solid phase peptide synthesis

1. 1
Submitted by:
K.KAVYA
1styr,M.pharmacy,
Pharmaceutical chemistry.

2. CONTENTS:
 INTRODUCTION
 SOLID PHASE PEPTIDE SYNTHESIS
 HISTORY
 PLANNING OF SOLID PHASE SYNTHESIS
 APPLICATIONS
 ADVANTAGES
 DIS ADVANTAGES
 SIDE REACTION BY PROTON ABSTRACTION
 SIDE REACTION INITIATED BY PROTONATION
 SIDE REACTION BY OVERACTIVATION
 SIDE REACTIONS RELATED TO INDIVIDUAL AMINO
ACID
 CONCLUSION
 REFERENCE 2

3. Abbreviations
 Boc - tert-butoxycarbonyl
 DMF- N,N-dimethylformamide
 Fmoc - 9-fluorenylmethoxycarbonyl
 HF-Hydrofluoric acid
 HHPSR- Hybrite Hydrophilic Polystyrene Resin
 SPPS - solid-phase peptide synthesis
 PEG - polystyrene glycol
 tBu -tert-butyl
 TFA-trifluoroacetic acid
3

4. INTRODUCTION
 Peptides are the sequence of amino acids which are
formed by the condensation of two amino acids .
 In which a peptide bond is formed between a carbonyl group of
one amino acid and the amino group of another amino acid .
 Peptides are synthesized in two ways they are
1.solid phase synthesis
2.solution phase synthesis
4

5.  Solid Phase Synthesis
 Solution Phase Synthesis
(Reagent) purification
5
Substrate (product) + (reagent) (product)

6. SOLID PHASE PEPTIDE SYNTHESIS
In chemistry solid-phase synthesis is a method in which
molecules are bound on a bead and synthesized step-by-step in
a reactant solution; compared [with] normal synthesis in a
liquid state, it is easier to remove excess reactant or byproduct
from the product.
This method is used for the synthesis of peptides,
deoxyribonucleic acid (DNA), and other molecules that need to
be synthesized in a certain alignment.
 This method has also been used in drug discovery process.
6

7. Problems with Traditional Synthesis
 1 chemist 1 molecule
 Can only make one molecule at a time
 Each synthesis very time consuming
 Purification of products very time-consuming between steps.
 Yields can be low and produces very few molecules at a time
for testing
7

8. Solid Phase Peptide Synthesis Principle:
8

9. HISTORY
9
 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.

10. Planning Of Solid Phase Synthesis
 The Resin (Solid Support)
 The Linkers
 The Protective groups
• The Reaction monitoring
• Purification
• Automation
10

11. Resin (Solid support)
1. Resin act as a solid support for a solid phase synthesis
2. The term solid support used to denote the matrix upon which
chemical reaction is performed
3. Solid support must be inert
4. It always swell extensively in solvent.
11

12. Types of Resins
 Resins are two types they are:
1. Hydrophobic polystyrene resins
2. Hybrite Hydrophilic Polystyrene Resin (HHPSR)
Hydrophobic polystyrene resins:
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
12

13.  Types of Hydrophobic polystyrene resins:
1. Benzylic halides –
a) Merrifide resin.
b) Trityl chloride resin.
2. Benzylic amines –
a) Rink amide resin
b) Amino Ethyle polystyrene
3. Benzylic alcohols –
a) Wang resin
4. Aromatic aldehyde –
a) Backbone amide linker
b) Bal resin
13

14. Benzylic Halide Type
Merrifield Resin
Attachment- Carboxylic acid,
Alcohol,
Phenol,
Cleavage- strong acidic condition
Application- Peptide synthesis
Trityl chloride Resin
Attachment- Carboxylic acid,
Alcohol,
Amine.
Cleavage - Mild acidic condition
Application- Peptide synthesis,
Alcohol & amine synthesis
14
Cl

