WO2015122845A1 - Crosslinked polymers prepared from functional monomers having imidazolium, pyridinium, aryl-substituted urea or aryl-substituted thiourea groups and uses thereof - Google Patents
Crosslinked polymers prepared from functional monomers having imidazolium, pyridinium, aryl-substituted urea or aryl-substituted thiourea groups and uses thereof Download PDFInfo
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- WO2015122845A1 WO2015122845A1 PCT/SG2015/000043 SG2015000043W WO2015122845A1 WO 2015122845 A1 WO2015122845 A1 WO 2015122845A1 SG 2015000043 W SG2015000043 W SG 2015000043W WO 2015122845 A1 WO2015122845 A1 WO 2015122845A1
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- monomer
- solvent
- group
- polymer
- monomers
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- 239000000178 monomer Substances 0.000 title claims abstract description 196
- RAXXELZNTBOGNW-UHFFFAOYSA-O Imidazolium Chemical compound C1=C[NH+]=CN1 RAXXELZNTBOGNW-UHFFFAOYSA-O 0.000 title claims description 21
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 title claims description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title description 9
- 239000004202 carbamide Substances 0.000 title description 6
- 229920006037 cross link polymer Polymers 0.000 title description 3
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea group Chemical class NC(=S)N UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 title description 2
- 150000003672 ureas Chemical class 0.000 title description 2
- 229920000642 polymer Polymers 0.000 claims abstract description 122
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 82
- 239000010452 phosphate Substances 0.000 claims abstract description 78
- 150000001875 compounds Chemical class 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 54
- 239000000203 mixture Substances 0.000 claims abstract description 47
- -1 phosphate ester compounds Chemical class 0.000 claims abstract description 44
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 42
- 238000004132 cross linking Methods 0.000 claims abstract description 25
- 239000007790 solid phase Substances 0.000 claims abstract description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 180
- 239000002904 solvent Substances 0.000 claims description 65
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 54
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 42
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 39
- 239000012156 elution solvent Substances 0.000 claims description 37
- 150000002500 ions Chemical class 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 34
- 125000001072 heteroaryl group Chemical group 0.000 claims description 31
- 125000003118 aryl group Chemical group 0.000 claims description 30
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical group FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 27
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- 230000008569 process Effects 0.000 claims description 20
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 19
- 230000001476 alcoholic effect Effects 0.000 claims description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 125000005647 linker group Chemical group 0.000 claims description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 230000002378 acidificating effect Effects 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 239000000463 material Substances 0.000 claims description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 13
- 125000001188 haloalkyl group Chemical group 0.000 claims description 13
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 125000000217 alkyl group Chemical group 0.000 claims description 10
- 125000000623 heterocyclic group Chemical group 0.000 claims description 10
- 238000004949 mass spectrometry Methods 0.000 claims description 10
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 10
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 10
- 238000006116 polymerization reaction Methods 0.000 claims description 10
- 150000003254 radicals Chemical class 0.000 claims description 10
- 150000003014 phosphoric acid esters Chemical class 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 229910052736 halogen Inorganic materials 0.000 claims description 8
- 150000002367 halogens Chemical class 0.000 claims description 8
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 8
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 8
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 8
- 125000001054 5 membered carbocyclic group Chemical group 0.000 claims description 7
- 125000004008 6 membered carbocyclic group Chemical group 0.000 claims description 7
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- OKKRPWIIYQTPQF-UHFFFAOYSA-N Trimethylolpropane trimethacrylate Chemical compound CC(=C)C(=O)OCC(CC)(COC(=O)C(C)=C)COC(=O)C(C)=C OKKRPWIIYQTPQF-UHFFFAOYSA-N 0.000 claims description 6
- 125000003545 alkoxy group Chemical group 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 125000001424 substituent group Chemical group 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 5
- 150000007514 bases Chemical class 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 4
- 239000006184 cosolvent Substances 0.000 claims description 4
- IQIJRJNHZYUQSD-UHFFFAOYSA-N ethenyl(phenyl)diazene Chemical compound C=CN=NC1=CC=CC=C1 IQIJRJNHZYUQSD-UHFFFAOYSA-N 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 125000004438 haloalkoxy group Chemical group 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims description 4
- UOCLXMDMGBRAIB-UHFFFAOYSA-N 1,1,1-trichloroethane Chemical compound CC(Cl)(Cl)Cl UOCLXMDMGBRAIB-UHFFFAOYSA-N 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 3
- 239000002168 alkylating agent Substances 0.000 claims description 3
- 229940100198 alkylating agent Drugs 0.000 claims description 3
- 150000001450 anions Chemical class 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 2
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 2
- HIACAHMKXQESOV-UHFFFAOYSA-N 1,2-bis(prop-1-en-2-yl)benzene Chemical compound CC(=C)C1=CC=CC=C1C(C)=C HIACAHMKXQESOV-UHFFFAOYSA-N 0.000 claims description 2
- 229940044192 2-hydroxyethyl methacrylate Drugs 0.000 claims description 2
- DBCSOHBZRZWHQM-UHFFFAOYSA-N 3-[1-(3-amino-3-oxoprop-1-enyl)piperazin-2-yl]prop-2-enamide Chemical compound NC(=O)C=CC1CNCCN1C=CC(N)=O DBCSOHBZRZWHQM-UHFFFAOYSA-N 0.000 claims description 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 2
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims description 2
- 150000004820 halides Chemical class 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 125000001475 halogen functional group Chemical group 0.000 claims 3
- 125000004767 (C1-C4) haloalkoxy group Chemical group 0.000 claims 1
- 125000004765 (C1-C4) haloalkyl group Chemical group 0.000 claims 1
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 abstract description 35
- 235000021317 phosphate Nutrition 0.000 description 69
- 210000002381 plasma Anatomy 0.000 description 47
- 230000015572 biosynthetic process Effects 0.000 description 26
- 239000011347 resin Substances 0.000 description 26
- 229920005989 resin Polymers 0.000 description 26
- 238000003786 synthesis reaction Methods 0.000 description 25
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- 239000011521 glass Substances 0.000 description 17
- 239000012634 fragment Substances 0.000 description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 15
- 239000002585 base Substances 0.000 description 15
- 238000000605 extraction Methods 0.000 description 14
- 229910052739 hydrogen Inorganic materials 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 11
- 150000003904 phospholipids Chemical class 0.000 description 11
- 238000011068 loading method Methods 0.000 description 10
- 238000002552 multiple reaction monitoring Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
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- 210000004369 blood Anatomy 0.000 description 9
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- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 9
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 8
- 238000001360 collision-induced dissociation Methods 0.000 description 8
- 239000012043 crude product Substances 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 125000005843 halogen group Chemical group 0.000 description 8
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- 238000004885 tandem mass spectrometry Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000001257 hydrogen Substances 0.000 description 7
- 239000003999 initiator Substances 0.000 description 7
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- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
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- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 description 6
- ONDSBJMLAHVLMI-UHFFFAOYSA-N trimethylsilyldiazomethane Chemical compound C[Si](C)(C)[CH-][N+]#N ONDSBJMLAHVLMI-UHFFFAOYSA-N 0.000 description 6
- OSSNTDFYBPYIEC-UHFFFAOYSA-N 1-ethenylimidazole Chemical compound C=CN1C=CN=C1 OSSNTDFYBPYIEC-UHFFFAOYSA-N 0.000 description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 5
- 239000005864 Sulphur Substances 0.000 description 5
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 5
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- 230000000694 effects Effects 0.000 description 5
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 5
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 5
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- 230000002441 reversible effect Effects 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 0 CCN1*2C1CC2 Chemical compound CCN1*2C1CC2 0.000 description 4
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 4
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- 238000012512 characterization method Methods 0.000 description 4
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- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 3
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
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- ZQQLMECVOXKFJK-NXCSZAMKSA-N N-octadecanoylsphingosine 1-phosphate Chemical compound CCCCCCCCCCCCCCCCCC(=O)N[C@@H](COP(O)(O)=O)[C@H](O)\C=C\CCCCCCCCCCCCC ZQQLMECVOXKFJK-NXCSZAMKSA-N 0.000 description 3
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- RPACBEVZENYWOL-XFULWGLBSA-M sodium;(2r)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate Chemical compound [Na+].C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)[O-])CO1 RPACBEVZENYWOL-XFULWGLBSA-M 0.000 description 3
- XGRLSUFHELJJAB-JGSYTFBMSA-M sodium;[(2r)-2-hydroxy-3-[(z)-octadec-9-enoyl]oxypropyl] hydrogen phosphate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP(O)([O-])=O XGRLSUFHELJJAB-JGSYTFBMSA-M 0.000 description 3
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 3
- DUYSYHSSBDVJSM-KRWOKUGFSA-N sphingosine 1-phosphate Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)COP(O)(O)=O DUYSYHSSBDVJSM-KRWOKUGFSA-N 0.000 description 3
- 150000003512 tertiary amines Chemical class 0.000 description 3
- UWYZHKAOTLEWKK-UHFFFAOYSA-N 1,2,3,4-tetrahydroisoquinoline Chemical compound C1=CC=C2CNCCC2=C1 UWYZHKAOTLEWKK-UHFFFAOYSA-N 0.000 description 2
- LBUJPTNKIBCYBY-UHFFFAOYSA-N 1,2,3,4-tetrahydroquinoline Chemical compound C1=CC=C2CCCNC2=C1 LBUJPTNKIBCYBY-UHFFFAOYSA-N 0.000 description 2
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical compound C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 2
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- CTAPFRYPJLPFDF-UHFFFAOYSA-N isoxazole Chemical compound C=1C=NOC=1 CTAPFRYPJLPFDF-UHFFFAOYSA-N 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000002370 liquid polymer infiltration Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- CAAULPUQFIIOTL-UHFFFAOYSA-N methyl dihydrogen phosphate Chemical compound COP(O)(O)=O CAAULPUQFIIOTL-UHFFFAOYSA-N 0.000 description 1
- 239000012022 methylating agents Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- UMRZSTCPUPJPOJ-KNVOCYPGSA-N norbornane Chemical compound C1C[C@H]2CC[C@@H]1C2 UMRZSTCPUPJPOJ-KNVOCYPGSA-N 0.000 description 1
- AHHWIHXENZJRFG-UHFFFAOYSA-N oxetane Chemical compound C1COC1 AHHWIHXENZJRFG-UHFFFAOYSA-N 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- AERBNCYCJBRYDG-KSZLIROESA-N phytosphingosine Chemical compound CCCCCCCCCCCCCC[C@@H](O)[C@@H](O)[C@@H](N)CO AERBNCYCJBRYDG-KSZLIROESA-N 0.000 description 1
- 229940033329 phytosphingosine Drugs 0.000 description 1
- 150000003038 phytosphingosines Chemical class 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000005936 piperidyl group Chemical group 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- ZVJHJDDKYZXRJI-UHFFFAOYSA-N pyrroline Natural products C1CC=NC1 ZVJHJDDKYZXRJI-UHFFFAOYSA-N 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 239000012966 redox initiator Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000004137 sphingolipid metabolism Effects 0.000 description 1
- 108010014501 sphingosine 1-phosphate lyase (aldolase) Proteins 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- CXWXQJXEFPUFDZ-UHFFFAOYSA-N tetralin Chemical compound C1=CC=C2CCCCC2=C1 CXWXQJXEFPUFDZ-UHFFFAOYSA-N 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 238000001196 time-of-flight mass spectrum Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- 238000012418 validation experiment Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F12/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F12/02—Monomers containing only one unsaturated aliphatic radical
- C08F12/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F12/14—Monomers containing only one unsaturated aliphatic radical containing one ring substituted by hetero atoms or groups containing heteroatoms
- C08F12/16—Halogens
- C08F12/20—Fluorine
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F12/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F12/02—Monomers containing only one unsaturated aliphatic radical
- C08F12/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F12/14—Monomers containing only one unsaturated aliphatic radical containing one ring substituted by hetero atoms or groups containing heteroatoms
- C08F12/22—Oxygen
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F12/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F12/02—Monomers containing only one unsaturated aliphatic radical
- C08F12/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F12/14—Monomers containing only one unsaturated aliphatic radical containing one ring substituted by hetero atoms or groups containing heteroatoms
- C08F12/26—Nitrogen
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/10—Esters
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F226/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
- C08F226/06—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a heterocyclic ring containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F236/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds
- C08F236/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds the radical having only two carbon-to-carbon double bonds
- C08F236/04—Copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds the radical having only two carbon-to-carbon double bonds conjugated
- C08F236/10—Copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds the radical having only two carbon-to-carbon double bonds conjugated with vinyl-aromatic monomers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F279/00—Macromolecular compounds obtained by polymerising monomers on to polymers of monomers having two or more carbon-to-carbon double bonds as defined in group C08F36/00
- C08F279/02—Macromolecular compounds obtained by polymerising monomers on to polymers of monomers having two or more carbon-to-carbon double bonds as defined in group C08F36/00 on to polymers of conjugated dienes
- C08F279/06—Vinyl aromatic monomers and methacrylates as the only monomers
-
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
Definitions
- Crosslinked Polymers Prepared from Functional Monomers Having Imidazolium, Pyridinium, Aryl-substituted Urea or Aryl-substituted Thiourea Groups and Uses Thereof.
- the present invention relates to polymers which selectively bind phosphate ester- containing compounds, and to a method for isolating phosphate ester-containing
- phosphate diesters and/or other compounds such as lipids.
- the phosphate diesters and/or other compounds such as lipids.
- invention relates to a separation method in which long chain organic phosphate esters, for example phospholipids, are captured on a solid phase comprising a
- Lipids are small molecules with large structural and chemical diversity. Over the past two decades, technologies such as chromatography and mass spectrometry have
- Sphingosine-1 -phosphate is an example of such a
- LCBs Long-chain amino alcohols, generally referred to as long- chain bases or LCBs (Pruett, S . et al. J Lipid Res 49, 1621 -1639 (2008)), and their phosphorylated forms (LCB-P) display particularly diverse chemistries across
- S1 P is currently emerging as a valid biomarker for life-threatening illnesses such as multiple sclerosis, cardiovascular disease and cancer.
- the commercially available method to detect S1 P has the major disadvantages that only high S1 P concentrations can be analysed and that there is a relatively high risk of false results because of the cross-reactivity of the antibodies with similar molecules. There is therefore a need for improved methods of detection of S1 P.
- animal and plant cells and tissues comprise other LCB-Ps. Little is known about the existence of other LCB-Ps of different chemical and structural composition, despite the presence of various non- phosphorylated LCB precursors. In order to investigate the structures and functions of these compounds, it is necessary to isolate, quantify and characterize them.
- Animal and plant cells and tissues also comprise other mono-phosphorylated compounds such as phosphoinositides (PIPs), phosphatidic acid, lysophosphatidic acid and ceramide-1 -phosphate; phosphodiester compounds, for example phosphodiester lipids; and compounds such as lipids which contain neither phosphate mono-esters nor phosphate-diesters.
- PIPs phosphoinositides
- phosphatidic acid phosphatidic acid
- lysophosphatidic acid and ceramide-1 -phosphate phosphodiester compounds
- phosphodiester compounds for example phosphodiester lipids
- compounds such as lipids which contain neither phosphate mono-esters nor phosphate-diesters.
- phosphate ester- containing compounds such as phospholipids, particularly LCB-Ps, as well as phosphoinositides, phosphatitic acid, lysophosphatidic acid and ceramide-1 - phosphate in the tissues and body fluids of humans and other animal species, for example mammals; and also in plants and microorganisms.
- the present inventors have developed polymers which selectively bind phosphate esters.
- a polymer obtainable by radical co-polymerisation of a first monomer comprising a compound of general formula (I) or (I I) or a mixture thereof:
- each of B and B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom; or a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected from halo, C 1-6 haloalkyl or nitro; each of Y and Y' is a linking group comprising 1 -6 -CH 2 - units wherein a -CH 2 - unit is optionally replaced by a C 5 . 4 aryl or heteroaryl group optionally substituted with one or more substituents selected from H, halogen, NH 2 , C 1- alkyl, alkoxy, haloalkyl or Ci_ 4 haloalkoxy or
- each of A or A' and B or B' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected halo, d. 6 haloalkyl or nitro
- each R 20 is independently H, methyl or ethyl
- Z is O or S
- X " is a halide ion or a hydrophobic anion such as PF 6 " ; with a second, cross-linking monomer and optionally with one or more further co- monomers;
- the molar ratio of the first monomer to the sum of the second monomer and the one or more further co-monomers (if present) is less than or equal to 1 :5.
- cross linker is one of the following:
- the monomer may be copolymerised in the presence of ethylene glycol dimethacrylate (EGDMA).
- EGDMA ethylene glycol dimethacrylate
- this polymer differs from that of the present invention in that the ratio of the di-imidazolium monomer to EGDMA is 1 : 1 .
- the polymers of the present invention are prepared using a far higher proportion of the cross-linking monomer.
- CN102050919 relates to polymer nanoparticles prepared from a monomer similar to those described by Buscemi et al except that the linker is -(CH 2 ) 4 -, and EGDMA. These polymer nanoparticles are used for catalyzing the addition reaction of carbon dioxide to an epoxy ring compound. However, in these polymers the ratio of the imidazolium monomer to the cross-linking agent is from 2:1 to 10: 1 whereas the polymers of the present invention are prepared using a far higher proportion of the cross-linking monomer.
- Vinylimidazole-EGDMA copolymers are known from EP0162388, US 4,600,578 and US 4,649,048, which relate to the use of these polymers as bile acid sequestrating agents and for treating diarrhoea.
- the monomers from which these polymers are prepared differ in structure from the monomers used in the polymers of the present invention.
- the polymer of the invention is capable of complexing phosphate ester groups, for example the phosphate groups of phosphorylated long-chain base phosphate molecules. Therefore, the polymer is suitable for use in detecting compounds comprising phosphate esters, for example LCB-Ps, and isolating them from biological samples.
- the present invention has immediate relevance for our understanding of S1 P biology as well as its therapeutic targeting.
- S1 P biosynthesis, trafficking, receptor binding and degradation are highly controlled, and the targeting of S1 P receptors and metabolizing enzymes remains an active field of research and drug development.
- part of the LCB-P signaling machinery is influenced by the chemical nature of their aliphatic portion. This factor has so far been neglected, mainly because of the lack of information on S1 P/LCB-P diversity.
- LCB-P chain diversity affects receptor binding, and impacts S1 P/LCB-P gradients, which are known to be important underlying components of immune cell trafficking via the reversible de-phosphorylation of S1 P.
- alkyl refers to a straight or branched fully saturated hydrocarbon chain.
- d -6 alkyl groups have from one to six carbon atoms and examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n- pentyl and n-hexyl.
- C1.4 alkyl groups have from one to four carbon atoms.
- halo refers to fluoro, chloro, bromo or iodo and a halide ion may be a fluoride, chloride, bromide or iodide ion.
- haloalkyi refers to an alkyl group which is substituted by one or more halo atoms and includes any number of halo substituents up to perhalo substituted groups. Examples include trifluoromethyl; 1 ,1 -dibromoethyl.
