*Corresponding Author:
G. Mariappan
Himalayan Pharmacy Institute, Majhitar, Rangpo, East Sikkim-737 132, India.
E-mail: [email protected]
Date of Submission 18 February 2005
Date of Revision 08 March 2006
Date of Acceptance 02 July 2006
Indian J Pharm Sci, 2006, 68 (4): 420-424  

Abstract

Combinatorial chemistry is a novel synthetic strategy which leads to produce a large number of chemical libraries with predetermined structures. The advances in the research of combinatorial library synthesis and screening methods have enabled the medicinal chemists to identify highly active compounds rapidly. This method has revolutionized basic research and drug discovery. A number of combinatorial methods have been developed to boost the morale of medicinal chemists. Out of those, positional scanning synthesis plays a significant role to generate libraries from libraries. This approach is capable of producing infinite libraries. Our aim is to explore this method to understand the principle of synthesis and deconvolution screening methods to identify individual active compounds.

Combinatorial chemistry represents the product of matrix chemistry which represents the product of linear chemistry. It has caused a great cultural change by redefining the scientific methods. For more than a decade, medicinal chemists execute experiments, one at a time, with very careful control of parameters. But combinatorial chemistry changed all the steps of orthodox chemistry. Let us imagine an orthodox chemistry laboratory. Here the medicinal chemist produces one compound at a time by the standard reaction A+B→C. But in combinatorial chemistry laboratory, the combinatorial chemist produces infinite compounds by the reaction A1 to An and reacts with B1 to Bn. The combinatorial libraries are A1 B1, A2 B2, A3 B3,………. An Bn. Combinatorial chemistry started its life with amino acids. Mario Geysen, in the mid-1980, began synthesizing peptides by the hundreds, first in parallel fashion and latter as mixtures. The classic technique, i.e., solid phase organic synthesis (SPOS), developed in the 1960 by R. Bruce Merrifield at Rockefeller University, quickly became the key technique for production of entire range of proteins and enzymes during the past three decades. This technique has become the basis for the exploration of solid phase organic synthesis, whereby molecular diversity can be introduced by producing a nearly infinite variety of heterocycles, steroids and carbohydrates.

Combinatorial chemistry has many parallels in nature. Our immune system is able to produce hundreds of millions of different antibodies by recombining segments of a variable region and primary structure. Even deadly South Pacific cone snails appear to have been making mixtures for the past 50 million years or so. Bal do mero Olivera of Utah has found that these creatures have the ability to make the mixture of 100 or more deadly venoms, essentially produced by combinatorial scrambling of amino acids.

Characteristics of mixture-based synthetic combinatorial libraries

Synthetic combinatorial libraries (SCLs) represent systematically arranged mixtures of a large number of synthetic compounds [1,2]. The multiple solid-phase synthesis, also called ‘tea-bag approach,’ generates the synthetic combinatorial libraries. In this approach [3], compartmentalized resin-bound compounds are synthesized. SCLs are made up of individual defined building blocks at certain positions of the compound scaffold, while the remaining diverse positions are mixtures of building blocks. SCLs are cleaved from a solid support and are assayed in solution. The assay procedure allows each compound within each mixture to freely interact with the given receptor. The first SCLs were composed of peptides of various lengths and amino acids (L, D and unnatural). An approach known as ‘libraries from libraries’ was used in the development of SCLs, which is made up of peptidomimetics [4], polyamines [5] and heterocycles [6-9]. The existing peptide SCLs can be transformed chemically to produce new SCLs having different physical, chemical and biological properties with respect to the original peptide SCLs used as starting material. There are two different deconvolution methods that are used to identify individual compounds from mixture-based SCLs. The first method involves identification of active mixtures having defined positions; the remaining mixture positions are then defined, one diversity position at a time, through a synthesis and selection process until individual compounds are identified [10,11].

The second deconvolution method is known as positional scanning [12-14]. In this case, separate sub-libraries individually address each diversity position. The building blocks of the active mixtures at each diversity position are combined and the resulting individual compounds are synthesized and tested to determine their activities. Iterative deconvolution is not required in case of positional scanning format. In most cases, positional scanning format needs a single synthesis to obtain active individual compounds. This positional scanning method is used not only to prepare peptide libraries but also heterocyclic libraries using dipeptide SCLs as starting materials; examples include hydantoin, cyclic urea, thiourea, indole-pyrido-imidazole and bicyclic guanidine SCLs. At first, this method was successfully adopted to generate heterocyclic libraries, e.g., benzodiazepine library synthesis [15]. Then to generate libraries belonging to groups like hydantoins, antibacterials, antihypertensives, thrombin inhibitors and metalloprotease inhibitors [16], combinatorial approach has been successfully adopted. Recently, this technique was used to design some peptidomimetic inhibitors for Hepatitis C virus NS3 protease [17].

