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WO2005090379A1 - Composes et capteurs destines a la detection d'hydrocarbures aromatiques polycycliques - Google Patents

Composes et capteurs destines a la detection d'hydrocarbures aromatiques polycycliques Download PDF

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Publication number
WO2005090379A1
WO2005090379A1 PCT/GB2005/001118 GB2005001118W WO2005090379A1 WO 2005090379 A1 WO2005090379 A1 WO 2005090379A1 GB 2005001118 W GB2005001118 W GB 2005001118W WO 2005090379 A1 WO2005090379 A1 WO 2005090379A1
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WIPO (PCT)
Prior art keywords
compound
pah
formula
hereinbefore defined
binding
Prior art date
Application number
PCT/GB2005/001118
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English (en)
Inventor
Laura Cano-Lerida
Paul Walton
Martin Rose
Jonathan Tarbin
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The Central Science Laboratory, 'csl'
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Publication date
Priority claimed from GB0405988A external-priority patent/GB0405988D0/en
Priority claimed from GB0502521A external-priority patent/GB0502521D0/en
Application filed by The Central Science Laboratory, 'csl' filed Critical The Central Science Laboratory, 'csl'
Publication of WO2005090379A1 publication Critical patent/WO2005090379A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton

Definitions

  • the present invention relates to novel compounds for selective binding of a polycyclic aromatic hydrocarbon guest species, a method for binding thereof; a process for the preparation of novel compounds; a novel sensor for detecting a desired polycyclic aromatic hydrocarbon species by binding or assay, novel components thereof, a sensing method for detection of a desired polycyclic aromatic hydrocarbon species by binding or assay, and the use thereof in nutritional, environmental and industrial applications and a method for the control of nutritional, environmental and industrial processes for the monitoring and/or control of a desired polycyclic aromatic hydrocarbon species.
  • PAHs Polycyclic aromatic hydrocarbons
  • PAHs and their derivatives are widespread in the environment as a result of combustion processes, such as burning fossil fuels. They bind strongly to soil organic matter (humic acids) and their rate of degradation in soil and other environmental compartments is usually slow. In addition, PAHs reaching watercourses are rapidly transferred to sediment.
  • soil organic matter humic acids
  • PAHs are also formed during domestic and industrial combustion processes such as extracting vegetable oils, herbs, spices and other food materials, smoking and grilling food materials and the like.
  • PAHs are toxic and carcinogenic compounds. The control of their presence and levels is of growing importance in the context of food safety and health and safety regulations. Several of the effects of PAHs are enzyme induction, immunosuppression, teratogenecity, and tumour promotion.
  • PAHs Polycyclic aromatic hydrocarbons generated in nutritional and industrial processes present a particularly difficult challenge for accurate detection.
  • Testing sensitivity to meet regulatory requirements is of the order of 2 micrograms/kg for detection of benzo[a]pyrene and some other PAHs.
  • numerous different tests must be conducted to determine any of a number of PAHs which may be present.
  • PAHs are also usually well solvated in oils whereby it is a major problem to get them to associate with any sensor surface.
  • molecular tweezers approach in recognition of electron deficient organic guests such as picric acid via formation of inclusion complexes in which they associate via electron rich aromatic substituents of non- polar bile acids (aromatic guests through 7T-7r interactions).
  • aromatic substituents of the bile acid OH groups have a spatial arrangement maintained by the semi-rigid cholic acid skeleton, such that they can form a sandwich type structure with guest molecules through 7T- stacking.
  • Maitra et al (Dsouza, L. J.; Maitra, U., J. Org. Chem. 1996, 61, 9494-9502) have synthesised various bile acid semi-rigid molecular tweezers in which pyrene units were joined to 3 -alpha and 12-alpha hydroxy groups, and studied the binding properties of these tweezers with polynitroaromatic compounds.
  • Dsouza, and U have synthesised various bile acid semi-rigid molecular tweezers in which pyrene units were joined to 3 -alpha and 12-alpha hydroxy groups, and studied the binding properties of these tweezers with polynitroaromatic compounds.
  • Dsouza, and U have synthesised various bile acid semi-rigid molecular tweezers in which pyrene units were joined to 3 -alpha and 12-alpha hydroxy groups, and studied the binding properties of these tweezers with polynitroar
  • a bile acid-based tweezer compound modified by a selective PAH binding group which acts as a synthetic receptor for PAHs.
  • a sensor may be provided comprising such a compound which is anchored securely at the sensor surface and which selectively and reversibly bonds to the species to be detected thereby allowing detection, and optionally additionally identification and quantification thereof.
  • each X is a linking group; and each X X is a group Ar which is independently selected from a C 6 -i2o fully or partially unsaturated fused aromatic ring system comprising from 1 to 30 fused aromatic rings substituted by a total of from 1 up to 20 halogen atoms selected from fluoro, chloro and or bromo, optionally comprising one or more heteroatoms selected from N, S, O, and optionally substituted by hydrocarbyl or heteroatom containing groups, halo, hydroxy, cyano, amino, carbonyl, and the like; and each n is a whole number integer from 1 to 3 and the sum of n is 2 to 6;
  • A is a group or moiety selected from lipophilic groups such as C 4 - 2 o aliphatic or alicyclic saturated or unsaturated hydrocarbon groups; a reactive linking group; or a moiety derived from reactive linking to a support or reporting element T; and
  • T is an optional support or reporting element and m is 0 or 1; and k is 0 or is a whole number integer selected from 1 to 3; wherein the groups Ar are pendant to at least two different rings of the planar B skeleton and are located on the same face of the planar B skeleton forming, together with B, a pocket suitable for receiving and binding a guest PAH species.
  • a compound of formula I may be chiral or non-chiral.
  • the compound of formula I is suitable for binding a planar PAH species by 7T-stacking and arene - perfluoroarene interaction, or equivalent interaction in the case of chloro or iodo analogue.
  • the arene -perfluoroarene interaction is an electrostatic interaction between quadrupoles of different charge distribution, arene being +ve and perfluoroarene being -ve.
  • a compound of formula I is preferably adapted to bind a PAH in a reportable chemical event.
  • a reportable chemical event may be reported by means of a transducer effect which provides a difference in detectable property of the compound of formula I and/or PAH to be detected in its free state and bound state, or by the immobilisation of either species in a specific location and in detectable manner indicating occurrence of a chemical event.
  • a reporting element T is suitably adapted for reporting binding of PAH.
  • a reporting element T is a transducer which provides a difference in detectable property when linked to compound of formula I in its free state and when linked to the compound of formula I bound to PAH.
  • T is adapted to be received in and bind in the pocket of the compound of formula I in its free state but is expelled from the pocket by the binding of a PAH species to be detected, ie the PAH species binds competitively over a reporting element T, and T provides an enhanced transducing effect, ie an enhanced difference in detectable property when bound within the pocket of compound of formula I and when expelled therefrom but linked to the compound of formula I bound to PAH.
  • A is a C 10 - C 2 o long chain aliphatic group which enables T to interact with the pocket of the compound of formula I.