- Carbocyclyl and “carbocyclic” refer to a non-aromatic cyclic group having three to ten ring atoms (unless otherwise specified), which may be fully or partially saturated and which may be fused or bridged. Examples include cycloalkyl groups such as cyclopropyl, cyclopentyl and cyclohexyl as well as cycloalkenyl groups such as cyclopentenyl and cyclohexenyl. Further examples include fused or bridged ring systems such as norbornane.
- More suitable carbocyclic rings are 5- or 6-membered rings such as cyclopentyl, cyclohexyl or cyclohexenyl.
- heterocyclyl and “heterocyclic” refer to a cyclic group as defined above for carbocyclyl, except that one or more ring atoms is replaced by a hetero atom selected from N, O and S.
- hetero atom selected from N, O and S.
- examples include aziridine, azetidine, pyrrolidine, pyrroline, imidazoline, piperidine, piperazine, morpholine, oxetane, tetrahydrofuran and oxazoline.
- fused or bridged ring systems such as octahydroquinoline or nor-tropane.
- heterocyclic rings are 5- or 6-membered rings such as piperidyl, piperazinyl, morpholinyl and tetrahydrofuryl.
- aryl in the context of the present specification refers to a ring system with aromatic character having from 6 to 14 ring carbon atoms (unless otherwise specified) and containing one ring or two fused rings. Where an aryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of aromatic moieties are benzene, naphthalene, anthracene, phenanthrene, tetrahydronaphthalene, indane and indene. More suitably, the aryl group is a phenyl group.
- heteroaryl in the context of the specification refers to a ring system with aromatic character having from 5 to 10 ring atoms (unless otherwise specified), at least one of which is a heteroatom selected from N, O and S, and containing one ring or two fused rings. Where a heteroaryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of monocyclic heteroaryl groups include pyridine, pyrimidine, furan, thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole and isothiazole.
- bicyclic fully aromatic heteroaryl groups examples include quinoline, isoquinoline, indole, benzofuran, benzimidazole and benzothiazole.
- bicyclic heteroaryl groups in which one ring is not fully aromatic in character examples include dihydroquinolines, tetrahydroquinoline,
- heteroaryl groups are monocyclic 5- or 6-membered rings such as pyridyl, furyl or thienyl.
- phosphate monoester and “phospho monoester” refer to compounds based on phosphoric acid (H 3 P0 4 ) in which one of the hydrogen atoms is replaced by an organic group.
- phosphate diester and “phosphodiester” refer to compounds based on phosphoric acid (H 3 P0 4 ) in which two of the hydrogen atoms are replaced by organic groups.
- a long chain base phosphate molecule refers to a molecule having an alkyl, alkenyl or alkynyl chain of from 12 to 24 carbon atoms substituted with at least one phosphate group represented by the formula:
- a long chain organic phosphate molecule such as a phospholipid
- a long chain base phosphate contains one to four phosphate groups, more usually one to three phosphate groups and in many cases one or two phosphate groups.
- a long chain organic phosphate molecule such as a long chain base phosphate molecule or other phospholipid may contain additional functional groups.
- the chain may be substituted with one or more groups selected from OR 10 , NR 11 R 12 and N + R 1 R 12 R 13 , wherein each of R 10 , R 11 , R 12 and R 13 independently represents H or a C 1-4 alkyl group.
- each of R 10 , R 11 , R 12 and R 3 independently represents H, methyl or ethyl, particularly H or methyl.
- long chain base phosphate molecules are referred to by the length of the carbon chain, the number of double bonds and the number of hydroxy! groups in the parent lipid, where "d” indicates dihydroxy and "t” indicates trihydroxy.
- sphingosine is denoted as d18:1 and phytosphingosine is t18:0.
- the position of the double bond may be indicated by a superscript, i.e. 4-sphingenine is d18:1 A4f or 4E-d18: 1.
- the "d” or “t” relating to the parent lipid molecule is retained for the equivalent long chain base phosphate, where one of the OH groups has been replaced by the phosphate moiety.
- sphingosine-1 -phosphate is also denoted as d18: 1.
- polymers wherein A and B are both imidazolium groups, Y is -(CH 2 ) X -, where x is 1 -6 are excluded from the scope of the invention.
- polymers wherein A and B are both imidazolium groups, Y is -(CH 2 ) X -, where x is 1 -6 and wherein a -CH 2 - unit is optionally replaced by a phenyl group are excluded from the scope of the invention.
- the molar ratio of the first monomer of general formula (I) or general formula (II) to the sum of the second cross linking monomer and one or more further co-monomers (if present) is less than or equal to 1 :5.
- the molar ratio is from about 1 : 100 to 1 :5. More suitably, the molar ratio of monomer of general formula (I) or general formula (II) to cross linking monomer is from about 1 :80 to 1 :20; still more suitably from 1 :65 to 1 :25, or from 1 :50 to 1 :30. Typically the molar ratio is between 1 :35 and 1 :45, for example about 1 :40.
- the polymer is obtainable by a polymerisation process in which the solvent is selected from a water, an alcoholic solvent such as methanol, ethanol or isopropanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 , 1 -trichloroethane, chloroform (CHCI3), dichloromethane (DCM), toluene, dimethylsulfoxide (DMSO), isopropanol or any mixture of these solvents or other solvents.
- an alcoholic solvent such as methanol, ethanol or isopropanol
- THF tetrahydrofuran
- DMF dimethylformamide
- ACN acetonitrile
- 1 , 1 , 1 -trichloroethane chloroform
- CHCI3 dichloromethane
- DCM dichloromethane
- DMSO dimethylsulfoxide
- the solvent is a mixture of an alcoholic solvent and a non-polar organic co- solvent, for example toluene.
- the most suitable polymers for use in the present invention are those in which the total concentration of the first monomer of formula (I) or (II) and the second cross- linking monomer and one or more further co-monomers (if present) is at least 100 mg/ml of solvent. More usually, the concentration of the monomers is from 100 to 500 mg/ml of solvent, for example 100-400 mg/ml, 100-300 mg/ml or 200-300 mg/ml. Typically, the total concentration of the monomers is about 250 mg/ml of solvent.
- X " is suitably a halide or a PF 6 " ion.
- X is bromide
- the linking group Y is suitably bonded to the quaternary nitrogen moiety of the group A or A'.
- the group A or A' is a pyridinium or imidazolium ion.
- the group B or B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom and in this embodiment:
- linking group Y is suitably bonded to the quaternary nitrogen moiety
- the group B or B' may be a pyridinium or imidazolium ion.
- Monomers of formula (la) are particularly suitable for use in the present invention.
- the group B or B' is a 5- or 6-membered aryl or heteroaryl ring, i.e. an uncharged moiety.
- B may be, for example, a phenyl or pyridyl group.
- Y is a linker group selected from:
- each of R 1 , R 2 and R 3 is independently selected from H, halogen, NH 2 , C 4 alkyl, C-i_ 4 alkoxy, haloalkyl, haloalkoxy or R 1 and R 2 or R 2 and R 3 may together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring; each of R 4 , R 5 , R 6 and R 7 is independently selected from H, halogen, NH 2 ,
- d- alkyl, d- alkoxy, d-4 haloalkyl, d-4 haloalkoxy or R 4 and R 5 or R 6 and R 7 may together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring;
- R 8 is H, halogen, NH 2 , d.4 alkyl, d-4 alkoxy, d_ 4 haloalkyl, d-4 haloalkoxy.
- R 1 , R 2 , R 3 and R 8 are as defined above; are more suitable.
- R 1 , R 2 and R 3 are as defined above.
- the monomer is a monomer of formula (la).
- R 1 , R 2 and R 3 are hydrogen or R 1 and R 2 or R 2 and R 3 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
- R 1 , R 2 and R 3 are all hydrogen.
- a particularly suitable monomer of this embodiment is Monomer 1 , which has the structure:
- linker Y is:
- R 1 , R 2 and R 3 are as defined above.
- R , R 2 and R 3 are hydrogen or R 1 and R 2 or R 2 and R 3 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
- R 1 , R 2 and R 3 are all hydrogen.
- a particularly suitable monomer of this embodiment is Monomer 2, which has the structure:
- linker Y is:
- R 4 , R 5 , R 6 and R 7 are as defined above.
- R 4 , R 5 , R 6 and R 7 are all hydrogen or R 4 and R 5 or R 6 and R 7 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
- R 4 , R 5 , R 6 and R 7 are all hydrogen.
- a particularly suitable monomer of this embodiment is Monomer 3, which has the structure:
- X is as defined above but is most suitably Br '
- Y is:
- R 1 , R 2 , R 3 and R 8 are as defined above.
- R 1 , R 2 , R 3 and R 8 are all hydrogen.
- a particularly suitable monomer of this embodiment is Monomer 5, which has the structure:
- the linker Y is 1 -6 -CH 2 - units.
- Y may be -CH 2 -.
- the linker Y may be 1-6 -CH 2 - units and B may be a 5- or 6-membered heteroaryl ring, for example phenyl.
- Monomer 4 which has the structure:
- R 20 is H or methyl but especially H
- each of A or A' and each of B or B' is 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more halo, Ci. 6 haloalkyl or nitro substituents. More suitably, each of A or A' and each of B or B' is a phenyl group optionally substituted with one or more halo, d. haloalkyl or nitro substituents, for example fluoro, trifluoromethyl or nitro substituents.
- a particularly suitable example of a monomer of this type is Monomer 6:
- the polymer of the invention may be prepared from a single monomer of general formula (I) or general formula (II) or from two or more such monomers.
- Suitable monomers for use in such combinations are those described above.
- a monomer selected from Monomer 1 , Monomer 2, Monomer 3, Monomer 4 or Monomer 5 may be combined with Monomer 6.
- one of the monomers for the preparation of the polymer may exhibit a change in fluorescence on binding of a phosphate ester and this property may be carried through into the polymer so that binding of a phosphate ester to the polymer can be detected as a quenching or an enhancement of the fluorescence emission of the polymer.
- monomers are Monomer 5 and Monomer 6.
- Monomers of general formulae (I) and (II) are known and are readily available or may be prepared by methods familiar to those of skill in the art, for example the methods set out in Examples 1 , 6, 8, 10 and 1 1 below, or adaptations thereof which would be clear to a person of skill in the art.
- Cross linking monomers which are suitable for use in the present invention include di- and tri-methacrylate monomers, dialkenyl benzene monomers and bis acrylamide monomers.
- Specific examples of cross linking monomers include ethyleneglycol dimethacrylate (EGDMA), divinylbenzene (DVB), diisopropenylbenzene, (DIB),
- TAM trimethylolpropanetrimethacrylate
- PETA pentaerythritoltriacrylate
- EBA ethylenebisacrylamide
- PBA piperazinebisacrylamide
- MCA methylenebisacrylamide
- EGDMA is particularly suitable.
- the polymer will be the product of the monomer of general formula (I) or (II) without further co-monomers.
- one or more further co-monomers are added to the mixture of first monomer and second, crosslinking monomer.
- the particular co-monomer which is chosen will depend upon the nature of the cross linking monomer.
- the cross linking monomer is a di- or tri-methacrylate such as EGDMA, TRIM, or MBA
- the co-monomer may be also be a methacrylate monomer, for example methyl methacrylate, 2-hydroxyethylmethacrylate, methacrylamide or an alternative d. 6 alkyl methacrylate so that a methacrylate matrix is produced.
- cross linking monomer is a dialkyl benzene-based monomer such as DIB
- an alkenyl benzene monomer such as styrene
- an acrylamide co- monomer for use in combination with an acrylamide cross linking monomer.
- Polymers formed from Monomer 1 or combinations of Monomer 1 with other monomers of general formula (I) or general formula (II) have been shown to be particularly suitable, especially polymers formed from Monomer 1 co-polymerised with EGDMA.
- the inventors have demonstrated that imprinted resins formed from Monomer 1 copolymerised with EGDMA bind a greater proportion of a phosphate ester anion in a neutral buffer such as HEPES (4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid) buffer, pH 7, compared with similar imprinted resins formed from Monomers 2, 3 or 4.
- a neutral buffer such as HEPES (4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid) buffer, pH 7, compared with similar imprinted resins formed from Monomers 2, 3 or 4.
- HEPES 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid
- the polymerisation reaction may be conducted at a temperature of from 40°C to 90°C, more usually about 40°C to 80°C.
- the temperature is maintained at about 40°C to 60°C, for example about 50°C for a period of 12 to 48 hours, for example about 24 hours, following which it is raised to about 65°C to 75°C, for example about 70°C for a further period of 2 to 6 hours, for example about 4 hours.
- the binding polymer of the invention is suitably a macroporous polymer such that the structure contains large pores or channels.
- Macroporous polymers are highly cross- linked polymers which contains large interconnected pores or channels within their structure. Unlike hydrogels and similar polymers, which require hydration in order for pores to form, the pores and channels in a macroporous polymer are formed during the polymerisation process and are present at all times. Macroporous polymers are known in the art and are described, for example, by Arrua et al, (Materials 2009, 2, 2429-2466).
- the polymer after crushing and sieving, can be used as a binding polymer to extract phosphate esters, including phospholipids, from a biological sample.
- the polymer is obtainable by radical co-polymerisation and therefore in a further aspect of the invention there is provided a process for the preparation of a novel polymer as described above, the process comprising the radical co-polymerisation of a first monomer of general formula (I) or general formula (II) as defined above with a second, cross-linking monomer; characterised in that the ratio of the first monomer to the second monomer is less than or equal to 1 :5.
- the molar ratio of the first monomer of general formula (I) to the second cross linking monomer is as described above and the reaction temperature and conditions are also as described above for the first aspect of the invention.
- the polymerisation reaction will be initiated using a polymerisation initiator.
- Suitable polymerisation initiators include azo-N,N'- bisdimethylvaleronitrile (ABDV), azo-N,N'-bisisobutyronitrile (AIBN), 2,2'-azobis-(2,4- dimethyl-4-methoxyvaleronitrile) (V70) or any other azo-based initiator (available from Wako Pure Chemical Ind. Ltd. , (Japan); or redoxinitiators e.g.
- ammoniumpersulfate with or without added accelerator e.g.
- TEMED tetramethylethylenediamine
- the process may further include the addition of a co-monomer.
- the reaction may be conducted in any suitable solvent such as water, methanol, ethanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 , 1 -trichloroethane, chloroform (CHCI 3 ), dichloromethane (DCM), toluene, dimethylsulfoxide (DMSO), isopropanol or any mixture of these or other solvents.
- suitable solvent such as water, methanol, ethanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 , 1 -trichloroethane, chloroform (CHCI 3 ), dichloromethane (DCM), toluene, dimethylsulfoxide (DMSO), isopropanol or any mixture of these or other solvents.
- the reaction is conducted in a solvent comprising a mixture of an alcoholic solvent and a non-polar organic co-solvent.
- Suitable alcoholic solvents include methanol, while toluene is an example of a suitable co-solvent.
- the alcoholic solvent and the non-polar organic solvent are present in a ratio of 1 :5 to 5: 1 v/v, more suitably 1 : 1 v/v.
- the polymerisation reaction is conducted under an inert atmosphere, for example nitrogen or argon.
- the temperature conditions for the reaction are also as described above.
- the polymer can also be prepared in other formats such as nanofibers, nanotubes, monoliths, nanoparticles and their nanostructured counterparts such as core shell nanoparticles with optionally different functional properties such as magnetic or luminescent cores or labeled with probes allowing facile detection (e.g. electrochemiluminescent probes ECL). They can be tuned to exhibit maximum compatibility with the surrounding medium e.g. coatings to minimize protein adsorption.
- the polymer can be produced by any technique available for producing polymer particles including techniques relying on
- the polymerisation reaction is performed using controlled radical polymerisation (CRP), which differs from conventional radical polymerisation in respect of the life time of the propagating radicals.
- CRP controlled radical polymerisation
- this can be extended to hours which allows the preparation of polymers with predefined molecular weights, low polydispersity, controlled composition and functionality. This leads to polymers displaying higher binding capacity, affinity and faster association and dissociation of peptide.
- RAFT reversible addition fragmentation chain transfer polymerisation
- ATRP atom transfer radical polymerisation
- iniferter initiator, transfer, termination
- polymer particles by grafting a polymer film onto the surface of a preformed support.
- the grafting can be performed according to either the "grafting to” or the “grafting from” approach, both of which are well known to those of skill in the art. The latter approach improves the production process as well as the molecular recognition and kinetic properties of the materials (and is described in US 6,759,488).
- polymer particles according to the invention may be produced using a molecular imprinting approach in which a template resembling a phosphate ester target molecule in shape and functionality is added to a mixture of the first monomer and the second, cross-linking monomer prior to polymerization.
- the template will usually be a monomer resembling a target phosphate monoester compound such as a LCB-P.
- Suitable LCB-P templates include PL-CL14 or a salt thereof, for example a mono- or di-alkali metal salt, such as a mono- or di- lithium, sodium or potassium salt. A mono- or di-sodium salt is particularly useful.
- the template may be removed from the polymer to leave a structure which exhibits affinity for the target phosphate monoester compound or compounds.
- the polymer in another embodiment can be produced in the form of nanoparticles in a typical size range of 10nm-500nm.
- the engineering of nano-micrometer-sized polymer particles can be done by precipitation polymerisation (F. Wang, P.A.G. Cormack, D.C. Sherrington and E. Khoshdel, Angew. Chem. Int. Ed. (2003) 42(43), 5336-5338) or miniemulsion polymerisation procedures where uniform imprinted beads can be produced prepared by high dilution polymerisation in one step. Both aqueous and non-aqueous protocols can be used.
- nanoparticle synthesis can be performed by grafting (N. Perez-Moral, A. G. Mayes, Macromol. Rapid Commun. 2007, 28, 2170). This starts from a nonporous nanosized core containing radical initiator or chain transfer groups. Onto the core a thin polymer films can be grafted with minimal influence of the bead size, dispersity and morphology on the polymerisation conditions. Nanoparticles can thus be engineered which can capture their target in biological fluids. Paramagnetic, monosized polymer particles, are commonly used for this purpose. The technique is fast, mild and involves no centrifugation or chromatography. Paramagnetic particles may be prepared in the form of core/shell microgels by a two-stage "seed-and-feed" precipitation polymerisation or by grafting a polymer shell on a magnetic core bead.
- the polymers of the present invention are capable of selectively binding phosphate esters and therefore in a further aspect of the invention there is provided a method for isolating compounds comprising a phosphate ester group from a sample, the method comprising:
- the biological sample may be derived from any species of animal, plant or microorganism.
- the sample may be a tissue sample from a mammal, including a human. Suitable samples blood, plasma and lymph or other tissues, including brain tissue. Blood and tissue samples from other animal species may also be used or, in some cases, for example with small animals such as insects, the sample may comprise the entire organism.
- the sample may also be derived from a plant and may include material taken from any part of the plant, including leaves and seeds.
- the sample will be a microorganism, for example bacteria or yeast such as Saccharomyces cerevisiae.
- the solid phase may be incorporated into a separation device which itself forms a further aspect of the invention as will be discussed below.
- the first solvent comprises an alcoholic solvent such as methanol, ethanol, n-propanol or isopropanol, particularly methanol, although in some circumstances a chlorinated solvent such as dichloromethane or chloroform may also be used.
- the soluble compounds extracted by the first solvent include not only compounds comprising phosphate monoesters, for example long chain base phosphates, and phosphate diesters but also other many other types of compound, including the lipids from which many long chain base phosphate compounds are derived. Therefore it is necessary to separate phosphate monoester- and phosphate diester-containing compounds from one another and from these other compounds.
