• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Preparation of bioconjugates and antibodies for the immunodetection of anatoxin-a. The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, by different positions of the molecule, suitable for the production of antibodies of high affinity and specificity for anatoxin-a. Likewise, the present invention also relates to the use of bioconjugates of anatoxin-a and of labeled derivatives of anatoxin-a as test antigens. In addition, the present invention also relates to methods of analysis, concentration and extraction of anatoxin-a using the antibodies obtained, sometimes together with test antigens that are bioconjugated or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies to this cyanotoxin, sometimes in conjunction with test antigens that are bioconjugated or labeled derivatives of anatoxin-a. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2612751A2
    申请号:ES201531661
    申请日:2015-11-17
    公开日:2017-05-18
    发明作者:Antonio Abad Fuentes；Josep Vicent Mercader Badia；Antonio Abad Somovilla；Consuelo AGULLÓ BLANES；Guillermo QUIÑONES REYES
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universitat de Valencia；
    IPC主号:
    专利说明:

    5
    10
    fifteen
    twenty
    25
    30
    PREPARATION OF BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF ANATOXINA-A
    DESCRIPTION
    The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, by different positions of the molecule, suitable for the production of antibodies of high affinity and specificity for anatoxin-a. Likewise, the present invention also relates to the use of bioconjugates of anatoxin-a and of labeled derivatives of anatoxin-a as test antigens. In addition, the present invention also relates to methods of analysis, concentration and extraction of anatoxin-a using the antibodies obtained, sometimes together with test antigens that are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies against this cyanotoxin, sometimes together with test antigens that are bioconjugates or labeled derivatives of anatoxin-a.
    STATE OF THE TECHNIQUE
    Biotoxins are a type of pollutant whose presence in food, water and feed represents a real problem for human health and animal welfare, causing significant economic losses to the agri-food, agriculture and fish farming sectors, and can seriously affect water health. potable. Among the most relevant biotoxins due to their toxicity and prevalence, those produced by cyanobacteria stand out, especially the microcystin-LR, the cylindrospermopsin and the anatoxin-a.
    Anatoxin-a (2-acetyl-9-azabicyl [4.2.1] non-2-eno) is a bicyclic secondary amine that incorporates an unsaturated alpha-beta methylcarbonyl moiety.
    image 1
    3
    This toxin is produced by different species of cyanobacteria mainly belonging to the genus Anabaena, Microcystis, Aphanizomenon, Oscillatoria,
    5
    10
    fifteen
    twenty
    25
    30
    35
    Planktothrix and Cylindrosperum. Under certain environmental conditions, these prokaryotic microorganisms are capable of proliferating in an exacerbated manner, giving rise to sudden hatching, known in the scientific literature as massive algal blooms [Osswald et al., Environ. Int., 2007, 33, 1070-1089]. These outcrops or blooms, in Anglo-Saxon terminology, are currently considered more frequent and intense than before due to the eutrophication resulting from human activity and global warming [Taranu et al., Ecol. Lett., 2015, 18, 375384]. The main routes of exposure of the human and animal population to anatoxin-a are through contaminated water, either by intentional intake (drinking water from surface sources) or accidental (recreational and sports uses), and through the consumption of dietary supplements derived from algae that do not always have adequate controls and may therefore contain unwanted species of toxin-producing cyanobacteria [Gutierrez-Praena et al., Food Chem. Toxicol., 2013, 53, 139-152]. An exhibition route that has become more relevant in recent years and therefore constitutes an emerging risk is through the consumption of fish and bivalve molluscs, both from traditional fishing and fish farming, which have developed in bodies of water affected by cyanobacterial pests and therefore have been exposed to these biotoxins [Ibelings et al., Environ. Pollut., 2007, 150, 177-192; Miller et al., PLOS ONE, 2010, 5, e12576;].
    The first confirmed case of anatoxin-a poisoning occurred in Canada in the 1960s, where it caused the death of cattle, and at that time, due to its high toxicity, it was called as Very Fast Death Factor - injected intraperitoneally into these mice bite in less than 5 minutes [Araoz et al., Toxicon, 2010, 56, 813-828; Osswald et al., Environ. Int., 2007, 33, 1070-1089]. Its high potency as a neurotoxin (intraperitoneal LD50 in mice: 250 pg / kg) is due to the fact that it is an acetylcholine agonist, irreversibly joining the nicotlnic acetylcholine receptor at the neuromuscular level, which blocks the sodium channel and prevents membrane repolarization, causing muscle overstimulation that causes seizures, paralysis and finally death due to cardiorespiratory arrest. Anatoxin-a is one of the most frequently detected cyanotoxins, which together with toxicological and epidemiological criteria has led the US Environmental Protection Agency (USEPA) to consider it a priority pollutant, and to promote additional studies to assess risks and establish regulations and guidelines [Hudnell and Dortch, In Cyanobacterial Harmful Algal Blooms (Chapter 2), Ed. HK Hudnell, Springer, NY, USA. 2008]. In this same line, the
    5
    10
    fifteen
    twenty
    25
    30
    35
    EFSA recently advised to consider the possible presence of cyanotoxins in foods as an emerging risk, and published a call for the realization of a bibliographic report on this subject (OC / EFSA / SCER / 2014/04).
    A large number of fatal cases have been recorded due to anatoxin-a intoxication in domestic animals, livestock and wildlife as a result of the intake of contaminated water, where concentrations higher than 1 mg / L have been recorded, a value at least 100 times higher than recommended by different official bodies [Trainer et al., Toxins, 2015, 7, 1206-1234]. In many areas the problem is recurring and is repeated every year.
    As corresponds to a contaminant so widely distributed and relevant, there are several methods developed for its detection [Dimitrakopoulos et al., Anal.
    Bioanal Chem, 2010, 397, 2245-2252; Lemoine et al., Toxicon, 2013, 61, 164-174; Roy-Lachapelle et al., Talanta, 2015, 132, 836-844], although the HPLC-MS / MS technique is undoubtedly the most sensitive and widely accepted procedure, and the one used by the USEPA as the official method. There is, however, a broad consensus on the need to obtain antibodies against anatoxin-a and to develop fast and reliable analytical methods that allow to efficiently manage the crises that occur, avoiding damage to human health and ecosystems Already in 2005, in a Symposium on cyanotoxins, the development of an ELISA for anatoxin-a was identified as a priority research line, an objective that remains in force 10 years later [Pegram and Nichols, In Harmful Algal Blooms Cyanobacterial (Chapter 15), Ed. HK Hudnell, Springer, NY, USA. 2008]. The closest to an immunoanalytic method developed to date is an assay based on the utilization of the acetylcholine receptor from electrode membranes of the electric stripe (Torpedo sp.) [Araoz et al., Toxicon, 2010, 56, 813- 828]. A kit based on this procedure is commercially available (Abraxis LLC, Warminster,
    USA), although its sensitivity does not seem sufficient, and it is far from those exhibited by commercial ELISAs for other cyanotoxins. The only document attempting to synthesize a functionalized analogue of anatoxin-a with a view to generating antibodies was published in 2009, although the authors did not prove to have achieved the objective pursued, nor did this work have continuity later [Marc et al., Tetrahedron Lett ., 2009, 50, 4554-4557]. Immunoanalytic methods are based on selective, reversible and non-covalent binding between the substance to be detected (analyte) and an antibody that recognizes it with a high affinity. Depending on the analytical purpose, the antibodies can be adapted to different formats, such as
    5
    10
    fifteen
    twenty
    25
    30
    immunoaffinity columns, lateral flow assays and immunoreactive strips, biosensors, microarrays, and especially ELISA type tests.
    To date, the preparation of antibodies capable of recognizing anatoxin-a has not been described, so that no immunoanaltic methods of any kind have been developed that allow the determination, detection, concentration or extraction of anatoxin-a using antibodies. There is therefore a need, in particular for the food, agricultural, clinical and / or environmental industry, to develop analytical methods comprising at least one antibody to anatoxin-a, preferably by using a kit.
    DESCRIPTION OF THE INVENTION
    The present invention provides bioconjugates and labeled derivatives of anatoxin-a, and the use of bioconjugates for obtaining antibodies to anatoxin-a.
    Therefore, a first aspect of the present invention relates to a bioconjugate of general formula (I):
    [T-L-Z] n-P
    (I)
    where:
    T is selected from the group consisting of R-I, R-II and R-III;
    image2
    L is a hydrocarbon chain of 0 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises the substitution of 0 to 10 carbon atoms by heteroatoms, which are selected from the group consisting of in S, O and N; preferably L is a linear hydrocarbon chain of 0 to 20 carbon atoms and said hydrocarbon chain comprises between 0 and 4 heteroatoms selected from the group consisting of O and N, and
    5
    10
    fifteen
    twenty
    25
    more preferably L is a saturated linear hydrocarbon chain of 1 to 10 carbon atoms and optionally the hydrocarbon chain comprises between 1 and 4 heteroatoms selected from the group consisting of O and N; Y
    Z is a functional group selected from:
    - (C = O) NH-, -NH (C = O) -, - (C = O) S-, -S (C = O) -, - (C = O) O-, -O (C = O) -, -O (C = O) O- -O (S = O) O-, -O (SO2) O-, -NH (S = O) O-, -O (S = O) NH- , -NH (SO2) O -, - O (SO2) NH- - (SO2) NH-, -NH (SO2) -, -O (C = O) NH-, -NH (C = O) O-, -NH (C = O) NH-,
    -NH (C = S) NH-, -NH-, -N (alkyl) -, -S-, -SS-, -NH-NH-, -N = C-, -C = N-, -NH ( C = NH) -, -N = N- -O-, -CH = CH-,
    image3
    image4
    Y
    image5
    In a preferred embodiment, Z is selected from the group consisting of - (C = 0) NH- -NH (C = 0) -, -0 (C = 0) NH-, -NH (C = 0) 0-, -NH (C = 0) NH-, -NH-, -S-,
    image6
    image7
    Y
    N = N
    image8
    More preferably, Z is - (C = 0) NH-
    P is a natural or synthetic peptide or polypeptide of molecular weight greater than 2000 daltons. The peptide or polypeptide P may or may not be linked, by covalent, electrostatic or other interaction, to a support. Said support may be a synthetic polymer or not, or be composed of nanomaterials such as carbon nanotubes, zeolites or mesoporous silica.
    According to another preferred embodiment of the present invention, the bioconjugate of formula (I) described in this patent application is characterized in that P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, beta-galactosidase, peroxidase, phosphatase and oxidase. More preferably P is peroxidase or albumin, which can be egg albumin or serine albumin; Y
    n is a number with a value between 1 and 500; preferably n is a value between 1 and 100.
    The value of n indicates the degree of conjugation, that is, the molar ratio between the fraction derived from the compound of formula T-L-Z and P, in the resulting bioconjugate of formula (I).
    According to another preferred embodiment of the present invention, the bioconjugate of formula 5 (I) is a bioconjugate of formula (la)
    P
    image9
    (the)
    where:
    P and n have been defined above. Preferably P is albumin or peroxidase and n 10 is a value selected from 1 to 50.
    According to another preferred embodiment, the bioconjugate of formula (I) is a bioconjugate of formula (lb)
    15 where:
    /
    V
    (Ib)
    image10
    image11
    p
    P and n have been defined above. Preferably P is albumin or peroxidase and n is a value selected from 1 to 50.
    According to another preferred embodiment, the bioconjugate of formula (I) is a bioconjugate of formula (Ic)
    5
    10
    fifteen
    twenty
    25
    where:
    image12
    (Ic)
    image13
    image14
    image15
    P
    P and n have been defined above. Preferably P is albumin or peroxidase and n is a value selected from 1 to 50.
    The bioconjugate of formula (I) of the present invention can be obtained by a method comprising reacting a functionalized derivative (hapten) of the anatoxin-a with P, a natural or synthetic polypeptide of molecular weight greater than 2000 daltons, by methods widely known in the art.
    In another embodiment of the present invention, when the carrier material is a detectable non-isotopic marker, the derivative is a compound of formula (II):
    [T-L-Z] m-Q
    (II)
    where T, L and Z have the same meaning defined above for the bioconjugate of formula (I);
    Q is a detectable non-isotopic marker; Y
    m is a number with a value between 1 and 1000; preferably m is a value selected from 1 to 100.