  • a compound for binding an electron deficient polycyclic aromatic hydrocarbon comprising a "tweezer” having a planar hydrocarbon skeleton and a plurality of perhaloaromatic groups pendant thereto in cofacial or pseudocofacial orientation forming a pocket for binding a guest PAH, wherein the compound comprises a pendant long chain hydrocarbyl anchor anchoring a fluorescent transducer which is adapted to be give a measurable fluorescent signal on binding, preferably to be bound within the pocket and to be expelled by the guest PAH, and give a measurable fluorescent signal on expulsion.
  • PAH electron deficient polycyclic aromatic hydrocarbon
  • B comprises a fused alicyclic system as hereinbefore defined wherein all rings are of "chair” configuration whereby B is planar and substituents Ar are linked to the planar B skeleton in stereochemical fashion on the same face thereof, preferably in cofacial or pseudo cofacial fashion.
  • B comprises only fused rings, and lacks any spiro or bicyclo- structures.
  • B lacks any bulky pendant groups R which might obscure the pocket between groups Ar.
  • B is semi-rigid, whereby the conformation of the tweezer is maintained in manner to receive and bind PAH.
  • B is selected from a C 15 - 35 saturated fused alicyclic as hereinbefore defined preferably comprising from 3 to 6 fused 5 and/or 6 membered saturated hydrocarbon rings.
  • B comprises from 3 to 8, preferably from 3 to 6, more preferably 4 or 5 fused 5 and or 6 membered saturated hydrocarbon rings, preferably being fused along one -CH 2 -CH 2 - bond only of each ring.
  • B comprises a moiety
  • each o, p, q 0 or 1 and the sum thereof is from 2 to 3.
  • Preferably B comprises any natural or synthetic cyclopentano perhydrophenanthrene nucleus; or analogue thereof such as the cholesterol or lanosterol nucleus or a steroid nucleus such as the testosterone or ergosterol nucleus, optionally including heteroatoms or up to two double bonds or substituted as hereinbefore defined; or analogue thereof wherein the cyclopentano ring is absent or is replaced by a cyclohexano ring, ie a perhydrophenanthrene bile acid analogue or a cyclohexano perhydrophenanthrene bile acid analogue.
  • B may contain one or two unsaturated groups and preferably these are unconjugated to preserve the planarity of the nucleus B.
  • B is a readily available structure, whereby a cyclopentano perhydrophenanthrene bile acid nucleus is favoured.
  • Bile acids are preferred for their peculiar structural characteristics, with an array of hydroxyl groups lining one face, which provides excellent linking points for structural units to generate rigid molecular arrangements.
  • their "sticky" arm may be used for immobilisation
  • Each X is suitably a linking group, linking to B, selected from any 2 or more valent group, preferably any 2, 3 or 4 valent group including O, S, N, C and/or H atoms, more preferably selected from -0-, -OR x -, -R x O-, -S-, -NH-, -NR X -, - C(0)0-, -C(0)NH-, -C(0)NR x - and the like where R x is H or hydrocarbyl.
  • X is not -P- or -OP-.
  • each Ar comprises from 1 to 8 substituents R, wherein each R is independently selected from C ⁇ _ 25 saturated or unsaturated substituted or unsubstituted hydrocarbon, preferably each R is hydrocarbyl optionally substituted by hydroxy, ether, thioether, ester, cyano, amino, carbonyl and the like; or is any of hydroxy, ether, thioether, ester, cyano, amino, carbonyl and the like.
  • hydrocarbyl is C ⁇ - 8 alkyl or alkenyl, more preferably is methyl, ethyl, propyl or butyl.
  • halogen atoms are present in each Ar in a high concentration on a few rings, for example are present on 1, 2, 3, 4 or 5 up to 10 rings of each 1 to 30 ring structure.
  • each Ar comprises at least one di-halo, tri-halo, terra- halo or penta-halo aromatic ring, more preferably Ar is or comprises a tetrafluoro, pentafluoro, hexafluoro, heptafluoro or octafluoro naphthalene as part of the fused ring structure.
  • Different numbers of halogenated rings provide different strength of binding and sensitivity.
  • Ar groups are at equidistant separations spaced apart by B, or sufficiently spaced to part or fully receive a PAH molecule therebetween, ie to allow tweezing of the PAH species to be detected.
  • B nucleus is ideally suited to spacing apart of groups Ar for this purpose.
  • each Ar group is located at a different cyclic ring in the B nucleus, ie preferably only one Ar is pendant to any one B nucleus ring.
  • each Ar is substantially planar and preferably independently comprises a partially or fully perfluorinated 1 to 6 fused aromatic ring structure, alone or as part of a partially or fully saturated cyclic or alicyclic system; more preferably comprises a 1 to 6 fused aromatic ring structure which is optionally pendant to or substituted by aliphatic, alicyclic or aromatic groups optionally containing one or two heteroatoms or unsaturated bonds.
  • each Ar is selected from any perfluorinated 2 or more, more preferably 2, 3, 4, 5 or 6 fused aromatic ring system optionally alkyl substituted or having one or more end or intermediate fused 5 or 6 ring cycloalkyl units, preferably selected from perfluorinated single aromatic ring structures such as benzene and toluene; perfluorinated fused two aromatic ring structures such as naphthalene; perfluorinated fused three aromatic ring structures such as anthracene, phenanthrene and benz[a] anthracene; perfluorinated four aromatic ring structures such as pyrene and chrysene; or perfluorinated 5 or 6 ring structures; optionally substituted by one to three . ⁇ aliphatic, alicyclic or aromatic groups optionally perfluorinated and optionally containing one or two heteroatoms or unsaturated bonds.
  • each Ar independently is F 5 .
  • fluorobenzyl, fluorotoluyl, fluoronaphthyl, phenanthryl, anthracyl or pyrenyl most preferably is heptafluorobenzyl, heptafluoronaphthyl, heptafluorotoluyl, nonafluoro phenanthryl, nonafluoroanthracyl, octafluoropyrenyl or undecafluoropyrenyl.
  • the sum of all n is 2 to 3 whereby the compound of formula I comprises 2 to 3 groups Ar.
  • compound I comprises a compound of sub formula I 1
  • R and R are same or different groups R as hereinbefore defined and are selected from H or C 1-8 saturated or unsaturated hydrocarbon, optionally substituted as hereinbefore defined for R; and k is 0 or 1 and when k is 1,
  • A is C 4 - 20 branched carboxylic acid or ester, for example C 4 - ⁇ 0 providing a more rigid structure for example is 4-methoxy butanoic acid or 4-methoxy butanoic acid ester, or C ⁇ - 20 providing a more remote link to T as hereinbefore defined.
  • compound I comprises a compound of sub formula I ⁇ :
  • each Ar is selected from heptafluoronaphthyl and heptafluorotoluyl, wherein -X- is -0-, the sum of o, p and q is 2 to 3 and A, T and m are as hereinbefore defined.
  • the compound of formula I is selected to facilitate accommodation of the desired PAH, for example the number of groups Ar may influence the ease of access of a desired species.
  • PAHs are suitably for detection of electron deficient PAHs, however electron withdrawing (chlorinated and nitro) PAHs may also be detected by suitable modification.
  • the compounds of formula I and subformulae are stable to heat, solvents and light.
  • the compound comprises moderate PAH species binding affinity (Ka of the order of 5 mM “1 to 1 x 10 9 mM “1 , preferably 30 mM “1 to 8,000 mM “1 , more preferably 40 mM “1 to 7,000 mM “1 providing highly sensitive levels of detection.
