PROTEINS

Amino acids, peptides,

and proteins
Pharmaceutical Chemistry 126
Lecture 3
Introduction
Peptides and proteins - polymers of
amino acids linked together by amide
bonds
Repeating units are called amino acid
residues
Introduction
dipeptide - two amino acid residues
oligopeptide - contains three to 10
residues
polypeptide
proteins - naturally occurring
polypeptides made up of 40 to 4000
amino acid residues
Introduction
Functions
Keratin – component of hair, horns,
hooves, feathers, fur
Collagen – component of bones, muscles,
and tendons
Snake venoms and plant toxins – protect
their owners from other species
Introduction
Blood-clotting proteins – protect the vascular
system when it is injured
Antibodies and protein antibiotics – protect us
from disease
Enzymes – catalyze chemical reactions that
occur in living systems
Hormones
Physiological functions – transport and storage
of oxygen in the body and contraction of
muscles
Outline
Amino acids
1. Classification and nomenclature
2. Configuration
3. Acid-base properties
4. Separation techniques
 Amino acids
α-amino acids

Other functional group

R
O

Amino Group H3N+ Cα C Carboxyl group

O-
H
Standard Amino Acids
Essential Non-Essential

Arginine Arg R Alanine Ala A

Histidine His H Asparagine Asn N
Isoleucine Ile I Aspartate Asp D
Leucine Leu L Cysteine Cys C
Lysine Lys K Glutamate Glu E
Methionine Met M Glutamine Gln Q
Phenylalanine Phe F Glycine Gly G
Threonine Thr T Proline Pro P
Tryptophan Trp W Serine Ser S
Valine Val V Tyrosine Tyr Y
Classification
Classification Based on R-Groups
1. Nonpolar (hydrophobic)
2. Polar
a. Uncharged
b. Charged
1) negatively charged (acidic)
2) positively charged (basic)
 Hydrophobic Amino Acids
- -
O O CH3 - -
CH3
O O
CH3 CH3
O CH3
O O O CH3
+ +
NH3 NH3 + +
NH3 CH3 NH3
Alanine(A, Ala) Isoleucine (I, Ile) Leucine (L, Leu) Valine(V, Val)

- -
O - O
O O
H
S
O CH3 O O N
+ + OH
+ NH3
NH3 NH3 N
H

Methionine(M, Met) Phenylalanine(F,Phe) Tryptophan(W, Trp) Proline(P, Pro)

 Polar Uncharged
- -
- CH3 O
O O

O OH O OH O
+ +
+ NH3
NH3 NH3 OH

Serine (S, Ser) Threonine (T, Thr) Tyrosine (Y, Tyr)

- - -
O O NH2 O -
O
NH2 +
O NH3
O O O SH O
+ + +
NH3 O NH3 NH3 H

Asapragine(N, Asp) Glutamine(Q, Gln) Cysteine(C, Cys) Glycine(G, Gly)

Positively Charged
-
- NH2 O
O +
NH3
+ O
O NH NH2 +
+ NH3
NH3

Arginine (R, Arg) Lysine (K, Lys)

-
O H
O N
+
NH3 +
N
H

Histidine (His, H)
Negatively Charged

- -
- O
O O
-
O
O O O
+ +
NH3 O NH3

Aspartic Acid (D, Asp) Glutamic Acid (E, Glu)

Configuration
Optical activity
All AAs are optically active EXCEPT glycine
All AAs found in nature have L- configuration
All L-amino acids EXCEPT L-cysteine has S
configuration
L-alanine D-alanine
O
O
OH OH
H NH2
H2N H
CH3 CH3

S R
Priorities
NH2 > COOH> Alkyl> H
Optical activity
L-cysteine

O
OH
H2N H
SH

Priorities
NH2 > COOH> Alkyl> H
Acid-base properties
Isoelectric point
Isoelectric point
Separation techniques
1. Electrophoresis
Separation techniques
2. Paper and thin-layer chromatography
 Separation of Amino Acids
Electrophoresis Chromatography
+ anode
pI = 2.98 Least polar

pI = 6.02

- -
-O-
O
- pI = 10.76 OO O -
Most polar
O NH2
-
- C
CHH
O33
CH
O 3 + O
OO O NH NH2 OO
+
+ + + + +N H 3
NH NH NH
NH O3
3 3 CH
NH
3 3 O 3

O
O - cathode stationary phase: more polar
O O
OH
O H2O
N
mobile phase: less polar
+ H NH2
∆
O R O O
Separation techniques
3. Ion-exchange chromatography

