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)
D and L isomers are mirror images of one another but the meso has internal mirror symmetry and therefore lacks optical activity.

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)
The Fischer scheme can be ambiguous so an absolute nomenclature system was developed in 1956 by Cahn, Ingold and Prelog.

4. Cahn-Ingold-Prelog or (RS) System
So L-Glyceraldehyde is S-glyceraldehyde by the RS system representation.
The chiral C atom is represented by the large circle and H atom is located behind the plane of the screen.

5. Cahn-Ingold-Prelog or (RS) System
The Fischer scheme can be ambiguous so an absolute nomenclature system wad developed in 1956 by Cahn, Ingold and Prelog.

6. Cahn-Ingold-Prelog or (RS) System
Newman projection diagrams of the stereoisomer of threonine and isoleucine derived from proteins.
Here the C alpha - C beta bond is viewed end on.

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
The Fischer scheme can be ambiguous so an absolute nomenclature system wad developed in 1956 by Cahn, Ingold and Prelog.

8. Prochiral centers
The two H atoms are prochiral. If assigned a and b then Hb is pro-R because in sighting from C1 towards Ha the order is clockwise whereas Hb is pro-S because substitutents decrease in a counterclockwise direction
Planar objects with no rotational symmetry are also prochiral.

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
A represents the re face and b is the si face.
We are mostly going to use the DL convention of naming but the RS system is necessary for describing prochirality and stereospecific reactions.

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
Amino acid residues in polypeptides are named by dropping of the suffix -ine and replacying it by -yl
The C- terminus is given the name of the parent amino acid.
So this compound is called alanyltyrosylaspartylglycine. We can replace this by Ala-Tyr-Asp-Gly or AYDG

11. Nomenclature
Nonhydrogen atoms of the amino acid side chain are named in sequence with the Greek alphabet
Greek letter used to identify the atoms in the glutamyl and lysyl R groups

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

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 R C
NH2 R C
NH2 +
Resonance forms

14. 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). R C O
NH2  

15. Amide character in the peptide bond
Since the peptide bond is also an amide it also undergoes resonance. H C N R1
H3N + O R2 O-
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. 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. C
-OOC H
CH2
H3N +
S-S
COO-
Cystine C
-OOC H
CH2
H3N +
Cysteine
ox. SH
red.

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

23. Cysteine reactions + + C -OOC H CH2 H3N + S-S COO- HS CH2-CH-CH-CH2 SH
Cystine HS +
CH2-CH-CH-CH2 SH OH OH
Dithiothreitol
Dithioerythritol
Cleland’s reagent C
-OOC H
CH2
H3N + SH
Cysteine
Doesn’t smell as bad as the b-mercaptoethanol. Prevents the formation of the disulfide bond in the presence of air. HO S 2 + HO S

24. Cysteine reactions + H ICH2COO- -OOC C CH2 SH H3N + C -OOC H CH2 H3N +
R-group
ICH2COO- +
-OOC C
CH2 SH
Iodoacetate
H3N +
Cysteine C
-OOC H
CH2
H3N + S
CH2COO- HI
N-ethylmaleimide
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. Sanger’s reagent - (fluorodintrobenzene) FDNBH C R1 O N R2 O- ..
O2N F + H N
NO2 H
FDNB HF
base
polypeptide
The reaction with FDNB is an aromatic nucleophillic substitution reaction.
The reaction with FDNB is an aromatic nucleophillic substitution reaction.
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. H C R1 O N R2 O- 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. Reaction with Dansyl Chloride
H3C H C R1 O N R2 O- .. Cl + H N O H
Dansyl Chloride
HCl
base
polypeptide S O N
H3C
This reaction is similar to Sanger’s reagent but dansyl amino acid is now fluroescent (intense yellow color). Dansyl chloride will also react with primary amines. This can be used to sequence picomole amounts of material.
Both of these methods are used prior to hydrolysis H C R1 O N R2 O- H N O

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