    In the present invention, "marker" is understood as any molecule or fragment that results in a signal measurable by any type of analytical technique. In the present invention, Q identifies a fragment or a non-isotopic chemical detector, marker or tracer molecule.
    In a preferred embodiment, Q is an enzyme, biotin, a luminescent compound, a fluorophore, a label coupled to an indirect detection system, micro or nanoparticles or others. Preferably, Q is selected from the group consisting of peroxidase, alkaline phosphatase, biotin, fluorescell or any of its derivatives,
    5 a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyril, luciferin or any of its derivatives, an acridinium ester, quantum nanoparticles (in English quantum dots), and gold micro- or nanoparticles colloidal, carbon or latex.
    This compound of formula (II) can be used with an anatoxin-a antibody to determine or detect this cyanotoxin in a sample by immunoassay technology.
    According to a preferred embodiment, the derivative of formula (II) is a derivative of formula 15 (IIa)
    image16
    (IIa)
    where Q is selected from the group consisting of peroxidase, biotin, fluorescell or nanoparticles, and m is a value selected from 1 to 10.
    According to another also more preferred embodiment, the derivative of formula (II) is a derivative of formula (IIb)
    /
    V
    (IIb)
    image17
    image18
    Q
    5
    10
    fifteen
    twenty
    25
    where Q is selected from the group consisting of peroxidase, biotin, fluorescell or nanoparticles, and m is a value selected from 1 to 10.
    According to another, more preferred embodiment, the derivative of formula (II) is a derivative of formula (IIc)
    image19
    (IIc)
    where Q is selected from the group consisting of peroxidase, biotin, fluorescell or nanoparticles, and m is a value selected from 1 to 10.
    The labeled derivative of formula (II) of the present invention can be obtained by a method comprising reacting a functionalized derivative (hapten) of the anatoxin-a with Q, a non-isotopic marker, by methods widely known in the art.
    The bioconjugate of formula (I) of the present invention can be used for the production of antibodies, or together with an anatoxin-a antibody to determine or detect this cyanotoxin in a sample by immunoassay technology. In addition, the labeled derivatives of formula (II) can be used together with an anatoxin-a antibody to determine or detect this cyanotoxin in a sample by immunoassay technology.
    To obtain antibodies against anatoxin-a, functionalized derivatives of said cyanotoxin (haptens) have been prepared, that is, structural analogs of anatoxin-a that incorporate a functional group capable of being used for conjugation to a P carrier or Q marker. Functional group is separated from the skeleton of the anatoxin-a molecule by a spacer L. The position of incorporation of the functional group into the structure of anatoxin-a for conjugation is not an obvious aspect and can be decisive for the viability of bioconjugates of formula (I) as inducers of the production of suitable affinity and selectivity antibodies against anatoxin-a and even for the viability of
    5
    10
    fifteen
    twenty
    25
    30
    35
    bioconjugates of formula (I) or of labeled derivatives of formula (II) to act as competing molecules that allow the development of a sensitive and specific immunoassay for said cyanotoxin.
    In the context of this invention the term "antibody" refers to the immunoglobulin that the animal or a hybrid cell (such as a hybridoma) synthesizes, specifically against the immunogen of the invention (bioconjugate of the invention).
    Therefore, a third aspect of the present invention relates to an antibody (from now on antibody of the invention) generated in response to a bioconjugate of the invention, in particular to the bioconjugate of formula (I). More preferably the antibodies are generated in response to the bioconjugate of formula (Ia), (Ib) or (Ic), more preferably to the bioconjugate of formula (Ib).
    The method of obtaining the antibodies of the invention from bioconjugates of the invention can be carried out by immunization methods widely known in the art. The antibodies generated from a bioconjugate of the present invention may be polyclonal antibodies, monoclonal antibodies, recombinant antibodies or antibody fragments. The antibodies of the invention have high affinity and specificity towards anatoxin-a
    Another aspect of the present invention relates to an antiserum (hereinafter antiserum of the invention) comprising the antibodies of the invention.
    The term "antiserum" refers to a serum obtained after immunization of an animal with an immunogen. The antiserum comprises antibodies specific for said immunogen generated after the immune response produced in the animal. In the context of the present invention, the immunogen is the bioconjugate of the invention and the antiserum comprises specific antibodies generated against the bioconjugate of the invention, the antibodies of the invention.
    A fourth aspect of the present invention relates to a method of in vitro analysis of anatoxin-a in a sample comprising the following steps:
    a) contacting the sample with the antibody or antiserum of the invention;
    b) incubating the sample and the antibody (or antiserum) of step (a) for a suitable period of time for an immunochemical reaction to take place; Y
    c) determine the existence of immunochemical reaction after the incubation of stage (b).
    5
    10
    fifteen
    twenty
    25
    30
    35
    The method of the present invention allows quantitative determination or qualitative analysis of the content of the cyanootoxin anatoxin-a in a sample. Likewise, the method of the present invention allows analyzing the content of anatoxin-a in different types of samples, for example, food samples, environmental samples such as water, soil or surface, and isolated biological samples such as urine. Preferably, the present invention provides a method of in vitro analysis of anatoxin-a in water.
    According to a preferred embodiment, the determination of the immunochemical reaction in step (c) is performed by a competitive immunoassay, using as a competitor a bioconjugate of formula (I) or a labeled derivative of formula (II). Preferably, the competitive immunoassay is of the ELISA type.
    The term immunoassay refers to an analytical assay in which an immunochemical reaction occurs for the detection or quantification of an analyte. Competitive immunoassays are those in which the analyte competes with another molecule for binding with the antibody.
    The term "antigen" in this patent application refers to a molecule capable of interacting specifically with an antibody. The interaction or immunochemical reaction consists in the specific and non-covalent union between an antibody and an antigen, this being the analyte or a test antigen.
    Here, the term "test antigen", "enzyme antigen" or "tracer" refers to a bioconjugate of formula (I) or a labeled derivative of formula (II) that is used in the competitive assay.
    A fifth aspect of the present invention also relates to an anatoxin-a detection kit that uses at least one antibody of the invention. Additionally, the anatoxin-a detection kit may comprise a bioconjugate of formula (I) or a labeled derivative of formula (II) as described in the present patent application.
    5
    10
    fifteen
    twenty
    25
    30
    35
    A sixth aspect of the present invention also relates to a method of purification and / or concentration of anatoxin-a of a sample consisting of immobilizing at least one antibody of the invention on any support and passing a sample through said support. to retain the anatoxin-a present in said sample. The subsequent elution of the anatoxin-retained in the support by methods widely known in the art (change of pH, modification of ionic strength, utilization of chaiotropic agents) will allow its purification and / or concentration, in a system known as chromatography of immunoaffinity
    The terms "immunogenic" and "immunogenic" as used in the present invention refer to a substance that is recognized as foreign to the living organism and is therefore capable of producing or generating an immune response in a host. In the present invention the immunogen is a bioconjugate of formula (I).
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention.
    The following illustrates with some examples and figures the way in which the preparation of several functionalized derivatives of anatoxin-a (haptens) and the corresponding bioconjugates of formula (I), which are not intended to be limiting of the present invention, and which serve to show not only the way in which they can be prepared but also the importance that the structural nature of the bioconjugate of formula (I) may have for the production of antibodies of adequate affinity towards the analyte, suitable for development of an effective immunoanalytical method.
    BRIEF DESCRIPTION OF THE FIGURES
    Fig. 1. Structure of the anatoxin-a.
    Fig. 2. Scheme of the synthesis of the hapten NHS-ANA-1. Fig. 3. Scheme of the synthesis of the hapten NHS-ANA-2. Fig. 4. Scheme of the synthesis of the hapten NHS-ANA-3.
    5
    10
    fifteen
    twenty
    25
    Fig. 5. Scheme of the preparation of a bioconjugate of formula (I) from the corresponding functionalized derivative (hapten) of anatoxin-a.
    Fig. 6. Standard curve for anatoxin-a with monoclonal antibodies obtained in the indirect competitive ELISA format.
    Fig. 7. Standard curve for anatoxin-a with monoclonal antibodies obtained in the direct competitive ELISA format.
    Fig. 8. Enantiospecificity of monoclonal antibodies against anatoxin-a EXAMPLES
    The invention will be illustrated below by tests carried out by the inventors, which show the effectiveness of the bioconjugates of formula (I) for obtaining antibodies against anatoxin-a and the development of a high sensitivity immunoassay for the same. . Bold numbers refer to the corresponding structure shown in the schemes. These examples are presented by way of demonstration but in no way can they constitute a limit to the invention.
    1. Preparation of bioconjugates of formula (I)
    Example 1: Preparation of bioconjugates of formula (I) for T = RI, L = CH2CH2CH2, Z = - (C = O) NH- and P = BSA (bovine seoalbumine), OVA (ovalbumine) and HRP (horseradish peroxidase ).
    1.1. Preparation of N-hydroxysuccinimidyl ester of hapten ANA-1 (NHS-ANA-
    one).
    The starting material (1) was prepared from 1,2-epoxy-5-cyclooctene by adaptation of a previously published route [D. B. Kanne et al., J. Am. Chem. Soc. 1986, 108, 7864-7865].
    image20
    image21
    image22
    3
    Preparation of tert-butyl 2-oxo-9-azabicyclo [4.2.1] nonano-9-carboxylate (2). A mixture of 300 mg (1.308 mmol) of benzylamine 1.57 mg of 20% Pd (OH) 2 on
    5
    10
    fifteen
    twenty
    25
    30
    Carbon (50% humidity) and 1.12 equivalents (320 mg, 1,465 mmol) of di-tert-butyl carbonate in 3.2 mL of AcOEt was kept under stirring under a pressure of 60 psi of H2 at room temperature for 12 h. After this time, the mixture was filtered on celite, washing with AcOEt. The filtrate was washed with 20 mL of a saturated NaHCO3 solution and brine and dried over anhydrous MgSO4. The residue resulting from evaporation of the solvent was purified by silica gel chromatography, using hexane-AcOEt 9: 1 as eluent, to obtain protected N-Boc 2 amine as a colorless oil that solidifies at -20 ° C (238 mg , 76%).
    IR vmax / cm-1 2967, 2931, 1709, 1687, 1392, 1343, 1168, 1102. 1H NMR (300 MHz, CDCl3) (2 rotamers in proportion 2: 1; majority data are described) 5 4,584.18 (2H, m, H-1 and H-6), 2.59-2.47 (1H, m, H-3), 2.40-2.25 (2H, m, H'-3 and H- 7), 2.25-2.06 (2H, m, H-5 and H-8), 1.96-1.86 (1H, m, H'-7), 1.81-1.62 ( 4H, m, H-4, H'-5, H'- 8), 1.42 (9H, s, CMes), 2 * * * * * * * * * * 13C NMR (75 MHz, CDCh) 5 215.4 (C-2, C = O), 153.1 (C = O Boc) 80.4 (CMe3), 65.1 (C-6), 56.6 (C-1), 41.8 (C-3), 33.0 (C-5), 30.0 (C-7), 28.5 (CMea), 26.9 (C-8), 19.4 (C-4); HRMS (ES) m / z calculated for C13H22NO3 [M + H] + 240.1594, found 240.1587.
