Unit 13 Amino Acids and Peptides

UNIT 13
Amino Acids and Peptides

Structure
13.1 Introduction 13.5 Structure of Peptides
Expected Learning Outcomes 13.6 Synthesis of Peptides
13.2 Structure and Physical Protection and Deprotection of
Properties of Amino Acids Amino Group
Structure of Amino Acids Protection and Deprotection of
Carboxy Group
Zwitterionic Nature
Peptide Bond formation using
Isoelectric Point and
Carboxy Activating Groups
Electrophoresis
13.7 Merrifield Solid-Phase
Stereochemistry of Amino Acids:
Synthesis
Optical Activity
13.8 Lab Detection of Amino Acids
13.3 Synthesis of 2-Amino Acids
13.9 Summary
Gabriel Phthalimide Synthesis
13.10 Terminal Questions
Strecker Synthesis
13.11 Answers
From 2-Halo Acids
13.4 Reactions of Amino Acids

13.1 INTRODUCTION
This is the first unit of Block 4 of this course. In this Unit, you will study about
the structure and chemistry of amino acids and peptides.

Amino acids are the compounds which contain both an amino group and a
carboxy group in their molecules. They constitute a particularly important class
of bifunctional compounds as they are the building blocks of proteins. Here,
you will first study about the amino acids and then the structure and synthesis
of peptides will be described.

We will begin this unit with a discussion on the structure and physical
properties of common amino acids. Then, we will explain the zwitterionic
nature of amino acids. This will be followed by description of the synthesis
of the amino acids.

Then, the structure of peptides will be illustrated. The synthesis of peptides

using N-protection, C-protection and C-activating groups will be discussed in
5
Block 4 Peptides, Proteins and Carbohydrates

detail. You will also study about Merrifield solid - phase synthesis which is a
versatile technique for the synthesis of peptides. Finally, the methods of
laboratory detection of amino acids will be explained.

Expected Learning Outcomes

After studying this unit and having the experiments performed, you should be
able to:

 list some amino acids and write their structures;

 give the common and IUPAC names of amino acids;

 discuss the zwitterionic nature of amino acids;

 explain the isoelectric point and importance of electrophoresis;

 give various methods of preparation of 2- amino acids;

 discuss the reactions of amino acids;

 briefly explain the structure of peptides;

 describe the methods of synthesis of peptides using N-protection,

C-protection and C-activating groups;

 discuss Merrifield solid-phase synthesis and highlight its importance;

and

 explain the laboratory detection of amino acids.

13.2 Structure and Physical Properties of Amino

Acids
Before studying the chemistry of amino acids, let us first understand their
structure.

13.2.1 Structure of Amino Acids

The general structure of a 2-amino acid can be represented as follows:

NH2
R CHCOOH

These are more often referred as α-amino acids.

While several hundred different amino acids are known to occur naturally, 20
of them deserve special mention as they are mainly present in proteins. These
amino acids are listed in Table 13.1. As given in the Table, for amino acids
trivial names are commonly used.

The convention to use a three letter code, as an abbreviation, for each amino
acid is also given in the table. These abbreviations are particularly useful in
designating the sequence of amino acids in peptides and proteins. The last
column of the table given below also shows a single letter code to denote the
amino acids. You will study more about peptides and proteins in Unit 14.
6
Unit 13 Amino Acids and Peptides

Table 13.1: Physical Properties of Amino Acids

Amino Acid, Name Abbreviation Abbreviation

NH2 Three letter One letter

Code Code
R CHCOOH

NH2
glycine Gly G
H CHCOOH

NH2
alanine Ala A
CH3 CHCOOH

CH3 NH2 valine Val V

CH3CH CHCOOH

CH3 NH2
leucine Leu L
CH3CHCH2 CHCOOH

CH3 NH2 Ile I

isoleucine
CH3CH2CH CHCOOH

NH2
methionine Met M
CH3SCH2CH2 CHCOOH

CH2
NH
CH2 proline Pro P
CHCOOH
CH2

NH2
CH2 CHCOOH phenylalanine Phe F

NH2
CH2 CHCOOH
tryptophan Trp W
N
H

NH2
serine Ser S
HOCH2 CHCOOH

OH NH2
threonine Thr T
CH3CH CHCOOH

NH2
cysteine Cys C
HSCH2 CHCOOH
7
Block 4 Peptides, Proteins and Carbohydrates

NH2
HO CH2 CHCOOH tyrosine Tyr Y

O NH2
asparagine Asn N
H2NCCH2 CHCOOH

O NH2
glutamine Gln Q
H2NCCH2CH2 CHCOOH

O NH2
aspartic acid Asp D
HOCCH2 CHCOOH

O NH2
glutamic acid Glu E
HOCCH2CH2 CHCOOH

NH2
lysine Lys K
H2NCH2CH2CH2CH2 CHCOOH

NH2 NH2
arginine Arg R
H2NCNHCH2CH2CH2 CHCOOH

NH2
N
CH2 CHCOOH histidine His H
N
H

Amino acids can be classified as , , ,… etc., depending upon the location
of the amino group on the carbon chain containing the carboxy function. Some
examples are illustrated below:
2 1 3 2 1 4 3 2 1
H2 N CH2COOH H2N CH2CH2COOH H2N CH2CH2CH2COOH
2-aminoethanoic acid 3-aminopropanoic acid 4-aminobutanoic acid
(-amino acid) (-amino acid) (-amino acid)

Thus, the amino acids listed in Table 13.1 are -amino acids or 2-amino acids.
Now, answer the following SAQ.

