6.13.0
Introduction - the importance of amino acids
There are many amino acids essential to life and are found combined together
in proteins e.g tissue, enzymes.
Amino acids are the building blocks for these substances form long
chains called polypeptides or proteins.
Twenty amino acids are found in the structure of proteins in the
human body (see
list in 6.13.7).
Twelve can be synthesised from other amino acids, but eight cannot
and are referred to as essential amino acids and must be found in our
diet.
An
amino acid molecule contains at least one carboxylic acid group (-COOH)
and at least one amine group (-NH2).
The general formula for alpha amino acids is R-CH(NH2)-COOH,
where R can be H or a wide variety of organic groups.
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6.13.1 Molecular and ionic structure and physical properties of amino acids
Amino acid molecules have at least one amino/amino group (-NH2) and
one carboxylic acid group (COOH).
The primary suffix name for an aliphatic carboxylic acid
is based on the "longest carbon chain name *" for the -COOH bond
system e.g. ethanoic acid, propanoic acid etc. The amino group
-NH2, with its C-atom position number, is added as a prefix. [*
without the end 'e']
They are usually colourless crystalline solids at room temperature
with relatively high melting points for molecules of their relative
molecular mass and usually highly or moderately soluble in water.
Data on some of the simplest amino acids is quoted below.
(mpt. = melting point; bpt. = boiling point; dec. = decomposes
on heating;
sub. = sublimes on heating)
If the amine group is on the first carbon that can have a hydrogen atom
substituted by a another different atom of group it is known as an alpha (α)
amino acid.
This is the 2nd carbon atom in the chain next to the carboxylic acid
group.
If the amine is on the 3rd carbon it is a beta (β) amino acid.
If the amine is on the 4th carbon it is a beta (γ) amino acid.
Remember in the IUPAC systematic naming, the C or the COOH group is
carbon atom 1.
IUPAC systematic name of amino acid
(common trivial name) |
The unionised molecular form and the isomeric
ionic zwitterion
form (see notes below table) |
Mpt/oC |
Comments including solubility
in water Has a chiral/asymmetric Cabcd carbon atom and
exhibits R/S stereoisomerism unless otherwise stated. |
Aminoethanoic acid
'glycine' |
H2NCH2COOH
+H3NCH2COO- |
dec. 232 |
Very soluble in water, the simplest amino
acid,
an α amino acid. No
R/S isomerism |
2-aminopropanoic acid
'α-alanine' |
CH3CH(NH2)COOH
CH3CH(+NH3)COO- |
sub. 258 |
Very soluble in water,
an α amino acid.
α &
β alanine are structural positional isomers. |
3-aminopropanoic acid
'β-alanine' |
H2NCH2CH2COOH
+H3NCH2CH2COO- |
200 |
Very
soluble in water,
a β amino acid.
No R/S isomerism, |
4-aminobutanoic acid |
H2NCH2CH2CH2COOH
+H3NCH2CH2CH2COO- |
204 |
Very soluble in water, a γ amino acid.
No R/S isomerism |
2-amino-3-methylbutanoic acid
'Valine' |
(CH3)2CHCH(NH2)COOH
(CH3)2CHCH(NH3+)COO- |
298 |
An α amino acid. Soluble in water. |
2-amino-4-methylpentanoic acid
'Leucine' |
(CH3)2CHCH2CH(NH2)COOH
(CH3)2CHCH2CH(NH3+)COO- |
294 |
An α amino acid. Soluble in
water. |
2-amino-3-sulfanylpropanoic acid
'Cysteine' |
HSCH2CH(NH2)COOH
HSCH2CH(NH3+)COO- |
dec. 240 |
An α amino acid.
Soluble in water.
Note the presence of a sulfur
based functional group HS-. |
Notes on the
data table
(i) Melting points and
boiling points
For the size of the molecule (e.g. as measured in electrons) they have
relatively high melting points, at which they sometimes thermally degrade
and decompose - this indicates strong intermolecular bonding of some form
between the amino acid molecules.
The intermolecular forces, apart from the 'usual' instantaneous
dipole - induced dipole forces, are greatly increased by hydrogen
bonding or ionic attraction between zwitterions (see below for their
structure).
e.g. comparing aminoethanoic acid H2NCH2COOH
(glycine), molecular mass 75 and 40 electrons, with other molecules
of similar molecular mass.
Glycine is a crystalline solid at room temperature that
melts and decomposes at 232oC.
It consists of an ionic lattice of the zwitterions which
are strongly attracted together, effectively an ionic bond:
...+[H3NCHCOO]- .... +[H3NCHCOO]-
...