    Boc ^
    image23
    I 0
    , 0S02CF3
    image24
    image25
    2. 3
    Preparation of 2 - (((trifluoromethyl) sulfonyl) oxy) -9-azabicide [4.2.1] non-2-eno-9-
    tert-butyl carboxylate (3). A solution with 123 mg was prepared in a flask
    (0.514 mmol) of ketone 2 in 6.65 mL of anhydrous THF under an inert atmosphere. The
    solution was cooled to -78 ° C and 2.0 equivalents (2.05 mL, were added dropwise)
    1,027 mmol) of bis (trimethylsilyl) potassium amide (KHMDS) 0.5 M in toluene; the reaction
    it was allowed to advance for 1.5 h and subsequently 2.5 equivalents were added (504
    mg, 1,284 mmol) of N- (5-chloro-2-pyridinyl) bis (trifluoromethanesulfonamide) solution
    (Comins reagent) dissolved in 0.4 mL of anhydrous THF. This mixture was stirred for an additional 1 h while maintaining the reaction temperature at -78 ° C. Finally, 2 mL of a saturated solution of ammonium chloride at 0 ° C, the mixture of
    The reaction was diluted with water and extracted with AcOEt (3x15 mL). The organic phases
    together they are washed with brine and dried over anhydrous MgSO4. The residue obtained after removing the solvent was purified by column chromatography,
    using hexane-AcOEt 9: 1 as eluent to obtain enol triflate 3 as a colorless oil (174.0 mg, 91%). IR vmax / cm-1 2972, 1679, 1455, 1400, 1366, 1243,
    5
    10
    fifteen
    twenty
    25
    1201, 1168, 1138, 1072, 884. 1H NMR (300 MHz, CDCl3) (2 rotamers in proportion 3: 1, majority data are described) 5 5.79-5.72 (1H, m, H-3 ), 4.55 (1H, m, H-
    1), 4.44-4.23 (1H, m, H-6), 2.21 (2H, m, H-4), 2.20-1.64 (2H, m, H-5), 2.06 (2H, m, H-8), 2.04-1.67 (2H, m, H-7), 1.44 (9H, s, CMe3) 13C NMR (75 MHz, CDCl3) 5 154 , 6 (C-2), 153.1 (C = O Boc), 120.5 (C-3), 116.5 (CF3), 80.5 (CMe3), 58.9 (C-1), 54.9 (C-6), 32.4 (C-7), 31.0 (C-8), 30.5 (C-5), 28.3 (CMe3), 19.7 (C-4 ) 19F-NMR (282 MHz, CDCl3) 5 -74.4 (s), EMAR (ES) m / z calculated for C14H21F3NO5S [M + H] + 372.1009, found 372.1014.
    Boc,
    <3
    OSO2CF 3
    Bocn
    image26

    • TMS
    5 4
    4
    Preparation of 2 - ((trimetiisiiii) efmii) -9-azabicicio [4.2.1] non-2-eno-9 tert-butyl carboxylate (4). 4.0 equivalents (655 pL, 4.64 mmol) of trimethylsilylacetylene were added to a mixture prepared from 430 mg (1.16 mmol) of the enol triflate 3, 81.4 mg (0.116 mmol, 10% mol) of PdCl2 (PPh3) 2 and 11.0 mg (0.06 mmol, 5% mol) of CuI under an inert atmosphere in 6.2 mL of anhydrous DMF. The resulting solution was degassed by sonication under a stream of nitrogen, 484 pL (3.48 mmol, 3 equivalents) of Et3N was added and the mixture was stirred for 1 h at room temperature. After checking the completion of the reaction by TLC (hexane: AcOEt, 8: 2), the reaction mixture was diluted with 10 mL of water and extracted with Et2O (3x15 mL), the combined organic phases were washed with LiCl solution 1.5%, brine and dried over anhydrous MgSO4. The residue obtained after evaporation of the solvent was purified by silica column chromatography, using hexane: AcOEt 8: 2 as eluent, to obtain conjugated enino 4 as a colorless oil (348 mg, 94%). 1 H NMR (300 MHz, CDCl 3) (2 rotamers in 4: 1 ratio; majority data are described) 5.12 (1H, m, H-3), 4.58 (1H, m, H-1) , 4.42-4.10 (1H, m, H-6),
    2.26-2.14 (2H, m, H-4), 2.20-1.89 (2H, m, H-7), 2.15-1.63 (2H, m, H-8) , 2.12-1.70 (2H, m, H-5), 1.47 (9H, s, CMe3), 0.16 (9H, s, SiMe3); 13C NMR (75 MHz, CD3CO 5153.4 (C = O), 138.0 (C-3), 131.7 (C-2), 106.6 (C-1 '), 92.6 (C- 2 '), 79.6 (CMe3), 59.8 (C-1), 55.6 (C-6), 31.4 (C-7), 31.3 (C-8), 28.8 (C-5), 28.6 (CMe3), 24.3 (C-4), 0.15 (SiMe3), EMAR (ES) m / z calculated for C18H30NO2Si [M + H] + 320,2040, found 320,2042.
    5
    10
    fifteen
    twenty
    25
    image27
    image28
    image29
    Preparation of 2-ethinii-9-azabicice [4.2.1] non-2-ene-9-carboxylate of tert-butyium (5). A mixture formed from 254 mg (0.795 mmol) of the trimethylsilylated enino 4, 5.0 equivalents (550 mg, 3,978 mmol) of anhydrous K2CO3 in 10 mL of MeOH, was stirred at room temperature under N2 atmosphere, controlling its evolution by CCF (using as eluent hexane-AcOEt in proportion 8: 2). After 1 h, the reaction mixture was diluted with 30 mL of water and extracted with CH2Cl2 (3x25 mL), the organic phases were washed with brine and dried over anhydrous MgSO4. After evaporating the solvent, the conjugated enino 5 was obtained as a light yellow oil, practically pure by 1 H NMR, which did not require subsequent purification (190 mg, 97%). IR Vmax / cm-1 2973, 2928, 1685, 1404, 1362, 1248, 1168, 1106, 1007, 934, 852; 1 H NMR (300 MHz, CDCl 3) (mixture of 2 rotamers in 2: 1 ratio; majority data are described) 5 6.16 (1H, dd, J = 6.1, 6.1 Hz, H-3) , 4.59 (1H, m, H-1), 4.40-4.18 (1H, m, H-6), 2.89 (1H, s, H-2 '), 2.25 (2H , m, H-4), 2.20-2.12 (3H, m, H-5, H-7, H-8), 1.92- 1.81 (1H, m, H'-7) , 1.71-1.60 (2H, m, H'-5, H'-8), 1.46 (9H, s, CMe3); 13C NMR (75 MHz, CD3CO 5 155.6 (C = O), 138.3 (C-3), 130.1 (C-2), 85.0 (C-1 '), 79.7 (CMe3 ), 76.0 (C-2 '), 60.0 (C-1), 55.6 (C-6), 31.8 (C-8), 31.2 (C-7), 29, 2 (C-5), 28.6 (CMe3), 24.2 (C-4); EMAR (ES) m / z calculated for C15H22NO2 [M + H] + 248.1645, found 248.1645.
    image30
    image31
    image32
    Preparation of trifiuoroacetate 2-ethinii-9-azabicice [4.2.1] non-2-en-9-io (6). A solution was prepared from 0.5 mL of freshly distilled trifluoroacetic acid in 0.5 mL of dry CH2Cl2, which was added under an inert atmosphere to 31.0 mg (0.125 mmol) of the dwarf 5. The mixture was stirred at room temperature for 1 h and then the solvent and excess acid were removed under reduced pressure to obtain a brown oily residue corresponding to the salt with the trifluoroacetic acid of the azabicyclo-enino conjugate 6 (23.5 mg, 89%) which without subsequent stage of purification was taken directly to the next reaction stage. 1 H NMR (300 MHz, CDCl 3) 5 6.43
    5
    10
    fifteen
    twenty
    25
    (1H, m, H-3), 4.38 (2H, m, H-1), 4.28 (1H, m, H-6), 3.0 (1H, s, H-2 '), 2.45-1.77 (8H, m, H-5, H-8, H-7, H-4). 19F NMR (282 MHz, CDCl3) 5-76.4.
    image33
    6
    Br (CH2) 3C02tBu
    CsC03, MeCN, 50 ° C, 3 h 51%
    image34
    Preparation of tert-butyl 4- (2-ethynyl-9-azabicyclo [4.2.1] non-2-en-9-yl) butanoate (7). A solution was prepared from 81.0 mg (0.419 mmol) of the alkyne salt 6, 3.0 equivalents (1.26 mmol) of CsCO3 and 2.5 equivalents (1.04 mmol) of the 4-bromobutanoate of tert-butyl in 1.5 mL of anhydrous acetonitrile under N2 atmosphere. The resulting reaction mixture was stirred at 50 ° C for 3 h and at the end of the reaction, this was diluted in water and saturated NaHCO3 solution, extracted with CH2Cl2 (3x20 mL) and the combined organic phases were washed with brine and dried over anhydrous MgSO4. The resulting residue after evaporation of the solvent was purified by silica gel column chromatography, using CHCl3-MeOH 9: 1 as eluent, to obtain the W-alkylated alkyne 7 as a slightly yellow oil (61.0 mg , 51%). IR vmax / cm -1 3294.9, 2973.0, 2927.7, 1725.8, 1421.5, 1365.8, 1254.9, 1149.4, 951.4, 845.4; 1H NMR (300 MHz, CDCl3)
    (mixture of 2 rotamers in proportion 6: 1; majority data are described) 5 6.25 (1H, ddd, J = 8.2, 8.2, 0.6 Hz, H-3 '), 3, 80 (1H, m, H-1 '), 3.47 (1H, m, H-6'), 2.86 (1H, s, C = CH), 2.60 (2H, m, H-4 ), 2.36 and 1.80 (2H, m, H-8 'and H'-8'), 2.32-2.13 (2H, m,
    H-4 '), 2.26 (2H, m, H-2), 2.23-2.15 (2H, m, H-5'), 2.06 and 1.64 (2H, m, H -7 'and H'-7'), 1.80 (2H, m, H-3), 1.44 (9H, s, CMe3); 13C NMR (75 MHz, CD3CO 5 173.3 (C-1), 138.9 (C-3 '), 129.1 (C-2'), 86.8 (CeCH), 80.1 (CMe3) , 75.4 (CeCH), 64.4 (C-1 '), 60.5 (C-6'), 46.4 (C-4), 33.9 (C-2), 31.5 ( C-8 '), 28.3 (C-7'), 28.2 (CMe3) 25.24 (C-5 '), 24.54 (C-4'), 23.84 (C-3) ; EMAR (ES) m / z calculated for C18H28NO2 [M + H] + 290.2115 found 290.2119.
    5
    10
    fifteen
    twenty
    25
    image35
    image36
    tBu02C (CH2) 3.
    H 1. HgO, BF3.Et20, CI3CC02H cat MeOH, t.a., 1h;
    2. PTSA, acetone, 20 min, r.t.
    7 77% 8
    Preparation of tert-butyl 4- (2-acetyl-9-azabicyclo [4.2.1] non-2-en-9-yl) butanoate (8). 4.6 mL of a solution prepared from 2 mg of trichloroacetic acid and 50 pL of BF3Et2O in 10 mL of anhydrous MeOH were added, under an inert atmosphere, over a solution of alkyne 7 (170.0 mg, 0.587 mmol) and 0.5 equivalents of HgO (63.7 mg, 0.293 mmol) in 2.4 mL of anhydrous MeOH. The resulting reaction mixture was stirred at room temperature, controlling it by TLC (CHCl3-MeOH, 9-1), until its completion. After about 1 h, the reaction mixture was poured onto 15 mL of cold water and 5 mL of saturated NaHCO3 solution, extracted with CH2Cl2 (3x15 mL) and the organic phases were washed with brine and dried over anhydrous MgSO4 . After removal of the solvent, a yellow oil was obtained which was directly subjected to acid hydrolysis using 1.5 equivalents (167.2 mg, 0.88 mmol) of p-toluenesulfonic acid (PTSAH2O) in 3 mL of acetone; The reaction was stirred at room temperature for 20 min. Finally, the reaction mixture was processed by dilution with 10 mL of H2O and 5 mL of saturated NaHCO3 solution, extracted with CH2Cl2 (3x10 mL) and the combined organic phases were washed with brine and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure provides a slightly brown oily residue that is purified by column chromatography, using CHCl3 with 0.5% Et3N as eluent, to obtain the anatoxin-a-W-alkylated 8 as a colorless oil (139 mg , 77%). 1H NMR (300 MHz, CDCl3) 5 6.92 (1H, m, H-3 '), 4.52 (1H, apparent d, J = 10.6 Hz, H-1'), 3.49 (1H , m, H- 6 '), 2.48-2.35 (2H, m, H-4), 2.43-2.35 (2H, m, H-4'), 2.27 (3H, s, Me), 2.31 and 1.86 (2H, m, H-8 'and H'-8'), 2.10 and 1.63 (2H, m, H-7 'and H'-7 '), 2.23-2.17 (2H, m, H-2), 1.85-1.72 (2H, m, H-5') 1.73-1.63 (2H, m, H -3), 1.41 (9H, s, CMe3); 13C NMR (75 MHz, CDCl3) 5 199.2 (COMe), 173.2 (CO2), 148.6 (C-2 '), 143.2 (C-3'), 80.0 (CMe3), 60.6 (C-6 '), 56.7 (C-1'), 48.1 (C-4), 33.7 (C-2), 31.3 (C-8 '), 28, 6 (C-7 '), 28.2 (CMe3), 25.6 (MeCO), 25.5
    (C-5 ’), 25.0 (C-4’), 24.2 (C-3); HRMS (ES) m / z calculated for C18H30NO3 [M + H] + 308.2220 found 308.2234.
