PREPARATION OF NEW BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF OCRATOXIN A (Machine-tran

专利摘要:
Preparation of new bioconjugates and antibodies for the immunodetection of ochratoxin A. The present invention relates to bioconjugates and labeled derivatives of ochratoxin A by different positions of the molecule, suitable for the production of high affinity antibodies to ochratoxin A. Likewise, the present invention also relates to the use of ochratoxin A bioconjugates and Ochratoxin-A-labeled derivatives as test antigens. In addition, the present invention also relates to methods of analysis, concentration and extraction of ochratoxin A using the antibodies obtained, sometimes in conjunction with test antigens that are bioconjugated or labeled derivatives. This invention also provides a kit for analyzing ochratoxin A comprising antibodies to this mycotoxin, sometimes in conjunction with test antigens that are bioconjugated or labeled derivatives of ochratoxin A. (Machine-translation by Google Translate, not legally binding)
公开号:ES2672945A1
申请号:ES201631470
申请日:2016-11-17
公开日:2018-06-18
发明作者:Antonio Abad Fuentes；Josep Vicent Mercader Badia；Antonio Abad Somovilla；Consuelo AGULLÓ BLANES；Daniel LÓPEZ PUERTOLLANO
申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universitat de Valencia；
IPC主号:

专利说明:

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PREPARATION OF NEW BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF OCRATOXIN A
DESCRIPTION
The present invention relates to bioconjugates and labeled derivatives of ocratoxin A by different positions of the molecule, suitable for the production of high affinity antibodies for ocratoxin A. Likewise, the present invention also relates to the use of bioconjugates of ocratoxin A and labeled derivatives of ochratoxin A as test antigens. In addition, the present invention also relates to methods of analysis, concentration and extraction of ocratoxin A using the antibodies obtained, sometimes together with test antigens that are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing ochratoxin A comprising antibodies against this mycotoxin, sometimes together with test antigens that are bioconjugates or labeled derivatives of ochratoxin 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. The most frequently detected class of biotoxins in foods are those produced by fungi, or mycotoxins. Although more than 400 different mycotoxins have been identified with a great structural diversity, it is about the most toxicologically relevant ones on which there is a more defined regulation regarding the maximum limits allowed in foods.
Ochratoxin A is the most important mycotoxin in the ochratoxin family. Structurally it consists of a molecule of L-phenylalanine linked, through an amide bridge, to a p-chlorophenolic group that contains a dihydroisocumarin group (Figure 1). The main harmful effects of this mycotoxin are nephrotoxic, although hepatotoxic, neurotoxic, teratogenic, immunotoxic and carcinogenic activity has also been observed in different animal species. Ochratoxin A is suspected of being the cause of chronic fatal outcome diseases, such as
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endemic Balkan nephropathy (BEN) and Tunisian nephropathy (TCIN). In addition, since the consumption of food contaminated with OTA is associated with a higher incidence of tumors in the upper urinary tract in man, this mycotoxin has been classified as a possible carcinogen by the International Agency for Research on Cancer (IARC). Two species of the genus Penicillium produce ochratoxin A: P. verrucosum, the main source of contamination in stored grain, and P. nordicum, the largest producer found in meat products. However, most ocratoxigenic fungi are of the genus Aspergillus, and particularly one of the main producers is A. carbonarius, which is frequently found in vineyards. In order of contribution of total ochratoxins to the diet we find cereals (58%), wine (21%), grape juice (7%), coffee (5%) and pork (3%).
The analytical techniques for the determination of ochratoxin A in food are fundamentally of two types: chromatographic and molecular recognition. Among the former, the one with the most acceptance today is HPLC-MS / MS, while among the latter the majority employ antibodies as a detection element. Chromatographic techniques constitute the reference methodology, due to their high sensitivity, reproducibility and mainly due to their ability to determine several mycotoxins simultaneously (K. F. Nielsen et al., J. Agric. Food Chem. 2015, 63, 1029-1034). On the other hand, techniques based on the antibody-analyte interaction (immunoassays, affinity chromatography, immunoreactive strips) are considered the best option when it is necessary to perform a high number of analyzes in a short time and / or in poorly equipped environments. In fact, mycotoxins, and in particular ochratoxin A, are probably the group of pollutants where these rapid immunochemical methods have reached a greater degree of acceptance and implantation, with a large number of immunodiagnostic companies that market kit-type tests for detection of these contaminants (EP Meulenberg, Toxins 2012, 4, 244-266).
Immunoanalytical methods are based on selective, reversible and non-covalent binding between the substance to be detected (analyte) and an antibody that specifically recognizes it. Ochratoxin A, due to its low molecular weight, is not
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immunogenic, and therefore unable to generate an immune response on its own when injected into an experimental animal. In order to generate antibodies for ochratoxin A it is necessary to covalently couple it to a protein, so that the conjugate obtained is immunogenic and allows the production of antibodies against mycotoxin. In order to obtain these conjugates, it is often necessary to resort to the design and synthesis ex novo of a derivative, through strategies that allow the incorporation, in the desired position of the molecule, of a hydrocarbon chain with a terminal functional group, respecting its structure and characteristic chemical groups. This strategy makes it possible to present the molecule to the immune system in the most appropriate way to achieve antibodies of great affinity and specificity. However, ochratoxin A has in its structure a carboxylate group that allows direct anchoring to the protein, so, due to the synthetic difficulties involved in the total synthesis of potentially more structurally appropriate derivatives, all Antibodies obtained to date capable of recognizing ochratoxin A have been obtained using said carboxylate group to obtain the conjugates. The effectiveness of this strategy has been very variable (X. Li et al., Food Anal. Methods 2013, 6, 1433-1440). In any case, the generation of antibodies to ocratoxin A from haptens in which the carboxylate group is free and therefore can interact with the antibody, constitutes an unexplored route that could allow the generation of antibodies with different characteristics to those of those currently available, suitable not only for the development of more sensitive and specific immunoassays, but also for their implementation in new analytical platforms based on advanced technologies, such as biosensors of different types, multiplex assays and methods based on resonance energy transfer fluorescence (FRET).
There is therefore a need to obtain functionalized derivatives of ocratoxin A not previously explored that allow the generation of higher affinity antibodies than those produced to date. These improved antibodies obtained from innovative haptens will form the basis for developing new immunoanalytical methods for the determination, detection, concentration or extraction of ochratoxin A, preferably through the use of a kit that can be used by the food, agricultural, clinical and clinical industries. / or environmental.
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DESCRIPTION OF THE INVENTION
The present invention provides bioconjugates and labeled derivatives of ocratoxin A, and the use of bioconjugates for obtaining antibodies to ocratoxin 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 and R-II; HCX /.O
image 1
Preferably T is R-I.
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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 in S, O and N; preferably L is a linear hydrocarbon chain of 1 to 20 carbon atoms and said hydrocarbon chain comprises between 0 and 4 heteroatoms selected from the group consisting of O and N, and more preferably L is a saturated linear hydrocarbon chain of 1 to 10 atoms of carbon 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-,
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- (SO2) NH-, -NH (SO2) -, -O (C = O) NH-, -NH (C = O) O-, -NH (C = O) NH-,
-NH (C = S) NH-, -NH-, -N (CrCa alkyl) - -S-, -S-S-, -NH-NH-, -N = C-, -C = N-,
-NH (C = NH) -, -N = N-, -O-, -CH = CH-,
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N—
Y
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In a preferred embodiment, 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-,
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N—
Y
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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 serum 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 T-L-Z fragment and the P peptide or polypeptide, in the resulting bioconjugate of formula (I).
According to another preferred embodiment of the present invention, the bioconjugate of formula (I) is a bioconjugate of formula (la)
image7
(Ia)
where:
P and n have been defined above. Preferably P is albumin or peroxidase and n 5 is a value selected from 1 to 50.
According to another preferred embodiment, the bioconjugate of formula (I) is a bioconjugate of formula (Ib)
image8
10 where:
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 ochratoxin 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):
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[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 gives rise to a signal that can be measured 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 marker coupled to an indirect detection system, micro or nanoparticles or others. Preferably, Q is selected from the group consisting of peroxidase, alkaline phosphatase, biotin, fluorescein or any of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any one of its derivatives, an acridinium ester, quantum nanoparticles (in English quantum dots), and micro- or nanoparticles of colloidal gold, carbon or latex.
In the compound of formula (II), Z is selected from the group that O) NH-, -NH (C = O) -, -O (C = O) NH-, -NH (C = O) O-, - NH (C = O) NH-,
Preferably L, in the compound of formula (II), is a linear hydrocarbon chain of 1 to 20 carbon atoms and said hydrocarbon chain comprises between 0
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Preferably, it consists of - (C = -NH-, -S-,
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Y
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and 4 heteroatoms selected from the group consisting of O and N, and more preferably L is a linear saturated 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 ;
This compound of formula (II) can be used with an ocratoxin A antibody to determine or detect this mycotoxin in a sample by immunoassay technology.
According to a preferred embodiment, the derivative of formula (II) is a derivative of formula (IIa)
image11
where Q is selected from the group consisting of peroxidase, biotin, fluorescein or nanoparticles, and m is a value selected between 1 and 10.
According to another also more preferred embodiment, the derivative of formula (II) is a derivative of formula (IIb)
image12
where Q is selected from the group consisting of peroxidase, biotin, fluorescein or nanoparticles, and m is a value selected between 1 and 10.
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The labeled derivative of formula (II) of the present invention can be obtained by a method comprising reacting a functionalized derivative (hapten) of ochratoxin 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 ocratoxin A antibody to determine or detect this mycotoxin in a sample by immunoassay technology. In addition, the labeled derivatives of formula (II) can be used together with an ocratoxin A antibody to determine or detect this mycotoxin in a sample by immunoassay technology.
In order to obtain antibodies against ocratoxin A, functionalized derivatives of said mycotoxin (haptens) have been prepared, that is, structural analogs of ocratoxin A that incorporate a functional group capable of being used for conjugation to a P carrier or Q marker. This functional group it is separated from the skeleton of the ochratoxin A molecule by a spacer L. The position of incorporation of the functional group into the structure of ocratoxin A for conjugation is not an obvious aspect and can be decisive for the viability of the bioconjugates of formula (I ) as inducers of the production of appropriate affinity and selectivity antibodies against ocratoxin A, and even for the viability of the 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 mycotoxin.
In the context of this invention the term "antibody" refers to the immunoglobulin that an animal or a hybrid cell (such as a hybridoma) synthesizes specifically against the immunogen of the invention (bioconjugate of the invention).
Thus, a third aspect of the present invention relates to an antibody (hereinafter antibody of the invention) generated in response to a bioconjugate of the invention, in particular the bioconjugate of formula (I). More preferably the antibodies are generated in response to the bioconjugate of formula (Ia) or (Ib), more preferably to the bioconjugate of formula (Ia).
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A fourth aspect of the invention relates to the use of the bioconjugate described above for obtaining antibodies.
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, such as from the immunization of an animal. 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 ochratoxin 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 to 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 fifth aspect of the present invention relates to a method of in vitro analysis of ocratoxin A in a sample comprising the use of the antibody described above. Preferably, this method comprises 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 step (b).
The method of the present invention allows quantitative determination or qualitative analysis of the content of mycotoxin ocratoxin A in a sample. Likewise, the method of the present invention allows analyzing the content of ochratoxin A in different types of samples, for example, food samples, such as cereals, fruits and wines, environmental samples such as water, soil or surface, and
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Isolated biological samples such as urine. Preferably, the present invention provides a method of in vitro analysis of ocratoxin-A in wines, beers and juices.
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 immunochemical interaction or reaction consists of the specific and non-covalent binding between an antibody and an antigen, which may be 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 sixth aspect of the present invention also relates to an ocratoxin A detection kit that uses at least one antibody of the invention. Additionally, the ocratoxin A detection kit may comprise a bioconjugate of formula (I) or a labeled derivative of formula (II) as described in the present patent application.
