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Thursday, May 14, 2009

COMBINATORIAL CHEMISTRY


COMBINATORIAL CHEMISTRY


P. Hima Anto HH, Toji Tom, Vimal Mathew
National College of Pharmacy, Manassery, Calicut
Cite this: P. Hima Anto, Toji Tom, Vimal Mathew, "Combinatorial chemistry", B. Pharm Projects and Review Articles, Vol. 1, pp. 420-462, 2006. (http://farmacists.blogspot.in/)
 

 

INTRODUCTION



 
    From the beginning of modern synthetic organic chemistry, the goal of chemists has been to produce single compounds in as pure form as possible. The one by one synthesis of thousands of new compounds followed by their one by one testing made the drug discovery process a very tedious, time consuming and expensive task.

 
    Beginning in the eighties, several innovative paper were published which radically changed theories and practices in designing and preparing new drug substances for pharmaceutical research and other areas of application. The new synthetic & screening procedures and, also very important, the new way of thinking were introduced in those paper which founded a rapidly growing new scientific field, combinational chemistry. These procedures revolutionized the pharmaceutical research and are gradually expanding to other area within & outside chemistry. The most influential method are briefly described as

 
  • The multipin procedures introduced by Geysen and his collegues – was used for the parallel synthesis of arrays of peptides. It is now considered as the prototype of the powerful automatic machines now capable of preparing hundreds of different kinds of individual compounds in parallel.

 
  • The " portioning - mixing method invented by Furka & his collegues – was developed to enable the users to prepare millions of new peptides in only a couple of days; also for synthesizing organic libraries.

 
Several others techniques developed for synthesis of large new compounds at a time were the biological method of preparation of peptide libraries, the light-directed spatially addressable parallel chemical synthesis.
DEFINITION

 
Thus COMBINATORIAL CHEMISTRY, is one of the important new methodologies developed by researches in the pharmaceutical industry to reduce the time &costs associated with producing effective & Competitive new dugs.

 
Modern combinational chemistry involves both the synthesis & screening of large sets of compounds called libraries. The libraries themselves can be always of individual compounds or mixtures.

 
When two reactants, A &B combines, A is actually a mixtures of five components while B may be a composite of ten, so on complete of reaction, a mixture of 50 different compounds will be produced.

 
    Combinatorial chemistry is one of the important new methodologies developed by academics & researchers in the pharmaceutical, agrochemical and biotechnology industries to reduce the time & costs associated with producing effective, marketable, & competitive new dugs. Simply put, scientists use combinatorial chemistry to create large population of molecules, or libraries that can be screened efficiently en masse. By producing larger diverse compound libraries, companies increase the probability that they will find novel compounds of significant therapeutic & Commercial values. The field represents a convergence of chemistry & biology, made possible by fundamental advances in miniaturizations, robotics, and receptor development.

 
    While combinatorial chemistry can be explained simply, its application can take a variety of forms, each requiring a complex interplay of classical organic synthesis techniques rational drug design strategies, robotics, scientific information management.

 
SYNTHETIC METHODS IN COMBINATORIAL CHEMISTRY

 
The various methods as used for synthesis of compounds in combinatorial chemistry. They all
  • Parallel synthesis leading to individual compounds
  • Combinatorial synthesis of mixtures

 
Methods is which the no: of synthesized compounds remain constant in the consecutive series of Coupling steps. These methods termed parallel procedures, are suitable for preparing series of individual compounds.

 
Methods in which the no: of the synthesized compound is increasing exponentially with the no: of the executed series of , coupling steps there methods are often called "real combinatorial procedures & they can produce either mixtures or series of individual compounds.

 
    They includes
  1. Portioning mixing (PM) synthesis
  2. Parallel Synthesis methods

 
THE PORTIONING – MIXING SYNTHESIS

 
    The PM method (Often named " split" Synthesis)is based as Merrifield's solid phase procedure.

