Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B61/00—Other general methods
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
  • C07C311/16—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
  • C07C311/18—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms, not being part of nitro or nitroso groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
  • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
  • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
  • C07C311/42—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B20/00—Methods specially adapted for identifying library members
  • C40B20/08—Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
  • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B80/00—Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/11—Compounds covalently bound to a solid support
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

• This invention relates to chemical constructs for use in solid phase synthesis, and to methods of analysis of the products of solid phase synthesis using the constructs.
• Solid phase synthesis has been known for many years in the field of peptide synthesis and more recently has also been used increasingly for the synthesis of non-peptides.
• Solid phase synthesis has found particular application in the field of combinatorial chemistry and the preparation of chemical libraries as potential sources of new leads for drug discovery, see for example Anthony W. Czarnik, Analytical Chemistry News and Features, June 1, 1998, pp 378A-386A, and The Combinatorial Index, Barry A. Bunin, Academic Press, San Diego 1998.
• a feature of combinatorial chemistry methods is that they enable very large numbers of different compounds to be prepared from a relatively limited number of molecular building blocks in a relatively small number of reactions.
• Combinatorial chemistry makes use of the "split and pool” approach in which a suspension of chemical starting material tethered to a solid support is split into N portions, each of which is reacted with a different reagent.
• Each individual solid support e.g. a resin bead
• each of the reaction products can be separated and analysed or subjected to biological testing simply by isolating each single solid support and cleaving the product from the support.
• the large numbers of compounds generated by combinatorial methods means that it can be impracticable to identify and characterise each compound. Consequently, the compounds usually are first tested, either on the solid support or after cleaving from the support, and only those compounds, which show some biological activity, are subsequently identified.
• compounds can be tested in pools containing a predetermined number of compounds, the inactive pools being discarded and the active pools being subject to further investigation.
• the biological activities of the compounds can be analysed using high throughput automated assay techniques permitting large numbers of compounds to be analysed in a short time.
• the resin beads typically used in the synthesis of combinatorial libraries are derived from cross-linked polystyrene resin and are usually of the order of 90 ⁇ m and 250 ⁇ m in diameter (i.e. just visible to the naked eye).
• the number of beads in a given mass or volume of resin will depend on the average bead size but, for example, with a bead size of 150 ⁇ m diameter, there will be approximately 500 beads per milligram of bulk resin.
• Typical compound loadings on the beads are of the order of 0.1 to 0.4 mmol g ⁇ l and hence beads of the dimensions given above will tend to have individual compound loadings of about 1 nanomole of compound per bead.
• a coding tag can be built up in sequential steps on the solid support in parallel with the construction of the desired target compound, the coding tag reflecting the synthesis history of the product compound and being unique for each product compound.
• the coding tag is usually built up on the support using chemical reactions of a type which are orthogonal to the chemistry used to build up the product compound, thereby ensuring that the coding units and product compounds do not become confused.
• coding tags such as isotopic labels can be incorporated into inter alia a linker group between the solid support and the substrate.
• This approach has the advantage that it does not require the formation of a separate coding tag using orthogonal chemistry and thereby reduces the overall number of synthesis steps involved.
• a further problem facing the solid phase chemist is the difficulty in quantifying the products of a given reaction sequence.
• any solid phase synthesis procedure whether or not part of a combinatorial procedure, it is important to be able to determine the optimal conditions for a given reaction step in a series of steps. It is also important to be able to monitor reactions, for example so that it can be determined whether a particular reaction has gone to completion. This is particularly important in a multistage solid phase synthesis where the failure of a given stage to proceed to completion can lead to the formation of side products thereby complicating what is otherwise a relatively straightforward separation procedure.
• Geysen et al. address this problem by suggesting the introduction of a readily ionisable basic centre such as lysine into the linker, and further suggest that the basic centre could be quaternised by alkylation during work up in order to give a charged group which would sensitise the construct to mass spectrometric analysis.
• a readily ionisable basic centre such as lysine
• the basic centre could be quaternised by alkylation during work up in order to give a charged group which would sensitise the construct to mass spectrometric analysis.
• one drawback of this approach is the need to protect the amine group during the various synthesis steps and the consequent need to deprotect the amine group prior to analysis.
• Carrasco's method involves the formation of a construct comprising a resin bead linked via a first linker group to a group, which the authors refer to as an "ionisation tag".
• the "ionisation tag” in turn is linked via a second linker group to a substrate.
• the first and second linker groups are orthogonally cleavable; i.e. they can be selectively cleaved using different chemistries.
• the first linker group is photochemically cleavable whilst the second linker group is chemically cleavable.
• the "ionisation tag" used by Carrasco is a tetrapeptide chain having the sequence (reading from the C-terminus) Gly-Phe-Lys-Ala, and having an N-(2- trimethylammonium)-acetyl group linked to the lysine. The purpose of the ionisation tag is twofold.