- washing solvent or solvents may also comprise alcoholic solvents and, for example, may be selected from methanol, ethanol, n-propanol and isopropanol or mixtures thereof.
- the solid phase is washed firstly with isopropanol followed by a mixture of isopropanol and methanol in a ratio of 1 : 1 v/v.
- step (iv) the phosphate esters are eluted with either or both of a basic elution solvent and an acidic elution solvent.
- the acid elution solvent is useful for removing phosphate monoesters from the solid phase and in some cases, for example when the method is intended for the isolation of phosphate monoesters, for example phospholipids such as S1 P, the acidic elution solvent may be used alone without the basic elution solvent. Although the basic elution solvent may also be used alone, the process more suitably, includes its use in combination with the acidic elution solvent.
- the basic elution solvent may be used prior to the acidic elution solvent.
- the acidic elution solvent suitably comprises a mixture of a chlorinated solvent and an alcoholic solvent.
- the chlorinated solvent may be, for example, chloromethane, dichloromethane or chloroform, with chloroform being particularly suitable.
- suitable alcoholic solvents include methanol, ethanol, n- propanol and isopropanol, but especially methanol.
- the ratio of chlorinated solvent to alcoholic solvent in the elution solvents is 1 :1 v/v and thus one suitable example of an elution solvent is an acidified 1 : 1 v/v mixture of chloroform and methanol.
- Acidification of the acidic elution solvent may be achieved using an organic acid, for example formic acid or trifluoroacetic acid.
- an organic acid for example formic acid or trifluoroacetic acid.
- a particularly suitable acidic elution solvent is a mixture of chloroform/methanol/1 % trifluoroacetic acid. Suitably these are in a volume ratio of 49.5/49.5/1.
- the acidic elution solvent removes the phosphate monoester compounds from the binding polymer and the elution rate differs for different phosphate monoesters.
- the basic elution solvent may comprise an alcoholic solvent or mixture of alcoholic solvents together with a basic compound such as an amine, especially a tertiary amine, for example a trialkyl amine such as triethylamine or trimethylamine.
- a basic compound such as an amine, especially a tertiary amine, for example a trialkyl amine such as triethylamine or trimethylamine.
- Suitable basic elution solvents include mixtures of isopropanol and methanol, for example a 1 : 1 mixture, and a tertiary amine. Where the basic compound is a tertiary amine the solvent may be isopropanol/methanol/amine, suitably in a volume ratio of 49.5/49.5/1. Triethylamine is a particularly suitable basic compound for use in such mixtures.
- phosphate monoesters from phosphate diesters and other compounds and also to separate them from one another.
- the separated phosphate ester compounds may be further characterized, for example using mass spectrometry. Thus, even if full identification of the compounds is not possible, a mass can be obtained.
- Mass spectrometry either may be carried out simultaneously with the
- a standard solution may be added to the sample solution before step (ii).
- the standard solution may comprise one oV more known phosphate ester compounds, for example phosphate monoesters such as long chain base phosphate compounds, and may therefore assist with the identification of similar compounds, e.g. long chain base phosphate compounds, in the sample.
- phosphate monoesters such as long chain base phosphate compounds
- the amount as well as the identity of phosphate oester compounds in the standard is known and this will enable the quantitative detection of the long chain base phosphates and other phosphate ester compounds in the sample.
- the method comprises the additional step of derivatising the phosphate ester compounds of the sample, for example by reacting with an alkylating agent in order to replace one or more of the hydrogen atoms on a phosphate or an amine group with an alkyl group.
- This method is particularly useful for the separation of phosphate monoester compounds.
- Derivatisation is suitably carried out after contacting the sample with the solid phase.
- the phosphate ester compounds are derviatised after the elution step (iv) it is preferable to repeat steps (ii) to (iv) using a mixture of the derivatised phosphate ester compounds as the sample.
- the alkylating agent is a methylating agent, for example trimethylsilyl diazomethane (TMS-diazomethane).
- TMS-diazomethane trimethylsilyl diazomethane
- the derivatisation step is particularly suitable when the sample comprises two or more similar phosphate ester compounds, especially phosphate monoester compounds, and more especially LCB-Ps.
- the inventors have found that derivatisation led to a gain in sensitivity, possibly explained by a more effective ionization of the alkylated derivatives. This is particularly true for long chain base phosphate compounds containing an amine group, which, when alkylated, carries a permanent positive charge while the negative charge on the phosphate is
- This new detection method was further validated in various biological matrices for its accuracy and precision, reproducibility and linearity. Moreover, the number of species detected and their relative abundance was conserved after the derivatization reaction.
- the aforementioned characterizations involved nano-electrospray ionization and high-resolution MS after chromatographic separation of the mixtures by Hydrophilic Interaction Chromatography (HILIC) built into a microfluidic chip. This was important to improve the co-elution of S P standards and endogenous LCB-Ps for qualitative and quantitative characterizations 6 of new LCB-P species. Indeed, few high-resolution mass spectra of LCB-Ps have been published, possibly because of the low signal intensities that hamper the precise characterization of ion peaks.
- HILIC Hydrophilic Interaction Chromatography
- the solid phase material formed from the polymer of the invention represents a further aspect of the invention.
- the solid phase material is suitable for capturing and separating phosphate monoester compounds, the solid phase comprising a binding polymer as defined above.
- the polymer may be used alone or in combination with other components.
- the solid phase is typically in the form of particles and the polymer is a macroporous polymer, the solid phase material may be prepared by a process comprising the steps of crushing and sieving the polymer of the invention.
- particles may be formed by the methods described above.
- a separation device comprising the solid phase material.
- the separation device may comprise a chromatography column, suitably a column for use in liquid chromatography or gas chromatography.
- a kit for isolating phosphate ester compounds from a mixture comprising one or more such compounds the kit comprising a solid phase material as defined above and instructions for the use of the material.
- the kit may further comprise one or more standards, which may be known phosphoester compounds
- the one or more standards may be provided in known amounts.
- a kit for the detection of S1 P may comprise a known amount of S1 P as a standard.
- FIGURE 1 illustrates substantially improved detection of LCB-P via an analytical workflow using phospho-monoester capture and derivatization
- Quadrupole Time of Flight mass spectrometry in either positive (left panel) or negative (right panel) ionization modes.
- IMP selectively captures phospho-monoester lipids (Ceramide-1 -phosphate, Cer- 1 -P; sphingosine-1 -phosphate, S1 P; phosphatidic acid, PA; lysophosphatidic acid, LPA).
- Lipids with no phosphate (Ceramide, Cer; Glucosyl-Ceramide, Glu-Cer) and those with phosphorous bound in diester configuration (phosphatidylcholine, PC; sphingomyelin, SM; lyso-phosphatidylcholine, LPC; phosphatidylglycerol, PG, and phosphatidylinositol, PI) do not efficiently bind to IMP and are recovered in the flow through and wash fractions instead.
- FIGURE 2 illustrates the discovery, characterization and quantification of new LCB-P in complex mixtures from diverse biological species
- the former two fragments are expected to be invariant for LCB-P with different aliphatic compositions which is indeed the case as shown for corresponding product ion spectra of LCB-P d18:2 (b), LCB-P d18: 1 (c) and LCB-P d18:0 (d), all derived from extracts of human blood plasma, and d16:0 (e) from whole fly extract.
- the C fragment instead, is characteristic of individual species of LCB-P, thus allowing for identification by targeted tandem mass spectrometry.
- FIGURE 3 illustrates the production of an IMP resin.
- FIGURE 4 shows the molecular structures of standard d17: 1 S1 P before and after derivatization with TMS-diazomethane.
- FIGURE 5 shows the effect of IMP resin on LCB-Ps detection.
- Extracted ion chromatograms representing the LCB-Ps identified in human plasma and murine brain, before (a and c) and after (b and d) the IMP enrichment step. Note the different scales in intensities between panels a and c and b and d.
- IMP enrichment also eliminates co-eluting contaminants (panels a and c) and improves the peak shapes, essential for SA1 P and LCB-P d20: 1 quantification and the detection of LCB-P d16:1 in plasma.
- FIGURE 6 shows the effect of TMS derivatization on LCB-P species distribution in human plasma.
- FIGURE 7 shows elution peaks recorded by multiple reaction monitoring of derivatized LCB-Ps in human plasma.
- the elution profiles (normalized intensities) are represented, showing a retention time increase with increasing unsaturation and decreasing chain length of the molecules as expected for HILIC chromatography.
- FIGURE 8 shows S1 P d18: 1 for illustration purposes of the different fragments observed during tandem mass spectrometry. Fragments A and B are invariant and common to all LCB-P tested here. Fragment C is LCB-P species specific and recorded as the [M+HCOO]- adduct in negative mode (C**) .
- FIGURE 9 shows an estimation of the limit of detection (LOD) based on signal to noise ratio (S/N) in plasma before (panel a) and after (panel b) IMP enrichment. Shown are extracted MRM peaks (440.3/60.08, i.e. derivatized 13C2D2 S1 P) of 0.3 fmol 13C2D2 S1 P spiked into 10 ⁇ human plasma.
- FIGURE 10 shows calibration curves for the derivatized S1 P standard in human and mouse plasma.
- FIGURE 1 1 shows the lipid elution profile for a mixture of compounds including phosphate diester- containing lipids, S1 P (a phosphate monoester-containing lipid), other phosphate diesters and non-phosphorylated molecules using the polymer of Example 4a as the stationary phase and a first elution solvent which is isopropanol: methanol 1 .1 ; a second elution solvent which is isopropanol: methanol: triethylamine 49.5:49.5: 1 ; and a third elution solvent which is isopropanol: methanol: 1 % trifluoroacetic acid, 49.5:49.5: 1.
- N-vinylimidazole 2,6-bis-(bromomethyl) pyridine and ethylene glycol dimethacrylate (EGDMA) were purchased from Sigma Aldrich (St. Louis, MO). Dry acetonitriie used for synthesis was purchased from Merck. /V,/V ? -azo-bis-(2,4-dimethyl) valeronitrile (ABDV) was purchased from Wako. DMSO-c/6 was purchased from Deuterio-GmbH (Kastellaun, Germany).
- EGDMA was purified by the following procedure prior to use. The received material was washed consecutively with 10% aqueous NaOH, water and brine. After drying over MgS0 4 , the purified monomer was obtained by distillation under reduced pressure. All other reagents were used as received.
- Lipid standards D-ery//?ro-Sphingosine-1 -phosphate (S1 P d18:1 ) and isotope labeled standard D-eryfftro-Sphingosine-l -phosphate ( 13 C 2 D 2 -S1 P,) were purchased from Toronto Research Chemicals, D-ery ?/ -C17-Sphingosine-1 -phosphate (S1 P d17:1 ), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dimyristoyl-sn- glycero-3-phospho-(1 '-rac-glycerol) (DMPG), 1 ,2-dihexadecanoyl-sn-glycero-3- phosphate (DPPA), 1 -heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (LPA) N- octanoyl-cer
- Human blood for plasma was obtained from healthy volunteers (NUS-IRB Reference No. 04-1 15) using EDTA as anticoagulant. Blood samples were centrifuged at 2200xg for 15 min to isolate plasma and frozen at -80°C within 45 min of blood collection.
- Monomer 1 (134 mg, 0.2975 mmol) and EGDMA (2) (2.25 ml, 1 .925 mmol) were mixed into a 20 ml glass vial containing 10 ml of toluene/methanol 1/1.
- ABDV 25 mg, 0.097 mmol was added and the solution was degassed for 10 min with N 2 .
- the polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours.
- the solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- Monomer 1 54 mg, 0.119 mmol
- urea monomer 89 mg, 0.238mmol
- EGDMA 89 mg, 0.238mmol
- ABDV 10 mg, 0.0387 mmol
- the polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours.
- the solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- Example 4 Synthesis of Imidazolium based imprinted Polymers (i-IMP).
- Example 4a Synthesis of an imidazolium based polymer imprinted with a
- the solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- Example 4b Synthesis of an imidazolium based polymer imprinted with a
- the mono sodium salt of PL-C14 80 mg, 0.1 19 mmol
- Monomer 1 54 mg, 0.1 19 mmol
- EGDMA 0.900 ml, 4.77 mmol
- ABDV 10 mg, 0.0387 mmol
- the polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours.
- the solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- the solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- the solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours.
- the crude product was further crushed and sieved to 25-50 ⁇ and sub-25 ⁇ particles.
- the former fraction was used as sorbent for solid phase extraction.
- LC-MS/MS experiments were performed using Agilent 1200 series HPLC-Chip systems connected to Agilent 6540 or 6550 Q-TOF and to Agilent 6490 QQQ mass spectrometers.
- Two different types of chip were used for Reverse Phase (only for initial studies on pure standards) and HILIC separations:
- the Reverse Phase Ultra High capacity Chip included a 500 nl enrichment column (5 pm particle size, 80 A pore size) and a 75 pm * 50 mm C18 analytical column (Agilent Technologies Corp., Santa Clara CA).
- the Agilent 1200 series HPLC used a capillary pump for sample injection onto the Enrichment column and a Nano pump for separation.
- Solvents used for the Reverse Phase LC 5% water in 95 % methanol with 5 mM ammonium formate and 0.1 % formic acid (solvent A), 50% methanol in 50% isopropanol with 5 mM ammonium formate and 0.1 % formic acid (solvent B). Analytes were eluted with the following gradient: 5%B to 20%B from 0 to 2 min, 60% B from 2 to 8 mins, 100% B from 8 to 18 min, 100% B from 18 to 23 min, 5% B from 23.1 to 33 min. The chip cube was operated with back flush mode and samples were injected through the enrichment column at 4 ⁇ /min.
- the valve was switched after 1.5 min to place the enrichment column in line with analytical column at a flow rate of 400 nl/min.
- the Agilent 6540 (or 6550) quadrupole time-of-flight (QTOF) mass spectrometer was operated in positive ion mode; electrospray voltage was set to 1600 V (Vcap), temperature 300°C, drying gas 4 (or 2) l/min, fragmentor voltage 150 V.
- the instrument was operated in targeted MS/MS mode with MS acquisition rate of 1 spectra/sec and MS/MS acquisition rate of 2.0 spectra/sec. All the samples were resuspended in 50 or 100 ⁇ of mobile phase A before injecting 1 ⁇ of sample for LC-MS analysis.
- Solvents used for HILIC HPLC 50% acetonitrile in water containing 25 mM
- ammonium formate pH 4.6 The pH value was adjusted with formic acid. Analytes were eluted with the following gradient: 100% B from 0 to 1.5 min, 40% B from 1.5 to 8.5 min, 30% B from 8.5 to 10.5 min, 0% B from 1 1.5 to 13.0 min, 100% B from 13.1 to 19 min.
- the chip cube was operated with back flush mode and samples were injected through the enrichment column at 4 ⁇ l/min.
- the valve was switched 1.5 min after injection to place the enrichment column in line with the analytical column at a flow rate of 400 nl/min.
- the Agilent 6540 (or 6550) instrument was operated with the same parameters described previously, except for the gas temperature kept at 185°C and Vcap set at 1580 V. Spectra were acquired in targeted MS/MS mode with MS acquisition rate of 2 spectra/sec and MS/MS acquisition rate of 2 spectra/sec.
- M/z 60 was used as a 'quantifier' (due to its high intensity) and m/z 3 was used as a 'qualifier' (except in the case of phytosphingosines where m/z 1 13 was used as the quantifier). These ions were present after fragmentation of all LCB-P species.
- the qualifier at m/z 1 13 represents the mono-methylated phosphate and was used to discriminate between penta-methylated species generated by the derivatization reaction. Quantification was performed according to the internal standard method, comparing peak areas of the sample to the internal standard. The method shows linearity in the physiological range that has been reported for S1 P (Table 2 and Figure 10). All the samples were resuspended in 50 or 100 ⁇ of mobile phase B before injecting 1 pi of sample for LC-MS analysis. Table 2 - Intraday/interday precision and accuracy for S1 P in human plasma
- Main stock solutions of standards were diluted in methanol to a final concentration of 0.5 mg/ml for d18: 1 S1 P, 13 C 2 D 2 -S1 P (Internal Standard, IS) and d17: 1 S1 P.
- the stock solutions were further diluted in a concentration range of 50-1000 ng/ml for S1 P (d18:1 ) for validation experiments, while the internal standard stock was kept at a concentration of 200 ng/ml or 400 ng/ml.
- the plasma was transferred to 5ml cryovials (Practical Mediscience).
- the plasma was frozen at -80°C.
- 10 ⁇ plasma were mixed with 10 ⁇ IS and 90 ⁇ methanol.
- the solution was shaken on a thermomixer for 20 min at 4°C and centrifuged for 10 mins at 14,000 rpm.
- the supernatant could then be diluted in isopropanol for loading onto the IMP resin or directly derivattsed for analysis.
- mice Male Wild Type C57 BL/6 (WT) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a chow diet (18% protein and >5% fat, Harlan Teklad, Madison, Wl) post wean until 6 weeks of age. Diet was then switched to a Western-type (21.2% fat and 0.2% cholesterol, Harlan Teklad) until mice were sacrificed at 22-28 weeks of age. Mice were maintained under specific pathogen-free conditions with free access to food and water in the Animal Housing Unit of the National University of Singapore. All studies were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore. Blood was drawn from the mice via cardiac puncture immediately after sacrifice and mixed with 50 ⁇ of 100 mM EDTA.
- IACUC Institutional Animal Care and Use Committee
- the blood-EDTA mixture was then centrifuged at 14000 g, for 10 minutes at 4°C and the plasma was obtained from the supernatant for subsequent S1 P extraction. Special care was taken to minimize red blood cells and platelets lysis throughout the whole process, avoiding aspiration of blood through fine needles. All mice were fasted overnight prior to lymph collection to clear dietary lipids from the lymph fluid. 25 ⁇ of lymph fluid were crenulated from the cysterna chili with the use of extended length gel-loading pipette tips (Neptune Scientific) and collected into a new Eppendorf tube. The lymph samples were centrifuged at 14,000 g for 10 minutes at 4°C and the supernatant was obtained for subsequent S1 P extraction.
- 10 ⁇ plasma (5 ⁇ for Lymph) were mixed with 10 ⁇ IS (5 ⁇ IS for lymph) and 90 ⁇ methanol. Samples were then treated in the same way as for human plasma.
- the recoveries of IS were determined from the ratio of the mean peak area of the extracted samples to the mean peak area of samples where the internal standard was spiked after extraction.
- the recovery for IS at concentrations of 40 ng/ml and 80 ng/ml was 80.1 ⁇ 9.6% and 87.9 ⁇ 9.5%, respectively, in human plasma and 82.1 ⁇ 5.4% and 97.4 ⁇ 2.5% in mouse plasma.
