Contains preparation of peptide linkage, solid phase peptide synthesis etc

1. Peptides

2. Peptides
• Polyamides formed by the condensation of
amino group of one amino acid with the
carboxylic group of the other.
• They are in fact secondary amides having –CO-
NH- linkage(peptide linkage).
• The term polypeptide is reserved only for
those compounds having molar mass of
10,000u or less.

3. • The main structural feature is that they have a free
amino group on one end N-terminus and a free
carboxylic group on the other C-terminus.
• When the identities of the amino acids in a peptide are
known but their sequence is unknown, the amino acods
are written separated by commas.
glu,cys,his,val,ala
• When sequence is known, separated by hyphens-
glu-cys-his-val-ala
Glutamine is the N-terminus and alanine is the C-terminus.
• The amino acids are numbered starting with N-teminus.
• In naming them, adjective names (ending “yl”) are used
for all except the C-terminus. Thus the pentapeptide
named as glutamylcysteylhistylvalylalanyl

4. Peptide Linkage:
 The amide linkage that link amino acid residues are called
peptide peptide linkage.
 A peptide bond has ~40% double-bond character because of
electron delocalization. As a result, restricted free rotation
about the peptide bond.
 Steric hindrance causes the trans configuration to be more
stable than the cis one, so the α-carbons of adjacent amino
acids are trans to each other.
 The C & N atoms of the peptide bond and the two atoms to
which each is attached are held rigidly in a plane.

5.  This partial double bond character may be rationalized
on the basis of the following facts:
i. Resonance because of which there is restricted
rotation about amide bond, thus giving rise to cis,
trans isomerism.
ii. Amide group is flat and the carbonyl and amino
groups lie in one plane.
iii. Further the pioneering work of Linus Pauling on X-ray
crystallography of peptides revealed the C-N bond
length to be 132nm(shorter than usual C-N length
142nm showing some double bouble bond
character).

6. Determining the Primary Structure of
Protein
I. To reduce any disulfide bridges. A commonly used reducing agent
is 2-mercaptoethanol.
II. Determination of the no. and kinds of amino acids in the peptide
or protein. To do this, the sample is dissolved in 6N HCl and
heated at 373K for 24 hours. This treatment hydrolyzes all the
amide bonds in the protein, including the amide bonds of
asparagine and glutamine.
III. The mixture is then passed through an amino acid analyzer to
determine the number and kind f each amino acid in the peptide
or protein.
 In the acidic hydrolysis, tryptophan(a pyrrole derivative) gets
destroyed. The presence of this acid is determined by hydrolysis
with 2N alkali. This procedure however destroys arginine, cystine
etc and racimizes many others

7. Techniques in common use for quantitative
separation of amino acids:
i. Ion-exchange chromatography: The solution is
placed on the column of resin. By proton transfer,
amino acid is converted into ammonium ion and
the resin into sulphonate anions. The ammonium
ions of different acids are then adsorbed on the
surface of sulphonic acid resin with different
strength depending upon the relative acidity of the
acid form of the amino acids. Elution of the column
with a buffer results in separation of amino acids
which can be isolated and weighed.

8. ii. pH dependent precipitation: The difference in solubilities of
amino acids at pI has been used for their separation. In an
actual experiment, the pH of the peptide or protein
hydrolyzate is changed gradually which results in the
successive precipitation of amino cids at their respective pI.
iii. Electrophoresis: The technique is based on the principle of
migration of amino acids towards a particular electrode at a
different pH than pI. . A few drops of a solution of an amino acid
mixture are applied to the middle of a piece of filter paper.
When it is placed in a buffer between two electrodes and an
electric field is applied, an amino acid with a greater pI than the
pH of the solution will have an overall positive charge and will
migrate toward the cathode( negatively charged one). Farther
the difference between pI and pH, farther it will migrate.
A similar fashion is observed for those having pI lower than pH of
solution but in opposite direction.

