Like the split and pool method, it results in the production of multiple compounds at the same time. However, unlike split and pool, parallel synthesis gives individual compounds, not a mixture. Thus deconvolution is not an issue in this method.

(1) It creates the compounds individually and in their own vessel. Thus the identity of the product is already known
(2) No deconvolution is required.
(3) Each compound is substantially pure in its location
(4) Defined location provides the structure of a certain compound
(5) Easier biological evaluation 
(1) Applicable only for medium libraries (several thousand compounds) 
(2) The amount of vessels required for this process is large, and the number of reactions performed is even greater. 
[Leard L. & Hendry A. (2007)]13
[Pandeya S.N. and Thakkar D. (2004)]16

Methods for parallel synthesis 
Methods that can be used for parallel synthetic includes: 
(1) Multipin method
(2) Teabag method
(3) SPOTS membrane method

(1) Multipin method 
This method was published by Geysen and his colleagues in 1984 for synthesizing a series of peptides epitopes by using Multipin apparatus. The multipin apparatus has a block of wells (96 wells format in 12×8 way) serving as reaction vessels and cover plate with mounted polyethylene rods or pins (4×40 mm) fitting into well. The end of polyethylene rods (pins) are coated with derivatized polyacrilic acid (marked by black).

Figure-8. The Multipin apparatus

The protected amino acids used in building the peptides and the coupling reagents are dissolved and added to the wells. The coated ends of the pins are immersed into solution and kept there until the coupling reactions ended. 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. [Furka A. (2007)]5
[Hima P. et al. (2009)]7

The peptides are screened by means of ELISA method after deprotection without leaving them from the pins to determine the binding capability of covalently bound peptide to antibodies. [Pandeya S.N. and Thakkar D. (2004)]16

The formed peptides are attached to the pins.The first amino acid was attached to the end of polyethylene rods (pins) grafted with derivatized polyacrylic acid (marked by gray). [Furka A. (2007)]5
[Hima P. et al. (2009)]7

[Kim S. (2005b)]10

The most characteristic feature is that the no. of product formed during the synthetic process never exceeds the no. of starting samples. 
[Hima P. et al. (2009)]7

(2) SPOTS membrane method
This method introduced by Frank (1992) and his group also for preparing peptide arrays. The synthesis is carried out on cellulose paper membranes instead of polyethylene pins as the solid support for peptide synthesis. [Pandeya S.N. and Thakkar D. (2004)]16

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.

Figure- 10. The SPOT synthesis

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. 

(3) Teabag method (Compromise between parallel & split - pool synthesis)
This method was developed in 1985 by Houghten for multiple peptide synthesis. The beads of the solid support are enclosed in permeable polypropylene (plastic) bags (5 x 22 mm mesh packets), then placed for coupling into a reaction vessel containing the solution of amino acid & the coupling reagent. All operations, including removal of protecting groups, couplings, washings & even the cleavages are performed on solid supports enclosed in bags. At the end of synthesis, each bag contains a single peptide. This procedure has significant advantages.  [Hima P. et al. (2009)]7

(1) Greater Quantity of each compound is available at once (structural characterisation) (2) Labelling of the tea bags: Easier identification of each compound  [Rose C. (2010)]19

All those bags which needed the attachment of the same amino acid (eg. Alanine) are grouped together, placed into the same reaction vessel,& the coupling can be done in a single operation.  [Hima P. et al. (2009)]7

Figure-11. The Teabag synthesis
[Kim S. (2005b)]10

Miscellaneous methods for generating combinatorial libraries
The other approaches for generating combinatorial libraries includes :
(1) Light directed spatially addressable parallel chemical synthesis
(2) Biological method

(1) Light directed spatially addressable parallel chemical synthesis
A very remarkable combinatorial synthesis was developed by Fodor and his colleagues by combining the solid phase synthesis with the photolithographic procedure applied in the fabrication of the computer chips.