Positional scanning concept

Positional scanning synthetic combinatorial libraries (PS-SCLs) are composed of positional SCLs or sub-libraries. Here each diversity position is defined with a single building block. The remaining positions are composed of mixtures of building blocks. Each positional sub-library represents the same collection of individual compounds. The positional sub-libraries are assayed and the assay screening data yield information about the most important building block for every diversity position of the PS-SCL. Table 1 represents the tripeptide combinatorial libraries. Here four different amino acids are incorporated at each of the three diversity positions, resulting in a diversity of 64 (43) individual peptides. Only 12-peptide mixture (4 amino acids and 3 positions) needs to be synthesized when the same diversity is arranged as a PS-SCL. Each of the three positional sub-libraries, namely, OXX, XOX and XXO, contain the same diversity of peptides; they differ only in the location of the position defined with a single amino acid (Table 2).

AAA RAA TAA WAA
AAR RAR TAR WAR
AAT RAT TAT WAT
AAW RAW TAW WAW
ARA RRA TRA WRA
ARR RRR TRR WRR
ART RRT TRT WRT
ARW RRW TRW WRW
ATA RTA TTA WTA
ATR RTR TTR WTR
ATT RTT TTT WTT
ATW RTW TTW WTW
AWA RWA TWA WWA
AWR RWR TWR WWR
AWT RWT TWT WWT
AWW RWW TWW W WW

Table 1: Tri-Peptide Combinatorial Library – 64 Individual Peptides

O X X X O X X X O
AXX XAX XXA
RXX XRX XXR
TXX XTX XXT
W X X X W X X X W

Table 2: Positional Scanning – Synthetic Combinatorial Possible Sub-Libraries

Any of the four amino acids is represented by O position, while each of the remaining two positions is a mixture (X) of the same four amino acids. It is shown below that each mixture is the 16 peptides (42) that make up that mixture. Here it is assumed that ART is the only tripeptide in these libraries that is recognized by the receptor. It is found that each positional sub-library contains the same diversity of polypeptides; the ART tripeptide is present in all three positional sub-libraries (Table 3). The ART tripeptide is present only in the mixture with activity. The tripeptide ART is obtained by combination of these amino acids in their respective positions. This tripeptide is to be synthesized and tested for activity against the receptor. It is to be noted that the activity observed for each of the three mixtures (AXX, XRX and XXT) is due to the presence of the tripeptide ART within each mixture and not due to the individual amino acids (A, R and T) that occupy the defined position.

O X X X O X X X O
A X X R X X T X X W X X X A X X R X X T X X W X X X A X X R X X T X X W
AAA RAA TAA WAA AAA ARA ATA AWA AAA AAR AAT AAW
AAR RAR TAR WAR AAR ARR ATR AWR ARA ARR ART ARW
AAT RAT TAT WAT AAT ART ATT AWT ATA ATR ATT ATW
AAW RAW TAW WAW AAW ARW ATW AWW AWA AWR AWT AWW
ARA RRA TRA WRA RAA RRA RTA RWA RAA RAR RAT RAW
ARR RRR TRR WRR RAR RRR RTR RWR RRA RRR RRT RRW
ART RRT TRT WRT RAT RRT RTT RWT RTA RTR RTT RTW
ARW RRW TRW WRW RAW RRW RTW RWW RWA RWR RWT RWW
ATA RTA TTA WTA TAA TRA TTA TWA TAA TAR TAT TAW
ATR RTR TTR WTR TAR TRR TTR TWR TRA TRR TRT TRW
ATT RTT TTT WTT TAT TRT TTT TWT TTA TTR TTT TTW
ATW RTW TTW WTW TAW TRW TTW TWW TWA TWR TWT TWW
AWA RWA TWA WWA WAA WRA WTA WWA WAA WAR WAT WAW
AWR RWR TWR WWR WAR WRR WTR WWR WRA WRR WRT WRW
AWT RWT TWT WWT WAT WRT WTT WWT WTA WTR WTT WTW
AWW RWW TWW W WW WAW WRW WTW W WW WWA WWR WWT W WW

Table 3: Positional Scanning – Synthetic Combinatorial Libraries

Tripeptide PS-SCL synthesis

A predetermined ratio of amino acids is coupled to the resin-bound amino acid. It is necessary to establish a set of ratio for the amino acids that will be used in the coupling step [18]. The method of simultaneous multiple peptide synthesis (SMPS) [19], also known as the ‘tea-bag approach,’ is advantageous as it provides all wash and deprotection step in a common vessel. It is possible as the resin used for each peptide is enclosed in separate polypropylene mesh bags (tea bags). Individual tea bags are simply placed in the appropriate amino acids solution for the coupling reaction. It is necessary that sufficient solvent covers the tea bags and vigorous shaking be used for each step. For the method described here, methybenzhydrylamine (MBHA) polystyrene resin is used in conjunction with t-Boc chemistry [20].

