Combinatorial synthesis

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COMBINATORIAL SYNTHESIS Naureen Shehzadi M.Phil. ( Pharmaceutical chemistry) University college of pharmacy, University of the Punjab, Lahore, Pakistan





Introduction :

Introduction In solid-phase synthesis, the compound libraries are synthesized on solid phase such as resin bead, pins, or chips.

Requirements for solid-phase synthesis:

Requirements for solid-phase synthesis

Solid support:

Solid support It refers to a cross-linked insoluble polymeric material that is inert to the condition of synthesis. A solid support may be; Polymeric beads Array of wells Array of pins Spatial arrays on microchip Cellulose sheets

Polymeric beads:

Polymeric beads Polymeric beads are most commonly used in combinatorial synthesis. These beads are functionalized to allow attachment of linker and substrate molecules. Examples : Beads made up of; Cross linked polystyrene Polyamide resin Tenta gel resin

1- Cross-linked polystyrene:

1- Cross-linked polystyrene Lightly cross-linked gel type polystyrene (GPS) is mostly used . Advantages: Commonly available Inexpensive It has ability to absorb large relative volumes of certain organic solvents Example : Wang resin (4-Benzyloxybenzyl Alcohol Resin)

2- Polyamide resin:

2- Polyamide resin Polyacrylamide polymers are used commonly. Advantages : Closely mimic the properties of peptide chains Improved solvation properties in polar, aprotic solvents (e.g. DMF) Example : PEGA resin

3- Tenta gel resin:

3 - Tenta gel resin These are poly (styrene-oxyethylene) graft copolymers consisting of PEG attached to cross-linked PS. Advantages : Uniform swelling in variety of solvents from medium to high polarity (e.g. toluene to water) Example : Tenta Gel resin onto cross-linked polystyrene

Linker :

Linker It is the group that joins the substrate to the resin bead is an essential part of solid phase synthesis. Functions: To support the attachment of a synthetic target T he polymer is usually modified by equipping it with a linker.

Choice of linker:

Choice of linker Choice of linker depends on ; Nature of reactions used in synthetic pathway Ease of detaching the product the end of synthesis

Characteristics of an ideal linker:

Characteristics of an ideal linker The linker must not be affected by the chemistry used to modify or extend the attached compound. The best linker must allow attachment and cleavage in quantitative yield.

Examples :

Examples Carboxylic acid linkers Carboxamide linkers Alcohol linkers Carbamate and amine linkers Traceless linkers

1- Carboxylic acid linkers:

1 - Carboxylic acid linkers First class of linker used for carboxylic acid is Merrifield resin. Cleavage to regenerate the carboxylic acid is usually achieved by hydrogen fluoride.

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The second class of linker used for carboxylic acid is the Wang linker. The carboxylic acid product can be cleaved with TFA.

2- Carboxamide linkers:

2- Carboxamide linkers The first developed linking group that would generate Carboxamide in mild acidic conditions was the methyl benzhydryl amine (MBHA) linker on polystyrene for improved synthesis of peptides using the BOC protection strategy.

3- Alcohol linkers:

3- Alcohol linkers All type of alcohols readily add to dihydropyran and the resulting THP protecting group is stable to strong base, but easily cleaved with acid. This linker is attached to a Merrifield resin.

4- Carbamate and amine linkers:

4 - Carbamate and amine linkers Two types of linkers may be used; 1- Linker based on hydroxyl methyl benzoic acid that allows cleavage under strong acidic conditions 2- Linker based on hydroxyl methyl benzoic acid with an electron-donating group added that allows cleavage by TFA

5- Traceless linkers:

5- Traceless linkers These linkers show non-specific function after cleavage. Traceless linkers are so called because an examination of the final compound reveals no trace of the point of linkage to the solid phase. The first and most widely explored of this traceless linker is the silyl linker.

Protection strategy:

Protection strategy A chemical protection strategy is needed to allow selective protection and deprotection of reactive groups. The amino acid building blocks are protected at their amino terminus by protecting groups. The protection group ensures that in each step only a single building block is coupled to each growing peptide chain .

Most commonly used protection groups:

Most commonly used protection groups Most commonly used protecting groups are; FMOC BOC TBOC

Method of solid-phase synthesis :

Method of solid-phase synthesis Solid support is taken . A linker is attached to the solid support. The intended carboxy terminal amino acid is anchored to a solid support by means of linker.

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Activated amino acid is coupled to the first amino acid (attached to linker). In order to prevent further chain growth at this point, the amino acid, which is added, has its amino group blocked. After the coupling step, the block is removed from the primary amino group and the coupling reaction is repeated with the next amino acid.

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The process continues until the peptide or protein is completed. At the end, the molecule is cleaved from the solid support and any groups protecting amino acid side chains are removed. The peptide or protein is purified to remove partial products and products containing errors.


Applications Synthesis of 1,4-benzodiazepines Synthesis of Benzopyran derivatives Solid phase-supported synthesis of (±)- Epibatidine Synthesis of luteinizing hormone releasing hormone analogues etc .


Advantages Ease of isolation (usually by filtration ) Removal of unreacted reagents is possible so large excesses can be used to drive the reaction to completion

Limitations :

Limitations All libraries have a common functional group at the position corresponding to the one used to link the initial building block to the linker or bead . Synthesis are usually carried out using linear approach Requires especially modified reactions with high yields (>98%) if multistep synthesis are attempted.

