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High-Throughput Screening. Combinatorial Chemistry . Combinatorial Chemistry. -> Although combinatorial chemistry has only really been taken up by industry since the 1990s, its roots can be seen as far back as the 1960s when a researcher at Rockefeller University, Bruce

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High-Throughput Screening

Combinatorial Chemistry

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Combinatorial Chemistry

-> Although combinatorial chemistry has only really been taken up by industry since the 1990s, its roots

can be seen as far back as the 1960s when a

researcher at Rockefeller University, Bruce

Merrifield, started investigating the solid-state

synthesis of peptides

-> Bruce Merrifield won the Nobel prize in chemistry in 1984 for his work on solid-phase synthesis.

-> During this time, automated peptide synthesizer technology was in its infancy, and the preparation of individual peptides was a challenge.

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Combinatorial Chemistry

-> Combinatorial chemistry was first conceived about 24 years ago -

although it wasn't called that until the early 1990s.

-> Initially, the field focused primarily on the synthesis of peptide and

oligonucleotide libraries.

-> H. Mario Geysen, distinguished research scientist at Glaxo

Wellcome Inc.,helped jump-start the field in 1984 when his

group developed a technique for synthesizing peptides on

pin-shaped solid supports. (currently, a faculty of Univ. of Virginia, Chemistry Department)

-> At the Coronado conference, Geysen reported on his group's recent development of an encoding strategy in which

molecular tags are attached to beads or linker groups used

in solid-phase synthesis. After the products have been

assayed, the tags are cleaved and determined by mass

spectrometry (MS) to identify potential lead compounds.

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Combinatorial Chemistry

-> Another early pioneer was Árpád Furka

who introduced the commonly used split-and-

pool methods.

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

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Combinatorial Chemistry

-> In 1985 Richard Houghten introduced the “tea-bag”

method for rapid multiple peptide synthesis.

-> These and other advances in manual multiplepeptide synthesis fed the beginnings of a wave of rapid bioassays based on the developing area of molecular biology.“

“Tea-bag method”

Polyethylene bag with fine holes, similar to real tea-bag, are filled with resins and each bag is put in the different reaction vessels to carry out amino acid coupling reaction. After reactions, all the bags are collected and processed together for protecting group removing and washing resins to

reduce the amount of time and efforts. In this method, the bag takes the role of filter and preventing resin mixing between reactions, and by labeling each bag, the synthesized peptide structure can be identified. About 100 different peptides in 500 micromol quantity could be synthesized by this method, which demonstrate a practical approach to parallel synthesis despite the fact that synthesized peptide number is moderate.

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Combinatorial Chemistry

-> Comparatively few organic chemists undertook the preparation of ordinary organic substances on solid

phases because the work is rather more complex

when applied to non-oligomeric substances caused

by greater variety of reactants and conditions

required, and this work at first failed to develop a

significant following.

-> Solid phase organic chemistry was also comparatively underdeveloped and this held back the field. This changed in dramatic fashion after the publication of Bunin and Ellman’s seminal work on solid phase organic synthesis (SPOS) of

arrays of 1,4-benzodiazepine-2-ones in 1992. Soon

other laboratories published related work on this ring

system, and work on other drug-like molecules

followed in rapid order and the race was on.

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Combinatorial Chemistry

From 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 complexity commonly 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 towards quality 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 the

biomedical 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 systematic dissection of these interactions. By no means will this be a trivial undertaking! The development of biological assays towards understanding biomacromolecular interactions is equally challenging as the need for having access to useful small molecule chemical probes.

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Combinatorial Chemistry

  • Used extensively in relation with drug discovery

  • Principle of Combinatorial Chemistry:

  • -> Generation of Compound Libraries from Molecular Building blocks which are usually used in high-throughput screening.



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Combinatorial Chemistry

What is a combinatorial (compound) library ?

-> prepared from a large number of different compounds at the same time

- not conventional one-at-a-time manner synthesis.

