The document discusses various methods for synthesizing amino acids, including the Strecker synthesis and Gabriel phthalimide synthesis. It then covers topics related to the properties of amino acids such as their dipolar nature, reactions that support this nature, and electrophoresis. The document concludes by discussing peptide bond formation and the four levels of protein structure: primary, secondary, tertiary, and quaternary.

1. Dr. Satish S. Kola
(Assistant professor )
Department of chemistry
M.G. Arts, Science and Late N.P. Commerce
College Armori
Amino Acids, Peptides
and Proteins
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2. Preparation of Amino Acids:
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3. 1. STRECKER SYNTHESIS
 The first known synthesis of an amino acid occurred in 1850 in the
laboratory of Adolph Strecker in Tübingen, Germany.
 Strecker added acetaldehyde to an aqueous solution of ammonia and
HCN. The product was propionitrile, which Strecker hydrolyzed to
alanine.
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4.  Step 1: The aldehyde reacts with ammonia to form the imine.
Step 2: Cyanide ion attacks the aldimine.
Step 3: hydrolysis of the nitrile to gives an α- amino acid
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5. 2. GABRIEL PHTHALIMIDE SYNTHESIS OF 𝛼 − AMINO ACID:
 The potassium salt of phthalimide is reacted with 𝛼 −haloester to produce the addition
product which on hydrolysis further gives the amino acid.
 For example, when potassium phthalimide reacts with chloroethyl acetate the product formed
yields phthalic acid and glycine along with the ethanol on hydrolysis.
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6. : DIPOLAR NATURE OF AMINO ACID
 Amino acids possess the dipolar nature. It has been found that
amino acid exist as a dipole, having one positive and another
negative end. That means it contain positive charge in one part and
negative charge in another part. The dipolar ionic structure of amino
acid can be shown as below.
 This is also called as Zwitter ion or internal salt. Now in this ion we
ca n see that there is no free amino or carboxylic group is present in
the molecule.
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7.  1] Spectroscopic studies of amino acids do not show bands characteristics
of -NH2 and - COOH groups.
 2] They have dipole moments indicating polar nature of the molecule.
 3] Amino acids are insoluble in nonpolar solvents and soluble in polar
solvents like water. This behaviour is expected from polar substances.
 4] Dissociation constants Ka and Kb give us an idea about the acid and
base strengths. Amino acids have very low values of Ka and Kb indicating
that the molecule does not possess these groups in the normal forms.
 5] Amino acids are nonvolatile crystalline solids, which melt at high
temperature. This is quite like ionic substances which have high melting
points and unlike amines and carboxylic acids which have low melting
points
How can we prove the dipolar nature of amino acid? What are the
evidences? Let us study the evidences in support of the dipolar nature of
amino acid
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8. ESTERIFICATION OF THE CARBOXYL GROUP
 Monofunctional carboxylic acids, amino acids are esterified by treatment with a
large excess of an alcohol and an acidic catalyst (often gaseous HCl).
 Under these acidic conditions, the amino group is present in its protonated form,
so it does not interfere with esterification
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9.  In presence of base when amino acid reacts with acid chloride or acid
anhydride, Acetylation takes place and acetyl derivatives are obtained.
ACETYLATION OF THE AMINO GROUP: FORMATION OF AMIDES
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10. REACTION WITH METAL: COMPLEXATION WITH CU2+ IONS
 Amino acid reacts with copper metal ion to produce the
coloured coordinate compound (complex)
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11. 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
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12. ISOELECTRIC POINT
 Amino acids are polar in nature and so they show electrical properties. When we
apply the electrical field to the solution of amino acids, they migrate to one or the
other electrode.
 Now how they migrate or when they will go to positive and when to negative end that
depend upon some factors. The factors are:
 1] On passing an electric current through the solution of the amino acid, if it moves
towards the cathode(-ve charge) then the solution is acidic and the equilibrium lies
towards positively charged amino acids (NH3 +CHR-COOH).
 2] On passing electricity, the amino acid molecule which is in the form of an anion, if
it moves towards the anode (+ve charge) then the solution is alkaline and the
equilibrium is lying towards the negatively charged amino acid (NH2CHR-COO- ).
