Combinatorial chemistry: strengths and weaknesses
Combinatorial chemistry is a technique which allows large number of structurally related molecules to be synthesised or generated through computer simulation. Since its initiation, the process has become an integral part of organic synthesis. It has changed the way organic synthesis is carried out in laboratories across the world. The pharmaceutical industry has taken great advantage of this technique; dramatically changing the process of drug discovery and synthesis. It has since moved on to different areas of chemistry, and is applied in several other areas. The same principles as drug discovery are applied to develop new catalysts. Although new catalysts are harder to find, combinatorial chemistry speeds up the process; increasing efficiency and decreasing wasted effort. Materials science is the latest area of chemistry to embrace the technique, to prepare new materials. Along with saving time and effort, it provides a great economic value.
Organic synthesis and its divisions such as medicinal chemistry have long been using the process of constructing organic molecules by targeting one molecule at a time. However, a new concept has since been developed which targets a collection of molecules, and simultaneously produces a library of compounds instead of a single product. This strategy of simultaneous synthesis of large number of compounds is known as combinatorial chemistry. The principles of combinatorial chemistry had been in place before 1990s. It has since become increasingly prominent due to the pressures faced by the pharmaceutical industry to speed up drug invention and synthesis. It has been aided greatly by the introduction of high-throughput screening.
The basic principle of combinatorial chemistry is to prepare a large number of different compounds at the same time - instead of synthesising compounds in a conventional one at a time method. It is followed by identifying the most promising compound for further development by high-throughput screening. Combinatorial chemistry works by generating different compounds simultaneously under identical reaction conditions in a systematic manner. This allows products of all possible combinations of a given set of starting materials (termed building blocks) to be obtained at once. The collection of these finally synthesised compounds is referred to as a combinatorial library. The library is then screened for the property in question and the active compounds are identified.
Combinatorial chemistry has overcome the initial criticism and is now employed widely in academia and industry. It is used to synthesise libraries of either mixtures or single compounds. Combinatorial synthesis can be carried out in solution or on solid support. Although combinatorial chemistry is one of the most recent fields of chemistry, however, these principles have been used by nature since the beginning. Chemists' belief in rational design has previously kept them away from systematic explorations in chemical synthesis.
The advent of combinatorial chemistry has severely shaken the traditional one molecule at a time approach to drug discovery. The initial euphoria of the early 1990s, however, was based to a considerable extent, on faulty ground. Initially the idea of synthesising a myriad of compounds randomly, often as mixtures, seemed more like a dream. The so called dream came to reality when the principles of combinatorial chemistry for small organic molecules crystallised on a more pragmatic platform. The prevailing approach today is that based on both solution-phase and solid-phase chemistry applied in parallel or split-and-pool formats and directed at discrete and high-purity compounds. Combinatorial chemistry is initially applied to rapidly discover lead compounds; these are then subjected to lead optimisation to produce drug candidates. The last part of the process is the domain of the medicinal chemists, who may also practice combinatorial strategies to achieve their goals. Thus smaller focused libraries are carefully designed and synthesised, either in parallel or by the split-and-pool strategy using solution-or solid-phase chemistry. Combinatorial chemistry has, therefore, penetrated the laboratories of medicinal chemists who recognised its power in delivering the targeted compounds in a much faster way, and in acceptable quantities and purities. In similar ways, academic laboratories have adopted and refined combinatorial techniques in their quest for libraries of compounds needed for chemical biology studies .
The capability of combinatorial chemistry to produce large numbers of compounds rapidly is a powerful tool not only for chemical biology and drug discovery but also for a host of other research endeavours. Indeed, this philosophy and these combinatorial processes have been successfully applied to reaction optimisation, the discovery of new materials and the development of new catalysts. Reddington and Sapienza  reported in 1998 results from a highly parallel, optical screening method to discover novel electro catalysts. Such practices are currently gaining wide popularity in industry for the optimisation of process chemistry. The first report of a combinatorial approach to new high-technology materials came from Schultz and co-workers , who prepared a spatially addressable array of potential superconducting materials. Most significantly, combinatorial chemistry has proven itself to be useful in discovery of new catalytic systems.
The payoff of combinatorial chemistry to drug discovery is already becoming obvious to the industry in terms of a significant increase in the number of drug candidates and of decreases in time from target identification to drug candidates and manpower employed per drug candidate. Similar benefits are beginning to emerge in process chemistry, catalyst discovery and material science, where combinatorial chemistry techniques have also been implemented.
This review aims to start with the basis of the combinatorial process i.e. the solid and solution phase synthesis. One will endeavour to review the current combinatorial drug synthesis and the effect it has on the pharmaceutical industry. This review will also look at the expansion of combinatorial chemistry into catalysts and material chemistry.
1 Organic synthesis
Organic synthesis is the front runner when it comes to combinatorial chemistry; it has taken a leaf out of peptide chemistry. Merrifield  presented the techniques of solid-state peptide synthesis in 1963, which was the fundamental inspiration behind the designs of Geysen's multipin apparatus in 1984, [2, 3] and Houghton's tea-bag method in 1985 , further work was carried out and essentially the synthesis was made possible by the efforts of Furka; technique known as the Split-pool method in 1988 [5-8]. This is widely regarded as the starting point of combinatorial chemistry.
1.1.1 Split-pool Method
The split-pool method established by Furka and co-workers of large peptides is also known as divide, couple and recombine.