A seventh aspect of the present invention also relates to a method of purification and / or concentration of ochratoxin A from a sample comprising the use of the antibody described above. Particularly, this method is based on immobilizing at least one antibody of the invention on any support and passing a sample through said support to retain the ochratoxin A present in said sample. Subsequent elution of ochratoxin A retained in the
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Support by methods widely known in the art (pH change, ionic strength modification, use of chaiotropic agents) will allow its purification and / or concentration, in a system known as immunoaffinity chromatography. In a preferred embodiment, this method comprises the following steps:
a) immobilizing at least one antibody described in any of claims 12 or 13 on a support;
b) passing the sample through said support to retain the ochratoxin A present in said sample; Y
c) elute the retained ochratoxin A in the support.
The terms "immunogen" 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 features 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 how the preparation of several functionalized derivatives of ocratoxin A (haptens) and the corresponding bioconjugates of formula (I) can be carried out, which are not intended to be limiting of the present invention, and that they 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 the development of An effective immunoanalytical method.
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BRIEF DESCRIPTION OF THE FIGURES Fig. 1. Structure of ochratoxin-A.
Fig. 2. Scheme of the synthesis of the active ester of hapten OTA-1 (NHS-OTA-1).
Fig. 3. Scheme of the synthesis of the active ester of hapten OTA-2 (NHS-OTA-2).
Fig. 4. Scheme of synthesis of the active ester of hapten OTA-3 (NHS-OTA-3).
Fig. 5. Scheme of the preparation of a bioconjugate of formula (I) from the corresponding functionalized derivative (active ester) of ocratoxin A.
Fig. 6. Standard curves for ochratoxin A in the direct homologous competitive ELISA format obtained with the best monoclonal antibody produced from the BSA-OTA-1 bioconjugate (mAb Ia # 310b; triangles) and with the best monoclonal antibody produced from of the bioconjugate BSA-OTA-2 (mAb Ib # 115; circles).
EXAMPLES
Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness of the bioconjugates of formula (I) for obtaining antibodies against ochratoxin 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 = CH2CH2CH2CH2CH2, Z = - (C = O) NH- and P = BSA (bovine serum albumin), OVA (ovalbumin) and HRP (horseradish peroxidase ).
1.1. Preparation of the N-hydroxysuccinimidyl ester of hapten OTA-1 (NHS-OTA-1).
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Preparation of 4-chloro-2,6-diiodo-3-methylphenol (2). Absolute EtOH (31 mL) was added over a mixture of 4-chloro-3-methylphenol (1) (1.78 g, 12.5 mmol), iodine (6.93 g, 27.3 mmol, 2.2 equiv ) and AgSO4 (7.76 g 24.9 mmol, 2 equiv) and the mixture was kept under stirring for 3 hours at room temperature. Once the reaction was finished, the mixture was filtered to separate the salts, using CHCl3 to wash. The filtrate was washed with a 10% aqueous solution of Na2S2O3 and brine. After drying over anhydrous MgSO4 and evaporating the solvent in vacuo, the residue obtained was purified through a silica gel column, using hexane as eluent, to obtain compound 2 as a white solid (3.72 g, 75% ). Mp 91.0-92.0 ° C (cold hexane crystallized).
Spectroscopic data of 2 1 H-NMR (CDCl 3, 300 MHz): 5 (ppm) 2.59 (s, 3 H, Me-3), 5.83 (s, 1 H, OH), 7.72 (s, 1 H, H-5). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 26.8 (Me-3), 77.6 (C-6), 90.5 (C-2), 125.2 (C-4), 137.9 (C-5), 140.3 (C-3), 152.6 (C-1). EMAR (TOF 15 ESI-): calculated for C7H4ClI2O [M-H] "392.8046, found 392.8042.
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Preparation of dimethyl 5-chloro-2-hydroxy-4-methylbenzene-1,3-dicarboxylate (3). A mixture of phenol 2 (300 mg, 0.76 mmol) and PdCl2 (dppf) CH2Cl2 (64.0 mg, 0.08 mmol, 0.1 equiv) in anhydrous MeOH (9 mL) in a tiny Büchi reactor was purged thoroughly for 20 repeated cycles of vacuum-purge with argon, cooling to 0 ° C. After this, and under a stream of argon, Et3N (0.53 mL, 3.8 mmol, 5 equiv) was quickly added and the system was again subjected to purge cycles at 0 ° C, first with argon and then with CO. Then, the CO pressure of 90 psi was adjusted and the reaction mixture was stirred at 90 ° C for 2.5 hours. After this time the reactor was cooled and
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vented, the reaction mixture was transferred to a round bottom flask with the aid of CH2Cl2 and concentrated to dryness under reduced pressure. The residue was suspended in Et2O and filtered, the filtrate was washed with a 1M aqueous solution of HCl and brine, dried over anhydrous MgSO4 and the solvent was evaporated in vacuo. The obtained residue was purified by chromatography on silica gel, using hexane-AcOEt mixtures (95: 5 and 90:10) as eluent, to obtain compound 3 as a white solid (158.4 mg, 82%) . Mp 66.1-67.1 ° C (cold hexane crystallized).
3: 1H-NMR spectroscopic data (CDCl3, 300 MHz): 5 (ppm) 2.35 (s, 3H, Me-4), 3.96 (s, 6H, 2xOMe), 7.87 (s, 1H, H-6), 10.96 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 18.0 (Me-4), 52.7 (2xOCH3), 111.5 (C-3), 124.9 (C-5), 125.1 (C-1), 130.6 (C - 6), 141.5 (C-4), 156.8 (C-2), 166.8 (CO2-3), 169.1 (CO2-1). EMAR (TOF ESI +): calculated for C11H12ClO5 [M + H] + 259.0368, found 259.0356.
H3CO
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Preparation of methyl 5-chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxylate (4). A solution of dimethyl dicarboxylate 3 (82.4 mg, 0.32 mmol) in anhydrous THF (200 ^ L) was added dropwise, at -78 ° C under nitrogen, on a solution of lithium diisopropylamide (LDA) in THF, generated from diisopropylamine (130 ^ L, 0.88 mmol, 2.75 equiv), BuLi (500 ^ L of a 1.6 M solution in hexane, 0.8 mmol, 2.5 equiv) and THF anhydrous (1.15 mL). The mixture was kept under stirring for 30 minutes at -78 ° C and then acetaldehyde (150 ^ L, 2.55 mmol, 8 equiv) was added. The reaction mixture was stirred at the same temperature for 10 minutes and at 0 ° C for 1.5 hours. Once the reaction time was over, 1 mL of a 1: 2 solution of AcOH in Et2O was added at 0 ° C, diluted with AcOEt and washed with water and brine, dried over anhydrous MgSO4 and concentrated in vacuo to obtain a residue that was purified by column chromatography, using hexane-AcOEt-AcOH mixtures (100: 0: 0.3 and 95: 5: 0.3) as eluent to obtain compound 4 as a white semi-solid (51 , 7 mg, 82%).
Spectroscopic data of 4 (a racemic mixture): 1H-NMR (CDCI3, 300 MHz): 5 (ppm) 1.58 (d, J = 6.4 Hz, 3H, Me-3), 2.84 (dd, J = 17.4, 11.6 Hz , 1H, H-4), 3.27 (dd, J = 17.3, 3.2 Hz, 1H, H'-4), 3.95 (s, 3H, CO2CH3), 4.42-4.92 (m, 1H, H-3), 8.11 (s, 1H, H- 6), 12.19 (s, 1H, OH). 13C-NMR (CDCI3, 75 MHz): 5 (ppm) 20.6 (Me-3), 32.6 (C-4), 52.6 5 (OCH3), 75.1 (C-3), 111.2 (C-7), 118.4 ( C-8a), 121.7 (C-5), 138.0 (C-6), 142.2 (C-4a),
161.1 (C-8), 165.0 (C-1), 167.9 (CO2-7). EMAR (TOF ESI +): calculated for C12H12CIO5 [M + H] + 271.0368, found 271.0371.
H3CO
i. LiOH, THF-H20, 3.5h, reflux
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Preparation of 5-chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxylic acid (5). A solution of LiOH H2O (115.0 mg, 2.73 mmol, 10 equiv) in water (1.20 mL) was added on a suspension of dihydroisocoumarin 4 (74.0 mg, 0.27 mmol) in THF anhydrous (910 pL). The mixture was heated at reflux for 3.5 hours and then cooled to 0 ° C and acidified with a 1M aqueous solution of HCl (4.91 mL, 4.91 mmol, 18 equiv). The reaction mixture was stirred at room temperature for 2 hours, diluted with water and extracted with AcOEt. The combined organic phases were washed with brine, dried over anhydrous MgSO4 and concentrated under reduced pressure to obtain acid 5 (69.9 mg, 100%) as a light brown amorphous solid.
5: 1H-NMR spectroscopic data (DMSO-d6, 300 MHz): 5 (ppm) 1.44 (d, J = 6.2 20 Hz, 3H, Me-3), 2.88 (dd, J = 17.3, 11.6 Hz, 1H , H-4), 3.20 (dd, J = 17.3, 3.2 Hz, 1H, H'-4), 4.75 (ddd, J = 11.7, 6.2, 3.2 Hz, 1H, H-3), 7.99 (s, 1H , H-6). 13C-NMR (DMSO-da, 75 MHz): 5 (ppm) 20.1 (Me-3), 32.2 (C-4), 74.4 (C-3), 112.5 (C-7), 117.8 (C-8a) , 120.6 (C-5), 136.0 (C-6), 143.4 (C-4a), 160.5 (C-8), 165.4 (C-1), 167.3 (CO2H). EMAR (TOF ESI +): calculated for C11H10ClO5 [M + H] + 257.0211, found 257.0212.
image21
Preparation of tert-butyl 2- (5-chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) -3- (4- iodophenyl) propanoate (7). To a solution of acid 5 (56.3 mg, 0.22 mmol) in anhydrous DMF (1.5 mL) was added successively a solution of HATU 5 (125.5 mg, 0.33 mmol, 1.5 equiv) in anhydrous DMF (1.5 mL) and DIEA (80 ^ L, 0.44 mmol,
2 equiv). The reaction mixture was stirred for 2 hours at room temperature and then a solution of the amine 6 (153 mg, 0.44 mmol, 2 equiv) and DIEA (80 ^ L, 0.44 mmol, 2 equiv) was added in Anhydrous DMF and stirred at room temperature for 4 hours, after which the reaction was diluted with AcOEt and washed successively with aqueous solutions of HCl (1M), LiCl (1.5%), NaHCO3 (5%) and brine , dried over anhydrous MgSO4 and the solvent evaporated in the rotary evaporator. The obtained residue was purified by column chromatography, using hexane-AcOEt-AcOH mixtures (100: 0: 0.3, 90: 10: 0.3 and 80: 20: 0.3) as eluent to obtain compound 7 (107 mg, 83%) as a yellowish oil.
