Nonredundant split/pool synthesis of combinatorial libraries

Details Nonredundant split/pool synthesis of combinatorial libraries Nonredundant split/pool synthesis of combinatorial libraries

2001-02-02

2004-09-28

Brusca, John S. (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C435S283100

Reexamination Certificate

active

06799120

ABSTRACT:

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Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
Modern methods of identifying compounds having desired chemical or physical properties typically involve assembling libraries of compounds, which are then systematically screened for members with the desired properties. One method of assembling compound libraries involves the highly labor-intensive process of isolating and characterizing naturally occurring compounds. Another approach involves synthesizing libraries of compounds using combinatorial processes in which sets of compounds are prepared from sets of building blocks via multi-step synthesis. The libraries produced by the latter approach typically successfully emulate the structural characteristics of naturally occurring compounds. In addition, combinatorial libraries also generally provide more rapid access to larger collections of more diverse compounds that may incorporate optimized chemical or physical properties into their structures.
Numerous techniques have been devised for producing combinatorial libraries. Many of these techniques utilize solid supports to exploit efficient “split-and-pool,” or simply “split/pool,” synthesis methods to assemble all possible combinations of a set of building blocks. The split/pool method typically utilizes a pool of solid supports containing reactive moieties. This pool is initially split into a number of individual pools of solid supports. Each pool is then subjected to a first reaction or randomization that results in a different modification to the solid supports in each of the pools. After the reaction, the pools of solid supports are combined, mixed, and split again. Each split pool is subjected to a second reaction or randomization that again is different for each of the pools. The process is continued until a library of target compounds is formed. Split/pool synthesis is a very efficient method that allows synthesis of a library of n1×n2×n3 members with just n1+n2+n3 reactions. Split/pool combinatorial synthesis is described further in, e.g., Furka and Bennett (1999) “Combinatorial libraries by portioning and mixing,”
Comb. Chem. High Throughput Screening
2:105-122 and Lam et al. (1997) “The ‘one-bead-one-compound’ combinatorial library method,”
Chem. Rev.
97:411-448.
The relative simplicity of split/pool synthesis is achieved at the expense of losing information about the identity of individual compounds during synthesis. As a consequence, structural determinations of synthesized compounds are typically performed following synthesis. Two general categories of techniques have been developed to identify the structures of individual library members during mixture deconvolution, namely, coding and noncoding strategies. Coding methods provide structure determination for libraries through the reading of a code that represents unambiguously the series of steps that a given solid support was subjected to during synthesis. The coding entity may be chemical which relies on the iterative coupling of chemical tags (e.g., peptides, oligonucleotides, isotopes, binary molecule systems, or the like) to orthogonally functionalized beads during library synthesis, where the tag structure is read using various analytical techniques. See, e.g., Czarnik (1997) “Encoding methods for combinatorial chemistry,”
Curr. Opin. Chem. Biol.
1:60-66 and Barnes et al. (1998)
S. Rec. Res. Dev. Org. Chem.
2:367-379. Various nonchemical encoding techniques have also been developed which record the synthetic or chemical history of library members by physical methods. See generally, Xiao and Nova (1997)
Comb. Chem.
135-152. These techniques include, e.g., radiofrequency encoding (see, e.g., Nicolaou et al. (1995)
Angew. Chem. Int. Ed. Engl.
34:24-2479 and Moran et al. (1995)
J. Am. Chem. Soc.
117:10787-10788), and optical or color encoding (see, e.g., Xiao et al. (1997)
Angew. Chem. Int. Ed. Engl.
36:780-782), where solid-phase supports are encapsulated in an encoded porous container.
Noncoding methods of determining compound structure involve techniques which do not utilize additional encoding constructs associated with library members structures. These methods include, e.g., synthesis in a fixed array (parallel synthesis), where a compound's position within the array identifies the series of synthetic steps used to create the compound; direct deconvolution by pooling methods, where deconvolution of active structure is performed through selection of active pools from various synthetic cycles; and direct deconvolution by bioanalytical methods, where the chemical structure of active library components is determined by bioanalytical methods. See, e.g., U.S. Pat. No. 5,143,854 “LARGE SCALE PHOTOLITHOGRAPHIC SOLID PHASE SYNTHESIS OF POLYPEPTIDES AND RECEPTOR BINDING SCREENING THEREOF,” issued Sep. 1, 1992 to Pirrung et al., Pirrung (1997) “Spatially addressable combinatorial libraries,”
Chem. Rev.
97:473-488, DeWitt et al. (1993) “‘Diversomers’: an approach to nonpeptide, nonoligomeric chemical diversity,”
Proc. Natl. Acad. Sci. USA
90:6909-6913, Geysen et al. (1986) “A priori delineation of a peptide which mimics a discontinuous antigenic determinant,”
Mol. Immunol.
23:709-715, and Geysen et al. (1987)
J. Immun. Meth.
102:259-274 (parallel synthesis of peptides on rods or pins).
Both coding and noncoding approaches to determining the identity of structures following split/pool synthesis have significant disadvantages. Although encoding strategies allow the use of the most efficient form of split/pool synthesis, pooling of solid phase synthesis units during intermediate steps in synthesis, encoding inevitably records only the series of steps that the support was exposed to during synthesis, which should, but does not necessarily, lead to the desired products. Furthermore additional steps are often required during synthesis and decoding to assign structure accurately. Parallel synthesis does not allow the most efficient means of synthesis as, by design, the support is split and not pooled during synthesis in order to unambiguously predict a structure to be present at a location in an array of compounds. Finally, the time and labor intensive nature of the processes employed to decode the synthetic product limit the application of this method to a small portion of the total number of synthetic products—typically, those structures which show activity in high throughput screening assays. In this approach, valuable information about closely related but inactive structures is not obtained.
Combinatorial chemistry has advanced to the point that it is not enough to synthesize a desired set of compounds. It has now become equally important to consider the steps that immediately follow synthesis. For example, within the last several years there is a clear trend in combinatorial chemistry towards producing pure, characterized individual compounds. Consequently, compound analysis, to assess purity and confirm that the intended compounds were synthesized, is routinely conducted following synthesis of combinatorial libraries. Mass spectrometry (MS) is usually the method used for confirmation of structure. High performance liquid chromatography (HPLC) is most often used for purity assessment. Typically, components of a combinatorial library are subjected to HPLC/MS analysis for quality control which is independent of the way in which the relevant library was synthesized (by parallel synthesis, encoding, etc). Also, the format of the screening assays, which will be used for testing compounds originating from combinatorial synthesis, is relevant.
From the above, it is apparent that there is a substantial need for new methods which permit more

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