EP0521941A1 - Acides sialiniques fluorescents actives par cmp et leur procede de production - Google Patents

Acides sialiniques fluorescents actives par cmp et leur procede de production

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Publication number
EP0521941A1
EP0521941A1 EP91906420A EP91906420A EP0521941A1 EP 0521941 A1 EP0521941 A1 EP 0521941A1 EP 91906420 A EP91906420 A EP 91906420A EP 91906420 A EP91906420 A EP 91906420A EP 0521941 A1 EP0521941 A1 EP 0521941A1
Authority
EP
European Patent Office
Prior art keywords
cmp
group
neuac
neu
amino
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.)
Withdrawn
Application number
EP91906420A
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German (de)
English (en)
Inventor
Reinhard Brossmer
Hans Jürgen GROSS
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Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0521941A1 publication Critical patent/EP0521941A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/815—Enzyme separation or purification by sorption
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/819—Multifunctional antigen or antibody

Definitions

  • the present invention relates to a new process for the production of CMP-activated, fluorescent or absorbent and new, CMP-activated fluorescent or absorbent sialic acids.
  • Sialic acids also known as acylneuraminic acids
  • Sialic acids are components of many bacterial and animal glycoproteins and glycolipids. With inflammation, tissue-destroying processes and certain tumor diseases, higher concentrations of sialic acids, in particular 5-N-acetylneuraminic acid, and increased activities of so-called sialyltransferases are also found in the blood plasma.
  • the latter physiologically incorporate sialic acid into glycoconjugates intracellularly; they are glycoprotein enzymes which have a defined specificity for the glycan sequence of the glycoconjugate acceptor in which the sialic acid is incorporated and for the glycosidic binding type in which the incorporated sialic acid is chemically bound.
  • the described method can be coupled with CMP.
  • the fluorescein-labeled CMP-sialic acid produced in this way proves to be superior to the known radioactive-labeled CMP-sialic acids as an indicator.
  • this compound as well as other previously unknown CMP-activated, fluorescent sialic acid derivatives, can also be prepared by first linking 9-amino-5-N-acetylneuraminic acid to CMP, as described in the literature (cf. briefly et al. Eur. J. Biochem. (1987) 168, 595-602). This reaction proceeds with a high yield (95%) and the cleaning of the end product is also comparatively simple.
  • another acyl group can also be present. Free 5-amino groups are not suitable because these compounds are not stable.
  • the product thus obtained can now be coupled with an activated fluorescent compound, for example with fluorescein isothiocyanate.
  • an activated fluorescent compound for example with fluorescein isothiocyanate.
  • This reaction too, surprisingly proceeds with a very high yield and comparatively simple purification of the end products, although it could be assumed that numerous side reactions would occur due to the large number of possible reactive centers and that there would be a considerable decomposition of the CMP glycosides.
  • this method can be used not only for fluorescein compounds, but also that practically all other fluorescent compounds which contain a hydroxyl, amino, carboxy, triazinyl or sulfonic acid group which can be coupled to one another by means of suitable activators or coupling compounds CMP-activated 9-amino N-acetylneuraminic acid could be bound.
  • biochemical activity ie the enzyme kinetic data
  • the fluorescent group is bonded not via the amino group in the 9-position but via an amino group in the 5-position of the N-acylneuraminic acid is, in the latter case first an Omega-amino-acyl group, for example ⁇ -aminoacetyl, is condensed as a spacer onto the 5-amino group.
  • the process according to the invention consists of a 5-acylamido-9-amino-3,5,9-trideoxy- ⁇ -D-glycero-D-galactononulosonic acid or 5-arainoacylamido-3,5-dideoxy- ⁇ -D - glycero-D-galacto-nonulosonic acid of the formula I (hereinafter referred to as 9-amino-5-N-acyl-Neu or 5-aminoacyl-Neu), of the formula I.
  • R 1 is an amino group and R 2 is an acyl group or R 1 is a hydroxy group or acylamino group and R 2 is an aminoacyl group with cytidine triphosphate (CTP) in the presence of a CMP-sialic acid synthase to form CMP-activated sialic acids of the formula II
  • Fl - Sp - X (III) in the Fl is a fluorescent compound
  • X denotes an activated carboxy or thiocarbonyl, triazinyl or sulfonic acid group to convert to CMP-activated, fluorescent sialic acids of the formula IV
  • R 3 represents the group Fl - Sp - X '- NH
  • R 4 is an acyl group
  • R 3 is a hydroxy or acylamino group
  • R4 denotes the group Fl - Sp X 'NH acyl
  • X ' represents a -CO-, -CS-, -SO 2 - or triazinyl group.
  • the activated acid group X is a group which chemically reacts with an amino group, either to one Amide, for example an ester, acid chloride, acid azide, or to a urea or thiourea, for example an isocyanate,
  • Isothiocyanate or to an aminotriazine e.g. a
  • Triazinyl dichloride group Suitable Triazinyl dichloride group. Appropriate reactions are well known for the formation of acid amides. Equivalent groups should therefore also be included.
  • the group Sp is predominantly a valence, since the absorbing or fluorescent compounds often contain a couplable group X 'directly on the chromophore.
  • a side chain in particular an alkyl chain with 1-10, preferably 2-6, carbon atoms can also be interposed.
  • a haloalkyl carboxylic acid or haloalkyl sulfonic acid such a group can easily be linked together with a coupling group X to a hydroxyl or amino group of the chromophore.
  • the starting substance for the synthesis of CMP-activated fluorescent neurarain acid analogs is CMP-9-amino-N-acetyl-neuraminic acid or CMP-5-aminoacetamido-neuraminic acid;
  • the reaction is carried out by linking the starting materials via the primary amino group at position C-9 or C-5 with various reactive fluorescent or absorbent substances which easily react with an amino function.
  • Activated substituents can exist as
  • Isothiocyanates isocyanates, as N-hydroxysuccinimide esters, p-nitrophenyl esters, sulfonic acid chlorides, as triazine chlorides or as acid azides.
