Transcript So, for basic R-groups, we require higher pHs, and for

Third Year Organic Chemistry
CO-303 Natural Product Chemistry
Amino Acids, Peptides and Proteins
Convener : Dr. Fawaz Aldabbagh
Primary, Secondary, Tertiary and Quaternary
structures of Proteins.
Isoelectric Point. Prosthetic Group. Investigation of
amino acid structure of a protein. Peptide Synthesis
R
H2N
CH
CO2H
All DNA encoded aa are 
CHO
All are chiral,
except Glycine
R=H
H
OH
CHO
HO
CH2OH
CH2OH
D-
All DNA
encoded aa
are usually L-
H
LR
CHO
=
HO
CH2OH
H
(S) - Glyceraldehyde
(-) -
=
H2N
C
CO2H
H
(L) - Amino Acids
(-) -
Draw tetrahedral 3D structures for (R) and (S) valine
NH2
NH2
C
HOOC
(H 3C) 2HC
H
H
C
COOH
CH9(CH 3)2
(R) -Enantiomer
(S) -Enantiomer
Of the 20 aa, only proline is not a primary aa
O
O
R
OH
NH2
OH
N
H
Proline (Secondary aa)
aa are high melting point solids! Why?
Answer = aa are ionic compounds under normal conditions
LOW pH
NEUTRAL
C
O
O
O
R
HIGH pH
R
C
OH
NH3
ammonium Form
R
C
O
NH3
Zwitterion
O
NH2
Carboxylate Form
Isoelectric Point = concentration of zwitterion is at a
maximum and the concentration of cations and anions is equal
r aa with basic R-groups, we require higher pHs, and
for aa with acidic R-groups, we require lower pHs
to reach the Isoelectric Point
NH 3
CO2
(CH2)2
pH 7
CH
CH
H3 N
(CH2)2
CO2
Glu
H3 N
CO2
Lys
Isoelectric Point is the pH at which an aa or
peptide carries no net charge.
i.e. [RCOO-] = [RNH3+]
So, for basic R-groups, we require higher pHs,
and for acidic R-groups we require lower pHs
e.g. Isoelectric point for gly pH = 6.0
Asp pH = 3.0
Lys pH = 9.8
Arg pH = 10.8
Preparation of Amino Acids
O
H
C
CN
H2N
R
NH3
HCN
H
C
R
-aminonitrile
H3O+, H2O
Heat
COO
H3N
H
C
R
Preparation of Optically active Amino Acids
- (Asymmetric Synthesis)
Resolution
Prepare the target aa in racemic form, and separate the
enantiomers afterwards
1. Crystallisation with a chiral Counter-ion
Pairs of Enatiomers
N
Pairs of Diastereomers
Chiral ion
H
One salt preferentially
crystallizes out
H
H
N H
O
O
H
Strechnine
COO
COO
H3N
H
H
NH3
R
Enantiomers
L (S)- R
Ac2O
Ac2O
COOH
AcHN
H
NHAc
R
R
R* NH2
AcHN
H
R
*
R NH2
R*-NH3
COO
NHAc R*-NH3
H
R
Diastereomeric
ammonium salts
NaOH, H2O
separation
COO
COO
H3N
H
R
H3C
COOH
H
COO
O
D (S)-
H
NH3
R
O
O
CH3
= Ac2O
2. Form Diastereotopic Peptides
3. Chiral HPLC
4. Enzyme Resolution
Form the N-ethanoyl (acetyl) protected aa then treat with an
acylase enzyme.
COO
HNAc
COO
H
+
H
R
NHAc
R
Hog-kidney
acylase
COO
H3N
H
R
COO
H
NHAc
R
Free L-enantiomer
easily separated
Test for Amino Acids - Ninhydrin
O
O
O
H
- H2O
O
O
H
H2O
O
O
Ninhydrin
Indan-1,2,3-trione
O
C
O
O
N
C
O
Positive Test
aa are covalently linked by amide bonds
(Peptide Bonds)
The resulting molecules are called
Peptides & Proteins
R'
R'
N
C
O
R
N
C
R
O
Features of a Peptide Bond;
1. Usually inert
2. Planar to allow delocalisation
3. Restricted Rotation about the amide bond
4. Rotation of Groups (R and R’) attached to the amide
bond is relatively free
aa that are part of a peptide or protein are referred
to as residues.
Peptides are made up of about 50 residues, and do not
possess a well-defined 3D-structure
Proteins are larger molecules that usually contain at least 50
residues, and sometimes 1000. The most important feature of
proteins is that they possess well-defined 3D-structure.
