Bioconjugation complexes, methods for their production (options) , phenylboronic complexing reagents, phenylboronic cross-linking reagents, the kit or system, isolation of the desired cell population
The invention relates to a group of new bioconjugation complexes of the General formula And BAS-L-Bc-L'-(Bc'-L")n-BAS', where BAS, BAS', BAS"- bioactive ingredients, which may be identical or different; Su and Su' - complexes phenylboric acid; L, L', L" - linkers; n = 0 or 1, three methods for their production, intermediate products, used in their synthesis and representing phenylboronic complexing reagents and phenylboronic cross-linking reagents, the method of selection of the desired cell population, as well as set or system containing bioconjugate And, to scroll to the desired population of cells. 8 C. and 1 C.p. f-crystals. The invention relates to the field bioconjugation drugs, in particular to the class of complex compounds (complexes) phenylboric acid used in the conjugation of biological macromolecules, and to methods for obtaining and using such complexes.A descriptive term bioconjugate means linking chemical or biological means molecules of two or more species, of which at least one molecule is a biological macromolecule. This includes the conjugation of proteins, peptides, polysaccharides, VNIMI to bring useful properties, including radionuclides, toxins, haptens, inhibitors, fluorophores, ligands, etc. a Special kind of conjugation is the immobilization of biological macromolecules, when they are subjected to reversible or irreversible conjugation with an insoluble carrier. Bioconjugate widely used in research in the field of biochemistry, immunochemistry and molecular biology. Use bioconjugate numerous and include affinity chromatography, affinity cytochemistry, histochemistry, detection of abnormalities in tests, diagnostics, amplification of signals, immunoassay, hybridoma technology, technology batterbee, bioaffinity sensors, detection of genes in samples of cross-linking reagents, determination of affinity or its breach, the transport of drugs, reagents for embedding genes, immovable reagents, selective extraction, selective shutdown, flow cytometry and analysis of cytological samples.Existing methods of cooking bioconjugates include system Avidin-Biotin and Digoxigenin-anti-Digixigenin.So functional groups for conjugation listed bioactive ingredients are the following: (a) for proteins, it is known that the conjugates, basically, get with lysine and cysteine on the overall amino acid (D3-D5); b) for glycoproteins, including immunoglobulin, it is known that the conjugates can be obtained using protein groups, as described above, or by activation in relation to the hydrazide diol groups proteichisma intermediate hydrocarbon side chain of the macromolecule adjacent to the aldehyde residue (D6-D7, describing the covalent joining of glycoproteins to dihydroxyaluminum residue on the adjacent hydroxyl groups of glycoproteins); C) for peptides, it is known that the conjugates basically get the same as above conjugatum proteins by reaction with located on the side chain amine and thiol groups (D3); g) for polysaccharides, it is well known that the conjugates, mainly produced by interaction with neighboring diol groups of the molecule (D8); d) for toxins, ligands and haptens, including drugs and hormones, it is known that the conjugates, basically, get like described above, the conjugates of the protein through interaction with located on the side chain of the macromolecule amine and thiol groups (D3); and for nucleic acids, it is known that the conjugates, mainly get through modification of the oligonucleotides in the 5-position of the pyrimidine or 8 Polo is eficacia through condensation reactive groups of the protein (i.e., amine or thiol) on the phosphate group of liposomes (D8);
l) for radionuclides known that the nuclides can be done reactive with many functional groups, including amine and tirinya group proteins (D4);
m) for inhibitors of enzymes is well known that the conjugates basically get the same as above protein conjugates, through interaction of the side chain of the macromolecule amine and tylnej groups (D3);
about to fluorophores, it is known that fluorescent proteins attached similarly to proteins, all fluorophores are amines or modified content amines and can be conjugated to a similar proteins (D11, describes the properties of known fluorophores).p) for solid-phase substrate, it is known that the substrate can be reactivated for many functional groups, including amine and tirinya group proteins (D3).It is known that phenylboric acid interact with requisitioned properties of polar molecules of many species. It is known that complexes of different stability, containing 1,2-diols, 1,3-diols, 1,2-hydroxy acid, 1,3-hydroxy acid, 1,2-hydroxyamine, 1,3-hydroxyamine, 1,2-diketones and 1,3-diketones can be formed if the but (sorbents for chromatography can be used for selective extraction of samples of complex compounds of biological molecules, with requisitioning functional properties. Many important biological molecules, including carbohydrates, catecholamine prostaglandins, ribonucleotides and steroids, have requisitioned properties and, therefore, can be allocated in this way.Phenylboric acid like boric acid is a Lewis acid and ionizes not direct deprotonization and hydration, forming a tetrahedral phenylbutane (PKand=8,86). Phenylboric acid three times stronger than boric. Ionization phenylboric acid is an important factor in the formation of the complexes, because after ionization triangular configuration bretanion (with an average angle between the bonds 120oand average length of links 1,37 angstroms) enters the tetrahedral (with an average angle between the bonds 109othe average length of relationships
). Development of systems using phenylboric acid with values PKalower than the most common of its commercially available derivatives of (3-AMINOPHENYL)boronic acid (PKa8,75) would be desirable, as would make possible the fixation of various biomolecules under physiological conditions (pH of 7.2), significantly increasing kolichestvennoi acid, include:
Now it is known that compounds having a CIS - or coaxial 1,2-diol and 1,3-disfunctionally group and, in particular, carbohydrates can form complexes with the immobilized phenylbutanone in the form of cyclic ethers only in aqueous-alkaline medium. It is known that the oxidation of 1,2-diol and 1,3-diastereomer complexes releases diols, due presumably due to the tension of the hydrolysis cycle of five-membered cyclic ether phenylboric acid, including triangular coordination of the boron atom. Coplanar aromatic 1,3-diols, like 1,8-dihydroxynaphthalene known to form complexes even in the acidic environment due to the hydrolytic stability of the six-membered cyclic ethers phenylboric acid. It is also known that substituted phenols with side residues of 1,3-hydroxyamide, 1,3-hydroxyamine and 1,3-hydroxyxine come in aqueous-alkaline medium in the reversible complexation with borate buffer annah bioconjugate research and investment in this area, the selectivity of phenylboric acid was not still used for conjugation with biological macromolecules of one or several molecular types, can give products useful functional properties. Moreover, what is new is the use of capable of complexing immobilized components to obtain complexes with the remnants of phenylboric acid. Such use is of particular interest, for example, in cases where the balance of phenylboric acid is associated with such biological macromolecule, as the antibody, which may be due to complexing component by using the selectivity of the rest of phenylboric acid chelation.Used herein, the terms have the following meanings:
The term bioactive ingredient (Bioactive species BAS) means a compound selected from the group consisting of (but not limited to) proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes, cells, drugs, radionuclides, toxins, haptens, inhibitors, fluorophores, ligands and antibodies (e.g. monoclonal antibodies with specific effects on epitope-specific populations of cells, for example, krovetvornuu also belong solid-phase substrate. Generally speaking, the bioactive ingredients are the chemicals that give bioconjugation complexes of biological activity or detecting properties. When the bioactive ingredient is combined with polyconjugated or bioconjugation complex according to the invention (for example, corresponding BAS, BAS', BAS* or BAS*' XI, XII, XIV, XVI or XVII of the following formulas), for example, after the reaction with electrophilic or nucleophilic reactive radical (i.e., the corresponding "R" in the following formulas XI, XII, XIV, XVI, or XVII), it may further contain the residue referred to electrophilic or nucleophilic reactive radicals.The term solid-phase substrate means suitable for connection with phenylboronic complexing reagent or phenylboronic reagent insoluble solid surface or a particle in the following form (i.e., in the form of metal or plastic beads that can be covered, such as carbohydrate or protein, to allow connection with phenylboronic complexing reagent or phenylboronic reagent, as described below), are suitable, for example: for stand-alone use or in the system achiece monoclonal antibody as bioconjugation complex, as is described below.The term phenylboronic complexing reagent reagent means comprising complexing the rest of phenylboric acid and a reactive group for attaching a complexing rest of phenylboric acid to bioactive ingredient, or a solid-phase substrate.The term phenylboronic reagent reagent means, consisting of a balance of phenylboric acid and a reactive group capable of attaching the balance of phenylboric acid to bioactive ingredient.The term cross-linking phenylboronic reagent reagent means comprising two residues phenylboric acid with a spacer between them.The term phenylboronic complexing polyconjugated means bioactive ingredient, or a solid-phase substrate, with a side phenylboronic complexing residue and which is the reaction product of bioactive ingredient, or a solid-phase substrate with phenylboronic complexing reagent.The term phenylboronic polyconjugated means bioactive ingredient, with a side phenylboronic residue and which is the reaction product of bioactive ingredient with phenylboronic is radiant (which may be the same as, or different), or bioactive ingredient and solid-phase substrate, which are connected by at least one boron atom, for example: at least one complex phenylboric acid; in particular, the product obtained by the reaction phenylboronic complexing polyconjugated with phenylboronic polyconjugated, or the resulting reaction phenylboronic complexing polyconjugated cross-linking phenylboronic reagent.In General bioconjugation complexes according to the invention, as shown below, correspond to (below) the formulas (I) to (X), i.e. of General formula (A)
BAS-L-Bc-L'-(Bc'-L')n-BAS' (A)
where BAS and BAS' denote bioactive ingredients (which may be identical or different);
L, L' and L" represent linkers (which may be the same or different, for example, corresponding to the groups Z, Z', Z*, Z*', Y, Y* in formulas I-X);
Su and Su' denote complexes phenylboric acid (which may be the same or different) formula D-E or E-D, where D denotes the residue of phenylboric acid (preferably obtained from, for example, a derivative or analog of phenylboric acid) and E denotes the complex-forming residue foilborne.When BAS - solid substrate, preferably Su E-D, and/or n=1, and/or BAS' - antibody.Thus, according to the invention proposed a new class bioconjugation complexes derived complexes phenylboric acid, and methods of making and using such bioconjugation complexes. According to the invention instead of the known systems of Avidin-Biotin and Digoxigenin-anti-Digoxigenin for chemical conjugation of bioactive ingredients without the participation of intermediate biological macromolecules used complexes phenylboric acid. For example, formulas I-VI, bioconjugation complexes, linking the two bioactive ingredient, in which the boron complexion with nitrogen, which in turn is connected by a spacer with a bioactive ingredient.
