SIDE REACTIONS IN PEPTIDE SYNTHESIS

PRESENTED BY: SHILPA R

JAYASHRITHA R

M-PHARM (PHARMACEUTICAL CHEMISTRY)

 PEPTIDES:

• Peptides are short chain of amino acids linked by peptide bonds.

• A Peptide bond is a chemical bond formed between two molecules, when carboxylic acid of one
molecule reacts with amino group of other molecule releasing water molecule.
• The resulting CO-NH bond is called peptide bond and the molecule is an amide,
SIDE REACTIONS IN PEPTIDE SYNTHESIS:

Peptide synthesis involves condensation of two or more amino acids which seems to be easier but
requires specialized techniques.
Since all the amino acids have basic skeleton but vary in their side chains, and their nature such as acidic,
basic or neutral depending on the presence or absence of functional groups.
These side chains are prone to side reactions during the process of synthesis either due to interaction with
the solvent used for synthesis or during the process of the deprotection of the specific groups. Reactions
apart from coupling reaction in peptide synthesis called side reactions in peptide synthesis.
 TYPES OF SIDE REACTIONS:
1. Side reaction by peptide deletion
2. Side reaction initiated by proton abstraction
a) Racemization
b) Undesired cyclization
c) O-acylation
3. Side reaction initiated by protonation
a) Racemization
b) Undesired cyclization
c) Alkylation
d) Chain fragmentation
4. Side reactions due to overactivation
5. Side reactions related to individual amino acid residues.
SIDE REACTION BY PEPTIDE DELETION:
• Peptide deletion reactions are the ones in which a group, side-chain, one or multiple amino acids are
eliminated from the peptide sequence, as a result of the undesired side-reaction.
• Deletion reactions can cause synthetic and further, purification issues. Ultimately, deletion reactions
can impact the stability of peptide products upon processing and/or storage.
• Diketopiperazine (dkp) formation is one of the most problematic deletion reactions in peptide
synthesis. It most commonly occurs at the C-terminal amino acids during the Fmoc cleavage.
Nevertheless, diketopiperazine formation can strike other positions further down the peptide synthesis,
and it does not necessarily have to impact only C-terminal amino acids. For example, depsipeptides are
especially susceptible (X = O-Peptide) to diketopiperazine (DKP) formation. It is because the hydroxyl
derivatives are better leaving groups than the amino derivatives. The reaction can proceed either in
acidic or basic condition.
SIDE REACTION BY PEPTIDE DELETION
SIDE REACTION INITIATED BY PROTON ABSTARCTION:

Abstraction of acidic proton in presence of a base from carboxyl group results in carboxylate anion which
prevents the formation of another anionic ester at α-carbon. Therefore, this anion prevents the elongation
of peptide chain due to the absence of carboxyl group to form a peptide bond
TYPES:

1. Racemization
a) By Direct abstraction of α-proton
b) By forming azlactones

2. Cyclization
a) O – Acylation
1. Racemization

Mechanism of racemization was involved in different steps they are:

a) By Direct abstraction of α-proton:
When an amino acid which is attached with a protecting group, is treated with a base, proton
abstraction occurs at alpha- carbon resulting in the formation of carbanion which can be attacked by any
electrophile resulting in undesired reaction which changes the stereochemistry of the amino acid.
b) By forming azlactones:

• The keto group of the amide bond undergoes ketoenol tautomerism to form a hydroxyl group which
upon treatment with a base, abstracts a proton from the hydroxyl group resulting in formation of
negatively charged oxygen. This initiates the activating group (X) to leave the carboxylic end. Electron
rich oxygen attack on electron deficiency carbon result in formation if azlactones.
2. Cyclization

During peptide synthesis (solid phase), presence of benzyl ester can cause premature cleavage of
the chain from insoluble support. The esters formed upon cleavage, undergoes cyclization to form
diketopiperzines.

This cyclic compound, when subjected to hydrolysis, leads to amide bond cleavage, as a result, the
dipeptide is obtained with a different stereochemistry making it inactive.
a) O – Acylation:

• When an amino acid is treated with a base such as tertiary amine, it abstracts the proton and converts
alcohols or phenols to alcoholates or phenolates.

