Third year organic chemistry

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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 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 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 Form Diastereotopic Peptides Chiral HPLC 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 that are part of a peptide or protein are referred to as residues Peptides are made up of about 50 residues, and 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 - 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 RSH M HCl hydrolysis Ph (CH2)4NH2 O H N H2 N Lys + Cys + 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 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 Protecting COOacid or base hydrolysis O + CH3CH2OH , H HO EtO O C NH2 NH2 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 NH2 SN1 mechanism H+, H2O HEAT NH2 [...]... 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... 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... 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,
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