This is strong ionic bonding, rather than the usual
relatively weaker intermolecular forces between covalent
organic molecules, raises the melting point well above most
other organic molecules of comparable molecular mass e.g.
Ethanoic acid, Mr = 74, 40 electrons, CH3CH2COOH,
mpt -21oC, bpt. 141oC.
Strong hydrogen bonding between molecules, forms a dimer.
Butan-ol, Mr = 74, 42 electrons, CH3CH2CH2CH2OH,
mpt. -89oC, bpt. 117oC.
Intermolecular forces comprise relatively strong hydrogen
bonding plus permanent dipole - permanent dipole and
instantaneous dipole - induced dipole attractive forces.
Butan-1-amine, Mr = 73, 42 electrons, CH3CH2CH2CH2NH2,
mpt. -50oC, bpt. 78oC.
Intermolecular forces comprise moderately strong hydrogen
bonding plus permanent dipole - permanent dipole and
instantaneous dipole - induced dipole attractive forces.
Pentane. Mr = 72, 42 electrons, CH3CH2CH2CH2CH3,
mpt. -130oC, bpt. 36oC.
Intermolecular forces only comprise very weak
instantaneous dipole - induced dipole attractive forces.
(ii) Solubility in water
Many amino acids are quite soluble in water because they have two
groups that can hydrogen bond with water, or more likely, the
zwitterion ionic form can be solvated by water molecules in at least
two places in the molecule i.e. at
H3N+
or COO-
points in the zwitterion.
(iii) Relative acidity and
alkalinity (no data in the table, but see
section 6.13.3)
All the above have one -COOH group and one -NH2 group in
the molecule, for such molecules the aqueous solution will tend to be neutral ~pH 7
(often ~ pH 6).
BUT, if the number of acid groups exceeds the number of amine
base groups, the solution will be tend to be of <pH 7 i.e. acid,
AND, if the number of amine groups exceeds the number of acid
groups, the solution will tend to be of pH >7 i.e. alkaline.
See amino acid examples in
section 6.13.3 which
are not neutral.
(iv) The α and β (alpha and beta)
refer to the second and third carbon atoms to which the amino group can
be a substituent.
Amino acids and
zwitterions
Amino-acids in aqueous solution, or in the
crystalline state, exist as 'zwitterions' where the proton migrates from the
acidic carboxylic -COOH group to the basic -NH2 amino group to form
the ionic groups -NH3+ and -COO–
BUT within the same 'molecule'.
H2N-CHR-COOH
+H3N-CHR-COO–
For the functional groups present in the molecule, the carboxylic acid is
weakly acidic and amino group is weakly basic.
This results in an equilibrium between the neutral molecular form and
the zwitterion ionic
forms (shown above).
For example, shown as structural formulae of the molecule and
zwitterion AND the skeletal formulae of the molecule and zwitterion forms of ...
aminoethanoic acid
(glycine)
,
AND
,
2-aminopropanoic acid (alanine)
,
,
,
,
There is a strong electrical attraction between the oppositely charged ends of
the zwitterions
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6.13.2 A laboratory synthesis of amino acids from
halogenated carboxylic acids
(not a biosynthesis method)
This is the nucleophilic substitution by ammonia of the halide atom in
the carboxylic
e.g. chloroethanoic acid + ammonia ===> aminoethanoic
acid + ammonium chloride
ClCH2COOH
+ 2NH3 ===> H2NCH2COOH
+ NH4Cl
A two stage synthesis of the amino acid alanine
2-aminopropanoic acid (the amino acid
alanine) when extracted from broken down protein will show optical
activity because it will consist of only one of the optical isomers, as
it was produced, and used in protein formation, by stereospecific
enzymes. It can be produced in the laboratory/industry by a two stage
synthesis e.g.
(1)
CH3CH2COOH
+ Cl2 ===> CH3CHClCOOH + HCl
free radical chlorination of
propanoic acid (no optical isomers) with chlorine/uv gives
2-chloropropanoic acid which does exhibit optical isomerism (the
reaction also forms isomeric 3-chloropropanoic acid).
(2)
CH3CHClCOOH + 2NH3
===> CH3CH(NH2)COOH + NH4+
+ Cl-
treating 2-chloropropanoic acid
with excess conc. ammonia gives 2-aminopropanoic acid, which again,
can exhibit optical isomerism.
In practice this kind of laboratory synthesis yields
a racemic mixture, a 50 : 50 mixture of the R and S stereoisomers.
In stage (1) the chlorine
radical could abstract/substitute either of the two middle H's with
equal probability and therefore a racemic mixture is likely to result.
OR if stage (2) went via a
carbocation (with a trigonal planar bond arrangement, SN1
mechanism), substitution can take place by the NH3 molecule hitting
either side of the carbocation 'centre' with equal probability.
