MXPA99009189A - Trans-platinum compound, and diagnostic kit - Google Patents

Abstract

The present invention is concerned with a trans-platinum based compound for use in a method for labelling a bio-organic molecule, having formula (I), wherein A represents a reactive moiety for attachment to a label, D represents a reactive moiety for attachment to a bio-organic molecule, and X1 and X2 represent the same or different inert moieties.

Description

COMPOSITE OF TRANS-PLATINUM AND DIAGNOSTIC EQUIPMENT The invention relates to a compound based on trans-platinum, to a diagnostic equipment comprising the compound, and to a method for labeling a bio-organic molecule wherein the compound is used. Platinum (coordination) compounds have been considered interesting molecules for a very long time. For a review of these compounds and their uses reference is made to Reedijk et al.

(Structure and Bonding, 67, pp. 53-89, 1987).

Especially cis-platinum has received a lot of attention as an anti-tumor drug. This anti-tumor reactivity of the platinum compounds originates from its having at least two reactive groups (prefer cis-oriented towards each), which makes it possible to crosslink the DNA molecules, thereby inhibiting the replication of these DNA molecules. A different use of the cis-platinum compounds has been described in the European patent application No. 95201197.1. In the present, a method is described for linking the bio-organic molecules and the marks through the cis-platinum compounds, of which two coordination sites are occupied by two REF. 31549 ends of a stabilization bridge, such as a group of ethylene diamine. These known cis-platinum compounds are suit for binding the labels to various classes of bio-organic molecules. Examples are (poly) -peptides, polynucleic acids and nucleotides. The labeling of polynucleic acids, such as DNA molecules, is desir for applications in fields such as recombinant DNA technology. However, in many cases it is desired not to label the nucleic acid macromolecule, but to mark a certain nucleotide and to enzymatically construct this in a polynucleic acid. This way, the location of the tag in the resulting polynucleic acid can be influenced, which is not possible when a tag binds to the macromolecule, however, it has been found that the nucleotides bound to a tag by the linkers of cis-platinum do not form satisfactorily in the DNA molecules if it is by the DNA polymerase, therefore, in order to use a cis-platinum linker to obtain a labeled DNA molecule, the macromolecule must be marked. Avail alternatives for marking a nucleotide, which can be incorporated into a polynucleic acid, are the most conventional methods of labeling, however, these conventional methods also have a major disadvantage when they are not suit for marking any nucleotide. for example when only a few residues of a certain nucleotide are present in a certain polynucleic acid, or when the terminal nucleotide residue of a polynucleic acid must be marked, it is desired to be to mark any nucleotide. An example of such a conventional method has been described by Dale et al., Biochemistry, 14, (1975), 2447-2457, a method involving covalent, direct binding as a labeling technique. Dale and colleagues report that cytosine and uracil can be mercurized in their C5 position under mild conditions. However, they also report that negative results were obtained for baseline adenine, thymine and guanine. In this way, there is a need for a universal link system, which is excellent for linking bio-organic tags and molecules, including all different nucleotides, and which also makes it possible to form enzymes in any nucleotide labeled through the linkage system in a polynucleotide acid in an efficient manner.

The present invention provides such a universal link system. It has been found that a compound based on trans-platinum, has the formula: A ^ / X1 Pt (I) X2 D wherein A represents a reactive portion for the binding of a tag, D represents a reactive portion for binding to a bio-organic molecule, and XI and X2 represent the same or different inert portions, meet the above requirements for use in a method for "labeling a bio-organic molecule." Surprisingly, the linkage of the labels to various classes of bio-organic molecules through the The use of the compound of the invention is at least as efficient as the known methods In addition, it has been found that nucleotides linked to a tag by the compound of the invention can be formed within the polynucleotides very efficiently. Two conformations: the syn conformation and the anti conformation The definitions of these conformations can be found in Saenger, "Principles of Nucleic Acid Structure", Springer-Verlag Inc., 1984. It is believed that for a_ nucleotide to be satisfactorily formed within , for example a DNA molecule, has to be in an anti-configuration In the US patent application No. 4,711,955 it has been reported that alteration of the anti normal conformation of a nucleoside should be avoided, otherwise because the nucleotide derivatives are unacceptable as polymerase substrates. In this way, if the nucleotides have a syn conformation they will not be incorporated enzymatically into a polynucleotide in an efficient manner. In addition to this, it is known from the publication mentioned above by Saenger that the presence of a bulky or substituent group at the N7 position of a nucleotide, i.e. the position to which a metal coordinates most easily, induces a nucleotide to have a syn conformation In this way, it would be expected that a nucleotide having a platinum compound coordinated to its N7 position would be in a syn orientation. Accordingly, it would be expected that it is not possible to incorporate the nucleotide into a polynucleotide with good results.