  • the reaction takes place in a partially aqueous environment in the neutral or alkaline pH range (7.5 - 10; best the reaction takes place at pH 8.5 - 9) at 20 ° C or better 37 ° C, with the addition of organic solvent to solubilize the reactive substance (eg 30% - 80% methanol, DMF, DMSO, acetone or acetonitrile, or the like).
  • organic solvent eg 30% - 80% methanol, DMF, DMSO, acetone or acetonitrile, or the like.
  • the CMP glycoside starting material and the CMP-NeuAc analog formed remain stable in the pH range mentioned (less than 5% decomposition).
  • the excess of activated fluorescent agent can only be 2-fold in the case of isothiocyanates in order to achieve over 90% converted CMP glycoside; with N-hydroxysuccinimide esters it is better 3-5 times to achieve the same conversion; in principle, a 10-fold excess is beneficial for shortening the reaction time (for isothiocyanates generally 10 - 15 min at 37 ° C, for N-hydroxysuccinimide esters up to 20-30 min).
  • triazinyl dichloride as a reactive group, a higher excess (6 to 15 times) is recommended, the reaction time to complete conversion (90%) is at least 15-20 h at 37 ° C.
  • Uras rate of 90% and more so that the new method guarantees optimal yield.
  • the method is therefore economical, simple and time-saving, and enables a variety of CMP-NeuAc analogs to be prepared by the variety of possible, activated substituents.
  • the known instability of the CMP glycosides does not constitute an obstacle, since CMP-9-Amino-NeuAc in particular is surprisingly stable in the pH range described (pH 7.5 -9.5).
  • the new fluorescent or absorbent CMP glycosides were characterized by various methods after purification (see the following table).
  • the retention time in the analytical HPLC system differed from the CMP-9-Amino-NeuAc or CMP-5-Aminoacetamido-Neu, the absorption coefficient ( ⁇ ) of the new CMP glycosides in the HPLC system 275 nm was above 1.0 (based on ⁇ 275nm from CMP).
  • the new CMP glycosides were completely decomposed to CMP by mild acid hydrolysis (1N HCl, 45 min at RT) and subsequently determined by analytical HPLC (275 nm). After acidic hydrolysis, the peak of the released, fluorescent or absorbing NeuAc analog appears in the analytical HPLC system at 200 nm.
  • the new substrates according to the invention can be used for the following analytical methods:
  • the test is based on the incorporation of the fluorescent neuraminic acid derivatives from the corresponding CMP glycoside by a sialyltransferase into an acceptor, for example a glycoprotein or ganglioside, separation of the substituted and unsubstituted acceptor molecules from the excess of the reagents by gel filtration or precipitation and measurement of the acceptor-bound fluorescence by corresponding spectrometric method.
  • an acceptor for example a glycoprotein or ganglioside
  • a simple test normally requires 30 ⁇ l of reaction solution, or possibly up to only 10 ⁇ l of reaction solution.
  • This enzymatic reaction is carried out, for example, in a buffer with pH 6 or 6.5 (depending on the pH optimum of the sialyltransferase), with 0.1-10.0 mg / ml acceptor [(Asialo) glycoprotein or ganglioside] correspondingly 690-1,875 ⁇ M galactose or N-acetylgalactosamine acceptor sites, and 10-100 ⁇ M of the respective CMP-activated fluorescent N-acetylneuraminic acid reagents are added.
  • the reaction solution is generally kept at 37 ° C.
  • the extent of the reaction is quantified by means of a fluorescence measurement of the macromolecularly bound fluorescent neuraminic acid, and can optionally also be determined by means of a measurement of the unreacted fluorescent CMP glycoside.
  • test is also particularly suitable for measuring the low sialyltransferase activities in culture cell lines, surgical or biopsy material and in body fluids, in particular in blood plasma or serum.
  • the test also enables the creation of the enzyme kinetic data (Michaelis constant, V max ) for the respective acceptor.
  • V max enzyme kinetic data
  • the test allows for the first time a differentiation of two sialyltransferases, one with specificity for the N-linked terminal glycan sequence Gal ⁇ 1, 4GlcNAc, another for O-linked GalNAc residues.
  • the cell membranes With the indicators according to the invention in a simple manner, by allowing an appropriate reaction solution of the CMP-activated fluorescent sialic acids to act in the presence of sialin transferase, washing out the excess of the indicator and determining the surface properties of certain cells to be examined from the extent of the fluorescence of the isolated cells.
  • the cell can be selectively fluorescence-labeled in the glycan part without chemical heat treatment.
  • the measurement of surface-bound fluorescence is very simple in the flow cytometer, but can also be observed under the fluorescence microscope. IV.
  • the compounds according to the invention also prove to be advantageous if certain proteins, which are particularly sensitive in their spatial structure, are to be fluorescence-labeled in order to be able to follow them on the basis of fluorescence when used in a biological system.
  • Such labeling can be carried out relatively gently and completely enzymatically, leaves the actual amino acid sequence of the protein unmodified, takes place selectively in certain glycan sequences and makes it possible to easily separate the superfluous indicator substance again.
  • the procedure is basically the same as described under I.
  • CMP-9-TRITC-NeuAc this substance is practically only transferred from Gal ⁇ 1, 4GalNAc ⁇ 2, 6sialyltransferases to glycoproteins and could therefore be used for differentiation; also other excitation and emission (red-fluorescent).
  • V max / Kin is 6.5 times higher than for CMP-fluoresceinyl-NeuAc.
  • CMP-9-DAB-NeuAC excitation in the UV range; height
  • pH 9-9.5 e.g. 0.5 M NaHCO 3 / Na 2 CO 3
  • fluoresceinyl isothiocyanate is added so that pH 8.5-9 is reached.
  • the reaction of the amino group at C-9 of the NeuAc part of the CMP glycoside with the fluoresceinyl isothiocyanate is complete after 15 minutes at the latest (greater than 90%).
  • the reaction can be observed via analytical HPLC, the retention time of CMP-9-Amino-NeuAc is considerably shorter than that of the fluorescent CMP glycoside formed.
  • the resulting CMP-9-fluoresceinyl-NeuAc is purified by preparative HPLC and ethanol precipitation, as described (Groß and Brossner (1988), Eur. J.
  • the purification is carried out as described for CMP-9-Fluoresceinyl-NeuAc.

