GB2342350A - Solid supports containing oxazole scintillants - Google Patents

Abstract

A support for a chemical or biological application having chemically reactive sites, the support formed from at least one scintillant monomer by polymerisation or copolymerisation of the monomer. The monomers are preferably 2,5-diphenyl-4-substituted oxazoles where the substituent of the 4-position is vinyl, 4-vinylbenzyl, allyoxymethyl, 4-vinylphenoxymethyl or 4-vinylbenzyloxymethyl. The supports may be used for biological scintillation assays, but also find new uses in solid phase synthetic chemistry and combinatorial chemistry.

Description

2342350 SOLID SUPPORTS CONTAINING SCINTILLANT The present invention

relates to SC4 ntillant polymers.

Scintillant polymers have many applications. Examples are their uses as solid supports in chemical and biochemical applications and as solid supports for chemical and/or biological reactions. The uses o-f the scintillant polymersdisclosed herein are not limited --o the uses exemzlified.

Solid supports for use in chemical and biochemical applications are well known. Solid supports may be formed from a polymeric material such as a resin and, in such cases, will have the overall structure of a polymer matrix. Polymer resins of the type described above are termed solid supports since they contain covalent cross-links between their constituent polymer chains and are, therefore, insoluble in all solvents. Solid supports may be porous. They may be in the form of solid beads of any desired diameter, for example in the form of microspheres typically between 5-500,pm in diameter; films; or a surface layer disposed, for example, on a microtitre plate or multipin synthetic apparatus.

In particular the present invention relates to supports incorporating a chemical group that can scintillate, ern-tting visible or otherwise detectable radiation, so as to aid in monitoring molecular interactions in chemical or biological systems or the progress of chemical or biolooical reactions. The present invention also rela-es to a method for synthesis of such solid supports and their use in biological and chemical assays and for the synthesis and deconvolution of solid phase combinato-r- Jal chemistry libraries.

SC4 ntillation can be defined as a flash of light produced in I phosphor by an ioniS4 ng particle such as a beta particle or photon. The term. phosphor can be further defined as a phosphorescent or fluorescent molecule and in the text of the present application the terms "scintillant molecules" and -scintilllan-z moieties" will be used to define molecules that react in this way, or a molecule that has a functional group W 4 z-.at reacts in this way, and "scintillation" ill be taken to mean 1--ght produced by such a scinti-lant molecule.

-he _ion phenomenon 4S scintillat used in scintillation proximity assays (SPA-s), where the light. emitted by the scintillant rolecule is detected and quantified in an appropriate scintillation counter. The SPA may be used to determine whether or not two different molecules interact. For example, if a biological receptor molecu-e is attached to a support such as a SPA bead (a solid support that contains scintillant molecules) and then placed in an aqueous solution containing a rad 4 clabelled molecule, there may be binding of the radiolabelled molecule to the SPA labelled biological rece7oto--. If such a binding interaction occurs, t,) e rad-clabe- is brought into close proximity with the scintillant molecules contaned in the bead. Radioactivity produces -Jon-sing particles and the close proximity of the scin-:illant and ion-ising radia--ion results in emission of light. Tritium is used routineily as the radiolabel and emits 4onising radiation with a -very short path length; for example n water the average path length is 1.5 pm. If the distance between the tritium and scJLntillant molecules is areater than 1.5 '4m no significant sc-ntil'at-on will result.

Consequen--'y, _Jf there is no bin-_J_ng interaction between the SPA labelled receptor molecule and tritiated molecule, the ty of tritium remains ma_j or too remote from the scintillant molecules to cause scintillation. Thus scintillat=, or the a-mount thereof, can be used to determine the extent of binding between the scintillan-: -'ab-e-'ed recer:)tor molecule and 3 radiolabelled molecule. Such SPA results are quantitative, the degree of scintillation being dependent on the extent of the binding interaction.

European Patent 0 154 734 describes an immediate ligand detection assay marketed by Amersham as the Scintillation Proximity Assay, and describes a process for integrating fluorescent molecules, including 2,5diphenyloxazole, 'n-0 support bodies such as cyanogen bromide activated Sepharose 4B beads. The pores within the Sepharose beads are impregnated with fluorescent molecules via a precipitation process. Specifically, a DMSO solution of the fluorescent molecule is added to the beads so that the pores within the beads become filled with the solution. Addition of water to this system results in the DMSO being washed away from the beads whilst the fluorescent molecules (which are insoluble in aqueous solvents) are precipitated within the pores of the beads. The beads are then coated with a biological receptor molecule through either non- specific, non-covallent interactions or else through covalent bonds via the cyanogen bromide. Beads prepared in -:his manner are incubated with a radiolabelled ligand. The radiolabel is chosen so that it emits beta particles or auger electrons that have a short path length in water. If the receptor binds to the ligand a significant portion of the radioactivity is brought into close proximity with the fluorescent molecules within the pores of the beads, these become activated and emit light. The 1'ght emitted can be detected and quantified directly in an appropriate scintillation counter. Conversely, if the receptor does not bind the ligand the majority of the radioactivity remains too remote from the fluorescent molecules to cause significant amounts of light to be emitted.

A related Amersham patent (EP 0 650 396) describes an extension of the SPA, name-'y the Cytostar-T Scintillating 4 Micronlates. This approach ut-ises microtitre plates which incor-oorate scintillant molecules within the base of each wel w'thin the plate. The surfaces of the well- bases are coated w-'th whole cells. Aqueous solutcns contaning radiolabelled substances are added to each well. if the cell incorporates the radiolabelled substance, the radiolabel is brought into close proximity of the scintillant molecules Within the base of the well and light is emitted. The process may be adapted to srudy loss of sgnal wher radiolabeLled compounds are released from the Cells. 7he scintillating properties of the plates have also been used to develop an in situ assay for mRNA. Scintillant containing microtitre plates are also available from NEEEN Dupont (known by the mark Flashplates).

The receptor molecule can only be attached to the surface of such known scintillant beads using aqueous solvent systems since addition of organic solvents to the bead would result n the fluorescent molecules beina dissolved and removed from the pores within the beads. Where scintillant microplates are used, the plates themselves are constructed from chemically un-f-anctionalised polymeric materials which incorpcrate scinz-ilant and which are inccmpatitle with the use of most orc[aric solvents. This restriction to aaueous solvents limits the applications of these known scintillant beads and plates.

The scintillation proximity assays described above have been used to study many biological receptor/ligand interactions (PC-- Application Nos WO/98/158.4; WO/98/03654; WO/97/47750; WO/97/45745; WO/97/28281; WC/97/26332; WO 9-7/10502; wo/196/21, 156; WC/96/ 13, 258; WC/93/191'15; WQ/90/11524; Eurc-pean patent publication Ncs EP -/ E'I, 8 53; EP 656, 422; EP 378, 059;and US Patent 7pp_' cation No. -768, 652 in eac n case the assay procedure is necessarily carried out in aqueo',s so-vents to oreven-_ 'eaching c-' the scirtillan, molecules into the surrounding solvent. Even in aqueous solvents, leaching from the scintillant containing beads has been reported (Bosworth, Towers; Nature; 341; 67 (1989)).

Yttrium silicate doped with rare earth elements is an inorganic based scintillator, and has been used'in beads which may be coated with biological receptor molecules and derivatised. Beads of this type are now used in many SPA basect applications (Amersham Pharmacia Biotech Catalogue 1998). However, yttrium silicate is not used as a solid support for solid phase synthetic organic chemistry.

The use of presently available scintillation proximity assays in the study of biological receptor/ligand reactions is further restricted by the need for a radiolabelled ligand. if the SPA assay is to be used to screen a number of potential ligands for binding ability to a specific biological receptor, either each potential ligand must be available in radiolabelled form, or a known radiolabelled ligand must be already available for displacement studies. This is potentially very expensive and may be extremely difficult to achieve.

Further, not all receptor molecules may be at--ached to known SPA beads.

Presumably, because of these drawbacks and others, there are still only a relatively small number of biological receptor SPA assays commercially available (21 in the 1998 Amersham Pharmacia Biotech Catalogue).

Though there are hundreds of commercially available solid supports for use in solid phase synthetic chemistry and combinatorial chemistry, it is impossible to use known SPA supports (such as beads) in solid phase synthesis 6 applicatJons, because their use wth organic solvents wil' cause leaching of --he molecules, as outlined above.

Aecc)rding to the present invention there is provided a support .Z for a chemical application, the support comprising a polymer matrix with a scintillant moiety covalently bonded into the polymer matrix.