Therefore either step could give an
equimolar mixture of the possible optical isomers.
For more details on reaction (2) see
carbocation mechanisms of haloalkane substitution reactions,.
Most synthetic amino acids are produced by complex biosynthetic pathways.
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6.13.3 The multi-functional group chemistry of amino acids
The acidic and basic character of the -COOH and -NH2 functional
groups and isoelectric point
Amino acids are carboxylic acids
(like ethanoic acid, with the -COOH group) but commonly, one of the hydrogen atoms of the 2nd carbon atom is substituted with an
amino/amine group (a nitrogen + two hydrogens gives -NH2).
-COOH is a weakly acid carboxylic acid functional group in aqueous
solution.
R-COOH(aq)
+ H2O(l)
R-COO-(aq) + H3O+(aq)
-NH2 is a weakly basic functional group in aqueous
solution.
R-NH2(aq)
+ H2O(l)
R-NH3+(aq) + OH-(aq)
R is the 'rest of the molecule' and if the two functional groups are
present in the same molecule, they can cancel each
other out to give a ~neutral solution.
Another
hydrogen on the same 2nd carbon can be substituted with other groups of
atoms (R) to give a variety of amino acids.
or
The simplest is aminoethanoic acid or 'Glycine'
and
another amino acid called 2-aminopropanoic acid or 'Alanine'
All amino acids have the general structure H2N-CH(R)-COOH
(see diagram by 5b heading).
R can vary, think of it as the 'Rest of the
molecule!
R = H for Glycine, R = CH3 for
Alanine.
Amino acids have 2 functional groups:
-COOH carboxylic acid
and -NH2
amino group.
BUT, there is the added complication of zwitterion form of the amino acid
e.g. for alpha-amino acids.
H2N-CHR-COOH
H3N+-CHR-COO–
Amino acids can be classified
into three groups depending on their acid-base molecular structure
Neutral amino acids
Here the amino acid has one basic amino group and one acidic carboxylic acid
group.
These will tend to cancel each other out and dissolves to give a
neutral aqueous solution
e.g.
CH3CH(NH2)COOH
H3N+-CH(CH3)-COO–
2-aminopropanoic acid (Alanine) in
neutral solution
Acidic amino acids
Here the amino acid typically has one amino group and two
carboxylic acid groups.
The presence of the 2nd acid group gives an acidic aqueous
solution (overriding the single base group).
e.g.
HOOCCH2CH(NH2)COOH
2-aminobutane-1,4-dioic acid (Aspartic acid)
Basic (alkaline) amino
acids
Here the amino acid typically has two amino groups and one
carboxylic acid group.
The presence of the 2nd amino group gives an alkaline solution
(overriding the single acidic group).
e.g.
H2NCH2CH2CH2CH2CH(NH2)COOH
2,6-daminohexanoic acid (Lysine)
A flavour of the dual
chemistry - I'm giving the equations using both the 'molecular' and
'zwitterion' forms.
(i) Reaction with a stronger acid, amino acid acts as a base
RCH(NH2)COOH(aq)
+ H+(aq)
RCH(NH3+)COOH(aq)
RCH(NH2)COOH(aq)
+ HCl(aq)
[RCH(NH3+)COOH]Cl-(aq)
The amine group is protonated giving an alkylammonium ion
RCH(NH3+)COO-(aq)
+ H+(aq)
RCH(NH3+)COOH(aq)
Here the -COO- group acts as the conjugate of the
carboxylic acid -COOH.
You reverse the reaction by adding alkali
RCH(NH3+)COOH(aq)
+ OH-(aq)
RCH(NH2)COOH(aq)
or RCH(NH3+)COO-(aq)
+ H2O(l)
(ii) Reaction with a stronger base, amino acid acts as an acid
RCH(NH2)COOH(aq)
+ OH-(aq)
RCH(NH2)COO-(aq) + H2O(l)
RCH(NH2)COOH(aq)
+ NaOH(aq)
[RCH(NH2)COO-]Na+(aq) + H2O(l)
The carboxylate anion is formed i.e. the sodium salt of the amino
acid in this case.
RCH(NH3+)COO-(aq)
+ OH-(aq)
RCH(NH2)COO-(aq) + H2O(l)
Here you can consider the protonated zwitterion acting as a conjugate acid via the -NH3+
group.
(This is just like the ammonium ion, NH4+ which
is the conjugate acid of the base ammonia NH3).
You reverse the reaction by adding acid
RCH(NH2)COO-(aq)
+ H+(aq)
RCH(NH2)COOH(aq) or
RCH(NH3+)COO-(aq)
(iii) To summarise the amphoteric behaviour of amino acids,
molecule or zwitterion, (aq) omitted.