Therefore, it is very surprising that nucleotides linked to a tag by a trans-platinum-based compound according to the invention can be formed within the plinucleotides in a highly efficient manner. Portions XI and X2 in formula (I) are inert. This means that these portions are ligands that remain attached to the platinum core during the labeling reactions, that is, they are stable under physiological conditions. These can be different or the same and can be any group that does not show interference reactions between them and the brand, the bio-organic molecule or any other compound that is used in the application of marking techniques. In these portions XI and X2 lies another advantage of the invention compared to the cis-platinum linkers. In order to obtain a stable cis-platinum compound it is imperative that the compound comprises a stabilization bridge. For transplatin compounds such stabilization bridge is not necessary. Accordingly, possible variations in the structure for the trans-platinum compounds of the invention are more numerous. Thus, according to the invention, the skilled person has many instruments in this arrangement to obtain the desired properties of the compound. In a preferred embodiment, the XI and X2 are the same or different non-salient ligands. This means, that these are groups which have a very small outgoing group character. The most preferred examples thereof are the groups NH3, NH2R, NHRR ', and NRR'R ", wherein R, R' and R" represent either an alkyl group having from 1 to 6 carbon atoms, when these groups form very stable bonds for the platinum core. The particularly preferred embodiment is wherein XI and X2 are both a group N (CH3) 3. The presence of the methyl groups has a positive effect in that they prevent the conformational modifications of the substrate due to, for example, the hydrogen bonding of a brand or a bio-organic molecule to the inert portions. The reactive portions A and D are selected such that they are easily replaced by or substituted with a label, respectively a bio-organic molecule. It is preferred that A and D are selected from the group of acetate, N03 ~, HC03 ~ ^ C032_, S03 ~, Cl ", I" and other halogens. These groups are good outgoing groups and therefore are easily replaced.