By chemical application, it will be appreciated that any chemical, biochemical, or biological application is meant. The supports may be used in any application where a conventional support might be used. The supports may be used in, for example, monitoring molecu-1ar interactions in chemical or biological systems, monitoring the progress of chemical or biological reactions, bioassays and the like.

The scintillant moiety forms an integral part of the matrix of the support, as opposed to being grafted or tagged onto the surface cf the support as a layer. The scintillant moiety is preferably subszantially uniformly dist--'buted throughout the polymer matrix.

The amount of scintillant. may be, for example, 5-10 mol %, though it cou"- be higher. Much lower concentrations of sc.ntillant also give efficient scintillation. The resu-ting beads have a relatively large number of scn-illanz moieties and are sensitive to low levells of radioactivity. There is a further advantage in that the significance of scintillant cation of - s quenching eve-its in the appl- he supports r' ve7Y - e.a-- ow.

Supports acccrding to the -invention do not fall prone to leaching when used:'.n organic solvents.

By polymeric:-,atrix it is meant a large scale solid structure 7 made of polymer chains, these polymer chains being made up of smaller chemical units called monomers. There may be crosslinking, to varying degrees, between polymer chains, but this cross-linking is not essential. The chains and cross-links are not necessarily in defined or regular positions; the arrangement of monomers or the overall structure is not. necessarily regular, and no crystallinity is implied.

Preferably, the support of the present invention is formed by a polymerisat,..on reaction. The polymerisation may be achieved by any conventional polymerisation method.

The supports may be used as supports for chemical reactions, in which case the polymer matrix will also have chemically reactive site(s). By chemical reaction, it will be appreciated that any chemical, biochemical, or biological reaction is meant; it will similarly be appreciated that the term "chemically reactive site" may be taken to mean a site capable of binding/reacting with a chemical, a biological or biochemical molecule. It will be appreciated by one skilled in the art that- the present invention is not limited only to the purely chemical field and will find much use in associated biological, pharmaceutical and biochemical fields.

The chemically reactive site may be incorporated into the polymer matrix. The chemically reactive site may be present as an integral part of the polymer matrix. Preferably, the chemically reactive site may be distributed substantially uniformly throughout the polymer matrix. If a support is made by a polymerisation reaction, the chemically reactive site may be incorporated during a polymerisation reaction ster- Prior to the polymerisation, the chemically reactive site may be disposed on a monomer C'the chemcally functionalised monomer"). On polymerisation, the chemically functionalised monomer will copolymerise with any other 8 monomer(s) and reagent(s) present and the polymer matrix will thus be formed w-it^- integrated c:'-em-cal",v reactive sizes. Sites incorporated this way may be distributed substantially uniformly throughout the polymer matrix, or, if desired, the polymerisation reaction may be control-led so as to restrict/ localise lihe distribution of chemically reactive sites to one area, for example the surface of the matrix. A chemically functionalised moiety may also be incorporated subsequent to support formation using conventional solid phase synthetic techniques and reagents. It will be appreciated that on a molecular scalle, the surface of the support polymer matrix may include cavities, channels and pores which will increase the surface area of the matriix/support available as a reaction surface as this "internal" surface 's the surface that is accessible to solvents. The chemicallv reactive size may be formed at the surface of the matrix as a layer in the sense that it is disposed over the full surface area, that is disposed over both the exposed surface and the "internal" surface: the cavities, channels and pores. The chemically reactive sites may be formed as a discrete external layer over surface of the support.

These chemically reactive sites enable me-ecules to be bound covalently to the support, in contrast to the support of the invention with no chemically reactive site, which can be coated with molecules only through ncn-specific non-covalent Jnteractions such as hydrophil-ic/hvdro,-hob--c or electrostatic interactions. Chemically reactive supports may be used in receptor -Lmmob-1-isat-Jon, in sclid phase synthesis and i, combinatorial chemistry.

Preferably, further additives may be the support. These mav be -incorporated by polymerisation or any other conventional reac--icn. The additives may be, for example, porcgens and/Or templating molecules as described 9 more fully hereafter.

Preferably, the support is in the form of a bead. Various diameter beads may be formed, depending on the reaction conditions of the synthesis. The polymerisation process may synthesise an assortment of different size beads. These may then be collected and divided by sieving. The bead may be of any diameter. Preferably, the bead is of diameter in the range 5 1m to 1 cm. Typical diameters might be in the ranges 37-75 im, 7590 lim, 90150 pm, 150-300 lim and 300-500 pm. Alternatively, the polymerisation may produce a uniform bead size. Beads can also be produced of a size adequate for a single bead assay. The scintillant beads may have high levels of polymer cross linking within the matrix (>5% and typically 20%), or low levels of cross linking (<5%) The latter are termed gel type polymers.

The support may be disposed as a layer in a reaction vessel surface, such as a microtitre plate. For plastics materials including microtitre plates, the most preferable physical characteristics would be for the polymer to be highly crosslinked. Other possible applications are as films and multipin synthetic apparatus, and for these latter applications, gel type polymers and highly crosslinked macroporous plastics are desirable.

The supports of the invention may be used to examine the interactions between molecules which are either non-covalentlv or covalently linked to the surface of the sup-port, and molecules free in a solution which contacts the support. TI, i e molecules free in solution must contain some form of activator to cause scintillation on said intermolecular interaction, for example a radiolabel. The supports may also be used to detect interactions between the surface of the solid support itself and molecules free in a sol------n which will come into contact with the support, the -free molecules having an activator, f-Cr examcle a radiolabel, to cause scintillation on i4 nteraction with the support.

- n further applications of the invention, supports of the invention containing chemically reactive sites may be used as solid supports in solid -chase synthetic chemistry and solid phase combinatorial chemistry.

In a further application of the invention, the chemical modificat 14 on of a molecule covalently attached to a support according to the invention may be studied, provided that the said molecule contains a radioisotope or other label which may be used to activate or in any way alter the properties of the scintillant moieties.

In a further aspect of the invention, a support for a chemical or biological application is -formed from a scintillant monomer (a monomer comprising a scintillant moiety). Examples o4scintillant monomers are described below. The support may be formed by polymerisation of one or more scin-zillant monomer (s), or copolymerisation of one cr more scintillant monomer(s) with at least one additional- monomer.

The additional monomer may be a monomer which comprises a chemically functionalised site - "a chemically funct'cna'Lised monomer". Examples of chemically functionalised monomers are 6-chicrc.--Lethyl--,i--nylbenzene and:D-acetoxystyrene.

The aaditicnal monomer may be a monomer which increases the bulk volume of the Dolymer matrix formed in the polymer'sation reaction. Although such monomers react to form polymers or copolymers, they will not show a high decree of chemical reac-r-ivity once (co)polvmerisation has occur- red, and they will be termed hereafter "iner-:i mcnomers". Examples of such I I monomers are styrene and 4-ethvlvinvlbenzene.

Preferably, the support is formed by copolymerisation of a scintillant monomer and a chemically functionalised monomer. The support may also be formed by copolymerisation of a scintillant monomer and more than one chemically functionalised monomer. Instead, a support may be formed by copolymerisation of a scintillant monomer and an inert monomer. The support may also be formed by copolymerisation of a scintillant monomer and more than one inert monomer. A further preference is for a support formed by copolymerisation of a scintillant monomer, a chemically reactive monomer, and an inert monomer. Any number of monomers (of any type) may be polymerised with at least one scintillant monomer to form a support according to the invention.

On polymerisation, the scintillant monomer will copolymerise with any other monomer(s) and reagent(s) present and the polymer matrix will thus be formed with integrated scintillant sites. Sites incorporated in this way may be distributed substantially uniformly throughout the polymer matrix, or, if desired, the polymerisation reaction may be controlled so as to restrict/localise the distribution of scintillant sites to one area, for example the surface of the matrix.

A cross linking agent may be used in the polymerisation. The cross linking agent may be a monomer. An example of a cross linking agent is divinylbenzene. Increased cross linking will reduce the likelihood of the support dissolving in organic solvents. The polymerisation may be of the scintillant monomer only, but in this case it might be highly desirable to add a cross linking agent.

-he scintillant monomer may be chemically functionalised to produce a support with chemically reactive sites. For 12 example, any of the scintillant monomers described hereafter may be chemically JLunctionalised by substituting a chemically functionalised group onto one (or both) phenyl groups of the dir)henvloxazo-e mcietv.

The scintillant monomers may be used to construct specifical-,v shaped supports, or may be disposed as a layer on a support or on a reaction vessel surface.

According to the present invention in a further aspect, there is provided a scintillant monomer comprising a scintillant moiety and a separate polymerisable moiety. Preferably the separate polymeri sable moiety includes an alkene group. Preferably these are dis--ant from each other within the scintillant -.ionorrer, so as to prevent electron delocalisation within the scintillant moietv being disrupted by th e polymerisation reaction. Any such disruption during polymerisation may have a detrimental effect o r. the scIntillant activity of the support.