 
    In Solid phase synthesis, it is a mtd in which the molecules are bound on a bead & synthesized step by step in a reactant solution . In this method, building blocks are protected at all reactive functional gp. The two functional groups that are able to participate in the desired reaction
b/n building blocks in the solution & on the bead can be controlled by the order of deproctection. In the basic method of solid- phase synthesis, building blocks that have two functional groups as used. One of the functional gps of building block is usually protected by a protective gp. The starting material is a bead which binds to the building block. At first, this bead is added into the solution of the protected building block & stirred. After the reaction b/n the bead of the protected building block is completed, the solution is removed & the bead is washed. Then the protecting gp is removed & the above steps are repeated. After all steps are furnished the synthesized compound is cut off from the bead.

 
        In PM synthesis, a tetrapeptide library on solid support using only three different amino acids, which are represented in figure : by white, black & gray circles. The synthesis is executed by repetition of the following three simple operations
  • Dividing the solid support into equal parts.
  • Coupling each portion individually with only one of the different amino acids.
  • Homogenously mixing the portions.
    .
In the first round Fig1 a , the amino acids are coupled to equal portions of the resin and the final product of the combining & mixing the portions – is the mixture of the three amino acids bound to resin. In the 2nd cycle, this mixture is again divided into these equal portions & the amino acids are individually coupled to these mixture. In each coupling step, these different resin bound dipeptides as formed, so the end product is a mixture of 9 peptides. The divergent, vertical & Convergent arrows indicate portioning, coupling & mixing, respectively. A further portioning, coupling & mixing step leads to the formation of a mixture of 27resin bound tripeptides.

Features

 
  1. Efficiency
            It can be seen that starting with a single substance, the no :of compounds is tripled after each coupling step. First 3 x 1 = 3 resin bound dipeptides, then 3 x 9 = 27 resin bound tripeptides & finally 3 x 27 = 81 tetrapeptides are formed. If a 20 different amino acids are used in the synthesis, the no :of peptides in each coupling step is increased by a factor of 20. The total no: of the synthesized peptides can be expressed by a simple formula 20', when n is the no :of amino acids.

     
  2. Formation of all possible sequence combinations
        Consecutive execution of the three simple operations (portioning, coupling, mixing) ensures with mathematical accuracy – the formation of all possible sequence combinations of amino acid building blocks used in the synthesis. This combinatorial principle embodied in PM synthesis captured the imagination of many researchers & had a profound effect on the development of the field.

     
  3. Formation of compounds in one to one molarities
    It is very important to prepare libraries in which the constituents are present in equal molar quantities .Otherwise, a low activity component, if present in a large amount, may show a stronger effect than a highly active component present in lower quantity. The PM method was designed to comply with requirement of 1:1 molar ratio. Before each round of couplings, the resin is thoroughly mixed, then divided into homogenous equal portions. This ensures that the previously formed peptides are present in equimolar quantities in each portion.

     
        The PM synthesis determination of the structure of the various organic compds is not as simple as sequencing peptide , the bead are usually encoded. The building blocks of the encoding tags are attached to the beads in parallel with the organic building blocks of the library. Two types of encoding are

     
  4. Encoding with sequences
  5. Binary Coding

     
        When encoding by sequences, the encoding tags are peptides or oligo nucleotides. Their sequences encode both identity of the organic reagents coupled to the bead & the order of coupling.     The white, black, gray & white square encode the organic reagents represented by white, black & gray circles & their white – black. Gray – white coupling order.

     
        In binary encoding system the coding units are halobenzenes carrying a varying length hydrocarbon chain attached to the bead through a cleavable spaces. It is simply their presence which codes for the organic building blocker & their position.

     

    Disadvantages



     

    1. There is need of large qty of resin needed in the preparation of the libraries of the higher member peptides

     
    1. Before beginning a synthesis, the relation of the no. of expected peptides to the no. of beads of the resin are to be considered. Since only one peptides forms on each bead, the maximum No. of peptides is limited by the No. of beads.

