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Advanced Level Organic Chemistry: Amino acids: Structure, preparation and reactions

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Part 6. The Chemistry of  Carboxylic Acids and their Derivatives

Doc Brown's Chemistry Advanced Level Pre-University Chemistry Revision Study Notes for GCE IB advanced A level organic chemistry students US K12 grade 11 grade 12 organic chemistry the chemistry of amino acids structure and reactions 20 which amino acids are in proteins? acid-base chemistry zwitterions

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6.13 Amino acids - structure, preparation, reactions of two functional group chemistries including acidic and basic character, peptide formation and hydrolysis analysis

Sub-index for amino acids page

6.13.0 Introduction - the importance of amino acids

6.13.1 Molecular and ionic structure of amino acids, zwitterions and physical properties

6.13.2 A laboratory synthesis of amino acids from halogenated carboxylic acids

6.13.3 The multi-functional group chemistry of amino acids - acidic/basic character and isoelectric point

6.13.4 The formation of polypeptides and proteins

6.13.5 Amino acids from polypeptides/proteins, hydrolysis and R/S isomerism

6.13.6 Chromatography - a method of amino acid analysis

6.13.7 The multi-functional group chemistry of amino acids - other reactions of the carboxylic acid and amino (amine) functional groups (other than the acid-base chemistry described in section 6.13.3)

6.13.8 An alphabetical list of the 20 amino acids that make up proteins (structure and comments)

6.13.9 Some specific uses of amino acids

See also organonitrogen chemistry 8.8 Amino acids, peptides, polypeptides and structure and types of proteins

and Amino acids as a case study of R/S isomerism (optical isomers, enantiomer structure)


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).

zwitterions molecular structure of alpha amino acids aminoethanoic acid structural formula 2-aminopropanoic acid 3-aminopropanoic acid skeletal formula

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

-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) (c) doc b,   (c) doc b  AND   (c) doc b(c) doc b

2-aminopropanoic acid (alanine(c) doc b,   (c) doc b(c) doc b ,  (c) doc b,   (c) doc b

 

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.

 amino acid chemistry  molecular structure structural formula advanced level organic chemistry  amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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).

 amino acid chemistry  molecular structure structural formula advanced level organic chemistry Aspartic acid has an isoelectric point of pH 2.9, a pH well below 7 because of the 2nd carboxylic acid group.

 amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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).

formation of a peptide link joining amino acids together aminoethane glycine dipeptide

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.

formation of protein polypeptide by condensation of amino acids

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.

general diagram section of polypeptide or protein molecule sequence of amino acid residues peptide link bond angles 120 degrees advanced level organic chemistry biochemistry

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 (c) doc b RCH(+NH3)COO-

Even so, the zwitterions will also exhibit R/S stereoisomerism as well as the non-ionic molecular form.

structural formula diagram of R/S optical isomers of alpha-amino acids stereoisomers enantiomers stereoisomerism molecular structure R/S isomers of the non-ionic molecular form (R = CH3 for alanine)

skeletal diagram of R/S optical isomers of zwitterions of alpha-amino acids stereoisomers enantiomers stereoisomerism ionic structure 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)chromatography at start  (2)chromatogram at the end  (3)chromatography (4)diagram of paper/thin layer chromatography at the end 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).


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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-


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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 amino acid chemistry  molecular structure structural formula advanced level organic chemistry CH3CH(NH2)COOH, alanine (ala), 2-aminopropanoic acid,

2. arginine (arg), has extra amine groups amino acid chemistry  molecular structure structural formula advanced level organic chemistry arginine (arg), has extra amine groups,

3. asparagine (asn), has an extra amide group amino acid chemistry  molecular structure structural formula advanced level organic chemistry asparagine (asn), has an extra amide group,

4. HOOCCH2CH(NH2)COOH, aspartic acid (asp), has an extra carboxylic acid group amino acid chemistry  molecular structure structural formula advanced level organic chemistry HOOCCH2CH(NH2)COOH, aspartic acid (asp), has an extra carboxylic acid group,

5. HSCH2CH(NH2)COOH, cysteine (cys), has an extra -SH mercapto group amino acid chemistry  molecular structure structural formula advanced level organic chemistry HSCH2CH(NH2)COOH, cysteine (cys), has an extra -SH mercapto group

6. HOOCCH2CH2CH(NH2)COOH, glutamic acid (glu), has an extra carboxylic acid group amino acid chemistry  molecular structure structural formula advanced level organic chemistry HOOCCH2CH2CH(NH2)COOH, glutamic acid (glu), has an extra carboxylic acid group,

7. H2NCOCH2CH2CH(NH2)COOH, glutamine gln), has an extra amide group amino acid chemistry  molecular structure structural formula advanced level organic chemistry H2NCOCH2CH2CH(NH2)COOH, glutamine gln), has an extra amide group,

8. H2NCH2COOH, glycine (gly), aminoethanoic acid, the simplest amino acid amino acid chemistry  molecular structure structural formula advanced level organic chemistry H2NCH2COOH, glycine (gly), aminoethanoic acid, the simplest amino acid,

9. histidine (his), has an extra secondary amine group amino acid chemistry  molecular structure structural formula advanced level organic chemistry histidine (his), has an extra secondary amine group,

10. isoleucine (ile), 2-amino-3-methylpentanoic acid amino acid chemistry  molecular structure structural formula advanced level organic chemistry isoleucine (ile), 2-amino-3-methylpentanoic acid,

11. leucine (leu), 2-amino-4-methylpentanoic acid amino acid chemistry  molecular structure structural formula advanced level organic chemistry leucine (leu), 2-amino-4-methylpentanoic acid,

12. lysine (lys), 1,6-diaminohexanoic acid, has an extra primary amine group amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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) amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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 amino acid chemistry  molecular structure structural formula advanced level organic chemistry phenylalanine (phe), 2-amino-3-phenylpropanoic acid, has a benzene ring,

15. proline (pro), a secondary amine group amino acid chemistry  molecular structure structural formula advanced level organic chemistry proline (pro), a secondary amine group

16. serine (ser), 2-amino-3-hydroxypropanoic acid, has an extra primary alcohol group amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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 amino acid chemistry  molecular structure structural formula advanced level organic chemistry threonine (thr), 2-amino-3-hydroxypropanoic acid, has an extra secondary alcohol group,

18.  amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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 amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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 amino acid chemistry  molecular structure structural formula advanced level organic chemistry 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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structural formulae for the 20 twenty amino acids that make up proteins 3 three letter codes

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