WO2014071965A1 - Nucleic-acid binding compounds - Google Patents

Nucleic-acid binding compounds Download PDF

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WO2014071965A1
WO2014071965A1 PCT/EP2012/004690 EP2012004690W WO2014071965A1 WO 2014071965 A1 WO2014071965 A1 WO 2014071965A1 EP 2012004690 W EP2012004690 W EP 2012004690W WO 2014071965 A1 WO2014071965 A1 WO 2014071965A1
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polyamine
nucleic acids
metallates
layered
cation
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PCT/EP2012/004690
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French (fr)
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Sergey Britvin
Depmeier WULF
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Christian-Albrechts-Universität Zu Kiel
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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G23/00—Compounds of titanium
    • C01G23/003—Titanates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3234—Inorganic material layers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G33/00—Compounds of niobium
    • C01G33/006—Compounds containing, besides niobium, two or more other elements, with the exception of oxygen or hydrogen
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G35/00—Compounds of tantalum
    • C01G35/006—Compounds containing, besides tantalum, two or more other elements, with the exception of oxygen or hydrogen
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/20—Two-dimensional structures
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    • C01—INORGANIC CHEMISTRY
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    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
    • C01P2002/82—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
    • C01P2002/87—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by chromatography data, e.g. HPLC, gas chromatography
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/01—Particle morphology depicted by an image
    • C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2006/00—Physical properties of inorganic compounds
    • C01P2006/10—Solid density

Definitions

  • the present invention relates to a new family of Titanium, Niobium or Tantalum oxide-based compounds (titanates, niobates and tantalates) containing chemically bound functional multivalent polyamines and the methods for preparation thereof, and using these new compounds for surface binding (adsorption) of nucleic acids.
  • nucleic acids DNA, RNA
  • concentration, separation, isolation, purification, removal or filtering of nucleic acids is an essential part of biochemical procedures.
  • the typical examples include:
  • PCR polymerase chain reaction
  • LCR lidase chain reaction
  • RCR repair chain reaction
  • Isolation of nucleic acids is a fundamental primary step in most of DNA or RNA detection protocols.
  • PCR allows multiplication and thus detection even a single copy of target nucleotide in an arbitrary mixture
  • real techniques require a step of isolation and purification of nucleic acids prior to amplification itself.
  • the main reason for carrying out this procedure is that different inorganic and organic moieties in raw assays are capable to inhibit amplification yielding false negative results of the reaction.
  • Yet another reason for pre-isolation and purification is that in case of very low concentrations of target nucleotides in raw assay, small aliquots taken for amplification may not be representative of a bulk probe, i.e. may not contain target nucleotides.
  • Condensation (compaction) of nucleic acids is used as a method for purification of RNA and DNA presented in WO 03/082892 and WO06/083962.
  • Compaction agent could be multivalent polypeptides, polyamides or polyquaternary ammonium compounds. Note that cation-induced condensation of nucleic acids reported in the scientific and patent literature is always carried out with solutions of corresponding compounds in liquid (aqueous) media, resulting in formation of colloidal solutions of compacted nucleic acids. Thus these methods do not allow adsorption of nucleic acids onto a certain solid substrate.
  • nucleotide probe purification methods and devices using solid substances including modified resins or polymers (US5336412; US5378360; US 5496473; US6524480; US 6576133) or modified inorganic metal oxides or zeolites (DE4321904).
  • modified resins or polymers US5336412; US5378360; US 5496473; US6524480; US 6576133
  • modified inorganic metal oxides or zeolites DE4321904
  • the objective of the present invention is to provide a new group of solid, stable, insoluble adsorbents for nucleic acids, namely: titanates, niobates and tantalates of DNA- and RNA-compacting multivalent polyamines.
  • Titanates, niobates and tantalates of said polyamines are best suitable for biochemical applications for several reasons. It is known (see ref. 6) that Ti, Nb and Ta are biologically indifferent elements possessing negligible biotoxicity. The oxides and hydroxides of Ti, Nb and Ta have vanishingly low solubility in aqueous solutions, therefore the said titanates, niobates and tantalates can be used as stable solid carriers of corresponding anion- free polyamines.
  • the new compounds combine the following important properties inherited both from their oxide nature and from the presence of multivalent nucleic acid-binding polyamines in their composition: 1) Insolubility in aqueous solutions;
  • the chemical composition of the new compounds can be identified in terms of mole ratios of constituents as follows:
  • Niobates ⁇ / , ) m ⁇ i-20-(Nb 2 . q q ) ⁇ 2(05-wOH x F y )5-z- «H 2 0
  • (P) means polyamine or polyamine cation possessing nucleic-acid binding properties (see, for instance, ref. 1-5, 7), and titanates, niobates and tantalates of said polyamines possess nucleic acid-binding properties as well.
  • polyamine means an organic compound having two or more terminal primary amino groups - NH 2 , optionally with secondary amino groups.
  • nucleic acid-binding polyamines are spermine ( ⁇ , ⁇ '- bis(3 -aminopropyl)- 1 ,4-diaminobutane, NH 2 (CH 2 )3NH(CH 2 ) 4 NH(CH 2 ) 3 NH 2 ), spermidine (N-(3 -aminopropyl)- 1,4-diaminobutane, NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 ), and norspermine (N,N'-bis(3 -aminopropyl)- 1 ,3-diaminopropane,
  • any other polyamine which possesses nucleic acid-binding properties can be used for the preparation of corresponding titanate, niobate or tantalate compounds.
  • Cation A may also represent any organic or elementoorganic cation.