RCH(NH3+)COOH
<= dec. pH with inc. [H+] = RCH(NH2)COOH
= inc. pH with inc, [OH-] => RCH(NH2)COO-
or RCH(NH3+)COO- *
* mains
species at isoelectric point pH
Isoelectric point
The pH at which an amino acid is neutral overall in aqueous
solution is called the isoelectric point.
It varies considerable from amino acid to amino acid depending on
both the number and strength of the acidic and basic groups in the
molecule e.g.
Valine and glycine have isoelectric points of pH 6.0 and 6.1
respectively, both with an isoelectric point pH close to 7, typical for amino acids
with one carboxylic acid group and one amine base group. It suggests
the weak carboxylic acid is slightly more stronger than the strength
of weak
amine base (acting as a base).
Aspartic acid has an isoelectric point of pH 2.9, a pH well below 7
because of the 2nd carboxylic acid group.
Arginine has an isoelectric point of 10.8, a pH well above 7 because
of the 2nd amine group.
Amino acids can be separated using a technique called
electrophoresis.
Charged particles can be separated by their relative movement in
a uniform electric field created by applying a potential difference
across an aqueous solution.
In the case of amino acids, the relative separation depends on
their different isoelectric points
The amino acid
particles with a net positive charge will migrate toward the
negative electrode.
Those particles with a
negative net charge will move toward the positive electrode.
(iv) Things get a bit more tricky when there are two -COOH or two -NH2
groups in the molecule e.g. several in the table below
Common name of amino acid |
IUPAC systematic name of amino
acid (allowed alternative) |
Molecular form and the isomeric zwitterion
form |
Comments |
Aspartic acid |
2-aminobutane-1,4-dioic acid
(2-aminobutanedioic acid) |
HOOCCH2CH(NH2)COOH
HOOCCH2CH(+NH3)COO- |
Will be weakly acidic in aqueous
solution. |
Glutamic acid |
2-aminopentane-1,5-dioic acid
(2-aminopentanedioic acid) |
HOOCCH2CH2CH(NH2)COOH
HOOCCH2CH2CH(+NH3)COO- |
Will be weakly acidic in aqueous solution. |
Lysine |
2,6-diaminohexanoic acid |
H2NCH2CH2CH2CH2CH(NH2)COOH
H2NCH2CH2CH2CH2CH(+NH3)COO- |
Will be weakly alkaline in aqueous solution. |
|
|
|
|
|
|
|
|
If there is one -COOH group and one -NH2 group in the moleule,
the aqueous solution will tend to be neutral ~pH 7.
If there are two -COOH groups and one -NH2 group, the aqueous
solution will tend to be weakly acid with a pH <7
If there is one -COOH group and two -NH2 group, the aqueous
solution will tend to be weakly alkaline with a pH >7.
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6.13.4 The formation of polypeptides
Amino acids can polymerise together, by
condensation
polymerisation, forming polypeptides.
The peptide linkage is formed by
elimination of water between two amino acids.
The simplest amino acid is glycine H2NCH2COOH
and the polymerisation can be written as ...
n H2NCH2COOH ===>
(-NHCH2CO-)n + nH2O,
where n can be quite a large
number in the polymer.
These polymers are therefore 'polyamides' and usually called 'polypeptides'.
In general the polymerisation to form a
polypeptide/secondary amide link is ...
H2N-CH(R)-COOH
+ H2N-CH(R)-COOH ==> H2N-CH(R)-CO-HN-CH(R)-COOH
+ H2O ...
... to form one peptide linkage, so ...
n H2N-CH(R)-COOH
==> -NH-CH(R)-CO-NH-CH(R)-CO-NH-CH(R)-CO-NH-CH(R)-CO-
+ n H2O
etc. n units long and remember the R can be
lots of different groups.
... where R is variable chemical group, as there
over 20 known amino acids,
and so proteins are long chain polypeptides
and are natural
condensation polymers of amino acids.
Long chain polypeptides are what make the majority of the molecular
structure of proteins.
Each polypeptide, protein, enzyme etc. has its
own unique sequence of amino acids (all encoded for in an organism's DNA).
Diagram showing the formation of the
polyamide/polypeptide link as a water molecule is eliminated when the
carboxylic acid of one amino acid, and the amino group condense together to
give an polypeptide/amide link.
In
this case two amino acids have a formed the simplest possible polypeptide -
a simple dipeptide.
Note that at each end of the molecule,
the amino/amine
group (-NH2,
on left) and the carboxylic
acid group (-COOH,
on right) can both form a bond with another amino acid molecule by
further elimination of water molecules.
