[go: up one dir, main page]

WO2007102396A1 - Procede de production de sphingolipide marque - Google Patents

Procede de production de sphingolipide marque Download PDF

Info

Publication number
WO2007102396A1
WO2007102396A1 PCT/JP2007/053917 JP2007053917W WO2007102396A1 WO 2007102396 A1 WO2007102396 A1 WO 2007102396A1 JP 2007053917 W JP2007053917 W JP 2007053917W WO 2007102396 A1 WO2007102396 A1 WO 2007102396A1
Authority
WO
WIPO (PCT)
Prior art keywords
sphingolipid
labeled
fatty acid
reaction
present
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2007/053917
Other languages
English (en)
Japanese (ja)
Inventor
Makoto Ito
Ai Yoshida
Hatsumi Monjusyo
Nozomu Okino
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyushu University NUC
Takara Bio Inc
Original Assignee
Kyushu University NUC
Takara Bio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyushu University NUC, Takara Bio Inc filed Critical Kyushu University NUC
Publication of WO2007102396A1 publication Critical patent/WO2007102396A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/08—Sphingolipids

Definitions

  • the present invention relates to a method for producing labeled sphingolipid useful for medicine, carbohydrate engineering, cell engineering, and the like, and a labeled sphingolipid obtained by the production method.
  • Sphingolipid is a general term for lipids having long-chain base sphingoids, including glycosphingolipids, sphingophospholipids, and ceramides, and is widely distributed from lower animals to higher animals. In recent years, these sphingolipids have been revealed to play an important role in biological activities such as cell proliferation, differentiation induction, and apoptosis. Moreover, since it is a structural component of the cell surface layer, it is also being used as an additive to cosmetics and the like.
  • the sphingolipid has a ceramide structure in which a long-chain fatty acid having a heterogeneous chain length is bound to an amide group of a sphingoid as an amide bond as a common structure.
  • sphingolipids do not exhibit characteristic absorption in the ultraviolet region or visible region, and are therefore difficult to detect optically by themselves. Therefore, labeling sphingolipids with chromophores, fluorescent dyes, piotin, antibody epitopes (no, pentene), etc., will help understand the functions of sphingolipids and biological membranes, leading to the development of drug discovery and artificial biological membranes. Is very useful for.
  • a long-chain fatty acid of a sphingolipid is modified! /
  • a method for producing a substituted sphingolipid or a sphingolipid derivative is based on a lysosphingolipid that lacks an acid amide-bonded fatty acid to the amino group of the sphingolipid of the sphingolipid.
  • chemical and enzymatic synthesis methods are known.
  • a fatty acid or a fatty acid derivative is condensed to an amino group of a lyso form by the following method.
  • a method using a fatty acid active ester such as N-hydroxysuccinimide ester of fatty acid a method using a coupling reagent such as fatty acid and carbonyldiimidazole dicyclohexyl carpositimide, a method using a fatty acid anhydride, a fatty acid salt
  • a method using a bowl is known.
  • Non-patent Document 1, Patent Document 1 and Patent Documents use lysogandarioside as a lysate of acidic glycolipid. Reported in tribute 2.
  • Non-Patent Document 2 for the method power using sphingosyl phosphorylcholine (lysosphingomyelin) as a lyso form of sphingophospholipid.
  • sphingosyl phosphorylcholine lysosphingomyelin
  • side reactions such as O-acylation may occur, and in order to obtain a product that is selectively N-acylated, complicated operations are required for the use and purification of protecting groups.
  • a sphingoid such as ceramide lysogandarioside obtained by chemically dehydrating a sphingoglycolipid containing a ceramide syratin and an amino sugar, a kind of sphingophosphonolipid.
  • Patent Document 3 This method is a method in which condensation is carried out by lipase in an organic solvent, and a substantially anhydrous organic solvent is required, and the substrate is limited by the solubility of the substrate.
  • the force reaction that describes the enzymatic synthesis method of ceramide and hybrid ceramide is not specific, and the generation of O-acylates has been found. As in the case of having multiple amino groups, it is difficult to specifically act only on the amino group of the sphingoid.
  • Non-Patent Document 3 and Patent Document 4 also describe the use of sphingolipid ceramide N-deacylase (SCD ase), an enzyme that specifically hydrolyzes acid amide bonds between sphingolipids of sphingolipids and fatty acids. Describes a method for producing a labeled sphingolipid by introducing a chromophore-forming substance, a fluorescent substance, piotin, a radioisotope, or the like into the fatty acid portion of the phospholipid.