  • Metal is at least one element having valence 1 to 7 substituting for titanium, niobium or tantalum, preferably from the group: Li, Mg, Al, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Y, Zr, Nb, Mo, Sn, Sb, Hf, Ta, W, Ru, Rh, Pd, Os, Ir, Pt.
  • Nucleic acid-binding polyamines (as molecular polyamines or as polyamine cations), titanium (for titanates), niobium (for niobates), tantalum (for tantalates) and oxygen are essential constituents of the disclosed compounds.
  • the disclosed compounds contain: 1) Titanates: 1-30 wt.%, preferably 10-20 wt.% of polyamine; Ti0 2 ranges from
  • Niobates 1-40 wt.%, preferably 15-30 wt.% of polyamine; Nb 2 0 5 ranges from 50 to 95%, preferably 60-80 wt.%; 3) Tantalates: 1-30 wt.%, preferably 10-20 wt.% of polyamine; Ta 2 0 5 ranges from 50 to 95%, preferably 60-80 wt.%.
  • FIGURES shows examples of typical particle morphology and particle size of new titanates, niobates and tantalates.
  • Fig. 2 shows infrared spectra of free spermidine and some of new titanates, niobates and tantalates.
  • A spermidine, free base, liquid, attenuated total reflectance (ATR) cell;
  • B spermidine titanate, powder in KBr pellet;
  • C spermidine niobate, powder in KBr pellet;
  • D spermidine tantalate, powder in KBr pellet.
  • Fig. 3 shows infrared spectra of free spermine and some of new titanates, niobates and tantalates.
  • A spermine, free base, liquid, attenuated total reflectance (ATR) cell;
  • B spermine niobate, powder in KBr pellet (Example 1);
  • C spermine titanate, powder in KBr pellet. shows infrared spectra of free norspermine and new norspermine niobate.
  • A norspermine, free base, liquid, attenuated total reflectance (ATR) cell;
  • B norspermine niobate, powder in KBr pellet.
  • Fig. 1 shows the typical particle sizes and morphologies of the new titanates, niobates and tantalates. These new compounds have particle sizes in from 5 nm to 10 ⁇ , depending on thermal and chemical conditions of the syntheses.
  • the new compounds may have platy, lamellar (plates, sheets, lamella, flakes), tubular (tubes, scrolls, worms), fibrous (rods, needles, whiskers) or isometric (balls or uneven) morphology.
  • the habit of the titanate, niobate or tantalate particles depends on their compositions, thermal and chemical conditions of their synthesis.
  • the Infrared spectra of the new titanates, niobates and tantalates show absorption bands in the regions characteristic of internal vibration modes of corresponding polyamines (regions from 1800 to 1000 cm “1 ) and vibration modes characteristic of host titanate, niobate or tantalate oxide matrices (1000-400 cm “1 ).
  • the exact frequencies of absorption bands are different from those observed in free polyamine bases.
  • the new compounds are insoluble in water and aqueous solutions under near- physiological conditions (pH from 6 to 8).
  • the corresponding parent polyamines can be re-extracted in their pristine form, by acid leaching of corresponding titanate, niobate or tantalate in solutions of strong acids (for example, HC1 or H 2 PtCl 6 ).
  • the polyamines are thus extracted in the form of salts of polyamine cations (for example, hydrochlorides or chloroplatinates). This evidences that polyamines in the title compounds were not decomposed (i.e., deaminated) during the synthesis but retained chemically inchanged.
  • titanates, niobates and tantalates that, besides of polyamines and water, they do not contain other chemically active species (i.e., no anions or cations forming water-soluble species, no oxidizing ions, no reducing ions).
  • oxides and hydroxides of Ti, Nb and Ta contrary to other transition metal oxides, combine (1) very weak acid properties (but allowing them to form stable salts with cations), (2) complete insolubility in water and acid solutions (besides of HF), (3) complete redox inertness, and (4) biological indifference.
  • the combination of those properties makes oxides and hydroxides of Ti, Nb and Ta as excellent neutral insoluble matrices for preparations of polyamine- containing sorbents suitable for biochemical assays and preparations.
  • Thermogravimeteric data show that the new titanates, niobates and tantalates are relatively thermally stable compounds.
  • the thermal decomposition (pyrolysis) of new compounds in air occurs at the temperatures above 200 °C.
  • the new titanates, niobates and tantalates possess high density (specific gravity), due to the presence of Ti, Nb or Ta in their composition.
  • the solid pellets made by pressing of corresponding spermine compounds show the following measured densities: titanate - 2.1 g/cm 3 ; niobate - 2.5 g/cm 3 ; tantalate - 3.1 g/cm 3 .
  • Their high density allows easy gravitational separation of title compounds in liquid media, without necessity of centrifugation.
  • Fig. 5 shows an example of a layer of DNA (calf thymus DNA) precipitated onto the surface of spermine niobate (example 2). Nucleic acids are bound to the surface of titanates, niobates or tantalates by enveloping the nano- or micro-particles of corresponding compounds. The result of such binding is immediate flocculation and adsorption of nucleic acids on corresponding titanate, niobate or tantalate.
  • the resulting products are thus surface conjugates of nucleic acids with nano- or micro-particles of titanates, niobate or tantalates.
  • the resulting conjugates are appeared as dense white compact flake-like aggregates, typically 1-2 mm in size. They are easily separated from parent solutions by gravitational separation, without necessity of centrifugation, due to high density of parent titanates, niobates or tantalates.
  • the phosphorus content in nucleic acid/(titanate, niobate, tantalate) conjugates is 0.3-0.5 wt.%.