In bioconjugation complexes of the formula (I):
(function) the group Q is selected from O, S, NH, N-alkyl, N-aryl, NCH2-aryl, where alkyl is a hydrocarbon radical, for example, the length of C1to C4may up With6in which the chain can be branched and the aryl is an aromatic ring or substituted aromatic ring or condensed aromatic ring;
(function is R, from C2to C6and aryl is an aromatic ring or substituted aromatic ring or condensed aromatic ring;
BAS and BAS* are the same or different bioactive ingredients.In the preferred bioconjugation complexes of the formula (I): the group Q is selected from O, NH, NC6H5; group Y is from About or CH2and the groups Z and Z* can be independently selected from (CH2)n(n=l-5) and (CH2CH2O)n(n=2-4); BAS and BAS* various bioactive ingredients, which, for example, a solid-phase substrate, and the other antibody.
In bioconjugation complexes of formulas (II) and (III):
(functional) groups Q and Q' are independently selected from O, S, NH, N-alkyl, N-aryl, NCH2-aryl, where alkyl is a hydrocarbon radical, for example with chains C1-C4and possibly With6that may be branched and the aryl is an aromatic ring or substituted aromatic ring or condensed aromatic ring;
(functional) groups Y and Y' are independently selected from Oh, NH, CH2, alkyl and aryl, where alkyl is a hydrocarbon radical, for example, circuits With1-C6and aryl is an aromatic ring, or replace kilou or polyester (for example, polietilenglikoli) chain, the length of which is equivalent 1-16 (atoms) of carbon and which may optionally contain one or more amide and/or disulfide linkages;
the group Z* added two residues phenylboric acid;
BAS and BAS* are the same or different bioactive ingredients. Preferably, if BAS and BAS' are the same, then Q and Q', Y and Y', Z and Z' are also the same.In the preferred bioconjugation complexes of formulas (II) and (III):
the groups Q and Q' are independently selected from Oh, NH, N6H5; the groups Y and Y' are independently selected from Oh or CH2; group Z, Z' and Z* can be independently selected from (CH2)n(n=1-5) and (CH2CH2O)n(n=2-4); BAS and BAS* various bioactive ingredients, for example, one of them is a solid-phase substrate, and the other is not such a carrier and represents, in particular, the antibody.
In bioconjugation complexes of the formula (IV):
(functional group X is selected from N, CH3With6H5;
(functional group Y is selected from Oh, NH, CH2, alkyl and aryl, where alkyl is a hydrocarbon radical, for example, circuits With1-C6and aryl is an aromatic ring or substituted aromatic ring, or the outdoor is the spacers and contain alkyl or polyester (for example, polietilenglikoli) chain, whose length is equivalent 1-16 (atoms) of carbon and which may optionally contain one or more amide and/or disulfide linkages;
BAS and BAS* are the same or different bioactive ingredients.In the preferred bioconjugation complexes of the formula (IV):
group X selected (but not limited) from N or C6H5; group Y is selected (but not limited) from O or CH2; groups Z and Z* can be the same or different and are preferably selected (but are not limited to this choice) from (CH2)n(n= 1-5) and (CH2CH2O)n(n=2-4); BAS and BAS* various bioactive ingredients, for example, one of them is preferably a solid-phase substrate, and the other is not and it is, in particular, the antibody.
In bioconjugation complexes of formulas (V) and (VI):
(functional) groups X and X' are independently selected from H, CH3With6H5;
(functional) groups Y and Y' are independently selected from Oh, NH, CH2, alkyl and aryl, where alkyl is a hydrocarbon radical, for example, C1-C6and aryl is an aromatic or substituted aromatic ring, or kondensirovannye) chain, the length of which is equivalent 1-16 (atoms) of carbon and which may optionally contain one or more amide and/or disulfide linkages;
the group Z is linked to two remnants of phenylboric acid;
BAS and BAS* are the same or different bioactive ingredients. Preferably, when BAS and BAS' are the same, then X and X', Y and Y', Z and Z' are also the same.In the preferred bioconjugation complexes of formulas (V) and (VI):
groups X and X' is selected from O or CH2; group Z, Z' and Z* can be independently selected from (CH2)n(n=1-5) and (CH2CH2O)n(n=2-4); BAS and BAS* - bioactive ingredients, for example, a solid-phase substrate, and the other is not and it may be, for example, an antibody.
In bioconjugation complexes of the formula (VIII):
(functional group W is selected from O, NH, N-alkyl, N-aryl, N2-aryl, NC6H5, NCH2CH2OH, NCOCH2CH2OH, NOH, NO-alkyl, NOCH2-aryl, where alkyl is a hydrocarbon radical, for example, With1-C4and, it is possible to6in which the chain may be branched and the aryl is an aromatic or substituted aromatic ring or condensed aromatic ring;
(functional) group Q select the national) group Z, Z* and Z*' are the same or different spacers containing alkyl or polyester (for example, polietilenglikoli) circuit, which is equivalent 1-16 (atoms) of carbon and which may optionally contain one or more amide and/or disulfide linkages;
(functional) group Z is added to the two complexing remains of phenylboric acid;
BAS and BAS* are the same or different bioactive ingredients.In the preferred bioconjugation complexes of the formula (VIII):
the group W is selected from Oh, NH, NCH3That NC6H5, NCH2CH2OH, NCOCH2CH2OH, NOH, NOCH3; group Q is preferably About; group Z, Z* and Z*' (same or different) is preferably selected from (CH2)n(n=1-5) and (CH2CH2O)n(n=2-4); BAS and BAS* are the same or different bioactive ingredients, for example, one of them is a solid-phase substrate, and the other is not and it may be, for example, an antibody.
In bioconjugation complexes of formulas (IX) and (X):
(functional) groups W and W are independently selected from O, NH, N-alkyl, N-aryl, N2-aryl, NC6H5, NCH2CH2OH, NCOCH2CH2OH, NOH, NO-alkyl, DOWN2may be branched, and aryl is an aromatic or substituted aromatic ring or condensed aromatic ring;
(functional) group Z* added two residues phenylboric acid. If BAS and BAS* are equal, it is preferable that BAS and BAS', Q and Q', Y and Y', Z and Z' were also the same.In the preferred bioconjugation complexes of formulas (IX) and (X): group W and W' is selected from Oh, NH, N3That NC6H5, NCH2CH2OH, NCOCH2CH2OH, NOH, DOWN3; group Q preferably About; group Z, Z* and Z*' (same or different) is preferably selected from (CH2)n(n=1-5) and (CH2CH2O)n(n=2-4); BAS and BAS* are the same or different bioactive ingredients, for example, one solid phase substrate, the other is not and it may be, for example, an antibody.Bioconjugation complexes having only compound phenylboric acid, for example, as in formulas I, IV and VII are preferably for the conjugation of two different bioactive ingredients, for example, conjugation of an enzyme to an antibody for use in enzyme immunosorbent analysis for conjugation of the sample nucleic acid with fluoroform required to identify the sequence genoma. In particular, such bioconjugation complexes can be used for conjugation of antibodies (e.g. monoclonal antibodies capable of selective connection with the epitope of hematopoietic cells, for example, anti-CD 34 antibody with the solid phase (for example, metal or plastic balls, which may be organic, such as carbohydrate or protein coating), for example, when the antibody - BAS, and solid surface - BAS* or antibody BAS*, and solid surface - BAS.Bioconjugation complexes with two complex compounds phenylboric acid, for example, as in formulas II, III, V, VI, VIII, IX and X, are used preferably for the identical conjugation of bioactive ingredients of their cross-stitching in macromolecular aggregates of bioactive ingredients with side remains or complexing the remains of phenylboric acid. Units of this type, including enzymes, can be used to increase the resolution of enzyme immunosorbent and related analyses due to the significant increase in the effective concentration of the enzyme necessary for the conversion of a colorless substrate into easily detectable substance. Marked by fluoroform PR is epatirovanie to facilitate visual or spectrophotometric detection. Units with excessive residues of phenylboric acid can be further conjugated with other bioactive ingredients with side complexing remains of phenylboric acid (phenylboronate complexing polyconjugated). This General approach is similar to the preparation of products such as "sandwich" for quantitative analysis using system Avidin Biotin.Cross-linking phenylboronic reagents introduced into a large excess with subsequent removal of excess reagent can be used in reactions of transformation of bioactive ingredients with side complexing remnants of phenylboric acid (phenylboronate complexing polyconjugated) bioactive ingredients with side groups of phenylboric acid (phenylboronate polyconjugated), and Vice versa.Usually component-spacer connects the bioactive ingredient with phenyl phenylboric acid (for example, Z* or Z*' in formulas I, VII or VIII, or Z* XIV or XV, infra). However, in some cases bioactive ingredients can be configured, allowing for direct (without spacer) cross-linking with the phenyl through electric or nucleophilic moiety (R) in the formula XIV. Thus, in Obelix formulas (I) to(X) are obtained as aqueous buffer solutions, and in organic solvents and in aqueous solutions with the addition of organic solvents. The complexation takes a few minutes at room temperature. Education bioconjugation complex insensitive to significant fluctuations in ionic strength, temperature and the presence of chaotropes (denaturants protein), which is unacceptable in previously known systems in which to save the required binding properties requires the preservation of the structure of biological macromolecules. In most cases, limitations in obtaining bioconjugation complexes described here by way reduced to the choice of the desired pH and the imposition of additional restrictions dictated by the requirements of the viability of bioactive ingredients.In addition, according to the invention proposed the reagents required for the modification of bioactive ingredients for the introduction of complexing rest of phenylboric acid for subsequent conjugation with other bioactive ingredients with lateral remnants of phenylboric acid, for example, upon receipt bioconjugate formulas VII, VIII, IX or X.