• the construct provides an already ionised "sensitiser group" which enables the construct to be detected by matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry, and secondly, it adds mass to the construct thereby enabling substrate molecules to be detected without being swamped or masked by low molecular weight peaks in the mass spectrum.
• MALDI matrix-assisted laser-desorption ionisation
• the invention provides a method of analysis of a solid phase construct; which method comprises:
• the spectrophotometric analysis may be used to determine either the absolute amount of substrate R present in a given sample, or it may be used to determine the relative amount (e.g. in terms of a molar ratio) of substrate R present relative to another component (e.g. where more than one substrate R is present) in the sample.
• references to methods of quantifying the substrate R in this application include both absolute determination and relative determination, unless the context indicates otherwise.
• the chromophore is typically selected such that it has a substantial absorption band that distinguishes it from the substrate.
• the abso ⁇ tion band may be remote from any significant abso ⁇ tion bands in the substrate.
• the abso ⁇ tion b band in the chromophore may be of such an intensity that it effectively swamps any overlapping band in the substrate.
• the chromophore C u has a principal log E max value of at least 2.5. It is further preferred that the principal log E max value is at least 1.5 times greater than the principal log E max of the substrate R.
• the spectrophotometric analysis is carried out at a wavelength(s) in the ultraviolet region of the spectrum.
• the molar abso ⁇ tivity E is constant for a given compound at a given wavelength and is usually expressed as E max - the molar abso ⁇ tivity at an absorption band maximum.
• E or E ax are typically expressed in units of 10 ⁇ 2 m-2 mol" * ⁇ and, unless indicated otherwise, references to E or E max values herein will refer to such units. Since E or E max values can be very large, the logarithmic equivalent is often used and such logarithmic equivalents are referred to herein as log E max values.
• the chromophore C u preferably has a principle log E max value of at least 2.5 and typically has a principal log E max value which is at least 1.5 times greater than the principal E max of the substrate R. More typically however, the chromophore C u has a principal log E max that is at least 2, more usually 2.5, and preferably 3 times greater than the principal log E max of the substrate R.
• the wavelength at which the measurement is taken will depend on the precise abso ⁇ tion characteristics of the substrate R and the chromophore C u , and can be determined on a case by case basis.
• the chromophore C u may be distinguished from any chromophores in the substrate in that it may possess an abso ⁇ tion band which is remote from any significant abso ⁇ tion bands. By this is meant that the abso ⁇ tion band of the chromophore is remote from, and does not overlap to any significant extent (e.g.
• a significant abso ⁇ tion in the present context means an abso ⁇ tion having a log E value of 1 or more.
• the invention provides a chemical construct for use in solid phase synthesis comprising a solid support Q having linked thereto via a connecting group Y a substrate R; the connecting group Y having first and second cleavage sites which are orthogonally and selectively cleavable; the second cleavage site being selectively cleavable to release the substrate; and the first cleavage site being located at a position between the second cleavage site and the solid support and being selectively cleavable to release a fragment Fr u comprising the substrate and at least a portion of the connecting group Y, wherein the said portion contains a chromophore C u which facilitates analysis of the fragment Fr u by ultra violet, visible or fluorescence spectrophotometry (preferably ultraviolet spectrophotometry), the chromophore C u having a principal log E max value of at least 2.5 and wherein (i) the principal log E max value is at least 1.5 times greater than the principal log E max of the substrate R; or
• a UV chromophore in the construct enables quantitation of the products of a synthesis to be carried out.
• quantitation can be (i) absolute quantitation or (ii) relative quantitation, or both.
• absolute quantitation is meant that the absolute concentrations of a substrate molecule or fragment or construct containing the substrate molecule can be determined, whereas the term " relative quantitation "is used herein to mean the determination of the amount of a substrate (or fragment or construct containing the substrate) relative to another component (such as a side product or starting material or another substrate) in a reaction mixture.
• the UV chromophore is typically selected such that it has a very strong abso ⁇ tion at a unique or characteristic wavelength, which is usually distinct from the wavelengths at which the maximum absorbences of a typical substrate molecule might be found.
• the abso ⁇ tion of the chromophore is very large relative to the substrate or substrate R, e the overlapping of abso ⁇ tions due to the substrate and the chromophore may have a minimal impact on the accuracy of the measurement of the quantity of substrate.
• overlapping abso ⁇ tion bands should not prevent meaningful analyses to be carried out provided that any error introduced is less than about 10% and preferably no more than about 5%.
• the chromophore C, and its relationship with the abso ⁇ tion due to the substrate can be defined in terms of the degree of error arising from any overlap between the abso ⁇ tion bands of the substrate R and the chromophore.