  • nucleic acid-binding polyamines are spermine (N,N'-bis(3-aminopropyl)-l,4-diaminobutane,
  • any other polyamine which possesses nucleic acid-binding properties (see, for instance, ref. 7, 8) can be used for the preparation of corresponding titanate, niobate or tantalate compounds.
  • the method for the preparation of titanates, niobates or tanatlates disclosed in the present invention comprises mixing, in any sequence, of solutions having the following general compositions: (A) Solution containing titanium, niobium or tantalum
  • Concentration of titanium in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter.
  • Any ion forming stable complex ions with titanium preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
  • X F,Cl,Br,I,OH, 0 ⁇ « ⁇ 1); hydrofluorides A n+ F n -wHF (A is any cation of valence n, 0 ⁇ m ⁇ 4); oxycarboxylic acids or their salts, preferably oxalic, citric, malic, tartaric, tartronic acid or their salts.
  • Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter.
  • Mole ratio of complexing ion to titanium in solution A ranges between 0.1 and 100, preferably 0.5- 10.
  • Complexing ion is an essential modifying constituent in the synthesis of polyamine titanates. Solution for preparation of niobates:
  • niobium in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter.
  • Any ion forming stable complex ions with niobium preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
  • A is any cation of valence n
  • fluoroniobic or oxofluoroniobic acids or their salts
  • carboxylic or oxycarboxylic acids or their salts preferably oxalic, citric, malic, tartaric, tartronic acid or their salts.
  • Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter.
  • Mole ratio of complexing ion to niobium in solution A ranges between 0.1 and 100, preferably 0.5-10.
  • Complexing ion is an essential modifying constituent in the synthesis of polyamine niobates.
  • a Any compound of tantalum, (Ta0 2 ) or (
  • Concentration of tantalum in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter.
  • Any ion forming stable complex ions with tantalum preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
  • fluorides A n+ F n (A is any cation of valence «); fluorotantalic or oxofluorotantalic acids or their salts; carboxylic or oxycarboxylic acids or their salts, preferably oxalic, citric, malic, tartaric, tartronic acid or their salts.
  • Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter.
  • Mole ratio of complexing ion to tantalum in solution A ranges between 0.1 and 100, preferably 0.5-10.
  • Complexing ion is an essential modifying constituent in the synthesis of polyamine tantalates.
  • the polyamine concentration in solution B varies in between 0.1 and 30.5 mole per liter, preferably 1-15 mole per liter.
  • Solution B is alkaline (pH>7).
  • the necessary level of alkalinity is achieved by the presence of free polyamine base or by adding of any alkali, preferably of aqueous solution of NaOH, KOH or NH 3 .
  • Solutions (A) and (B) are mixed together in any sequence under following required conditions:
  • reaction temperature ranging between -10 and 130°C, preferably 50-60 °C;
  • Post-reaction thermal treatment of the resultant mixture is not essential condition for synthesis of polyamine titanates, niobates or tantalates, but it results in increasing of their particle size and removing traces of fluorine from their composition.
  • the new titanates, niobates and tantalates can be used as polyamine-containing sources for many technical purposes including:
  • the present invention is illustrated below by way of typical examples. However, the present invention is not limited to concentrations, compositions and synthesis conditions described in the examples.
  • the chemical composition of synthesized spermine niobate (as determined by TGA with infrared screening, fluorine by electron microprobe): spermine 26.9 wt.%; Nb 2 Os 61.2; F 0.3 wt.%; H 2 0 11.9 wt.%; Total 100.3 wt.%.
  • spermine niobate powder 10 mg is suspended in 1 mL of deionised water (18 ⁇ ) in plastic syringe.
  • Precipitation of DNA To 10 mL of DNA solution in cacodylate buffer, 1 mL of suspension of spermine niobate is added with shaking. Flocculation of resultant mixture is observed immediately, resulting in formation of white, flake-like, 1 -2 mm in size conjugates of spermine niobate with DNA.
  • EFTEM Energy-filtered transmission electron microscopy

Abstract

The present invention relates to Titanium, Niobium or Tantalum oxide-based compounds containing chemically bound functional multivalent polyamines for surface binding (absorption) of nucleic acids. The chemical composition of the new metallates is as follows: Formula (1) or Formula (2) or Formula (3) wherein k, m, q, w, x, y, z are coefficients of at most 1 n is an integer, wherein 0 ≤ n ≤ 10 A is at least one cation of valence 1 to 3 M is at least one metal having valence 1 to 7 P is a chemically bounded polyamine or polyamine cation.

Description

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Nucleic-acid binding compounds
The present invention relates to a new family of Titanium, Niobium or Tantalum oxide-based compounds (titanates, niobates and tantalates) containing chemically bound functional multivalent polyamines and the methods for preparation thereof, and using these new compounds for surface binding (adsorption) of nucleic acids. REFERENCES
[1] Gosule, L.C. and Schellman, J.A. (1976) Compact form of DNA induced by spermidine. Nature, 259, 333-335. [2] Eickbush,T.H. and Moudrianakis,E.N. (1978) The compaction of DNA
helices into either continuous supercoils or folded-fiber rods and toroids. Cell, 13, 295-306.
[3] Wilson, R.W. and Bloomfield, V.A. (1979) Counterion-induced condensation of deoxyribonucleic acid. A light scattering study. Biochemistry, 18, 2192-
2196.
