2.

0 Synthesis of Aminoacids
Naturally occurring amino acids can be obtained by hydrolyzing proteins and separating the
amino acid mixture. However, some unusual amino acid or unnatural enantiomer maybe needed,
and it must be synthesized. Also it is less expensive to synthesize the pure amino acid. Herein,
we consider four methods for making amino acids which are extensions of already studied
reactions.
2.1 Reductive Amination
Reductive amination of ketones and aldehydes is one of the best methods for synthesizing
amines. It also be extended to amino acids. When an -ketoacid is treated with ammonia, the
ketone forms an imine. The imine is then reduced to an amine by hydrogen and a palladium
catalyst. Under these conditions, the carboxylic acid will not be reduced.

This synthesis is accomplished in one step by treating the -ketoacid with ammonia and
hydrogen in the presence of a palladium catalyst. However, the product will be a racemic -
amino acid. For example the synthesis of racemic phenylalanine from 3-phenyl-2-oxopropanoic
acid is shown.

Note that, reductive amination is a biomimetic (“mimicking the biological process”) synthesis. It
resembles the biological synthesis of amino acids for which the biosynthesis begins with
reductive amination of -ketoglutaric acid (an intermediate in the metabolism of carbohydrates),
using ammonium ion as the aminating agent and NADH as the reducing agent. The product of
this enzyme-catalyzed reaction is the pure L enantiomer of glutamic acid.
2.2 Amination of an α-Halo Acid
The Hell–Volhard–Zelinsky reaction is an effective method for introducing bromine at the α-
position of a carboxylic acid. The racemic acid is converted to a racemic -amino acid by direct
amination, using a large excess of ammonia.

2.3 The Gabriel–Malonic Ester Synthesis

One of the best methods of amino acid synthesis is a combination of the Gabriel synthesis of
amines with the malonic ester synthesis of carboxylic acids. The conventional malonic ester
synthesis involves alkylation of diethyl malonate, followed by hydrolysis and decarboxylation to
give an alkylated acetic acid.

To adapt this synthesis to making amino acids, we begin with a malonic ester that contains an -
amino group. The amino group is protected as a non-nucleophilic amide to prevent it from
attacking the alkylating agent (RX).
The Gabriel–malonic ester synthesis begins with N-phthalimidomalonic ester. The amino group
protected as an amide (phthalimide) to keep it from acting as a nucleophile. The acid is protected
as an ethyl ester, and the - position is further activated by the additional (temporary) ester group
of diethyl malonate. i.e for glycine
When the alkylated N-phthalimidomalonic ester is hydrolyzed, the phthalimido group is
hydrolyzed along with the ester groups. The product is an alkylated aminomalonic acid.
Decarboxylation gives a racemic a-amino acid.

The Gabriel–malonic ester synthesis is used to make many amino acids that cannot be formed by
direct amination of haloacids. Eg

Also;
Amidomalonate Synthesis:

2.4 The Strecker Synthesis

Adolph Strecker (Germany 1850) added acetaldehyde to an aqueous solution of ammonia and
HCN. The product was -amino propionitrile, which was hydrolyzed to racemic alanine.
The Strecker synthesis can form a large number of amino acids from appropriate aldehydes.
The mechanism is as follows;
Step 1: The aldehyde reacts with ammonia to form the imine

Step 2: Cyanide ion attacks the imine.

In a separate step, hydrolysis of the -amino nitrile gives -amino acid.

OR:

Recall reductive amination and Cyanohydrin formation

Ref: Amino Acids_Ch24
3.0: Reactions of Amino Acids.

Amino acids will undergo reactions characteristic of the amino (amide formation) and carboxylic
acid (ester formation) groups. Conditions must however be carefully selected, so that the amino
group does not interfere with a carboxyl group reaction, and vice versa. These reactions are often
used to protect either the carboxyl group or the amino group while the other group is being
modified or coupled to another amino acid. Amino acids also undergo reactions that are specific
to the -amino acid structure like that with ninhydrin.

3.1 Esterification of the Carboxyl Group

Amino acids are esterified with a large excess of alcohol and acidic catalyst (often gaseous HCl).
Under these acidic conditions, the amino group is present in its protonated (-NH3+) form, so it
does not interfere with esterification. E.g

Esters of amino acids are often used as protected derivatives to prevent the carboxyl group from
reacting in some undesired manner. Methyl, ethyl, and benzyl esters are the most common
protecting groups. Aqueous acid hydrolyzes the ester and regenerates the free amino acid.