SAQ 1
Give one example each for a 2-amino acid which contains the following in
the side chain:
i) Sulphur
ii) An aromatic ring
iii) A carboxyl group
8
Unit 13 Amino Acids and Peptides

13.2.2 Zwitterionic Nature

Because amino acids contain both carboxy and amino groups in their
molecules, they are amphoteric in nature, i.e. they behave both as acids and
bases. Amino acids actually exist as inner salts, called zwitterions. A
zwitterionic structure is possible for amino acids because the amino group is
basic in nature and can accept a proton from the acidic carboxy group. A
zwitterion can be represented as shown below.

COOH COO

H2 N H H3 N+ H

R R
a zwitterion

The highly polar nature of zwitterion allows the formation of strong crystal
lattices similar to the ionic compounds. Amino acids, therefore, resist
conversion from solid to liquid state and do not melt but decompose on
heating.

The zwitterionic nature is also reflected in their higher solubility in water and
low solubility in nonpolar solvents. In addition to the above observations, large
dipole moments also indicate the zwitterionic nature of amino acids.

13.2.3 Isoelectric Point and Electrophoresis

Let us now study the zwitterionic form of amino acids in more detail. You can
see in the zwitterion shown above that the amino group is protonated and the
carboxy group exists as the carboxylate anion. Thus, the acidic group is a
substituted ammonium ion and the basic group is the carboxylate anion. As a
result, in strongly acidic medium i.e., at low pH, the carboxylate group will be
protonated to yield the following species, I.

COO COOH
+
H
H3 N+ H H3 N+ H
+
H
R R
a zwitterion I
species present in
strongly acidic medium

Let us next consider the species present in strongly basic medium, i.e. at
higher pH of the solution. Under these conditions, the proton will be removed
from the +NH3 group to yield the following species.

COO COO
H+
H3 N+ H H2 N H
+ H+
R R
a zwitterion II
species present in
strongly basic conditions
9
Block 4 Peptides, Proteins and Carbohydrates

Thus, we can write a combined equation for the acid-base behavior of the
acids as shown below.
COOH COO COO
H+ H+
H3 N +
H H3 N+ H H2 N H
+ H+ + H+
R R R
I a zwitterion II
low pH high pH
(acidic conditions) (basic conditions)

You can see that at low pH, species I has a net positive charge and has two
acidic sites (+NH3 and COOH). On the other hand, at high pH, species II has a
net negative charge and has two basic sites (NH2 and COO).

It is clear from the above equation that the amino group will be first protonated
and then the carboxylate anion. Also at some intermediate pH, the amino acid
exists as a zwitterion with no net charge. The pH at which this occurs is known
as isoelectric point, pHi of the amino acid. At this pH, the amino acid is
stationary in an electric field, i.e., it migrates neither to the negative pole nor to
the positive pole because the charges on it are balanced.

At low pH, there are two acidic sites in the amino acid I and therefore, it has
two pKa values. The pKa value corresponding to the more acidic site is referred
to as pKa1 and that corresponding to the less acidic site as pKa2. Thus, for the
simplest amino acid, glycine, we can write the two equilibria as follows:
+ pKa1 = 2.4 +
H3NCH2COOH + H2O H3NCH2COO + H3O+

+ pKa2 = 9.8
and H3NCH2COO + H2O H2NCH2COO + H3O+

At this stage you can compare the pKa1, with pKa of ethanoic acid which is
equal to 4.76. This leads to the conclusion that due to the electron withdrawing
nature of the protonated amino group, the acidity of amino acid is increased as
compared to ethanoic acid. Table 13.2 lists the pKa values and pHi of some
amino acids.

Table 13.2: pKa and pHi values of some amino acids

Amino acid pKa1 pKa2 pHi
Glycine 2.34 9.60 5.97
Alanine 2.34 9.69 6.00
Valine 2.32 9.62 5.96
Leucine 2.36 9.60 5.98
Isoleucine 2.36 9.60 6.02
Methionine 2.28 9.21 5.74
Proline 1.99 10.60 6.30
Phenylalanine 1.83 9.13 5.48
Tryptophan 2.83 9.39 5.89
Asparagine 2.02 8.80 5.41
Glutamine 2.17 9.13 5.65
Serine 2.21 9.15 5.68
Threonine 2.09 9.10 5.60
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Unit 13 Amino Acids and Peptides

You can verify from Table 13.2 that

pKa1  pKa 2
pHi 
2

The amino acids having acidic and basic side chains are characterised by
three pKa values. The third pKa value, i.e., pKa3 reflects the nature of the
functional group present in the side chain.