[4] Marquet, R., Houssier, C. and Fredericq E. (1985) An electro-optical study of the mechanisms of DNA condensation induced by spermine. Biochimica et Biophysica Acta, 825, 365-374. Bloomfield, V.A. (1991) Condensation of DNA by multivalent cations: considerations and mechanism. Biopolymers, 31, 1471-1481.
Zardiackas, L.D., Kraay, MJ. and Freese, H.L. (eds.) Titanium, Niobium, Zirconium, and Tantalum for Medical and Surgical Applications. ASTM STP1741, PA 2006.
Saminathan, M., Thomas, T., Shirahata, A., Pillai, C.K.S. and Thomas, T.J. (2002) Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics. Nucleic Acids Research, 30, 3722-3731
Vijayanathan, V., Thomas, T., Antony, T., Shirahata, A. and Thomas ,T.J. (2004) Formation of DNA nanoparticles in the presence of novel polyamine analogues: a laser light scattering and atomic force microscopic study. Nucleic Acids Research, 32, 127-134.
Concentration, separation, isolation, purification, removal or filtering of nucleic acids (DNA, RNA) is an essential part of biochemical procedures. The typical examples include:
Industrial production of nucleic acids and their derivatives;
Preparation of assays for different amplification protocols (polymerase chain reaction (PCR), lidase chain reaction (LCR), self-sustained sequence replication (3SR), repair chain reaction (RCR), etc.);
Removal of polluting nucleic acids from air and water systems in biochemical laboratories and plants;
Extraction of nucleic acids from air, aerosols, aqueous solutions or water systems for subsequent analysis for potentially hazardous nucleotide sequences;
Separation of nucleic acids by liquid chromatography.
Isolation of nucleic acids is a fundamental primary step in most of DNA or RNA detection protocols. Although in theory, for instance, PCR allows multiplication and thus detection even a single copy of target nucleotide in an arbitrary mixture, real techniques require a step of isolation and purification of nucleic acids prior to amplification itself. The main reason for carrying out this procedure is that different inorganic and organic moieties in raw assays are capable to inhibit amplification yielding false negative results of the reaction. Yet another reason for pre-isolation and purification is that in case of very low concentrations of target nucleotides in raw assay, small aliquots taken for amplification may not be representative of a bulk probe, i.e. may not contain target nucleotides.
The ability of some polyamines (spermine, spermidine and some of synthetic polyamines) and inorganic cations (hexaamminecobalt (III) Co(NH3)63+, Cr3+, Tb3+) to cause condensation (compaction) of nucleic acids is a well studied phenomenon (see, for instance, ref. 1-5). The process of condensation is explained by covalent binding of positively charged cations to negatively charged phosphate sites of nucleic acids. Noteworthy that condensation requires certain degree (~ 90 %) of nucleic acid charge neutralization (see ref. 3) which can be achieved only with tri- and tetravalent cations. Condensation (compaction) of nucleic acids is used as a method for purification of RNA and DNA presented in WO 03/082892 and WO06/083962. Compaction agent could be multivalent polypeptides, polyamides or polyquaternary ammonium compounds. Note that cation-induced condensation of nucleic acids reported in the scientific and patent literature is always carried out with solutions of corresponding compounds in liquid (aqueous) media, resulting in formation of colloidal solutions of compacted nucleic acids. Thus these methods do not allow adsorption of nucleic acids onto a certain solid substrate. There are a number of patents reporting nucleotide probe purification methods and devices using solid substances, including modified resins or polymers (US5336412; US5378360; US 5496473; US6524480; US 6576133) or modified inorganic metal oxides or zeolites (DE4321904). However, none of these patents reports on solid-supported compounds containing polyamines capable of cation-induced condensation of nucleic acids.
The objective of the present invention is to provide a new group of solid, stable, insoluble adsorbents for nucleic acids, namely: titanates, niobates and tantalates of DNA- and RNA-compacting multivalent polyamines. Titanates, niobates and tantalates of said polyamines are best suitable for biochemical applications for several reasons. It is known (see ref. 6) that Ti, Nb and Ta are biologically indifferent elements possessing negligible biotoxicity. The oxides and hydroxides of Ti, Nb and Ta have vanishingly low solubility in aqueous solutions, therefore the said titanates, niobates and tantalates can be used as stable solid carriers of corresponding anion- free polyamines. The new compounds combine the following important properties inherited both from their oxide nature and from the presence of multivalent nucleic acid-binding polyamines in their composition: 1) Insolubility in aqueous solutions;
2) Stability in aqueous solutions under near physiological conditions (pH between 6 and 8);
3) Stability in air;
4) Biological indifference of oxide matrices;
5) Micrometer- to nanometer-sized particles;
6) Chemically bound anion-free multivalent polyamines;
7) Absence of oxidizing, reducing, soluble anions in their composition; 7) Ability to surface binding (adsorption) of nucleic acids;
8) High density, allowing separation of nucleic acids without need of centrifugation.