-> The characteristic of combinatorial synthesis is that different compounds are generated simultaneously under identical

reaction conditions in a systematic manner, so that ideally the products of all possible combinations of a given set of starting materials (termed building blocks) will be obtained at once

-> The collection of these finally synthesized compounds is

referred to as a combinatorial library.

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Combinatorial Chemistry

The game with the large numbers !!!

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Combinatorial Chemistry

The game with the large numbers !!!

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Combinatorial Chemistry

  • 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

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Combinatorial Chemistry

Drug Discovery

  • 1991-2003;

    ~2500 libraries

  • ”Unbiased libraries”; 1-2 million compounds

  • -> Screening does not always result in hits.

  • ”Directed libraries” build on a privileged structure”

    • -> Libraries based on a modelling.

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Combinatorial Chemistry

  • Solid-Phase Organic Synthesis

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

  • Solution-Phase Organic Synthesis

    -> The compound library have been synthesized in solvent in the reaction flask

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Combinatorial Chemistry

Solid Phase Synthesis

  • 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 glycol resins has been developed to improve the yield)

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Combinatorial Chemistry

Solution Phase Synthesis

  • Reaction proceeds in Solution

    + Easy characterization of intermediates as well as end pruduct, No limitations in attachment point -> Faster validation times relative to solid phase synthesis (Standard analytical protocols can be used to characterize products between each reaction step)

    - Difficult to drive the reaction towards the product, extensive purification is needed

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Combinatorial Chemistry

Polymer-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

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Combinatorial Chemistry

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Combinatorial Chemistry

Preparation 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 -> small libraries

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Combinatorial Chemistry

Preparation of Libraries

  • Pool/Split Synthesis

    • Good to generate large libraries

    • Labeling required to keep track of each compound

    • Beads (resin) are split into different


    • Then reacted, shuffled, and

      split again.

    • 1000 compund library prepared

      from 10 building blocks in each

      step  30 reaction steps.

      (1110 steps for parallel synthesis)

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Combinatorial Chemistry

Keeping Track of the Reactions

  • Radio Frequency (RF) tagging

    • Transponder tags incase in porous glass beads with a loading capacity of 30-300 mg of resin beads

  • Nano tagging

    • One reacent development in the labeling of beads is the nano-reactors -> labled with 2D-barcodes making it possible to keep track of libraries with up to 100,000

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Combinatorial Chemistry

Extraction 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 are insoluble.

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Combinatorial Chemistry

Library Formats

  • Combinatorial Libraries vary in size, amount, purity and structual complexity

  • The libraries can be devided into 3 groups

    • 1: One-bead one-compound

    • 2: Preencoded libraries

    • 3: Spatially addressable libraries

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Combinatorial Chemistry

Library Formats

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Combinatorial Chemistry

Library Formats

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Combinatorial Chemistry


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

-> library consists of a set of mixtures in which one of the variable positions is defined in each mixture

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

-> process repeated -> in each case a different variable position is probed

-> each library is smaller than the one preceding as the number of optimized positions increases

-> in the end yields individual structure (peptide sequence)

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Combinatorial Chemistry

Example: Scanning peptide libraries

-> generate a sublibrary for each variable position in sequence

-> in each sublibrary a specific position is fixed/defined

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

-> investigate significance of properties of AA at each position

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

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Combinatorial Chemistry

Example: 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

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Combinatorial Chemistry

Example: Scanning peptide libraries

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

-> requires target that recognizes concensus sequence (-> antibody, receptor)

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

-> based on performance establish concensus sequence

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Combinatorial Chemistry

Drug Discovery - Lead Identification

  • By screening pool/split solid-phase library of 128 000 2-arylindoles (1) split into 320 pools of 400 compounds and screened against 16 G-protein coupled receptor targets

    • Some pools both active and selective

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

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Combinatorial Chemistry

Drug 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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Combinatorial Chemistry

Drug Discovery - Lead Optimization

  • Solid phase synthesis with RF tagging

  • Screening of ~650 000 compounds

  • 28; active in a human erythropoietin (EPO) assay and have phosphodiesterase 3 activity

  • 32; treatment of anemia

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