3. Solid-Phase Synthesis
Who get it started
Merrifield 1963 – solid-phase
– Revolution in synthesis
– Reducing of temporial and technical effort
– Nobel prize in 1985
– Base of automation and Combinatorial Chemistry
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4. Multiple solid-phase synthesis
Stäbchen
Development of parallel synthesis
• Polyethylene skewer with a amino function carrier
• 40 mm lenght; 4 mm diameter
• 96 Skewers in 8 rows fits in a microtiterplate
• Coupling of aminoacids in the plate
• Chemistry analog merrifield
• Peptides stay on the skewers
• Method for investigation of antibodies
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5. Split-Mix-Synthesis
Lam, 1991
Method to eliminate the problems with the coupling
tendency of aminoacids
The polymere are little beads, diameter 100-200 μm
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7. Arrays
Fodor, 1991
• Solid phase synthesis + Photo Lithography and Photocemistry
• Synthesis on a functionalised glass plates
• Photolabile α-amino protecting group
• Masks + light with a specific wavelenght
• Control of the sequence
• Screening with antibodies
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8. Introduction
1) Solid Supports
Insoluble in solvents
Not to react with the reagents
Form of small resin beads, polymer or glass beads, rods, sheets
etc ( that may or may not swell in the solvents)
Composed of two parts
1. The core
2. The linker
CORE Linkers Start compound
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9. The core
1) Crosslinked polystyrene
2) Polyethylene glycol (PEG) grafted supports
3) Inorganic supports
4) Non-bead form supports
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10. 1) Crosslinked polystyrene
• Most commonly used supports
• Synthesized from styrene and divinylbenzene
• By suspension polymerization in the form of small beads
• The ratio of divinylbenzene to styrene determines the density
of cross links.
Higher crosslink density
Increases the mechanical stability of the beads
Lowering the crosslink density
• Increases swelling
• Increases the accessibility of the functional groups
Ratio: 1-2% divinylbenzene
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11. Specification of bead
Functional groups can be introduced into the resin
• Post functionalization of the aromatic rings of polystyrene
• functionalized styrene in polymerization
The reactions are faster when small beads are used
• Diameter of the Beads or by the inversely proportional mesh
size
• 200-400 mesh (35-75micron)
• 100-200 mesh (75-150 micron)
• Narrow bead size distribution is advantageous
• Capacity of the Polystyrene beads is around 0.5 mmol/g.
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12. 2) Polyethylene glycol (PEG) grafted supports
Structure:
Tentagel
Where X is Br, OH, SH or NH2 for attachment of the substrate
• Aromatic rings & polyethylene glycol chains are covalently
attached
• Ratio: 1-2% crosslinked polystyrene
Advantage
• flexible
• chain is more accessible to reagents
• behaves like being in a solution-like environment
• gives a hydrophilic character to the resin
• swells well in water and methanol 12
13. 3) Inorganic supports
Glass beads
functionalized
controlled pore size
Commercially available
mechanical stability
do not swell in solvents
Functionalized ceramics can also be used as supports
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14. 4) Non-bead form supports
Microscopic beads
Surface of macroscopic objects can be functionalized with
groups
Can serve as anchors to hold the substrate
Styrene or polyolefin chains can be grafted by radiation into
the Surface of the objects
The chains can be functionalized
SynPhase lanterns are commercially available in different sizes
at Mimotopes
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15. Linkers
Covalently attached to the solid support
Bifunctional molecule
One functional group for irreversible attachment to the core
resin
Second functional group for forming a reversible covalent
bond with the initial building block of the product
Also called anchor
+ =Resin Linker Resin Linker
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16. 1) Merrifield resin
Used to attach carboxylic acids to the resin
cleaved from the resin in carboxylic acid form using HF
2) Trityl chloride resin
much more reactive than the Merrifield resin
used for attachment of a vide variety of compounds like
carboxylic acids, alcohols, phenols, amines & thiols.
cleaved under mild conditions using a solution of
trifluoroacetic acid (TFA) in varying concentrations (2-50%)
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17. 3) Hydroxymethyl resin
Applied for attachment of activated carboxylic acids
Cleavage conditions resemble that of the Merrifield resin (HF)
4) Wang resin
Used to bind carboxylic acids
Ester linkage formed has a good stability
Cleavage by 95% TFA is applied
Frequently used in peptide synthesis
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18. 5) Aminomethyl resin
Carboxylic acids in their activated form can be attached to the
resin
Formed amide bond is resistant to cleavage
Resin is used when the synthesized products are not cleaved
from the support
6) Rink amide resin
Designed to bind carboxylic acids
Cleave the product in carboxamide form under mild conditions
Amino group in the resin is usually present in protected form
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19. 7) Photolabile anchors
• Photolabile anchors have been developed that allow cleavage
of the product from the support by irradiation without using
any chemical reagents
• 2-nitrobenzhydrylamine
• Usually contain nitro group that absorbs UV light
8) Traceless anchors
• Block of a multi-step solid phase synthesis
• It may happen that In the end product this group is
unnecessary and needs to be removed
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20. Parallel synthesis
• Synthesis of compound arrays based on saving reaction time
• food cooking processes
• She is doing different activities in parallel in order to be more
effective
Standard microtiter plate: 8 rows and 12 columns 20
21. Principle of Parallel Synthesis
• Chemical reactions takes time
• During that time not only one but a series of reactions can be
realized
• Each synthetic reaction is started in a different reaction vessel
• All the necessary operations are executed in parallel
Ex. Tripeptides in parallel 21
22. • Number of reaction vessels is the same as the number of
compounds
• Number of operations is practically the same
• Solvents and reagents have to be serially transported into each
reaction vessel
• The real advantage is that the reaction time for the in
synthesizing the 5 compounds is about the same as preparing a
single one
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23. Advantages
Each compound is substantially pure in this location
Define location provides the structure of certain compound
Easier biological evaluation
No Deconvolution is required
No risk of synergistic effect
Disadvantages
Applicable only for medium libraries
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24. Types of Parallel Synthesis
1. The multipin metod of Geysen
2. The Spot technique of Frank
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25. The multipin metod of Geysen
• The first example of parallel synthesis was published by
Geysen and his colleagues
• Synthesized series of peptide
• Used the microtiter plate
• Cover plate with mounted polyethylene rods
• End of polyethylene rods (pins) were coated with derivatized
polyacrilic acid
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26. The Spot technique of Frank
• The synthesis is carried out on cellulose paper membranes
• Small droplets of solutions of protected amino acids dissolved
in low volatility solvents
• Coupling reagents are pipetted onto predefined positions of the
membrane
• The spots thus formed can be considered as reaction vessels
where the conversion reactions of the solid phase synthesis
take place
• Many as 2000 peptides can be made on an 8x12 cm paper
sheet
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27. The temperature of the reaction mixtures could be controlled by
heating or cooling the reaction block
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28. The split-mix synthesis
• Based on Merrifield's solid phase procedure
• Using only three different protected amino acids as building blocks
• by red, yellow and blue circles
• The synthesis is executed by repetition of the following three simple
operations that form a cycle
1. Dividing the solid support into equal portions;
2. Coupling each portion individually with only one of the different amino
acids
3. Mixing and homogenizing the portions.
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30. In the first round
The amino acids are coupled to equal portions of the resin
The final product - after recombining and mixing the portions -
is the mixture of the three amino acids bound to resin.
In the second cycle
This mixture is again divided into three equal portions
The amino acids are individually coupled to these mixtures
In each coupling step three different resin bound dipeptides are
formed, so the end product is a mixture of 9 dipeptides
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