The chemical composition of the new compounds can be identified in terms of mole ratios of constituents as follows:
1) Titanates:
Figure imgf000006_0001
2) Niobates: ^/,) m^i-20-(Nb2.q q)∑2(05-wOHxFy)5-z-«H20
3) Tanatlates: ^( )'W^ i-20-(Ta2.q q)∑2(05-wOHxFy)5-z-«H20
The term "∑1" only confirms that the sum of the coefficients in the preceding brackets, that is 1-q+q, is 1. The term "∑2" only confirms that the sum of the coefficients in the preceding brackets, that is 2-q+q, is 2. In the present formulae, (P) means polyamine or polyamine cation possessing nucleic-acid binding properties (see, for instance, ref. 1-5, 7), and titanates, niobates and tantalates of said polyamines possess nucleic acid-binding properties as well. The term polyamine means an organic compound having two or more terminal primary amino groups - NH2, optionally with secondary amino groups. At present knowledge, preferably used nucleic acid-binding polyamines are spermine (Ν,Ν'- bis(3 -aminopropyl)- 1 ,4-diaminobutane, NH2(CH2)3NH(CH2)4NH(CH2)3NH2), spermidine (N-(3 -aminopropyl)- 1,4-diaminobutane, NH2(CH2)3NH(CH2)4NH2), and norspermine (N,N'-bis(3 -aminopropyl)- 1 ,3-diaminopropane,
NH2(CH2)3NH(CH2)3NH(CH2)3NH2). However, any other polyamine which possesses nucleic acid-binding properties (see, for instance, ref. 7, 8) can be used for the preparation of corresponding titanate, niobate or tantalate compounds.
In the present formulae, k,m,q,w,x,y,z are coefficients of at most 1 ; 0<«<10. Cation A is at least one cation of valence 1 to 3, preferably from the group: H, Li, Na, K, Rb, Cs, Tl, NR4 (ammonium or its organic derivatives where R = H, alkyl, alkenyl, alkinyl or aryl), Ni?3Oi? (hydroxylammonium or its organic derivatives where R = H, alkyl, alkenyl, alkinyl or aryl), N2R4 (hydrazinium or its organic derivatives where R - H, alkyl, alkenyl, alkinyl or aryl), H30 (hydronium), Ag, Au, Mg, Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, Hg, Sn, Pb, Ca, Sr, Ba. Cation A may also represent any organic or elementoorganic cation. Metal is at least one element having valence 1 to 7 substituting for titanium, niobium or tantalum, preferably from the group: Li, Mg, Al, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Y, Zr, Nb, Mo, Sn, Sb, Hf, Ta, W, Ru, Rh, Pd, Os, Ir, Pt. Nucleic acid-binding polyamines (as molecular polyamines or as polyamine cations), titanium (for titanates), niobium (for niobates), tantalum (for tantalates) and oxygen are essential constituents of the disclosed compounds.
The disclosed compounds contain: 1) Titanates: 1-30 wt.%, preferably 10-20 wt.% of polyamine; Ti02 ranges from
50 to 95%, preferably 60-70 wt.%;
2) Niobates: 1-40 wt.%, preferably 15-30 wt.% of polyamine; Nb205 ranges from 50 to 95%, preferably 60-80 wt.%; 3) Tantalates: 1-30 wt.%, preferably 10-20 wt.% of polyamine; Ta205 ranges from 50 to 95%, preferably 60-80 wt.%.
SHORT DESCRIPTION OF THE FIGURES shows examples of typical particle morphology and particle size of new titanates, niobates and tantalates. (a) spermine niobate; (b) spermine tantalate; (c) norspermine niobate; (d) spermidine niobate; (e) spermine titanate. TEM bright field images.
Fig. 2 shows infrared spectra of free spermidine and some of new titanates, niobates and tantalates. (A) spermidine, free base, liquid, attenuated total reflectance (ATR) cell; (B) spermidine titanate, powder in KBr pellet; (C) spermidine niobate, powder in KBr pellet; (D) spermidine tantalate, powder in KBr pellet.
Fig. 3 shows infrared spectra of free spermine and some of new titanates, niobates and tantalates. (A) spermine, free base, liquid, attenuated total reflectance (ATR) cell; (B) spermine niobate, powder in KBr pellet (Example 1); (C) spermine titanate, powder in KBr pellet. shows infrared spectra of free norspermine and new norspermine niobate. (A) norspermine, free base, liquid, attenuated total reflectance (ATR) cell; (B) norspermine niobate, powder in KBr pellet. shows thin layer of calf thymus DNA enveloping nanoparticles of spermine niobate (example 2). (a) Energy filtered TEM (EFTEM) micrograph showing nanospheres of spermine niobate coated by thin layer of DNA. (b) the same area mapped by use of EFTEM elemental mapping (using phosphorus Z-edge), light area correspond to DNA coating. Fig. 1 shows the typical particle sizes and morphologies of the new titanates, niobates and tantalates. These new compounds have particle sizes in from 5 nm to 10 μπι, depending on thermal and chemical conditions of the syntheses. The new compounds may have platy, lamellar (plates, sheets, lamella, flakes), tubular (tubes, scrolls, worms), fibrous (rods, needles, whiskers) or isometric (balls or uneven) morphology. The habit of the titanate, niobate or tantalate particles depends on their compositions, thermal and chemical conditions of their synthesis.
The Infrared spectra of the new titanates, niobates and tantalates (Fig. 2-4) show absorption bands in the regions characteristic of internal vibration modes of corresponding polyamines (regions from 1800 to 1000 cm"1) and vibration modes characteristic of host titanate, niobate or tantalate oxide matrices (1000-400 cm"1). The exact frequencies of absorption bands, however, are different from those observed in free polyamine bases. These observations lead to the following conclusions: (1) the new titanates, niobates and tantalates contain corresponding polyamines and (2) the new substances are chemical compounds of corresponding polyamines but not mechanical mixtures of parent polyamines with Ti, Nb or Ta hydroxides.
The new compounds are insoluble in water and aqueous solutions under near- physiological conditions (pH from 6 to 8). The corresponding parent polyamines, however, can be re-extracted in their pristine form, by acid leaching of corresponding titanate, niobate or tantalate in solutions of strong acids (for example, HC1 or H2PtCl6). The polyamines are thus extracted in the form of salts of polyamine cations (for example, hydrochlorides or chloroplatinates). This evidences that polyamines in the title compounds were not decomposed (i.e., deaminated) during the synthesis but retained chemically inchanged.