  • SCD ase sphingolipid ceramide N-deacylase
  • the ⁇ -amino group of a ⁇ -amino fatty acid protected by ⁇ -trifluoroacetylation is protected with lysosaccharide using SCDase derived from Pseudomonas bacteria.
  • SCDase derived from Pseudomonas bacteria.
  • Sphingolipid ceramide N-deacylase is known to be SCDase (Patent Document 5, Non-Patent Document 4) derived from the marine bacterium, Shewanella alga G8. Only C14 labeled fatty acids have been identified.
  • Patent Document 1 JP-A-2-200697
  • Patent Document 2 Japanese Patent Laid-Open No. 7-309888
  • Patent Document 3 International Publication No. 94Z26919 Pamphlet
  • Patent Document 4 Pamphlet of International Publication No.98Z03529
  • Patent Document 5 International Publication No. 2002Z026963 Pamphlet
  • Non-Patent Document 1 Methods in Enzymology, 138, 319-341 (1987)
  • Non-Patent Document 2 Journal of Lipid Research, 28th, 710-718 (1987)
  • Non-Patent Document 3 Journal of Biochemistry, Vol. 126, pp. 604-611 (1999)
  • Non-Patent Document 4 Journal of Biologi cal Chemistry, 277, 17300-17307 (2002)
  • an object of the present invention is to provide a production method for specifically and simply synthesizing a labeled sphingolipid obtained by modifying or substituting a lysosphingolipid or a long-chain fatty acid that binds to the sphingoid from the sphingolipid.
  • Another object of the present invention is to provide a labeled sphingolipid obtained by the production method.
  • the present inventors have conducted a study on a method for synthesizing a sphingolipid having a label, Binding of fatty acid with label to lysosphingolipid sphingoid amino group, or substitution power of fatty acid with acid amide bond to sphingoid of sphingolipid and fatty acid with other label Sphingoid acid amide bond of sphingoid
  • SCDase an enzyme that acts on lysosphingolipids and hydrolyzes into fatty acids.
  • ⁇ -amino fatty acid having a protecting group is introduced into sphingolipids or lysosphingolipids using the above enzyme, followed by deprotection and labeling. Helped to react.
  • the present inventors can directly introduce a fatty acid having a label on the sphingoid amino group of lysosphingolipid under mild conditions in an aqueous solution by using a specific SCDase, Alternatively, the present inventors have found that a labeled sphingolipid can be synthesized by a simple operation by directly exchanging a fatty acid having an acid amide bond with a sphingolipid of a sphingolipid with a fatty acid having another label.
  • a sufingolipid ceramide N-deacylase a sphingolipid or lysosphingolipid and a chromophore-forming substance, a fluorescent substance, an aliphatic carboxylic acid having a label selected from the group consisting of piotin and stencil A labeled sphingolipid, characterized in that the labeled sphingolipid is obtained,
  • the present invention relates to a labeled sphingolipid obtained by the production method according to any one of [1] to [5].
  • the invention's effect is to be applied to any one of [1] to [5].
  • the present invention provides a method for producing labeled sphingolipids efficiently in a simple process.
  • INDUSTRIAL APPLICABILITY The present invention can directly introduce a fatty acid having a label into sphingolipid or lysosphingolipid, and is useful for industrial production of labeled sphingolipid.
  • labeled sphingolipid refers to a labeled sphingolipid.
  • the sphingolipid is a simple substance or a mixture of natural or synthetic products having a long-chain base sphingoid, including glycosphingolipid, sphingophospholipid, and ceramide. Can be mentioned.
  • lysosphingolipid or lysozyme of sphingolipid refers to an N-deacyl form of sphingolipid lacking a fatty acid bonded with an acid amide to the amino group of sphingoid.
  • the sphingolipid that can be used in the present invention is not particularly limited to the present invention.
  • celebrity mouthside GlcCer, GalCer, etc.
  • Gandario series GM1, GM2, GDI, etc.
  • lacto series ratatotetra
  • Neolacto series Neolatatotetraosylceramide etc.
  • Sphingoglycolipids such as Globo series
  • Sphingolin lipids such as Sphingomyelin
  • the aliphatic carboxylic acid includes a saturated fatty acid, an unsaturated fatty acid, and a hydrocarbon chain having a functional group such as a halogen, a substituted or unsubstituted amino group, an oxo group, a hydroxyl group, and a thiol group.
  • Carboxylic acids having aliphatic properties such as substituted saturated or unsaturated fatty acids, or saturated or unsaturated fatty acids in which the hydrocarbon chain has elements such as oxygen, sulfur and nitrogen are included.