Electrophoresis

It is a technique which involves the migration of charged particles using

an electric field. The charged particles have different mobilities
depending upon their shape, size and charge. Thus, different amino
acids and in turn, the peptides and proteins which are made up of these
amino acids, can be separated using electrophoresis. Since different
amino acids and peptides (or proteins) have different acidic and/or basic
groups, they have different isoelectric points. Thus, at a particular pH, in a
solution, they move towards the cathode or the anode at different rates.

In this way, electrophoresis can be used for the separation and analysis
of various amino acids, peptides or proteins when present in a mixture.

13.2.4. Stereochemistry of Amino Acids:

Optical Activity
With the exception of 2-aminoethanoic acid (glycine), the 2-amino acids have
at least one chiral centre.

According to the older D, L system of specifying the configuration (discussed

in Sec. 11.3, Unit 11, Block 3 of BCHCT-131 Course), the 2-amino acids
derived from animals or higher plants were found to have L configuration, i.e.,
they have the same relative configuration as L-glyceraldehyde. Thus, we can
write the following Fischer projection formula of an L amino acid.

COO Enzymes use up one

+ enantiomer
H 3N H preferentially.

R
an L amino acid

On the basis of your knowledge about assigning the absolute configuration,

can you attempt the following SAQ?

SAQ 2
What is the absolute configuration (R or S) of the following amino acids?

COO COO
+ +
a) H3 N H b) H3 N H

CH2OH CH2SH
L-serine L-cysteine
11
Block 4 Peptides, Proteins and Carbohydrates

Having learnt about some general aspects of the structure of amino acids, let
us now focus our attention on the synthesis of 2-amino acids.

13.3 SYNTHESIS OF 2-AMINO ACIDS

2-Amino acids can be synthesised by using the methods given in Table 13.3.

Table 13.3: Methods of Synthesis of 2-Amino Acids

1. By N-alkylation of 1,2-benzenedicarboxylic imide (phthalimide) anion

O
COOCH2CH3 COOH
- +
NK +H C Br CH2 NH2
COOCH2CH3
O diethyl 2-bromopropanedioate

2-substituted amino acids can also be prepared.

2. From aldehydes : Strecker Synthesis

NH2
R CHO + NH3 + HCN R CH COOH

3. From 2-halo acids

X NH2

R CHCOOH + NH3 R CHCOOH

13.3.1 Gabriel Phthalimide Synthesis

This method is a modification of the Gabriel synthesis of amines which has
been discussed in Unit 11, Sec. 11.4.

It involves the N-alkylation of 1,2-benzenedicarboxylic imide (phthalimide)

anion with diethyl 2-bromopropanedioate as shown below:

O O
COOCH2CH3
COOCH2CH3
 +
HCBr + :N: K N CH
KBr
COOCH2CH3 COOCH2CH3
O O
diethyl 2-bromo- potassium 1,2-benzene- N-alkylated product
propanedioate dicarboxylic imide (85%)
(diethyl 2-bromo- (potassium phthalimide)
malonate)

The advantage of this method is that the alkylated product obtained in the
above reaction can be further alkylated to yield a variety of substituted amino
12 acids by the following sequence of reactions.
Unit 13 Amino Acids and Peptides

O O
COOCH2CH3  +
COOCH2CH3 Diethyl
1. CH3CH2O Na
N CH 2. RX N C R 2-bromopropanedioate
COOCH2CH3 COOCH2CH3 can be prepared by the
O O bromination of diethyl
propanedioate.
H+, H2O, D
2 CH3CH2OH

O O
H H COOH
+
H , H2O CO2
H 2N C R N C R N C R
COOH COOH
COOH
O O
2-amino acid

Thus, we can get a variety of amino acids depending upon the nature of R.

A variation of the above mentioned method utilises diethyl

2-(N-ethanoylamino)propanedioate instead of the imide derivative. The
sequence of reactions involved is shown below:

O COOCH2CH3 O COOCH2CH3
1. Na+ OC2H5
CH3CHNCH 2. RX CH3CHNCH C R
COOCH2CH3 COOCH2CH3
diethyl 2-(N-ethanoylamino)propanedioate
H+, H2O, D

O COOH
CH3COH + H2N C R
COOH
(not isolated)

CO2

H
H2 N C R
COOH
2-amino acid

13.3.2 Strecker Synthesis

It was pointed out in sub-Sec. 14.4.1, Unit 14, Block 3 of BCHCT-133 Course
that aldehydes on reaction with hydrogen cyanide yield a cyanohydrin. But,
when the same reaction is carried out in the presence of ammonia, the first
step is probably the initial formation of an imine from the reaction of the
aldehyde with ammonia. The addition of hydrogen cyanide to the imine
furnishes the corresponding 2-amino nitrile which on acidic or basic hydrolysis
yields the 2-amino acid. This is also known as Strecker synthesis. The
sequence of reactions involved in this synthesis is given below.