Using this procedure allows combinatorial chemistry to take place in just a few reaction vessels, (Fig. 1.1). The initial step involves a quantity of resin beads being split into multiple, equally sized segments in separate reaction vessels, each of them being coupled with a single building block. Upon completion of the first reaction step, the resin-bound compounds from all reaction vessels are pooled together in one vessel. Common steps such as resin washing and de-protection are carried out in this vessel. The resin-bound compounds are distributed into the required number of separate reaction vessels. The second solid-phase reaction provides compounds which incorporate all of the possible permutations of the two sets of building blocks. These split and pool methods are continued until the required combinatorial library has been assembled. Ideally, through this process, each resin bead in the library ends up with just one single compound being attached to it. Combinatorial libraries resulting from split-pool synthesis are known as “one-bead-one-compound” libraries [9-11]. The library consists of separate compounds with similar attributes as long as they are resin-bound.
At present the split-pool method is the most popular technique for the synthesis of large combinatorial libraries of compound mixtures, comprising thousands to hundred thousands of compounds. As we know the split-pool procedure is usually carried out on resin beads, there are certain limitations when generating mixtures of compounds. Due to the statistical distribution of the solid support at each splitting step, the synthesis will lead to over and under representation within the library. In order to ensure that 95% of all possible compound members of the library are included with a probability greater than 99% [12,13], the split-pool synthesis should be carried out with an approximately threefold amount of resin beads (termed 3-fold redundancy). For the commonly used resins (about 100 μm diameter bead), 1gram of the support material corresponds to several million resin beads, so that from a statistical point of view, libraries of the order of >105 different compounds are possible in practice [13-15]. Depending on the loading capacity of the resin bead, quantities of about 200 pmol (0.1 mg compound Mr = 500) can be obtained per resin bead.
1.1.2 Tea-bag method
In the so called tea-bag method, originated in 1984 by Houghten et al  for multiple peptide synthesis, the split-pool protocol occur batch wise on 15x22 mm polypropylene mesh packets with μm-sized pores known as tea bags, sealed with resin beads for solid-phase synthesis. This method offers the advantage that a greater quantity of each compound of the library is available at once (up to 500 μmol), which is enough for a complete biological and structural characterization, furthermore, labelling the tea-bags preserves the identity of each compound synthesised during the split-pool method.
More recently, Nicalou and Xiao , as well as Moran , developed a radiofrequency
Encoding system, which essentially involves a microchip capable of receiving, storing and emitting radiofrequency signals [18, 19] being placed in a porous polypropylene capsules along with resin beads. This development allows each of the capsules (tea-bags) to undergo the split-pool synthesis in a very precise manner. In essence, the device can be scanned to record the identity of a compound attached to each batch of resin beads.
1.1.3 Parallel synthesis
Combinatorial libraries can also be prepared by parallel synthesis , the method involves compounds being synthesised in parallel, using ordered arrays of spatially separated reaction vessels, sticking to a traditional “one vessel-one compound” philosophy (Fig1.2). This offers the advantage that each compound, when evaluated for some desired performance, is substantially ‘pure' in its local area, provided that the synthesis has proceeded with high efficiency in each stage. Furthermore in parallel synthesis the defined location of the compound in the array provides the structure of the compound, a commonly used format for parallel synthesis is the 96-well microtiter plate.
In general combinatorial libraries comprising hundreds to thousands of compounds are synthesised by parallel synthesis, often in a preset fashion. A number of different solid supports and uniquely designed reaction vessels are adopted for the parallel synthesis of organic compound libraries. The yields of the individual compounds synthesised vary widely from nanomoles to millimoles. Unlike split-pool synthesis, which requires a solid support, parallel synthesis can be done either on solid phase or in solution.
Combinatorial chemistry has expanded to encompass many variations on a theme. In one of its simplest forms, parallel synthesis, libraries of polymers and core scaffolds with points of randomization are created by varying the chemical nature of the building blocks A-E. In deep well plates randomization of a scaffold would be carried out by coupling A1 to all of the compounds in plate A1, and this is repeated to the Nth plate. On each plate, variants B1 to BN are added along the columns, and variants C1 to CN are added along the rows.
In 1984, Geysen and co-workers introduced an apparatus that allowed individual peptides to be produced in parallel in microplates containing 96 wells [2,3]. The apparatus works by using an array of polyethylene pins which are about 40mm long and 4mm in diameter. They are spaced in such a way that they fit in a conventional 96-deep-well polypropylene microtiter plate. Polyacrylamide- or polystyrene- grafted polypropylene pins  are functionalised with variety of linkers to allow flexibility in solid-phase synthesis. The loading levels per pin can range from about 100 nmol to 50 millimol of one compound, permitting quantities up to 25 mg compound (Mr=500) to be prepared. This apparatus, marketed as Multipin, has been adopted by Chiron in Australia and applications are preliminary in the use of immunology .
The execution of the parallel synthesis of up to 96 single compounds by the Multipin method involves pipetting the reactants to each well of a 96-well microtiter plate. The pin array is then placed on top of the plate and the resin is allowed to incubate with the reactants to perform the coupling step. The reaction temperature can be raised by 90C by placing the reaction block into an incubator. Following each reaction step, the pin array is removed and treated in batch to wash the solid support. These operations are repeated until the desired combinatorial synthesis is completed. The resulting compounds can then be removed from the pins into individual wells on a microtiter plate, each of which ideally contains one single compound.
In principle, combinatorial synthesis can be performed both in solution and on solid phase. Although chemistry in solution has the advantage of being familiar and well-established as the method of choice in conventional organic synthesis, to date the majority of the compound libraries have been synthesised on solid phases such as resin beads, pins, or chips.