  • C6-C26 preferably C10-C22, more preferably a saturated fatty acid having a carbon chain length of C12-C18, about C6-C26, preferably C10-C22, and more.
  • Fatty acids such as unsaturated fatty acids having a carbon chain length of C12 to C18 and 2-hydroxy fatty acids are exemplified as the aliphatic rubonic acid used in the present invention. By using those fatty acids labeled with these fatty acids, labeled sphingolipids can be produced.
  • the label used in the present invention means a substance capable of facilitating detection of a substance to which the label is added, and does not particularly limit the present invention, but forms a chromophore. It means substances, fluorescent substances, piotin, haptens, etc.
  • any commercially available chromogenic reagent can be used as the substance that forms a chromophore. Examples of such a chromophore include 4-phenol-trophenol, 2-phenol 1-phenol and 2-phenol. And naphthalene derivatives such as amino naphthalene.
  • a commercially available fluorescent substance can be used as the fluorescent substance.
  • Examples include 7—nitrobenz— 2—oxa— 1, 3 — diazole (NBD), 4, 4— difluor— 4— bora— 3a, 4a— diaza— s— indacene (B ODIPY (registered trademark)), fluorescein Not only these, but also a wide variety of fluorescent materials can be used.
  • the hapten is not particularly limited as long as an antibody that recognizes the hapten is available.
  • digoxigenin can be used in the present invention.
  • labeled fatty acid for example, NBD-labeled fatty acid, BODIPY-labeled fatty acid, or the like
  • labeled fatty acid for example, NBD-labeled fatty acid, BODIPY-labeled fatty acid, or the like
  • NBD-labeled fatty acid for example, NBD-labeled fatty acid, BODIPY-labeled fatty acid, or the like
  • These labels can be easily introduced by reacting a derivative of a labeled compound having reactivity with an amino group or thiol group with a fatty acid having an amino group or thiol group.
  • fluorescent substances and fluorescently labeled fatty acids that can be used in the present invention can be found in Invitrogen's Handbook of Fluorescent Probes and Research Products, Molecular Probes (Handbook of Fluorescent Prooes and Research Products, Molecular Probes). And it is self-loaded.
  • an enzyme that acts on an amide bond of a sphingoid of a sphingolipid and specifically hydrolyzes into a lysosphingolipid and a fatty acid that is, a sphingolipid ceramide N deacylase (SCDase) is used.
  • SCDase sphingolipid ceramide N deacylase
  • the ocean is an enzyme that acts widely on sphingolipids including sphingoglycolipids (gandariosides, neutral glycolipids) and sphingophospholipids (sphingomyelin).
  • a force including SCDase (Non-Patent Document 4, Patent Document 5) derived from the strain Shewanella alga G8 is not limited thereto.
  • the gene encoding SCDase derived from Siebanella alga G8 strain is obtained from, for example, a DNA fragment inserted in a plasmid held by Escherichia coli JM109 ZpSE5 deposited under Patent Document 5 under the accession number FERM BP-7717. can do. Therefore, a recombinant SCDase can be produced using the above gene. Furthermore, recombinant SC Dase described in Patent Document 5 and Non-Patent Document 4 from which the C-terminal polypeptide is deleted can also be suitably used in the present invention.
  • a culture solution or a crude product containing the enzyme as long as a desired reaction can be catalyzed.
  • An extract can also be used.
  • sphingolipid or lysosphingolipid and a labeled fatty acid may be added to the culture solution to produce labeled sphingolipid.
  • the labeled sphingolipid is produced by the method of the present invention in a buffer solution containing a raw sphingolipid or lysosphingolipid, a labeled aliphatic carboxylic acid, and SCDase.
  • the amount of these raw materials used is not particularly limited and can be used up to the saturation amount.
  • it is desirable that the aliphatic carboxylic acid is excessively present.
  • the reaction proceeds even if the molar ratio of the sphingolipid or lysosphingolipid to the aliphatic carboxylic acid is 1: 1. Or lysosphingolipid may be present in excess.
  • the molar ratio of the sphingolipid or lysosphingolipid to the aliphatic carboxylic acid is 2: 1 to 1:10.
  • the amount of the enzyme used is not particularly limited, and a wide range of force can be selected as appropriate. For example, per 1 mL of the starting solution, usually 0.1 lmU or more, preferably 0.5 mU to 200 mU, more preferably lmU to 10 mU, or The amount of sphingolipid or lysosphingolipid used is about 2 U to 20 U per lnmol.
