Lightdirected Synthesis

In 1991, Fodor and colleagues combined photolithography, photochemistry, and solid-phase synthesis in a new technology [19-22] called light-directed, spatially addressable parallel synthesis or VLSIPS (very-large-scale immobilized polymer synthesis). The principal points of the technology are illustrated in Scheme 5.10.

The synthesis occurs on a flat glass surface modified with an appropriate linker (e.g. 3-aminopropyl-triethoxysilane) to allow for the covalent attachment of protected amino acids. The entire synthesis area of the slide is derivatized with a photolabile protecting group (PG). At the first step of the synthesis, selected sites of the synthesis area (typically three squares per slide, 1.28 cm x 1.28 cm each) are exposed to UV light through photolithographic mask A. The variety of patterns available for photolithography is essentially unlimited. The exposure to light causes removal of the photolabile groups, thus elaborating amino functionality. At the next step, the synthesis area is treated with the reagents necessary for the elongation of the peptide chain. Only the sites that were previously photodeprotected will participate in a coupling reaction; the rest of the synthesis area remains protected and intact. Synthesis continues by illuminating another part of the surface through photolithographic mask B, followed by the next chain elongation reaction. By repeating the photodeprotection and coupling steps, highly dense arrays, each consisting of thousands of peptides, can by synthesized. Importantly, the primary structure of each peptide in the array is sufficiently defined by the sequence of coupling and photolysis steps, and by photolithography mask patterns. Therefore, the structure can be easily deduced from the (x, y) coordinates of the peptide on the slide. This eliminates the need for encoding-decoding procedures required by some other combinatorial technologies. After completion of the synthesis, the synthesis area is exposed to reagents necessary for the elimination of side-chain protecting groups. To assess the binding properties of all synthesized peptides, the entire array is incubated with a fluorescently labeled target molecule and scanned using a stage-scanning confocal fluorescence microscope. Sites, containing peptides that bind to the target, become fluorescent. Affinity data on all peptides in the entire array are obtained in one step.

The consumption of chemical reagents required for the synthesis of thousands of peptides composing the array, together with the biological reagents necessary for bioassay, is very small, because the capacity of the flat glass surface is only 520 pmol cm-2. Biological reagents used in this technology are recoverable and can be reused. Moreover, after performing an assay with one target molecule, the bound target can be easily dissociated from the array (e.g. by treating it with 6 M guanidine hydrochloride), making the array available for subsequent screening with other targets. These arrays are reusable for at least 6 months.

With special (orthogonal) masking strategies the number of synthetic regions on the glass surface can be increased until the limit of photolithographic resolution is achieved (10-20 mm). With this resolution, 250,000-1,000,000 compounds can be synthesized in 1 cm2. Routinely, 50-mm resolution is practiced and allows for the production of 40,000 compounds in the same area.

Light-directed, spatially addressable synthesis is a powerful technology for generating chemical diversity. Unfortunately, the technique is limited to peptides, oligonucleotides and other linear oligomeric structures.

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