The measured Kb refers to the basicity of its carboxylate ion RCO 2- which is a
much weaker base than a typical aliphatic amine

Acid-base properties
Amphoteric nature of amino acids
-

Amino acids are amphoteric (have both acidic and basic properties) since
they contain both acidic and basic groups
The acidic part of an amino acid molecule is the NH 3+ group
The basic part of an amino acid molecule is the CO 2- group

The -CO2H is more acidic than the side-chain CO 2H, as the latter is attached to an
electron-donating alkyl chain which de-stabilises the corresponding CO 2
conjugate base ion by intensifying its negative charge

Isoelectric point
pH
Acidic

pI

Amino acids are always charged in an aqueous solution e.g. glycine

Predominant form
-CO2- group becomes protonated
yielding a cationic form of the
amino acid (gly+)
Zwitterion (gly0)

Effect of electric field

[gly+]>[gly-]
Net migration of glycine towards
cathode (negative electrode)
Gly0 has no net charge
No migration
15

Basic

-NH3+ group becomes

deprotonated, yielding an
anionic form of glycine (gly-)

[gly-]>[gly+]
Net migration of glycine towards
anode (positive electrode)

Isoelectric point, pI, of an amino acid: the pH at which the amino acid does not
migrate under the influence of an electric field
At pI,
-

[zwitterion] is at its maximum

[cation]=[anion]
Solubility of amino acid is at its minimum
Buffering effect of the amino acid solution is also at its minimum

Each amino acid has a characteristic isoelectric point.

Differences in isoelectric points can be used to separate mixtures of amino
acids by electrophoresis

Separation of amino acids by electrophoresis

-

Electrophoresis: separation technique based on the movement of charged

ions under the influence of a strong electric field
Primarily used for the separation of amino acids and polypeptides on the
basis of their net charge which in turn is affected by the pH of the solution
Combining the principles of electrolysis and paper or thin layer
chromatography, separation is effected because no amino acids have
o Different isoelectric points
o Different rates of migration due to their different sizes and net charges
in the buffered solution
The amino acids migrate in the electric field at a rate which depends on their
charge to mass ratio.
Their final positions are determined using staining agents like ninhydrin which
form violet compounds with otherwise colourless amino acids.

Amino acid

pH

Mr

A
B

pH= pI
pH<pI

m
m

Predominant
form
Zwitterion
Anion

pH>pI

Cation

pH>pI

2m

cation

Observation
No net migration
Net migration
towards the anode
Net migration
towards the
cathode
As Mrc<Mrd, C
migrates more
quickly and is
closer to the
cathode than D
Net migration
16

towards the
cathode

Amino acids as buffers

-

At certain pH, amino acid solutions can function as buffers

Around the two pKa values
At pKa 1, amino acids exist as the following two species in equal proportion
and can function as an acidic buffer

When small amount of H+ is added,

Excess H+ is removed by the conjugate base (zwitterion) as the acid (cation). Thus
pH of solution remain approximately constant
When a small amount of OH- is added,

Excess OH- is removed by the acid (cation) as the conjugate base (zwitterion). Thus
pH of solution remains approximately constant.

pH titration graph of a neutral amino acid

-

At low pH, an amino acid is fully protonated. It has at least two dissociable H +
ions: one on the -NH3+ group and another on the -CO2H group.
Diprotic weak acid: graph shows two plateaus which represent two buffering
regions

17

Equivalents of OH-: moles of OH- per mole of protonated acid

The completed titration occurs in two steps:

Step 1:

Step 2:

The first equivalence point corresponds to the completion of step 1. pH at this

point corresponds to the isoelectric point, pI, of alanine where

pI =

pK a 1 + pK a 2
2

The second equivalence point corresponds to the completion of step 2.

Point
A

pH
0

Predominant specie

Remarks
Exists as a fully protonated
form

pKa1

[cation]=[zwitterion[
Mixture is a buffer solution
of maximum buffering
capacity

pI

The first equivalence point

of the titration is reached
Amino acid exists mainly
as a zwitterion

pKa2

[zwitterion]=[anion]
Mixture is a buffer solution
of maximum buffering
capacity

pH at
final

At high pH, amino acid is

fully deprotonated.
18

equivale
nce point
and
above

Peptide formation
-

In the laboratory, an amide can be prepared by mixing a carboxylic acid and

an amine, and then heating the mixture to drive off water.
In a similar fashion, the NH2 of an amino acid molecule can condense with
the COOH of another amino acid molecule to form an amide linkage under
suitable conditions

If the amide bond is formed between the -COOH group of one amino acid
with the -NH2 group of another amino acid molecule, the resulting CONH
bond is known as a peptide bond or peptide linkage and the amide product is
called a peptide.
Peptide linkage biochemical term. Amide bond/linkage chemical term
Any number of amino acids can undergo condensation to form a peptide. As
water molecules are lost in the process, the resulting peptide is said to be
made up of amino acid residues.
2 amino acid residues per peptide molecule: dipeptide. 3: tripeptide. 4:
tetrapeptide, many: polypeptide
By convention polypeptides of molecular weights above 10000 (~50 amino
acids) are known as proteins.