It is an essential advantage of the invented titanates, niobates and tantalates that, besides of polyamines and water, they do not contain other chemically active species (i.e., no anions or cations forming water-soluble species, no oxidizing ions, no reducing ions). It is known that oxides and hydroxides of Ti, Nb and Ta, contrary to other transition metal oxides, combine (1) very weak acid properties (but allowing them to form stable salts with cations), (2) complete insolubility in water and acid solutions (besides of HF), (3) complete redox inertness, and (4) biological indifference. The combination of those properties makes oxides and hydroxides of Ti, Nb and Ta as excellent neutral insoluble matrices for preparations of polyamine- containing sorbents suitable for biochemical assays and preparations. Thermogravimeteric data show that the new titanates, niobates and tantalates are relatively thermally stable compounds. The thermal decomposition (pyrolysis) of new compounds in air occurs at the temperatures above 200 °C.
The new titanates, niobates and tantalates possess high density (specific gravity), due to the presence of Ti, Nb or Ta in their composition. As examples, the solid pellets made by pressing of corresponding spermine compounds show the following measured densities: titanate - 2.1 g/cm3; niobate - 2.5 g/cm3; tantalate - 3.1 g/cm3. Their high density allows easy gravitational separation of title compounds in liquid media, without necessity of centrifugation.
The new titanates, niobates and tantalates possess ability to bind nucleic acids, as a consequence of presence of corresponding functional polyamines in their composition. Fig. 5 shows an example of a layer of DNA (calf thymus DNA) precipitated onto the surface of spermine niobate (example 2). Nucleic acids are bound to the surface of titanates, niobates or tantalates by enveloping the nano- or micro-particles of corresponding compounds. The result of such binding is immediate flocculation and adsorption of nucleic acids on corresponding titanate, niobate or tantalate. The resulting products are thus surface conjugates of nucleic acids with nano- or micro-particles of titanates, niobate or tantalates. The resulting conjugates are appeared as dense white compact flake-like aggregates, typically 1-2 mm in size. They are easily separated from parent solutions by gravitational separation, without necessity of centrifugation, due to high density of parent titanates, niobates or tantalates. By results of ICP-MS analyses, the phosphorus content in nucleic acid/(titanate, niobate, tantalate) conjugates is 0.3-0.5 wt.%. Taking into account that calf thymus DNA (Sigma-Aldrich) contains 5-9 wt.% of phosphorus, the bulk DNA content in resultant conjugates can be estimated as 4-7 wt.% of total mass. Thus, the ability of invented titanates, niobates and tantalates to bind nucleic acids is a property inherited from the presence of nucleic-acid binding polyamines in their composition. At present, preferably used nucleic acid-binding polyamines are spermine (N,N'-bis(3-aminopropyl)-l,4-diaminobutane,
NH2(CH2)3NH(CH2)4NH(CH2)3NH2), spermidine (N-(3-aminopropyl)-l,4- diaminobutane, NH2(CH2)3NH(CH2)4NH2), and norspermine (N,N'-bis(3- aminopropyl)- 1 ,3 -diaminopropane, NH2(CH2)3NH(CH2)3NH(CH2)3NH2). However, any other polyamine which possesses nucleic acid-binding properties (see, for instance, ref. 7, 8) can be used for the preparation of corresponding titanate, niobate or tantalate compounds.
The method for the preparation of titanates, niobates or tanatlates disclosed in the present invention comprises mixing, in any sequence, of solutions having the following general compositions: (A) Solution containing titanium, niobium or tantalum
Solution for preparation of titanates:
The solution contains water and the following essential constituents: a. Any compound of titanium or titanyl (TiO)2+ as source of titanium, preferably halogenides TiX4 (X=F,Cl,Br,I); titanyl salts T1OX2 (X is any anion of valence n); salts or complexes of trivalent titanium Ti3+; halogeno titanic acids and their salts A2/mTi[Xi-n(OH)n]6 (A is any cation of valence m, X=F,Cl,Br,I, 0<«<1); any complex compounds of titanium; any titanoorganic compounds, preferably aryloxides or alkoxides of titanium Ti(OR)4-nXn, where R is any organic or metalloorganic radical, preferably methyl (CH3), ethyl (C2H5), propyl isomers (C3H7), butyl isomers (C4H9), X=F,Cl,Br,I. Concentration of titanium in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter. b. Any ion forming stable complex ions with titanium, preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
fluorides An+Fn (A is any cation of valence n); hexafluorotitanic acid H2TiF6;
hexahalogenotitanates A2/mTi(F1.nXn)6 (A - any cation of valence m,
X=F,Cl,Br,I,OH, 0<«<1); hydrofluorides An+Fn-wHF (A is any cation of valence n, 0<m<4); oxycarboxylic acids or their salts, preferably oxalic, citric, malic, tartaric, tartronic acid or their salts. Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter. Mole ratio of complexing ion to titanium in solution A ranges between 0.1 and 100, preferably 0.5- 10. Complexing ion is an essential modifying constituent in the synthesis of polyamine titanates. Solution for preparation of niobates:
The solution contains water and the following essential constituents: a. Any compound of niobium, (Nb02)+ or (NbO)2+ as source of niobium, preferably halogenides NbX5 (X=F,Cl,Br,I); oxohalogenides Nb02X or NbOX3 (X=F,Cl,Br,I); halogenoniobic and oxohalogenoniobic acids and their salts; any complex compounds of niobium; any organic compounds of niobium, preferably aryloxides or alkoxides of niobium Nb(OR)5-nXn, where R is any organic or metalloorganic radical, preferably methyl (CH3), ethyl (C2¾), propyl isomers (C3H7), butyl isomers (C4H9), X=F,Cl,Br,I. Concentration of niobium in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter. b. Any ion forming stable complex ions with niobium, preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
fluorides An+Fn (A is any cation of valence n); fluoroniobic or oxofluoroniobic acids or their salts; carboxylic or oxycarboxylic acids or their salts, preferably oxalic, citric, malic, tartaric, tartronic acid or their salts. Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter. Mole ratio of complexing ion to niobium in solution A ranges between 0.1 and 100, preferably 0.5-10. Complexing ion is an essential modifying constituent in the synthesis of polyamine niobates.
