WO1991010908A1 - Oligosaccharide derivates linked to lanthanide chelates and to a reactive group - Google Patents

Abstract

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 compounds of the invention have the structure Am - B - Cn, where B is an oligosaccharide, cyclic or non-cyclic, Am is an exactly known number of groups, same or different, covalently linked to B, capable of binding biologically interesting compounds, Cn is a plurality of lanthanide chelates, covalently linked to B, m is an integer 1 - 6 and n is an integer 2 - 40.

Description

Oligosaccharide derivates linked to lanthanide chelates and to a reactive group.

FIELD OF INVENTION

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 nόn- 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. Moreover, 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

In im unoassays time-resolved fluorescence spectroscopy using monomeric lanthanide chelates is well known (e.g. E.Soini et al. , U.S.Pat. 4,374,120; I.Hemmila et al. , U.S.Pat. 4,565,790; H.Mikola et al., U.S.Pat. 4,808,541; R.Evangelista et al., EPA 171 978; G.Mathis et al., French Pat. Appl. 2 570 703). In competitive fluoroimmunoassays only one chelate is usually coupled to the hapten molecule (G.Barnard et al., Clin.Chem. 3_.5, 555 (1989); I.Hemmila et al., Clin.Chem., 34_., 2320 (1988); G.Barnard et al. ,

J.Biolumin.Chemilumin. , 4., 177 (1989)) . Because of the very low concentration of some haptens (e.g. many steroids, vitamin B12 and free thyroid hormones) the use of one chelate per hapten limits the sensitivity of the immunoassay. In noncompetitive fluoroimmunoassays (e.g. M.Suonpaa et al. , Clin.Chem. Acta, 145, 341 (1985); H.Siitari et al., Nature, 301. (5897) , 258 (1983)) labelled antibodies (antigens) usually contain 1-20 monomeric chelates per molecule. 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.

Some papers have been published discussing experiments where more than one detectable group has been coupled to a carrier molecule which also contains the molecule (e.g. hapten) to be determined by appropriate assay. In all of these experiments a polymer has been used as the carrier molecule. Usually this polymer is a protein or a polyamino acid (e.g. G.Rowley et al. , Clin.Chem., 33_, 1563 (1987); Y.Manabe et al., Biochi .Biophys.Acta, 883, 460 (1987); E.Reichstein et al., Anal.Chem., 6O, 1069 (1988); A.Oser et al. , Nucleic Acids Res., 16_, 1181 (1988); D.Exley et al. , J.Steroid Biochemistry, 14_., 1297 (1981)) . Some other polymers have also been used (e.g. T.Hirschfeld et al., Applied Optics, 15, 2965 (1976)) . The disadvantage of all these methods is the inaccurate number of both biologically active compounds and detectable groups. Hence it follows that there are three variables with different deviations, i.e. the molecular weight of the polymer, the number of detectable groups and the number of biologically active compounds.

The deviation of one parameter can be controlled by using an oligomer with exact length. Examples of 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)) .

The labelling of sugar moieties with detectable groups has been described in several articles (e.g. T.T.Ngo, Biotechnology, 4_, 134 (1986) ; Excited States of Proteins and Nucleic Acids, ed. by R.F.Steiner and I.Weinryb, London 1971, 200-213; A.N. De Belder, Carbohydrate Research, 30_.,

375 (1973); M.Wilchek et al. , Applied Biochemistry and Bio¬ technology, 11_., 191 (1985); M.Wilchek et al. , Methods in Enzymology, 138, 429 (1987)) .

From the above mentioned considerations it follows that the compound to be used in the immunoassay will be much more homogeneous than molecules based on polymers. Moreover, when we have a more exact chemical structure, the variation from batch to batch can be limited. THE INVENTION

As neutral molecules 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) . 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.

The compounds of the invention have the common structure given in formula I.

Am - B - Cn Formula I parent compound

In formula I, m is an integer 1-6, preferably 1 and n is an integer 2-40.