Saybana preferably from H, HE, NH2, N3, NHOH, N3;
(function) the group Q is selected from O, S, NH;
(functional) groups Z and Z* - spacers independently selected from alkyl or polyester (for example, polietilenglikoli) circuits, the length of which is equivalent 1-16 (atoms) of carbon and which optionally may contain one or more amide and/or disulfide linkages;
(functional) group R in the formula XI - reactive electrophilic or nucleophilic moiety capable of complexing reaction between the reagent phenylboric acid and bioactive ingredient.Preferred reagents of formulas X and XI include: group W is selected from HE, NHOH, N3; group Q is O; Z and Z*, usually choose from (CH2)n(n=1-5), (CH2CH2O)n(n=2-4).From the reagents of formula XI are preferred those in which the radical R is selected (but without limitation only by them), amino or hydrazide groups, N-hydroxysuccinimidyl ether, N-hydroxyandrostenedione ether, isothiocyanate, bromoacetamide, iodoacetamide, maleimide and thiol. When the reaction reagent of General formula XI with a bioactive ingredient get polyconjugated formula XIII with side (one or more) complexor is Hecny defined for formula VII.
Similar phenylboronic reagents (e.g., such as according to the formula (XIV):
where Z* denotes a spacer, the same as defined for any of formulas I, IV, VII or VIII, and
R is a reactive electrophilic or nucleophilic moiety is the same as defined for formula XI can be attached to the bioactive ingredients to obtain polyconjugated formula XV with one or more lateral remnants of phenylboric acid:
Here the symbol BAS* indicates a bioactive ingredient, denoted by identical BAS (or different), and the group Z* has been defined for formula VII.Note that polyconjugated formula XV and others described here phenylboronic reagents and polyconjugated have either tetrahedral phenylbutane in alkaline or phenylboric acid triangular configuration in a neutral or acidic environment, for example:
moreover, both forms are included in the scope of the invention.Cross-linking phenylboronic reagents correspond to the formula (XVI):
where Z* denotes the linker set is defined for any of formulas III, VI, X.To obtain polyconjugated formula VII polyconjugated formula XIII prepared from bioactive ingredient BAS and having side groups of complexing rest of phenylboric acid, can be introduced into the reaction of complexation with polyconjugated formula XV, made from the second bioactive ingredient BAS* and having side groups of the remainder of phenylboric acid. In this way biological macromolecules can be conjugated to each other and with other functional groups to achieve beneficial properties.Similarly, the reagent of formula XII can be introduced into the reaction of complexation with polyconjugated formula XIV is prepared from bioactive ingredient BAS*, and to get in the end bioconjugate formula VIII. This method can be conjugated to two or more identical bioactive ingredient BAS*. This method can be used to prepare aggregates of enzymes for highly sensitive detection of the enzyme immunosorbent assay.Bioconjugate receive buffer aqueous or aqueous-organic solutions. They are formed for a few minutes at room temperature. The stability of the specific bioconjugate at a given pH depends on weddnig solutions with a pH below 4.5. Bioconjugate formulas VII and VIII, in which the group W is selected from NH and NCH, and the group Q from O and S, which are stable in buffered aqueous-alkaline solutions with a pH in the range of about from 8.5 to 11.5. Similarly, bioconjugate formulas VII and VIII, in which both groups W and Q presents NH, are stable in buffered aqueous-alkaline solutions with a pH in the range of about from 8.5 to 11.5. Bioconjugate formulas VII and VIII, in which the group W is selected from NOH and NOCH, and the group Q from O and S, which are stable in buffered aqueous solutions with a pH in a wide range of about from 2.5 to 11.5.The reaction bioconjugate (education complexes phenylboric acid) is insensitive to significant fluctuations in ionic strength, temperature, presence of organic solvents and chaotropic (denaturants protein), which is unacceptable in known systems in which to save the required binding properties requires the preservation of the structure of biological macromolecules. In most cases, limitations in obtaining bioconjugation complexes described here by way reduced to the choice of the desired pH and additional restrictions dictated by the requirements of the viability of bioactive ingredients.In the following embodiment according to the invention is provided Benevolenskaya balance phenylboric acid, for example, to get bioconjugation complexes of formulas I, II, III, IV, V or VI), i.e., the reagents of the General formula (XVIII) or (XIX):
In the formula (XVIII) X, Y, and Z are defined as for formula IV, V or VI. For formula (XVIII) X is preferably selected from N, CH2With6H5and Y is from About and CH2. For the formula (XIX) Q, Y and Z set the same as for formulas I, II, or III, and group Y is preferably selected from O and CH2and Q is O. In both formulas (XVII and XIX) R is an electrophilic or nucleophilic moiety capable of reaction phenylboronic complexing reagent with a bioactive ingredient.For General formula XVIII is most preferable to select a group X from N and CH3and for the group Y to take Acting To the General formula XVI as Y is preferable to choose Acting In both formulas XVIII and XIX as Z is preferable to choose either alkyl or polyester (for example, polietilenglikoli) chain equivalent to the length 1-16, preferably 2-12 carbon atoms, and the chain may contain one or more amide and/or disulfide functional groups, preferably (CH)nfor n=2-6, or - (CH2CH2O)nwhen n=2-4. In both formulas XV is cinemediapromo ether, N-hydroxyandrostenedione ether, isothiocyanate, ether of imidate, 2,2,2-cryptgethashparam, iodoacetamide, maleimide and 2-Tianeti-m,M-diisopropylphosphoramidite ether.Reagents of formula XVIII or XIX react with the bioactive ingredients BAS (or BAS*) or, respectively, form polyconjugated formula (XX) and (XXI):
where X, Y and Z are defined as for formula XVIII.
where X, Y and Z are defined as for formula XIX.Thus, according to the invention suggested:
1. Bioconjugation complexes, described above, for example, formulas (I) to (X);
2. Phenylboronic complexing polyconjugated described above, for example, formula XII, XX and XXI;
3. Phenylboronic polyconjugated according to the formula XV;
4. Phenylboronic complexing reagents described above, for example, formula XI, XVIII, and XIX.5. Phenylboronic reagents described above, for example, of the formula XIV;
6. Cross-linking phenylboronic reagents (crosslinker), described above, for example, formula XII, XVI and XVII.In addition, according to the invention the methods of use of the produced compounds in any of the described applications for b and, and ways to use get polyconjugated, crosslinkers and reagents (e.g., of formulas XI-XXI) to obtain bioconjugation complexes (e.g., formulas I-X).In a particular embodiment according to the invention proposed a set of tools or system for separation or purification of cells, for example, hematopoietic cells, in particular cells CD 34+consisting of bioconjugation complex (corresponding to any of formulas I-X) connecting the first and second bioactive ingredients, of which the first solid phase substrate, for example, metal or plastic ball (possibly), covered with a carbohydrate, protein or other organic matter, providing the reactivity and the ability to establish communication with the reagent according to the invention, for example, R in formula XI, XIV, XVIII and XIX, and the second bioactive ingredient the antibody can, for example, to recognize and to gain a foothold in the epitope present in a specific population of cells, for example, cells CD 34+; proposed above the isolation and purification of cells, including:
the contact carrier of the required cells with bioconjugation complex according to the invention containing a second bioactive igrok from the media, and
if necessary, the Department selected cells from bioconjugation complex.Bioconjugation complexes of formula I, IV or VII get in three stages:
(1) Phenylboronic complexing reagent, preferably obtained from salicylic acid, aminosalicylic acid or dithiosalicylic acid, condense together with a bioactive ingredient to obtain phenylboronic complexing polyconjugated;
(2) Phenylboronic reagent derived from (3-AMINOPHENYL) boronic acid, or from (4-carboxyphenyl) boric acid (or other connection), corresponding, for example, the formula XIV, condense together with a bioactive ingredient to obtain phenylboronic polyconjugated;
(3) Phenylboronic complexing polyconjugated obtained in stage (1), and phenylboronic polyconjugated obtained at stage (2), is introduced into the reaction for obtaining bioconjugation complex, for example, of formula I, IV or VII.Bioconjugation complexes of formulas II, V or IX get in two stages:
(1) Phenylboronic complexing reagent of formula XI, XVIII, or XIX condense together with a bioactive ingredient to obtain phenylboronic complexion, the lead reacts with the cross-linking phenylboronic reagent, preferably obtained from (3-AMINOPHENYL) boronic acid, for example, according to the formula XVI.Bioconjugation complexes of formulas III, VI or X get in two stages:
(1) Phenylboronic complexing reagent of formula XI, XVIII, or XIX enter into reaction with a bioactive ingredient to obtain phenylboronic complexing polyconjugated;
(2) Phenylboronic complexing polyconjugated prepared in stage (1), enter into reaction with cross-linking phenylboronic reagent, preferably obtained from (4-carboxymethyl) boric acid, for example, according to the formula XVII, to obtain the desired complex.Bioconjugation complexes of the formula VIII get in two stages:
(1) Phenylboronic reagent of formula XIV is introduced into reaction with a bioactive ingredient to obtain polyconjugated formula XV;