1.2.1 Solid phase
Solid Phase organic synthesis really began in 1963 when Merrifield  used polystyrene resin beads to aid the synthesis of peptides. This was followed in the 1970s by investigations on solid-phase synthesis towards organic compounds by Leznoff, Camps, Frechet, Rapaport and others [23-25]
Throughout the solid-phase synthesis the compound under construction is covalently attached to a swollen insoluble solid support (usually a resin bead) by a linker that can cleaved under specific conditions with an appropriate reagent to give the target compound in solution later on (e.g. for assessment of purity, analytical characterisation, biological evaluation). The reaction can be accelerated and driven to completion by using a relatively large molar excess of reagents, resulting in reduced reaction time and higher yields. The support matrix in particular facilitates all steps of a synthesis protocol, such as addition of reagent solutions, filtration and washing. Thus, solid-phase synthesis enables full automation, even for multistep synthesis, where the building blocks are added respectively to build up the desired final compound.
In combinatorial synthesis the reaction must operate with reliable yield on a structurally broad set of building blocks to provide a multitude of almost pure final compounds under identical conditions. In the most time -and labour intensive step, selected building blocks are “rehearsed” individually through reactions in the solid-phase format, under conditions mimicking those that will be used faithfully in the final combinatorial synthesis. As it will often be impracticable to examine every member of the desired library to confirm its presence, building blocks combinations that are anticipated to represent worst case scenarios (e.g. with respect to steric and/or electronic factors) are studied and optimised with problematic building blocks being excluded from the library construction.
1.2.2 Solution phase
Combinatorial chemistry is slowly making its way into solution-phase synthesis; it has so far played a considerably lesser role than its solid-phase counterpart. This is probably due to the main problem of solution-phase combinatorial synthesis i.e. to obtain pure products. In solid-phase synthesis, components such as auxiliary reagents and non-reactive starting materials can be easily separated from the desired products by simple washing procedures since both reside in different phases. In solution-phase synthesis, all components occur in the same phase so that purification becomes a much more demanding task. With respect to side products derived from the resin-bound reaction component, the purification problems are, however, the same in both solution and solid phase synthesis.
However, most of the groundbreaking work through combinatorial chemistry is carried out in solid-state synthesis. This idea has now been used to carry out several classical organic reactions, which we will look at further in this review. The biggest challenge faced by organic chemists is developing high yielding reactions with minimal steps; this is an area where the combinatorial chemistry has been of great help to organic chemistry.
1.2.3 Comparison of solid and solution based methods
Both solid and solution based methods have their own advantages and disadvantages and are used widely in building several compound libraries. Solid-phase chemistry has distinct advantages over solution-phase, for example in solid-phase synthesis, large excesses of reagents can be used to accelerate reactions and to drive them to completion; these reagents can then conveniently be removed at the end. Due to the ease in separation, solution-phase has a better scope of integrating robotic aid compared to the solution-phase. Most importantly, solid-phase chemistry can be applied to the elegant and powerful “split-and-pool” synthesis strategy for combinatorial chemistry. Despite its dramatic contribution to increasing efficient over traditional methods, high-throughput parallel synthesis remains a laborious task. Thus, combinatorial chemist quickly realised the benefits of automation as a crucial component of combinatorial chemistry. Apart from just the synthesis, purification and characterisation of compounds are also important aspects of combinatorial chemistry. Development of analytical methods such as high-throughput chromatography and mass spectrometry provide a great foil for automated parallel synthesis as it enables the collection of the desired product.
The analytical methods for determining structures, synthesised through combinatorial chemistry have to be highly sensitive, since the amount of compounds bound to a single resin bead is generally of the order of several hundred picomoles . In spite of the progress in NMR and IR spectroscopy, the sort of sensitivity in determining the structures can only be achieved by mass spectrometry, especially electro-spray mass spectrometry.
When the combinatorial libraries consist of compound mixtures, analytical characterisation becomes complicated. Mass spectroscopy is still used but it is based on the prediction of mass distribution of the library. On occasions computer-generated distribution profiles are used to compare with the distribution profile acquired from the compound library [27,28], the process works by identifying synthetic problems based on 1) Incomplete coupling, which shows a shift towards lower molecular masses 2) Incomplete de-protection, or superfluous library modification, which shows a shift towards higher molecular masses. Having said this, for a library with thousands of compound mixtures, it is expected that many different compounds will have the same molecular mass; this complicates matters immensely in terms of structural determination. Reliable results of compound mixtures can however be obtained by combining mass spectrometry with HPLC or capillary electrophoresis, this combination allows analysis of mixtures of several hundred compounds.
There are other approaches to determining structures, which are incredibly valuable when the library in question is particularly large. Most of these approaches are more useful when dealing with biological compounds however; On-Bead Screening is useful regardless of the characteristics of the compound.
The approach is relatively simple and because the compounds are attached to the resin beads or onto a surface, the solid support and its linkers have to be soluble in water and for quantitative results the beads must be homogeneous in both size and substitution.
The solid-bound library is treated with a labelled soluble biological target. A fluorescent label is employed as a standard because of the high sensitivity of fluorescence detection. The labelled receptors bind to those resin beads, which are synthesised with compounds that have the highest affinity to the biological receptor. The labelled beads are then selected, followed by structural clarification of the support-bound compound. The identity of the bioactive substances is then limited to a few alternative structures by using mass spectrometry to determine their molecular mass. 
This on-bead screening is extremely useful when the size of the library is incredibly large; several thousand to a million compounds, and the isolation of a few bioactive compounds from many inactive ones. By using an on-bead analysis, the screening of a library of 107 resin-bound compounds can be accomplished routinely by one person in one day, which evidently is very efficient and would help to cut costs of an operation. As well as being efficient, another advantage of this method is that, once the library has been prepared and assayed, the remaining compounds can be reused for different biological assays, which makes this technique environmentally friend and economically viable.
When the size of the combinatorial library reaches thousands of separate single compounds or compound mixtures, manual synthesis and testing against a biological target will hardly be manageable in an acceptable time frame. Therefore, the advent of combinatorial chemistry for the high-throughput synthesis of compounds has driven the advancement of automated methods for synthesis [30,31] as well as requisite pre- and-post synthesis operations i.e. resin loading, reagent and resin delivery, compound isolation, purification, and analysis.
