Determination of Primary structure of Peptides Topic 2

what are peptides

Brief Classification of Peptides

Nomenclature of Peptides

Determination of Primary Structutre of Protein

Methods of synthesis of Peptides

Problems for Practice

Course Content

Developed by

Rakesh Singh

Associate Professor in Chemistry , Govt. Degree College, Kathua

Department

Department of Higher Education UT of Jammu and Kashmir

Paper

Organic Chemistry

Class

Semester 3

(UG-CBCS, General)

University/College

Jammu University, Cluster University, Autonomous College- GCW Parade

Syllabus

Determination of Primary structure of Peptides by degradation Edmann degradation (N-terminal) and C–

terminal (thiohydantoin and with carboxypeptidase enzyme). Synthesis of simple peptides (upto

dipeptides) by N-protection (t-butyloxycarbonyl and phthaloyl) & C-activating groups and Merrifield solid-

phase synthesis. Note : Overview of Primary, Secondary, Tertiary and Quaternary Structures of Proteins

is in syllabus of Cluster University and GCW Parade.

Learning Objectives

Unit : Peptides and Proteins

Topic 1 : Determination of Primary structure of Peptides

To Understand

Topic 2 : Methods of synthesis of Peptides

Peptides and Proteins are fundamental components of cells which perform important

biological functions. Structurally, proteins and peptides are quite similar, as both are made

up of chains of α-amino acids that are held together by peptide bonds (also called amide

bonds). The basic distinguishing factors are size and structure. Peptides are smaller than

proteins. Traditionally, peptides are defined as molecules that consist of between 2 and 50

amino acids, whereas proteins are made up of 50 or more amino acids. In addition, peptides

tend to be less well defined in structure than proteins, which can adopt complex

conformations known as secondary, tertiary, and quaternary structures. All proteins are

polypeptides but not all polypeptides are proteins.

Structure of α- amino acid

1.2 Peptides:

Peptides are defined as condensation products of two or more amino acids formed by

reaction between amino group of one amino acid molecule with carboxylic group of other

amino acid molecule. In peptide, amino acids are linked together by amide bonds. The amide

bond between the amino group of one amino acid and the carboxyl of another is called a

peptide bond. For example , when two molecules of glycine combine , it results in formation

of dipeptide along with release of water as condensation by-product.

The amide linkage

is called peptide bond or peptide linkage.

1.1 Peptides and Proteins

COO

+ NH

COO

CONH CH

COO

+ H

Glycine Glycine Glycylglycine(Dipeptide) + Water

1.3 Classification of Peptides:

Depending upon number of amino acids involved in peptide formation, peptides have been

classified into following types :

a) Dipeptides : A peptide formed by condensation of two amino acids is called dipeptide.

b) Tripeptide : A peptide formed by condensation of three amino acids is called tripeptide.

c) Tetrapeptide : A peptide formed by condensation of four amino acids is called tetrapeptide.

d) Polypeptide : A peptide formed by condensation of more than four amino acids is called

polypeptide.

For example :

• Peptides possess a free H

-- group on one end and a free –COO

group at the other end.

• The amino acid having free H

-- group is called N-terminal amino acid residue

• The amino acid having free -COO

group is called C-terminal amino acid residue.

• While writing formula of peptide, we proceed from left with the N-terminal amino acid

residue to the right with C-terminal amino acid residue. So it is a convention to write N-

terminal amino acid on left hand side and C-terminal amino acid of right hand side.

1.4 Nomenclature of Peptides:

The names of amino acids which constitute a peptide are written from N-terminal i.e. starting

from L.H.S to C-terminal i.e. R.H.S. While writing the names , the suffix “ine” of all amino

acids, except the C-terminal amino acids is replaced by “yl” . Sometimes three letter

abbreviations of amino acids are also used for writing name of polypeptide.