- Intra-day/inter-day precision and accuracy were determined by analyzing quality control samples (QC) consisting of human plasma spiked with standard S1 P (d18: 1 ) at three different concentration levels, Low-QC (5 ng/ml), Middle-QC (40 ng/ml) and High-QC (80 ng/ml), and a fixed IS concentration of 40 ng/ml. Five replicates for each control were analysed while a plasma lipid extract spiked with IS was used as blank, to subtract the area of the endogenous S1 P when calculating precision and accuracy. Sample extraction from murine tissues
- Lymph nodes (1 lymph node/sample) and brain samples (2.5 or 5 mg) from individual mice were resuspended in 190 ⁇ of methanol + 10 ⁇ of IS. Samples were sonicated in water bath at 4°C for 30 min. After centrifuging at 4°C for 10 min at 14,000 rpm, the supernatant was collected for enrichment onto IMP resin.
- Pre-cultures of S. cerevisiae were grown in YPD medium (BD) for 24 h at 30°C. Then YPD was inoculated to a starting OD 60 o of 0.1 and cells were grown at 30°C until they reached OD 1 . Cells were then harvested and lyophilized. 2.5 mg of lyophilized yeast were resuspended in 500 ⁇ methanol and after addition of 5 ⁇ IS (400 ng/ml), homogenised with glass beads by vortexing for 2 min. After addition of 500 ⁇ methanol, the samples were sonicated for 1 h at 4°C. After centrifugation at 14,000 rpm, 4°C, for 0 min, supernatant was collected and dried in speedvac.
- BD YPD medium
- the IMP resin wells can be reused after regenerating with 3 x 1 ml methanol/acetic acid/water (60/30/10) and equilibrating with 3 x 1 ml loading solution.
- the obtained fractions were dried and reconstituted in 100 ⁇ of methanol. 10 ⁇ of TMS-Diazomethane (2M in hexane) were added and the sample incubated for 20 min at room temperature under gentle mixing at 750 rpm. The reaction was stopped by adding 1 ⁇ of acetic acid. The derivatized samples were dried in speedvac and reconstituted in 100 ⁇ of mobile phase before injecting into the LC-MS system.
- a brain tissue extract obtained as described above was spiked with standard DMPC (250 ng), DMPG(100 ng), Glu-Cer (100 ng),SM (50 ng), LPC (50 ng), Cer (50 ng), C8-PI (15 ng), S1 P ISTD (4 ng), DPPA(50 ng), Cer-1 P (50 ng), LPA (50 ng) before homogenisation and loaded onto the IMP resin in 1 ml loading solution.
- the lipid content of the different fractions was measured by LC-MS before (for phosphate diesters and non-phosphorylated species detection) and after (for monoesters) derivatization with TMS. Recovery in flow through+wash and eluate was estimated as % of the lipids present in the loaded fraction.
- CID collision-induced dissociation
- derivatized d18: 1 13C2D2- S1 P standard yielded three product ions, denoted ions A-C.
- both A and B ions are expected to be invariant for different LCB-Ps compared with ion C (m/z 328), which originated from the aliphatic portion. Indeed, when LCB-Ps from plasma were analyzed, the C fragment yielded m/z values as predicted for d18:2 ( Figure 1 b), d18:1 ( Figure 2c), d18:0 ( Figure 2d) and d16:0 for a lipid extract from fly ( Figure 2e, Figure 6).
- FIG. 8 shows S1 P d18: 1 for illustration purposes of the different fragments observed during tandem mass spectrometry. Fragments A and B are invariant and common to all LCB-P tested here. Fragment C is LCB-P species specific and recorded as the [M+HCOO]- adduct in negative mode (C**) .
- Tables 3a (positive ion mode) and 3b (negative ion mode) are a summary of the main fragments generated by CID fragmentation in both positive and negative mode for each LCB-P molecular species reported in this study (See Figure 8). All data were collected using Quadrupole Time of Flight (QToF) mass spectrometry except # which were acquired on a triple quadrupole mass spectrometer.
- QToF Quadrupole Time of Flight
- S1 P lyase is the key enzyme involved in the irreversible degradation of S1 P; its impaired function in D. melanogaster (sply05091 ) leads to an accumulation of LCB- Ps 7.
- the versatility of the polymer of the invention allows the isolation of different types of phospholipids.
- different types of compounds in addition to phosphate monoesters can be separated or isolated.
- polymer is the polymer of Example 4a and the conditions used are:
- the specificity of the polymer can be changed accordingly so that not only phosphate monoester containing lipids but also other types of lipids can be enriched by the polymer material (see Figure 1 1 ).
- Elution solvent 2 (basic) Isopropanol: methanol: triethylamine (49.5:49.5: 1 )
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Abstract
The invention relates to a polymer obtainable by radical co-polymerisation of a first monomer of general formula (I) or (II) or a mixture thereof: wherein A, A', B, B' X, Y, Y' and n are as defined herein; with a second, cross-linking monomer and optionally with one or more further co-monomers; wherein the molar ratio of the first monomer to other monomers is less than or equal to 1:5. The polymers selectively bind to phosphate ester compounds and can be used as a solid phase in a method for isolating compounds comprising a phosphate ester group from a mixture comprising one or more phosphate monoesters and/or phosphate diesters and/or other compounds such as lipids.
Description
Crosslinked Polymers Prepared from Functional Monomers Having Imidazolium, Pyridinium, Aryl-substituted Urea or Aryl-substituted Thiourea Groups and Uses Thereof.
The present invention relates to polymers which selectively bind phosphate ester- containing compounds, and to a method for isolating phosphate ester-containing
5 compounds from mixtures comprising one or more phosphate monoesters,
phosphate diesters and/or other compounds such as lipids. In particular, the
invention relates to a separation method in which long chain organic phosphate esters, for example phospholipids, are captured on a solid phase comprising a
polymer of the invention and can be separately eluted. Other aspects of the
10 invention relate to solid phases comprising the polymers and to separation devices
comprising the solid phase.
Lipids are small molecules with large structural and chemical diversity. Over the past two decades, technologies such as chromatography and mass spectrometry have
15 driven the biochemical analyses of complex lipid mixtures, tremendously advancing
our knowledge of lipid diversity. Lipid extraction (preceding analysis), however, is still largely based on partitioning procedures developed in the 1950s. Although
appropriate for many abundant components, these approaches result in variable recovery of the less-abundant and highly charged lipids, including phosphorylated
20 signaling lipids.
Phosphate is common amongst biological lipids and present in either monoester and/or diester configurations, with most biologically active lipids containing at least one phospho-monoester. Sphingosine-1 -phosphate (S1 P) is an example of such a
25 biologically active lipid. Long-chain amino alcohols, generally referred to as long- chain bases or LCBs (Pruett, S . et al. J Lipid Res 49, 1621 -1639 (2008)), and their phosphorylated forms (LCB-P) display particularly diverse chemistries across
biological species and tissues. In human plasma, the most abundant LCB-P is S1 P, the phosphorylated derivative of [(2S, 3R, 4E)-2-aminooctadec-4-ene-1 ,3-diol], which
30 has the structure:
S1 P is currently emerging as a valid biomarker for life-threatening illnesses such as
multiple sclerosis, cardiovascular disease and cancer. The commercially available method to detect S1 P (antibody-based 'ELISA' kit) has the major disadvantages that only high S1 P concentrations can be analysed and that there is a relatively high risk of false results because of the cross-reactivity of the antibodies with similar molecules. There is therefore a need for improved methods of detection of S1 P. In addition to the more common LCB-Ps such as S1 P, animal and plant cells and tissues comprise other LCB-Ps. Little is known about the existence of other LCB-Ps of different chemical and structural composition, despite the presence of various non- phosphorylated LCB precursors. In order to investigate the structures and functions of these compounds, it is necessary to isolate, quantify and characterize them.
Animal and plant cells and tissues also comprise other mono-phosphorylated compounds such as phosphoinositides (PIPs), phosphatidic acid, lysophosphatidic acid and ceramide-1 -phosphate; phosphodiester compounds, for example phosphodiester lipids; and compounds such as lipids which contain neither phosphate mono-esters nor phosphate-diesters.
There is therefore a need to isolate, quantify and characterize phosphate ester- containing compounds such as phospholipids, particularly LCB-Ps, as well as phosphoinositides, phosphatitic acid, lysophosphatidic acid and ceramide-1 - phosphate in the tissues and body fluids of humans and other animal species, for example mammals; and also in plants and microorganisms.
The present inventors have developed polymers which selectively bind phosphate esters.
Therefore, in a first aspect of the invention there is provided a polymer obtainable by radical co-polymerisation of a first monomer comprising a compound of general formula (I) or (I I) or a mixture thereof:
(I) (II) wherein each of A and A' is a 5- or 6-membered positively charged heteroaryl ring
containing a quaternary nitrogen atom or when Y or Y' is -NR -(C=Z)-N-R -, A or A' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with halo, C,.e haloalkyl or nitro;
each of B and B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom; or a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected from halo, C1-6 haloalkyl or nitro; each of Y and Y' is a linking group comprising 1 -6 -CH2- units wherein a -CH2- unit is optionally replaced by a C5. 4 aryl or heteroaryl group optionally substituted with one or more substituents selected from H, halogen, NH2, C1- alkyl, alkoxy, haloalkyl or Ci_4 haloalkoxy or
when each of A or A' and B or B' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected halo, d.6 haloalkyl or nitro, each of Y and Y' may be -NR20-(C=Z)-N-R20-, wherein
each R20 is independently H, methyl or ethyl; and
Z is O or S;
n is 1 or 2 or when Y and Y' is -NR 0-(C=Z)-N-R20-, n is 0;. and
X" is a halide ion or a hydrophobic anion such as PF6 "; with a second, cross-linking monomer and optionally with one or more further co- monomers;
wherein the molar ratio of the first monomer to the sum of the second monomer and the one or more further co-monomers (if present) is less than or equal to 1 :5.
Polymers similar to the polymers of the present invention. For example Buscemi et al, ChemPlusChem, 79, 421 -426 (2014) discloses palladium scavengers which are polymers prepared from monomers of the formula:
where the cross linker is one of the following:
and wherein the monomer may be copolymerised in the presence of ethylene glycol dimethacrylate (EGDMA). However, this polymer differs from that of the present invention in that the ratio of the di-imidazolium monomer to EGDMA is 1 : 1 . In contrast, the polymers of the present invention are prepared using a far higher proportion of the cross-linking monomer.
CN102050919 relates to polymer nanoparticles prepared from a monomer similar to those described by Buscemi et al except that the linker is -(CH2)4-, and EGDMA. These polymer nanoparticles are used for catalyzing the addition reaction of carbon dioxide to an epoxy ring compound. However, in these polymers the ratio of the imidazolium monomer to the cross-linking agent is from 2:1 to 10: 1 whereas the polymers of the present invention are prepared using a far higher proportion of the cross-linking monomer. Vinylimidazole-EGDMA copolymers are known from EP0162388, US 4,600,578 and US 4,649,048, which relate to the use of these polymers as bile acid sequestrating agents and for treating diarrhoea. However, the monomers from which these polymers are prepared differ in structure from the monomers used in the polymers of the present invention.
The polymer of the invention is capable of complexing phosphate ester groups, for example the phosphate groups of phosphorylated long-chain base phosphate molecules. Therefore, the polymer is suitable for use in detecting compounds comprising phosphate esters, for example LCB-Ps, and isolating them from biological samples.
Although low molecular weight bis-imidazolium hosts have been studied for the recognition of inorganic phosphate (Kim, S.K. et al. Org Lett 5, 2083-2086 (2003)), a polymer capable of binding long chain base phosphates has not previously been developed, nor have such hosts been used for the recognition and isolation of phospholipids.
The present invention has immediate relevance for our understanding of S1 P biology as well as its therapeutic targeting. S1 P biosynthesis, trafficking, receptor binding and degradation are highly controlled, and the targeting of S1 P receptors and
metabolizing enzymes remains an active field of research and drug development. It is conceivable that part of the LCB-P signaling machinery is influenced by the chemical nature of their aliphatic portion. This factor has so far been neglected, mainly because of the lack of information on S1 P/LCB-P diversity. It is likely that LCB-P chain diversity affects receptor binding, and impacts S1 P/LCB-P gradients, which are known to be important underlying components of immune cell trafficking via the reversible de-phosphorylation of S1 P. Irreversible cleavage of S1 P by S1 P lyase, on the other hand, is a critical step that links sphingolipid metabolism with that of other lipids. The results from the sply-/- mutant demonstrate the unexpected accumulation of LCB-Ps when this single degradation step is impaired. Finally, it is possible that some of the chemically diverse LCBs (i.e., the non-phosphoryated precursor of the LCB-P described here) found in fungi, plants and insects could be substrates for kinases (in the respective organism, or in other organisms as a result of infection/diet) leading to even greater diversity among LCB-Ps. Our improved method for LCB-P analysis is therefore expected to have a profound impact in a variety of fields. Moreover, this technique can be also applied for the study of other low abundant signalling molecules (phosphate monoesters), broadening the number of its applications. In the present invention the term "alkyl" refers to a straight or branched fully saturated hydrocarbon chain. d-6 alkyl groups have from one to six carbon atoms and examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n- pentyl and n-hexyl. C1.4 alkyl groups have from one to four carbon atoms. The term "halo" refers to fluoro, chloro, bromo or iodo and a halide ion may be a fluoride, chloride, bromide or iodide ion.
The term "haloalkyi" refers to an alkyl group which is substituted by one or more halo atoms and includes any number of halo substituents up to perhalo substituted groups. Examples include trifluoromethyl; 1 ,1 -dibromoethyl.
The terms "carbocyclyl" and "carbocyclic" refer to a non-aromatic cyclic group having three to ten ring atoms (unless otherwise specified), which may be fully or partially saturated and which may be fused or bridged. Examples include cycloalkyl groups such as cyclopropyl, cyclopentyl and cyclohexyl as well as cycloalkenyl groups such
as cyclopentenyl and cyclohexenyl. Further examples include fused or bridged ring systems such as norbornane.
More suitable carbocyclic rings are 5- or 6-membered rings such as cyclopentyl, cyclohexyl or cyclohexenyl.
The terms "heterocyclyl" and "heterocyclic" refer to a cyclic group as defined above for carbocyclyl, except that one or more ring atoms is replaced by a hetero atom selected from N, O and S. Examples include aziridine, azetidine, pyrrolidine, pyrroline, imidazoline, piperidine, piperazine, morpholine, oxetane, tetrahydrofuran and oxazoline. Further examples include fused or bridged ring systems such as octahydroquinoline or nor-tropane.
More suitable heterocyclic rings are 5- or 6-membered rings such as piperidyl, piperazinyl, morpholinyl and tetrahydrofuryl.
The term "aryl" in the context of the present specification refers to a ring system with aromatic character having from 6 to 14 ring carbon atoms (unless otherwise specified) and containing one ring or two fused rings. Where an aryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of aromatic moieties are benzene, naphthalene, anthracene, phenanthrene, tetrahydronaphthalene, indane and indene. More suitably, the aryl group is a phenyl group. The term "heteroaryl" in the context of the specification refers to a ring system with aromatic character having from 5 to 10 ring atoms (unless otherwise specified), at least one of which is a heteroatom selected from N, O and S, and containing one ring or two fused rings. Where a heteroaryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of monocyclic heteroaryl groups include pyridine, pyrimidine, furan, thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole and isothiazole. Examples of bicyclic fully aromatic heteroaryl groups include quinoline, isoquinoline, indole, benzofuran, benzimidazole and benzothiazole. Examples of bicyclic heteroaryl groups in which one ring is not fully aromatic in character include dihydroquinolines, tetrahydroquinoline,
tetrahydroisoquinoline, chromene, chromane, benzimidazoline, benzomorpholine,
isoindoline and indoline.
More suitable heteroaryl groups are monocyclic 5- or 6-membered rings such as pyridyl, furyl or thienyl.
In the present invention, the terms "phosphate monoester" and "phospho monoester" refer to compounds based on phosphoric acid (H3P04) in which one of the hydrogen atoms is replaced by an organic group. The terms "phosphate diester" and "phosphodiester" refer to compounds based on phosphoric acid (H3P04) in which two of the hydrogen atoms are replaced by organic groups.
A long chain base phosphate molecule refers to a molecule having an alkyl, alkenyl or alkynyl chain of from 12 to 24 carbon atoms substituted with at least one phosphate group represented by the formula:
or a salt thereof. Typically, a long chain organic phosphate molecule such as a phospholipid, for example a long chain base phosphate contains one to four phosphate groups, more usually one to three phosphate groups and in many cases one or two phosphate groups. In addition to the phosphate group, a long chain organic phosphate molecule such as a long chain base phosphate molecule or other phospholipid may contain additional functional groups. For example, the chain may be substituted with one or more groups selected from OR10, NR11R12 and N+R1 R12R13, wherein each of R10, R11, R12 and R13 independently represents H or a C1-4 alkyl group.
More suitably each of R10, R11, R12 and R 3 independently represents H, methyl or ethyl, particularly H or methyl.
In the present application, long chain base phosphate molecules are referred to by the length of the carbon chain, the number of double bonds and the number of hydroxy! groups in the parent lipid, where "d" indicates dihydroxy and "t" indicates trihydroxy. Thus, sphingosine is denoted as d18:1 and phytosphingosine is t18:0.
The position of the double bond may be indicated by a superscript, i.e. 4-sphingenine is d18:1 A4f or 4E-d18: 1.The "d" or "t" relating to the parent lipid molecule is retained for the equivalent long chain base phosphate, where one of the OH groups has been replaced by the phosphate moiety. Thus, sphingosine-1 -phosphate is also denoted as d18: 1.
In some cases, polymers wherein A and B are both imidazolium groups, Y is -(CH2)X-, where x is 1 -6 are excluded from the scope of the invention.
In some cases, polymers wherein A and B are both imidazolium groups, Y is -(CH2)X-, where x is 1 -6 and wherein a -CH2- unit is optionally replaced by a phenyl group are excluded from the scope of the invention. In some cases, polymers wherein Y or Y' is -NR20-(C=Z)-N-R20- and A or A' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with halo, haloalkyl or nitro are excluded from the scope of the invention.
In the polymers of the invention, the molar ratio of the first monomer of general formula (I) or general formula (II) to the sum of the second cross linking monomer and one or more further co-monomers (if present) is less than or equal to 1 :5.
Typically the molar ratio is from about 1 : 100 to 1 :5. More suitably, the molar ratio of monomer of general formula (I) or general formula (II) to cross linking monomer is from about 1 :80 to 1 :20; still more suitably from 1 :65 to 1 :25, or from 1 :50 to 1 :30. Typically the molar ratio is between 1 :35 and 1 :45, for example about 1 :40.
Suitably, the polymer is obtainable by a polymerisation process in which the solvent is selected from a water, an alcoholic solvent such as methanol, ethanol or isopropanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 , 1 -trichloroethane, chloroform (CHCI3), dichloromethane (DCM), toluene,
dimethylsulfoxide (DMSO), isopropanol or any mixture of these solvents or other solvents.
Typically, the solvent is a mixture of an alcoholic solvent and a non-polar organic co- solvent, for example toluene.
The most suitable polymers for use in the present invention are those in which the total concentration of the first monomer of formula (I) or (II) and the second cross- linking monomer and one or more further co-monomers (if present) is at least 100 mg/ml of solvent. More usually, the concentration of the monomers is from 100 to 500 mg/ml of solvent, for example 100-400 mg/ml, 100-300 mg/ml or 200-300 mg/ml. Typically, the total concentration of the monomers is about 250 mg/ml of solvent.
In monomers of general formula (I) or (II), X" is suitably a halide or a PF6 " ion.