Automated combinatorial chemistry demands computer control of processing instrumentation. A serial processing system performs all operations on one sample before proceeding to the next. On the other hand, automated synthesisers that process samples in parallel perform the same operation on multiple samples before proceeding to the next operation. Parallel processing significantly improves the throughput and efficiency of automated synthesis systems. Furthermore, efficient software tools are necessary to program the synthesis run, to retain records of the synthetic operations or the biological testing, and to handle the huge amount of compounds and corresponding data with respect to compound searches or data interpretation .
In principal, combinatorial synthesis does not create analytically pure compounds even though the applied chemistry might be optimised. With respect to structure-activity relationships, the compounds evaluated in lead optimisation should be as pure as possible. In high-throughput screening, for lead finding, the compounds are usually tested without purification. If the purity becomes a severe problem, it must be considered that all compounds be purified before screening, or that only the bioactive molecules be purified.
2 Drug syntheses
Combinatorial chemistry has revolutionised the way drugs are discovered and has had a major effect on the pharmaceutical industry. Working together with genomics and proteomics, combinatorial chemistry is able to provide new and better medications. Utilising the ground breaking research in genomics, combinatorial chemistry provides the uncanny ability of producing tailor-made molecules. These molecules can then rapidly enter the drug development pipeline. The simultaneous dramatic increase in both the number of targets and potential hit compounds is the underlining reason for the change in paradigm in drug discovery . However, to take full advantage of these new techniques, the way drugs are synthesised needs to change. Screening fifty chemical compounds against a particular target is not efficient, combinatorial approaches often require that thousands be screened. However, because of the preponderance of targets, that screening must take place in days rather than months. High- throughput screening is a key factor in realising the potential of new drug discovery strategies by building a bridge between the increased number of targets and the vast number of compounds to be screened.
Increasing the speed of screening throughput need to be increased, but novel assay techniques and detection technologies need to be employed, in order to provide more information about the suitability of specific compounds earlier in the screening process. This combination of quality and quantity must be achieved in order to take advantage of the increased potential for target identification offered by genomics and proteomics and the potential for direct, rapid access to novel chemical compounds offered by combinatorial chemistry  Figure below shows this:
The new methods for the synthesis of discrete molecules have enabled modern combinatorial chemistry to surpass the capacity of traditional screening strategies in the search for new active substances. Regardless of the type of library being screened, ranging from libraries of high diversity - such as combinatorial peptide and organic compound libraries, or large libraries of individual natural or rationally designed compounds, modern screening strategies dictate that 105-106 compounds are screened with a single assay system. Using 96-well mircotiter plate automated screening technology; this procedure would approximately require a month to screen a diversity of 105 molecules at a rate of 50 plates per day. This is far too long for the competition faced by the pharmaceutical research labs and would need to be cut by roughly a factor of ten for a research laboratory to remain competitive.
Several factors are connected with the necessary increase in screening throughput in light of combinatorial strategies. At the screening rates enabled by high-and ultra-high-throughput applications, reagent consumption is a critical factor. Miniaturisation during both synthesis and screening phases can lead to significant savings over current methods, i.e. decreasing the volume of reagent required for synthesis and the amount of compound required for analysis . As the number of compounds which require screening tends to be relatively high and often of similar chemistry, it is wise to use novel assay techniques and detection technologies to provide better characterization of specific compounds earlier in the screening process. A collection of synthetic compounds is likely to be used in several different assays - another reason to keep reagent consumption to a minimum. Multiplexing strategies, whereby multiple parameters can be measured by employing multiple assays and/or read-outs in a single sample, will also improve screening efficiency. This combination of quality and quantity must be achieved in order to take full advantage of the increased potential for target identification offered by genomics and proteomics and the potential for direct, rapid access to novel chemical compounds offered by combinatorial chemistry . High-and ultra-high-throughput screening (uHTS) not only allows realisation of the potential of these new technologies, it also enables better usage of natural compound libraries - forming a primary backbone of drug discovery. Screening, therefore, is the link between biology (targets) and chemistry (drug like molecules). And in the age of genomics and combinatorial chemistry, that link must achieve high throughput. The unification of targets, compounds and assays in screening application is shown below:
The effective use of high throughput screening for combinatorial application depends on effective synergy between the traditional disciplines of drug discovery, biology, biochemistry and chemistry, the technologies associated with combinatorial chemistry , and such diverse disciplines as information technology, robotics, physics and fluidics. Screening was once the exclusive province of giants in the pharmaceutical industry. Today, screening is increasingly characterised by alliances between biotech ventures devoted to developing screening strategies, and research laboratories, whether they be large pharmaceutical firms or other biotech ventures, who provide a specific screening target. As these alliances are forged, the appropriate union between target, assay method and screening technology is of primary importance.
Natural product libraries, shelf compounds, libraries from chemically modified natural products and combinatorial compounds collections, including peptides, peptidomimetics and small organic molecules, are well established as sources for new lead structures. Solution-phase or solid-phase synthesis with a variety of chemistries and scaffolds allows the experienced chemist to produce libraries directed at almost any class of targets. Combinatorial chemistry is used in structure based drug design, for lead optimisation, and to generate highly diverse compound collections for random screening. Methods for their reproducible, effective and fully automated synthesis and analysis have been established and are dependent on the heterogeneity of the mixtures . As the number of products accessible from a given set of components increases exponentially, synthesis strategies had to be developed that allow the highly parallel and simultaneous production of compounds which were generated and tested as mixture or as individuals. These strategies are supported by new methodologies for diversity measurement and compound selection. Combinatorial and high-throughput synthesis has boosted the generation of compounds in many pharmaceutical companies 10- to 50-fold.