15 Spectroscopic data of 7 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.43 (two s, 4.5H each, CMe3 of each diastereoisomer), 1.61 (two d, J = 6.4, 1.5H each, Me-3 'of each diastereoisomer), 2.84 and 2.89 (each dd, J = 17.4, 11.6 Hz, 0.5H each, H-4' of each diastereoisomer), 3.07-3.24 (m, 2H, H2-3), 3.30 (dd, J = 17.4, 3.5 Hz, 1H, H'-4 '), 4.77 (m, 1H, H-3'), 4.94 (dt, J = 20 7.3 , 6.0 Hz, 1H, H-2), 6.96 (br d, J = 7.9 Hz, 2H, H-2 '' and H-6 ''), 7.56-7.62 (m, 2H, H-3 '' and
H-5 ’’), 8.44 (s, 1H, H-6 ’), 8.57 (m, 1H, NH), 12.78 (wide s, 1H, OH). 13C-NMR (CDCh, 75 MHz): 5 (ppm) 20.7 (Me-3 ’), 28.0 (CMe3), 32.3 (C-4’), 37.6 (C-3), 54.3 (C-2), 75.9
(C-3 '), 82.6 (CMea), 92.4 (C-4' '), 110.0 (C-8a), 120.7 (C-7'), 123.1 (C-5 '), 131.5 (C-2' 'and C-6' '), 136.1 (C-1' '), 137.4 (C-3' 'and C-5' '), 138.9 (C-6'), 140.7 (C-4a), 159.0 ( C-8 '), 162.1 (CONH), 169.7 (C-1'), 170.1 (C-1). EMAR (TOF ESI +): calculated for C24H26NClIO6 [M + H] + 586.0488, found 586.0459.
image22
OCH,
5
Preparation of 6- (4- (3- (tert-butoxy) -2- (5-chloro-8-hydroxy-3-methyl-1-oxochromoxane-7-carboxamido) -3-oxopropyl) phenyl) hex-5- methyl innate (9). A mixture of the iodinated derivative 7 (36.5 mg, 0.062 mmol), methyl hex-5-ionate (8) (27.4 mg, 0.217 mmol, 3.5 equiv), PdCl2 (Ph3P) 2 (5.1 mg, 7.310-3 mmol, 0.12 equiv) and CuI (2.2 mg, 11.510-3 mmol, 10 0.18 equiv) was purged with repeated cycles of vacuum-nitrogen while cooling to 0
° C. Next, anhydrous DMF (730 pL) was added and the system was purged again, Et3N (540 pL, 3.87 mmol, 62 equiv) was added and the system was purged again at low temperature. The reaction mixture was stirred at room temperature for 21 hours, after which it was diluted with AcOEt and washed successively with aqueous solutions of HCl (1M), LiCl (1.5%), NaHCO3 (5%) and brine. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to obtain a residue that was purified by column chromatography on silica gel, using hexane-AcOEt mixtures as eluent (80:20, 70:30 and 50:50 ), to obtain the alkyne 9 (22.5 mg, 62%) as a yellowish oil.
Spectroscopic data of 9 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCI3, 300 MHz): 5 (ppm) 1.43 (wide s, 9H, CMe3 of the two diastereoisomers), 1.60 (two d, J = 6.3 Hz, 1.5H each, Me-3 '' of each diastereoisomer), 1.87-1.98 (m, 2H, H2-3), 2.50 (m, 4H, H2-2 and H2-4), 2.84 and 2.89 ( two dd, J = 17.5, 11.5 Hz, 0.5H each, H-4 '' '5 of each diastereoisomer), 3.19 (m, 2H, H2-1' '), 3.30 (dd, J = 17.4, 3.5 Hz , 1H, H'-4 '' '), 3.68 (s, 3H, CH3O), 4.71-4.82 (m, 1H, H-3' ''), 4.96 (dt, J = 7.3, 6.0 Hz, 1H, H-2 ''), 7.14 (d, J = 7.9 Hz, 2H, H-3 'and H-5'), 7.30 (dd, J = 8.2, 1.4 Hz, 2H, H-2 'and H-6 '), 8.45 (s, 1H, H-6' ''), 8.56 (m, 1H, NH), 12.76 (s, 1H, OH). 13C-NMR (CDCI3, 75 MHz): 5 (ppm) 18.9 (C-4), 20.7 (Me-3``), 23.9 (C-3), 28.0 (CMe3), 32.3 (C-4 '' '), 32.9 (C-2), 38.0 (C-1' '), 51.6 10 (CH3O), 54.5 (C-2' '), 75.8 (C-3' ''), 81.3 (C-6) , 82.5 (CMe3), 88.8 (C-5), 110.1 (C-8a), 120.8 (C-7 ''), 122.3 (C-4 '), 123.1 (C-5' ''), 129.4 ( C2 'and C-6'), 131.5 (C-3 'and C-5'), 136.0 (C-1 '), 139.0 (C-6' ''), 140.6 (C-4a), 159.1 (C -8 '' '), 162.1 (CONH), 169.7 (C-1' ''), 170.2 (C-3 ''), 173.6 (C-1). EMAR (TOF ESI +): calculated for C31H35CINO8 [M + H] + 584.2046, found 584.2029.
fifteen
image23
OH OR
image24
76%
RhCI (PPh3) 3 H2 (4 atm) THF, 28h, ta
9
image25
image26
Preparation of methyl 6- (4- (3- (tert-butoxy) -2- (5-chloro-8-hydroxy-3-methyl-1-oxoisochroman-7- carboxamido) -3-oxopropyl) phenyl) hexanoate ( 10). A solution of alkyne 9 (17.6 mg, 0.03 mmol) and RhCl (PPh3) 3 (4.2 mg, 4.510-3 mmol, 0.15 equiv) in anhydrous THF (1.3 mL) contained in a tiny Büchi reactor was purged with hydrogen. The hydrogen pressure was adjusted to 4 atm and maintained with stirring at temperature for 28 hours. The reactor was then vented and the reaction mixture was
concentrated to dryness in vacuo to obtain a residue that was purified by column chromatography, using hexane-AcOEt mixtures (90:10, 80:20 and 60:40) as eluent to obtain compound 10 (13.4 mg, 76 %) as a yellowish oil.
5 Spectroscopic data of 10 (a 1: 1 mixture of diastereoisomers): 1 H-NMR
(CDCl3, 300 MHz): 5 (ppm) 1.31-1.38 (m, 2H, H2-4), 1.42 (wide s, 9H, CMe3 of
both diastereoisomers), 1.54-1.71 (m, 4H, H2-3, H2-5), 1.60 (two d, J = 6.2 Hz, 1.5H each, Me-3 '' 'of each diastereoisomer), 2.30 (t , J = 7.5 Hz, 2H, H2-2), 2.57 (t, J = 7.6 Hz, 2H, H2-6), 2.83 and 2.88 (two dd, J = 17.4, 11.6, 0.5H each, H-4 ''' decade
10 diastereoisomer), 3.17 (d, J = 6.0 Hz, 2H, H2-1 ’), 3.29 (dd, J = 17.4, 3.5 Hz, 1H, H’-
4 '' '), 3.66 (s, 3H, OMe), 4.69-4.82 (m, 1H, H-3' ''), 4.94 (dt, J = 7.3, 6.0 Hz, 1H, H-2 '') , 7.03-7.16 (m, 4H, H-2 ', H-3', H-5 'and H-6'), 8.46 (s, 1H, H-6 '' '), 8.54 (m, 1H, NH), 12.73 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 20.7 (Me-3``), 24.8 (C-5), 28.0 (CMe3), 28.7 (C-4), 31.0 (C-3), 32.3 (C-4 '' '), 34.0 (C-2), 35.3 (C-6), 37.8 (C-1' '), 51.4 15 (OMe), 54.7 (C-2' '), 75.8 ( C-3 '' '), 82.2 (CMe3), 110.0 (C-8a), 121.0 (C-7' ''), 123.0 (C-5 ''), 128.4 (C-2 'and C-6 '), 129.4 (C-3' and C-5 '), 133.5 (C-1'), 139.0 (C-6 '' '), 140.5 (C-4a), 141.1 (C-4'), 159.1 (C-8 '' '), 162.1 (CONH), 169.8 (C-1' ''), 170.5 (C-3 ''), 174.2 (C-1). EMAR (TOF ESI +): calculated for C31H39ClNO8 [M + H] + 588.2359, found 588.2358.
twenty
image27
5
10
fifteen
twenty
25
Preparation of 6- (4- (3- (tert-butoxy) -2- (5-chloro-8-hydroxy-3-methyl-1-oxo-isochroman-7-carboxamido) -3-oxopropyl) phenyl) hexanoic acid (11 ). A suspension of methyl ester 10 (13.4 mg, 0.023 mmol) and Candida antarctica lipase immobilized in macroporous acrylic resin (Novozyme 435, 26.4 mg) in THF (160 pL) in a phosphate buffer solution with pH = 7, 4 (630 pL) was gently stirred at room temperature for 7 hours, after which it was filtered and the resin and reactor were washed with water and AcOEt. The aqueous phase was acidified with a 1M aqueous solution of HCl to pH = 2 and extracted with AcOEt. The combined organic phases were washed with brine, dried over anhydrous MgSO4 and concentrated to dryness under reduced pressure to obtain an oily residue that was purified by column chromatography on silica gel, using CHCl3-MeOH mixtures as eluent (100: 0, 95: 5 and 90:10), to obtain acid 11 (9.9 mg, 75%) as a yellowish oil.
Spectroscopic data of 11 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.31-1.37 (m, 2H, H2-4), 1.43 (s, 9H, CMe3), 1.54-1.70 (m, 4H, H2-3, H2-5), 1.60 (two d, J = 6.2 Hz, 1.5H each, Me-3 '' 'of each diastereoisomer), 2.32 (t, J = 7.3 Hz, 2H, H2-2), 2.57 (t, J = 7.5 Hz, 2H, H2-6), 2,782.92 (m, 1H, H-4 '' '), 3.17 (m, 2H, H2-1 ''), 3.29 (dd, J = 17.4, 3.3 Hz, 1H, H'-4 '' '), 4,684.82 (m, 1H, H-3' ''), 4.88-5.00 (m, 1H, H-2 ''), 7.02-7.16 (m, 4H, H-2 ', H-3', H-5 'and H-6'), 8.45 (s, 1H, H-6 '' '), 8.56 (m, 1H, NH), 12.73 (broad s, 1H, OH). 13C-NMR (CDCI3, 125 MHz): 5 (ppm) 20.7 (Me-3``), 24.5 (C-3), 28.0 (CMe *), 28.4 (C-4), 30.9 (C-5) , 32.3 (C- 4 '' '), 33.8 (C-2), 35.2 (C-6), 37.8 (C-1' '), 54.7 (C-2' '), 75.9 (C-3' ' '), 82.3 (CMe3), 110.0 (C-8a), 121.0 (C-7' ''), 123.0 (C-5 '' '), 128.4 (C-2' and C-6 '), 129.4 ( C-3 'and C-5'), 133.5 (C-1 '), 139.0 (C-6' ''), 140.6 (C-4a), 141.1 (C-4 '), 159.1 (C-8' ''), 162.2 (CONH), 169.8 (C-1 '' '), 170.5 (C-3' '), 179.3 (CO2H). EMAR (TOF ESI +): calculated for C30H37NCO [M + H] + 574.2202, found 574.2220.
HO
image28
OR
H
image29
eleven
OH
r
Cl
image30
Preparation of 6- (4- (3- (tert-butoxy) -2- (5-chloro-8-hydroxy-3-methyl-1-oxochromochrome-7- carboxamido) -3-oxopropyl) phenyl) hexanoate of 2, 5-dioxopyrrolidin-1-yl (12). A solution of acid 11 (10.2 mg, 0.018 mmol), NHS (2.2 mg, 0.019 mmol, 1.1 equiv) and 5 EDCHCl (3.7 mg, 0.019 mmol, 1.1 equiv) in anhydrous CH3CN (1.0 mL) was stirred at room temperature for 24 hours under a nitrogen atmosphere. After this time, the reaction mixture was diluted with AcOEt and washed successively with water, a 5% aqueous solution of NaHCO3 and brine. After evaporating to dryness, the obtained residue was passed through a small column of 10 silica gel, using CH2Cl2 as eluent, to obtain N-hydroxysuccinimidyl ester 12 (9.6 mg, 79%) as a yellowish oil.