Solution for preparation of tantalates:
The solution contains water and the following essential constituents: a. Any compound of tantalum, (Ta02) or (TaO) as source of tantalum, preferably halogenides TaX5 (X=F,Cl,Br,I); oxohalogenides Ta02X or TaOX3 (X=F,Cl,Br,I); halogenotantalic and oxohalogenotantalic acids and their salts; any complex compounds of tantalum; any organic compounds of tantalum, preferably aryloxides or alkoxides of tantalum Ta(OR)5-nXn, where R is any organic or metalloorganic radical, preferably methyl (CH3), ethyl ( H5), propyl isomers (C3H7), butyl isomers (C4H9), X=F,Cl,Br,I. Concentration of tantalum in solution A is varying in between 0.01 and 6 mole per liter, preferably 0.1-1 mole per liter. b. Any ion forming stable complex ions with tantalum, preferably from the group: fluoride ion in form of any compound, preferably as HF (hydrofluoric acid);
fluorides An+Fn (A is any cation of valence «); fluorotantalic or oxofluorotantalic acids or their salts; carboxylic or oxycarboxylic acids or their salts, preferably oxalic, citric, malic, tartaric, tartronic acid or their salts. Concentration of complexing ion in solution A is varying in between 0.01 and 30 mole per liter, preferably 0.1-1 mole per liter. Mole ratio of complexing ion to tantalum in solution A ranges between 0.1 and 100, preferably 0.5-10. Complexing ion is an essential modifying constituent in the synthesis of polyamine tantalates.
(B) Polyamine Solid or liquid polyamine or an aqueous solution containing polyamine or salts of polyamine in any soluble form. The polyamine concentration in solution B varies in between 0.1 and 30.5 mole per liter, preferably 1-15 mole per liter. Solution B is alkaline (pH>7). The necessary level of alkalinity is achieved by the presence of free polyamine base or by adding of any alkali, preferably of aqueous solution of NaOH, KOH or NH3. Solutions (A) and (B) are mixed together in any sequence under following required conditions:
- the resultant reaction mixture should have alkaline reaction (pH>7);
- reaction temperature ranging between -10 and 130°C, preferably 50-60 °C;
- in an atmosphere of any gas, preferably in air;
- under vacuum or gas pressure of 0-100 bar, preferably under atmospheric pressure.
Post-reaction thermal treatment (boiling or hydrothermal treatment) of the resultant mixture is not essential condition for synthesis of polyamine titanates, niobates or tantalates, but it results in increasing of their particle size and removing traces of fluorine from their composition.
The new titanates, niobates and tantalates can be used as polyamine-containing sources for many technical purposes including:
1) Sorption and accumulative pre-concentration of nucleic acids from different solutions, including solutions containing trace amounts of nucleic acids, as for forensic applications;
2) Sorption and accumulative pre-concentration of nucleic acids from gases and aerosols;
3) Preparation of "cut-off filters for removal of nucleic acids from aqueous solutions;
4) Preparation of "cut-off filters for removal of nucleic acids from gases and aerosols, as in airflow systems in biochemical laboratories and manufactures;
5) Preparation of lysate-impregnated air filters for in-situ, on-filter lysis of bacterial cells and/or viral plasmids, with simultaneous binding of nucleic acids obtained by lysis. This can allow continuous screening of air for potentially dangerous DNA and/or RNA sequences (as in airports, railway stations, trade centers, etc.).
6) Fabrication of thin layers of functional polyamines and /or nucleic acids on surfaces of metallic titanium, niobium, tantalum or their alloys, for possible applications in biocompatible implants;
7) Fabrication of thin layer micro- and nano-patterns composed by functional polyamines and /or nucleic acids on surfaces of metallic titanium, niobium, tantalum or their alloys;
8) Fabrication of thin layers of functional polyamines and /or nucleic acids on surfaces of compounds of titanium, niobium, tantalum (i.e., piezoelectric oxides), for applications in biosensors;
9) Fabrication of thin layer micro- and nano-patterns composed by functional polyamines and /or nucleic acids on surfaces of compounds of titanium, niobium, tantalum (i.e., piezoelectric oxides), for applications in micro- or nano- biosensor arrays;
10) Chromatographic separation and/or purification of nucleic acids
The applications above are listed as typical examples. However, the present invention is not limited to above mentioned examples; layered hydrazine titanates may also be used in other industrial or scientific applications.
The present invention is illustrated below by way of typical examples. However, the present invention is not limited to concentrations, compositions and synthesis conditions described in the examples.
