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)

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)

Cahn-Ingold-Prelog or (RS) System

Cahn-Ingold-Prelog or (RS) System

Cahn-Ingold-Prelog or (RS) System

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

Prochiral centers

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

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

Nomenclature • Nonhydrogen atoms of the amino acid side chain are named in

sequence with the Greek alphabet

Peptide bonds • Proteins are sometimes called polypeptides since they contain many peptide bonds

H

C

R1

H3N+

C

O

OH NH

H

C

R2

O-C

OH

+

H

C N

R1

H3N+

C

O

H H

C

R2

O-C

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

R C

O

NH2 R C

O

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+

C

O

H H

C

R2

O-C

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+

Cysteine

C-OOC

H

CH2

H3N+

S-S

C COO-

H

CH2

H3N+

Cystine

SHox.

red.

S-CH2-CH2-OH

Cysteine reactions

HS +-mercaptoethanol

S-CH2-CH2-OH

CH2 CH2 OH C-OOC

H

CH2

H3N+

S-S

C COO-

H

CH2

H3N+

Cystine

2

+C-OOC

H

CH2

H3N+

SH

Cysteine

2

Cysteine reactions

HS +

DithiothreitolDithioerythritol

Cleland’s reagent

CH2-CH-CH-CH2

OH

SH

OH C-OOC

H

CH2

H3N+

S-S

C COO-

H

CH2

H3N+

Cystine

C-OOC

H

CH2

H3N+

SH

Cysteine

+HO

HO S

S2

Cysteine reactions

ICH2COO- + C-OOC

H

CH2

H3N+

SH

Cysteine

R-group

Iodoacetate

C-OOC

H

CH2

H3N+

S CH2COO- + HI

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 N

HNO2

FO2N

FDNB

+

HFbase

NO2

O2N N

H

polypeptide

H

C

R1

C

O

N

H

C

R2

H

C

O

O-

H

C

R1

C

O

N

H

C

R2

H

C

O

O-

..

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.

Reaction with Dansyl Chloride

H N

HDansyl Chloride

+

HClbase

N

H

Cl

O

S

ON

H3C

H3C

S

ON

H3C

H3C

O

H

C

R1

C

O

N

H

C

R2

H

C

O

O-

H

C

R1

C

O

N

H

C

R2

H

C

O

O-

polypeptide

..

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