Role Of Transgenic Animal In Target Validation-1.pptx
ödev Peptit Sentezleri- FKM620- Leyla hoca.pptx
1. Second semester 2022-2023
(İkinci Dönemi 2022-2023)
Sağlık Bilimleri Enstitüsü
Farmasötik Kimya Anabilim Dalı
“Noor El-huda” Khaled Daoud 1*
TC: 99726717132
1 Anadolu Üniversitesi, Sağlık Bilimleri Enstitüsü, Farmasötik Kimya Anabilim Dalı (Doktora Programi), Eskişehir, Türkiye.
* nourekd@anadolu.edu.tr
30 November 2022 (30 Kasım 2022)
Introduction to Peptide Synthesis*
*Fields, G. B. (2001). Introduction to peptide synthesis. Current protocols in protein
science, 26(1), 18-1.
Supervisor
Dr. Leyla Yurttaş
Peptide Synthesis Course
(Peptit Sentezleri- FKM620)
2. 1. Introduction
2. Development of solid-phase peptide-synthesis
methodology
3. The solid support
4. Coupling reagents
5. Synthesis of modified residues and structures
6. Protein synthesis
7. Side-reactions
8. Purification and analysis of synthetic peptides
9. Conclusion
10. References
Second semester 2022-2023
2 2 May 2023
5. What we will discuss?
We will provide an overview of:
The field of synthetic peptides and proteins
Discussing selecting the solid support and
common coupling reagents… etc.
Additional information on common side
reactions
Purification and analysis of synthetic
peptides
5 Fields, G. B. (2001). Introduction to peptide synthesis. Current protocols in protein science, 26(1), 18-1. 2 May 2023
7. What are Peptide Bonds?
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7
Peptide bonds: are amide
bonds that form between the
α-carboxyl group (-COOH) of
one amino acid and the α-
amino group (-NH2) of another
another amino acid.
This bond formation results in
the release of a molecule of
water and the formation of a
covalent bond between the two
amino acids.
Fields, G. B. (2001). Introduction to peptide synthesis. Current protocols in protein science, 26(1), 18-1.
8. What are Peptide Synthesis?
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8
Peptide synthesis:
is the process of creating
peptides, which are short
chains of amino acids linked
linked by peptide bonds.
Peptides play important roles
roles in biology, including as
hormones, neurotransmitters,
neurotransmitters, and
signaling molecules.
Fields, G. B. (2001). Introduction to peptide synthesis. Current protocols in protein science, 26(1), 18-1.
9. What are the methods of peptide synthesis?
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10
There are two main methods of peptide synthesis:
Chemical synthesis: involves linking amino acids one by one using
chemical reactions. There is two methods:
Solid phase synthesis
Solution phase synthesis
Biological synthesis: involves using living cells or organisms to
produce peptides.
The most widely used method of chemical peptide synthesis is solid-
phase peptide synthesis (SPPS).
SPPS: involves linking amino acids to a solid support and then using
using chemical reactions to remove the amino protecting groups
and form peptide bonds between the amino acids.
10. What is the applications of synthetic peptides
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11
Peptides play important roles in biology, including as hormones,
neurotransmitters, and signaling molecules.
12. Brief history of solid-phase peptide synthesis
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13
1963:
Merrifield introduces the concept of solid-phase peptide synthesis
(SPPS)
1969:
First automated peptide synthesizer developed by Bruce
Merrifield
1970s:
Development of new protecting groups and coupling reagents to
improve SPPS
13. Brief history of solid-phase peptide synthesis
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14
1980s:
Introduction of Fmoc-based SPPS, which is now the most widely used
method
1990s:
Advances in resin technology and automation lead to increased
efficiency and scalability of SPPS
Today:
SPPS is a widely used method for the chemical synthesis of peptides,
with over 400 peptides having entered clinical studies so far.
14. Solid-phase peptide synthesis methodology
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15
Solid phase synthesis was invented by Bruce Merrifield in
1963 and became very quickly the routine tool for preparation
of peptides and later small proteins and earned him the Nobel
Prize in 1984.
