Chapter 21

Amino Acids, Peptides, and Proteins

Proteins come in a wide variety of shapes and sizes. They all are
polymers of α−amino acids. The amino acid units are held together
with amide bonds. Proteins are polyamides.
amide bonds
R R R' R'
CH CH CH CH

:
N

:
H2N: CO2H C-N C-N C

=
H OH OH O
an α-amino acid

an amino acid unit

a polyamide structure

The α−amino acids differ in the structure of the R group. There are
20
22 different important amino acids in nature. In a protein, the
sequence of amino acids along the protein structure is called the
primary structure. The intrinsic folding of the polyamide chain and
intra-structural attractive interactions are responsible for the
secondary and tertiary structures. The latter will be discussed later.
5
 20 a-amino acids

pH = 7.0
6
Stereocenters

Except for glycine (R=H), all the natural amino acids

have the L configuration (Fischer notation).

CHO COOH COOH

C C
HO H H2N H H2N H
CH2OH R
R
L-glyceraldehyde L-α-amino acid
(S) (usually S)

cysteine is the exception

8
Amino Acids as Dipolar Ions
The properties of amino acids indicate they exist as dipolar ions
(zwitterions) in the solid state and in solution in water.

• Amino acids are nonvolatile, crystalline solids with

high melting points.

• They are soluble in water,insoluble in nonpolar solvents.

• Aqueous solutions of amino acids behave like solutions of

high ionic strength.

• Acidity and basicity constants (Ka and Kb) are consistent

with the zwitterionic structures.

9
Amino Acids as Dipolar Ions

The structure of the species in solution depends on the pH:

+ -H+ + - -H+
H3NCHCO2H H3NCHCO2 H2NCHCO2-
R +H+ +H+ R
R
cation dipolar ion anion
(no net charge)
in strong acid in strong base
pH < 3 pH > 10

Isoelectric Point
In the presence of an electric field, chemical species with a net charge
migrate towards the pole of the electric field with opposite charge.
The moving charges through an aqueous solution conduct a current.
+ -H+ + - -H+
H3NCHCO2H H3NCHCO2 H2NCHCO2-
R +H+ +H+ R
R
cation dipolar ion anion
migrates towards cathode (no net charge) migrates towards anode

10
The Experiment

The isoelectric point experiment measures the current conducted by

an aqueous solution of an amino acid (the ability to transport
charge) as the pH is varied.

The isoelectric point is the pH where there is

minimal current flow. This minimal current results
from a low concentration of chemical species with a
net charge. The dominant species in solution at this
pH will have no net charge.

11
Example: Alanine CH3CHCOOH (2-aminopropanoic acid)
NH2
At very low pH, the dominant species is the cation:

CH3CHCOOH + H2O CH3CHCO2- + H3O+

NH3 NH3
+ +
pKa = 2.3 The positive charge increases the
acidity of the carboxylic acid.

Near pH = 7, the dominant species is the zwitterion: Above pH = 12, the

CH3CHCO2- + H3O+ dominant species is
CH3CHCO2- + H2O
NH3 NH2 the anion:
+
zwitterion CH3CHCO2-
pKa = 9.7 NH2

The isoelectric point (pI) of an pKa pK a

+
amino acid with a single amino and pI =
a single carboxyl function is the 2
average of the pKa and the pKa.
At pH = 6.0, there will be minimal
2.3 + 9.7 current flow because the dominant
For alanine, pI = = 6.0 species in solution is the zwitterion
2
that does not have a net charge. 12
The concentrations of the three species of the alanine system vary
according to the pH of the solution, as shown below.
+ CH3 -H+ + CH3 - -H+ CH3 -
H3NCHCO2H H3NCHCO2 H2NCHCO2
+H+ +H+
pKa = 2.3 pKa = 9.7

cation zwitterion anion

100

At the half neutralization

point of an acid,
percentage of species

• [acid]
50 • pKa = pH + Log
pKa = pH pKa = pH [conj. base]
pKa = pH + Log(1) = pH + 0

isoelectric point thus, pKa = pH

1 2 3 4 5 6 7 8 9 10 11 12 13
pH
Some Other General Observations on Isolectric Points
Lysine has two amino and one carboxyl functions. At low pH, it has
three acid functions that deprotonate as HO- is added in order of their
acid strengths.
+ HO- + HO- +
H3N(CH2)4CHCOOH H3N(CH2)4CHCO2- H3N(CH2)4CHCO2-
NH3 H3O+ NH H3O+ NH2
+ + 3
dicationic form of lysine monocation form of lysine zwitterionic form of lysine
(pKa1 = 2.2) (pKa2 = 9.0) (pKa3 = 10.5)

H3O+ HO-
The isoelectric point of lysine is
the average of pKa2 and pKa3:
H2N(CH2)4CHCO2-
pI = 9.0 + 10.5 = 9.8
2 NH2
anionic form of lysine

At pH = 9.8, the dominant lysine species

in solution is the neutral zwitterion.