Example 1. Preparation of spermine niobate
Solution of oxofluoroniobic acid (i^ bOFs) - 100 mL of 0.3 aqueous solution of oxofluoroniobic acid, H2NbOFs. Preparation: 4 g (0.015 mole) of commercial niobium oxide, Nb205, is dissolved in 10 mL of 40% hydrofluoric acid HF in Teflon- lined autoclave (140 °C, 12 hours). After cooling, the volume of resultant clear solution is adjusted to 100 mL by adding water. Preparation of spermine niobate
10 mL of 0.3 aqueous solution of oxofluoroniobic acid is heated on hot plate to 60 °C in a Teflon beaker. To that solution, 1 g of solid spermine base (Sigma-Aldrich) is added under continuous stirring by magnetic stirrer. The resultant mixture is heated to boiling point under stirring, and then boiled for 15 min. The obtained white precipitate is washed by adding distilled water and decantation (5 times repeat), then filtered off on paper filter and dried under ambient conditions. The chemical composition of synthesized spermine niobate (as determined by TGA with infrared screening, fluorine by electron microprobe): spermine 26.9 wt.%; Nb2Os 61.2; F 0.3 wt.%; H20 11.9 wt.%; Total 100.3 wt.%.
Example 2. Precipitation of DNA by spermine niobate
Preparation of 10 mM cacodylate buffer. 214 mg of sodium cacodylate trihydrate (Sigma-Aldrich) is dissolved in 100 mL of deionised water (18 ΜΩ) under room temperature. To that solution, 18.6 mg (0.5 mM) of Na-EDTA (Sigma-Aldrich) is added and dissolved. The pH of resultant solution is adjusted to 7.2 by gentle adding of 0.1 M HC1. Preparation of DNA solution. 1 mg of calf thymus DNA, sodium salt (readily soluble, Sigma-Aldrich) is dissolved in 10 mL of cacodylate buffer, without further sonication. The solution is gently stirred by magnetic stirrer for 2 hours, until complete dissolution of DNA is achieved. Preparation of aqueous suspension of spermine niobate. 10 mg of spermine niobate powder (Example 1) is suspended in 1 mL of deionised water (18 ΜΩ) in plastic syringe. Precipitation of DNA. To 10 mL of DNA solution in cacodylate buffer, 1 mL of suspension of spermine niobate is added with shaking. Flocculation of resultant mixture is observed immediately, resulting in formation of white, flake-like, 1 -2 mm in size conjugates of spermine niobate with DNA. Energy-filtered transmission electron microscopy (EFTEM) of synthesized conjugate reveals that DNA is bound to the surface of niobate particles, forming thin layer enveloping spermine niobate (Fig. 5). Phosphorus content in resultant conjugate is determined as 0.3 wt.% (by ICP analysis) corresponding to ~4 wt. % of DNA.

Claims

1. Layered metallates of the formula ^( )-^1-20-(Ti1-q q)∑1(02.wOHxFy)2.z-«H20 (1) or ^Tw^!^O-CNb^ ^iOs-wOH^s-z-rt^O
(2)
or ^( )-^i-20-(Ta2.q q)∑2(05-wOHxFy)5-z-«H20 (3)
wherein
k,m,q,w,x,y,z are coefficients of at most 1
n is an integer, wherein 0 < n < 10
A is at least one cation of valence 1 to 3
M is at least one metal having valence 1 to 7
P is a chemically bounded polyamine or polyamine cation 2. Layered metallates according to claim 1 , characterized in that A is selected from the group: H, Li, Na, K, Rb, Cs, Tl, Ni?4 (ammonium or its organic derivatives where R = H, alkyl, alkenyl, alkinyl or aryl), Ni?3OJ?
(hydroxylammonium or its organic derivatives where R - H, alkyl, alkenyl, alkinyl or aryl), N2/?4 (hydrazinium or its organic derivatives where R = H, alkyl, alkenyl, alkinyl or aryl), H30 (hydronium), Ag, Au, Mg, Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, Hg, Sn, Pb, Ca, Sr, Ba.
3. Layered metallates according to any one of the preceding claims, characterized in that M is selected from the group: Li, Mg, Al, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Y, Zr, Nb, Mo, Sn, Sb, Hf, Ta, W, Ru, Rh, Pd, Os, Ir, Pt.
4. Layered metallates according to any one of the preceding claims, characterized in that P is a polyamine or polyamine cation possessing nucleic-acid binding properties.
5. Layered metallates according to any one of the preceding claims, characterized in that P is selected from the group: spermine (N,N'-bis(3- aminopropyl)- 1 ,4-diaminobutane, NH2(CH2)3NH(CH2)4NH(CH2)3NH2), spermidine (N-(3-aminopropyl)- 1 ,4-diaminobutane, NH2(CH2)3NH(CH2)4NH2), and
norspermine (N,N'-bis(3-aminopropyl)- 1 ,3-diaminopropane,
NH2(CH2)3NH(CH2)3NH(CH2)3NH2).
6. Layered metallates according to any one of the preceding claims, characterized in that the weight composition is as follows:
(1) Titanates (1): 1-30 wt.%, preferably 10-20 wt.% of polyamine; Ti02 ranges from 50 to 95%, preferably 60-70 wt.%;
(2) Niobates (2): 1 -40 wt.%, preferably 15-30 wt.% of polyamine; Nb205 ranges from 50 to 95%, preferably 60-80 wt.%;
(3) Tantalates (3): 1 -30 wt.%, preferably 10-20 wt.% of polyamine; Ta205 ranges from 50 to 95%, preferably 60-80 wt.%.
7. Use of layered metallates according to any one of the preceding claims as an adsorbent for nucleic acids.
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