     
    There difficulties can be circumvented by:

     
  6. Reducing the No. of the varied amino acids
  7. Reducing the No. of varied positions

     
    THE MIXED REAGENT METHOD OF THE SYNTHESIS OF COMBINATORIAL LIBERTIES

     
        Combinatorial peptide libraries can be prepared by using mixture of amino acids in the acylation step of the solid phase synthesis. Although the method is even more efficient than PM procedure, it has serious disadvantages. The one to one molar ratio of the formed compound cannot be assured due to the differences in the coupling rates of amino acids. There differences can in part be compensated by proper adjustment of the concentration of amino acids in the coupling mixtures. The method has been applied mainly in preparation of peptides libraries, but non peptide libraries have also been sythesized. Because mixtures of reactants are used in couplings, at the end of the synethsis of all library components are present on every bead. This means that only mixtures can be prepared by their method.

     


    THE BIOLOGYCAL METHOD



     

        The biological method of creating peptide libraries is briefly exemplified by phase display libraries. First on oligo nucleotide library is synthesized chemically by a series of coupling with equimolar nucleotide mixtures. The formed oligonucleotides are then inserted into the DNA of phages. In the next stage the phages infect the host bacterium and their DNA replicate together with the inserted "foreign" DNA segment. A library of phages clone forms. Each clone carrier in its DNA a different "foreign" sequence segment which is expressed as a partial sequence of its coat protein. Every page particle carries a couple of thousand identical coat protein molecules with the same peptides sequence fused to the Outer end. In this respect the pages resemble the bead in PM synthesis, with each containing an individual compound. The DNA of the phage can be considered as an encoding tag since the sequence of the peptide can be determined by sequencing the proper portion of the DNA.
    .
    THE LIGHT – DIRECTED, SPATIALLY ADDRESSABLE PARALLEL CHEMICAL SYNTHESIS

     
        The light directed method makes it possible to prepare an array of peptides or other kinds of molecular on the surface of a glass slide. The surface of the glass is functionalized with amino alkyl gps protected by the photo- labile 6 – nitro Vera tryloxycarbonyl (NVOC) gps. The amino acid used in the synthesis are also protected by NVOC. group.

     

     
    Figure 1. The
    light – directed synthesis of nine dipeptides


     
        Nine dipeptides are synthesized from amino acids A, G & K. Before each coupling step one or more area of the slide is irradiated through a mask to remove the protecting g.p. then submitted to coupling with the indicated protected amino acid coupling occurs only in the irradiated area. After completing through f cyder of irradiation & coupling , nine dipeptide sequence are found in locations.

     
        By irradiating through marks h, i, j & coupling with amino acids A, G, & K , 27 tripeptides will form. All these are individual compound which are formed is an efficient way resembling PM synthesis.

     
    Advantages
        The sequence are defined by their locations on the slide.

    RESINS FOR SOLID PHASE SYNTHESIS



     
        In all the above solid phase synthesis, the solid support is generally based on a polystyrene resin. The most commonly used resin supports for SPS include spherical beads of lightly cross linked gel type polystyrene (1-2% divinyl benzene) & poly (styrene oxyethylene) graft copolymers which are functionalised to allow attachment of linkers& substrate molecules.

     

    GOSS LINKED POLYSTYRENE



     

        Lightly cross-linked gel type polystyrene (GPS) has been most widely used due to its common availability & inexpensive cost. GPS beads which are functionalized with chloromethyl- amino methyl -, & a variety of linkers are commercially available from a variety of sources. A prominent characteristic of GPS beads is their ability to large relative volume of certain organic solvents (swelling). This swelling causes a phase change of the bead from a solid to a solvent – swollen gel, & there for, the reactive sites are accessed by diffusion of reactants through a solvent – swollen get network.