In a highly preferred embodiment, a spacer is used, such as an oligolysin or a polylysine. According to this embodiment, A and / or D in formula (I) comprises a "separator, separator comprising a chain having at least four atoms, a chain comprising an electron donor portion at one end and a reactive portion at the other end, where the chain is attached to the platinum through the electron donor portion.The separator can bind a bio-organic mark or molecule by a reaction with the end of the chain which is the most distant end The use of a separator has the advantage that the distance between the mark and the bio-organic molecule can be optimized, so that the spherical factors can not be an obstacle to an efficient labeling reaction. The electrons of the separator are preferably comprised of an amine group or a thiolate anion, because it has been found that these groups are capable of forming very strong bonds for platinum. and preferred, the chain of the separator comprises at least one heteroatom, preferably an oxygen atom, as this positively produces the hydrophilicity of the chain, which is an important aspect in the phiosiological systems. It is also possible to use a separator comprising a platinum atom. In this embodiment, the invention provides a bis-platinum compound for binding the labels and the bio-organic molecules. The second platinum can have an orientation either cis or trans. Preferentially, the separator comprises no more than 20 carbon atoms in the chain, which is preferably an unbranched chain, which does not cause in this way a steric hindrance. The reasons for this will be clear. A highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, in the present referred to as Dadoo. Dadoo is a very flexible separator which has two primary amine groups and a size which makes it very suitable for use as a separator according to the invention. The labels that "can be linked to a bio-organic molecule by the compound of the invention are not critical." In principle, all the labels that can be attached to a bio-organic molecule and used to date can be used. These labels may be radioactive labels, enzymes (which require reaction with a substrate to be detected), components of specific binding pairs such as avidin, streptavidin or biotin, biocytin, iminobiotin, colloidal dye substrates, fluorochromes (rhodamine, etc.) including Cy® dyes, reducing substances (eosin, erythrosin, etc.), lasoles (with color), digoxigenin, metals (ruthenium), metal sols or other particulate sols (selenium, carbon and the like), dansyl lysine , Infrared Dyes, coumarins (amino methyl coumarin), antibodies, protein A, protein G, etc. The invention has greater benefits with the bulkier brands such as biotin, avidin, streptavidin and digoxy genina Cy® dyes are preferred labels to be linked to bio-organic molecules by the compound of the invention. These marks can be used very appropriately for a technique referred to as multi-color marking. The reason for this is that different dyes of this kind, while having different colors, are very similar in chemical structure. Almost every bioorganic molecule which contains an accessible sulfur or nitrogen atom can be linked to a label by the present compounds. The very suitable compounds to be labeled are proteins, peptides, DNA molecules, RNA molecules, and (oligo) -nucleotides. - Platinum binds very easily to the N7 position of nucleotides. This way, the DNA or RNA molecules are single stranded or otherwise can be easily detected. It also allows the production of assays for hybridization techniques where unlabeled DNA / RNA molecules hydridize the labeled assay. If the platinum compounds do little interference with hybridization. The platinum compounds of the invention are also very suitable for the binding of bio-organic molecules to solid surfaces such as micellulose, nylon filters or microtiter plates. Modified nucleotides by using a linker according to the invention and oligo- and polynucleotides in which the nucleotides have been formed, or oligo- and polynucleotides that have been directly modified using these novel platinum compounds can be used as assays in the biomedical research, clinical diagnostics and recombinant DNA technology.

The wide variety of utilities are based on the ability of platinum compounds to form stable complexes with polypeptides which in turn can be detected either by means of the detectable portions which bind to or interact with the polypeptide. Some -use include detecting and identifying the etiological agents that contain nucleic acid, for example bacteria and viruses, select bacteria for resistance to antibiotics, select animals for genetic disorders in relation to pharmaceutical effects, diagnose genetic disorders, for example trisomy 21, sickle cell anemia: chromosomal karyotype, and identify tumor cells The invention also encompasses a diagnostic kit for detecting, determining and / or locating the biological substances of interest, comprising the platinum compound of the invention, optionally together with another medium suitable for detection. Platinum in the team can comprise one or more of a spacer, a brand and a bio-organic molecule. Of course, the invention also encompasses a device, wherein these components are present separately, that is to say in unbound form.

In a different embodiment, the invention is directed to a method for labeling bio-organic molecules wherein the compounds described hereinabove are used. In a method for labeling a bio-organic molecule according to this embodiment, a reactive portion of a compound based on trans-platinum, having the formula: A XI \ / Pt \ íl) X2 D wherein A, D, XI and X2 have the above meanings, is reacted with a mark; and the other reactive portion of the compound based on trans-platinum is reacted with a bio-organic molecule or vice versa. In a preferred method for labeling a bio-organic molecule, the label and / or bio-organic molecule are connected to the platinum through a separator, separator comprising a chain having at least four atoms, chain comprising a portion electron donor at one end and a reactive portion at the other end, where the chain binds to platinum through the electron donating portion. This separator can be any of the separators described hereinabove. Of course, the separator (s), the brand, the bio-organic molecule and the trans-platinum can be linked together in any order. For example, the separator (s) can first be bound to the platinum followed by - reacting the compound obtained with the brand and the bio-organic molecule, but it is also possible to first join the separator (s) to the brand and / or bio-organic molecule before the reaction with the platinum compound. In a highly preferred embodiment, the invention provides a method as described above wherein the bio-organic molecule is a nucleotide. The invention will be elucidated by the following non-restrictive examples.

