JP2005511701A - ホスホリパーゼa2の活性を測定するための化合物 - Google Patents
ホスホリパーゼa2の活性を測定するための化合物 Download PDFInfo
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- JP2005511701A JP2005511701A JP2003549360A JP2003549360A JP2005511701A JP 2005511701 A JP2005511701 A JP 2005511701A JP 2003549360 A JP2003549360 A JP 2003549360A JP 2003549360 A JP2003549360 A JP 2003549360A JP 2005511701 A JP2005511701 A JP 2005511701A
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- SUHOQUVVVLNYQR-MRVPVSSYSA-O glycerylphosphorylcholine Chemical compound C[N+](C)(C)CCO[P@](O)(=O)OC[C@H](O)CO SUHOQUVVVLNYQR-MRVPVSSYSA-O 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000002011 intestinal secretion Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- IFGBWZMAYOBIGC-UHFFFAOYSA-N methyl 4-pyren-1-ylbutanoate Chemical compound C1=C2C(CCCC(=O)OC)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 IFGBWZMAYOBIGC-UHFFFAOYSA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical group CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001433 sodium tartrate Substances 0.000 description 1
- 229960002167 sodium tartrate Drugs 0.000 description 1
- 235000011004 sodium tartrates Nutrition 0.000 description 1
- RNVYQYLELCKWAN-UHFFFAOYSA-N solketal Chemical compound CC1(C)OCC(CO)O1 RNVYQYLELCKWAN-UHFFFAOYSA-N 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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Abstract
【化1】
Description
エム イー ブランクら(M.E.Blank et al),J.Bio l.Chem.、1981年、第256巻、第175〜178頁。 エイチ エス ヘンドリックソンら(H.S.Hendrickson e t al)、Anal.Biochem.、1999年、第276巻、第27〜35頁。
Fは発蛍光団としての非置換または置換ピレンであり;
Qは消光物質であり;
L2は、C(O)−L1またはC(O)−L1−NHであり、このL1は上記定義の通りである。
−3a,4a−ジアザ−s−インダセン)と比べると、極性が低く、消光波長と発光波長との間隔が大きく、水溶液中で機能する能力を有し、細胞中で有利に分配され、蛍光消光せずに膜に組込むことができ、かつPH非依存性蛍光であるという利点を有する。ピレンのさらなる有利な性質は、高濃度でエキシマーを形成し、同時に発光波長を長波長へとシフトすることである。エキシマー形成により、発蛍光団の濃度を評価することができる。
(O)−(CH2)5−NHを表す。これらのリンカーは、一方では、CH2鎖が代謝的に安定であるために、この位置での消光物質の不要な切断が回避され、他方では、PLA2、特にPAF−AHのエステル基−C(O)−O−がこれらの酵素によって意図的かつ選択的に切断されるという理由で特に好適である。
特に好ましい実施形態において、本発明の化合物は下記式(2)の構造を有する。
得る。そのような連続的な測定により、数分から数時間、特に5分から0.5時間という長い時間にわたって、蛍光を連続的に測定することができる。
・例えば、以下のような場合における、ヒトおよび動物の多種多様な生物学的サンプル(鼻分泌物、***、肺分泌物(BAL)、血液またはその成分、単離細胞集団、生検組織、胆嚢分泌物、腸分泌物、糞便検体を鑑別診断するための診断薬として:
