Grafted cross-linked polyolefin substrates for peptide synthesis

Details Grafted cross-linked polyolefin substrates for peptide synthesis Grafted cross-linked polyolefin substrates for peptide synthesis

1995-12-18

1999-03-23

Mullis, Jeffrey

Synthetic resins or natural rubbers -- part of the class 520 ser

Synthetic resins

Mixing of two or more solid polymers; mixing of solid...

5253333, 525374, 436518, 436531, 436532, 436535, C08F25502, C12Q 100, G01N 33545

Patent

active

058861041

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention concerns a solid support for the solid-phase synthesis of, in particular, peptides or proteins in high yield and in high purity. The support is well suited both to the synthesis of a single peptide or protein and to the parallel and substantially simultaneous synthesis of a plurality thereof. More specifically, the invention relates to a solid support based on a cross-linked polyolefin substrate, especially a cross-linked polyethylene substrate, to which are grafted polymer chains such as polystyrene chains, the polymer chains being functionalized with a chemical functionality facilitating the formation of an anchoring linkage between the polymer moiety and another chemical species, the polymer chains such as polystyrene chains optionally further bearing substituents which are not reactive under the conditions prevailing in the synthesis.
Syntheses employing the substrate of the invention can make use of established chemical methodology, and the substrate is well suited to syntheses on an analytical (microgram) scale or on a preparative (milligram or larger) scale. Furthermore, the substrate of the invention can be employed in both batchwise and continuous-flow procedures which are performed manually, semi-automatically or fully automatically.
In addition, the solid supports of the invention are particularly well suited for use in solid-phase biosystems, notably bioassays, such as immunoassays, DNA hybridization assays or PCR amplification.


BACKGROUND OF THE INVENTION

Until quite recently, solid-phase methods for the synthesis of peptides or proteins have to a large extent been based on the original methodology developed by Merrifield, employing a functionalized cross-linked styrene/divinylbenzene copolymer, the cross-linked copolymer having been formed by the polymerization of styrene monomer to which has been added a few per cent (typically about 2%) of divinylbenzene. This copolymer is generally provided in the form of beads or particles, often with a dominant particle size of 20-80 .mu.m. The functionalization originally functionalization of the aromatic rings of the copolymer with chloromethyl groups, introduced via reaction of the solid copolymer with SnCl.sub.4 /chloromethyl methyl ether, although a number of other functionalities, including aminomethyl, .alpha.-aminobenzyl and .alpha.-amino-4-methylbenzyl, have subsequently been employed. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminal of the first amino acid which it is desired to couple to the solid support. Later refinements of the Merrifield methodology have included the further introduction, between a functionality (e.g. one of the above-mentioned functionalities) on the polystyrene chains and the C-terminal of the first amino acid which is to be coupled, of a bifunctional "spacer" or "handle" group whose reactivity is tailored inter alia to meet desired requirements with respect both to the coupling of the first amino acid to the solid support and/or to the ease with which the completed, synthesized peptide or protein chain is cleaved from the solid support. Examples of such spacer groups include the phenylacetamidomethyl Protein Res. 30, 705-739 (1987)! have reviewed the development of solid-phase peptide synthesis methodology from its introduction by Merrifield up to about 1986.
The advances in biotechnology which have been made in the last decade or so, particularly in the area of recombinant DNA, have produced a situation in which vast numbers of new protein sequences with undefined or unknown function and/or unknown biological activity have become available. In this connection, detailed structural analysis by site-directed mutagenesis or similar molecular engineering techniques has provided a useful approach to the study of the roles of amino acid residues in active sites of proteins.
However, specific information concerning biologically active functional subunits (peptides) containin

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patent: 4908405 (1990-03-01), Bayer et al.
patent: 5133878 (1992-07-01), Gsell et al.
patent: 5258454 (1993-11-01), Berg et al.

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