• the invention provides a chemical construct for use in solid phase synthesis comprising a solid support Q having linked thereto via a connecting group Y a substrate R; the connecting group Y having first and second cleavage sites which are orthogonally and selectively cleavable; the second cleavage site being selectively cleavable to release the substrate; and the first cleavage site being located at a position between the second cleavage site and the solid support and being selectively cleavable to release a fragment Fr u comprising the substrate and at least a portion of the connecting group Y, wherein the said portion contains a chromophore C u which facilitates analysis of the fragment Fr u by ultra violet, visible or fluorescence spectroscopy, wherein the abso ⁇ tion characteristics of the chromophore C u and the substrate R are such that at a given measurement wavelength, any errors in measurement of the quantity of substrate R (or any fragment or construct containing the fragment) arising from any overlap between
• chromophores are groups containing an aryl group, preferably a fused polycyclic aryl group, e.g. a C - C30 polycyclic aryl group in which one or more (e.g. 1, 2, or 3) ring carbon atoms are optionally replaced by a heteroatom such as nitrogen, sulphur or oxygen.
• polycyclic aryl groups are polycyclic hydrocarbons such as naphthyl, phenanthrenyl and anthracenyl groups, and polycyclic heteroaryl groups such as acridine, or phenanthroline.
• Such aryl groups or polycyclic groups can be optionally substituted, preferably with a substituent or substituents which is or are inert and/or non- interfering with regard to the reaction conditions employed in a given reaction scheme.
• substituent groups include alkyl and alkoxy (e.g. alkyl and alkoxy having from 1 to 20 carbon atoms, preferably from 1 to 6 carbons), each optionally containing one or more unsaturated linkages, and being optionally interrupted by one or more heteroatoms selected from oxygen, nitrogen and sulphur; amino (preferably protected amino or mono-or disubstituted amino, e.g.
• alkylthio e.g. C]_20 alkylthio, preferably C ⁇ .
• chromophores include highly conjugated aryl or non-aryl systems, provided that such compounds are inert under the synthetic conditions employed in the solid phase chemistry, and so-called "push-pull" systems of the type G-J-M wherein G is an electron-donating group (e.g. amino or alkoxy), J is an conjugated array of multiple, e.g. double, bonds, and M is an electron withdrawing group (e.g. nitro or sulphonyl), the groups G and M being electronically linked through the conjugated array of multiple bonds.
• G is an electron-donating group (e.g. amino or alkoxy)
• J is an conjugated array of multiple, e.g. double, bonds
• M is an electron withdrawing group (e.g. nitro or sulphonyl)
• G and M being electronically linked through the conjugated array of multiple bonds.
• chromophore is the dansyl (l-dimethylamino-5-naphthylsulphonyl)
• the precise nature of the chromophore, its chemical properties and its absorbance characteristics, can be determined on a case by case basis depending upon the type of chemistry to be used in a given synthesis. It is most preferred however, that the chromophore is inert to the reactants and reaction conditions employed in the synthesis.
• chromophores include anthracenyl and dansyl (5-dimethylamino-l- naphthylsulphonyl groups, anthracenyl being particularly preferred.
• the quantitation of the substrate R can be either absolute quantitation or relative quantitation in which the relative amounts of the substrate R and another component in the synthesis mixture (e.g. another substrate, or starting material, or a side product for example) are determined.
• Relative quantitation makes use of the fact that the chromophore C u confers on each substrate a common set of characteristic absorbances that can be used as the basis for the spectrophotometric analysis.
• the analysis is carried out on a single bead, or a small number of beads, for example from 1 to 20 beads, more usually less than ten beads.
• Such beads typically have an average diameter in the range from 90 ⁇ m to 250 ⁇ m and a compound loading of between 0.1 mmol g-1 and 0.4 mmol g-1 average loading of an individual bead is less than 10 nanomoles, for example less than 5 nanomoles, e.g. approximately 1 nanomole.
• the invention provides a method of analysis of a solid phase construct; which method comprises:
• a chemical construct comprising a solid support Q (e.g. a resin bead having an average diameter in the range from 90 ⁇ m to 250 ⁇ m) having linked thereto via a connecting group Y a substrate R, the substrate R being present on each solid support in an amount of no more than 10 nanomoles, for example less than 5 nanomoles, e.g.
• a solid support Q e.g. a resin bead having an average diameter in the range from 90 ⁇ m to 250 ⁇ m
• the substrate R being present on each solid support in an amount of no more than 10 nanomoles, for example less than 5 nanomoles, e.g.
