Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-02-08
2003-03-04
Ponnaluri, Padmashri (Department: 1627)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S289100, C435S294100, C435S091500, C435S091500, C435S091500, C536S023100, C536S025100, C530S333000, C530S339000
Reexamination Certificate
active
06528324
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to apparatus and methods for carrying out multiple different operations on multiple articles and establishing the operations on each article by its position in the array.
The present invention relates to apparatus and method useful in creating combinatorial chemistry libraries. More particularly, the present invention relates to apparatus and methods for synthesizing spatially-dispersed positionally encoded combinatorial chemistry libraries of oligomer whereby the synthesis is carried out on a plurality of solid supports which in turn are distributed in the form of a series of arrays. The position of each solid support in each array determines the exact identity of the oligomer.
BACKGROUND OF THE INVENTION
The screening of chemical libraries to identify compounds which have novel pharmacological and material science properties is a common practice. These chemical libraries may be a collection of structurally related oligopeptides, oligonucleotides, small or large molecular weight organic or inorganic molecules. Those practiced in the art of combinatorial chemistry can accomplish the synthesis of combinatorial chemical libraries using two general methods. These methods are known to those skilled in the art as “spatially-addressable” methods and “split-pool” methods. It is common to practice these methods using solid support chemical synthesis techniques as discussed by Gordon, et al.
A common feature to the spatially-addressable combinatorial library methods is that a unique combination of monomers is reacted to form a single oligomer or compound or, alternately, set of oligomer or compounds at a predefined unique physical location or address in the synthesis process. An example of the spatially-addressable method is provided by Geysen et al. and involves the generation of peptide libraries on an array of immobilized polymeric pins (a solid support) that fit the dimensions of a 96-well microtiter plate. A two-dimensional matrix of combinations is generated in each microtiter plate experiment, where n×m unique oligomer or compounds are produced for a combination of n+rn parallel monomer addition steps. The structure of each of the individual library members is determined by analyzing the pin location and the monomers employed at that address during the sequence of reaction steps in the synthesis.
An advantage of this method is that individual oligomer or compound products can be released from the polymeric pin surface in a spatially-addressable manner to allow isolation and screening of each discrete member of the library. Another advantage of this method is that the number of solid supports required is equal to, i.e. no larger than, the number of library members to be synthesized. Thus, relatively large quantities, i.e. micromolar quantities, of individual library members are synthesized in a practical manner using this method.
Related to the Geysen pin method are the parallel synthesis methods which use a reaction vessel system such as that practiced by Cody, et al. This is the practice of distributing a quantity of solid supports, such as chemically-derivatized polymeric resin beads (namely those of the composition polystyrene, polystyrene grafted with polyethylene glycol, or polyacrylimide, etc.) in a two dimensional matrix of n×m individual reaction vessels allowing the parallel addition of a set of n×m reactive monomers to produce a set of n×m oligomer; or compounds. This spatially-addressable method has advantages similar to that of Geysen, et al. Thus, individual oligomer or compound products can be released from the solid support in a spatially-addressable manner to allow isolation and screening of each discrete member of the library. Additionally, the number of solid supports required is equal to, i.e. no larger than, the number of library members to be synthesized. Thus, relatively large quantities, i.e. micromolar to millimolar quantities, of individual library members also are synthesized in a practical manner using this method.
Another example of a spatially-addressable method is the photo lithographic method for synthesizing a collection oligomer or compounds on the chemically-derivatized surface of a chip (a solid support) provided by Fodor et al. A variety of masking strategies can be employed to selectively remove photochemically-labile protecting groups thus revealing reactive functional groups at defined spatial locations on the chip. The functional groups are reacted with a monomer by exposing the chip surface to appropriate reagents. The sequential masking and reaction steps are recorded, thus producing a pre-defined record of discrete oligomer or compounds at known spatial addresses in an experiment. An advantage of this method is that binary masking strategies can be employed to produce a unique oligomer or compounds for n masking and monomer addition cycles. Two important disadvantages of this method are that a) relatively minute quantities are produced on the surface of the chip and; b) release and isolation of individual library members is not technically feasible.
Split-pool combinatorial library methods differ from spatially addressable methods in that the physical location of each unique oligomer or compound is not discrete. Instead, pools of library members are manipulated throughout the experiment. There are two major categories of split-pool methods currently in practice. These are: 1) deconvolution method S7 pioneered by Furka et al. and Houghten, et al. and 2) encoded methods by Gallop et al., Still, et al. and others.
It is common in the practice to employ solid support-based chemistry for these methods. A collection of solid supports are split into individual pools. These pools are then exposed to a series of reactive monomers, followed by a recombination step, in which the position of all solid supports is randomized. The solid supports are then split into a new set of individual pools, exposed to a new series of reactive monomers, followed by a second recombination step. By repeating this split, react and recombine process all possible combinations of oligomer or compounds from the series of monomers employed are obtained, providing a large excess of solid supports are utilized.
The number of oligomer or compounds obtained in an experiment is equal to the product of the monomers employed, however, the number of chemical transformation steps required is only equal to the sum of the monomers employed. Therefore, a geometric amplification of oligomer or compounds is realized relative to the amount of chemical transformation steps employed. For instance, only nine (9) transformation steps were employed using three (3) amino acid monomers in a three step process for the combinatorial synthesis of 27 peptide oligomer.
The prior art split-pool methods produce pools of oligomer or compounds as a product of the experiment. Therefore, the identification of a specific member of the library is typically found by screening the pools for a desired activity, biological or otherwise. The disadvantages of the deconvolution split-pool methods are that (a) the technique always requires that large mixtures of oligomer are screened in bioassays, (b) sequential rounds of resynthesis and bioassay are always required to deconvolute a library, and (c) since single oligomer are not produced a library is always stored as a mixture, requiring later deconvolution.
In the practice of encoded split-pool methods physical separation of the solid support is required to accomplish two tasks: first, to physically isolate the individual library member after screening and, second, to de-code the identity of the tag and thus deduce the chemical structure of the member. A disadvantage specific to the chemically encoded split-pool methods is that chemical tags introduce potential side reactions and failures both with orthogonal linkers and with tags, thus requiring compatibility between the tag chemistry and the chemistry utilized to synthesize the combinatorial library.
In practice, both categories
Baiga Thomas J.
Maiefski Romaine R.
Moran Edmund J.
Baker Freling E.
Brown Jay M.
Ontogen Corporation
Ponnaluri Padmashri
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