     
    GPS has good swelling characteristics in solvents of low to medium polarity ranging from aliphatic hydrocarbons to dichloromethane. Polar, protic solvents, such as alcohols & water do not swell GPS resin, & accessibility to all reaction sites may be compromised. Hence GPS support are most suitable for chemistry performed in solvent of low to medium polarity.

     
    POLYAMIDE RESIN


     

    It was expected that these polymers would more closely mimic the properties of the peptide chain themselves & have greatly improved solvation properties in polar, aprotic solvents the protecting gp chosen was the fluorenyl meth- oxycarbonyls (fmoc)which can be removed by base ( usually piperidine )

     
    TENTA GEL RESINS


     

        Poly (sty rene – oxy ethylene ) graft copolymes are another class of widely used supports for as organic synthesis. As with poly acrylamide resins, in order to produce a polar rxn that is closer to the solvents generally used by solution synthetic chemists grafted polymer beads have been prepared. The most pre-eminent of these is Tentagel resin which consists of polyethylene glycol attached to cross linked polystyrene through an ether links. Combines the benefit of the soluble polyethylene glycol support with the insolubility& handling characteristics of the polystyrene bead. The resin was originally prepared by the polymerisation of ethylene oxide on cross linked polyethylene already derives with tetra ethylene glycol to give polyethylene glycol chains.

     

    Disadvantages



     

        Relatively low functional gp loading compared with Gps; the potential for the PEG chain to complex lewis acids:
  • The potential instability of PEG
  • The presence of linear PEG impurities found in the small molecule products after cleavage from the resin
    • The tendency for resins to become sticky & difficult to handle as the synthesis progress.

LINKERS



 


 

    The gp that joints the substrate to the resin bead is an essential part of solid phase synthesis. The linker is a specialized protecting gp, in that much of the time the linker will lie up a functional group, only for it to reappear at the end of synthesis.

 

CARBOXYLIC ACID LINKERS



 

    The first linking gp used for peptide synthesis bears the name of the father of solid phase synthesis. Merrifield resin in cross linked polystyrene functionalized with a chloromethyl gp. The carbonyl gp is attached by the nucleophilic displacement of the chloride with a cesium carboxylate salt in DMF. Cleavage to regenerate the carboxylic acid is usually achieved by HF.

 
    The 2nd class of linker used for carboxylic acid is Wang linkers. The linker is generally attached to cross – linked polystyrene, Tentagel & polyacrylamide to form wang resin – It was designed for the synthesis of peptide carboxylic acid using the Fmoc- protection strategy, & due to the activated benzoyl alcohol design, The carboxyl acid product can be cleaved with T.F.A

 
    A more acid –labile form of the Wang resin has been developed the SASRIN resin has the same structure at the Wang linker but with the addition of a methoxy gp to stabilize the carbonium ion formed during a catalyzed leavage

 

CARBOXAMIDE LINKER



 

    The Wang ests linker can be cleaved with NH3 to generate 10 Carboxamide, but there is a difficult rxn, that is very slow with sterically 100 amino ā such as valine A prolonged treatment with NH3 could lead to a recemisatioin of chiral peptide.

 

Amino linkers



 

    Carbamaties linkers has been used for the synthesis of a combinatorial library of 5 + 6 polyamines prepared in the search of inhibitors of trypanosomal parastic infections. Two linkers was investigated. One based on hydroxy methyl benzoic acid ,and the other one ,an electron donating group has been added. The last one allowed cleavage by TFA while the first one could be cleaved with strong acidic condition.
PARALLEL SYNTHETIC METHOD

 


 

Two methods can be used for parallel synthetic methods they are
  1. The multipin method
  2. The teabag method

     
    THE MULTIPIN METHOD

     
        In parallel procedures an array of different substances are simultaneously prepared. The first example of parallel synthesis was published by Geysen his colleagues. They synthesized a series of peptides epitopes in an apparatus developed for this purpose. The multipin apparatus had a block of wells serving as reaction vessels and cover plate with mounted polyethylene rods fitting into well. The first amino acid was attached to the end of polyethylene rods (pins) grafted with derivatized polyacrylic acid (marked by gray) The solutions of protected amino a coupling reagent were added to the wells (dark gray). The peptides formed on the pins immersed into solutions. The sequence of peptides depended on the order of amino acids of added to the wells. The peptides were screened after deprotection without leaving them from the pins.

