EP0511201A1 - Derives d'oligosaccharide relies a des chelates de lanthanides et a un groupe reactif - Google Patents

Derives d'oligosaccharide relies a des chelates de lanthanides et a un groupe reactif

Info

Publication number
EP0511201A1
EP0511201A1 EP19900901604 EP90901604A EP0511201A1 EP 0511201 A1 EP0511201 A1 EP 0511201A1 EP 19900901604 EP19900901604 EP 19900901604 EP 90901604 A EP90901604 A EP 90901604A EP 0511201 A1 EP0511201 A1 EP 0511201A1
Authority
EP
European Patent Office
Prior art keywords
compound
oligosaccharide
groups
cyclic
lanthanide
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Withdrawn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
EP19900901604
Other languages
German (de)
English (en)
Inventor
Veli-Matti Mukkala
Harri Takalo
Pertti Hurskainen
Jyrki Ylikoski
Jouko Kankare
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.)
Wallac Oy
Original Assignee
Wallac Oy
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 Wallac Oy filed Critical Wallac Oy
Publication of EP0511201A1 publication Critical patent/EP0511201A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages

Definitions

  • Oligosaccharide derivates linked to lanthanide chelates and to a reactive group.
  • This invention pertains to water soluble oligosaccharide which is covalently linked to several lanthanide chelates and to a reactive group or groups through which the entire molecule can be introduced by covalent binding to biologically interesting compounds.
  • the fluorescent or non-fluorescent macromolecules of our invention are useful as probes in time-resolved fluorescence spectroscopy giving a better sensitivity than compounds containing only one luminescent group. Besides, when n ⁇ n- fluorescent lanthanide chelates are used, there is no concentration quenching as in cases of other fluorophores. Contrary to other known macromolecules which are based on polymeric materials as carriers of luminescent groups, the small-size macromolecules of this invention involve advantages of better binding to antigens or antibodies because the covalent binding becomes more difficult as the size of the macromolecule is increased i.e. less steric hindrance.
  • the macromolecules of the invention contain a known number, preferably one, reactive group for covalent coupling to biologically interesting compounds, i.e. there is an exact specific binding site and a chemical structure or a concise molecular weight distribution in those macromolecules. This implies that they have some very important extra advantages over polymeric macromolecules. There are not so many possibilities for side reaction like cross-linking as in the case of polymers. Besides, a high analytical sensitivity (i.e. the number of lanthanide chelates in the macromolecules of the present invention) and the biological activity of the antibodies (antigens) and nucleic acids are not decreased. This is due to the fact that only a few reactive groups in the antibodies (antigens) and nucleic acids are labelled with the compounds. DESCRIPTION OF THE PRIOR ART
  • the number of chelates coupled to antibodies (antigens) has to be adjusted to the level which preserves the biological activity of antibodies (antigens) and on the other hand makes sensitive immunoassays possible.
  • the compromise in labelling with chelates results in inadequate sensitivity in instances where the amount of the biological molecule to be measured is extremely low.
  • the deviation of one parameter can be controlled by using an oligomer with exact length.
  • suitable oligomers are e.g. cyclodextrines and reducing sugars such as • maltoheptose and inulin.
  • the deviation of the number of biologically active compounds can be controlled by having only one reactive spacer because then exactly one biologically active compound can be coupled to the carrier molecule.
  • the coupling of only one spacer to an oligosaccharide is well known art (e.g. E.Kallin et al . , Glycoconjugate J. , 3., 311 (1986); H.Essien, J.Med.Chem., 31 . ' 898, (1988); I.Tabushi et al. , J.A .Chem.Soc. , 7100 (1977); F.Nanjo et al., J.Carbohydrate Chemistry, 1_, 67 (1988)) .
  • oligosaccharides demonstrate very low unspecific binding to antibodies. As being very soluble in water they make highly aromatic chelates more hydrophilic. Moreover, the labelled compounds can be more easily purified because there is less adsorption to column material and plastics. In immunoassays oligosaccharides also decrease unspecific binding to a solid phase.
  • the macromolecules of our invention would also have applications in other methods apart from immunoassay, e.g. in fluorescence microscopy. Because of the paramagnetic properties of lanthanides, these compounds would also be useful as sensitive probes in magnetic resonance imaging (MRI) .
  • MRI magnetic resonance imaging
  • the radioactive isotopes of the metals such as indium and stable chelating ligands on the macromolecules offer a possibility to use these compounds in treatment of diseases like cancer.
  • n is an integer 2-40.
  • A represents a group, which is capable of binding biologically interesting compounds (D) such as antigens (haptens) , the homologous antibody active components, drugs, proteins (such as enzymes) , receptors, antibodies, oligonucleotides, nucleic acids (RNA, DNA) , lectins, carbohydrate structures (such as dextran) , protein A and IgG etc.
  • D biologically interesting compounds
  • A can be divided into an inert and stable bridge (E) and a functional group (F) or a residue of a biologically interesting compound (D') after it has been coupled covalently to the parent compound.
  • inert means that the group or bridge characterized by this adjective does not have any efficient chelating heteroatom closer than at a distance of four atoms from the heteroatoms participating in the chelation of a joint metal ion. In actual practice this means that the four atoms are normally represented by a four-carbon chain.
  • “Stable” means that the bridge E does not deteriorate when the compounds of the invention are used, for instance the bridge does not easily undergo hydrolysis.
  • the carbon chain may be purely aliphatic or purely aromatic (including phenyl, naphthyl, quinolyl , pyridyl and bipyridyl) , and other inert functional groups not participating in the chelation mentioned above.
  • the symbol R in the substituted amide above represents prefera ⁇ bly hydrogen but may be alkyl , for instance an alkyl having less than 5 carbon atoms.
  • F may be a functional group so selected that it can be made to react chemically with a functionally group of a biologically interesting compound (D' 1 ) so as to form a covalent linkage between D and a parent compound of formula I.