The scintillant monomer comprises a molecule of structure:

R-Y; wherein R is a scint-lant group; and Y is a group which includes a polvmerisable moietv.

Preferably R --s a 2,5-d_Jphenyloxazcle group. Preferacly Y -s a substituted or unsubstituted aliphatic or aromatic grout); or an ether.

Preferably the scin--illant monomer has one of the following structures (-,, -:5) 1 13 ^'//11 X 0 N 0 N- 0 0 0 A 3 A A 2 N 0 0 4 According to the invention in a further aspect there is provided a scintillant polymer formed by polymerisation or copolymer i sat ion of at least one scintillant monomer. The scintillant monomer comprises a scintillant moiety and a separate pclymerisable moietv. Preferably, the scintillant monomer is one of structure (1) to (5).

According to the present invention in a further aspect there is provided a method of preparing a scintil'ant polymer comprising the steps of [a] taking at least one scintillant monomer, and [b) effecting a polymerisation step.

The scintillant polymer may instead be formed by 14 copolymerisation of a scint_J..'ant monomer and at 'east one additional monomer.

Jon According to 7-he present invent M a further aspect there is provided a method of preparing a scintillant polymer comprising the steps of [a] taking at least one scintiilan- [b] effecting mcnomer and at east one additional monomer, and a polymerisation step.

Anv conventiona- polymerisation process may be used to produce -he scintillant polymer: bulk, susnension, emulsion and solution reactions are all suitable.

structure Preferably, the sc ntillant monomer is one o to (5). The additional monomer may be a chemically functonalsec monomer. The additional monomer may be a monomer that increases the bulk volume of the polymer matrix formed in the polymerisation reaction: "inert monomers", as described hereinbefore. Examples of chemically funczionalised monomers are 4- chloromethylvinylbenzene and p-acetoxystyrene. Examples of fnert. monomers are styrene, 4-ethylvinylbbenzene and divi4nylbenzene.

Scintillant monomers of the invention, and the methods of produ=g scintiant polymers and scintillant supports according to the inven-r_on, may be used to prepare a support for a chemica" aDi:)--icat4on or a chemical reaction.

-he support may be in the form of a bead. The bead may be of diameter 5im, to lcm. The suppor- may be a gel- type Polyl-I.er support. The support may have macro-ocrous structure.

Any conventional polymerisation process may be used to produce the scintil-lant support: bulk, suspension, emulsion and solution polyMerisation reactions are all suitable.

Consequently, scintillant supports with a variety of physical properties and forms can be produced. Beads may be formed by a suspension copolymerisation in which one of the monomers is a scintillant monomer.

Preferably, the support is formed by copolymerisation of a scintillant monomer and a chemically functionalised monomer. The support may also be formed by copolymerisation of a scintillant monomer and more than one chemically functionalised monomer. A support may instead be formed by monomer and an inert copolymerisation of a scint-L monomer. The support may also be formed by copolymerisation of a scintillant monomer and more than one inert monomer. A further preference is for a support formed by copolymerisation of a scintillant monomer, a chemically reactive monomer, and an inert monomer. Any number of monomers (of any type) may be polymerised with at least one scintillant monomer to form supports according to the invention.

Preferably, the scintillant monomer is one of structure (1) to (5). Examples of chemically functionallised monomers are 4chloromethylvinylbenzene and p-acetoxystyrene. Examples of inert monomers are styrene, 4-ethylvinylbbenzene and div-inylbenzene.

-he polymerisation reactions described above may be carried out either in the presence or absence of chemical cross-linking agents such as divinylbenzene. If no cross-linking agent is used, the resultant polymers may be soluble -n organic solvents. However, if a cross-l-inking agent is used, the resultant cross-linked polymers contain covalent linkages between their constituent polymer chains. The degree of crosslinking in a solid support varies its properties considerably. A solid support W4 th a low degree of cross-linking (typically <59.) --may swell considerably in some organic solvents (but not 16 act.ially disso ve) and is termed a ge -type solid support.

d supporzs do not swell cc7.tras-:, hign-y cross-linked sol i n crganic solve-i--s.

A porogen may sc.-aetimes be added to a polymerisation reaction. An example of a porogen molecule is toluene. The poroger, is chemically inert to the polymerisation react'-on conditions and is used to introduce pores into the product polymer. After the polymer-sation reaction, the pcrogen _Js easily removed from the product polymer, by, for example soxhlet extraction. Solid supports constructed in the presence of a porogen are said to be macroporcus. The resultant polymers scintillate in the presence of 140-4 ,sing radiatLcn.

A template molecule may be added to the polymerisation reaction. This is done to imprin7 locations /environments with known electronic and/or structural identitv (that is, the identity of the template) into the support during the polymerisation. Once polymerised, the template is incorporated into the suiDDort. The template may be bound into the support in a covalent manner or non-covalently. To leave the template f known electronic and/or structural identity, the locations of template is removed. An example of a template molecule would be cho-esterol. The template molecule may added as a free molecule, or as a de-rivat-ve. If the template is added as a free molecule, it may be removed by washing. A derivat-ve may comprise the temp-ate mo-ecule and a pollymerisable site. In is cova the latter case, the template lently bound into the support, and w-J-1 have to be removed by ceaving the template molecule from the polymerisab e s_ze.

7 n a further applicaticn of the in-rentiion supports comprising sc-int-llant monomers and chemically reactive sites may be used as solid suppcrts in solid phase synthetic chemiszry and sol-d Phase combinatoriachemis--rv.

17 According to the invention in a still further aspect, there is provided an assay incorporating the steps of:

[a] providing a support for a chemical applicaton comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scintillant moiety being covalently bonded into the polymer matrix; m'xing the support with a molecule compris;ng an activating group and a site which may react with the reactive site on the support; and [c] measuring the scintillation produced by the scintillant moiety.

The activating group may be, for example, an a emitter, emitter, or an Auger electron emitter. Preferably the Jonising group is a radiolabelled group, such as a tritiated group or a group labelled with 1151, I'S or 33P.

Throughout the specification, mention is made of causina scintillation by the close proximity of a radiolabelled molecule. The present invention is not limited to the use of radioisotope labelling; anything which activates the scintillant molecule or in any way alters its fluorescent or scintillant properties may be employed.

Preferably, the support is a bead. The assay may be performed using a single bead, or many beads.

The supports of the invention are particularly suitable for use in solidphase synthetic chemistry. Solid phase synthetic chemistry has been known for many years. Conventionally, a sol'd support is used as a support for a stepwise synthesis 18 of a molecule. As a first step, a "base"-reactant molecule 4s c-,valently bound to the support at one position and remains so bound during each step of --he synthesis scheme. The desired reaction takes p-ace a, one or more chemicall-,; react've location(s) elsewhere on the reactant molecule. Th4s covalenz attachment between the base molecule and the support means that after each successive chemical react-on, the reaction solvent, anv unreacted reagents and any reaction byproducts can be removed simply by washing the solid suppor-with an appropriate organic solvent, while the reacted base molecule remains bound. All reagents can be employed in excess and thus all of the chemical reactions can be driven,o completion.

C field's resin, Wang

Conventional solid supports include Merri resin, Rink res-,.n, Sieber resin and PEG polystyrene resin.

th SPA techniques as they These are not suitable for use w. 'llant moiecontain no scint ies.

According --o the invention in a still further aspect there is provided a suppor-- for use in solid phase synthetic chemistry comprising a polymer matrix wizh at least one scintillant moiezy ccvalenty bonded in-:o the polymer matrix, and at least one chemically react-ve site.

It w--,-' be possible to attach virz,,.:allY any molecule zo these supports by 1-nks which are specific and covalent 'n nature.

Preferably the support is in -he form of a bead. The bead may be of diameter -= to lc,-,i. The bead may be a gel type po' yn.ier. The bead may have macroporous structure. The s-=cture mav be highly polymer crosslinked.

The syn-E.re--ic chemistry may be performed using a single bead, 19 or many beads.

The support may be a gel type polymer.

The supports of the invention may be used in combinatorial chemistry.

This is a branch of synthetic chemistry which centres on the simultaneous production of large numbers of structurally related compounds: "a library". The library may include positionally fixed components. The compounds within the library are screened simultaneously to determine if one or more compounds exhibits a desired property, for example, the ability to bind to a biological receptor molecule. -If this is found to be the case, it is then necessary to identify the chemical structure of the 'active' compound (s). The process whereby the chemical structure of the active compound(s) is deduced is termed deconvolution, and is the crux to any successful combinatorial chemistry strategy.