NH CH

–

N- Terminus Polypeptide (n =2-50) C-Terminus

Glycylalanine (Gly-Ala)

Glycylalanylvaline

1.5.1 Primary Structure : It refers to the sequence in which various amino acids are present

in a polypeptide or protein are linked to each other. For example, the hormone insulin has

two polypeptide chains, A and B, shown in diagram below (image credit :OpenStax Biology)

Importance of Primary Structure:

It must be noted that sequence of amino acid is very important because change of just one

amino acid in a protein’s sequence can affect the protein’s overall structure and biological

functions. e.g. change of just one amino acid in polypeptide sequence of haemoglobin causes

a disease called sickle cell anaemia. In sickle cell anemia, one of the polypeptide chains that

make up hemoglobin, the protein that carries oxygen in the blood, has a slight sequence

change. The glutamic acid that is normally the sixth amino acid of the hemoglobin β chain

(one of two types of protein chains that make up hemoglobin) is replaced by a valine. What

is most remarkable to consider is that a hemoglobin molecule is made up of two α chains and

two β chains, each consisting of about 150 amino acids, for a total of about 600 amino acids

in the whole protein. The difference between a normal hemoglobin molecule and a sickle cell

molecule is just 2 amino acids out of the approximately 600.

1.5 Overview of Primary, Secondary, Tertiary and Quaternary Structures of Proteins

1.5.2 Secondary structure:

Secondary structure refers to the conformation, which a polypeptide chain assumes due to

hydrogen bonding. Depending upon size of side chain R- attached to alpha carbon, the

proteins show two types of secondary structure as :

i) α-helix (by Linus Pauling in 1951): If size of R-group is quite large , then proteins possess

α-helix structure at secondary level. Alpha helix structure arises due to intramolecular H-

bonding between , the carbonyl (C=O) of one amino acid residue to the N-H of forth amino

acid residue. This causes the polypeptide chain to coil up into a spiral structure (helical

structure) that resembles a curled ribbon, with each turn of the helix containing 3.6 amino

acids. The R groups of the amino acids point outward from the α-helix, where they are free

to interact. The α-helix may be right handed or left handed. However, Moffitt in 1956 ,

theoretically proved that for L-amino acid , right handed helix is more stable. Hence, proteins

always possess right handed helical structure. i.e. polypeptide chain turns clockwise to form

α-helix.

ii) β-pleated sheet structure (Linus Pauling in 1951): If size of R-groups is small or of moderate

size, then In a β-pleated sheet structure, two or more segments of a polypeptide chain line

up side by side, forming a sheet-like structure held together by hydrogen bonds, between

carbonyl groups of chain and amino groups of other chain, while the R groups extend above

and below the plane of the sheet. The strands of a β pleated sheet may be parallel, pointing

in the same direction (meaning that their N- and C-termini match up), or antiparallel, pointing

in opposite directions (meaning that the N-terminus of one strand is positioned next to the C-

terminus of the other).

Many proteins contain both α helices and β pleated sheets, though some contain just one

type of secondary structure.

(Image credit: OpenStax Biology)

1.5.3 Tertiary structure :

The overall three-dimensional structure of a polypeptide is called its tertiary structure. At normal p

and temperature, each protein molecule acquires tertiary structure which is most stable. For example

, fibrous proteins which have almost same secondary structure through out their length possess rod

like or rope like as its tertiary structure. (e.g. α-keratin, the major protein of hair and wool). On the

other hand, globular proteins, don’t have same secondary structure through out their length. Some

parts may have α-helix, some other parts may have β-pleated sheet, whereas some other parts may

have random coils. In such cases, the entire protein molecule may fold up to give spherical shape to

protein molecule. So, tertiary structure of globular protein is spherical.

The tertiary structure is primarily due to various interactions like hydrogen bonding, ionic bonding,

dipole-dipole interactions, and London dispersion forces, due to which protein molecule acquires a

particular three dimensional shape. Also important to tertiary structure are hydrophobic interactions,

between nonpolar, hydrophobic R groups. In addition the disulfide bond, i.e. covalent linkages

between the sulfur-containing side chains of cysteines, are much stronger than the other types of

bonds that contribute to tertiary structure. They act like molecular "safety pins," keeping parts of the

polypeptide firmly attached to one another.