Spectroscopic data of 12 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 500 MHz): 5 (ppm) 1.42 (broad s, 9H, CMe3 of both diastereoisomers), 1.43-1.49 (m, 2H, H2 -5), 1.58-1.67 (m, 2H, H2-4), 1.60 (two d, 1.5H each, J = 6.4, 15 Me-3 '' 'of each diastereoisomer), 1.77 (quint, J = 7.6 Hz, 2H, H2-3), 2.59 (m, 4H, H2-2
and H2-6), 2.79-2.90 (m, 5H, H-4 '' ', COCH2CH2CO), 3.11-3.22 (m, 2H, H2-1' '), 3.29 (dd, J = 17.4, 3.5 Hz, 1H, H-4 '' '), 4.69-4.81 (m, 1H, H-3' ''), 4.89-4.97 (m, 1H, H-2 ''), 7.06-7.16 (m, 4H, H -2 ', H-3', H-5 'and H-6'), 8.45 (s, 1H, H-6 '' '), 8.54 (m, 1H, NH), 12.73 (wide s, 1H, OH). 13C-NMR (CDCl3, 125 MHz): 5 (ppm) 20.7 (Me-3``), 24.4 (C-3), 25.6 20 (COCH2CH2CO), 28.0 (CMe3), 28.4 (C-4), 30.8 (C-5), 30.9 (C-2), 32.3 (C-4 '' '), 35.2 (C-
6), 37.8 (C-1 ''), 54.7 (C-2 ''), 75.9 (C-3 '' '), 82.2 (CMe3), 110.0 (C-8a), 121.0 (C-7' ' '), 123.0 (C-5' ''), 128.4 (C-2 'and C-6'), 129.5 (C-3 'and C-5'), 133.6 (C-1 '), 139.0 (C -6 '' '), 140.5
(C-4a), 140.9 (C-4 '), 159.1 (C-8' ''), 162.1 (CONH), 168.6 (C-1), 169.1 (COCH2CH2CO), 169.8 (C-1 '' ') , 170.5 (C-3``).
image31
image32
1: 1 CF3Ca2H-CH2Cl2 ta, 2h
image33
NHS-OTA-1
Preparation of 2- (5-Chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) -3-5 (4- (6 - ((2,5-dioxopyrrolidin-1-yl) oxy) acid) -6-oxohexyl) phenyl) propanoic acid (N- ester
Hydroxysuccimidyl of hapten OTA-1, NHS-OTA-1). Trifluoroacetic acid (370 pL, 4.8 mmol, 370 equiv) was added dropwise over a solution of tert-butyl ester 12 (9.0 mg, 0.013 mmol) in anhydrous CH2Cl2 (760 pL) and the resulting mixture was maintained with stirring for 2 hours at room temperature. After this, the reaction mixture was concentrated to dryness under reduced pressure to provide the NHS-OTA-1 ester (7.9 mg, 99%) as a resinous oil and brownish color.
Spectroscopic data of NHS-OTA-1 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 500 MHz): 5 (ppm) 1.42 (m, 2H, H2-2 ''), 1.57-1.68 (m , 2H, H2-3 ''), 1.75 (m, 2H, H2-4 ''), 1.60 (two d, 1.5H each, J = 6.4, Me-3 '' 'of each diastereoisomer), 15 2.59 (m, 4H, H2-1 '' and H2-5 ''), 2.78-2.91 (m, 5H, H-4 '' ', COCH2CH2CO), 3.16-3.34 (m,
3H, H2-3 and H'-4 '' '), 4.77 (m, 1H, H-3' ''), 5.00-5.04 (m, 1H, H-2), 7.06-7.18 (m, 4H, H-2 ', H-3', H-5 'and H-6'), 8.42 (m, 1H, H-6 '' '), 8.51 and 8.58 (two m, 0.5H each, NH of each diastereoisomer). EMAR (TOF ESI +): calculated for C30H32ClN2O10 [M + H] + 615.1740, found 615.1732.
twenty
10
image34
1.2. Preparation of a bioconjugate of hapten OTA-1 with BSA (BSA-OTA-1). 100 µL of the NHS-OTA-1 active ester solution (50 mM in DMF) obtained in the previous reaction was added slowly and with constant stirring over 0.9 mL of a BSA solution (15 mg / mL) in buffer 50 mM carbonate, pH 9.6. The conjugation reaction was incubated for 2 hours 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.
5
To determine the haptic load (n) obtained in the conjugate, a 100 ^ L aliquot of the purified BSA-OTA-1 bioconjugate was dialyzed (dialysis against 5 L of deionized water 15 with at least 2 to 3 water changes every 24 ha 4 ° C); finally, the dialyzed product was lyophilized and the number of hapten molecules (OTA-1) conjugated per BSA molecule was determined by MALDI-TOF-MS (n = 15.2, see Table 1, entry 1).
5
10
image35
1.3. Preparation of a bioconjugate of hapten OTA-1 with OVA (OVA-OTA-1). From a 50 mM solution of the activated hapten NHS-OTA-1 in DMF, 90 pL were taken and added slowly and with constant stirring to a volume of 1.7 mL of an OVA solution (15 mg / mL) in 50 mM carbonate buffer, pH 9.6. After 2 h of reaction under gentle stirring 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 aliquot of the newly obtained OVA-OTA-1 conjugate was dialyzed and lyophilized to calculate the efficiency of conjugation in terms of the number of hapten molecules (OTA-1) coupled to the protein by MALDI-TOF-MS (n = 9, 6, see Table 1, entry 2).
5
10
image36
1.4. Preparation of a bioconjugate of hapten OTA-1 with HRP (HRP-OTA-1). From a 5 mM solution of the activated hapten NHS-OTA-1 in DMF, 80 pL were taken and added slowly and with constant and gentle stirring 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 BSA and OVA bioconjugates, and was brought to a concentration of 400 pg / mL in PBS buffer with 1% (w / v) BSA and 0.02% thimerosal (p / v) and stored at 4 ° C. An aliquot of the newly obtained HRP-OTA-1 conjugate was dialyzed and lyophilized to calculate the efficiency of conjugation in terms of the number of hapten molecules (OTA-1) coupled to the protein by MALDI-TOF-MS (n = 1, 1, see Table 1, entry 3).
TABLE 1. Values of the haptic load of the hapten OTA-1 protein conjugates determined by MALDI-TOF-MS
 RMo m / z reference protein m / z A (m / z) Am / hapten n
 one  BSA-OTA-1 24 6,380 73,988 7,608 500 15.2
 2  OVA-OTA-1 10 2,397 # 28,235 # 4,838 500 9.6
 3  HRP-OTA-1 8 43.884 44.437 553 500 1.1
RM0: initial hapten / protein molar ratio used for conjugation n: hapten / protein 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 # corresponding to the double charged ion ([M + 2H] 2+)
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 N-hydroxysuccinimidyl ester of hapten OTA-2 (NHS-OTA-2).
image37
OCH, i- LDA, THF
image38
5
Preparation of methyl 3- (5- (benzyloxy) pentyl) -5-chloro-8-hydroxy-1-oxoxychroman-7-carboxylate (14). A solution of diester 3 (117.0 mg, 0.45 mmol) in anhydrous THF (280 pL) was added dropwise over a solution of LDA in THF, generated from diisopropylamine (185 pL, 1.25 mmol, 2.75 equiv) and 1.6 M BuLi in hexane (710 pL, 10 0.14 mmol, 2.5 equiv) in THF (1.4 mL), at -78 ° C under nitrogen. The mixture remained
at the same temperature for 30 minutes and then a solution was added
of aldehyde 13 (150 mg, 0.73 mmol, 1.6 equiv) in anhydrous THF (150 pL). The reaction mixture was stirred at -78 ° C for 15 minutes and then allowed to warm slowly to -5 ° C (approximately 3 hours). After this time, the reaction mixture was treated with a 1: 2 mixture of AcOH-Et2O and stirred for 10 minutes at 0 ° C, diluted with AcOEt and washed with water, brine and dried over
Anhydrous MgSO4 to obtain, after evaporating the solvent in vacuo, a residue that was purified by chromatography, using hexane-AcOEt-AcOH mixtures as eluent (100: 0: 0.3, 95: 5: 0.3 and 85:15 : 0.3) to obtain compound 14 (109.0 mg, 67%) as yellowish oil.
10 Spectroscopic data of 14 (a racemic mixture): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.30-1.93 (m, 8H, H2-1 ', H2-2', H2-3 'and H2- 4 '), 2.77 (dd, J = 17.3, 11.7 Hz, 1H, H- 4), 3.16 (dd, J = 17.3, 3.4 Hz, 1H, H'-4), 3.43 (t, J = 6.3 Hz, 3H, H2-5 '), 3.89 (s, 3H, OMe), 4.44 (s, 2H, OCH2), 4.45-4.58 (m, 1H, H-3), 6.98-7.46 (m, 5H, Ph), 8.05 (s, 1H, H-6), 12.14 (s, 1H, OH). 13C-NMR (CDCh, 75 MHz): 5 (ppm) 24.5 (C-2 ’), 25.9 (C-3’),
15 29.5 (C-4 ’), 30.9 (C-4), 34.6 (C-1’), 52.5 (OMe), 70.0 (C-5 ’), 72.8 (OCH2), 78.5 (C-3),
111.4 (C-7), 118.2 (C-8a), 121.7 (C-5), 127.5 (C-4 ''), 127.6 (C-2 '' 'and C-6' '), 128.3 (C -3 '' 'and C-5' ''), 137.9 (C-1 '' '), 138.5 (C-6), 142.3 (C-4a), 161.0 (C-8), 165.0 (C-1 ), 167.9 (CO2). EMAR (TOF ESI +): calculated for C23H2aCl Oa [M + H] + 433.1412, found 433.1415.
image39
97%
i. LiOH, THF-H20, 3.5h, refl.
ii. 1M HCI-H20, 2h, 0 ° C
image40
image41
Preparation of 3- (5- (benzyloxy) pentyl) -5-chloro-8-hydroxy-1-oxoisocroman-7- carboxylic acid (15). A solution of LiOH H2O (135.0 mg, 3.21 mmol, 10 equiv) in water (1.40 mL) was added over a solution of dihydroisocoumarin 14 (139.0 mg,
0.32 mmol) in anhydrous THF (1.30 mL) and the mixture was heated at reflux for 3.5 hours. The reaction mixture was then cooled in an ice bath and treated with a 1M aqueous solution of HCl (5.80 mL, 5.80 mmol, 18 equiv), stirred at 0 ° C for 2 hours, diluted with water and extracted with AcOEt. The combined organic phases were washed with brine, dried over anhydrous MgSO4 and concentrated to dryness under reduced pressure to obtain acid 15 (130.5 mg, 97%) as a yellowish semi-solid, whose 1 H NMR shows that it has a purity high enough to be used in the next stage without further purification.
10 Spectroscopic data of 15 (a racemic mixture): 1H-NMR (MeOD, 300 MHz): 5 (ppm) 1.42-1.91 (m, 8H, H2-1 ', H2-2', H2-3 'and H2- 4 '), 2.87 (dd, J = 17.3, 11.7 Hz, 1H, H-4), 3.21-3.29 (m, 1H, H'-4), 3.52 (t, J = 6.4 Hz, 2H, H2-5 '), 4.50 (s, 2H, OCH2), 4.56 (m, 1H, H-3), 7.21-7.37 (m, 5H, Ph), 8.12 (s, 1H, H-6). 13C-NMR (MeOD, 75 MHz): 5 (ppm) 25.8 (C-2 '), 27.2 (C-3'), 30.7 (C-4 '), 32.5 (C-4), 35.7 (C-1) '), 71.4 (C-5'), 74.0
15 (OCH2), 79.7 (C-3), 114.0 (C-7), 118.6 (C-8a), 122.8 (C-5), 128.8 (C-4 ’), 129.0 (C-2’ and
C-6``), 129.5 (C-3 '' and C-5``), 138.2 (C-1 ''), 140.0 (C-6), 145.4 (C-4a), 163.0 (C- 8), 167.5 C-1), 169.8 (CO2H). EMAR (TOF ESI +): calculated for C22H24 CO [M + H] + 419.1256, found 419.1251.
image42
image43
image44
Cl
Preparation of tert-butyl 2- (3- (5- (benzyloxy) pentyl) -5-chloro-8-hydroxy-1-oxoisocromano-7-carboxa mido-3-phenylpropanoate (17). A solution of HATU ( 123.5 mg, 0.33 mmol, 1.5 equiv) in anhydrous DMF (1.5 mL) and DIEA (80 pL, 0.44 mmol, 2 equiv) were
they added over a solution of acid 15 (91.0 mg, 0.22 mmol) in anhydrous DMF (1.5 mL) under nitrogen and the resulting mixture was stirred at room temperature for 2 hours. Then, a solution of hydrochloride 16 (108 mg, 0.44 mmol, 2 equiv) and DIEA (80 pL, 0.44 mmol, 2 equiv) in anhydrous DMF (1.1 mL) was added and stirred 5 to at the same temperature for 4 hours, the reaction mixture was diluted with AcOEt and washed successively with aqueous solutions of HCl (1M), LiCl (1.5%), NaHCO3 (5%) and brine, dried over anhydrous MgSO4 and concentrated to dryness under reduced pressure. The obtained residue was purified by chromatography on silica gel, using hexane-AcOEt-AcOH mixtures (100: 0: 0.3, 90: 10: 0.3, and 80: 20: 0.3) as eluent. obtain amide 17 as a yellowish oil (87 mg, 65%).