− 他の局所性および全身性炎症症状;
− アレルギー性および/または免疫性疾患、例えば、花粉アレルギーおよびラテックスアレルギー、糸球体腎炎;
− 中毒発生疾患、例えば、中毒性肺不全(toxic lung failure)または中毒症状;
− 冠動脈疾患、例えば、急性冠症候群、狭心症、プリンツメタルアンギナ、心筋梗塞、ならびに、動脈硬化症、急性脳発作;
− 他の症状、例えば、移植、手術、他の侵襲性処置後の症状;
− 上記医学的症候群に関する健康診断関連において;
− 多様な代謝機能、例えば、高脂血症などの不特定症状、酸化ストレス亢進症状の検出;
− 急性炎症性疾患、例えば、敗血症、ARDS(肺不全)、および孤立性器官の炎症、特に創傷治癒障害の診断;
− 慢性炎症症状、例えば、関節リウマチや、リウマチ型の他の疾患、潰瘍性大腸炎、クローン病の検出;
・以下のような医学的処置の療法モニタリングとして:
− 特に上述の治療適応分野における、あらゆる種類の医学的介入、例えば、組換え酵素を用いた治療、および対応酵素の活性の変化を伴うあらゆるタイプの医学的介入;
− 特に上述の治療適応分野における、侵襲的処置、例えば、炎症原因の除去手術、心肺機能の代替または機械的支持を含む手術、肝臓手術および多様な移植;
− 上記の組み合わせ。
ロ−3−ホスホコリンの調製
M(C2H4 78Br35Cl2O2 31P)=239g/mol
EI−MS: m/z(%)=243(0.5)[M],241(0.9)[M],239(0.6)[M],163(25),161(41),149(18),147(30),137(36),135(55),119(23),117(34),109(64),108(100),107(69),106(100),99(18),47(27).1H−NMR(CDCl3):δ(ppm)=4.59(2H,m,CH2Br,3JHH=6.26Hz,3JHP=10.34Hz),3.13(2H,m,CH2O,3JHH=6.26Hz,4JHP=1.05Hz).13C−NMR(CDCl3):δ(ppm)=69.88(1 CH2O,2JCP=8.39Hz),27.32(1
CH2Br,3JCP=10.29Hz).IR(フィルム):v(cm−1)=3033w,2971w,2883w,1453m,1423m,1385w,1306s,1233m,1180m,1071s,1016s,963m,908m,845w,759m.
M(C20H14O3)=302g/mol
EI−MS:m/z(%)=303(10)[MH+],302(42)[M],230(16),229(100),202(11),201(65),200(24).HR−EI−MS(C20H14O3):計算値:302.0943;実測値:302.0943.1H−NMR([D6]−DMSO):δ(ppm)=8.81−8.12(9H,m,CH芳香族),3.52(2H,t,COOH2,3JHH=6.22Hz),2.82(2H,t,CH2CO2H,3JHH=6.22Hz).13C−NMR([D6]−DMSO):δ(ppm)=203.05(1 CO),173.87(1 CO2H),132.97−123.41(9 CH芳香族,7 C芳香族),36.87(
1 COCH2),28.52(1 CH2CO2H).IR(KBr):v(cm−1)=3426m,3039m,2984m,2929m,2677w,2594w,1695s,1665s,1507w,1404w,1212m,1123w,1075w,842m,713w.
M(C20H16O2)=288g/mol
EI−MS:m/z(%)=289(16)[MH+],288(62)[M],216(20),215(100),213(11).HR−EI−MS(C20H16O2):計算値:288.1150;実測値:288.1150.1H−NMR([D6]−DMSO):δ(ppm)=8.50−7.92(9H,m,CH芳香族),3.36(2H,m,CH2C16H9),2.41(2H,m,CH2CO2H),2.04(2H,m,CH2).1H−NMR(CDCl3):δ(ppm)=8.32−7.86(9H,m,CH芳香族),3.42(2H,t,CH2C16H9,3JHH=7.57Hz),2.51(2H,t,CH2CO2H,3JHH=7.00Hz),2.23(2H,m,CH2,3JHH=7.57Hz,3JHH=7.00Hz).13C−NMR([D6]−DMSO):δ(ppm)=174.74(1 CO),136.39−123.39(9 CH芳香族,7 C芳香族),33.64−26.98(1 CH2CO2H,2CH2).13C−NMR(CDCl3):δ(ppm)=177.48(1
CO),135.25−123.04(9 CH芳香族,7 C芳香族),33.21−26.46(1 CH2CO2H,2 CH2).IR(KBr):v(cm−1)=3447w,3037w,2950m,2934w,2874w,1695s,1431w,1275m,1206m,918w,846s,711w.
