Third year organic chemistry

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 α
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
(H3C)2 HC

H

H

C

COOH
CH9 (CH3)2

(R) -Enantiomer

(S) -Enantiomer

Of the 20 aa, only proline is not a primary aa
O
R

O
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

OH

NH3
ammonium Form

R

C

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


CO2
(CH2)2
H3 N

CH

NH3

pH 7

CO2

Glu

(CH2)2
H3 N

CH

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

H2N
R

NH3

HCN

H

CN

C

R

α-aminonitrile
H3O+, H2O

Heat

H3N
H

COO
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

Pairs of Diastereomers
Chiral ion

N
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

H

+

COO
H
NHAc

R

R
Hog-kidney
acylase

COO
H3N

H

COO
H

R

NHAc
R

Free L-enantiomer
easily separated


Test for Amino Acids - Ninhydrin
O

O
O

H

- H2O
O

O

H

H2O

O

O
Indan-1,2,3-trione

Ninhydrin
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'

C
O

N

R'
R

C

N

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
H 3N

CH

H3N

C

O

C

H3 N

C

O

O

O

Serine

Alanine

Valine

- 2 H 2O
CH3

O
H
N

H3 N

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

Cysteine residues create
Disulfide Bridges
between chains

1. RSH

2. 6 M HCl hydrolysis

Ph
(CH2)4NH2

O

H
N
H2 N

Lys + 2 Cys
+ 2 Phe + Ser

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
RS H

Oxidation

RS SR

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

α-helix

β-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 α-helix
strands are wound into a “superhelix”. The superhelix makes one
complete turn for each 35 turns of the α-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 α and β globins.
The α and β chains are very similar but distinguishable in both
primary structure and folding



R

C

O

H

O
+
OH

carboxylic acid

N

H

C

NH4
R
O
ammonium carboxylate salt
(solid)

H
ammonia
O

OH

H2 N

OH

Gly O

NH2 Leu
Activate the Acid
O
X

NH2

O

NH2

N
H

OH
O

Dipeptide - LeuGly


O
O
2 X H2 N

R
X
If X= F, Cl, Br, I

R

Unprotected Coupling Three Competing Nucleophiles
O

NH
HN

R
O

Diketopiperazine

X
NH2

O
X
NH2
OH

H2 N
O

OH

H2N
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

N

N

H

H

O

O
OR
H
N

H
C

N

O
Dicyclohexylurea (DCU)


Protecting Groups
CH3

Protecting NH2

O

H3N
O

O
O

PROTECT

O

CH3
H 3C

C

O

C

CH3

O

O

(Boc)2O
Di-tert-butyl dicarbonate (Boc-anhydride)
CH3

CH3

tBoc

OH

N

=

H

Leu

O

H

O
PEPTIDE SYNTHESIS

De-PROTECT
mild acid and neutralize
CH3

H
N

H 3N
O

COO

OH

N
O


Protecting NH2
O

CH3

Cbz-Cl
CH3

Ph

O

O

H
CH3

O

H3 N

Ph
O

O

O

N

Cl

Benzyl Chloroformate

Cbz

O
O

N
H

O

H2, PtO2
De-Protect

CH3

O
O

H3 N

Fmoc-Cl

O

O

CH3

Base

O
O

Cl

=

Fmoc-Cl

N
H

O
O