1. Combinatorial Chemistry
Monica Sharfin Rahman
Lecturer, Department of Pharmacy
BRAC University

2. Drug Discovery Process
The identification of a chemical structure that has both the desired potency against a nominated biological target and also a suitable bioavailability and efficacy in an appropriate animal model of the targeted disease.
1st phase: finding a lead compound or structure that has some degree of affinity for the biological target.
2nd phase: identifying the drug development candidate by improving the leads structure.
When receptors and enzymes are therapeutic target a synthesis process is needed for supplying thousands and millions of compounds rapidly. In these consequences combinatorial chemistry develops.

3. COMBINATORIAL CHEMISTRY
Combinatorial chemistry is a technology through which large numbers of structurally distinct molecules may be synthesized in a time and resource effective manner and then be efficiently used for a verity of application.
Combinatorial chemistry is a synthetic strategy which utilizing different technique aims at the rapid synthesis of large collections of compounds.

4. Combinatorial chemistry offers a way by which many compounds
can be synthesized very quickly in parallel to optimize a lead
compounds activity in the absence of any binding model. It provides
a way of rapidly exploring SAR.
A+B  A-B  A-B-C
E.g. Compound A and Compound B react to produce AB, further react with C to produce ABC, which is isolated after reaction and purification through crystallization, distillation or chromatography in Orthodox synthesis.
Where as in combinatorial chemistry every possible of compound A1 to An with B1 to Bn can be produced parallelly by solid phase or solution phase synthesis.
E.g. a two stage synthesis with 10 starting compounds and 10different building blocks at each stage would yield 1000 compounds.

5. Design of Combinatorial synthesis
Two general strategies are usually followed:
The sequential attachment of building blocks:
Grow in one direction
b. Non-sequential attachment of building blocks:
Grow in different directions from an initial building block (Template)

6. The reactions used in designing a combinatorial sequence
should satisfy certain criteria:
From a bond between the building blocks.
Specific and high yielding.
Suitable for use in automated equipment.
Allow the formation of wide range of structure and all possible stereoisomer’s.
Easy availability of building blocks.
High diversity of building blocks for utilizing all types of bonding.
Accurately determinable structures of the final products.

7. General techniques for Combinatorial Synthesis:
Solid phase synthesis
Solution phase synthesis
Difference Involved:
Reagents
Purification
Automation
Flexibility
Scale-up

8. Solid phase synthesis
Combinatorial Synthesis is based on solid phase chemistry. It uses filtration as a separation and purification technique. The easy removal of unwanted substance by a simple filtration is the core of library synthesis.
In this synthesis substituted resin beads are used as a solid phase which is actually a gel like matrix of connected polymeric molecules distended by the access of solvent molecules.
Fig: Synthesis of dimer XY on resin beads using excess monomer Y and reagent R, which can be easily removed by filtration.

9. Resins beads The use of solid support for organic synthesis requires:
A cross-linked, insoluble, but solvent swellable polymeric material that is inert to the conditions of synthesis.
Some means of linking the substrate to this solid phase that permits selective cleavage of the product from the solid support during synthesis.
A synthetic procedure compatable with the linker and the solid.

10. Preparation of Resin Beads
The addition and dispersion of an organic phase of monomer and cross linker in an aqueous solution dissolving a free radical initiator in organic phase and rising of temperature starts polymerization and produces small solid spherical resin beads.
Size: µm usually.

11. A. Cross-linked polystyrene
Gel type polymer due to polymerization of styrene. 1% divinylbenzene acts as cross-linker. Its use is limited for high electronic reagents and above 130ₒ C.
Fig: The molecular structure of polystyrene, Where X could be any suitable functionally.

12. B. Polyamide resins
It is polyacrylamide polymers, usually formed by using N,N-dimethylacrylamide as backbone monomer, N,N-bisacryloyethyleneamine, as cross linker and is functionalized through N-acryloyl-N-Boc-ẞ-alaninylhexamethylenediamine.