M(C21H18O2)=302g/mol
EI−MS:m/z(%)=303(13)[MH+],302(57)[M],228(15),216(22),215(100),213(12),108(13).HR−EI−MS(C21H18O2):計算値:302.1307;実測値:302.1306.1H−NMR(CDCl3):δ(ppm)=8.25−7.70(9H,m,CH芳香族),3.63(3H,s,CH3),2.36(2H,t,CH2CO,3JHH=7.20Hz),2.11(2H,m,CH2,3JHH=7.60Hz).13C−NMR(CDCl3):δ(ppm)=173.73(1 CO),135.51−123.15(9 CH芳香族,7,C芳香族),51.38(1 CH3),33.52−26.64(1 CH2CO,2 CH2).IR(KBr):v(cm−1)=3033w,2949w,2876w,1734s,1429m,1209m,1161m,842s.
M(C20H18O)=274g/mol
EI−MS:m/z(%)=275(10)[MH+],274(53)[M],216(17),215(100).HR−EI−MS(C20H18O):計算値:274.1358;実測値:274.1357.1H−NMR(CDCl3):δ(ppm)=8.23−7.79(9H,m,CH芳香族),3.64(2H,t,CH2O,3JHH=6.39Hz),3.31(2H,t,CH2C16H9,3JHH=7.60Hz),1.87(2H,m,CH2,3JHH=7.70Hz,3JHH=7.60Hz),1.69(2H,m,CH2,3JHH=7.70Hz、3JHH=6.39Hz).13C−NMR(CDCl3):δ(ppm)=136.65−123.35(9 CH芳香族,7 C芳香族),62.77(1 CH2O),33.17−27.93(3 CH2).IR(KBr):v〜(cm−1)=3423s,幅広,3038m,2927m,2859m,1181w,1030w,982w,841s.
黄色固体を得た。
M(C30H37O78Br)=492g/mol
EI−MS:m/z(%)=495(10)[MH+],494(31)[M],493(10)[MH+],492(33)[M],412(13),228(22),215(100),55(18),41(13).HR−EI−MS(C30H37O78Br):計算値:492.2028;実測値:492.2028.1H−NMR(CDCl3):δ(ppm)=8.30−7.85(9H,m,CH芳香族),3.49−3.45(2H,t,CH2Br,3JHH=6.44Hz),3.41−3.34(6H,m,CH2C16H9,2 CH2O,3JHH=7.08Hz,3JHH=6.81Hz),1.96−1.90(2H,m,CH2,3JHH=7.08Hz,3JHH=6.44Hz),1.83−1.73(4H,m,2 CH2,3JHH=6.81Hz),1.55−1.53(2H,m,CH2),1.25(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=136.72−123.30(9 CH芳香族,7 C芳香族),70.93−70.55(2 CH2O),33.94−26.15(1 CH2Br,11 CH2).IR(KBr):v〜(cm−1)=3037w,2924s,2851s,1499m,1448m,1429w,1369w,1243w,1184w,1117s,1063w,965w,849m,841s,710s.
EI−MS:m/z(%)=545(22)[MH+],544(62)[M],257(22),215(100),101(18),55(11),43(14).HR−EI−MS(C36H48O4):計算値:544.3553;実測値:544.3553.1H−NMR(CDCl3):δ(ppm)=8.29−7.83(9H,m,CH芳香族),4.26−4.21(2H,m,CHCH2O),4.07−4.00(1H,m,CHO),3.75−3.31(10H,m,4 OCH2,CH2C16H9),1.95−1.86(2H,m,CH2),1.78−1.72(2H,m,CH2),1.55−1.53(4H,m,CH2),1.41および1.35(6H,s,2 CH3),1.26(12H,m,6CH2).13C−NMR(CDCl3):δ(ppm)=136.92−123.48(9 CH芳香族,7 C芳香族),109.33(1 C(CH3)2),74.77(1 CHO),71.85−70.64(4 CH2O),66.94(1 CH2O),33.33−25.89(11 CH2),26.77および25.43(2 CH3).IR(KBr):v〜(cm−1)=3059m,2952s,2890m,2866s,1524w,1467m,1369w,1289m,11121m,1058m,837w,684s.