(2) Polyconjugated formula XV is introduced into reaction with cross-linking phenylboronic reagent according to the formula XII, preferably derived from salicylic acid, aminosalicylic or dithiosalicylic acid to obtain the desired complex.Reagents of formula XIV are obtained from derivatives and is a selection from the group consisting of (3-AMINOPHENYL) boronic acid, (4-carboxyphenyl)boric acid, N-(6-nitro-3-dihydroxybiphenyl)amino amber acid, (3-isothiocyanates) boric acid, (5-carboxy-3-isothiocyanates) boric acid, (3-iodoacetamide) boric acid, (3-maleimide) boric acid, Succinimidyl ether (3-dihydroxybiphenyl) succinic acid hydrazide (3-dihydroxybiphenyl) of succinic acid, which may be purchased or synthesized by methods described (or similar to that described, for example, Linder, K. E., Wen, M. D., Nowotnik, D. P., M. F. Malley, Gougoutas, J. Z., A. D. Nunn and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170, and R. Linder, T., Wen, M. D., Nowotnik, D. P., Ramalingam K. , Sharkey, R. M., Yost, P., Narra R. K., Nunn, A. D. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 407-415.Phenylboronic reagents of formula XVI is obtained by condensation of (3-AMINOPHENYL) boronic acid in the presence of an activated dicarboxylic acid, preferably selected (but without limitation, from the group consisting of succinylcholine, editorchoice, adipic acid, diisobutylamine, suberoylanilide, 3,3'-dithiopyrophosphate, 3,6,9-trioxa-undecidability and diisobutylamine 3,6,9-trioxadecyl acid, similar to the method described Burnett T. J., Peebles C and H. Hageman J. H. (1980) Biochem. Biophis. Research Commun., 96, 157-162.FeNi is imidom with subsequent condensation in the presence of a diamine, preferably selected (but without limitation) from the group consisting of 1,4-butanediamine, 1,6-hexanediamine and 2,2'-dithiodipyridine (H2NCH2CH2SSCH2CH2NCH2).Bioconjugation complexes receive buffer aqueous solutions of compounds, preferably selected (but without limitation this choice) from the group consisting of acetate, citrate, phosphate and carbonate buffers. You should not use borate and Tris buffers due to their ability to complex formation, respectively, with complexing remnants of phenylboric acid and the remnants of phenylboric acid. Bioconjugation complex is formed within 1-15 minutes at room temperature. The reaction is insensitive to fluctuations in ionic strength in the range from 0.01 to 2 Modernista. The stability of the complex increases with increasing temperature and is limited only by the volatility of the buffer. The addition of organic solvents, including acetonitrile, methanol, ethanol, isopropanol, butanol, N,N-dimethylformamide and dimethylsulfoxide, contributes to the stabilization bioconjugates. Chaotrope reagents (denaturant protein) including urea, guanidine hydrochloride and formamid, also contribute to further stabilization bioconjugates, e is W ith a salting out, dialysis, chromatography classification by size and by electrophoresis. Bioconjugation complexes are stable during storage after removal of water and can be dried.Ionization phenylboric acid is an important factor in education bioconjugation complexes, for after her baratarian change the configuration with a triangular (with an average value of the angle between the bonds 120oand average length of ties
) tetrahedral (with an average value of the angle between the bonds 109oand average length of ties
). Phenylboric acid have PKavarying in the range of about from 5.2 to 9.2. Bioconjugation complexes of General formulas I, III, IV, VI, VII, VIII and IX, obtained from (4-carboxyphenyl) of boric acid are the values of the PKain the range of about 6.5 to 7.5. Bioconjugation complexes of General formulas I, II, IV, V, VI, VII and VIII, obtained from (3-AMINOPHENYL) boronic acid matter PKawithin approximately 8 to 9. Complexes of General formulas I, II, IV, V, VII and IX, obtained from (3-amino-2-nitrophenyl)boronic acid, (3-amino-5-nitrophenyl)boronic acid or (3-amino-6-nitrophenyl) boronic acid have intermediate values PKa. As a rule, RK
20 ml) and dried in vacuum over night.1.26 g (0,01 mol) of (3-AMINOPHENYL) boronic acid are dissolved in 50 ml saturated NaHCO3when briefly heated the mixture on a hot tile. The solution is cooled in an ice bath to about 0oAnd with vigorous stirring, added 1.55 g (0,01 mol) N-methoxycarbonylamino. After 10 min the solution was diluted with 200 ml of water and stirred at room temperature for 30-40 min the pH was adjusted to about 5.5 by the addition of 1 mol H2SO4and the precipitate is filtered off, cotnam pellets of NaOH, receiving a result of 1.39 g (yield 64%) of (3-maleimide) of boric acid. The structure is confirmed by 'H NMR spectroscopy at 300 MHz in DMSO.Example IV. The use of a reagent of General formula XIV, reactive towards thiol.Proteins containing disulfide bonds (cystine residues) or cysteine residues can be modified reactive towards thiol phenylboronate reagents of formula XIV. First, if required, by reaction with 2-mercaptoethanol or dithiothreitol in aqueous-alkaline buffer to restore the disulfide bonds. The excess of the reducing reagent is removed by dialysis or by salting out, and the protein is introduced intoreaction with (3-maleimide) boric acid in 25-100 M phosphate buffer at pH of 7.0 to 7.5 for 1 h at a temperature of 4oFrom getting in the end polyconjugated with lateral balance of phenylboric acid, covalently bound to protein. Proteins without tylnej groups can be given functional properties through reaction with Teollisuuden reagent with subsequent modification, as described above. Tiliroside reagents, which, as installed, can be used for this purpose include N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate, N-hydroxybiphenyl)amino amber acid of General formula XIV, reactive relative to the aldehyde.10 ml of methanol cooled in an ice bath to about 0oWith and slowly add 1 ml of thionyl chloride. To the resulting solution while stirring add to 1.25 g (0,005 mol) of N-(3-dihydroxybiphenyl)amino amber acid, obtained as in example I, and conducting the reaction while stirring over night at room temperature. The solution was concentrated in a rotary evaporator, obtaining in the end a white crystalline substance, which is twice evaporated with methanol (2
10 ml) to remove residual thionyl chloride. The substance is dissolved in 5 ml of methanol with the addition of 1 ml of hydrazine hydrate is added. The resulting solution is stirred over night at room temperature. After a few hours, a precipitate, which is filtered off, washed with cold methanol and dried in vacuum for days over NaOH pellets, getting in the end of 1.11 g (yield 88%) hydrazide, N-(3-dihydroxybiphenyl)amino amber acid. The structure is confirmed by 'H NMR spectroscopy at 300 MHz in DMSO.Example VI. The use of the reagent of formula XIV, reactive relative to the aldehyde.Glycoproteins, in particular antibodies can be conjugated to hydrazide penalba is the atrium NaIO4(in quantities of from 5 to 20 mmol) within 30 min to 4 h at a temperature of 0oWith in (0,1-0,5) M buffer solution of sodium acetate with a pH from 5 to 6, containing 0.2 M NaCl. Excess periodate removed by salting out, and activated protein C adjacent side aldehyde groups introduced in the result of the oxidation periodata residues of carbohydrates having a 1,2-diol components condense in the presence of hydrazide reagent for 1 to 24 h at room temperature, obtaining in the end polyconjugated side with the remnants of phenylboric acid, covalently linked to a protein core communication Schiff (imine type). The communication stability with protein, if desired, can be increased by restoring the Schiff base weak Laboratorium to the appropriate alkylamine. It is important to note that the oxidation of the glycoprotein periodata activates the protein to react with the hydrazide reagent type, while removing the formed naturally by complexing the remains of phenylboric acid (coaxial 1,2-diols), associated with the glycoproteins.Example VIa (original number VI, re - approx. the translator). Synthesis phenylboronic complexing reagents of General formula 4-aminosalicylic, either 5-aminosalicylic acid. Accordingly, 4 - or 5-aminosalicylic acid first etherification to obtain or methyl -, 4-, or methyl-5-aminosalitsilata. The ester is neutralized and then lidiruyut reaction with an amine, selected from the group consisting of ammonia, methylamine and hydroxylamine, receiving the result, 4 - or 5-aminosalicylate formula (XXII):
where R' is H, W is selected from NH2, MSN3, NHOH, and Q is N.This compound is then condensed in the presence of an activated carboxylic acid, preferably (but without limitation) selected from the group consisting of succinic anhydride, methylsuccinate, maleic anhydride, N-methoxycarbonylmethyl, 3-bromopropionitrile, 3-iodopropionic, iodotetrachloride, bromocatechol and chlorocatechol, and receive appropriate 4 - or 5-aminosalicylate, in which Q is O, W is - NH2, NHCH3or NHOH, a R becomes amidol formula Z ' -CO-, where Z is CH2CH2COOH, CH2CH2SOON3CH= SSOON, CH2CH2Br, CH2CH2I, CH2I, CH2Sh or CH2Cl.Compounds in which Z is CH2I or CH2VG, can be used as reactive towards thiol reagents, with the side tirinya group. Compounds in which Z is CH=SSOON, can be further functionalized by shorting ring, so get reactive towards thiol maleimide reagent capable of binding complexing the remainder of phenylboric acid with bioactive ingredients with side tirinya group. If Z is CH2CH2SOON2such connection may be further functionalized by reaction with hydrazinehydrate eventually get what hydrazide reagent, in which Z is CH2CH2CONHNH2capable of connecting the complexing the remainder of phenylboric acid with a bioactive ingredient that has side aldehyde group (arisen in the result of oxidation of the carbohydrate residue by periodate). If Z" is selected from the group consisting of CH2CH2Sh and CH2CH2I, such a connection may be further functionalized by reaction with thioacetate potassium, with the result that receive the intermediate compound from which deprotonization get tiradera connection with Z - CH2CH2SH. Tiradera connection can be activated by reaction with a reagent, preferably (but without limitation) selected from the group consisting of 2,2'-dithiodipyridine, able to bind split by a disulfide bond complexing the remainder of phenylboric acid having a side tirinya group bioactive ingredient.If the formula XXII W - NH2or N3and the group Z is CH2CH2COOH, further functionalization of possible handling dicyclohexylcarbodiimide (DCC) and a reagent, preferably (but without limitation) selected from the group consisting of N-hydroxysuccinimide (NHS) and N-hydroxysultaine (SNHS), eventually activated ether reagent capable of binding complexing the remainder of phenylboric acid having lateral amino groups of the bioactive ingredient. Activated esters of formula XXII, where W is NH2or NHCH3a Z - CH2CH2CO-NHS, can be used as intermediate compounds for the synthesis of reagents of formula XI, where the group Z is an alkyl or polietilenglikolya circuit equivalent to the length of at least 6 (atoms) of carbon.Compounds where W is NHOH, cannot be directly used to obtain the water-activated essential residues because of problems arising from the activation of the carboxyl group in the presence of simultaneously NHS and benzohydroxamic-because of the prevalence of N-hydroxysuccinimide reagents and the fact that only reagents of formula XI, where W is NHOH or or NHOCH3form stable complex compounds in a wide range of pH. To remove this limitation, to obtain activated essential reagents of formula XI using another method of synthesis, for which the group W - NHOCH3a Q - Acting 4 - or 5-aminosalicylic acid condense with methylsuccinimide with obtaining the compounds of formula XXII, where W is