19

Proteins
Introduction
-

Proteins are macromolecules made up of one or more polyamides derived

from the same set off 20 -amino acid monomers

Classification & function

-

2 broad classes:
Fibrous proteins: insoluble in water
Globular proteins: soluble in water, aqueous solutions of acids, bases or salts
Differences in molecular and intermolecular structure determine their
solubility in water and the kind of function they perform

Fibrous protein

Globular protein

Collagen

Myoglobin
Molecular structure
- Molecules are long and thread-like
- They tend to lie side by side to
form fibres
- Each fibrous protein has its unique
amino acid sequence that favours
a particular kind of secondary
structure
- E.g. the secondary structure of keratin is composed of
predominantly -helices while that
of -keratin contains mainly pleated sheets
Inter-molecular structure
Held together at many points by strong
hydrogen bonds

Molecules are often folded into

compact spheroidal units
Secondary structure is
composed of both helices and
-pleated sheets

The folding turns hydrophobic

parts inward toward each other
and away from water while
hydrophilic parts e.g. ionic
groups are oriented outwards to
be in contact with water
Hydrogen bonding is chiefly
intramolecular
20

Solubility in water
- weak intermolecular vdW
attraction formed between A and
solvent cannot displace the strong
intermolecular hydrogen/polar
bonds in water for solvation to
occur
- Fibrous proteins are insoluble in
water

functions
- Due to their water-insoluble and
fibre-forming tendencies, fibrous
proteins serve as structural
materials of animal tissue
examples
- Keratin in skin, hair, nails, wool,
horn and feathers
- Collagen n tendons
- Myosin in muscle
- Fibroin in silk

Areas of contact between

molecules are small
Intermolecular forces are
comparatively weak
Thus globular proteins can
dissolve in water by forming
favourable interactions with
water
Because of the large size of
protein molecules, these
solutions are not true solutions
but colloidal
Globular proteins serve
functions that require mobility
and solubility
These relate to maintenance
and regulation of life processes
All enzymes
Many hormones e.g. Insulin,
thyroglobulin
Antibodies responsible for
allergies and for defence
against foreign organisms
Haemoglobin which transports
oxygen from the lungs to the
tissues
Fibrinogen which is converted
into the insoluble fibrous
protein fibrin during the clotting
of blood

Protein structure
Representing peptide structures
-

Amino acid residue with free -NH2 group (N-terminus) is drawn on the left
Amino acid residue with free COOH group (C-terminus) is drawn on the right
Peptide chain is represented using three-letter amino acid abbreviations
separated by a hyphen - e.g. gly-ala-phe

21

Primary structure
-

Linear sequence of amino acids held together by peptide linkages in its

polypeptide chain.
The Polypeptide chain is also known as the backbone of a protein while the R
groups are known as the side chains
N-terminus of a peptide is distinct from the C-terminus
Peptide composed of the same amino acids can have several primary
structures
E.g. asp-phe =/= phe-asp

Reaction
Complete hydrolysis
Acid hydrolysis
1.
2.
3.
4.

Reagent and: HCl 6mol dm-3

Conditions: heat for 24h at 110C
Type of reaction: hydrolysis
Equation:

Basic hydrolysis
22

1.
2.
3.
4.

Reagent and: NaOH or KOH 2mol dm-3

Conditions: heat at 100C
Type of reaction: hydrolysis
Equation:

Acidic conditions generally destroy fewer amino acids completely and is hence
preferred over alkaline hydrolysis
Complete hydrolysis of a protein does not provide any information as to the order in
which amino acids are joined together in the primary structure.
Partial hydrolysis
- Used to determine the sequence of amino acids on the polypeptide chain
Mild acidic hydrolysis
1. Reagent: 6 mol dm-3 HCl
2. Condition: room temperature, several days
3. Type of reaction: hydrolysis

Peptide bonds between certain amino acid residues hydrolyse faster than
others
Partial hydrolysis of the peptide chain and the formation of peptide fragments
dipeptides, tripeptides etc.

Enzymatic hydrolysis
-

Enzymes cleave the polypeptide chain

Is more selective
E.g. trypsin, papain

Secondary structure
-

Within a long polypeptide chain, there are regions in which the chain is
organised into regular structures known as alpha-helices and beta-pleated
sheets. This forms the secondary structure of protein
These structures are formed and stabilised by the hydrogen bond between
the N-H of one peptide linkage and the C=O of another peptide linkage in the
polypeptide backbone.