The diversity accessible through the application of combinatorial chemistry places significant demands on screening strategies. An ideal screening system must provide sufficient flexibility to allow a very large number of individual compounds to be tested against a variety of targets. To boil that statement down to numbers: at least 50000 compounds per day. The goal must also include the wringing of as much information as possible out of a single library synthesis. Due to the desire to use a library a number of times to maximise the use of each synthesised compound per round, it is also necessary that HTS systems be able to operate with small amounts of compound. As stated earlier, miniaturisation is prerequisite of an efficient HTS system.
Libraries used for lead finding, are, by nature, diverse, but screening places the added demand this diversity e describable: we need to be able to register and store a variety of characteristics associated with each individual compound, as well as collate chemical and biological information. Not only is it necessary to keep track of each compound, the results of screening must also be tracked for a variety of assays. In the future, efficient integrated synthesis and screening systems will be able to take the stream of compounds directly from synthesis to screening without the need for interim storage. Hits will be re-synthesised in scaled-up quantities for downstream studies based on screening and synthesis information alone.
The initial design phase for combinatorial compound collections focuses on computer generation of a virtual library and selecting a subset of compounds for chemical synthesis on the basis of specific characteristics, such as maximum diversity, desired lipophilicity, and lack of toxic and reactive functionality . When combinatorial compounds are used for lead optimisation, collections can be designed on the basis of a reference structure with the collection chosen to represent the desired degree of diversity. Software for the design of such combinatorial reaction is commercially available. An alternative approach to generate a diverse compound collection is selection from an existing database of compounds . Similarity-based selection of test compounds from an existing compound collection increases the effectiveness of lead finding and lead optimisation.
The construction of a fully automated laboratory where all manual steps - from chemical synthesis to product characterisation - are managed by robots is no longer a futuristic dream. Currently about 20 companies are specialised in the design of chemistry robots of different sizes and levels of automation . The robotized laboratory for chemical synthesis has to be connected with databases for building blocks, for reaction information and for library information. The tools for data analysis should be accessible to the operator to design libraries and lead optimisation in cooperation with specialists for screening, bioinformatics database management, computational and medicinal chemistry, and for compound characterisation.
Today most combinatorial and high throughput synthesis strategies are calculated for 10mg, 10-20 micromolar yield. In the first generation of microreactors, single compound beads or spots on functionalised surfaces have been introduced for the synthesis of pico to nanomolar amounts of product . As they come into wide use, microreactors will bring about a fundamental change in combinatorial synthesis and screening strategies.
Microreactors will be directly connected to microscale purification units and highly sensitive analytical devices . Nanotiter plates are already being used for direct collection from microfluidic separation units, transferring samples directly to biological assay units. Such integrated labs-on-a-chip combing microreactors, microscale purification and separation units, and highly sensitive analytical devices - are likely to make compound storage systems obsolete. Test compounds will move directly from synthesis to screening. Furthermore, high-throughput miniaturised synthesis will be designed for parallel synthesis, as well as for the initial scale-up process. To fulfil all of these requirements, all components of integrated microreactors, mixers, valves and process-control sensors, must be stable to aggressive, highly reactive chemicals.
Compound collections and libraries generated by the combination of different chemical reaction and building blocks are valuable tools in the search for novel ligands. Such combinatorial synthesis strategies results in residues at either defined or degenerate positions on defined chemical scaffolds. Compound mixtures can be designed for screening either in solution, or with compounds immobilized on solid supports, such as polypropylene pins or resin beads. Pharmaceutical procedures have started to establish a database for published and proprietary procedures for solid-phase organic synthesis which can be downloaded to their automated laboratory for chemical synthesis.
The “split-and-pool” method was developed to generate resin beads that are structurally homogenous and is characterised by coupling different building blocks in separate vessels, batch de-protection with pooling of all resins with redistribution prior to the next coupling. Polymers offering a hydrophilic surface are especially preferred for testing polymer-bound compounds with soluble receptors .
For screening, compounds are removed from the solid support at the point of attachment by cleaving a chemical linker. A variety of chemical linkers have been described, all with the common trait that enables release of the bead surface compounds under mild conditions. For identification of specific compounds, several methods for on or off bead analysis are available: sequencing of peptides on single beads , mass spectrometry of the compounds from one isolated bead , chemical tags for decoding by gas chromatography  and new tagging strategies, such as DNA-tags  or radio-tags .
EVOTEC, a leader in the discovery and development of novel small molecule drugs, has developed a system for the homogenous analysis of bead-surface interactions. This system allows compounds to be screened without the need to remove them from the surface of the bead. The system also enables direct recovery of single beads for further analysis. By enabling the rapid, on-bead analysis of thousands of beads, the EVOTEC systems eliminates a critical bottleneck, namely the need for de-convolution of solid-phase diversity. 
Solution-phase combinatorial chemistry overcomes the restrictions of solid-phase synthesis caused by the need to attach educts to, and release products from, the solid support. The adaptation of standard synthesis procedures to solid-phase chemistry is not necessary. On the other hand, the chemist must be careful to ensure that educts have reacted almost completely, and that excess reagents are removed after each reaction step: this represents a challenge for automation and process control. A core molecule as a template with several reactive groups has been used to generate xanthenene  or piperazine  libraries. An iterative process for identification can be carried out by the deletion of one of the building blocks.