Benzyl esters are the most useful protecting groups because they can be removed either by acidic
hydrolysis or neutral hydrogenolysis (“breaking apart by addition of hydrogen”). Catalytic
hydrogenation cleaves the benzyl ester, converting the benzyl group to toluene and leaving the
deprotected amino acid.
3.2 Acylation of the Amino Group (Formation of Amides)
An acylating agent converts the amino group to an amide. It is often done to protect the amino
group from unwanted nucleophilic reactions. A wide variety of acid chlorides and anhydrides are
used for acylation. Benzyl chloroformate acylates the amino group to give a benzyloxycarbonyl
derivative, often used as a protecting group in peptide synthesis.

The amino group of the N-benzyloxycarbonyl derivative is protected as the amide half of a
carbamate ester (a urethane), which is more easily hydrolyzed than most other amides. In
addition, the ester half of this urethane is a benzyl ester that undergoes hydrogenolysis. Catalytic
hydrogenolysis of the N-benzyloxycarbonyl amino acid gives an unstable carbamic acid that
quickly decarboxylates to give the deprotected amino acid. i.e,

3.3 Reaction with Ninhydrin

Ninhydrin is a common reagent for visualizing spots or bands of amino acids that have been
separated by chromatography or electrophoresis. When ninhydrin reacts with an amino acid, one
of the products is a deep violet, resonance-stabilized anion called Ruhemann’s purple. Ninhydrin
produces this same purple dye regardless of the structure of the original amino acid. The side
chain of the amino acid is lost as an aldehyde.
Reaction of an amino acid with ninhydrin

Note: The reaction of amino acids with ninhydrin can detect amino acids on a wide variety of
substrates. For example, if a kidnapper touches a ransom note with his fingers, the dermal ridges
on his fingers leave traces of amino acids from skin secretions.
Look:

3.4: Some Biochemical Reactions of Amino Acids.

Many enzymes involved in amino acid biosynthesis, metabolism and catabolism are pyridoxal
phosphate (vitamin B6) dependent.
Ref: Amino Acids_Ch24
4.0: Peptides.

Proteins and peptides are polymers made up of amino acid units (residues) that are linked
together through the formation of amide bonds (peptide bonds) from the amino group of one
residue and the carboxylate of a second residue

By convention,
peptide sequences are written left to right from the N-terminus to the C-terminus

backbone

The amide (peptide) bond has C=N double bond character due to resonance resulting in a planar
geometry
4.1: Introduction to Peptide Structure Determination.
Protein Structure:
primary (1°) structure: the amino acid sequence
secondary (2°): frequently occurring substructures or folds
tertiary (3°): three-dimensional arrangement of all atoms in asingle polypeptide chain
quaternary (4°): overall organization of non-covalently linked subunits of a functional protein.
1. Determine the amino acids present and their relative ratios
2. Cleave the peptide or protein into smaller peptide fragments and determine their
sequences
3. Cleave the peptide or protein by another method and determine their sequences. Align
the sequences of the peptide fragments from the two methods
4.2: Amino Acid Analysis.

Automated method to determine the amino acid content of a peptide or protein

Reaction of primary amines with ninhydrin

4.3: Partial Hydrolysis of Peptides.

Acidic hydrolysis ofpeptides cleave the amide bonds indiscriminately.
Proteases (peptidases): Enzymes that catalyzed the hydrolysis of the amide bonds of peptides
and proteins.
Enzymatic cleavage of peptides and proteins at defined sites:
• trypsin: cleaves at the C-terminal side of basic residues,
Arg, Lys but not His

•
chymotrypsin: cleaves at the C-terminal side of aromatic residues
Phe, Tyr, Trp

Trypsin and chymotrypsin are endopeptidases

Carboxypeptidase: Cleaves the amide bond of the C-terminal amino acid (exopeptidase)

4.4: End Group Analysis.

The C-terminal AA is identified by treating with peptide with carboxypeptidase, then analyzing
by liquid chormatography (AA Analysis).
N-labeling: The peptide is first treated with 1-fluoro-2,4-dinitrobenzene (Sanger’s reagent),
which selectively reacts with the N-terminal amino group. The peptide is then hydrolyzed to
their amino acids and the N-terminal amino acid identified as its N-(2,4-dinitrophenyl) derivative
(DNP).

