Combinatorial chemistry allows for the rapid synthesis of large collections of compounds through various techniques. It has applications in drug discovery by enabling the efficient synthesis and screening of thousands of compounds. Solid phase synthesis is a common method, using resin beads as a solid support onto which compounds are built up through sequential addition of reagents. Merrifield solid phase peptide synthesis pioneered this approach, using insoluble polymer beads and filtration to remove excess reagents at each step. Parallel synthesis is also used, involving the simultaneous synthesis of many compounds in an array of individual reaction vessels to efficiently generate compound libraries for screening.

1. School Of Studies in Pharmaceutical
Science
Jiwaji University Gwalior
(474011)
Assignment On -
COMBINATORIAL
CHEMISTRY
SUBMITTED TO -
Mrs. Prachi Awasthi
Ma'am
SUBMITTED BY -
Piyush Lodhi
B-Pharma (6th.Sem.)

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

6. 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.

7. 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.

8. 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: 80-200 µm usually.

9. 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.

10. ❑ 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.

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

12. 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 1971. 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.

13. 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.

14. 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.

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

16. 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.

17. ✓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.

18. 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.

19. 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.

20. ✓ 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.

21. 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

22. 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.

23. ❑ 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.