Typically X is bromide.
In monomers of general formula (I) or (II) the linking group Y is suitably bonded to the quaternary nitrogen moiety of the group A or A'. Suitably, the group A or A' is a pyridinium or imidazolium ion.
When A or A' is a pyridinium ion, the group -CH=CH2 may be connected at the pyridinium 4-position. When A or A' is an imidazolium ion, the group -CH=CH2 may be linked to the non- quaternary ring nitrogen atom.
In one embodiment, the group B or B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom and in this embodiment:
the linking group Y is suitably bonded to the quaternary nitrogen moiety;
the group B or B' may be a pyridinium or imidazolium ion.
When B is a pyridinium ion, the group -CH=CH2 may be connected at the pyrid' 4-position;
when B is an imidazolium ion, the group -CH=CH2 may be linked to the non-
quaternary ring nitrogen atom.
In some suitable compounds A and B are the same and the monomer of general formula (I) is a compound of general formula (la) or (lb):
(la) (lb) wherein X and Y are as defined above.
Monomers of formula (la) are particularly suitable for use in the present invention.
In an alternative embodiment, the group B or B' is a 5- or 6-membered aryl or heteroaryl ring, i.e. an uncharged moiety.
In this embodiment, B may be, for example, a phenyl or pyridyl group.
In suitable monomers of general formulae (I) and (II), Y is a linker group selected from:
wherein
each of R1, R2 and R3 is independently selected from H, halogen, NH2, C 4 alkyl, C-i_4 alkoxy, haloalkyl, haloalkoxy or R1 and R2 or R2 and R3 may together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring;
each of R4, R5, R6 and R7 is independently selected from H, halogen, NH2,
d- alkyl, d- alkoxy, d-4 haloalkyl, d-4 haloalkoxy or R4 and R5 or R6 and R7 may together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring; R8 is H, halogen, NH2, d.4 alkyl, d-4 alkoxy, d_4 haloalkyl, d-4 haloalkoxy.
Monomers of general formula (I) or general formula (II) in which Y is
Monomers of general formula (I) or (II), and especially monomers of general formaul (la) above, in which Y is:
wherein each of R1, R2, R3 and R8 is as defined above are particularly suitable. In one embodiment, in the first monomer of general formula (I) or (II), the linker Y
wherein R1 , R2 and R3 are as defined above.
Most suitably the monomer is a monomer of formula (la).
In this embodiment, suitably, R1, R2 and R3 are hydrogen or R1 and R2 or R2 and R3 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
More suitably, R1, R2 and R3 are all hydrogen.
A particularly suitable monomer of this embodiment is Monomer 1 , which has the structure:
wherein R1, R2 and R3 are as defined above.
In this embodiment, suitably, R , R2 and R3 are hydrogen or R1 and R2 or R2 and R3 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
More suitably, R1, R2 and R3 are all hydrogen.
where X is as defined above but is most suitably Br
In an alternative embodiment, the linker Y is:
wherein R4, R5, R6 and R7 are as defined above.
In this embodiment, suitably, R4, R5, R6 and R7 are all hydrogen or R4 and R5 or R6 and R7 together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring.
More suitably, R4, R5, R6 and R7 are all hydrogen.
A particularly suitable monomer of this embodiment is Monomer 3, which has the structure:
where X" is as defined above but is most suitably Br' In another embodiment, Y is:
wherein R1, R2, R3 and R8 are as defined above.
In this embodiment, suitably, R1, R2, R3 and R8 are all hydrogen.
A particularly suitable monomer of this embodiment is Monomer 5, which has the structure:
In a further alternative embodiment, the linker Y is 1 -6 -CH2- units. In particular, Y may be -CH2-.
In the monomer of general formula (II), the linker Y may be 1-6 -CH2- units and B may be a 5- or 6-membered heteroaryl ring, for example phenyl.
An example of a monomer of this embodiment is Monomer 4, which has the structure:
In another embodiment, the monomer of general formula (I) or general formula (II) may be a compound in which Y or Y' is -NR20-(C=Z)-N-R20-. In this embodiment, more suitably, independently or in combination:
R20 is H or methyl but especially H; and/or
Z is O.
In such compounds each of A or A' and each of B or B' is 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more halo, Ci.6 haloalkyl or nitro substituents. More suitably, each of A or A' and each of B or B' is a phenyl group optionally substituted with one or more halo, d. haloalkyl or nitro substituents, for example fluoro, trifluoromethyl or nitro substituents.
A particularly suitable example of a monomer of this type is Monomer 6:
The polymer of the invention may be prepared from a single monomer of general formula (I) or general formula (II) or from two or more such monomers. In one embodiment, a monomer of general formula (I) or (II) in which Y or Y' is -
NR20-(C=Z)-N-R20-, may be combined with another monomer of general formula (I) or (II) in which Y or Y' is other than is -NR20-(C=Z)-N-R20-.
Suitable monomers for use in such combinations are those described above. In particularly suitable combinations, a monomer selected from Monomer 1 , Monomer 2, Monomer 3, Monomer 4 or Monomer 5 may be combined with Monomer 6.
In some cases, one of the monomers for the preparation of the polymer may exhibit a change in fluorescence on binding of a phosphate ester and this property may be carried through into the polymer so that binding of a phosphate ester to the polymer can be detected as a quenching or an enhancement of the fluorescence emission of the polymer.
In some cases, the monomer which exhibits this property may be the monomer of general formula (I) or (II), for example, a monomer in which the linker Y or Y' is an anthracene group or a monomer in which the linker Y or Y' is -NR20-(C=Z)-N-R20-. Examples of monomers are Monomer 5 and Monomer 6.
Monomers of general formulae (I) and (II) are known and are readily available or may be prepared by methods familiar to those of skill in the art, for example the methods set out in Examples 1 , 6, 8, 10 and 1 1 below, or adaptations thereof which would be clear to a person of skill in the art.
Cross linking monomers which are suitable for use in the present invention include di- and tri-methacrylate monomers, dialkenyl benzene monomers and bis acrylamide monomers. Specific examples of cross linking monomers include ethyleneglycol dimethacrylate (EGDMA), divinylbenzene (DVB), diisopropenylbenzene, (DIB),
trimethylolpropanetrimethacrylate (TRIM), pentaerythritoltriacrylate (PETA), ethylenebisacrylamide (EBA), piperazinebisacrylamide (PBA), and
methylenebisacrylamide (MBA).
EGDMA is particularly suitable.
In some cases, the polymer will be the product of the monomer of general formula (I) or (II) without further co-monomers.
In other cases, however, one or more further co-monomers are added to the mixture of first monomer and second, crosslinking monomer. The particular co-monomer which is chosen will depend upon the nature of the cross linking monomer. Thus, when the cross linking monomer is a di- or tri-methacrylate such as EGDMA, TRIM, or MBA, the co-monomer may be also be a methacrylate monomer, for example methyl methacrylate, 2-hydroxyethylmethacrylate, methacrylamide or an alternative d.6 alkyl methacrylate so that a methacrylate matrix is produced.
When the cross linking monomer is a dialkyl benzene-based monomer such as DIB, it is more appropriate to select an alkenyl benzene monomer such as styrene as the co-monomer in order to produce a suitable matrix. Similarly, an acrylamide co- monomer for use in combination with an acrylamide cross linking monomer.
Polymers formed from Monomer 1 or combinations of Monomer 1 with other monomers of general formula (I) or general formula (II) have been shown to be
particularly suitable, especially polymers formed from Monomer 1 co-polymerised with EGDMA. The inventors have demonstrated that imprinted resins formed from Monomer 1 copolymerised with EGDMA bind a greater proportion of a phosphate ester anion in a neutral buffer such as HEPES (4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid) buffer, pH 7, compared with similar imprinted resins formed from Monomers 2, 3 or 4. Thus the copolymer formed from Monomer 1 bound 89% of the available phosphate compared with 58% and 34% respectively for polymers formed from Monomers 2 and 3. The polymerisation reaction may be conducted at a temperature of from 40°C to 90°C, more usually about 40°C to 80°C. In one embodiment, the temperature is maintained at about 40°C to 60°C, for example about 50°C for a period of 12 to 48 hours, for example about 24 hours, following which it is raised to about 65°C to 75°C, for example about 70°C for a further period of 2 to 6 hours, for example about 4 hours.
The binding polymer of the invention is suitably a macroporous polymer such that the structure contains large pores or channels. Macroporous polymers are highly cross- linked polymers which contains large interconnected pores or channels within their structure. Unlike hydrogels and similar polymers, which require hydration in order for pores to form, the pores and channels in a macroporous polymer are formed during the polymerisation process and are present at all times. Macroporous polymers are known in the art and are described, for example, by Arrua et al, (Materials 2009, 2, 2429-2466).
The polymer, after crushing and sieving, can be used as a binding polymer to extract phosphate esters, including phospholipids, from a biological sample.
As discussed above, the polymer is obtainable by radical co-polymerisation and therefore in a further aspect of the invention there is provided a process for the preparation of a novel polymer as described above, the process comprising the radical co-polymerisation of a first monomer of general formula (I) or general formula (II) as defined above with a second, cross-linking monomer; characterised in that the ratio of the first monomer to the second monomer is less than or equal to 1 :5.
Suitably, the molar ratio of the first monomer of general formula (I) to the second cross linking monomer is as described above and the reaction temperature and conditions are also as described above for the first aspect of the invention. Usually, the polymerisation reaction will be initiated using a polymerisation initiator. Examples of suitable polymerisation initiators include azo-N,N'- bisdimethylvaleronitrile (ABDV), azo-N,N'-bisisobutyronitrile (AIBN), 2,2'-azobis-(2,4- dimethyl-4-methoxyvaleronitrile) (V70) or any other azo-based initiator (available from Wako Pure Chemical Ind. Ltd. , (Japan); or redoxinitiators e.g.
ammoniumpersulfate with or without added accelerator (e.g.
TEMED=tetramethylethylenediamine).. The choice of initiator is not critical and any known polymerisation initiator could be used.
As described above, the process may further include the addition of a co-monomer.
The reaction may be conducted in any suitable solvent such as water, methanol, ethanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 , 1 -trichloroethane, chloroform (CHCI3), dichloromethane (DCM), toluene, dimethylsulfoxide (DMSO), isopropanol or any mixture of these or other solvents.
More suitably, the reaction is conducted in a solvent comprising a mixture of an alcoholic solvent and a non-polar organic co-solvent. Suitable alcoholic solvents include methanol, while toluene is an example of a suitable co-solvent. Suitably in this case, the alcoholic solvent and the non-polar organic solvent are present in a ratio of 1 :5 to 5: 1 v/v, more suitably 1 : 1 v/v.
Suitably, the polymerisation reaction is conducted under an inert atmosphere, for example nitrogen or argon.
The temperature conditions for the reaction are also as described above.
In addition to macroporous polymers, however, the polymer can also be prepared in other formats such as nanofibers, nanotubes, monoliths, nanoparticles and their nanostructured counterparts such as core shell nanoparticles with optionally different
functional properties such as magnetic or luminescent cores or labeled with probes allowing facile detection (e.g. electrochemiluminescent probes ECL). They can be tuned to exhibit maximum compatibility with the surrounding medium e.g. coatings to minimize protein adsorption. The polymer can be produced by any technique available for producing polymer particles including techniques relying on
polymerisations in liquid-liquid two phase systems by suspension polymerisation or emulsion polymerisation or any variant of these (e.g. miniemulsion polymerisation)., or in one liquid phase by dispersion polymerisation or precipitation polymerisation. In one embodiment, the polymerisation reaction is performed using controlled radical polymerisation (CRP), which differs from conventional radical polymerisation in respect of the life time of the propagating radicals. In CRP this can be extended to hours which allows the preparation of polymers with predefined molecular weights, low polydispersity, controlled composition and functionality. This leads to polymers displaying higher binding capacity, affinity and faster association and dissociation of peptide. Common CRP techniques are RAFT (reversible addition fragmentation chain transfer polymerisation), ATRP (atom transfer radical polymerisation) and iniferter (initiator, transfer, termination) polymerisation. In some cases, it is possible to produce polymer particles by grafting a polymer film onto the surface of a preformed support. The grafting can be performed according to either the "grafting to" or the "grafting from" approach, both of which are well known to those of skill in the art. The latter approach improves the production process as well as the molecular recognition and kinetic properties of the materials (and is described in US 6,759,488).
In other cases, polymer particles according to the invention may be produced using a molecular imprinting approach in which a template resembling a phosphate ester target molecule in shape and functionality is added to a mixture of the first monomer and the second, cross-linking monomer prior to polymerization. In the present case, the template will usually be a monomer resembling a target phosphate monoester compound such as a LCB-P. Suitable LCB-P templates include PL-CL14 or a salt thereof, for example a mono- or di-alkali metal salt, such as a mono- or di- lithium, sodium or potassium salt. A mono- or di-sodium salt is particularly useful. After polymerization, the template may be removed from the polymer to leave a structure
which exhibits affinity for the target phosphate monoester compound or compounds.
In another embodiment the polymer can be produced in the form of nanoparticles in a typical size range of 10nm-500nm. The engineering of nano-micrometer-sized polymer particles can be done by precipitation polymerisation (F. Wang, P.A.G. Cormack, D.C. Sherrington and E. Khoshdel, Angew. Chem. Int. Ed. (2003) 42(43), 5336-5338) or miniemulsion polymerisation procedures where uniform imprinted beads can be produced prepared by high dilution polymerisation in one step. Both aqueous and non-aqueous protocols can be used.
Alternatively, nanoparticle synthesis can be performed by grafting (N. Perez-Moral, A. G. Mayes, Macromol. Rapid Commun. 2007, 28, 2170). This starts from a nonporous nanosized core containing radical initiator or chain transfer groups. Onto the core a thin polymer films can be grafted with minimal influence of the bead size, dispersity and morphology on the polymerisation conditions. Nanoparticles can thus be engineered which can capture their target in biological fluids. Paramagnetic, monosized polymer particles, are commonly used for this purpose. The technique is fast, mild and involves no centrifugation or chromatography. Paramagnetic particles may be prepared in the form of core/shell microgels by a two-stage "seed-and-feed" precipitation polymerisation or by grafting a polymer shell on a magnetic core bead.
The polymers of the present invention are capable of selectively binding phosphate esters and therefore in a further aspect of the invention there is provided a method for isolating compounds comprising a phosphate ester group from a sample, the method comprising:
i. contacting the sample with a first solvent so as to extract soluble compounds into a sample solution, wherein the first solvent is capable of dissolving phosphate ester compounds;
ii. contacting the sample solution with a solid phase comprising a polymer according to the first aspect of the invention;
iii. washing the solid phase with one or more washing solvents to remove compounds not comprising a phosphate ester group; and
iv. eluting compounds comprising a phosphate ester group with either or both of a basic elution solvent and an acidic elution solvent.
In the method of the invention, the biological sample may be derived from any species of animal, plant or microorganism. The sample may be a tissue sample from a mammal, including a human. Suitable samples blood, plasma and lymph or other tissues, including brain tissue. Blood and tissue samples from other animal species may also be used or, in some cases, for example with small animals such as insects, the sample may comprise the entire organism. The sample may also be derived from a plant and may include material taken from any part of the plant, including leaves and seeds. In some cases, the sample will be a microorganism, for example bacteria or yeast such as Saccharomyces cerevisiae.
The solid phase may be incorporated into a separation device which itself forms a further aspect of the invention as will be discussed below.
Suitably, the first solvent comprises an alcoholic solvent such as methanol, ethanol, n-propanol or isopropanol, particularly methanol, although in some circumstances a chlorinated solvent such as dichloromethane or chloroform may also be used. The soluble compounds extracted by the first solvent include not only compounds comprising phosphate monoesters, for example long chain base phosphates, and phosphate diesters but also other many other types of compound, including the lipids from which many long chain base phosphate compounds are derived. Therefore it is necessary to separate phosphate monoester- and phosphate diester-containing compounds from one another and from these other compounds.
Once the sample has been loaded onto the solid phase or device containing the polymer of the invention, molecules which are non-specifically bound to the solid phase can be eluted using the one or more washing solvents. The washing solvent or solvents may also comprise alcoholic solvents and, for example, may be selected from methanol, ethanol, n-propanol and isopropanol or mixtures thereof. In one embodiment, the solid phase is washed firstly with isopropanol followed by a mixture of isopropanol and methanol in a ratio of 1 : 1 v/v.
In step (iv), the phosphate esters are eluted with either or both of a basic elution solvent and an acidic elution solvent.
The acid elution solvent is useful for removing phosphate monoesters from the solid
phase and in some cases, for example when the method is intended for the isolation of phosphate monoesters, for example phospholipids such as S1 P, the acidic elution solvent may be used alone without the basic elution solvent. Although the basic elution solvent may also be used alone, the process more suitably, includes its use in combination with the acidic elution solvent.
When a basic elution solvent and an acidic elution solvent are employed, the basic elution solvent may be used prior to the acidic elution solvent.
In step (iv) the acidic elution solvent suitably comprises a mixture of a chlorinated solvent and an alcoholic solvent. The chlorinated solvent may be, for example, chloromethane, dichloromethane or chloroform, with chloroform being particularly suitable. Examples of suitable alcoholic solvents include methanol, ethanol, n- propanol and isopropanol, but especially methanol.
Suitably, the ratio of chlorinated solvent to alcoholic solvent in the elution solvents is 1 :1 v/v and thus one suitable example of an elution solvent is an acidified 1 : 1 v/v mixture of chloroform and methanol.
Acidification of the acidic elution solvent may be achieved using an organic acid, for example formic acid or trifluoroacetic acid. One example of a particularly suitable acidic elution solvent is a mixture of chloroform/methanol/1 % trifluoroacetic acid. Suitably these are in a volume ratio of 49.5/49.5/1.
The acidic elution solvent removes the phosphate monoester compounds from the binding polymer and the elution rate differs for different phosphate monoesters.
The basic elution solvent may comprise an alcoholic solvent or mixture of alcoholic solvents together with a basic compound such as an amine, especially a tertiary amine, for example a trialkyl amine such as triethylamine or trimethylamine.
Suitable basic elution solvents include mixtures of isopropanol and methanol, for example a 1 : 1 mixture, and a tertiary amine. Where the basic compound is a tertiary amine the solvent may be isopropanol/methanol/amine, suitably in a volume ratio of 49.5/49.5/1. Triethylamine is a particularly suitable basic compound for use in such
mixtures.
Using the method of the present invention it is possible to separate the phosphate monoesters from phosphate diesters and other compounds and also to separate them from one another. The separated phosphate ester compounds may be further characterized, for example using mass spectrometry. Thus, even if full identification of the compounds is not possible, a mass can be obtained.
Mass spectrometry either may be carried out simultaneously with the
chromatography step, for example using an LC-MS apparatus, or after the chromatography step.
In some cases a standard solution may be added to the sample solution before step (ii). The standard solution may comprise one oV more known phosphate ester compounds, for example phosphate monoesters such as long chain base phosphate compounds, and may therefore assist with the identification of similar compounds, e.g. long chain base phosphate compounds, in the sample. Usually, the amount as well as the identity of phosphate oester compounds in the standard is known and this will enable the quantitative detection of the long chain base phosphates and other phosphate ester compounds in the sample.
