Amino Acids, Peptides and Proteins

Convener : Dr. Fawaz Aldabbagh

Third Year Organic Chemistry

CO-303 Natural Product Chemistry

Primary, Secondary, Tertiary and Quaternary structures of Proteins.Isoelectric Point. Prosthetic Group. Investigation of amino acid structure of a protein. Peptide Synthesis

CHO

H OH

CH2OH

CHO

HO H

CH2OH

D - L -

=

R

C CO2HH

H2N

(L) - Amino Acids(-) -

=

CHO

CH2OHH

HO

(S) - Glyceraldehyde(-) -

R

CHH2N CO2H

All DNA encoded aa are

All are chiral, except GlycineR = HAll DNA

encoded aa are usually L-

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

NH2

C

(H3C)2HC

H

NH2

C COOHHCH9(CH3)2

(S) -Enantiomer (R) -Enantiomer

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

HOOC

O

OHR

NH2

O

OH

NH

Proline (Secondary aa)

aa are high melting point solids! Why?

Answer = aa are ionic compounds under normal conditions

C

O

OHR

NH3

C

O

OR

NH3

C

O

OR

NH2

LOW pH

Zwitterion

NEUTRAL

Carboxylate Form

HIGH pH

ammonium 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

(CH2)2

CHCO2H3N

CO2

Glu

(CH2)2

CHCO2H3N

NH3

Lys

pH 7

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

CH R

CH R

H2N CN

CH R

H3N COO

NH3 HCN

-aminonitrile

H3O+, H2OHeat

Preparation of Optically active Amino Acids- (Asymmetric Synthesis)

Prepare the target aa in racemic form, and separate theenantiomers afterwards

Resolution

Pairs of Enatiomers Pairs of Diastereomers

One salt preferentially crystallizes out

Chiral ion

1. Crystallisation with a chiral Counter-ion

N

O

O

N

H

H

H

H

H

Strechnine

COO

H3N H

R

COO

H NH3

R O

H3C O

O

CH3= Ac2O

Ac2O Ac2O

COOH

AcHN H

R

COOH

H NHAc

R

R NH2*

COO

AcHN H

R

R NH2*

R*-NH3COO

H NHAc

R

R*-NH3

Diastereomeric ammonium salts

Enantiomers

separation

COO

H3N H

R

COO

H NH3

R

NaOH, H2O

L (S)- D (S)-

2. Form Diastereotopic Peptides3. Chiral HPLC4. Enzyme Resolution

Form the N-ethanoyl (acetyl) protected aa then treat with an Form the N-ethanoyl (acetyl) protected aa then treat with an acylase enzyme.acylase enzyme.

COO

HNAc H

R

+

COO

H NHAc

R

Hog-kidneyacylase

COO

H3N H

R

Free L-enantiomer

COO

H NHAc

R

easily separated

Test for Amino Acids - Ninhydrin

O

O

O

O

H

H

Indan-1,2,3-trione

- H2OO

O

O

Ninhydrin

H2O

C

O

O

N C

O

O

Positive Test

aa are covalently linked by amide bonds (Peptide Bonds)

The resulting molecules are called Peptides & Proteins

NC R

R'

O

NC R

R'

O

Features of a Peptide Bond;1. Usually inert2. Planar to allow delocalisation3. Restricted Rotation about the amide bond4. 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

Strong Acid Required to hydrolyse peptide bonds

CH3

H3N CO

O

CH

H3N CO

O

CH2OH

H3N CO

O

Alanine SerineValine

CH3

H3N C

HN

O CH2OH

CNH

O

CO

O

Tripeptide : Ala . Ser. Val

- 2 H2O

(CH2)4NH2

H2N C

HN

O

S

CNH

O

C

Ph

OH

O

S

CNH

O

OHC

O

HN

HOO

H2N

Ph

Lys. Cys. Phe

Phe. Ser. Cys

1. RSH

2. 6 M HCl hydrolysis

Lys + 2 Cys + 2 Phe + Ser

Cysteine residues create Disulfide Bridges between chains

This does not reveal Primary Structure

RS H RS SROxidation

Reduction

REVERSIBLE DENATURING

Dr. Frederick Sanger, Nobel Prize for Chemistry1958 and 1980Peptide sequencing

                               

Prof. R. B. MerrifieldNobel Prize for Chemistry 1984Automated Peptide Synthesis

                               

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 propertiesBays or pockets in proteins are called Active SitesEnzymes are Stereospecific and possess Geometric Specificity

Emil Fischer formulated the lock-and-key mechanism for enzymes

The range of compounds that an enzyme excepts varies from a particular functional group to a

specific compound

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

CR OH

N H

H

H

+

O

CR O NH4

ammonium carboxylate salt(solid)

carboxylic acid ammonia

O

OH

NH2

O

X

NH2

Activate the Acid

Leu O

OHH2N

Gly

O

NH2

Dipeptide - LeuGly

NH

O

OH

O

X

R

H2NHN

NH

O

O

R

R

2 X

Diketopiperazine

If X= F, Cl, Br, I

O

X

NH2 O

OHH2N

O

X

NH2

O

OHH2N

Unprotected Coupling Three Competing Nucleophiles

Three Criteria for a Good Protecting Group?

What is the best way to activate the Carboxyl group?

CH3

N

H

Boct

H2N

O

OR+

N C N

Dicyclohexylcarbodiimide (DCC)

CH3

N

H

Boct

O

N

H O

OR

N C N

H H

O

Dicyclohexylurea (DCU)

O

OH

Protecting Groups

Protecting NH2

CH3

N

H

Boct

O

OH

=

CH3

N

H

C

O

OH

O

OCH3C

CH3

CH3

(Boc)2O

O

O O

O

O

Di-tert-butyl dicarbonate (Boc-anhydride)

CH3

H3N

O

O

PEPTIDE SYNTHESIS

PROTECT

De-PROTECT

mild acid and neutralize

CH3

H3N

O

N

H

COO

Leu

CH3

H3N

O

O

Protecting NH2

O

O ClPh

Benzyl Chloroformate

CH3

N

O

O

H

O

OPh

CH3

N

O

O

H

Cbz

Cbz-Cl

H2, PtO2

De-Protect

CH3

H3N

O

O Fmoc-Cl

Base

CH3

NH

O

OO

O

O

O

Cl Fmoc-Cl=

Protecting COO-

NH2

CH3CH2OH , H+

NH2

C

O

HO

O

EtO

acid or base hydrolysis

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

OR

NH2

O

HO

H

NH2

O

O

isobutene in sulfuric acid

H+, H2OHEAT

SN1 mechanism