Lecture 4: Amino Acids

1. Lecture 4: Amino Acids • For the quiz on Wed. (9/7)-NH3+ ~ 9.0, -COO- ~ 2.0, you must know pKs of side chain groups! • Introduction to amino acid structure (continued) • Amino acid chemistry

2. Diastereomers • Special case: 2 asymmetric centers are chemically identical (2 asymmetric centers are mirror images of one another) • A molecule that is superimposable on its mirror image is optically inactive (meso form)

3. Cahn-Ingold-Prelog or (RS) System • The 4 groups surrounding a chiral center a ranked as follows: Atoms of higher atomic number bonded to a chiral center are ranked above those of lower atomic number. • Priorities of some common functional groups SH > OH > NH2 > COOH > CHO > CH2OH > C6H5 > CH3 > 2H > 1H • Prioritized groups are assigned letters W, X, Y, Z, so that W > X > Y > Z • Z group has the lowest priority (usually H) and is used to establish the chiral center. • If the order of the groups W X Y is clockwise, as viewed from the direction of Z, the configuration is (R from the latin rectus, right) • If the order of the groups W X Y is counterclockwise, as viewed from the direction of Z, the configuration is (S from the latin sinister, left)

4. Cahn-Ingold-Prelog or (RS) System

5. Cahn-Ingold-Prelog or (RS) System

6. Cahn-Ingold-Prelog or (RS) System

7. Prochiral centers have distinguishable substituents • Prochiral molecules can be converted from an achiral to chrial molecule by a single substitution • Molecules can be assigned a right side and left side for two chemically identical substituents. • True for tetrahedral centered molecules • Example is ethanol

8. Prochiral centers

9. Planar objects can also be prochiral • Stereospecific additions in enzymatic reactions • If a trigonal carbon is facing the viewer so that the substituents decrease in a clockwise manner it is the re face • If a trigonal carbon is facing the viewer so that the substituents decrease in a counterclockwise manner it is the si face • Acetaldehyde example

10. Nomenclature • Glx can be Glu or Gln • Asx can be Asp or Asn • Polypeptide chains are always described from the N-terminus to the C-terminus

11. Nomenclature • Nonhydrogen atoms of the amino acid side chain are named in sequence with the Greek alphabet

12. + + O R2 O R1 H H3N H3N O- C C OH C C H N H H O R2 O R1 O- C C C N C H H H + H2O Peptide bonds • Proteins are sometimes called polypeptides since they contain many peptide bonds +

13. Structural character of amide groups • Understanding the chemical character of the amide is important since the peptide bond is an amide bond. • These characteristics are true for the amide containing amino acids as well (Asn, Gln) • Amides will not ionize but will undergo resonance - O O NH2 R C NH2 R C + Resonance forms

14.  O  NH2 R C Amide has partial charge & double bond • We can also look at the partial charge and double bond of an amide as shown below. • Since the free electrons of the N atom are tied up in forming the partial double bond, the N atom can not accept a proton (H+). • This N also has a partial positive charge which will repel protons and prevent them from binding to the nitrogen (thus no ionization).

15. + H3N O R2 O R1 O- C C C N C H H H Amide character in the peptide bond • Since the peptide bond is also an amide it also undergoes resonance. • Therefore, peptides are rigid due to resonance around the amide bond, having ≈ 40% double-bond character. • This restricts the rotation due to delocalization of electrons and overlap of the O-C-N  orbitals.

16. Amide character in the peptide bond • The double bond character results in a planar form around the peptide bond.

17. Structural hierarchy in proteins • Primary structure (1º structure)-for a protein is the amino acid sequence of its polypeptide chain(s). • Secondary structure (2º structure)-the local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformations of their side chains. • Tertiary structure (3º structure)-refers to the 3-dimensional structure of an entire polypeptide (close to secondary structure). • Quaternary structure (4º structure)-The spatial arrangement of a protein’s subunits • Most protein is made up of two or more polypeptide chains (subunits) associated through noncovalent interactions.