    5
    10
    fifteen
    twenty
    25
    ‘BuC ^ Chya
    image37
    8 9 Preparation of the 2,2,2-trifluoroacetate 4- (2-acetyl-9- (3-carboxypropyl) -azabicyclo [4.2.1] non-2-en-9-io (Haptene ANA-1, 9) 30.5 mg (99.0 pmol) of the tert-butyl ester 8 were treated with 1 mL of a 1: 1 mixture of CF3CO2H-CH2Cl2 The resulting solution was stirred at room temperature for 1 h. At the end of the reaction, The solvent was evaporated under reduced pressure to dryness, obtaining the hapten ANA-1 (9) as a salt of the trifluoroacetic acid as a colorless oil (36 mg, 99%). IR Vmax / cm -1 2915.9, 2848, 3, 1669.1, 1418.4, 1198.0, 1132.8, 754; 1H NMR (300 MHz, CD3OD) 5.50 (1H, m, H-3 '), 5.19 (1H, apparent t, J = 11.5 Hz, H-1 '), 4.22 (1H, m, H- 6'), 3.16 (2H, m, H-4), 2.65-2.55 ( 2H, m, H-4 '), 2.47 (2H, m, H-2), 2.35 (3H, s, Me), 2.23 and 2.03 (2H, m, H-8' and H'-8 '), 2.19 and 1.85 (2H, m, H-7' and H'-7 '), 2.03 (2H, m, H-3), 1.89-1 , 79 (2H, m, H-5); 13C NMR (75 MHz, CDCh) 5 196.0 (COMe), 176.0 (CO2), 161.4 (F3CCO2-), 149.0 (C-2 '), 148.0 (C-3'), 64.3 (C -6 '), 57.0 (C-1'), 49.0 (C-4), 32.9 (C-2), 32.9 (C-8 '), 29.3 (C-7 '), 26.8 (C-5'), 25.4 (Me), 23.5 (C-4 '), 20.7 (C-3); 19F NMR (282 MHz, CD3OD) 5-77.73; HRMS (ES) m / z calculated for C14H22NO3 [M + H] + 252.1594, found 252.1596.
    H02C (CH2) 3v, h or
    image38
    5 '4'
    hapten AN / - 1 ■ HCI (10)
    Preparation of 4- (2-acetyl-9-azabicyclo [4.2.1] non-2-en-9-yl) butanoic acid hydrochloride (ANA-1, 10 hapten hydrochloride). 80 mg of amberlite IRA-400 resin, previously conditioned, was added to a solution prepared from 36 mg (0.098 mmol) of the salt of the trifluoroacetic acid of hapten ANA-1 (9) in 1 mL of Milli-Q H2O and The suspension was left with gentle agitation overnight. The reaction mixture was filtered to separate the resin and the aqueous filtrate was lyophilized to obtain a very hygroscopic foamy solid corresponding to the ANA-1 hapten hydrochloride (10) (24.9 mg, 88%). 1 H NMR (300 MHz, CDCl 3) 5 7.33 (1H, apparent t, J = 5.9 Hz, H-3 '), 5.13 (1H, apparent d, J = 7.9 Hz, H-1 '), 4.14 (1H, m, H-6'), 3.01 (2H, m, H-4), 2.59 (2H, m, H-4 '), 2.42 (2H, m, H-2), 2.42 and 1.96 (2H, m, H-8 'and H'-8'), 2.35 (3H, s, Me), 2.32 and 1.96 ( 2H, m, H-7 'and H'-7'), 1.96 (2H, m, H-3), 1.81-1.72 (2H, m, H-5 '); 13C NMR (75 MHz, CDCl3) 5 196.0 (COMe), 176.0 (CO2), 149.0 (C-2 ’), 148.0 (C-3’), 64.3
    5
    10
    fifteen
    twenty
    25
    (C-6 '), 57.0 (C-1'), 49.0 (C-4), 33.0 (C-2), 32.9 (C-8 '), 29.3 (C -7 '), 26.8 (C-5'), 25.4 (Me), 23.5 (C-4 '), 20.7 (C-3); HRMS (ES) m / z calculated for C14H22NO3 [M + H] + 252.1594, found 252.1597.
    image39
    image40

    ° <3
    hapten ANA-1 ■ HCI (10) NHS-ANA-1 (11)
    Preparation of the hapten N-hydroxysuccinimidyl ester ANA-1 (11). A solution was prepared from 13.2 mg (0.046 mmol) of ANA-1 hapten hydrochloride (10), 1.5 equivalents (7.9 mg, 0.069 mmol) of N-hydroxysuccinimide and 1.5 equivalents (13 , 2 mg, 0.069 mmol) of EDCHCl in 900 pL of dry CH2Cl2 under N2 atmosphere. The reaction mixture was stirred at room temperature for 17 h. After this time it was confirmed by TLC, using 9: 1 chloroform-acetone as eluent, the completion of the reaction, after which the reaction mixture was concentrated to dryness under vacuum and the residue obtained was dissolved in DMF to obtain a solution approximately 50 mM of the active ester NHS-ANA-1 (11), which was directly used for the preparation of the corresponding bioconjugates.
    image41
    NHS-ANA-1 (11)
    1.2. Preparation of a bioconjugate of hapten ANA-1 with BSA (BSA-ANA-1). 200 pL of the solution of the active ester NHS-ANA-1 (11) in DMF obtained in the previous reaction (approximately 50 mM) was added slowly and with constant stirring on 1.8 mL of a BSA solution (15 mg / mL ) in 50 mM carbonate buffer, pH 9.6. The conjugation reaction was incubated for 2 h with gentle stirring at room temperature. After this time, the conjugates were purified by molecular exclusion in 3 HiTrap Desalting Columns chromatographic columns of 5 mL each, serially coupled, using 100 mM sodium phosphate elution buffer, pH 7.4. Finally, after the purification process, the collected fractions containing the BSA bioconjugate were brought to a final concentration of 1 mg / mL with elution buffer and stored at -20 ° C.
    To determine the haptenic charge (n) obtained in the conjugate, an allcuota of 100 pL of the purified BSA-ANA-1 bioconjugate was dialyzed (dialysis against 5 L of deionized water with at least 2 to 3 water changes for 24 h at 4 ° C); finally, the dialyzed product was lyophilized and the number of hapten molecules conjugated per 5 BSA molecule was determined by MALDI-TOF-MS (n = 8, see Table 1, entry 2).
    TABLE 1, Values of haptenic load of protein conjugates determined by MALDI-TOF-MS
     RMo m / z A (m / z) Am / hapten n
     one  BSA - 66431.0 - - -
     2  BSA-ANA-1 24 68316.2 1885.2 233.3 8.0
     3  BSA-ANA-2 24 69810.5 3379.5 233.3 14.5
     4  BSA-ANA-3 24 69952.8 3522.5 220.1 16.0
     5  OVA - 42749.0 - - -
     6  OVA-ANA-1 8 43504.0 755.0 233.3 3.2
     7  OVA-ANA-2 8 44715.0 1966.0 233.3 8.4
     8  OVA-ANA-3 8 44500.0 1751.0 220.1 7.9
     9  HRP - 43973.5 - - -
     10  HRP-ANA-1 10 n.d. n.d. n.d. n.d.
     eleven  HRP-ANA-2 10 44937.4 963.5 233.3 4.1
     12  HRP-ANA-3 10 44529.0 555.5 220.1 2.5
    RM0: initial hapten / proline molar ratio used for conjugation n: hapten / proline molar ratio obtained for each conjugate A (m / z): (m / z conjugate) - (m / z reference protein)
    Am / hapten: mass increase for each molecule of conjugated hapten n.d .: not determined
    5
    10
    fifteen
    twenty
    J3 *
    ° N °
    i
    OR
    nh2
    n H, N-OVA-l
    r> | «
    image42
    image43
    NHS-ANA-1 (11)
    1.3. Preparation of a bioconjugate of hapten ANA-1 with OVA (OVA-ANA-1). From a 50 mM solution in DMF of the activated hapten NHS-ANA-1 (11), 100 pL were taken and added slowly and with constant stirring to a volume of 1.9 mL of an OVA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After 2 h of reaction under gentle agitation and at room temperature, the bioconjugate was purified as described above for the BSA conjugate. The collected fractions were brought to a final concentration of 1 mg / mL in elution buffer with 0.01% thimerosal (v / v) and stored at -20 ° C. An allcuot of the newly obtained OVA-ANA-1 conjugate was dialyzed and lyophilized to calculate the efficiency of the conjugation in terms of the number of hapten molecules (11) coupled to the prothene by MALDI-TOF-MS (n = 3.2, see Table 1, entry 6).

    Xil ^ X ^ N _ / X-CH3 h2n-hrp-nh2 HRP
    0 <3 -
    NHS-ANA-1 (11)
    DMF
    50 mM carbonate buffer pH 9.6, 2h, t.a
    image44
    1.4. Preparation of a bioconjugate of hapten ANA-1 with HRP (HRP-ANA-1). From a 5 mM solution of the activated hapten NHS-ANA-1 (11) in DMF, 100 pL were taken and added slowly and with constant and gentle agitation over 0.9 mL of a HRP solution at a concentration of 2 , 5 mg / mL in 50 mM carbonate buffer, pH 7.4. The conjugation reaction was incubated for 2 h at room temperature. Subsequently, the bioconjugate was purified following the procedure previously described for the bioconjugates of BSA and OVA, and was carried out at known concentrations of between 250-650 pg / mL in PBS buffer with 1% BSA (w / v) and thimerosal at 0 , 02% (w / v) and stored at 4 ° C.
    5
    10
    fifteen
    twenty
    25
    30
    Example 2: Preparation of bioconjugates of formula (I) for T = R-II, L = CH2CH2CH2CH2, Z = - (C = O) NH- and P = BSA, OVA and HRP.
    2.1. Preparation of the hapten N-hydroxysuccinimidyl ester ANA-2 (NHS-ANA-2).
    Bocn
    0S02CF3

    image45
    image46
    co2ch3
    Preparation of 2- (6-methoxy-6-oxohex-1-in-1-yl) -9-azabicyclo [4.2.1] non-2-en-9-
    methyl carboxylate (12). To a mixture prepared from 108 mg (0.291 mmol) of the enol triflate 3, 20.4 mg (10% mol, 0.029 mmol) of PdCl2 (PPh3) 2 and 2.7 mg (5% mol, 0.014 mmol) of CuI under the atmosphere of N2, 1.5 mL of anhydrous DMF and 4.0 equivalents (153 pL, 1,167 mmol) of commercial methyl 5-hexinoate were added. The resulting solution was degassed by vacuum cycles under a stream of nitrogen and 3.0 equivalents (122 pL, 0.875 mmol) of Et3N were added. After 1 h of stirring at room temperature, it was checked by TLC that the starting triflate has been completely consumed. The reaction mixture was diluted with 10 mL of water and extracted with Et2O (3x15 mL); The organic phases were washed successively with 1.5% LiCl solution and brine and dried over anhydrous MgSO4. The residue obtained after evaporation of the solvent was purified by silica gel column chromatography, using hexane-AcOEt 9: 1 as eluent, to first obtain the alkyne coupling product of the alkyne (10.9 mg, 11%) , followed by the conjugate enino cross-coupling product, 12, as a colorless oil (86.4mg, 85%). IR v ^ x / cm "1 2953, 2400, 2350, 1733, 1690, 1402.5, 1365.1, 1164.7; 1 H NMR (300 MHz, CDCl3) (mixture of 2 rotamers in proportion 2: 1; se describe the majority data) 5 5.94 (1H, dd, J = 11.6, 5.5 Hz, H-3), 4.51 (1H, m, H-1), 4.39-4, 13 (1H, m, H-6), 3.66 (3H, s, CO2Me), 2.43 (2H, t, J = 7.4 Hz, H-5 '), 2.31
    (2H, t, J = 6.9 Hz, H-3 '), 2.25-2.18 (2H, m, H-4), 2.16-2.04 (3H, m, H-8 , H-7), 1.83 (2H, dt, J = 6.8 Hz, H-4 '), 1.83-1.68 (3H, m, H-5, H'-7), 1 , 44 (9H, s, CMe3 Boc); 13C NMR (75 MHz, CDCl3) 5 173.1 (CO2CH3), 153.6 (C = O Boc), 135.2 (C-3), 130.9 (C-2), 87.4 (C- 2 '), 82.6 (C-1'), 79.4 (CMe3 Boc), 60.5 (C-1), 55.1 (C-6), 51.7 (CO2CH3), 32.9 (C-5 '), 31.3 (C-7), 30.2 (C-8), 29.4 (C-5), 28.6 (CMe3 Boc), 24.1 (C-4' ), 23.6 (C-4), 18.9 (C-3 '), EMAR (ES) m / z calculated for C20H30NO4 [M + H] + 348.2169, found 348.2168.