Protocol for the synthesis of a tripeptide PS-SCL

Polypropylene tea bags are used to prepare PS-SC libraries. These tea bags are numbered from 1 to 60. To each bag, 100 mg of MBHA polypropylene resin is added and sealed. The first coupling step involves coupling of the 20-proteinogenic amino acids individually to resin-filled bags 41-60. To bags 1-40, 19 amino acid mixtures are coupled by using a predetermined ratio of amino acids. The bags are neutralized before the next process of coupling. Cysteine is excluded to avoid disulfide aggregates in 19 amino acid mixtures. The second coupling step involves coupling of 20 amino acids individually to bags 21-40. Then 19 amino acid mixtures are coupled to bags 1-20 and 41-60. The N-terminal BOC is removed and bags are neutralized for the next coupling process. In the third coupling step, 20 amino acids are coupled individually to bags 1-20. The N-terminal BOC is removed to have an N-terminal amine. Further, the N-terminal amino groups of the peptide mixture can be acetylated if needed.

PS-SCL deconvolution

In order to identify the most active individual compound from the PS-SCLs, the synthesis and testing of the individual compound that corresponds to the combination of building blocks defined in the most active mixtures at each position are needed. The minimum number of building blocks is to be selected from each position that will be used to synthesize individual compounds. For example, if two amino acids were selected from each position of a hexapeptide PS-SCL, one needs to synthesize 64 peptides; and if three amino acids were selected at each position, 729 would be required. It should be noted that different number of building blocks could be chosen from each position, depending upon the activity of the mixtures.

Since PS-SCLs are composed of separate positional SCLs, each one can be considered independent of the others. Thus each positional SCL can be independently screened and pursued using an iterative synthesis and selection process [21]. Deconvolution of active individual compounds from mixture-based libraries depends on reproducible screening data and clear dose response activities of the most active mixtures. Dose response curve is to be determined for the most active mixtures and the activities based on IC50 values are used to select the building blocks that will be included in the synthesis of individual compounds. It has been noted that the building blocks of similar chemical character will yield similar activities at a given position.

One aspect of screening PS-SCL is that the activity of a mixture with a given building block in the defined position can be due to one or more families of compounds having that same building block at its respective position. When the combination of the most active building blocks are synthesized and tested as individual compounds, it becomes clear whether the activities of the mixtures between positions are connected. Several strategies can be used for deconvolution. The activities of the mixtures of the library are due to the activities of individual compounds and they can be deconvoluted. In order to identify the highly active mixture, the connectivity of the active mixture at each position of diversity is the most important step.

Assay optimization

PS-SCLs have been used in a number of biological assays. The knowledge of the various assay parameters, such as signal to noise ratio, variability and sensitivity, is required for successful identification of active compounds from libraries. Table 4 illustrates various assays in which PS-SCLs have been used and the signal ranges obtained.

Assay Readout Range Ratio
Radio receptor Counts/min 200-1000 5
T cell proliferation Counts/min 100-500 5
Micro dilution Optical density 0.1-0.5 5
Enzyme inhibition Optical density 0.1-0.8 8
ELISA Optical density 0.1-2.0 20

Table 4: Biological Assays With Various Parameters

All these parameters are inherent to the assay and are not influenced with respect to whether complex mixtures are being tested as individual compounds. The parameter used as a control for an assay system is the variability. While screening a complex mixture, it is critical that the assay variability is known. For an assay with low variability, one has confidence that a 5-10-fold difference in observed activity between mixtures is significant. For an assay with high variability, the variation between replicates for a given mixture may obscure real differences in activities from other mixtures. The use of repeated experiments and averaged data ensures accurate deconvolution (selective of truly active mixtures), which results in the identification of individual compounds having significant activity.

Limitations

The major limitations include the following: 1. the chemistry for SPOS is limited; 2. the laboratory should be completely sophisticated to carry out SPOS; 3. side chain de-protection and cleavage is the mandatory last stage; 4. molecules should have polar groups, such as NH2, OH, to bind with resins; 5. The resins introduce other problems, such as poor homogeneity; swelling, low loading capacity and these factors may hamper reaction rate and site accessibility; 6. High Throughput Positional Scanning (HTPS) is needed to screen these libraries; 7. analytical difficulties are inevitable, i.e., 100 000 NMR is needed to study 100 000 libraries; and finally, 8. physical requirement is inevitable, i.e., to make 10 000 compounds, 10 000 vials or wells are needed.

Conclusion

We have dealt with an elegant method, that is, PS-SCL, to generate libraries from libraries. This method is amenable to the production of a huge number of libraries from commercially available building blocks. Nowadays, combinatorial chemistry has become popular in academia and industry, but it is still a relatively young technology whose value remains unproven. The PS-SCL will certainly enrich the efforts of medicinal chemists towards discovery of effective drug candidates for the benefits of mankind.

References