O NH NH2 NH2
NH3 HCN H+, H2O
CH3CH CH3CH CH3 C CN CH3CHCOOH
H2O
H
ethanal imine 2-aminopropane nitrile 2-aminopropanoic acid
(55%)
13
Block 4 Peptides, Proteins and Carbohydrates

13.3.3 From 2-Halo Acids

In Unit 9, Sec. 9.5, you have studied that 2-halo acids can be obtained from
carboxylic acids using the Hell-Volhard-Zelinsky reaction. These 2-halo acids
on nucleophilic substitution by NH3 yield 2-amino acids as shown below:

H2O
CH3CHCOOH + 2 NH3 CH3CHCOOH + NH4Br
Br NH2

2-bromopropanoic acid 2-aminopropanoic acid

(65-70%)

Enzymes preferably It is also worthwhile to mention here that the amino acids obtained by
use one enantiomer synthesis using the methods discussed above are racemic mixtures.
Enantiomerically pure amino acids can be obtained by resolution of the
racemic mixtures or by chiral synthesis using enzymes.

SAQ 3
Which alkyl halide will you use for the synthesis of methionine in the Gabriel
phthalimide synthesis?

13.4 REACTIONS OF AMINO ACIDS

Amino acids undergo many of the reactions characteristic of the amino and
carboxy groups. For example, a typical reaction of carboxy group is
esterification and that of the amino functional group is alkanoylation. Let us
now study these reaction in detail.

1. Esterification

The carboxy group of an amino acid can be esterified in the normal way using
Methyl, ethyl and
benzyl esters are excess of an alcohol under acidic conditions.
used as intermediates
in the synthesis of + +
peptides. NH3 NH3
HCl
CH3CHCOO + CH3CH2OH CH3CHCOOCH2CH3Cl
(90-95%)
hydrochloride salt
of amino acid ester

Neutralisation of the hydrochloride with alkali yields the ester.

Esters of amino acids undergo intermolecular cyclisation to yield cyclic amides

shown below:
O H 2N O
C OC2H5 CH2 C NH
+ 2 C2H5OH CH2
CH2 C2 H5 O C CH2
O
NH C
NH2 O
2,5-diazacyclohexane-1,4-dione
(diketopiperazine)
14
Unit 13 Amino Acids and Peptides

2. Alkanoylation of Amino Acids

Alkanoylation of the amino group of an amino acid is carried out under basic
conditions so that the free amino form is present in substantial concentration.
Alkanoylation can be carried out by alkanoyl halides (acid chlorides) or
carboxylic acid anhydrides. The product is finally obtained by acidifying the
reaction mixture.
O
+
NH3 O NHCC6H5
 1. OH
(CH3)2CHCHCOO + C6H5CCl (CH3)2CHCHCOOH
H2O
valine benzenecar- 2 hr, 277 K N-benzenecarbonylvaline
bonyl 2. H+, H2O (N-benzoylvaline)
chloride (80%)
(benzoyl
chloride)

O O O
+
H3NCH2COO + CH3COCCH3 CH3CNHCH2COOH + CH3COOH

glycine ethanoic N-ethanoylglycine

anhydride (89-92%)

3. Reaction with Ninhydrin

When the aqueous solution of a 2-amino acid is treated with triketohydrindene

hydrate (ninhydrin), a blue-violet colour is obtained.

O
OH pH=9
2 + H3N+ CH CO2
OH
R
O
ninhydrin

O& O Ninhydrin test is given

by amino acids
N + R CH O + CO2 + H+ containing primary
amino group.
O O
Ruhemann's purple max = 570 nm

The blue-violet coloured compound formed is also known as Ruhemann’s

purple.

This is an important reaction used in the detection of small amounts of amino

acids.

4. Formation of Lactones

Some amino acids undergo cyclisation to yield cyclic amides, called lactams.
See Sec. 10.7, Unit 10 for nomenclature of lactams.
O
O O
+ D
H2NCH2CH2CH2CO H2NCH2CH2CH2COH :NH + HOH

H
(86%)
a lactam
15
Block 4 Peptides, Proteins and Carbohydrates

5. Formation of Peptides

In addition to the above reactions, amino acids constitute the structural units of
peptides and proteins about which you will study in the forthcoming sections.

But before studying the formation of peptides, you must be curious to know
about the structure of peptides.

13.5 STRUCTURE OF PEPTIDES

Peptides are biologically important polymers in which 2-amino acids are joined
by the amide linkages, formed by the reaction of the carboxy group of one
amino acid with the amino group of another amino acid. These amide linkages
are also called peptide bonds. The general structure of a peptide is shown
The detailed structure
below:
of peptides will be 2
O R O
dealt in Unit 14. 12 o 153 pm

12
... NH C ...
1
C pm CH pm NH
5
o
114o 7 2
14 13
CH NH C CH

124 pm
123o 123o
1 3
R O R

peptide bonds

General Peptide Structure

Peptides can be classified as dipeptides, tripeptides and tetrapeptides,

depending on whether the number of amino acids is two, three or four,
respectively. Peptides containing up to 50 amino acids are called
polypeptides. Bradykinin is an important naturally occurring nonapeptide which
is present in blood plasma and is involved in the regulation of blood pressure.
Remember that a
three letter code to Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
represent the amino bradykinin
acids was given in
Table 13.1, Sec. 13.2
of this Unit. 13.6 SYNTHESIS OF PEPTIDES
The synthesis of peptides poses a challenge because even if you want to
synthesise a dipeptide from the starting amino acids A and B, we get a mixture
of following four dipeptides:

A-A, B-B, A-B and B-A

For example, the reaction of glycine and alanine will give the following four
The amino acids in a dipeptides:
peptide are also
known as amino acid Glycyl glycine , Alanyl alanine, Glycyl alanine and Alanyl glycine
residues.
Here, the convention is to write the N- terminal amino acid on the left and the
C- terminal amino acid on the right.