Image credit OpenStax Biology

1.5.4 Quaternary structure:

Many proteins are made up of a single polypeptide chain and have only three levels of structure i.e.

Primary, secondary and tertiary. However, some proteins are made up of multiple polypeptide chains,

also known as subunits. When these subunits come together, they give the protein its quaternary

structure. For example, haemoglobin, which carries oxygen in the blood and is made up of four

polypeptide chains or subunits [two each of the α (each chain with 141 amino acids )and β (each chain

with 146 amino acid residues ) types]. Another example is DNA polymerase, an enzyme that

synthesizes new strands of DNA and is composed of ten subunits -

In general, the same types of interactions that contribute to tertiary structure (mostly weak

interactions, such as hydrogen bonding and London dispersion forces) also hold the subunits together

to give quaternary structure. So quaternary structure of protein refers to determination of number of

subunits and their overall arrangement in aggregate protein molecule as a whole. For example in

haemoglobin , the four subunits lie at the vertices of regular tetrahedron.

Credit : Image modified from OpenStax Biology's modification of work by the National Human

Genome Research Institute.

1.6.1 Step 1: Determination of Amino acid Composition :

• The given polypeptide/protein is completely hydrolysed to its constituent amino acids.

• Hydrolysis is preferably done by 6N HCl at 373-393K or by enzymes.

• However, alkaline hydrolysis is not preferred because it causes racemization.

• The mixture of amino acids thus obtained is separated and individually identified by either

ion-exchange or gas chromatography.

• The weights of each of the amino acid is noted .

• From their weights, the number of moles of each of the amino acid is determined.

• Hence number of each type of amino acid present in given polypeptide or protein is then

calculated.

However, now a days the whole process is automated and is done with the help of an

instrument called amino acid analyser.

It requires very small amount of peptide (10

-5

to 10

-7

1.6.2 Step 2 : Sequencing of amino acids present in given polypeptide or protein :

After determination of amino acids composition (i.e. number of each type of amino acid

present in polypeptide), the next and the most important step is to determine sequence of

amino acids in given polypeptide/protein. This is done by terminal residue analysis and partial

hydrolysis.

1.6.3 Terminal Residues analysis : The terminal amino acid residue written on extreme left

of polypeptide chain possesses free amino group and is called N-terminal amino acid, where

as the amino acid residue present on extreme right of polypeptide chain possesses free -

COOH group and is called C-terminal amino acid.

1.6 Determination of Primary Structure of Peptide

Sequance of Amino Acids present in a given plypeptide or protein

Determination of Aminoacid Composition

Steps involved in determination of Primary Structure

The whole process of terminal residue analysis involves treating a given polypeptide with a

suitable reagent, which selectively reacts with either N-terminal amino acid or C-terminal

amino acid. As a result the N-terminal or C-terminal amino acid gets labelled which is then

selectively removed by partial hydrolysis of polypeptide and hence identified. The resulting

degraded peptide is then again subjected to same treatment and one by one , the whole

sequence of amino acids in the given polypeptide is determined.

The sequencing of amino acids can be done by following two methods:

a) N-Terminal residue analysis b) C-terminal residue analysis

a) N-terminal residue analysis :

It is done by following methods:

(i) Edman’s method (Edman’s degradation)

This method was developed by Pehr Edman. The Edman degradation reaction was automated

in 1967 by Edman and Beggs. In this process

• The given peptide is reacted with phenyl isothiocyanate (PITC) under mildly alkaline

conditions when the , -NH

group of N-terminus of polypeptide reacts to give a phenyl

thiocarbamoyl derivative (PTC-peptide).

• The PTC-peptide upon mild hydrolysis with HCl, selectively removes, the N-terminal amino

acid as phenyl thiohydantoin (PTH), while rest of polypeptide chain remains intact.

• The PTH so obtained is then separated from reaction mixture and then identified

chromatographically. This is done by comparing with PTC obtained from known amino

acids. Thus, the N-terminal amino acid get identified.