HE, and the group Z is CH2CH2SOON3. Subsequent reaction with N,N-carbonyl diimidazol and adding methoxyamphetamine give a compound in which W is NHOCH3and Z' is CH2CH2SOON3. Alkaline hydrolysis of the ester group gives compound with a free carboxyl group. Further, the activation of the carboxyl group processing DCC and reagent, preferably (but without limitation) selected from NHS and SNHS, network activated essential reagent in which W - NHOCH3and Z - Succinimidyl and which is suitable for joining complexing rest of phenylboric acid to having amino groups of the bioactive ingredient. Such N-Succinimidyl esters serve as intermediate reactants for the synthesis of reagents of formula XI, in which Z contains alkyl or esters can be further functionalized by treatment with a reagent, preferably (but without limitation) selected from the group consisting of 6-aminohexanoic or 4-aminobutanoic acid, N-tert-butoxycarbonyl-1,6-diaminohexane, (N-BOC-1,6-diaminohexane) and N-BOC-1,4-diaminobutane, with the result that after removal (if necessary) protective Treatment group receive a connection to an elongated spacer and the terminal or carboxyl, or amino group. The above reagents with lateral carboxyl groups can be used for the preparation of NHS-ester, SNHS-ester and hydrazide-containing reagents with long spacers that are useful when Troubleshooting steric problems, as is well known characteristic of biomolecules with high molecular weight. Similarly, the above-mentioned reagents with lateral amino groups can be used to obtain iodoacetamide, maleimide and activated disulfide containing compounds with long spacers. In addition, the above-mentioned reagents with lateral carboxyl or amino groups can be used as intermediate compounds in the preparation of solid-phase substrates.Example VII. Synthesis phenylboronic complexing reagents of formula XII.Reagents of formula XII, where the group W is selected from NH2, N3and NHOH, and Q is About what rmula XI. 4 - or 5-aminosalicylate, prepared as described above, condense in the presence of an activated dicarboxylic acid, preferably (but without limitation) selected from the group consisting of succinylated, adipocere, diisobutylamine adipic acid, suberoylanilide, 3,3'-dithiopyrophosphate, 3,6,9-trioxadecyl, and diisobutylamine 3,6,9-trioxadecyl acid, receiving as a result, the compound of the formula XII, in which the group W is selected from NH2, NHCH3and NHOH, group Q, and the group Z* (but without limitation) - (CH2)2, (CH2)4, (CH2)6, (CH2)2SS(CH2)2and CH2(Och2CH2)2Och2.Reagents of formula XII, where the group W - NHOCH2and the group Q - O get an alternative way similar to obtaining a reagent of formula XI, where W - NHCOH3. 4 - or 5-aminosalicylic acid condense in the presence of an activated dicarboxylic acid, preferably (but without limitation) selected from the group consisting of succinylated, adipocere, diisobutylamine adipic acid, suberoylanilide, 3,3'-dithiopyrophosphate, 3,6,9-trioxadecyl, and diisobutylamine 3,6,9-trioxadecyl (but do not limit choice) of the elements (CH2)2(CH2)4, (CH2)6, (CH2)2SS(CH2)2and CH2(Och2CH2)2Och2.Subsequent reaction of compounds of formula XII, where W is HE, Q, and Z* is selected (but without limitation) of (CH2)2, (CH2)4, (CH2)6, (CH2)2SS(CH2)2and CH2(Och2CH2)2Och2with CDI followed by the addition of methoxyacetanilide gives compound in which W is NHOCH3and groups Q and Z* meet certain in advance.Example VIII. Preparation of 4-amino-2-hydroxyanthraquinones.100 ml of absolute methanol and sizecontentelement H2SO4gently mixed in a 250 ml round bottom flask under constant stirring (exothermic process). Adding 10.0 g, (65,4 mmole) of 4-aminosalicylic acid, get a dark solution, which is heated under reflux for 6 hours resulting substance is allowed to cool and then evaporated in a rotary evaporator to reduce the volume to about half. After that you receive a solid residue. The concentrate is poured into 400 ml of water and the resulting suspension is titrated to a pH of about 3 by adding at the beginning (to pH 6.5) with 5 N NaOH, then a solid Na2oC. the Structure is confirmed by 'H-NMR spectrometry in d6-acetone.4.0 g NaOH in 16 ml of water is carefully added to 2.8 g (40 mmol) of hydroxylaminopurine and 20 g of ice. Once dissolved, add first, 0.4 g of Na2SO3then at 3.35 g (20 mmol) of methyl-4-aminosalitsilata. The resulting solution was stirred for 24 h at room temperature, checking the course of the reaction every few hours using HPLC. The resulting solution was cooled in ice and acidified with addition of 25% H2SO4. The precipitate formed initially at pH approximately equal to 6. Finally the pH was adjusted to 4, and light brown precipitate are filtered. The resulting material is dried in vacuum over P2About5getting up 3.0 g (yield 89%) of 4-amino-2-hydroxyanthraquinones (tPL= 180-181oC). The structure is confirmed by 'H NMR spectroscopy at 300 MHz in DMSO.4-amino-2-hydroxyanthraquinones is a key intermediate compound in the preparation of reagents of both formulas I and II, in which the group X IS NHOH, and group Y Is O. Reagents with the remnants of the 2-hydroxyanthraquinones can obree IX. Preparation phenylboronic complexing reagents of formula XI, reactive relative to the aldehyde.To prepared as described above, cooled on ice and containing 7.0 g (0,02 mol) NaHCO3to a solution of 8.4 g (0.05 mol) 4-amino-2-hydroxyanthraquinones in 150 ml of water with stirring dropwise within 15 min added 9.0 g (0,06 mol) 3-carbomethoxyamino. After stirring for 1 h at a temperature of 0-5oWith the solution acidified with cold 5 N Hcl. The precipitate was separated and dried in vacuum over granuloma NaOH, resulting in total of 13.5 g (yield 96%) of crude N-4-(3-carbomethoxyamino)-2-hydroxyanthraquinones, which is used without further purification.To a solution of 10 g (or 0.035 mole) of N-4-(3-carbomethoxyamino)-2-hydroxyanthraquinones in 50 ml of methanol, add 12 ml of hydrazine hydrate is added. The reaction is continued overnight at room temperature. The resulting material is filtered off, washed with ether, twice recrystallized from dimethylformamide and up to 7.3 g (yield 78%) of N-4-(3-carbomethoxyamino)-2-hydroxyanthraquinones.Example X. the Application phenylboronic complexing reagents, reaktsionnosposobnykh against aldehyde-hydrazide phenylboronate complexing reagents after processing of the protein by 5-20 mmol of metaperiodate sodium (NaIO4), containing up to 0.2 moles NaCl 0.1-0.5 M buffer solution of sodium acetate at pH 5 to 6 and a temperature of 0oC for from 30 minutes to 4 hours, the Excess periodate removed by dialysis or by salting out, and activated protein C on the adjacent side of the aldehyde groups formed by the oxidation of carbohydrate residues by periodate, and adjacent coaxial 1,2-diol residues condense with hydrazide reagent for 1 to 24 h at room temperature, obtaining in the end polyconjugated with side complexing remnants of phenylboric acid, covalently linked to a protein with a link type of Schiff base (imine type).The stability of the connection with the protein can be increased by restoring the Schiff base to the appropriate alkylamine weak lamborghini.com sodium. It is important to note that the oxidation of the glycoprotein metaperiodate sodium activates the protein to react with the hydrazide reagent type, while removing the formed naturally by complexing the remains of phenylboric acid (coaxial 1,2-diols), associated with the glycoproteins.Example XI. Preparation of complexing reagents phenylboric acid of formula XI, reactiondoxycycline in 150 ml of water, containing 42,0 g (0.5 mole) Panso3within 15 min was added dropwise to 18.4 g (of 0.09 mole) of educationed. After stirring for 1 h at a temperature of 0-5oWith the solution acidified with cold 6 N Hcl. The precipitate was separated and dried in vacuum over granuloma NaOH, resulting in total of 21.3 g (yield 96%) of crude 5-(iodoacetamide)salicylamide.Example XII. The use of complexing reagents phenylboric acid reactive towards thiol.Proteins containing disulfide bonds (cystine residues) or cysteine residues may be modified such reactive towards thiol complexing reagent phenylboric acid, 5-(iodoacetamide)salicylamide. In the beginning, if you want, disulfide bonds is reactivated by treatment with 2-mercaptoethanol or dithiothreitol in carefully obezvojennom aqueous-alkaline solution. Excess reductant reagent is removed by dialysis or by salting out, and the protein is introduced into the reaction with the alkylating reagent in neutral aqueous solution for 1 h at 4oFrom getting in the end polyconjugated side complexing the remainder of phenylboric acid, covalently bound to protein. Upon completion of the reaction, the excess reagent is removed actionspane against Amin.To prepared as described above, cooled on ice containing 7.0 g (0,02 mol) Panso3to a solution of 7.7 g (0.05 mol) of 4-aminosalicylic acid in 150 ml of water with stirring dropwise within 15 min added 9.0 g (0,06 mol) 3-carbomethoxyamino. After stirring for 1 h at a temperature of 0-5oWith the solution acidified with cold 6 N Hcl. The precipitate was separated and dried in vacuum over NaOH pellets, getting up 11.9 g (yield 89%) of crude N-4-(3-carbomethoxyamino) salicylic acid, which is used without purification.To a vigorously stirred solution of 10.0 g (being 0.036 mole) of N-4-(3-carbomethoxyamino) salicylic acid in 50 ml of tetrahydrofuran are added in several portions of 5.84 g (being 0.036 mole) of 1,1'-carbonyldiimidazole and 3.0 g (being 0.036 mole) of methoxyacetanilide. Vessel supply drying tube and the reaction is carried out under vigorous stirring for 30 min at room temperature. Released in the reaction imidazolidinone filtered. The filtrate is evaporated in a rotary evaporator to obtain an amber oil, which was dissolved in 10 ml of warm tetrahydrofuran and then added to 150 ml of 2 N H2SO4. The precipitate is filtered off, washed with 2 N H2SO4and water and dried over benzohydroquinone.of 7.4 g (0,025 mol) of N-4-(3-carbomethoxyamino)-2-hydroxy-O-methylbenzhydrylamine dissolved in 25 ml of 0.2 N methanolic LiOH. The solution is stirred over night at room temperature under nitrogen. The methanol is removed in a rotary evaporator, and the residue is dissolved in 150 ml of water. The solution is acidified with 2 N H2SO4to about pH 2 and extracted with 100 ml of ether. After repeated extraction with a mixture of ether extracts dried over anhydrous Na2SO4. The resulting material was concentrated in a rotary evaporator, then dried over night in vacuum over P2About5getting up 6,28 g (yield 89%) of N-4-(3-carbomethoxyamino)-2-hydroxy-O-methylbenzhydrylamine.the 5.65 g (0,02 mol) of N-4-(3-carbomethoxyamino)-2-hydroxy-O-methylbenzhydrylamine dissolved in 50 ml of hot dimethylformamide and allowed to cool to room temperature. To the solution was added with stirring first 2.3 g (0,02 mol) of