14
Aspartic acid has one amino and two carboxyl functions. At low pH, it
has three acid functions that deprotonate as HO- is added in order of
their acid strengths.
HO- HO- -
HOOCCH2CHCOOH HOOCCH2CHCO2- O2CCH 2CHCO2-
NH3 H3O+ NH3 H3O+ NH3
+ + +
cationic form of aspartic acid
zwitterionic form anionic form
(pKa1 = 2.1)
(pKa2 = 3.9) (pKa3 = 9.8)

The isoelectric point of aspartic acid

H3O+ HO-
is the average of pKa1 and pKa2:
-
pI = 2.1 + 3.9 = 3.0 O2CCH 2CHCO2-
2 NH2
At pH = 3.0, the dominant form of dianionic form
aspartic acid is the neutral zwitterion.

Summary of Observations on pI Values

+
CHCO2- HOOC CHCO2- H2N CHCO2-
+
NH3 NH3+ NH3
pI 6-7 ~3 ~9
15
 Synthesis of α-Amino Acids

There are several standard syntheses of α-amino acids.

Direct Ammonolysis of α-Halo Acids

(i) Br2, P
CH3CH2COOH CH3CHCOOH excess NH3 CH3CHCO-2
(ii) H2O NH3
Br +
propanoic acid 2-bromopropanoic acid alanine
(as a racemic form)

16
The Malonic Ester Synthetic Route
COOEt COOEt COOH
(i) NaOEt, EtOH (i) HO- , H2O
CH2 CH-CH2C6H5 CHCH2C6H 5
COOEt (ii) C6H5CH2Cl (ii) H3O+
COOEt COOH
diethyl malonate
bromination occurs via
the enol of the acid:
Br2

: : : :
HO COOH ether, heat
C=C
HO CH2C6H 5
COOH
C6H5CH2CHCO2- excess NH3 C H CH CHCOOH heat, >100oC Br-CCH2C6H 5
6 5 2
NH3 (-CO2) COOH
+ Br
phenylalanine
(as a racemic form)

17
The Gabriel Synthesis: Potassium Phthalimide

O O O
alkylation

N H KOBu-t N- K+ ClCH2COOEt N CH2COOEt

O O O
(97%)
phthalimide potassium phthalimide
(i) KOH, H2O hydrolysis
(pKa = 8.3)
(ii) HCl neutralization

COOH
+
+ H3NCH2CO2-
COOH
phthalic acid glycine

18
The Malonic Ester Variation of the Gabriel Synthesis
N-alkylation
O O
CO2Et CO2Et
N- K+ + BrCH N C-H
CO2Et CO2Et
O O
potassium phthalimide diethyl bromomalonate
(i) NaOEt
C-alkylation
(ii) BrCH2CHCH 3
COOH CH3
hydrolysis O
COOH CO2Et
heat (i) NaOH, H2O
phthalic acid N C-CH2CH(CH3)2
(-CO2) (ii) HCl CO2Et
+
O
(CH3) 2CHCH2CHCO2-
NH3
+
leucine
(as a racemic form)

19
The Strecker Synthesis
α-Amino acids may be prepared by reaction of an aldehyde, ammonia and
hydrogen cyanide, followed by hydrolysis of the α-amino nitrile product to
an amino acid. This procedure is called the Strecker synthesis in
recognition of the early work of Adolph Strecker (1822-71) who discovered
the reaction in the laboratory of Justus von Liebig in 1850.
O RCHCO2-
RCHCN H3O+
=

RCH + NH3 + HCN NH3+

NH2 heat
α-amino nitrile α-amino acid

A Mechanism for the Strecker Synthesis

O addition O- OH (-H2O)
RCH RCH=NH
=

RCH + :NH3 RCH

NH3+ NH2
imine
addition product

addition
- - +H+
RCH=NH + :C N: RCH-NH RCH-NH2
CN CN
α-amino nitrile
20
 Resolution of DL-Amino Acids
All of the synthetic methods presented so far yield
racemic forms of chiral α-amino acids.