The idea:
15. What is solid-phase peptide-synthesis methodology
16
The Solid-Phase Peptide-Synthesis Methodology refers to the process
of synthesizing peptides using solid-phase techniques.
The concept of solid-phase peptide synthesis (SPPS) is to retain
chemistry that has been proven in solution but to add a covalent
attachment step that links the nascent peptide chain to an insoluble
polymeric support (resin).
Subsequently, the anchored peptide is extended by a series of
addition cycles. It is the essence of the solid-phase approach that
reactions are driven to completion by the use of excess soluble
reagents, which can be removed by simple filtration and washing
without.
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16. The development of solid-phase techniques
17
The next step in the development of solid-phase techniques includes :
Applications for peptides containing non-native amino acids, post-
translationally modified amino acids, and pseudoamino acids, as well
well as for organic molecules in general.
The solid support must be versatile so that a great variety of solvents
solvents can be used, particularly for organic-molecule applications.
Coupling reagents must be sufficiently rapid so that sterically hindered
hindered amino acids can be incorporated.
Construction of peptides that contain amino acids bearing post-
translational modifications should take advantage of the solid-phase
approach.
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17. Planning of solid phase synthesis
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18
The Resin (solid support)
The Linkers
The protective group
Reaction monitoring
Purification
Automation
18. SPPS in steps
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19
1st Step: In this the stating molecule to an inert solid.
Typically inert polymers or resin are used.
These are commercially available.
2nd Step: Here, Solid are deep into solution containing subsequent
reagent next they can be removed from the dip and placed into a
ash solution.
3rd Step: After all reactions are don the product is still attached
to the insoluble bead Product
Can be washed in a reaction well excess solvent is washed out
finally the product is cleaved from the bead and isolated.
Washing: After each synthetic step and prior to cleavage the solid
support must be exhaustically washed with large vol. of solvent.
Purifications: purification is done by semi preparative HPLC utilizing
new developments in column technology to cut down run times to less
than 10 mins.
21. The Resin (solid support)
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22
Can be functionalized;
Chemical stability (it must be inert to all applied chemicals);
Mechanical stability (it shouldn’t brake under stirring);
It must swell extensively in the solvents used for the synthesis;
Peptide-resin bond should be stable during the synthesis;
Peptide-resin bond can be cleaved effectively at the end of the
synthesis
22. The Resin (solid support)
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23
Earliest form of resin (used by Merrifield) – polystyrene beads –
styrene cross-linked with 1% divinylbenzene.
Derivatized with a chloromethyl group (anchor/linker) – amino acids
can be coupled via an ester group.
This ester group is stable to reaction conditions but cleaved at end
of synthesis using acids (e.g., HF).
23. Types of Resins
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24
Disadvantages of polystyrene beads:
Growing peptide chain is hydrophobic,
So not solvated and folds itself & forms internal H bonds thus,
Hinders access of further amino acids to growing chain
Resins are two types they are:
A) Hydrophobic polystyrene resins
B) Hybrite Hydrophilic Polystyrene Resin (HHPSR)
24. Types of Resins
A) Hydrophobic polystyrene resins
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25
Polystyrene resin beads under class Gelatinous solid support
Cross linked with 1-2% divinylbenzene
Particle size 90-200µm
Used in large number of reaction sites
They are cheap & commercially available with any functional groups
Examples of resins
1. Benzylic halids :
a) Merrifide resin.
b) Trityl chloride resin.
2. Benzylic amines:
a) Rink amide resin
b) Amino Ethyle polystyrene
3. Benzylic alcohls :
a) Wangresin
4. Aromatic aldehyde:
a) Backbone amide linker
b) Bal resin
25. Types of Resins
B) Hybrite Hydrophilic Polystyrene Resin (HHPSR)
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26
A major drawback of hydrophilic PS resin is poor swelling in protic
solvent. Thus support is prepared by grafting hydrophilic mono-
functional or bi-functional polystyrene glycol (PEG).
Examples:
ChemMatriex
1) It has chemical and thermal stability
2) Compatible with microwave
3) High degree of swelling in acetonitrile, DMF, TFA 4 Used for
synthesis of difficult and long peptides.