M(C33H44O4)=504g/mol
EI−MS:m/z(%)=505(32)[MH+],504(100)[M],257(17),228(21),216(19),215(89),55(14).HR−EI−MS(C33H44O4):計算値:504.3240;実測値:504.3238.1H−NMR(CDCl3):δ(ppm)=8.29−7.83(9H,m,CH芳香族),3.87−3.59(3H,m,CHOH,CH2OH),3.47−3.31(10H,m,4 OCH2,CH2C16H9),1.94−1.89(2H,m,CH2),1.77−1.72(2H,m,CH2),1.54−1.51(4H,m,2 CH2),1.24(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=136.94−123.48(9 CH芳香族,7 C芳香族),72.03−70.65(4 CH2O),70.50(1 CHOH),63.81(1 CH2OH),33.31−25.97(11 CH2).IR(KBr):v〜(cm−1)=3419s,幅広,3058w,2932m,2851m,1437m,1433m,1265m,1205m,1186m,1138m,1112m,1048m,843m.
M(C52H58O4)=746g/mol
FAB−MS(FAB+,NBA):m/z=746.4[M],504.3,244.
1,215.1.HR−FAB(C52H58O4):計算値:746.4335;実測値:746.4375.HR−FAB(C52H58O4Na):計算値:769,4233;実測値:769.4304.1H−NMR(CDCl3):δ(ppm)=8.31−7.85(9H,m,CH芳香族),7.45−7.19(15H,m,CH芳香族),3.95(1H,m,CH),3.55−3.33(10H,m,4 OCH2,CH2C16H9),3.24−3.18(2H,m,CH2OC(C6H5)3),1.97−1.87(2H,m,CH2),1.81−1.73(2H,m,CH2),1.55−1.51(4H,m,2 CH2),1.25(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=146.90−123.51(24 CH芳香族,10 C芳香族),86.65(1 C(C6H5)3),72.95−70.67(4 CH2O),69.87(1 CH),64.65(1 CH2OC(C6H5)3),33.35−26.97(11 CH2).IR(KBr):v〜(cm−1)=3429m,3056w,2927s,2855s,1602w,1490m,1448m,1319w,1213m,1113m,1077m,1033m,899w,846m,746m,706s.
M(C59H62O5)=850g/mol
FAB−MS(FAB+,NBA):m/z=873.4[M+Na],850.4[M],608.3,591.3,391.3,307.1,243.1,215.1.1H−NMR(CDCl3):δ(ppm)=8.30−7.86(9H,m,CH芳香族),7.45−7.21(20H,m,CH芳香族),5.46−5.41(1H,m,CH),3.79−3.34(12H,m,4 OCH2O,CH2C16H9,CH2OC(C6H5)3),1.97−1.89(2H,m,CH2),1.80−1.72(2H,m,CH2),1.55−1.51(4H,m,CH2),1.25(12H,m,CH2).13C−NMR(CDCl3):δ(ppm)=166.00(1 CO),146.88−123.51(29 CH芳香族,11 C芳香族),86.47(1
C(C6H5)3),72.65(1 CH),71.59−69.47(4 CH2O),62.86(1 CH2OC(C6H5)3),33.35−26.01(11 CH2).IR(KBr):v〜(cm−1)=3057w,2927s,2854m,1718s,1448m,1384m,1272m,1212w,1177w,1158w,1112m,1032w,899w,846m,763m,703m.