  • a buffer solution having a pH of about 5 to 9 may be used in the present invention. It is desirable to carry out the production in a buffer solution of acid buffer or Tris-HCl buffer having a pH of around 6.5-9.
  • a surfactant may be usually added to the buffer solution for enzyme activity or substrate solubility.
  • the surfactant the present invention is not particularly limited, but a bile acid surfactant, a nonionic surfactant, or the like can be used.
  • the amount of surfactant to be added is particularly limited if it is set to an amount that activates the enzyme, dissolves the substrate, or is effective in obtaining the product efficiently.
  • a nonionic surfactant it is preferably added in the range of 0.01% to 2% by weight. More preferably, it is added in the range of 0.05% by weight to 0.5% by weight, and more preferably in the range of 0.05% by weight to 0.25% by weight.
  • Metal salts may be added to the buffer to activate the enzyme! /.
  • the metal salt for example, a divalent metal salt can be used, and for example, magnesium chloride, salt-manganese, salt-calcium and the like are preferably used.
  • the addition amount of the metal salt is not particularly limited as long as the activity of the enzyme is recognized or the product can be obtained efficiently, but it is preferably in the range of 1 mM to 10 mM.
  • an organic solvent may be added to these reaction solutions.
  • the organic solvent at this time may be a water-soluble organic solvent, or the reaction may be carried out in a two-phase system formed using a water-insoluble organic solvent.
  • the amount of organic solvent added is not particularly limited as long as the enzyme is not deactivated and the product can be obtained efficiently.
  • the labeled sphingolipid thus produced can be confirmed, for example, by thin layer chromatography.
  • the sphingolipid obtained according to the present invention is an unreacted sphingolipid or lysosphingolipid, and labeled aliphatic carboxylic acid by a purification means generally used for organic compounds, for example, extraction using an organic solvent or various chromatography. It can be separated and isolated and purified.
  • the present invention provides a method for producing labeled sphingolipid in which a label is added to the fatty acid of sphingolipid.
  • the desired labeled sphingolipid can be industrially advantageously produced using the production method.
  • Natural sphingolipids generally have a variety of long chain fatty acid chain lengths, and it has been difficult to obtain sphingolipids having a uniform long chain fatty acid chain length.
  • a sphingolipid having a chromophore-forming substance, a fluorescent substance, a marker such as piotin and the like, and a long-chain fatty acid homogenized Can be easily obtained.
  • Plasmid pSCDl was obtained from Escherichia coli JM109ZpSE5, which is a microorganism deposited under the accession number FERM BP-7717 described in Patent Document 5.
  • PETSCD-del a plasmid containing DNA encoding SCDase, which contains the DNA encoding SCDase contained in this plasmid and has a C-terminal polypeptide deleted in accordance with the procedure described in Patent Document 5. It was constructed.
  • Sphingolipid ceramide N-deacylase (SCDase) was purified according to the following series of procedures described in Patent Document 5, using bacterial cells recovered from 15 liters of culture broth as a starting material.
  • substrate solution [10 nmol gandarioside GMla, 5 mM MgCl, 5 mM Mn
  • Decomposition rate (%) ([area of free lysate GMla] Z [area of free lyso GMla + area of unreacted GMla]) X 100
  • one unit (U) of SCDase was defined as the amount of enzyme that decomposes 1 ⁇ mol of GMla per minute.
  • the optimum pH of the obtained SCDase was 5.5 to 6.5, and the molecular weight was 75 kDa.
  • Gandarioside GMla (Sigma) 325 nmol (approx. 500 ⁇ g) and SC Dase 8.4 mU prepared in Reference Example 2.5 mM MgCl, 2.5 mM MnCl, 2.5 mM CaCl and 0.1
  • Mia was confirmed in the ammonia elution fraction.
  • the solvent of this elution fraction was concentrated and removed to obtain purified lyso GMla.
  • FIG. 1 shows the results of detecting reaction products by fluorescence under a UV lamp.
  • Figure 2 shows the results of glycolipid detection for TLC plates using an orcinol sulfate reagent according to a conventional method.
  • Lanes 1 and 2 in Figure 1 and 2 are samples applied to the column, lane 2 is a flow-through fraction, lane 3 is a hydrochloric acid wash fraction, lane 4 is a methanol elution fraction, lane 5 is aqueous ammonia Z methanol elution fraction, lane 6 Is an SCDase digest (marker) of Gandarioside GMla. It was confirmed that NBD-labeled GMla (C12—NBD-GMla) was recovered in the methanol elution fraction (lane 4).