In Solid Phase Combinatorial Chemistry, library compounds are synthesised on the accessible surfaces of a chemically reactve solid support, such as a polymeric resin bead.

By Jar the most common solid phase combinatorial chemistry strategy employs resin beads as the solid support and uses a 'split and mix' method to synthesise the library compounds.

Figure 1A shows a schematic representation of the 'sD-t and mix' method.

The 'split and mix' method also called the 'one bead, one compound' approach enables libraries that contain large numbers 0.0 f compounds to be constructed extremely rapidly. AA -er library synthesis, each resin bead bears multiple copies c--- the same library compound, with different beads bearing different ibrary compounds. The ''brary of compounds is then screened en masse for a desired properity such as the ability to bind to a b-clogical receptor.

In a typical convent-Lonal assay procedure, the beads bear'ng ibrary comDounds are h he 1- incutated wit a dye labelled receptor molecule. Should a library compound bind to the biological receptor, the bead bearing that compound will appear more coloured than the other beads. -Assuming this to be the casef the most intensely co"oured bead is physically removed from the other beads.

It is then necessary to determine the identity of the 'active' library compound attached to this bead. Two commonly used methods are i) positionally fixed library synthesis and ii) encoded library synthesis.

Figure 13 shows a schematic representation of posit"onally fixed library syrthesis.

This approach, is widely used in solid phase combinatorial cheraistry. A library of the type X-X-X-X shown in figure 12, has each position w1th-Ln the library fully randomised and may be for example be one of six different amino acids (A-F",. Rather than svnthesise a single library where each position --s ferent sets of six 'positionally fu-1v random-ised, four diff rst set of sx fixed' sublibrar-es are svnthesised. In the f--' su'-!braries position 1 is fixed as each of the six amino acids in z-,. :rn, whi-Ist p3s-t'Lons 2, 1 and 4 are fully randcm'sed. In the second set, position 2 is fixed as each of the six amino 4s -n turn whils positions 1, 3 and 4 are ful-v randomised. ac-'-- In the third set of six sublitraries, position 3 --'.s fixed and in the final set, position 4 is -fixed. All 24 sunlibraries are then screened for a specific activity. Again, in a typ-Jcal assa-; crocedure, the beads bearing the library --cmpo-.:nds are 21 incubated with a dye labelled receptor molecule, and the assay completed as described above. The most active sublibrary within each set of six indicates the identity of the optimuir. amino acid at the fixed position. The identity of the optimum compound within the library may thus be deduced (in this case C-D-F-B).

In Encoded Library Synthesis, at each stage of library synthesis, a coding molecule is attached to the resin beads. once a bead bearing an active compound has been identified, again, typically by using a dye based assay, as described above, the coding molecule(s) attached to the bead are analysed to allow the code to be deciphered -and thus enable the identity of the active compound to be determined.

Scintillation assays as used in combinatorial chemistry strategies as outlined above are problematic.

Conventional SPA's cannot be used readily with positionally fixed library synthesis or encoded library synthesis, since, in either strategy, all of the solid phase synthetic chemistry steps utilised to construct the library compounds require the use of organic solvents. Conventional SPA beads and scintillant microplates are incompatible with solid phase synthetic chemistry as, for example, they are incompatible with the use of most organic solvents.

in order to use a conventional scintillation type assay with either positionally fixed library synthesis or encoded library synthesis the library compounds must be cleaved from the sol-d support into spatially addressable vessels. Additionally, each library compound itself has to be synthesised in radiolabelled form, (which can be expensive and difficult to accomplish), or a known radiolabelled substrate for the target receptor must be available or synthesised for displacement studies (which again can be expens-ve and difficult to accomclish).

According to the present invention in a further aspect there Is ded a support for use 4n combinatorial chemistry prov- compr-is-ng a polymer matrix with at least one scintillant moJety covalently bonded into the polymer matrix, and at least one biochemically/chemically reactve site.

Preferably the support is in the form of a bead. The bead may be of diameter 5im to 1cm. The bead may be a gel type polymer.

The bead polymer Lhe bead may have macroporous structure. matrix structure may be highly polymer crosslinked.

-he svnthetic chemistry may be performed using a single bead, or many beads.

The support may be a gel type -polymer.

Preferably the combinatorial chemistry strategy includes at!east one step wherein a rad-olabelled receptor molecule is incubated with a support. Preferably, the receptor molecule is biologically active. Preferably the support 's in the form of beads, and each --ead bearing a potentiall 1gand for a receptor:-,.)lecul'e, with different beads bearing different ligands. A-fter --ncubation. the biologically active molecule, having great affinity for the ligand(s) attached to the bead(s), wi-1 be bound to the bead(,s) bearing the 1"gand(s). The bindinc of the radiolabelleed recenter molecule to the bea-(s', incorporating scint'llant moieties will result in activai-on of these scintillant moieties. T'.e beads bearing the most active ligands w-11 thus d-splay the most scint,' llatonal fluorescence. A main library and positionally fixed s,,,iblibraries may be synthesised and the scint'lllation emitted by each 1-brary and sub-11brary used to show: 1) that the mai-n 23 library contains one or more molecules that bind to the receptor molecule; and 2) identify the most active sublibraries and thus identify the most active library compound directly. In a different strategy, it is possible to use a scintillation based assay to identify the most active bead, and deduce the identity of the compound on that bead by employing convent 14 onal encoding strategies.

This application of the supports of the present invent-ion has the advantage that it can be the ligand which is on the support and this is added to a solution containing the radiolabelled biologically active molecule. This is in direct contrast to the use of conventional SPA beads, in which the biological receptor is linked to SPA beads and these SPA beads are added to a solution containing a potential ligand which is radiolabelled. By use of the present invention, a single radiolabelled receptor compound may be screened against as many potential ligands on the support as required. Radiolabelling is thus kept to a minimum and there is no requirement for a known ligand to be available in radiolabelled form.

The supports of the present invention have the flexibility to be used in conventional SPA style assays too, in which the receptor is linked to the support and the radiolabel is to be found in the ligand Ln solution.

The scintillant supports of the present invention permit simultaneous assay and deconvolution for libraries of compounds synthesised on the supports. Sublibraries may be positionally fixed and the assay procedure may be by direct scintillation counting to detect binding interactions or by scintillation counting after a washing or dilution procedure.

In a further aspect, the present invention provides a method -or determining how many chemically reactive sites there are on 24 or within a scintillant solid support, incorporatina the steps o f support for a chemical [a] providing a known amount o reaction comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scint-.-lant moiety being covalently bonded into the polymer matrix; Ibl mixing the support with a molecule comprising a sIte which may bind/react with the chemically reactive site on the support and an activating group; and [c] measuring the scintillation produced by the scintillant moiety.

By activating group it is meant a group which will activate the 'llate.

scintillant moiety and cause it to scint -he method can be used to determine the number of reactive sites per unit volume, per unit area or per unit mass.

A method of monitoring the progress of a chemical reaction comprises the steps of:

[a] -;:rovidin--- an activating group and a known amount of suppc--- a chemical reaction compr-sing a polymer -,,,atr-x having at least one scintillant mo-ety and at least one chemica';ly react-'ve site, the scintillant moiety being covaler.--'y bonded into the polymer matrix, the chemicallv reactive site being bound to a reacta= mc-ecu'e comprising a site which binds with the chemically reactive site on the support; meas,-r'n-, the scintillation produced by the scintillant moiety; r c] subjecting the support to reaction conditions whereby the activating group is removed from the reactant molecule such that the activating group is removed from the support; and [d] measuring the scintillation produced by the scintillant moiety.

By activating group it is meant a group which will activate the scintillant moiety and cause it to scintillate.

In a still a further embodiment of the invention, a method of monitoring the progress of a chemical reaction comprises the steps of:

[a] providing a known amount of support for a chemical reaction comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scintillant moiety being covalently bonded into the polymer matrix; [b] mixing the support with a molecule comprising a site which may bind/react with the chemically reactive site on the support and an activating group; and [c] measu=g the scintillation produced by the scintillant moiety.

Embodiments of the invention will now be described. 'It w-'--'! be appreciated that embodiments of the invention can be used according to the methods described in the drawings, in which:

Figure 1A is a schematic representation of the 'split and mix' 26 met,'-)od; and Ficure 1B is a schemat--c representation of positionally fixed library synthesis.

Examples 1-5: Synthesis of Scintillant Monomers Monomers (1-5) contain the 2,5-diphenvloxazole moiety. 2,5Diphenyloxazo- le -s a well known scinti-lant and these monomers are termed 'scintillant monomers'. The synthetic --oute to each of these monomers is outl-'ned in scheme 1.