EXAMPLE Preparation of trans-diaminodichloro-platinum (II) (trans- [Pt (NH3) 2C12] One gram of potassium tetrachloroplatinate was dissolved in 20 ml of Milli-Q and 0.6 ml of concentrated HCl was added. The mixture was heated to boiling and 2.5 ml of a 25% solution of NH4OH with swirl was added dropwise. The resulting solution was evaporated to approximately 10 ml. To the resulting pale yellow residue, 100 ml of 6N HCl was added, and the mixture was evaporated as above to a volume of 15-20 ml.The trans-diaminodichloroplatinum (II) was deposited as a yellow, fine powder. After cooling with ice, the mixture was filtered, washed with three 10 ml portions of water cooled with ethanol and ether, and dried with air.The trans-diaminodichloroplatinum (II) was crystallized from a minimum amount of Boiling 0.1 N HCl The product was characterized by infrared spectroscopy.

EXAMPLE 2: Preparation of trans-platinum diamine-DigDadoo chloride (trans- [Pt (NH3) 2 (DigDadoo-NH2) Cl] (N03)) mg of the trans- [Pt (NH3) 2C12] (0.1 mmol) was suspended in 25 ml of -N, N '-dimethylformamide and 17 ml (0.1 mmol) of AgN03 was added to the mixture which was reacted for 16 hours. hours at room temperature protected from light. The precipitated AgCl was filtered and 3.4 ml of the filtrate (3.9 mg (0.014 mmol) of trans- [Pt (NH3) 2 (Cl) (N03)]) was taken and incubated with 8 mg of DigDadoo (digoxigenin-1). , 8-diamino-3,6-dioxaoctane, 0.014 mmole, purchased from Boehringer Mannheim) for 48 hours at 40 ° C, protected from light. The N, N'-dimethylformamide was removed in vacuo and the remainder was stirred in CH2C12 after which a yellowish powder was precipitated. The precipitated powder was filtered, washed with CH2C12 and dried. The product was characterized by high resolution NMR-H.

EXAMPLE Preparation of diamine-DigDadoo-2 '-deoxyGuanosine-5'-trans-platinumtriphosphate (trans- [Pt (NH3) 2 (DigDadoo-NH2) (dGTP-N7)] 12 mg of trans- [Pt (NH3) 2 (DigDadoo-NH2) Cl] (N03) (0.014 mmol) were dissolved in 2 ml of Milli-Q and 3 mg (0.006 mmol) of 2'-deoxyguanosine- were added. 5 '-triphosphate. The pH of the mixture was adjusted to ~6 with 0.1 N NaOH and the reaction was carried out for 24 hours at 50 ° C protected from light. The mixture was lyophilized and the residue was applied to a preparative ion exchange chromatography column (Q-Sepharose 26/10, Pharmacia BioTech) and a gradient was applied starting with 100% Milli-Q at 100% NH4CH03. The appropriate fraction was collected, concentrated and lyophilized to remove the NH4HC03. The product was analyzed by high resolution 1H and 31P NMR and analytical FPLC methods, ion exchange (MonoQ 5/57 Pharmacia BioTech) and reverse phase (PepRPC 5/5, Pharmacia BioTech). The product was converted to its tetra-lithium salt by passing it over a Dowex cation exchange column.

EXAMPLE 4: Preparation of diamine-Cy5-transplatin chloride (trans- [Pt (NH3) 2 (Cy5®-NH2) Cl] (N03)) 7.9 mg of monofunctional reactive dye Cy5 (0.01 _ mmoles) was dissolved in 5 ml of dry N, N 'dimethylformamide and reacted with an excess of ethylenediamine for 4 hours at room temperature in an inert atmosphere (argon). The N, N'-dimethylformamide and the excess ethylenediamine were removed in vacuo. The residue was redissolved in 10 ml of N, N'-dimethylformamide and 3 mg (0.009 mmol) of trans- [Pt (NH3) 2 (Cl) (N03)]) were added. The mixture was stirred for 16 hours at 50 ° C, protected from light. The N, N'-dimethylformamide was removed in vacuo and the residue was stirred in. CH2C12, after which the product was precipitated. The precipitated product was filtered and dried in vacuo. The product was characterized by high resolution 1 H-NMR.