13. C. Controlled pore glass
It is rigid, glass derived bead material, compatible with any type of solvent, stable to aggressive reagents and extreme of pressure and temp. Due to ability of continuous solvent flow through macro pores it is suitable for continuous flow synthesis.
D. Tenta Gel Resin
Consists of polyethylene glycol attached to cross-linked polystyrene through ether link. It is obtained by polymerization of ethylene oxide on cross linked polystyrene. Due to polarity it facilitates the release of products or screening of the beads in an aqueous environment.
Fig: Polyethylene glycol chain grafted onto a cross-linked polystyrene.

14. E. Magnetic beads
Nitration of polyvinylbenzene and reducing the nitro group with ferrous sulfate hexahydrate result the incorporation of Fe +2 and Fe+3 within the bead.
Thus the bead contain 24-32% iron by weight and can be manipulated by bar magnet.

15. Merrifield solid support peptide synthesis
Solid-phase peptide synthesis (SPPS), pioneered by Merrifield, resulted in a paradigm shift within the peptide synthesis community.
It is now the accepted method for creating peptides and proteins in the lab in a synthetic manner. SPPS allows the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.

16. Small beads are treated with linkers on which peptide chains can be built. The synthesis beads will retain strong bondage to the peptides until cleaved by a reagent such as trifluoroacetic acid. The beads create a synthesis environment in which the peptide chains being created will not pass through a filter material while the reagents used to create them will.
The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield, if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents
There are two majorly used forms of SPPS - Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by these two groups and added onto a deprotected amino acid chain.
Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.
SPPS is limited by yields, and typically peptides and proteins in the range of 70~100 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.

17. Merrifield solid support peptide synthesis
Figure: The solid phase peptide synthesis (SPPS) principle

18. Merrifield solid support peptide synthesis
Boc SPPS
When R. B. Merrifield invented SPPS in 1963, it was according to the tBoc method. t-Boc (or Boc) stands for tert-Butyloxycarbonyl.
To remove Boc from a growing peptide chain, acidic conditions are used (usually neat TFA).
Removal of side-chain protecting groups and the peptide from the resin at the end of the synthesis is achieved by incubating in hydrofluoric acid (which can be dangerous); for this reason Boc chemistry is generally disfavored.
However for complex syntheses Boc is favourable.
When synthesizing nonnatural peptide analogs which are base-sensitive (such as depsi-peptides), Boc is necessary.
Fmoc SPPS
This method was introduced by R.C. Sheppard in Fmoc stands for 9-Fluorenylmethoxycarbonyl which describes the Fmoc protecting group.
To remove an Fmoc from a growing peptide chain, basic conditions (usually 20% piperidine in DMF) are used.
Removal of side-chain protecting groups and peptide from the resin is achieved by incubating in trifluoroacetic acid (TFA).
Fmoc deprotection is usually slow because the anionic nitrogen produced at the end is not a particularly favorable product, although the whole process is thermodynamically driven by the evolution of carbon dioxide.
The main advantage of Fmoc chemistry is that no hydrofluoric acid is needed. It is therefore used for most routine synthesis.

19. Merrifield solid support peptide synthesis
Solid supports  
                        
The physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of crosslinking, as well as the linker and handle being used.
1. Polystyrene resin
This is a versatile resin, which is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in DCM.
2. Polyamide resin
This too is a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers, if the wells are too small.

20. Protective groups:
Due to amino acid excesses used to ensure complete coupling during each synthesis step, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protective groups are used. This adds additional deprotection phases to the synthesis reaction, creating a repeating design flow as follows:
Protective group is removed from trailing amino acids in a deprotection reaction
Deprotection reagents washed away to provide clean coupling environment
Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) are combined with coupling reagents are pumped through the synthesis column
Coupling reagents washed away to provide clean deprotection environment
Currently, two protective groups (Fmoc, Boc) are commonly used in solid-phase peptide synthesis. Their lability is caused by the carbamate group which readily releases CO2 for an irreversible decoupling step.

