It is the presentation for Combinatorial Chemistry. this presentation should be helpful for B. Pharm students. It includes introduction, types, applications, advantages and disadvantages.
2. Introduction
• Combinatorial chemistry comprises chemical synthetic methods that make it
possible to prepare a large number (tens to thousands or even millions) of
compounds in a single process. These compounds are then tested for
pharmacological activity.
• The key of combinatorial chemistry is that a large range of analogues is synthesised
using the same reaction conditions, the same reaction vessels. In this way, the
chemist can synthesise many hundreds or thousands of compounds in one time
instead of preparing only a few by simple methodology.
• The collection of these finally synthesized compounds is referred to as a
“combinatorial library”.
4. Combinatorial library
Def: Collection of finally synthesized compounds
Size: depends on the number of building blocks used per reaction and the number of reaction
steps, in which a new building block is introduced
Typical: 102 up to 105 compounds
5.
6. Application:
• Applications of combinatorial chemistry are very wide. Scientists use
combinatorial chemistry to create large populations of molecules that
can be screened efficiently.
• By producing larger, more diverse compound libraries, companies
increase the probability that they will find novel compounds of
significant therapeutic and commercial value.
• Provides a stimulus for robot-controlled and immobilization
strategies that allow high-thrughput and multiple parallel approaches
to drug discovery.
7. Applications continued
• Finding the right combination of drug molecules.
• It provides fresh and promising leads for medicinal chemistry.
• Synthesis of small molecular libraries.
• Application of antibody libraries obtained.
• Development of enzyme linked inhibitors.
• As a tool for lead optimization.
• Quantitative and qualitative characterization of drug database.
8. Advantages
• Easily removed from reactions by filtration.
• Excess reagents can be used to drive reactions to completion.
• Economical, environmentally sound and efficient.
• Easy to handle.
• Safe to handle.
• Very good selectivity.
9. Advantages:
Fast
Combinatorial approach can give rise to million of compound in same time as it will take to
produce one compound by traditional method of synthesis .
Economical
A negative result of mixture saves the effort of synthesis, purification & identification of each
compound
Easy
Isolation, purification & identification of active molecule from combinatorial library is
relatively easy.
Drug Discovery
Mixed Combinatorial synthesis produces chemical pool. Probability of finding a molecule in a
random screening process is proportional to the number of molecules subjected to the
screening process
Drug Optimization
Parallel synthesis produces analogues with slight differences which is required for lead
optimization
10. Disadvantages:
1. Some reagents may not interact with solid support.
2. Ability of recycled reagents is not assured on solid support
3. Due to diffusional constraints reactions may run more slowly.
4. Can be very expensive to prepare specific polymeric support materials.
5. Under harsh reaction conditions, stability of the support material can
be poor.
6. Sometimes side reactions with polymer support.
7. Efficiency is highly affected by compound's size, solubility and function
group.
8. Compounds produced tend to be Achiral of Racemic mixture.
11. Solid Phase synthesis
• In this method the compound library is synthesized on solid phase such as resin bead, pins or chips etc.
• It was first introduced by Merrifield in the year 1963 for which he was awarded Nobel prize.
• Merrifield developed a series of chemical reactions that can be used to synthesise proteins. The intended
carboxy terminal amino acid is anchored to a solid support. Then, the next amino acid is coupled to the first one.
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. The process continues until the peptide or protein is completed. Then, the
molecule is cleaved from the solid support and any groups protecting amino acid side chains are removed.
Finally, the peptide or protein is purified to remove partial unwanted products.
12. 1. SOLID PHASE TECHNIQUES
• Reactants are bound to a polymeric surface and modified whilst still attached. Final product is released at the end of the
synthesis
• Specific reactants can be bound to specific beads
• Beads can be mixed and reacted in the same reaction vessel
• Products formed are distinctive for each bead and physically distinct
• Excess reagents can be used to drive reactions to completion
• Excess reagents and by products are easily removed
• Reaction intermediates are attached to bead and do not need to be isolated and purified
• Individual beads can be separated to isolate individual products
• Polymeric support can be regenerated and re-used after cleaving the product
• Automation is possible
13. The use of solid support for organic synthesis relies on three interconnected
requirements:
1) A cross linked, insoluble polymeric material that is inert to the condition of synthesis
2) Some means of linking the substrate to this solid phase that permits selective cleavage of
some or all of the product from the solid support during synthesis for analysis of the extent of
reaction(s), and ultimately to give the final product of interest
3) A chemical protection strategy to allow selective protection and deprotection of reactive
groups.
