Solid phase peptide synthesis

MAPS (Multiple Antigenic Peptide System) and Solid phase peptide synthesis

1. MAP - SPPS
BY-
Abhishek Sirsikar
Sameer Hadawale

2. • More than 40 marketed peptides worldwide
• 270 peptides in clinical trials
• 400 peptides in advanced preclinical phases
Peptides as Drugs Today
The polymers of amino acids that are linked by peptide bonds. The
protein is a polypeptide.
Formation of a peptide bond by condensation

3. Before starting….
•Choose the C-terminal protecting group
•Choose the N-terminal protecting group
•Choose the coupling reagent
Mainly peptide synthesis are two types
1. Solution phase peptide synthesis
2. Solid phase peptide synthesis

4. H
2N
O
OH
R
1.C-terminal protecting group:
H
2N
O
O
H
2N
O
O
H
2N
O
O
C
2H
5
Alanine methyl ester Alanine allyl esterAlanine ethyl ester
• The most common acid protecting group used is the methyl ester.
•It is stable in most coupling reaction and deprotection reaction conditions.

5. H
2N
O
OH
R
2.N-terminal protecting group
There are two standard types of N-protecting groups used,
1. Boc Group


2. Fmoc group
O
O
CH
3
CH
3
CH
3
O
O

6. 3.Coupling Reagents
N-(3-Dimethylaminopropyl)-N’-ethylcarbonate
(EDC)
NCN
NCN NNCN N
N,N’-Dicyclohexylcarbodimide
(DCC)
N
N
N
O
Me
2N NMe
2
PF
6 (BF
4 ) N
N
N
N
O
Me
2N NMe
2
HBTU (TBTU) HATU
PF
6
N
N
N
OH
N
N
N
N
OH
HOBt
HOAt

7. Solution Phase Peptide Synthesis
H
2N COOMe
BocNH COOH
BocNH C
O
H
NCH
2COOMe
H
2N C
H
NCH
2COOMe
O
acid protectionamine protection
coupling
amine deprotection
BocNH COOH
amine protection
coupling
N
H
C
H
NCH
2COOMe
O
C
O
BocNH

8. The Merrifield Method
Synthesized a nonapeptide (bradykinin) in
1962 in 8 days in 68% yield.
Synthesized ribonuclease (124 amino acids)
in 1969.
369 reactions; 11,391 steps
Nobel Prize in chemistry: 1984

9. •Laboratory
•Industrial


Peptide
synthetiser
(500L)
Solid Phase
Peptide Synthesizers
2.Solid-Phase Peptide Synthesis
Nobel prize in 1984

10. In solid-phase synthesis, the starting
material is bonded to an inert solid support
Reaction occurs at the interface between
the solid and the solution. Because the
starting material is bonded to the solid, any
product from the starting material remains
bonded as well.
Purification involves simply washing the
byproducts from the solid support.

11. •First step is to attach the starting
molecule to an inert solid.
–Typically inert polymers or resins are
used.
–These are commercially available
Since the molecule is attached to a solid,
any other chemicals added or products can
be removed by filtration.
After all reactions are done the product is
still attached to the insoluble bead.
Finally, the product is cleaved from the
bead and isolated.

12. Solid phase peptide synthesis (SPPS)
Resin
O
AA1HNFmoc
Fmoc
P1
Resin
O
AA1H
2N
P1
O
AA2HNFmoc
P2
OH
O
AA2HNFmoc
P2
A
A
Resin
O
AA1NH
P1
O
AA2HNFmoc
P2
A
activation deblocking
coupling
repeat steps
for each amino
acid in peptide,
then deblock,
deprotect,
cleave off resin
Resin
Fmoc
P2P1
AA2AA1
A
solid supportsolid support
fmoc protecting groupfmoc protecting group
protecting groupsprotecting groups
for side chainsfor side chains
1st and 2nd1st and 2nd
amino acidsamino acids
carbonyl activatingcarbonyl activating
groupgroup

13. Resin
O
AA1NH
P1
O
AA2HNFmoc
P2
Fmoc
final
deblocking
Resin
O
AA1NH
P1
O
AA2H
2N
P2
deprotection and
cleavageP1P2Resin
O
AA1NH
O
AA2H
2N
OH
at the end a final
deblocking is done
followed by removal
of the side-chain
protecting groups
and cleavage from
the resin to recover
the peptide
SPPS using Fmoc can
be used to make
peptides up to 70-
100
residues in length
(chemical ligation
can be used to make
longer ones)

15. Applications of
synthetic peptides
Immune peptides:
synthetic antigens;
vaccines
diagnostic tools
immunostimulator peptides;
muramyl dipeptide
tuftsin derivatives
Hormones:
oxytocin
vasopressin
insulin
somatostatin
GnRH
etc.
Neuropeptides:
substance P
cholecystokinin
neurotensin
Antibiotics:
tachikinin
gramicidine S
Toxins:
conotoxins
spider toxins
snake toxins
ionchanel blockers
Enzymes and
enzyme inhibitors:
Ribonuclease A
Carriers:
templates
miniproteins
Peptides
for structural studies:
turn mimicking cyclic peptides
Transporter peptides:
penetratin
oligoarginine
HIV-Tat protein

16. • time consuming;
• side chain protecting groups for
• Lys, Asp, Glu, (Cys);
• coupling: less than 90% conversion;
• purification after each steps;
• large scale;
• cheap.
• fast;
• side chain protecting groups for
• all functional groups
• coupling: over 99.5% conversion;
• purification at the end;
• rather small scale;
• expensive.
Solution phase peptide synthesisSolid phase peptide synthesis
(SPPS)

17. MAPS (Multiple Antigenic Peptide System)
MAPS is a method for producing high-titer
anti-peptide antibodies and synthetic peptide
vaccines. This system utilizes the α- and ε-
amino functional groups of lysine to form a
backbone to which multiple peptide chains
are attached. Depending on the number of
lysine tiers (2, 4, 8, etc.), different numbers of
peptide branches can be synthesized. Using
this new technology, our customers have
successfully produced high-titer antibodies.

18. Thank youThank you