     

     
    Figure 2. Multipin Apparatus
        The most characteristic feature is that the no. of product formed during the synthetic process never exceeds the no. of starting samples The multipin method is still used,$ the multiple apparatus is commercially available product. The multipin procedure was applied by Ellman & his Colleagues pioneering the preparation of organic libraries by parallel synthesis. Derivatives of 1,4- Benzodiazepines were constructed from 2 – aminobenzophenones, amino acid and alkylating agents. The Fmoc protected 2 – amino benzophenones were first attached to an acid labile linker (L), to the then through the linker to the pins (P). After removal of the protecting group it was coupled with a protected amino acid (!). This was followed by the removal of the Fmoc protecting gp & eycligation (2), then by alkylation of the ring nitrogen to introduce R4(3).Finally the product was cleaved from the support(4)

     

    THE TEABAG METHOD



     
        A different version of parallel synthesis was developed in 1985 by Houghten for preparing array of peptides. The beads of the solid support were enclosed in permeable plastic bags, then placed for coupling into a reaction vessel containing the solution of amino acid & the coupling reagent. All operations, including removal of protuting groups, couplings, washings & even the cleavages were performed on solid supports enclosed in bags. This procedure has significant advantages.

     
        All those bags which needed the attachment of the same amino acid (eg. Alanine) were grouped together, placed into the same reaction vessel,& the coupling could be done in a single operation could be done in a single operation.

    THE SPOT TECHNIQUE



     

        The SPOT method introduced by Frank and his group was also developed for preparing peptide arrays. The synthesis is carried out on cellulose paper membranes derivatised to serve as anchors for the first amino acids of the sequences to be prepared. Small droplets of solutions of protected amino acids dissolved in low volatility solvents & coupling reagents are pipetted on to predefined positions of the membrane. The spots thus formed can be considered as reactors when the conversion reactions of the solid phase synthesis take place.

     
        An array of as many as 2000 peptides can be made on a 8 x 12 cm paper sheet . The peptides can be screened on the paper after removing the protecting groups. The method was also used to make minutes in the spots.

     

    OTHER DEVICES FOR PARALLEL SYNTHESIS



     

        De Witt & coworkers also developed an apparatus for parallel synthesis. It was designed for the synthesis of small organic molecules. The solid support were placed into porous tubes immersed in vials containing solutions of reagents which differed into tubes. The temperature of the reaction mixture could be controlled by heating or cooling the reaction block.

     
        Another inexpensive device was described by Meyers et al. Beckman polypropylene deep wells plates were modified by drilling a small hole in the bottom of each well. A porous polyethylene frit was fitted into the bottom of the well to allow removal of the solutions & solvents by vacuum. At the bottom, a rubber gasket prevented the leakage from the well during the reactions.
    SCREENING METHODS

     

     
        A reliable high throughput assay is essential to successfully screen a combinatorial library. For screening assays have been developed for the one bead one – compound combinational library method. In the solid phase assays, the ligands are still covalently attached to the solid support $ the assay involve either (1) direct binding of molecular target to the bead – bound ligand or (ii) detection of functional properties of the bead – bound ligand such as identifying phosphorylation or proteolytic substances (substrates).

     
        Solution phase assays require cleavable linkers so that the ligands can be released while the beads are still spatially separated $ the positive beads from which positive ligands are released can be identified. Two solution phase assays have been developed.

