INTRODUCTION:

Combinatorial Chemistry plays very important role in the lead discovery and hit optimization processes in the pharmaceutical world as well as in other areas of discovery. R. Bruce Merrifield of Rockefeller University reported first time a New Approach to the Continuous, Stepwise Synthesis of Peptides” records the first formal expression of solid-phase synthesis (SPS). At the end of 1962, Bruce had demonstrated feasibility for solid-phase synthesis by preparing a tetrapeptide utilizing the carbobenzoxy group for amino protection and HBr/HOAc for deprotecting the amino group of the growing peptide chain^1-3. It was obvious to Bruce that the simplicity of the steps involved in SPS could be automated as only introduction of solvent/reagent, shaking and filtering were involved. He had witnessed the automation of amino acid analysis by Prof. Moore and Stein at Rockefeller, and saw the dramatic improvement in time savings and reproducibility. In 1992 by Bunin and Ellman workon the synthesis of a combinatorial library of benzodiazepines, benzodiazepines synthesis by solid phase synthesis was a turning point in acceptance of the overall approach^4-5. The groundwork for combinatorial chemistry had already been set in peptide chemistry by Geysen with his pin approach^6-8 and Houghten with his “teabag” approach to epitope mapping⁹.

Both approaches used physical separation of polymers to control reaction sequence and thus peptide products.

ADVANTAGES OF SOLID PHASE SYNTHESIS

Solid phase chemistry has some advantages over the solution-phase. First, in solid-phase synthesis, large excesses of reagents can be used to drive reactions to completion; these excess reagents can then be removed at the end of the reactions by filtration and washing. Second, easy separation of reagents and products and solid-phase chemistry can be automated more easily than solution chemistry.

PROPERTIES OF SOLID SUPPORT USED IN SOLID PHASE SYNTHESIS^10-12

Most solid state combinatorial chemistry is conducted by using polymer beads ranging from 10 to 750 µm in diameter. The solid support must have the following characteristics for an efficient solid-phase synthesis:

1) Physical stability and of the right dimensions to allow for liquid handling and filtration.

2) Chemical inertness to all reagents involved in the synthesis.

3) An ability to swell while under reaction conditions to allow permeation of solvents and reagents to the reactive sites within the resin.

4) Derivatization with functional groups to allow for the covalent attachment of an appropriate linker or first monomeric unit.

SOLID SUPPORT USED IN SOLID PHASE SYNTHESIS

1. Polystyrene resins- In this Polystyrene is cross linked with divinyl benzene (about 1% cross linking).polystyrene resin are suitable for nonpolar solvents.

2. Tenta Gel resins- Polystyrene in which some of the phenyl groups have polyethylene glycol (PEG) groups attached in the para position. The free OH groups of the PEG allow the attachment of compounds to be synthesized. PEG containing resins are suitable for use in polar solvents.

3. Polyacrylamide resins- like super blue these resin swell better in polar solvent, since they contain amide bonds, more closely resemble biological materials.

Solid-phase chemistry cannot simply be taken from the existing solution literature without modification like changes in media polarity, solvation and steric constraints within the bead or changes in the partitioning of compounds between the different environments. Characterization of basic resin properties such as their behaviour in different solvents, the influence of solvation on polymer-supported reactions and understanding of resin based kinetics is important in order to understand the solid phase synthesis. Bradley have carried out one such series of studies, in which the distribution of the functional sites predict the desirable properties of new resins within a number of commercial resins (polystyrene [PS] and Tenta-Gel[TG]) was studied by using confocal Raman spectroscopy (a non-fluorescent method) by the coupling reaction of amino methylated PS and TG resins with 4-cyanobenzoic acid. The reaction was quenched after different times to give a variety in site loading¹³. Influence of the cross-linking of polystyrene-divinylbenzene (PS-DVB) on solid-phase chemistry efficiency, such as peptide synthesis and Suzuki coupling and it was seen that the optimal resin for solid-phase synthesis depended greatly upon the chemistry being carried out. Diffusion into the polymeric matrix can become rate-limiting with the higher cross-linked materials. Solvent also plays a very important role in solid phase synthesis. During peptide synthesis, a higher cross-linked resin surprisingly gave higher yields and purity¹⁴. The pore structure of PS-DVB resins is prepared by <10 mol% DVB collapsed to clear glassy particles on drying from dichloromethane or toluene. However, on drying these resins from acetone, methanol and scCO₂ (supercritical CO₂), some opacity was retained. Those resins containing ≤6 mol % DVB and dried from the former two solvents systems had negligible surface area and superficially looks like gel-type resins¹⁵.