EXAMPLE Enzymatic incorporation of trans [Pt (NH3) 2 (DigDadoo-NH2) '(dGTP)] into pBR 322 DNA Enzymatic incorporation was performed by incubating the following mixture overnight at 37 ° C: Denatured DNA (1 μg) _ 8.0 μl dTTP (5.0 mM) 0.8 μl dCTP (5.0 mM) 0.8 μl dATP (5.0 mM) - 0.8 μl dGTP (1.0 mM) 2.6 μl trans- [Pt (NH3) 2 (DigDadoo-NH2 ) (dGTP)] (1 mM) 1.4 μl Very High Quality Mixture = 4.0 μl Demineralized water, sterile 1.6 μl Total 20 μl The Very High Quality Blend contained the Klenow polymerase and hexanucleotides in an optimized buffer. "" The incubation stopped when adding 2 μl of 0.2 M EDTA and the volume of the mixture was adjusted to 40 μl by adding demineralized, sterile water. A filter hybridization was performed at a test concentration of 25 ng / ml, using homologous, denatured DNA labeled at spots on the filter strips with the following dilution range: 100 pg, 30 pg, 3 pg, 1 pg , 0.3 pg, 0.1 pg, 0.03 pg, 0.01 pg and 0 pg. Detection was carried out with anti-Dig-AP (Fab fragments of a sheep anti-digoxigenin antibody conjugated with alkaline phosphatase (AP) (1: 10,000) and CDP-Star (chemiluminescent substrate). t observed 0.3 pg of hybridization.

EXAMPLE Aggregation of trans- [Pt (NH3) 2 (DigDadoo-NH2) (dGTP)] Oligo using Transferase Terminal The aggregation reaction was performed by incubating the next mixture for 15 minutes at 37 ° C. 7A Reaction vessel: potassium cacodylate 4 μl 1M, Tris 0.125 M-HC1, 1.25 mg / m; bovine serum albumin pH 6.6 (25 ° C) CoCl2 solution (25 mM) 4 μl oligo (20 pmol / μl) 5 μl trans- [Pt (NH3) z (DigDadoo-NH2) (dGTTP)] / DATO * 1 μl Transferase Terminal 50 U / μl 1 μl Sterile demineralized water 5 μl Total 10 μl ^ Mixture of 9 μl of -trans- [Pt (NH3) 2 (DigDadoo-NH2) (dGTP)] (1 mMO and 1 μl dATP (100 mM) Incubation was stopped by adding 2 μl of a mixture of 1 μl of glycogen and 200 μl of 0.2 M EDTA having a pH of 8.0. Subsequently, a purification was performed by ethanol precipitation and an hybridization was performed analogous to the filter hybridization described in Example 5. As a result, 10 pg of hybridization was observed.

EXAMPLE 7: Marking of normal DNA using trans- [Pt (NH3) 2 (DigDadoo-NH2) Cl] (N0) From a normal QC procedure, testing a range of different relationships, the following protocol was performed. A mixture of 2 μl of DNA (2 μg), 2 μl of trans- [Pt (NH3) 2 (DigDadoo-NH2) Cl] (N03) (0.1 mg / ml) and 16 μl of sterile, demineralized water was incubated for 30 minutes at 85 ° C. The incubation was stopped by adding 5 μl of diethyldithiocarbamic acid 1% sodium. In a filter hybridization, which was performed analogous to the hybridization described in Example 5, 3 pg of hybridization was observed.

EXAMPLE 8: Marking in DNA relationships by mixing dye assays that have been independently labeled The identical dyeing assays were labeled in independent reactions using Fluorescein (Fl) and Rhodamine (Rho). After labeling, the independent assays were mixed using a ratio of 1: 9, 1: 5, 1: 1, 5: 1 and 9: 1, respectively, and the spot analyzed and by filter hybridization. Detection was performed by antibodies conjugated with alkaline phosphatase raised against Flu and Rho, respectively. After detection, the relationships of the labeled dye assays appeared to be identical when compared to the ratio of the input marker molecule.

EXAMPLE 9: Labeling in DNA ratios using a mixed, defined entry of complex trans- [Pt- (NH3) 2 (Dadoo-NH2) C1] N03 markers in a reaction.