18. Structural hierarchy in proteins

19. Primary structure (1º structure) of proteins • Primary structure (1º structure)-for a protein is the amino acid sequence of its polypeptide chain(s). • Amino acid sequence of a protein determines • three-dimensional conformation. • Resulting functional specificity (molecular mechanism of action) • Sequence comparisons among analogous proteins are important in comparing how proteins function and have indicated evolutionary relationships among proteins • Amino acid sequence analyses have important clinical applications because many diseases are caused by mutations that lead to an amino acid change in a protein. • Therefore, amino acid sequence analysis is an important tool for research.

20. General approach for the analysis of the amino acid sequence of a protein • Purify protein to homogeneity • Break disulfide bonds • Determine the aa composition • Identify the N-terminal sequence • Identify the C-terminal sequence • Break the polypeptide into fragments by internal cleavage (Trypsin, chymotrypsin, pepsin, CNBr). • Determine the amino acid sequence of each fragment. • Repeat using different enzymes or CNBr. • Overlap and align fragments.

21. + + + H3N H3N H3N H COO- C H CH2 C CH2 -OOC H S-S C CH2 -OOC Cystine Cysteine Breaking disulfide bonds • Recall that cysteine (Cys-SH HS-Cys) can convert to cystine (Cys-S-S-Cys) in the presence of air (oxidation) and will convert back if reduced. • We can also prevent the formation of the disulfide bond by modifying the SH group of Cys. ox. SH red.

22. H + + + H3N H3N H3N SH C CH2 -OOC Cysteine COO- C H CH2 H S-S C CH2 -OOC S-CH2-CH2-OH S-CH2-CH2-OH Cystine Cysteine reactions 2 + OH CH2 CH2 HS -mercaptoethanol 2 +

23. H + + + H3N H3N H3N SH C CH2 -OOC Cysteine COO- C H CH2 H S-S C CH2 -OOC Cystine Cysteine reactions + SH HS CH2-CH-CH-CH2 OH OH Dithiothreitol Dithioerythritol Cleland’s reagent HO S + 2 S HO

24. + + H3N H3N H S C CH2 -OOC + HI CH2COO- Cysteine reactions H R-group + ICH2COO- SH C CH2 -OOC Iodoacetate Cysteine Carboxymethylcysteine

25. General approach for the analysis of the amino acid sequence of a protein • Purify protein to homogeneity • Break disulfide bonds • Determine the aa composition • Identify the N-terminal sequence • Identify the C-terminal sequence • Break the polypeptide into fragments by internal cleavage (Trypsin, chymotrypsin, pepsin, CNBr). • Determine the amino acid sequence of each fragment. • Repeat using different enzymes or CNBr. • Overlap and align fragments.

26. N-terminus identification • Sanger’s reagent - (fluorodintrobenzene) FDNB • Dansylation - (1-dimethyl-amino-naphthalene-5-sulfonyl chloride) Dansyl Chloride • Edman degradation • Invented by Pehr Edman • Phenylisothiocyanate (PITC, Edman’s Reagent)

27. O O O O R1 R1 R2 R2 O- O- N N C C C C C C C C H H H H H H Sanger’s reagent - (fluorodintrobenzene) FDNB .. O2N F + H N NO2 H FDNB HF base polypeptide The reaction with FDNB is an aromatic nucleophillic substitution reaction. H O2N N NO2 Sanger’s reagent will also react with other amino groups (epsilon amino group in-lysine). But only one alpha amino group will be labeled by this reagent. Aromatic amino groups are more stable than the peptide bond.

28. H3C H3C N N O O O O O O R1 R1 R2 R2 H3C H3C S S O- O- N N C C C C C C C C H H H H H H Reaction with Dansyl Chloride .. Cl + N H O H Dansyl Chloride HCl base polypeptide H N O

29. From this we know the N-terminal amino acid and the amino acid composition but not the sequence.