4. New PS-PEG and PEG-based resins- The most common resins that are used today based on PS-DVB. By the grafting of polyethylene glycol (PEG) to cross-linked polystyrene has been used to improve its poor swelling in polar solvents, but has the inherent limitation of reducing its substitution level. An alternative approach to the grafting after polymerization is the introduction of polar monomers into the polymer formulation. Several new polar co-monomers for the preparation of Merrifield-type resins have been synthesized. Styrene PEG derivatives were prepared on a large-scale and used as co-monomers in the synthesis of a series of polymer supports¹⁶. New polystyrene- PEG acrylate resins have been prepared by polymerization of styrene with a new cross-linker, O, O’-bis(2-acrylamidopropyl)polyethylene glycol₁₉₀₀. These resins showed excellent swelling properties in a broad range of solvents and proved to be chemically inert to various reagents used in solid-phase peptide synthesis. They were compared with commercial supports such as those of Merrifield and Sheppard or Rapp (TG) resins and demonstrated high efficiency during peptide synthesis¹⁷. PEG-based resins are especially suitable for solid-phase enzymatic chemistry. A number of new members of this resin type have been synthesized. One such type of resin was prepared with two functional groups (OH and NH2) present throughout the polymer matrix, prepared by reductive amination of a mixture of mono-aldehyde and di-aldehyde PEG1500 and the branched cross-linker tris(2 aminoethyl) amine. The resin loading was varied (0.33–0.80 mmol g^–1 OH and 0.24–0.88 mmol g^–1 NH2) by changing the monomer composition in the polymerization mixture. The resin was stable towards strongly acidic and basic conditions for weeks and is fully permeable to a 27 kDa protease¹⁸. Several novel PEG-based resins that exhibit some advantages for application in solid-phase organic synthesis like solid-phase enzymatic reaction, and on-bead screening assays¹⁹.

Numerous techniques have been used to study the resins commonly utilized in solid-phase synthesis to allow a greater understanding of the chemical nature and the physical properties of the supports. In addition, to overcome some of the drawbacks of existing materials, several new resins and new methods of handling solid supports have been developed. New methodologies have also been introduced to simplify the preparation of solid supports.

LINKERS USED IN SOLID PHASE SYNTHESIS:

Broadly applicable linkers groups that are stable under a variety of reaction conditions and enable the release of target compounds from polymeric supports under the mildest conditions is a major goal in combinatorial chemistry. Several important parameters need to be defined for a successful solid-phase synthesis, including the correct choice of solid support and the mode of attachment and cleavage of materials from the resin matrix. Efficiency in anchoring and removing from an adequate linker system relies on the correct choice of linker group: this is crucial when planning a synthetic strategy. The resin–(spacer)–linker unit should be chemically stable during synthesis and cleavage should be takes place under appropriate conditions. Although numerous linkers have been developed over the past 15 years, many have the disadvantage that they are often themselves labile to common chemical reagents that are used in solid-phase synthesis.

There are many type of linkers that are used in solid phase synthesis, some of the linkers used in solid phase synthesis are

1. Selenyl linker:

A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide. Dehydropeptides, containing α, β-unsaturated amino acid (or dehydroamino acid) moiety, are typical abnormal peptides. Their structures are rigid and reactive because they have a double bond conjugated with a peptide linkage. In the design of artificial peptides, the introduction of dehydroamino acid residues into normal peptides induced folded conformations based on structural factors. To avoid these problems, a new selenyl linker was designed. Main purposes required an aryl selenocyanate with a direct carboxylic acid precursor. Thus, acid treatment had been to expose the carboxylic acid, whose condition is orthogonal to Fmoc (N-terminal) and allyl (C-terminal) deprotection^{20- 23}.