The method was published in 1991 under the title “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 groups protected by the photolabile 6–nitro Vera tryloxy carbonyl (NVOC) groups. The amino acid used in the synthesis are also protected by NVOC. group. [Furka A. (2007)]5

The basic principle of the method is that each set of building blocks contains a photolabile protecting group (NVOC) and only the building blocks which have been exposed to light can be coupled with another building block. Thus, pattern of masks and the sequence of protecting groups define the final structure of the compounds synthesized.

Figure- 12. Basic principle of light directed synthesis
[Rose C. (2010)]19

Example: Nine di-peptides 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 groups from those areas. Then the slide is submitted to coupling with the indicated protected amino acid. This can be done by immersing the slide into the solvent containing the protected amino acid and the coupling reagent. Although the full slide is submitted to coupling reaction, coupling occurs only in the irradiated area where the free amino acids are found. By completing a coupling cycle the full area of the slide becomes again protected. Before the next coupling cycle a new area have to be irradiated in order to produce free amino groups. The synthesis of 9 di-peptides is completed in 6 cycles irradiation and coupling (a to f).

Figure- 13. The light – directed synthesis of nine dipeptides

[Furka A. (2007)]5

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.

The sequence are defined by their locations on the slide ie. Each member of the library is synthesized at a specific location. 

(2) Biological method
In 1990 three different research groups introduced a new biological approach for producing peptide sequence libraries. This approach of creating peptide libraries is briefly exemplified by phage display libraries. First, an oligonucleotide 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 phage particle carries a couple of thousand identical coat protein molecules with the same peptides sequence fused to the Outer end. In this respect the phages 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 (after amplification) by sequencing the proper portion of the DNA.  [Hima P. et al. (2009)]7

Screening is the process of determining whether compounds in a chemical library have a desired chemical or biological activity, without necessarily identifying the precise chemical nature of the compound(s) being screened.  There are a range of options for testing the libraries in a biological assay. These include:

(i)Test mixture in solution:  All the compounds are cleaved from the beads and tested in solution. If the resin beads were intimately mixed, it is not possible to test the products separately, but rather as a mixture. If activity in a pharmacological screen is observed it is not possible to say which compound or compounds are active. In order to identify the most active component, it is necessary to resynthesize the compounds individually and thereby find the most potent. This iterative process of resynthesis and screening is one of the most simple and successful methods for identifying active compounds from libraries.

(ii)Test individual compounds in solution:  A second method is to separate the beads manually into individual wells and cleave the compounds from the solid-phase. These compounds can now be tested as individual entities. 

(iii)Test compounds on the beads:  A third method for screening is testing on the beads, using a colorimetric or fluorescent assay technique. If there are active compounds, the appropriate beads can be selected by color or fluorescence, ‘picked’ out by micromanipulation and the product structure, if a peptide, determined by sequencing on the bead. Non-peptide structures would need to be identified by one of the tagging methods. Screening on the bead may be an inappropriate method for drug discovery, as the bead and linker present conformational restrictions that may prevent binding to the receptor. Furthermore for pharmaceutical applications compounds will be invariably need to act, and thus ideally need to be tested in solution. [Leard L. & Hendry A. (2007)]13

By the process of screening the number of libraries that has “desirable properties” are sorted out. It is now very important to learn the identity of “winning” library member. The process of identification of active compound in a mixture of compounds is known as Encoding.
For identification of active compound following types of encoding methods are used:
(1) Positional encoding or deconvolution (iterative resynthesis and rescreening)
(2) Chemical encoding (Tagging)
(3)  Electronic encoding

(1) Positional encoding or deconvolution (iterative resynthesis and rescreening)
In this method, the resynthesis and rescreening is carried out to know the identity of the active compound. In other terms, it is a process of optimizing an activity of interest by fractionating (normally by resynthesis, or by elaborating a partial library) a pool with some level of the desired activity to give a set of smaller pools. Repeating this strategy leads to single members with (ideally) a high level of activity and is termed iterative deconvolution. [Pandeya S.N. and Thakkar D. (2004)]16

In split-pool method, once active pools have been identified, and last step leading to these active pools is known.  One now must work backwards to determine the order of reagent addition leading to the active compound(s).  To do this, the reaction is begun a new, but when the last step is reached, the beads are not pooled and split, but separately reacted with the reagent that led to the active pool.  These pools are now tested for activity.  From the active pool found here, the second-last reagent added to give the active compound is now determined.  This process is repeated again, until the order of reagents, and thus the active compound, has been identified.