Therefore, for the selective synthesis of a particular peptide, we have to

16 protect
Unit 13 Amino Acids and Peptides

i) the N-terminal of one amino acid and

ii) the C-terminal of the other amino acid, and then

iii) combine or react the two protected amino acids to give the desired
dipeptide using the carboxy group activation .
activating group
protecting group
O O O O
X&NHCH &C& Z + H 2N&CHC &OY X &NHCH &C&NH &CH &C&OY+ HZ
R1 R2 R1 R2
protecting group amino acid 1 amino acid 2
peptide bond
dipeptide

The higher peptides can be synthesised by extending the chain of the

dipeptide either from the N-terminus side or the C-terminus side. For that we
have to deprotect and remove the protecting groups X or Y and repeat the
above sequence using the third amino acid.

Let us now understand the protection and deprotection of amino- and

carboxy- groups.

13.6.1 Protection and Deprotection of the Amino Group

The protection of amino group reduces its nucleophilicity. The following
reagents and groups are used for the protection of the amino group:

i) N-protection by Benzyloxycarbonyl and tert-Butoxycarbonyl

groups

Reagent Protecting group

O O
CH 2O&C&
CH 2OCCl

Phenylmethyl chloroformate Benzyloxycarbonyl

(Benzyloxycarbonyl chloride) (Cbz group or & Z group in older literature)

O O O

(CH3)3 COCOCC(CH 3)3 (CH3)3 COC &

tert-butoxycarbonyl
bis (1,1-dimethylethyl) dicarbonate
(Boc-group)
di-tert-butyl dicarbonate

The protection of the amino group of an amino acid by benzyloxycarbonyl

group can be represented as shown below:

O O
O
+ &
CH 2CCl + H 3NCHC &O 1. NaOH CH 2OC &NHCHCOOH
2. HCl, H2O R
R
III
(Z-protected amino acid)
(a carbamate) 17
Block 4 Peptides, Proteins and Carbohydrates

Similarly, we can write for the protection by Boc group and get the
Boc- protected amino acid.
O
(CH 3)3COC &NHCHCOOH
R

(Boc-protected amino acid)

When required, after the formation of the required dipeptide, the deprotection
of the protected amino group can be carried out as follows:

1. By hydrogenolysis

The Boc benzyloxycarbonyl group can be removed from the Cbz- protected
amino acid by reaction with H2 in the presence of a transition metal catalyst.
The reaction gives toluene and the carbamic acid which on instantaneous
decarboxylation gives the unprotected amino group in the peptide.
CH 2H
O O
O O
H 2,Pd-C
CH 2OC &NH CHCONHCHCY
+ HOCNH & CHCONHCHCY + CO2
R1 R2
R1 R2
toluene
(Benzyloxycarbonyl protected peptide) O
carbamic
acid part H2N-CHCONHCHCY
R1 R2

2. By treatment with acid in mild conditions

The Boc carbamate group is stable in dilute base but it can be removed in mild
acidic conditions which do not affect the peptide bond. The deprotection of
Boc- protected amino group can be done by treatment with HBr in CH3COOH
or HCl.

Thus,
O
HBr +
(CH3)3COCNH - peptide CH2 = C(CH 3)2 + CO2 + H3N - peptide
CH3COOH

ii) N-protection by Phthaloyl (Phth) Group

The reaction of 1, 2-dicarboxylic acid anhydrides with amino acids yields

imides. Imides being stable to acids and hydrogenolysis, are good protecting
groups as they can be used in variety of synthetic conditions. Phthalic acid
derivatives can, thus, be used for N-protection.

Earlier, phthalic anhydride was used as phthaloylating agent which involved

fusion with the amino acid. These conditions were harsh and caused
racemisation. Even the use of solvents such as benzene, dioxane etc. could
not eliminate the problem of racemisation.