A hybrid between combinatorial synthesis on solid and in solution is the liquid-phase method characterised by the application of carrier polymers completely soluble in one solvent and insoluble in another solvent. [52, 53]
Bioassays are influenced by protein-reactive compounds and tolerate only limited numbers of non related molecules. False-positive and false-negative results from screening of compound mixtures have been reported. For example, octa-and hexapeptide mixture significantly depress binding of the hormone neuropeptide Y to its receptor when measure in a standard competition assay 
Many pharmaceutical companies own sample collections of one to a million individual natural or synthetic substances. Combinatorial libraries will enlarge the collections to several million compounds. This explosion in the number of samples and the associated assay costs have forced most screening strategies to resort to a pooling strategy whereby up to thousand of samples are mixed in a single well for primary screening in traditional 96-well formats. This pooling strategy makes the results of screening difficult to interpret but it is currently the best way to meet necessary throughput levels with conventional assay format. This is the primary reason behind the drive to miniaturization and increased throughput. The screening systems of the future must not only be faster, they must provide more reliable data and be more economical.
Pharmaceutical industry has taken full advantage of the combinatorial chemistry for many years and this approach has now been implemented in catalysts. Catalysts play a big part in modern synthesis processes in the chemical industry. They contribute substantially to the efficiency and thus to the profitability. Therefore any improvements in catalysts usually translates directly into substantial savings, hence, there is no surprise that tremendous efforts are invested by industries and academics worldwide to not only improve existing catalysts but also develop novel ones.
Improving or developing new catalysts is an incredibly time-consuming process. As the process is often driven by trial and error - combinatorial synthesis will be of great help as it would speed up the process considerably. Optimisation and incremental improvements of catalysts are usually carried out using a very well-developed set of tools, but completely new formulations or a totally novel application of a known formulation are found rather by serendipity than by rational design. Combinatorial approach has not completely substituted the conventional process of catalyst development; they are fundamentally used to discover new active compounds.
There is a strong economic driving force to apply combinatorial techniques in catalysis. There are several processes in the chemical industry which do not have any useful catalysts at all, for example the direct oxidations of propene to propene oxide, the synthesis of alpha olefins from alkanes, or direct activation processes for methane at moderate temperatures. These processes would be operated at a scale of several hundred thousand tons per year with a suitable catalyst, and the possible annual revenues involved would be in millions.
Catalysts development face the same concerns as drug discovery, libraries are required to be adequately diverse to ensure a broad range of parameters are scanned. The parameter space for a catalyst formulation is much larger compared to that for molecular entities. Along with the chemical diversity with respect to the constituting elements, diversity in the methods of preparation is also necessary, for example catalyst prepared by a sputtering or CVD technique will constitute of different properties when compared to one synthesised by precipitation or impregnation. An advanced laboratory for combinatorial catalyst development should therefore have access to automated synthesis of materials by all means which are used nowadays in catalysts synthesis.
Two extreme strategies are proposed when choosing suitable chemical compositions of the libraries, one which focuses fully on elements in the periodic table which are known to be active for a certain class of reactions. For a partial oxidation, it is possible to use vanadium, molybdenum, bismuth etc. However, for the interesting catalytic processes in which a combinatorial approach would be most useful, a lot of time and effort has already been spent in the development thought without any real success. This means that many of the obvious combination of elements have already been tested. A systematic scanning of the composition range might lead to active catalysts, but the chance to find a fundamentally new catalyst seems to be limited. The other is approach is rather unconventional, where it is proposed to neglect any prior knowledge of catalysis and to randomly scan combinations of elements in the periodic table. However, this approach does not seem to be very useful, since by this procedure one would, for instance, combine elements that form volatile compounds under reaction conditions which would then ruin the whole library and will be of very little use under industrial conditions. Economic factors might be an issue too. 
The best approach seems to be the one which combines both approaches. In most cases testing should be based on elements known to be active in catalysts in a broad sense, however, by adding non-obvious components it is possible to create non-obvious formulations. This approach will smooth the progress of creating the chemical diversity which is required to increase the chances of discovering something fundamentally new.
Similar approaches exist in the testing of catalysts as do in drug discovery. It would be possible to screen the different catalysts at one time (cocktail screening), and then to isolate the active ones by dividing the catalysts; alternatively, they could be screened one at a time (parallel screening)
In most cases in heterogeneous catalysis - at least in gas-phase reactions- parallel screening is the method of choice. A catalyst bed with difference catalyst pellets over which the same feed is passed, is only useful in reactions where (i) there is only one or few products; (ii) most catalysts are totally inactive and ;(iii) the product dies no react further. If one of these conditions is not fulfilled, the test would give such complex results that further conclusions would be almost impossible. [56, 57]
Although at present the bottleneck in the combinatorial approach to catalysis is most probably that of testing to obtain meaningful catalytic data, high-throughput synthesis of potential catalyst materials is an important aspect of the whole process. In order to exploit fully the whole parameter space, one cannot rely on only one method of synthesis, but most acquire capabilities to produce possible catalyst materials by all routes used conventionally, but now adapted to the high-throughput mode, and probably also additional routes designed especially for the combinatorial process.
Using the evaporation/sputtering techniques with adjustable marks, it is possible to prepare an array of different compounds on a support material . Using such techniques, different compounds could be evaporated simultaneously or subsequently, and converted to a desired compound in subsequent steps. In a variation of this approach, one does not deposit a certain composition in a desired location on a substrate, but uses a gradient sputtering technique from different sources. By correctly placing the sources of the precursors, compositional gradients are achieved on a substrate. Gradient sputtering to synthesise a range of compositions in one step has been used in many laboratories for some time before combinatorial approaches were introduced and have been found useful to produce in a simple way of variety of materials. In combinatorial experiments, it is not even necessary to know the exact composition of each of the locations on a substrate, only regions which appear to have promising properties can then be analysed to determine their exact composition.
