6.13.2 A laboratory synthesis of amino acids from halogenated carboxylic acids

6.13.3 The multi-functional group chemistry of amino acids (focus on acidic and basic character)

6.13.4 The formation of polypeptides

6.13.5 Amino acids from proteins

6.13.5 Chromatography - a method of amino acid analysis

6.13.6 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)

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

and Amino acids as a case study of R/S isomerism

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.

Data on some of the simplest amino acids is quoted below.

(Mpt. = melting point;  dec. = decomposes;  sub. = sublimes)

Notes on the data table

(i) 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.

(ii) 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)..

(iii) The α and β (alpha and beta) refer to the second and third carbon atoms to which the amino group can be a substituent.

(iv) All the above have one -COOH group and one -NH₂ group in the molecule, so the aqueous solution will tend to be neutral ~pH 7. See amino acid examples in section 6.13.3 which are not neutral.

 

There 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.

Twenty amino acids are found in the structure of proteins in the human body.

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.

 

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

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, S_N1 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.

6.13.3 The multi-functional group chemistry of amino acids

The acidic and basic character of the -COOH and -NH₂ functional groups

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-NH_2(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 amino group and one carboxylic acid group.

These will tend to cancel each other out and dissolves to give a neutral aqueous solution

e.g. CH₃CH(NH₂)COOH  2-aminopropanoic acid (Alanine)

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.

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

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)

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)

The carboxylate anion is formed

RCH(NH₃⁺)COO^-_(aq)  +  OH^-_(aq)    RCH(NH₂)COO^-_(aq)   +  H₂O_(l)

Here the protonated zwitterion acts 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) 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

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.

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  ===>  (-NHCH₂COO-)n + nH₂O,

where n can be quite a large number in the polymer.

These polymers are therefore 'polyamides' as well as '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-CO-CH(R)-NH-CO-CH(R)-NH-CO-CH(R)-NH-CO-CH(R)- etc. n units long

... 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.

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

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,

6.13.5 Amino acids from proteins and R/S stereoisomerism

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 no impossible, using very sophisticated 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 or enantiomers.

(see carbocation mechanisms of haloalkane substitution reactions, addition reactions of aldehydes/ketones.

However, since the 1990's the problem is being tackled by the use of chiral auxiliary molecules.

6.13.6 Chromatography - a method of amino acid analysis

Hydrolysis means breaking down a molecule with water to form two or more products.

Hydrolysis is accelerated if the substance is heated with acid or alkali solutions.

When proteins are heated with strong aqueous mineral acid they are hydrolysed to amino acids.

(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 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 placed in solvent, but below start line of pencil.

(2) The solvent, the mobile phase called the eluent, rises up paper/plate.

(3) When solvent near top of paper, 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.

(4) You can then measure the R_f 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 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 R_f value (a simple ratio) and has a value of 0.0 (not moved - no good), to 1.0 (too soluble - no good either).

 R_f ratio values between 0.1 and 0.9 can be useful for analysis and identification of the amino acids.

R_f = distance moved by amino acid spot / distance moved by solvent

 

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.

Theory of paper and thin layer chromatography

6.13.6 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

 

(b) -

 

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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