• the connecting group Y having first and second cleavage sites which are orthogonally and selectively cleavable; the second cleavage site being selectively cleavable to release the substrate; and the first cleavage site being located at a position between the second cleavage site and the solid support and being selectively cleavable to release a fragment Fr u comprising the substrate and at least a portion of the connecting group Y, wherein the said portion contains a chromophore C u which facilitates analysis of the fragment Fr u by ultra violet, visible or fluorescence spectrophotometry;
• UV chromophore-containing constructs of the present invention can be used to provide quantitative information on very small amounts of substrate compounds, for example amounts of the order of 1 nanomolar typically available from single resin beads of the size used in split and mix combinatorial methods.
• substrate compounds for example amounts of the order of 1 nanomolar typically available from single resin beads of the size used in split and mix combinatorial methods.
• combinatorial libraries formed by the split and mix method each bead typically will contain only a single substrate compound, but a library will contain a plurality of beads bearing different substrates. The analysis of mixtures of beads bearing different substrates is problematical and hence it is generally necessary to carry out analyses on single beads.
• the constructs of the present invention enable analysis to carried out efficiently and accurately at the single bead level, and in particular with beads of a size of the order of 90 ⁇ m to 250 ⁇ m in diameter with loadings of the order of 0.1- 0.4 mmol g-1.
• the inco ⁇ oration of a chromophore into the connecting group Y enables the substrate to be subjected to quantitative analysis.
• the fragment Fr u also contains a mass spectroscopic sensitising group G as well as the chromophore C u in order to facilitate qualitative analysis of the substrate as well as quantitative analysis.
• the sensitising group G renders the fragment Fr, and hence indirectly the substrate R, more sensitive to analysis by mass spectrometric techniques.
• the sensitising group can be a group which is readily ionisable under the conditions encountered in a mass spectrometer, and in particular electrospray mass spectrometry, to afford a strong signal.
• the inclusion of ionisable sensitising groups serves to ensure that the fragment is ionised sufficiently in the mass spectrometer to give a strong response. This overcomes a problem inherent in many molecules synthesised by solid phase methods where a suitable ionising group is not present and analysis by high throughput mass spectral techniques is problematical.
• the ionisable group can be for example a basic amino group or a carboxylate group but preferably it is a basic amino group. It will be appreciated that the term "basic amino group” as used herein refers in particular to an amino group, which is readily protonated.
• the basic amino group can be a primary amino group, a secondary amino group, or a tertiary amino group.
• the basic amino group can be for example, a cyclic amino group such as piperidino, piperazino, pyrrolidino, or mo ⁇ holino, piperidino or piperazino (e.g.N-methylpiperazino) being presently preferred.
• the chemical construct is configured such that cleavage at the first cleavage site forms or introduces on the chemical fragment Fr u at the first cleavage site a moiety comprising the mass spectral or other sensitising group G.
• the sensitising group G is formed or introduced by cleavage of the "skeleton" of the construct, and not by cleavage of a side chain or removal of a protecting group from a pendant sensitising group.
• the atoms or functional group making up the sensitising group G can be present in a masked form in the construct before cleavage of the fragment Fr u from the resin, the cleavage conditions merely serving to unmask the sensitising group G.
• the atoms or functional group making up or containing the sensitising group G can be introduced at the cleavage site during the cleavage reaction.
• the sensitising group is a basic amino group
• the nitrogen atom of the amino group can be present in the construct before cleavage, or it can be introduced during the cleavage reaction.
• the sensitising group G When the sensitising group G is introduced from an external source during the cleavage reaction, it can form part of a larger chemical moiety.
• the group G can be introduced as part of a group X-G wherein X is the residue of a nucleophile or electrophile, for example a nitrogen or sulphur-based nucleophilic group, e.g. a group of the formula G-Alk- Nuc wherein Alk is an alkylene group (e.g. a C2-20 an ⁇ ⁇ preferably C2-6 alkylene group) optionally interrupted by one or more heteroatoms selected from oxygen, nitrogen and sulphur, and Nuc is a nucleophile, such as an amine (e.g. NH or NR' where R' is a C ⁇ _6 alkyl group) or a thiolate group.
• a nucleophile or electrophile for example a nitrogen or sulphur-based nucleophilic group
• Alk is an
• the chemical fragment Fr u contains a means for imparting a characteristic signature to the mass spectrum of the fragment.
• the signature can advantageously be provided by inco ⁇ orating into the fragment a "peak splitting" isotopic label.
• the peak splitting isotopic label comprises at least one atom that exists in a number of stable isotopic forms.
• the mass spectrum will show the molecular ion as a characteristic doublet in which the peaks are of approximately equal height.
• the pu ⁇ ose of the peak splitting atom(s) is to provide a characteristic pattern, which will characterise any peak in the mass spectrum originating from the analytical fragment Fr, thereby distinguishing such peaks from those due to extraneous materials.