    5
    10
    fifteen
    twenty
    25
    30
    image47
    Boc_
    image48
    4 ^ / ^ 5 ^ C020H3
    Preparation of methyl 2- (6-methoxy-6-oxohexanoyl) -9-azabicyclo [4.2.1] non-2-en-9-carboxylate (13). At a solution prepared from 80 mg of the alkyne 12 (0.230 mmol) and 0.5 equivalents of HgO (25 mg, 0.115 mmol) in 900 pL of anhydrous MeOH were added dropwise, under the atmosphere of N2, 1.8 mL of a solution prepared from 2 mg of trichloroacetic acid and 50 pL of BF3Et2O in 10 mL of anhydrous MeOH. The resulting reaction mixture was stirred at room temperature until the alkyne disappeared, monitoring the reaction by TLC using hexane-AcOEt 7: 3 as eluent. Approximately after 1 h, the reaction mixture was poured into cold water, basified with a saturated NaHCO3 solution, extracted with CH2Cl2 (3x15 mL) and the combined organic phases were washed with brine and dried over anhydrous Na2SO4. Evaporation of the solvent in vacuo gave a yellow oil that was purified by column chromatography, using hexane-AcOEt 8: 2 as eluent, obtaining conjugated dwarf 13 as a slightly yellow oil (65 mg, 64%). 1 H NMR (300 MHz, CDCl 3) (mixture of 2 rotamers in proportion 2: 1; majority data are described) 5 6.79 (1H, m, H-3), 5.13 (1H, m, H- 1), 4.42-4.28 (1H, m, H-6), 3.65 (3H, s, CO2Me), 2.65 (2H, m, H-2 '), 2.42 (2H , m, H-5 '), 2.33 (3H, m, H-4', H-4), 2.17-2.07 (2H, m, H-8, H'-4), 1 , 65 (7H, m, H-3 ', H-5, H-7, H'-8), 1.39 (9H, s, CMe3 Boc); 13C NMR (75 MHz, CDCh) 5 199.7 (C-1 ', C = O), 173.0 (CO2Me), 153.2 (C = O Boc), 150.0 (C-2), 141 , 1 (C-3), 79.4 (CMe3 Boc), 55.7 (C-6), 53.3 (C-1), 51.7 (CO2CH3), 36.9 (C-5 ') , 35.5 (C-2 '), 31.7 (C-8), 30.5 (C-7), 28.9 (C-5), 28.2 (CMe3 Boc), 24.9 ( C-3 '), 24.4 (C-4'), 24.3 (C-4); HRMS (ES) m / z calculated for C20H32NO5 [M + H] + 366.2275, found 366.2274.
    image49
    C02CH3
    LiOH
    THF-H2O, 30 min
    98%
    image50
    Preparation of 6- (9- (tert-butoxycarbonyl) -9-azabicyclo [4.2.1] non-2-en-2-yl) -6-oxo hexanoic acid (14). A solution of 97 mg (0.27 mmol) of the metallic ester 13 in 2 mL of THF was prepared, cooled in an ice bath and a 2 mL cold solution of an aqueous solution 2 was added dropwise with stirring. 7 M (5.4 mmol, 10 final equivalents) of LiOH. After stirring at room temperature for 30 min, the reaction mixture was poured onto ice-water and acidified to pH 2-3 with a solution.
    5
    10
    fifteen
    twenty
    25
    30
    1 M aqueous KHSO4 fry. The mixture was extracted with AcOEt (3x15 mL) and the combined organic phases were washed with brine and dried over anhydrous Na2SO4. Removal of the solvent in vacuo provided carboxylic acid 14 as a colorless oil (93 mg, 98%), which by NMR showed to be practically pure and therefore was used in the next stage without further purification. IR vmax / cm'1 2973, 2931.6, 2356.8, 1662.4, 1409, 1365.1, 1246.5, 1216, 1166.9, 1115.8, 931.7, 750; 1 H NMR (300 MHz, CDCl 3) (a mixture of 2 rotamers in a 2: 1 ratio; the majority data is described), 5 6.79 (1H, m, H-3 '), 5.13 (1H, apparent d, J = 8.9 Hz, H-1 '), 4.44-4.25 (1H, m, H-6'), 2.65 (2H, m, H-5), 2.42 ( 5H, m, H-2, H-4 ', H-8'), 2.10 (4H, m, H-3, H'-8 ', H-7'), 1.65 (5H, m , H-4, H-5 ', H'-7'), 1.39 (9H, s, CMe3 Boc); 13C NMR (75 MHz, CDCl3) 5 199.6 (C = O, C-6), 178.9 (CO3H), 157.6 (C = O Boc), 149.9 (C-2 '), 141 , 2 (C-3 '), 79.6 (CMe3 Boc), 55.1 (C-6'), 53.3 (C-1 '), 36.7 (C-5), 33.6 ( C-2), 31.5 (C-8 '), 31.1 (C-7'), 30.5 (C-4), 28.9 (C-5 '), 28.5 (CMe3 Boc ), 24.5 (C-4 '), 24.3 (C-3); HRMS (ES) m / z calculated for C19H30NO5 [M + H] + 352.2118, found 352.2131.
    image51
    C02H edc-HCI, NHS
    image52
    image53
    14 5 “15
    Preparation of 2- (6 - ((2,5-dioxopyrrolidin-1-yl) oxy) -6-oxohexanoyl) -9-azabicyclo [4.2.1] non-2-en-9-tert-butyl carboxylate (15 ). A solution was prepared from 80 mg (0.228 mmol) of acid 14, 1.5 equivalents (39.3 mg, 0.341 mmol) of N-hydroxysuccinimide and 1.5 equivalents (65.4 mg, 0.341 mmol) of EDCHCl in 3 mL of dry CH2Cl2 under N2 atmosphere. The reaction mixture was stirred at room temperature for 17 h. After this time, the completion of the reaction was confirmed by TLC using 9: 1 chloroform-acetone as eluent. The reaction mixture was diluted with CH2Cl2, 5 mL of a saturated NH4Cl solution was added and extracted with the same solvent (3x10 mL). The combined organic phases were washed with brine and dried with anhydrous Na2SO4. After evaporating the solvent, a colorless oil was obtained which was purified by column chromatography, using as eluent CHCl3, to obtain the N-hydroxysucccimidyl ester 15 (77 mg, 75%). 1 H NMR (300 MHz, CDCl3) (a mixture of 2 rotamers in 2: 1 ratio; the majority data are described), 5 6.79 (1H, m, H-3), 5.13 (1H, m, H-1), 4.44-4, 25 (1H, m, H-6), 2.83 (4H, wide s, COCH2CH2CO), 2.65 (2H, m, H-2 '), 2.42 (5H, m, H-5', H-4, H-8), 2.10 (4H, m, H-3 ', H'-8, H-7), 1.65 (5H, m, H-4', H-5, H '-7), 1.39 (9H, s, CMe3 Boc); 13C NMR (75 MHz, CDCh) 5 199.6 (C = O, C-1 ’), 168.9 (COCH2CH2CO),
    165.5 (C = O, C-6 '), 157.6 (C = O Boc), 149.9 (C-2), 141.2 (C-3), 79.6 (CMe3 Boc), 55.1
    5
    10
    fifteen
    twenty
    25
    (C-6), 53.3 (C-1), 36.7 (C-2 '), 33.6 (C-5'), 31.5 (C-8), 31.1 (C- 7), 30.5 (C-4 '), 28.9 (C-5), 28.5 (CMe3 Boc), 25.6 (COCH2CH2CO), 24.5 (C-4), 24.3 ( C-3 '); HRMS (ES) m / z calculated for C23H33N2O7 [M + H] + 449.2282, found 449.2281.
    OR
    image54
    NHS-ANA-2 ■ CF3C02H (16) Preparation of 2,5-dioxopyrrolidin-1-yl-6- (9-azabicyclo [4.2.1] non-2-en-2-il) -6-
    oxohexanoate as the salt of trifluoroacetic acid (16). A solution of 25 mg (0.055 mmol) of the N-hydroxysuccinimidyl ester 15 in 1 mL of a 1: 1 mixture of CF3CO2H-CH2Cl2 was stirred at room temperature under N2 atmosphere for 1 h. The solvent and excess of CF3CO2H were evaporated under reduced pressure to provide the salt with the trifluoroacetic acid of the active ester NHS-ANA-2 (16) as a colorless oil (24.4 mg, 96%). 1H NMR (300 MHz, THF-d8) 7.26 (1H, dd, J = 8.0, 3.7 Hz, H- 3 '), 5.13 (1H, apparent d, J = 9.2 Hz , H-1 '), 4.30 (1H, m, H-6'), 2.75 (4H, wide s, COCH2CH2CO), 2.75-2.70 (2H, m, H-5), 2.64-2.59 (4H, m, H-2, H-4 '), 2.51-2.40 (2H, m, H-8'), 2.36-2.27 (1H, m, H-7 '), 2.10 (1H, m, H-5'), 1.98 (1H, m, H-7 '), 1.95-1.82 (3H, m, H- 4, H-5 '), 1.70 (2H, m, H-3), 19 F NMR (282 MHz, THF-d8) 5-76.86.
    image55
    BSA
    DMF
    50 mM carbonate buffer NHS-ANA-2 ■ CF3C02H (16) pH 9.6, 2h, t.a
    J n
    BSA-ANA-2 bioconjugate
    2.2. Preparation of a bioconjugate of hapten ANA-2 with BSA (BSA-ANA-2). Prepared as described above for the BSA-ANA-1 bioconjugate from 200 pL of a 50 mM DMF solution of the NHS-ANA-2 activated hapten (16) and 1.8 mL of a BSA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg / mL in elution buffer and stored at -20 ° C. The number of molecules of (16) conjugated per BSA molecule, determined by MALDI-TOF-MS, was n = 14.5 (see Table 1, entry 3).
    image56
    OVA
    10
    DMF
    50 mM carbonate buffer NHS-ANA-2 ■ CF3C02H (16) pH 9.6, 2h, t.a
    2.3. Preparation of a bioconjugate of hapten ANA-2 with OVA (OVA-ANA-2). Prepared as described above for the bioconjugate OVA-ANA-1 from 100 pL of a 50 mM DMF solution of the activated hapten NHS-ANA-2 (16) and 1.9 mL of an OVA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg / mL in elution buffer with 0.01% thimerosal (v / v) and stored at -20 ° C. The number of molecules of (16) conjugated for each molecule of OVA, determined by MALDI-TOF-MS, was n = 8.4 (see Table 1, entry 7).
    fifteen
    image57
    HRP
    DMF
    50 mM carbonate buffer NHS-ANA-2 ■ CF3C02H (16) pH 9.4, 2h, t.a
    2.4. Preparation of a bioconjugate of hapten ANA-2 with HRP (HRP-ANA-2). Prepared from 100 pL of a 5 mM solution of the activated hapten NHS-ANA-2 (16) in DMF and 0.9 mL of a solution of HRP (2.5 mg / mL) in 50 mM carbonate buffer, pH 7 ,4. After chromatographic purification, the fractions obtained containing the bioconjugate were brought to known concentrations of between 250650 pg / mL in PBS buffer with 1% BSA (w / v) and thimerosal 0.02% (w / v) and stored at 4 ° C. The number of molecules of (16) conjugated for each molecule of HRP, determined by MALDI-TOF-MS, was n = 4.1 (see Table 1, entry 11).