  • the methanol elution fraction was further purified using Sep-Pak C18 and Sep-Pak plus Silica (Waters) and subjected to mass spectrometry.
  • Mass spectrometry was performed in negative ion mode using API-3000 (Applied Biosystems).
  • Gandarioside GMla As glycolipid substrate, 1) Gandarioside GMla 2) Gandarioside Mitsutya 1) Galactosylceramide (GalCer) 4) Psychocin (Psy, galactosylsphingosine) was used. Gandarioside mistatia was prepared according to the method described in Methods in Enzymology, pages 14 and 660-664. GalCer (derived from bovine brain) and Psy were manufactured by Sigma-Aldrich.
  • a final concentration of 250 M glycolipid substrate and fluorescently labeled fatty acid were prepared in the reference example, and SCDase was added to a final concentration of 0.5 mM Triton X-100 containing 25 mM Tris-HCl.
  • the reaction was allowed to proceed for 16 hours at 37 ° C in buffer pH 7.5.
  • the final volume of the reaction liquid was 20 ⁇ .
  • the developed plate was observed under a UV lamp.
  • Gandarioside GM 1a NBD-labeled GM 1a and BODIPY-labeled GM 1a were produced.
  • Gandarioside Mitastia was used. Is the ability to produce each NBD-labeled Gandarioside and BODIPY-labeled Gandarioside NB D-labeled GalCer and BODIPY-labeled GalCer-generated power
  • Psy NBD-labeled GalCer and BODIPY-labeled GalCer Generation was confirmed.
  • the amount of fluorescent-labeled sphingoglycolipid produced increased as the amount of enzyme used increased.
  • the sphingolipid in addition to the condensation reaction of the fluorescent labeled fatty acid acyl group directly with the amino group of the sphingosine base of the lysosulfoglycolipid, the sphingolipid is also exchanged by a reaction in which the fatty acid portion of the sphingolipid and the fluorescent labeled fatty acid are exchanged.
  • a fluorescent group could be introduced into the glycolipid. That is, using either lysosphingoglycolipid or glycosphingolipid as a starting material (acceptor) It was also possible to introduce fluorescently labeled fatty acids (donors) into glycosphingolipids.
  • lysogandarioside GMla was used as the acceptor glycolipid
  • the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid.
  • the mixture was reacted at 37 ° C for 16 hours.
  • the final volume of the reaction solution was 20.
  • the buffer used was ⁇ 4-6: sodium acetate buffer; pH 6-7.5: phosphate buffer; pH 7.5-9.5: Tris-HCl buffer.
  • the reaction solution was treated at 100 ° C for 5 minutes.
  • CaCl 5: 4: l (vZvZv)
  • the fluorescent lipid was quantified with OOPC (manufactured by Shimadzu Corporation). The results are shown in Figure 3.
  • the condensation reaction rate was good in the range of pH 7.0 force to pH 9.0.
  • lysogandarioside GMla was used as the acceptor glycolipid
  • the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid.
  • the mixture was reacted at 37 ° C for 16 hours in H7.5.
  • the final volume of the reaction solution was 20.
  • the surfactants used were Triton X-100 (nonionic), sodium cholate (anionic), sodium taurodeoxycholate (TDC, anionic).
  • TDC sodium taurodeoxycholate
  • the final concentration was changed to 0%, 0.05% by weight, 0.1% by weight, 0.25% by weight, 0.5% by weight, 1% by weight, and 2% by weight.
  • the product was analyzed by the method described in Example 4. As a result, in the reaction to which Triton X-100 was added, a good condensation reaction rate was obtained when the concentration was in the range of 0.05 wt% to 0.25 wt%.
  • lysogandarioside GMla is used as an acceptor glycolipid, and a fluorescent standard is used.
  • the fatty acid the C12-NBD-fatty acid used in Example 2 was used.
  • the final concentration of 0.1% by weight of Triton X-100 and the final concentration of 5 mM of various metal salts were prepared by adding SCDase 80 ⁇ U prepared in a reference example with a glycolipid with a final concentration of 250 ⁇ each and a fluorescently labeled fatty acid.
  • the reaction was carried out at 37 ° C for 16 hours in 25 mM Tris-HCl buffer, pH 7.5.
  • the final volume of the reaction solution was 20 ⁇ m.
  • the metal salts used are CaCl, MnCl, MgCl, CuCl, ZnCl.
  • lysogandarioside GMla was used as the acceptor glycolipid
  • the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid.
  • MgCl final concentration 2 50 M final concentration 0.1 wt 0/0 was added
  • the reaction was carried out in hydrochloric acid buffer, pH 7.5, at 37 ° C for 16 hours.
  • the final volume of the reaction solution was 20.
  • the amount of enzyme used was 2000 U, 80 U, 40 U, 20 U, 10 ⁇ U, 5 U, 2.5 U, and 1.25 U, respectively. These are 400 U, 16 U, 8 U, 4 U, 2 U, 1 U, 0.5 U, and 0.25 U per lnmol of glycolipid, respectively.