• Examples of atoms that can be used as isotopic peak splitting labels include 'H/ ⁇ H (D), 79 Br /8lBr, 12 C /13 C , 14 N /15 N and 16 0 /18 0 . I 2>
• the fragment Fr u can contain a single peak splitting isotopic label or more than one such label.
• the isotopic label can be a single bromine atom in which case the peak for the molecular ion of the analytical fragment Fr u liberated following cleavage from the solid support will appear as a doublet.
• a second or subsequent peak splitting label(s) By introducing a second or subsequent peak splitting label(s), a more complex peak pattern will be produced for the molecular ion.
• the isotopic peak splitting label(s) preferably is/are located between the first and second cleavage sites.
• the first and second cleavage sites can be defined by first and second linker groups L ⁇ and L-2.
• a "spacer group” A can be interposed between the two linker groups L ⁇ and b , the spacer group A containing or having linked thereto the UV chromophore C u .
• the group A typically also contains an isotopic peak splitting label.
• the connecting group Y has the formula L ⁇ -A-L ⁇ ; wherein L ⁇ is a first linker group defining the first cleavage site; A is a chemical group (the spacer group) containing or having linked thereto the UV chromophore C u , and optionally containing a peak splitting isotopic label; and b is a second linker group defining the second cleavage site.
• the first and second cleavage sites are orthogonally and selectively cleavable; i.e. the conditions used to effect cleavage at one of the cleavage sites will not cleave the other.
• a wide variety of different types of cleavage reaction can be used, examples being reactions selected from acid catalysed cleavage, base catalysed cleavage, oxidative cleavage, reductive cleavage, nucleophilic displacement, electrophilic displacement, and thermal, photochemical and enzymatic cleavage.
• Either the first or second cleavage sites/linkers, or both, can be of the "safety catch" variety; i.e.
• the cleavage site or linker group must be chemically modified in a first step before it can be subjected to cleavage in a second step.
• An advantage of such an arrangement is that it prevents or significantly reduces the possibility of cleavage taking place inadvertently.
• One example of a "safety catch" mechanism involves oxidation of a functional group in a first step, the oxidation serving to make the functional group more amenable to displacement by a nucleophile in a subsequent cleavage step.
• the nucleophile can vary considerably in structure.
• the nucleophile can be an amino group-containing nucleophile), the amino group participating in the nucleophilic displacement action such that the amino group is attached directly to the cleavage site.
• the amino group (or other sensitising group) can be present in a group (e.g. a dialkylaminoalkyl-thiolate anion) containing another nucleophile such as a sulphur nucleophile, which becomes attached to the cleavage site.
• a group e.g. a dialkylaminoalkyl-thiolate anion
• another nucleophile such as a sulphur nucleophile
• Linkers of the "Rink” or " norr” type typically comprise an N-protected 1 -amino- 1,1-diphenylmethane moiety, the amino group when deprotected allowing attachment to a substrate, one of the phenyl rings being substituted for example with dimethoxy groups and the other having a carboxyalkyloxy substituent providing a second point of attachment. Cleavage with TFA gives rise to a substrate compound having a terminal carboxamido group.
• Linkers of the "Wang" type typically contain a substituted phenoxyacetyl group, the acetyl group providing one point of attachment, and a benzylic hydroxyl group on the phenyl ring forming a second point of attachment.
• Esters can be formed between a carboxyl group of a substrate and the benzylic hydroxyl group, the ester groups being subsequently cleavable with TFA to release a substrate compound having a terminal carboxylate group.
• DBU 1,8- diazobicyco[5.4.0]undec-7-ene
• groups that can be cleaved by nucleophilic displacement include mercaptopyrimidine-based "safety catch" linkers such as 5-carboxy-2-mercaptopyrimidine, where cleavage can be effected by reacting under oxidising conditions to generate a sulphoxide or sulphone linkage, followed by reaction with a nucleophilic amino group to form a 2-aminopyrimidine.
• linkers can be cleaved, for example, by reaction with a cyclic amine such as piperidine or an N- substituted piperazine (e.g.N-methylpiperazine), or an amino group-containing thiolate nucleophile (e.g.
• dimethylaminoethylthiolate after first oxidising with an oxidising agent, preferably a realtively mild oxidising agent such as a per-acid or an inorganic per-salt such as potassium peroxymonosulphate (e.g. OxoneTM).
• an oxidising agent preferably a realtively mild oxidising agent such as a per-acid or an inorganic per-salt such as potassium peroxymonosulphate (e.g. OxoneTM).
• the first cleavage site (which can be , for example, defined by a sulphonamide linker) is selectively cleavable by nucleophilic displacement and the second cleavage site (which can be, for example, defined by a Rink linker) is selectively cleavable under acidic conditions.
• the first cleavage site (which can be defined by, for example, an ester linkage) is selectively cleavable by base and the second cleavage site (which can be, for example, defined by a Rink linker) is selectively cleaved under acidic conditions.