• The degraded peptide is then subjected to same process again and in this way the whole

sequence of amino acids is determined.

NH CH

–

N- Terminus Polypeptide (n =2-50) C-Terminus

Note : Theoretically this process can be can be repeated over and over again till whole

sequence is determined, but practically , it is possible only up to 20 amino acids. However,

nowadays, the automated Edman degradation (the protein sequenator) is used widely, and it

can sequence peptides up to 50 amino acids.

(ii) Sanger’s method or DNFB method : In this method

• The given peptide is treated with 2,4-dinitrofluorobenzene (DNFB), commonly known as

Sanger’s reagent.

• DNFB is very reactive in nucleophilic displacements with amines but not amides.

• The reagent reacts with free amino group of the N-terminal amino acid of peptide to form

N-2,4-dinitrophenyl (DNP) derivative.

• The DNP derivative of polypeptide upon hydrolysis with dilute HCl gives 2,4-dinitrophenyl

(DNP) derivative of N-terminal amino acid along with mixture of amino acids.

• The 2,4-dinitrophenyl (DNP) derivative of N-terminal amino acid is isolated and identified

chromatographically.

Demerits : By this method, only terminal amino acids can be identified.

b) C-terminal residue analysis :

The C-terminal residue is determined by the use of either a chemical reagent or the

enzyme carboxypeptidase. The two commonly used methods are as :

i) Enzymatic Method ( with carboxypeptidase enzyme) :

• The carboxypeptidase enzyme (obtained from pancreas) specifically hydrolyses the

peptide bond adjacent to free carboxyl group.

• Hence when given polypeptide is treated with carboxypeptidase enzyme, it selectively

separates the C-terminal amino acid along with formation of degraded peptide.

• The C-terminal amino acid so set free is identified. The process is repeated on degraded

peptide and in this way the whole sequence of amino acids is determined.

Polypeptide

NH CH

NHCH

NH CH

N NHCH

Mixture of Aminoacids

2,4-dinitrofluorobenzene

DNP derivative of amino acids

NH CH

Free C-terminal aminoacid

Polypeptide

Carboxypeptidase

NH CH

Degraded peptide

ii) Thiohydantoin method : In this method,

• First, side-chains of polypeptide having carboxyl groups and hydroxyl groups are protected as

amides or esters.

• Then, the C-terminal carboxyl group is activated as an anhydride and reacted with thiocyanate.

• The C-terminal acyl thiocyanate peptide product automatically rearranges to a thiohydantoin

incorporating the penultimate C-terminal unit.

• The peptidyl thiohydantoin so formed upon hydrolysis with acid gives thiohydantoin of C-terminal

amino acid and degraded peptide. The of C-terminal amino acid is then identified.

• Thus, repetitive analyses is conducted on the degraded peptide and one by one whole sequence

of peptide is determined.

iii) Hydrazinolysis Method :

• In this method, the given peptide is treated with anhydrous hydrazine at 373 K.

• The hydrazine forms aminoacyl hydrazide with every residue except the C-terminal amino

acid.

• The C-terminus is thus readily identified by chromatographic procedures.

• The disadvantage of hydrazinolysis is that the entire sample is used to determine just one

residue.

Note 1 : The first ever primary structure of protein was determined by Fredric Sanger, a British

Chemist and he was awarded Nobel Prize in 1958 for the same.

Note 2 : In practice it is not feasible to determine the sequence of all the residues in a long

peptide chain by the stepwise removal of terminal residues. Instead, the chain is subjected to

partial hydrolysis (acidic or enzymatic), and the fragments formed dipeptides, tripeptides, and

so on-are identified, with the aid of terminal residue analysis. When enough of these smaller

fragments have been identified. it is possible to work out the sequence of residues in the

entire chain.

Synthesis of polypeptides is of great significance. In recent times scientists have been

successful in synthesizing some important polypeptides in laboratory. E.g. the insulin used for

the treatment of diabetes was earlier obtained by extraction from the pancreas glands of

cows and pigs. However, since the early 1980s, the “synthetic” insulin prepared by

recombinant DNA technology has replaced “natural” insulin. Synthetic insulin is not only

identical to human insulin, it is safer as well as less expensive than insulin obtained from

animals.