  • the product was analyzed by the method described in Example 4. The results are shown in FIG. The condensation reaction rate was good when 20 ⁇ U to 80 ⁇ U SCDase was used.
  • the following 1-11 sphingolipids were used as acceptors, and the reaction was carried out using the C 12 -NBD-fatty acid used in Example 2 as the fluorescently labeled fatty acid.
  • a final concentration of 250 M glycolipids and fluorescently labeled fatty acids were prepared in the reference examples, and SCDase 40 U was added to a final concentration of 0.1 mM Triton X-100 and a final concentration of 5 mM CaCl containing 25 mM Tris HCl.
  • the reaction was carried out at 37 ° C. for 16 hours in a buffer, PH 7.5.
  • the final volume of the reaction solution was 20 L.
  • Sphingolipids used were 1) Sphingomyelin (derived from chicken egg yolk, manufactured by Sigma Aldrich), 2) Darcosylceramide (GlcCer, manufactured by Sigma-Aldrich), 3) Ratatosyl ceramide (LacCer, manufactured by Sigma-Aldrich) 4) Gandarioside GM3 (from Ushi brain) 5) Gandarioside GM2 (Matreya), 6) Gandarioside GMla (from Ushi brain), 7) Gandarioside GDla (from Ushi brain), 8) Gandarioside GTlb (from Ushi brain), 9) Ganglioside GQ lb 10) Crude Gandarioside Mixture (from porcine brain).
  • the product was subjected to TLC analysis by the method described in Example 4, and fluorescently labeled sphingolipid was detected under UV light. As a result, it was confirmed that all the sphingolipids used were fluorescently labeled.
  • Rhizogandarioside GMla was used as the acceptor glycolipid
  • C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid.
  • the mixture was reacted at 37 ° C for 16 hours in a squirrel hydrochloric acid buffer, pH 7.5.
  • the final volume of the reaction solution was 600 ⁇ m.
  • the reaction solution was treated at 100 ° C for 5 minutes, and labeled GMla was separated using an Oasis MCX column (manufactured by Waters).
  • the Oasis MCX column was conditioned with methanol and equilibrated with water according to the manual supplied with the product. Hydrochloric acid was added to the reaction solution after heat treatment to a final concentration of 0.1 N, and then applied to the column.
  • the developed NBD detection plate was subjected to fluorescence detection using a chromatographic scanner CS9300PC according to a conventional method.
  • the glycolipid detection plate was quantified by using orcinol sulfate as a coloring reagent.
  • the condensation rate of the above reaction was about 40%
  • the purification recovery rate of the reaction product C12-NBD-GMla was about 90%
  • the obtained C12-NBD-GMla was about 50 nmol.
  • Sphingomyelin (SM, Sigma-Aldrich) was used as the acceptor lipid.
  • the reaction was carried out in a hydrochloric acid buffer, pH 7.5, at 37 ° C for 16 hours. The final volume of the reaction solution was 20.
  • the production method of the present invention has made it possible to efficiently and inexpensively prepare any labeled sphingolipid containing sphingoglycolipid and sphingomyelin.
  • the labeled sphingolipid obtained by the method of the present invention is useful for the development of medical technologies such as drug discovery and artificial biomembranes, as well as research in the fields of physiology and biochemistry such as elucidation of functions of sphingolipids and biomembranes.
  • FIG. 1 is a diagram showing the results (fluorescence signal) of TLC analysis of NBD-labeled GMla produced by the method of the present invention.
  • FIG. 2 is a diagram showing the results (sugar staining) of TLC analysis of NBD-labeled GMla produced by the method of the present invention.
  • FIG. 3 is a graph showing the results of examination of buffer solution and pH.
  • FIG. 4 is a diagram showing the results of study on metal salts.
  • FIG. 5 is a diagram showing the results of examination of the amount of enzyme.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production d'un sphingolipide marqué, comprenant la réaction d'un sphingolipide ou d'un lysosphingolipide avec un acide carboxylique aliphatique ayant une étiquette choisie dans le groupe constitué par une substance capable de former un chromophore, une substance fluorescente, une biotine et un haptène en utilisant une sphingolipide céramide N-déacylase de façon à produire le sphingolipide marqué. Le procédé permet de préparer n'importe quel sphingolipide marqué y compris un sphingoglycolipide marqué et une sphingomyéline marquée avec un rendement élevé et à bas coût.
PCT/JP2007/053917 2006-03-02 2007-03-01 Procede de production de sphingolipide marque Ceased WO2007102396A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006056753 2006-03-02
JP2006-056753 2006-03-02