 3] At a certain pH of the solution, the anionic and cationic structures will be in equal
concentration. On passing electricity, we shall observe that there is no movement of
the amino acid in such case. The pH at which a particular amino acid does not
migrate under the influence of the electrical field is called isoelectric point.
 For example, Glycine has an isoelectric point at pH 6.1.
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13. ELECTROPHORESIS OF AMINO ACID
 Electrophoresis uses differences in isoelectric points to separate mixtures of
amino acids.
 A streak of the amino acid mixture is placed in the center of a layer of acrylamide
gel or a piece of filter paper wet with a buffer solution.
 Two electrodes are placed in contact with the edges of the gel or paper, and a
potential of several thousand volts is applied across the electrodes.
 Positively charged (cationic) amino acids are attracted to the negative electrode
(the cathode), and negatively charged (anionic) amino acids are attracted to the
positive electrode (the anode).
 An amino acid at its isoelectric point has no net charge, so it does not move.
 Example:- consider a mixture of alanine, lysine, and aspartic acid in a buffer
solution at pH 6. Alanine is at its isoelectric point, in its dipolar zwitter ionic form
with a net charge of zero.
 A pH of 6 is more acidic than the isoelectric pH for lysine (9.7), so lysine is in the
cationic form. Aspartic acid has an isoelectric pH of 2.8, so it is in the anionic form
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14.  Structure at pH 6
 When a voltage is applied to a mixture of alanine, lysine, and aspartic acid at
pH 6, alanine does not move. Lysine moves toward the negatively charged
cathode, and aspartic acid moves toward the positively charged anode.
 After a period of time, the separated amino acids are recovered by cutting the
paper or scraping the bands out of the gel.
 If electrophoresis is being used as an analytical technique (to determine the
amino acids present in the mixture)
 The paper or gel is treated with a reagent such as ninhydrin to make the bands
visible. Then the amino acids are identified by comparing their positions with
those of standards.
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15. Electrophoresis of amino acid
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16. Electrophoresis of amino acid
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17. PEPTIDE BOND AND PROTEIN
 Proteins are known to break down into peptides in stomach and duodenum
under the influence of enzymes, pepsin being one of them which is secreted by
stomach.
 Polypeptides are further broken down to ∝-amino acids. This implies that
proteins are formed by connecting ∝-amino acids to each other. The bond that
connects ∝-amino acids to each other is called peptide bond
Two amino acid units are linked by a peptide bond 17
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18.  Combination of a third molecule of an ∝-amino acid with a dipeptide would
result in formation of a tripeptide. Similarly linking of four, five or six ∝-amino
acids results in formation of tetrapeptide, pentapeptide or hexapeptide
respectively.
 When the number of ∝-amino acids linked by peptide bonds is more than four,
the products are called polypeptides.
 The -CHR- units linked by peptide bonds are referred to as ‘amino acid
residues’. Proteins are polypeptides having more than hundred amino acid
residues linked by peptide bonds.
The two ends of a polypeptide chain of protein are not identical. The end having
free carboxyl group is called C-terminal
while the other end having free amino group is called N-terminal.
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19. Types of proteins :-
Globular proteins : Molecules of globular proteins have spherical shape. This
shape results from coiling around of the polypeptide chain of protein. Globular
proteins are usually soluble in water.
 For example : insulin, egg albumin, serum albumin, legumelin (protein in pulses).
 Fibrous proteins : Molecules of fibrous proteins have elongated, rod like
shape. This shape is the result of holding the polypeptide chains of protein
parallel to each other. Hydrogen bonds and disulfide bonds are responsible for
this shape. Fibrous proteins are insoluble in water.
 For example : keratin (present in hair, nail, wool), myosin (protein of muscles)
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20. CLASSIFICATION OF PROTEINS ON THE BASIS OF
HYDROLYSIS PRODUCT
Simple Proteins : - On hydrolysis yield only Amino Acids
egg albumin, tisuue Globuline, Wheat
Gluteline
Conjugated Proteins :-Proteineous and non-Proteineous
part ( Prosthetic part)
These are further classifies into : Nucleoproteins Prostehetic part
(Nucleic acids), Glycoproetins (Carbohydrate),and
Chromoproteins (Chorophyll) ,
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21. STRUCTURE OF PROTEINS :
Proteins are responsible for a variety of functions in organisms. Proteins
of hair, muscles, skin give shape to the structure, while enzymes are
proteins which catalyze physiological reactions. These diverse
functions of proteins can be understood by studying the four level
structure of proteins
1. Primary structure of proteins,
2. Secondary structure of proteins,
3. Tertiary structure of proteins
4. Quaternary structure of proteins.
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22. PRIMARY STRUCTURE OF PROTEINS
 Primary structure of proteins is the sequence of constituent ∝-amino acid
residues linked by peptide bonds.