• first and second cleavage sites e.g. as defined by the first and second linker groups orthogonally cleavable
• the chemist can subject the construct to conditions suitable for cleaving off the analytical fragment Fr u thereby allowing analysis to be carried out to determine the outcome of each test reaction.
• a preparative reaction e.g.
• quality control can be carried out by removing a lb number of individual solid supports from the reaction vessel, cleaving the constructs at the first cleavage site and analysing the resulting fragment Fr u to see whether a particular reaction step has worked.
• the substrate R is released from the solid support.
• an advantage of the constructs of the present invention is that they can be used both at an experimental level to optimise a particular process step or for QC purposes, and also at a preparative level to release exclusively the substrate for screening pu ⁇ oses, without modifying the linker groups.
• the fragment Fr u and more preferably the spacer group A contains an alkylene diamine group or aminoalkoxy group.
• the precise size of the alkylene group and its degree of substitution is not currently considered to be important, but by way of example the chain could be from 2 to 30 carbon atoms in length, for example up to 20 carbon atoms, more typically less than 10 carbon atoms, particular examples being ethylene or propylene diamine or amino alcohol groups which may be substituted or unsubstituted.
• the alkylene diamine group or amino alcohol typically contains a peak splitting isotopic label as hereinbefore defined.
• the two amino groups can each be bonded to respectively the first and second linker groups.
• the alkylene diamine group can be substituted by an aryl group, for example an N-aryl group such as an N- benzyl group.
• the N-aryl group may optionally be substituted with one or more substituent groups.
• the chromophore C u may be formed as an integral part of the backbone or skeleton of the fragment Fr u (e.g. as part of the spacer group A), or it can be a pendant group.
• it when it is a pendant group, it can be linked to A either directly or via an alkylene chain (e.g. of 1 to 6 carbon atoms) optionally interrupted by one or more oxygen, nitrogen or sulphur atoms, or by means of an ether, thioether, amide, sulphonamide or ester linkage.
• the chromophore C u can, for example, be linked (directly or via an optionally interrupted alkylene chain as hereinbefore defined) to the nitrogen atom of the, or one the, amino groups.
• the chromophore C u can be linked to the nitrogen atom by means of an ethylene or propylene chain or by a sulphonyl group.
• the spacer group contains one or more mass-spectral peak splitting isotopic labels, these can be located either in the alkylene chain or in a substituent ' T- group attached to the alkylene chain.
• an N-benzyl group bonded to one of the two amino groups in an alkylenediamine can have a methylene group, which is substituted with the peak splitting atom deuterium.
• an aryl ring e.g. an N-benzyl group
• present in a substituent on the alkylene diamine can be substituted with a peak splitting bromine atom, one particular example of an aryl group being an N- ⁇ -bromobenzyl group.
• the chromophore C u can be linked, directly or indirectly, to the other of the amino groups.
• the chromophore C u is a polycyclic aryl group such as an anthracenyl or phenanthrenyl group linked to a nitrogen atom at one end of the diamine spacer group.
• spacer groups can be formed from hydrocarbon chains containing up to thirty or more carbon atoms in the chain which can be optionally interrupted with one or more heteroatoms such as oxygen, nitrogen or sulphur.
• the spacer can be for example a peptide chain containing one or more amino acids. The precise nature and length of the spacer is not currently considered to be important provided that the spacer does not interfere with the chemistry of the construct.
• the solid support Q can be any type of solid support suitable for use in solid phase synthesis, and in particular combinatorial chemistry.
• the solid support can take the form of beads, a solid surface, solid substrates, particles, pellets, discs, capillaries, hollow fibres, needles, solid fibres, or organic or inorganic gels such as silica gels, and insoluble organic particles such as particles formed from fullerenes.
• beads are polymeric beads such as cellulose beads or resin beads, particular examples of materials from which resin beads can be prepared including functionalised polymer resins such as polystyrene resins, polyacrylamide resins and dimethylacrylamide resins.
• suitable supports are listed in The Combinatorial Index by Barry A. Bunin, referred to above.
• the invention also provides a method of analysing the constructs hereinbefore defined; the method comprising cleaving the construct at the first cleavage site to release the chemical fragment Fr u and subjecting the fragment to UV, visible or fluorescence spectrometric analysis, for example quantitative UV analysis.
• ⁇ UV, visible or fluorescence spectrometric analysis
• the cleavage products can be subjected to chromatography, preferably a liquid chromatography technique such as HPLC, using a UV detector which can be set up to provide a means of determining concentrations of the various compounds as they elute from the chromatography column.
• the chromatography column can in turn be linked to another instrumental means of analysis for the pu ⁇ ose of identifying the substrate.