15. 2.Benzylic Amine Type resin
Attachment - Carboxylic acid , alcohol , Phenol.
Cleavage – Mild acidic condition.
Application - Peptide synthesis( Aryl & Alkyl
carboxamide)
3. Benzylic alcohol Type Resin
Attachment - Carboxylic acid.
Cleavage – Acidic condition.
Application - Peptide synthesis.
15
NH2
O
OMe
MeO
Rink amide resin
OH
O
Wang resin

16. 4.Aromatic Aldehyde Type Resin
Attachment – Primary Amine.
Cleavage – Mild acidic condition.
Application - Peptide synthesis.
16
O
CHO
OMeMeO
Bal resin

17. Hybrite Hydrophilic Polystyrene Resin (HHPSR):
 A major drawback of hydrophilic PS resin is poor swelling in protic
solvent. Thus support is prepared by grafting hydrophilic
monofunctional 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.
17

18. THE LINKER
 Linker referred as a handle to attach the small
molecule onto polymeric resin Linkers has many
similarities to protecting group in solid phase
synthesis.
18
Resin Linker
X
Attachment
Resin Linker Molecule
Resin Linker Molecule
Cleavage
Molecule

19.  Properties of linkers which may assist Solid phase
synthesis are:
 Stable to the reaction conditions
 Cleaved selectively at the end of synthesis
 Re-useable
 Facilitate reactions monitoring
 Sequential / Partial release
 Easy to prepared
 It should be highly selective to one or most small
number of specific cleavage reagents/ conditions.
19

20. Types of Linkers
 Linkers are different types they are :
1 Acid-Cleavable Anchors and Linker:
SPS of peptides was developed using this type of
linkers
20
Acid labile, commercialy available SP Linker
OH
O
R
R
OH
O
OCH3 R
O
dec
SPS
R OH
O
300
RT
dec

21. 2.Base /Nucleophil-Liable linkers:
 The commonly used Fmoc peptide coupling protocols
required Fmoc deprotection under basic conditions
during synthesis.
21
Base/ Nucleophilic Laibale Linker
OH
NH
NH
R
O
O
OCH3 R
O
dec
R OH
O
dec
RCOOR
sps
N
H
O
20%DMF
2hr,RT

22. 3)Photo Labile Linkers:
 The light is used to break the bond between the
intermediate and the linker and releases pure
compound from the SPS into solution without
interference from side produced .
22
photo labile SP Linker
O2N
NH
R
CH2
Br
NO2CH3
OR
O
dec
Rdec COOH
RCOOH
350 nm
meOH
24hr,RT

23. 4.Safety-Catch Linkers:
 It is totally stable during the synthetic procedure & labile
after a process known as activation.
 It is very popular and allows the support and release of
many different functionalities.
 Examples : sulfonamide based safety catch linkers, phenol
based safety linkers, acid labile safety catch linkers,
imidazole safety linkers.
23
Slphonamide Based
SCL
Safety catch (SC) Linker
N
S
R
O
O
CH3
CH2
Rdec Nu
Rdec
O
RCOOH
NH2
S
R
O
O
sps Nucleophilic

24.  Examples of linkers:
Ellman linker
Trialkylsillyl chloride (or triflate ) generated from
ethanophenylsane.
 Attachment of alcohols ketones
24
+anisole
Function of linkers:
1) As a functional group
2) As a linker releases another functional group
SiH2
OH
O
CH3
HCl
Cl Cl
SiH2Cl

25. PROTECTING GROUPS
A species conjugated 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)
25

26. 26

27. 27

28. 28

29. 29

30. 30

31. SPS procedure
 In this the stating molecule to an inert solid.
Typically inert polymers or resin are used.
These are commercially available.
 Solid are deep into solution containing subsequent reagent next
they can be removed from the dip and placed into a ash
solution.(Tea bags also used)
 After all reactions are done 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.
31
Washing: After each synthetic step and prior to cleavage
the solid support must be exhaustically washed with large
vol. of solvent.