Spectroscopic data of 17 (a 1: 1 mixture of diastereomers): 1 H-NMR (CDCl 3, 300 MHz): 5 (ppm) 1.28-1.91 (m, 8H, H2-1 '', H2-2 '', H2- 3``, H2-4 ''), 1.36 (s, 9H, CMe3), 2.69-2.86 (m, 1H, H-4 '), 3.08-3.23 (m, 3H, H'-4' and H2- 3), 3.43 (t, J = 6.3 Hz, 2H, H2-5 ''), 4.44 (s, 2H, OCH2), 4.47-4.59 (m, 1H, H-3 '), 4.86-4.96 (m, 1H, H-2), 7.04-7.32 (m, 10H, 15 Ph and PhBn), 8.39 (s, 1H, H-6 '), 8.50 (d, J = 5.3 Hz, 1H, NH), 12.66 (s , 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 24.5 (C-2``), 25.9 (C-3 ''), 27.9 (CMe3), 29.5 (C-4``), 30.6 (C- 4 '), 34.6 (C-1' '), 38.1 (C-3), 54.6 (C-2), 70.0 (C-5' '), 72.9 (OCH2), 79.2 (C-3'), 82.2 (CMe3), 110.2 (C-8a), 120.8 (C-7 '), 123.0 (C-5'), 126.9 (C-4 '' '), 127.5 (C-4 PhBn), 127.6 (C-2 and C-6 PhBn), 128.3 (C-3 '' 'and C-5' ''), 129.0 (C-3 and C-5 PhBn), 129.5 (C-2 '' 'and C-6' ' '), 20 136.3 (C-1' ''), 138.5 (C-1 PhBn), 138.9 (C-6 '), 140.6 (C-4a), 159.0 (C-8'), 162.1
(CONH), 169.7 (C-1 ’), 170.3 (C-1). EMAR (TOF ESI +): calculated for C35H41CINO7 [M + H] + 622.2566, found 622.2570.
image45
Preparation of tert-butyl 2- (5-chloro-8-hydroxy-3- (5-hydroxypentyl) -1-oxoisocromano-7-carbox amido) -3-phenylpropanoate (18). A suspension of Pd (OH) 2 (44 mg, 0.04 mmol, 0.3 equiv) and benzyl ether 17 (85 mg, 0.14 mmol) in AcOEt (4.8 mL) was purged with repeated cycles of vacuum-hydrogen and then stirred vigorously under a pressure of H2 of 1 atm (balloon) at room temperature for 6 hours. The reaction mixture was filtered on a small silica gel column, using AcOEt to wash, and the filtrate and washings were concentrated to dryness to provide alcohol 18 (65 mg, 90%) as an oil, whose NMR. 1H shows the presence of a small percentage (5-6%) of the hydrogenolysis product of the C-Cl bond.
Spectroscopic data of 18 (a 1: 1 mixture of diastereomers): 1 H-NMR (CDCl 3, 300 MHz): 5 (ppm) 1.40-1.98 (m, 8H, H2-1 '', H2-2 '', H2- 3``, H2-4 ''), 1.42 (s, 9H, CMe3), 2.78-2.94 (two dd, J = 17.4, 11.8 Hz, 1H, H-4 'of each diastereoisomer), 3.14-3.33 (m , 3H, H-4 'and H2-3), 3.67 (t, J = 6.4 Hz, 2H, H2-5' '), 4.51-4.69 (m, 1H, H-3'), 4.96 (m, 15 1H, H-2), 7.10-7.35 (m, 5H, Ph), 8.44 (s, 1H, H-6 '), 8.50-8.63 (two d, J = 7.4 Hz, 0.5H
each, NH of each diastereomer), 12.72 (two s, 0.5H each, OH of each diastereomer). 13C-NMR (CDCI3, 75 MHz): 5 (ppm) 24.5 (C-2``), 25.5 (C-3 ''), 27.9 (CMe3), 30.6 (C-4 '), 32.4 (C-4 ''), 34.6 (C-1 ''), 38.1 (C-3), 54.6 (C-2), 62.6 (C-5 ''), 79.3 (C-3 '), 82.3 (CMe3), 110.1 (C-8a), 120.8 (C-7 '), 123.1 (C-5'), 126.9 (C-4 '' '), 128.3 (C-2' '' 20 and C-6 '' '), 129.5 (C-3 '' 'and C-5' ''), 136.2 (C-1 '' '), 138.9 (C-6'), 140.6 (C-4a), 159.0 (C-8 '), 162.1 (CONH), 169.7 (C-1 '), 170.4 (C-1). EMAR (TOF ESI +): calculated for C28H35CINOt [M + H] + 532.2097, found 532.2060.
image46
Preparation of tert-butyl 2- (5-chloro-8-hydroxy-1-oxo-3- (5-oxopentyl) isochromano-7-carboxamido) -3-phenylpropanoate (19). A suspension of periodinan Dess-Martin (DMP) (78.0 mg, 0.18 mmol, 1.5 equiv) and NaHCO3 (82 mg, 0.98 mmol, 8.0 equiv) in CH2CI2 (1.0 mL) A mixture of alcohol 18 (65.0 mg, 0.12 mmol) and 5 NaHCO3 (82 mg, 0.98 mmol, 8.0 equiv) in CH2Cl2 (4 mL) cooled to 0 ° C was added. The resulting mixture was stirred 10 minutes at 0 ° C and 1 hour at room temperature, diluted with AcOEt and washed successively with aqueous solutions of Na2S2O3 (10%), saturated NaHCO3 and brine, dried over anhydrous MgSO4 and concentrated to dryness under reduced pressure to obtain aldehyde 19 (59.6 mg, 92%) as a yellowish oil.
Spectroscopic data of 19 (a 1: 1 mixture of diastereomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.42 (s, 9H, CMe3), 1.48-1.99 (m, 6H, H2-1 '' , H2-2 '' and H2-3 ''), 2.52 (dt, J = 7.0, 1.1 Hz, 2H, H2-4 ''), 2.87 (two dd, J = 17.3, 11.7, 0.5H each, H-4 'of each diastereomer), 3.14-3.31 (m, 3H, H'-4' and H2-3), 4.60 (ddt, J = 11.7, 7.7, 4.0 Hz, 15 1H, H-3 '), 4.92-5.01 (dt, J = 7.3, 6.1 Hz, 1H, H-2), 7.16-7.32 (m, 5H, Ph), 8.45 (s, 1H,
H-6 ’), 8.56 (two d, J = 7.3 Hz, 0.5H each, NH of each diastereoisomer), 9.80 (t, J = 1.4 Hz, 1H, CHO), 12.69 (s, 1H, OH). 13C-NMR (CDCfe, 75 MHz): 5 (ppm) 21.6 (C-3 ''), 24.3 (C-2 ''), 27.9 (CMe *), 30.6 (C-4 '), 34.5 (C- 1 ''), 38.1 (C-3), 43.6 (C-4 ''), 54.6 (C-2), 79.0 (C-3 '), 82.3 (CMe3), 110.1 (C-8a), 120.9 ( C-7 '), 123.1 (C-5'), 126.9 (C-4 '' '), 20 128.3 (C-2' '' and C-6 '' '), 129.5 (C-3' '' and C-5 '' '), 136.3 (C-1' ''), 139.0 (C-6 '), 140.5 (C-4a),
159.0 (C-8 ’), 162.3 (CONH), 169.6 (C-1’), 170.4 (C-1), 201.9 (C-5 ’). EMAR (TOF ESI +): calculated for C28H33ClNO7 [M + H] + 530.1940, found 530.1920.
image47
image48
Preparation of 5- (7 - ((1- (tert-butoxy) -1-oxo-3-phenylpropan-2-yl) carbamoyl) -5-chloro-8-hydroxy-1-oxoisocromano-3-yl) pentanoic acid (twenty). A solution of NaH2PO4 (92.7 mg, 0.67 mmol, 6.0 equiv) and NaClO2 (40.5 mg, 0.45 mmol, 4 equiv) in water (620 ^ L) was added over a solution of the aldehyde 19 (59.6 mg, 0.11 mmol) and 2-methylbut-2-ene 5 (175 ^ L, 1.57 mmol, 14 equiv) in tBuOH (1.5 mL) and the mixture was stirred at temperature
atmosphere for 1 hour. Then, a 1M aqueous solution of HCl (1.5 mL) was added and stirred for 3 minutes at room temperature, diluted with AcOEt, washed with brine and concentrated to dryness in vacuo to obtain acid 20 (62 , 6 mg, 94%) as a yellowish oil.
10 Spectroscopic data of 20 (a 1: 1 mixture of diastereomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.41 (two s, 4.5H each, CMe3 of each diastereoisomer), 1.53
2.00 (m, 6H, H2-3, H2-4 and H2-5), 2.42 (t, J = 7.10 Hz, 2H, H2-2), 2.80-2.92 (m, 1H, H-4 '), 3.13 -3.31 (m, 3H, H'-4 'and H2-3' '), 4.55-4.65 (m, 1H, H-3'), 4.97 (dt, J = 7.3, 6.1 Hz, 1H, H-2 ''), 7.11-7.31 (m, 5H, Ph), 8.44 (s, 1H, H-6 '), 8.57-8.62 (m, 1H, NH), 12.71 (s, 1H, 15 OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 24.2 (C-4), 27.9 (CMe3), 29.6 (C-3), 30.6 (C- 4 '), 33.6 (C-2), 34.3 ( C-5), 38.1 (C-3 ''), 54.6 (C-2 ''), 79.0 (C-3 '), 82.4 (CMe3), 110.1 (C- 8a), 120.7 (C-7') , 123.1 (C-5 '), 126.9 (C-4' ''), 128.3 (C-2 '' 'and C-6' '), 129.5 (C-3' '' and C-5 '' '), 136.2 (C-1' ''), 139.0 (C-6 '), 140.6 (C-4a), 159.0 (C-8'), 162.2 (CONH), 169.6 (C-1 '), 170.3 (C-1``), 178.6 (C-1). EMAR (TOF ESI +): calculated for C28H33ClNO8 [M + H] + 20 546.1889, found 546.1867.
image49
Preparation of 5- (7- ((1- (tert-butoxy) - 1-oxo-3-phenylpropan-2-yl) carbamoyl) -5-chloro-
2,5-dioxopyrrolidin-1-yl-8-hydroxy-1-oxoisocromano-3-yl) pentanoate (21). A
solution of acid 20 (31.8 mg, 0.058 mmol), NHS (7.3 mg, 0.064 mmol, 1.1 equiv) and EDC HCl (13.4 mg, 0.07 mmol, 1.2 equiv) in CH3CN Dry (1.6 mL) was stirred at room temperature for 24 hours under nitrogen, after which it was diluted with AcOEt and washed successively with water, 5% aqueous NaHCO3 solution and 5 brine. After drying over anhydrous MgSO4 and removing the solvent under reduced pressure, the N-hydroxysuccinimidyl ester 21 (33.0 mg, 90%) was obtained as an oil.