• a preferred means of instrumental analysis is mass spectroscopic analysis, and thus the method and constructs of the invention afford a means of means of achieving both identification and quantitation of the substrate in a single linked series of analytical procedures
• the mass spectrometric analysis is a " soft" mass spectrometric technique such as MALDI or electrospray or ion cyclotron mass spectrometry.
• the construct typically contains a mass spectrometric sensitising group as hereinbefore defined.
• the construct is configured such that cleavage at the first cleavage site generates on the chemical fragment Fr u a mass spectrometric sensitising group (e.g. a group which is ionisable under mass spectroscopic conditions), and then subjecting the chemical fragment to mass spectrometry, e.g. electrospray mass spectrometry.
• a mass spectrometric sensitising group e.g. a group which is ionisable under mass spectroscopic conditions
• the analysis of the fragment Fr u provides information on the reaction history of the construct. Thus, by mass spectrometric analysis, it can readily be determined whether or not the desired substrate has been formed in a given reaction sequence. Analysis of the fragment Fr u can therefore be used not only to characterise the substrate or product of the solid phase reaction sequence, but also to follow the progress of the reactions. Quantitation of the reaction products is achieved by means of the UV chromophore.
• the invention provides an intermediate chemical construct for use preparing a chemical construct as hereinbefore defined, the intermediate construct having the formula Q-Y' wherein Y' is a reactive or protected form of the group Y, and Q and Y are as hereinbefore defined.
• the invention provides an intermediate construct of the formula Q-L ⁇ -AP wherein Q and L ⁇ are as hereinbefore defined and AP is a reactive or protected form of the spacer group A containing a chromophore C u and optionally a splitting isotopic label.
• the intermediate construct has the general formula Q-L'-N(Alk- C u )-Alk-NH-X' wherein Alk is an alkylene group and ⁇ l is hydrogen or an aralkyl group.
• the intermediate construct is preferably isotopically labelled with a peak splitting combination of atoms such as 1H/H2 (D), 79 Br /8lBr, 12 C/13c, 1 >J/15N and I60/I80.
• the invention further provides a method of identifying a pharmaceutically useful substrate comprising preparing a library containing a plurality of chemical constructs as hereinbefore defined and subjecting the library to biological testing to identify biologically active substrates.
• the method may include the further step of formulating a biologically active substrate thus identified with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
• a construct containing an anthracenyl UV chromophore was prepared according to the synthetic route shown in Schemes 1, 2 and 3 below.
• the construct was provided with a sulphonamide linker at the first cleavage site and a Rink linker at the second cleavage site, an ethylene diamine chain connecting the two linkers and the two nitrogen atoms of the diamine chain serving as the points of attachment of the UV chromophore and either a peak splitting deuterium labelled benzyl group or an unlabelled benzyl group.
• Scheme 2 illustrates the attachment of the construct of Scheme 1 to a resin support and subsequent reactions on the resin
• Scheme 3 illustrates the attachment of different substrate groups to the constructs and subsequent cleavage experiments.
• the compound numbers used relate to the compounds identified in Schemes 1, 2 and 3. 10
• Dry aminomethyl Argogel® (0.87 g, 0.35 mmol) ), having an average bead diameter of 150 ⁇ m and a loading of 0.4 mmol g-1, in a reaction vessel flushed with dry nitrogen was swollen with dry dichloromethane (5 ml).
• the benzoic acid (1) (0.67 g, 0.93 mmol)
• PyBOP 0.728 g, 1.4 mmol
• 1 -hydroxy-benzotriazole (0.19 g, as
• Resin 2 (0.35 mmol) was swollen in dichloromethane (3 ml) and phenol (66 mg, 0.7 mmol) added, followed by trifluoroacetic acid (4.5 ml). The resin was gently agitated for 5 minutes using a stream of nitrogen then drained and washed with dichloromethane (2x5 ml). The process was repeated and, after draining, the resin washed (dichloromethane (3x8 ml), 10% diisopropylamine in dichloromethane (2x6 ml) and dichloromethane (4x8 ml)) and dried in vacuo. An analytical sample gave a positive Kaiser test.
• a sample of resin 3 (50 mg, 14 ⁇ mol) was treated with 20% v/v piperidine in 1 : 1 dichloromethane/dimethylformamide (2 ml) for 15 minutes.
• the resin was drained and washed (dichloromethane (3x2 ml), dimethylformamide (3x2 ml) and dichloromethane (3x2 ml)) then dried in vacuo, an analytical sample giving a positive bromophenol blue test.
• the reaction vessel containing the resin was flushed with nitrogen and dry 1 : 1 dichloromethane /dimethylformamide (0.5 ml) added.
• Resin 3 (214 mg, 59 ⁇ mol) was treated with 20% v/v piperidine in 1: 1 dichloromethane/dimethylformamide (3 ml) for 60 minutes.
