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

All are chiral, except Glycine R=H All DNA encoded aa are usually L=
HO H CHO H OH CH2OH HO CHO H CH2OH

DCHO CH2OH

LR

H2N

C H

CO2H

(S) - Glyceraldehyde (-) -

(L) - Amino Acids (-) -

Draw tetrahedral 3D structures for (R) and (S) valine

NH 2 C HOOC (H 3C) 2HC H H NH 2 C COOH CH9(CH 3)2

(S) -Enantiomer

(R) -Enantiomer

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

O R OH NH2 N H OH O

Proline (Secondary aa)

aa are high melting point solids! Why?

Answer = aa are ionic compounds under normal conditions

LOW pH O R C NH 3 ammonium Form

NEUTRAL O R OH NH 3 Zwitterion C

HIGH pH O R C NH 2 Carboxylate Form

Isoelectric Point = concentration of zwitterion is at a

maximum and the concentration of cations and anions is equal

For 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 CH H3N CO2

NH3

pH 7
H3N

(CH2)2 CH CO2

Glu

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 R NH3 HCN

H2N H C

CN R

-aminonitrile Heat H3O+, H2O COO C R

H3N H

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 H H H N H O H O
One salt preferentially crystallizes out

Pairs of Diastereomers Chiral ion

Strechnine

COO H3N
L (S)- R Ac2O

COO H
Enantiomers

NH3 R
D (S)-

O H3C

O O CH3 = Ac2O

Ac2O

COOH AcHN R R* NH2 H H

COOH NHAc R
* R NH2

COO AcHN R H

R*-NH 3
Diastereomeric ammonium salts separation

COO H R
NaOH, H2O

NHAc R*-NH 3

COO H3N R H H

COO NH3 R

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 R Hog-kidney acylase H + COO H NHAc R

COO H3N H R
Free L-enantiomer

COO H R NHAc

easily separated

Test for Amino Acids - Ninhydrin

O O O O Ninhydrin O C O N C O O H H O

- H2O
O

H2O
O Indan-1,2,3-trione

Positive Test

aa are covalently linked by amide bonds (Peptide Bonds) The resulting molecules are called Peptides & Proteins
R' R C O N R

R' C 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

CH3 O H3 N C O Alanine H3 N

CH2OH O C O Serine - 2 H2 O H3 N

CH O C O Valine

CH3 H N H3 N C O

O C O N H C O

CH2OH

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

H2 N

(CH2)4NH2 H N C O S S O H N C N H O HO

O C OH N H C O

This does not reveal Primary Structure

Ph

OH C O

H2 N

REVERSIBLE DENATURING Oxidation RS H Reduction RS SR

Dr. Frederick Sanger, Prof. R. B. Merrifield Nobel Prize for Chemistry Nobel Prize for Chemistry 1984 1958 and 1980 Automated Peptide Synthesis Peptide sequencing

Prof. Linus Pauling

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

O C R OH +

H N H ammonia
O

O H NH4 R O ammonium carboxylate salt (solid)

OH OH NH2 Leu Activate the Acid O X NH2 O OH N H NH2 O Dipeptide - LeuGly H2N Gly O

carboxylic acid

O O
2 X H2 N

R NH X HN R O
Diketopiperazine

If X= F, Cl, Br, I

Unprotected Coupling Three Competing Nucleophiles O X O X NH2 OH H2N O NH2 OH H2 N O

Three Criteria for a Good Protecting Group?

What is the best way to activate the Carboxyl group?

CH3 t Boc N H O OH + H2 N O OR

Dicyclohexylcarbodiimide (DCC) CH3 t Boc N H O H N OR H N C O Dicyclohexylurea (DCU) H N O

Protecting Groups
CH3

Protecting NH2

O H3 N O O O O (Boc)2O Di-tert-butyl dicarbonate (Boc-anhydride) O CH3 H3 C C O C N H O PEPTIDE SYNTHESIS De-PROTECT mild acid and neutralize CH3 H3 N O H N COO CH3 OH = t Boc N H O CH3 OH O O

PROTECT

CH3 Leu

Protecting NH2
O Cbz-Cl CH3 O H3 N O Ph O Cl Cbz N H H2, PtO2 De-Protect O Benzyl Chloroformate O Ph O N H CH3 O O CH3 O

CH3 O H3 N O Base Fmoc-Cl O

O N H

CH3 O O

O O Cl = Fmoc-Cl

Protecting COOO HO NH2 acid or base hydrolysis

+

O C

CH3CH2OH , H

EtO NH2

Much Milder Conditions are required to Break an ester as compared to an amide bond.

OR isobutene in sulfuric acid O HO NH2 SN1 mechanism H+, H2O HEAT O H O NH2