Theoretically , it seems easy to join amino acids one by one starting from one terminal, but in

actual practice , it is not so simple as, there are certain difficulties involved . The two

important methods which are used for synthesis of polypeptides are as:

i) Classical peptide synthesis ii) Merrifield Solid phase peptide synthesis

1.7: Synthesis of Peptides

NH CH

Heat

NH NH

+ NH

NH NH

Hydrazide

Free terminal aminoacid

Polypeptide

It is also known as solution phase synthesis of peptides. The objective of peptide synthesis is

to join amino acids in a specific sequence by peptide bond . Scientists have designed very

effective methods and reagents for peptide bond formation, so that the joining of amino acids

by amide linkages is not difficult. The real difficulty is in ensuring, the desired sequence. For

example, If we want to synthesize a dipeptide, say Glycylalanine, by treating, glycine with

alanine in presence of some dehydrating agent, there is equal chance that amino group of

either of amino acid can react with -COOH group of other amino acid as well as of its own acid

, thus forming four products instead of one.

Glycine + Alanine Gly-Gly + Gly-Ala + Ala-Ala + Ala-Gly + H

To overcome this difficulty, the amino group of glycine and the carboxyl group of alanine

must be protected so that they cannot react under the conditions of peptide bond formation.

The formation of peptide bond with formation of desired dipeptide can be represented with

the help of following equation. (where X and Y are -NH

and -COOH protecting groups,

respectively)

Thus, dipeptide synthesis requires following sequence of four steps :

Step 1: Protection of the -NH

group of the N-terminal amino acid and the -COOH group of

the C-terminal amino acid.

Step 2 : Activation of -COOH group of N-protected amino acid.

Step 3 : Reaction between two protected amino acids, so as to join them via amide bond or

peptide bond formation.

Step 4 : Deprotection of the -NH

group of the N-terminus and the -COOH group of the

C-terminus.

The same procedure can be further extended for synthesizing the higher peptides by using

same logic as outlined for the synthesis of dipeptides.

1.7.1 : Classical peptide synthesis

The protecting group used for protecting amino group of N-terminal amino acid and

carboxylic acid group of C-terminal amino acid must possess following characteristics:

(a) It should be easy to introduce.

(b) It should stable under experimental conditions.

Let us discuss, the synthesis of a dipeptide say Glycylalanine (Gly-Ala) :

Step 1a : Protection of amino group of N-terminal amino acid i.e. Glycine

The two most widely groups which are used for protection of amino group are

i) tert-butoxycarbonyl (Boc) group

ii) benzyloxycarbonyl (Z) group

The four reagents which are quite often used for protection of amino group are benzyl

chloroformate, di-tert-butyl dicarbonate (sometimes abbreviated Boc2O, where Boc stands

for tert-butyloxycarbonyl), N-ethoxycarbonylphthalimide and 9-fluorenylmethyl

chloroformate:

i) Protection by tert-butoxycarbonyl (Boc) group :

tert-butoxycarbonyl group { (CH

)

C-OCO-} is abbreviated as Boc. Protection by Boc is done

by treating the amino acid with di-tert-butyldicarbonate i.p.o. triethylamine as:

ii) Protection by benzyloxycarbonyl (Z) group :

Benzyloxycarbonyl group has been given symbol Z as per IUPAC system. Protection by Z is

done by treating the amino acid with bezyloxycarbonyl chloride or Benzyl chloroformate

( earlier known by name carbobenzyloxy chloride) i.p.o. NaOH at 5

iii) Protection by phthaloyl group :

Protection by Phthaloyl group is done by treating the amino acid with N-

ethoxycarbonylphthalimide i.p.o. aq Na

as depicted below.