The protection of NH2 group can be done by the following phthaloylating

18 reagents:
Unit 13 Amino Acids and Peptides
O O
O
OH
N C OCH 2CH 3
OCH 2CH 3
O
O
monoethylphthalate
N-(ethoxycarbonyl) phthalimide
O
OCH 3
Cl P
OCH 3
O

3-chloro-3-(dimethoxyphosphoryl)isobenzofuran-1(3H)-one

The phthaloylation using the above reagents can be carried out under mild
conditions and the racemisation does not occur under these conditions. This is
shown below:
R R
O O
O
aq. Na 2CO 3
N C OCH 2CH 3 + o N COOH
H2N COOH 0 C,5 min or RT, 30 min
O O

Deprotection

The phthaloyl group can be removed by the following methods:

i) By hydrazinolysis

It involves treatment with hydrazine hydrate under reflux conditions

using methanol or ethanol as the solvent.

ii) Reductive opening of the ring followed by acid- catalysed lactonisation

of the formed hydroxyl compound.
R
R
O O

1. NaBH 4, isopropanol / H 2O, 24 hr NH CO

N
o
2. AcOH, 80 C, 2 hr
CH 2 OH
O

R
O

O + NH CO
2
CH 2

13.6.2 Protection and Deprotection of the Carboxyl

Group
The carboxyl group of the amino acids can be protected by converting it to an
ester. The methyl, ethyl or benzyl esters can be prepared. 19
Block 4 Peptides, Proteins and Carbohydrates

Remember that methyl and ethyl esters can be conveniently prepared by

Fischer esterification.

O O
+
H ,D
H2N&CH &C&OH + R" OH H2N&CH &C&OR"

R' R'
R"= CH 3&, C2H5& ester

or CH 2 &

Deprotection: The ester group can be converted back to the carboxyl group
by the following methods at appropriate stage of the peptide synthesis.

i) Hydrolysis using aqueous base

O O

H2N&CH &C&OR" 1. aq. NaOH H2N&HC &C&OH + R"OH

+
2. H ,H2O R
R

But this method is not preferred because base can cause racemisation.

ii) The benzyl esters can be also deprotected by the following methods:

a) Hydrogenolysis with H2 using Pt or Pd as the catalyst, or

b) Treatment with HBr in acidic conditions in acetic acid.

13.6.3 Peptide Bond formation using Carboxy

Activating Groups
The most commonly used reagent for this purpose is dicyclohexylcarbodimide
(DCC).

DCC is the anhydride of N, N-dicyclohexylurea (DCU). DCC on reaction with

water gets converted to DCU
O
N C N + H2O NH C NH

1, 3-Dicyclohexylcarbodiimide N, N'-Dicyclohexylurea
DCC DCU

DCC activates the carbonyl group of the N-protected amino acid by taking
away a proton in the first step.

Step 1

O O
.. .. &
BocNH &CH &C&O &H + C6H11&N C N&C6H11 BocNH &CH &C&O
R R
+ ..
+ C6H11&N C N&C6H11
20 H
Unit 13 Amino Acids and Peptides

Step 2

In the next step, the carboxylate ion attacks the carbon of the C=N bond to
yield an O-acyl isourea.
C6H11

O O NH
& +
BocNH &CH &C&O + C6H11&N &
&C &
& N-C 6H11 BocNH &CH &C&O&C
H R
R N
C6H11
O-acyl isourea
Step 3

Nucleophilic addition of amino group of the carboxyl protected amino acid to

the carbonyl group of O-acyl isourea.

C6H11 C6H11
&
O NH O O NH
..
BocNH &CH &C&O&C + + H2N&CH &C&OR" BocNH &CH &C&O&C
+
R N R NH 2 N
R'
C6H11 CH-R' C6H11
O-acyl isourea

O C
OR"

tetrahedral intermediate
Step 4

Regeneration of carbonyl group and displacement of DCC as DCU.

C 6H 11
&
O NH O C 6H 11
BocNH &CH &C &O &C BocNH &CH &C NH
+
R HN H N R NH + O C
CH-R' C 6H 11 CH-R' HN
O C O C C 6H 11
OR" OR"
tetrahedral intermediate dipeptide DCU

The tetrahedral intermediate breaks down to give the dipeptide and

N, N’-dicyclohexylurea. Hence, we have synthesised a dipeptide which is
having both the amino end and the carboxy end protected with their respective
protective groups.

If we want of synthesise a tripeptide from this, we have to deprotect the amino

group and react it with another N-protected amino acid and repeat the rest of
the steps. Alternatively, to get a dipeptide from it, we can deprotect both the
protecting groups present at the amino end and the carboxy end. 21
Block 4 Peptides, Proteins and Carbohydrates

Having understood the above sequence of steps to be followed for the

synthesis of a dipeptide, answer the following SAQ.

SAQ 4
Write the steps for the preparation of the peptide Gly-Ala.

SAQ 5
What could be the major problems associated with the above discussed
method of synthesis of peptides?

SAQ 6
Write all the possible structures of the dipeptides formed by the amino
acids glycine and valine.

13.7 MERRIFIELD SOLID PHASE SYNTHESIS

A major advancement in the syntheses of polypeptides was introduced by
R. Brace Merrifield in 1962. This technique is also known as polymer
supported synthesis and uses a polymer. Merrifield used a polystyrene in
which about 5% of the phenyl groups carried a chloromethyl (-CH2Cl) group in
their para positions. This functionalisation of the phenyl ring can be done by
Friedel -Crafts alkylation as given below:
Robert Bruce
Merrifield
(15th July, 1921- &CH &CH 2&CH &
14th May, 2006)

He was awarded ClCH 2OCH 2CH 3, SnCl 4

Nobel Prize in
Chemistry for the &CH 3CH 2OH
year 1984.
polystyrene

Polystyrene is a &CH &CH 2& CH &

polymer of styrene
which is
ethenylbenzene.

CH CH 2
CH 2Cl
functionalised polystyrene

The chloromethyl groups are reactive towards nucleophilic substitution

styrene reactions.

22 The solid phase synthesis of a dipeptide involves the following steps:

Unit 13 Amino Acids and Peptides

Step 1: The amino protected amino acid is attached to the polystyrene chain.
O R
&
(CH 3)3COC &NHCHCOO + Cl&CH 2 polystyrene chain

O R O
(CH 3)3COC &NHCHC &OCH 2 polystyrene chain

Step 2: The deprotection of the amino terminus is done by treatment with

trifluoroacetic acid.
O R O
(CH 3)3COC &NHCHC &OCH 2 polystyrene chain

CF 3COOH, CH 2Cl 2

R O
NH 2CHC &OCH 2 polystyrene chain

Step 3: Coupling with second N-protected amino acid is done using DCC.

The peptide (or amide) bond formation takes place in solution similar
to the previous method discussed in last sub-section. Here, the
difference is that the dipeptide formed is attached to the insoluble
resin.
R O O R'
NH 2CHC &OCH 2 polystyrene chain + (CH 3)3COC &NHCHCOOH

DCC

O R' O R O
(CH 3)3COCNHCHCNHCHCOCH 2 polystyrene chain + DCU

The DCU formed is removed by washing.

Step 4: Deprotection of the amino terminus of dipeptide

O R' O R O
(CH 3)3COCNHCHCNHCHCOCH polystyrene chain
2

CF 3COOH, CH 2Cl 2

R' O R O
NH 2CHCNHCHCOCH polystyrene chain
2
23
Block 4 Peptides, Proteins and Carbohydrates

Step 5: If we want to stop at the dipeptide stage and do not wish to synthesise
higher peptides, the benzyl ester bond is broken using HF to release
the dipeptide from the polymer chain.
R' O R O
NH 2CHCNHCHCOCH polystyrene chain
2

HF

R' O R
+ &
H3NCHCNHCHCOO + FCH 2 polystyrene chain

dipeptide

Alternatively, if we wish to synthesise higher peptides, then coupling with third

N-protected amino acid is done using DCC .This is followed by deprotection of
the amino protected group, as explained above in Steps 3 and 4, respectively.
These steps, i.e. Steps 3 and 4, are repeated till we get the desired peptide;
and then finally, Step 5 is performed to get the product.

You have now studied two methods of synthesis of peptides. At this stage, you
may be curious to know which one is more advantageous. Obviously, the solid
phase synthesis has the following advantages over the previous method.

The polymer beads carrying the peptide chain are insoluble in the solvents
used in the synthesis. Hence, the product can be purified by simply filtering
and washing to remove the excess reagent and the byproducts.

In 1969, the enzyme ribonuclease was synthesised by Merrifield using solid

support. He designed a machine by which the steps required for the synthesis
could be performed automatically. This made the synthesis of peptides much
simpler and less time consuming.

The synthesis of ribonuclease required the addition of 124 N-protected

amino-acids and coupling reactions and 11,931 operations. The automated
machine made it possible in much shorter time and the intermediate stages of
isolation were also not required.

Similarly, synthesis of insulin involving 51 amino acids in two separate chains

comprising more than 5000 operations was achieved only in few days using
the automated procedure.

Automation in the protein synthesis has helped in the confirmation of the

structure of polypeptides obtained by degradation of chains. Also, new
proteins and polypeptides can be easily syntheised which are useful and have
better activity as compared to the ones available in nature. These new
synthetic options find applications in the field of medicine and biology.

13.8 LAB DETECTION OF AMINO ACIDS

The amino acids can be tested in the laboratory by the following methods.

1. Complexation with Cu2+ Ions

Aqueous solution of amino acids on addition of copper sulphate solution gives

24 a deep blue colour. The complex formed has the following structure:
Unit 13 Amino Acids and Peptides
H2N
O CO
RCH
Cu CHR
CO O
H2N
2. Ninhydrin Test

In Sec. 13.4 above, you have read about the reaction of amino acids with
ninhydrin.

Ninhydrin was accidentally discovered by Siegfried Ruhemann in 1910. He

was an German- English chemist .The reaction of amino acids and ninhydrin
was observed by him in the same year.

Ninhydrin is a white solid soluble in water, ethanol, and acetone etc. It reacts
with ammonia, primary and secondary amines, and peptides to form a blue-
purple coloured compound which is also known as Ruhemann's purple.

The mechanism of the formation of the blue colour is given below: Siegfried Ruhemann
th
4 January,1859–
O September, 1943)
O
C
C OH
&H2O N&CHCOOH
C
+ H2N&CHCOOH C This is a very
C R sensitive test and is
C OH also given by some
O
O β-amino acids
&CO 2
ninhydrin (3- amino acids) and
O some peptides as well
O
C as proteins, especially
H2O C on warming.
CH&NH 2 + CHO
CH &N CH
C
R C R
O
ninhydrin &H2O O Ninhydrin is also used
as a reagent for the
O O detection of latent
O& O fingerprints.
C C
+ C C
CH &N C &H
C &N C
C C
C C
O O
O O
blue-violet

13.9 SUMMARY
In this Unit, you have studied that

 20 amino acids occurring in proteins are L amino acids.

 The convention to use a three letter code, as an abbreviation, for each

amino acid. The single letter code are also used to denote the amino
acids.

 Amino acids exist as inner salts called zwitterions.

 At isoelectric point, pHi , the amino acid is stationary in an electric field,

i.e., it migrates neither to the negative pole nor to the positive pole
because the charges on it are balanced. 25
Block 4 Peptides, Proteins and Carbohydrates

 2- Amino acids can be synthesised from potassium

1,2-benzenedicarboxylic imide, aldehydes (Strecker synthesis) and
2-halo carboxylic acids.

 The reactions of amino acids include the usual reactions of the carboxy
and the amino group. For example, they undergo esterification
(characteristic of the carboxy group) and alkanoylation (characteristic of
the amino group) reactions.

 Amino acids give a blue-violet color with ninhydrin.

 In peptides, 2-amino acids are joined by the amide linkages which are
formed by the reaction of the carboxy group of one amino acid with the
amino group of another amino acid. These amide bonds are called peptide
bonds.

 For the selective synthesis of a particular peptide, we have to protect i) the

N-terminal of one amino acid and ii) the C-terminal of the other amino acid,
and then iii) combine or react the two protected amino acids to give the
desired dipeptide using the carboxy group activation.

 Various protecting groups such as benzyloxycarbonyl, tert-butoxycarbonyl

and phthaloyl groups are used for protecting the amino group of the amino
acids.

 The polymer supported solid phase synthesis is advantageous because

the product can be purified by simply filtering and washing to remove the
excess reagent and the byproducts.

 Amino acids can be detected in the laboratory by complexation with

Cu2+ ions and ninhydrin test.

13.10 TERMINAL QUESTIONS

1. Which properties of amino acids indicate their zwitterionic nature?

2. Write the structures of leucine and isoleucine.

3. Name the amino acids which contain a basic group in the side chain.

4. Outline the Strecker synthesis of tyrosine.

5. Which protecting groups can be used for the protection of amino group of
the amino acid?

6. How can benzyloxycarbonyl group be removed from the N-

protected amino acid/peptide?

13.11 ANSWERS
Self-Assessment Questions
1. i) Methionine or cysteine

ii) Phenylalanine or Tyrosine or Tryptophan

26 iii) Glutamic acid or Aspartic acid

Unit 13 Amino Acids and Peptides

2. a) The order of priority of substituents is

+
H3N > COO > CH2OH > H

The given Fischer projection can be converted to another Fischer

projection given below by interchanging twice two substituents.

N + H3

OOC CH2OH

Overlooking H and moving from the substituents of highest priority

to lower priority, the direction is anticlockwise, so the configuration
is S.

b) Similarly, we can arrive at the configuration by following the steps

given in 2 a) above and get the configuration as R.

3. ClCH2CH2SCH3

O CH 3 O

4. (CH 3)3COCNHCH 2COOH + H2N CH &COCH 2C6H5 DCC

Boc &Gly Ala &OCH 2C6H5

O O CH 3 O

(CH 3)3COCNHCH 2CNHCH &COCH 2C6H5

Boc &Gly &Ala &OCH 2C6H5
+
1. H , H2O
2. H2, Pd&C
O CH 3
+ & + C6H5&CH 3 + CO2 + CH2 C(CH 3)2
H3NCH 2CNHCHCOO
Gly &Ala

5. It involves a large number of steps such as protection, activation,

coupling and deprotection. This is time consuming and involves
purification at each step. The overall yield obtained is also low.

6. i) C(CH 3)2 ii) H2N&CHCO&NH &CH2COOH

H2NCH 2CONH &CHCOOH C(CH3)2

iii) H2NCH2CONH&CH2COOH iv) H2N&CHCONH &CHCOOH

C(CH 3)2 C(CH 3)2

Terminal Questions
1. i) They form strong crystal lattices like ionic compounds.

ii) They decompose and not melt on heating. 27

Block 4 Peptides, Proteins and Carbohydrates

iii) They are more soluble in water as compared to non-polar

solvents.

iv) They have large dipole moments.

2. Structures of leucine and isoleucine are given below.

CH3 NH2
CH3 NH2
CH3CH2CH CHCOOH
CH3CHCH2 CHCOOH

leucine isoleucine

3. Lysine, arginine and histidine

O NH2
NH3, HCN H+, H2O
4. HO CH 2CH HO CH 2CHCN

NH2
HO CH 2CHCOOH

tyrosine

5. Benzyloxycarbonyl, tert-butoxycarbonyl and phthaloyl groups are some

such protecting groups.

6. By hydrogenolysis i.e. reaction with H2 in the presence of a transition

metal catalyst.

28