CH 24 AMINO ACIDS, PEPTIDES & PROTEINS

- peptides & proteins are arguably the most versatile & important molecules found in nature
- amino acids are the monomeric building blocks of peptides & proteins
proteins

H , H2O,

(polymer)

peptides
(oligomer)

AA AAn AA

AA AAn AA

102 's units

2 - 100 units

H , H2O,

-amino acids
(monomer)
AA
1 unit

I. AMINO ACIDS
- the characteristics of amino acids determine the overall structure & function of peptides & proteins

A. STRUCTURE
- although any carboxylic acid with an amino group substitutent is considered an amino acid,
peptides & proteins are constructed from alpha-amino acids (-AAs)
R

CH COOH

R = alkyl or aryl

NH2

R can contain hydroxyl, sulfhydryl , carboxyl, amino, amido,...

alpha-amino acid
-AA

- standard amino acids & the twenty alpha amino acids used in the synthesis of peptides & proteins
- essential amino acids & not synthesized in the body, must be obtained from diet

- the twenty standard amino acids are shown below:

The Standard Amino Acids

The Standard Amino Acids contd

- know the names & structures of the following amino acids:

Name

Abbreviation

R Group

Glycine

Gly

hydrogen

Alanine

Ala

methyl

Valine

Val

isopropyl

Phenylalanine

Phe

benzyl

Stereochemistry
- the alpha carbon of every amino acid (except glycine) is an asymmetric carbon; thus, all amino
acids are chiral (except glycine)
- the D/L convention derived from carbohydrates is used to distinguish the possible enantiomeric
amino acid stereoisomers
CHO
H

CHO
H

HO

OH

CH2OH

CH2OH
D-glyceraldehyde

L-glyceraldehyde

- the carboxyl group is placed at the top of the Fischer projection & the R group is placed on the
bottom the horizontal position of the amino group (right or left) determines the configuration
COOH
H

COOH

NH2

H2N

H
R

D-amino acid

L-amino acid

- most naturally occurring amino acids possess the L-series configuration

B. ACID-BASE PROPERTIES
- because they contain both an acid & base functionality, amino acids exist primarily in an ionized
form which is the result of an intramolecular proton transfer
R

CH COO
NH3

dipolar ion or zwitterion

- owing to their zwitterionic nature, amino acids exhibit the properties of ionic compounds L they
are high-melting, extremely polar, crystalline solids which are highly water-soluble & only sparingly
soluble in organic solvents
- amino acids are also amphoteric L they behave as both acids & bases
- note that the acidic moiety is the ammonium group (-NH3+) & the basic moiety is the carboxylate
group (-COO-)
- the amphoteric nature of amino acids is manifested in three unique pH-dependent forms:
R

OH

CH COOH

NH3

OH

CH COO

NH3

CH COO
NH2

acidic form

zwitterionic form

basic form

cationic (+1)

neutral (0)

anionic (-1)

low pH

high pH

pHI

Isoelectric Point, pHI A

- the pH at which the neutral zwitterionic form of the amino acid predominates
- since the overall charge on the zwitterion is zero, an amino acid at its isoelectric point does not

migrate in an electric field

- each amino acid has its own unique & characteristic isoelectric point value (pHI values can be used
to identify an amino acid)
- the migratory behavior in an electric field of a molecular species whose charge is pH-dependent
is the basis for the separation technique known as electrophoresis

C. SYNTHESIS
- amino acids can be prepared using several already established methodologies
1) Reductive Amination
- alpha-ketoacids are converted to alpha-amino acids using ammonia & a reducing agent:
O
R

COOH

-ketoacid

NH3
RA

NH2
R

CH COOH

-amino acid

RA = H2/Pd mostly, others possible

Review CH 19-19
Mechanism
- the overall transformation proceeds through the imine, as examined previously
EX.
COOH

NH3

COOH

H2/Pd

NH2
valine

- synthesis of amino acids using reductive amination is known as a biomimetic reaction L it

approximates the biosynthetic method used by organisms

2) Amination of Alpha-Haloacids
- alpha-bromination of a carboxylic acid using the Hell-Volhard-Zelinsky reaction, followed by
treatment with excess ammonia affords the alpha-amino acid (as its ammonium carboxylate salt)

CH2

COOH

acid

Br2

H2O

PBr3

NH3
(xs)

NH2
R

CH

COOH

-amino acid

Review CH 22-4 & CH 19-12

Mechanism
- alkylation by the -bromoacid in excess ammonia provides a good yield of the primary amine

EX.
CH3 S CH2CH2CH2 COOH

Br2
PBr3

H2O

NH3
(xs)

NH2
CH3 S CH2CH2 CH
methionine

COOH

3) Gabriel-Malonic Ester Synthesis

- in a variation of the malonic ester synthesis, the alpha-carbon with a protected amino group is
alkylated; then the diester is hydrolyzed & decarboxylated as usual to yield an alpha-amino acid
O

O
C OCH2CH3

N CH
O

H
H2O,

C OCH2CH3
O

COOH
H2N CH R

COOH
+ 2 CH3CH2OH + CO2

+
COOH

-amino acid

N-phthalimidomalonic ester
B

= OEt , .....