M(C40H48O5)=608g/mol
FAB−MS(FAB+,NBA):m/z=608.3[M],591.3,469.3,329.1,257.2,215.1.HR−FAB(C40H48O5):計算値:608.3502;実測値:608.3519.HR−FAB(C40H38O5Na):計算値:631.3399;実測値:631.3424.1H−NMR(CDCl3):δ(ppm)=8.29−7.84(9H,m,CH芳香族),7.62−7.34(5H,m,CH芳香族),5.27−5.22(1H,m,CH),3.99−3.34(12H,m,4 CH2O,CH2C16H9,CH2OH),1.97−1.88(2H,m,CH2),1.80−1.72(2H,m,CH2),1.57−1.51(4H,m,2 CH2),1.25(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=166.38(1 CO),136.94−123.51(14 CH芳香族,8 C芳香族),73.81(1 CH),71.94−68.95(4 CH2O),62.87(1 CH2OH),33.80−26.19(11 CH2).IR(KBr):v〜(cm−1)=3431m,3038m,2930s,2859m,1718s,1603m,1585m,1453m,1435m,1317m,1274s,1179m,1113m,1027m,936m,846m,709s.
a2SO4で脱水し、15mlのCHCl3、10mlのアセトニトリルおよび5mlのイソプロパノール中で回転させて溶かした。次いで、1.5gのトリメチルアミン溶液(エタノール中33%)を加えた。反応フラスコを緊密に密閉し、50℃で一晩攪拌した。反応溶液を回転させ、5mlのCHCl3で抽出し、それぞれ4mlのギ酸/MeOH(5:7)、4mlの酢酸ナトリウム(0.1M)/MeOH(5:7)および3mlの塩化ナトリウム(1M)/MeOH(5:7)の各々を2回ずつ振盪した。NaSO4で脱水し、有機相をカラムクロマトグラフィーにかけて、99mg(38%)の所望の化合物を得た。
M(C45H60N2O8 31P)=773g/mol
FAB−MS(FAB+,NBA):m/z=796.3[M+Na],774.4[MH+],601.5,569.4,531.4,413.3,215.1.HR−FAB(C45H61O8NP):計算値:774.4145;実測値:774.4135.HR−FAB(C45H60O8NPNa):計算値:796.3954;実測値:796.3951.1H−NMR(CD3OD):δ(ppm)=8.34−7.89(9H,m,CH芳香族),7.73−7.45(5H,m,CH芳香族),5.41−5.37(1CH,m,CH),4.20−4.17(2H,m,CH2N),4.05−3.35(14H,m,4 OCH2,CH2OP,POCH2,CH2C16H9),3.16(9H,s,3 CH3),1.96−1.90(2H,m,CH2),1.81−1.72(2H,m,CH2),1.52−1.48(4H,m,2 CH2),1.28(12H,m,6 CH2).13C−NMR(CD3OD):δ(ppm)=167.78(1 CO),138.30−124.60(14 CH芳香族,8 C芳香族),74.27(1 CH),72.81−70.48(4 CH2O),68.87,66.90,60.95(1 CH2N,1 CH2OP,1 POCH2),54.90(3 CH3),34.38−27.29(11 CH2).
M(C38H56NO7 31P)=669g/mol
FAB−MS(FAB+,NBA):m/z=692.3[M+Na],670.4[MH+],669.4[M],584.3,242.3,289.1,215.1.HR−FAB(C38H57NO7 31P):計算値:670.3873;実測値:670.3881.HR−FAB(C38H56NO7 31PNa):計算値:692.3692;実測値:692.3693.1H−NMR(CD3OD):δ(ppm)=8.23−7.88(9H,m,CH芳香族),4.43−4.38(2H,m,CH2N),4.0
2−3.36(15H,m,4 OCH2,OCH,CH2OP,POCH2,CH2C16H9),3.22(9H,s,3 CH3),1.96−1.87(2H,m,CH2),1.77−1.66(2H,m,CH2),1.55−1.47(4H,m,2 CH2),1.27(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=170.46(1 CO),138.16−124.48(9 CH芳香族,7 C芳香族),72.90−71.47(4 CH2O),71.03(1 CH),67.31,66.63,59.33(1 CH2OP,1 CH2N,1 POCH2),54.78(3 CH3),33.36−24.59(10 CH2).IR(BKr):v〜(cm−1)=3418s,3039m,2852m,1635m,1490m,1467m,1375w,1230m,1111m,1087m,1051m,970m,928w,850m.