In the parent compound B is an oligosaccharide, cyclic or non-cyclic, having an exactly known number, one or more, of groups A, same or different, which are capable of binding biologically interesting compounds, covalently linked to B, and also a plurality of analytically indicatable group C, same or different lanthanide chelate, covalently linked to B. 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. This type of biologically active molecules are often called target substances (target molecules) . Usually 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. The term "inert" above 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.

E may contain at least one structural element selected from among the following: -NR- (secondary and tertiary amine) , -CONR- and -NRCO- (substituted amide) , -S-S- (aliphatic disulfide) , -S- (aliphatic thioether) , -0- (ether) , -COO- and -OOC- (ester) , -N=N- (diaza) and pure hydrocarbon chain which may be straight, branched or cyclic and contain from 1 to 12 carbon atoms. 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. 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

b) chemically convert one of the groups of the pair to a group of the opposite type. An example of the latter case is the activation with bifunctional coupling reagents (also called activation reagents) .

If F is nucleophilic and D' ' electrophilic or vice versa these two 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) .

Examples of 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. Other functional groups are familiar to those skilled in the art.

In a compound according to the invention it is imperative that all the groups mentioned can coexist. However, the reactive group F does not necessarily have to coexist with a chelating group of a lanthanide chelate of the invention. For some purposes 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

(1981)) . 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 α- or/3 -glycosidic bonds. That is, glycosidic bonds may be formed by condensation between α- 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.

Examples of suitable oligosaccharides are α-, 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. Good examples of 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.

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 1. Europium chelate of N'-[p-(dichlorotriazinyl- amino)benzyl]diethylenetriamine-N* ,N' ' ,N' ' ' ,N" ' '-tetraacetic acid (2) .

The preparation of europium chelate of N'- (p-aminobenzyl) - diethylenetriamine-N' ,N* ' ,N' ' ' ,N' ' '-tetraacetic acid (1) has been described earlier (U.S.Pat. 4,808,541) . Compound 1 (0.2 m ol) was dissolved in destilled water (4 ml) and cooled to 0°C. An equivalent amount of trichlorotriazine dissolved in acetone (2 ml, cooled) was dropped vigorously stirring into the mixture so that pH was kept at 6.0-7.5 by adding 5 M

NaOH. Compound 2 was precipitated with acetone and dried in a vacuum dessiccator.

Example 2. Labelling of 4-nitrophenyl-α-D-hepta- (l-*4) - glucopyranoside (3) with compound 2.

Compound 3 (30 μmol) dissolved in destilled water (1 ml) was stirred together with compound 2. The mixture was stirred at room temperature and pH was adjusted to 10 with 5 M NaOH. The reaction was monitored by TLC using ethyl acetate/ace¬ tic acid/methanol/water (6/3/3/2 by vol.) as eluent. The reaction was over after 3 h. The product (4) was purified by fractional precipitation with ethanol. The purity was checked by gel filtration chromatography (Sephadex G 15, Pharmacia) and fluoro eter (Arcus' , Pharmacia Wallac) . Example 3. Reduction of the nitro group of compound 4.

Compound 4 (25 μmol) was dissolved in destilled water (1 ml) and nitrogen was passed through the solution (5 in) . Palladium on carbon (30 g, 10% Pd) was added and hydrogen was passed through the mixture for 3 h. After centrifugation the product (5) was precipitated with ethanol .

Example 4. Conversion of the amino derivative (5) to the corresponding isothiocyanate (6) .

Compound 5 (25 μmol) dissolved in buffer-ethanol mixture (1 ml, O.i M phosphate, pH 7.5 , 0.1 M NaCl and 0.5 ml ethanol) was cooled to 0°C. Thiophosgene (10 μl) dissolved in acetone (0.5 ml) was dropped in vigorously stirring and adjusting pH to 7.5. After 30 min the mixture was extracted 3 times with diethylether. The product (6) was precipitated from ethanol and dried in a vacuum dessiccator.