Mechanism
- after alkylation, the imide & both ester groups are hydrolyzed, yielding an aminomalonic acid
which decarboxylates upon heating

EX.
O

O
C OCH2CH3

N CH
O

C OCH2CH3
O

Br

CH2CH2

COOH

EtO

H
H2O

COOH
H2N CH CH2CH2
glutamic acid

COOH

D. REACTIONS
- the carboxyl & amino group react in the usual fashion under the proper conditions
1) Esterification of the Carboxyl Group
- a direct or Fischer esterification of the carboxyl group with an alcohol
O
R

CH C OH

+ R' OH

NH2

CH C OR'

H OH

NH2

amino acid

amino acid ester

- under acidic conditions, the amino group is protonated (as -NH3+) & is unreactive
EX.
HO

CH2

CH COOH

CH2

OH

NH2

O
HO

CH2

CH C O CH2
NH2

tyrosine

tyrosine benzylester

2) Acylation of the Amino Group

- the amino group is acylated with a carboxylic acid derivative
O
NH2
R

CH COOH

O
+

R'

Lv

NH C R'

CH COOH

Lv

N-acyl amino acid

amino acid
Lv = Cl, OCOR, OR, ........
B

= OH , CO32 , OAc , ......

- under basic conditions, the carboxyl group is deprotonated (as -COO-) & is unreactive

EX.

HO CH2

CH COOH
NH2

serine

CH2

O C Cl

OH

HO CH2

CH COOH
NH C O CH2
O

N-benzyloxycarbonyl serine

II. PEPTIDES
- oligomeric chains of amino acids linked together by amide or peptide bonds:
from amino grp of one AA

from carboxyl grp of another AA

CH NH C

CH

peptide bond

A. STRUCTURE & CLASSIFICATION

- a peptide has a regular repeating pattern reflective of its amino acid monomer units
O

H3N CH C NH CH C NH CH C (NH CH C)n NH CH C O

R

N-terminus

peptide

Rn
C-terminus

- the R groups are referred to as residues L the residue defines the amino acid monomer at that
position in the peptide chain
- note that the peptide chain is drawn from the N-terminal residue on the left to the C-terminal
residue on the right & each amino acid repeating unit has its amino nitrogen left & its carboxyl
carbon right

- peptides are classified according to the number of amino acid monomer units contained therein

Number of Amino Acids

Class of Peptide

two

dipeptide

three

tripeptide

four

tetrapeptide

ten to twenty

oligopeptides

more than thirty

polypeptides

Peptide Bond
- the amide bond is a heteroatomic delocalized pi () electron system

O
C

O
N

N
H

N
H

resonance hybrid

resonance contributors

- this delocalization results in a coplanar arrangement of the carbon, oxygen, nitrogen, hydrogen
& attached atoms
- the carbon-nitrogen bond is rigid (no rotation) & the nitrogen is not basic
- hydrogen bonding exists between the carbonyl group oxygen of one peptide bond & the
amino group hydrogen of another (between chains or within a chain)
C O

H N

HB

Disulfide Bond
- a covalent bond interaction within a peptide chain or between peptide chains
- cysteine (cys) is an amino acid whose side chain contains a sulfhydryl group (-SH)
- two cysteine residues can be oxidized to form a disulfide bond known as a cystine residue

- the disulfide bridge forms a cross-link between two separate peptide chains or a loop within
a single peptide chain

B. NOMENCLATURE
- peptides are named in order of their amino acid residues (as drawn) from the leftmost N-terminal
residue to the rightmost C-terminal residue
- each amino acid residue is named with a yl ending, except for the C-terminus, whose name is
unchanged
- abbreviations are also used
- peptides must be drawn & named consistently (left to right), because the slightest change in the
order of the residues will create a dramatic change in the structure & function of the molecule
EX. Two dipeptides
O