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  • Cell Biology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention concerne un procédé permettant de produire des acides sialiniques marqués par indicateur de fluorescence et activés par CMP de formule (IV) dans laquelle ou bien R3 représente le groupe Fl-Sp-X'-NH- et R4 représente un groupe acyle, ou bien R3 représente un groupe hydrolyle ou acylamino et R4 représente Fl-Sp-X'-NH-acyle, Fl représente un groupe fluorescent, Sp représente une valence ou un groupe d'espacement assurant le couplage et X' représente un groupe -CO-, -CS-, -SO2- ou triazynile. L'invention concerne également les nouveaux acides sialiniques marqués par indicateur de fluorescence et activés par CMP ainsi que leur utilisation pour a) la détermination de l'activité de sialyltransférases, b) la détermination de la spécificité de l'accepteur et la détermination des données cinétiques de l'accepteur, c) le marquage par fluorescence de surfaces de cellules, d) le marquage de glycoprotéines et de gangliosides.
EP91906420A 1990-03-26 1991-03-19 Acides sialiniques fluorescents actives par cmp et leur procede de production Withdrawn EP0521941A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4009630 1990-03-26
DE4009630A DE4009630C2 (de) 1990-03-26 1990-03-26 CMP-aktivierte fluoreszierende Sialinsäuren sowie Verfahren zu ihrer Herstellung

Publications (1)

Publication Number Publication Date
EP0521941A1 true EP0521941A1 (fr) 1993-01-13

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EP91906420A Withdrawn EP0521941A1 (fr) 1990-03-26 1991-03-19 Acides sialiniques fluorescents actives par cmp et leur procede de production

Country Status (5)

Country Link
US (1) US5405753A (fr)
EP (1) EP0521941A1 (fr)
JP (1) JPH05507191A (fr)
DE (1) DE4009630C2 (fr)
WO (1) WO1991014697A1 (fr)

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DE4009630C2 (de) 1995-09-28
US5405753A (en) 1995-04-11

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