So, both functional groups are involved in the condensation
to form the polymer.
So, if the process continues, as shown
below), you build up a long chain polymer - known as a polyamide,
polypeptide or a protein - they are all the same here.
Proteins have the same
(amide) linkages as nylon but with different units.
In the case of natural polypeptides/proteins the
HN-C=O
link is referred to as
peptide linkage.
A generalised diagram of a section of a polypeptide or
section of a protein molecule, but only in terms of amino acid residues.
A sequence of 7
amino acid residues is shown. In biochemistry or molecular biology, a
residue refers to a single unit that makes up a polymer, such as an
amino acid in a polypeptide or protein.
Note the
peptide linkage formed by loss of H from the H2N
group and OH from the COOH group i.e. the loss of H2O between two amino
acids to give the HN-C=O
link.
Also, note for the peptide link, the ~120o bond angles for C-C=O,
C-C-N and O=C-N giving a trigonal planar arrangement of bonds around
the carbon atom of the peptide linkage HN-CO.
Proteins are an important component of
tissue structure and enzymes (powerful biological chemical catalysts) are
also protein molecules.
Proteins tend to adopt a particular three dimensional shape (3D) which aids
its function.
Apart from the structural proteins in you body e.g. muscle
tissue, enzymes are protein molecules wrapped into a specific 3D shape to carry
out their catalytic function.
For more detailed notes see
Enzymes and Biotechnology
and detailed notes on
Proteins
structure (primary, secondary, tertiary and quaternary), enzyme structure and function
and metalloenzymes
When proteins are heated with aqueous
hydrochloric acid or sodium hydroxide solution they are hydrolysed to amino acids.
see
chromatography
below, about how amino acids are identified in proteins.
There are 20 amino acids that make up the proteins in the
human body.
12 can be synthesised by us from other amino acids,
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6.13.5 Amino acids from proteins, hydrolysis and R/S
stereoisomerism
(a) Hydrolysis
Hydrolysis
means breaking down a
molecule with water to form two or more products, in this case proteins or
polypeptides into amino acids.
Hydrolysis is usually accelerated if the substance is heated with acid or alkali solutions.
The basic equation is the reverse of the condensation
polymerisation described above in
section 6.13.4.
-NH-CH(R)-CO-NH-CH(R)-CO-NH-CH(R)-CO-NH-CH(R)-CO- + n H2O
==> n H2N-CH(R)-COOH
The polypeptide/protein is n units long and remember the
R 'side-chain' can be lots of different groups e.g. from the 20 amino
acids that make up proteins.
The hydrolysis equation be expressed in abbreviated ways
e.g. refluxing protein under (aq) different conditions
(-NH-CH(R)-CO-)n
+ nH2O
==> n[H3N+-CH(R)-COO-]
Very slow in water to give the zwitterion form of
the amino acids.
(-NH-CH(R)-CO-)n
+ nH+ + nH2O
==> n [H3N+-CH(R)-COOH]
Much faster with mineral acid e.g. HCl(aq)
to give the cationic form of the amino acids.
(-NH-CH(R)-CO-)n
+ nOH-
==> n [H2N-CH(R)-COO-]
Much faster with a strong base/alkali e.g. NaOH(aq))
to give the anionic form of the amino acids.
For the acid and alkaline hydrolysis you use
concentrated reagent and heated under reflux for several
hours.
Some simple equations for a dipeptide with R and R'
groups respectively
i.e. H2N-CH(R)-CO-NH-CH(R')-COOH in molecular
form, but watch out for formation of ionic forms.
H2N-CH(R)-CO-NH-CH(R')-COOH
+ H2O ===> H2N-CH(R)-COOH +
H2N-CH(R')-COOH
H2N-CH(R)-CO-NH-CH(R')-COOH
+ 2H+ + H2O ===> [H3N+-CH(R)-COOH]
+ [H3N+-CH(R')-COOH
H2N-CH(R)-CO-NH-CH(R')-COOH
+ 2NaOH ===> [H2N-CH(R)-COO-]Na+
+ [H2N-CH(R')-COO-]Na+
+ H2O
or
H2N-CH(R)-CO-NH-CH(R')-COOH
+ 2OH- ===> [H2N-CH(R)-COO-]
+ [H2N-CH(R')-COO-]
+ H2O
Note! the isomeric dipeptide H2N-CH(R')-CO-NH-CH(R)-COOH
will give the same hydrolysis products!
(b) R/S isomerism - a form of stereoisomerism (optical
isomerism)

The alpha carbon atom of alpha amino acids (except H2NCH2COOH)
is an asymmetric or chiral carbon because it is bonded to
4 different atoms/groups.