N-hydroxysuccinimide, and then freshly prepared solution of 4.1 g (0,02 mol) dicyclohexylcarbodiimide in 10 ml of dimethylformamide. The resulting suspension is stirred over night at room temperature is filtered off from the solution dicyclohexylamine, and the filtrate koncentriruiutsia and 2-propanol and briefly dried in vacuum over P2About5getting up 5.6 g (yield 74%) Succinimidyl ether N-4-(3-carbomethoxyamino)-2-hydroxy-O-methylbenzhydrylamine, which is stored in the freezer at -15oC.Example XIV. The use of complexing reagents phenylboric acid reactive towards amine.Proteins can be conjugated with complexing reagents phenylboric acid reactive towards amine, by reaction with the e-amino group lysine residues in the side chain with the receipt of polyconjugated with side complexing remnants of phenylboric acid, covalently linked to protein a stable amide bonds. For removal of hydrogen from the amino group while minimizing hydrolysis of the NHS-ester should be used slightly alkaline aqueous buffers with a pH range from 7.8 to 8.8 except for buffers containing primary amines, including Tris and glycine, to avoid cross-reactions. Solid-phase substrate with the side of the primary amino groups can be functionalized in a similar way by reaction with phenylboronic complexing reagent with the receipt of a solid-phase substrates with komplyeksoobrazuyusciye formula XII.6.4 g (of 0.038 mol) of 4-amino-2-hydroxyanthraquinones dissolved in 50 ml of dry dichloromethane. Add to 5.3 ml (of 0.038 mole) of triethylamine, and then for 2 h was added dropwise a solution of 5.0 g is 0.019 mole) of 3,6,9-trioxadecyl in 50 ml of dry dichloromethane. Triarylmethane removed by filtration, the filtrate washed with water (2
100 ml), then saturated Panso3(2100 ml) and saturated NaCl (100 ml) and dried over anhydrous Na2SO4. The solvent is removed in a rotary evaporator, and the residue will recrystallized from 100 ml of methanol, receiving 6.0 g (yield 61%) of colorless crystals. The structure is confirmed by 'H NMR spectroscopy in DMSO.Example XVI. The use of complexing reagent phenylboric acid of formula XII.Proteins conjugated with phenylboric acid, can be cross linked reagents of formula XII. This can be particularly useful in the preparation of protein aggregates which are used to modify the properties of the protein, as stability and solubility. In addition, aggregates of enzymes obtained cross-stitching, used to increase the sensitivity of enzyme immunosorbent assay. This principle, which we initially used to produce high molecular weight complex with Avidya (cross stitching), and then enter this complex enzyme immunosorbent analysis for highly sensitive detection of biotinylated antibodies.Example XVII. Total synthesis phenylboronic complexing reagents of formula XVIII.Reagents of formula XVIII, for example, in which X is selected from the group N, CH3and C6H5, a Y - Oh, obtained by condensation of N-hydroxyphthalimide connection with the General formula R1-Z-R2where R1selected from Br, Cl and I (preferably Br), R2- from Br, Cl, I, CO2N and CO2CH3(preferably from Br, CO2N and CO2CH3), a Z are defined as in formula XVIII, i.e., preferably as a spacer, in the form of either alkyl or polyether chain, which is equivalent to the length of 2-12 carbon atoms and may have intermediate amide functional group, i.e. preferably chain (CH2)n(n=1-5) or (CH2CH2O)n(n=2-4).(a) initial reactions the substance of the General formula R1-Z-R2warm up in dimethylforamide with one equivalent of N-hydroxyphthalimide at a temperature of from 60oWith up to 100oTo obtain the solution. Then the solution is allowed to cool to room temperature, adding one equivalent of triethylamine and getting ito the ur for 1 to 4 days, checking the course of the reaction by thin-layer chromatography (VGH). Upon completion of the reaction, water is added to cause precipitation, the precipitate washed with water and dried at room temperature, obtaining in the end a matter in which R1- phthalimido, a R2as defined previously. Substance in which R2selected from Br, Cl and I, condense together with the reagent, preferably selected (but without limitation) from C6H4(CO)2NK, CH3COONa and CH2COSK. Conditions can be changed depending on the choice of the desired product, but usually you need to add 1.1 equivalent of one of the compounds C6H4(CO)2NK, CH3COONa, CH2COSK in polar solvent selected from the group consisting of acetic acid, dimethylformamide, methanol or ethanol, at the boiling under reflux for 1 to 24 hours(b) the Intermediate from (a) is subjected to acid catalytic hydrolysis by phthalimide group so that R1turns into NH2, R2azetiliruetsa to R2Ac, where R2Ac selected from N(CO)2With6H4OSON3and S3a Z is as defined previously. Acid catalytic hydrolysis by phthalimide group provided that R2- CO2CH3dailymed group treated with concentrated Hcl, or concentrated Hcl in acetic acid, or 30% Nug, or 48% Nug under reflux for from 15 to 60 minutes In each case, after cooling to room temperature, phthalic acid as a by-product is filtered off from the resulting solution, after cooling to room temperature. Volume reduce and neutralize the substance or NaOH, or Panso3or Paso3. The final product produce by extraction with ether or ethyl acetate followed by concentration in vacuo.(c) Intermediate (b), condensed with a reagent which is either salicylaldehyde or 2-hydroxyacetophenone or 2-hydroxydiphenylamine to obtain the corresponding carbonimidoyl balance R1. This condensation is carried out in an environment of methanol, or 90% methanol boiling under reflux for 4 to 12 h at 60oFrom watching the reaction by TLC. The product was concentrated in vacuo and then dried in a desiccator over night.(d) the product from (C) remove the protection alkaline catalytic hydrolysis in a warm water solution To2CO3or NaOH for 8 to 24 h, resulting in the end of the intermediate of General formula R5 in which R3and Q are as previously defined. None vacuum. The protective group is removed by reaction with hydrazinehydrate in the environment of ethanol by boiling under reflux for 12 to 24 hours Dropped phthalhydrazide filtered from the solution, which is concentrated. The product is extracted with ethyl acetate, dried over anhydrous MgSO4and concentrated in vacuo.(e) the Final product is produced by activation of amino, hydroxyl, tylnej or carboxyl groups associated with products. The amino group can be activated by treatment with a reagent, preferably (but without limitation) selected from the group consisting of bromoxynil anhydride, iuxury anhydride and maleic anhydride. Hydroxyl groups can be activated by treatment with a reagent, preferably (but without limitation) selected from the group consisting of 2,2,2-triftoratsetofenona, pentafluorobenzenesulfonyl and 2-Tianeti-N,N-diisopropylchlorophosphoramidite. Tirinya group can be activated by treatment with a reagent, preferably (but without limitation) selected from the group consisting of 2-dipyrido, 4-dipyrido and 3-nitro-2-mercaptopyridine. Carboxyl groups can be activated by treatment with a reagent, preferably selected from dicyclohexylcarbodiimide, and the frame of the group, containing N-hydroxysuccinimide and N-hydroxysultaine. On the other hand, the carboxyl group can be tarifitsirovana alcohol, preferably methanol or ethanol, and then functionalized by reaction with either hydrazinehydrate, or with hydroxylamine.Substances of General formula XVIII, in which X is selected from N, CH3and C6H5, a Y - CH2receive, as described previously, with the substitution of N-hydroxyphthalimide phthalimido sodium in the first stage of the synthesis.Example XVIII. Total synthesis phenylboronic complexing reagents of formula XIX.(a) Reagents of General formula XIX, where Y Is Oh, get a way similar to that described above, and in the stages (a) and (b) the synthesis is carried out exactly as described in the previous example. The product obtained in stage (b) in the preceding example, condensation reagent, preferably (but without limitation) selected from the group consisting of 2-acetoxybenzoic and 2-benzyloxybenzoate, receiving a result corresponding amide. The condensation products obtained in conjunction with the reagent, preferably (but without limitation) selected from the group consisting of 2-acetoxybenzoic and 2-benzyloxybenzoate, carried out in dichloromethane, content and using TLC. From the solution is filtered triarylmethane, the filtrate is washed with water, dried over anhydrous MgSO4and concentrated in vacuo.(b) products of the previous stage remove the protection (i.e. decelerat R2) alkaline catalytic hydrolysis of a warm solution of K2CO3or NaOH for 8 to 24 hours resulting substance oxidizes Hcl, extracted with ethyl acetate, dried over anhydrous MgSO4and concentrated in vacuo. When preparing product for protecting the phenolic hydroxyl group was used acetosella group, it will also be removed, which will eliminate the need for subsequent stages of the synthesis. The protective group is removed by reaction with hydrazinehydrate (N2H2H2O) in ethanol by boiling under reflux for 12 to 48 hours Dropped phthalhydrazide filtered from the solution, which is concentrated. The product is extracted with ethyl acetate, dried over anhydrous MgSO4and concentrated in vacuo.(c) Further product stage (b) is removed, if necessary, protection, removing the protective benzyloxy catalytic hydrogenation, which is carried out with palladium charcoal catalyst in betweensome.