29. The Linker
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30
Linker referred to a molecular unit covalently attached to polymer
chain making up solid support
Contains a reactive functional group with which starting material
can react and attach to the resin
Resulting link – stable to reaction conditions but cleavable to
release final product
Most linkers – in interior of polymer beads, so swelling is important
30. Properties of linkers
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31
Stable to the reaction conditions
Cleaved selectively at the end of synthesis
Re-useable
Facilitate reactions monitoring
Easy to prepared
It should be highly selective to one or most small number of
specific cleavage reagents/ conditions.
Types:
1. Acid-Cleavable Anchors and Linker
2. Base / Nucleophil-Laibale linkers
3. Photo Labile Linkers
31. How choice linker
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32
depends on
Functional group present on starting material
Functional group to be present on final product upon release
32. The solid support with linker
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Resins of different linkers have different names:
Wang resin – has a linker suitable for attachment and release of
carboxylic acids
Rink resin – for attachment of carboxylic acids and release of
carboxamides
Dihydropyran-derivatized resin – suitable for attachment and
release of alcohols
33. The solid support with linker
Wang resin
Used in peptide synthesis where N-protected amino acid – linked to
resin by means of ester link.
Ester link – remains stable to coupling and deprotection steps in
synthesis and cleaved using trifluoroacetic acid (TFA) to release
final product.
34.
35. The solid support with linker
Rink resin
Attach starting material (with carboxylic
acid) via amide link
When reaction is complete, treatment
with TFA releases final product with
primary amide group.
37. Protecting groups
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A fragment bound to a functional group that block the reactivity of
that group.
Good protective group are easily attached and removed using
reaction.
It mainly two types of protecting groups are used they are:
Fmoc (9-fluorenylmethoxycarbonyl)
t-Boc (tert-butoxycarbonyl)
38. Protecting groups
A) Amine Protection
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43
There are two standard types of N-protecting groups used, the Boc
and Fmoc group.
39. Protecting groups:
B) Protection of the R-group
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44
Some of the different R-groups that must be protected before
coupling are:
hydroxyl groups (Ser)
thiol groups (Cys)
amines (Lys)
carboxylic acids (Asp).
41. Coupling Reagents
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46
In the coupling reactions of peptide synthesis the carboxyl group of
the acylating amino acid is activated.
The care should be taken in selecting the activation method to
avoid racemization.
N
N
N
OH
N N
N
N
OH
HOBt HOAt
N
N
N
O
Me2N NMe2
PF6 (BF4 ) N N
N
N
O
Me2N NMe2
HBTU (TBTU) HATU
PF6
HOBt: (N- Hydroxy benztriazole)
HOAt: (1- Hydroxy-7-aza- benztriazole)
HBTU: (O-benztriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro phosphate)
HATU: 2-(1H-7-Azabenzotriazol-1-yl)--1,1,3,3-tetramethyl uranium hexafluorophosphate
43. Deprotecting Groups
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48
It is the cleavage of the side-chain protecting groups.
For protecting group such as Boc using Trifluoroacetic acid (TFA)
the deprotecting groups like HF and Trifluoromethanesulfonic acid
(TFMSA) have been used.
Mild Acids :
e.g. Trifluoroacetic acid.
Hydrochloric acid.
Methanesulfonic Acid.
Alkaline condition :
e.g. Piperidine in dimethylformami
45. Reaction monitoring
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50
Chromatography is the first monitor (i.e TLC).
Non destructive methods such as infrared spectroscopy, Nuclear
magnetic resonance are also used in reaction monitoring.
46. Advantages of Solid Phase Synthesis
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51
Synthetic intermediates don’t have to be isolated.
Quick process.
Reagents simply washed away each step.
Can be automated with robots!!
47. Disadvantages to Solid Phase Synthesis
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All the synthesis can’t be done on solid phase.
Typically, kinetics not the same.
Unsuitable for solvent assisted chemical reaction.
High viscosity in reactant system.
Insufficient purity if reaction steps are incomplete.