Figure- 14. Identification of active compound by iterative resynthesis and rescreening

This process is easiest when only a small number of compounds are expected to show any activity, as in the early stages of drug designs.  Deconvolution is a time consuming method, taking longer than the original synthesis of the library.  It also uses up reagents, thus it can also be an expensive process.   Thus methods of tagging the resins, which allow for even simpler determination of the active compound, are generally employed now.   [Leard L. & Hendry A. (2007)]13

(2)  Chemical encoding (Tagging)
The most common approach to encoding solid phase libraries is to attach a chemical tag to the resin beads as the target molecule gets synthesized. Typically, at each step in the reaction, a tag is attached that is unique for the given step. For example, if we are creating a tripeptide and we have 10 possible amino acids at each position, we need to attach either a single tag that says “the tripeptide on this bead has amino acid Ala at position 1, Phe at position 2 and Gly at position 3” or we need to attach three different tags, one for each position. [Henry D.R. (2004)]6

After cleavage of the compound, the tags are cleaved (ie. decoding) from the solid support and analyzed by following analytical microtechniques:
* Polynucleotides (polymerase chain reaction-PCR), amino acids (Edman degradation & HPLC),
*Electrophoric tags (halocarbon molecules determined after silylation by gas chromatography),
* Amines (analyzed by HPLC) etc.
[Kom C. (2009)]11

Figure- 15. Chemical encoding (tagging) [Kim S. (2005b)]10

(3) Electronic encoding
This technique uses a micro electronic device called a radio frequency (RF) memory tag. The tag measuring 13×3 mm is encased in heavy walled glass and contains the following:
-   A silicon chip ,onto which laser etched a binary code,
-   A rectifying circuit with which absorbed RF energy is converted to D.C. electrical energy,
-   A transmitter/receiver circuit,
-  An antenna, through which energy is received and RF signals are both received and sent.
[Pandeya S.N. and Thakkar D. (2004)]16

Figure-16. Radio frequency (RF) memory chip  [Furka A. (2007)]5

The combinatorial chemistry first shows its presence in synthesis of peptide libraries. The peptide plays varying role in body. By the use of combinatorial chemistry we can generate vast peptide, which may be active. Biologically active peptide hormones play an important role in regulating a multitude of human physiological response and many low molecular weight bioactive peptides can act as a hormone receptor against or antagonists. In addition, peptide structure commonly is found in molecules designed to inhibit enzymes that catalyze proteolysis, phosphorylation and other past translational protein modification that may play important role in pathologies of various disease states. 

A few examples of the application of combinatorial chemistry in lead optimization and drug discovery are given below.

(a) Synthesis of peptoids
Some of the polypeptides of polypeptide libraries were found to be potent inhibitors for enzyme like kinases and proteases useful in treatment of AIDS and cancer, but these peptides have a poor bio-availability and unfavorable pharmacokinetic properties. So, the focus has been shifted on developing synthetic peptido mimetic like peptoides, one of the synthetic diversities has been developed by Simon et. al.

This group has created a basic set of monomers N-substituted glycine units, each bearing a nitrogen substitute similar to natural α-amino acid side chain. The formal polymerization of these monomers resulted in a class of polymeric diversity which was termed as ‘PEPTOIDS”. Peptoides may be synthesized either by “Full monomer” oligomers synthesis “Sub monomer” oligomers synthesis.

(a) Full monomer oligomers synthesis

(b) Sub monomer oligomers synthesis

Figure- 17. Schematic representation of synthesis of peptoides

Comparison of peptide and peptoid backbone

Various biological activities have been established for specific peptoid synthesized, including inhibition of α-amylase and the hepatitis-A viruses 3C protease, binding to that fat RNA of HIV, antagonism at α1-adrenergic receptor.



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