Publications (1)

Publication Number Publication Date
WO2007102396A1 true WO2007102396A1 (fr) 2007-09-13

Family

ID=38474827

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/053917 Ceased WO2007102396A1 (fr) 2006-03-02 2007-03-01 Procede de production de sphingolipide marque

Country Status (1)

Country Link
WO (1) WO2007102396A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148125A1 (fr) * 2015-03-16 2016-09-22 国立大学法人大阪大学 Nouveau composé de sphingomyéline marqué par fluorescence et utilisation de celui-ci

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1081655A (ja) * 1996-07-19 1998-03-31 Takara Shuzo Co Ltd スフィンゴ脂質及びスフィンゴ脂質誘導体の製造方法
JP2002058507A (ja) * 2000-08-22 2002-02-26 Akiyama Shigeju 万歩計（登録商標）付きバックル
WO2002026963A1 (fr) * 2000-09-26 2002-04-04 Takara Bio Inc. Gene de ceramide deacylase de shingolipide
WO2003093287A1 (fr) * 2002-05-03 2003-11-13 Institut Pasteur Nouveau procede de preparation d'alpha-glycosylceramides, nouveaux derives alpha-glycosylceramide et leurs applications.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1081655A (ja) * 1996-07-19 1998-03-31 Takara Shuzo Co Ltd スフィンゴ脂質及びスフィンゴ脂質誘導体の製造方法
JP2002058507A (ja) * 2000-08-22 2002-02-26 Akiyama Shigeju 万歩計（登録商標）付きバックル
WO2002026963A1 (fr) * 2000-09-26 2002-04-04 Takara Bio Inc. Gene de ceramide deacylase de shingolipide
WO2003093287A1 (fr) * 2002-05-03 2003-11-13 Institut Pasteur Nouveau procede de preparation d'alpha-glycosylceramides, nouveaux derives alpha-glycosylceramide et leurs applications.