2. Thermally-Cleavable Linker:

The development of a thermally cleavable linker that is cleaved by thermal promotion of retro-Diels–Alder (rDA) reactions, and a demonstration of its use in solid-phase organic synthesis like Oxabicyclo[2.2.1]norbornenes(A) constitute a convenient and readily cleaved linker for solid-phase organic synthesis. Some other furfuryl-substituted resin (B) can capture and release dienophiles such as maleimides to facilitate the synthesis²⁴.

3. Dde.based primary amine linkers:

1-(4, 4-Dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), developed in laboratories, has shown promise in the assembly of atypical peptides and biomolecules . The value of this new protecting group is increases due to its complete stability towards a variety of acid conditions, compatibility with uronium type coupling reagents and can be removed by 2% v/v hydrazine hydrate in DMF. It is also sufficiently stable to base and used orthogonally with Fmoc protection in peptide synthesis²⁵.

4. Aryl Hydrazides linker:

Aryl hydrazide linker is versatile linker which is readily prepared in high yield and easily attached to functionalized solid supports. It is stable to acidic and basic conditions and is cleaved with high specificity under mild oxidative conditions in the presence of base and a nucleophile, yielding a range of C-terminal functionalities. An aryl hydrazide linker is formed by coupling a carboxylic acid to an aryl hydrazine. This may be cleaved oxidatively by a range of reagents including Fehling's solution, Tollen's reagent and N-bromosuccinimide/pyridine .The linker may be used to synthesize fully or partially protected peptides for fragment coupling²⁶.

5. Linker for side-chain anchoring of arginine:

A new linker based on a chroman system is described for the side-chain anchoring of Arg and other guanidine-containing molecules. The system is compatible with the Fmoc/tBu solid-phase strategy, because the release of the final product is achieved by treatment with TFA in the presence of scavengers²⁷.

6. Substituted benzyl amide linker:

It is prepare by adding methoxy groups to the benzene, by increasing the length of the spacer that separates the electron-donating oxygen para to the anchored amino acid from the electron-withdrawing alkyl amide function linking the handle to the support, increased the acid liability of the linker. A model peptide (H-Tyr-Gly-Gly-Phe-Met-NH2) was prepared and the cleavage yield was about 80% with 90% purity. This linker is suitable to use with Fmoc protected amino acids²⁸.

7. Rink Linker:

N-Substituted hydroxamic peptide acids were synthetized using this linker with the yields of 80-90%. This peptides are isolated from microorganisms are known to be potent and selective inhibitors of many metallo proteases. N-Substituted hydroxamic peptide acids were synthetized using this linker in yields of 80-90%. A modified Fmoc strategy was used to synthesize this peptide using this linker. A 9-residue peptide consisting of arginine and tryptophan was prepared by using rink linker with high purity and yield²⁹.

8. Benzyloxy Dimethoxytrityl Amine (BDMTA) Resin

In these two benzene rings have methoxy groups para to the benzylic carbon attached to the three phenyl groups. This linker was used to prepare Phe-Thr-Pro-Arg-Leu-NH2 which can be cleaved with dilute TFA. Purity of 90% (HPLC) and satisfactory yields 80% based on resin loading were reported³⁰.

Some other linkers which are used commonly in solid phase synthesis are summarized below³.

COMMON PROTECTING GROUPS AND CLEAVAGE METHODS USED IN SOLID PHASE SYNTHESIS:

Primary function of protecting group is to protect the portion of the molecule that is not covalently bound to the resin must be protected to avoid subsequent³ polymerization of excess monomers in solution nonreactive side of linkers.