32.  Purifications: purification is done by semi preprative
HPLC utilizing new developments in column technology to
cut down run times to less than 10 mins.
32

33. Solid phase synthesis strategy
33

34. Reaction monitoring
Chromatography is the first monitor (i.e TLC).
Non destructive methods such as infrared
spectroscopy, Nuclear magnetic resonance are also
used in reaction monitoring.
34

35. APPLICATION:
 Combinatorial synthesis of solid phase
 Peptide synthesis
 In DNA synthesis
 In various reactions
 Clainsan rearrangement
 Beckmann rearrangement
 Organic synthesis
35

36. ADVANTAGES
 Over all process is quick
 Purification of each product can be achieved in one step. Only
purification technique is filtration.
 Purification in each step is possible.
 Synthetic intermediates don’t have to be isolated.
 Can be automated with Roberts.
 Use of solvent are minimized.
 Less health hazards and eco- friendly and hence follows the
principle of GREEN CHEMISTRY.
36

37. DISADVANTAGES
 High chemical consume.
 Expensive.
 Low reaction rates.
 Special substances needed.
 It produces very few molecules at a time for testing.
 Solid phase synthesis without using any solvents.
37

38. SIDE REACTION IN PEPTIDE SYNTHESIS
38

39. SIDE REACTION BY PROTON ABSTRACTION
 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 .
 This reaction in which glycine when treated by a base
is taken as an example.
39
O
NH2
OH
O
NH2
O
-
O
NH2
C
-
O
-
H
Glycine
Base (OH-
)
Base (OH-
)
carboxylate anion Not possible
Formation of carboxylate anion when treated with a base

40. Racemization
o Mechanism of racemization was involved in different steps
they are:
a)Direct abstraction of α-proton:
When an amino acid which is attached with a protecting
group (Z), 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.
40
Racemization by direct abstraction of
proton
NH
O
Xz C:
NH
O
Xz
Base
Phenyl glycine
Phenyl glycine carbanion

41. 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.
• As a result, the carbon holding the keto group gets
positive charge on it.
• the carbon now has deficient of electrons, and the oxygen
with excess of electrons, a bond is formed between the
carbon and the oxygen, and therefore forms an azlactone
ring.
41
Racemization by forming of azlactone
Glycylalanine
Azlactone derivatives
O
NH2
NH
X O
CH3
OH
NH2
N
X O
CH3
O
-
NH2
NH
X O
CH3
O
-
NH2
NH
O
CH3
O
N
O
CH3
NH2

42.  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 .
 Formation of azlactones is better explained by
considering Benzoyl L-leucine-p-Nitrophenol, in
which, the carboxyl and amino groups are protected
by p-Nitrophenol and Benzoyl group respectively.
42
Resonating structures of azlactones

43.  This when treated with a basic solvent (tertiary
amine), it results in formation of azlactone.
43
Formation of azlactone in Benzoyl L-
leucine-p-Nitrophenol

44.  Most of the racemization occurs through azlactones only and
rarely by direct abstraction .
 The Factors that affect racemization through azlactones are:
i. Nature of amino acid involved
ii. Solvent used in reaction
iii. Presence of tertiary amine.
 When stability aspect of anion produced by proton abstraction
through azlactones is considered, it is enhanced by electron
withdrawing effects in acyl groups .
 methyl azlactone is less stable when compared to phenyl
azlactone.
 This is so, as aryl azlactone has more resonating structures than
alkyl azlactone.
44Order of stability of azlactones
O
N
O
R
CH3
CH3
CH3
O
N
O
R
CH3
CH3
O
N
O
R
CH3
O
N
O
R
CH3
Phenyl azlactone
>
O
N
O
R
>
> >
Tertiary azlactone secondary azalactone primary azlactone methyl azlactone

45. 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
45

46. 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 OAcylation.
46

47.  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 pNitrophenol.
47
(a) Formation of tyrosine phenolate (b) Formation of carbocation
(c) Acylation of Tyrosine

48.  It can also occur in coupling reactions mediated by
carbonyldiimidazole with alcohols when tertiary
amine is absent.
 Since, imidazole acts as proton abstractor; it mediates
the abstraction of proton from alcohols.
48
NNH
+ OH R NHNH
+ O
-
R