(d) the Final product is produced by activation of amino, hydroxyl, tylnej or carboxyl groups associated with intermediates. The amino group can be activated with a reagent, preferably (but without limitation) selected from the group consisting of bromoacetamide, iodoacetamide and maleic anhydride. Hydroxyl groups can be activated with a reagent, preferably (but without limitation) selected from the group consisting of 2,2,2-triftoratsetofenona, pentafluorobenzenesulfonyl and 2-Tianeti-N,N-diisopropylchlorophosphoramidite. Tirinya group can be activated by reaction with a reagent, preferably (but without limitation) selected from the group consisting of 2-dipyrido, 4-dipyrido and 3-nitro-2-mercaptopyridine. Carboxyl groups can be activated by reaction with a reagent, preferably either dicyclohexylcarbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of the reagent preferably (but without limitation) selected from the group consisting of N-hydroxysuccinimide and N-hydroxysultaine. On the other hand, the carboxyl group can be tarifitsirovana alcohol, or preferably methanol or ethanol, and then functionalizing the ub>2receive, as described previously, with the substitution of N-hydroxyphthalimide phthalimido sodium at the initial stage of synthesis.Example XIX. Preparation of complexing reagents phenylboric acid of formula XVIII, reactive relative to the aldehyde.In the initial stage of synthesis of methyl 6-bromhexina condense N-hydroxyphthalimide with stirring in dimethylformamide containing 1 equivalent of triethylamine, within 24 hours of the Obtained semi-product is precipitated by pouring into water, filtered off, washed with water, dried in a vacuum dessicator and used without further purification.In the second stage of synthesis of the obtained crude product is briefly refluxed in a mixture of acetic acid and concentrated hydrochloric acid. After cooling, the precipitated precipitated phthalic acid is filtered off, the filtrate is concentrated and then is evaporated several times from a small amount of water to remove traces of acid. Next, the resulting aminooxyacetic neutralize NaHCO3, extracted with ethyl acetate, dried over MgSO4and concentrated in vacuo.At the third stage of synthesis obtained aminooxyacetic condense in the presence of 1 equivalent of 2-hydroxybenzaldehyde km.Finally, the obtained aldoxime treated in methanol with excess hydrazine hydrate is added with stirring over night. Fallen in sediment hydrazinolysis cooled in an ice bath, filtered from the solution, re-dissolved in methanol and then concentrated in vacuo.Example XX. Application phenylboronic complexing reagents reactive with respect to the aldehyde.Glycoproteins and, in particular, monoclonal antibodies can be conjugated to hydrazide phenylboronic reactive with respect to the aldehyde complexing reagent after treatment of the protein metaperiodate sodium vodolazna solution within 1 to 12 hours Excess periodate removed by dialysis or by salting out, and activated protein having the adjacent side of the aldehyde groups formed by the oxidation periodata carbohydrate residues adjacent coaxial 1,2-diol residues condense with hydrazide reagent for 1 h at room temperature, obtaining in the end polyconjugated with side complexing remnants of phenylboric acid, covalently linked to a protein with a link type of Schiff bases. The communication stability of protein per mark, oxidation of the glycoprotein metaperiodate sodium activates the protein to react with the hydrazide reagent type, while removing the formed most natural way kompleksoobrazuyushchim residues phenylboric acid (coaxial 1,2-diols) associated with glycoproteins.Example XXI. Preparation phenylboronic complexing reagents of General formula XIX, reactive towards thiol.At the initial stage of synthesis of 1,2-bis-(2-iodoethane)ethane condense N-hydroxyphthalimide by boiling under reflux in dimethylformamide containing 1 equivalent of triethylamine for 3 days. The resulting material is then precipitated by pouring into water, filtered off, washed with water, dried in a vacuum dessicator and used without further purification.In the second stage of the synthesis, the resulting product is treated with excess thioacetate sodium in absolute ethanol and the resulting yellow suspension is heated under reflux for 1 h the Mixture is cooled, filtered, concentrated in vacuo and then divided between ethyl acetate and water. An ethyl acetate layers washed with saturated aqueous solution of NaHCO3and water, dried over anhydrous MgSO4and the oxygen is in a mixture of acetic acid and concentrated hydrochloric acid. After cooling, the precipitated precipitated phthalic acid is filtered off, the filtrate concentrated, and then evaporated several times from a small amount of water to remove traces of acid. Next, the resulting aminooxyacetic neutralize Panso3, extracted with ethyl acetate, dried over MgS4and concentrated in vacuo.In the fourth stage of the synthesis aminooxyacetic condense with 2 acetoxybenzoic under stirring in dichloromethane containing 1 equivalent of triethylamine for 1 h at room temperature, checking the progress of the reaction using TLC. From the solution is filtered triarylmethane, the filtrate is washed with water, dried over anhydrous MgS4and concentrated in vacuo.At the fifth stage of synthesis of the obtained 2-atsetoksimetilnitraminov in absolute methanol carefully obzharivayut nitrogen, is treated with 1 equivalent of dehydrated K2CO3and the resulting yellow suspension is vigorously stirred for 12 h, the Suspension is filtered and concentrated in vacuo. Finally, mercapto-2-hydroxyanthraquinone treated with solution (methoxycarbonyl)sulphonylchloride dry obezvojennom methanol, while stirring at 0oSole and treated with one equivalent of 3-nitro-2-mercaptopyridine, stirring at room temperature for 12 hours by Filtration from the mixture to remove unreacted 2-nitro-2-mercaptopyridine, and the resulting material was concentrated in vacuo.Example XXII. Application phenylboronic complexing reagent that is reactive towards thiol.Proteins containing disulfide bonds can be conjugated with reactive towards thiol phenylboronate reagents. First restore the disulfide bonds using the reaction with 2-mercaptoethanol or dithiothreitol in carefully obezvojennom aqueous-alkaline solution. The excess reducing agent is removed by dialysis or by salting out, and the protein is introduced into reaction with phenylboronic reactive towards thiol reagents for 1 h at a temperature of 4oFrom getting in the end polyconjugated with lateral balance of phenylboric acid, covalently associated with a protein disulfide bonds. Excess reagents are removed by salting out or tileable chromatography. Complexing the remains of phenylboric acid can be derived from polyconjugated by restoring a disulfide bond as described above. This way you can split bioconjug reagents.Example XXIII. Preparation phenylboronic complexing reagents of formula XIX, reactive towards amine.In the first stage of synthesis of 2-[2-(2-chloroethoxy)ethoxy]ethanol condense N-hydroxyphthalimide by boiling under reflux in dimethylformamide containing one equivalent of triethylamine in 2 days. The resulting material is then precipitated by pouring into water, filtered, rinsing with water, dried in a vacuum dessicator and used without further purification.In the second stage of synthesis of the obtained crude product is briefly refluxed in a mixture of acetic acid and concentrated hydrochloric acid. After cooling, the precipitated precipitated phthalic acid is filtered off, the filtrate is concentrated and then is evaporated several times from a small amount of water to remove traces of acid. Finally, aminooxyacetic neutralize NaHCO3, extracted with ethyl acetate, dried over MgS4and concentrated in vacuo.At the third stage of synthesis obtained aminooxyacetic condense with 1 equivalent of 2-benzyloxybenzaldehyde under stirring for 1 h at room temperature in dichloromethane containing 1 equivalent is washed with water, dried over anhydrous MgSO4and concentrated in vacuo.In the fourth stage of the synthesis of the obtained hydroxy-2-benzyloxybenzophenone condensed with one equivalent of 2,2,2-triftoruranmetilidina under stirring for 1 h in acetonitrile containing 1 equivalent of triethylamine at room temperature. Triarylmethane filtered off, the filtrate is washed with water, dried over anhydrous MgS4and concentrated in vacuo.Finally, catalytic hydrogenation with palladium charcoal catalyst dehydrated in absolute ethanol for 8 h to remove the protective benzyloxy. The catalyst is filtered off, and the resulting material was concentrated in vacuo.Example XXIV. Application phenylboronic complexing reagent that is reactive towards amine.Proteins can be conjugated with complexing phenylboronic reactive towards amine reagent by reaction with the e-amino group lysine residues in the side chain to obtain polyconjugated with lateral remnants of phenylboric acid, covalently associated with a protein stable sulfonamidnuyu links. For removal of water is including Tris and glycine, to avoid cross-reactions. Solid-phase substrate having a side of the primary amino group may be functionalized by reaction with phenylboronic complexing reagents with the receipt of a solid-phase substrates with lateral complexing remnants of phenylboric acid.Example XXV. Preparation phenylboronic complexing reagents of formula XIX, reactive against the oligonucleotides.In the first stage of synthesis of 2-[2-(2-chloroethoxy)ethoxy]ethanol condense N-hydroxyphthalimide by boiling under reflux in dimethylformamide containing one equivalent of triethylamine in 2 days. The resulting material is then precipitated by pouring into water, filtered off, washed with water, dried in a vacuum dessicator and used without further purification.In the second stage of synthesis of the obtained crude product is briefly refluxed in a mixture of acetic acid and concentrated hydrochloric acid. After cooling, the precipitated precipitated phthalic acid is filtered off, the filtrate is concentrated and then is evaporated several times from a small amount of water to remove traces of acid. Finally, aminooxyacetic not settled At the third stage of synthesis obtained aminooxyacetic condense with 1 equivalent of 2-benzyloxybenzaldehyde under stirring for 1 h at room temperature in dichloromethane, containing 1 equivalent of triethylamine, checking the progress of the reaction using TLC. Triarylmethane filtered off, the filtrate is washed with water, dried over anhydrous MgS4and concentrated in vacuo.In the fourth stage of the synthesis of the obtained hydroxy-2-benzyloxybenzophenone condensed with one equivalent of 2-Tianeti-N,N-diisopropylchlorophosphoramidite under stirring for 1 h in acetonitrile containing 1 equivalent of triethylamine at room temperature. Triarylmethane filtered off, the filtrate is washed with water, dried over anhydrous MgS4and concentrated in vacuo.At the fifth stage of synthesis of the obtained 2-Tianeti-N,N-diisopropylchlorophosphoramidite-2-atsetoksimetilnitraminov dissolved in acetonitrile, bring in the auxiliary tank automatic synthesizer oligonucleotides and condense with the free 5'-OH ends of immobilized synthetic oligonucleotide synthesized pyridine-catalytic reaction with 2-Tianeti-N,N-diisopropylchlorophosphoramidite reagent in acetonitrile. In this way complete the synthesis of the solid phase. At the final stage of the synthesis product otscheplaut from solid glass Podo from the solid-phase substrate, and also remove all acyl protective group, including acetochlor associated with the functional group 2-atsetoksimetilnitraminov. The resulting substance concentrate, removing ammonia in a high-speed vacuum apparatus, and then clear back-phase high-performance liquid chromatography (HPLC).Example XXVI. Application phenylboronic complexing reagents of formula XIX, reactive against the oligonucleotides.At the final stage of the automatic synthesis of synthetic oligonucleotides can be conjugated with 2-Tianeti-N,N-diisopropylchlorophosphoramidite phenylboronate complexing reagents to form eventually a synthetic oligonucleotides with 5'-side complexing remnants of phenylboric acid.Below are the experimental data, confirming that kompleksoobrazuyuthie conjugates phenylboric acid are effective.