In one embodiment, the method comprises the additional step of derivatising the phosphate ester compounds of the sample, for example by reacting with an alkylating agent in order to replace one or more of the hydrogen atoms on a phosphate or an amine group with an alkyl group. This method is particularly useful for the separation of phosphate monoester compounds.
Derivatisation is suitably carried out after contacting the sample with the solid phase. However, if the phosphate ester compounds are derviatised after the elution step (iv) it is preferable to repeat steps (ii) to (iv) using a mixture of the derivatised phosphate ester compounds as the sample.
Suitably the alkylating agent is a methylating agent, for example trimethylsilyl diazomethane (TMS-diazomethane).
The derivatisation step is particularly suitable when the sample comprises two or more similar phosphate ester compounds, especially phosphate monoester compounds, and more especially LCB-Ps. In this case, the inventors have found that derivatisation led to a gain in sensitivity, possibly explained by a more effective ionization of the alkylated derivatives. This is particularly true for long chain base phosphate compounds containing an amine group, which, when alkylated, carries a permanent positive charge while the negative charge on the phosphate is
neutralised. By introducing and optimizing the above two steps (phospho-monoester capture via the binding polymer and derivatization with TMS), the inventors have:
(i) improved the signal intensity of LCB-Ps derived from plasma by -60-fold compared with LC-MS alone;
(ii) reduced matrix interference; and
(iii) eliminated the carry-over effects commonly encountered with LC-MS of native LCB-Ps (see Berdyshev, E.V., Gorshkova, I.A., Garcia, J.G., Natarajan, V. &
Hubbard, W.C. Anal Biochem 339, 129-136 (2005)).
This enhanced workflow led to the discovery of previously undescribed LCB-P species unable to be detected/quantified by LC-MS alone.
This new detection method was further validated in various biological matrices for its accuracy and precision, reproducibility and linearity. Moreover, the number of species detected and their relative abundance was conserved after the derivatization reaction. The aforementioned characterizations involved nano-electrospray ionization and high-resolution MS after chromatographic separation of the mixtures by Hydrophilic Interaction Chromatography (HILIC) built into a microfluidic chip. This was important to improve the co-elution of S P standards and endogenous LCB-Ps for qualitative and quantitative characterizations 6 of new LCB-P species. Indeed, few high-resolution mass spectra of LCB-Ps have been published, possibly because of the low signal intensities that hamper the precise characterization of ion peaks.
The solid phase material formed from the polymer of the invention represents a further aspect of the invention.
The solid phase material is suitable for capturing and separating phosphate monoester compounds, the solid phase comprising a binding polymer as defined above. In the solid phase material, the polymer may be used alone or in combination with other components.
The solid phase is typically in the form of particles and the polymer is a macroporous polymer, the solid phase material may be prepared by a process comprising the steps of crushing and sieving the polymer of the invention.
This process forms a further aspect of the invention.
In other cases, particles may be formed by the methods described above.
There is further provided a separation device comprising the solid phase material. The separation device may comprise a chromatography column, suitably a column for use in liquid chromatography or gas chromatography. In a further aspect of the invention there is provided a kit for isolating phosphate ester compounds from a mixture comprising one or more such compounds, the kit comprising a solid phase material as defined above and instructions for the use of the material. The kit may further comprise one or more standards, which may be known phosphoester compounds Suitably, the one or more standards may be provided in known amounts. For example, a kit for the detection of S1 P may comprise a known amount of S1 P as a standard. Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. "Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.
Throughout the description and claims of this specification, the singular
encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
The invention will now be described in greater detail with reference to the Examples and to the drawings in which: FIGURE 1. illustrates substantially improved detection of LCB-P via an analytical workflow using phospho-monoester capture and derivatization
(a) Methanol extracts from different types of starting materials were loaded onto an imidazolium polymer (IMP) after protein precipitation.
(b) Extracted ion chromatograms (EIC) of the load, flow-through/wash and eluate fractions as analyzed by liquid chromatography-nanoelectrospray ionization
Quadrupole Time of Flight mass spectrometry in either positive (left panel) or negative (right panel) ionization modes.
(c) IMP selectively captures phospho-monoester lipids (Ceramide-1 -phosphate, Cer- 1 -P; sphingosine-1 -phosphate, S1 P; phosphatidic acid, PA; lysophosphatidic acid, LPA). Lipids with no phosphate (Ceramide, Cer; Glucosyl-Ceramide, Glu-Cer) and those with phosphorous bound in diester configuration (phosphatidylcholine, PC; sphingomyelin, SM; lyso-phosphatidylcholine, LPC; phosphatidylglycerol, PG, and phosphatidylinositol, PI) do not efficiently bind to IMP and are recovered in the flow through and wash fractions instead.
(d) Structure of d17:1 S1 P standard before and after derivatization with TMS- diazomethane. The 4Met-S1 P derivative (95% of the reaction product) is drawn
based on results from high-resolution product ion spectra, (e) Two-step analytical enhancement using IMP enrichment (—2-fold increase in signal intensity, and importantly, removal of matrix interference and TMS derivatization (~30-fold increase in signal intensity) leading to substantially improved detection of long chain base phosphates (LCB-P) with different aliphatic compositions (e.g. the di-ene form of S1 P, LCB-P d18:2) in human blood plasma with excellent analytical precision and linearity (Table 2).
FIGURE 2. illustrates the discovery, characterization and quantification of new LCB-P in complex mixtures from diverse biological species
(a) Collision-induced dissociation product ion mass spectra (in positive ionisation mode) of TMS-derivatized synthetic stable isotope standard (d 8: 1 13C2D2-S1 P, m/z 440, diamond symbol) yielded expected ions at m/z 60 (fragment A), m/z 1 13 (fragment B) and m/z 328 (fragment C). The former two fragments are expected to be invariant for LCB-P with different aliphatic compositions which is indeed the case as shown for corresponding product ion spectra of LCB-P d18:2 (b), LCB-P d18: 1 (c) and LCB-P d18:0 (d), all derived from extracts of human blood plasma, and d16:0 (e) from whole fly extract. The C fragment, instead, is characteristic of individual species of LCB-P, thus allowing for identification by targeted tandem mass spectrometry. Targeted mass spectrometric analysis based on scanning for neutral loss of the methylated amine fragment (m/z 60) allows for determination of LCB-P pattern in extracts derived from human plasma (f) and D. melanogaster (g and h). Sphingosine- phosphate lyase (sply in Drosophila) is the key enzyme in the irreversible
degradation of LCB-P. Deletion of this gene in Drosophila leads to an accumulation of a multitude of LCB-Ps, many of which have not been described before (g,).
Quantification using multiple reaction monitoring of LCB-P in mammalian blood plasma (i) and lymphatic system (j), as well as extracts from model organisms (k-m), led to the discovery of new LCB-Ps (Table 4). (n) LCB-P species detected in this study. Dark blue, new LCB-P not described so far in the literature; light blue, LCB-P not known to exist in the respective biological species/tissue; grey, LCB-P previously described and measured with comparable results in this study (see Table 5).
FIGURE 3 illustrates the production of an IMP resin.
FIGURE 4 shows the molecular structures of standard d17: 1 S1 P before and after derivatization with TMS-diazomethane.
(a) Two different methylated derivatized structures are generated, one containing four methyl groups (top) and one containing five methyl groups (bottom). Methylated moieties are highlighted on red background color.
(b) Chromatographic peaks of S1 P d17: 1 before (left) and after derivatization (right) recorded with the same LC-MS (positive mode) conditions. After derivatization, the tetra-methylated form represents 95% of the reaction product judged by the relative intensities of the ion chromatograms.
(c) High resolution product ion analysis (using collision induced dissociation) of the tetra-methylated (top) and penta-methylated (bottom) forms. The precursor m/z is labeled with the diamond symbol.
FIGURE 5 shows the effect of IMP resin on LCB-Ps detection. Extracted ion chromatograms representing the LCB-Ps identified in human plasma and murine brain, before (a and c) and after (b and d) the IMP enrichment step. Note the different scales in intensities between panels a and c and b and d. IMP enrichment also eliminates co-eluting contaminants (panels a and c) and improves the peak shapes, essential for SA1 P and LCB-P d20: 1 quantification and the detection of LCB-P d16:1 in plasma. . FIGURE 6 shows the effect of TMS derivatization on LCB-P species distribution in human plasma. Full scan Quadrupole-Time of Flight mass spectrum of LCB-P species found in the same human plasma sample before (black; lower trace) and after (red; upper trace) derivatization with TMS-diazomethane (both samples were IMP purified). The number of species and their relative abundance is conserved during derivatization.
FIGURE 7 shows elution peaks recorded by multiple reaction monitoring of derivatized LCB-Ps in human plasma. In the inset, the elution profiles (normalized intensities) are represented, showing a retention time increase with increasing unsaturation and decreasing chain length of the molecules as expected for HILIC
chromatography.
FIGURE 8 shows S1 P d18: 1 for illustration purposes of the different fragments observed during tandem mass spectrometry. Fragments A and B are invariant and common to all LCB-P tested here. Fragment C is LCB-P species specific and recorded as the [M+HCOO]- adduct in negative mode (C**) .
FIGURE 9 shows an estimation of the limit of detection (LOD) based on signal to noise ratio (S/N) in plasma before (panel a) and after (panel b) IMP enrichment. Shown are extracted MRM peaks (440.3/60.08, i.e. derivatized 13C2D2 S1 P) of 0.3 fmol 13C2D2 S1 P spiked into 10 μΙ human plasma.
FIGURE 10 shows calibration curves for the derivatized S1 P standard in human and mouse plasma.
FIGURE 1 1 shows the lipid elution profile for a mixture of compounds including phosphate diester- containing lipids, S1 P (a phosphate monoester-containing lipid), other phosphate diesters and non-phosphorylated molecules using the polymer of Example 4a as the stationary phase and a first elution solvent which is isopropanol: methanol 1 .1 ; a second elution solvent which is isopropanol: methanol: triethylamine 49.5:49.5: 1 ; and a third elution solvent which is isopropanol: methanol: 1 % trifluoroacetic acid, 49.5:49.5: 1.
EXAMPLES
Materials
N-vinylimidazole; 2,6-bis-(bromomethyl) pyridine and ethylene glycol dimethacrylate (EGDMA) were purchased from Sigma Aldrich (St. Louis, MO). Dry acetonitriie used for synthesis was purchased from Merck. /V,/V?-azo-bis-(2,4-dimethyl) valeronitrile (ABDV) was purchased from Wako. DMSO-c/6 was purchased from Deuterio-GmbH (Kastellaun, Germany).
EGDMA was purified by the following procedure prior to use. The received material was washed consecutively with 10% aqueous NaOH, water and brine. After drying over MgS04, the purified monomer was obtained by distillation under reduced pressure.
All other reagents were used as received.
1H NMR spectra were recorded at 300 MHz unless otherwise mentioned. Elemental microanalyses were performed using a CHN-rapid HERAEUS Analyzer.
All solvents for LC-MS analysis were LC-MS grade and were purchased from Fisher Scientific and Merck Millipore.
Lipid standards: D-ery//?ro-Sphingosine-1 -phosphate (S1 P d18:1 ) and isotope labeled standard D-eryfftro-Sphingosine-l -phosphate (13C2D2-S1 P,) were purchased from Toronto Research Chemicals, D-ery ?/ -C17-Sphingosine-1 -phosphate (S1 P d17:1 ), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dimyristoyl-sn- glycero-3-phospho-(1 '-rac-glycerol) (DMPG), 1 ,2-dihexadecanoyl-sn-glycero-3- phosphate (DPPA), 1 -heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (LPA) N- octanoyl-ceramide-1 -phosphate (Cer-1 P), D-glucosyl-β-Ι , Ι ' N-octanoyl-D-e/yf 7/Ό- sphingosine (Glu-Cer), N-heptadecanoyl-D-e/yfftro-sphingosine (Cer), N-lauroyl-D- e/yi 7ro-sphingosylphosphorylcholine (SM) were purchased from Avanti polar lipids (Alabaster, AL, USA) Dioctanoyl Phosphatidylinositol (PI) was purchased from Echelon Biosciences (USA). Acetic acid, ammonium formate (HPLC grade), formic acid and (Trimethylsilyl) diazomethane solution was purchased from Sigma-Aldrich (St. Louis, MO).
Human blood for plasma was obtained from healthy volunteers (NUS-IRB Reference No. 04-1 15) using EDTA as anticoagulant. Blood samples were centrifuged at 2200xg for 15 min to isolate plasma and frozen at -80°C within 45 min of blood collection.
Example 1 - Synthesis of Monomer 1.
The synthesis of Monomer 1 was carried out according to Scheme 1 below.
Scheme 1
A solution of 2,6-bis-(bromomethyl) pyridine (Aldrich, 0.5 g, 1 .88 mmol) and N- vinylimidazole (Aldrich, 0.35 ml, 3.76 mmol) in acetonitrile (Merck, 25 ml) was refluxed overnight. To prevent polymerisation, a spatula tip of sulphur was added to the reaction mixture. After cooling to room temperature, the solvent was evaporated to dryness under vacuum. The resulting solid was then redissolved in ethanol and precipitated out with the addition of diethyl ether, to afford the desire product (0.71 g) as a solid (83%).
1H NMR (300 MHz, DMSO) δ 5.43-5.47 (m, 2H), 5.64 (s, 4H), 5.98-6.04 (m, 2H), 7.35-7.43 (m, 2H), 7.55- 7.57 (d, 2H), 7.87-7.88 (m, 2H), 7.97- 8.02 (t, 1 H), 8.24-8.25 (t, 2H), 9.67 (s, 2H); 13C NMR (75 MHz, DMSO) δ 52.73, 108.92, 1 18.70, 122.24, 124,1 1 , 128.76, 136.16, 138.82, 153.15.
Calculated C 45,06 %, H 4,23 % , N 15,45%; found C 43,26 %, H 4,81 %, N 15,17%.
Example 2 - Synthesis of Imidazolium Polymer (IMP) Resin.
The synthesis of the IMP resin was carried out as illustrated in Scheme 2.
Scheme 2
Monomer 1 (134 mg, 0.2975 mmol) and EGDMA (2) (2.25 ml, 1 .925 mmol) were
mixed into a 20 ml glass vial containing 10 ml of toluene/methanol 1/1. ABDV (25 mg, 0.097 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μηη and sub-25 μηη particles. The former fraction was used as sorbent for solid phase extraction.
Example 3 - Synthesis of Imidazolium co diaryi urea Polymer (IMUP) Resin
Scheme 3
IMP resin
Monomer 1 (54 mg, 0.119 mmol), urea monomer (89 mg, 0.238mmol) and EGDMA (0.900 ml, 4.77 mmol) were mixed into a 20 ml glass vial containing 4 ml of toluene/methanol 1/1 . ABDV (10 mg, 0.0387 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μηη and sub-25 μιη particles. The former fraction was used as sorbent for solid phase extraction.
Example 4 - Synthesis of Imidazolium based imprinted Polymers (i-IMP). Example 4a: Synthesis of an imidazolium based polymer imprinted with a
phospholipid dianion
The synthesis of the IMP was carried out as illustrated in Scheme 4 using PL-CL14
template with imdazolium monomer. After subsequent polymerization and removal of template, imprinted polymer was produced.
Scheme 4
Na salt of PL-C14 (80 mg, 0.1 19 mmol), 0.120 ml of 1 M tetrabutyl ammonium hydroxide (in MeOH), Monomer 1 (54 mg, 0.1 19 mmol) and EGDMA (0.900 ml, 4.77 mmol) were mixed into a 20 ml glass vial containing 3.880 ml of toluene/methanol 1/1 . ABDV (10 mg, 0.0387 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μηη and sub-25 μΐη particles. The former fraction was used as sorbent for solid phase extraction.
Example 4b: Synthesis of an imidazolium based polymer imprinted with a
phospholipid mono anion
The mono sodium salt of PL-C14 (80 mg, 0.1 19 mmol), Monomer 1 (54 mg, 0.1 19 mmol) and EGDMA (0.900 ml, 4.77 mmol) were mixed into a 20 ml glass vial containing 3.880 ml of toluene/methanol 1/1. ABDV (10 mg, 0.0387 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and
subsequently at 70 °C for 4 hours. The solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μηι and sub-25 μΐτι particles. The former fraction was used as sorbent for solid phase extraction.
Example 5 - Synthesis of Imidazolium co diaryl urea based imprinted Polymer (i-IMUP).
Na salt of PL-C14 (80 mg, 0.1 19 mmol), 0.120 ml of 1 M tetrabutyl ammonium hydroxide (in MeOH), Monomer 1 (54 mg, 0.1 9 mmol), urea monomer (89 mg, 0.238mmol) and EGDMA (0.900 ml, 4.77 mmol) were mixed into a 20 ml glass vial containing 3.880 ml of toluene/methanol 1/1. ABDV (10 mg, 0.0387 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid polymer was removed from the glass vial, roughly crushed and Soxhiet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μΐη and sub-25 μηι particles. The former fraction was used as sorbent for solid phase extraction.
Example 6 - Synthesis of Monomer 2.
The synthesis of Monomer 2 was carried out according to Scheme 5 below.
Scheme 5
A solution of 1 ,3-bis(bromomethyl)benzene (Aldrich, 2g, 7.56mmol) and
vinyiimidazole (Aldrich, 1.4ml, 5.04 mmol) in acetonitrile (Merck, 60ml) was refluxed over night. To prevent polymerisation, a spatula tip of sulphur was added to the reaction mixture. After cooling to room temperature, the solvent was evaporated to dryness under vacuum. The resulting solid was then redissolved in ethanol and precipitated out with the addition of diethyl ether, to afford the desire product (2.41 g) as a solid (72 %).
Ή NMR (400 MHz, DMSO) δ 5.42-5.45 (m, 2H), 5.43, 5.45, 5.45, 5.52 (s, 4H), 5.99- 6.04 (m, 2H), 7.33-7.39 (m, 2H), 7.50 (s, 3H), 7.68 (s, 1 H), 8.01 (s, 2H), 8.29 (s, 2H), 9.80 (S, 2H); 13C NMR (101 MHz, DMSO) δ 51.84, 108.89, 1 19.45, 123.31 , 128.80, 128.86, 128.96, 129.70, 135.10, 135.62.
Calculated C 47,81 %, H 4,46% , N 12,39%; found C 46,08%, H 4,96 %, N 12.28%. Example 7 - Synthesis of Imidazolium Polymer (IMP2) Resin.
The synthesis was carried out according to Scheme 6 below.
Scheme 6
Monomer 2 (134.5 mg, 0.2975 mmol) and EGDMA (2.25 ml, 1 1.925 mmol) were mixed into a 20 ml glass vial containing 10 ml of toluene/methanol 1/1. ABDV (25 mg, 0.097 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μΐη and sub-25 μιτι particles. The former fraction was used as sorbent for solid phase extraction. Example 8 - Synthesis of Monomer 3.
The synthesis of Monomer 3 was carried out according to Scheme 7 below.
Scheme 7
A solution of 1 ,4-bis(bromomethyl)benzene (Acros, 1.3498 g, Smmol) and
vinylimidazole (Aldrich, 0.45ml, 5mmol) in acetonitrile (Merck, 25ml) was refluxed over night To prevent polymerisation, a spatula tip of sulphur was added to the reaction mixture. After cooling to room temperature, the solvent was evaporated to dryness under vacuum. The resulting solid was then redissolved in ethanol and precipitated out with the addition of diethyl ether, to afford the desire product 40% as a solid .