Primary Structure is the order (or sequence) of amino acid residues
Peptides are always written and named
with the amino terminus on the left and
the carboxy terminus on the right
CH2OH
CH3
O
O
H3N
CH
H3 N
C
O
C
H3N
C
O
O
O
Serine
Alanine
Valine
- 2 H2O
CH3
O
H
N
H3N
C
C
O
CH2OH
O
N
H
C
O
Tripeptide : Ala . Ser. Val
Strong Acid Required to hydrolyse peptide bonds
Lys. Cys. Phe
Phe. Ser. Cys
1. RSH
2. 6 M HCl hydrolysis
Lys + 2 Cys
+ 2 Phe + Ser
Ph
Cysteine residues create
Disulfide Bridges
between chains
(CH2)4NH2
O
H
N
H2 N
C
C
OH
N
H
O
O
This does not reveal
Primary Structure
S
S
Ph
O
H
N
H2 N
C
OH
N
H
O
C
O
HO
C
REVERSIBLE DENATURING
Oxidation
RS SR
RS H
Reduction
Prof. Linus Pauling
Dr. Frederick Sanger,
Prof. R. B. Merrifield
Nobel Prize for Chemistry Nobel Prize for Chemistry 1984
1958 and 1980
Automated Peptide Synthesis
Peptide sequencing
Secondary Structure
The Development of Regular patterns of Hydrogen
Bonding, which result in distinct folding patterns
-helix
-pleated sheets
Tertiary Structure
This is the 3D structure resulting from further regular
folding of the polypeptide chains using H-bonding, Van
der Waals, disulfide bonds and electrostatic forces –
Often detected by X-ray crystallographic methods
Globular Proteins – “Spherical Shape” , include Insulin,
Hemoglobin, Enzymes, Antibodies
---polar hydrophilic groups are aimed outwards towards water,
whereas non-polar “greasy” hydrophobic hydrocarbon portions
cluster inside the molecule, so protecting them from the hostile
aqueous environment ----- Soluble Proteins
Fibrous Proteins – “Long thin fibres” , include Hair,
wool, skin, nails – less folded ----- e.g. keratin - the -helix strands
are wound into a “superhelix”. The superhelix makes one
complete turn for each 35 turns of the -helix.
In globular proteins this tertiary structure or
macromolecular shape determines biological properties
Bays or pockets in proteins are called Active Sites
Enzymes are Stereospecific and possess Geometric Specificity
The range of compounds that an enzyme excepts varies
from a particular functional group to a specific compound
Emil Fischer formulated the lock-and-key mechanism for enzymes
All reactions which occur in living cells are mediated by enzymes and
are catalysed by 106-108
Some enzymes may require the presence of a Cofactor.
This may be a metal atom, which is essential for its redox activity.
Others may require the presence of an organic molecule, such as
NAD+, called a Coenzyme.
If the Cofactor is permanently bound to the enzyme, it is called a
Prosthetic Group.
For a protein composed of a single polypeptide molecule, tertiary
structure is the highest level of structure that is attained
Myoglobin and hemoglobin were the first proteins to be
successfully subjected to completely successful X-rays
analysis by J. C. Kendrew and Max Perutz (Nobel Prize for
Chemistry 1962)
Quaternary Structure
When multiple sub-units are held together in
aggregates by Van der Waals and electrostatic
forces (not covalent bonds)
Hemoglobin is tetrameric myglobin
For example, Hemoglobin has four heme units, the protein
globin surrounds the heme – Takes the shape of a giant
tetrahedron – Two identical  and  globins.
The  and  chains are very similar but distinguishable in both
primary structure and folding
+
C
R
O
H
O
OH
carboxylic acid
N
H
C
NH 4
R
O
ammonium carboxylate salt
(solid)
H
ammonia
O
OH
H2 N
OH
Gly O
NH 2 Leu
Activate the Acid
O
X
NH 2
O
OH
N
H
NH 2
O
Dipeptide - LeuGly
O
O
R
NH
2 X H2N
X
If X= F, Cl, Br, I
R
Unprotected Coupling Three Competing Nucleophiles
O
HN
R
O
Diketopiperazine
X
NH 2
O
X
NH 2
OH
H2 N
O
OH
H2 N
O
Three Criteria for a Good
Protecting Group?
What is the best way to activate the Carboxyl group?
CH3
tBoc
OH
N
H
+
OR
H2 N
O
O
N
C
N
Dicyclohexylcarbodiimide
(DCC)
CH3
t
Boc
H
O
N
N
H
OR
O
H
N
H
C
N
O
Dicyclohexylurea (DCU)
Protecting Groups
CH3
Protecting NH2
O
H3 N
O
O
O
PROTECT
O
CH3
H3 C
C
CH3
O
CH3
CH3
tBoc
OH
N
=
H
Leu
O
(Boc)2O
Di-tert-butyl dicarbonate (Boc-anhydride)
C
O
O
OH
N
H
O
PEPTIDE SYNTHESIS
De-PROTECT
mild acid and neutralize
CH3
H
N
H3 N
O
COO
O
Protecting NH2
O
CH3
Cbz-Cl
CH3
O
Ph
O
O
N
H
CH3
O
H3 N
Ph
O
O
Cl
Benzyl Chloroformate
O
O
Cbz
N
H
O
H2, PtO2
De-Protect
CH3
O
O
CH3
Fmoc-Cl
O
H3 N
O
O
N
H
O
Base
O
O
Cl
=
Fmoc-Cl
Protecting COOacid or base hydrolysis
O
+
CH3CH2OH , H
HO
O
C
EtO
NH 2
NH 2
Much Milder Conditions are required
to Break an ester as compared to an amide bond.
OR
isobutene in sulfuric acid
O
O
H
O
HO
NH 2
SN1 mechanism
H+, H2O
HEAT
NH 2