49. SIDE REACTION INITIATED BY PROTONATION
Racemization :
• 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.
• 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 .
49

50. Racemization through protonation
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 .
 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
50

51. 51
Cyclization by protonation

52. 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.
52

53.  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 .
53
Alkylation by electrophilic substitution

54. 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 .
54

55.  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.
55

56. SIDE REACTION BY 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.
56

57. Reaction showing complete overactivation
57

58.  Imidazole containing amino acids such as tryptophan
react with carbodiimide and forms substituted
guanidine and similar is the case with that of
histidine.
58
Formation of substituted guanidine in (a) Tryptophan (b) Histidine

59.  However, the O-Acylation or substituted guanidine
side reaction that occurred can be revered by acid
catalyzed methanolysis.
59

60. SIDE REACTIONS RELATED TO INDIVIDUAL AMINO ACID
 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) followed by
ureides.
60

61. CH3
CH3
NH2
COOH
+
CH3
CH2 CH3
CH3
NH2
O
O
CH3
NHR
NR
Isoleucine Carbodiimide
O-Acylisourea of isoleucine
CH3
NH2
O
O
CH3
NHR
NR
CH3
NH2
O
N
CH3
NHR
O
R
CH3
NH2
O
NHR
CH3
Ureides O-Acylisourea of isoleucine
peptide bond
Fast Reaction Slow Reaction
61
Formation of ureides (left) and peptide bond (right) from O -
Acylisourea

62.  β - Carbon branching also interferes with other
reactions such as alkaline hydrolysis and
hydrazinolysis of alkyl esters.
 Alkylcarbonic mixed anhydride, which is a second
acylation product (urethane), is formed as a result of
coupling of valine or isoleucine since the nucleophile
has better chance to attack on the undesired carbonyl
group.
62

63.  This causes the reaction to occur at other position
rather than the desired position.
 when isoleucine is treated with trimethyl acetyl
chloride, alkylcarbonic mixed anhydride is formed
which upon treatment with a primary amine, undergo
hydrolysis at the undesired carbonyl group rather than
the desired carbonyl group.
 This is because of the bulkiness of isoleucine that
prevents the hydrolysis at first carbonyl carbon.
63

64.  Despite forming alcoholates in presence of a base,
alcoholic hydroxyls, under neutral conditions are
reactive to undergo intramolecular reactions to get
acylated at the carbodiimide activated carboxyl
group and produce lactone which upon further
treatment with another amino acid forms a peptide
bond.
 Such reactions are seen in amino acids containing a
hydroxyl group such as serine and threonine.
64

65.  the reaction of threonine forming lactone followed by
a peptide bond which is similar for serine as well.
65
•In case of tyrosine, the phenolate ion formed after proton
abstraction acts as an excellent nucleophile and gets acylated
easily to form esters which can be later deacylated by treating
with ammonia, hydrazine or hydroxylamine.

66. CONCLUSION
 Peptide synthesis involves different robust techniques .
 In sps is used in the peptide, DNA and combinatiorial
synthesis.
 Solid phase synthesis is green chemistry.
 In spps temporary and permanent protecting groups are
used.
 The side chains in the peptides are prone to side
reactions which can degrade the amino acid or stop the
peptide synthesis.
66

67. REFERENCE:
1.Introduction to medicinal chemistry 3rd edition by Patric.
2.Solid phase synthesis & combinatorial technologics
P.Seneci,Wiley 2000.
3.Organic synthesis on solid phase,F.Z.Dorwald Wiley-VCH2000.
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.
6. R.B. Merriffield , G. Barany, W.L. Cosand, M. Engelhard and S.
Mojsov, pept. Am pept. Symp. 5 th edittion 1997, page no 48-55.
7. Bodanszky M, Kwei JZ. Side reactions in peptide synthesis. Chemical
Biology & Drug Design. 1978; 12(2):69-74.
8.www.google.com
67

68. 68