• The formed alcoholate/phenolate, then reacts with an acylating agent and facilitates acylation at the
electron rich oxygen atom. Since the acylation occurs at the nucleophile (O- ), the reaction is named as
O-Acylation.
• p-Hydroxy alanine (tyrosine) is treated with a tertiary amine which acts as proton abstractor, and also
with p-Nitrophenyl ester, as a result, the acylated product p-Acyl oxy phenyl alanine (Acyl tyrosine)
is formed along with p-nitrophenol.
SIDE REACTION INITIATED BY PROTONATION:

a) Racemization
b) Undesired cyclization
c) Alkylation
a) Racemization

• It is an acid catalyzed reaction which involve protonation of carbonyl oxygen that results in the
formation of a carbocation.
• Proton abstraction then occurs at the adjacent carbon next to carbocation and therefore forms a double
bond by sharing the electrons
• This produces enolized products which do not retain their stereochemistry and lose their chiral purity.It
requires a strong acid as protonation does not occur with weak acids. Racemization by protonation
occurs during the process of deprotection of the groups using strong acids like HB or HF resulting in
loss of chirality
b) undesired cyclization

• The products obtained by cyclization via protonation are same as that of products obtained by
cyclization via proton abstraction. The only difference is that, former occurs in presence of acids where
as latter occurs in the presence of the base
• Eg: By taking dipeptide (Aspartyl glycine) of which carboxylic acid end of aspartic acid is protected
by oxy benzyl group. In the resulting products, the protecting group leaves as hydroxy toluene and the
dipeptide forms a cyclic molecule which is a succinamide derivative
c) alkylation

• Formation of carbocation is the general step during the removal of protecting groups from amino acids
in presence of an acid.
• These carbocations then act as alkylating agent to any nucleophilic centers and undergo intramolecular
rearrangement to form the alkylated amino acid.

Alkylation by intramolecular rearrangement

d) Chain fragmentation

Chain Fragmentation The amide bonds linking amino acids to each other to create the backbone of a peptide chains
are stable enough to withstand the usual rigors of peptide synthesis.
Under the influence of strong acids, an acyl group attached to the nitrogen atom of a serine residue migrates to its
hydroxyl oxygen. Such an N O shift takes place also when the acyl group is a part of a peptide chain.
This reaction, which in all likelihood proceeds via cyclic intermediates, is easily reversed by treating the product
with aqueous sodium bicarbonate but partial hydrolysis of the sensitive ester bond will lead to fragmentation of the
chain.
The reaction of the fragmentation in which a dipeptide (serine and alanine) forms cyclic intermediate in presence of
acid followed by acyl group attached to the nitrogen atom of serine residue shift to its hydroxyl oxygen and its
hydrolysis to form two different amino acids.
Reaction showing the fragmentation of pepetides
4. SIDE REACTIONS DUE TO OVERACTIVATION
• Overactivation occurs in the process of acylation of amino acid where the carboxyl component is too
powerful to be acylated. Therefore, acylation occurs primarily at the amino group which is exposed
for peptide bond formation followed by acylation of hydroxyl group of the carboxylic component.
• Sometimes, during coupling of amino acids, using a coupling agent like N, N’- disubstituted
carbodiimide, subtle intermediates are formed such as O-Acylisourea which give rise to symmetrical
anhydrides and azlactones and can also undergo rearrangement to N-acylurea derivatives.

Reaction showing complete overactivation

5. SIDE REACTIONS RELATED TO INDIVIDUAL AMINO ACID
RESIDUES
• Amino acids with no functional side chains are not involved in side reactions which can be possible
only in case of alanine and leucine as there is no side chain in alanine whereas in leucine, branching is
at γ carbon which is far away from the α-carbon to undergo a side reaction. In case of valine and
isoleucine, branching at β-carbon atom leads to steric hindrance which lowers the rate of coupling
reaction and therefore, cause an increase in the extent of unimolecular side reactions. the condensation
reaction with carbodiimide where isoleucine undergo a unimolecular side reaction (higher rate of
reaction) by forming acylated intermediate (O-Acylisourea) [38] followed by ureides. However, the
same O-Acylisourea when treated with a primary amine (amino acid) forms a peptide bond and has
lower rate of reaction [9] [44] . Due to higher rate of reaction, formation of ureides is dominated over
peptide bond formation.
Formation of ureides (left) and peptide bond (right) from O - Acylisourea