Spectroscopic data of 21 (a 1: 1 mixture of diastereomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.41 (s, 9H, CMe3), 1.50-1.91 (m, 6H, H2-3, H2 -4 and H2-5), 2.67 (t, J =
7.0 Hz, 2H, H2-2), 2.83 (s, 4H, COCH2CH2CO), 2.88-2.94 (m, 1H, H-4 '), 3.14-3.24 (m, 10 2H, H2-3' '), 3.27 (dd, J = 17.2, 3.3 Hz, H'-4 '), 4.54-4.68 (m, 1H, H-3'), 4.90-5.01 (m, 1H,
H-2 ''), 7.12-7.34 (m, 5H, Ph), 8.44 (s, 1H, H-6 '), 8.55 (two dd, J = 7.4 Hz, 0.5H each, NH of each diastereomer) , 12.69 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 24.2 (C-4), 25.6 (COCH2CH2CO), 27.9 (CMe3), 29.6 (C-3), 30.6 (C-2), 30.7 (C- 4 '), 34.1 (C-5), 38.1 (C-3' '), 54.6 (C-2' '), 78.9 (C-3'), 82.3 (CMe3), 110.1 (C-8a), 120.8 15 (C-7 '), 123.1 (C-5'), 126.9 (C-4 '' '), 128.3 (C-2' '' and C-6 '' '), 129.5 (C-3' '' and C-5 '' '), 136.2 (C-
1 '' '), 138.9 (C-6'), 140.5 (C-4a), 159.0 (C-8 '), 162.1 (CONH), 168.3 (C-1), 169.1 (COCH2CH2CO), 169.6 (C- 1 '), 170.3 (C-1' '). EMAR (TOF ESI +): calculated for C32H3aClN2O10 [M + H] + 643.2053, found 643.2050.
image50
image51
OR
image52
1: 1 CF3CO2H-CH2CI2 2h, ta
image53
image54
Preparation of 2- (5-Chloro-3- (5 - ((2,5-dioxopyrrolidin-1-yl) oxy) -5-oxopentyl) -8- hydroxy-1-oxoisocroman-7-carboxamido) -3- phenylpropanoic (N- ester
hydroxy succinimidyl of hapten OTA-2, NHS-OTA-2). A solution of the tert-butyl ester 21 (33.0 mg, 0.051 mmol) and CF3CO2H (1.5 mL, 19.0 mmol, 370 equiv) in anhydrous CH2Cl2 (2.3 mL) was stirred for 2 hours at room temperature low nitrogen Then, the solvents were removed to dryness under reduced pressure to obtain NHS-OTA-2 (29.8 mg, 99%) as a residue of resinous appearance and brownish color.
Spectroscopic data of NHS-OTA-2 (a 1: 1 mixture of diastereomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.54-1.93 (m, 6H, H2-1 '', H2-2 ' 'and H2-3' '), 2.68 (t, J = 6.69 Hz, 2H, H2-4' '), 2.85 (wide s, 4H, COCH2CH2CO), 2.92 (m, 1H, H-4'), 3.17 -3.43 10 (m, 3H, H2-3 and H'-4 '), 4.61 (m, 1H, H-3'), 5.03 (m, 1H, H-2), 7.18-7.33 (m, 5H, Ph), 8.39
(s, 1H, H-6 ’), 8.56 (wide s, 1H, NH), 12.73 (wide s, 1H, OH). EMAR (TOF ESI +): calculated for C28H28ClN2O10 [M + H] + 587.1427, found 587.1425.
image55
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image56
image57
image58
image59
BSA-OTA-2
image60
2.2. Preparation of a bioconjugate of hapten OTA-2 with BSA (BSA-OTA-2,). Prepared as described above for the BSA-OTA-1 bioconjugate from 200 pL of a 50 mM DMF solution of the NHS-OTA-2 activated hapten 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 conjugated OTA-2 molecules per BSA molecule, determined by MALDI-TOF-MS, was n = 11.8 (see Table 2, entry 1).
image61
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image62
image63
image64
image65
OVA-OTA-2
image66
2.3. Preparation of a bioconjugate of hapten OTA-2 with OVA (OVA-OTA-2). Prepared as described above for the OVA-OTA-1 bioconjugate from 100 pL of a 50 mM DMF solution of the NHS-OTA-2 activated hapten 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 conjugated OTA-2 molecules per each OVA molecule, determined by MALDI-TOF-MS, was n = 10 3.1 (see Table 2, entry 2).
image67
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image68
2.4. Preparation of a bioconjugate of hapten OTA-2 with HRP (HRP-OTA-2). Prepared from 80 pL of a 5 mM solution of the NHS-OTA-2 activated hapten in DMF and 0.9 mL of a HRP solution (2.5 mg / mL) in 5 mM 50 carbonate buffer, pH 7.4 . After chromatographic purification, the fractions obtained containing the bioconjugate were brought to a concentration of 460 pg / mL in PBS buffer with 1% BSA (w / v) and thimerosal 0.02% (w / v) and stored at 4 ° C The number of conjugated OTA-2 molecules per each HRP molecule, determined by MALDI-TOF-MS, was n = 0.6 (see Table 2, entry 3).
10
TABLE 2. Values of the haptic load of the hapten OTA-2 protein conjugates determined by MALDI-TOF-MS
 RMo m / z reference protein m / z A (m / z) Am / hapten n
 one  BSA-OTA-2 24 66,428 71,982 5,554 472 11.8
 2  OVA-OTA-2 8 21,340 22,071 731 472 3.1
 3  HRP-OTA-2 8 43,928 44,200 272 472 0.6
RM0, n, A (m / z) and Am / hapten have the same meaning as in Table 1.
Example 3: Preparation of comparative bioconjugates based on protein anchoring through the OTA carboxylate group where L = CH2CH2CH2CH2CH2, Z = - (C = O) NH- and P = BSA, OVA and HRP.
3.1. Preparation of the N-hydroxysuccinimidyl ester of hapten OTA-3 (NHS-OTA-5 3).
image69
HATU, DIEA
OR
image70
Cl
image71
22
Preparation of tert-butyl 2- (5-chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) -3-phenylpropanoate (22). To a solution of acid 5 (40 mg, 0.16 mmol) in anhydrous DMF (1.1 mL) was added a solution of HATU (91.3 mg, 0.24 mmol, 1.5 10 equiv) in anhydrous DMF (1.1 mL) and DIEA (60 pL, 0.32 mmol, 2 equiv). The reaction mixture was stirred for 2 hours at room temperature and then a solution of hydrochloride 16 (82.5 mg, 0.32 mmol, 2 equiv) and DIEA (60 pL, 0.324 mmol, 2 equiv) in anhydrous DMF was added (1.1 mL). It was stirred at room temperature for 4 hours, after which the reaction mixture was diluted with AcOEt and washed successively with aqueous solutions of HCl (1M), LiCl (1.5%), NaHCO3 (5%) and brine, dried over anhydrous MgSO4 and concentrated to dryness on the rotary evaporator. The obtained residue was purified by chromatography on silica gel, using hexane-AcOEt-AcOH mixtures (100: 0: 0.3, 90: 10: 0.3 and 80: 20: 0.3) as eluent. compound 22 (67 mg, 65%) as a yellowish oil.
20 Spectroscopic data of 22 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.46 (s, 9H, CMe3), 1.62-1.64 and 1.63-1.65 (two d, J = 6.3,
I. 5H each, Me-3 'of each diastereoisomer), 2.84-2.89 and 2.90-2.95 (two dd, J =
II. 7, 3.4 Hz, 0.5H each, H-4 'of each diastereoisomer), 3.25 (m, 2H, H2-3), 3.33 (dd, J = 17.3, 3.4 Hz, 1H, H'-4'), 4.80 (m, 1H, H-3 '), 5.01 (dt, J = 7.4, 6.0 Hz, 1H, H-2), 7.20-7.38 (m, 5H, Ph), 8.49 (s, 1H, H-6 '), 8.58-8.62 (two d, J = 6.9 Hz, 0.5H each,
5 NH of each diastereoisomer), 12.77 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 20.6 (Me-3 '), 27.9 (CMe3), 32.2 (C-4'), 38.1 (C-3), 54.5 (C-2), 75.8 (C-3 '), 82.3 (CMe3), 110.0 (C-8a), 120.8 (C-7'), 123.0 (C-5 '), 126.9 (C-4' '), 128.3 (C-2' 'and C-6' '), 129.5 (C-3' 'and C-5' '), 136.2 (C-1' '), 138.9 (C-6'), 140.5 (C-4a), 159.0 ( C-8 '), 162.1 (CONH), 169.7 (C-1'), 170.3 (C-1). EMAR (TOF ESI +): calculated for C24H27CINO6 [M + H] + 460.1521, 10 found 460.1522.
image72
Preparation of 2- (5-Chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) -3-phenylpropanoic acid (23, OTA). Trifluoroacetic acid (1.27 mL, 16.28 mmol, 370 equiv) was added over a solution of ester 22 (20.5 mg, 0.045 mmol) in anhydrous CH2Cl2 (2 mL) and the mixture was kept under stirring for 2h at room temperature. The reaction mixture was concentrated to dryness under reduced pressure to obtain 23 (17.6 mg, 99%) as a cream-colored solid.
Spectroscopic data of 23 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.60 (d, J = 6.2 Hz, 3H, Me-3 '), 2.82 and 2.88 (two dd, J = 20 11.5, 3.2 Hz, 0.5H each, H-4 'of each diastereoisomer), 3.16-3.40 (m, 3H, H2-3 and
H'-4 '), 4.75 (m, 1H, H-3') 4.99-5.08 (m, 1 H, H-2) 7.10-7.37 (m, 5H, Ph) 8.42 (s, 1H, H- 6 '), 8.50 (two d, J = 6.8 Hz, 0.5H each, NH of each diastereoisomer), 12.74 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 20.6 (Me-3 ’), 32.2 (C-4’), 37.3 (C-3), 54.3
(C-2), 75.9 (C-3 '), 110.0 (C-8a), 120.2 (C-7'), 123.2 (C-5 '), 127.3 (C-4' '), 128.3 (C- 2 '' and C-6 ''), 129.3 (C-3 '' and C-5 ''), 135.7 (C-1 ''), 138.9 (C-6 '), 141.0 (C-4a), 159.0 (C-8 '), 163.1 (CONH), 169.7 (C-1'), 175.1 (C-1). EMAR (TOF ESI +): calculated for C20H19ClNO6 [M + H] + 404.0895, found 404.0901.
5
image73
Preparation of methyl 6- (2- (5-chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) -3-phenylpropanamido) hexanoate (25). On a solution of acid 23 (18 mg, 0.044 mmol) and 6-methoxy-6-oxohexan-1-ammonium chloride (24.9.4 mg, 0.052 mmol, 1.1 equiv) in anhydrous DMF (600 pL) DIEA (25 pL, 0.133 mmol, 10 3.25 equiv) was added under nitrogen, the resulting mixture was stirred for 10 minutes and then
added a solution of PyAOP (34 mg, 0.065 mmol, 1.5 equiv) in anhydrous DMF (600 pL). After 4 hours of stirring at room temperature, the mixture was diluted with AcOEt and washed with aqueous solutions of LiCl (1.5%) and NaHCO3 (5%) and brine, dried over anhydrous MgSO4. The residue obtained after evaporation of the solvent under reduced pressure was purified by chromatography on silica gel, using as eluent mixtures of CHCl3-MeOH (100: 0 and 99: 1) to obtain amide 25 (16.8 mg, 72 %) as a yellowish oil.