15. Cross linked Polystyrene resins,
Polyacrylamide resins, Polyamide resins,
Tenta gel
Carboxylic acid linkers, Wang linker,
carboxamide linker, Carbamate linkers
FMOC(Fluro methoxy carbonyl benzyl ester),
TBOC (Tertiary butyloxy carbonyl)
Solid
support
Linkers
Protecting
groups
16. Starting material,
reagents and solvent
Swelling
Linkers
1. SOLID PHASE TECHNIQUES
• Beads must be able to swell in the solvent used, and remain
stable
• Most reactions occur in the bead interior
Resin bead
17. 1. SOLID PHASE TECHNIQUES
Anchor or linker
• A molecular moiety which is covalently attached to the solid support, and which contains a
reactive functional group
• Allows attachment of the first reactant
• The link must be stable to the reaction conditions in the synthesis but easily cleaved to
release the final compound
• Different linkers are available depending on the functional group to be attached and the
desired functional group on the product
• Resins are named to define the linker e.g. Merrifield, Wang, Rink
18.
19. Solid phase synthesis: protecting groups
A few protecting groups used in solid phase synthesis.
For amines.
Boc ( t-butoxycarbonyl )
Fmoc (9-fluorenylmetoxy carbonyl)
Tmsec (2 [ trimethylsilyl ] ethoxycarbonyl)
For carboxylic acids.
Tert Bu ester(t-butyl ester)
Fm ester(9-fluronyl methyl ester)
Tmse ester(2 [trimethylsilyl] ethyl) 19 19
20. Deprotection
O
aa1aa2aa3
O
aan NH2
Merrifield resin for peptide synthesis (chloromethyl group)
O
O
R
NHBoc
H
O
O
R
NH2
H
HO2C NHBoc
R2
H
coupling
O
O
R
NH
H
O
NHBoc
R2
H
= resin bead
Cl HO2C NHBoc
R H
+
Linker
HF
OH
aa1aa2aa3 aanHO2C NH2
Peptide
Release from
solid support
22. 2. Parallel Synthesis
Aims:
• To use a standard synthetic route to produce a range of analogues, with a different
analogue in each reaction vessel, tube or well
• The identity of each structure is known
• Useful for producing a range of analogues for SAR or drug optimisation
24. • Each tea bag contains beads and is labelled
• Separate reactions are carried out on each tea bag
• Combine tea bags for common reactions or work up procedures
• A single product is synthesised within each teabag
• Different products are formed in different teabags
• Economy of effort - e.g. combining tea bags for workups
• Cheap and possible for any lab
• Manual procedure and is not suitable for producing large quantities of different
products
2. Parallel Synthesis
2.1 Houghton’s Tea Bag Procedure
22
25. 2. Parallel Synthesis
Automated parallel synthesis
Wells
• Automated synthesisers are available with 42, 96 or 144 reaction vessels or wells
• Use beads or pins for solid phase support
• Reactions and work ups are carried out automatically
• Same synthetic route used for each vessel, but different reagents
• Different product obtained per vessel
27. 27 TRIPEPTIDES
27 VIALS
2. Parallel Synthesis
Automated parallel synthesis of all 27 tripeptides from 3 amino acids
28. 3. Mixed Combinatorial Synthesis
Aims
• To use a standard synthetic route to produce a large variety of different analogues where
each reaction vessel or tube contains a mixture of products
• The identities of the structures in each vessel are not known with certainty
• Useful for finding a lead compound
• Capable of synthesising large numbers of compounds quickly
• Each mixture is tested for activity as the mixture
• Inactive mixtures are stored in combinatorial libraries
• Active mixtures are studied further to identify active component
29. Synthesis of all possible tri peptides using 3 amino acids
3. Mixed Combinatorial Synthesis
The Mix and Split Method
52. Solution phase synthesis
• This method is carried out without the aid of solid support.