 Cation-exchange resin
Separation techniques
Separation techniques
Synthesis of amino acids
Hell-Volhard-Zellinski reaction
Synthesis of amino acids
Show how you could prepare the
following α-amino acids from the
appropriate carboxylic acids:
1. Phenylalanine
2. Valine
Synthesis of amino acids
α-amino acids can be prepared by treating an
aldehyde with ammonia and hydrogen
cyanide, followed by acid-catalyzed hydrolysis
1. Give the structures of the two intermediates
formed in this reaction.
2. What amino acid is formed when the
aldehyde that is used is 3-methylbutanal?
3. What aldehyde would be needed to prepare
valine?
Synthesis of amino acids
Amidomalonate synthesis
Synthesis of amino acids
What alkyl halides would you use to
prepare the following α-amino acids by
the amidomalonate method?
1. Leucine
2. Histidine
3. Tryptophan
4. Methionine
Synthesis of amino acids
Reductive amination of α-keto acids
Resolution of racemic mixtures
Kinetic resolution
Outline
Peptides
1. Peptide and disulfide bonds
2. Peptide synthesis
Peptide bonds
Peptide bonds
Peptide bonds
 Peptide bonds
1. Name the following peptides:
HO O
NH2 O
O
O
H2N O NH
NH NH
NH O
OH NH
CH3 CH3

HO H3C

2. Draw the following peptides:

P-H-A-R-M
C-H-E-M
Peptide bonds
Peptide bonds
Disulfide bonds
Disulfide bonds
Disulfide bonds
Peptide bond synthesis
Outline
Proteins
1. Protein structure
2. Protein denaturation
Protein structure
Levels of protein structure:
1. Primary structure
◦ Sequence of amino acids in the chain and
location of all disulfide bridges
2. Secondary structure
◦ Describes regular conformation assumed by
segments of the protein’s backbone
◦ Describes how local regions of the backbone
fold
Protein structure
Levels of protein structure:

3. Tertiary structure
◦ Describes 3D structure of the entire
polypeptide
4. Quaternary structure
◦ Applicable if a protein has more than one
polypeptide chain
◦ Describes the way the individual protein
chains are arranged with respect to each
other
Protein structure
Fibrous Globular
Determination of primary structure
1. Reduce any disulfide bridge present
◦ 2-mercaptoethanol
◦ Iodoacetic acid
Determination of primary structure
2. Determine the number and kinds of
amino acids present in the peptide or
protein

 Hydrolyzes all amide bonds including

those of asparagine and glutamine
Determination of primary structure
Mixture of amino acids is then passed
through an amino acid analyzer
# of Asp residues = # of Asp + Asn
residues
# of Glu residues = # of Glu + Gln residues
Acid hydrolysis destroys Trp residues; Trp
content can be determined by basic
hydrolysis
Determination of primary structure
3. Determine N-terminal residue
 Phenyl isothiocyanate (PITC) or Edman’s
reagent
 N-terminus + PITC = thiazolinone
derivative
 Thiazolinone derivative is cleaved from
protein, extracted into organic solvent,
rearranges to a phenylthiohydantoin
Determination of primary structure
The particular PTH formed can be
identified by chromatography using
known standards
Sequenator – automated instrument
which allows 50 successive Edman
degradations to be carried out
Determination of primary structure
4. Determine C-terminal residue
◦ Carboxypeptidase A
 Cleaves off C-terminal amino acid as long as it is
not Arg or Lys
◦ Carboxypeptidase B
 Cleaves off C-terminal amino acid only if it is Arg
or Lys
Determination of primary structure
5. Hydrolyze protein with dilute acid
◦ Partial hydrolysis
◦ Fragments are separated, amino acid
composition of each is determined
◦ N-terminal and C-terminal amino acids of
each fragment is identified
◦ Entire sequence  line up peptides and look
for points of overlap
Determination of primary structure
A nonapeptide a. Pro, Ser
undergoes partial b. Gly, Glu
hydrolysis to give
c. Met, Ala, Leu
peptides whose amino
acid compositions are d. Gly, Ala
shown. Reaction of the e. Glu, Ser, Val, Pro
intact nonapeptide with f. Glu, Pro, Gly
Edman’s reagent g. Met, Leu
releases PTH-Leu. What
h. His, Val
is the sequence of the
nonapeptide?
Determination of primary structure
A decapeptide a. Ala, Trp
undergoes partial b. Val, Pro, Asp
hydrolysis to give
c. Pro, Val
peptides whose amino
acid compositions are d. Ala, Glu
shown. Reaction of the e. Trp, Ala, Arg
intact decapeptide with f. Arg, Gly
Edman’s reagent g. Glu, Ala, Leu
releases PTH-Gly. What
h. Met, Pro, Leu, Glu
is the sequence of the
decapeptide?
Determination of primary structure
Determination of primary structure
Trypsin
Determination of primary structure
Trypsin – cleaves at C-side of only
arginine or lysine residues