     
    1) the 96 – will two stage approach.
    2) the insitu – releasable solution phase assay.
        Two methods are adopted for screening of compounds. They are.
    A) On – bead screening
    B) Solution phase screening
    C) Combination of on - bead and solution phase screening assay

     
    A) ON BEAD SCREENING

     
            In this assay system, the ligands are still covalently attached to the solid support. They require aqueous media. Thus solid support $ its linkers must be water compatible. Two common solid support fit this criterion.
    1)    The polyethylene glycol grafted polystyrene bead (eg: Tentagel)
    2)    The polydimethyl acrylamide bead.
            The beads should be uniform in both size $ substitution .Unfortunately, although the size distribution for tentagel is relatively uniform, its functional substitution is not. In contrast, the substitution of the polydimethyl acrylamide bead is fairly uniform. But they are more sticky $ have a tendency to clump together. There is a need for non sticky beads with both uniform size $ substitution that are fully compatible with aqueous media.

     
            The two approaches to screening bead – bound ligand libraries involve the detection of
  • target binding to the ligand
  • functional properties of the ligand.

 
1. Binding Assay

 
    The binding of target to bead-bound ligand can be detected either by direct visualisation (eg. Colour target such as dye or a larger target such as cell) or indirectly by using a reporter group such as an enzyme, a radio nuclides, a fluorescent probe, or a colour dye covalently attached to a target. The enzyme linked colourimetric assay first described in 1991 is simple & highly efficient. The screening of a library of 107 bead bound ligands can early be accomplished by one person in one day.

 
    Non specific binding or specific binding to an undesirable site of the receptor could be a significant problem in screening a library of million of beads. It can be usually be eliminated by using a high ionic strength buffer (eg: 0.3 – 0.4 Nacl.) with nonionic detergent & blocking proteins. The chromogenic substrate used in the intial screening system with alkaline phosphatase coupled target were a combination of nitroblue teratzolium (NBT) & 5 – bromo – 4- chloro – 3. indolyl phospate (BCIP). Alkaline phospatase hydrolyses the phoshate & BCIP in then oxidised into terquoice colour with indigo dye. Inturn NBT is reduced to formazan, a dark purple insoluble precipitate deposited on the surface of the positive bead. The addition of NBT to the BCIP substrate greatly amplifies the sensitivity of the assay.
Ohlmeyer at all used a dye-labeled target to screen bead bound peptide libraries. A dual colour screening method using two different colour dyes to detail specific binding has also been designed.

 
Chen et al used a fluorescently labeled SH3 domain to screen a peptides bead library & isolated the beads with a fluorescent microscope.

 
Radio nuclides – labeled targets have been used to screen bead libraries Kassaaiyian et al. used a 1 125 labeled anti – beta endorphin to probe a random peptide library & used the autoradiographic detection method to localize the positive beads. This detection method appear more tedious when compared to the enzyme linked colourimetric approach, particularly when macromolecular targets were used.

 
For molecular targets that are intrinsically coloured or fluorescent, a bead library can be secured directly. Lam at all of other have used colour dyes to directly screen combinatorial peptides libraries & have isolated peptides that interact with the dye molecule.

 
With use of this method, peptides that bind to the surface integrin of a prostate cancer are cell line were identified. In principle, the one – bead. one compound library method can also be applied to the intact viral particle, bacteria or yeast. Since these organisms are so small, a reporting group of some sort may be needed for identification of positive beads.

 
2.    Functional assay

 
        Specific functional assays have been developed for detecting ligands that are covalently attached to beads. Lam and Wu reported the use of one – bead – one- compound library method to identify peptide substrate motifs for post translational modifications such as protein phosphorylation. In this assay, a random peptide- bead library is incubated with ( Gama – 32 p) ATP and protein kinase. After incubation, the beads are washed thoroughly and heated to 100 c for five minutes. With .1 M HCl in order to hydrolyze the ( Gama 32 P) ATP thus eliminating any non specific binding of ( Gama 32 P) ATP to the positively charged peptide beads. The beads are then immobilized on glass plate with .5 % molten low – gelling temperature agarose . After drying, the immobilized beads are exposed to an X-ray film. Autoradiography is then used for localization of the positive beads. Because of the limited resolution of autoradiography, precise localization of the positive bead in the primary screen is impossible. The beads collected at or around the dark spot excised, heated to 100c , diluted with additional molten agarose, and immobilized on a second glass plate and then the autoradiography repeted . With this secondary screen, individual positive beads can be precisely located and isolated for structure determination.