• the resin was drained, washed (dichloromethane (3x3 ml), dimethylformamide (3x3 ml) and dichloromethane 93x3 ml)) and dried in vacuo, an analytical sample giving a positive bromophenol blue test.
• the reaction vessel was then flushed with nitrogen and the resin swollen with dry 1 : 1 dichloromethane- dimethylformamide (2 ml).
• a single bead of 7 was then selected and cleaved in the same way.
• the ratio of the components was again measured using HPLC.
• a solid phase construct was formed on hydroxylated polystyrene resin by coupling together a base cleavable ester group, a diaminoethyl spacer group, an acid cleavable Rink linker and a dansyl group as shown in Scheme 4 below. 3 ⁇
• the resulting resin 15 was filtered, washed with dimethylformamide (2 x 10ml), dichloromethane (5 x 10ml), methanol (2 x 10ml), dichloromethane (5 x 10ml) and dried under reduced pressure.
• UV analysis to detect the Fmoc group revealed that the cleavage reaction to release the primary amine 15 had been quantitative. The presence of a primary amine group was confirmed by a positive Kaiser test.
• a second construct including a dansyl group as the chromophore was prepared.
• the sequence of steps leading to the construct is shown in Scheme 5 below.
• the construct is analogous to the construct of Example 1 in that a sulphonamide linker group provides a first cleavage site to enable release of analytical fragment, and a Rink linker provides a second cleavage site to enable release of substrate.
• the chromophore rather than being linked to the sulphonamide nitrogen, is attached to a lysine group positioned between the ethylene diamine chain and the Rink linker. .
• the ester 3a (0.40 g, 0.80 mmol) in methanol (2 ml) was treated with IM aqueous sodium hydroxide (1.60 ml, 1.60 mmol) and stirred at room temperature for 1 hour. The reaction was then concentrated in vacuo and the residue dissolved in water (10 ml) then acidified with IM hydrochloric acid (1.80 ml, 1.80 mmol) at 0°C. The resulting white precipitate was extracted with dichloromethane (3 x 10 ml).
• the ester 3b (0.40 g, 0.80 mmol) in methanol (2 ml) was treated with IM aqueous sodium hydroxide (1.60 ml, 1.60 mmol) and stirred at room temperature for 1 hour. The reaction was then concentrated in vacuo and the residue dissolved in water (10 ml) then acidified with IM hydrochloric acid (1.80 ml, 1.80 mmol) at 0°C. The resulting white precipitate was extracted with dichloromethane (3 x 10 ml).
• the protected resin 5 (0.85 g, 0.34 mmol) was suspended in dichloromethane (2 ml) and treated with trifluoroacetic acid (2 x 4 ml) for 5 min. Solvents were removed by filtration and the resin washed with 10%) DIPEA solution in DMF (3 x 5 ml), dichloromethane (6 x 5 ml), DMF (6 MO x 5 ml), dichloromethane (6 x 5 ml), ether (2 x 5 ml). The amine resin was then suspended in
• Resin (6) (0.93 g, 0.28 mmol) was treated with 20% Piperidine in DMF solution (2 x 5 ml) for 10 min. Solvents were removed by filtration and the resin washed with DMF (6 x 5 ml), dichloromethane (6 x 5 ml), and ether (2 x 4 ml). The resin was suspended in DMF (5 ml) and dichloromethane (5 ml) and the Rink acid linker (0.92 g, 1.62 mmol) was added, followed by PyBOP (0.84 g, 1.62 mmol) and HOBT (0.22 g, 1.62 mmol). After 4 min. Hunig's base (0.42g, 3.24 mmol) was added.
• Resin (7) (70 mg, 0.02 mmol) was treated with 20% piperidine in DMF solution (2 x 1 ml) for 10 min. Solvents were removed by filtration and the resin washed with DMF (6 x 1 ml), dichloromethane (6 x 1 ml), ether (2 x 1 ml). The resin was suspended in DMF (0.5 ml) and dichloromethane (0.5 ml) and the appropriate acid (8a-d) (0.15 mmol) was added, followed by PyBOP (0.08 g, 0.15 mmol) and HOBT (0.02 g, 0.15 mmol). After 4 min. Hunig's base (0.04 g, 0.30 mmol) was added.
• Resin (9a-d) (ca. 70 mg, 0.02 mmol) was treated with 10% hydrazine in DMF solution (3 x 1 ml) for 30 min. Solvents were removed by filtration and each resin washed with DMF (6 x 1 ml), dichloromethane (6 x 1 ml), ether (2 x 1 ml). To each resin portion was then added dansyl chloride (40 mg, 0.15 mmol) and triethylamine (15 mg, 0.15 mmol) in dichloromethane (1 ml).

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