49. Side-reactions
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54
all the amino acids have basic skeleton
but vary in their side chains, and their
nature such as acidic, basic or neutral.
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.
50. Side-reactions
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During peptide synthesis, several side reactions can occur, which
can affect the yield, purity, and quality of the final product. Some
of the common side reactions in peptide synthesis include:
1. Racemization: Racemization occurs when an amino acid in the
peptide changes from the L- to the D-configuration. This can
occur during coupling reactions or deprotection steps, and can
lead to the formation of diastereomers, which can be difficult
separate and purify.
2. Deamidation: Deamidation occurs when an amide bond in the
is hydrolyzed, leading to the formation of an acidic or basic
This can occur during acidic or basic conditions, high temperature,
or prolonged reaction times.
51. Side-reactions
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56
3. Epimerization: Epimerization occurs when the stereochemistry of an
amino acid in the peptide changes, leading to the formation of a
different stereoisomer. This can occur during coupling reactions or
deprotection steps and can lead to the formation of diastereomers.
52. Side-reactions
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57
To minimize the occurrence of side reactions, several strategies can
be employed, such as optimizing reaction conditions, using
protected amino acids, and using purification techniques such as
HPLC or gel filtration chromatography.
54. Side reaction by (A) proton abstraction
From carboxyl group: stop reaction
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59
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
55. Side reaction by (A) proton abstraction
From α-carbon in esters: Racemization
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60
In esters, electron withdrawing forces present in the activating
group (X) enhance the activity of α- Hydrogen abstraction that
leads to the formation of carbanion which results in total or
partial loss of chiral purity resulting in irreversible racemization.
Thus it is necessary to avoid such formation which result into
racemization of compounds by cyclization.
56. Side reaction by (A) proton abstraction
Direct abstraction of α-proton: Racemization
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When an amino acid which is attached with a protecting group
(Y), is treated with a base, proton abstraction occurs at α-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
57. Side reaction by (A) proton abstraction
From (OH) of enol from amid group: Azlactone.
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62
The keto group of the amide bond undergoes keto- enol
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 deficient carbon result in
formation of azlactones.
58. Side reaction by (A) proton abstraction
From (OH) of enol from amid group: Azlactone.
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Due to presence of unsaturation in the azlactone, and upon
treating with a base leads to the abstraction of proton at α- carbon,
which results in a total of three resonating structures.
Therefore, the resonance stabilized structure forms the carbanion.
59. Side reaction by (A) proton abstraction
From (OH) of enol from amid group: Azlactone.
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This when treated with a basic solvent (tertiary amine), it results in
formation of azlactone.
Example:
Formation of azlactone in Benzoyl L- leucine-p-Nitrophenol
60. Side reaction by (A) proton abstraction
From amide (N) of acyl amino group: Cyclization
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In a peptide chain, due to amide bond, proton abstraction does not
occur at the α-carbon but occurs at the amide nitrogen of acyl
amino acid. This is due to presence of lone pair of electrons at ‘N’.
When amide bond, in presence of an acid, undergoes proton
abstraction, the abstracted proton leaves the Nitrogen atom
retaining its electrons result in cyclization.
61. Side reaction by (A) proton abstraction
From amide (N) of acyl amino group: Cyclization
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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
62. Side reaction by (A) proton abstraction
From (-OH) of R/Ar-OH: O–Acylation
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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
63. Side reaction by (A) proton abstraction
From (-OH) of R/Ar-OH: O–Acylation
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Example:
p-Hydroxy alanine (tyrosine) is treated with a tertiary amine which
which acts as proton abstractor, and also with p- Nitrophenyl ester, as
as a result, the acylated product p-Acyl oxy phenyl alanine (Acyl
tyrosine) is formed along with p-Nitrophenol.
(a) Formation of tyrosine phenolate
(b) Formation of carbocation
(c) Acylation of Tyrosine
64. Side reaction by (B) Protonation
to (o) of carbonyl group: Racemization
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It is an acid catalyzed reaction
involving protonation of
carbonyl oxygen resulting 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 as
shown in figure.