  • Detection levels vary according to PAH to be detected, by virtue of the nature of binding to the compound of formula I, for example typical detection levels (see example) are: -50 mM "1 Cholic acid-OFT (CDC1 3 ); -600 mM "1 Cholic acid- OFN (CDC1 3 ); -6000 mM "1 Chenodeoxycholic-OFN (CDC1 3 ).
  • the compounds of formula I have an attraction affinity for the species to be detected thereby enhancing sensitivity of detection.
  • Binding of a PAH by a compound of formula I is preferably a reportable chemical event.
  • a reportable chemical event may be transduced to a difference in property of the compound of formula I and or PAH to be detected, in free state and binding or bound state, or may be identified by the immobilisation of either species in a specific location indicating occurrence of a chemical event.
  • the compound of formula I preferably comprises a reporting element T as hereinbefore defined, for example is labelled with a label as known in the art having a property enabling reporting of binding and detection or visualisation thereof, or the compound may comprise no reporting element T but reporting of binding and detection or visualisation may be enabled through a property of the PAH species to be detected.
  • a property enabling reporting is suitably selected from optical, electrical or spectral property such as luminescence eg fluorescence, surface particle resonance, visible colour and the like.
  • a reporting element T comprises a chromophore, or a fluorophore which is adapted to fluoresce on binding at a wavelength which is distinct from any natural background fluorescence of the compound or the PAH, or T comprises visible colour provided in the form of a coloured support.
  • At least one m is 1 and T is selected from a polymer or glass support such as a latex bead, a supporting polymer body or supporting glass body or the like, or from a chromophore or fluorophore or moiety, or a combination thereof.
  • a polymer or glass support such as a latex bead, a supporting polymer body or supporting glass body or the like, or from a chromophore or fluorophore or moiety, or a combination thereof.
  • T is a coloured latex bead or a well plate for multihost detection by assay, or alternatively or additionally is a fluorophore for fluorescence detection.
  • a latex label may be coloured enabling direct visualisation as known in the art of solid phase or supported chemistry, as are methods of supporting compounds thereon.
  • a group or moiety A as hereinbefore defined may be any group suitable for anchoring or linking to a support surface or reporting means T, or A may be any moiety derived from anchoring or linking to such support surface or reporting means T.
  • Chemical anchoring groups may include any chemical modifying groups which are known for anchoring chemical substrates to metal, glass, composite or polymer or like surfaces.
  • Physical linking groups A include groups providing electrostatic attraction to supports, or providing steric interaction and association.
  • A is selected from lipophilic groups such as C - 20 aliphatic or alicyclic saturated or unsaturated hydrocarbon groups for example C 8 octyl groups and the like, more preferably selected from C . 20 branched or straight chain alkyl carboxylic acid, ester, acid chloride, amide or ether, or carbonyl moiety derived therefrom, optionally including one or more unsaturated double or triple bonds, and optionally substituted by one or more hydroxy, halo, cyano, and the like groups, suitable for reactive linking to or reactively linked to a support or reporting element T as hereinbefore defined.
  • lipophilic groups such as C - 20 aliphatic or alicyclic saturated or unsaturated hydrocarbon groups for example C 8 octyl groups and the like, more preferably selected from C . 20 branched or straight chain alkyl carboxylic acid, ester, acid chloride, amide or ether, or carbonyl moiety derived therefrom, optionally including one or more unsatur
  • a group or moiety A suitable for covalent anchoring to a polymer for example suitably comprises a polymerisable moiety or comprises a reactive group capable of chain terminating a polymer chain.
  • a polymerisable moiety or polymer reactive group is selected from the group of acrylics, alcohols, allyls, amines, anhydrides, carboxylic acids, epoxides, isocyanates, silicones, styrenics, vinyl, vinyl esters, polyurethanes and the like.
  • a fluorophore may be selected from any fluorophore which fluoresces at a wavelength which is distinguishable from any background fluorescence, for example selected from fluorophores as disclosed in "Fluorescent tagging of physiologically important carboxylic acids including fatty acids, for their detection in liquid chromatography", Toyo'oka T, Anal. Chim. Acta. 465 (2002) 111-130, the contents of which are incorporated herein by reference, or equivalent reactive fluorophores for tagging amides, ethers, acid chlorides and the like.
  • Preferred fluorophores include but are not limited to the coumarins, the dansyls, the quinoxalines, the benzoxazoles, diazoles and triazoles, the oxacenes (eg Nile Blue), anthracenes and carbazoles.
  • a polymer or glass supporting body is suitably a well plate for use in assay of a number of samples or with a number of analytes.
  • Any desired multi well plate may be employed, preferably a polymer well plate, more preferably a polycarbonate well plate as is readily commercially available.
  • compound of formula I or its subformulae is attached by covalent or static attraction to the surface of the well plate T.
  • a plurality of same or different compounds of formula I may be anchored to different wells in the well plate, enabling assay of different samples for a particular PAH, or assay of one sample for different PAH's, or for one PAH in different concentrations.
  • a polymer support is a recognition polymer in which cavities are provided in the form of the PAH species to be detected and in which compound of formula I is immobilised in a binding configuration about the cavities in manner to facilitate binding of PAH entering the cavity.
  • the polymer thereby provides for selective detection of desired PAHs, and exclusion of non desired PAHs or spatially dissimilar species.
  • the compound of formula I concentrates PAH at the polymer surface and in the case of a recognition polymer, this facilitates PAH entering the cavities and binding.
  • immobilising compound of formula I minimises degree of movement and adds spatial constraints to non desired species to avoid false indication of detection.
  • a support T in the form of a polymer or recognition polymer may comprise any suitable polymer for immobilising compounds and is preferably selected from carboxylic acid polymers, polyurethanes, epoxy polymers, silicone polymers, and the like, preferably carboxylic acids such as carboxymethyl cellulose, sodium salt, acrylics such as polyacrylamide, acrylate esters, polyacrylic acid, polyacrylonitrile, maleic acid/esters, methacrylate esters, methacrylic acids such as poly(ethylene-co-methacrylic acid, amines such as vinylpyridines i.e. poly(4- vinylpyridine, vinylpyrrolidones i.e. poly(vinylpyrrolidone), amides such as nylon, epoxides such as poly(bisphenol A-co-epichlorohydrin), silicones such as poly(methylhydrosyloxane and the like.
  • carboxylic acids such as carboxymethyl cellulose, sodium salt
  • acrylics such as polyacrylamide, acryl
  • a polymer support may additionally comprise a reporting element T as hereinbefore defined, for example may be labelled with a label as known in the art for reporting of binding and detection or visualisation, or reporting of binding and detection or visualisation may be enabled through a property of the compound of formula I or the PAH species.
  • a support comprising immobilised compound of formula I or its subformulae as hereinbefore defined.
  • a support is a glass plate such as a multiwell plate, preferably a 96 well plate, or is a polymer body such as a latex bead or a molecularly imprinted polymer as hereinbefore defined.
  • B, X, A, T, m, k and n are as hereinbefore defined and each L is a leaving group or is a reactive group or is hydrogen
  • X X is as hereinbefore defined and L' is a leaving group or is a reactive group or is hydrogen.