 
         This approach can be applied to many other post translational modifications such as methylation, glycosylation, adenylation , sulfation and hydroxylation.

 
B. SOLUTION PHASE SCREENING

 
        Solution phase assays, usually in the 96-well plate format, have been used in mass screening for most drug discovery programmes. There are many solution phase assays available. Eg. Competetive receptor binding assays with radiolabelled ligands, various enzymatic assays, cell based signal transduction assays , antibacterial assays, antiviral assays, anticancer assays. All these solution-phase assays, in principle can be adapted to combinatorial library. Because the number of compounds mixture of compounds generated by combinatorial methods are enormous, the current trend is to miniaturize and automate. These solutions- phase assays .

 
        There are two general approaches to screen one bead –one compound library with the solution phase.

 
  1. The 96- well two stage release assays and
  2. The insitu – releasable solution phase assay with immobilized beads

     
        In both approaches, ligands are attached to the solid support via cleavable linker. The ligands are done released from each bead into solution phase where the biological assays take place. The bead of origin of the positive releasate can subsequently be identified, and isolated for structure determination.

     
    The 96 – well two stage releasable assays

     
        In this assays, double orthogonally cleavable linkers are incorporated into the preparation of the library. Approximately 100 to 500 beads are added in to each well of a 96 – well filtration plate. Upon neutrilization, the first the linker is cleaved with the formation of a diketopiperazine molecule on the bead. After incubation overnight suction is applied so that the filtrates are collected in a 96 – well placed beneath the filtration plate. The filtrates are then tested for biological activity. Beads from the positive wells are then redistributed into filtration plates. With one bead per well. With alkali treatment eg gaseous ammonia, the second linker is cleaved, and the filtrate from each well are then tested for biological activity. The beads the correspond to the positive wells are then identified and isolated for structure determination. This two stage release assay is needed if a higher number of beads are assayed. If the number of beads to be assayed is limited, a single release assay with one bead per well may be sufficient particularly if the transfer of individual bead into each well can be automated. For a 100 micrometre bead ,approximately. 100 p. mole of compound can , in principle, be recovered giving a final conc. of 1 micrometre . In order to increase the conc. of the recovered compound, one may (1) miniaturize the assay volume, (2) use bigger beads, or (3) use beads with higher substitution.

     
        However, if the bead is too big, the efficiency of the compound extraction from a bead may greatly diminish.

     
    Insitu solution phase releasable assay

     
        Here be use soft agar to immobilize beads. After the linker has been cleaved the compounds will be released and diffuse into the surrounding agar, where the solution phase assay takes place. Another method is that they first immobilized the bead the library on a thin film of polyethylene and exposed the library to gaseous trifluoroacetic acid for 10 hour at room temperature. After neutrilization with gaseous ammonia, beads were layered on the surface of a dish of melanocytes growing in soft agar. As a result of pigment dispersion, the cell located underneath and around the positive beads with MSH agonist activity turned dark within 15 minutes. This elegant assay system has also been adapted to another G- protein coupled receptors by transfecting those receptors into a cell line with melanocyte background.

     
        The insitu- releasable assay is highly efficient and in principle, only a single cleavable linker is needed since the beads are already spatially separated ; the two stage release assay as described in the 96 –well releasable method is not needed. The insitu assay has an advantage in that the conc. of the released compound could be rather –high in close proximity to the bead and the potency of the compound could be estimated on the basis of the size of the activity ring surrounding each positive bead . Currently, not all solution phase assays can be adapted to their assay format.

