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High-ThroughputScreening

Combinatorial Chemistry




-> Although combinatorial chemistry has only really beentaken up by industry since the 1990s, its rootscan be seen as far back as the 1960s when aresearcher at Rockefeller University, BruceMerrifield, started investigating the solid-statesynthesis of peptides

-> Bruce Merrifield won the Nobel prize in chemistry in1984 for his work on solid-phase synthesis.

-> During this time, automated peptide synthesizertechnology was in its infancy, and the preparation ofindividual peptides was a challenge.






-> Another early pioneer was Árpád Furkawho introduced the commonly used split-and-pool methods.

-> Dr. Árpád Furka is considered to be one ofthe fathers of combinatorial synthesispublishing in 1982 the first split-mix synthesiswork in the area of peptide synthesis .




-> In 1985 Richard Houghten introduced the “tea - bag” method for rapid multiple peptide synthesis.

-> These and other advances in manual multiplepeptidesynthesis fed the beginnings of a wave of rapid bioassaysbased on the developing area of molecular biology.“

“Tea -bag method” Polyethylene bag with fine holes, similar to real tea-bag, arefilled with resins and each bag is put in the different reactionvessels to carry out amino acid coupling reaction. Afterreactions, all the bags are collected and processed together for protecting group removing and washing resins toreduce the amount of time and efforts. In this method, the bag

takes the role of filter and preventing resin mixing betweenreactions, and by labeling each bag, the synthesized peptidestructure can be identified. About 100 different peptides in 500micromol quantity could be synthesized by this method, whichdemonstrate a practical approach to parallel synthesis despitethe fact that synthesized peptide number is moderate.





Combinatorial ChemistryFrom a historical perspective -> classical combinatorial chemistry can be briefly outlined in three phases:

The 1st Phase: In the early 1990s, the initial efforts in the combinatorial chemistry

-> driven by the improvements made in high-throughput screening (HTS) technologies-> demand for access to a large set of compounds for biological screening.-> the molecules in the first phase were simple peptides (or peptide-like) and lacked the structural complexitycommonly found in modern organic synthesis literature.

The 2nd Phase: In the late 1990s, when chemists became aware that it is not just about numbers;

-> but something was missing in compounds produced in a combinatorial fashion. Emphasis was thus shifted towardsquality rather than quantity.

The 3rd Phase: As the scientific community moved into the post-genomic chemical biology age

-> there was a growing demand in understanding the role of newly discovered proteins and their interactions with

other bio-macromolecules (i.e. other proteins and DNA or RNA). For example, the early goals of thebiomedical research community were centered on the identification of small molecule ligands for biological targets,such as G-protein-coupled receptors (GPCRs) and enzymes.-> current challenges are moving in the direction of understanding bio-macromolecular (i.e. protein-protein,protein- DNA/RNA) interactions and how small molecules could be utilized as useful chemical probes in systematicdissection of these interactions. By no means will this be a trivial undertaking! The development of biologicalassays towards understanding biomacromolecular interactions is equally challenging as the need for having accessto useful small molecule chemical probes.



Combinatorial Chemistry Used extensively in relation with drug discovery

Principle of Combinatorial Chemistry:

-> Generation of Compound Libraries fromMolecular Building blocks which are usuallyused in high-throughput screening.

HTS Library




-> prepared from a large number ofdifferent compounds at the same time- not conventional one-at-a-time mannersynthesis.

-> The characteristic of combinatorialsynthesis is that different compounds are generated simultaneously under identicalreaction conditions in a systematic manner ,so that ideally the products of all possiblecombinations of a given set of startingmaterials (termed building blocks) will beobtained at once

-> The collection of these finally

synthesized compounds isreferred to as a combinatorial library.

What is a combinatorial (compound) library ?




The game with the large numbers !!!




The game with the large numbers !!!




Establishment of Libraries

◦ Unbiased libraries (Random libraries) Typically a common chemical core (starting

point scaffold) Large number of building blocks (highly

diverse) Many targets Generating ”lead” structures

> 5.000 compounds Solid phase synthesis (one bead screening if

possible)

◦ Directed libraries Again a common chemical core Limited number of building blocks (structural

similar) Directed towards a specific target

Used to optimize ”lead” structures << 5.000 compounds Solid phase synthesis, synthesis in solution



Combinatorial ChemistryDrug Discovery

1991-2003;~2500 libraries

”Unbiased libraries”; 1-2million compounds-> Screening does not alwaysresult in hits.

”Directed libraries” buildon a privileged structure”

-> Libraries based on amodelling.