  • Reaction is suitably conducted with excess of compound of formula III whereby the maximum substitution takes place.
  • Reaction products may include a mixture of mono, di, tri, terra, hepta and/or hexa ) substituted compounds which may be separated in known manner.
  • the reaction may be conducted in manner to introduce a desired number of groups ( T " ) in desired location.
  • reaction may be controlled by means of differential reactivities of the different XL groups or protection of desired XL groups as known in the art to direct the substitution.
  • a compound II is ofthe formula II 1'
  • contacting is under reflux.
  • contacting is in the presence of solvent such as DMF, C1 2 CH 2 and the like.
  • solvent such as DMF, C1 2 CH 2 and the like.
  • a template or catalyst is present such as CsF 2 .
  • the product comprises almost pure compound or mixture of compounds of formula (I) or its subformulae in which groups A ""N are located on the same side of the B ring system core, with a small amount of side product comprising a single group A X .
  • Undesired compounds of formula I or starting material may be recycled for further substitution or separated for example by column chromatography.
  • the compound II 1 may comprise residual reactive groups L which may be selected from groups R as hereinbefore defined or may be further reacted as known in the art to derivatise to substituents R, preferably to a group selected from alkyl, ether, halo, cyano, amino, carbonyl and the like.
  • a product compound of formula I may be further derivatised to interconvert groups R to alternative groups as hereinbefore defined, using methods as known in the art.
  • a product compound of formula I may be further reacted as known in the art to introduce at A a support or reporting element T as hereinbefore defined.
  • a polymerisable moiety or polymer reactive group A as hereinbefore defined may be introduced suitably by the reaction of a compound of formula I with a polymerisable monomer or suitable substrate such as a reactive acrylic, alcohol, allyl, amine, anhydride, carboxylic acid, epoxide, isocyanate, silicone, styrenic, vinyl ester, and the like.
  • a polymerisable monomer or suitable substrate such as a reactive acrylic, alcohol, allyl, amine, anhydride, carboxylic acid, epoxide, isocyanate, silicone, styrenic, vinyl ester, and the like.
  • Introduction of polymerisable moiety or polymer reactive group may be performed before or after perfluorination.
  • the compound incorporating a support T such as a latex label as hereinbefore defined may be prepared by known means and reference is made to the general knowledge relating to solid phase compound libraries and the like.
  • the compound of formula I incorporating a reporting element T in the form of a fluorophore as hereinbefore defined may be prepared by known means for example as disclosed in Toyo'oka above, suitably by reaction of a compound of formula I comprising a reactive linking group or moiety A as hereinbefore defined, with a reactive fluorophore.
  • a reactive fluorophore is preferably a hydrazine reagent, amine reagent, bromide, hydroxyl reagent or sulfonate (OTf).
  • the compound of formula I where T is a fluorophore and A is a moiety as hereinbefore defined is suitably prepared by a process comprising contacting the compound of formula I where m is 0 and A comprises a reactive group as hereinbefore defined with an activator, and coupling to a reactive fluorophore.
  • the compound of formula I where T is a fluorophore and A is derived from a reactive carboxy anchor is prepared by a process comprising contacting with an activator for the carboxy group, such as chloropyridinium iodide, oxalyl chloride, diisopropyl carbodiimide, phosphonitrilic chloride, or the like, optionally with a further reactive intermediate group such as provided by n-OH succinimide or the like, and coupling to the reactive fluorophore.
  • an activator for the carboxy group such as chloropyridinium iodide, oxalyl chloride, diisopropyl carbodiimide, phosphonitrilic chloride, or the like
  • a further reactive intermediate group such as provided by n-OH succinimide or the like
  • the preparation of a compound of formula I as hereinbefore defined immobilised on a polymer or glass plate may be performed by any method as known in the art. Suitably immobilisation is to a cation exchange matrix, which is negatively charged, or by alternative means as known in the art.
  • the preparation of a compound of formula I as hereinbefore defined immobilised in a polymer such as a recognition polymer comprises polymerising an amount of monomers capable of reacting with a compound of formula I or its subformulae as hereinbefore defined, in the presence of a compound of formula I as hereinbefore defined.
  • the method is a method for preparing a recognition polymer as hereinbefore defined, it comprises additionally providing an amount of a recognition element comprising a PAH template to be detected, in the presence of reacting monomers and one or more compounds of formula I, polymerising and removing the PAH template.
  • the compound of formula I binds to a recognition element comprising template PAH in the monomer solution, and polymerisation fixes this conformation, with subsequent removal of PAH to leave a selective binding cavity.
  • monomers comprise any monomer which is capable of reacting with the compound of formula I and which provides desired polymer properties, for example selected from reactive acrylic, alcohol, allyl, amine, anhydride, carboxylic acid, epoxide, isocyanate, silicone, styrenic, vinyl ester, and the like.
  • Reaction includes for example the reaction of a carboxylic acid and amine, an isocyanide and amide, transesterification or the like
  • a recognition polymer is prepared by either the pre-organised (or covalent) procedure, where the aggregates in solution prior to polymerisation are maintained by covalent bonds (reversible, in the extraction of the template phase), or the self-assembly (or non-covalent) procedure, where the pre- arrangement between the template molecule and the functional monomers is formed by non-covalent or metal coordination interactions.
  • the polymerisation is conducted with a compromise between a too cross-linked network, that does not allow mass transfer (removing the template PAH and rebinding of analyte PAH), and one that is not cross- linked enough, and would lose the cavity shape upon removal of template.
  • the method for preparing recognition polymer comprises:
  • Porogen dissolves components, encouraging formation of pores and drives reactions but does not interfere with the reaction (or selectivity), or recognition and binding.
  • Porogen may be selected from any solvent for the monomer, PAH template or compound of formula I, and is more preferably selected from oils such as vegetable oils, halogenated solvents, C 5 - 10 alkanes, alcohols, acetonitriles, ethers, ketones, aromatics and water, such as dichloromethane, trichloromethane tetrachloromethane, pentane, hexane, methanol, ethanol, DMF, PET-ether, acetone, DMSO, benzene, toluene, water and deuterated analogues.
  • Some porogens such as chloroform create large pores whilst others such as DMSO generate a glassy not very porous surface.
  • porogen may include any swelling agent as solvent, to generate porosity.
  • porogen is a polar solvent. This will enhance the non-polar interactions ( ⁇ - ⁇ mainly) between the template PAH and the monomers allowing them to prearrange.
  • the polymerisation is activated, with the addition of the cross-linkers and initiators, the three dimensional favourable arrangement of the components will be locked inside the polymeric network.
  • Cross linkers are selected from vinyl monomers, amide and acrylamide monomers, acrylate and mefhacrylate monomers, carboxylate monomers and the like; preferably cross linkers are selected from 4-divinylbenzene, N,N' -methylene - bisacrylamide, N,N' - phenylene bisacrylamide, 2,6-biacrylamidopyridine, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, and mixtures thereof, structures of which are shown in Figure 2. Extraction may be by any means which disrupts the non covalent interactions of the complex, for example washing, removal of porogen, resuspending in low polarity or non polar solvent or the like
  • pulverisation employs particle size reduction and sieving, and sedimentation. These processes are very important for example if the polymer is to be used for applications such as to pack a chromatographic column.
  • a method for detecting a PAH as hereinbefore defined comprising combining a sample to be detected for the presence of an amount of a PAH in fluid form or present in solvent, with the compound of formula I or its subformulae, supported or unsupported, as hereinbefore defined and detecting the presence or absence of a reportable chemical event indicative of binding.
  • the PAH is of known structure and present in unknown amount.
  • a reportable chemical event may be any optical, electrical or spectral or like change as known in the art and as hereinbefore defined.
  • An event may be a positive or negative result depending on the nature thereof. Accordingly no event may indicate that binding has in fact occurred.
  • the method comprises preparing a sample to be analysed for presence of PAH, and adding compound of formula I or its subformulae, optionally in the presence of a competition species capable of binding compound of formula I or of binding PAH to be detected and detecting for a reportable chemical event.
  • the compound of formula I or a PAH competition species or both comprises reporting element T as hereinbefore defined or comprises other inherent reporting means as hereinbefore defined.
  • the PAH competition species may be immobilised on a support or compound of formula I or its subformulae may be immobilised on a support.
  • a PAH competition species is selected from the PAH to be detected or a PAH analogue or PAH template or the like.
  • a compound of formula I or its subformulae as hereinbefore defined is adapted to selectively and substantially reversibly bind the PAH species to be detected, in manner that the binding thereof causes a detectable signal to be emitted or prevents a detectable signal from being generated or causes a localised concentration of PAH species, enhancing the existing detectable signal.
  • the compound is adapted to release the species at a characteristic voltage or give an absorbance measurement at a characteristic optical wavelength, or to emit a fluorescence, luminescence, or radioactive signal, chromophoric signal or spectral change, eg by NMR titration; or compound labelled with a reporting element is adapted to bind to sample PAH preventing binding to an immobilised test line of PAH competition species, thereby preventing immobilising reporting element at the immobilised test line and preventing detection of reporting element at the immobilised test line; or immobilised compound is adapted to preferentially bind to sample PAH preventing binding to PAH competition species labelled with a reporting element, present in the sample, thereby preventing immobilising reporting element at the immobilised test line and preventing detection of reporting element at the immobilised test line.
  • a reporting element such as latex operates by being attached to individual molecules and being visibly coloured, whereby the location of the attached molecule is indicated.
  • NMR titrations look at the chemical shift of a proton around the binding site and follow the chemical shift change when the host is bound to the guest.
  • a series of NMR spectra may be taken of a number of solutions screening the range of molar fractions of the host from 0 to 1.0. These spectra may then be analysed to find any shifting peaks. These shifting peaks represent nuclei close to the binding site, whose environment are influenced by the binding process. Converting to a value for binding constant indicates characteristic binding and thereby the nature of the bound species.
  • Luminescence eg fluorescence may be emitted when one of the compound of formula I or the species to be detected, ie the PAH, is luminescent, eg is a fluorescent probe.
  • Excimer fluorescence is widely used to determine dissociation constants, especially in systems involving detection of pyrene, perylene, dansyl or the like moieties or incorporating as a group Ar in a compound of formula I as hereinbefore defined, as pyrene is, along with perylene and dansyl, one of the most popular fluorescent probes.
  • Excimers are excited state complexes formed between two identical species, one of which is in the excited state and the other in the ground state. Chromophore emission such as fluorescent light emission is detected by intensity as number of photons, or strength of radiosignal from radioisotopes, and converting to nanomoles of PAH using a suitable chart or conversion index.
  • Surface plasmon resonance employs a polarised light source and detects light at the surface of a sensor of the invention, light coming from the side of higher refractive index is partly reflected and partly refracted. Above a certain critical angle of incidence, no light is refracted across the interface, and total internal reflection is observed. While incident light is totally reflected the electromagnetic field component penetrates a short (tens of nanometers) distance into a medium of a lower refractive index creating an exponentially detenuating evanescent wave.
  • the interface between the media is coated with a thin layer of metal (gold), and light is monochromatic and p-polarized, the intensity of the reflected light is reduced at a specific incident angle producing a sharp shadow (called surface plasmon resonance) due to the resonance energy transfer between evanescent wave and surface plasmons.
  • the resonance conditions are influenced by the material adsorbed onto the thin metal film. Any chemical receptor can be adapted to SPR technique provided it has sufficient selectivity for the analytes or the samples are of such a nature that only the specie of interest is going to become attached to the receptor.
  • the resonance conditions are influenced by the material absorbed onto the thin metal film. Satisfactory linear relationship is found between resonance energy and mass concentration of biochemically relevant molecules such as proteins, sugars and DNA.
  • the SPR signal which is expressed in resonance units is therefore a measure of mass concentration at the sensor chip surface. This means that the analyte and ligand association and dissociation can be observed and ultimately rate constants as well as equilibrium constants can be calculated.
  • the compounds of formula (I) or the PAH to be detected are strongly fluorescent molecules and are conducive to fluorescence detection methods.
  • a fluorescently tagged compound of formula I exhibits a reportable change in fluorescence intensity on binding a PAH.
  • the detectable fluorescence is that of the PAH, typically in the range ⁇ 500nm, fluorescence intensity typically decreases on binding.
  • the detectable fluorescence is that of a fluorophore T in a compound of formula I as hereinbefore defined fluorescence intensity typically increases on binding or is activated on binding.
  • Immobilisation of compound or PAH or PAH competition species indicates location of fluorescent species and therefore whether binding has or has not taken place.
  • a reporting element T is present in the form of latex beads as hereinbefore defined or the like which show a visible colour, and immobilisation of compound or PAH competition species, for example as mentioned above, indicates location of reporting element and therefore whether binding has or has not taken place.
  • a method for detecting a PAH as hereinbefore defined comprises combining a plurality of samples with a plurality of same or different compound of formula I comprising fluorophore reporting element T and support means T in the form of a well plate as hereinbefore defined, each sample being introduced into a different well.
  • each well comprises a different host compound of formula I
  • a plurality of different samples may bind differently to each compound. Detection of fluorescence by means of a fluorescence micro array spectrometer or a multi channel fluorescence spectrometer generates a fingerprint for the amounts and type of PAH bound in each well, giving a one stop full speciation. A full concentration analysis may be derived with use of suitable algorithms.
  • the method employs from 3 to 30 different host compounds of formula I more preferably from 3 to 12 such as 6 to 8.
  • the method may be used to co ⁇ ect for solvent by introducing a sample blank into one or more wells.
  • the method may comprise repriming of well plates in known manner.
  • the compound may be suitable for binding any desired PAH species, for example any non functionalised electron deficient PAH, preferably any C ⁇ _ 30 fused ring unsubstituted planar PAH or PAH substituted by 2D-substituents including simple PAH metabolites, preferably a 2 to 6 ring PAH as listed below or electron deficient analogues, or simple metabolites which do not destroy the planarity of the PAH, and which include C1-, OH- and CH 3 O- substituted PAHs such as:
  • Binding may be in any suitable stoichiometry and is preferably compound: PAH 1 :1 or 2:1.
  • PAH 1 :1 or 2:1 the size of the PAH species or of the binding pocket may affect the stoichiometry.
  • Binding may also be in any binding affinity.
  • differential binding of different PAH's allows for speciation, for example using multi algorithms
  • the compound may be useful in binding PAHs present in any environment, for example pesticides, oil residues, contaminated water, land or crops or grass, heat treated or dried or smoked products such as vegetable oils, dried spices or herbs, smoke or flame cooked foods and the like.
  • the solvent is selected from polar and non polar solvents and oils.
  • the compounds of formula I bind PAH present in polar and non-polar solvents alike.
  • the binding affinity of the compounds is so favourable that they attract a PAH from its solvent or medium.
  • a solvent is water based and is modified to enable extraction of PAH into the solvent, preferably a solvent is polar and is a solvent for both the PAH and the compound of formula I or subformulae.
  • a solvent is more preferably selected from oils such as vegetable oils, halogenated solvents, C 5 . 10 alkanes, alcohols, acetonitriles, ethers, ketones, aromatics and water, such as dichloromethane, trichloromethane tetrachloromethane, pentane, hexane, methanol, ethanol, DMF, PET-ether, acetone, DMSO, benzene, toluene, water and deuterated analogues.
  • the binding constant of a species typically varies in different media, and selection of suitable solvent is therefore made as a balance between sufficiently low polarity to achieve species availability and yet at a sufficient polarity to achieve a detectable binding level. For example binding in CHC1 3 is ofthe order 10 less than that in water.
  • Measurement may be of liquid samples directly or of analyte extracted from soil or from industrial process streams or effluent and made up as a sample.
  • Sample preparation is known in the art and includes grinding, macerating, extracting etc the sample to be analysed, and purifying extracts from other components.
  • Oil phase samples may be used neat or diluted. Extracts may be conveniently obtained by subjecting to ultrasound to break down signal distorting contaminants, and to break down polymerised species to detectable form.
  • the analyte is chromatographically mobile or comprises a chromatographic transport facilitating agent.
  • the affinity for PAH species of the compounds of formula I and subformulae is so high that the PAHs are effectively extracted from their solvent and concentrated at the compound of formula I, for example at the surface of a sensor comprising the compound of formula I.
  • the method is suitable for the measurement of PAH as hereinbefore defined down to 0.1 nanomoles of PAH.
  • the method and compound of the invention are ideally suited to detecting PAHS from as low as 0.01 microg / kg, with no upper limit of detection. If a sample comprises too high a level of PAH for detection or quantification, sample dilution enables detection and quantification, and reverse calculation to determine sample concentration. Accordingly if no signal is obtained or saturation point is reached, changing solvent may affect the range of detection, facilitating or discouraging binding, as known in the art.
  • binding is rapid and characterised by kinetics of binding of the order of seconds, in the case of both host and guest being in free start, to minutes in the case of one of host and guest being in immobilised state.
  • Speciation to verify the nature ofthe guest PAH species is suitably by any known means such as detecting wavelength of fluorescence, or detecting the binding constant of a Host-Guest complex, if a species characteristic binding constant is known. Characteristic binding constants may be determined, if not known, using a sample known to contain the PAH which it is desired to detect. Before any determination of association constant is performed it is necessary to determine the stoichiometry of the complex (method of continuous variations). With these set of spectra, and using a graphical method, the Scatchard (Foster-Fyfe) method, it is possible to determine the K a . Subsequent removal and rebinding experiments may be conducted to provide evidence for the selectivity of this material in binding PAHs with regard to structural analogues. Removal is suitably as hereinbefore defined by washing or resuspending in non polar solvent.
  • a sensor comprising a contact surface for contacting with a sample to be detected, the contact surface comprising immobilised compound of formula I or its subformulae as hereinbefore defined or comprising means for immobilising or accumulating said compound of formula I or its subformulae or comprising means for inhibiting accumulation thereof.
  • the contact surface acts to accumulate PAH, if present in a sample, at the contact surface comprising immobilised compound of formula I; or acts to accumulate complex of compound of formula I and PAH, if PAH is present in a sample, at the contact surface, by the presence of means for immobilising the complex; or acts to inhibit accumulation of compound of formula I at the contact surface, if PAH is present in a sample, by the presence of means for immobilising free compound of formula I at the contact surface but not for immobilising complex of compound of formula I and PAH at the contact surface or comprises a competition species.
  • a contact surface comprises compound of formula I for example as a coating or impregnant or layer which may be across the entire surface or part thereof for example as a cross flow stripe, or is designed to accumulate compound of formula I at the surface, for example comprises a means for binding compound of formula I such as a reactive group on the compound or on a reporting element as hereinbefore defined or such as immobilised PAH competition species for example as a coating or impregnant or layer which may be across the entire surface or part thereof for example as a cross flow stripe.
  • a sensor may be suitable for direct or competition type assay, whereby the immobilised compound of formula I is specific for the accumulation of a desired PAH, and the PAH in a sample may be bound by compound of formula I giving a change indicative thereof as hereinbefore defined; alternatively the compound of formula I may competitively accumulate PAH in a sample also containing bulkier PAH competition species, giving a change indicative of one and not the other or a different change depending on which binding event occurs; alternatively free state PAH in a sample may compete for binding of compound of formula I with immobilised PAH competition species thereby preventing accumulation of compound of formula I at the sensor surface.
  • a sensor may comprise immobilised compound of formula I, immobilised PAH competition species, or means for immobilising any of compound of formula I, sample PAH, PAH competition species or complex thereof.
  • references to a method for detection using a compound of formula I are to be read to include a method for detection using a sensor comprising compound of formula I or means for immobilising compound of formula I.
  • the sensor suitably comprises or is used with detection means for detecting binding.
  • Detecting means may comprise any electrical, optical, mass, or other known detector, for example in the form of an electrode or the like to accumulate charge generated by binding or dissociation; an optical source and/or detector to generate or detect an absorbance or fluoresence signal; a surface to immobilise a species thereby giving a visual signal, a mass spectrometer or other analytical apparatus to detect mass, structure or the like.
  • a PAH or a compound of formula I or its subformulae as hereinbefore defined may comprise or be labelled with a radiolabel, chromophore or fluorophore or spectrally detectable atom or the like, suitable for detection by means of radiation detection, spectrophotometric detection, spectral detection or visually detectable structure or label, for example for fluorescence detection or detection by the naked eye. Fluorescence detection may be by the naked eye or by intensity measurement using a suitable detector. A label may be visually detectable or example by colour.
  • the senor comprises a display which clearly displays the result of binding, for example a fluorescence or mass sensor indicates detection in a numerical display, or comprises a template on a chip as a means of transducing a signal from a binding event, or is adapted to be received in a reader, which reads and converts a sensor result into a semi-quantitative result.
  • a display which clearly displays the result of binding
  • a fluorescence or mass sensor indicates detection in a numerical display
  • a template on a chip as a means of transducing a signal from a binding event
  • a reader which reads and converts a sensor result into a semi-quantitative result.
  • the sensor may provide for a signal to be generated or diminished or enhanced on binding, for example if the PAH is fluorescent and is accumulated by the compound of Formula I, this may be detectable over the background stream of non concentrated (non accumulated) PAH.
  • the contact surface comprises a polymer or a recognition polymer as hereinbefore defined or comprises a PAH competition species as hereinbefore defined or polymer form thereof.
  • the sensor thereby provides binding sites in cavity walls for selective sensing of desired PAHs, and exclusion of non desired PAHs or spatially dissimilar species.
  • the sensor may be multi use or may be for disposable single use.
  • the sensor may be suited for immersing in a sample cell, or may be in the form of a probe for inserting directly into material to be sampled, for example mounted in a product or effluent stream to provide continuous measurement of PAH concentrations or in a reaction vessel to provide continuous measurement of PAH concentrations in the course of a production reaction.
  • the sensor may be provided as an array or as a single contact sensor.
  • the sensor may comprise any known sensor type, and preferably comprises a lateral flow device, a BIAcore ® device, a template on a chip for performing a competitive binding assay, or the like.
  • the senor comprises a test kit or device in the form of a lateral flow kit or device comprising a lateral flow membrane linking an application zone for applying analyte with a downstream binding zone comprising a compound of formula I or its subformulae as hereinbefore defined or having means for accumulating or inhibiting accumulation of compound of formula I or its subformulae, for example comprising a PAH competition species as hereinbefore defined, and optionally and preferably additionally comprising a control zone downstream thereof for indicating the presence of a control substrate in a sample to be detected.
  • Lateral flow devices are known for example from EP291194.
  • the application zone comprises an absorbent material portion and is in capillary flow communication with a solid phase membrane comprising along the length thereof or at one or more portions thereof a compound of formula I or its subformulae, forming a binding zone or zones.
  • the binding zone comprises compound of formula I directly coated or coated as a recognition polymer as hereinbefore defined, or comprises immobilised PAH competition species, as hereinbefore defined, or may comprise other means for competitive assay of PAH using any method as known in the art.
  • the control zone suitably comprises one component of a two component reaction, the second component being suited for incorporating in sample to be detected, the reaction product thereof being detectable by same or different means to that used for detecting the binding of PAH at the binding zone.
  • the control zone and sample may each comprise one or other component selected from an antigen and antibody, one of which, suitably the sample component, is suitably inherently detectable, for example is fluorescent, or is labelled with a reporting element as hereinbefore defined, the reaction product formed thereof at the control zone being visible by fluorescence or visually detectable, for example by colour.
  • Control reactions and reagents are known in the art. In a particular advantage a control reaction is semi-quantitative and can be used as a zero reference for the PAH binding or non-binding.
  • the compound of formula I or PAH competition species and the control reagent, where present may be provided by impregnating or otherwise coating the lateral flow membrane at the desired zone on the lateral flow membrane as a coating strip from one side of the membrane to the other perpendicular to the lateral flow direction.
  • the other of compound of formula I and PAH competition species is provided in an analyte medium supported on a reporting element such as polymer beads or particles, typically latex beads. The analyte medium is then suitable for combining with sample to be detected for presence of PAH, whereby a competitive assay may be performed.
  • the lateral flow sensor may be suited for detection of one or more PAHs, by means of one or more binding zones comprising one or more different compounds of Formula I or one compound of Formula I present on one or more different recognition polymers, or comprising one or more different PAH competition species.
  • the lateral flow sensor suitably provides for detection for example by visualisation at the binding zone of binding or non binding of PAH, for example in the case of competition assay, in which binding of sample PAH inhibits binding of labelled compound of formula I or PAH competition species, by means as hereinbefore defined, but may provide for additional detection of binding or non binding or level of binding downstream thereof.
  • the binding zone is present on a non-absorbent, preferably a glass fibre material.
  • a non-absorbent preferably a glass fibre material.
  • the solid phase membrane provides for lateral flow as known in the art, and prevents reverse flow. Suitable materials for the membrane include nitrocellulose and nylon.
  • a sensor comprising immobilised PAH competition species, for use in a method for detecting a PAH species as hereinbefore defined, the sensor being adapted to accumulate compound of formula I in the event that the sample is negative, and to accumulate no species in the event that the sample comprises PAH.
  • a method for the manufacture of a sensor as hereinbefore defined comprising providing a contact surface comprising a compound of formula I or its subformulae as hereinbefore defined or comprising means for accumulating a compound of formula I or its subformulae or comprising PAH competition species as hereinbefore defined.
  • the method may include providing a reporting element for detecting binding of PAH if appropriate. Detection means may be integral with the binding surface or separate therefrom.
  • the compound of formula I is provided by anchoring by chemical means, or means for in situ anchoring are provided.
  • the sensor surface is provided in the form of a layer of polymer or recognition polymer, or immobilising means therefor specifically PAH competition species as hereinbefore defined.
  • a method for PAH detection with use of a sensor as hereinbefore defined comprising contacting a sample to be detected in fluid phase with a sensor as hereinbefore defined and detecting a signal or lack or signal indicating the presence of a PAH species, optionally with conversion thereof to indicate nature and/or concentration of species.
  • a sample of a solid phase material to be investigated is provided in fluid phase by fluid phase extraction.
  • Contact may be by means of immersion or of application of an amount of sample to a sample zone or otherwise to the sensor surface.
  • the method is operated as a screen which is suited for rapidly screening many compounds, and gives a positive or negative result, without speciation of PAH nor quantification.
  • a more complex method will allow speciation and quantification, and may even provide for separation of PAH.
  • the method using a lateral flow device as hereinbefore defined comprises spotting a sample of analyte, comprising sample and analyte medium comprising compound of formula I or PAH competition species associated with a detection element such as latex as hereinbefore defined, onto the application zone of a lateral flow applicator, allowing sample to migrate or diffuse over the solid phase lateral flow membrane as hereinbefore defined to the binding zone comprising compound of formula I as hereinbefore defined or immobilisation means therefor, optionally in the form of polymer or recognition polymer, or PAH competition species as hereinbefore defined and detecting binding or lack of binding, for example by placing the device under a lamp and detecting presence or absence of fluorescence, and optionally intensity of fluorescence at the binding zone, or by visual inspection.
  • a detection element such as latex as hereinbefore defined
  • a control zone may be present downstream ofthe binding zone as hereinbefore defined, and shows as a separate signal on contact with analyte, irrespective of PAH content, verifying that analyte has reached and passed the binding zone, in the case that no binding signal is detected.
  • a method for the regeneration or priming of a sensor as hereinbefore defined comprising disrupting the complex with PAH, for example by means as hereinbefore defined. The method may be useful for changing the specificity of the sensor to detect a different species, or simply regenerating the existing sensor for prolonged lifetime.
  • a method for effluent control or contaminant detection or the control of nutritional, environmental and industrial processes for the monitoring and/or control of a desired polycyclic aromatic hydrocarbon species using the compound or sensor of the invention as hereinbefore defined.
  • the method is a method for detection or process control of an industrial process known to generate PAH species as an undesired byproduct or on malfunction of the method, for example by operating at excessive heat, pressure or the like.
  • the method comprises monitoring PAH with use of the detection or sensing method and compound or sensor as hereinbefore defined, in situ in real time operation, optionally periodically obtaining values for the concentration of PAH in a given location and deriving parameters to maintain or alter the process controls for continued efficient operation.
  • the method involves the use of suitable software and instrumentation to receive and process signals and relate to a calibration logarithm to produce concentration values.
  • a method or sensor as hereinbefore defined in nutritional, environmental and industrial applications such as nutrient growth and development, food preparation and manufacture and the like, contaminant detection or remediation and the like.
  • Typical uses are in relation to monitoring or detecting PAHs as a result of combustion processes, such as burning fossil fuels, including growing crops in the vicinity of combustion sources or in proximity to roads or other poluting thoroughfares, or growing on land affected thereby, or in environments affected by such combustion processes or their effluents, including growing crops on land contaminated by PAHs in environments affected by pesticides, during domestic and industrial combustion processes such as extracting vegetable oils, herbs, spices, beans (eg coffee, cocoa) and other food materials, smoking and grilling food materials (fish, meat) and the like.
  • the use is in binding and detecting PAHs in solids such as soil, food and the like, liquid extracts thereof, or fluids such as oils, aqueous fluids and extracts and the like.
  • cyclopentano perhydrophenanthrene bile acid is commercially available (Avocado or Sigma) and octafluoronaphthalene and octafluorotoluene are commercially available (Sigma)
  • the target compound was separated from the crude by column chromatography
  • the target compound was separated from the crude by column chromatography [Fluka Silica Gel 60, eluting solvent system: AcOEt/Acetone (9:1)] giving 0.43g (25.8 %) of a pale yellow solid.
  • R f 0.64 in AcOEt/Acetone (1: 1) , fluorescent both in short and long wavelength and developed with phosphomolybdic acid.
  • the target compound was separated from the crude by column chromatography [Fluka Silica Gel 60, eluting solvent system: Petroleum ether 40-60/AcOEt (10:1) ] giving as the second product eluted 1.48 g (60.0 %) of a very sticky yellow solid .
  • the target compound was separated from the crude by column chromatography [Fluka Silica Gel 60, eluting solvent system: AcOEt/Acetone (4:1)] giving 0.934 g (34.75 %) of a yellow solid .
  • the target compound was separated from the crude by column chromatography [Fluka Silica Gel 60, eluting solvent system: Petroleum ether 40-60/AcOEt (4:1) for the elution of OFN and (3:1) for the elution ofthe target compound] giving as the second product eluted 0.674 g (25.1 %) of a very sticky yellow solid.
  • R f 0.66 in Pet.ether 40-60/AcOEt (4:1), fluorescent both in short and long wavelength and developed with phosphomolybdic acid.
  • the target compound was separated from the crude by column chromatography [Fluka Silica Gel 60, eluting solvent system: Petroleum ether 40-60/AcOEt (3:1) for the elution of OFN and (2:1) for the elution of the target compound ] giving as the second product eluted 0.88g (40.0%) of a very sticky yellow solid .
  • R f 0.5 in Pet.ether 40-60/AcOEt (2:1), non fluorescent and developed with phosphomolybdic acid.
  • a sample of PAH phenanthrene was combined with samples of compound of Example 1.1 to 1.6 above in C1 3 CD. Samples were left for a few minutes to allow binding and then tic plates shown against plates of the starting samples. A spot was shown at different elution to the starting samples and detection of fluorescence wavelength allowed the PAH to be determined, indicating this had bound to the compound of the Example.
  • 1H NMR showed shifts in aromatic H of the guest PAH at 6 - 8 or 19 F NMR showed shifts in aromatic F of the host compound of formula I, indicating binding had taken place.
  • Typical detection levels are: -50 mM "1 Cholic acid-OFT (CDC1 3 ); ⁇ 6oo mM "1 Cholic acid-OFN (CDC1 3 ); ⁇ 6oo0 mM "1 Chenodeoxycholic-OFN (CDC1 3 ).
  • Example 3 Determination of binding affinity of Compounds of Formula I
  • Example 4.2 Methodology for Preparation of fluorescent compound of formula I - Method 2 Using this method (J.Am.Chem.Soc. 1996, 118,1573-1574, Regen et al) a fluorescent non-fluorinated cholic acid was prepared. This method was successful and suggested that the fluorinated analogue might be prepared.
  • the tweezer-PAH complexes of Example 2 are suitable for polymerising into a polymeric matrix and preparation of a recognition polymer sensor for the determination of PAHs.
  • Bisphenol A (0.01 moles, 2.28 g), phloroglucinol (0.02 moles, 2.52 g), 4,4- Methylenebis (phenyl isocyanate) (0.01 moles, 2.50 g) and phenanthrene (5wt% , 0.46 g) were dissolved in dried THF.
  • the freshly prepared mixture containing 5wt% of the bile acid derivative- phenanthrene complex (not shown in the previous scheme) was polymerised under nitrogen at 25 °C. Normally, polymerisation requires aggressive initiators, such as peroxides, to get the process going. However in this case, the polymerisation started as soon as just a few minutes after mixing the monomers and bubbling with N 2 without any initiator present.
  • Product comprised polyurethane having immobilised fluorinated bile acid of Example 1 to 6 in a prea ⁇ anged position/location to re-bind another PAH of equal or similar characteristics to the templating one.
  • the PAH was washed away in work up as it is complexed by the bile acid through non-covalent interactions.

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Abstract

L'invention porte sur un composé permettant de lier un hydrocarbure aromatique polycyclique déficient en électrons (PAH), qui comprend une « pince » de la formule (1) dans laquelle B est un quelconque squelette plan C15-45 composé d'un système de 3 à 8 noyaux alicycliques condensés saturés ; chaque X est un groupe de liaison ; et chaque (symbole ovale) désigne un groupe Ar substitué par un total de 1 à 20 atomes d'halogène ; chaque n est un nombre entier compris entre 1 et 3 et la somme de n est 2 à 6 ; A est un groupe ou un fragment choisi parmi des groupes lipophiles tels que des groupes hydrocarbures aliphatiques ou alicycliques C4-20 saturés ou insaturés ; T est un élément de support ou de détection facultatif et m est 0 ou 1 ; et k est 0 ou est un nombre entier choisi de 1 à 3 ; les groupes Ar étant latéraux par rapport à au moins deux noyaux différents du squelette B plan et étant situés sur le même côté du squelette B plan, formant dès lors avec B une poche apte à recevoir et à lier une espèce PAH hôte ; sur un procédé de préparation de ce dernier ; sur un procédé permettant de détecter un PAH à l'aide d'un composé de la formule (1) ; sur un capteur de PAH comprenant un composé de la formule (1) ; sur un procédé permettant de détecter un PAH à l'aide de ce dernier ; et sur des utilisations de ces derniers.
PCT/GB2005/001118 2004-03-17 2005-03-16 Composes et capteurs destines a la detection d'hydrocarbures aromatiques polycycliques WO2005090379A1 (fr)

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US7820821B2 (en) 2006-02-10 2010-10-26 Transtech Pharma, Inc. Benzazole derivatives, compositions, and methods of use as aurora kinase inhibitors
US8377983B2 (en) 2006-02-10 2013-02-19 Transtech Pharma, Inc. Benzazole derivatives, compositions, and methods of use as aurora kinase inhibitors
CN100389123C (zh) * 2006-07-21 2008-05-21 中国科学院上海有机化学研究所 一类26-溴代-16,22-二氧代-胆甾醇化合物的合成方法
CN108246271A (zh) * 2018-02-23 2018-07-06 合肥学院 一种用于2，4，6-三硝基苯酚检测的分子印迹聚合物微球的制备方法
CN108246271B (zh) * 2018-02-23 2021-05-25 合肥学院 一种用于2，4，6-三硝基苯酚检测的分子印迹聚合物微球的制备方法

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