SCREENING SYSTEM USED FOR SOLID PHASE SYNTHESIS:

Combinatorial chemistry refers to techniques to fabricate, test, and store the resulting data for a material library containing tens, hundreds or even thousands different materials or compounds. Combinatorial investigations require rapid screening techniques to test and evaluate variations of composition, structure and property within a material library. X-ray diffraction is one of the most suitable screening techniques because abundant information can be obtained from the diffraction pattern. X-ray diffraction method is fast and non-destructive in nature. A two-dimensional X-ray diffraction system designed for rapid screening for Combinatorial Screening. X-ray diffraction, especially two-dimensional x-ray diffraction, can be used to measure the structural information of a material library with high speed and high accuracy.

An XRD system is designed for the rapid screening of combinatorial libraries. This system is based on two-dimensional X-ray diffraction (XRD²) theory. A two-dimensional multiwire area detector can collect a large area of a diffraction pattern with high speed, high sensitivity, low noise, and in a real time mode. A 2D-diffraction pattern contains information about the structure, quantitative phase contents, crystal orientation and deformation. The laser/video system ensures that each sample is aligned accurately on the instrument center. The x-ray beam is collimated to various sizes from 1000 to 50µm. The GADDS software helps to select and save a record of the screening area and steps. The diffraction results are processed and mapped to the screening grid based on the user selected parameters³⁹.

Two-Dimensional X-Ray Diffraction (XRD²):

A two-dimensional X-ray diffraction (XRD²) system has both the capability of acquiring diffraction patterns in 2D space simultaneously, and analyzing the 2D diffraction data accordingly. An XRD² system consists of at least one 2D detector, x-ray source and optics, sample positioning stage, sample alignment and monitoring device as well as corresponding computer control and data reduction and analysis software. The diffraction pattern from a polycrystalline powder consists of a series of diffraction cones if a large number of crystals (oriented randomly in the space) are irradiated by the incident X-ray beam. The diffraction measurement in the conventional diffractometer is confined within a plane, referred to as diffractometer plane. A point detector makes a 2q scan along a detection circle. If a one-dimensional position sensitive detector (PSD) is used in the diffractometer, it will be mounted on the detection circle. Since the variation of the diffraction pattern in the direction (Z) perpendicular to the diffractometer plane is not considered in the conventional diffractometer, the corresponding structure information will be either ignored, or has to be measured by various additional sample rotations.

When we use a 2D detector, diffraction data is no longer limited to measurement only in the diffractometer plane. Instead, the whole, or a large portion of the diffraction rings can be measured simultaneously, depending on the detector size and position. Since the variations of diffraction intensity in all directions are equally important, a point focus beam is normally used for X-ray diffraction (XRD²) ^39-40.

Diffraction patterns in 3D space from a powder sample and the diffractometer plane of a conventional q-2q diffractometer.

CONCLUSION:

Solid phase synthesis continues to provide an important technique particularly to the medicinal chemist engaged in lead optimization work. Combinatorial chemistry and parallel synthesis can greatly benefit by their unique features offered by new synthetic technology. These include the possibilities of high-speed parallel processing of chemical transformations in the context of library production, and the rapid optimization of reaction conditions. Among the solid and solution phase synthesis Solid-phase organic synthesis (SPOS) is the most important method for the production of combinatorial libraries because all the synthetic transformations successfully applied to solid phase and with the development of high-throughput screening, libraries are widespread in pharmaceutical and agricultural chemistry.

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Protecting Group	Structure	Cleavage Method
N^α-Protecting Groups
Fluorenylmethoxycarbonyl (Fmoc)		Base-catalyzed (20% Piperidine in DMF)
2-(4- nitrophenylsulfonyl) ethoxycarbonyl (Nsc)		Base catalyzed (20% piperidine in DMF
Allyloxycarbonyl (Alloc)		Hydrogenolysis (Pd/C; ethanol)
5-Methyl-1,3,4 thiadiazole-2- sulfonyl (Ths)		Zn-Acetic Acid Al-Hg/THF/H₂O
Benzothiazole-2-sulfonyl (Bts)		Zn-Acetic Acid Al-Hg/THF/H₂O, Na₂S₂O₄
Side-Chain Protecting Groups
t-Butyl		Acidolysis (TFA)
Dimethoxytrityl (Dmt)		Acidolysis (Weak Acid)
Benzyloxycarbonyl (Z)		Catalytic Hydrogenation Acidolysis