This carbon atom is the chiral centre of the
molecule which can exhibit R/S isomerism (a form of
stereoisomerism, once called 'optical isomerism').
The molecule can therefore exist in two
non-superimposable mirror image forms called enantiomers ('optical
isomers').
Apart from glycine, all naturally occurring amino acids
are R/S isomers, and apart from cysteine they are the S isomer (L
or laevorotatory in old nomenclature).
The diagram below puts R/S isomerism in its
stereoisomeric context compared to other forms of isomerism in organic
molecules.
(c) Amino acids from proteins
All the alpha-amino acids obtained from proteins are
optically active except glycine (aminoethanoic acid), that is they exhibit
R/S stereoisomerism (pin-pointed in the diagram above) where you have two
mirror image forms that cannot be superimposed on each other (as with
your right and left hands!).
R/S stereoisomerism page
2-aminoethanoic acid, H2NCH2COOH,
is not an optically active molecule because it
has no chiral/asymmetric carbon atom
because there is no carbon with 4 different groups attached, but all the
other alpha-amino acids have four different groups attached to the 2nd
carbon atom i.e. R-CH(NH2)COOH
In aqueous solution, and in the
solid state, they predominantly exist as zwitterions, the ionic form
derived from proton transfer from the carboxylic group onto the amino
group.
RCH(NH2)COOH
RCH(+NH3)COO-
Even so, the zwitterions will also exhibit R/S
stereoisomerism as well as the non-ionic molecular form.
R/S isomers of the non-ionic molecular form (R = CH3
for alanine)
R/S isomers of the ionic zwitterion form (R = CH3
for alanine)
need zwitterion form too
Comparing 'natural' and
'laboratory' synthesised amino acids (other than
biosynthetic routes)
When molecules capable of exhibiting
optical isomerism are obtained from natural sources, they usually
consist of one of the possible isomers (one of the enantiomers).
On extraction, purification and isolation, they show optical
activity (that is rotating the plane of polarised light in a polarimeter
tube).
This is due to the need for stereospecific structures from
enzymes to proteins.
The '3D' stereospecificity of enzyme
sites is discussed in section 6.
However, when the same compound is
synthesised in the laboratory by a non-biosynthetic route, it often consists of an
equimolar
mixture of the two optical isomers (R/S isomer enantiomers).
This is known as a racemic
mixture and it is optically inactive due to one isomer cancelling
out the optical effect of the other.
Warning:
It is wrong to say that R/S optical isomers are not, or cannot be formed,
in a laboratory synthesis!
Its difficult, but not
impossible, using very sophisticated stereospecific synthesis techniques.
The most common explanation for the
production of a racemic mixture lies in understanding the mechanisms of
the laboratory synthesis reactions.
For example, if a carbocation is
formed, which has three C-R bonds in a trigonal planer arrangement, the
reagent molecule or ion (electron pair donor) can attack on either side
with equal probability.
So when a possible chiral carbon molecule is
formed in many a laboratory synthesis, it tends to be an equimolar
mixture of the two spatial possibilities, R/S isomers (enantiomers).
See also
carbocation mechanisms of haloalkane substitution reactions
and addition reactions of aldehydes/ketones
However, since the 1990's the problem
is being tackled by the use of
chiral auxiliary
molecules.
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6.13.6
Chromatography - a method of amino acid
analysis
Hydrolysis
means breaking down a
molecule with water to form two or more products (see
section 6.13.5)
and is accelerated if the substance is heated with acid or alkali solutions.
The resulting mixture can then be analysed by an appropriate technique e.g.
paper or thin-layer chromatography.
(1)
(2)
(3)
(4)
paper chromatography
Paper chromatography or thin layer chromatography (TLC) is used to separate
coloured compounds (illustrated above).
Thin layer chromatography (TLC) uses a stationary
phase (immobile phase) of either silica gel or aluminium oxide immobilised on a flat
inert surface that can be made of a glass or plastic plate.
In the procedure:
(1) Samples spotted onto start
line of the paper/plate which is carefully placed in solvent, but below
start line of pencil - there should NOT be any immediate contact of the
spots with the solvent, which must rise up to carry the solutes along
without dissolving them in the bulk solvent.
Although not shown, the whole system should be in a
larger sealed/covered container to avoid evaporation of
solvent from the paper or T.L.C coating.
(2) The solvent, the moving or mobile phase called the
eluent, rises up the paper or plate carrying the mixture of
solutes.
The chromatographic separation occurs because the
different solutes (e.g. amino acids) are attracted/solvated to
different extents by the immobile phase and the mobile solvent
phase.
Paper chromatography
Water is held by the cellulose fibres of the
paper and the solvent can be water, organic solvents like
alcohols or a mixture of water and organic solvent.