N N 0 N 0 0 3 2 N 0 N 0 0 4 T5 27 The synthetic details required to synthesise the starting materials (aldehyde 6, alcohol 7 and bromide 8) are published at Tetrahedron Lett., 1997, 38, 52, 9061.

SYNTHESIS OF SCINTILLANT MONOMERS AND PRECURSORS 2,5-Diphenyl-4-hydroxymethyloxazole (alcohol 7) Lithium borohydride (1M solution in THF, 280 ml, 280 mmol) was added, over a 0.5 h period, to a stirred solution of ethyl 2,5-diphenyloxazole-4- carboxylate (78.6 9, 0.27 mol) in THF (100 ml) at 01C under an atmosphere of nitrogen. Lithium triethylborohydride (1M solution in THF, 28.0 mI, 28. 0 mmol) was added and the mixture was stirred at room temperature for a further 3 h. Hydrochloric acid (2M) was added cautiously until no further effervescence was observed, and then an aqueous solution of sodium hydroxide (2M, 200 ml) was added to make the aqueous phase slightly basic. The organic layer was separated and the aqueous phase was extracted with ethyl acetate (200 rr.1). The combined organic phases were washed with a saturated aqueous solution of ammonium chloride (200 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to furnish 2,5-di)henyl-hydroxymethyloxazole (66.1 g, 98%) as a yellow solid; M.Pt. 126-1280C, V.,a,. (cm-') 3 2 5 2 (b r, m), 3 0 5 6 (w), 2 90 2 5 (w), 2 875 (w), 1 589 (w), 1 548 (w), 1 486 (m), 1 446 (w), 1 026 (m), 1 007 (m), 776 (m), 704 (s), 688 (s); 6,. (270 MHz, CDC1-,).86 (2 H s), 7.377.51 (6 H m), 7.7 (2 H dd j 8.3, 1.6,, 8.03-8.07 (2 H m); 6. (67.5 MHz, C-DC1,) 57.1, 126.1, 12 6.. 4, 127.0, 128.1, 128.6, 128.8, 128. 9, 130.5, 136.1, 147.3, 1160.0; MS (APC I, mle) 2 52. 5 (M + 1).

4-Bromomethyl-2,5-diphenyloxazole (bromide 8) Phosphorous tribromide (1.40 ml, 14.7 mmol) was added to a 28 solution of 2,5-dii-D'ne.nyl-4-hvdroxymethvioxazole (10.0 a, 39.8 rrir.ol) in dichloromethane (200 The resultant mixture was stirred at room temperature for 2 h before a small portion of brine was added to auench any excess phosphorous tribromJde.

The,-rganic phase was then washed with brine (200 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure -0 furnish -bromomethyl-2,5 diphenvloxazole (10.3 g, 82%) as a pale yellow solid; M.Pt.

160-161C, v,,,, (em-,) 3 413 (w), 3 032 (w), 2 976 (w), 1 592 (w), 1 552 (w), 1 484 (m), 1 446 (m), 1 212 (m), 1 068 (w,), 90-7 kw), '769 (m), '701 (s), 686 (s), 665 (s); &, (270 MHz, CDC1A 4. 67 (2 H s), 7.39-7. 56 (6 H m), 7. 79 (2 H dd J 3. 6, 1.7), 8. 08-8.12 (2 H m) 8_ (67. 5 MHz, CDC1-) 25. 5, 126. 2, 126.5, 1-26.9, 127.77, 128.8, 129.0, 129.1, 130.7, 133.6, 147.7, 160.j.; MS m/e) 314.6 (M+ 1), 316.7 ',M,+ 1).

4-Carboxaldehyde2,5-diphenyloxazole(aldehyde 6) 2-Iodoxybenzoic acid (2.67 g, 9.27 mmol) was added to a solution of 4 - hydroxT.-,ie thyl - 2, 5 -diphenyloxa zo le (2.00 g, 7.97 nnol in DYISO (40 ml). The resultant mixture was st-"rred for 3 h before water (160 ml) was added. The product was extracted intc diezhv- ether (3x 50 ml). The combined organic extracts were washed wth an aqueous solution of sodium hy-4--cxide (2M, 2x 101' ml), wa.er (5x 50 m"), brine (2x 50 ml), 4= dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to furnish carboxa-.dehyde-2,5-,-.pheny'oxazole (1.84 g, 93%); M.Pt. 1C2- 1 03 '' 3 060 (w), 2 837 (w), 2 766 'w), 1 695 (s), v,,, (em--, 1 563 1m), 1 548 (m), 1 90 (m), 1 448 (m), 1 069 (w), 1 025 (w) 7 / (s), -10 (s), 688 (s) (270 MHz, CDC13) -7. 51-7. 55 (6 H m), 8.12-8.16 (At H m), 110.16 '1 H s); 6,(6-7.5 MHz, CDCl,) 126.2, 1-26.2, 126.8, 127.7, 128.9, 129.0, 131.2, 131.3, 135.5, 155.9, 160.3, 185.1; MS (APC--,, m/e) 250.5 (M+ 1).

29 2,5-Diphenyl-4-vinyloxazole (monomer 1) A solution of methyltriphenylphosphonium bromide (3.17 g, 8.87 mmol) and sodium methoxide (0.48 g, 8.89 mmol) in THF (60 m!) and DMF (10 ml) was cooled to OOC and stirred for 4 h under an atmosphere of nitrogen. The solution was allowed to warm to room temiDerature and a solution of 4carboxaldehyde-2,5diphenyloxazole (1.84 g, 7.39 mmol) in THF (30 ml) was added. The resultant solution was then stirred for 14 h at room temperature before being poured into brine (50 ml). The organic layer was separated and the aqueous phase extracted with diethyl ether (2x 50 ml). The combined organic extracts were then washed with brine (5x 50 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to furnish the crude product as a brown solid (4.72 g). Purification by column chromatography (gradient elution; 0-3% ethyl acetate/light petroleum) gave 2,5-diphenyl-4vinyloxazole (1.37 g, 74%) as a white solid; M.Pt. 7071'C, Vrax (cm- 3 051 (w), 1 830 (w), 1 636 (w), 1 598 (m), 1 558 (m), 1 488 (s), 1 444 (s), 1 411 (w), 1 357 (w), 1 245 (w), 1 084 (m), 1 070 (m), 1 027 (m), 979 (m), 945 (m), 912 (m), 7-78 (m), 703 (m), 648 (m); 6, (270 MHz, CDC1,) 5.47 (1 h dd J 10.9, 2.0), 6.28 (1 H dd J 17.2, 2.0), 6.97 (1 H dd J 16.8f 10.10, 7.30-7.48 (6 H m), 7. 64-7. 69 (2 H -,i), 8. 09-8.14 (2 H m);6, (67.5 MHz, CDC13) 117.5, 125.8, 126. 4, 126.6, 127.2, 128.4, 128.6, 128.7, 128.8, 130.4, 135.2, 146.1, 160.0; MS (APC i, mle) 2 4 8. 2 (M + 1).

Allyl-2,5-diphenyloxazole-4-methylether (monomer 2) Sodiw-, hydride (55% dispersion in oil; 0.35 g, 8.02 mmol) was washed with light petroleum (2x 5 ml) under a ni-zrogen atmosphere. A solution of 2,5-diphenyl-4-hydroxymethyloxazole (I - 00 g, 3. 98 mmol) in THF (4 0 ml) was added to the sodium hydride and the mixture was stirred at room temperature for 2 h. Allyl bromide (0.56 ml, 6.03 mmol) was then added and the resulting sc-'utJLon was szirred at room temperature for a further 14 h. An aqueous solution of saturated ammonium chloride (30 ml) was added cautious-y, the organic layer was seiDarated and the aqueous phase extracted with diethyl ether (30 ml). The combined organic extracts were washed with brine (30 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to furnish a yellow solid (1.89 g). Purification bv column chromatography (gradient elution; 0-20% ethyl acetate/light petroleum) gave ally.'-2,5diphenyloxazole-methylether (0.91 g, 78% vield) as a white so',id; M.Pt. 7172'C, V,,, (em') 3 432 (w), 3 064 (,w), 3 014 (w), 2 9 19 (w), 2 8 4 0 (w), 1 6 4 6 (w), 1 5 9 0 (w), 1 5 4 7 (m), 1 494 (m), 1 48 (m), 1 280 (w), 1 146 (w), 1 084 (S), 1 057 (s), 912 (s), 778 (s), 759 (s), -708 (s), 688 (s); 6H (270 MHz, CDC13) 4. 16-4.19 (2 H m), 4. 66 (2 H s), 5. 25 (1 H dd J 10.2, 1. 6), 5. 38 (1 H dd J 17.5, 1. 6), 5. 94-6. 08 (1 H m), 7. 35-7. 51 (6 H m), 7.80 (2 H dd J 8.2, 11.31,, 8.10-8.15 (2 H m);6,, (67. 5 MHz, CDC1,) 64. 4, 71.5, 117.8, 126-3, 126.4, 127.3, 128.2, 128.6, 128.8, 128.9, 130.4, 134.0, 134.4, 18.9, 159.8; MS (APC7, mle) 291.9 (M + -), 292.9 (M + 2).

