Developing support in solid-phase chemistry

Reactions in solvent often leave behind complex mixtures that usually require a high degree of purifying before the product can be used. However, as Sarah Houlton explains, the use of solid support resins can improve the situation, making product isolation much easier for the chemist to handle

Standard solution-phase chemistry is well established and understood. For centuries, chemists have been mixing potions, bubbling solutions and isolating their final products from the liquids. But solution-phase chemistry has its drawbacks. Only a few, very lucky reactions crash perfect crystals out as they form, ready for filtration and washing. Most reaction mixtures are a complex mixture of product, solvent, starting material, reagents and by-products. Solid-phase chemistry has great appeal, as it makes product isolation much simpler.

Automated solid-phase chemistry has its roots in Merrifield's procedure for peptide synthesis.¹ He attached the substrates to a polystyrene resin, and several different amino acids can be added to the chain sequentially without the need for purification between steps. The method was subsequently applied to the synthesis of polypeptides, oligonucleotides and oligosaccharides, and the repetitive nature of the procedure meant that the obvious next step was the introduction of robots to automate the procedure. Hence, the synthesis of complex amino acid derivatives such as modified peptides was made routine and straightforward. And the large numbers of different compounds that could be made easily by these automated means led to the development of huge combinatorial libraries of peptides and other large molecules.

However, the potential for exploiting these libraries in the search for new drugs is limited. Peptides and the like are not ideal as drugs, as they are rarely orally-available, though they can give clues about what functional groups may be needed for activity. But as the potential of rapid parallel synthesis was realised, there has been a huge explosion in the research effort being put into discovering new methods of automated solid phase synthesis to make small-molecule drugs to satisfy the voracious appetites of modern high-throughput screening programmes.

Solid-phase versions of many solution-phase reactions have been developed in recent years. One of the earliest examples of successful application of solid-phase chemistry to small molecule libraries were the straightforward and general routes to benzodiazepines published in the early 1990s by Ellman.² and Hobbs DeWitt.³ The basic principle behind solid-phase synthesis is immobilisation. A substrate is attached to a polymer by some form of covalent linker, and a spacer molecule can also be inserted between the polymer and the linker. A chemical reaction is then performed at a different site on the substrate. Because the substrate is tightly bound to the polymer, as in Merrifield's peptide synthesis, excess reagents and their by-products are simply washed away. Further chemical elaboration of the product can then be performed, and ultimately it is cleaved away from the support, leaving a clean compound. In addition, it is usually possible to regenerate the support polymer so it can be re-used. Suitable supports for this purpose include chloromethylated PS/DVB resin, otherwise known as Merrifield resin, chlorotrityl-PS/DVB resin, and hydroxymethylated PS/DVB resins.

Picking the right support and the best linking group are essential, and the differences in reactivities of substrates and tolerability to reaction conditions mean that no single polymer or linker will ever be useable for all purposes. Not only must both the support and the linking group be inert to the chemical steps that are to be performed, but it must be possible to cleave the covalent bond that attaches the molecule to the linker under mild conditions that do not affect the rest of the molecule. The linking groups that were developed for the early work on peptides are usually stable in the presence of both bases and weak acids, and generally are only suitable for immobilising carboxylic acids. Chlorotrityl-PS/DVB resin can be used for most nucleophilic substrates. The disadvantage of this is that it only takes a very weak acid to cleave the trityl anchoring bond.

The linking group can be thought of as a double-ended protecting group, being attached to the substrate by a relatively easily cleaved bond, and to the support by a much stronger one. Unsurprisingly, many of the linking groups that have been developed in recent years are essentially based on standard protecting group chemistry, such as silyl ethers or esters, and carbamates. More recently, however, many more have been created that bear little resemblance to traditional protecting groups.

The perfect linking group is readily-available and cheap, easy to attach to the substrate in high yield, stable to reaction conditions, and simple to cleave again once the product needs to be removed from the polymer support. Most linking groups leave a 'trace' behind, i.e. the functional group where molecule and linker were attached to each other. Historically, many of these are carboxyl groups or amides, which are necessary in peptide products, but not necessarily desirable in non-peptides because of their polarity.

It is possible to use 'traceless' linkers that leave no residue on the cleaved molecule, as a new carbon;carbon or carbon;hydrogen bond is formed during cleavage. Probably the most commonly used molecules of this type are the aryl-silyl groups used in the attachment of aryl groups, which give a new carbon;hydrogen bond on cleavage, scheme 1.

The most commonly used polymer support is polystyrene that has been cross-linked with 1;2% divinylbenzene. Functional groups are introduced onto some of the benzene rings to allow the attachment of linkers. These resins are widely used because they are stable in a variety of commonly used solvents, both polar and apolar, such as alcohols, tetrahydrofuran, dichloromethane and dimethyl formamide. However, care has to be taken in stirring to avoid damaging the resin. Mechanical stirring is particularly likely to cause problems, so other methods such as orbital shaking or magnetic stirring are preferable.

Many reagents can be used without harming the resin, including acids like TFA, alkyl lithium bases, oxidising agents, transition metal catalysts and Lewis acids. And it is tolerant of temperatures down to at least ;78°C and over 150°C.

For the most part, the polymer beads need to swell and increase the size of the pores within them to operate successfully. In microporous polymers, the polymer particles are manufactured with a low level of crosslinker, typically 1% DVB, which ensures the resin can swell in apolar solvents to allow rapid permeation and access to all active sites within a bead.