9. Sequencing: End group analysis:
Edman’s reagent: One of the most widely used way to
identify N-terminus is to treat the peptide/protein with
pheny isocyanate(PITC) also called Edman’s reagent. It
reacts with the N-terminus and the resulting thiazoline
derivative cleaved under mildly acidic conditions. The
derivative is extracted with organic solvent and in the
presence of acid, reaarranges to give more stable
phenylthiohydantoin(PTH).
Each amino acid forms a different PTH which
can be identified by chromatography using known stds.

11. Sanger’s Reagent: 2,4-dinitrofluorobemzene(DNFB)
The reagent,because of strong electron withdrawing
nitro groups undergoes facile nucleophilic
displacement reaction with the terminal –NH2 group
giving rise to a tagged peptide. Hydrolysis of this,
with mineral acid yields a mixture containing 2,4-
dinitrophenyk substituted amino acid along with
other amino acids. The compound finally formed
being yellow in colour can be easily identified
spectroscopically.

13. Dansyl chloride: 5-dimethlamino-1-
napthalenesulphonyl chloride
The free amino group is converted into the corresponding
sulphonamide. These derivatives are better than the previous
ones as they are resistant to the action of acids as compared
to the corresponding dinitrophenyl substituted amino acids
and can be analyzed spectroscopically even at lower conc.

14. • The C-terminal amino acid can be identified by
treating the protein with carboxypeptidaseA
which cleaves off the C-terminus unless its
arginine or lysine. Carboxypeptidase B, on the
other hand, cleaves it off only if its arginine or
lysine.
• Carboxypeptodases are exopeptidases( an
enzyme that catalyzes the hydrolysis of a
peptide bond at the end of a peptide chain.

15. Partial hydrolysis:
Once the N- and C- terminus amino acids have
been identified, a sample is hydrolyzed with
dil.acid. This treatment, called so, hydrolyzes
only some of the peptide bonds. The resulting
fragments are saperated, and the amino acid
composition of each is determined. The
sequence of the original protein can then be
determined by lining up the peptides.

16. • Enzymatic partial hydrolysis: Endopeptidase is
an enzyme which catalysis the hydrolysis of a peptide
bond i.e., not at the end.

17. Synthesis of peptide bond:
N-protecting and C-protecting
• The reagent most often used to protect the
amino group of an amino acid is di-tert-butyl
dicarbonate( t-BOC).

18. • Carboxylic acids are generally activated by being
converted into acyl chloride.
• The preferred method for activating the carboxyl group
of an N-protected amino acid is to convert it into an
imidate using dicyclohexylcarbodimide(DCC).
• DCC activates a carboxyl group by putting a good
leaving group on the carbonyl carbon.
• After the amino acid has its N-terminal group
protected and its C-terminal group activated, the
second amino acid is added to form the new peptide
bond. The C-O bond of the tetrahedral intermediate is
easily broken because the bonding electrons are
delocalized, forming dicyclohexylurea, a stable
diamide.

20. Synthesis of peptide bond:

21. • t-BOC is an ideal protecting group because it
can be removed by washing with
trifluoroacetic acid and methylene chloride,
the reagents that will’nt break any other
covalent bonds. The protecting group is
removed by an elimination reaction, forming
isobutylene and carbon dioxide. Because
these products are gases, the escape, driving
the reaction to completion.

22. Solid Phase peptide synthesis
• By using this technique, Merrifield and his
coworkers succeeded in synthesizing enzyme
ribonuclease(containing 124 amino acid residues)
in the time span of only six weeks. That’s why it is
also referred as protein making machine.
• Uses insoluble polymer supports such as
halomethyl derivatives of polystyrene in the form
of small beads made by treating polystyrene with
CH3OCH3X in presence of SnX4.

23. Solid Phase peptide synthesis

24. Advantages:
1. Larger molecules difficult to synthesize using
BOC, DCC but easily and efficiently prepared by
this.
2. After the new amino acid unit is added the
reagents and byproducts can be easily washed
away eith negligeble loss of the desired
productand the growing peptide is easily
available for next round of reactions
3. Automated process