2,5-d.iphenyl-4-methyloxazole (4-formylphenyl) ether (precursor to monomer 3) A mixture of -'--romome-hyl-2,5-diiDhenyloxazole (4.30 g, 13.7 mmol), 4-hydroxybenzaldehvde (1.67 Q, 13.71 mmol) and potassium ,uxed in butanone (200 carbonate (2.08 g, 15.1 mmo., were ref ml) for h. zfter cooling to room:emerature, the mixture was filtered t.rrough a pad of celite and concentrated under reduced pressure to furnish a dark brown solid (4.40 c).

Purification by coluirm chromatography (gradient eluton; ethyl acetate/light petro"eum) gave 2, 5-dip'.-ienyl-4 methyloxazole'4-formylphenyl) ether (1.16 g, 24%) as a wh--te solid; M.Pt. l',9-1l,'2C; V.Max (em-) 3 0577 (w), 2 840 (w), 2 752 (w,', 1 692 (s), 1 604 (s), 1 578 (s), -- 551 (m), 1 48 (m), 31 1 446 (m), 1 391 (m), 1 313 (m), 1 247 (s), 1 164 (s), 1 091 (w), 1 068 (w), 997 (s), 868 (s), 839 (m), 744 (m), 702 s), 688 (s); 6, (270 MHz, CDC13) 5.27 (2 H s), 7. 21 (2 H d J 8.9), 7.41-7.51 (6 H m) 7.73-7.77 (2 H m), 7.87 (2 H d 1 8.6), 8.10-8.14 (2 H m) 9. 90 (1 H s);8, (67. 5 MHz, CDC13) 63. 1, 115.3, 126.3, 126.5, 127.0, 127.7, 128.9, 129.1, 130.3, 130. 7, 131.7, 132.0, 149-8, 160.1, 163.4, 190.8; MS (APCI, mle) 356.3 (M + 1).

2,5-Diphenyl4-methyloxazole(4-vinylphenyl) ether (monomer 3) A solution of methyltriphenylphosphonium bromide (4.28 g, 12.0 mmol) and sodium methoxide (0.65 g, 12.0 mmol) in THF (30 ml) and DMF (20 ml) was cooled to O'C and stirred for 4 h under an atmosphere of nitrogen. The solution was allowed to warm to room temperature and a solution of 2,5diphenyl-4methyloxazole(4formylphenyl) ether (3.55 g, 10.0 mmol) in THF (30 ml) was added. The resultant solution was stirred at room temperature for 14 h before being poured into brine (50 M1). The organic layer was separated and the aqueous phase extracted with diethyl ether (2x 50 ml). The combined organic extracts were washed with brine (5x 50 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under d reduced pressure to furnish a brown sol (6.15 g).

Puri-fication by column chromatography (gradient elution; 5-15% ethvl acetate/light petroleum) gave 2,5-diphenyl-4 methyloxazole(4-vinylphenyl) ether (1.57 g, 44%) as a white solid; M.Pt. 112-113'C; vm,,. ( cm-) 3 4 3 3 (w), 3 0 5 6 M, 2 9 2 6 (w), 2 856 (w), 1 546 (m), 1 498 (m) 1 456 (m), 1 378 (w) 1 279 (w), 1 076 (s), 987 (m), 905 (m) 824 (m), 780 (m), 7759 (m), 706 (s), 688 (s); 6, (270 MHz, WC13) 5.17 (1 H dd J 10. 9, 1. 0), 5. 19 (2 H s), 5. 67 (1 H dd J 17. 8, 1. 0), 6. 7 3 (1 H dd j 17.5, 10.9), 7.06 (2 H dd J 6.9, 2.3), 7.35-7.51 (8 H m), 7 -78 (2 H dd J 6. 6, 1. 6), 8. 098. 16 (2 H m); 3, (67.5 MHz, 32 DC13) 62. 9, 111.111, 115. 0, 126. 3, 126. 5, 127. 2, 127.4, 127. 9, 128. 8, 128.9, 129.0, 130.5, 131.0, 132.5, 136.2, l9.6, 158.2; MS (APC!, mle 354. -- (M + 1).

2,5-Diphenyl-4-methyloxazole (4-vinylbenzyl) ether (monomer 4) Sodiur-., hydride (55% disDersion in oil; 1. 00 g, 22.9 mmol) was washed with 1, ight petroleum (2x 5 ml) under a nitrogen atmosphere. A soluti, on of 4-hvd-roxymethyl-2, 5-diphenv-oxazole (2. 00 g, 7. 97 in THF (50 ml) was added to the sodium, hydride and the mixture was stirred at room temperature for 2 h. 4-Vinylbenzylchloride (1.35 mI, 9.58 mmol) was then added -ng solution was stirred at room temperature for and the result.

14 h. DYF (30 ml) was added and stirring continued 'or a further 24 h at room temperature. An aqueous solution of saturazed ammoni.= chlor'de (33 m!) was added cautiously, the organic layer was separated and the acueous phase extracted with diethyl ether (2x 50 mI,). The combined organic extracts were washed w'th brine (5x 50 ml), dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to furnish a yellow solid (4.00 g). Purif-cazion by column chromatography (gradient elution; 0-10% ethvl acezate/light petroleum) gave 2-1,5-diphenyl-4-methy-loxazole vinylbenzyl) e--.ier as a white solid (2.21 g, -16%); M.Pt. 97 9 8'C;; V,-,ax 3 432 (w), 2 926 (w), 1 605 (m) 1 509 (s) 2 4 0 (s), 1 1 - 5 (m), 1 0 0 3 (m), 8 9 7 (m), 8 3 4 (m,, 7 0 9 (m) 685 (m), 668 ',m,; 6, (270 M11-1z, CDC1-) 4. 68 (4 H s) 5. 27 (1 H dd J 11.9, 11 -0', 5.79 (1 H dd j 11.5, 1.0), 6.77 (1 H dd -7 1.5, 10.9), 7.34-7.50 1,6 H m), 7.71-7.74 (2 H m), 8.09-8.16 (2 1H m);8, (67.5 MHZ, CKI-J 64.2, 113.8, 113.9, 126.1, 126.2, 126.4, 127.3, '-2e.1, 128.5, 12E.7, 128.8, 13C., 133.9, 137.11, 137.4,:,fS (APC.1, m.le) 368 (M + 1 33 N 0 N J\ -0 a), - y 0 0 Reaction conditions: a) Ph3P'CH3 Br, NaOMe, THF, DMF Monomer 2 IN OH N( H b) 0 7 2 Reaction conditions: b) NaH, THF Monomer3 O'Cr IN Br C) N Z", I O 0 8 d) C-11, C 3 0 // n,; Reaction conditions: c) HO \Y--, K2CO3, Butanone, - d) Ph3PCH3 Br', NaOMe, THF, DMF Monomer IN OH e) D 7 OD 0 4 7 Reaction condiluions e) Cl - //, NaH, THF Monomer 5 Br IN 0 0 D 5 B 5 Reaction conditions: BU3Sn Pd(Ph3)4, CHCJ3 Cl>-f - Scheme I 34 Chemically inert scintillant solid supports may be constructed by co- polymerising a scint-Ilant. monomer (1-5) with a chemically un- functionali3et monomer or a chemica-,ly funct-Jonalised monomer. The covalent incorporation of scintillant molecules into the poly-mer matrices of these so-id supports enables their use in all solvents, w- thout leaching of the scintil-'ant molecules. Consequently, these supports will retain the abil-ty to scintillate strona'y in the presence of ionising radiation (for example P particles and aucer electrcns), even if they have been used previously to carry out solid phase synthetic chemistry._ Example 6: Synthesis of Scintillant Merrifield's Resin

Merrifield's resin is a commercially available polystyrene based, beaded form of solid support, used widely in solid phase synthetic chemistry. Chemically reactive benzy7 chloride groups are distributed randomly throughout each resin bead. When the beads are used for solid ohase synthes's, each solvent-accessible benzyl chlo-r-de group reacts 'n identical Fashion. Merrifield's resn -s constructed 'n a free radical suspension cc-polymerisation reaction of styrene, chloromethylviny'---nzene and div4nvibenzene.