    5
    twenty
    5
    10
    fifteen
    twenty
    25
    Example 3: Preparation of bioconjugates of formula (I) for T = R-III, L = = N-O-CH2, Z = - (C = O) NH- and P = BSA, OVA and HRP.
    3.1. Preparation of the hapten N-hydroxysuccinimidyl ester ANA-3 (NHS-ANA-
    3).
    image58
    image59
    image60
    Preparation of tert-butyl 2-acetyl-9-azabicyclo [4.2.1] non-2-en-9-carboxylate (17). A solution prepared from 1 mg of trichloroacetic acid, 22 pL of BF3Et2O in 1.9 mL of methanol was added dropwise onto a mixture prepared from 140 mg (0.569 mmol) of alkyne 5 (0.283 mmol) and 0 , 5 equivalents of HgO (62 mg, 0.284 mmol) in 3.6 mL of anhydrous MeOH under an inert atmosphere. The resulting reaction mixture was stirred for 1 h at room temperature. After this time, 300 pL of distilled water was added to the reaction mixture and stirring was continued for an additional 15 min. After the indicated time, the reaction mixture was poured onto 10 mL of cold water and 5 mL of saturated aqueous NaHCO3 solution, extracted with CH2Cl2 (3x15 mL) and the combined organic phases were washed with brine, dried over Na2SO4 and they were concentrated under reduced pressure to provide a yellow oil. Purification by column chromatography, using as eluent hexane-AcOEt 9: 1, provided N-Boc anatoxin-a (17) as a colorless oil (134 mg, 90%). IR v ^ x / cm "1 2978.5, 2925.8, 2853.6, 1690.6, 1662.9, 1404.7, 1390, 1363.2, 1337.5, 1231, 1168, 1108.1, 991.3; 1H NMR (300 MHz, CDCh) (mixture of 2 rotamers in proportion 3: 1; majority data are described) 5 6.81 (1H,
    m, H-3), 5.12 (1H, m, H-1), 4.44-4.23 (1H, m, H-6), 2.52-2.43 (2H, m, H -4), 2.29 (3H, s, Me), 2.22-2.0 (3H, m, H-8, H-7), 1.69-160 (3H, m, H-5, H-7), 1.37 (9H, s, CMe3); 13C NMR (75 MHz, CDCh) 5 197.9 (COMe), 150.5 (C = O Boc), 142.2 (C-2), 141.4 (C-3),
    79.5 (CMe3 Boc), 55.8 (C-6), 53.2 (C-1), 31.6 (C-8), 30.5 (C-7), 29.1 (C- 5), 28.9 and 28.6 (CMe3 Boc), 25.5 (Me), 24.3 (C-4); HRMS (ES) m / z calculated for C15H24NO3 [M + H] + 266,1751, found 266.1747.
    5
    10
    fifteen
    twenty
    25
    Boc, HoC
    image61
    Boc
    image62

    image63
    Preparation of acid 2 - ((((E) -1 - (- 9- (tert-butoxycarbonyl) -9-azabicyclo [4.2.1] non-2-en-2 yl) ethylidene) amino) oxy) acetic acid (18 ). 46 pL of pyridine were added to a solution of 50 mg (0.178 mmol) of N-Boc anatoxin-a (17) and 3.0 equivalents (0.534 mmol) of the amino-oxyacetic acid hydrochloride in 3.2 mL of dry ethanol under argon atmosphere. The mixture was stirred at room temperature for 2 h, at the end of which it was confirmed by TLC (using CHCl3-CH3CO2H-MeOH as eluent in proportion 92: 4: 4) the consumption of all starting material. The reaction mixture was poured onto a mixture of 15 mL of ice-water and 10 mL of 1M HCl, extracted with CHCl3 (4x10 mL) and the organic phases were washed with brine and dried over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave a slightly yellow oil corresponding to the oxime 18 (59 mg, 98%) which, for 1 H NMR, proved to be practically pure and did not require further purification. IR vmax / cm-1 2972.6, 2929.2, 2539.5, 1739.9, 1674.0, 1593.5, 1476.5, 1408.4, 1364, 1336.8, 1257.2, 1168, 8, 1,109.8, 1066.4; 1 H NMR (300 MHz, CDCl 3) (mixture of 2 rotamers in 2: 1 ratio; majority data are described) 5 6.07 (1H, m, H-3 '), 5.15 (1H, m, H -1 '), 4.66 (2H, m, H-2), 4.36 (1H, m, H-6'), 2.39-2.25 (2H, m, H-4 '), 2.22-2.12 (3H, m, H-7 ', H-8'), 2.01 (3H, s, Me-C = N), 1.78-1.51 (3H, m, H-5 ', H-7'), 1.38 (9H, s, CMe3 Boc) 13C NMR (75 MHz, CDCh) 5 172.9 (C-1), 157.7 (C = NO), 153 , 7 (CO2 Boc), 144.9 (C-2 '), 132.1 (C-3'), 79.1 (CMe3 Boc), 68.6 (C-2), 55.5 (C- 6 '), 55.2 (C-1'), 34.3 (C-8 '),
    31.5 (C-7 ’), 28.6 (CMe3 Boc), 24.2 (C-4’), 11.3 (Me-C = N); HRMS (ES) m / z calculated for C17H27N2O5 [M + H] + 339.1914, found 339.1912.
    OR,
    image64
    , 0 ^ '
    C02H
    image65
    Preparation of 2 - ((E) -1 - ((2 - ((- 1-yl) oxy) -2-oxoethoxy) imino) ethyl-9-azabicyclo [4.2.1] non-2- en-9-carboxylate of tert-butyl (19) A solution of 86.6 mg (0.256 mmol) of acid 18, 1.5 equivalents (44.2 mg, 0.384 mmol) of N-hydroxysuccinimide and 1.5 equivalents (73.6 mg 0.384 mmol) of EDCHCl in 6.0 mL of dry CH2Cl2 was stirred at room temperature under N2 atmosphere and the reaction progress was monitored by TLC using 9: 1 chloroform-acetone as eluent. confirm the
    5
    10
    fifteen
    twenty
    25
    30
    disappearance of the starting material, the reaction mixture was diluted with CH2Cl2, then 2 mL of a saturated NH4Cl solution was added and extracted with the same solvent (3x10 mL). The combined organic phases were washed with brine and dried over anhydrous Na2SO4. After evaporating the solvent in vacuo, a colorless oil was obtained which was purified by column chromatography, using as eluent CHCl3, obtaining the ester of W-hydroxysuccinimidyl 19 (59 mg, 53%). IR vmax / cm-1 2973, 2928.6, 1826.7, 1787.8, 1737.8, 1682.6, 1407.4, 1363.4, 1199.6, 1169.3, 1109.8, 1071, 4, 861, 750.7; 1 H NMR (300 MHz, CDCl 3) (a mixture of 2 rotamers in 2: 1 ratio; majority data is described) 5 6.05 (1H, ddd, J = 6.1, 6.1, 0.5 Hz , H-3), 5.15 (1H, m, H-1), 4.97 (2H, m, H-2 '), 4.31 (1H, m, H-6), 2.84 ( 4H, wide s, COCH2CH2CO), 2.37-2.20 (3H, m, H2-4 and H-8), 2.20-2.03 (2H, m, H-5 and H-7), 2.00 (3H, s, Me), 1.80-1.55 (3H, m, H'-8, H'-7 and H'-5), 1.37 (9H, s, Me3C-Boc ); 13C NMR (75 MHz, CDCl3) 5 168.8 (COCH2CH2CO), 165.5 (C-1 '), 157.7 (C = NO), 153.7 (CO2-Boc), 144.9 (C- 2), 132.1 (C-3), 79.1 (Me3C-Boc), 68.6 (C-2 '), 55.5 (C-6), 55.2 (C-1), 31 , 9 (C-5), 31.5 (C-8), 29.8 (C-7), 28.6 (Me3C-Boc), 25.7 (COCH2CH2CO), 24.2 (C-4) , 11.3 (Me); HRMS (ES) m / z calculated for C21H30N3O7 [M + H] + 436,2078 found
    436,2083.
    image66
    5 4
    NHS-ANA-3 CF3C02H (20)
    Preparation of 2,5-dioxopyrrolidin-1-yl 2 - (((((E) -1 - (- 9-azabicyclo [4.2.1] non-2-en-2-
    il) ethilidene) amino) oxy) acetate as the salt of trifluoroacetic acid (NHS-ANA-3, 20). A solution of 28.0 mg (0.064 mmol) of the W-hydroxysuccinimidyl ester 19 in 1 mL of a 1: 1 mixture of CF3CO2H-CH2Cl2 was stirred for 1 h at room temperature under N2 atmosphere. The course of the reaction was monitored by CFF, using 8: 2 chloroform-acetone as eluent, to verify the absence of the starting material. After completion of the reaction, the solvent and excess CF3CO2H were evaporated under reduced pressure, to provide a colorless oil corresponding to the active ester NHS-ANA-3 as salt of the trifluoroacetic acid (20) (27.7 mg, 96%). IR Vmax / cm-1 2948.1, 2359.6, 1783.8, 1737.4f, 1705.2, 1668.8, 1429.5, 1191.0, 1132.7,
    1074.1, 720.1; 1H NMR (300 MHz, CDCh) 5 6.45 (1H, ddd, J = 6.0, 6.0, 0.5 Hz, H-3),
    5.43 (1H, m, H-1), 4.92 (2H, apparent s wide, H-2 '), 4.37 (1H, m, H-6), 2.82 (4H, wide s , COCH2CH2CO), 2.56 (3H, m, H2-4 and H-8), 2.34 (1H, m, H-5), 2-06-1.98 (3H, m,
    10
    fifteen
    twenty
    H’-8, H’-5 and H-7), 2.01 (3H, s, Me), 1.88 (1H, m, H’-7); 13C NMR (75 MHz, CDCl3) 5 169.4 (COCH2CH2CO), 165.3 (C-1 '), 156.6 (C = NO), 140.3 (C-2), 136.3 (C- 3), 69.4 (C-2 '), 59.6 (C-6), 54.4 (C-1), 30.1 (C-8), 28.8 (C-7), 27 , 9 (C-5), 25.1 (COCH2CH2CO),
    23.3 (C-4), 10.8 (Me); 19 F NMR (282 MHz, CDCl 3) 5-76.34 (s); HRMS (ES) m / z calculated for C16H22N3O5 [M + H] + 336,1554, found 336.1565.
    image67
    NHS-ANA-3 ■ CF3C02H (20)
    DMF
    50 mM carbonate buffer pH 9.6, 2h, t.a
    OR
    CL Jl l / BSA
    H N
    N U H
    BSA-ANA-3 bioconjugate
    3.2. Preparation of a bioconjugate of hapten ANA-3 with BSA (BSA-ANA-3). Prepared as described above for the BSA-ANA-1 bioconjugate from 200 pL of a 50 mM DMF solution of the activated hapten NHS-ANA-3 (20) and 1.8 mL of a BSA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg / mL in elution buffer and stored at -20 ° C. The number of molecules of (20) conjugated per BSA molecule, determined by MALDI-TOF-MS, was n = 16 (see Table 1, entry 4).
    image68
    OR
    .o> o'N
    NHS-ANA-3 ■ CF3C02H (20)
    image69
    OR
    , CL Ji> OVA
    H N
    N LI H
    CH3
    OVA-ANA-3 bioconjugate
    3.3. Preparation of a bioconjugate of hapten ANA-3 with OVA (OVA-ANA-3). Prepared as described above for the OVA-ANA-1 bioconjugate from 100 pL of a 50 mM DMF solution of the NHS-ANA-3 activated hapten (20) and 1.9 mL of an OVA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg / mL in elution buffer with 0.01% thimerosal (v / v) and stored at -20 ° C. The number of molecules of (20) conjugates
    5
    10
    fifteen
    twenty
    25
    30
    for each molecule of OVA, determined by MALDI-TOF-MS, it was n = 7.9 (see Table 1, entry 8).