M(C50H69N4O12 31P)=948g/mol
FAB−MS(FAB+,NBA):m/z=971.4[M+Na],949.4[MH+],884.4,862.4,721.4,243.2,215.1.HR−FAB(C50H70O12N4P):計算値:949.4725;実測値:949.4755.HR−FAB(C50H69O12N4PNa):計算値:971.4545;実測値:971.4547.1H−NMR(CDCl3):δ(ppm)=9.00−6.67(12H,m,CH芳香族),5.13−5.10(1H,m,CH),4.34−4.28(2H,m,CH2N),4.08−3.23(16H,m,4 OCH2,POCH2,CH2OP,CH2N,CH2C16H9),3.18(9H,s,3 CH3),2.38−2,31(2H,m,CH2CO),1.95−1.86(4H,m,CH2),1.80−1.72(2H,m,CH2),1.56−1.41(8H,m,4 CH2),1.25(12H,m,6 CH2).13C−NMR(CDCl3):δ(ppm)=170.46(1 CO),148.66−113.98(12 CH芳香族,10 C芳香族),73.86(1 CH),72.81−69.12(4 CH2O),67.31,66.63,60.30(1 CH2OP,1 CH2N,1 POCH2),54.78(3 CH3),43.27(1 CH2N),33.36−24.59(1 CH2CO,14 CH2).IR(BKr):v〜(cm−1)=3365m,3107w,3042w,2928m,2853m,1729m,1623s,15
89m,1525m,1500w,1475w,1425m,1369w,1337s,1312m,1280m,1245m,1187m,1144m,1094m,1060w,1032w,923w,832w,745w.
実施例2:本発明の1−O−{10−[4−(ピレン−1−イル)ブトキシ]デシル}−2−(6−(2,4−ジニトロフェニル)アミノヘキソイル)−rac−グリセロ−3−ホスホコリンを用いたPAF−AH活性の測定
検査用に、実施例1の本発明化合物の10mM DMSOストック溶液を調製し、次いで、使用緩衝液(0.1M トリス塩酸(TRIS HCl)、0.1% Tween(登録商標)20、pH7.4)で、それぞれ各試験に望ましい濃度に希釈した。
ヘルマ社(Helma)〔独国ミュルハイム(Mullheim)/バーデン(Baden)所在〕の1ml容量石英キュベット(1cm×1cm)を使用する。
・ 動力学モジュールにおいて:励起波長(nm)=344.00;蛍光波長(nm)=377.00;励起スリット(nm)=5;蛍光スリット(nm)=5;平均化時間(s)=0.1000;滞留時間(s)=0.1000;サイクル時間(分)=0.2500;励起フィルター=自動;蛍光フィルター=オープン;マルチセルホルダー=マルチセル;PMT電圧(V)=中;温度制御。
(マイクロタイタープレートでの測定)
パーキンエルマー社(Perkin Elmer)〔独国ユーバリンゲン(Uberlingen)所在〕の白色96ウエルタイタープレートおよびグライナー社(Greiner)〔独国フリケンハウゼン(Frikenhausen)所在〕の黒色96ウエルFIAプレートを用いた。
・ 動力学モジュールにおいて:励起波長(nm)=344.00;蛍光波長(nm)=377.00;励起スリット(nm)=5;蛍光スリット(nm)=5;平均化時間(s)=0.1000;サイクル時間(分)=0.0000;励起フィルター=自動;蛍光フィルター=オープン;PMT電圧(V)=中;ウエルプレート=ON;読取位置=ウエル中心。
(血漿およびヒトLDLの調製および純度検査)
使用緩衝液:PBS緩衝液(150mM NaCl、40mM リン酸、1mM EDTA、pH=7.4)。この緩衝液を15分間アルゴンで飽和した。
3,000rpmのヘルムレ(Hermle)遠心分離機を用いて、100mlの採取したてのヘパリン添加血液を2×50ml管中室温下で遠心した。各上清から20mlを取ってプールし、これに、400μlの0.1M EDTA溶液(pH=7.45)を加え、アルゴン雰囲気とした(mit Argon ueberschichtet) 。
32mlの血漿サンプルに12.2112gのKBr p.a.を加え、アルゴン雰囲気とした。KBrを低速振動により溶解させた。ベックマン社(Beckman)〔独国ミュンヒェン(Munich)所在〕の6×4ml遠心分離管中で2mlのPBS溶液を調製し、KBrを添加した血漿サンプルを加えて層を形成した。次いで、これを、4℃、真空下37,000rpmで2時間遠心した。この後、ロータから遠心分離管を慎重に抜出した。所望のLDLは、遠心分離管の底から約1/3離れた位置の黄色帯域として区別可能である。先ず、上部の白色層を取り出して廃棄した。黄色層は厚さ約1mmである。この層を、先の丸い曲った針を用いて取り出した。全6本の遠心分離管中に含まれていたLDLをプールし、アルゴン雰囲気にした。KBrを除去するために、得られた溶液のうち300μlを、PBS緩衝液を用いるセファデックス−G25ミニカラムを用いて色層分析にかけた。
溶液A:1% Na2BCA、2% Na2CO3H2O、0.16% 酒石酸ナトリウム、0.4% NaOH、0.95% NaHCO3、pH=11.25
溶液B:4% CuSO4・5H2O
標準としてBSAを用いた。すべての測定は2重測定として実施した。1種の調製物の総量は300μl/ウエルで、たいてい2.5μlの測定サンプル(場合により希釈後)を用いた。これを水で75μlまで満たし、これに、225μlの溶液Aと溶液Bの50:1混合物を加えた。インキュベーション時間は室温下で2時間であった。次いで、サンプルを562nmで光度測定した。
検査はサブマリン型ミニゲル電気泳動を用いて実施した。
・ ゲル組成:20mlのバルビタール緩衝液(50mM、pH=8.6)、0.3% Separide(商標)(60mg)
・ 単離LDLの調製:10μl(0.5〜1μg)の円柱状LDL、0.5μlの9−ジエチルアミノ−5H[α]フェノキサゼン−5−オン(ナイルレッド)(0.4mg/ml)、10μlの使用緩衝液(TBE緩衝液、30% グリセロール、1% SDS、ブロムフェノールブルーのスパチュラチップ)
・ TBE緩衝液(89mM トリス塩酸、89mM B(OH)3、2mM EDTA、pH=8.5)
・ 電気泳動時間:室温下で(56Vの一定Uで)1時間
・ 固定液:25% iPrOH、10% 酢酸、65% 水
・ 染色液:50% MeOH、10% 酢酸、40% 水、0.05% クーマシーブリリアントブルー R250
・ 脱色液:5% MeOH、7% 酢酸、88% 水
(LDLからのPAF−AHの単離)
緩衝液A:25mM トリス塩酸、0.1% Tween20、2mM EDTA、pH7.6
緩衝液B:25mM トリス塩酸、0.1% Tween20、2mM EDTA、50〜200mM NaCl、pH7.6(線形勾配)
すべての作業は4℃で実施した。
Amicon YM30)で濃縮した。次いで、濃縮留分を、アマーシャム・ファルマシアバイオテク社(独国フライブルグ所在)の予備平衡(緩衝液A)Blue Sepharoseョ 6FFクロマトグラフィーカラムにピペットで移し、6倍カラム充填量で
洗浄して、タンパク質を含まない溶離液を得た。次いで、緩衝液A+0.5M NaClでタンパク質を溶出させた。最高のPAF−AH活性を有する留分をプールし、限外ろ過(Amicon YM30)して濃縮した。
Claims (10)
- 消光物質が2,4−ジニトロフェニル残基である、請求項1に記載の化合物。
- R1およびR2がそれぞれ独立に(CH2)nであり、nが2〜12の整数である、請求項1または2に記載の化合物。
- L1が(CH2)4−O−(CH2)10を表す、請求項1乃至3のいずれか1項に記載の化合物。
- Rが(CH2)oであり、oが1〜20の整数である、請求項1または2に記載の化合物。
- L2が、C(O)−(CH2)pまたはC(O)−(CH2)p−NHであり、pが1〜20の整数である、請求項1乃至5のいずれか1項に記載の化合物。
- L2がC(O)−(CH2)5−NHである、請求項6に記載の化合物。
- ホスホリパーゼA2の活性を測定するための請求項1乃至8のいずれか1項に記載の化合物の使用。
- 前記ホスホリパーゼA2が血小板活性化因子アセチルヒドロラーゼである、請求項11に記載の使用。
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EP01129137A EP1318154B1 (de) | 2001-12-07 | 2001-12-07 | Verbindungen zur Bestimmung der Aktivität von Phospholipase A2 |
PCT/EP2002/011852 WO2003048172A1 (de) | 2001-12-07 | 2002-10-23 | Verbindungen zur bestimmung der aktivität von phospholipase a¿2? |
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JP2009505956A (ja) * | 2005-07-27 | 2009-02-12 | テクニッシェ ウニヴェルシテート グラーツ | フルオロフォアの部分を含んでいる酸化リン脂質、および抗動脈硬化作用を有している酵素の存在を決定するための使用 |
JP2010528615A (ja) * | 2007-05-31 | 2010-08-26 | サノフィ−アベンティス | NAPE−PLD又はAbh4の活性を修飾する薬剤を同定するためのアッセイ方法 |
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EP1633864B1 (en) | 2003-05-28 | 2010-04-07 | Glaxo Group Limited | High throughput assay of lp-pla2 activity |
WO2005005977A2 (en) * | 2003-06-30 | 2005-01-20 | Applera Corporation | Fluorescent phospholipase assays and compositions |
DK1718967T3 (da) | 2004-02-03 | 2013-06-03 | Diadexus Inc | Metoder til at detektere Lp-PLA2 aktivitet |
WO2005113797A2 (en) | 2004-04-16 | 2005-12-01 | Glaxo Group Limited | METHODS FOR DETECTING Lp-PLA2 ACTIVITY AND INHIBITION OF Lp-PLA2 ACTIVITY |
US20150017671A1 (en) | 2004-04-16 | 2015-01-15 | Yaping Shou | Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity |
US9957546B2 (en) | 2008-03-04 | 2018-05-01 | The Trustees Of The University Of Pennsylvania | Vivo detection of phospholipase activation |
US20140283157A1 (en) | 2013-03-15 | 2014-09-18 | Diadexus, Inc. | Lipoprotein-associated phospholipase a2 antibody compositions and methods of use |
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US6146625A (en) * | 1993-10-06 | 2000-11-14 | Icos Corporation | Platelet-activating factor acetylhydrolase |
JPH1017600A (ja) * | 1996-06-28 | 1998-01-20 | Suntory Ltd | 血小板活性化因子アセチルヒドロラーゼおよびその遺伝子 |
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JP2009505956A (ja) * | 2005-07-27 | 2009-02-12 | テクニッシェ ウニヴェルシテート グラーツ | フルオロフォアの部分を含んでいる酸化リン脂質、および抗動脈硬化作用を有している酵素の存在を決定するための使用 |
KR101299614B1 (ko) * | 2005-07-27 | 2013-08-23 | 테크니쉐 우니베르지테트 그라츠 | 발색단 모이어티를 포함하는 산화된 인지질 및 항아테롬성활성을 가진 효소의 존재 결정을 위한 이의 용도 |
JP2010528615A (ja) * | 2007-05-31 | 2010-08-26 | サノフィ−アベンティス | NAPE−PLD又はAbh4の活性を修飾する薬剤を同定するためのアッセイ方法 |
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ATE269342T1 (de) | 2004-07-15 |
US20050064532A1 (en) | 2005-03-24 |
EP1318154B1 (de) | 2004-06-16 |
WO2003048172A1 (de) | 2003-06-12 |
JP4150675B2 (ja) | 2008-09-17 |
EP1318154A1 (de) | 2003-06-11 |
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AU2002346871A1 (en) | 2003-06-17 |
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