An analogous suspension pcly-nerisation reaction which additionally contains one of the scintillant monomers (11-5, vields a Merrifield's resin,,h-ch also contains sc-inti'lant mo'ecu'es incorporated cova-e.-tly 'nto the polymer matrix of each resin bead. --his can be termed 'scintillant Merrifielci's res-in'. Even after sc-ntillant!,err-Jfield's res--r, beads have been used for so!-d phase svn--het-,c chemistry, the covallent incorporation of scintillant ensures that they retain the ability to sc--tilla-e in the presence of fonising radiation even after Prolonged exposure to organic solvents. As a suppc--t for carrying out solid phase synthetic chemistry, Merrifield's resin and scintillant Merrifield's resin are essentially interchangeable.

Scintillant monomer (1) is co-polymerised with 4ethylvinylbenzene, divinylbenzene and 4-vinylbenzyl chloride. AIBN (2,21 azobisisobutyronitrile) was used as a free radical initiator-to start the reaction and toluene was used as a porogen. A standard suspension polymerisation procedure was carried out to produce a gel type, highly cross-linked, Merrifield's resin. Unreacted monomers and any impurities were removed from the product polymer by exhaustive soxhlet extraction.

The reaction scheme is shown in Scheme 2 and full experimental details of the reaction are given below.

Scintillant monomer (1) = 2.60 g, 10.5 mmol 4-Ethylvinylbenzene (9) (co-monomer) = 4.91 ml, 4.49 g, 34.0 mmol Divinylbenzene (10) = 6.00 ml, 5.48 g, 42.1 mmol (used as a cross-linking agent) 4-vinylbenzyl chloride (11) = 2.62 ml, 2.84 g, 18.6 mmol (functionalised monomer) Toluene (12) (used as a porogen) 13.53 ml (equal to the - combined monomer volume.) AIBN (used as a radical initiator) = 0.250 g Each of the reagents listed above were added to a bulk acrueous phase (250 ml) that contained 87-899. hydrolysed polyvinylalcohol (2.5 g) (acts as a droplet stabiliser).

Synthesis ol'Scintilfan! Merrifield's Resin

Ph 0 Ph Scintillant 4-Ethylvinyl Divinylbenzene 10 4-Chloromethyl Toluene12 Monomer I benzene 9 vinylbenzene 11 suspension polymerisaon AIBN Cl Ph Sc Ph Ph Ph Sc Ph Sc 11 _= Sc Ph Sc Ph Sc C Cl C1 (Sc = 2,5-diphenyloxazolel, Scheme 2 The mixture was placed under a nitrogen atmosphere, stirred at 500 rpm and rner. heated to 80'C to initiate thermal decomposition of the radical initiator and thus start the polymerisation reaction. Stirring and heating were maintained for a further twelve hours, after which tIme all of ihe organic drop-ets In the bulk aqueous phase had solidified. 'he mixture was cooled to room temperature, filtered and io washed (water 3x 200 ml followed by ethanol 3x 200 mil furnish 3.911 a c-' a beaded oroduc. of between 300-500 im in diameter. The beads were then soxhIet, extracted for two successive periods c-' 3 hours w-th tetrahydrofuran (250 ml) - 37 Example 7: Evaluation of Scintillant Merrifield's Resin by Scintillation Counting

Portions of scintillant Merrifield's resin obtained in Example in 6 were placed in 0.5 ml Eppendorf tubes. To each portion of beads, an aliquot of a stock solution of. 'C labelled hexadecane in toluene was added. Each tube was then monitored in a scintillation counter. As a control experiment, the tozal amount of radioactivity (maximum counts per minute (CPM)) per aliquot was determined by counting in Ultima Gold (RTM), a commercial scintillation cocktail. The results obtained are tabulated below and indicate that even after prolonged periods of soxhlet extraction the scintillant molecules remain within the resin beads. This finding indicates that the scintillant molecules are covalently incorporated into the polymer matrix, and that the support may subsequently be used in organic solvents without leaching of the scintillant molecules occurring.

EXPT. Mass of TOTAL CPM CPM /mg resin scintillant Merrifield's resin used /mg Soxhlet time 5.05 36 472 7 222 Ohrs 7.18 43 044 5 995 7.31 46 582 6 372 average 6 530 Soxhlet time 7.19 35 462 4 932 8hrs 6.64 33 052 4 977 7.65 37 433 4 893 38 average 4 934 Soxhlet time 5.82 35 535 6 016 16hrs 7.32 40 923 5 591 7.06 40 418 5 725 average 807 Ultima Gold 122 632 (represents 120 887 UM possible ounts) 121 432 average = 121 650 Example 8:Synthesis of Scintillant Wang Resin Scintillant Merrifield's res--In obtained in Example 6 has been derivatised into scintillant Wang resin. The following experimental - procedure was employed, and is outlined in scheme 3:

Svnthesis of Scintillant Wang Resin OH rc Ow C1 NaGlyle, 601C 0-&) dimethylacetamiae Scheme 3 Hydroxybenzy! alcoholl ',0.45 a, 3.62 mmol) and sod-',.rr, methoxide (0. 19 a, 3-12 rm-nol', were place-- uncier an atmosphere of ni,rogen 39 and dissolved in dime thyl ace tamide (25 ml) After stirring this solution for 30 minutes it was transferred into a second flask containing scintillant Merrifield's resin (300-500im -n diameter) of Example 6, (1. 00 g, approx. 1.2 n-Lmol) The resulting mixture was stirred at 60'C for 14 hrs. Af ter cooling to room temperature, the beads were collected by filtration and washed with dioxane (100 ml), dioxane: water (3: 1, 10 0 ml), dioxane (10 0 ml) and methanol (10 0 ml). The beads were then dried under vacuum to yield 1.07 g of scLntillant Wang resin.

In a non-quantified experiment, an aliquot of - 'C labelled hexadecane in toluene was added to a portion of the scintillant Wang resin and the mixture counted in a scintillation counter. The resin scintillated strongly, indicating that the scintillant molecules remain covalently incorporated into the polymer matrix of the resin.

Example 9: Reaction of Scintillant Wang Resin of Example 8 with Fmoc-(Gly)-COOH The reaction pathway is shown in scheme 4a.

Rea.,IiOn viiih Fmoc-(Gly)-COOH 0 CSC OH FmocGly)-COOH Sc and either, DCCI, DMAP, DMF NH moc or Cl C col 1 Ryddine, DMF cl Schem 4a Scintillant Wang resin of Examcle 8 has been coupled to an Fmoc protected amino acid. The success of this reaction demonstrates that scintillant resins can be coupled to amino acids, and indicates that it is possible to use scintillant resins to carry out solid phase synthesis. The full experimental details are given below.

Scintillant Wang resin (0.20 g, approx. 0.2 mmol) and FmocGIVcine (0.178 g, 0.6 irmol) were placed under an a--mosphere of nitrogen and s-irred In DMF (5 ml). After 30 minutes, 2,6dich-lorobenzoyl chloride '0.085 ml, 0.6 mmol) and pyridine (0.100 ml, 1.24 mmoL) were added to the mixture. Stirring was continued for a further 14 hours after which time the beads were collected by fil-ration. The resin beads were washed in situ with dichloromethane (30 ml) followed by methanol (30 ml). The beads were then dried under vacuum to yield 0.210 g of the derivatised resin.

-he beads were ':'nen subjected to standard Fmoc cleavage conditions (scheme 4b) (piperidine in DMF, NOVA BioChem 97/98 Catalogue page S3-/) and the average loading of the Fmoc-Glycine on the resin calculated. The resn loading was 0.15 mmol cram of resin beads.

41 0 piperidine OSC DMF g70 0 NH NH2 Fmoc Ckaved Frnoc group detedled and quandfied by UVIvis spectroscopy Scheme 4b Example 10: Reaction of Scintillant Wang Resin of Example 8 with Tritiated Acetic Anhydride The reaction pathway is shown in scheme 5.

