Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1999-08-24
2003-07-29
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S333000, C530S335000, C530S336000, C530S337000
Reexamination Certificate
active
06600016
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to high-speed parallel synthesis of combinatorial libraries and more particularly to a multifunctionalized solid support resin and to a method for the synthesis of combinatorial libraries using a multi-functional solid support resin.
BACKGROUND OF THE INVENTION
The use of solid phase synthesis techniques for the synthesis of polypeptides and oligonucleotides are well known in the art. More recently, the use of solid phase techniques for the synthesis of small organic molecules has become a major focus of research. Of prime importance has been the ability of solid phase techniques to be automated, with an attendant increase in compound throughput and efficiency in research. This has been exploited with great vigor in the area of pharmaceutical research where it has been estimated that 10,000 compounds must be synthesized and tested in order to find one new drug (Science, 259, 1564, 1993). The focus on combinatorial chemistry techniques to increase compound throughput has now become almost universal in the pharmaceutical and agricultural industries.
An additional aspect relates to the chemical diversity of the compound stocks that are available for screening in pharmaceutical companies in the search for new lead structures. These have tended to be limited to the classes of compounds previously investigated through medicinal chemical techniques within each company. Therefore the availability of new classes of molecules for screening has become a major need.
Combinatorial chemistry involves both the synthesis and screening of large sets of compounds, called libraries. The libraries themselves can be arrays of individual compounds or mixtures. Therefore, the synthetic approaches are also classified into two categories, including combinatorial synthesis of mixtures and parallel synthesis leading to individual compounds. For screening purposes it is also important that the formed compounds be synthesized in 1 to 1 molar ratios.
In the first approach to creating molecular diversity, the combinatorial synthesis comprises multiple reactions in one reaction vessel resulting in the generation of all possible product combinations from a set of reactants. The simplest manifestation of the approach is to allow several reagents to react in solution at the same time to form all possible products. Among the examples is the synthesis of a library of over 97,000 members by reaction of a mixture of amines with 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid tetrachloride (Carell, T; et al. Angew. Chem. Int. Ed. Engl. 33, 2059). However, this approach is usually unproductive unless the reagents are few and their reactivities are well matched to approach formation of the various compounds in 1 to 1 mol ratios.
Another approach is the use of the portioning-mixing method or the split synthesis (Furka, A; et al. Int. J. Pept. Protein Res. 37, 487, 1991). The synthesis is executed by repetition of three simple operations, including dividing a monofunctional solid support resin into equal portions, reacting each portion individually with one of the building blocks and then homogeneously mixing the portions. Starting with a single substance the number of compounds is tripled after each coupling step. For example, in the preparation of trimers, 27 different compounds can be prepared in three pools. These compounds can be cleaved into solution and screened as soluble pools, or the ligands can remain attached to the beads and screened in immobilized form. However, biological screens performed on such large mixtures of soluble compounds can be ambiguous since the observed activity could be due to a single compound or to a combination of compounds acting either collectively or synergistically. The subsequent identification of specific biologically active members is challenging, since the numbers of compounds present in the pools and their often limited concentration deter their isolation and re-assay. Because of this, the identification of individual active compounds from the library requires the repetitive re-synthesis and re-testing of the most active smaller subsets of the library until activity data are obtained on homogenous compounds. There is no direct method available to elucidate the chemical structures of large libraries of mixtures. However many methods have been developed to aid and accelerate the deconvolution process, including recursive deconvolution and multiple encoding approaches. There still remain a number of critical issues in screening libraries consisting of large mixtures of compounds.
By contrast, many other practitioners are using a method called parallel, or robotic, synthesis. This practice simply involves performing a series of individual reactions in separate vessels. Using traditional manual organic synthesis a chemist can synthesize only about 50 compounds per year. By the use of robots, which can perform multiple reactions simultaneously, this procedure can be made more efficient.
One of earliest examples of the parallel method for the synthesis of compounds is the multi-pin method developed by Geysen et al., for combinatorial solid-phase peptide synthesis (Geysen et al.; J. Immunol. Meth. (1987) 102:259-274). According to this method, a series of 96 pins are mounted on a block in an arrangement and spacing which correspond to a 96-well microtiter reaction plate, and the surface of each pin is derivatized to contain terminal linker functional groups. The pin block is then lowered into a series of reaction plates to immerse the pins in the wells of the plates where coupling occurs at the terminal linker functional groups, and a plurality of further reactions are carried out in a similar fashion. Reagents varying in their substituent groups occupy the wells of each plate in a predetermined array, to form a unique product on each pin. By using different combinations of substituents, one achieves a large number of different compounds with an array of central core structures.
Another type of solid phase parallel synthesis method is the diversomer approach from Park-Davis group (DeWitt, S. H.; et al. Proc. Natl. Acad. Sci. USA, 90, 6909, 1993). It was designed for the synthesis of small organic molecules. The solid support resin was placed into porous tubes immersed into tubes containing the various reagents which pass through the porous walls to contact the solid phase support resin.
A related method of synthesis uses porous polyethylene bags (Tea Bag method) containing the functionalized solid phase resins (Houghton, R. A., et al., Nature, 354, 84-86, 1991). These bags of resin can be moved from one reaction vessel to another in order to undergo a series of reaction steps for the synthesis of libraries of products.
As a consequence of the development of the efficient automation equipment and processes, the parallel synthesis technique has now become the most extensively used method in combinatorial chemistry. However, the libraries created using the parallel method (one compound per vessel) usually require more steps than those created using other combinatorial syntheses. As a result, more time is required to synthesize a comparable size library than would be required using other combinatorial techniques, such as the portioning-mixing method discussed above.
In view of the above, the field of pharmaceutical and agricultural research has a strong need for highly flexible technologies to generate a large number of novel classes of compounds for screening and clinical testing.
Solid Support Resins:
Solid support resin synthesis is carried out on a substrate consisting of a polymer, cross-linked polymer, functionalized polymeric pin, or other insoluble material. These polymers or insoluble materials have been described in literature and are known to those who are skilled in the art of solid phase synthesis (Stewart J M, Young J. D.; Solid Phase Peptide Synthesis, 2nd Ed; Pierce Chemical Company: Rockford. Ill., 1984). Some of them are based on polymeric organic substrates such as polyethylene, polystyrene, polypropylene, polyethyl
Campian Eugene
Lu Boliang
Zhang Jinfang
Advanced Syntech LLC
Low Christopher S. F.
Lukton David
Stites & Harbison PLLC
Vanderburgh John E.
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