Paper chromatography is good for separating food
dyes and coloured plant extracts.
Thin layer chromatography
T.L.C uses a stationary phase coating of
aluminium oxide ('alumina', Al2O3) or
silica powder (silicon dioxide, SiO2).
(3) When the solvent front reaches near top of
paper/coating, remove paper to dry.
However, amino acids are colourless,
but can still be separated in this way, and made visible!
For colourless amino acids, you spray the paper
with ninhydrin and gently heated, which gives a purple spot for each amino acid.
You measure the vertical distance travelled by each
spot.
In the case of T.L.C you can actually o it on a larger scale
than paper and actually scrape off individual molecules for further
investigation - you extract them individually with a solvent and filter off
the alumina/silica.
(4) You can then measure the Rf
values to identify amino acids in the mixture.
To illustrate the method I've
described the separation of coloured dye molecules to represent different
amino acids.
1 to 5 represent five pure compounds, 6 is a
mixture.
Red, brown and blue make up the mixture because its spots
horizontally line up with the three known colours.
The substances (solutes)
to be analysed must dissolve in the solvent, which is called the mobile phase because it moves.
The solvent may be water or other suitable solvent that
the amino acids will dissolved in - can vary the pH of the eluent
solvent - the liquid that moves up the paper or TLC plate.
The solvent may incorporate, buffer, acid or alkaline
solutions to control the pH - optimisation.
The paper or thin layer of
material on which the separation takes place is called the stationary or
immobile phase because it doesn't move.
The distance a substance
moves, compared to the distance the solvent front moves (top of grey area on
diagram 2) is called the reference or Rf value (a simple
ratio) and has a
value of 0.0 (not moved - no good), to 1.0 (too soluble - no good either).
Rf ratio values between 0.1 and 0.9 can be useful for analysis and
identification of the amino acids.
Rf =
distance moved by amino acid spot / distance moved by solvent
(5) Two-dimensional chromatography
This is an alternative extra step in the
chromatography separation process.
You take the first separation paper/coating using
solvent 1, dry it, and turn it through 90o and
expose it in the same way as described in (1) to a different
solvent 2.
Solvent 1 might not separate some materials with the
same Rf value, but these same materials are like to have
different Rf values with another solvent - 'trial and
error' sorts this out!
This gives a better 2D dispersal of the individual
'spots' of the different molecules (amino acids here).
For colourless materials you identify them from the
2D Rf values after treatment with e.g. ninhydrin or other
developing chemical reagent or using uv light to get a fluorescent
effect from the spots.
Thin layer or paper chromatography
can still used
to separate and identify the products of hydrolysis of
proteins because you make them coloured by using another
chemical reagent.
The hydrolysis can be done by boiling-refluxing the protein with hydrochloric acid.
The hydrolysed mixture is then 'spotted'
onto the pencil base line of the chromatography paper or the TLC plate.
Known amino acids are also
spotted onto the base line too.
The prepared paper is then placed
vertically in a suitable solvent, which rises up the paper/TLC plate.
Since the products are colourless, the
dried chromatogram is treated with another chemical to produce a
coloured compound.
A reagent spray of Ninhydrin produces purple spots with
amino acids
Using known amino acid samples, you can then tell which amino acids made
up the protein that you hydrolysed.
The number of different spots tells
you how many different amino acids made up the protein or peptide.
Spots which horizontally match the
standard known molecule spots confirm identity their identity.
Uses of paper and T.L.C chromatography
This type of chromatography has been used to
investigate:
Protein structure - to determine the amino
acid residue sequences in polypeptides
Identifying the intermediate compounds in
photosynthesis - from water and carbon dioxide to glucose.
More on the theory of paper and thin layer
chromatography
Reminder of other amino acid analysis techniques
Electrophoresis, in which the ionic forms of the amino acids are
separated by movement in a buffered aqueous gel medium under the
influence of an applied electric field (from d.c. voltage electrodes).
The different amino acid mobilities depend on the average total +ve or
-ve charge in a particular buffer.
The amino acids form bands which can
be detected-analysed by using staining techniques or uv
light fluorescence. The technique can also be applied to the analysis
of protein or nucleic acid mixtures, and the latter can be detected using a radioactive phosphorus tracer
32P (in
the laboratory you should only deal with stable 31P).
TOP OF PAGE
and sub-index
6.13.7
The multi-functional group chemistry of amino
acids
Other
reactions of the carboxylic acid and amino (amine) functional groups
The acidic and basic character of the -COOH and -NH2 groups
has been dealt with in
section 6.13.3
Other reactions of the carboxylic acid group
(a) Esterification
Amino acids can be reacted with alcohols to form
esters using a strong acid catalyst like concentrated sulfuric acid
e.g.