Reaction with Tritiated Acetic Anhydride 0 om Tribated (CH3CO)20 C)-( 0 0 CHrr3 Pyridine. DMAP, DCM A I tritiated rnethyl group Scheme 5 Scintillant Wang resin of ExampLe 8 (0.123 a), 42 dimethylamin.opyridine (DMAP) (catalytic amount) and pyridine m!) were st'o-rred _n d.;_--h-,cromethane (5 ml) To zh mixture, tritiated acetic anhydride (0.100 mI, 1.06 r-rLmol, 10 nours kIC:i) was added. The reaction m-xture was stirred for six at room temperature. After this time, the resin beads were ccilect-ed by filzration. The beads were washed in situ, extensively with successive portions of dichloromethane (3x 10 m!), dichloromethane:methano'l,. (50:50, 3x 10 ml), methanol (3x ml), dichloromethane: methanol (50: 50,3x 10 ml) and f -nally dichloromethane (3x 10 ml). The beads were then left to dry in situ for 12 hours. Approximately 7 mg portions of the beads were placed in scinti'lation vials before being counted in the scintillation counter. The -following counts per minute (CPM) per mg of resin were measured as shown in column 4 of the following table.

When U-tima Gold (10 ml), a commercial scintillation cocktail, was added to each tube, the CPM per mg of resin increased (see column 5 of the table).

Tube Mass of cpm cpm / mg cpm / tritiated of resin mg of resin mg resin in Ultim a Gold 1 6.39 3 396 _532 10-72 2 8.17 4 699 575 11086 3 4 287 586 995 4 7.78 3011 841 C -7.33 3 90^1 532 867 This gave an average cpm / mg of rritiated resin of 555 cpm / mg and an average epm / mg in Ultima Geld of 972. The beads 43 are thus 57% as efficient at counting as the commercial scintillation cocktail Ultima Gold. When these figures are used to work out the degree of loading on the precursor scintillant Wang resin a loading of 0.137 mmol / g of resin is obtained. This value is in excellent agreement with the value of 0.15 mmol / g obtained by standard methods, described in Example 9.

it is thus demonstrated that covalent attachment of a radiolabel to a scintillant resin according to the present invention causes scintillation, with an efficiency of scintillation counting of 57% relative to a commercial scintillation cocktail. The scintillation based method to detect and quantify binding interactions by scintillation counting is thus viable.

Example 11: Synthesis of a tetrapeptide on a scintillant solid support The tetrapeptide (Gly-Gly-Tyr-Arg) has been synthessed on scintillant Wang resin of Example 6 according to scheme 6. The tetrapeptide is a known micromolar inhibitor of the cysteine protease papain. Papain with a radiolabel (such as tritium) is incubated with the said Scintillant Wang resin and the system monitored with a scintillation counter. Scintillation counting in this manner enables the amount and kinetics of binding of papain to the tetrapeptide to be measured direct-v.

44 i) Fmoc-Arg(Pb,-OH, DMF OH ii) Diisopropylcarbodiimide, DCM 0-Arg(Pb-Fmoc iii) DMAP, DMF NB. 8--\ (R-\ a) OH 0-Arg(Pb-H Ill OH O-W 0-Arg(Pb-Tyr(tBu)-Fmoc a) Scintillant Wang Resin 0-Arg(Pb-Tyr(tBu)-H Reagents and Conditions c) a) 20% piperidine, DMF @-\ 0-Arg(Pb-Tyr(tBu)-Gly-Fmoc b) HO-Tyr(tBu)-Fmoc, PyBOP, DMF C) HO-Gly-Fmoc, PyBOP, DMF d) TFA, H20 a) 0- Arg(Pb-Tyr(tBu)-Gly-H c) 0-Arg-Tyr-Gly-Gly 0-Arg(Pb-Tyr(tBu)-Gly-Gly-Fmo, Scheme 6

Claims (34)

C L A I M S:

1. A scintillant polymer formed by polymerisation or copolymerisation of a scintillant monomer, the scintillant monomer comprising a molecule of structure R-Y, in which R is a scintillant group and Y is a group comprising a polymerisable moiety.

2. A scintillant monomer comprising a molecule of structure R-Y; in which R is a scintillant group and Y is a grouP comprising a polymerisable moiety.

3. A scintillant monomer according to claim 2 in which R is 2,5diphenyloxazole.

4. A scintillant monomer according to claim 2 or 3 in which Y is a substituted or unsubstituted aliphatic or aromatic group or an ether.

5. A scintillant monomer of one of the following structures f..,Specspib'\398 18,98 46 N 0 N OT \N 0 0 1 0 1 /,., /v 3 1 CI/ 2 N

6. A support for a chemical application formed from at least one scinti- Ilant mnomer, optionally by polymer--saticn or copolymerisation of the monomer.

7. T- support for a chemical application formed from at least one scintillant monomer of claims 2-5.

8. A support according to claim 6 or 7 formed from at least one additional, chemically functionalised, monomer.

f:',spe cs p i b,,3

9 918.98 47 9. A support according to claim 6, 7 or 8 formed from at least one additional, inert, monomer.

10. A support according to claim 6,7,8 or 9 for use in so-lid phase synthetic chemistry or solid phase combinator-al chemistry.

11. A support according to any of claims 6 to 10 havirg polymer crosslinking.

12. A support according to any of claims 6 to 11 in the form of a bead, with a diameter in the range 5-500 micrometers.

13. A bead according to claim 12 which has a porous or macroporous structure.

14. A support according to any of claims 6 to 10 or 13 in the form of a gel type polymer.

15. A support for a chemical reaction accord-ina to any of claims 8-14.

" 6. A suzoo--t for a chemical application compris'ng a polymer matrix with a scintillant moiety covalently bonded into the polymer matrix.

17. A support according to claim 16 in which the scintillant moiety is distributed substantially uniformly throughout the polymer matrix.

i8. A support according to claim 16 or 17 in which the po.Lymer matrLx further comprises at least one chemically fAspecspib39818.98 48 reactive site.

i9. A support according to claim 18 in whicn the chemically reactive site is an integral part of, and distributed substantially uniformly throughout, the polymer matrix.

' v

20. A support according to claim 18 in which the chem ca reactive site is formed at the surface of the matrix as a ayer.

21. A support according to any of claims 16-20 in the form of a bead, with a diameter in the range 5 micrometers to 1 centimeter.

22. A supporz- according to any of claims 16-21 which has porous or macroporous structure.

23. A support according to any of claims 16-20 disposed as a laver on a substrate.

24. A support according to any of cla=s 16-24 formed by polymerisat-Jon of a monomer comprising the scinzillant moiety.

An assay incorporatrg the steps of:

providing a support for a chemical application comprising a polymer matrix having at least one scint"I'ant moiety and at least one chemically reactive site, the scintillan-7- moiety being covalently bonded into the polymer mazrx; b', mixing the support with a molecule comprising an fi'specspib'39818.98 49 activating group and a site which may react with he reactive site on the support; and [c] measuring the scintillation produced by the scintillant moiety.

26. A support for use in solid phase synthetic chernistry comprising a polymer matrix with a scintillant mo-iety covalently bonded into the polymer matrix and a chemically reactive site.

27. A support for use in combinatorial chemistry comprising a polymer matrix with a scintillant moiety covalently bonded into the polymer matrix and a chemically reactive site.

28. A support according to claim 26 or 27 in the form of a bead, with a diameter in the range 5-500 micrometers.

29. A support according to claim 26, 27 or 28, in which the polymer matrix has more than 20% polymer- cross linking.

30. A support according to claim 26, 27, 28 or 29 having porous or macroporous structure.

31. A support according to claim 26 or 27 in the for-,-,, of a gel type polymer.

32. A method for determining how many chemically reactive sites there are on or within a scintillant solid suiDf)c)r7:, incorporating the steps of:

[a] providing a known amount of support for a cheinical f.\specspib'39818 98 reaction comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scintillant moiety being covalently bonded into the polymer mar-rix; b mixing the support with a molecule comprising a site which may bind with the chemically reactive site on the support and an acz-ivatina group; and [c] measuring the sc-intilla.:iion produced by the scintillant moiety.

33. A method of monitoring the progress of a chemical reaction comprising the steps of:

[a, providing an activating group and a known amount of support for a chemIcal reaction comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scintillant moiety being covalent-y bonded -nto the polymer matrix, the chemically reactive site being bound to a reactant molecule comprising a site which binds or reacts with the chemically react-ve site on the support; !bl measuring the scintillation produced by the scintillant moiety; 1 c] subjecting the support to reaction conditions whereby the activating group Is removed from the reactant molecule such that the activating group is removed from the support; and f:\specspib'J98 18 98 51 [d] measuring the scintillation produced by the sc- - ntJllant moiety.

34. A method of monitoring the progress of a chemical reaction comprising the steps of:

[a] providing a known amount of support for a chemical reaction comprising a polymer matrix having at least one scintillant moiety and at least one chemically reactive site, the scintillant moiety being covalently bonded into the polymer matrix; [b] mixing the support with a molecule comprising a site which may bind with the chemically reactive site on the support and an activating group; and [c] measuring the scintillation produced by the scintillant moiety.

f.\spccspib'.398 18.98