The assays were labeled using similar ratios as described in Example 8, with the exception that the test DNA was incubated with a mixture of the complexes (Flu and Rho) trans-markers [Pt (NH3) 2 (Dadoo-NH2 ) C1] N03 in a reaction tube. After detection by the conjugated antibodies, it appears that the distribution of the label occurred equimolar and that the different molecules of the marker did not interfere with each other, nor did they negatively affect each other. All the results were - confirmed by microscopic analysis.

It is noted that in relation to this date, the best method known to the applicant to bring the said invention to practice is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

Claims (19)

1. A method for labeling a bio-organic molecule, characterized in that a reactive portion of a compound based on trans-platinum, having the formula: A XI \ / Pt / \ (I) X2 D wherein A and D represent the same or different reactive portions, and XI and X2 represent the same or different inert portions, are reacted with a label, and wherein the other reactive portion of the compound based on trans-platinum is reacted with a bio-organic molecule, in any order.

2. A method according to claim 1, characterized in that XI and X2 are the same or different non-salient ligands.

3. A method according to any of the preceding claims, characterized in that XI and X2 are selected from the groups NH3, NH2R, NHRR ', NRR'R ", wherein R, R' and R" ^ represent an alkyl group having 1 to 6 carbon atoms.

4. A method according to any of the preceding claims, characterized in that XI and X2 both represent a group N (CH3) 3.

5. A method according to any of the preceding claims, characterized in that A and / or D are selected from the group of acetate, N03 ~, HC03 ~, C032 ~, S03 ~ Cl-, I ", and other halogens.

6. A method according to any of the preceding claims, characterized in that the label and / or bio-organic molecule are connected to the platinum through a separator, separator comprising a chain having at least four atoms, chain comprising a portion electron donor at one end and a reactive portion at the other end, wherein one or both reactive portions of the trans-platinum-based compound are reacted with the electron donor portion of the separator (s).

7. A compound according to claim 6, characterized in that the electron donor portion is an amine group or a thiolate anion.

8. A compound according to claim 6 or 7, characterized in that the chain also comprises at least one heteroatom.

9. A compound according to any of claims 6-8, characterized in that the spacer comprises no more than 20 carbon atoms in the chain and wherein the chain is essentially not branched.

10. A compound according to claim 9, characterized in that A and / or D is 1,8-diamino-3,6-dioxaoctane.

11. The compound according to any of the preceding claims, characterized in that it has a label attached thereto.

12. A compound according to claim 11, characterized in that the label is selected from the group of biotin, avidin, streptavidin, digoxigenin and fluorochromes that include Cy® dyes.

13. A compound according to any of the preceding claims, characterized in that it has a bio-organic molecule attached thereto.

14. A compound according to claim 13, characterized in that the bio-organic molecule is selected from the group of proteins, peptides, DNA molecules, RNA molecules and (oligo) -nucleotides.

15. A compound according to claim 14, characterized in that the bio-organic molecule is a nucleotide.

16. A diagnostic equipment for detecting, determining and / or locating the biological substances of interest, characterized in that it comprises a compound according to any of claims 1-15, optionally together with another means suitable for detection.

17. A method for labeling a bio-organic molecule, characterized in that a reactive portion of a compound based on trans-platinum has the formula: I) X2 XX (D wherein A and D represent the same or different reactive portions, and XI and X2 represent the same or different inert portions, are reacted with a label, and wherein the other reactive portion of the compound based on trans-platinum is reacted with a bio-organic molecule, in any order.

18. A method according to claim 17, characterized in that the label and / or the bio-organic molecule are connected to the platinum through a separator, separator comprising a chain having at least four atoms, the chain comprising one - electron donating portion at one end and a reactive portion at the other end, wherein one or both reactive portions of the compound based on trans-platinum are reacted with the electron donor portion of the separator (s) and wherein the reactive portion of the separator (s) is reacted with the bio-organic label and / or molecule, in any order.

19. A method according to claim 17 or 18, characterized in that the bio-organic molecule is a nucleotide.