  • D' 1 a biologically interesting compound
  • the selection of F depends on D' ' and vice versa, but it is believed that any artisan can make the proper selection of mutually reactive groups.
  • F and D' 1 may be selected from among electrophilic and nucleophilic groups. If they are a pair of electrophilic groups or a pair of nucleophilic groups, it is possible for instance to
  • a) employ oxidative coupling for forming the bond (e.g. -SH + HS- S-S-) or
  • nucleophilic groups can usually be reacted with each other without any preceding activation.
  • Most nucleophilic groups comprise a heteroato having an electron pair available for reaction with an electron deficient atom (electrophilic group) .
  • Suitable functional groups include isothiocyanato, bro oacetamido, iodoacetamido, succinamido, pyridyldithio, mercapto, carboxyl and its active esters (e.g. N-hydroxy-succinimido or p-nitrophenyl) , hydroxyl, aldehyde, amino, diazonium, tosyl, mesytylyl, trexyl, phosphodiester or phosphotriester.
  • active esters e.g. N-hydroxy-succinimido or p-nitrophenyl
  • Other functional groups are familiar to those skilled in the art.
  • the reactive group F does not necessarily have to coexist with a chelating group of a lanthanide chelate of the invention.
  • the chelating part of the molecule may be temporarily protected e.g. in the form of an ester so that the protected ligand will be coupled to the target molecule, and after deblocking may finally form the desired labelled product.
  • the protective group is chosen in accordance with know principles (see for instance Protective Groups in Organic Synthesis; Greene TN; John Wiley & Sons Inc; USA
  • Factors to be taken into account when the group is chosen are inter alia the stability of the compound with which F is to be reacted, the reactivity of F and the type of structure formed upon reaction of F and the compound intended.
  • B represents a cyclic or noncyclic branched or nonbranched oligosaccharide having 2-40 same or different monosaccharide units. These units can be linked by ⁇ - or/3 -glycosidic bonds. That is, glycosidic bonds may be formed by condensation between ⁇ - or 3-hydroxyls of the anomeric carbon of one monosaccharide unit and alcoholic hydroxyl groups at carbon 2, 3, 4 or 6 of a second unit. The preferred bonds are 1,4 and 1,6.
  • the oligosaccharide may be branched so that two units have glycosidic linkages to different alcoholic hydroxyls of a third unit.
  • the most common monosaccharides are glucose, fructose, galactose, mannose, arabinose and xylose. Also modified derivatives of the above mentioned monosaccharides such as amino- and carboxylmethylated sugars can be used.
  • oligosaccharides are ⁇ -, 3- or Y ⁇ cyclodextrines, maltopentaose, maltohexaose, maltoheptaose, stachyose, verbascose and inulin.
  • C represents a fluorescent or non-fluorescent lanthanide chelate where the lanthanide is Eu, Tb, Dy, Sm and Gd.
  • suitable chelates have been mentioned e.g. in H.Mikola et al. , U.S.Pat. 4,808,541; J.Kankare et al. EP- A-203,047; J.Kankare et al. Swedish Pat. Appl. SE 8802575-4; M.Kwiatkowski et al. Swedish Pat. Appl. SE 8702824-7.
  • fig. 1 One possible way to activate lanthanide chelate is shown in fig. 1 in the scheme "synthesis of compound 2".
  • the other structures and synthetic routes employed in the experimental part are shown in fig. 2 in the general scheme.
  • the general scheme shows two alternative pathways of synthesis of wanted products, i.e. first labelling oligosaccharide with lanthanide chelate and then coupling the new molecule to a biologically active compound or vice versa.
  • the labelling of oligosaccharide can also be performed by two different methods i.e. either by activating first the oligosaccharide or the lanthanide chelate.
  • Example 5 Coupling of isothiocyanate (6) to DNA containing amino groups to get compound 7, where biologically active compound is ⁇ DNA.
  • Lambdaphage DNA purchased from Pharmacia was transa inated as taught by R.Viscidi et al .
  • the transamination reaction yields DNA which contains aliphatic amino groups attached to cytosine residues.
  • the conditions during transamination were adjusted so that 5-6 % of all nucleotides (about 20-25 % of cytosine residues) in J ⁇ DNA were modified.
  • Transaminated DNA was purified by dialysis.
  • Transaminated ⁇ DNA (75 ⁇ g in 75 ⁇ l H2O) was denaturated by boiling for 10 min. ⁇ DNA solution was cooled on ice and 8.0 mg (1.2 ⁇ mol) of the isothiocyanate analog of europium labelled maltoheptose (6) was added. The reaction mixture was brought to pH 9.8 by adding 8 ⁇ l of 1 M NaHCOs , pH 9.8. The reaction was allowed to proceed for 18 hours at room temperature.
  • Example 6 An alternative method of synthesis of compound 7, where biologically active compound is ⁇ DNA.
  • Lambdaphage DNA ( ⁇ DNA) was transaminated as described in example 5.
  • Isothiocyanate-derivative of maltoheptose was synthesized according to the procedure described in the examples 3 and 4 using 4-nitrophenyl- ⁇ -D-hepta-(1 ⁇ 4) - glucopyranoside (3) as starting material.
  • Transaminated ⁇ DNA which contains primary aliphatic amino groups, was denatured (100°C, 10 min.) and then reacted with compound 9. Fifty ⁇ g of transaminated ⁇ DNA was mixed with 110 ⁇ g of compound 9 and 1 M sodium carbonate, pH 9.8, was added to give the final concentration of 0.1 M. The reaction was allowed to proceed overnight at room temperature.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Saccharide Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention se rapporte à un oligosaccharide hydrosoluble, qui est lié par covalence à plusieurs chélates de lanthanides et à un ou à des groupes réactifs, par l'intermédiaire desquels toute la molécule peut être introduite par liaison covalente dans des composés biologiquement intéressants. Les composés décrits dans l'invention se caractérisent par la structure Am - B - Cn, où B représente un oligosaccharide, cyclique ou non cyclique, Am représente un nombre exactement connu de groupes identiques, ou différents, reliés par covalence à B et capables de se lier à des composés biologiquement intéressants, Cn représente une pluralité de chélates de lanthanides, reliés par covalence à B, m est égal à un nombre entier compris entre 1 et 6 et n est égal à un nombre entier compris entre 2 et 40.
EP19900901604 1990-01-17 1990-01-17 Derives d'oligosaccharide relies a des chelates de lanthanides et a un groupe reactif Withdrawn EP0511201A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/FI1990/000020 WO1991010908A1 (fr) 1990-01-17 1990-01-17 Derives d'oligosaccharide relies a des chelates de lanthanides et a un groupe reactif