    CF3coP

    NHS-ANA-3 ■ CF3CO2H (20)
    image70
    , 0. JL> HRP
    H N
    N LI H
    J n
    bioconjugate HRP-ANA-3
    3.4. Preparation of a bioconjugate of the hapten ANA-3 with HRP (HRP-ANA-3) Prepared from 100 pL of a 5 mM solution of the activated hapten NHS-ANA-3 (20) in DMF and 0.9 mL of a solution HRP (2.5 mg / mL) in 50 mM carbonate buffer, pH 7.4. After chromatographic purification, the fractions obtained containing the bioconjugate were brought to known concentrations of between 250,650 pg / mL in PBS buffer with 1% BSA (w / v) and thimerosal 0.02% (w / v) and stored at 4 ° C. The number of molecules of (20) conjugated for each molecule of HRP, determined by MALDI-TOF-MS, was n = 2.5 (see Table 1, entry 12).
    2. ELISA procedure
    96-well polystyrene plates were used. Each antibody was evaluated in the two classic formats of competitive ELISA (the one of antigen or conjugate immobilized with indirect detection and the one of immobilized antibody with direct detection) using homologous test antigens, that is, a test antigen from the same bioconjugate of formula (I) than the one used to obtain the immunogen but in which P = OVA or HRP. After each incubation stage, the plates were washed four times with a wash solution, using a 96-channel ELx405 washer (Biotek Instruments, Winooski, USA). The signal produced by the peroxidase used as a marker was revealed with 100 pL per well of a 2 mg / mL solution of o-phenylenediamine in 25 mM citrate buffer, 62 mM phosphate, pH 5.4, containing 0.012% (v / v) of H2O2. This development was developed for 10 min at room temperature and stopped using 100 pL per well of 2.5 M sulfuric acid. At the end of the tests, the absorbance of each well was read at 492 nm using a reference wavelength of 650 nm in a PowerWave HT microplate reader (Biotek Instruments, Winooski, USA). The sigmoid pattern curves obtained by representing the absorbance versus analyte concentration were adjusted to a
    5
    10
    fifteen
    twenty
    25
    30
    35
    four-parameter logistic equation using the SPSS SigmaPlot software package (Chicago, USA).
    The affinity of the antibody (IC50) was estimated as the concentration of free analyte capable of halving the maximum signal (Amax).
    2.1. Competitive ELISA assays in immobilized antigen or conjugate format with indirect detection (indirect assay)
    The plates were upholstered with 100 pL per well of a test antigen solution which is a bioconjugate of formula (I) where P is OVA, at 0.01 or 0.1 pg / mL in 50 mM carbonate buffer, pH 9.6, by incubation overnight at room temperature. After washing the plates, 50 pL per well of a complete standard analyte curve in PBS was dispensed in each column followed by 50 pL per well of a given antibody diluted in PBST (0.05% Tween 20). The immunochemical reaction was carried out for 1 h at room temperature and then the plates were washed. Next, each well received 100 pL of a 1/2000 dilution of RAM-HRP (rabbit anti-mouse immunoglobulins labeled with peroxidase) in PBST. This reaction was left at room temperature for 1 h. After washing the plates, the retained peroxidase activity was revealed and the absorbance at 492 nm was read as described above.
    2.2. Competitive ELISA assays in immobilized antibody format with direct detection (direct assay)
    The plates were upholstered with 100 pL per well of an antibody dilution in 50 mM carbonate buffer, pH 9.6, by incubation overnight at room temperature. After washing the plates, 50 pL per well of a complete standard analyte curve in PBS was dispensed in each column followed by 50 pL per well of a specific dilution in enzymatic bioconjugate PBST which is a bioconjugate of formula (I) where P It's HRP
    The same reagent distribution was repeated for each plate with a different antibody. The immunochemical reaction was carried out for 1 h at room temperature and then the plates were washed. Finally, the retained peroxidase activity was revealed and the absorbance at 492 nm was read as described.
    5
    10
    fifteen
    twenty
    25
    30
    35
    3. Production of mouse monoclonal antibodies
    3.1. Immunization of mice
    For immunization, the bioconjugates of formula (I) were used in which P is BSA (immunizing conjugates) obtained in the previous examples. BALB / c mouse females were used, with an age at the beginning of the process between 6 and 8 weeks.
    In each dose, 100 pg of bioconjugate per mouse were administered intraperitoneally, the total volume being administered 200 pL. In the first immunization the bioconjugate was supplied in an emulsion prepared with complete Freund's adjuvant (1: 1, v / v). At 3-week intervals, the mice received two additional immunizations, in these cases emulsifying the bioconjugates with incomplete Freund's adjuvant. Four days before each cell fusion, the selected mouse received a final dose of 100 pg of the corresponding bioconjugate diluted in PBS.
    3.2. Cellular fusions to obtain hybridomas
    The fusions with the immunized mice were carried out basically following previously described methodologies and well established in the state of the art. Immediately after the sacrifice of the mice, the spleen was removed, which was homogenized with the embolus of a sterile syringe. After lysing the red blood cells by osmotic shock with 1 mL of lysis buffer for one minute in the fried, the lymphocytes were washed twice with complete medium (with serum) and filtered to remove the clots formed.
    The myeloma line P3-X63-Ag8.653 was cultured the days before fusion in DMEM supplemented medium [2 mM L-alanine-L-glutamine, 1 mM non-essential amino acids, 25 pg / mL gentamicin, fetal bovine serum ( SBF) 10% (v / v)], keeping the cells in exponential growth phase, so that the merger day had a sufficient number of them.
    After two washes with serum-free medium, both cell populations joined a lymphocyte: myeloma 4: 1 ratio. Then, the cells were centrifuged, to
    5
    10
    fifteen
    twenty
    25
    30
    35
    immediately after the cell fusion. For this, the chemical fusing agent PEG1500 (1 mL per spleen, 1 min) was used, which partially dissolving the membranes allows the fusion of the cells. Once both populations were fused, the cells were resuspended in supplemented DMEM medium [SBF 15% (v / v)] and seeded in 96-well culture plates (100 pL per well) at a cell density of 150 * 103 lymphocytes per well, and incubated at 37 oC in an atmosphere with 5% CO2 and 95% humidity. 24 h after fusion, 100 pL per well of HAT medium was added for hybridoma selection [DMEM supplemented with 100 pM hypoxanthine, 0.4 pM aminopterin, 16 pM thymidine, and 20% (v / v) SBF] containing HFCS (High Fusion and Cloning Supplement) at 1% (v / v).
    3.3. Selection, cloning and conservation of hybridomas
    Approximately 10-12 days after cell fusion, the evaluation of the supernatants of the seeded wells was carried out, in order to identify which ones contained antibody secreting hybridomas capable of recognizing anatoxin-both in its conjugated and free form (clones competitors). Previously, the fusion efficiency was determined by visual inspection, defined as the percentage of wells that had at least one clone clearly visible to the microscope.
    In order to carry out the identification of competing clones, the culture supernatants were analyzed by means of the differential ELISA technique, which consists in parallel analysis in adjacent wells of each supernatant in the absence of analyte and in the presence of a predetermined concentration of analyte, usually 100 nM. To do this, the plates were upholstered with the homologous conjugate, which is a bioconjugate of formula (I) in which P is OVA, at a concentration of 0.1 pg / mL, and the test was carried out by adding 50 pL of the culture supernatant. The conditions for the indirect ELISA format are detailed in section 2.1.
    Subsequently, those wells containing antibody producing hybridomas capable of providing an absorbance signal equal to or greater than 0.5 in the assay in the absence of anatoxin-a and inhibition of the signal equal to or greater than 80% in the assay were selected in the presence of anatoxin-a. Additionally, for all positive wells a second more thorough screening was conducted in a competitive two-dimensional mode in order to select the best hybridomas with greater certainty. For this, the supernatant of each hybridoma was tested at 4 dilutions
    5
    10
    fifteen
    twenty
    25
    30
    35
    (1/8, 1/32, 1/128 and 1/512) on upholstered plates with the bioconjugate homologous at 0.01 and 0.1 pg / ml, and using as an competitor anatoxin-a at 5 and 50 nM test). So,
    200 pL of the culture supernatant was diluted in 600 pL of PBST and the following dilutions were made serially from this first. The test was performed by adding 50 pL per well of the corresponding supernatant dilution and 50 pL of the anatoxin-a solution in PBS at the concentration of 100, 10 and 0 nM.
    The cells of the finally selected wells were cloned by the limit dilution method, sowing from each well a new 96-well plate to 2 cells per well in HT medium, of the same composition as the HAT but without aminopterin, and containing HFCS at 1% (v / v).
    Generally, 7-10 days after the first cloning, wells containing a single clone were identified by visual inspection, the culture supernatant being reassessed in the same manner as previously described for fusion supernatants. This process was carried out as many times as necessary (at least two) to ensure the monoclonality of the selected hybridomas, as well as their stability. Finally, the selected cell lines were expanded, progressively cultivating the hybridoma in larger containers. Once the clone was grown, the cells were frozen in liquid nitrogen at a concentration of 107 cells per vial (2-4 vials for each hybridoma) in a solution of SBF with 10% DMSO (v / v) as a cryoprotective agent. The vials were kept at -80 oC inside a polystyrene box for 24 h before being transferred to the liquid nitrogen container.
    3.4. Production and purification of monoclonal antibodies
    In the last phase of the hybridoma cell expansion, these were progressively divided into culture plates until a final volume between 100 and 200 mL of supernatant was reached. The cells were allowed to grow until the confluence was reached, and once the nutrients from the culture medium were exhausted, the contents of the plates were collected. The collected volume was centrifuged to remove cell debris and the supernatant was precipitated by adding a volume of a saturated solution of ammonium sulfate, keeping at 4 ° C until purification.
    5
    10
    fifteen
    twenty
    25
    30
    35
    The purification of the antibodies was performed by affinity chromatography with protelna G columns following the manufacturer's instructions. For this purpose, the precipitated antibody was centrifuged for 20 min at 5000 rpm (4000 * g) and the supernatant was discarded. The precipitate containing the antibodies was redissolved with 20 mM sodium phosphate buffer, pH 7.4 and filtered with nitrocellulose membranes (pore diameter 0.45 pm) to remove suspended particles. Elution of the antibody from the column was performed with 100 mM sodium citrate buffer, pH 2.5. Fractions containing the antibody were identified by UV spectrophotometry and collected. The solution was neutralized by adding 1M Tris-HCl, pH 9.5. Finally, the concentration of the purified antibody [A280 (1 mg / mL igG) = 1.4] was determined by UV spectrophotometry and a working solution was prepared at a concentration of 500 pg / mL in PBS with 1% BSA (p / v) and 0.01% thimerosal (w / v), which was stored at 4 oC. The remaining solution was precipitated with saturated ammonium sulfate [1: 1, (v / v)], which guarantees its stability at 4 ° C for years.
    4. Results
    4.1. Generation of hybridomas producing monoclonal antibodies against anatoxin-a
    From mice immunized with bioconjugates of formula (I) are those that P is BSA, cell fusions aimed at hybridoma generation were carried out. The bioconjugates of formula (I) in which T is R-I or R-III were not able to induce the generation of monoclonal antibody secreting hybridomas with a high affinity towards anatoxin-a. On the contrary, from the bioconjugate of formula (I) in which T is R-II, it was possible to obtain 5 monoclonal antibody producing cell lines with high affinity towards anatoxin-a. Monoclonal antibodies obtained from said hybridomas have been named for the purposes of the present invention and the examples here included: mAb # 38, mAb # 44, mAb # 325, mAb # 417 and mAb # 39. This result shows that the bioconjugate of formula (I) most suitable for obtaining antibodies against anatoxin-a is not an obvious and obvious issue. The results also demonstrate the suitability of a bioconjugate of formula (I) in which T is R-II to induce the obtaining of antibodies capable of recognizing anatoxin-a.