One company that specialises in the development of polymers and resins as supports for combinatorial chemistry is Rapp Polymere of Tuebingen in Germany. It makes a variety of membranes, films and slides from cellulose, acrylamide-grafted polypropylene and polystyrene-grafted PTFE. Its supports are functionalised from 0.1 to 10µmol/cm2, with or without PEG spacers, and available functionalities include amines, carboxylic acids and thiols. Its TentaGel resins are grafted copolymers that have a low crosslinked polystyrene matrix, onto which polyethylene glycol has been grafted. The simplest way to graft the PEG onto the resin is to couple it via one of its terminal hydroxyl groups to chloromethylated polystyrene using a classical ether synthesis procedure, or to use other bifunctional PEGs to couple on to the solid support. But when long-chain PEGs, with a molecular weight over 800, are used, the observed yields are too low.

The PEGs have a propensity to react at both ends, giving cyclic poly(ethylene glycols), and hence additional uncontrollable crosslinking. The resulting reduction in free functional groups means the copolymer's capacity is reduced. Rapp Polymere has found that by using an anionic graft copolymerisation procedure, chains with molecular weights over 20,000 can be immobilised on functionalised cross-linked polystyrenes. The graft copolymers are pressure stable, and can be used in batch processes, as well as under continuous flow conditions. As the copolymer contains about 50;70% PEG, its properties are dominated by those of PEG rather than those of the polystyrene matrix.

Another of Rapp Polymere's support products, HypoGel, is a hydrophilic polystyrene gel-type resin. Based on a low crosslinked polystyrene matrix (1% DVB), oligoethylene glycols are grafted on to form a high-loaded hydrophilic resin. The reactive centres are located at the terminus of the glycol spacers, and they are available in a wide variety of different reactive groups.

Another company that specialises in resins and supports for combinatorial chemistry is Polymer Laboratories. Its StratoSpheres particles are claimed to give high chemical product purities, reproducible results in synthesis and high resin loadings to enable non-destructive on-bead analysis by FTIR and NMR. They include conventional microporous solid phase synthesis supports, high-load scavenger resins and macroporous supports with rapid mass transfer. They have tightly-controlled particle size distributions to improve filtration rates and highly-controlled swell characteristics. The company makes polymeric particles in sizes of less than 1µm up to over 500µm in a purpose-designed facility. It also offers a confidential contract manufacturing service to produce particles designed to meet customers' specific requirements. The company says its resin products are guaranteed to within 10% of the nominal loading and, typically, the variation is much lower. This is important to ensure comparable results in repeat syntheses.

Polystyrene supports

Argonaut Technologies also manufactures polystyrene supports for solid-phase synthesis. As well as standard 1% crosslinked PS-DVB, it produces grafted (polyethylene glycol)polystyrene (PEG-PS) under the trade name ArgoGel, and a highly crosslinked macroporous polystyrene, ArgoPore. ArgoGel is based on a novel grafted PEG-PS copolymer. Its flexible PEG grafts provide a solution-like environment for bound molecules, which mean high resolution ¹H and ¹³C NMR spectra are possible. The beads have very low reachable PEG impurities, higher loading and greater stability as a result of its bifurcated linkage ; benzylic ether linkages are more labile. They are compatible with a wide range of solvents, including water.

ArgoPore is based on a highly crosslinked PS framework that does not require bead swelling to allow access to reaction sites. The company says it gives low, predictable swelling in all solvents, giving rapid diffusional access of reagents to reactions sites, and the removal of by-products using virtually any solvent. Reactions with low solubility intermediates are possible, as are reactions at low temperatures. The free-flowing beads do not stick to glass reaction vessels.

A completely different form of resin for solid-phase organic synthesis is cross-linked ethoxylate acrylate resins (CLEAR), available from Peptides International. They have an inert matrix, good swelling properties and are easily filtered. They are said to promote rapid reaction rates and are suitable for batch or continuous flow synthesis. As there are no aromatic rings in the base resin, there are no large blocks of hydrophobic structure that can cause undesirable aggregation with lipophilic substrates.

Most of the core structure is made up of ethylene glycol units, and the only other constituents in its structure are the cross-linking unit that makes it solid, and an amine-containing moiety, which provides the handle for further structural elaboration in solid phase synthesis.

The resins, developed at the University of Minnesota⁴, retain the solvation properties of polyethylene glycol and PEG-linked products, but are easier to use. The highly crosslinked resins are produced in bead form, and swell in a wide range of solvents, including dichloromethane, water and dimethylformamide. They are also compatible with a variety of more nonpolar solvents like dioxane and tetrahydrofuran.

The company says that, as the particles are not grafted onto polystyrene, they mimic the solubility properties of polyethylene glycol and PEG-linked products, but are easier to use. The resin does not swell in water alone, but if it is pretreated with an organic solvents like DMF before the water concentration is gradually increased, it can be used in aqueous reaction mixtures.

Libris Discovery

The growth in importance of combinatorial libraries has also led to an explosion in the number of companies offering off-the-shelf libraries for rent to collaborators which, without automated solid-phase chemistry techniques, would be almost impossible to create cost-effectively. Many of these companies specialise in one specific area of chemistry. One that focuses on carbohydrates is Libris Discovery, a joint-venture between Dextra Laboratories of Reading, UK, and The Technology Partnership of Royston, Herts, UK. It exploits Dextra Laboratories" expertise in carbohydrate chemistry with The Technology Partnership's advanced chemical synthesis technology. It follows two approaches in its creation of carbohydrate-containing libraries: exploiting the inherent diversity of carbohydrate scaffolds, and building libraries around them; and glycosylating non-carbohydrate compounds. It emphasises the production of libraries of small-molecule, drug-like compounds. Many existing drugs contain sugar moieties, including cardiac glycosides and a number of antivirals. Dextra has been building up its collection of scaffolds, building blocks and reagents over the past decade. One example of its approach, the combinatorial elaboration of an aglycone via carbohydrate scaffold, is shown in scheme 2.