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

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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

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

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  

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.

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

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.

Structural hierarchy in proteins

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.

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.

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.

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

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

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

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.

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)

Sanger’s reagent - (fluorodintrobenzene) FDNB H 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.

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

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