 Any change in the sequence of amino acid residuce results in a different protein.
 Primary structure of proteins is represented by writing the three letter symbols of
amino acid residuces as per their sequence in the concerned protein.
 symbols are separated by dashes. According to the convention, the N-terminal
amino acid residue as written at the left end and the C-terminal amino acid
residue at the right end
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23. SECONDARY STRUCTURE OF PROTEINS
 The three-dimensional arrangement of localized regions of a protein
chain is called the secondary structure of protein. Hydrogen bonding
between N-H proton of one amide linkage and C=O oxygen of another
gives rise to the secondary structure.
 Two types of secondary structures commonly found in proteins are ∝-
helix and β-pleated sheet
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24.  ∝-Helix : The ∝-helix forms when a polypeptide chain twists into a right
handed or clockwise spiral Some characteristic features of ∝-helical structure of
protein are:
 Each turn of the helix has 3.6 amino acids.
 A C=O group of one amino acid is hydrogen bonded to N-H group of the fourth
amino acid along the chain.
 Hydrogen bonds are parallel to the axis of helix while R groups extend outward
from the helix core. Myosin in muscle and ∝-keratin in hair are proteins with
almost entire ∝-helical secondary structure.
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25.  β-Pleated sheet : The secondary structure is called β-pleated sheet when
two or more polypeptide chains, called strands, line up side-by-side. The β-
pleated sheet structure of protein consists of extended strands of polypeptide
chains held together by hydrogen bonding. The characteristics of β-pleated
sheet structure are :
 The C=O and N-H bonds lie in the planes of the sheet.
 Hydrogen bonding occurs between the N-H and C=O groups of nearby amino
acid residues in the neighbouring chains.
 The R groups are oriented above and below the plane of the sheet. The β-
pleated sheet arrangement is favoured by amino acids with small R groups.
 Most proteins have regions of ∝-helix and β-pleated sheet, in addition to other
random regions that cannot be characterised by either of these secondary
structures.
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26. TERTIARY STRUCTURE OF PROTEINS
 The three-dimensional shape adopted by the entire polypeptide chain of a
protein is called its tertiary structure.
 It is the result of folding of the chain in a particular manner that the structure is
itself stabilized and also has attractive interaction with the aqueous
environment of the cell.
 The globular and fibrous proteins represent two major molecular shapes
resulting from the tertiary structure.
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27. TERTIARY STRUCTURE OF PROTEINS
The forces that stabilize a particular tertiary structure include hydrogen
bonding, dipole-dipole attraction (due to polar bonds in the side chains),
electrostatic attraction (due to the ionic groups like -COO , NH3 ⊕ in the side
chain) and also London dispersion forces.
 Finally, disulfide bonds formed by oxidation of nearby -SH groups (in cysteine
residues) are the covalent bonds which stabilize the tertiary structure
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28. QUATERNARY STRUCTURE OF PROTEINS
 When two or more polypeptide chains with folded tertiary structures come
together into one protein complex, the resulting shape is called quaternary
structure of the protein.
 Each individual polypeptide chain is called a subunit of the overall protein.
 For example: Haemoglobin consists of four subunits called haeme held
together by intermolecular forces in a compact three dimensional shape.
 Haemoglobin can do its function of oxygen transport only when all the four
subunits are together. summerizes the four levels of protein structure
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30. DENATURATION OF PROTEINS
 High temperature, acid, base and even agitation can disrupt the
noncovalent interactions responsible for a specific shape of protein.
This is denaturation of protein. Denaturation is the process by which
the molecular shape of protein changes without breaking the
amide/peptide bonds that form the primary structre.