21. Fmoc protective group
The Fmoc (9-fluorenylmethyl carbamate) is currently a widely used protective group that is generally removed from the N terminus of a peptide in the iterative synthesis of a peptide from amino acid units.
The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions. This allows mild acid labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide.
This orthogonal protecting group strategy is common in the art of organic synthesis.
Fmoc cleavage

22. Boc protective group
Before the Fmoc group became popular, the Boc group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthogonal strategies.
It retains usefulness in reducing aggregation of peptides during synthesis. Boc groups can be added to amino acids with boc anhydride and a suitable base.
Boc cleavage

23. Parallel synthesis
This technique is normally used to prepare combinatorial libraries that consist of separate compounds. It is not suitable for the production of libraries containing thousands to millions of compounds. In parallel synthesis the compounds are prepared in separate reaction vessels but at the same time, that is, in parallel.
The array of individual reaction vessels often takes the form of either a grid of wells in a plastic plate or a grid of plastic rods called pins attached to a plastic base plate (Fig. 5.7) that fits into a corresponding set of wells.
In the former case the synthesis is carried out on beads placed in the wells whilst in the latter case it takes place on so-called plastic ‘crowns’ pushed on to the tops of the pins, the building blocks being attached to these crowns by linkers similar to those found on the resin beads. Both the well and pin arrays are used in the same general manner; the position of each synthetic pathway in the array and hence the structure of the product of that pathway is usually identified by a grid code.

24. Figure :The reaction of amino acids with isocyanates to form hydantoins

25. The technique of parallel synthesis is best illustrated by means of an example.
Consider the general theoretical steps that would be necessary for the preparation of a combinatorial library of hydantoins by the reaction of isocyanates with amino acids using a 96-well array. At each stage in this synthesis the product would be purified by washing with suitable reagents.

26. Eight N-protected amino acids (X1, X2_ _ _X8) are placed in the well array so that only one type of amino acid occupies a row, that is, row A will only contain amino acid X1, row B will only contain amino acid X2, and so on (Fig.5.9a).
Beads are added to each well and the array placed in a reaction environment that will join the X compound to the linker of the bead.
The amino acids are deprotected by hydrogenolysis and 12 isocyanates (Y1, Y2_ _ _Y8) added to the wells so that each numbered row at right angles to the lettered rows contains only one type of isocyanate.
In otherwords, compound Y1 is only added to rowone, compound Y2 is only added to row two, and so on (Fig. 5.9b).
The isocyanates are allowed to react to form substituted ureas.
Each well is treated with 6M hydrochloric acid and the whole array heated to simultaneously form the hydantoins and release them from the resin.
Although it is possible to simultaneously synthesise a total of 96 different hydantoins (Z1–Z26, Fig.5.9c) by this technique, in practice it is likely that some of the reactions will be unsuccessful and a somewhat smaller library of compounds is normally obtained.

27. Fodor’s method for parallel synthesis:
In theory almost any solid material can be used as the solid support for parallel combinatorial synthesis. Fodor et al. (1991) produced peptide libraries using a form of parallel synthesis that could be performed on a glass plate. The plate is treated so that its surface is coated with hydrocarbon chains containing a terminal amino group. These amino groups are protected by the UV-labile 6-nitroveratryloxycarbonyl (NVOC) group.

28. Figure 5.10: A schematic representation of the Fodor approach to parallel synthesis. X represents an NVOC protected amino group attached to the glass plate. The other letters correspond to the normal code used for amino acids. Each of these amino acids is in its NVOC-protected form.