-helix
Drawing:
-

Helical structure
R groups pointing outwards and are perpendicular to the axis of the helix
Two hydrogen bonds
23

o
o
o

- lone pair of electrons on O in C=O group

Dipole
Hydrogen bond

Description:
-

In the alpha-helix, the polypeptide chain is coiled to

forma helical arrangement with 3.6 amino acids
per turn of helix
The structure is stabilise by hydrogen bonds
formed between peptide C=O and another peptide N-H group four amino acid
residues away along the backbone
The side-chain of each amino acid residue points outwards from the helix and
is perpendicular to the main axis of the helix

- Pleated sheets
Drawing:
-

Parallel or anti-parallel beta-pleated sheets

One hydrogen bond
o lone pair of electrons on O in C=O group
o Dipole
o Hydrogen bond

Description:
-

In a beta pleated sheet, the backbone of the polypeptide chain is folded so

that segments of the chain lie alongside each other
These segments can either be parallel or anti-parallel, with adjacent strands
being stabilised by hydrogen bonding between N-H of one peptide linkage
and the C=O of another peptide linkage along the polypeptide backbone

24

Tertiary structure
-

Overall 3-dimensional shape of its polypeptide chain, formed and stabilised

by interactions between the R-groups of its amino acids
4 types of R group interactions that hold the tertiary structure in its
necessary shape:
i.
Van der Waals forces of attraction between non-polar groups
ii.
Ionic bonds (aka salt bridges) between oppositely-charged groups
iii.
Hydrogen bonds between polar groups with O-H and N-H bonds
iv.
Disulphide bridges which form when SH side chains of two cysteine
residues are oxidised to form an S-S bond.

25

The nature of the R group in an amino acid is of crucial importance

When the -NH2 and the -COOH group of amino acids are combined to form
a polypeptide chain, the R group is the remaining feature which distinguishes
the amino acids
Interactions between the different R groups profoundly influence the folding
of the polypeptide chain, and hence the shape of the final protein

26

Quaternary structure
-

Consists of more than one polypeptide chain coming together to form the
complete protein maintained by the same four types of R group interactions
that hold the tertiary structure in its necessary shape
Van der Waals forces of attraction between non-polar groups
Ionic bonds (aka salt bridges) between oppositely-charged groups
Hydrogen bonds between polar groups with O-H and N-H bonds
Disulphide bridges which form when SH side chains of two cysteine residues
are oxidised to form an S-S bond.

Note:
-

Proteins must contain two or more polypeptide chains to have quaternary

structure
Each polypeptide chain in the quaternary structure is called a subunit
Two subunit: dimer e.g. insulin
Four subunits: tetramer e.g. haemoglobin

Separation & analysis of protein mixtures by electrophoresis

-

While all proteins contain the polypeptide backbone, each protein has its own
characteristic sequence of R groups.
Different proteins have different proportions of positively charged acidic and
negatively charged basic R groups
The rate at which a protein migrates in an electric field depends on
o The relative number of positive and negative charges on the R groups,
which in turn are affected by the pH of the solution
o Molecular size and shape as well as overall charge of the protein (i.e.
charge to mass ratio)
The difference in behaviour in an electric field is the basis of separation and
analysis of protein mixtures by electrophoresis
pH
<pI

=pI

>pI

Positive charges
exceed negative
charges on the
protein molecule
Positive charges
and negative
charges are
exactly balanced
on the protein
molecule
Negative charges
exceed positive
charges on the
protein molecule

Predominant form
Net cationic

observation
Net migration
towards the
cathode

Net zero charge

No net migration

Net anionic

Net migration
towards the anode

27

Haemoglobin
-

Haemoglobin is made up of four polypeptide chains i.e. two -subunits and

two -subunits with each being dative bonded to a haem group that contains
a central Fe2+ ion that can bind with oxygen

Quaternary structure
-

Spatial arrangement of these 4 polypeptide chains into a specific globular

structure held together by the same four types of R group interactions that
hold the tertiary structure in its necessary shape

Tertiary structure
-

The tertiary structure of haemoglobin is the overall 3-dimensional shape of

each polypeptide chain formed and stabilised by four types of interactions
between the R-groups of its amino acids
i.
Van der Waals forces of attraction between non-polar groups
ii.
Ionic bonds (aka salt bridges) between oppositely-charged groups
iii.
Hydrogen bonds between polar groups with O-H and N-H bonds
iv.
Disulphide bridges which form when SH side chains of two cysteine
residues are oxidised to form an S-S bond.