Using sputtering or evaporation techniques will normally results in materials which are rather different from most conventional catalysts in that they expose only their geometric surface area and usually will not have any porosity. Therefore, synthesis approaches have been developed which allow the preparation of materials which are closer to a conventional catalyst material. The easiest approach is simply to deliver precursor solutions manually from a pipette to the substrate. This approach has been chosen by Reddington et al to create the smaller libraries used in their study 
Such a procedure is impractical is the library size exceeds a certain threshold, or if the amounts of catalyst to be deposited become extremely small. A multipurpose synthesis system has been developed which allows all type of impregnation techniques to be carried out, as well as ion exchange and precipitation or co-precipitation. The system is based on a Gilson automated dispenser which has been modified to suit the systems needs (page 470 figure).In particular, the precipitation step is somewhat difficult to implement, as it normally involves relatively large amounts of liquids. Precipitation reactions area therefore carried out in relatively large vessels which are continuously shaken to prevent settling of the precipitate. The suspension is then pipette onto a 16-well filtering unit which has the same dimensions as the catalytic reactor and washed after the mother liquor has been filtered off. The solids thus prepared can be post-treated after synthesis and the simultaneously filled into the reactor for the catalytic test. Currently, this system is being used to synthesise high-activity, gold-based catalysts for oxidation reactions, in which the synthesis and precipitation conditions strongly affect the catalytic behaviour[60, 61].
Catalyst testing is the most crucial problem in any combinatorial approach to catalysis, as in many reactions a certain amount of catalyst seems to be necessary to obtain meaningful catalytic data. Thus, it must always be established that any trends observed in a high throughput experiment are reproduced in a conventional unit. The ultimate proof that a catalyst which appears promising in a combinatorial test must be obtained in the conventional manners, as the final performance of a catalyst in a process is governed by an intimate interplay between catalyst, reaction conditions and reactor - and all three factors cannot be simultaneously optimized in a high throughput experiment.
Similarly to the pharmaceutical industry, where the procedure in discovering new leads is a multistage process, where the first stage consists of testing a highly unselective (through rapid) test to identify compounds which warrant closer inspection. The next screening stage is then more selective, although more time consuming, and so on until a potential drug reaches the stage of clinical trials, or even commercialization. A similar approach is needed in the development of novel catalysts through combinatorial process. In the first stage a very broad - but probably relatively unselective - screening will be necessary. The second stage will resemble a conventional catalytic experiment, but it will be parallelised on a large scale. This is followed by the test set-ups as in miniplants, or a catalytic microreactor which currently is the first step in industrial catalyst development. So in the combinatorial process, two additional steps are applied before the conventional development process, this means an increased number of possible combinations will be available for further testing, increasing the probability of developing a novel catalyst.
The very first method used to analyse catalytic activity in a parallel fashion employed IR thermography , here the conventionally prepared catalyst pellets were placed on a supporting wafer that was located in a reaction chamber. The top of the reaction chamber was sealed with an IR-transparent window and a hydrogen/oxygen mixture was passed over the catalysts. Catalytic activity was detected via the heat of this highly exothermic reaction, which was analysed using a thermographic imaging device. Using this technique, the noble metal-containing samples for which high activity was expected were indeed found to be the most active.
Other variations of the thermographic techniques have been used in a rather elaborate fashion; the heat of the reaction released in an acylation reaction was analysed . The catalysts in this case were heterogenised homogenous catalysts on polymer beads, the reaction being carried out in solution. One problem of themography in solution is the fact, that the bulk of the solution screens the heat released at an individual catalyst bead, this issues can be eliminated by using chloroform as the solvent. As the polymer beads are less dense than the chloroform, the beads float on the surface of the solution and the thermographic image thus represents the actual heat released by the reaction. Individual tests on the hottest beads revealed that highly active acylation catalysts were bound to these beads.
There is no denying that combinatorial chemistry, coupled with high-throughput screening and integrated data management systems, has forever changed the way drugs are discovered. With rising economic pressures and the increase in efficiency in other areas of research and development, it is not surprising that a similar paradigm is taking hold in the chemical industry as a whole. We are now witnessing combinatorial synthesis and sophisticated screening technologies being applied to the discovery of more efficient materials, which would consequently result in reduced research and development costs.
with these new technologies come the promise of faster commercialisation rates and reduced research and development costs.
The combinatorial process aims at efficiently exploring the large parameter space that controls the properties of a material through the application of rapid parallel or combinatorial synthesis and subsequent high-throughput characterization for a given application. The procedures of synthesis and screening developed in the pharmaceutical industry can be adapted to the new areas of research. However, unlike in the pharmaceutical industry where certain aspects such as solvent, temperature, and additives are held constant to eliminate assay variability. These conditions are varied when searching for new materials, this variation is a critical component of the combinatorial search. Variation coupled with the reaction conditions, combined with parallel synthesis, results in an exponential increase in the total number of experiments, dramatically increasing the chances of identifying a new material.
Complex interactions involving the host structure, dopants, defects and interfaces are often precursors of properties of solid-state materials . Therefore, they depend sensitively on both composition and processing conditions. Few general principles have emerged that allow the prediction of structure beyond binary systems and the resulting properties of such solid-state compounds. Conventional “one at a time” synthesis and characterisation can be a long and expensive process, and combinatorial materials science holds great promise in facilitating the materials in discovery and optimisation enterprises.
Conventional materials research typically begins with a decision on a general phase space which targets a property of interest and which is based on a set of physical or chemical constraints, many of which may be empirically or intuitively grounded. This parameter space is then divided into discrete compositions that must be synthesised and screened for properties of interest. Chemical additions, substitutions, and modifications of synthesis and processing conditions allow the researcher to optimise the properties of a given system. This process is typically long and laborious, and may or may not lead to a promising material. The integrated application of rapid synthesis, high-throughput screening, and sophisticated data analysis allows for a promising alternative to the time-consuming classical methodology. However, a well planned experiment design of the experiments is required to reduce the number of samples that will be necessary to define sample spaces within the experimental universe or to direct screening to other spaces.