 Denaturation results in disturbing the secondary, tertiary or quaternary
structure of protein. This causes change in properties of protein and the
biological activity is often lost
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31. SYNTHESIS OF SIMPLE PEPTIDES
 Total synthesis of peptides is rarely an economical method for their
commercial production. Important peptides are usually derived from biological
sources.
 example, insulin for diabetics was originally taken from pork pancreas
 Now, recombinant DNA techniques have improved the quality and availability of
peptide pharmaceuticals. Amide formation is not so easy with amino acids,
however. Each amino acid has both an amino group and a carboxyl group.
 If we activate the carboxyl group, it reacts with its own amino group. some
amino acids have side chains that might interfere with peptide formation.
 example, glutamic acid has an extra carboxyl group, and lysine has an extra
amino group.
 As a result, peptide synthesis always involves both activating reagents to form
the correct peptide bonds and protecting groups to block formation of incorrect
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32.  Chemists have developed many ways of synthesizing peptides, falling into
two major groups.
 1. solution-phase method : - Involves adding reagents to solutions of growing
peptide chains and purifying the products as needed.
 2. Solid-phase method :- involves adding reagents to growing peptide chains
bonded to solid polymer particles.
 In 1962, Robert Bruce Merrifield of Rockefeller University developed a method
for synthesizing peptides without having to purify the intermediates. He did this
by attaching the growing peptide chains to solid polystyrene beads.
 After each amino acid is added, the excess reagents are washed away by
rinsing the beads with solvent.
 Merrifield built a machine that can add several amino acid units while running
unattended. Using this machine,
 Merrifield synthesized ribonuclease (124 amino acids) in just six weeks,
obtaining an overall yield of 17%.
 Merrifield’s work in solid-phase peptide synthesis won the Nobel Prize in
Chemistry in 1984.
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33.  The Solid Phase Peptide Synthesis is carried out
cyclically.
 The first step is attaching an amino acid to the
polymer;
 the second step is protection;
 the third step is coupling;
 the fourth step is deprotection ,
 the last step is polymer removal.
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34. SOLID-PHASE PEPTIDE SYNTHESIS
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35. PROTECTION OF AMINO GROUP :
 Using the tert-Butyloxycarbonyl (Boc) Protecting Group :- The N-protecting
group used in the Merrifield procedure is the tert-butyloxycarbonyl group,
abbreviated Boc or t-Boc.
 The Boc group is similar to the Z group, except that it has a tert-butyl group in
place of the benzyl group. Like other tert-butyl esters, the Boc protecting
group is easily removed under acidic conditions.
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36. PROTECTION OF AMINO GROUP :
 The Boc group is easily cleaved by brief treatment with trifluoroacetic acid
(TFA), CF3COOH.
 Loss of a relatively stable tert-butyl cation from the protonated ester gives an
unstable carbamic acid.
 Decarboxylation of the carbamic acid gives the deprotected amino group of
the amino acid.
 Loss of a proton from the tert-butyl cation gives isobutylene.
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37.  we will consider the synthesis of the same tripeptide we made using the
solution-phase method.
 Ala-Val-Phe
 The solid-phase synthesis is carried out in the direction opposite that of the
solution-phase synthesis.
 The first step is attachment of the N-protected C-terminal amino acid (Boc-
phenylalanine) to the polymer.
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38. Trifluoroacetic acid (TFA) cleaves the Boc protecting group of phenylalanine so
that its amino group can be coupled with the next amino acid. group with the
free ¬ NH2 group of phenylalanine.
The second amino acid (valine) is added in its N-protected Boc form so that it
cannot couple with itself. Addition of DCC couples the valine carboxyl
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39.  To couple the final amino acid (alanine), the chain isfirst deprotected by
treatment with trifluoroacetic acid. Then the N-protected Boc-alanine and
DCC are added.
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40.  If we were making a longer peptide, the addition of each subsequent amino
acid would require the repetition of two steps:
 1. Use trifluoroacetic acid to deprotect the amino group at the end ofthe
growing chain.
 2. Add the next Boc-amino acid, using DCC as a coupling agent. Once the
peptide is completed, the final Boc protecting group must be removed, and the
peptide must be cleaved from the polymer.
 Anhydrous HF cleaves the ester linkage that bonds the peptide to the polymer,
and it also removes the Boc protecting group. In our example, the following
reaction occurs:

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42. THANK YOU
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