Example 5. Coupling of isothiocyanate (6) to DNA containing amino groups to get compound 7, where biologically active compound is λDNA.

Lambdaphage DNA (λDNA) purchased from Pharmacia was transa inated as taught by R.Viscidi et al .

(J.Clin.Microbiol. 23_. (2) 1986, 311-317) . 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 λDNA (75 μg in 75 μl H2O) was denaturated by boiling for 10 min. λDNA solution was cooled on ice and 8.0 mg (1.2 μmol) of the isothiocyanate analog of europium labelled maltoheptose (6) was added. The reaction mixture was brought to pH 9.8 by adding 8 μl of 1 M NaHCOs , pH 9.8. The reaction was allowed to proceed for 18 hours at room temperature.

Purification of the labelled DNA was done in Sephacryl S-400 column (0.7 x 48 cm) equilibrated and run in 10 niM Tris-HCl pH 7.5, 0.1 M NaCl. Fractions coming in the void volume contained the europium labelled λDNA.

Example 6. An alternative method of synthesis of compound 7, where biologically active compound is λDNA.

Lambdaphage DNA (ΛDNA) 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-α-D-hepta-(1→4) - glucopyranoside (3) as starting material.

Transaminated λDNA, which contains primary aliphatic amino groups, was denatured (100°C, 10 min.) and then reacted with compound 9. Fifty μg of transaminated λDNA was mixed with 110 μg 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.

Subsequently λDNA with maltoheptose groups covalently attached and unreacted maltoheptose were reacted with compound 2. Three mg of compound 2 was dissolved in the reaction mixture from the first step. After overnight reaction Eu-labelled λDNA was purified from unreacted chelate and free Eu-labelled maltoheptose in a Sephacryl S- 400 (Pharmacia) column equilibrated and eluted with 10 mM Tris-HCl, pH 7.5, 0.1 M sodium chloride.

Example 7. Synthesis of compound 7, where biologically active compound is progesterone.

Coupling of isothiocyanate (6) to progesterone containing an amino group was performed analogically to example 5.

Claims

1. Compound having the structure

Am — B — Cu ,

where B is an oligosaccharide, cyclic or non-cyclic, Am is an exactly known number of groups, same or different, covalently linked to B, capable of binding biologically interesting compounds, Cn is a plurality of lanthanide chelates, covalently linked to B, is an integer 1 - 6 and n is an integer 2 - 40.

2. The compound of claim 1, characterized in that A- is an alkyl, an aryl, an alkylaryl or an arylalkyl group or a group containing aryl and alkylene parts in each of which the alkylene part contains 2 to 8 carbon atoms or from 0 to 4 other atoms such as 0, S, N and P and each of the mentioned groups contain amino, aminoxy, carboxyl, hydroxy or ercapto groups or an activated form made from them such as isothiocyanate, isocyanate, active esters, pyridyldithio for the binding to said biologically interesting compounds.

3. The compound of claim 1, characterized in that B is an oligosaccharide, cyclic or noncyclic, having 2 - 40 saccharide parts, same or different.

4. The compound of claim 1, characterized in that C is a fluorescent or nonfluorescent lanthanide chelate where the lanthanide is Eu, Tb, Dy, S or Gd.

5. The compound of claim 3 characterized in that the oligosaccharide B is cyclodextrin.

6. The compound of claim 3 characterized in that the oligosaccharide B is oligomaltose.

7. The compound of claim 1 characterized in that B is maltoheptose, A is p-aminophenyl, m is one and C is europium or terbium chelate.

8. The compound of any of claims 1 - 7 characterized in that the biologically interesting compound is an antigen (hapten) or a drug or a protein such as enzyme, receptor, antibody, protein A and IgG or oligonucleo- tide or DNA or RNA or lectins or a carbohydrate such as dextran.

9. The use of the compound in any of claims 1 - 3 in the time-resolved fluoroim unoassay.

10. The use of any compound in any of claims 1 - 8 in the nucleic acid hybridization based assays.