1H NMR (300 MHz, DMSO) δ 5.39 -5.42 (d, 2H), 5.53 (s, 4H), 6.00-6.06 (m, 2H),
7.33-7.42 (m, 2H), 7.58 (s, 4H), 8.04 (s, 2H), 8.31 (s, 2H), 10.03 (s, 2H);
13C NMR (75 MHz, DMSO) δ 51 .65, 108.78, 1 19.50, 123.27, 128.92, 129.26, 135.1 1 ,
135.73.
Calculated C 59, 51 %, H 5.55 % , N 15,42%; found C 53,27%, H 6,65 %, N 13,82%.
Example 9 - Synthesis of Imidazolium Polymer (IMP3) Resin.
The synthesis of the IMP3 resin was carried out as illustrated in Scheme 8.
Scheme 8
Monomer 3(134 mg, 0.2975 mmol) and EGDMA (2) (2.25 ml, 1 1.925 mmol) were
mixed into a 20 ml glass vial containing 10 ml of toluene/methanol 1/1. ABDV (25 mg, 0.097 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μηη and sub-25 μηη particles. The former fraction was used as sorbent for solid phase extraction.
Example 10 - Synthesis of Monomer 4.
The synthesis of Monomer 4 was carried out according to Scheme 9 below.
Scheme 9
A solution of bromomethylbenzene (Acros, 0.86, 5mmol) and vinylimidazole (Aldrich, 0.45ml, 5mmol) in acetonitrile (Merck, 25ml) was refluxed over night. To prevent polymerisation, a spatula tip of sulphur was added to the reaction mixture. After cooling to room temperature, the solvent was evaporated to dryness under vacuum. The resulting solid was then redissolved in ethanol and precipitated out with the addition of diethyl ether, to afford the desire product (1 g) as a solid (81 %). H-NMR (CDCI3, 500 MHz) δ : 5.35 (d, 1 H), 5.66 (s, 2H), 5.97 (dd, 1 H), 7.26-7.37 (m, 4H), 7.49-7.53 (m, 3H), 7.77 (s, 1 H), 1 1.10 (s, 1 H).
13C NMR (CD3OD, 101 MHz) δ 54.43, 99.93, 106.3, 1 10.05, 120.92, 124.28, 127.24, 129.83, 130.49, 134.76.
Example 11 - Synthesis of Monomer 5.
The synthesis of Monomer 5 was carried out according to SchemelO below.
Scheme 10
A solution of 1 ,8 bis (bromomethyl) anthracene (Aidrich 5mmol ) and vinylimidazole (Aidrich, 5mmol) in acetonitrile (Merck, 25ml) was refluxed over night. To prevent polymerisation, a spatula tip of sulphur was added to the reaction mixture. After cooling to room temperature, the solvent was evaporated to dryness under vacuum. The resulting solid was then redissolved in ethanol and precipitated out with the addition of diethyl ether, to afford the desire product as a solid. Example 12 Synthesis of an imidazolium based polymer from monomer 5 imprinted with a phospholipid dianion
Na salt of PL-CL14 (Scheme 4) (80 mg, 0.119 mmol), 0.120 ml of 1 M tetrabutyl ammonium hydroxide (in MeOH), Monomer 5 (67 mg, 0.1 19 mmol) and EGDMA (0.900 ml, 4.77 mmol) were mixed into a 20 ml glass vial containing 3.880 ml of toluene/methanol 1/1. ABDV (10 mg, 0.0387 mmol) was added and the solution was degassed for 10 min with N2. The polymerisation tube was sealed and the mixture was kept at 50 °C using a water bath for 24 hours and subsequently at 70 °C for 4 hours. The solid imprinted polymer was removed from the glass vial, roughly crushed and Soxhlet extracted with methanol for 48 hours. The crude product was further crushed and sieved to 25-50 μιτι and sub-25 μηη particles. The former fraction was used as sorbent for solid phase extraction.
Example 13
Liquid chromatography (LC) and mass spectrometry (MS)
All LC-MS/MS experiments were performed using Agilent 1200 series HPLC-Chip systems connected to Agilent 6540 or 6550 Q-TOF and to Agilent 6490 QQQ mass spectrometers. Two different types of chip were used for Reverse Phase (only for initial studies on pure standards) and HILIC separations:
(1 ) The Reverse Phase Ultra High capacity Chip included a 500 nl enrichment column (5 pm particle size, 80 A pore size) and a 75 pm * 50 mm C18 analytical column (Agilent Technologies Corp., Santa Clara CA). The Agilent 1200 series HPLC used a capillary pump for sample injection onto the Enrichment column and a Nano pump for separation. Solvents used for the Reverse Phase LC: 5% water in 95 % methanol with 5 mM ammonium formate and 0.1 % formic acid (solvent A), 50% methanol in 50% isopropanol with 5 mM ammonium formate and 0.1 % formic acid (solvent B). Analytes were eluted with the following gradient: 5%B to 20%B from 0 to 2 min, 60% B from 2 to 8 mins, 100% B from 8 to 18 min, 100% B from 18 to 23 min, 5% B from 23.1 to 33 min. The chip cube was operated with back flush mode and samples were injected through the enrichment column at 4 μΙ/min. The valve was switched after 1.5 min to place the enrichment column in line with analytical column at a flow rate of 400 nl/min. The Agilent 6540 (or 6550) quadrupole time-of-flight (QTOF) mass spectrometer was operated in positive ion mode; electrospray voltage was set to 1600 V (Vcap), temperature 300°C, drying gas 4 (or 2) l/min, fragmentor voltage 150 V. The instrument was operated in targeted MS/MS mode with MS acquisition rate of 1 spectra/sec and MS/MS acquisition rate of 2.0 spectra/sec. All the samples were resuspended in 50 or 100 μΙ of mobile phase A before injecting 1 μΙ of sample for LC-MS analysis.
(2) A customised HILIC-chip containing Amide-80 stationary phase (Tosoh
Bioscience, LLC. Montgomeryville, PA, 5 pm particle size, 80 A pore size) was also prepared, including a 160 nl trapping column and a 75 pm χ 150 mm analytical column (Agilent Technologies Corp., Santa Clara CA).
Solvents used for HILIC HPLC: 50% acetonitrile in water containing 25 mM
ammonium formate pH 4.6 (solvent A), 95% acetonitrile containing 25 mM
ammonium formate pH 4.6. The pH value was adjusted with formic acid. Analytes were eluted with the following gradient: 100% B from 0 to 1.5 min, 40% B from 1.5 to 8.5 min, 30% B from 8.5 to 10.5 min, 0% B from 1 1.5 to 13.0 min, 100% B from 13.1 to 19 min.
The chip cube was operated with back flush mode and samples were injected through the enrichment column at 4 μ l/min. The valve was switched 1.5 min after
injection to place the enrichment column in line with the analytical column at a flow rate of 400 nl/min. The Agilent 6540 (or 6550) instrument was operated with the same parameters described previously, except for the gas temperature kept at 185°C and Vcap set at 1580 V. Spectra were acquired in targeted MS/MS mode with MS acquisition rate of 2 spectra/sec and MS/MS acquisition rate of 2 spectra/sec.
For the validation of the method, murine and human plasma samples were spiked with known amounts of standard S1 P d18: 1 and ISTD (S1 P-13C2D2) in the concentrations reported below. The Agilent 6490 triple quadrupole (QQQ) mass spectrometer was operated in positive mode for MRM (see Table 1 for instrument parameters and MRM transitions) and negative mode for neutral loss (NL) scan mode (for NL of 60 m/z, electrospray voltage was set to 1580 V (Vcap), temperature 185°C, drying gas 12 l/min and collision energy of 25 V). In positive ion MRM mode, two product ions were monitored after CID of the LCB-P precursors.
Table 1 - Multiple reaction monitoring (MRM) parameters for LCB-P
Product ion m/z 60.08 was used as Quantifier and Product ion m/z 1 13 was used as Qualifier for accurate identification and quantification of LCB-P species
Compound Group Compound Name Precursor Ion Product ion t20 LCB-P t20:0 482.3 1 13.0
482.3 60.1
LCB-P 120:1 480.3 1 13.0
480.3 60.1
d20 LCB-P d20:0 466.3 1 13.0
466.3 60.1
LCB-P d20: 1 464.3 1 13.0
464.3 60.1
LCB-P d20:2 462.3 1 13.0
462.3 60.1
d19 LCB-P d19:0 452.3 1 13.0
452.3 60.1
LCB-P d19: 1 450.3 1 13.0
450.3 60.1
LCB-P d19:2 448.3 1 13.0
Compound Group Compound Name Precursor Ion Product ion
448.3 60.1 t18 LCB-P t18:0 454.3 113.0
454.3 60.1
LCB-P t18:1 452.3 113.0
452.3 60.1 d18 13C2D2-S1P d18:1 440.3 113.0
440.3 60.1
SA1Pd18:0 438.3 113.0
438.3 60.1
S1Pd18:1 436.3 113.0
436.3 60.1
LCB-P d18:2 434.3 113.0
434.3 60.1 d17 LCB-P d17:0 424.3 113.0
424.3 60.1
LCB-P d17:1 422.3 113.0
422.3 60.1
LCB-P d17:2 420.3 113.0
420.3 60.1 d16 LCB-P d16:0 410.3 113.0
410.3 60.1
LCB-P d16:1 408.3 113.0
408.3 60.1
LCB-P d 16:2 406.3 113.0
406.3 60.1 d15 LCB-P d15:0 396.3 113.0
396.3 60.1
LCB-P d15:1 394.3 113.0
394.3 60.1
LCB-P d15:2 392.3 113.0
392.3 60.1 d14 LCB-P d14:0 382.3 113.0
Compound Group Compound Name Precursor Ion Product ion
382.3 60.1
LCB-P d14: 1 380.3 1 13.0
380.3 60.1
LCB-P d14:2 378.3 1 13.0
378.3 60.1
Dwell time (msec) 20
Fragmentor 380
Collision Energy 25
Cell Accelerator voltage 7
Polarity Positive
Quantification peak areas of LCB-P compared to peak area of internal standard (IS)
M/z 60 was used as a 'quantifier' (due to its high intensity) and m/z 3 was used as a 'qualifier' (except in the case of phytosphingosines where m/z 1 13 was used as the quantifier). These ions were present after fragmentation of all LCB-P species. The qualifier at m/z 1 13 represents the mono-methylated phosphate and was used to discriminate between penta-methylated species generated by the derivatization reaction. Quantification was performed according to the internal standard method, comparing peak areas of the sample to the internal standard. The method shows linearity in the physiological range that has been reported for S1 P (Table 2 and Figure 10). All the samples were resuspended in 50 or 100 μΙ of mobile phase B before injecting 1 pi of sample for LC-MS analysis. Table 2 - Intraday/interday precision and accuracy for S1 P in human plasma
Cone n Intraday Interday
(ng/ml) Mean ± Accuracy Precision Mean ± Accuracy Precision
SD (%) (%) SD (%) (%)
Low 5 5 5.26±0.26 105.29 5.18 4.95+0.25 99.04 13.14 QC
Middle 40 4 39.64±0.96 99.12 2.4 39.72+1.3 100.35 3.59 QC
High 80 5 80.92±2.43 101.16 3.04 82.54±4.66 104.58 5.92 QC
Preparation of standards
Main stock solutions of standards were diluted in methanol to a final concentration of 0.5 mg/ml for d18: 1 S1 P, 13C2D2-S1 P (Internal Standard, IS) and d17: 1 S1 P. The stock solutions were further diluted in a concentration range of 50-1000 ng/ml for S1 P (d18:1 ) for validation experiments, while the internal standard stock was kept at a concentration of 200 ng/ml or 400 ng/ml.
Sample extraction from human plasma
All healthy human plasma samples were collected from a representative cohort of the Singaporean population in accordance to ethical guidelines and protocols approved by the National Health Group Institutional Review Board, Singapore. All participants who were approached and agreed to participate in this study were required to provide written consent by signing on consent forms. Participants were required to fast overnight for at least 8 hours and samples were collected between 8 to 10 am to minimize changes in metabolite profile due to circadian rhythm. 10 ml of whole blood samples were obtained in BD Vacutainer® plastic plasma tubes with EDTA as anticoagulant by venipuncture. Plasma was processed by spinning 10 ml of fresh blood collected at 2,200 g for 15 mins at 4°C using a swing-out bucket rotor centrifuge. Subsequently the plasma was transferred to 5ml cryovials (Practical Mediscience). The plasma was frozen at -80°C. For LCB-P extraction 10 μΐ plasma were mixed with 10 μΙ IS and 90 μΙ methanol. The solution was shaken on a thermomixer for 20 min at 4°C and centrifuged for 10 mins at 14,000 rpm. The supernatant could then be diluted in isopropanol for loading onto the IMP resin or directly derivattsed for analysis.
Sample extraction from murine plasma and lymph
Male Wild Type C57 BL/6 (WT) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a chow diet (18% protein and >5% fat, Harlan Teklad, Madison, Wl) post wean until 6 weeks of age. Diet was then switched to a Western-type (21.2% fat and 0.2% cholesterol, Harlan Teklad) until mice were sacrificed at 22-28 weeks of age. Mice were maintained under specific pathogen-free conditions with free access to food and water in the Animal Housing Unit of the National University of Singapore. All studies were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore. Blood was drawn from the mice via cardiac puncture immediately after sacrifice and mixed with 50 μΙ of 100 mM EDTA. The
blood-EDTA mixture was then centrifuged at 14000 g, for 10 minutes at 4°C and the plasma was obtained from the supernatant for subsequent S1 P extraction. Special care was taken to minimize red blood cells and platelets lysis throughout the whole process, avoiding aspiration of blood through fine needles. All mice were fasted overnight prior to lymph collection to clear dietary lipids from the lymph fluid. 25 μΙ of lymph fluid were crenulated from the cysterna chili with the use of extended length gel-loading pipette tips (Neptune Scientific) and collected into a new Eppendorf tube. The lymph samples were centrifuged at 14,000 g for 10 minutes at 4°C and the supernatant was obtained for subsequent S1 P extraction.
10 μΙ plasma (5 μΙ for Lymph) were mixed with 10 μΙ IS (5μΙ IS for lymph) and 90 μΙ methanol. Samples were then treated in the same way as for human plasma.
Method validation
Recovery studies. The extraction recovery of S1 P was determined by spiking the Internal Standard (IS) 13C2D2-S1 P in the plasma samples at two different
concentrations (40 and 80 ng/ml, n=3) before lipid extraction. The recoveries of IS were determined from the ratio of the mean peak area of the extracted samples to the mean peak area of samples where the internal standard was spiked after extraction. The recovery for IS at concentrations of 40 ng/ml and 80 ng/ml was 80.1 ±9.6% and 87.9±9.5%, respectively, in human plasma and 82.1 ±5.4% and 97.4±2.5% in mouse plasma.
Calibration Curves. Calibration curves (Figure 10) were constructed using the standard addition method using a double blank (extracted plasma samples without IS), a blank (extracted plasma sample with IS) and 8 standards containing 5, 7.5, 10, 20, 40, 60, 80, 100 ng/ml concentration of S1 P (d18:1 ) spiked in plasma together with IS (40 ng/ml). The results were fitted by a linear equation from 5 to100 ng/ml and the resulting mean regression equations for S1 P in human plasma (y=0.032x-0.128) and in mouse plasma (y=0.024x-0.038) showed R2 values of 0.9993 and 0.9972 respectively (Table 2).
Precision and Accuracy. Intra-day/inter-day precision and accuracy were determined by analyzing quality control samples (QC) consisting of human plasma spiked with standard S1 P (d18: 1 ) at three different concentration levels, Low-QC (5 ng/ml), Middle-QC (40 ng/ml) and High-QC (80 ng/ml), and a fixed IS concentration of 40
ng/ml. Five replicates for each control were analysed while a plasma lipid extract spiked with IS was used as blank, to subtract the area of the endogenous S1 P when calculating precision and accuracy. Sample extraction from murine tissues
Lymph nodes (1 lymph node/sample) and brain samples (2.5 or 5 mg) from individual mice were resuspended in 190 μΙ of methanol + 10 μΙ of IS. Samples were sonicated in water bath at 4°C for 30 min. After centrifuging at 4°C for 10 min at 14,000 rpm, the supernatant was collected for enrichment onto IMP resin.
Sample extraction from D. melanogaster
Adult flies (up to 2 days old), fed on standard Bloomington semi-defined Drosophila medium, of the genotypes +/+ (homozygous w 118), sply0509 /sply05091 homozygotes and sply05091/+ heterozygotes (resulting from a cross between sply05091 and w1118) were collected, flash frozen in liquid nitrogen, and stored at -80 C for up to one week before lipid extraction. Extractions of LCB-Ps were done in triplicate. For each experiment 5 flies were weighed and, after addition of 5 μΙ IS (40 ng/ml),
homogenised in 500 μΙ methanol by using a pellet pestle (Sigma). After addition of 500 μΙ methanol, the samples were sonicated for 1 h at 4°C. After centrifugation at 14000 rpm, 4°C, for 10 min, supernatant was collected and dried in speedvac.
Samples were resuspended in 100 μΙ of methanol prior enrichment onto IMP resin and derivatization.
Sample extraction from S. cetevisiae
Pre-cultures of S. cerevisiae (W303) were grown in YPD medium (BD) for 24 h at 30°C. Then YPD was inoculated to a starting OD60o of 0.1 and cells were grown at 30°C until they reached OD 1 . Cells were then harvested and lyophilized. 2.5 mg of lyophilized yeast were resuspended in 500 μΙ methanol and after addition of 5 μΙ IS (400 ng/ml), homogenised with glass beads by vortexing for 2 min. After addition of 500 μΙ methanol, the samples were sonicated for 1 h at 4°C. After centrifugation at 14,000 rpm, 4°C, for 0 min, supernatant was collected and dried in speedvac.
Samples were resuspended in 100 μΙ of methanol prior enrichment onto IMP resin and derivatization.
Example 14 - Selective binding and elution of S1 P
Different protocols were optimized for selective capture of S1 P and related species (as described later) with different solvents for conditioning, loading, washing, eluting and regenerating the resin in an offline format. To do so, 10 (for plasma and lymph) and 20 (for tissues) mg of the IMP resin were dry packed in an empty 96 well plate (Agilent) with polypropylene filters at both bottom and top of the resin.
To investigate the selectivity of the IMP resin for LCB-Ps, standard lipids were loaded and eluted in different conditions. The effect of various percentages of basic and acidic solutions was investigated (data not shown). Best selectivity and recovery were obtained when loading the resuspended sample in 1 ml isopropanol and washing the resin sequentially with 1 ml isopropanol, 1 ml isopropanol/methanol (50/50). Elution was obtained with 2 x 0.8 ml chloroform/methanol/1 % trifluoroacetic acid (49.5/49.5/1). This enrichment protocol was applied to all the analysed biological samples.