With the exception of glycine, all the natural α-amino acids

have a stereocenter of the L-configuration:

- * CO2-
CH2-CO2 RCH-CO 2
-
CO2-
NH3+ NH3+ + + C
H3N H H3N H
glycine chiral α-amino acid R R
L-configuration

21
Enzymatic Resolution
Racemic mixtures of α-amino acids may be resolved by enzymes called
deacylases. These enzymes catalyze the hydrolysis of N-acylamino
acids of the L-configuration. The strategy involves acetylating both
the D- and L-amino acids, and then selectively hydrolyzing the
L-amino acid derivative with a deacylase.
acetylation
O O
=
DL-RCHCO2 - =
CH3COCCH3 DL-RCHCO2H
NH3+ NHCCH3

=
racemic mixture O

deacylase

CO2-
CO2-
+ H NHCCH3
CH3COOH + H3N H

=
R O
R
L-amino acid D-N-acetyl amino acid

separable

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 Amino Acid Sequence of Polypeptides and Proteins

Structure Determination

The different levels of structural information of a polypeptide or

protein are similar to those of small molecules:

(1) The molecular weight (amu or Daltons) may be determined by

several physical measurements such as ultracentrifugation, light
scattering, gel electrophoresis, or mass spectroscopy. Important
breakthroughs in techniques in recent years (such as electrospray
ionization) have made mass spectroscopy an almost routine, but
powerful, methodology for molecular weight determination.

(2)The amino acid composition (analogous to elemental

composition for small molecules) may be determined by classical
chemical methods (complete hydrolysis and separation).

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The Primary Structure
(3) The sequence of amino acids may be determined by older
automated chemical, or newer mass spectroscopic, methods. The
amino acid sequence of a polypeptide is the primary structure.

The primary structure is written left to right starting with the

N-terminal residue and ending with the C-terminal residue.

example: a tripeptide of glycine, alanine and phenylalanine

O O
+ C-terminal
=
=

N-terminal H3NCH 2CNHCHCNHCHCO2-

residue
residue CH3 CH2C6H 5

Gly.Ala.Phe

Sequence information of a polypeptide is given by writing the

amino acid residues in order from left to right as shown.
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The Primary Structure of Polypeptides and Proteins O

=
CH(CH3)2 C-NH2
CH2 CH2
O O O O O

=
=

=
H2NCCH2NHCCHNHC N C CHNHCCHNH
H2NGly Leu Pro Cys Asn Gln CH2 C=O
O
S

=
CHCH2CH2CNH2
oxytocin Cys Ile S
Tyr NH
CH2
C=O
H2N CH CHCHCH2CH3
O=C O CH3

=
NH CHC NH
CH2

OH
NH O

=
CH2CH2NHCNH2 C-NH2
CH2 CH2
H2NGly Arg Pro Cys Asn Gln O O O O

=
=

=
H2NCCH2NHCCHNHC N CHNHCCHNH
vasopressin Cys Phe
CH2
Tyr C=O
O
S

=
CHCH2CH2CNH2
S
NH
CH2
C=O
H2N CH O CH-CH2-

=
O=C NH CHC NH
CH2

25
OH
Insulin
In 1953, Sanger completed the sequencing of Bovine insulin. It contains
51 amino acid residues and differs from Human insulin in only 3 amino
acid residues. Insulin in all species regulates carbohydrate metabolism.
Insulin from other species can be used by humans.

Sequence Differences in Insulin

Insulin is a protein hormone produced in the pancreas. It is
essential for the regulation of carbohydrate metabolism. Insulin
from other mammalian species differ from Human insulin only in
positions 8,9, and 10 of the A chain. In addition, Human insulin
differs from all others at position 30 of the B chain where a
threonine replaces the alanine.

26
 The Primary Structure of Polypeptides and Proteins

A chain 21

Bovine insulin Ser-Leu-Tyr-Gln-Leu-Gln-Asn-Tyr-Cys-Asn-COOH

Val Ser 9 Cys
Cys 10 Val Gly
Ala 8 Leu Gln
1
Tyr Arg
H2 N-Gly-Ile-Val-Glu-Gln-Cys-Cys
Leu Gly
B chain Ala Phe
1
H2 N-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln Phe
Tyr
Sequence Differences in Mammalian Insulin Thr
Pro
species amino acid positions Lys
8 9 10
30 Ala
cow Ala Ser Val COOH
sheep Ala Gly Val
horse Thr Gly Ile
human Thr Ser Ile Note the disulfide bridges ( )
pig Thr Ser Ile connecting the A and B chains.
whale Thr Ser Ile
dog Thr Ser Ile

27