Chymotrypsin – cleaves at C-side of

amino acids that contain aromatic six-
membered rings (Phe, Tyr, Trp)
Determination of primary structure
Elastase – cleaves at C-side of small
amino acids (Gly, Ala)

None of the mentioned will catalyze the

hydrolysis of an amide bond if proline is
at the hydrolysis site
Determination of primary structure
Cyanogen bromide
◦ Causes hydrolysis of amide bond on the C-side
of a methionine residue
◦ Will still cleave the peptide bond if proline is
at the cleavage site
Determination of primary structure
6. Determinine location of any disulfide
bonds
 Hydrolysis of protein with intact
disulfide bonds
 Determine amino acids in Cys-containing
fragments
Determination of primary structure
Determination of primary structure
Secondary structure of proteins
Describes conformation of segments of
the backbone chain of a peptide or
protein
α-helix
β-pleated sheet
 α-helix

Secondary structure: α-helix

 α-helix

Secondary structure: α-helix

Secondary structure of proteins
α-helix
◦ Backbone of the polypeptide coils around the
long axis of the protein molecule
◦ Stabilized by H bonds
◦ Each hydrogen attached to an amide nitrogen
is H-bonded to carbonyl oxygen of an amino
acid four residues away
◦ R groups protrude outward from the helix,
minimizing steric hindrance
Secondary structure of proteins
α-helix
◦ Right-handed helix
 Rotates in a clockwise direction as it spirals down
 Because of L-amino acids
◦ Each turn of the helix contains 3.6 amino acid
residues
◦ Pro residue forces a bend in the helix
 Bond between Pro nitrogen and α-carbon cannot
rotate
 α-helix

Secondary structure: β-pleated sheet

 α-helix

Secondary structure: β-pleated sheet

 α-helix

Secondary structure: β-pleated sheet

 α-helix

Secondary structure: β-pleated sheet

Secondary structure of proteins
β-pleated sheet
◦ polypeptide backbone is extended in a zigzag
structure resembling a series of pleats
◦ almost fully extended
◦ H-bonding occurs between neighboring
peptide chains
◦ adjacent hydrogen-bonded peptide chains can
run in the same direction (parallel) or in
opposite (antiparallel) directions
◦ R groups – close to each other; small
Tertiary
structur
e of
protein
s
Tertiary
structur
e of
protein
s
Tertiary structure of proteins
Overall 3D shape of a protein
Forces that stabilize structure:
1. Covalent bonds (disulfide links)
2. Ionic bonds
3. Hydrogen bonds
4. Van der Waals or hydrophobic
interactions
Tertiary structure of proteins
Covalent bond
Tertiary structure of proteins
Ionic bond
Tertiary structure of proteins
Hydrogen bond
Tertiary structure of proteins
Van der Waals interaction
Tertiary
structur
e of
protein
s
Tertiary structure of proteins
Determination of structure
◦ X-ray crystallography
◦ Magnetic Resonance Imaging
◦ 2-D NMR Spectroscopy
Quaternary structure of proteins
Oligomers – proteins that have more
than one peptide chain
Subunits – individual chains
Monomer – protein with a single subunit
Dimer – two subunits
Trimer – three subunits
Tetramer – four subunits

ex. Hemoglobin
Quaternary structure of proteins
Subunits are held together by hydrophobic
interactions, H-bonding, and ionic attractions

Quaternary structure describes the way

subunits are arranged in space
Quaternary structure of proteins
Possible arrangements of the six subunits
of a hexamer:
Protein denaturation
Destruction of tertiary structure
Bonds responsible for maintaining the 3D
shape of the protein are broken
Random coil
Protein denaturation
Change in pH: alters charges on side chains;
disrupts electrostatic attractions and H-bonds
Urea and guanidine HCl: forms H-bonds to protein
groups that are stronger than the H-bonds formed
between the groups
Detergents (sodium dodecyl sulfate): associates
with nonpolar groups of the protein; interferes with
normal hydrophobic interactions
Organic solvents: disrupts hydrophobic interactions
Heat and agitation: Increase molecular motion;
disrupts attractive forces
Applications to the pharmaceutical
sciences
Proteins as Drug Targets
Enzymes
Receptors
Carrier Proteins
Structural Proteins

Proteins or Peptides as Drugs

Polypeptide hormones
Immunoglobulins and antigens
Polypeptide antibiotics
Natural toxins
End of lecture 