Spectroscopic data of 25 (a 1: 1 mixture of diastereoisomers): 1H-NMR (CDCl3, 300 MHz): 5 (ppm) 1.11-1.28 (m, 2H, H2-4), 1.31-1.44 (m, 2H, H2 -5), 1.49-1.63 20 (m, 2H, H2-3), 1.61 (two d, J = 6.4 Hz, 1.5H each, Me-3 '' 'of each of the
diastereoisomers), 2.17-2.34 (t, J = 7.4 Hz, 2H, H2-2), 2.86 (two dd, J = 11.6, 1.6 Hz, 0.5H each, H-4 '' of each diastereoisomer), 3.04 -3.37 (m, 5H, H2-6, H2-3 'and H'-
4 '' '), 3.66 (s, 3H, OMe), 4.62-4.89 (m, 2H, H-3' '' and NH), 5.83 (m, 1H, H-2 '), 7.12-7.40 (m , 5H, Ph), 8.41 (s, 1H, H-6 '' '), 8.59 (d, J = 7.4 Hz, 1H, NH), 12.81 (s, 1H, OH). 13C-NMR (CDCl3, 75 MHz): 5 (ppm) 20.7 (Me-3``), 24.4 (C-3), 26.2 (C-4), 28.9 (C-5), 32.2 (C-4 '' '), 33.8 (C-2), 38.4 (C-3'), 39.2 (C-6), 51.5 (OMe), 55.7 (C-2 '), 75.9 (C-3' ''), 5 110.1 (C-8a), 120.6 (C-7 '' '), 123.1 (C-5' '), 127.0 (C-4' '), 128.6 (C-2' 'and C-6' ' ), 129.3
(C-3 '' and C-5 ''), 136.8 (C-1 ''), 138.8 (C-6 ''), 140.8 (C-4a), 159.0 (C-8 ''), 162.7 (CONH), 169.7 (C-1 ''), 170.4 (C-1 '), 173.9 (C-1). EMAR (TOF ESI +): calculated for C27H32CIN2O7 [M + H] + 531.1893, found 531.1878.
H3CO
image74
10 Preparation of 6- (2- (5-Chloro-8-hydroxy-3-methyl-1-oxoisocromano-7-carboxamido) 3-phenylpropanamido) hexanoic acid (26). A solution of LiOH H2O (13 mg, 0.31 mmol, 10 equiv) in water (700 pL) was added over a solution of methyl ester 25 (16.6 mg, 0.031 mmol) in THF (700 pL) and the mixture stirred at room temperature for 40 minutes. Then, the reaction mixture was cooled in an ice bath and acidified with a 1M aqueous solution of KHSO4 to pH = 2, diluted with AcOEt, washed with brine and dried over anhydrous MgSO4 to obtain, after evaporating. the solvent under reduced pressure, a residue that was dissolved in anhydrous THF (1.2 mL) onto which a drop of 4M HCl in dioxane was added. After 30 min of stirring at room temperature, the solvent was removed in vacuo to dryness to obtain acid 26 (15.7 mg, 98%) as a yellowish semisolid.
Spectroscopic data of 26 (a 1: 1 mixture of diastereoisomers): 1 H-NMR (DMSO-da, 500 MHz): 5 (ppm) 1.18-1.28 (m, 2H, H2-4), 1.30-1.40 (m, 2H , H2-5), 1,421.52 (m, 2H, H2-3), 1.46-1.47 (two d, J = 6.4 Hz, 1.5H each, Me-3 '' 'of each of the diastereoisomers), 2.14-2.20 (t, J = 7.4 Hz, 2H, H2-2), 2.88-3.08 (m, 6H, H-4 '', 5 H2-3 ', H2-6 and NH), 3.22 (dd, J = 17.2, 3.1 Hz, 1H, H'-4 '' '), 4.69-4.76 (m, 1H, H-3' ''), 4.84
(m, 1H, H-2 '), 7.15-7.28 (m, 5H, Ph), 8.08 and 8.09 (two s, 0.5H each, H-6' 'of each diastereoisomer), 8.14 and 8.60 (two m, 0.5H each, NH of each diastereoisomer). 13C-NMR (DMSO-da, 125 MHz): 5 (ppm) 20.0 (Me-3``), 24.2 (C-3), 25.9 (C-4), 28.6 (C-5), 31.6 (C -4 '' '), 33.56 (C-2), 38.1 (C-3'), 38.4 (C-6), 54.5 (C-2 '), 75.4 (C-3' ''), 111.3 10 ( C-8a), 120.2 (C-7 ''), 121.4 (C-5 '' '), 126.4 (C-4' '), 128.1 (C-2' 'and C-6' '), 129.2 (C-3 '' and C-
5``), 136.0 (C-1 ''), 139.2 (C-6``), 141.6 (C-4a), 158.4 (C-8 ''), 162.54 (CONH), 169.9 (C- 1 '' '), 169.9 (C-1'), 174.3 (C-1). EMAR (TOF ESI +): calculated for C26H30CIN2O7 [M + H] + 517.1736, found 517.1731.
HO
image75
OR
H
image76
Preparation of 2,5-dioxopyrrolidin-1-yl 6- (2- (5-chloro-8-hydroxy-3-methyl-1-oxochromochano-7-carboxamido) -3-phenyl propanamide) hexanoate (W ester -hydroxysucccimidyl of hapten OTA-3, NHS-OTA-3). A solution of EDCHCl (7.0 mg, 0.037 mmol, 1.2 equiv) in anhydrous DMF (0.5 mL) was added over a solution of acid 26 (16 mg, 0.031 mmol) and NHS (3.9 mg, 0.034 mmol, 1.1 equiv) in anhydrous DMF (0.5 mL) under 20 nitrogen. The mixture was kept under stirring at room temperature for 24 hours and then treated with a saturated NH4Cl solution, diluted with AcOEt and washed with aqueous solutions of LiCl (1.5%), NaHCO3 (5%) and brine.
After drying over anhydrous MgSO4 and evaporating the solvent under reduced pressure, a residue was obtained which was purified by chromatography on silica gel, using as eluent mixtures of CH2Cl2-acetone (90:10, 85:15 and 80:20), to obtain the NHS-OTA-3 ester (8.5 mg, 45%) as yellowish oil.
5 NHS-OTA-3 spectroscopic data (a 1: 1 mixture of diastereoisomers): 1 H-NMR (DMSO-da, 500 MHz): 5 (ppm) 1.29-1.37 (m, 2H, H2-4), 1.39- 1.47 (m, 2H, H2-5), 1.60 (d, J = 6.4 Hz, 3H, Me-3 '' '), 1.64-1.78 (m, 2H, H2-3), 2.56 (t, J = 6.8 Hz, 2H, H2-2), 2.76-2.93 (m, 5H, H-4 '' ', COCH2CH2CO), 3.06-3.37 (m, 5H, H'-4' '', H2-3 'and H2- 6), 4,684.91 (m, 2H, H-3 '' 'and NH), 6.04 (m, 1H, H-2'), 7.16-7.35 (m, 5H, Ph), 8.38 (s, 1H, H-6 '' '), 10 8.60 (d, J = 7.0 Hz, 1H, NH), 12.79 (s, 1H, OH). EMAR (TOF ESI +): calculated for
C30H33N3ClO9 [M + H] + 614.1900, found 614.1892.
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image77
Cl - ■ n
BSA-OTA-3 bioconjugate
3.2. Preparation of a bioconjugate of hapten OTA-3 with BSA (BSA-OTA-3,). Prepared as described above for the BSA-OTA-1 bioconjugate from 178 pL of a 50 mM DMF solution of the NHS-OTA-3 activated hapten and 1.6 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 conjugated OTA-3 molecules per BSA molecule, determined by MALDI-TOF-MS, was n = 14.4 (see Table 3, entry 1).
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image78
Ci - ■ n
OVA-OTA-3 bioconjugate
3.3. Preparation of a bioconjugate of hapten OTA-3 with OVA (OVA-OTA-3). Prepared as described above for the bioconjugate OVA-OTA-1 from 100 pL of a 50 mM DMF solution of the activated hapten NHS-OTA-3 5 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 conjugated OTA-3 molecules per OVA molecule, determined by MALDI-TOF-MS, was n = 10 3.0 (see Table 3, entry 2).
DMF
50 mM carbonate buffer pH 9.6, 2h, t.a
image79
C! - ■ n
bioconjugate HRP-OTA-3
3.4. Preparation of a bioconjugate of hapten OTA-3 with HRP (HRP-OTA-3,). Prepared from 80 pL of a 5 mM solution of the NHS-OTA-5 3 activated hapten in DMF and 0.9 mL of a HRP solution (2.5 mg / mL) in carbonate buffer 50
mM, pH 7.4. After chromatographic purification, the fractions obtained containing the bioconjugate were brought to a concentration of 470 pg / mL in PBS buffer with 1% BSA (w / v) and thimerosal 0.02% (w / v) and stored at 4 ° C The number of conjugated OTA-3 molecules per each HRP molecule, determined by MALDI-10 TOF-MS, was n = 0.9 (see Table 3, entry 3).
TABLE 3. Values of the haptic load of the hapten OTA-3 protein conjugates determined by MALDI-TOF-MS
 RMo m / z reference protein m / z A (m / z) Am / hapten n
 one  BSA-OTA-3 24 66,428 73,642 7,214 500 14.4
 2  OVA-OTA-3 8 21.340 # 22.079 # 739 500 3.0
 3  HRP-OTA-3 8 43,928 44,382 454 500 0.9
RM0, n, A (m / z), Am / hapten and # have the same meaning as in Table 1.
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2. ELISA procedure
96-well polystyrene plates were used. Each antibody was evaluated in the two classic competitive ELISA formats (the one for antigen or conjugate immobilized with indirect detection and the one for immobilized antibody with direct and pre-capped detection) using homologous test antigens, that is, a test antigen from it. bioconjugate of formula (I) or comparative bioconjugate than that used to obtain the immunogen but in which P = OVA or HRP. After each incubation step, 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 standard curves obtained by representing absorbance versus analyte concentration were adjusted to a 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 that is a bioconjugate of formula (I) or comparative bioconjugate where P is OVA, at various concentrations in 50 mM carbonate buffer, pH 9.6, by incubation during 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 led to
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out for 1 h at room temperature and then the plates were washed. Then, 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 a dilution of capture antibody in 50 mM carbonate buffer, pH 9.6, by incubation overnight at room temperature. After washing the plates, 100 pL per well of antibody or antiserum in PBST was added at the concentration or dilution considered optimal, and incubated at room temperature for 1 h. After washing the plates again, in each column 50 pL per well of a complete standard analyte curve in PBS was dispensed followed by 50 pL per well of a specific dilution in PBST of enzymatic bioconjugate which is a bioconjugate of formula (I) or comparative bioconjugate where P is HRP. 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.
3. Immunization of rabbits
Two rabbit females of the New Zealand breed were immunized following standardized protocols with each bioconjugate of formula (I) or comparative bioconjugate where P is BSA. Each animal received 0.3 mg of one of the bioconjugates of formula (I) or the comparative bioconjugate dissolved in 1 mL of a 1: 1 mixture of PB buffer and complete Freund's adjuvant. Immunization continued with the inoculation of a booster dose every 21 days with the same amount of conjugate but using incomplete Freund's adjuvant. Ten days after the fourth injection, the animals were bled and the blood obtained was allowed to clot at 4 ° C overnight. The next day, sera were recovered by centrifugation, diluted to% with cold PBS and a volume of a saturated solution of
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ammonium sulfate The resulting protein precipitate from each serum was collected by centrifugation and redissolved in cold PBS buffer. Finally, the proteins were reprecipitated as before and stored in this state at 4 ° C. This precipitate contains an indeterminate mixture of proteins that we call antiserum, polyclonal antibody or simply antibody. Two antibodies were obtained from each bioconjugate of formula (I) and the comparative bioconjugate where P is BSA, identified as # 1 and # 2.
4. Production of mouse monoclonal antibodies
4.1. Mouse immunization
For immunization, the bioconjugates of formula (Ia) and (Ib) in which P is BSA (immunizing conjugates) obtained in the previous examples were used. 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 was administered intraperitoneally per mouse, 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 mice received a final dose of 100 pg of the corresponding bioconjugate diluted in PBS.
4.2. Cellular fusions to obtain hybridomas
Mergers with immunized mice were basically carried out 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 plunger of a sterile syringe. After lysing the red blood cells by osmotic shock with 1 mL of lysis buffer for one minute in cold, the lymphocytes
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they were washed twice with complete medium (with cold serum) and filtered to remove the formed clots.