The enolized product does not
retain theire chiral purity.
65. Side reaction by (B) Protonation
to (o) of carbonyl group: Cyclization
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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 .
The mechanism is explained 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.
66. Side reaction by (B) Protonation
to (o) of carbonyl group: Cyclization
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Cyclization by protonation
67. Side reaction by (B) Protonation
to removal of protecting group: Alkylation
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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.
68. Side reaction by (B) Protonation
to removal of protecting group: Alkylation
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Sometimes, the
carbocations formed also
react with the solvent
surrounding them and
form a better alkylating
agent and act by
electrophilic substitution
reaction.
Alkylation in tyrosine
occurs only at -ortho
position to hydroxyl group
and not at -meta position
due to steric hindrance by
the bulkiness of the amino
acid skeleton .
Alkylation by electrophilic substitution
69. Side reaction by (B) Protonation
N →O shift: Chain Fragmentation
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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
bond will lead to fragmentation of the chain
70. Side reaction by (B) Protonation
N →O shift : Chain Fragmentation
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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.
71. Side reaction by (C) over-activation
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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.
72. Side reaction by (C) over-activation
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Sometimes, during coupling of amino acids, using a coupling agent
like N, N’- disubstituted carbodiimide,
subtle intermediates are formed such as O-Acyl isourea which give
rise to symmetrical anhydrides and azlactones and can also
undergo rearrangement to N-acylurea derivatives.
73. Side reaction by (C) over-activation
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Reaction showing
complete overactivation
74. Side reaction by (C) over-activation
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Imidazole containing amino acids such as tryptophan react with
carbodiimide and forms substituted guanidine and similar is the
case with that of histidine .
Formation of substituted guanidine in
(a) Tryptophan (b) Histidine
75. Side reaction by (C) over-activation
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However, the O-Acylation or substituted guanidine side reaction
that occurred can be revered by acid catalyzed methanolysis.
77. Purification and analysis of synthetic peptides
The most common methods used for the purification and analysis of
synthetic peptides are:
1) Reversed-phase high-performance liquid chromatography (RP-
HPLC)
2) Ion-exchange HPLC
3) Gel-filtration HPLC
These techniques can be used either alternatively or in tandem to
isolate desired peptide products.
Mass spectroscopy (MS) used for identification of synthetic peptides.
89 2 May 2023
78. Purification and analysis of synthetic peptides
RP-HPLC is the most commonly used method for peptide
purification, as it has high resolving power and can remove many of
the systematic low-level by-products that accrue during chain
assembly and upon cleavage.
Ion-exchange HPLC and gel-filtration HPLC are also useful for
peptide purification, depending on the specific properties of the
peptide being synthesized.
90 2 May 2023
82. 94
solid-phase peptide synthesis (SPPS) is a widely used
method for the chemical synthesis of peptides.
The process involves retaining chemistry that has been
proven in solution but adding a covalent attachment step that
links the nascent peptide chain to an insoluble polymeric
support (resin).
Subsequently, the anchored peptide is extended by a
series of addition cycles.
The purification and analysis of synthetic peptides can be
achieved using various methods, including reversed-phase
high-performance liquid chromatography (RP-HPLC), ion-
exchange HPLC, and gel-filtration HPLC.
These techniques can be used either alternatively or in tandem
to isolate desired peptide products.
Conclusion
2 May 2023
83. References
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95
1. Fields, G. B. (2001). Introduction to peptide synthesis. Current
protocols in protein science, 26(1), 18-1.
2. Muzaffar-Ur-Rehman, M., Jabeen, A., & Mariya, M. (2018). Side
reactions in peptide synthesis: An overview. International Journal of
Pharmacy Research & Technology (IJPRT), 8(1), 1-11.
3. Solid phase synthesis & combinatorial technologics P.Seneci, Wiley
2000.
4. Solid phase organic synthesis, A.R.Vanio, K.D.Janda, J.Comb.
Chem. 2000.
5. Combinatorial peptide and non peptide libraries-A Handbook, Jung,
G.Ed.VCH Weinheim,1996.