RCH(NH2)COOH + R'OH
RCH(NH2)COOR'
+ H2O
The strong acid ensures the amino acid is protonated
and the carboxylic acid group is free to undergo esterification.
The zwitterion form of the amino acid cannot be
esterified.
Technically it is the protonated form that is
esterified and the ester freed on adding alkali
e.g. preparing ethyl 2-aminopropanoate in 2 stages
(i) Esterification under acid conditions
CH3CH(NH3+)COOH +
CH3CH2OH
CH3CH(NH3+)COOCH2CH3
+ H2O
(ii) Addition of alkali
CH3CH(NH3+)COOH +
OH-
CH3CH(NH2)COOCH2CH3
+ H2O
Other
reactions of the amine/amino acid group
(a) Acylation
Amino acids will react with acid chlorides and acid
anhydrides to replace one of the hydrogens on the amino group with
an acyl group (R-C=O, RCO) e.g.
RCH(NH2)COOH + R'COCl
RCH(NHOCR')COOH
+ H2O
The product is a secondary amide, but still with a
carboxylic acid group.
(b) Acting as a nucleophile in nucleophilic substitution reactions
The zwitterion nature of amino acids means that the
lone pair of electrons on the nitrogen is not available to allow the
amino acid to act as a nucleophile unless the pH is much higher than
7 i.e. via reaction (i) below in alkaline conditions
(i) RCH(NH3+)COO-
+ OH-
RCH(:NH2)COO- + H2O
The allows the amine group to acts as the 'front
end' of an electron pair donating nucleophile (: in the
right-hand formula).
The amino acid can then react with halogenoalkanes
to yield a secondary amine, albeit with a carboxylic acid group
retained. (X = Cl, Br or I)
(ii)
2RCH(NH2)COO- + R'X +
2H2O ===>
RCH(NHR')COOH + RCH(NH3+)COOH
+ X- + 2OH-
TOP OF PAGE
and sub-index
6.13.8 An alphabetical list of the 20 amino acids that
make up proteins
Their molecular structure, (three letter abbreviation code),
names and extra comments.
They are known as alpha amino acids because the amine
group is on the first carbon atom on which a hydrogen atom can be
substituted with another atom or group.
Except for aminoethanoic acid (glycine), they all
exhibit R/S stereoisomerism.
Except for proline, they all have at least one
carboxylic acid group and one primary amine base group, therefore in the
comments, other functional groups are pointed out.
1.
CH3CH(NH2)COOH, alanine (ala), 2-aminopropanoic acid,
2.
arginine (arg), has extra amine groups,
3.
asparagine (asn), has an extra amide group,
4.
HOOCCH2CH(NH2)COOH, aspartic acid (asp), has an extra
carboxylic acid group,
5.
HSCH2CH(NH2)COOH, cysteine (cys), has an extra -SH
mercapto group
6.
HOOCCH2CH2CH(NH2)COOH, glutamic acid (glu),
has an extra carboxylic acid group,
7.
H2NCOCH2CH2CH(NH2)COOH,
glutamine gln), has an extra amide group,
8.
H2NCH2COOH, glycine (gly), aminoethanoic acid, the
simplest amino acid,
9.
histidine (his), has an extra secondary amine group,
10.
isoleucine (ile), 2-amino-3-methylpentanoic acid,
11.
leucine (leu), 2-amino-4-methylpentanoic acid,
12.
lysine (lys), 1,6-diaminohexanoic acid, has an extra primary amine group
13.
methionine (met), has a disulfide linkage (analogous to a C-O-C ether
linkage),
14.
phenylalanine (phe), 2-amino-3-phenylpropanoic acid, has a benzene ring,
15.
proline (pro), a secondary amine group
16.
serine (ser), 2-amino-3-hydroxypropanoic acid, has an extra primary alcohol
group,
17.
threonine (thr), 2-amino-3-hydroxypropanoic acid, has an extra secondary
alcohol group,
18.
tryptophan (trp), has an extra cyclic secondary amine group connected to a
benzene ring,
19.
tyrosine (tyr), 2-amino-3-(4-hydroxylphenyl)propanoic acid, has a benzene
ring with a phenol group,
20.
valine (val), 2-amino-3-methylbutanoic acid,
6.13.9 Some specific uses of amino acids
H2NCH2CH2CH2COOH
4-aminobutanoic acid is an important amino acid, though not in protein
structures.
It is a crucial neurotransmitter molecule by blocking nerve
impulses from one nerve cell to another to reduce overload of the CNS.
Epilepsy can be treated with drugs that stimulate the brain to increase the
production of it and reduce brain activity.
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