The IMP resin wells can be reused after regenerating with 3 x 1 ml methanol/acetic acid/water (60/30/10) and equilibrating with 3 x 1 ml loading solution. Derivatization Step
The obtained fractions were dried and reconstituted in 100 μΙ of methanol. 10 μΙ of TMS-Diazomethane (2M in hexane) were added and the sample incubated for 20 min at room temperature under gentle mixing at 750 rpm. The reaction was stopped by adding 1 μΙ of acetic acid. The derivatized samples were dried in speedvac and reconstituted in 100 μΙ of mobile phase before injecting into the LC-MS system.
To test the recovery rate from the IMP resin with the reported enrichment protocol, a brain tissue extract obtained as described above was spiked with standard DMPC (250 ng), DMPG(100 ng), Glu-Cer (100 ng),SM (50 ng), LPC (50 ng), Cer (50 ng), C8-PI (15 ng), S1 P ISTD (4 ng), DPPA(50 ng), Cer-1 P (50 ng), LPA (50 ng) before homogenisation and loaded onto the IMP resin in 1 ml loading solution. The lipid content of the different fractions (flow through+wash and eluate) was measured by LC-MS before (for phosphate diesters and non-phosphorylated species detection) and after (for monoesters) derivatization with TMS. Recovery in flow through+wash and eluate was estimated as % of the lipids present in the loaded fraction.
When fragmented by collision-induced dissociation (CID), derivatized d18: 1 13C2D2- S1 P standard (m/z 440) yielded three product ions, denoted ions A-C. Originating from the methylated amine group (fragment A at m/z 60; Figure 2a) and methyl- phosphate (fragment B at m/z 1 13), both A and B ions are expected to be invariant for different LCB-Ps compared with ion C (m/z 328), which originated from the aliphatic portion. Indeed, when LCB-Ps from plasma were analyzed, the C fragment yielded m/z values as predicted for d18:2 (Figure 1 b), d18:1 (Figure 2c), d18:0 (Figure 2d) and d16:0 for a lipid extract from fly (Figure 2e, Figure 6).
The product ion analysis of derivatised LCB-Ps was obtained using collision induced dissociation (CID). Figure 8 shows S1 P d18: 1 for illustration purposes of the different fragments observed during tandem mass spectrometry. Fragments A and B are invariant and common to all LCB-P tested here. Fragment C is LCB-P species specific and recorded as the [M+HCOO]- adduct in negative mode (C**) .
Tables 3a (positive ion mode) and 3b (negative ion mode) are a summary of the main fragments generated by CID fragmentation in both positive and negative mode for each LCB-P molecular species reported in this study (See Figure 8). All data were collected using Quadrupole Time of Flight (QToF) mass spectrometry except # which were acquired on a triple quadrupole mass spectrometer.
Table 3a
LCB-P m/z precursor Product ions (m/z)
A B C* d14 0 382.270 60.080 112.999 270.277
1 380.254 60.080 112.999 268.261
2 378.238 60.080 12.999 266.247 d15 0 396.284 60.080 112.998 284.292
1 394.269 60.079 112.999 282.275
2 392.255 60.080 nd nd d16 0 410.301 60.083 112.999 298.309
1 408.286 60.080 112.999 296.293
2 406.270 60.080 112.999 294.278 d17 0 424.313 60.080 12.999 312.324
1 422.299 60.080 112.999 310.308
2 nd nd nd Nd d18 0 438.332 60.084 112.999 326.321
1 436.316 60.084 112.999 324.307
13C2D2 440.334 60.080 112.999 328.325 d18:1
2 434.298 60.081 112.999 322.310 t18 0 454.321 60.079 112.998 342.331 d19 0 452.342 60.080 112.999 340.282
1 450.324 60.086 112.999 338.340 d20 0 466.362 60.079 112.999 354.368
1 464.343 60.079 112.999 352.351 t20 0 482.332 60.080 113.001 370.369
1 480.336 60.079 112.999 368.348
Table 3b
This product ion information, collected using a high-resolution Time of flight mass spectrometer with high mass accuracy (<5 ppm), provides a firm basis for targeted approaches using tandem-MS. Scanning for neutral loss of m/z 60 (methylated amine group, fragment A) using a triple quadrupole mass spectrometer (with otherwise comparable analytical conditions) is a convenient mode to rapidly reveal
LCB-Ps. Human plasma gave rise to strong signals of the d18 series (Figure 2f), including the newly discovered 18:2 derivative (HCCO adduct at m/z 478.1 , Figure 2f) not known to have been described previously to our best knowledge. We next extended our analysis to various biological species known to have different LCB (and thus possibly also LCB-P) inventories. Thus, the process was repeated using the samples extracted from human plasma, murine plasma and lymph, D. melanogaster and S. cerevisiae as described above. S1 P lyase is the key enzyme involved in the irreversible degradation of S1 P; its impaired function in D. melanogaster (sply05091 ) leads to an accumulation of LCB- Ps 7. Here, with a starting sample of only five flies, we demonstrated a 200-fold accumulation in not only d14 and d16 (the major LCB-Ps in Drosophila), but also d15, d17, d18, d19 and d20 LCB-Ps in sply05091/sply05091 when compared with wild type (Figure 2g, h). Genetic background effects were controlled for by also analyzing changes in heterozygous animals that resulted from a cross between sply05091 and a control strain, w1 1 18 (wild type for sply). Heterozygous sply05091/+ showed much less extreme changes than sply/sply, suggesting that the differences in sply/sply were indeed due to the mutation rather than the genetic background.
Finally, we established multiple reaction monitoring (MRM) conditions for
quantification of LCB-P in extracts from different biological origins (Figure 2i-m, Table 4) with a limit of detection of 0.3 fmol on the column at a signal-to-noise (S/N) ratio of 120 and 20 with and without IMP, respectively, in human plasma (Figure 9). The overall levels measured for d18: 1 S1 P and 18:0 LCB-P in murine and human plasma corresponded well with published reports. Further, d18:2 LCB-P was present at levels comparable to SA1 P (Figure 2i). Lymph node (but not lymph fluid) is devoid of this form of LCB-P (Figure 2j), which could be biologically relevant for the regulation of immune cell function in gradients of LCB-P 9. We show for the first time that baker's yeast (S. cerevisiae) contains LCB-P with double bonds (Figure 2m) in addition to the reported fully saturated LCB-P.
Collectively, we have identified, characterized (co-elution with standard, exact mass, product ion analysis) and quantified ten LCB-P species not described previously and six LCB-P not known to be present in the tested biological samples, roughly doubling the number of known LCB-Ps (Figure 2n, Table 5).
Table 4 - LCB-P concentrations in different biological systems
Table 5 - literature references for LCB-P quantification
LCB-P
double bonds
Example 15 - Enrichment of Phosphate Diesters in a Mixture
The versatility of the polymer of the invention allows the isolation of different types of phospholipids. Thus, by changing the composition of the loading, washing and eluting solutions, different types of compounds (in addition to phosphate monoesters) can be separated or isolated.
In this example our data clearly show the possibility of separating different phosphate diesters containing lipids (like phosphatidylinositol, PI, and phosphatidylglycerol, DMPG) from phosphate monoesters (S1 P), from other diesters (SM. DMPC, LPC) and non phosphorylated molecules (Cer, GluCer).
In this case the polymer is the polymer of Example 4a and the conditions used are:
Loading: isopropanol
- Elution solvent 1 : isopropanol/methanol
Elution solvent 2: Isopropanol/methanol/basic compound
Elution solvent 3: chloroform/methanol/acidic compound
By fine-tuning the composition of loading, washing and eluting solutions the specificity of the polymer can be changed accordingly so that not only phosphate monoester containing lipids but also other types of lipids can be enriched by the polymer material (see Figure 1 1 ).
Loading Isopropanol 100%
Elution solvent 1 (washing) Isopropanol: methanol
Elution solvent 2 (basic) Isopropanol: methanol: triethylamine (49.5:49.5: 1 )
Elution solvent 2 (acidic) Chloroform: methanol: 1 % trifluoroacetic acid
(TFA) (49.5:49.5: 1 )
Claims
1. A polymer obtainable by radical co-polymerisation of a first monomer
(l) (II)
wherein each of A and A' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom or when Y or Y' is -NR20-(C=Z)-N-R20-, A or A' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with halo, Ci-6 haloalkyl or nitro;
each of B and B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom; or a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected from halo, C-i.6 haloalkyl or nitro; each of Y and Y' is a linking group comprising 1-6 -CH2- units wherein a -CH2- unit is optionally replaced by a C5-14 aryl or heteroaryl group optionally substituted with one or more substituents selected from H, halogen, NH2, C1-4 alkyl, C -4 alkoxy, C1- haloalkyl or C -4 haloalkoxy or
when each of A or A' and B or B' is a 5- or 6-membered aryl or heteroaryl ring optionally substituted with one or more substituents selected halo, C.i.e haloalkyl or nitro, each of Y and Y' may be -NR20-(C=Z)-N-R20-, wherein
each R20 is independently H, methyl or ethyl; and
Z is O or S;
n is 1 or 2; or when Y and Y is -NR20-(C=Z)-N-R20-, n is 0;
X" is a haiide ion or a hydrophobic anion such as PF6 "; with a second, cross-linking monomer and optionally with one or more further co- monomers;
wherein the molar ratio of the first monomer to the sum of the second monomer and the one or more further co-monomers (if present) is less than or equal to 1 :5.
2. A polymer according to claim 1 wherein, in the monomer of general formulae (I) or (II), X" is a halide or a PF6 " ion.
3. A polymer according to claim 1 or claim 2 wherein, in the monomer of general formulae (I) or (II) the linking group Y is bonded to the quaternary nitrogen moiety of the group A or A'.
4. A polymer according to any one of claims 1 to 3 wherein, in the monomer of general formula (I) or (II), the group A or A' is a pyridinium or imidazolium ion wherein:
when A or A' is a pyridinium ion, the group -CH=CH2 is connected at the pyridinium 4-position; and
when A or A' is an imidazolium ion, the group -CH=CH2 is linked to the non- quaternary ring nitrogen atom.
5. A polymer according to any one of claims 1 to 4 wherein, in the monomer of general formula (I) or (II), the group B or B' is a 5- or 6-membered positively charged heteroaryl ring containing a quaternary nitrogen atom wherein the linking group Y is suitably bonded to the quaternary nitrogen moiety.
6. A polymer according to claim 5 wherein the group B or B' is a pyridinium or imidazolium ion.
7. A polymer according to claim 6 wherein B is a pyridinium ion and the group - CH=CH2 is connected at the pyridinium 4-position; or
B is an imidazolium ion and the group -CH=CH2 is linked to the non-quaternary ring nitrogen atom.
8 A polymer according to any one of claims 1 to 7, in the monomer of general formula (I), wherein A and B are the same and the monomer of general formula (I) is a compound of general formula (la) or (lb):
(la) (lb) wherein X and Y are as defined in claim 1 .
9. A polymer according to any one of claims 1 to 8 wherein the monomer of general formual (I) is a monomer of formula (la) as defined in claim 8.
10. A polymer according to any one of claims 1 to 4 wherein, in the monomer of general formula (I) or (II), the group B or B' is a 5- or 6-membered aryl or heteroaryl ring.
1 1 . A polymer according to any one of claims 1 to 10 wherein, in the monomer of general formulae (I) or (II), Y is a linker group selected from:
each of R1 , R2 and R3 is independently selected from H, halogen, NH2, Ci. alkyl, alkoxy, haloalkyl, C1-4 haloalkoxy or R1 and R2 or R2 and R3 may together form a
5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring;
each of R4, R5, R6 and R7 is independently selected from H, halogen, NH2,
d.4 alkyl, Cv4 alkoxy, d.4 haloalkyl, d.4 haloalkoxy or R4 and R5 or R6 and R7 may together form a 5- or 6-membered carbocyclic, heterocyclic, aryl or heteroaryl ring;
R8 is H, halogen, NH2, d-4 alkyl, C1 4 alkoxy, C1 4 haloalkyl, d-4 haloalkoxy.
12. A polymer according to claim 1 1 wherein, in the monomer of general formula (I) or (II), Y is:
wherein each of R , R , R and R is as defined in claim 15.
13. A polymer according to claim 1 wherein the monomer of general formula (I) or formula (II) is Monomer 1 , which has the structure:
where X is as defined in claim 1 but is most suitably Br"; or
Monomer 2, which has the structure:
where X is as defined in claim 1 but is most suitably Br"; or Monomer 3, which has the structure:
Monomer 5, which has the structure:
where X is as defined above but is most suitably Br"; or Monomer 4, which has the structure:
Monomer 6 which has the structure:
14. A polymer according to any one of claims 1 to 13 wherein the second cross linking monomer is selected from di- and tri-methacrylate monomers, dialkenyl benzene monomers and bis acrylamide monomers.
15. . A polymer according to claim 14 wherein the second cross linking monomer is ethyleneglycol dimethacrylate (EGDMA), divinylbenzene (DVB),
diisopropenylbenzene, (DIB), trimethylolpropanetrimethacrylate (TRIM),
pentaerythritoltriacrylate (PETA), ethylenebisacrylamide (EBA),
piperazinebisacrylamide (PBA), or methylenebisacrylamide (MBA).
16. A polymer according to claim 1 wherein the first monomer is Monomer 1 or a combination of Monomer 1 with one or more other monomers of general formula (I) or general formula (II); and the second monomer is EGDMA.
17. A process for the preparation of a polymer according to any one of claims 1 to 6, the process comprising the radical co-polymerisation of a first monomer of general formula (I) or general formula (II) as defined in any one of claims 1 to 13 with a second, cross-linking monomer as defined in claim 14 or claim 15 and optionally with one or more further co-monomers; characterised in that the molar ratio of the first monomer to the sum of the second monomer and the one or more further co- monomers (if present) is less than or equal to 1 :5.
18. A polymer according to any one of claims 1 to 16 or a process according to claim 17 wherein the molar ratio of the first monomer of general formula (I) or general formula (II) to the sum of the second monomer and the one or more further co- monomers (if present) is from 1 :80 to 1 :20.
19. A polymer or a process according to any one of claims 1 to 18 wherein the polymerisation solvent is selected from water, an alcoholic solvent such as methanol, ethanol or isopopanol, tetrahydrofuran (THF), acetone, dimethylformamide (DMF), acetonitrile (ACN), 1 , 1 ,1 -trichloroethane, chloroform (CHCI3), dichloromethane (DCM), toluene, dimethylsulfoxide (DMSO), isopropanol or any mixture of these solvents.
20. A polymer or a process according to claim 19 wherein the solvent is a mixture of an alcoholic solvent and a non-polar organic co-solvent, for example toluene.
21. A polymer or a process according to any one of claims 1 to 20 wherein the total concentration of the first monomer of formula (I) or (II), the second cross-linking monomer and the one or more further co-monomers (if present) is at least 100 mg/ml of solvent.
22. A polymer or a process according to any one of claims 1 to 21 whereinthe second, cross linking monomer is a di- or tri-methacrylate such as EGDMA, TRIM, or MBA and the one or more further co-monomers are selected from methacrylate monomers, for example methyl methacrylate, 2-hydroxyethylmethacrylate, methacrylamide or an alternative C^6 alkyl methacrylate; or
the second, cross linking monomer is a dialkyl benzene-based monomer such as DIB, and the one or more further co-monomer are selected from alkenyl benzene monomers such as styrene.
23. A process or a polymer according to any one of claims 17 to 22, wherein the process further comprises the steps of:
adding a template to a mixture of the first monomer and the second, cross-linking monomer prior to polymerization, wherein the template is a monomer resembling a target phosphate monoester compound such as a LCB-P in shape and functionality; and
after polymerization, removing the template from the polymer to leave a structure which exhibits affinity for the target phosphate monoester compound or compounds.
24. A method for isolating compounds comprising a phosphate ester group from a sample, the method comprising:
i. contacting the sample with a first solvent so as to extract soluble compounds into a sample solution, wherein the first solvent is capable of dissolving phosphate ester compounds;
ii. contacting the sample solution with a solid phase comprising a polymer according to any one of claims 1 to 23;
iii. washing the solid phase with one or more washing solvents to remove compounds not comprising a phosphate ester group; and
iv. eluting compounds comprising a phosphate ester group with either or both of a basic elution solvent and an acidic elution solvent.
25. A method according to claim 24 wherein, independently or in any
combination:
the first solvent comprises an alcoholic solvent such as methanol, ethanol, n- propanol or isopropanol; and
the washing solvent or solvents comprise alcoholic solvents, for example methanol, ethanol, n-propanol and isopropanol or mixtures thereof.
26. A method according to claim 24 or claim 25 wherein, in step (iv), the phosphate esters are eluted with an acidic elution solvent.
27. A method according to claim 24 or claim 25 wherein, in step (iv), the phosphate esters are eluted with a basic elution solvent and an acidic elution solvent.
28. A method according to any one of claims 24 to 27 wherein the acidic elution solvent comprises a mixture of a chlorinated solvent and an alcoholic solvent acidified with an organic acid, for example formic acid or trifluoroacetic acid.
29. A method according to any one of claims 24, 25, 27 and 28 wherein the basic elution solvent comprises an alcoholic solvent or mixture of alcoholic solvents together with a basic compound such as an amine, for example triethylamine or trimethylamine.
30. A method according to any one of claims 24 to 29 further including one or more of the steps of:
characterising the separated phosphate esters using mass spectrometry; and/or adding a standard solution to the sample solution before step (ii); and/or
derivatising the phosphate ester compounds of the sample, for example by reacting with an alkylating agent in order to replace one or more of the hydrogen atoms on a phosphate or an amine group with an alkyl group.
31. A solid phase material comprising a polymer according to any one of claims 1 to 23.
32. Process for preparing a solid phase material, the process comprising crushing and sieving a polymer according to any one of claims 1 to 23.
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US15/118,225 US10005870B2 (en) | 2014-02-12 | 2015-02-12 | Crosslinked polymers prepared from functional monomers having imidazolium, pyridinium, aryl-substituted urea or aryl-substituted thiourea groups and uses thereof |
SG11201606529QA SG11201606529QA (en) | 2014-02-12 | 2015-02-12 | Crosslinked polymers prepared from functional monomers having imidazolium, pyridinium, aryl-substituted urea or aryl-substituted thiourea groups and uses thereof |
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WO2018049315A1 (en) * | 2016-09-12 | 2018-03-15 | Dana-Farber Cancer Institute, Inc. | Acrylamide copolymerization for sequestration and production of single-stranded nucleic acid |
EP3262412A4 (en) * | 2015-02-27 | 2018-10-24 | Portland State University | Enrichment of lysophosphatidic acids with templated polymeric materials |
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DE4301801A1 (en) | 1993-01-23 | 1994-07-28 | Basf Ag | Crosslinked N-vinyl]-imidazolium salt polymers |
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WO2006041370A1 (en) * | 2004-09-10 | 2006-04-20 | Mip Technologies Ab | Imprinting using dendrimers as templates |
WO2009064245A1 (en) * | 2007-11-12 | 2009-05-22 | Mip Technologies Ab | Imprinted polymers with affinity for phosphorylated peptides and proteins |
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EP3262412A4 (en) * | 2015-02-27 | 2018-10-24 | Portland State University | Enrichment of lysophosphatidic acids with templated polymeric materials |
US10773238B2 (en) | 2015-02-27 | 2020-09-15 | Portland State University | Enrichment of lysophosphatidic acids with templated polymeric materials |
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