The myeloma line P3-X63-Ag8.653 was cultured the days prior to fusion in supplemented DMEM 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 on the day of fusion a sufficient number of them were available.
After two washes with serum-free medium, both cell populations joined a lymphocyte: myeloma 4: 1 ratio. Next, the cells were centrifuged, to immediately then carry out cell fusion. For this, the chemical fusing agent PEG1500 (1 mL per spleen, 1 min) was used, which partially dissolves 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 ° C 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).
4.3. Selection, cloning and conservation of hybridomas
Approximately 10-12 days after cell fusion, the evaluation of supernatants from seeded wells was carried out, in order to identify which ones contained antibody secreting hybridomas capable of recognizing ochratoxin A both in its conjugated and free form (competing clones) . Previously, fusion efficiency was determined by visual inspection, defined as the percentage of wells that had at least one clone clearly visible under a microscope.
To carry out the identification of competing clones, the culture supernatants were analyzed using the differential ELISA technique, which consists in analyzing
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in parallel in adjacent wells each supernatant in the absence of analyte and in the presence of a predetermined concentration of analyte, usually 100 nM. For 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.
Next, those wells containing hybridoma producing antibodies capable of providing an absorbance signal equal to or greater than 0.5 in the assay in the absence of ochratoxin A and inhibition of the signal equal to or greater than 80% in the assay were selected presence of ochratoxin 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 more safely. For this, the supernatant of each hybridoma was tested at 4 dilutions (1/8, 1/32, 1/128 and 1/512) on upholstered plates with the homologous bioconjugate at 0.01 and 0.1 pg / mL, and using as a competitor ocratoxin A at 10 and 100 nM (in trial). Thus, 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 ocratoxin 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, planting from each well a new 96-well plate at 2 cells per well in HT medium, of the same composition as 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 performed 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 growing the hybridoma in larger containers
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volume. 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 ° C inside a polystyrene box for 24 h before passing them to the liquid nitrogen container.
4.4. 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.
Purification of the antibodies was performed by affinity chromatography with G protein 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 ° C. The remaining solution was precipitated with saturated ammonium sulfate [1: 1, (v / v)], which guarantees its stability at 4 ° C for years.
5. Results
5.1. Immune response and determination of affinity of antisera
Each of the obtained antisera was tested against its homologous test antigen 5 using the competitive ELISA type assay in the antibody format
immobilized. Different concentrations of test antigen against different antibody concentrations were tested using as a competitor several concentrations of ochratoxin A prepared by serial dilution. The three immunogens produced adequate immune responses, with high and usual titers for this type of analyte. However, while the antisera
from animals immunized with the immunogens of formula Ia and Ib had a high affinity towards ocratoxin A, with IC50 values close to or less than 1 nM, the antisera obtained from the comparative immunogen showed clearly lower affinities towards ochratoxin A (higher IC50) . This result confirms that some structures are more suitable than others for the purpose
which is pursued, and that the haptens functionalized through alternative positions to the carboxylate group of ochratoxin A, and therefore leaving said group free, give rise to antibodies of greater affinity towards the target mycotoxin. The values of the maximum signal, the IC50 and the slope of the resulting inhibition curve 20 for each antiserum with homologous test antigen have been included in Table 4.
TABLE 4 Result of tests in competitive ELISA antiserum format
immobilized with direct detection
 Antiserum &  Tracer (ng / mL) Dilution As. (X103) A ^ max Slope IC50 (nM)
 Ia # 1  10 36 1,632 0.541 0.735
 Ia # 2  30 36 1,472 0.442 1,889
 Ib # 1  30 12 1,523 0.408 0.445
 Ib # 2  10 36 1,050 0.709 0.361
 Comp. # 1  10 12 1,574 0,394 9,842
 Comp. # 2  100 36 1,225 0,359> 100
& Rabbit antisera; immunization with the conjugates BSA-OTA-1 (la), BSA-OTA-2 (Ib) and BSA-OTA-3 (Comp.),
5.2. Generation of hybridomas producing monoclonal antibodies against ocratoxin-A
5 To demonstrate more conclusively the suitability of the bioconjugates of formula (Ia) and (Ib) for obtaining anti-ocratoxin A antibodies, mice were immunized with both conjugates of formula (Ia) and (Ib) in which P is BSA and cell fusions aimed at hybridoma generation were carried out. It was possible to obtain 4 monoclonal antibody producing cell lines with high 10 affinity towards ocratoxin A from the bioconjugate of formula (Ia), and 7 cell lines from the bioconjugate of formula (Ib). Monoclonal antibodies obtained from said hybridomas have been named for the purposes of the present invention and the examples included herein: mAb Ia # 39, mAb Ia # 41, mAb Ia # 310a and mAb Ia # 310b (monoclonal antibodies from bioconjugate of formula Ia); and 15 mAb Ib # 16, mAb Ib # 21, mAb Ib # 27, mAb Ib # 111, mAb Ib # 114, mAb Ib # 115 and mAb Ib # 118 (monoclonal antibodies from the bioconjugate of formula Ib). These results show that the bioconjugates of formula (Ia) and (Ib) are suitable for obtaining very high affinity monoclonal antibodies against ocratoxin A.
5.3. Determination of antibody affinity
Once the 11 monoclonal antibodies obtained were purified by immunoaffinity chromatography, their affinity towards ocratoxin A was determined by direct homologous competitive ELISA 5. Each monoclonal antibody was tested at various concentrations (100, 300 and 1000 ng / mL) against different concentrations of enzyme tracer (300, 100, 30 and 10 ng / mL), which is a labeled derivative of formula II where Q is peroxidase . The Amax, slope and IC50 values for each monoclonal antibody shown in Table 5 correspond to the optimal combination, that is, the concentration of immunoreactive agents generated by the calibration curve with a lower IC50 value, and therefore a higher affinity for ocratoxin A. In said test format the antibodies showed IC50 values for ocratoxin A between 0.071 and 1,457 nM, the antibody with the highest affinity being among those produced from the bioconjugate of formula Ia mAb 15 Ia # 310b (IC50 = 0.071 nM), and the antibody with the highest affinity among those produced at
from the bioconjugate of formula Ib the mAb Ib # 115 (IC50 = 0.169 nM).
TABLE 5 Result of tests in competitive ELISA format
immobilized antibody homolog with direct detection
 Monoclonal antibody  Tracer (ng / mL) mAb (ng / mL) A ^ max Slope IC50 (nM)
 Ia # 38  10 100 1,880 1,309 0.504
 Ia # 39  30 100 1,907 0,891 1,457
 Ia # 310a  30 100 1,013 1,116 0,088
 Ia # 310b  30 100 1,054 1,324 0,071
 Ib # 16  30 100 1,739 2,003 0,397
 Ib # 25  30 100 1,639 1,468 0,394
 Ib # 27  30 100 1,830 1,128 1,114
 Ib # 111  30 100 1,679 2,219 0,321
 Ib # 114  30 100 1,958 1,442 0.623
 Ib # 115  100 100 1,245 1,277 0,169
 Ib # 118  100 100 2,499 1,646 0,357

权利要求:
Claims (22)
[1]
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1. A bioconjugate of formula (I):
[T-L-Z] n-P
(I)
where
T is selected from the group consisting of R-I and R-II;
image 1
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 (Cr alkyl
Ca) -, -S-, -S-S-, -NH-NH-, -N = C- -C = N-, -NH (C = NH) -, -N = N-, -O-,
- (
S
image2
Y
N = N
image3
P is a natural or synthetic peptide or polypeptide of molecular weight greater than 2000 daltons where the peptide or polypeptide P may or may not be linked, by covalent, electrostatic or other interaction, to a support and where said support can 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 1 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-,
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Y
image5
[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)
image6
20 where P is selected from the group consisting of albumin or peroxidase, and n is a value selected from 1 to 50.
[6]
6. The bioconjugate of formula (I) according to any of claims 1 to 4, with the formula (Ib)
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image7
where P is selected from the group consisting of albumin or peroxidase, and n is a value selected between 1 and 50.
[7]
7. A labeled derivative of formula (II):
[T-L-Z] m-Q
(II)
where
T, L and Z are as defined in claim 1; Q is a detectable non-isotopic marker and m is a number with a value between 1 and 1000.
[8]
8. The labeled derivative of formula (II) according to claim 7, wherein Q is selected from the group consisting of enzymes, biotin, fluorescein or any one of its derivatives, 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 micro- or nanoparticles of colloidal gold, carbon or latex.
[9]
9. The labeled derivative of formula (II) according to any one of claims 7 or 8 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-
image8
_ Y
image9
[10]
10. The labeled derivative of formula (II) according to any one of claims 7 to 9 characterized in that L is a linear hydrocarbon chain of 1 to 20 atoms
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of carbon and said hydrocarbon chain comprises between 0 and 4 heteroatoms selected from the group consisting of O and N.
[11]
11. The labeled derivative of formula (II) according to any of claims 7 to 10, with the formula (Ila)
image10
(lla)
where Q is selected from the group consisting of peroxidase, biotin, fluorescein 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 7 to 10, with formula (IIb)
image11
(IIb)
where Q is selected from the group consisting of peroxidase, biotin, fluorescein or nanoparticles, and m is a value selected between 1 and 10.
[13]
13. An antibody that specifically recognizes a bioconjugate as described in any one of claims 1 to 6.
[14]
14. The antibody according to claim 13, wherein the antibody is selected from monoclonal, polyclonal and recombinant.
[15]
15. Use of a bioconjugate as described in any one of claims 1 to 6 for obtaining an antibody.
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35
[16]
16. Method of in vitro analysis of ocratoxin A in a sample comprising the use of an antibody as described in any one of claims 13 or 14.
[17]
17. Method according to claim 16 comprising the following steps:
a) contacting the sample with the antibody described in any of claims 13 or 14;
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 step (b).
[18]
18. A method according to claim 17, wherein the determination of the immunochemical reaction in step (c) is performed by a competitive assay, using as a competitor a bioconjugate as described in any one of claims 1 to 6.
[19]
19. Method according to claim 17, wherein the determination of the immunochemical reaction in step (c) is performed by a competitive assay, using as a competitor a labeled derivative as described in any of claims 7 to 12.
[20]
20. Ochratoxin A detection and / or determination kit, comprising at least one antibody as described in any of claims 13 or 14 together with a bioconjugate described in any one of claims 1 to 6 or together with a labeled derivative described in any of claims 7 to 12.
[21]
21. Method of purification and / or concentration of ochratoxin A of a sample comprising the use of an antibody as described in any one of claims 13 or 14.
[22]
22. Method according to claim 21 performed by affinity chromatography comprising the following steps:
a) immobilizing at least one antibody described in any of claims 13 or 14 on a support;
b) passing the sample through said support to retain the ochratoxin A present in said sample; Y
c) elute the retained ochratoxin A in the support.

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同族专利:

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公开号 | 公开日

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ES2672945B1|2019-04-26|

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WO2018091756A1|2018-05-24|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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US8003766B2|2009-04-17|2011-08-23|Chung Shan Medical University|Monoclonal antibody specific to ochratoxin A|

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CN103575886B|2012-07-20|2016-09-21|北京勤邦生物技术有限公司|The enzyme linked immunological kit of detection ochratoxin A and application thereof|

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CN104569399B|2013-10-22|2016-09-21|北京勤邦生物技术有限公司|A kind of test strips detecting ochratoxin A and application thereof|CN110850091A|2019-11-13|2020-02-28|中国科学院生态环境研究中心|Fluorescent probe and reagent set for detecting ochratoxin A|

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ES201631470A|ES2672945B1|2016-11-17|2016-11-17|PREPARATION OF NEW BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF OCRATOXIN A|ES201631470A| ES2672945B1|2016-11-17|2016-11-17|PREPARATION OF NEW BIOCONJUGATES AND ANTIBODIES FOR THE IMMUNODETECTION OF OCRATOXIN A|

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PCT/ES2017/070759| WO2018091756A1|2016-11-17|2017-11-17|Preparation of new bioconjugates and antibodies for the immunodetection of ochratoxin a|