The combinatorial process relies on the implementation and coupling of high-speed synthesis and high-throughput screening techniques. These methods facilitate more efficient explorations of a given composition space and offer a valuable tool for the investigation of ternary and higher order systems. However, it is often impossible to rapidly synthesise materials the physical features of which (e.g. Composition, microstructure, grain size, and density) are exactly the same as materials made using the final production process. Similarly, screening of desired properties is often very slow. Thus, combinatorial studies are based on the predictive capabilities of synthesis and screening tools, and the challenge of the combinatorial process is to implement appropriate synthesis and screening techniques. Rather than comparing the properties of a few specific compositions within a phase space, entire phase spaces can now be examined in a single experiment. The first library of compounds is often a broad compositional search covering an entire phase space, e.g. an entire ternary composition diagram. (page 1021)
(Nature Materials 3, 429 - 438 (2004)
doi:10.1038/nmat1157 Combinatorial solid-state chemistry of inorganic materials Hideomi Koinuma1 and Ichiro Takeuchi2)
A primary screen specifically developed to evaluate the very large number of compositions within that library identifies a particular composition or range of compositions that is of further interest. Primary screens are typically designed to eliminate a large fraction of the composition studies in the first library, while secondary and follow-up libraries examine a more narrow range of compositions as well as additional chemical substitutions and processing conditions. Optical and electronic properties such as capacitance and luminescence are examples of physical properties that may be efficiently examined in high-throughput primary screens.
At the follow-up level, more detailed information can be obtained from high-throughput secondary screens because the number of compounds that must be screened has been greatly reduced. This process of synthesis, screening, and optimisation continues until a manageable number of compositions have been reached. Theses selected compositions, all of which have passed previous screens, can now be studied using conventional methods to obtain the more precise chemical and physical data necessary to characterise a material completely. Combinatorial methods can thus acts as an efficient filter for conventional methods by selecting only the best candidates for further, more detailed study.
Some synthesis techniques have been developed for combinatorial materials library formation. Some materials can be made using solution deposition methods, while others are more suited for thin-film deposition [33-38]. By modifying technologies similar to those used to make integrated circuit (IC) chips, materials libraries or integrated materials (IM) chips were first developed and utilised by Schultz and co-workers . The choice on synthesis technique is based on both the material being prepared as well as the primary screen employed after synthesis. Unlike the case of drug discovery, however, the synthesis of solid-state materials often relies on processing temperatures in excess of 400C. High-temperature reaction conditions have been addressed though the creation of two-dimensional, spatially addressable arrays of samples deposited on thermally stable substrates.
Vapor deposition techniques
Vapor deposition is commonly used in the semiconductor industry to deposit thin films of material onto a substrate. Vapor deposition techniques that have been utilised in combinatorial library synthesis include sputtering and thermal evaporation, electron-beam evaporation, pulsed laser ablation, ion-beam implantation, molecular-beam epitaxy and chemical vapor deposition.
One of the most straightforward thin-film approaches is the continuous composition spread [CCS] technique, which uses two or three off-axis sources to co-deposit material on a substrate . This technique relies on the non-uniform deposition of materials formed by the geometric arrangement between the sources and the substrate. The relative concentration of each component at a specific location on the substrate decreases with the distance from the source. As materials spread from the sources, they mix in the vapor and are deposited on the substrate creating atomic-level mixing that reduces or eliminates the need for high-temperature post processing of the library. This technique is also amenable to the isolation of metastable or low-temperature phases that are crystalline on deposition. The lack of precise stoichiometric control and limited compositional range has relegated this technique primarily to optimisation and exploration of systems with only two independent variables.
A typical vacuum deposition system for combinatorial materials science has several source materials and is used in conjunction with masking techniques to deposit different materials, sequentially or simultaneously, in particular areas of the substrate. The design on the masks and the sequence in which they are employed determine which materials are deposited at any given location on the substrate. By altering the sequence, time, and rate of deposition, it is possible to control the exact chemical composition of each element in the library.
We find ourselves at the dawn of a new age of materials discovery and optimisation. As this review demonstrates, significant first steps in that direction have been taken in various areas of materials science, and a multitude of tools are now available using combinatorial technologies to accommodate the new tasks and requirements for combinatorial accelerated materials research. Rapid serial and parallel adaptations of conventional analytical techniques will become increasingly important in the characterisation of materials properties, as will the development and implementation of new and unconventional high throughput screening tools.
Since the initial application of combinatorial methods to materials science discovery research, tremendous advances in this rapidly growing field have been made in the academic, private and public sectors.
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a, The reaction coordinate of the generalized scheme of materials synthesis where a desired product (P) is obtained from reactants (R). Energy (E) can be supplied in various forms. b, Merrifield synthesis can be viewed as a process where steps (1) to (3) in a are integrated. Sequences of different reactants (R) are attached to the bead (B). c, Microchemistry. Reactants and a medium (M) are mixed in microreactors. Steps (1) to (4) take place in a continuous flow. d, Combinatorial chemistry is a parallel integration of Merrifield synthesis. e, Combinatorial solid-state technology: solid formation is carried out in parallel in a spatially addressable library.
Combinatorial chemistry has come a long way since it was pioneered by Merrifield. It has become an integral part of chemistry in general, compared to its origin in organic synthesis. Organic synthesis still remains the biggest benefactor of combinatorial chemistry, which in turn has revolutionised drug discovery and drug synthesis. Pharmaceutical industry has taken full advantage of combinatorial chemistry; it has allowed several companies to stay in front by employing this technique to consistently improve and synthesising new drugs. The time span for developing new drugs has decreased substantially, perhaps the biggest gain from combinatorial chemistry. Although pharmaceutical industry tends to use the technique to maintain their status quo, it has nonetheless benefited society in general.
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