     
    C. COMBINATION OF ON BEAD AND SOLUTION PHASE SCREENING ASSAY

     
         In some instances, it may be advantageous to combine solution phase assays with on – bead assays to screen a specific target . Positive beads isolated by this approach are more likely to be true positives Eg. The compound beads are partitioned in to 1000 beads per well and a portion of the compound on each bead is released into the solution for biological testing.

     
        The 1000 beads from a positive well can then be recycled and an on – bead binding assay performed to identify single positive bead. Using this approach SALMON et al , successfully isolated ligands that bind to an anti – beta endorphin monoclonal antibody.

     
    Alternatively, an on – bead binding assay can be performed. Positive beads can then be collected for a releasable functional solution – phase assay to identify true positive bead. Eg. The beads that bind to a protein kinase can first be identified and isolated by an enzyme – linked colorimetric assay .

     
    Compounds from each positive bead can then be released and tested for protein –kinase inhibitory activity.

     
    USES

     
        Combinatorial chemistry reduces the time and cost associated with producing effective and competitive new drugs. This method is having a profound effect on all branches of chemistry, but especially on drug discovery. It is now possible to produce libraries of small molecules to screen for novel bio activities. This powerful new technology has begun to help pharmaceutical companies to find new drugs candidate quickly, same significant money in preclinical development costs and ultimately change their fundamental approach to drug discovery .

     
        While combinatorial chemistry can be explained simply, its application can take a variety of forms, each requiring a complex interplay of classical organic synthesis techniques , rational drug design strategies, robotics and scientific information management .
    CONCLUSION

     
        The last five years has seen an explosion in the exploration and adoption of combinatorial techniques. Indeed, it is difficult to identify any other topic in chemistry that has ever caught the imagination of chemists with such fervor. For pharmaceutical chemists, at least, the reason for this change is not hard to fathom. Two decade ago, the market for pharmaceuticals was growing at around 10% per annum, but more recently, rate of the market growth has declined. At the same time, cost constraints on pharmaceutical research have forced the investigation on methods that offer higher productivity at lower expenses. The belief that combinatorial chemistry will allow the productive and cost-efficient generation of both compounds and drug molecules has fuelled enormous investment in this area. This technique has definitely decreased the cost involved in new drug research and increased the chances of finding new lead molecules.

     
        Combinatorial chemistry represents a broad spectrum of techniques that are rapidly becoming a standard part of the medicinal chemist's tool kit. Combinatorial chemistry as a technique for the rapid synthesis of drug like compounds will continue to make a major impact on the way drug molecules are discovered. The development of combinatorial chemistry is timely and undoubtedly will contribute to the discovery of new drugs that can benefit mankind.
    BIBLIOGRAPHY

     
    1. Muralidhar, P. and Sastry, B.S., Combinatorial Chemistry - A Novel Tool For Drug Discovery, Indian J. Pharm. Sci., 63, 2001, 279-285.

 
  1. Patrick, M., Introduction to Medicinal Chemistry, Combinatorial Synthesis, 2nd Ed, Oxford university Press, New York 2001, 289-302.

 
  1. Scott, J.K. and Smith, G.P., Searching for Peptide Ligands with an Epitope Library Science, 249, 1990, 386-390.

 
  1. Atherton, E., Clive, P.L.J. and Sheppard, R.C., Polyamide Supports for Polypeptide Synthesis, J. Am. Chem. Soc., 97,1975, 6584-6585.

 
  1. http://www.combichem.net

 

 

 

 

 

 

 

 

 

 

 
Cite this: P. Hima Anto, Toji Tom, Vimal Mathew, "Combinatorial chemistry", B. Pharm Projects and Review Articles, Vol. 1, pp. 420-462, 2006. (http://farmacists.blogspot.in/)
 

 

 

 

 

 

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