Insulin. Insulin has two peptide chains (the A chain has 21 amino acids and the B chain has 30
amino acids) held together by two disulfide linkages.
Pepsin: cleaves at the C-terminal side of Phe, Tyr, Leu; but not at Val or Ala.

4.5: The Edman Degradation and Automated Peptide

Sequencing. Chemical method for the sequential cleavage and identification of the amino acids
of a peptide, one at a time starting from the N-terminus.
Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)
4.5.1 Peptide sequencing by Edman degradation:
• Cycle the pH to control the cleavage of the N-terminal amino acid by PITC.
• Monitor the appearance of the new N-phenylthiohydantoin for each cycle.
• Good for peptides up to ~ 25 amino acids long.
• Longer peptides and proteins must be cut into smaller fragments before Edman sequencing.
Tandem mass spectrometry has largely replaced Edman degradation for peptide sequencing.
5.0: The Strategy for Peptide Synthesis:
Chemical synthesis of peptide:
1. Solution phase synthesis
2. Solid-phase synthesis

The need for protecting groups

5.1: Amino Group Protection.
The -amino group is protected as a carbamate.

5.2: Carboxyl Group Protection.

Protected as a benzylester; removed by hydrogenolysis

5.3: Peptide Bond Formation.

Amide formation from the reaction of an amine with a carboxylic acid is slow. Amide bond
formation (peptide coupling) can be accelerated if the carboxylic acid is activated. Reagent:
dicyclohexylcarbodiimide (DCC)
O O C6H11 C6H11
O O
H NH NH
R O R O R O C R O C
+ +N
N R' H N
C6H11 N C N C6H11 + H C6H11
C6H11 N C N C6H11 C6H11
(DCC) H ••
R'-NH2 "activated acid"
C6H11 O O
O
NH R' + C6H11 C6H11
R O C R N N N
NH H H H
R' HN
+ C6H11 Amide DCU
• In order to practically synthesize peptides and proteins, time consuming purifications steps
must be avoided until the very end of the synthesis.
• Large excesses of reagents are used to drive reactions forward and accelerate the rate of
reactions.
• How are the excess reagents and by-products from the reaction, which will interfere with
subsequent coupling steps, removed without a purification step?
5.4: Solid-Phase Peptide Synthesis: The Merrifield Method.
Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer
support. Purification is conveniently accomplished after each step by a simple wash and
filtration.
The solid support (Merrifield resin): polystyrene polymer

Solid-phase peptide synthesis

Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNA
Solid-phase synthesis of RNase A:
Synthetic RNase A: 78 % activity
0.4 mg was synthesized
2.9 % overall yield
average yield ~ 97% per coupling step

R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:

“for his development of methodology for chemical synthesis on a solid matrix.”

6.0: Secondary Structures of Peptides and Proteins.

-sheet: Two or more extended peptide chain, in which the amide backbones are associated by
hydrogen bonded
6.1: Tertiary Structure of polypeptides and Proteins.
Fibrous. Polypeptides strands that “bundle” to form elongated fibrous assemblies; insoluble.
Globular. Proteins that fold into a “spherical” conformation.
Hydrophobic effect. Proteins will fold so that hydrophobic amino acids are on the inside
(shielded from water) and hydrophilic amino acids are on the outside (exposed to water)

Enzymes: proteins that catalyze biochemical reactions.

• by bringing the reactive atoms together in the optimal geometry for the reaction.
• lowering the activation energy (G‡) by stabilizing the transition state and/or high energy
intermediate.
• many enzymes use the functional groups of the amino acid side chain to carry out the reactions
Proteases (peptidases): catalyzes the hydrolysis of peptide bonds

Four classes of proteases:

Serine (trypsin): aspartate-histidine-serine
Aspartyl (HIV protease, renin): two aspartates
Cysteine (papain, caspase): histidine-cysteine
Metallo (Zn2+) (carboxypeptidase, ACE): glutamate

Mechanism of carboxylpeptidase, metalloprotease

Mechanism of a serine protease (trypsin, chymotrypsin):

6.3: Coenzymes. Some reactions require additional organic molecules or metal ions. These
are referred to as cofactors or coenzymes.
6.4: Protein Quaternary Structure: Hemoglobin. (read)
6.5: G-Coupled Protein Receptors. (read)
Assignment:
1. Explain seven protein functions (class, example and function)
2. Explain the amphoteric nature of proteins
3. Explain the functional role of a protein in its isoelectric state
4. Explain the role of electrophoresis in protein Chemistry
5. Explain transamination