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BERGSTROEM F. ET AL.: "Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, no. 2, 2002, pages 196 - 204 *
BOLDYREV I.A. ET AL.: "A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY)-labeled lipids", RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY, vol. 32, no. 1, January 2006 (2006-01-01), pages 78 - 83 *
GEGE C. ET AL.: "Synthesis of fluorescence labeled sialyl Lewisx glycosphingolipids", TETRAHEDRON LETTERS, vol. 42, no. 3, 2001, pages 377 - 380 *
HE X. ET AL.: "A fluorescence-based high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease", ANALYTICAL BIOCHEMISTRY, vol. 314, no. 1, 2003, pages 116 - 120 *
KLAR R. ET AL.: "Synthesis of pyrenesulfonylamido-sphingomyelin and its use as substrate for determining sphingomyelinase activity and diagnosing Niemann-Pick disease", CLINICA CHIMICA ACTA, vol. 176, no. 3, 1988, pages 259 - 267 *
MARCHESINI S. ET AL.: "Synthesis, spectral properties and enzymic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes", CHEMISTRY AND PHYSICS OF LIPIDS, vol. 53, no. 2-3, 1990, pages 165 - 175 *
NAKAGAWA T. ET AL.: "Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins", JOURNAL OF BIOCHEMISTRY (TOKYO), vol. 126, no. 3, 1999, pages 604 - 611 *
RON I. ET AL.: "Use of fluorescent substrates for characterization of Gaucher disease mutations", BLOOD CELLS, MOLECULES & DISEASES, vol. 35, no. 1, 2005, pages 57 - 65 *
SALVAYRE R. ET AL.: "Use of mixed dispersion of fluorescent galactosylceramide and sodium dodecylsulfate for assaying galactosylceramide-beta-galactosidase and diagnosing Krabbe disease", ENZYME, vol. 33, no. 3, 1985, pages 175 - 180 *
SUMIDA T. ET AL.: "Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12", JOURNAL OF BIOCHEMISTRY (TOKYO, JAPAN), vol. 132, no. 2, 2002, pages 237 - 243 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148125A1 (fr) * 2015-03-16 2016-09-22 国立大学法人大阪大学 Nouveau composé de sphingomyéline marqué par fluorescence et utilisation de celui-ci
JPWO2016148125A1 (ja) * 2015-03-16 2017-07-13 国立大学法人大阪大学 新規蛍光標識スフィンゴミエリン及びその利用

Similar Documents

Publication Publication Date Title
Gelb et al. Do membrane-bound enzymes access their substrates from the membrane or aqueous phase: interfacial versus non-interfacial enzymes
KR100468306B1 (ko) 스핑고지질 및 스핑고지질 유도체의 제조방법
Bruzik et al. Phospholipids chiral at phosphorus. Part 3. Preparation and spectral properties of chiral thiophospholipids
Karbarz et al. Purification and characterization of the lipid A 1-phosphatase LpxE of Rhizobium leguminosarum
WO2007102396A1 (fr) Procede de production de sphingolipide marque
Oskolkova et al. Fluorescent organophosphonates as inhibitors of microbial lipases
JP3625244B2 (ja) リゾスフィンゴ脂質の製造方法
US6428999B1 (en) Sphingolipid ceramide N-deacylase, methods for producing sphingolipids and sphingolipid derivatives, and spingolipid ceramide N-deacylase gene
JP3669595B2 (ja) スフィンゴ脂質セラミドデアシラーゼ
JP2657274B2 (ja) リン脂質の製造法
US20110224096A1 (en) Fluorescent phosphonic ester library
JP3868052B2 (ja) スフィンゴ脂質セラミドｎ−デアシラーゼ
KR100585471B1 (ko) 리소스핑고리피드의 제조 방법
JPH0761276B2 (ja) 酵素法リン脂質−ｄ−セリン誘導体の製造法
JPH0253498A (ja) ３‐メルカプト‐２‐アルキル‐プロピオン酸のラセミエステル誘導体の光学異性体の酵素分割法
den Kamp et al. Two isomers of glucosaminylphosphatidylglycerol. Their occurrence in Bacillus megaterium, structural analysis, and chemical synthesis
JPH03123493A (ja) ジアシルグリセロリン脂質の加水分解法
Riedy et al. The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine
Huang et al. A facile method for controlling the reaction equilibrium of sphingolipid ceramide
JP2000300287A (ja) トランスグルタミナーゼを用いた糖鎖導入方法
JP3970898B2 (ja) 光学活性α−メチルアルカンジカルボン酸−ω−モノエステル及びその対掌体ジエステルを製造する方法
JPH0656856A (ja) リン脂質誘導体
Lombardo et al. Rate-deter111ining Step in Phospholipase A2 Mechanislll
JP2000270857A (ja) ホスホリパーゼｄおよびその製造法
EP1605056A2 (fr) Procédé de récupération de la sérine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07737597

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP