WO2002002570A1 - Chromogenic compound - Google Patents
Chromogenic compound Download PDFInfo
- Publication number
- WO2002002570A1 WO2002002570A1 PCT/US2000/019547 US0019547W WO0202570A1 WO 2002002570 A1 WO2002002570 A1 WO 2002002570A1 US 0019547 W US0019547 W US 0019547W WO 0202570 A1 WO0202570 A1 WO 0202570A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- formula
- assay
- phospholipase
- deoxy
- Prior art date
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- 239000003593 chromogenic compound Substances 0.000 title description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 109
- 238000000034 method Methods 0.000 claims abstract description 45
- 238000003556 assay Methods 0.000 claims description 72
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 47
- 102000015439 Phospholipases Human genes 0.000 claims description 43
- 108010064785 Phospholipases Proteins 0.000 claims description 43
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 33
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol Substances OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 19
- 102000019267 Hepatic lipases Human genes 0.000 claims description 17
- 108050006747 Hepatic lipases Proteins 0.000 claims description 17
- 150000003839 salts Chemical class 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 230000002255 enzymatic effect Effects 0.000 claims description 13
- 230000007062 hydrolysis Effects 0.000 claims description 13
- 238000006460 hydrolysis reaction Methods 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 10
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 9
- 230000002452 interceptive effect Effects 0.000 claims description 8
- 229940086609 Lipase inhibitor Drugs 0.000 claims description 6
- 125000003342 alkenyl group Chemical group 0.000 claims description 6
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- 125000000304 alkynyl group Chemical group 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000000865 phosphorylative effect Effects 0.000 claims description 5
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims description 5
- 150000001768 cations Chemical class 0.000 claims description 3
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- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 2
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 54
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- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 22
- 150000003904 phospholipids Chemical class 0.000 description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 20
- 238000005481 NMR spectroscopy Methods 0.000 description 20
- YDSCLGYBUIDZNF-SEHARXJXSA-N [(2r)-3-hexadecoxy-2-[(5e,8e,11e,14e)-icosa-5,8,11,14-tetraenoyl]sulfanylpropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCOC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)SC(=O)CCC\C=C\C\C=C\C\C=C\C\C=C\CCCCC YDSCLGYBUIDZNF-SEHARXJXSA-N 0.000 description 18
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 17
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
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- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 15
- 238000000921 elemental analysis Methods 0.000 description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 12
- 150000003573 thiols Chemical class 0.000 description 12
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- 239000000203 mixture Substances 0.000 description 10
- -1 oxy ester Chemical class 0.000 description 10
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- 238000006243 chemical reaction Methods 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000000434 field desorption mass spectrometry Methods 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 238000004040 coloring Methods 0.000 description 7
- 235000019439 ethyl acetate Nutrition 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000002585 base Substances 0.000 description 6
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- 239000000377 silicon dioxide Substances 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- 0 C1C2C1CC*C2 Chemical compound C1C2C1CC*C2 0.000 description 4
- 102000015779 HDL Lipoproteins Human genes 0.000 description 4
- 108010010234 HDL Lipoproteins Proteins 0.000 description 4
- 108010058864 Phospholipases A2 Proteins 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
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- 210000002381 plasma Anatomy 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
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- DNJIEGIFACGWOD-UHFFFAOYSA-N ethanethiol Chemical compound CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 3
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- 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 3
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- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
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- UHBAPGWWRFVTFS-UHFFFAOYSA-N 4,4'-dipyridyl disulfide Chemical compound C=1C=NC=CC=1SSC1=CC=NC=C1 UHBAPGWWRFVTFS-UHFFFAOYSA-N 0.000 description 2
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- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000006140 methanolysis reaction Methods 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229940042880 natural phospholipid Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 150000007530 organic bases Chemical class 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
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 229940074439 potassium sodium tartrate Drugs 0.000 description 1
- AFNBMGLGYSGFEZ-UHFFFAOYSA-M potassium;ethanethioate Chemical compound [K+].CC([S-])=O AFNBMGLGYSGFEZ-UHFFFAOYSA-M 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- KOUKXHPPRFNWPP-UHFFFAOYSA-N pyrazine-2,5-dicarboxylic acid;hydrate Chemical compound O.OC(=O)C1=CN=C(C(O)=O)C=N1 KOUKXHPPRFNWPP-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 239000003774 sulfhydryl reagent Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- LZTRCELOJRDYMQ-UHFFFAOYSA-N triphenylmethanol Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(O)C1=CC=CC=C1 LZTRCELOJRDYMQ-UHFFFAOYSA-N 0.000 description 1
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/14—Esters of phosphoric acids containing P(=O)-halide groups
Definitions
- This invention is directed to a chromogenic compound, a thiophospholipid enzyme substrate, specifically thiophosphatidyl ethyleneglycol (thioPEG) , which is useful as an indicator compound in an analytical test system.
- the present invention relates to a novel chromogenic substrate compound, its preparation and use in an assay and for the spectrophotometric detection of enzymes in a liquid test sample.
- All biological membranes have the same classes of chemical compounds and a number of properties in common. These membranes are very dynamic structures with a movement that permits the cell as well as subcellular structures to adjust their shape and to move.
- the chemical components of membranes which include lipids and protein, are well suited for the dynamic movement of membranes.
- membranes control the composition of the space they enclose by their ability to exclude a variety of molecules and via selective transport systems which permit the movement of molecules from one side to another.
- membranes modulate the concentration of substances, thereby exerting a strong influence on the body's metabolic pathways.
- Lipids are a major component of membranes.
- the three major lipid components of cell membranes are phosphoglycerides, sphingolipids, and cholesterol.
- the phosphoglycerides and sphingomyelin, a sphingolipid containing phosphate, are classified as phospholipids .
- Phospholipids which are waxy solids, are found almost exclusively in cellular membranes and in the lipoproteins of blood plasma. Phospholipids thus serve primarily as structural elements and are never stored in large amounts. As their name implies, this group of lipids contains phosphorus in the form of phosphoric acid.
- the major phospholipids found in cells contain two fatty acid molecules which are esterified to the first and second hydroxyl groups of glycerol. The third hydroxyl group, at carbon atom 3, is esterified with phosphoric acid.
- Phospholipids contain a second alcohol which is also esterified to the phosphoric acid to form a phosphodiester; the second alcohol group is thus located on the polar head of the phospholipid molecule.
- RO- denotes the second alcohol group.
- R alkyl, alkenyl or alkynyl group
- phospholipids Different types are named according to the second alcohol at their polar heads .
- the most abundant phospholipids are the closely related phosphatidylethanolamine (also called cephalin) and phosphatidylcholine (also called lecithin) , which contain ethanolamine and choline, respectively, at their heads. Each of these can occur in different forms depending on the fatty acids they contain.
- Phospholipids readily undergo hydrolysis, catalyzed by acids, bases, or enzymes. Dilute base removes the two fatty acid groups of phosphatidylcholine, leaving the rest of the molecule intact. Strong base causes cleavage of both the fatty acids as well as the choline, leaving glycerol 3-phosphate, which can then be cleaved to yield glycerol and phosphoric acid by boiling with hydrochloric acid.
- phospholipases Different types are categorized based on the specific linkage for which they catalyze hydrolysis in the phospholipid molecule. Sites of action of phospholipases A ] _, A2 , C and D on phosphatidylcholine are shown below.
- V 2 alkyl, alkenyl or alkynyl group
- phospholipase enzymatic activity may be measured.
- the determination of phospholipase enzymes via assay is important in a variety of fields such as biochemical research, environmental and industrial testing, and medical diagnostics.
- the quantitation of enzyme levels in body fluids such as serum and plasma provides very useful information to the physician in diagnosing disease states and their treatment.
- enzymes can also serve as detection reagents in a variety of analytical systems such as immunoassays and nucleic acid hybridization techniques. In such systems, enzymes are useful directly or indirectly as indicators to monitor the extent of antigen-antibody binding or nucleic acid hydridization that occurs.
- optical indicator compounds for use in the detection and measurement of the activity of such enzymes.
- optical indicator compounds comprise a detectable chemical group, such as a fluorogen or a chromogen, which has been derivatized with an enzyme cleavable substrate group specific for the enzyme of interest.
- Such optical indicator compounds exhibit an optical signal which is different from the optical signal which is provided by the cleaved native form of the fluorogen or chromogen.
- the enzyme cleaves the indicator compound to liberate the chromogen in the form of a distinctly fluorescent or colored product to provide a change in fluoroescence or color which is proportional to the amount of enzyme present which, in turn, can be correlated to the amount of analyte present in a liquid test sample.
- Phospholipases are capable of cleaving thio ester bonds of an unnatural substrate.
- phospholipase A2 hydrolyzes and cleaves an sn-2 thio ester.
- the fact that hydrolysis releases a free thiol group has been utilized as the basis for a spectrophotometric assay shown below in Scheme A. See also, Lin Yu and Edward A. Dennis, Methods in Enzyology, Vol. 197, 65-75 (1991).
- phospholipase Ai hydrolyzes and cleaves an sn-1 thio ester.
- R an al yl, alkenyl or al ynyl group
- Phospholipase A2 cleaves the sn-2 oxy ester of phospholipids; it will also hydrolyze an sn-2 thio ester.
- the liberated thiol is allowed to react with a thiol- ⁇ ensitive reagent, and the formation is measured continuously by monitoring the increase in absorption associated with its production.
- DTNB is used to detect the free thiol group. This reagent is commercially available from Aldrich (Milwaukee, I) . DTNB is preferable because it is sufficiently soluble in buffer such that stock solutions are aqueous .
- phospholipases The ability of phospholipases to cleave the thio ester bonds of unnatural substrates has also been utilized to develop continuous spectrophotometric assays for phospholipase A2 (PLA2) , phospholipase Al (PLAl) , phospholipase C (PLC) , lysophospholipase and lipase.
- the detection methods available for phospholipase assays include titra etric, acidimetric, radiometric, nuclear magnetic resonance and others, including the thio assay.
- the thio assay possesses many characteristics that recommend it as a general assay for phospholipases. The most important are that it is a continuous, spectrophotometric assay which is very convenient, it directly detects one of the products liberated upon hydrolysis, it is one of the more sensitive assays, and it is also suitable for detailed kinetic studies.
- the thio assay can be used for phospholipases A ⁇ and A2 and with appropriate modification of the substrate would be applicable to other phospholipases.
- the thio assay has not been used extensively.
- thiophospholipids have been synthesized as substrates, including stereospecific didecanoyl-thio-phosphatidylcholine (thioPC) and its analog, didecanoyl-thio-phosphatidylethanolamine (thioPE) .
- thioPC stereospecific didecanoyl-thio-phosphatidylcholine
- thioPE didecanoyl-thio-phosphatidylethanolamine
- the free thiol which is formed on hydrolysis of the thio ester bond is detected by the thio assay upon addition of either 5, 5 ' -dithiobis (2-nitrobenzoic acid) (DTNB) or 4, 4 ' -dithiobispyridine (DTP) as a thiol coloring reagent.
- DTNB 5, 5 ' -dithiobis (2-nitrobenzoic acid)
- DTP 4 ' -dithiobispyridine
- the thiol coloring reagents, DTP and DTNB, shown below, are routinely used to detect free thiol groups in the thio assay.
- thiol coloring reagent is more advantageous to use in the thio assay depends on pH, solubility, the effect on the extinction coefficient, and the effect on the enzyme. Another important consideration in choice of which thiol coloring reagent to use in an assay is whether or not either of the above thiol reagents affects the activity of an enzyme .
- the thio assay is a method used to measure phospholipase activity in an assay.
- the thio assay is incompatible with free thiols or any other substance which would significantly reduce the diaryl disulfide bond in the thiol coloring reagent and, as a consequence, is generally limited to the measurement of pure phospholipases.
- the thio assay is convenient and reproducible when compared to other types of assays.
- the thio assay has a lower limit of detection of 1 nmol/min. Recently, interest has grown in the study of assay strategies and methods for phospholipases including the thio assay.
- thioPC and thioPE are effective substrates in the phospholipase assay.
- thioPC and thioPE are several limitations in the use of thioPC and thioPE as substrates.
- a lengthy synthesis is required for the preparation of these substrates.
- thioPC requires additional portions of enzyme in order to obtain a detectable color.
- thioPC hydrolyzes slowly and can thus cause low sensitivity within the assay.
- thioPE is increasingly unavailable due to its complicated synthesis method.
- the present invention an unnatural substrate, is like thioPC substrates because it allows DTNB to be present throughout assay hydrolysis; and, thus, color is produced as the substrate is hydrolyzed. Therefore, only one incubation period is required compared to the two or more incubation periods required for thioPC, which further requires additional enzyme for color. Further, when the product of the reagent of the present invention reacts with a thio coloring agent, it does not cause an extraneous effect on the activity of the enzyme. Given the number of factors involved in choosing a thio coloring reagent for the thio assay, it is advantageous to minimize extraneous effect on the enzyme to the greatest extent possible. The invention is also an improvement over thioPE because its synthesis is much less complicated and should therefore be more generally available.
- the present invention relates to a novel chromogenic substrate which is a useful indicator compound in an analytical test system for the spectrophotometric detection of an enzyme in a liquid test sample.
- it relates to a better chromogenic substrate in the previously discussed phospholipase 3 , assay which is useful for spectrophotometric identification of potent inhibitors for treatment of phospholipase j . disorder.
- this invention relates to a new chromogenic substrate in the phospholipase assay, the process of preparing the substrate and the use of the substrate in a phospholipase assay to identify a potent inhibitor for a phospholipase related disorder.
- the present invention is directed to a compound of formula I :
- each of Q and M is independently C5-C19 alkyl, alkenyl or alkynyl;
- Y is (CH2)2 ⁇ H or CH 2 CH (OH) CH 2 OH; or a non-interfering salt thereof.
- the present invention is also directed to a compound of formula (I) in which each of Q and M is independently C5-C2 . 9 n-alkyl, n-alkenyl or n-alkynyl .
- each of M and Q is independently pentyl, hexyl, heptyl, nonyl, or tridecanyl; and Y is (CH2)2 ⁇ H.
- a more particular compound of formula (I) is one in which:
- Another particular compound of formula (I) is one in which: each of Q and M is nonyl; and Y is (CH2)2 OH - (This particular compound is ThioPEG, or a salt thereof, as exemplified in Example 1)
- Alkenyl means an aliphatic hydrocarbon group containing carbon-carbon double bonds.
- Alkyl means an aliphatic hydrocarbon group which is straight or branched.
- Alkynyl means an aliphatic hydrocarbon group containing carbon-carbon triple bonds.
- a compound of the present invention or an equivalent expression, is meant to embrace a compound of general formula (I) as hereinbefore described, which expression includes a salt, where the context so permits.
- reference to an intermediate, whether or not it itself is claimed is meant to embrace a salt, where the context so permits.
- This invention provides, in addition, a process for preparing a compound of formula I (or a non-interfering salt thereof) comprising:
- Another particular compound is the compound of formula I which is (l-decanoylthio-l-deoxy-2-decanoyl-sn-glycero-3- phosphoryl ) ethylene glycol .
- Another particular compound is the compound of formula I which is l-decanoylthio-l-deoxy-2-decanoyl-sn-glycero-3- phosphoryl-1 ' -sn-glycerol .
- An additional aspect of the present invention is directed to a compound of formula III:
- a process for preparing a novel compound of formula I as provided in any of the above descriptions which is selected from any of those described in the examples.
- This includes phosphorylation of the deprotected hydroxy group of a compound of formula II using commonly known phosporylating agents.
- the preferred phosphorylating agent when Y is (CH2)2 OH ' i- s 2-chloro- phospholane-2-oxide.
- the preferred temperature for this reaction is from about 10 °C to about 50 °C. The most preferred temperature for this reaction is about ambient temperature .
- the present invention further provides a method of using the compound of formula I as an indicator in an analytical test system for the measurement of enzymatic activity in a liquid test sample.
- the invention further provides for a method of using the compound of formula I as an indicator compound in analytical test systems for the measurement of enzymatic activity in a liquid test sample for the treatment of diseases.
- the invention also provides for a method of using the compound of formula I in a phospholipase A]_ or B assay to spectrophoto etrically identify an inhibitor for the treatment of a phospholipase A ⁇ related disorder.
- the invention further provides for a method of using the compound of formula I in the phospholipase A]_ assay to identify a hepatic lipase inhibitor.
- the present invention relates to a novel chromogenic substrate for use in an assay to afford identification of enzymatic activity in a screen.
- the present invention further relates to a chromogenic substrate which may be used in an assay and will allow identification of a potent inhibitor such as a hepatic lipase inhibitor.
- the present invention also relates to a chromogenic substrate which may be used in a phospholipase A ⁇ _ assay and which will allow the identification of a potent inhibitor for the treatment of a phospholipase A]_ related disorder (s).
- the present invention also relates to a chromogenic substrate which may be used in a phospholipase A3 , assay and which will allow the identification of a potent inhibitor for the treatment of a phospholipase A ⁇ related disorder such as a hepatic lipase inhibitor to be used as a cardiovascular protective agent for raising plasma high- density lipoprotein (HDL) levels.
- a phospholipase A ⁇ related disorder such as a hepatic lipase inhibitor to be used as a cardiovascular protective agent for raising plasma high- density lipoprotein (HDL) levels.
- this compound is a chromogenic substrate in the hepatic lipase inhibition assay in the presence of DTNB (Ellman's Reagent), replacing thioPC.
- Another special embodiment of the method of the present invention is the use of a compound of formula I in a phospholipase 3 , assay to spectrophotometrically identify an inhibitor for the treatment of a phospholipase A3 , related disorder.
- Another special embodiment of the method of the present invention is the use of a substrate of formula I in a phospholipase A ⁇ assay to identify a hepatic lipase inhibitor. It is a further object of the invention to provide a kit or test device which may be effectively utilized for carrying out the novel uses of the invention.
- kits or test devices comprises a compound of forumula I in a suitable container.
- a kit according to the invention can further include an additional or additional containers that contain, for example, a control reagent, a coloring agent and other reagents suitable for running an assay of interest.
- the kit or test device for measurement of enzymatic activity contains a carrier matrix incorporated with the compound of formula I.
- a novel chromogenic substrate which has valuable properties .
- the substrate may be particularly useful in an assay for identification of an inhibitor of an enzyme.
- the inhibitor in turn, may be useful for treatment of a phospholipase A ⁇ related disorder.
- the substrate could be used in an assay to identify a hepatic lipase inhibitor, for example, which could then be used as cardiovascular protective agents for raising plasma high-density lipoprotein (HDL) levels.
- HDL high-density lipoprotein
- a compound of formula I may be prepared according to the route outlined in Scheme B below, and described in the Examples, in which each of M and Q, respectively, represents a value defined for the groups M and Q.
- values of M and Q may be different.
- the epoxide of formula 2 is ring opened with a thio fatty acid nucleophile of formula MCOS " where M is as previously defined, to form compound 8.
- An example of a thio fatty acid nucleophile is thio decanoyl acid sodium salt.
- the compounds of formula 5, II, and I are formed using methods similar to those described above in Scheme B and in the Examples.
- compound II may be phosphorylated with POCI3 to form a compound of formula III.
- the compound of formula III may be utilized to prepare the compounds of formula I wherein Y is CH 2 CH 2 OH (compound 9) or CH 2 CH 2 OHCH 2 OH (compound 10) .
- Y is CH 2 CH 2 OH (compound 9) or CH 2 CH 2 OHCH 2 OH (compound 10) .
- ethylene glycol is treated with the compound of formula III, followed by an acid hydrolysis wash.
- Compound 10 may similarly be formed via reaction of a compound of formula III with glycerol followed by acid hydrolysis wash.
- the necessary starting materials for the preparation of a compound of formula I may be prepared by procedures which are selected from standard techniques of organic chemistry, from techniques which are analogous to the syntheses of known, structurally similar compounds, and techniques which are analogous to the above described procedures or procedures described in the Examples. It will be clear to one skilled in the art that a variety of sequences is available for the preparation of the starting materials. Starting materials which are novel provide another aspect of the invention.
- the invention includes acceptable salts of the thioPEG substrate compounds defined by the above formula I.
- Acceptable salts are those that do not interfere with analytical methods and procedures.
- Such salt may be made with a base which affords an acceptable cation, which includes alkali metal salts (especially sodium and potassium) , alkaline earth metal salts, aluminum salts and ammonium salts, as well as salts made from organic bases such as triethylamine, morpholine, piperidine and triethanolamine.
- alkali metal salts especially sodium and potassium
- alkaline earth metal salts especially aluminum salts and ammonium salts
- organic bases such as triethylamine, morpholine, piperidine and triethanolamine.
- the potassium and sodium salt forms are particularly preferred.
- Fig. 1 shows the hydrolysis of thioPEG and thioPC substrates by Hepatic Lipase.
- Hepatic Lipase enzyme was diluted 1, 32 or 64 fold for thioPC and 100, 500, 1000 or 5000 fold for thioPEG. Hydrolysis was measured by the papain thiol detection system (discussed below) .
- the Y-axis shows the Optical Density at 410 nM.
- the X-axis shows the Relative Enzyme Concentration.
- Data curve I represents 0.5 mM thioPEG and Data Curve II represents 1.0 inM thioPC.
- thioPEG is an improvement over substrates currently used in the phospholipase assay, such as thioPC.
- thioPEG is hydrolyzed much faster than thioPC. Since the enzyme hydrolyzes thioPC much more slowly, there is much less product formed in the reaction and thus, less sensitivity for the assay. Due to increased enzymatic activity with thioPEG, therefore, sensitivity of the assay is increased.
- the invention also has the additional advantage of requiring fewer incubation periods in order to obtain detectable color.
- thioPC substrate requires a reaction coupled with an enzymatic detection process for the liberated thiol- lysophospholipid which has two additional incubation periods in the papain system.
- L-BAPNA is an abbreviation for N-benzoyl-L-arginine, p-nitroanilide, hydrochloride.
- thioPC is combined with hepatic lipase and incubated for about 30 minutes yielding a free- thiol lysophospholipid.
- the free-thiol lysophospholipid is combined with inactive papain-S-S-CH3 and incubated for 60 minutes, yielding activated papain-SH enzyme.
- Activated papain-SH enzyme is combined with a coloring agent, L-BAPNA, and incubated for 60 minutes yielding a colored product, p-nitro aniline.
- ThioPEG substrate is an improvement since it requires only a single incubation period to yield detectable color.
- Papain-S-S-CH inactive
- Papain-SH active
- ThioPEG is more direct because the assay has DTNB present throughout the hydrolysis so that color is produced as the substrate is hydrolyzed. As the procedural Scheme F shows below, there is only one incubation time for thioPEG.
- Using ThioPEG as a substrate has an additional advantage because the DTNB system is a chemical reaction as opposed to an enzymatic reaction. Since the DTNB system is a chemical reaction, there is a decreased chance for an inhibitor, such as hepatic lipase, to interfere with the detection system itself.
- Substrate Buffer A 100 mM Hepes, pH 8.3 at 37 °C
- Substrate Buffer B 100 mM Hepes, pH 8.3 at 37 °C with 6.83 mM Triton X100
- ThioPEG Molecular wt . of 540 Recombinant Hepatic Lipase Thiophospholipid: about 0.42 mM thiophospholipid in chloroform
- DTNB Solution about 50 nM DTNB in DMSO (dimethyl sulfoxide)
- Hepes Buffer A there is 2.4 g Hepes/100 mL water. Therefore 36 grams of Hepes is dissolved in 1500 mL of water. The mix solution's pH is adjusted to pH83 at 37° C and brought up to 1500 mL with water. 500 mL of Buffer A is retained for the Protein Buffer.
- thioPEG/mL of Substrate Buffer B For 0.42 mM substrate stock, use 0.227 mg of thioPEG/mL of Substrate Buffer B. Approximately 20 mg of sn-1 thiol substituted Phosphatidyl Ethylene Glycol (see Examples for preparation method) is weighed into a vial, such as a scintillation vial. Enough chloroform should be added to make a 2.043 mg/mL solution. Sonicate the solution briefly until well dissolved. Next, pipette 1 mL of chloroform/substrate solution into each scintillation vial. This should give enough substrate for one full 96 well plate.
- Each vial is dried with nitrogen until solvent removed, swirling each vial simultaneously such that a thin film of substrate will be easily reconstituted in each buffer. Each vial is then frozen.
- Daily stock preparation is performed for 9 mL of substrate (one icrotiter plate) .
- the substrate vial is removed from the freezer and combined with 9 mL of pre-warmed (37 °C) substrate buffer (the final concentration is 0.227 mg/mL). Place the buffer in a 37°C water bath. Sonicate for 5 minutes or vortex until solution is clear before use.
- Enzyme Solution The enzyme is stored at -80 °C in 100 or 50 ⁇ L portions. A 0.406 mg/mL recombinant hepatic lipase stock requires a 50 fold dilution. Therefore, to a 50 ⁇ l or 100 ⁇ l enzyme aliquot, 2450 ⁇ l or 4900 ⁇ l, respectively, of substrate Buffer A (protein buffer) should be added. The enzyme should then be stored on ice until ready to use. The protein concentration of enzyme is about 0.406 mg/mL.
- DTNB dimethyl sulfoxide
- Table 1 below shows final assay volumes and concentrations of various components used following the above procedure .
- test compound is dissolved in pure DMSO at 1 ⁇ M (1000 nM) .
- assay concentrations are 10, 1, 0.1, 0.33, 0.011, 0.0037, 0.0012 and 0.00041 ⁇ M.
- Table 2 shows the assay concentrations and the corresponding volume of stock and 10% DMSO for each concentration.
- DTNB is used as a thiol coloring reagent with an incubator temperature of 37 °C.
- Substrate Buffer B is placed in a 37 °C water bath to pre-warm. The substrate is removed from the freezer and 9 mL of substrate Buffer B, 100 mM Hepes, 6.83 mM Tx-100) is added, sonicated for 5 min. and then kept in a 37 °C water bath. Dilutions of the test compound are next made in preparation for assay. 10 ⁇ l of the diluted test compound are transferred via pipette into the wells. Control wells receive 10 ⁇ l each of 10% DMSO and enzyme solution, while blank wells receive 10 microliters of 10% DMSO and 10 microliters of saline (no enzyme) .
- DTNB is weighed and diluted to 20 mg/mL with DMSO. The DTNB is then diluted 10 fold with the substrate Buffer B. 540 ⁇ l of diluted DTNB is added to 9 ml of ThioPEG and mixed well.
- the stock enzyme is diluted with Buffer A. Next, 10 microliters of protein solution is added to each well except the blank, and the wells mixed. The stock solution and test compounds are incubated at 37 °C for 10 min. At 10 minutes, 80 microliters of substrate are added to each well. The plate is then placed in the spectrometer and read at 412 nM every 2 minutes for 30 minutes.
- PPA polyphosphoric acid
- Si ⁇ 2 silica gel
- Triethylsilane (29.4 mL, 182 mmol) and trifluoroacetic acid (5.90 mL, 75.9 mmol) were sequentially added to a stirred solution of l-decanoylthio-l-deoxy-2-decanoyl-3- ( triphenylmethyl) -sn-glycerol (10.0 g, 15.2 mmol) in anhydrous 1, 2-dichloroethane (100 mL) at -32 °C (ethylene glycol/methanol/dry ice) under a nitrogen atmosphere.
- the resultant yellow solution was allowed to warm to -25 °C where it was stirred for an additional 1.5 h.
- Example 1 title compound was obtained from the compound of Example 1, Part C, in a 65% yield.
- Example 1 title compound was obtained from 1-heptanoylthio- l-deoxy-2-he ⁇ tanoyl-sn-glycerol in a 48% yield.
- IR (CHCI3) 3300 (br) , 1733, 1691 cur 1 ;
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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EP00948748A EP1305323A1 (en) | 2000-07-25 | 2000-07-25 | Chromogenic compound |
PCT/US2000/019547 WO2002002570A1 (en) | 2000-07-25 | 2000-07-25 | Chromogenic compound |
AU2000262205A AU2000262205A1 (en) | 2000-07-03 | 2000-07-25 | Chromogenic compound |
Applications Claiming Priority (1)
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PCT/US2000/019547 WO2002002570A1 (en) | 2000-07-25 | 2000-07-25 | Chromogenic compound |
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WO2002002570A1 true WO2002002570A1 (en) | 2002-01-10 |
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PCT/US2000/019547 WO2002002570A1 (en) | 2000-07-03 | 2000-07-25 | Chromogenic compound |
Country Status (3)
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EP (1) | EP1305323A1 (en) |
AU (1) | AU2000262205A1 (en) |
WO (1) | WO2002002570A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3583226A4 (en) * | 2017-02-20 | 2020-04-15 | Regents of the University of California | Amplification technology using dual enzyme cascade detection |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0002062A1 (en) * | 1977-11-22 | 1979-05-30 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Cyclic organophosphorus compounds, method for their preparation and their use |
EP0331167A2 (en) * | 1988-03-04 | 1989-09-06 | Roche Diagnostics GmbH | Substrates for phospholipases |
WO1995005479A1 (en) * | 1993-08-17 | 1995-02-23 | The Regents Of The University Of California | Assay and substrate for arachidonoyl-specific phospholipase a¿2? |
-
2000
- 2000-07-25 WO PCT/US2000/019547 patent/WO2002002570A1/en not_active Application Discontinuation
- 2000-07-25 EP EP00948748A patent/EP1305323A1/en not_active Withdrawn
- 2000-07-25 AU AU2000262205A patent/AU2000262205A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0002062A1 (en) * | 1977-11-22 | 1979-05-30 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Cyclic organophosphorus compounds, method for their preparation and their use |
EP0331167A2 (en) * | 1988-03-04 | 1989-09-06 | Roche Diagnostics GmbH | Substrates for phospholipases |
WO1995005479A1 (en) * | 1993-08-17 | 1995-02-23 | The Regents Of The University Of California | Assay and substrate for arachidonoyl-specific phospholipase a¿2? |
Non-Patent Citations (2)
Title |
---|
DECKELBAUM R.J.: "Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins.", BIOCHEMISTRY., vol. 31, no. 36, - 15 September 1992 (1992-09-15), AMERICAN CHEMICAL SOCIETY. EASTON, PA., US, pages 8544 - 8551, XP002163344, ISSN: 0006-2960 * |
KUCERA G.L. ET AL.: "Hydrolysis of thioester analogs by rat liver phospholipase a1", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 263, no. 26, - 15 September 1988 (1988-09-15), AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD., US, pages 12964 - 12969, XP002163343, ISSN: 0021-9258 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3583226A4 (en) * | 2017-02-20 | 2020-04-15 | Regents of the University of California | Amplification technology using dual enzyme cascade detection |
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EP1305323A1 (en) | 2003-05-02 |
AU2000262205A1 (en) | 2002-01-14 |
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