    4.2. Determination of antibody affinity
    Once affinity purified the 5 monoclonal antibodies obtained (see 4.1, mAb # 38, mAb # 44, mAb # 325, mAb # 417 and mAb # 39) antibodies, their affinity towards anatoxin-a was determined by competitive homologous ELISA. In indirect format 5 (Fig. 6) the antibodies showed IC50 values for anatoxin-a between 3.15 nM and 11.00 nM. In the direct format (Fig. 7) the antibodies showed IC50 values for anatoxin-a between 2.18 nM and 9.49 nM.
    4.3. Determination of antibody specificity
    10 Anatoxin-a exists chemically in 2 enantiomeric forms. Natural cyanotoxin is exclusively dextrogira (+), while the levogira (-) form can be obtained by organic synthesis. To verify the extent to which the monoclonal antibodies obtained were really specific, they were tested in the indirect homologous ELISA format against the two enantiomers of the anatoxin-a. As can be seen in Fig. 8, all antibodies recognized (+) - anatoxin-a with an affinity at least 100 times higher than that shown for their enantiomer, (-) - anatoxin-a. This assay reliably demonstrates the enormous specificity and discriminatory ability of the monoclonal antibodies described in this invention, and their great ability therefore to determine the natural enantiomer of anatoxin-a in samples, even in the presence of other possible contaminating compounds.
    权利要求:
    Claims (20)
    [1]
    5
    10
    fifteen
    twenty
    25
    1. A bioconjugate of formula (I):
    [T-L-Z] n-P
    (I)
    where
    T is selected from the group consisting of R-I, R-II and R-III;
    image 1
    image2
    R-ll
    R-II
    image3
    L is a hydrocarbon chain of 0 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises between 0 and 10 heteroatoms that are selected from the group consisting of S, O and N;
    Z is a functional group selected from - (C = O) NH-, -NH (C = O) -, - (C = O) S-, - S (C = O) -, - (C = O) O -, -O (C = O) -, -O (C = O) O-, -O (S = O) O-, -O (SO2) O- -NH (S = O) O-, -O (S = O) NH-, -NH (SO2) O -, - O (SO2) NH-, - (SO2) NH-, -NH (SO2) -, -O (C = O) NH-, -NH (C = O) O-, -NH (C = O) NH-, -NH (C = S) NH- -NH-, -N (alkyl) -,
    -S-, -SS-, -NH-NH-, -N = C-, -C = N-, -NH (C = NH) -, -N = N-, -O-, -CH = CH- ,
    image4
    image5
    Y
    image6
    P is a natural or synthetic peptide or polypeptide of molecular weight greater than 2000 daltons. The peptide or polypeptide P may or may not be linked, by covalent, electrostatic or other interaction, to a support. Said support may be a synthetic polymer or not, or be composed of nanomaterials such as carbon nanotubes, zeolites or mesoporous silica.
    n is a number with a value between 1 and 500.
    [2]
    2. The bioconjugate of formula (I) according to claim 1 characterized in that L is a linear hydrocarbon chain of 0 to 20 carbon atoms and said hydrocarbon chain comprises between 0 and 4 heteroatoms selected from the group consisting of O and N.
    5
    [3]
    3. The bioconjugate of formula (I) according to any one of claims 1 or 2 characterized in that Z is selected from the group consisting of - (C = O) NH-, - NH (C = O) -, -O (C = O) NH-, -NH (C = O) O-, -NH (C = O) NH-, -NH-, -S-,
    10
    fifteen
    image7
    Y
    image8
    [4]
    4. The bioconjugate of formula (I) according to any one of claims 1 to 3, characterized in that P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, beta-galactosidase, peroxidase, phosphatase and oxidase.
    [5]
    5. The bioconjugate of formula (I) according to any of claims 1 to 4, with formula (la)
    P
    NHOC
    image9
    (the)
    20 where P is selected from the group consisting of albumin or peroxidase, and n is a value selected between 1 and 50.
    [6]
    6. The bioconjugate of formula (I) according to any of claims 1 to 4, with the formula (lb)
    5
    10
    fifteen
    twenty
    25
    image10
    where P is selected from the group consisting of albumin or peroxidase, and n is a value selected between 1 and 50.
    [7]
    7. The bioconjugate of formula (I) according to any of claims 1 to 4, with formula (Ic)
    image11
    (Ic)
    where P is selected from the group consisting of albumin or peroxidase, and n is a value selected between 1 and 50.
    [8]
    8. A labeled derivative of formula (II):
    [T-L-Z] m-Q
    (II)
    where
    T and L have been defined according to any one of claims 1 to 4; Z is defined in claims 1 and 3; Q is a detectable non-isotopic marker and m is a number with a value between 1 and 1000.
    [9]
    9. The labeled derivative of formula (II) according to claim 8, wherein Q is selected from the group consisting of enzymes, biotin, fluorescell or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore , a ruthenium bipyril, luciferin or any of its
    5
    10
    fifteen
    twenty
    25
    derivatives, an acridinium ester, quantum nanoparticles (in English quantum dots), and micro- or nanoparticles of colloidal gold, carbon or latex.
    [10]
    10. The labeled derivative of formula (II) according to any one of claims 8 or 9 characterized in that Z is selected from the group consisting of - (C = O) NH-, - NH (C = O) -, -O ( C = O) NH-, -NH (C = O) O-, -NH (C = O) NH-, -NH-, -S-,
    image12
    _ Y
    image13
    [11]
    11. The labeled derivative of formula (II) according to any of the 10, with formula (IIa)
    claims 8 to
    image14
    (IIa)
    where Q is selected from the group consisting of peroxidase, biotin, fluorescema or nanoparticles, and m is a value selected between 1 and 10.
    [12]
    12. The labeled derivative of formula (II) according to any of claims 8 to 10, with formula (IIb)
    image15
    (IIb)
    where Q is selected from the group consisting of peroxidase, biotin, fluorescema or nanoparticles, and m is a value selected between 1 and 10.
    5
    10
    fifteen
    twenty
    25
    30
    [13]
    13. The labeled derivative of formula (II) according to any of claims 8 to 10, with formula (IIc)
    image16
    (IIc)
    where Q is selected from the group consisting of peroxidase, biotin, fluorescell or nanoparticles, and m is a value selected from 1 to 10.
    [14]
    14. An antibody obtained by utilizing a bioconjugate as described in any one of claims 1 to 7.
    [15]
    15. The antibody according to claim 14, wherein the antibody is selected from monoclonal, polyclonal and recombinant.
    [16]
    16. Method of in vitro analysis of anatoxin-a in a sample comprising the following steps:
    a) contacting the sample with the antibody described in any of claims 14 or 15;
    b) incubating the sample and the antibody of step (a) for a suitable period of time for an immunochemical reaction to take place; Y
    c) determine the existence of immunochemical reaction after the incubation of stage (b).
    [17]
    17. Method according to claim 16, wherein the determination of the immunochemical reaction in step (c) is performed by a competitive test, using a bioconjugate as a competitor as described in any one of claims 1 to 7.
    [18]
    18. Method according to claim 16, wherein the determination of the immunochemical reaction in step (c) is performed by a competitive assay, using
    as a competitor a derivative labeled as described in any one of claims 8 to 13.
    [19]
    19. Anatoxin-a detection and / or determination kit, comprising at least one antibody as described in any of claims 14 or 15 together
    with a bioconjugate described in any of claims 1 to 7 or together with a labeled derivative described in any of claims 8 to 13.
    [20]
    20. Method of purification and / or concentration of anatoxin-a of a sample by means of affinity chromatography comprising the following steps:
    a) immobilizing at least one antibody described in any of claims 14 or 15 on a support;
    b) passing the sample through said support so that it retains the anatoxin present in said sample; Y
    15 c) elute the retained anatoxin-a in the support.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP6971927B2|2021-11-24|Antibodies to risperidone hapten and their use
    JP2018118972A|2018-08-02|Antibodies to paliperidone haptens and use thereof
    ES2374942T3|2012-02-23|RELEASE REAGENT FOR VITAMIN D COMPOUNDS.
    ES2739383T3|2020-01-30|Tell us about paliperidone
    ES2524603T3|2014-12-10|Immunoassays for topiramate
    US5654178A|1997-08-05|Immunoassay for tetrachloroisophthalonitrile |, its derivatives and breakdown products
    JP5695049B2|2015-04-01|Imatinib immunoassay
    JP4435305B2|2010-03-17|Topiramate immunoassay and analogs and antibodies
    ES2828097T3|2021-05-25|Methods to detect symmetric dimethylarginine
    ES2612751B1|2018-05-10|PREPARATION OF BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF ANATOXINA-A
    ES2672945B1|2019-04-26|PREPARATION OF NEW BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF OCRATOXIN A
    AU652809B2|1994-09-08|Haptens, tracers, immunogens and antibodies for immunoassays for cotinine
    CN103288965B|2015-01-14|Polychlorobiphenyl monoclonal antibody preparation method
    EP0043285B1|1984-01-04|Method for determination of valproic acid and reagents therein
    Abad Fuentes et al.2017|Preparation of bioconjugues et d'anticorps pour l'immunodetection d'anatoxine a
    ES2421056B1|2014-11-11|Haptenes, conjugates and antibodies to the fungicide pyrimethanil
    ES2849398A1|2021-08-17|BIOCONJUGATES AND ANTIBODIES FOR THE DERIVATIZATION-ASSISTED IMMUNODETECTION OF PATULIN MYCOTOXIN |
    US7038021B2|2006-05-02|Anti-dioxins monoclonal antibody suitable for assaying dioxins in environment and hybridoma producing the same
    WO1994029478A1|1994-12-22|Immunoassay reagents and methods for detecting brequinar and analogs
    WO2014060623A1|2014-04-24|Functionalised and immunoreactive derivatives for the fungicide fludioxonil
    WO2013135930A1|2013-09-19|Compounds derived from doxycycline such as haptens, conjugates and antibodies thereof, and immunochemical method for detecting doxycycline
    AU2019284029B2|2022-03-10|Antibodies to paliperidone haptens and use thereof
    KR100876430B1|2008-12-31|Antibody and hapten-protein conjugate utilized in an enzyme-linked immunosorbent assay | for herbicide metamifop residues
    JP2005035893A|2005-02-10|Alachlor hapten and antibody against alachlor and immunoassay using the same
    ES2293924T3|2008-04-01|ANTIURACILO MONOCLONAL ANTIBODY.
    同族专利:
    公开号 | 公开日
    ES2612751B1|2018-05-10|
    ES2612751R1|2017-07-06|
    WO2017085339A1|2017-05-26|
    CA3005506A1|2017-05-26|
    US20190265233A1|2019-08-29|
    US11054415B2|2021-07-06|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20120088312A1|2010-10-08|2012-04-12|Salamone Salvatore J|Vincristine immunoassay|
    ES2461415B1|2012-10-16|2015-03-16|Consejo Superior De Investigaciones Científicas |FUNCTIONALIZED DERIVATIVES AND IMMUNORREACTIVES FOR THE FUNGICIDE FLUDIOXONIL|CN110559271A|2019-08-25|2019-12-13|南京理工大学|Quintuple stimulation response type nano carrier with imaging function and preparation method thereof|
    法律状态:
    2018-05-10| FG2A| Definitive protection|Ref document number: 2612751 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180510 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201531661A|ES2612751B1|2015-11-17|2015-11-17|PREPARATION OF BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF ANATOXINA-A|ES201531661A| ES2612751B1|2015-11-17|2015-11-17|PREPARATION OF BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF ANATOXINA-A|
    PCT/ES2016/070656| WO2017085339A1|2015-11-17|2016-09-21|Preparation of bioconjugates and antibodies for the immunodetection of anatoxin-a|
    CA3005506A| CA3005506A1|2015-11-17|2016-09-21|Preparation of bioconjugates and antibodies for the immunodetection of anatoxin-a|
    US15/776,814| US11054415B2|2015-11-17|2016-09-21|Preparation of bioconjugates and antibodies for the immunodetection of anatoxin-a|
    [返回顶部]