Publications (1)

Publication Number Publication Date
EP0511201A1 true EP0511201A1 (fr) 1992-11-04

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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4210970C2 (de) * 1992-04-02 1996-10-17 Markus Dipl Chem Sauer Verfahren zur simultanen optischen qualitativen und quantitativen Erfassung von verschiedenen mit Fluorochromen oder Fluorogenen markierten Molekülen eines Gemisches mittels Laserspektroskopie
US5792330A (en) * 1995-05-31 1998-08-11 Board Of Regents, The University Of Texas System Lanthanide metal cations for concurrent detection and separation in capillary electrophoresis

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4735907A (en) * 1985-03-18 1988-04-05 Eastman Kodak Company Stabilized fluorescent rare earth labels and labeled physiologically reactive species
US4719182A (en) * 1985-03-18 1988-01-12 Eastman Kodak Company Fluorescent labels and labeled species and their use in analytical elements and determinations
SE454781B (sv) * 1986-10-17 1988-05-30 Wallac Oy Hybridiseringsforfarande for detektion av polynukleotidsekvens
SE8703682L (sv) * 1987-09-24 1989-03-25 Wallac Oy Homogen bestaemningsmetod som utnyttjar affinitetsreaktioner

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9110908A1 *

Also Published As

Publication number Publication date
WO1991010908A1 (fr) 1991-07-25

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