Protection by Phthaloyl group can also be done by treating the amino acid with monoethyl

phthalate as:

iv) Protection by 9-fluorenylmethoxycarbonyl (Fmoc) group :

Protection by 9-fluorenylmethoxycarbonyl is done by treating the amino acid with N-9-

fluorenylmethyl chloroformate i.p.o. sodium carbonate and dioxane at 273K . E.g. protection

of amino group with Fmoc group is done as depicted below.

Step 1b : Protection of -COOH group of C-terminal amino acid i.e. Alanine:

This is done by converting the -COOH group into its methyl, ethyl, or benzyl ester. e.g In our

example , of synthesis Glycylalanine, Alanine is C-terminal amino acid and hence, its -COOH

group is protected as shown below :

Step 2 : Activation of the carboxyl Group of N-protected amino acid : It is done by following

methods:

i) By converting -COOH group into acid chloride : In earlier methods, carboxyl group of N-

protected amino acid was activated by converting it to an acyl chloride This is done by treating the

N-protected amino acid with SOCl

or PCl

5 .

ii) By converting into mixed anhydride : The acid chlorides as formed in above method are actually

more reactive than necessary. Due to which their use leads to complicating side reactions Hence, a

much better method is to convert the carboxyl group of the N-protected amino acid to a mixed

anhydride using ethyl chloroformate as described below.

iii) By converting -COOH group into a reactive p-nitrophenyl ester: This is done by treating N-

protected amino acid (Boc-glycine in our example) with p-nitrophenol i.p.o. N,N

’

dicyclohexylcarbodiimide (DCC) under mild conditions as:

–

ClH

Alanine

Methyl ester of Alanine

Benzyl ester of Alanine

Step 3 : Formation of Peptide Bond :

The N- protected and C- activated amino acid is then treated with C-protected amino acid having free

amino group, when we get desired N- and C-terminal protected dipeptide. For example, for synthesising

Glycylalanine, a dipeptide, the N-protected and C-activated glycine is treated with C-protected Alanine,

when it results in formation of peptide bond as:

Step 4 : Removal of Protecting Groups :

Finally, the protecting groups are removed, to obtain the desired dipeptide. Depending upon the type of

protecting groups, following reagents and methods are commonly used as described below:

a) Removal of N-protecting groups : N- protecting groups are removed by following methods as ;

i) Removal of benzyloxycarbonyl group : Benzyloxycarbonyl (Boc) group is removed either by

catalytic hydrogenation with H

/Pd or by hydrolysis with HBr in cold CH

COOH.

ii) Removal of t-Butoxycarbonyl group : It can be removed by treating the N-protected dipeptide with

HBr/CH

COOH or by treatment with CF

COOH

iii) Removal of phthaloyl group : The Phthaloyl group is removed by means of hydrazinolysis

by treating the N-protected dipeptide with hydrazine hydrate in refluxing MeOH as :

b) Removal of Carboxyl or C-protecting groups :

The C-protecting groups are removed by hydrolysis with an aqueous base or acid under mild conditions.

Under mild conditions, only the ester get hydrolysed, where as amide bond remains intact. This is

because esters are more reactive than amides.

N- & C- terminal protected Glycylalanine (a dipeptde)

N CH

CH C

, H

Reflux for 1hr

CH C

C-protected glycylalanine

2,3-dihydrophthalazine-1,4-dione

N- & C- terminal protected Glycylalanine (a dipeptde)

HBR/CH

COOH

or CF

COOH

C-protected Glycylalanine

CH C

+ C

Benzyl esters can also be removed by catalytic hydrogenation with H

/Pd i.e. by hydrogenolysis.

1.7.2: Limitations of Classical Peptide Synthesis :

The classical peptide synthesis involves condensation of amino acid molecules in a step wise

manner. Hence, it can be presumed that this method cab used for thesis of polypeptides of any

length. However, in actual practice, it has been found that this method can be used to

synthesize polypeptides containing up to 10 amino acid residues. However, for the synthesis

of polypeptides with larger number of amino acids, this method can’t be used because of

following limitations :

i) Time consuming: The classical method of peptide synthesis requires several steps for each

new peptide bond formed, like protection, activation, condensation and deprotection .

Moreover, the new peptide synthesized at each step needs to be purified before repeating the

procedure . Hence, it requires lot amount of time, which makes this method quite time

consuming, even for a small peptide.

ii) Low yield. Since , classical peptide synthesis involves number of steps, and each step needs

purification of main product before proceeding to next step, the overall yield goes on

decreasing after each step even though each individual step has an excellent yield of 80%. For

example, if the yield of a dipeptide formed is 80% , then the yield of the tripeptide will be

80% of 80 i.e 64%. Similarly, the yield of tetrapeptide would be 80% of 64% i.e. 51.2%. If a

dodecapeptide is to be synthesized, the overall yield would be around 8.576%. Hence, it is

quite evident that the classical method is not useful for the synthesis of higher peptides.

CH C

–

Glycylalanine

(Dipeptide)

Benzyl ester of Glycylalanine

Toluene

/ Pd

The classical method of peptide synthesis has been used to synthesize a number of

polypeptides, including ones as large as insulin. However, the difficulties encountered in the

classical peptide synthesis make the method less useful as at every stage, the peptides need

to be purified. Furthermore, with each isolation and purification stage, significant loss of

peptide may occur, thus decreasing substantially the yield of final polypeptide. The difficulties

encountered in the classical peptide synthesis were overcome by R.B. Merrifield (Rockefeller

University, USA) . He devised a brilliant method for peptide synthesis called the solid phase

peptide synthesis in 1964. He was awarded Nobel Prize in Chemistry in 1984 for developing

solid-phase peptide synthesis method.

Principle : The principle involved in this is method is to synthesize the peptide residue by

residue while one end of the peptide remains attached to an insoluble solid support. The

insoluble support is a polymer of styrene crossed linked with about 2% p-divinylbenzene. In

addition, 5 % of the benzene rings carry chloromethyl group. The chloromethylated

polystyrene polymer is used in the form of small beads, which are porous. It is important to

note that protecting groups and other reagents are still necessary, but because the peptide

being synthesized is attached to a solid support, hence by-products, excess reagents, and

solvents can simply be rinsed off. When the desired polypeptide get synthesized, it is cleaved

from the polymer support and finally purified by HPLC.

Let us explain the main steps involved in Solid Phase Peptide synthesis

Step 1 : Protection of Amino group of C-terminal amino acid : The amino group of C-

terminal acid of desired peptide is protected by suitable protecting group. E.g. tert-

butoxycarbonyl chloride (Boc) can be used as :

Step 2 : Attachment of N-protected C-terminal amino acid to Polymer : The Boc protected

C- terminal amino acid is then attached to the polystyrene polymer via benzyl ester linkage by

1.7.3: Merrifield Solid-Phase Peptide Synthesis

heating it in presence of triethyl amine in a suitable solvent . The excess reagents are removed

by washing with suitable solvent.

Step 3 : Removal of Boc protecting group : The Boc protecting Polymer bound amino acid is then

removed by treating with trifluoro acetic acid and the salt of polymer bound amin acid formed is

converted into free form by treating with excess of triethylamine. Finally, the Polymer bound amino

acid is purified by washing.

Step 4 : Addition of next Boc-protected amino acid : The next Boc protected amino acid is

then linked to polymer bound amino acid by condensation reaction with the help of

dehydrating agent diisopropylcarbodiimide (DCC). The excess reagents are removed by

washing.

Step 5 : Removal of Boc-protecting group : The Boc-protecting group is removed by treating with

trifluoro acetic acid as :

Step 6 : Repetition of steps 4 & 5 : The steps 4 and 5 are repeated to add desired amino acids . By

repeating these two steps, we can add as many amino acids as we want and hence can synthesize the

desired polypeptide.

Step 7 : Separation of polypeptide chain from Polymer : When the desired polypeptide has been

synthesised, it is then separated from polymer by treating the Polymer bound polypeptide with

anhydrous HF as :

Note : Fmoc is preferably used instead of Boc as protecting group because with each new

addition of amino acid residue , the repetitive application of the acidic conditions to remove

Boc-protecting groups from each new residue slowly interfere with the synthesis by

prematurely cleaving some peptide molecules from the solid support and deprotecting some

of the side chains. The basic conditions for Fmoc removal eliminates the probability of these

problematic side reactions.

Advantages of Solid Phase Peptide Synthesis(SPPS) :

• The great advantage of solid-phase peptide synthesis is that purification of the peptide at

each stage involves simply rinsing the plymer beads (solid support) so to wash away excess

reagent, by-products, and solvents.

• Furthermore, the peptide being attached to a concrete solid support during the synthesis,

facilitates the all the steps involved in the synthesis of desired peptide to be carried out

by a machine in repeated cycles. Hence polypeptide synthesis can be carried out

Automated peptide synthesizers that can complete one cycle in 40 min and can perform

unattended operation of 45 cycles. So this saves lot of time. Though, it is not as efficient

as protein synthesis in the body, where enzymes directed by DNA can synthesize a protein

with 150 amino acids in about 1 min.

• An example automated peptide synthesis was the synthesis of ribonuclease, a protein with

124 amino acid residues. The synthesis involved 369 chemical reactions and 11,930

automated steps—all carried out without isolating an intermediate. The synthetic

ribonuclease not only had the same physical characteristics as the natural enzyme, it

possessed the identical biological activity as well. The overall yield was 17%, which means

that the average yield of each individual step was greater than 99%.

Problems for Practice :

Problem 1: What is meant by protection of group ? Name the conditions which a group must

satisfy to act as protecting group.

Problem 2: Illustrate various methods used for protecting N-terminal of Amino acid.

Problem 3: Illustrate various methods used for protecting C-terminal of Amino acid.

Problem 4: Write down various steps involved in Classical Peptide synthesis.

Problem 5: How solid phase peptide synthesis is superior to classical peptide synthesis.

Problem 6: Using Fmoc group as N-protecting group, describe synthesis of Alanylvaline by

classical peptide synthesis method.

(Hint : In this dipeptide, Alanine is N-terminal amino acid and Valine is C-terminal amino acid)

Problem 7: Using Boc group as N-protecting group, describe synthesis of Valylleucine by Solid

Phase peptide synthesis method.

(Hint: In this case Valine is N-terminal amino acid and Leucine is C- terminal amino acid.

General reaction given. Write all the steps.)

Alanine

Valine

CH C

Reference Books :

• Kotz, J.C., Treichel, P.M. & Townsend, J.R. General Chemistry, Cengage Learning India

Pvt. Ltd.: New Delhi (2009).

• Mahan, B.H. University Chemistry, 3rd Ed. Narosa (1998).

• Petrucci, R.H. General Chemistry, 5th Ed., Macmillan Publishing Co.: New York (1985).

• Morrison, R. T. & Boyd, R. N. Organic Chemistry, Dorling Kindersley (India) Pvt. Ltd.

(Pearson Education).

• Finar, I. L. Organic Chemistry (Volume 1), Dorling Kindersley (India) Pvt. Ltd. (Pearson

Education).

• Finar, I. L. Organic Chemistry (Volume 2), Dorling Kindersley (India) Pvt. Ltd. (Pearson

Education). ·

• Nelson, D. L. & Cox, M. M. Lehninger’s Principles of Biochemistry 7 th Ed., W. H.

Freeman.

• Berg, J.M., Tymoczko, J.L. & Stryer, L. Biochemistry, W.H. Freeman, 2002.

• Graham Solomons, T. W., & Fryhle, C. B. (2011). Organic chemistry 10th edition.

Some Important Links where students can have more e-resources:

1. Link for Vidya-mitra, Integrated E-Content Portal : http://vidyamitra.inflibnet.ac.in/

2. Link for e-PG Pathshala – Inflibnet : https://epgp.inflibnet.ac.in/

3. Link for INFLIBNET Centre Gandhinagar : https://inflibnet.ac.in/