Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-08
2004-08-24
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S025300, C536S026600, C435S006120
Reexamination Certificate
active
06780981
ABSTRACT:
This invention concerns reagents of the kind which comprise a product which is built up using stepwise reactions often chemical reactions, and associated tag moieties which track the synthetic pathway and/or the reagents used. The product will often be an oligomer and the tags define the identity and position of at least one monomer residue in the oligomer. Such reagents are useful in assay methods in which they can generate much more information than can be generated by a simple labelled analyte. Sets and libraries of such reagents can be created by combinatorial chemistry and are valuable for screening large numbers of compound e.g. for biological activity. In preferred systems according to the invention, positively charged tag groups are generated for analysis by mass spectrometry by cleavage, e.g. photocleavage of neutral molecules.
WO 95/04160 describes a reagent which comprises:
a) an analyte moiety comprising at least two analyte residues, and linked to
b) a tag moiety comprising one or more reporter groups adapted for detection by mass spectrometry, wherein a reporter group designates an analyte residue, and the reporter group at each position of the tag moiety is chosen to designate an analyte residue at a defined position of the analyte moiety. A plurality of such reagents, each comprising a different analyte moiety, provides a library of reagents which may be used in assay methods involving a target substance. Analysis of the tag moieties indicates the nature of the analyte moiety bound to the target substance.
WO 94/08051 describes a system used to make simultaneously a library of all oligomers each attached to a bead. Any individual bead made by a split and mix process carries a unique chemical product, and this is true of each bead which goes through the same synthetic pathway. Two coupling steps are used at each point in the process: one step affects the synthon or ligand; the other alters the structure of a tag which is also carried on the bead. Tags are designed to identify the steps through which the bead has been taken.
It is an object of this invention to provide a set or library of labelled compounds which may be synthesised on a support and may be used either attached to or separated from that support.
In one aspect the invention provides a method of making a set of labelled compounds, by the use of a support and a set of labels, which method comprises the steps:
a) at least one first or intermediate step comprising dividing the support into lots, performing a different chemical reaction on each lot of the support so as either to modify that lot of the support or to couple a chemical moiety to that lot of the support, tagging a fraction of each lot of the support with a different label, and combining the said lots of the support, and
b) at least one intermediate or final step comprising dividing the support into lots, performing a different chemical reaction on each lot of the support, so as either to modify that lot of the support or to couple a chemical moiety to that lot of the support, tagging a fraction of each lot of the support with a different label, whereby each different label is linked to a chemical moiety coupled to the support in a different step and forms with that chemical moiety a labelled compound which is separable from the support, and combining the said lots of the support.
The method uses a support which is repeatedly divided into lots which are then recombined. The support may be a massive support e.g. a flat sheet or silicon chip or microtitre plate which is divided e.g. by masking into regions for performing the different chemical reactions. The support may be a polymeric material which is soluble in some solvents and not in others, and which is separated into lots or recombined e.g. by precipitation or dissolution. Most usually the support will be particulate, e.g. pins or fibres or capillaries or preferably beads. Derivatised beads for performing combinatorial chemistry by a split and mix strategy are commercially available and can be used here. A preferred particulate support comprises beads having cleavable linkers, wherein each cleavable linker has one group for defined chemical procedures e.g. oligomer synthesis and another group for labelling. By this means it is possible at the end of the synthesis, to recover the labelled chemical products e.g. oligomers into solution.
The method of the invention involves performing at least one step a) and at least one step b), most usually at least three steps in all. Each step involves performing a reaction, generally but not necessarily a chemical reaction. An example of such a reaction might be the removal of a protective group so as to leave a primary amine or hydroxyl or carboxylic acid group. Most usually, the chemical reaction involves coupling a chemical moiety to the support. The chemical moiety will usually be an organic chemical group, for example as described in WO 94/08051. While successive chemical moieties may be attached to the support through separate linkers, more usually, successive chemical moieties will be joined to each other to form a chain extending from the support. Preferably the chemical moieties are monomer units which are built up to form oligomer chains.
In a preferred method according to the invention, the set of labelled compounds is a library of n
s
labelled oligomers, where n is the number of different monomer units and s is the number of monomer units in each labelled oligomer, wherein step a) is performed once to couple a different monomer unit to each lot of the support, and step b) is performed s−1 times.
The oligomer may be for example an oligonucleotide or an oligopeptide. When the oligomer is an oligonucleotide or analogue, then n is generally 4. When the oligomer is an oligopeptide, then n is generally about 20 when only natural amino acids are used. But the principles of the invention are equally applicable to other oligomers formed from other polymerisable monomers. The value of s is not critical, and may typically be from 2-100 e.g. 3-20 or more.
The fraction of each lot that is tagged in each step is generally less than 50%. Preferably from 0.25% to 25% of each lot of the support is tagged in each step with a different label. Preferably the support has cleavable linkers, wherein each cleavable linker has at least one group for chemical reaction e.g. chemical synthesis and another group for labelling. Preferably each resulting labelled compound comprises a single label and at least one chemical moiety.
The method involves the use of a set of up to and including n×s different labels. Although the nature of the labels is not critical, it is a preferred feature of the invention that each different label be distinguishable by the analytical procedure used to detect the labels. Groups used as labels should be much more stable to acidic (or other chemical) treatment involved in oligomer synthesis compared to the protecting groups commonly used (e.g. DMT groups to provide 5′ or 3′-protection in nucleotide synthons). Preferred labels are those in which a charged group, preferably a positively charged group is generated by cleavage e.g. photocleavage of a neutral molecule for analysis by mass spectrometry. Examples of such preferred labels are discussed below.
In a preferred embodiment, a split and mix strategy requires a solid support carrying cleavable linkers with three arms—one to attach to the solid support through a cleavable bond; one to initiate synthesis of a chemical product e.g. oligomer; and a third for attachment of the tags. The sites for coupling of synthon and tag monomers will optionally be protected by removable groups. The process can be illustrated by the synthesis of oligomers on a particulate solid support.
At each stage in the synthetic route, the particles of the support are first combined and mixed, and then divided into n lots, where n is the number of different monomers—4 in the case of natural nucleotides—and each monomer is coupled to its site on one lot of the support. A unique tag representing th
Elder John Kenneth
Hamilton Alan Lewis
Housby John Nicholas
Shchepinov Mikhail Sergeevich
Southern Edwin Mellor
Oxford Gene Technology IP Limited
Riley Jezia
Smith DeAnn F.
Zacharakis Maria L.
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