H3N CH C NH CH C O

H3N CH C NH CH C O
CH2

CH3

CH3

phenylalanylalanine

CH2

alanylphenylalanine

PheAla

AlaPhe

EX. Draw valinylglycylvaline

O

H2N CH C NH CH2 C NH CH C OH
CH CH3

CH CH3

CH3

CH3
a tripeptide

C. SYNTHESIS
- laboratory preparations of peptides are carried out for the purpose of structure proof, biological
activity testing, drug discovery & the like
- because they are difunctional molecules, protection of one of the functional groups on each amino
acid reactant is required to avoid the undesired side reactions
- therefore, the amino group of one reactant & the carboxyl group of the other must be protected
for every peptide bond forming step
- a protect-react-deprotect sequence must be employed for each new amino acid added to the
peptide chain

EX. Synthesize AA1-AA2

Incorrect Method:
AA1 AA2 + AA2 AA1 + AA1 AA1 + AA2 AA2

AA1 + AA2

Correct Method:
P

+ AA1

AA2 +

N-Pro
C-Pro

P
AA2

AA1

couple

(react)

AA1 AA2

P
N-Depro

AA1 AA2

C-Depro

AA1 AA2

- applying the conventional laboratory approach to peptide synthesis is very tedious, timeconsuming & expensive
- a vastly superior automated methodology has been developed:

Solid-Phase Peptide Synthesis

- the initial amino acid & ensuing peptide chain is anchored to an insoluble polymer matrix resin
bead which renders each reaction & product isolation step a simple matter of filtering & washing
- the synthesis proceeds in a backwards fashion L the peptide is constructed from its C-terminal
residue to its N-terminal residue
- the entire process can be programmed & monitored by a machine using the following basic
chemical components:
1) Solid-Phase Anchor & C-Protecting Group A
- a derivatized polystyrene polymer matrix (formed into resin beads) serves both purposes:
CH2

CH2

CH
+ CH3O CH2 Cl

CH

SnCl4
becomes C-protecting grp

polystyrene

CH2 Cl
solid-phase anchor

2) N-Protecting Group A
- converting the amino group to an amide using a carboxylic acid derivative affords protection
- an especially reactive, easily removable N-protecting group is tert-butoxycarbonyl or BOC
CH3

CH3 C O C
CH3

t-butoxycarbonyl grp
B OC

- to N-protect, the amino acid is treated with either the acyl chloride (less stable) or the anhydride
(more useful) in the presence of base:
CH3

CH3

CH3 C O C O C O C
CH3

CH3

CH3

H2N CH COOH
R

t-butoxycarbonic anhydride
(BOC)2O

amino acid
AA

Et3N

CH3

CH3 C O C NH CH COOH
CH3

t-butoxycarbonylamino acid
BOC-AA

3) Coupling Agent A
- recall that direct amide formation requires removal of water from between the ammonium cation
(-NH3+) & the carboxylate anion (-COO-)
- peptide bond formation is achieved efficiently using dicyclohexylcarbodiimide (DCC) as the
dehydrative coupling reagent:
N C N
dicyclohexylcarbodiimide
DCC

- the two protected reactants are combined in the presence of dicyclohexylcarbodiimide to

produce the peptide bond & dicyclohexylurea (DCU)

H
+

HN CH C OH

H N CH C

N C N

amino acid 2

amino acid 1
AA1

dicyclohexylcarbodiimide
DCC

AA2

O
HN CH C

N CH C

H O H
+

N C N

R
peptide
AA1 AA2

Mechanism

dicyclohexylurea
DCU

4) N-Deprotection A
- mild, nonaqueous hydrolysis with trifluoroacetic acid (TFA) removes the tert-butoxycarbonyl
(BOC) N-protecting group without breaking any of the peptide bonds
CF3

COOH

trifluoroacetic acid
TFA

- the tert-butoxycarbonyl protecting group fragments as it leaves:

O
C

CH3

CH NH C O C
R

CH3

TFA

O
C

CH3

CH NH2

+ CO2

CH3
+

CH2

CH3

t-butoxycarbonylamino acid

amino acid

BOC-AA

AA

5) C-Deprotection A
- mild, nonaqueous hydrolysis with hydrofluoric acid (HF) removes the polymer matrix anchor &
C-protecting group without degrading the peptide
CH2

CH

CH2

CH
O

HF

H O C

O
CH2 O C

CH NH
R

CH NH

CH2

R
anchored & C-protected peptide

EX. Solid-phase peptide synthesis using general formulas for amino acids
H2N CH
Rn

COOH

free peptide