Solid-Phase Organic Synthesis

-> The compound library have been synthesized on solid phasesuch as resin bead, pins, or chips

Solution-Phase Organic Synthesis

-> The compound library have been synthesized in solvent in thereaction flask




Product is linked to a Solid Support

+ Easy purification -> Easy removal of excess reagents through filtration

- Low yield, Tagged at the point of attachment, Dificult to apply standard

characterization methods on intermediates (Dendrimer and poly ethylene glycolresins has been developed to improve the yield)

Solid Phase Synthesis




Solution Phase Synthesis

Reaction proceeds in Solution

+ Easy characterization of intermediates as well as end pruduct, No limitationsin attachment point -> Faster validation times relative to solid phase

synthesis (Standard analytical protocols can be used to characterizeproducts between each reaction step)

- Difficult to drive the reaction towards the product, extensive purification isneeded



Combinatorial ChemistryPolymer-supported reagents and scavangers

(Polymer assisted solution phase synthesis – PASP)

Hybrid between solid and solution phase synthesis

Reagents and scavangers are brougth to the reaction on solid supports

Excess reagent or by-products can be removed by filtration




Solid Phase CC Solution Phase CC PASP

Use of excess

reagent √ √

Easy of work up √

√

Minimalpurification √

√

Easy of reaction

monitoring √

√

Simple

chemistry set up √

√

Large libraries √

High quantities √ √



Combinatorial ChemistryPreparation of Libraries

Parallel Synthesis

Each compund is prepared in a specific vessel (on pins or Tea-bags)

Array of reaction vessels (96 well plates -> each well other compound)

Automated control of reactions -> easy to keep track of each compound

High yields Useful for epitope mapping

Just applicable when small number of positions are being varied -> smalllibraries



Combinatorial ChemistryPreparation of Libraries

Pool/Split Synthesis

◦ Good to generate large libraries

◦ Labeling required to keep track of eachcompound

◦ Beads (resin) are split into different

vessels

◦ Then reacted, shuffled, and

split again.

◦ 1000 compund library prepared

from 10 building blocks in eachstep 30 reaction steps.

(1110 steps for parallel synthesis)





Combinatorial ChemistryExtraction Techniques for Purification

-> Liquid-Liquid extraction Extensivley used for solution-phase combinatorial synthesis.

Automated by freezing liquid phase. Fails when;

◦ Emulsions formed

◦

The impurities have the same solubility properties

-> Fluorous phase technique Attach a insoluble perfluorinated moiety to the compound.

Retain the molecules from fluorous solvent.

-> Solid-phase extraction◦ Based on adsorption to a suitable surface.

◦ Impurities are washed away with a solvent in which the compound areinsoluble.



Combinatorial ChemistryLibrary Formats

Combinatorial Libraries vary in size, amount, purityand structual complexity

The libraries can be devided into 3 groups

1: One-bead one-compound

2: Preencoded libraries 3: Spatially addressable libraries









Combinatorial ChemistryDeconvolution

-> general method to determine active sequenceor order (+ nature) of attached groups

-> library consists of a set of mixtures in whichone of the variable positions is defined in eachmixture

-> library assayed and the optimalresidue(s)/groups at that position determined

-> process repeated -> in each case a differentvariable position is probed

-> each library is smaller than the one preceding

as the number of optimized positions increases

-> in the end yields individual structure (peptidesequence)





Combinatorial ChemistryExample: Scanning peptide libraries

-> screen mixture in each library to identify which AA at the first position is best

-> requires target that will recognize consensus sequence



Combinatorial ChemistryExample: Scanning peptide libraries

-> screen mixture in each library to identifywhich AA at fixed position is best

-> requires target that recognizes concensussequence (-> antibody, receptor)

-> in example library: 4 classes of AA(Hydrophobic, basic, acidic, neutral)

-> based on performance establish concensussequence



Combinatorial ChemistryDrug Discovery - Lead Identification

By screening pool/split solid-phase library of 128 000 2-arylindoles (1) split into320 pools of 400 compounds and screened against 16 G-protein coupled receptortargets Some pools both active and selective

• Compound 2 higly selective for Natural Killer Cell receptors,therefore viable lead for medical chemistry



Combinatorial ChemistryDrug Discovery - Lead Optimization

Lead Identification vs. Lead Optimization

Lead identification libraries < 10 000

Lead optimization libraries 1000-2000 Lead optimization via focussed libraries based on a privileged

structure

Both solution and solid-phase synthesis



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