Claims
BAS-L-Bc-L'-(Bc'-L')n-BAS; (A)
selected from formulas (I) - (X)
c="https://img.russianpatents.com/img_data/58/583835.gif">
where Q and Q' are independently selected from O, S, NH, N-alkyl, N-aryl and N3-aryl;
Y and Y1independently of one another selected from O, NH, N-alkyl, alkyl and aryl;
Z, Z', Z* and Z*' - spacers independently selected from alkyl or polyether chains, the length of which is equivalent 1-16 carbon atoms and which may contain intermediate amide and disulfide bond;
Vases, BAS', S*, S*' - bioactive ingredients, which may be identical or different;
X and X' are independently selected from H, CH3and C6H5;
W and W' are independently selected from O, NH, N-alkyl, NC6H5N-aryl, N2-aryl, N2CH2HE, N2CH2HE, NOH, NO-alkyl and DOWN2-aryl, where, if it is not given to other definitions, the alkyl is a hydrocarbon radical of up to C6and aryl is an aromatic ring or substituted aromatic ring or condensed aromatic ring.2. The method of obtaining bioconjugation complexes according to p. 1, characterized in that for obtaining bioconjugation complexes use phenylboronic polyconjugated formula (XV)
<br /><img src=)
where Z* is defined in paragraph 1;
R - electrophilic or nucleophilic residue can react with the bioactive ingredients.4. Phenylboronic complexing reagents selected from formula (XI), (XVIII) and (XIX)
in which Q, X, Y, and Z are defined in paragraph 1;
W is selected from the group including H, HE, NH2, NHCH3, NHOH and N3;
R - electrophile or nucleophilic residue can react with the bioactive ingredients.5. Phenylboronic cross-linking reagents of the formula (XII)
where Q and Z* is defined in paragraph 1;
W* is defined in paragraph 4.6. The method of obtaining bioconjugation complexes according to p. 1, characterized in that for obtaining bioconjugation complexes use phenylboronic cross-linking reagents of the formula (XVI) or (XVII)
where Z* is defined in paragraph 1.7. Bioconjugation complexes or Polycom.8. A set of tools and the system to scroll to the desired population of cells containing bioconjugate or polyantha under item 7.9. The method of selection of the desired population of cells, comprising introducing into contact containing cell means with bioconjugation complex in which the antibody recognizes and binds to an epitope characteristic of a desired population of cells, and separation of cells from the funds.
Same patents:
A method for predicting diabetic nephropathy // 2200328
The invention relates to medicine, in particular to internal medicine
The invention relates to medicine and veterinary medicine, and is intended for the quantitative determination of hemoglobin in human blood or animal
The invention relates to medicine, namely to forensic medicine
The invention relates to medicine, in particular for orthopedics and clinical laboratory diagnostics
The invention relates to medicine, in particular to intensive care, Pediatrics
The invention relates to medicine, namely, Hepatology, and can be used to predict the development of internal biliary fistulas in patients with gall-stone disease
The invention relates to medicine, namely to Pediatrics, and can be used for early preclinical diagnosis of rejection of the state of health of children and adolescents
The invention relates to medicine and biochemistry
The invention relates to medicine, namely cardiology, and can be used in cardiology and internal medicine hospitals, in clinics for the diagnosis tolerance (addiction) to nitrate in diseases of the cardiovascular system, such as coronary heart disease (CHD)
The invention relates to medicine, namely to Oncology, and can be used for planning effective methods of treatment for cancer of the vulva in women, depending on the disease
The invention relates to medicine, specifically to Allergology, and relates to methods of diagnosis of bronchial asthma (BA)
The predictor of ventricular septal defect // 2201596
The invention relates to medicine and can be used for prediction of ventricular septal defect in children
The invention relates to medicine, in particular to infectious diseases
The method of assessing the functional state of immune cells in patients with thermal injury // 2200956
The invention relates to medicine, in particular to immunology
The invention relates to veterinary medicine and can be used to identify patients with leukemia bovine animals
The invention relates to medicine, in particular to gastroenterology and Hematology
The invention relates to medicine, in particular to ophthalmology
The invention relates to medicine, in particular to gastroenterology, surgery, and can be used in predicting complications of peptic ulcer of the duodenum
The invention relates to new biologically active compounds, namely spiroheterocyclic heterocyclic compounds of the formula I
< / BR>where n is 0 or 1;
m is 0 or 1;
p is 0;
X represents oxygen or sulfur;
Y represents CH, N or NO;
W represents oxygen or H2;
And represents N or C(R2);
G represents N or C(R3);
D represents N or C(R4)
provided that not more than one of A, G and D represents nitrogen, but at least one of Y, a, G, and D represents nitrogen or NO;
R1represents hydrogen or C1-C4-alkyl;
R2, R3and R4are independently hydrogen, halogen, C1-C4-alkyl, C2-C4alkenyl,2-C4-quinil, aryl, heteroaryl, including five - or six-membered aromatic ring with 1 or 2 nitrogen atoms, as well as furyl or morpholyl, HE OS1-C4-alkyl, CO2R1, -CN, -NO2, -NR5R6or R2and R3or R3and R4accordingly, together with part a and G or G and D southwest a hydrogen, WITH1-C4-alkyl, C(O)R7C(O)OTHER8WITH(O)OR9, SO2R10, -NR5R6, (CH3)3Si and phenyl, or may together represent (CH2)jQ(CH2)kwhere Q represents a bond; j is 2 and k is 0 to 2;
R7, R8, R9, R10and R11are independently C1-C4-alkyl, NH2, aryl or its enantiomer,
and their pharmaceutically acceptable salts, and methods for their preparation, intermediate compounds and pharmaceutical compositions, which has an activating effect against nicotine
7-acetylcholine receptors and can be used for the treatment and prevention of psychotic disorders and disorders of the type of lower intellectual
< / BR>where n is 0 or 1;m is 0 or 1;
p is 0;
X represents oxygen or sulfur;
Y represents CH, N or NO;
W represents oxygen or H2;
And represents N or C(R2);
G represents N or C(R3);
D represents N or C(R4)
provided that not more than one of A, G and D represents nitrogen, but at least one of Y, a, G, and D represents nitrogen or NO;
R1represents hydrogen or C1-C4-alkyl;
R2, R3and R4are independently hydrogen, halogen, C1-C4-alkyl, C2-C4alkenyl,2-C4-quinil, aryl, heteroaryl, including five - or six-membered aromatic ring with 1 or 2 nitrogen atoms, as well as furyl or morpholyl, HE OS1-C4-alkyl, CO2R1, -CN, -NO2, -NR5R6or R2and R3or R3and R4accordingly, together with part a and G or G and D southwest a hydrogen, WITH1-C4-alkyl, C(O)R7C(O)OTHER8WITH(O)OR9, SO2R10, -NR5R6, (CH3)3Si and phenyl, or may together represent (CH2)jQ(CH2)kwhere Q represents a bond; j is 2 and k is 0 to 2;
R7, R8, R9, R10and R11are independently C1-C4-alkyl, NH2, aryl or its enantiomer,
and their pharmaceutically acceptable salts, and methods for their preparation, intermediate compounds and pharmaceutical compositions, which has an activating effect against nicotine
7-acetylcholine receptors and can be used for the treatment and prevention of psychotic disorders and disorders of the type of lower intellectual