Secondary structure
-

Within each polypeptide chain, there are regions in which the chain is
organised into regular structures known as alpha-helices and beta-pleated
sheets.
These structures are formed and stabilised by hydrogen bonding between the
N-H of one peptide linkage and the C=O of another peptide linkage in the
polypeptide backbone

Primary structure
-

The primary structure of haemoglobin is the linear sequence of amino acids

within each polypeptide chain held together by peptide linkages

Biological function
-

Some protein molecules contain a non-peptide portion called a prosthetic

group
Haemoglobin: prosthetic group is haem
One haem group is held to each of the four polypeptide subunits (globin) of
the protein by a combination of forces
o Dative covalent bonding between the iron (II) ion and N atom of the
protein
o Hydrogen bonding and van der Waals forces between non-polar parts
of the ahem and protein molecule
Each haem group forms a reversible oxygen-haem complex that enables
haemoglobin to carry oxygen from the lungs to the rest of the body, i.e. the
tissues, where oxygen is released and used to convert nutrients to energy
needed to power the functions of the human body
28

Only one O2 group can be coordinated to the central Fe(II) ion in each haem
group. Thus each haemoglobin molecule carries a maximum of four O2
molecules

Denaturation
-

Loss of its quaternary, tertiary or secondary structure due to the disruption of

non-covalent interactions (i.e. van der Waals forces, ionic bonds and
hydrogen bonding) and/or disulphide bridges that leaves peptide linkages and
thus its primary structure intact.

Quaternar
y
Tertiary

Spatial arrangement of subunits is disrupted as R group

interactions are disrupted
- Some or all R group interactions that stabilise the tertiary
structure may be disrupted
i.
Van der Waals forces of attraction between non-polar groups
ii.
Ionic bonds (aka salt bridges) between oppositely-charged
groups
iii.
Hydrogen bonds between polar groups with O-H and N-H
bonds
iv.
Disulphide bridges which form when SH side chains of two
cysteine residues are oxidised to form an S-S bond.
Secondary
- Hydrogen bonds between N-H and C-O in peptide linkages that
stabilise the -helices and -pleated sheets may be disrupted.
- The resulting polypeptide chain is a random coil
Primary
- Denaturation methods leave covalent peptide linkages
between amino acid residues in the primary structure intact.
- Since its overall 3-D shape is destroyed, the protein can no longer perform its
biological function.
- Its physical and chemical properties are also changed in the process, e.g.
solubility of the protein decreases and precipitation occurs
- Many denaturation processes are irreversible
-

Factors leading to protein denaturation

Metal ions
- Metal ions compete with positively charged groups e.g. NH3+ for attraction
to negatively charged groups e.g. CO2- resulting in the disruption of the
original ionic linkages in the protein molecule
- E.g.
- Salt solutions such as sodium chloride and ammonium sulfate are used in a
protein separation technique called salting out. Ions present in these salt
solutions bind to ionic R groups and disrupt the original salt bridges that
stabilise the quaternary and tertiary protein structures

29

Heavy metal ions like Ag+, Pb2+ and Hg 2+ attack SH groups of cysteine
residues to form stable metal-sulphur salt bridges

High temperature
- Heat increases thermal vibrations of the protein molecule, disrupting weaker
interactions like hydrogen bonds and van der Waals interactions
- During the process, hydrogen bonds that stabilise the -helices and -pleated
sheets in the secondary structure are broken, and hydrophobic R groups that
are originally oriented inward (away from water) become exposed to the
aqueous medium
- This results in the coagulation of protein
- E.g. raw egg whites are transparent and liquid as they contain mainly egg
albumins in water.
- When cooked by heating, the albumins become denatured and the egg
whites turn opaque, forming a solid mass
Changes in pH
- At low pH (acidic conditions), basic R groups become protonated

At high pH (basic conditions), acidic R groups become deprotonated

These extreme pH changes disrupt salt bridges or hydrogen bonds that

stabilise the quaternary and tertiary structure of the protein.

Oxidising and reducing agents

- Reducing agents such as 2-mercaptoethanol (HOCH 2CH2SH) can break
disulphide bridges, reducing them to SH groups

Other chemical means

- 70%%% ethanol or isopropanol solution is used as a disinfectant on the skin.
- At this concentration, the alcohol is able to penetrate the bacterial cell wall
and denature the proteins and enzymes inside of the cell

30

Alcohol denatures bacteria proteins by forming hydrogen bonds with polar R

groups, thereby disrupting original hydrogen bonds that maintains the
tertiary and secondary protein structures

Mechanical agitation
- Actions such as stirring, whipping or shaking can also disrupt weaker
interactions like hydrogen bonds and van der Waals interactions that
maintain the tertiary and secondary structure of the protein

31