29. A photolithography mask (M1) is placed over the plate so that only a specific area of the plate can be irradiated with UV light (Fig. 5.10).
This results in removal of the NVOC protecting group from the amino groups in the irradiated area.
The entire plate is exposed to the first activated NVOC-protected amino acid.
However, it will only bond to the amino groups exposed in the irradiated area (Step A).
The process is repeated using a new mask (M2) and a second activated NVOC-protected amino acid attached to the exposed amino groups (Step B).
This process is repeated using different masks (M3, etc.) until the desired library is obtained, the structure of the peptide occupying a point on the plate depending on the masks used and the activated NVOC-protected amino acid used at each stage in the synthesis.
The technique is so precise that it has been reported that each compound occupies an area of about 50 mm × 50 mm.
A record of the way in which the masks are used will determine both the order in which the amino acids are added and, as a result, the structures of each of the peptides at specific coordinates on the plate.

30. Split and Mix Synthesis
Also known as :
Split synthesis
One-bead-one compound synthesis
Selectide synthesis
Proton mixing synthesis
Divide-couple recombine synthesis
Originated in peptide synthesis
Simple efficient chemistry
Long linear sequence of reactions
Solid Phase approaches
Figure:
No of reagents=10
No of reactions=steps reagents; 5• 10= 50
No of products=reagents 105=10000

31. Encoding Method: (Sequential Chemical Tagging
Building Block: Oligonucletide
Glycine: CACATG
Methionine: ACGGTA

32. Selected Solid Phase Chemistry
Stille reaction:
Here aryl-aryl bonds are formed by palladium catalyzed rxn between resin bound aryl iodides and alkenyl stannanes.
Fig.
The palladium acetate catalyzed rxn of alkynes with resin bound o-iodoamines is used to synthesize indole.

33. Mitsunobu couplings:
Aryl ether forming reaction under very mild condition on Tenta Gel resin bead using triphenylphosphine and diethylazodicarboxylate (DEAD). A series of primary and secondary alcohols may be coupled to synthesize a combinatorial library.

34. Heterocyclic synthesis:
Benzodiazepines:
A range of independently synthesized Fmoc-protected 2-amino benzophenones were linked to the HMP linker on polystyrene resin through a phenolic carboxylic acid residue which were acylated with a set of Fmoc protected α-amino acid fluorides. After deprotection acid catalyzed eyelization gives Benzodiazepines. Further functionalization is achived by N-alkylation of the anilide.

35. Quinolones:
Wang resin is derivavatized with 2,4,5-trifluorobenzoylacetic acid in the first step Activation and addition of cyclopropylamine and formation of the quinolone template was achived by cyclization under tetramethylguanidine catalysis. Nucleophilic substitution with piperazine gives resin bound Ciprofloxacin.
Fig.

36. 1. Carboxylic acid linkers:
By neucleophilic displacement of chloride of Merrifield resin
with the help of caesium carboxylate salt in DMF.
Fig.
Can also be done on Wang linker
Benzodiazepines
Hydantoins
Diketopiperazines

37. 2.Carboxamide linkers:
The MBHA (methyl BenzHydrylAmine) linker on polystyrene resin beads.
Fig.
For the synthesis of peptide amides.
Rink linkers can be used for synthesis of 1ᵒ and 2ᵒ sulphonamides.

38. 3. Alcohol linkers:
THP (Tetrahydropyranyl) based alcohol linkers are developed on Merrifield resin by reaction of Merrifield resin with the Na-salt of hydroxymethyldihydropyran. Stirring the THP-linked resin with alcohols results alcohol linkers.
Fig.
Mercaptoketones.

39. 4. Amine linkers:
The carbamate linkers are prepared by the reaction of amine with a Polystyrene resin bound chloroformate. The resultant linkers contain amine (may be 1ᵒ and 2ᵒ).
Fig.
For Polyamines useful for Enzyme Inhibitors

40. 5. Traceless linkers:
Silyl Linkers
For Benzodiazepinone
6. Light-cleavable linkers:
Nitro-substituted benzydrylamine linker for generation of Carboxamide (e.g. Thiazolidinone)
Synthesis of Hydantoin using carbamate linkers.

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