Substrate
Reagent
Product Excess
reagent
Scavenger
Product
Filter
Product Scavenger
+
excess reagent
57. 5.1 Recursive Deconvolution:
• Method of identifying the active component in a mixture
• Quicker than separately synthesising all possible components
• Need to retain samples before each mix and split stage
Example:
Consider all 27 tripeptides synthesised by the mix and split strategy
from glycine, alanine and valine
5. Identification of structures from mixed combinatorial
synthesis
60. 5. Identification of structures from mixed combinatorial
synthesis
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Val
Val
Val
Val
Val
Val
Val
Val
Val
Mixture
Inactive
Mixture
Inactive
Mixture
Active
• 9 Possible tripeptides in active mixture
• All end in valine
• Add valine to the three retained dipeptide mixtures
5.1 Recursive Deconvolution
63. NH2
NH2
RCHBrCO2H
Step 1
NH2
NH
O
R
Br
amino acid(aa 1)
Tag 1
HN
NH
O
R
Br
aa1
NH2
R'NH2
Step 2
HN
NH
O
R
NHR'
aa1
NH2
amino acid(aa 2)
Tag 2
HN
NH
O
R
NHR'
aa1
aa2
NH2
R"COCl
Step 3
HN
NH
O
R
NR'COR"
aa1
aa2
NH2
amino acid(aa 3)
Tag 3
HN
NH
O
R
NR'COR"
aa1
aa2
aa3 NH2
5.2 Tagging
Example
64. N H X N H X N H X N H X N H X
N H XN H XN H XN H XN H XN H X
N H XN H XN H XN H XN H XN H X
6. Identification of structures from combinatorial synthesis
6.2 Photolithography - example
N H XN H XN H XN H XN H XN H X
M A S K 1
Mask
LIGHT
LIGHT
N H XN H XN H XN H X
N H XN H XN H XN H XN H XN H X
N H X
C O 2 H
coupling
N H X N H X
N H X N H X N H X
N H X N H XN H X N H X N H 2 N H 2 N H 2
N H XN H XN H XN H 2N H 2N H X
N H XN H XN H XN H XN H XN H X
Deprotection
65. Y Y Y
repeat
6. Identification of structures from combinatorial synthesis
6.2 Photolithography - example
Y
amino acids
O M e
O M eO
O 2 N
O
X= Nitroveratryloxycarbonyl
fluorescent tag
Target receptor
Y
66. SEPARATIONAND ANALYSISOF COMBINATORIAL LIBRARIES
Separation and analysis of combinatorial libraries places a high
demand on existing analytical techniques because
The quantities to be analysed are very small
The analysis should be non destructive and allow the recovery
of the product
The methods must be suitable for rapid parallel analysis
68. Screening
Screening is the process performed to identify the biologically active
compound among all the compounds synthesised.
The most commonly used screening methods in combinatorial chemistry
are
1. Virtual screening
2. High throughput screening
69. Virtual screening
Virtual screening refers to the use of
computers to predict whether a
compound will show a desired activity
or not on the basis of its two
dimensional and three dimensional
structure.
70. High-throughput screening(HTS)
HTS involves the process of finding an active compound against a chosen target using
robotics, data processing and control software, liquid handling devices, and sensitive
detectors, High-throughput screening allows a researcher to quickly conduct millions of
chemical, genetic, or pharmacological tests.
The results of these experiments provide starting points for drug design and for
understanding the interaction or role of a particular biochemical process in biology.
71. Assay plate preparation
Reaction observation
Automation systems
Hit selection
Experimental design and data analysis
Quality control
Steps involved in HTS
73. Conclusion
The last ten 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 fervour.
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.
For pharmaceutical chemists at least the reason for this change is not hard to fathom. 20
years ago the market for pharmaceuticals was growing at around 10% per annum but more
recently the rate of the market growth as decline. At the same time, cost constraints on
pharmaceutical research have forced the investigation of 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.
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.