Limitations :

Limitations Requires additional synthesis steps to attach the initial building block to and remove the product from the support . Final product is contaminated with fragments of the product formed by incomplete reaction at different stages and often needs an additional purification steps.

Techniques in solid-phase synthesis:

Techniques in solid-phase synthesis

Parallel synthesis:

Parallel synthesis Parallel synthesis is based on “One vessel - one compound philosophy” (i.e. multiple reactions are carried out, each in a separate vessel, at once instead of in series.)

Basic principle:

Basic principle Compounds are synthesized in parallel using spatially separated compartments.

Method :

Method Each starting material is reacted with each building block separately. After each reaction step, the product is split into “n” partitions before it is reacted with next building blocks.

Approaches :

Approaches Conventional Technique: Using resin beads Reaction vessels: 96 well Microtiter plate (MTP) or an array of chromatography tubes etc . Houghten’s tea bag procedure Geysen’s multi-pin technique Fodor’s method of parallel synthesis (Light Directed Spatially Addressable Parallel Chemical Synthesis)

Advantages :

Advantages Parallel synthesis gives individual compounds, not a mixture. Thus deconvolution is not required . No risk of synergistic effects leading to false positive results during screening Each compound is substantially “pure “ in its location Defined location provides the structure of a certain compound Easier biological evaluation

Limitations :

Limitations The amount of vessels required for this process is large, and the number of reactions performed is even greater. So, this process is better suited to lead optimization rather than library generation . Applicable only for medium libraries (several thousand compounds)

Split-mix synthesis:

Split-mix synthesis The synthesis is based on “One bead - one compound” library philosophy

Basic principle:

Basic principle The starting material is split into “n” portions, reacted with “n” building blocks, and recombined in one flask for the second step. The whole procedure is repeated unless desired product is obtained.

Method :

Method Resins are divided equally into wells. The number of pools needed is dictated by the number of reagents that will be used during the reaction and the overall number of beads used must be at least equal to the total number of compounds which will be generated by the process. Each individual well is reacted with a different reagent.

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The products from these reactions are isolated via filtration and pooled together to give an equimolar mixture. This mixture is then equally divided amongst the wells, and reacted again with reagents. The process is repeated as many times as necessary. The end result is equimolar library of compounds representing all possible combinations of reagents.

Advantages :

Advantages Only few reaction vessels required Method of choice for large libraries (up to 10 5 compounds)

Limitations :

Limitations Threefold amount of resin beads is necessary Complex mixtures are formed Deconvolution or tagging is required The amount of product made is very small often in such small numbers that one compound is attached to one bead.

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Synergistic effects may be observed during screening leading to false positive results . Biological assays performed on mixtures are sometimes deceiving as well, as one product which is extremely biologically active could be masked by many inactive compounds in the same pool. A chemist could be fooled by identifying a pool containing a large number of moderately active molecules as a pool containing a few largely active molecules.



Introduction :

Introduction It involves conducting chemical reaction simultaneously, preferably in well-ordered sets (arrays) of reaction vessels in solution.

Method (formation of libraries of mixtures):

Method (formation of libraries of mixtures) These are formed by separately reacting each of members of a set of similar compounds with the same mixture of all members of the second set of compounds.

Combinatorial library of amides:

Combinatorial library of amides Set of 5 acid chlorides(A1-A5) is treated with ten amines(B1-B10 ) Each of five acid chlorides (A1-A5) is reacted with an equimolar mixture of all 10 amines and each of the amine is reacted with an equimolar mixture of all the acid chlorides. Two sub-libraries are formed (one sub-library consisting of a set of 5 mixtures based on individual acid halides and the other consisting of ten mixtures based on individual amines .


Applications Parallel synthesis of anti-inflammatory drug “fanetizole” in solution Libraries produced using resin-bound scavenging agents Libraries formed using fluorocarbon reagents

Function of scavengers:

Function of scavengers These are used to separate the product from the reagent used in the reaction easily. If the reagent is electrophilic, use a nucleophilic scavenger! If the reagent is nucleophilic, use an electrophilic scavenger!


Advantages Libraries do not have a common functional group at the position corresponding to the one used to link the initial building block to the linker or bead . Synthesis may be possible by linear and convergent approach. Unmodified traditional organic reactions may be used .


Advantages Does not require additional synthesis steps to attach the initial building block to and remove the product from the support . Final product is not likely to be contaminated with truncated intermediates.

Limitations :

Limitations Solution phase synthesis leads to formation of mixture of products. So, biggest challenge with solution phase synthesis is isolation of the product, and ways to automate this. Difficulty in removing unwanted material . Purification in each step is necessary.


COMPARISON BETWEEN SOLID AND SOLUTION PHASE SYNTHESIS PARAMETERS SOLID PHASE SYNTHESIS SOLUTION PHASE SYNTHESIS Reagent Excess Optimum (unless purification done) Purification Easy Can be difficult Automation Easy Difficult


COMPARISON BETWEEN SOLID AND SOLUTION PHASE SYNTHESIS PARAMETERS SOLID PHASE SYNTHESIS SOLUTION PHASE SYNTHESIS Reaction Suitable for few substances Suitable for any organic reaction Scale-up Expensive Easy and inexpensive Dependence of reaction development Mainly on Supports Linkers Time

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